US20110183855A1

US20110183855A1 - Somatic hypermutation systems

Info

Publication number: US20110183855A1
Application number: US12/972,328
Authority: US
Inventors: Robert A. Horlick; Andrew B. Cubitt; Peter M. Bowers
Original assignee: Anaptysbio Inc
Current assignee: Anaptysbio Inc
Priority date: 2007-02-20
Filing date: 2010-12-17
Publication date: 2011-07-28
Also published as: EP2126087A4; US20140094392A1; EP2126105A4; NZ579010A; US20090093024A1; JP5675109B2; US20110287485A1; BRPI0807952A2; EP2126087A1; JP2010520748A; WO2008103475A1; US20120028301A1; US20140170705A1; US8603950B2; WO2008103474A1; US9637556B2; JP2010518859A; EP2126105A1; CA2678451A1; US8685897B2

Abstract

The present application relates to somatic hypermutation (SHM) systems and synthetic genes. Synthetic genes can be designed using computer-based approaches to increase or decrease susceptibility of a polynucleotide to somatic hypermutation. Genes of interest are inserted into the vectors and subjected to activation-induced cytidine deaminase to induce somatic hypermutation. Proteins or portions thereof encoded by the modified genes can be introduced into a SHM system for somatic hypermutation and proteins or portions thereof exhibiting a desired phenotype or function can be isolated for in vitro or in vivo diagnostic or therapeutic uses.

Description

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional Application No. 60/902,414 (Attorney docket no. 33547-705.101), filed Feb. 20, 2007, U.S. Provisional Application No. 60/904,622 (Attorney docket no. 33547-706.101), filed Mar. 1, 2007, U.S. Provisional Application No. 61/020,124 (Attorney docket no. 33547-706.102), filed Jan. 9, 2008, and U.S. Provisional Application No. 60/995,970 (Attorney docket no. 33547-708.101), filed Sep. 28, 2007, each of which applications is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

The market for the use of recombinant protein therapeutics has increased steadily for the last quarter century. In 2005, six of the top 20 drugs were proteins, and overall, biopharmaceutical drugs accounted for revenues of approximately $40 billion, of which approximately $17 billion was based on the sales of monoclonal antibodies.
Monoclonal antibodies represent a distinct class of biotherapeutics with a great deal of promise. The antibody scaffold is well tolerated in the clinic, and glycosylated IgG molecules have favorable pharmacokinetic and pharmacodynamic properties. There is the potential for rapidly selecting new drug candidates that vary little from currently marketed drugs. Issues relating to non-mechanism based toxicity, and the manufacturing and formulation of antibody products are known and consistent across the therapeutic group, which reduces the potential failure rate associated with this class of drug candidates as compared to small molecule therapeutics.
In contrast to traditional small molecule based approaches, therapeutic antibodies have significant advantages, including the facts that (i) they can be generated and validated quickly; (ii) they exhibit fewer side effects and have improved safety profiles, (iii) they have well understood pharmacokinetic characteristics, and can be optimized to create long half-life products with reduced dosing frequency; iv) they are versatile and exhibit flexibility in drug function; v) scale-up and manufacturing processes are robust and well-understood; vi) they have a proven track record of clinical and regulatory success; and vii) the current regulatory environment makes the introduction of competitive generic, or bioequivalent, antibodies both difficult and costly.
Even given the success of monoclonal antibodies, the antibody-as-drug modality is continuing to evolve, and subject to inefficiency. Further, intrinsic biological bias within the native immune system often works against the more rapid development of improved therapeutics. These limitations include, i) the long development time for the isolation of biologically active antibodies with affinity constants of therapeutic caliber, ii) the inability to raise antibodies to certain classes of protein targets (intractable targets), and iii) the intrinsic affinity ceiling inherent in immune system based affinity selection.
Specifically there is a need for methods to more rapidly develop antibodies with improved pharmacokinetics, cross-reactivity, safety profiles and superior dosing regimens. Central to this need is the development of methods that enable the systematic analysis of potential epitopes with a protein, and enable the selective development of antibodies with the desired selectivity profiles.
There are several existing well-established methods of developing monoclonal antibodies; however, many of these technologies have specific disadvantages that limit their ability to rapidly evolve the best clinical candidates. These technological limitations include: i) mouse immunization and hybridoma technology cannot be used iteratively and often fails to yield an antibody with desired characteristics due to antigen intractability; ii) phage display or panning often fails to yield monoclonal antibodies with affinity constants of therapeutic caliber, and cannot easily be used to select and co-evolve entire heavy and light chains; and iii) rational design strategies often provides an incomplete solution, and are based solely on existing knowledge.
An approach used includes the use of random or semi-random mutagenesis (for example the use of error prone PCR), in conjunction with in vitro molecular evolution. This approach is based on the creation of random changes in protein structure and the generation of large libraries of mutant polynucleotides that are subsequently screened for improved variants, usually through the expression of the encoded proteins within a living cell. From these libraries, a few improved proteins can be selected for further optimization.
Such in vitro mutation approaches can be limited by the inability to systematically search a portion of any given sequence, and by the relative difficulty of detecting very rare improvement mutants out of a large number of mutations. This fundamental problem arises because the total number of possible mutants for a reasonably sized protein is large. For example, a 100 amino acid protein has a potential diversity of 20¹⁰⁰different sequences of amino acids, while existing high throughput screening methodologies are, in some cases, limited to a maximum screening capacity of 10⁷-10⁸samples per week. Additionally, such approaches are relatively inefficient because of redundant codon usage, in which up to around 3¹⁰⁰of the nucleotide sequences possible for a 100 amino acid residue protein actually encode for the same amino acids and protein, (Gustafsson et al. (2004) Codon Bias and heterologous protein expression Trends. Biotech. 22 (7) 346-353).
A more sophisticated approach uses a mixture of random mutagenesis with recombination between protein domains in order to select for improved proteins (Stemmer Proc. Natl. Acad. Sci. (1994) 91 (22) 10747-51). This approach exploits natural design concepts inherent in protein structures across families of proteins, but again requires significant recombinant DNA manipulation and screening capacity of a large number of sequences to identify rare improvements. Both approaches require extensive follow-up mutagenesis and analysis to understand the significance of each mutation, and to identify the best combination of the many thousands or millions of mutants identified.

SUMMARY OF THE INVENTION

The present invention is based on the development of a system to design and make or generate SHM susceptible and SHM resistant DNA sequences, within a cell or cell-free, environment. The present invention is further based on the development of a SHM system that is stable over a suitable time period to reproducibly maintain increased and/or decreased rates of SHM without affecting structural portions or polypeptides or structural proteins, transcriptional control regions and selectable markers. The system allows for stable maintenance of a mutagenesis system that provides for high level targeted SHM in a polynucleotide of interest, while sufficiently preventing non-specific mutagenesis of structural proteins, transcriptional control regions and selectable markers.
In part, the present system is based upon the creation of a more stable version of activation induced cytidine deaminase (AID) that can provide for high level sustained SHM.
In the present application, in vitro somatic hypermutation (SHM) systems involve the use of in vitro SHM in conjunction with directed evolution and bioinformatic analysis to create integrated systems that include, but are not limited to, optimized, controlled systems for codon design and usage, library design, screening, selection and integrated systems for the data mining. These systems include:
I. An expression system designed to create SHM susceptible and or SHM resistant DNA sequences, within a cell or cell-free, environment. The system enables the stable maintenance of a mutagenesis system that provides for high level targeted SHM in a polynucleotide of interest, while significantly preventing non-specific mutagenesis of structural proteins, transcriptional control regions and selectable markers.
II. Polynucleotide libraries that are focused in size and specificity, and are enriched for those functions of interest and are efficient substrates for SHM to seed in situ diversity creation upon exposure to AID.
III. A process based on computational analysis of protein structure, intra-species and inter-species sequence variation, and the functional analysis of protein activity for selecting optimal epitopes that provide for the selection of antibodies with superior selectivity, cross species reactivity, and blocking activity.
Provided herein is a method to design polynucleotide sequences to either maximize or minimize the tendency of a polynucleotide to undergo SHM, while at the same time maximizing protein expression, RNA stability, and the presence of conveniently located restriction enzyme sites.
Also provided herein are synthetic or semi-synthetic versions of a polynucleotide that are optimized to either enhance, or decrease the impact of SHM on the rate of mutagenesis of that polynucleotide compared to its wild type's susceptibility to undergo SHM (i.e., SHM susceptible or SHM resistant).
SHM susceptible sequences enable the rapid evolution of polynucleotides which are designed based on codon usage to be more susceptible to SHM; optimized polynucleotides can be exposed to AID and expressed as polypeptides. Conversely, SHM resistant sequences enable the rapid evolution of polynucleotides which are designed based on codon usage to be less susceptible (resistant) to SHM; optimized polynucleotides can be exposed to AID and expressed as polypeptides. Modified versions of the encoded polypeptides can be selected for improved function or increased stability and resistance to SHM.
The system described herein combines the power of rational design with accelerated random mutagenesis and directed evolution.
Also included in the invention are SHM resistant polynucleotide sequences that enable important conserved domains to be spared from mutagenesis, while simultaneously targeting desired sequences.
Polynucleotides for which these methods are applicable include any polynucleotide sequence that can be transcribed and a functional activity devised for screening.
The overall result of the integration of these approaches is an integrated system for creating targeted diversity in situ, and for the automated analysis and selection of proteins with improved traits.
In certain embodiments, the present invention is based in part on an improved understanding of the context of multiple rounds of SHM within the reading frame of a polynucleotide sequence, and the underlying logic relationships of codon usage patterns.
Provided herein is a SHM susceptible synthetic gene encoding a protein, or a portion thereof, wherein one or more first SHM motifs in an unmodified polynucleotide sequence encoding said protein or portion thereof has been replaced by one or more second SHM motifs having a higher probability of SHM, wherein said SHM susceptible synthetic gene exhibits a higher rate of activation induced cytidine deaminase (AID)-mediated mutagenesis compared to said unmodified polynucleotide sequence. Preferred hot spot SHM codons or preferred hot spot SHM motifs are for, example, a codon including, but not limited to codons AAC, TAC, TAT, AGT and AGC. Such sequences may be potentially embedded within the context of a larger SHM motif, recruit SHM mediated mutagenesis and generate targeted amino acid diversity at that codon.
The present invention also contemplates that a SHM susceptible synthetic gene can be created in a step-wise or sequential fashion such that some modifications are made to the gene and then a subsequent round of modification is made to the gene. Such sequential or step-wise modifications are contemplated by the present invention and are one way of carrying out the process and one way of producing the genes claimed herein.
In one embodiment, the SHM susceptible synthetic gene encodes a protein or portion thereof having about 99%, about 95%, about 90% amino, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, or any percentage between about 50% and about 100% identity to an unmodified gene.
In one embodiment, the SHM susceptible synthetic gene exhibits a higher rate of AID-mediated mutagenesis including, but not limited to, 1.05-fold, 1.1-fold, 1.25-fold, 1.5-fold, 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold or more, or any range therebetween.
In one embodiment, the SHM susceptible synthetic gene exhibits a rate of AID-mediated mutagenesis at a level which is at least about 101%, at least about 105%, at least about 110%, at least about 115%, at least about 120%, at least about 125%, at least about 130%, at least about 135%, at least about 1140%, at least about 145%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 450%, at least about 5000%, or higher of that exhibited by an unmodified gene.
In one embodiment, provided herein is a SHM susceptible gene encoding a protein or a portion thereof wherein one or more first SHM motifs in an unmodified polynucleotide sequence encoding said protein or portion thereof has been replaced by one or more second SHM motifs having a higher probability of SHM, said synthetic gene having a greater density of hot spot motifs than said unmodified polynucleotide sequence.
In yet another non-limiting aspect, the said synthetic gene includes one or more amino acid mutations that introduce preferred SHM hot spot motifs.
Provided herein is a SHM resistant synthetic gene encoding a protein, or a portion thereof, wherein one or more first SHM motifs in an unmodified polynucleotide sequence encoding said protein or portion thereof has been replaced by one or more second SHM motifs having a lower probability of SHM, wherein said SHM resistant synthetic gene exhibits a lower rate of AID-mediated mutagenesis compared to said unmodified polynucleotide sequence.
The present invention also contemplates that a SHM resistant synthetic gene can be created in a step-wise or sequential fashion such that some modifications are made to the gene and then a subsequent round of modification is made to the gene. Such sequential or step-wise modifications are contemplated by the present invention and are one way of carrying out the process and one way of producing the genes claimed herein.
In one embodiment, the SHM resistant synthetic gene encodes a protein or portion thereof having about 95%, about 90% amino, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, or any percentage between about 50% and about 100% identity to an unmodified gene.
In one embodiment, the SHM resistant synthetic gene exhibits a lower rate of AID-mediated mutagenesis including, but not limited to, 1.05-fold, 1.1-fold, 1.2-fold, 1.5-fold, 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold or less, or any range therebetween.
In one embodiment, the SHM resistant synthetic gene exhibiting a rate of AID-mediated mutagenesis at a level which is less than about 99%, less than about 95%, less than about 90%, less than about 85%, less than about 80%, less than about 75%, less than about 70%, less than about 65%, less than about 60%, less than about 55%, or less than about 50%, of that exhibited by an unmodified gene.
In another embodiment, provided herein is a SHM resistant synthetic gene encoding a protein or a portion thereof wherein one or more first SHM motifs in an unmodified polynucleotide sequence encoding said protein or portion thereof has been replaced by one or more second SHM motifs having a lower probability of SHM, said synthetic gene having a greater density of cold spots than said unmodified polynucleotide sequence.
Provided herein is a selectively targeted, SHM optimized synthetic gene encoding a protein, or a portion thereof, wherein one or more first SHM motifs in an unmodified polynucleotide sequence encoding said protein or portion thereof has been replaced by one or more second SHM motifs having a higher probability of SHM; and one or more third SHM motifs in said unmodified polynucleotide sequence encoding said protein or portion thereof has been replaced by one or more fourth SHM motifs having a lower probability of SHM; wherein said selectively targeted, SHM optimized synthetic gene exhibits targeted AID-mediated mutagenesis. In such an embodiment, the selectively targeted, SHM optimized synthetic gene has portions that exhibit a higher rate of AID-mediated mutagenesis and other portions that exhibit a lower rate of AID-mediated mutagenesis.
In yet another embodiment, provided herein is a selectively targeted, SHM optimized synthetic gene encoding a protein or portion thereof, wherein one or more first SHM motifs in an unmodified polynucleotide sequence encoding said protein or portion thereof has been replaced by one or more second SHM motifs having a higher probability of SHM, said synthetic gene having a greater density of hot spot motifs than said unmodified polynucleotide sequence; and one or more third SHM motifs in said unmodified polynucleotide sequence encoding said protein or portion thereof has been replaced by one or more fourth SHM motifs having a lower probability of SHM, said synthetic gene having a greater density of cold spots than said unmodified polynucleotide sequence; wherein said selectively targeted, SHM optimized synthetic gene exhibits targeted AID-mediated mutagenesis. In one embodiment, the selectively targeted, SHM optimized synthetic gene has portions that exhibit a higher rate of AID-mediated mutagenesis and other portions that exhibit a lower rate of AID-mediated mutagenesis.
The present invention also contemplates that a selectively targeted SHM optimized synthetic gene can be created in a step-wise or sequential fashion such that some modifications are made to the gene and then a subsequent round of modification is made to the gene. Such sequential or step-wise modifications are contemplated by the present invention and are one way of carrying out the process and one way of producing the genes claimed herein.
In one non-limiting aspect, the synthetic gene includes one or more conservative or semi-conservative amino acid mutations to modulate hot spot or cold spot density and said synthetic gene encodes a protein or portion thereof having about 90% or greater amino acid sequence identity compared to said unmodified gene.
In another non-limiting aspect, the synthetic gene includes one or more conservative or semi-conservative amino acid mutations to modulate hot spot or cold spot density, and said synthetic gene encodes a protein or portion thereof having about 70% or greater amino acid sequence identity compared to said unmodified gene.
In another non-limiting aspect, the synthetic gene includes one or more conservative or semi-conservative amino acid mutations to modulate hot spot or cold spot density, and said synthetic gene encodes a protein or portion thereof having about 50% or greater amino acid sequence identity compared to said unmodified gene.
In yet another non-limiting aspect, the said synthetic gene includes one or more amino acid mutations that introduce preferred SHM hot spot motifs.
In one non-limiting example, a synthetic gene does not include genes comprising a stop motif inserted into an open reading frame.
In one aspect, a protein or portion thereof encoded by a synthetic gene is selected from among, but not limited to, antibodies or antigen-binding fragments thereof, selectable markers, reporter proteins, cytokines, chemokines, growth factors, hormones, neurotransmitters, hormones, cytokines, chemokines, enzymes, receptors, structural proteins, toxins, co-factors and transcription factors.
Provided herein is an expression vector, comprising at least one synthetic gene. In one aspect, the expression vector is an integrating expression vector. When the expression vector is an integrating expression vector, the expression vector can further comprise one or more sequences to direct recombination. In another aspect, the expression vector is an episomal expression vector. In yet another aspect, the expression vector is a viral expression vector.
Provided herein is a eukaryotic cell comprising a synthetic gene as described herein. In one aspect, the eukaryotic cell is a mammalian cell or a yeast cell.
Provided herein is a prokaryotic cell comprising a synthetic gene as described herein. In one aspect, the prokaryotic cell is an Escherichia coli cell.
Provided herein is a method for preparing a gene product having a desired property, comprising: a) preparing a synthetic gene encoding a gene product which exhibits increased SHM; b) expressing said synthetic gene in a population of cells; wherein said population of cells express AID, or can be induced to express AID via the addition of an inducing agent; and c) selecting a cell or cells within the population of cells which express a mutated gene product having the desired property. In one aspect, the method, optionally, further comprises activating or inducing the expressing AID in said population of cells. In another aspect, the method, optionally, further comprises establishing one or more clonal populations of cells from the cell or cells identified in (c). In yet another aspect of the method, at least one synthetic gene is located in an expression vector such as any one of the vectors described elsewhere herein. In one aspect of the method, the cell is a cell as described elsewhere herein.
Provided herein is a method for preparing a gene product having a desired property, comprising: a) expressing said gene product in a population of cells; wherein said population of cells comprises at least one synthetic gene which exhibits decreased SHM; and wherein said population of cells express an AID, or can be induced to express AID via addition of an inducing agent; and b) selecting a cell or cells within the population of cells which express a mutated gene product (a polypeptide encoded by the mutated synthetic gene, the gene having one or more mutations) having the desired property. In one aspect, the method, optionally, further comprises activating or inducing the expressing AID in said population of cells. In another aspect, the method, optionally, further comprises establishing one or more clonal populations of cells from the cell or cells identified in (b). In yet another aspect of the method, at least one synthetic gene is located in an expression vector such as any one of the vectors described elsewhere herein. In one aspect of the method, the cell is a cell as described elsewhere herein.
Provided herein is an in vitro hypermutation system, comprising: a) a polynucleotide comprising a synthetic gene; b) a recombinant AID; and c) an in vitro expression system. The in vitro system can further comprise a polymerase to amplify nucleic acids after transcription. The in vitro system can further comprise an in vitro translation system. In one aspect, the polynucleotide is located in an expression vector such as any one of the vectors described elsewhere herein. The in vitro system can further comprise a cell population of a cell as described elsewhere herein.
Provided herein is a kit for in vitro mutagenesis, comprising: a) a recombinant AID protein; b) one or more reagents for in vitro transcription; and c) instructions for design or use of a synthetic gene. The kit can further comprise one or more reagents for in vitro translation. The kit can further comprise comprising an expression vector such as, for example, any one of the expression vectors as described herein. The kit can further comprise a cell population of a cell as described elsewhere herein.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

A better understanding of the features and advantages of the present invention can be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

FIG. 1 and FIG. 2 show the 20 most common codon transitions observed in complementarity determining regions (CDRs) and framework regions (FWs or FRs) during SHM-mediated affinity maturation and demonstrate how simple frame shifts can determine the two radically different patterns of mutagenesis seen in CDRs and FWs. These observations lead directly to a hypothesis that both functional selection during affinity maturation and the reading frame context determines the amino acid diversity generated at SHM hot spot codons.

FIG. 1 Shows that within CDRs, (the codons AGC, TAT, and TAC which encode tyrosine and serine amino acids), feed a directed flow of primary, secondary and tertiary SHM events generating amino acid diversity. Within CDRs, the most common codon transition observed is AGC to AAC (785 instances), leading to a serine to asparagine conversion. While that transition is also common in framework regions (354 instances), a simple frame shift of the same mutation in the same hotspot motif ( . . . TACAGCTAT . . . ; SEQ ID NO: 42) context leads to a CAG to CAA silent mutation that is common in framework regions (288 instances) but not commonly observed in CDRs.

FIG. 2 Shows that in contrast to FIG. 1, the most commonly observed codon (amino acid) transition events in frame work regions generate silent mutations.

FIG. 3 Provides all of the motifs encoded by the WAC library, given in any of the three possible reading frames, produce a concatenation of hot spots and compares these motifs with all other possible 4096 6-mer nucleotide combinations for their ability to recruit SHM-mediated machinery. Longer assemblies result in the same high density of SHM “hot spots” with no “cold spots.” This assembly of degenerate codons (WACW) results in a subset of possible 4-mer hot spots described by Rogozin et al. (WRCH), where R=A or G, H=A or C or T, and W=T or A.

FIG. 4 Preferred SHM hot spot codons AAC and TAC, which are the basis for a synthetic library, can result in a set of primary and secondary mutation events that create considerable amino acid diversity, as judged by equivalent SHM mutation events observed in Ig heavy chains antibodies. From these two codons, basic amino acids (histidine, lysine, arginine), an acidic amino acid (Aspartate), hydrophilic amino acids (serine, threonine, asparagine, tyrosine), hydrophobic amino acids (Alanine, and phenylalanine), and glycine are generated as a result of SHM events.

FIG. 5 The distribution of all 4096 6-mer nucleotide z-scores describing the hotness or coldness of the motif to SHM-mediated mutation. The z-scores for all permutations of 6-mers in the WRC synthetic library are superimposed on this distribution, with the dashed line denoting the top 5% of all possible motifs.

FIG. 6 The series of mutation events that lead to the creation of amino acid diversity, starting from “preferred SHM hot spot codons” AGC and TAC, as observed in affinity matured IGV heavy chain sequences. 4200 primary and secondary mutation events, starting from codons encoding asparagine and tyrosine, lead to a set of functionally diverse amino acids.

FIG. 7 Illustrates the convergence of sequence optimization with progressive iterations of replacement using the program SHMredesign. The figure shows both optimization towards an idealized hot and cold sequence, in this case starting with unmodified canine AID nucleotide sequence.

FIG. 8 Provides the amino acid (A; SEQ ID NO: 294), and polynucleotide sequence (B; SEQ ID NO: 26) of unmodified blasticidin gene. Also shown is the initial analysis of hot spots (C), cold spots (D) and occurrences of CpGs (E) as illustrated by bold capital letters.

FIG. 9 Provides the amino acid (A; SEQ ID NO: 294), and polynucleotide sequence (B; SEQ ID NO: 295) of a synthetic, SHM resistant version of the blasticidin gene. Also shown is the analysis of hot spots (C), cold spots (D) and occurrences of CpGs (E) in the synthetic sequence as illustrated by bold capital letters.

FIG. 10 Provides the amino acid (A; SEQ ID NO: 294), and polynucleotide sequence (B; SEQ ID NO: 296) of a synthetic, SHM susceptible version of the blasticidin gene. Also shown is the analysis of hot spots (C), cold spots (D) and occurrences of CpGs (E) in the synthetic sequence as illustrated by bold capital letters.

FIG. 11 Provides the polynucleotide sequence (A; SEQ ID NO: 297) of the unmodified form of the hygromycin gene. Also shown is the initial analysis of hot spots (B) and cold spots (C) as illustrated by bold capital letters. 102 CpG methylation sites were present (data not shown).

FIG. 12 Provides the polynucleotide sequence (A; SEQ ID NO: 298) of a synthetic, SHM resistant (cold) form of the hygromycin gene. Also shown is the analysis of hot spots (B) and cold spots (C) as illustrated by bold capital letters. 41 CpG methylation sites were present (data not shown).

FIG. 13 Provides the polynucleotide sequence (A; SEQ ID NO: 299) of a synthetic, SHM susceptible (hot) form of the hygromycin gene. Also shown is the analysis of hot spots (B) and cold spots (C) as illustrated by bold capital letters. 32 CpG methylation sites were present (data not shown).

FIG. 14 Provides the polynucleotide sequence (A; SEQ ID NO: 300) of a unmodified form of the Teal Fluorescent Protein (TFP). Also shown is the analysis of hot spots (B) and cold spots (C) as illustrated by bold capital letters. 40 CpG methylation sites were present (data not shown).

FIG. 15 Provides the polynucleotide sequence (A; SEQ ID NO: 301) of a synthetic SHM susceptible (hot) form of the Teal Fluorescent Protein (TFP). Also shown is the analysis of hot spots (B) and cold spots (C) as illustrated by bold capital letters. 14 CpG methylation sites were present (data not shown).

FIG. 16 Provides the polynucleotide sequence (A; SEQ ID NO: 302) of a synthetic SHM resistant (cold) form of the Teal Fluorescent Protein (TFP). Also shown is the analysis of hot spots (B) and cold spots (C) as illustrated by bold capital letters. 21 CpG methylation sites were present (data not shown).

FIG. 16D shows the mutations for a representative segment of the hot and cold TFP constructs. The central row shows the amino acid sequence of TFP (residues 59 thru 87) in single letter format (SEQ ID NO: 378), and the “hot” and “cold” starting nucleic acid sequences encoding the two constructs are shown above (hot; SEQ ID NO: 379) and below (cold) the amino acid sequence (SEQ ID NO: 380). Mutations observed in the hot sequence are aligned and stacked top of the gene sequences, while mutations in the cold TFP sequence are shown below. The results illustrate how “silent” changes to the coding sequences generate dramatic changes in observed AID-mediated SHM rates, demonstrating that engineered sequences can be effectively optimized to create fast or slow rates of SHM.

FIG. 16E shows that the spectrum of mutations generated by AID in the present in vitro tissue culture system mirror those observed in other studies and those seen during in vivo affinity maturation. FIG. 16E shows the mutations generated in the present study (Box (i) upper left, n=118), and compares them with mutations observed by Zan et al. (box (ii) upper right, n=702), Wilson et al. (lower left, n=25000; box (iii)), and a larger analysis of IGHV chains that have undergone affinity maturation (lower right, n=101,926; box (iv)). The Y-axis in each chart indicates the starting nucleotide, the X-axis indicates the end nucleotide, and the number in each square indicates the percentage (%) of time that nucleotide transition is observed. In the present study, the frequency of mutation transitions and transversions was similar to those seen in other data sets. Mutations of C to T and G to A are the direct result of AID activity on cytidines and account for 48% of all mutation events. In addition, mutations at bases A and T account for ˜30% of mutation events (i.e., slightly less than frequencies observed in other datasets).

FIG. 16F shows that mutation events are distributed throughout the SHM optimized nucleotide sequence of the hot TFP gene, with a maximum instantaneous rate of about 0.08 events per 1000 nucleotides per generation centered around 300 nucleotides from the beginning of the open reading frame. Stable transfection and selection of a gene with AID (for 30 days) produces a maximum rate of mutation of 1 event per 480 nucleotides. As a result, genes may contain zero, one, two or more mutations per gene.

FIG. 16G Illustrates the distribution of SHM-mediated events observed in hot TFP sequenced genes compared to the significantly reduced pattern of mutations seen in cold TFP (FIG. 16H).

FIG. 17 Provides a sequence comparison of activation-induced cytidine deaminase (AID) from Homo sapiens (human; SEQ ID NO: 303), Mus musculus (mouse; SEQ ID NO: 304), Canis familiaris (dog; SEQ ID NO: 305), Rattus norv (rat; SEQ ID NO: 306) and Pan troglodyte (chimpanzee; SEQ ID NO: 307). Variations between the species are represented by bold amino acids.

FIG. 18 Provides the amino acid (A; SEQ ID NO: 308), and polynucleotide sequence (B; SEQ ID NO: 309) of canine cytidine deaminase (AID) (L198A) Also shown is the analysis of hot spots (C), cold spots (D) and occurrences of CpGs (E) in the unmodified sequence as illustrated by bold capital letters.

FIG. 19 Provides the polynucleotide sequence (A; SEQ ID NO: 310) of a synthetic SHM susceptible form of canine AID. Also shown is the analysis of hot spots (B), cold spots (C) and occurrences of CpGs (D) as illustrated by bold capital letters.

FIG. 20 Provides the polynucleotide sequence (A; SEQ ID NO: 311) of a synthetic SHM resistant form of canine AID. Also shown is the analysis of hot spots (B), cold spots (C) and occurrences of CpGs (D) as illustrated by bold capital letters.

FIG. 21 Provides a sequence comparison of genomic Canis familiaris (dog; SEQ ID NO: 312) and SHM-resistant (cold) Canis familiaris (dog; SEQ ID NO: 313), Homo sapiens (human; SEQ ID NO: 314) and Mus musculus (mouse; SEQ ID NO: 315) mRNA activation-induced cytidine deaminase (AID) sequences. GAG sequences are illustrated by bold, underlining. Variations between the species are represented by bold amino acids.

FIG. 22 FIG. 22A Shows the predicted effect of AID activity on reversion frequency using a protein containing a mutable stop codon such as a fluorescent protein. FIG. 22B shows the actual rates of loss of fluorescence achieved (shown as GFP extinction) with cells transfected with two different concentrations of an expression vector capable of expressing AID, and stably expressing GFP. FIG. 22C shows the initial rates of GFP reversion with comparing directly, wild type human AID, and cold canine AID. Also shown is the effect of Ig enhancers on reversion rate.

FIG. 23 Provides the amino acid (A; SEQ ID NO: 316), and polynucleotide sequence (B; SEQ ID NO: 317) of unmodified Pol eta as well as the analysis of the number of hot spots, cold spots and CpGs (C).

FIG. 24 Provides the polynucleotide sequence (A; SEQ ID NO: 318) of a synthetic SHM resistant (cold) form of Pol eta well as the analysis of the number of hot spots, cold spots and CpGs (B).

FIG. 25 Provides the polynucleotide sequence (A; SEQ ID NO: 319) of a synthetic SHM susceptible (hot) form of Pol eta well as the analysis of the number of hot spots, cold spots and CpGs (B).

FIG. 26 Provides the amino acid sequence of unmodified Pol theta (SEQ ID NO: 320).

FIG. 27 Provides the polynucleotide sequence of unmodified Pol theta (SEQ ID NO: 321).

FIG. 28 Provides the polynucleotide sequence of a synthetic SHM resistant (cold) form of Pol theta (SEQ ID NO: 322).

FIG. 29 Provide the polynucleotide sequence of a synthetic SHM susceptible (hot) form of Pol theta (SEQ ID NO: 323).

FIG. 30 Provides the amino acid (A; SEQ ID NO: 324), and polynucleotide sequence (B; SEQ ID NO: 325) of unmodified uracil DNA glycosylase well as the analysis of the number of hot spots, cold spots and CpGs (C).

FIG. 31 Provides the polynucleotide sequence of a synthetic SHM resistant (cold) form of uracil DNA glycosylase (A; SEQ ID NO: 326) and a synthetic SHM susceptible (hot) form of uracil glycosylase (B; SEQ ID NO: 327).

FIG. 32 Provides a schematic of Vector Formats 1 (A) & 2 (B).

FIG. 33 Provides a schematic of Vector Format 3 (A) & 4 (B).

FIG. 34 Provides a schematic of Vector Format 5.

FIG. 35 Provides a schematic of Vectors F1 (A) and F7 (B).

FIG. 36 Provides schematics of Vector AB102. Restriction sites and genetic elements are illustrated in 36A and fragments used in construction of the vector are illustrated in 36B.

FIG. 37 Provides a schematic of Vectors AB184 (A) and F10 (B).

FIG. 38 Provides a schematic of Vector ANA209.

FIG. 39 Provides the polynucleotide sequence of unmodified NisB (A; SEQ ID NO: 328) and the initial analysis of hot spots, cold spots and CpG content (B).

FIG. 40 Provides the polynucleotide sequence of a SHM resistant (cold) form of NisB (A; SEQ ID NO: 329) and the initial analysis of hot spots, cold spots and CpG content (B).

FIG. 41 Provides the polynucleotide sequence of unmodified NisP (A; SEQ ID NO: 330) and the initial analysis of hot spots, cold spots and CpG content (B).

FIG. 42 Provides the polynucleotide sequence of a SHM resistant (cold) form of NisP (A; SEQ ID NO: 331) and the initial analysis of hot spots, cold spots and CpG content (B).

FIG. 43 Provides the polynucleotide sequence of unmodified NisT (A; SEQ ID NO: 332) and SHM resistant (cold) form of NisT (B; SEQ ID NO: 333).

FIG. 44 Provides the polynucleotide sequence of unmodified NisA (A; SEQ ID NO: 334), as well as the initial analysis of hot spots (B) and cold spots (C). Also shown is a synthetic form of NisA (SEQ ID NO: 335) showing areas of SHM resistant sequence (D; underlined) and SHM susceptible sequence (D; non-underlined), and the analysis of hot spots (E) and cold spots (F).

FIG. 45 Provides the polynucleotide sequence of unmodified NisC (A; SEQ ID NO: 336), as well as the initial analysis of hot spots (B) and cold spots (C).

FIG. 46 Shows a synthetic resistant (cold) form of NisC (A; SEQ ID NO: 337) showing the analysis of hot spots (B) and cold spots (C).

FIG. 47 Provides a diagram of the synthesis and maturation of Nisin (47A). FIG. 47B illustrates a backbone trace of the protein NisC, as described in the pdb structure 2G0D (rcsb.org/pdb/), with residues in the binding pocket highlighted. A zinc metal and several cysteine, histidine and aspartate residues are the residues that carry out cyclization of NisA. Here, residues within 10 Angstroms (Å) of the catalytic site are labeled and shown with a surface representation.

FIG. 48 FIG. 48A shows cells expressing the 30 pM HyHEL antibody (dark gray) or no antibody (light gray) after selection by incubating with streptavidin microparticles conjugated to the mature Hen Egg Lysozyme (HEL) protein (Protein), HEL peptide monomer (Monomer), tandem HEL dimer (Tandem), HEL MAPS dimer (MAPS) or naked unconjugated streptavidin microparticles (Naked). FIG. 48B shows cells expressing the 800 pM HyHEL antibody (dark gray) or no antibody (light gray) were selected by incubating with tosylactivated microparticles conjugated to either the mature HEL protein (Protein) or naked unconjugated tosylactivated microparticles (Naked).

FIG. 49 FIG. 49A Shows cells expressing the 30 pM HyHEL antibody were selected by incubating with streptavidin microparticles conjugated to the mature HEL protein (Protein), HEL peptide monomer (Monomer), tandem HEL dimer (Tandem), HEL MAPS dimer (MAPS) or naked unconjugated streptavidin microparticles (Naked). FIG. 49B shows cells expressing the 800 pM HyHEL antibody were selected by incubating with tosylactivated microparticles conjugated to either the mature HEL protein (Protein) or naked unconjugated tosylactivated microparticles (Naked).

FIG. 50 shows the 20 most hot spot codon hypermutation transition events within the FR and CDR regions of heavy chain antibodies, where the numbers labeling the arrows indicate how often a codon transition event was observed. The codons AGC and AGT (Serine), and to a lesser extent TAC and TAT (Tyrosine), account for ˜50% of the originating mutations observed in affinity matured antibodies. Use of these hot spot codons within the correct reading frame, combined with affinity maturation leads to many fewer observed silent mutations within CDRs compared to framework regions (highlighted by dotted circles in the figure).

FIG. 51 HEK-293 cells transfected with a low affinity anti-HEL antibody (comprising the light chain mutation N31G) and an constitutive AID expression vector either after stable transfection and selection (panels A and C) or transiently with the addition of re-transfected AID expression vector (panels B and D) were incubated with either 50 pM HEL-FITC (A and B) or 500 pM HEL-FITC C and D) and living HEL-FITC-binding cells were sorted and expanded in culture for another round of selection and sequence analysis.

FIG. 52 Previously sorted HEK-293 cells expressing anti-HEL antibodies and constitutive canine AID either after stable transfection and selection (A and C) or transiently with the addition of re-transfected AID expression vector (panels B and D) were incubated with either 50 pM HEL-FITC (A and B) or 500 pM HEL-FITC C and D) and living HEL-FITC-binding cells were sorted and expanded in culture for another round of selection and sequence analysis.

FIG. 53 HEK-293 cells transfected with a low affinity anti-HEL antibody and evolved over 4 rounds of selection and evolution were analyzed by incubation with 50 pM HEL-FITC, as described in Example 13. Panel A shows that over 4 rounds of evolution, a clear increase in positive cells is evident in both the FACS scatter plot (panel A), as well as total number of positive cells gated (panel B).

FIG. 54 Panel A shows a selection of amino sequences around the HyHEL10 light chain CDR1, showing the evolved sequence around the site of the Asn 31 mutation introduced in the starting constructs (SEQ ID NOS: 367, 368 and 369). Panel B shows the corresponding nucleic acid sequences (SEQ ID NOS: 370, 371 and 372), and panel C shows a representation of the measured affinity of the evolved mutants.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

As used herein and in the appended claims, the terms “a,” “an” and “the” can mean, for example, one or more, or at least one, of a unit unless the context clearly dictates otherwise. Thus, for example, reference to “an antibody” includes a plurality of such antibodies and reference to “a variable domain” includes reference to one or more variable domains and equivalents thereof known to those skilled in the art, and so forth. In the event that there is a plurality of definitions for a term herein, those in this section prevail unless stated otherwise.
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges can independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.
The term “specific binding member” describes a member of a pair of molecules which have binding specificity for one another. The members of a specific binding pair can be naturally derived or wholly or partially synthetically produced. One member of the pair of molecules has an area on its surface, or a cavity, which specifically binds to and is therefore complementary to a particular spatial and/or polar organization of the other member of the pair of molecules. Thus, the members of the pair have the property of binding specifically to each other. Examples of types of specific binding pairs include antigen-antibody, Avimer™-substrate, biotin-avidin, hormone-hormone receptor, receptor-ligand, protein-protein, and enzyme-substrate.
The term “antibody” describes an immunoglobulin whether natural or partly or wholly synthetically produced. The term also covers any polypeptide or protein having a binding domain which is, or is homologous to, an antigen-binding domain. CDR grafted antibodies are also contemplated by this term.
“Native antibodies” and “native immunoglobulins” are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is, in some cases, linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (“V_H”) followed by a number of constant domains (“C_H”). Each light chain has a variable domain at one end (“V_L”) and a constant domain (“C_L”) at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light-chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light- and heavy-chain variable domains.
The term “variable domain” refers to protein domains that differ extensively in sequence among family members (i.e. among different isoforms, or in different species). With respect to antibodies, the term “variable domain” refers to the variable domains of antibodies that are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called hypervariable regions both in the light chain and the heavy chain variable domains. The more highly conserved portions of variable domains are called the “framework region” or “FR.” The variable domains of unmodified heavy and light chains each comprise four FRs (FR1, FR2, FR3 and FR4, respectively), largely adopting a β-sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the β-sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991), pages 647 669). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.
The term “hypervariable region” when used herein refers to the amino acid residues of an antibody which are responsible for antigen-binding. The hypervariable region comprises amino acid residues from three “complementarity determining regions” or “CDRs,” which directly bind, in a complementary manner, to an antigen and are known as CDR1, CDR2, and CDR3 respectively.
In the light chain variable domain, the CDRs correspond to approximately residues 24-34 (CDRL1), 50-56 (CDRL2) and 89-97 (CDRL3), and in the heavy chain variable domain the CDRs correspond to approximately residues 31-35 (CDRH1), 50-65 (CDRH2) and 95-102 (CDRH3); Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) and/or those residues from a “hypervariable loop” (i.e., residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk J., Mol. Biol. 196:901-917 (1987)).
As used herein, “variable framework region” or “VFR” refers to framework residues that form a part of the antigen binding pocket and/or groove that may contact antigen. In some embodiments, the framework residues form a loop that is a part of the antigen binding pocket or groove. The amino acids residues in the loop may or may not contact the antigen. In an embodiment, the loop amino acids of a VFR are determined by inspection of the three-dimensional structure of an antibody, antibody heavy chain, or antibody light chain. The three-dimensional structure can be analyzed for solvent accessible amino acid positions as such positions are likely to form a loop and/or provide antigen contact in an antibody variable domain. Some of the solvent accessible positions can tolerate amino acid sequence diversity and others (e.g. structural positions) can be less diversified. The three-dimensional structure of the antibody variable domain can be derived from a crystal structure or protein modeling. In some embodiments, the VFR comprises, consists essentially of, or consists of amino acid positions corresponding to amino acid positions 71 to 78 of the heavy chain variable domain, the positions defined according to Kabat et al., 1991. In some embodiments, VFR forms a portion of Framework Region 3 located between CDRH2 and CDRH3. Preferably, VFR forms a loop that is well positioned to make contact with a target antigen or form a part of the antigen binding pocket.
Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chain constant domains (Fc) that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.
The “light chains” of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa or (“κ”) and lambda or (“λ”), based on the amino acid sequences of their constant domains.
The terms “antigen-binding portion of an antibody,” “antigen-binding fragment,” “antigen-binding domain,” “antibody fragment” or a “functional fragment of an antibody” are used interchangeably in the present invention to mean one or more fragments of an antibody that retain the ability to specifically bind to an antigen (see, e.g., Holliger et al., Nature Biotech. 23 (9): 1126-1129 (2005)). Non-limiting examples of antibody fragments included within, but not limited to, the term “antigen-binding portion” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the V_L, V_H, C_Land C_H1domains; (ii) a F(ab′)₂fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the V_Hand C_H1domains; (iv) a Fv fragment consisting of the V_Land V_Hdomains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a V_Hdomain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, V_Land V_H, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the V_Land V_Hregions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; and Osbourn et al. (1998) Nat. Biotechnol. 16:778). Such single chain antibodies are also intended to be encompassed within the term “antigen-binding portion” of an antibody. Any V_Hand V_Lsequences of specific scFv can be linked to human immunoglobulin constant region cDNA or genomic sequences, in order to generate expression vectors encoding complete IgG molecules or other isotypes. V_Hand V_Lcan also be used in the generation of Fab, Fv or other fragments of immunoglobulins using either protein chemistry or recombinant DNA technology. Other forms of single chain antibodies, such as diabodies are also encompassed.
“F(ab′)₂” and “Fab′” moieties can be produced by treating immunoglobulin (monoclonal antibody) with a protease such as pepsin and papain, and includes an antibody fragment generated by digesting immunoglobulin near the disulfide bonds existing between the hinge regions in each of the two H chains. For example, papain cleaves IgG upstream of the disulfide bonds existing between the hinge regions in each of the two H chains to generate two homologous antibody fragments in which an L chain composed of V_L(L chain variable region) and C_L(L chain constant region), and an H chain fragment composed of V_H(H chain variable region) and C_Hγ1(γ1 region in the constant region of H chain) are connected at their C terminal regions through a disulfide bond. Each of these two homologous antibody fragments is called Fab′. Pepsin also cleaves IgG downstream of the disulfide bonds existing between the hinge regions in each of the two H chains to generate an antibody fragment slightly larger than the fragment in which the two above-mentioned Fab′ are connected at the hinge region. This antibody fragment is called F(ab′)₂.
The Fab fragment also contains the constant domain of the light chain and the first constant domain (C_H1) of the heavy chain. Fab′ fragments differ from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain C _H1 domain including one or more cysteine(s) from the antibody hinge region. Fab′-SH is the designation herein for Fab′ in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab′)₂antibody fragments originally were produced as pairs of Fab′ fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
“Fv” is the minimum antibody fragment which contains a complete antigen-recognition and antigen-binding site. This region consists of a dimer of one heavy chain and one light chain variable domain in tight, non-covalent association. It is in this configuration that the three hypervariable regions of each variable domain interact to define an antigen-binding site on the surface of the V_H-V_Ldimer. Collectively, the six hypervariable regions confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three hypervariable regions specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
“Single-chain Fv” or “sFv” antibody fragments comprise the V_Hand V_Ldomains of an antibody, wherein these domains are present in a single polypeptide chain. In some embodiments, the Fv polypeptide further comprises a polypeptide linker between the V_Hand V_Ldomains which enables the sFv to form the desired structure for antigen binding. For a review of sFv molecules, see, e.g., Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994).
The term “Avimer™” refers to a new class of therapeutic proteins that are from human origin, which are unrelated to antibodies and antibody fragments, and are composed of several modular and reusable binding domains, referred to as A-domains (also referred to as class A module, complement type repeat, or LDL-receptor class A domain). They were developed from human extracellular receptor domains by in vitro exon shuffling and phage display, (Silverman et al., 2005, Nat. Biotechnol. 23:1493-94; Silverman et al., 2006, Nat. Biotechnol. 24:220). The resulting proteins can comprise multiple independent binding domains that can exhibit improved affinity (in some cases sub-nanomolar) and specificity compared with single-epitope binding proteins. See, for example, U.S. Patent Application Publ. Nos. 2005/0221384, 2005/0164301, 2005/0053973 and 2005/0089932, 2005/0048512, and 2004/0175756, each of which is hereby incorporated by reference herein in its entirety.
Each of the known 217 human A-domains comprises ˜35 amino acids (˜4 kDa) and domains are separated by linkers that average five amino acids in length. Native A-domains fold quickly and efficiently to a uniform, stable structure mediated primarily by calcium binding and disulfide formation. A conserved scaffold motif of only 12 amino acids is needed for this common structure. The end result is a single protein chain containing multiple domains, each of which represents a separate function. Each domain of the proteins binds independently and that the energetic contributions of each domain are additive. These proteins were called “Avimers™” from avidity multimers.
As used herein, “natural” or “naturally occurring” antibodies or antibody variable domains, refers to antibodies or antibody variable domains having a sequence of an antibody or antibody variable domain identified from a non-synthetic source, for example, from a germline sequence, or differentiated antigen-specific B cell obtained ex vivo, or its corresponding hybridoma cell line, or from the serum of an animal. These antibodies can include antibodies generated in any type of immune response, either natural or otherwise induced. Natural antibodies include the amino acid sequences, and the nucleotide sequences that constitute or encode these antibodies, for example, as identified in the Kabat database.
The term “synthetic polynucleotide” or “synthetic gene” means that the corresponding polynucleotide sequence, or amino acid sequence, is derived, in whole or part, from a sequence that has been designed, or synthesized de novo, or modified as compared to an equivalent unmodified sequence. Synthetic genes can be prepared in whole or part, via chemical synthesis, or amplified via PCR, or similar enzymatic amplification systems. Synthetic genes are, in some embodiments, different from unmodified genes, either at the amino acid, or polynucleotide level, (or both) and are, for example, located within the context of synthetic expression control sequences. Synthetic gene sequences can include amino acid or polynucleotide sequences that have been changed, for example, by the replacement, deletion, or addition, of one or more, amino acids or nucleotides, thereby providing an amino acid sequence, or a polynucleotide coding sequence that is different from the source sequence. Synthetic gene polynucleotide sequences may not necessarily encode proteins with different amino acids, compared to the unmodified gene, for example, they can also encompass synthetic polynucleotide sequences that incorporate different codons or motifs, but which encode the same amino acid(s); i.e., the nucleotide changes can represent silent mutations at the amino acid level. In one embodiment, synthetic genes exhibit altered susceptibility to SHM compared to the unmodified gene. Synthetic genes can be iteratively modified using the methods described herein and, in each successive iteration, a corresponding polynucleotide sequence or amino acid sequence, is derived, in whole or part, from a sequence that has been designed, or synthesized de novo, or modified, compared to an equivalent unmodified sequence.
The terms “semi-synthetic polynucleotide” or “semi-synthetic gene,” as used herein, refer to polynucleotide sequences that consist in part of a nucleic acid sequence that has been obtained via polymerase chain reaction (PCR) or other similar enzymatic amplification system which utilizes a natural donor (i.e., peripheral blood monocytes) as the starting material for the amplification reaction. The remaining “synthetic” polynucleotides, i.e., those portions of semi-synthetic polynucleotide not obtained via PCR or other similar enzymatic amplification system can be synthesized de-novo using methods known in the art including, but not limited to, the chemical synthesis of nucleic acid sequences.
The term “synthetic variable regions” refers to synthetic polynucleotide sequences within a synthetic gene that are substantially comprised of optimal SHM hot spots and hot codons or motifs that, when combined with the activity of AID and one or more error-prone polymerases, generates a broad spectrum of potential amino acid diversity at each position. Synthetic variable regions can encode antibody or non-antibody polypeptides and can be separated by synthetic frame work sequences that encompass codons or motifs that are not specifically targeted for, or susceptible to, SHM, or that are resistant to SHM.
The term “diabodies” refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy chain variable domain (V_H) connected to a light chain variable domain (V_L) in the same polypeptide chain (V_H-V_L). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are described more fully in, for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993).
Antibodies of the present invention also include heavy chain dimers, such as antibodies from camelids and sharks. Camelid and shark antibodies comprise a homodimeric pair of two chains of V-like and C-like domains (neither has a light chain). Since the V_Hregion of a heavy chain dimer IgG in a camelid does not have to make hydrophobic interactions with a light chain, the region in the heavy chain that normally contacts a light chain is changed to hydrophilic amino acid residues in a camelid. V_Hdomains of heavy-chain dimer IgGs are called V_HHdomains. Shark Ig-NARs comprise a homodimer of one variable domain (termed a V-NAR domain) and five C-like constant domains (C-NAR domains).
In camelids, the diversity of antibody repertoire is determined by the complementary determining regions (CDR) 1, 2, and 3 in the V_Hor V_HHregions. The CDR3 in the camel V_HHregion is characterized by its relatively long length averaging 16 amino acids (Muyldermans et al., 1994, Protein Engineering 7(9): 1129). This is in contrast to CDR3 regions of antibodies of many other species. For example, the CDR3 of mouse V_Hhas an average of 9 amino acids.
Libraries of camelid-derived antibody variable regions, which maintain the in vivo diversity of the variable regions of a camelid, can be made by, for example, the methods disclosed in U.S. Patent Application Ser. No. 20050037421, published Feb. 17, 2005.
“Humanized” forms of non-human (e.g., murine) antibodies are chimeric antibodies which contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which hypervariable region residues of the recipient are replaced by hypervariable region residues from a synthetic, or non-human source, such as mouse, rat, rabbit or non-human primate (donor antibody) having the desired specificity, affinity, and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies can comprise residues which are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. A humanized antibody can comprise substantially all of at least one (and in some cases two) variable domain(s), in which all or substantially all of the hypervariable regions correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody, optionally, also can comprise at least a portion of an immunoglobulin constant region (Fc), such as that of a human immunoglobulin. For further details, see Jones et al., Nature 321:522-525 (1986); Reichmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).
A “humanized antibody” of the present invention includes semi-synthetic antibodies prepared by genetic engineering and specifically includes a monoclonal antibody in which the CDR3 of the heavy and light chain is derived from a non-human monoclonal antibody (e.g. murine monoclonal antibody), or region is derived from synthetic variable region polynucleotides, as described herein (both heavy and light chain) and the constant region is derived from the synthetic human constant region templates likewise described herein and in commonly owned, priority U.S. Provisional Patent Application Nos. 60/904,622 and 61/020,124.
The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that can be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations, which can include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention can be made by the hybridoma method first described by Kohler et al., Nature 256:495 (1975), or can be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). In certain embodiments, the “monoclonal antibodies” can also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol. 222:581-597 (1991), for example.
In other embodiments, monoclonal antibodies can be isolated and purified from the culture supernatant or ascites mentioned above by saturated ammonium sulfate precipitation, euglobulin precipitation method, caproic acid method, caprylic acid method, ion exchange chromatography (DEAE or DE52), or affinity chromatography using anti-immunoglobulin column or protein A column.
A polyclonal antibody (antiserum) or monoclonal antibody can be produced by known methods. Namely, mammals, preferably, mice, rats, hamsters, guinea pigs, rabbits, cats, dogs, pigs, goats, horses, or cows, or more preferably, mice, rats, hamsters, guinea pigs, or rabbits are immunized, for example, with an antigen mentioned above with Freund's adjuvant, if necessary. The polyclonal antibody can be obtained from the serum obtained from the animal so immunized. The monoclonal antibodies are produced as follows. Hybridomas are produced by fusing the antibody-producing cells obtained from the animal so immunized and myeloma cells incapable of producing auto-antibodies. Then the hybridomas are cloned, and clones producing the monoclonal antibodies showing the specific affinity to the antigen used for immunizing the mammal are screened.
As used herein, an “intrabody or fragment thereof” refers to antibodies that are expressed and function intracellularly. Intrabodies, in some embodiments, lack disulfide bonds and are capable of modulating the expression or activity of target genes through their specific binding activity. Intrabodies include single domain fragments such as isolated V_Hand V_Ldomains and scFvs. An intrabody can include sub-cellular trafficking signals attached to the N or C terminus of the intrabodies to allow them to be expressed at high concentrations in the sub-cellular compartments where a target protein is located. Upon interaction with the target gene, an intrabody modulates target protein function, and/or achieves phenotypic/functional knockout by mechanisms such as accelerating target protein degradation and sequestering the target protein in a non-physiological sub-cellular compartment. Other mechanisms of intrabody-mediated gene inactivation can depend on the epitope to which the intrabody is directed, such as binding to the catalytic site on a target protein or to epitopes that are involved in protein-protein, protein-DNA or protein-RNA interactions. In one embodiment, an intrabody is a scFv.
The “cell producing an antibody reactive to a protein or a fragment thereof” of the present invention means any cell producing any of the above-described antibodies of the present invention.
The term “germline gene segments” refers to the genes from the germline (the haploid gametes and those diploid cells from which they are formed). The germline DNA contain multiple gene segments that encode a single immunoglobulin heavy or light chain. These gene segments are carried in the germ cells but cannot be transcribed and translated into heavy and light chains until they are arranged into functional genes. During B-cell differentiation in the bone marrow, these gene segments are randomly shuffled by a dynamic genetic system capable of generating more than 108 specificities. Most of these gene segments are published and collected by the germline database.
As used herein, “library” refers to a plurality of polynucleotides, proteins, or cells comprising two or more non-identical members. A “synthetic library” refers to a plurality of synthetic polynucleotides, or a population of cells that comprise said plurality of synthetic polynucleotides. A “semi-synthetic library” refers to a plurality of semi-synthetic polynucleotides, or a population of cells that comprise said plurality of semi-synthetic polynucleotides. A “seed library” refers to a plurality of one or more synthetic or semi-synthetic polynucleotides, or cells that comprise said polynucleotides, that contain one or more sequences or portions thereof, that have been modified to increase or decrease susceptibility to SHM, e.g., AID-mediated SHM, and that are capable, when acted upon by somatic hypermutation, to create a library of polynucleotides, proteins or cells in situ.
As used herein, the term “antigen” refers to substances that are capable, under appropriate conditions, of inducing an immune response to the substance and of reacting with the products of the immune response. For example, an antigen can be recognized by antibodies (humoral immune response) or sensitized T-lymphocytes (T helper or cell-mediated immune response), or both. Antigens can be soluble substances, such as toxins and foreign proteins, or particulates, such as bacteria and tissue cells; however, only the portion of the protein or polysaccharide molecule known as the antigenic determinant (epitopes) combines with the antibody or a specific receptor on a lymphocyte. More broadly, the term “antigen” refers to any substance to which an antibody binds, or for which antibodies are desired, regardless of whether the substance is immunogenic. For such antigens, antibodies can be identified by recombinant methods, independently of any immune response.
As used herein, the term “affinity” refers to the equilibrium constant for the reversible binding of two agents and is expressed as Kd. Affinity of a binding protein to a ligand such as affinity of an antibody for an epitope can be, for example, from about 100 nanomolar (nM) to about 0.1 nM, from about 100 nM to about 1 picomolar (pM), or from about 100 nM to about 1 femtomolar (fM). As used herein, the term “avidity” refers to the resistance of a complex of two or more agents to dissociation after dilution.
“Epitope” refers to that portion of an antigen or other macromolecule capable of forming a binding interaction that interacts with the variable region binding pocket of a binding protein. Such binding interaction can be manifested as an intermolecular contact with one or more amino acid residues of a CDR. Antigen binding can involve a CDR3 or a CDR3 pair. An epitope can be a linear peptide sequence (i.e., “continuous”) or can be composed of noncontiguous amino acid sequences (i.e., “conformational” or “discontinuous”). A binding protein can recognize one or more amino acid sequences; therefore an epitope can define more than one distinct amino acid sequence. Epitopes recognized by binding protein can be determined by peptide mapping and sequence analysis techniques well known to one of skill in the art. A “cryptic epitope” or a “cryptic binding site” is an epitope or binding site of a protein sequence that is not exposed or substantially protected from recognition within an unmodified polypeptide, but is capable of being recognized by a binding protein of a denatured or proteolyzed polypeptide Amino acid sequences that are not exposed, or are only partially exposed, in the unmodified polypeptide structure are potential cryptic epitopes. If an epitope is not exposed, or only partially exposed, then it is likely that it is buried within the interior of the polypeptide. Candidate cryptic epitopes can be identified, for example, by examining the three-dimensional structure of an unmodified polypeptide.
The term “specific” is applicable to a situation in which one member of a specific binding pair will not show any significant binding to molecules other than its specific binding partner(s). The term is also applicable where e.g. an antigen binding domain is specific for a particular epitope which is carried by a number of antigens, in which case the specific binding member carrying the antigen binding domain will be able to bind to the various antigens carrying the epitope.
The term “binding” refers to a direct association between two molecules, due to, for example, covalent, electrostatic, hydrophobic, and ionic and/or hydrogen-bond interactions, including interactions such as salt bridges and water bridges.
The term “adjuvant” refers to a compound or mixture that enhances the immune response, particularly to an antigen. An adjuvant can serve as a tissue depot that slowly releases the antigen and also as a lymphoid system activator that non-specifically enhances the immune response (Hood et al., Immunology, Second Ed., 1984, Benjamin/Cummings: Menlo Park, Calif., p. 384). Often, a primary challenge with an antigen alone, in the absence of an adjuvant, will fail to elicit a humoral or cellular immune response. Previously known and utilized adjuvants include, but are not limited to, complete Freund's adjuvant, incomplete Freund's adjuvant, saponin, mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil or hydrocarbon emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvant such as BCG (Bacille Calmette-Guerin) and Corynebacterium parvum. Mineral salt adjuvants include but are not limited to: aluminum hydroxide, aluminum phosphate, calcium phosphate, zinc hydroxide and calcium hydroxide. Preferably, the adjuvant composition further comprises a lipid of fat emulsion comprising about 10% (by weight) vegetable oil and about 1-2% (by weight) phospholipids. Preferably, the adjuvant composition further optionally comprises an emulsion form having oily particles dispersed in a continuous aqueous phase, having an emulsion forming polyol in an amount of from about 0.2% (by weight) to about 49% (by weight), optionally a metabolizable oil in an emulsion-forming amount of up to 15% (by weight), and optionally a glycol ether-based surfactant in an emulsion-stabilizing amount of up to about 5% (by weight).
As used herein, the term “immunomodulator” refers to an agent which is able to modulate an immune response. An example of such modulation is an enhancement of antibody production. Another example of such modulation is an enhancement of a T cell response.
An “immunological response” to a composition or vaccine comprised of an antigen is the development in the host of a cellular- and/or antibody-mediated immune response to the composition or vaccine of interest. Usually, such a response consists of the subject producing antibodies, B cells, helper T cells, suppressor T cells, and/or cytotoxic T cells directed specifically to an antigen or antigens included in the composition or vaccine of interest.
The term “nucleotide” as used herein refers to a monomeric unit of a polynucleotide that consists of a heterocyclic base, a sugar, and one or more phosphate groups. The naturally occurring bases, (guanine, (G), adenine, (A), cytosine, (C), thymine, (T), and uracil (U)) are derivatives of purine or pyrimidine, though it should be understood that naturally and non-naturally occurring base analogs are also included. The naturally occurring sugar is the pentose (five-carbon sugar) deoxyribose (which forms DNA) or ribose (which forms RNA), though it should be understood that naturally and non-naturally occurring sugar analogs are also included. Nucleic acids are linked via phosphate bonds to form nucleic acids, or polynucleotides, though many other linkages are known in the art (such as, though not limited to phosphorothioates, boranophosphates and the like).
The terms “nucleic acid” and “polynucleotide” as used herein refer to a polymeric form of nucleotides of any length, either ribonucleotides (RNA) or deoxyribonucleotides (DNA). These terms refer to the primary structure of the molecules and, thus, include double- and single-stranded DNA, and double- and single-stranded RNA. These terms include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs and modified polynucleotides such as, though not limited to, methylated and/or capped polynucleotides.
A “DNA molecule” refers to the polymeric form of deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in its either single stranded form or a double-stranded helix. This term refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear DNA molecules (e.g., restriction fragments), viruses, plasmids, and chromosomes. In discussing the structure of particular double-stranded DNA molecules, sequences can be described herein according to the normal convention of giving only the sequence in the 5′ to 3′ direction along the non-transcribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA).
A DNA “coding sequence” or “coding region” is a double-stranded DNA sequence which is transcribed and translated into a polypeptide in vivo when placed under the control of appropriate expression control sequences. The boundaries of the coding sequence (the “open reading frame” or “ORF”) are determined by a start codon at the 5′ (amino) terminus and a translation stop codon at the 3′ (carboxyl) terminus. A coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and synthetic DNA sequences. A polyadenylation signal and transcription termination sequence is, usually, be located 3′ to the coding sequence. The term “non-coding sequence” or “non-coding region” refers to regions of a polynucleotide sequence that are not translated into amino acids (e.g. 5′ and 3′ un-translated regions).
The term “reading frame” refers to one of the six possible reading frames, three in each direction, of the double stranded DNA molecule. The reading frame that is used determines which codons are used to encode amino acids within the coding sequence of a DNA molecule.
As used herein, an “antisense” nucleic acid molecule comprises a nucleotide sequence which is complementary to a “sense” nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule, complementary to an mRNA sequence or complementary to the coding strand of a gene. Accordingly, an antisense nucleic acid molecule can hydrogen bond to a sense nucleic acid molecule.
The term “base pair” or (“bp”): a partnership of adenine (A) with thymine (T), or of cytosine (C) with guanine (G) in a double stranded DNA molecule. In RNA, uracil (U) is substituted for thymine.
As used herein a “codon” refers to the three nucleotides which, when transcribed and translated, encode a single amino acid residue; or in the case of UUA, UGA or UAG encode a termination signal. Codons encoding amino acids are well known in the art and are provided for convenience herein in Table 1.

TABLE 1

Codon Usage Table

Codon	Amino acid	AA	Abbr.	Codon	Amino acid	AA	Abbr.

