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WO2017204277A1 - ANTI-TGF-beta3 ANTIBODY AND USE THEREOF - Google Patents

ANTI-TGF-beta3 ANTIBODY AND USE THEREOF Download PDF

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WO2017204277A1
WO2017204277A1 PCT/JP2017/019457 JP2017019457W WO2017204277A1 WO 2017204277 A1 WO2017204277 A1 WO 2017204277A1 JP 2017019457 W JP2017019457 W JP 2017019457W WO 2017204277 A1 WO2017204277 A1 WO 2017204277A1
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seq
antibody
cdr3
variable region
chain variable
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PCT/JP2017/019457
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French (fr)
Japanese (ja)
Inventor
圭志 藤尾
僚久 岡村
一彦 山本
眞璃子 井上
俊彦 駒井
信広 坂
達也 野中
慎也 石井
博子 村田
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国立大学法人東京大学
中外製薬株式会社
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Publication of WO2017204277A1 publication Critical patent/WO2017204277A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology

Definitions

  • the present invention relates to an anti-TGF-beta3 antibody and use thereof, and more specifically to an inhibitor of B cell activation and a therapeutic agent for autoimmune diseases using the anti-TGF-beta3 antibody.
  • Autoantibodies are known to induce various autoimmune diseases such as systemic lupus erythematosus (SLE) characterized by severe inflammation in multiple organ systems (Non-patent Document 1). .
  • SLE systemic lupus erythematosus
  • the main origin of high-affinity autoantibodies is self-reactive B cells that have undergone somatic hypermutation at the germinal center (GC) (Non-patent Document 2).
  • GC germinal center
  • TGF-beta3 produced by regulatory T cells (LAG3 + Treg) defined by CD4 positive CD25 negative LAG3 positive was found to suppress B cell activation (Non-patent literature) 3).
  • the present invention provides an anti-TGF-beta3 antibody and a method for using the same.
  • the present invention provides the following.
  • [1] A non-neutralizing antibody that specifically binds to TGF-beta3.
  • An antibody comprising a light chain variable region having CDR2 described in SEQ ID NO: 107 and CDR3 described in SEQ ID NO: 107 (2) CDR1 described in SEQ ID NO: 141, CDR2 described in SEQ ID NO: 142, described in SEQ ID NO: 143
  • An antibody (3) comprising a heavy chain variable region having CDR3, and a light chain variable region having CDR1 set forth in SEQ ID NO: 145, CDR2 set forth in SEQ ID NO: 146, CDR3 set forth in S
  • [4] The antibody according to any one of [1] to [3], wherein the suppression of TGF-beta3 physiological activity is 20% or less.
  • [5] The antibody according to any one of [1] to [4], wherein the binding value to TGF-beta1 and TGF-beta2 relative to the binding value to TGF-beta3 is less than 10%.
  • [6] The antibody according to any one of [1] to [5], which is a monoclonal antibody.
  • [7] The antibody according to any one of [1] to [6], which is a human antibody, a humanized antibody, or a chimeric antibody.
  • An inhibitor of B cell activation comprising the antibody according to any one of [1] to [9].
  • a therapeutic agent for autoimmune disease comprising the antibody according to any one of [1] to [9].
  • the autoimmune disease is systemic lupus erythematosus, pemphigus, multiple sclerosis, neuromyelitis optica, ANCA-related vasculitis, rheumatoid arthritis, transplant organ rejection, Sjogren's syndrome, juvenile dermatomyositis, myasthenia gravis, or [11]
  • the therapeutic agent according to [11] which is any of autoimmune thyroid diseases such as Graves' disease and Hashimoto's disease.
  • an “acceptor human framework” is a light chain variable domain (VL) framework or heavy chain variable domain derived from a human immunoglobulin framework or human consensus framework as defined below ( VH) A framework containing the amino acid sequence of the framework.
  • Acceptor human frameworks derived from human immunoglobulin frameworks or human consensus frameworks may contain those same amino acid sequences or may contain amino acid sequence changes. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less.
  • the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or human consensus framework sequence.
  • Bind refers to the total strength of non-covalent interactions between a binding site of a molecule (eg, an antibody) and a binding partner (eg, an antigen) of the molecule.
  • binding affinity refers to intrinsic binding affinity that reflects a 1: 1 interaction between members of a binding pair (eg, an antibody and an antigen).
  • the affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by conventional methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.
  • An “affinity matured” antibody is one or more modifications that result in improved affinity of the antibody for the antigen in one or more hypervariable regions (HVRs) compared to the parent antibody without the modifications.
  • anti-TGF-beta3 antibody or "antibody that binds to TGF-beta3” is an antibody that can bind to TGF-beta3 with sufficient affinity so that the antibody targeted TGF-beta3. It refers to antibodies that are sometimes useful as inhibitors of B cell activation and / or as therapeutic agents for autoimmune diseases.
  • the extent of binding of the anti-TGF-beta3 antibody to an irrelevant non-TGF-beta3 protein is determined by the binding of the antibody to TGF-beta3, as measured (eg, by radioimmunoassay (RIA)). Less than about 10%.
  • the antibody that binds to TGF-beta3 is ⁇ 1 ⁇ M, ⁇ 100 nM, ⁇ 10 nM, ⁇ 1 nM, ⁇ 0.1 nM, ⁇ 0.01 nM, or ⁇ 0.001 nM (eg, 10 ⁇ 8 M or less, such as 10 ⁇ A dissociation constant (Kd) of 8 M to 10 -13 M, for example, 10 -9 M to 10 -13 M).
  • the anti-TGF-beta3 antibody binds to an epitope of TGF-beta3 that is conserved among TGF-beta3 from different species.
  • antibody is used in the broadest sense and is not limited thereto as long as it exhibits a desired antigen-binding activity, but is not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, Various antibody structures are included, including bispecific antibodies) and antibody fragments.
  • Antibody fragment refers to a molecule other than the complete antibody, including a portion of the complete antibody that binds to the antigen to which the complete antibody binds.
  • Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab ′, Fab′-SH, F (ab ′) 2 ; diabodies; linear antibodies; single chain antibody molecules (eg, scFv And multispecific antibodies formed from antibody fragments.
  • the “competing antibody” or “antibody that binds to the same epitope” described in the present invention refers to the first antibody and the first antibody according to the evaluation method of Example 6 (epitope competition assay by in-tandem method using OctetHTX system).
  • the binding response value of the second antibody is less than 60%, preferably 40% or less, more preferably 20% or less.
  • chimeric antibody is one in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remaining portion of the heavy and / or light chain is derived from a different source or species. Refers to an antibody.
  • Class of an antibody refers to the type of constant domain or constant region provided in the heavy chain of the antibody.
  • IgA immunoglobulin
  • IgD immunoglobulin D
  • IgE immunoglobulin D
  • IgG immunoglobulin G
  • IgM immunoglobulin M
  • “Effector function” refers to a biological activity resulting from the Fc region of an antibody, depending on the antibody isotype.
  • Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (antibody-dependent cell) -mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (eg, B cell receptors); and B cell activation.
  • an “effective amount” of an agent refers to the amount at a required dose and over a required period of time that is effective to achieve a desired therapeutic or prophylactic result.
  • Fc region is used herein to define the C-terminal region of an immunoglobulin heavy chain that includes at least a portion of the constant region.
  • the term includes native sequence Fc regions and variant Fc regions.
  • the human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain.
  • lysine (Lys447) or glycine-lysine (Gly446-Lys447) at the C-terminal of the Fc region may or may not be present.
  • the numbering of amino acid residues in the Fc region or constant region is Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, follow the EU numbering system (also called EU index) described in MD 1991.
  • “Framework” or “FR” refers to variable domain residues other than hypervariable region (HVR) residues.
  • the FR of a variable domain usually consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, HVR and FR sequences usually appear in VH (or VL) in the following order: FR1-H1 (L1) -FR2-H2 (L2) -FR3-H3 (L3) -FR4.
  • full-length antibody “complete antibody”, and “total antibody” are used interchangeably herein and have a structure substantially similar to or defined herein.
  • host cell refers to a cell (including the progeny of such a cell) into which a foreign nucleic acid has been introduced.
  • Host cells include “transformants” and “transformed cells”, including the primary transformed cell and progeny derived from that cell regardless of the passage number.
  • the progeny may not be completely identical in nucleic acid content with the parent cell, and may contain mutations. Also included herein are mutant progeny that have the same function or biological activity as was used when the original transformed cells were screened or selected.
  • a “human antibody” is an antibody comprising an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell or an antibody derived from a non-human source using a human antibody repertoire or other human antibody coding sequence. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen binding residues.
  • “Human consensus framework” is a framework showing the most commonly occurring amino acid residues in a selected group of human immunoglobulin VL or VH framework sequences. Usually, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Usually, the subgroups of sequences are those in KabatKaet al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3. In one embodiment, for VL, the subgroup is subgroup ⁇ I according to Kabat et al. In one embodiment, for VH, the subgroup is subgroup III by Kabat et al.
  • Humanized antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs.
  • a humanized antibody comprises substantially all of at least one, typically two variable domains, in which all or substantially all HVRs (eg, CDRs) are non- It corresponds to that of a human antibody and all or substantially all FRs correspond to those of a human antibody.
  • a humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody.
  • a “humanized form” of an antibody (eg, a non-human antibody) refers to an antibody that has undergone humanization.
  • hypervariable region or “HVR” as used herein is hypervariable in sequence (“complementarity determining region” or “CDR”) and / or structurally determined. Refers to each region of the variable domain of an antibody that forms a loop (“hypervariable loop”) and / or contains antigen contact residues (“antigen contact”). Usually, an antibody contains 6 HVRs: 3 in VH (H1, H2, H3) and 3 in VL (L1, L2, L3).
  • Exemplary HVRs herein include the following: (a) at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) The resulting hypervariable loop (Chothia and Lesk, J. Mol. Biol. 196: 901-917 (1987)); (b) at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) The resulting CDR (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.
  • HVR residues and other residues in the variable domains are numbered herein according to Kabat et al., Supra.
  • “Individual” or “subject” is a mammal. Mammals include, but are not limited to, domestic animals (eg, cattle, sheep, cats, dogs, horses), primates (eg, humans and non-human primates such as monkeys), rabbits, and , Including rodents (eg, mice and rats). In certain embodiments, the individual or subject is a human.
  • an “isolated” antibody is one that has been separated from its original environmental components.
  • the antibody is, for example, by electrophoresis (eg, SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatograph (eg, ion exchange or reverse phase HPLC). Measured and purified to a purity greater than 95% or 99%.
  • electrophoresis eg, SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis
  • chromatograph eg, ion exchange or reverse phase HPLC
  • isolated nucleic acid refers to a nucleic acid molecule that has been separated from its original environmental components.
  • An isolated nucleic acid includes a nucleic acid molecule contained within a cell that normally contains the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or on a chromosome different from the original chromosomal location. Exists in position.
  • isolated nucleic acid encoding an anti-TGF-beta3 antibody refers to one or more nucleic acid molecules encoding the heavy and light chains (or fragments thereof) of an antibody, either a single vector or separate Nucleic acid molecules carried on vectors and nucleic acid molecules present at one or more locations in the host cell.
  • the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies. That is, the individual antibodies that make up the population are mutated antibodies that can occur (eg, mutated antibodies that contain naturally occurring mutations, or mutated antibodies that occur during the production of monoclonal antibody preparations. Are present in the same amount and / or bind to the same epitope. In contrast to polyclonal antibody preparations that typically include different antibodies to different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is against a single determinant on the antigen.
  • 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 used in accordance with the present invention include, but are not limited to, hybridoma methods, recombinant DNA methods, phage display methods, transgenic animals containing all or part of a human immunoglobulin locus.
  • Such methods and other exemplary methods for making monoclonal antibodies can be made by a variety of techniques, including methods utilizing the methods described herein.
  • Natural antibodies refer to immunoglobulin molecules with various naturally occurring structures.
  • a native IgG antibody is a heterotetrameric glycoprotein of approximately 150,000 daltons composed of two identical light chains and two identical heavy chains that are disulfide bonded.
  • each heavy chain From the N-terminus to the C-terminus, each heavy chain has a variable region (VH) ⁇ ⁇ , also called variable heavy chain domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3).
  • VH variable region
  • VL variable region
  • CL constant light chain
  • the light chain of an antibody may be assigned to one of two types, called kappa ( ⁇ ) and lambda ( ⁇ ), based on the amino acid sequence of its constant domain.
  • package insert is usually included in commercial packages of therapeutic products and includes information about indications, usage, dosages, administration methods, combination therapies, contraindications, and / or warnings regarding the use of such therapeutic products. Used to refer to instructions for use.
  • Percent (%) amino acid sequence identity relative to a reference polypeptide sequence is any conservative substitution after aligning the sequences and introducing gaps, if necessary, to obtain maximum percent sequence identity. Defined as the percentage ratio of amino acid residues in a candidate sequence that are identical to amino acid residues in a reference polypeptide sequence, when not considered part of the identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved by various methods within the skill in the art, such as BLAST, BLAST-2, ALIGN, Megalign® (DNASTAR) ® software, or GENETYX® (stocks). This can be achieved by using publicly available computer software such as Company Genetics. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • the ALIGN-2 sequence comparison computer program is a work of Genentech, whose source code was submitted to the US Copyright Office (US Copyright Office, Wasington DC, 20559) along with user documentation, and was registered under US copyright registration number TXU510087. It is registered.
  • the ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, and may be compiled from source code.
  • the ALIGN-2 program is compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.
  • the% amino acid sequence identity of a given amino acid sequence A to, or with, or against a given amino acid sequence B (or a given amino acid sequence) A given amino acid sequence A, which has or contains some% amino acid sequence identity to, with or to B) is calculated as follows: fraction X / Y Hundredfold. Where X is the number of amino acid residues scored by the sequence alignment program ALIGN-2 as identical matches in the A and B alignments of the program, and Y is the total number of amino acid residues in B .
  • pharmaceutical formulation refers to a preparation that is in a form such that the biological activity of the active ingredient contained therein can be effective and is unacceptable to the subject to which the formulation is administered. Refers to a preparation that does not contain any additional toxic elements.
  • “Pharmaceutically acceptable carrier” refers to an ingredient other than the active ingredient in a pharmaceutical preparation that is non-toxic to a subject.
  • Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.
  • TGF-beta is from any vertebrate source, including mammals such as primates (eg, humans) and rodents (eg, mice and rats), unless otherwise indicated. Any natural type of TGF-beta.
  • the term encompasses TGF-beta that has not been "full length” processed, as well as any form of TGF-beta that results from processing in the cell.
  • the term also encompasses naturally occurring variants of TGF-beta, such as splice variants and allelic variants.
  • treatment is clinical that is intended to alter the natural course of the individual being treated. Means intervention and can be carried out either for prevention or during the course of clinical pathology. Desirable effects of treatment include but are not limited to prevention of disease occurrence or recurrence, reduction of symptoms, attenuation of any direct or indirect pathological effects of the disease, prevention of metastasis, disease Includes reduced rate of progression, recovery or alleviation of disease state, and remission or improved prognosis.
  • the antibodies of the invention are used to delay the onset of disease or slow the progression of disease.
  • an “autoimmune disease” refers to a non-malignant disease or disorder that arises from and is directed to the individual's own tissue.
  • an autoimmune disease is one that specifically excludes a malignant or cancerous disease or condition, particularly B-cell lymphoma, acute lymphoblastic leukemia (ALL), chronic lymphocytes Excludes chronic lymphocytic leukemia (CLL), hairy cell leukemia, and chronic myeloblastic leukemia.
  • ALL acute lymphoblastic leukemia
  • CLL chronic lymphocytic leukemia
  • hairy cell leukemia and chronic myeloblastic leukemia.
  • autoimmune diseases or disorders include, but are not limited to: inflammatory reactions such as inflammatory skin diseases including psoriasis and dermatitis (eg, atopic dermatitis); Scleroderma and sclerosis; reactions associated with inflammatory bowel disease (eg, Crohn's disease and ulcerative colitis); respiratory distress syndrome (including adult respiratory distress syndrome; including ARDS); dermatitis; Meningitis; encephalitis; uveitis; colitis; glomerulonephritis; allergic conditions such as eczema and asthma and other conditions with T cell infiltration and chronic inflammatory response; atherosclerosis; leukocyte adhesion failure; Rheumatism; systemic lupus erythematosus (SLE) (including but not limited to lupus nephritis, cutaneous lupus); diabetes (eg, type I diabetes or insulin-dependent diabetes) ); Multiple sclerosis; Raynaud's syndrome; autoimmune thyroiditis; Hashimoto'sclerosis
  • variable region refers to the heavy or light chain domain of an antibody involved in binding the antibody to an antigen.
  • Natural antibody heavy and light chain variable domains (VH and VL, respectively) are typically similar, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR). It has a structure. (See, for example, Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007)).
  • One VH or VL domain will be sufficient to confer antigen binding specificity.
  • antibodies that bind to a particular antigen may be isolated by screening complementary libraries of VL or VH domains, respectively, using VH or VL domains from antibodies that bind to the antigen. For example, see Portolano et al., J. Immunol. 150: 880-887 (1993); Clarkson et al., Nature 352: 624-628 (1991).
  • vector refers to a nucleic acid molecule that can multiply another nucleic acid to which it has been linked.
  • the term includes vectors as self-replicating nucleic acid structures and vectors that are integrated into the genome of the host cell into which it has been introduced. Certain vectors can provide for the expression of nucleic acids to which they are operably linked. Such vectors are also referred to herein as “expression vectors”.
  • Anti-TGF-beta3 antibody and use thereof focused on the effect of inhibiting B cell activation by TGF-beta3, and conducted extensive research to develop a therapeutic agent for autoimmune diseases that utilizes this effect. It was. As a result, it was found that antibodies having a predetermined function (profile) that recognize TGF-beta3 suppress B cell activation and are useful as a therapeutic agent for autoimmune diseases.
  • the anti-TGF-beta3 antibody of the present invention has the following profile: (1) Non-neutralizing for TGF-beta3; (2) It binds specifically to TGF-beta3 among TGF-beta1, 2, 3; (3) (Optional) Binds not only to the Mature body but also to the Latent body of TGF-beta3.
  • non-neutralizing with respect to TGF-beta3 means that the physiological activity of TGF-beta3 under the condition of an antibody concentration of 400 ng / mL by the evaluation method described in Example 4.
  • the suppression efficiency of TGF-beta3 is more preferably 25% or less under the antibody concentration of 2000 ng / mL, more preferably under the condition of the antibody concentration of 2000 ng / mL. It means that the suppression efficiency of the physiological activity of TGF-beta3 is 20% or less.
  • a non-neutralizing antibody can also be defined as an antibody that binds so as not to inhibit the binding of TGF-beta3 to the TGF-beta receptor.
  • a non-neutralizing antibody that binds to TGF-beta3 can also be expressed as “an antibody that binds to TGF-beta3 in such a manner that TGF-beta3 can bind to the TGF-beta receptor”. From another point of view, non-neutralizing antibodies do not inhibit TGF-beta3 binding to the TGF-beta receptor so that it does not inhibit the biological effects on cells expressing the TGF-beta receptor. It can also be defined as an antibody that binds to beta3.
  • a non-neutralizing antibody that binds to TGF-beta3 can also be expressed as “an antibody that binds to TGF-beta3 in such a manner that TGF-beta3 can activate cells expressing the TGF-beta receptor”.
  • a non-neutralizing antibody should be defined as an antibody that binds to TGF-beta3 so that the activation of cells expressing TGF-beta receptor does not inhibit the physiological activity of the Smad signal. You can also.
  • a non-neutralizing antibody that binds to TGF-beta3 can also be expressed as “an antibody that binds to TGF-beta3 in such a manner that TGF-beta3 can produce a Smad signal”.
  • a non-neutralizing antibody can also be defined as an antibody that binds to TGF-beta3 so as not to inhibit the action of suppressing B cell activation by the entry of a Smad signal. Therefore, “a non-neutralizing antibody that binds to TGF-beta3” can also be expressed as “an antibody that binds to TGF-beta3 in such a manner that TGF-beta3 can suppress the activation of B cells”.
  • TGFb3-specific specifically binds to TGF-beta3
  • TGFb3-specific means that the binding activity to TGF-beta3 is stronger than TGF-beta1, 2, specifically, According to the evaluation method described in Example 5, the binding value to TGF-beta1 and TGF-beta2 for binding to TGF-beta3 is less than 10%, preferably less than 1%, more preferably less than 0.1%. Point to.
  • the anti-TGF-beta3 antibody of the present invention does not have a neutralizing activity to TGF-beta3, the antibody of the present invention binds to TGF-beta3 to thereby bind TGF-beta3. It is expected that the half-life of beta3 will be prolonged, and consequently the blood concentration of TGF-beta3 will be increased, and as a result, the inhibitory effect of BGF activation by TGF-beta3 will be enhanced.
  • the anti-TGF-beta3 antibody of the present invention has a binding activity specific to TGF-beta3, unlike the antibody that also binds to TGF-beta1, 2, the effect of binding to TGF-beta3 is diminished. There is no fear of side effects such as fibrosis due to binding to TGF-beta1 and causing lung adenocarcinoma. Furthermore, the anti-TGF-beta3 antibody of the present invention can preferably bind not only to the mature body but also to the Latent body of TGF-beta3.
  • the antibody of the present invention which has the binding activity of the Latent body to TGF-beta3, exhibits a higher inhibitory effect on B cell activation. be able to.
  • the antibody of the invention is a Bispecific antibody (bispecific antibody) capable of binding to TGF-beta3 in one variable region and binding to a B cell surface marker in the other variable region.
  • Bispecific antibody bispecific antibody
  • TGF-beta3 By binding to the two types of antigens, TGF-beta3 can be efficiently delivered to the target B cell.
  • the antibody is not a bispecific antibody but a normal antibody but does not have neutralizing activity to TGF-beta3
  • the half-life of TGF-beta3 can be increased by binding and the blood concentration can be increased. Therefore, enhancement of the inhibitory effect on B cell activation is expected. Therefore, the antibody of the present invention is sufficient as long as it satisfies the predetermined profile, and is not particularly limited to a Bispecific antibody.
  • the present invention provides an inhibitor of B cell activation comprising an anti-TGF-beta3 antibody.
  • the B cell is preferably a self-reactive B cell, and the suppression of B cell activation includes, for example, suppression of antibody production by the B cell.
  • the suppressor can be used for a therapeutic agent for autoimmune diseases.
  • TGF-beta3 (TGF ⁇ 3 / TGFb3) includes those having the amino acid sequences listed in SEQ ID NOs: 1 (latent body) to 2 (mature body), and similar B cell activity by altering and modifying part of these amino acid sequences Including those having an inhibitory effect on oxidization.
  • Autoimmune diseases include SLE, Pemphigus, Multiple Sclerosis, Optic myelitis (NMO), ANCA-related vasculitis, Rheumatoid arthritis, Transplant rejection, Sjogren's syndrome (Sjogren's syndrome), Juvenile Examples include dermatomyositis (Juvenile dermatomyositis), myasthenia gravis, or autoimmune thyroid disease including Graves' disease or Hashimoto's thyroiditis and the like.
  • the present invention provides a therapeutic agent for an autoimmune disease comprising an antibody or antibody fragment that binds to TGF-beta3.
  • An antibody fragment means one in which antibody components such as a variable region or Fc region are separated.
  • the antibody or antibody fragment may have a variable region recognizing B cell.
  • Recognition of B cells can be performed using, for example, any one or more of CD19, CD20, CD40, CD22, IL21R, BAFF-R, BCMA, TACI, CD27, or CD138 as a marker.
  • the present invention provides a therapeutic agent for autoimmune diseases comprising a multispecific antibody comprising a first variable region recognizing TGF-beta3 and a second variable region recognizing B cells. To do.
  • the present invention provides a medical kit containing the therapeutic agent.
  • the present invention provides isolated antibodies that bind to TGF-beta3.
  • the anti-TGF-beta3 antibody is an antibody that specifically binds to TGF-beta3 among TGF-beta1, 2, 3 and is non-neutralizing to TGF-beta3.
  • the anti-TGF-beta3 antibody binds to the mature and latent bodies of TGF-beta3.
  • the present invention provides a non-neutralizing antibody that specifically binds to TGF-beta3, as described in any of (1) to (29) below: (1) A heavy chain variable region having CDR1 described in SEQ ID NO: 101, CDR2 described in SEQ ID NO: 102, CDR3 described in SEQ ID NO: 103, and CDR1 described in SEQ ID NO: 105, SEQ ID NO: 106 An antibody comprising a light chain variable region having CDR2 described in SEQ ID NO: 107 and CDR3 described in SEQ ID NO: 107 (2) CDR1 described in SEQ ID NO: 141, CDR2 described in SEQ ID NO: 142, described in SEQ ID NO: 143 An antibody (3) comprising a heavy chain variable region having CDR3, and a light chain variable region having CDR1 set forth in SEQ ID NO: 145, CDR2 set forth in SEQ ID NO: 146, CDR3 set forth in SEQ ID NO: 147 161, CDR2 of SEQ ID NO: 162,
  • the anti-TGF-beta3 antibody is a monoclonal antibody, including a chimeric, humanized, or human antibody.
  • the anti-TGF-beta3 antibody is an antibody fragment, such as, for example, an Fv, Fab, Fab ′, scFv, diabody, or F (ab ′) 2 fragment.
  • the anti-TGF-beta3 antibody has FcRn binding ability, for example, an Fc region containing an FcRn binding region.
  • the antibody is a full length antibody.
  • the anti-TGF-beta3 antibody may incorporate any of the features described in items 1-7 below, alone or in combination.
  • an antibody provided herein has a ⁇ 1 ⁇ M, ⁇ 100 nM, ⁇ 10 nM, ⁇ 1 nM, ⁇ 0.1 nM, ⁇ 0.01 nM or ⁇ 0.001 nM (eg, 10 ⁇ 8 M or less, For example, it has a dissociation constant (Kd) of 10 ⁇ 8 M to 10 ⁇ 13 M, such as 10 ⁇ 9 M to 10 ⁇ 13 M.
  • Kd dissociation constant
  • Kd is measured by a radiolabeled antigen binding assay (RIA).
  • RIA is performed using the Fab version of the antibody of interest and its antigen.
  • Fab binding affinity of an antigen to an antigen is such that the Fab is equilibrated with a minimal concentration of ( 125I ) -labeled antigen in the presence of increasing series of unlabeled antigen, and then the bound antigen is coated with an anti-Fab antibody. Measured by catching on a plate. (See, eg, Chen et al., J. Mol. Biol. 293: 865-881 (1999)).
  • MICROTITER® multiwell plates (Thermo Scientific) were coated overnight with 5 ⁇ g / ml capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), then Block with 2% (w / v) bovine serum albumin in PBS for 2-5 hours at room temperature (approximately 23 ° C.).
  • a non-adsorbed plate (Nunc # 269620), 100 pM or 26 pM [ 125 I] -antigen (see, for example, the anti-VEGF antibody Fab- in Presta et al, Cancer Res.
  • Kd is measured using a BIACORE® surface plasmon resonance assay.
  • a measurement method using BIACORE (registered trademark) -2000 or BIACORE (registered trademark) -3000 (BIAcore, Inc., Piscataway, NJ) is used to obtain a CM5 having about 10 response units (RU) of immobilized antigen. Performed at 25 ° C using a chip.
  • a carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc.) is prepared according to the supplier's instructions with N-ethyl-N ′-(3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) and N Activated with -hydroxysuccinimide (NHS).
  • EDC N-ethyl-N ′-(3-dimethylaminopropyl) -carbodiimide hydrochloride
  • NHS N Activated with -hydroxysuccinimide
  • the antigen is brought to 5 ⁇ g / ml (approximately 0.2 ⁇ M) using 10 mM sodium acetate, pH 4.8 before being injected at a flow rate of 5 ⁇ l / min to achieve approximately 10 reaction units (RU) of protein binding. Diluted. After the injection of antigen, 1M ethanolamine is injected to block unreacted groups.
  • a 2-fold serial dilution of Fab (0.78 nM) in PBS (PBST) containing 0.05% polysorbate 20 (TWEEN-20 TM) surfactant at 25 ° C. and a flow rate of approximately 25 ⁇ l / min. ⁇ 500nM) is injected.
  • the association rate (k on ) and dissociation rate (k off ) can be determined by simultaneously fitting the association and dissociation sensorgrams using a simple one-to-one Langmuir association model (BIACORE® evaluation software version 3.2). Calculated.
  • the equilibrium dissociation constant (Kd) is calculated as the ratio k off / k on . See, for example, Chen et al., J. Mol. Biol.
  • the on-rate exceeds 10 6 M -1 s -1 by the surface plasmon resonance assay described above, the on-rate is measured using a spectrophotometer (eg, a stop flow spectrophotometer (Aviv Instruments) or a 8000 series SLM- Fluorescence emission intensity at 25 ° C.
  • a spectrophotometer eg, a stop flow spectrophotometer (Aviv Instruments) or a 8000 series SLM- Fluorescence emission intensity at 25 ° C.
  • the antibodies provided herein are antibody fragments.
  • Antibody fragments include, but are not limited to, Fab, Fab ′, Fab′-SH, F (ab ′) 2 , Fv, and scFv fragments, as well as other fragments described below.
  • Fab fragment antigen
  • Fab′ fragment antigen binding domain
  • a diabody is an antibody fragment with two antigen binding sites, which may be bivalent or bispecific.
  • a single domain antibody is an antibody fragment comprising all or part of an antibody heavy chain variable domain or all or part of a light chain variable domain.
  • the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, eg, US Pat. No. 6,248,516 B1).
  • Antibody fragments include, but are not limited to, various forms of proteolytic digestion of complete antibodies, as described herein, including production by recombinant host cells (eg, E. coli or phage). It can be made by the method of.
  • a chimeric antibody comprises a non-human variable region (eg, a variable region derived from a non-human primate such as a mouse, rat, hamster, rabbit, or monkey) and a human constant region.
  • a chimeric antibody is a “class switch” antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies also include antigen binding fragments thereof.
  • the chimeric antibody is a humanized antibody.
  • non-human antibodies are humanized in order to reduce human immunogenicity while maintaining the specificity and affinity of the parent non-human antibody.
  • a humanized antibody comprises one or more variable domains, in which the HVR (eg, CDR (or portion thereof)) is derived from a non-human antibody and FR (or portion thereof) is derived from a human antibody sequence. Derived from.
  • a humanized antibody optionally comprises at least a portion of a human constant region.
  • some FR residues in the humanized antibody are non-human antibodies (eg, antibodies derived from HVR residues, eg, to restore or improve antibody specificity or affinity). ) With the corresponding residue from
  • Human framework regions that can be used for humanization have been selected using, but not limited to: the “best fit” method (see Sims et al. J. Immunol. 151: 2296 (1993)). Framework regions; framework regions derived from consensus sequences of human antibodies of a particular subgroup of light or heavy chain variable regions (Carter et al. Proc. Natl. Acad. Sci. USA, 89: 4285 (1992)) Presta et al. J. Immunol., 151: 2623 (1993)); human maturation (somatic mutation) framework region or human germline framework region (eg Almagro and Fransson, Front. Biosci.
  • the antibodies provided herein are human antibodies.
  • Human antibodies can be produced by various techniques known in the art. Human antibodies are reviewed in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20: 450-459 (2008).
  • Human antibodies may be prepared by administering an immunogen to a transgenic animal that has been modified to produce fully human antibodies or fully antibodies with human variable regions in response to an antigen challenge (load).
  • Such animals typically include all or part of a human immunoglobulin locus, which all or part of the human immunoglobulin locus replaces the endogenous immunoglobulin locus or is extrachromosomal or It exists in a state of being randomly incorporated in the chromosome of the animal. In such transgenic mice, the endogenous immunoglobulin locus is usually inactivated.
  • Human antibodies can also be made by methods based on hybridomas. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have already been described. (Eg, Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp.51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et , J. Immunol., 147: 86 (1991).) Human antibodies generated via human B cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103: 3557- It is stated in 3562 (2006).
  • Additional methods include, for example, US Pat. No. 7,189,826 (describing the production of monoclonal human IgM antibodies from hybridoma cell lines), and Ni, Xiandai Mianyixue, 26 (4): 265-268 (2006) (human) -Describe human hybridomas).
  • Human hybridoma technology (trioma technology) is also available from Vollmers and Brandlein, Histology and Histopathology, 20 (3): 927-937 (2005) and Volllmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27 (3): 185-91 (2005).
  • Human antibodies can also be generated by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences can then be combined with the desired human constant domain. A technique for selecting a human antibody from an antibody library is described below.
  • Antibodies of the present invention may be isolated by screening combinatorial libraries for antibodies with one or more desired activities. For example, various methods are known in the art for generating phage display libraries and screening such libraries for antibodies with the desired binding properties. Such methods are reviewed in Hoogenboom et al. In Methods in Molecular Biology 178: 1-37 (O'Brien et al., Ed., Human Press, Totowa, NJ, 2001), and further e.g. McCafferty et al., Nature 348: 552-554; Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol.
  • phage display methods repertoires of VH and VL genes are cloned separately by polymerase chain reaction (PCR) and randomly recombined in a phage library, which is linked to a Winter library. ., Ann. Rev. Immunol., 12: 433-455 (1994) ⁇ ⁇ can be screened for antigen-binding phages. Phages typically display antibody fragments, either as single chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high affinity antibodies to the immunogen without the need to construct hybridomas.
  • scFv single chain Fv
  • the na ⁇ ve repertoire can be cloned (eg, from human) without extensive immunization and without immunization. It is also possible to provide a single source of antibodies to self antigens.
  • the na ⁇ ve library was used to clone the pre-rearranged V-gene segment from stem cells as described in Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992) It can also be made synthetically by using PCR primers that encode the region and contain random sequences to achieve reconstitution in vitro.
  • Patent documents describing human antibody phage libraries include, for example: US Pat. No.
  • an antibody or antibody fragment isolated from a human antibody library is regarded as a human antibody or a human antibody fragment.
  • the antibodies provided herein are multispecific antibodies (eg, bispecific antibodies).
  • Multispecific antibodies are monoclonal antibodies that have binding specificities at at least two different sites.
  • one of the binding specificities is for TGF-beta3 and the other is for any other antigen (eg, a B cell surface marker).
  • the bispecific antibody may bind to two different epitopes of TGF-beta3.
  • Bispecific antibodies may be used to localize TGF-beta3 to cells that express a B cell surface marker.
  • Bispecific antibodies can be prepared as full length antibodies or as antibody fragments.
  • Multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (Milstein and Cuello, Nature 305: 537 (1983), WO 93/08829, and Traunecker et al., EMBO J. 10: 3655 (1991)), and the knob-in-hole technology (see, eg, US Pat. No. 5,731,168).
  • Multispecific antibodies can manipulate electrostatic steering effects to create Fc heterodimeric molecules (WO2009 / 089004A1); bridge two or more antibodies or fragments (US patents) No.
  • Modified antibodies with three or more functional antigen binding sites including “Octopus antibodies” are also included herein (see, eg, US Patent Application Publication No. 2006/0025576 A1).
  • an antibody or fragment also includes a “dual-acting Fab” or “DAF” that contains one antigen-binding site that binds TGF-beta3 to another different antigen (eg, US Patent Application Publication No. 2008 / 0069820).
  • DAF dual-acting Fab
  • amino acid sequence variants of the antibodies provided herein are also contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody.
  • Amino acid sequence variants of the antibody may be prepared by introducing appropriate modifications to the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletion from the amino acid sequence of the antibody, and / or insertion into the amino acid sequence of the antibody, and / or substitution of residues in the amino acid sequence of the antibody. Given that the final construct is provided with the desired characteristics (eg, antigen binding), any combination of deletions, insertions, and substitutions can be made to arrive at the final construct.
  • antibody variants having one or more amino acid substitutions are provided.
  • Target sites for substitutional mutagenesis include HVR and FR.
  • Conservative substitutions are shown under the heading “Preferred substitutions” in Table 1. More substantial changes are provided under the heading “Exemplary substitutions” in Table 1 and are detailed below with reference to the class of amino acid side chains.
  • Amino acid substitutions may be introduced into the antibody of interest and the product may be, for example, retained / improved antigen binding, decreased immunogenicity, decreased neutralizing activity, improved antigen specificity, or improved blood It may be screened for a desired activity, such as retention.
  • Amino acids can be grouped according to common side chain properties: (1) Hydrophobicity: norleucine, methionine (Met), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile); (2) Neutral hydrophilicity: cysteine (Cys), serine (Ser), threonine (Thr), asparagine (Asn), glutamine (Gln); (3) Acidity: Aspartic acid (Asp), glutamic acid (Glu); (4) Basicity: histidine (His), lysine (Lys), arginine (Arg); (5) Residues that affect chain orientation: Glycine (Gly), Proline (Pro); (6) Aromaticity: Tryptophan (Trp), Tyrosine (Tyr), Phenylalanine (Phe). Non-conservative substitutions refer to exchanging one member of these classes for another class.
  • substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (eg, a humanized or human antibody). Usually, the resulting mutants selected for further study are modified (eg, improved) in specific biological properties compared to the parent antibody (eg, increased affinity, decreased immunogenicity). And / or substantially retain certain biological properties of the parent antibody.
  • An exemplary substitution variant is an affinity matured antibody, which can be made as appropriate using, for example, phage display-based affinity maturation techniques (eg, those described herein). Briefly, one or more HVR residues are mutated and the mutated antibody is displayed on a phage and screened for a specific biological activity (eg, binding affinity).
  • HVRs Modifications (eg, substitutions) can be made in HVRs, for example, to improve antibody affinity.
  • modifications are HVR “hot spots”, ie, residues encoded by codons that mutate frequently during the somatic maturation process (eg, Chowdhury, Methods Mol. Biol. 207: 179-196 (See 2008)) and / or in residues that contact antigen, and the resulting mutant VH or VL can be tested for binding affinity.
  • Affinity maturation by construction and reselection from secondary libraries is described, for example, by Hoogenboom et al.
  • variable genes selected for maturation by any of a variety of methods (eg, error-prone PCR, chain shuffling or oligonucleotide-directed mutagenesis).
  • a secondary library is then created. This library is then screened to identify any antibody variants with the desired affinity.
  • Another way to introduce diversity involves an HVR-oriented approach that randomizes several HVR residues (eg, 4-6 residues at a time). HVR residues involved in antigen binding can be specifically identified using, for example, alanine scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are often targeted.
  • substitutions, insertions, or deletions can be made in one or more HVRs as long as such modification does not substantially reduce the ability of the antibody to bind to the antigen.
  • conservative modifications that do not substantially reduce binding affinity eg, conservative substitutions as provided herein
  • modifications can be, for example, outside of the antigen contact residues of HVR.
  • each HVR is unaltered or contains as few as 1, 2 or 3 amino acid substitutions.
  • a useful method for identifying antibody residues or regions that can be targeted for mutagenesis is described by Cunningham and Wells (1989) Science, 244: 1081-1085, ⁇ Alanine scanning mutagenesis ''. It is what is called.
  • a residue or group of target residues eg, charged residues such as arginine, aspartic acid, histidine, lysine, and glutamic acid
  • neutral or negatively charged amino acids eg, alanine or Polyalanine
  • the crystal structure of the antigen-antibody complex may be analyzed to identify contact points between the antibody and the antigen. Such contact residues and neighboring residues may be targeted as substitution candidates or excluded from substitution candidates. Variants can be screened to determine if they contain the desired property.
  • Amino acid sequence insertions are within the length of a polypeptide comprising from 1 to 100 residues or more at the amino and / or carboxyl terminus. Includes fusion.
  • Examples of terminal insertions include antibodies with a methionyl residue at the N-terminus.
  • Other insertional variants of the antibody molecule include those in which the N- or C-terminus of the antibody is fused to an enzyme (eg, for ADEPT) or a polypeptide that increases the plasma half-life of the antibody.
  • Fc region variants In certain embodiments, one or more amino acid modifications may be introduced into the Fc regions of the antibodies provided herein, thereby generating Fc region variants.
  • An Fc region variant may comprise a human Fc region sequence (eg, a human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification (eg, substitution) at one or more amino acid positions.
  • Fc receptor “Fc receptor” or “FcR” refers to a receptor that binds to the Fc region of an antibody.
  • the FcR is native human FcR.
  • the FcR is one that binds to an IgG antibody (gamma receptor) and forms Fc ⁇ RI, Fc ⁇ RII, and Fc ⁇ RIII subclass receptors by allelic variants and alternative splicing of these receptors Including, including.
  • Fc ⁇ RII receptors include Fc ⁇ RIIA (“activating receptor”) and Fc ⁇ RIIB (“inhibitory receptor”), which have similar amino acid sequences that differ primarily in their cytoplasmic domains.
  • Activating receptor Fc ⁇ RIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain.
  • the inhibitory receptor Fc ⁇ RIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain.
  • ITAM immunoreceptor tyrosine-based activation motif
  • ITIM immunoreceptor tyrosine-based inhibition motif
  • FcR is, for example, Ravetch and Kinet, Annu. Rev. Immunol 9: 457-92 (1991); Capel et al., Immunomethods 4: 25-34 (1994); and de Haas et al., J. Lab. Clin. Med 126: 330-41 (1995).
  • Other FcRs including those identified in the future, are also encompassed by the term “FcR” herein.
  • Fc receptor or “FcR” also refer to the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117: 587 (1976) and Kim et al., J. Immunol. 24: 249 (1994)) as well as the neonatal receptor FcRn, which is responsible for the regulation of immunoglobulin homeostasis. Methods for measuring binding to FcRn are known (eg, Ghetie and Ward., Immunol. Today 18 (12): 592-598 (1997); Ghetie et al., Nature Biotechnology, 15 (7): 637- 640 (1997); Hinton et al., J. Biol. Chem. 279 (8): 6213-6216 (2004); WO2004 / 92219 (Hinton et al.)).
  • human FcRn high affinity binding polypeptides can be administered, for example, in transgenic mice expressing human FcRn or transfected human cell lines or administered with a polypeptide with a mutated Fc region. Can be measured in primates.
  • WO2000 / 42072 (Presta) describes antibody variants with increased or decreased binding to FcR. See, for example, Shields et al. J. Biol. Chem. 9 (2): 6591-6604 (2001).
  • Fc region-containing antibody refers to an antibody comprising an Fc region.
  • the C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) or the C-terminal glycine-lysine of the Fc region (residues 446-447) can be used, for example, during antibody purification or of the nucleic acid encoding the antibody. It can be removed by recombination operations. Therefore, a composition comprising an antibody having an Fc region according to the present invention comprises an antibody with G446-K447, an antibody with G446 without K447, an antibody with G446-K447 completely removed, or the above three types of antibodies May be included.
  • antibody variants with some, but not all, effector functions are also within the scope of the present invention, which effector functions with respect to its in vivo half-life, Certain effector functions (such as complement and ADCC) are good candidates for application when they are unnecessary or harmful.
  • In vitro sputum and / or in vivo sputum cytotoxicity measurements can be performed to confirm a decrease / deficiency of CDC and / or ADCC activity.
  • Fc receptor (FcR) binding measurements can be performed to ensure that antibodies lack Fc ⁇ R binding (and thus are more likely to lack ADCC activity) while maintaining FcRn binding ability.
  • NK cells which are primary cells that mediate ADCC, express only Fc ⁇ RIII, whereas monocytes express Fc ⁇ RI, Fc ⁇ RII, and Fc ⁇ RIII.
  • the expression of FcR on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9: 457-492 (1991).
  • in vitro assays for assessing ADCC activity of a molecule of interest include US Pat. No. 5,500,362 (eg, Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA). 83: 7059-7063 (1986)) and Hellstrom, I et al., Proc.
  • non-radioactive assays may be used (eg, ACT1 TM non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA); and CytoTox 96® non-radioactive cytotoxicity assays (see Promega, Madison, WI). Effector cells useful for such measurement methods include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells.
  • PBMC peripheral blood mononuclear cells
  • NK natural killer
  • the ADCC activity of the molecule of interest is assessed in vivo in an animal model as described, for example, in Clynes et al. Proc. Nat'l Acad. Sci. USA 95: 652-656 (1998). May be.
  • C1q binding measurements may be performed to confirm that the antibody cannot bind to C1q and thus lacks CDC activity. See, for example, the C1q and C3c binding ELISA in WO2006 / 029879 and WO2005 / 100402.
  • CDC measurements may also be performed to assess complement activation (eg, Gazzano-Santoro et al., J. Immunol.
  • Antibodies with reduced effector function include those with one or more substitutions of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (US Pat. No. 6,737,056).
  • Fc variants include amino acid positions 265, 269, 270, 297, and 327, including so-called “DANA” Fc variants (US Pat. No. 7,332,581) with substitution of residues 265 and 297 to alanine.
  • DANA DANA
  • modified ie, increased
  • CDC complement dependent cytotoxicity selection
  • FcRn maternal IgGs to the fetus
  • Kim et al. J. Antibodies with increased binding to Immunol. 24: 249 (1994)
  • FcRn maternal IgGs to the fetus
  • These antibodies comprise an Fc region with one or more substitutions therein that increase the binding of the Fc region to FcRn.
  • Such Fc variants include Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382 , 413, 424, or 434 at one or more substitutions (eg, substitution of Fc region residue 434 (US Pat. No. 7,371,826)).
  • the antibodies provided herein may be further modified to include additional non-protein moieties known in the art and readily available. Suitable moieties for antibody derivatization include, but are not limited to, water soluble polymers.
  • Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly 1,3 Dioxolane, poly1,3,6, trioxane, ethylene / maleic anhydride copolymer, polyamino acid (either homopolymer or random copolymer), and dextran or poly (n-vinylpyrrolidone) polyethylene glycol, polypropylene glycol homopolymer, Polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (eg, glycerol), polyvinyl alcohol, and mixtures thereof.
  • PEG polyethylene glycol
  • ethylene glycol / propylene glycol copolymers carboxymethyl cellulose
  • dextran polyvinyl alcohol
  • polyvinyl pyrrolidone poly
  • Polyethylene glycol propionaldehyde may be advantageous in manufacturing due to its water stability.
  • the polymer may have any molecular weight and may or may not be branched.
  • the number of polymers added to the antibody can vary, and if more than one polymer is added, they can be the same molecule or different molecules. In general, the number and / or type of polymers used for derivatization is not limited to these, but is specific to the antibody's particular characteristics or function to be improved, under conditions where the antibody derivative is under defined conditions. It can be determined based on considerations such as whether or not to use in therapy.
  • Antibodies can be produced using recombinant methods and configurations, for example, as described in US Pat. No. 4,816,567.
  • an isolated nucleic acid encoding an anti-TGF-beta3 antibody described herein is provided.
  • Such a nucleic acid may encode an amino acid sequence comprising an antibody VL and / or an amino acid sequence comprising a VH (eg, an antibody light and / or heavy chain).
  • one or more vectors eg, expression vectors
  • host cells comprising such nucleic acids are provided.
  • the host cell comprises (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the antibody VL and an amino acid sequence comprising the antibody VH, or (2) an amino acid comprising the antibody VL.
  • a first vector comprising a nucleic acid encoding sequence and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the antibody VH are included (eg, transformed).
  • the host cell is eukaryotic (eg, Chinese hamster ovary (CHO) cells) or lymphoid cells (eg, Y0, NS0, Sp2 / 0 cells)).
  • a method for producing an anti-TGF-beta3 antibody is provided, which comprises recovering from the medium).
  • nucleic acid encoding the antibody (eg, as described above) is isolated and one or more for further cloning and / or expression in a host cell. Insert into vector.
  • nucleic acids will be readily isolated and sequenced using conventional procedures (eg, oligonucleotide probes that can specifically bind to the genes encoding the antibody heavy and light chains). By using).
  • Suitable host cells for cloning or expression of antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein.
  • antibodies may be produced in bacteria, particularly where glycosylation and Fc effector function are not required. See, eg, US Pat. Nos. 5,648,237, 5,789,199, and 5,840,523 for expression of antibody fragments and polypeptides in bacteria. (In addition, see also Charlton, Methods Molecular Biology, Vol.BK248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp.245-254, which describes the expression of antibody fragments in E. coli. ) After expression, the antibody may be isolated from the bacterial cell paste in a soluble fraction and can be further purified.
  • true species such as filamentous fungi or yeast, including fungal and yeast strains in which the glycosylation pathway is “humanized”, resulting in the production of antibodies with partial or complete human glycosylation patterns.
  • Nuclear microorganisms are suitable cloning or expression hosts for antibody-encoding vectors. See Gerngross, Nat. Biotech. 22: 1409-1414 (2004) and Li et al., Nat. Biotech. 24: 210-215 (2006).
  • invertebrate and vertebrate organisms are also suitable host cells for the expression of glycosylated antibodies.
  • invertebrate cells include plant and insect cells.
  • a number of baculovirus strains have been identified that are used for mating with insect cells, particularly for transformation of Spodoptera frugiperda cells.
  • Plant cell cultures can also be used as hosts. See, for example, US Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (described PLANTIBODIES TM technology for producing antibodies in transgenic plants).
  • Vertebrate cells can also be used as hosts.
  • mammalian cell lines adapted to grow in suspension will be useful.
  • Other examples of useful mammalian host cell lines include monkey kidney CV1 strain (COS-7) transformed with SV40; human embryonic kidney strain (Graham et al., J. Gen Virol. 36:59 (1977 293 or 293 cells as described in); pup hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells as described in Mather, Biol. Reprod.
  • monkey kidney cell (CV1 ); African green monkey kidney cell line (VERO-76); Human cervical cancer cell line (HELA); Canine kidney cell line (MDCK); Buffalo rat liver cell line (BRL 3A); Human lung cell line (W138); Hep G2); mouse breast cancer (MMT 060562); TRI cells (for example, described in Mather et al., Annals NY Acad. Sci. 383: 44-68 (1982)); MRC5 cells; and FS4 cells.
  • Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR- CHO cells (Urlaub et al., Proc. Natl. Acad. Sci.
  • Y0 Includes myeloma cell lines such as NS0 and Sp2 / 0.
  • myeloma cell lines such as NS0 and Sp2 / 0.
  • the anti-TGF-beta3 antibodies provided herein can be identified, screened, or revealed for physical / chemical properties and / or biological activity by various assays known in the art May be.
  • the antibodies of the present invention are tested for their antigen binding activity by known methods such as ELISA, Western blot, and the like.
  • a competition assay can be used to assess the competition of multiple anti-TGF-beta3 antibodies for binding to TGF-beta3.
  • antibodies that are evaluated to compete with each other can be evaluated to bind to the same epitope (eg, a linear or conformational epitope).
  • epitope eg, a linear or conformational epitope.
  • Detailed exemplary methods for mapping the epitope to which an antibody binds are provided in Morris (1996) "Epitope Mapping Protocols," in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ).
  • immobilized TGF-beta3 is tested for the ability to compete with the first labeled antibody that binds to TGF-beta3 and the first antibody for binding to TGF-beta3. Incubate in solution containing 2 unlabeled antibodies. The second antibody can be present in the hybridoma supernatant. As a control, immobilized TGF-beta3 is incubated in a solution containing the first labeled antibody but no second unlabeled antibody. After incubation under conditions that allow binding of the first antibody to TGF-beta3, excess unbound antibody is removed and the amount of label bound to immobilized TGF-beta3 is measured.
  • the expression “substantially reduced” or “substantially different” means between two numbers (usually for one molecule and for a reference / comparison molecule). The difference between the two numbers is considered sufficiently statistically to be considered statistically significant in terms of the biological characteristics measured by the number (eg, Kd value). It ’s big.
  • a measurement method for identifying an anti-TGF-beta3 antibody having a predetermined function is provided.
  • the function (profile) is, for example, (1) non-neutralizing against TGF-beta3 (Non-Neutralizing); (2) TGF-beta1, 2, 3 binding specific to TGF-beta3 It may include activity (TGFb3-specific); and optionally (3) ability to bind not only to the mature form but also to the TGF-beta3 of the Latent form (Latent-Binder).
  • An antibody having such a function (profile) in vivo and / or in vitro is also provided.
  • the antibodies of the invention are tested for such a function (profile).
  • Exemplary test methods include, but are not limited to, the methods described in the examples.
  • the anti-TGF-beta3 antibody pharmaceutical formulations described herein can be prepared by administering an antibody having a desired purity to one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol , A. Ed. (1980)) in the form of a lyophilized formulation or an aqueous solution.
  • Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed, including but not limited to the following: phosphate, citric acid Buffers such as acid salts and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, Or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); small molecule (less than about 10 residues) polypeptide; serum albumin, gelatin, or Proteins such as immunoglobulins; polyvinyl Hydrophilic polymers such as loridone; amino acids such as glycine, glutamine, asparagine, histidine, argin
  • Exemplary pharmaceutically acceptable carriers herein further include a soluble neutral active hyaluronidase glycoprotein (sHASEGP) (eg, rHuPH20 (HYLENEX®, Baxter International, Inc.) PH-20 hyaluronidase glycoprotein) and other interstitial drug dispersants.
  • sHASEGP soluble neutral active hyaluronidase glycoprotein
  • rHuPH20 HYLENEX®, Baxter International, Inc.
  • PH-20 hyaluronidase glycoprotein interstitial drug dispersants.
  • sHASEGPs and their methods of use including rHuPH20
  • sHASEGP is combined with one or more additional glycosaminoglycanases such as chondroitinase.
  • Aqueous antibody formulations include those described in US Pat. No. 6,171,586 and WO2006 / 044908, the latter formulations containing histidine-acetate buffer.
  • formulations herein may contain more than one active ingredient as necessary for the particular indication being treated. Those with complementary activities that do not adversely affect each other are preferred.
  • the active ingredients are incorporated into microcapsules prepared by, for example, droplet formation (coacervation) techniques or by interfacial polymerization (for example, hydroxymethylcellulose or gelatin microcapsules and poly (methyl methacrylate) microcapsules, respectively).
  • it may be incorporated into colloidal drug delivery systems (eg, liposomes, albumin spherules, microemulsions, nanoparticles, and nanocapsules) or macroemulsions.
  • colloidal drug delivery systems eg, liposomes, albumin spherules, microemulsions, nanoparticles, and nanocapsules
  • Such a technique is disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).
  • sustained release preparations may be prepared.
  • suitable examples of sustained release formulations include a semi-permeable matrix of solid hydrophobic polymer containing antibodies, which matrix is in the form of shaped articles such as films or microcapsules.
  • the preparations used for in vivo administration are usually sterile. Aseptic conditions are easily achieved, for example, by filtration through sterile filtration membranes.
  • anti-TGF-beta3 antibodies may be used in therapeutic methods.
  • anti-TGF-beta3 antibodies are provided for use as pharmaceuticals.
  • anti-TGF-beta3 antibodies are provided for use in the treatment of autoimmune diseases.
  • anti-TGF-beta3 antibodies are provided for use in therapeutic methods.
  • the present invention provides a method for treating an individual having TGF-beta3, comprising the step of administering to the individual an effective amount of an anti-TGF-beta3 antibody, for use in the method. Provide beta3 antibody.
  • the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent (eg, as described below).
  • the present invention provides an anti-TGF-beta3 antibody for use in inhibiting B cell activation.
  • the present invention is a method of inhibiting B cell activation in an individual, comprising administering an effective amount of an anti-TGF-beta3 antibody to the individual to inhibit B cell activation
  • An anti-TGF-beta3 antibody for use in is provided.
  • An “individual” according to any of the above embodiments is preferably a human.
  • the present invention provides the use of an anti-TGF-beta3 antibody in the manufacture or preparation of a medicament.
  • the medicament is for the treatment of an autoimmune disease.
  • the medicament is for use in a method of treating an autoimmune disease comprising administering an effective amount of the medicament to an individual having an autoimmune disease.
  • the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent (eg, as described below).
  • the medicament is for suppression of B cell activation.
  • the medicament is for use in a method of inhibiting B cell activation in an individual, the method comprising administering an effective amount of the medicament to the individual to inhibit B cell activation. It is.
  • An “individual” according to any of the above aspects may be a human.
  • the present invention provides a method for treating an autoimmune disease.
  • the method comprises administering an effective amount of anti-TGF-beta3 antibody to an individual having such an autoimmune disease.
  • the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent (as described below).
  • An “individual” according to any of the above aspects may be a human.
  • the present invention provides a method for inhibiting B cell activation in an individual.
  • the method comprises administering to the individual an effective amount of an anti-TGF-beta3 antibody to inhibit B cell activation.
  • an “individual” is a human.
  • the present invention provides a pharmaceutical formulation comprising any of the anti-TGF-beta3 antibodies provided herein (eg, for use in any of the therapeutic methods described above). .
  • the pharmaceutical formulation comprises any of the anti-TGF-beta3 antibodies provided herein and a pharmaceutically acceptable carrier.
  • the pharmaceutical formulation comprises any of the anti-TGF-beta3 antibodies provided herein and at least one additional therapeutic agent (eg, as described below).
  • the antibody of the present invention can be used in therapy either alone or in combination with other agents.
  • an antibody of the invention may be co-administered with at least one additional therapeutic agent.
  • Combination therapy as described above includes combination administration (two or more therapeutic agents included in the same or separate formulations) and individual administration, where administration of the antibody of the present invention is an additional treatment. Prior to, simultaneously with and / or subsequent to administration of the agent.
  • the antibodies (and any additional therapeutic agent) of the invention may be administered by any suitable means, including parenteral, pulmonary, and nasal administration, and intralesional administration if desired for local treatment.
  • Parenteral injection includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.
  • Dosing can be by any suitable route, such as by injection, such as intravenous or subcutaneous injection, depending in part on whether administration is short or long term.
  • Various dosing schedules are within the scope of this specification, including, but not limited to, single doses or repeated doses over various time points, bolus doses, and pulse infusions.
  • the antibody of the present invention is formulated, dosed and administered in a manner consistent with good medical practice. Factors to be considered in this regard are the specific disorder being treated, the particular mammal being treated, the clinical symptoms of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the administration Schedule, and other factors known to healthcare professionals.
  • the antibody need not be, but is optionally formulated with one or more agents currently used to prevent or treat the disorder in question.
  • the effective amount of such other agents depends on the amount of antibody present in the formulation, the type of disorder or treatment, and other factors discussed above. These are usually at the same dose and route of administration as described herein, or at about 1 to 99% of the doses described herein, or any dose determined empirically / clinically appropriate. And in any route.
  • an appropriate dose of the antibody of the present invention (when used alone or in combination with one or more other additional therapeutic agents) will determine the type of disease being treated, It will depend on the type, severity and course of the disease, whether the antibody is administered for prophylactic or therapeutic purposes, drug history, patient clinical history and response to antibodies, and the discretion of the attending physician .
  • the antibody is suitably administered to the patient at one time or over a series of treatments.
  • about 1 ⁇ g / kg to 15 mg / kg (eg, 0.1 mg / kg to 10 mg) antibody may be the first candidate dose for administration to a patient.
  • One typical daily dose may vary from about 1 ⁇ g / kg to over 100 mg / kg, depending on the factors described above. For repeated administrations over several days or longer, depending on the situation, treatment is usually maintained until a desired suppression of disease symptoms occurs.
  • One exemplary dose of antibody is in the range of about 0.05 mg / kg to about 10 mg / kg.
  • one or more doses (or any combination thereof) of about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg, or 10 mg / kg may be administered to the patient.
  • Such doses may be administered intermittently, such as every week or every 3 weeks (eg, so that the patient receives about 2 to about 20, or such as about 6 doses of antibody).
  • One or more low doses may be administered after the high initial loading dose. The progress of this therapy is easily monitored by conventional techniques and measurements.
  • products that include equipment useful for the treatment, prevention, and / or diagnosis of the disorders described above.
  • the product includes a container and a label on the container or package insert attached to the container.
  • Preferred containers include, for example, bottles, vials, syringes, IV solution bags and the like.
  • the containers may be formed from various materials such as glass and plastic.
  • the container may hold the composition alone, in combination with another composition that is effective for the treatment, prevention, and / or diagnosis of symptoms, and with a sterile access port.
  • the container may be a solution bag or vial for intravenous administration having a stopper that can be pierced by a hypodermic needle).
  • At least one active ingredient in the composition is an antibody of the invention.
  • the label or package insert indicates that the composition is to be used to treat the selected condition.
  • the product further comprises: (a) a first container with a composition comprising an antibody of the present invention contained therein; and (b) a second container, A second container may be included with the composition containing an additional cytotoxic agent or otherwise therapeutic agent contained therein.
  • the product in this aspect of the invention may further include a package insert indicating that the composition can be used to treat a particular condition.
  • the product further comprises a second (or pharmaceutically acceptable buffer, such as water for injection (BWFI), phosphate buffered saline, Ringer's solution, and dextrose solution.
  • BWFI water for injection
  • phosphate buffered saline such as phosphate buffered saline, Ringer's solution, and dextrose solution.
  • a third) container may be included.
  • Other buffers, diluents, filters, needles, and syringes may further include equipment desirable from other commercial
  • Example 1 Production of TGF-beta3 (1) Production of Latent mouse TGF-beta3 (latent mTGF-beta3) Equivalent amounts of latent mTGF-beta3 expression vector and soluble human Furin expression vector were expressed as Expi293F cells Thermo Fisher) and transiently expressed latent mTGF-beta3. The obtained culture supernatant was added to an Anti FLAG M2 column (Sigma), and after washing the column, the protein adsorbed on the column was eluted with 0.1 mg / mL FLAG peptide (Sigma).
  • the fraction containing Latent mTGF-beta3 was concentrated with an ultrafiltration membrane, and this was separated and collected with a Superdex 200 increase column (GE Healthcare) to obtain purified latent mTGF-beta3 (SEQ ID NO: 1).
  • the fraction containing the target molecule was solvent-substituted with D-PBS ( ⁇ ) using an ultrafiltration membrane.
  • the latent mTGF-beta3 was biotinylated using EZ-Link NHS-PEG4-Biotin, No-Weigh Format (Thermo Fisher), concentrated with an ultrafiltration membrane, and then separated with a Superdex 200 increase column (GE Healthcare). By collecting, purified biotinylated latent mTGF-beta3 was obtained.
  • TGF-beta3 (hereinafter mature h (m) TGF-beta3)
  • Latent mTGF-beta3 expression vector was introduced into Expi293F cells (Thermo Fisher) and transiently transientd mTGF-beta3 was expressed.
  • the obtained culture supernatant was added to an Anti FLAG M2 column (Sigma), and after washing the column, the protein adsorbed on the column was eluted with 0.1 M Glycine-HCl, pH 2.8.
  • a mature body is formed from the Latent body, and a fraction containing mature h (m) TGF-beta3 is concentrated with an ultrafiltration membrane, and then 0.1 M Glycine-HCl, 150 mM NaCl, pH 2. It added to the Superdex 200pg column (GE Healthcare) equilibrated in 5, and isolate
  • the fraction containing Mature h (m) TGF-beta3 was added to a Vydac C4 column (Vydac) equilibrated with 0.1% trifluoroacetic acid. After washing the column, the protein bound to the column was removed by the acetonitrile concentration gradient. Eluted.
  • TGF-beta3 (SEQ ID NO: 2)
  • SEQ ID NO: 2 mature h (m) TGF-beta3
  • the mature body of human TGF-beta3 and mouse TGF-beta3 have complete homology, they can be regarded as the same.
  • Example 2 Preparation of anti-TGF-beta3 antibody from antibody library (1) Preparation of human antibody naive library Antibody heavy chain by PCR using poly-A RNA prepared from human PBMC or commercially available human poly-A RNA as a template The variable region and antibody light chain variable region gene libraries were amplified. The prepared antibody heavy chain variable region gene library and antibody light chain variable region gene library were combined and inserted into a phagemid vector to construct a human antibody phage display library displaying a Fab domain comprising human antibody sequences. Methods Mol Biol., (2002) 178, 87-100 was referred as a construction method.
  • the linker part that connects the phagemid Fab and the phage pIII protein and the sequence of the phage display library in which a trypsin cleavage sequence is inserted between the N2 domain and CT domain of the helper phage pIII protein gene are used. It was.
  • Phage was produced from Escherichia coli holding the constructed phagemid for phage display.
  • a phage library solution was obtained by diluting the precipitated phage population with PBS by adding 2.5M NaCl / 10% PEG to the culture solution of Escherichia coli that carried out the phage production.
  • BSA and isopropanol were added to the phage library solution to prepare a final concentration of 4% BSA and 2.5% isopropanol.
  • a panning method a panning method using an antigen immobilized on magnetic beads, which is a general method, was referred to (J. Immunol.
  • the phage library solution was added at room temperature to 60 ° C. Contacted with antigen for minutes. The beads were washed 3 times with 1 mL of 2.5% isopropanol / PBST (PBST containing 2.5% isopropanol), and then further washed twice with 1 mL of 2.5% isopropanol / PBS (PBS containing 2.5% isopropanol). Thereafter, 0.5 mL of 1 mg / mL trypsin was added to the beads and suspended at room temperature for 15 minutes.
  • the beads were immediately separated using a magnetic stand, and the phage solution was recovered.
  • the recovered phage solution was added to 10 mL of E. coli strain ER2738 in the logarithmic growth phase (OD600: 0.4-0.7).
  • E. coli was infected with the phage by gently stirring the E. coli at 37 ° C. for 1 hour. Infected E. coli were seeded on 225 mm x 225 mm plates.
  • a phage library solution was prepared by recovering the phage from the seeded E. coli culture solution and used for the second panning.
  • a phage library solution was prepared by adding 200 pmol of biotin-labeled antigen to the prepared phage library solution and performing the same operation as in the first panning.
  • a third panning was performed using this preparation.
  • 100 pmol of biotin-labeled antigen was added to the prepared phage library solution, and the same operations as in the first and second panning were performed to recover the seeded E. coli.
  • the DNA encoding the antibody light chain variable region was recombined with the DNA encoding k0MT (SEQ ID NO: 12), which is a human kappa light chain constant region.
  • the ligation product was used to transform E. coli DH5 ⁇ (TOYOBO, DNA-903), and a full-length antibody plasmid for animal cell expression was extracted from the resulting single colony.
  • Antibody expression was carried out using the following method. Suspend human fetal kidney cell-derived FreeStyle 293-F strain (Thermo Fisher) in FreeStyle 293 Expression Medium medium (Thermo Fisher), and add 400 ⁇ L to each well of a 96-well deep well plate at a cell density of 3.2 ⁇ 10 5 cells / well. Sowing.
  • Each prepared plasmid was introduced into the cells by the lipofection method.
  • the cells were cultured for 5 days in a CO 2 incubator (37 ° C., 8% CO 2 ), and the antibody was secreted into the culture supernatant.
  • Secreted antibodies in the culture supernatant were purified using Multi screen HTS GV (Millipore, MSGVN2250).
  • mature mouse TGF-beta2 (hereinafter mature mTGF-beta2, 7346-B2 / CF, R & D systems) were applied to measure the binding response between the antibody immobilized on the biosensor and the antigen. did. As a result, a large number of antibodies confirmed to have specific binding to mature h (m) TGF-beta3 were obtained.
  • Example 3 Production of anti-TGF-beta3 antibody from rabbit / mouse Anti-TGF-beta3 antibody was prepared, selected and assayed as follows. 10-16 week old NZW rabbits and 7-18 week old BALB / c mice were immunized with mouse TGFb3 mature form (15-30 ⁇ g / dose / rabbit). Rabbits were immunized by intradermal administration and mice were immunized by subcutaneous administration. Immunization at this dose was repeated 4-5 times over 2 months. One week after the final immunization, spleen, blood and lymph nodes were collected from the immunized animals.
  • Antigen-specific B cells are stained with labeled antigen (mouse TGFb3 mature form or mouse TGFb3 latent form), sorted with a cell sorter (FACS aria III, BD), and at a density of 1 cell / well, 25,000 cells / well. Seeded in 96-well plate with EL4 cells (European Collection of Cell Cultures) and 20-fold diluted activated rabbit T cell conditioned medium, cultured for 7-17 days, and used secreted antibody in its B cell culture supernatant It was used for antibody screening. EL4 cells were treated with mitomycin C (Sigma, catalog number M4287) for 2 hours and washed 3 times in advance.
  • Activated rabbit T-cell conditioned medium contains rabbit thymocytes, RPMI-1640 containing phytohemagglutinin M (Roche, catalog number 11082132001), phorbol 12-myristate 13-acetate (Sigma, catalog number P1585) and 2% FBS. Prepared by culturing in. After culture, B cell culture supernatant was collected for further analysis and cell pellets were stored frozen.
  • ELISA assay was used to test the specificity of the antibodies in the B cell culture supernatant.
  • Mature mTGF-beta3, mature mTGF-beta1, and mature mTGF-beta2 were coated on 384-well High Bind Microplate (Corning, Catalog No. 3700) at 8-16nM in PBS for 1 hour at room temperature, then D-PBS (-) Blocked with a solution of BSA (SIGMA, catalog number A7030) diluted to 1% with Latent mTGF-beta3 was first labeled with NHS-PEG4-biotin (PIERCE, catalog number 21329).
  • the OctetHTX system (Pall Life Sciences) was used to evaluate the antibody-antigen reaction in a manner that did not solidify the antigen on the plate.
  • the antibody secreted into the rabbit B cell culture supernatant is bound on the Protein A biosensor (Pall Life Sciences), and the antibody secreted into the mouse B cell culture supernatant is the Anti-Mouse IgG IgG Fc Capture Bio (AMC) Bio After binding on a sensor (Pall Life Sciences), it was immersed in a 100 nM mouse TGFb3 form form solution to evaluate the binding reaction between the antibody and the antigen.
  • RNA of selected cell lines was purified from cryopreserved cell pellets using the ZR-96 Quick-RNA kit (ZYMO RESEARCH, catalog number R1053).
  • DNA encoding the antibody heavy chain variable region in the selected cell line was amplified by reverse transcription PCR and recombined with DNA encoding the human IgG1 heavy chain constant region (SEQ ID NO: 10).
  • DNA encoding the antibody light chain variable region is amplified by reverse transcription PCR, and in the case of a rabbit derived antibody light chain variable region, it encodes k0MTC (SEQ ID NO: 11), which is a modified human kappa light chain constant region.
  • a DNA encoding the heavy chain variable region (VH) is fused in-frame to a DNA encoding the human IgG1 heavy chain constant region (SEQ ID NO: 13), and a DNA encoding the light chain variable region (VL) is obtained. It was fused in-frame to DNA encoding the light chain constant region of k0MT (SEQ ID NO: 14). Each fused coding sequence was cloned into an expression vector. Antibodies are expressed in FreeStyle (TM) 293-F cells (Invitrogen), purified from the culture supernatant, and evaluated for binding specificity between TGF-beta family molecules and binding to Latent TGF-beta3 as described above And functional activity was evaluated. The sequences of the anti-TGF-beta3 antibodies prepared in Examples 2 and 3 (SEQ ID NO: 100 to SEQ ID NO: 867 represented by Kabat Numbering) are summarized in Table 2.
  • Example 4 Evaluation of the neutralizing activity of the obtained anti-TGF-beta3 antibody
  • the neutralizing activity was evaluated using a Japanese antibody (GC1008-hIgG1), mature h (m) TGF-beta3 recombinant protein and HEK-Blue_TGFb cells (hkb-tgfb, InvivoGen).
  • About the evaluation method of the physiological activity of mature h (m) TGF-beta3 we verified the input of Smad signal by evaluating the activity of secretory alkaline phosphatase (SEAP).
  • SEAP secretory alkaline phosphatase
  • Each antibody is serially diluted from 2000 ng / mL to 3.2 ng / mL, and the final concentration is 0.32 ng / mL using mature h (m) TGF-beta3 recombinant protein dissolved in 100 ⁇ g / mL with 4 mM HCl. Dispense into a flat-bottom 96-well plate to make mL, add HEK-Blue_TGFb cell suspension to 2 ⁇ 10 5 cells / well, and overnight in a CO 2 incubator at 37 ° C. (20 hrs) was cultured.
  • Example 5 Evaluation of Binding Specificity of Anti-TGF-beta3 Antibody to Various TGF-beta
  • the binding specificity of anti-TGF-beta3 antibody to TGF-beta1-3 was evaluated.
  • one of the substances (ligands) for observing the interaction is fixed on the gold thin film of the sensor chip and light is applied from the back side of the sensor chip so that it is totally reflected at the interface between the gold thin film and the glass, part of the reflected light A portion (SPR signal) where the reflection intensity is reduced is formed.
  • the mass of the immobilized ligand molecule increases and the refractive index of the solvent on the sensor chip surface changes. To do. This change in refractive index shifts the position of the SPR signal (conversely, when the bond dissociates, the signal position returns).
  • the Biacore system takes the shift amount, that is, the mass change at the sensor chip surface on the vertical axis, and displays the time change of mass as measurement data (sensorgram).
  • the amount of analyte binding to the ligand captured on the sensor chip surface from the sensorgram (the amount of change in response on the sensorgram before and after the interaction of the analyte) is determined. However, since the amount of binding also depends on the amount of ligand, it is necessary to compare under the condition that the amount of ligand can be regarded as essentially the same amount.
  • the binding activity of the antibodies obtained in Examples 2 and 3 to various TGF-betas was measured at 25 ° C. using HIA-EP + as a running buffer using Biacore T200 (GE Healthcare, Control Software Version 2.0). HBS-EP + was prepared by diluting HBS-EP + x10 (GE Healthcare) 10 times with ultrapure water collected from Milli-Q Advantage (Merck).
  • Binding was performed at a flow rate of 60 ⁇ l / min for 1 minute and then dissociated for 1 minute. The response 5 seconds before the end of binding was defined as the binding value of TGF-beta.
  • Sensor chip CM4 was regenerated using 25 mM NaOH and repeatedly used. This measurement result was obtained by first subtracting the buffer response as a blank using Biacore T200 Evaluation Software Version 2.0 (GE Healthcare), and then dividing the TGF-beta binding value by the amount of antibody captured. TGF-beta binding values were calculated. In addition, when the value was less than 0.01, it was considered that there was no coupling
  • TGF-beta3 has been reported to be expressed in prostate cancer and other cancer cell lines, and is thought to be involved in cancer progression such as metastasis and invasion by enhancing the motility of cancer cells Yes.
  • TGF-beta3 is known to be expressed at high frequency in the tumor tissue of patients with clinical invasive breast cancer, and the expression level and blood concentration of TGF-beta3 in the tumor tissue are related to the overall survival of breast cancer patients. It is reported to show an inverse correlation. (Anticancer Res. 2000 Nov-Dec; 20 (6B): 4413-8.) Thus, since TGF-beta3 is considered to have functions related to cancer progression and poor prognosis, the neutralizing antibody that specifically binds to TGF-beta3 obtained in the present invention is a cancer. Expected to be applicable to treatment.
  • Example 6 Competition Evaluation of Anti-TGF-beta3 Antibody The anti-TGF-beta3 antibody prepared in Examples 2 and 3 was selected for further analysis. By subjecting the selected antibodies to epitope binning experiments using the in-tandem method using the OctetHTX system, it is possible to determine whether antibodies with various binding properties bind to the same or overlapping epitopes of the TGF-beta3 protein. evaluated. The selected antibodies were classified into Group 1-7 by classifying the following three criteria: TGF-beta3-specific binding, binding to Latent TGF-beta3, and neutralizing activity (Table 5).
  • Neutralizing activity A: TGF-beta3 suppression efficiency within 20% at antibody concentration of 2000 ng / mL in Example 4 B: Specificity other than A: A: mature hTGF-beta1, 2 / mature mTGF-beta1, 2 binding at 100% ratio of mature h (m) TGF-beta3 in Example 5 B: other than A Latent binding: A: 10% or more of binding to latent mTGF-beta3 at 100% ratio of mature h (m) TGF-beta3 in Example 5 B: Other than A Group1: Neutralizing activity (A), specificity (A), Latent binding (A) Group2: Neutralizing activity (B), specificity (A), Latent binding (A) Group3: Neutralizing activity (A), specificity (B), Latent binding (A) Group4: Neutralizing activity (A), specificity (A), Latent binding (B) Group5: Neutralizing activity (B), specificity (A), Latent binding (B) Group6: Neutralizing activity (A
  • TGF-beta3 monomer is a small protein with a molecular weight of about 12.5 kDa, but it forms a higher-order structure with three disulfide bonds in the monomer molecule and one in the monomer molecule.
  • Epitope binning which is an epitope analysis method using epitope competition as an index, was performed.
  • TGF-beta3 protein forms a homodimer, but generally, sandwich ELISA is not effective for such targets, so the in tandem method using the OctetHTX system (http: // www.fortebio.com/interactions/Autumn_2010/page2.html).
  • the binding of the primary antibody saturated (Table 6 minutes at an antibody concentration of 15 or 50 ⁇ g / mL depending on the type of antibody as described in 5).
  • IC17-hIgG1 which is an antibody against KLH
  • the binding response value of the second antibody to be evaluated original binding of the second antibody
  • Response value / Original response by 2nd Ab is defined as 100%
  • the binding response value of the second antibody when each anti-TGFb3 antibody is used as the first antibody was calculated and used as a criterion for epitope competition.
  • Group 1 which is a non-neutralizing antibody that specifically binds to TGF-beta3 (neutralizing activity (A), specificity (A), latency binding ( All of the antibodies belonging to A)) compete with each other for binding to TGF-beta3, and antibodies belonging to Group 4 (neutralizing activity (A), specificity (A), patent binding (B)) also Both compete with each other for binding to TGF-beta3. From these results, it was confirmed that the non-neutralizing antibodies that specifically bind to TGF-beta3 prepared in the present invention compete with each other in binding to TGF-beta3.
  • these antibodies are thought to recognize common or similar nearby epitopes of TGF-beta3.
  • the epitope competition pattern of these non-neutralizing antibodies that specifically bind to TGF-beta3 is different from that of antibodies that have neutralizing activity or antibodies that are inferior to TGF-beta3-specific binding. Show. Considering such a series of results, it is considered that the antibody group produced in the present invention widely covers epitopes characteristic of TGF-beta3 antibodies having desirable binding characteristics classified into Group 1 and Group 4.
  • Example 7 Inhibition of human B cell proliferation by immuno-complex (IC) of mature h (m) TGF-beta3 and anti-TGF-beta3 specific non-neutralizing antibody Obtained in Examples 2 and 3, Anti-TGF-beta3 antibody (hereinafter referred to as “anti-TGF-beta3-specific non-neutralizing antibody”), which was confirmed to be non-neutralizing and TGF-beta3-specific, in vivo with TGF-beta3 antigen In order to confirm whether it retains the effect of suppressing the proliferation of B cells when bound, it was examined using human PBMC (PB005F, Lot. A4811, AllCells) purchased from AllCells, LLC.
  • PBMC PB005F, Lot. A4811, AllCells
  • Fractionate B cells negative fraction
  • Human B cells isolation kits II (130-091-151, Miltenyi Biotec)
  • Medium RPMI / 10% FBS / 55 ⁇ M 2-ME
  • IL-21 (200-21, Peprotech) was mixed and added. After culturing in a CO 2 incubator at 37 ° C for 3 days, the amount of viable cells grown using Cell titer glo (G7572, Promega) is measured, so that it is specific for mature h (m) TGF-beta3 and anti-TGF-beta3 Inhibitory effects of human non-neutralizing antibodies on the proliferation of human B cells were examined. Stimulation was started at the same timing using an anti-KLH antibody (IC17-hIgG1) and an anti-pan-TGFbeta neutralizing antibody (GC1008-hIgG1) as standards.
  • Example 8 Suppressive effect of anti-dsDNA antibody production in CD4 + CD25 ⁇ T cell transfer model to BALB / c nu / nu mice As one of the typical symptoms of autoimmune diseases including systemic lupus erythematosus (SLE), An increase in serum antinuclear antibody titer can be mentioned.
  • SLE systemic lupus erythematosus
  • MRL-Faslpr / lpr mice and NZB / W F1 mice as model animals for SLE, all of which are associated with increased serum antinuclear antibody titers. It is known to take a long time of nearly one year from the moon.
  • anti-antinuclear antibody titer of IgG type is increased within one month by transferring BALB / c CD4 + CD25 - T cells into BALB / c nu / nu mice.
  • the in vivo efficacy of TGF-beta3 antibody was examined.
  • mice Eight weeks old female BALB / c mice purchased from Japan SLC were euthanized by whole blood collection under isoflurane anesthesia, spleen was collected and hemolyzed with ACK lysing buffer (A10492-01, Gibco) After collecting CD4 positive cells using CD4 + T Cell Isolation Kit, mouse (130-104-454, Miltenyi Biotec), CD4 using CD25 MicroBead Kit, mouse (130-091-072, Miltenyi Biotec) BALB / c mouse spleen cell-derived CD4 + CD25 ⁇ T cells were prepared by recovering + CD25 ⁇ cells.
  • ACK lysing buffer A10492-01, Gibco
  • Blood collection of EDTA-2Na was carried out before the first administration of the antibody (Pre) and on Day 12, Day 20, and Day 28 after administration, and mouse anti-dsDNA IgG ELISA (Levis anti-dsDNA-mouse ELISA kit: AKRDD-061, Shibayagi) Antibody titers in various plasmas were measured using Smith antibody (Mouse anti-Smith antibody ELISA Kit: MBS705621, MyBioSource).
  • the anti-dsDNA antibody titer in plasma on Day 28 was 131.3 Units / mL in the IC17-hIgG1 administration group, whereas the anti-TGF-beta3-specific non-neutralizing antibody of the present invention, TTA0003 antibody, TTA0035 antibody, TTB0019 In the anti-TGFbeta3 antibody administration group of the antibody, TTB0043 antibody, and F3NKB2-3_062 antibody, it was confirmed that there was a tendency to suppress with 78.5 Units / mL, 48.3 Units / mL, 40.2 Units / mL, 88.3 Units / mL, 43.8 Units / mL, respectively It was. (Fig. 6A)

