Monoclonal antibody against the β-chain region of human TRBV9

Abstract

The present invention relates to monoclonal humanized antibodies or antigen-binding fragments thereof that specifically bind to the TRBV9 family of human T-cell receptors. The present invention also relates to nucleic acids encoding said antibodies or antigen-binding fragments thereof, expression vectors, methods for preparing said antibodies and the use of said antibodies in the treatment of diseases or disorders associated with said family of human T-cell receptors. The present invention relates to the production of antibodies that can be used to treat, inter alia, ankylosing spondylitis (AS or Bechterev's disease), celiac disease and blood cancers, whose pathogenesis involves T-cell receptors of the TRBV9 family.

Description

Technical Field

[0001] The present invention relates to the fields of biotechnology and biomedicine, and in particular to antibodies or antigen-binding fragments thereof, and to uses thereof. More specifically, the present invention relates to monoclonal humanized antibodies that specifically bind to the human T cell receptor family. The present invention also relates to nucleic acids encoding the antibodies or antigen-binding fragments thereof, expression vectors, methods for preparing the antibodies, and uses of the antibodies in treating diseases or disorders associated with the human T cell receptor family. Background Art

[0002] Autoimmune diseases are caused by autoreactive T lymphocytes (Haroon N et al., Arthritis Rheum. 2013 Oct; 65(10):2645-54., Duarte J. et al., PloS One 2010 May 10; 5(5):e10558; Konig M. et al., Front Immunol 2016 Jan 25; 7:11). The prior art discloses that T cell receptor (TCR) sequences are markers that allow identification of T-lymphocyte clones involved in the pathogenesis of autoimmune diseases. Structurally, the subunits of the T-cell receptor are members of the immunoglobulin superfamily and are formed by several gene segments. The TCR variable regions form the TCR antigen binding site. This means that they are clone-specific, i.e., they differ in that they are T lymphocytes that respond to unique antigens.

[0003] According to the amino acid homology of the variable (V) gene segment in the TCR variable domain, T cell receptor is divided into different families. According to the IMGT nomenclature, β-chain is classified into 26 unique families, and α chain is classified into 41 families (Turner SJ et al., Nature Reviews Immunology 2006, V.6, 883-894). In order to determine the TCR chain family, people use the multiple comparison of the test amino acid sequence and the known TCR chain sequence, and the information about the known TCR chain sequence is summarized in the IMGT database (" The international ImMunoGeneTics information system ", Lefranc MP., Nucl Acids Res 2001; 29:207-209), which can be obtained on the Internet at http: / / www.imgt.org. Multiple comparisons and determination of TCR chain families can be performed using the IgBlast software package.

[0004] WO9006758 discloses monoclonal antibodies W112 and 2D1 directed against the β-chain region of the variable domain of the human T cell receptor belonging to the TRBV5-3 and TRBV8-1 families, which are proposed as diagnostic and therapeutic tools for rheumatoid arthritis. The monoclonal antibodies recognize 0.3-5% of peripheral T lymphocytes with TRBV5-3 and 0.5-13% of peripheral T lymphocytes with TRBV8-1, respectively. The results of many studies confirming the involvement of T lymphocytes in the pathogenesis of rheumatoid arthritis have led to the application of monoclonal antibodies specific to the β-chain region of the T receptor. Specifically, Brennan et al., Clin ExpImmunol. 1988 September; 73(3): 417-423 have confirmed the elevated percentage of T lymphocytes with TRBV5 and TRBV8 in synovial fluid samples of patients suffering from rheumatoid arthritis compared with healthy individuals. WO9405801 discloses monoclonal antibodies for the diagnosis and treatment of rheumatoid arthritis that interact with an epitope of the VB3.1 variable region of the human T-cell receptor, which interacts with the TCR V(β)3.1 subfamily.

[0005] Monoclonal antibodies specifically recognizing the 13th family β-chain of rat TRC have also been described. Animal models have confirmed that, with the help of these antibodies, a small group of T cells (VB13+ T cells) whose T receptors contain VB13β-chains can be preventively removed, and it has been confirmed that such a procedure protects against the occurrence of type I diabetes in rats with a tendency to diabetes, and also significantly reduces the risk of occurrence of virus-induced diabetes (Zhijun Liu et al., Diabetes.2012 May; 61(5): 1160-1168.). Meanwhile, the removal results of T cells whose T receptors contain a unique β-chain family (VB16) are not different from those of the control group. Importantly, it is noted that even the first administration of the monoclonal antibody for VB13 results in a 60% decrease in the number of VB13+ T cells in the spleen of rats.

[0006] A consistent variant of an autoimmune TCR has been described in patients with ankylosing spondylitis (AS or Bechterev's disease), which has been shown to be present in the synovial fluid and peripheral blood of patients with AS and absent in the same depth analysis of healthy donors, regardless of their HLA*B27 allele status (Faham M. et al., ArthritisRheumatol. 2017;69(4):774-784; Komech Е et al. 12th EJI-EFIS Tatra ImmunologyConference; September 3-7, 2016; Strbske Pleso, Slovakia. Abstract book p. 39). The TCR is a member of the TRBV9 family (according to the IMGT nomenclature). It has been shown that T cell receptors with TRBV9 family β-chains are also involved in the development of autoimmune diseases such as celiac disease (Petersen J et al., J Immunol. 2015; 194(12):6112-22). They are also present on the surface of T cells undergoing progression in T cell lymphomas and T cell leukemias, including T-cell lymphomas caused by Epstein-Barr virus (EBV) (Toyabe S et al., Clin Exp Immunol. 2003; 134(1): 92-97).

[0007] Application No. RU2017145662 has recently described chimeric monoclonal antibodies capable of specifically binding to the β-chain region of the TRBV9 family of human T receptors, which can be used to treat autoimmune and oncological diseases whose pathogenesis involves TCRs belonging to the TRBV9 family, such as AS, celiac disease and some T-cell lymphomas and T-cell leukemias.

[0008] The antibodies are the only currently known antibodies that can be used to eliminate T cells with TRBV9 family TCRs. The main disadvantage of the antibodies is the relatively low degree of humanization, i.e. they contain human-like constant regions and structural components, but have rat-like variable domains. The degree of humanization of the variable fragment of the heavy chain of the antibody is 72%, while the degree of humanization of the variable fragment of the light chain is 69%.

[0009] The above parent monoclonal antibodies include:

[0010] 1) The variable domain of their heavy chain (VH), which contains three hypervariable regions HCDR1, HCDR2 and HCDR3, wherein

[0011] HCDR1 (according to the Kabat numbering scheme) has the amino acid sequence of SEQ ID NO: 1,

[0012] HCDR2 has the amino acid sequence of SEQ ID NO: 2

[0013] HCDR3 has the amino acid sequence of SEQ ID No: 3;

[0014] 2) The variable domain of their light chain (VL), which comprises three hypervariable regions LCDR1, LCDR2 and LCDR3, wherein:

[0015] LCDR1 has the amino acid sequence of SEQ ID NO: 4,

[0016] LCDR2 has the amino acid sequence of SEQ ID NO: 5,

[0017] LCDR3 has the amino acid sequence of SEQ ID NO: 6.

[0018] The above parent monoclonal antibody includes variable domains of heavy and light chains, which have the amino acid sequences shown in SEQ ID NOs: 8 and 10.

[0019] The above parent monoclonal antibody includes a light chain having the amino acid sequence shown in SEQ ID No. 12 and an antibody heavy chain having the amino acid sequence of SEQ ID No. 14.

[0020] Examples of nucleotide sequences encoding the amino acid sequences of the heavy chain and light chain of the above parent antibody are shown in SEQ ID NOs: 13 and 11.

[0021] The present invention relates to the preparation of monoclonal antibodies, which can be used to eliminate T cells with TRBV9 family TCRs, in particular for the treatment of AS, celiac disease and malignant blood diseases whose pathogenesis involves TRBV9 family TCRs, and are characterized by a high degree of humanization. At the same time, humanization often leads to a critical decrease in antibody affinity and / or solubility. Therefore, the preparation of humanized functional antibodies is a relevant task. Summary of the invention

[0022] The present invention relates to a humanized monoclonal antibody and an antigen-binding fragment thereof, which has the ability to specifically bind to the TRBV9 family β-chain region of human T receptors with high affinity. The antibody according to the present invention can be used as a drug for treating autoimmune and oncological diseases whose pathogenesis involves TCR belonging to the TRBV9 family, for example, AS, celiac disease and some T cell lymphomas and T cell leukemias.

[0023] In a preferred embodiment, an antibody of the invention comprises a heavy chain variable domain (VH) having three hypervariable regions.

[0024] 1) HCDR 1 (according to the Kabat numbering scheme) has the amino acid sequence of SEQ ID NO: 1,

[0025] 2) HCDR 2 has the amino acid sequence of SEQ ID NO: 2

[0026] 3) HCDR 3 has the amino acid sequence of SEQ ID No: 3;

[0027] 2) a light chain variable domain (VL) having three hypervariable regions LCDR1, LCDR2 and LCDR3, wherein:

[0028] LCDR 1 has the amino acid sequence of SEQ ID NO: 4,

[0029] LCDR 2 has the amino acid sequence of SEQ ID NO: 5,

[0030] LCDR 3 has the amino acid sequence of SEQ ID NO: 6.

[0031] Unless specifically stated otherwise, the well-known Kabat numbering scheme is used hereinafter to identify the CDRs of antibodies.

[0032] Thus, the antibody heavy and light chain variable domains contain amino acid substitutions in the FR segments of the heavy and light chain variable domains that increase the degree of humanization of the antibody compared to the parent antibody.

[0033] In a preferred embodiment, the variable domain of the heavy chain of the antibody of the present invention has the sequence shown in SEQ ID No: 16, and differs from the variable domain of the heavy chain of the parent antibody whose amino acid sequence is shown in SEQ ID No: 8 in that there are humanizing amino acid substitutions.

[0034] In certain embodiments, the variable domain of the heavy chain of an antibody of the invention contains other amino acid substitutions that do not alter the specificity of the antibody.

[0035] In a preferred embodiment, the variable domain of the light chain of the antibody of the present invention has the sequence shown in SEQ ID NO: 18, and differs from the variable domain of the light chain of the parent antibody whose amino acid sequence is shown in SEQ ID No: 10 in that there are humanizing amino acid substitutions.

[0036] In certain embodiments, the variable domain of the light chain of an antibody of the invention contains other amino acid substitutions that do not alter the specificity of the antibody.

[0037] In certain embodiments, the monoclonal antibodies of the invention are fully human IgG antibodies, e.g., IgG1 or IgG2 or IgG3 or IgG4.

[0038] In certain embodiments, an antibody of the invention comprises a heavy chain whose amino acid sequence is at least 85% identical, or at least 90% identical, or at least 91% identical, or at least 92%, or at least 93% identical, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 20.

[0039] In certain embodiments, an antibody of the invention comprises a light chain whose amino acid sequence is at least 85% identical, or at least 90% identical, or at least 91% identical, or at least 92%, or at least 93% identical, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 22.

[0040] In certain embodiments, the antibody has a light chain whose amino acid sequence is shown in SEQ ID NO: 22 and a heavy chain whose amino acid sequence is shown in SEQ ID NO: 20.

[0041] Also provided are nucleic acids encoding the variable domains of the heavy and light chains of the antibodies according to the invention, nucleic acids encoding the heavy and light chains of the antibodies according to the invention and functional fragments thereof.

[0042] Also provided are expression cassettes and expression vectors comprising nucleic acids of the present invention and regulatory elements necessary for expression of the nucleic acids in selected host cells. The vector or expression cassette may be present in the host cell as an extrachromosomal element, or may be integrated into the cell genome as a result of the introduction (by transfection) of the expression cassette or vector into the cell.

[0043] Furthermore, cells and stable cell lines comprising the nucleic acid, vector or expression cassette of the invention are provided, as well as methods for their preparation.

[0044] A method for producing the above antibody or antigen-binding fragment thereof is also provided, the method comprising culturing the above host cell in a culture medium under conditions that ensure the production of the antibody. In certain embodiments, the method comprises subsequently isolating and purifying the resulting antibody.

[0045] Also provided is a pharmaceutical composition for preventing or treating a disease or disorder mediated by the TRBV9 family β-chain region of human T receptors, comprising the above antibody or antigen-binding fragment thereof in combination with one or more pharmaceutically acceptable excipients.

[0046] In one embodiment, the pharmaceutical composition is intended to prevent or treat a disease or disorder selected from the group consisting of ankylosing spondylitis, celiac disease, T-cell leukemia, T-cell lymphoma.

[0047] Also provided is a pharmaceutical combination for preventing or treating a disease or disorder mediated by a human T cell receptor with a TRBV9 family β-chain, comprising the above antibody or antigen-binding fragment thereof and at least one other therapeutically active compound.