UUU	Phenylalanine	Phe	F	UCU	Serine	Ser	S
UUC	Phenylalanine	Phe	F	UCC	Serine	Ser	S
UUA	Leucine	Leu	L	UCA	Serine	Ser	S
UUG	Leucine	Leu	L	UCG	Serine	Ser	S

CUU	Leucine	Leu	L	CCU	Proline	Pro	P
CUC	Leucine	Leu	L	CCC	Proline	Pro	P
CUA	Leucine	Leu	L	CCA	Proline	Pro	P
CUG	Leucine	Leu	L	CCG	Proline	Pro	P

AUU	Isoleucine	Ile	I	ACU	Threonine	Thr	T
AUC	Isoleucine	Ile	I	ACC	Threonine	Thr	T
AUA	Isoleucine	Ile	I	ACA	Threonine	Thr	T
AUG	Methionine	Met	M	ACH	Threonine	Thr	T

GUU	Valine	Val	V	GCU	Alanine	Ala	A
GUC	Valine	Val	V	GCC	Alanine	Ala	A
GUA	Valine	Val	V	GCA	Alanine	Ala	A
GUG	Valine	Val	V	GCG	Alanine	Ala	A

UAU	Tyrosine	Tyr	Y	UGU	Cysteine	Cys	C
UAC	Tyrosine	Tyr	Y	UGC	Cysteine	Cys	C
UUA		Stop		UGA		Stop
UAG		Stop		UGG	Tryptophan	Trp	W

CAU	Histidine	His	H	CGU	Arginine	Arg	R
CAC	Histidine	His	H	CGC	Arginine	Arg	R
CAA	Glutamine	Gln	Q	CGA	Arginine	Arg	R
CAG	Glutamine	Gln	Q	CGG	Arginine	Arg	R

AAU	Asparagine	Asn	N	AGU	Serine	Ser	S
AAC	Asparagine	Asn	N	AGC	Serine	Ser	S
AAA	Lysine	Lys	K	AGA	Arginine	Arg	R
AAG	Lysine	Lys	K	AGG	Arginine	Arg	R

GAU	Aspartate	Asp	D	GGU	Glycine	Gly	G
GAC	Aspartate	Asp	D	GGC	Glycine	Gly	G
GAA	Glutamate	Glu	E	GGA	Glycine	Gly	G
GAG	Glutamate	Glu	E	GGG	Glycine	Gly	G

AA: amino acid; Abbr: abbreviation. It should be understood that the codons specified above are for RNA sequences. The corresponding codons for DNA have a T substituted for U. Optimal codon usage is indicated by codon usage frequencies for expressed genes, for example, as shown in the codon usage chart from the program “Human—_High.cod” from the Wisconson Sequence Analysis Package, Version 8.1, Genetics Computer Group, Madison, Wis. Codon usage is also described in, for example, R. Nussinov, “Eukaryotic Dinucleotide Preference Rules and Their Implications for Degenerate Codon Usage,” J. Mol. Biol. 149: 125-131 (1981). The codons which are most frequently used in highly expressed human genes are presumptively the optimal codons for expression in human host cells and, thus, form the bases for constructing a synthetic coding sequence.
As used herein, a “wobble position” refers to the third position of a codon. Mutations in a DNA molecule within the wobble position of a codon, in some embodiments, result in silent or conservative mutations at the amino acid level. For example, there are four codons that encode Glycine, i.e., GGU, GGC, GGA and GGG, thus mutation of any wobble position nucleotide, to any other nucleotide, does not result in a change at the amino acid level of the encoded protein and, therefore, is a silent substitution.
Accordingly a “silent substitution” or “silent mutation” is one in which a nucleotide within a codon is modified, but does not result in a change in the amino acid residue encoded by the codon. Examples include mutations in the third position of a codon, as well in the first position of certain codons such as in the codon “CGG” which, when mutated to AGG, still encodes Arg.
The phrase “conservative amino acid substitution” or “conservative mutation” refers to the replacement of one amino acid by another amino acid with a common property. A functional way to define common properties between individual amino acids is to analyze the normalized frequencies of amino acid changes between corresponding proteins of homologous organisms (Schulz, G. E. and R. H. Schirmer, Principles of Protein Structure, Springer-Verlag). According to such analyses, groups of amino acids can be defined where amino acids within a group exchange preferentially with each other, and therefore resemble each other most in their impact on the overall protein structure (Schulz, G. E. and R. H. Schirmer, Principles of Protein Structure, Springer-Verlag).
Examples of amino acid groups defined in this manner include: a “charged/polar group,” consisting of Glu, Asp, Asn, Gln, Lys, Arg and His; an “aromatic, or cyclic group,” consisting of Pro, Phe, Tyr and Trp; and an “aliphatic group” consisting of Gly, Ala, Val, Leu, Ile, Met, Ser, Thr and Cys.
Within each group, subgroups can also be identified, for example, the group of charged/polar amino acids can be sub-divided into the sub-groups consisting of the “positively-charged sub-group,” consisting of Lys, Arg and His; the negatively-charged sub-group,” consisting of Glu and Asp, and the “polar sub-group” consisting of Asn and Gln.
The aromatic or cyclic group can be sub-divided into the sub-groups consisting of the “nitrogen ring sub-group,” consisting of Pro, His and Trp; and the “phenyl sub-group” consisting of Phe and Tyr.
The aliphatic group can be sub-divided into the sub-groups consisting of the “large aliphatic non-polar sub-group,” consisting of Val, Leu and Ile; the “aliphatic slightly-polar sub-group,” consisting of Met, Ser, Thr and Cys; and the “small-residue sub-group,” consisting of Gly and Ala.
Examples of conservative mutations include amino acid substitutions of amino acids within the sub-groups above, for example, Lys for Arg and vice versa such that a positive charge can be maintained; Glu for Asp and vice versa such that a negative charge can be maintained; Ser for Thr such that a free —OH can be maintained; and Gln for Asn such that a free —NH₂can be maintained.
“Semi-conservative mutations” include amino acid substitutions of amino acids with the same groups listed above, that do not share the same sub-group. For example, the mutation of Asp for Asn, or Asn for Lys all involve amino acids within the same group, but different sub-groups.
“Non-conservative mutations” involve amino acid substitutions between different groups, for example Lys for Leu, or Phe for Ser etc.
The term “amino acid residue” refers to the radical derived from the corresponding alpha-amino acid by eliminating the OH portion of the carboxyl group and the H-portion of the alpha amino group. For the most part, the amino acids used in the application are those naturally occurring amino acids found in proteins, or the naturally occurring anabolic or catabolic products of such amino acids which contain amino and carboxyl groups. Alternatively, un-natural amino acids can be incorporated into proteins to facilitate the chemical conjugation to other proteins, toxins, small organic compounds or anti-cancer agents (Datta et al., J Am Chem Soc. 2002; 124(20):5652-3). The abbreviations used herein for designating the amino acids and the protective groups are based on recommendations of the IUPAC-IUB Commission on Biochemical Nomenclature (see Biochemistry (1972) 11: 1726-1732). The term “amino acid residue” also includes analogs, derivatives and congeners of any specific amino acid referred to herein, as well as C-terminal or N-terminal protected amino acid derivatives (e.g., modified with an N-terminal or C-terminal protecting group). For example, the present invention contemplates the use of amino acid analogs wherein a side chain is lengthened or shorted while still providing a carboxyl, amino or other reactive precursor functional group for cyclization, as well as amino acid analogs having variant side chains with appropriate functional groups).
The term “amino acid side chain” is that part of an amino acid exclusive of the —CH—(NH₂)COOH portion, as defined by K. D. Kopple, “Peptides and Amino Acids,” W.A. Benjamin Inc., New York and Amsterdam, 1996, pages 2 and 33; examples of such side chains of the common amino acids are —CH₂CH₂SCH₃(the side chain of methionine), —CH₂(CH₃)—CH₂CH₃(the side chain of isoleucine), —CH₂CH(CH₃)₂(the side chain of leucine) or H—(the side chain of glycine).
The amino acid residues described herein are preferred to be in the “L” isomeric form. However, residues in the “D” isomeric form can be substituted for any L-amino acid residue, as long as the desired functional property of antibody (immunoglobulin)-binding is retained by the polypeptide. NH₂refers to the free amino group present at the amino terminus of a polypeptide. COOH refers to the free carboxy group present at the carboxy terminus of a polypeptide.
An “amino acid motif” is a sequence of amino acids, optionally a generic set of conserved amino acids, associated with a particular functional activity.
As used herein, the terms “protein,” “peptide” and “polypeptide” are used interchangeably to refer to polymers of amino acid residues of any length connected to one another by peptide bonds between the alpha-amino group and carboxy group of contiguous amino acid residues. Polypeptides, proteins and peptides can exist as linear polymers, branched polymers or in circular form. These terms also include forms that are post-translationally modified in vivo or chemically modified during synthesis.
It should be noted that all amino acid residue sequences are represented herein by formulae whose left and right orientation is in the conventional direction of amino-terminus to carboxy terminus. Furthermore, it should be noted that a dash at the beginning or end of an amino acid residue sequence indicates a peptide bond to a further sequence of one or more amino-acid residues.
The terms “gene,” “recombinant gene” and “gene construct” as used herein, refer to a DNA molecule, or portion of a DNA molecule, that encodes a protein or a portion thereof. The DNA molecule can contain an open reading frame encoding the protein (as exon sequences) and can further include intron sequences. The term “intron” as used herein, refers to a DNA sequence present in a given gene which is not translated into protein and is found in some, but not all cases, between exons. It can be desirable for the gene to be operably linked to, (or it can comprise), one or more promoters, enhancers, repressors and/or other regulatory sequences to modulate the activity or expression of the gene, as is well known in the art.
As used herein, a “complementary DNA” or “cDNA” includes recombinant polynucleotides synthesized by reverse transcription of mRNA and from which intervening sequences (introns) have been removed.
The term “operably linked” as used herein, describes the relationship between two polynucleotide regions such that they are functionally related or coupled to each other. For example, a promoter (or other expression control sequence) is operably linked to a coding sequence if it controls (and is capable of effecting) the transcription of the coding sequence. Although an operably linked promoter can be located upstream of the coding sequence, it is not necessarily contiguous with it.
“Expression control sequences” are DNA regulatory sequences, such as promoters, enhancers, polyadenylation signals, terminators, internal ribosome entry sites (IRES) and the like, that provide for the expression of a coding sequence in a host cell. Exemplary expression control sequences are described in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990).
A “promoter” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3′ direction) coding sequence. As used herein, the promoter sequence is bounded at its 3′ terminus by the transcription initiation site and extends upstream (5′ direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. A transcription initiation site (conveniently defined by mapping with nuclease S1) can be found within a promoter sequence, as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. Eukaryotic promoters can often, but not always, contain “TATA” boxes and “CAT” boxes. Prokaryotic promoters contain Shine-Dalgarno sequences in addition to the −10 and −35 consensus sequences.
A large number of promoters, including constitutive, inducible and repressible promoters, from a variety of different sources are well known in the art. Representative sources include for example, viral, mammalian, insect, plant, yeast, and bacterial cell types), and suitable promoters from these sources are readily available, or can be made synthetically, based on sequences publicly available on line or, for example, from depositories such as the ATCC as well as other commercial or individual sources. Promoters can be unidirectional (i.e., initiate transcription in one direction) or bi-directional (i.e., initiate transcription in either a 3′ or 5′ direction). Non-limiting examples of promoters include, for example, the T7 bacterial expression system, pBAD (araA) bacterial expression system, the cytomegalovirus (CMV) promoter, the SV40 promoter, the RSV promoter. Inducible promoters include the Tet system, (U.S. Pat. Nos. 5,464,758 and 5,814,618), the Ecdysone inducible system (No et al., Proc. Natl. Acad. Sci. (1996) 93 (8): 3346-3351; the T-RE_x™ system (Invitrogen Carlsbad, Calif.), LacSwitch® (Stratagene, (San Diego, Calif.) and the Cre-ER^Ttamoxifen inducible recombinase system (Indra et al. Nuc. Acid. Res. (1999) 27 (22): 4324-4327; Nuc. Acid. Res. (2000) 28 (23): e99; U.S. Pat. No. 7,112,715; and Kramer & Fussenegger, Methods Mol. Biol. (2005) 308: 123-144) or any promoter known in the art suitable for expression in the desired cells.
As used herein, a “minimal promoter” refers to a partial promoter sequence which defines the transcription start site but which by itself is not capable, if at all, of initiating transcription efficiently. The activity of such minimal promoters depends on the binding of activators such as a tetracycline-controlled transactivator to operably linked binding sites.
The terms “IRES” or “internal ribosome entry site” refer to a polynucleotide element that acts to enhance the translation of a coding sequence encoded with a. polycistronic messenger RNA. IRES elements, mediate the initiation of translation by directly recruiting and binding ribosomes to a messenger RNA (mRNA) molecule, bypassing the 7-methyl guanosine-cap involved in typical ribosome scanning. The presence of an IRES sequence can increase the level of cap-independent translation of a desired protein. Early publications descriptively refer to IRES sequences as “translation enhancers.” For example, cardioviral RNA “translation enhancers” are described in U.S. Pat. No. 4,937,190 to Palmenberg, et al. and U.S. Pat. No. 5,770,428 to Boris-Lawrie.
The terms “nuclear localization signal” and “NLS” refer to a domain, or domains capable of mediating the nuclear import of a protein or polynucleotide, or retention thereof, within the nucleus of a cell. A “strong nuclear import signal” represents a domain or domains capable of mediating greater than 90% subcellular localization in the nucleus when operatively linked to a protein of interest. Representative examples of NLSs include but are not limited to, monopartite nuclear localization signals, bipartite nuclear localization signals and N and C-terminal motifs. N terminal basic domains usually conform to the consensus sequence K-K/R-X-K/R which was first discovered in the SV40 large T antigen and which represents a monopartite NLS. One non-limiting example of an N-terminal basic domain NLS is PKKKRKV (SEQ ID NO: 340). Also known are bipartite nuclear localization signals which contain two clusters of basic amino acids separated by a spacer of about 10 amino acids, as exemplified by the NLS from nucleoplasmin: KR[PAATKKAGQA]KKKK (SEQ ID NO: 366). N and C-terminal motifs include, for example, the acidic M9 domain of hnRNP A1, the sequence KIPIK (SEQ ID NO: 381) in yeast transcription repressor Mata2 and the complex signals of U snRNPs. Most of these NLSs appear to be recognized directly by specific receptors of the importin β family.
The term “enhancer” as used herein, refers to a DNA sequence that increases transcription of, for example, a gene or coding sequence to which it is operably linked. Enhancers can be located many kilobases away from the coding sequence and can mediate the binding of regulatory factors, patterns of DNA methylation or changes in DNA structure. A large number of enhancers, from a variety of different sources are well known in the art and available as or within cloned polynucleotides (from, e.g., depositories such as the ATCC as well as other commercial or individual sources). A number of polynucleotides comprising promoters (such as the commonly-used CMV promoter) also comprise enhancer sequences. Operably linked enhancers can be located upstream, within, or downstream of coding sequences. The term “Ig enhancers” refers to enhancer elements derived from enhancer regions mapped within the Ig locus (such enhancers include for example, the heavy chain (mu) 5′ enhancers, light chain (kappa) 5′ enhancers, kappa and mu intronic enhancers, and 3′ enhancers, (see, e.g., Paul W E (ed) Fundamental Immunology, 3^rdEdition, Raven Press, New York (1993) pages 353-363; U.S. Pat. No. 5,885,827).
“Terminator sequences” are those that result in termination of transcription. Termination sequences are known in the art and include, but are not limited to, poly A (e.g., Bgh Poly A and SV40 Poly A) terminators. A transcriptional termination signal will typically include a region of 3′ untranslated region (or “3′ ut”), an optional intron (also referred to as intervening sequence or “IVS”) and one or more poly adenylation signals (“p(A)” or “pA.” Terminator sequences may also be referred to as “IVS−pA,” “IVS+p(A),” “3′ ut+p(A)” or “3′ ut/p(A).” Natural or synthetic terminators can be used as a terminator region.
The terms “polyadenylation,” “polyadenylation sequence,” “polyadenylation signal,” “Poly A,” “p(A)” or “pA” refer to a nucleic acid sequence present in a RNA transcript that allows for the transcript, when in the presence of the polyadenyl transferase enzyme, to be polyadenylated. Many polyadenylation signals are known in the art. Non-limiting examples include the human variant growth hormone polyadenylation signal, the SV40 late polyadenylation signal and the bovine growth hormone polyadenylation signal.
The term “splice site” as used herein refers to polynucleotides that are capable of being recognized by the spicing machinery of a eukaryotic cell as suitable for being cut and/or ligated to a corresponding splice site. Splice sites allow for the excision of introns present in a pre-mRNA transcript. In one example, the 5′ portion of the splice site is referred to as the splice donor and the 3′ corresponding splice site is referred to as the acceptor splice site. The term splice site includes, for example, naturally occurring splice sites, engineered splice sites, for example, synthetic splice sites, canonical or consensus splice sites, and/or non-canonical splice sites, for example, cryptic splice sites.
A “signal sequence” can be included before the coding sequence. This sequence encodes a signal peptide, N-terminal to the polypeptide, that communicates to the host cell to direct the polypeptide to the cell surface or secrete the polypeptide into the media, and this signal peptide is clipped off by the host cell before the protein leaves the cell. Signal sequences can be found associated with a variety of proteins native to prokaryotes and eukaryotes.
“Post-translational modification” can encompass any one of or combination of modification(s), including covalent modification(s), which a protein undergoes after translation is complete and after being released from the ribosome or on the nascent polypeptide co-translationally. Posttranslational modification includes but is not limited to phosphorylation, myristylation, ubiquitination, glycosylation, coenzyme attachment, methylation, 5-nitrosylation and acetylation. Posttranslational modification can modulate or influence the activity of a protein, its intracellular or extracellular destination, its stability or half-life, and/or its recognition by ligands, receptors or other proteins. Post-translational modification can occur in cell organelles, in the nucleus or cytoplasm or extracellularly.
The term “primer” as used herein refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product, which is complementary to a nucleic acid strand, is induced, i.e., in the presence of nucleotides and an inducing agent such as a DNA polymerase and at a suitable temperature and pH. The primer can be either single-stranded or double-stranded and must be sufficiently long to prime the synthesis of the desired extension product in the presence of the inducing agent. The exact length of the primer can depend upon many factors, including temperature, source of primer and use of the method. For example, for diagnostic applications, depending on the complexity of the target sequence, an oligonucleotide primer can contain about 15 to about 25 or more nucleotides, although it can contain fewer nucleotides.
The primers herein are selected to be “substantially” complementary to different strands of a particular target polynucleotide sequence. This means that the primers must be sufficiently complementary to hybridize with their respective strands. Therefore, the primer sequence need not reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment can be attached to the 5′ end of the primer, with the remainder of the primer sequence being complementary to the strand. Alternatively, non-complementary bases or longer sequences can be interspersed into the primer, provided that the primer sequence has sufficient complementarity with the sequence of the strand to hybridize therewith and thereby form the template for the synthesis of the extension product.
As used herein, the terms “restriction endonucleases” and “restriction enzymes” refer to bacterial enzymes, each of which cut double-stranded DNA at or near a specific nucleotide sequence.
The term “multiple cloning site” as used herein, refers to a segment of a vector polynucleotide which can recognize several different restriction enzymes.
A “replicon” is any genetic element (e.g., plasmid, episome, chromosome, YAC, virus) that functions as an autonomous unit of DNA replication in vivo; i.e., capable of replication under its own control, and containing autonomous replicating sequences.
A “vector” or “cloning vector” is a replicon, such as plasmid, phage or cosmid, to which another polynucleotide segment can be introduced so as to bring about the replication of the inserted segment. Vectors can exist as circular, double stranded DNA, and range in size form a few kilobases (kb) to hundreds of kb. Preferred cloning vectors have been modified from naturally occurring plasmids to facilitate the cloning and recombinant manipulation of polynucleotide sequences. Many such vectors are well known in the art; see for example, by Sambrook (In. “Molecular Cloning: A Laboratory Manual,” second edition, edited by Sambrook, Fritsch, & Maniatis, Cold Spring Harbor Laboratory, (1989)), Maniatis, In: Cell Biology: A Comprehensive Treatise, Vol. 3, Gene Sequence Expression, Academic Press, NY, pp. 563-608 (1980).
The term “expression vector” as used herein, refers to a vector used for expressing certain polynucleotides within a host cell or in-vitro expression system. The term includes plasmids, episomes, cosmids retroviruses or phages; the expression vector can be used to express a DNA sequence encoding a desired protein and in one aspect includes a transcriptional unit comprising an assembly of expression control sequences. The choice of promoter and other regulatory elements can vary according to the intended host cell, or in-vitro expression system.
As used herein, a “recombination system” refers to one which allows for recombination between a vector of the present application and a chromosome for incorporation of a gene of interest. Recombination systems are known in the art and include Cre/Lox systems and FLP-IN systems.
As used herein an “in vitro expression system” refers to cell free systems that enable the transcription, or coupled transcription and translation of DNA templates. Such systems include for example the classical rabbit reticulocyte system, as well as novel cell free synthesis systems, (J. Biotechnol. (2004) 110 (3): 257-63; Biotechnol Annu. Rev. (2004) 10:1-30).
As used herein, a “Cre/Lox” system refers to one such as described by Abremski et al., Cell, 32: 1301-1311 (1983) for a site-specific recombination system of bacteriophage P1. Methods of using Cre-Lox systems are known in the art; see, for example, U.S. Pat. No. 4,959,317, which is hereby incorporated in its entirety by reference. The system consists of a recombination site designated loxP and a recombinase designated Cre. In methods for producing site-specific recombination of DNA in eukaryotic cells, DNA sequences having first and second lox sites can be introduced into eukaryotic cells and contacted with Cre, thereby producing recombination at the lox sites.
As used here, “FLP-IN” recombination refers to systems in which a polynucleotide activation/inactivation and site-specific integration system has been developed for mammalian cells. The system is based on the recombination of transfected sequences by FLP, a recombinase derived from Saccharomyces. In several cell lines, FLP has been shown to rapidly and precisely recombine copies of its specific target sequence. FLP-IN systems have been described in, for example, U.S. Pat. Nos. 5,654,182 and 5,677,177).
The term “transfection,” “transformation,” or “transduction” as used herein, refers to the introduction of one or more exogenous polynucleotides into a host cell by using one or physical or chemical methods. Many transfection techniques are known to those of ordinary skill in the art including but not limited to calcium phosphate DNA co-precipitation (see Methods in Molecular Biology, Vol. 7, Gene Transfer and Expression Protocols, Ed. E. J. Murray, Humana Press (1991)); DEAE-dextran; electroporation; cationic liposome-mediated transfection; tungsten particle-facilitated microparticle bombardment (Johnston, S. A., Nature 346: 776-777 (1990)); and strontium phosphate DNA co-precipitation (Brash D. E. et al. Molec. Cell. Biol. 7: 2031-2034 (1987). Phage, or retroviral vectors can be introduced into host cells, after growth of infectious particles in packaging cells that are commercially available.
The terms “cells,” “cell cultures,” “cell line,” “recombinant host cells,” “recipient cells” and “host cells” are often used interchangeably and will be clear from the context in which they are used. These terms include the primary subject cells and any progeny thereof, without regard to the number of transfers. It should be understood that not all progeny are exactly identical to the parental cell (due to deliberate or inadvertent mutations or differences in environment), however, such altered progeny are included in these terms, so long as the progeny retain the same functionality as that of the originally transformed cell. For example, though not limited to, such a characteristic might be the ability to produce a particular recombinant protein. A “mutator positive cell line” is a cell line containing cellular factors that are sufficient to work in combination with other vector elements to affect hypermutation. The cell line can be any of those known in the art or described herein. A “clone” is a population of cells derived from a single cell or common ancestor by mitosis.
A “reporter gene” refers to a polynucleotide that confers the ability to be specifically detected (or detected and selected), when expressed with a cell of interest. Numerous reporter gene systems are known in the art and include, for example, alkaline phosphatase (Berger, J., et al., Gene 66: 1-10 (1988); Kain, S R., Methods Mol. Biol. 63: 49-60 (1997)), beta-galactosidase (U.S. Pat. No. 5,070,012), chloramphenicol acetyltransferase (Gorman et al., Mol. Cell. Biol. 2: 1044-51 (1982)), beta glucuronidase, peroxidase, beta lactamase (U.S. Pat. Nos. 5,741,657, 5,955,604), catalytic antibodies, luciferases (U.S. Pat. Nos. 5,221,623; 5,683,888; 5,674,713; 5,650,289; 5,843,746) and naturally fluorescent proteins (Tsien, R Y, Annu. Rev. Biochem. 67 509-544 (1998)). The term “reporter gene,” also includes any peptide which can be specifically detected based on the use of one or more, antibodies, epitopes, binding partners, substrates, modifying enzymes, receptors, or ligands that are capable of, or desired to (or desired not to), interact with the peptide of interest to create a detectable signal. Reporter genes also include genes that can modulate cellular phenotype.
The term “selectable marker gene” as used herein, refers to polynucleotides that allow cells carrying the polynucleotide to be specifically selected for or against, in the presence of a corresponding selective agent. Selectable markers can be positive, negative or bifunctional. Positive selectable markers allow selection for cells carrying the marker, whereas negative selectable markers allow cells carrying the marker to be selectively eliminated. The selectable marker polynucleotide can either be directly linked to the polynucleotides to be expressed, or introduced into the same cell by co-transfection. A variety of such marker polynucleotides have been described, including bifunctional (i.e., positive/negative) markers (see, e.g., WO 92/08796, published May 29, 1992, and WO 94/28143, published Dec. 8, 1994), hereby incorporated in their entirety by reference herein. Specific examples of selectable markers of drug-resistance genes include, but are not limited to, ampicillin, tetracycline, blasticidin, puromycin, hygromycin, ouabain or kanamycin. Specific examples of selectable markers are those, for example, that encode proteins that confer resistance to cytostatic or cytocidal drugs, such as the DHFR protein, which confers resistance to methotrexate (Wigler et al., Proc. Natl. Acad. Sci. USA, 77:3567 (1980); O'Hare et al., Proc. Natl. Acad. Sci. USA, 78:1527 (1981)); the GPF protein, which confers resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA, 78:2072 (1981)), the neomycin resistance marker, which confers resistance to the aminoglycoside G-418 (Colberre-Garapin et al., J. Mol. Biol., 150:1 (1981)); the hygromycin protein, which confers resistance to hygromycin (Santerre et al., Gene, 30:147 (1984)); murine Na+, K+-ATPase alpha subunit, which confers resistance to ouabain (Kent et al., Science, 237:901-903 (1987); and the Zeocin™ resistance marker (available commercially from Invitrogen). In addition, the herpes simplex virus thymidine kinase (Wigler et al., Cell, 11:223 (1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA, 48:2026 (1962)), and adenine phosphoribosyltransferase (Lowy et al., Cell, 22:817 (1980)) can be employed in tk⁻, hgprt⁻ or aprt⁻ cells, respectively. Glutamine synthetase permits the growth of cells in glutamine(GS)-free media (see, e.g., U.S. Pat. Nos. 5,122,464; 5,770,359; and 5,827,739). Other selectable markers encode, for example, puromycin N-acetyl transferase or adenosine deaminase.
“Homology” or “identity” or “similarity” refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology and identity can each be determined by comparing a position in each sequence which can be aligned for purposes of comparison. When an equivalent position in the compared sequences is occupied by the same base or amino acid, then the molecules are identical at that position; when the equivalent site occupied by the same or a similar amino acid residue (e.g., similar in steric and/or electronic nature), then the molecules can be referred to as homologous (similar) at that position. Expression as a percentage of homology/similarity or identity refers to a function of the number of identical or similar amino acids at positions shared by the compared sequences. A sequence which is “unrelated” or “non-homologous” shares less than 40% identity, less than 35% identity, less than 30% identity, or less than 25% identity with a sequence of the present invention. In comparing two sequences, the absence of residues (amino acids or nucleic acids) or presence of extra residues also decreases the identity and homology/similarity.
The term “homology” describes a mathematically based comparison of sequence similarities which is used to identify genes or proteins with similar functions or motifs. The nucleic acid and protein sequences of the present invention can be used as a “query sequence” to perform a search against public databases to, for example, identify other family members, related sequences or homologs. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and BLAST) can be used (See www.ncbi.nlm.nih.gov).
As used herein, “identity” means the percentage of identical nucleotide or amino acid residues at corresponding positions in two or more sequences when the sequences are aligned to maximize sequence matching, i.e., taking into account gaps and insertions. Identity can be readily calculated by known methods, including but not limited to those described in (Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988). Methods to determine identity are designed to give the largest match between the sequences tested. Moreover, methods to determine identity are codified in publicly available computer programs. Computer program methods to determine identity between two sequences include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA (Altschul, S. F. et al., J. Molec. Biol. 215: 403-410 (1990) and Altschul et al. Nuc. Acids Res. 25: 3389-3402 (1997)). The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990). The well known Smith Waterman algorithm can also be used to determine identity.
A “heterologous” region of a DNA sequence is an identifiable segment of DNA within a larger DNA sequence that is not found in association with the larger sequence in nature. Thus, when the heterologous region encodes a mammalian gene, the gene can usually be flanked by DNA that does not flank the mammalian genomic DNA in the genome of the source organism. Another example of a heterologous coding sequence is a sequence where the coding sequence itself is not found in nature (e.g., a cDNA where the genomic coding sequence contains introns or synthetic sequences having codons or motifs different than the unmodified gene). Allelic variations or naturally-occurring mutational events do not give rise to a heterologous region of DNA as defined herein.
The term “activation-induced cytidine deaminase” or (“AID”) refers to members of the AID/APOBEC family of RNA/DNA editing cytidine deaminases capable of mediating the deamination of cytosine to uracil within a DNA sequence. (See, e.g., Conticello et al., Mol. Biol. Evol. 22 No 2 367-377 (2005), “Evolution of the AID/APOBEC Family of Polynucleotide (Deoxy)cytidine Deaminases” and U.S. Pat. No. 6,815,194). Suitable AID enzymes include all vertebrate forms of the enzyme, including, for example, primate, rodent, avian and bony fish. Representative examples of AID enzymes include without limitation, human (GenBank Accession No. NP_—065712), rat, chicken, canine and mouse (GenBank Accession No. NP_—033775) forms. The term “AID homolog” refers to the enzymes of the Apobec family and include, for example, Apobec and, in particular, can be selected from Apobec family members such as Apobec-1, Apobec3C or Apobec3G (described, for example, by Jarmuz et al., (2002) Genomics, 79: 285-296) (2002)). AID and AID homologs also include, without limitation, modified polypeptides (e.g. mutants or muteins) that retain the ability to deaminate a polynucleotide sequence. The term “AID activity” includes activity mediated by AID and AID homologs.
The term “transition mutations” refers to base changes in a DNA sequence in which a pyrimidine (cytidine (C) or thymidine (T) is replaced by another pyrimidine, or a purine (adenosine (A) or guanosine (G) is replaced by another purine.
The term “transversion mutations” refers to base changes in a DNA sequence in which a pyrimidine (cytidine (C) or thymidine (T) is replaced by a purine (adenosine (A) or guanosine (G), or a purine is replaced by a pyrimidine.
The term “base excision repair” refers to a DNA repair pathway that removes single bases from DNA such as uridine nucleotides arising by deamination of cytidine. Repair is initiated by uracil glycosylase that recognizes and removes uracil from single- or double-stranded DNA to leave an abasic site.
The term “mismatch repair” refers to the repair pathway that recognizes and corrects mismatched bases, such as those that arise from errors of chromosomal DNA replication.
The term “pol eta” (also called PolH, RAD30A, XPV, XP-V) refers to a low-fidelity DNA polymerase that plays a role in replication through lesions, for instance, replication through UV-induced thymidine dimers. The gene for pol eta is defective in Xeroderma pigmentosum variant type protein, XPV. On non-damaged DNA, pol eta mis-incorporates incorrect nucleotides at a rate of approximately 3 per 100 bp, and is especially error-prone when replicating through templates containing WA dinucleotides (W=A or T) (Gearhart and Wood, 2001). Pol eta has been shown to play an important role as an A/T mutator during SHM in immunoglobulin variable genes (Zeng et al., 2001). Representative examples of pol eta include without limitation, human (GenBank Accession No. BAA81666), rat (GenBank Accession No. XP_—001066743), chicken (GenBank Accession No. NP_—001001304), canine (GenBank Accession No. XP_—532150) and mouse (GenBank Accession No. NP_—109640) forms.
The term “pol theta” (also called PolQ) refers to a low-fidelity DNA polymerase that may play a role in crosslink repair (Gearhart and Wood, Nature Rev Immunol 1: 187-192 (2001)) and contains an intrinsic ATPase-helicase domain (Kawamura et al., Int. J. Cancer 109(1):9-16 (2004)). The polymerase is able to efficiently replicate through an abasic site by functioning both as a mispair inserter and as a mispair extender (Zan et al., EMBO Journal 24, 3757-3769 (2005)). Representative examples of pol theta include without limitation, human (GenBank Accession No. NP_—955452), rat (GenBank Accession No. XP_—221423), chicken (GenBank Accession No. XP_—416549), canine (GenBank Accession No. XP_—545125) and mouse (GenBank Accession No. NP_—084253) forms. Pol ete and Pol theta are sometimes referred to collectively as “error prone polymerases.”
As used herein, the term “SHM hot spot” or “hot spot” or “hot spot motif” refers to a polynucleotide sequence or motif of 3-6 nucleotides (i.e., 1-2 codons) that exhibits an increased tendency to undergo SHM, as determined via a statistical analysis of SHM mutations in antibody genes (see Tables 2 and 3 which provide a relative ranking of various motifs for SHM, and Table 7 which lists canonical hot spots and cold spots). The statistical analysis can be extrapolated to analysis of SHM mutations in non-antibody genes as described elsewhere herein. For the purposes of graphical representations of hot spots in Figures, the first nucleotide of a canonical hot spot is represented by the letter “H.”
Likewise, as used herein, a “SHM coldspot” or “cold spot” or “cold spot motif” refers to a polynucleotide or motif of 3-6 nucleotides (i.e., 1-2 codons) that exhibits a decreased tendency to undergo SHM, as determined via a statistical analysis of SHM mutations in antibody genes (see Tables 2 and 3 which provide a relative ranking of various motifs for SHM, and Table 7 which lists canonical hot spots and cold spots). The statistical analysis can be extrapolated to analysis of SHM mutations in non-antibody genes as described elsewhere herein. For the purposes of graphical representations of cold spots in Figures, the first nucleotide of a canonical cold spot is represented by the letter “C.”
The term “somatic hypermutation motif” or “SHM motif” refers to a polynucleotide sequence that includes, or can be altered to include, one or more hot spots and/or cold spots, and which encodes a defined set of amino acids. SHM motifs can be of any size, but are conveniently based around polynucleotides of about 2 to about 20 nucleotides in size, preferred SHM motifs range from about 3 to about 9 nucleotides in size. SHM motifs can include any combination of canonical hot spots and/or cold spots, or may lack both canonical hot spots and/or cold spots.
The term “preferred SHM motif” refers to one or more preferred SHM codons (see Table 7 infra).
The terms “preferred hot spot SHM codon,” “preferred hot spot SHM motif,” “preferred SHM hot spot codon” and “preferred SHM hot spot motif,” all refer to a codon including, but not limited to codons AAC, TAC, TAT, AGT and AGC. Such sequences may be potentially embedded within the context of a larger SHM motif, recruit SHM mediated mutagenesis and generate targeted amino acid diversity at that codon.
A polynucleotide sequence has been “optimized for SHM” if the polynucleotide, or a portion thereof has been altered to increase or decrease the frequency and/or location of hot spots and/or cold spots within the polynucleotide. A polynucleotide that has been made “susceptible to SHM” if the polynucleotide, or a portion thereof, has been altered to increase the frequency (density) and/or location of hot spots within the polynucleotide or to decrease the frequency and/or location of cold spots within the polynucleotide. Conversely, a polynucleotide that has been made “resistant to SHM” if the polynucleotide, or a portion thereof, has been altered to decrease the frequency and/or location of hot spots and/or has been altered to increase the frequency (density) and/or location of cold spots within the polynucleotide. In one embodiment, a sequence can be prepared that has a greater or lesser susceptibility (or rate) to undergo SHM-mediated mutagenesis by altering the codon usage, and/or the amino acids encoded by polynucleotide sequence relative to the unmodified polynucleotide.
Optimization of a polynucleotide sequence refers to modifying about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 20%, about 25%, about 50%, about 75%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, 100% or any range therein of the nucleotides in the polynucleotide sequence. Optimization of a polynucleotide sequence also refers to modifying about 1, about 2, about 3, about 4, about 5, about 10, about 20, about 25, about 50, about 75, about 90, about 95, about 96, about 97, about 98, about 99, about 100, about 200, about 300, about 400, about 500, about 750, about 1000, about 1500, about 2000, about 2500, about 3000 or more, or any range therein of the nucleotides in the polynucleotide sequence such that some or all of the nucleotides are optimized for SHM-mediated mutagenesis. Reduction in the frequency (density) of hot spots and/or cold spots refers to reducing about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 20%, about 25%, about 50%, about 75%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, 100% or any range therein of the hot spots or cold spots in a polynucleotide sequence. Increasing the frequency (density) of hot spots and/or cold spots refers to increasing about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 20%, about 25%, about 50%, about 75%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, 100% or any range therein of the hot spots or cold spots in a polynucleotide sequence.
The position or reading frame of a hot spot or cold spot is also a factor governing whether SHM mediated mutagenesis that can result in a mutation that is silent with regards to the resulting amino acid sequence, or causes conservative, semi-conservative or non conservative changes at the amino acid level. As discussed below, these design parameters can be manipulated to further enhance the relative susceptibility or resistance of a nucleotide sequence to SHM. Thus both the degree of SHM recruitment and the reading frame of the motif are considered in the design of SHM susceptible and SHM resistant polynucleotide sequences.
As used herein, “somatic hypermutation” or “SHM” refers to the mutation of a polynucleotide initiated by, or associated with the action of activation-induced cytidine deaminase, uracil glycosylase and/or error prone polymerases on that polynucleotide sequence. The term is intended to include mutagenesis that occurs as a consequence of the error prone repair of the initial lesion, including mutagenesis mediated by the mismatch repair machinery and related enzymes.
As used herein, the term “UDG” refers to uracil DNA glycosylase, one of several DNA glycosylases that recognize different damaged DNA bases and remove them before replication of the genome. DNA glycosylases can remove DNA bases that are cytotoxic or cause DNA polymerase to introduce errors, and are part of the base excision repair pathway for DNA. Uracil DNA glycosylase recognizes uracil in DNA, a product of cytidine deamination, leading to its removal and potential replacement with a new base.
The term “isolated” refers to the state in which polypeptides or polynucleotides of the invention will be in accordance with the present invention. Polypeptides or polynucleotides will be free or substantially free of material with which they are naturally associated such as other polypeptides or polynucleotides with which they are found in their natural environment, or the environment in which they are prepared (e.g. cell culture) when such preparation is by recombinant DNA technology practiced in vitro or in vivo. Polypeptides or polynucleotides can be formulated with diluents or adjuvants and still for practical purposes be isolated—for example, the polypeptides or polynucleotides can be mixed with gelatin or other carriers if used to coat microtitre plates for use in immunoassays, or can be mixed with pharmaceutically acceptable carriers or diluents when used in diagnosis or therapy. Polypeptides or polynucleotides can be glycosylated, either naturally or by systems of heterologous eukaryotic cells, or they can be (for example, if produced by expression in a prokaryotic cell) unglycosylated.
The term “selection” refers to the separation of one or more members, such as polynucleotides, proteins or cells from a library of such members. Selection can involve both detection and selection, for example where cells are selected by use of a fluorescence activated cell sorter (FACS) that detects a reporter gene and then sorts the cells accordingly.
As used herein, “pg” means picogram, “ng” means nanogram, “ug” or “μg” mean microgram, “mg” means milligram, “g” means gram “ul” or “μl” mean microliter, “ml” means milliliter, “l” means liter, “kb” means kilobases, “nM” means nanomolar, “pM” means picomolar, “fM” means femtomolar, and “M” means molar.
The phrase “pharmaceutically acceptable” refers to molecular entities and compositions that are physiologically tolerable and do not produce an unsafe reaction.

II. Somatic Hypermutation

During the generation of antibodies, in vivo point mutations occur within the variable region (V-region) coding sequence of the antigen receptor, and the rate of mutation observed, called SHM, is about a million times greater than the spontaneous mutation rate in other genes. Bachl et al., Increased transcription levels induce higher mutation rates in a hypermutating cell line. J Immunol. 2001 Apr. 15; 166(8):5051-7; Martin and Scharff, AID and mismatch repair in antibody diversification. Nat Rev Immunol. 2002; 2(8):605-14.
In humans and mice, after the primary repertoire of antibody specificity is created by V-D-J rearrangement, and following antigen encounter, the rearranged V genes in those B cells that have been triggered by the antigen are subjected to two further types of genetic modification: SHM which triggers the diversification of the variable region of immunoglobulin genes, generating the secondary repertoire thereby allowing affinity maturation of the humoral response, and Class Switch Recombination (CSR) which involves the specific non-homologous recombination process which leads to isotype changes in the constant region of the expressed antibody. In chickens and rabbits (but not man or mouse) an additional mechanism, gene conversion, is a major contributor to V gene diversity.
AID is expressed within activated B cells and is an essential protein factor for SHM, CSR and gene conversion (Muramatsu et al., 2000; Revy et al., 2000). AID belongs to a family of enzymes, the APOBEC family, which share certain features with the metabolic cytidine deaminases but differs from them in that AID deaminates nucleotides within single stranded polynucleotides, and cannot utilize free nucleotide as a substrate. Other enzymes of the AID/APOBEC family can also act to deaminate cytidine on single stranded RNA or DNA (Conticello et al., (2005)).
The human AID protein comprises 198 amino acids and has a predicted molecular weight of 24 kDa. The human AID gene is located at locus 12p13, close to APOBEC-1. The AID protein has a cytidine/deoxycytidine deaminase motif, is dependent on zinc, and can be inhibited by tetrahydrouridine (THU) which is a specific inhibitor of cytidine deaminases.
Even prior to the discovery of unmodified AID, it was noted that SHM occurs more frequently in cytidines that are within the context of WRCY (AT/GA/C/AT) motifs. There is now accumulating evidence that this motif for SHM likely represents a composite of this hot spot motif for AID deamination and for initiating error prone repair by the DNA polymerases pol eta and pol theta (Rogozin et al. (2004); Zan et al. (2005)).
High levels of DNA transcription have been shown necessary but alone are not sufficient for AID-mediated mutagenesis. In vivo, SHM begins about 80 to about 100 nucleotides from the transcription start site, but decreases in frequency as a function of distance from the promoter. Native AID has been shown in vitro to interact directly with the transcriptional elongation complex, but not the transcriptional initiation complex, and this interaction may be dependent upon the dissociation of the initiation factors, that occurs as the transcriptional initiation complex converts to the fully processive, elongation-competent transcription elongation complex (Besmer et al., 2006).
Since AID is only able to deaminate cytidines on single stranded DNA, it is likely that the requirement for transcription reflects the generation of single stranded regions by transcription bubbles. Studies with purified unmodified AID in vitro, however, suggest that AID binding is sequence independent, potentially allowing a scanning mode for hot spot capture that is driven by active transcription of the gene. In vitro studies suggest that unmodified AID has an apparent Kd for single stranded DNA in the range of 0.3 to 2 nM, and that the complex has a half life of 4-8 minutes. The turnover number of purified unmodified AID on single stranded DNA is approximately one deamination every 4 minutes, (Larijani et al., (2006)).
AID acts on DNA to deaminate cytidine residues to uracil residues on either strand of the transcribed DNA molecule. If the initial (C→U) lesion is not further modified prior to, or during DNA replication, then an adenosine (A) can be inserted opposite the U nucleotide, ultimately resulting in C→T or G→A transition mutations. The significance of this change at the amino acid level depends upon the location of the nucleotide within the codon within the reading frame. If this mutation occurs in the first or second position of the codon, the result is likely to be a non conservative amino acid substitution. By contrast, if the change occurs at the third position of the codon reading frame, within the wobble position, the practical effect of the mutation at the amino level will be slight because the effect of the nucleotide change will be silent or result in a conservative amino acid substitution.
Alternatively, the C→U lesion, and potentially the neighboring bases can be acted upon by DNA repair machinery, which in SHM, leads to repair in an error prone fashion. Studies in knock out mice have established that base excision repair via uracil DNA glycosylase (UDG), plays a role in mediating the mutation of A and T residues close to hot spot motifs; (Shen et al (2006)). Additionally there is increasing evidence that the creation of abasic sites by UDG recruits error prone polymerases, such as pol eta and pol theta, and that these polymerases introduce additional mutations at all base positions in the surrounding sequence (Watanabe et al. (2004); Neuberger et al (2005)). It is believed that pol eta is central to the creation of A mutations during SHM and is particularly error prone for coding strand adenosines proceeded by A or T (W/A) that are preferentially mutated to G.
It has been observed that in antibody genes, codon usage and precise concomitant hot spot/cold spot targeting of AID activity and pol eta errors in the CDRs and FRs, respectively, has evolved under selective pressure to maximize mutations in the variable regions and minimize mutations in the framework regions (Zheng et al., JEM 201(9): 1467-1478 (2005)). For example, Zheng et al. observed that the precise alignment of C and G nucleotides within the codons preferentially used within an antibody gene causes most C to T and G to A mutations to be silent or conservative. Juxaposed on the precise placement of Cs and Gs, Zheng et al., also observed the preferential placement of As and Ts in hot spots of pol eta in the variable regions and the exclusion from these sites in the framework regions.
The regulation of SHM in vivo and the determinants that direct and limit SHM to the Ig locus has been the subject of intense debate and experimental research. The rate of SHM observed in vivo has been shown to be at least partially dependent upon, for example, the following factors: 1) the AID expression levels and AID activity levels within a particular cell type; (Martin et al. (2002), Rucci et al., (2006)), 2) the degree of AID post translational modification and degree of nuclear localization; (McBride et al. (2006), Pasqualucci et al. (2006), Muto et al. (2006)), 3) the presence of immune locus specific enhancer regions, E-box motifs, or associated cis acting binding factors; (Komori et al. (2006), Schoetz, et al. (2006)), 4) the proximity of the targeted sequence to the transcriptional initiation site/promoter region; (Rada et al., (2001)), 5) the rate of transcription of the target sequence; (Storb et al., (2001)), 6) the degree of target gene methylation; (Larijani et al (2005)), 7) the genomic context of the target gene, if integrated into the cell's genomic DNA; 8) the presence or absence of auxiliary factors, such as Pol Eta, MSH2; (Shen et al. (2006)), 9) the existence of hotspot or coldspot sequences within the target sequence; (Zheng et al. (2005)), 10) the existence of inhibitory factors; (Santa-Marta, et al. (2006)), 11) rate of DNA repair within the cell type of interest, (Poltoratsky (2006)), 12) the formation of local DNA or RNA hairpins structures; (Steele et al. (2006)), and 13) the phosphorylation state of histone H2B (Odegard et al. (2005)).
The present invention is based, in part, upon the optimization of some of the factors above to create both a temporally and spatially controlled system for hypermutation.

III. Identification and Analysis of Polynucleotides for Somatic Hypermutation

Previous analyses of antibody sequences, see, for example, Zheng et al. J. Exp. Med. (2005); 201(9):1467-1478; and Wang and Wabl, J. Immunol. 2005; 174(9):5650-4, have been based primarily on identifying the polynucleotide sequence motifs involved in SHM rather than elaborating the underlying logical operations that connect multiple rounds of SHM during protein evolution within a polynucleotide sequence.
As a first step to developing such an improved understanding of the context of one or more rounds of SHM within the reading frame of a polynucleotide sequence, and the underlying logic of relationships within codon usage patterns, the present applicants have used a statistical approach to identify consensus hot spots and cold spots. Such a statistical approach can also be used to functionally track the consequences of those mutations at the nucleotide level and to structural consequences at the amino acid level following SHM by AID.
Starting from any given polynucleotide sequence, this approach can be used to generate polynucleotide sequences that rapidly converge to structural consequences at the amino acid level a small number of possible sequences that are optimized for the properties described herein.
Polynucleotides sequences of the present application include fully synthetic polynucleotides, fully synthetic genes, semi-synthetic polynucleotides and semi-synthetic genes. “Semi-synthetic polynucleotides” and “semi-synthetic genes” as used herein, refer to a polynucleotide sequences that consists in part of a nucleic acid sequence that has been obtained via polymerase chain reaction (PCR) or other similar enzymatic amplification system which utilizes a natural donor (i.e., peripheral blood monocytes) as the starting material for the amplification reaction. The remaining “synthetic” polynucleotides, i.e., those portions of semi-synthetic polynucleotide not obtained via PCR or other similar enzymatic amplification system can be synthesized de-novo using methods known in the art including, but not limited to, the chemical synthesis of nucleic acid sequences.
In the present application, where the observed number of SHM mutations at a polynucleotide motif is conditioned on the underlying frequency of observing that motif, the degree to which a polynucleotide sequence or motif is a SHM “hot spot” or “cold spot” was derived from analysis of SHM mutations identified in approximately 50,000 antibody sequences. One measure of the statistical significance of a SHM “hot” or “cold” motif compares the number of times a motif is observed (Ns) at the site of a SHM mutation event, with how often it would be expected at random (Nps) (where N equals the total number of observed mutations in the dataset and ps is the background probability of observing the motif). A Markov chain was used to calculate the background frequency of the observing any motif at the site of mutation, as described previously (Tompa, 1999), using di-nucleotide transition probabilities taken directly from human germline IGHV sequences, as shown below.
$ij = ⌊ \begin{matrix} 0.169 & 0.270 & 0.381 & 0.179 \\ 0.289 & 0.287 & 0.101 & 0.321 \\ 0.239 & 0.219 & 0.314 & 0.227 \\ 0.155 & 0.278 & 0.413 & 0.154 \end{matrix} ⌋ where i, j \in {A, C, T, G}$
The difference in the number of observed:expected motifs occurrences at the site of mutation is given by Ns−Nps, where √{square root over (Np_s(1−p_s))} represents the standard deviation of Nps, and the z-score for each motif is given by:
M _s=(N _s −Np _s)/√{square root over (Np _s(1−p _s))}
where Ms is the number of standard deviations by which the observed number of motif occurrences (at the site of mutation) exceeds the expected value. This metric can been used to rank all possible SHM “hot spot” and “cold spot” motifs, and to characterize the degree to which any motif is “hot” or “cold” to SHM mediated mutagenesis. For instance, those 3-mer, 4-mer, 5-mer, or 6-mer polynucleotide motifs having rank-ordered z-scores in the top 5% or 10% of all equivalent length polynucleotide motifs can be considered SHM “hot spots.” Likewise, those 3-mer, 4-mer, 5-mer or 6-mer polynucleotide motifs having rank-ordered z-scores in the bottom 5% or 10% of all equivalent length motifs can be considered SHM “cold spots.” In one aspect, the hot spot can be, for example, a preferred hot spot SHM codon or motif or a more preferred hot spot SHM codon or motif. Rank-ordered tables of the top 3-mer, 4-mer and 6-mer nucleotide sequences with their corresponding SHM mutation z-scores, describing their propensity to attract SHM-mediated mutagenesis (i.e., be more susceptible to SHM), are provided below in Tables 2 and 3.

TABLE 2

3-	3-mer		4-mer		4-mer		4-mer		4-mer
mer	z-score	4-mer	z-score	4-mer	z-score	4-mer	z-score	4-mer	z-score