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Abstract

The present invention provides an anti-TGF-beta3 antibody and a method of using the antibody. The anti-TGF-beta3 antibody according to the present invention has a binding activity to TGF-beta3 but does not have a neutralizing activity to TGF-beta3, and is useful as an inhibitor for the activation of B-cells and as a therapeutic agent for autoimmune diseases.

Description

抗ＴＧＦ－ｂｅｔａ３抗体およびその使用Anti-TGF-beta3 antibody and use thereof
　本発明は、抗TGF-beta3抗体およびその使用、より詳細には抗TGF-beta3抗体を用いたB細胞活性化の抑制剤および自己免疫疾患の治療剤に関する。 The present invention relates to an anti-TGF-beta3 antibody and use thereof, and more specifically to an inhibitor of B cell activation and a therapeutic agent for autoimmune diseases using the anti-TGF-beta3 antibody.
　自己抗体は、複数の臓器系での重篤な炎症によって特徴付けられる全身性エリテマトーデス（systemic lupus erythematosus: SLE）などの様々な自己免疫疾患を誘導する事が知られている（非特許文献１）。高親和性の自己抗体の主な起源は、胚中心（germinal center : GC）で体細胞超変異を受けた自己反応性B細胞である（非特許文献２）。最近になって、CD4陽性CD25陰性LAG3陽性で規定されるレギュラトリーT細胞（LAG3+ Treg）により産生されたTGF-beta3によりB細胞活性化が抑制されることが明らかとなった（非特許文献３）。 Autoantibodies are known to induce various autoimmune diseases such as systemic lupus erythematosus (SLE) characterized by severe inflammation in multiple organ systems (Non-patent Document 1). . The main origin of high-affinity autoantibodies is self-reactive B cells that have undergone somatic hypermutation at the germinal center (GC) (Non-patent Document 2). Recently, TGF-beta3 produced by regulatory T cells (LAG3 + Treg) defined by CD4 positive CD25 negative LAG3 positive was found to suppress B cell activation (Non-patent literature) 3).
WO 2005097832WO 2005097832
　本発明は、抗TGF-beta3抗体およびその使用方法を提供する。 The present invention provides an anti-TGF-beta3 antibody and a method for using the same.
　具体的には、本発明は以下のものを提供する。
〔１〕TGF-beta3に特異的に結合する非中和抗体。
〔２〕〔１〕の抗体であって、以下（１）から（２９）のいずれかに記載の抗体：
（１）配列番号：１０１に記載のCDR１、配列番号：１０２に記載のCDR２、配列番号：１０３に記載のCDR３を有する重鎖可変領域、および配列番号：１０５に記載のCDR１、配列番号：１０６に記載のCDR２、配列番号：１０７に記載のCDR３を有する軽鎖可変領域を含む抗体
（２）配列番号：１４１に記載のCDR１、配列番号：１４２に記載のCDR２、配列番号：１４３に記載のCDR３を有する重鎖可変領域、および配列番号：１４５に記載のCDR１、配列番号：１４６に記載のCDR２、配列番号：１４７に記載のCDR３を有する軽鎖可変領域を含む抗体
（３）配列番号：１６１に記載のCDR１、配列番号：１６２に記載のCDR２、配列番号：１６３に記載のCDR３を有する重鎖可変領域、および配列番号：１６５に記載のCDR１、配列番号：１６６に記載のCDR２、配列番号：１６７に記載のCDR３を有する軽鎖可変領域を含む抗体
（４）配列番号：１７１に記載のCDR１、配列番号：１７２に記載のCDR２、配列番号：１７３に記載のCDR３を有する重鎖可変領域、および配列番号：１７５に記載のCDR１、配列番号：１７６に記載のCDR２、配列番号：１７７に記載のCDR３を有する軽鎖可変領域を含む抗体
（５）配列番号：１８１に記載のCDR１、配列番号：１８２に記載のCDR２、配列番号：１８３に記載のCDR３を有する重鎖可変領域、および配列番号：１８５に記載のCDR１、配列番号：１８６に記載のCDR２、配列番号：１８７に記載のCDR３を有する軽鎖可変領域を含む抗体
（６）配列番号：１９１に記載のCDR１、配列番号：１９２に記載のCDR２、配列番号：１９３に記載のCDR３を有する重鎖可変領域、および配列番号：１９５に記載のCDR１、配列番号：１９６に記載のCDR２、配列番号：１９７に記載のCDR３を有する軽鎖可変領域を含む抗体
（７）配列番号：２０１に記載のCDR１、配列番号：２０２に記載のCDR２、配列番号：２０３に記載のCDR３を有する重鎖可変領域、および配列番号：２０５に記載のCDR１、配列番号：２０６に記載のCDR２、配列番号：２０７に記載のCDR３を有する軽鎖可変領域を含む抗体
（８）配列番号：２１１に記載のCDR１、配列番号：２１２に記載のCDR２、配列番号：２１３に記載のCDR３を有する重鎖可変領域、および配列番号：２１５に記載のCDR１、配列番号：２１６に記載のCDR２、配列番号：２１７に記載のCDR３を有する軽鎖可変領域を含む抗体
（９）配列番号：２４１に記載のCDR１、配列番号：２４２に記載のCDR２、配列番号：２４３に記載のCDR３を有する重鎖可変領域、および配列番号：２４５に記載のCDR１、配列番号：２４６に記載のCDR２、配列番号：２４７に記載のCDR３を有する軽鎖可変領域を含む抗体
（１０）配列番号：２６１に記載のCDR１、配列番号：２６２に記載のCDR２、配列番号：２６３に記載のCDR３を有する重鎖可変領域、および配列番号：２６５に記載のCDR１、配列番号：２６６に記載のCDR２、配列番号：２６７に記載のCDR３を有する軽鎖可変領域を含む抗体
（１１）配列番号：２９１に記載のCDR１、配列番号：２９２に記載のCDR２、配列番号：２９３に記載のCDR３を有する重鎖可変領域、および配列番号：２９５に記載のCDR１、配列番号：２９６に記載のCDR２、配列番号：２９７に記載のCDR３を有する軽鎖可変領域を含む抗体
（１２）配列番号：３０１に記載のCDR１、配列番号：３０２に記載のCDR２、配列番号：３０３に記載のCDR３を有する重鎖可変領域、および配列番号：３０５に記載のCDR１、配列番号：３０６に記載のCDR２、配列番号：３０７に記載のCDR３を有する軽鎖可変領域を含む抗体
（１３）配列番号：３６１に記載のCDR１、配列番号：３６２に記載のCDR２、配列番号：３６３に記載のCDR３を有する重鎖可変領域、および配列番号：３６５に記載のCDR１、配列番号：３６６に記載のCDR２、配列番号：３６７に記載のCDR３を有する軽鎖可変領域を含む抗体
（１４）配列番号：３７１に記載のCDR１、配列番号：３７２に記載のCDR２、配列番号：３７３に記載のCDR３を有する重鎖可変領域、および配列番号：３７５に記載のCDR１、配列番号：３７６に記載のCDR２、配列番号：３７７に記載のCDR３を有する軽鎖可変領域を含む抗体
（１５）配列番号：３９１に記載のCDR１、配列番号：３９２に記載のCDR２、配列番号：３９３に記載のCDR３を有する重鎖可変領域、および配列番号：３９５に記載のCDR１、配列番号：３９６に記載のCDR２、配列番号：３９７に記載のCDR３を有する軽鎖可変領域を含む抗体
（１６）配列番号：４０１に記載のCDR１、配列番号：４０２に記載のCDR２、配列番号：４０３に記載のCDR３を有する重鎖可変領域、および配列番号：４０５に記載のCDR１、配列番号：４０６に記載のCDR２、配列番号：４０７に記載のCDR３を有する軽鎖可変領域を含む抗体
（１７）配列番号：４８１に記載のCDR１、配列番号：４８２に記載のCDR２、配列番号：４８３に記載のCDR３を有する重鎖可変領域、および配列番号：４８５に記載のCDR１、配列番号：４８６に記載のCDR２、配列番号：４８７に記載のCDR３を有する軽鎖可変領域を含む抗体
（１８）配列番号：４９１に記載のCDR１、配列番号：４９２に記載のCDR２、配列番号：４９３に記載のCDR３を有する重鎖可変領域、および配列番号：４９５に記載のCDR１、配列番号：４９６に記載のCDR２、配列番号：４９７に記載のCDR３を有する軽鎖可変領域を含む抗体
（１９）配列番号：５１１に記載のCDR１、配列番号：５１２に記載のCDR２、配列番号：５１３に記載のCDR３を有する重鎖可変領域、および配列番号：５１５に記載のCDR１、配列番号：５１６に記載のCDR２、配列番号：５１７に記載のCDR３を有する軽鎖可変領域を含む抗体
（２０）配列番号：５２１に記載のCDR１、配列番号：５２２に記載のCDR２、配列番号：５２３に記載のCDR３を有する重鎖可変領域、および配列番号：５２５に記載のCDR１、配列番号：５２６に記載のCDR２、配列番号：５２７に記載のCDR３を有する軽鎖可変領域を含む抗体
（２１）配列番号：５３１に記載のCDR１、配列番号：５３２に記載のCDR２、配列番号：５３３に記載のCDR３を有する重鎖可変領域、および配列番号：５３５に記載のCDR１、配列番号：５３６に記載のCDR２、配列番号：５３７に記載のCDR３を有する軽鎖可変領域を含む抗体
（２２）配列番号：５６１に記載のCDR１、配列番号：５６２に記載のCDR２、配列番号：５６３に記載のCDR３を有する重鎖可変領域、および配列番号：５６５に記載のCDR１、配列番号：５６６に記載のCDR２、配列番号：５６７に記載のCDR３を有する軽鎖可変領域を含む抗体
（２３）配列番号：５７１に記載のCDR１、配列番号：５７２に記載のCDR２、配列番号：５７３に記載のCDR３を有する重鎖可変領域、および配列番号：５７５に記載のCDR１、配列番号：５７６に記載のCDR２、配列番号：５７７に記載のCDR３を有する軽鎖可変領域を含む抗体
（２４）配列番号：６８１に記載のCDR１、配列番号：６８２に記載のCDR２、配列番号：６８３に記載のCDR３を有する重鎖可変領域、および配列番号：６８５に記載のCDR１、配列番号：６８６に記載のCDR２、配列番号：６８７に記載のCDR３を有する軽鎖可変領域を含む抗体
（２５）配列番号：７２１に記載のCDR１、配列番号：７２２に記載のCDR２、配列番号：７２３に記載のCDR３を有する重鎖可変領域、および配列番号：７２５に記載のCDR１、配列番号：７２６に記載のCDR２、配列番号：７２７に記載のCDR３を有する軽鎖可変領域を含む抗体
（２６）配列番号：７４１に記載のCDR１、配列番号：７４２に記載のCDR２、配列番号：７４３に記載のCDR３を有する重鎖可変領域、および配列番号：７４５に記載のCDR１、配列番号：７４６に記載のCDR２、配列番号：７４７に記載のCDR３を有する軽鎖可変領域を含む抗体
（２７）配列番号：７９１に記載のCDR１、配列番号：７９２に記載のCDR２、配列番号：７９３に記載のCDR３を有する重鎖可変領域、および配列番号：７９５に記載のCDR１、配列番号：７９６に記載のCDR２、配列番号：７９７に記載のCDR３を有する軽鎖可変領域を含む抗体
（２８）上記（１）～（２７）のいずれかに記載の抗体とTGF-beta3への結合において競合する抗体
（２９）上記（１）～（２７）のいずれかに記載の抗体が結合するTGF-beta3のエピトープと同じエピトープに結合する抗体
〔３〕TGF-beta3のLatent体および／またはMature体に結合する〔１〕または〔２〕の抗体。
〔４〕TGF-beta3の生理活性の抑制が20％以下である〔１〕－〔３〕のいずれかの抗体。
〔５〕TGF-beta3への結合値に対するTGF-beta1およびTGF-beta2への結合値が10％未満である〔１〕－〔４〕のいずれかの抗体。
〔６〕モノクローナル抗体である、〔１〕－〔５〕のいずれかの抗体。
〔７〕ヒト抗体、ヒト化抗体、またはキメラ抗体である、〔１〕－〔６〕のいずれかの抗体。
〔８〕B細胞を認識する可変領域を更に含む二重特異性抗体である〔１〕－〔７〕のいずれかの抗体。
〔９〕前記可変領域はCD19、CD20、CD40、CD22、IL21R、BAFF-R、BCMA、TACI、CD27、又はCD138に含まれるいずれか1つを認識する〔８〕の抗体。
〔１０〕〔１〕－〔９〕のいずれかの抗体を含むB細胞活性化の抑制剤。
〔１１〕〔１〕－〔９〕のいずれかの抗体を含む自己免疫疾患の治療剤。
〔１２〕前記自己免疫疾患は全身性エリテマトーデス、天疱瘡、多発性硬化症、視神経脊髄炎、ANCA関連血管炎、関節リウマチ、移植臓器拒絶、シェーグレン症候群、若年性皮膚筋炎、重症筋無力症、又はバセドウ病、橋本病等の自己免疫性甲状腺疾患のいずれかである〔１１〕の治療剤。
〔１３〕〔１〕－〔９〕のいずれかの抗体を投与する工程を含む、B細胞活性化を抑制する方法または自己免疫疾患を治療する方法。
〔１４〕B細胞活性化の抑制または自己免疫疾患の治療において用いるための、〔１〕－〔９〕のいずれかの抗体。
〔１５〕B細胞活性化の抑制剤または自己免疫疾患の治療剤の製造のための、〔１〕－〔９〕のいずれかの抗体の使用。 Specifically, the present invention provides the following.
[1] A non-neutralizing antibody that specifically binds to TGF-beta3.
[2] The antibody according to [1], wherein the antibody is any one of (1) to (29) below:
(1) A heavy chain variable region having CDR1 described in SEQ ID NO: 101, CDR2 described in SEQ ID NO: 102, CDR3 described in SEQ ID NO: 103, and CDR1 described in SEQ ID NO: 105, SEQ ID NO: 106 An antibody comprising a light chain variable region having CDR2 described in SEQ ID NO: 107 and CDR3 described in SEQ ID NO: 107 (2) CDR1 described in SEQ ID NO: 141, CDR2 described in SEQ ID NO: 142, described in SEQ ID NO: 143 An antibody (3) comprising a heavy chain variable region having CDR3, and a light chain variable region having CDR1 set forth in SEQ ID NO: 145, CDR2 set forth in SEQ ID NO: 146, CDR3 set forth in SEQ ID NO: 147 161, CDR2 of SEQ ID NO: 162, heavy chain variable region having CDR3 of SEQ ID NO: 163, CDR1 of SEQ ID NO: 165, CDR2 of SEQ ID NO: 166, sequence Antibody comprising light chain variable region having CDR3 of No. 167 (4) CDR1 of SEQ ID NO: 171; CDR2 of SEQ ID NO: 172; heavy chain variable having CDR3 of SEQ ID NO: 173 An antibody comprising a region and a light chain variable region having CDR1 set forth in SEQ ID NO: 175, CDR2 set forth in SEQ ID NO: 176, CDR3 set forth in SEQ ID NO: 177 (5) CDR1 set forth in SEQ ID NO: 181, SEQ ID NO: 182 CDR2, SEQ ID NO: 183 heavy chain variable region having CDR3, and SEQ ID NO: 185 CDR1, SEQ ID NO: 186 CDR2, SEQ ID NO: 187 Antibody comprising a light chain variable region having CDR3 (6) CDR1 set forth in SEQ ID NO: 191, CDR2 set forth in SEQ ID NO: 192, heavy chain variable region having CDR3 set forth in SEQ ID NO: 193, and SEQ ID NO: An antibody comprising a light chain variable region having CDR1 described in 95, CDR2 described in SEQ ID NO: 196, CDR3 described in SEQ ID NO: 197 (7) CDR1 described in SEQ ID NO: 201, described in SEQ ID NO: 202 And a heavy chain variable region having the CDR3 of SEQ ID NO: 203, a CDR1 of the SEQ ID NO: 205, a CDR2 of the SEQ ID NO: 206, and a light chain variable having the CDR3 of the SEQ ID NO: 207 Antibody comprising region (8) CDR1 set forth in SEQ ID NO: 211, CDR2 set forth in SEQ ID NO: 212, heavy chain variable region having CDR3 set forth in SEQ ID NO: 213, and CDR1 set forth in SEQ ID NO: 215, An antibody comprising a light chain variable region having CDR2 set forth in SEQ ID NO: 216 and CDR3 set forth in SEQ ID NO: 217 (9) CDR1 set forth in SEQ ID NO: 241, CDR2 set forth in SEQ ID NO: 242, SEQ ID NO: 24 An antibody comprising a heavy chain variable region having CDR3 according to 3, and a CDR1 according to SEQ ID NO: 245, a CDR2 according to SEQ ID NO: 246, and a light chain variable region having CDR3 as SEQ ID NO: 247 (10 ) CDR1 described in SEQ ID NO: 261, CDR2 described in SEQ ID NO: 262, heavy chain variable region having CDR3 described in SEQ ID NO: 263, and CDR1 described in SEQ ID NO: 265, described in SEQ ID NO: 266 An antibody comprising a light chain variable region having CDR3 described in SEQ ID NO: 267 (11) CDR1 described in SEQ ID NO: 291, CDR2 described in SEQ ID NO: 292, CDR3 described in SEQ ID NO: 293 An antibody (12) comprising a heavy chain variable region having a light chain variable region having a CDR1 described in SEQ ID NO: 295, a CDR2 described in SEQ ID NO: 296, a CDR3 described in SEQ ID NO: 297; CDR1, described in SEQ ID NO: 302, heavy chain variable region having CDR3 described in SEQ ID NO: 303, CDR1 described in SEQ ID NO: 305, CDR2 described in SEQ ID NO: 306, SEQ ID NO: Antibody comprising a light chain variable region having CDR3 described in 307 (13) CDR1 described in SEQ ID NO: 361, CDR2 described in SEQ ID NO: 362, heavy chain variable region having CDR3 described in SEQ ID NO: 363 And an antibody comprising a light chain variable region having CDR1 as set forth in SEQ ID NO: 365, CDR2 as set forth in SEQ ID NO: 366, CDR3 as set forth in SEQ ID NO: 367 (14) CDR1, set forth in SEQ ID NO: 371 SEQ ID NO: 372 CDR2, SEQ ID NO: 373 heavy chain variable region having CDR3, SEQ ID NO: 375 CDR1, SEQ ID NO: 376 CDR2, SEQ ID NO: 377 Antibody comprising a light chain variable region having the CDR3 of (15) CDR1 set forth in SEQ ID NO: 391, CDR2 set forth in SEQ ID NO: 392, heavy chain variable region having CDR3 set forth in SEQ ID NO: 393, and SEQ ID NO: Antibody comprising a light chain variable region having CDR1 described in SEQ ID NO: 396, CDR2 described in SEQ ID NO: 396, CDR3 described in SEQ ID NO: 397 (16) CDR1 described in SEQ ID NO: 401, SEQ ID NO: 402 A heavy chain variable region having the CDR2 of SEQ ID NO: 403 and a light chain having a CDR3 of SEQ ID NO: 405, a CDR2 of SEQ ID NO: 406, a light chain having a CDR3 of SEQ ID NO: 407 Antibody comprising variable region (17) CDR1 described in SEQ ID NO: 481, CDR2 described in SEQ ID NO: 482, heavy chain variable region having CDR3 described in SEQ ID NO: 483, and SEQ ID NO: 485 An antibody comprising a light chain variable region having CDR3 of SEQ ID NO: 487, CDR3 of SEQ ID NO: 487, CDR1 of SEQ ID NO: 491, CDR2 of SEQ ID NO: 492, A heavy chain variable region having the CDR3 of SEQ ID NO: 493, a CDR1 of SEQ ID NO: 495, a CDR2 of SEQ ID NO: 496, a light chain variable region having the CDR3 of SEQ ID NO: 497 Antibody (19) CDR1 set forth in SEQ ID NO: 511, CDR2 set forth in SEQ ID NO: 512, heavy chain variable region having CDR3 set forth in SEQ ID NO: 513, and CDR1 set forth in SEQ ID NO: 515, SEQ ID NO: An antibody comprising a light chain variable region having CDR2 described in 516 and CDR3 described in SEQ ID NO: 517 (20) CDR1 described in SEQ ID NO: 521, CDR2 described in SEQ ID NO: 522, SEQ ID NO: 523 The antibody (21) sequence comprising the heavy chain variable region having the CDR3 and the CDR1 described in SEQ ID NO: 525, the CDR2 described in SEQ ID NO: 526, and the light chain variable region having the CDR3 described in SEQ ID NO: 527 SEQ ID NO: 531, CDR2 set forth in SEQ ID NO: 532, heavy chain variable region having CDR3 set forth in SEQ ID NO: 533, and CDR1 set forth in SEQ ID NO: 535, CDR2 set forth in SEQ ID NO: 536 An antibody comprising a light chain variable region having CDR3 described in SEQ ID NO: 537 (22) CDR1 described in SEQ ID NO: 561, CDR2 described in SEQ ID NO: 562, and a heavy chain having CDR3 described in SEQ ID NO: 563 An antibody (23) comprising a chain variable region and a light chain variable region having CDR1 set forth in SEQ ID NO: 565, CDR2 set forth in SEQ ID NO: 566, CDR3 set forth in SEQ ID NO: 567 (23) in SEQ ID NO: 571 The CDR1, the CDR2 described in SEQ ID NO: 572, the heavy chain variable region having the CDR3 described in SEQ ID NO: 573, the CDR1 described in SEQ ID NO: 575, the CDR2 described in SEQ ID NO: 576, the SEQ ID NO: An antibody comprising a light chain variable region having CDR3 according to 577 (24), CDR1 according to SEQ ID NO: 681, CDR2 according to SEQ ID NO: 682, heavy chain variable region having CDR3 according to SEQ ID NO: 683, And an antibody comprising a light chain variable region having CDR1 described in SEQ ID NO: 685, CDR2 described in SEQ ID NO: 686, CDR3 described in SEQ ID NO: 687 (25) CDR1 described in SEQ ID NO: 721, SEQ ID NO: : CDR2 described in 722, heavy chain variable region having CDR3 described in SEQ ID NO: 723, CDR1 described in SEQ ID NO: 725, CDR2 described in SEQ ID NO: 726, CDR described in SEQ ID NO: 727 Antibody comprising a light chain variable region having 3 (26) CDR1 set forth in SEQ ID NO: 741, CDR2 set forth in SEQ ID NO: 742, heavy chain variable region having CDR3 set forth in SEQ ID NO: 743, and SEQ ID NO: An antibody comprising a light chain variable region having CDR1 described in 745, CDR2 described in SEQ ID NO: 746, CDR3 described in SEQ ID NO: 747 (27) CDR1 described in SEQ ID NO: 791, SEQ ID NO: 792 And a heavy chain variable region having the CDR3 of SEQ ID NO: 793, a CDR1 of SEQ ID NO: 795, a CDR2 of SEQ ID NO: 796, and a light chain variable having a CDR3 of SEQ ID NO: 797 An antibody comprising a region (28) an antibody that competes with TGF-beta3 for binding to the antibody according to any of (1) to (27) above (29) according to any of (1) to (27) above TGF-bet to which antibody binds An antibody that binds to the same epitope as the epitope of a3 [3] The antibody of [1] or [2] that binds to the Latent body and / or the Mature body of TGF-beta3.
[4] The antibody according to any one of [1] to [3], wherein the suppression of TGF-beta3 physiological activity is 20% or less.
[5] The antibody according to any one of [1] to [4], wherein the binding value to TGF-beta1 and TGF-beta2 relative to the binding value to TGF-beta3 is less than 10%.
[6] The antibody according to any one of [1] to [5], which is a monoclonal antibody.
[7] The antibody according to any one of [1] to [6], which is a human antibody, a humanized antibody, or a chimeric antibody.
[8] The antibody of any one of [1]-[7], which is a bispecific antibody further comprising a variable region that recognizes B cells.
[9] The antibody according to [8], wherein the variable region recognizes any one of CD19, CD20, CD40, CD22, IL21R, BAFF-R, BCMA, TACI, CD27, or CD138.
[10] An inhibitor of B cell activation comprising the antibody according to any one of [1] to [9].
[11] A therapeutic agent for autoimmune disease comprising the antibody according to any one of [1] to [9].
[12] The autoimmune disease is systemic lupus erythematosus, pemphigus, multiple sclerosis, neuromyelitis optica, ANCA-related vasculitis, rheumatoid arthritis, transplant organ rejection, Sjogren's syndrome, juvenile dermatomyositis, myasthenia gravis, or [11] The therapeutic agent according to [11], which is any of autoimmune thyroid diseases such as Graves' disease and Hashimoto's disease.
[13] A method for suppressing B cell activation or a method for treating an autoimmune disease, comprising a step of administering the antibody according to any one of [1] to [9].
[14] The antibody of any one of [1]-[9] for use in inhibiting B cell activation or treating an autoimmune disease.
[15] Use of the antibody according to any one of [1] to [9] for the manufacture of an inhibitor of B cell activation or a therapeutic agent for autoimmune diseases.
TGF-beta3のエピトープ・ビニングの実験デザインを示す図である。It is a figure which shows the experimental design of the epitope binning of TGF-beta3. 抗原には結合しないコントロール抗体を第一抗体として用いた場合の、「in-tandem法」に依るエピトープ・ビニングの典型的なセンサーグラムを示す図である。It is a figure which shows the typical sensorgram of the epitope binning by the "in-tandem method" at the time of using the control antibody which does not couple | bond with an antigen as a 1st antibody. 各抗TGFb3抗体を第一抗体として用いた場合の、「in-tandem法」に依るエピトープ・ビニングの典型的なセンサーグラムを示す図である。It is a figure which shows the typical sensorgram of the epitope binning by the "in-tandem method" at the time of using each anti- TGFb3 antibody as a 1st antibody. 取得された抗TGF-beta3抗体の競合を示す図である。It is a figure which shows the competition of the acquired anti- TGF-beta3 antibody. Mature h(m)TGF-beta3と抗TGF-beta3特異的非中和抗体のimmuno-complex (IC)によるヒトB細胞増殖抑制効果を示す図である。It is a figure which shows the human B cell proliferation inhibitory effect by mature-h (m) TGF-beta3 and immuno-complex (IC) of the anti- TGF-beta3 specific non-neutralizing antibody. BALB/c nu/nuマウスへのCD4+CD25-T細胞移入モデルにおける抗dsDNA抗体および抗Smith抗体産生抑制効果を示す図である。(A) Day12, 20, 28における血漿中の抗dsDNA抗体価の推移を示す。(B) Day28における血漿中の抗Smith抗体価を示す。エラーバーは標準誤差を示す。IC17-hIgGに対する有意差検定はStudent-t testを用いた。(N=3～5, *; p ＜ 0.05, **; p ＜ 0.01)BALB / c nu / nu mice of CD4 + CD25 - a diagram showing the anti-dsDNA and anti-Smith antibody production suppressive effect on T cell transfer model. (A) Changes in the anti-dsDNA antibody titer in plasma on Days 12, 20, and 28. (B) shows the anti-Smith antibody titer in plasma on Day 28. Error bars indicate standard error. Student-t test was used for the significant difference test for IC17-hIgG. (N = 3-5, *; p <0.05, **; p <0.01)
Ｉ．定義
　本明細書の趣旨での「アクセプターヒトフレームワーク」は、下で定義するヒト免疫グロブリンフレームワークまたはヒトコンセンサスフレームワークに由来する、軽鎖可変ドメイン (VL) フレームワークまたは重鎖可変ドメイン (VH) フレームワークのアミノ酸配列を含む、フレームワークである。ヒト免疫グロブリンフレームワークまたはヒトコンセンサスフレームワークに由来するアクセプターヒトフレームワークは、それらの同じアミノ酸配列を含んでもよいし、またはアミノ酸配列の変更を含んでいてもよい。いくつかの態様において、アミノ酸の変更の数は、10以下、9以下、8以下、7以下、6以下、5以下、4以下、3以下、または2以下である。いくつかの態様において、VLアクセプターヒトフレームワークは、VLヒト免疫グロブリンフレームワーク配列またはヒトコンセンサスフレームワーク配列と、配列が同一である。 I. Definitions For purposes of this specification, an “acceptor human framework” is a light chain variable domain (VL) framework or heavy chain variable domain derived from a human immunoglobulin framework or human consensus framework as defined below ( VH) A framework containing the amino acid sequence of the framework. Acceptor human frameworks derived from human immunoglobulin frameworks or human consensus frameworks may contain those same amino acid sequences or may contain amino acid sequence changes. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or human consensus framework sequence.
　「アフィニティ」は、分子（例えば、抗体）の結合部位1個と、分子の結合パートナー（例えば、抗原）との間の、非共有結合的な相互作用の合計の強度のことをいう。別段示さない限り、本明細書で用いられる「結合アフィニティ」は、ある結合対のメンバー（例えば、抗体と抗原）の間の１：１相互作用を反映する、固有の結合アフィニティのことをいう。分子XのそのパートナーYに対するアフィニティは、一般的に、解離定数 (Kd) により表すことができる。アフィニティは、本明細書に記載のものを含む、当該技術分野において知られた通常の方法によって測定され得る。結合アフィニティを測定するための具体的な実例となるおよび例示的な態様については、下で述べる。 “Affinity” refers to the total strength of non-covalent interactions between a binding site of a molecule (eg, an antibody) and a binding partner (eg, an antigen) of the molecule. Unless otherwise indicated, “binding affinity” as used herein refers to intrinsic binding affinity that reflects a 1: 1 interaction between members of a binding pair (eg, an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by conventional methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.
　「アフィニティ成熟」抗体は、改変を備えていない親抗体と比較して、1つまたは複数の超可変領域 (hypervariable region: HVR) 中に抗体の抗原に対するアフィニティの改善をもたらす1つまたは複数の改変を伴う抗体のことをいう。 An “affinity matured” antibody is one or more modifications that result in improved affinity of the antibody for the antigen in one or more hypervariable regions (HVRs) compared to the parent antibody without the modifications. An antibody with
　用語「抗TGF-beta3抗体」または「TGF-beta3に結合する抗体」は、充分なアフィニティでTGF-beta3と結合することのできる抗体であって、その結果その抗体がTGF-beta3を標的化したときにB細胞活性化抑制剤および／または自己免疫疾患の治療剤として有用であるような抗体のことをいう。一態様において、無関係な非TGF-beta3タンパク質への抗TGF-beta3抗体の結合の程度は、（例えば、放射免疫測定法 (radioimmunoassay: RIA) により）測定したとき、抗体のTGF-beta3への結合の約10％未満である。特定の態様において、TGF-beta3に結合する抗体は、≦1μM、≦100nM、≦10nM、≦1nM、≦0.1nM、≦0.01nM、または≦0.001nM（例えば、10-8M以下、例えば10-8M～10-13M、例えば、10-9M～10-13M）の解離定数 (Kd) を有する。特定の態様において、抗TGF-beta3抗体は、異なる種からのTGF-beta3間で保存されているTGF-beta3のエピトープに結合する。 The term "anti-TGF-beta3 antibody" or "antibody that binds to TGF-beta3" is an antibody that can bind to TGF-beta3 with sufficient affinity so that the antibody targeted TGF-beta3. It refers to antibodies that are sometimes useful as inhibitors of B cell activation and / or as therapeutic agents for autoimmune diseases. In one embodiment, the extent of binding of the anti-TGF-beta3 antibody to an irrelevant non-TGF-beta3 protein is determined by the binding of the antibody to TGF-beta3, as measured (eg, by radioimmunoassay (RIA)). Less than about 10%. In certain embodiments, the antibody that binds to TGF-beta3 is ≦ 1 μM, ≦ 100 nM, ≦ 10 nM, ≦ 1 nM, ≦ 0.1 nM, ≦ 0.01 nM, or ≦ 0.001 nM (eg, 10 −8 M or less, such as 10 − A dissociation constant (Kd) of 8 M to 10 -13 M, for example, 10 -9 M to 10 -13 M). In certain embodiments, the anti-TGF-beta3 antibody binds to an epitope of TGF-beta3 that is conserved among TGF-beta3 from different species.
　本明細書で用語「抗体」は、最も広い意味で使用され、所望の抗原結合活性を示す限りは、これらに限定されるものではないが、モノクローナル抗体、ポリクローナル抗体、多重特異性抗体（例えば、二重特異性抗体）および抗体断片を含む、種々の抗体構造を包含する。 As used herein, the term “antibody” is used in the broadest sense and is not limited thereto as long as it exhibits a desired antigen-binding activity, but is not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, Various antibody structures are included, including bispecific antibodies) and antibody fragments.
　「抗体断片」は、完全抗体が結合する抗原に結合する当該完全抗体の一部分を含む、当該完全抗体以外の分子のことをいう。抗体断片の例は、これらに限定されるものではないが、Fv、Fab、Fab'、Fab'-SH、F(ab')2；ダイアボディ；線状抗体；単鎖抗体分子（例えば、scFv）；および、抗体断片から形成された多重特異性抗体を含む。 “Antibody fragment” refers to a molecule other than the complete antibody, including a portion of the complete antibody that binds to the antigen to which the complete antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab ′, Fab′-SH, F (ab ′) 2 ; diabodies; linear antibodies; single chain antibody molecules (eg, scFv And multispecific antibodies formed from antibody fragments.
　本発明に記載する「競合する抗体」あるいは「同じエピトープに結合する抗体」とは、実施例６（OctetHTXシステムを用いたin-tandem法に依るエピトープ競合アッセイ）の評価手法により第一抗体と第二抗体との結合レスポンスを測定した場合に、第二抗体の結合レスポンス値が60％未満、好ましくは40％以下、より好ましくは20％以下となものを指す。 The “competing antibody” or “antibody that binds to the same epitope” described in the present invention refers to the first antibody and the first antibody according to the evaluation method of Example 6 (epitope competition assay by in-tandem method using OctetHTX system). When the binding response to the two antibodies is measured, the binding response value of the second antibody is less than 60%, preferably 40% or less, more preferably 20% or less.
　用語「キメラ」抗体は、重鎖および／または軽鎖の一部分が特定の供給源または種に由来する一方で、重鎖および／または軽鎖の残りの部分が異なった供給源または種に由来する抗体のことをいう。 The term “chimeric” antibody is one in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remaining portion of the heavy and / or light chain is derived from a different source or species. Refers to an antibody.
　抗体の「クラス」は、抗体の重鎖に備わる定常ドメインまたは定常領域のタイプのことをいう。抗体には5つの主要なクラスがある：IgA、IgD、IgE、IgG、およびIgMである。そして、このうちいくつかはさらにサブクラス（アイソタイプ）に分けられてもよい。例えば、IgG1、IgG2、IgG3、IgG4、IgA1、およびIgA2である。異なるクラスの免疫グロブリンに対応する重鎖定常ドメインを、それぞれ、α、δ、ε、γ、およびμと呼ぶ。 “Class” of an antibody refers to the type of constant domain or constant region provided in the heavy chain of the antibody. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM. Some of them may be further divided into subclasses (isotypes). For example, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.
　「エフェクター機能」は、抗体のFc領域に起因する、抗体のアイソタイプによって異なる生物学的活性のことをいう。抗体のエフェクター機能の例には次のものが含まれる：C1q結合および補体依存性細胞傷害（complement dependent cytotoxicity:CDC）；Fc受容体結合；抗体依存性細胞介在性細胞傷害（antibody-dependent cell-mediated cytotoxicity: ADCC）；貪食作用；細胞表面受容体（例えば、B細胞受容体）の下方制御；および、B細胞活性化。 “Effector function” refers to a biological activity resulting from the Fc region of an antibody, depending on the antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (antibody-dependent cell) -mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (eg, B cell receptors); and B cell activation.
　ある剤（例えば、薬学的製剤）の「有効量」は、所望の治療的または予防的結果を達成するために有効である、必要な用量におけるおよび必要な期間にわたっての、量のことをいう。 An “effective amount” of an agent (eg, a pharmaceutical formulation) refers to the amount at a required dose and over a required period of time that is effective to achieve a desired therapeutic or prophylactic result.
　本明細書で用語「Fc領域」は、少なくとも定常領域の一部分を含む免疫グロブリン重鎖のC末端領域を定義するために用いられる。この用語は、天然型配列のFc領域および変異体Fc領域を含む。一態様において、ヒトIgG重鎖Fc領域はCys226から、またはPro230から、重鎖のカルボキシル末端まで延びる。ただし、Fc領域のC末端のリジン (Lys447) またはグリシン‐リジン（Gly446-Lys447）は、存在していてもしていなくてもよい。本明細書では別段特定しない限り、Fc領域または定常領域中のアミノ酸残基の番号付けは、Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD 1991 に記載の、EUナンバリングシステム（EUインデックスとも呼ばれる）にしたがう。 The term “Fc region” is used herein to define the C-terminal region of an immunoglobulin heavy chain that includes at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. However, lysine (Lys447) or glycine-lysine (Gly446-Lys447) at the C-terminal of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Follow the EU numbering system (also called EU index) described in MD 1991.
　「フレームワーク」または「FR」は、超可変領域 (HVR) 残基以外の、可変ドメイン残基のことをいう。可変ドメインのFRは、通常4つのFRドメイン：FR1、FR2、FR3、およびFR4からなる。それに応じて、HVRおよびFRの配列は、通常次の順序でVH（またはVL）に現れる：FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4。 “Framework” or “FR” refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain usually consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, HVR and FR sequences usually appear in VH (or VL) in the following order: FR1-H1 (L1) -FR2-H2 (L2) -FR3-H3 (L3) -FR4.
　用語「全長抗体」、「完全抗体」、および「全部抗体」は、本明細書では相互に交換可能に用いられ、天然型抗体構造に実質的に類似した構造を有する、または本明細書で定義するFc領域を含む重鎖を有する抗体のことをいう。 The terms “full-length antibody”, “complete antibody”, and “total antibody” are used interchangeably herein and have a structure substantially similar to or defined herein. An antibody having a heavy chain containing an Fc region.
　用語「宿主細胞」、「宿主細胞株」、および「宿主細胞培養物」は、相互に交換可能に用いられ、外来核酸を導入された細胞（そのような細胞の子孫を含む）のことをいう。宿主細胞は「形質転換体」および「形質転換細胞」を含み、これには初代の形質転換細胞および継代数によらずその細胞に由来する子孫を含む。子孫は、親細胞と核酸の内容において完全に同一でなくてもよく、変異を含んでいてもよい。オリジナルの形質転換細胞がスクリーニングされたまたは選択された際に用いられたものと同じ機能または生物学的活性を有する変異体子孫も、本明細書では含まれる。 The terms “host cell”, “host cell line”, and “host cell culture” are used interchangeably and refer to a cell (including the progeny of such a cell) into which a foreign nucleic acid has been introduced. . Host cells include “transformants” and “transformed cells”, including the primary transformed cell and progeny derived from that cell regardless of the passage number. The progeny may not be completely identical in nucleic acid content with the parent cell, and may contain mutations. Also included herein are mutant progeny that have the same function or biological activity as was used when the original transformed cells were screened or selected.
　「ヒト抗体」は、ヒトもしくはヒト細胞によって産生された抗体またはヒト抗体レパートリーもしくは他のヒト抗体コード配列を用いる非ヒト供給源に由来する抗体のアミノ酸配列に対応するアミノ酸配列を備える抗体である。このヒト抗体の定義は、非ヒトの抗原結合残基を含むヒト化抗体を、明確に除外するものである。 A “human antibody” is an antibody comprising an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell or an antibody derived from a non-human source using a human antibody repertoire or other human antibody coding sequence. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen binding residues.
　「ヒトコンセンサスフレームワーク」は、ヒト免疫グロブリンVLまたはVHフレームワーク配列の選択群において最も共通して生じるアミノ酸残基を示すフレームワークである。通常、ヒト免疫グロブリンVLまたはVH配列の選択は、可変ドメイン配列のサブグループからである。通常、配列のサブグループは、Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3におけるサブグループである。一態様において、VLについて、サブグループは上記のKabatらによるサブグループκIである。一態様において、VHについて、サブグループは上記のKabatらによるサブグループIIIである。 “Human consensus framework” is a framework showing the most commonly occurring amino acid residues in a selected group of human immunoglobulin VL or VH framework sequences. Usually, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Usually, the subgroups of sequences are those in KabatKaet al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3. In one embodiment, for VL, the subgroup is subgroup κI according to Kabat et al. In one embodiment, for VH, the subgroup is subgroup III by Kabat et al.
　「ヒト化」抗体は、非ヒトHVRからのアミノ酸残基およびヒトFRからのアミノ酸残基を含む、キメラ抗体のことをいう。ある態様では、ヒト化抗体は、少なくとも1つ、典型的には2つの可変ドメインの実質的にすべてを含み、当該可変領域においては、すべてのもしくは実質的にすべてのHVR（例えばCDR）は非ヒト抗体のものに対応し、かつ、すべてのもしくは実質的にすべてのFRはヒト抗体のものに対応する。ヒト化抗体は、任意で、ヒト抗体に由来する抗体定常領域の少なくとも一部分を含んでもよい。抗体（例えば、非ヒト抗体）の「ヒト化された形態」は、ヒト化を経た抗体のことをいう。 “Humanized” antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody comprises substantially all of at least one, typically two variable domains, in which all or substantially all HVRs (eg, CDRs) are non- It corresponds to that of a human antibody and all or substantially all FRs correspond to those of a human antibody. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. A “humanized form” of an antibody (eg, a non-human antibody) refers to an antibody that has undergone humanization.
　本明細書で用いられる用語「超可変領域」または「HVR」は、配列において超可変であり（「相補性決定領域」または「CDR」(complementarity determining region)）、および／または構造的に定まったループ（「超可変ループ」）を形成し、および／または抗原接触残基（「抗原接触」）を含む、抗体の可変ドメインの各領域のことをいう。通常、抗体は6つのHVRを含む：VHに3つ（H1、H2、H3）、およびVLに3つ（L1、L2、L3）である。本明細書での例示的なHVRは、以下のものを含む：
　(a) アミノ酸残基26-32 (L1)、50-52 (L2)、91-96 (L3)、26-32 (H1)、53-55 (H2)、および96-101 (H3)のところで生じる超可変ループ (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987))；
　(b) アミノ酸残基24-34 (L1)、50-56 (L2)、89-97 (L3)、31-35b (H1)、50-65 (H2)、 および95-102 (H3)のところで生じるCDR (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991))；
　(c) アミノ酸残基27c-36 (L1)、46-55 (L2)、89-96 (L3)、30-35b (H1)、47-58 (H2)、および93-101 (H3) のところで生じる抗原接触 (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996))；ならびに、
　(d) HVRアミノ酸残基46-56 (L2)、47-56 (L2)、48-56 (L2)、49-56 (L2)、26-35 (H1)、26-35b (H1)、49-65 (H2)、93-102 (H3)、および94-102 (H3)を含む、(a)、(b)、および／または(c)の組合せ。
　別段示さない限り、HVR残基および可変ドメイン中の他の残基（例えば、FR残基）は、本明細書では上記のKabatらにしたがって番号付けされる。 The term “hypervariable region” or “HVR” as used herein is hypervariable in sequence (“complementarity determining region” or “CDR”) and / or structurally determined. Refers to each region of the variable domain of an antibody that forms a loop (“hypervariable loop”) and / or contains antigen contact residues (“antigen contact”). Usually, an antibody contains 6 HVRs: 3 in VH (H1, H2, H3) and 3 in VL (L1, L2, L3). Exemplary HVRs herein include the following:
(a) at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) The resulting hypervariable loop (Chothia and Lesk, J. Mol. Biol. 196: 901-917 (1987));
(b) at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) The resulting CDR (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991));
(c) at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) Resulting antigen contact (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996));
(d) HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49 A combination of (a), (b), and / or (c), including -65 (H2), 93-102 (H3), and 94-102 (H3).
Unless indicated otherwise, HVR residues and other residues in the variable domains (eg, FR residues) are numbered herein according to Kabat et al., Supra.
　「個体」または「被験体」は哺乳動物である。哺乳動物は、これらに限定されるものではないが、飼育動物（例えば、ウシ、ヒツジ、ネコ、イヌ、ウマ）、霊長類（例えば、ヒト、およびサルなどの非ヒト霊長類）、ウサギ、ならびに、げっ歯類（例えば、マウスおよびラット）を含む。特定の態様では、個体または被験体は、ヒトである。 “Individual” or “subject” is a mammal. Mammals include, but are not limited to, domestic animals (eg, cattle, sheep, cats, dogs, horses), primates (eg, humans and non-human primates such as monkeys), rabbits, and , Including rodents (eg, mice and rats). In certain embodiments, the individual or subject is a human.
　「単離された」抗体は、そのもともとの環境の成分から分離されたものである。いくつかの態様において、抗体は、例えば、電気泳動（例えば、SDS-PAGE、等電点分離法 (isoelectric focusing: IEF)、キャピラリー電気泳動）またはクロマトグラフ（例えば、イオン交換または逆相HPLC）で測定して、95%または99%を超える純度まで精製される。抗体の純度の評価のための方法の総説として、例えば、Flatman et al., J. Chromatogr. B 848:79-87 (2007) を参照のこと。 An “isolated” antibody is one that has been separated from its original environmental components. In some embodiments, the antibody is, for example, by electrophoresis (eg, SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatograph (eg, ion exchange or reverse phase HPLC). Measured and purified to a purity greater than 95% or 99%. For a review of methods for assessing antibody purity, see, for example, Flatman et al., J. Chromatogr. B B: 848: 79-87 (2007).
　「単離された」核酸は、そのもともとの環境の成分から分離された核酸分子のことをいう。単離された核酸は、その核酸分子を通常含む細胞の中に含まれた核酸分子を含むが、その核酸分子は染色体外に存在しているかまたは本来の染色体上の位置とは異なる染色体上の位置に存在している。 “Isolated” nucleic acid refers to a nucleic acid molecule that has been separated from its original environmental components. An isolated nucleic acid includes a nucleic acid molecule contained within a cell that normally contains the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or on a chromosome different from the original chromosomal location. Exists in position.
　「抗TGF-beta3抗体をコードする単離された核酸」は、抗体の重鎖および軽鎖（またはその断片）をコードする1つまたは複数の核酸分子のことをいい、1つのベクターまたは別々のベクターに乗っている核酸分子、および、宿主細胞中の1つまたは複数の位置に存在している核酸分子を含む。 “Isolated nucleic acid encoding an anti-TGF-beta3 antibody” refers to one or more nucleic acid molecules encoding the heavy and light chains (or fragments thereof) of an antibody, either a single vector or separate Nucleic acid molecules carried on vectors and nucleic acid molecules present at one or more locations in the host cell.
　本明細書でいう用語「モノクローナル抗体」は、実質的に均一な抗体の集団から得られる抗体のことをいう。すなわち、その集団を構成する個々の抗体は、生じ得る変異抗体（例えば、自然に生じる変異を含む変異抗体、またはモノクローナル抗体調製物の製造中に発生する変異抗体。そのような変異体は通常若干量存在している。）を除いて、同一でありおよび／または同じエピトープに結合する。異なる決定基（エピトープ）に対する異なる抗体を典型的に含むポリクローナル抗体調製物とは対照的に、モノクローナル抗体調製物の各モノクローナル抗体は、抗原上の単一の決定基に対するものである。したがって、修飾語「モノクローナル」は、実質的に均一な抗体の集団から得られるものである、という抗体の特徴を示し、何らかの特定の方法による抗体の製造を求めるものと解釈されるべきではない。例えば、本発明にしたがって用いられるモノクローナル抗体は、これらに限定されるものではないが、ハイブリドーマ法、組換えDNA法、ファージディスプレイ法、ヒト免疫グロブリン遺伝子座の全部または一部を含んだトランスジェニック動物を利用する方法を含む、様々な手法によって作成されてよく、モノクローナル抗体を作製するためのそのような方法および他の例示的な方法は、本明細書に記載されている。 As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies. That is, the individual antibodies that make up the population are mutated antibodies that can occur (eg, mutated antibodies that contain naturally occurring mutations, or mutated antibodies that occur during the production of monoclonal antibody preparations. Are present in the same amount and / or bind to the same epitope. In contrast to polyclonal antibody preparations that typically include different antibodies to different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is against a single determinant on the antigen. Thus, the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention include, but are not limited to, hybridoma methods, recombinant DNA methods, phage display methods, transgenic animals containing all or part of a human immunoglobulin locus. Such methods and other exemplary methods for making monoclonal antibodies can be made by a variety of techniques, including methods utilizing the methods described herein.
　「天然型抗体」は、天然に生じる様々な構造を伴う免疫グロブリン分子のことをいう。例えば、天然型IgG抗体は、ジスルフィド結合している2つの同一の軽鎖と2つの同一の重鎖から構成される約150,000ダルトンのヘテロ四量体糖タンパク質である。N末端からC末端に向かって、各重鎖は、可変重鎖ドメインまたは重鎖可変ドメインとも呼ばれる可変領域 (VH) を有し、それに3つの定常ドメイン（CH1、CH2、およびCH3）が続く。同様に、N末端からC末端に向かって、各軽鎖は、可変軽鎖ドメインまたは軽鎖可変ドメインとも呼ばれる可変領域 (VL) を有し、それに定常軽鎖 (CL) ドメインが続く。抗体の軽鎖は、その定常ドメインのアミノ酸配列に基づいて、カッパ（κ）およびラムダ（λ）と呼ばれる、2つのタイプの1つに帰属させられてよい。 “Natural antibodies” refer to immunoglobulin molecules with various naturally occurring structures. For example, a native IgG antibody is a heterotetrameric glycoprotein of approximately 150,000 daltons composed of two identical light chains and two identical heavy chains that are disulfide bonded. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH) 呼 ば, also called variable heavy chain domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL) 呼 ば, also called a variable light chain domain or light chain variable domain, followed by a constant light chain (CL) domain. The light chain of an antibody may be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.
　用語「添付文書」は、治療用品の商用パッケージに通常含まれ、そのような治療用品の使用に関する、適応症、用法、用量、投与方法、併用療法、禁忌、および／または警告についての情報を含む使用説明書のことをいうために用いられる。 The term “package insert” is usually included in commercial packages of therapeutic products and includes information about indications, usage, dosages, administration methods, combination therapies, contraindications, and / or warnings regarding the use of such therapeutic products. Used to refer to instructions for use.
　参照ポリペプチド配列に対する「パーセント (％) アミノ酸配列同一性」は、最大のパーセント配列同一性を得るように配列を整列させてかつ必要ならギャップを導入した後の、かつ、いかなる保存的置換も配列同一性の一部と考えないとしたときの、参照ポリペプチド配列中のアミノ酸残基と同一である候補配列中のアミノ酸残基の、百分率比として定義される。パーセントアミノ酸配列同一性を決める目的のアラインメントは、当該技術分野における技術の範囲内にある種々の方法、例えば、BLAST、BLAST-2、ALIGN、Megalign (DNASTAR) ソフトウェア、またはGENETYX（登録商標）（株式会社ゼネティックス）などの、公に入手可能なコンピュータソフトウェアを使用することにより達成することができる。当業者は、比較される配列の全長にわたって最大のアラインメントを達成するために必要な任意のアルゴリズムを含む、配列のアラインメントをとるための適切なパラメーターを決定することができる。 “Percent (%) amino acid sequence identity” relative to a reference polypeptide sequence is any conservative substitution after aligning the sequences and introducing gaps, if necessary, to obtain maximum percent sequence identity. Defined as the percentage ratio of amino acid residues in a candidate sequence that are identical to amino acid residues in a reference polypeptide sequence, when not considered part of the identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved by various methods within the skill in the art, such as BLAST, BLAST-2, ALIGN, Megalign® (DNASTAR) ® software, or GENETYX® (stocks). This can be achieved by using publicly available computer software such as Company Genetics. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