[0048] In one embodiment, the pharmaceutical combination is intended to prevent or treat a disease or disorder selected from the group consisting of ankylosing spondylitis, celiac disease, T-cell leukemia, T-cell lymphoma.

[0049] In one embodiment, the pharmaceutical combination or composition comprises an additional therapeutically active compound selected from a small molecule, an antibody or a steroid hormone, such as a corticosteroid.

[0050] Also provided is a method for inhibiting the biological activity of a T cell receptor whose β-chain belongs to the TRBV9 family in a subject in need of such inhibition, the method comprising administering an effective amount of the above antibody or antigen-binding fragment thereof to the subject.

[0051] Also provided is a method for treating a disease or disorder mediated by a human T cell receptor with a TRBV9 family β-chain, the method comprising administering the above antibody or antigen-binding fragment thereof or the pharmaceutical composition in a therapeutically effective amount to a subject in need of such treatment.

[0052] In one embodiment of the method for treating a disease or disorder, the disease or disorder is selected from the group consisting of ankylosing spondylitis, celiac disease, T-cell leukemia, T-cell lymphoma.

[0053] Also provided is the use of the above antibody or antigen-binding fragment thereof or the above pharmaceutical composition for treating a disease or disorder mediated by a human T cell receptor with a TRBV9 family β-chain in a subject in need of such treatment.

[0054] In one embodiment of the use, the disease is selected from the group consisting of ankylosing spondylitis, celiac disease, T-cell leukemia, and T-cell lymphoma.

[0055] The technical result of the present invention is to obtain antibodies with a high degree of humanization, which specifically bind with high affinity to TCRs whose β-chains belong to the TRBV9 family and can be used to treat autoimmune and oncological diseases whose pathogenesis involves TCRs whose β-chains belong to the TRBV9 family.

[0056] In a preferred embodiment, the antibody heavy chain variable fragment is characterized by a degree of humanization of 84%. In a preferred embodiment, the antibody light chain variable fragment is characterized by a degree of humanization of 85%. DETAILED DESCRIPTION

[0057] The present invention relates to isolated monoclonal antibodies and functional fragments thereof having the ability to specifically bind to the β-chain region of the TRBV9 family of human T receptors, which have an increased degree of humanization compared to analogs. Also provided are nucleic acids encoding the antibodies and fragments thereof of the present invention, including expression cassettes and expression vectors comprising the nucleic acids of the present invention and regulatory elements necessary for expression of the nucleic acids in selected host cells. In addition, cells and stable cell lines comprising the nucleic acids, vectors or expression cassettes of the present invention are provided. Also provided is a method for producing a monoclonal antibody or a functional fragment thereof, a pharmaceutical composition and a drug combination comprising an effective amount of the antibodies of the present invention in combination with one or more pharmaceutically acceptable excipients, diluents or carriers, and a method for diagnosing and treating AS and other diseases using the antibodies of the present invention.

[0058] definition

[0059] The present invention will be more easily understood by first defining some terms.

[0060] It should be understood that the materials and methods provided herein are not limited to specific compositions and method steps, as these may vary.It must be noted that as used herein and in the appended claims, the singular form includes the corresponding plural form unless the context clearly dictates otherwise.

[0061] The human "T cell receptor" (also referred to as "TCR", "T receptor") is a heterodimeric protein complex found on the surface of T lymphocytes. T receptors are only present on T lymphocytes. The main function of TCR is to specifically recognize processed antigens bound to molecules of the major histocompatibility complex (HLA).

[0062] Human TCR is made up of 2 subunit α and β chains or γ and δ chains connected by disulfide bonds and embedded in the cell membrane. Each TCR chain has an N-terminal variable (V) domain, a connecting domain and a constant (C) domain connected to a transmembrane domain, which anchors the receptor in the T lymphocyte plasma membrane. The length of the constant domain of α and β-chains is 91 and 129 amino acid residues respectively. The connection of α chain and the length of transmembrane domain are 30 and 17 amino acid residues (AAR), and the connection of β-chain and the length of transmembrane domain are 21 and 22 AAR. The length of T receptor variable domains varies between 104-125 AAR.

[0063] A small fraction of T lymphocytes have receptors of the gamma / delta type. They are arranged similarly to the alpha / beta receptors, but differ in their primary structure and have many functional characteristics. They exhibit much lower variability (limited clonal specificity) and they recognize antigens in complex with "non-classical" (non-MHC) antigen-presenting molecules or even free antigens.

[0064] The T receptor reacts with the MHC / antigen complex through six complementarity determining regions (CDRs): three α-chain regions and three β-chain regions. These CDRs are the hypervariable regions, loops of the variable domain of the T cell receptor, Vα and Vβ.

[0065] The term "TRBV9" or "TRBV9 family" refers to the ninth family of β-chains of T cell receptors distinguished according to the IMGT nomenclature, characterized in that the amino acid sequence of its variable domains comprises the unique motifs of CDR1 (amino acid sequence is SGDLS) and CDR2 (amino acid sequence is YYNGEE). The term "TRBV9 family TCR" refers to a T cell receptor whose β-chain belongs to the TRBV9 family.

[0066] The term "pathological" with respect to a T lymphocyte or a TCR means that such TCR or a T lymphocyte carrying the TCR is associated with a disease or pathology and / or causes the disease and / or contributes to the occurrence of a disease.

[0067] The term "autoimmune" with respect to a TCR means that such TCR is involved in the development of an autoimmune disease.

[0068] The term "antibody" as used herein is intended to mean an immunoglobulin molecule consisting of four polypeptide chains (two heavy (H) chains and two light (L) chains) connected by disulfide bonds. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon; they determine the antibody isotype, such as IgG, IgM, IgA, IgD, and IgE, respectively, and several of them can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Each heavy chain type is characterized by a specific constant region.

[0069] Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain, CL. The VH and VL regions can be further subdivided into regions of high variability, called complementarity determining regions (CDRs), which are surrounded by more conservative regions, called framework regions (FRs). Each VH and VL consists of 3 CDRs and 4 FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0070] In this application, the three heavy chain CDRs are referred to as "HCDR1, HCDR2 and HCDR3", while the three light chain CDRs are referred to as "LCDR1, LCDR2 and LCDR3". The CDRs contain most of the residues that specifically interact with the antigen. Unless otherwise indicated, the CDR-amino residues within the HCVR and LCVR of the antibodies according to the invention are numbered and positioned according to the well-known Kabat numbering scheme. This application includes the conventional letter codes for amino acids, unless otherwise indicated.

[0071] The terms "anti-TRBV9 antibody", "antibody against TRBV9", "antibody that specifically binds to the TRBV9 family β-chain" and "antibody against the TRBV9 family β-chain" are interchangeable in the context of the present application and relate to an antibody that specifically binds to an epitope of the TRBV9 family β-chain of the human T cell receptor.

[0072] In addition, the "monoclonal antibody" used in the present application may be a single-chain Fv-fragment, which can be obtained by binding a linker sequence to DNA encoding LCVR and HCVR (see Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, eds., Springer-Verlag, New York, pp. 269-315, 1994). It is contemplated that the term "antibody" used in the present application includes such fragments or portions as well as single-chain forms, regardless of whether a fragment or portion is mentioned. As long as the protein retains its ability to specifically or preferentially bind to its target (e.g., epitope or antigen), it is covered by the term "antibody". Antibodies may be glycosylated or non-glycosylated and still be within the scope of the present invention.

[0073] For the purpose of this application, the terms "antibody" and "monoclonal antibody" refer to monoclonal antibodies against TRBV9 family TCR. "Monoclonal antibody" as used herein refers to antibodies of rodents, primates or camelids, preferably mice, monkeys, camels or llamas, chimeric antibodies, humanized antibodies or fully human antibodies, unless otherwise indicated.

[0074] A population of "monoclonal antibodies" refers to a homogeneous or substantially homogeneous population of antibodies (i.e., at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, more preferably at least about 97% or 98%, or even more preferably at least 99% of the antibodies in the population will compete for the same antigen / epitope in an ELISA, or more preferably the antibodies are identical in their amino acid sequence). Antibodies may be glycosylated or non-glycosylated and still be within the scope of the invention. Monoclonal antibodies may be homogeneous if they have the same amino acid sequence, although they may differ in post-translational modifications (e.g., glycosylation pattern).

[0075] The variable regions of each pair of light / heavy chains form the antigen binding site of the antibody. As used in this application, "antigen binding portion" or "antigen binding region" or "antigen binding domain" or "antigen binding site" interchangeably refers to the portion of an antibody molecule that contains amino acid residues that interact with an antigen and provide the antibody with specificity and affinity for the antigen. This portion of the antibody includes the "framework" amino acid residues required to maintain the proper conformation of the antigen binding residues.

[0076] The term "human antibody" used herein means an antibody in which the sequence of the variable domains and the constant domains are derived from human sequences. Human antibodies according to the present invention may include atypical amino acid residues of the people (e.g., mutations introduced by in vitro non-directional or site-specific mutagenesis or in vivo somatic mutation), e.g., in CDR, and specifically, in CDR3.

[0077] The term "humanized" when used in reference to antibodies is used to denote antibodies that are characterized by the presence of human-like constant regions and structural components, but have typical complementarity determining regions (CDRs) of immunoglobulins of other origins, or of corresponding fragments of modified antibodies.

[0078] As used in this application, a "parent" antibody is an antibody encoded by the amino acid sequence used to obtain a variant. The parent antibody may be from a rodent, llama, chimeric, humanized or human antibody.

[0079] The term "humanization degree" in relation to antibodies is used to indicate the percent identity of the framework region sequences of a humanized antibody to the original human acceptor framework regions that are used to generate the humanized antibody and that are available from a human library. Preferably, the antibodies of the invention comprise framework regions that are at least 80% identical, usually at least 82%, more often at least 83%, for example at least 84% or at least 85% or at least 86% or at least 87% identical to the framework regions obtained from a human library.

[0080] The term "humanizing substitutions" refers to amino acid substitutions that increase the degree of humanization of an antibody or fragment thereof.

[0081] The term "chimeric" with respect to the antibodies of the present invention is used to denote antibodies that are characterized by human-like constant regions, but have variable regions of other origins. In such antibodies, the variable domains of the light and / or heavy chains of non-human origin (e.g., rat origin) are operably linked to the constant domains of the corresponding chains of human origin.

[0082] When used to describe antibodies, the term "operably linked" and the like refers to polypeptide sequences that are placed in physical (covalent, unless otherwise specified) and functional association with each other. In the most preferred embodiment, the function of the polypeptide component of the chimeric molecule is unchanged compared to the functional performance of the isolated polypeptide component. When used to describe nucleic acids, the term "operably linked" and the like refers to that the nucleic acids are covalently linked so that there is no frame shift and stop codon at their point of attachment. As will be apparent to those skilled in the art, the nucleotide sequence encoding the chimeric protein comprising "operably linked" components (proteins, polypeptides, linker sequences, protein domains, etc.) is composed of fragments encoding the components, wherein the fragments are covalently linked so that a full-length chimeric protein is produced during translation and transcription of the nucleotide sequence, e.g., a chimeric antibody according to the present invention.

[0083] As used herein, the term "isolated" refers to a molecule or cell that is in a specific environment that is different from the environment in which the molecule or cell is found in vivo.

[0084] In a preferred embodiment, the antibody of the present invention is a recombinant, i.e., obtained using recombinant DNA technology. The term "recombinant antibody" used herein includes all antibodies obtained, expressed, established or separated by recombinant means, such as antibodies expressed using a recombinant expression vector introduced into a host cell, antibodies separated from a group of known recombinant combinatorial human antibody libraries, antibodies separated from transgenic animals of human immunoglobulin genes (see, e.g., Taylor LD et al. (1992) Nucl. Acids Res. 20: 6287-6295). In certain embodiments, the recombinant human antibody is subjected to in vitro mutagenesis (or, when a transgenic animal of a human Ig sequence is used, in vivo somatic mutagenesis is performed), and thus the amino acid sequence of the VH and VL regions of the recombinant antibody is a sequence that, although it is derived from and is related to human germline VH and VL sequences, it is impossible to naturally exist in the human antibody germline pedigree in vivo.

[0085] The term "specifically binds" used in this application refers to a situation in which one member of a specific binding pair does not significantly bind to molecules other than its specific binding partner. The term is also applicable in cases where, for example, the antigen binding domain of an antibody of the present invention is specific for a particular epitope carried by many antigens; in this case, a specific antibody comprising an antigen binding domain will be able to specifically bind to a variety of antigens carrying the epitope. Thus, the monoclonal antibody of the present invention specifically binds to an epitope of the TRBV9 family β-chain of the human T cell receptor, however it does not specifically bind to the TCR β-chain and TCR α chain of other families.