ATA	271.09	AATA	249.23	TACC	92.73	ACGA	19.69	CTGG	−55.05

AGC	185.10	AGCA	225.50	GAAA	89.97	TTTT	17.21	CGGA	−56.07

TAT	178.79	ATAT	224.06	CTGC	88.23	TTCT	16.95	ACGG	−58.65

CAG	176.52	AACA	215.78	CCAA	87.55	GATC	16.55	GCCT	−61.62

ACA	161.58	ATAA	213.14	TATC	86.83	TGTA	15.70	CGCC	−62.50

CCA	156.43	ATCA	193.93	CCCA	86.81	CCCC	14.29	CTTG	−63.02

ATT	128.07	TACA	190.78	GCTA	84.30	TTCC	8.07	AGTG	−64.08

AAT	123.91	CACA	183.94	CTTA	83.60	CGCA	7.95	GGAC	−66.33

CAC	113.31	ACAA	182.20	GCAA	83.41	CCTG	6.44	CCCG	−68.14

CAT	106.72	ATTA	174.57	ATCC	82.88	AAGT	6.21	GTGA	−69.31

GCT	99.04	CAGA	172.86	GAAT	82.09	GTTA	5.83	TTGT	−70.87

TCA	92.35	AACT	171.38	ATTC	80.57	GTAA	5.54	GCGA	−71.78

TAC	90.32	AGAT	167.36	AGCC	79.90	GACT	5.46	GTTT	−73.35

ACT	84.63	ACAG	165.72	CTCA	78.97	TCCT	4.16	GGGA	−75.77

ATC	82.30	CAAC	163.72	CCAG	78.46	GACC	2.64	CGTA	−76.30

AGA	78.69	TATA	159.43	AGTA	78.05	GGAT	−0.62	TCGA	−76.40

CTA	71.32	ATAC	157.31	TAGC	76.80	TCTG	−1.62	CGAG	−78.05

GCA	70.80	ACTA	152.17	ATTT	74.50	GCTG	−2.06	AGGG	−81.46

GAT	68.06	CAGC	148.78	ACTG	74.10	GATG	−2.19	GAGT	−82.94

CTG	67.83	ACCA	146.54	TCAC	71.95	ACCG	−2.66	CCGG	−85.06

ACC	65.99	AAGC	145.36	CTGA	68.58	TTTC	−4.30	GAGG	−85.74

GAA	59.03	AGAA	144.62	CCTA	67.05	TAGT	−4.65	GTTG	−86.35

TGA	56.50	AAAA	136.44	TCTA	66.67	CGCT	−5.54	TCCG	−88.86

ATG	52.18	ACAT	135.69	AATG	66.07	AGCG	−5.58	GTTC	−89.62

CAA	48.79	AGCT	134.58	GCAT	65.56	CCCT	−7.38	CGGC	−90.00

AAA	39.39	CAAT	133.12	ACCC	62.47	CCTC	−7.50	GCGC	−91.60

AAC	37.15	GATA	131.74	TCAT	61.22	TGGA	−8.79	CTCG	−92.05

TTA	35.04	ACAC	130.35	TGCT	61.11	CTGT	−10.50	TGGC	−92.93

TAA	31.78	ATCT	128.86	CTAG	59.03	GTAT	−10.53	TCGC	−96.14

AAG	24.73	CACC	125.86	ACTT	58.98	TATG	−13.14	TGTG	−96.30

CTT	17.61	CATA	125.75	AGAG	58.81	AAGG	−13.25	TTGG	−100.73

TTC	16.92	ATAG	121.65	TTAC	57.51	CCGC	−13.98	GGTT	−102.17

GTA	15.61	TAAT	121.29	TTTA	56.94	ATGG	−13.99	GCCG	−104.21

TAG	13.84	CAAA	121.00	TCAG	56.45	CGAA	−14.21	CCGT	−105.94

GGA	11.44	TATT	120.42	ATGC	54.70	TCTT	−15.45	GTCT	−108.78

TTT	6.80	CTAA	119.93	AGAC	53.01	TGAC	−16.19	GGCC	−110.06

AGT	2.60	CATC	118.61	TGAT	51.51	CCTT	−16.61	GACG	−112.93

CTC	−1.47	TTCA	117.73	GCAC	51.04	CACG	−19.16	TGGT	−115.42

TCC	−5.22	AAAC	116.35	AGGA	50.16	GGCA	−21.99	GTGC	−117.74

CCT	−5.42	TTAT	114.64	TAAG	49.76	TCCC	−23.02	TTCG	−118.98

CCC	−7.09	AAAT	114.43	CAGT	49.09	AACG	−26.20	ACGT	−121.92

GAG	−8.26	CCAT	113.51	ACTC	46.69	CGAT	−27.41	GCGG	−124.24

TGC	−14.70	ACCT	111.92	AGTT	45.47	AGGT	−29.09	TGCG	−126.58

TCT	−18.88	TAAC	111.26	CAAG	43.20	TCTC	−29.53	TGGG	−127.63

GAC	−23.11	CTAT	110.83	CTCC	43.07	TTGC	−29.86	GTCC	−128.75

AGG	−27.85	TAAA	110.30	GTAC	42.84	CCGA	−32.32	GGGC	−132.40

GCC	−38.10	CCAC	110.05	GAAC	42.62	TGAG	−34.69	GGGG	−133.41

TGG	−40.97	AATT	109.92	GAGC	41.24	ATGT	−34.90	TCGT	−135.34

TTG	−43.86	TGCA	107.12	GCCA	40.88	TAGG	−37.28	GGTG	−135.80

ACG	−61.29	CATT	106.83	GCTT	39.88	GGCT	−38.30	CGTT	−136.77

GTT	−62.25	TCAA	104.12	CAGG	37.16	GCCC	−40.66	TGTC	−137.57

CGA	−62.60	AAAG	103.76	GATT	35.99	GGAG	−44.01	GTGT	−142.24

TGT	−64.56	TACT	101.53	GACA	35.71	TGTT	−44.49	CGGT	−144.04

GGC	−70.30	AAGA	100.90	CTTC	34.67	CGAC	−45.06	GTGG	−149.24

CGC	−82.93	CACT	100.32	CTCT	33.87	GGTA	−46.07	CGTC	−155.95

CCG	−85.43	AACC	99.86	GAAG	31.97	AGGC	−46.08	GGTC	−158.84

GGG	−97.46	GCAG	99.17	TTGA	31.29	TACG	−46.78	TCGG	−159.56

GTG	−110.90	ATGA	98.38	CTTT	28.94	AGTC	−46.82	CGGG	−159.99

GGT	−112.41	CTAC	95.93	TTAG	27.86	ACGC	−47.10	GGGT	−162.17

CGG	−116.32	TCCA	95.63	GGAA	26.38	ATCG	−48.15	GGCG	−171.27

GCG	−118.80	AATC	95.61	ATTG	25.55	GTCA	−52.15	CGCG	−172.40

TCG	−125.83	TGAA	93.81	CATG	24.39	TTTG	−52.48	CGTG	−180.34

GTC	−126.67	TTAA	93.67	GCTC	22.00	GTAG	−53.73	GCGT	−194.57

CGT	−130.10	TAGA	93.03	GAGA	21.55	TGCC	−54.56	GTCG	−207.74

	TABLE 3

		6-mer
	6-mer	z-score

	ACAGCT	266.45

	ATTAAT	248.7

	ATAATA	227

	CAGCTA	223.27

	AATATA	220.6

	AATACA	215.65

	AGCTAC	211.24

	AGATAT	211.07

	AGCTAA	210.24

	ATATAT	209.3

	AATACT	203.19

	ATATAC	192.44

	ATAACT	190.78

	ATATTA	189.76

	ATAGCA	186.89

	ATACCA	186.58

	ATACAA	181.41

	GCAGCT	180.69

	ATTACA	180.46

	CAGCTC	180.29

	ATAGCT	180.08

	AATAAT	179.41

	AGCTAT	178.14

	CAGCTT	176.31

	ATATCT	174.41

	AGCTGC	169

	CAGCTG	167.78

	AGCTGA	167.41

	AATAAA	167.35

	ACTACA	167.11

	AACAGC	167.08

	ATTATT	166.89

	AAGCTA	166.44

	ACTACT	164.71

	AATACC	164.29

	TATTAT	164.1

	ACAGCA	161.72

	AGCAGA	160.66

	AGCAAT	159.61

	TAATAC	159.28

	AATCCA	156.67

	AATAGA	156.3

	TATACA	155.5

	AGCTCC	153.55

	CATATA	152.22

	ATACAT	151.77

	TATATT	150.71

	TAATAT	150.37

	ATTACT	150.2

	TCAGCT	149.79

	AACTAC	149.11

	AAAGCT	148.88

	CAGCAT	147.47

	ATACAC	147.42

	ATAGAT	147.33

	ATCAGC	147.06

	AGATAC	146.34

	AGCACA	146.01

	CAGATA	145.75

	TAGCTA	145.22

	TTAGCT	144.8

	AAGCTG	143.55

	CACAGC	141.38

	ACAACT	140.89

	CATACA	139.87

	AGCAGC	139.64

	ACTATT	139.36

	CCAGCT	137.43

	GATACA	136.87

	AGCTTC	136.64

	AGCTCA	136.52

	ACCAGC	136.02

	AAATAC	135.35

	AGCTTA	135.22

	AGAGCT	134.71

	TAACTA	134.57

	TACTAC	134.52

	AACTAT	133.79

	ATAAAC	132.79

	TAGATA	132.74

	AACACA	131.7

	CTAATA	131.46

	AATAGC	130.99

	GAGCTA	130.78

	ATACTA	130.56

	ATATCA	130.47

	CTACTA	130.24

	ATACAG	129.95

	CCAGCA	129.73

	CAGCAG	129.37

	AATGCA	128.88

	ACTAAT	128.87

	AGCTTT	128.11

	ATCCAC	128.11

	GAAGCT	126.98

	CAGCAA	126.51

	ACCACC	126.44

	GCTACA	126.36

	AGCTGT	126.35

	ATAACA	126.34

	AGTTAT	125.56

	TTACTA	125.4

	AATTAC	124.76

	AATTCA	123.97

	CAGCAC	123.54

	ACAGCC	123.25

	TTAATA	122.8

	AGTATT	122.69

	CAACTA	122.15

	CAATAA	121.87

	AGCAAC	121.8

	ATCTAC	121.63

	TACACC	121.61

	AGCACC	121.59

	ATAGCC	120.05

	TAGCTG	119.3

	AAAACA	119.25

	ATTATA	119.17

	AGTACT	118.38

	CACCAT	117.87

	ATCTAT	116.19

	ACCATT	115.23

	TACTAT	115.17

	TCAGCA	115.13

	AGCATA	114.84

	TATTAA	114.69

	CAAGCT	113.83

	AGATGA	113.27

	GATATA	112.88

	TAGCTT	112.54

	TATTAC	111.72

	AGCTCT	111.46

	TCACCA	111.34

	ATAGTA	110.66

	ATACCT	110.48

	AGCATC	109.68

	TATCTA	109.46

	TACAAC	108.83

	GCAGCA	108.59

	AGTAAT	108.57

	TGCACA	108.53

	TTTATT	108.51

	ATGATA	108.34

	CAAATA	108.12

	ACAATA	107.6

	AATAGT	107.19

	AACAAC	107.08

	CACCAG	107.01

	TAGCTC	106.68

	TACAGC	106.65

	AACTGA	106.63

	GCATAT	106.63

	GAGCTG	106.39

	ATTCAC	106.22

	AAATAA	105.92

	TAGCAA	105.71

	CCAGAT	105.22

	ACCATC	105.14

	AATAAC	105.1

	TACCAT	104.92

	AGAACA	104.85

	ATCATA	104.56

	ATCACC	104.5

	AGAAAT	104.29

	ATATAA	104.19

	CATATC	103.97

	ATTCCA	103.78

	GGAGCT	102.99

	TACAGA	102.58

	TACTAA	102.18

	ATCACT	102.01

	ATATGA	101.89

	AAACAG	101.82

	ACACAG	101.77

	ACACCA	101.38

	ACAACC	101.23

	TAAGCT	100.84

	CAATAG	100.69

	CTATTA	100.61

	TTACCA	100.56

	AGTACA	100.42

	AACCAC	100.39

	CCACCA	100.19

	AAACAC	99.94

	ATAAAT	99.38

	GCTATA	99.35

	GTAGCT	99.14

	CAGCCA	99.11

	TTCAGC	99

	AGACAC	98.97

	AGCACT	98.85

	CCAATA	98.8

	AAACCA	98.68

	CAGCCT	98.34

	AAGCAC	98.34

	ACTGCA	98.25

	AGAAGC	98.23

	CCATCA	98.1

	CAACCA	97.53

	CAACTG	97.51

	ATTAGC	97.37

	AATATT	96.98

	ACCACA	96.82

	ATATGC	96.53

	GTATTA	96.49

	CATAGC	96.33

	GTATAT	96.2

	ACCAAC	96.14

	CAGATC	96.05

	AACATA	96.05

	AGATCC	95.89

	CTACCA	95.82

	GATCCA	95.8

	ATTGCT	95.61

	ACCATA	95.61

	CATCTA	95.61

	CCAGCC	95.4

	ACCTAC	95.39

	TCAACT	95.32

	ATGCAC	95.22

	GAAATA	95.07

	TATAGC	94.95

	TACCAC	94.81

	AGCTAG	94.59

	CCATAT	94.32

	TATATA	94.2

	CATATT	94.16

	TAATAA	94.05

	AGAACT	93.81

	TATCAC	93.66

	CACCAC	93.38

	AAAGCC	93.36

	CTACAG	93.16

	GCAGAT	93.16

	AGATCA	93.03

	ACTTCA	92.78

	ACACAC	91.91

	ACCACT	91.48

	AAGCTT	91.27

	ACCAAT	90.89

	CTAGCT	90.83

	ATTTAT	90.72

	CAGTTA	90.71

	CATAGA	90.61

	ATACTG	90.19

	ATTACC	90

	TATCAT	89.91

	ACTATA	89.16

	TACACA	89.01

	GCTGAA	88.67

	CCATTA	88.62

	TGCTAT	88.19

	TACATA	88.12

	CACCAA	88.08

	ATAGTT	87.88

	CACCTA	87.77

	GCACCA	87.64

	CTATCA	87.58

	GCTATT	87.58

	TATTAG	87.34

	CCACCT	87.28

	AGAACC	87.26

	ACTACC	87.25

	TATAAT	87.06

	ATTTCA	86.86

	TAGCAG	86.76

	AAGCTC	86.67

	AACCAA	86.61

	AATATC	86.37

	TAGTAA	86.29

	GCTGAT	86.25

	TATATC	86.21

	TAATTA	86.14

	AACCAT	86.06

	ATAGAC	86.03

	CCATCT	85.84

	TTATTA	85.75

	TCAGCC	85.73

	ACATAC	85.65

	ACATAG	85.6

	CACAAT	85.55

	GTAATA	85.54

	GAAGCA	85.45

	TCATAT	85.24

	CAGCCC	85.03

	ACCTAT	84.68

	AGCCAC	84.68

	CAGTAA	84.62

	CCAACA	84.17

	AAAAGC	84.12

	AACTGC	83.95

	CCAACT	83.78

	ATCATT	83.47

	AGAGCA	83.38

	GATACT	83.35

	CCACAG	83.35

	ATAATT	83.26

	TAAACA	83.21

	ACATAT	82.99

	GCTACT	82.86

	CAGTAT	82.76

	ATCACA	82.36

	TCAACA	82.34

	AGCCCA	82.25

	AATTAT	82.21

	ATCATC	82.17

	TGCTAC	81.84

	GCTTCA	81.55

	CCACTA	81.49

	GCTGCA	81.44

	TAGTTA	80.97

	AATCAA	80.92

	CAATTA	80.84

	CTGCTA	80.71

	ATATAG	80.66

	TGCACC	80.52

	AAGACA	80.5

	TAATAG	80.31

	TGCAGC	80.23

	CCTCCA	80.17

	GATGCA	80.15

	AACTCC	80.09

	TCCAGC	80.02

	ACACTG	79.79

	TATAAC	79.77

	TTATAA	79.58

	CAACAA	79.5

	GCTAAT	79.35

	TGATAC	79

	AGATCT	78.63

	ATAACC	78.57

	AGAAAC	78.2

	ATTGCA	78.18

	AACACC	78.06

	TGCATT	78

	CAACTC	77.9

	GTACTA	77.86

	ACTCCA	77.83

	CAGATG	77.71

	TGCAGA	77.69

	AAGAAA	77.67

	TCCACC	77.66

	TAACCA	77.39

	TAACAG	77.34

	TTATAT	77.04

	TCTATT	76.92

	ACACTA	76.75

	CACTAA	76.68

	GTAGCA	76.59

	AGCCAT	76.52

	TCATCT	76.5

	CACTAT	76.28

	CAATAT	76.05

	CACAGA	76.03

	AGTTAC	75.97

	ATACTC	75.91

	TATATG	75.77

	CACTAC	75.68

	ATTTCT	75.56

	TACCAA	75.44

	GCAATA	75.24

	ATCTCA	74.72

	ACAGAT	74.63

	TCACCT	74.58

	CATCAG	74.49

	TCAGAT	74.33

	AGTAAC	74.08

	CTACAC	73.7

	AATGAT	73.53

	ATTAGT	73.5

	TAGTAC	73.49

	TAACTG	73.35

	AAAATA	73.29

	AAAACT	73.19

	ATTTAC	72.97

	ATCTGA	72.97

	ATCCAT	72.95

	ATACCC	72.75

	AACTTC	72.62

	AATACG	72.39

	AAATCA	72.22

	TTCACA	72.18

	CAGATT	72.08

	CAGAAA	71.97

	ACACAT	71.91

	AAGATA	71.91

	CTGCAG	71.63

	GCAACT	71.57

	GATATT	71.57

	AGATTC	71.53

	ACCAGA	71.47

	CTATAT	71.38

	TGATAT	71.06

	AAGAGC	70.89

	ATACGC	70.65

	CTGATA	70.47

	GATAAA	70.39

	ACATCC	70.36

	AAACTA	70.26

	ATCAAT	70.13

	GAAACA	70.11

	CATCAT	70.01

	AGCTTG	70.01

	TGAGCT	69.96

	CTATAA	69.96

	ATTCAT	69.85

	TACTGC	69.83

	CAGAGA	69.69

	CATTTA	69.68

	AGCTGG	69.06

	GAATCA	68.99

	TTATTT	68.98

	ATCTGC	68.96

	TAGCAC	68.84

	ATGCTA	68.58

	TATACT	68.54

	TCATCA	68.5

	AGATGC	68.48

	ATAGCG	68.46

	CATACT	68.15

	TAGCAT	68.15

	TACAAA	68.02

	TACCTA	67.99

	CATCTT	67.88

	ATCAAC	67.83

	ACCTTC	67.82

	TTAGCA	67.82

	AGTAGC	67.72

	TTGCTA	67.61

	TAAGCA	67.57

	AATATG	67.49

	TCACTA	67.42

	CATTAA	67.2

	AGCAAA	67.17

	GGCTAT	67.15

	ATGCAA	67.06

	ACACCC	67.05

	GCAGTA	67.04

	AGTAAA	67

	TTCACC	66.71

	GATACC	66.69

	CTACAA	66.54

	CTGAAA	66.27

	ATGTAT	66.24

	CACCTT	66.08

	ACCCAG	65.77

	ATATCC	65.64

	CAAAGC	65.58

	ACAGTA	65.5

	CATACC	65.47

	TGAATT	65.43

	TATTCA	65.2

	GATATC	65.15

	ACAAAT	65.04

	CCATTT	64.91

	AAAAAC	64.81

	GCTCCA	64.64

	AAGCCA	64.61

	CCTTCA	64.45

	GAGCTT	64.45

	ATAGAA	64.31

	TGAAGC	64.22

	GAACCA	64.2

	ACAGAC	64.16

	ACAGAG	64.14

	TGTATA	64

	TGAACC	63.94

	TTATCA	63.94

	AACAGA	63.94

	GATTCA	63.93

	ATGAAT	63.83

	GCTGCT	63.71

	CACACA	63.58

	GCAGCC	63.54

	TAGCCA	63.4

	GAGCTC	63.35

	AACTCA	63.19

	GTATCA	63.01

	CATAAT	62.96

	TCCACA	62.68

	CAGAAG	62.65

	CCCAGC	62.57

	CGCTAT	62.55

	CCTACT	62.52

	CAATAC	62.45

	CAACTT	62.28

	AGAATC	62.21

	GAGCAC	62.17

	TCTGCA	62.09

	CAATCC	61.99

	AGAATT	61.72

	CATTAC	61.65

	ACTGCT	61.63

	AACACT	61.62

	GTAACA	61.62

	TATCAG	61.58

	ATGAAC	61.56

	CAACAT	61.55

	TCAATA	61.47

	TGCATC	61.37

	GCACAG	61.24

	AGAGCC	61.12

	AGTATA	61.1

	GTAGAT	60.86

	TACACT	60.8

	TATCCA	60.75

	AGCATT	60.65

	ATTAAA	60.65

	ACAAGC	60.61

	ACTGAT	60.54

	CAACAG	60.42

	ATGCTG	60.37

	TATCAA	60.3

	AGTTGA	60.16

	TTTACT	60.02

	CTTCAC	59.96

	GAAGAT	59.8

	CATCTG	59.68

	ATCCCA	59.65

	CAACAC	59.49

	AACATC	59.39

	AAGCAG	59.37

	CATCAC	59.3

	ACTAGC	59.24

	ACAACA	59.21

	CATAAC	59.02

	TATTTC	58.98

	CCATAA	58.89

	CACCCT	58.6

	ACACCG	58.31

	TACTAG	58.31

	TGAATA	58.12

	ACAATC	58.11

	AGGAGC	58.09

	TGAGCA	57.87

	TATGAT	57.78

	TATACC	57.77

	GATATG	57.64

	TCTGCT	57.47

	AGTAGT	57.38

	ACCAAA	57.17

	TGTAAT	57.16

	CAGCGA	57.12

	AAGCAT	57.06

	GATGCT	57.03

	CATTTC	56.98

	AAGATG	56.93

	ATCCAG	56.88

	CATATG	56.87

	TGGATT	56.83

	TGCAAC	56.76

	CACCTC	56.75

	CAGACT	56.73

	ATGCAG	56.72

	GTAACT	56.7

	AGTAGA	56.45

	TATGCA	56.42

	GGAATA	56.3

	AGTATC	56.23

	CATTAG	56.19

	CAGTAC	56.18

	TACATC	56.14

	AAAGCA	56.13

	TCTCCA	56.01

	ACAGAA	55.96

	GGAGCA	55.88

	CAGCCG	55.8

	CTGCAC	55.6

	AGCAGT	55.46

	CACATA	55.45

	TATCTG	55.37

	TACTCA	55.36

	CTTATA	55.34

	GACACA	55.17

	TGTATT	55.14

	GAATCT	55.12

	AACAGT	55.1

	ATCAGA	55.06

	GCATCT	54.8

	AACTAA	54.79

	CAGCGC	54.76

	ACACAA	54.74

	TAACAA	54.73

	TGCATA	54.73

	TTACAG	54.68

	GAAGCC	54.6

	AAGAAC	54.37

	TTACTG	54.36

	GTTTAT	54.25

	ACCAGT	54.25

	AATCCT	54.22

	ACAAAG	54.18

	TCACAG	54.18

	ACTATG	54.15

	GATGAT	54.08

	TGCAAT	54.03

	GTAATT	53.95

	TTAGTA	53.95

	CATGAA	53.93

	CATCTC	53.89

	AGCCTC	53.8

	CACATT	53.79

	AATTAA	53.78

	GCACAT	53.76

	ATTGAT	53.75

	AAAACC	53.75

	TACCAG	53.61

	ACTAGT	53.57

	AAAGAT	53.54

	CTCCAA	53.42

	CACACT	53.37

	CCACAA	53.24

	TACAAT	53.13

	CTATTG	53.01

	TAGTAG	52.94

	GATCAT	52.84

	AATCAT	52.81

	ATTCAG	52.71

	AGTACC	52.64

	AAAAAT	52.58

	CAGAAC	52.37

	ACAGTT	52.35

	TGAAAT	52.33

	GAGATC	52.3

	CATTCA	52.24

	CGAGCT	52.22

	GATAGC	52.17

	TCATTA	52.11

	CTCCAG	52.03

	CAGAGC	51.98

	TGCTGA	51.92

	CCAAGA	51.92

	ATAAGC	51.86

	TTACAC	51.85

	AGATGG	51.72

	TCTACT	51.69

	TTACAA	51.68

	TGCAAA	51.62

	TAGTAT	51.42

	TTTATC	51.26

	CCCAGA	51.25

	GACTAC	51.19

	ATTCTA	51.19

	CAAAAA	51.15

	ATACTT	51.15

	ATACGA	51.08

	ATCTTC	51.06

	ACATCA	51.04

	AACCCA	51

	CATAAA	50.95

	TGAAGA	50.88

	TAGATG	50.83

	CTGCAT	50.78

	CAAGCA	50.65

	AAATCC	50.5

	GAACTA	50.47

	CTATGA	50.36

	ACTTAT	50.3

	CCAAAT	50.25

	CCTGCA	50.24

	TACTCC	50.15

	GAGCAG	50.07

	TACCCA	50.02

	ACCTCC	49.97

	GTTATA	49.88

	CATCAA	49.87

	TGATAA	49.86

	AATCAC	49.84

	ATTAGA	49.71

	CATCCC	49.63

	GTATTT	49.61

	ACCTGA	49.59

	ACTGAA	49.51

	CATCCA	49.5

	TAACAC	49.46

	AGAGAT	49.39

	AGCATG	49.33

	CAACCC	49.27

	ACTTCT	49.23

	ATGATC	49.2

	GATAGA	49.19

	GAACAG	48.99

	CCAAAA	48.88

	GAAACT	48.8

	GACAGC	48.76

	CAATGA	48.7

	ACAAGA	48.64

	CTCAGA	48.55

	AGATAA	48.54

	CTAGCA	48.43

	ATCAAA	48.36

	TCTTCA	48.34

	GATGAA	48.34

	ATCCAA	48.27

	AACCAG	48.27

	CACATC	48.25

	TCCAAC	48.16

	TAAAGC	48.1

	AGACCC	48.09

	CAGGAA	48.07

	TTAACA	48.04

	TTATTG	48

	CATGGA	47.99

	CTTCCA	47.96

	CAGTTG	47.94

	ATATGG	47.86

	GTATCT	47.79

	CTTCAA	47.73

	GAGAAC	47.72

	TTCACT	47.71

	AAAGAA	47.71

	ACACCT	47.51

	AGTTCA	47.47

	ACCTGC	47.45

	TATGCT	47.44

	TTGTAT	47.43

	ACAGGC	47.42

	TCCATA	47.27

	TATTCC	47.17

	GGCTGA	47.15

	TGCTAA	47.05

	ACCCCA	46.96

	GTAGTA	46.89

	ATCCTA	46.79

	CGCATA	46.68

	AATTCT	46.54

	GGATCT	46.23

	TTATAG	46.2

	ACTAAA	46.2

	CAGACA	46.2

	GTACCA	46.16

	CAAAGA	46.13

	ACTCCT	46.11

	CACAGT	46.1

	AAACCT	46.05

	CGCTGA	46.02

	AATGAA	45.98

	GTTACT	45.95

	TACAAG	45.86

	AGGAAT	45.81

	ACTCAA	45.79

	ATGACA	45.7

	ACCATG	45.69

	CATAGT	45.61

	ATATTG	45.6

	AGGTAT	45.57

	CTCAGC	45.54

	ATATTC	45.46

	CTACTC	45.36

	TACAGG	45.33

	CCTCAG	45.33

	CACTGC	45.24

	GCACCT	45.13

	ACTATC	45.05

	CTGCTG	44.96

	AGCCTT	44.9

	GGTATT	44.89

	TAAATA	44.79

	TTCCAC	44.78

	CAAAAG	44.78

	TTTCAG	44.77

	TAATGA	44.74

	TTACAT	44.73

	AACCCC	44.73

	ATGGTA	44.66

	CACTGA	44.64

	CAAATC	44.64

	CATGCT	44.62

	GCTTCT	44.61

	TCCATC	44.59

	TCAGTT	44.56

	ACTGCC	44.54

	CTTCAT	44.49

	TGCTCA	44.45

	TGGAAT	44.41

	CTTCAG	44.4

	ACATCT	44.4

	CACCTG	44.39

	ATGCAT	44.36

	CCAACC	44.33

	CATTAT	44.25

	CTAGTA	44.22

	TACAGT	44.18

	TACTGA	44.12

	CTACTG	44.1

	TAGAAT	44.07

	ACAGCG	44.06

	ATGGAT	44.04

	TTCATA	43.92

	ATAAAA	43.84

	ACTCAG	43.83

	CTGCAA	43.65

	CAGGCT	43.52

	TGATAG	43.5

	AGAGAC	43.5

	CCATGA	43.49

	CTACTT	43.4

	ACATTA	43.36

	GAATAG	43.29

	GCAGTT	43.25

	CACAAA	43.25

	TGAACT	43.25

	TGAGAT	43.21

	CACTAG	43.13

	CCCCAT	43.06

	CTAACA	42.92

	CCAGTA	42.86

	CTCCAT	42.76

	CAAGAT	42.74

	GAACCC	42.71

	CCAGAA	42.65

	TTCATC	42.62

	AACCTG	42.6

	AGCCCC	42.52

	CCTACA	42.47

	GGATAT	42.47

	TCCACT	42.41

	ATTACG	42.39

	AAGATC	42.32

	AGCCTA	42.29

	ACACGG	42.21

	CTGAAT	42.18

	CTATTC	42.04

	ACAATG	42.01

	TCATAA	42

	TGAATC	41.89

	ATCAGT	41.74

	GATTTA	41.74

	AATCTG	41.72

	GCTGGA	41.71

	AGCGAT	41.68

	TATTTT	41.67

	GAATCC	41.64

	TTTACC	41.63

	AGCAGG	41.62

	AAATAT	41.58

	ATTATC	41.55

	GAGATA	41.47

	CCAGGA	41.41

	TCATAG	41.39

	GCTTTT	41.33

	ATGACT	41.26

	GAACTG	41.19

	CTGAAC	41.19

	GGCTAC	41.14

	AGCTCG	41.12

	ACCCAC	41.04

	CAATCA	41.01

	AGCGCA	40.99

	ACTCCC	40.96

	CTCCAC	40.95

	AATCTA	40.93

	GCATCA	40.9

	ATTTTT	40.87

	TGAAAA	40.84

	TCACAT	40.84

	ATTCCT	40.83

	TTGATA	40.69

	CACAAC	40.69

	TATTGA	40.61

	AGGCTG	40.57

	AATGCT	40.53

	TATTTG	40.53

	CAGGTA	40.51

	CATGCA	40.5

	AAACTG	40.46

	AACAAA	40.38

	CTTTCA	40.38

	CAAACT	40.38

	TATTTA	40.37

	GGAACA	40.37

	GCCACT	40.35

	CGCAGC	40.24

	TAAATC	40.2

	AGGTAC	40.19

	ACTGTA	40.17

	GAAGGA	40.16

	CAGTTC	40.09

	TTTTAC	40.04

	TGAACA	40

	GCTATC	39.99

	GCTTTA	39.98

	ATTAAC	39.98

	GAATAT	39.96

	CCATCC	39.94

	TACCTG	39.93

	CAAACC	39.91

	CACTTC	39.84

	TTATAC	39.76

	TTGCAT	39.73

	CTGTAT	39.67

	GAAACC	39.64

	AGTGAT	39.53

	CAAGCC	39.3

	AGGATT	39.29

	CAGTAG	39.29

	AGAATA	39.23

	ATGCCA	39.23

	GTGATA	39.2

	AATCCC	39.2

	AACAAT	39.16

	GAAGAA	39.02

	TAACAT	39

	CAAACA	38.97

	AGGATA	38.8

	AAATGG	38.8

	TTTAAT	38.75

	TTTACA	38.66

	GACACC	38.6

	CTTACT	38.54

	TAAAAC	38.52

	TCAGCG	38.41

	TTTGCA	38.37

	ACAAAC	38.35

	GATCTC	38.32

	TGGATC	38.23

	AAAAAA	38.16

	CACGAT	38.16

	TTTTCA	38.15

	AAACAA	38.11

	AATCAG	38.1

	ATGAGA	38.04

	CCAATT	38.03

	CTATAC	37.99

	AGGACA	37.98

	GAACAA	37.98

	TCCAAA	37.84

	TTTCCA	37.82

	ACTGGA	37.81

	AAGCAA	37.77

	ATGAAG	37.77

	ACAAGG	37.76

	AAGCCC	37.72

	GCTCCT	37.68

	ACACGA	37.64

	AGCCGA	37.6

	CCAGCG	37.57

	ATCCCC	37.48

	TGTAGC	37.33

	AGCCGC	37.29

	TCAGAA	37.28

	TAAAAA	37.16

	GATAAT	37.15

	TCCTAC	37.13

	TACTTC	37.09

	GAAATG	36.99

	ATATTT	36.91

	GAACTC	36.81

	CTAATG	36.79

	AACAGG	36.76

	AAGGCT	36.76

	TCCAAT	36.72

	TATGAC	36.67

	ACCTCA	36.63

	TGATGA	36.62

	AAGCCT	36.59

	GAGACA	36.59

	ATGATT	36.47

	CCACCC	36.46

	GCAATT	36.27

	CCCACA	36.26

	TACTTA	36.25

	TGACCA	36.23

	CCATAG	36.13

	ATTCCC	36.08

	CCCACT	36.08

	AAACCC	35.99

	GAACCT	35.97

	GTTATT	35.96

	CCATAC	35.9

	TTCTAC	35.9

	ATGAGC	35.85

	GATCAG	35.85

	TATGAA	35.79

	CAAGAA	35.7

	TATAAG	35.62

	ATCTCC	35.59

	ACTACG	35.54

	GAACAC	35.49

	TATTGC	35.48

	TAAATG	35.47

	ATGAAA	35.43

	GATCTG	35.38

	TATAAA	35.37

	ATACGG	35.34

	ATTATG	35.3

	CAAGGA	35.22

	AAATAG	35.19

	AAGACT	35.13

	ACCCCC	35.07

	AGATTT	35.05

	GAGCAT	35.02

	CCCCAA	35.02

	AAATGC	35

	TGATCA	34.95

	GAGCCC	34.9

	ATCTGG	34.82

	AGAAGT	34.81

	ACTAAC	34.76

	TGGAGA	34.73

	TAATCA	34.7

	CAACCT	34.69

	GACCAC	34.64

	GTAAAA	34.56

	TCTACC	34.54

	GATTAC	34.54

	CCAGTT	34.52

	ACCAGG	34.5

	GCAACC	34.48

	ACATTT	34.47

	ACTTCC	34.46

	AAGTAC	34.43

	ACCTTA	34.43

	TAATTG	34.26

	CACCCA	34.26

	ATCTTT	34.13

	TTAATT	34.07

	TTGCAC	34.06

	CACCCC	34.06

	CATGAT	34.02

	ATAGGT	33.92

	GCTACC	33.92

	ATAGAG	33.86

	AGTTCT	33.81

	TGCTTA	33.8

	GCTGTT	33.73

	AAGAAT	33.68

	GATTCT	33.67

	ACCGCC	33.57

	ACAGGG	33.56

	CAAGAC	33.52

	CCACTG	33.47

	AAGTAA	33.38

	TGTACT	33.36

	CTGAAG	33.36

	AGACCT	33.33

	ACTAGA	33.32

	AAATCT	33.23

	GCTATG	33.22

	TTGATT	33.18

	TGCTGC	33.18

	AGAAGA	33.16

	AATGGA	33.11

	TTCCCA	33.1

	AATGGT	33.08

	GTTACA	33.07

	TCAGGA	33.04

	TACACG	32.96

	TTACTT	32.93

	TAAAGA	32.93

	CACTTT	32.87

	AACTGG	32.82

	CTCACC	32.81

	ACATGC	32.79

	AGCCTG	32.79

	TCCCAG	32.78

	ACATGG	32.77

	CACTTA	32.69

	CCCCCA	32.63

	ATGATG	32.59

	GCAGAG	32.58

	ACATAA	32.53

	AAAGTA	32.47

	AAAAGA	32.46

	GAACAT	32.46

	CAATTC	32.4

	CCACTT	32.39

	GGCTTT	32.37

	TTCAAC	32.34

	GCTTAT	32.32

	CAGGAT	32.32

	AGCCCT	32.3

	CAATGC	32.26

	TGTATC	32.2

	TGATCT	32.2

	CTGTTA	32.12

	ACAATT	32.12

	TATCTT	32.05

	ATTCAA	32.04

	TTCAAA	32.03

	CAGACC	31.98

	ACATGA	31.9

	CTAAGC	31.75

	CTAAGA	31.7

	ATAAAG	31.69

	AACTAG	31.56

	GTACCT	31.55

	AGATAG	31.51

	CAAAAT	31.5

	GTGAAT	31.48

	AGCCAA	31.4

	GAGATG	31.33

	GGAGAA	31.29

	AATTGC	31.29

	ATGGCT	31.23

	GCAAAT	31.22

	TAGAAC	31.2

	ATGGAA	31.19

	GATGGA	31.15

	CTGCTC	31.09

	CCAGAC	31.09

	ACTCAT	31.09

	CGAACA	31.02

	AGCCAG	31.01

	GGATAC	31.01

	GCAGAA	30.98

	GTAAAT	30.95

	TTTATA	30.85

	TGCTTC	30.8

	CTCAAC	30.7

	AAAGAC	30.65

	GCTCAA	30.56

	ACAGTC	30.55

	CACAAG	30.53

	TGGATA	30.52

	GCATAG	30.51

	ACCTGG	30.5

	CTCCCA	30.43

	TGATTC	30.33

	GCTGTA	30.33

	GCATAC	30.26

	TCAAGC	30.25

	CAGAAT	30.22

	TCATAC	30.18

	CATCCT	30.14

	TGAAAC	30.04

	AAACTC	30

	GCATTT	29.91

	AAGGAC	29.86

	ACAAAA	29.84

	GAGTAT	29.79

	AAATGA	29.74

	AGCGGA	29.72

	GAATTA	29.71

	AGTGAA	29.7

	AACAAG	29.69

	TCAAGA	29.63

	AACCTT	29.53

	GAATAA	29.53

	CTCACA	29.49

	TCACAA	29.46

	CCCATC	29.46

	TGTGCA	29.41

	ATTGGA	29.27

	ATTGAA	29.23

	ATAATG	29.22

	CCTTTA	29.21

	GGAACT	29.21

	TTCAGA	29.18

	GCAACA	29.12

	ATAATC	29.11

	CTCATA	29.07

	GAATAC	29

	CTGATC	29

	ACCAAG	28.96

	CACAGG	28.94

	ATTTCC	28.86

	GCATAA	28.83

	TCCCAC	28.82

	GAGCAA	28.81

	TCCAGA	28.65

	TTCCAT	28.63

	GGCACA	28.6

	TTTCTT	28.55

	TAAACC	28.53

	AAATTA	28.46

	CTTGCA	28.46

	ACCTCT	28.41

	TCAGTA	28.39

	GAAGTT	28.37

	TACATT	28.33

	GACCCA	28.32

	GACCAT	28.29

	CCACAT	28.23

	CATTTT	28.22

	ATCGCT	28.15

	AAGGAA	28.11

	TATAGT	27.92

	TAACTT	27.89

	CTTAGC	27.87

	CTTAAG	27.83

	CCTGCT	27.78

	GATACG	27.7

	TAGACA	27.69

	GGTTCA	27.68

	ATGCTT	27.68

	TTCATT	27.66

	TAATCC	27.62

	ATATGT	27.59

	CCACGA	27.56

	AAAATC	27.56

	GAAGTA	27.52

	TGCTCC	27.5

	CCCATA	27.47

	TTAACC	27.43

	TAGAGC	27.38

	AGGCTA	27.34

	GCAAAA	27.32

	GCTCAT	27.31

	AGGACC	27.3

	AGACTA	27.27

	CCATTC	27.24

	ACGACT	27.24

	AGGAAA	27.08

	TTCCAG	27

	TCACCC	26.94

	AAATTC	26.9

	AACTGT	26.84

	TTCTAT	26.75

	TAATGG	26.71

	ACAACG	26.66

	AGGTGA	26.64

	AGGAAC	26.59

	TGGTAT	26.57

	AAACAT	26.53

	AGTTGC	26.52

	CAGTGA	26.47

	GATTCC	26.47

	AGCGAC	26.44

	ATCAAG	26.44

	ACCCCT	26.4

	CCCCAG	26.4

	CGTATT	26.39

	TACTTT	26.39

	AGACAG	26.37

	TTATGA	26.36

	CAAGAG	26.32

	TGCAGT	26.31

	AGGAGA	26.3

	CCATGC	26.27

	GAAAGC	26.23

	ACGATA	26.23

	CAAGGC	26.22

	CTTTAT	26.22

	CATTCC	26.22

	GAAAAT	26.2

	CATTGC	26.13

	TATACG	26.08

	GTAGAA	26.03

	GGACCA	26.02

	GCTCTT	25.97

	TGTTAC	25.87

	TCCCCA	25.78

	TCCATT	25.78

	AGAAAA	25.72

	CCCAAG	25.69

	GTGCAT	25.62

	TTTTAT	25.58

	ACCTTT	25.53

	CTACGA	25.52

	CCTTAA	25.52

	GGCATA	25.52

	GAAAAC	25.47

	AGTTTT	25.42

	GAATTC	25.36

	GATCAC	25.35

	CACACC	25.27

	AAGCCG	25.26

	ACTGAG	25.25

	ATCTAA	25.24

	AGACTG	25.18

	AAGTTA	25.15

	TCACTG	25.11

	ATCGCA	25.08

	CGATAT	25.02

	GTCATA	24.99

	AACCCT	24.98

	TTAATG	24.97

	ACTTTT	24.96

	ACGCAG	24.82

	ATTTAA	24.79

	TGATTT	24.76

	CTGATG	24.75

	ATCTTA	24.75

	TATGTA	24.71

	GAAGAC	24.69

	TTACCT	24.69

	TAGATT	24.68

	ATAAGT	24.67

	CGGATA	24.54

	CTTTTA	24.43

	ACCACG	24.41

	ACAGGA	24.4

	TATGGA	24.4

	TTACTC	24.37

	GCAAAG	24.34

	GAGGCT	24.32

	ATCATG	24.24

	TGTTAT	24.2

	GCAAGA	24.19

	CTGGAA	24.11

	CTATTT	24.06

	TCCATG	24.06

	AGTGCT	24.05

	AGCGCC	24.04

	CTGTAA	24.03

	GAGCCT	24.03

	ACCCAT	24.03

	TGGAGC	23.99

	ATGGAC	23.95

	CAGCGG	23.91

	TAAGAA	23.9

	GCATTA	23.88

	AGTCAT	23.86

	GGAACC	23.86

	CCCTCA	23.86

	AACCTA	23.83

	CTTACA	23.77

	GGTAAT	23.77

	GGAGCC	23.69

	CCCACC	23.65

	GGAGAT	23.63

	GTAGTT	23.62

	CTGAGC	23.61

	TTTCAC	23.61

	CTGAGA	23.59

	CATAGG	23.58

	TTTCAT	23.55

	AAGTAT	23.48

	AATTCC	23.45

	TACATG	23.39

	GGAAAT	23.35

	TGACCT	23.35

	CGCACA	23.34

	TACGAC	23.32

	ATTTTC	23.32

	CCTGAA	23.3

	ACAGTG	23.28

	AATCGA	23.28

	ATCTCT	23.2

	GACATG	23.19

	AAGTAG	23.18

	ATACCG	23.16

	GGCAGC	23.07

	TCTACA	23.02

	CTAAAA	23

	ACACGC	23

	ACCCTG	22.98

	TGAAAG	22.87

	CACATG	22.71

	CCTGTA	22.67

	TGGTAA	22.66

	CAGAGT	22.64

	CCGCTA	22.64

	GGAATC	22.63

	TTCAAT	22.52

	CTGCTT	22.49

	CCTATT	22.49

	GGTGCA	22.48

	CAGGAG	22.48

	CCCCAC	22.46

	AGGCTC	22.43

	CTAACT	22.4

	CCAAGC	22.4

	GCAGAC	22.36

	CCAGGT	22.36

	ACTGTT	22.3

	ACCCTC	22.25

	CTATGC	22.23

	TCTAAT	22.15

	TGGAAA	22.14

	CAGTTT	22.08

	TAATTC	22.08

	TCACTT	22.06

	TTTTTA	22.01

	CCTTCC	21.92

	ATCGAT	21.89

	AAAATG	21.87

	GCACAA	21.78

	TGCACT	21.71

	AAGACC	21.69

	AATTGA	21.68

	GCATCC	21.65

	CACTGT	21.65

	GAAAAA	21.64

	GCTCAG	21.6

	AACACG	21.59

	GTTGCA	21.57

	GCCCCA	21.54

	GACTAT	21.53

	GACCAG	21.52

	GTTCAT	21.39

	GAGAAT	21.24

	TAAAAG	21.2

	GAATTT	21.15

	CACCGT	21.13

	GATTAT	21.11

	TTTCAA	21.05

	ATCCTC	21.03

	CTGGAT	21

	CCTATA	20.97

	ATAGGA	20.97

	TAGGTA	20.96

	GGATTT	20.93

	ACTCAC	20.88

	CGACTA	20.85

	GGATCA	20.8

	CTACCC	20.78

	ACTTAC	20.74

	GATAAC	20.71

	GATCCC	20.66

	TACGCA	20.62

	GCCACC	20.56

	AGACTC	20.56

	GACTCA	20.5

	CCTTAT	20.39

	TAGGAT	20.38

	AACATT	20.37

	ATGCTC	20.32

	ACTCTA	20.3

	CTGCCA	20.29

	TGGCTA	20.29

	AGTCCA	20.26

	CAGTCA	20.24

	TTCCAA	20.24

	GACATA	20.22

	TCTATC	20.15

	TCCTGA	20.13

	ATGGCA	20.05

	GTAGCC	20.05

	CCTGGA	20

	CTTAGA	20

	AACGCT	19.94

	CGCTAC	19.9

	CTGTAG	19.87

	CACTCA	19.87

	CTTCTA	19.83

	TCCTTC	19.8

	CAAGTA	19.73

	ATCAGG	19.71

	TATTGG	19.66

	AGTTCC	19.66

	ACACTC	19.6

	AATTTA	19.59

	ACATTG	19.58

	GAAATC	19.45

	TGAAGT	19.45

	GTACAT	19.44

	CTTTAA	19.44

	CATTGA	19.38

	GGCTTC	19.38

	CACGAA	19.33

	TATCCT	19.28

	ATGGAG	19.27

	AATAGG	19.25

	GTATAA	19.24

	AATAAG	19.23

	GGATTC	19.19

	TCTATG	19.13

	ACCCTT	19.09

	ACTTTA	19.01

	CCAATC	19

	TCTGTA	18.99

	GCTCTA	18.93

	GATCTT	18.92

	GGATTA	18.85

	CGTATA	18.83

	ACGAAC	18.75

	ATTCTT	18.75

	AGGTCA	18.72

	TAGAAA	18.72

	CGTAAT	18.7

	GTACAG	18.63

	ATGTAA	18.6

	TTCATG	18.6

	AGTTTC	18.56

	TAGTTG	18.52

	TGGACA	18.5

	ATTTGC	18.49

	CACCGC	18.45

	CTCTAT	18.44

	CAATCT	18.42

	GAGAAG	18.39

	ACATTC	18.38

	ATTTGA	18.37

	TTGCAA	18.35

	AAGATT	18.34

	AAAGGA	18.34

	ATTGTA	18.33

	TTAAAA	18.28

	ATATCG	18.27

	ATAGTG	18.25

	GAGACT	18.19

	GCTTAA	18.18

	TGATTA	18.16

	GGATCC	18.16

	AGCACG	18.12

	AACCGC	18.1

	TTGCTG	18.05

	CCAAGG	17.94

	AGGCTT	17.91

	CGCAAA	17.91

	CCGATA	17.87

	TCAAAT	17.85

	CCGAGA	17.85

	GCCATT	17.84

	GCCATA	17.82

	GCACTA	17.75

	ACTCTG	17.67

	AGTAAG	17.64

	CGCTCA	17.58

	TATCCC	17.54

	AACTCT	17.47

	TCCACG	17.46

	GGAGAC	17.43

	CTTGAA	17.42

	TCTCAT	17.31

	TAGCGA	17.31

	CTAAAG	17.28

	CACTCC	17.24

	CCGTAT	17.21

	GAGAAA	17.2

	AACTTT	17.19

	CACTCT	17.18

	GACTCC	17.16

	GCACCC	17.12

	TTATCT	17.12

	TAGCCT	17.07

	CCTACC	16.97

	TAAGAT	16.95

	GCAATG	16.95

	GGTAAA	16.95

	AAAATT	16.92

	AACGGC	16.9

	CTATCT	16.81

	TATCTC	16.81

	GCTCCC	16.8

	CTGACA	16.79

	CATGGC	16.78

	GACCTC	16.77

	CCTTGA	16.76

	CTCATC	16.72

	CACGGA	16.69

	CTATGT	16.65

	TAGAAG	16.62

	CATAAG	16.58

	GGAAGC	16.58

	CGCAGA	16.48

	AACGCA	16.48

	CGAAGA	16.41

	TAACCT	16.4

	CTGATT	16.33

	CAGGCA	16.28

	GAAAAG	16.25

	CCCAAC	16.24

	TAGTGA	16.23

	TTGCAG	16.2

	TGAAGG	16.18

	TTTGAA	16.15

	TACCTT	16.14

	GCACAC	16.12

	ATGACC	15.97

	TTAAGC	15.91

	GTTGCT	15.9

	CATGTA	15.9

	ACGACC	15.86

	CAGGTT	15.84

	AAAAGT	15.82

	AGACCA	15.79

	GCTTGA	15.71

	GATGTA	15.67

	TGACAT	15.66

	TTCTCC	15.65

	TTAGAA	15.63

	TTAGAT	15.61

	ATTTTA	15.6

	TTAAAT	15.52

	GGTACA	15.49

	CATCGC	15.48

	GCCATC	15.39

	AATTTT	15.39

	TCAATC	15.38

	ACCCAA	15.38

	CTGTTT	15.36

	CCAGAG	15.35

	AGAAGG	15.33

	TCATTT	15.32

	CCAGTC	15.26

	AGTAGG	15.25

	TGCAAG	15.23

	AGGATC	15.22

	GACAAC	15.19

	TCCTCC	15.19

	TCAATT	15.18

	TCAAAA	15.15

	CCTGAT	15.13

	ATCCGC	15.08

	GACCTT	15.07

	TTATTC	15.07

	GCTAAG	15.01

	CTCAAG	14.96

	CAGGCC	14.89

	ATGTAC	14.83

	CTTCTG	14.71

	AGACAT	14.69

	TAAGTA	14.61

	TTGAAG	14.6

	ATGTTA	14.54

	TGGAAC	14.52

	GGCTCC	14.47

	ATAAGG	14.45

	CTTATT	14.45

	ATCCTG	14.42

	TGTTTA	14.41

	TGAGAA	14.39

	CACGCC	14.39

	CCATGT	14.39

	ACGCTG	14.36

	TCCAGT	14.34

	CTACAT	14.31

	AGTGCA	14.28

	AATCTT	14.25

	GGCTCA	14.24

	CCCTAT	14.21

	CCAGGC	14.21

	CTGGAG	14.2

	ACCCGC	14.16

	GGTATA	14.14

	GACTTC	14.11

	AAGAGA	14.08

	GCTTCC	14

	AGCGCT	14

	AGACTT	13.99

	AAACGC	13.99

	TCACCG	13.98

	CACGCA	13.93

	CCCAGG	13.91

	CTCTGC	13.88

	CGAGAA	13.83

	TATAGA	13.82

	AAAGCG	13.82

	GAGTAA	13.8

	GATTGA	13.77

	TTGAAA	13.74

	TAATTT	13.71

	AGTTGT	13.57

	GGAGTA	13.54

	TAAACG	13.52

	CCGCTG	13.48

	GGCTGC	13.46

	GGTACT	13.42

	GTGCAA	13.36

	TCTGAA	13.23

	TCCAGG	13.15

	CTTTAC	13.11

	GGAAAA	13.07

	ATCCTT	13.06

	GAAGGT	13.04

	GATTAA	13

	CAATTG	12.98

	CATGCC	12.96

	TCTTTA	12.95

	GATTTT	12.9

	TTTGAT	12.87

	CCACTC	12.84

	TGTACA	12.83

	TATGCC	12.83

	GCTGCC	12.82

	ATGGTT	12.82

	GTTCCA	12.79

	ATCCCT	12.79

	ACTAAG	12.76

	ATTCTC	12.75

	AACCTC	12.75

	CCTATG	12.71

	GAATGA	12.69

	ACAAGT	12.63

	TACTGT	12.62

	AGGTAA	12.62

	AACGAC	12.6

	TCCGCT	12.59

	TCAAAC	12.55

	GCACTT	12.49

	AATGCC	12.48

	ACGCTT	12.45

	CAACGC	12.44

	TAACTC	12.43

	TCTTAC	12.42

	CTTCCC	12.42

	ACACTT	12.38

	TTTTAA	12.23

	GAACCG	12.23

	GGGAAT	12.21

	TTCTCA	12.16

	TGCTCT	12.15

	GTACAC	12.13

	TTTTTT	12.1

	GTTTCA	12.07

	CCCAAT	12.04

	TTCAAG	12.03

	TTGAAT	12

	AGTATG	11.99

	TAGTTT	11.98

	CGACCA	11.98

	GCATGA	11.98

	CAGGAC	11.97

	GCCTCA	11.96

	GTCTAT	11.95

	CTATCC	11.89

	TGCCAT	11.88

	CGATCA	11.82

	AAGGAT	11.76

	GTGGAT	11.71

	CCATGG	11.69

	TCAACC	11.69

	TCCCAA	11.68

	GCTGAC	11.66

	TCAAAG	11.63

	GACACT	11.61

	TCCAAG	11.61

	CGGCTA	11.53

	GCCATG	11.51

	GCCCAT	11.46

	GAGCCA	11.41

	GAAAGA	11.4

	GCGTAT	11.39

	AAACGG	11.38

	CCCAGT	11.36

	ACACGT	11.35

	TTCCCC	11.35

	GGCACC	11.33

	AGCCGG	11.32

	TTAAAG	11.31

	CTATAG	11.27

	ATCTTG	11.27

	TACTGG	11.23

	CTCAAT	11.2

	GCTAAA	11.18

	GGTTAT	11.16

	TGCCAC	11.14

	GAGACC	11.07

	GTTACC	11.04

	AGGAGT	11.04

	CCGCAG	11.03

	CAAATT	11.02

	CTTCTC	10.99

	TATGTT	10.99

	AATTTC	10.99

	ACCGCT	10.99

	CCCGCT	10.9

	CGATTA	10.87

	ACATCG	10.86

	CCGGCT	10.85

	TAGATC	10.82

	AAGTTG	10.82

	CTTGAT	10.79

	TACCGC	10.78

	AAAGGC	10.74

	GATCTA	10.72

	TCCCCT	10.64

	GATAGT	10.62

	GGATAA	10.61

	TGAGTA	10.57

	GGAGTT	10.54

	ACGCAC	10.52

	CCCATT	10.51

	TGTAAC	10.49

	GATTTC	10.48

	TAACCC	10.46

	AATGTA	10.46

	ACGGCC	10.46

	TGCAGG	10.44

	CTGTAC	10.44

	AACATG	10.43

	ACTGGT	10.38

	AAGGCC	10.36

	TAAAGG	10.29

	TATTGT	10.25

	GGAGGA	10.19

	AAGTGA	10.18

	ATTTGG	10.18

	TGTTTT	10.17

	CAAAGT	10.16

	AGTCAC	10.14

	CTGAGG	10.12

	CTAGAT	10.11

	AATTGG	10.08

	GGAAGA	10.08

	CTCTTA	10.04

	CTCTCA	9.99

	GAACTT	9.97

	AGAGAA	9.94

	GAGGAT	9.93

	GGGAAA	9.93

	CCCTGA	9.92

	CCAATG	9.9

	TCATGA	9.89

	CCTTCT	9.88

	TCATTG	9.81

	TACCCC	9.78

	TTCTGA	9.75

	AGAACG	9.72

	ACGCTA	9.69

	CTCCTA	9.69

	TCCGCA	9.59

	TTCACG	9.57

	CGAATA	9.54

	ATTTTG	9.43

	GCCACA	9.39

	CCTAGA	9.37

	TTATCC	9.33

	AGGCAC	9.29

	GGCAAA	9.28

	AACCCG	9.28

	GTTAAT	9.27

	AATGGC	9.23

	GTATAC	9.16

	CAGTCT	9.12

	CTCAGT	9.12

	TTTATG	9.09

	TGAGCC	9.05

	GGTGAA	9.04

	TAAAAT	9.04

	CACACG	9.02

	GTACTT	9.02

	TTACCG	9

	GCCAGA	8.99

	TCGCTA	8.97

	GGCTCT	8.95

	GACAGA	8.93

	GGAATT	8.9

	TATTCT	8.89

	CCGCAC	8.89

	TGCCAA	8.87

	GCCAAC	8.84

	GATCCT	8.82

	ACGCCA	8.74

	AAAAAG	8.73

	CCAAAC	8.69

	TAACCG	8.68

	TTGAGC	8.68

	GCATTG	8.65

	CACTGG	8.65

	GTAAGA	8.62

	GACAAA	8.62

	CCCTTC	8.61

	TTAATC	8.61

	GTACAA	8.54

	ATAAGA	8.53

	AATCCG	8.5

	TTCTTC	8.39

	CATGAG	8.38

	GAAATT	8.32

	CATACG	8.31

	TCTGAT	8.28

	GACCAA	8.27

	TAAGAG	8.24

	GGATTG	8.2

	CAAATG	8.17

	CCACGG	8.17

	GAGAGA	8.12

	GCTTAG	8.08

	CAGCGT	8.07

	GTGCTA	8.07

	TTAACT	8.05

	TGATCC	8.04

	AATGAC	8.04

	GTAACC	8.01

	CTCAGG	8

	CGATAC	7.98

	CTTTTC	7.89

	TTCAGT	7.81

	CCCCTT	7.75

	TGCACG	7.71

	TTCTTA	7.71

	TAATGC	7.7

	CCTGAG	7.69

	TATCCG	7.64

	GACATC	7.64

	GACCCC	7.61

	CTTTGA	7.6

	TTAAGA	7.56

	CACGAC	7.55

	TAAATT	7.54

	ATTGAC	7.51

	AGAAAG	7.5

	TTTGCT	7.5

	CCAAAG	7.46

	CACGGC	7.4

	GTTTTT	7.39

	TGTGAA	7.37

	GTAATC	7.37

	CGTATC	7.36

	TACGAT	7.3

	GGACAT	7.28

	CCCTTA	7.23

	GATTTG	7.22

	ATTTGT	7.2

	ACATGT	7.19

	CACGCT	7.18

	TGCTGG	7.14

	CACCGA	7.05

	ATCCGA	7.01

	TAGTTC	6.93

	CTGGAC	6.9

	CCTCAC	6.9

	TGAATG	6.89

	GCCCAG	6.83

	CGGCTG	6.82

	CTTGTA	6.77

	AATCTC	6.73

	AAGAAG	6.68

	GAATTG	6.67

	AAGGAG	6.63

	TAGGCT	6.62

	TTTGTA	6.58

	TTCTAA	6.55

	TCTCAG	6.51

	ACCCTA	6.51

	TTATGC	6.47

	CTGGGA	6.46

	TTTGGA	6.43

	CTTTGC	6.39

	GGAAAC	6.38

	AACCGA	6.33

	ACGATG	6.33

	GCTACG	6.32

	CTTTAG	6.28

	GCAGGC	6.25

	CTGCCC	6.22

	TTCTTT	6.2

	GCACTG	6.19

	ATAGTC	6.11

	GCTCAC	6.11

	ATTGGT	6.09

	GTACTG	6.09

	GGTATC	6.07

	CCCAAA	6.05

	CATTGT	5.96

	GTGCAC	5.86

	GTTTTA	5.81

	GCAAAC	5.79

	CGCACC	5.79

	CTACCG	5.78

	GGGATA	5.77

	ACAGGT	5.76

	GCTGAG	5.75

	AAATGT	5.7

	TGTAGT	5.67

	TGATGG	5.64

	ATGCCC	5.63

	TTTCCC	5.63

	GCCAAT	5.59

	AAGGTA	5.58

	GTATCC	5.56

	TGGACC	5.48

	AGGCAT	5.46

	GATGGT	5.44

	TTCCTT	5.44

	TGGAAG	5.39

	CCTATC	5.33

	CGGACA	5.31

	AGGGCT	5.22

	TTTAAC	5.22

	TTGTAA	5.21

	ATAGGC	5.18

	TGTTAA	5.15

	TGACTA	5.12

	CCCCTA	5.11

	AGATGT	5.1

	GACAAT	5.09

	GATCAA	5.07

	GCCAGC	5.05

	TCATCC	5.04

	AGTTAA	4.96

	TCTCAC	4.95

	ACGGCT	4.94

	TCTATA	4.87

	GTAGGA	4.85

	TTTCTA	4.85

	CAGAGG	4.84

	TTTTTG	4.77

	TCCTAT	4.76

	GAAGGC	4.74

	TCAGAC	4.73

	GCAGCG	4.71

	AGTGGA	4.7

	CCACGC	4.69

	TTGTTA	4.62

	CTTAAA	4.62

	ACTGCG	4.61

	GTTCAC	4.59

	TCAAGG	4.58

	AGGATG	4.56

	CCCTGT	4.46

	CAAAGG	4.45

	TTTAAA	4.39

	TTATGG	4.38

	CTAGAA	4.37

	CCGTAA	4.36

	TAGCCG	4.36

	ACTTTG	4.36

	GACTGA	4.33

	TCACAC	4.31

	GGTAGA	4.27

	GACTGC	4.25

	AGATTG	4.24

	CGGCTT	4.23

	ATGTCA	4.23

	TCTTGA	4.2

	CTTTTG	4.2

	TGTAAA	4.2

	GCTTTG	4.19

	CCAAGT	4.16

	TGTACC	4.15

	AAAGTT	4.14

	ACCGTA	4.1

	TACGAA	4.04

	CTTATC	3.94

	CCTCAA	3.94

	ACCCGA	3.93

	GTTGAT	3.93

	TGCTGT	3.92

	GTTCAG	3.91

	TGGTTA	3.91

	AAAACG	3.88

	GCGCAG	3.86

	CCTTTC	3.85

	TCTCAA	3.85

	ATCTAG	3.83

	GAGATT	3.8

	ACGACA	3.75

	TAGACT	3.73

	TGTATG	3.7

	GCTAGT	3.7

	TAAGCC	3.7

	AAAGGT	3.68

	CTAAAT	3.65

	CAGTGT	3.61

	GAGTTC	3.56

	AGGGCA	3.54

	CGCTTC	3.53

	TACCGA	3.51

	TCCTCA	3.51

	AGCAAG	3.5

	GAAGCG	3.49

	GCCTTA	3.43

	TTAGTT	3.4

	ACCGAC	3.39

	GCAGGA	3.39

	ATGCGA	3.38

	ACGAGC	3.35

	GCAGGT	3.33

	AGGGAT	3.33

	CAGGGC	3.29

	AAGGGA	3.26

	AGCGGC	3.25

	GACCCT	3.25

	CGCCAT	3.18

	GTGAAA	3.17

	AGAGGA	3.16

	GGGATT	3.16

	ACGGAT	3.13

	TGCTAG	3.1

	TATGCG	3.06

	GACCTG	3

	TTGGAT	2.99

	TACTTG	2.98

	GACAAG	2.95

	TATGAG	2.93

	GACTCT	2.87

	GTTGTA	2.85

	GTCACC	2.84

	CATGTC	2.82

	TGGTAC	2.78

	CTCCTT	2.78

	ATCTGT	2.78

	AGGACT	2.76

	GGTAAC	2.76

	TCCCAT	2.75

	CAATTT	2.73

	GCTGGT	2.69

	ACGATT	2.63

	CGAACT	2.6

	GACACG	2.58

	ATGTGA	2.58

	CCTAAA	2.57

	TGGCAT	2.49

	CTGGTA	2.48

	ACTTTC	2.47

	GAGTAG	2.46

	TTTCCT	2.4

	CCACAC	2.39

	TGTTCA	2.38

	AACTTA	2.38

	TGTTGA	2.35

	GAAAGG	2.33

	ACGGCA	2.33

	GAGCCG	2.32

	TCTTAG	2.32

	CAATGT	2.29

	GTCCAT	2.28

	ACCGCA	2.24

	CTCCTG	2.22

	CTAGAG	2.19

	TCATTC	2.19

	AAGGCA	2.18

	CCCTTT	2.15

	AGGTTC	2.11

	CTTAAC	2.1

	TTGACC	2.07

	GCTTTC	2.06

	AGACAA	2.06

	TTTCTG	2.02

	GGTGAT	2.01

	CCTCAT	1.99

	GAGAGC	1.95

	GCCTTC	1.91

	TGATGC	1.88

	AGAGGC	1.87

	GATGAC	1.87

	GTTTCT	1.83

	TAACGA	1.8

	CTTACC	1.79

	ACTGAC	1.72

	ACGCAA	1.7

	CGAATC	1.69

	GGACAG	1.64

	GCCGAT	1.64

	TGGGAA	1.62

	AGACGC	1.6

	TTACCC	1.58

	CAACCG	1.55

	CCCTCC	1.51

	TTCAGG	1.48

	TCACGA	1.48

	TGCTTT	1.44

	AGGGGA	1.42

	ACGGAC	1.41

	CTCCCC	1.38

	ACCTTG	1.35

	AGAGTA	1.3

	GCCAAA	1.29

	AAAGTG	1.28

	CCCCTG	1.21

	TTGAAC	1.21

	GATGAG	1.21

	GCGCTG	1.2

	TCAATG	1.17

	CTTGGA	1.16

	AGGGAA	1.14

	GTTGAA	1.14

	AGAGTT	1.08

	AGACGG	1.08

	TTGGAA	1.05

	TCTCCC	1.02

	CTCTAA	1.01

	TCTGAG	1

	TCGATT	0.95

	ACGAAT	0.83

	TGGAGG	0.82

	CATGGT	0.82

	GAAGAG	0.81

	TTCCTG	0.78

	CGCTTT	0.75

	CGGAGA	0.75

	GATAAG	0.72

	GGCATT	0.71

	GGCAGT	0.67

	ATTCGA	0.67

	CATTTG	0.59

	TCTTAA	0.58

	ATTGAG	0.55

	TTTTCC	0.54

	CAAAAC	0.47

	AGTGAC	0.47

	GCCTCC	0.45

	GACGCT	0.39

	CATCCG	0.39

	CTATGG	0.38

	TCATGG	0.37

	GGGACA	0.36

	CCTGCC	0.36

	CAGGGA	0.34

	TTCGCA	0.32

	AAGGTG	0.25

	GATGTT	0.2

	TTTTAG	0.18

	TGGTGA	0.16

	CTGTGA	0.14

	GGCAGA	0.11

	GTGTAT	0.1

	CCCTAA	0.09

	TCTCCT	0.06

	ACTCGA	0.05

	TACCTC	0

	AATCGC	−0.05

	ACTTAA	−0.05

	CTCAAA	−0.06

	GCCCCC	−0.1

	GGTTAA	−0.11

	GCGAAA	−0.15

	CTAGTT	−0.16

	TCCCCC	−0.21

	AACTTG	−0.22

	CTCCGC	−0.27

	AAACGA	−0.29

	TGCCCC	−0.34

	CGCTGC	−0.35

	AAAAGG	−0.35

	TGCATG	−0.38

	CAGACG	−0.39

	TGACAC	−0.39

	CGATGA	−0.4

	TTAAGG	−0.41

	TTGGAG	−0.41

	GCCCAA	−0.41

	AGGTTA	−0.42

	ATTTAG	−0.45

	AGATTA	−0.46

	AGGTTT	−0.49

	GCCTAT	−0.53

	TCATGC	−0.55

	CTCATG	−0.58

	CAGTCC	−0.59

	GTATGA	−0.64

	CCTCTA	−0.65

	CATTCT	−0.65

	CCGACA	−0.73

	AGTTAG	−0.81

	GCCAAG	−0.86

	ATTCTG	−0.86

	GAGTTG	−0.88

	AAAGAG	−0.91

	TGTGCT	−0.96

	TCTAAG	−0.96

	AAACTT	−1.01

	GCGGCT	−1.04

	TTAGAC	−1.04

	TTAAAC	−1.08

	AAGGTT	−1.14

	AGTTGG	−1.15

	AGAGGT	−1.2

	CCCTAG	−1.2

	CCGCTC	−1.21

	GCATGG	−1.24

	GCTAGA	−1.26

	ACGATC	−1.27

	CGTGCA	−1.27

	TTTAGC	−1.32

	CTCATT	−1.33

	CGCAGT	−1.35

	AATTGT	−1.36

	TGACAG	−1.37

	ATGCCT	−1.38

	AAGTTC	−1.41

	CTTGAC	−1.45

	TTTTGA	−1.48

	ATAACG	−1.48

	GCATTC	−1.49

	ATCGGC	−1.51

	GTAATG	−1.54

	TAAACT	−1.55

	GAATGG	−1.56

	AATTTG	−1.6

	CCTCCC	−1.61

	CGGATT	−1.62

	TTGAGA	−1.64

	GTGAAG	−1.66

	GCCCCT	−1.7

	CGTTTA	−1.73

	GAGGAA	−1.76

	CGTTCA	−1.77

	TTCGAA	−1.81

	ATCGAC	−1.83

	TTTTTC	−1.87

	TGCGCA	−1.89

	ACCGAA	−1.9

	CTGCGC	−1.93

	AAGTCA	−1.93

	TTACGA	−2

	TGGACT	−2.05

	TACGCT	−2.06

	GAGGCA	−2.1

	TTGATG	−2.12

	ACCGAT	−2.13

	TACTCT	−2.17

	CGCCAG	−2.18

	GAGTTA	−2.18

	CACGTT	−2.2

	CTGCGA	−2.22

	GTGCTT	−2.23

	AATGAG	−2.24

	AGTGTA	−2.25

	CTTATG	−2.26

	TCTCTG	−2.27

	CCTAAT	−2.29

	GGAATG	−2.29

	CCATTG	−2.34

	CGATAA	−2.35

	ACTTGA	−2.35

	TTGGTA	−2.35

	TAGAGA	−2.36

	GACATT	−2.38

	GGGAAC	−2.38

	TGACAA	−2.38

	GTGCAG	−2.42

	CGGCTC	−2.43

	ATTGTT	−2.45

	ATGAGT	−2.46

	GGATGA	−2.48

	GTTCTA	−2.49

	GTTAAA	−2.5

	ATGTTC	−2.57

	CCTAGC	−2.61

	CCCTAC	−2.61

	AGAATG	−2.65

	CGAAGC	−2.7

	CGGTAA	−2.71

	CTAATC	−2.72

	ACCTAA	−2.76

	GCGCCA	−2.8

	GTCCCA	−2.83

	CGAGCA	−2.88

	TCAGGT	−2.9

	AGAGTC	−2.92

	GAGGAC	−2.92

	ACGAAA	−2.95

	AGGCAG	−2.97

	GGACCT	−2.98

	TCACTC	−3.01

	GACTGG	−3.03

	CTTGAG	−3.03

	CGAGCC	−3.07

	GGCTGT	−3.1

	GCCGCT	−3.11

	GGACAA	−3.11

	TACCCT	−3.12

	GTCAGC	−3.12

	CTGTTC	−3.18

	CCGAAT	−3.21

	AGAGCG	−3.21

	ATGGTG	−3.29

	TCCTTT	−3.3

	CATGAC	−3.31

	TAGACC	−3.31

	GGACTC	−3.32

	CCCTGC	−3.32

	GGAAGG	−3.35

	GGTTCT	−3.38

	GCAATC	−3.41

	AGTCTT	−3.46

	TACGGA	−3.49

	CGCACT	−3.51

	GCCTGC	−3.57

	GGACCC	−3.57

	GCCTTT	−3.58

	TTTAGT	−3.6

	GGTGCT	−3.6

	CGACTC	−3.65

	GGATAG	−3.69

	GGATGC	−3.7

	ACTCTT	−3.73

	ATTGCC	−3.84

	TGAACG	−3.84

	CTTTTT	−3.89

	GAATGC	−3.91

	TATAGG	−3.92

	GTATAG	−3.93

	GAGCGC	−3.96

	ATTGTG	−3.97

	TCAGAG	−3.97

	GGGATC	−3.98

	CCGCCA	−4

	TGTCCA	−4.01

	TGTTCT	−4.03

	AGGCCA	−4.04

	CCTTAC	−4.05

	TTTTCT	−4.07

	CATCGA	−4.09

	AGCGAA	−4.12

	AAAGGG	−4.12

	GGGAGA	−4.13

	CTGAGT	−4.13

	GAAGTC	−4.15

	CGTAGC	−4.16

	CGGCAC	−4.18

	TGCGAA	−4.19

	TCTTTT	−4.21

	ACGGAA	−4.22

	CCGACT	−4.25

	ACCTGT	−4.26

	ATCGTA	−4.29

	TATGGT	−4.29

	TAATCG	−4.31

	CGATTC	−4.32

	GGGAGC	−4.38

	CTCTAC	−4.38

	CGTACA	−4.41

	CAAGTT	−4.42

	TAAGGC	−4.46

	AAGCGA	−4.46

	GGTACC	−4.48

	GACAGT	−4.49

	CCGCAA	−4.53

	GCTAAC	−4.61

	TCCCTA	−4.62

	CAGGTG	−4.63

	CAATGG	−4.64

	TAGTCA	−4.67

	TAGACG	−4.67

	CGTGAA	−4.7

	AGACGA	−4.7

	AAGCGT	−4.74

	TGGGAT	−4.81

	CCGAAG	−4.83

	CGAAAA	−4.87

	AGCCCG	−4.93

	GGCTGG	−4.96

	GTCACT	−4.99

	CAACGA	−4.99

	TGACCC	−5

	GCCGCA	−5.04

	GTTCAA	−5.06

	TCGCTG	−5.07

	GTGAAC	−5.11

	CCTTAG	−5.16

	ATAGGG	−5.17

	CAGTGC	−5.18

	AGGCGA	−5.2

	CGAACC	−5.2

	ACTCCG	−5.21

	CTCCTC	−5.24

	GGTCCA	−5.25

	AAATTG	−5.27

	CAAGTC	−5.27

	TACCCG	−5.28

	CTTTCC	−5.29

	GCACTC	−5.29

	TTGGCA	−5.3

	ACTTGC	−5.32

	AGTCCC	−5.32

	TGGCAC	−5.33

	GTGGAA	−5.33

	GGCCAT	−5.36

	GCGGAT	−5.39

	GCGCAT	−5.4

	GGGGAA	−5.4

	TCTAGA	−5.4

	ACTTGG	−5.44

	TGCGAT	−5.45

	GCGATA	−5.45

	TGCCCA	−5.45

	TGGCTT	−5.48

	AGAGAG	−5.48

	TTGCTT	−5.51

	AATGTG	−5.57

	TTACGG	−5.57

	AAGGTC	−5.59

	TGAGAC	−5.62

	GACTGT	−5.69

	TTAGTG	−5.71

	CATTGG	−5.71

	CAGGTC	−5.73

	TCCCTT	−5.8

	CGAATT	−5.82

	AATGTT	−5.82

	GGCAAT	−5.83

	TAGGAC	−5.91

	TACGGC	−5.94

	TCTTCT	−5.95

	GGGCTC	−5.97

	TCGCAT	−5.98

	CTAGGC	−5.98

	CCTTTT	−5.99

	CCAGTG	−6

	CACGAG	−6.01

	TCCTAA	−6.03

	TAGGCA	−6.08

	TCTAAC	−6.1

	CACCCG	−6.13

	CTACGG	−6.14

	AGGTGC	−6.16

	CCCATG	−6.17

	ACGCCC	−6.17

	CGATCC	−6.18

	GAAACG	−6.2

	ATGTGC	−6.21

	GCAAGC	−6.24

	AAATCG	−6.25

	CCTCTC	−6.29

	ACCGGC	−6.31

	TTTAGA	−6.33

	CGACTG	−6.33

	AGGCAA	−6.33

	GGACAC	−6.35

	TAGCCC	−6.37

	TCTGGA	−6.37

	TAAAGT	−6.37

	TGAGTT	−6.37

	AAACCG	−6.45

	ACCCGG	−6.51

	CCTGAC	−6.51

	AAATTT	−6.52

	AACTCG	−6.52

	AAGGGC	−6.52

	TTTTGC	−6.54

	GGAAGT	−6.61

	GGTTAC	−6.66

	TCGTAT	−6.68

	GTTCTC	−6.7

	GGAAAG	−6.72

	TCCTTG	−6.72

	GCGAAT	−6.75

	AGTCTC	−6.77

	GGCACT	−6.8

	GCTCTG	−6.8

	CTACCT	−6.8

	TTGACA	−6.81

	AGCGTA	−6.81

	AGCGTT	−6.83

	TCGCAG	−6.85

	CGAAAT	−6.88

	GCCCTT	−6.9

	CATCGT	−6.91

	AATTAG	−7

	GACGAT	−7

	AACCGG	−7.03

	TTGCCA	−7.04

	CTAGCC	−7.1

	CACGGT	−7.11

	CTCTTC	−7.13

	AACGCC	−7.15

	GTTATG	−7.15

	ACTGTG	−7.15

	TAATGT	−7.16

	CCAACG	−7.21

	GCCTTG	−7.21

	CCTTGT	−7.23

	TTCTGC	−7.23

	TAAGAC	−7.23

	GCTGTG	−7.24

	CCCCTC	−7.25

	GACTAA	−7.25

	CGCTCC	−7.27

	GCGACT	−7.27

	TTCCCT	−7.28

	CGCCCA	−7.29

	TGCGCT	−7.33

	CCCCCC	−7.34

	TTAGAG	−7.34

	CCTGTT	−7.36

	TCTTCC	−7.38

	CCATCG	−7.38

	TCTAAA	−7.39

	CTAATT	−7.42

	AAGCGC	−7.42

	CTCTGT	−7.48

	AGGCCT	−7.48

	TAGGAA	−7.51

	GTTCTT	−7.54

	GTATTC	−7.63

	ACGAGA	−7.68

	ATGTTT	−7.68

	GGGTAT	−7.76

	ACTAGG	−7.77

	CGGAAA	−7.79

	ATGGCC	−7.82

	GTTATC	−7.85

	TCGACT	−7.87

	CTTCTT	−7.9

	AACGAT	−7.91

	GATCGA	−7.94

	CTCTAG	−8

	CTAACC	−8.08

	CTAGGA	−8.08

	GATTGT	−8.1

	CCCGCA	−8.1

	CGAGTA	−8.11

	TGGGCT	−8.15

	GGCTTA	−8.19

	TCGGCT	−8.24

	GATGGC	−8.25

	ACGCAT	−8.3

	CCGCTT	−8.3

	TGGCTG	−8.32

	ACTCTC	−8.35

	GCCCAC	−8.37

	CGCTGG	−8.37

	TTGCTC	−8.38

	TGGTAG	−8.38

	CTCTGA	−8.49

	TACTCG	−8.59

	TGAGAG	−8.6

	GCACCG	−8.61

	ATGGGA	−8.69

	TGACTG	−8.7

	CGATTT	−8.72

	CGGAGC	−8.74

	CGGATC	−8.78

	AGTCAA	−8.79

	TTCCTA	−8.79

	CCTAGG	−8.79

	GTTGGA	−8.82

	AGTCTG	−8.83

	CAAGGT	−8.85

	AATTCG	−8.91

	ATTCGC	−8.93

	GAAAGT	−8.99

	CTAGAC	−9

	AACGAA	−9.02

	CGACAA	−9.03

	GCCTAG	−9.04

	AAGTGC	−9.06

	GGTGTA	−9.06

	GATAGG	−9.08

	TTTGCC	−9.1

	TTTAAG	−9.1

	CCCCCT	−9.1

	CGATAG	−9.13

	ATCCCG	−9.15

	GTCACA	−9.21

	GTCCAG	−9.24

	CAAACG	−9.25

	AGGCCC	−9.29

	AGGGAC	−9.3

	CTGACC	−9.3

	GCTGCG	−9.34

	TTTCTC	−9.36

	CGACAG	−9.37

	TGAGGA	−9.41

	CCAGGG	−9.43

	AGTCTA	−9.43

	GCCGAA	−9.48

	TCCCTC	−9.49

	AAGTCT	−9.51

	AGGGCC	−9.52

	GCAGTC	−9.54

	ATGTTG	−9.55

	GTAAAC	−9.56

	GAGTTT	−9.58

	ATGCGC	−9.6

	CTCCCT	−9.65

	TTTTGG	−9.67

	GTCAAT	−9.69

	TAGGAG	−9.7

	CTTCGA	−9.71

	AGTTTA	−9.73

	GTAAAG	−9.78

	CGCCAC	−9.81

	GACAGG	−9.82

	AGGAAG	−9.83

	ACGTAT	−9.85

	GAACGC	−9.88

	AAGAGT	−9.91

	CACTTG	−9.92

	GCGATT	−9.92

	CGCCAA	−9.93

	GCTTAC	−9.94

	TGACTT	−9.94

	CATGTT	−9.95

	TGATTG	−9.97

	TCACGG	−9.98

	TCGAAT	−9.98

	CTCTTG	−10.02

	GTGATT	−10.03

	GAACGA	−10.03

	TGTTCC	−10.05

	TGTTTC	−10.07

	TCTTAT	−10.08

	GAGACG	−10.09

	CGGTTA	−10.12

	GCATGT	−10.13

	GGATGT	−10.15

	CCTTGG	−10.18

	GAATCG	−10.2

	GGGCTG	−10.21

	TAGAGT	−10.25

	TAGCGG	−10.25

	GCAGTG	−10.25

	GTCCAC	−10.28

	GAGTAC	−10.33

	CCACCG	−10.36

	CGACAT	−10.37

	GGGGAT	−10.38

	CGCTAA	−10.4

	CCGTTT	−10.41

	TCTAGC	−10.42

	GGGATG	−10.45

	CTGTGC	−10.48

	CTAAGG	−10.48

	TTGATC	−10.5

	ATTGGC	−10.52

	AGCCGT	−10.56

	ACTGGG	−10.56

	CTGGCT	−10.58

	ACGCCT	−10.59

	ATACGT	−10.63

	GGTAGC	−10.65

	TGTCAT	−10.65

	GATGCC	−10.66

	GGTTTA	−10.7

	GTGCTC	−10.84

	TAAGGA	−10.86

	CTTAAT	−10.91

	GATCCG	−10.94

	CGAGAT	−11

	GGCGAA	−11.02

	CCGCAT	−11.03

	GGCGCT	−11.04

	GCACGA	−11.04

	TGCCGA	−11.07

	GGCATC	−11.1

	TCGGCA	−11.1

	GATTAG	−11.14

	TCCTTA	−11.15

	CTAAAC	−11.17

	CGGAAG	−11.23

	CTTTGT	−11.26

	TTAGGA	−11.27

	CCGGAT	−11.36

	ATTAAG	−11.38

	GTGCTG	−11.41

	CTCTCC	−11.45

	TATTCG	−11.47

	GCCCTG	−11.51

	TCGCCA	−11.54

	TGTAGA	−11.62

	CTAGTG	−11.62

	CCGCCC	−11.66

	CAAGCG	−11.66

	GGTGGA	−11.74

	ATTAGG	−11.75

	GCCTAC	−11.77

	CTCACT	−11.78

	AAGCGG	−11.79

	AACCGT	−11.81

	AGATCG	−11.85

	TGACTC	−11.92

	TTCTTG	−11.94

	ATCGCC	−11.99

	ATCGAA	−11.99

	GGTTTT	−12.02

	TGGCAA	−12.04

	CGCCTT	−12.06

	TTGTAG	−12.07

	ACTTGT	−12.08

	TGGTTT	−12.08

	ACTCGG	−12.09

	TATGGC	−12.1

	TTGGTT	−12.12

	GCGATG	−12.19

	CAGGGT	−12.2

	AGTTTG	−12.24

	TAATCT	−12.24

	AAACGT	−12.25

	CGCAAT	−12.28

	CCCTCT	−12.28

	GGGCAT	−12.33

	AGTGGC	−12.33

	GCCAGG	−12.34

	TAAGGT	−12.35

	GGCCTT	−12.37

	GGGAAG	−12.37

	TGCCTA	−12.4

	CCGTCA	−12.43

	GTATTG	−12.44

	GTGACA	−12.48

	CGGCAG	−12.51

	TGTGAT	−12.53

	GACGCA	−12.56

	CAAGGG	−12.58

	GAGTCA	−12.63

	GCCGAG	−12.66

	CTTTCT	−12.68

	GACTTT	−12.69

	GGTCAA	−12.72

	TCGCAC	−12.75

	TCTTGC	−12.82

	CCTTTG	−12.82

	TTCGCC	−12.88

	TGGTCA	−12.91

	GCGCTC	−12.95

	GAAGTG	−12.95

	GCCTCT	−12.96

	AGGTGG	−12.96

	CAGTGG	−13

	GTACCC	−13.02

	TTCCTC	−13.04

	TCGACA	−13.05

	TGGCAG	−13.07

	CCGAAA	−13.09

	CTGCCT	−13.11

	ATGGGC	−13.12

	ACCGAG	−13.13

	CGTAGA	−13.16

	GGGCTT	−13.18

	CCGAGC	−13.19

	GACTTG	−13.23

	CTGACT	−13.26

	GAGGGA	−13.28

	AGTCGA	−13.32

	CCCGAG	−13.32

	CTTCCT	−13.32

	TCACGC	−13.37

	TAGGTG	−13.39

	CCTCTG	−13.41

	GCGACA	−13.46

	GCTAGC	−13.46

	TCATCG	−13.48

	CCCGCC	−13.49

	GTCCAA	−13.5

	TGGAGT	−13.55

	ACGAGT	−13.6

	CCCGGC	−13.6

	ACGGTA	−13.65

	TCCTGG	−13.65

	CGATCT	−13.73

	CAATCG	−13.76

	CTACGC	−13.79

	ATCACG	−13.84

	CGCTCT	−13.89

	CCCGAT	−13.92

	CGGTAT	−13.94

	AAGTCC	−13.95

	GGCATG	−14

	ATGAGG	−14.05

	AGGTCT	−14.05

	CTTAGT	−14.06

	ACTCGC	−14.08

	ACGAAG	−14.09

	GGGACT	−14.1

	AAGACG	−14.11

	TCCTGC	−14.11

	GCTCTC	−14.12

	TCTACG	−14.14

	TTGAGT	−14.15

	TCGAAC	−14.16

	CCCTTG	−14.27

	GTTCCT	−14.28

	GTCTCC	−14.29

	ACCGTC	−14.35

	TCTTTG	−14.39

	GGTTCC	−14.39

	GTTCCC	−14.42

	CGCTGT	−14.51

	CAACGT	−14.53

	CAGGCG	−14.56

	TACGCC	−14.59

	CGAAAC	−14.6

	TCTTTC	−14.65

	TGCCCT	−14.67

	GCCCTA	−14.68

	GTTTTC	−14.68

	GTATGC	−14.7

	GAAGGG	−14.72

	CGAAAG	−14.79

	GATTCG	−14.79

	CGATGC	−14.9

	TGAGCG	−14.92

	ACGCGG	−14.93

	CTCGAG	−14.94

	TGCGGA	−14.95

	ATGTCC	−15.01

	CGGCCA	−15.02

	ACCTAG	−15.05

	GTCAAA	−15.06

	GTGCCA	−15.08

	CCCCGA	−15.11

	CTGGCA	−15.12

	AAGGCG	−15.13

	GATTGC	−15.14

	TTTGAC	−15.14

	GTAGGT	−15.16

	GTTGTT	−15.17

	CCTAAC	−15.17

	GGACTT	−15.18

	CGTAAA	−15.2

	TCATGT	−15.21

	GGGACC	−15.22

	GGGCAG	−15.23

	CTGGTG	−15.25

	GGATGG	−15.27

	CCGTTA	−15.31

	GACGCC	−15.32

	CGCATC	−15.33

	ACGCTC	−15.33

	AAAGTC	−15.35

	GGGGCA	−15.38

	CTCGCA	−15.38

	GCACGG	−15.39

	AGCGAG	−15.4

	ACTGGC	−15.44

	CTGTCA	−15.51

	AGCGTC	−15.52

	GAGGAG	−15.53

	GTGTAA	−15.58

	TTGTAC	−15.6

	TCAGTG	−15.65

	GGCGCA	−15.71

	GCGAAC	−15.71

	TCTCTA	−15.73

	CCCGAA	−15.75

	TGAGGC	−15.76

	CCCCGG	−15.78

	CCTCGA	−15.83

	TATCGG	−15.85

	ATCCGT	−15.86

	AGCGGG	−15.87

	CCCACG	−15.87

	ACTGTC	−15.88

	GTTTAA	−15.92

	TAGTGG	−15.97

	AATGGG	−15.99

	ATCGAG	−15.99

	GTCCTT	−16.01

	AACGTG	−16.03

	CGCAAG	−16.03

	GGCCCT	−16.05

	CACGTA	−16.06

	TAGGGA	−16.09

	CGGCAA	−16.12

	CCTAAG	−16.15

	TCGAGA	−16.16

	GCCTGA	−16.16

	GACCCG	−16.19

	GTTAGA	−16.27

	TGCTTG	−16.27

	TCGAGC	−16.29

	ACGGTG	−16.32

	TCGATC	−16.34

	CAGGGG	−16.36

	GAATGT	−16.41

	TTGACT	−16.46

	TCAGTC	−16.47

	GCTCGA	−16.48

	AATCGT	−16.48

	GCCCTC	−16.49

	GACGGA	−16.49

	AAGAGG	−16.52

	CGTTAC	−16.52

	ATCCGG	−16.55

	TTATGT	−16.55

	CTTCGC	−16.56

	GAGTCC	−16.57

	GAGAGG	−16.6

	TGTCTT	−16.61

	AGAGTG	−16.64

	ACCCCG	−16.65

	TAACGG	−16.65

	CTCGGC	−16.66

	TAGAGG	−16.67

	CTTCCG	−16.75

	AACGGA	−16.76

	AAGTTT	−16.76

	GCCTGT	−16.77

	AGTCCT	−16.79

	GAACGG	−16.8

	GGCAAC	−16.84

	CTCGGA	−16.85

	TCGATA	−16.85

	ATGGGG	−16.85

	GGAGGC	−16.88

	CCGCCT	−16.93

	CCTCCT	−16.95

	AAGGGG	−16.95

	ACCGTG	−17

	GCCTAA	−17.04

	TGGGAC	−17.08

	TGGATG	−17.08

	TATCGC	−17.09

	GGACTA	−17.1

	CGAAGG	−17.11

	TCTAGT	−17.14

	GTCAAC	−17.15

	TTCTAG	−17.16

	CGAGAC	−17.19

	AAGGGT	−17.2

	GCGAAG	−17.21

	GCAAGT	−17.22

	CGGCCC	−17.26

	ATTTCG	−17.3

	GTGGTA	−17.33

	TGGTTC	−17.37

	GCATCG	−17.37

	GTACTC	−17.39

	ACGTAA	−17.4

	CTTGTT	−17.4

	GGACTG	−17.41

	GCCGTA	−17.43

	CCTGGC	−17.44

	AACGAG	−17.52

	CGCAGG	−17.55

	TCTTGG	−17.58

	AGACCG	−17.62

	TGCGAC	−17.65

	CAGTCG	−17.66

	GCCGTT	−17.66

	TAACGC	−17.67

	CGACAC	−17.69

	CCCGGA	−17.72

	GTCATT	−17.72

	ATCGGA	−17.74

	CCGAGT	−17.76

	GGTTTC	−17.8

	CGCATT	−17.82

	CCTTGC	−17.83

	TCTGCC	−17.83

	GCAAGG	−17.84

	CCCTGG	−17.85

	GTTTAC	−17.87

	AGGTCC	−17.91

	GCTTGT	−17.93

	CCGATC	−17.95

	TCGAAA	−17.95

	CTTGCC	−17.99

	TCCGAC	−18

	TATCGA	−18

	GATTGG	−18.08

	CGTTAT	−18.09

	TATCGT	−18.16

	TTTCGC	−18.18

	AAGTGG	−18.22

	GGCCCC	−18.22

	GGCCCA	−18.24

	ACCGGA	−18.25

	TCAGGC	−18.26

	CGTCTA	−18.28

	GTCATC	−18.3

	GACCTA	−18.31

	TTGTTC	−18.38

	TCCTAG	−18.4

	ACCCGT	−18.48

	ATCGTG	−18.49

	TTGGAC	−18.49

	CGGAAT	−18.51

	CAACGG	−18.61

	ACCGTT	−18.62

	CCGTAG	−18.63

	TGCCAG	−18.65

	TGTTAG	−18.77

	CGACCC	−18.77

	TTGTTT	−18.77

	TCGCAA	−18.77

	ATGGTC	−18.81

	CGTGGA	−18.81

	TCCTCT	−18.83

	TCGCCT	−18.84

	TCGGAT	−18.89

	GCGACC	−18.9

	ACGTTT	−18.91

	TTAGCC	−18.92

	CTCTTT	−18.92

	ACTTCG	−18.95

	CTATCG	−18.96

	GCGCAC	−18.96

	TCGAAG	−18.97

	TTATCG	−19.01

	TAAGTG	−19.03

	TGATCG	−19.03

	CTCGAT	−19.04

	CTCGAA	−19.13

	CTCACG	−19.18

	GGCTTG	−19.19

	CGCCGA	−19.19

	CTTGCT	−19.24

	GTAGTC	−19.28

	CACCGG	−19.31

	TTTGTT	−19.31

	TCTGTT	−19.32

	TTTACG	−19.34

	GTCCCC	−19.35

	CGAGGA	−19.35

	CGGATG	−19.35

	CCGATG	−19.37

	CATCGG	−19.38

	GGTAGT	−19.38

	CCGTGA	−19.41

	TCCGCC	−19.41

	TCTCTT	−19.42

	GGAGAG	−19.43

	CATTCG	−19.47

	CGAATG	−19.54

	TCTCTC	−19.56

	GGCCAA	−19.57

	GCTGTC	−19.65

	ACCGCG	−19.66

	GTGAGA	−19.68

	GGCCAC	−19.7

	CCTAGT	−19.71

	TCTTCG	−19.73

	GTGATC	−19.73

	ATGTAG	−19.77

	GTGACT	−19.79

	GACGGC	−19.8

	AGGGGC	−19.83

	ATTCCG	−19.84

	GTTTCC	−19.85

	GGCAAG	−19.96

	CGGCAT	−19.96

	TCCCGC	−19.96

	AGTGTT	−19.97

	GCCGAC	−19.99

	CCGATT	−20.01

	ATTCGG	−20.03

	TACCGT	−20.03

	TCAGGG	−20.08

	GTTTGA	−20.11

	GCTCCG	−20.13

	CCTGGT	−20.17

	CCTCTT	−20.18

	ACGTGA	−20.22

	GTCTAA	−20.25

	TAAGGG	−20.27

	TCCCCG	−20.29

	CACGTC	−20.32

	GGCAGG	−20.33

	CGTAAC	−20.35

	GAGGCC	−20.36

	TAGTCT	−20.36

	AGGGAG	−20.39

	ACTCGT	−20.39

	CGCTTA	−20.4

	GCGGAA	−20.46

	GGCTAA	−20.5

	CCTTCG	−20.52

	TAAGTT	−20.52

	TTGGCT	−20.53

	CCGGAG	−20.53

	ACGCCG	−20.58

	GTCTCT	−20.59

	CCGAAC	−20.66

	AGGGTT	−20.69

	GGTCAC	−20.72

	AGTGGT	−20.73

	TGTCAC	−20.75

	CCCCCG	−20.77

	TTCGAT	−20.79

	CGTAGT	−20.82

	GCGGCA	−20.82

	TCCGAG	−20.86

	TCAAGT	−20.87

	CCGTCT	−20.88

	GGAGGT	−20.93

	CTGACG	−20.94

	TGCCTC	−20.94

	AGTCAG	−20.95

	TTCTCT	−20.97

	CGGTTC	−20.97

	TGTCTA	−21

	TCTCCG	−21.04

	CACTCG	−21.05

	TGACGA	−21.15

	GTCTCA	−21.17

	GTCAAG	−21.27

	CTTGGC	−21.28

	ACGTCC	−21.28

	CGGTGA	−21.32

	TTGGGA	−21.4

	TCGTAA	−21.4

	CGGAAC	−21.42

	GGTATG	−21.43

	ACCGGT	−21.5

	CCGGAA	−21.51

	TCGTTA	−21.53

	AATGTC	−21.55

	CATGTG	−21.55

	GCGAGA	−21.58

	TTTAGG	−21.6

	GAGGTA	−21.69

	CCGGCC	−21.71

	TGTGGA	−21.72

	CTCTCT	−21.73

	GTGGCT	−21.74

	GCCGCC	−21.76

	GACCGA	−21.76

	GGTCAT	−21.8

	TCCCTG	−21.84

	GCGCTA	−21.87

	TCCGTA	−21.87

	TTGTTG	−21.9

	GTCCTA	−21.93

	GCCACG	−21.95

	TGCGTA	−21.97

	TCCGAA	−21.99

	GCCGGA	−22.01

	GAGCGG	−22.07

	TTCTCG	−22.07

	GACGAA	−22.08

	CTGCCG	−22.11

	CTGGTT	−22.11

	AGGCCG	−22.12

	GAGTCT	−22.25

	ATGGCG	−22.26

	GGGCAC	−22.28

	AGTCGC	−22.31

	GCGGAG	−22.37

	TCTCGA	−22.4

	GACCGC	−22.5

	CTCGAC	−22.51

	ACGGGC	−22.53

	GCGCAA	−22.56

	CTCCCG	−22.58

	GTATGT	−22.6

	GGGCAA	−22.61

	ATCTCG	−22.63

	AGTGCC	−22.66

	GTCTTC	−22.66

	CGGGAA	−22.68

	CGATGT	−22.69

	GACTTA	−22.7

	CGCGCA	−22.71

	GACGAC	−22.71

	GGGGCT	−22.72

	TCCCGA	−22.78

	TCAACG	−22.81

	CGTCAA	−22.81

	GATGGG	−22.81

	TGCCTT	−22.82

	TACGGT	−22.84

	TTTGCG	−22.87

	CGCCCC	−22.92

	GAGGTC	−22.93

	ATTGGG	−22.97

	CGGACT	−22.99

	AACGTA	−23.02

	ACGTTC	−23.04

	GACTCG	−23.1

	CTGTTG	−23.16

	GCTGGG	−23.19

	CGTTTT	−23.21

	TACGAG	−23.26

	GCCAGT	−23.28

	TTCGTA	−23.29

	CCTCCG	−23.29

	TTCTGG	−23.3

	GGGGAC	−23.32

	GATCGC	−23.32

	CCCGAC	−23.33

	CGGGAT	−23.34

	GTGTTA	−23.34

	GTAGAC	−23.35

	GCAGGG	−23.38

	AGAGGG	−23.41

	ACGGTT	−23.42

	CGCCTA	−23.43

	GGGCTA	−23.43

	GTACGA	−23.43

	TTTGAG	−23.44

	TACGTA	−23.44

	GTGACC	−23.47

	CTCGCT	−23.49

	ATTGTC	−23.58

	TTAAGT	−23.58

	TTACGC	−23.64

	GTTAAG	−23.65

	CCGGCA	−23.74

	AACGGT	−23.77

	CGAGTT	−23.8

	GGCCGA	−23.81

	GCGGTA	−23.82

	GCAACG	−23.84

	GCGATC	−23.9

	CTCCGA	−23.94

	CGGCCT	−23.97

	TCCGAT	−23.99

	AGACGT	−24.01

	TTTCGA	−24.02

	TTTTGT	−24.03

	ATTCGT	−24.04

	TCACGT	−24.05

	CCTGGG	−24.05

	TGTAAG	−24.09

	AATGCG	−24.13

	CGTTCT	−24.15

	CCGAGG	−24.17

	TCTAGG	−24.2

	TGGGTA	−24.22

	GTGTTT	−24.23

	TGATGT	−24.25

	TAGGTT	−24.27

	ACTTAG	−24.29

	AACGTC	−24.31

	AGGTTG	−24.34

	GTAGCG	−24.4

	GTTAAC	−24.41

	TATGGG	−24.43

	TAGCGC	−24.44

	CGTCAC	−24.48

	TTCCGA	−24.5

	GACTAG	−24.54

	TGGGGA	−24.57

	GCGCTT	−24.58

	TTCTGT	−24.59

	GGAGTC	−24.6

	CGCCTG	−24.62

	CGATTG	−24.63

	GGTGAC	−24.68

	TCGTAG	−24.68

	TGTCAA	−24.69

	GGGTTC	−24.7

	TTCGAC	−24.76

	TGTGTA	−24.79

	GAGTGA	−24.81

	GACGAG	−24.82

	CTAGGG	−24.83

	GTTGAG	−24.87

	TGACGC	−24.87

	CGCAAC	−24.94

	CGCCTC	−24.96

	GAGCGA	−24.96

	CAAGTG	−25.01

	TGGTGC	−25.01

	ACGGCG	−25.02

	CGAGGC	−25.03

	TACGCG	−25.05

	CATGGG	−25.06

	CTGTCC	−25.07

	GTAAGC	−25.1

	CGTGTA	−25.17

	ATGCCG	−25.2

	ACGTAC	−25.24

	TTCCGC	−25.28

	GTCTTT	−25.31

	TCCGGA	−25.33

	TTGTGA	−25.35

	AGTTCG	−25.37

	AGCGGT	−25.47

	GCCCGA	−25.51

	CTGGGC	−25.54

	TAGTGC	−25.55

	TTGCCC	−25.57

	TCTTGT	−25.63