　ALIGN-2配列比較コンピュータプログラムは、ジェネンテック社の著作であり、そのソースコードは米国著作権庁 (U.S. Copyright Office, Wasington D.C., 20559) に使用者用書類とともに提出され、米国著作権登録番号TXU510087として登録されている。ALIGN-2プログラムは、ジェネンテック社 (Genentech, Inc., South San Francisco, California) から公に入手可能であるし、ソースコードからコンパイルしてもよい。ALIGN-2プログラムは、Digital UNIX V4.0Dを含むUNIXオペレーティングシステム上での使用のためにコンパイルされる。すべての配列比較パラメーターは、ALIGN-2プログラムによって設定され、変動しない。
　アミノ酸配列比較にALIGN-2が用いられる状況では、所与のアミノ酸配列Aの、所与のアミノ酸配列Bへの、またはそれとの、またはそれに対する％アミノ酸配列同一性（あるいは、所与のアミノ酸配列Bへの、またはそれとの、またはそれに対する、ある％アミノ酸配列同一性を有するまたは含む所与のアミノ酸配列A、ということもできる）は、次のように計算される：分率X／Yの100倍。ここで、Xは配列アラインメントプログラムALIGN-2によって、当該プログラムのAおよびBのアラインメントにおいて同一である一致としてスコアされたアミノ酸残基の数であり、YはB中のアミノ酸残基の全数である。アミノ酸配列Aの長さがアミノ酸配列Bの長さと等しくない場合、AのBへの％アミノ酸配列同一性は、BのAへの％アミノ酸配列同一性と等しくないことが、理解されるであろう。別段特に明示しない限り、本明細書で用いられるすべての％アミノ酸配列同一性値は、直前の段落で述べたとおりALIGN-2コンピュータプログラムを用いて得られるものである。 The ALIGN-2 sequence comparison computer program is a work of Genentech, whose source code was submitted to the US Copyright Office (US Copyright Office, Wasington DC, 20559) along with user documentation, and was registered under US copyright registration number TXU510087. It is registered. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, and may be compiled from source code. The ALIGN-2 program is compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.
In situations where ALIGN-2 is used for amino acid sequence comparison, the% amino acid sequence identity of a given amino acid sequence A to, or with, or against a given amino acid sequence B (or a given amino acid sequence) A given amino acid sequence A, which has or contains some% amino acid sequence identity to, with or to B) is calculated as follows: fraction X / Y Hundredfold. Where X is the number of amino acid residues scored by the sequence alignment program ALIGN-2 as identical matches in the A and B alignments of the program, and Y is the total number of amino acid residues in B . It will be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, then the% amino acid sequence identity of A to B is not equal to the% amino acid sequence identity of B to A. Let's go. Unless stated otherwise, all% amino acid sequence identity values used herein are those obtained using the ALIGN-2 computer program as described in the immediately preceding paragraph.
　用語「薬学的製剤」は、その中に含まれた有効成分の生物学的活性が効果を発揮し得るような形態にある調製物であって、かつ製剤が投与される被験体に許容できない程度に毒性のある追加の要素を含んでいない調製物のことをいう。 The term “pharmaceutical formulation” refers to a preparation that is in a form such that the biological activity of the active ingredient contained therein can be effective and is unacceptable to the subject to which the formulation is administered. Refers to a preparation that does not contain any additional toxic elements.
　「薬学的に許容される担体」は、被験体に対して無毒な、薬学的製剤中の有効成分以外の成分のことをいう。薬学的に許容される担体は、これらに限定されるものではないが、緩衝液、賦形剤、安定化剤、または保存剤を含む。 “Pharmaceutically acceptable carrier” refers to an ingredient other than the active ingredient in a pharmaceutical preparation that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.
　本明細書でいう用語「TGF-beta」は、別段示さない限り、霊長類（例えば、ヒト）およびげっ歯類（例えば、マウスおよびラット）などの哺乳動物を含む、任意の脊椎動物供給源からの任意の天然型TGF-betaのことをいう。この用語は、「全長」のプロセシングを受けていないTGF-betaも、細胞中でのプロセシングの結果生じるいかなる形態のTGF-betaも包含する。この用語はまた、自然に生じるTGF-betaの変異体、例えば、スプライス変異体や対立遺伝子変異体も包含する。 The term “TGF-beta” as used herein is from any vertebrate source, including mammals such as primates (eg, humans) and rodents (eg, mice and rats), unless otherwise indicated. Any natural type of TGF-beta. The term encompasses TGF-beta that has not been "full length" processed, as well as any form of TGF-beta that results from processing in the cell. The term also encompasses naturally occurring variants of TGF-beta, such as splice variants and allelic variants.
　本明細書で用いられる「治療」（および、その文法上の派生語、例えば「治療する」、「治療すること」など）は、治療される個体の自然経過を改変することを企図した臨床的介入を意味し、予防のためにも、臨床的病態の経過の間にも実施され得る。治療の望ましい効果は、これらに限定されるものではないが、疾患の発生または再発の防止、症状の軽減、疾患による任意の直接的または間接的な病理的影響の減弱、転移の防止、疾患の進行速度の低減、疾患状態の回復または緩和、および寛解または改善された予後を含む。いくつかの態様において、本発明の抗体は、疾患の発症を遅らせる、または疾患の進行を遅くするために用いられる。 As used herein, “treatment” (and grammatical derivatives thereof, eg, “treat”, “treating”, etc.) is clinical that is intended to alter the natural course of the individual being treated. Means intervention and can be carried out either for prevention or during the course of clinical pathology. Desirable effects of treatment include but are not limited to prevention of disease occurrence or recurrence, reduction of symptoms, attenuation of any direct or indirect pathological effects of the disease, prevention of metastasis, disease Includes reduced rate of progression, recovery or alleviation of disease state, and remission or improved prognosis. In some embodiments, the antibodies of the invention are used to delay the onset of disease or slow the progression of disease.
自己免疫疾患
　「自己免疫疾患」は、その個体自身の組織から生じかつその個体自身の組織に対して向けられる非悪性疾患または障害のことをいう。本明細書で、自己免疫疾患は、悪性またはがん性の疾患または状態を明確に除外するものであり、特にB細胞リンパ腫、急性リンパ芽球性白血病 (acute lymphoblastic leukemia: ALL)、慢性リンパ球性白血病 (chronic lymphocytic leukemia: CLL)、ヘアリー細胞白血病、および慢性骨髄芽球性白血病を除外する。自己免疫疾患または障害の例は、これらに限定されるものではないが、以下のものを含む：乾癬および皮膚炎（例えば、アトピー性皮膚炎）を含む炎症性皮膚疾患などの炎症性反応；全身性強皮症および硬化症；炎症性腸疾患に関連する反応（例えば、クローン病および潰瘍性大腸炎）；呼吸窮迫症候群（成人呼吸窮迫症候群；adult respiratory distress syndrome: ARDSを含む）；皮膚炎；髄膜炎；脳炎；ブドウ膜炎；大腸炎；糸球体腎炎；例えば湿疹および喘息ならびにT細胞の浸潤および慢性炎症反応を伴う他の状態などのアレルギー性状態；アテローム硬化；白血球接着不全症；関節リウマチ；全身性エリテマトーデス (SLE) （ループス腎炎、皮膚ループスを含むがこれらに限定されない）；糖尿病（例えば、I型糖尿病またはインスリン依存性糖尿病）；多発性硬化症；レイノー症候群；自己免疫性甲状腺炎；橋本甲状腺炎；アレルギー性脳脊髄炎；シェーグレン症候群；若年発症糖尿病；ならびに典型的に結核、サルコイドーシス、多発性筋炎、肉芽腫症、および血管炎において見られるサイトカインおよびTリンパ球によって媒介される急性および遅延型過敏症に関連する免疫反応；悪性貧血（アジソン病）；白血球の漏出を伴う疾患；中枢神経系 (central nervous system: CNS) 炎症性障害；多臓器損傷症候群；溶血性貧血（クリオグロブリン血症またはクームス陽性貧血を含むがこれらに限定されない）；重症筋無力症；抗原‐抗体複合体介在性疾患；抗糸球体基底膜疾患；抗リン脂質症候群；アレルギー性神経炎；バセドウ病；ランバート‐イートン筋無力症候群；水疱性類天疱瘡；天疱瘡；自己免疫性多腺性内分泌障害；ライター病；スティフマン症候群；ベーチェット病；巨細胞性動脈炎；免疫複合体性腎炎；IgA腎症；IgM多発性ニューロパシー；免疫性血小板減少性紫斑病（immune thrombocytopenic purpura: ITP）または自己免疫性血小板減少症。 Autoimmune disease An “autoimmune disease” refers to a non-malignant disease or disorder that arises from and is directed to the individual's own tissue. As used herein, an autoimmune disease is one that specifically excludes a malignant or cancerous disease or condition, particularly B-cell lymphoma, acute lymphoblastic leukemia (ALL), chronic lymphocytes Excludes chronic lymphocytic leukemia (CLL), hairy cell leukemia, and chronic myeloblastic leukemia. Examples of autoimmune diseases or disorders include, but are not limited to: inflammatory reactions such as inflammatory skin diseases including psoriasis and dermatitis (eg, atopic dermatitis); Scleroderma and sclerosis; reactions associated with inflammatory bowel disease (eg, Crohn's disease and ulcerative colitis); respiratory distress syndrome (including adult respiratory distress syndrome; including ARDS); dermatitis; Meningitis; encephalitis; uveitis; colitis; glomerulonephritis; allergic conditions such as eczema and asthma and other conditions with T cell infiltration and chronic inflammatory response; atherosclerosis; leukocyte adhesion failure; Rheumatism; systemic lupus erythematosus (SLE) (including but not limited to lupus nephritis, cutaneous lupus); diabetes (eg, type I diabetes or insulin-dependent diabetes) ); Multiple sclerosis; Raynaud's syndrome; autoimmune thyroiditis; Hashimoto's thyroiditis; allergic encephalomyelitis; Sjogren's syndrome; juvenile-onset diabetes; and typically tuberculosis, sarcoidosis, multiple myositis, granulomatosis, and Immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T lymphocytes found in vasculitis; pernicious anemia (Addison's disease); disease with leukocyte leakage; central nervous system (CNS) Inflammatory disorders; multiple organ injury syndrome; hemolytic anemia (including but not limited to cryoglobulinemia or Coombs-positive anemia); myasthenia gravis; antigen-antibody complex-mediated disease; antiglomerular basement membrane disease Antiphospholipid syndrome; allergic neuritis; Graves'disease; Lambert-Eaton myasthenia syndrome; bullous pemphigoid; Autoimmune multi-endocrine disorders; Reiter's disease; Stiffman syndrome; Behcet's disease; Giant cell arteritis; Immune complex nephritis; IgA nephropathy; IgM polyneuropathy; Immune thrombocytopenic purpura : ITP) or autoimmune thrombocytopenia.
　用語「可変領域」または「可変ドメイン」は、抗体を抗原へと結合させることに関与する、抗体の重鎖または軽鎖のドメインのことをいう。天然型抗体の重鎖および軽鎖の可変ドメイン（それぞれVHおよびVL）は、通常、各ドメインが4つの保存されたフレームワーク領域 (FR) および3つの超可変領域 (HVR) を含む、類似の構造を有する。（例えば、Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007) 参照。）1つのVHまたはVLドメインで、抗原結合特異性を与えるに充分であろう。さらに、ある特定の抗原に結合する抗体は、当該抗原に結合する抗体からのVHまたはVLドメインを使ってそれぞれVLまたはVHドメインの相補的ライブラリをスクリーニングして、単離されてもよい。例えばPortolano et al., J. Immunol. 150:880-887 (1993)； Clarkson et al., Nature 352:624-628 (1991) 参照。 The term “variable region” or “variable domain” refers to the heavy or light chain domain of an antibody involved in binding the antibody to an antigen. Natural antibody heavy and light chain variable domains (VH and VL, respectively) are typically similar, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR). It has a structure. (See, for example, Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007)). One VH or VL domain will be sufficient to confer antigen binding specificity. In addition, antibodies that bind to a particular antigen may be isolated by screening complementary libraries of VL or VH domains, respectively, using VH or VL domains from antibodies that bind to the antigen. For example, see Portolano et al., J. Immunol. 150: 880-887 (1993); Clarkson et al., Nature 352: 624-628 (1991).
　本明細書で用いられる用語「ベクター」は、それが連結されたもう1つの核酸を増やすことができる、核酸分子のことをいう。この用語は、自己複製核酸構造としてのベクター、および、それが導入された宿主細胞のゲノム中に組み入れられるベクターを含む。あるベクターは、自身が動作的に連結された核酸の、発現をもたらすことができる。そのようなベクターは、本明細書では「発現ベクター」とも称される。 As used herein, the term “vector” refers to a nucleic acid molecule that can multiply another nucleic acid to which it has been linked. The term includes vectors as self-replicating nucleic acid structures and vectors that are integrated into the genome of the host cell into which it has been introduced. Certain vectors can provide for the expression of nucleic acids to which they are operably linked. Such vectors are also referred to herein as “expression vectors”.
ＩＩ．抗TGF-beta3抗体およびその使用
　本発明者らは、TGF-beta3によるB細胞活性化の抑制の作用に着目し、当該作用を活用する自己免疫疾患の治療剤を開発するために鋭意研究を行った。その結果、TGF-beta3を認識する抗体のうち、所定の機能（プロファイル）を有するものが、B細胞活性化を抑制し、ひいては自己免疫疾患の治療剤として有用であることを見出した。 II. Anti-TGF-beta3 antibody and use thereof The inventors of the present invention focused on the effect of inhibiting B cell activation by TGF-beta3, and conducted extensive research to develop a therapeutic agent for autoimmune diseases that utilizes this effect. It was. As a result, it was found that antibodies having a predetermined function (profile) that recognize TGF-beta3 suppress B cell activation and are useful as a therapeutic agent for autoimmune diseases.
　具体的には、本発明の抗TGF-beta3抗体は、以下のプロファイルを有する：
（１）TGF-beta3に対し非中和である；
（２）TGF-beta1, 2, 3の中でTGF-beta3に特異的に結合する；
（３）（任意）Mature体だけでなく、Latent体のTGF-beta3にも結合する。 Specifically, the anti-TGF-beta3 antibody of the present invention has the following profile:
(1) Non-neutralizing for TGF-beta3;
(2) It binds specifically to TGF-beta3 among TGF-beta1, 2, 3;
(3) (Optional) Binds not only to the Mature body but also to the Latent body of TGF-beta3.
　本明細書において、TGF-beta3に対し「非中和（Non-Neutralizing）」であるとは、実施例４に記載の評価手法により、抗体濃度400ng/mLの条件下でTGF-beta3の生理活性の抑制効率が20％以下であることを指し、より好ましくは抗体濃度2000ng/mLの条件下でTGF-beta3の生理活性の抑制効率25％以下、より好ましくは抗体濃度2000ng/mLの条件下でTGF-beta3の生理活性の抑制効率が20％以下であることを指す。
　別の観点から見ると、非中和抗体とは、TGF-beta3のTGF-beta受容体への結合を阻害しないように結合する抗体と定義することもできる。したがって、「TGF-beta3に結合する非中和抗体」は、「TGF-beta3がTGF-beta受容体に結合し得る態様でTGF-beta3に結合する抗体」と表すこともできる。
　また別の観点から見ると、非中和抗体は、TGF-beta3のTGF-beta受容体への結合によりTGF-beta受容体を発現する細胞に与える生物学的作用を阻害しないように、TGF-beta3に結合する抗体と定義することもできる。したがって、「TGF-beta3に結合する非中和抗体」は、「TGF-beta3がTGF-beta受容体を発現する細胞を活性化し得る態様でTGF-beta3に結合する抗体」と表すこともできる。
　また別の観点から見ると、非中和抗体は、TGF-beta受容体を発現する細胞の活性化によりSmadシグナルが入る生理活性を阻害しないように、TGF-beta3に結合する抗体と定義することもできる。したがって、「TGF-beta3に結合する非中和抗体」は、「TGF-beta3がSmadシグナルを出し得る態様でTGF-beta3に結合する抗体」と表すこともできる。
　また別の観点から見ると、非中和抗体は、Smadシグナルが入ることによりB細胞活性化が抑制される作用を阻害しないように、TGF-beta3に結合する抗体と定義することもできる。したがって、「TGF-beta3に結合する非中和抗体」は、「TGF-beta3によりB細胞の活性化を抑制し得る態様でTGF-beta3に結合する抗体」と表すこともできる。 In the present specification, “non-neutralizing” with respect to TGF-beta3 means that the physiological activity of TGF-beta3 under the condition of an antibody concentration of 400 ng / mL by the evaluation method described in Example 4. The suppression efficiency of TGF-beta3 is more preferably 25% or less under the antibody concentration of 2000 ng / mL, more preferably under the condition of the antibody concentration of 2000 ng / mL. It means that the suppression efficiency of the physiological activity of TGF-beta3 is 20% or less.
From another point of view, a non-neutralizing antibody can also be defined as an antibody that binds so as not to inhibit the binding of TGF-beta3 to the TGF-beta receptor. Therefore, “a non-neutralizing antibody that binds to TGF-beta3” can also be expressed as “an antibody that binds to TGF-beta3 in such a manner that TGF-beta3 can bind to the TGF-beta receptor”.
From another point of view, non-neutralizing antibodies do not inhibit TGF-beta3 binding to the TGF-beta receptor so that it does not inhibit the biological effects on cells expressing the TGF-beta receptor. It can also be defined as an antibody that binds to beta3. Therefore, “a non-neutralizing antibody that binds to TGF-beta3” can also be expressed as “an antibody that binds to TGF-beta3 in such a manner that TGF-beta3 can activate cells expressing the TGF-beta receptor”.
From another point of view, a non-neutralizing antibody should be defined as an antibody that binds to TGF-beta3 so that the activation of cells expressing TGF-beta receptor does not inhibit the physiological activity of the Smad signal. You can also. Therefore, “a non-neutralizing antibody that binds to TGF-beta3” can also be expressed as “an antibody that binds to TGF-beta3 in such a manner that TGF-beta3 can produce a Smad signal”.
From another point of view, a non-neutralizing antibody can also be defined as an antibody that binds to TGF-beta3 so as not to inhibit the action of suppressing B cell activation by the entry of a Smad signal. Therefore, “a non-neutralizing antibody that binds to TGF-beta3” can also be expressed as “an antibody that binds to TGF-beta3 in such a manner that TGF-beta3 can suppress the activation of B cells”.
　本明細書において、「TGF-beta3に特異的（TGFb3-specific）に結合する」とは、TGF-beta1, 2よりもTGF-beta3への結合活性が強いことを意味し、具体的には、実施例５に記載の評価手法により、TGF-beta3への結合に対するTGF-beta1およびTGF-beta2への結合値が１０％未満、好ましくは１％未満、より好ましくは０．１％未満であることを指す。 In the present specification, “specifically binds to TGF-beta3 (TGFb3-specific)” means that the binding activity to TGF-beta3 is stronger than TGF-beta1, 2, specifically, According to the evaluation method described in Example 5, the binding value to TGF-beta1 and TGF-beta2 for binding to TGF-beta3 is less than 10%, preferably less than 1%, more preferably less than 0.1%. Point to.
　特定の理論に拘束されるものではないが、本発明の抗TGF-beta3抗体はTGF-beta3への中和活性を有さないため、本発明の抗体がTGF-beta3に結合することによってTGF-beta3の半減期が長くなり、ひいてはTGF-beta3の血中濃度が高められ、結果としてTGF-beta3によるB細胞活性化の抑制効果が増強されることが期待される。
　また、本発明の抗TGF-beta3抗体はTGF-beta3に特異的な結合活性を有するため、TGF-beta1, 2にも結合する抗体とは異なり、TGF-beta3へ結合することによる効果が薄れることがなく、TGF-beta1への結合により線維化が起こり肺腺癌を引き起こす等の副作用の恐れがない。
　さらに、本発明の抗TGF-beta3抗体は好ましくは、Mature体だけでなくLatent体のTGF-beta3にも結合することができる。TGF-beta3はMature体よりもLatent体の方が血中濃度は高いため、Latent体のTGF-beta3への結合活性を有する本発明の抗体は、より高いB細胞活性化の抑制効果を発揮することができる。 Without being bound by any particular theory, since the anti-TGF-beta3 antibody of the present invention does not have a neutralizing activity to TGF-beta3, the antibody of the present invention binds to TGF-beta3 to thereby bind TGF-beta3. It is expected that the half-life of beta3 will be prolonged, and consequently the blood concentration of TGF-beta3 will be increased, and as a result, the inhibitory effect of BGF activation by TGF-beta3 will be enhanced.
In addition, since the anti-TGF-beta3 antibody of the present invention has a binding activity specific to TGF-beta3, unlike the antibody that also binds to TGF-beta1, 2, the effect of binding to TGF-beta3 is diminished. There is no fear of side effects such as fibrosis due to binding to TGF-beta1 and causing lung adenocarcinoma.
Furthermore, the anti-TGF-beta3 antibody of the present invention can preferably bind not only to the mature body but also to the Latent body of TGF-beta3. Since TGF-beta3 has a higher blood concentration in the Latent body than in the Mature body, the antibody of the present invention, which has the binding activity of the Latent body to TGF-beta3, exhibits a higher inhibitory effect on B cell activation. be able to.
　本発明の特定の態様において、本発明の抗体は、一方の可変領域でTGF-beta3に結合し、他方の可変領域でB細胞表面マーカーに結合することができるBispecific抗体（二重特異性抗体）であり、当該二種の抗原に結合することにより、TGF-beta3を効率的に目的とするB細胞へ届けることができる。しかし、Bispecific抗体ではなく、通常抗体であっても、TGF-beta3への中和活性を有さない抗体であれば、結合によりTGF-beta3の半減期が長くなり血中濃度を高めることができるため、B細胞活性化の抑制効果の増強が期待される。したがって、本発明の抗体は前記の所定のプロファイルを満たすものであれば足り、特にBispecific抗体等に限定されない。 In a particular embodiment of the invention, the antibody of the invention is a Bispecific antibody (bispecific antibody) capable of binding to TGF-beta3 in one variable region and binding to a B cell surface marker in the other variable region. By binding to the two types of antigens, TGF-beta3 can be efficiently delivered to the target B cell. However, if the antibody is not a bispecific antibody but a normal antibody but does not have neutralizing activity to TGF-beta3, the half-life of TGF-beta3 can be increased by binding and the blood concentration can be increased. Therefore, enhancement of the inhibitory effect on B cell activation is expected. Therefore, the antibody of the present invention is sufficient as long as it satisfies the predetermined profile, and is not particularly limited to a Bispecific antibody.
　本発明は、抗TGF-beta3抗体を含むB細胞活性化の抑制剤を提供する。B細胞は好ましくは自己反応性B細胞であり、B細胞活性化の抑制としては、例えばB細胞による抗体産生の抑制があげられる。また抑制剤は自己免疫疾患の治療剤の用途で用いることができる。 The present invention provides an inhibitor of B cell activation comprising an anti-TGF-beta3 antibody. The B cell is preferably a self-reactive B cell, and the suppression of B cell activation includes, for example, suppression of antibody production by the B cell. In addition, the suppressor can be used for a therapeutic agent for autoimmune diseases.
　TGF-beta3（TGFβ3/TGFb3）は、配列番号１（latent体）～２（mature体）にあげるアミノ酸配列を有するものを含み、またこれらアミノ酸配列の一部を改変、修飾し同様のB細胞活性化の抑制作用を有するものを含む。自己免疫疾患としてはSLE、天疱瘡(Pemphigus)、多発性硬化症(Multiple Sclerosis)、視神経脊髄炎（NMO）、ANCA関連血管炎、関節リウマチ、移植臓器拒絶、シェーグレン症候群(Sjogren's syndrome)、若年性皮膚筋炎(Juvenile dermatomyositis)、重症筋無力症(Myasthenia gravis)、又はバセドウ病、橋本病等の自己免疫性甲状腺疾患(Autoimmune thyroid disease including Graves' disease or Hashimoto's thyroiditis)等が挙げられる。 TGF-beta3 (TGFβ3 / TGFb3) includes those having the amino acid sequences listed in SEQ ID NOs: 1 (latent body) to 2 (mature body), and similar B cell activity by altering and modifying part of these amino acid sequences Including those having an inhibitory effect on oxidization. Autoimmune diseases include SLE, Pemphigus, Multiple Sclerosis, Optic myelitis (NMO), ANCA-related vasculitis, Rheumatoid arthritis, Transplant rejection, Sjogren's syndrome (Sjogren's syndrome), Juvenile Examples include dermatomyositis (Juvenile dermatomyositis), myasthenia gravis, or autoimmune thyroid disease including Graves' disease or Hashimoto's thyroiditis and the like.
　別の観点においては、本発明は、TGF-beta3に結合する抗体又は抗体断片を含む自己免疫疾患の治療剤を提供する。抗体断片は可変領域又はFc領域等の抗体の構成要素が分離されたものを意味する。 In another aspect, the present invention provides a therapeutic agent for an autoimmune disease comprising an antibody or antibody fragment that binds to TGF-beta3. An antibody fragment means one in which antibody components such as a variable region or Fc region are separated.
　前記抗体又は抗体断片はB細胞を認識する可変領域を含む構成をとることもできる。B細胞を認識は、例えばCD19、CD20、CD40、CD22、IL21R、BAFF-R、BCMA、TACI、CD27、又はCD138に含まれるいずれか1つまたは複数をマーカーとして行うことができる。 The antibody or antibody fragment may have a variable region recognizing B cell. Recognition of B cells can be performed using, for example, any one or more of CD19, CD20, CD40, CD22, IL21R, BAFF-R, BCMA, TACI, CD27, or CD138 as a marker.
　別の観点においては、本発明は、TGF-beta3を認識する第一の可変領域と、B細胞を認識する第二の可変領域とを含む多重特異性抗体を含む自己免疫疾患の治療剤を提供する。 In another aspect, the present invention provides a therapeutic agent for autoimmune diseases comprising a multispecific antibody comprising a first variable region recognizing TGF-beta3 and a second variable region recognizing B cells. To do.
　別の観点においては、本発明は、前記治療剤を含む医療用キットを提供する。 In another aspect, the present invention provides a medical kit containing the therapeutic agent.
　なお、本明細書において引用された全ての先行技術文献は、参照として本明細書に組み入れられる。 Note that all prior art documents cited in this specification are incorporated herein by reference.
Ａ．例示的抗TGF-beta3抗体
　一局面において、本発明はTGF-beta3に結合する、単離された抗体を提供する。特定の態様において、抗TGF-beta3抗体は、TGF-beta1, 2, 3の中でTGF-beta3に特異的に結合し、TGF-beta3に対し非中和である抗体である。特定の態様において、抗TGF-beta3抗体は、TGF-beta3のMature体およびLatent体に結合する。 A. Exemplary Anti-TGF-beta3 Antibodies In one aspect, the present invention provides isolated antibodies that bind to TGF-beta3. In a particular embodiment, the anti-TGF-beta3 antibody is an antibody that specifically binds to TGF-beta3 among TGF-beta1, 2, 3 and is non-neutralizing to TGF-beta3. In certain embodiments, the anti-TGF-beta3 antibody binds to the mature and latent bodies of TGF-beta3.
　別の局面において、本発明は、以下（１）から（２９）のいずれかに記載の、TGF-beta3に特異的に結合する非中和抗体を提供する：
（１）配列番号：１０１に記載のCDR１、配列番号：１０２に記載のCDR２、配列番号：１０３に記載のCDR３を有する重鎖可変領域、および配列番号：１０５に記載のCDR１、配列番号：１０６に記載のCDR２、配列番号：１０７に記載のCDR３を有する軽鎖可変領域を含む抗体
（２）配列番号：１４１に記載のCDR１、配列番号：１４２に記載のCDR２、配列番号：１４３に記載のCDR３を有する重鎖可変領域、および配列番号：１４５に記載のCDR１、配列番号：１４６に記載のCDR２、配列番号：１４７に記載のCDR３を有する軽鎖可変領域を含む抗体
（３）配列番号：１６１に記載のCDR１、配列番号：１６２に記載のCDR２、配列番号：１６３に記載のCDR３を有する重鎖可変領域、および配列番号：１６５に記載のCDR１、配列番号：１６６に記載のCDR２、配列番号：１６７に記載のCDR３を有する軽鎖可変領域を含む抗体
（４）配列番号：１７１に記載のCDR１、配列番号：１７２に記載のCDR２、配列番号：１７３に記載のCDR３を有する重鎖可変領域、および配列番号：１７５に記載のCDR１、配列番号：１７６に記載のCDR２、配列番号：１７７に記載のCDR３を有する軽鎖可変領域を含む抗体
（５）配列番号：１８１に記載のCDR１、配列番号：１８２に記載のCDR２、配列番号：１８３に記載のCDR３を有する重鎖可変領域、および配列番号：１８５に記載のCDR１、配列番号：１８６に記載のCDR２、配列番号：１８７に記載のCDR３を有する軽鎖可変領域を含む抗体
（６）配列番号：１９１に記載のCDR１、配列番号：１９２に記載のCDR２、配列番号：１９３に記載のCDR３を有する重鎖可変領域、および配列番号：１９５に記載のCDR１、配列番号：１９６に記載のCDR２、配列番号：１９７に記載のCDR３を有する軽鎖可変領域を含む抗体
（７）配列番号：２０１に記載のCDR１、配列番号：２０２に記載のCDR２、配列番号：２０３に記載のCDR３を有する重鎖可変領域、および配列番号：２０５に記載のCDR１、配列番号：２０６に記載のCDR２、配列番号：２０７に記載のCDR３を有する軽鎖可変領域を含む抗体
（８）配列番号：２１１に記載のCDR１、配列番号：２１２に記載のCDR２、配列番号：２１３に記載のCDR３を有する重鎖可変領域、および配列番号：２１５に記載のCDR１、配列番号：２１６に記載のCDR２、配列番号：２１７に記載のCDR３を有する軽鎖可変領域を含む抗体
（９）配列番号：２４１に記載のCDR１、配列番号：２４２に記載のCDR２、配列番号：２４３に記載のCDR３を有する重鎖可変領域、および配列番号：２４５に記載のCDR１、配列番号：２４６に記載のCDR２、配列番号：２４７に記載のCDR３を有する軽鎖可変領域を含む抗体
（１０）配列番号：２６１に記載のCDR１、配列番号：２６２に記載のCDR２、配列番号：２６３に記載のCDR３を有する重鎖可変領域、および配列番号：２６５に記載のCDR１、配列番号：２６６に記載のCDR２、配列番号：２６７に記載のCDR３を有する軽鎖可変領域を含む抗体
（１１）配列番号：２９１に記載のCDR１、配列番号：２９２に記載のCDR２、配列番号：２９３に記載のCDR３を有する重鎖可変領域、および配列番号：２９５に記載のCDR１、配列番号：２９６に記載のCDR２、配列番号：２９７に記載のCDR３を有する軽鎖可変領域を含む抗体
（１２）配列番号：３０１に記載のCDR１、配列番号：３０２に記載のCDR２、配列番号：３０３に記載のCDR３を有する重鎖可変領域、および配列番号：３０５に記載のCDR１、配列番号：３０６に記載のCDR２、配列番号：３０７に記載のCDR３を有する軽鎖可変領域を含む抗体
（１３）配列番号：３６１に記載のCDR１、配列番号：３６２に記載のCDR２、配列番号：３６３に記載のCDR３を有する重鎖可変領域、および配列番号：３６５に記載のCDR１、配列番号：３６６に記載のCDR２、配列番号：３６７に記載のCDR３を有する軽鎖可変領域を含む抗体
（１４）配列番号：３７１に記載のCDR１、配列番号：３７２に記載のCDR２、配列番号：３７３に記載のCDR３を有する重鎖可変領域、および配列番号：３７５に記載のCDR１、配列番号：３７６に記載のCDR２、配列番号：３７７に記載のCDR３を有する軽鎖可変領域を含む抗体
（１５）配列番号：３９１に記載のCDR１、配列番号：３９２に記載のCDR２、配列番号：３９３に記載のCDR３を有する重鎖可変領域、および配列番号：３９５に記載のCDR１、配列番号：３９６に記載のCDR２、配列番号：３９７に記載のCDR３を有する軽鎖可変領域を含む抗体
（１６）配列番号：４０１に記載のCDR１、配列番号：４０２に記載のCDR２、配列番号：４０３に記載のCDR３を有する重鎖可変領域、および配列番号：４０５に記載のCDR１、配列番号：４０６に記載のCDR２、配列番号：４０７に記載のCDR３を有する軽鎖可変領域を含む抗体
（１７）配列番号：４８１に記載のCDR１、配列番号：４８２に記載のCDR２、配列番号：４８３に記載のCDR３を有する重鎖可変領域、および配列番号：４８５に記載のCDR１、配列番号：４８６に記載のCDR２、配列番号：４８７に記載のCDR３を有する軽鎖可変領域を含む抗体
（１８）配列番号：４９１に記載のCDR１、配列番号：４９２に記載のCDR２、配列番号：４９３に記載のCDR３を有する重鎖可変領域、および配列番号：４９５に記載のCDR１、配列番号：４９６に記載のCDR２、配列番号：４９７に記載のCDR３を有する軽鎖可変領域を含む抗体
（１９）配列番号：５１１に記載のCDR１、配列番号：５１２に記載のCDR２、配列番号：５１３に記載のCDR３を有する重鎖可変領域、および配列番号：５１５に記載のCDR１、配列番号：５１６に記載のCDR２、配列番号：５１７に記載のCDR３を有する軽鎖可変領域を含む抗体
（２０）配列番号：５２１に記載のCDR１、配列番号：５２２に記載のCDR２、配列番号：５２３に記載のCDR３を有する重鎖可変領域、および配列番号：５２５に記載のCDR１、配列番号：５２６に記載のCDR２、配列番号：５２７に記載のCDR３を有する軽鎖可変領域を含む抗体
（２１）配列番号：５３１に記載のCDR１、配列番号：５３２に記載のCDR２、配列番号：５３３に記載のCDR３を有する重鎖可変領域、および配列番号：５３５に記載のCDR１、配列番号：５３６に記載のCDR２、配列番号：５３７に記載のCDR３を有する軽鎖可変領域を含む抗体
（２２）配列番号：５６１に記載のCDR１、配列番号：５６２に記載のCDR２、配列番号：５６３に記載のCDR３を有する重鎖可変領域、および配列番号：５６５に記載のCDR１、配列番号：５６６に記載のCDR２、配列番号：５６７に記載のCDR３を有する軽鎖可変領域を含む抗体
（２３）配列番号：５７１に記載のCDR１、配列番号：５７２に記載のCDR２、配列番号：５７３に記載のCDR３を有する重鎖可変領域、および配列番号：５７５に記載のCDR１、配列番号：５７６に記載のCDR２、配列番号：５７７に記載のCDR３を有する軽鎖可変領域を含む抗体
（２４）配列番号：６８１に記載のCDR１、配列番号：６８２に記載のCDR２、配列番号：６８３に記載のCDR３を有する重鎖可変領域、および配列番号：６８５に記載のCDR１、配列番号：６８６に記載のCDR２、配列番号：６８７に記載のCDR３を有する軽鎖可変領域を含む抗体
（２５）配列番号：７２１に記載のCDR１、配列番号：７２２に記載のCDR２、配列番号：７２３に記載のCDR３を有する重鎖可変領域、および配列番号：７２５に記載のCDR１、配列番号：７２６に記載のCDR２、配列番号：７２７に記載のCDR３を有する軽鎖可変領域を含む抗体
（２６）配列番号：７４１に記載のCDR１、配列番号：７４２に記載のCDR２、配列番号：７４３に記載のCDR３を有する重鎖可変領域、および配列番号：７４５に記載のCDR１、配列番号：７４６に記載のCDR２、配列番号：７４７に記載のCDR３を有する軽鎖可変領域を含む抗体
（２７）配列番号：７９１に記載のCDR１、配列番号：７９２に記載のCDR２、配列番号：７９３に記載のCDR３を有する重鎖可変領域、および配列番号：７９５に記載のCDR１、配列番号：７９６に記載のCDR２、配列番号：７９７に記載のCDR３を有する軽鎖可変領域を含む抗体
（２８）上記（１）～（２７）のいずれかに記載の抗体とTGF-beta3への結合において競合する抗体
（２９）上記（１）～（２７）のいずれかに記載の抗体が結合するTGF-beta3のエピトープと同じエピトープに結合する抗体。 In another aspect, the present invention provides a non-neutralizing antibody that specifically binds to TGF-beta3, as described in any of (1) to (29) below:
(1) A heavy chain variable region having CDR1 described in SEQ ID NO: 101, CDR2 described in SEQ ID NO: 102, CDR3 described in SEQ ID NO: 103, and CDR1 described in SEQ ID NO: 105, SEQ ID NO: 106 An antibody comprising a light chain variable region having CDR2 described in SEQ ID NO: 107 and CDR3 described in SEQ ID NO: 107 (2) CDR1 described in SEQ ID NO: 141, CDR2 described in SEQ ID NO: 142, described in SEQ ID NO: 143 An antibody (3) comprising a heavy chain variable region having CDR3, and a light chain variable region having CDR1 set forth in SEQ ID NO: 145, CDR2 set forth in SEQ ID NO: 146, CDR3 set forth in SEQ ID NO: 147 161, CDR2 of SEQ ID NO: 162, heavy chain variable region having CDR3 of SEQ ID NO: 163, CDR1 of SEQ ID NO: 165, CDR2 of SEQ ID NO: 166, sequence Antibody comprising light chain variable region having CDR3 of No. 167 (4) CDR1 of SEQ ID NO: 171; CDR2 of SEQ ID NO: 172; heavy chain variable having CDR3 of SEQ ID NO: 173 An antibody comprising a region and a light chain variable region having CDR1 set forth in SEQ ID NO: 175, CDR2 set forth in SEQ ID NO: 176, CDR3 set forth in SEQ ID NO: 177 (5) CDR1 set forth in SEQ ID NO: 181, SEQ ID NO: 182 CDR2, SEQ ID NO: 183 heavy chain variable region having CDR3, and SEQ ID NO: 185 CDR1, SEQ ID NO: 186 CDR2, SEQ ID NO: 187 Antibody comprising a light chain variable region having CDR3 (6) CDR1 set forth in SEQ ID NO: 191, CDR2 set forth in SEQ ID NO: 192, heavy chain variable region having CDR3 set forth in SEQ ID NO: 193, and SEQ ID NO: An antibody comprising a light chain variable region having CDR1 described in 95, CDR2 described in SEQ ID NO: 196, CDR3 described in SEQ ID NO: 197 (7) CDR1 described in SEQ ID NO: 201, described in SEQ ID NO: 202 And a heavy chain variable region having the CDR3 of SEQ ID NO: 203, a CDR1 of the SEQ ID NO: 205, a CDR2 of the SEQ ID NO: 206, and a light chain variable having the CDR3 of the SEQ ID NO: 207 Antibody comprising region (8) CDR1 set forth in SEQ ID NO: 211, CDR2 set forth in SEQ ID NO: 212, heavy chain variable region having CDR3 set forth in SEQ ID NO: 213, and CDR1 set forth in SEQ ID NO: 215, An antibody comprising a light chain variable region having CDR2 set forth in SEQ ID NO: 216 and CDR3 set forth in SEQ ID NO: 217 (9) CDR1 set forth in SEQ ID NO: 241, CDR2 set forth in SEQ ID NO: 242, SEQ ID NO: 24 An antibody comprising a heavy chain variable region having CDR3 according to 3, and a CDR1 according to SEQ ID NO: 245, a CDR2 according to SEQ ID NO: 246, and a light chain variable region having CDR3 as SEQ ID NO: 247 (10 ) CDR1 described in SEQ ID NO: 261, CDR2 described in SEQ ID NO: 262, heavy chain variable region having CDR3 described in SEQ ID NO: 263, and CDR1 described in SEQ ID NO: 265, described in SEQ ID NO: 266 An antibody comprising a light chain variable region having CDR3 described in SEQ ID NO: 267 (11) CDR1 described in SEQ ID NO: 291, CDR2 described in SEQ ID NO: 292, CDR3 described in SEQ ID NO: 293 An antibody (12) comprising a heavy chain variable region having a light chain variable region having a CDR1 described in SEQ ID NO: 295, a CDR2 described in SEQ ID NO: 296, a CDR3 described in SEQ ID NO: 297; CDR1, described in SEQ ID NO: 302, heavy chain variable region having CDR3 described in SEQ ID NO: 303, CDR1 described in SEQ ID NO: 305, CDR2 described in SEQ ID NO: 306, SEQ ID NO: Antibody comprising a light chain variable region having CDR3 described in 307 (13) CDR1 described in SEQ ID NO: 361, CDR2 described in SEQ ID NO: 362, heavy chain variable region having CDR3 described in SEQ ID NO: 363 And an antibody comprising a light chain variable region having CDR1 as set forth in SEQ ID NO: 365, CDR2 as set forth in SEQ ID NO: 366, CDR3 as set forth in SEQ ID NO: 367 (14) CDR1, set forth in SEQ ID NO: 371 SEQ ID NO: 372 CDR2, SEQ ID NO: 373 heavy chain variable region having CDR3, SEQ ID NO: 375 CDR1, SEQ ID NO: 376 CDR2, SEQ ID NO: 377 Antibody comprising a light chain variable region having the CDR3 of (15) CDR1 set forth in SEQ ID NO: 391, CDR2 set forth in SEQ ID NO: 392, heavy chain variable region having CDR3 set forth in SEQ ID NO: 393, and SEQ ID NO: Antibody comprising a light chain variable region having CDR1 described in SEQ ID NO: 396, CDR2 described in SEQ ID NO: 396, CDR3 described in SEQ ID NO: 397 (16) CDR1 described in SEQ ID NO: 401, SEQ ID NO: 402 A heavy chain variable region having the CDR2 of SEQ ID NO: 403 and a light chain having a CDR3 of SEQ ID NO: 405, a CDR2 of SEQ ID NO: 406, a light chain having a CDR3 of SEQ ID NO: 407 Antibody comprising variable region (17) CDR1 described in SEQ ID NO: 481, CDR2 described in SEQ ID NO: 482, heavy chain variable region having CDR3 described in SEQ ID NO: 483, and SEQ ID NO: 485 An antibody comprising a light chain variable region having CDR3 of SEQ ID NO: 487, CDR3 of SEQ ID NO: 487, CDR1 of SEQ ID NO: 491, CDR2 of SEQ ID NO: 492, A heavy chain variable region having the CDR3 of SEQ ID NO: 493, a CDR1 of SEQ ID NO: 495, a CDR2 of SEQ ID NO: 496, a light chain variable region having the CDR3 of SEQ ID NO: 497 Antibody (19) CDR1 set forth in SEQ ID NO: 511, CDR2 set forth in SEQ ID NO: 512, heavy chain variable region having CDR3 set forth in SEQ ID NO: 513, and CDR1 set forth in SEQ ID NO: 515, SEQ ID NO: An antibody comprising a light chain variable region having CDR2 described in 516 and CDR3 described in SEQ ID NO: 517 (20) CDR1 described in SEQ ID NO: 521, CDR2 described in SEQ ID NO: 522, SEQ ID NO: 523 The antibody (21) sequence comprising the heavy chain variable region having the CDR3 and the CDR1 described in SEQ ID NO: 525, the CDR2 described in SEQ ID NO: 526, and the light chain variable region having the CDR3 described in SEQ ID NO: 527 SEQ ID NO: 531, CDR2 set forth in SEQ ID NO: 532, heavy chain variable region having CDR3 set forth in SEQ ID NO: 533, and CDR1 set forth in SEQ ID NO: 535, CDR2 set forth in SEQ ID NO: 536 An antibody comprising a light chain variable region having CDR3 described in SEQ ID NO: 537 (22) CDR1 described in SEQ ID NO: 561, CDR2 described in SEQ ID NO: 562, and a heavy chain having CDR3 described in SEQ ID NO: 563 An antibody (23) comprising a chain variable region and a light chain variable region having CDR1 set forth in SEQ ID NO: 565, CDR2 set forth in SEQ ID NO: 566, CDR3 set forth in SEQ ID NO: 567 (23) in SEQ ID NO: 571 The CDR1, the CDR2 described in SEQ ID NO: 572, the heavy chain variable region having the CDR3 described in SEQ ID NO: 573, the CDR1 described in SEQ ID NO: 575, the CDR2 described in SEQ ID NO: 576, the SEQ ID NO: An antibody comprising a light chain variable region having CDR3 according to 577 (24), CDR1 according to SEQ ID NO: 681, CDR2 according to SEQ ID NO: 682, heavy chain variable region having CDR3 according to SEQ ID NO: 683, And an antibody comprising a light chain variable region having CDR1 described in SEQ ID NO: 685, CDR2 described in SEQ ID NO: 686, CDR3 described in SEQ ID NO: 687 (25) CDR1 described in SEQ ID NO: 721, SEQ ID NO: : CDR2 described in 722, heavy chain variable region having CDR3 described in SEQ ID NO: 723, CDR1 described in SEQ ID NO: 725, CDR2 described in SEQ ID NO: 726, CDR described in SEQ ID NO: 727 Antibody comprising a light chain variable region having 3 (26) CDR1 set forth in SEQ ID NO: 741, CDR2 set forth in SEQ ID NO: 742, heavy chain variable region having CDR3 set forth in SEQ ID NO: 743, and SEQ ID NO: An antibody comprising a light chain variable region having CDR1 described in 745, CDR2 described in SEQ ID NO: 746, CDR3 described in SEQ ID NO: 747 (27) CDR1 described in SEQ ID NO: 791, SEQ ID NO: 792 And a heavy chain variable region having the CDR3 of SEQ ID NO: 793, a CDR1 of SEQ ID NO: 795, a CDR2 of SEQ ID NO: 796, and a light chain variable having a CDR3 of SEQ ID NO: 797 An antibody comprising a region (28) an antibody that competes with TGF-beta3 for binding to the antibody according to any of (1) to (27) above (29) according to any of (1) to (27) above TGF-bet to which antibody binds An antibody that binds to the same epitope as a3.
　本発明のさらなる局面において、上述の態様の任意のものによる抗TGF-beta3抗体は、キメラ、ヒト化、またはヒト抗体を含む、モノクローナル抗体である。一態様において、抗TGF-beta3抗体は、例えば、Fv、Fab、Fab'、scFv、ダイアボディ、またはF(ab')2断片などの、抗体断片である。一態様において、抗TGF-beta3抗体はFcRn結合能を有し、例えば、FcRn結合領域を含むFc領域を有する。別の態様において、抗体は全長抗体である。 In a further aspect of the invention, the anti-TGF-beta3 antibody according to any of the above embodiments is a monoclonal antibody, including a chimeric, humanized, or human antibody. In one embodiment, the anti-TGF-beta3 antibody is an antibody fragment, such as, for example, an Fv, Fab, Fab ′, scFv, diabody, or F (ab ′) 2 fragment. In one embodiment, the anti-TGF-beta3 antibody has FcRn binding ability, for example, an Fc region containing an FcRn binding region. In another embodiment, the antibody is a full length antibody.
　さらなる局面において、上述の態様の任意のものによる抗TGF-beta3抗体は、単独または組み合わせで、以下の項目1～7に記載の任意の特徴を取り込んでもよい。  In a further aspect, the anti-TGF-beta3 antibody according to any of the above embodiments may incorporate any of the features described in items 1-7 below, alone or in combination.
１．抗体のアフィニティ
　特定の態様において、本明細書で提供される抗体は、≦1μM、≦100nM、≦10nM、≦1nM、≦0.1nM、≦0.01nMまたは≦0.001nM（例えば、10-8M以下、例えば10-8M～10-13M、例えば10-9M～10-13M）の解離定数 (Kd) を有する。 1. Antibody Affinity In certain embodiments, an antibody provided herein has a ≦ 1 μM, ≦ 100 nM, ≦ 10 nM, ≦ 1 nM, ≦ 0.1 nM, ≦ 0.01 nM or ≦ 0.001 nM (eg, 10 −8 M or less, For example, it has a dissociation constant (Kd) of 10 −8 M to 10 −13 M, such as 10 −9 M to 10 −13 M.
　一態様において、Kdは、放射性標識抗原結合測定法 (radiolabeled antigen binding assay: RIA) によって測定される。一態様において、RIAは、目的の抗体のFabバージョンおよびその抗原を用いて実施される。例えば、抗原に対するFabの溶液中結合アフィニティは、非標識抗原の漸増量系列の存在下で最小濃度の (125I) 標識抗原によりFabを平衡化させ、次いで結合した抗原を抗Fab抗体でコーティングされたプレートにより捕捉することによって測定される。（例えば、Chen et al., J. Mol. Biol. 293:865-881(1999) を参照のこと）。測定条件を構築するために、MICROTITER（登録商標）マルチウェルプレート (Thermo Scientific) を50mM炭酸ナトリウム (pH9.6) 中5μg/mlの捕捉用抗Fab抗体 (Cappel Labs) で一晩コーティングし、その後に室温（およそ23℃）で2～5時間、PBS中2％ (w/v) ウシ血清アルブミンでブロックする。非吸着プレート (Nunc #269620) において、100 pMまたは26 pMの [125I]-抗原を、（例えば、Presta et al., Cancer Res. 57:4593-4599 (1997) における抗VEGF抗体、Fab-12の評価と同じように）目的のFabの段階希釈物と混合する。次いで、目的のFabを一晩インキュベートするが、このインキュベーションは、平衡が確実に達成されるよう、より長時間（例えば、約65時間）継続され得る。その後、混合物を、室温でのインキュベーション（例えば、1時間）のために捕捉プレートに移す。次いで溶液を除去し、プレートをPBS中0.1％のポリソルベート20（TWEEN-20（登録商標））で8回洗浄する。プレートが乾燥したら、150μl/ウェルのシンチラント（MICROSCINT-20（商標）、Packard）を添加し、TOPCOUNT（商標）ガンマカウンター (Packard) においてプレートを10分間カウントする。最大結合の20％以下を与える各Fabの濃度を、競合結合アッセイにおいて使用するために選択する。 In one embodiment, Kd is measured by a radiolabeled antigen binding assay (RIA). In one embodiment, RIA is performed using the Fab version of the antibody of interest and its antigen. For example, Fab binding affinity of an antigen to an antigen is such that the Fab is equilibrated with a minimal concentration of ( 125I ) -labeled antigen in the presence of increasing series of unlabeled antigen, and then the bound antigen is coated with an anti-Fab antibody. Measured by catching on a plate. (See, eg, Chen et al., J. Mol. Biol. 293: 865-881 (1999)). To establish the measurement conditions, MICROTITER® multiwell plates (Thermo Scientific) were coated overnight with 5 μg / ml capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), then Block with 2% (w / v) bovine serum albumin in PBS for 2-5 hours at room temperature (approximately 23 ° C.). In a non-adsorbed plate (Nunc # 269620), 100 pM or 26 pM [ 125 I] -antigen (see, for example, the anti-VEGF antibody Fab- in Presta et al, Cancer Res. 57: 4593-4599 (1997) Mix with serial dilutions of the desired Fab (as in the 12 assessment). The Fab of interest is then incubated overnight, which can be continued for a longer time (eg, about 65 hours) to ensure that equilibrium is achieved. The mixture is then transferred to a capture plate for incubation at room temperature (eg, 1 hour). The solution is then removed and the plate is washed 8 times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. When the plate is dry, 150 μl / well scintillant (MICROSCINT-20 ™, Packard) is added and the plate is counted for 10 minutes in a TOPCOUNT ™ gamma counter (Packard). The concentration of each Fab that gives 20% or less of maximum binding is selected for use in the competitive binding assay.
