CN110551216A

CN110551216A - Multivalent anti-OX 40 antibodies and uses thereof

Info

Publication number: CN110551216A
Application number: CN201910459910.9A
Authority: CN
Inventors: 刘军建; 周帅祥
Original assignee: Xinda Biopharmaceutical (suzhou) Co Ltd
Current assignee: Xinda Biopharmaceutical (suzhou) Co Ltd
Priority date: 2018-05-31
Filing date: 2019-05-29
Publication date: 2019-12-10
Anticipated expiration: 2039-05-29
Also published as: CN110551216B

Abstract

The present invention relates to a multivalent antibody comprising an Fc domain and at least 4 antigen binding fragments, in particular to novel multivalent antibodies having at least 4 antigen binding fragments that specifically bind to OX 40. The invention also relates to compositions comprising said multivalent antibodies, nucleic acids encoding said antibodies, and host cells comprising said nucleic acids. Furthermore, the invention relates to the therapeutic and diagnostic use of these novel multivalent antibodies, compositions comprising the same, nucleic acids encoding the same, and host cells comprising said nucleic acids.

Description

Multivalent anti-OX 40 antibodies and uses thereof

The present invention relates to a multivalent antibody having at least four antigen binding sites, and in particular to a novel multivalent antibody having at least four antigen binding sites that specifically binds OX 40. The invention also relates to compositions comprising said multivalent antibodies, nucleic acids encoding said antibodies, and host cells comprising said nucleic acids. Furthermore, the invention relates to the therapeutic and diagnostic use of these novel multivalent antibodies, compositions comprising said multivalent antibodies, encoding nucleic acids and host cells comprising said nucleic acids.

Background

Multivalent antibodies

Most natural antibodies are bivalent antibodies with four variable domains, two VH domains and two VL domains. Naturally occurring monoclonal antibodies can exist as multimeric structures such as IgM that increase affinity for a target antigen on a fixed surface. Therefore, the multivalent antibody has certain functions in increasing the affinity of the antibody and the antigen and enhancing the biological function of the antibody.

In recent years, a wide variety of recombinant multispecific antibody formats have been developed, such as tetravalent bispecific antibodies by fusing an IgG antibody format and a single chain domain (see, e.g., Coloma, M.J. et al, Nature Biotech.15(1997) 159-; WO 2001/077342; and Morrison, S.L., Nature Biotech.25(2007) 1233-1234. bispecific antibodies can be made by chemically coupling two different mAbs (see Staerz, U.D. et al, Nature, 1985.314 (6012): pages 628-31.) other methods use chemically coupling two different monoclonal antibodies or smaller antibody fragments (see Brennan, M. et al, Science 1985.229 (4708): pages 81-3).

In a recent study, tandem scFv against CD28 and melanoma-associated proteoglycans were reported by expression in vivo in transgenic rabbits and cattle (see Gracie, J.A. et al, J Clin Invest, 1999.104 (10): page 1393-. In this construct, the two scFv molecules were linked by a CH1 linker and serum concentrations of bispecific antibody were found to be as high as 100 mg/L. A few studies have reported the expression of soluble tandem scFv molecules in bacteria using either the very short Ala3 linker or a longer glycine/serine rich linker (see Leung, B.P. et al, J Immunol, 2000.164 (12): pp 6495-502; Ito, A. et al, J Immunol, 2003.170 (9): pp 4802-9; Karni, A. et al, JNeurommunol, 2002.125 (1-2): pp 134-40).

Bivalent antibodies fused to Fc produce molecules more similar to Ig, called bivalent antibodies (see Lu, D. et al, J Biol Chem, 2004.279 (4): pages 2856-65). In addition, multivalent antibody constructs comprising two Fab repeats in the heavy chain of IgG and capable of binding four antigenic molecules have also been described (see U.S. Pat. No. 8,722,859B2, and Miller, K. et al, JImmunol, 2003.170 (9): pages 4854-61).

Other examples are tetravalent IgG single chain variable fragment (scFv) fusions (Dong J et al, 2011MAbs 3: 273-288; Coloma MJ, Morrison SL 1997Nat Biotechnol 15: 159;. Lu D et al, 2002J Immunol methods 267: 213-.

Furthermore, us patent 8,722,859B2 describes multivalent antibody constructs comprising two Fab repeats in the heavy chain of IgG and capable of binding four antigen molecules.

CN106459182A provides multivalent and multispecific tandem Fab immunoglobulins (FIT-Ig) capable of binding two or more antigens, or two or more epitopes.

OX40

OX40 (also known as CD134, TNFRSF4 and ACT35) WAs originally described as a T cell activation marker on rat CD 4T cells (Paterson DJ, Jefferies WA, Green JR, Brandon MR, Corthesy P, Puklavec M, Williams AF. inhibitors of activated T lymphocytes encoding a module 50,000Mr protected only on CD4positive T blast. mol Immunol. 1987; 24: 1281. supplement 1290) and WAs subsequently shown to be up-regulated in TCR recruitment (Mallett S, Fossum S, BarclayAN. mutagenesis of the C40 antigen of activated CD4positive Tcell 1068. EMBROn. 1068. model 1068). OX40 signaling can promote costimulatory signaling to T cells, leading to enhanced cell proliferation, survival, effector function and migration (Gramaglia I, WeinbergAD, Lemon M, Croft M.Ox-40 ligand: a pore timing molecular fbr stabilizing primary CD 4T JIell responses. MMunol.1998; 161: 6510-.

The ligand of OX40, OX40L, is expressed primarily on Antigen Presenting Cells (APCs) and its expression can be induced by CD40 and mast cell signaling, toll-like receptors (TLRs), and inflammatory cytokines. In addition to APCs, non-hematopoietic cells such as smooth muscle and vascular endothelial cells may also express OX 40L. In transgenic mice overexpressing OX40L, there was increased T-cell activation and when immunized, these mice produced an enhanced T-cell response (Murata K, Nose M, Ndhlovu LC, Sato T, Sugamura K, Ishii N.structural OX40/OX40ligand interaction indexes. J Immunol.2002; 169: 4628. sup. an. Sato T, IshiiN, Murata K, Kikuchi K, Nakagawa S, Ndhlovu LC, Sugarura K.sequences of OX40-OX40ligand interaction in cell function: enhanced connectivity sensitivity proteins 40L-3332; Eubacterium J.2002: 3332). This data indicates that OX40L expression is a limiting factor for OX40 signaling in T cells.

In tumor-bearing mice, in vivo ligation of mouse OX40 (via soluble mouse OX 40L-immunoglobulin fusion protein or mouse OX40L mimetics, such as anti-mouse CD134 specific antibody) enhanced anti-tumor immunity, resulting in tumor-free survival in mouse models of various mouse malignant tumor cell lines, such as lymphoma, melanoma, sarcoma, colon cancer, breast cancer, and glioma (Sugamura et al Nature Rev Imm 2004; 4: 420-431).

In Severe Combined Immunodeficiency (SCID) mice, in vivo ligation of human OX40 (via anti-human OX40 specific antibodies that interact with the OX40L binding domain on human OX 40; US2009/0214560a1) enhances anti-tumor immunity, which results in tumor growth inhibition of various human malignant tumor cell lines such as lymphoma, prostate, colon, and breast cancers.

Thus, there remains a need to develop new multivalent antibodies (e.g., anti-OX 40 antibodies) with at least four antigen binding sites that have better agonist activity than corresponding conventional antibodies (e.g., anti-OX 40 antibodies) and thus are better used to treat or delay various cancers, immune related diseases, and T cell dysfunctional diseases.

In poorly immunogenic tumors, single anti-OX 40 treatment failed to provide sufficient anti-tumor immunogenicity. Since agonism of TNFRs requires receptor aggregation, there remains a need in the art for single molecule entities with multivalent antigen binding functions for the TNF family (particularly the OX40 molecule) with improved aggregation and receptor agonism to meet increasing health and medical needs. The present invention meets these and other needs.

Summary of The Invention

The applicants have surprisingly found that multivalent antibodies having at least four antigen binding sites as provided by the present invention bind antigen efficiently and have better agonist activity than corresponding bivalent antibodies known in the art. Preferably, the antibody or fragment thereof is capable of activating T cells, for example enhancing the immunostimulatory/effector function of T effector cells and/or proliferating these cells and/or down regulating the immunosuppressive function of T regulatory cells. Thus, multivalent antibodies of the invention having at least four antigen binding sites can be used to treat or delay various cancers, immune-related diseases, and T cell dysfunctional diseases. In one embodiment, the multivalent antibody having at least four antigen binding sites is an anti-TNF antibody. The multivalent antibody having at least four antigen binding sites is an antibody against OX 40.

The present invention provides multivalent antibodies having at least four antigen binding sites. In one embodiment, the multivalent antibody comprises a dimerization domain and at least four antigen binding sites. Preferred dimerization domains contain (or consist of) an Fc domain and/or a hinge region.

In one embodiment, the invention provides an isolated antibody comprising a dimerization domain and at least four antigen binding sites at its amino terminus. The invention further provides isolated antibodies comprising an Fc domain and having at least four antigen binding sites at the amino terminus of the Fc domain. In one embodiment, the at least four antigen binding sites bind different antigens. In another embodiment, the at least four antigen binding sites bind the same antigen. In one embodiment, the at least four antigen binding sites are four Fv fragments that are the same or different. In a further preferred embodiment, the at least four antigen binding sites are four different Fab fragments. In another embodiment, the Fab fragments are the same Fab fragment.

In one embodiment, the invention provides an isolated antibody comprising an Fc domain and at least 4 antigen-binding fragments, wherein the antigen-binding fragments are in tandem, each fused at the amino terminus of the Fc domain, either directly or through a linker.

In another embodiment of the invention, an isolated antibody is provided wherein the Fc domain and 2 antigen-binding fragments comprise a full-length antibody having a valency of 2, and wherein the 2 additional antigen-binding fragments are tandemly linked at the amino terminus of the heavy chain of said full-length antibody, and wherein in another embodiment said 2 additional antigen-binding fragments are fused to the amino terminus of the heavy chain of said full-length antibody by a linker.

In a preferred embodiment, wherein said additional 2 antibodiesThe primary binding fragment is a 2 Fv fragment or a Fab fragment, preferably said Fab fragment is fused via its CH1 end (the Fab fragment is referred to as the distal Fab fragment, or as the outer Fab), respectively, to the amino terminus of the 1 heavy chain of said full-length antibody, either directly or via a linker (e.g., a linker of several amino acids). In a preferred embodiment, the Fab fragment is fused to the amino terminus of the 1 heavy chain of the full length antibody, either directly or via a linker, through amino acid residue H113(Kabat numbering) of its CH1 fragment, respectively, to form a fusion polypeptide of VH-CH 1-heavy chain (i.e., Fab-heavy chain). In one embodiment, the fusion polypeptide formed has the structure VH-CH 1-linker-heavy chain (i.e., Fab-linker-heavy chain). In a preferred embodiment, the linker is (GGGGS)_nWherein n is 0 to 4, and in a further preferred embodiment, the linker is GGGGS (SEQ ID NO: 57).

In one embodiment, the multivalent antibody comprises 2 heavy chain fusion polypeptides, wherein each heavy chain fusion polypeptide contains 2 or more heavy chain variable regions. In a preferred embodiment, the multivalent antibody comprises two identical heavy chain fusion polypeptides and four identical light chains, wherein each heavy chain fusion polypeptide contains 2 variable regions.

In one embodiment, the multivalent antibody comprises the structure of Fv 1-linker 1-Fv 2-linker 2-Fc, wherein Fv1 is the first Fv fragment and Fv2 is the second Fv fragment, wherein Fv1 is the same as or different from Fv2, and in a preferred embodiment, linker 2 is the hinge region of a native antibody.

In a further preferred embodiment, the multivalent antibody has the structure of Fab 1-linker 1-Fab 2-linker 2-Fc, wherein Fab1 is the same or different from Fab 2. In a more preferred embodiment, linker 1 is (GGGGS)_nWherein n-0-4 and linker 2 is the hinge region connecting CH1 and CH2 in a natural antibody. In another preferred embodiment, the multivalent antibody has 4 identical or different Fab fragments.

In a preferred embodiment, the multivalent antibody has 4 identical Fab fragments, wherein the heavy chain H113(Kabat numbering) of one Fab fragment is linked to the amino terminus of the heavy chain of the other Fab fragment by a linker. Is better atIn selected embodiments, the linker is (GGGGS)_nWherein n is 0 to 4. In one embodiment, the Fab is an anti-TNF Fab. In a preferred embodiment, the Fab is an anti-OX 40 Fab.

In one embodiment, the invention provides a multivalent anti-OX 40 antibody, wherein the multivalent anti-OX 40 antibody comprises one, two, or three CDRs (preferably three CDRs), or variants thereof, of a VH region sequence of any one of the antibodies shown in table C. In other embodiments, an antibody of the invention comprises one, two, or three CDRs (preferably three CDRs), or variants thereof, of the VL region sequence of any of the antibodies shown in table C. In some embodiments, an antibody of the invention comprises the 6 CDR region sequences of any one of the antibodies shown in tables a-E and I, or a variant thereof.

In one embodiment, the invention provides a multivalent anti-OX 40 antibody, wherein the multivalent anti-OX 40 antibody comprises at least four identical Fab fragments comprising 3 heavy chain Complementarity Determining Regions (CDRs) HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1. 2, 3, 59, 66, 67, 68 and 69, or relative to an amino acid sequence comprising NO more than 5 amino acid changes thereto, HCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4. 5, 6 and 60, or an amino acid sequence comprising NO more than 5 amino acid changes relative thereto, and HCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 7. 8, 9, 70, 71 and 72, or an amino acid sequence comprising no more than 5 amino acid changes relative thereto. In one embodiment, the alteration is an amino acid substitution (e.g., a conservative substitution), deletion, or insertion.

In one embodiment, the invention provides a multivalent anti-OX 40 antibody, wherein the multivalent anti-OX 40 antibody comprises at least four identical Fab fragments comprising a light chain variable region (VL), wherein the LCVR comprises complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10 and 11, or relative to an amino acid sequence comprising NO more than 5 amino acid changes thereto, LCDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 and 13, or relative to an amino acid sequence which comprises no more than 5 amino acid changes, LCDR3 comprising an amino acid sequence selected from the group consisting of SEQ id nos: 14. 15 and 16, or an amino acid sequence comprising no more than 5 amino acid changes relative thereto. In one embodiment, the alteration is an amino acid substitution (e.g., a conservative substitution), deletion, or insertion.

