Monoclonal antibody that specifically binds to CD20

Abstract

The present invention relates to biotechnology and provides monoclonal antibodies that specifically bind to CD20. The present invention also relates to DNA encoding said antibodies, corresponding expression vectors, and methods for their production, as well as methods of treatment using said antibodies.

Description

[Technical field]

[0001] The present invention relates to the field of biotechnology, in particular to an anti-CD20 antibody or an antigen-binding fragment thereof, and the use of said antibody or fragment for the treatment of diseases associated with B cells. More specifically, the present invention relates to a monoclonal antibody that specifically binds to CD20 (B lymphocyte antigen CD20). The present invention also relates to a nucleic acid encoding said antibody or an antigen-binding fragment thereof, an expression vector, a method for producing the antibody, and the use of the antibody for the treatment of diseases or disorders associated with B cells, in particular with the B lymphocyte antigen CD20. [Background technology]

[0002] Lymphocytes are one of the many types of white blood cells; lymphocytes specifically recognize and respond to foreign antigens. The three main types of lymphocytes are B lymphocytes (B cells), T lymphocytes (T cells), and natural killer (NK) cells. B lymphocytes are cells involved in antibody production and humoral immunity. B cells mature in the bone marrow and leave the bone marrow expressing antigen-bound antibodies on the cell surface. When a previously unexposed B cell first encounters an antigen for which the membrane-bound antibody is specific, the cell begins to divide rapidly, and its progeny differentiate into memory B cells and effector cells called "plasma cells." Memory B cells have a longer life span and continue to express membrane-bound antibodies with the same specificity as the original parent cell. Plasma cells do not produce membrane-bound antibodies, but instead produce secretable forms of antibodies. Secreted antibodies are the primary effector molecules of humoral immunity.

[0003] The antigen CD20 (also called human B-lymphocyte-restricted differentiation antigen, Bp35) is a hydrophobic transmembrane protein with a molecular weight of approximately 35 kDa located on pre-B and mature B-lymphocytes (Valentine et al., J. Biol. Chem. 264(19), 1989, p.11282-11287; and Einfeld et al., EMBO J. 7(3), 1988, p.711-717). The antigen is also expressed on the cell surface of more than 90% of B cells in non-Hodgkin's lymphoma (HXJI) (Anderson et al., Blood 63(6), 1984, p.1424-1433), but has not been detected on hematopoietic stem cells, pro-B cells, normal plasma cells or other normal tissues (Tedder et al., J. Immunol. 135(2), 1985, p.973-979). CD20 appears to regulate an early step(s) in the activation process of cell cycle initiation and differentiation (Tedder et al., supra), and possibly functions as a calcium ion channel (Tedder et al. J. Cell. Biochem. 14D, 1990, p.195).

[0004] Given that CD20 is expressed in B-cell lymphomas, this antigen may be a valuable therapeutic target in the treatment of said lymphomas. The antibody rituximab (RITUXAN, MabThera, Acerbia) is a genetically engineered chimeric murine / human monoclonal antibody against the human antigen CD20 and is indicated for the treatment of patients with relapsed or refractory low-grade or follicular CD20-positive B-cell non-Hodgkin's lymphoma. Rituximab is an antibody designated "C2B8" in US 5,736,137 and US 5,776,456. In vitro mode of action studies have shown that rituximab binds human complement and lyses B-lymphocyte lineages through complement-dependent cytotoxicity (CDC) (Reff et al., Blood 83(2), 1994, p.435-445). Furthermore, the antibody has significant activity in antibody-dependent cellular cytotoxicity (ADCC) assays. In vivo preclinical studies have shown that rituximab depletes B cells from the peripheral blood, lymph nodes and bone marrow of cynomolgus monkeys (Macaca fascicularis), likely through complement and cell-mediated processes (Reff et al., Blood 83(2), 1994, p.435-445).

[0005] Furthermore, anti-CD20 antibodies, such as rituximab, were later found to be effective therapeutic agents in the treatment of various autoimmune diseases, such as rheumatoid arthritis (patents, RU2358762, RU2489166 and RU2457860) or Wegener's granulomatosis (patent, RU2326127).

[0006] A major limitation of the use of murine antibodies in human therapy is the formation of human anti-mouse antibodies (HAMA) (see, for example, Miller RA et al., "Monoclonal antibody therapeutic trials in seven patients with T-cell lymphoma," Blood, 62, 1983, p.988-995; and Schroff RW et al., "Human anti-murine immunoglobulin response in patients receiving monoclonal antibody therapy," Cancer Res., 45, 1985, p.879-885). Even chimeric molecules in which the variable (V) domain of a rodent antibody is fused to a human constant (C) region can still elicit a significant immune response (HACA, human anti-chimeric antibody response) (Neuberger et al., Nature (London), 314, 1985, p.268-270).

[0007] A powerful approach to overcome these limitations in the clinical use of monoclonal antibodies is the "humanization" of murine antibodies or antibodies derived from non-human species (Jones et al., Nature (London), 321, 1986, p. 522-525; Riechman et al., Nature (London), 332, 1988, p. 323-327). [Prior art documents] [Patent documents]

[0008] [Patent Document 1] US 5,736,137 [Patent Document 2] US 5,776,456 [Patent Document 3] RU2358762 [Patent Document 4] RU2489166 [Patent Document 5] RU2457860 [Patent Document 6] RU2326127

Non-licensed literature

[0009] [Non-licensed document 1] Valentine, J. Biol. Chem. 264(19), 1989, p.11282-11287 [Non-licensed document 2] Einfeld, EMBO J. 7(3), 1988, p.711-717 [Non-licensed document 3] Andersonら, Blood 63(6), 1984, p.1424-1433

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[0010] It would therefore be beneficial to produce therapeutic antibodies against the CD20 antigen that would produce minimal or no antigenicity when administered to a patient and that are intended primarily for use in long-term therapy. The present invention solves this problem. [Means for solving the problem]

[0011] Monoclonal antibody BCD-132 selectively and specifically binds to the CD20 antigen and is a potent inhibitor of the CD20 antigen; also, the antibodies of the invention have minimal antigenicity when administered to a patient.

[0012] BRIEF SUMMARY OF THE INVETION In one aspect, the present invention provides an antibody that specifically binds CD20 and is 1) a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:6; 2) A light chain variable domain comprising the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 8. The present invention relates to a monoclonal antibody or an antigen-binding fragment thereof, comprising:

[0013] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof is: 1) a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:2; 2) A light chain variable domain comprising the amino acid sequence shown in SEQ ID NO:4. Includes.

[0014] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof is: 1) a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:6; 2) A light chain variable domain comprising the amino acid sequence shown in SEQ ID NO:8. Includes.

[0015] In some embodiments, the monoclonal antibody is: 1) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:5; 2) A light chain comprising the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 7. Includes.

[0016] In some embodiments, the monoclonal antibody is: 1) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:1; 2) A light chain comprising the amino acid sequence shown in SEQ ID NO:3. Includes.

[0017] In some embodiments, the monoclonal antibody is: 1) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:5; 2) A light chain comprising the amino acid sequence shown in SEQ ID NO:7. Includes.

[0018] In some embodiments, the monoclonal antibody that specifically binds to CD20 is a full-length IgG antibody. In some embodiments, the monoclonal antibodies are of the human IgG1, IgG2, IgG3, IgG4 isotype.

[0019] In some embodiments, the monoclonal antibodies are of the human IgG1 isotype. In one aspect, the invention relates to a nucleic acid encoding the above-mentioned antibody.

[0020] In some embodiments, the nucleic acid is DNA. In one aspect, the present invention relates to an expression vector comprising the above-mentioned nucleic acid. In one aspect, the invention relates to a method for the production of a host cell for producing said antibody, comprising transformation of said cell with said vector.

[0021] In one aspect, the invention relates to a host cell for the production of the above-mentioned antibody, said host cell comprising the above-mentioned nucleic acid. In one aspect, the invention relates to a method for the production of said antibodies, comprising culturing said host cells in a culture medium under conditions sufficient to produce said antibody, and, if necessary, thereafter isolating and purifying the obtained antibody.

[0022] In one aspect, the invention relates to a pharmaceutical composition for use in the treatment of a disease or disorder mediated by CD20, comprising a therapeutically effective amount of the antibody or antigen-binding fragment thereof in combination with one or more pharma- ceutically acceptable excipients.

[0023] In some embodiments, the pharmaceutical composition is intended for use for the treatment of a disease or disorder, wherein the disease or disorder is: a) a neoplastic disease or disorder; or b) an autoimmune disease or disorder is selected from.

[0024] In some embodiments, the pharmaceutical composition is intended to be used for the treatment of a neoplastic disease or disorder selected from the group comprising: B-cell lymphoma or leukemia. In some embodiments, the pharmaceutical composition is intended for the treatment of a B-cell lymphoma selected from: non-Hodgkin's lymphoma (NHL) or Hodgkin's disease (Hodgkin's lymphoma).

[0025] In some embodiments, the pharmaceutical composition is intended for the treatment of a leukemia selected from: chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL). In some embodiments, the pharmaceutical composition is intended for the treatment of an autoimmune disease or disorder selected from the group comprising: rheumatoid arthritis, juvenile rheumatoid arthritis (Still's disease), systemic lupus erythematosus (SLE), lupus nephritis, ulcerative colitis, Wegener's disease, inflammatory bowel disease, idiopathic thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura (TTP), autoimmune thrombocytopenia, multiple sclerosis, psoriasis, IgA nephropathy, IgM polyneuropathy, myasthenia gravis, vasculitis, ANCA vasculitis, parenchymal organ transplant rejection, graft versus host disease (GvHD), diabetes, Raynaud's syndrome, Sjogren's syndrome, and glomerulonephritis.

[0026] In one aspect, the present invention relates to a pharmaceutical combination for the prevention or treatment of a disease or disorder mediated by CD20, comprising said antibody or antigen-binding fragment thereof and at least one therapeutically active compound.

[0027] In some embodiments, the pharmaceutical combination comprises different therapeutically active anti-tumor compounds selected from a chemotherapeutic agent, an antibody, or an anti-hormonal agent. In one aspect, the invention relates to a method for inhibiting the biological activity of CD20 in a subject in need of such inhibition, comprising administration of an effective amount of said antibody or antigen-binding fragment thereof.

[0028] In one aspect, the invention relates to a method for the treatment of a disease or disorder mediated by CD20, comprising administration of a therapeutically effective amount of said antibody or antigen-binding fragment thereof, or said pharmaceutical composition to a subject in need of such treatment.

[0029] In some embodiments, a method for treating a disease or disorder comprises: a) a neoplastic disease or disorder; or b) an autoimmune disease or disorder The present invention relates to a disease or disorder selected from the group consisting of

[0030] In some embodiments, the method for the treatment of a disease or disorder relates to a neoplastic disease or disorder selected from the group comprising: B-cell lymphoma or leukemia. In some embodiments, the methods for the treatment of a disease or disorder relate to a B-cell lymphoma selected from: non-Hodgkin's lymphoma (NHL) or Hodgkin's disease (Hodgkin's lymphoma).

[0031] In some embodiments, the methods for treatment of a disease or disorder relate to leukemia selected from: chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL). In some embodiments, the method for the treatment of a disease or disorder relates to an autoimmune disease or disorder selected from the group comprising: rheumatoid arthritis, juvenile rheumatoid arthritis (Still's disease), systemic lupus erythematosus (SLE), lupus nephritis, ulcerative colitis, Wegener's disease, inflammatory bowel disease, idiopathic thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura (TTP), autoimmune thrombocytopenia, multiple sclerosis, psoriasis, IgA nephropathy, IgM polyneuropathy, myasthenia gravis, vasculitis, ANCA vasculitis, parenchymal organ graft rejection, graft versus host disease (GvHD), diabetes, Raynaud's syndrome, Sjogren's syndrome, and glomerulonephritis.

[0032] In one aspect, the invention relates to the use of said antibody or antigen-binding fragment thereof, or said pharmaceutical composition, for the treatment of a disease or disorder mediated by CD20 in a subject in need of such treatment.

[0033] In some embodiments, the antibody or antigen-binding fragment thereof is used for the treatment of a disease or disorder, wherein the disease or disorder is: a) a neoplastic disease or disorder; or b) an autoimmune disease or disorder is selected from.

[0034] In some embodiments, the antibody or antigen-binding fragment thereof is used for the treatment of a neoplastic disease or disorder selected from the group including: B-cell lymphoma or leukemia. In some embodiments, the antibody or antigen-binding fragment thereof is used for the treatment of a B-cell lymphoma selected from: non-Hodgkin's lymphoma (NHL) or Hodgkin's disease (Hodgkin's lymphoma).

[0035] In some embodiments, the antibody or antigen-binding fragment thereof is used for the treatment of a leukemia selected from: chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL).

[0036] In some embodiments, the antibody or antigen-binding fragment thereof is used for the treatment of an autoimmune disease or disorder selected from the group including: rheumatoid arthritis, juvenile rheumatoid arthritis (Still's disease), systemic lupus erythematosus (SLE), lupus nephritis, ulcerative colitis, Wegener's disease, inflammatory bowel disease, idiopathic thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura (TTP), autoimmune thrombocytopenia, multiple sclerosis, psoriasis, IgA nephropathy, IgM polyneuropathy, myasthenia gravis, vasculitis, ANCA vasculitis, parenchymal organ transplant rejection, graft versus host disease (GvHD), diabetes, Raynaud's syndrome, Sjogren's syndrome, and glomerulonephritis. [Brief description of the drawings]

[0037] [Figure 1] Map of the expression vector pEE CK. [Diagram 2] Map of the expression vector pEE HC. [Diagram 3] Schematic diagram of the IgG1 format. [Figure 4] Schematic of the synthesis of human naive combinatorial libraries. [Diagram 5] Map of the phagemid for cloning the Fab phage display library. [Figure 6] Map of the expression plasmid pLL for culturing Fab. [Figure 7] Analysis of BCD132L candidate-biotinylated CD20 peptide interactions on Forte Bio Octet RED 386. [Figure 8] Analysis of BCD132L-026 candidate-biotinylated CD20 peptide interactions on Forte Bio Octet RED 386. [Figure 9] Analysis of BCD132L-028 candidate-biotinylated CD20 peptide interactions on Forte Bio Octet RED 386. [Figure 10] Analysis of BCD132L-075 candidate-biotinylated CD20 peptide interactions on Forte Bio Octet RED 386. [Figure 11] Analysis of BCD132L-077 candidate-biotinylated CD20 peptide interactions on Forte Bio Octet RED 386. [Figure 12] Analysis of BCD132L-028 candidate-biotinylated CD20 peptide interactions on Forte Bio Octet RED 386. [Figure 13] Analysis of BCD132L-077(1,2) candidate-biotinylated CD20 peptide interactions on Forte Bio Octet RED 386. [Figure 14] Analysis of BCD132L-077(3,4) candidate-biotinylated CD20 peptide interactions on Forte Bio Octet RED 386. [Figure 15] Analysis of BCD132L-077(5,6) candidate-biotinylated CD20 peptide interactions on Forte Bio Octet RED 386. [Figure 16] Analysis of candidate BCD132L-028-FcγRIIIa-158F interactions on Forte Bio Octet RED 386. [Figure 17] Analysis of candidate BCD132L-077(1,2)-FcγRIIIa-158F interactions on Forte Bio Octet RED 386. [Figure 18] Analysis of candidate BCD132L-077(3,4)-FcγRIIIa-158F interactions on Forte Bio Octet RED 386. [Figure 19]Analysis of candidate BCD132L-077(5,6)-FcγRIIIa-158F interactions on Forte Bio Octet RED 386. [Figure 20] Analysis of candidate BCD132L-028-FcγRIIIa-158V interactions on Forte Bio Octet RED 386. [Figure 21] Analysis of candidate BCD132L-077(1,2)-FcγRIIIa-158V interactions on Forte Bio Octet RED 386. [Figure 22] Analysis of candidate BCD132L-077(3,4)-FcγRIIIa-158V interactions on Forte Bio Octet RED 386. [Diagram 23] Analysis of candidate BCD132L-077(5,6)-FcγRIIIa-158V interactions on Forte Bio Octet RED 386. [Figure 24] Map of the expression vector pSX. [Diagram 25] Specific binding of BCD-132-L-028 and BCD-132-L-077 to the CD20 receptor on the WIL2-S cell line measured by flow cytometry in comparison to MabThera. [Figure 26] Measurement of complement-dependent cytotoxicity of BCD-132-L-028 and BCD-132-L-077 in comparison with MabThera. BCD-132-L-028 ● MabThera ■ BCD-132-L-028 ▼ BCD-132-L-077. [Figure 27] Measurement of complement-dependent cytotoxicity of BCD-132-L-028 and BCD-132-L-077 in comparison with MabThera. BCD-132-L-077 ● MabThera ■ BCD-132-L-028 ▼ BCD-132-L-077. [Figure 28]Antibody-dependent cellular cytotoxicity measurements of BCD-132-L-028 and BCD-132-L-077 compared to MabThera. Low affinity CD16 reporter cell line, target WIL2-S cell line. BCD-132-L-028 ● MabThera ■ BCD-132-L-028 ▼ BCD-132-L-077. [Figure 29] Antibody-dependent cellular cytotoxicity measurements of BCD-132-L-028 and BCD-132-L-077 compared to MabThera. Low affinity CD16 reporter cell line, target WIL2-S cell line. BCD-132-L-077 ● MabThera ■ BCD-132-L-028 ▼ BCD-132-L-077. [Diagram 30] Antibody-dependent cellular cytotoxicity measurements of BCD-132-L-028 and BCD-132-L-077 compared to MabThera. High affinity CD16 reporter cell line, target WIL2-S cell line. BCD-132-L-028 ● MabThera ■ BCD-132-L-028 ▼ BCD-132-L-077. [Diagram 31] Antibody-dependent cellular cytotoxicity measurements of BCD-132-L-028 and BCD-132-L-077 compared to MabThera. High affinity CD16 reporter cell line, target WIL2-S cell line. BCD-132-L-077 ● MabThera ■ BCD-132-L-028 ▼ BCD-132-L-077. [Diagram 32] Measurement of CD19+ B cell depletion induced by antibodies BCD-132-L-028 and BCD-132-L-077 compared to MabThera in whole blood from healthy donors of FF (A), FV (B) and VV (C) allotypes. [Diagram 33] Measurement of antibody-dependent cellular cytotoxicity (ADCC) of BCD-132-L-028 and BCD-132-L-077 compared to the commercially available antibody, rituximab. [Diagram 34] Inflammatory response severity (BCD132-L-077). [Diagram 35]Demyelination severity (BCD132-L-077). [Diagram 36] Smoothed concentration curves for primate serum under repeated intravenous administration of BCD132-L-077 product at doses of 22.0 mg / kg, 44.0 mg / kg, and 88.0 mg / kg. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0038] Definitions and general methods Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0039] Further, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. Typically, the classification and methods of cell culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, medical and pharmaceutical chemistry, and protein and nucleic acid hybridization and chemistry described herein are well known and widely used by those of skill in the art. Enzymatic reactions and purification methods are performed according to manufacturer's instructions as common in the art or as described herein.

[0040] Antibody Definitions CD20, or B-lymphocyte antigen CD20, is a protein coreceptor found on the surface of B-lymphocytes. CD20 is the product of the human MS4A1 gene. The exact function of this protein is still unknown, but it is believed to play a role in the activation and proliferation of B-lymphocytes.

[0041] The MS4A1 gene is a member of the MS4A (transmembrane 4A) gene family, which consists of at least 25 other genes. The genes of this family are clustered at the human chromosomal locus 11q12-13. The corresponding proteins appear to have a similar spatial structure: they have a four-transmembrane topology with N- and C-terminal cytoplasmic domains (JANAS E. et al., Functional role of lipid rafts in CD20 activity?, Biochem Soc Symp. 2005;(72):165-75).