　別の態様によれば、Kdは、BIACORE（登録商標）表面プラズモン共鳴アッセイを用いて測定される。例えば、BIACORE（登録商標）-2000またはBIACORE（登録商標）-3000 (BIAcore, Inc., Piscataway, NJ) を用いる測定法が、およそ10反応単位 (response unit: RU) の抗原が固定されたCM5チップを用いて25℃で実施される。一態様において、カルボキシメチル化デキストランバイオセンサーチップ (CM5、BIACORE, Inc.) は、供給元の指示にしたがいN-エチル-N'- (3-ジメチルアミノプロピル)-カルボジイミドヒドロクロリド (EDC) およびN-ヒドロキシスクシンイミド (NHS) を用いて活性化される。抗原は、およそ10反応単位 (RU) のタンパク質の結合を達成するよう、5μl/分の流速で注入される前に、10mM酢酸ナトリウム、pH4.8を用いて5μg/ml（およそ0.2μM）に希釈される。抗原の注入後、未反応基をブロックするために1Mエタノールアミンが注入される。キネティクスの測定のために、25℃、およそ25μl/分の流速で、0.05％ポリソルベート20（TWEEN-20（商標））界面活性剤含有PBS (PBST) 中のFabの2倍段階希釈物 (0.78nM～500nM) が注入される。結合速度 (kon) および解離速度 (koff) は、単純な1対1ラングミュア結合モデル（BIACORE（登録商標）評価ソフトウェアバージョン3.2）を用いて、結合および解離のセンサーグラムを同時にフィッティングすることによって計算される。平衡解離定数 (Kd) は、koff/kon比として計算される。例えば、Chen et al., J. Mol. Biol. 293:865-881 (1999) を参照のこと。上記の表面プラズモン共鳴アッセイによってオン速度が106M-1s-1を超える場合、オン速度は、分光計（例えばストップフロー式分光光度計 (Aviv Instruments) または撹拌キュベットを用いる8000シリーズのSLM-AMINCO（商標）分光光度計 (ThermoSpectronic)）において測定される、漸増濃度の抗原の存在下でのPBS、pH7.2中20nMの抗抗原抗体（Fab形態）の25℃での蛍光発光強度（励起=295nm；発光=340nm、バンドパス16nm）の増加または減少を測定する蛍光消光技術を用いることによって決定され得る。 According to another embodiment, Kd is measured using a BIACORE® surface plasmon resonance assay. For example, a measurement method using BIACORE (registered trademark) -2000 or BIACORE (registered trademark) -3000 (BIAcore, Inc., Piscataway, NJ) is used to obtain a CM5 having about 10 response units (RU) of immobilized antigen. Performed at 25 ° C using a chip. In one embodiment, a carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc.) is prepared according to the supplier's instructions with N-ethyl-N ′-(3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) and N Activated with -hydroxysuccinimide (NHS). The antigen is brought to 5 μg / ml (approximately 0.2 μM) using 10 mM sodium acetate, pH 4.8 before being injected at a flow rate of 5 μl / min to achieve approximately 10 reaction units (RU) of protein binding. Diluted. After the injection of antigen, 1M ethanolamine is injected to block unreacted groups. For kinetic measurements, a 2-fold serial dilution of Fab (0.78 nM) in PBS (PBST) containing 0.05% polysorbate 20 (TWEEN-20 ™) surfactant at 25 ° C. and a flow rate of approximately 25 μl / min. ~ 500nM) is injected. The association rate (k on ) and dissociation rate (k off ) can be determined by simultaneously fitting the association and dissociation sensorgrams using a simple one-to-one Langmuir association model (BIACORE® evaluation software version 3.2). Calculated. The equilibrium dissociation constant (Kd) is calculated as the ratio k off / k on . See, for example, Chen et al., J. Mol. Biol. 293: 865-881 (1999). When the on-rate exceeds 10 6 M -1 s -1 by the surface plasmon resonance assay described above, the on-rate is measured using a spectrophotometer (eg, a stop flow spectrophotometer (Aviv Instruments) or a 8000 series SLM- Fluorescence emission intensity at 25 ° C. (excitation) of 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2 in the presence of increasing concentrations of antigen as measured in an AMINCO ™ spectrophotometer (ThermoSpectronic)) = 295 nm; emission = 340 nm, bandpass 16 nm) can be determined by using a fluorescence quenching technique that measures the increase or decrease.
２．抗体断片
　特定の態様において、本明細書で提供される抗体は、抗体断片である。抗体断片は、これらに限定されるものではないが、Fab、Fab'、Fab'-SH、F(ab')2、Fv、および scFv断片、ならびに、後述する他の断片を含む。特定の抗体断片についての総説として、Hudson et al. Nat. Med. 9:129-134 (2003) を参照のこと。scFv断片の総説として、例えば、Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp.269-315 (1994)；加えて、WO93/16185；ならびに米国特許第5,571,894号および第5,587,458号を参照のこと。サルベージ受容体結合エピトープ残基を含みインビボ (in vivo) における半減期の長くなったFabおよびF(ab')2断片についての論説として、米国特許第5,869,046号を参照のこと。 2. Antibody Fragments In certain embodiments, the antibodies provided herein are antibody fragments. Antibody fragments include, but are not limited to, Fab, Fab ′, Fab′-SH, F (ab ′) 2 , Fv, and scFv fragments, as well as other fragments described below. For a review of specific antibody fragments, see Hudson et al. Nat. Med. 9: 129-134 (2003). For reviews of scFv fragments, see, for example, Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994); 16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458. See US Pat. No. 5,869,046 for a discussion of Fab and F (ab ′) 2 fragments that contain salvage receptor binding epitope residues and have increased in vivo half-life.
　ダイアボディは、二価または二重特異的であってよい、抗原結合部位を2つ伴う抗体断片である。例えば、EP404,097号; WO1993/01161; Hudson et al., Nat. Med. 9:129-134 (2003); Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993) 参照。トリアボディ (triabody) やテトラボディ (tetrabody) も、Hudson et al., Nat. Med. 9:129-134 (2003) に記載されている。 A diabody is an antibody fragment with two antigen binding sites, which may be bivalent or bispecific. For example, EP404,097; WO1993 / 01161; Hudson et al., Nat. Med. 9: 129-134 (2003); Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993 See. Triabody and tetrabody are also described in Hudson et al., Nat. Med. 9: 129-134 2003 (2003).
　シングルドメイン抗体は、抗体の重鎖可変ドメインのすべてもしくは一部分、または軽鎖可変ドメインのすべてもしくは一部分を含む、抗体断片である。特定の態様において、シングルドメイン抗体は、ヒトシングルドメイン抗体である（Domantis, Inc., Waltham, MA；例えば、米国特許第6,248,516号B1参照）。 A single domain antibody is an antibody fragment comprising all or part of an antibody heavy chain variable domain or all or part of a light chain variable domain. In certain embodiments, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, eg, US Pat. No. 6,248,516 B1).
　抗体断片は、これらに限定されるものではないが、本明細書に記載の、完全抗体のタンパク質分解的消化、組み換え宿主細胞（例えば、大腸菌 (E. coli) またはファージ）による産生を含む、種々の手法により作ることができる。 Antibody fragments include, but are not limited to, various forms of proteolytic digestion of complete antibodies, as described herein, including production by recombinant host cells (eg, E. coli or phage). It can be made by the method of.
３．キメラおよびヒト化抗体
　特定の態様において、本明細書で提供される抗体は、キメラ抗体である。特定のキメラ抗体が、例えば、米国特許第4,816,567号；および、Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984) に記載されている。一例では、キメラ抗体は、非ヒト可変領域（例えば、マウス、ラット、ハムスター、ウサギ、またはサルなどの非ヒト霊長類に由来する可変領域）およびヒト定常領域を含む。さらなる例において、キメラ抗体は、親抗体のものからクラスまたはサブクラスが変更された「クラススイッチ」抗体である。キメラ抗体は、その抗原結合断片も含む。 3. Chimeric and humanized antibodies In certain embodiments, the antibodies provided herein are chimeric antibodies. Certain chimeric antibodies are described, for example, in US Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984). In one example, a chimeric antibody comprises a non-human variable region (eg, a variable region derived from a non-human primate such as a mouse, rat, hamster, rabbit, or monkey) and a human constant region. In a further example, a chimeric antibody is a “class switch” antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies also include antigen binding fragments thereof.
　特定の態様において、キメラ抗体は、ヒト化抗体である。典型的には、非ヒト抗体は、親非ヒト抗体の特異性およびアフィニティを維持したままでヒトへの免疫原性を減少させるために、ヒト化される。通常、ヒト化抗体は1つまたは複数の可変ドメインを含み、当該可変ドメイン中、HVR（例えばCDR（またはその部分））は非ヒト抗体に由来し、FR（またはその部分）はヒト抗体配列に由来する。ヒト化抗体は、任意で、ヒト定常領域の少なくとも一部分を含む。いくつかの態様において、ヒト化抗体中のいくつかのFR残基は、例えば、抗体の特異性またはアフィニティを回復または改善するために、非ヒト抗体（例えば、HVR残基の由来となった抗体）からの対応する残基で置換されている。 In certain embodiments, the chimeric antibody is a humanized antibody. Typically, non-human antibodies are humanized in order to reduce human immunogenicity while maintaining the specificity and affinity of the parent non-human antibody. Usually, a humanized antibody comprises one or more variable domains, in which the HVR (eg, CDR (or portion thereof)) is derived from a non-human antibody and FR (or portion thereof) is derived from a human antibody sequence. Derived from. A humanized antibody optionally comprises at least a portion of a human constant region. In some embodiments, some FR residues in the humanized antibody are non-human antibodies (eg, antibodies derived from HVR residues, eg, to restore or improve antibody specificity or affinity). ) With the corresponding residue from
　ヒト化抗体およびその作製方法は、Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)において総説されており、また、例えば、Riechmann et al., Nature 332:323-329 (1988)； Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989)； 米国特許第5,821,337号、第7,527,791号、第6,982,321号、および第7,087,409号；Kashmiri et al., Methods 36:25-34 (2005)（特異性決定領域 (specificity determining region: SDR) グラフティングを記載）；Padlan, Mol. Immunol. 28:489-498 (1991) （リサーフェイシングを記載）； Dall'Acqua et al., Methods 36:43-60 (2005) （FRシャッフリングを記載）；ならびに、Osbourn et al., Methods 36:61-68 (2005) およびKlimka et al., Br. J. Cancer, 83:252-260 (2000) （FRシャッフリングのための「ガイドセレクション」アプローチを記載） において、さらに記載されている。 Humanized antibodies and methods for their production are reviewed in Almagro and Fransson, Front. Biosci. 13: 1619-1633 ま た (2008), and for example, Riechmann et al., Nature 332: 323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86: 10029-10033 (1989); U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36 : 25-34 (2005) (specificity determination region (specificity determining region: SDR) indicates grafting); Padlan, Mol. Immunol. 28: 489-498 (1991) (describing resurfacing); Dall'Acqua et al., Methods 36: 43-60 (2005) (with FR shuffling); and Osbourn et al., Methods 36: 61-68 (2005) and Klimka et al., Br. J. Cancer, 83: 252-260 (2000) (describes the “guide selection” approach for FR shuffling) is further described.
　ヒト化に使われ得るヒトフレームワーク領域は、これらに限定されるものではないが：「ベストフィット」法（Sims et al. J. Immunol. 151:2296 (1993) 参照）を用いて選択されたフレームワーク領域；軽鎖または重鎖可変領域の特定のサブグループのヒト抗体のコンセンサス配列に由来するフレームワーク領域（Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992) および Presta et al. J. Immunol., 151:2623 (1993) 参照）；ヒト成熟（体細胞変異）フレームワーク領域またはヒト生殖細胞系フレームワーク領域（例えば、Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008) 参照）；および、FRライブラリのスクリーニングに由来するフレームワーク領域（Baca et al., J. Biol. Chem. 272:10678-10684 (1997) および Rosok et al., J. Biol. Chem. 271:22611-22618 (1996) 参照）を含む。 Human framework regions that can be used for humanization have been selected using, but not limited to: the “best fit” method (see Sims et al. J. Immunol. 151: 2296 (1993)). Framework regions; framework regions derived from consensus sequences of human antibodies of a particular subgroup of light or heavy chain variable regions (Carter et al. Proc. Natl. Acad. Sci. USA, 89: 4285 (1992)) Presta et al. J. Immunol., 151: 2623 (1993)); human maturation (somatic mutation) framework region or human germline framework region (eg Almagro and Fransson, Front. Biosci. 13: 1619 -1633 (2008)); and framework regions derived from FR library screening (Baca et al., J. Biol. Chem. 272: 10678-10684 (1997) and Rosak et al., J J Biol. See Chem. 271: 22611-22618 (1996). Including the.
４．ヒト抗体
　特定の態様において、本明細書で提供される抗体は、ヒト抗体である。ヒト抗体は、当該技術分野において知られる種々の手法によって製造され得る。ヒト抗体は、van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) および Lonberg, Curr. Opin. Immunol. 20:450-459 (2008) に、概説されている。 4). Human Antibodies In certain embodiments, the antibodies provided herein are human antibodies. Human antibodies can be produced by various techniques known in the art. Human antibodies are reviewed in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20: 450-459 (2008).
　ヒト抗体は、抗原チャレンジ（負荷）に応答して完全ヒト抗体またはヒト可変領域を伴う完全抗体を産生するように改変されたトランスジェニック動物へ免疫原を投与することにより、調製されてもよい。そのような動物は、典型的にはヒト免疫グロブリン遺伝子座の全部もしくは一部分を含み、ヒト免疫グロブリン遺伝子座の全部もしくは一部分は、内因性の免疫グロブリン遺伝子座を置き換えるか、または、染色体外にもしくは当該動物の染色体内にランダムに取り込まれた状態で存在する。そのようなトランスジェニックマウスにおいて、内因性の免疫グロブリン遺伝子座は、通常不活性化されている。トランスジェニック動物からヒト抗体を得る方法の総説として、Lonberg, Nat. Biotech. 23:1117-1125 (2005) を参照のこと。また、例えば、XENOMOUSE（商標）技術を記載した米国特許第6,075,181号および第6,150,584号；HUMAB（登録商標）技術を記載した米国特許第5,770,429号；K-M MOUSE（登録商標）技術を記載した米国特許第7,041,870号；ならびに、VELOCIMOUSE（登録商標）技術を記載した米国特許出願公開第2007/0061900号を、併せて参照のこと。このような動物によって生成された完全抗体からのヒト可変領域は、例えば、異なるヒト定常領域と組み合わせるなどして、さらに修飾されてもよい。 Human antibodies may be prepared by administering an immunogen to a transgenic animal that has been modified to produce fully human antibodies or fully antibodies with human variable regions in response to an antigen challenge (load). Such animals typically include all or part of a human immunoglobulin locus, which all or part of the human immunoglobulin locus replaces the endogenous immunoglobulin locus or is extrachromosomal or It exists in a state of being randomly incorporated in the chromosome of the animal. In such transgenic mice, the endogenous immunoglobulin locus is usually inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23: 1117-1125 (2005). Also, for example, US Pat. Nos. 6,075,181 and 6,150,584 describing XENOMOUSE ™ technology; US Pat. No. 5,770,429 describing HUMAB® technology; US Patent No. describing KM MOUSE® technology See also 7,041,870; and US Patent Application Publication No. 2007/0061900 describing the VELOCIMOUSE® technology. Human variable regions from intact antibodies produced by such animals may be further modified, for example, in combination with different human constant regions.
　ヒト抗体は、ハイブリドーマに基づいた方法でも作ることができる。ヒトモノクローナル抗体の製造のための、ヒトミエローマおよびマウス‐ヒトヘテロミエローマ細胞株は、既に記述されている。（例えば、Kozbor J. Immunol., 133: 3001 (1984)；Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp.51-63 (Marcel Dekker, Inc., New York, 1987)；およびBoerner et al., J. Immunol., 147: 86 (1991) 参照。）ヒトB細胞ハイブリドーマ技術を介して生成されたヒト抗体も、Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006) に述べられている。追加的な方法としては、例えば、米国特許第7,189,826号（ハイブリドーマ細胞株からのモノクローナルヒトIgM抗体の製造を記載）、および、Ni, Xiandai Mianyixue, 26(4):265-268 (2006)（ヒト－ヒトハイブリドーマを記載）に記載されたものを含む。ヒトハイブリドーマ技術（トリオーマ技術）も、Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) およびVollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005)に記載されている。 Human antibodies can also be made by methods based on hybridomas. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have already been described. (Eg, Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp.51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et , J. Immunol., 147: 86 (1991).) Human antibodies generated via human B cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103: 3557- It is stated in 3562 (2006). Additional methods include, for example, US Pat. No. 7,189,826 (describing the production of monoclonal human IgM antibodies from hybridoma cell lines), and Ni, Xiandai Mianyixue, 26 (4): 265-268 (2006) (human) -Describe human hybridomas). Human hybridoma technology (trioma technology) is also available from Vollmers and Brandlein, Histology and Histopathology, 20 (3): 927-937 (2005) and Volllmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27 (3): 185-91 (2005).
　ヒト抗体は、ヒト由来ファージディスプレイライブラリから選択されたFvクローン可変ドメイン配列を単離することでも生成できる。このような可変ドメイン配列は、次に所望のヒト定常ドメインと組み合わせることができる。抗体ライブラリからヒト抗体を選択する手法を、以下に述べる。 Human antibodies can also be generated by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences can then be combined with the desired human constant domain. A technique for selecting a human antibody from an antibody library is described below.
５．ライブラリ由来抗体
　本発明の抗体は、所望の1つまたは複数の活性を伴う抗体についてコンビナトリアルライブラリをスクリーニングすることによって単離してもよい。例えば、ファージディスプレイライブラリの生成や、所望の結合特性を備える抗体についてそのようなライブラリをスクリーニングするための、様々な方法が当該技術分野において知られている。そのような方法は、Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001) において総説されており、さらに例えば、McCafferty et al., Nature 348:552-554；Clackson et al., Nature 352: 624-628 (1991)； Marks et al., J. Mol. Biol. 222: 581-597 (1992)； Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003)； Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004)； Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004)； Fellouse, Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004)；およびLee et al., J. Immunol. Methods 284(1-2): 119-132(2004) に記載されている。 5). Library-derived antibodies Antibodies of the present invention may be isolated by screening combinatorial libraries for antibodies with one or more desired activities. For example, various methods are known in the art for generating phage display libraries and screening such libraries for antibodies with the desired binding properties. Such methods are reviewed in Hoogenboom et al. In Methods in Molecular Biology 178: 1-37 (O'Brien et al., Ed., Human Press, Totowa, NJ, 2001), and further e.g. McCafferty et al., Nature 348: 552-554; Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Marks and Bradbury, in Methods in Molecular Biology 248: 161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338 (2): 299-310 (2004); Lee et al., J. Mol. Biol. 340 (5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101 (34): 12467-12472 (2004); and Lee et al. , J. Immunol. Methods 284 (1-2): 119-132 (2004).
　特定のファージディスプレイ法において、VHおよびVL遺伝子のレパートリーは、ポリメラーゼ連鎖反応 (polymerase chain reaction: PCR) により別々にクローニングされ、無作為にファージライブラリ中で再結合され、当該ファージライブラリは、Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994) に述べられているようにして、抗原結合ファージについてスクリーニングされ得る。ファージは、典型的には、単鎖Fv (scFv) 断片としてまたはFab断片としてのいずれかで、抗体断片を提示する。免疫化された供給源からのライブラリは、ハイブリドーマを構築することを要さずに、免疫源に対する高アフィニティ抗体を提供する。あるいは、Griffiths et al., EMBO J, 12: 725-734 (1993) に記載されるように、ナイーブレパートリーを（例えば、ヒトから）クローニングして、免疫化することなしに、広範な非自己および自己抗原への抗体の単一の供給源を提供することもできる。最後に、ナイーブライブラリは、Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992) に記載されるように、幹細胞から再編成前のV-遺伝子セグメントをクローニングし、超可変CDR3領域をコードしかつインビトロ (in vitro) で再構成を達成するための無作為配列を含んだPCRプライマーを用いることにより、合成的に作ることもできる。ヒト抗体ファージライブラリを記載した特許文献は、例えば：米国特許第5,750,373号、ならびに、米国特許出願公開第2005/0079574号、2005/0119455号、第2005/0266000号、第2007/0117126号、第2007/0160598号、第2007/0237764号、第2007/0292936号、および第2009/0002360号を含む。 In certain phage display methods, repertoires of VH and VL genes are cloned separately by polymerase chain reaction (PCR) and randomly recombined in a phage library, which is linked to a Winter library. ., Ann. Rev. Immunol., 12: 433-455 (1994) 得 る can be screened for antigen-binding phages. Phages typically display antibody fragments, either as single chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high affinity antibodies to the immunogen without the need to construct hybridomas. Alternatively, as described in Griffiths et al., EMBO J, 12: 725-734 (1993), the naïve repertoire can be cloned (eg, from human) without extensive immunization and without immunization. It is also possible to provide a single source of antibodies to self antigens. Finally, the naïve library was used to clone the pre-rearranged V-gene segment from stem cells as described in Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992) It can also be made synthetically by using PCR primers that encode the region and contain random sequences to achieve reconstitution in vitro. Patent documents describing human antibody phage libraries include, for example: US Pat. No. 5,750,373, and US Patent Application Publication Nos. 2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007. / 0160598, 2007/0237764, 2007/0292936, and 2009/0002360.
　ヒト抗体ライブラリから単離された抗体または抗体断片は、本明細書ではヒト抗体またはヒト抗体断片と見なす。 In the present specification, an antibody or antibody fragment isolated from a human antibody library is regarded as a human antibody or a human antibody fragment.
６．多重特異性抗体
　特定の態様において、本明細書で提供される抗体は、多重特異性抗体（例えば、二重特異性抗体）である。多重特異性抗体は、少なくとも2つの異なる部位に結合特異性を有する、モノクローナル抗体である。特定の態様において、結合特異性の1つは、TGF-beta3に対するものであり、もう1つは他の任意の抗原（例えば、B細胞表面マーカー）へのものである。特定の態様において、二重特異性抗体は、TGF-beta3の異なった2つのエピトープに結合してもよい。二重特異性抗体は、B細胞表面マーカーを発現する細胞にTGF-beta3を局在化するために使用されてもよい。二重特異性抗体は、全長抗体としてまたは抗体断片として調製され得る。 6). Multispecific Antibodies In certain embodiments, the antibodies provided herein are multispecific antibodies (eg, bispecific antibodies). Multispecific antibodies are monoclonal antibodies that have binding specificities at at least two different sites. In certain embodiments, one of the binding specificities is for TGF-beta3 and the other is for any other antigen (eg, a B cell surface marker). In certain embodiments, the bispecific antibody may bind to two different epitopes of TGF-beta3. Bispecific antibodies may be used to localize TGF-beta3 to cells that express a B cell surface marker. Bispecific antibodies can be prepared as full length antibodies or as antibody fragments.
　多重特異性抗体を作製するための手法は、これらに限定されるものではないが、異なる特異性を有する2つの免疫グロブリン重鎖－軽鎖ペアの組み換え共発現（Milstein and Cuello, Nature 305: 537 (1983)、WO93/08829、およびTraunecker et al., EMBO J. 10: 3655 (1991) 参照）、およびknob-in-hole技術（例えば、米国特許第5,731,168号参照）を含む。多重特異性抗体は、Fcヘテロ二量体分子を作製するために静電ステアリング効果 (electrostatic steering effects) を操作すること (WO2009/089004A1)；2つ以上の抗体または断片を架橋すること（米国特許第4,676,980号およびBrennan et al., Science, 229: 81 (1985)参照）；ロイシンジッパーを用いて2つの特異性を有する抗体を作成すること（Kostelny et al., J. Immunol., 148(5):1547-1553 (1992) 参照）；「ダイアボディ」技術を用いて二重特異性抗体断片を作製すること（Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993) 参照）；および、単鎖Fv (scFv) 二量体を用いること（Gruber et al., J. Immunol., 152:5368 (1994) 参照）；および、例えばTutt et al. J. Immunol. 147: 60 (1991) に記載されるように三重特異性抗体を調製すること、によって作製してもよい。 Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (Milstein and Cuello, Nature 305: 537 (1983), WO 93/08829, and Traunecker et al., EMBO J. 10: 3655 (1991)), and the knob-in-hole technology (see, eg, US Pat. No. 5,731,168). Multispecific antibodies can manipulate electrostatic steering effects to create Fc heterodimeric molecules (WO2009 / 089004A1); bridge two or more antibodies or fragments (US patents) No. 4,676,980 and Brennan et al., Science, 229: 81 (1985)); making antibodies with two specificities using leucine zippers (Kostelny et al., J. Immunol., 148 (5 ): 1547-1553 (1992) ；); using the “diabody” technique to generate bispecific antibody fragments (Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993)）); and using single chain Fv (scFv) dimers (see Gruber et al., J. Immunol., 152: 5368 (1994)); and, for example, Tutt et al. J. Immunol It may be made by preparing a trispecific antibody as described in 147: 60 (1991).
　「オクトパス抗体」を含む、3つ以上の機能的抗原結合部位を伴う改変抗体も、本明細書では含まれる（例えば、米国特許出願公開第2006/0025576号A1参照）。 Modified antibodies with three or more functional antigen binding sites, including “Octopus antibodies” are also included herein (see, eg, US Patent Application Publication No. 2006/0025576 A1).
　本明細書で抗体または断片は、TGF-beta3と別の異なる抗原とに結合する1つの抗原結合部位を含む、「デュアルアクティングFab」または「DAF」も含む（例えば、米国特許出願公開第2008/0069820号参照）。 As used herein, an antibody or fragment also includes a “dual-acting Fab” or “DAF” that contains one antigen-binding site that binds TGF-beta3 to another different antigen (eg, US Patent Application Publication No. 2008 / 0069820).
７．抗体変異体
　特定の態様において、本明細書で提供される抗体のアミノ酸配列変異体も、考慮の内である。例えば、抗体の結合アフィニティおよび／または他の生物学的特性を改善することが、望ましいこともある。抗体のアミノ酸配列変異体は、抗体をコードするヌクレオチド配列に適切な修飾を導入すること、または、ペプチド合成によって、調製されてもよい。そのような修飾は、例えば、抗体のアミノ酸配列からの欠失、および／または抗体のアミノ酸配列中への挿入、および／または抗体のアミノ酸配列中の残基の置換を含む。最終構築物が所望の特徴（例えば、抗原結合性）を備えることを前提に、欠失、挿入、および置換の任意の組合せが、最終構築物に至るために行われ得る。 7). Antibody Variants In certain embodiments, amino acid sequence variants of the antibodies provided herein are also contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibody may be prepared by introducing appropriate modifications to the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletion from the amino acid sequence of the antibody, and / or insertion into the amino acid sequence of the antibody, and / or substitution of residues in the amino acid sequence of the antibody. Given that the final construct is provided with the desired characteristics (eg, antigen binding), any combination of deletions, insertions, and substitutions can be made to arrive at the final construct.
ａ）置換、挿入、および欠失変異体
　特定の態様において、1つまたは複数のアミノ酸置換を有する抗体変異体が提供される。置換的変異導入の目的部位は、HVRおよびFRを含む。保存的置換を、表１の「好ましい置換」の見出しの下に示す。より実質的な変更を、表１の「例示的な置換」の見出しの下に提供するとともに、アミノ酸側鎖のクラスに言及しつつ下で詳述する。アミノ酸置換は目的の抗体に導入されてもよく、産物は、例えば、保持／改善された抗原結合性、減少した免疫原性、減少した中和活性、改善した抗原特異性、または改善した血中滞留性などの、所望の活性についてスクリーニングされてもよい。 a) Substitution, insertion and deletion variants In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Target sites for substitutional mutagenesis include HVR and FR. Conservative substitutions are shown under the heading “Preferred substitutions” in Table 1. More substantial changes are provided under the heading “Exemplary substitutions” in Table 1 and are detailed below with reference to the class of amino acid side chains. Amino acid substitutions may be introduced into the antibody of interest and the product may be, for example, retained / improved antigen binding, decreased immunogenicity, decreased neutralizing activity, improved antigen specificity, or improved blood It may be screened for a desired activity, such as retention.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　アミノ酸は、共通の側鎖特性によって群に分けることができる：
(1) 疎水性：ノルロイシン、メチオニン (Met)、アラニン (Ala)、バリン (Val)、ロイシン (Leu)、イソロイシン (Ile)；
(2) 中性の親水性：システイン (Cys)、セリン (Ser)、トレオニン (Thr)、アスパラギン (Asn)、グルタミン (Gln)；
(3) 酸性：アスパラギン酸 (Asp)、グルタミン酸 (Glu)；
(4) 塩基性：ヒスチジン (His)、リジン (Lys)、アルギニン (Arg)；
(5) 鎖配向に影響する残基：グリシン (Gly)、プロリン (Pro)；
(6) 芳香族性：トリプトファン (Trp)、チロシン (Tyr)、フェニルアラニン (Phe)。
非保存的置換は、これらのクラスの1つのメンバーを、別のクラスのものに交換することをいう。 Amino acids can be grouped according to common side chain properties:
(1) Hydrophobicity: norleucine, methionine (Met), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile);
(2) Neutral hydrophilicity: cysteine (Cys), serine (Ser), threonine (Thr), asparagine (Asn), glutamine (Gln);
(3) Acidity: Aspartic acid (Asp), glutamic acid (Glu);
(4) Basicity: histidine (His), lysine (Lys), arginine (Arg);
(5) Residues that affect chain orientation: Glycine (Gly), Proline (Pro);
(6) Aromaticity: Tryptophan (Trp), Tyrosine (Tyr), Phenylalanine (Phe).
Non-conservative substitutions refer to exchanging one member of these classes for another class.
　置換変異体の1つのタイプは、親抗体（例えば、ヒト化またはヒト抗体）の1つまたは複数の超可変領域残基の置換を含む。通常、その結果として生じ、さらなる研究のために選ばれた変異体は、親抗体と比較して特定の生物学的特性における修飾（例えば、改善）（例えば、増加したアフィニティ、減少した免疫原性）を有する、および／または親抗体の特定の生物学的特性を実質的に保持しているであろう。例示的な置換変異体は、アフィニティ成熟抗体であり、これは、例えばファージディスプレイベースのアフィニティ成熟技術（例えば本明細書に記載されるもの）を用いて適宜作製され得る。簡潔に説明すると、1つまたは複数のHVR残基を変異させ、そして変異抗体をファージ上に提示させ、特定の生物学的活性（例えば、結合アフィニティ）に関してスクリーニングを行う。 One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (eg, a humanized or human antibody). Usually, the resulting mutants selected for further study are modified (eg, improved) in specific biological properties compared to the parent antibody (eg, increased affinity, decreased immunogenicity). And / or substantially retain certain biological properties of the parent antibody. An exemplary substitution variant is an affinity matured antibody, which can be made as appropriate using, for example, phage display-based affinity maturation techniques (eg, those described herein). Briefly, one or more HVR residues are mutated and the mutated antibody is displayed on a phage and screened for a specific biological activity (eg, binding affinity).
　改変（例えば、置換）は、例えば抗体のアフィニティを改善するために、HVRにおいて行われ得る。そのような改変は、HVRの「ホットスポット」、すなわち、体細胞成熟プロセスの間に高頻度で変異が起こるコドンによってコードされる残基（例えば、Chowdhury, Methods Mol. Biol. 207:179-196 (2008) を参照のこと）および／または抗原に接触する残基において行われ得、得られた変異VHまたはVLが結合アフィニティに関して試験され得る。二次ライブラリからの構築および再選択によるアフィニティ成熟が、例えば、Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)) に記載されている。アフィニティ成熟のいくつかの態様において、多様性は、任意の様々な方法（例えば、エラープローンPCR、チェーンシャッフリングまたはオリゴヌクレオチド指向変異導入）によって成熟のために選択された可変遺伝子に導入される。次いで、二次ライブラリが作製される。次いで、このライブラリは、所望のアフィニティを有する任意の抗体変異体を同定するためにスクリーニングされる。多様性を導入する別の方法は、いくつかのHVR残基（例えば、一度に4～6残基）を無作為化するHVR指向アプローチを含む。抗原結合に関与するHVR残基は、例えばアラニンスキャニング変異導入またはモデリングを用いて、具体的に特定され得る。特に、CDR-H3およびCDR-L3がしばしば標的化される。 Modifications (eg, substitutions) can be made in HVRs, for example, to improve antibody affinity. Such modifications are HVR “hot spots”, ie, residues encoded by codons that mutate frequently during the somatic maturation process (eg, Chowdhury, Methods Mol. Biol. 207: 179-196 (See 2008)）) and / or in residues that contact antigen, and the resulting mutant VH or VL can be tested for binding affinity. Affinity maturation by construction and reselection from secondary libraries is described, for example, by Hoogenboom et al. In Methods in Molecular Biology 178: 1-37 (O'Brien et al., Ed., Human Press, Totowa, NJ, (2001 )) It is described in. In some embodiments of affinity maturation, diversity is introduced into variable genes selected for maturation by any of a variety of methods (eg, error-prone PCR, chain shuffling or oligonucleotide-directed mutagenesis). A secondary library is then created. This library is then screened to identify any antibody variants with the desired affinity. Another way to introduce diversity involves an HVR-oriented approach that randomizes several HVR residues (eg, 4-6 residues at a time). HVR residues involved in antigen binding can be specifically identified using, for example, alanine scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are often targeted.
　特定の態様において、置換、挿入、または欠失は、そのような改変が抗原に結合する抗体の能力を実質的に減少させない限り、1つまたは複数のHVR内で行われ得る。例えば、結合アフィニティを実質的に減少させない保存的改変（例えば、本明細書で提供されるような保存的置換）が、HVRにおいて行われ得る。そのような改変は、例えば、HVRの抗原接触残基の外側であり得る。上記の変異VHおよびVL配列の特定の態様において、各HVRは改変されていないか、わずか1つ、2つ、もしくは3つのアミノ酸置換を含む。 In certain embodiments, substitutions, insertions, or deletions can be made in one or more HVRs as long as such modification does not substantially reduce the ability of the antibody to bind to the antigen. For example, conservative modifications that do not substantially reduce binding affinity (eg, conservative substitutions as provided herein) can be made in HVRs. Such modifications can be, for example, outside of the antigen contact residues of HVR. In certain embodiments of the above mutated VH and VL sequences, each HVR is unaltered or contains as few as 1, 2 or 3 amino acid substitutions.
　変異導入のために標的化され得る抗体の残基または領域を同定するのに有用な方法は、Cunningham and Wells (1989) Science, 244:1081-1085によって記載される、「アラニンスキャニング変異導入」と呼ばれるものである。この方法において、一残基または一群の標的残基（例えば、荷電残基、例えばアルギニン、アスパラギン酸、ヒスチジン、リジン、およびグルタミン酸）が同定され、中性または負に荷電したアミノ酸（例えば、アラニンもしくはポリアラニン）で置き換えられ、抗体と抗原の相互作用が影響を受けるかどうかが決定される。この初期置換に対して機能的感受性を示したアミノ酸位置に、さらなる置換が導入され得る。あるいはまたは加えて、抗体と抗原の間の接触点を同定するために、抗原抗体複合体の結晶構造を解析してもよい。そのような接触残基および近隣の残基を、置換候補として標的化してもよく、または置換候補から除外してもよい。変異体は、それらが所望の特性を含むかどうかを決定するためにスクリーニングされ得る。 A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is described by Cunningham and Wells (1989) Science, 244: 1081-1085, `` Alanine scanning mutagenesis ''. It is what is called. In this method, a residue or group of target residues (eg, charged residues such as arginine, aspartic acid, histidine, lysine, and glutamic acid) are identified and neutral or negatively charged amino acids (eg, alanine or Polyalanine) to determine whether the antibody-antigen interaction is affected. Additional substitutions can be introduced at amino acid positions that have shown functional sensitivity to this initial substitution. Alternatively or additionally, the crystal structure of the antigen-antibody complex may be analyzed to identify contact points between the antibody and the antigen. Such contact residues and neighboring residues may be targeted as substitution candidates or excluded from substitution candidates. Variants can be screened to determine if they contain the desired property.
　アミノ酸配列の挿入は、配列内部への単一または複数のアミノ酸残基の挿入と同様、アミノ末端および／またはカルボキシル末端における1残基から100残基以上を含むポリペプチドの長さの範囲での融合も含む。末端の挿入の例は、N末端にメチオニル残基を伴う抗体を含む。抗体分子の他の挿入変異体は、抗体のN-またはC-末端に、酵素（例えば、ADEPTのための）または抗体の血漿半減期を増加させるポリペプチドを融合させたものを含む。 Amino acid sequence insertions, as well as insertions of single or multiple amino acid residues within the sequence, are within the length of a polypeptide comprising from 1 to 100 residues or more at the amino and / or carboxyl terminus. Includes fusion. Examples of terminal insertions include antibodies with a methionyl residue at the N-terminus. Other insertional variants of the antibody molecule include those in which the N- or C-terminus of the antibody is fused to an enzyme (eg, for ADEPT) or a polypeptide that increases the plasma half-life of the antibody.
b）Fc領域変異体
　特定の態様において、本明細書で提供される抗体のFc領域に1つまたは複数のアミノ酸修飾を導入して、それによりFc領域変異体を生成してもよい。Fc領域変異体は、1つまたは複数のアミノ酸ポジションのところでアミノ酸修飾（例えば、置換）を含む、ヒトFc領域配列（例えば、ヒトIgG1、IgG2、IgG3、またはIgG4のFc領域）を含んでもよい。 b) Fc region variants In certain embodiments, one or more amino acid modifications may be introduced into the Fc regions of the antibodies provided herein, thereby generating Fc region variants. An Fc region variant may comprise a human Fc region sequence (eg, a human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification (eg, substitution) at one or more amino acid positions.
Fc受容体
　「Fc受容体」または「FcR」は、抗体のFc領域に結合する受容体のことをいう。いくつかの態様において、FcRは、天然型ヒトFcRである。いくつかの態様において、FcRは、IgG抗体に結合するもの（ガンマ受容体）であり、FcγRI、FcγRII、およびFcγRIIIサブクラスの受容体を、これらの受容体の対立遺伝子変異体および選択的スプライシングによる形態を含めて、含む。FcγRII受容体は、FcγRIIA（「活性化受容体」）およびFcγRIIB（「阻害受容体」）を含み、これらは主としてその細胞質ドメインにおいて相違する類似のアミノ酸配列を有する。活性化受容体FcγRIIAは、その細胞質ドメインに免疫受容体チロシン活性化モチーフ (immunoreceptor tyrosine-based activation motif: ITAM) を含む。阻害受容体FcγRIIBは、その細胞質ドメインに免疫受容体チロシン阻害モチーフ（immunoreceptor tyrosine-based inhibition motif: ITIM）を含む。（例えば、Daeron, Annu. Rev. Immunol. 15:203-234 (1997) を参照のこと。）FcRは、例えば、Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991)；Capel et al., Immunomethods 4:25-34 (1994)；およびde Haas et al., J. Lab. Clin. Med 126:330-41 (1995)において総説されている。将来同定されるものを含む他のFcRも、本明細書の用語「FcR」に包含される。 Fc receptor “Fc receptor” or “FcR” refers to a receptor that binds to the Fc region of an antibody. In some embodiments, the FcR is native human FcR. In some embodiments, the FcR is one that binds to an IgG antibody (gamma receptor) and forms FcγRI, FcγRII, and FcγRIII subclass receptors by allelic variants and alternative splicing of these receptors Including, including. FcγRII receptors include FcγRIIA (“activating receptor”) and FcγRIIB (“inhibitory receptor”), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (See, for example, Daeron, Annu. Rev. Immunol. 15: 203-234 (1997).) FcR is, for example, Ravetch and Kinet, Annu. Rev. Immunol 9: 457-92 (1991); Capel et al., Immunomethods 4: 25-34 (1994); and de Haas et al., J. Lab. Clin. Med 126: 330-41 (1995). Other FcRs, including those identified in the future, are also encompassed by the term “FcR” herein.
　用語「Fc受容体」または「FcR」はまた、母体のIgGの胎児への移動（Guyer et al., J. Immunol. 117:587 (1976)およびKim et al., J. Immunol. 24:249 (1994)）ならびに免疫グロブリンのホメオスタシスの調節を担う、新生児型受容体FcRnを含む。FcRnへの結合を測定する方法は公知である（例えば、Ghetie and Ward., Immunol. Today 18(12):592-598 (1997); Ghetie et al., Nature Biotechnology, 15(7):637-640 (1997); Hinton et al., J. Biol. Chem. 279(8):6213-6216 (2004); WO2004/92219 (Hinton et al.)を参照のこと）。 The terms “Fc receptor” or “FcR” also refer to the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117: 587 (1976) and Kim et al., J. Immunol. 24: 249 (1994)) as well as the neonatal receptor FcRn, which is responsible for the regulation of immunoglobulin homeostasis. Methods for measuring binding to FcRn are known (eg, Ghetie and Ward., Immunol. Today 18 (12): 592-598 (1997); Ghetie et al., Nature Biotechnology, 15 (7): 637- 640 (1997); Hinton et al., J. Biol. Chem. 279 (8): 6213-6216 (2004); WO2004 / 92219 (Hinton et al.)).
　インビボでのヒトFcRnへの結合およびヒトFcRn高アフィニティ結合ポリペプチドの血漿半減期は、例えばヒトFcRnを発現するトランスジェニックマウスもしくはトランスフェクトされたヒト細胞株においてまたは変異Fc領域を伴うポリペプチドが投与される霊長類において測定され得る。WO2000/42072 (Presta) は、FcRに対する結合が増加したまたは減少した抗体変異体を記載している。例えば、Shields et al. J. Biol. Chem. 9(2):6591-6604 (2001) も参照のこと。 In vivo binding to human FcRn and the plasma half-life of human FcRn high affinity binding polypeptides can be administered, for example, in transgenic mice expressing human FcRn or transfected human cell lines or administered with a polypeptide with a mutated Fc region. Can be measured in primates. WO2000 / 42072 (Presta) describes antibody variants with increased or decreased binding to FcR. See, for example, Shields et al. J. Biol. Chem. 9 (2): 6591-6604 (2001).
Fc領域含有抗体
　用語「Fc領域含有抗体」は、Fc領域を含む抗体のことをいう。Fc領域のC末端リジン（EUナンバリングシステムにしたがえば残基447）またはFc領域のC末端グリシン－リジン（残基446-447）は、例えば抗体の精製の間にまたは抗体をコードする核酸の組み換え操作によって除去され得る。したがって、本発明によるFc領域を有する抗体を含む組成物は、G446-K447を伴う抗体、G446を伴いK447を伴わない抗体、G446-K447が完全に除去された抗体、または上記3つのタイプの抗体の混合物を含み得る。 Fc region-containing antibody The term “Fc region-containing antibody” refers to an antibody comprising an Fc region. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) or the C-terminal glycine-lysine of the Fc region (residues 446-447) can be used, for example, during antibody purification or of the nucleic acid encoding the antibody. It can be removed by recombination operations. Therefore, a composition comprising an antibody having an Fc region according to the present invention comprises an antibody with G446-K447, an antibody with G446 without K447, an antibody with G446-K447 completely removed, or the above three types of antibodies May be included.