	TGCGCC	−25.65

	CGAGAG	−25.69

	TATGTC	−25.72

	TGTCCT	−25.75

	AATCGG	−25.77

	TTTCCG	−25.78

	TATGTG	−25.8

	TGGGCA	−25.88

	GCTTGC	−26.03

	TCGACC	−26.05

	TTAGCG	−26.06

	CCGTTC	−26.08

	CTAACG	−26.09

	GGCGAT	−26.11

	GTTAGC	−26.11

	GTGGCA	−26.14

	CCGGGA	−26.15

	GCCTGG	−26.17

	CTTAGG	−26.18

	AACGCG	−26.19

	CGCGAA	−26.21

	ATCGTC	−26.24

	CTTGGG	−26.27

	GCACGC	−26.29

	GAGAGT	−26.3

	GCATGC	−26.3

	ATCGTT	−26.33

	GAGGTG	−26.36

	TTAACG	−26.36

	CTGCGG	−26.38

	ACGGGA	−26.47

	GGCCTG	−26.49

	CCTGCG	−26.49

	AGGGTA	−26.49

	GAACGT	−26.5

	TTTGGT	−26.53

	ACGGAG	−26.54

	GGAGCG	−26.73

	CCGCCG	−26.75

	CCTACG	−26.76

	GTAACG	−26.81

	CCCGTA	−26.81

	GCTTCG	−26.82

	TAGTCG	−26.83

	CGTCCA	−26.91

	TGAGTC	−27.01

	CTCTGG	−27.01

	ATTGCG	−27.01

	CGATGG	−27.05

	GCTAGG	−27.14

	GGGAGT	−27.16

	ATGTCT	−27.2

	CTGGGT	−27.21

	GGACGA	−27.23

	CGTTTC	−27.24

	ATGACG	−27.27

	TTCGCT	−27.27

	AGGGTG	−27.33

	CTTCGG	−27.4

	CGAAGT	−27.41

	TTGCCT	−27.41

	GGATCG	−27.41

	AGGCGC	−27.45

	GGGTTA	−27.47

	ACGCGC	−27.48

	TTGTCA	−27.57

	TAGTGT	−27.58

	GAGGTT	−27.6

	TGTCTC	−27.6

	GTGATG	−27.65

	GGTCCT	−27.65

	CGGACC	−27.65

	TCGCTT	−27.66

	TCGGAA	−27.69

	ACGTCA	−27.74

	TTCCCG	−27.84

	GCACGT	−27.87

	GTCGCA	−27.88

	CGTTAA	−27.93

	ACCTCG	−27.95

	TGGGAG	−27.96

	CTGTGT	−27.97

	TAGCGT	−28.06

	AGGACG	−28.08

	GGCCTC	−28.1

	AGTACG	−28.14

	TAAGCG	−28.21

	CTGCGT	−28.23

	TGTGTT	−28.25

	GGGTAA	−28.26

	TTTTCG	−28.33

	GCGTTT	−28.33

	TCTCGG	−28.34

	GCGGAC	−28.36

	CGACTT	−28.38

	CGACGA	−28.4

	GTTAGT	−28.44

	CCTCGC	−28.53

	TTGCGA	−28.62

	GTCGCT	−28.65

	GTTCTG	−28.7

	CGCGGA	−28.75

	GACGTA	−28.8

	ATGTGT	−28.81

	CCGCGT	−28.84

	TTAGGC	−28.88

	CTTTGG	−28.94

	TACCGG	−29

	GTAAGT	−29.01

	ACGAGG	−29.02

	ACGTAG	−29.02

	TGGCTC	−29.02

	GCTTGG	−29.05

	ACGTTA	−29.06

	AGGAGG	−29.07

	TGACGG	−29.11

	CCACGT	−29.16

	CGTATG	−29.17

	CGGGCA	−29.21

	AACGGG	−29.25

	CTCCGG	−29.27

	GGGCCA	−29.28

	CGTACC	−29.28

	CCGTAC	−29.41

	CGTACT	−29.46

	CTGTCT	−29.48

	TGCCTG	−29.64

	CTGTGG	−29.64

	TGGTTG	−29.7

	GGTTGA	−29.72

	GAGGGC	−29.76

	TTCGGC	−29.83

	GGTTGC	−29.89

	TCTGGT	−29.9

	CCTCGG	−29.96

	GTTGAC	−30

	TTGACG	−30.03

	AACGTT	−30.07

	CCGACC	−30.12

	GGGTTT	−30.13

	GTCTAC	−30.13

	ACGACG	−30.19

	CGGGCT	−30.2

	GTAGAG	−30.23

	GGAACG	−30.3

	GTCTTA	−30.3

	GCTGGC	−30.31

	CGTGCT	−30.39

	CTACGT	−30.41

	CTTTCG	−30.42

	TGTCCC	−30.46

	CGGTAG	−30.52

	TCTGCG	−30.54

	TCGATG	−30.54

	TCGGAC	−30.58

	TCCGGC	−30.61

	TTCGAG	−30.64

	CCCTCG	−30.66

	CCGCGA	−30.67

	ACGTGC	−30.67

	GGCCTA	−30.68

	CCGGAC	−30.7

	GCGTAA	−30.77

	GTCCTC	−30.77

	TTCGGA	−30.82

	CCCCGC	−30.82

	AGCGCG	−30.84

	CTCGCC	−30.85

	GGCTAG	−30.87

	CTTACG	−30.96

	GATGTC	−30.96

	GGACGC	−30.98

	ACGTCT	−30.99

	TGTCAG	−31

	ACGCGA	−31.01

	GTTCGA	−31.06

	TTGAGG	−31.08

	TCGTAC	−31.09

	TTAGGG	−31.1

	TGCCGC	−31.12

	TAGGCC	−31.12

	CCCGGG	−31.15

	CGACCT	−31.16

	CGAGTC	−31.3

	TCTGAC	−31.36

	GTCCCT	−31.46

	TCTGGG	−31.48

	CGCGTA	−31.55

	TGAGGG	−31.55

	CGGGGA	−31.59

	CGACGC	−31.63

	TGAGGT	−31.63

	TTTGGC	−31.64

	CGTCAG	−31.68

	GATGTG	−31.69

	TGGCGA	−31.75

	GTGAGC	−31.75

	GTCGTA	−31.76

	TCTGGC	−31.78

	GTGTCT	−31.81

	GCGTCA	−31.86

	GCGCCT	−31.88

	CCTGTG	−31.89

	AGTCGT	−31.89

	TCGGTA	−31.95

	CCGGTT	−31.95

	CGCGCT	−31.96

	CTTGGT	−31.98

	TTACGT	−32.02

	GTGTAC	−32.06

	CGCTTG	−32.07

	CCGACG	−32.12

	CCGTGT	−32.13

	GTATCG	−32.13

	TTAGTC	−32.13

	TCGGCC	−32.14

	CATGCG	−32.19

	GTCAGA	−32.21

	ACGTTG	−32.23

	CGCATG	−32.23

	TCCTGT	−32.37

	GCGAGC	−32.37

	ACGTGT	−32.41

	ATGCGG	−32.41

	TGGTCC	−32.44

	ATGGGT	−32.58

	CGTCTC	−32.61

	TGTGCC	−32.64

	CTTGTC	−32.65

	GGCGGA	−32.72

	GTCTGA	−32.74

	CTGGTC	−32.79

	GGGGGA	−32.83

	AGGGTC	−32.86

	TAGTCC	−32.91

	CGGGTA	−32.94

	GCGTTA	−32.98

	GACCGT	−33

	GATCGT	−33.15

	ATCGGT	−33.22

	CACGGG	−33.24

	GACGTT	−33.25

	CACGCG	−33.27

	GGTAAG	−33.36

	GTCGAT	−33.37

	GATGCG	−33.38

	GGACCG	−33.38

	GCCCCG	−33.46

	GCGGGA	−33.56

	GGTCCC	−33.6

	GTATGG	−33.62

	CCCGTT	−33.63

	CGCGAT	−33.69

	CCGTGC	−33.75

	GACGGT	−33.89

	CGACCG	−33.91

	CCTGTC	−33.96

	GTAGTG	−34.01

	GGGTCA	−34.05

	TAGGGC	−34.19

	GTTACG	−34.32

	AGGTAG	−34.33

	GGCCGC	−34.45

	GCGGCC	−34.5

	GCCTCG	−34.53

	CGAACG	−34.71

	GTCGAA	−34.72

	CGTCCC	−34.81

	CTAAGT	−34.82

	CCGGTA	−34.83

	GTCTGC	−34.87

	TCGTGA	−34.87

	CGGAGT	−34.91

	GGGTAC	−34.91

	GTGGAC	−34.93

	ACGGTC	−34.94

	CTCGTA	−34.95

	TCGAGT	−35

	TCTGTG	−35

	GGTTGT	−35.09

	AGGGGT	−35.15

	TACGTT	−35.18

	TCGTCA	−35.34

	AAGTGT	−35.39

	TGTAGG	−35.44

	GCGGTT	−35.48

	TACGTC	−35.52

	TGTTGC	−35.56

	TTGGTG	−35.57

	AGCGTG	−35.58

	CTGGCG	−35.58

	TGTACG	−35.8

	CGTCAT	−35.87

	TCCTCG	−36.02

	GGGCCT	−36.04

	CTAGTC	−36.07

	TGTTGG	−36.07

	GTGGAG	−36.09

	GGCCGT	−36.15

	GTTTGC	−36.17

	CCCCGT	−36.18

	GTGGTT	−36.22

	CGCCCT	−36.23

	TCGCCC	−36.23

	GATCGG	−36.23

	TGACCG	−36.25

	GGGTGA	−36.29

	TTCCGT	−36.3

	ATCGGG	−36.36

	TCCCGG	−36.42

	TGGCCA	−36.53

	GTGTAG	−36.53

	ATGCGT	−36.65

	GCCCGC	−36.69

	TGGCGC	−36.74

	GTGGGA	−36.74

	TGTTCG	−36.88

	TGGCCT	−36.92

	GGTCTA	−36.94

	TGCGGC	−36.96

	CGTGAC	−37

	TAACGT	−37.18

	TCGTTT	−37.19

	CGCTAG	−37.2

	CGGCCG	−37.2

	CTTGCG	−37.21

	AGGCGG	−37.21

	CGTTGA	−37.28

	TGTTTG	−37.33

	GTAAGG	−37.38

	CGGACG	−37.41

	CGCCGT	−37.43

	CGGAGG	−37.44

	CGTTCC	−37.45

	TGCGAG	−37.46

	GTTGGT	−37.56

	TTTGTC	−37.57

	GAGGGT	−37.59

	TAAGTC	−37.59

	GGCTCG	−37.63

	GACGCG	−37.63

	GGGTCT	−37.66

	TCGCTC	−37.67

	TCTCGC	−37.72

	TTTGTG	−37.74

	ATCGCG	−37.75

	GGGGTT	−37.76

	GTCACG	−37.82

	GGTCTT	−37.95

	CCCGTC	−37.96

	CTAGCG	−37.99

	CGCACG	−38.02

	TTAGGT	−38.03

	CGGGAG	−38.06

	GTTTAG	−38.11

	GCCCGT	−38.11

	GTCCGA	−38.11

	AGTGAG	−38.2

	CTTGTG	−38.23

	TCGAGG	−38.24

	TTGGCC	−38.25

	AGTCCG	−38.38

	CGGTTT	−38.45

	TGCGTT	−38.48

	CGTGAT	−38.53

	GCGTTC	−38.53

	TTGGGG	−38.54

	GGTTTG	−38.55

	CGGTAC	−38.57

	TGGCCC	−38.57

	GCTCGC	−38.62

	ACGCGT	−38.63

	TGTGAC	−38.71

	GACCGG	−38.72

	GCGCCC	−38.73

	ACCGGG	−38.81

	GGTGCC	−38.81

	TTGTCC	−38.88

	TTGCCG	−38.89

	ACGGGT	−38.92

	ATGTGG	−39

	GGTCTC	−39.04

	CGTAAG	−39.11

	TTCGTT	−39.12

	TACGGG	−39.13

	GTCATG	−39.17

	GGACGT	−39.21

	CGGGAC	−39.25

	TGGGTT	−39.32

	GAGTGC	−39.35

	CTCTCG	−39.4

	CGCGAC	−39.42

	TAGGGG	−39.5

	GGCACG	−39.52

	CCGCGC	−39.55

	TGGACG	−39.58

	GGCGAC	−39.6

	CTGGGG	−39.69

	CGGGTT	−39.73

	GTGCCT	−39.76

	TTGGGC	−39.77

	GCCGTC	−39.8

	GGCCAG	−39.84

	CCGTCC	−39.9

	GCGCGA	−39.9

	CGCGGC	−39.95

	TCGGGA	−39.98

	GTCTTG	−40.04

	AGTGGG	−40.12

	CCGGGG	−40.16

	TTTGGG	−40.17

	CTTCGT	−40.23

	CGGTCA	−40.24

	CACGTG	−40.28

	GGTGAG	−40.43

	GTCGAC	−40.48

	TGCTCG	−40.49

	TGGGGC	−40.62

	GGAGGG	−40.68

	TGTGAG	−40.75

	GGGGCC	−40.89

	GGTGGT	−40.9

	AGGCGT	−40.91

	TCCGTC	−40.92

	TCCGCG	−40.92

	GTACCG	−41.02

	AGGTGT	−41.07

	GCTCGT	−41.08

	TTTCGT	−41.14

	TGCCCG	−41.17

	CGATCG	−41.22

	CGTCTT	−41.34

	TTCCGG	−41.5

	GTTTTG	−41.52

	GCGAGT	−41.58

	GGGGAG	−41.63

	CTAGGT	−41.64

	CCGTGG	−41.64

	GAGGCG	−41.68

	CCGCGG	−41.7

	TTGTGC	−41.74

	TTTCGG	−41.75

	GCGTAC	−41.83

	GTACGC	−41.88

	GAGTCG	−41.9

	TCCGTG	−42.01

	CGGCGA	−42.01

	CTCCGT	−42.07

	TTGCGC	−42.08

	GTGCCC	−42.11

	GCGTGA	−42.12

	GTAGGC	−42.16

	CTCGTT	−42.19

	GTGCGA	−42.23

	AGTGCG	−42.39

	CGCCCG	−42.43

	GGCGTA	−42.43

	GAGCGT	−42.48

	TCGTTC	−42.53

	AAGTCG	−42.67

	GTCAGG	−42.73

	CGTTAG	−42.84

	TCGGTT	−42.92

	TCGCGA	−42.92

	GGGAGG	−42.93

	GGGACG	−42.94

	GTCAGT	−43.2

	TGCCGT	−43.2

	GGGGTA	−43.2

	GCGTCT	−43.23

	GCCGCG	−43.26

	AGTCGG	−43.28

	TCCGTT	−43.36

	CTCGGG	−43.37

	GGGCCC	−43.5

	TAGGCG	−43.53

	GGTCAG	−43.58

	GGGTAG	−43.61

	TACGTG	−43.67

	GTCCTG	−43.69

	CGCCGC	−43.8

	CTGGCC	−43.85

	TAGGGT	−43.88

	GCCGGG	−43.92

	GGTTAG	−43.96

	CCGGGT	−44.07

	CTCGTC	−44.16

	GTCTAG	−44.19

	GGTGTT	−44.21

	CCGGGC	−44.24

	AGTGTG	−44.25

	CGAGGG	−44.3

	GTTGGC	−44.3

	CGGCGC	−44.3

	AGTGTC	−44.31

	CGTTGT	−44.33

	GTTCGC	−44.35

	GTTGCC	−44.36

	GGCGGT	−44.48

	TTCGCG	−44.51

	TTGCGG	−44.57

	GTGTTC	−44.64

	ATGTCG	−44.73

	GGCGGC	−44.81

	TCGGAG	−44.82

	GACGGG	−44.82

	CGTCCT	−44.86

	TCGACG	−44.94

	GGACGG	−44.99

	TGGTGG	−44.99

	TCCCGT	−45.06

	TGTCGA	−45.08

	GCTCGG	−45.1

	GGGCCG	−45.15

	GTTTGT	−45.27

	GAGGGG	−45.32

	TTCGTG	−45.45

	GCGAGG	−45.47

	CCTCGT	−45.53

	GCCCGG	−45.6

	GTCTGT	−45.62

	TTGTCT	−45.66

	CGGTGT	−45.71

	CGTTTG	−45.74

	GGTAGG	−45.84

	GTCCGC	−45.88

	GCCGGC	−45.88

	CGCGTT	−45.93

	ACGGGG	−45.94

	CCGTTG	−45.97

	TCTGTC	−46.05

	GGGCGA	−46.06

	GACGTG	−46.08

	TTGGTC	−46.16

	GCCGGT	−46.32

	TTGCGT	−46.32

	GGGTTG	−46.34

	GGCGAG	−46.43

	CGTGTT	−46.44

	GGGTCC	−46.51

	TGGGCC	−46.53

	GCGTTG	−46.58

	CGACGG	−46.68

	AGGGGG	−46.69

	GTGTCA	−46.75

	GCGTAG	−46.76

	TAGGTC	−46.77

	CGCGAG	−46.79

	TGAGTG	−47.04

	GACGTC	−47.04

	GTCGGA	−47.14

	GGTTGG	−47.18

	TCGCGC	−47.26

	GCGCCG	−47.28

	TGTCTG	−47.32

	GCCGTG	−47.35

	CGTTGC	−47.38

	TCGTCT	−47.39

	GGCGCC	−47.47

	GGGGGC	−47.53

	TTGTGG	−47.6

	CGGTCC	−47.61

	CGCGCC	−47.71

	TTCGGT	−47.79

	TGACGT	−47.8

	TGTCCG	−47.88

	TGTTGT	−47.91

	CCCGGT	−48.15

	GCGTCC	−48.17

	TCCGGG	−48.25

	CCCGCG	−48.5

	TCGTCC	−48.56

	GTTCGT	−48.56

	GTTCCG	−48.59

	TTGGCG	−48.69

	TGGCCG	−48.69

	GCGACG	−48.74

	GGAGTG	−48.78

	GTTAGG	−49.18

	GGCCGG	−49.22

	CGGTTG	−49.22

	TCTCGT	−49.23

	CGAGCG	−49.24

	CGAGGT	−49.43

	CGTCTG	−49.43

	CTCGGT	−49.55

	TTCGTC	−49.6

	GGCGTT	−49.72

	TCGGCG	−49.79

	CGTCGA	−49.86

	GTGACG	−49.87

	CGACGT	−49.9

	GGTACG	−49.96

	CGGTGC	−50.01

	GTACGG	−50.02

	CGTGAG	−50.26

	CGGCGG	−50.36

	TTGTGT	−50.37

	GAGTGG	−50.58

	TTCGGG	−50.66

	TGTGTC	−50.68

	TGGGTC	−50.7

	GGTCGA	−50.8

	GTTTGG	−50.88

	CCCGTG	−51.09

	GTTTCG	−51.17

	CGAGTG	−51.21

	GAGTGT	−51.21

	TGGTGT	−51.29

	TCGGGC	−51.42

	TGCGCG	−51.46

	TCGCCG	−51.51

	CCGGTC	−51.68

	CGCGTC	−51.71

	GTCGTT	−51.72

	TGGTCT	−51.83

	CGCGGT	−51.85

	GGTCTG	−51.86

	CTCGCG	−51.88

	CTCGTG	−51.94

	CGGGCC	−52.44

	GTACGT	−52.73

	AGGGCG	−52.77

	GTGCCG	−52.81

	GTGAGT	−52.89

	TGTGGT	−52.99

	CGGTCT	−53.11

	TCGTGG	−53.14

	CGGGGC	−53.26

	TCGTTG	−53.27

	ACGTGG	−53.35

	GGTTCG	−53.38

	ACGTCG	−53.48

	GGCCCG	−53.53

	CGTGCC	−53.55

	TGGGGG	−53.57

	CGGCGT	−53.63

	CGTAGG	−53.63

	GTCTGG	−53.69

	GTGAGG	−53.7

	CGTACG	−53.71

	GTTGTC	−53.73

	TTGGGT	−53.74

	CGTCCG	−53.8

	TGCCGG	−53.82

	TCGTGC	−53.92

	CGGGTC	−54

	GTCGAG	−54.01

	CGTTGG	−54.19

	CCGGCG	−54.27

	TCCGGT	−54.32

	GCGGGT	−54.37

	TCGTGT	−54.38

	CGCCGG	−54.53

	CGCTCG	−54.55

	GTCCGT	−54.62

	GGTGGC	−54.7

	TGCGTC	−54.83

	GGGTGC	−54.96

	GTCGCC	−55.39

	TGTGCG	−55.49

	CGTGTC	−55.5

	GGCGTC	−55.61

	GCGCGC	−55.66

	CTGTCG	−55.74

	GTCCCG	−56.36

	GCGGGC	−56.49

	GTAGGG	−56.76

	TGTCGC	−56.8

	TCGCGG	−56.94

	TGGCGT	−57.03

	GTGCGC	−57.04

	TTGTCG	−57.09

	GTGTTG	−57.15

	TGGGGT	−57.19

	GGTCGC	−57.25

	CGTGGC	−57.9

	GGGCGC	−58.19

	TGCGGT	−58.27

	TGGCGG	−58.3

	GGGGGT	−58.38

	TCGGGT	−58.51

	CCGGTG	−58.6

	CGTTCG	−58.67

	TCGGTC	−58.82

	GTCGGC	−58.88

	GTGGTC	−58.88

	GTGTGA	−59.14

	CGTGGT	−59.24

	GTGGCC	−59.29

	GCGGTC	−59.3

	GCGCGT	−59.36

	AGGTCG	−59.5

	GTCTCG	−59.51

	GGTGTC	−59.7

	TGGTCG	−59.72

	GCGGTG	−60.02

	TGCGTG	−60.04

	GTGTGT	−60.05

	GGGGTC	−60.15

	CGCGCG	−60.19

	TGGGCG	−60.25

	GCGTGT	−60.27

	GTTGGG	−60.36

	TGCGGG	−60.39

	TGTGGC	−60.71

	GCGCGG	−60.73

	CGTCGC	−60.8

	CCGTCG	−60.85

	GTGGTG	−60.86

	GTTCGG	−61.52

	GGGCGG	−61.53

	TCGCGT	−61.64

	GTGTCC	−61.73

	GGGTGT	−61.79

	GGGGGG	−62.06

	TGTGTG	−62.08

	GCGTGC	−62.32

	CGGGGG	−62.44

	CGGGCG	−62.52

	GGCGTG	−62.89

	TCGGTG	−63.03

	GGCGGG	−63.07

	GTTGTG	−63.22

	GGTCGT	−63.3

	TCGGGG	−63.6

	GTTGCG	−64.3

	GGGCGT	−64.62

	TCGTCG	−64.83

	GGTCCG	−64.88

	GCGGCG	−64.99

	GTGCGG	−65.11

	GGTGCG	−65.21

	GCGTGG	−65.85

	GGGGCG	−66.57

	CGCGTG	−66.73

	GTGTGC	−66.98

	GTCCGG	−67.1

	GTGCGT	−67.14

	TGTCGT	−67.26

	TGTGGG	−67.31

	CGGTCG	−67.35

	CGGGGT	−67.36

	CGCGGG	−67.6

	TGTCGG	−67.61

	CGTCGG	−68.18

	GGCGCG	−68.24

	GGGGTG	−68.68

	CGTCGT	−68.69

	GTCGGT	−68.84

	TGGGTG	−69.08

	GTCGTC	−69.14

	GCGTCG	−69.26

	CGGGTG	−69.69

	GGGTGG	−69.98

	GTGGGC	−70.27

	CGTGTG	−71.38

	CGGTGG	−71.52

	CGTGCG	−71.83

	GCGGGG	−72.46

	GTGGGT	−73.21

	GTCGCG	−73.55

	GTCGTG	−73.94

	GTGGCG	−73.94

	GTGGGG	−74.96

	GGTGGG	−75.37

	CGTGGG	−75.74

	GGGTCG	−76.6

	GTCGGG	−80.38

	GGTCGG	−81.93

	GGTGTG	−82.57

	GTGTCG	−84.85

	GTGTGG	−90.52

Another application of the rank ordering of all motifs for their ability to increase or decrease SHM-mediated mutagenesis is the creation of gene constructs that are colder or hotter relative to wildtype sequence. Any sequence position in a gene can be evaluated for an equivalent sequence with a hotter or colder (relative to the starting or unmodified sequence) motif consistent with the amino acid according to the z-score shown in Tables 2 and 3. So while no best preferred hot spot or cold spot motif from Table 7 may be found to substitute at a particular sequence position, a relative improvement in the SHM properties of the replacement motif may almost always be made where there is an underlying degeneracy in the codon sequence. A log-odds based score of the observed to expected mutation frequencies at each motif might also be used to score a tile-library for a polynucleotides cumulative hotness or coldness to SHM.
Thus, one can appreciate that the replacement of any SHM motif with one that has a greater probability of SHM, mediated mutagenesis can result in a sequence that is more susceptible to somatic hypermutation, and that the replacement of any SHM motif with one with a lower probability of SHM mediated mutagenesis can result in a sequence that is more resistant to somatic hypermutation.
Tables 2 and 3 shows the 3-mer, 4-mer, and 6-mer motifs ranked by z-score for their ability to attract SHM-mediated mutation. Another possible representation of hot and cold hot spot motifs can be made by constructing a position specific matrix from the assembly of motifs represented amongst the highest and lowest scoring z-scores. Below is a single example of a 6-mer motif overrepresented amongst the top scores motif “hot spots,” given as a position specific matrix in Table 4:

TABLE 4

Position	Position
1	2	Position 3	Position 4	Position 5	Position 6

A	0.0	1.0	0.0	0.0	0.2	0.5
C	0.75	0.0	0.0	1.0	0.0	0.0
G	0.0	0.0	1.0	0.0	0.0	0.5
T	0.25	0.0	0.0	0.0	0.8	0.0

In one non-limiting example, the term “preferred hot spot SHM codon” or “preferred hot spot SHM motif” refers to a codon or motif, including but not limited to codons TAC, TAT, or AGT, AGC, potentially embedded within the context of a larger hot spot motif which recruits AID-mediated mutagenesis and generates targeted amino acid diversity at that position. SHM introduces specific nucleotide transitions at each position of a “hot spot” motif with a frequency that can quantified. This spectrum of nucleotide transitions results in different possible silent or non-silent amino acid transitions is dependent on which of the three possible reading frames is used. By defining the most likely codon transitions mediated by SHM and the sequential flow mutation events, “preferred hot spot SHM codons” or “preferred hot spot SHM motifs” can be chosen in such a way as to generate a specific panel of amino acid transitions that suit the functionality of a library at each amino acid position, as described in Section V.

IV. Polynucleotide Design Strategies for SHM

Provided herein is a method to design nucleotide templates to either maximize or minimize the tendency of a polynucleotide to undergo SHM, while at the same time maximizing protein expression, RNA stability, and the presence of conveniently located restriction enzyme sites.
Also provided herein are synthetic versions of a polynucleotide that are altered to either enhance, or decrease the impact of SHM on the rate of mutagenesis of that polynucleotide compared to its wild type's susceptibility to undergo SHM (i.e., SHM susceptible or SHM resistant).
The SHM susceptible sequences facilitate the rapid evolution and selection of improved mutant versions of proteins and the system combines the power of rational design with accelerated random mutagenesis and directed evolution.
Conversely, the SHM resistant sequences facilitate the rapid evolution and selection of improved mutant versions of proteins and the system combines the power of rational design with decreased random mutagenesis and directed evolution
Also included in the invention are SHM resistant polynucleotide sequences that allow for conserved domains to be resistant to SHM-mediated mutagenesis, while simultaneously targeting desired sequences for increased susceptibility to SHM-mediated mutagenesis.
Polynucleotides for which these methods are applicable include any polynucleotide sequence that can be transcribed and a functional assay devised for screening. Preferred polynucleotide sequences include those encoding proteins, polypeptides and peptides such as, for example, specific binding members, antibodies or fragment thereof, an antibody heavy chain or portion thereof, an antibody light chain or portion thereof, an intrabodies, selectable marker genes, enzymes, receptors, peptide growth factors and hormones, co-factors, and toxins.
Other non-limiting examples of molecules for use herein include polynucleotides that have enzymatic or binding activity without the need for translation into a protein or peptide sequence, such polynucleotides including for example, enzymatic nucleic acids, antisense nucleic acids, triplex forming oligonucleotides, 2,5-A chimeras, RsiNA, dsRNA, allozymes, abd aptamers.
Biologically active molecules of the invention also include molecules capable of modulating the pharmacokinetics and/or pharmacodynamics of other biologically active molecules, for example, lipids and polymers such as polyamines, polyamides, polyethylene glycol and other polyethers. For example, polypeptides are those such as, for example, VEGF, VEGF receptor, Diptheria toxin subunit A, B. pertussis toxin, CC chemokines (e.g., CCL1-CCL28), CXC chemokines (e.g., CXCL1-CXCL16), C chemokines (e.g., XCL1 and XCL2) and CX₃C chemokines (e.g., CX₃CL1), IFN-gamma, IFN-alpha, IFN-beta, TNF-alpha, TNF-beta, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, TGF-beta, TGF-alpha, GM-CSF, G-CSF, M-CSF, TPO, EPO, human growth factor, fibroblast growth factor, nuclear co-factors, Jak and Stat family members, G-protein signaling molecules such as chemokine receptors, JNK, Fos-Jun, NF-κB, I-κB, CD40, CD4, CD8, B7, CD28 and CTLA-4.
One strategy for altering the ability of a polynucleotide to undergo SHM is through modulating the frequency and location of hot stops within the polynucleotide sequence of interest. The position or reading frame of a hot spot or cold spot is also an important factor governing whether SHM mediated mutagenesis can result in a mutation that is silent with regards to the resulting amino acid sequence. Both the degree of SHM recruitment and the reading frame of the motif are considered in the design of hot and cold spots.
An optimized polynucleotide sequence has been made “susceptible for SHM” if the polynucleotide sequence, or a portion thereof, has been altered, or designed, to increase the frequency and/or location of hot spots within the open reading frame and/or has been altered, or designed, to decrease the frequency and/or location of cold spots within the open reading frame of the polynucleotide sequence compared to the wild type polynucleotide sequence.
Conversely, an optimized polynucleotide sequence has been made “resistant to SHM” if the polynucleotide sequence, or a portion thereof, has been altered to decrease the frequency and/or location of hot spots within the open reading frame of the polynucleotide sequence, and/or has been altered, or designed, to increase the frequency and/or location of cold spots within the open reading frame of the polynucleotide sequence compared to the wild type polynucleotide sequence.
One can target specific regions of a polynucleotide for optimization of either hot spots, cold spots or both. In one embodiment, regions of a polynucleotide can be made hot (e.g., ligand binding, enzymatic activity, etc.) while other regions (e.g., those needed for structural folding, conformation, etc.) of a polynucleotide can be made cold. For example, it has been observed that in antibody genes, the codon usage and precise concomitant hot spot/cold spot targeting of AID activity and pol eta errors has evolved under selective pressure to maximize mutations in the variable regions and minimize mutations in the framework regions.
A polynucleotide sequence can be prepared that has a greater or lesser propensity to undergo SHM mediated mutagenesis by selectively altering the codon usage to modulate SHM hot spot and/or cold spot density. Based on this information, it is possible to optimize particular regions of a polynucleotide that appear to be directly involved in a functional attribute of a protein encoded by the polynucleotide. In one non-limiting example, nucleotides to be optimized can encode amino acids that can lie within, or within about 5 Å of a specific functional or structural attribute of interest. Specific examples include, but are not limited to, amino acids within CDRs of antibodies, binding pockets of receptors, catalytic clefts of enzymes, protein-protein interaction domains, of co-factors, allosteric binding sites, etc.
SHM hot spot and cold spot motifs have been described elsewhere herein such as, for example, in Tables 2, 3 and 7. Non-limiting examples of 4-mer nucleotide hot spot motifs that can be used for this purpose, include for example, nucleotide sequences of TACC, TACA, TACT, TGCC, TGCA, TGCT, AACC, AACA, AACT, AGCC, AGCA, AGCT, GGTA, TGTA, AGTA, GGCA, TGCA, AGCA, GGTT, TGTT, AGTT, GGCT, TGCT and AGCT, and their complementary sequences encoded on the alternative DNA strand. Exemplary 3-mer cold spot motifs include, for example, nucleotide sequences of CCC, CTC, GCC, GTC, GGG, GAG, GGC and GAC.
An additional consideration is the reading frame of the hot or cold spot. Hot spot motifs can be situated relative to the reading frame such that SHM-mediated mutation is more likely to occur at the wobble position of a codon (the third position), making a silent mutation more likely to result. Conversely, a hot spot motif can be situated relative to the reading frame such that a non-silent mutations result from changes in codons (data not shown). As discussed below, these design parameters can be conveniently optimized using an iterative computer algorithm.
In addition to optimizing hot spot and/or cold spot motif density, it can also desirable to consider the following characteristics such that optimized polynucleotides are efficiently translated, and stable in a host system.
The density of CpG dinucleotides motifs: Excessive CG motifs can result in gene methylation leading to gene silencing, and can be normalized to the density found in highly transcribed gene in the host system in question (see for example, Kameda et al., Biochem. Biophys. Res. Commun. (2006) 349(4): 1269-1277).
The ability of single stranded sequences to form stem-loop structures: the formation of stem-loop structures can result inefficient transcription and or translation, particularly when located near the 5′ region of the coding frame (see, e.g., Zuker M., Mfold web server for nucleic acid folding and hybridization prediction. Nucl. Acid Res. (2003); 31(13): 3406-3415). Stem loop structure formation can be minimized by avoiding repetitive or palindromic stretches of greater than 6 nucleotides, for example, near the 5′ end. Alternatively, longer stems are acceptable if the loop contains greater than about 25 nucleotides (nt).
Codon Usage: Appropriate codon usage, i.e., the use of codons that encode for more common and frequently used tRNAs, rather than very rare tRNAs, is important to enable efficient translation in the expression system being used (see generally Nakamura et al., Nuc. Acid. Res. (2000) 28 (1): 292, “Codon usage tabulated from international DNA sequence databases: status for the year 2000;” which includes codon frequency tables of each of the complete protein sequences in the GenBank DNA sequence database as of 2000). Generally codon usage is more important near the 5′ end of the gene where transcription of the polynucleotide begins, and rare codons should be avoided in this region where ever possible. Preferred is the elimination of about 80% or more, of the codons that are used less than 10% of the time within the coding frame of the expressed genes in the organism of interest.
GC content: Generally this should be matched, to the GC content of highly expressed genes in the host organism, for example in mammalian systems GC content should be less than about 60%.
Restriction sites: Restriction sites should be placed judiciously where desired. Similarly, important restriction sites (i.e. those that are intended to be used to clone the entire gene, or other genes) within a polynucleotide should be removed where not desired by altering wobble positions.
Stretches of the same nucleotide: Minimize or eliminate stretches of the same nucleotide to less six (6) contiguous nucleotides.
In addition, expression can be further optimized by including a Kozak consensus sequence [i.e., (a/g)cc(a/g)ccATGg] at the start codon. Kozak consensus sequences useful for this purpose are known in the art (Mantyh et al. PNAS 92: 2662-2666 (1995); Mantyh et al. Prot. Exp. & Purif. 6,124 (1995)).
Avoid, or minimize the usage of certain codons (“Non preferred SHM codons”) that can be mutated in one step to create a stop codon. “Non preferred codons” include, UGG (Trp), UGC (Cys), UCA (Ser), UCG (Ser), CAA, (Q) GAA (Glu) and CAG (Gln).
Beyond sequence specific constraints within the coding sequence of the polynucleotide of interest, additional design criteria for engineering a polynucleotide sequence with altered susceptibility to SHM can include the following factors:
The choice of promoter; a strong promoter will generally induce a higher rate of transcription resulting a higher overall rate of mutagenesis compared to a weaker promoter. Further, an inducible promoter, such as the tet-promoter enables expression, and hence SHM, to be inducibly controlled, to switch on, or off, transcription and mutagenesis of the polynucleotide of interest. Gossen and Bujard, Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci USA. 1992 Jun. 15; 89(12):5547-51; Gossen et al., Transcriptional activation by tetracyclines in mammalian cells. Science. 1995 Jun. 23; 268(5218):1766-9.
The location of the coding sequence relative to the transcriptional start point; generally for high level mutagenesis, the polynucleotide of interest should be located between about 50 nucleotides, and 2 kb of the transcriptional start site.
One convenient approach to optimizing a polynucleotide sequence to SHM, involves analyzing the corresponding amino acid sequence of interest via a computer algorithm that compares and scores (according to the parameters above) possible alternative polynucleotides sequences that can be used, via alternative codon usage to encode for the amino acid sequence of interest. By iteratively replacing codons, or groups of codons (tiles or SHM motifs) with progressively preferred sequences it is possible to computationally evolve a polynucleotide sequence with desired properties. Specifically, for example, a sequence that is susceptible to SHM, or that is resistant to SHM, and yet also exhibits reasonable translational efficiency, stability, minimizes restriction sites and avoids rare codons in the particular organism of interest.
Using this approach, a library of files can be generated that is based on the starting amino acid or polynucleotide sequence. In one non limiting example of the analysis and optimization strategy, the library can be created based on the analysis of groups of 9 nucleotides, corresponding to 3 codons (a “tile” or a “SHM motif”). Each tile can be scored for the attributes described above, to create an initial library data set of tiles, containing hundreds of thousands of 9-mer permutations, and their respective scores.
A representative sample of a section of the library file is shown in Table 5 which shows the potential diversity in nucleotide sequences arising from alternative codon usage for just the three amino acids, Serine (S), Arginine (R) and Leucine (L). A person of skill in the art readily appreciates that a complete set of files can be readily assembled for all possible amino acid combinations using known codon usage patterns. Sequence identifiers are next to each sequence in parenthesis.

TABLE 5

Representative polynucleotide diversity encoding a three
amino acid sequence (Ser Arg Leu)

3-mer
AA	Potential nucleotides	Hotspots	Coldspots	CpG	MaxNt	Log(πp(AA))

SRL	AGTCGACTT (43)	0	2	1	1	−5

SRL	AGTCGACTG (44)	0	2	1	1	−3

SRL	AGTCGATTA (45)	0	1	1	2	−5

SRL	AGTCGACTA (46)	0	2	1	1	−5

SRL	AGTCGACTC (47)	0	3	1	1	−4

SRL	AGTCGATTG (48)	0	1	1	2	−5

SRL	AGTAGGCTT (49)	2	0	0	2	−4

SRL	AGTAGGCTG (50)	2	0	0	2	−2

SRL	AGTAGGTTA (51)	2	0	0	2	−4

SRL	AGTAGGCTA (52)	2	0	0	2	−4

SRL	AGTAGGCTC (53)	2	1	0	2	−3

SRL	AGTAGGTTG (54)	2	0	0	2	−4

SRL	AGTCGTCTT (55)	0	2	1	1	−5

SRL	AGTCGTCTG (56)	0	2	1	1	−3

SRL	AGTCGTTTA (57)	0	1	1	3	−5

SRL	AGTCGTCTA (58)	0	2	1	1	−5

SRL	AGTCGTCTC (59)	0	3	1	1	−4

SRL	AGTCGTTTG (60)	0	1	1	3	−5

SRL	AGTAGACTT (61)	1	1	0	1	−4

SRL	AGTAGACTG (62)	1	1	0	1	−2

SRL	AGTAGATTA (63)	1	0	0	2	−4

SRL	AGTAGACTA (64)	1	1	0	1	−4

SRL	AGTAGACTC (65)	1	2	0	1	−3

SRL	AGTAGATTG (66)	1	0	0	2	−4

SRL	AGTCGGCTT (67)	1	1	1	2	−4

SRL	AGTCGGCTG (68)	1	1	1	2	−2

SRL	AGTCGGTTA (69)	1	1	1	2	−4

SRL	AGTCGGCTA (70)	1	1	1	2	−4

SRL	AGTCGGCTC (71)	1	2	1	2	−3

SRL	AGTCGGTTG (72)	1	1	1	2	−4

SRL	AGTCGCCTT (73)	0	2	1	2	−4

SRL	AGTCGCCTG (74)	0	2	1	2	−2

SRL	AGTCGCTTA (75)	0	1	1	2	−4

SRL	AGTCGCCTA (76)	0	2	1	2	−4

SRL	AGTCGCCTC (77)	0	3	1	2	−3

SRL	AGTCGCTTG (78)	0	1	1	2	−4

SRL	TCACGACTT (79)	0	1	1	1	−5

SRL	TCACGACTG (80)	0	1	1	1	−3

SRL	TCACGATTA (81)	0	0	1	2	−5

SRL	TCACGACTA (82)	0	1	1	1	−5

SRL	TCACGACTC (83)	0	2	1	1	−4

SRL	TCACGATTG (84)	0	0	1	2	−5

SRL	TCAAGGCTT (85)	1	0	0	2	−4

SRL	TCAAGGCTG (86)	1	0	0	2	−2

SRL	TCAAGGTTA (87)	1	0	0	2	−4

SRL	TCAAGGCTA (88)	1	0	0	2	−4

SRL	TCAAGGCTC (89)	1	1	0	2	−3

SRL	TCAAGGTTG (90)	1	0	0	2	−4

SRL	TCACGTCTT (91)	0	1	1	1	−5

SRL	TCACGTCTG (92)	0	1	1	1	−3

SRL	TCACGTTTA (93)	0	0	1	3	−5

SRL	TCACGTCTA (94)	0	1	1	1	−5

SRL	TCACGTCTC (95)	0	2	1	1	−4

SRL	TCACGTTTG (96)	0	0	1	3	−5

SRL	TCAAGACTT (97)	0	1	0	2	−4

SRL	TCAAGACTG (98)	0	1	0	2	−2

SRL	TCAAGATTA (99)	0	0	0	2	−4

SRL	TCAAGACTA (100)	0	1	0	2	−4

SRL	TCAAGACTC (101)	0	2	0	2	−3

SRL	TCAAGATTG (102)	0	0	0	2	−4

SRL	TCACGGCTT (103)	1	0	1	2	−4

SRL	TCACGGCTG (104)	1	0	1	2	−2

SRL	TCACGGTTA (105)	1	0	1	2	−4

SRL	TCACGGCTA (106)	1	0	1	2	−4

SRL	TCACGGCTC (107)	1	1	1	2	−3

SRL	TCACGGTTG (108)	1	0	1	2	−4

SRL	TCACGCCTT (109)	0	1	1	2	−4

SRL	TCACGCCTG (110)	0	1	1	2	−2

SRL	TCACGCTTA (111)	0	0	1	2	−4

SRL	TCACGCCTA (112)	0	1	1	2	−4

SRL	TCACGCCTC (113)	0	2	1	2	−3

SRL	TCACGCTTG (114)	0	0	1	2	−4

SRL	AGCCGACTT (115)	1	2	1	2	−5

SRL	AGCCGACTG (116)	1	2	1	2	−3

SRL	AGCCGATTA (117)	1	1	1	2	−5

SRL	AGCCGACTA (118)	1	2	1	2	−5

SRL	AGCCGACTC (119)	1	3	1	2	−4

SRL	AGCCGATTG (120)	1	1	1	2	−5

SRL	AGCAGGCTT (121)	2	0	0	2	−4

SRL	AGCAGGCTG (122)	2	0	0	2	−2

SRL	AGCAGGTTA (123)	2	0	0	2	−4

SRL	AGCAGGCTA (124)	2	0	0	2	−4

SRL	AGCAGGCTC (125)	2	1	0	2	−3

SRL	AGCAGGTTG (126)	2	0	0	2	−4

SRL	AGCCGTCTT (127)	1	2	1	2	−5

SRL	AGCCGTCTG (128)	1	2	1	2	−3

SRL	AGCCGTTTA (129)	1	1	1	3	−5

SRL	AGCCGTCTA (130)	1	2	1	2	−5

SRL	AGCCGTCTC (131)	1	3	1	2	−4

SRL	AGCCGTTTG (132)	1	1	1	3	−5

SRL	AGCAGACTT (133)	1	1	0	1	−4

SRL	AGCAGACTG (134)	1	1	0	1	−2

SRL	AGCAGATTA (135)	1	0	0	2	−4

SRL	AGCAGACTA (136)	1	1	0	1	−4

SRL	AGCAGACTC (137)	1	2	0	1	−3

SRL	AGCAGATTG (138)	1	0	0	2	−4

SRL	AGCCGGCTT (139)	2	1	1	2	−4

SRL	AGCCGGCTG (140)	2	1	1	2	−2

SRL	AGCCGGTTA (141)	2	1	1	2	−4

SRL	AGCCGGCTA (142)	2	1	1	2	−4

SRL	AGCCGGCTC (143)	2	2	1	2	−3

SRL	AGCCGGTTG (144)	2	1	1	2	−4

SRL	AGCCGCCTT (145)	1	2	1	2	−4

SRL	AGCCGCCTG (146)	1	2	1	2	−2

SRL	AGCCGCTTA (147)	1	1	1	2	−4

SRL	AGCCGCCTA (148)	1	2	1	2	−4

SRL	AGCCGCCTC (149)	1	3	1	2	−3

SRL	AGCCGCTTG (150)	1	1	1	2	−4

SRL	TCGCGACTT (151)	0	1	2	1	−6

SRL	TCGCGACTG (152)	0	1	2	1	−4

SRL	TCGCGATTA (153)	0	0	2	2	−6

SRL	TCGCGACTA (154)	0	1	2	1	−6

SRL	TCGCGACTC (155)	0	2	2	1	−5

SRL	TCGCGATTG (156)	0	0	2	2	−6

SRL	TCGAGGCTT (157)	1	1	1	2	−5

SRL	TCGAGGCTG (158)	1	1	1	2	−3

SRL	TCGAGGTTA (159)	1	1	1	2	−5

SRL	TCGAGGCTA (160)	1	1	1	2	−5

SRL	TCGAGGCTC (161)	1	2	1	2	−4

SRL	TCGAGGTTG (162)	1	1	1	2	−5

SRL	TCGCGTCTT (163)	0	1	2	1	−6

SRL	TCGCGTCTG (164)	0	1	2	1	−4

SRL	TCGCGTTTA (165)	0	0	2	3	−6

SRL	TCGCGTCTA (166)	0	1	2	1	−6

SRL	TCGCGTCTC (167)	0	2	2	1	−5

SRL	TCGCGTTTG (168)	0	0	2	3	−6

SRL	TCGAGACTT (169)	0	2	1	1	−5

SRL	TCGAGACTG (170)	0	2	1	1	−3

SRL	TCGAGATTA (171)	0	1	1	2	−5

SRL	TCGAGACTA (172)	0	2	1	1	−5

SRL	TCGAGACTC (173)	0	3	1	1	−4

SRL	TCGAGATTG (174)	0	1	1	2	−5

SRL	TCGCGGCTT (175)	1	0	2	2	−5

SRL	TCGCGGCTG (176)	1	0	2	2	−3

SRL	TCGCGGTTA (177)	1	0	2	2	−5

SRL	TCGCGGCTA (178)	1	0	2	2	−5

SRL	TCGCGGCTC (179)	1	1	2	2	−4

SRL	TCGCGGTTG (180)	1	0	2	2	−5

SRL	TCGCGCCTT (181)	0	1	2	2	−5

SRL	TCGCGCCTG (182)	0	1	2	2	−3

SRL	TCGCGCTTA (183)	0	0	2	2	−5

SRL	TCGCGCCTA (184)	0	1	2	2	−5

SRL	TCGCGCCTC (185)	0	2	2	2	−4

SRL	TCGCGCTTG (186)	0	0	2	2	−5

SRL	TCCCGACTT (187)	0	2	1	3	−5

SRL	TCCCGACTG (188)	0	2	1	3	−3

SRL	TCCCGATTA (189)	0	1	1	3	−5

SRL	TCCCGACTA (190)	0	2	1	3	−5

SRL	TCCCGACTC (191)	0	3	1	3	−4

SRL	TCCCGATTG (192)	0	1	1	3	−5

SRL	TCCAGGCTT (193)	1	0	0	2	−4

SRL	TCCAGGCTG (194)	1	0	0	2	−2

SRL	TCCAGGTTA (195)	1	0	0	2	−4

SRL	TCCAGGCTA (196)	1	0	0	2	−4

SRL	TCCAGGCTC (197)	1	1	0	2	−3

SRL	TCCAGGTTG (198)	1	0	0	2	−4

SRL	TCCCGTCTT (199)	0	2	1	3	−5

SRL	TCCCGTCTG (200)	0	2	1	3	−3

SRL	TCCCGTTTA (201)	0	1	1	3	−5

SRL	TCCCGTCTA (202)	0	2	1	3	−5

SRL	TCCCGTCTC (203)	0	3	1	3	−4

SRL	TCCCGTTTG (204)	0	1	1	3	−5

SRL	TCCAGACTT (205)	0	1	0	2	−4

SRL	TCCAGACTG (206)	0	1	0	2	−2

SRL	TCCAGATTA (207)	0	0	0	2	−4

SRL	TCCAGACTA (208)	0	1	0	2	−4

SRL	TCCAGACTC (209)	0	2	0	2	−3

SRL	TCCAGATTG (210)	0	0	0	2	−4

SRL	TCCCGGCTT (211)	1	1	1	3	−4

SRL	TCCCGGCTG (212)	1	1	1	3	−2

SRL	TCCCGGTTA (213)	1	1	1	3	−4

SRL	TCCCGGCTA (214)	1	1	1	3	−4

SRL	TCCCGGCTC (215)	1	2	1	3	−3

SRL	TCCCGGTTG (216)	1	1	1	3	−4

SRL	TCCCGCCTT (217)	0	2	1	3	−4

SRL	TCCCGCCTG (218)	0	2	1	3	−2

SRL	TCCCGCTTA (219)	0	1	1	3	−4

SRL	TCCCGCCTA (220)	0	2	1	3	−4

SRL	TCCCGCCTC (221)	0	3	1	3	−3

SRL	TCCCGCTTG (222)	0	1	1	3	−4

SRL	TCTCGACTT (223)	0	2	1	1	−5

SRL	TCTCGACTG (224)	0	2	1	1	−3

SRL	TCTCGATTA (225)	0	1	1	2	−5

SRL	TCTCGACTA (226)	0	2	1	1	−5

SRL	TCTCGACTC (227)	0	3	1	1	−4

SRL	TCTCGATTG (228)	0	1	1	2	−5

SRL	TCTAGGCTT (229)	1	0	0	2	−4

SRL	TCTAGGCTG (230)	1	0	0	2	−2

SRL	TCTAGGTTA (231)	1	0	0	2	−4

SRL	TCTAGGCTA (232)	1	0	0	2	−4

SRL	TCTAGGCTC (233)	1	1	0	2	−3

SRL	TCTAGGTTG (234)	1	0	0	2	−4

SRL	TCTCGTCTT (235)	0	2	1	1	−5

SRL	TCTCGTCTG (236)	0	2	1	1	−3

SRL	TCTCGTTTA (237)	0	1	1	3	−5

SRL	TCTCGTCTA (238)	0	2	1	1	−5

SRL	TCTCGTCTC (239)	0	3	1	1	−4

SRL	TCTCGTTTG (240)	0	1	1	3	−5

SRL	TCTAGACTT (241)	0	1	0	1	−4

SRL	TCTAGACTG (242)	0	1	0	1	−2

SRL	TCTAGATTA (243)	0	0	0	2	−4

SRL	TCTAGACTA (244)	0	1	0	1	−4

SRL	TCTAGACTC (245)	0	2	0	1	−3

SRL	TCTAGATTG (246)	0	0	0	2	−4

SRL	TCTCGGCTT (247)	1	1	1	2	−4

SRL	TCTCGGCTG (248)	1	1	1	2	−2

SRL	TCTCGGTTA (249)	1	1	1	2	−4

SRL	TCTCGGCTA (250)	1	1	1	2	−4

SRL	TCTCGGCTC (251)	1	2	1	2	−3

SRL	TCTCGGTTG (252)	1	1	1	2	−4

SRL	TCTCGCCTT (253)	0	2	1	2	−4

SRL	TCTCGCCTG (254)	0	2	1	2	−2

SRL	TCTCGCTTA (255)	0	1	1	2	−4

SRL	TCTCGCCTA (256)	0	2	1	2	−4

SRL	TCTCGCCTC (257)	0	3	1	2	−3

SRL	TCTCGCTTG (258)	0	1	1	2	−4

Each polynucleotide sequence is ranked based on the following attributes; number of SHM hot and cold motifs, number of CpG motifs, MaxNt (maximum number of nucleotides in a single stretch) and codon usage frequency of the host cell to be used. The term “Log(πp(AA))” contained in the final column of Table 5 was calculated as the log of the product of the individual probabilities of observing each of the amino acids in the trimer, given by the formula:
Log(πp(AA)=ln(p(codon_i−1|amino acid_i−1)*p(codon_i|amino acid_i)*p(codon_i+1|amino acid₁₊₁).
Individual probabilities for each amino acid were based on published codon usage patterns in the organism of interest, in this case, for mammalian cells. (See generally Nakamura et al., Nucleic Acid Res. (2000) 28 (1): 292 Codon usage tabulated from international DNA sequence databases: status for the year 2000).
As can be readily seen from above, codon usage diversity alone enables polynucleotide sequences to be created that vary widely in their susceptibility to somatic hypermutation, as measured by the number (density) of hot or cold spot motifs present within the sequence.
This analysis readily identifies potential combinations of codons or motifs that are optimized for SHM and minimize CpGs and use optimal codons for efficient translation. For example, the sequences listed below in Table 6 represent top ranking hot sequences because they comprise the maximum number of hot spots and no cold spots. Sequence identifiers are next to each sequence in parenthesis.

TABLE 6

Top Hot Spot Sequences

SRL	AGTAGGCTT (259)	2	0	0	2	−4

SRL	AGTAGGCTG (260)	2	0	0	2	−2

SRL	AGTAGGTTA (261)	2	0	0	2	−4

SRL	AGTAGGCTA (262)	2	0	0	2	−4

SRL	AGCAGGCTT (263)	2	0	0	2	−4

SRL	AGCAGGCTG (264)	2	0	0	2	−2

SRL	AGCAGGTTA (265)	2	0	0	2	−4

SRL	AGCAGGCTA (266)	2	0	0	2	−4

SRL	AGCAGGTTG (267)	2	0	0	2	−4

SRL	AGTAGGTTG (268)	2	0	0	2	−4

Of these, the sequences AGTAGGCTG and AGCAGGCTG are preferred because they encompass codons with a higher frequency of use in mammalian cells.
Having defined and scored all possible 9-mer nucleotide tiles, it is possible to scan through a starting amino acid or nucleotide template, identifying positions in the gene/protein that can be improved by substitution from the tile library. This process can be conveniently completed using a computer algorithm, such as the perl program SHMredesign.pl; the code of which is shown below:
In addition to the file of potential 3 amino acid tiles shown above, the program also calls upon a file of hot spots and cold spot motifs as outlined below in Table 7, and a listing of the genetic code to translate amino acid sequences to polynucleotide sequences:

TABLE 7

Canonical Hot and Cold Spot Motifs

	Coldspots	Hotspots

CCC	TACC	GGTA

CTC	TACA	TGTA

GCC	TACT	AGTA

GTC	TGCC	GGCA

GGG	TGCA	TGCA

GAG	TGCT	AGCA

GGC	AACC	GGTT

GAC	AACA	TGTT

	AACT	AGTT

	AGCC	GGCT

	AGCA	TGCT

	AGCT	AGCT

One can recognize that there are many potential approaches, and computational methods which can be used to find the best codon usage to maximize hot spot or cold spot density, and that the invention is not intended to be limited to any one specific method of determining the optimum sequence.
When a starting amino acid template is given (for instance when the underlying DNA sequence may not be known), the algorithm begins by first generating a DNA nucleotide sequence that is consistent with both the given amino acid sequence and known codon usage in that organism. The starting nucleotide template contains an additional line that instructs the perl program SHMredesign.pl as to whether HOT or COLD sites should be incorporated at a given position, making it possible to silence or minimize SHM in portions of evolving proteins, while simultaneously directing SHM to areas for targeting, for instance, the CDRs of an antibody molecule. A given 9-mer SHM motif in the polynucleotide can be compared with all other possible nonameric oligonucleotides that would encode the same three amino acids at that position.
If a sequence, or portion thereof, is susceptible to SHM (made “hot”), an exhaustive search of all nucleotide sequences consistent with the amino acid sequence is made, and the nucleotide sequence of the evolving construct is replaced by a new nucleotide sequence if the following conditions are met: (1) the new 9-mer SHM motif contains more hot spot motifs that the existing sequence, (2) the new 9-mer contains a number of cold spotmotifs equal to or less than the evolving sequence, (3) the new 9-mer contains a number of CpG sequence motifs equal to or less than the evolving sequence, (4) the evolving sequence has a codon usage score that equals or improves known aggregate codon usage at the position, and (5) the sequence does not contain a stretch of any one nucleotide greater than 4 residues.
If a sequence, or portion thereof, is being made resistant to SHM (being made “cold”), an exhaustive search of all nucleotide sequences consistent with the amino acid sequence is made, and the nucleotide sequence of the evolving construct is replaced by a new nucleotide sequence if the following conditions are met: (1) the new 9-mer SHM motif contains more cold spot motifs that the existing sequence, (2) the new 9-mer contains a number of hot spot motifs equal to or less than the evolving sequence, (3) the new 9-mer contains a number of CpG sequence motifs equal to or less than the evolving sequence, (4) the evolving sequence has a codon usage score that equals or improves known aggregate codon usage at the position, and (5) the new 9-mer nucleotide sequence does not contain a stretch of any one nucleotide greater than 4 residues.
If a sequence is being optimized for other factors other than SHM (being made “neutral”), an exhaustive search of all nucleotide sequences consistent with the amino acid sequence is made, and the nucleotide sequence of the evolving construct is replaced by the new nucleotide sequence if the following conditions are met: (1) the new 9-mer contains a number of CpG sequence motifs equal to or less than the evolving sequence, (2) the evolving sequence has a codon usage score that equals or improves known aggregate codon usage at the position, and (3) the new 9-mer nucleotide sequence does not contain a stretch of any one nucleotide greater than 4 residues.
Starting from any given polynucleotide sequence, this approach can be used to generate polynucleotide sequences that rapidly converge to a small number of possible sequences that are optimized for the properties described herein (Example 1).
Following computational analysis, a final optimized polynucleotide can be synthesized using standard methodology and sequenced to confirm correct synthesis. Once the sequence of the polynucleotide has been confirmed, the polynucleotide can be inserted into a vector. The vector can be introduced into a host cell as described herein and tested for expression, activity, or increased and/or decreased susceptibility to SHM.

V. Construction of Synthetic Libraries for SHM Mediated Diversification

Synthetic polynucleotide libraries can be used for the directed evolution and selection of proteins with novel phenotypes by exploiting the diversity generating and targeting properties of SHM.
In the case of antibodies, this means targeted diversification of complementarity determining regions (CDRs) that can bind new or altered epitopes. Simplified CDR libraries containing four and even 2 amino acid alphabets (serine and tyrosine) have also been described and were found to be capable of binding antigens with high affinity and selectivity. See, e.g., Fellouse F A, Li B, Compaan D M, Peden A A, Hymowitz S G, Sidhu S S Molecular recognition by a binary code. J Mol Biol. (2005) 348:1153-62; and Fellouse F A, Wiesmann C, Sidhu S S Synthetic antibodies from a four-amino-acid code: a dominant role for tyrosine in antigen recognition. Proc Natl Acad Sci USA. (2004) 101:12467-72.
Synthetic polynucleotide libraries can also be used the case of non-antibody polypeptides such as enzymes and other protein classes, this refers to targeting diversification to regions of the enzyme or protein of interest which regulate the biological activity of said enzyme or protein, such as binding specificity, enzymatic function, fluorescence, or other properties. Libraries are usually combined with one or more selection strategies as disclosed below, which provide for the identification and/or separation of the improved, or functional members of the library from the non-functional members of the library.
Static libraries are, in some embodiments, limited in their size and scope. Phage display libraries, for example can display as many as 10¹²members, and ribosomal libraries have been constructed that potentially contain ˜10¹⁶members. Libraries presented on the surface of bacterial and mammalian cells are not usually this complex, with fewer than about 10⁹members. In addition, robust library construction and selection usually requires that libraries contain several fold redundancy, which further limits this theoretical complexity, and makes screening the entire library slow, expensive, and in some cases in-practical.
Despite these levels of complexity, such static libraries can explore only a small fraction of possible sequence space. In one non-limiting example, a heavy chain IgG sequence can contain more than 30 amino acids within the CDR1, CDR2, and CDR3 complementarity regions, giving this single chain more than 20³⁰possible permutations, dwarfing even the largest of potential libraries. Because of this limitation, researchers have explored methodologies for evolving protein sequences and libraries. SHM, as addressed in the present application, uses AID and error-prone polymerases as the mechanism for evolving antibody sequences undergoing affinity maturation. A system that would facilitate SHM-mediated mutagenesis and selection at each position of interest within a polynucleotide library of a given gene would permit the selective exploration of functional sequence space. Such a search strategy enables a much larger sequence diversity to be explored, making this method very attractive for the rapid development of new functionalities and therapeutics. For instance, a library composed of only a small number of hot spot codons at each coding position of a stretch of 10 amino acids (2¹⁰permuations=1.6*10⁴), where each position is capable of evolving under SHM to a diverse panel of resulting amino acids, represents a vast simplication of the complexity/diversity needed to encode an equivalent static library of all 20 amino acids at each of the ten library positions (20¹⁰permutations=1.6*10¹⁴).
In one aspect, the present invention includes a synthetic dynamic library that is capable of rapid evolution through SHM-mediated mutagenesis. Such a synthetic library has the following properties: i) The library is easy to synthesize and is based around a limited number of discrete functional sequences; ii) The library contains synthetic polynucleotide sequences that comprises one or more synthetic variable regions that are targeted for selective mutagenesis and includes a high density of SHM hot spots; iii) The library contains synthetic polynucleotide sequences that comprise one or more synthetic framework regions that are resistant to SHM mediated mutagenesis and include a low density of SHM hot spots; iv) The library does not contain, or contains a minimum number of, certain codons (“non preferred codons”) that can be mutated to stop codons in one step through SHM, including, UGG (Trp), UGC (Cys), UCA (Ser), UCG (Ser), CAA (Gln), GAA (Glu) and CAG (Gln); and v). From the starting set of codons, AID-mediated mutagenesis produces a large potential diversity at each position for further evolution and selection of function (“preferred SHM hot spot codons” or “preferred SHM hot spot motifs”).
A method for SHM optimization of gene sequences as described herein uses a previously scored library of 9-mer tiles (SHM motifs) that can be substituted to modify a sequence in order to generate a ‘hotter’ or ‘colder’ sequence as described above, while still maintaining the amino acid sequence and other desirable properties. It should be noted that the tiles, while chosen to be 9 nucleotides in this application, can be of any length, as long as they permit the accurate assessment of sequence based properties. Likewise, the hot and cold SHM motifs can be used to score the 9-mer tile libraries for substitution, it is possible to use any other sized or defined SHM hot or cold spot motifs in combination with this approach; for example based on hot and cold scores derived from statistical analyses, to make improved constructs. Tiles can be scored based on their use of in-frame SHM hot spot motifs as a way of seeding not just mutagenesis but the resulting amino acid diversity (or lack of same) at various positions throughout the construct. Tiles can be scored based on their use of in-frame SHM cold spot motifs as a way of stabilizing a polynucleotide against AID-mediated mutagenesis at various positions throughout the construct.
One can recognize that there are many potential approaches, and computational methods which could be used to find the best codon usage to maximize hot spot or cold spot motif density, and that the invention is not intended to be limited to any one specific method of determining the optimum sequence.

VI. Library Design

A synthetic library around a specific protein of interest can be designed in light of any known pre-existing information regarding structure activity relationships, homology between different species, and x-ray or NMR structural information of the protein or protein family in question, if available.
In one aspect, initial library design can involve the following steps:
1. The amino acid sequence of the protein of interest is identified, and the corresponding polynucleotide sequence determined or reverse transcribed.
2. Any relevant structural information on the protein of interest, and related proteins, or on homologous proteins of interest is obtained.
3. A sequence comparison is preformed on the protein of interest compared to all other proteins from closely related species, and known isoforms, and in certain embodiments, a sequence alignment can be created to identify conserved, and variable amino acid sequences between species.
This information can be used to establish whether a specific amino acid or protein domain is likely to be important in a functional, or structural, attribute of the protein of interest, and whether it is conserved or variant across functional isoforms of the protein across protein families.
Based on this information, it is possible to establish particular regions of interest that appear to be directly involved in functional or structural attributes of the protein of interest. For example, these amino acids can lie within, or within about 5 Å of a specific functional or structural attribute of interest. Specific examples include, but are not limited to, amino acids within CDRs of antibodies, binding pockets of receptors, catalytic clefts of enzymes, protein-protein interaction domains, of co-factors, allosteric binding sites etc.
Based on the structural and sequence analysis as set forth above, one or more polynucleotides can be designed to increase or decrease SHM-mediated mutagenesis using the parameters described above. Furthermore, the design can incorporate one or more of the following concepts:
i) Highly conserved amino acids, or amino acids known, or believed to directly contribute key binding energy can be initially conserved, and the codon usage within their immediate vicinity changed to either create a cold spot motif, or altered to promote mostly conservative amino acid changes during SHM as described herein.
ii) Amino acid domains that appear to be involved in maintaining the core structural framework of the protein can be initially conserved, and their codon usage changed to promote mostly conservative amino acid changes during SHM Amino acid residues in particularly important frame work regions can be altered to use a higher percentage of cold spots, and utilize codons or motifs that are resistant to SHM, or result in silent mutations during SHM.
iii) Amino acids in regions of interest can be varied to incorporate synthetic variable regions enabling high efficiency SHM, as described below.
iv) Amino acids that are not identified as playing clearly identified roles with respect to a function or structure of a polypeptide can be modified to enable effective SHM, i.e., the frequency of SHM hot spots can be maximized and the frequency of SHM cold spots can be minimized.

VII. The Design of Synthetic Variable Regions

The rank ordering of susceptibility to mutagenicity of all SHM hot spots for AID and error prone polymerases was presented in the Section III. We further identified a reading frame context that is critical for generation of silent vs. non-silent mutations. Herein we describe a synthetic library approach that includes the use of a high-density of preferred SHM hot spot codons or motifs that lead to generation of diverse amino acids at each library position which is desired to be mutated. Such high density hot spot motifs are particularly important at the boundary of synthetic variable regions to ensure efficient mutagenesis.
A. WAC Based Motifs
Polynucleotide sequences comprising only the sequence WAC (WAC, where W=A or T is encoded in equal proportions, and where the reading frame of reference places C at the wobble or 3^rdposition of each codon) provides for a high density of hot spots.
This simple pattern would produce only 4 potential 6-mer nucleotide patterns containing only two codons (AAC and TAC) encoding the 2 amino acids, Asparagine (Asn) and Tyrosine (Tyr). Sequence identifiers are next to each sequence in parenthesis.