[0042] Amplification of this gene and / or overexpression of its protein has been observed in many cancers or autoimmune diseases, including in: a) neoplastic diseases or disorders from the group including non-Hodgkin's lymphoma (NHL), Hodgkin's disease (Hodgkin's lymphoma), chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL), b) Autoimmune diseases or disorders from the group including rheumatoid arthritis, juvenile rheumatoid arthritis (Still's disease), systemic lupus erythematosus (SLE), lupus nephritis, ulcerative colitis, Wegener's disease, inflammatory bowel disease, idiopathic thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura (TTP), autoimmune thrombocytopenia, multiple sclerosis, psoriasis, IgA nephropathy, IgM polyneuropathy, myasthenia gravis, vasculitis, ANCA vasculitis, parenchymal organ graft rejection, graft versus host disease (GvHD), diabetes mellitus, Raynaud's syndrome, Sjogren's syndrome and glomerulonephritis.

[0043] The term "binding molecule" includes antibodies and immunoglobulins. The term "antibody" or "immunoglobulin" (Ig), as used herein, includes complete antibodies and any antigen-binding fragment (i.e., "antigen-binding portion") or single chain. The term "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or antigen-binding portion. Each heavy chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Five types of mammalian Ig heavy chains are known, designated by the Greek letters: α, δ, ε, γ, and μ. The type of heavy chain defines the antibody class: these chains are found in IgA, IgD, IgE, IgG, and IgM antibodies, respectively. Different heavy chains differ in size and composition; α and γ contain approximately 450 amino acids, while μ and ε have approximately 550 amino acids. Each heavy chain has two regions, the constant region and the variable region. The constant region is identical in all antibodies of the same isotype, but differs in antibodies of different isotypes. Heavy chains gamma, alpha and delta have a constant region, composed of three constant domains CH1, CH2 and CH3 (in line), as well as a hinge region to add flexibility (Woof J., Burton D., Nat Rev Immunol 4, 2004, cc.89-99); heavy chains μ and ε have a constant region composed of four constant domains, CH1, CH2, CH3 and CH4. In mammals, only two types of light chains are known, designated lambda (λ) and kappa (κ). Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The approximate length of the light chain is 211-217 amino acids. Preferably, the light chain is a kappa (κ) light chain, and the constant domain CL is preferably C kappa (κ).

[0044] An "antibody" according to the present invention may be of any class (e.g., IgA, IgD, IgE, IgG, and IgM, preferably IgG) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2, preferably IgG1).

[0045] The VL and VH regions may be further divided into hypervariable regions, termed complementarity determining regions (CDRs), interspersed between more conserved regions, termed framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0046] The term "antigen-binding portion" or "antigen-binding fragment" of an antibody (or simply "antibody portion" or "antibody fragment"), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment consisting of the VH / VHH domains (Ward et al. (1989) Nature 341:544-546); and (vi) extracted complementarity determining regions (CDRs). Furthermore, the two regions of the Fv fragment, VL and VH, are encoded by separate genes and can be linked using recombinant methods with a synthetic linker that allows them to accept a single protein chain, where the VL and VH regions pair to form a monovalent molecule (known as single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain molecules are also assumed to be included within the term "antigen-binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened in the same manner as intact antibodies.

[0047] Preferably, the CDRs of the antigen-binding moieties or full antibody antigen-binding moieties of the invention are derived from mouse, llama or human donor libraries or are substantially of human origin, and specific amino acid residues have been modified, e.g. replaced with different amino acid residues, to optimize certain antibody properties, e.g. KD, koff, IC50, EC50, ED50. Preferably, the framework regions of the antibodies of the invention are of human origin or substantially of human origin (at least 80, 85, 90, 95, 96, 97, 98 or 99% human origin).

[0048] In other embodiments, the antigen-binding portions of the invention may be derived from other non-human species, including, but not limited to, mouse, llama, rabbit, rat or hamster. Alternatively, the antigen-binding region may be derived from a human species.

[0049] The term "variable domain" refers to the fact that certain portions of the variable domains are highly variable in sequence among antibodies. The V domains mediate antigen binding and determine the specificity of each particular antibody for its particular antigen. However, the variability is not uniformly distributed across the 110 amino acid span of the variable domain. Instead, the V region consists of invariant segments of 15-30 amino acids called framework regions (FRs) separated by shorter regions of hypervariability called "hypervariable regions" or CDRs. Naturally occurring heavy and light chain variable domains each contain four FRs, which adopt a predominantly beta-sheet configuration and are linked by three hypervariable regions that form loops that connect, and in some cases form part of, the beta-sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, together with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of the antibody. The constant domains are not directly involved in the binding of the antibody to the antigen, but exhibit various effector functions, such as the participation of the antibody in antibody-dependent cellular cytotoxicity (ADCC).

[0050] The term "hypervariable region" as used herein refers to the amino acid residues of an antibody which are involved in antigen binding. Hypervariable regions generally comprise amino acid residues from the "complementarity determining regions" or "CDRs" and / or residues from the "hypervariable loops".

[0051] In certain cases, it may also be desirable to modify one or more CDR amino acid residues to improve binding affinity to the target epitope. This is known as "affinity maturation" and may be carried out, optionally, in conjunction with humanization, for example, in situations where antibody humanization leads to a decrease in binding specificity or affinity and back mutation alone is not sufficient to improve binding specificity or affinity. A variety of affinity maturation methods are known in the art, such as the in vitro scanning saturation mutagenesis method described by Burks et al., Proc Natl Acad Sci USA, 94:412-417 (1997), and the stepwise in vitro affinity maturation method by Wu et al., Proc Natl Acad Sci USA 95:6037 6042 (1998).

[0052] "Framework Regions" (FR) are those residues of the variable domains that are different from the CDR residues. Each variable domain typically has four FRs identified as FR1, FR2, FR3 and FR4. When the CDRs are defined according to Kabat, the light chain FR residues are located at approximately residues 1-23 (LCFR1), 35-49 (LCFR2), 57-88 (LCFR3), and 98-107 (LCFR4), and the heavy chain FR residues are located at approximately residues 1-30 (HCFR1), 36-49 (HCFR2), 66-94 (HCFR3), and 103-113 (HCFR4) in the heavy chain. When a CDR comprises amino acid residues from a hypervariable loop, the light chain FR residues are located at approximately residues 1-25 (LCFR1), 33-49 (LCFR2), 53-90 (LCFR3), and 97-107 (LCFR4) in the light chain, and the heavy chain FR residues are located at approximately residues 1-25 (HCFR1), 33-52 (HCFR2), 56-95 (HCFR3), and 102-113 (HCFR4) in the heavy chain. In some cases where a CDR comprises amino acids from both a CDR and one from a hypervariable loop as defined by Kabat, the FR residues will be adjusted accordingly. For example, if CDRH1 comprises amino acids H26-H35, the heavy chain FR1 residues are at positions 1-25 and the FR2 residues are at positions 36-49.

[0053] The fragment crystallizable region of an immunoglobulin ("Fc region, Fc") is the "tail" region of an immunoglobulin that interacts with cell surface Fc receptors, as well as with some proteins of the complement system. This property allows the antibody to activate the immune system. In IgG, IgA and IgD antibody isotypes, the Fc region is composed of two identical protein fragments, derived from the second and third constant domains of the two heavy chains, respectively; in IgM and IgE isotypes, the Fc region contains three heavy chain constant domains (CH domains 2-4) in each polypeptide chain.

[0054] The antibody of the present invention "binds" to a target antigen refers to an antibody that can bind to the antigen with sufficient affinity so that it can be used as a diagnostic and / or therapeutic agent to target proteins or cells expressing said antigen, and that only cross-reacts to a small extent with other proteins. In such embodiments, the degree of antibody binding to non-target proteins is less than 10% of that of antibody binding to a specific target protein, according to analytical methods: fluorescence activated cell sorting (FACS), radioimmunoassay (RIA) or ELISA. With respect to the binding of an antibody to a target molecule, the term "specific binding" or "specifically binds" or "is specific" to a particular polypeptide or epitope on a particular polypeptide target means binding that is significantly (measurably) different from non-specific interactions (e.g., in the case of bH1-44 or bH1-81, non-specific interactions are binding to bovine serum albumin, casein, fetal bovine serum, or neutravidin).

[0055] For example, specific binding may be measured by determining the binding of a molecule compared to the binding of a control molecule. For example, specific binding may be determined by competition with a control molecule similar to the target, such as an excess of unlabeled target. In this case, specific binding is indicated if the binding of the labeled target to the probe is competitively inhibited by an excess of unlabeled target. As used herein, the term "specific binding" or "specifically binds" or "is specific" for a particular polypeptide or epitope on a particular polypeptide target can be described by a molecule having a Kd for the target of at least about 200nM, or at least about 150nM, or at least about 100nM, or at least about 60nM, or at least about 50nM, or at least about 40nM, or at least about 30nM, or at least about 20nM, or at least about 10nM, or at least about 8nM, or at least about 6nM, or at least about 4nM, or at least about 2nM, or at least about 1nM, or more. In one embodiment, the term "specific binding" refers to binding where a molecule binds to a particular polypeptide or an epitope on a particular polypeptide without substantial binding to any other polypeptides or polypeptide epitopes.

[0056] The term "Ka," as used herein, refers to the on-rate of a particular antibody-antigen interaction. The term "Kd," as used herein, refers to the dissociation (off) rate of a particular antibody-antigen interaction.

[0057] "Binding affinity" generally refers to the total strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, "binding affinity" refers to the intrinsic (characteristic, true) binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its binding partner Y can generally be represented by the dissociation constant (Kd). Preferred Kd values ​​are about 200nM, 150nM, 100nM, 60nM, 50nM, 40nM, 30nM, 20nM, 10nM, 8nM, 6nM, 4nM, 2nM, 1nM, or less. Affinity may be measured by common methods known in the art, including those described herein. Low affinity antibodies generally bind antigens slowly and tend to dissociate easily, whereas high affinity antibodies generally bind antigens more quickly and tend to remain bound longer. A variety of methods for measuring binding affinity are known in the art, and any of these methods may be used for the purposes of the present invention.

[0058] In one embodiment, "Kd" or "Kd value" is measured at 25°C, ∼10 response units (RU), with immobilized antigen CM5 chip, using BIAcore TMThe binding activity is measured by using a surface plasmon resonance assay using a BIAcore®-2000 or BIAcore®-3000 (BIAcore, Inc., Piscataway, NJ). Briefly, a carboxymethylated dextran biosensor chip (CM5, BIAcore, Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the manufacturer's instructions. The antigen is diluted to 5 μg / ml (~0.2 μM) in 10 mM sodium acetate, pH 4.8, and then injected at a flow rate of 5 μl / min to achieve approximately 10 relative units (RU) of bound protein. After antigen administration, 1 M ethanolamine solution is administered to block unreacted groups. For kinetic measurements, twofold serial dilutions of Fab (e.g., 0.78 nM to 500 nM) are injected in PBS containing 0.05% Tween 20 (PBST) at 25° C. with a flow rate of approximately 25 μl / min. On-rates (kon) and off-rates (koff) are calculated using a simple one-to-one Langmuir binding model (BIAcore evaluation software version 3.2) by simultaneously fitting the association and dissociation sensorgrams. Equilibrium dissociation constants (Kd) are calculated as the ratio koff / kon. See, e.g., Chen, Y. et al. (1999) J. Mol. Biol. 293:865-881. The surface plasmon resonance assay described above determined that the on-rate was 10 6 M -1 s -1 above 100 nm, the on-rate may be determined by using a fluorescence quenching technique measuring the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nM, 16 nm bandpass) of a 20 nM anti-antigen antibody solution (Fab form) in PBS, pH 7.2 at 25°C in the presence of increasing concentrations of antigen as measured in a spectrophotometer, e.g., a stop-flow eqipped spectrophotometer (Aviv Instruments) or an 8000 series SLM-Aminco spectrophotometer with stirred cuvette (ThermoSpectronic).

[0059] The term "koff" refers to the off-rate constant of a particular interaction between a binding molecule and an antigen. TM The system may be used to measure the off-rate constant, koff, using biolayer interferometry.

[0060] In addition, BIAcore was performed using immobilized antigen CM5 chips at 25°C with ~10 relative units (response units, RU). TM The "on-rate" or "kon" according to the present invention may be measured by using the above-mentioned surface plasmon resonance assay using a BIAcore®-2000 or BIAcore®-3000 (BIAcore, Inc., Piscataway, NJ). Briefly, a carboxymethylated dextran biosensor chip (CM5, BIAcore, Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the manufacturer's instructions. Antigen is diluted to 5 μg / ml (~0.2 μM) in 10 mM sodium acetate, pH 4.8, and then injected at a flow rate of 5 μl / min to achieve approximately 10 response units (RU) of bound protein. After antigen administration, 1 M ethanolamine solution is administered to block unreacted groups.

[0061] Unless otherwise specified, the terms "biologically active" and "biological activity" and "biological properties" with respect to the polypeptides of the present invention mean having the ability to bind to biological molecules.

[0062] The term "biological molecule" refers to nucleic acids, proteins, carbohydrates, lipids, and combinations thereof. In one embodiment, the biological molecule is naturally occurring. Antibody fragments, such as Fab and F(ab')2 fragments, may be prepared from intact antibodies using conventional techniques, such as papain or pepsin digestion of intact antibodies. Additionally, antibodies, portions thereof, and immunoadhesion molecules may be prepared using standard recombinant DNA techniques, such as those described herein.

[0063] The term "recombinant antibody" is intended to refer to an antibody that is expressed in a cell or cell line that contains a nucleotide sequence(s) encoding an antibody, where said nucleotide sequence(s) is not naturally associated with the cell.

[0064] As used herein, the term "variant antibody" is intended to refer to an antibody having an amino acid sequence that differs from that of its "parent" antibody due to the addition, deletion and / or substitution of one or more amino acid residues when compared to the sequence of the parent antibody. In preferred embodiments, the variant antibody contains at least one or more (e.g., 1 to 12, e.g., 2, 3, 4, 5, 6, 7, 8 or 9, 10, 11 or 12; in some embodiments, the variant antibody contains from 1 to about 10) additions, deletions, and / or substitutions of amino acids when compared to the parent antibody. In some embodiments, such additions, deletions and / or substitutions are made in the CDRs of the variant antibody. Identity or homology with respect to the sequence of a variant antibody is defined herein as the percentage of amino acid residues in the variant antibody sequence that are identical to those of the parent antibody residues after aligning the sequences and introducing gaps if necessary to achieve the maximum percentage of sequence identity. A variant antibody retains the ability to bind to the same antigen, and preferably to the same epitope, as the parent antibody; and in some embodiments, at least one property or biological activity is superior to that of the parent antibody. For example, a variant antibody may have, for example, a stronger binding affinity, a longer half-life, a lower IC50, or an enhanced ability to inhibit antigen biological activity, as compared to the parent antibody. Variant antibodies of particular interest herein are those that exhibit at least a 2-fold (preferably at least a 5-fold, 10-fold, or 20-fold) enhanced biological activity as compared to the parent antibody.

[0065] The term "bispecific antibody" refers to an antibody having an antigen-binding domain or domains capable of specifically binding to two distinct epitopes on a single biological molecule or capable of specifically binding to epitopes on two separate biological molecules. Bispecific antibodies are also referred to herein as having "dual specificity" or as being "bispecific" antibodies.

[0066] In a broad sense, the term "chimeric antibody" refers to an antibody that contains one or more regions of one antibody and one or more regions of one or several other antibodies, typically an antibody that is partly human and partly non-human, i.e. partly derived from a non-human animal, e.g. mouse, rat or similar vermin, or from the Camelidae family, e.g. llamas and alpacas. Chimeric antibodies are generally preferred over non-human antibodies, because they reduce the risk of human anti-antibody immune reactions, e.g. human anti-mouse antibody immune reactions in the case of murine antibodies. A typical example of a chimeric antibody is one in which the variable region sequences are murine sequences, while the constant region sequences are human. In the case of chimeric antibodies, the non-human portions may be subjected to further modifications in order to humanize the antibody.

[0067] The term "humanized" refers to the fact that when an antibody has a completely or partially non-human origin, for example a mouse or llama antibody obtained by immunizing a mouse or llama with an antigen of interest, or a chimeric antibody based on such an antibody of a mouse or llama, it is possible to replace certain amino acids, especially those in the framework regions and constant domains of the heavy and light chains, in order to avoid or minimize immune reactions in humans. Antibodies interact with the target antigen mainly through amino acid residues located in the six heavy and light chain CDRs. For this reason, the amino acid sequences within the CDRs are much more diverse between individual antibodies than those outside the CDRs. Since the CDR sequences are involved in the majority of the antibody-antigen interaction, it is also possible to express recombinant antibodies that mimic the properties of a specific naturally occurring antibody, or more generally any specific antibody that contains said amino acid sequences, for example by constructing an expression vector that expresses the CDR sequences from a specific antibody and the framework sequences from a different antibody. As a result, it is possible to "humanize" a non-human antibody and retain to a large extent the binding specificity and affinity of the original antibody. Although it is not possible to accurately predict the immunogenicity, and therefore the human anti-antibody response, of a particular antibody, non-human antibodies are typically more immunogenic than human antibodies. Chimeric antibodies, in which foreign (e.g. vermin or camelid) constant regions are replaced by sequences of human origin, generally show less immunogenicity than antibodies of completely foreign origin, and in therapeutic antibodies there is a trend to use humanized or fully human antibodies. Chimeric or other antibodies of non-human origin may therefore be humanized to reduce the risk of human anti-antibody responses.

[0068] For chimeric antibodies, humanization typically involves modification of the framework regions of the variable region sequences. Amino acid residues that are part of the complementarity determining regions (CDRs) are not modified for humanization in most cases, but in some cases modification may be desirable to modify individual amino acid residues of the CDRs, for example to remove glycosylation sites, deamidation sites, aspartic acid isomerization sites, or to delete undesired cysteine ​​or methionine residues. N-linked glycosylation occurs by attaching an oligosaccharide chain to an asparagine residue in the tripeptide sequence Asn-X-Ser or Asn-X-Thr, where X may be any amino acid except Pro. Removal of N-glycosylation sites may be achieved by mutating either the Asn or Ser / Thr residue with a different residue, preferably by conservative substitution. Deamidation of asparagine and glutamine residues may occur depending on factors such as pH and surface exposure. Asparagine residues are particularly susceptible to deamidation, especially when present in Asn-Gly sequences, and to a lesser extent in other dipeptide sequences, such as Asn-Ala. If a CDR sequence contains such a deamidation site, especially Asn-Gly, it may be preferable to remove this site, typically by a conservative substitution that deletes one of the involved residues.

[0069] Many methods for humanizing antibody sequences are known in the art. One commonly used method is CDR grafting. CDR grafting may be based on the CDR definition by Kabat, although the final version (Magdelaine-Beuzelin et al., Crit Rev. Oncol Hematol. 64:210 225(2007)) suggests that the IMGT® (international ImMunoGeneTics information system®, www.imgt.org) definition may improve humanization results (see Lefranc et al., Dev. Comp Immunol. 27:55-77(2003)). In some cases, CDR grafting can reduce the binding specificity and affinity, and thus the biological activity, of the CDR-grafted non-human antibody when compared to the parent antibody from which the CDRs were obtained. To restore the binding specificity and affinity of the parent antibody, back mutations (sometimes referred to as "framework region restoration") may be introduced at selected positions of the CDR-grafted antibody, typically in the framework regions. Information available in the literature and antibody databases may be used to identify positions for possible back mutations. Candidate amino acid residues for back mutations are typically located on the surface of the antibody molecule, while buried or less surface-exposed residues will usually not be altered. An alternative humanization technique to CDR grafting and back mutation is resurfacing, in which surface residues are altered to human residues while non-surface-exposed residues of non-human origin are retained.