　特定の態様において、すべてではないがいくつかのエフェクター機能を備える抗体変異体も、本発明の考慮の内であり、当該エフェクター機能は、抗体を、そのインビボでの半減期が重要であるが、特定のエフェクター機能（補体およびADCCなど）は不要または有害である場合の適用に望ましい候補とするものである。CDCおよび／またはADCC活性の減少／欠乏を確認するために、インビトロ および／またはインビボ の細胞傷害測定を行うことができる。例えば、Fc受容体（FcR）結合測定は、抗体がFcγR結合性を欠く（よってADCC活性を欠く蓋然性が高い）一方でFcRn結合能を維持することを確かめるために行われ得る。ADCCを媒介するプライマリ細胞であるNK細胞はFcγRIIIのみを発現するが、一方単球はFcγRI、FcγRII、FcγRIIIを発現する。造血細胞上のFcRの発現は、Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991) の第464頁のTable 3にまとめられている。目的の分子のADCC活性を評価するためのインビトロ測定法（アッセイ）の非限定的な例は、米国特許第5,500,362号（例えば、 Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986) 参照）および Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985)；米国特許第5,821,337号（Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987) 参照）に記載されている。あるいは、非放射性の測定法を用いてもよい（例えば、ACT1（商標）non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA)；および、CytoTox 96（登録商標）non-radioactive cytotoxicity assays 法 (Promega, Madison, WI) 参照）。このような測定法に有用なエフェクター細胞は、末梢血単核細胞 (peripheral blood mononuclear cell: PBMC) およびナチュラルキラー (natural killer: NK) 細胞を含む。あるいはまたは加えて、目的の分子のADCC活性は、例えば、Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998) に記載されるような動物モデルにおいて、インビボで評価されてもよい。また、抗体がC1qに結合できないこと、よってCDC活性を欠くことを確認するために、C1q結合測定を行ってもよい。例えば、WO2006/029879 および WO2005/100402のC1qおよびC3c結合ELISAを参照のこと。また、補体活性化を評価するために、CDC測定を行ってもよい（例えば、Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996)；Cragg, M.S. et al., Blood 101:1045-1052 (2003)；およびCragg, M.S. and M.J. Glennie, Blood 103:2738-2743 (2004) 参照）。さらに、FcRn結合性およびインビボでのクリアランス／半減期の決定も、当該技術分野において知られた方法を用いて行い得る（例えばPetkova, S.B. et al., Int'l. Immunol. 18(12):1759-1769 (2006) 参照）。 In certain embodiments, antibody variants with some, but not all, effector functions are also within the scope of the present invention, which effector functions with respect to its in vivo half-life, Certain effector functions (such as complement and ADCC) are good candidates for application when they are unnecessary or harmful. In vitro sputum and / or in vivo sputum cytotoxicity measurements can be performed to confirm a decrease / deficiency of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding measurements can be performed to ensure that antibodies lack FcγR binding (and thus are more likely to lack ADCC activity) while maintaining FcRn binding ability. NK cells, which are primary cells that mediate ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. The expression of FcR on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9: 457-492 (1991). Non-limiting examples of in vitro assays (assays) for assessing ADCC activity of a molecule of interest include US Pat. No. 5,500,362 (eg, Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA). 83: 7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82: 1499-1502 (1985); U.S. Patent No. 5,821,337 (Bruggemann, M. et al. , J. Exp. Med. 166: 1351-1361 (1987)). Alternatively, non-radioactive assays may be used (eg, ACT1 ™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA); and CytoTox 96® non-radioactive cytotoxicity assays (see Promega, Madison, WI). Effector cells useful for such measurement methods include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or in addition, the ADCC activity of the molecule of interest is assessed in vivo in an animal model as described, for example, in Clynes et al. Proc. Nat'l Acad. Sci. USA 95: 652-656 (1998). May be. Also, C1q binding measurements may be performed to confirm that the antibody cannot bind to C1q and thus lacks CDC activity. See, for example, the C1q and C3c binding ELISA in WO2006 / 029879 and WO2005 / 100402. CDC measurements may also be performed to assess complement activation (eg, Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996); Cragg, MS et al., Blood 101 : 1045-1052 (2003); and Cragg, MS MS and MJ Glennie, Blood 103: 2738-2743 (2004)). Furthermore, determination of FcRn binding and in vivo clearance / half-life can also be performed using methods known in the art (eg, Petkova, SB et al., Int'l. Immunol. 18 (12): 1759-1769 (2006) IV).
　減少したエフェクター機能を伴う抗体は、Fc領域残基238、265、269、270、297、327、および329の1つまたは複数の置換を伴うものを含む（米国特許第6,737,056号）。このようなFc変異体は、残基265および297のアラニンへの置換を伴ういわゆる「DANA」Fc変異体（米国特許第7,332,581号）を含む、アミノ酸ポジション265、269、270、297、および327の2つ以上の置換を伴うFc変異体を含む。 Antibodies with reduced effector function include those with one or more substitutions of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (US Pat. No. 6,737,056). Such Fc variants include amino acid positions 265, 269, 270, 297, and 327, including so-called “DANA” Fc variants (US Pat. No. 7,332,581) with substitution of residues 265 and 297 to alanine. Includes Fc variants with two or more substitutions.
　FcRsへの増加または減少した結合性を伴う特定の抗体変異体が、記述されている。（米国特許第6,737,056号；WO2004/056312、およびShields et al., J. Biol. Chem. 9(2): 6591-6604 (2001) を参照のこと。） Specific antibody variants with increased or decreased binding to FcRs have been described. (See US Pat. No. 6,737,056; WO2004 / 056312 and Shields et al., J. Biol. Chem. 9 (2): 6591-6604 (2001)).
　いくつかの態様において、例えば米国特許第6,194,551号、WO99/51642、およびIdusogie et al. J. Immunol. 164: 4178-4184 (2000) に記載されるように、改変された（つまり、増加したか減少したかのいずれかである）C1q結合性および／または補体依存性細胞傷害 (CDC) をもたらす改変が、Fc領域においてなされる。 In some embodiments, modified (ie, increased) as described, for example, in US Pat. No. 6,194,551, WO99 / 51642, and Idusogie et al. J. Immunol. 164: 4178-4184 (2000). Modifications are made in the Fc region that result in C1q binding and / or complement dependent cytotoxicity selection (CDC) selection, which is either reduced.
　増加した半減期、および新生児型Fc受容体（FcRn：母体のIgG類を胎児に移行させる役割を負う（Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)））に対する増加した結合性を伴う抗体が、米国特許出願公開第2005/0014934号A1(Hinton et al.) に記載されている。これらの抗体は、Fc領域のFcRnへの結合性を増加する1つまたは複数の置換をその中に伴うFc領域を含む。このようなFc変異体は、Fc領域残基：238、256、265、272、286、303、305、307、311、312、317、340、356、360、362、376、378、380、382、413、424、または434の1つまたは複数のところでの置換（例えば、Fc領域残基434の置換（米国特許第7,371,826号））を伴うものを含む。 Increased half-life and role in transferring neonatal Fc receptors (FcRn: maternal IgGs to the fetus (Guyer et al., J. Immunol. 117: 587 (1976) and Kim et al., J. Antibodies with increased binding to Immunol. 24: 249 (1994))) are described in US Patent Application Publication No. 2005/0014934 A1 (Hinton et al.). These antibodies comprise an Fc region with one or more substitutions therein that increase the binding of the Fc region to FcRn. Such Fc variants include Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382 , 413, 424, or 434 at one or more substitutions (eg, substitution of Fc region residue 434 (US Pat. No. 7,371,826)).
c）抗体誘導体
　特定の態様において、本明細書で提供される抗体は、当該技術分野において知られておりかつ容易に入手可能な追加の非タンパク質部分を含むように、さらに修飾されてもよい。抗体の誘導体化に好適な部分は、これに限定されるものではないが、水溶性ポリマーを含む。水溶性ポリマーの非限定的な例は、これらに限定されるものではないが、ポリエチレングリコール (PEG)、エチレングリコール／プロピレングリコールのコポリマー、カルボキシメチルセルロース、デキストラン、ポリビニルアルコール、ポリビニルピロリドン、ポリ１,3ジオキソラン、ポリ1,3,6,トリオキサン、エチレン／無水マレイン酸コポリマー、ポリアミノ酸（ホモポリマーまたはランダムコポリマーのいずれでも）、および、デキストランまたはポリ（n-ビニルピロリドン）ポリエチレングリコール、ポリプロピレングリコールホモポリマー、ポリプロピレンオキシド／エチレンオキシドコポリマー、ポリオキシエチル化ポリオール類（例えばグリセロール）、ポリビニルアルコール、および、これらの混合物を含む。ポリエチレングリコールプロピオンアルデヒドは、その水に対する安定性のために、製造において有利であるだろう。ポリマーは、いかなる分子量でもよく、枝分かれしていてもしていなくてもよい。抗体に付加されるポリマーの数には幅があってよく、1つ以上のポリマーが付加されるならそれらは同じ分子であってもよいし、異なる分子であってもよい。一般的に、誘導体化に使用されるポリマーの数および／またはタイプは、これらに限定されるものではないが、改善されるべき抗体の特定の特性または機能、抗体誘導体が規定の条件下での療法に使用されるか否か、などへの考慮に基づいて、決定することができる。 c) Antibody Derivatives In certain embodiments, the antibodies provided herein may be further modified to include additional non-protein moieties known in the art and readily available. Suitable moieties for antibody derivatization include, but are not limited to, water soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly 1,3 Dioxolane, poly1,3,6, trioxane, ethylene / maleic anhydride copolymer, polyamino acid (either homopolymer or random copolymer), and dextran or poly (n-vinylpyrrolidone) polyethylene glycol, polypropylene glycol homopolymer, Polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (eg, glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may be advantageous in manufacturing due to its water stability. The polymer may have any molecular weight and may or may not be branched. The number of polymers added to the antibody can vary, and if more than one polymer is added, they can be the same molecule or different molecules. In general, the number and / or type of polymers used for derivatization is not limited to these, but is specific to the antibody's particular characteristics or function to be improved, under conditions where the antibody derivative is under defined conditions. It can be determined based on considerations such as whether or not to use in therapy.
Ｂ．組み換えの方法および構成
　例えば、米国特許第4,816,567号に記載されるとおり、抗体は組み換えの方法や構成を用いて製造することができる。一態様において、本明細書に記載の抗TGF-beta3抗体をコードする、単離された核酸が提供される。そのような核酸は、抗体のVLを含むアミノ酸配列および／またはVHを含むアミノ酸配列（例えば、抗体の軽鎖および／または重鎖）をコードしてもよい。さらなる態様において、このような核酸を含む1つまたは複数のベクター（例えば、発現ベクター）が提供される。さらなる態様において、このような核酸を含む宿主細胞が提供される。このような態様の1つでは、宿主細胞は、(1)抗体のVLを含むアミノ酸配列および抗体のVHを含むアミノ酸配列をコードする核酸を含むベクター、または、(2)抗体のVLを含むアミノ酸配列をコードする核酸を含む第一のベクターと抗体のVHを含むアミノ酸配列をコードする核酸を含む第二のベクターを含む（例えば、形質転換されている）。一態様において、宿主細胞は、真核性である（例えば、チャイニーズハムスター卵巣 (CHO) 細胞）またはリンパ系の細胞（例えば、Y0、NS0、Sp2/0細胞））。一態様において、抗TGF-beta3抗体の発現に好適な条件下で、上述のとおり当該抗体をコードする核酸を含む宿主細胞を培養すること、および任意で、当該抗体を宿主細胞（または宿主細胞培養培地）から回収することを含む、抗TGF-beta3抗体を作製する方法が提供される。 B. Recombinant Methods and Configurations Antibodies can be produced using recombinant methods and configurations, for example, as described in US Pat. No. 4,816,567. In one aspect, an isolated nucleic acid encoding an anti-TGF-beta3 antibody described herein is provided. Such a nucleic acid may encode an amino acid sequence comprising an antibody VL and / or an amino acid sequence comprising a VH (eg, an antibody light and / or heavy chain). In further embodiments, one or more vectors (eg, expression vectors) comprising such nucleic acids are provided. In a further aspect, host cells comprising such nucleic acids are provided. In one such embodiment, the host cell comprises (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the antibody VL and an amino acid sequence comprising the antibody VH, or (2) an amino acid comprising the antibody VL. A first vector comprising a nucleic acid encoding sequence and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the antibody VH are included (eg, transformed). In one embodiment, the host cell is eukaryotic (eg, Chinese hamster ovary (CHO) cells) or lymphoid cells (eg, Y0, NS0, Sp2 / 0 cells)). In one embodiment, culturing a host cell comprising a nucleic acid encoding the antibody as described above under conditions suitable for expression of the anti-TGF-beta3 antibody, and optionally, the antibody is transformed into a host cell (or host cell culture). A method for producing an anti-TGF-beta3 antibody is provided, which comprises recovering from the medium).
　抗TGF-beta3抗体の組み換え製造のために、（例えば、上述したものなどの）抗体をコードする核酸を単離し、さらなるクローニングおよび／または宿主細胞中での発現のために、1つまたは複数のベクターに挿入する。そのような核酸は、従来の手順を用いて容易に単離および配列決定されるだろう（例えば、抗体の重鎖および軽鎖をコードする遺伝子に特異的に結合することができるオリゴヌクレオチドプローブを用いることで）。 For recombinant production of an anti-TGF-beta3 antibody, a nucleic acid encoding the antibody (eg, as described above) is isolated and one or more for further cloning and / or expression in a host cell. Insert into vector. Such nucleic acids will be readily isolated and sequenced using conventional procedures (eg, oligonucleotide probes that can specifically bind to the genes encoding the antibody heavy and light chains). By using).
　抗体をコードするベクターのクローニングまたは発現に好適な宿主細胞は、本明細書に記載の原核細胞または真核細胞を含む。例えば、抗体は、特にグリコシル化およびFcエフェクター機能が必要とされない場合は、細菌で製造してもよい。細菌での抗体断片およびポリペプチドの発現に関して、例えば、米国特許第5,648,237号、第5,789,199号、および第5,840,523号を参照のこと。（加えて、大腸菌における抗体断片の発現について記載したCharlton, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ, 2003), pp.245-254も参照のこと。）発現後、抗体は細菌細胞ペーストから可溶性フラクション中に単離されてもよく、またさらに精製することができる。 Suitable host cells for cloning or expression of antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies may be produced in bacteria, particularly where glycosylation and Fc effector function are not required. See, eg, US Pat. Nos. 5,648,237, 5,789,199, and 5,840,523 for expression of antibody fragments and polypeptides in bacteria. (In addition, see also Charlton, Methods Molecular Biology, Vol.BK248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp.245-254, which describes the expression of antibody fragments in E. coli. ) After expression, the antibody may be isolated from the bacterial cell paste in a soluble fraction and can be further purified.
　原核生物に加え、部分的なまたは完全なヒトのグリコシル化パターンを伴う抗体の産生をもたらす、グリコシル化経路が「ヒト化」されている菌類および酵母の株を含む、糸状菌または酵母などの真核性の微生物は、抗体コードベクターの好適なクローニングまたは発現宿主である。Gerngross, Nat. Biotech. 22:1409-1414 (2004)および Li et al., Nat. Biotech. 24:210-215 (2006) を参照のこと。 In addition to prokaryotes, true species such as filamentous fungi or yeast, including fungal and yeast strains in which the glycosylation pathway is “humanized”, resulting in the production of antibodies with partial or complete human glycosylation patterns. Nuclear microorganisms are suitable cloning or expression hosts for antibody-encoding vectors. See Gerngross, Nat. Biotech. 22: 1409-1414 (2004) and Li et al., Nat. Biotech. 24: 210-215 (2006).
　多細胞生物（無脊椎生物および脊椎生物）に由来するものもまた、グリコシル化された抗体の発現のために好適な宿主細胞である。無脊椎生物細胞の例は、植物および昆虫細胞を含む。昆虫細胞との接合、特にSpodoptera frugiperda細胞の形質転換に用いられる、数多くのバキュロウイルス株が同定されている。 Those derived from multicellular organisms (invertebrate and vertebrate organisms) are also suitable host cells for the expression of glycosylated antibodies. Examples of invertebrate cells include plant and insect cells. A number of baculovirus strains have been identified that are used for mating with insect cells, particularly for transformation of Spodoptera frugiperda cells.
　植物細胞培養物も、宿主として利用することができる。例えば、米国特許第5,959,177号、第6,040,498号、第6,420,548号、第7,125,978号、および第6,417,429号（トランスジェニック植物で抗体を産生するための、PLANTIBODIES（商標）技術を記載）を参照のこと。 Plant cell cultures can also be used as hosts. See, for example, US Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (described PLANTIBODIES ™ technology for producing antibodies in transgenic plants).
　脊椎動物細胞もまた宿主として使用できる。例えば、浮遊状態で増殖するように適応された哺乳動物細胞株は、有用であろう。有用な哺乳動物宿主細胞株の他の例は、SV40で形質転換されたサル腎CV1株 (COS-7)；ヒト胎児性腎株（Graham et al., J. Gen Virol. 36:59 (1977) などに記載の293または293細胞）；仔ハムスター腎細胞 (BHK)；マウスセルトリ細胞（Mather, Biol. Reprod. 23:243-251 (1980) などに記載のTM4細胞）；サル腎細胞 (CV1)；アフリカミドリザル腎細胞 (VERO-76)；ヒト子宮頸部癌細胞 (HELA)；イヌ腎細胞 (MDCK)；Buffalo系ラット肝細胞 (BRL 3A)；ヒト肺細胞 (W138)；ヒト肝細胞 (Hep G2)；マウス乳癌 (MMT 060562)；TRI細胞（例えば、Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982) に記載）；MRC5細胞；および、FS4細胞などである。他の有用な哺乳動物宿主細胞株は、DHFR－ CHO細胞 (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)) を含むチャイニーズハムスター卵巣 (CHO) 細胞；およびY0、NS0、およびSp2/0などの骨髄腫細胞株を含む。抗体産生に好適な特定の哺乳動物宿主細胞株の総説として、例えば、Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003) を参照のこと。 Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to grow in suspension will be useful. Other examples of useful mammalian host cell lines include monkey kidney CV1 strain (COS-7) transformed with SV40; human embryonic kidney strain (Graham et al., J. Gen Virol. 36:59 (1977 293 or 293 cells as described in); pup hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells as described in Mather, Biol. Reprod. 23: 243-251 (1980) ；); monkey kidney cell (CV1 ); African green monkey kidney cell line (VERO-76); Human cervical cancer cell line (HELA); Canine kidney cell line (MDCK); Buffalo rat liver cell line (BRL 3A); Human lung cell line (W138); Hep G2); mouse breast cancer (MMT 060562); TRI cells (for example, described in Mather et al., Annals NY Acad. Sci. 383: 44-68 (1982)); MRC5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR- CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77: 4216 (1980)); and Y0, Includes myeloma cell lines such as NS0 and Sp2 / 0. For a review of specific mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003) See.
Ｃ．測定法（アッセイ）
　本明細書で提供される抗TGF-beta3抗体は、当該技術分野において知られている種々の測定法によって、同定され、スクリーニングされ、または物理的／化学的特性および／または生物学的活性について明らかにされてもよい。 C. Measurement method (assay)
The anti-TGF-beta3 antibodies provided herein can be identified, screened, or revealed for physical / chemical properties and / or biological activity by various assays known in the art May be.
１．結合測定法およびその他の測定法
　一局面において、本発明の抗体は、例えばELISA、ウエスタンブロット等の公知の方法によって、その抗原結合活性に関して試験される。 1. Binding Assays and Other Assays In one aspect, the antibodies of the present invention are tested for their antigen binding activity by known methods such as ELISA, Western blot, and the like.
　別の局面において、TGF-beta3への結合に関して複数種の抗TGF-beta3抗体同志の競合を評価するために、競合アッセイが使用され得る。特定の態様において、互いに競合すると評価された抗体は、いずれも同じエピトープ（例えば、線状または立体構造エピトープ）に結合すると評価することができる。抗体が結合するエピトープをマッピングする、詳細な例示的方法は、Morris (1996) "Epitope Mapping Protocols," in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ)に提供されている。 In another aspect, a competition assay can be used to assess the competition of multiple anti-TGF-beta3 antibodies for binding to TGF-beta3. In certain embodiments, antibodies that are evaluated to compete with each other can be evaluated to bind to the same epitope (eg, a linear or conformational epitope). Detailed exemplary methods for mapping the epitope to which an antibody binds are provided in Morris (1996) "Epitope Mapping Protocols," in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ).
　例示的な競合アッセイにおいて、固定化されたTGF-beta3は、TGF-beta3に結合する第1の標識された抗体およびTGF-beta3への結合に関して第1の抗体と競合する能力に関して試験される第2の未標識の抗体を含む溶液中でインキュベートされる。第2の抗体は、ハイブリドーマ上清に存在し得る。対照として、固定化されたTGF-beta3が、第1の標識された抗体を含むが第2の未標識の抗体を含まない溶液中でインキュベートされる。第1の抗体のTGF-beta3に対する結合を許容する条件下でのインキュベーションの後、余分な未結合の抗体が除去され、固定化されたTGF-beta3に結合した標識の量が測定される。固定化されたTGF-beta3に結合した標識の量が対照サンプルと比較して試験サンプルにおいて実質的に減少している場合、それは第2の抗体がTGF-beta3への結合に関して第1の抗体と競合していることを示す。Harlow and Lane (1988) Antibodies:  A Laboratory Manual ch.14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) を参照のこと。 In an exemplary competition assay, immobilized TGF-beta3 is tested for the ability to compete with the first labeled antibody that binds to TGF-beta3 and the first antibody for binding to TGF-beta3. Incubate in solution containing 2 unlabeled antibodies. The second antibody can be present in the hybridoma supernatant. As a control, immobilized TGF-beta3 is incubated in a solution containing the first labeled antibody but no second unlabeled antibody. After incubation under conditions that allow binding of the first antibody to TGF-beta3, excess unbound antibody is removed and the amount of label bound to immobilized TGF-beta3 is measured. If the amount of label bound to the immobilized TGF-beta3 is substantially reduced in the test sample compared to the control sample, it indicates that the second antibody is different from the first antibody for binding to TGF-beta3. Indicates that there is a conflict. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch.14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).
実質的に減少した
　本明細書で用いられる表現「実質的に減少した」または「実質的に異なる」は、2つの数値の間（通常、ある分子に関するものと、参照／比較用分子に関するものの間）の差が、当業者がそれら2つの数値の間の差が該数値（例えば、Kd値）によって測定される生物学的特徴の観点で統計学的に有意であるとみなす程度に、充分に大きいことをいう。 Substantially reduced As used herein, the expression “substantially reduced” or “substantially different” means between two numbers (usually for one molecule and for a reference / comparison molecule). The difference between the two numbers is considered sufficiently statistically to be considered statistically significant in terms of the biological characteristics measured by the number (eg, Kd value). It ’s big.
２．機能測定法
　一局面において、所定の機能（プロファイル）を有する抗TGF-beta3抗体のそれを同定するための測定法が提供される。当該機能（プロファイル）は、例えば、（１）TGF-beta3に対し非中和であること（Non-Neutralizing）；（２）TGF-beta1, 2, 3の中でTGF-beta3に特異的な結合活性を有すること（TGFb3-specific）；および任意で（３）Mature体だけでなく、Latent体のTGF-beta3にも結合できること（Latent-Binder）を含んでよい。また、このような機能（プロファイル）をインビボおよび／またはインビトロで有する抗体が、提供される。 2. Functional measurement method In one aspect, a measurement method for identifying an anti-TGF-beta3 antibody having a predetermined function (profile) is provided. The function (profile) is, for example, (1) non-neutralizing against TGF-beta3 (Non-Neutralizing); (2) TGF-beta1, 2, 3 binding specific to TGF-beta3 It may include activity (TGFb3-specific); and optionally (3) ability to bind not only to the mature form but also to the TGF-beta3 of the Latent form (Latent-Binder). An antibody having such a function (profile) in vivo and / or in vitro is also provided.
　特定の態様において、本発明の抗体は、このような機能（プロファイル）について試験される。例示的な試験方法としては、実施例に記載する方法が挙げられるが、これらに限定されない。 In a particular embodiment, the antibodies of the invention are tested for such a function (profile). Exemplary test methods include, but are not limited to, the methods described in the examples.
Ｄ．薬学的製剤
　本明細書に記載の抗TGF-beta3抗体の薬学的製剤は、所望の純度を有する抗体を、1つまたは複数の任意の薬学的に許容される担体 (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)) と混合することによって、凍結乾燥製剤または水溶液の形態で、調製される。薬学的に許容される担体は、概して、用いられる際の用量および濃度ではレシピエントに対して非毒性であり、これらに限定されるものではないが、以下のものを含む：リン酸塩、クエン酸塩、および他の有機酸などの緩衝液；アスコルビン酸およびメチオニンを含む、抗酸化剤；保存料（オクタデシルジメチルベンジル塩化アンモニウム；塩化ヘキサメトニウム；塩化ベンザルコニウム；塩化ベンゼトニウム；フェノール、ブチル、またはベンジルアルコール；メチルまたはプロピルパラベンなどのアルキルパラベン；カテコール；レソルシノール；シクロヘキサノール；3-ペンタノール；およびm-クレゾールなど）；低分子（約10残基未満）ポリペプチド；血清アルブミン、ゼラチン、または免疫グロブリンなどのタンパク質；ポリビニルピロリドンなどの親水性ポリマー；グリシン、グルタミン、アスパラギン、ヒスチジン、アルギニン、またはリジンなどのアミノ酸；グルコース、マンノース、またはデキストリンを含む、単糖、二糖、および他の炭水化物；EDTAなどのキレート剤；スクロース、マンニトール、トレハロース、ソルビトールなどの、砂糖類；ナトリウムなどの塩形成対イオン類；金属錯体（例えば、Zn-タンパク質錯体）；および／またはポリエチレングリコール (PEG) などの非イオン系表面活性剤。本明細書の例示的な薬学的に許容される担体は、さらに、可溶性中性活性型ヒアルロニダーゼ糖タンパク質 (sHASEGP)（例えば、rHuPH20 (HYLENEX（登録商標）、Baxter International, Inc.) などのヒト可溶性PH-20ヒアルロニダーゼ糖タンパク質）などの間質性薬剤分散剤を含む。特定の例示的sHASEGPおよびその使用方法は（rHuPH20を含む）、米国特許出願公開第2005/0260186号および第2006/0104968号に記載されている。一局面において、sHASEGPは、コンドロイチナーゼなどの1つまたは複数の追加的なグリコサミノグリカナーゼと組み合わせられる。 D. Pharmaceutical Formulations The anti-TGF-beta3 antibody pharmaceutical formulations described herein can be prepared by administering an antibody having a desired purity to one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol , A. Ed. (1980)) in the form of a lyophilized formulation or an aqueous solution. Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed, including but not limited to the following: phosphate, citric acid Buffers such as acid salts and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, Or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); small molecule (less than about 10 residues) polypeptide; serum albumin, gelatin, or Proteins such as immunoglobulins; polyvinyl Hydrophilic polymers such as loridone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; Nonionic surfactants such as sugars; salt-forming counterions such as sodium; metal complexes (eg, Zn-protein complexes); and / or polyethylene glycol (PEG), such as mannitol, trehalose, sorbitol. Exemplary pharmaceutically acceptable carriers herein further include a soluble neutral active hyaluronidase glycoprotein (sHASEGP) (eg, rHuPH20 (HYLENEX®, Baxter International, Inc.) PH-20 hyaluronidase glycoprotein) and other interstitial drug dispersants. Certain exemplary sHASEGPs and their methods of use (including rHuPH20) are described in US Patent Application Publication Nos. 2005/0260186 and 2006/0104968. In one aspect, sHASEGP is combined with one or more additional glycosaminoglycanases such as chondroitinase.
　例示的な凍結乾燥抗体製剤は、米国特許第6,267,958号に記載されている。水溶液抗体製剤は、米国特許第6,171,586号およびWO2006/044908に記載のものを含み、後者の製剤はヒスチジン－アセテート緩衝液を含んでいる。 An exemplary lyophilized antibody formulation is described in US Pat. No. 6,267,958. Aqueous antibody formulations include those described in US Pat. No. 6,171,586 and WO2006 / 044908, the latter formulations containing histidine-acetate buffer.
　本明細書の製剤は、治療される特定の適応症のために必要であれば1つより多くの有効成分を含んでもよい。互いに悪影響を与えあわない相補的な活性を伴うものが好ましい。 The formulations herein may contain more than one active ingredient as necessary for the particular indication being treated. Those with complementary activities that do not adversely affect each other are preferred.
　有効成分は、例えば液滴形成（コアセルベーション）手法によってまたは界面重合によって調製されたマイクロカプセル（それぞれ、例えば、ヒドロキシメチルセルロースまたはゼラチンマイクロカプセル、およびポリ（メタクリル酸メチル）マイクロカプセル）に取り込まれてもよいし、コロイド状薬剤送達システム（例えば、リポソーム、アルブミン小球体、マイクロエマルション、ナノ粒子、およびナノカプセル）に取り込まれてもよいし、マクロエマルションに取り込まれてもよい。このような手法は、Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980) に開示されている。 The active ingredients are incorporated into microcapsules prepared by, for example, droplet formation (coacervation) techniques or by interfacial polymerization (for example, hydroxymethylcellulose or gelatin microcapsules and poly (methyl methacrylate) microcapsules, respectively). Alternatively, it may be incorporated into colloidal drug delivery systems (eg, liposomes, albumin spherules, microemulsions, nanoparticles, and nanocapsules) or macroemulsions. Such a technique is disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).
　徐放性製剤を調製してもよい。徐放性製剤の好適な例は、抗体を含んだ固体疎水性ポリマーの半透過性マトリクスを含み、当該マトリクスは例えばフィルムまたはマイクロカプセルなどの造形品の形態である。 Sustained release preparations may be prepared. Suitable examples of sustained release formulations include a semi-permeable matrix of solid hydrophobic polymer containing antibodies, which matrix is in the form of shaped articles such as films or microcapsules.
　生体内 (in vivo) 投与のために使用される製剤は、通常無菌である。無菌状態は、例えば滅菌ろ過膜を通して濾過することなどにより、容易に達成される。 The preparations used for in vivo administration are usually sterile. Aseptic conditions are easily achieved, for example, by filtration through sterile filtration membranes.
Ｅ．治療的方法および治療用組成物
　本明細書で提供される抗TGF-beta3抗体のいずれも、治療的な方法において使用されてよい。
　一局面において、医薬品としての使用のための、抗TGF-beta3抗体が提供される。さらなる局面において、自己免疫疾患の治療における使用のための、抗TGF-beta3抗体が提供される。特定の態様において、治療方法における使用のための、抗TGF-beta3抗体が提供される。特定の態様において、本発明は、TGF-beta3を有する個体を治療する方法であって、当該個体に抗TGF-beta3抗体の有効量を投与する工程を含む方法における使用のための、抗TGF-beta3抗体を提供する。このような態様の1つにおいて、方法は、当該個体に少なくとも1つの（例えば後述するような）追加治療剤の有効量を投与する工程を、さらに含む。さらなる態様において、本発明は、B細胞活性化の抑制における使用のための抗TGF-beta3抗体を提供する。特定の態様において、本発明は、個体においてB細胞活性化を抑制する方法であって、B細胞活性化を抑制するために当該個体に抗TGF-beta3抗体の有効量を投与する工程を含む方法における使用のための、抗TGF-beta3抗体を提供する。上記態様の任意のものによる「個体」は、好適にはヒトである。 E. Therapeutic methods and therapeutic compositions Any of the anti-TGF-beta3 antibodies provided herein may be used in therapeutic methods.
In one aspect, anti-TGF-beta3 antibodies are provided for use as pharmaceuticals. In a further aspect, anti-TGF-beta3 antibodies are provided for use in the treatment of autoimmune diseases. In certain embodiments, anti-TGF-beta3 antibodies are provided for use in therapeutic methods. In certain embodiments, the present invention provides a method for treating an individual having TGF-beta3, comprising the step of administering to the individual an effective amount of an anti-TGF-beta3 antibody, for use in the method. Provide beta3 antibody. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent (eg, as described below). In a further aspect, the present invention provides an anti-TGF-beta3 antibody for use in inhibiting B cell activation. In certain embodiments, the present invention is a method of inhibiting B cell activation in an individual, comprising administering an effective amount of an anti-TGF-beta3 antibody to the individual to inhibit B cell activation An anti-TGF-beta3 antibody for use in is provided. An “individual” according to any of the above embodiments is preferably a human.
　さらなる局面において、本発明は医薬品の製造または調製における抗TGF-beta3抗体の使用を提供する。一態様において、医薬品は、自己免疫疾患の治療のためのものである。さらなる態様において、医薬品は、自己免疫疾患を治療する方法であって、自己免疫疾患を有する個体に医薬品の有効量を投与する工程を含む方法における使用のためのものである。このような態様の1つにおいて、方法は、当該個体に少なくとも1つの（例えば後述するような）追加治療剤の有効量を投与する工程を、さらに含む。さらなる態様において、医薬品は、B細胞活性化の抑制のためのものである。さらなる態様において、医薬品は、個体においてB細胞活性化を抑制する方法であって、B細胞活性化を抑制するために当該個体に医薬品の有効量を投与する工程を含む方法における使用のためのものである。上記態様の任意のものによる「個体」は、ヒトであってもよい。 In a further aspect, the present invention provides the use of an anti-TGF-beta3 antibody in the manufacture or preparation of a medicament. In one aspect, the medicament is for the treatment of an autoimmune disease. In a further aspect, the medicament is for use in a method of treating an autoimmune disease comprising administering an effective amount of the medicament to an individual having an autoimmune disease. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent (eg, as described below). In a further embodiment, the medicament is for suppression of B cell activation. In a further aspect, the medicament is for use in a method of inhibiting B cell activation in an individual, the method comprising administering an effective amount of the medicament to the individual to inhibit B cell activation. It is. An “individual” according to any of the above aspects may be a human.
　さらなる局面において、本発明は自己免疫疾患を治療する方法を提供する。一態様において、方法は、そのような自己免疫疾患を有する個体に抗TGF-beta3抗体の有効量を投与する工程を含む。そのような態様の1つにおいて、方法は、当該個体に少なくとも1つの（後述するような）追加治療剤の有効量を投与する工程を、さらに含む。上記態様の任意のものによる「個体」は、ヒトであってもよい。 In a further aspect, the present invention provides a method for treating an autoimmune disease. In one embodiment, the method comprises administering an effective amount of anti-TGF-beta3 antibody to an individual having such an autoimmune disease. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent (as described below). An “individual” according to any of the above aspects may be a human.
　さらなる局面において、本発明は個体においてB細胞活性化を抑制するための方法を提供する。一態様において、方法は、B細胞活性化を抑制するために個体に抗TGF-beta3抗体の有効量を投与する工程を含む。一態様において、「個体」は、ヒトである。 In a further aspect, the present invention provides a method for inhibiting B cell activation in an individual. In one embodiment, the method comprises administering to the individual an effective amount of an anti-TGF-beta3 antibody to inhibit B cell activation. In one embodiment, an “individual” is a human.
　さらなる局面において、本発明は、本明細書で提供される抗TGF-beta3抗体の任意のものを含む、薬学的製剤を提供する（例えば上述の治療的方法の任意のものにおける使用のための）。一態様において、薬学的製剤は、本明細書で提供される抗TGF-beta3抗体の任意のものと、薬学的に許容される担体とを含む。別の態様において、薬学的製剤は、本明細書で提供される抗TGF-beta3抗体の任意のものと、少なくとも1つの（例えば後述するような）追加治療剤とを含む。 In a further aspect, the present invention provides a pharmaceutical formulation comprising any of the anti-TGF-beta3 antibodies provided herein (eg, for use in any of the therapeutic methods described above). . In one embodiment, the pharmaceutical formulation comprises any of the anti-TGF-beta3 antibodies provided herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical formulation comprises any of the anti-TGF-beta3 antibodies provided herein and at least one additional therapeutic agent (eg, as described below).
　本発明の抗体は、療法において、単独または他の剤との組み合わせのどちらでも使用され得る。例えば、本発明の抗体は、少なくとも1つの追加の治療剤と同時投与されてもよい。 The antibody of the present invention can be used in therapy either alone or in combination with other agents. For example, an antibody of the invention may be co-administered with at least one additional therapeutic agent.
　上述したような併用療法は、併用投与（2つ以上の治療剤が、同じまたは別々の製剤に含まれる）、および個別投与を包含し、個別投与の場合、本発明の抗体の投与が追加治療剤の投与に先立って、と同時に、および／または、続いて、行われ得る。 Combination therapy as described above includes combination administration (two or more therapeutic agents included in the same or separate formulations) and individual administration, where administration of the antibody of the present invention is an additional treatment. Prior to, simultaneously with and / or subsequent to administration of the agent.
　本発明の抗体（および、任意の追加治療剤）は、非経口投与、肺内投与、および経鼻投与、また局所的処置のために望まれる場合は病巣内投与を含む、任意の好適な手段によって投与され得る。非経口注入は、筋肉内、静脈内、動脈内、腹腔内、または皮下投与を含む。投薬は、投与が短期か長期かに一部応じて、例えば、静脈内注射または皮下注射などの注射によるなど、任意の好適な経路によってなされ得る。これらに限定されるものではないが、単回投与または種々の時点にわたる反復投与、ボーラス投与、および、パルス注入を含む、種々の投薬スケジュールが本明細書の考慮の内である。 The antibodies (and any additional therapeutic agent) of the invention may be administered by any suitable means, including parenteral, pulmonary, and nasal administration, and intralesional administration if desired for local treatment. Can be administered. Parenteral injection includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, such as by injection, such as intravenous or subcutaneous injection, depending in part on whether administration is short or long term. Various dosing schedules are within the scope of this specification, including, but not limited to, single doses or repeated doses over various time points, bolus doses, and pulse infusions.
　本発明の抗体は、優良医療規範 (good medical practice) に一致したやり方で、製剤化され、投薬され、また投与される。この観点から考慮されるべきファクターは、治療されているその特定の障害、治療されているその特定の哺乳動物、個々の患者の臨床症状、障害の原因、剤を送達する部位、投与方法、投与のスケジュール、および医療従事者に公知の他のファクターを含む。抗体は、必ずしもそうでなくてもよいが、任意で、問題の障害を予防するまたは治療するために現に使用されている1つまたは複数の剤とともに、製剤化される。そのような他の剤の有効量は、製剤中に存在する抗体の量、障害または治療のタイプ、および上で論じた他のファクターに依存する。これらは通常、本明細書で述べたのと同じ用量および投与経路で、または本明細書で述べた用量の約1から99%で、または経験的／臨床的に適切と判断される任意の用量および任意の経路で、使用される。 The antibody of the present invention is formulated, dosed and administered in a manner consistent with good medical practice. Factors to be considered in this regard are the specific disorder being treated, the particular mammal being treated, the clinical symptoms of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the administration Schedule, and other factors known to healthcare professionals. The antibody need not be, but is optionally formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents depends on the amount of antibody present in the formulation, the type of disorder or treatment, and other factors discussed above. These are usually at the same dose and route of administration as described herein, or at about 1 to 99% of the doses described herein, or any dose determined empirically / clinically appropriate. And in any route.
　疾患の予防または治療のために、本発明の抗体の適切な用量（単独で用いられるときまたは1つまたは複数の他の追加治療剤とともに用いられるとき）は、治療される疾患のタイプ、抗体のタイプ、疾患の重症度および経過、抗体が予防的目的で投与されるのか治療的目的で投与されるのか、薬歴、患者の臨床歴および抗体に対する応答、ならびに、主治医の裁量に依存するだろう。抗体は、患者に対して、1回で、または一連の処置にわたって、好適に投与される。疾患のタイプおよび重症度に応じて、例えば、1回または複数回の別々の投与によるにしても連続注入によるにしても、約1μg/kgから15 mg/kg（例えば、0.1mg/kg～10mg/kg）の抗体が、患者に対する投与のための最初の候補用量とされ得る。1つの典型的な1日用量は、上述したファクターに依存して、約1μg/kgから100mg/kg以上まで、幅があってもよい。数日またはより長くにわたる繰り返しの投与の場合、状況に応じて、治療は通常疾患症状の所望の抑制が起きるまで維持される。抗体の1つの例示的な用量は、約0.05mg/kg から約 10mg/kgの範囲内である。よって、約0.5mg/kg、2.0mg/kg、4.0mg/kg、もしくは 10mg/kgの1つまたは複数の用量（またはこれらの任意の組み合わせ）が、患者に投与されてもよい。このような用量は、断続的に、例えば1週間毎にまたは3週間毎に（例えば、患者が約2から約20、または例えば約6用量の抗体を受けるように）、投与されてもよい。高い初回負荷用量の後に、1回または複数回の低用量が投与されてもよい。この療法の経過は、従来の手法および測定法によって、容易にモニタリングされる。 For the prevention or treatment of disease, an appropriate dose of the antibody of the present invention (when used alone or in combination with one or more other additional therapeutic agents) will determine the type of disease being treated, It will depend on the type, severity and course of the disease, whether the antibody is administered for prophylactic or therapeutic purposes, drug history, patient clinical history and response to antibodies, and the discretion of the attending physician . The antibody is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, for example, about 1 μg / kg to 15 mg / kg (eg, 0.1 mg / kg to 10 mg), either by one or more separate doses or by continuous infusion / kg) antibody may be the first candidate dose for administration to a patient. One typical daily dose may vary from about 1 μg / kg to over 100 mg / kg, depending on the factors described above. For repeated administrations over several days or longer, depending on the situation, treatment is usually maintained until a desired suppression of disease symptoms occurs. One exemplary dose of antibody is in the range of about 0.05 mg / kg to about 10 mg / kg. Thus, one or more doses (or any combination thereof) of about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg, or 10 mg / kg may be administered to the patient. Such doses may be administered intermittently, such as every week or every 3 weeks (eg, so that the patient receives about 2 to about 20, or such as about 6 doses of antibody). One or more low doses may be administered after the high initial loading dose. The progress of this therapy is easily monitored by conventional techniques and measurements.
Ｆ．製品
　本発明の別の局面において、上述の障害の治療、予防、および／または診断に有用な器材を含んだ製品が、提供される。製品は、容器、および当該容器上のラベルまたは当該容器に付属する添付文書を含む。好ましい容器としては、例えば、ボトル、バイアル、シリンジ、IV溶液バッグなどが含まれる。容器類は、ガラスやプラスチックなどの、様々な材料から形成されていてよい。容器は組成物を単体で保持してもよいし、症状の治療、予防、および／または診断のために有効な別の組成物と組み合わせて保持してもよく、また、無菌的なアクセスポートを有していてもよい（例えば、容器は、皮下注射針によって突き通すことのできるストッパーを有する静脈内投与用溶液バッグまたはバイアルであってよい）。組成物中の少なくとも1つの有効成分は、本発明の抗体である。ラベルまたは添付文書は、組成物が選ばれた症状を治療するために使用されるものであることを示す。さらに製品は、(a)第一の容器であって、その中に収められた本発明の抗体を含む組成物を伴う、第一の容器；および、(b)第二の容器であって、その中に収められたさらなる細胞傷害剤またはそれ以外で治療的な剤を含む組成物を伴う、第二の容器を含んでもよい。本発明のこの態様における製品は、さらに、組成物が特定の症状を治療するために使用され得ることを示す、添付文書を含んでもよい。あるいはまたは加えて、製品はさらに、注射用制菌水 (BWFI)、リン酸緩衝生理食塩水、リンガー溶液、およびデキストロース溶液などの、薬学的に許容される緩衝液を含む、第二の（または第三の）容器を含んでもよい。他の緩衝液、希釈剤、フィルター、針、およびシリンジなどの、他の商業的観点またはユーザの立場から望ましい器材をさらに含んでもよい。 F. Products In another aspect of the invention, products are provided that include equipment useful for the treatment, prevention, and / or diagnosis of the disorders described above. The product includes a container and a label on the container or package insert attached to the container. Preferred containers include, for example, bottles, vials, syringes, IV solution bags and the like. The containers may be formed from various materials such as glass and plastic. The container may hold the composition alone, in combination with another composition that is effective for the treatment, prevention, and / or diagnosis of symptoms, and with a sterile access port. (For example, the container may be a solution bag or vial for intravenous administration having a stopper that can be pierced by a hypodermic needle). At least one active ingredient in the composition is an antibody of the invention. The label or package insert indicates that the composition is to be used to treat the selected condition. The product further comprises: (a) a first container with a composition comprising an antibody of the present invention contained therein; and (b) a second container, A second container may be included with the composition containing an additional cytotoxic agent or otherwise therapeutic agent contained therein. The product in this aspect of the invention may further include a package insert indicating that the composition can be used to treat a particular condition. Alternatively or in addition, the product further comprises a second (or pharmaceutically acceptable buffer, such as water for injection (BWFI), phosphate buffered saline, Ringer's solution, and dextrose solution. A third) container may be included. Other buffers, diluents, filters, needles, and syringes may further include equipment desirable from other commercial or user standpoints.
実施例１　TGF-beta3の作製
（１）Latent体のマウスTGF-beta3（以下latent mTGF-beta3）の作製
　等量のlatent mTGF-beta3の発現ベクターと可溶型ヒトFurinの発現ベクターをExpi293F細胞（Thermo Fisher社）に共に導入し、一過性にlatent mTGF-beta3を発現させた。得られた培養上清をAnti FLAG M2カラム（Sigma社）に添加し、当該カラムの洗浄の後、0.1 mg/mL FLAGペプチド（Sigma社）によりカラムに吸着したタンパクを溶出した。Latent mTGF-beta3を含む画分を限外ろ過膜で濃縮し、これをSuperdex 200 increaseカラム（GE Healthcare社）で分離、回収することで精製latent mTGF-beta3を得た（配列番号：１）。 Example 1 Production of TGF-beta3 (1) Production of Latent mouse TGF-beta3 (latent mTGF-beta3) Equivalent amounts of latent mTGF-beta3 expression vector and soluble human Furin expression vector were expressed as Expi293F cells Thermo Fisher) and transiently expressed latent mTGF-beta3. The obtained culture supernatant was added to an Anti FLAG M2 column (Sigma), and after washing the column, the protein adsorbed on the column was eluted with 0.1 mg / mL FLAG peptide (Sigma). The fraction containing Latent mTGF-beta3 was concentrated with an ultrafiltration membrane, and this was separated and collected with a Superdex 200 increase column (GE Healthcare) to obtain purified latent mTGF-beta3 (SEQ ID NO: 1).
