Monoclonal antibodies directed against the beta chain region of human TRBV9

文档序号：246112 发布日期：2021-11-12 浏览：32次中文

阅读说明：本技术 针对人TRBV9的β链区域的单克隆抗体 (Monoclonal antibodies directed against the beta chain region of human TRBV9 ) 是由 O·V·布里塔诺娃 D·B·斯塔罗韦罗夫 A·V·艾福斯特拉特娃 A·K·米索林 T·A·尼于 2019-12-24 设计创作，主要内容包括：本发明涉及特异性地结合人T细胞受体的TRBV9家族的单克隆人源化抗体或其抗原结合片段。本发明也涉及编码所述抗体或其抗原结合片段的核酸、表达载体、用于制备所述抗体的方法和所述抗体在治疗与人T细胞受体的所述家族有关的疾病或障碍中的用途。本发明涉及产生抗体,所述抗体可以用于治疗尤其是强直性脊柱炎(AS或别赫捷列夫氏病)、乳糜泻和血液癌症,其发病机制涉及TRBV9家族的T细胞受体。(The present invention relates to monoclonal humanized antibodies of the TRBV9 family or antigen-binding fragments thereof that specifically bind to human T cell receptors. The invention also relates to nucleic acids encoding the antibodies or antigen binding fragments thereof, expression vectors, methods for making the antibodies, and the use of the antibodies in treating diseases or disorders associated with the family of human T cell receptors. The present invention relates to the production of antibodies which can be used for the treatment of, inter alia, ankylosing spondylitis (AS or alloheirlichiosis), celiac disease and hematological cancers, the pathogenesis of which involves T cell receptors of the TRBV9 family.)

1. A monoclonal antibody or antigen-binding fragment thereof that specifically binds to the TRBV-9 family β -chain region of the human T cell receptor comprises 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. The monoclonal antibody of any one of claim 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 1-3, 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 comprising the nucleic acid of claim 4.

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

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

8. A method of obtaining an antibody or antigen-binding fragment thereof according to any one of claims 1-3, said method comprising culturing the host cell of claim 7 in a culture medium under conditions that ensure production of said antibody, followed by isolation and purification of the resulting antibody.

9. A pharmaceutical composition for preventing or treating a disease or disorder mediated by the TRBV9 family β -chain region of human T receptor, the pharmaceutical composition comprising a therapeutically effective amount of the antibody or antigen-binding fragment thereof of any one of claims 1-3 in combination with one or more pharmaceutically acceptable excipients.

10. The pharmaceutical composition of claim 9, wherein the disease or disorder is selected from the group consisting of: ankylosing spondylitis, celiac disease, T-cell leukemia, and T-cell lymphoma.

11. A pharmaceutical composition for preventing or treating a disease or disorder mediated by a human T cell receptor bearing a TRBV9 family beta-chain, the pharmaceutical composition comprising a therapeutically effective amount of the antibody or antigen-binding fragment thereof of any one of claims 1-3 and at least a therapeutically effective amount of one other therapeutically active compound.

12. The pharmaceutical composition of claim 11, wherein the disease or disorder is selected from the group consisting of: ankylosing spondylitis, celiac disease, T-cell leukemia, and T-cell lymphoma.

13. The pharmaceutical composition according to any one of claims 11-12, wherein the further therapeutically active compound is selected from a small molecule, an antibody or a steroid hormone.

14. 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 to the subject an effective amount of the antibody or antigen-binding fragment thereof of any one of claims 1-3.

15. A method for treating a disease or disorder mediated by a human T cell receptor bearing a TRBV9 family β -chain, the method comprising administering to a subject in need of such treatment a therapeutically effective amount of an antibody or antigen-binding fragment thereof according to any one of claims 1-3 or a pharmaceutical composition according to claims 9-13.

16. A method for treating a disease or disorder according to claim 17, wherein the disease or disorder is selected from: ankylosing spondylitis, celiac disease, T-cell leukemia, and T-cell lymphoma.

17. 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 9-13 for the treatment of a disease or disorder mediated by a human T cell receptor bearing a TRBV9 family β -chain in a subject in need of such treatment.

18. The use according to claim 17, wherein the disease is selected from: ankylosing spondylitis, celiac disease, T-cell leukemia, and T-cell lymphoma.

Technical Field

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

Background

Autoimmune diseases are caused by autoreactive T lymphocytes (Haroon N et al, Arthritis Rheum. 2013, 10 months; 65(10):2645-54, Duarte J. et al, PloS One 2010, 5 months 10 days; 5(5): e10558; Konig M. et al, Front Immunol 2016, 1 months 25 days; 7: 11). The prior art discloses that T Cell Receptor (TCR) sequences are markers that allow the 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 from several gene segments. The TCR variable region forms a TCR antigen binding site. This means that they are clone-specific, i.e. differ in the T lymphocytes responding to the unique antigen.

T cell receptors are divided into different families based on amino acid homology of the variable (V) gene segments within the TCR variable domain. According to IMGT nomenclature, the β -strands are classified into 26 distinct families and the α -strands into 41 families (Turner SJ et al, Nature Reviews Immunology 2006, V.6, 883-. To determine The TCR chain family, one uses multiple alignments of The test amino acid sequences and known TCR chain sequences, and information about known TCR chain sequences is summarized in The IMGT database ("The International ImmunoGeneTiCs information system", Lefranc M-P., Nucl Acids Res 2001; 29: 207-. Multiple alignments and determinations of the TCR chain family can be performed using the IgBlast software package.

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, proposed as diagnostic and therapeutic tools for rheumatoid arthritis. The monoclonal antibodies recognized 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 demonstrating the involvement of T lymphocytes in the pathogenesis of rheumatoid arthritis led to the use of monoclonal antibodies specific for the β -chain region of the T receptor. In particular, Brennan et al, Clin Exp Immunol, 1988, 9 months, 73(3): 417-. WO9405801 discloses monoclonal antibodies for the diagnosis and treatment of rheumatoid arthritis which interact with epitopes of the V beta 3.1 variable region of the human T-cell receptor, which interact with the TCR V (β)3.1 subfamily.

Monoclonal antibodies that specifically recognize the 13 th family β -chain of rat TRC have also been described. Animal models have demonstrated that with the aid of these antibodies, a small population of T cells whose T receptors comprise the VB13 β -chain (VB13+ T cells) can be preventively removed, and that such a procedure would protect against the development of type I diabetes in diabetes-prone rats and also significantly reduce the risk of the development of virus-induced diabetes (Zhijun Liu et al diabetes 2012, month 5; 61(5): 1160 1168.). At the same time, the removal of T cells whose T receptors comprise a unique β -chain family (VB16) did not differ from the control. It is important to note that even the first administration of a monoclonal antibody against VB13 resulted in a 60% reduction in the number of VB13+ T cells in the rat spleen.

A consensus variant of the autoimmune TCR in patients with ankylosing spondylitis (AS or Abelian disease) has been described, which has been shown to be present in synovial fluid and peripheral blood of patients with AS and absent in the same depth analysis of healthy donors, regardless of their HLA x B27 allelic status (Faham M. et al, Arthritis Rheumatotol. 2017;69(4): 774-. The TCR is a member of the TRBV9 family (according to IMGT nomenclature). T cell receptors with TRBV9 family beta-chains have also been shown to be 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 exacerbation 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).

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 for the treatment of autoimmune and oncological diseases whose pathogenesis involves TCRs belonging to the TRBV9 family, such AS, celiac disease and some T-cell lymphomas and T-cell leukemias.

The antibodies are the only antibodies currently known that can be used to eliminate T cells bearing a TRBV9 family TCR. The main drawback of said antibodies is the relatively low degree of humanization, i.e. they comprise 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 was 72%, while that of the variable fragment of the light chain was 69%.

The parent monoclonal antibodies comprise:

1) the variable domain (VH) of their heavy chain, comprising 3 hypervariable regions HCDR1, HCDR2 and HCDR3, wherein

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

HCDR2 has the amino acid sequence of SEQ ID NO 2

HCDR3 has the amino acid sequence of SEQ ID No. 3;

2) the variable domain (VL) of their light chain comprising 3 hypervariable regions LCDR1, LCDR2 and LCDR3, wherein:

LCDR1 has the amino acid sequence of SEQ ID NO. 4,

LCDR2 has the amino acid sequence of SEQ ID NO. 5,

LCDR3 has the amino acid sequence of SEQ ID NO 6.

The above parent monoclonal antibodies include the variable domains of the heavy and light chains having the amino acid sequences shown in SEQ ID NOS: 8 and 10.

The above parent monoclonal antibody includes a light chain having an amino acid sequence shown in SEQ ID number 12 and an antibody heavy chain having an amino acid sequence of SEQ ID number 14.

Examples of nucleotide sequences encoding the amino acid sequences of the heavy and light chains of the above parent antibodies are shown in SEQ ID NOS: 13 and 11.

The present invention relates to the preparation of monoclonal antibodies which can be used to eliminate T cells bearing a TRBV9 family TCR, in particular for the treatment of AS, celiac disease and hematological malignancies whose pathogenesis involves a TRBV9 family TCR, and which are characterized by a high degree of humanization. At the same time, humanization often results in a critical decrease in antibody affinity and/or solubility. Thus, the preparation of humanized functional antibodies is a related task.