[0070] Two techniques may be used to generate fully human antibodies: using in vitro harvested phage libraries or by in vivo immunization of humanized animals (mice, rats, etc.).

[0071] The construction of a combinatorial phage antibody library begins with the selection of a gene repertoire source, according to which several antibody library types can be distinguished: naive, immune and synthetic. Naive and immune libraries are constructed using naturally rearranged genes encoding the variable immunoglobulin domains of healthy donors or donors immunized with a specific antigen, respectively. mRNA from an antibody-producing lymphoid cell line is isolated for this purpose. Peripheral blood lymphocytes are mainly used, but spleen cells [Sheets MD, Amersdorfer P, Finnern R, Sargent P, Lindquist E, Schier R, et al. Efficient construction of a large nonimmune phage antibody library: the production of high-affinity human single-chain antibodies to protein antigens. Proc Natl Acad Sci USA 1998,95:6157-6162 and de Haard HJ, van Neer N, Reurs A, Hufton SE, Roovers RC, Henderikx P, et al. A large non-immunized human Fab fragment phage library that permits rapid isolation and kinetic analysis of high affinity antibodies. J Biol Chem 1999,274:18218-18230.], tonsil cells or bone marrow lymphocytes [Vaughan TJ, Williams AJ, Pritchard K, Osbourn JK, Pope AR, Earnshaw JC et al. Human antibodies with sub-nanomolar affinities isolated from a large non-immunized phage display library. Nat Biotechnol 1996,14:309-314.] have also been used.cDNA can then be synthesized against the bases of the mRNA, and using both oligo-dT primers and statistically designed hexanucleotides, cDNA copies of all possible gene variants encoding the variable domains of the antibody can be generated [Ulitin AB, Kapralova MV, Laman AG, Shepelyakovskaya AO, Bulgakova EB, Fursova KK, et al. The library of human miniantibodies in the phage display format: Designing and testing DAN: Izd-vo "Nauka"; 2005.].

[0072] One or several primers may be used simultaneously to limit the range of genes amplified here at the cDNA level to one or several variable domain gene families or antibody isotypes [Marks JD, Hoogenboom HR, Bonnert TP, McCafferty J, Griffiths AD, Winter G. Bypassing immunization. Human antibodies from V-gene libraries displayed on phage. J Mol Biol 1991,222:581-597]. The primers used to amplify immunoglobulin-encoding genes are complementary to their most conserved regions. The sequences are selected from gene collections organized in databases such as the Kabat or V BASE databases. The primer design also provides internal restriction sites for cloning the PCR products into appropriate vectors.

[0073] The construction of synthetic libraries is based on the replacement of the natural CDRs with a set of random sequences, in which case it is possible to generate a large variety of antigen binding sites. Phage display is one of the most powerful and widely used in vitro antibody screening techniques. In 1985, Smith found that foreign DNA sequences could be cloned into the filamentous bacteriophage M13 and expressed on the phage particle surface as fusion proteins (Smith GP: Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 1985, 228:1315-1317.). Thus, fusion proteins of interest could be selected based on their ability to bind to other proteins. This discovery, combined with PCR amplification techniques, allowed the cDNA repertoire of immunoglobulin genes to be cloned to generate diverse phage libraries containing variable domains, which could be used to rapidly screen target-specific monoclonal antibodies. The phage library repertoire reflects the B cell antibody repertoire of each human or animal whose blood was used to generate the library. In 1995, two papers reported the generation of genetically engineered mice expressing a fully human antibody repertoire comparable to that produced by hybridoma technology (Lonberg N, Taylor LD, Harding FA, Trounstine M, Higgins KM, Schramm SR, Kuo CC, Mashayekh R, Wymore K, McCabe JG, et al.: Antigen-specific human antibodies from mice comprising four distinct genetic modifications. Nature 1994, 368:856-859). In these animals, their own endogenous heavy and kappa light immunoglobulin chain genes were purposefully disrupted and then transgenes that were segments of human heavy and kappa light chain genes were introduced.It has been found that the mouse immune system is capable of using the human gene repertoire to produce high-specificity and high-affinity antibodies against a greater variety of antigens. Despite the fact that the transgenic mice express B cell receptors that are essentially hybrids of mouse and human components (human immunoglobulins, mouse Igα, Igβ and other signaling molecules), their B cells develop and mature normally.

[0074] The term "monoclonal antibody" or "mAb" refers to an antibody synthesized and isolated by a distinct clonal population of cells. The clonal population may be a clonal population of immortalized cells. In some embodiments, the immortalized cells in the clonal population are hybrid cells, hybridomas, typically produced by fusion of individual B lymphocytes from an immunized animal with individual cells from a lymphocytic tumor. Hybridomas are an engineered type of cell and do not exist in nature.

[0075] A "native antibody" is usually a heterotetrameric glycoprotein of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced interchain disulfide bridges. Each heavy chain has a variable domain (VH) at one end followed by several constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at the other end. The constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Certain amino acid residues are believed to form an interface between the light and heavy chain variable domains.

[0076] The term "isolated" as used herein to describe various antibodies refers to an antibody that has been identified and separated and / or regenerated from the cell or cell culture in which it was expressed. Impurities (contaminating components) from the natural environment are substances that would interfere with diagnostic or therapeutic uses of the polypeptide and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In a preferred embodiment, the antibody is purified (1) to a sufficient degree to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequencer (Edman sequencer), or (2) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie Brilliant Blue or preferably silver stain. An isolated antibody includes an antibody in situ in a recombinant cell, since at least one component of the polypeptide's natural environment will not be present. An isolated polypeptide is typically prepared by at least one purification step.

[0077] An "isolated" nucleic acid molecule is one that is identified and separated from at least one nucleic acid molecule impurity associated with the antibody nucleic acid in its natural source. An isolated nucleic acid molecule is different from the type or set found under natural conditions. Thus, an isolated nucleic acid molecule is different from the nucleic acid molecule present in a cell under natural conditions. However, an isolated nucleic acid molecule includes the nucleic acid located in the cell in which the antibody is normally expressed, for example, when the nucleic acid molecule has a chromosomal location that is different from its location in the cell under natural conditions.

[0078] The term "epitope" is intended herein to refer to a portion (determinant) of an antigen that specifically binds to a binding molecule (e.g., an antibody or related molecule, e.g., a bispecific binding molecule). Epitope determinants usually consist of chemically active surface groups of a molecule, such as amino acids or carbohydrate or sugar side chains, and typically include specific three-dimensional structural characteristics, as well as specific charge characteristics. Epitopes may be either "linear" or "conformational". In a linear epitope, all of the points of interaction between a protein (e.g., an antigen) and an interacting molecule (e.g., an antibody) occur linearly along the primary amino acid sequence of the protein. In a conformational epitope, the points of interaction occur across amino acid residues on the protein that are separated from one another in the primary amino acid sequence. Once a desired epitope of an antigen has been determined, it is also possible to generate antibodies against this epitope, using techniques well known in the art. Furthermore, the generation and characterization of antibodies or other binding molecules may elucidate information about the desired epitope. From this information, it is then possible to competitively screen for antibodies that bind to the same or identical epitope, for example by performing competition studies to find binding molecules that compete with each other for binding to the antigen.

[0079] The term "peptide linker" is intended herein to mean any peptide capable of combining domains, having a length according to the domains to be linked together and containing any amino acid sequence. Preferably, the peptide linker has a length of more than 5 amino acids and consists of any set of amino acids selected from G, A, S, P, E, T, D, K.

[0080] The term "in vitro" refers to a biological substance, biological process, or biological reaction under artificial conditions, outside the body. For example, cells grown in vitro shall be understood as cells grown in an environment outside the body, such as in a test tube, culture vial, or microtiter plate.

[0081] The term “IC50 "(50% inhibitory concentration)" as used herein refers to the drug concentration that inhibits a measurable activity or response, e.g., the growth / proliferation of cells, such as tumor cells, by 50%. Using appropriate dose-response curves and specialized statistical software for curve fitting, the IC 50 The value may be calculated.

[0082] The term “IC 50 "(50% inhibitory concentration or half-maximal inhibitory concentration) refers to the drug concentration and indicates the volume of inhibitor required to inhibit a biological process by 50%. Using appropriate dose-response curves and special statistical software for curve fitting, the IC 50 The value may be calculated.

[0083] The term GI50 (50% growth inhibition) refers to the concentration of a drug that inhibits the growth of cells, such as tumor cells, by 50%. The term "ED50" (EC50) (50% effective dose / concentration) refers to the concentration of a drug that produces 50% of its biological effect, which may include cytotoxicity.

[0084] The term "antiproliferative activity" is intended to refer to arresting or inhibiting the proliferation of cells, eg, cancer cells. The term "effector functions" of an antibody refers to biological activities that can be attributed to the Fc region of an antibody (either a native Fc region sequence or an Fc region amino acid variant), and vary with antibody isotype. Examples of antibody effector functions include: C1 q These include: binding and complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor, BCR) and B cell activation.

[0085] "Antibody-dependent cellular cytotoxicity" or "ADCC" refers to a cell-mediated reaction in which non-specific cytotoxic cells expressing Fc receptors (FcR), such as natural killer (NK) cells, neutrophils, and macrophages, recognize bound antibodies on target cells and subsequently cause lysis or phagocytosis of the target cells. The primary cells mediating ADCC, NK cells, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9: 457-92 (1991). To assess the ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Pat. No. 5,500,362 or 5,821,337, may be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).

[0086] "Human effector cells" are leukocytes that express one or more FcRs and perform effector functions. Preferably, the cells express at least FcγRIII and perform ADCC effector functions. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils; PBMCs and NK cells are preferred. Effector cells may be isolated from their native source, for example from blood or PBMCs, as described herein.

[0087] The term "Fc receptor" or "FcR" is used to describe a receptor that binds to the Fc region of an antibody. A preferred FcR is a native sequence human FcR. Additionally, a preferred FcR is one that binds an IgG antibody (gamma receptor), and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibitory receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain (see review in Daeron, Annu. Rev. Immunol. 15:203-234(1997)). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92(1991). Other FcRs, including those that will be identified in the future, are included in the term "FcR" herein. The term also includes the neonatal receptor FcRn, which is involved in the transfer of maternal IgG to the fetus.

[0088] "Complement-dependent cytotoxicity" or "CDC" refers to the ability of a molecule to lyse a target in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (C1q) to a molecule (e.g., an antibody) complexed with a cognate antigen. To assess complement activation, a CDC assay, e.g., as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), may be performed.

[0089] The term "identity" or "homology" is taken to mean the percentage of amino acid residues in a candidate sequence that are identical to the residues of the corresponding sequence being compared, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity for the entire sequence, and without considering any conservative substitutions as part of the sequence identity. Any N- or C-terminal extensions or insertions will not be considered to reduce the identity or homology. Methods and computer programs for alignment are well known in the art. Sequence identity may be measured using sequence analysis software (e.g., Sequence Analysis Software Package, Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Ave., Madison, WI 53705). This software matches similar sequences by assigning a degree of homology to various substitutions, deletions (removals), and other modifications.

[0090] The term "homologous" with respect to a polypeptide sequence of an antibody shall refer to an antibody exhibiting at least 70%, preferably 80%, more preferably 90% and most preferably 95% sequence identity to the polypeptide sequence. The term with respect to a nucleic acid sequence shall refer to a nucleotide sequence exhibiting at least 85%, preferably 90%, more preferably 95% and most preferably 97% sequence identity to the nucleic acid sequence.

[0091] Amino acid sequence modification(s) of the antibody described herein are provided. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibody are prepared by introducing appropriate nucleotide changes into the antibody nucleic acid or by peptide synthesis. Such modifications include, for example, deletion, and / or insertion and / or substitution of residues within the amino acid sequence of the antibody. Any combination of deletion, insertion and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired properties. Amino acid changes may also modify post-translational processes of the antibody, for example, changing the number or position of glycosylation sites.

[0092] Variants of antibody amino acid sequence modification using amino acid substitution. Such variants are substitutions of at least one amino acid residue in antibody molecule with a different residue. The most interesting sites for substitution mutagenesis include hypervariable regions or CDRs, but FR or Fc modifications are also contemplated. Conservative substitutions are shown in Table A under "preferred substitutions". If such substitutions cause alteration of biological activity, further substantial changes, referred to as "exemplary substitutions" in Table A, or modifications further described below when describing classes of amino acids, may be introduced, and the products may also be screened.

[0093] [Table 1]

[0094] The terms "nucleic acid", "nucleic sequence", "nucleic acid sequence", "polynucleotide", "oligonucleotide", "polynucleotide sequence" and "nucleotide sequence" are used interchangeably in this description and refer to a precise sequence of nucleotides, modified or not, determining a fragment or region of a nucleic acid, containing or not containing non-naturally occurring nucleotides, and being either double-stranded DNA or RNA, single-stranded DNA or RNA, or a transcription product of said DNA.

[0095] It must also be included herein that the present invention is not associated with nucleotide sequences in their natural chromosomal environment, i.e. in their natural state. The sequences of the present invention are isolated and / or purified, i.e. they are sampled directly or indirectly, for example by copying, and their environment is at least partially modified. Thus, isolated nucleic acids obtained by recombinant genetics, for example by a host cell, or obtained by chemical synthesis must also be mentioned herein.

[0096] A reference to a nucleotide sequence includes its complement unless otherwise specified. Thus, a reference to a nucleic acid having a particular sequence should be understood to include its complementary strand, along with its complementary sequence.

[0097] The expression "control sequence" refers to a DNA sequence necessary for the expression of an operably linked coding sequence in a particular host organism. Control sequences suitable for prokaryotes include, for example, promoters, optionally operator sequences, and ribosome binding sites. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0098] A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader sequence is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; and a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading frame. However, enhancers need not be contiguous.

[0099] The term "vector" as used herein refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. In some embodiments, a vector is a plasmid, i.e., a circular double-stranded piece of DNA into which additional DNA segments can be ligated. In some embodiments, a vector is a viral vector, where additional DNA segments can be ligated into the viral genome. In some embodiments, a vector is capable of autonomous replication in a host cell into which it is introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). In further embodiments, a vector (e.g., non-episomal mammalian vectors) can be integrated into the genome of the host cell upon introduction into the host cell, and thereby replicated along with the host genes. In addition, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors").

[0100] The term "recombinant host cell" (or simply "host cell") is intended herein to refer to a cell into which a recombinant expression vector has been introduced. The present invention relates to a host cell that may contain, for example, a vector according to the present invention described above. The present invention also relates to a host cell that contains, for example, a nucleotide sequence encoding a heavy chain or an antigen-binding portion thereof, a light chain encoding nucleotide sequence or an antigen-binding portion thereof, or both, of the first binding domain and / or the second binding domain of a binding molecule of the present invention. It should be understood that "recombinant host cell" and "host cell" are intended to refer not only to the particular subject cell, but also to the progeny of such a cell. Such progeny may not in fact be identical to the parent cell, since modifications may occur in successive generations, either due to mutation or environmental influences, but such cells are still included within the scope of the term "host cell" herein.

[0101] The term "excipient" is used herein to describe any ingredient other than the compound(s) of the invention. The term "CD20-mediated disease or disorder" refers to any disease or disorder associated with CD20, either directly or indirectly, including the etiology, development, progression, persistence or pathology of the disease or disorder. "Treate", "treating" and "treatment" refer to a method of alleviating or abrogating at least one of a biological disorder and / or its associated symptoms. As used herein, "alleviating" a disease, disorder or condition means reducing the severity and / or frequency of occurrence of the symptoms of the disease, disorder or condition. Additionally, as used herein, reference to "treatment" includes reference to curative, palliative and prophylactic treatment.

[0102] In one aspect, the subject or patient of the treatment is a mammal, preferably a human subject. The subject may be either male or female of any age. The term "disorder" means any condition that would benefit from treatment with the compounds of the invention. It refers to chronic and acute disorders or diseases, including pathological conditions that predispose a mammal to the disorder in question.

[0103] The terms "cancer" and "cancerous" refer to or describe a physiological state in a mammal that is typically characterized by uncontrolled growth / proliferation of cells. The definition includes both benign and malignant cancerous diseases. Examples of cancerous diseases include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific examples of such cancerous diseases include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma and lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastric cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney or renal cancer, prostate cancer, vaginal cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, melanoma, and various head and neck cancers.

[0104] The terms "immune response", "autoimmune response" and "autoimmune inflammation" refer to the action of, for example, lymphocytes, antigen-presenting cells, phagocytic cells, granulocytes and soluble macromolecules produced by said cells or hepatocytes, including antibodies, cytokines and complement, that are produced as a result of selective damage, destruction or elimination of invasive pathogens, pathogen-infected cells or tissues, cancer cells, or, in the case of autoimmune or pathological inflammation, normal cells or tissues from the body.

[0105] The terms "immune response", "autoimmune response" and "autoimmune inflammation" refer to the action of, for example, lymphocytes, antigen-presenting cells, phagocytic cells, granulocytes and soluble macromolecules produced by said cells or hepatocytes, including antibodies, cytokines and complement, that are produced as a result of selective damage, destruction or elimination of invasive pathogens, pathogen-infected cells or tissues, cancer cells, or, in the case of autoimmune or pathological inflammation, normal cells or tissues from the body.

[0106] As used herein, the term "autoantibody" refers to the antibody directed against autoantigen.Autoantigen includes, but is not limited to, nucleic acid (e.g. double-stranded DNA or RNA, single-stranded DNA or RNA, or combination thereof), nuclear protein (e.g. SS-A(Ro), SS-B(La), Scl-70, centromere, Jo-1, histidyl-tRNA synthetase, threonyl-tRNA synthetase, PM-1, Mi-2, histone, and chromatin), cell receptor (e.g. acetylcholine receptor, thyroid stimulating hormone receptor), cell protein (e.g. cardiolipin, β2GP1), cell membrane protein (e.g. aquaporin, desmoglein), RNA protein complex (e.g. RNP and Sm), erythrocyte and platelet receptor glycoprotein.