（２）ビオチン化latent mTGF-beta3の作製
　Latent mTGF-beta3の発現ベクターをExpi293F細胞（Thermo Fisher社）に導入し、一過性にlatent mTGF-beta3を発現させた。得られた培養上清をAnti FLAG M2カラム（Sigma社）に添加し、当該カラムの後、0.1 mg/mL FLAGペプチド（Sigma社）によりカラムに吸着したタンパクを溶出した。Latent mTGF-beta3を含む画分をMono Qカラム（GE Healthcare社）に添加し、当該カラムの洗浄の後、NaClの濃度勾配により蛋白を溶出した。目的分子を含む画分を限外濾過膜によりD-PBS(-)に溶媒置換した。EZ-Link NHS-PEG4-Biotin, No-Weigh Format（Thermo Fisher社）を用いてlatent mTGF-beta3をビオチン化し、限外ろ過膜で濃縮した後、Superdex 200 increaseカラム（GE Healthcare社）で分離・回収することで精製ビオチン化latent mTGF-beta3を得た。 (2) Preparation of biotinylated latent mTGF-beta3 An expression vector for Latent mTGF-beta3 was introduced into Expi293F cells (Thermo Fisher) to transiently express latent mTGF-beta3. The obtained culture supernatant was added to an Anti FLAG M2 column (Sigma), and then the protein adsorbed on the column was eluted with 0.1 mg / mL FLAG peptide (Sigma). A fraction containing Latent mTGF-beta3 was added to a Mono Q column (GE Healthcare), and after washing the column, the protein was eluted with a NaCl concentration gradient. The fraction containing the target molecule was solvent-substituted with D-PBS (−) using an ultrafiltration membrane. The latent mTGF-beta3 was biotinylated using EZ-Link NHS-PEG4-Biotin, No-Weigh Format (Thermo Fisher), concentrated with an ultrafiltration membrane, and then separated with a Superdex 200 increase column (GE Healthcare). By collecting, purified biotinylated latent mTGF-beta3 was obtained.
（３）Mature体のヒト（マウス）TGF-beta3（以下mature h(m)TGF-beta3）の作製
　Latent mTGF-beta3の発現ベクターをExpi293F細胞（Thermo Fisher社）に導入し、一過性にlatent mTGF-beta3を発現させた。得られた培養上清をAnti FLAG M2カラム（Sigma社）に添加し、当該カラムの洗浄の後、0.1 M Glycine-HCl, pH2.8によりカラムに吸着した蛋白を溶出した。低いpHを維持することでLatent体からMature体が形成され、mature h(m)TGF-beta3を含む画分を限外ろ過膜で濃縮した後、0.1 M Glycine-HCl, 150 mM NaCl, pH2.5で平衡化されたSuperdex 200pgカラム（GE Healthcare社）に添加し、同液で分離した。Mature h(m)TGF-beta3を含む画分を0.1%トリフルオロ酢酸で平衡化したVydac C4カラム（Vydac社）に添加し、当該カラムの洗浄後、アセトニトリルの濃度勾配によりカラムに結合したタンパクを溶出した。目的分子を含む画分を回収することでmature h(m)TGF-beta3（配列番号：２）を得、乾燥させて-80℃にて保管した。
　なお、ヒトTGF-beta3およびマウスTGF-beta3のMature体は完全な相同性を有しているため、同一のものとみなすことができる。 (3) Production of mature human (mouse) TGF-beta3 (hereinafter mature h (m) TGF-beta3) Latent mTGF-beta3 expression vector was introduced into Expi293F cells (Thermo Fisher) and transiently transientd mTGF-beta3 was expressed. The obtained culture supernatant was added to an Anti FLAG M2 column (Sigma), and after washing the column, the protein adsorbed on the column was eluted with 0.1 M Glycine-HCl, pH 2.8. By maintaining a low pH, a mature body is formed from the Latent body, and a fraction containing mature h (m) TGF-beta3 is concentrated with an ultrafiltration membrane, and then 0.1 M Glycine-HCl, 150 mM NaCl, pH 2. It added to the Superdex 200pg column (GE Healthcare) equilibrated in 5, and isolate | separated with the same liquid. The fraction containing Mature h (m) TGF-beta3 was added to a Vydac C4 column (Vydac) equilibrated with 0.1% trifluoroacetic acid. After washing the column, the protein bound to the column was removed by the acetonitrile concentration gradient. Eluted. The fraction containing the target molecule was collected to obtain mature h (m) TGF-beta3 (SEQ ID NO: 2), dried and stored at −80 ° C.
In addition, since the mature body of human TGF-beta3 and mouse TGF-beta3 have complete homology, they can be regarded as the same.
（４）ビオチン化mature h(m)TGF-beta3の作製
　乾燥させたmature h(m)TGF-beta3に2 mM 塩酸を添加し、これに1/35容量のイソプロパノールと1/9容量の10x D-PBS(-)を加えた。EZ-Link NHS-PEG4-Biotin, No-Weigh Format（Thermo Fisher社）を用いてmature h(m)TGF-beta3をビオチン化し、これを0.1%トリフルオロ酢酸で平衡化したVydac C4カラム（Vydac社）に添加した。当該カラムの洗浄後、アセトニトリルの濃度勾配によりカラムに吸着したタンパクを溶出した。目的分子を含む画分を回収することでビオチン化mature h(m)TGF-beta3を得、乾燥させて-80℃にて保管した。 (4) Preparation of biotinylated mature h (m) TGF-beta3 2 mM hydrochloric acid was added to dried mature h (m) TGF-beta3, to which 1/35 volume of isopropanol and 1/9 volume of 10x D were added. -PBS (-) was added. Vydac C4 column (Vydac) equilibrated with 0.1% trifluoroacetic acid using biotinylated mature h (m) TGF-beta3 using EZ-Link NHS-PEG4-Biotin, No-Weigh Format (Thermo Fisher) ). After washing the column, the protein adsorbed on the column was eluted with a gradient of acetonitrile. The fraction containing the target molecule was collected to obtain biotinylated mature h (m) TGF-beta3, dried and stored at −80 ° C.
実施例２　抗体ライブラリからの抗TGF-beta3抗体の作製
（１）ヒト抗体ナイーブライブラリの作製
　ヒトPBMCから作成したポリA RNAや、市販されているヒトポリA RNAなどを鋳型としてPCR法により抗体重鎖可変領域および抗体軽鎖可変領域の遺伝子ライブラリを増幅した。
　作製した抗体重鎖可変領域の遺伝子ライブラリと抗体軽鎖可変領域の遺伝子ライブラリを組合せ、ファージミドベクターへ挿入し、ヒト抗体配列からなるFabドメインを提示するヒト抗体ファージディスプレイライブラリを構築した。構築方法として、Methods Mol Biol., (2002) 178, 87-100を参考にした。上記ライブラリの構築に際しては、ファージミドのFabとファージpIIIタンパク質をつなぐリンカー部分、および、ヘルパーファージpIIIタンパク遺伝子のN2ドメインとCTドメインの間にトリプシン切断配列が挿入されたファージディスプレイライブラリの配列が使用された。 Example 2 Preparation of anti-TGF-beta3 antibody from antibody library (1) Preparation of human antibody naive library Antibody heavy chain by PCR using poly-A RNA prepared from human PBMC or commercially available human poly-A RNA as a template The variable region and antibody light chain variable region gene libraries were amplified.
The prepared antibody heavy chain variable region gene library and antibody light chain variable region gene library were combined and inserted into a phagemid vector to construct a human antibody phage display library displaying a Fab domain comprising human antibody sequences. Methods Mol Biol., (2002) 178, 87-100 was referred as a construction method. When constructing the above library, the linker part that connects the phagemid Fab and the phage pIII protein and the sequence of the phage display library in which a trypsin cleavage sequence is inserted between the N2 domain and CT domain of the helper phage pIII protein gene are used. It was.
（２）合成ヒト抗体ライブラリの構築
　当業者公知の方法により、10種類の重鎖germline配列, 7種類の軽鎖germline配列を用いた合成ヒト抗体ファージディスプレイライブラリを構築した。用いたGermline配列は、ヒトB細胞レパートリーにおける出現頻度、可変領域ファミリーでの物理化学的性質を指標にし、VH1-2, VH1-69, VH3-23, VH3-66, VH3-72, VH4-59, VH4-61, VH4-b, VH5-51, VH6-1, Vκ1-39, Vκ2-28, Vκ3-20, Vλ1-40, Vλ1-44, Vλ2-14, Vλ3-21を選択した。ヒトB細胞の抗体のレパートリーを模した形で、合成抗体ライブラリの抗原認識部位に多様性を持たせた。 (2) Construction of synthetic human antibody library A synthetic human antibody phage display library using 10 types of heavy chain germline sequences and 7 types of light chain germline sequences was constructed by a method known to those skilled in the art. The Germline sequences used were based on the frequency of appearance in the human B cell repertoire and the physicochemical properties of the variable region family as indicators. , VH4-61, VH4-b, VH5-51, VH6-1, Vκ1-39, Vκ2-28, Vκ3-20, Vλ1-40, Vλ1-44, Vλ2-14, and Vλ3-21 were selected. Diversified antigen recognition sites in the synthetic antibody library were modeled on the antibody repertoire of human B cells.
（３）ビーズパンニングによるヒト抗体ナイーブライブラリからのTGF-beta3に結合する抗体断片の取得
　構築したファージディスプレイ用ファージミドを保持した大腸菌からファージ産生を行った。ファージ産生を行った大腸菌の培養液に2.5M NaCl/10% PEGを添加することによって、沈殿させたファージの集団をPBSにて希釈することによりファージライブラリ液を得た。次に、ファージライブラリ液にBSAおよびイソプロパノールを添加することによって終濃度4% BSA, 2.5% イソプロパノールとなるよう調製した。パンニング方法としては、一般的な方法である磁気ビーズに固定化した抗原を用いたパンニング方法を参照した（J. Immunol. Methods., (2008) 332 (1-2), 2-9、J. Immunol. Methods., (2001) 247 (1-2),191-203、Biotechnol. Prog., (2002) 18(2) 212-20、Mol. Cell Proteomics, (2003) 2 (2), 61-9）。磁気ビーズとして、NeutrAvidin coated beads（FG beads NeutrAvidin）もしくはStreptavidin coated beads（Dynabeads MyOne Streptavidin T1）を用いた。 (3) Acquisition of antibody fragment binding to TGF-beta3 from human antibody naive library by bead panning Phage was produced from Escherichia coli holding the constructed phagemid for phage display. A phage library solution was obtained by diluting the precipitated phage population with PBS by adding 2.5M NaCl / 10% PEG to the culture solution of Escherichia coli that carried out the phage production. Next, BSA and isopropanol were added to the phage library solution to prepare a final concentration of 4% BSA and 2.5% isopropanol. As a panning method, a panning method using an antigen immobilized on magnetic beads, which is a general method, was referred to (J. Immunol. Methods., (2008) 332 (1-2), 2-9, J. Immunol. Methods., (2001) 247 (1-2), 191-203, Biotechnol. Prog., (2002) 18 (2) 212-20, Mol. Cell Proteomics, (2003) 2 (2), 61- 9). NeutrAvidin coated beads (FG beads NeutrAvidin) or Streptavidin coated beads (Dynabeads MyOne Streptavidin T1) were used as magnetic beads.
　具体的には、BSAでブロッキングした磁気ビーズに前述の500 pmolのビオチン化mature h(m)TGF-beta3を加え、さらに調製したファージライブラリ液を加えることによって、当該ファージライブラリ液を室温にて60分間抗原と接触させた。ビーズを1mLの2.5% isopropanol/PBST（2.5% isopropanolを含むPBST）にて3回洗浄した後、1 mLの2.5% isopropanol/PBS（2.5% isopropanolを含むPBS）にてさらに2回洗浄した。その後、ビーズに1mg/mLのトリプシン0.5 mLを加え、室温で15分懸濁した後、即座に磁気スタンドを用いてビーズを分離し、ファージ溶液を回収した。回収したファージ溶液を、対数増殖期（OD600が0.4-0.7）となった10 mLの大腸菌株ER2738に添加した。37℃で1時間緩やかに上記大腸菌の攪拌培養を行うことによって、ファージを大腸菌に感染させた。感染させた大腸菌は、225 mm x 225 mmのプレートへ播種した。次に、播種した大腸菌の培養液からファージを回収することによって、ファージライブラリ液を調製し、2回目のパンニングに利用した。
　2回目のパンニングでは、調製したファージライブラリ液に200 pmolのビオチン標識抗原を加え、1回目のパンニングと同様の操作を行うことにより、ファージライブラリ液を調製した。この調製液を利用して、3回目のパンニングを行った。
　3回目および4回目のパンニングでは、調製したファージライブラリ液に100 pmolのビオチン標識抗原を加え、1、2回目のパンニングと同様の操作を行い、播種された大腸菌を回収した。 Specifically, by adding the above-described 500 pmol biotinylated mature h (m) TGF-beta3 to magnetic beads blocked with BSA, and adding the prepared phage library solution, the phage library solution was added at room temperature to 60 ° C. Contacted with antigen for minutes. The beads were washed 3 times with 1 mL of 2.5% isopropanol / PBST (PBST containing 2.5% isopropanol), and then further washed twice with 1 mL of 2.5% isopropanol / PBS (PBS containing 2.5% isopropanol). Thereafter, 0.5 mL of 1 mg / mL trypsin was added to the beads and suspended at room temperature for 15 minutes. The beads were immediately separated using a magnetic stand, and the phage solution was recovered. The recovered phage solution was added to 10 mL of E. coli strain ER2738 in the logarithmic growth phase (OD600: 0.4-0.7). E. coli was infected with the phage by gently stirring the E. coli at 37 ° C. for 1 hour. Infected E. coli were seeded on 225 mm x 225 mm plates. Next, a phage library solution was prepared by recovering the phage from the seeded E. coli culture solution and used for the second panning.
In the second panning, a phage library solution was prepared by adding 200 pmol of biotin-labeled antigen to the prepared phage library solution and performing the same operation as in the first panning. A third panning was performed using this preparation.
In the third and fourth panning, 100 pmol of biotin-labeled antigen was added to the prepared phage library solution, and the same operations as in the first and second panning were performed to recover the seeded E. coli.
（４）抗体断片から完全長抗体への変換、発現および精製
　3回目もしくは4回目のパンニング時に回収した大腸菌から、NucleoBond Xtra Midi Plus (MACHEREY-NAGEL, 740412.50)によってファージミドの抽出を行った。その後、制限酵素処理によって抗体の可変領域部分を切出し、EF1プロモーターとEBNA1の複製開始点であるOriPを保持するベクターに抗体定常領域が導入されているカセットベクターに、ライゲーションを行った。抗体重鎖可変領域をコードするDNAはhuman IgG1(配列番号：１０)重鎖定常領域をコードするDNAと再結合させた。抗体軽鎖可変領域をコードするDNAはヒトκ型軽鎖定常領域であるk0MT(配列番号：１２)をコードするDNAと再結合させた。ライゲーション産物を用いて、大腸菌DH5α(TOYOBO, DNA-903)を形質転換し、得られたシングルコロニーから動物細胞発現用の完全長抗体プラスミドの抽出を行った。
　抗体の発現は以下の方法を用いて行った。ヒト胎児腎細胞由来FreeStyle 293-F株（Thermo Fisher）をFreeStyle 293 Expression Medium培地（Thermo Fisher）に懸濁し、3.2×105細胞/wellの細胞密度で96ウェルディープウェルプレートの各ウェルへ400μLずつ播種した。調製したそれぞれのプラスミドを、リポフェクション法によって細胞に導入した。CO2インキュベーター（37℃、8% CO2）中で5日間培養し、培養上清中に抗体を分泌させた。培養上清中の分泌抗体は、Multi screen HTS GV (Millipore, MSGVN2250)を用いて精製した。 (4) Conversion, expression and purification from antibody fragment to full-length antibody Phagemid was extracted from E. coli collected at the third or fourth panning by NucleoBond Xtra Midi Plus (MACHEREY-NAGEL, 740412.50). Thereafter, the variable region portion of the antibody was excised by restriction enzyme treatment, and ligation was performed on a cassette vector in which the antibody constant region was introduced into the vector holding the EF1 promoter and OriP, which is the replication origin of EBNA1. DNA encoding the antibody heavy chain variable region was recombined with DNA encoding human IgG1 (SEQ ID NO: 10) heavy chain constant region. The DNA encoding the antibody light chain variable region was recombined with the DNA encoding k0MT (SEQ ID NO: 12), which is a human kappa light chain constant region. The ligation product was used to transform E. coli DH5α (TOYOBO, DNA-903), and a full-length antibody plasmid for animal cell expression was extracted from the resulting single colony.
Antibody expression was carried out using the following method. Suspend human fetal kidney cell-derived FreeStyle 293-F strain (Thermo Fisher) in FreeStyle 293 Expression Medium medium (Thermo Fisher), and add 400 μL to each well of a 96-well deep well plate at a cell density of 3.2 × 10 5 cells / well. Sowing. Each prepared plasmid was introduced into the cells by the lipofection method. The cells were cultured for 5 days in a CO 2 incubator (37 ° C., 8% CO 2 ), and the antibody was secreted into the culture supernatant. Secreted antibodies in the culture supernatant were purified using Multi screen HTS GV (Millipore, MSGVN2250).
（５）取得した抗TGF-beta3抗体の結合能の確認
　調製した抗体培養上清を用い、mature h(m)TGF-beta3への結合能の確認を、Octet RED384 もしくはOctetHTXシステム(Pall Life Sciences)を用いて行った。具体的には、Protein G Biosensor (Pall Life Sciences)に抗体を固相化し、続いてmature h(m)TGF-beta3, mature体のマウスTGF-beta1（以下mature mTGF-beta1。7666-MB/CF, R&D systems　）, 及びmature体のマウスTGF-beta2（以下mature mTGF-beta2。7346-B2/CF, R&D systems）をアプライし、バイオセンサーに固相化された抗体と抗原との結合レスポンスを測定した。
　その結果mature h(m)TGF-beta3への特異的結合が確認された抗体を多数取得した。
(5) Confirmation of binding ability of the obtained anti-TGF-beta3 antibody Using the prepared antibody culture supernatant, confirmation of binding ability to mature h (m) TGF-beta3 was performed using Octet RED384 or OctetHTX system (Pall Life Sciences). It was performed using. Specifically, the antibody was immobilized on Protein G Biosensor (Pall Life Sciences), followed by mature h (m) TGF-beta3, mature mouse TGF-beta1 (hereinafter mature mTGF-beta1, 7766-MB / CF). , R & D systems), and mature mouse TGF-beta2 (hereinafter mature mTGF-beta2, 7346-B2 / CF, R & D systems) were applied to measure the binding response between the antibody immobilized on the biosensor and the antigen. did.
As a result, a large number of antibodies confirmed to have specific binding to mature h (m) TGF-beta3 were obtained.
実施例３　ウサギ/マウスからの抗TGF-beta3抗体の作製
　抗TGF-beta3抗体を次のように調製し、選択し、アッセイした。
　10～16週齢のNZWウサギおよび7～18週齢のBALB/cマウスをマウスTGFb3 mature form (15～30μg/用量/ウサギ)で免疫化した。ウサギは皮内投与にて、マウスは皮下投与にて免疫化を実施した。この用量での免疫化を2ヶ月間にわたって4～5回繰り返した。最終免疫化の1週間後、脾臓と血液とリンパ節を免疫化動物から採取した。抗原特異的B細胞を標識抗原(マウスTGFb3 mature formまたはマウスTGFb3 latent form)で染色し、セルソーター(FACS aria III、BD)で選別し、細胞1個/ウェルの密度で、細胞25,000個/ウェルのEL4細胞(European Collection of Cell Cultures)および20倍希釈した活性化ウサギT細胞馴化培地と共に96ウェルプレートに播種し、7～17日間培養して、そのB細胞培養上清中の分泌抗体を使った抗体スクリーニングに供した。EL4細胞は、マイトマイシンC(Sigma、カタログ番号M4287)で2時間処理し、事前に3回洗浄しておいたものを用いた。活性化ウサギT細胞馴化培地は、ウサギ胸腺細胞を、フィトヘマグルチニンM(Roche、カタログ番号11082132001)、ホルボール12-ミリステート13-アセテート(Sigma、カタログ番号P1585)および2％ FBSを含有するRPMI-1640中で培養することにより、調製した。培養後、B細胞培養上清をさらなる分析のために回収し、細胞のペレットを凍結保存した。 Example 3 Production of anti-TGF-beta3 antibody from rabbit / mouse Anti-TGF-beta3 antibody was prepared, selected and assayed as follows.
10-16 week old NZW rabbits and 7-18 week old BALB / c mice were immunized with mouse TGFb3 mature form (15-30 μg / dose / rabbit). Rabbits were immunized by intradermal administration and mice were immunized by subcutaneous administration. Immunization at this dose was repeated 4-5 times over 2 months. One week after the final immunization, spleen, blood and lymph nodes were collected from the immunized animals. Antigen-specific B cells are stained with labeled antigen (mouse TGFb3 mature form or mouse TGFb3 latent form), sorted with a cell sorter (FACS aria III, BD), and at a density of 1 cell / well, 25,000 cells / well. Seeded in 96-well plate with EL4 cells (European Collection of Cell Cultures) and 20-fold diluted activated rabbit T cell conditioned medium, cultured for 7-17 days, and used secreted antibody in its B cell culture supernatant It was used for antibody screening. EL4 cells were treated with mitomycin C (Sigma, catalog number M4287) for 2 hours and washed 3 times in advance. Activated rabbit T-cell conditioned medium contains rabbit thymocytes, RPMI-1640 containing phytohemagglutinin M (Roche, catalog number 11082132001), phorbol 12-myristate 13-acetate (Sigma, catalog number P1585) and 2% FBS. Prepared by culturing in. After culture, B cell culture supernatant was collected for further analysis and cell pellets were stored frozen.
　ELISA測定法を用いて、B細胞培養上清中の抗体の特異性を試験した。Mature mTGF-beta3、mature mTGF-beta1、mature mTGF-beta2は384ウェルHigh Bind Microplate (Corning、カタログ番号3700)上にPBS中8～16nMにて室温で1時間コーティングした後に、D-PBS(-)にて1%に希釈したBSA(SIGMA、カタログ番号A7030)の溶液でブロッキングした。
　Latent mTGF-beta3は、まずNHS-PEG4-ビオチン(PIERCE、カタログ番号21329)で標識した。384ウェルHigh Bind Microplateにストレプトアビジン(GeneScript、カタログ番号Z02043)をPBS中50nMにて室温で1時間コーティングした後に1% BSAでブロッキングしたELISAプレートを用意し、上述のBiotin化標識したマウスLatent mTGF-beta3をPBS中8nMにて添加し、1時間インキュベートして洗浄した。
　B細胞培養上清をELISAプレートに添加し、1時間インキュベートして洗浄した。結合の検出は、ウサギB細胞培養上清はマウス抗ウサギIgG-西洋ワサビペルオキシダーゼ(SouthernBiotech、カタログ番号4090-05)を用いて、マウスB細胞培養上清はヤギ抗マウスIgG-西洋ワサビペルオキシダーゼ(Sigma、 カタログ番号A2554)に続いてABTS ELISA HRP Substrate(KPL、カタログ番号50-66-06)を添加することによる発色反応で検出した。 An ELISA assay was used to test the specificity of the antibodies in the B cell culture supernatant. Mature mTGF-beta3, mature mTGF-beta1, and mature mTGF-beta2 were coated on 384-well High Bind Microplate (Corning, Catalog No. 3700) at 8-16nM in PBS for 1 hour at room temperature, then D-PBS (-) Blocked with a solution of BSA (SIGMA, catalog number A7030) diluted to 1% with
Latent mTGF-beta3 was first labeled with NHS-PEG4-biotin (PIERCE, catalog number 21329). Prepare an ELISA plate prepared by coating streptavidin (GeneScript, catalog number Z02043) with 384-well High Bind Microplate at 50 nM in PBS for 1 hour at room temperature and blocking with 1% BSA. Beta3 was added at 8 nM in PBS and washed by incubation for 1 hour.
B cell culture supernatant was added to the ELISA plate and washed by incubation for 1 hour. Detection of binding was performed using mouse anti-rabbit IgG-horseradish peroxidase (SouthernBiotech, Cat.No. 4090-05) as the rabbit B cell culture supernatant and goat anti-mouse IgG-horseradish peroxidase (Sigma). , Catalog number A2554) followed by ABTS ELISA HRP Substrate (KPL, catalog number 50-66-06).
　OctetHTXシステム(Pall Life Sciences)を使用して、抗原をプレートに固相しない様式での抗体抗原反応を評価した。ウサギB細胞培養上清中に分泌された抗体はProtein Aバイオセンサー(Pall Life Sciences)上に結合させ、マウスB細胞培養上清中に分泌された抗体はAnti-Mouse IgG Fc Capture (AMC)バイオセンサー(Pall Life Sciences)上に結合させた後に、100nMのマウスTGFb3 mature form溶液中に浸漬させて、抗体と抗原との結合反応を評価した。 The OctetHTX system (Pall Life Sciences) was used to evaluate the antibody-antigen reaction in a manner that did not solidify the antigen on the plate. The antibody secreted into the rabbit B cell culture supernatant is bound on the Protein A biosensor (Pall Life Sciences), and the antibody secreted into the mouse B cell culture supernatant is the Anti-Mouse IgG IgG Fc Capture Bio (AMC) Bio After binding on a sensor (Pall Life Sciences), it was immersed in a 100 nM mouse TGFb3 form form solution to evaluate the binding reaction between the antibody and the antigen.
　ウサギ由来のB細胞を3960種、マウス由来のB細胞を10560種、合計14520種のB細胞の中から、ELISAまたはOctetHTXによる結合評価においてmature h(m)TGF-beta3に結合するものを選択して、ウサギ由来をTTA0001～0188、マウス由来をTTB0001～0188と命名した。 Select from 3960 rabbit B cells and 10560 mouse B cells, a total of 14520 B cells that bind to mature 結合 h (m) TGF-beta3 in ELISA or OctetHTX binding evaluation. The rabbit origin was named TTA0001-0188, and the mouse origin was named TTB0001-0188.
　選択された細胞株のRNAを、ZR-96 Quick-RNAキット(ZYMO RESEARCH、カタログ番号R1053)を用いて凍結保存細胞ペレットから精製した。選択された細胞株中の抗体重鎖可変領域をコードするDNAを逆転写PCRにより増幅し、human IgG1重鎖定常領域(配列番号：１０)をコードするDNAと再結合させた。抗体軽鎖可変領域をコードするDNAを逆転写PCRにより増幅し、ウサギ由来の抗体軽鎖可変領域の場合は改変型のヒトκ型軽鎖定常領域であるk0MTC(配列番号：１１)をコードするDNAと、マウス由来の抗体軽鎖可変領域の場合はヒトκ型軽鎖定常領域であるk0MT(配列番号：１２)をコードするDNAと再結合させた。
　これとは別に、実験における対照サンプルとして用いる目的で、既知のpan-TGFb抗体であるGC1008の重鎖可変領域(VH)および軽鎖可変領域(VL)のアミノ酸配列(WO 2014/164709 A2に記載されているSEQ ID NO: 1およびSEQ ID NO:2)をコードする遺伝子を合成した。重鎖可変領域(VH)をコードするDNAを、human IgG1重鎖定常領域をコードするDNA(配列番号：１３)にインフレームで融合させ、かつ軽鎖可変領域(VL)をコードするDNAを、k0MTの軽鎖定常領域をコードするDNA(配列番号：１４)にインフレームで融合させた。融合したコード配列の各々を発現ベクターにクローニングした。抗体をFreeStyle(商標)293-F細胞(Invitrogen)において発現させ、培養上清から精製して、前述と同様にTGF-betaファミリー分子間の結合特異性評価、Latent TGF-beta3への結合性評価、および機能的活性を評価した。
　以上、実施例２、３で作製した抗TGF-beta3抗体の配列（Kabat Numberingで表示した配列番号：１００～配列番号：８６７）を表２にまとめる。
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-I000003
Figure JPOXMLDOC01-appb-I000004
RNA of selected cell lines was purified from cryopreserved cell pellets using the ZR-96 Quick-RNA kit (ZYMO RESEARCH, catalog number R1053). DNA encoding the antibody heavy chain variable region in the selected cell line was amplified by reverse transcription PCR and recombined with DNA encoding the human IgG1 heavy chain constant region (SEQ ID NO: 10). DNA encoding the antibody light chain variable region is amplified by reverse transcription PCR, and in the case of a rabbit derived antibody light chain variable region, it encodes k0MTC (SEQ ID NO: 11), which is a modified human kappa light chain constant region. In the case of a mouse-derived antibody light chain variable region, the DNA was recombined with DNA encoding k0MT (SEQ ID NO: 12), which is a human kappa light chain constant region.
Separately, the amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) of GC1008, a known pan-TGFb antibody, for use as a control sample in experiments (described in WO 2014/164709 A2 The genes encoding SEQ ID NO: 1 and SEQ ID NO: 2) were synthesized. A DNA encoding the heavy chain variable region (VH) is fused in-frame to a DNA encoding the human IgG1 heavy chain constant region (SEQ ID NO: 13), and a DNA encoding the light chain variable region (VL) is obtained. It was fused in-frame to DNA encoding the light chain constant region of k0MT (SEQ ID NO: 14). Each fused coding sequence was cloned into an expression vector. Antibodies are expressed in FreeStyle (TM) 293-F cells (Invitrogen), purified from the culture supernatant, and evaluated for binding specificity between TGF-beta family molecules and binding to Latent TGF-beta3 as described above And functional activity was evaluated.
The sequences of the anti-TGF-beta3 antibodies prepared in Examples 2 and 3 (SEQ ID NO: 100 to SEQ ID NO: 867 represented by Kabat Numbering) are summarized in Table 2.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-I000003
Figure JPOXMLDOC01-appb-I000004
実施例４　取得した抗TGF-beta3抗体の中和活性の評価
　実施例２、３で取得した抗TGF-beta3抗体、抗KLH（キーホールリンペットヘモシアニン）抗体（IC17-hIgG1）、pan-TGFb中和抗体（GC1008-hIgG1）、およびmature h(m)TGF-beta3リコンビナントタンパクおよびHEK-Blue_TGFb細胞（hkb-tgfb, InvivoGen）を用いて中和活性能を評価した。mature h(m)TGF-beta3の生理活性の評価方法については、Smadシグナルの入力を分泌型アルカリフォスファターゼ（SEAP）の活性を評価することで検証した。
　HEK-Blue_TGFb細胞のGrowth medium（DMEM: D5796; Sigma, 10% FBS: SFBS-C; Bovogen, 2mM L-Glutamine: 25030-081; Invitrogen, 1×Penicilin-Streptomicin: 15140-122; Invitrogen, 100μg Normocin: ant-nr-1; InvivoGen, 30μg/mL Blasticidin: ant-bl-1; InvivoGen, 200μg/mL HygroGold: ant-hg-1; InvivoGen, 100μg/mL Zeocin: 46-0072; Invitrogen）およびTest medium（DMEM: D5796; Sigma, 0.1%w/w BSA: A9576; Sigma, 2mM L-Glutamine: 25030-081; Invitrogen, 1×Penicilin-Streptomicin: 15140-122; Invitrogen, 100μg Normocin: ant-nr-1; InvivoGen）はInvivoGenの条件にしたがって調製した。
　各種抗体は最終濃度が2000ng/mLから3.2ng/mLまで段階希釈となるように、また4mM HClで100μg /mLに溶解したmature h(m)TGF-beta3リコンビナントタンパクを用いて最終濃度0.32ng/mLとなるようにflat-bottom 96-well plateに分注し、HEK-Blue_TGFb細胞懸濁液を各2×105 cells/wellとなるように添加し、37℃のCO2インキュベーターにて一晩（20hrs）培養した。翌日、20μLの細胞培養上清に対して80μLのQUANTI-Blue （rep-qb1, InvivoGen） 発色基質を添加し、37℃、CO2インキュベーターで1時間反応後、VersaMaxスペクトロメーター（Molecular devices）にてOD620nmで分泌型アルカリフォスファターゼ（SEAP）活性を測定した。
　最終濃度0.32ng/mLのmature h(m)TGF-beta3リコンビナントタンパクの生理活性が2000ng/mLのGC1008-hIgG1にて完全に抑制されるOD値を100%抑制、GC1008-hIgG1が不含のときのOD値を0%抑制と定義することによって、各種抗体のTGF-beta3の生理活性の抑制効果を評価した。
その結果、中和活性を若干でも保有する抗体が確認される中で、抗TGFbeta3抗体の濃度が2000ng/mLの存在下においても、全くTGF-beta3の生理活性を抑制しない抗体の存在が確認された。その結果を表３に示す。
尚、本中和活性評価系において、抗KLH抗体であるIC17-hIgG1の2000ng/mLにおいて10～20%程度の抑制効率が確認されることから、本中和活性評価系において、少なくとも抗TGF-beta3抗体の濃度が2000ng/mLの存在下におけるTGF-beta3の生理活性の抑制効率で20%以内の抗体は本発明における非中和抗体であるものと評価した。実験は全てN=4で実施した。市販抗体のうち、TGF-beta3への結合が確認できているIgGタイプのモノクローナル抗体（R&D systems #44922、R&D systems #20724、Santa Cruz #sc-166833）の結果を表の最後にレファレンスとして示す。
表３の結果は、非中和抗体の取得が成功していることを示している。
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-I000006
Figure JPOXMLDOC01-appb-I000007
Figure JPOXMLDOC01-appb-I000008
Figure JPOXMLDOC01-appb-I000009
Figure JPOXMLDOC01-appb-I000010
Figure JPOXMLDOC01-appb-I000011
Figure JPOXMLDOC01-appb-I000012
Figure JPOXMLDOC01-appb-I000013
Figure JPOXMLDOC01-appb-I000014
Figure JPOXMLDOC01-appb-I000015
Figure JPOXMLDOC01-appb-I000016
Figure JPOXMLDOC01-appb-I000017
Figure JPOXMLDOC01-appb-I000018
Example 4 Evaluation of the neutralizing activity of the obtained anti-TGF-beta3 antibody The anti-TGF-beta3 antibody, anti-KLH (keyhole limpet hemocyanin) antibody (IC17-hIgG1) obtained in Examples 2 and 3 and pan-TGFb The neutralizing activity was evaluated using a Japanese antibody (GC1008-hIgG1), mature h (m) TGF-beta3 recombinant protein and HEK-Blue_TGFb cells (hkb-tgfb, InvivoGen). About the evaluation method of the physiological activity of mature h (m) TGF-beta3, we verified the input of Smad signal by evaluating the activity of secretory alkaline phosphatase (SEAP).
Growth medium of HEK-Blue_TGFb cells (DMEM: D5796; Sigma, 10% FBS: SFBS-C; Bovogen, 2mM L-Glutamine: 25030-081; Invitrogen, 1 × Penicilin-Streptomicin: 15140-122; Invitrogen, 100μg Normocin: ant-nr-1; InvivoGen, 30 μg / mL Blasticidin: ant-bl-1; InvivoGen, 200 μg / mL HygroGold: ant-hg-1; InvivoGen, 100 μg / mL Zeocin: 46-0072; Invitrogen) and Test medium (DMEM) : D5796; Sigma, 0.1% w / w BSA: A9576; Sigma, 2mM L-Glutamine: 25030-081; Invitrogen, 1 × Penicilin-Streptomicin: 15140-122; Invitrogen, 100μg Normocin: ant-nr-1; InvivoGen) Was prepared according to InvivoGen conditions.
Each antibody is serially diluted from 2000 ng / mL to 3.2 ng / mL, and the final concentration is 0.32 ng / mL using mature h (m) TGF-beta3 recombinant protein dissolved in 100 μg / mL with 4 mM HCl. Dispense into a flat-bottom 96-well plate to make mL, add HEK-Blue_TGFb cell suspension to 2 × 10 5 cells / well, and overnight in a CO 2 incubator at 37 ° C. (20 hrs) was cultured. The next day, add 20 μL of QUANTI-Blue (rep-qb1, InvivoGen) chromogenic substrate to 20 μL of cell culture supernatant, react for 1 hour in a CO 2 incubator at 37 ° C, and use VersaMax spectrometer (Molecular devices). Secreted alkaline phosphatase (SEAP) activity was measured at OD620nm.
When the final concentration of mature h (m) TGF-beta3 recombinant protein at 0.32 ng / mL is completely suppressed by 2000 ng / mL of GC1008-hIgG1, the OD value is 100% suppressed, and when GC1008-hIgG1 is not included By defining OD value of 0% as 0% inhibition, the effect of inhibiting the physiological activity of TGF-beta3 of various antibodies was evaluated.
As a result, the presence of antibodies that did not suppress the physiological activity of TGF-beta3 at all even in the presence of 2000 ng / mL of anti-TGFbeta3 antibody concentration was confirmed, while antibodies having even a slight neutralizing activity were confirmed. It was. The results are shown in Table 3.
In this neutralization activity evaluation system, suppression efficiency of about 10 to 20% was confirmed at 2000 ng / mL of IC17-hIgG1 which is an anti-KLH antibody. Therefore, in this neutralization activity evaluation system, at least anti-TGF- Antibodies with a TGF-beta3 suppression efficiency of 20% or less in the presence of a beta3 antibody concentration of 2000 ng / mL were evaluated as non-neutralizing antibodies in the present invention. All experiments were performed with N = 4. Among the commercially available antibodies, the results of IgG type monoclonal antibodies (R & D systems # 44922, R & D systems # 20724, Santa Cruz # sc-166833) that have been confirmed to bind to TGF-beta3 are shown as references at the end of the table.
The results in Table 3 indicate that the acquisition of non-neutralizing antibodies has been successful.
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-I000006
Figure JPOXMLDOC01-appb-I000007
Figure JPOXMLDOC01-appb-I000008
Figure JPOXMLDOC01-appb-I000009
Figure JPOXMLDOC01-appb-I000010
Figure JPOXMLDOC01-appb-I000011
Figure JPOXMLDOC01-appb-I000012
Figure JPOXMLDOC01-appb-I000013
Figure JPOXMLDOC01-appb-I000014
Figure JPOXMLDOC01-appb-I000015
Figure JPOXMLDOC01-appb-I000016
Figure JPOXMLDOC01-appb-I000017
Figure JPOXMLDOC01-appb-I000018
実施例５　抗TGF-beta3抗体の各種TGF-betaに対する結合特異性の評価
　次に、抗TGF-beta3抗体のTGF-beta1-3に対する結合特異性を評価した。
　相互作用を観察する物質の一方（リガンド）をセンサーチップの金薄膜上に固定し、センサーチップの裏側から金薄膜とガラスの境界面で全反射するように光を当てると、反射光の一部に反射強度が低下した部分（SPRシグナル）が形成される。相互作用を観察する物質の他方（アナライト）をセンサーチップの表面に流しリガンドとアナライトが結合すると、固定化されているリガンド分子の質量が増加し、センサーチップ表面の溶媒の屈折率が変化する。この屈折率の変化により、SPRシグナルの位置がシフトする（逆に結合が解離するとシグナルの位置は戻る）。Biacoreシステムは上記のシフトする量、すなわちセンサーチップ表面での質量変化を縦軸にとり、質量の時間変化を測定データとして表示する（センサーグラム）。センサーグラムからセンサーチップ表面に捕捉したリガンドに対するアナライトの結合量（アナライトを相互作用させた前後でのセンサーグラム上でのレスポンスの変化量）が求められる。ただし、結合量はリガンドの量にも依存するため、比較する際にはリガンドの量を本質的に同じ量にしたとみなせる条件下で比較する必要がある。
　実施例２、３で取得した抗体の各種TGF-betaに対する結合活性測定は、Biacore T200 (GE　Healthcare, Control Software Version 2.0)を用いてHBS-EP+をランニング緩衝液として25℃で行った。なおHBS-EP+ は、HBS-EP+ x10 (GE Healthcare)をMilli-Q Advantage（Merck）より採取した超純水で10倍希釈して作製した。TGF-betaの非特異結合を低減するためにSensor chip CM4（GE Healthcare）の前処理として、チップ表面にあるカルボキシル基をNHSおよびEDC（GE Healthcare, Amine Coupling kit, type 2付属品）で活性化してEthanolamine-HCl（同付属品）でブロッキングした。その後、アミンカップリング法によりMabselect SuRe Protein A（GE Healthcare）をSensor chip CM4に固定化した。Mabselect SuRe Protein Aに500 Resonance Units (RU)キャプチャー（capture/捕捉）されるように濃度を調整した各抗体を添加した後に、HBS-EP+で50 nMに希釈したmature hTGF-beta1(100-21C, Peprotech。)、mature hTGF-beta2 (100-35B, Peprotech。）、mature h(m)TGF-beta3、mature mTGF-beta1 (7666-MB/CF, R&D systems)、mature mTGF-beta2(7346-B2/CF, R&D systems)、 latent mTGF-beta3、を結合させた。結合は流速60 μl/minで1分間行った後に1分間解離させた。結合終了5秒前のレスポンスをTGF-betaの結合値とした。Sensor chip CM4は25 mM NaOHを用いて再生し、繰り返し使用した。
　本測定結果は、まずBiacore T200 Evaluation Software Version 2.0 (GE Healthcare) を用いて、ブランクとしてbufferのレスポンスを差し引きした後に、captureした抗体量で、TGF-beta結合値を割り、1 RUの抗体あたりのTGF-beta結合値を算出した。なお、その値が0.01 未満の場合には、結合がなかったものとみなし、値を0とした。次に、mature h(m)TGF-beta3に対する結合の値で、各TGF-betaに対する結合の値を割り、算出された値に100を掛け、mature h(m)TGF-beta3に対する結合を100%としたときの相対的な結合量を計算した。この解析結果のまとめを表４に示した。
表４の中で、mature h(m)TGF-beta3 100％に対し、mature hTGF-beta1, 2/ mature mTGF-beta1, 2への結合が10％未満、好ましくは1％未満、より好ましくは0.1％未満の抗体はTGF-beta3へ特異的に結合する抗体であると評価した。この結果、作製した抗TGF-beta3抗体の中には、TGF-beta3へ特異的に結合する抗体が多数確認された。
更に、mature h(m)TGF-beta3 100％に対し、latent mTGF-beta3への結合が10％以上、好ましくは30％以上、より好ましくは50％以上、更に好ましくは100％以上の抗体はTGF-beta3のmature体とlatent体の両方に結合する抗体であると評価し、このような抗体も多数確認された。 Example 5 Evaluation of Binding Specificity of Anti-TGF-beta3 Antibody to Various TGF-beta Next, the binding specificity of anti-TGF-beta3 antibody to TGF-beta1-3 was evaluated.
When one of the substances (ligands) for observing the interaction is fixed on the gold thin film of the sensor chip and light is applied from the back side of the sensor chip so that it is totally reflected at the interface between the gold thin film and the glass, part of the reflected light A portion (SPR signal) where the reflection intensity is reduced is formed. When the other substance (analyte) that observes the interaction is flowed to the surface of the sensor chip and the ligand and the analyte bind, the mass of the immobilized ligand molecule increases and the refractive index of the solvent on the sensor chip surface changes. To do. This change in refractive index shifts the position of the SPR signal (conversely, when the bond dissociates, the signal position returns). The Biacore system takes the shift amount, that is, the mass change at the sensor chip surface on the vertical axis, and displays the time change of mass as measurement data (sensorgram). The amount of analyte binding to the ligand captured on the sensor chip surface from the sensorgram (the amount of change in response on the sensorgram before and after the interaction of the analyte) is determined. However, since the amount of binding also depends on the amount of ligand, it is necessary to compare under the condition that the amount of ligand can be regarded as essentially the same amount.
The binding activity of the antibodies obtained in Examples 2 and 3 to various TGF-betas was measured at 25 ° C. using HIA-EP + as a running buffer using Biacore T200 (GE Healthcare, Control Software Version 2.0). HBS-EP + was prepared by diluting HBS-EP + x10 (GE Healthcare) 10 times with ultrapure water collected from Milli-Q Advantage (Merck). In order to reduce non-specific binding of TGF-beta, as a pretreatment of Sensor chip CM4 (GE Healthcare), the carboxyl group on the chip surface is activated with NHS and EDC (GE Healthcare, Amine Coupling kit, type 2 accessory) Then, it was blocked with Ethanolamine-HCl (same accessory). Thereafter, Mabselect SuRe Protein A (GE Healthcare) was immobilized on Sensor chip CM4 by an amine coupling method. After adding each antibody whose concentration was adjusted to capture 500 Resonance Units (RU) to Mabselect SuRe Protein A, mature hTGF-beta1 (100-21C, diluted to 50 nM with HBS-EP +) Peprotech.), Mature hTGF-beta2 (100-35B, Peprotech.), Mature h (m) TGF-beta3, mature mTGF-beta1 (7666-MB / CF, R & D systems), mature mTGF-beta2 (7346-B2 / CF, R & D systems) and latent mTGF-beta3. Binding was performed at a flow rate of 60 μl / min for 1 minute and then dissociated for 1 minute. The response 5 seconds before the end of binding was defined as the binding value of TGF-beta. Sensor chip CM4 was regenerated using 25 mM NaOH and repeatedly used.
This measurement result was obtained by first subtracting the buffer response as a blank using Biacore T200 Evaluation Software Version 2.0 (GE Healthcare), and then dividing the TGF-beta binding value by the amount of antibody captured. TGF-beta binding values were calculated. In addition, when the value was less than 0.01, it was considered that there was no coupling | bonding and the value was set to 0. Next, divide the value of binding to each TGF-beta by the value of binding to mature h (m) TGF-beta3, multiply the calculated value by 100, and bind 100% to mature h (m) TGF-beta3. The relative binding amount was calculated. A summary of the analysis results is shown in Table 4.
In Table 4, for 100% mature h (m) TGF-beta3, binding to mature hTGF-beta1, 2 / mature mTGF-beta1, 2 is less than 10%, preferably less than 1%, more preferably 0.1% Less than% antibody was evaluated as an antibody that specifically binds to TGF-beta3. As a result, a large number of antibodies specifically binding to TGF-beta3 were confirmed among the prepared anti-TGF-beta3 antibodies.
Furthermore, 10% or more, preferably 30% or more, more preferably 50% or more, and even more preferably 100% or more of the antibody binding to latent mTGF-beta3 is 100% of mature h (m) TGF-beta3. We evaluated that this antibody binds to both mature and latent bodies of -beta3, and many such antibodies were confirmed.
Figure JPOXMLDOC01-appb-T000019
Figure JPOXMLDOC01-appb-I000020
Figure JPOXMLDOC01-appb-I000021
　以上の実施例４，５の結果から、表２に記載した抗体のうち、少なくともNo.1, 5, 7-12, 15, 17, 20, 21, 27, 28, 30, 31, 39, 40, 42-44, 47, 48, 59, 63, 65, 70の抗体がTGF-beta3へ特異的に結合する非中和抗体であることを確認した。
　尚、実施例４、５の評価では、TGF-beta3に特異的に結合し、かつ強い中和活性を示す抗体も確認された。
　TGF-beta3は前立腺癌などの癌細胞株において発現していることが報告されており、癌細胞の運動能を亢進させることで転移や浸潤などの癌の増悪に関与していると考えられている。(Clinical & Experimental Metastasis, January 2013, Volume 30, Issue 1, pp 13-23)
　またTGF-beta3は臨床の浸潤性乳がん患者の腫瘍組織においても高頻度で発現していることが知られ、TGF―beta3の腫瘍組織における発現レベルや血中濃度は、乳がん患者の全生存期間と逆相関を示すと報告されている。(Anticancer Res. 2000 Nov-Dec;20(6B):4413-8.)
　このように、TGF-beta3が癌の増悪や予後不良と関係する機能を有していると考えられることから、本発明において得られたTGF-beta3に特異的に結合する中和抗体が、癌治療に適用できることが期待される。
Figure JPOXMLDOC01-appb-T000019
Figure JPOXMLDOC01-appb-I000020
Figure JPOXMLDOC01-appb-I000021
From the results of Examples 4 and 5 above, at least No. 1, 5, 7-12, 15, 17, 20, 21, 27, 28, 30, 31, 39, 40 of the antibodies listed in Table 2 were used. , 42-44, 47, 48, 59, 63, 65, 70 were confirmed to be non-neutralizing antibodies that specifically bind to TGF-beta3.
In the evaluations of Examples 4 and 5, antibodies that specifically bound to TGF-beta3 and showed strong neutralizing activity were also confirmed.
TGF-beta3 has been reported to be expressed in prostate cancer and other cancer cell lines, and is thought to be involved in cancer progression such as metastasis and invasion by enhancing the motility of cancer cells Yes. (Clinical & Experimental Metastasis, January 2013, Volume 30, Issue 1, pp 13-23)
TGF-beta3 is known to be expressed at high frequency in the tumor tissue of patients with clinical invasive breast cancer, and the expression level and blood concentration of TGF-beta3 in the tumor tissue are related to the overall survival of breast cancer patients. It is reported to show an inverse correlation. (Anticancer Res. 2000 Nov-Dec; 20 (6B): 4413-8.)
Thus, since TGF-beta3 is considered to have functions related to cancer progression and poor prognosis, the neutralizing antibody that specifically binds to TGF-beta3 obtained in the present invention is a cancer. Expected to be applicable to treatment.
実施例６　抗TGF-beta3抗体の競合評価
　実施例２，３で作製した抗TGF-beta3抗体をさらなる解析のために選択した。選択された抗体をOctetHTXシステムを用いたin-tandem法に依るエピトープ・ビニング実験に供することで、様々な結合特性を有する抗体がTGF-beta3タンパク質の同一のエピトープまたは重複するエピトープに結合するかを評価した。
　選択された抗体はTGF-beta3特異的結合性、Latent TGF-beta3に対する結合性、中和活性の3つの項目を以下の基準により分類しGroup1-7とした（表５）。