[0086] The term "epitope" refers to that portion of a molecule that can be recognized and bound by an antibody at one or more of the antigen binding regions of an antibody. An epitope often consists of a chemically active surface cluster of molecules (such as amino acids or sugar side chains) and has specific three-dimensional structural characteristics as well as specific charge characteristics.

[0087] The term "epitope" as used herein refers in particular to a polypeptide fragment having antigenic activity and / or immunogenic activity in an animal, preferably in a mammal such as a mouse, rat or human. The term "antigenic epitope" as used in this application is a polypeptide fragment that can specifically bind to an antibody and can be detected by any technique well known from the prior art (e.g., by a standard immunoassay). Antigenic epitopes are not necessarily immunogenic; however, they can be immunogenic. An "immunogenic epitope" as used herein is defined as a polypeptide fragment that causes an antibody response in an animal, as determined by any method known from the prior art. A "non-linear epitope" or "conformational epitope" is a non-adjacent polypeptide (or amino acid) contained within an antigenic protein that binds an epitope-specific antibody.

[0088] The phrases "biological performance" or "biological characteristics" or the terms "activity" or "biological activity" with respect to the antibodies or functional fragments thereof of the present invention are used interchangeably in this application and include, but are not limited to, epitope / antigen affinity and specificity, the ability to neutralize or antagonize the activity of TCRs including the β-chain belonging to the TRBV9 family.

[0089] Other identifiable biological properties of antibodies include, for example, cross-reactivity (i.e., generally, with non-human homologs of the target peptide, or with other proteins or tissues), and the ability to maintain high protein expression levels in mammalian cells. The aforementioned properties or characteristics can be observed, measured and / or evaluated using techniques recognized in the art, including, but not limited to, ELISA, competitive ELISA, BIACORE or KINEXA surface plasmon resonance analysis, in vitro or in vivo inhibition assays (but not limited to), receptor binding assays, cytokine or growth factor production and / or secretion assays, and signal transduction and immunohistochemistry of tissue sections obtained from various sources (including humans, primates or any other source).

[0090] The terms "inhibit" or "neutralize" used in this application with respect to the activity of the antibodies of the present invention refer to the ability to substantially antagonize, block, prevent, limit, slow down, destroy, eliminate, stop, reduce, for example, the progression or severity of the object to be inhibited (including, but not limited to, the biological activity of the antibody, or properties, diseases or disorders as above).

[0091] The term "mutant" or "variant" as used herein refers to an antibody disclosed in the present invention, wherein one or more amino acids are added and / or replaced and / or deleted and / or inserted into the N-terminus and / or C-terminus of the antibody of the present invention or its fragment and / or in the native amino acid sequence. The term "mutant" as used herein also refers to a nucleic acid molecule encoding a mutant protein. In addition, the term "mutant" refers to any variant that is shorter or longer than a protein or nucleic acid.

[0092] The term "homology" is used to describe the relationship of a nucleotide or amino acid sequence to other nucleotide or amino acid sequences based on the degree of identity and / or similarity between the sequences being compared.

[0093] As used herein, an amino acid or nucleotide sequence is "substantially similar" or "substantially identical" to a reference sequence if the amino acid or nucleotide sequence has at least 85% identity to a specified sequence within the region selected for comparison. Thus, substantially similar sequences include those having, for example, at least 90% identity, or at least 91% identity, or at least 92% identity, or at least 93% identity, or at least 94% identity, or at least 95% identity, or at least 96% identity, or at least 97% identity, or at least 98% identity, or at least 99% identity. Two sequences that are identical to each other are also substantially similar.

[0094] Sequence identity is determined based on reference sequence. Algorithms for sequence analysis are known in the art, such as IgBLAST described in Ye et al. Nucleic Acids Res. 2013, W34-40. For purposes of the present invention, in order to determine the level of identity and similarity between nucleotide sequence and amino acid sequence, gapped comparisons of nucleotide and amino acid sequences can be used with the help of the IgBLAST software package provided by the National Center for Biotechnology Information (https: / / www.ncbi.nlm.nih.gov / igblast / ) with standard parameters. In order to calculate the identity percentage, the reference sequence such as the full length of the variable region is used.

[0095] Reference to a nucleotide sequence "encoding" a polypeptide refers to the generation of a polypeptide from the nucleotide sequence during translation and transcription of the mRNA. Thus, a coding strand identical to the mRNA and commonly used in sequence listings and a complementary strand serving as a transcription template may be indicated. As will be apparent to those skilled in the art, the term also includes any degenerate nucleotide sequence encoding the same amino acid sequence. Nucleotide sequences encoding polypeptides include sequences containing introns.

[0096] Antibody

[0097] As described above, the present invention relates to isolated monoclonal humanized antibodies and functional fragments thereof having the ability to specifically bind to the TRBV9 family β-chain region of human T receptors.

[0098] The antibody according to the present invention may be a chimeric, humanized or human antibody or an antigen-binding fragment thereof, and may be used as a drug for treating AS and other diseases whose pathogenesis involves TCRs belonging to the TRBV9 family (eg, celiac disease or T-cell lymphoma).

[0099] Antibodies according to the present invention are monoclonal. Monoclonal antibodies of the present invention can be produced using, for example, hybridoma technology well known in the art, and recombinant technology, phage display technology, synthetic technology or a combination of such technology or other technology well known in the art. The term "monoclonal antibody" used in this application means an antibody obtained from a single copy or clone (including, for example, any eukaryotic, prokaryotic or phage clone), rather than representing its production method.

[0100] Humanized and chimeric antibodies can be prepared by peptide synthesis or using recombinant DNA techniques as described in the "Nucleic Acids" section below.

[0101] In certain embodiments, the antibodies of the invention are chimeric and are characterized in that they have variable domains of light and heavy chains of non-human origin (eg, rat or murine origin), and constant domains of human origin.

[0102] The antibodies of the present invention comprise a heavy chain variable domain (VH) having three hypervariable regions

[0103] 1) HCDR1 (according to the Kabat numbering scheme) has the amino acid sequence of SEQ ID NO: 1,

[0104] 2) HCDR2 has the amino acid sequence of SEQ ID NO: 2,

[0105] 3) HCDR3 has the amino acid sequence of SEQ ID No: 3;

[0106] 2) a light chain variable domain (VL) having three hypervariable regions LCDR1, LCDR2 and LCDR3, wherein:

[0107] LCDR 1 has the amino acid sequence of SEQ ID NO: 4,

[0108] LCDR 2 has the amino acid sequence of SEQ ID NO: 5,

[0109] LCDR 3 has the amino acid sequence of SEQ ID NO: 6.

[0110] Unless specifically stated otherwise, the well-known Kabat numbering scheme is used hereinafter to identify the CDRs of antibodies.

[0111] In all embodiments, the variable domains of the light and heavy chains of the antibodies of the invention are humanized and differ from those of the parent antibody in terms of humanized amino acid substitutions, wherein the variable domains of the heavy and light chains of the antibodies comprise amino acid substitutions in the FR fragments of the variable domains of the heavy and light chains, thereby increasing the degree of humanization of the antibody compared to the parent antibody.

[0112] In a preferred embodiment, the variable domain of the heavy chain of the antibody of the present invention has the amino acid sequence shown in SEQ ID No: 16, and differs from the variable domain of the heavy chain of the parent antibody whose amino acid sequence is shown in SEQ ID No: 8 in that there is a humanizing amino acid substitution.

[0113] In certain embodiments, the variable domain of the heavy chain of an antibody of the invention contains other amino acid substitutions that do not alter the specificity of the antibody.

[0114] In a preferred embodiment, the variable domain of the light chain of the antibody of the present invention comprises amino acid substitutions and has the sequence shown in SEQ ID NO: 18, and differs from the variable domain of the light chain of the parent antibody whose amino acid sequence is shown in SEQ ID No: 10 in that there are humanizing amino acid substitutions.

[0115] In certain embodiments, the variable domain of the light chain of an antibody of the invention contains other amino acid substitutions that do not alter the specificity of the antibody.

[0116] In certain embodiments, the monoclonal antibodies of the invention are fully human IgG antibodies, e.g., IgG1 or IgG2 or IgG3 or IgG4.

[0117] In certain embodiments, an antibody of the invention comprises a heavy chain whose amino acid sequence is at least 85% identical, or at least 90% identical, or at least 91% identical, or at least 92%, or at least 93% identical, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 20.

[0118] In certain embodiments, an antibody of the invention comprises a light chain whose amino acid sequence is at least 85% identical, or at least 90% identical, or at least 91% identical, or at least 92%, or at least 93% identical, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 22.

[0119] In certain embodiments, the antibody has a light chain whose amino acid sequence is shown in SEQ ID NO: 22 and a heavy chain whose amino acid sequence is shown in SEQ ID NO: 20.

[0120] As known from the prior art, mutations can be introduced into antibody sequences, including variable domains, which will not substantially change the ability of the antibody to bind antigens. The antibody according to the present invention may also contain other mutations, which will not result in the loss of the ability of the antibody to bind the TRBV9 family β-chain of the TCR, but may result in a decrease in antibody-dependent cell-mediated cytotoxicity or an increase in the affinity or other biological properties of the antibody. Specifically, it is well known from the prior art that conservative amino acid substitutions can be made in the antibody sequence. "Conservative substitution" in the context of the present application means such a substitution: one of the amino acid residues is replaced by another amino acid residue with a similar side chain. Families of amino acid residues with similar side chains are well known in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Preferably, the CDR3 region in the VL and / or VH domain includes no more than 5 conservative amino acid substitutions, more often no more than 3 conservative substitutions. Typically, conservative substitutions are not made at amino acid positions that are critical for binding to the epitope of the TRBV9 family β-chain.

[0121] The above variants (mutants) of the antibodies according to the present invention can be obtained by peptide synthesis or using recombinant DNA techniques as described in the "Nucleic Acids" section below.

[0122] In a preferred embodiment, the antibody comprises a constant region of a heavy chain, such as a constant region of human IgG1, IgG2, IgGS, IgG4, IgA, IgE, IgM, IgD. Preferably, the heavy chain constant region is a human IgG1 heavy chain constant region. In addition, the antibody may comprise a light chain constant region or a light chain kappa constant region or a light chain lambda constant region. Preferably, the antibody comprises a light chain kappa constant region.

[0123] In a preferred embodiment, the antibody heavy chain variable fragment is characterized by a degree of humanization of 84%. In a preferred embodiment, the antibody light chain variable fragment is characterized by a degree of humanization of 85%.

[0124] Antigen-binding fragments of the antibodies of the present invention are also provided. The term "antigen-binding fragment of an antibody" (or "functional fragment of an antibody" or "active fragment of an antibody") as used herein refers to one or more antibody fragments that retain the ability to specifically bind to an antigen. It has been demonstrated that the antigen-binding function of an antibody can be performed by a fragment of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody include: (a) a Fab fragment, i.e., a monovalent fragment consisting of a VL, VH, CL, and CH1 domain; (b) a F(ab)2 fragment, i.e., a bivalent fragment comprising two Fab fragments, the Fab fragments being linked by a disulfide bond in the hinge region; (c) a Fd fragment consisting of a VH and CH1 domain; (d) a Fv fragment consisting of a single-arm VL and VH domain of an antibody; (e) a dAb fragment consisting of a VH domain (Ward et al. (1989) Nature 341:544-546), and (f) an isolated complementarity determining region (CDR). In addition, although the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be connected by synthetic joints using recombinant methods, which enable them to be prepared as a single protein chain, wherein the VL and VH regions are paired to form a monovalent molecule (called single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 242: 423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883). Such single-chain antibodies are also intended to be included in the term "antigen-binding fragment" of an antibody. They also include other forms of single-chain antibodies, such as diabodies. Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but with a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementary domains of another chain and create two antigen-binding sites (see, e.g., Holliger Р. et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak RJ et al. (1994) Structure 2:1121-1123).

[0125] Antibody fragments, such as Fab and F(ab')2, can be obtained from intact antibodies using conventional techniques, such as papain or pepsin digestion of intact antibodies, respectively. In addition, antibodies, antibody fragments and immunoadhesion molecules can be obtained using standard recombinant DNA techniques.

[0126] Antibodies or their antigen-binding portions can be parts of larger immunoadhesion molecules formed by covalent or non-covalent association of antibodies or antibody fragments with one or more proteins or peptides. Examples of such immunoadhesion molecules include the use of streptavidin core regions for preparing tetrameric scFv molecules (Kipriyanov SM et al. (1995) Human Antibodies and Hybridomas 6:93-101) and cysteine ​​residues, marker peptides and C-terminal polyhistidine tags for preparing divalent and downsized scFv biomolecules (Kipriyanov SM et al. (1994) Mol. Immunol., 31:1047-1058). Other chemical bonds between antibody fragments are also well known from the prior art.