[0107] The term "autoimmune disease" as used herein means a non-malignant disease or disorder arising from and directed against an individual's own (self) antigens and / or tissues. The term includes, but is not limited to, rheumatoid arthritis, juvenile chronic arthritis, septic arthritis, Lyme osteoarthritis, psoriatic arthritis, reactive arthritis, spondyloarthropathies, systemic lupus erythematosus, Crohn's disease, ulcerative colitis, inflammatory bowel disease, diabetes, thyroiditis, asthma, allergic diseases, psoriasis, atopic dermatitis, scleroderma, "graft versus host" reaction, organ transplant rejection, acute or chronic immune disorders associated with organ transplantation, sarcoidosis, Kawasaki disease, Graves' disease, nephrotic syndrome, chronic fatigue syndrome, Wegener's granulomatosis, Henoch-Schonlein purpura, microscopic renal vasculitis, chronic active hepatitis, uveitis, ita), septic shock, toxic shock syndrome, septic syndrome, cachexia, acquired immune deficiency syndrome, acute transverse myelitis, Huntington's chorea, Parkinson's disease, Alzheimer's disease, stroke, primary biliary cirrhosis, hemolytic anemia, adult (acute) respiratory distress syndrome, alopecia, focal alopecia, seronegative arthropathy, arthropathy, Reiter's disease, psoriatic arthropathy associated with ulcerative colitis arthropathy, atopic allergy, autoimmune bullous disease, pemphigus vulgaris, pemphigus foliaceus, pemphigoid disease, linear IgA, IgG-related disease, autoimmune hemolytic anemia, Coombs' positive hemolytic anemia, pernicious anemia, juvenile pernicious anemia, giant cranial arteritis (cranial giant arteritis), arthritis, primary sclerosing hepatitis A, idiopathic autoimmune hepatitis, pulmonary fibrotic disease, idiopathic fibrosing alveolitis, postinflammatory interstitial lung disease, interstitial pneumonia, chronic eosinophilic pneumonia, postinfectious interstitial lung disease, gouty arthritis, autoimmune hepatitis, autoimmune hepatitis type I (classical autoimmune hepatitis or lupoid), autoimmune hepatitis type II, osteoarthritis, primary sclerosing cholangitis, psoriasis, idiopathic leukopenia, autoimmune neutropenia, renal NOS disease, glomerulonephritis, microscopic renal vasculitis, discoid lupus erythematosus, idiopathic or male NOS infertility [autoimmune against sperm], multiple sclerosis (all subtypes), sympathetic ophthalmia, pulmonary hypertension secondary to connective tissue disease, Goodpasture's syndrome, nodular polyarthritisnodosa), acute rheumatic fever, rheumatic spondylitis, ankylosing spondylitis, Still's disease, systemic sclerosis, localized sclerosis, Sjögren's syndrome, Sjögren's disease, Behçet's disease, ankylosing spondylitis, spondyloarthritis, axial enthesitis, relapsing polychondritis, Takayasu's disease, autoimmune thrombocytopenia, idiopathic thrombocytopenia, autoimmune thyroiditis, autoimmune gastritis, polyglandular autoimmune syndrome, hyperthyroidism, Hashimoto's disease, autoimmune atrophic hypothyroidism, primary myxedema, lens-induced uveitis, primary vasculitis, vitiligo, acute Liver disease, chronic liver disease, allergies, asthma, psychiatric disorders (including depression and schizophrenia), Th2 / Th1 mediated disorders, conjunctivitis, allergic contact dermatitis, allergic rhinitis, alpha-I antitrypsin deficiency, amyotrophic lateral sclerosis, anemia, cystic fibrosis, disorders associated with cytokine therapy, demyelinating diseases, dermatitis, iridocyclitis / uveitis / optic neuritis, ischemia-reperfusion injury, ischemic stroke, juvenile rheumatoid arthritis, autoimmune enteropathy, autoimmune hearing loss, autoimmune lymphoproliferative syndrome, autoimmune myocarditis, autoimmune rheumatoid arthritis, autoimmune enteropathy, autoimmune lymphoproliferative syndrome, autoimmune myocarditis, autoimmune rheumatoid arthr ... Autoimmune cardiomyopathy, Coxsackie myocarditis, Dressler syndrome, Lupus nephritis, Angioedema including hereditary angioedema, Urticaria, Hidradenitis suppurativa, Lichen planus, Lichen sclerosus, Pityriasis parvum, Vitiligo, Addison's disease, Autoimmune polyendocrine syndrome, Autoimmune pancreatitis, Celiac disease, Microscopic colitis, Antiphospholipid syndrome, Autoimmune lymphoproliferative syndrome, Cold agglutinin disease, Essential cryoglobulinemia, Evans syndrome, Pernicious anemia, Red blood cell aplasia, CREST syndrome, Eosinophilic fasciitis, Felty syndrome, Overlap syndrome, Chronic Lyme disease, The antibodies may also be used to treat any combination of the above disorders.

[0108] A "therapeutically effective amount" is intended to refer to that amount of the therapeutic agent being administered that will relieve to some extent one or more of the symptoms of the disorder being treated. The term "chronic" use refers to continuous (uninterrupted) use of an agent(s), as opposed to acute (transient) routes of administration, such that the initial therapeutic effect (activity) is maintained for an extended period of time.

[0109] "Intermittent" use refers to treatment that is not performed consistently without interruption, but rather is periodic in nature. As used herein, the words "comprise," "have," "include," or variations such as "comprises," "comprising," "has," "having," "includes," or "including" and all grammatical variations thereof, are understood to indicate the inclusion of a reference integer or group of integers, but are not intended to exclude any other integers or groups of integers.

[0110] Disclosure of the Invention antibody The present invention relates to a monoclonal antibody that specifically binds to CD20.

[0111] In one aspect, the present invention relates to a monoclonal antibody or antigen-binding fragment thereof that specifically binds to CD20, and comprises: 1) Amino acid sequence

[0112] [ka]

[0113] a heavy chain variable domain comprising: 2) Amino acid sequence

[0114] [ka]

[0115] A light chain variable domain comprising Includes. In some embodiments, the monoclonal antibody or antigen-binding fragment thereof is: 1) Amino acid sequence

[0116] [ka]

[0117] a heavy chain variable domain comprising: 2) Amino acid sequence

[0118] [ka]

[0119] A light chain variable domain comprising Includes. In some embodiments, the monoclonal antibody or antigen-binding fragment thereof is: 1) Amino acid sequence

[0120] [ka]

[0121] a heavy chain variable domain comprising: 2) Amino acid sequence

[0122] [ka]

[0123] A light chain variable domain comprising Includes. In some embodiments, the monoclonal antibody is: 1) Amino acid sequence

[0124] [ka]

[0125] a heavy chain; 2) Amino acid sequence

[0126] [ka]

[0127] Light chain containing Includes. In some embodiments, the monoclonal antibody that specifically binds to CD20 is BCD132-077.

[0128] The monoclonal antibody BCD132-077: 1) Amino acid sequence

[0129] [ka]

[0130] a heavy chain comprising; 2) Amino acid sequence

[0131] [ka]

[0132] Light chain containing Includes. In some embodiments, the monoclonal antibody that specifically binds to CD20 is BCD132-L-028.

[0133] The monoclonal antibody BCD132-L-028: 1) Amino acid sequence

[0134] [ka]

[0135] a heavy chain comprising; 2) Amino acid sequence

[0136] [ka]

[0137] Light chain containing Includes. In some embodiments, the monoclonal antibody that specifically binds to CD20 is a full-length IgG antibody.

[0138] In some embodiments, the monoclonal antibodies are of the human IgG1, IgG2, IgG3, IgG4 isotype. In some embodiments, the monoclonal antibodies are of the human IgG1 isotype.

[0139] nucleic acid molecule The present invention also relates to nucleic acid molecules, in particular sequences encoding monoclonal antibodies that specifically bind to CD20 according to the invention as described herein, optionally including any peptide linker sequence linked thereto.

[0140] Reference to a nucleotide sequence includes its complement unless otherwise indicated. Thus, reference to a nucleic acid having a particular sequence should be understood to include its complementary strand as well as its complementary sequence. The term "polynucleotide" as used herein refers to a polymeric form of nucleotides, either ribonucleotides or deoxyribonucleotides, or modified forms of either type of nucleotide, of at least 10 bases in length. The term includes single-stranded and double-stranded forms.

[0141] In one aspect, the present invention relates to a nucleic acid molecule comprising a nucleotide sequence encoding an amino acid sequence selected from SEQ ID NOs: 1 to 8. The nucleic acid molecule may also comprise any combination of said nucleotide sequences.

[0142] In one aspect, the present invention provides an antibody that specifically binds CD20 and is 1) a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:6; 2) A light chain variable domain comprising the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 8. The present invention relates to a nucleic acid molecule comprising a nucleotide sequence encoding a monoclonal antibody or an antigen-binding fragment thereof, comprising:

[0143] In some embodiments, the nucleic acid molecule is: 1) a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:2; 2) A light chain variable domain comprising the amino acid sequence shown in SEQ ID NO:4 The present invention includes a nucleotide sequence encoding a monoclonal antibody or antigen-binding fragment thereof comprising:

[0144] In some embodiments, the nucleic acid molecule is: 1) a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:6; 2) A light chain variable domain comprising the amino acid sequence shown in SEQ ID NO:8. The present invention includes a nucleotide sequence encoding a monoclonal antibody or antigen-binding fragment thereof comprising:

[0145] In some embodiments, the nucleic acid molecule is: 1) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:5; 2) A light chain comprising the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 7 The present invention includes a nucleotide sequence encoding a monoclonal antibody comprising:

[0146] In some embodiments, the nucleic acid molecule is: 1) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:1; 2) A light chain comprising the amino acid sequence shown in SEQ ID NO:3. The present invention includes a nucleotide sequence encoding a monoclonal antibody comprising:

[0147] In some embodiments, the nucleic acid molecule is: 1) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:5; 2) A light chain comprising the amino acid sequence shown in SEQ ID NO:7. The present invention includes a nucleotide sequence encoding a monoclonal antibody comprising:

[0148] In any of the above aspects, the nucleic acid molecule may be isolated. The nucleic acid molecules of the invention may be isolated from any source that produces monoclonal antibodies that specifically bind to CD20. In certain embodiments, the nucleic acid molecules of the invention may be synthesized rather than isolated.

[0149] In one embodiment, a nucleic acid molecule encoding a VH (SEQ ID NO:2 or SEQ ID NO:6) or VL (SEQ ID NO:4 or SEQ ID NO:8) domain is converted into a full-length antibody gene by insertion into an expression vector already encoding a heavy chain constant (CH) or light chain constant (CL) domain, such that the VH segment is operably linked to the CH segment(s) in the vector and / or the VL segment is operably linked to the CL segment in the vector, respectively. In another embodiment, a nucleic acid molecule encoding a VH and / or VL domain is converted into a full-length antibody gene by linking, e.g., ligating, the nucleic acid molecule encoding the VH and / or VL domain to the nucleic acid molecule encoding the CH and / or CL domain using standard molecular biology techniques. These may then be expressed from a cell into which the full-length nucleic acid molecules encoding the heavy and / or light chains have been introduced.

[0150] The nucleic acid molecules may be used to express large amounts of recombinant monoclonal antibodies that specifically bind to CD20. vector In another aspect, the invention relates to vectors suitable for the expression of any of the nucleotide sequences described herein.

[0151] The present invention relates to vectors comprising a nucleic acid molecule encoding any amino acid sequence of a monoclonal antibody or portion thereof (e.g., the heavy chain sequence of the first binding domain and / or the heavy and / or light chain sequence of the second binding domain) that specifically binds to CD20 as described herein. The present invention further provides vectors comprising a nucleic acid molecule encoding a fusion protein, a modified antibody, an antibody fragment.

[0152] In some embodiments, the monoclonal antibody specifically binding to CD20 according to the present invention is expressed by inserting DNA partially or completely encoding the sequence of the first or second binding domain (e.g., the light and heavy chain sequences in the case where the binding domain comprises light and heavy chain sequences) obtained as described above in an expression vector such that the gene is operably linked to the necessary expression control sequences, e.g., transcription and translation control sequences. Expression vectors include plasmids, retroviruses, adenoviruses, adeno-associated viruses (AAV), plant viruses such as cauliflower mosaic virus, tobacco mosaic virus, cosmids, YACs, EBV-derived episomes, and the like. The DNA molecule may be ligated into the vector such that the transcription and translation control sequences in the vector perform the intended function of controlling the transcription and translation of the DNA. The expression vector and expression control sequences may be selected to be compatible with the expression host cell used. The DNA molecules partially or completely encoding the sequence of the first and second binding domain (e.g., the heavy and light chain sequences in the case where the binding domain comprises heavy and light chain sequences) may be introduced into each vector. In one embodiment, any combination of the DNA molecules is introduced into the same expression vector. The DNA molecules may be introduced into the expression vector by standard methods (e.g., ligation of complementary restriction sites on the antibody gene fragment and the vector, or blunt end ligation if no restriction sites are present).

[0153] A suitable vector is one that encodes a functionally complete human CH or CL immunoglobulin sequence with appropriate restriction site manipulation so that any VH or VL sequence can be easily inserted and expressed as described above. The HC and LC coding genes in such vectors may contain intron sequences that stabilize the corresponding mRNA, thereby resulting in an increase in the overall antibody protein yield. The intron sequence is flanked by splice donor and splice acceptor sites that determine where RNA splicing occurs. The location of the intron sequence may be in either the variable or constant region of the antibody chain, or in both the variable and constant regions when multiple introns are used. Polyadenylation and transcription termination may occur at the native chromosomal site downstream of the coding region. The recombinant expression vector may also encode a signal peptide that facilitates secretion of the antibody chain from the host cell. The antibody chain gene may be cloned into the vector such that the signal peptide is linked in frame to the amino terminus of the immunoglobulin chain. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (ie, a signal peptide from a non-immunoglobulin protein).

[0154] In addition to the antibody chain genes, the recombinant vector expression of the invention may carry regulatory sequences that control the expression of the antibody chain genes in a host cell. One of skill in the art will understand that the design of the expression vector, including the selection of regulatory sequences, may depend on the choice of the host cell to be transformed, the desired protein expression level, and the like. Preferred regulatory sequences for mammalian expression host cells include viral elements that ensure high levels of protein expression in mammalian cells, such as retroviral LTRs, cytomegalovirus (CMV) (e.g., CMV promoter / enhancer), simian virus 40 (SV40) (e.g., SV40 promoter / enhancer), adenovirus (e.g., major late promoter adenovirus (AdMLP)), polyoma virus, and strong mammalian promoters, such as the native immunoglobulin promoter or actin promoter. For further description of viral regulatory elements and sequences thereof, see, e.g., U.S. Patent Nos. 5,168,062, 4,510,245, and 4,968,615. Methods for expressing binding molecules, such as antibodies, in plants are known in the art, including promoters and vectors, and plant transformation descriptions.See, for example, U.S. Patent No. 6,517,529.Methods for expressing polypeptides in bacterial or fungal cells, such as yeast cells, are also well known in the art.

[0155] In addition to the antibody chain genes and control sequences, the recombinant expression vector of the present invention may carry additional sequences, such as sequences that control replication of the vector in a host cell (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of a host cell into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and 5,179,017). For example, typically the selectable marker gene confers resistance to pharmaceutical agents, such as G418, hygromycin or methotrexate, on the host cell into which the vector has been introduced. For example, selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr- host cells during methotrexate selection / amplification), the neo gene (for G418 selection), and the glutamate synthetase gene.

[0156] The term "expression control sequences" is intended herein to refer to polynucleotide sequences necessary to affect the expression and processing of a coding sequence to which it is linked. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals, such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., Kozak consensus sequences); sequences that enhance protein stability; and sequences that enhance protein secretion, if desired. The nature of such control sequences will vary depending on the host organism; in prokaryotes, such control sequences generally include a ribosome binding site, promoter, and transcription termination sequences; in eukaryotes, such control sequences typically include a promoter and transcription termination sequences. The term "control sequences" includes at least all components whose presence is essential for expression and processing, and may also include additional components whose presence is beneficial, such as leader sequences and fusion partner sequences.

[0157] host cell A further aspect of the present invention relates to a method for producing a monoclonal antibody that specifically binds to CD20 according to the present invention. One embodiment of the present invention relates to a method for producing a monoclonal antibody that specifically binds to CD20 as defined herein, comprising the steps of producing a recombinant host cell capable of expressing a monoclonal antibody that specifically binds to CD20, culturing said host cell under conditions suitable for the expression / production of a monoclonal antibody that specifically binds to CD20, and isolating the resulting monoclonal antibody that specifically binds to CD20. The monoclonal antibody that specifically binds to CD20 produced by such expression in such a recombinant host cell is referred to herein as a "recombinant monoclonal antibody that specifically binds to CD20". The present invention also relates to the progeny of cells derived from such host cells, and to the monoclonal antibody that specifically binds to CD20 produced as well.

[0158] Nucleic acid molecules encoding monoclonal antibodies specifically binding to CD20 according to the present invention and vectors containing these nucleic acid molecules may be used to transfect suitable mammalian or cells thereof, plant or cells thereof, bacterial or yeast host cells. Transformation may be by any known technique for introducing polynucleotides into host cells. Methods for administering heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, cationic polymer-nucleic acid complex transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, encapsulation of polynucleotide(s) in liposomes, and direct microinjection of DNA into the nucleus. Additionally, nucleic acid molecules may be introduced into mammalian cells by viral vectors. Methods for transfecting cells are well known in the art. See, for example, U.S. Patent Nos. 4,399,216, 4,912,040, 4,740,461 and 4,959,455. Methods for transforming plant cells are well known in the art and include, for example, Agrobacterium-mediated transformation, biolistic transformation, direct injection, electroporation and viral transformation. Methods for transforming bacterial and yeast cells are also well known in the art.

[0159] Mammalian cell lines used as hosts for transformation are well known in the art and include many immortalized cell lines available. These include, for example, Chinese hamster ovary (CHO) cells, NS0 cells, SP2 cells, HEK-293T cells, FreeStyle 293 cells (Invitrogen), NIH-3T3 cells, HeLa cells, baby hamster kidney (BHK) cells, African green monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., HepG2), A549 cells, and many other cell lines. A cell line is selected by determining which cell line has high expression levels and provides the necessary characteristics for the protein to be produced. Other cell lines that can be used are insect cell lines, such as Sf9 or Sf21 cells. When a recombinant expression vector encoding a monoclonal antibody that specifically binds to CD20 is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a period of time sufficient to allow expression of the antibody in the host cell, or more preferably, secretion of the antibody into the medium in which the host cell is grown. Monoclonal antibodies that specifically bind to CD20 may be reconstituted from the culture medium using standard protein purification techniques. Plant host cells include, for example, Nicotiana, Arabidopsis, duckweed, corn, wheat, potato, etc. Bacterial host cells include Escherichia and Streptomyces species. Yeast host cells include Schizosaccharomyces pombe, Saccharomyces cerevisiae, and Pichia pastoris.

[0160] Furthermore, many known techniques may be used to enhance the production level of the monoclonal antibody that specifically binds to CD20 according to the present invention from the production cell line. For example, the glutamine synthetase gene expression system (GS system) is a common approach to enhance expression under certain conditions. The GS system is discussed in whole or in part in connection with EP Nos. 0216846, 0256055, 0323997 and 0338841.

[0161] Monoclonal antibodies that specifically bind to CD20 expressed by various cell lines or in transgenic animals will likely have different glycosylation profiles when compared to each other. However, monoclonal antibodies that specifically bind to CD20, encoded by the nucleic acid molecules described herein or comprising the amino acid sequences provided herein, are part of the present invention, regardless of glycosylation of the binding molecule, and generally regardless of the presence or absence of post-translational modifications.

[0162] Preparation of antibodies The present invention also relates to methods and processes for producing monoclonal antibodies and antigen-binding fragments thereof that specifically bind to CD20.

[0163] Monoclonal antibodies The monoclonal antibodies may be prepared using the hybridoma method first described by Kohler et al. Nature 256, 1975, p. 495, or may be made using recombinant DNA methods (US 4,816,567).

[0164] When using the hybridoma method, a mouse or other suitable host animal, such as a hamster, is immunized according to the method described above to induce lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used in immunization. According to another embodiment, lymphocytes may be produced by in vitro immunization. After immunization, the lymphocytes are fused with a myeloma cell line using a suitable fusing agent, such as polyethylene glycol, to produce hybridoma cells.

[0165] Hybridoma cells produced in the above manner may be cultured in an appropriate medium, preferably containing one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the medium for the hybridomas will typically contain hypoxanthine, aminopterin, and thymidine (HAT medium), substances that prevent the growth of HGPRT-deficient cells.

[0166] The preferred cells used as components of myeloma cell fusion are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to the medium in which the unfused parent cells are selected. Preferred myeloma cell lines are murine myeloma cell lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, Calif., USA, and SP-2 or X63-Ag8-653 cells available from the American Type Culture Collection, Rockville, Md., USA. Human myeloma and mouse-human heteromyeloma cell lines for producing monoclonal antibodies have also been described (Kozbor, J. Immunol, 133, 1984, p.3001).

[0167] Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

[0168] The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis described in Munson et al., Anal. Biochem., 107:220 (1980).

[0169] Once hybridoma cells that produce antibodies of the desired specificity, affinity, and / or activity are identified, the clones may be subcloned by limiting dilution and grown by standard methods. Suitable media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, hybridoma cells may be grown in vivo as ascites tumors in animals, for example, by intraperitoneal (ip) injection of the cells into mice.

[0170] The monoclonal antibodies secreted by the subclones may be separated from the culture medium, ascites fluid, or serum by conventional antibody purification techniques, such as affinity chromatography (e.g., with Protein A or Protein G-Sepharose) or ion exchange chromatography, hydroxylapatite chromatography, gel electrophoresis, dialysis, etc.