Disclosure of Invention

The present invention relates to a humanized monoclonal antibody and antigen binding fragments thereof, which has the ability to specifically bind with high affinity to the TRBV9 family β -chain region of human T receptor. The antibodies according to the invention can be used AS medicaments for the treatment of autoimmune and oncological diseases, the pathogenesis of which involves TCRs belonging to the TRBV9 family, such AS, for example, AS, celiac disease and some T-cell lymphomas and T-cell leukemias.

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

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

2) HCDR2 has the amino acid sequence of SEQ ID NO 2

3) HCDR3 has the amino acid sequence of SEQ ID No. 3;

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

LCDR1 has the amino acid sequence of SEQ ID NO. 4,

LCDR2 has the amino acid sequence of SEQ ID NO 5,

LCDR3 has the amino acid sequence of SEQ ID NO 6.

Unless otherwise specifically stated, the well-known Kabat numbering scheme is used hereinafter to determine the CDRs of an antibody.

Thus, antibody heavy and light chain variable domains comprise amino acid substitutions in the FR fragments of the heavy and light chain variable domains which increase the degree of humanization of the antibody compared to the parent antibody.

In certain embodiments, the variable domain of the heavy chain of an antibody of the invention comprises at least 10 humanized amino acid substitutions as compared to the variable domain of the heavy chain of the parent antibody whose amino acid sequence is shown in SEQ ID No. 8.

In a preferred embodiment, the variable domain of the heavy chain of the antibody of the invention has the sequence shown in SEQ ID No. 16.

In certain embodiments, the variable domain of the heavy chain of an antibody of the invention comprises additional amino acid substitutions that do not alter the specificity of the antibody.

In certain embodiments, the variable domain of the light chain of the antibody of the invention comprises at least 10 humanized amino acid substitutions as compared to the variable domain of the light chain of the parent antibody whose amino acid sequence is shown in SEQ ID No. 10.

In a preferred embodiment, the variable domain of the light chain of the antibody of the invention has the sequence shown in SEQ ID NO 18.

In certain embodiments, the variable domain of the light chain of an antibody of the invention comprises additional amino acid substitutions that do not alter the specificity of the antibody.

In certain embodiments, the monoclonal antibody of the invention is a full length human IgG antibody, e.g., IgG1 or IgG2 or IgG3 or IgG 4.

In certain embodiments, an antibody of the invention comprises a heavy chain having an amino acid sequence that 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.

In certain embodiments, the antibodies of the invention comprise a light chain having an amino acid sequence 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.

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.

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.

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

In addition, cells and stable cell lines comprising the nucleic acids, vectors or expression cassettes of the invention, and methods of making the same are provided.

Also provided is a method for producing the above antibody or antigen-binding fragment thereof, comprising culturing the above host cell in a culture medium under conditions that ensure production of the antibody. In certain embodiments, the methods comprise subsequent isolation and purification of the resulting antibody.

Also provided are pharmaceutical compositions for preventing or treating a disease or disorder mediated by the TRBV9 family β -chain region of human T receptor comprising the above antibody or antigen-binding fragment thereof in combination with one or more pharmaceutically acceptable excipients.

In one of the embodiments, the pharmaceutical composition is intended for the prevention or treatment of a disease or disorder selected from the group consisting of: ankylosing spondylitis, celiac disease, T-cell leukemia, and T-cell lymphoma.

Also provided is a pharmaceutical combination for the prevention or treatment of a disease or disorder mediated by a human T cell receptor carrying a TRBV9 family β -chain comprising the above antibody or antigen binding fragment thereof and at least one other therapeutically active compound.

In one of the embodiments, the pharmaceutical combination is intended for the prevention or treatment of a disease or disorder selected from the group consisting of: ankylosing spondylitis, celiac disease, T-cell leukemia, and T-cell lymphoma.

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

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

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

In one of the embodiments 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, and T-cell lymphoma.

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

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.

The technical result of the present invention consists in obtaining an antibody with a high degree of humanization, which specifically binds with high affinity to T C Rs whose beta-chain belongs to the TRBV9 family, and which can be used for the treatment of autoimmune and oncological diseases whose pathogenesis involves TCRs whose beta-chain belongs to the TRBV9 family.

In a preferred embodiment, the antibody heavy chain variable fragment is characterized by a degree of humanization of 87%. In a preferred embodiment, the antibody light chain variable fragment is characterized by a degree of humanization of 85%.

Drawings

FIG. 1 shows the results of sorting T lymphocytes using antibody MA-042.

FIG. 2 shows the results of flow cytometry of T lymphocytes after determination of the cytotoxic activity in the presence of the antibody MA-042 at a concentration of 1 ng/ml (right) and 1. mu.g/ml (left). The rectangle shows the population of CD45+ CD3+ TRBV9 +.

Figure 3 shows the number of dead T lymphocytes as a function of MA-042 concentration to determine the half effective concentration of MA-042 (EC50) in the cytotoxicity assay.

Detailed Description

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

Definition of

The present invention will be more readily understood by first defining certain terms.

It is to be understood that the materials and methods provided herein are not limited to particular compositions and method steps, as these may vary. It must be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

The human "T cell receptor" (also known as "TCR", "T receptor") is a heterodimeric protein complex found on the surface of T lymphocytes. T receptors are present only on T lymphocytes. The primary function of TCRs is to specifically recognize processed antigens that bind to molecules of the major histocompatibility complex (HLA).

Human TCRs consist of 2 subunits, alpha and beta chains, or gamma and delta chains, linked by disulfide bonds and embedded in the cell membrane. Each TCR chain has an N-terminal variable (V) domain, a linking domain, and a constant (C) domain linked to a transmembrane domain that anchors the receptor in the T lymphocyte plasma membrane. The constant domains of the alpha and beta-strands are 91 and 129 amino acid residues in length, respectively. The alpha chain linking and transmembrane domain is 30 and 17 Amino Acid Residues (AAR) in length, and the beta chain linking and transmembrane domain is 21 and 22 AAR in length. The length of the T receptor variable domain varies between 104-125 AARs.

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

The T receptor reacts with the MHC/antigen complex via 6 regions (CDRs) that determine its complementarity: 3 alpha chain region and 3 beta chain region. These CDRs are the hypervariable regions, loops of the variable domain of the T cell receptor, V α and V β.

The terms "TRBV 9" or "TRBV 9 family" denote the ninth family of beta-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 (the amino acid sequence is S-G-D-L-S) and CDR2 (the amino acid sequence is Y-N-G-E). The term "TRBV 9 family TCR" denotes a T cell receptor whose β -chain belongs to the TRBV9 family.

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

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

The term "antibody" as used herein is intended to mean an immunoglobulin molecule consisting of 4 polypeptide chains (2 heavy (H) chains and 2 light (L) chains) linked by disulfide bonds. Light chains are classified as either kappa or lambda. Classifying the heavy chain as gamma, mu, alpha, delta or epsilon; they determine antibody isotypes such as IgG, IgM, IgA, IgD, and IgE, respectively, and several of them can be further divided into subclasses (isotypes), for example IgG1, IgG2, IgG3, IgG4, IgA1, and IgA 2. Each heavy chain type is characterized by a specific constant region.

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 CH 3. 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 a domain CL. The VH and VL regions can be further subdivided into regions of high variability, called Complementarity Determining Regions (CDRs), surrounded by more conserved regions, called Framework Regions (FRs). Each VH and VL consists of 3 CDRs and 4 FRs, arranged in the following order from amino-terminus to carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4.

In this application, the 3 heavy chain CDRs are referred to as "HCDR 1, HCDR2 and HCDR 3" and the 3 light chain CDRs are referred to as "LCDR 1, LCDR2 and LCDR 3". The CDRs contain most of the residues that interact specifically with the antigen. Unless otherwise indicated, CDR-amino residues within the HCVR and LCVR of an antibody according to the invention are numbered and positioned according to the well-known Kabat numbering scheme. The present application includes the conventional letter codes for amino acids, unless otherwise indicated.

The terms "anti-TRBV 9 antibody", "antibody against TRBV 9", "antibody specifically binding to the TRBV9 family β -chain" and "antibody against the TRBV9 family β -chain" are interchangeable within the context of the present application and relate to antibodies that specifically bind to an epitope of the TRBV9 family β -chain of the human T cell receptor.

In addition, The "Monoclonal antibody" used in The present application may be a single-chain Fv-fragment which can be obtained by binding DNA encoding LCVR and HCVR to a linker sequence (see Pluckthun, The pharmaceutical of Monoclonal Antibodies, Vol.113, eds. Rosenburg and Moore, Springer-Verlag, New York, p. 269-315, 1994). It is contemplated that the term "antibody" as used in this application includes such fragments or portions as well as single chain forms, whether or not reference is made to the fragment or portion. As long as the protein retains its ability to specifically or preferentially bind its target (e.g., an epitope or antigen), it is encompassed by the term "antibody". Antibodies may be glycosylated or unglycosylated and still be within the scope of the invention.