	TABLE 8

	Codons	Amino acids

	AACTAC (269)	Asn Tyr

	AACAAC (270)	Asn Asn

	TACTAC (271)	Tyr Tyr

	TACAAC (272)	Tyr Asn

All of the motifs encoded by the WAC library, given in any of the three possible reading frames, produce a concentration of hot spots. FIG. 3 compares these motifs with all other possible 4096 6-mer nucleotide combinations for their ability to recruit SHM-mediated machinery. Longer assemblies result in the same high density of SHM “hot spots” with no “cold spots.” It is also worth noting that this assembly of degenerate codons (WACW) results in a subset of possible 4-mer hot spots described by Rogozin et al. (WRCH), where R=A or G, H=A or C or T, and W=T or A.
As seen in FIG. 4, the preferred SHM hot spot codons AAC and TAC, which are the basis for this synthetic library, can result in a set of primary and secondary mutation events that create considerable amino acid diversity, as judged by equivalent SHM mutation events observed in Ig heavy chains antibodies. From these two codons, basic amino acids (histidine, lysine, arginine), an acidic amino acid (aspartate), hydrophilic amino acids (serine, threonine, asparagine, tyrosine), hydrophobic amino acids (alanine, and phenylalanine), and glycine are generated as a result of SHM events.
B. WRC Based Motifs
A second potential synthetic high density SHM motif, termed here the WRC motif, is one that contains two possible codons: AGC and TAC encoding the 2 amino acids, Serine (Ser) and Tyrosine (Tyr). In this embodiment, the four possible 6-mer nucleotides include:
TABLE 9

Codons Amino acids

AGCTAC (273) Ser Tyr

AGCAGC (274) Ser Ser

TACAGC (275) Tyr Ser

TACTAC (271) Tyr Tyr

Sequence identifiers are next to each sequence in parentheses.
The distribution of all 4096 6-mer nucleotide z-scores describing the hotness or coldness of the motif to SHM-mediated mutation is illustrated in FIG. 5. The z-scores for all permutations of 6-mers in the WRC synthetic library are superimposed on this distribution, with the dashed line denoting the top 5% of all possible motifs.
The series of mutation events that lead to the creation of amino acid diversity, starting from “preferred SHM hot spot codons” AGC and TAC, as observed in affinity matured IGV heavy chain sequences is illustrated in FIG. 6. 4200 primary and secondary mutation events, starting from codons encoding asparagine and tyrosine, lead to a set of functionally diverse amino acids.
Again this motif results in an unusually high density of optimal SHM hot spots and hot codons, as visualized in FIG. 5, when compared with all other 6-mer nucleotide motifs. Like the WAC synthetic motif, the WRC synthetic motif presents preferred SHM hot spot codons that, when combined with the activity of SHM, AID and one or more error-prone polymerases, generates a broad spectrum of potential amino acid diversity at each position (FIG. 6).
Thus in one aspect, such synthetic variable regions can be targeted to specific regions of interest within a polynucleotide sequence that encode specific domains, or sub domains of interest and for which a high degree of diversity is desired.
In another aspect WAC or WRC motifs can be inserted systematically throughout the open reading frame of the protein of interest. For example, for a 100 amino acid residue protein, 300 discrete polynucleotides could be generated in which a WAC or WRC motif was separately introduced once into every possible position within the protein. Each of these 100 polynucleotides could then be screened, either separately, or after being pooled into a library, to identify optimal amino acid substitutions at each position. The improved mutations at each position could then be re-combined to create a next generation construct comprising all of best individual amino acids identified at each position.
C. Region Mutagenesis
To provide for effective mutagenesis within larger regions, codons or motifs can be modified as discussed previously to increase the density of hot spots throughout one or more regions of interest. This approach has the advantage of needing no preconceived idea of where SHM should be targeted, or what specific amino acids are essential for activity.
For regions in which efficient SHM is desired, a synthetic variable region can be created by both changing codon usage and by making conservative amino acid substitutions so as to insert codons or motifs that have an improved hot spot density. Suitable amino acid substitutions can be selected from those listed below in Table 10, while observing the same overall criteria for stable gene creation, and domain structure.

TABLE 10

Preferred SHM Codons

Codons	Amino Acid	Group/Sub group	Use in place of:

AGC/AGU	Ser	Aliphatic/Slightly non polar	Thr/Cys

GGU	Gly	Aliphatic/Small residue	Ala

GCU/GCA	Ala	Aliphatic/Small residue	Gly

CUA/UUG/CUU	Leu	Aliphatic/Large	Val/Met

Charged

AAA/AAG	Lys	Charged/Positive	Arg

CAU	His	Charged/Positive	Arg/Phe

GAU	Asp	Charged/Negative	Glu

GAG	Glu	Charged/Negative	Asp

Charged/Polar

CAG	Gln	Charged/Polar	Asn

AAU/AAC	Asn	Charged/Polar	Gln

Aromatic/Phenyl

UAU/UAC	Tyr	Aromatic/Phenyl	Trp

UUU/UUA/UUC	Phe	Aromatic/Phenyl	Trp/Phe

In some embodiments, the amino acids Trp, Pro and Gly are conserved where their location suggests a functional or structural role. Other than these amino acids, if an amino acid to be optimized is not listed, an amino acid from the same sub-group as listed below is selected.
Such synthetic variable regions can be interspersed with framework regions containing primarily SHM resistant sequences, which can be designed as described previously.
Depending on the amount of information available, a number of distinct library design strategies can be employed, ranging from a very aggressive targeted approach based on the use of WAC or WRC motifs, to a more conservative strategy of using fairly selective amino acid replacements, to a cautious strategy in which only codon usage is changed. An advantage of the present invention is that each approach results in the generation of only a limited number of distinct nucleotide sequences; thus all of these strategies can be subjected to SHM mediated diversity in parallel without significant additional burden.

VIII. Methods for Monitoring SHM Activity

Art-recognized methods for monitoring SHM in antibody and non-antibody proteins using various vectors and cell lines are known (Rückerl et al. (Mol. Immunol. 43 (2006); 1645-1652), Bacl et al. (J. Immunol., 2001, 166: 5051-5057), Cumbers et al. (Nat Biotechnol. 2002; 20(11): 1129-1134), Wang, et al. (Proc Natl Acad Sci USA. 2004; 101(19):7352-7356)). In addition, various methods for directly measuring cytidine deamination are known in the art; see, e.g., Genetic and In vitro assays of DNA deamination, Coker et al., Meth. Enzymol. 408: 156-170 (2006).
Such methods provide rapid means for evaluating the rate of on-going SHM. The methods include, for example, the use of reporter genes, or selectable marker genes that have been modified to include a stop codon within the coding frame which can be mutated in the presence of AID activity.
As AID acts on a population, it can produce mutations that restore or improve function (of a selectable marker, for instance), or mutations that reduce or eliminate function. The balance in these two rates generates early and rare mutation events that restore function, followed by secondary and ternary mutation events that destroy function in these proteins. The net effect of these competing rates on the observation of gain-of-function events in a population. Given three different assumptions regarding number of inactivating mutations needed to silence GFP, one would expect to observe three very different profiles of reversion events as a function of time, dependent on the rate of enzymatic activity of the AID.
Additionally, polynucleotides subjected to SHM activity can be sequenced to determine if the nucleotide sequence of has been modified and to what degree. Polynucleotides can be rescued from culture to determine SHM at various time points. Methods of isolating and sequencing genes are well known in the art, and include, the use of standard techniques such as, for example, Reverse Transcription—Polymerase Chain Reaction (RT-PCR). Briefly, the polynucleotide can be reverse transcribed and subjected to PCR using appropriate primers. Clones can be sequenced using automated DNA sequences from companies such as Applied Biosystems (ABI-377 or ABI 3730 DNA sequencers). Sequences can be analyzed for frequency of nucleotide modifications. The polynucleotide can be compared with the polynucleotide from the starting material and analyzed for sequence modifications.

IX. Somatic Hypermutation (SHM) Systems

A. Synthetic Polynucleotide Sequences
The development of a practical system for the use of SHM requires that mutations be directed to specific genes (polynucleotides) or regions of interest (made “hot”), and be directed away from structural or marker genes that are functionally required within the cell or episome, to maintain overall system functionality and/or stability (made “cold”). In certain embodiments, a synthetic gene is one that does naturally undergo SHM when expressed in a B cell (i.e., an antibody gene). In other embodiments, a synthetic gene is one that does not naturally undergo SHM when expressed in a B cell (i.e., a non-antibody gene).
The present invention is based on the development of a system to design and make or generate SHM susceptible and SHM resistant polynucleotide sequences within a cell or cell-free, environment. The present invention is further based on the development of a SHM system that is stable over a suitable time period to reproducibly maintain increased and/or decreased rates of SHM without affecting structural portions or polypeptides or structural proteins, transcriptional control regions and selectable markers. The system allows for stable maintenance of a mutagenesis system that provides for high level targeted SHM in a polynucleotide of interest, while sufficiently preventing non-specific mutagenesis of structural proteins, transcriptional control regions and selectable markers.
In part, the present system is based around the creation of a more stable version of cytidine deaminase that can provide for high level sustained SHM. High level over-expression of wild type AID in mammalian cells does not necessarily lead to a stable increase in SHM activity because the enzyme itself can either accumulate inactivating mutations through SHM of its own DNA sequence, or be silenced through post-translational modifications (Ronai et al., PNAS USA 2005; 102(33): 11829-34; Iglesias-Ussel et al., J. Immunol. Methods. 2006, 316: 59-66). The present SHM system, therefore, includes a synthetic AID gene (SEQ ID NO: 22) that is resistant to SHM (cold) and exhibits a reduced rate of self mutation.
Thus, in one aspect, the present invention includes a synthetic AID gene that has been altered at the polynucleotide level to have a reduced number of hot spots, and/or increased number of cold spots. In one embodiment of this synthetic gene, the gene also has a modified content of CpG methylation sites. In another aspect, the synthetic gene has been optimized for SHM codon usage specific to the organism in which the synthetic gene is to be expressed or mutated.
Additionally, there are a variety of other component nucleotide sequences, such as coding sequences and genetic elements that can make up the core system that one would, in some embodiments, prefer not to hypermutate to maintain overall system integrity. These component nucleotide sequences include without limitation, i) selectable markers such as neomycin, blasticidin, ampicillin, etc; ii) reporter genes (e.g. fluorescent proteins, epitope tags, reporter enzymes); iii) genetic regulatory signals, e.g. promoters, inducible systems, enhancer sequences, IRES sequences, transcription or translational terminators, kozak sequences, splice sites, origin of replication, repressors; iv) enzymes or accessory factors used for high level enhanced SHM, or it's regulation, or measurement, such as AID, pol eta, transcription factors, and MSH2; v) signal transduction components (kinases, receptors, transcription factors) and vi) domains or sub domains of proteins such as nuclear localization signals, transmembrane domains, catalytic domains, protein-protein interaction domains, and other protein family conserved motifs, domains and sub-domains.
Thus, in another aspect of the present invention, the SHM system described herein can include any synthetic gene that has been altered, at the polynucleotide level, either in whole, or part, to have a reduced number of hot spots, and/or an increased number of cold spots using the methods described herein. In one embodiment, the synthetic gene also has a modified content of CpG methylation sites.
Provided herein is an expression vector, comprising at least one synthetic gene. In one aspect, the expression vector is an integrating expression vector. When the expression vector is an integrating expression vector, the expression vector can further comprise one or more sequences to direction recombination. In another aspect, the expression vector is an episomal expression vector. In yet another aspect, the expression vector is a viral expression vector.
In another aspect, the synthetic gene has been optimized for SHM codon usage specific to the organism in which the synthetic gene is to be expressed and/or mutated.
Provided herein is a SHM resistant synthetic gene encoding a protein, or a portion thereof, wherein one or more first SHM motifs in an unmodified polynucleotide sequence encoding said protein or portion thereof has been replaced by one or more second SHM motifs having a lower probability of SHM, wherein said SHM resistant synthetic gene exhibits a lower rate of AID-mediated mutagenesis compared to said unmodified polynucleotide sequence.
The present invention also contemplates that a SHM resistant synthetic gene can be created in a step-wise or sequential fashion such that some modifications are made to the gene and then a subsequent round of modification is made to the gene. Such sequential or step-wise modifications are contemplated by the present invention and are one way of carrying out the process and one way of producing the genes claimed herein.
In one embodiment, the SHM resistant synthetic gene encodes a protein or portion thereof having about 95%, about 90% amino, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, or any percentage between about 50% and about 100% identity to the unmodified gene.
In one embodiment, the SHM resistant synthetic gene exhibits a lower rate of AID-mediated mutagenesis including, but not limited to, 1.05-fold, 1.1-fold, 1.2-fold, 1.5-fold, 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold or less, or any range therebetween.
In one embodiment, the SHM resistant synthetic gene exhibiting a rate of AID-mediated mutagenesis at a level which is less than about 99%, less than about 95%, less than about 90%, less than about 85%, less than about 80%, less than about 75%, less than about 70%, less than about 65%, less than about 60%, less than about 55%, or less than about 50%, of that exhibited by an unmodified gene.
In other embodiments, a high rate of SHM mediated mutagenesis targeted to a polynucleotide of interest is desirable to direct rapid directed evolution of the polynucleotide.
Provided herein is a SHM susceptible synthetic gene encoding a protein, or a portion thereof, wherein one or more first SHM motifs in an unmodified polynucleotide sequence encoding said protein or portion thereof has been replaced by one or more second SHM motifs having a higher probability of SHM, wherein said SHM susceptible synthetic gene exhibits a higher rate of AID-mediated mutagenesis compared to said unmodified polynucleotide sequence.
The present invention also contemplates that a SHM susceptible synthetic gene can be created in a step-wise or sequential fashion such that some modifications are made to the gene and then a subsequent round of modification is made to the gene. Such sequential or step-wise modifications are contemplated by the present invention and are one way of carrying out the process and one way of producing the genes claimed herein.
In one embodiment, the SHM susceptible synthetic gene encodes a protein or portion thereof having about 95%, about 90% amino, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, or any percentage between about 50% and about 100% identity to the unmodified gene.
In one embodiment, the SHM susceptible synthetic gene exhibits a higher rate of AID-mediated mutagenesis including, but not limited to, 1.05-fold, 1.1-fold, 1.25-fold, 1.5-fold, 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold or more, or any range therebetween.
In one embodiment, the SHM susceptible synthetic gene exhibits a rate of activation induced cytidine deaminase (AID)-mediated mutagenesis at a level which is at least about 101%, at least about 105%, at least about 110%, at least about 115%, at least about 120%, at least about 125%, at least about 130%, at least about 135%, at least about 1140%, at least about 145%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 450%, at least about 5000%, or higher of that exhibited by an unmodified gene.
Provided herein is a selectively targeted, SHM optimized synthetic gene encoding a protein, or a portion thereof, wherein one or more first SHM motifs in an unmodified polynucleotide sequence encoding said protein or portion thereof has been replaced by one or more second SHM motifs having a higher probability of SHM; and one or more third SHM motifs in said unmodified polynucleotide sequence encoding said protein or portion thereof has been replaced by one or more fourth SHM motifs having a lower probability of SHM; wherein said selectively targeted, SHM optimized synthetic gene exhibits targeted AID-mediated mutagenesis. In such an embodiment, the selectively targeted, SHM optimized synthetic gene has some portions that exhibit a higher rate of AID-mediated mutagenesis and some portions that exhibit a lower rate of AID-mediated mutagenesis.
The present invention also contemplates that a selectively targeted SHM optimized synthetic gene can be created in a step-wise or sequential fashion such that some modifications are made to the gene and then a subsequent round of modification is made to the gene. Such sequential or step-wise modifications are contemplated by the present invention and are one way of carrying out the process and one way of producing the genes claimed herein.
Further provided herein is a system that enables high level somatic hypermutation that can be targeted to specific polynucleotides (e.g., synthetic genes or areas within synthetic genes) of interest, while avoiding the non-specific mutagenesis of components involved in the maintenance of the mutagenesis and expression system. Polynucleotides which can be mutated in the present system include any polynucleotide sequence that can be expressed and modified through AID-mediated mutagenesis. Such polynucleotides include those that encode polypeptides including, for example, specific binding members, enzymes, receptors, neurotransmitters, hormones, cytokines, chemokines, structural proteins, co-factors, toxins, or any other polypeptide or protein of interest or portion thereof. In one aspect such polynucleotides encode immunoglobulin polypeptides (antibodies) such as variable heavy chains or light chains or portions thereof.
Provided herein is a SHM system, e.g. one or more vectors (e.g., expression vectors) for SHM comprising at least one component (e.g. polynucleotide, gene, nucleic acid sequence, coding sequence, genetic element, a portion thereof, etc.) that includes, but is not limited to, a polynucleotide that has been altered from wild type to either positively or negatively influence the rate of SHM experienced by that component, or portion thereof.
In one aspect of the SHM system, at least one component of the expression system comprises a polynucleotide that has been altered, in whole, or part, to negatively influence the rate of SHM experienced by that component. In one aspect of this system, the component is a polynucleotide encoding a protein such as, but not limited to, an AID, or an AID homolog, Pol eta, a selectable marker, a fluorescent protein, EBNA1, or a represser (or transactivator) protein.
In one aspect of the SHM system, at least one component of the expression system comprises a polynucleotide that has been altered, in whole, or part, from wild-type to positively influence the rate of SHM experienced by that component; or comprises a polynucleotide that has a high rate of SHM, such as, for example, a hypervariable region of an antibody gene, the DNA binding domain of a transcription factor, the active site of an enzyme, the binding domain of a receptor, or a sub domain or domain of interest.
In another aspect, the SHM system comprises; i) at least one polynucleotide that can be a polynucleotide that has been altered in whole or part, from wild type to positively influence the rate of SHM experienced by that polynucleotide, or a polynucleotide that has a naturally high frequency of hot spots, ii) and the expression system also includes a polynucleotide that has been altered in whole or part, from wild-type to negatively influence the rate of SHM.
In one embodiment, provided herein is a SHM susceptible gene encoding a protein or a portion thereof wherein one or more first SHM motifs in an unmodified polynucleotide sequence encoding said protein or portion thereof has been replaced by one or more second SHM motifs having a higher probability of SHM, said synthetic gene having a greater density of hot spot motifs than said unmodified polynucleotide sequence.
In another embodiment, provided herein is a SHM resistant synthetic gene encoding a protein or a portion thereof wherein one or more first SHM motifs in an unmodified polynucleotide sequence encoding said protein or portion thereof has been replaced by one or more second SHM motifs having a lower probability of SHM, said synthetic gene having a greater density of cold spots than said unmodified polynucleotide sequence.
In yet another embodiment, provided herein is a selectively targeted, SHM optimized synthetic gene encoding a protein or portion thereof, wherein one or more first SHM motifs in an unmodified polynucleotide sequence encoding said protein or portion thereof has been replaced by one or more second SHM motifs having a higher probability of SHM, said synthetic gene having a greater density of hot spot motifs than said unmodified polynucleotide sequence; and one or more third SHM motifs in said unmodified polynucleotide sequence encoding said protein or portion thereof has been replaced by one or more fourth SHM motifs having a lower probability of SHM, said synthetic gene having a greater density of cold spots than said unmodified polynucleotide sequence; wherein said selectively targeted, SHM optimized synthetic gene exhibits targeted AID-mediated mutagenesis.
In yet another non-limiting aspect, the said synthetic gene includes one or more amino acid mutations that introduce preferred SHM hot spot motifs.
Thus, one aspect the present invention includes a method of creating SHM resistant (“cold”) or SHM susceptible (“hot”) genes wherein the hot spot or cold spot density is altered from the unmodified density, while maintaining translational efficiency of the synthetic protein (i.e. the ability to be successfully expressed in a mammalian cell).
In one aspect, a synthetic SHM resistant gene of the present invention has an average hot spot density of 7, or less, hot spots per 100 nucleotides. In another aspect, said synthetic SHM resistant gene has an average cold spot density of greater than 16 cold spots per 100 nucleotides.
In a preferred aspect, a synthetic SHM resistant gene of the present invention has an average hot spot density of less than 6 hot spots per 100 nucleotides. In another aspect, said synthetic SHM resistant gene has an average cold spot density of greater than 18 cold spots per 100 nucleotides. In another aspect, said synthetic SHM resistant gene has an average cold spot density of greater than 20 cold spots per 100 nucleotides. In another aspect, said synthetic SHM resistant gene has an average cold spot density of greater than 22 cold spots per 100 nucleotides.
In one aspect, a synthetic SHM susceptible gene of the present invention has an average cold spot density of 13 or less cold spots per 100 nucleotides. In another aspect, said synthetic SHM susceptible gene has an average hot spot density of greater than 10 hot spots per 100 nucleotides.
In a preferred aspect, a synthetic SHM susceptible gene of the present invention has an average cold spot density of less than 11 cold spots per 100 nucleotides. In another aspect, said synthetic SHM susceptible gene has an average hot spot density of greater than 13 hot spots per 100 nucleotides. In another aspect, said synthetic SHM susceptible gene has an average hot spot density of greater than 14 hot spots per 100 nucleotides. In another aspect, said synthetic SHM susceptible gene has an average hot spot density of greater than 16 hot spots per 100 nucleotides
In another aspect, a synthetic SHM susceptible polynucleotide has an average hot spot density of greater than 20 hot spots per 100 nucleotides over at least 12 contiguous nucleotides. In another aspect, a synthetic SHM susceptible polynucleotide has an average hot spot density of greater than 20 hot spots per 100 nucleotides over at least 15 contiguous nucleotides. In another aspect, a synthetic SHM susceptible polynucleotide has an average hot spot density of greater than 20 hot spots per 100 nucleotides over at least 18 contiguous nucleotides. In another aspect, a synthetic SHM susceptible polynucleotide has an average hot spot density of greater than 20 hot spots per 100 nucleotides over at least 30 contiguous nucleotides.
In one non-limiting example, a synthetic gene does not include genes comprising a stop motif inserted into an open reading frame.
In each of the SHM systems described herein, the systems can further include one or more of the following additional elements: i) an inducible system to regulate the expression of AID, or an AID homolog, ii) one or more enhancers, iii) one or more E-boxes, iv) one or more auxiliary factors for SHM, or v) one or more factors for stable episomal expression, such as EBNA1, or EBP2.
In another aspect of the SHM systems, the system includes two polynucleotides in which both polynucleotides are located in proximity to a promoter. In one aspect of the system, the promoter can be a bi-directional promoter; such as a bi-directional CMV promoter.
In another aspect, the polynucleotide can encode antibody chains. For example, heavy or light chains can be inserted into a vector for expression. In other aspects the polynucleotide can encode any protein, i.e. a wild-type polypeptide, a non-wild-type polypeptide, a synthetic polypeptide, a recombinant polypeptide or any portion thereof such as, without limitation, antibody heavy chains or fragments thereof, antibody light chains or fragments thereof, enzymes, receptors, structural proteins, co-factors, synthetic peptides, intrabodies, or toxins.
Provided herein is a method for preparing a gene product having a desired property, comprising: a) preparing a synthetic gene encoding a gene product which exhibits increased somatic hypermutation; b) expressing said synthetic gene in a population of cells; wherein said population of cells express activation induced cytidine deaminase (AID), or can be induced to express AID via the addition of an inducing agent; and c) selecting a cell or cells within the population of cells which express a mutated gene product having the desired property. In one aspect, the method, optionally, further comprises activating or inducing the expressing AID in said population of cells. In another aspect, the method, optionally, further comprises establishing one or more clonal populations of cells from the cell or cells identified in (c). In yet another aspect of the method, at least one synthetic gene is located in an expression vector such as any one of the vectors described elsewhere herein. In one aspect of the method, the cell is a cell as described elsewhere herein.
Provided herein is a method for preparing a gene product having a desired property, comprising: a) expressing said gene product in a population of cells; wherein said population of cells comprises at least one synthetic gene which exhibits decreased somatic hypermutation; and wherein said population of cells express an activation induced cytidine deaminase (AID), or can be induced to express AID via addition of an inducing agent; Provided herein is a method of generating SHM-susceptible or SHM-resistant polynucleotides by modifying hot and/or cold spots in a polynucleotide encoding a polypeptide. The method of generating a SHM-susceptible or SHM-resistant polypeptide by includes the following: a) identifying a polypeptide or portion thereof; b) generating a polynucleotide sequence that codes for the identified polypeptide sequence, c) changing the codon usage in the polynucleotide sequence to increase or decrease the frequency, or location with respect to the reading frame of hot spots and/or cold spots within that polynucleotide sequence, without substantially changing the amino acids encoded by the polynucleotide sequence; d) selecting a polynucleotide sequence in which the frequency and b) selecting a cell or cells within the population of cells which express a mutated gene product (e.g., a polypeptide encoded by the mutated synthetic gene, the gene having one or more mutations) having the desired property. In one aspect, the method, optionally, further comprises activating or inducing the expressing AID in said population of cells. In another aspect, the method, optionally, further comprises establishing one or more clonal populations of cells from the cell or cells identified in (b). In yet another aspect of the method, at least one synthetic gene is located in an expression vector such as any one of the vectors described elsewhere herein. In one aspect of the method, the cell is a cell as described elsewhere herein.
In one aspect, a protein encoded by a synthetic gene is selected from among antibodies or antigen-binding fragments thereof, selectable markers, fluorescent proteins, cytokines, chemokines, growth factors, hormones, enzymes, receptors, structural proteins, toxins, co-factors and transcription factors.
Provided herein is a method of generating SHM-susceptible or SHM-resistant polynucleotides by modifying hot and/or cold spots in a polynucleotide encoding a polypeptide. The method of generating a SHM-susceptible or SHM-resistant polypeptide includes the following: a) identifying a polypeptide or portion thereof; b) generating a polynucleotide sequence that codes for the identified substantially changing the amino acids encoded by the polynucleotide sequence; d) selecting a polynucleotide sequence in which the frequency of hot and/or cold spots has been altered to the desired degree. In one aspect the frequency of hot spots can be altered by about 0% to about 25%, in another aspect, by about 25% to about 50%. In still another aspect by about 50% to about 75%, and in yet another aspect by about 75% to about 100% of all possible hot spots or cold spots.
Provided herein is a method for preparing a gene product having a desired property, comprising: (a) expressing a synthetic gene in a population of cells; wherein said population of cells express AID, or can be induced to express AID via the addition of an inducing agent; and (b) selecting a cell or cells within the population of cells which express a modified gene product having the desired property.
In another aspect of the present invention, the polynucleotide sequence can also be altered by the substitution of non preferred codons or motifs for more preferred codons or motifs.
In another aspect of the present invention, the polynucleotide sequence can also be altered by the substitution or replacement of nucleotides to further alter the frequency and or location of hot spots. In one aspect, these nucleotide substitutions can be conservative amino acid replacements, or be located in variable regions across protein families.
Provided herein is a method of optimizing a sequence for SHM by making it susceptible or resistant to SHM, comprising the steps of; a) identifying a polynucleotide sequence; b) changing the codon usage in the polynucleotide sequence to alter the frequency and/or location of hot spots, or cold spots within the polynucleotide sequence and at least two parameters selected from the group of optimization factors consisting of, CpG dinucleotide frequency, the predicted formation of step-loop structures; restriction site frequency, mammalian codon usage; limiting global GC content to less than about 60%; minimizing or eliminatinstretches greater than (>) 6 of same nucleotide; wherein said SHM susceptible or SHM resistant sequence exhibits altered susceptibility to SHM, and is translated into protein within a cell at a level that is equivalent to an unmodified polynucleotide sequence.
In one aspect of this method, step b) involves the alteration of at least three parameters from the group of optimization factors.
In one aspect of this method, step b) involves the alteration of at least four parameters from the group of optimization factors.
In one aspect of this method, step b) involves the alteration of at least five parameters from the group of optimization factors.
In one aspect of this method, step b) involves the alteration of at least six parameters from the group of optimization factors.
In one aspect, step b) is conducted by using a perl program SHMredesign.pl (www.PERL.COM/DOWNLOAD.CSP).
These methods can be used for generating SHM-susceptible or SHM-resistant polynucleotides of any sequence. In either case, the polynucleotide can include an open reading frame of at least 18 nucleotides, and can be operatively linked to regulatory elements to enable the polynucleotide of interest to be efficiently transcribed into RNA. The polynucleotides can be cloned using standard, art-recognized techniques into a vector. In one aspect, the vector is a vector described herein.
Non-limiting examples of polynucleotides to be modified in whole, or part, using such methods include wild-type polynucleotides, synthetic polynucleotides, recombinant polynucleotides or any portion thereof. The polynucleotide can encode a protein or polypeptide sequence.
Provided herein are SHM resistant polynucleotide sequences encoding various genes generated using the methods described herein. In one non-limiting embodiment, the gene is an enzyme involved in SHM, including without limitation, activation-induced cytidine deaminase (AID), Pol eta, and UDG made by the methods described herein.
In one embodiment, the SHM resistant polynucleotide that encodes an enzyme involved in SHM is derived from a vertebrate. In one aspect the gene is derived from a mammal, in one aspect from a mammal selected from the group consisting of rat, dog, human, mouse, cow, and primate.
In one embodiment the SHM resistant enzyme is AID having a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 22.
In one embodiment, the SHM resistant gene is Pol eta having a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 23.
In one embodiment, the SHM resistant gene is UDG having a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 24.
In one embodiment, the cold AID is a mutant form of the enzyme which exhibits increased mutator activity. Mutant forms of AID can contain a strong nuclear import signal (NLS), a mutation that alters the activity of the nuclear export signal or both.
In one aspect, the mutated AID contains a modified nuclear export sequence made by one or more mutations independently selected at positions 180 to 198 of AID (SEQ ID NO: 311), which one or more mutations enhance mutator activity of the modified AID.
In one embodiment of the mutated AID, the modified nuclear export sequence contains one or more mutations at amino acid residue positions Leucine 181 (L181), Leucine 183 (L183), Leucine 189 (L189), Leucine 196 (L196) and/or Leucine 198 (L198). In one non-limiting example, each of the leucine residues in the nuclear export sequence can be mutated to an alanine.
In one embodiment, each of Leucines 181, 183, 189, 196 and 198 can be independently substituted with glycine, alanine, isoleucine, valine, serine, threonine, aspartate or lysine. In another embodiment, each Leucine can be independently substituted with glycine, alanine or serine. In one embodiment, the mutant protein comprises at least one, at least two, at least three or at least four mutations selected from L181A, L183A, L189A, L196A and L198A.
In one embodiment of the mutated AID, the modified nuclear export sequence comprises a mutation at one or more of amino acid residue positions aspartate 187 (D187), aspartate 188 (D188), aspartate 191 (D191) and/or threonine 195 (T195). In one non-limiting example, the modified nuclear export sequence comprises a D187E mutation. In one non-limiting example, each of the aspartate residues in the nuclear export sequence can be mutated to a glutamate. In another non-limiting example, threonine 195 can be mutated to isoleucine.
In one embodiment, each of aspartates 187, 188, and 191 can be independently substituted with serine, threonine, glutamate, asparagine or glutamine. In another embodiment, each aspartate can be independently substituted with glutamate or asparagine. In one aspect, the mutant protein comprises at least one, at least two, at least three or at least four mutations selected from D187E, D188E, D191E, T1951 and L198A.
Mutated AID polypeptides can also contain a nuclear localization signal which can be N-terminal or C-terminal. In one non-limiting example, a mutated AID can contain a strong nuclear localization signal such as, but not limited to PKKKRKV (SEQ ID NO: 340). In another non-limiting example, the NLS can be a sequence conforming to the motif K-K/R-X-K/R.
In another aspect, the mutated AID contains both a strong NLS and a modified nuclear export sequence. In one non-limiting example, the modified nuclear export sequence contains one or more of the following mutations: L181A, L183A, L189A, L196A and L198A. In another non-limiting example, the modified nuclear export sequence contains one or more of the following mutations: D187E, D188E, D191E, T1951 and L198A.
In any of these mutant forms of AID, the gene may be SHM resistant, SHM susceptible, or can include the appropriate optimal codon usage for expression of the AID in the host cell of choice, without regard for SHM susceptibility. When used in an expression system to target SHM to a protein of interest, the mutant form of AID can be SHM resistant.
In another embodiment, the SHM resistant gene is a selectable marker gene. In one-non-limiting embodiment the selectable marker gene is selected from the group consisting of tetracycline, ampicillin, blasticidin, puromycin, hygromycin, kanamycin DHFR neomycin, Zeocin™, thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase, adenine phosphoribosyltransferase and adenosine deaminase.
In one aspect, the SHM resistant gene is the tetracycline resistance gene having a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 25.
In one aspect, the SHM resistant gene is the blasticidin resistance gene having a nucleic acid sequence that is at least 90-95% identical to SEQ ID NO: 26.
In one aspect, the SHM resistant gene is the puromycin resistance gene having a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 27.
In one aspect, the SHM resistant gene is the hygromycin resistance gene having a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 28.
In one aspect, the SHM resistant gene is the neomycin resistance gene having a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 29.
In one aspect, the SHM resistant gene is the Zeocin resistance gene having a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 30.
In one aspect, the SHM resistant gene is thymidine kinase having a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 31.
Also included in the present invention are optimized versions of particular genes of interest that are specifically susceptible to SHM mediated inactivation, and for which the activation of SHM can be used to selectively knock out the function of the genes. For example, the gene of interest can include one or more hot spot motifs that introduce a stop codon as a result of SHM mediated mutagenesis.
Also provided herein are SHM optimized polynucleotide sequences, or portions thereof, encoding various proteins of interest that enable improved versions of the proteins to be rapidly evolved using the SHM systems of the present invention. For example, the proteins, or portions thereof, encoded by said polynucleotides includes antibody genes that can be iteratively evolved to higher affinity, selectivity, stability or solubility.
Other exemplary polynucleotide sequences to be optimized by SHM include, but are not limited to, the following proteins toxins, growth factors, neurotransmitters, co-factors, transcription factors (e.g., zinc finger binding proteins), receptors (e.g., an Fc receptor) all of which can be evolved using the present invention. Specific proteins of interest, and their evolution to improved forms, are discussed in detail in Section X.
B. Vector Systems
Provided herein are replicons for use in any of the SHM systems described herein to facilitate the selective modification of target nucleic acid sequences, genes, or portions thereof, while repressing the mutagenesis of structural proteins, resistance markers and other factors for SHM.
Such replicons can include at least one synthetic polynucleotide sequence that is resistant to SHM. In one aspect the replicon can include at least one unmodified, or synthetic polynucleotide sequence of interest that is designed, or contains, nucleotide sequences that are optimized for SHM. In some embodiments the replicon can include expression control sequences to enable the expression of one or more polynucleotides of interest in the mutator cell line.
Suitable replicons can be based on any known viral, or non-viral vector or an artificial chromosome. An expression system can include any combination of different replicons which can be used in sum to create a coordinated system for SHM.
In one aspect, a replicon suitable for the present invention is created by the insertion, or replacement of at least one polynucleotide sequence with a synthetic polynucleotide sequence that is resistant to SHM.
In another aspect a replicon suitable for the present invention is created by the insertion, or replacement of at least one polynucleotide sequence with a synthetic polynucleotide sequence that is optimized for SHM.
In another aspect a replicon suitable for the present invention is created by the insertion, or replacement of at least one polynucleotide sequence with a synthetic polynucleotide sequence that is optimized for SHM, and said replicon also includes at least one polynucleotide sequence with a synthetic polynucleotide sequence that is resistant to SHM. In a preferred embodiment, the replicon is capable of expression of one or more synthetic polynucleotide sequences within the mutator cell line.
Suitable, vectors can be based on any known episomal vector or integrating vector, including those described herein, known in the art, or discovered or designed in the future.
Representative commercially available viral expression vectors include, but are not limited to, the adenovirus-based systems, such as the Per.C6 system available from Crucell, Inc., lentiviral-based systems such as pLP1 from Invitrogen, and retroviral vectors such as Retroviral Vectors pFB-ERV and pCFB-EGSH from Stratagene.
An episomal expression vector is able to replicate in the host cell, and persists as an extrachromosomal episome within the host cell in the presence of appropriate selective pressure. (See for example, Conese et al., Gene Therapy 11: 1735-1742 (2004)). Representative commercially available episomal expression vectors include, but are not limited to, episomal plasmids that utilize Epstein Barr Nuclear Antigen 1 (EBNA1) and the Epstein Barr Virus (EBV) origin of replication (oriP), specific examples include the vectors pREP4, pCEP4, pREP7 from Invitrogen. The host range of EBV based vectors can be increased to virtually any eukaryotic cell type through the co-expression of EBNA1 binding protein 2 (EPB2) (Kapoor et al., EMBO. J. 20: 222-230 (2001)), vectors pcDNA3.1 from Invitrogen, and pBK-CMV from Stratagene represent non-limiting examples of an episomal vector that uses T-antigen and the SV40 origin of replication in lieu of EBNA1 and oriP.
An integrating expression vector can randomly integrate into the host cell's DNA, or can include a recombination site to enable the specific recombination between the expression vector and the host cells chromosome. Such integrating expression vectors can utilize the endogenous expression control sequences of the host cell's chromosomes to effect expression of the desired protein. Examples of vectors that integrate in a site specific manner include, for example, components of the flp-in system from Invitrogen (e.g., pcDNA™5/FRT), or the cre-lox system, such as can be found in the pExchange-6 Core Vectors from Stratagene. Examples of vectors that integrate into host cell chromosomes in a random fashion include, for example, pcDNA3.1 (when introduced in the absence of T-antigen) from Invitrogen, pCI or pFN10A (ACT) Flexi® from Promega.
Alternatively, the expression vector can be used to introduce and integrate a strong promoter or enhancer sequences into a locus in the cell so as to modulate the expression of an endogenous gene of interest (Capecchi M R. Nat Rev Genet. (2005); 6 (6):507-12; Schindehutte et al., Stem Cells (2005); 23 (1):10-5). This approach can also be used to insert an inducible promoter, such as the Tet-On promoter (U.S. Pat. Nos. 5,464,758 and 5,814,618), in to the genomic DNA of the cell so as to provide inducible expression of an endogenous gene of interest. The activating construct can also include targeting sequence(s) to enable homologous or non-homologous recombination of the activating sequence into a desired locus specific for the gene of interest (see for example, Garcia-Otin & Guillou, Front Biosci. (2006) 11:1108-36). Alternatively, an inducible recombinase system, such as the Cre-ER system, can be used to activate a transgene in the presence of 4-hydroxytamoxifen (Indra et al. Nuc. Acid. Res. (1999) 27 (22): 4324-4327; Nuc. Acid. Res. (2000) 28(23): e99; and U.S. Pat. No. 7,112,715).
Elements to be included in an expression vector are well known in the art, and any existing vector can be readily modified for use in the present invention, for example, through the insertion or replacement of one or more polynucleotide sequences with synthetic polynucleotide sequences as described above.
An expression vector of the present invention can include one or more of the following elements operatively linked together, either on a single replicon, or within multiple replicons; expression control sequences, polynucleotides comprising an open reading frame of a gene of interest, transcription termination signals, origin of replication, and selectable marker genes.
Expression vectors can also include, one or more internal ribosomal entry sites, one or more tags for ease of purification of the protein encoded by the gene of interest (e.g., VSV tag, HA tag, 6×His tag, FLAG tag), one or more reporter genes, EBNA1 (in cases where episomal replication is desired for a OriP base vector and or EBP2; T-antigen can also be used in conjunction with the SV40 on as an alternative to EBNA1 plus oriP), one or more moieties for copy analysis such as portions of a gene that can be used to verify copy number of the vector relative to the host cell's chromosomal DNA, (e.g. glucose-6-phosphate dehydrogenase (hG6PDH) (or variants thereof), one or enzymes or factors for SHM, (e.g. AID, pol eta, UDG, enhancer sequences).
Expression vectors can also include anti-sense, ribozymes or siRNA polynucleotides to reduce the expression of target sequences such as, for example, to reduce the level of Pol beta (See, e.g., Sioud M, & Iversen, Curr. Drug Targets (2005) 6 (6):647-53; Sandy et al., Biotechniques (2005) 39 (2):215-24).
It may be desirable in some instances to convert a surface displayed protein into a secreted protein for further characterization. Conversion can be accomplished through the use of a specific linker that can be cleaved by incubation with a selective protease such as factor X, thrombin or any other selective proteolytic agent. It is also possible to include polynucleotide sequences that enable the genetic manipulation of the encoded protein in the vector (i.e., that allow excision of a surface attachment signal from the protein reading frame). For example, the insertion of one or more unique restriction sites, or cre/lox elements, or other recombination elements that enable the selective removal of an attachment signal and subsequent intracellular accumulation (or secretion) of the protein of interest at will. Further examples include the insertion of flanking loxP sites around an attachment signal (such as a transmembrane domain) allowing for efficient cell surface expression of a protein of interest. However, upon expression of the cre recombinase in the cell, recombination occurs between the LoxP sites resulting in the loss of the attachment signal, and thus leading to the secretion of the protein of interest.
A plasmid encoding the cre recombinase protein (open reading from synthesized by DNA2.0 and inserted into an expression vector) can be transiently transfected (or virally transduced) into a cell population of interest. Action by the expressed cre recombinase protein leads to the in situ removal of the transmembrane portion of the coding region resulting in the translation and production of a secreted form of the protein in the transfected cell population, which can then be used for further studies.
The order and number of elements can be determined by one of ordinary skill in the art.
Example 5 below provides a brief description of the vectors that could be used in any of the SHM systems provided herein.
Briefly, in one aspect, provided herein is an episomal hypermutation competent expression vector comprising: one or more origins (e.g., a prokaryotic on, such as colE1, and one or more eukaryotic origins, such as oriP, or SV40-ori, or both oriP and SV40 on), one or more selectable markers (e.g., an ampicillin resistance gene), one or more b-actin or G6PDH fragments or variants thereof, one or more promoters (e.g., a pCMV promoter) at least one of which drives the transcription of a gene or genes in which hypermutation is desired, one or more restriction sites for insertion of a nucleic acid sequence or gene, optionally including one or more secretion signals, attachment signals, purification tags (e.g., a hemagglutinin (HA) tag) for the gene(s) of interest, an internal ribosomal entry site (IRES), one or more puromycin genes, and one or more transcriptional termination signals. The transcriptional termination signal can include a region of 3′ untranslated region, an optional intron (also referred to as intervening sequence or IVS) and one or more poly adenylation signals (p(A)). In one non-limiting example, episomal expression vectors can have the nucleic acid sequence for a fluorescent protein, inserted into the vector for SHM, to increase or decrease fluorescence or to alter wavelength of absorption or emission.
In another aspect, provided herein is an integrating expression vector comprising a recombination system, and one or more of the following elements operatively linked together; expression control sequences, polynucleotides comprising an open reading frame of a gene of interest, transcription termination signals, origin of replication, and selectable marker genes.
Expression vectors can also include, one or more internal ribosomal entry sites, one or more tags for ease of purification of the protein encoded by the gene of interest (e.g., VSV tag, HA tag, 6×His tag, FLAG tag), one or more reporter genes, one or more moieties for copy analysis, one or enzymes or factors for SHM, (e.g. AID, pol eta, UDG, Ig enhancer sequences)
In one non-limiting embodiment, the expression vector is designed to enable the insertion of a gene of interest into a genomic locus, for example an Ig gene locus.
C. Systems for Transcription and Hypermutation.
1. In Vitro Expression and Hypermutation Systems
In vitro expression and hypermutation systems include cell free systems that enable the transcription, or coupled transcription and translation of DNA templates and on-going mutagenesis via SHM. In one embodiment, such in vitro translation and hypermutation systems can be used in combination with ribosome display to enable the ongoing mutagenesis and selection of proteins.
In vitro translation systems include, for example, the classical rabbit reticulocyte system, as well as novel cell free synthesis systems, (J. Biotechnol. (2004) 110 (3) 257-63; Biotechnol Annu. Rev. (2004) 10 1-30). Systems for ribosome display are described for example in Villemagne et al., J. Imm. Meth. (2006) 313 (1-2) 140-148).
In one aspect, an in vitro hypermutation system can comprise a polynucleotide, or library of polynucleotides, that include an expression cassette for the expression of a gene of interest. The gene of interest can be a synthetic or semi-synthetic gene comprising a sequence that has been optimized for SHM. For ribosome display, the polynucleotide can lack a stop codon so that it remained attached to the ribosome after translation.
To effect transcription and or translation of the gene of interest the system would include purified or semi-purified components for in vitro transcription and translation, for example via the use of recombinant factors with purified 70S ribosomes. To enable on-going SHM, the system would further include recombinant, or purified AID and/or other factors for SHM/DNA repair. Optimized proteins would be selected via functional selection as described for surface displayed proteins, and then the associated ribosomes sequenced to determine the identity of favorable mutations.
Provided herein is an in vitro hypermutation system, comprising: a) a polynucleotide comprising a synthetic gene; b) a recombinant AID; and c) an in vitro expression system. In one aspect the synthetic gene has been optimized for SHM. The in vitro system can further comprise a polymerase to amplify nucleic acids after transcription. The in vitro system can further comprise an in vitro translation system. In one aspect, the polynucleotide is located in an expression vector such as any one of the vectors described elsewhere herein. The in vitro system can further comprise a cell population of a cell as described elsewhere herein.
Provided herein is a kit for in vitro mutagenesis, comprising: a) a recombinant AID protein; b) one or more reagents for in vitro transcription; and c) instructions for design or use of a synthetic gene. The kit can further comprise one or more reagents for in vitro translation. The kit can further comprise comprising an expression vector such as, for example, any one of the expression vectors as described herein. The kit can further comprise a cell population of a cell as described elsewhere herein.
2. Cell Expression and Hypermutation Systems
Cell based expression and hypermutation systems include any suitable prokaryotic or eukaryotic expression systems. Preferred systems are those that can be easily and reliably grown, have reasonably fast growth rates, have well characterized expression systems and can be transformed or transfected easily and efficiently.
a. Prokaryotic Expression Systems
Within these general guidelines, useful microbial hosts include, but are not limited to, bacteria from the genera Bacillus, Escherichia (such as E. coli), Pseudomonas, Streptomyces, Salmonella, Erwinia, Bacillus subtilis, Bacillus brevis, the various strains of Escherichia coli (e.g., HB101, (ATCC NO. 33694) DH5α, DH10 and MC1061 (ATCC NO. 53338)).
b. Yeast
Many strains of yeast cells known to those skilled in the art are also available as host cells for the expression of polypeptides including those from the genera Hansenula, Kluyveromyces, Pichia, Rhino-sporidium, Saccharomyces, and Schizosaccharomyces, and other fungi. Preferred yeast cells include, for example, Saccharomyces cerivisae and Pichia pastoris.
c. Insect Cells
Additionally, where desired, insect cell systems can be utilized in the methods of the present invention. Such systems are described, for example, by Kitts et al., Biotechniques, 14:810-817 (1993); Lucklow, Curr. Opin. Biotechnol., 4:564-572 (1993); and Lucklow et al. (J. Virol., 67:4566-4579 (1993). Preferred insect cells include Sf-9 and HI5 (Invitrogen, Carlsbad, Calif.).
d. Mammalian Expression Systems
A number of suitable mammalian host cells are also known in the art and many are available from the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209. Examples include, but are not limited to, mammalian cells, such as Chinese hamster ovary cells (CHO) (ATCC No. CCL61) CHO DHFR-cells (Urlaub et al., Proc. Natl. Acad. Sci. USA, 97:4216-4220 (1980)), human embryonic kidney (HEK) 293 or 293T cells (ATCC No. CRL1573), or 3T3 cells (ATCC No. CCL92). The selection of suitable mammalian host cells and methods for transformation, culture, amplification, screening and product production and purification are known in the art. Other suitable mammalian cell lines are the monkey COS-1 (ATCC No. CRL1650) and COS-7 cell lines (ATCC No. CRL1651), and the CV-1 cell line (ATCC No. CCL70). Further exemplary mammalian host cells include primate cell lines and rodent cell lines, including transformed cell lines. Normal diploid cells, cell strains derived from in vitro culture of primary tissue, as well as primary explants, are also suitable. Candidate cells can be genotypically deficient in the selection gene, or can contain a dominantly acting selection gene. Other suitable mammalian cell lines include, but are not limited to, mouse neuroblastoma N2A cells, HeLa, mouse L-929 cells, 3T3 lines derived from Swiss, Balb-c or NIH mice, BHK or HaK hamster cell lines, which are available from the ATCC. Each of these cell lines is known by and available for protein expression.
Also of interest are lymphoid, or lymphoid derived cell lines, such as a cell line of pre-B lymphocyte origin. Specific examples include without limitation RAMOS(CRL-1596), Daudi (CCL-213), EB-3 (CCL-85), DT40 (CRL-2111), 18-81 (Jack et al., PNAS (1988) 85 1581-1585), Raji cells, (CCL-86) and derivatives thereof.
For use in an SHM system, any of the vectors described herein can be co-transfected into a host cell with a separate vector containing the nucleic acid sequence of AID. In one aspect, the vectors described herein can be transfected into a host cell that contains endogenous AID. In another aspect, the vectors described herein can be co-transfected into a host cell that contains endogenous AID with a separate vector containing the nucleic acid sequence of AID such that AID is over-expressed in the cell. In yet another aspect, the vectors described herein can be modified to include the sequence of AID for transfection into a host cell that does, or does not, contain endogenous AID. In a preferred embodiment the AID is a synthetic AID that comprises a polynucleotide sequence that is SHM resistant.
In one embodiment, the SHM system comprises one or more of the following selected from among: i) at least one polynucleotide that comprises either a polynucleotide that has been altered in whole or part, from wild type polynucleotide to positively influence the rate of SHM experienced by that polynucleotide, or a polynucleotide that has a naturally high percentage of hot spots prior to any modification; and ii) at least one component of the expression system comprises a polynucleotide that has been altered in whole or part, to negatively influence the rate of SHM.
In one aspect, the SHM system comprises one or more polynucleotides that have been altered from wild-type to negatively influence the rate of SHM. The polynucleotides can encode, for example, one or more of factors for SHM (e.g. AID, Pol eta, UDG), one or more selectable marker genes, or one or more reporter genes.
In another aspect, the SHM system comprises one or more polynucleotides that have been altered in whole, or part, from wild-type to positively influence the rate of SHM. The polynucleotide can be, for example, a polynucleotide of interest encoding an enzyme, receptor, transcription factor, structural protein, toxin, co-factor, specific binding protein of interest.
In yet another aspect, the SHM system comprises a polynucleotide having an intrinsically high rate of SHM such as, for example, a polynucleotide of interest encoding an immunoglobulin heavy chain or an immunoglobulin light chain, or a hypervariable region of an antibody gene.
An SHM system as described herein can further comprise one or more of the following additional elements selected from among: i) an inducible system to regulate the expression of AID, or AID homolog, ii) one or more Ig enhancers, iii) one or more E-boxes, iv) one or more auxiliary factors for SHM, v) one or more factors for stable episomal expression, such as EBNA1, EBP2 or ori-P, vi) one or more selectable marker genes, one or more secondary vectors containing the gene for AID and vii) a combination thereof.
In one aspect, the system includes two polynucleotides of interest in which both polynucleotides are located in proximity to a promoter, and expressed and co-evolved in the same cell simultaneously. In one embodiment, the promoter is a bi-directional promoter such as a bi-directional CMV promoter. In another embodiment, the two polynucleotides of interest are placed in front of two uni-directional promoters. The two promoters can be the same promoter or different promoters. The two polynucleotides of interest can be in the same vector or on different vectors.
Following recombinant introduction of one or more polynucleotides of interest into an expression vector, the vector can be amplified, purified, introduced into a host cell using standard transfection techniques and characterized using standard molecular biological techniques. Purified plasmid DNA can be introduced into a host cell using standard transfection/transformation techniques and the resulting transformants/transfectants grown in appropriate medium containing antibiotics, selectable agents and/or activation/transactivator signals (e.g. inducible agents such as doxycycline) to induce expression of the polynucleotides of interest. If the host cell endogenously expresses AID, the vector containing the one or more polynucleotides of interest can be introduced alone into the host cell. Alternatively, if the cell does not endogenously express AID, then the AID gene, or more preferably, a synthetic AID gene that is more resistant to SHM than wild-type can be transfected into the host cell. Thus, AID expression can be achieved by either using the same, or a different expression vector, as described above for the polynucleotide of interest.
Enhancers (e.g., Ig enhancers) can be inserted into a vector to increase expression, and/or targeting of SHM to the polynucleotide of interest.
If an inducible system is used, such as the Tet-controlled system, doxycycline can be added to the medium to induce expression of the polynucleotide of interest, or AID for a period of time (e.g., 1 hour (hr), 2 Ins, 4 hrs, 6 hrs, 8 hrs, 10 hrs, 15 hrs, 20 hrs, 24 hrs or any other time) prior to analysis by an appropriate assay. The cells can be allowed to grow for a certain time to provide for on-going diversification, for example, for 1-3 cell generations, or in certain cases 3-6 generations, or in some cases 6 to 10 generations, or longer.
Cells can be iteratively grown, assayed and selected as described herein to selectively enrich those cells that express a polynucleotide of interest exhibiting a desired property. Suitable assay and enrichment strategies (e.g., fluorescent activated cell sorting (FACS); affinity separation, enzyme activity, toxicity, receptor binding, growth stimulation, etc.) are described below.
Once a population of cells has been obtained that is of interest, the polynucleotides of interest can be rescued and the corresponding mutations sequenced and identified. For example, total mRNA, or extrachromosal plasmid DNA can be amplified by co-expression of SV40 T antigen (J. Virol. (1988) 62 (10) 3738-3746) and/or can be extracted from cells and used as a template for polymerase chain reaction (PCR) or reverse transcriptase (RT)-PCR to clone the modified polynucleotide using appropriate primers. Mutant polynucleotides can be sub-cloned into a vector and expressed in E. coli. A tag (e.g., His-6 tag) can be added to the carboxy terminus to facilitate protein purification using chromatography.

X. Proteins of Interest

As used herein, the term “proteins of interest” relates to proteins, or portions thereof, for which it is desired that the polynucleotide encoding the protein is optimized for SMH by AID in order to rapidly create, select and identify improved variants of that protein. Such optimized polynucleotide can be made more susceptible to SHM as a result of codon usage, thereby inducing amino acid changes when the polynucleotide is subjected to AID, and screened for improved function. Conversely, such optimized polynucleotide can be made more resistant to SHM, thereby decreasing amino acid changes when the polynucleotide is subjected to AID as a result of codon usage, and screened for improved function.
It should be understood however that the present invention also includes compositions and methods that relate to polynucleotide sequences that encode proteins that are resistant to SHM, or comprise codons that can be converted to stop codons. Such synthetic genes confer certain advantages in the present invention and are specifically disclosed, for example, in Section IX.
Any protein for which the amino acid, or corresponding nucleotide sequence is known, or available (e.g. can be cloned into a vector of the present invention) and a phenotype or function can be improved is a candidate for use in the vectors and SHM systems provided herein. Proteins of interest include, for example, surface proteins, intracellular proteins, membrane proteins and secreted proteins from any unmodified or synthetic source. Exemplary, but non-limiting types of proteins for use in the vectors and SHM systems provided herein include an antibody heavy chain or portion thereof, an antibody light chain or portion thereof, an enzyme, a receptor, a structural protein, a co-factor, a polypeptide, a peptide, an intrabody, a selectable marker, a toxin, growth factor, peptide hormone, and any other protein which can be optimized, is intended to be included.
Biologically active proteins (molecules) also include molecules capable of modulating the pharmacokinetics and/or pharmacodynamics of other biologically active proteins (molecules), for example, lipids and polymers such as polyamines, polyamides, polyethylene glycol and other polyethers. For example, polypeptides are those such as, for example, VEGF, VEGF receptor, Diptheria toxin subunit A, B. pertussis toxin, CC chemokines (e.g., CCL1-CCL28), CXC chemokines (e.g., CXCL1-CXCL16), C chemokines (e.g., XCL1 and XCL2) and CX₃C chemokines (e.g., CX₃CL1), IFN-gamma, IFN-alpha, IFN-beta, TNF-alpha, TNF-beta, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, TGF-beta, TGF-alpha, GM-CSF, G-CSF, M-CSF, TPO, EPO, human growth factor, fibroblast growth factor, nuclear co-factors, Jak and Stat family members, G-protein signaling molecules such as chemokine receptors, JNK, Fos-Jun, NF-κB, I-κB, CD40, CD4, CD8, B7, CD28 and CTLA-4.
Additionally, there are a variety of other component nucleotide sequences, such as coding sequences and genetic elements that can make up the core system that one would, in some embodiments, prefer not to hypermutate to maintain overall system integrity. These component nucleotide sequences include without limitation, i) selectable markers such as neomycin, blasticidin, ampicillin, etc; ii) reporter genes (e.g. fluorescent proteins, epitope tags, reporter enzymes); iii) genetic regulatory signals, e.g. promoters, inducible systems, enhancer sequences, IRES sequences, transcription or translational terminators, kozak sequences, splice sites, origin of replication, repressors; iv) enzymes or accessory factors used for high level enhanced SHM, or it's regulation, or measurement, such as AID, pol eta, transcription factors, and MSH2; v) signal transduction components (kinases, receptors, transcription factors) and vi) domains or sub domains of proteins such as nuclear localization signals, transmembrane domains, catalytic domains, protein-protein interaction domains, and other protein family conserved motifs, domains and sub-domains.
One could, based on the present application, select a protein of interest as a suitable candidate for optimization, and devise a suitable assay to monitor the desired trait of the protein of interest.
Depending on the nature of the protein of interest, and amount of information available on the protein of interest, a practitioner can follow any combination of the following strategies prior to mutagenesis to create the optimized polynucleotide.
1. No optimization: Although it can be desirable to enhance the number of hot spots within the polynucleotide sequence encoding a protein of interest, it should be noted that any unmodified protein is expected to undergo a certain amount of SHM, and can be used in the present invention without optimization, or any specific knowledge of the actual sequence. Additionally certain proteins, for example antibodies, naturally comprise polynucleotide sequences which have evolved suitable codon usage, and do not require codon modification. Alternatively, it can be desirable to enhance the number of cold spots within the polynucleotide sequence encoding a protein of interest (e.g., framework regions of antibodies or fragments thereof).
2. Global Hot spot optimization: In some aspects, the number of hotspots in a polynucleotide encoding a protein can be increased, as described herein. This approach can be applied to the entire coding region of the gene, thereby rendering the entire protein more susceptible to SHM. As discussed herein, this approach can be preferred if relatively little is known about structure activity relationships within the protein, or between related protein isotypes.
3. Selective hot spot modification: Alternatively, as discussed herein, a polynucleotide sequence encoding the protein of interest can be selectively, and or systematically modified through the targeted replacement of regions of interest with synthetic variable regions, which provide for a high density of hot spots and seed maximal diversity through SHM at specific loci.
One of ordinary skill in the art would understand, based on the teaching provided herein, that any or all of the above approaches can be undertaken using the present invention. Approaches relating to global hot spot optimization, and selective hot spot modification, are however likely to lead to faster and more efficient optimization of protein function.
Following the design of an optimized polynucleotide encoding the protein of interest, it can be synthesized using standard methodology and sequenced to confirm correct synthesis. Once the sequence of the polynucleotide has been confirmed, the polynucleotide can be inserted into a vector of the present invention, and the vector then introduced into a host cell as described herein to effect mutagenesis.
Once introduced into a suitable host cell, cells can be induced to express AID, and/or other factors to initiate SHM, thereby inducing on-going sequence diversification of the protein of interest. After an appropriate period of time, (e.g., 2-10 cell divisions) the resulting host cells, including variants of the protein of interest can be screened and improved mutants identified and separated for the cell population. This process can be iteratively repeated to selectively improve the properties of the protein of interest.
A cell-surface displayed protein can be created through the creation of a chimeric molecule of a protein of interest coupled in frame to a suitable transmembrane domain. In the case of mammalian cell expression, for example, a MHC type 1 transmembrane domain such as that from H2kk (including peri-transmembrane domain, transmembrane domain, and cytoplasmic domain; NCBI Gene Accession number AK153419) can be used. Likewise the surface expression of proteins in prokaryotic cells (such as E. coli and Staphylococcus) insect cells, and yeast is well established in the art. For reviews, see for example Winter, G. et al., Annu. Rev. Immunol. (1994) 12:433-55; Plückthun, A., (1991) Bio/Technology 9: 545-551; Gunneriusson et al., (1996) J. Bacteriol 78 1341-1346; Ghiasi et al., (1991) Virology 185 187-194; Boder and Wittrup, (1997) Nat. Biotechnol. 15 553-557; and Mazor et al., (2007) Nat. Biotech. 25(5) 563-565.
Surface displayed antibodies or proteins can be created through the secretion and then binding (or association) of the secreted protein on the cell surface. Conjugation of the antibody or protein to the cell membrane can occur either during protein synthesis or after the protein has been secreted from the cell. Conjugation can occur via covalent linkage, by binding interactions (e.g., mediated by specific binding members) or a combination of covalent and non-covalent linkage.
In yet another aspect, proteins can be coupled to a cell through the creation of an antibody or binding protein fusion protein comprising a first specific binding member that specifically binds to a target of interest fused to a second binding member specific for display on a cell surface (e.g., in the case of exploiting the binding of protein A and a Fc domain: protein A is expressed on and attached to a cell surface and binds to, and localizes, a secreted antibody (or a protein of interest expressed as an Fc fusion protein)).
Transfection of appropriate expression vectors containing the corresponding polynucleotide sequences into suitable mutator positive cells can be performed using any art recognized or known transfection protocol. An exemplary surface expressed library of proteins is described in Examples 4 and 5 of priority U.S. Application Nos. 60/904,622 and 61/020,124.
Cells expressing a plurality of antibodies or binding proteins from the transfections above can, optionally, be characterized to select cells expressing specific ranges of surface expression of the protein on the cell surface using conventional assays including, but not limited to, FACS.
Staining of light and heavy chain expression can be accomplished, for example, by using commercially available fluorescein Isothiocyanate (FITC) or R-Phycoerythrin (R-PE) conjugated rat anti-mouse Ig, kappa light chain, and FITC or R-PE conjugated rat anti-mouse Ig G1 monoclonal antibodies (BD Pharmingen). Staining can be performed using the manufacture's suggested protocols, usually via incubation of the test cells in the presence of labeled antibody for 30 minutes on ice. Expression levels of cellular antigen expression can be quantified using Spherotech rainbow calibration particles (Spherotech, IL).
Transfected cell populations exhibiting specific ranges of expression can be selected. For example, cells with a surface copy number of greater than about 10,000, about 50,000, about 100,000, or about 500,000 proteins per cell can be selected, and can then be used for efficient affinity profiling.
Populations of stably transfected cells can be created via, for example, growth for 2 to 3 weeks in the presence of appropriate selectable agents; the resulting cell library can be frozen and stored as a cell bank. Alternatively, cells can be transiently transfected and used within a few days of transfection.
It may be desirable in some instances to convert a surface displayed protein into a secreted protein for further characterization. Conversion can be accomplished through the use of a specific linker that can be cleaved by incubation with a selective protease such as factor X, thrombin or any other selective proteolytic agent. It is also possible to include polynucleotide sequences that enable the genetic manipulation of the encoded protein in the vector (i.e., that allow excision of a surface attachment signal from the protein reading frame). For example, the insertion of one or more unique restriction sites, or cre/lox elements, or other recombination elements that enable the selective removal of an attachment signal and subsequent intracellular accumulation (or secretion) of the protein of interest at will. Further examples include the insertion of flanking loxP sites around an attachment signal (such as a transmembrane domain) allowing for efficient cell surface expression of a protein of interest. However, upon expression of the cre recombinase in the cell, recombination occurs between the LoxP sites resulting in the loss of the attachment signal, and thus leading to the secretion of the protein of interest.
Once a polypeptide has been optimized to a determined degree, the cell or population of cells expressing an optimized polypeptide of interest can be isolated or enriched and the phenotype (function) of the optimized polypeptide can be assayed using art-recognized assays.
Cells can then be re-grown, SHM re-induced, and re-screened over a number of cycles to effect iterative improvements in the desired function. At any point, the polynucleotide sequence encoding the protein of interest can be rescued and/or sequenced to monitor on-going mutagenesis.
For example, episomal plasmid DNA can be extracted (or amplified by co-expression with SV40 T Antigen (J. Virol. (1988) 62 (10) 3738-3746)) and then extracted and amplified by PCR using DNA primers that are specific for the polynucleotide or interest or flanking regions, using standard methodology. Alternatively, total RNA can be isolated from various cell populations that have been isolated by flow cytometry or magnetic beads; episomal DNA and/or total RNA and can be amplified by RT-PCR using primers that are specific for the polynucleotide or interest or flanking regions using standard methodology. Clones can be sequenced using automated DNA sequences from companies such as Applied Biosystems (ABI-377 or ABI 3730 DNA sequencers). Sequences can be analyzed for frequency of nucleotide insertions and deletions compared to the starting sequence.
A. Antibodies and Fragments Thereof
With respect to antibodies, the present invention provides the ability to bypass the need for immunization in vivo to select antibodies that bind to key surface epitopes that are aligned with producing the most robust biological effects on target protein function. Additionally, mammalian antibodies intrinsically process optimal codon usage patterns for targeted SHM, greatly simplifying template design strategies. For certain antigens, in vivo immunization leads to epitope selection that does not impact target function, thereby hindering the selection of potent and efficacious antibody candidates. In still other embodiments, the present invention can provide for the rapid evolution of site-directed antibodies that have potent activity by nature of the role of that epitope in determining target protein function. This provides the ability to scan target proteins for optimal epitope position and produce best in class antibodies drugs for use in the clinic.
As described herein, all naturally occurring germline, affinity matured, synthetic, or semi-synthetic antibodies, as well as fragments thereof, may be used in the present invention. In general, such antibodies can be altered through SHM to improve one or more of the following functional traits: affinity, avidity, selectivity, thermostability, proteolytic stability, solubility, folding, immunotoxicity and expression. Depending upon the antibody format, antibody libraries can comprise separate heavy chain and light chain libraries which can be co-expressed in a host cell. In certain embodiments, full length antibodies can be secreted, and/or surface displayed at the plasma membrane of the host cell. In still other embodiments, heavy and light chain libraries can be inserted in to the same expression vector, or different expression vectors to enable simultaneous co-evolution of both antibody chains.
In one embodiment, increasing the hotspot density in specific sub domains of antibodies or fragments thereof (e.g., F(ab′)₂, Fab′, Fab, Fv, scFv, dsFv, dAb or a single chain binding polypeptide) can result in an improvement in a characteristic such as one or more of increased binding affinity, increased binding avidity and/or decreased non-specific binding. In another embodiment, the use of synthetic antibodies with increased hotspots in the constant domain (e.g., Fc) can result in increased binding affinity for an Fc receptor (FcR), thereby modulating signal cascades. Heavy chains and light chains, or portions thereof, can be simultaneously modified using the procedures described herein.
Intrabodies used in the methods provided herein can be modified to improve or enhance folding of the heavy and/or light chain in the reducing environment of the cytoplasm. Alternatively, or in addition, a sFv intrabody can be modified to stabilize frameworks that could fold properly in the absence of intradomain disulfide bonds. Intrabodies can also be modified to increase, for example, one or more of the following characteristics: binding affinity, binding avidity, epitope accessibility, competition with endogenous proteins for the target epitope, half-life, target sequestration, post-translational modification of the target protein, etc. Because intrabodies act within the cell, their activity is more analogous to assay methodologies for enzyme activity assays, which are discussed below in section B.
Polynucleotide Identification and Design
Methods for designing and creating targeted antibody libraries, as well as methods for identifying optimal epitopes that provide for the selection of antibodies with superior selectivity, cross species reactivity, and blocking activity are known in the art and described herein. Such methods are disclosed in sections IV and V of the present specification, as well as commonly owned priority U.S. Patent Application Nos. 60/904,622 and 61/020,124.
Screening Methodology
Specific screens to detect and select surface exposed or secreted antibodies with improved traits, are well known in the art, and are described in detail in section XI. Such screens can involve several rounds of selection based on the simultaneous selection of multiple parameters, for example, affinity, avidity, selectivity and thermostability in order to evolve the overall best antibody.
Once an antibody or fragment thereof has been optimized using SHM, the phenotype/function of the optimized antibody or fragment thereof can be further analyzed using art-recognized assays. Assays for antibodies or fragments thereof include, but are not limited to enzyme-linked immunosorbant assays (ELISA), enzyme-linked immunosorbent spot (ELISPOT assay), gel detection and fluorescent detection of mutated IgH chains, Scatchard analysis, BIACOR analysis, western blots, polyacrylamide gel (PAGE) analysis, radioimmunoassays, etc. which can determine binding affinity, binding avidity, etc. Such assays are more fully described in Section XI below.
Once optimized antibodies have been identified, episomal DNA can be extracted (or amplified by co-expression with SV40 T Antigen (J. Virol. (1988) 62 (10) 3738-3746)) and then extracted and subjected to PCR using variable heavy chain (V_H) leader region and/or variable light chain (V_L) leader region specific sense primers and isotype specific anti-sense primers. Alternatively, total RNA from selected sorted cell populations can be isolated subjected to RT-PCR using variable heavy chain (V_H) leader region and/or variable light chain (V_L) leader region specific sense primers and isotype specific anti-sense primers. Clones can be sequenced using standard methodologies and the resulting sequences can be analyzed for frequency of nucleotide insertions and deletions, receptor revision and V gene selection. The resulting data can be used to populate a database linking specific amino acid substitutions with changes in one or more of the desired properties. Such databases can then be used to recombine favorable mutations or to design next generation polynucleotide library with targeted diversity in newly identified regions of interest, e.g. nucleic acid sequences which encode a functional portion of a protein.
B. Enzymes
Enzymes and pro-enzymes present another category of polypeptides which can be readily improved, and for which SHM is useful. Of particular interest is the application of the present invention to the co-evolution of multiple enzymatic pathways, involving the simultaneous mutation of two or more enzymes. Enzymes and enzyme systems of particular note include, for example, enzymes associated with microbiological fermentation, metabolic pathway engineering, protein manufacture, bio-remediation, and plant growth and development.
Specific high throughput screening systems to measure, select and evolve enzymes with improved traits, are well known in the art, and are outlined in Section XI. Such screens can involve several rounds of selection based on the simultaneous selection of multiple parameters, for example, pH stability, Km, Kcat, thermostability, solubility, proteolytic stability, substrate specificity, co-factor dependency, and tendency for hetero or homo dimerization.
Polynucleotide Identification and Design
As described previously, the starting point for mutagenesis is either a cDNA clone of the gene of interest, or it's amino acid or polynucleotide sequence. To maximize the effectiveness of SHM, the starting polynucleotide sequence can be modified to maximize the density of hot spots and to reduce the density of cold spots. Such methods are disclosed in sections IV and V of the present specification, as well as commonly owned priority U.S. Patent Application Nos. 60/904,622 and 61/020,124.
If particular regions of interest have been defined in the protein or proteins of interest, these areas can be targeted with preferred hot spot motifs, alternatively a scanning approach can be used to systematically insert hot spot motifs throughout the reading frame of the enzyme or enzymes of interest, as described previously.
For the co-evolution of a particular enzymatic pathway involving multiple enzymes, a particular advantage of the present invention is the ability to co-evolve all the enzymes simultaneously with a single cell. This approach exploits the ability to identify mutations that only confer an advantage to the overall system when all of the members of the system are present. For example, mutations that induce heterodimerization between enzymes involved in consecutive enzymatic reactions.
Screening Methodology
Many high throughput screening approaches are well known in the art and can be readily applied to identify and select improved enzymes (see, e.g., Olsen et al., Methods. Mol. Biol. (2003) 230: 329-349; Turner, Trends Biotechnol. (2003) 21 (11): 474-478; Zhao et al., Curr. Opin. Biotechnol. (2002) 13 (2): 104-110; and Mastrobattista et al., Chem Biol. (2005) 12 (12): 1291-300). The screening modality used can depend on the nature of the enzyme and whether the enzyme of interest is intracellular, or extracellular, and further whether it is membrane associated or freely secreted.
Initial screens that provide useful quantitative information over a wide dynamic window, and which have a high screening capacity, are preferred. Representative screening approaches include, for example, assays based on the altered ability, or speed of growth of improved cells, and/or based on the sorting of cells using a flow cytometer. FACS based approaches can detect the presence of intracellular fluorogenic reaction products or altered reporter gene expression, and specific protocols for the FACS based optimization of enzyme activity are reviewed in the following references; Farinas et al., Comb. Chem. High Throughput Screen (2006) 9(4): 321-8; Becker et al., Curr. Opin. Biotechnol. (2004) 15(4): 323-9; Daugherty et al., J. Immunol. Methods (2000) 243 (1-2): 211-227.
Once an enzyme or set of enzymes has been optimized using SHM, a complete biochemical analysis of the optimized enzyme(s) can be further analyzed using art-recognized assays. Additionally as previously discussed, once optimized enzymes have been identified, episomal DNA can be extracted or amplified by co-expression with SV40 T Antigen (J. Virol. (1988) 62 (10) 3738-3746), then extracted and subjected to PCR using specific primers. Alternatively, total RNA can be obtained from selected cell populations and subjected to RT-PCR using specific primers. Clones can be sequenced using standard methodologies and the resulting sequences can be analyzed for the frequency of nucleotide mutations. The resulting data can be used to populate a database linking specific amino acid substitutions with changes in one or more of the desired properties. Such databases may then be used to recombine favorable mutations, or to design next generation polynucleotide library with targeted diversity in newly identified regions of interest, e.g. nucleic acid sequences which encode a functional portions of a protein.
C. Receptors
Receptors bind ligands and encompass a broad genus of unmodified and synthetic polypeptides encoding specific binding members, including, but not limited to, cell-bound receptors such as antibodies (B cell receptors), T cell receptors, Fc receptors, G-coupled protein receptors, cytokine receptors, carbohydrate receptors, and Avimer™ based receptors.
In one embodiment, such receptors can be altered through SHM to improve one or more of the following traits; affinity, avidity, selectivity, thermostability, proteolytic stability, solubility, dimerization, folding, immunotoxicity, coupling to signal transduction cascades and expression.
Polynucleotide Identification and Design
As described previously, the starting point for mutagenesis can be either a cDNA clone of the gene of interest, or its amino acid or polynucleotide sequence. To maximize the effectiveness of SHM, the starting polynucleotide sequence can be modified to maximize the density of hot spots and to reduce the density of cold spots. Such methods are disclosed in sections W and V of the present specification.
Such receptors possess clearly defined domains that can be either targeted for mutagenesis through the use of SHM optimized sequences, or conserved during mutagenesis through the use of SHM resistant sequences. Domains (regions) targeted for mutagenesis include, but are not limited to, sites of post-translational modification, surface exposed loop domains, positions of variation between species, protein-protein interaction domains, and binding domains. Domains (regions) conserved during mutagenesis include transmembrane domains, invariant amino acid positions, signal sequences, and intracellular trafficking domains. Alternatively, a scanning approach can be used to systematically insert hot spot motifs throughout the reading frame of the receptor of interest, as described previously.
Screening Methodology
Many high throughput screening approaches are well known in the art and can be readily applied to identify and select improved receptors. Representative screening approaches include, for example, binding assays, growth assays, reporter gene assays and FACS based assays.
Once an enzyme or set of enzymes has been optimized using SHM, a complete pharmacological analysis of the optimized receptor can be further analyzed using art-recognized assays. Additionally as previously discussed, once an optimized receptor has been identified, episomal DNA can be extracted or amplified by co-expression with SV40 T Antigen (J. Virol. (1988) 62 (10) 3738-3746), then extracted and subjected to PCR using specific primers. Alternatively, total RNA can be obtained from selected cell populations and subjected to RT-PCR using specific primers. Clones can be sequenced using standard methodologies and the resulting sequences can be analyzed for the frequency of nucleotide mutations. The resulting data can be used to populate a database linking specific amino acid substitutions with changes in one or more of the desired properties. Such databases may then be used to recombine favorable mutations or to design next generation polynucleotide library with targeted diversity in newly identified regions of interest, e.g., nucleic acid sequences which encodes functional portions of a protein.