In one embodiment, the invention provides a multivalent anti-OX 40 antibody, wherein the multivalent anti-OX 40 antibody comprises at least four identical Fab fragments comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the HCVR comprises 3 Complementarity Determining Regions (CDRs) HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1. 2, 3, 59, 66, 67, 68 and 69, or relative to an amino acid sequence comprising NO more than 5 amino acid changes thereto, HCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4. 5, 6 and 60, or an amino acid sequence comprising NO more than 5 amino acid changes relative thereto, and HCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 7. 8, 9, 70, 71 and 72, or an amino acid sequence comprising NO more than 5 amino acid changes relative thereto, wherein the LCVR comprises LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence of SEQ ID NO:10 and 11, or relative to an amino acid sequence which comprises NO more than 5 amino acid changes, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO:12 and 13, or relative to an amino acid sequence which comprises NO more than 5 amino acid changes, LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 14. 15 and 16, or an amino acid sequence comprising no more than 5 amino acid changes relative thereto. In one embodiment, the alteration is an amino acid substitution (e.g., a conservative substitution), deletion, or insertion.

In one embodiment, the invention provides a multivalent anti-OX 40 antibody, wherein the multivalent anti-OX 40 antibody comprises at least four identical Fab fragments comprising a heavy chain variable region (VH), wherein the HCVR comprises 3 Complementarity Determining Regions (CDRs) HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1. 2, 3, 59, 66, 67, 68 and 69, or consists of said amino acid sequence; HCDR2 comprises a sequence selected from SEQ ID NO: 4. 5, 6 and 60, or consists of said amino acid sequence; HCDR3 comprises a sequence selected from SEQ ID NO: 7. 8, 9, 70, 71 and 72 or consists of said amino acid sequence.

In one embodiment, the invention provides a multivalent anti-OX 40 antibody, wherein the multivalent anti-OX 40 antibody comprises at least four identical Fab fragments comprising a light chain variable region (VL), wherein the LCVR comprises complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10 and 11 or consists of said amino acid sequence; LCDR2 comprising a nucleotide sequence selected from SEQ ID NO:12 and 13 or consists of said amino acid sequence; LCDR3 comprising a nucleotide sequence selected from SEQ ID NO: 14. 15 and 16 or consists of said amino acid sequence.

in one embodiment, the invention provides a multivalent anti-OX 40 antibody, wherein the multivalent anti-OX 40 antibody comprises at least four identical Fab fragments comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the HCVR comprises 3 Complementarity Determining Regions (CDRs) HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1. 2, 3, 59, 66, 67, 68 and 69, or consists of said amino acid sequence; HCDR2 comprises a sequence selected from SEQ ID NO: 4. 5, 6 and 60, or consists of said amino acid sequence; HCDR3 comprises a sequence selected from SEQ ID NO: 7. 8, 9, 70, 71 and 72 or consists of said amino acid sequence; wherein the LCVR comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:10 and 11 or consists of said amino acid sequence; LCDR2 comprising a nucleotide sequence selected from SEQ ID NO:12 and 13 or consists of said amino acid sequence; LCDR3 comprising a polypeptide selected from seq id NO: 14. 15 and 16 or consists of said amino acid sequence.

In one embodiment, the invention provides a multivalent anti-OX 40 antibody, wherein the multivalent anti-OX 40 antibody comprises at least four identical Fab fragments comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the HCVR comprises 3 Complementarity Determining Regions (CDRs) HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises SEQ ID NO:1, or consists of said amino acid sequence; HCDR2 comprises SEQ ID NO:4, or consists of said amino acid sequence; HCDR3 comprises SEQ ID NO:7 or consists of said amino acid sequence; wherein LCDR1 comprises SEQ ID NO:10, LCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO:12, LCDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:14 or consists thereof.

In one embodiment, the multivalent anti-OX 40 antibody of the invention comprises at least four identical Fab fragments comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the HCVR comprises 3 Complementarity Determining Regions (CDRs) HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 2, or consists of the amino acid sequence shown in the specification; HCDR2 comprises SEQ ID NO:5, or consists of said amino acid sequence; HCDR3 comprises SEQ ID NO: 8 or consists of said amino acid sequence; wherein LCDR1 comprises SEQ ID NO: 11, LCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 13, LCDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 15 or consists thereof.

In one embodiment, the multivalent anti-OX 40 antibody of the invention comprises at least four identical Fab fragments comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the HCVR comprises 3 Complementarity Determining Regions (CDRs) HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO:3, or consists of said amino acid sequence; HCDR2 comprises SEQ ID NO:6, or consists of said amino acid sequence; HCDR3 comprises SEQ ID NO: 9 or consists of said amino acid sequence; wherein LCDR1 comprises SEQ ID NO:10, LCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO:12, LCDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 16 or consists thereof.

In one embodiment, the multivalent anti-OX 40 antibody of the invention comprises at least four identical Fab fragments comprising a heavy chain variable region VH, wherein the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 36. 37, 38, and 61, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity thereto, or an amino acid sequence comprising no more than 10, no more than 5 amino acid changes relative thereto.

In one embodiment, the multivalent anti-OX 40 antibody of the invention comprises at least four identical Fab fragments comprising a light chain variable region VL comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 39. 40 and 41, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity thereto, or an amino acid sequence comprising no more than 10, no more than 5 amino acid changes relative thereto.

In one embodiment, the multivalent anti-OX 40 antibody of the invention comprises at least four identical Fab fragments comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 36. 37, 38, and 61, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity thereto, or an amino acid sequence comprising no more than 10, no more than 5 amino acid changes relative thereto; the light chain variable region VL comprises an amino acid sequence selected from SEQ ID NOs: 39. 40 and 41, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity thereto, or an amino acid sequence comprising no more than 10, no more than 5 amino acid changes relative thereto.

In one embodiment, the alteration is an amino acid substitution (e.g., a conservative substitution), deletion, or insertion.

In one embodiment, the multivalent anti-OX 40 antibody of the invention comprises at least four identical Fab fragments comprising a heavy chain variable region VH comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 36. 37, 38 and 61 or consists thereof.

In one embodiment, the multivalent anti-OX 40 antibody of the invention comprises at least four identical Fab fragments comprising a light chain variable region VL comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 39. 40 and 41 or consists thereof.

In one embodiment, the multivalent anti-OX 40 antibody of the invention comprises at least four identical Fab fragments comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 36. 37, 38 and 61 or consists thereof; the light chain variable region VL comprises an amino acid sequence selected from SEQ ID NOs: 39. 40 and 41 or consists thereof.

In a preferred embodiment, the multivalent anti-OX 40 antibody of the invention comprises at least four identical Fab fragments comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region VH comprises the amino acid sequence of SEQ ID NO:36 or consists thereof; the light chain variable region VL comprises SEQ ID NO:39 or consists thereof.

In a preferred embodiment, the multivalent anti-OX 40 antibody of the invention comprises at least four identical Fab fragments comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region VH comprises the amino acid sequence of SEQ ID NO: 37 or consists of the amino acid sequence shown in seq id no; the light chain variable region VL comprises SEQ ID NO: 40 or consists thereof.

In a preferred embodiment, the multivalent anti-OX 40 antibody of the invention comprises at least four identical Fab fragments comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region VH comprises the amino acid sequence of SEQ ID NO: 38 or consists thereof; the light chain variable region VL comprises SEQ ID NO: 41 or consists thereof.

In a preferred embodiment, the multivalent anti-OX 40 antibody of the invention comprises at least four identical Fab fragments comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region VH comprises the amino acid sequence of SEQ ID NO: 61 or consists of the amino acid sequence shown in seq id no; the light chain variable region VL comprises SEQ ID NO: 40 or consists thereof.

In one embodiment, the multivalent anti-OX 40 antibody of the invention comprises a heavy chain or heavy chain fusion polypeptide, wherein the heavy chain or heavy chain fusion polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 42. 43, 45, 46, 48, 49, 51, 52, 53, 54, 55, 56, 62, 63, 64 and 65, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more identity thereto, or an amino acid sequence comprising no more than 10, no more than 5 amino acid changes relative thereto.

In some embodiments, the multivalent anti-OX 40 antibodies of the invention comprise a light chain, wherein the light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 44. 47, 50, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity thereto, or an amino acid sequence comprising no more than 10, no more than 5 amino acid changes relative thereto.

In some embodiments, the multivalent anti-OX 40 antibodies of the invention comprise a heavy chain fusion polypeptide and a light chain, wherein the heavy chain fusion polypeptide comprises a heavy chain sequence selected from the group consisting of SEQ ID NOs: 51. 52, 53, 54, 55, 56, 64, 65, or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more identical thereto, or an amino acid sequence comprising no more than 10, no more than 5 amino acid changes relative thereto; the light chain comprises an amino acid sequence selected from SEQ ID NOs: 44. 47, 50, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity thereto, or an amino acid sequence comprising no more than 10, no more than 5 amino acid changes relative thereto.

In a preferred embodiment, the multivalent anti-OX 40 antibody of the invention comprises a heavy chain fusion polypeptide and a light chain, wherein the heavy chain fusion polypeptide comprises a heavy chain sequence selected from the group consisting of SEQ ID NOs: 51. 52 or consists thereof; the light chain comprises SEQ id no:44 or consists thereof.

In a preferred embodiment, the multivalent anti-OX 40 antibody of the invention comprises a heavy chain fusion polypeptide and a light chain, wherein the heavy chain fusion polypeptide comprises a heavy chain sequence selected from the group consisting of SEQ ID NOs: 53. 54 or consists thereof; the light chain comprises SEQ id no:47 or consists thereof.

In a preferred embodiment, the multivalent anti-OX 40 antibody of the invention comprises a heavy chain fusion polypeptide and a light chain, wherein the heavy chain fusion polypeptide comprises a heavy chain sequence selected from the group consisting of SEQ ID NOs: 55. 56 or consists thereof; the light chain comprises SEQ id no:50 or consists thereof.

In a preferred embodiment, the multivalent anti-OX 40 antibody of the invention comprises a heavy chain fusion polypeptide and a light chain, wherein the heavy chain fusion polypeptide comprises a heavy chain sequence selected from the group consisting of SEQ ID NOs: 64. 65 or consists of the amino acid sequence shown in seq id no; the light chain comprises SEQ id no:47 or consists thereof.

In some embodiments, the antibodies of the invention also encompass variants of the amino acid sequence of the anti-OX 40 antibody, as well as antibodies that bind the same epitope as any of the antibodies described above.

In some embodiments, the multivalent anti-OX 40 antibody of the invention is an IgG₁Formal antibodies (denoted g1 in antibody nomenclature) or IgG₂Antibody in the form (denoted g2 in the antibody nomenclature). In some embodiments, the anti-OX 40 antibody is a monoclonal antibody. In some embodiments, the anti-OX 40 antibody is humanized. In some embodiments, the anti-OX 40 antibody is a human antibody. In some embodiments, at least a portion of the framework sequence of the anti-OX 40 antibody is a human consensus framework sequence.

In one aspect, the invention provides multivalent anti-OX 40 antibodies with agonist activity that are capable of activating T cells (e.g., CD4+ T cells).

In some embodiments, the agonist activity of the multivalent anti-OX 40 antibody is assessed by the level of cytokine released upon T cell activation. Thus, the invention provides multivalent anti-OX 40 antibodies that increase cytokine production by CD4+ T cells compared to cytokine production by CD4+ T cells treated with IgG control. In some embodiments, the cytokine is an inflammatory cytokine, such as IL-2 or IFNg.

In one aspect, the invention provides a nucleic acid encoding any of the above anti-OX 40 antibodies or fragments thereof. In one embodiment, a vector comprising the nucleic acid is provided. In one embodiment, the vector is an expression vector. In one embodiment, a host cell comprising the vector is provided. In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from a yeast cell, a mammalian cell (e.g., a CHO cell or 293 cell), or other cell suitable for production of an antibody or antigen-binding fragment thereof. In another embodiment, the host cell is prokaryotic.

In one embodiment, the invention provides a method of making a multivalent anti-OX 40 antibody, wherein the method comprises, in cloning a construct comprising VH-CH1 and a construct comprising VL-CL into an expression vector, transfecting a host cell, and culturing the host cell under conditions suitable for expression of a nucleic acid encoding the multivalent antibody, and optionally isolating the antibody. In a certain embodiment, the method further comprises recovering the anti-multivalent anti-OX 40 antibody from the host cell.

In some embodiments, the invention provides compositions, preferably pharmaceutical compositions, comprising any of the multivalent anti-OX 40 antibodies described herein. In one embodiment, the composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition is for treating cancer, preferably lung cancer (e.g., non-small cell lung cancer), liver cancer, gastric cancer, colon cancer, and the like.

In some embodiments, the invention provides immunoconjugates comprising any of the multivalent anti-OX 40 antibodies provided herein and a cytotoxic agent. In some embodiments, the immunoconjugates are used to treat cancer, preferably lung cancer (e.g., non-small cell lung cancer), liver cancer, gastric cancer, colon cancer, and the like.

In one aspect, the invention relates to a method of activating T cells or inducing a T cell-mediated anti-tumor activity or enhancing an immune response in a subject, comprising administering to the subject an effective amount of any of the multivalent anti-OX 40 antibodies described herein. The invention also relates to the use of any of the multivalent anti-OX 40 antibodies described herein for the preparation of a composition or medicament for activating T cells or inducing T cell-mediated anti-tumor activity or enhancing an immune response in a subject.

In another aspect, the invention relates to a method of treating cancer in a subject, comprising administering to the subject an effective amount of any of the multivalent anti-OX 40 antibodies described herein. In one embodiment, the cancer is lung cancer (e.g., non-small cell lung cancer), liver cancer, gastric cancer, colon cancer, and the like. In another aspect, the invention also relates to the use of any of the multivalent anti-OX 40 antibodies or fragments thereof described herein for the preparation of a medicament for treating cancer in a subject. In one embodiment, the cancer is lung cancer (e.g., non-small cell lung cancer), liver cancer, gastric cancer, colon cancer, and the like.

In some embodiments, the methods described herein further comprise co-administering to the subject an effective amount of a second drug or active agent, wherein the multivalent anti-OX 40 antibody described herein is the first drug. In some embodiments, the second drug or active agent is selected from chemotherapeutic agents. In some embodiments, the second drug or active agent is selected from a PD-1 axis binding antagonist (e.g., an anti-PD-1 antibody or an anti-PD-L1 antibody or an anti-PD-L2 antibody) or an anti-angiogenic agent (e.g., bevacizumab).

In some embodiments, the subject or individual is a mammal, preferably a human.