[0171] DNA encoding a monoclonal antibody can be easily isolated and sequenced using conventional methods (e.g., by using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of a murine antibody). Hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA can be placed into an expression vector, which is then transfected into host cells, such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce antibody protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.

[0172] In further embodiments, monoclonal antibodies or antibody fragments may be isolated from antibody phage libraries generated using the techniques described in McCafferty et al., Nature, 348:552-554 (1990). Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991) describe the isolation of murine and human antibodies, respectively, using phage libraries. Subsequent publications describe the production of high affinity (nM range) human antibodies by chain shuffling (Marks et al., Bio / Technology, 10:779-783 (1992)), as well as combinatorial infection and in vivo recombination as strategies for constructing very large phage libraries (Waterhouse et al., Nucl. Acids. Res. 21:2265-2266 (1993)). Therefore, these techniques are promising alternatives to traditional monoclonal antibody hybridoma techniques for the isolation of monoclonal antibodies.

[0173] For example, to produce chimeric or fusion antibody polypeptides, DNA encoding an antibody may be modified, for example, by substituting heavy and light chain (CH and CL) constant region sequences for homologous murine sequences (US 4816567 and Morrison et al., Proc. Natl. Acad. Sci. USA: 81:6851 (1984)), or by covalently fusing an immunoglobulin coding sequence to all or part of the coding sequence of a non-immunoglobulin polypeptide (heterologous polypeptide). Non-immunoglobulin polypeptide sequences may be substituted for the constant regions of an antibody, or they may be substituted for the variable domains of the antigen-binding center of the antibody to generate chimeric bivalent antibodies containing one antigen-binding site with specificity for an antigen and another antigen-binding site with specificity for a different antigen.

[0174] Phage display library-based human antibodies and methodologies It is now possible to produce transgenic animals (e.g. mice) that can produce the full range of human antibodies after immunization without endogenous immunoglobulin production. For example, it has been described that the homozygous deletion of antibody heavy chain joining region (JH) genes in chimeric and germline mutant mice results in complete inhibition of endogenous antibody production. The transfer of human germline immunoglobulin gene arrays into these germline mutant mice results in the production of human antibodies after antigen exposure (US 5545806, 5569825, 5591669 (all GenPharm); 5545807; and WO 97 / 17852).

[0175] Alternatively, human antibodies and antibody fragments may be produced in vitro from immunoglobulin variable (V) region gene repertoires derived from the bodies of immunized donors using phage display technology (McCafferty et al., Nature, 348:552-553 (1990)). According to this technology, antibody V region genes are cloned in frame with either the major or minor coat protein genes of a filamentous bacteriophage, such as M13 or fd, and displayed as functional antibody fragments on the surface of the phage particle. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the functional properties of the antibody also result in selection of the gene encoding the antibody exhibiting said properties. Thus, the phage mimics some of the properties of B cells. Phage display may be performed in a variety of formats. Several sources of V gene segments may be used for phage display. Clackson et al., Nature, 352:624-628 (1991) isolated a diverse array of anti-oxazolone antibodies from a small random combinatorial library of V genes derived from the spleens of immunized mice. Essentially following the techniques described in Marks et al., J. Mol. Biol. 222:581-597 (1991), one can construct a repertoire of V genes from immunized human donors and isolate antibodies to a diverse array of antigens, including self-antigens.

[0176] Human antibodies may also be generated by in vitro activated B cells, as discussed above (see US Pat. Nos. 5,567,610 and 5,229,275). antibody fragment In certain circumstances it is advantageous to use antibody fragments rather than whole antibodies: the small size of fragments contributes to their rapid clearance and may contribute to better penetration into dense tumors.

[0177] A variety of techniques have been developed for the production of antibody fragments. Traditionally, these fragments were obtained through proteolytic digestion of intact antibodies. However, now these fragments may be obtained directly by recombinant host cells. Fab, Fv and ScFv antibody fragments may be expressed in and secreted from E. coli, thereby facilitating the production of large amounts of these fragments. Antibody fragments may be isolated from the antibody phage libraries described above. According to another embodiment, Fab'-SH fragments may be directly isolated from E. coli and chemically coupled to form F(ab')2 fragments (Carter et al., Bio / Technology 10:163-167 (1992)). According to another approach, F(ab')2 fragments may be directly isolated from recombinant host cell cultures. Fab and F(ab')2 fragments with increased in vivo half-life while retaining epitope binding receptor residues are described in US 5,869,046. Other techniques for producing antibody fragments will be apparent to those skilled in the art. In other embodiments, the antibody of choice is a single chain Fv fragment (scFv) (see WO 93 / 16185; US 5571894 and US 5587458). Fv and scFv are the only species that do not contain constant regions and contain intact binding sites; as a result, they are suitable for reducing non-specific binding during in vivo use. scFv fusion proteins may be constructed to result in fusion of effector proteins at either the N- or C-terminus of the scFv. The antibody fragment may also be a "linear antibody" as described, for example, in US 5641870. Such linear antibody fragments may be monospecific or bispecific.

[0178] Pharmaceutical Compositions In another aspect, the present invention provides a pharmaceutical composition comprising as an active ingredient (or as the only active ingredient) a monoclonal antibody that specifically binds to CD20.

[0179] The pharmaceutical composition may include at least one monoclonal antibody that specifically binds to CD20, and at least one component selected from the group consisting of a pharma- ceutically acceptable and pharmacologically compatible excipient.

[0180] The pharmaceutical composition may include at least one monoclonal antibody that specifically binds to CD20 and one or more additional binding molecules (e.g., antibodies) that target one or more corresponding surface receptors. In some embodiments, the composition is intended to ameliorate, prevent, or treat a disorder that may be associated with CD20.

[0181] "Pharmaceutical composition" means a composition comprising a monoclonal antibody specifically binding to CD20 according to the present invention and at least one component selected from the group consisting of pharma- ceutically acceptable and pharmacologically compatible excipients, such as fillers, solvents, diluents, carriers, adjuvants, dispensing agents, delivery agents, preservatives, stabilizers, emulsifiers, suspending agents, thickening agents, sustained delivery control agents, the selection and proportion of which depend on the type and route of administration and dosing. The pharmaceutical composition of the present invention and its preparation method will be clearly apparent to those skilled in the art. The pharmaceutical composition should preferably be manufactured in compliance with GMP (Good Manufacturing Practice) requirements. The composition may include a buffer composition, an isotonic agent, a stabilizer and a solubilizer. Prolonged action of the composition may be achieved by agents that delay absorption of the active pharmaceutical ingredient, such as aluminum monostearate and gelatin. Examples of suitable carriers, solvents, diluents and delivery agents include water, ethanol, polyalcohols and mixtures thereof, oils, and organic esters for injection.

[0182] A "pharmaceutical product" is a compound or mixture of compounds as tablets, capsules, powders, lyophilisates, injections, infusions, ointments and other ready-to-use pharmaceutical compositions intended for the restoration, improvement or modification of physiological functions, for the treatment and prevention of diseases, for diagnosis, anesthesia, contraception, cosmetology, etc. in humans and animals. Any method for administering peptides, proteins or antibodies accepted in the art may be suitably used for the monoclonal antibodies that specifically bind CD20 according to the present invention.

[0183] The term "pharmaceutical acceptable" refers to one or more compatible liquid or solid components suitable for administration in a mammal, preferably a human. The term "excipient" as used herein describes any other component of the present invention. These are inorganic or organic substances used in pharmaceutical manufacturing to impart the necessary physicochemical properties to the pharmaceutical product.

[0184] The terms "buffer", "buffer composition", and "buffer" refer to a solution that is capable of resisting changes in pH by the action of its acid-base conjugate components and that allows a monoclonal antibody drug that specifically binds to CD20 to resist changes in pH. Generally, the pharmaceutical composition preferably has a pH in the range of 4.0 to 8.0. Examples of buffers that may be used include, but are not limited to, acetate, phosphate, citrate, histidine, succinate, and the like buffer solutions.

[0185] The terms "isotonicity agent", "osmolyte" or "osmotic pressure regulator" as used herein refer to excipients that can increase the osmotic pressure of a liquid antibody formulation. An "isotonic" agent is one that has an osmotic pressure equivalent to that of human blood. An isotonic agent typically has an osmotic pressure of about 250-350 mOsm / kg. Isotonicity agents that may be used include, but are not limited to, polyols, sugars and sucrose, amino acids, metal salts such as sodium chloride, and the like.

[0186] "Stabilizer" refers to an excipient or a mixture of two or more excipients that provide physical and / or chemical stability of an active agent.Stabilizers 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 its copolymers (trade name: Poloxamer), Pluronic, sodium dodecyl sulfate (SDS); antioxidants, such as but not limited to methionine, acetylcysteine, ascorbic acid, monothioglycerol, sulfite, etc.; chelating agents, such as but not limited to ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), sodium citrate, etc.

[0187] A pharmaceutical composition is "stable" if the active agent retains its physical and / or chemical stability and / or biological activity during the specified shelf life at storage temperatures, e.g., 2-8°C. Preferably, the active agent retains both physical and chemical stability, as well as biological activity. The shelf life is adjusted based on the results of stability tests under accelerated or natural aging conditions.

[0188] The pharmaceutical composition of the present invention may be manufactured, packaged, or widely sold in the form of a single unit dose or a plurality of single unit doses in the form of a ready-to-use formulation.The term "single unit dose" as used herein refers to a discrete amount of a pharmaceutical composition that contains a predetermined amount of active ingredient.The amount of active ingredient is typically equal to the dose of the active ingredient to be administered to a subject, or a suitable fraction of such a dose, such as a half or a third of such a dose.

[0189] The pharmaceutical compositions according to the present invention are suitable for parenteral administration, typically as sterile formulations intended to be administered into the human body by injection, infusion and implantation, bypassing the gastrointestinal tract and through a breach in the skin or mucosal barrier.For example, parenteral administration includes, among others, subcutaneous, intraperitoneal, intramuscular, intrasternal, intravenous, intraarterial, intrathecal, intraventricular, intraurethral, ​​intracranial, intrasynovial, transdermal injection or infusion; and kidney dialysis infusion techniques.Intratumoral delivery, such as intratumoral injection, may also be used.Local perfusion is also provided.Preferred embodiments include intravenous and subcutaneous routes.Any method for administering peptides or proteins that is accepted in the art may be appropriately used for the monoclonal antibody that specifically binds to CD20 according to the present invention.

[0190] Injectable formulations may be prepared, packaged, or sold in unit dosage form, for example, without limitation, in ampoules, vials, plastic containers, prefilled syringes, autoinjection devices, etc. Formulations for parenteral administration include suspensions, solutions, emulsions in oily or aqueous bases, pastes, etc., among others.

[0191] In another embodiment, the present invention provides compositions for parenteral administration, including pharmaceutical compositions provided in dry (i.e., powder or granule) form for reconstitution with a suitable base (e.g., sterile, pathogen-free water) prior to administration. Such formulations may be prepared, for example, by the lyophilization process, known in the art as freeze-drying, and involving freezing the product followed by removal of the solvent from the frozen material.

[0192] The monoclonal antibodies that specifically bind to CD20 according to the present invention may also be administered intranasally or by inhalation, either alone or in admixture with suitable pharma- ceutically acceptable excipients, from an inhalation device, such as a pressurized aerosol container, pump, spray, atomizer or nebulizer, with or without a suitable propellant, or as nasal drops, or in a spray.

[0193] Dosage forms for parenteral administration may be formulated to be immediate or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted and programmed release.

[0194] Therapeutic Uses of Monoclonal Antibodies Specifically Binding to CD20 According to the Invention In one aspect, monoclonal antibodies that specifically bind to CD20 according to the invention are useful for the treatment of disorders associated with (mediated by) CD20 activity.

[0195] In one aspect, the subject is a mammal, preferably a human subject. The subject may be either male or female and may be of any age. In the case of tumors (e.g., cancer), the antibody or fragment thereof (e.g., an antibody or fragment thereof that specifically binds to CD20) may reduce the number of cancer cells; reduce the initial tumor size; inhibit (i.e., slow to some extent and preferably stop) cancer cell invasion into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; inhibit tumor growth to some extent; and / or alleviate to some extent one or more symptoms associated with the disorder. The antibody or fragment thereof may prevent to some extent the proliferation of cancer cells and / or kill existing cancer cells, which may be cytostatic and / or cytotoxic. With respect to cancer therapy, in vivo efficacy may be measured, for example, by assessing survival, time to tumor progression (TTP), tumor response rate (RR) to treatment, duration of response, and / or quality of life.

[0196] As used herein, the terms "co-administration," "co-administered," and "in combination with," referring to a monoclonal antibody that specifically binds to CD20 and one or more distinct therapeutic agents, are intended to mean, refer to, or include: 1) simultaneous administration of such a combination of a monoclonal antibody that specifically binds to CD20 according to the present invention and a therapeutic agent to a patient in need of treatment, where such components are formulated together in a single dosage form that releases said components to said patient substantially simultaneously; 2) simultaneous administration of such a combination of a monoclonal antibody that specifically binds to CD20 according to the present invention and a therapeutic agent to a patient in need of such treatment, where such components are formulated separately from one another in separate dosage forms that are taken by the patient at substantially the same time, whereby the components are released to the patient at substantially the same time; 3) sequential administration of such a combination of a monoclonal antibody that specifically binds to CD20 according to the present invention and a therapeutic agent to a patient in need of treatment, where such components are formulated separately from one another in separate dosage forms that are taken by the patient at successive times with a sufficient time interval between each administration, such that upon administration, the components are released to the patient at substantially different times; and 4) Sequential administration of such a combination of a monoclonal antibody that specifically binds to CD20 according to the present invention and a therapeutic agent to a patient in need of treatment, where such components are formulated together in a single dosage form which releases said components in a controlled manner and upon release to said patient at the same and / or different time points, simultaneously, sequentially or conjointly, wherein each part may be administered either by the same or different routes.

[0197] The monoclonal antibody specifically binding to CD20 according to the present invention may be administered without additional therapeutic treatment, i.e. as an independent therapy. Furthermore, the treatment with the monoclonal antibody specifically binding to CD20 according to the present invention may include at least one additional therapeutic treatment (combination therapy). In some embodiments, the monoclonal antibody specifically binding to CD20 may be administered in combination or formulated with another pharmaceutical product / preparation for the treatment of cancer or autoimmune disease.

[0198] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents the function of cells and / or causes destruction of cells. The term includes radioactive isotopes (e.g., At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 and radioactive isotopes of Lu), chemotherapeutic agents, and toxins of bacterial, fungal, plant or animal origin, including fragments and / or variants thereof, such as small molecule toxins or enzymatically active toxins.

[0199] A "chemotherapeutic agent" is a compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN®); alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; acetogenins (e.g., bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol; colchicine; betulinic acid; camptothecin (synthetic analog topotecan (HYCAMTIN®)); Camptothecins, such as erythropoietin, scopolectin, and 9-aminocamptothecin; bryostatins; kallistatins; CC-1065 (including its adozelesin, carzelesin, and bizelesin synthetic analogs); podophyllotoxins; podophyllic acid; teniposide; cryptophycins (e.g., cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (including the synthetic analogs KW-2189 and CB1-TM1); eleutherobin; pancratistatins; sarcodictins; spongiostatins; nitrogen mustards, such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride oxide hydrochloride), melphalan, novembitine, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine and ranimustine;Antibiotics, such as enediyne antibiotics (e.g., the calicheamicins, e.g., calicheamicin gamma II and calicheamicin omega II (e.g., Agnew, Chem. Intl. Ed. Engl., 33:183-186 (1994)); the dynemicins, including dynemicin A; esperamicin; and the neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores, aclacinomycin, actinomycin, authramicin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (ADRIAMYCIN®, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin HCl liposome injection (DOXOL®), liposomal doxorubicin TLC D-99 (including MYOCET®, PEGylated liposomal doxorubicin (including CAELYX®, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins, such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptomycin, glythin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate, gemcitabine (GEMZAR®), tegafur (UFTORAL®), capecitabine (XELODA®), epothilones, and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine;Pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmophor, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as floric acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; eniluracil; amsacrine; bestravcil; bisantrene; edatraxate; defofamine mine); demecolcine; diaziquone; elfornitine; elliptinium acetate; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids, such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidamol; nitraerine; pentostatin; phenamet; pirarubicin; rosoxantrone; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oregon); razoxane; rhizoxin; schizofiran; spirogermanium; tenuazonic acid; triazicon; 2,2',2"-trichlorotriethylamine; trichothecenes (e.g., T-2 toxin, veraculin A, roridin A, and anguidine); urethanes; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); thiotepa; taxoids such as paclitaxel (TAXOL®), albumin engineered nanoparticle formulations of paclitaxel (ABRAXANETM), and doxorubicin. cetaxel (TAXOTERE®); chlorambucil; 6-thioguanine; mercaptopurine; methotrexate; platinum agents, such as cisplatin, oxaliplatin, and carboplatin; vincas, which prevent tubulin polymerization to form microtubules, including vinblastine (VELBAN®), vincristine (ONCOVIN®), vindesine (ELDISINE®), FILDESIN®, and vinorelbine (NAVELBINE®); etoposide (VP16); ifosfamide; mitoxantrone; leucovorin;Novantrone; edatrexate; daunomycin; aminopterin; ibandronate; the topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid, including bexarotene (TARGRETIN®); bisphosphonates, such as asclodronate (e.g., BONEFOS® or OSTAC®), ethilodonate (DIDROCAL®), NE-58095, zoledronic acid / zoledronate (ZOMETA®), alendronate (FOSAMAJX®), pamidronate (AREDIA®), tiludronate (SKELID®), or risedronate (ACTONEL®); troxacitabine (1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, In particular, those that inhibit the expression of genes in signal transduction pathways involved in abnormal cell proliferation, such as PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines, such as THERATOOPE® vaccine and gene therapy vaccines, such as ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; topoisomerase 1 inhibitors (e.g. LURTOTECAN®); rmRH (e.g. ABARELIX®); BAY439006 (sorafenib; Bayer); SU-11248 (Pfizer); perifosine, COX-2 inhibitors (e.g. celecoxib or etoricoxib), proteosome inhibitors (e.g. PS341); bortezomib (VELCADE®); CCI-779; tipifarnib (RI 1577); orafenib, ABT510; Bcl-2 inhibitors, such as oblimersen sodium (GENASENSE®); pixantrone; EGFR inhibitors (see definition below); tyrosine kinase inhibitors (see definition below); and pharmaceutically acceptable salts, acids or derivatives of any of the above;and combinations of two or more of the above, such as CHOP, an abbreviation for the combination therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone, and FOLFOX, an abbreviation for treatment with oxaliplatin in combination with 5-FU and leucovorin (ELOXATIN™);

[0200] Also included in this definition are antihormonal agents, such as antiestrogens with a mixed agonist / antagonist profile, which act to regulate or inhibit hormone action on tumors, including tamoxifen (NOLVADEX®), 4-hydroxytamoxifen, toremifene (FARESTON®), idoxifene, droloxifene, raloxifene (EVTSTA®), trioxyfene, ketoxifene, and selective estrogen receptor modulators (SERMs), For example, SERM3; pure antiestrogens without agonistic properties, such as fulvestrant (FASLODEX®), and EM800 (such agents may also block estrogen receptor (ER) dimerization, inhibit DNA binding, increase ER turnover, and / or suppress ER levels); steroidal aromatase inhibitors, such as formestane and exemestane (AROMASIN®), and nonsteroidal aromatase inhibitors, such as anastrazole (AREVIIDEX®). aromatase inhibitors, including vorozole (RIVISOR®), megestrol acetate (MEGASE®), fadrozole, and other aromatase inhibitors including the imidazoles; luteinizing hormone-releasing hormone agonists, including leuprolide (LUPRON® and ELIGARD®), goserelin, buserelin, and tripterelin; progestins, such as megestrol acetate and medoxomil acetate; sex steroids including roxyprogesterone, estrogens such as diethylstilbestrol and premarin, and androgens / retinoids such as floxymesterone, all-trans retinoic acid and fenretinide; onapristone; antiprogesterones; estrogen receptor downregulators (ERDs); antiandrogens such as flutamide, nilutamide and bicalutamide; testolactone; and pharma- ceutically acceptable salts, acids or derivatives of any of the above; and combinations of two or more of the above.