For the purposes of this application, the terms "antibody" and "monoclonal antibody" refer to a monoclonal antibody directed against a TCR of the TRBV9 family. As used herein, "monoclonal antibody" relates to a rodent, primate or camelid antibody, preferably to a murine, monkey, camel or vicuna antibody, a chimeric antibody, a humanized antibody or a fully human antibody, unless otherwise specified.

A population of "monoclonal antibodies" means 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 sequences). Antibodies may be glycosylated or unglycosylated while remaining within the scope of the invention. Monoclonal antibodies may be homogeneous if they have identical amino acid sequences, although they may differ in post-translational modifications (e.g., glycosylation patterns).

The variable regions of each pair of light/heavy chains form the antigen binding site of the antibody. As used herein, "antigen binding portion" or "antigen binding region" or "antigen binding domain" or "antigen binding site" refers interchangeably to such portions of an antibody molecule: which comprises 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.

The term "human antibody" as used herein denotes antibodies in which the sequences of the variable and constant domains are derived from human sequences. Human antibodies according to the invention may comprise human atypical amino acid residues (e.g., mutations introduced by in vitro non-directed or site-specific mutagenesis or in vivo somatic mutation), for example, in the CDRs, and in particular, in CDR 3.

The term "humanized" when used with respect to an antibody is used to denote an antibody that: characterized by the presence of human-like constant regions and structural components, but having Complementarity Determining Regions (CDRs) typical of immunoglobulins of other origin, or of corresponding fragments of modified antibodies.

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

The term "degree of humanization" in relation to an antibody is used to indicate the percentage of identity of the framework region sequence of a humanized antibody to the original human acceptor framework region used to generate the humanized antibody and available from a human library. Preferably, the antibody of the invention comprises a framework region that is at least 80% identical, typically at least 82%, more often at least 83%, e.g. at least 84% or at least 85% or at least 86% or at least 87% identical to a framework region obtained from a human library.

The term "humanized substitution" refers to an amino acid substitution that increases the degree of humanization of an antibody or fragment thereof.

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

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

The term "isolated" as used herein 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 present in vivo.

In a preferred embodiment, the antibodies of the invention are recombinant, i.e., obtained using recombinant DNA techniques. The term "recombinant antibody" as used herein includes all antibodies obtained, expressed, established or isolated by recombinant means, such as antibodies expressed using recombinant expression vectors introduced into host cells, antibodies isolated from a set of known recombinant combinatorial human antibody libraries, antibodies isolated from transgenic animals for human immunoglobulin genes (see, e.g., Taylor L.D. et al (1992) Nucl. Acids Res. 20: 6287-. In certain embodiments, the recombinant human antibodies are subjected to in vitro mutagenesis (or, when a transgenic animal using human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are such that: it is unlikely to occur naturally within the human antibody germline repertoire in vivo, despite being derived from and related to human germline VH and VL sequences.

The term "specifically binds" as used in the present application denotes the situation: wherein one member of the specific binding pair does not significantly bind to a molecule other than its specific binding partner. The term is also applicable where, for example, the antigen binding domain of an antibody of the invention is specific for a particular epitope carried by a number of antigens; in this case, a specific antibody comprising an antigen binding domain will be capable of specifically binding to various antigens carrying the epitope. Thus, the monoclonal antibody of the invention specifically binds to an epitope of the TRBV9 family β -chain of the human T cell receptor, whereas it does not specifically bind to the TCR β -chain and TCR α chain of the other families.

The term "epitope" refers to the portion of a molecule that is capable of being recognized and bound by an antibody at one or more of its antigen binding regions. Epitopes often consist of chemically active surface clusters of molecules, such as amino acids or sugar side chains, and have specific three-dimensional structural characteristics as well as specific charge characteristics.

The term "epitope" as used herein denotes in particular a polypeptide fragment having antigenic and/or immunogenic activity in an animal, preferably in a mammal such as a mouse, a rat or a human. The term "antigenic epitope" as used herein 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 standard immunoassay). Epitopes need not be immunogenic, however, they may be immunogenic. As used herein, an "immunogenic epitope" is defined as a polypeptide fragment that elicits an antibody response in an animal, as determined by any method known from the prior art. A "nonlinear epitope" or "conformational epitope" comprises a non-contiguous polypeptide (or amino acid) within an antigenic protein that binds an epitope-specific antibody.

The phrases "biological property" or "biological characteristic" or the terms "activity" or "biological activity" in reference 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 β -chains belonging to the TRBV9 family.

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 foregoing properties or characteristics may be observed, measured and/or assessed using art-recognized techniques including, but not limited to, ELISA, competitive ELISA, BIACORE or KINEXA surface plasmon resonance assays, 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.

The term "inhibit" or "neutralize" as used herein with respect to the activity of an antibody of the invention refers to the ability to substantially antagonize, prevent, limit, slow, destroy, eliminate, halt, reduce the progression or severity of, for example, a subject to be inhibited (including, but not limited to, the above, the biological activity, or the nature, disease or disorder of the antibody).

The term "mutant" or "variant" as used herein denotes an antibody disclosed in the present invention, wherein one or more amino acids are added and/or substituted and/or deleted and/or inserted at the N-terminus and/or C-terminus and/or within the native amino acid sequence of the antibody or fragment thereof of the present invention. The term "mutant" as used herein also refers to nucleic acid molecules encoding the mutant proteins. Furthermore, the term "mutant" denotes any variant that is shorter or longer than the protein or nucleic acid.

The term "homology" is used to describe the association of a nucleotide or amino acid sequence with other nucleotide or amino acid sequences, depending on the degree of identity and/or similarity between the sequences being compared.

An amino acid or nucleotide sequence as used herein is "substantially similar" or "substantially identical" to a reference sequence if it is at least 85% identical to the specified sequence within the region selected for comparison. Thus, substantially similar sequences include those that are, for example, at least 90% identical, or at least 91% identical, or at least 92% identical, or at least 93% identical, or at least 94% identical, or at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical. The 2 sequences that are identical to each other are also substantially similar.

Sequence identity is determined based on the 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 the purposes of the present invention, gapped alignments with standard parameters of nucleotide and amino acid sequences can be used with the aid of the IgBLAST software package provided by the national center for Biotechnology information (https:// www.ncbi.nlm.nih.gov/IgBLAST /) to determine the level of identity and similarity between nucleotide and amino acid sequences. To calculate percent identity, the full length of the reference sequence, e.g., the variable region, is used.

Reference to a nucleotide sequence that "encodes" a polypeptide refers to the production of the polypeptide from the nucleotide sequence during translation and transcription of the mRNA. Thus, the coding strand and the complementary strand that serve as transcription templates that are identical to the mRNA and are commonly used in sequence listings 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 that include introns.

Antibodies

As noted 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 receptor.

The antibodies according to the invention may be chimeric, humanized or human antibodies or antigen-binding fragments thereof and may be used AS medicaments for the treatment of AS and other diseases whose pathogenesis involves TCRs belonging to the TRBV9 family (e.g. celiac disease or T cell lymphoma).

The antibodies according to the invention are monoclonal. Monoclonal antibodies of the invention can be produced using, for example, hybridoma techniques, as well as recombinant techniques, phage display techniques, synthetic techniques, or a combination of such techniques, or other techniques well known in the art. The term "monoclonal antibody" as used in this application refers to an antibody obtained from a single copy or clone (including, for example, any eukaryotic, prokaryotic, or phage clone), and not to the method of production thereof.

Humanized and chimeric antibodies can be prepared by peptide synthesis or using recombinant DNA techniques as described in the "nucleic acid" section below.

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

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

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

2) HCDR2 has the amino acid sequence of SEQ ID NO. 2,

3) HCDR3 has the amino acid sequence of SEQ ID No. 3;

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

LCDR1 has the amino acid sequence of SEQ ID NO. 4,

LCDR2 has the amino acid sequence of SEQ ID NO 5,

LCDR3 has the amino acid sequence of SEQ ID NO 6.

Unless otherwise specifically stated, the well-known Kabat numbering scheme is used hereinafter to determine the CDRs of an antibody.

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

In a preferred embodiment, the variable domain of the heavy chain of the antibody of the invention has the amino acid sequence shown in SEQ ID No. 16.

In certain embodiments, the variable domain of the heavy chain of an antibody of the invention comprises additional amino acid substitutions that do not alter the specificity of the antibody.

In a preferred embodiment, the variable domain of the light chain of the antibody of the invention comprises amino acid substitutions and has the sequence shown in SEQ ID NO. 18.

In certain embodiments, the variable domain of the light chain of an antibody of the invention comprises additional amino acid substitutions that do not alter the specificity of the antibody.

In certain embodiments, the monoclonal antibody of the invention is a full length human IgG antibody, e.g., IgG1 or IgG2 or IgG3 or IgG 4.

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.

As is known from the prior art, mutations can be introduced into antibody sequences, including variable domains, which do not substantially alter the antibody's ability to bind antigen. The antibodies according to the invention may also contain other mutations that do not result in loss of the antibody's ability to bind the TCR TRBV9 family β -chain, 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. In particular, it is well known from the prior art that conservative amino acid substitutions may be made in an antibody sequence. "conservative substitutions" in the context of the present application mean such substitutions: wherein one amino acid residue is replaced with another amino acid residue having 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), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-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 domains 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 an epitope of the TRBV9 family β -chain.