In one aspect, the invention relates to a method of detecting OX40 in a sample, the method comprising (a) contacting the sample with any multivalent anti-OX 40 antibody described herein; and (b) detecting the formation of a complex between the multivalent anti-OX 40 antibody and OX 40. In one embodiment, the multivalent anti-OX 40 antibody is detectably labeled.

In some embodiments, the invention relates to a kit or article of manufacture comprising any of the multivalent anti-OX 40 antibodies described herein. In some embodiments, the kit or article of manufacture comprises a multivalent anti-OX 40 antibody described herein and optionally a pharmaceutically acceptable carrier. In some embodiments, the kit or article of manufacture further comprises instructions for administering the medicament to treat cancer.

The invention also encompasses any combination of any of the embodiments described herein. Any of the embodiments described herein, or any combination thereof, are applicable to any and all of the anti-OX 40 antibodies, methods, and uses of the invention described herein.

Drawings

FIG. 1 shows that antibodies of the invention produced in CHO cells block the binding of OX40L to OX40 expressed on CHO cells.

FIG. 2 stimulation of the exacerbation group OX40L with IgG produced in CHO cells by anti-CD 3/anti-CD 28₁Or IgG₂Forms of the antibodies of the invention were detected in a luciferase reporter gene in Jurkat stably transfected with human OX40 and the NFkB promoter-luc.

Figure 3 assesses agonist activity of the antibodies of the invention on OX40 signaling. IgG produced in CHO cells and with S.aureus Enterotoxin E (SEE) (1ng/ml)₁Or IgG₂Forms of the DC co-culture assay of OX40 antibodies of the invention were assayed.

Figure 4 shows the structure of a 4x Fab mAb containing 4 identical fabs linked to a1 x GS linker through the CH1 domain on the C-terminus of the outer Fab and the N-terminus of the inner Fab contained in the conventional mAb structure.

FIG. 5 shows size exclusion chromatography on human tetravalent OX40 mAb in the form of IgG 2: measurement of ADI-20057-g2-4xFab

FIG. 6 shows size exclusion chromatography on human tetravalent OX40 mAb in the form of IgG 2: measurement of ADI-23515-g2-4xFab

FIG. 7 shows size exclusion chromatography on human tetravalent OX40 mAb in the form of IgG 1: measurement of ADI-20112-g1-4xFab

Figure 8 shows the agonist activity of each antibody. IgG production in 293 cells stimulated by anti-CD 3/anti-CD 28₁、IgG₂And 4xFab forms of the antibody of the invention, detected with the luciferase reporter gene in Jurkat stably transfected with human OX40 and the NFkB promoter-luc.

Figure 9 shows the agonist activity of each antibody. IgG production in 293 cells stimulated by anti-CD 3/anti-CD 28₁、IgG₂And 4xFab form of the antibody of the invention, mixed Raii cells, detected with the luciferase reporter gene in Jurkat stably transfected with human OX40 and NFkB promoter-luc.

FIG. 10 assesses the blocking activity of anti-OX 40 antibodies of the invention. Stimulation of IgG production by anti-CD 3/anti-CD 28 and recombinant OX40L and 293 cells₁、IgG₂And 4xFab forms of the antibody of the invention, detected with the luciferase reporter gene in Jurkat stably transfected with human OX40 and the NFkB promoter-luc. Wherein 4xF is an abbreviation for 4xFab in the figure.

Detailed Description

Definition of

Before the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodologies, protocols, and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

For the purpose of interpreting this specification, the following definitions will be used, and terms used in the singular may also include the plural and vice versa, as appropriate. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

The term "about," when used in conjunction with a numerical value, is intended to encompass a numerical value within a range having a lower limit that is 5% less than the stated numerical value and an upper limit that is 5% greater than the stated numerical value.

The term "and/or" should be understood to mean either one of the selectable items or both of the selectable items.

As used herein, the term "comprising" or "comprises" is intended to mean including the stated elements, integers or steps, but not excluding any other elements, integers or steps. When the term "comprising" or "includes" is used herein, unless otherwise specified, it also encompasses the presence of stated elements, integers or steps. For example, when referring to an antibody variable region "comprising" a particular sequence, it is also intended to encompass antibody variable regions consisting of that particular sequence.

As used herein, the terms "anti-OX 40 antibody," "anti-OX 40," "OX 40 antibody," or "antibody that binds to OX 40" refer to an antibody that is capable of binding to a human or cynomolgus monkey OX40 protein or fragment thereof with sufficient affinity such that the antibody can be used as a diagnostic and/or therapeutic agent in targeting human or cynomolgus monkey OX 40. In one embodiment, the anti-OX 40 antibody binds to a non-human or non-cynomolgus monkey OX40 protein to a degree that is less than about 10% of the binding of the antibody to human or cynomolgus monkey OX40 as measured, for example, by Radioimmunoassay (RIA) or biophotonic interferometry or MSD assay. In some embodiments, the anti-OX 40 antibody has a dissociation constant (Kd) of less than or equal to 100nM, less than or equal to 10nM, or less than or equal to 1nM (e.g., 10 nM)^-7M or less, e.g. 10^-7M to 10^-10M, e.g. 10^-8M to 10^-9M)。

"affinity" or "binding affinity" refers to the intrinsic binding affinity that reflects the interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be determined by the equilibrium dissociation constant (K)_D) To express. Equilibrium dissociation constants are the dissociation and association rate constants (k respectively)_disAnd k_on) The ratio of (a) to (b). Affinity can be measured by common methods known in the art, including those known in the art and described herein.

As used herein, "monoclonal antibody" or "mAb" refers to an antibody derived from a single copy or clone, e.g., of a eukaryote, prokaryote, or phage clone, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope except for possible variant antibodies (e.g., variant antibodies containing natural mutations or produced during the production of a monoclonal antibody preparation) that are typically present in small amounts. 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. Monoclonal antibodies can be produced, for example, by hybridoma techniques, recombinant techniques, phage display techniques, synthetic techniques such as CDR grafting, or a combination of these or other techniques known in the art.

As will be understood by those skilled in the art, a "whole antibody" (used interchangeably herein with "full length antibody", "whole antibody" and "whole antibody") comprises at least two heavy chains (H) and two light chains (L). Each heavy chain consists, from N to C terminus, of a heavy chain variable region (also referred to as the variable heavy domain or heavy chain variable domain, abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of 3 domains, CH1, CH2, and CH 3. Each light chain consists of a light chain variable region (also referred to as a variable light domain or light chain variable domain, abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain CL. The VH and VL regions may be further subdivided into hypervariable regions (being Complementarity Determining Regions (CDRs) interspersed with more conserved regions (being Framework Regions (FRs)). Each VH and VL consists of three CDRs and 4 FRs arranged in the order from amino terminus to carboxy terminus FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. the variable regions are domains of the heavy or light chain of an antibody that are involved in the binding of the antibody to its antigen. the constant regions are not directly involved in the binding of the antibody to the antigen, but exhibit multiple effector functions. The additional peptide sequence is a Fab fragment, thereby forming a fusion polypeptide of VH-CH 1-heavy chain (i.e., Fab-heavy chain).

"Natural antibody" refers to a naturally occurring immunoglobulin molecule having a different structure. A "native sequence Fc domain" comprises an amino acid sequence that is identical to the amino acid sequence of an Fc domain found in nature. Native sequence human Fc domains include, for example, native sequence human IgG₁Fc domains (non a and a allotypes); native sequence human IgG₂An Fc domain; native sequence human IgG3Fc domain; and a native sequence human IgG4Fc domain; and naturally occurring variants thereof.

The "binding site" or "antigen-binding site" of an antibody is the region of the antibody molecule that actually binds to an antigen. In a preferred embodiment, the antigen binding site is comprised of a VH/VL pair consisting of an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH).

An "antigen-binding fragment" is a portion or fragment of an intact or complete antibody having fewer amino acid residues than the intact or complete antibody, which is capable of binding to an antigen or competing with the intact antibody (i.e., the intact antibody from which the antigen-binding fragment is derived) for binding to an antigen. Examples of antigen binding fragments include, but are not limited to, Fv, Fab '-SH, F (ab')₂Double-stranded antibodies (diabodies), single domain antibodies (sdabs), single-chain antibody molecules (e.g., scFv), triple-chain antibodies (triabodies), quadruple-chain antibodies (tetrabodies), minibodies, single domain antibodies (sdabs). The Fab fragment is a monovalent fragment consisting of the VL, VH, CL and CH1 domains, and can be obtained, for example, by papain digestion of whole antibodies. The light chain (L chain) and the heavy chain (H chain) of a Fab can be fused by means of a linker to a single polypeptide chain, i.e. a single chain Fab (scfab) (see e.g. US20070274985a 1). Digestion of the complete antibody by pepsin below the disulfide bond in the hinge region yields F (ab')₂Which is a dimer of Fab' and is a bivalent antibody fragment. F (ab')₂Can be reduced under neutral conditions by disrupting disulfide bonds in the hinge region, from F (ab')₂The dimer is converted to Fab' monomer. The Fab' monomer is essentially a Fab fragment with a hinge region. Fab '-SH is a Fab' in which the cysteine residues of the constant domains carry a free thiol group. Others of antibody fragmentsChemical couplings are also known. The Fv fragment consists of the VL and VH domains of a single arm of an antibody. Alternatively, genes independently encoding the two domains of the Fv fragment, VL and VH, can be recombinantly expressed using recombinant methods, joined together by a nucleic acid sequence encoding a linker peptide (linker) to form a single chain Fv or "scFv" in which the VL and VH regions pair to form the desired structure.

It is known in the art that antibodies can be classified into five major classes based on the heavy chain constant region amino acid sequence: IgA, IgD, IgE, IgG and IgM, and in addition, several of these antibodies may be further divided into subclasses (isotypes), e.g., IgG₁，IgG₂，IgG₃，IgG₄，IgA₁And IgA₂. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ and μ, respectively. Antibody light chains can be classified into one of two types, called kappa (κ) and lambda (in), based on the light chain constant region amino acid sequence.

By "antibody in IgG form" is meant that the heavy chain constant region of the antibody is the gamma chain. The gamma chain has various subclasses, e.g., IgG₁、IgG₂、IgG₃、IgG₄. For IgG herein₁Antibodies of class are suffixed to-g 1 in the nomenclature of antibodies, herein for IgG₂Antibodies of class are suffixed with-g 2 in the antibody name.

The term "diabodies" refers to antibody fragments having two antigen-binding sites, which fragments comprise a VL and a VH connected by a short linker in the same polypeptide chain. In a diabody, because the linker is too short, the two VH and VL domains on the same chain cannot pair but are forced to pair with the complementary domains of the other chain to create two antigen binding sites. Diabodies can be bivalent or bispecific. Diabodies are more fully described in e.g. EP 404, 097; WO 1993/01161; hudson et al, nat. med.9: 129-134 (2003); and Hollinger et al, proceedings of the national academy of sciences of the united states (proc.natl.acad.sci.usa) 90: 6444- > 6448 (1993). Three-and four-chain antibodies are also described in Hudson et al, nat. med.9: 129-134(2003) and shore glory et al (eds.), antibody drug research and applications, national health press (2013). Single domain antibodies (sdabs) generally refer to antibodies in which a single variable domain (e.g., a heavy chain variable domain (VH) or light chain variable domain (VL), a heavy chain variable domain derived from a camelid heavy chain antibody, a VH-like single domain (v-NAR) derived from a fish IgNAR) can confer antigen binding without the need to interact with another variable domain to recognize the target antigen. (for a more detailed description of antibody fragments see also: basic Immunology, edited by W.E.Paul, Raven Press, N.Y. (1993)).

The term "Fc domain" is used herein to define the C-terminal region of an immunoglobulin heavy chain, which region comprises at least a portion of a constant region. The term includes native sequence Fc domains and variant Fc domains. For example, in natural antibodies, the Fc domain consists of two identical protein fragments derived from the second and third constant domains of the two heavy chains of an antibody in the IgG, IgA, and IgD isotypes; the IgM and IgE Fc domains contain three heavy chain constant domains (CH domains 2-4) in each polypeptide chain. In certain embodiments, the Fc domain of a human IgG heavy chain extends from Cys226 or Pro230 to the carbonyl end of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc domain may or may not be present. Unless otherwise indicated, the numbering of amino acid residues in the Fc domain or constant region is according to the Kabat EU numbering system, also known as the Kabat EU index, as described in Kabat et al, Sequences of Proteins of Immunological Interest, 5th ed.

A "functional Fc domain" possesses the "effector function" of a native sequence Fc domain. Exemplary "effector functions" include C1q binding, CDC, Fc receptor binding, ADCC, phagocytosis, down-regulation of cell surface receptors (e.g., B cell receptor, BCR), and the like. Such effector functions generally require that an Fc domain be associated with a binding domain (e.g., an antibody variable domain) and can be evaluated using a variety of assays, such as those disclosed in the definitions herein.

The term "valency" antibody refers to the number of antigen binding sites present in an antibody molecule. "bivalent," "trivalent," and "tetravalent" antibodies refer to the presence of 2 antigen binding sites, 3 binding sites, and 4 binding sites, respectively, in an antibody molecule. In one embodiment, the antigen binding sites may bind different epitopes, and in another embodiment, the antigen binding sites bind the same epitope. In one embodiment, the antibody reported herein is "tetravalent".

By "multivalent antibody" is meant an antibody having several but at least four variable domains. Advantageously the multivalent antibody has four or eight variable domains, i.e. it is bivalent or tetravalent. Furthermore, the variable domains may be identical or different from each other, such that the antibody construct recognizes one or several antigens or epitopes. Multivalent antibodies preferably recognize one or two antigens, i.e., they are monospecific or bispecific, respectively.

The term "variant" in relation to an antibody refers herein to an antibody comprising a region of the antibody of interest (e.g. a heavy chain variable region or a light chain variable region or a heavy chain CDR region or a light chain CDR region) that has been altered in amino acid by at least 1, e.g. 1-30, or 1-20 or 1-10, e.g. 1 or 2 or 3 or 4 or 5 amino acid substitutions, deletions and/or insertions, wherein the variant substantially retains the biological properties of the antibody molecule prior to the alteration. In one aspect, the invention encompasses variants of any of the antibodies described herein. In one embodiment, an antibody variant retains at least 60%, 70%, 80%, 90%, or 100% of the biological activity (e.g., antigen binding capacity) of the antibody prior to alteration. In some embodiments, the alteration does not result in the antibody variant losing binding to the antigen, but optionally may confer properties such as increased antigen affinity and different effector functions. It will be appreciated that the variable heavy or light chain regions, or the respective CDR regions, of the antibody may be altered individually or in combination. In some embodiments, the amino acid change in one or more or all three heavy chain CDRs is no more than 1, 2, 3, 4, 5, 6,7, 8, 9, or 10. Preferably, the amino acid change is an amino acid substitution, preferably a conservative substitution.