[0127] The antibodies and functional fragments thereof according to the present invention are present in an isolated form, i.e. this means that such a protein is substantially free of the presence of other proteins or other naturally occurring biomolecules (such as oligosaccharides, nucleic acids and fragments thereof, etc.), wherein the term "substantially free" in this case means that less than 70%, usually less than 60% and more often less than 50% of the composition comprising the isolated protein is other naturally occurring biomolecules. In certain embodiments, the protein is present in a substantially purified form, wherein the term "substantially purified form" refers to a purity equal to at least 95%, usually equal to at least 97% and more often equal to at least 99%.

[0128] Methods for purifying antibodies obtained by recombinant or hybridoma technology are well known in the art, for example, purification can be performed by chromatography (e.g., ion exchange chromatography, affinity chromatography, particularly affinity for specific antigenic protein A or protein G, and size column chromatography), centrifugation, solubility differences, or any other standard technique for purifying proteins. In addition, antibodies or fragments thereof obtained by the techniques according to the present invention can be fused to heterologous polypeptide sequences (e.g., histidine tags) to facilitate purification.

[0129] Antibody affinity can be determined using standard assays by determining the dissociation constant (KD). KD is calculated using the equation KD = kdis / kon, where kdis is the experimentally calculated dissociation rate constant of the antibody-antigen complex and kon is its experimentally calculated association rate constant.

[0130] Preferred antibodies are those that bind to human antigens with a KD value of no more than about 1×10 -7 M; preferably not more than about 1×10 -8 M; more often not more than about 1 × 10 -9 M; more preferably not more than about 1×10 -10M, and most preferably no more than about 1×10 -11 M, for example, does not exceed about 1×10 -12 M.

[0131] Antibodies and fragments thereof that can be used in the compositions and methods of the present invention are biologically active antibodies and fragments, ie, they are capable of binding to a desired antigenic epitope and exerting a biological effect, either directly or indirectly.

[0132] The antibodies and functional fragments thereof according to the present invention are capable of specifically binding to the epitope (region) of the TRBV9 family β-chain. In a preferred embodiment, as a result of its specific binding to the β-chain of the TRBV9 family, the activity of the TCR comprising the β-chain is inhibited. Typically, the inhibition amount is preferably at least about 20%, or 30%, or 40%, or 50%, or 60%, or 70%, or 80%, or 90%, or 95% or higher.

[0133] In certain embodiments, antibodies or fragments thereof directed against TRBV9 family β-chains according to the present invention can eliminate T cells with TCRs comprising TRBV9 family β-chains. In certain embodiments, antibodies or fragments thereof according to the present invention can provide at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or about 100% elimination of T lymphocytes.

[0134] In a preferred embodiment of the present invention, the antibody is antibody MA-043.

[0135] Antibody MA-043 includes variable domains of heavy and light chains having the amino acid sequences shown in SEQ ID NOs: 16 and 18.

[0136] Antibody MA-043 includes a heavy chain and a light chain having the amino acid sequences shown in SEQ ID NOs: 20 and 22, respectively.

[0137] Nucleic Acids

[0138] The present invention provides nucleic acid molecules encoding the heavy and light chains of the antibodies of the present invention, their functional fragments and variable domains, which can be used to obtain chimeric antibodies, including the variable domains of the present invention operably fused to known constant domains of human antibodies.

[0139] In a preferred embodiment, the nucleic acid of the invention encodes an antibody heavy chain whose variable domain contains three hypervariable regions HCDR1, HCDR2 and HCDR3, wherein

[0140] HCDR1 (according to the Kabat numbering scheme) has the amino acid sequence of SEQ ID No: 1;

[0141] HCDR2 has the amino acid sequence of SEQ ID No: 2;

[0142] HCDR3 has the amino acid sequence of SEQ ID No: 3.

[0143] In a preferred embodiment, the nucleic acid of the invention encodes an antibody light chain whose variable domain comprises three hypervariable regions LCDR1, LCDR2 and LCDR3, wherein:

[0144] LCDR1 has the amino acid sequence of SEQ ID No: 4;

[0145] LCDR2 has the amino acid sequence of SEQ ID No: 5;

[0146] LCDR3 has the amino acid sequence of SEQ ID No: 6.

[0147] In a preferred embodiment, the nucleic acids of the invention encode antibody heavy and light chain variable domains containing amino acid substitutions in the FR segments of the variable domains of the heavy and light chains which increase the degree of humanization of the antibody compared to the parent antibody.

[0148] Nucleic acid molecules encoding homologs and mutants of said antibody chains, functional fragments and domains thereof are also within the scope of the invention.

[0149] In certain embodiments, the nucleic acid encodes an antibody heavy chain whose variable domain has the amino acid sequence of SEQ ID No: 16.

[0150] In certain embodiments, the nucleic acid encodes an antibody light chain whose variable domain has the amino acid sequence of SEQ ID No: 18.

[0151] In certain embodiments, the nucleic acid encodes an antibody heavy chain whose amino acid sequence is at least 85% identical, or at least 90% identical, or at least 91% identical, or at least 92%, or at least 93% identical, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 20.

[0152] In certain embodiments, the nucleic acid encodes an antibody light chain whose amino acid sequence is at least 85% identical, or at least 90% identical, or at least 91% identical, or at least 92%, or at least 93% identical, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 22.

[0153] Examples of nucleic acids encoding the light and heavy chains of the present invention are shown in SEQ ID Nos: 19 and 21.

[0154] Nucleic acids encoding the variable domains of the light and heavy chains of antibodies are also of interest. Nucleic acids encoding the variable domains of the light and heavy chains of antibodies can be used to operably fuse with nucleic acids encoding the corresponding constant domains of antibodies.

[0155] In certain embodiments, the nucleic acid encodes the variable domain of the heavy chain of the antibody, the amino acid sequence of which is shown in SEQ ID No: 16.

[0156] In certain embodiments, the nucleic acid encodes the variable domain of the light chain of the antibody, the amino acid sequence of which is shown in SEQ ID No: 18.

[0157] Examples of nucleic acids encoding the variable domains of the heavy and light chains of antibodies are shown in SEQ ID NOs: 15 and 17.

[0158] As used herein, "nucleic acid molecule" or "nucleic acid" is a DNA molecule, such as a genomic DNA molecule or a cDNA molecule, or an RNA molecule, such as an mRNA molecule. In certain embodiments, a nucleic acid molecule of the invention is a DNA (or cDNA) molecule containing an open reading frame encoding an antibody or antibody fragment of the invention and capable of being used for expression in a heterologous expression system under suitable conditions (e.g., physiological intracellular conditions).

[0159] In certain embodiments, nucleic acid molecules of the present invention are produced by genetic engineering methods. The method of producing nucleic acid is well known in the art. For example, the availability of amino acid sequence information or nucleotide sequence information enables the preparation of isolated nucleic acid molecules of the present invention by oligonucleotide synthesis. In the case of amino acid sequence information, many nucleic acids different from each other due to degenerate codes can be synthesized. The method of selecting codon variants for the desired host is well known in the art.

[0160] The oligonucleotide synthesized can be prepared by the aminophosphite method, and can be according to methods well known in the art, such as high performance liquid chromatography (HPLC) or other methods described in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, (1989) Cold Spring Harbor Press, Cold Spring Harbor, NY, and under the guidance of, for example, United States Dept. of HHS, National Institute of Health (NIH) Guidelines for Recombinant DNA Research, the construct obtained can be purified. Long double-stranded DNA molecules of the present invention can be synthesized in the following manner: synthesize several smaller fragments with appropriate complementarity, which include appropriate ends that can bond to adjacent fragments. Adjacent fragments can be connected using DNA ligase or based on PCR methods.

[0161] The nucleic acid molecules of the invention may also be cloned from biological sources.

[0162] The present invention also includes nucleic acids that are homologous, substantially identical, identical, or derived from nucleic acids encoding polypeptides of the present invention.

[0163] Nucleic acids of the invention are present in an environment other than that in which they naturally occur, for example, they are isolated, present in increased amounts, present or expressed in an in vitro system or in a cell or organism other than those in which they naturally occur.

[0164] The variation or difference of the nucleotide sequence between the closely related nucleic acid sequences may represent the nucleotide variation in the sequence produced in the normal replication or repetitive process. Other variations can be specially designed for a specific purpose and introduced into the sequence, such as in order to change the codon of the specific amino acid or nucleotide sequence in the regulatory region. Such special variation can be made in vitro using multiple mutagenesis techniques, or is placed in a host organism under the specific selection condition of inducing or selecting these variations to produce. Such special sequence variants obtained can be referred to as "mutants" or "derivatives" of the original sequence.

[0165] By modifying, deleting or adding one or more nucleotides in the template sequence, or a combination thereof, to obtain a variant of the template nucleic acid, a mutant or derivative nucleic acid can be obtained on a template nucleic acid selected from the above nucleic acids. Modifications, additions or deletions can be performed by any method known in the art (see, e.g., Gustin et al., Biotechniques (1993) 14: 22; Barany, Gene (1985) 37: 111-123; and Colicelli et al., Mol. Gen. Genet. (1985) 199: 537-539, Sambrook et al., Molecular Cloning: A Laboratory Manual, (1989), CSH Press, pp. 15.3-15.108), including error-prone PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sex PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-specific mutagenesis, random mutagenesis, gene reassembly, gene site saturation mutagenesis (GSSM), synthetic ligation reassembly (SLR), or a combination thereof. Modifications, additions or deletions can also be performed by methods comprising recombination, recursive sequence recombination, phosphothioate-modified DNA mutagenesis, uracil-containing template mutagenesis, gapped duplex mutagenesis, point mismatch repair mutagenesis, repair-deficient host strain mutagenesis, chemical mutagenesis, radiogenic mutagenesis, deletion mutagenesis, restriction-selection mutagenesis, restriction-purification mutagenesis, artificial gene synthesis, global mutagenesis, chimeric nucleic acid multimer generation and combinations thereof.

[0166] Also provided are degenerate variants of nucleic acids encoding proteins of the present invention. Degenerate variants of nucleic acids include codons that replace nucleic acids with other codons encoding the same amino acids. Specifically, degenerate variants of nucleic acids are prepared to increase expression in host cells. In this embodiment, codons that are over-expressed in the encoding sequence in the host cell gene are replaced by codons that are non-preferred or sub-preferred nucleic acids in the host cell gene, wherein the replaced codons encode the same amino acid.

[0167] The above modifications will not substantially change the performance of the antibody or its functional fragment, but can promote the protein folding in the host cell, reduce the aggregation capacity, or regulate other biochemical properties of the protein, for example, half-life. In certain embodiments, these modifications will not change the biochemical properties of the protein. In certain embodiments, these modifications result in reduced antibody immunogenicity. All types of modifications and mutations noted above are performed at the nucleic acid level.

[0168] The disclosed nucleic acids can be isolated and prepared in a substantially purified form. By substantially purified form it is meant that the nucleic acid is at least about 50% pure, typically at least about 90% pure, and typically is "recombinant," i.e., is flanked by one or more nucleotides that are not normally associated with the nucleotides on the chromosome in which it naturally occurs in its natural host organism.

[0169] Also provide the nucleic acid of the fusion rotein that coding comprises protein of the present invention or its fragment, they are discussed in more detail below.The nucleic acid of encoding variable domain of the present invention can be operably connected to the nucleic acid of the corresponding constant domain of the light chain of encoding antibody and heavy chain.The nucleic acid of the light chain of encoding antibody and heavy chain can be operably connected to the nucleic acid of encoding leading (signal) peptide, and described leading peptide promotes the transportation of expression product from host cell.Remove leading peptide subsequently in the maturation process of polypeptide.

[0170] Carrier

[0171] Also provided are vectors and other nucleic acid constructs comprising the disclosed nucleic acids. The term "vector" means a nucleic acid molecule capable of transporting another nucleic acid to which it has been operably connected. Certain vectors can replicate autonomously in the host cells into which they are introduced, while other vectors can be integrated into the host cell genome and replicated together with the host genome. In addition, some vectors can direct the expression of genes to which they have been operably connected. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"), and exemplary vectors are well known from the prior art. Suitable vectors include viral and non-viral vectors, plasmids, clays, phages, etc., preferably plasmids, and are used for cloning, amplification, expression, transfer to appropriate hosts, etc. of the nucleic acid sequences of the present invention. The selection of suitable vectors is obvious to those skilled in the art. Usually, by means of DNA ligase and the connection of the restriction enzyme site cut in the vector, the full-length nucleic acid or its portion is inserted into the vector. Alternatively, by homologous recombination in vivo, usually by connecting the homology region to the flank of the desired nucleotide sequence on the vector, the desired nucleotide sequence can be inserted. By the connection of oligonucleotide, or by the polymerase chain reaction using primers, add homology regions, the primers include for example the parts of homology regions and the desired nucleotide sequence.Usually, the carrier has a replication origin that guarantees that it is replicated in the host cell due to being introduced into the cell as an extrachromosomal element.The carrier can also include regulatory elements, which guarantee the expression of nucleic acid in the host cell and the generation of target polypeptide.In the expression vector, the nucleic acid is operably connected to a regulatory sequence, which can include a promoter, an enhancer, a terminator, an operator, a repressor and an initiator and a polypeptide.In certain embodiments, nucleic acid of the present invention is further operably connected to a leader peptide, which guarantees that the expression product is separated into the extracellular space from the host cell.