XI. Screening and Enrichment Systems

Polypeptides generated by the expression of the synthetic libraries, semi-synthetic libraries, or seed libraries of polynucleotides described herein can be screened for improved phenotype using a variety of standard physiological, pharmacological and biochemical procedures. Such assays include for example, biochemical assays such as binding assays, fluorescence polarization assays, solubility assays, folding assays, thermostability assays, proteolytic stability assays, and enzyme activity assays (see generally Glickman et al., J. Biomolecular Screening, 7 No. 1 3-10 (2002); Salazar et al., Methods. Mol. Biol. 230 85-97 (2003)), as well as a range of cell based assays including signal transduction, motility, whole cell binding, flow cytometry and fluorescent activated cell sorting (FACS) based assays. Cells expressing polypeptide of interest encoded by a synthetic or semi-synthetic library as described herein can be enriched any art-recognized assay including, but not limited to, methods of coupling peptides to microparticles.
Many FACS and high throughput screening systems are commercially available (see, e.g., Zymark Corp., Hopkinton, Mass.; Air Technical Industries, Mentor, Ohio; Beckman Instruments Inc., Fullerton, Calif.; Precision Systems, Inc., Natick, Mass.) that enable these assays to be run in a high throughput mode. These systems typically automate entire procedures, including all sample and reagent pipetting, liquid dispensing timed incubations, and final readings of the microplate in detector(s) appropriate for the assay. These configurable systems provide high throughput and rapid start up as well as a high degree of flexibility and customization. The manufacturers of such systems provide detailed protocols for various high throughput systems. Thus, for example, Zymark Corp. provides technical bulletins describing screening systems for detecting the modulation of gene transcription, ligand binding, and the like.
A. Cell-Based Methods to Measure Activities.
1. Signal Transduction Based Assays
Proteins such as, for example, growth factors, enzymes, receptors and antibodies can influence signal transduction within a cell or cell population, and thereby influence transcriptional activity that can be detected using a reporter gene assay. Such modulators can behave functionally as full or partial agonists, full or partial antagonists, or full or partial inverse agonists.
Thus in one assay format, signal transduction assays can be based on the use of cells comprising a reporter gene whose expression is directly or indirectly regulated by the protein of interest, which can be measured by a variety of standard procedures.
Reporter plasmids can be constructed using standard molecular biological techniques by placing cDNA encoding for the reporter gene downstream from a suitable minimal promoter (that is, any sequence that supports transcription initiation in eukaryotic cells) that sits 5′ to the coding sequence of the reporter gene. A minimal promoter can be derived from a viral source such as, for example: SV40 early or late promoters, cytomegalovirus (CMV) immediate early promoters, or Rous Sarcoma Virus (RSV) early promoters; or from eukaryotic cell promoters, for example, beta actin promoter (Ng, Nuc. Acid Res. 17:601-615, 1989; Quitsche et al., J. Biol. Chem. 264:9539-9545, 1989), GADPH promoter (Alexander, M. C. et al., Proc. Nat. Acad. Sci. USA 85:5092-5096, 1988, Ercolani, L. et al., J. Biol. Chem. 263:15335-15341, 1988), TK-1 (thymidine kinase) promoter, HSP (heat shock protein) promoters, or any eukaryotic promoter containing a TATA box.
A reporter plasmid also typically includes an element 5′ to the minimal promoter that contains a consensus recognition sequence, usually repeated 2 to 7 times in a concatenate, to the appropriate branch of the signal transduction pathway for which monitoring is desired. Examples include, but are not limited to: cyclic AMP response elements (CRE, which responds to changes in intracellular cAMP concentrations, available from Stratagene in phagemid vector pCRE-Luc, Cat. No. 219076), serum response elements (SRE, Stratagene phagemid vector pSRE-Luc. Cat. No. 219080), nuclear factor B response elements (NF-kB, Stratagene phagemid vector pNFKB-Luc Cat. No. 219078), activator protein 1 response elements (AP-1, Stratagene phagemid vector pAP-1-Luc, Cat. No. 219074), serum response factor response elements (Stratagene phagemid vector pSRF-Luc, Cat. No. 219082), or p53 binding sites.
Numerous reporter gene systems are known in the art and include, for example, alkaline phosphatase Berger, J., et al. (1988) Gene 66 1-10; Kain, S. R. (1997) Methods. Mol. Biol. 63 49-60), .beta.-galactosidase (See, U.S. Pat. No. 5,070,012, issued Dec. 3, 1991 to Nolan et al., and Bronstein, I., et al., (1989) J. Chemilum. Biolum. 4 99-111), chloramphenicol acetyltransferase (See Gorman et al., Mol Cell Biol. (1982) 2 1044-51), .beta.-glucuronidase, peroxidase, beta-lactamase (U.S. Pat. Nos. 5,741,657 and 5,955,604), catalytic antibodies, luciferases (U.S. Pat. Nos. 5,221,623; 5,683,888; 5,674,713; 5,650,289; 5,843,746) and naturally fluorescent proteins (Tsien, R. Y. (1998) Annu. Rev. Biochem. 67 509-44).
Alternatively, intermediate signal transduction events that are proximal to gene regulation can also be observed, such as, by measuring fluorescent signals from reporter molecules that respond to intracellular changes including, but not limited to, fluctuations in calcium concentration due to release from intracellular stores, alterations in membrane potential or pH, increases in inositol triphosphate (IP₃) or cAMP concentrations, or release of arachidonic acid.
As used herein, agonists refer to modulators that stimulate signal transduction and can be measured using various combinations of the construct elements listed above. As used herein, partial agonists refer to modulators able to stimulate signal transduction to a level greater than background, but less than 100% as compared to a full agonist. A superagonist is able to stimulate signal transduction to greater than 100% as compared to a full agonist reference standard.
As used herein, antagonists refer to modulators that have no influence on signal transduction on their own, but are able to inhibit agonist- (or partial agonist-) induced signaling. As used herein, partial antagonists refer to modulators that have no influence on signal transduction on their own, but are able to inhibit agonist- (or partial agonist-) induced signaling to an extent that is measurable, but less than 100%.
As used herein, inverse agonists refer to modulators that are able to inhibit agonist- (or partial agonist-) induced signaling, and are also able to inhibit signal transduction when added alone.
2. Motility Assays
Agonistic activity on several categories of cell surface molecules (e.g., GPCR's such as chemokine receptors, histamine H4, cannabinoid receptors, etc.) can lead to cell movements. Thus, partial or full agonist or antagonist activities of test molecules can be monitored via effects on cell motility, such as in chemotaxis assays (Ghosh et al., (2006) J Med Chem. May 4; 49(9):2669-2672), chemokinesis (Gillian et al., (2004) ASSAY and Drug Development Technologies. 2(5): 465-472) or haptotaxis (Hintermann et al., (2005) J. Biol. Chem. 280(9): 8004-8015).
3. Whole Cell Binding Assays
Binding assays that utilize receptors, membrane associated antibodies, and cell surface proteins can be performed using whole cells (as opposed to membrane preparations) in order to monitor activity or binding selectivity of proteins of interest. Such assays can also be used to directly select desired cell populations via the use of FACS. (Fitzgerald et al., (1998) J Pharmacol Exp Ther. 1998 November; 287(2):448-456; Baker, (2005) Br J Pharmacol. February; 144(3):317-22)
A large number of fluorescently tagged compounds are available to perform whole cell binding assays. In addition, specific peptides can be readily labeled in order to profile the binding affinity and selectivity of membrane associated antibodies. In general peptides can be conjugated to a wide variety of fluorescent dyes, quenchers and haptens such as fluorescein, R-phycoerythrin, and biotin. Conjugation can occur either during peptide synthesis or after the peptide has been synthesized and purified.
Biotin is a small (244 kilodaltons) vitamin that binds with high affinity to avidin and streptavidin proteins and can be conjugated to most peptides without altering their biological activities. Biotin-labeled peptides are easily purified from unlabeled peptides using immobilized streptavidin and avidin affinity gels, and streptavidin or avidin-conjugated probes can be used to detect biotinylated peptides in, for example, ELISA, dot blot or Western blot applications.
N-hydroxysuccinimide esters of biotin are the most commonly used type of biotinylation agent. N-hydroxysuccinimide-activated biotins react efficiently with primary amino groups in physiological buffers to form stable amide bonds. Peptides have primary amines at the N-terminus and can also have several primary amines in the side chain of lysine residues that are available as targets for labeling with N-hydroxysuccinimide-activated biotin reagents. Several different N-hydroxysuccinimide esters of biotin are available, with varying properties and spacer arm length (Pierce, Rockford, Ill.). The sulfo-N-hydroxysuccinimide ester reagents are water soluble, enabling reactions to be performed in the absence of organic solvents.
Alternatively, peptides can be conjugated with R-Phycoerythrin, a red fluorescent protein. R-Phycoerythrin is a phycobiliprotein isolated from marine algae. There are several properties that make R-Phycoerythrin ideal for labeling peptides, including an absorbance spectra that includes a wide range of potential excitation wavelengths, solubility in aqueous buffers and low nonspecific binding. R-Phycoerythrin also has a high fluorescence quantum yield (0.82 at 578 nanometers) that is temperature and pH independent over a broad range. Conjugating peptides with R-Phycoerythrin can be accomplished using art-recognized techniques described in, for example, Glazer, A N and Stryer L. (1984). Phycofluor probes. Trends Biochem. Sci. 9:423-7; Kronick, M N and Grossman, P D (1983) Immunoassay techniques with fluorescent phycobiliprotein conjugates. Clin. Chem. 29:1582-6; Lanier, L L and Loken, M R (1984) Human lymphocyte subpopulations identified by using three-color immunofluorescence and flow cytometry analysis: Correlation of Leu-2, Leu-3, Leu-7, and Leu-11 cell surface antigen expression. J Immunol, 132:151-156; Parks, D R et al. (1984) Three-color immunofluorescence analysis of mouse B-lymphocyte subpopulations. Cytometry 5:159-68; Hardy, R R et al. (1983) demonstration of B-cell maturation in X-linked immunodeficient mice by simultaneous three-color immunofluorescence. Nature 306:270-2; Hardy R R et al. (1984) J. Exp. Med. 159:1169-88; and Kronick, M N (1986) The use of phycobiliproteins as fluorescent labels in immunoassay. J Immuno Meth. 92:1-13.
A number of cross-linkers can be used to produce phycobiliprotein conjugates including, but not limited to, N-Succinimidyl 3-[2-pyridyldithio]-propionamido, (Succinimidyl 6-(3-[2-pyridyldithio]-propionamido)hexanoate, or (Sulfosuccinimidyl 6-(3-[pyridyldithio]-propianamido)hexanoate. Such cross-linkers react with surface-exposed primary amines of the phycobiliprotein and create pyridyldisulfide group(s) that can be reacted with peptides that contain either free sulfhydryl groups or primary amines.
Another option is to label peptides with fluorescein isothiocyanate (molecular weight 389). The isothiocyanate group on the fluorescein will cross-link with amino, sulfhydryl, imidazoyl, tyrosyl or carbonyl groups on peptides, but generally only derivatives of primary and secondary amines yield stable products. Fluorescein isothiocyanate has an excitation and emission wavelengths at 494 and 520 nanometers respectively and a molar extinction coefficient of 72,0000 M⁻¹cm⁻¹in an aqueous buffer at pH 8 (Der-Balian G, Kameda, N and Rowley, G. (1988) Fluorescein labeling of Fab while preserving single thiol. Anal. Biochem. 173:59-63).
4. Whole Cell Activity Assays
Many proteins, including enzymes, intrabodies and receptors can be directly assayed within a living cell, or when surface displayed on the surface. Typically for successful FACS based screening a fluorescent or fluorogenic membrane permeant substrate is required, many such reagents are commercially available, for example from Molecular Probes (Invitrogen, CA). An increase in enzyme activity typically results in increased production of a fluorescent product that is trapped within the cell resulting in cells with more fluorescence which can be separated from less fluorescent cells, for example by FACS. Additionally many high throughput microplate screens exist for screening of protein libraries that exploit virtually any existing assay of enzymatic activity, see generally, Geddie, et al., Meth. Enzymol. 388 134-145 (2004).
5. Cell Growth Assays
The expression, or activity of a variety of proteins such as, for example, growth factors, enzymes, receptors and antibodies can influence the rate of growth of a host cell which be exploited either as an assay, or as a means of separating improved proteins.
Thus in one assay format, cells can be diluted to a limiting dilution and cells which grow more rapidly detected and selected. In one aspect such growth based assays can involve the ability to grow in the presence of a new substrate for which an improved enzymatic pathway of metabolism is required, for example a new carbon source. In another embodiment, growth assays can involve selection in the presence of a toxin, where a de-activation mechanism for the toxin is required. In another case, growth can be desired in response to the presence of a specific ligand, where high affinity binding of the ligand is required.
B. Selection and Enrichment Strategies
1. Flow Cytometry and FACS
Flow cytometry and the related flow sorting (also known as fluorescence activated cell sorting, or FACS) are methods by which individual cells can be quantitatively assayed for the presence of a specific component or component variant based upon staining with a fluorescent reporter. Flow cytometry provides quantitative, real time analysis of living cells, and can achieve efficient cell sorting rates of 50,000 cells/second, and is capable of selecting individual cells or defined populations. Many commercial FACS systems are available, for example BD Biosciences (CA), Cytopeia (Seattle, Wash.) Dako Cytomation (Australia).
A FACS can be equipped with a variety of lasers, which can produce a wide range of available wavelengths for multiple parameter analysis, and for use with different fluorophores. Classically the water cooled ion lasers using argon, krypton, or a mix of both can produce several specific lines; 408 nm, 568 nm, and 647 nm for example are major emission lines for Krypton; 488 nm, 457 nm, and others are argon lines. These lasers require high voltage multiphase power and cooling water, but can produce high power outputs. Additionally tunable and non tunable diode lasers exist, for example a 408 nm line can be stably created via a light emitting diode (LED) and this can be easily added to a sorter. Additionally dye lasers can be used to further extend the range of available wavelengths available for FACS analysis.
During FACS analysis, cells are stained with the specific reporter and then hydrodynamically focused into a single cell steam for interrogation with a laser which excites the fluorescent moiety. Fluorescent emission is detected through a wavelength restricted optical pathway and converted to numeric data correlated to an individual cell. In the case of flow sorting, predefined subsets of emission criteria can be met and the cells of interest diverted into a collection receptacle for further use by electrostatic repulsion or mechanical action (Herzenberg L A, Sweet R G, Herzenberg L A: Fluorescence activated cell sorting, Sci Amer 234(3):108, March 1976).
FACS based approaches are compatible with signal transduction based assays, activity based assays, and binding assays, and with a wide variety of proteins of interest, including for example, antibodies, receptors, enzymes and any surface displayed protein. FACS can be efficiently applied to most mammalian, yeast and bacterial cells, as well as fluorescently tagged beads.
In one embodiment, FACS can be used to screen a library of cells expressing surface displayed proteins (e.g., surface displayed antibodies) that are undergoing, or have undergone, SHM mediated diversity. In this approach, a cell surface displayed library is used and the displayed proteins are first incubated with fluorescently tagged antigen in solution. The FACS instrument is able to separate the high affinity protein members of the library, which have greater fluorescence intensity, from the lower affinity members. The use of optimized binding protocols in conjunction with FACS based selection has been shown to be capable of evolving antibodies with up to femtomolar affinities, See, e.g., Boder et al. PNAS, (2000) 97: 10701-10705; Boder et al., (2000) Meth. Enzymol. (2000) 328: 430-444; VanAntwerp et al., Biotechnol. Prog. (2000) 16: 31-37).
In order to effectively select and rapidly evolve, the antibodies and binding proteins which have high affinity to an antigen of interest, protocols can be established that can facilitate the isolation of antibodies with a broad range of affinities to the antigens of interest, and yet eliminate proteins that bind to labeling or coupling reagents. These protocols involve both a progression in the stringency of the cell population selected, and a decrease in the concentration and density of the target antigen presented to the cells.
With respect to the stringency or fraction of the total cell population collected during each round of selection, initial screens will generally use relatively low discrimination factors in order to capture as many proteins as possible that possess small incremental improvements in binding characteristics. For example, a typical initial sort may capture the top 10%, top 5% or top 2% of all cells that bind a target. Large improvements in affinity may be the result of combinations of mutations, each of which contribute small additive effects to overall affinity. (Hawkins et al., (1993) J. Mol. Biol. 234: 958-964). Therefore, recovery of all library clones with even marginally improved affinities (2-3 fold) is desirable during the early stages of library screening, and sorting gates can be optimized to recover as many clones as possible with minimum sacrifice in enrichment.
These selected cells can subsequently be allowed to recover and grown using standard culture conditions for a number of days until the population has reached a reasonable number to allow for a subsequent round of FACS sorting, analysis, mutagenesis, cell banking, or to determine sequence information. As discussed below, subsequent rounds of selection to identify higher affinity binders can be achieved by progressively decreasing the density and concentration of labeled binding peptide used in the preincubation steps prior to FACS analysis.
Following a successful first round of sorting, the collected cells can be re-grown to amplify the population and then resorted. At this, and subsequent stages of sorting, greater enrichments are possible since more copies of each desirable clone are present within the examined cell population. For example only about the top 1%, top 0.5%, top 0.2%, or top 0.1% of the cells in the population may be selected in order to identify significantly improved clones. With respect to establishing optimal binding and selection strategies, first generation hits, including germline antibodies, typically have low affinities and relatively rapid off rates. For example, Sagawa et al. (Mol. Immunology, 39: 801-808 (2003)) observed that the apparent affinity for germline Abs is typically in the range of 2×10⁴to 5×10⁶M⁻¹, but that this affinity increases to around 10⁹M⁻¹during affinity maturation (i.e., an effect that is mediated primarily by decreasing the off rate (K_off)).
The binding characteristics of weak binding antibodies may slow the screening of early generation, non-optimized libraries because specific, but low affinity binding antibodies typically have rapid off rates and tend therefore tend to be lost during wash steps. Loss of these specific binders may result in the isolation of antibodies that bind non-specifically to components used in the selection process (Cumbers et al., Nat. Biotechnol. 2002 November; 20(11): 1129-113).
To maximize the selection of proteins with relatively low affinities (i.e., having a Kd greater than about 500 nM), binding interactions are stabilized to prevent the dissociation of binding peptides during the screening process, and include appropriate blocking reagents to eliminate binding to coupling reagents and support matrices. To achieve this goal, initial screens should use fluorescently tagged beads loaded with a high density of antigens to exploit avidity effects, based on the use of multiple binding interactions to increase the binding strength of low affinity interactions, while also including pre-incubations with coupling and labeling reagents such as streptavidin, avidin, and naked beads etc., to eliminate non-specific binding (see generally, Aggarwal et al., (2006) Bioconjugate Chem. 17 335-340; Wrighton et al., (1996) Science 273 458-64; Terskikh et al. (1997) PNAS 94 1663-8; Cwirla et al., (1997) Science 276 1696-9; and Wang et al. (2004) J. Immunological methods 294 23-35).
By careful control of bead loading density, washing and pre-incubation conditions it has been demonstrated that even such low affinity binding interactions can be reproducibly monitored, (Werthen et al., (1993) BBA 326-332). Importantly these improvements to binding efficiency have been demonstrated to occur without any significant increase in non-specific reactivity (Giordano et al., (2001) Nat. Med. 7 1249-53). As discussed above, selections generally will also be based on using a relatively low stringency cut off during FACS to ensure that all of these weak binding library members are selected.
To further eliminate non-specific members of the library (i.e., those that bind to the beads, or coupling reagents, rather than the binding peptides), the resultant cell populations are screened directly with either polymeric binding peptide or intact polymeric antigen using distinct coupling reagents (e.g., via the use of biotinylated antigen coupled to streptavidin-fluorophore conjugate to form an antigen-streptavidin fluorescent complex). Coupling or labeling of the binding peptide to biotin or fluorophores can be achieved using standard, art-recognized protocols, as described herein and in the Examples.
Streptavidin binds biotin with femtomolar affinity and forms tetramers in physiological conditions, thereby generating a tetravalent complex when preincubated with singly biotinylated antigen (which is subsequently termed a streptavidin microaggregate as described below). Streptavidin pre-loading can increase the effective antigen concentration up to 500-fold, and is useful for isolating weak antigen binders that bind specifically to the antigen. Employment of streptavidin microaggregates is useful for isolating antibodies ranging in affinity from very weak to moderate (Kd greater than about 200 nM) affinities. Furthermore, biotinylated epitopes can be pre-reacted with streptavidin-fluorophore at room temperature for 10 to 15 minutes in order to create microaggregates prior to contacting cell populations. The microaggregates are subsequently allowed to contact cells simultaneously for 15 to 30 minutes prior to addition of secondary reagents, such as anti-human IgG-fluorophore conjugates. In one experimental approach, cells are centrifuged at 1500×g for 5 minutes and resuspended in a small volume (typically 500 μL to 1 mL) of DAPI (PBS, 1% BSA, 2 μg/mL DAPI). In a second approach termed “homogeneous assay conditions,” cells are resuspended directly in DAPI into which antigen-streptavidin microaggregate and goat-anti-human IgG-fluorophore are added. This second approach is particularly desirable for more weakly interacting antibodies (Kd greater than about 200 nM), where minimizing dissociation time may be more relevant.
At higher affinities (with Kd>10 nM, but less than about 100 nM), libraries are more easily screened directly for improved affinity by incubating the library with monomeric binding peptide or full length target protein under equilibrium binding conditions at a concentration of binding peptide that is ideally less than the Kd of the starting (wild type) interaction (apparent Kds can be readily determined by a series of analytical FACS experiments conducted with a range of antigen concentrations, ahead of a sort). Under these conditions, cells that possess antibodies and binding proteins with higher affinities will possess significantly more fluorescently labeled binding peptide than weaker binders, allowing the most fluorescent cells in the population to be easily selected for further optimization. Typically, FACS sorting gates can be established that select about the top 0.5% to about 0.1% of cells. In one non-limiting method, about the top 0.2% of cells are selected.
As recognized by Boder and Wittrup (Biotechnol. Prog. (1998) 14 55-62), the screening of very high affinity protein-ligand interactions (Kd<10 nM) can be accomplished by screening for decreased off-rate rather than directly for affinity. In this approach, cells are labeled to saturation with fluorescent binding peptide, followed by addition of an excess of non-fluorescent ligand. Cell associated fluorescence decays exponentially with time approaching a background level and the dissociation reaction is stopped after a fixed duration, usually by extensive dilution with cold buffer. The duration of the competition reaction determines the difference in observed fluorescence for different library clones and, thus, determines the range of kinetic improvements likely to be selected from the library. For a competitive dissociation reaction, the presence of excess non-fluorescent ligand can yield an effective forward reaction rate of zero. Mean fluorescence intensity at a given time after the initiation of the competition reaction is a function of the off-rate (K_off). (VanAntwerp & Wittrup (2000) Biotechnol. Prog. 16 31-37; Boder et al. (2000) PNAS 97 10701-10705; and Foote and Eisen (2000) PNAS 97 10679-10681). Cells in the population that express antibodies with improved affinities and more stable binding can be systematically identified by progressively increasing the length of time for the competition reaction, and then selecting the most fluorescent cells remaining in the population for further optimization.
Under these conditions, cells that possess surface displayed antibodies and binding proteins with higher affinities will exhibit significantly more bead or streptavidin-biotinylated antigen microaggregate binding compared to cells that express proteins with little or no binding. The most fluorescently labeled cells (displaying proteins with the highest affinity) can then be separated from the rest of the cells in the population using standard FACS sorting protocols, as described, for example, in Example 9.
Once a selected cell population has been created that expresses a protein that exhibits reproducible binding to a binding peptide, it can be characterized with two or more intact proteins to confirm that the antibodies or binding proteins exhibit the desired pattern of cross-reactivity and/or specificity (e.g., to both mouse and human variants of the protein of interest), or to two different members of a related gene family, but not to an unrelated, or more distantly related, protein.
In one embodiment, this can be accomplished using multi-parameter FACS using two or more proteins species labeled with two differently colored detectable tags (e.g., FITC and phycoerythrin) which can be simultaneously analyzed in a flow cytometer. Using this approach, it is possible to identify cells that display binding to only one protein, or are capable of binding to both proteins. The population of cells that exhibits the required dual specific binding can be selected by the FACS operator based upon the number of cells sorted and the percentage of cells identified that exhibit polyspecificity. As described previously, these selected cells can subsequently be allowed to recover and grown using standard culture conditions for a number of days until the population has reached a reasonable number to enable either a subsequent round of FACS sorting, analysis, cell banking, or to determine sequence information.
Selected binders from the library can be further characterized as described herein, and the sequence of the antibody or binding protein determined after PCR of cellular DNA, RT-PCR of RNA isolated from the selected cell population, or episome rescue.
Candidate antibodies and binding proteins can be iteratively subjected to rounds of hypermutation and selection in order to evolve populations of cells expressing antibodies or binding proteins with enhanced binding properties as described herein. Cells that preferentially and/or selectively bind to the binding peptide with a higher affinity are selected and allowed to expand. If needed, another round of mutagenesis is repeated and, again, cells that exhibit improved, selective, and high affinity binding, are retained for further propagation and growth. The new improved variants obtained can be further characterized as described herein, and the sequence of the heavy and light chains determined after RT-PCR or episome rescue.
Mutations that are identified in the first one, two or three rounds of hypermutation/selection can be recombined combinatorially into a set of new templates within the original parental backbone context, and all, or a subset of the resulting templates, can be subsequently transfected into cells which are then selected by FACS sorting. The best combination(s) of mutations are thus isolated and identified, and either used in a subsequent round of hypermutation/selection, or if the newly identified template(s) demonstrate sufficiently potent affinity, are used instead in experiments for further functional characterization.
In another embodiment, FACS can be used to screen a library of cells expressing intracellular proteins that are undergoing, or have undergone, SHM mediated diversity creation. In this approach, a membrane permeable fluorogenic, or florescent reagent is used and first pre-incubated with the library of cells to allow uptake and conversion of the reagent. The FACS instrument is able to separate the high activity protein members of the library, which are able to convert a greater percentage of the reagent and are more fluorescent than cells comprising lower activity members. (See, e.g., Farinas, Comb. Chem. High Throughput Screen. (2006) 9: (4) 321-328).
Fluorescent moieties to be detected include, but are not limited to, compounds such as fluorescein (commonly called FITC), phycobiliproteins such as phycoerythrin (PE) and allophycocyanin (APC) (Kronick, M. N. J. Imm. Meth. 92:1-13 (1986)), fluorescent semiconductor nanocrystals such as Quantum dot (QDot) bioconjugates for ultrasensitive nonisotopic detection (Chan W C, Nie S. Science 281: 2016-8 (1998)), and coumarin derivatives such as Fluorescent Acylating Agents derived from 7-Hydroxycoumarin.
Fluorescence can also reported from fluorescent proteins such as Teal Fluorescent Protein (TFP), from chemical stains of cellular components such as DAPI bound to DNA, from fluorescent moieties covalently conjugated to antibodies that recognize cellular products, from fluorescent moieties covalently conjugated to ligands of cellular receptors, and from fluorescent moieties covalently conjugated to substrates of cellular enzymes.
Cells stained with membrane impermeant reporters, such as antibodies, can be sorted for subsequent processing to recover components such as genes, episomes, or proteins of interest. Cells stained for surface expression components or stained with cell membrane permeant reporters can also be sorted intact for propagation.
2. Affinity Separation
Affinity separation based on the use microparticles enables the separation of surface displayed proteins based on affinity to a specific compound or sequence of interest. This approach is rapid, can easily be scaled up, and can be used iteratively with living cells.
Paramagnetic polystyrene microparticles are commercially available (Spherotech, Inc., Libertyville, Ill.; Invitrogen, Carlsbad, Calif.) that couple compounds or peptides to microparticle surfaces that have been modified with functional groups or coated with various antibodies or ligands such as, for example, avidin, streptavidin or biotin.
In one aspect paramagnetic beads can be used in which the paramagnetic property of microparticles allows them to be separated from solution using a magnet. The microparticles can be easily re-suspended when removed from the magnet thereby enabling the selective separation of cells that find to the attached probe.
In one embodiment, peptides can be coupled to paramagnetic polystyrene microparticles coated with a polyurethane layer in a tube. The hydroxyl groups on the microparticle surface are activated by reaction with p-toluensulphonyl chloride (Nilsson K and Mosbach K. “p-Toluenesulfonyl chloride as an activating agent of agarose for the preparation of immobilized affinity ligands and proteins.” Eur. J. Biochem. 1980:112: 397-402). The resulting sulphonyl ester can subsequently react covalently with peptide amino or sulfhydryl groups. The peptides are quickly absorbed onto the surface of the activated microparticles followed by the formation of covalent amine bonds with further incubation. The microparticles (2⁰⁹microparticles/milliliter) are washed two times by placing the tube containing 1 milliliter (ml) of microparticles on a magnet, allowing the microparticles to migrate to the magnet side of the tube, removing the supernatant, and re-suspending the microparticles in 1 ml of 100 millimolar (mM) borate buffer, pH 9.5. After washing, the microparticles are re-suspended in 100 mM borate buffer, pH 9.5 at a concentration of 1⁰⁹microparticles/ml. Eleven nanomoles of peptide are added to the microparticles and the microparticle/peptide mixture is vortexed for 1 minute to mix. The microparticles are incubated with peptides at room temperature for at least 48 hours with slow tilt rotation. To ensure an optimal orientation of the peptide on the microparticles, bovine serum albumin (BSA) is added to the microparticle/peptide mixture to a final concentration of 0.1% (weight/volume) after incubation has proceeded for 10 minutes. After incubation, the tube containing the microparticle/peptide mixture is placed on the magnet until the microparticles migrate to the magnet side of the tube. The supernatant is removed and the microparticles are washed four times with 1 ml phosphate buffered saline solution (PBS), pH 7.2 containing 1% (weight/volume) BSA. Finally, the microparticles are re-suspended in 1 ml PBS solution, pH 7.2 containing 1% (weight/volume) BSA.
Alternatively, paramagnetic polystyrene microparticles containing surface carboxylic acid can be activated with a carbodiimide followed by coupling to a peptide, resulting in a stable amide bond between a primary amino group of the peptide and the carboxylic acid groups on the surface of the microparticles (Nakajima N and Ikade Y, Mechanism of amide formation by carbodiimide for bioconjugation in aqueous media, Bioconjugate Chem. 1995, 6(1), 123-130; Gilles M A, Hudson A Q and Borders C L Jr, Stability of water-soluble carbodiimides in aqueous solution, Anal Biochem. 1990 Feb. 1; 184(2):244-248; Sehgal D and Vijay I K, a method for the high efficiency of water-soluble carbodiimide-mediated amidation, Anal Biochem. 1994 April; 218(1):87-91; Szajani B et al, Effects of carbodiimide structure on the immobilization of enzymes, Appl Biochem Biotechnol. 1991 August; 30(2):225-231). The microparticles (2⁹microparticles/milliliter) are washed twice with 1 ml of 25 mM 2-[N-morpholino]ethane sulfonic acid, pH 5 for 10 minutes with slow tilt rotation at room temperature. The washed microparticles are re-suspended in 700 microliters (μL) 25 mM 2-[N-morpholino]ethane sulfonic acid, pH 5 followed by the addition of 21 nanomoles of peptide re-suspended in 25 mM 2-[N-morpholino]ethane sulfonic acid, pH 5 to the microparticle solution. The microparticle/peptide mixture is mixed by vortexing and incubated with slow tilt rotation for 30 minutes at room temperature. After this first incubation, 300 μL of ice-cold 100 milligram (mg)/mL 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride re-suspended in 25 mM 2-[N-morpholino]ethane sulfonic acid, pH 5 is added to the peptide/microparticle mixture and incubated overnight at 4° Celsius with slow tilt rotation. The peptide-coupled microparticles are washed four times with 1 ml 50 mM Tris pH 7.4/0.1% BSA for 15 minutes at room temperature with slow tilt rotation. After washing, the peptide-coupled microparticles are re-suspended at a concentration of 1⁹microparticles/ml in PBS solution, pH 7.2 containing 1% (weight/volume) BSA.
Another option is to couple biotinylated peptides to paramagnetic polystyrene microparticles whose surfaces have been covalently linked with a monolayer of streptavidin. Briefly, one ml of the streptavidin microparticles are transferred to a microcentrifuge tube and washed four times by placing the tube on a magnet and allowing the microparticles to collect on the magnet side of the tube. The solution is then removed and the microparticles are gently re-suspended in 1 ml of PBS solution, pH 7.2 containing 1% (weight/volume) BSA. After the final wash, the microparticles are re-suspended in 1 ml of PBS solution, pH 7.2 containing 1% (weight/volume) BSA; and 33 picomoles of biotinylated peptide are added to the microparticle solution. The microparticle/peptide solution is incubated for 30 minutes at room temperature with slow tilt rotation. After coupling, the unbound biotinylated peptide is removed from the microparticles by washing four times with PBS solution, pH 7.2 containing 1% (weight/volume) BSA. After the final wash, the microparticle/peptide mixture is re-suspended to a final bead concentration of 1⁹microparticles/ml. (Argarana C E, Kuntz I D, Birken S, Axel R, Cantor C R. Molecular cloning and nucleotide sequence of the streptavidin gene. Nucleic Acids Res. 1986; 14(4):1871-82; Pahler A, Hendrickson W A, Gawinowicz Kolks M A, Aragana C E, Cantor C R. Characterization and crystallization of core streptavidin. J Biol Chem 1987:262(29):13933-7)
The identification, selection and use of specific peptide sequences for use in the present inventions is disclosed in commonly owned priority application No. 60/995,970 (Attorney docket no. 33547-708.101), filed Sep. 28, 2007.

XII. Databases

The invention includes methods of producing computer-readable databases comprising the sequence and identified mutations of certain proteins, including, but not limited to, sequences of binding domains, or active sites, as well as their binding characteristics, activity, stability characteristics and three-dimensional molecular structure. Specifically included in the present invention is the use of such a database to aid in the design and optimization of a protein of interest, based on a database of mutations created from the protein of interest, or related proteins or portions thereof.
In other embodiments, the databases of the present invention can comprise mutations of a protein or proteins that have been identified by screening to bind to a specific target, or other representations of such proteins such as, for example, a graphic representation or a name.
By “database” is meant a collection of retrievable data. The invention encompasses machine readable media embedded with or containing information regarding the amino acid and nucleic structure of a protein or proteins, such as, for example, its sequence, structure, and the activity or binding activity, as described herein. Such information can pertain to subunits, domains, and/or portions thereof such as, for example, portions comprising active sites, accessory binding sites, and/or binding pockets in either liganded (bound) or unliganded (unbound) forms.
Alternatively, the information can be that of identifiers which represent specific structures found in a protein. As used herein, “machine readable medium” refers to any medium that can be read and accessed directly by a computer or scanner. Such media can take many forms, including but not limited to, non-volatile, volatile and transmission media. Non-volatile media, i.e., media that can retain information in the absence of power, includes a ROM. Volatile media, i.e., media that cannot retain information in the absence of power, includes a main memory.
Transmission media includes coaxial cables, copper wire and fiber optics, including the wires that comprise the bus. Transmission media can also take the form of carrier waves; i.e., electromagnetic waves that can be modulated, as in frequency, amplitude or phase, to transmit information signals. Additionally, transmission media can take the form of acoustic or light waves, such as those generated during radio wave and infrared data communications.
Such media also include, but are not limited to: magnetic storage media, such as floppy discs, flexible discs, hard disc storage medium and magnetic tape; optical storage media such as optical discs or CD-ROM; electrical storage media such as RAM or ROM, PROM (i.e., programmable read only memory), EPROM (i.e., erasable programmable read only memory), including FLASH-EPROM, any other memory chip or cartridge, carrier waves, or any other medium from which a processor can retrieve information, and hybrids of these categories such as magnetic/optical storage media. Such media further include paper on which is recorded a representation of the amino acid or polynucleotide sequence, that can be read by a scanning device and converted into a format readily accessed by a computer or by any of the software programs described herein by, for example, optical character recognition (OCR) software. Such media also include physical media with patterns of holes, such as, for example, punch cards and paper tape.
Specifically included in the present invention is the transmission of data from the data base via transmission media to third party site to aid in the design and optimization of a protein of interest.
A variety of data storage structures are available for creating a computer readable medium having recorded thereon the amino acid or polynucleotide sequences of the invention or portions thereof and/or activity data. The choice of the data storage structure can be based on the means chosen to access the stored information. All format representations of the amino acid or polynucleotide sequences described herein, or portions thereof, are contemplated by the present invention. By providing computer readable medium having stored thereon the sequences of the invention, one can routinely access the SHM mediated changes in amino acid or polynucleotide sequence and related information for use in modeling and design programs, to create improved proteins.
A computer can be used to display the sequence of the protein or peptide structures, or portions thereof, such as, for example, portions comprising active sites, accessory binding sites, and/or binding pockets, in either liganded or unliganded form, of the present invention. The term “computer” includes, but is not limited to, mainframe computers, personal computers, portable laptop computers, and personal data assistants (“PDAs”) which can store data and independently run one or more applications, i.e., programs. The computer can include, for example, a machine readable storage medium of the present invention, a working memory for storing instructions for processing the machine-readable data encoded in the machine readable storage medium, a central processing unit operably coupled to the working memory and to the machine readable storage medium for processing the machine readable information, and a display operably coupled to the central processing unit for displaying the structure coordinates or the three-dimensional representation.
The computers of the present invention can also include, for example, a central processing unit, a working memory which can be, for example, random-access memory (RAM) or “core memory,” mass storage memory (for example, one or more disk drives or CD-ROM drives), one or more cathode-ray tube (“CRT”) display terminals or one or more LCD displays, one or more keyboards, one or more input lines, and one or more output lines, all of which are interconnected by a conventional bi-directional system bus. Machine-readable data of the present invention can be inputted and/or outputted through a modem or modems connected by a telephone line or a dedicated data line (either of which can include, for example, wireless modes of communication). The input hardware can also (or instead) comprise CD-ROM drives or disk drives. Other examples of input devices are a keyboard, a mouse, a trackball, a finger pad, or cursor direction keys. Output hardware can also be implemented by conventional devices. For example, output hardware can include a CRT, or any other display terminal, a printer, or a disk drive. The CPU coordinates the use of the various input and output devices, coordinates data accesses from mass storage and accesses to and from working memory, and determines the order of data processing steps. The computer can use various software programs to process the data of the present invention. Examples of many of these types of software are discussed throughout the present application.

EXAMPLES

Elements of the present application are illustrated by the following examples, which should not be construed as limiting in any way.

Example 1

Creation of Synthetic Polynucleotides Encoding Blasticidin

By decreasing the likelihood of somatic hypermutation in a vector element, such as a selectable marker, an enzyme involved in SHM, or a reporter gene, the vector and system for exerting and tracking SHM becomes more stable, thereby enabling somatic hypermutation to be more effectively targeted to a polynucleotide of interest.
A. Polynucleotide Design
In general, sequences are engineered for SHM using the teaching described herein, and as elaborated in sections III and IV. In the following examples, the sequence optimization is based on the hot spot and cold spot motifs listed in Table 7, and using the computer program SHMredesign.pl as described above.
Using this program, every position within the sequence is annotated with either a ‘+’, ‘−’, or ‘.’ symbol to designate whether it is desired to obtain a hotter, colder, or neutral changes in SHM susceptibility at that specific position. Where ‘+’ designates a hot spot, ‘−’ cold spot, and ‘.’ a neutral position. For example, the following input sequence for blasticidin is used to identify SHM resistant versions at every position of the blasticidin gene.

(SEQ ID NO: 26)

>ATGGCCAAGCCTTTGTCTCAAGAAGAATCCACCCTCATTGAAAGAGCAACGGCTACAATCAACAGCATCCCCAT

CTCTGAAGACTACAGCGTCGCCAGCGCAGCTCTCTCTAGCGACGGCCGCATCTTCACTGGTGTCAATGTATATCA

TTTTACTGGGGGACCTTGCGCAGAACTCGTGGTGCTGGGCACTGCTGCTGCTGCGGCAGCTGGCAACCTGACTTG

TATCGTCGCGATCGGAAATGAGAACAGGGGCATCTTGAGCCCCTGCGGACGGTGCCGACAGGTTCTTCTCGATCT

GCATCCTGGGATCAAAGCCATAGTGAAGGACAGTGATGGACAGCCGACGGCAGTTGGGATTCGTGAATTGCTGCC

CTCTGGTTATGTGTGGGAGGGCTAA

<−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−

−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−

−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−

−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−

−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−

−−−−−−−−−−−−−−−−−−−−−−−−−

By comparison, the following input file, is used to identify hotter versions of the blasticidin gene that are more susceptible to SHM at every position of the gene.

(SEQ ID NO: 26)

>ATGGCCAAGCCTTTGTCTCAAGAAGAATCCACCCTCATTGAAAGAGCAACGGCTACAATCAACAGCATCCCCAT

CTCTGAAGACTACAGCGTCGCCAGCGCAGCTCTCTCTAGCGACGGCCGCATCTTCACTGGTGTCAATGTATATCA

TTTTACTGGGGGACCTTGCGCAGAACTCGTGGTGCTGGGCACTGCTGCTGCTGCGGCAGCTGGCAACCTGACTTG

TATCGTCGCGATCGGAAATGAGAACAGGGGCATCTTGAGCCCCTGCGGACGGTGCCGACAGGTTCTTCTCGATCT

GCATCCTGGGATCAAAGCCATAGTGAAGGACAGTGATGGACAGCCGACGGCAGTTGGGATTCGTGAATTGCTGCC

CTCTGGTTATGTGTGGGAGGGCTAA

<++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++

+++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++

+++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++

+++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++

+++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++

+++++++++++++++++++++++++

As described previously, during this process, all nucleotide sequences over a 9 base region consistent with the wild type protein's amino acid sequence are enumerated and scored for the number of hot spot motifs, cold spot motifs, CpG motifs, codon usage, and stretches of the same nucleotide. The program then determines whether it is possible to replace any random sequence with either a hotter, colder, or neutral polynucleotide tile.
As shown in FIG. 7, this approach, as applied to canine AID quickly, (within a few hundred tile substitutions), converges to identify a cold optimized canine AID new sequence, which differs from the original sequence through the substitution 15-20% of the nucleotide sequence. The majority of changes occur early in the iterative cycle and are usually complete after about 500 iterations. As one might expect, larger genes require a larger number of iterations to reach a fully optimized sequence. Routinely the use of 2000 to 3000 iterations is more than sufficient for the majority of genes.
Analysis of a number of unmodified genes at random demonstrates that most mammalian genes use codons that create on average about 9 to 15 cold spots per 100 nucleotides, and with a median density of about 13.8 cold spots/100 nucleotides, and have a hot spot density of between about 7 to 13 hot spots per 100 nucleotides, with a median density of about 8.3 hot spots per 100 nucleotides.
The initial starting sequence, as well as the frequency of hot spots, cold spots and CpGs for the unmodified, blasticidin gene are shown in FIG. 8.
1. Cold Blasticidin
An optimized sequence for a SHM resistant (cold) version of blasticidin created using this approach is shown in FIG. 9, together with the resulting changes in frequency of hot spots and cold spots. Optimization of the blasticidin sequence to make the sequence more resistant to somatic hypermutation resulted in an increase of 188% in number of cold spots (an increase of 73), and reduced the number of hot spots by 57% (a decrease of 15). Overall the frequency of cold spots increased to an average density of about 28 cold spots per 100 nucleotides from an initial density of about 15 cold spots per 100 nucleotides, and the overall frequency of hot spots decreased from about 9 hot spots per 100 nucleotides, in the unmodified gene to about 5 hot spots per 100 nucleotides in the SHM resistant form.
2. Hot Blasticidin
An optimized sequence for a SHM susceptible version of blasticidin created using this approach is shown in FIG. 10, together with the resulting changes in frequency of hot spots and cold spots. Optimization of the blasticidin sequence to make the sequence more susceptible to somatic hypermutation resulted in an increase of about 197% in number of hot spots (an increase of 34), and reduced the number of cold spots by about 56% (a decrease of 26). Overall the frequency of hot spots increased to an average density of about 17 hot spots per 100 nucleotides from an initial density of about 9 hot spots per 100 nucleotides, and the overall frequency of cold spots decreased from about 15 cold spots per 100 nucleotides in the unmodified gene to about 9 cold spots per 100 nucleotides in the SHM susceptible form.
B. Cloning and Analysis
After final review to ensure that the synthetic polynucleotide sequence is free of extraneous restriction sites, the complete polynucleotide sequence is synthesized (DNA 2.0, Menlo Park, Calif.), cloned into one of DNA2.0's cloning vectors (see Table 11 below), and sequenced to confirm correct synthesis.

TABLE 11

	DNA2.0 source	restriction sites	Vector that insert
Construct	plasmid	(5′, 3′)	is cloned into

cold TFP	pJ15	Sac1, BsrG1	AB136
hot TFP	pJ15	Sac1, BsrG1	AB102
GFP* stop	pJ31	Sac1, BsrG1	AB105
(Y82stop)
cold hygromycin	pJ2	NgoMIV, Xba1	AB179, AB163
unmodified	pJ51	NgoMIV, Xba1	AB150, AB161
puromycin
cold blasticidin	pJ13	NgoMIV, Xba1	AB102, AB153
cold AID	pJ45	Sac1, BsrG1	AB135, AB174
Human AID
Kappa enhancers	PCR
	amplification
	of genomic DNA

Other elements, for example E-box motifs or Ig enhancer elements are created by either oligo synthesis or PCR amplification as described in Example 5 below.
To test functionality of the new synthetic inserts, coding regions are excised from DNA2.0 source vectors using restriction enzymes as listed in Table 11 above, and inserted into expression vectors (Table 11) using standard recombinant molecular biological techniques. Insertion of selection markers (i.e., cold blasticidin, cold hygromycin, and unmodified puromycin) into the AB series of vectors places them down stream of the EMCV IRES sequence (AB150, AB102, AB179; see FIG. 33A) or downstream of the pSV promoter (AB161, AB153, AB163; see FIG. 33B).
To test functional activity of the optimized synthetic genes, Hek 293 cells are plated at 4×10⁵/well, in 6-well microtiter dish. After 24 hours, transfections are performed using Fugene6 reagent from Roche Applied Sciences (Indianapolis, Ind.) at a reagent-to-DNA ratio of 3 μL:1 μg DNA per well. This ratio is also maintained for transfections with multiple plasmids. Transfections are carried out in accordance with manufacturer's protocol.
To determine the relative stability/susceptibility of each construct to somatic hypermutation, stable cell lines of each transfected cell population are created, and tested to determine the relative speed by which they accumulate SHM mediated mutations. Because the majority of these mutations result in a loss of function, relative mutagenesis load are conveniently measured as a loss of fluorescence via FACS (see below and Example 4).
FACS Analysis. Prior to FACS analysis, cells are harvested by trypsinization, washed twice in PBS containing 1% w/v BSA, and re-suspended in 200 μl PBS/1% BSA containing 2 ng/ml DAPI. Cells are analyzed in the Cytopeia Influx with 200 mW 488 nm and 50 mW 403 nm laser excitation. Up to one million cells per sample are acquired. DAPI fluorescence is measured through a 460/50 bandpass filter. GFP fluorescence is measured through a 528/38 bandpass filter. Percent GFP expression is reported as percentage of DAPI excluding live cells with no detectable GFP fluorescence above cellular background.
Reversion assays to test for function of the canine AID gene. GFP* (GFP with a stop codon introduced by site directed mutagenesis at position 82 [Y82stop]) is co-transfected with AB174 (cold canine AID), and cells are analyzed by flow cytometry 3 days post transfection, placed under antibiotic selection and analyzed further by flow cytometry every other day for 13-15 days.
Antibiotic selections. Antibiotic concentrations used in the selection of Hek 293 cells are determined empirically by performing a kill curve (i.e., determining the minimal concentration of antibiotic that kills all un-transfected—and thus antibiotic sensitive—cells). At 3 days post transfection, cells are plated at 4×10⁵/well and selected at the following concentrations: 1.5 μg/ml puromycin (Clontech, Mountain View, Calif.); 16 μg/mL blasticidin (Invitrogen, Carlsbad, Calif.); and 360 μg/mL hygromycin (Invitrogen, Carlsbad, Calif.).
Resistance marker genes are tested to determine functionality by transfection of the appropriate expression plasmid (i.e. AB102 for blasticidin, AB179 for hygromycin) in Hek 293 cells based on their ability to promote drug resistance cell growth in the presence of 16 μg/mL blasticidin (Invitrogen, Carlsbad, Calif.); and 360 μg/mL hygromycin (Invitrogen, Carlsbad, Calif.) for two weeks.
Transfection of the AB 102 containing cold blasticidin resulted in the creation of drug resistant colonies of transfected hek 293 cells at comparable rates as the wild type gene.

Example 2

Creation of Synthetic Polynucleotides Encoding Hygromycin

A. Polynucleotide Design
The starting sequence for unmodified hygromycin is shown in FIG. 11, together with the initial analysis of hot spot and cold spot frequency.
As described for Example 1, sequence optimization is completed using the computer program SHMredesign, based on the hot spot and cold spot motifs listed in Table 7.
1. Cold Hygromycin
From iteration 1 to iteration 2000, an additional 71 cold spots are inserted into the gene, 12 existing hot spots are removed, and 61 CpG sites are removed making the gene sequence less susceptible to somatic hypermutation. No further beneficial changes are observed upon further iterations.
An optimized sequence for a SHM resistant version of hygromycin created using this approach is shown in FIG. 12, together with the resulting changes in frequency of hot spots and cold spots. Optimization of the hygromycin sequence to make the sequence more resistant to somatic hypermutation resulted in an increase of 144% in number of cold spots (an increase of 71), and reduced the number of hot spots by about 17% (a decrease of 12). Overall the frequency of cold spots increased to an average density of about 22 cold spots per 100 nucleotides from an initial density of about 15 cold spots per 100 nucleotides, and the overall frequency of hot spots decreased from about 7 hot spots per 100 nucleotides, in the unmodified gene to about 5 hot spots per 100 nucleotides in the SHM resistant form.
Transfection of the AB179 containing cold hygromycin resulted in the creation of drug resistant colonies of transfected Hek 293 cells at comparable rates as the wild type gene.
2. Hot Hygromycin
Conversely, by increasing the probability of somatic hypermutation in a vector element such as a selectable marker, one is able to “reclaim” a marker for future use.
In the case of the hot hygromycin construct, the designed nucleotide sequence is maximized for hot spots, minimized for cold spots, minimized for CpG repeats and is rendered consistent with known mammalian optimized codon usage.
Optimization of the hygromycin sequence to make the sequence more susceptible to somatic hypermutation resulted in an increase of about 183% in number of hot spots (an increase of 61), and reduced the number of cold spots by about 42% (a decrease of 35) (FIG. 13). Overall the frequency of hot spots increased to an average density of about 13 hot spots per 100 nucleotides from an initial density of about 7 hot spots per 100 nucleotides, and the overall frequency of cold spots decreased from about 15 cold spots per 100 nucleotides in the unmodified gene to about 12 cold spots per 100 nucleotides in the SHM susceptible form.
B. Cloning and Analysis
After final review to ensure that a synthetic polynucleotide sequence is free of extraneous restriction sites, the complete polynucleotide sequence is synthesized (DNA 2.0, Menlo Park, Calif.), cloned into one of DNA2.0's cloning vectors (see Table 11; Example 1), sequenced to confirm correct synthesis and tested for activity as described above for Example 1.
To determine the relative stability/susceptibility of each of the constructs, to somatic hypermutation, stable cell lines of each transfected cell population re created, and tested to determine the relative speed by which they accumulate SHM mediated mutations. Because the majority of these mutations result in a loss of function, relative mutagenesis load are conveniently measured as a loss of fluorescence via FACS (see Example 4).

Example 3

Creation of Synthetic Polynucleotides Encoding Reporter Genes
A. Polynucleotide Design
The starting sequence for unmodified Teal Fluorescent Protein (TFP) is shown in FIG. 14, together with the initial analysis of hot spot and cold spot frequency.
1. Hot TFP
As described for Example 1, sequence optimization is completed using the computer program SHMredesign, based on the hot spot and cold spot motifs listed in Table 7; the resulting hot and cold versions of TFP are shown in FIGS. 15 and 16, respectively.
Optimization of the TFP sequence to make the sequence more susceptible to somatic hypermutation resulted in an increase of about 170% in number of hot spots (an increase of 28), and reduced the number of cold spots by about 26% (a decrease of 27). Overall the frequency of hot spots increased to an average density of about 10 hot spots per 100 nucleotides from an initial density of about 6 hot spots per 100 nucleotides, and the overall frequency of cold spots decreased from about 15 cold spots per 100 nucleotides in the unmodified gene to about 11 cold spots per 100 nucleotides in the SHM susceptible form.
2. Cold TFP
Optimization of the TFP sequence to make the sequence more resistant to somatic hypermutation resulted in an increase of 120% in number of cold spots (an increase of 21), and reduced the number of hot spots by about 10% (a decrease of 4). Overall the frequency of cold spots increased to an average density of about 18 cold spots per 100 nucleotides from an initial density of about 15 cold spots per 100 nucleotides, and the overall frequency of hot spots decreased from about 6 hot spots per 100 nucleotides, in the unmodified gene to about 5 hot spots per 100 nucleotides in the SHM resistant form.
B. Cloning and Analysis
After final review to ensure that the synthetic polynucleotide sequence is free of extraneous restriction sites, the complete polynucleotide sequence is synthesized (DNA 2.0, Menlo Park, Calif.), cloned into one of DNA2.0's cloning vectors (see Table 11; Example 1), sequenced to confirm correct synthesis and tested for activity as described below.
Hek 293 cells are transfected with the expression vectors (AB102 and 136 as described above in Example 1) containing either hot or cold versions of TFP driven for expression by an identical CMV promoter. Selection for stable expression began 3 days post transfection. Prior to FACS analysis, cells are harvested by trypsinization, ished twice in PBS containing 1% w/v BSA, and re-suspended in 2000 PBS/1% BSA containing 2 ng/ml DAPI. Cells are analyzed in the Cytopeia Influx with 200 mW 488 nm and 50 mW 403 nm laser excitation. Up to one million cells per sample are acquired. DAPI fluorescence is measured through a 460/50 bandpass filter. GFP fluorescence is measured through a 528/38 bandpass filter. Percent GFP expression is reported in Table 12A as percentage of DAPI excluding live cells with no detectable GFP fluorescence above cellular background.

TABLE 12A

Expression analysis of “hot” and “cold” versions of TFP

	% TFP			Fold
	Expressing	TFP	Control	over
Construct	cells	Fluorescence	Fluorescence	control

Hot TFP	63.74	189.33	20.61	9
(SHM susceptible)
Cold TFP	66.92	429.72	19.93	22
(SHM resistant)
Hot TFP	48.39	183.21	20.09	9
(SHM susceptible)
Cold TFP	51.20	656.06	20.26	32
(SHM resistant)

These results show good expression above background of both hot and cold versions of TFP. In this case, making the sequence “cold” produced the surprising result that relative expression of the protein is improved. Such improved expression provides an additional benefit to the SHM resistant synthetic genes.
To determine the relative stability/susceptibility of each construct to somatic hypermutation, stable cell lines of each transfected cell population are created, and tested to determine the relative speed by which they accumulate SHM mediated mutations. Because the majority of these mutations result in a loss of function, relative mutagenesis load are conveniently measured as a loss of fluorescence via FACS (see Example 4).
Episomal expression constructs carrying either a SHM optimized coding sequence for hot TFP or cold TFP were individually stably co-transfected with AID into HEK 293 cells and allowed to expand and grow for 3 weeks (the cold canine AID used in these experiments contains the NES-inactivating L198A mutation; SEQ ID NO: 22). Cell stocks were then frozen, and one vial each of hot TFP and cold TPF were thawed, grown in culture for 4 days, and then pulsed with supplemental AID by transiently transfecting the 4 day post-thaw culturing with an additional aliquot of the original AID expression construct (termed “AID pulsing”). Cells were harvested by trypsinization nine days following the AID pulse, pelleted at 1150×g for 5 min., and frozen for later use.
Cell pellets were subsequently thawed and TFP ORFs were recovered by PCR using oligonucleotide (oligo) primers GTGGGAGGTCTATATAAGCAGAGC (SEQ ID NO: 339) and GATCGTCTCACGCGGATTGTAC (SEQ ID NO: 377). The former oligo amplifies from near the 3′ end of the CMV promoter used for driving expression of TFP mRNA, which lies 142 nt 5′ to the TFP start codon, and the latter oligo matches sequences ending 1 nt 3′ to the TFP stop codon.
Each PCR reaction (total volume of 50 μL) was run 35 cycles under the following conditions: 95° C. for 5 min, 35 cycles of (95° C. for 30 sec, 55° C. for 30 sec, 68° C. for 45 sec), followed by 1 min at 68° C. before cooling to 4° C. PCR amplified products were cloned into the TOPO® TA cloning vector (Invitrogen, Carlsbad, Calif.), and inserts were sequenced. A total of 166 hot and 111 cold TFP ORFs were rescued, sequenced and compared the resulting spectrum of mutations. Global statistics for the mutations observed in the two sets of sequences are shown in Table 12B.

TABLE 12B

Mutation metrics for cold- and hot-TFP

	# ORFs	#	total # nt	kb per	templates per
template	sequenced	mutations	sequenced	mutation	mutation

coldTFP

111

	18	61050	3391	6.1
hotTFP	166	100	88500	885	1.6

The mutation frequency is approximately 3.8-fold greater in the TFP template version with maximized hotspots vs. the cold TFP sequence with minimized hotspots. The data demonstrates that SHM optimization of polynucleotide sequences can be used to either increase or decrease the frequency of mutations experienced by a polynucleotide encoding a protein of interest.
FIG. 16D shows the mutations for a representative segment of the hot and cold TFP constructs. The central row shows the amino acid sequence of TFP (residues 59 thru 87) in single letter format, and the “hot” and “cold” starting nucleic acid sequences encoding the two constructs are shown above (hot) and below (cold) the amino acid sequence. Mutations observed in the hot sequence are aligned and stacked top of the gene sequences, while mutations in the cold TFP sequence are shown below. The results illustrate how “silent” changes to the coding sequences generate dramatic changes in observed AID-mediated SHM rates, demonstrating that engineered sequences can be effectively optimized to create fast or slow rates of SHM.
FIG. 16E shows that the spectrum of mutations generated by AID in the present in vitro tissue culture system mirror those observed in other studies and those seen during in vivo affinity maturation. FIG. 16E shows the mutations generated in the present study (Box (i) upper left, n=118), and compares them with mutations observed by Zan et al. (box (ii) upper right, n=702), Wilson et al. (lower left, n=25000; box (iii)), and a larger analysis of IGHV chains that have undergone affinity maturation (lower right, n=101,926; box (iv)). The Y-axis in each chart indicates the starting nucleotide, the X-axis indicates the end nucleotide, and the number in each square indicates the percentage (%) of time that nucleotide transition is observed. In the present study, the frequency of mutation transitions and transversions was similar to those seen in other data sets. Mutations of C to T and G to A are the direct result of AID activity on cytidines and account for 48% of all mutation events. In addition, mutations at bases A and T account for ˜30% of mutation events (i.e., slightly less than frequencies observed in other datasets).
FIG. 16F shows that mutation events are distributed throughout the SHM optimized nucleotide sequence of the hot TFP gene, with a maximum instantaneous rate of about 0.08 events per 1000 nucleotides per generation centered around 300 nucleotides from the beginning of the open reading frame. Stable transfection and selection of a gene with AID (for 30 days) produces a maximum rate of mutation of 1 event per 480 nucleotides. As a result, genes may contain zero, one, two or more mutations per gene. The distribution of SHM-mediated events observed in hot TFP sequenced genes can be seen in FIG. 16G, compared to the significantly reduced pattern of mutations seen in cold TFP (FIG. 16H).
Thus the present study demonstrates that the creation of non-synonymous versions of genes such as Teal-fluorescent protein (TFP) that do not normally undergo somatic hypermutation can be used to target such genes for high rates of somatic hypermutation. Additionally, the creation of SHM resistant genes (while encoding for the same amino acids) can lead to proteins that have a reduced number of somatic hypermutation hot-spots and, thus, experience a dramatically reduced level of AID mediated hypermutation. In each instance of SHM optimization, mammalian codon usage and other factors effecting gene expression levels were considered in generating the engineered sequences, leading to proteins that also exhibit reasonable levels of translation and expression. The results, therefore, demonstrate that the present methods of SHM optimization (i) can be successfully used to target the activity of AID to specific regions of an expressed gene; (ii) can be used to speed or slow the rate of SHM, (iii) demonstrate that the spectrum of mutations generated by AID using this methodology is equivalent to that observed in vivo; (iv) and demonstrate that SHM optimization can be successfully performed on a gene of interest to either positively or negatively impact its rate of AID-mediated SHM without significantly negatively impacting its expression.

Example 4

Creation of Synthetic Polynucleotides Encoding Enzymes Involved in SHM

The systems and methods described herein can be applied to enzymes such as, for example, AID, Pol eta, Pol theta and UDG.
A. Activation Induced Cytidine Deaminase (AID)
Analysis of sequence variations in cytidine deaminase (AID) between mammalian species (e.g., rat, chimpanzee, mouse, human, dog, cow, rabbit, chicken, frog, zebra fish, fugu and tetraodon (puffer fish)) as compared to humans demonstrates that organisms as distantly related as human and frog display a surprisingly high (70%) sequence identity, and >80% sequence similarity. In addition, it has been shown that AID from other organisms can be substituted for human AID in somatic hypermutation (SHM), and that all mammalian species of AID are functionally equivalent.
Shown in FIG. 17 is a comparison of human AID with other terrestrial AIDs in order to identify a promising beginning construct for SHM in vivo. The figure provides a sequence alignment of AID from human (H_sap/1-198), mouse (M_musc/1-198), canine (C_fam/1-198), rat (R_norv/1-199), and chimpanzee (P_trog/1-199). FIG. 21 illustrates the sequence identity between human, canine and mouse AID proteins.
Canine AID has overall 94% amino acid identity to human and mouse AID and, thus, is selected as the starting point for codon optimization. To optimize codon usage, the canine amino acid sequences are reverse translated and then iteratively optimized.
AID is known to contain a nuclear export signal, which is contained within the C-terminal 10 amino acids (McBride et al., J Exp Med. 2004 Can 3; 199(9):1235-44; Ito et al., PNAS 2004 Feb. 17; 101(7):1975-80). For purposes of the experiments described below, the canine AID contains a leucine to alanine mutation at position 198, while the human AID construct retains the unmutated, intact nuclear export signal.
As described in Example 1, SHM sequence optimization is completed using the computer program SHMredesign, based on the hot spot and cold spot motifs listed in Table 7; the resulting hot and cold versions of canine AID are shown in FIGS. 19 and 20, respectively. The starting sequence for canine AID is shown in FIG. 18, together with the initial analysis of hot spot and cold spot frequency.
1. Hot AID
Optimization of the AID sequence to make the sequence more susceptible to somatic hypermutation resulted in an increase of about 200% in number of hot spots (an increase of 43), and reduced the number of cold spots by about 30% (a decrease of 23). Overall the frequency of hot spots increased to an average density of about 14 hot spots per 100 nucleotides from an initial density of about 7 hot spots per 100 nucleotides, and the overall frequency of cold spots decreased from about 13 cold spots per 100 nucleotides in the unmodified gene to about 9 cold spots per 100 nucleotides in the SHM susceptible form.
2. Cold AID
Optimization of the canine AID sequence to make the sequence more resistant to somatic hypermutation resulted in an increase of 186% in number of cold spots (an increase of 68), and reduced the number of hot spots by about 35% (a decrease of 14). Overall the frequency of cold spots increased to an average density of about 25 cold spots per 100 nucleotides from an initial density of about 13 cold spots per 100 nucleotides, and the overall frequency of hot spots decreased from about 7 hot spots per 100 nucleotides, in the unmodified gene to about 5 hot spots per 100 nucleotides in the SHM resistant form.
After final review to ensure that the synthetic polynucleotide sequence is free of extraneous restriction sites, the complete polynucleotide sequence is synthesized (DNA 2.0, Menlo Park, Calif.), cloned into one of DNA2.0's cloning vectors (see Table 11; Example 1), sequenced to confirm correct synthesis and tested for activity as described below and in Example 1.
To determine canine AID activity, the cold or wild type versions of canine AID are co-transfected with expression vectors expressing the GFP* construct that contains a stop codon within it's coding region (as described in Example 1) either in the presence or absence of Ig enhancer elements within the target vector sequence. Mutation of the stop codon by AID results in the creation of a functional fluorescent protein that is a direct indicator of AID activity.
In this experiment, cells are harvested by trypsinization, washed twice in PBS containing 1% w/v BSA, and re-suspended in 200 μl PBS/1% BSA containing 2 ng/ml DAPI. Cells are analyzed in the Cytopeia Influx with 200 mW 488 nm and 50 mW 403 nm laser excitation. Up to one million cells per sample are acquired and revertants are determined as percentage of DAPI excluding live cells with detectable GFP fluorescence above cellular background.
FIG. 22A shows the predicted effect of AID activity on protein function, in this type of assay. Of note is the observation that mutagenesis can produce mutations that both initially restore or improve function and later reduce or eliminate function. The balance in these two rates generates early and rare mutation events that restore function, followed by secondary and ternary mutation events that destroy function in these proteins. The net effect of these competing rates on the observation of gain-of-function events in a population can be seen in FIG. 22B. Given three different assumptions regarding number of inactivating mutations needed to silence GFP, one would expect to observe three very different profiles of reversion events as a function of time, dependent on the rate of enzymatic activity of the AID.
Thus although initial reversion rates can provide an accurate assessment of AID activity, long term studies of activity require an analysis of the rate of extinction of activity, rather than reversion of fluorescence.
To test this possibility, a cell line that is stably expressing a fluorescent protein is transfected with 2 concentrations of expression vector containing cold canine AID. Cells are stably maintained in culture and sample assayed for total fluorescence after the indicated periods of time.
Prior to FACS analysis, cells are harvested by trypsinization, washed twice in PBS containing 1% w/v BSA, and re-suspended in 2000 PBS/1% BSA containing 2 ng/ml DAPI. Cells are analyzed in the Cytopeia Influx with 200 mW 488 nm and 50 mW 403 nm laser excitation. DAPI fluorescence is measured through a 460/50 bandpass filter. GFP fluorescence is measured through a 528/38 bandpass filter. Percent GFP expression is reported as percentage of DAPI excluding live cells with no detectable GFP fluorescence above cellular background.
The results, shown in FIG. 22C, show a steady and sustained progressive, dose dependent decrease in GFP expression (shown as increasing GFP extinction) with time when co-expressed with increasing amounts of cold AID. The data are consistent with the hypothesis that cold AID is able to introduce multiple mutations into a target gene, and is both functional and stable when expressed in a “cold form” for many days.
To directly compare the ability of cold canine AID to exert mutagenesis, initial reversion assays are set up comparing cold canine AID with wild type human AID. Hek 293 cells are transfected with the expression vectors (as described above in Example 1) containing either the GFP* as described above, or GFP* with the Kappa E3 and intronic enhances inserted 5′ to the CMV promoter, together with either human or cold canine AID. Selection for stable expression began 3 days post transfection. Prior to FACS analysis, cells are harvested by trypsinization, washed twice in PBS containing 1% w/v BSA, and re-suspended in 2000 PBS/1% BSA containing 2 ng/ml DAPI. Cells are analyzed in the Cytopeia Influx with 200 mW 488 nm and 50 mW 403 nm laser excitation. Up to one million cells per sample are acquired. DAPI fluorescence is measured through a 460/50 bandpass filter. GFP fluorescence is measured through a 528/38 bandpass filter. Percent GFP expression is reported as percentage of DAPI excluding live cells with no detectable GFP fluorescence above cellular background.
The results show (FIG. 22C) that canine AID exhibited significantly enhanced reversion activity compared to human AID. Also in this experiment is shown the effect of the kappa 3′E and intronic enhancers on the rate of reversion experienced by the target gene when these are included in the expression vector. As shown inclusion of the enhancer elements further enhanced reversion frequency.
B. Pol eta
The starting unmodified sequence for Pol eta is shown in FIG. 23, together with the initial analysis of hot spot and cold spot frequency.
As described above, sequence optimization is completed using the computer program SHMredesign, based on the hot spot and cold spot motifs listed in Table 7; the resulting cold and hot versions of Pol eta are shown in FIGS. 24 and 25, respectively. Changes in hot and cold spot density are summarized below in Table 13:

TABLE 13

Summary of hot spot and cold spot changes

				Cold Spot
Gene	No. Hot Spots	No. Cold Spots	Hot Spot Density	Density

Native	177	307	8	9
Cold	98	606	5	28
Hot	359	185	17	8

In Table 13, “Hot Spot Density” or “Cold Spot Density,” refers to the total number of hot or cold spot motifs in the ORF, divided by the number of nucleotides in the ORF, multiplied by 100, rounded to the nearest whole number.
C. Pol theta
The starting amino acid and nucleic acid sequences for Pol theta are shown in FIG. 26 and in FIG. 27, respectively, together with the initial analysis of hot spot and cold spot frequency.
As described above, sequence optimization is completed using the computer program SHMredesign, based on the hot spot and cold spot motifs listed in Table 7; the resulting cold and hot versions of Pol theta are shown in FIG. 28 and in FIG. 29, respectively. Changes in hot and cold spot density (per 100 nucleotides) are summarized below in Table 14:

TABLE 14

Summary of hot spot and cold spot changes

				Cold
Gene	Hot Spots #	Cold Spots #	Hot Spot Density	Spot Density

Native	597	1054	8	14
Cold	349	1847	4	24
Hot	1053	700	13	9

In Table 14, “Hot Spot Density” or “Cold Spot Density” refers to the total number of hot or cold spots in the ORF, divided by the number of nucleotides in the ORF, multiplied by 100, rounded to the nearest whole number.
D. UDG
The starting amino acid and nucleic acid sequences for UDG are shown in FIGS. 30A and 30B, respectively, together with the initial analysis of hot spot and cold spot frequency (FIG. 30C).
As described above, sequence optimization is completed using the computer program SHMredesign, based on the hot spot and cold spot motifs listed in Table 7; the resulting cold and hot versions of UDG are shown in FIGS. 31A and 31C, respectively, together with the initial analysis of hot spot and cold spot frequency (FIGS. 31B and 31D), respectively. Changes in hot and cold spot density (per 100 nucleotides) are summarized below in Table 15:

TABLE 15

Summary of hot spot and cold spot changes

				Cold
Gene	Hot Spots #	Cold Spots #	Hot Spot Density	Spot Density

Native	70	140	8	15
Cold	44	269	5	29
Hot	145	115	16	13

In Table 15, “Hot Spot Density” or “Cold Spot Density,” refers to the total number of hot or cold spots in the ORF, divided by the number of nucleotides in the ORF, multiplied by 100, rounded to the nearest whole number.