[0201] In the treatment of the above-mentioned autoimmune disease or related autoimmune condition, the monoclonal antibody specifically binding to CD20 as provided herein may be administered to the patient using a multi-drug regimen in combination with different therapeutic agents, such as immunosuppressants, anti-inflammatory agents, systemic hormonal agents, anti-neoplastic and immunomodulatory agents or others. The monoclonal antibody specifically binding to CD20 may be administered simultaneously, sequentially or alternately with the different therapeutic agents, or may be administered after resistance to the different therapies is demonstrated. The different therapeutic agents may be administered at the same or lower dosages compared to those used in the art. When selecting a preferred different therapeutic agent, many factors must be considered, including the type of disease to be treated and the medical record of the patient.

[0202] As used herein, the term "immunosuppressant" in add-on therapy refers to a substance that suppresses or masks the patient's immune system. Such a substance may be a substance that inhibits cytokine production, downregulates or suppresses self-antigen expression, or masks major histocompatibility complex (MHC) antigens. Examples of such agents include steroids, e.g. glucocorticoids, such as prednisone, methylprednisolone and dexamethasone; 2-amino-6-aryl-5-substituted pyrimidines (see US 4,665,077), azathioprine (or cyclophosphamide if there is an adverse reaction to azathioprine); bromocriptine; glutaraldehyde (which masks MHC antigens as described in US 4,120,649); anti-idiotypic antibodies to MHC antigens and MHC fragments; cyclosporin A; cytokine and cytokine receptor antagonists, including interferon-gamma, -beta, or -alpha antibodies; anti-tumor necrosis factor antibodies; anti-interleukin-2 antibodies and anti-IL-2 receptor antibodies; anti-L3T4 antibodies, xenogeneic antilymphocyte globulin, pan-T antibodies, preferably anti-CD3 or anti-CD4 / CD4a antibodies; soluble peptides containing the LFA-3 binding domain (WO 90 / 08187, published June 26, 1990); streptokinase; TGF-p:streptodomase; host DNA / RNA; FK506; RS-61443; deoxyspergualin; rapamycin; T cell receptors (US 5,114,721); T cell receptor fragments (Offner et al., Science 251:430-432 (1991); WO 90 / 11294; and WO 91 / 01133); and T cell receptor antibodies (ЕР 340109), such as T10B9.

[0203] In the treatment of rheumatoid arthritis, the patient may be administered a monoclonal antibody that specifically binds to CD20 according to the present invention, either alone or in combination with one or more of the following drugs: DMARDs (basic anti-inflammatory drugs, e.g., methotrexate, leflunomide, sulfasalazine), NSAIDs (non-steroidal anti-inflammatory drugs, e.g., cyclooxygenase inhibitors), corticosteroids (e.g., prednisolone, budesonide). Exemplary DMARDs used in the treatment of RA are hydroxychloroquine, sulfasalazine, methotrexate, leflunomide, azathioprine, D-penicillamine, gold-based preparations (oral), gold-based preparations (intramuscular), minocycline, cyclosporine, Staphylococcus obtained by protein A immunoadsorption. Conventional methods for treating RA are described, for example, in JA Singh et al., 2015 American College of Rheumatology Guideline for the Treatment of Rheumatoid Arthritis. Arthritis Care Res (Hoboken) 68, 1-25 (2016).

[0204] It is meant that the monoclonal antibodies that specifically bind to CD20 according to the present invention may be used in therapeutic methods as described above, may be used in treatments as described above, and / or may be used in the manufacture of medicaments for treatments as described above.

[0205] Dosage and Route of Administration The monoclonal antibodies that specifically bind to CD20 according to the present invention will be administered in an amount effective to treat the condition in question, i.e., at a dosage and for a period of time necessary to achieve the desired result. The therapeutically effective amount may vary according to factors such as the particular condition being treated, the age, sex, and weight of the patient, and whether the monoclonal antibodies that specifically bind to CD20 are administered as a stand-alone therapy or in combination with one or more additional drugs or treatments.

[0206] Dosage regimes may be adjusted to provide an optimal response. For example, a single bolus may be administered, or several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. For ease of administration and uniformity of dosage, it is particularly preferred to formulate parenteral compositions in unit dosage forms. As used herein, unit dosage forms are intended to refer to physically discrete units suitable as unitary dosages for the patient / subject to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect, in association with the desired pharmaceutical carrier. The specification of unit dosage forms of the present invention is typically dictated by and directly dependent on (a) the unique characteristics of the chemotherapeutic agent and the particular therapeutic or prophylactic effect to be achieved, and (b) the limits of subject sensitivity inherent in the art of compounding such active compounds for treatment.

[0207] Thus, those skilled in the art will recognize that, based on the disclosure provided herein, doses and dosing regimens will be adjusted according to methods well known in the therapeutic field. That is, maximum tolerated doses can be readily established, and effective amounts that provide a detectable therapeutic effect on the patient can also be determined, as can the temporal requirements for administering each agent to provide a detectable therapeutic effect on the patient. Thus, although specific doses and dosing regimens are exemplified herein, these examples are not in any way limiting of the doses and dosing regimens that can be provided to a patient in the practice of embodiments of the present invention.

[0208] It should be noted that dosage values ​​may vary depending on the type and severity of the condition to be alleviated, and may include single or multiple doses. Furthermore, it should be understood that for any particular subject, a particular dosage regimen should be adjusted over time according to the individual's needs and the judgment of the medical professional administering or monitoring the administration of the composition, 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. Furthermore, dosage regimens including the compositions of the present invention may be based on a variety of factors, including the type of disease, the patient's age, weight, sex, medical condition, the severity of the condition, the route of administration, and the particular monoclonal antibody that specifically binds to CD20 used. Thus, dosage regimens may vary widely, but are routinely determinable using standard methods. For example, dosages 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 includes intrapatient dose escalation, as determined by one of skill in the art. Methods for determining appropriate dosages and regimens are well known in the art and would be understood by one of ordinary skill in the art once armed with the ideas disclosed herein.

[0209] Examples of suitable dosing regimes are provided above. Suitable doses of the monoclonal antibodies specifically binding to CD20 according to the present invention will be in the range of 0.1 to 200 mg / kg, preferably 0.1 to 100 mg / kg, including about 0.5 to 50 mg / kg, such as about 1 to 20 mg / kg. For example, the monoclonal antibodies specifically binding to CD20 may be administered at a dose of at least 0.25 mg / kg, such as at least 0.5 mg / kg, including at least 1 mg / kg, such as at least 1.5 mg / kg, such as at least 2 mg / kg, including at least 3 mg / kg, including at least 4 mg / kg, such as at least 5 mg / kg; and for example up to 50 mg / kg, including up to 30 mg / kg, including up to 20 mg / kg, including up to 15 mg / kg. Administration is typically repeated at appropriate time intervals, for example once weekly, once every two weeks, once every three weeks, or once every four weeks, and for as long as deemed appropriate by the attending physician, who may in some cases increase or decrease the dosage if necessary.

[0210] Diagnostic Uses and Compositions The monoclonal antibodies specifically binding to CD20 according to the present invention can also be used in diagnostic processes (e.g., in vitro, ex vivo). For example, the monoclonal antibodies specifically binding to CD20 according to the present invention can be used to detect or measure the level of CD20 in a sample obtained from a patient (e.g., a tissue sample or a body fluid sample, such as inflammatory exudate, blood, serum, interstitial fluid, saliva, or urine). Suitable methods for detection and measurement include immunoassays, such as flow cytometry, enzyme-linked immunosorbent assay (ELISA), chemiluminescence assays, radioimmunoassays, and immunohistology. The present invention further includes kits, such as diagnostic kits, that include the monoclonal antibodies specifically binding to CD20 described herein.

[0211] The following examples are provided for a better understanding of the present invention. These examples are for illustrative purposes only and are not to be construed as limiting the scope of the invention in any manner.

[0212] All publications, patents, and patent applications cited herein are hereby incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example, for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art that, in view of the teachings of the invention, certain changes and modifications may be made without departing from the spirit and scope of the accompanying embodiments. EXAMPLES

[0213] The following examples are provided for a better understanding of the present invention. These examples are for illustrative purposes only and are not to be construed as limiting the scope of the invention in any manner.

[0214] All publications, patents, and patent applications cited herein are hereby incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example, for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art that, in view of the teachings of the invention, certain changes and modifications may be made without departing from the spirit and scope of the accompanying embodiments.

[0215] Materials and General Methods General information regarding the nucleotide sequences of human immunoglobulin light and heavy chains is provided in: Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991). The amino acids of antibody chains are numbered and designated according to the EU numbering system (Edelman, GM et al., Proc. Natl. Acad. Sci. Natl. Acad. Sci. USA 63(1969)78-85; Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, (1991)).

[0216] Recombinant DNA Technology Standard methods were used to manipulate DNA as described in Sambrook, J. et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. Molecular biology reagents were used according to the manufacturers' instructions.

[0217] Gene synthesis The desired gene segments were prepared from oligonucleotides made by chemical synthesis. Gene segments of 300-4000 kb in length flanked by single restriction sites were assembled by annealing and ligation of oligonucleotides, including PCR amplification, and subsequently cloned through the indicated restriction sites. The DNA sequences of the subcloned gene fragments were confirmed by DNA sequencing.

[0218] DNA sequencing DNA sequences were determined by Sanger sequencing. DNA and protein sequence analysis and sequence data management Infomax's Vector NT1 Advance suite version 8.0 was used for sequence generation, mapping, analysis, annotation and illustration.

[0219] Expression vector For expression of the described antibodies and antigens, variants of expression plasmids intended for expression in prokaryotic cells (E. coli), transient expression in eukaryotic cells (e.g. in CHO cells) were applied. In addition to the antibody expression cassette, the vectors contained: an origin of replication allowing replication of said plasmid in E. coli, genes conferring resistance to various antibiotics in E. coli (e.g. to ampicillin and kanamycin).

[0220] Fusion genes containing the described antibody chains as described below were generated by PCR and / or gene synthesis and assembled using known recombinant methods and techniques, for example by ligation of matching nucleic acid segments using unique restriction sites in the corresponding vectors. Subcloned nucleic acid sequences were verified by DNA sequencing. Larger quantities of plasmids were prepared by plasmid preparation from transformed E. coli cultures for transient transfection.

[0221] Example 1. Production of recombinant control antibodies in mammalian cell suspension cultures Rituximab, an antibody with a published sequence, was used as a control. Genes for the heavy / light variable domains of the antibodies were synthesized and cloned into the SalI / NheI and SalI / BstWI restriction sites, respectively, of vectors pEE-HC, pEE-CK intended for protein production in mammalian cells (Figures 1, 2).

[0222] The required amount of the plasmid was grown in E. coli cells and purified using a Qiagen kit. The control antibody was produced in an established cell line (CHO-T line) derived from Chinese hamster ovary cells. Suspension cultures were performed in flasks on an orbital incubator shaker using serum-free medium (HyCell TransFx-C) supplemented with 8 mM L-glutamine and 1 g / l Pluronic 68. For transient expression, cells (2–2.2 x 10 6 Cells / ml) were transfected with linear polyethylenimine (PEI MAX, Polysciences). DNA / PEI ratios ranged from 1:3 to 1:10. Nine days after transfection, the culture medium was separated from the cells by filtration through a 0.5 / 0.22 μm depth filter. Target proteins were isolated from the culture medium by affine HPLC using bacterial protein protein A.

[0223] The purity of the resulting protein solution was assessed by reducing and non-reducing SDS gel electrophoresis. Example 2. Construction of naive human antibody FAB-library MeganLib™ Total RNA of B lymphocytes from blood samples from over 1000 individual human donors was isolated using the RNeasy mini kit according to the suggested protocol (QIAGEN). RNA concentration assays were performed using the Nanovue kit (GE Healthcare) and the quality of the isolated RNA was examined by 1.5% agarose gel electrophoresis.

[0224] Reverse transcription reactions were carried out using the MMLV RT kit (Evrogen) according to the recommended protocol, using MMuLV reverse transcriptase and random hexamer oligonucleotides as primers.

[0225] The reverse transcriptase was used as a matrix in a two-step polymerase chain reaction to generate genes for the variable domains flanked by restriction sites; the reaction was carried out using an oligonucleotide kit according to the protocol by [J Biol Chem. 1999 Jun 25; 274(26): 18218-30].

[0226] The resulting DNA preparation VL-CK-VH (Figure 4) was treated with NheI / Eco91I restriction endonucleases and ligated into the original plasmid pH5 (Figure 5). The ligation product was transformed into SS320 electrocompetent cells prepared according to the protocol [Methods Enzymol. 2000;328:333-63.]. The repertoire of the combinatorial phage Fab display library MeganLibTM was 1011 transformants. The Fab library phage products were prepared according to a previously described method [J Mol Biol. 1991 Dec 5;222(3):581-97].

[0227] Example 3. Selection of FAB libraries by phage display Specific anti-CD20 human phage Fab antibodies were obtained from the combinatorial phage Fab display library MeganLibTM. Biopanning was performed against human CD20-expressing eukaryotic cells by phage display [Nat Biotechnol. 1996 Mar;14(3):309-14; J Mol Biol.1991 Dec 5;222(3):581-97] using magnetic beads and a KingFisher Flex device, as this technology allows for up to 96 different biopanning schemes and variations to be performed simultaneously.

[0228] During biopanning, biotinylated eukaryotic cells were immobilized on the surface of streptavidin magnetic beads by incubating the beads with the cells for 20 min on a rotating device. The beads were then washed with PBS (pH 7.4) and then blocked with a 2% nonfat milk solution in PBS (pH 7.4) for 1 h. The phage solution pre-incubated with antigen-negative cells in PBS (pH 7.4) supplemented with 2% skim milk was then added to the magnetic beads with bound cells. The mixture was incubated for 40 min with stirring. Unbound phages were removed by washing the magnetic beads with several cycles of PBS solution (pH 7.4) supplemented with 0.1% Tween-20. The number of washing cycles was increased from cycle to cycle (10 washing cycles in the first cycle, 30 washing cycles in the second and third cycles). The phages bound to the antigens on the magnetic bead surface were eluted from the beads with 100 mM Gly-HCl solution (pH 2.2) for 15 min with stirring, and then the solution was neutralized with 1 M Tris-HCl (pH 7.6). The resulting phages were used to infect E. coli TG1 bacteria, in which the phages were cultured, isolated, and used in the next selection cycle. After 3-4 cycles, DNA (phagemid) was isolated from the phages, and the antibody variable domain genes were cloned into an expression vector (Figure 6) for producing Fab in E. coli cells.

[0229] Example 4. Library screening Initial Screening Fabs were produced according to standard techniques: bacterial cells were transformed with an expression vector containing the Fab gene, while during cultivation of the resulting transformants, expression of the Fab was induced by subsequent addition to the medium of an inducer that induces transcription of the lac operon.

[0230] We then performed an ELISA for binding of Fab to the support-immobilized CD20 peptide. Antigen-binding Fab was detected using an anti-human Fab HRP-conjugated secondary antibody (Pierce-ThermoScientific).

[0231] The rituximab Fab sequence inserted into the expression plasmid pLL was used as a positive control (Figure 6). As a result of the first screening, we selected clones capable of binding to the target CD20 peptide. The material was transferred for a second screening.

[0232] Secondary Screening The secondary screening aimed to select Fab-producing clones that interacted with the CD20 peptide and not with other antigens, IL6R-Fc, PCSK9-VG-FE, PD-1-Fc.

[0233] Fabs were produced according to standard techniques, and we then performed ELISA for binding of the Fabs to various support-immobilized antigens according to standard methods. As a result of the secondary screening, Fab-producing clones that specifically bound only to the target CD20 peptide were selected.

[0234] Example 5. Optimization of leader candidates The selected candidates were optimized to increase humanization. The Humanizer tool of YLab software package (developed by BIOCAD) was used to select substitution points. Germline functional V, D, J segments from various biological species were obtained from IMGT database and used as data source. Human segments were used as positive reference, while those from rat and mouse were used as negative reference. Based on this data, the tool suggested the positions to be substituted.

[0235] For further selection, we generated 1000 candidates, including multiple subsets of the selected substitution set. The resulting candidates were modeled based on the crystal structure of the initial candidate-target CD20 complex. The models were generated using BENDER (developed by BIOCAD) and BioLuminate (developed by Schrodinger, from Schrodinger Suite software). The resulting models were evaluated by calculating the average MM-GBSA along 100ns molecular dynamics trajectory using the OPLS 2005 force field. The molecular dynamics trajectory was obtained using Desmond (developed by Schrodinger, Schrodinger Suite). In the obtained results, we clearly distinguished 133 candidate clusters, which were suggested for further synthesis together with control candidates.

[0236] Example 6. Preparation of full-length antibodies in IgG1 format The 133 candidates prepared were codon-optimized for CHO cells using the OligoDesigner tool of the Ylab software package (BIOCAD). Optimized sequences of the heavy and light chain variable domains were de novo synthesized and cloned into the Sal1 / Nhe1 and Sal1 / BsiW1 restriction sites of vectors pEE-HC and pEE-CK (IgG1 format), respectively (Figures 1 and 2). A schematic diagram of the IgG1 format is shown in Figure 3.

[0237] The resulting gene constructs were used to transform a CHO-T cell line. Proteins were isolated and purified according to standard methods by affinity chromatography on bacterial protein A as described in Example 1. Electrophoresis was performed in a denaturing 7.5% PAGE. The production performance of 22 candidates was below the threshold level (50 mg / l); therefore, they were not isolated and purified.

[0238] Example 7. Sequencing of high affinity clones The variable domain genes of positive clones were sequenced and analyzed according to standard protocols on an Applied Biosystems 3130 Genetic Analyzer (Applied Biosystems).

[0239] Example 8. Determination of affinity of full length antibodies on Forte Bio Octert RED 384 The KD values ​​of the resulting full length candidates were determined on a Forte Bio Octet RED 384.

[0240] SAX biosensor and biotin-modified CD20 peptide (Sigma Aldrich) were used in the study. The antibody, rituximab, was used as a control. The SAX biosensor was immersed in a solution containing biotinylated CD20 peptide at a concentration of 20 μg / ml, where the peptide was immobilized. Further analysis was performed at 30° C. using PBS containing 0.1% Tween 20 and 0.1% BSA as the working buffer.

[0241] After recording a baseline in buffer solution, the sensor was immersed in a well containing an antibody solution at a concentration of 10 μg / ml for 150 seconds, where the complexes associated, and then complex dissociation in buffer solution was detected for 300 seconds.

[0242] Binding curves after subtraction of the reference signal were analyzed using a 1:1 interaction model according to standard methods using Octet data analysis software (version 9.0). 116 candidates were transferred for analysis. Of these, 67 did not show any binding to the peptide. The remaining 49 candidates interacted with the peptide with nanomolar and micromolar affinities (Figure 7 and Table 1).

[0243] Table 1

[0244] [Table 2-1]

[0245] [Table 2-2]

[0246] [Table 2-3]

[0247] [Table 2-4]

[0248] [Table 2-5]

[0249] Based on the results of the above analyses, candidates BCD132L-026, BCD132L-028, BCD132L-075 and BCD132L-077 were selected. Example 9. Determination of affinity of final candidates after transient production to CD20 using Forte Bio Octert RED 384 SAX biosensor and biotin-modified CD20 peptide (Sigma Aldrich) were used in the study. The antibody, rituximab, was used as a control. The SAX biosensor was immersed in a solution containing biotinylated CD20 peptide at a concentration of 20 μg / ml, where the peptide was immobilized. Further analysis was performed at 30° C. using PBS containing 0.1% Tween 20 and 0.1% BSA as the working buffer.