[0172] Also provided is an expression cassette or system, which is particularly used to obtain a disclosed polypeptide based on them (e.g., the light chain and heavy chain of an antibody of the present invention, or the variable domains of the light chain and heavy chain of an antibody of the present invention), or for duplicating a disclosed nucleic acid molecule. As a result of the introduction of the expression cassette into a cell, the expression cassette can exist as an extrachromosomal element, or can be integrated into the cell genome. Regarding expression, the protein product encoded by the nucleic acid of the present invention is expressed in any convenient expression system, including, for example, bacterial systems, yeast, insects, amphibians or mammalian cells. In the expression cassette, the target nucleic acid is operably connected to a regulatory sequence, which may include a promoter, an enhancer, a terminator sequence, an operator, a repressor and an inducer, and the start codon of a polypeptide. In certain embodiments, the nucleic acid of the present invention is further operably connected to a leader peptide, which ensures the separation of the expression product from the host cell to the extracellular space. The method of obtaining an expression cassette or system capable of expressing a desired product is known to those skilled in the art.

[0173] Host cells

[0174] The above expression system can be used in prokaryotic or eukaryotic hosts. Host-cells, such as Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, insect cells in combination with baculovirus vectors, or cells of higher organisms (which are not human embryonic cells) such as yeast, plants, vertebrates, for example, CHO cells (e.g. ATCC CRL-9096), NSO cells, SP2 / 0 cells, HEK293 cells, COS cells (e.g. ATCC CRL-1650, CRL-1651) and HeLa (e.g. ATCC CCL-2), can be used to obtain the protein.

[0175] In order to produce the antibody of the present invention, host cells are co-transformed with an expression vector comprising a nucleic acid encoding an antibody light chain and an expression vector comprising a nucleic acid encoding an antibody heavy chain. In certain embodiments, a single expression vector is used, wherein nucleic acids encoding light and heavy chains of an antibody are introduced.

[0176] In order to express the light chain and the heavy chain, the expression vector encoding the heavy chain and the light chain is transformed (co-transformed) into the host cell so that the light chain and the heavy chain are expressed in the host cell and preferably secreted into the culture medium in which the host cell is cultured, and the antibody can be separated from the culture medium. The various interpretations of the term "conversion" are intended to include a wide range of methods commonly used to introduce foreign DNA into prokaryotic or eukaryotic host cells, for example, electroporation, calcium phosphate precipitation, DEAE-dextran transfection, etc., as described in Sambrook, Fritsch and Maniatis (eds.) Molecular Cloning; A Laboratory Manual, 2nd edition, Cold Spring Harbor, NY (1989; Ausubel FM et al. (eds.) Current Protocols in Molecular Biology, Green Publishing Associates (1989).

[0177] When a recombinant expression vector containing a nucleic acid of an antibody is introduced into a host cell, the antibody is obtained as follows: the host cell is cultured for a period of time sufficient to express the antibody in the host cell or (more preferably) to secrete the antibody into a culture medium in which the host cell is cultured. The antibody can be separated from the culture medium using standard protein purification techniques. Cell culture conditions are well known to those skilled in the art and are described in Current Protocols in Cell Biology, Bonifacino J.S., Dasso M., Harford J.V., Lippincott-Schwartz J. and Yamada KM (eds.), published by John Wiley & Sons, Inc. in 2000.

[0178] If use is suitable for the above host cell of nucleic acid of the present invention or any one of other host cell or biology, the nucleic acid of replication, the albumen or polypeptide of expression obtained so are within the scope of the present invention as the product of host cell or biology.Can separate described product by suitable technology known in the art.

[0179] Cell lines stably expressing the proteins of the invention can be selected by methods known in the art (e.g., co-transfection with selection markers such as dhfr, gpt, neomycin, hygromycin, which allows identification and isolation of transfected cells containing the gene integrated into the genome).

[0180] Nucleic acid molecules of the present invention can also be used to determine the gene expression in biological samples. The method of checking cells for the presence of specific nucleotide sequences (such as genomic DNA or RNA) is well known in the art. In short, DNA or mRNA is separated from cell samples. mRNA can be amplified by RT-PCR, wherein a complementary DNA chain is formed using reverse transcriptase, and then a primer specific to the subject DNA sequence is used to carry out polymerase chain reaction amplification. Alternatively, the mRNA sample is separated by gel electrophoresis, transferred to a suitable carrier, such as nitrocellulose, nylon, etc., and then the fragment of the subject DNA is used as a probe for detection. Other techniques can also be used, such as oligonucleotide connection assays, in situ hybridization and hybridization with a DNA probe immobilized on a solid chip. The detection of the mRNA hybridized with the subject sequence indicates the gene expression in the sample.

[0181] Therapeutic uses of the antibodies of the invention

[0182] In one aspect, the antibodies or active fragments thereof of the present invention are used to treat disorders associated with the activity of pathological T lymphocytes with surface TRBV9 family TCRs, for example, autoimmune T lymphocyte activity in AS, celiac disease, and T cell lymphoma.

[0183] The term "patient" as used in this application refers to a mammal, including, but not limited to, mice, monkeys, humans, livestock mammals, sports mammals, and pet mammals; preferably the term is applicable to humans. In a specific embodiment, the patient is further characterized by a disease or disorder or condition mediated by the presence of a TCR in his body, the β-chain of which belongs to the TRBV9 family. As known from the prior art, TCRs whose β-chains belong to the TRBV9 family are associated with AS and celiac disease. In addition, TCRs whose β-chains belong to the TRBV9 family may be associated with the occurrence of many blood diseases (such as T-cell lymphomas caused by Epstein-Barr virus).

[0184] As used herein, the terms "co-administered," "co-administered," and "in combination with" referring to an antibody and one or more other therapeutic agents are intended to mean, represent, and include the following:

[0185] 1) when such components are formulated together in a single dosage form, said dosage form releases said components to said patient substantially simultaneously, administering such combination of an antibody of the invention and a therapeutic agent simultaneously to a patient in need of treatment,

[0186] 2) when such components are formulated separately from one another in separate dosage forms, said dosage forms are taken by said patient substantially simultaneously, after which said components are released to said patient substantially simultaneously, and such combination of an antibody of the invention and a therapeutic agent is administered simultaneously to a patient in need of treatment,

[0187] 3) when such components are formulated separately from one another in separate dosage forms, said dosage forms are taken sequentially by said patient with a significant time interval between each administration, after which said components are released to said patient at substantially different times, and such combination of an antibody of the invention and a therapeutic agent is administered sequentially to a patient in need of treatment; and

[0188] 4) When such components are formulated together in a single dosage form, the dosage form releases the components in a controlled manner, whereafter they are released to the patient in parallel, sequentially and / or overlappingly at the same and / or different times, wherein each part can be administered by the same or different routes, and such combination of the antibody of the present invention and the therapeutic agent is administered sequentially to a patient in need of treatment.

[0189] The antibodies of the invention may be administered without further therapeutic treatment, i.e. as an independent therapy. In addition, the treatment of the antibodies of the invention may include at least one additional therapeutic treatment (combination therapy). In certain embodiments of the invention, the antibodies may be co-administered or formulated with another agent / drug for an autoimmune or oncological disease, the pathogenesis of which involves a TCR comprising a TRBV9 β-chain, e.g., AC, celiac disease, T-cell lymphoma, T-cell leukemia.

[0190] Dosage and route of administration

[0191] The antibodies of the invention will be administered in an amount effective to treat the target disorder, i.e., at the dosage and duration required to achieve the desired result. The therapeutically effective amount may vary with factors such as the specific disorder to be treated, the age, sex and weight of the patient, and whether the antibody is administered alone or in combination with one or more additional immunosuppressive or anti-inflammatory therapeutic techniques.

[0192] The dosage regimen may be adjusted to provide the most suitable answer. For example, a single bolus delivery may be administered, several divided doses may be administered over time, or the dosage may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is particularly advantageous to formulate the parenteral composition in unit dosage form for ease of administration and uniformity of dosage. Standard dosage forms as used herein are intended to represent physically discrete units suitable as unit doses for the patient / subject to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect in combination with a desired pharmaceutical carrier. The specifications of the standard dosage forms of the present invention are generally determined and directly dependent on: (a) the unique characteristics of the chemotherapeutic agent and the specific therapeutic or preventive effect to be achieved, and (b) inherent limitations in the art of formulation, such as the active compound used to treat the sensitivity of the subject.

[0193] Thus, it will be understood by a skilled artisan based on the disclosure provided herein that dosage and dosage regimens are adjusted according to methods well known in the therapeutic field. That is, the maximum tolerated dose can be easily established, and the effective amount that provides a detectable therapeutic benefit to the patient can also be determined, and the time requirement for applying each medicament to provide a detectable therapeutic benefit to the patient can also be determined. Thus, although some dosages and dosage regimens are given as examples in this document, these examples in no way limit the dosage and regimen that may be necessary for the patient in the practice of the present invention.

[0194] It should be noted that the dosage value may vary with the type and severity of the disease to be alleviated, and may include single or multiple doses. In addition, it should be understood that for any particular subject, the specific dosage regimen should be adjusted over time according to individual needs and the judgment of the medical professional who applies the composition or supervises the application of the composition, and the dosage ranges set forth herein are merely exemplary and are not intended to limit the scope or practice of the claimed composition. In addition, the dosage regimen using the composition of the present invention can be based on a variety of factors, including the type of disease, age, weight, sex, health status of the patient, severity of the disease, route of administration, and specific antibodies used. Thus, the dosage regimen can vary widely, but can be routinely determined using standard methods. For example, the dosage can be adjusted based on pharmacokinetic or pharmacodynamic parameters, which may include clinical effects such as toxic effects and / or laboratory values. Thus, the present invention includes intra-patient dose escalation determined by those skilled in the art. Methods for determining appropriate dosages and regimens are well known in the art, and are understood by skilled technicians after learning the ideas disclosed herein.

[0195] Examples of suitable methods of administration are provided above.

[0196] It is foreseen that suitable doses of the antibodies of the invention are in the range of 0.1-200 mg / kg, preferably 0.1-100 mg / kg, including about 0.5-50 mg / kg, for example about 1-20 mg / kg. The antibody can be administered, for example, at a dose of at least 0.25 mg / kg, such as at least 0.5 mg / kg, including at least 1 mg / kg, for example at least 1.5 mg / kg, such as at least 2 mg / kg, for example at least 3 mg / kg, including at least 4 mg / kg, for example at least 5 mg / kg; and for example a maximum of up to 50 mg / kg, including a maximum of up to 30 mg / kg, for example a maximum of up to 20 mg / kg, including a maximum of up to 15 mg / kg. It is usually repeated at appropriate time intervals, such as once a week, once every 2 weeks, once every 3 weeks, or once every 4 weeks, as long as the responsible physician deems it appropriate, and in some cases, if necessary, the responsible physician may increase or decrease the dose.

[0197] Pharmaceutical composition

[0198] The antibody of the present invention can be incorporated into a pharmaceutical composition suitable for administration to a patient. The antibody of the present invention can be administered individually or in combination with a pharmaceutically acceptable carrier, diluent and / or excipient in a single dose or multiple doses. The pharmaceutical composition for administration is designed to be suitable for the selected mode of administration, and a pharmaceutically acceptable diluent, carrier and / or excipient, such as a dispersant, a buffer, a surfactant, a preservative, a solubilizing agent, an isotonic agent, a stabilizer, etc., is used when appropriate. The composition is designed according to conventional techniques, such as, for example, in Remington, The Science and Practice of Pharmacy, the 19th edition, Gennaro, ed., Mack Publishing Co., Easton, PA 1995, as generally known to practitioners, which provides various techniques for obtaining compositions.

[0199] "Medicament (drug)" - is a compound or a mixture of compounds, which is a pharmaceutical composition in the form of tablets, capsules, powders, lyophilized powders, injections, infusions, ointments and other ready-made forms, intended for restoring, improving or modifying the physiological functions of humans and animals, and also for treating and preventing diseases, for diagnosis, anesthesia, contraception, cosmetology, etc. Any method accepted in the art for administering peptides, proteins or antibodies can be appropriately used for the antibodies of the present invention.

[0200] The term "pharmaceutically acceptable" means one or more compatible liquid or solid components that are suitable for administration in a mammal, preferably a human.

[0201] The term "excipient" is used herein to describe any ingredient other than the above ingredients of the present invention. These are substances of inorganic or organic nature, which are used in the preparation of medicaments in order to provide the necessary physicochemical properties to the drug product.