The above variants (mutants) of the antibodies according to the invention can be obtained by peptide synthesis or using recombinant DNA techniques described in the "nucleic acid" section below.

In a preferred embodiment, the antibody comprises a heavy chain constant region, 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.

Antigen-binding fragments of the antibodies of the 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 fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody include: (a) fab fragments, i.e., monovalent fragments consisting of the VL, VH, CL and CH1 domains; (b) f (ab)2 fragments, i.e. bivalent fragments comprising 2 Fab fragments linked by a disulfide bond at the hinge region; (c) an Fd fragment consisting of the VH and CH1 domains; (d) (ii) an Fv fragment consisting of the VL and VH domains of a single arm 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). Furthermore, although the 2 domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined using recombinant methods by a synthetic linker that enables them to be prepared as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see, e.g., 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 encompassed within the term "antigen-binding fragment" of an antibody. They also include other forms of single chain antibodies, such as diabodies. A diabody is a bivalent bispecific antibody in which VH and VL domains are expressed on a single polypeptide chain, but a linker is used which is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of the other chain and generating two antigen binding sites (see, e.g., Holliger R. et al (1993) Proc. Natl. Acad. Sci. USA 90: 6444-.

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

An antibody or antigen-binding portion thereof can be part of a larger immunoadhesion molecule formed by covalent or non-covalent association of an antibody or antibody fragment with one or more proteins or peptides. Examples of such immunoadhesion molecules include the use of the streptavidin core region for the preparation of tetrameric scFv molecules (Kipriyanov S.M. et al (1995) Human Antibodies and hybrids 6:93-101) and the use of cysteine residues, tag peptides and C-terminal polyhistidine tags for the preparation of bivalent and reduced size scFv biomolecules (Kipriyanov S.M. et al (1994) mol. Immunol., 31: 1047-1058). Other chemical bonds between antibody fragments are also well known from the prior art.

The antibodies and functional fragments thereof according to the present invention are present in isolated form, i.e. this means that such proteins are substantially free of other proteins or other naturally occurring biomolecules (such as oligosaccharides, nucleic acids and fragments thereof etc.) present, wherein the term "substantially free" in this case means that less than 70%, typically 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%, typically equal to at least 97%, and more often equal to at least 99%.

Methods for purifying antibodies obtained by recombinant or hybridoma techniques are well known in the art, and purification may be performed, for example, by chromatography (e.g., ion exchange chromatography, affinity chromatography, in particular affinity for the particular antigen protein a or protein G, and size column chromatography), centrifugation, differential solubility, or any other standard technique for purifying proteins. Furthermore, antibodies or fragments thereof obtained by the techniques according to the present invention may be fused to heterologous polypeptide sequences (e.g., histidine tags) to facilitate purification.

By determining the dissociation constant (KD), antibody affinity can be determined using standard assays. KD was 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.

Preferred antibodies are those that bind to human antigens with the following KD values: not more than about 1X 10^-7M; preferably no more than about 1 x 10^-8M; more often no more than about 1 x 10^-9M; more preferably no more than about 1 × 10^-10M, and most preferably no more than about 1X 10^-11M, e.g., not more than about 1X 10^-12 M。

Preferred antibodies include the antibody MA-042 described in detail in the experimental section below.

Antibodies and fragments thereof that may be used in the compositions and methods of the invention are biologically active antibodies and fragments, i.e., they are capable of binding a desired antigenic epitope and exhibit a biological effect, either directly or indirectly.

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

In certain embodiments, antibodies or fragments thereof directed to TRBV9 family β -chains according to the present invention may eliminate T cells bearing TCRs comprising TRBV9 family β -chains. In certain embodiments, an antibody or fragment thereof according to the 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.

In a preferred embodiment of the invention, the antibody is the antibody MA-042.

Antibody MA-042 comprises the variable domains of the heavy and light chains having the amino acid sequences shown in SEQ ID NOS: 16 and 18.

Antibody MA-042 includes heavy and light chains having the amino acid sequences set forth in SEQ ID NOS: 20 and 22, respectively.

Nucleic acids

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

In a preferred embodiment, the nucleic acid of the invention encodes an antibody heavy chain, the variable domain of which comprises 3 hypervariable regions HCDR1, HCDR2 and HCDR3, wherein

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

HCDR2 has the amino acid sequence of SEQ ID No. 2;

HCDR3 has the amino acid sequence of SEQ ID No. 3.

In a preferred embodiment, the nucleic acid of the invention encodes an antibody light chain, the variable domain of which comprises 3 hypervariable regions LCDR1, LCDR2 and LCDR3, wherein:

LCDR1 has the amino acid sequence of SEQ ID No. 4;

LCDR2 has the amino acid sequence of SEQ ID No. 5;

LCDR3 has the amino acid sequence of SEQ ID No. 6.

In a preferred embodiment, the nucleic acid of the invention encodes antibody heavy and light chain variable domains that contain amino acid substitutions in the FR fragments of the variable domains of the heavy and light chains that increase the degree of humanization of the antibody compared to the parent antibody.

Nucleic acid molecules encoding homologues and mutants of said antibody chains, functional fragments and domains thereof are also within the scope of the present invention.

In certain embodiments, the nucleic acid encodes the variable domain of the heavy chain of the antibody of the invention, which contains at least 10 humanized amino acid substitutions as compared to the variable domain of the heavy chain of the parent antibody whose amino acid sequence is shown in SEQ ID No. 8.

In certain embodiments, the nucleic acid encodes an antibody heavy chain, the variable domain of which has the amino acid sequence of SEQ ID No. 16.

In certain embodiments, the nucleic acid encodes an antibody light chain whose variable domain comprises at least 10 humanized amino acid substitutions as compared to the variable domain of the light chain of the parent antibody whose amino acid sequence is shown in SEQ ID No. 10.

In certain embodiments, the nucleic acid encodes an antibody light chain, the variable domain of which has the amino acid sequence of SEQ ID No. 18.

In certain embodiments, the nucleic acid encodes an antibody heavy chain having an amino acid sequence 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.

In certain embodiments, the nucleic acid encodes an antibody light chain having an amino acid sequence 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.

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

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 an antibody can be used in operable fusions with nucleic acids encoding the corresponding constant domains of an antibody.

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.

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

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.

As used herein, a "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, the nucleic acid molecules of the invention are DNA (or cDNA) molecules containing an open reading frame that encodes an antibody or antibody fragment of the invention and is capable of being used for expression in a heterologous expression system under suitable conditions (e.g., physiological intracellular conditions).

In certain embodiments, the nucleic acid molecules of the invention are produced by genetic engineering methods. Methods for producing nucleic acids are well known in the art. For example, the availability of amino acid sequence information or nucleotide sequence information enables the preparation of the isolated nucleic acid molecules of the invention by oligonucleotide synthesis. In the case of amino acid sequence information, a plurality of nucleic acids which differ from one another by virtue of the degenerate code can be synthesized. Methods for selecting codon variants for a desired host are well known in the art.

Synthetic oligonucleotides can be prepared by the amino phosphite method and the resulting construct can be purified 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, 2 nd edition, (1989) Cold Spring Harbor Press, Cold Spring Harbor, NY, under Guidelines described, for example, in United States depth of HHS, National Institute of Health (NIH) Guidelines for Recommendant DNA Research. The long double-stranded DNA molecules of the invention can be synthesized in the following manner: several smaller fragments of appropriate complementarity are synthesized, containing appropriate ends capable of binding to adjacent fragments. The adjacent fragments may be ligated using DNA ligase or PCR based methods.

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

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

The nucleic acids of the invention are present in an environment other than that in which they are naturally found, e.g., 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 are naturally found.

Changes or differences in nucleotide sequence between closely related nucleic acid sequences may represent nucleotide changes in the sequence that occur during normal replication or repetition. Other changes may be specifically designed and introduced into the sequence for specific purposes, such as to alter the codons for specific amino acids or nucleotide sequences in the regulatory regions. Such particular changes can be made in vitro using a variety of mutagenesis techniques, or can be produced in a host organism placed under specific selection conditions to induce or select for such changes. Such specifically derived sequence variants may be referred to as "mutants" or "derivatives" of the original sequence.

Mutant or derivative nucleic acids may be obtained on a template nucleic acid selected from the above nucleic acids by modification, deletion or addition of one or more nucleotides in the template sequence, or a combination thereof, to obtain a variant of the template nucleic acid. 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, PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-specific mutagenesis, random mutagenesis, Gene reassembly, Gene site-saturated mutagenesis (GSSM), Synthetic Ligation Reassembly (SLR), or combinations thereof. Modifications, additions, or deletions may also be performed by methods comprising recombination, recursive sequence recombination, phosphorothioate-modified DNA mutagenesis, uracil-containing template mutagenesis, gapped duplex mutagenesis, point mismatch repair mutagenesis, repair-deficient host strain mutagenesis, chemical mutagenesis, radiation mutagenesis, deletion mutagenesis, restriction-selection mutagenesis, restriction-purification mutagenesis, artificial gene synthesis, ensemble mutagenesis, chimeric nucleic acid multimer generation, and combinations thereof.