Example 5

Creation of Vectors for Somatic Hypermutation

Vectors are constructed from sub-fragments that are each synthesized by DNA2.0 (Menlo Park, Calif.). Vectors are able to simultaneously express multiple open reading frames and are capable of stable, episomal replication in mammalian cells that are naturally permissive or rendered to be permissive (i.e., via co-expression of human EBP2 (Habel et al., 2004; Kapoor et al., 2001) for replication of Epstein Barr Virus (EBV) origin of replication (oriP) containing vectors.
Plasmids are rendered highly modular through the strategic placement of one or more restriction endonuclease recognition sequences (restriction sites) between discreet fragments throughout the vector.
A. Vectors Formats.
In the first format (FIG. 32A); vectors contained an internal ribosome entry site (IRES) from the encephalomyocarditis virus (EMCV). Elements contained within the vectors are operably linked together as shown in FIG. 32A and, in some cases, included the following functional elements (numbers refer to corresponding sequence information found further below in this section): 1) CMV promoter; 2) Multicloning sites; 3) Gene of interest; 4) IRES; 5) Eukaryotic selectable marker such as blasticidin S deaminase (bsd), hygromycin phosphotransferase (hyg) or puromycin-N-acetyl-transferase; 6) Terminator sequences, (3′ untranslated region, small intron and polyA signals from SV40 (“IVS pA”)); 7) Epstein Barr Virus (EBV) origin of replication (oriP) (preceded by optional intergenic spacer region); 8) Prokaryotic origin of replication ColE1; 9) Prokaryotic selectable marker such as beta lactamase (bla) gene or kanamycin (kan); 10) gene fragment for copy number determination (such as beta actin or glucose-6-phosphate dehydrogenase (G6PDH), and Ig enhancers.
In a second format, (FIG. 32B), the expression vectors are made without an IRES, but contained instead an independent expression cassette for expressing a selectable marker gene. This expression cassette included, 11) the SV40 immediate early promoter (pSV) and eukaryotic selectable marker, and IVS pA as described above. Elements contained within the vectors are operably linked together as shown in FIG. 32 and, in some cases, included the following functional elements: CMV promoter, multicloning sites, gene of interest, IVS pA, Epstein Barr Virus (EBV) origin of replication (oriP), pSV, selectable marker, IVS pA, prokaryotic origin of replication ColE1, prokaryotic selectable marker such as beta lactamase (bla) gene, or kanamycin (kan), gene fragment for copy number determination, Ig enhancers, and multicloning sites.
In a third format, (FIG. 33A) vectors contained a bidirectional promoter that drives expression of 2 different genes oriented in opposite directions. This vector also contained IRES sequences to generate 1 or 2 bi- or tri-cistronic messages Elements contained within the vectors are operably linked together as shown in FIG. 33 using the same functional elements as described previously.
In a fourth format, (FIG. 33B) vectors contain a bidirectional promoter, one or more IRES sequences that express bi- or tri-cistronic messages, and an independent, cis-linked cassette from which a eukaryotic selectable marker is expressed.
Any of the vectors can be interchanged with each other to form hybrids. In addition, any of the strong constitutive eukaryotic promoters contained on the episomal vector can be substituted with an inducible promoter (i.e., the reverse tetracycline transactivator promoter system [prtTA]) to achieve conditional expression of a desired gene. In this case, one of the other genes of interest should encode the transactivating protein, which can be expressed in cis on the same episome (as shown in FIG. 34), or supplied in trans on a second, transfected episomal vector.
The orientations for the prokaryotic selectable marker and colEI origin of replication provided in sections 8 and 9 below (SEQ ID NOS: 9-11), and in FIGS. 33-35 are not absolute and can be reversed with respect to the remainder of the vector. Similarly, the orientation of the independent expression cassette (pSV—selectable marker (or other gene of interest)—IVS pA) can also be reversed with respect to the remainder of the vector (i.e. transcribing toward the oriP instead of the current portrayal of transcription away from the oriP). Additionally Enhancer elements, such as Ig enhancers can be placed either 5′ or 3′ to the gene of interest, or can excluded.
B. Representative Sequences of Functional Elements
1. A strong transcriptional promoter that works in eukaryotic cells. In FIGS. 32-33, the CMV promoter is used and the sequence is provided as SEQ ID NO: 1 (the TATA box sequence is shown underlined). The CMV promoter is altered to remove SacI and BsrGI sites.

(SEQ ID NO: 1)

AGCTTGGCCCATTGCATACGTTGTATCCATATCATAATATCTACATTTAT

ATTGGCTCATGTCCAACATTACCGCCATGTTGACATTGATTATTGACTAG

TTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGG

AGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCC

AACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAAC

GCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAA

CTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCT

ATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACAT

GACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCG

CTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAG

CGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGG

GAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACA

ACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTC

TATATAAGCAGAGCTGGTTTAGTGAACCGTCAGATCGCCTA.

2. A region encoding multiple restriction sites termed a multicloning site (mcs) region:

(SEQ ID NO: 2)

TTCCCTGCAGGATTGTTTAAACACCAGATCTGCTTGAATCCGCGGATAAG

AGGACTAGTATTCGTCTCACTAGGGAGAGCTCCTA.

3. A gene of interest can be, for example, specific binding member, antibody or fragment thereof, antibody heavy or light chain, enzyme, receptor, peptide growth hormone or transcription factor.
4. An internal ribosome entry site (IRES), in FIG. 32-34 from the encephalomyocarditis virus (EMCV)-permits the concomitant bicistronic expression of two open reading frames (ORF's): one 5′ to itself, and a second 3′ to itself. A region containing 2 restriction sites (BsrGI and AscI) is shown 5′ to the IRES (lower case letters). The 3′ end of the IRES includes an NgoMIV site.

(SEQ ID NO: 3)

tgtacaatccgcgtgagacgatcggcgcgccCGCCCCTCTCCCTCCCCCC

CCCCTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTT

GTCTATATGTTATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGC

CCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCC

CTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTT

CCTCTGGAAGCTTCTTGAAGACAAACAACGTCTGTAGCGACCCTTTGCAG

GCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCAC

GTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTG

AGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAA

CAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATC

TGGGGCCTCGGTGCACATGCTTTACATGTGTTTAGTCGAGGTTAAAAAAA

CGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACACG

ATGATAATATGGCCGGC.

5. The open reading frame (ORF) for a mammalian selectable marker gene, such as, for example, blasticidin S deaminase (bsd) (SEQ ID NO: 4), hygromycin phosphotransferase (hyg) (SEQ ID NO: 5), or puromycin-N-acetyl-transferase (SEQ ID NO: 6). Start and stop codons are underlined. 3′ to each ORF is an XbaI site (TCTAGA) used in the cloning step.
Blasticidin S deaminase (bsd; cold spot optimized)

(SEQ ID NO: 4)

ATGGCCAAGCCTTTGTCTCAAGAAGAATCCACCCTCATTGAAAGGGCCAC

TGCTACAATCAACAGCATCCCCATCTCTGAAGACTACTCTGTCGCCAGCG

CAGCTCTCTCCTCTGACGGGAGAATCTTCACTGGTGTCAATGTATATCAT

TTTACTGGGGGACCTTGCGCAGAGCTTGTGGTCCTGGGGACTGCTGCTGC

TGCTGCAGCCGGAAACCTGACTTGTATCGTCGCCATAGGGAATGAGAACA

GAGGCATCTTGAGCCCCTGTGGGAGATGCAGACAAGTCCTCCTGGACCTC

CATCCTGGGATCAAAGCCATAGTGAAGGACAGTGATGGACAGCCCACAGC

CGTTGGGATCAGGGAGTTGCTGCCATCTGGTTATGTGTGGGAGGGCTAAT

CTAGA.

Hygromycin phosphotransferase (hyg; cold spot optimized)

(SEQ ID NO: 5)

ATGAAAAAGCCTGAACTGACTGCCACCTCTGTTGAGAAGTTTTTAATAGA

GAAGTTTGACTCTGTGTCAGACCTCATGCAGCTTTCTGAGGGAGAGGAGT

CTAGAGCCTTTAGCTTTGATGTGGGGGGGAGAGGCTATGTCCTGAGAGTC

AATAGCTGTGCAGATGGTTTCTACAAAGATAGGTATGTCTATAGACATTT

TGCATCCGCCGCCCTCCCCATTCCAGAGGTCCTTGACATTGGGGAATTCT

CAGAGAGCCTGACCTATTGCATTTCCCGGAGAGCCCAGGGTGTGACTCTT

CAAGACCTGCCTGAGACAGAACTCCCTGCAGTGCTCCAGCCCGTCGCCGA

GGCCATGGATGCAATCGCCGCCGCAGACCTCAGCCAGACCTCGGGGTTTG

GGCCCTTTGGCCCCCAGGGGATAGGCCAATACACTACATGGAGAGATTTC

ATATGCGCTATTGCTGACCCCCATGTGTATCACTGGCAAACTGTGATGGA

CGACACAGTCTCAGCCTCTGTCGCACAAGCCCTGGACGAGCTGATGCTTT

GGGCCGAGGACTGCCCAGAGGTCAGACATCTCGTCCATGCCGACTTTGGG

TCAAACAATGTCCTGACGGACAATGGGAGAATCACTGCTGTCATTGACTG

GAGCGAGGCCATGTTTGGGGACTCCCAATACGAGGTCGCCAACATCTTCT

TCTGGAGACCCTGGTTGGCTTGTATGGAGCAGCAGACCCGTTACTTTGAG

AGGAGGCATCCAGAGCTCGCTGGGAGCCCTAGATTGAGGGCCTATATGCT

CAGGATAGGGCTTGACCAACTCTATCAGAGCTTGGTTGACGGCAATTTTG

ATGACGCAGCTTGGGCTCAGGGGAGATGCGACGCCATAGTGAGGAGTGGG

GCCGGGACTGTCGGGAGAACTCAGATCGCCAGGAGGTCAGCTGCCGTCTG

GACTGACGGCTGTGTAGAAGTCTTAGCCGACTCTGGGAACAGGAGACCCA

GCACTCGTCCAGAGGCCAAGGAATGATCTAGA.

Puromycin-N-acetyl-transferase (Pur; wild type sequence).
Contains a Kozak consensus sequence immediately 5′ to the start codon (underlined). Stop codon is also underlined.

(SEQ ID NO: 6)

CACCATGACCGAGTACAAGCCCACGGTGCGCCTCGCCACCCGCGACGACG

TCCCCCGGGCCGTTCGCACCCTCGCCGCCGCGTTCGCCGACTACCCCGCC

ACGCGCCACACCGTGGACCCGGACAGGCACATCGAGCGGGTCACCGAGCT

GCAAGAACTCTTCCTCACGCGCGTCGGGCTCGACATCGGCAAGGTGTGGG

TCGCGGACGACGGCGCCGCTGTGGCGGTCTGGACCACGCCGGAGAGCGTC

GAAGCGGGGGCGGTGTTCGCCGAGATCGGCCCGCGCATGGCCGAGTTGAG

CGGTTCCCGGCTGGCCGCGCAGCAACAGATGGAAGGCCTCCTGGCGCCGC

ACCGGCCCAAGGAGCCCGCGTGGTTCCTGGCTACCGTCGGAGTCTCGCCC

GACCACCAGGGCAAGGGTCTGGGCAGCGCCGTCGTGCTCCCCGGAGTGGA

GGCTGCCGAGCGTGCCGGGGTGCCCGCCTTCCTCGAGACCTCCGCGCCCC

GCAACCTCCCCTTCTACGAGCGGCTCGGCTTCACCGTCACCGCCGACGTC

GAGGTGCCCGAAGGACCGCGCACCTGGTGCATGACCCGCAAGCCCGGTGC

CTGATCTAGA.

6. Terminator sequences, IVS-pA (shown with 3′ BamH I).

(SEQ ID NO: 7)

GGATCTTTGTGAAGGAACCTTACTTCTGTGGTGTGACATAATTGGACAAA

CTACCTACAGAGATTTAAAGCTCTAAGGTAAATATAAAATTTTTAAGTGT

ATAATGTGTTAAACTACTGATTCTAATTGTTGTGGTATTTTAGATTCCAA

CCTATGGAACTTATGAATGGGAGCAGTGGTGGAATGCCTTTAATGAGGAA

AACCTGTTTTGCTCAGAAGAAATGCCATCTAGTGATGATGAGGCTACTGC

TGACTCTCAACATTCTACTCCTCCAAAAAAGAAGAGAAAGGTAGAAGACC

CCAAGGACTTTCCTTCAGAATTGGTAAGTTTTTTGAGTCATGCTGTGTTT

AGTAATAGAACTCTTGCTTGCTTTGCTATTTACACCACAAAGGAAAAAGC

TGCACTGCTATACAAGAAAATTATGGAAAAATATTTGATGTATAGTGCCT

TGACTAGAGATCATAATCAGCCATACCACATTTGTAGAGGTTTTACTTGC

TTTAAAAAACCTCCCACACCTCCCCCTGAACCTGAAACATAAAATGAATG

CAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAA

AGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTATCACTGCATTC

TAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGAT

CC.

7. Sequence of EBV oriP. This element permits episomal replication in EBV oriP permissive cells that express Epstein Barr Nuclear Antigen 1 (EBNA1). The oriP sequence is preceded by an optional intergenic spacer region (small letters):

(SEQ ID NO: 8)

actgtcttctttatcatgcaactcgtaggacaggtgccctggccgggtc

cGCAGGAAAAGGACAAGCAGCGAAAATTCACGCCCCCTTGGGAGGTGG

CGGCATATGCAAAGGATAGCACTCCCACTCTACTACTGGGTATCATAT

GCTGACTGTATATGCATGAGGATAGCATATGCTACCCGGATACAGATT

AGGATAGCATATACTACCCAGATATAGATTAGGATAGCATATGCTACC

CAGATATAGATTAGGATAGCCTATGCTACCCAGATATAAATTAGGATA

GCATATACTACCCAGATATAGATTAGGATAGCATATGCTACCCAGATA

TAGATTAGGATAGCCTATGCTACCCAGATATAGATTAGGATAGCATAT

GCTACCCAGATATAGATTAGGATAGCATATGCTATCCAGATATTTGGG

TAGTATATGCTACCCAGATATAAATTAGGATAGCATATACTACCCTAA

TCTCTATTAGGATAGCATATGCTACCCGGATACAGATTAGGATAGCAT

ATACTACCCAGATATAGATTAGGATAGCATATGCTACCCAGATATAGA

TTAGGATAGCCTATGCTACCCAGATATAAATTAGGATAGCATATACTA

CCCAGATATAGATTAGGATAGCATATGCTACCCAGATATAGATTAGGA

TAGCCTATGCTACCCAGATATAGATTAGGATAGCATATGCTATCCAGA

TATTTGGGTAGTATATGCTACCCATGGCAACATTAGCCCACCGTGCTC

TCAGCGACCTCGTGAATATGAGGACCAACAACCCTGTGCTTGGCGCTC

AGGCGCAAGTGTGTGTAATTTGTCCTCCAGATCGCAGCAATCGCGCCC

CTATCTTGGCCCGCCCACCTACTTATGCAGGTATTCCCCGGGGTGCCA

TTAGTGGTTTTGTGGGCAAGTGGTTTGACCGCAGTGGTTAGCGGGGTT

ACAATCAGCCAAGTTATTACACCCTTATTTTACAGTCCAAAACCGCAG

GGCGGCGTGTGGGGGCTGACGCGTGCCATCACTCCACAATTTCAAGAG

AAAGAGTGGCCACTTGTCTTTGTTTATGGGCCCCATTGGCGTGGAGCC

CCGTTTAATTTTCGGGGGTGTTAGAGACAACCAGTGGAGTCCGCTGCT

GTCGGCGTCCACTCTCTTTCCCCTTGTTACAAATAGAGTGTAACAACA

TGGTTCACCTGTCTTGGTCCCTGCCTGGGACACATCTTAATAACCCCA

GTATCATATTGCACTAGGATTATGTGTTGCCCATAGCCATAAATTCGT

GTGAGATGGACATCCAGTCTTTACGGCTTGTCCCCACCCCATGGATTT

CTATTGTTAAAGATATTCAGAATGTTTCATTCCTACACTAGGATTTAT

TGCCCAAGGGGTTTGTGAGGGTTATATTGGTGTCATAGCACAATGCCA

CCACTGAACCCATCGTCCAAATTTTATTCTGGATGCGTCACCTGAAAC

CTTGTTTTCGAGCACCTCACATACACCTTACTGTTCACAACTCAGCAG

TTATTCTATTAGCTAAACGAAGGAGAATGAAGAAGCAGGCGAAGATTC

AGGAGAGTTCACTGCCCGCTCCTTGATCTTCAGCCACTGCCCTTGTGA

CTAAAATGGTTCACTACCCTCGTGGAATCCTGACCCCATGTAAATAAA

ACCGTGACAGCTCATGGGGTGGGAGATATCGCTGTTCCTTAGGACCCT

TTTACTAACCCTAATTCGATAGCATATGCTTCCCGTTGGGTAACATAT

GCTATTGAATTAGGGTTAGTCTGGATAGTATATACTACTACCCGGGAA

GCATATGCTACCCGTTTAGGGTTAACAAGGGGGCCTTATAAACACTAT

TGCTAATGCCCTCTTGAGGGTCCGCTTATCGGTAGCTACACAGGCCCC

TCTGATTGACGTTGGTGTAGCCTCCCGTAGTCTTCCTGGGCCCCTGGG

AGGTACATGTCCCCCAGCATTGGTGTAAGAGCTTCAGCCAAGAGTTAC

ACATAAAGG.

8. Sequence of Escherichia coli origin of replication colEI, derived from vector pJ15 and pJ31 from DNA2.0 (Menlo Park, Calif.): colE1

(SEQ ID NO: 9)

AAAAGGGGCCCGAGCTTAAGACTGGCCGTCGTTTTACAACACAGAAAG

AGTTTGTAGAAACGCAAAAAGGCCATCCGTCAGGGGCCTTCTGCTTAG

TTTGATGCCTGGCAGTTCCCTACTCTCGCCTTCCGCTTCCTCGCTCAC

TGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCA

CTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGG

AAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAA

GGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCA

TCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACT

ATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCC

TGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTC

GGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTC

GGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGT

TCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAA

CCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAG

GATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTG

GTGGGCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGC

TCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATC

CGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCA

GCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTT

TTCTACGGGGTCTGACGCTCAGTGGAACGACGCGCGCGTAACTCACGT

TAAGGGATTTTGGTCATGAGCTTGCGCCGTCCCGTCAAGTCAGCGTAA

TGCTCTG.

9A. Sequence of beta lactamase (bla) gene for resistance. The open reading frame (ORF) is shown in reverse orientation.

(SEQ ID NO: 10)

CTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATT

TCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATA

CGGGAGGGCTTACCATCTGGCCCCAGCGCTGCGATGATACCGCGAGAAC

CACGCTCACCGGCTCCGGATTTATCAGCAATAAACCAGCCAGCCGGAAG

GGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCT

ATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTT

TGCGCAACGTTGTTGCCATCGCTACAGGCATCGTGGTGTCACGCTCGTC

GTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTT

ACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTC

CGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTAT

GGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTT

TCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGC

GGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCC

ACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGG

CGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAAC

CCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGT

TTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATA

AGGGCGACACGGAAATGTTGAATACTCATATTCTTCCTTTTTCAATATT

ATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGA

ATGTATTTAGAAAAATAAACAAATAGGGGTCAGTGTTACAACCAATTAA

CCAATTCTGAACATTATCGCGAGCCCATTTATACCTGAATATGGCTCAT

AACACCCCTTGCAGTGCGACTAACGGCATGAAGCTCGTCGGGGCGTACG.

9B. Sequence of kanamycin (kan), derived from vector pJ31 from DNA2.0 (Menlo Park, Calif.). The open reading frame (ORF) is shown in reverse orientation.

(SEQ ID NO: 11)

CTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATAT

CAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAG

GAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATC

GGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCC

CCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGA

CTGAATCCGGTGAGAATGGCAAAAGTTTATGCATTTCTTTCCAGACTT

GTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAA

CCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGGCGAAATACGC

GATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAGTGCAACCGGC

GCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGAT

ATTCTTCTAATACCTGGAACGCTGTTTTTCCGGGGATCGCAGTGGTGA

GTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAA

GTGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAA

CATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCG

CATCGGGCTTCCCATACAAGCGATAGATTGTCGCACCTGATTGCCCGA

CATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGG

AATTTAATCGCGGCCTCGACGTTTCCCGTTGAATATGGCTCATATTCT

TCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGA

GCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTCA

GTGTTACAACCAATTAACCAATTCTGAACATTATCGCGAGCCCATTTA

TACCTGAATATGGCTCATAACACCCCTTGCAGTGCGACTAACGGCATG

AAGCTCGTCGGGGAAATAATGATTTTATTTTGACTGATAGTGACCTGT

TCGTTGCAACAAATTGATAAGCAATGCTTTCTTATAATGCCAACTTTG

TACAAGAAAGCTGGGTTTTTTTTTTAGCCTGCTTTTTTGTACAAAGTT

GGCATTATAAAAAAGCATTGCTCATCAATTTGTTGCAACGAACAGGTC

ACTATCAGTCAAAATAAAATCATTATTT.

10. A moiety used for determination of episomal copy number per cell. Ideally, the moiety contains a sequence that exists uniquely in the genome. Shown below are 2 fragments, beta actin and G6PDH that can be used in vectors known in the art or described herein. Each fragment is bounded by a BsiWI and a Cla I site.
beta actin moiety

(SEQ ID NO: 12)

CGTACGTACTCCTGCTTGCTGATCCACATCTGCTGGAAGGTGGACAGCGA

GGCCAGGATGGAGCCGCCGATCCACACGGAGTACTTGCGCTCAGGAGGAG

CAATGAAGCTTATCTGAGGAGGGAAGGGGACAGGCAGTGAGGACCCTGGA

TGTGACAGCTCCAAGCTTCCACACACCACAGGACCCCACAGCCGACCTGC

CCAGGTCAGCTCAGGCAGGAAAGACACCCACCTTGATCTTCATTGTGCTG

GGTGCCAGGGCAGTGATCTCCTTCTGCATCCTGTCATCGAT.

Human glucose-6-phosphate dehydrogenase (hG6PDH) moiety

(SEQ ID NO: 13)

CGTACGAGGTGAGGCTGCAGTTCCATGATGTGTCCGGCGACATCTTCCAC

CAGCAGTGCAAGCGCAACGAGCTGGTGATCCGCGTGCAGCCCAACGAGGC

CGTGTACCAGAGAAGGAGCAGTGTGGAGGGTGGGCGGCCTGGGCCCGGGG

GACTCCACATGGTGGCAGGCAGTGGCATCAGCAAGACACTCTCTCCCTCA

CAGAACGTGAAGCTCCCTGACGCCTACGAGCGCCTCATCCTGGACGTCTT

CTGCGGGAGCCAGATGCACTTCGTGCGCAGGAATCGAT.

11. pSV, immediate early promoter from SV40. The sequence is preceded by a BstBI site and followed by an NgoMIV site.

(SEQ ID NO: 14)

TTCGAAGCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCT

CCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACC

AGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCAT

GCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCC

CGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTA

ATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATT

CCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGC

TCTGACCCCTCACAAGGAGCCGGC.

Ig Enhancers. Representative Ig enhancer sequences include the heavy or light chain enhancers. The Kappa 3′ enhancer region (Ek3′) (See Meyer, K. B. and Neuberger, M. S., EMBO J. 8 (7), 1959-1964 (1989)), and Kappa intronic enhancer region, Eki LOCUS L80040 7466 by ROD 2 Sep. 2003 are shown below by way of example. At least 1 major active element within the enhancer regions is the E box sequence: CAGGTG(N)₁₃CAGGTG [core sequence: CANNTG] Storb et al., Immunity 19:235-242, 2003). The Ek3′ and Eki enhancer elements are obtained from Dr. Neuberger (MRC, UK). The Ek3′ sequence is amplified by PCR from Neuberger plasmid identification #1352, using the following primers, which contain an XhoI and EcoRI site, respectively, that are used for cloning: GACTACCTCGAGccagcttaggctacacagag (SEQ ID NO: 276) and GTAGTCGAATTCCCACATGTCTTACATGGTATATG (SEQ ID NO: 277).
The Eki enhancer sequence is amplified from Dr. Neuberger's vector (identification #Me123) using oligonucleotides GACTACGAATTCtcctgaggacacagtgatag (SEQ ID NO: 278) and GTAGTCGCGGCCGCCTAGTTCCTAGCTACTTCTTTA (SEQ ID NO: 279), which encode an EcoRI and NotI restriction site, respectively. Resulting fragments are digested with the appropriate restriction enzyme, and cloned sequentially into mcs2 (described in below): Ek3′ is cloned into the XhoI and EcoRI sites of mcs2, and the resulting plasmid is then digested with EcoRI plus NotI into which the Eki fragment is subsequently ligated to generate vector AB156.
As described above, E boxes are known to be present in the kappa enhancer region. Consequently, a synthetic cassette consisting of 3 tandemly arrayed E boxes is synthesized using the complementary oligonucleotides AATTCaggtgctggggtagggagcaggtgctacactgcagaccaggtgctGC (SEQ ID NO: 280) and ggccgcagcacctggtctgcagtgtagcacctgctccctaccccagcacctg (SEQ ID NO: 281), which when annealed contain EcoRI and NotI overhangs. The annealed oligo product is thus cloned into the EcoRI and NotI sites of mcs2 to generate vector AB157.
A representative Ig-kappa locus 3′ enhancer element is listed below. (Accession number X15878)

(SEQ ID NO: 282)

CCAGCTTAGGCTACACAGAGAAACTATCTAAAAAATAATTACTAACTACT

TAATAGGAGATTGGATGTTAAGATCTGGTCACTAAGAGGCAGAATTGAGA

TTCGAAGCCAGTATTTTCTACCTGGTATGTTTTAAATTGCAGTAAGGATC

TAAGTGTAGATATATAATAATAAGATTCTATTGATCTCTGCAACAACAGA

GAGTGTTAGATTTGTTTGGAAAAAAATATTATCAGCCAACATCTTCTACC

ATTTCAGTATAGCACAGAGTACCCACCCATATCTCCCCACCCATCCCCCA

TACCAGACTGGTTATTGATTTTCATGGTGACTGGCCTGAGAAGATTAAAA

AAAGTAATGCTACCTTATTGGGAGTGTCCCATGGACCAAGATAGCAACTG

TCATAGCTACCGTCACACTGCTTTGATCAAGAAGACCCTTTGAGGAACTG

AAAACAGAACCTTAGGCACATCTGTTGCTTTCGCTCCCATCCTCCTCCAA

CAGCCTGGGTGGTGCACTCCACACCCTTTCAAGTTTCCAAAGCCTCATAC

ACCTGCTCCCTACCCCAGCACCTGGCCAAGGCTGTATCCAGCACTGGGAT

GAAAATGATACCCCACCTCCATCTTGTTTGATATTACTCTATCTCAAGCC

CCAGGTTAGTCCCCAGTCCCAATGCTTTTGCACAGTCAAAACTCAACTTG

GAATAATCAGTATCCTTGAAGAGTTCTGATATGGTCACTGGGCCCATATA

CCATGTAAGACATGTGG.

A representative Kappa intronic enhancer region, Eki is presented below:

(SEQ ID NO: 283)

TCCTGAGGACACAGTGATAGGAACAGAGCCACTAATCTGAAGAGAACAGA

GATGTGACAGACTACACTAATGTGAGAAAAACAAGGAAAGGGTGACTTAT

TGGAGATTTCAGAAATAAAATGCATTTATTATTATATTCCCTTATTTTAA

TTTTCTATTAGGGAATTAGAAAGGGCATAAACTGCTTTATCCAGTGTTAT

ATTAAAAGCTTAATGTATATAATCTTTTAGAGGTAAAATCTACAGCCAGC

AAAAGTCATGGTAAATATTCTTTGACTGAACTCTCACTAAACTCCTCTAA

ATTATATGTCATATTAACTGGTTAAATTAATATAAATTTGTGACATGACC

TTAACTGGTTAGGTAGGATATTTTTCTTCATGCAAAAATATGACTAATAA

TAATTTAGCACAAAAATATTTCCCAATACTTTAATTCTGTGATAGAAAAA

TGTTTAACTCAGCTACTATAATCCCATAATTTTGAAAACTATTTATTAGC

TTTTGTGTTTGACCCTTCCCTAGCCAAAGGCAACTATTTAAGGACCCTTT

AAAACTCTTGAAACTACTTTAGAGTCATTAAGTTATTTAACCACTTTTAA

TTACTTTAAAATGATGTCAATTCCCTTTTAACTATTAATTTATTTTAAGG

GGGGAAAGGCTGCTCATAATTCTATTGTTTTTCTTGGTAAAGAACTCTCA

GTTTTCGTTTTTACTACCTCTGTCACCCAAGAGTTGGCATCTCAACAGAG

GGGACTTTCCGAGAGGCCATCTGGCAGTTGCTTAAGATCAGAAGTGAAGT

CTGCCAGTTCCTCCCAGGCAGGTGGCCCAGATTACAGTTGACCTGTTCTG

GTGTGGCTAAAAATTGTCCCATGTGGTTACAAACCATTAGACCAGGGTCT

GATGAATTGCTCAGAATATTTCTGGACACCCAAATACAGACCCTGGCTTA

AGGCCCTGTCCATACAGTAGGTTTAGCTTGGCTACACCAAAGGAAGCCAT

ACAGAGGCTAATACCAGAGTATTCTTGGAAGAGACAGGAGAAAATGAAAG

CCAGTTTCTGCTCTTACCTTATGTGCTTGTGTTCAGACTCCCAAACATCA

GGAGTGTCAGATAAACTGGTCTGAATCTCTGTCTGAAGCATGGAACTGAA

AAGAATGTAGTTTCAGGGAAGAAAGGCAATAGAAGGAAGCCTGAGAATAT

CTTCAAAGGGTCAGACTCAATTTACTTTCTAAAGAAGTAGCTAGGAACTA

G.

C. Vector Construction
Vector Format 1. The functional elements described below are ordered as 7 synthetic DNA fragments, each cloned into vector pJ2 or pJ15 from DNA2.0 (Menlo Park, Calif.). Both pJ vectors contain a colE1 E. coli origin of replication and a selectable marker (amp for pJ15, kan for pJ2); the sequences of each have been altered by DNA2.0 to minimize restriction sites. The pJ vector inserts are designed to contain one or more of the genetic elements listed below for the final vector construct bounded by restriction sites that allowed for the correct assembly of fragments in the desired order.
Vector F1, a covalently closed circular plasmid, contains (in order): DNA2.0 vector pJ15 (which contains the colE1 on and amp resistance marker), restriction sites BsiWI, Sad, BsrGI, NgoMIV, XbaI, BamHI, MluI, approximately 800 bp of the EBV oriP, and AflII (see FIG. 35A).
Insert F2 contains approximately 880 bp of the oriP, including a highly repetitive portion, from the natively encoded restriction sites Mlu I to Nsi I. This fragment is recovered from a clone purchased from the American Type Culture Collection (catalog number ATCC 59562) using restriction sites Mlu I to Nsi I.
Insert F3 is reclaimed from pJ2 and encodes the SV40 3′ untranslated region and poly adenylation signals (3′ ut/pA), a BamHI restriction site, and the remaining portion of the oriP. The fragment is bounded by NsiI and XbaI sites.
Insert F4 is reclaimed from pJ2 and contains the eukaryotic antibiotic resistance marker puromycin-N-acetyl-transferase (pur) bounded by restriction sites NgoMIV and XbaI.
Insert F5 is reclaimed from pJ15 and contains the EMCV IRES sequence bounded by restriction sites NgoMIV and BsrGI.
Insert F6 is reclaimed from pJ15 and contains a synthetic version of the teal fluorescent protein open reading frame (hotTFP) that has been altered to render it more susceptible to somatic hypermutation (SHM), and thus the appellation “hot.” The fragment is delineated by BsrGI and SacI restriction sites.
Vector F7, a covalently closed circular plasmid, contains (in order): DNA2.0 vector pJ15 (which contains the amp resistance marker and colE1 on), restriction sites AflII, BamHI, XbaI, NgoMIV, BsrGI, SacI, multicloning site region 1 (mcs1), CMV immediate early promoter, multicloning site region 2 (mcs2), beta actin (β actin) moiety for determination of episomal copy number per cell, BsiWI (see FIG. 35B).
The Mcs1 contains the following restriction sites: (5′) SbfI, PmeI, BglII, SacII, SpeI, BsmBI, and SacI (3′). Mcs2 contains (5′) ClaI, XhoI, BclI, EcoRI, AgeI, NotI, and NheI (3′).
The prototypic vector AB102 is assembled as described below using standard molecular biological manipulations: All final constructs are confirmed by sequence analysis.
1. A triple ligation is performed to incorporate inserts F5 (reclaimed with BsrG I and NgoM IV) and F6 (reclaimed with Sac I and BsrG I) into vector F7 to make vector ANA113.
2. A second triple ligation is performed to integrate inserts F2 (Mlu I to Nsi I) and F3 (Nsi I to Xba I) into vector F1 to generate vector ANA110.
3. A double ligation is performed to include insert F4 (from Xba I to NgoMIV) in vector ANA110, thus generating vector ANA119.
4, Finally, a double ligation is performed to assimilate the insert from ANA113 above (BsiW I to NgoM IV) into the BsiW I and NgoM IV cut vector ANA119 to generate AB102.
The judicious placement of restriction sites renders AB102 highly modular (see FIGS. 36A and 36B). Consequently, most subsequent vector formats (i.e. vector formats 2-4) are, in some cases, generated by simple fragment exchange. For example, the open reading frames for blasticidin S deaminase (cold bsd) and hygromycin phosphotransferase (cold hyg) are synthesized at DNA2.0 using codons that are SHM resistant, as described herein (thus the designation “cold”). Both of these markers are bounded by restriction sites NgoMIV and XbaI and are therefore easily cloned in place of pur (also delineated by NgoMIV and XbaI) to generate versions of the vectors that confer resistance to bsd and hyg, respectively.
Similarly, other genes of interest are synthesized so as to be bordered on the 5′ side by one of the unique restriction sites in mcs1 plus BsrGI or AscI at the 3′ end. Such genes cloned into the position initially occupied by TFP (FIGS. 36A and 36B) included other fluorescent proteins (GFP, and “GFP*” [a GFP that contains an in frame stop codon in lieu of tyr82]), immunoglobulins such as antibody heavy or light chains, activation induced cytidine deaminase (AID, also known as AICDA), and the reverse tetracycline transactivatable protein (rtTA).
There are several versions of beta actin and G6PDH, differing by length, that permit the simultaneous identification and quantification of more than one episomal vector. Each version is cloned into the same location using the BsiWI and ClaI sites,
Different varieties of the CMV immediate early promoter (i.e. versions in which the Sad, and BsrGI restriction sites have been eliminated, and versions that include more or fewer nucleotides at the 5′ end of the enhancer region are swapped in and out of AB vector series constructs by using NheI and SbfI, which are unique restriction sites found in mcs2 (5′) and mcs1 (3′), respectively.
Vector Format 5. AB184, a derivative of AB102, is an archetypical example of a vector in which expression of a gene of interest can be induced by addition of doxycycline (dox) (FIG. 37A). AB184 differs from AB102 in the following ways:
The TFP open reading frame is replaced with a cold codon version of the rtTA coding region (see, e.g., Gossen and Bujard, Proc Natl Acad Sci USA. (1992) 89(12):5547-51; Gossen et al., Science (1995) 268 (5218):1766-9). The rtTA gene is synthesized at DNA2.0 in vector pJ2 and is reclaimed by PCR amplification such that BglII is included as a cloning site on the 5′ (sense) primer and AscI included on the 3′ (antisense) primer. The PCR fragment is cloned into the BglII (found in mcs1) and AscI sites of AB102 (FIG. 38).
The hyg open reading frame is synthesized with cold codon usage, and with 5′ NgoMIV and 3′ XbaI sites in pJ2 by DNA2.0) to make vector ANA112. The hyg insert from this vector replaced pur as the selectable marker (FIG. 38) in AB184.
ANA112 is also used to supply the prokaryotic resistance marker kan. A BsiWI site is added 5′ to the kan marker by site directed mutagenesis (SDM) to generate ANA136. The contiguous kan and colE1 fragment is reclaimed from ANA136 using BsiWI and AflII and replaced the homologous amp-colE1 fragment in AB102 to generate the kan resistant precursor to AB184.
A BstBI site is added next to the unique AflII site (order of elements: kan-colE1-AflII-BstBI . . . . )
Finally, a dox-inducible cold canine AID expression cassette is created and added to the vector at the AflII site found between the oriP and colE1 on. The steps to do this are described below.
Vector F10 is synthesized at DNA2.0 and contains the following elements (in order): i. colE1 ori and kan marker from pJ2; ii. SacI site; iii. the human growth hormone (hGH) minimal promoter (sequence ACAGTGGGAGAGAAGGGGCCAGGGTATAAAAAGGGCCCACAAGAGACCAGCTCAAGGATTCCAA GGC; SEQ ID NO: 284); iv. SpeI site; v. canine AID with cold codon usage, and a single mutation (L198A) that disables the nuclear export signal; and vi. AgeI site.
Vector F10 contained an unwanted AflII site 3′ to the AgeI site (FIG. 37B). This site is removed by site directed mutagenesis. An AflII site is added by site directed mutagenesis 5′ to the SacI site to generate ANA165.
A 7-fold repeat of the tet operator elements, including the minimal hGH promoter sequence listed in (a) above is synthesized at DNA2.0 in plasmid pJ51 to generate vector 7xprtTA. The insert order is (5′) AflII—7x prtTA operator elements—SacI—hGH promoter—and SpeI (3′). The 7× repeat is reclaimed from pJ51 with AflII and SpeI and cloned into the cognate sites of ANA165 to generate ANA209 (FIG. 38).
To complete the expression cassette, a triple ligation is performed that included (1) AflII-cut and CIP (calf intestinal phosphatase) treated vector pJ15; plus (2) Insert 7xprtTA-coldCanine AIDnes(−) from Age I to Afl II; plus (3) PCR amplified 3′ ut/pA fragment (originally synthesized as insert F3, in which Age I (5′) and Afl II (3′) restriction sites are incorporated into the PCR primers. This created vector ANA285.
Finally, the entire inducible expression cassette (which contained in order the following genetic markers: AflII-7xprtTA-canine AID-3′ ut/pA-AflII) is reclaimed from ANA285 by PCR using oligos that include BstBI sites and span the AflII site on the 5′ side. The reclaimed insert thus had the following genetic elements: AflII-BstBI-7xprtTA-canine AID-3′ ut/pA-AflII-BstBI. This PCR amplified product is digested with BstBI and is cloned into the unique BstBI site in the AB184 precursor described in step (3) above to generate AB184 (FIG. 37A). (Note: the AflII site at the 3′ end of the cassette is part of the cloning vector and is not carried into the final construct by the PCR amplified insert).

Example 6

Application of SHM to Generation of Novel Lantibiotic Synthesis

The evolution of bacteria with resistance to existing therapeutic regimens has sparked interest in the discovery and development of novel antibiotics. Ideal candidates for further research are those that act via multiple modes of action, making resistance significantly more difficult to attain. One such antibiotic is Nisin.
Nisin is a natural product of Lactococcus lactic, a lantibiotic with a broad spectrum of activity against Gram-positive bacteria, commonly used in food preservation against such pathogens as Listeria monocytogenes and Clostridium botulinum. (BAVIN et al., Lancet. 1952 Jan. 19; 1(3):127-9) Nisin is a ribosomally translated and post-translated peptide, which despite decades of use by the food industry, has not seen the induction of common resistance mechanisms. This finding is likely a result of two facts: one, the mode of action of Nisin biocidal activity comes from its binding to Lipid II and secondary induction of pore formation, (Breukink et al., Nat Rev Drug Discov. 2006 April; 5(4):321-32). Lipid II is a bacterial cell-wall component that is not easily modified by Gram-positive bacteria and whose use forms a rate-limiting step in the generation of the bacterial cell wall. Nisin also acts to inhibit spore formation.
Nisin is currently in preclinical development for the treatment of several bacterial pathogens. It displays a spectrum of activity towards several pathogens, including multi drug-resistant Streptococcus pneumoniae, vancomycin-resistant Enterococcus faecium, and Streptococcus pyogenes, all areas where new therapeutics are desperately needed (Goldstein et al., J. Antimicrob. Chemother. 1998 August; 42(2):277-8). In one study, Nisin was shown to be 8-16 times more potent in the treatment of S. pneumoniae (in mice) than vancomycin (Brumfitt et al., J Antimicrob Chemother. 2002 November; 50(5):731-4).
Despite these promising features, Nisin and other lantibotics suffer from several important limitations. Bacteria, even closely related (isogenic) species, display a significant variation in their sensitivity to Nisin and other lantibiotics. Secondly, Nisin is cleared quickly from mammalian circulatory system. For Nisin to become a truly efficacious therapeutic, it will need to have improved pharmacodynamic properties with a broad spectrum of biocidal activity. Here we discuss application of SHM to engineer a Nisin with improved qualities.
Biosynthesis of bioactive Nisin has been to shown to be dependent on only five L. lactis proteins, NisA, NisB, NisC, NisP, and NisT (Kuipers et al., J Biol Chem. 2004 Can 21; 279(21):22176-82; Rink et al., Biochemistry. 2005 Jun. 21; 44(24):8873-82). NisA encodes for a precursor peptide which is dehydrated at several serine and threonine positions by NisB, leading to a modified peptide that is cyclized at five positions by NisC. Finally the pro-antibiotic has its leader peptide cleaved by protease NisP, and is excreted to the media by transporter NisT (see FIG. 47A). The five thioester rings, each catalyzed by NisC, are termed lanthionines, and define the lantibiotic family of modified peptide antibiotics.
The modular nature of this pathway, easy assay for bioactivity, broad specificity and activity of the dehydratase and cyclase NisB and NisC, make this an ideal target for SHM driven co-evolution to produce novel antibiotic constructs. In one approach such a strategy could be based on making certain genes, or portions of genes more susceptible to SHM, while making other genes, or portions of those genes, resistant to SHM.
The amino acid sequences of the 5 genes involved in Nisin biosynthesis are shown below: In these sequences, bold residues indicate those positions to be made hot to SHM, while underlined residues are those to be made cold to SHM.
NisA, Native Gene>NisA|gi|530218|gb|AAA26948.1|nisin [Lactococcus lactis]

(SEQ ID NO: 285)

MSTKDFNLDLVSVSKKDSGASPRITSISLCTPGCKTGALMGCNMKTATCH

CSIHVSK

NisC, Native Gene>NisC|gi|44045|emb|CAA48383.1|nisC [Lactococcus lactis]

(SEQ ID NO: 286)

MRIMMNKKNIKRNVEKIIAQWDERTRKNKENFDFGELTLSTGLPGIILML

AELKNKDNSKIYQKKIDNYIEYIVSKLSTYGLLTGSLYSGAAGIALSILH

LREDDEKYKNLLDSLNRYIEYFVREKIEGFNLENITPPDYDVIEGLSGIL

SYLLLINDEQYDDLKILIINFLSNLTKENNGLISLYIKSENQMSQSESEM

YPLGCLNMGLAHGLAGVGCILAYAHIKGYSNEASLSALQKIIFIYEKFEL

ERKKQFLWKDGLVADELKKEKVIREASFIRDAWCYGGPGISLLYLYGGLA

LDNDYFVDKAEKILESAMQRKLGIDSYMICHGYSGLIEICSLFKRLLNTK

KFDSYMEEFNVNSEQILEEYGDESGTGFLEGISGCILVLSKFEYSINFTY

WRQALLLFDDFLKGGKRK

NisB, Native Gene>gi|473018|emb|CAA79468.1|NisB protein [Lactococcus lactis]

(SEQ ID NO: 287)

MIKSSFKAQPFLVRNTILSPNDKRSFTEYTQVIETVSKNKVFLEQLLLAN

PKLYNVMQKYNAGLLKKKRVKKLFESIYKYYKRSYLRSTPFGLFSETSIG

VFSKSSQYKLMGKTTKGIRLDTQWLIRLVHKMEVDFSKKLSFTRNNANYK

FGDRVFQVYTINSSELEEVNIKYTNVYQIISEFCENDYQKYEDICETVTL

CYGDEYRELSEQYLGSLIVNHYLISNLQKDLLSDFSWDTFLTKVEAIDED

KKYIIPLKKVQKFIQEYSEIEIGEGIEKLKEIYQEMSQILENDNYIQIDL

ISDSEINFDVKQKQQLEHLAEFLGNTTKSVRRTYLDDYKDKFIEKYGVDQ

EVQITELFDSTFGIGAPYNYNHPRNDFYESEPSTLYYSEEEREKYLSMYV

EAVKNHNVINLDDLESHYQKMDLEKKSELQGLELFLNLAKEYEKDIFILG

DIVGNNNLGGASGRFSALSPELTSYHRTIVDSVERENENKEITSCEIVFL

PENIRHANVMHTSIMRRKVLPFFTSTSHNEVQLTNIYIGIDEKEKFYARD

ISTQEVLKFYITSMYNKTLFSNELRFLYEISLDDKFGNLPWELIYRDFDY

IPRLVFDEIVISPAKWKIWGRDVNNKMTIRELIQSKEIPKEFYIVNGDNK

VYLSQENPLDMEILESAIKKSSKRKDFIELQEYFEDENIINKGQKGRVAD

VVVPFIRTRALGNEGRAFIREKRVSVERREKLPFNEWLYLKLYISINRQN

EFLLSYLPDIQKIVANLGGKLFFLRYTDPKPHIRLRIKCSDLFLAYGSIL

EILKRSQKNRIMSTFDISIYDQEVERYGGFDTLELSEAIFCADSKIIPNL

LTLIKDTNNDWKVDDVSILVNYLYLKCFFQNDNKKILNFLNLVSPKKVKE

NVNEKIEHYLKLLKVDNLGDQIFYDKNFKELKHAIKNLFLKMIAQDFELQ

KVYSIIDSIIHVHNNRLIGIERDKEKLIYYTLQRLFVSEEYMK

NisP, Native Gene>gi|730155|sp|Q07596|NISP_LACLA Nisin leader peptide-processing serine protease nisP precursor

(SEQ ID NO: 288)

MKKILGFLFIVCSLGLSATVHGETTNSQQLLSNNINTELINHNSNAILSS

TEGSTTDSINLGAQSPAVKSTTRTELDVTGAAKTLLQTSAVQKEMKVSLQ

ETQVSSEFSKRDSVTNKEAVPVSKDELLEQSEVVVSTSSIQKNKILDNKK

KRANFVTSSPLIKEKPSNSKDASGVIDNSASPLSYRKAKEVVSLRQPLKN

QKVEAQPLLISNSSEKKASVYTNSHDFWDYQWDMKYVTNNGESYALYQPS

KKISVGIIDSGIMEEHPDLSNSLGNYFKNLVPKGGFDNEEPDETGNPSDI

VDKMGHGTEVAGQITANGNILGVAPGITVNIYRVFGENLSKSEWVARAIR

RAADDGNKVINISAGQYLMISGSYDDGTNDYQEYLNYKSAINYATAKGSI

VVAALGNDSLNIQDNQTMINFLKRFRSIKVPGKVVDAPSVFEDVIAVGGI

DGYGNISDFSNIGADAIYAPAGTTANFKKYGQDKFVSQGYYLKDWLFTTA

NTGWYQYVYGNSFATPKVSGALALVVDKYGIKNPNQLKRFLLMNSPEVNG

NRVLNIVDLLNGKNKAFSLDTDKGQDDAINHKSMENLKESRDTMKQEQDK

EIQRNTNNNFSIKNDFHNISKEVISVDYNINQKMANNRNSRGAVSVRSQE

ILPVTGDGEDFLPALGIVCISILGILKRKTKN

NisT, Native Gene>gi|44044|emb|CAA48382.1|nisT [Lactococcus lactis]

(SEQ ID NO: 289)

MDEVKEFTSKQFFYTLLTLPSTLKLIFQLEKRYAIYLIVLNAITAFVPLA

SLFIYQDLINSVLGSGRHLINIIIIYFIVQVITTVLGQLESYVSGKFDMR

LSYSINMRLMRTTSSLELSDYEQADMYNIIEKVTQDSTYKPFQLFNAIIV

ELSSFISLLSSLFFIGTWNIGVAILLLIVPVLSLVLFLRVGQLEFLIQWQ

RASSERETWYIVYLLTHDFSFKEIKLNNISNYFIHKFGKLKKGFINQDLA

IARKKTYFNIFLDFILNLINILTIFAMILSVRAGKLLIGNLVSLIQAISK

INTYSQTMIQNIYHYNTSLFMEQLFEFLKRESVVHKKIEDTEICNQHIGT

VKVINLSYVYPNSNAFALKNINLSFEKGELTAIVGKNGSGKSTLVKIISG

LYQPTMGIIQYDKMRSSLMPEEFYQKNISVLFQDFVKYELTIRENIGLSD

LSSQWEDEKIIKVLDNLGLDFLKTNNQYVLDTQLGNWFQEGHQLSGGQWQ

KIALARTFFKKASIYILDEPSAALDPVAEKEIFDYFVALSENNISIFISH

SLNAARKANKIVVMKDGQVEDVGSHDVLLRRCQYYQELYYSEQYEDNDE

NisB, NisP and NisT
As described above, the creation of SHM resistant (cold) versions of the essential genes NisP and NisT means that these genes will tend to mutate at a lower rate than SHM-susceptible genes that are targeted for diversity generation. Both NisP and NisT currently have broad specificity for the Nisin and do not add to the potential diversity of the post-translationally modified peptide. In this initial example, NisB is also made SHM-resistant; however, it could also be selectively mutated following the same guidelines outlined below for NisA. Native and SHM-resistant polynucleotides of NisB are provided in FIGS. 39A and 40A, respectively, together with the initial analysis of hot spot and cold spot frequency (FIGS. 439B and 40B), respectively. Native and SHM-resistant polynucleotides of NisP are provided in FIGS. 41A and 42A, respectively, together with the initial analysis of hot spot and cold spot frequency (FIGS. 41B and 42B), respectively.
NisA Peptide
As shown above, the majority of the leader peptide region of the NisA peptide can be made resistant to AID-mediated mutagenesis because this sequence is absolutely necessary for substrate recognition by NisBCPT. The bulk of the remainder of the NisA peptide sequence can be made susceptible to AID-mediated mutagenesis, or alternatively, as shown above key residues involved in the generation of the lanthionines can be made SHM-resistant thereby reducing the rate of their mutagenesis.
Corresponding unmodified and SHM-resistant (cold) polynucleotide versions of the NisA polynucleotide sequence are shown in FIGS. 44A and 44D, respectively. The initial analysis of the unmodified hot spot and cold spot frequency (FIGS. 44B and 44C), respectively, are compared to the SHM-resistant hot spot and cold spot frequency (FIGS. 44E and 44F), respectively. Codon optimization of NisA results in the creation of 20 cold spots and elimination of all but one hot spot in the leader sequence, and the creation of 17 hot spots, compared to 8 hot spots in the wild type sequence, in the rest of the molecule.
NisC Protein
Regions of NisC involved in substrate recognition and cyclization, such as those outlined above as bold residues, and as shown in FIG. 47B, can be made hot (susceptible) to AID-mediated mutation, so that they have a greater probability of generating mutants with alternate activities and specificities thereby creating mature Nisin molecules with altered modifications and bioactivity. Structural areas that govern only stability of the protein can be made cold. Corresponding unmodified and SHM-resistant (cold) polynucleotide versions of the NisC polynucleotide sequence are shown in FIGS. 45A and 46A, respectively. The initial analysis of the unmodified hot spot and cold spot frequency (FIGS. 45B and 45C), respectively, are compared to the SHM-resistant hot spot and cold spot frequency (FIGS. 46B and 46C), respectively.
A specific example of the creation of a targeted hot spot in this gene is shown below:
In this example, using SHMredesign, an additional hot spot has been inserted into the region of interest (LSTG) and a cold spot has been removed. Additionally the flanking sequence has been made significantly more SHM resistant.

(SEQ ID NO: 290)

..N..F..D..F..G..E..L..T.. L..S..T..G ..L..P..G amino acid sequence

Native polynucleotide sequence:
HhhhhhhhhhhhhhhhHhhhhhhhhhhhhHhhhhhhhhhhhhHhh hot spots

cccccccCcccccCCcCccccCcCcCcCccccccCCccccccccc cold spots

Optimized polynucleotide sequence:
HhhhhhhhhhhhhhhhHhhhhhhhhhhHhHhhhhhhhhhhhhhh hot spots

ccccccCcccccCCcCccCcccCcccCcccccccCcCcCCccCc cold spots

After final review to ensure that the synthetic polynucleotide sequence is free of extraneous restriction sites, the complete synthetic polynucleotide sequences can be synthesized (DNA 2.0, Menlo Park, Calif.), and cloned appropriate cloning vectors and sequenced to confirm correct synthesis.
Synthetic genes can then be introduced into expression vectors and transformed into an appropriate bacterial strain, for example a Lactococcus lactis strains as previously described (Mota-Meira et al., FEBS Lett. 1997 Jun. 30; 410(2-3):275-9) together with AID, (Besmer et al., Mol Cell Biol. 2006 June; 26(11):4378-85) or an AID homolog such as an Apobec-1 enzyme.
Screening can be accomplished by allowing the SHM mediated generated diversity to evolve L. lactis co-cultured with Gram positive bacterial targets that are currently poorly targeted by Nisin. Eventually strains of L. lactis will evolve that comprise mutated Nisin genes with enhanced activity against the chosen bacterial target.
Mass spectroscopy of the supernatant of evolved cell-cultures can be used to assess the progress of the process (i.e., identified novel lantibiotics with improved activity to a pathogen).

Example 7

Application of SHM to the Generation of Improved Receptors

Receptors bind ligands and encompass a broad genus of polypeptides including, but not limited to, cell-bound receptors such as antibodies (B cell receptors), T cell receptors, Fc receptors, G-coupled protein receptors, cytokine receptors, etc.
Fc receptors (FcR) are a family of related receptors that are specific for the Fc portion of immunoglobulin (Ig) molecules. Receptors have been defined for each of the immunoglobulin classes and, as such are defined by the class of Ig of which they bind: Fc gamma receptors (FcγR) bind gamma immunoglobulin (IgG), Fc epsilon receptors (FcεR) bind epsilon immunoglobulin (IgE), Fc alpha receptors (FcαR) bind alpha immunoglobulin (IgA).
Fcγ receptors are expressed on most hematopoietic cells and, via binding of IgG, play an important role in homeostasis of the immune system and unmodified host protection against infection. Three subfamily members of FcγR receptors have been identified: (1) FcγRI, which is a high affinity receptor for IgG; (2) FcγRII, which is a low affinity receptor for IgG that avidly binds to aggregates of immune complexes; and (3) FcγRIII, which is a low affinity receptor that binds to immune complexes. In spite of being structurally related, the receptors perform different functions.
Three subclasses of FcγR have been identified: FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16). Because each FcγR subclass is encoded by two or three genes, and alternative RNA spicing leads to multiple transcripts, a broad diversity in FcγR isoforms exists. The three genes encoding the FcγRI subclass (FcγRIA, FcγRIB and FcγRIC) are clustered in region 1q21.1 of the long arm of chromosome 1; the genes encoding FcγRII isoforms (FcγRIIA, FcγRIIB and FcγRIIC) and the two genes encoding FcγRIII (FcγRIIIA and FcγRIIIB) are all clustered in region 1q22. These different FcR subtypes are expressed on different cell types (reviewed in Ravetch and Kinet, Annu. Rev. Immunol 9.457-492 (1991)). For example, in humans, FcγRIIIB is found only on neutrophils, whereas FcγRIIIA is found on macrophages, monocytes, natural killer (NK) cells, and a subpopulation of T-cells. Notably, FcγRIIIA is the only FcR present on NK cells, one of the cell types implicated in ADCC.
FcγRI, FcγRII and FcγRIII are immunoglobulin superfamily (IgSF) receptors; FcγRI has three IgSF domains in its extracellular domain, while FcγRII and FcγRIII have only two IgSF domains in their extracellular domains.
Another type of Fc receptor is the neonatal Fc receptor (FcRn). FcRn is structurally similar to major histocompatibility complex (MHC) and consists of an α-chain non-covalently bound to 132-microglobulin.
The binding site on human and murine antibodies for FcγR have been previously mapped to the so-called “lower hinge region” consisting of residues 233-239 (EU index numbering as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Woof et al. Molec. Immunol 23:319-330 (1986); Duncan et al. Nature 332:563 (1988); Canfield and Morrison, J. Exp. Med. 173:1483-1491 (1991); Chappel et al., Proc. Natl. Acad. Sci USA 88:9036-9040 (1991).
Other amino acid residues considered to be involved in binding to FcγR include: G316-K338 (human IgG) for human FcγRI (Woof et al. Molec. Immunol 23:319-330 (1986)); K274-R301 (human IgG1) for human FcγRIII (based on peptides) (Sarcan et al. Molec. Immunol 21:43-51 (1984)); Y407-R416 (human IgG) for human FcγRIII (based on peptides) (Gergely et al. Biochem. Soc. Trans. 12:739-743 (1984)); as well as N297 and E318 (murine IgG2b) for murine FcγRII (Lund et al., Molec. Immunol, 29:53-59 (1992)). In one case, Pro331 in IgG3 is mutated to Ser, and the affinity of this modified IgG3 to target cells analyzed: the affinity is found to be six fold lower than that of unmutated IgG3, indicating the involvement of Pro331 in FcγRI binding. Morrison et al., Immunologist, 2:119-124 (1994); and Canfield and Morrison, J. Exp. Med. 173:1483-91 (1991).
Compounds that interfere with the dimerization interface between two FcγRII proteins can affect cellular signal transduction through one or both of the FcR proteins. Specifically, amino acid residues 117-131 and 150-164 of FcγRII are thought to be the interfacial area of the FcγIIa dimer, and compounds which can mimic or bind to these regions are considered to be good binding modulators (see U.S. Pat. No. 6,835,753).
In one aspect, synthetic polynucleotides encoding at least a portion of the Fc regions of a plurality of IgG molecules can be modified to increase susceptibility of that portion to somatic hypermutation using the methods described herein to create modified IgG molecules exhibiting increased binding affinity for this region of FcγRIIa.
In another aspect, polypeptides encoded by such SHM-modified polynucleotides are considered herein. In one embodiment, the SHM-modified IgG includes a synthetic polynucleotide encoding the hexapeptide sequence Phe121 to Ser126 or shorter segments spanning a region with significant hydrogen bonding interactions, in which the polynucleotide has been optimized for SHM. Such Ig molecules are suitable modulators of dimerization between two FcγRIIa molecules.
The upper portion of the FG loop of FcR has been shown to be involved in Ig binding as demonstrated by mutagenesis studies. The FG peptide strand contains an extended β-sheet which projects the amino acid side chains in the FG loop in a defined orientation such described in U.S. Pat. No. 6,675,105, entitled “3 Dimensional Structure and Models of Fc Receptors and Uses Thereof.” Molecules which can act as β-turn mimics and which present their side chains at the top of the FG loop in the same way as those in the receptor have also been found to be effective in modulating the FcR receptor activities.
In one aspect, polynucleotides encoding a PF peptide strand containing an extended β-sheet which projects the amino acid side chains in the FG loop in a defined orientation can be modified to increase susceptibility to somatic hypermutation using the methods described herein to create modified polypeptides exhibiting increased binding affinity for this region of FcR. In another aspect, polypeptides encoded by such SHM-modified polynucleotides are considered herein.
Fc receptors play roles in normal immunity and resistance to infection and provide the humoral immune system with a cellular effector arm. Binding of an Ig gamma (IgG) to FcγR can lead to disease indications that are associated with regulation by FcγR. For example, the autoimmune disease thrombocytopenia purpura involves tissue (platelet) damage resulting from FcγR-dependent IgG immune complex activation of platelets or their destruction by FcγR+ phagocytes. In addition, various inflammatory diseases are known to involve IgG immune complexes (e.g., rheumatoid arthritis and systemic lupus erythematosus), including type II and type III hypersensitivity reactions. Type II and type III hypersensitivity reactions are mediated by IgG, which can activate either complement-mediated or phagocytic effector mechanisms, leading to tissue damage.
Due to the role of FcRs in a variety of biological mechanisms, there is a need for compounds which affect the binding of immunoglobulins to FcR and which can be used to treat a variety of illnesses associated with regulation by FcRs.
Fc receptor modulators (modulators of Fc receptor binding of immunoglobulins) can modulate FcαR, FcεR and FcγR polypeptides. Polynucleotides encoding Fc receptor modulators of FcαR, FcεR and FcγR polypeptides can be modified to increase and/or decrease susceptibility of one or more portions of the polypeptide to somatic hypermutation using the methods described herein. Modified Fc receptor modulators made using such methods can be assayed to identify modulators exhibiting modified binding and activity. In one aspect, the FcR modulator interacts with a FcγRI, a FcγRII and/or a FcγRIII. In a further aspect, the FcR modulator interacts with a FcγRIIa, a FcγRIIb and/or a FcγRIIc. Fc receptor modulators provided herein can be used in a variety of applications including treatment or diagnosis of any disease where aggregates of antibodies are produced and where immune complexes are produced by contact of antibody with intrinsic or extrinsic antigen. Non-limiting treatments and diagnosis applicable by the Fc receptor modulators include immune complex diseases; autoimmune diseases including but not limited to rheumatoid arthritis, systemic lupus erythematosus, immune thrombocytopenia, neutropenia, hemolytic anemias; vasculitities including but not limited to polyarteritis nodosa, systemic vasculitis; xenograft rejection; and infectious diseases where FcR uptake of virus enhances infection including but not limited to flavivirus infections such as Dengue virus-dengue hemorrhagic fever and measles virus infection. The Fc receptor modulators can also be used to reduce IgG-mediated tissue damage and to reduce inflammation.
The SHM-modified Fc receptor modulators presented herein can also enhance leukocyte function by enhancing FcR function such as antibody dependent cell mediated cytotoxicity, phagocytosis and release of inflammatory cytokines. Treatments and diagnosis for enhanced FcR function include any infection where normal antibodies are produced to remove the pathogen; and any disease requiring FcR function where unmodified or recombinant antibodies can be used in treatment such as cancer and infections. For example, an immunoglobulin (e.g., normal Ig or SHM-modified Ig) can be administered in combination with a Fc receptor modulator (e.g., a SHM modified modulator) to enhance the effect of the immunoglobulin treatment.
In another aspect, FcR are involved in the complement pathway via C1q Binding. C1q and two serine proteases (C1r and C1s) form the complex C1, the first component of the Complement Dependent Cytotoxicity (CDC) pathway. To activate the complement cascade, it is necessary for C1q to bind to at least two molecules of IgG1, IgG2, or IgG3, but only one molecule of IgM, attached to an antigenic target (Ward and Ghetie, Therapeutic Immunology 2:77-94 (1995) at page 80). Based upon the results of chemical modifications and crystallographic studies, Burton et al. (Nature, 288:338-344 (1980)) proposed that the binding site for the complement subcomponent C1q on IgG involves the last two (C-terminal) β-strands of the C _H2 domain. Burton later proposed (Molec. Immunol, 22(3):161-206 (1985)) that the region including amino acid residues 318 to 337 might be involved in complement fixation. In one aspect provided herein are SHM-modified Ig molecules (e.g., IgG1, IgG2, or IgG3 or IgM) that have been modified such that they exhibit increased affinity for the antigenic target, increased binding to C1q, or both. Such modified Ig molecules can be tested in one or more art-recognized assays to evaluate changes in binding and/or biological activity compared to the starting Ig.
Assays for testing a SHM-modified Fc receptor modulator include those known in the art for testing compounds that modulate Fc receptor activity such as, for example, binding assays, platelet aggregation inhibition assays, assessment of ADCC activity, assessment of C1q binding, as well as other assays to test binding and function as described below. Binding and activity of SHM-modified Fc receptor modulators can be compared to control Ig molecules.
Binding Assay
In one example, the interaction between recombinant soluble FcγRIIa and human immunoglobulin in the presence of SHM-modified Fc receptor modulators are investigated using a BIAcore 2000 biosensor (Pharmacia Biotech, Uppsala, Sweden) at 22° C. in Hepes buffered saline [HBS: 10 mM Hepes (pH 7.4), 150 mM NaCl, 3.4 mM EDTA, 0.005% Surfactant P20 (Pharmacia)]. Monomeric human IgG1, IgG3, and IgE (50 μg/mL) (non-specific binding control) are covalently coupled to the carboxymethylated dextran surface of the CM-5 sensor-chip (BIAcore, Uppsala, Sweden) using the amine coupling protocol (BIAcore, Uppsala, Sweden). An additional channel is chemically treated using the coupling protocol. Recombinant soluble FcγRIIa is used at a concentration of 125 μg/mL, which is equivalent to 50% binding capacity. Recombinant soluble FcγRIIa is preincubated with each of the SHM-modified Fc receptor modulators at room temperature for 30 minutes before being injected over the sensor-chip surface for 1 minute at 10 μL/min followed by a 3 minute dissociation phase. All surfaces are regenerated with 50 mM diethylamine (about pH 11.5), 1 M NaCl between each of the compounds being analyzed. The maximum response for each interaction is measured. Non-specific binding responses (IgE channel) are subtracted from binding to IgG1 and IgG3. Measurements are corrected for differences in buffer composition between the SHM-modified Fc receptor modulators and receptor.
Platelet Aggregation Inhibition
Platelet aggregation inhibition can be tested using art-recognized assays such as described herein. Briefly, the procedure involves adding a test compound to a mixture of the platelets and HAGG compared to a control compound. This procedure illustrates the ability of the test compound to inhibit platelet aggregate formation as well as its ability to break apart the platelet aggregates which have formed prior to the addition of-the compound compared to controls.
Platelets express a single class of gamma receptors, FcγRIIa. Following the cross-linking of FcγRIIa, platelets undergo a variety of biochemical and cellular modifications that culminate in aggregation. The capacity of the compounds to inhibit platelet activation is measured using an assay that specifically measures platelet aggregation.
Briefly, platelets are isolated as follows: 30 mL of fresh whole blood is collected into citrated collection vials and centrifuged at 1000 rpm for ten minutes. The platelet rich plasma is separated and centrifuged at 2000 rpm for five minutes in four tubes. The supernatants are removed and the platelets are gently re-suspended in 2 mL of Tyrodes buffer-per tube (137 mM NaCl, 2.7 mM KCl, 0.36 mM NaH₂PO₄, 0.1% dextrose, 30 mM sodium citrate, 1.0 mM MgCl₂.6H₂O, pH 6.5) and centrifuged again at 2000 rpm for five minutes. The supernatants are again removed and platelets are re-suspensed in 0.5 mL of Hepes containing Tyrodes buffer per tube (137 mM NaCl, 2.7 mM KCl, 0.36 mM NaH₂PO₄, 0.1% dextrose, 5 mM Hepes, 2 mM CaCl₂1.0 mM MgCl₂.6H₂O, pH 7.35). The platelet count is determined using a haematolog analyzer (Coulter) and adjusted to a concentration of approximately 100×10⁵platelets/mL using the Hepes containing Tyrodes buffer.
For each aggregation experiment, a mixture of 50 μL of a Fc receptor agonist, heat aggregated gamma globulin (“HAGG,” 200 μg/mL) or collagen (2 μg/mL) is incubated with 50 μL of phosphate buffered saline (“PBS”: 3.5 mM NaH₂PO₄, 150 mM NaCl) or BRI compound (5 mg/mL in PBS) for 60 minutes at room temperature. The assay is then performed using a two cell aggregometer at 37° C. as follows: glass cuvettes are placed in holders and pre-warmed to 37° C. and 400 μL of the platelet suspension added. After a stable baseline is reached, 100 μL of HAGG:PBS, HAGG:BRI compound or collagen:PBS, collagen:BRI compound are added to the platelet suspension. The subsequent aggregation of the platelets is monitored for 15 minutes or until aggregation is complete. The rate of aggregation is determined by measuring the gradient of the aggregation slope.
Assessment of ADCC Activity
To assess ADCC activity of a SHM-modified Fc receptor modulator, an in vitro ADCC assay can be performed using varying effector:target ratios. Useful “effector cells” for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the polypeptide variant can be assessed in vivo, e.g., in a animal model such as that disclosed by Clynes et al. PNAS (USA) 95:652-656 (1998).
For example, to prepare chromium 51-labeled target cells, tumor cell lines are grown in tissue culture plates and harvested using sterile 10 mM EDTA in PBS. SK-BR-3 cells, a 3+ HER2-overexpressing human breast cancer cell line, are used as targets in all assays. The detached cells are washed twice with cell culture medium. Cells (5×10⁶) are labeled with 200 μCi of chromium⁵¹(New England Nuclear/DuPont) at 37° C. for one hour with occasional mixing. Labeled cells are washed three times with cell culture medium, then are re-suspended to a concentration of 1×10⁵cells/mL. Cells are used either without opsonization, or are opsonized prior to the assay by Incubation with rhuMAb HER2 wild-type (HERCEPTIN™) or SHM-modified FcR modulators in PBMC assay or in NK assay.
Peripheral blood mononuclear cells are prepared by collecting blood on heparin from normal healthy donors and dilution with an equal volume of phosphate buffered saline (PBS). The blood is then layered over LYMPHOCYTE SEPARATION MEDIUM™ (LSM: Organon Teknika) and centrifuged according to the manufacturer's instructions. Mononuclear cells are collected from the LSM-plasma interface and are washed three times with PBS. Effector cells are suspended in cell culture medium to a final concentration of 1×10⁷cells/mL.
After purification through LSM, natural killer (NK) cells are isolated from PBMCs by negative selection using an NK cell isolation kit and a magnetic column (Miltenyi Biotech) according to the manufacturer's instructions. Isolated NK cells are collected, washed and re-suspended in cell culture medium to a concentration of 2×10⁶cells/mL. The identity of the NK cells is confirmed by flow cytometric analysis.
Varying effector:target ratios are prepared by serially diluting the effector (either PBMC or NK) cells two-fold along the rows of a microtiter plate (100 μL final volume) in cell culture medium. The concentration of effector cells ranges from 1.0×10⁷/mL to 2.0×10⁴/mL for PBMC and from 2.0×10⁶/mL to 3.9×10³/mL for NK. After titration of effector cells, 100 μL of chromium 51-labeled target cells (opsonized or non-opsonized) at 1×10⁵cells/mL are added to each well of the plate. This results in an initial effector:target ratio of 100:1 for PBMC and 20:1 for NK cells. All assays are run in duplicate, and each plate contains controls for both spontaneous lysis (no effector cells) and total lysis (target cells plus 100 μL) 1% sodium dodecyl sulfate, 1 N sodium hydroxide). The plates are incubated at 37° C. for 18 hours, after which the cell culture supernatants are harvested using a supernatant collection system (Skatron Instrument, Inc.) and counted in a Minaxi auto-gamma 5000 series gamma counter (Packard) for one minute. Results are then expressed as percent cytotoxicity using the formula: % Cytotoxicity=(sample cpm−spontaneous lysis)/(total lysis−spontaneous lysis)×100 Four-parameter curve-fitting is then used to evaluate the data (KaleidaGraph 3.0.5).
C1q Binding
The ability of the variant to bind C1q and mediate complement dependent cytotoxicity (CDC) can be assessed. To determine C1q binding, a C1q binding ELISA can be performed. Briefly, assay plates are coated overnight at 4° C. with SHM modified Fc receptor modulator or control polypeptide in coating buffer. The plates are then be washed and blocked. Following washing, an aliquot of human C1q is added to each well and incubated for 2 hrs at room temperature. Following a further wash, 1000 of a sheep anti-complement C1q peroxidase conjugated antibody is added to each well and incubated for 1 hour at room temperature. The plate is then washed with wash buffer and 100 μl of substrate buffer containing OPD (O-phenylenediamine dihydrochloride (Sigma)) is added to each well. The oxidation reaction, observed by the appearance of a yellow color, is allowed to proceed for 30 minutes and stopped by the addition of 100 μl of 4.5 NH₂SO₄. The absorbance is then read at (492-405) nm.
Binding and Biological Activity
The SHM modified Fc receptor modulator can then be subjected to one or more assays to evaluate any change in binding and biological activity compared to the starting polypeptide.
In one example, the SHM modified Fc receptor modulator essentially retains the ability to bind receptor compared to the non-modified polypeptide, i.e. the binding capability is no worse than about 20 fold, e.g. no worse than about 5 fold of that of the non-modified polypeptide. The binding capability of the SHM modified Fc receptor modulator is determined using techniques such as fluorescence activated cell sorting (FACS) analysis or radioimmunoprecipitation (RIA), for example.
To determine receptor binding, a polypeptide comprising at least the binding domain of the receptor of interest (e.g. the extracellular domain of an α subunit of an FcR) is coated on solid phase, such as an assay plate. The binding domain of the receptor alone or a receptor-fusion protein is coated on the plate using standard procedures. Examples of receptor-fusion proteins include receptor-glutathione S-transferase (GST) fusion protein, receptor-chitin binding domain fusion protein, receptor-hexaHis tag fusion protein (coated on glutathione, chitin, and nickel coated plates, respectively). Alternatively, a capture molecule is coated on the assay plate and used to bind the receptor-fusion protein via the non-receptor portion of the fusion protein. Examples include anti-hexaHis F(ab′)₂coated on the assay plate used to capture receptor-hexaHis tail fusion or anti-GST antibody coated on the assay plate used to capture a receptor-GST fusion. In other embodiments, binding to cells expressing at least the binding domain of the receptor is evaluated. The cells can be naturally occurring hematopoietic cells that express the FcR of interest or can be transformed with nucleic acid encoding the FcR or a binding domain thereof such that the binding domain is expressed at the surface of the cell to be tested.
The immune complex described herein above is added to the receptor-coated plates and incubated for a sufficient period of time such that the polypeptide binds to the receptor. Plates are then washed to remove unbound complexes, and binding of the analyte is detected according to known methods. For example, binding is detected using a reagent (e.g. an antibody or fragment thereof which binds specifically to the analyte, and which is optionally conjugated with a detectable label—detectable labels and methods for conjugating them to polypeptides are described below in the section entitled “Non-Therapeutic Uses for the Polypeptide Variant”).
Low Affinity Receptor Binding Assay
Binding of an IgG Fc region to recombinant FcγRIIA, FcγRIIB and FcγRIIIA α subunits expressed as His6-glutathione S transferase (GST)-tagged fusion proteins can be determined. Since the affinity of the Fc region of IgG1 for the FcγRI is in the nanomolar range, the binding of SHM-modified IgG1 Fc can be measured by titrating monomeric IgG and measuring bound IgG with a polyclonal anti-IgG in a standard ELISA format. The affinity of the other members of the FcγR family, i.e. FcγRIIA, FcγRIIB and FcγRIIIA for IgG, is however in the micromolar range and binding of monomeric IgG1 for these receptors can not be reliably measured in an ELISA format.
FcγR Binding ELISAs
FcγRI α subunit-GST fusion is coated onto Nunc F96 maxisorb plates (cat no. 439454) by adding 100 of receptor-GST fusion at 1 μg/ml in PBS and incubated for 48 hours at 4° C. Prior to assay, plates are washed 3× with 250 μl of wash buffer (PBS pH 7.4 containing 0.5% TWEEN 20) and blocked with 250 μl of assay buffer (50 mM Tris buffered saline, 0.05% TWEEN 20, 0.5% RIA grade bovine albumin (Sigma A7888), and 2 mM EDTA pH 7.4). Samples diluted to 10 μg/ml in 1 ml of assay buffer are added to FcγRI α subunit coated plates and incubated for 120 minutes at 25° C. on an orbital shaker. Plates are washed 5× with wash buffer to remove unbound complexes and IgG binding is detected by adding 100 μl horse radish peroxidase (HRP) conjugated goat anti-human IgG heavy chain specific (Boehringer Mannheim 1814249) at 1:10,000 in assay buffer and incubated for 90 min at 25° C. on an orbital shaker. Plates are washed 5× with wash buffer to remove unbound HRP goat anti-human IgG and bound anti-IgG is detected by adding 100 μl of substrate solution (0.4 mg/ml o-phenylenedaimine dihydrochloride, Sigma P6912, 6 mM H₂O₂in PBS) and incubating for 8 min at 25° C. Enzymatic reaction is stopped by the addition of 10004.5N NH₂SO₄and calorimetric product is measured at 490 nm on a 96 well plate densitometer (Molecular Devices). Binding of SHM-modified FcR modulators is expressed as a percent of the parent molecule (i.e., wild-type or previously modified).
FcRn Binding ELISA
For measuring FcRn binding activity of IgG variants, ELISA plates are coated with 2 μg/ml streptavidin (Zymed, South San Francisco) in 50 mM carbonate buffer, pH 9.6, at 4° C. overnight and blocked with PBS-0.5% BSA, pH 7.2 at room temperature for one hour. Biotinylated FcRn (prepared using biotin-X-NHS from Research Organics, Cleveland, Ohio and used at 1-2 μg/ml) in PBS-0.5% BSA, 0.05% polysorbate 20, pH 7.2, is added to the plate and incubated for one hour. Two fold serial dilutions of IgG standard (1.6-100 ng/ml) or variants in PBS-0.5% BSA, 0.05% polysorbate 20, pH 6.0, are added to the plate and incubated for two hours. Bound IgG is detected using peroxidase labeled goat F(ab′)₂anti-human IgG F(ab′)₂in the above pH 6.0 buffer (Jackson ImmunoResearch, West Grove, Pa.) followed by 3,3′,5,5′-tetramethyl benzidine (Kirgaard & Perry Laboratories) as the substrate. Plates are washed between steps with PBS-0.05% polysorbate 20 at either pH 7.2 or 6.0. Absorbance is read at 450 nm in a Vmax plate reader (Molecular Devices, Menlo Park, Calif.). Titration curves are fit with a four-parameter nonlinear regression curve-fitting program (KaleidaGraph, Synergy software, Reading, Pa.). Concentrations of IgG variants corresponding to the mid-point-absorbance of the titration curve of the standard are calculated and then divided by the concentration of the standard corresponding to the mid-point absorbance of the standard titration curve.