[0202] The terms "buffer", "buffer composition", "buffer" refer to a solution that is able to resist pH changes caused by the action of its acid-base conjugate components and that allows the antibody drug to tolerate pH changes. Typically, the pharmaceutical composition preferably has a pH in the range of 4.0-8.0. Examples of buffers used include, but are not limited to, acetate, phosphate, citrate, histidine, succinate and other buffer solutions.

[0203] As used herein, the terms "tonicity agent", "osmotic agent" or "osmotic agent" refer to excipients that can increase the osmotic pressure of a liquid antibody formulation. An "isotonic" drug is a drug that has an osmotic pressure equal to that of human blood. Isotonic drugs typically have an osmotic pressure of about 250-350 mOsm / kg. Isotonic agents that can be used include, but are not limited to, polyols, sugars and sucrose, amino acids, metal salts, e.g., sodium chloride, etc.

[0204] "Stabilizer" means an excipient or a mixture of two or more excipients that provides physical and / or chemical stability to the active agent. Stabilizers that can be used include amino acids, such as, but not limited to, arginine, histidine, glycine, lysine, glutamine, proline; surfactants, such as, but not limited to, polysorbate 20 (commercial name: Tween 20), polysorbate 80 (commercial name: Tween 80), polyethylene-polypropylene glycol and copolymers thereof (commercial names: poloxamer, Pluronic, sodium dodecyl sulfate (SDS); antioxidants, such as, but not limited to, methionine, acetylcysteine, ascorbic acid, monothioglycerol, sulfites, etc.; chelating agents, such as, but not limited to, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), sodium citrate, etc.

[0205] A pharmaceutical composition is "stable" if the active agent maintains its physical stability and / or chemical stability and / or biological activity over a specified shelf life at a storage temperature of, for example, 2-8° C. Preferably, the active agent maintains physical and chemical stability, as well as biological activity. The storage period is selected based on the results of stability testing under accelerated or natural aging conditions.

[0206] Compositions containing the monoclonal antibodies of the invention can be administered to a patient exhibiting a condition as described herein using standard administration techniques, including oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration.

[0207] The pharmaceutical compositions of the present invention preferably contain or are a "therapeutically effective amount" of an antibody of the present invention. The term "therapeutically effective amount" is intended to mean an amount that is effective at the dosage and duration required to achieve the desired therapeutic result. The therapeutically effective amount of an antibody may vary with factors such as the disease state, age, sex, and weight of the subject, and the ability of the antibody or portion thereof to elicit a desired response in the subject. A therapeutically effective amount is also an amount in which the therapeutically beneficial effects of the antibody outweigh any toxic or deleterious effects. A "prophylactically effective amount" is intended to mean an amount that is effective at the dosage and duration required to achieve the desired prophylactic result. Because prophylactic doses are prescribed for individuals prior to disease or in the early stages of disease, the prophylactically effective amount may generally be less than the therapeutically effective amount.

[0208] A therapeutically effective amount or a prophylactically effective amount is at least the minimum therapeutically beneficial dose, which is less than a toxic dose of the active agent. On the other hand, a therapeutically effective amount of an antibody of the invention is an amount that reduces the biological activity of an autoimmune clone in a mammal, preferably a human, for example by binding to a TCR whose β-chain belongs to the TRBV9 family, wherein the presence of the clone causes or promotes an undesirable pathological effect, or by reducing the TCR whose β-chain belongs to the TRBV9 family, resulting in a beneficial therapeutic effect in a mammal, preferably a human.

[0209] The route of administration of the antibodies of the present invention can be oral, parenteral, inhaled or topical. Preferably, the antibodies of the present invention can be included in a pharmaceutical composition acceptable for parenteral administration. The term "parenteral" as used in this application includes intravenous, intramuscular, subcutaneous, rectal, vaginal or intraperitoneal administration. Intravenous, intraperitoneal or subcutaneous injection is a preferred route of administration. Acceptable pharmaceutical carriers for such injections are well known from the prior art.

[0210] As described in the appropriate guidelines, the pharmaceutical composition should be sterile and stable under the conditions of production and storage in the container, which is provided by, for example, a hermetically sealed vial (ampoule) or syringe. Thus, the pharmaceutical composition can be filter-sterilized after the composition is prepared, or can be made microbiologically suitable by any other technique. A typical composition for intravenous infusion may include 250-1000 ml of fluid such as sterile Ringer's solution, saline, dextrose solution or Hank's salt solution, and a therapeutically effective dose (e.g., 1-100 mg / ml or more) of antibody concentrate. The dose may vary with the type and severity of the disease. It is well known from the prior art in the medical field that the dose for any patient depends on a variety of factors, including the patient's size, body surface area, age, specific compound to be administered, sex, duration and route of administration, general health status, and other drugs administered simultaneously. A typical dose may be, for example, in the range of 0.001-1000 μg; however, doses lower and higher than this exemplary range are foreseen, particularly in view of the above-mentioned parameters. The daily parenteral dosing regimen can be 0.1 μg / kg to 100 μg / kg total body weight, preferably 0.3 μg / kg to 10 μg / kg, and more preferably 1 μg / kg to 1 μg / kg, even more preferably 0.5-10 μg / kg body weight per day. The course of treatment can be monitored by regularly assessing the patient's health status. Regarding repeated administration for several days or longer, depending on the patient's condition, the treatment is repeated until the desired response or suppression of disease symptoms. However, other dosing regimens not described herein can also be applied. Depending on the pharmacokinetic disintegration desired by the practitioner, the desired dose can be administered by single bolus or multiple bolus administration or by means of continuous infusion of the antibody.

[0211] These estimated amounts of antibodies are largely dependent on the physician's judgment. The desired effect is a key factor in selecting an appropriate dosage and regimen. Factors considered herein include the disease to be treated, the mammal to be treated, the clinical condition of a patient, the cause of the disease, the site of antibody administration, the specific antibody type, the route of administration, the administration regimen, and other factors well known in the medical field.

[0212] The therapeutic agent of the present invention can be frozen or lyophilized and reconstituted in an appropriate sterile carrier before administration. Freeze drying and reconstitution can result in some loss of activity of the antibody. The dosage can be adjusted to compensate for this loss. In general, a pharmaceutical composition pH value between 6-8 is preferred.

[0213] Finished products and test kits

[0214] Another embodiment of the present invention is a manufactured product containing a product for treating autoimmune diseases and related disorders and hematological malignancies whose pathogenesis involves TCRs with TRBV9 family β-chains. Such diseases include, for example, AS, celiac disease, T-cell leukemia, T-cell lymphoma, etc.

[0215] The finished product is a container with a label and a package insert, which may be in a blister and / or package. Suitable containers include, for example, vials, ampoules, syringes, etc. The container may be made of various materials, such as glass or polymeric materials. The container contains a composition that is effective for treating a certain condition and may have a sterile access port. At least one active ingredient in the composition is an antibody according to the present invention. The label and package insert indicate that the drug is intended for treating a certain condition. The label and / or package insert additionally contain instructions for administering the antibody composition in a patient, including the indications, frequency, dosage, route of administration, contraindications, and / or warnings for such a therapeutic product. In one embodiment, the package insert indicates that the composition is intended for treatment.

[0216] Additionally, the article of manufacture may include, but is not limited to, other products necessary for commercial purposes or desirable to the consumer, such as solvents, diluents, filters, needles, and syringes.

[0217] The present invention also relates to a kit, which can be used for a variety of purposes, for example, for evaluating the ability to kill T cells with TRBV9 family TCRs, for purifying or immunoprecipitating TRBV9 receptors from cells. For separation and purification, the kit can contain antibodies coupled to beads (e.g., agarose beads). The kit comprises a container, a label, and a package insert.

[0218] Diagnostic Uses

[0219] The antibodies of the present invention are also used for diagnostic purposes (e.g., in vitro, ex vivo). For example, the antibody can be used to detect or measure the level of T lymphocytes containing TRBV9 family TCR in a sample obtained from a patient (e.g., a tissue sample or a body fluid sample, such as inflammatory exudate, blood, intestinal fluid, saliva or urine). Suitable detection and measurement methods include immunoassays, such as flow cytometry, enzyme-linked immunosorbent assay (ELISA), chemiluminescence assay, radioimmunoassay and immunohistology. The present invention also includes a kit, for example, a diagnostic kit comprising the antibody described herein.

[0220] The following examples are provided for a better understanding of the present invention. These examples are for illustrative purposes only and should not be construed as limiting the scope of the present invention in any way.

[0221] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference. Although the above invention has been described in more detail by way of examples and examples in order to avoid ambiguous interpretations, it will be very clear to those skilled in the art based on the ideas disclosed in the present invention that certain changes and modifications can be made without departing from the essence and scope of the embodiments included in the present invention.

[0222] Experimental Section

[0223] Example 1. In silico humanization of antibody variable domain sequences

[0224] As parent (reference) sequences, the variable domain sequences of the heavy and light chains of the anti-TRBV9-2 antibody were used, whose amino acid sequences are shown in SEQ ID NOs: 8 and 10.

[0225] The amino acid sequences of the variable domains of the heavy and light chains were compared with a collection of germline sequences of variable domains of human immunoglobulins, germline sequences of variable domains of rat immunoglobulins, and sequences of mature human and rat antibodies obtained from donor libraries provided by open sources and Biocad (Russia). The Ylab software package was used for analysis (Biocad, Russia).

[0226] The positions and their combinations determined by the analysis are the most animal-like and non-human-like in the variable domain sequences of the test antibodies. At the same time, the amino acid combinations most frequently represented in human antibodies at these positions are determined. Based on the data obtained, artificial sequences of the variable domains are designed, which contain substitutions that increase the degree of humanization of the antibodies.

[0227] Furthermore, the nucleotide sequences encoding the subject amino acid variants were codon optimized for expression of humanized antibodies in CHO cell lines. Humanized nucleotide sequences of HCVR and LCVR (SEQ ID NOs: 15 and 17) were synthesized de novo and cloned into pEE-HC, pEE-CK vectors, and IgG1 format at the SalI / NheI and SalI / BsiWI restriction sites, respectively (Xu et al. Front. Chem. Sci. Eng. 2015, 9(3): 376-380).

[0228] The resulting nucleic acid sequences of the light and heavy chains were verified by Senger sequencing. Antibody MA-043 was selected for further study, and the amino acid and nucleotide sequences of its light and heavy chains are shown in SEQ ID NOs: 19-22.

[0229] Antibody MA-043 includes variable domains of heavy and light chains having the amino acid sequences shown in SEQ ID NOs: 16 and 18.

[0230] The degree of humanization of the variable domain of the heavy chain of antibody MA-043 is 84%, while the degree of humanization of the variable domain of the light chain is 85% (Table 1). The heavy chain constant domain is represented by the IgG1 format, Gm3 allotype.

[0231] Table 1. Comparison of the degree of humanization between the variable domains of the parental antibodies and antibody MA-043.

[0232] Antibody Degree of humanization of HCVR, % Degree of humanization of LCVR, % TRBV9-2 72 69 MA-043 84 85

[0233] Example 2. Preparation of recombinant antibodies and determination of their affinity

[0234] The vector obtained as described in Example 1 and comprising the nucleic acid encoding the light and heavy chains of the antibody MA-043 was amplified in E. coli cells and purified using a plasmid DNA purification kit from Qiagen (Germany) and used for transfection of the CHO-Fut8 cell line using linear polyethyleneimine (PEI "MAX", "Polysciences", USA) according to the manufacturer's instructions. The resulting reaction mixture was incubated at 37°C on a shaker. Nine days after transfection, the culture fluid was separated from the cells by filtration through a 0.5 / 0.22 µm filter. After filtration, the culture fluid was used for isolation of the antibodies using affinity chromatography on a 0.2 ml PreDictor RoboColumn MabSelect SuRe column (GE Healthcare, USA) equilibrated with phosphate-buffered saline (PBS, pH 7.4). The column was then washed with 5 volumes of PBS. The vector-bound proteins were eluted using 0.1 M glycine buffer рН3. We collected the main peak containing the protein and adjusted the pH to neutral with 1 M Tris buffer (pH 7.5). All stages were performed at a flow rate of 110 cm / h. The protein was then transferred to PBS (pH 7.4) using dialysis, filtered through a 0.22 μm filter, and transferred to a new sterile tube. The separation quality was evaluated using 12% PAGE under denaturing conditions.

[0235] The separation quality was evaluated using 12% PAGE under denaturing conditions. Quantitative evaluation was performed by measuring the NanoDrop 2000 microspectrophotometer at 280 A. The separated proteins were stored at -70 °C.