中和活性：
　Ａ：実施例４における抗体濃度2000ng/mLでのTGF-beta3の抑制効率が20%以内
　Ｂ：Ａ以外
特異性： 
　Ａ：実施例５におけるmature h(m)TGF-beta3 100％比でのmature hTGF-beta1, 2/mature mTGF-beta1, 2結合が10％未満
　Ｂ：Ａ以外
Latent結合性：
　Ａ：実施例５におけるmature h(m)TGF-beta3 100％比でのlatent mTGF-beta3への結合が10％以上
　Ｂ：Ａ以外

Group1:中和活性(Ａ),特異性(Ａ),Latent結合性(Ａ)
Group2:中和活性(Ｂ),特異性(Ａ),Latent結合性(Ａ)
Group3:中和活性(Ａ),特異性(Ｂ),Latent結合性(Ａ)
Group4:中和活性(Ａ),特異性(Ａ),Latent結合性(Ｂ)
Group5:中和活性(Ｂ),特異性(Ａ),Latent結合性(Ｂ)
Group6:中和活性(Ａ),特異性(Ｂ),Latent結合性(Ｂ)
Group7:中和活性(Ｂ),特異性(Ｂ),Latent結合性(Ｂ)
Example 6 Competition Evaluation of Anti-TGF-beta3 Antibody The anti-TGF-beta3 antibody prepared in Examples 2 and 3 was selected for further analysis. By subjecting the selected antibodies to epitope binning experiments using the in-tandem method using the OctetHTX system, it is possible to determine whether antibodies with various binding properties bind to the same or overlapping epitopes of the TGF-beta3 protein. evaluated.
The selected antibodies were classified into Group 1-7 by classifying the following three criteria: TGF-beta3-specific binding, binding to Latent TGF-beta3, and neutralizing activity (Table 5).