Example 8

Use of AID for Enzymatic Pathway Optimization

Using the SHM systems described herein, polynucleotides encoding one or more enzymes can be simultaneously modified via somatic hypermutation to increase the speed or efficiency of metabolic conversions. Enzymes involved in pathways of interest include, for example, those associated with yeast fermentation, antibiotics and clean-up of oil spills (see, for example, Ho, N. W. Y., Chen Z. and A. Brainard. 1998. Appl. and Environ. Microbiol. 64:1852-1859; and Sonderegger M. and U. Sauer. 2003. Appl. And Environ. Microbiol. 69:1990-1998).
In one aspect, to develop a commercially viable yeast fermentation system for converting plant-based cellulosic biomass to ethanol, Saccharomyces cerevisiae that have been genetically engineered to use xylose as a substrate can be further modified by step-wise induction of somatic hypermutation followed by selection for the ability to grow anaerobically using xylose as the sole carbon source present in the growth medium.
One advantage of ethanol is that it is a non-fossil biofuel that produces less pollution than gasoline. Another advantage is that ethanol can be produced from readily available, renewable cellulosic biomass from plant material. Cellulosic biomass is composed of pentose sugars (mainly glucose and xylose). A major obstacle to developing a commercial process for converting cellulosic biomass to ethanol, however, is that Saccharomyces species of yeast (the microorganisms currently used for large scale industrial production of ethanol from glucose) are currently unable to ferment ethanol from xylose with high yields and specific rates.
Metabolic engineering has been used successfully to develop strains of Saccharomyces cerevisiae that can ferment both xylose and arabinose to ethanol. However, none of the recombinant strains or any other naturally-occurring yeast strains have been able to grow anaerobically on xylose alone.
Provided herein is a method of somatic hypermutation of one or more of the genes of the xylose utilization pathway to create recombinant strains of anaerobic xylose-utilizing eukaryotes such as Saccharomyces cerevisiae that can grow anaerobically on xylose alone and ferment xylose and arabinose to ethanol.
In one aspect, step-wise somatic hypermutation can be used to develop a yeast strain that is capable of anaerobic growth on xylose alone. In one non-limiting embodiment, a strain of Saccharomyces cerevisiae that over-expresses the three enzymes from Pichia stipitis that act sequentially to convert xylose to xylulose 5-phosphate (a substrate that Saccharomyces species are able to ferment). The resulting xylose-utilizing yeast strain has utility for significantly improving ethanol fermentations and commercially viable ethanol production from plant-based cellulosic biomass.
Briefly, an inducible activation-induced cytidine deaminase (AID) SHM-resistant polynucleotide sequence is introduced into the starting yeast strain by stable chromosomal insertion and then step-wise somatic hypermutation by AID is induced with monitoring of culture growth and xylose utilization between steps. The growth rate of the culture is monitored by measuring optical density at 600 nanometers. Xylose utilization is monitored using a commercially available enzymatic kit (Medichem, Steinenbronn, Germany) to measure xylose in the culture medium.
The next step is initiated when the culture growth rate and xylose utilization increase, for example, about 2-fold. An aerobic chemostat culture containing 5 grams xylose per liter and 1 gram glucose per liter is prepared. AID expression is induced for about 10, about, 15, or about 20 generations (i.e., approximately 6-12 days). The number of generations for induction of AID expression can be determined based on reversion data, DT40 screening data (Cumbers et al. Nat. Biotechnol. 2002 November; 20(11): 1129-1134)) and the yeast growth rate of 1.73 generations/day. At the point where the culture growth rate increases and the xylose is consumed, a culture aliquot is withdrawn and a new aerobic chemostat culture containing 5 grams per liter xylose as the sole carbon source is inoculated. AID expression is induced in this culture for about 10, about, 15, or about 20 generations. Again, the culture is monitored for growth rate and xylose concentration. When a growing population that consumes the xylose is obtained, the aeration rate is dropped to less than 1 milliliter per minute and AID expression is again induced for about 10, about, 15, or about 20 generations. When the growth rate of the culture stabilizes, a culture aliquot is withdrawn and a new aerobic chemostat culture containing 5 grams per liter xylose as the sole carbon source is inoculated and grown in the absence of any aeration and with induction of AID expression for about 10, about, 15, or about 20 generations. The culture growth rate and xylose utilization are monitored.
When the growth rate of the culture starts to increase and the xylose concentration decreases, a culture aliquot is withdrawn and a strict anaerobic batch culture in a 17 milliliter Pyrex glass tube sealed with butyl rubber septa and plastic screw cap is inoculated with xylose as the sole carbon source. AID expression is induced for about 10, about, 15, or about 20 generations and the culture is monitored for growth rate and xylose utilization. When the culture growth rate increases and the xylose concentration decreases an aliquot of the population is plated on anaerobic minimal medium agar plates containing 20 grams per liter xylose as the sole carbon source. The plates are incubated at 30° Celsius in sealed jars using, for example, the GasPack Plus System (Becton Dickinson) to provide an anaerobic atmosphere. The anaerobic atmosphere is monitored using indicator strips (Becton Dickinson).
The fermentation performance of the parental strain along with the evolved populations, and 15 clones isolated from the anaerobic xylose plates are compared in anaerobic batch cultures with 50 grams per liter of glucose and 50 grams of xylose per liter. The growth rate, xylose and glucose utilization of the cultures are monitored. Glucose utilization is determined using a commercial kit (Beckman).
The last step of SHM is to take the clone that has the best growth and xylose utilization characteristics, induce AID expression, and grow in multiple serial batch cultures for about 15 generations. Twenty clones isolated from this culture are then grown on xylose as the sole carbon source in strictly anaerobic culture conditions. The performance of clones with the fastest growth rates are further evaluated in anaerobic xylose batch cultures.

Example 9

Application of SHM for Affinity Maturation of Antibodies

As described previously, antibodies provide an unmodified template through which SHM can be applied to create mutant proteins with enhanced properties. Such improved antibodies can be selected based upon affinity selection, for example via FACS or via binding to magnetic beads.
Antibodies directed towards hen egg lysozyme (HyHel) represent an extremely well characterized system that enables the testing and optimization of the mutation and selection systems of the present invention. Specifically the HyHel antibodies enable the testing of a number of highly related antibodies that exhibit a well defined range of affinities and have characterized sequences and binding properties. For example, the following antibodies, and sequence variants thereof (muteins) have fully defined sequences starting from the germline sequence to the fully affinity mature antibody, see, e.g., Pons et al., (1999) Protein Science 8:958-68; and Smith-Gill et al., (1984) J. Immunology 132:963.

TABLE 16

Hen Egg Lysozyme antibody constructs (HyHEL)

Construct	Published		Light Chain
Name	Affinity	Heavy Chain Identity	Identity

HyHEL10	0.03 nM	Heavy Chain (“wild type”)	Light Chain
			(“wild type”)
Mutein 1	66 nM	Heavy Chain (“wild type”)	Light Chain
			(Y50A)
Mutein 2	167 nM	Heavy Chain (“wild type”)	Light Chain
			(N32A)
Mutein 3	460 nM	Heavy Chain (“wild type”)	Light Chain
			(N31E)
Mutein 4	800 nM	Heavy Chain (Y33A)	Light Chain
			(“wild type”)
Mutein 5	7,000 nM	Heavy Chain (Y50A)	Light Chain
			(“wild type”)
Germline	unknown	(Germline sequence)	(Germline
			sequence)

To further optimize the system of the present invention, a range of high and low affinity HyHel constructs are made and cloned into the expression vectors of the present invention. These vectors are then transfected into mutator cells expressing AID and selected using magnetic beads. Wild type HyHEL constructs are used as positive controls to optimize binding conditions and validate assay methodology.
A. Synthesis and Cloning of (“Wild Type”) HyHEL10 Heavy and Light Chain Constructs.
The prototypic HyHEL10 heavy chain and light chain expression vectors are created starting from the expression vector AB102, (as described previously), using standard molecular genetic manipulations as follows:
The puromycin resistance marker in AB102 is replaced with cold bsd or cold hyg using the NgoMIV and XbaI restriction sites, to generate the vectors AB187 and AB185, respectively.
A slightly longer, transcriptionally more robust version of the CMV promoter is exchanged for the original sequence found in AB102 using NheI (the mcs2 restriction site most proximal to the CMV promoter) and SbfI (the most CMV-proximal mcs1 site). The original AB102 CMV promoter included 553 bp of the unmodified CMV sequence upstream from the first T of the TATA box, while the AB187 version includes 645 bp upstream from the first T of the TATA box.
The nucleotide sequences for the “wild type” HyHEL heavy and light chains (as disclosed above) are synthesized at DNA 2.0, (Menlo Park, Calif.). For cloning purposes, the heavy chain is bordered by BglII and AscI, and the light chain is bounded by restriction sites SacI and AscI.
In order to express HyHEL10 IgG and its muteins on the cell surface, the heavy chain is created as a chimeric molecule with the following features:
Kozak consensus sequence; HyHEL10 heavy chain variable region; full-length murine IgG1 constant region; XhoI site; Murine H2kk (MHC type I) peri-transmembrane domain, transmembrane domain and cytoplasmic domain. The H2kk sequences is determined from accession number AK153419 at the National Center for Biotechnology Information (NCBI) nucleotide database.
The nucleotide sequence of the full length chimeric, cell-surface associated HyHEL10 heavy chain is as listed below:
In this sequence, the BglII site is underlined; Kozak sequence is underlined and italicized; stop codon is underlined and bolded; XhoI site is indicated by boxed nucleotides; Double underlined sequences are derived from H2kk. The AscI cloning site 3′ to the TGA stop codon is indicated by italicized nucleotides.
The amino acid sequence of the chimeric, cell-surface associated HyHEL10 heavy chain is as listed below. The 2 amino acids (leu-glu) encoded by the synthetic XhoI site are marked by bold-and-underline; the bold-underline Glu also represents the most amino proximal amino acid of the H2kk domain; double underline indicates the putative transmembrane domain; asterisk indicates stop codon.

(SEQ ID NO: 292)

MNKLLCCALVFLDISIKWTTQDVQLQESGPSLVKPSQTLSLTCSVTGDSI

TSDYWSWIRKFPGNRLEYMGYVSYSGSTYYNPSLKSRISITRDTSKNQYY

LDLNSVTTEDTATYYCANWDGDYWGQGTLVTVSAAKTTPPSVYPLAPGSA

AQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSS

SVTVPSSPRPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSS

VFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQT

QPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKT

KGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAE

NYKNTQPIMNTNGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEK

SLSHSPGK LE PPPSTVSNMATVAVLVVLGAAIVTGAVVAFVMKMRRRNTG

GKGGDYALAPGSQTSDLSLPDCKVMVHDPHSLA*.

The amino acid and nucleotide sequence of the (“wild type”) HyHEL kappa light chain.
Amino acid sequence of the HyHEL kappa light chain. Asterisk indicates stop codon.

(SEQ ID NO: 338)

MNKLLCCALVFLDISIKWTTQDIVLTQSPATLSVTPGNSVSLSCRASQSI

GNNLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGTDFTLSINSVE

TEDFGMYFCQQSNSWPYTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGG

ASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTL

TLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC*.

The nucleotide sequence of the HyHEL kappa light chain. Start and stop codons are underlined. SacI and AscI cloning sites are bolded.

(SEQ ID NO: 293)

GAGCTCACCACAATGAACAAGTTGCTGTGCTGCGCGCTCGTGTTTCTGGA

CATCTCCATTAAGTGGACCACCCAGGATATTGTGCTAACTCAGTCTCCAG

CCACCCTGTCTGTGACTCCAGGAAATAGCGTCAGTCTTTCCTGCAGGGCC

AGCCAAAGTATTGGCAACAACCTACACTGGTATCAACAAAAATCACATGA

GTCTCCAAGGCTTCTCATCAAGTATGCTTCCCAGTCCATCTCTGGGATCC

CCTCCAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACTCTCAGTATC

AACAGTGTGGAGACTGAAGATTTTGGAATGTATTTCTGTCAACAGAGTAA

CAGCTGGCCTTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGGG

CTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTA

ACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAA

AGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCG

TCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATG

AGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTA

TACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCT

TCAACAGGAATGAGTGTTGA GGCGCGCC.

Muteins of the “wild type” heavy and light chains, as well as the germline sequence, as described below in Table 17, were created using site directed mutagenesis and their sequence confirmed by sequencing.

TABLE 17

Hen Egg Lysozyme antibody constructs with measured affinities

Mutations	DNA Sequence	Kd	koff	kon

wt LC/wt HC	GGC30-AAC31-AAC32-CTA33	3.93E−11	8.6E−05	2.2E+06

Light chain variants

LC G30(silent)N31A/wt	GGA30-GCT31-AAC32-CTA33	1.48E−09	8.29E−03	5.61E+06
HC

N31G LC/wt HC	GGC30-GGT31-AAC32-CTA33	2.78E−09	1.21E−02	4.33E+06

N31S LC/wt HC	GGC30-AGC31-AAC32-CTA33	7.10E−10	9.70E−04	1.40E+06

N32S LC/wt HC	GGC30-AAC31-AGC32-CTA33	1.00E−10	1.90E−04	1.90E+06

N32G LC/wt HC	GGC30-AAC31-GGT32-CTA33	6.29E−10	2.85E−03	4.53E+06

N31SN32S/wt HC	GGC30-AGC31-AGC32-CTA33	2.50E−09	6.10E−03	2.40E+06

LC L33(silent)/wt HC	GGC30-AAC31-AAC32-TTA33	5.96E−11	9.33E−05	1.56E+06

N31D LC/wt HC	GGC30-GAT31-AAC32-CTA33	1.1E−10

Heavy chain variants

wt LC/Y50A HC	GGG49-GCC50-GTA51	Not detectable

wt LC/Y33A HC	GAT32-GCC33-TGG34	2.0E−08	4.45E−02	2.13E+06

Mixed heavy and light chain variants

LC N31G/Y33A HC	see above	7.0E−06

LC N32G/Y33A HC	see above	2.00E−08

Nucleotides in bold represent codons in which defined mutations were made to introduce SHM optimized codons to increase somatic hypermutation compared to the “wild type” (HyHEL10) sequence (“wt”), as defined below. LC=Light Chain; HC=Heavy Chain. Also shown are the measured affinities of each mutant, obtained via BIACORE analysis.
These positions have been previously shown to be important for binding and to have been naturally mutated from the corresponding germline sequence during somatic hypermutation. Specifically, the light chain sequence of HyHEL10 contains the residue Asn31 located within CDR1 that makes a thermodynamically important contact to the HEL antigen residue Lys96. The Gly31 mutant (codon GGT) of HyHEL10 has a measured dissociation constant of around 2.5 nM, whereas the Asp31 (codon GAT) mutant of HyHEL10 has a measured dissociation constant of around 110 pM, and the wild-type Asn31 (codon AAC) of HyHEL10 has a measured dissociation constant of around 30 pM.
B. Transfection of Cells
Hek 293 cells are plated at 4×10⁵/well, in 6-well microtiter dish. After 24 hrs., transfections are performed using Fugene6 reagent from Roche Applied Sciences (Indianapolis, Ind.) at a reagent-to-DNA ratio of 3 μL:1 μg DNA per well with the expression vectors AB187 and AB185 that comprised the HyHEL heavy and light chains and conferred blasticidin and hygromycin resistance respectively. Transfections are carried out in accordance with manufacturer's protocol.
C. Selection of Peptides
An unlabeled and biotinylated monomeric peptide sequence that comprises the majority of the hen egg lysozyme (HEL) binding surface is synthesized. Two dimeric peptide sequences are also synthesized to compare whether presenting the peptide as a dimer would enhance antibody binding by increasing the avidity of the antibody-peptide interaction. A tandem dimer and a branched multiple antigenic peptide (MAP) dimer are also tested. Peptides as well as biotinylatd or unlabeled HEL protein are coupled to paramagnetic polystyrene microparticle surfaces that had been modified with functional groups or coated with streptavidin (Invitrogen, 1600 Faraday Ave., PO Box 6482, Carlsbad, Calif. 92008).
D. Coupling HEL Protein and Peptides to Tosylactivated Microparticles
The HEL protein and peptides are coupled to 2.8 micron Tosylactivated paramagnetic polystyrene microparticles in a 1.5 ml microcentrifuge tube (Nilsson K and Mosbach K. “p-Toluenesulfonyl chloride as an activating agent of agarose for the preparation of immobilized affinity ligands and proteins.” Eur. J. Biochem. 1980:112: 397-402). The microparticles (2e09 microparticles/milliliter) are washed and re-suspended in 100 mM borate buffer, pH 9.5 at a concentration of 1e09 microparticles/ml. Eleven nanomoles of peptide or 6 ug/ml HEL are added to the microparticles and the microparticle/peptide mixture is incubated at room temperature for at least 48 hours with slow tilt rotation. After incubation, the supernatant is removed and the microparticles are washed with 1 ml phosphate buffered saline solution (PBS), pH 7.2 containing 1% (weight/volume) BSA. Finally, the microparticles are re-suspended in 1 ml PBS solution, pH 7.2 containing 1% (weight/volume) BSA.
E. Coupling Biotinylated HEL Protein and Peptides to Streptavidin-Conjugated Microparticles
Another option is to couple biotinylated peptides to paramagnetic polystyrene microparticles whose surfaces have been covalently linked with a monolayer of streptavidin. Briefly, the streptavidin microparticles are washed, re-suspended in 1 ml PBS solution, pH 7.2 containing 1% (weight/volume) BSA and 33 picomoles of biotinylated peptide or approximately 10 μg/ml biotinylated HEL are then added to the microparticle solution. The microparticle/peptide solution is incubated for 30 minutes at room temperature with slow tilt rotation. After coupling, the microparticles are washed and re-suspended to a final microparticle concentration of 1e09 microparticles/ml. (Argarana C E, Kuntz I D, Birken S, Axel R, Cantor C R. Molecular cloning and nucleotide sequence of the streptavidin gene. Nucleic Acids Res. 1986; 14(4):1871-82; Pahler A, Hendrickson W A, Gawinowicz Kolks M A, Aragana C E, Cantor C R. Characterization and crystallization of core streptavidin. Biol Chem 1987:262(29):13933-7).
F. Cell Selection
Transfected HEK 293 cells expressing the 30 pM and 800 nM affinity HyHEL antibody heavy and light chains are screened in order to isolate cells that bind to the peptide-conjugated paramagnetic microparticles. A similar control cell line that did not express antibody is used as a negative control for the selections.
The cells are washed with an equal volume of PBS solution, pH 7.2 and re-suspended in PBS solution, pH 7.2 containing 1% (weight/volume) BSA to a final cell concentration of 1e07 cells/ml. The cells are pre-cleared by adding 1e06 naked microparticles to the cells and incubating on a rotator at 4° C. for 30 minutes. The unbound cells are gently transferred to a new tube. Peptide-conjugated or naked microparticles (1e07) are transferred into the tube with the cells and the cell:microparticle mixture is incubated on a rotator at 4° C. for 30 minutes. The unbound cells are then removed and the microparticle:cell mixture is washed with cold PBS/1% BSA. The microparticles and attached cells are re-suspended in 100 μl cell culture medium and grown initially in one well of a 96-well plate. The number of microparticle-bound cells is determined and the cells are expanded until the next round of selection. The number of microparticle-bound cells selected on the peptide-conjugated microparticles is compared with cells bound to the naked microparticles and to the cells that do not express antibody.
FIG. 48A shows cells expressing the 30 pM HyHEL antibody (dark gray) or no antibody (light gray) after selection by incubating with streptavidin microparticles conjugated to the mature HEL protein (Protein), HEL peptide monomer (Monomer), tandem HEL dimer (Tandem), HEL MAPS dimer (MAPS) or naked unconjugated streptavidin microparticles (Naked). The whole HEL protein- and HEL peptide monomer-conjugated microparticles are effective in isolating cells expressing the 30 pM HyHEL antibody in this experiment.
In FIG. 48B, cells expressing the 800 pM HyHEL antibody (dark gray) or no antibody (light gray) are selected by incubating with tosylactivated microparticles conjugated to either the mature HEL protein (Protein) or naked unconjugated tosylactivated microparticles (Naked). The whole HEL protein-conjugated microparticles are effective in isolating cells expressing the 800 pM HyHEL antibody in this experiment.
In FIG. 49A, cells expressing the 30 pM HyHEL antibody are selected by incubating with streptavidin microparticles conjugated to the mature HEL protein (Protein), HEL peptide monomer (Monomer), tandem HEL dimer (Tandem), HEL MAPS dimer (MAPS) or naked unconjugated streptavidin microparticles (Naked). The whole HEL protein- and HEL peptide monomer-conjugated microparticles are effective in isolating cells expressing the 30 pM HyHEL antibody in this experiment.
In FIG. 49B, cells expressing the 800 pM HyHEL antibody are selected by incubating with tosylactivated microparticles conjugated to either the mature HEL protein (Protein) or naked unconjugated tosylactivated microparticles (Naked). The whole HEL protein-conjugated microparticles are effective in isolating cells expressing the 800 pM HyHEL antibody in this experiment.
G. In Vitro Affinity Maturation
A clonal population of HyHEL10 Gly31 (GGT) mutants (presented on the surface of HEK293 cells) was subjected to iterative rounds of FACS based selection against 50 pM FITC-HEL in the presence of SHM as described below to determine how effectively somatic hypermutation could restore the affinity of a relatively weakly binding mutant.
1. Transfection of Cells
A stable HEK-293 cell line expressing the [N31G LC/wt HC] anti-HEL immunoglobulin and AID activity was generated by seeding a T75 culture flask with 3×10⁶HEK-293 cells in 10 mL DMEM medium containing 10% FBS (Invitrogen Corporation, Carlsbad, Calif.). The following day, 500 μL OptiMEM (Invitrogen Corporation, Carlsbad, Calif.), 20 μL HD-Fugene (Roche Diagnostics Corporation, Indianapolis, Ind.), 1 μg of the optimized AID expression vector (Example 5), and 1.5 μg each of the heavy and light chain expression vectors were mixed and incubated for approximately 25-30 minutes at room temperature. After incubation, this mixture was added drop-wise to the cell culture medium.
Approximately three days post-transfection, the cell growth medium was exchanged with 10 mL DMEM medium containing 10% FBS, 50 μg/mL Geneticin, 10 μL/mL Antibiotic-Antimycotic Solution, 1.5 μg/mL puromycin, 15 μg/mL blasticidin, and 350 μg/mL hygromycin (Invitrogen Corporation, Carlsbad, Calif.) and the cells were incubated for approximately four weeks with periodic re-seeding and exchange of the cell culture medium. At the end of the selection period, the cell culture was expanded, archived and a T75 cell culture flask was seeded with 3×10⁶HEK-293 cells that expressed the [N31G LC/wt HC] anti-HEL immunoglobulin and AID activity in 10 mL DMEM medium containing 10% FBS (Invitrogen Corporation, Carlsbad, Calif.). The following day, 500 μL OptiMEM (Invitrogen Corporation, Carlsbad, Calif.), 204 HD-Fugene (Roche Diagnostics Corporation, Indianapolis, Ind.), and 3 μg of the AID expression vector DNA described above were mixed and incubated for approximately 25-30 minutes at room temperature. After incubation, this mixture was added drop-wise to the cell culture medium. After approximately one week of incubation, the original stable HEK-293 cell line expressing the [N31G LC/wt HC] anti-HEL immunoglobulin and AID as well as the culture that has been transiently transfected with additional AID expression vector were prepared for cell sorting.
2. Selection of Higher Affinity Mutants:
The HEK-293 cell line expressing the [N31G LC/wt HC] anti-HEL immunoglobulin and AID activity and the culture that had been transiently transfected with additional AID expression vector were prepared for cell sorting by collecting the cells, washing with an equal volume of PBS solution, pH 7.2 and resuspending 1e07 cells from each culture in ice-cold PBS solution, pH 7.2 containing 1% (weight/volume) BSA and either 50 pM or 500 pM HEL-FITC at a final cell concentration of 2e05 cells/mL.

Round 1

Hen Egg lysozyme (Sigma Aldrich, MO) was labeled with fluorescein iosthiocyanate (FITC) using the EZ-Label™ FITC protein labeling kit (Pierce, Rockford, Ill.) following the manufacturers directions.
Following incubation for 30 minutes at 4° C., the cells were pelleted by centrifugation and the volume was reduced to 200 μL. After transfer to sterile 3-mL tubes, a 1:500 dilution of PE-conjugated goat-anti-mouse immunoglobulin was added to the cells and cells were incubated at 4° C. for 30 minutes. The cells were then pelleted by centrifugation and resuspended in 1 mL of sterile ice-cold PBS solution, pH 7.2 containing 1% (weight/volume) BSA plus 2 nanograms/milliliter DAPI. Live IgG-positive cells that were positive for FITC (excitation with a 150 mW 488 nm laser, collection through a 528/38 filter) were isolated by fluorescence activated cell sorting (FACS) using a Cytopiea Influx Cell Sorter at a flow rate of approximately 10,000 events/second (FIG. 51). FACS windows were calibrated to ensure that higher affinity clones could be discriminated using this approach using HyHEL expressing cells.
The results show a small population of cells that, in all cases, is clearly separated from the main bulk of non-mutated cells. In cells that have been newly transfected with the AID expression (panels B and D of FIG. 51), this population of cells is consistently larger than in the populations of cells that did not receive additional AID expression vector (panels A and C of FIG. 51). These cells were cultured as described below.
Sorted cells were placed in 3 mL DMEM medium containing 10% FBS, 50 μg/mL Geneticin, 10 μL/mL Antibiotic-Antimycotic solution, 1.5 μg/mL puromycin, 15 μg/mL blasticidin, and 350 μg/mL hygromycin (Invitrogen Corporation, Carlsbad, Calif.) in one well of a 6-well plate. The cells were cultured until confluent and then archived and re-seeded in one well of a 6-well plate at a cell density of 4×10⁵cells/mL. The next day, 100 μL OptiMEM (Invitrogen Corporation, Carlsbad, Calif.), 4 μL Fugene6 (Roche Diagnostics Corporation, Indianapolis, Ind.), and 1 μg of the AID expression vector plasmid DNA were mixed and incubated for approximately 25-30 minutes at room temperature. After incubation, this mixture was added drop-wise to the cell culture medium and the cells were cultured and expanded for approximately 7 days. Samples of cells were also taken for sequence analysis.

Round 2

Cells selected using FITC-HEL in the first round, as described above, were then subjected to the same selection conditions (i.e., incubation with either 50 or 500 pM FITC-labeled HEL) in a second round of FACS sorting. Fifty milliliters (1e07 cells) of the cells selected from the first round were incubated in an ice-cold PBS solution, pH 7.2 containing 1% (weight/volume) BSA and either approximately 50 pM or 500 pM HEL-FITC for 30 minutes at 4° C. The cell mixture was pelleted, the volume was reduced to 200 μL and the cells were transferred to sterile 3 ml tubes. A 1:500 dilution of PE-conjugated goat-anti-mouse Ig was added to the cells and the cells were incubated at 4° C. for 30 minutes. The cells were then pelleted and resuspended 1 mL of an ice-cold PBS solution, pH 7.2 containing 1% (weight/volume) BSA plus 2 nanograms/milliliter DAPI. Live IgG-positive cells that were positive for FITC (excitation with a 150 mW 488 nm laser, collection through a 528/38 filter) were isolated by fluorescence activated cell sorting using a Cytopiea Influx Cell Sorter at a flow rate of approximately 10,000 events/second (FIG. 52).
The results of the second sort show a significantly larger population of cells exhibiting high affinity HEL binding, consistent with the formation of higher affinity mutants by SHM during growth and culture. In cells that have been newly transfected with the AID expression vector and then incubated with 500 pM HEL (panel D of FIG. 52), this is clearly a much larger population of highly fluorescent cells (25.9% of the population versus 6.88% compared cells that did not receive additional AID expression vector; panel C in FIG. 52). These results demonstrate that re-transformation with the AID expression vector is effective in promoting a significant improvement in mutagenesis rate.
Continuing this process for 2 additional rounds of mutation with stringent gating on the selected cells (shown in FIG. 53, panel A) resulted in a profound and significant shift in the binding properties of the selected cells (FIG. 53, panel B).
3. Production of Secreted Immunoglobulins for Functional Analysis
Heavy and light chains of interest may be produced in a secreted form for further functional analysis as described below. In the case of heavy chains obtained from the surface displayed libraries, these are processed as described in Example 5 of priority U.S. Application Nos. 60/904,622 and 61/020,124 (i.e., by digestion with XhoI, followed by re-ligation), to remove the transmembrane domain allowing for direct secretion of the antibody into the media.
Approximately one day prior to transfection, 3×10⁶HEK-293 cells were seeded in 10 mL DMEM/10% FBS medium in a T75 culture flask and incubated overnight at 37° C. and 5% CO₂. On the day of transfection, 500 μL OptiMEM (Invitrogen Corporation, Carlsbad, Calif.), 20 μL HD-Fugene (Roche Diagnostics Corporation, Indianapolis, Ind.) and 1.5 μg of each heavy and light chain expression vectors were mixed and incubated for approximately 25-30 minutes at room temperature. After incubation, this mixture was added drop-wise to the cell culture medium.
Approximately three days post-transfection, the cell growth medium was exchanged with 10 mL Freestyle medium (Invitrogen Corporation, Carlsbad, Calif.) and the cells were incubated for an additional 7 days. At the end of the incubation period, the cell culture supernatants were harvested and filtered through a sterile 0.2 μm filter. The secreted immunoglobulins were isolated via standard protein A affinity column chromatography prior to BIACORE analysis as described below.
HEL is immobilized onto a research grade CM5 sensor chip using standard amine coupling. Each of three surfaces is first activated for seven minutes using a 1:1 mixture of 0.1 mM N-hydroxysuccinimide (NHS) and 0.4 mM 1-ethyl-3-(3-dimethylaminopropyl)-carbodimide (EDC). Then, the HEL sample is diluted 1- to 50-fold in 10 mM sodium acetate, pH 4.0, and exposed to the activated chip surface for different lengths of time (ten seconds to two minutes) to create three different density surfaces of HEL. Each surface is then blocked with a seven-minute injection of 1 M ethanolamine, pH 8.2. Alternatively biotinylated HEL is diluted 100-fold and injected for different amounts of time to be captured at three different surface densities (60 RU, 45 RU, 12 RU; Response Unit (RU) is termed by Biacore and relates to target molecule per surface area) onto a streptavidin-containing sensor chip. All experiments are performed on a Biacore® 2000 or T100 optical biosensor. Anti-HEL antibodies are supplied at 100 μg/mL and tested in a 3-fold dilution series in sample running buffer over HEL conjugated surfaces. Bound anti-HEL antibody is removed using a five-second pulse with sensor regeneration solution. All data is collected at a temperature-controlled 20° C. The kinetic responses for the antibody injections are analyzed using the non-linear least squares analysis program CLAMP (Myszka, D. G. and Morton, T. A. (1998) Trends Biochem. Sci., 23: 149-150).
4. Sequence Analysis
Sequences of the heavy and light chains isolated in the first sort were determined by PCR amplification of heavy and light chains as described below.
At least 50,000 cells taken from populations of interest were pelleted at 1100×g for 5 min. at 4° C. Pelleted cells were resuspended in 15 μL distilled H₂O and either used immediately in PCR reactions or were frozen for later processing.
PCR reactions consisting of 27.6 μL H₂O, 5 μL, 10× Pfx buffer, 1 μL cells from above, 8 μL of 2.5 pM of each primer (listed below), and 0.4 μL Pfx polymerase (Invitrogen Corp., Carlsbad, Calif.) for a total of 50 μL were run using the following format: 1 cycle of 95° C.×2 min., followed by 35 cycles of 95° C.×30 sec, 55° C. for 30 sec, 68° C. for 45 sec, followed by 1 cycle of 68° C. for 1 min PCR primers used to amplify the open reading frames are:
Oligo 540: GTGGGAGGTCTATATAAGCAGAGC (SEQ ID NO: 362), which is a forward primer which maps at the 3′ end of a CMV promoter region, approximately 140 nucleotides 5′ to the ATG start codon for both heavy and light chain open reading frames.
Oligo 554: CAGAGGTGCTCTTGGAGGAGGGT (SEQ ID NO: 363), which is a heavy chain-specific reverse primer which maps in the IgG gamma chain constant region.
Oligo 552: ACACAACAGAGGCAGTTCCAGATT (SEQ ID NO: 364), which is a kappa light chain-specific reverse primer that maps near the amino end of the kappa constant region.
Oligo 577: AGTGTGGCCTTGTTGGCTTGAA (SEQ ID NO: 365), which is a lambda light chain-specific reverse primer that maps to an N-proximal constant region sequence shared by all five functional human lambda genes (IgL1, 2, 3, 6, and 7).
To amplify the heavy chain, oligos 540+554 were used.
To amplify the light chains from a population of cells (in which there was likelihood that a mixture of both kappa and lambda light chains would be present), oligos 540, 552 and 577 were used simultaneously. In this case, the volume of water in the PCR reaction mix was adjusted to 19.6 μL.
Following PCR, 5 μL of sample was removed for analysis on an agarose gel. Reactions for bands which were visualized on the gel and were then subjected to further PCR in the presence of Taq polymerase (Invitrogen) using the following conditions: added directly to the remaining 45 μL of PCR reaction were 2 μL H₂O, 0.5 μL Taq, 0.2 μL dNTPs at 2.5 mM each, and 1.5 μL×50 mM MgCl₂for a total of 50 μL (or alternatively, 1 μL of 10×Taq buffer was used in place of MgCl₂while adjusting the H₂O to maintain 50 μL final volume). PCR cycling was run as follows: 1 cycle of 95° C.×2 min., followed by 2 cycles of 95° C.×30 sec, 55° C. for 30 sec, 72° C. for 45 sec, followed by 1 cycle of 72° C. for 1 min.
Reactions for bands which were either not visualized on the gel or were otherwise judged to be too weak to continue, were supplemented with 1 μL Pfx buffer, 3.7 μL H₂O, and 0.3 μL Pfx polymerase and subjected to 1 cycle of 95° C.×2 min., followed by 10 cycles of 95° C.×30 sec, 55° C. for 30 sec, 68° C. for 45 sec, followed by 1 cycle of 68° C. for 1 min.
PCR reactions for bands which were visible following analysis on an agarose gel were cloned using a TOPO® cloning kit from Invitrogen following the manufacturer's suggested protocol. In brief, 4 μL PCR reaction was added to 1 μL salt solution (provided in the TOPO® kit) plus 1 μL TOPO® cloning vector. Following a 20 min. incubation at room temp., 1 or 2 μL were used to transform 100 μL XL1 blue as per the manufacturer's suggested protocol.
Reading frames from templates whose sequences were of further interest were recovered as follows: heavy chain templates were recovered by digesting the TOPO® clones with SgrAI and NheI, which are both present in all of the original heavy chain sequences. The resulting approximately 500 bp fragments (which contain the entire variable region including all of CDR3), were cloned into the cognate sites of an expression vector already comprising the heavy chain constant region to generate an intact, contiguous heavy chain open reading frame. One version of this vector also contains the transmembrane domain and cytoplasmic domain from the murine H2kk gene as an in-frame fusion with the IgG1 constant region to permit retention of the final IgG molecule on the cell surface, as described in Example 9. The alternative version of the expression vector has the transmembrane deleted to allow for direct secretion of the antibodies of interest.
Similarly, light chain templates of interest were removed from their TOPO® cloning vectors using SbfI and MunI for kappa or SbfI and AclI for lambda, all of which sites are present in the original sequences. The resulting 350-400 bp fragments (which contain the entire light chain variable region including CDR3), were cloned into the cognate sites of the expression vector to generate an intact, contiguous light chain open reading frame.
The results demonstrated that in approximately 23% of the sequenced clones, there was at least one mutation within the CDR of the light chain resulting in the mutation of Glycine 31 to Aspartate (G31D). Based on the crystal structure of HyHEL 10 bound to HEL (Pons et al., (1999) Protein Science 8:958-68), this mutation would be predicted to result in the formation of an additional hydrogen bond interaction during antigen binding, which accounts for the increase in binding observed in the presence of 500 pM HEL in FIG. 52 and Biacore measurements. The type of mutations observed (FIGS. 54A and B) followed the predicted pattern of mutations for SHM mediated mutation (as shown in FIG. 50), and did not result in widespread non-specific mutation of the entire coding regions of the heavy and light chains. These results, therefore, demonstrate the ability of the system to provide good affinity discrimination, selection of improved variants of the antibodies and binding proteins of the present invention, and the ability to provide for both sustained and pulsed hypermutation directed to specific regions of interest within one or more target proteins. Furthermore, a handful of additional mutations were identified that, when recombined into a single antibody construct, improved upon the affinity of the wild-type protein from 30 pM to better than 4 pM (FIG. 54C). This example demonstrates how a single sequence or library under selective pressure and in the presence of SHM can quickly generate higher affinity mutants, and how this flow of mutational events can be predicted exactly by the computational algorithms outlined above.
The data presented herein demonstrate that the disclosed systems and seed polynucleotides for somatic hypermutation are capable of high level targeted mutagenesis of a target protein of interest. The system is capable of iterative rounds of mutagenesis and selection enabling the directed evolution of favorable mutations while reducing the accumulation of neutral and harmful mutations, both within the protein of interest and within the expression system.
5. Episomal Rescue
As episomal vectors remain unintegrated and easily separable from a host cell's chromosomal material, plasmids can be recovered by the method of Hirt (Hirt, 1967; Kapoor and Frappier, 2005; Yates et al., 1984), transformed into competent bacteria and further manipulated to verify the sequence, identity and/or properties of the encoded polypeptides.
Using an estimate of an average of 3 resident episomes of 8000 base pairs (bp) each per cell, one can expect a yield of approximately 30 picogram (pg) per million cells (see, e.g., Formula I). Assuming a transformation efficiency into electrocompetent E. coli of 10⁷colonies per μg of relaxed circle DNA, one can expect approximately 300 E. coli colonies, each representing a single recovered episome, to result per million mammalian cells.
(10⁶cells×3 episomes/cell)×(660 g/mol/bp)×(8000 bp/episome)×(10⁶colonies/μg)×(10⁶μg/g)÷(6×10²³episomes/mol)=2.6×10⁻¹¹g (DNA per 10⁶cells) Formula 1
Plasmids can also be recovered using a standard alkaline lysis procedure, e.g., as per a protocol from Qiagen, Inc. (for procedure, see e.g., wwwl.qiagen.com/literature/handbooks/PDF/PlasmidDNAPurification/PLS_QP_Miniprep/1034641_HB_QIAp rep_—112005.pdf; and Wade-Martins et al., Nuc Acids Res 27:1674-1682 (1999)). In one aspect, transfected mammalian cells are treated the same way as the E. coli described in the Qiagen protocol. Episomes present in the final eluate are transformed into competent E. coli as described above. Using either the Hirt supernatant or alkaline lysis method requires beginning with a significant cell population for isolating resident episomes. In one non-limiting example, starting with 50,000 clonally derived cells, one might expect to obtain 10 to 20 recovered episomes as manifested in colonies of transformed E. coli.
Additionally, expression of the SV40 T antigen provides for the rapid amplification of vectors containing an SV400n, thus providing for a method to amplify vector number prior to episome rescue. To achieve this amplification, the SV40 T-antigen was cloned into an expression vector (as described herein) and was transiently transfected into 6.3×10e5 HEK 293 cells that were stably harboring HyHEL10 HC and LC episomal vectors. Samples were taken at time 0 (immediately prior to transfection), and at day 1, 2 and 3. Cells were harvested by trypsinization and episomal DNA was extracted using a Qiagen miniprep kit. Extracted DNA was transformed into E. coli, which were grown on carbenicillin plates overnight, and colonies were counted the next morning (Table 18).

TABLE 18

Number of colonies resulting from HyHEL10-expressing cell
population before and following transient transfection with T-antigen.

day	# cells	# colonies

0	6.3 × 10⁵	0
1	6.3 × 10⁵	35
2	6.3 × 10⁵	800-900
3	6.3 × 10⁵	>5000

Another standard method to characterize transfected genes, whether episomal or integrated, involves performing a Polymerase Chain Reaction (PCR) reaction directly on the relevant cell population followed by cloning and characterizing individual resulting PCR fragments. This method has the advantage of not requiring a large starting population of cells. PCR amplification of the resident active antibody open reading frame can successfully be performed on as little as a single cell. This has the effect of foreshortening the time from isolation of a cell of interest to the point of sequencing the responsible open reading frame.
Another option is to perform RT-PCR on the isolated cells thus identifying and characterizing the resident polypeptide(s) via expressed mRNA.

Example 10

Engineering Enhanced Mutants of AID

Activation induced cytidine deaminase (AID) is the primary enzyme responsible for initiating somatic hypermutation (SHM), class switch recombination (CSR) and gene conversion (GC) events during affinity maturation by the immune system. The enzyme has been especially well conserved during evolution, with the human, rat, cow, mouse and chicken orthologs exhibiting 94.4%, 93.9%, 93.9%, 92.4% and 89.4% identity to the canine (dog) amino acid sequence, respectively.
AID contains several predicted protein—protein interaction domains, post-translational modification sites and subcellular targeting motifs, one of which is a nuclear export signal (NES) that is localized in the carboxy terminal amino acids of the enzyme. The question as to whether or not a nuclear localization signal (NLS) is present within AID remains controversial with some groups claiming such a signal exists (Ito et al., PNAS 2004 Feb. 17; 101(7):1975-80) while others maintain that no functional NLS is present (Brar et al., J. Biol. Chem. 2004 Jun. 8; 279(25):26395-401; McBride et al., J. Exp. Med. 2004 May 3; 199(9):1235-44).
Native AID is found primarily in the cytoplasmic compartment of cells, as demonstrated by cell fractionation, western blotting and immunohistochemistry. Removal or disabling of the NES tends to permit higher steady-state resident concentrations of AID in the nucleus, higher levels of SHM, but also impaired or absent CSR (Brar et al, Id.; Durandy et al., Hum. Mutat. 2006 December; 27(12):1185-91; Ito et al, Id.; McBride et al, Id.).
Example 2 above describes the design and construction of an SHM resistant form of AID (SEQ ID NO. 341) comprising a mutation in the NES (L198A) designed to disable nuclear export thereby promoting nuclear retention. To further enhance nuclear localization and, thus, the mutator activity of AID, further engineered versions of the enzyme were created by inserting the strong nuclear localization signal (NLS; PKKKRKV; SEQ ID NO: 340) derived from the SV40 T antigen (Kalderon et al, (1984). Cell 39, 499-509) near the amino terminus. To track AID expression, a FLAG epitope tag was also inserted to create (SEQ ID NO. 342) which contains both a strong NLS and the mutant NES sequence.
Additional engineered versions of AID were also created by further modifying the C-terminal NES to reduce nuclear export. These constructs were prepared with and without the SV40 T antigen NLS.
In the first pair of NES mutants, polynucleotide sequences of SEQ ID NO: 341 (without NLS) and SEQ ID NO: 342 (with NLS) were modified such that amino acid residues L181, L183, L189, L196 and L198 encoded by the polynucleotide sequences were mutated to Alanine resulting in polynucleotide sequences of SEQ ID NO: 344 (without NLS) and SEQ ID NO: 346 (with NLS), respectively, and amino acid sequences of SEQ ID NO: 345 (without NLS) and SEQ ID NO: 347 (with NLS), respectively.
Muteins were generated by PCR, and then treated with Dpn1 to remove parental DNA.
To generate the alanine containing muteins, the following oligos were used:

(SEQ ID NO: 352)

	CAGCTCAGGAGAATCCTCGCCCCCGCTTATGAGGTCGACGACCTC
	and

(SEQ ID NO: 353)

GAGGTCGTCGACCTCATAAGCGGGGGCGAGGATTCTCCTGAGCTG.

Two separate PCR reactions were set up using vectors containing polynucleotide sequences set forth as SEQ ID NO: 341 or SEQ ID NO: 342 as template DNA, using Pfu Taq polymerase (Invitrogen) with the manufacturers kit buffers and 2.5 uM of each deoxynucleotide (Roche). PCR was performed with the following cycle conditions: 1 cycle of 95° C. for 3 min, followed by 20 cycles of [95° C. for 45 sec, 55° C. for 45 sec, 68° C. for 17 min], followed by 1 cycle of 68° C. for 5 min. After completion, 5 μl of the PCR reaction was run on a 1% agarose gel to confirm a successful reaction. The PCR reaction mix was then treated with Dpn1 (New England Biolabs) for at least 4 hrs at 37° C. to remove the parental DNA.
Five (5)₄of the Dpn1-treated PCR reaction was added to 100 μL of XL1-Blue super competent cells (Invitrogen) and transformed per the manufacturer's suggested protocol. Following sequence verification, the resulting DNA (which contained 2 of the 4 desired mutations; i.e., 181 and 183), was used as a template with oligos CCGCTTATGAGGTCGACGACGCCAGAGATGCCTTCCGGACCG (SEQ ID NO: 354) and AGGGTCCGGAAGGCATCTCTGGCGTCGTCGACCTCATAAGCGG (SEQ ID NO: 355) in the same protocols listed above to introduce the third of four mutations (i.e., 189). Finally, oligos CCAGAGATGCCTTCCGGACCGCCGGGGCTTGATGTACAATC (SEQ ID NO: 356) and GATTGTACATCAAGCCCCGGCGGTCCGGAAGGCATCTCTGG (SEQ ID NO: 357) were used to incorporate the fourth and final mutation (i.e., 196).
The final set of alanine-containing mutein products were digested using Sac1 and BsrG1 and ligated into vector backbones cut with the cognate restriction enzymes to generate SEQ. ID. NO. 344 (without NLS) and SEQ. ID. NO. 346 (with NLS), respectively.
In a second pair of muteins: polynucleotide sequences of SEQ. ID. NO. 341 (without NLS) and SEQ. ID. NO. 342 (with NLS) were modified such that amino acid residues Asp187, Asp188 and Asp191 encoded by the polynucleotide sequences were mutated to Glutamate and amino acid residue Thr195 encoded by the polynucleotide sequences was mutated to Isoluecine, thereby creating polynucleotide sequences SEQ. ID. NO. 348 (without NLS) and SEQ. ID. NO. 350 (with NLS), respectively, and amino acid sequences of SEQ. ID. NO. 349 (without NLS) and SEQ. ID. NO. 351 (with NLS), respectively.
The same set of procedures described above with respect to the alanine muteins was repeated to generate the glutamate containing muteins of AID SEQ. ID. NO. 348 and SEQ. ID. NO. 350, except that the following oligos: TCCTCCCCCTCTATGAGGTCGAAGAACTCAGAGAAGCCTTCCGGACCCTCGGGGC (SEQ ID NO: 358) and GCCCCGAGGGTCCGGAAGGCTTCTCTGAGTTCTTCGACCTCATAGAGGGGGAGGA (SEQ ID NO: 359) were used in place of the first pair of oligos, and the following oligos: AACTCAGAGAAGCCTTCCGGATCCTCGGGGCTTGATGTACAAT (SEQ ID NO: 360) and ATTGTACATCAAGCCCCGAGGATCCGGAAGGCTTCTCTGAGTT (SEQ ID NO: 361) were used in lieu of the second pair of oligos (no third PCR reaction was needed in this case). Products were treated as described above to generate SEQ. ID. NO. 348 (without NLS) and SEQ. ID. NO. 350 (with NLS).
Results and Discussion.
The six resulting AID constructs were subsequently tested for activity in a green fluorescent protein (GFP) reversion assay, and for frequency of mutations on an immunoglobulin IgG heavy chain (HC) template.
To perform the GFP reversion assay, the TAC codon for tyrosine 82 was altered to a TAG stop codon (GFP*). GFP* was cloned into an Anaptys episomal expression vector and stably transfected into HEK 293 (note: this cell line expresses EBNA1 from an integrated copy of the gene). Each AID construct in turn was transfected into the stably transfected GFP* cell line, and cells were placed under selection (blasticidin for GFP* and hygromycin for each of the AID constructs) by day 2 post transfection. Reversion of the stop codon back to tyrosine caused the episome-harboring cell to fluoresce green. The frequency of GFP reversion was measured by fluorescence-activated cell sorter (FACS) analysis at 3, 6, and 10 days post selection.

TABLE 19

Functional competence of AID muteins as gauged by FACS
analysis of GFP revertant cells gated on days 3, 6, and 10.
Table 19

	% gated	% gated	% gated
Vector(s)/AID variants	day	3	day 6	day 10

GFP* alone	0.04%	0.02%	0.01%
GFP* + expression of (SEQ ID. NO. 341)	0.44%	0.35%	0.39%
GFP* + expression of (SEQ ID. NO. 342)	0.31%	0.37%	0.19%
GFP* + expression of (SEQ ID. NO. 344)	0.19%	0.26%	0.21%
GFP* + expression of (SEQ ID. NO. 346)	0.36%	0.35%	0.32%
GFP* + expression of (SEQ ID. NO. 348)	0.37%	0.30%	0.41%
GFP* + expression of (SEQ ID. NO. 350)	0.18%	0.26%	0.21%

The results indicate that co-transfection with each of the six AID constructs consistently yielded GFP revertants significantly above background, indicating that all 6 muteins of AID are functional.
Because the GFP reversion assay requires both the initial activity of AID and subsequent action by error prone polymerase in order to generate a positive, reverted cell, the results can provide a qualitative yes/no for function. In order to determine actual reversion rates, a more precise template mutagenesis experiment was also conducted. Thus, in addition to the GFP reversion assay, 2 of the AID constructs (SEQ ID. NO. 341; containing the L198A mutation in the NES) and SEQ ID. NO. 342, (containing the L198A NES mutation and the SV40 NLS)) were tested for their ability to induce mutations in the HC of HyHEL10 IgG (Pons et al, (1999) Protein Science 8:958-68; Smith-Gill et al. (1984) J. Immunology 132:963). Episomal expression constructs (as described previously) encoding the HC of HyHEL10, an N31G mutein of the HyHEL10 light chain (LC), and either an expression vector containing SEQ ID NO: 341 or the same vector backbone containing SEQ ID NO: 342, were co-transfected into HEK 293 cells. Antibiotic selective pressure was added to the transfected cell population (i.e., blasticidin, puromycin and hygromycin for HC, LC and AID, respectively), and cells were harvested following 2 months of culture. A total of 83 IgG HC templates were sequenced from cells transfected with an expression vector comprising SEQ ID NO. 341, and 61 templates were sequences from cells transfected with an expression vector comprising SEQ ID NO. 342. The percentage of mutations per template vs. form of AID is shown in Table 20, below. The mutation frequency calculated from the sequencing data is 1 mutation per 1438 bp generated by SEQ ID NO: 341, and 1 mutation per 1059 bp generated by SEQ ID NO: 342.

TABLE 20

Percentage of HyHEL10 IgG templates identified with mutations
observed after co-expression of AID muteins SEQ ID NO. 341 or
SEQ ID NO. 342
Table 20

# Mutations per heavy
chain template	SEQ ID. No. 341	SEQ ID. No. 342

0	71%	72%
1	26%	20%
2	2.4%	6.8%
3	0	1.6%
4	0	1.6%

The results indicate that the version of AID that contains the NLS (SEQ ID. NO. 342) induced a greater number of mutations in the HyHEL10 HC IgG template (1 per 1059 bp vs 1 per 1438 for the non-NLS containing homolog), and similarly resulted in a greater number of templates containing multiple mutations (10% of templates by AID+NLS vs 2.4% for AID−NLS).
Sequences
Cold canine AID. Nuclear export signal was abrogated by altering the unmodified CTT (Leu198) codon to GCT (ala, shown underlined below).

(SEQ ID NO: 341)

ATGGACTCTCTCCTCATGAAGCAGAGAAAGTTTCTCTACCACTTCAAGAA

CGTCAGATGGGCCAAGGGGAGACATGAGACCTATCTCTGTTACGTCGTCA

AGAGGAGAGACTCAGCCACCTCTTTCTCCCTCGACTTTGGGCATCTCCGG

AACAAGTCTGGGTGTCATGTCGAACTCCTCTTCCTCCGCTATATCTCAGA

CTGGGACCTCGACCCCGGGAGATGCTATAGAGTCACTTGGTTTACCTCTT

GGTCCCCCTGTTATGACTGCGCCAGACATGTCGCCGACTTCCTCAGGGGG

TATCCCAATCTCTCCCTCCGCATATTCGCCGCCCGACTCTATTTTTGTGA

GGACAGGAAAGCCGAGCCCGAGGGGCTCAGGAGACTCCACCGGGCCGGGG

TCCAGATCGCCATCATGACATTTAAGGACTATTTCTATTGTTGGAATACA

TTTGTCGAGAATCGGGAGAAGACTTTCAAAGCCTGGGAGGGGCTCCATGA

GAACTCTGTCAGACTCTCTAGGCAGCTCAGGAGAATCCTCCTCCCCCTCT

ATGAGGTCGACGACCTCAGAGATGCCTTCCGGACCCTCGGGGCTTGA.

Features of the polynucleotide sequences (or amino acid sequences) are in 5′ to 3′ (or N- to C-terminal where appropriate) as follows:
SacI restriction site used for cloning, boxed letters; Kozak consensus, underlined; ATG start codon (bold capital letters); FLAG epitope tag (single underline); NLS (double-underline); cold canine AID; TGA stop codon (bold capital letters); BsrGI and AscI restriction sites used for cloning (boxed letters). * indicates stop codon in protein sequence.
Flag-NLS-AID.
The 4 underlined-and-capitalized GCC codons (ala) were changed from the original sequence (CTC encoding Leu) by site directed mutagenesis.

(SEQ ID NO: 344)

gagctcctaaccaccATGgactctctcctcatgaagcagagaaagtttct

ctaccacttcaagaacgtcagatgggccaaggggagacatgagacctatc

tctgttacgtcaagaggagagactcagccacctctttctccctcgacttt

gggcatctccggaacaagtctgggtgtcatgtcgaactcctcttcctccg

ctatatctcagactgggacctcgaccccgggagatgctatagagtcactt

ggtttacctcttggtccccctgttatgactgcgccagacatgtcgccgac

ttcctcagggggtatcccaatctctccctccgcatattcgccgcccgact

ctatttttgtgaggacaggaaagccgagcccgaggggctcaggagactcc

accgggccggggtccagatcgccatcatgacatttaaggactatttctat

tgttggaatacatttgtcgagaatcgggagaagactttcaaagcctggga

ggggctccatgagaactctgtcagactctctaggcagctcaggagaatcc

tcGCCcccGCCtatgaggtcgacgacGCCagagatgccttccggaccGCC

ggggctTGAtgtaca.

(SEQ ID NO: 345)

MDSLLMKQRKFLYHFKNVRWAKGRHETYLCYVVKRRDSATSFSLDFGHLR

NKSGCHVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRG

YPNLSLRIFAARLYFCEDRKAEPEGLRRLHRAGVQIAIMTFKDYFYCWNT

FVENREKTFKAWEGLHENSVRLSRQLRRILAPAYEVDDARDAFRTAGA*.

The 4 underlined-and-capitalized GCC codons (ala) were changed from the original sequence (CTC encoding Leu) by site directed mutagenesis. Boxes and underlines are as described above.
The 3 underlined-and-capitalized GAA codons (Glu) were changed from the original sequence (Aspartate encoding codons). One additional mutation, T195I, (ACC to ATC) was also generated.

(SEQ ID NO: 348)

gagctcctaaccaccATGgactctctcctcatgaagcagagaaagtttct

ctaccacttcaagaacgtcagatgggccaaggggagacatgagacctatc

tctgttacgtcgtcaagaggagagactcagccacctctttctccctcgac

tttgggcatctccggaacaagtctgggtgtcatgtcgaactcctcttcct

ccgctatatctcagactgggacctcgaccccgggagatgctatagagtca

cttggtttacctcttggtccccctgttatgactgcgccagacatgtcgcc

gacttcctcagggggtatcccaatctctccctccgcatattcgccgcccg

actctatttttgtgaggacaggaaagccgagcccgaggggctcaggagac

tccaccgggccggggtccagatcgccatcatgacatttaaggactatttc

tattgttggaatacatttgtcgagaatcgggagaagactttcaaagcctg

ggaggggctccatgagaactctgtcagactctctaggcagctcaggagaa

tcctcctccccctctatgaggtcGAAGAActcagaGAAgccttccggATC

ctcggggctTGAtgtaca.

(SEQ ID NO: 349)

MDSLLMKQRKFLYHFKNVRWAKGRHETYLCYVVKRRDSATSFSLDFGHLR

NKSGCHVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRG

YPNLSLRIFAARLYFCEDRKAEPEGLRRLHRAGVQIAIMTFKDYFYCWNT

FVENREKTFKAWEGLHENSVRLSRQLRRILLPLYEVEELREAFRILGA*.

The 3 underlined-and-capitalized GAA codons (Glu) were changed from the original sequence (Aspartate encoding codons). One additional mutation, T195I (ACC to ATC) was also generated. Boxes and underlines are as described above.
While preferred embodiments of the present invention have been shown and described herein, such embodiments are provided by way of example only. It should be understood that various alternatives to the embodiments of the invention described herein can be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

REFERENCES

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Claims

1.-41. (canceled)

42. A method for preparing a gene product having a desired property, which method comprises:

(a) expressing a first polynucleotide sequence encoding a gene product of interest in a cell that expresses AID activity or can be induced to express AID activity via the addition of an inducing agent, whereupon expression of AID activity in the cell produces one or more mutated first polynucleotide sequences comprising one or more mutations;

(b) sequencing at least one mutated first polynucleotide sequence produced in step (a), and identifying one or more mutations in the one or more mutated first polynucleotide sequences,

(c) preparing one or more second polynucleotide sequences, each of which differs from the first polynucleotide sequence and contains one or more mutations of the one or more mutated first polynucleotide sequences identified in step (b),

(d) expressing the second polynucleotide sequences in a cell,

(e) identifying one or more second polynucleotide sequences that express a gene product having a desired property and differing from the gene product of interest, and

(f) optionally iteratively repeating steps (a)-(e), wherein an identified second polynucleotide sequence in step (e) becomes the first polynucleotide sequence in step (a).

43. The method of claim 42, wherein the second polynucleotide sequence has an altered density of hot spot motifs as compared to the first polynucleotide sequence.

44. The method of claim 42, wherein the second polynucleotide sequence exhibits an altered rate of AID mediated mutagenesis as compared to the first polynucleotide sequence.

45. The method of claim 42, wherein the gene product of interest is an antibody or antigen-binding fragment thereof, a selectable marker, a reporter protein, a neurotransmitter, a hormone, a cytokine, a chemokine, an enzyme, a receptor, a structural protein, a toxin, a co-factor, or a transcription factor.

46. The method of claim 42, wherein the gene product having a desired property and differing from the gene product of interest is an antibody or antigen-binding fragment thereof, a selectable marker, a reporter protein, a neurotransmitter, a hormone, a cytokine, a chemokine, an enzyme, a receptor, a structural protein, a toxin, a co-factor, or a transcription factor.

47. The method of claim 46, wherein the gene product having a desired property and differing from the gene product of interest is an antibody or antigen-binding fragment thereof.

48. The method of claim 47, wherein the desired property is selected from the group consisting of affinity, cross-reactivity, specificity, expression, stability, and solubility.

49. The method of claim 42, wherein step (f) is carried out at least one time.

50. An isolated gene product having a desired property, which gene product is produced by a method comprising:

(d) expressing the second polynucleotide sequences in a cell,

51. The isolated gene product of claim 50, wherein the second polynucleotide sequence has an altered density of hot spot motifs as compared to the first polynucleotide sequence.

52. The isolated gene product of claim 50, wherein the second polynucleotide sequence exhibits an altered rate of AID mediated mutagenesis as compared to the first polynucleotide sequence.

53. The isolated gene product of claim 50, wherein the gene product of interest is an antibody or antigen-binding fragment thereof, a selectable marker, a reporter protein, a neurotransmitter, a hormone, a cytokine, a chemokine, an enzyme, a receptor, a structural protein, a toxin, a co-factor, or a transcription factor.

54. The isolated gene product of claim 50, wherein the gene product having a desired property and differing from the gene product of interest is an antibody or antigen-binding fragment thereof, a selectable marker, a reporter protein, a neurotransmitter, a hormone, a cytokine, a chemokine, an enzyme, a receptor, a structural protein, a toxin, a co-factor, or a transcription factor.

55. The isolated gene product of claim 54, wherein the gene product having a desired property and differing from the gene product of interest is an antibody or antigen-binding fragment thereof.

56. The isolated gene product of claim 55, wherein the desired property is selected from the group consisting of affinity, cross-reactivity, specificity, expression, stability, and solubility.

57. The isolated gene product of claim 50, wherein step (f) is carried out at least one time.