Degenerate variants of nucleic acids encoding the proteins of the present invention are also provided. Degenerate variants of a nucleic acid include substitutions of codons of the nucleic acid with other codons that encode the same amino acid. In particular, degenerate variants of the nucleic acids are prepared to increase expression in a host cell. In this embodiment, codons of the nucleic acid that are non-preferred or less preferred in the host cell gene are replaced with codons that are over-represented in the coding sequence in the host cell gene, wherein the replaced codons encode the same amino acid.

The above modifications do not substantially alter the properties of the antibody or functional fragment thereof, but may promote protein folding in the host cell, reduce aggregation capacity, or modulate other biochemical properties of the protein, e.g., half-life. In certain embodiments, these modifications do not alter the biochemical properties of the protein. In certain embodiments, these modifications result in reduced immunogenicity of the antibody. All types of modifications and mutations indicated above are performed at the nucleic acid level.

The disclosed nucleic acids can be isolated and prepared in 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 is typically "recombinant," i.e., flanked by one or more nucleotides that are not normally associated with the nucleotide on the chromosome as it naturally occurs in its natural host organism.

Also provided are nucleic acids encoding fusion proteins comprising a protein of the invention or a fragment thereof, which are discussed in more detail below. Nucleic acids encoding the variable domains of the invention may be operably linked to nucleic acids encoding the corresponding constant domains of the light and heavy chains of an antibody. The nucleic acids encoding the light and heavy chains of the antibody may be operably linked to nucleic acids encoding a leader peptide that facilitates transport of the expression product from the host cell. The leader peptide is subsequently removed during maturation of the polypeptide.

Carrier

Vectors and other nucleic acid constructs comprising the disclosed nucleic acids are also provided. The term "vector" denotes a nucleic acid molecule capable of transporting another nucleic acid to which it has been operably linked. Certain vectors are capable of autonomous replication in a host cell into which they are introduced, while other vectors are capable of integration into the host cell genome and replication together with the host genome. In addition, some vectors are capable of directing the expression of genes to which they have been operably linked. 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, cosmids, phages and the like, preferably plasmids, and are used for cloning, amplification, expression, transfer to a suitable host and the like of the nucleic acid sequences of the invention. The selection of an appropriate carrier will be apparent to those skilled in the art. The full length nucleic acid or portion thereof is typically inserted into the vector by means of ligation of a DNA ligase to the cleaved restriction enzyme sites in the vector. Alternatively, the desired nucleotide sequence may be inserted by in vivo homologous recombination, typically by flanking the region of homology to the desired nucleotide sequence on the vector. The regions of homology are added by ligation of oligonucleotides, or by polymerase chain reaction using primers that comprise, for example, the regions of homology and portions of the desired nucleotide sequence. Typically, the vector has an origin of replication that ensures its replication in the host cell by virtue of its introduction into the cell as an extrachromosomal element. The vector may also contain regulatory elements which ensure expression of the nucleic acid in the host cell and production of the target polypeptide. In an expression vector, the nucleic acid is operably linked to regulatory sequences, which may include promoters, enhancers, terminators, operators, repressors and inducers, and the start codon for the polypeptide. In certain embodiments, the nucleic acids of the invention are further operably linked to a leader peptide, which ensures isolation of the expression product from the host cell into the extracellular space.

Also provided are expression cassettes or systems, particularly for obtaining the disclosed polypeptides based thereon (e.g., the light and heavy chains of the antibodies of the invention, or the variable domains of the light and heavy chains of the antibodies of the invention), or for replicating the disclosed nucleic acid molecules. As a result of the introduction of the expression cassette into the cell, the expression cassette may be present as an extrachromosomal element, or may be integrated into the genome of the cell. With respect to expression, the protein product encoded by a nucleic acid of the invention is expressed in any convenient expression system, including, for example, a bacterial system, yeast, insect, amphibian, or mammalian cell. In an expression cassette, the target nucleic acid is operably linked to regulatory sequences, which may include promoters, enhancers, termination sequences, operators, repressors and inducers, and the start codon of the polypeptide. In certain embodiments, the nucleic acids of the invention are further operably linked to a leader peptide that ensures isolation of the expression product from the host cell into the extracellular space. Methods for obtaining expression cassettes or systems capable of expressing the desired product are known to those skilled in the art.

Host cell

The above expression systems may be used in prokaryotic or eukaryotic hosts. Host-cells such as E.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, e.g., CHO cells (e.g., ATCC CRL-9096), NS0 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 proteins.

To produce the antibodies of the invention, host cells are co-transformed with an expression vector comprising nucleic acid encoding the light chain of the antibody and an expression vector comprising nucleic acid encoding the heavy chain of the antibody. In certain embodiments, a single expression vector is used into which nucleic acids encoding the light and heavy chains of an antibody are introduced.

For expression of the light and heavy chains, the expression vectors encoding the heavy and light chains are transformed (co-transformed) into the host cell such that the light and heavy chains are expressed in the host cell and preferably secreted into the medium in which the host cell is cultured, and the antibody can be isolated from the medium. The various interpretations of the term "transformation" are intended to include a wide range of methods commonly used to introduce foreign DNA into prokaryotic or eukaryotic host cells, e.g., electroporation, calcium phosphate precipitation, DEAE-dextran transfection, etc., as described in Sambrook, Fritsch and Maniatis (eds.) Molecular Cloning, A Laboratory Manual, 2 nd edition, Cold Spring Harbor, N.Y. (1989; Ausubel F.M. et al (eds.) Current Protocols in Molecular Biology, Green Publishing Associates (1989).

When a recombinant expression vector containing 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) secrete the antibody into the medium in which the host cell is cultured. Antibodies can be isolated 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.B., Lippincott-Schwartz J.and Yamada K.M. (eds.), John Wiley & Sons, Inc. published in 2000.

If any of the above host cells or other host cells or organisms suitable for replicating and/or expressing the nucleic acids of the invention are used, then the resulting replicated nucleic acids, expressed proteins or polypeptides as products of the host cells or organisms are within the scope of the invention. The product may be isolated by suitable techniques known in the art.

Cell lines stably expressing the protein 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 for the identification and isolation of transfected cells containing the gene integrated into the genome).

The nucleic acid molecules of the invention can also be used to determine gene expression in a biological sample. Methods in which cells are examined for the presence of a particular nucleotide sequence, such as genomic DNA or RNA, are well known in the art. Briefly, DNA or mRNA is isolated from a cell sample. mRNA can be amplified by RT-PCR, in which complementary DNA strands are formed using reverse transcriptase, followed by polymerase chain reaction amplification using primers specific for the subject DNA sequence. Alternatively, the mRNA sample is separated by gel electrophoresis, transferred to a suitable carrier such as nitrocellulose, nylon, or the like, and then probed with a fragment of the subject DNA as a probe. Other techniques such as oligonucleotide ligation assays, in situ hybridization, and hybridization to DNA probes immobilized on a solid chip may also be used. Detection of mRNA that hybridizes to the subject sequence is indicative of gene expression in the sample.

Therapeutic uses of the antibodies of the invention

In one aspect, the antibodies of the invention or active fragments thereof are used to treat disorders associated with the activity of pathological T lymphocytes bearing surface TRBV9 family TCR, e.g., exhibiting autoimmune T lymphocyte activity in AS, celiac disease, T cell lymphoma.

The term "patient" as used in this application means a mammal, including, but not limited to, mice, monkeys, humans, livestock mammals, sports mammals, and pet mammals; preferably the term applies to humans. In a particular embodiment, the patient is further characterized by a disease or disorder or condition mediated by the presence in their body of a TCR whose β -chain belongs to the TRBV9 family. AS is known from the prior art, TCRs whose β -chain belongs to the TRBV9 family are associated with AS and celiac disease. In addition, TCRs whose β -chain belongs to the TRBV9 family may be involved in the development of many hematological diseases such as T cell lymphoma caused by epstein-barr virus.

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

1) when such components are formulated together in a single dosage form that releases the components to the patient substantially simultaneously, such combination of an antibody of the invention and a therapeutic agent is administered to a patient in need of treatment,

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

3) when such components are formulated separately from each other in separate dosage forms that are administered sequentially by the patient with a significant time interval between each administration after which the components are released to the patient at substantially different times, such combinations of the antibody of the present invention and the therapeutic agent are administered sequentially to the patient in need of treatment; and

4) when such components are formulated together in a single dosage form that releases the components in a controlled manner, after which they are released to the patient at the same and/or different times in parallel, consecutively and/or overlapping, wherein each part may be administered by the same or different routes, such combinations of the antibody of the invention and the therapeutic agent are administered sequentially to the patient in need of treatment.

The antibodies of the invention may be administered without further therapeutic treatment, i.e., as a stand-alone therapy. Furthermore, the treatment with the antibody of the invention may comprise at least one additional therapeutic treatment (combination therapy). In certain embodiments of the invention, the antibody may be co-administered or formulated with another agent/drug for autoimmune or oncological diseases whose pathogenesis involves a TCR comprising the TRBV9 β -chain, e.g., AC, celiac disease, T cell lymphoma, T cell leukemia.

Dosage and route of administration

The antibodies of the invention will be administered in an amount effective to treat the condition of interest, i.e., in the dosage and duration required to achieve the desired result. The therapeutically effective amount may vary depending on factors such as the particular condition being 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.