In some embodiments, the antibody variant has at least 80%, 90% or 95% or 99% or more amino acid identity with the parent antibody over the region of the antibody sequence of interest. In another embodiment, an antibody of the invention has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity in the heavy chain variable region as compared to any of the antibodies listed in tables a-E. In yet another embodiment, an antibody of the invention has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity in the light chain variable region as compared to any of the antibodies listed in tables a-E. In yet another embodiment, an antibody of the invention has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity over the heavy chain variable region and/or the light chain variable region as compared to any of the antibodies listed in tables a-E. In yet another embodiment, an antibody of the invention has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity over the heavy chain (or heavy chain fusion polypeptide) and/or light chain as compared to any of the antibodies listed in tables a-E.

The expression "linker" refers to a molecule suitable for linking Fv fragments, or Fab fragments to each other. In one embodiment, the molecule may be a natural peptide or a synthetic peptide. The peptide linker may contain any amino acid, preferably the amino acids glycine (G), serine (S) and proline (P). The peptide linkers may be the same or different from each other. Non-limiting examples of peptide linkers are disclosed in Shen et al, anal. chem.80 (6): 1910- & 1917(2008), WO2012/138475 and WO2014/087010, the contents of which are incorporated by reference in their entirety. Further, the peptide linker may have a length of about 0 to 10 amino acids. In the former case, the peptide linker is simply a peptide bond from the COOH residue of one of the variable domains and the NH 2 residue of the other variable domain. The peptide linker preferably comprises the amino acid sequence GG. The peptide linker may also have a length of about 3 to 10 amino acids, particularly 5 amino acids, most particularly the amino acid sequence GGGGS, which is used to achieve the linkage of Fv fragments or Fab fragments. In the construction of multivalent antibodies, preferably, the linker will facilitate pairing of the VH and VL without interfering with the formation of a functionally effective antigen binding site for the VH and VL pairs.

Examples of peptide linkers that may be used in the present invention include: glycine polymer (G) n; glycine-serine Polymer (G)_1-5S_1-5) n, wherein n is an integer of at least 1, 2, 3, 4, or 5; glycine-alanine polymer; alanine-serine polymers; and other flexible joints known in the art. It will be appreciated by those skilled in the art that in some embodiments the linker between VH and VL may consist entirely of flexible connecting peptide, or the linker may consist of a flexible connecting peptide portion and one or more portions that confer a less flexible structure.

Multivalent antibodies according to the invention can be produced by conventional methods. A method is advantageous in which DNA encoding a peptide linker is linked to DNA encoding the Fv fragment, the Fab fragment of a multivalent antibody, such that the peptide linker links the Fv fragment, the Fab fragment to each other, and the resulting DNA molecule is expressed in an expression plasmid.

The DNA encoding the multivalent antibody according to the invention is also a subject of the invention.

another subject of the invention relates to a kit comprising:

(a) Multivalent antibody constructs according to the invention, and/or

(b) An expression plasmid, a plasmid-containing vector or a host cell comprising a vector according to the invention, and

(c) Conventional auxiliaries, such as buffers, solvents and controls.

The multivalent antibodies according to the invention are well suited for use not only for diagnostic but also for therapeutic purposes.

an "antibody that binds to the same epitope" as a reference antibody refers to an antibody that blocks binding of 50% or more of the reference antibody to its antigen in a competition assay, whereas a reference antibody blocks binding of 50% or more of the antibody to its antigen in a competition assay.

"antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a cytotoxic form in which secreted immunoglobulins that bind to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., NK cells, neutrophils, and macrophages) enable these cytotoxic effector cells to specifically bind to antigen-bearing target cells, followed by killing of the target cells with cytotoxins. The main cell mediating ADCC, NK cells, expresses only Fc γ RIII, whereas monocytes express Fc γ RI, Fc γ RII and Fc γ RIII. Ravatch and Kinet, annu.rev.immunol.9: 457-92(1991) 464 Page table 3 summarizes FcR expression on hematopoietic cells. To assess ADCC activity of a molecule of interest, an in vitro ADCC assay may be performed, such as described in U.S. Pat. No.5,500,362 or 5,821,337 or U.S. Pat. No.6,737,056 (Presta). Effector cells useful in such assays include PBMC and NK cells. Alternatively/additionally, ADCC activity of a molecule of interest may be assessed in vivo, for example in animal models, such as Clynes et al, pnas (usa) 95: 652-. An exemplary assay for assessing ADCC activity is provided in the examples herein.

As used herein, "agonist activity of an antibody" refers to the biological activity of an antibody that activates the antigen to which it binds.

"immunogenicity" refers to the ability of a particular substance to elicit an immune response. Tumors are immunogenic and enhancing tumor immunogenicity aids in the elimination of tumor cells by immune response.

"Effector function" refers to those biological activities attributable to the Fc domain of an antibody and which vary with the antibody isotype. Examples of antibody effector functions include: c1q binding and Complement Dependent Cytotoxicity (CDC); fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down-regulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

"human effector cells" refer to leukocytes which express one or more fcrs and which exert effector function. In certain embodiments, the cell expresses at least Fc γ RIII and performs ADCC effector function. Examples of human leukocytes that mediate ADCC include Peripheral Blood Mononuclear Cells (PBMCs), Natural Killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils. The effector cells may be isolated from their natural source, e.g., blood.

suitable "antibodies and antigen-binding fragments thereof" for use in the present invention include, but are not limited to, polyclonal, monoclonal, monovalent, bispecific, heteroconjugate, multispecific, recombinant, heterologous, heterohybrid, chimeric, humanized (particularly CDR grafted), deimmunized, or human antibodies, Fab fragments, Fab 'fragments, F (ab')₂Fragments, fragments produced by Fab expression libraries, Fd, Fv, disulfide linked Fv (dsfv), single chain antibodies (e.g., scFv), diabodies or tetrabodies (Holliger p. et al (1993) proc. natl. acad. sci. u.s.a.90(14), 6444-6448), nanobodies (also known as single domain antibodies), anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies directed against an antibody of the invention), and epitope-binding fragments of any of the foregoing.

The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding of the antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies typically have similar structures, with each domain comprising four conserved Framework Regions (FRs) and three Complementarity Determining Regions (CDRs). (see, e.g., Kindt et al Kuby Immunology, 6^thed., page 2007, w.h.freeman and co.91). A single VH or VL domain may be sufficient to confer antigen binding specificity. In addition, VH or VL domains from antibodies that bind to a particular antigen can be used to isolate antibodies that bind the antigen to screen libraries of complementary VL or VH domains, respectively. See, e.g., Portolano et al, j.immunol.150: 880- & ltwbr & gt 887 & gt (1993); clarkson et al, Nature 352: 624-628(1991).

"complementarity determining regions" or "CDR regions" or "CDRs" (used interchangeably herein with hypervariable region "HVRs") are regions of amino acids in an antibody variable region that are primarily responsible for binding to an epitope of an antigen. The CDRs of the heavy and light chains are commonly referred to as CDR1, CDR2, and CDR3, numbered sequentially from the N-terminus. The CDRs located within the antibody heavy chain variable domain are referred to as HCDR1, HCDR2 and HCDR3, while the CDRs located within the antibody light chain variable domain are referred to as LCDR1, LCDR2 and LCDR 3.

Various schemes are known in the art for determining the CDR sequences of a given VH or VL amino acid sequence: kabat Complementarity Determining Regions (CDRs) are determined based on sequence variability and are the most commonly used (Kabat et al, sequence of Proteins of Immunological Interest, 5th edition, Public Health Service, national institutes of Health, Bethesda, Md. (1991)), while Chothia refers to the position of structural loops (Chothia et al, (1987) J.mol.biol.196: 901-. The residues of each of these HVRs/CDR are as follows, according to different CDR determination schemes.

In one embodiment, the HVRs of an antibody of the invention are HVR sequences located according to the numbering system of Kabat at:

Positions 24-34(LCDR1), positions 50-56(LCDR2), and positions 89-97(LCDR3) in the VL, and positions 27-35B or 31-35B (HCDR1), positions 50-65(HCDR2), and positions 93-102 or 95-102(HCDR3) in the VH.

Positions 24-34(LCDR1), positions 50-56(LCDR2), and positions 89-97(LCDR3) in the VL, and positions 27-35B (HCDR1), positions 50-65(HCDR2), and positions 93-102(HCDR3) in the VH.

In one embodiment, the HVRs of an antibody of the invention are HVR sequences (CDRs as defined by Kabat) located according to the numbering system of Kabat at:

Positions 24-34(LCDR1), positions 50-56(LCDR2), and positions 89-97(LCDR3) in the VL, and positions 31-35B (HCDR1), positions 50-65(HCDR2), and positions 95-102(HCDR3) in the VH.

HVRs can also be determined based on having the same Kabat numbered position as a reference CDR sequence (e.g., any of the exemplary CDRs of the invention).

Unless otherwise indicated, in the present invention, the terms "CDR" or "CDR sequence" or "HVR sequence" encompass HVRs or CDR sequences determined in any of the ways described above.

Unless otherwise indicated, in the present invention, when referring to residue positions in the variable region of an antibody (including heavy chain variable region residues and light chain variable region residues), reference is made to the numbering positions according to the Kabat numbering system (Kabat et al, Sequences of proteins of Immunological Interest, 5th ed. public Health Service, national institutes of Health, Bethesda, Md. (1991)).

In one embodiment, the CDR sequences of the invention are shown in table I below.

Table I:

In one embodiment, the CDRs of the antibodies claimed herein are from the CDRs of the VH and/or VL regions listed in table C.

Antibodies with different specificities (i.e., different binding sites for different antigens) have different CDRs. However, although CDRs vary from antibody to antibody, only a limited number of amino acid positions within a CDR are directly involved in antigen binding. Using at least two of the Kabat, Chothia, AbM, and Contact methods, the region of minimum overlap can be determined, thereby providing a "minimum binding unit" for antigen binding. The minimum binding unit may be a sub-portion of the CDR. As will be appreciated by those skilled in the art, the residues in the remainder of the CDR sequences can be determined by the structure and protein folding of the antibody. Thus, the present invention also contemplates variants of any of the CDRs given herein. For example, in a variant of one CDR, the amino acid residue of the smallest binding unit may remain unchanged, while the remaining CDR residues according to Kabat or Chothia definition may be replaced by conserved amino acid residues.

In some embodiments, an antibody of the invention comprises at least one, two, three, four, five, or six CDRs that are identical to corresponding CDRs of any of the antibodies listed in tables a-E, or variants thereof. In some embodiments, an antibody of the invention comprises at least one, two, or three HCDRs, or variants thereof, identical to the corresponding heavy chain CDRs of any of the antibodies listed in tables a-E. In some embodiments, an antibody of the invention comprises at least one, two, or three HCDRs, or variants thereof, identical to the corresponding light chain CDRs of any of the antibodies listed in tables a-E. Herein, "corresponding CDRs" refer to CDRs at substantially similar positions in the variable region amino acid sequence. Herein, a CDR variant is a CDR that has been modified by at least one, e.g., 1 or 2 or 3 amino acid substitutions, deletions, and/or insertions, wherein the antigen binding molecule comprising the CDR variant substantially retains the biological properties of the antigen binding molecule comprising the unmodified CDR, e.g., retains at least 60%, 70%, 80%, 90%, or 100% of the biological activity (e.g., antigen binding capacity). It is understood that each CDR can be modified individually or in combination. Preferably, the amino acid modification is an amino acid substitution, in particular a conservative amino acid substitution, such as the preferred conservative amino acid substitutions listed in the specification.

"framework" or "F R" refers to variable domain residues other than the hypervariable region (HVR) (e.g., complementarity determining region) residues. The FRs of a variable domain typically consist of four FR domains: FR1, FR2, FR3 and FR 4. Thus, HVR and FR sequences typically occur in the following sequences of the heavy chain variable domain (VH) (or light chain variable domain (VL)): FR1-H1(L1) -FR2-H2(L2) -FR3-H3(L3) -FR 4.

Unless otherwise indicated, the numbering of residues in each domain of an antibody is according to the EU numbering system, also known as the EU index, as described in Kabat et al, Sequences of Proteins of Immunological Interest, 5th ed.

"Fv" is the smallest antibody fragment that contains the entire antigen-binding site. In one embodiment, a two-chain Fv species consists of dimers of one heavy chain variable domain and one light chain variable domain in tight, non-covalent association. In the single chain Fv (scFv) species, one heavy chain variable domain and one light chain variable domain may be covalently linked by a flexible peptide linker so that the light and heavy chains may associate in a "dimeric" structure similar to that of a two-chain Fv species. In this configuration, the three HVRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. In summary, six HVRs confer antigen binding specificity to antibodies. However, even a single variable domain (or half of an Fv comprising only three HVRs specific for an antigen) has the ability to recognize and bind antigen, although with less affinity than the entire binding site. For reviews on scFv see, for example, Pluckthun in The Pharmacology of monoclonal antibodies, Vol 113, Rosenburg and Moore eds, (Springer-Verlag, New York, 1994), pp.269-315.

"human antibody" refers to an antibody having an amino acid sequence corresponding to that of an antibody produced by a human or human cell or derived from a non-human source using a human antibody library or other human antibody coding sequence. This definition of human antibodies specifically excludes humanized antibodies comprising non-human antigen binding residues.

"human consensus framework" refers to a framework that represents the most commonly occurring amino acid residues in the selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a subset of variable domain sequences. In general, a subtype of this sequence is a subtype as in Kabat et al, Sequences of Proteins of immunological Interest, fifth edition, NIH Publication 91-3242, Bethesda MD (1991), volumes 1-3. In one embodiment, for VL, this subtype is subtype kappa I as in Kabat et al (supra). In one embodiment, for the VH, this subtype is subtype III as in Kabat et al (supra).

