WO2017204277A1 - Anticorps anti-tgf-β3 et utilisation associée - Google Patents

Anticorps anti-tgf-β3 et utilisation associée 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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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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Chugai Pharmaceutical Co Ltd
University of Tokyo NUC
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Chugai Pharmaceutical Co Ltd
University of Tokyo NUC
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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 [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IG], 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

La présente invention concerne un anticorps anti-TGF-β3 et une méthode d'utilisation de l'anticorps. L'anticorps anti-TGF-β3 selon la présente invention présente une activité de liaison au TGF-β3 mais n'a pas d'activité neutralisante vis-à-vis du TGF-bêta3, et est utile en tant qu'inhibiteur pour l'activation de lymphocytes B et en tant qu'agent thérapeutique pour des maladies auto-immunes.
PCT/JP2017/019457 2016-05-27 2017-05-25 Anticorps anti-tgf-β3 et utilisation associée Ceased WO2017204277A1 (fr)

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020095104A1 (fr) * 2018-11-05 2020-05-14 Ludwig Institute For Cancer Research Ltd. ANTICORPS SPÉCIFIQUES DE TGF-β3 VARIANTS ET PROCÉDÉS ET UTILISATIONS ASSOCIÉS
WO2021021991A1 (fr) 2019-08-01 2021-02-04 Vaccinex,Inc. Inhibition combinée de sémaphorine-4d et tgfb, et compositions associées
EP3922644A3 (fr) * 2020-05-20 2022-04-13 The Texas A&M University System Compositions et utilisation d'un motif de liaison au fibrinogène en présence d'efb et de coa pour agents thérapeutiques et vaccins contre le staphylococcus aureus
CN115380050A (zh) * 2020-02-07 2022-11-22 新加坡科技研究局 针对alppl2和/或alpp的抗原结合分子及其用途
WO2023039612A1 (fr) * 2021-09-13 2023-03-16 The Board Of Regents Of The University Of Texas System Protéines de liaison à l'antigène trem2 et leurs utilisations
CN118530349A (zh) * 2024-06-03 2024-08-23 华中农业大学 一株可中和猪轮状病毒不同血清型毒株的单克隆抗体
TWI867190B (zh) * 2020-03-19 2024-12-21 美商建南德克公司 同功型選擇性抗-TGF-β抗體及其使用方法
WO2025145087A1 (fr) * 2023-12-29 2025-07-03 Eureka Therapeutics, Inc. Constructions ciblant des complexes peptide de msln/cmh et leurs utilisations

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5055289A (en) * 1983-09-01 1991-10-08 Hybritech Incorporated Interferon antibody therapeutic compositions having an extended serum half-life
EP0536275B1 (fr) * 1990-06-25 1998-04-22 Oncogene Science, Inc. Inhibiteurs de croissance de tumeurs derives de tissus, procedes de preparation et utilisations desdits inhibiteurs

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5055289A (en) * 1983-09-01 1991-10-08 Hybritech Incorporated Interferon antibody therapeutic compositions having an extended serum half-life
EP0536275B1 (fr) * 1990-06-25 1998-04-22 Oncogene Science, Inc. Inhibiteurs de croissance de tumeurs derives de tissus, procedes de preparation et utilisations desdits inhibiteurs

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
FUJIO K. ET AL.: "Therapeutic potential of regulatory cytokines that target B cells", INT. IMMUNOL., vol. 28, no. 4, 2015, pages 189 - 195, XP055442133 *
HAWORTH R.S. ET AL.: "Differential changes in transforming growth factor-beta isoform expression during postnatal cardiac growth", BIOCHEM. BIOPHYS. RES. COMMUN., vol. 245, no. 3, 1998, pages 923 - 927, XP055442130 *
HOFFMANN E. ET AL.: "PK modulation of haptenylated peptides via non-covalent antibody complexation", J. CONTROL. RELEASE, vol. 171, no. 1, 2013, pages 48 - 56, XP055114266 *
METZ S. ET AL.: "Bispecific digoxigenin-binding antibodies for targeted payload delivery", PROC. NATL. ACAD. SCI. U.S.A., vol. 108, no. 20, 2011, pages 8194 - 8199, XP055345937 *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020095104A1 (fr) * 2018-11-05 2020-05-14 Ludwig Institute For Cancer Research Ltd. ANTICORPS SPÉCIFIQUES DE TGF-β3 VARIANTS ET PROCÉDÉS ET UTILISATIONS ASSOCIÉS
CN113613726A (zh) * 2018-11-05 2021-11-05 路德维格癌症研究所有限公司 人源化和变体TGF-β3特异性抗体及其方法和用途
CN113613726B (zh) * 2018-11-05 2025-07-15 路德维格癌症研究所有限公司 人源化和变体TGF-β3特异性抗体及其方法和用途
WO2021021991A1 (fr) 2019-08-01 2021-02-04 Vaccinex,Inc. Inhibition combinée de sémaphorine-4d et tgfb, et compositions associées
CN115380050A (zh) * 2020-02-07 2022-11-22 新加坡科技研究局 针对alppl2和/或alpp的抗原结合分子及其用途
TWI867190B (zh) * 2020-03-19 2024-12-21 美商建南德克公司 同功型選擇性抗-TGF-β抗體及其使用方法
EP3922644A3 (fr) * 2020-05-20 2022-04-13 The Texas A&M University System Compositions et utilisation d'un motif de liaison au fibrinogène en présence d'efb et de coa pour agents thérapeutiques et vaccins contre le staphylococcus aureus
WO2023039612A1 (fr) * 2021-09-13 2023-03-16 The Board Of Regents Of The University Of Texas System Protéines de liaison à l'antigène trem2 et leurs utilisations
WO2025145087A1 (fr) * 2023-12-29 2025-07-03 Eureka Therapeutics, Inc. Constructions ciblant des complexes peptide de msln/cmh et leurs utilisations
CN118530349A (zh) * 2024-06-03 2024-08-23 华中农业大学 一株可中和猪轮状病毒不同血清型毒株的单克隆抗体

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