The dosage regimen may be adjusted to provide the optimum response. For example, a single bolus delivery may be administered, several divided doses may be administered over time, or the doses may be proportionally reduced or increased as indicated by the exigencies of the treatment situation. It is particularly advantageous to formulate parenteral compositions in unit dosage form for ease of administration and uniformity of dosage. As used herein, standard dosage form is intended to mean a physically discrete unit suitable as a unit dose for the patient/subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the desired pharmaceutical carrier. The specification for the standard dosage forms of the invention generally depends on and directly depends on: (a) the unique characteristics of chemotherapeutic agents and the particular therapeutic or prophylactic effect to be achieved, and (b) limitations inherent in the art of formulation, such as active compounds used to treat sensitivity in a subject.

Thus, the skilled artisan will appreciate, based on the disclosure provided herein, that dosages and dosage regimens are adjusted according to methods well known in the therapeutic arts. That is, the maximum tolerated dose can be readily established, and the effective amount to provide a detectable therapeutic benefit to the patient can also be determined, as can the time requirement for administration of each agent to provide a detectable therapeutic benefit to the patient. Thus, while some dosages and dosage regimens are given in this document as examples, these examples in no way limit the dosages and regimens that a patient may have to administer in the practice of the invention.

It should be noted that dosage values may vary with the type and severity of the condition to be alleviated, and may include single or multiple doses. Further, it is to be understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the judgment of the medical professional administering the compositions or supervising the administration of the compositions, and that the dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions. In addition, the dosage regimen using the compositions of the present invention can be based on a variety of factors including the type of disease, age, weight, sex, health of the patient, severity of the condition, route of administration and the particular antibody used. Thus, the dosage regimen may vary widely, but may be determined routinely using standard methods. For example, the dosage may 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 encompasses the dose escalation in patients as determined by one skilled in the art. Methods for determining appropriate dosages and schedules are well known in the art and will be understood by the skilled artisan after learning the ideas disclosed herein.

Examples of suitable methods of administration are provided above.

It is contemplated that suitable dosages 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 may 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 e.g. a maximum of at most 50 mg/kg, including a maximum of at most 30 mg/kg, such as a maximum of at most 20 mg/kg, including a maximum of at most 15 mg/kg. Administration is usually repeated at appropriate intervals, such as 1 time per week, 1 time per 2 weeks, 1 time per 3 weeks or 1 time per 4 weeks, as long as deemed appropriate by the attending physician who may increase or decrease the dosage in some cases, if necessary.

Pharmaceutical composition

The antibodies of the invention may be incorporated into pharmaceutical compositions suitable for administration to a patient. The antibodies of the invention may be administered alone or in combination with pharmaceutically acceptable carriers, diluents and/or excipients in single or multiple doses. The pharmaceutical compositions for administration are designed to suit the mode of administration selected and pharmaceutically acceptable diluents, carriers and/or excipients such as dispersing agents, buffers, surfactants, preservatives, solubilizers, isotonicity agents, stabilizers and the like are used as appropriate. The compositions are designed according to conventional techniques, as described, for example, in Remington, The Science and Practice of Pharmacy, 19 th edition, Gennaro, eds., Mack Publishing co., Easton, PA 1995, as is generally known to practitioners, which provide various techniques for obtaining compositions.

"medicament (drug)" -is a compound or a mixture of compounds intended for restoring, improving or modifying the physiological functions of humans and animals, and also for the treatment and prevention of diseases, for diagnosis, anesthesia, contraception, cosmetic surgery, etc., as a pharmaceutical composition in the form of tablets, capsules, powders, lyophilized powders, injections, infusions, ointments and other ready-to-use forms. Any method accepted in the art for administering peptides, proteins or antibodies may be suitably used for the antibodies of the invention.

The term "pharmaceutically acceptable" denotes one or more compatible liquid or solid components suitable for administration in a mammal, preferably a human.

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 pharmaceuticals to provide the necessary physicochemical properties to the drug product.

The terms "buffer", "buffer composition", "buffer" denote a solution that is resistant to pH changes caused by the action of its acid-base conjugate components and which allows the antibody drug to tolerate pH changes. Generally, 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 like buffer solutions.

The term "tonicity agent", "osmotic agent" or "osmotic agent" as used herein denotes an excipient that can increase the osmotic pressure of a liquid antibody formulation. An "isotonic" drug is a drug having 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 may be used include, but are not limited to, polyols, sugars and sugars, amino acids, metal salts, e.g., sodium chloride, and the like.

By "stabilizer" is meant an excipient or a mixture of two or more excipients that provides physical and/or chemical stability to the active agent. Stabilizers that may 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 (trade name: Tween 20), polysorbate 80 (trade name: Tween 80), polyethylene-polypropylene glycol and copolymers thereof (trade name: 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.

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

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.

The pharmaceutical compositions of the invention preferably contain or are "therapeutically effective amounts" of the antibodies of the invention. The term "therapeutically effective amount" is intended to mean an amount effective at the dosage and duration required to achieve the desired therapeutic result. A 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 of: wherein the therapeutic benefit of the antibody outweighs any toxic or deleterious effects. A "prophylactically effective amount" is intended to mean an amount effective at the dosage and duration required to achieve the desired prophylactic result. Since prophylactic doses are prescribed to an individual prior to or at an early stage of disease, a prophylactically effective amount can generally be less than a therapeutically effective amount.

The therapeutically effective amount or prophylactically effective amount is at least a minimum therapeutically beneficial dose, which is less than the toxic dose of the active agent. In another aspect, a therapeutically effective amount of an antibody of the invention is an amount which reduces the biological activity of an autoimmune clone in a mammal, preferably a human, for example by binding a TCR whose β -chain belongs to the TRBV9 family, wherein the presence of said clone causes or contributes to an undesired pathological effect, or causes a beneficial therapeutic effect in a mammal, preferably a human, by reducing a TCR whose β -chain belongs to the TRBV9 family.

The route of administration of the antibodies of the invention may be oral, parenteral, inhalation or topical. Preferably, the antibodies of the invention may be comprised in a pharmaceutical composition acceptable for parenteral administration. The term "parenteral" as used herein includes intravenous, intramuscular, subcutaneous, rectal, vaginal or intraperitoneal administration. Intravenous, intraperitoneal or subcutaneous injection is the preferred route of administration. Acceptable pharmaceutical carriers for such injections are well known from the prior art.

As described in appropriate guidelines, the pharmaceutical compositions should be sterile and stable under the conditions of manufacture and storage in containers provided, for example, by hermetically sealed vials (ampoules) or syringes. Thus, the pharmaceutical composition may be filter sterilized after preparation of the composition, or may be made microbiologically suitable by any other technique. A typical composition for intravenous infusion may comprise 250-1000 ml of a fluid such as sterile ringer's solution, physiological saline, dextrose solution, or hank's saline solution, and a therapeutically effective dose (e.g., 1-100 mg/ml or more) of antibody concentrate. The dosage may vary with the type and severity of the disease. It is well known from the prior art in the medical field that the dosage for any patient depends on a variety of factors including the size of the patient, the body surface area, the age, the particular compound to be administered, the sex, the duration and route of administration, the general health and other drugs being administered simultaneously. Typical doses may be, for example, in the range of 0.001-1000 μ g; however, lower and higher doses than this exemplary range are contemplated, particularly in view of the parameters described above. The daily parenteral dosing regimen may be 0.1 to 100 mug/kg of total body weight, preferably 0.3 to 10 mug/kg, and more preferably 1 to 1 mug/kg, even more preferably 0.5 to 10 mug/kg of body weight per day. By periodically assessing the health of the patient, the course of treatment can be monitored. With respect to repeated administrations over several days or longer, depending on the condition of the patient, the treatment is repeated until the desired response or suppression of disease symptoms. However, additional dosing regimens not described herein may also be applied. Depending on the pharmacokinetic disintegration desired by the practitioner, the desired dose may be administered by single or multiple bolus administration or by continuous infusion with the aid of an antibody.

These estimates of antibodies are largely dependent on the judgment of the physician. The desired effect is a key factor in the selection of the appropriate dose and regimen. Factors to be considered herein include the particular disease to be treated, the particular mammal to be treated, the clinical condition of the particular patient, the cause of the condition, the site of antibody administration, the type of specific antibody, the route of administration, the administration regimen, and other factors well known in the medical arts.

The therapeutic agents of the present invention may be frozen or lyophilized and reconstituted in a suitable sterile vehicle prior to administration. Lyophilization 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 pH of the pharmaceutical composition between 6 and 8 is preferred.

Manufactured article (product) and kit

Another embodiment of the invention is an article of manufacture comprising a product for treating autoimmune diseases and related conditions and hematological malignancies, the pathogenesis of which involves TCRs with TRBV9 family β -chains. Such diseases include, for example, AS, celiac disease, T cell leukemia, T cell lymphoma, and the like.

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 and the like. The container may be made of various materials, such as glass or polymeric materials. The container contains a composition effective to treat a condition and may have a sterile access port. At least one active ingredient in the composition is an antibody according to the invention. The label and package insert indicate that the medication is intended for treatment of a certain condition. The label and/or package insert additionally contains instructions for administration of the antibody composition in a patient, including indications, frequency, dosage, route of administration, contraindications, and/or warnings of such therapeutic products. In one embodiment, the package insert indicates that the composition is intended for use in therapy.