Multivalent antibodies vary according toThe components and the construction modes of the method can be divided into a plurality of types. Various multivalent antibody designs have various advantages and disadvantages, for example, although Blinatumomab can be produced by recombinant Chinese Hamster Ovary (CHO) cells in large-scale culture, it is easy to form aggregates, has a short half-life in vivo, and requires an additional continuous infusion device for practical use; the Catumaxomab production process is complex and murine alloantibodies are relatively prone to immunogenic problems in humans. Furthermore, unwanted pairing of unrelated heavy and light chains in a four-chain immunoglobulin (Ig) -like multispecific antibody leads to the formation of inactive antigen binding sites and/or other non-functional unwanted byproducts, which is also a problem in clinical scale production and therapeutic applications of antibodies (Klein, c. et al, Progress in overlapping the chainaassiaction assay in biochemical diagnostic IgG antibodies, mAbs, 2012, 4: 653-. Theoretically, two heavy chains can associate in four different combinations, and each of these heavy chains can associate with the light chain in a random manner, resulting in 2⁴(-16 in total) possible chain combinations. Of these 16 theoretically possible combinations, 10 combinations were actually found, but only one of them corresponded to the desired functional multispecific antibody. The difficulty in isolating the desired one of the multispecific antibodies from a complex mixture and the inherently poor yield of up to 12.5% in theory render the production of four-chain Ig-like multispecific antibodies in a cellular expression system difficult.

The present application provides a novel multivalent antibody having 4 Fab fragments, which can be expressed in high purity and yield in a cell, preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% purity and/or yield.

The term "dysfunction" in the context of immune dysfunction refers to a state of reduced immune responsiveness to antigenic stimulation. As used herein, the term "dysfunction" also includes an inability to sense or respond to antigen recognition, in particular, an impaired ability to translate antigen recognition into downstream T cell effector functions, such as proliferation, cytokine production (e.g., gamma interferon) and/or target cell killing.

By "activating T cells" is meant inducing, causing or stimulating effector or memory T cells to have renewed, sustained or amplified biological function. Examples of enhancing T cell function include: elevated levels from CD8 relative to pre-intervention such levels⁺Secretion of gamma-interferon (e.g., IFNg) or interleukin (e.g., IL-2) by effector T cells, elevated levels of interferon-gamma from CD4⁺Increased secretion of interferon-gamma (e.g., IFNg) or interleukins (e.g., IL-2) by memory and/or effector T cells, increased CD4⁺increased CD8 in response to and/or memory T cell proliferation⁺Effector T cells proliferate, increased antigen responsiveness (e.g., clearance). In one embodiment, the level of enhancement is at least 50%, or 60%, 70%, 80%, 90%, 100%, 120%, 150%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold. The manner of measuring this enhancement is known to those of ordinary skill in the art.

"tumor immunity" refers to the process by which a tumor evades immune recognition and clearance. As such, as a therapeutic concept, tumor immunity is "treated" when such evasion is diminished, and the tumor is recognized and attacked by the immune system. Examples of tumor recognition include tumor binding, tumor shrinkage and tumor clearance.

"anti-angiogenic agent" refers to a compound that blocks or interferes to some extent with vascular development. The anti-angiogenic agent can be, for example, a small molecule or antibody that binds to a growth factor or growth factor receptor involved in promoting angiogenesis. In one embodiment, the anti-angiogenic agent is an antibody that binds Vascular Endothelial Growth Factor (VEGF), such as bevacizumab (AVASTIN).

The term "PD-1 axis binding antagonist" refers to a molecule that inhibits the interaction of a PD-1 axis binding partner with one or more of its binding partners, thereby removing T cell dysfunction resulting from signaling on the PD-1 signaling axis, one result being restoration or enhancement of T cell function (e.g., proliferation, cytokine production, target cell killing). As used herein, PD-1 axis binding antagonists include PD-1 binding antagonists (e.g., anti-PD-1 antibodies, such as MDX-1106(nivolumab), MK-3475(pembrolizumab), CT-011(pidilizumab), or AMP-224 disclosed in WO 2015/095423), PD-L1 binding antagonists (e.g., anti-PD-L1 antibodies, in a particular aspect, anti-PD-L1 antibodies are yw243.55.s70, MDX-1105, MPDL3280A, or MEDI4736 disclosed in WO 2015/095423), and PD-L2 binding antagonists (e.g., anti-PD-L2 antibodies, immunoadhesins).

As used herein, the term "OX 40" refers to any native OX40 from any vertebrate source, including mammals such as primates (e.g., humans, cynomolgus monkeys) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses "full length," unprocessed OX40 as well as any form of OX40 that results from processing in a cell. The term also encompasses naturally occurring variants of OX40, such as splice variants or allelic variants.

"OX 40 activation" refers to the activation of OX 40. Generally, OX40 activation results in signal transduction.

The term "cytotoxic agent" as used herein refers to a substance that inhibits cell function and/or causes cell death or destruction. Examples of cytotoxic agents are disclosed in WO2015/153513, including but not limited to: a radioactive isotope; a chemotherapeutic agent or drug; a growth inhibitor; enzymes and fragments thereof, such as nucleolytic enzymes; (ii) an antibiotic; toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof; and various anti-tumor or anti-cancer agents.

"chemotherapeutic agents" include chemical compounds useful in the treatment of cancer. Examples of chemotherapeutic agents are disclosed in WO 2015/153513. Chemotherapeutic agents also include (i) anti-hormonal agents that act to modulate or inhibit the action of hormones on tumors, such as anti-estrogens and Selective Estrogen Receptor Modulators (SERMs); (ii) aromatase inhibitors which inhibit aromatase which regulates estrogen production in the adrenal gland; (iii) anti-androgens; (iv) protein kinase inhibitors; (v) a lipid kinase inhibitor; (vi) antisense oligonucleotides, particularly antisense oligonucleotides that inhibit gene expression in signaling pathways involved in abnormal cell proliferation, such as, for example, PKC-alphaRalf and H-Ras; (vii) ribozymes, such as VEGF expression inhibitors (e.g.And inhibitors of HER2 expression; (viii) vaccines, such as gene therapy vaccines; and (ix) and pharmaceutically acceptable salts, acids and derivatives of any of the foregoing. Chemotherapeutic agents also include antibodies. Many commercially available antibodies can be used in combination with the compounds of the present invention.

The term "cytokine" is a generic term for proteins released by one cell population that act on another cell as intercellular mediators. Examples of such cytokines are lymphokines, monokines; interleukins (IL), such as IL-1, IL-1 α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, IL-15; tumor necrosis factors such as TNF- α or TNF- β; and other polypeptide factors, including LIF and Kit Ligand (KL) and interferon gamma. As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the natural sequence cytokines, including small molecule entities produced by artificial synthesis, and pharmaceutically acceptable derivatives and salts thereof.

The term "effective amount" refers to an amount or dose of an antibody or fragment of the invention that produces the desired effect in the treated patient upon administration to the patient in a single or multiple dose. An effective amount can be readily determined by the attending physician, as one skilled in the art, by considering a number of factors: species such as mammals; its size, age and general health; the specific diseases involved; the degree or severity of the disease; the response of the individual patient; the specific antibody administered; a mode of administration; bioavailability characteristics of the administered formulation; a selected dosing regimen; and the use of any concomitant therapies.

The terms "host cell", "host cell line" and "host cell culture" are used interchangeably and refer to a cell into which an exogenous nucleic acid is introduced, including progeny of such a cell. Host cells include "transformants" and "transformed cells," which include primarily transformed cells and progeny derived therefrom, regardless of the number of passages. Progeny may not be identical in nucleic acid content to the parent cell, but may contain mutations. Included herein are mutant progeny that have the same function or biological activity as screened or selected in the originally transformed cell.

The terms "cancer" and "cancerous" refer to or describe a physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include, but are not limited to, squamous cell cancer (e.g., epithelial squamous cell cancer), lung cancer (including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), cancer of the peritoneum, hepatocellular cancer, gastric cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urinary tract cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, melanoma, superficial spreading melanoma, lentigo malignant melanoma, acromegaly melanoma, nodular melanoma, multiple myeloma and B-cell lymphoma, Chronic Lymphocytic Leukemia (CLL), Acute Lymphoblastic Leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with scarring nevus (phakomatases), edema (such as associated with brain tumors) and Meigs (Meigs) syndrome, brain tumors and cancers, as well as head and neck cancers, and associated metastases. In certain embodiments, cancers suitable for treatment by the antibodies of the invention include lung cancer (e.g., non-small cell lung cancer), liver cancer, gastric cancer, or colon cancer, including metastatic forms of those cancers.

The terms "cell proliferative disorder" and "proliferative disorder" refer to a disorder associated with a degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder is cancer.

The term "tumor" refers to all neoplastic (neoplastic) cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer," "cancerous," "cell proliferative disorder," "proliferative disorder," and "tumor" are not mutually exclusive when referred to herein.

An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules (including but not limited to cytotoxic agents).

An "individual" or "subject" includes a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human.

An "isolated" antibody is one that has been separated from components of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC). For a review of methods for assessing antibody purity, see, e.g., Flatman et al, j.chromatogr.b848: 79-87(2007).

An "isolated" nucleic acid is a nucleic acid molecule that has been separated from components of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but which is present extrachromosomally or at a chromosomal location different from its natural chromosomal location.

An "isolated nucleic acid encoding a multivalent anti-OX 40 antibody" refers to one or more nucleic acid molecules encoding antibody heavy and light chains, including such nucleic acid molecules in a single vector or separate vectors, as well as such nucleic acid molecules present at one or more locations in a host cell.

"percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are the same as the amino acid residues in the reference polypeptide sequence, after the sequences are aligned (and gaps introduced, if necessary) to obtain the maximum percent sequence identity, and no conservative substitutions are considered as part of the sequence identity. Sequence alignments can be performed using various methods in the art to determine percent amino acid sequence identity, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or megalign (dnastar) software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms required to obtain maximum alignment over the full length of the sequences being compared.

When referring to percentages of sequence identity in the present application, these percentages are calculated over the full length of the longer sequence, unless otherwise specifically indicated. The full length calculation relative to the longer sequence applies to both nucleic acid and polypeptide sequences.

The term "pharmaceutical composition" refers to a formulation that is present in a form that allows the biological activity of the active ingredient contained therein to be effective, and that does not contain additional ingredients that have unacceptable toxicity to the subject to which the formulation is administered.

The term "pharmaceutically acceptable carrier" refers to a diluent, adjuvant (e.g., freund's adjuvant (complete and incomplete)), excipient, or vehicle with which the therapeutic agent is administered.

As used herein, "treating" refers to slowing, interrupting, arresting, alleviating, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease.

The term "vector" when used herein refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors which are self-replicating nucleic acid structures as well as vectors which are incorporated into the genome of a host cell into which they have been introduced. Some vectors are capable of directing the expression of a nucleic acid to which they are operably linked. Such vectors are referred to herein as "expression vectors".

"subject/patient sample" refers to a collection of cells or fluids obtained from a cancer patient or cancer subject. The source of the tissue or cell sample may be a solid tissue, like from a fresh, frozen and/or preserved organ or tissue sample or biopsy sample or punch sample; blood or any blood component; body fluids such as cerebrospinal fluid, amniotic fluid (amniotic fluid), peritoneal fluid (ascites), or interstitial fluid; cells from a subject at any time of pregnancy or development. Tissue samples may contain compounds that are not naturally intermixed with tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like. Examples of tumor samples herein include, but are not limited to, tumor biopsies, fine needle aspirates, bronchial lavage, pleural fluid (pleural fluid), sputum, urine, surgical specimens, circulating tumor cells, serum, plasma, circulating plasma proteins, ascites, primary cell cultures or cell lines derived from tumors or exhibiting tumor-like properties, and preserved tumor samples, such as formalin-fixed, paraffin-embedded tumor samples or frozen tumor samples.

The term "package insert" is used to refer to instructions for use typically contained in commercial packages of therapeutic products that contain information regarding the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings relating to the use of such therapeutic products.

Examples

The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA technology, genetics, immunology and cell biology within the skill of the art. These methods can be described, for example, in Sambrook et al, Molecular Cloning: a laboratory manual (3 rd edition, 2001); sambrook et al, Molecular Cloning: a Laboratory Manual (2 nd edition, 1989); maniatis et al, Molecular Cloning: a Laboratory Manual (1982); ausubel et al, Current Protocols in Molecular Biology (John Wiley and Sons, 2008, 7 month update); short protocols in Molecular Biology: a Complex of Methods from Current protocols Molecular Biology, Greene pub.associates and Wiley-Interscience; glover, DNACloning: a Practical Approach, vol.I & II (IRL Press, Oxford, 1985); anand, Techniques for the Analysis of Complex genoms, (Academic Press, New York, 1992); transcription and transformation (b.hames & s.higgins, eds., 1984); perbal, A Practical Guide to Molecular Cloning (1984); harlow and Lane, Antibodies, (Cold Spring Harbor laboratory Press, Cold Spring Harbor, n.y., 1998) Current Protocols in immunology q.e.coli, a.m.kruisbeam, d.h.margulies, e.m.show and w.strober, eds., 1991); annual Review of Immunology; and journal works such as Advances in Immunology.

Example 1 preparation of anti-OX 40 monoclonal antibodies

Antibody production and purification

The amino acid sequences of the CDR regions, the light and heavy chain variable regions, the light and heavy chains, and the corresponding nucleotide sequences of the exemplary antibodies of the invention are set forth in tables a-E and "sequence listing" sections of the present application. The light chain variable region and the heavy chain variable region of the above-described exemplary antibody of the present invention have the sequence numbers shown in the following table.

TABLE 1

Name of antibody	VH	yL
			ADI-20112	SEQ ID NO：38	SEQ ID NO：41
ADI-20078	SEQ ID NO：61	SEQ ID NO：40
			ADI-23515	SEQ ID NO：37	SEQ ID NO：40
ADI-20057	SEQ ID NO：36	SEQ ID NO：39

The following anti-OX 40 antibodies of the invention were expressed and purified in yeast or CHO-S cells or HEK293 cells

For yeast material:

yeast clones expressing the corresponding antibodies were grown to saturation on appropriate media, then shake-cultured at 30 ℃ and induced with IPTG for 48 hours. After induction, yeast cells were pelleted and supernatants harvested for antibody purification. IgG was purified using a protein a column and eluted with acetic acid (PH 2.0) according to the manufacturer's instructions. Purified antibodies were digested with papain to generate the corresponding Fab fragments and the fabs were purified by kappaselect (ge healthcare life sciences) according to the manufacturer's instructions.

For CHO-S cells treatment:

Used according to the manufacturer's instructionsThe kit (Invitrogen) generated an expression CHO-S cell line. For mAb expression, the DNA sequences encoding the heavy and light chains were inserted together into the pCHO1.0 plasmid (Invitrogen), with the heavy chain upstream of the light chain, the heavy chain inserted into the pCHO1.0 plasmid at the cleavage site AvrII/Bstz17, and the light chain inserted into the pCHO1.0 plasmid at the cleavage site EcorV/PacI. The full length human OX40 CDS sequence (Sinobiology) was inserted into the pCHO1.0 vector for the generation of stable over-expressed cell lines.