Neutralizing activity:
A: TGF-beta3 suppression efficiency within 20% at antibody concentration of 2000 ng / mL in Example 4 B: Specificity other than A:
A: mature hTGF-beta1, 2 / mature mTGF-beta1, 2 binding at 100% ratio of mature h (m) TGF-beta3 in Example 5 B: other than A
Latent binding:
A: 10% or more of binding to latent mTGF-beta3 at 100% ratio of mature h (m) TGF-beta3 in Example 5 B: Other than A

Group1: Neutralizing activity (A), specificity (A), Latent binding (A)
Group2: Neutralizing activity (B), specificity (A), Latent binding (A)
Group3: Neutralizing activity (A), specificity (B), Latent binding (A)
Group4: Neutralizing activity (A), specificity (A), Latent binding (B)
Group5: Neutralizing activity (B), specificity (A), Latent binding (B)
Group6: Neutralizing activity (A), specificity (B), Latent binding (B)
Group7: Neutralizing activity (B), specificity (B), Latent binding (B)
Figure JPOXMLDOC01-appb-T000022
Figure JPOXMLDOC01-appb-I000023
Figure JPOXMLDOC01-appb-T000022
Figure JPOXMLDOC01-appb-I000023
　エピトープの解析は、標的抗原タンパク質由来の重複ペプチドアレイを用いたエピトープ・マッピングがよく用いられるが、高次立体構造を形成する標的抗原タンパク質のエピトープ解析には有効ではない場合がある。
　TGF-beta3単量体は分子量約12.5kDaの小さなタンパク質でありながら、単量体分子内に3箇所、単量体分子間に1箇所のジスルフィド結合を有する高次立体構造を形成することから、エピトープ競合を指標にしたエピトープ解析方法であるエピトープ・ビニングを実施した。
　TGF-beta3タンパク質はホモ二量体を形成するが、一般的にこのような標的に対してはsandwich ELISA法などの手法は有効ではないため、OctetHTXシステムを用いたin tandem法(http://www.fortebio.com/interactions/Autumn_2010/page2.html)を採用した。Streptavidinバイオセンサー(Pall Life Sciences)上にビオチン標識したmature TGF-beta3を2μg/mLにて5分間結合させた後に、第一抗体(飽和抗体/Saturating Ab/1stAb)の結合を飽和させた(表５に記すように抗体の種類に依り15または50μg/mLの抗体濃度にて6分間)。第一抗体の結合が飽和しているバイオセンサーに、続いて第二抗体(競合抗体/Competing Ab/2ndAb)を反応させて(第一抗体と同様に、抗体の種類に依り15または50μg/mLの抗体濃度にて6分間)結合反応が確認されるかを評価することで、抗体間のエピトープの競合を評価した。全ての抗TGF-beta3抗体は、第一抗体と第二抗体の両方として使用し、網羅的に対にした。（図１） For epitope analysis, epitope mapping using a duplicate peptide array derived from a target antigen protein is often used, but it may not be effective for epitope analysis of a target antigen protein forming a higher-order conformation.
TGF-beta3 monomer is a small protein with a molecular weight of about 12.5 kDa, but it forms a higher-order structure with three disulfide bonds in the monomer molecule and one in the monomer molecule. Epitope binning, which is an epitope analysis method using epitope competition as an index, was performed.
TGF-beta3 protein forms a homodimer, but generally, sandwich ELISA is not effective for such targets, so the in tandem method using the OctetHTX system (http: // www.fortebio.com/interactions/Autumn_2010/page2.html). After binding biotin-labeled mature TGF-beta3 on Streptavidin biosensor (Pall Life Sciences) at 2 μg / mL for 5 minutes, the binding of the primary antibody (saturated antibody / Saturating Ab / 1stAb) was saturated (Table 6 minutes at an antibody concentration of 15 or 50 μg / mL depending on the type of antibody as described in 5). A biosensor in which the binding of the first antibody is saturated, and then the second antibody (competitive antibody / Competing Ab / 2ndAb) is reacted (similar to the first antibody, 15 or 50 μg / mL depending on the type of antibody) The epitope competition between the antibodies was evaluated by evaluating whether the binding reaction was confirmed (for 6 minutes at the antibody concentration). All anti-TGF-beta3 antibodies were used as both primary and secondary antibodies and were paired exhaustively. (Figure 1)
　第一抗体としてTGF-beta3には結合しないアイソタイプコントロール抗体であり、KLHに対する抗体であるIC17-hIgG1を用いた場合の、評価対象である第二抗体の結合レスポンス値（第二抗体の本来の結合レスポンス値/Original response by 2nd Ab）を100%と定義し、各抗TGFb3抗体を第一抗体に用いたときの第二抗体の結合レスポンス値（第二抗体による追加の結合レスポンス値/Acquired response by 2nd Ab）の割合を算出し、これをエピトープ競合の判断基準とした。一部の解離速度の速い抗体を第一抗体に用いた場合には、第一抗体の解離に伴う第二抗体への置き換わりが発生するため、結合反応開始から30秒後の結合レスポンス値の割合で評価した。（図２，３）
　ただし、結合反応開始から30秒後の結合レスポンス値の割合で評価しても、第一抗体の解離に伴う第二抗体への置き換わりの影響を完全に抑えることはできないため、20%以下の結合レスポンス値の割合はエピトープの競合があるものと判断した。また、エピトープの競合がなかったとしても第一抗体の存在下では第二抗体の結合速度や結合量が多少は影響を受ける可能性を考慮し、60%以上の結合レスポンス値の割合はエピトープの競合がないものと判断した。各抗体間の組み合わせから得られた結合レスポンス値の割合を、ヒートマップ形式にて図４に示した。20%以下は白色で示し、60%以上は黒色で示し、20%から60%の間はその数値の大きさに準じた色付けを行った。 This is an isotype control antibody that does not bind to TGF-beta3 as the first antibody. When using IC17-hIgG1, which is an antibody against KLH, the binding response value of the second antibody to be evaluated (original binding of the second antibody) Response value / Original response by 2nd Ab) is defined as 100%, and the binding response value of the second antibody when each anti-TGFb3 antibody is used as the first antibody (additional response value by the second antibody / Acquired response by 2nd Ab) was calculated and used as a criterion for epitope competition. When some antibodies with high dissociation rate are used for the first antibody, the replacement with the second antibody due to the dissociation of the first antibody occurs, so the ratio of the binding response value 30 seconds after the start of the binding reaction It was evaluated with. (Figures 2 and 3)
However, even if it is evaluated by the ratio of the binding response value 30 seconds after the start of the binding reaction, the effect of the replacement with the second antibody due to the dissociation of the first antibody cannot be completely suppressed. The ratio of response values was judged to have epitope competition. In addition, even if there is no epitope competition, in consideration of the possibility that the binding rate and binding amount of the second antibody may be affected somewhat in the presence of the first antibody, the ratio of the binding response value of 60% or more Judged that there was no competition. The ratio of the binding response value obtained from the combination between each antibody is shown in FIG. 4 in a heat map format. 20% or less is shown in white, 60% or more is shown in black, and coloring between 20% and 60% was performed according to the numerical value.
　図４から分かるように、望ましい結合特性を持つ抗体群、即ちTGF-beta3に特異的に結合する非中和抗体であるGroup1(中和活性(Ａ),特異性(Ａ),Latent結合性(Ａ))に属する抗体はいずれもTGF-beta3への結合において互いに競合しており、更にGroup4(中和活性(Ａ),特異性(Ａ),Latent結合性(Ｂ))に属する抗体もまたいずれもTGF-beta3への結合において互いに競合している。
　これらの結果から、本発明で作製したTGF-beta3に特異的に結合する非中和抗体は、TGF-beta3への結合において互いに競合することを確認した。更に、これらの抗体はTGF-beta3の共通した、あるいは類似した近傍のエピトープを認識すると考えられる。
　また、これらのTGF-beta3に特異的に結合する非中和抗体のエピトープ競合パターンは、中和活性を有している抗体群やTGF-beta3特異的結合性に劣る抗体群とは異なるパターンを示している。このような一連の結果を考慮すると、本発明で作製した抗体群は、 Group1およびGroup4に分類された望ましい結合特性を持つTGF-beta3抗体に特徴的なエピトープを広くカバーしていると考えられる。 As can be seen from FIG. 4, a group of antibodies having desirable binding characteristics, that is, Group 1 which is a non-neutralizing antibody that specifically binds to TGF-beta3 (neutralizing activity (A), specificity (A), latency binding ( All of the antibodies belonging to A)) compete with each other for binding to TGF-beta3, and antibodies belonging to Group 4 (neutralizing activity (A), specificity (A), patent binding (B)) also Both compete with each other for binding to TGF-beta3.
From these results, it was confirmed that the non-neutralizing antibodies that specifically bind to TGF-beta3 prepared in the present invention compete with each other in binding to TGF-beta3. Furthermore, these antibodies are thought to recognize common or similar nearby epitopes of TGF-beta3.
In addition, the epitope competition pattern of these non-neutralizing antibodies that specifically bind to TGF-beta3 is different from that of antibodies that have neutralizing activity or antibodies that are inferior to TGF-beta3-specific binding. Show. Considering such a series of results, it is considered that the antibody group produced in the present invention widely covers epitopes characteristic of TGF-beta3 antibodies having desirable binding characteristics classified into Group 1 and Group 4.
実施例７　Mature h(m)TGF-beta3と抗TGF-beta3特異的非中和抗体のimmuno-complex (IC)によるヒトB細胞増殖抑制効果
　実施例２、３で取得し、実施例４，５で非中和であること、TGF-beta3特異的であることを確認した抗TGF-beta3抗体（以下、「抗TGF-beta3特異的非中和抗体」）について、生体内においてTGF-beta3抗原と結合した時にB細胞の増殖を抑制する効果を保持しているかどうかを確認するために、AllCells, LLCより購入したヒトPBMC（PB005F, Lot. A4811, AllCells）を用いて検討した。 Example 7 Inhibition of human B cell proliferation by immuno-complex (IC) of mature h (m) TGF-beta3 and anti-TGF-beta3 specific non-neutralizing antibody Obtained in Examples 2 and 3, Anti-TGF-beta3 antibody (hereinafter referred to as “anti-TGF-beta3-specific non-neutralizing antibody”), which was confirmed to be non-neutralizing and TGF-beta3-specific, in vivo with TGF-beta3 antigen In order to confirm whether it retains the effect of suppressing the proliferation of B cells when bound, it was examined using human PBMC (PB005F, Lot. A4811, AllCells) purchased from AllCells, LLC.
　Human B cell isolation kit II (130-091-151, Miltenyi Biotec)によってB細胞 (negative fraction)を分画した後、Medium (RPMI/10%FBS/55μM 2-ME)で2回洗浄を行い10,000 cells/wellとなるようにround-bottom 96-well plateに分注した。 Fractionate B cells (negative fraction) with Human B cells (isolation kits II (130-091-151, Miltenyi Biotec), then wash twice with Medium (RPMI / 10% FBS / 55μM 2-ME) to 10,000 cells Dispensed into round-bottom 96-well plate to be / well.
　最終濃度が0.32ng/mLのMature TGF-beta3と2000, 400, 80, 16, 3.2, 0.64, 0.128 ng/mLの抗TGF-beta3特異的非中和抗体となるように調製し、室温で1時間反応させることでimmuno-complex (IC)を形成させ、1×104個のB細胞と混合した。さらに細胞増殖の刺激として最終濃度5 μg/mL のanti-human IgM (109-006-129, Jackson ImmunoResearch)、1 μg/mL のanti-CD40 (AF632, R&D Systems)、33 ng/mL のrecombinant human IL-21 (200-21, Peprotech)を混合して加えた。3日間37℃のCO2インキュベーターにて培養後、Cell titer glo（G7572, Promega）を用いて増殖した生細胞量の測定を行うことで、mature h(m)TGF-beta3と抗TGF-beta3特異的非中和抗体のICによるヒトB細胞増殖抑制効果を検証した。標準品として、抗KLH抗体 (IC17-hIgG1)と抗pan-TGFbeta中和抗体(GC1008-hIgG1)を用いて同じタイミングで刺激を開始した。 Prepare a final concentration of 0.32 ng / mL of Mature TGF-beta3 and 2000, 400, 80, 16, 3.2, 0.64, 0.128 ng / mL of anti-TGF-beta3 specific non-neutralizing antibody. An immuno-complex (IC) was formed by reacting for a period of time and mixed with 1 × 10 4 B cells. In addition, the final concentration of 5 μg / mL of anti-human IgM (109-006-129, Jackson ImmunoResearch), 1 μg / mL of anti-CD40 (AF632, R & D Systems), and 33 ng / mL of recombinant human were also used to stimulate cell growth. IL-21 (200-21, Peprotech) was mixed and added. After culturing in a CO 2 incubator at 37 ° C for 3 days, the amount of viable cells grown using Cell titer glo (G7572, Promega) is measured, so that it is specific for mature h (m) TGF-beta3 and anti-TGF-beta3 Inhibitory effects of human non-neutralizing antibodies on the proliferation of human B cells were examined. Stimulation was started at the same timing using an anti-KLH antibody (IC17-hIgG1) and an anti-pan-TGFbeta neutralizing antibody (GC1008-hIgG1) as standards.
　結果として、mature h(m)TGF-beta3によるB細胞増殖抑制効果と同様に、mature h(m)TGF-beta3と抗TGF-beta3特異的非中和抗体のICによるヒトB細胞増殖抑制効果が確認された。一方で、mature h(m)TGFbeta3とGC1008-hIgG1のIC添加条件においてB細胞増殖抑制効果は確認されず、B細胞増殖刺激単独と変わらない細胞の増殖が確認された。以上の結果は、血中半減期の非常に短いことが知られているTGF-beta3と抗TGF-beta3特異的非中和抗体とのIC形成によって、そのヒトB細胞増殖抑制の生理活性が保持されることを示している。（図５） As a result, human B cell growth inhibitory effect by IC of mature h (m) TGF-beta3 and anti-TGF-beta3 specific non-neutralizing antibody is similar to that of mature h (m) TGF-beta3. confirmed. On the other hand, B cell proliferation inhibitory effect was not confirmed under the conditions of IC addition of maturemh (m) TGFbeta3 and GC1008-hIgG1, and cell proliferation was confirmed to be the same as B cell proliferation stimulation alone. These results indicate that the formation of IC between TGF-beta3, which is known to have a very short blood half-life, and anti-TGF-beta3-specific non-neutralizing antibody retains its biological activity in inhibiting human B cell proliferation. It is shown that. (Fig. 5)
実施例８　BALB/c nu/nuマウスへのCD4+CD25-T細胞移入モデルにおける抗dsDNA抗体産生抑制効果
　全身性エリスマトーデス（SLE）を含む自己免疫疾患の代表的な症状の一つとして、血清抗核抗体価の上昇が挙げられる。SLEのモデル動物としてMRL-Faslpr/lpr（MRL/lpr）マウスやNZB/W F1マウスなどがありいずれも血清抗核抗体価の上昇を伴うモデルであるが、発症・病態の進展までに数か月から1年近くの長期間の時間がかかることが知られている。本試験においては、BALB/c nu/nuマウスにBALB/cのCD4+CD25-T細胞を移入することによって、１ヶ月以内にIgGタイプの抗核抗体価が上昇するモデルを用いることで、抗TGF－beta3抗体のin vivo薬効を検討した。
　日本SLCより購入した8週齢・メスのBALB/c マウスをイソフルラン麻酔下全採血による安楽死処置を行い、脾臓を採取してACK lysing buffer（A10492-01, Gibco）にて溶血処理を行い、CD4+ T Cell アイソレーションキット, マウス（130-104-454, Miltenyi Biotec）を用いてCD4陽性細胞を回収した後に、CD25 マイクロビーズキット, マウス（130-091-072, Miltenyi Biotec）を用いてCD4+CD25-細胞を回収することにより、BALB/cマウス脾細胞由来CD4+CD25-T細胞を調製した。
　日本SLCより購入した8週齢・メスのBALB/c nu/nuマウスに対して、1匹あたり2×106 cellsのBALB/cマウス脾細胞由来CD4+CD25-T細胞の腹腔内投与を行った。細胞移入の直前に抗TGFbeta3抗体 10 mg/kgの尾静脈内投与を実施し、その後週1回の頻度で計4回尾静脈内投与を実施した。キャピジェクトEDTA-2Na採血を抗体の初回投与前（Pre）、および投与後Day12, Day20, Day28で行い、mouse anti-dsDNA IgG ELISA（レビス抗dsDNA-マウスELISA kit: AKRDD-061, Shibayagi）、抗Smith抗体（Mouse anti-Smith antibody ELISA Kit: MBS705621, MyBioSource）を用いて各種血漿中の抗体価を測定した。 Example 8 Suppressive effect of anti-dsDNA antibody production in CD4 + CD25 − T cell transfer model to BALB / c nu / nu mice As one of the typical symptoms of autoimmune diseases including systemic lupus erythematosus (SLE), An increase in serum antinuclear antibody titer can be mentioned. There are MRL-Faslpr / lpr (MRL / lpr) mice and NZB / W F1 mice as model animals for SLE, all of which are associated with increased serum antinuclear antibody titers. It is known to take a long time of nearly one year from the moon. In this study, anti-antinuclear antibody titer of IgG type is increased within one month by transferring BALB / c CD4 + CD25 - T cells into BALB / c nu / nu mice. The in vivo efficacy of TGF-beta3 antibody was examined.
Eight weeks old female BALB / c mice purchased from Japan SLC were euthanized by whole blood collection under isoflurane anesthesia, spleen was collected and hemolyzed with ACK lysing buffer (A10492-01, Gibco) After collecting CD4 positive cells using CD4 + T Cell Isolation Kit, mouse (130-104-454, Miltenyi Biotec), CD4 using CD25 MicroBead Kit, mouse (130-091-072, Miltenyi Biotec) BALB / c mouse spleen cell-derived CD4 + CD25 − T cells were prepared by recovering + CD25 − cells.
Eight weeks old female BALB / c nu / nu mice purchased from Japan SLC were intraperitoneally administered with 2 x 10 6 cells of BALB / c mouse splenocyte-derived CD4 + CD25 - T cells per mouse It was. Immediately before cell transfer, 10 mg / kg of anti-TGFbeta3 antibody was administered into the tail vein, and then administered once a week for a total of 4 times in the tail vein. Blood collection of EDTA-2Na was carried out before the first administration of the antibody (Pre) and on Day 12, Day 20, and Day 28 after administration, and mouse anti-dsDNA IgG ELISA (Levis anti-dsDNA-mouse ELISA kit: AKRDD-061, Shibayagi) Antibody titers in various plasmas were measured using Smith antibody (Mouse anti-Smith antibody ELISA Kit: MBS705621, MyBioSource).
　Day28における血漿中の抗dsDNA抗体価は、IC17-hIgG1投与群で131.3 Units/mLであったのに対し、本発明の抗TGF-beta3特異的非中和抗体であるTTA0003抗体・TTA0035抗体・TTB0019抗体・TTB0043抗体・F3NKB2-3_062抗体の抗TGFbeta3抗体投与群においてそれぞれ78.5 Units/mL・48.3 Units/mL・40.2 Units/mL・88.3 Units/mL・43.8 Units/mLと抑制傾向があることが確認された。（図６A） The anti-dsDNA antibody titer in plasma on Day 28 was 131.3 Units / mL in the IC17-hIgG1 administration group, whereas the anti-TGF-beta3-specific non-neutralizing antibody of the present invention, TTA0003 antibody, TTA0035 antibody, TTB0019 In the anti-TGFbeta3 antibody administration group of the antibody, TTB0043 antibody, and F3NKB2-3_062 antibody, it was confirmed that there was a tendency to suppress with 78.5 Units / mL, 48.3 Units / mL, 40.2 Units / mL, 88.3 Units / mL, 43.8 Units / mL, respectively It was. (Fig. 6A)
　さらにまた、Day28における血漿中の抗Smith抗体価を測定したところ、IC17-hIgG1投与群で266.4 ng/mLであったのに対し、TTA0003抗体・TTA0035抗体・TTB0019抗体・TTB0043抗体・TTB0048抗体・F3NKB2-3_062抗体の抗TGFbeta3抗体投与群において、それぞれ140.2 ng/mL・180.3 ng/mL・132.5 ng/mL・177.2 ng/mL・159.2 ng/mL・115.5 ng/mLと抗dsDNA抗体価の抑制傾向と同様に抗Smith抗体価においても抑制効果があることが確認された。（図６B） Furthermore, when anti-Smith antibody titer in plasma on Day 28 was measured, it was 266.4 ng / mL in the IC17-hIgG1 administration group, whereas TTA0003 antibody, TTA0035 antibody, TTB0019 antibody, TTB0043 antibody, TTB0048 antibody, F3NKB2 In the anti-TGFbeta3 antibody administration group of -3_062 antibody, the suppression tendency of anti-dsDNA antibody titer was 140.2 ng / mL, 180.3 ng / mL, 132.5 ng / mL, 177.2 ng / mL, 159.2 ng / mL, 115.5 ng / mL, respectively. Similarly, the anti-Smith antibody titer was confirmed to have an inhibitory effect. (Fig. 6B)
　これらの結果は、抗TGF-beta3特異的非中和抗体の投与によって生体内で産生されるTGF-beta3の血中濃度が上昇することでIgG産生を伴う自己免疫疾患のモデルマウスにおける自己抗体産生を抑制する効果を発揮した結果であると考えられ、抗TGF-beta3特異的非中和抗体の自己免疫疾患の治療剤としての応用可能性を示唆している。（図６） These results show that the production of autoantibodies in autoimmune disease model mice accompanied by IgG production due to increased blood levels of TGF-beta3 produced in vivo by administration of anti-TGF-beta3-specific non-neutralizing antibodies. This is thought to be the result of exerting an inhibitory effect, suggesting the possibility of application of anti-TGF-beta3-specific non-neutralizing antibody as a therapeutic agent for autoimmune diseases. (Fig. 6)

Claims (12)

  1. TGF-beta3に特異的に結合する非中和抗体。 Non-neutralizing antibody that specifically binds to TGF-beta3.
  2. 請求項１に記載の抗体であって、以下（１）から（２９）のいずれかに記載の抗体：
    （１）配列番号：１０１に記載のCDR１、配列番号：１０２に記載のCDR２、配列番号：１０３に記載のCDR３を有する重鎖可変領域、および配列番号：１０５に記載のCDR１、配列番号：１０６に記載のCDR２、配列番号：１０７に記載のCDR３を有する軽鎖可変領域を含む抗体
    （２）配列番号：１４１に記載のCDR１、配列番号：１４２に記載のCDR２、配列番号：１４３に記載のCDR３を有する重鎖可変領域、および配列番号：１４５に記載のCDR１、配列番号：１４６に記載のCDR２、配列番号：１４７に記載のCDR３を有する軽鎖可変領域を含む抗体
    （３）配列番号：１６１に記載のCDR１、配列番号：１６２に記載のCDR２、配列番号：１６３に記載のCDR３を有する重鎖可変領域、および配列番号：１６５に記載のCDR１、配列番号：１６６に記載のCDR２、配列番号：１６７に記載のCDR３を有する軽鎖可変領域を含む抗体
    （４）配列番号：１７１に記載のCDR１、配列番号：１７２に記載のCDR２、配列番号：１７３に記載のCDR３を有する重鎖可変領域、および配列番号：１７５に記載のCDR１、配列番号：１７６に記載のCDR２、配列番号：１７７に記載のCDR３を有する軽鎖可変領域を含む抗体
    （５）配列番号：１８１に記載のCDR１、配列番号：１８２に記載のCDR２、配列番号：１８３に記載のCDR３を有する重鎖可変領域、および配列番号：１８５に記載のCDR１、配列番号：１８６に記載のCDR２、配列番号：１８７に記載のCDR３を有する軽鎖可変領域を含む抗体
    （６）配列番号：１９１に記載のCDR１、配列番号：１９２に記載のCDR２、配列番号：１９３に記載のCDR３を有する重鎖可変領域、および配列番号：１９５に記載のCDR１、配列番号：１９６に記載のCDR２、配列番号：１９７に記載のCDR３を有する軽鎖可変領域を含む抗体
    （７）配列番号：２０１に記載のCDR１、配列番号：２０２に記載のCDR２、配列番号：２０３に記載のCDR３を有する重鎖可変領域、および配列番号：２０５に記載のCDR１、配列番号：２０６に記載のCDR２、配列番号：２０７に記載のCDR３を有する軽鎖可変領域を含む抗体
    （８）配列番号：２１１に記載のCDR１、配列番号：２１２に記載のCDR２、配列番号：２１３に記載のCDR３を有する重鎖可変領域、および配列番号：２１５に記載のCDR１、配列番号：２１６に記載のCDR２、配列番号：２１７に記載のCDR３を有する軽鎖可変領域を含む抗体
    （９）配列番号：２４１に記載のCDR１、配列番号：２４２に記載のCDR２、配列番号：２４３に記載のCDR３を有する重鎖可変領域、および配列番号：２４５に記載のCDR１、配列番号：２４６に記載のCDR２、配列番号：２４７に記載のCDR３を有する軽鎖可変領域を含む抗体
    （１０）配列番号：２６１に記載のCDR１、配列番号：２６２に記載のCDR２、配列番号：２６３に記載のCDR３を有する重鎖可変領域、および配列番号：２６５に記載のCDR１、配列番号：２６６に記載のCDR２、配列番号：２６７に記載のCDR３を有する軽鎖可変領域を含む抗体
    （１１）配列番号：２９１に記載のCDR１、配列番号：２９２に記載のCDR２、配列番号：２９３に記載のCDR３を有する重鎖可変領域、および配列番号：２９５に記載のCDR１、配列番号：２９６に記載のCDR２、配列番号：２９７に記載のCDR３を有する軽鎖可変領域を含む抗体
    （１２）配列番号：３０１に記載のCDR１、配列番号：３０２に記載のCDR２、配列番号：３０３に記載のCDR３を有する重鎖可変領域、および配列番号：３０５に記載のCDR１、配列番号：３０６に記載のCDR２、配列番号：３０７に記載のCDR３を有する軽鎖可変領域を含む抗体
    （１３）配列番号：３６１に記載のCDR１、配列番号：３６２に記載のCDR２、配列番号：３６３に記載のCDR３を有する重鎖可変領域、および配列番号：３６５に記載のCDR１、配列番号：３６６に記載のCDR２、配列番号：３６７に記載のCDR３を有する軽鎖可変領域を含む抗体
    （１４）配列番号：３７１に記載のCDR１、配列番号：３７２に記載のCDR２、配列番号：３７３に記載のCDR３を有する重鎖可変領域、および配列番号：３７５に記載のCDR１、配列番号：３７６に記載のCDR２、配列番号：３７７に記載のCDR３を有する軽鎖可変領域を含む抗体
    （１５）配列番号：３９１に記載のCDR１、配列番号：３９２に記載のCDR２、配列番号：３９３に記載のCDR３を有する重鎖可変領域、および配列番号：３９５に記載のCDR１、配列番号：３９６に記載のCDR２、配列番号：３９７に記載のCDR３を有する軽鎖可変領域を含む抗体
    （１６）配列番号：４０１に記載のCDR１、配列番号：４０２に記載のCDR２、配列番号：４０３に記載のCDR３を有する重鎖可変領域、および配列番号：４０５に記載のCDR１、配列番号：４０６に記載のCDR２、配列番号：４０７に記載のCDR３を有する軽鎖可変領域を含む抗体
    （１７）配列番号：４８１に記載のCDR１、配列番号：４８２に記載のCDR２、配列番号：４８３に記載のCDR３を有する重鎖可変領域、および配列番号：４８５に記載のCDR１、配列番号：４８６に記載のCDR２、配列番号：４８７に記載のCDR３を有する軽鎖可変領域を含む抗体
    （１８）配列番号：４９１に記載のCDR１、配列番号：４９２に記載のCDR２、配列番号：４９３に記載のCDR３を有する重鎖可変領域、および配列番号：４９５に記載のCDR１、配列番号：４９６に記載のCDR２、配列番号：４９７に記載のCDR３を有する軽鎖可変領域を含む抗体
    （１９）配列番号：５１１に記載のCDR１、配列番号：５１２に記載のCDR２、配列番号：５１３に記載のCDR３を有する重鎖可変領域、および配列番号：５１５に記載のCDR１、配列番号：５１６に記載のCDR２、配列番号：５１７に記載のCDR３を有する軽鎖可変領域を含む抗体
    （２０）配列番号：５２１に記載のCDR１、配列番号：５２２に記載のCDR２、配列番号：５２３に記載のCDR３を有する重鎖可変領域、および配列番号：５２５に記載のCDR１、配列番号：５２６に記載のCDR２、配列番号：５２７に記載のCDR３を有する軽鎖可変領域を含む抗体
    （２１）配列番号：５３１に記載のCDR１、配列番号：５３２に記載のCDR２、配列番号：５３３に記載のCDR３を有する重鎖可変領域、および配列番号：５３５に記載のCDR１、配列番号：５３６に記載のCDR２、配列番号：５３７に記載のCDR３を有する軽鎖可変領域を含む抗体
    （２２）配列番号：５６１に記載のCDR１、配列番号：５６２に記載のCDR２、配列番号：５６３に記載のCDR３を有する重鎖可変領域、および配列番号：５６５に記載のCDR１、配列番号：５６６に記載のCDR２、配列番号：５６７に記載のCDR３を有する軽鎖可変領域を含む抗体
    （２３）配列番号：５７１に記載のCDR１、配列番号：５７２に記載のCDR２、配列番号：５７３に記載のCDR３を有する重鎖可変領域、および配列番号：５７５に記載のCDR１、配列番号：５７６に記載のCDR２、配列番号：５７７に記載のCDR３を有する軽鎖可変領域を含む抗体
    （２４）配列番号：６８１に記載のCDR１、配列番号：６８２に記載のCDR２、配列番号：６８３に記載のCDR３を有する重鎖可変領域、および配列番号：６８５に記載のCDR１、配列番号：６８６に記載のCDR２、配列番号：６８７に記載のCDR３を有する軽鎖可変領域を含む抗体
    （２５）配列番号：７２１に記載のCDR１、配列番号：７２２に記載のCDR２、配列番号：７２３に記載のCDR３を有する重鎖可変領域、および配列番号：７２５に記載のCDR１、配列番号：７２６に記載のCDR２、配列番号：７２７に記載のCDR３を有する軽鎖可変領域を含む抗体
    （２６）配列番号：７４１に記載のCDR１、配列番号：７４２に記載のCDR２、配列番号：７４３に記載のCDR３を有する重鎖可変領域、および配列番号：７４５に記載のCDR１、配列番号：７４６に記載のCDR２、配列番号：７４７に記載のCDR３を有する軽鎖可変領域を含む抗体
    （２７）配列番号：７９１に記載のCDR１、配列番号：７９２に記載のCDR２、配列番号：７９３に記載のCDR３を有する重鎖可変領域、および配列番号：７９５に記載のCDR１、配列番号：７９６に記載のCDR２、配列番号：７９７に記載のCDR３を有する軽鎖可変領域を含む抗体
    （２８）上記（１）～（２７）のいずれかに記載の抗体とTGF-beta3への結合において競合する抗体
    （２９）上記（１）～（２７）のいずれかに記載の抗体が結合するTGF-beta3のエピトープと同じエピトープに結合する抗体     The antibody according to claim 1, wherein the antibody according to any one of (1) to (29) below:
    (1) A heavy chain variable region having CDR1 described in SEQ ID NO: 101, CDR2 described in SEQ ID NO: 102, CDR3 described in SEQ ID NO: 103, and CDR1 described in SEQ ID NO: 105, SEQ ID NO: 106 An antibody comprising a light chain variable region having CDR2 described in SEQ ID NO: 107 and CDR3 described in SEQ ID NO: 107 (2) CDR1 described in SEQ ID NO: 141, CDR2 described in SEQ ID NO: 142, described in SEQ ID NO: 143 An antibody (3) comprising a heavy chain variable region having CDR3, and a light chain variable region having CDR1 set forth in SEQ ID NO: 145, CDR2 set forth in SEQ ID NO: 146, CDR3 set forth in SEQ ID NO: 147 161, CDR2 of SEQ ID NO: 162, heavy chain variable region having CDR3 of SEQ ID NO: 163, CDR1 of SEQ ID NO: 165, CDR2 of SEQ ID NO: 166, sequence Antibody comprising light chain variable region having CDR3 of No. 167 (4) CDR1 of SEQ ID NO: 171; CDR2 of SEQ ID NO: 172; heavy chain variable having CDR3 of SEQ ID NO: 173 An antibody comprising a region and a light chain variable region having CDR1 set forth in SEQ ID NO: 175, CDR2 set forth in SEQ ID NO: 176, CDR3 set forth in SEQ ID NO: 177 (5) CDR1 set forth in SEQ ID NO: 181, SEQ ID NO: 182 CDR2, SEQ ID NO: 183 heavy chain variable region having CDR3, and SEQ ID NO: 185 CDR1, SEQ ID NO: 186 CDR2, SEQ ID NO: 187 Antibody comprising a light chain variable region having CDR3 (6) CDR1 set forth in SEQ ID NO: 191, CDR2 set forth in SEQ ID NO: 192, heavy chain variable region having CDR3 set forth in SEQ ID NO: 193, and SEQ ID NO: An antibody comprising a light chain variable region having CDR1 described in 95, CDR2 described in SEQ ID NO: 196, CDR3 described in SEQ ID NO: 197 (7) CDR1 described in SEQ ID NO: 201, described in SEQ ID NO: 202 And a heavy chain variable region having the CDR3 of SEQ ID NO: 203, a CDR1 of the SEQ ID NO: 205, a CDR2 of the SEQ ID NO: 206, and a light chain variable having the CDR3 of the SEQ ID NO: 207 Antibody comprising region (8) CDR1 set forth in SEQ ID NO: 211, CDR2 set forth in SEQ ID NO: 212, heavy chain variable region having CDR3 set forth in SEQ ID NO: 213, and CDR1 set forth in SEQ ID NO: 215, An antibody comprising a light chain variable region having CDR2 set forth in SEQ ID NO: 216 and CDR3 set forth in SEQ ID NO: 217 (9) CDR1 set forth in SEQ ID NO: 241, CDR2 set forth in SEQ ID NO: 242, SEQ ID NO: 24 An antibody comprising a heavy chain variable region having CDR3 according to 3, and a CDR1 according to SEQ ID NO: 245, a CDR2 according to SEQ ID NO: 246, and a light chain variable region having CDR3 as SEQ ID NO: 247 (10 ) CDR1 described in SEQ ID NO: 261, CDR2 described in SEQ ID NO: 262, heavy chain variable region having CDR3 described in SEQ ID NO: 263, and CDR1 described in SEQ ID NO: 265, described in SEQ ID NO: 266 An antibody comprising a light chain variable region having CDR3 described in SEQ ID NO: 267 (11) CDR1 described in SEQ ID NO: 291, CDR2 described in SEQ ID NO: 292, CDR3 described in SEQ ID NO: 293 An antibody (12) comprising a heavy chain variable region having a light chain variable region having a CDR1 described in SEQ ID NO: 295, a CDR2 described in SEQ ID NO: 296, a CDR3 described in SEQ ID NO: 297; CDR1, described in SEQ ID NO: 302, heavy chain variable region having CDR3 described in SEQ ID NO: 303, CDR1 described in SEQ ID NO: 305, CDR2 described in SEQ ID NO: 306, SEQ ID NO: Antibody comprising a light chain variable region having CDR3 described in 307 (13) CDR1 described in SEQ ID NO: 361, CDR2 described in SEQ ID NO: 362, heavy chain variable region having CDR3 described in SEQ ID NO: 363 And an antibody comprising a light chain variable region having CDR1 as set forth in SEQ ID NO: 365, CDR2 as set forth in SEQ ID NO: 366, CDR3 as set forth in SEQ ID NO: 367 (14) CDR1, set forth in SEQ ID NO: 371 SEQ ID NO: 372 CDR2, SEQ ID NO: 373 heavy chain variable region having CDR3, SEQ ID NO: 375 CDR1, SEQ ID NO: 376 CDR2, SEQ ID NO: 377 Antibody comprising a light chain variable region having the CDR3 of (15) CDR1 set forth in SEQ ID NO: 391, CDR2 set forth in SEQ ID NO: 392, heavy chain variable region having CDR3 set forth in SEQ ID NO: 393, and SEQ ID NO: Antibody comprising a light chain variable region having CDR1 described in SEQ ID NO: 396, CDR2 described in SEQ ID NO: 396, CDR3 described in SEQ ID NO: 397 (16) CDR1 described in SEQ ID NO: 401, SEQ ID NO: 402 A heavy chain variable region having the CDR2 of SEQ ID NO: 403 and a light chain having a CDR3 of SEQ ID NO: 405, a CDR2 of SEQ ID NO: 406, a light chain having a CDR3 of SEQ ID NO: 407 Antibody comprising variable region (17) CDR1 described in SEQ ID NO: 481, CDR2 described in SEQ ID NO: 482, heavy chain variable region having CDR3 described in SEQ ID NO: 483, and SEQ ID NO: 485 An antibody comprising a light chain variable region having CDR3 of SEQ ID NO: 487, CDR3 of SEQ ID NO: 487, CDR1 of SEQ ID NO: 491, CDR2 of SEQ ID NO: 492, A heavy chain variable region having the CDR3 of SEQ ID NO: 493, a CDR1 of SEQ ID NO: 495, a CDR2 of SEQ ID NO: 496, a light chain variable region having the CDR3 of SEQ ID NO: 497 Antibody (19) CDR1 set forth in SEQ ID NO: 511, CDR2 set forth in SEQ ID NO: 512, heavy chain variable region having CDR3 set forth in SEQ ID NO: 513, and CDR1 set forth in SEQ ID NO: 515, SEQ ID NO: An antibody comprising a light chain variable region having CDR2 described in 516 and CDR3 described in SEQ ID NO: 517 (20) CDR1 described in SEQ ID NO: 521, CDR2 described in SEQ ID NO: 522, SEQ ID NO: 523 The antibody (21) sequence comprising the heavy chain variable region having the CDR3 and the CDR1 described in SEQ ID NO: 525, the CDR2 described in SEQ ID NO: 526, and the light chain variable region having the CDR3 described in SEQ ID NO: 527 SEQ ID NO: 531, CDR2 set forth in SEQ ID NO: 532, heavy chain variable region having CDR3 set forth in SEQ ID NO: 533, and CDR1 set forth in SEQ ID NO: 535, CDR2 set forth in SEQ ID NO: 536 An antibody comprising a light chain variable region having CDR3 described in SEQ ID NO: 537 (22) CDR1 described in SEQ ID NO: 561, CDR2 described in SEQ ID NO: 562, and a heavy chain having CDR3 described in SEQ ID NO: 563 An antibody (23) comprising a chain variable region and a light chain variable region having CDR1 set forth in SEQ ID NO: 565, CDR2 set forth in SEQ ID NO: 566, CDR3 set forth in SEQ ID NO: 567 (23) in SEQ ID NO: 571 The CDR1, the CDR2 described in SEQ ID NO: 572, the heavy chain variable region having the CDR3 described in SEQ ID NO: 573, the CDR1 described in SEQ ID NO: 575, the CDR2 described in SEQ ID NO: 576, the SEQ ID NO: An antibody comprising a light chain variable region having CDR3 according to 577 (24), CDR1 according to SEQ ID NO: 681, CDR2 according to SEQ ID NO: 682, heavy chain variable region having CDR3 according to SEQ ID NO: 683, And an antibody comprising a light chain variable region having CDR1 described in SEQ ID NO: 685, CDR2 described in SEQ ID NO: 686, CDR3 described in SEQ ID NO: 687 (25) CDR1 described in SEQ ID NO: 721, SEQ ID NO: : CDR2 described in 722, heavy chain variable region having CDR3 described in SEQ ID NO: 723, CDR1 described in SEQ ID NO: 725, CDR2 described in SEQ ID NO: 726, CDR described in SEQ ID NO: 727 Antibody comprising a light chain variable region having 3 (26) CDR1 set forth in SEQ ID NO: 741, CDR2 set forth in SEQ ID NO: 742, heavy chain variable region having CDR3 set forth in SEQ ID NO: 743, and SEQ ID NO: An antibody comprising a light chain variable region having CDR1 described in 745, CDR2 described in SEQ ID NO: 746, CDR3 described in SEQ ID NO: 747 (27) CDR1 described in SEQ ID NO: 791, SEQ ID NO: 792 And a heavy chain variable region having the CDR3 of SEQ ID NO: 793, a CDR1 of SEQ ID NO: 795, a CDR2 of SEQ ID NO: 796, and a light chain variable having a CDR3 of SEQ ID NO: 797 An antibody comprising a region (28) an antibody that competes with TGF-beta3 for binding to the antibody according to any of (1) to (27) above (29) according to any of (1) to (27) above TGF-bet to which antibody binds An antibody that binds to the same epitope as that of a3
  3. TGF-beta3のLatent体および／またはMature体に結合する請求項１または２に記載の抗体。 The antibody according to claim 1 or 2, which binds to a Latent body and / or a Mature body of TGF-beta3.
  4. TGF-beta3の生理活性の抑制が20％以下である請求項１－３のいずれかに記載の抗体。 The antibody according to any one of claims 1 to 3, wherein the inhibition of physiological activity of TGF-beta3 is 20% or less.
  5. TGF-beta3への結合値に対するTGF-beta1およびTGF-beta2への結合値が10％未満である請求項１－４のいずれかに記載の抗体。 The antibody according to any one of claims 1 to 4, wherein the binding value to TGF-beta1 and TGF-beta2 relative to the binding value to TGF-beta3 is less than 10%.
  6. モノクローナル抗体である、請求項１－５のいずれかに記載の抗体。 The antibody according to any one of claims 1 to 5, which is a monoclonal antibody.
  7. ヒト抗体、ヒト化抗体、またはキメラ抗体である、請求項１－６のいずれかに記載の抗体。 The antibody according to any one of claims 1 to 6, which is a human antibody, a humanized antibody, or a chimeric antibody.
  8. B細胞を認識する可変領域を更に含む二重特異性抗体である請求項１－７のいずれかに記載の抗体。 The antibody according to any one of claims 1 to 7, which is a bispecific antibody further comprising a variable region recognizing B cells.
  9. 前記可変領域はCD19、CD20、CD40、CD22、IL21R、BAFF-R、BCMA、TACI、CD27、又はCD138に含まれるいずれか1つを認識する請求項８に記載の抗体。 The antibody according to claim 8, wherein the variable region recognizes any one of CD19, CD20, CD40, CD22, IL21R, BAFF-R, BCMA, TACI, CD27, or CD138.
  10. 請求項１－９のいずれかに記載の抗体を含むB細胞活性化の抑制剤。 An inhibitor of B cell activation comprising the antibody according to any one of claims 1-9.
  11. 請求項１－９のいずれかに記載の抗体を含む自己免疫疾患の治療剤。 The therapeutic agent of an autoimmune disease containing the antibody in any one of Claims 1-9.
  12. 前記自己免疫疾患は全身性エリテマトーデス、天疱瘡、多発性硬化症、視神経脊髄炎、ANCA関連血管炎、関節リウマチ、移植臓器拒絶、シェーグレン症候群、若年性皮膚筋炎、重症筋無力症、又はバセドウ病、橋本病等の自己免疫性甲状腺疾患のいずれかである請求項１１に記載の治療剤。 The autoimmune disease is systemic lupus erythematosus, pemphigus, multiple sclerosis, optic neuromyelitis, ANCA-related vasculitis, rheumatoid arthritis, transplant organ rejection, Sjogren's syndrome, juvenile dermatomyositis, myasthenia gravis, or Graves' disease, The therapeutic agent according to claim 11, which is any of autoimmune thyroid diseases such as Hashimoto's disease.
PCT/JP2017/019457 2016-05-27 2017-05-25 ANTI-TGF-beta3 ANTIBODY AND USE THEREOF WO2017204277A1 (en)

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Cited By (4)

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WO2023039612A1 (en) * 2021-09-13 2023-03-16 The Board Of Regents Of The University Of Texas System Trem2 antigen binding proteins and uses thereof

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