[0250] After recording a baseline in buffer solution, the sensor was immersed for 210 seconds in a well containing an antibody solution at a concentration of 10 μg / ml, where the complexes associated, and then complex dissociation in buffer solution was detected for 100 seconds.

[0251] Binding curves after subtraction of the reference signal were analyzed using Octet data analysis software (version 9.0) according to standard methods using a 1:1 interaction model (see Figures 8-11).

[0252] [Table 3]

[0253] Based on the results of the above analysis, candidates BCD132L-028 and BCD132L-077 were selected. Example 10. Preparation of cell lines stably producing IgG1-format antibodies Based on the above assay results, BCD132-L-028, BCD132-L-077 showed the best performance. These heavy and light chain sequences were cloned into the HindIII, XbaI sites of vector pSX (Figure 24). The resulting plasmids were cultured in E. coli cells and 600-700 μg were isolated using BenchPro. The plasmids were linearized overnight by PvuI endonuclease and then reprecipitated with ethanol to a final concentration of 900-1100 ng / μl.

[0254] The CHO-K1-S cell line was cultured in S.3.87 MM medium (FBS-free synthetic medium developed by BIOCAD) + 6 mM glutamine. Transfection with gene constructs containing sequences encoding the BCD132-L-028 and BCD132-L-077 candidate chains was performed using a Nucleofector according to the manufacturer's protocol. TM (Lonza) was used for electroporation.

[0255] The day after transfection, the transfected cultures were selected for 24 days by adding puromycin (7.2 μg / ml final concentration) and hygromycin B (640 μg / ml final concentration) to the medium. The selected cell populations were cloned. Based on the analysis results of target protein level / structure homogeneity, taking into account the proliferation rate, population homogeneity, and the absence of morphological changes, cell clones expressing BCD132-L-028 and BCD132-L-077, respectively, were selected, while candidates BCD132-L-026 and BCD-132-L-075 were eliminated.

[0256] Example 11. Determination of affinity of final candidates cultured in stable cell lines for CD20 on Forte Bio Octert RED 384 SAX biosensor and biotin-modified CD20 peptide (Sigma Aldrich) were used in the study. The antibody, rituximab, was used as a control. The SAX biosensor was immersed in a solution containing biotinylated CD20 peptide at a concentration of 20 μg / ml, where the peptide was immobilized. Further analysis was performed at 30° C. using PBS containing 0.1% Tween 20 and 0.1% BSA as the working buffer.

[0257] After recording a baseline in buffer solution, the sensor was immersed in a well containing an antibody solution at a concentration of 10 μg / ml for 150 seconds, where the complexes associated, and then complex dissociation in buffer solution was detected for 300 seconds.

[0258] Binding curves after subtraction of the reference signal were analyzed using a 1:1 interaction model according to standard methods using Octet data analysis software (version 9.0) (Figures 12-15).

[0259] [Table 4]

[0260] Example 12. Determination of affinity of final candidates cultured in stable cell lines for FcγRIIIa-158F on Forte Bio Octert RED 384 SAX biosensor and biotin-modified FcγRIIIa-158F protein (Sigma Aldrich) were used in the study. The SAX biosensor was immersed in a solution containing biotinylated protein at a concentration of 5 μg / ml, where the protein was fixed at a signal level of 0.5 nm. Further analysis was performed at 30° C. using PBS containing 0.1% Tween 20 and 0.1% BSA as the working buffer.

[0261] After recording a baseline, the sensor was immersed for 90 seconds in wells containing antibody solutions of various concentrations, where the complexes associated, and then complex dissociation in a buffer solution was detected for 150 seconds.

[0262] Binding curves after subtraction of the reference signal were analyzed using a 1:1 interaction model according to standard methods using Octet data analysis software (version 9.0) (Figures 16-19).

[0263] [Table 5]

[0264] Example 13. Determination of affinity of final candidates cultured in stable cell lines for FcγRIIIa-158V on Forte Bio Octert RED 384 SAX biosensor and biotin-modified FcγRIIIa-158V protein (Sigma Aldrich) were used in the study. The SAX biosensor was immersed in a solution containing biotinylated protein at a concentration of 5 μg / ml, where the protein was fixed at a signal level of 0.5 nm. Further analysis was performed at 30° C. using PBS containing 0.1% Tween 20 and 0.1% BSA as the working buffer.

[0265] After recording a baseline, the sensor was immersed for 90 seconds in wells containing antibody solutions of various concentrations, where the complexes associated, and then complex dissociation in a buffer solution was detected for 150 seconds.

[0266] Binding curves after subtraction of the reference signal were analyzed using a 1:1 interaction model according to standard methods using Octet data analysis software (version 9.0) (Figures 20-23).

[0267] [Table 6]

[0268] Example 14. Measurement of specific binding of BCD-132-L-028 and BCD-132-L-077 to the CD20 receptor on the WIL2-S cell line using flow cytometry.

[0269] MabThera (rituximab) was used as a control antibody. Samples and control antibodies were diluted to a concentration of 200 μg / ml and titrated in increments of 4 in staining buffer (PBS, 0.5% BSA, 0.1% NaN3). 6WIL2-S (ATCC® CRL8885) cell suspensions at a concentration of 100 μl / ml were incubated with a titer of standard and test sample solutions. The suspensions were vortexed and incubated in ice for 30 minutes. After the incubation time, the plates were centrifuged, the supernatants were collected, 100 μl of staining buffer was added, the mixture was resuspended, and centrifuged. The supernatants were collected and the precipitates were resuspended in a conjugated fluorescent anti-human Fc-PE antibody (Jackson Immunoresearch, 109-115-098) solution in staining buffer. The plates were incubated in the dark in ice for 30 minutes. After the incubation time, the plates were centrifuged, the supernatants were collected, 100 μl of staining buffer was added, the mixture was resuspended, and centrifuged. The supernatants were collected and the precipitates were resuspended in 150 μl of staining buffer, and assayed by a flow cytometer Guava12HT (Merck Millipore). Data were analyzed using the InCyte module of guavaSoft 3.1.1 software.

[0270] The specific binding levels of the tested antibodies BCD-132-L-028 and BCD-132-L-077 to the CD20 receptor on the WIL2-S cell line are identical to that of MabThera (rituximab). The results are shown in FIG.

[0271] Example 15. Measurement of complement-dependent cytotoxicity of BCD-132-L-028 and BCD-132-L-077 WIL2-S (ATCC® CRL-8885 TM ) cell lines were used in complement-dependent cytotoxicity assays.

[0272] The assay was performed in RPMI-1640 supplemented with 2 mM glutamine, 0.1% bovine serum albumin, and 50 μg / ml gentamicin. Test antibodies BCD-132-L-028, BCD-132-L-077, and MabThera (rituximab) were serially diluted from a concentration of 50 μg / ml. The resulting solution was added to a 96-well plate at 50 μl / well. 1x10 6A WIL2-S cell suspension of cells / ml was prepared and added to plate wells at 50 μl / well. A working solution of complement (Quidel, A113) was prepared and added to culture plates at 50 μl / well.

[0273] Plates were incubated for 2 hours at 37°C with 5% CO2. After the incubation period, 15 μl / well of Alamar Blue dye was added to the plate wells and the plates were incubated at 37°C with 5% CO2 until gradient staining was observed. Fluorescence was measured using an Infinite M200Pro plate reader at excitation / emission wavelengths of 544 / 590 nm.

[0274] The levels of complement-dependent cytotoxicity of BCD-132-L-028 and BCD-132-L-077 are comparable to that of MabThera (rituximab). The results are shown in Figures 26 and 27.

[0275] Example 16. Measurement of antibody-dependent cellular cytotoxicity of BCD-132-L-028 and BCD-132-L-077 using the reporter strain Jurkat-NFAT-CD16. WIL-2S (ATCC® CRL-8885 TM ) as a target cell line to measure antibody-dependent cellular cytotoxicity. As effector cells, we used reporter lines Jurkat-NFAT-CD16high (high affinity allotype of CD16, V158) and Jurkat-NFAT-CD16low (low affinity allotype of CD16, F158), which stably express the cell surface FcγRIIIa (CD16a) receptor and carry a luciferase-encoding gene under the control of NFAT response elements.

[0276] We cultured 0.5×10 cells in RPMI 1640 supplemented with 2 mM L-Gln, 4% (v / v) low IgG FBS, and 5 μg / ml gentamicin. 6 A WIL-2S cell suspension was prepared at 100 cells / ml. 25 μl / well of the target cell suspension was added to a white-walled culture plate.

[0277] We tested BCD-132-L-028 or BCD-132-L-077 and MabThera (rituximab) at a titer of 5 μg / ml in increments of 5 (25 μl / well) and 3x10 6 A suspension of cells / ml (25 μl / well) was added. Plates were agitated and incubated at 37° C., 5% CO2 for 4-8 hours.

[0278] After the incubation period, we added 75 μl / well of Bio-Blo luciferase assay reagent (Promega) and measured luminescence using an Infinite M200Pro with a 100 ms integration time.

[0279] BCD-132-L-028 and BCD-132-L-077 showed significantly higher ADCC activity when compared to that of MabThera: activity was 3-4 fold higher when using the high affinity CD16 arotype and reporter strain, and 12-16 fold higher when using the low affinity CD16 arotype and reporter strain. The results are shown in Figure 28; Figure 29 shows the results with the low affinity CD16 arotype and reporter strain, and Figures 30 and 31 show the results with the high affinity CD16 arotype and reporter strain.

[0280] Example 17. Measurement of BCD-132-L-028- and BCD-132-L-077-induced CD19+ B cell depletion using whole blood from healthy donors. The activity of the test samples was measured ex vivo using whole blood from healthy donors carrying the following CD16a receptor allotypes: FF (low affinity receptor), FV (heterozygote), and VV (high affinity receptor).

[0281] MabThera (rituximab) was used as a control antibody. Control and test antibodies were titrated in triplicate in 96-well plates. Blood from healthy donors was collected in vacuum tubes containing Li-heparin. The tubes were incubated at room temperature for 30 minutes. 10 μl of prepared antibody solution and 190 μl of whole blood were added to a 96-well plate (Eppendorf) with side grooves. 5 ml of DPBS was added to the side grooves of the 96-well plate. The plates were agitated at 600 rpm on an orbital shaker (2 mm) for 2 minutes and then incubated in a CO2 incubator for 22 hours.

[0282] After the incubation period, samples were stained with fluorescently labeled antibodies against CD45, CD3 / CD19 (BD Pharmingen) for 1 hour. We fixed the cells and lysed the red blood cells using BD Pharmingen lysis buffer, washed twice with lysis buffer in staining buffer (DPBS, 0.1% NaN3, 0.5% BSA), and measured the number of CD45+CD3+ and CD45+CD19+ events (at least 10,000 events within the gate CD45+). Data were analyzed using the InCyte module of guavaSoft 3.1.1 software.

[0283] The B cell / T cell ratio of antibody-free points was used to measure relative B cell depletion (B cell count taken as 100% = 0% B cell depletion) using the following formula:

[0284] [ka]

[0285] The B cell / T cell ratio was calculated using the following formula: The following formula:

[0286] [ka]

[0287] The percentage of B cell depletion was calculated using the formula: The four-parameter curves were plotted using the statistical environment R with the extended package drc.

[0288] The tested antibodies BCD-132-L-028 and BCD-132-L-077 show significantly higher activity when compared to MabThera (Rituximab). Thus, when using donor blood of allotype FF, BCD-132-L-028 and BCD-132-L-077 induce approximately 50% depletion of CD19+ cells, whereas MabThera (Rituximab) induces approximately 20% depletion. The ED50 value of MabThera (Rituximab) is significantly higher than that of BCD-132-L-028 and BCD-132-L-077 when using donor blood of CD16 allotypes FV and VV. The level of B cell depletion of BCD-132-L-028 and BCD-132-L-077 is independent of donor CD16 allotype. The results are shown in Figure 32.

[0289] Example 18. Antibody-dependent cellular cytotoxicity (ADCC) activity assay of anti-CD20 antibody candidates against Ramos cell line using human peripheral blood mononuclear cells (PBMC). CD20-expressing Ramos cell line and PBMCs from healthy donors were used for the ADCC assay. Ramos cells were cultured at 37°C and 5% CO2 in RPMI-1640 medium supplemented with 10% FBS (fetal bovine serum), and cells were stained with the fluorescent dye calcein AM, which can escape only from cells with damaged cell walls. 5 A cell suspension at a density of 100 cells / ml was prepared in RPMI-1640 medium supplemented with 10% FBS.

[0290] Ficoll density gradient separation (1.077g / cm 3 PBMCs were isolated from venous blood from healthy donors by the method described above. * 10 6 A cell suspension at a density of 100 cells / ml was prepared in RPMI-1640 medium supplemented with 10% FBS.

[0291] For the ADCC assay, 50 μl / well of the antibody dilutions were added to wells of a 96-well plate. 100 μl / well of Ramos suspension and 50 μl / well of PBMC suspension were added to these. The plates were incubated for 4 hours at 37°C with 5% CO2. 30 minutes before the end of the incubation, 10 μl / well of 10% Triton X-100 was added to the maximum lysis wells. After incubation, 100 μl / well of the cell solution was transferred to a new plate without harvesting the cells. Fluorescence was measured at excitation / emission wavelengths of 485 / 538 nm.

[0292] The following formula:

[0293] [ka]

[0294] The ADCC efficiency was calculated using the following formula: Based on the ADCC as a function of antibody concentration, we determined the dependence described by a four-parameter equation and calculated the half-maximal effective concentration (EC50) using the GraphPad Prism 6.0 software package.

[0295] According to the ADCC assay, the anti-CD20 antibody candidates BCD-132-L-028 and BCD-132-L-077 show better performance than that of rituximab. The results are shown in Figure 33.

[0296] Example 19. Activity study of the BCD132-L-077 monoclonal antibody product after repeated intravenous administration in cynomolgus monkeys against a model of experimental autoimmune encephalomyelitis (EAE).

[0297] The study was carried out in male cynomolgus monkeys. A total of 12 animals were involved in the experiment, with each group containing 4 monkeys. We used two product doses in the experiment; 5 mg / kg; 22 mg / kg, and the control group of animals received a placebo product. The data of the animal experimental groups are shown in Table 2.

[0298] Table 2. Animal groups

[0299] [Table 7]

[0300] To induce experimental autoimmune encephalomyelitis in cynomolgus monkeys, we used a modified technique described in several publications. To sensitize primates, we used JSC <biocad>The recombinant protein used is the extracellular domain of human myelin oligodendrocyte protein (rhMOG, amino acids 1-125).

[0301] Each animal was injected three times with an emulsion containing 400 μg of protein in 400 μl of phosphate-buffered saline mixed with 400 μl of Freund's complete adjuvant. Immediately after the first dose of rhMOG, the monkeys were injected with heat-inactivated B. pertussis vaccine.

[0302] First administration of rhMOG To prepare the emulsion, 10 mg of rhMOG was dissolved in 10 ml of phosphate buffered saline. To the resulting solution, 10 ml of Freund's complete adjuvant was added. The resulting emulsion was administered subcutaneously by eight point 100 μl injections (total dose volume per monkey is 800 μl): 4 injections in the spinal cord area (between the scapulae, 2 on the right side and 2 on the left side of the spinal cord); Two injections in the groin (one on the right and one on the left); Two injections into the axillary space (one on the right and one on the left); After the first dose of rhMOG, 10 10 Inactivated Bordetella pertussis particles were administered intravenously. The interval between the first and second immunizations was 28 days.

[0303] Second administration of rhMOG To prepare the emulsion, 10 mg of rhMOG was dissolved in 10 ml of phosphate buffered saline. To the resulting solution, 10 ml of Freund's complete adjuvant was added. The resulting emulsion was administered subcutaneously by eight point 100 μl injections (total dose volume per monkey is 800 μl): 4 injections in the spinal cord area (between the scapulae, 2 on the right side and 2 on the left side of the spinal cord); Two injections in the groin (one on the right and one on the left); Two injections into the axillary space (one on the right and one on the left); The interval between the second and third immunizations was 14 days.

[0304] 3. Third Administration of rhMOG To prepare the emulsion, 10 mg of rhMOG was dissolved in 10 ml of phosphate buffered saline. To the resulting solution, 10 ml of Freund's complete adjuvant was added. The resulting emulsion was administered subcutaneously by eight point 100 μl injections (total dose volume per monkey is 800 μl): 4 injections in the spinal cord area (between the scapulae, 2 on the right side and 2 on the left side of the spinal cord); Two injections in the groin (one on the right and one on the left); Two injections into the axillary space (one on the right and one on the left);

[0305] Evaluating the efficacy of BCD132-L-077 products in models of experimental autoimmune encephalomyelitis (EAE) Histological examination of brain and spinal cord tissues was performed to measure product activity. The severity of inflammatory reaction in spinal cord and brain tissues was scored on a 3-point scale according to Table 3.

[0306] Table 3. Inflammatory Response Severity Scale

[0307] [Table 8]

[0308] The severity of degenerative changes in primate spinal cord and brain tissue was scored on the scale shown in Table 4.

[0309] Table 4. Spinal Cord / Brain Tissue Demyelination Severity Scale

[0310] [Table 9]

[0311] The results of the inflammation severity measurements are shown in Figure 34. It has been shown that there is a reduction in the total group score when compared to the control group (sham control) at the doses of 5.0 mg / kg and 22.0 mg / kg used in the study. The changes detected were not reliable and there were no significant differences between the experimental groups. Therefore, it can be said that there is a comparable anti-inflammatory efficacy for the two tested doses of the product.

[0312] The results of measuring the severity of degenerative changes in nervous tissue are shown in Figure 35. When using the test product BCD132-L-077 at the minimum dose of 5.0 mg / kg, the inventors observed a significant reduction in the demyelination score compared to that of the control.

[0313] The group of animals administered the product at a dose of 22.0 mg / kg also showed a decrease in the parameter values ​​in question, but not reliably. There were no significant differences between the values ​​of the experimental groups; therefore, it may be concluded that the activity levels of the two doses were comparable.

[0314] Studies have shown that at doses of 5.0 mg / kg and 22.0 mg / kg, the product exhibits comparable anti-inflammatory effects in terms of efficacy and reduces the level of demyelination to a similar extent in the nervous tissue of experimental primates. Based on the above data, the dose of 5.0 mg / kg can be established as the pharmacologically active dose (FAD).

[0315] Example 20. Toxicity and main pharmacokinetic parameters (toxicokinetics) study of BCD132-L-077 after multiple subcutaneous administration in cynomolgus monkeys. The study was carried out in male cynomolgus monkeys. After quarantine, the animals were divided into four experimental groups, each containing three males, according to the dose of the product to be administered; body weight was used as the criterion for distributing the monkeys into groups. We used three product doses in the experiment: 44 mg / kg; 88 mg / kg; 176 mg / kg, and the animals in the control group received a placebo product. The condition of the animals, the number of dead animals and the timing of their death were used as evaluation criteria. The animals were observed 8 hours after injection and then daily for 42 days. Data regarding animal study groups and product doses are shown in Table 5:

[0316] Table 5. Animal groups in efficacy studies of BCD132-L-077 products

[0317] [Table 10]

[0318] The condition of the animals, the number of dead animals and the timing of their death were used as evaluation criteria. Within the study, clinical examinations were performed 8 hours after injection and then daily; in addition, we assessed the following: Animal weight; Body temperature; · Urine testing; Complete blood analysis for the following parameters: red blood cell count, white blood cell count, hemoglobin concentration; · Serum biochemical analysis for the following parameters: lactate dehydrogenase, total bilirubin, total protein, glucose, aspartate aminotransferase, alanine aminotransferase; · Product concentration in serum.