[0236] Antibody affinity was determined on the OctetRed 96 system (ForteBio, USA). Antigens at a concentration of 20 μg / ml were immobilized on the surface of an AR2G sensor (ForteBio) according to standard protocols and manufacturer's instructions. PBS containing 0.1% Tween 20 and 0.1% BSA was used as working buffer and analyzed at 30°C. After baseline recording, the sensor was immersed in a well containing the antibody solution for 300 seconds, where the complex was associated. The complex dissociation in the buffer solution was then detected for 600 seconds.

[0237] Binding curves after subtraction of the reference signal were analyzed using Octet Data Analysis (version 9.0) software according to standard procedures and using a 1:1 global interaction model. The data obtained (Table 2) showed that the antibody binds to human antigens specifically and with high affinity.

[0238] Table 2. Affinity characteristics of antibody MA-043.

[0239] Antibody KD (M) kon(1 / Ms) kdis(1 / s) Full R^2 MA-043 <1.0E-12 3.29E+05 <1.0E-07 0.955

[0240] Example 3. Preparation of a cell line that stably produces antibodies in the IgG1 form and production of antibodies

[0241] The sequences of the heavy and light chains of antibody MA-043 were prepared as described in Example 1 and cloned into the pSX vector at the HindIII, XbaI restriction sites. The resulting plasmid was cultured in E. coli cells and 600-700 μg was isolated using BenchPro (LifeTechnologies, США) according to the manufacturer's instructions. The plasmid was linearized overnight using PvuI endonuclease, then ethanol precipitated and the precipitate was dissolved in water at a concentration of 900-1100 ng / μl in the final volume.

[0242] CHO-K1-S cell line was cultured in Ham's F12 Gibco medium (Thermo, USA) and transfected with a gene construct containing the coding sequence of the 3 chain of MA-043 using electroporation on a Nucleofector™ (Lonza, Switzerland) according to the manufacturer's protocol.

[0243] One day after transfection, the transfected cells were subjected to antibiotic selection for 24 days by adding puromycin (7.2 μg / ml final concentration) and hygromycin B (640 μg / ml final concentration) to the culture medium. Antibiotic-resistant cell clones that were homogeneous in expressing high levels of MA-043 constructs were then selected.

[0244] In order to cultivate the CHO-K1-S expressing MA-043, the serum-free medium Ham's F12 Gibco (Thermo, USA) supplemented with 25-100 uM 2-deoxy-2-fluoro-L-fucose (CarboSynth, UK) was used. The monoclonal antibody MA-043 for preclinical research was produced in a 50 L HyClone disposable bioreactor fermentor (Thermo Fisher Scientific). Millistak COHC depth filter (Merck-Millipore, USA) was used to remove the production cells from the culture fluid. The initial purification of the antibody from the clean culture medium was carried out on the protein A affinity adsorbent. The target protein was specifically eluted with the glycine buffer of pH3.3-3.8 under acidic conditions. For the purpose of virus inactivation, the eluate collected was exposed to acidic pH 30-60 min, and then neutralized to pH 6.5-7.0 with 1M Tris-HCl solution. Final chromatographic purification was performed in flow-through mode using CaptoAdhere adsorbent (GE Healthcare LifeSciences) to remove possible impurities (DNA, producer cell proteins, released ligands of the affinity adsorbent, aggregates and antibody fragments). Thus, the protein solution was subjected to low conductivity (< 3 msec / cm 2 ) was flowed through the prepared adsorbent equilibrated with Tris buffer at pH 6.5-7.0. The purified protein was then filtered to remove viruses using the Viresolve PRO filtration kit (Millipore, USA), concentrated, and diafiltered against a final buffer containing acetate buffer (pH 5.0-5.5) and trehalose.

[0245] Example 4. Use of antibodies for specific binding to TRBV9 family β-chain fragments of T cell receptors

[0246] The monoclonal antibody MA-043 obtained as described in Example 3 was used to sort lymphocyte subsets. The antibody was labeled with fluorescein using fluorescein isothiocyanate reagent (Sigma, USA) according to the manufacturer's protocol. The number of fluorophores reacting with the antibody molecule was controlled by the absorption spectrum ratio at a wavelength of 495 / 280 nm.

[0247] Obtain T lymphocytes from the peripheral blood from healthy donors.Blood is collected in EDTA Vacuette test tubes (each 2x9 ml), according to the standard procedure described (Kovalchuk LV et al. Immunology: Workshop -2010. - 176 p.) separates the mononuclear fraction.After separation, cells are transferred to phosphate buffered saline (PBS) containing 0.5% bovine serum albumin (BSA) and 2mM EDTA.By the trypan blue staining method described by Lang NR (Stimulation of lymphocytes M.: Medicine , 1976.-288 p.), determine the total number of cells and their viability.An isopyknic 0.1% trypan blue solution is added to the cell suspension, and then the (dead) and unstained blue cells of dyeing are counted in the Goryaev chamber.Based on these data, determine the percentage of dead cells in the test sample.

[0248] To confirm the selectivity of the binding of MA-043 to a target population of T-lymphocytes with a membrane TCR belonging to the TRBV9 family, 500,000 cell aliquots of the mononuclear fraction were added to a PBS buffer containing 0.5% bovine serum albumin (BSA), 2 mM EDTA pH 8, antibody MA-043 labeled with FITC, CD3-eFluor405 (T lymphocyte marker) (eBioscience, USA) and 100 ng / ml concentration of CD45-PC5 (eBioscience, USA) (total leukocyte marker). The 50 μl reaction mixture was incubated at room temperature for 30 min, after which the cells were washed with a PBS buffer supplemented with 0.5% BSA, 2 mM EDTA. After the staining procedure, the cells were used for sorting using flow cytometry (FACSARIA III, USA). The use of these markers in staining makes it possible to separate target population cells carrying surface TRBV9+, CD3+, CD45+, as well as to obtain negative population cells, i.e. those corresponding to the immunophenotype "TRBV9-, CD3+, CD45+". TRBV9+ and TRBV9- population cells from 2 replicates were used for the isolation of total RNA and sequencing of TCRβ-chains. The resulting cell fractions were placed in RLT buffer (Quagen, Germany) and RNA was isolated from them using Quiagen RNAeasy mini kit #217004 reagent kit (Quagen) according to the manufacturer's protocol. cDNA was synthesized on the isolated RNA template, and a fragment of the T receptor β-chain was amplified using the Mint cDNA synthesis kit (Eurogen, Russia) according to the protocol described by Britanova et al. (JImmunol, 2016, 196(12) 5005-5013). Illumina adapters (USA) were connected to the produced amplicons and sequencing was performed on the MiSeq Illumina platform according to the sequencer manufacturer's protocol. Sequencing data were analyzed using MiGEC, MiXCR and VDJtools software available on the Internet at https: / / milaboratory.com. Analysis of the resulting TCR β-chain repertoire showed that the library obtained by sorting with antibody MA-043 was enriched with 91% of the sequences encoded by the TRBV9 gene segment, while no sequences containing TRBV9 were detected in the repertoire of the "TRBV9-" negative fraction β-chain.

[0249] Example 5. Preparation of a pharmaceutical composition comprising the antibody of the present invention

[0250] Pharmaceutical compositions are obtained by standard techniques known in the art.

[0251] The components of the pharmaceutical composition are shown in Table 3.

[0252] Table 3. Concentrations of components of pharmaceutical compositions.

[0253] Components concentration Antibody MA-043 10-50 mg / ml 10 mM citrate buffer to pH 6.0-7.0 Sodium chloride 50-150 mM Sucrose, trehalose 0.3-0.5% Water for injection Make up to 1 ml.

[0254] Example 6. Kits containing pharmaceutical compositions containing antibodies

[0255] To produce a kit containing a dosage form comprising the antibody MA-043 composition, the pharmaceutical composition prepared according to Example 5 is sealed in a 1 ml ampoule or syringe under sterile conditions, labeled, and packaged into a plastic or cardboard container.