In addition, the article of manufacture may contain, but is not limited to, other products necessary for commercial purposes or for the consumer, such as solvents, diluents, filters, needles, and syringes.

The invention also relates to kits that may be used for a variety of purposes, for example, to assess the ability to kill T cells bearing a TCR of the TRBV9 family, to purify or immunoprecipitate the TRBV9 receptor from cells. For isolation and purification, the kit may contain an antibody coupled to a bead (e.g., an agarose bead). The kit comprises a container, a label, and a package insert.

Diagnostic use

The antibodies of the invention are also useful for diagnostic purposes (e.g., in vitro, ex vivo). For example, the antibodies may be used to detect or measure the level of T lymphocytes comprising a TRBV9 family TCR in a sample (e.g., a tissue sample or a bodily fluid sample, such as inflammatory exudate, blood, intestinal fluid, saliva, or urine) obtained from a patient. Suitable detection and measurement methods include immunoassays, such as flow cytometry, enzyme-linked immunosorbent assays (ELISA), chemiluminescent assays, radioimmunoassays, and immunohistology. The invention also includes kits, e.g., diagnostic kits, comprising the antibodies described herein.

In order to better understand the present invention, the following examples are given. These examples are for illustrative purposes only and should not be construed as limiting the scope of the invention in any way.

All publications, patents and patent applications mentioned in this specification are herein incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example to avoid obscuring the explanation, it will be readily apparent to those of ordinary skill in the art in view of the teachings of this disclosure that certain changes and modifications may be made without departing from the spirit and scope of the included embodiments of the present invention.

Experimental part

Example 1 in silico environmental humanization of antibody variable domain sequences

As the parent (reference) sequence, the variable domain sequences of the heavy and light chains of the anti-TRBV 9-2 antibody, the amino acid sequences of which are shown in SEQ ID NO: 8 and 10, were used.

The amino acid sequences of the variable domains of the heavy and light chains were compared to the germline sequences of the variable domains of human immunoglobulins, the germline sequences of the variable domains of rat immunoglobulins, and the collection of sequences of mature human and rat antibodies obtained from donor libraries provided by open source and Biocad (russia). The YLab software package was used for the analysis (Biocad, Russia).

Analytically determined positions and combinations thereof are most animal-like and not 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 were determined. Based on the data obtained, artificial sequences of variable domains were designed which contained substitutions that increased the degree of humanization of the antibodies.

Also, the nucleotide sequence encoding the subject amino acid variant was codon optimized for expression of the humanized antibody in a CHO cell line. Humanized nucleotide sequences for the variable domains of the heavy and light chains were synthesized de novo (SEQ ID NOS: 15 and 17) and cloned into the pEE-HC, pEE-CK vector, IgG1 format at the SalI/NheI and SalI/BsiWI restriction sites, respectively (Xu et al Front. chem. Sci. Eng. 2015, 9(3): 376-380).

The resulting nucleic acid sequences of the light and heavy chains were verified by Senger sequencing. Antibody MA-042 was selected for further studies, and the amino acid and nucleotide sequences of its light and heavy chains are shown in SEQ ID NOS: 19-22.

Antibody MA-042 comprises the variable domains of the heavy and light chains having the amino acid sequences shown in SEQ ID NOS: 16 and 18.

The degree of humanization of the variable domain of the heavy chain of antibody MA-042 was 87%, while that of the light chain was 85% (Table 1). The heavy chain constant domain is represented by the IgG1 format, Gm3 allotype.

TABLE 1 comparison of the degree of humanization between the variable domains of the parent antibody and antibody MA-042.

Antibodies	Degree of humanization of the heavy chain variable domain%	Degree of humanization of the light chain variable domain%
			TRBV9-2	72	69
MA-042	87	85

Example 2 preparation of recombinant antibodies and determination of affinity thereof

The vector containing the nucleic acid encoding the light and heavy chains of antibody MA-042, obtained as described in example 1, 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 polyethylenimine (PEI "MAX", "Polysciences", USA) according to the manufacturer's instructions. The resulting reaction mixture was incubated at 37 ℃ on a shaker. 9 days after transfection, the culture broth was separated from the cells by filtration through a 0.5/0.22 μm filter. After filtration, the culture broth was used for isolation of the antibody using affinity chromatography on a 0.2 ml PreDictor robococolumn 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 carrier-bound protein was eluted using 0.1M glycine buffer ph 3. We collected the major peak containing the protein and adjusted the pH to neutral with 1M Tris buffer (pH 7.5). All stages were carried out 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 quality of separation was assessed using 12% PAGE under denaturing conditions. Quantitative evaluation was performed by measuring a NanoDrop2000 microspectrophotometer at 280A. The isolated protein was stored at-70 ℃.

Antibody affinity was determined on the OctetRed 96 system (ForteBio, USA). Antigen at a concentration of 20 μ g/ml was immobilized on the surface of an AR2G sensor (ForteBio) according to standard protocols and manufacturer's instructions. The assay was performed at 30 ℃ using PBS containing 0.1% tween 20 and 0.1% BSA as working buffer. After baseline recording, the sensor was immersed in the well containing the antibody solution for 300 seconds, where the complex was associated. The complex dissociation in buffer solution was then detected for 600 seconds.

The binding curves after subtraction of the reference signal were analyzed using Octet Data Analysis (version 9.0) software according to standard protocols and using a 1:1 global interaction model. The data obtained (table 2) show that the antibodies bind to human antigens specifically and with high affinity.

TABLE 2 antibody MA-042 affinity characteristics.

Antibodies	KD (M)	kon(1/Ms)	kdis(1/s)	Full R2
					МА-042	<1.0E-12	3.19E+05	<1.0E-07	0.9758

Example 3 preparation of cell lines stably producing antibodies in the form of IgG1 and production of antibodies

The sequences of the heavy and light chains of antibody MA-042 were prepared as described in example 1 and cloned into the pSX vector at HindIII, XbaI restriction sites. The resulting plasmid was cultured in E.coli cells and 600-700. mu.g was isolated using BenchPro (Life Technologies, C.A.) 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/. mu.l in the final volume.

The CHO-K1-S cell line was cultured in Ham' S F12 Gibco medium (Thermo, USA). Electroporation using a Nucleofector (Lonza, switzerland) was performed according to the manufacturer's protocol using a gene construct containing the coding sequence of the melam-042 chain.

One day after transfection, the transfected cells were subjected to antibiotic selection for 24 days by adding puromycin (final concentration of 7.2. mu.g/ml) and hygromycin B (final concentration of 640. mu.g/ml) to the medium. Clones of antibiotic-resistant cells that are homogeneous in their structure expressing high levels of MA-042 are then selected.

For the cultivation of CHO-K1-S expressing MA-042, serum-free medium Ham' S F12 Gibco (Thermo, USA) supplemented with 25-100 uM 2-deoxy-2-fluoro-L-fucose (CarboSynth, UK) was used. Monoclonal antibody MA-042 was produced in a 50L HyClone disposable bioreactor fermentor (Thermo Fisher Scientific) for preclinical studies. The producer cells were removed from the culture broth using a Millistak C0HC depth filter (Merck-Millipore, USA). Primary purification of the antibody from the clean medium was performed on a protein a affinity adsorbent. The target protein is specifically eluted under acidic conditions with a glycine buffer of pH 3.3-3.8. The collected eluate is exposed to acidic pH for 30-60 min for virus inactivation and then neutralized to pH 6.5-7.0 with 1M Tris-HCl solution. The final chromatographic purification was performed in flow-through mode using CaptoAdhere adsorbents (GE Healthcare LifeSciences) to remove possible impurities (DNA, production cell proteins, released ligands of affinity adsorbents, aggregates and antibody fragments). Thus, the protein solution is brought to a low conductivity (< 3msec/cm²) The lower flow was through the prepared adsorbent equilibrated with Tris buffer pH 6.5-7.0. The purified protein was then virus-free filtered using a Viresolve PRO filtration kit (Millipore, USA), concentrated, and diafiltered against a final buffer containing acetate buffer (pH 5.0-5.5) and trehalose.

Example 4 use of antibodies to specifically bind to a TRBV9 family TCR

Monoclonal antibody MA-042 obtained as described in example 3 was used to sort subpopulations of lymphocytes. The antibodies were labeled with fluorescein isothiocyanate reagent (Sigma, USA) according to the manufacturer's protocol. The number of fluorophores reacted with the antibody molecule was controlled by the ratio of the absorption spectra at a wavelength of 495/280 nm.

T lymphocytes were obtained from peripheral blood from healthy donors. Blood was collected in EDTA Vacuette tubes (2 × 9 ml each) and mononuclear fractions were separated according to the standard protocol described (Kovalchuk l.v. et al. Immunology: works hop-2010-176 p.). After isolation, cells were transferred to Phosphate Buffered Saline (PBS) containing 0.5% Bovine Serum Albumin (BSA) and 2mM EDTA. The total number of cells and their viability were determined by the trypan blue staining method described by Lang N.R. (Stimulation of lymphocytes M.: Medicine, 1976.-288 p.). Equal volumes of 0.1% trypan blue solution were added to the cell suspension, and stained (dead) and unstained blue cells were counted in a Goryaev chamber. Based on these data, the percentage of dead cells in the test sample was determined.