Treatment of HEK293 cells:

For transient expression of proteins in HEK293 cells, the vector PTT5 was used, wherein DNA encoding the heavy and light chains of the antibody was cloned into separate vectors via cleavage sites XhoI/NotI, respectively. Transfection of HEK293 cells with pei (polysciences) was performed using standard procedures routine in the art; after 7 days of culture, the supernatants were collected and the corresponding antibodies expressed in the supernatants were purified on the AKTA system (GE).

The four exemplary antibodies described above were obtained.

The control antibody is

pogalizumab
	Hu106-222
11D4
	tavolixizumab

As used herein, pogalizumab is a human IgG transiently expressed in HEK293 cells₁OX40 antibody which utilises antibodies from INN: list 114 (see alsohttp：//www.who.int/medicines/publications/ druginformation/innlists/PL114.pdf) The heavy and light chain sequences of (a). As used herein, Hu106-222 is a humanized IgG transiently expressed in HEK293 cells₁An OX40 antibody which utilises the heavy and light chain sequences from US 9006399. As used herein, 11D4 is a humanized IgG transiently expressed in HEK293 cells₁An OX40 antibody which utilises the heavy and light chain sequences from US 8236930. As used herein, tavolixizumab is a humanized IgG transiently expressed in HEK293 cells₁OX40 antibody which utilises antibodies from INN: list 115 (see alsohttp：//www.who.int/medicines/ publications/druginformation/innlists/PL115.pdf) The heavy and light chain sequences of (a).

Exemplary sequences herein are found in tables a-E below:

Example 2: binding kinetics and affinity assays for anti-OX 40 antibodies

The present application uses a biophotonic interferometry (BLI, ForteBio) assay to determine the kinetic and equilibrium dissociation constant (K) for binding of the antibodies of the invention to human OX40_D)。

BLI KD assay (Bio-optical interferometry)

The BLI affinity assay is carried out according to the known methods (Estep, P et al, High throughput solution Based measurement of antibody-antibody affinity and epitope binding. MAbs, 2013.5 (2): p.270-8). Briefly, the sensor was equilibrated in assay buffer for 30 minutes under the line, then the baseline was established by on-line detection for 60 seconds, and affinity measurements were performed on an AHQ sensor (ForteBio) on-line loading of purified antibody obtained as described above. The sensor with the loaded IgG was exposed to 100nM of OX40 antigen for an additional 5 minutes before transferring the sensor to assay buffer for dissociation for 5 minutes for dissociation rate measurements. Kinetic analysis was performed using a 1: 1 binding model.

In the implementation as described in the assayIn the assay, ADI-20057, ADI-20112 and ADI-23515 (IgG expressed in yeast)₁Form Fab of anti-OX 40 antibody) at a monovalent K in the less than two-digit nM range_DBinding to human OX40_ Fc (human OX40 bound to the Fc part of an antibody, purchased from R)&D Systems). ADI-20112, ADI-23515, and ADI-20057 (as IgG) when the antibody is on the sensor tip₁Forms and expressed in yeast) in the single digit nanomolar to subnanomolar range as bivalent (avid) K_DValues bind to human OX40_ Fc, ADI-20112, ADI-23515 and ADI-20057 (as IgG)₁Forms and expressed in yeast) bind cynomolgus monkey OX40_ Fc (cyno OX40_ Fc, available from AcroBiosystems) at the nanomolar level of single digits. ADI-23515 (as IgG) when antibody is on the sensor tip₁Format and expressed in yeast) bound mouse OX40_ Fc (purchased from Acro Biosystems) at 8.98E-09nM (table 2). 11D4, a positive control, exhibited similar binding affinity.

Table 2: measurement of IgG by Bio-optical interferometry₁Binding of forms of the antibodies of the invention

Example 3: binding of anti-OX 40 antibodies of the invention to human OX40

Binding to human OX40 on CHO cells

Binding of the antibodies of the invention to human OX40 can be measured in a flow cytometry-based assay.

cDNA encoding human OX40(Sinobiologics) was cloned into pCHO1.0 vector (Invitrogen) by means of a multiple cloning site (AvrII/PacI), and CHO cells overexpressing human OX40 (CHO-hOX40 cells) were then generated and screened by conventional transfection methods. CHO-hOX40 cells (cell density: 0.2X 10)⁶) Incubate 100nM of the test antibody in PBS with 0.1% BSA on ice for 30 min. The cells were then washed at least twice and incubated with a secondary antibody (phycobilichrome, pe (southern biotech)) labeled at a final concentration of5 μ g/ml) in PBS containing 0.1% BSA on ice (protected from light) for 30 minutes. Cells were washed at least twice and analyzed by flow cytometry. In CantoFlow cytometry was performed on the II system (BD Biosciences) and MFI calculated accordingly.

The experimental results show that: in this assay, ADI-20057, ADI-23515, and ADI-20112 had high binding strength to CHO cells overexpressing human OX40, with over 1500-fold difference in MFI values compared to CHO cells not expressing OX40 (Table 3). 11D4, as a positive control, also showed > 1500-fold difference.

Table 3: IgG of the invention produced in yeast by flow cytometry₁Forms of the antibody bind to CHO cells expressing human OX40

Example 4 pharmacokinetics of OX40 antibodies produced in CHO cells

Pharmacokinetic assay

Pharmacokinetic data were evaluated after administration of the antibodies of the invention ADI-20078-IgG1, ADI-20078-IgG2, ADI-20112-IgG1, ADI-20112-igg2i.v. to female Balb/C mice at a dose of 10 mg/kg. The corresponding dose volume was injected into mice based on body weight.

The timing of blood collection was started at the beginning of the drug administration. For i.v. administration, time points included 0.083 hours (5 minutes) until day 21. Blood was collected from three mice at each time point. Approximately 100. mu.l of blood was collected in a microcentrifuge tube. Blood samples were stored on ice at a speed setting of 3000RPM for at least 10 minutes to obtain approximately 40-50ul serum.

The serum concentration of the antibody administered to the mice was determined by immunoassay using a sandwich ELISA method. Briefly, microtiter plates (Nunc, cat #442404) were coated overnight with lug/mL of OX40(ACRO, Cat #1044-5CIS1-AF) in 0.2M Carbonate Buffer (CBS) (pH9.4) at 4 ℃. After washing, the plates were then blocked with 5% skim milk for 1.5 hours at ambient temperature and mouse serum samples were applied to the coated plates for 1.5 hours at room temperature. Standard curves ranging from 0.315 to 80ng/mL were prepared in normal mouse serum with anti-OX 40 antibody diluted.

Bound anti-OX 40 antibody was detected using horseradish peroxidase-labeled goat anti-human Fc antibody (Bethyyl, cat # A80-104P-84) diluted 1: 100,000 in 0.05% PBST (0.05% Tween in PBS), 5% BSA. After the washing step, the plates were developed with 3, 3 ', 5, 5' -tetramethylbenzidine hydrochloride solution (TMB) for 10-15 minutes at room temperature and after 5 minutes stopped by adding 2N sulfuric acid. The optical density was measured at 450nm using a Thermo ELISA plate reader (Multiskan FC), minus the 620nm reference wavelength. Quantitation was based on a four parameter logistic (1/Y2) regression of a standard curve prepared using Skanit software3.1 (Thermo).

PK parameters (Cmax, AUC, t1/2, C1 and Vss) were analyzed by PKSolver software based on a non-compartmental model (non-comparative model) with mean values for each set of concentrations.

TABLE 4

Example 5 blocking of human OX40L interaction with OX40 by antibodies of the invention

1. Blocking of human OX40L interaction with OX40 on CHO cells by antibodies of the invention

The ability of an antibody of the invention to block the binding of human OX40 to OX40L can be measured by flow cytometry.

CHO cells (density: 0.2X 10)⁶) Incubate experimental antibody (100nM) in PBS containing 0.1% BSA on ice for 30 min. The cells were then washed 3 times and incubated with OX40L (. about.10. mu.g/ml) fused to mouse Fc (. about.10. mu.g/ml) in PBS containing 0.1% BSA for 30 min on ice (protected from light). Cells were washed 3 times and subsequently incubated with anti-murine FC-FITC secondary antibody on ice for 30 minutes in PBS containing 0.1% BSA. After washing the cells were analyzed by flow cytometry. Flow cytometry was performed on the Accuri C6 system (BD Biosciences) and MFI was calculated on the C6 software.

Results of the experimentShows that: in the experiment, ADI-20057-g2, ADI-23515-g2 and ADI-20112-g1 (IgG, respectively)₂(g2) And IgG₁(g1) Form, and expressed in CHO cells) blocks binding of human OX40L (60 μ g/ml) to OX40(ACRO, cat #1044-5CIS1-AF), showing lower blocking activity compared to Pogalizumab at higher concentrations, but at similar levels to OX40L (fig. 1). ADI-20057-g2 showed very low blocking levels, similar to the IgG control (FIG. 1).

2. OX40 blockade assay based on luciferase reporter genes

The blocking activity of an anti-OX 40 antibody of the invention can be assessed by measuring the ability of the antibody to block OX 40L-mediated activation of OX 40. Jurkat cells overexpressing human OX40 and NFkB-luciferase constructs were activated using anti-CD 3 (2. mu.g/ml, Biolegend), anti-CD 28 (2. mu.g/ml, Biolegend) and recombinant OX40L (60. mu.g/ml; Acro Biosystems) in solution with increasing concentrations of anti-OX 40 antibody for 18 hours, followed by cell lysis and substrate addition for detection and absorbance measurements.

As a result, it was found that: pogalizumab readily blocks OX 40L-based activation at concentrations greater than about 0.08nM or higher, whereas human IgG produced in CHO cells₁Type anti-OX 40 antibody ADI-20112-g1 and human IgG₂Type anti-OX 40 antibody ADI-23515-g2 blocked OX 40L-based activation at concentrations of 20nM and above (FIG. 2). In addition, anti-OX 40 antibody ADI-20057-g2 was found to increase luciferase levels, suggesting that it has non-OX 40L blocking properties and contributes to OX40 aggregation (FIG. 1).

Example 6 agonist Activity of anti-OX 40 antibodies of the invention

Agonist activity of the anti-OX 40 antibodies of the invention can be assessed by measuring inflammatory cytokines released by T cells following T cell activation.

1.Mixed lymphocyte reaction/DC-T cell co-culture assay

Agonist activity of the antibodies of the invention against OX40 signaling can be assessed by measuring the release of IL-2 during a mixed lymphocyte reaction or a DC-T cell co-culture assay. 2 x 10 to⁶PBMC were plated in each well of a 6-well tissue culture plateMedium or T25 tissue culture flasks in complete T cell Medium, 37 ℃, 5% CO₂Culturing in an atmosphere. Cells were incubated for 2-3 hours to allow monocyte PBMCs to attach. If the adhesion is insufficient, serum-free medium is used. Non-attached cells were removed by gently vortexing the flasks with 3 × fresh medium (X-VIVO 15, Lonza).

Monocytes (1X 10) from PBMC were cultured in X-VIVO 15 medium containing 1% AB serum, 10mM HEPES, 50. mu.M beta-Me, IL-4(1000U/ml) and GM-CSF (1000U/ml), or 25-50ng/ml each⁶Individual cells/ml) produced immature bone marrow DC cells. After 2 days, fresh medium X-VIVO 15 supplemented with IL-4 and GM-CSF was added. On day 5, cells were frozen or frozen by addition of a solution containing rTNFa (1000U/ml), IL-1b (5ng/ml), IL-6(10ng/ml) and 1. mu. MPGE₂Stimulation mixture of (2) at 3X 10⁵Cell density of individual cells/ml for 2 days induced maturation. T cell isolation was performed according to the manufacturer's instructions in the unouched CD4+ T cell isolation kit (Invitrogen). A magnet fitted with a 1.5ml tube rack was used to remove unwanted magnetic beads (QIAGEN).

100,000-200,000 isolated T cells were mixed with 10,000-20,000 allogeneic MODCs in a total volume of 200. mu.l in 96-round bottom tissue culture plates for 4-5 days at 37 ℃. Staphylococcus aureus enterotoxin E (SEE for short) (1ng/ml) was added to increase T cell activation. Test antibody was added at the beginning of the mixed lymphocyte experiment (MLR) and incubated throughout the culture period. The detection of IL-2 was carried out according to the manufacturer's (eBioscience) instructions. OD measurements were determined on Multiskan FC system (Thermo).

The results show that, in this assay, IgG is expressed in CHO cells₂Human antibodies in the form ADI-23515-g2 and human IgG₁Formal antibody ADI-20112-g1 and human IgG expressed in CHO cells₂Form of antibody ADI-20057-g2, increased IL-2 secretion at concentrations of-13.3. mu.g/ml, -4.4. mu.g/ml and-1.48. mu.g/ml, with the ability to increase IL-2 secretion equal to or better than pogalizumab, 11D4, Hu106-222 and tavolixizumab (FIG. 3). Pogalizumab had poor agonist activity at all concentrations, 40. mu.g11D4, Hu106-222, tavolixizumab, ADI-23515-g1, and ADI-20112-g1 at ml showed lower agonist activity at the highest OX40 antibody concentration of 40 μ g/ml (FIG. 3). Only 40. mu.g/ml ADI-20057-g2 had equal or better activation of IL-2 secretion compared to lower concentrations.

Example 7: construction, expression, purification and analysis of tetravalent (4xFab) mAbs

1. Construction and expression of tetravalent (4xFab) mAb

Naturally occurring monoclonal antibodies can exist as multimeric structures such as IgM that increase affinity for their target antigen on a fixed surface. Because agonism of TNFR requires receptor aggregation, tetravalent or hexavalent monoclonal antibodies may provide increased aggregation and receptor agonism. The invention is a mAb comprising four identical Fab regions with the C-terminal CH1 region of the distal Fab linked to the N-terminus of the Fab on a conventional mAb either through or without a linker, such as a GGGGS linker. Briefly, to generate tetravalent mabs, 2 constructs (construct #1 and construct #2) were cloned into the expression vector pTT5 of HEK293 cells by conventional methods using XhoI and NotI cleavage sites, respectively. In particular, construct #1 comprises the gene sequence encoding the test antibody VH-CH1, wherein the test antibody VH-CH1 (from H1 to H113, Kabat numbering) is either directly or via a linker (GGGGS)_n(where N may be an integer from 0 to 4, and N ═ 1 was used in this example) fused to the N-terminus of the heavy chain of the same antibody (fig. 4). Construct #2 was the normal VL-CL (from L1 to L110, Kabat numbering) chain of the antibody. The 2 constructs were co-transfected in HEK293 cells, screened for expressed and secreted 4xFab mAb molecules, and the correct molecules were selected for further experiments.