[0256] Also, an insert is included in the ampoule container. <110> biocade united ag <120> Monoclonal antibody against the β-chain region of human TRBV9 <130> MA043.docx <160> twenty two <170> PatentIn Version 3.5 <210> 1 <211> 5 <212> PRT <213> Artificial <220> <223> HCDR1 sequence of parent antibody TRBV9-2 <400> 1 Asp Tyr Leu Val His 1 5 <210> 2 <211> 17 <212> PRT <213> Artificial <220> <223> HCDR2 sequence of parent antibody TRBV9-2 <400> 2 Trp Ile Asn Thr Tyr Thr Gly Thr Pro Thr Tyr Ala Asp Asp Phe Glu 1 5 10 15 Gly <210> 3 <211> 13 <212> PRT <213> Artificial <220> <223> HCDR3 sequence of parent antibody TRBV9-2 <400> 3 Ser Trp Arg Arg Gly Leu Arg Gly Ile Gly Phe Asp Tyr 1 5 10 <210> 4 <211> 11 <212> PRT <213> Artificial <220> <223> LCDR1 sequence of parental antibody TRBV9-2 <400> 4 Lys Ala Ser Lys Ser Ile Asn Lys Tyr Leu Ala 1 5 10 <210> 5 <211> 7 <212> PRT <213> Artificial <220> <223> LCDR2 sequence of parental antibody TRBV9-2 <400> 5 Asp Gly Ser Thr Leu Gln Ser 1 5 <210> 6 <211> 9 <212> PRT <213> Artificial <220> <223> LCDR3 sequence of parent antibody TRBV9-2 <400> 6 Gln Gln His Asn Glu Tyr Pro Pro Thr 1 5 <210> 7 <211> 366 <212> DNA <213> Artificial <220> <223> Nucleotide sequence of the variable domain of the heavy chain of parent antibody TRBV9-2 <400> 7 caaatacaac tggtgcagag cgggccagaa ttgagagaac ccggagaatc tgtgaagctg 60 agttgtaagg ccagcggata cactttcact gactatctcg tgcactgggt gaaacaggct 120 cccggtaagg gattgaaatg gatgggatgg atcaatactt ataccggcac acctacatat 180 gcagacgatt tcgaggggcg atttgtgttc agtttggagg cctctgccag cacggcgaac 240 ctgcagatat cgaatctcaa gaatgaggac accgccacgt atttctgcgc tagatcttgg 300 agacgcggat tgagaggtat cggattcgac tactggggac aaggcgtctt cgtgactgta 360 tcatcc 366 <210> 8 <211> 122 <212> PRT <213> Artificial <220> <223> Amino acid sequence of the variable domain of the heavy chain of parent antibody TRBV9-2 <400> 8 Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Arg Glu Pro Gly Glu 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Leu Val His Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met 35 40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Thr Pro Thr Tyr Ala Asp Asp Phe 50 55 60 Glu Gly Arg Phe Val Phe Ser Leu Glu Ala Ser Ala Ser Thr Ala Asn 65 70 75 80 Leu Gln Ile Ser Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95 Ala Arg Ser Trp Arg Arg Gly Leu Arg Gly Ile Gly Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Val Phe Val Thr Val Ser Ser 115 120 <210> 9 <211> 321 <212> DNA <213> Artificial <220> <223> Nucleotide sequence of the light chain variable domain of parent antibody TRBV9-2 <400> 9 gatgtacaga tgacacaatc accctacaac cttgctgctt cccctgggga aagtgtcagt 60 atcaattgca aggcatcgaa gtcgatcaac aagtatcttg cgtggtatca gcagaagcca 120 ggaaagccca acaagctcct gatctatgac ggctctacac tgcaatctgg cataccttcg 180 cggttttctg gctcggggtc cgggactgac ttcactctta caatacgagg acttgaaccc 240 gaagacttcg gcctgtatta ctgccagcag cacaatgagt atccacctac cttcggggct 300 ggcaccaagt tggagcttaa g 321 <210> 10 <211> 107 <212> PRT <213> Artificial <220> <223> Amino acid sequence of the light chain variable domain of parent antibody TRBV9-2 <400> 10 Asp Val Gln Met Thr Gln Ser Pro Tyr Asn Leu Ala Ala Ser Pro Gly 1 5 10 15 Glu Ser Val Ser Ile Asn Cys Lys Ala Ser Lys Ser Ile Asn Lys Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Pro Asn Lys Leu Leu Ile 35 40 45 Tyr Asp Gly Ser Thr Leu Gln Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Arg Gly Leu Glu Pro 65 70 75 80 Glu Asp Phe Gly Leu Tyr Tyr Cys Gln Gln His Asn Glu Tyr Pro Pro 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 <210> 11 <211> 639 <212> DNA <213> artificial <220> <223> Nucleotide sequence of the light chain of parental antibody TRBV9-2 <400> 11 gacgtgcaga tgacccagtc cccctacaac ctggccgcct cccccggcga gtccgtgtcc 60 atcaactgca aggcctccaa gtccatcaac aagtacctgg cctggtacca gcagaagccc 120 ggcaagccca acaagctgct gatctacgac ggctccaccc tgcagtccgg catcccctcc 180 aggttctccg gctccggctc cggcaccgac ttcaccctga ccatcagggg cctggagccc 240 gaggacttcg gcctgtacta ctgccagcag cacaacgagt acccccccac cttcggcgcc 300 ggcaccaagc tggagctgaa gcgaactgtg gctgcaccat ctgtcttcat cttcccgcca 360 tctgatgagc agttgaaatc tggaactgcc tctgtcgtgt gcctgctgaa taacttctat 420 cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 480 gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg 540 ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 600 ctgtcctcgc ccgtcacaaa gagcttcaac aggggagag 639 <210> 12 <211> 213 <212> PRT <213> Artificial <220> <223> Amino acid sequence of the light chain of parent antibody TRBV9-2 <400> 12 Asp Val Gln Met Thr Gln Ser Pro Tyr Asn Leu Ala Ala Ser Pro Gly 1 5 10 15 Glu Ser Val Ser Ile Asn Cys Lys Ala Ser Lys Ser Ile Asn Lys Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Pro Asn Lys Leu Leu Ile 35 40 45 Tyr Asp Gly Ser Thr Leu Gln Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Arg Gly Leu Glu Pro 65 70 75 80 Glu Asp Phe Gly Leu Tyr Tyr Cys Gln Gln His Asn Glu Tyr Pro Pro 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu 210 <210> 13 <211> 1365 <212> DNA <213> Artificial <220> <223> Nucleotide sequence of the heavy chain of parent antibody TRBV9-2 <400> 13 cagatccagc tggtgcagtc cggccccgag ctgagggagc ccggcgagtc cgtgaagctg 60 tcctgcaagg cctccggcta caccttcacc gactacctgg tgcactgggt gaagcaggcc 120 cccggcaagg gcctgaagtg gatgggctgg atcaacacct acaccggcac ccccacctac 180 gccgacgact tcgagggcag gttcgtgttc tccctggagg cctccgcctc caccgccaac 240 ctgcagatct ccaacctgaa gaacgaggac accgccacct acttctgcgc caggtcctgg 300 aggaggggcc tgaggggcat cggcttcgac tactggggcc agggcgtgtt cgtgaccgtg 360 tcctccgcct ccaccaaggg cccatcggtc ttccccctgg caccctcctc caagagcacc 420 tctgggggca cagcggccct gggctgcctg gtcaaggact acttccccga accggtgacg 480 gtgtcgtgga actcaggcgc cctgaccagc ggcgtgcaca ccttcccggc tgtcctacag 540 tcctcaggac tctactccct cagcagcgtg gtgactgtgc cctctagcag cttgggcacc 600 cagacctaca tctgcaacgt gaatcacaag cccagcaaca ccaaggtgga caagaaagtt 660 gagcccccga aatcttgtga caaaactcac acatgcccac cgtgcccagc acctgaactc 720 ctggggggac cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc 780 cggacccctg aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag 840 ttcaactggt acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag 900 cagtacaaca gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg 960 aatggcaagg agtacaagtg caaggtctcc aacaaagccc tcccagcccc catcgagaaa 1020 accatctcca aagccaaagg gcagccccga gaaccacagg tgtacaccct gcccccatcc 1080 cgggatgagc tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatccc 1140 agcgacatcg ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg 1200 cctcccgtgc tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag 1260 agcaggtggc agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac 1320 cactacacgc agaagagcct ctccctgtct ccgggtaaat gataa 1365 <210> 14 <211> 453 <212> PRT <213> Artificial <220> <223> Amino acid sequence of the heavy chain of parental antibody TRBV9-2 <400> 14 Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Arg Glu Pro Gly Glu 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Leu Val His Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met 35 40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Thr Pro Thr Tyr Ala Asp Asp Phe 50 55 60 Glu Gly Arg Phe Val Phe Ser Leu Glu Ala Ser Ala Ser Thr Ala Asn 65 70 75 80 Leu Gln Ile Ser Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95 Ala Arg Ser Trp Arg Arg Gly Leu Arg Gly Ile Gly Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Val Phe Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Pro Lys 210 215 220 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 225 230 235 240 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260 265 270 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 275 280 285 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 290 295 300 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 305 310 315 320 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 325 330 335 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 340 345 350 Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 355 360 365 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375 380 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 385 390 395 400 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 405 410 415 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 420 425 430 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 435 440 445 Leu Ser Pro Gly Lys 450 <210> 15 <211> 366 <212> DNA <213> Artificial <220> <223> Nucleotide sequence of the variable heavy chain of antibody MA-043 <400> 15 caggtgcaac ttgttcagtc ggggcctgaa cttaagaagc caggggaaag tgtaaaagtg 60 agctgcaaag cctcaggcta cacgtttacc gattatcttg tgcattgggt tagacaggct 120 ccaggtaagg gactggaatg gatgggatgg atcaatacct atacagggac acccacatat 180 gccgatgact ttgagggacg gtttgtcttc tcacttgata ccagtgcgtc cactgctaac 240 ctccagatat gcagcctgaa gaatgaggac accgccacct attactgcgc ccgatcatgg 300 aggagaggcc tacgaggaat cggattcgat tactggggtc agggcacttt agtcactgtc 360 tctagc 366 <210> 16 <211> 122 <212> PRT <213> Artificial <220> <223> Amino acid sequence of the variable domain of the heavy chain of parent antibody MA-043 <400> 16 Gln Val Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Leu Val His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Thr Pro Thr Tyr Ala Asp Asp Phe 50 55 60 Glu Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Ala Ser Thr Ala Asn 65 70 75 80 Leu Gln Ile Cys Ser Leu Lys Asn Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Arg Ser Trp Arg Arg Gly Leu Arg Gly Ile Gly Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 17 <211> 321 <212> DNA <213> Artificial <220> <223> Nucleotide sequence of the variable light chain of antibody MA-043 <400> 17 gacatacaga tgactcaaag cccttattcg ctcagtgcgt cggtcgggga cagagtaacc 60 atcacctgca aggcgtcaaa gtcaatcaat aagtatctgg cgtggttcca gcagaagcca 120 ggaaagccta acaagctatt aatatacgat gggtctaccc tccaatccgg ggtcccttca 180 cgattttctg gaagcggctc aggaaccgat ttcacgctga ccatcagtag cttggagcct 240 gaggactttg ccacttatta ttgccagcag cacaacgagt atcctcccac cttcggacag 300 ggtacaaaac tggagatcaa g 321 <210> 18 <211> 107 <212> PRT <213> Artificial <220> <223> Amino acid sequence of the variable light chain of antibody MA-043 <400> 18 Asp Ile Gln Met Thr Gln Ser Pro Tyr Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Lys Ser Ile Asn Lys Tyr 20 25 30 Leu Ala Trp Phe Gln Gln Lys Pro Gly Lys Pro Asn Lys Leu Leu Ile 35 40 45 Tyr Asp Gly Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Asn Glu Tyr Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 19 <211> 1356 <212> DNA <213> Artificial <220> <223> Nucleotide sequence of the heavy chain of antibody MA-043 <400> 19 caggtgcaac ttgttcagtc ggggcctgaa cttaagaagc caggggaaag tgtaaaagtg 60 agctgcaaag cctcaggcta cacgtttacc gattatcttg tgcattgggt tagacaggct 120 ccaggtaagg gactggaatg gatgggatgg atcaatacct atacagggac acccacatat 180 gccgatgact ttgagggacg gtttgtcttc tcacttgata ccagtgcgtc cactgctaac 240 ctccagatat gcagcctgaa gaatgaggac accgccacct attactgcgc ccgatcatgg 300 aggagaggcc tacgaggaat cggattcgat tactggggtc agggcacttt agtcactgtc 360 tctagcgcta gcaccaaggg cccatcggtc ttccccctgg caccctcctc caagagcacc 420 tctgggggca cagcggccct gggctgcctg gtcaaggact acttccccga accggtgacg 480 gtgtcgtgga actcaggcgc cctgaccagc ggcgtgcaca ccttcccggc tgtcctacag 540 tcctcaggac tctactccct cagcagcgtg gtgaccgtgc cctccagcag cttgggcacc 600 cagacctaca tctgcaacgt gaatcacaag cccagcaaca ccaaggtgga caagagagtt 660 gagcccaaat cttgtgacaa aactcacaca tgcccaccgt gcccagcacc tgaactcctg 720 gggggaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg 780 acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc 840 aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag 900 tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 960 ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc cagcccccat cgagaaaacc 1020 atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg 1080 gaggagatga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc 1140 gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 1200 cccgtgctgg actccgacgg ctccttcttc ctctatagca agctcaccgt ggacaagagc 1260 aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac 1320 tacacgcaga aaagcctctc cctgtccccg ggtaaa 1356 <210> 20 <211> 452 <212> PRT <213> Artificial <220> <223> Amino acid sequence of the heavy chain of antibody MA-043 <400> 20 Gln Val Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Leu Val His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Thr Pro Thr Tyr Ala Asp Asp Phe 50 55 60 Glu Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Ala Ser Thr Ala Asn 65 70 75 80 Leu Gln Ile Cys Ser Leu Lys Asn Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Arg Ser Trp Arg Arg Gly Leu Arg Gly Ile Gly Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445 Ser Pro Gly Lys 450 <210> twenty one <211> 642 <212> DNA <213> Artificial <220> <223> Nucleotide sequence of the light chain of antibody MA-043 <400> 21 gacatacaga tgactcaaag cccttattcg ctcagtgcgt cggtcgggga cagagtaacc 60 atcacctgca aggcgtcaaa gtcaatcaat aagtatctgg cgtggttcca gcagaagcca 120 ggaaagccta acaagctatt aatatacgat gggtctaccc tccaatccgg ggtcccttca 180 cgattttctg gaagcggctc aggaaccgat ttcacgctga ccatcagtag cttggagcct 240 gaggactttg ccacttatta ttgccagcag cacaacgagt atcctcccac cttcggacag 300 ggtacaaaac tggagatcaa gcgtacggtg gctgcaccat ctgtcttcat cttcccgcca 360 tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 420 cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 480 gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg 540 ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 600 ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gt 642 <210> 22 <211> 214 <212> PRT <213> Artificial <220> <223> Amino acid sequence of the light chain of antibody MA-043 <400> 22 Asp Ile Gln Met Thr Gln Ser Pro Tyr Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Lys Ser Ile Asn Lys Tyr 20 25 30 Leu Ala Trp Phe Gln Gln Lys Pro Gly Lys Pro Asn Lys Leu Leu Ile 35 40 45 Tyr Asp Gly Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Asn Glu Tyr Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210

Claims

1. A monoclonal antibody or an antigen-binding fragment thereof that specifically binds to the TRBV-9 family β-chain region of the human T cell receptor, comprising a heavy chain variable domain whose amino acid sequence is shown in SEQ ID No: 16 and a light chain variable domain whose amino acid sequence is shown in SEQ ID No:

18. 2 . The monoclonal antibody according to claim 1 , comprising a heavy chain having the amino acid sequence of SEQ ID No: 20 and a light chain having the amino acid sequence of SEQ ID No:

22. 3 .

3. The monoclonal antibody according to any one of claims 1-2, which is a full-length IgG antibody.

4. A nucleic acid encoding the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the antibody or antigen-binding fragment thereof specifically binds to the TRBV9 family β-chain region of human T receptor.

5. An expression vector containing the nucleic acid according to claim 4.

6. A method for obtaining a host cell for producing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, the method comprising transforming the cell with the vector according to claim 5.

7. A host cell for obtaining the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the host cell comprises the nucleic acid according to claim 4.

8. A method for obtaining the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, the method comprising culturing the host cell according to claim 7 in a culture medium under conditions that ensure the production of the antibody, and subsequently isolating and purifying the obtained antibody.

9. A pharmaceutical composition for preventing or treating a disease or disorder mediated by the β-chain region of the TRBV9 family of human T receptors, the pharmaceutical composition comprising a therapeutically effective amount of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 in combination with one or more pharmaceutically acceptable excipients, wherein the disease or disorder is selected from: ankylosing spondylitis, celiac disease, T-cell leukemia, T-cell lymphoma.

10. A pharmaceutical composition for preventing or treating a disease or disorder mediated by a human T-cell receptor with a TRBV9 family β-chain, the pharmaceutical composition comprising a therapeutically effective amount of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 and a therapeutically effective amount of at least another therapeutically active compound, wherein the disease or disorder is selected from: ankylosing spondylitis, celiac disease, T-cell leukemia, T-cell lymphoma.

11. The pharmaceutical composition according to claim 10, wherein the other therapeutically active compound is selected from an antibody or a steroid hormone.

12. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1-3 or a pharmaceutical composition according to any one of claims 9-11 in the preparation of a medicament for treating a disease or disorder mediated by a human T-cell receptor with a TRBV9 family β-chain, wherein the disease is selected from: ankylosing spondylitis, celiac disease, T-cell leukemia, T-cell lymphoma.