To demonstrate the selectivity of MA-042 binding to a target population of T-lymphocytes bearing membrane TCRs belonging to the TRBV9 family, a 500,000 cell aliquot of the mononuclear fraction was added to PBS buffer containing 0.5% Bovine Serum Albumin (BSA), 2mM EDTA pH8, the antibodies MA-042 labeled with FITC, CD3-eFluor405 (T lymphocyte marker) (eBioscience, USA) and CD45-PC5 (eBioscience, USA) (total leukocyte marker) at a concentration of 100 ng/ml. 50 μ l of the reaction mixture was incubated at room temperature for 30 min, after which the cells were washed with PBS buffer supplemented with 0.5% BSA, 2mM EDTA. Following the staining protocol, the cells were used for sorting using flow cytometry (FACSARIA III, USA, fig. 1). The use of these markers in staining allowed the isolation of target population cells carrying surface TRBV9+, CD3+, CD45+, and the obtainment of negative population cells, i.e. those corresponding to the immunophenotype "TRBV 9-, CD3+, CD45 +". Cells from the TRBV9+ and TRBV 9-populations of 2 replicates were used for isolation of total RNA and sequencing of TCR β -chains. The resulting cell fraction was placed in RLT buffer (Quagen, germany) and RNA was isolated therefrom using the queagen RNAeasy mini kit #217004 reagent kit (Quagen) according to the manufacturer's protocol. cDNA was synthesized on an isolated RNA template and fragments of the T receptor beta-strand were amplified using the Mint cDNA synthesis kit (Eurogen, Russia) according to the protocol described by Britanova et al (J Immunol, 2016, 196(12) 5005-5013). The Illumina linker (USA) was ligated to the amplicons produced and sequencing was performed on the MiSeq Illumina platform according to the sequencer manufacturer's protocol. Sequencing data were analyzed using MiGEC, migcr and vdjsols software available on the internet at https:// laboratory. Analysis of the resulting TCR β -chain repertoire revealed that the library obtained by sorting using antibody MA-042 was enriched in 93% of the sequences encoded by the TRBV9 gene segment, whereas no sequences comprising TRBV9 were detected in the repertoire of the "TRBV 9-" negative fraction β -chains.

Example 5 in vitro functional Activity of antibody MA-042

Monoclonal antibody MA-042 was obtained as described in example 3. Mononuclear fractions of human blood were obtained as described in example 4. The cytotoxic activity of MA-042 was determined using a cytofluorimetric assay. Aliquots of cells from the mononuclear fraction were used to calculate the total number of cells and viability was determined by the ability to stain with trypan blue. To assess the cytotoxic efficiency, 3-4X 10⁶Individual cells were incubated for 1 hour in PBS buffer with the following concentrations of antibody MA-042: 20 ng/ml, 40 ng/ml, 100 ng/ml, 200 ng/ml, 500 ng/ml and 1. mu.g/ml, for the "zero control", cells were incubated without addition of antibody. After incubation, cells were washed 2 times with PBS, transferred to RPMI medium containing 10% human serum, and incubated in CO₂Incubate in incubator for 72 hours. The cells were then centrifuged and stained with the antibodies CD4-PE, CD3-eFluor405 (eBioscience, USA) and MA-042-FITC. Stained cell samples were used in cell count analysis on a FacsAria III cell sorter (BD, USA). Cytotoxic effects were assessed by gradually decreasing the proportion of TRBV 9-positive cells in the CD3+ lymphocyte population (reduction in the number of target cells associated with an increase in antibody MA-042 concentration until complete elimination of the target population). Complete elimination of the target population after MA-042 staining was detected at an antibody concentration of 500 ng/ml. In the "zero control", the percentage of TRBV 9T lymphocytes remained unchanged. Figure 2 shows typical results of flow cytometry. This gave an EC50 value (half the maximal effective concentration means the concentration of antibody that induced half the maximal effect of a given antibody after the specified period of time), which for antibody MA-042 amounts to 100 ng/ml (fig. 3).

Example 6 in vivo functional Activity of antibody MA-042

Monoclonal antibody MA-042 was obtained as described in example 3. MA-042 on rhesus monkey (Macaca mulatta) To evaluate specific activity and basic pharmacokinetic parameters.Experiments were performed on sexually mature male rhesus monkeys of 4-10 kg body weight, as supplied by the Federal State Budget Scientific organization of Medical principles. After delivery, animals were quarantined for 30-days.

The fractional amounts of TRBV9+ lymphocytes in peripheral blood were preliminarily estimated to form a cohort of experimental and control animals. Venous blood from the animals was collected at 4 ml/tube into EDTA vacuum tubes (Vacuette, Greiner Bio-One, Austria). The mononuclear cell fraction was then separated by Ficoll gradient (1.077 g/cm3 PanEco, Russia). For immunophenotyping, we used 100,000 cells, which were supplemented with 1. mu.l of commercially available anti-CD 8 PE/Cy5 (clone RPA-T8) antibody (BioLegend, USA), anti-CD 4-Alexa Fluor 488 (clone S3.5) antibody (Thermo Fisher, USA) and anti-CD 2-PerCP Cy 5.5 (clone RPA-2.10) antibody (BioLegend, USA), anti-TcRV β 1 (TRBV9) -PE antibody (Beckman Coulter, USA), incubated for 20 minutes at room temperature and washed 2 times with an equal volume of Henkel' S solution. Samples were analyzed using a FACSAria III cell sorter (USA).

With respect to the experimental time, selected animals were kept in individual metal cages equipped with feedbox, label holder, 1 animal per cage. The diet consists of all-in-one feed, fruits, vegetables according to average feeding standards. The animals received water from a central water feeder.

Animals were divided into three groups, 4 animals/group, including control group, based on the results of cytometric analysis with antibodies against major lymphocyte surface determinants (CD4, CD8, CD 2). Animals in the control group received human immunoglobulin intravenously ("Immunovenin", Microgen, russia).

About rhesus monkey which varies with the product dose (Macaca mulatta) Comparative study of the percentage (%) of TRBV9+ T cells in peripheral blood, MA-042 was administered to 2 experimental groups at doses of 1 and 10 mg per animal, respectively. "Immunovenin" was diluted with sterile water according to the instructions and administered at a concentration of 10 mg per animal. The MA-042 product was diluted with calcium and magnesium free Dulbecco's Phosphate Buffered Saline (DPBS). The product is administered to the right in a volume of no more than 5 ml per injectionIn the elbow vein of the forelimb.

The observation period lasted 42 days. Blood samples were selected as indicated in table 3. Immunophenotyping and analysis of samples were performed as described above.

Table 3 whole blood sampling protocol for determining the percentage (%) of TRBV9+ in T cells.

Week (week)	Sky	Hour(s)	Remarks for note
				1	1	0	Background (before first application)
1	4	72	72 hours after application
				1	7	144	144 hours after application
3	15	336	336 hours after application
				4	25	576	576 hours after administration
6	42	984	984 hours after application

As a result, animals showed almost complete elimination of TRBV9+ in peripheral blood at both concentrations 72 hours after MA-042 administration. At a concentration of 1 mg/animal, a fraction of TRBV9+ lymphocytes was detected 336 hours after product administration. In animals receiving a 10 mg dose of product, no TRBV9+ lymphocytes were detected. No cases of TRBV9+ lymphocytes were detected in the experimental group at 42 days post-administration; the control group showed the same level of TRBV9+ lymphocytes as before the experiment.

Example 8 preparation of pharmaceutical compositions comprising the antibodies of the invention

The pharmaceutical compositions are obtained by standard techniques known in the art.

The components of the pharmaceutical composition are shown in table 4.

Table 4. concentration of components of the pharmaceutical composition.

Components	Concentration of
		Antibody M-042	10-50 mg/ml
10 mM citrate buffer to pH	6.0-7.0
		Sodium chloride	50-150 mM
Sucrose and trehalose	0.3-0.5%
		Water for injection	Make up to 1 ml.

Example 9 kit comprising a pharmaceutical composition comprising an antibody

To produce a kit containing dosage forms comprising the antibody MA-042 composition, the pharmaceutical composition prepared according to example 5 is sealed under sterile conditions in 1 ml ampoules or syringes, labeled, and packaged into plastic or cardboard containers.

Also, an insert is included in the ampoule.

<110> Betaka-Okade Union-stockings

<120> monoclonal antibody against beta chain region of human TRBV9

<130> MA043.docx

<160> 22

<170> PatentIn 3.5 edition

<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 parent 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 parent 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 variable domain of 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 the 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 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 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 light chain of parent 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 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 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 heavy chain of parent 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 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 the 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 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 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 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 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> 21

<211> 642

<212> DNA

<213> Artificial

<220>

<223> nucleotide sequence of 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 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

41页详细技术资料下载

上一篇：一种医用注射器针头装配设备

下一篇：癌症靶向的、病毒编码的、可调节的T细胞(CATVERT)或NK细胞(CATVERN)接头

Monoclonal antibodies directed against the beta chain region of human TRBV9

相关技术

网友询问留言