The protein expression process is as follows:

1) HEK293 cells (Invitrogen) were passaged according to the desired transfection volume, and the cell density was adjusted to 1X 10 the day before transfection⁶And/ml. Cell density at day of transfection was approximately 2X 10⁶/ml；

2) Taking a final volume of 1/10F 17(Gibco, A13835-01) culture medium as a transfection buffer solution, adding a proper plasmid, and uniformly mixing;

3) AddingThe appropriate PEI (Polysciences, 23966) was added to the plasmid and mixed well and incubated for 10 minutes at room temperature. The mixture was poured gently into the cells at 36.5 ℃ with 8% CO₂Culturing;

4) After overnight, each was supplemented with 200G/L FEED (Sigma, H6784-100G) and 200G/L glucose master solution in a transfection volume of 1/50, and after 20 hours VPA (Gibco, 11140-050) was added to 2 mM/L;

5) Continuously culturing for 7 days or collecting cell supernatant for purification when cell viability is less than or equal to 60%.

2. Purification and analysis of tetravalent (4xFab) mAb

A. Purification and Size Exclusion Chromatography (SEC)

The 4xFab mAb molecule obtained above was used in HiTrap^TMMabSelect SuRe^TMpurification was carried out on a 5ml column (GE 11-0034-93) according to the manufacturer's instructions, using 20mM Tris +150mM sodium chloride (pH7.2) as binding buffer. The antibody was eluted with elution buffer (0.1M sodium citrate, pH 3.5) and the pH of the eluted antibody solution was adjusted to 6 with 1M Tris-HCl. Each 4xFab mAb molecule, for example ADI-20112-g1-4xFab, was further purified by ion exchange (CEX) using a Mono S5/50 column (GE 17-5168-01) according to the manufacturer' S instructions.

SEC analysis methods can be used to test the aggregation propensity of purified 4xFab antibodies. Rapid SEC analysis of mabs was performed using a TSKgel SuperSW3000 column (18675) at a rate of 0.35mL/min with a cycle time of 15 minutes/run. A solution of 20mM sodium phosphate and 150mM sodium chloride was adjusted to pH 6.8 and used as a mobile phase. The injection volume was 20. mu.l and the UV signal was detected at 280 nm.

The results show that IgG is a tetravalent molecule₂Forms of human OX40 antibodies ADI-20057-g2-4xFab, ADI-23515-g2-4xFab (FIG. 4), IgG as tetravalent molecule₁Forms of the human OX40 antibody ADI-20112-g1-4xFab (fig. 4), which can be expressed and purified in high purity after a single protein a purification step, are 98.72% (fig. 5) and 98.51% (fig. 6) pure for ADI-20057-g2-4xFab and ADI-23515-g2-4xFab, respectively. ADI-20112-g1-4xFab was purified to 99.22% purity in three steps of affinity chromatography + ion exchange + molecular sieve (FIG. 7).

Example 8 agonist Activity of anti-OX 40 antibodies of the invention

Luciferase reporter T cell activation assay

Agonist activity of the anti-OX 40 antibodies of the invention can be assessed by measuring the promotion of NFkB-mediated transcriptional activation in a luciferase reporter assay. Jurkat cells overexpressing human OX40(Sinobiologics) and NFkB-luciferase (Promega) constructs were constructed by conventional methods, followed by activation of Jurkat cells overexpressing human OX40 and NFkB-luciferase constructs with anti-CD 3 (2. mu.g/ml; Biolegend) and anti-CD 28 (2. mu.g/ml; Biolegend) antibodies, followed by lysis of the cells according to conventional methods known to those skilled in the art, addition of substrate (purchased from Promega corporation) to the cell lysate and initiation of bioluminescence, followed by testing on a detection apparatus (Molecular Devices) indicating the relative values of luciferase induced expression.

In experiments conducted as described above in assays, Raji cells were added to provide co-stimulatory signals and FcgRIIb-expressing cell surfaces for IgG cross-linking, and the following antibodies of the invention were used: IgG expressed in CHO cells₂Human anti-OX 40 antibody in the form ADI-23515-g2, IgG₁Forms of the human anti-OX 40 antibody ADI-20112-g1 and IgG₂A form of the human anti-OX 40 antibody ADI-20057-g 2.

The results show that the anti-OX 40 antibodies of the invention all have good agonist activity using anti-CD 3/anti-CD 28 antibodies as T cell activators. For example, human anti-OX 40 antibodies ADI-23515-g2, ADI-20112-g1, and ADI-20057-g2 have good agonist activity.

The 4xFab molecule of the invention, human anti-OX 40 antibody ADI-23515-g2-4xFab (IgG) was transiently expressed in HEK293 cells according to the methods and procedures disclosed above₂Format), human anti-OX 40-4xFab antibody ADI-20112-g1-4xFab (IgG)₁Format) and the human anti-OX 40 antibody ADI-20057-g2-4xFab (IgG)₂Forms). The 4xFab molecules of the invention human anti-OX 40 antibodies were then evaluated for agonist activity.

the experimental results show that at lower concentrations, all 4xFab anti-OX 40 antibodies had better agonist activity than the corresponding conventional IgG counterpart of each 4xFab molecule (FIG. 8). In addition, the agonist activity of the above 4xFab molecule is high compared to the tavolixumab and pogalizumab benchmarks which show little or no agonist activity in this assay.

Tavolixizumab, pogalizumab and ADI-20112-g1 all had increased agonist activity at higher concentrations when Raji cells were introduced into the experiment compared to no Raji cells (fig. 9).

Example 9 OX40 blockade assay based on luciferase reporter

The blocking activity of an anti-OX 40 antibody of the invention can be assessed by measuring the ability of the antibody to block OX 40L-mediated activation of OX 40. Jurkat cells overexpressing human OX40 and NFkB-luciferase constructs were activated using anti-CD 3 (2. mu.g/ml; Biolegend), anti-CD 28 (2. mu.g/ml; Biolegend) and recombinant OX40L (60. mu.g/ml; Acro Biosystems) in solution with increasing concentrations of anti-OX 40 antibody for 18 hours, followed by cell lysis and substrate addition for detection and absorbance measurements.

Experiments were performed in assays essentially as described above, in which human anti-OX 40 antibody ADI-23515-g2, human anti-OX 40 antibody ADI-20112-g1, human anti-OX 40 antibody ADI-20057-g2 were expressed in CHO cells. Human anti-OX 40 antibody ADI-23515-g2-4xFab, human anti-OX 40IgG1-4xFab antibody ADI-20112-g1-4xFab, and human anti-OX 40 antibody ADI-20057-g2-4xFab were transiently expressed in HEK293 cells.

The results show that ADI-20112-g1 and ADI-20112-g1-4xFab have low OX40L blocking function. ADI-23515-g2 showed OX40L blockade at concentrations above-0.5 ug/ml, while ADI-23515-g2-4xFab showed increased signal at-0.18 ug/ml or above (FIG. 10). All concentrations of ADI-20057-g2 and ADI-20057-g2-4xFab showed increased activation of OX40 (FIG. 10). Since ADI-20057-g2 is a non-ligand blocker (FIG. 2), this explains how the addition of ADI-20057-g2 and ADI-20057-g2-4xFab to an experiment can potentiate OX 40L-based activation of its receptor. This was also observed in DC-T cell co-culture experiments (FIG. 3).

Sequence listing

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130 135 140

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

145 150 155 160

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

165 170 175

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

180 185 190

Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val

195 200 205

Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Ala Glu Pro Lys

210 215 220

Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu

225 230 235 240

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

245 250 255

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

260 265 270

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

275 280 285

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

290 295 300

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

305 310 315 320

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

325 330 335

Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

340 345 350

Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln

355 360 365

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

370 375 380

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

385 390 395 400

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

405 410 415

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

420 425 430

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

435 440 445

Leu Ser Pro Gly Lys

450

<210> 43

<211> 448

<212> PRT

<213> Artificial sequence

<220>

<223> antibody/antibody fragment sequences

<400> 43

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp His Ala Ser Ser Ser Trp Tyr Thr Thr His Leu Asp Leu

100 105 110

Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly

115 120 125

Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser

130 135 140

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

145 150 155 160

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

165 170 175

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

180 185 190

Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val

195 200 205

Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys

210 215 220

Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val

290 295 300

Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 44

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> antibody/antibody fragment sequences

<400> 44

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Ser Ala Asn Tyr Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 45

<211> 452

<212> PRT

<213> Artificial sequence

<220>

<223> antibody/antibody fragment sequences

<400> 45

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Arg Ser Gly

20 25 30

Ala Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Asp Gly Gln Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Asp Val Gly Tyr Pro His Tyr Tyr Gly Met Asp Val Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Ala Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro Gly Lys

450

<210> 46

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> antibody/antibody fragment sequences

<400> 46

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Arg Ser Gly

20 25 30

Ala Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Asp Gly Gln Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Asp Val Gly Tyr Pro His Tyr Tyr Gly Met Asp Val Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys

210 215 220

Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val

290 295 300

Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 47

<211> 215

<212> PRT

<213> Artificial sequence

<220>

<223> antibody/antibody fragment sequences

<400> 47

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Glu Arg Ser Pro

85 90 95

Phe Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala

100 105 110

Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser

115 120 125

Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu

130 135 140

Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser

145 150 155 160

Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu

165 170 175

Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val

180 185 190

Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys

195 200 205

Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 48

<211> 454

<212> PRT

<213> Artificial sequence

<220>

<223> antibody/antibody fragment sequences

<400> 48

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Arg Pro Trp Tyr Ser Glu Thr Gly Thr Ser Ala Phe Asp

100 105 110

Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys

115 120 125

Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly

130 135 140

Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

145 150 155 160

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

165 170 175

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

180 185 190

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn

195 200 205

Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Ala Glu Pro

210 215 220

Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

225 230 235 240

Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

245 250 255

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

260 265 270

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

275 280 285

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn

290 295 300

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

305 310 315 320

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro

325 330 335

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

340 345 350

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn

355 360 365

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

370 375 380

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

385 390 395 400

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

405 410 415

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

420 425 430

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

435 440 445

Ser Leu Ser Pro Gly Lys

450

<210> 49

<211> 449

<212> PRT

<213> Artificial sequence

<220>

<223> antibody/antibody fragment sequences

<400> 49

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Arg Pro Trp Tyr Ser Glu Thr Gly Thr Ser Ala Phe Asp

100 105 110

Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys

115 120 125

Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu

130 135 140

Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

145 150 155 160

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

165 170 175

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

180 185 190

Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn

195 200 205

Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg

210 215 220

Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg

290 295 300

Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly

<210> 50

<211> 213

<212> PRT

<213> Artificial sequence

<220>

<223> antibody/antibody fragment sequences

<400> 50

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Ser Asp His Tyr Pro Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> 51

<211> 688

<212> PRT

<213> Artificial sequence

<220>

<223> antibody/antibody fragment sequences

<400> 51

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp His Ala Ser Ser Ser Trp Tyr Thr Thr His Leu Asp Leu

100 105 110

Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly

115 120 125

Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly

130 135 140

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

145 150 155 160

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

165 170 175

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

180 185 190

Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val

195 200 205

Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys

210 215 220

Ser Cys Asp Lys Thr His Thr Gly Gly Gly Gly Ser Gln Val Gln Leu

225 230 235 240

Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val

245 250 255

Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Tyr Met His Trp

260 265 270

Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Ile Ile Asn

275 280 285

Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe Gln Gly Arg Val

290 295 300

Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr Met Glu Leu Ser

305 310 315 320

Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp His

325 330 335

Ala Ser Ser Ser Trp Tyr Thr Thr His Leu Asp Leu Trp Gly Arg Gly

340 345 350

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

355 360 365

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

370 375 380

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

385 390 395 400

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

405 410 415

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

420 425 430

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

435 440 445

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

450 455 460

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

465 470 475 480

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

485 490 495

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

500 505 510

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

515 520 525

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

530 535 540

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

545 550 555 560

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

565 570 575

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

580 585 590

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

595 600 605

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

610 615 620

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

625 630 635 640

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

645 650 655

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

660 665 670

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

675 680 685

<210> 52

<211> 684

<212> PRT

<213> Artificial sequence

<220>

<223> antibody/antibody fragment sequences

<400> 52

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp His Ala Ser Ser Ser Trp Tyr Thr Thr His Leu Asp Leu

100 105 110

Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly

115 120 125

Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly

130 135 140

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

145 150 155 160

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

165 170 175

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

180 185 190

Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val

195 200 205

Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys

210 215 220

Ser Cys Asp Lys Thr His Thr Gly Gly Gly Gly Ser Gln Val Gln Leu

225 230 235 240

Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val

245 250 255

Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Tyr Met His Trp

260 265 270

Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Ile Ile Asn

275 280 285

Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe Gln Gly Arg Val

290 295 300

Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr Met Glu Leu Ser

305 310 315 320

Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp His

325 330 335

Ala Ser Ser Ser Trp Tyr Thr Thr His Leu Asp Leu Trp Gly Arg Gly

340 345 350

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

355 360 365

Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu

370 375 380

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

385 390 395 400

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

405 410 415

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

420 425 430

Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro

435 440 445

Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu

450 455 460

Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu

465 470 475 480

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

485 490 495

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln

500 505 510

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

515 520 525

Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu

530 535 540

Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

545 550 555 560

Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

565 570 575

Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

580 585 590

Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

595 600 605

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

610 615 620

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly

625 630 635 640

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

645 650 655

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

660 665 670

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

675 680

<210> 53

<211> 687

<212> PRT

<213> Artificial sequence

<220>

<223> antibody/antibody fragment sequences

<400> 53

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Arg Ser Gly

20 25 30

Ala Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Asp Gly Gln Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Asp Val Gly Tyr Pro His Tyr Tyr Gly Met Asp Val Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Gly Gly Gly Gly Ser Gln Val Gln Leu Gln

225 230 235 240

Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln Thr Leu Ser Leu Thr

245 250 255

Cys Thr Val Ser Gly Gly Ser Ile Arg Ser Gly Ala Tyr Tyr Trp Ser

260 265 270

Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Trp Ile Gly Tyr Ile

275 280 285

Tyr Tyr Asp Gly Gln Thr Tyr Tyr Asn Pro Ser Leu Lys Ser Arg Val

290 295 300

Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser

305 310 315 320

Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp Val

325 330 335

Gly Tyr Pro His Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr

340 345 350

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

355 360 365

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

370 375 380

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

385 390 395 400

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

405 410 415

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

420 425 430

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

435 440 445

Thr Lys Val Asp Lys Lys Ala Glu Pro Lys Ser Cys Asp Lys Thr His

450 455 460

Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val

465 470 475 480

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

485 490 495

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

500 505 510

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

515 520 525

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

530 535 540

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

545 550 555 560

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

565 570 575

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

580 585 590

Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

595 600 605

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

610 615 620

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

625 630 635 640

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

645 650 655

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

660 665 670

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

675 680 685

<210> 54

<211> 682

<212> PRT

<213> Artificial sequence

<220>

<223> antibody/antibody fragment sequences

<400> 54

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Arg Ser Gly

20 25 30

Ala Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Asp Gly Gln Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Asp Val Gly Tyr Pro His Tyr Tyr Gly Met Asp Val Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Gly Gly Gly Gly Ser Gln Val Gln Leu Gln

225 230 235 240

Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln Thr Leu Ser Leu Thr

245 250 255

Cys Thr Val Ser Gly Gly Ser Ile Arg Ser Gly Ala Tyr Tyr Trp Ser

260 265 270

Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Trp Ile Gly Tyr Ile

275 280 285

Tyr Tyr Asp Gly Gln Thr Tyr Tyr Asn Pro Ser Leu Lys Ser Arg Val

290 295 300

Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser

305 310 315 320

Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp Val

325 330 335

Gly Tyr Pro His Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr

340 345 350

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

355 360 365

Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys

370 375 380

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

385 390 395 400

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

405 410 415

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn

420 425 430

Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn

435 440 445

Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro

450 455 460

Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro

465 470 475 480

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

485 490 495

Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn

500 505 510

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

515 520 525

Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val

530 535 540

Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

545 550 555 560

Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys

565 570 575

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu

580 585 590

Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

595 600 605

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

610 615 620

Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe

625 630 635 640

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

645 650 655

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

660 665 670

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

675 680

<210> 55

<211> 691

<212> PRT

<213> Artificial sequence

<220>

<223> antibody/antibody fragment sequences

<400> 55

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Arg Pro Trp Tyr Ser Glu Thr Gly Thr Ser Ala Phe Asp

100 105 110

Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys

115 120 125

Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly

130 135 140

Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

145 150 155 160

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

165 170 175

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

180 185 190

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn

195 200 205

Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Ala Glu Pro

210 215 220

Lys Ser Cys Asp Lys Thr His Thr Gly Gly Gly Gly Ser Gln Val Gln

225 230 235 240

Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu Arg

245 250 255

Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Gly Met His

260 265 270

Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Val Ile

275 280 285

Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys Gly Arg

290 295 300

Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met

305 310 315 320

Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly

325 330 335

Arg Pro Trp Tyr Ser Glu Thr Gly Thr Ser Ala Phe Asp Ile Trp Gly

340 345 350

Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

355 360 365

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

370 375 380

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

385 390 395 400

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

405 410 415

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

420 425 430

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

435 440 445

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Ala Glu Pro Lys Ser Cys

450 455 460

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

465 470 475 480

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

485 490 495

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

500 505 510

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

515 520 525

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

530 535 540

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

545 550 555 560

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

565 570 575

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

580 585 590

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

595 600 605

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

610 615 620

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

625 630 635 640

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

645 650 655

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

660 665 670

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

675 680 685

Pro Gly Lys

690

<210> 56

<211> 686

<212> PRT

<213> Artificial sequence

<220>

<223> antibody/antibody fragment sequences

<400> 56

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Arg Pro Trp Tyr Ser Glu Thr Gly Thr Ser Ala Phe Asp

100 105 110

Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys

115 120 125

Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly

130 135 140

Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

145 150 155 160

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

165 170 175

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

180 185 190

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn

195 200 205

Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Ala Glu Pro

210 215 220

Lys Ser Cys Asp Lys Thr His Thr Gly Gly Gly Gly Ser Gln Val Gln

225 230 235 240

Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu Arg

245 250 255

Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Gly Met His

260 265 270

Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Val Ile

275 280 285

Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys Gly Arg

290 295 300

Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met

305 310 315 320

Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly

325 330 335

Arg Pro Trp Tyr Ser Glu Thr Gly Thr Ser Ala Phe Asp Ile Trp Gly

340 345 350

Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

355 360 365

Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala

370 375 380

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

385 390 395 400

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

405 410 415

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

420 425 430

Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His

435 440 445

Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys

450 455 460

Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val

465 470 475 480

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

485 490 495

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

500 505 510

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

515 520 525

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser

530 535 540

Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

545 550 555 560

Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile

565 570 575

Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

580 585 590

Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

595 600 605

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

610 615 620

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser

625 630 635 640

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

645 650 655

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

660 665 670

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

675 680 685

<210> 57

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> antibody/antibody fragment sequences

<400> 57

Gly Gly Gly Gly Ser

1 5

<210> 58

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> antibody/antibody fragment sequences

<400> 58

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly

20

<210> 59

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> antibody/antibody fragment sequences

<400> 59

Gly Ser Ile Ser Ser Gly Ser Tyr Tyr Trp Ser

1 5 10

<210> 60

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> antibody/antibody fragment sequences

<400> 60

Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 61

<211> 122

<212> PRT

<213> Artificial sequence

<220>

<223> antibody/antibody fragment sequences

<400> 61

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Ser Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Asp Val Gly Tyr Pro His Tyr Tyr Gly Met Asp Val Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 62

<211> 452

<212> PRT

<213> Artificial sequence

<220>

<223> antibody/antibody fragment sequences

<400> 62

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Ser Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Asp Val Gly Tyr Pro His Tyr Tyr Gly Met Asp Val Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Ala Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro Gly Lys

450

<210> 63

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> antibody/antibody fragment sequences

<400> 63

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Ser Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Asp Val Gly Tyr Pro His Tyr Tyr Gly Met Asp Val Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys

210 215 220

Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val

290 295 300

Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 64

<211> 687

<212> PRT

<213> Artificial sequence

<220>

<223> antibody/antibody fragment sequences

<400> 64

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Ser Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Asp Val Gly Tyr Pro His Tyr Tyr Gly Met Asp Val Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Ala Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Gly Gly Gly Gly Ser Gln Val Gln Leu Gln

225 230 235 240

Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln Thr Leu Ser Leu Thr

245 250 255

Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly Ser Tyr Tyr Trp Ser

260 265 270

Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Trp Ile Gly Tyr Ile

275 280 285

Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Ser Arg Val

290 295 300

Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser

305 310 315 320

Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp Val

325 330 335

Gly Tyr Pro His Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr

340 345 350

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

355 360 365

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

370 375 380

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

385 390 395 400

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

405 410 415

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

420 425 430

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

435 440 445

Thr Lys Val Asp Lys Lys Ala Glu Pro Lys Ser Cys Asp Lys Thr His

450 455 460

Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val

465 470 475 480

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

485 490 495

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

500 505 510

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

515 520 525

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

530 535 540

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

545 550 555 560

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

565 570 575

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

580 585 590

Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

595 600 605

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

610 615 620

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

625 630 635 640

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

645 650 655

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

660 665 670

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

675 680 685

<210> 65

<211> 682

<212> PRT

<213> Artificial sequence

<220>

<223> antibody/antibody fragment sequences

<400> 65

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Ser Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Asp Val Gly Tyr Pro His Tyr Tyr Gly Met Asp Val Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Ala Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Gly Gly Gly Gly Ser Gln Val Gln Leu Gln

225 230 235 240

Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln Thr Leu Ser Leu Thr

245 250 255

Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly Ser Tyr Tyr Trp Ser

260 265 270

Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Trp Ile Gly Tyr Ile

275 280 285

Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Ser Arg Val

290 295 300

Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser

305 310 315 320

Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp Val

325 330 335

Gly Tyr Pro His Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr

340 345 350

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

355 360 365

Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys

370 375 380

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

385 390 395 400

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

405 410 415

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn

420 425 430

Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn

435 440 445

Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro

450 455 460

Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro

465 470 475 480

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

485 490 495

Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn

500 505 510

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

515 520 525

Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val

530 535 540

Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

545 550 555 560

Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys

565 570 575

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu

580 585 590

Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

595 600 605

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

610 615 620

Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe

625 630 635 640

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

645 650 655

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

660 665 670

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

675 680

<210> 66

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> antibody/antibody fragment sequences

<400> 66

Ser Tyr Tyr Met His

1 5

<210> 67

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> antibody/antibody fragment sequences

<400> 67

Ser Gly Ala Tyr Tyr Trp Ser

1 5

<210> 68

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> antibody/antibody fragment sequences

<400> 68

Ser Tyr Gly Met His

1 5

<210> 69

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> antibody/antibody fragment sequences

<400> 69

Ser Gly Ser Tyr Tyr Trp Ser

1 5

<210> 70

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> antibody/antibody fragment sequences

<400> 70

Asp His Ala Ser Ser Ser Trp Tyr Thr Thr His Leu Asp Leu

1 5 10

<210> 71

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> antibody/antibody fragment sequences

<400> 71

Asp Val Gly Tyr Pro His Tyr Tyr Gly Met Asp Val

1 5 10

<210> 72

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> antibody/antibody fragment sequences

<400> 72

Gly Arg Pro Trp Tyr Ser Glu Thr Gly Thr Ser Ala Phe Asp Ile

1 5 10 15

Claims

1. an isolated antibody comprising an Fc domain and 4 Fab fragments, wherein the Fc domain and 2 Fab fragments constitute a full length antibody of valency 2 and the other 2 Fab fragments are fused to the amino terminus of the 1 heavy chain of the full length antibody, either directly or by means of a linker, respectively, through amino acid residue H113(Kabat numbering) of its CH1 fragment, thereby forming a fusion polypeptide of the structure "VH-CH 1- (linker) -heavy chain", preferably wherein the 4 Fab fragments are identical Fab fragments, preferably the Fab fragment is a Fab fragment of anti-OX 40 comprising the 3 heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3, respectively, from any of the heavy chain variable regions (VH) shown in table C and the 3 light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, respectively, from any of the light chain variable regions (VL) shown in table C.

2. the antibody of claim 1, wherein the HCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 1, 2, 3, 59, 66, 67, 68 and 69, or an amino acid sequence comprising NO more than 5 amino acid changes relative thereto; HCDR2 comprises an amino acid sequence selected from SEQ ID NOs 4, 5, 6 and 60, or an amino acid sequence comprising NO more than 5 amino acid changes relative thereto; the HCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 7, 8, 9, 70, 71 and 72, or an amino acid sequence comprising NO more than 5 amino acid changes relative thereto, the LCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 10 and 11, or an amino acid sequence comprising NO more than 5 amino acid changes relative thereto, the LCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 12 and 13, or an amino acid sequence comprising NO more than 5 amino acid changes relative thereto, the LCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 14, 15 and 16, or an amino acid sequence comprising NO more than 5 amino acid changes relative thereto.

3. The antibody of claim 1 or 2, wherein the 4 Fab fragments comprise a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence having at least 90% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs 36, 37, 38, and 61; the light chain variable region comprises an amino acid sequence having at least 90% identity to an amino acid sequence selected from the group consisting of those set forth as SEQ ID NOs 39, 40, and 41.

4. The antibody of any one of claims 1-3, wherein the fusion polypeptide of the heavy chain comprises an amino acid sequence having at least 90% identity to an amino acid sequence selected from the group consisting of SEQ ID NO 51, 52, 53, 54, 55, 56, 64, 65; the light chain comprises an amino acid sequence having at least 90% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs 44, 47, 50.

5. The antibody of any one of claims 1-4, which is a monoclonal antibody against OX40, preferably the monoclonal antibody is a humanized antibody, more preferably the monoclonal antibody is a human antibody.

6. The antibody of any one of claims 1-5, wherein the antibody is a multivalent anti-OX 40 antibody having agonist activity.

7. The antibody of any one of claims 1-6, wherein the linker is (GGGGS)_nWherein n is 0 to 4, preferably, the linker is GGGGS (SEQ ID NO: 57).

8. An isolated multivalent anti-OX 40 antibody comprising a heavy chain fusion polypeptide comprising an amino acid sequence selected from the group consisting of those set forth as SEQ ID NOs 51, 52, and a light chain comprising an amino acid sequence set forth as SEQ ID NO 44.

9. An isolated multivalent anti-OX 40 antibody comprising a heavy chain fusion polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO 53, 54, and a light chain comprising an amino acid sequence set forth in SEQ ID NO 47.

10. An isolated multivalent anti-OX 40 antibody comprising a heavy chain fusion polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO 55, 56 and a light chain comprising an amino acid sequence set forth in SEQ ID NO 50.

11. An isolated multivalent anti-OX 40 antibody comprising a heavy chain fusion polypeptide comprising an amino acid sequence selected from the group consisting of those set forth as SEQ ID NO 64, 65, and a light chain comprising an amino acid sequence set forth as SEQ ID NO 47.

12. A nucleic acid encoding the antibody of any one of claims 1-11.

13. A vector comprising the nucleic acid of claim 12, preferably said vector is an expression vector.

14. A host cell comprising the nucleic acid of claim 12 or the vector of claim 13, preferably the host cell is a prokaryotic host cell or a eukaryotic host cell, more preferably the host cell is selected from the group consisting of a yeast cell, a mammalian cell (e.g. a CHO cell or 293 cell) or other cell suitable for the production of an antibody or antigen binding fragment thereof.

15. A method of making the antibody of any one of claims 1-11, wherein the method comprises the steps of:

a) Cloning the construct comprising VH-CH1 and the construct comprising VL-CL into an expression vector,

b) Transfecting a host cell and culturing said host cell under conditions suitable for expression of a nucleic acid encoding said antibody, optionally,

c) Isolating the antibody.

16. A composition comprising the antibody of any one of claims 1-11, preferably the composition is a pharmaceutical composition, more preferably the composition further comprises a pharmaceutically acceptable carrier.

17. An immunoconjugate comprising the antibody of any one of claims 1-11 and a cytotoxic agent.

18. Use of the composition of claim 16 or the immunoconjugate of claim 17 in the manufacture of a medicament for the treatment of cancer, wherein the cancer is preferably lung cancer (e.g. non-small cell lung cancer), liver cancer, stomach cancer, colon cancer.

19. A method of activating T cells or inducing a T cell-mediated anti-tumor activity or enhancing an immune response in a subject, the method comprising administering to the subject an effective amount of the antibody of any one of claims 1-11.

20. The method of claim 19, wherein the subject or individual is a mammal, preferably a human.

21. A method of detecting OX40 in a sample, the method comprising:

(a) contacting the sample with an antibody of any one of claims 1-11; and

(b) Detecting the formation of a complex between the multivalent antibody and OX40, preferably the multivalent anti-OX 40 antibody is detectably labeled.

22. A kit or article of manufacture comprising the antibody of any one of claims 1-11.