[0319] According to the study, the test product did not induce death in cynomolgus monkeys after a single intravenous administration and the administration process was well tolerated by the experimental animals. The product did not show any effect on the integrated toxicity index and, according to the parameters in question, also on the functional status of the organs. In the selected dose range, the product shows linear pharmacokinetics.

[0320] Example 21. Pharmacokinetic and immunogenicity study of repeated intravenous administration of the BCD132-L-077 product in cynomolgus monkeys for 4 weeks, followed by a 2-week recovery period. The pharmacokinetics and immunogenicity after repeated intravenous administration were studied using doses of 22.0, 44.0 and 88.0 mg / kg. A total of 18 sexually mature monkeys, 9 female and 9 male (cynomolgus) monkeys aged 4-7 years, were included in the study. The animals were divided into 3 groups based on the dose of the product to be administered.

[0321] Table 6. Animal groups

[0322] [Table 11]

[0323] Levels of BCD132-L-077 were measured in primate serum by enzyme-linked immunosorbent assay. During the study, the immunogenicity of the BCD132-L-077 product was measured to establish the possible effect of the formation of product-binding antibodies on pharmacokinetic parameters. We also used the ratio AUC SS168(336~504) :AUC 0~168 The accumulation coefficient was calculated from

[0324] AUC 0~168 To calculate the values, serum was collected immediately before the first administration of the product and then 0.25, 24, 72 and 168 hours after administration; AUCss 168(336~504) Values ​​were calculated, serum was taken immediately before the fourth dose of product, and pharmacokinetic parameters were calculated based on data from only animals where serum was taken at 0.25, 24, 72 and 168 hours after dosing but no detectable reaction was detected. Data from animals showing an immune response to the product were therefore excluded from the calculation of pharmacokinetic parameters. Product accumulation was measured by an accumulation index; for this purpose, AUC 0~168 and AUCss (336~504) value is determined and calculated according to the following formula:

[0325] [ka]

[0326] In the formula, AUCss 168(336~504) is the equilibrium value of the area under the product concentration-time curve under repeated dosing over a period corresponding to the dosing interval (168 hours); AUC 0~168 is the area under the product concentration-time curve from the moment the product is introduced into the body to 168 hours after the first dose The index was calculated by

[0327] To analyze the levels of antibody-bound BCD132-L-077 products, we used primate serum. Study samples were taken before the first dose and then at weeks 4 and 7 of the study.

[0328] Example 22. Comparison of pharmacokinetic parameters under repeated intravenous administration of increasing doses (22.0 mg / kg, 44.0 mg / kg, 88.0 mg / kg) of BCD132-L-077 product.

[0329] Figure 36 shows the concentration-time curves of the smoothed BCD132-L-077 product for primate serum. Table 7 shows the mean values ​​of the main pharmacokinetic parameters for the groups. Table 7. Comparative data on the main pharmacokinetic parameters under administration of increasing doses of the BCD132-L-077 product (steady-state (ss) values ​​were calculated using a period of 336 to 504 hours (168 hours) corresponding to the dosing interval).

[0330] [Table 12]

[0331] The pharmacokinetic parameters of the BCD132-L-077 product were calculated using steady-state AUC values ​​during the dosing period (168 hours) 336-504 hours after product administration. The initial AUC calculated during the first dosing interval (1-168 hours) was directly related to the dose used. In the lowest dose group, this parameter was 13,435.59 ± 11,159.80 (μg / ml) hours; in the average dose group: 13,301.04 ± 8,719.10 (μg / ml) hours; and in the highest dose group: 34,145.90 ± 16,765.50 (μg / ml) hours. The AUC calculated at steady state (336-504 hours) also depended on the dose used. The AUCss168(336-504) was 26,227.47 ± 16,819.40 (μg / ml) hours in the group of animals that received the product at a dose of 22 mg / kg; in the intermediate dose group, the value was 22,366.93 ± 10,370.60 (μg / ml) hours, and in the maximum dose group, it was 97,088.33 ± 94,675.60 (μg / ml) hours. The half-life value (T1 / 2) was 94.76 ± 50.30 hours in the group of animals that received the product at a dose of 22 mg / kg, 155.38 ± 111.20 hours in the intermediate dose group, and 471.00 ± 782.50 hours in the maximum dose group. Clearance was 0.00924±0.004 l / h in the lowest dose group, 0.01873±0.015 l / h in the intermediate dose group, and 0.01228±0.005 l / h in the highest dose group. The mean residence time (MRT) of the product was 66.37±13.60 h in the lowest dose group, 68.30±17.50 h in the intermediate dose group, and 71.51±9.20 h in the intermediate dose group. The steady-state volume of distribution (V dss ) were 521.65 ± 186.90 ml / kg, 1,639.68 ± 1,170.50 ml / kg, and 2,384.08 ± 2,213.30 ml / kg in the lowest, middle, and highest dose groups, respectively. ss ) were 156.12 ± 100.10 μg / ml, 133.14 ± 61.70 μg / ml, and 577.91 ± 563.50 μg / ml in the lowest, middle, and highest dose groups, respectively. ssmax ) were 326.89 ± 241.70 μg / ml, 427.03 ± 266.50 μg / ml, and 1,212.72 ± 830.90 μg / ml. ssmin ) were 82.64±75.20 μg / ml, 84.58±63.90 μg / ml, and 162.88±74.70 μg / ml. The data obtained indicate that the product concentrations in primate serum are directly related to the BCD132-L-077 dose used in the study.

[0332] For the group of animals receiving the product at the lowest dose of 22.0 mg / kg, the accumulation index (R) was 1.95. For the group of animals receiving BCD-132 at the intermediate dose of 44.0 mg / kg, the accumulation index R was 1.68. For the group of animals receiving the product at the highest dose (88.0 mg / kg), the index R was 2.84. The experimental data obtained show that the accumulation index value is independent of the dose used.

[0333] During the study, the immunogenicity of the BCD132-L-077 product was measured to establish the possible impact of the formation of product-binding antibodies on the pharmacokinetic parameters. Experimental data show the presence of BAbs in 11.11% of all animals involved in the study. No gender dependency was observed; BAbs were observed in one male and one female. Product-binding antibodies were found in the mid- and maximum dose groups, with only one animal in each group. Pharmacokinetic parameters may be evaluated in all animal groups, provided that experimental data on animals with established presence of binding antibodies are excluded from processing.

[0334] Example 23. Study of toxicity and local irritation effects after repeated intravenous administration of the BCD132-L-077 product in cynomolgus monkeys for 4 weeks, followed by a 2-week recovery period. A total of 30 cynomolgus monkeys, 15 males and 15 females, aged 4-7 years, were involved in the study. Each group contained 6 monkeys. Product toxicity was studied under repeated administration at doses of 22 mg / kg, 44 mg / kg and 88 mg / kg. According to the dose of the product to be administered and the time to euthanasia, the animals were divided into 5 groups: BCD132-L-077 product at a minimum dose of 22 mg / kg (3 females and 3 males); BCD132-L-077 product at an intermediate dose of 44 mg / kg (3 females and 3 males); BCD132-L-077 product at a maximum dose of 88 mg / kg, euthanized after completion of dosing (3 females and 3 males); BCD132-L-077 product at a maximum dose of 88 mg / kg, euthanasia after completion of the recovery period (3 females and 3 males); and sham controls (3 females and 3 males).

[0335] Data regarding the animal study groups are shown in Table 8. Table 8. Animal groups

[0336] [Table 13]

[0337] Within the study, clinical tests were performed daily; in addition, the inventors investigated: Animal weight; Body temperature; · Urine testing; ·ECG; · Hemostatic parameters: activated partial thromboplastin time, fibrinogen concentration, prothrombin time; Complete blood analysis for the following parameters: red blood cell count, white blood cell count, hemoglobin concentration, lymphocytes, monocytes, neutrophils, eosinophils, basophils; · Serum biochemical analysis for the following parameters: lactate dehydrogenase, total bilirubin, total protein, glucose, aspartate aminotransferase, alanine aminotransferase, cholesterol, triglycerides, urea, creatinine (createnine), sodium, potassium, alkaline phosphatase; -Pathomorphological and histological studies.

[0338] Local irritant effects were evaluated based on laboratory data and histological examination results. Administration site tissues and draining lymph nodes were selected for histological examination. According to histological examination, the test product does not show any local irritant effect.

[0339] JSC <biocad>The data obtained in the toxicity study of the BCD132-L-077 therapeutic monoclonal antibody product produced by Pharmacia show that the test product does not show toxic effects on the major organs and organ systems of experimental animals under repeated weekly intravenous administration for 4 weeks. As established in this study, the absence of observed adverse effects level (NOAEL) corresponds to the maximum dose of the test product BCD132-L-077 and amounts up to 88 mg / kg.

[0340] Example 24. Study of the antitumor activity of BCD132-L-028 products after repeated intraperitoneal administration in immunodeficient hIL15-NOG mice humanized with human NK cells against a subcutaneous xenograft model using the Raji human lymphoma cell line.

[0341] In this study, we plan to use seven groups of immunodeficient transgenic hIL15-NOG mice that constitutively express human IL-15 and weigh between 15.0 and 25.0 g. A total of 84 female animals were involved in the study. Data regarding the groups are shown in Table 9.

[0342] Table 9. Animal groups

[0343] [Table 14]

[0344] Animals are weighed prior to administration of the tumor cell lines and then twice weekly throughout the experiment. Following tumor cell administration, tumor nodules are measured twice weekly throughout the experiment. The volume of the tumor nodule is calculated by the following formula: V=π / 6xLxWxH, where L, W, H are the tumor linear dimensions.

[0345] The efficacy of the test products is evaluated by the Tumor Growth Inhibition Index (TGI) versus the Tumor Growth Index (I). The index is calculated according to the following formula:

[0346] [ka]

[0347] In the formula, V k and V о are the median tumor volumes (MM) in the control and treatment groups, respectively. 3 ). I i =V i / V o where I is the tumor growth index, i is the day of the experiment, and V is the tumor volume per day.

[0348] Example 25. Study of toxicity and main pharmacokinetic parameters (toxicokinetics) of the BCD132-L-028 monoclonal antibody product after a single intravenous dose in cynomolgus monkeys. In the study, we plan to use four experimental groups of cynomolgus monkeys, each group containing three males. The monkeys will be kept in individual cages containing animal number indications according to the experiment.

[0349] Animals are randomly assigned to groups according to the dose of material to be administered, using body weight as a criterion. Table 10. Animal groups

[0350] [Table 15]

[0351] The product is planned to be administered intravenously in the ulnar vein as a sterile isotonic saline solution. A control group of animals receives isotonic saline (placebo) using the same volume and method as used in the experimental group.

[0352] Within the study, clinical tests are performed daily; in addition, we examine: 1. Animal weight; 2. Body temperature; 3. Urine testing; 4. Complete blood analysis for the following parameters: red blood cell count, white blood cell count, hemoglobin concentration; 5. Serum biochemical analysis for the following parameters: lactate dehydrogenase, total bilirubin, total protein, glucose, aspartate aminotransferase, alanine aminotransferase; 6. Study of product concentrations in serum, calculation of main pharmacokinetic parameters, and evaluation of pharmacokinetic linearity.

[0353] Example 26. Pharmacokinetic and immunogenicity study of repeated intravenous administration of BCD132-L-028 monoclonal antibody product in cynomolgus monkeys for 13 weeks followed by a 30-day recovery period. We plan to use 18 cynomolgus monkeys (9 males and 9 females) in a pharmacokinetic and immunogenicity study after 13 weeks of repeated intravenous administration followed by a 30 day recovery period.

[0354] The animals were assigned to three groups (Table 11) according to the dose of substance to be administered, each group containing 3 females and 3 males: the lowest dose group (6 mg / kg), the middle dose group (20 mg / kg) and the highest dose group (60 mg / kg).

[0355] Table 11. Animal groups

[0356] [Table 16]

[0357] In the study, the inventors: 1. Evaluate test product concentrations in serum of experimental animals; 2. Evaluate test product accumulation under increasing doses; 3. Assess the levels of bound antibodies under repeated intravenous administration of the test product Plan accordingly.

[0358] Example 27. Study of toxicity and local irritation effects after repeated intravenous administration of BCD132-L-028 monoclonal antibody product in cynomolgus monkeys for 13 weeks, followed by a 30 day recovery period.

[0359] In the study, we planned to use five experimental groups of cynomolgus monkeys, each group including three males and three females: a minimum dose group (6 mg / kg, no necropsy), a mid-dose group (20 mg / kg, no necropsy), a satellite maximum dose group (60 mg / kg, necropsy at the end of the product administration period), a main maximum dose group (60 mg / kg, necropsy at the end of the recovery period) and a sham control group (necropsy at the end of the recovery period).

[0360] Table 12. Animal groups ( * -Satellite group of animals)

[0361] [Table 17]

[0362] Clinical examination of each animal will be performed daily; in addition, we will assess: Animal weight; · body temperature (before dosing and then weekly until the end of the study); Effects on the cardiovascular system using Poly-Spectr electrocardiography; · Urine testing; · Complete blood analysis for the following parameters: red blood cell count, white blood cell count, hemoglobin concentration, lymphocyte count, monocyte count, neutrophil count, eosinophil count, basophil count; · Evaluation of the effect on the coagulation system with regard to the following parameters: activated partial thromboplastin time, fibrinogen concentration, prothrombin time; · Serum biochemical analysis for the following parameters: sodium, potassium, creatinine, urea, alkaline phosphatase, lactate dehydrogenase, total bilirubin, total protein, glucose, triglycerides, aspartate aminotransferase, alanine aminotransferase, total cholesterol; At the end of the product administration period (week 14), we plan to euthanize 3 males and 3 females from each animal group. The remaining 2 males and 2 females from each group will be euthanized at the end of the recovery period (week 18). Local irritation effects will be evaluated based on necropsy data and histological examination results. For histological examination, we will select sections of the ulnar vein, administration site tissues and draining lymph nodes. Without being limited thereto, the present invention includes the following aspects. [Aspect 1] A monoclonal antibody or antigen-binding fragment thereof that specifically binds to CD20: 1) a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:6; 2) A light chain variable domain comprising the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 8. The monoclonal antibody or antigen-binding fragment thereof comprising: [Aspect 2] 1) the heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NO:2; 2) the light chain variable domain comprises the amino acid sequence shown in SEQ ID NO:4 2. The monoclonal antibody or antigen-binding fragment thereof according to embodiment 1. [Aspect 3] 1) the heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NO:6; 2) the light chain variable domain comprises the amino acid sequence shown in SEQ ID NO:8 2. The monoclonal antibody or antigen-binding fragment thereof according to embodiment 1. [Aspect 4] 1) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:5; 2) A light chain comprising the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 7. 2. The monoclonal antibody of embodiment 1, comprising: [Aspect 5] 1) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:1; 2) A light chain comprising the amino acid sequence shown in SEQ ID NO:3. 5. The monoclonal antibody of embodiment 4, comprising: [Aspect 6] 1) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:5; 2) A light chain comprising the amino acid sequence shown in SEQ ID NO:7. 5. The monoclonal antibody of embodiment 4, comprising: [Aspect 7] The monoclonal antibody of embodiment 1, wherein the antibody that specifically binds to CD20 is a full-length IgG antibody. [Aspect 8] 8. The monoclonal antibody of embodiment 7, wherein the full length IgG antibody is related to the human IgG1, IgG2, IgG3, IgG4 isotype. [Aspect 9] 9. The monoclonal antibody of embodiment 8, wherein the full length IgG antibody is related to the human IgG1 isotype. [Aspect 10] A nucleic acid encoding the antibody or antigen-binding fragment thereof according to any one of aspects 1 to 9. [Aspect 11] 11. The nucleic acid of embodiment 10, wherein the nucleic acid is DNA. [Aspect 12] An expression vector comprising the nucleic acid according to any one of aspects 10 to 11. [Aspect 13] A method for producing a host cell for producing the antibody or antigen-binding fragment thereof according to any one of aspects 1 to 9, comprising the step of transforming the cell with the vector according to aspect 12. [Aspect 14] A host cell for producing the antibody or antigen-binding fragment thereof according to any one of aspects 1 to 9, comprising the nucleic acid according to any one of aspects 10 to 11. [Aspect 15] A method for producing an antibody or an antigen-binding fragment thereof according to any one of aspects 1 to 9. 15. The method of claim 14, comprising the step of culturing a host cell of embodiment 14 in a culture medium under conditions sufficient to produce the antibody or antigen-binding fragment thereof, and thereafter, if necessary, isolating and purifying the antibody or antigen-binding fragment thereof produced. [Aspect 16] 10. A pharmaceutical composition for the prevention or treatment of a disease or disorder mediated by CD20, comprising an antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 9, in combination with one or more pharma- ceutically acceptable excipients. [Aspect 17] A pharmaceutical composition for the prevention or treatment of embodiment 16, wherein the disease or disorder is: a) a neoplastic disease or disorder; or b) an autoimmune disease or disorder The pharmaceutical composition selected from the group consisting of [Aspect 18] The pharmaceutical composition according to embodiment 17, wherein the neoplastic disease or disorder is selected from the group comprising: B-cell lymphoma or leukemia. [Aspect 19] 20. The pharmaceutical composition of embodiment 18, wherein the B cell lymphoma is selected from: non-Hodgkin's lymphoma (NHL) or Hodgkin's disease (Hodgkin's lymphoma). [Aspect 20] The pharmaceutical composition of embodiment 18, wherein the leukemia is selected from: chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL). [Aspect 21] 18. The pharmaceutical composition of embodiment 17, wherein the autoimmune disease or disorder is selected from the group comprising: rheumatoid arthritis, juvenile rheumatoid arthritis (Still's disease), systemic lupus erythematosus (SLE), lupus nephritis, ulcerative colitis, Wegener's disease, inflammatory bowel disease, idiopathic thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura (TTP), autoimmune thrombocytopenia, multiple sclerosis, psoriasis, IgA nephropathy, IgM polyneuropathy, myasthenia gravis, vasculitis, ANCA vasculitis, parenchymal organ graft rejection, graft versus host disease (GvHD), diabetes mellitus, Raynaud's syndrome, Sjogren's syndrome and glomerulonephritis. [Aspect 22] 10. A pharmaceutical combination for the prevention or treatment of a disease or disorder mediated by CD20, comprising an antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 9, and at least one different therapeutically active compound. [Aspect 23] 23. The pharmaceutical combination according to embodiment 22, wherein the different therapeutically active anti-tumor compounds are selected from a chemotherapeutic agent, an antibody or an anti-hormonal agent. [Aspect 24] 10. A method for inhibiting a biological activity of CD20 in a subject in need of such inhibition, the method comprising administration of an effective amount of an antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 9. [Aspect 25] A method for the treatment of a disease or disorder mediated by CD20, comprising the step of administering to a subject in need of such treatment a therapeutically effective amount of the antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 9, or the pharmaceutical composition according to embodiment 16. [Aspect 26] If the disease or disorder is: a) a neoplastic disease or disorder; or b) an autoimmune disease or disorder 26. The method for treating a disease or disorder according to embodiment 25, selected from the group consisting of: [Aspect 27] 27. The method for the treatment of a disease or disorder according to embodiment 26, wherein the neoplastic disease or disorder is selected from the group comprising: B-cell lymphoma or leukemia. [Aspect 28] 28. The method for the treatment of a disease or disorder according to embodiment 27, wherein the B cell lymphoma is selected from: non-Hodgkin's lymphoma (NHL) or Hodgkin's disease (Hodgkin's lymphoma). [Aspect 29] 28. The method for the treatment of a disease or disorder according to embodiment 27, wherein the leukemia is selected from: chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL). [Aspect 30] 27. The method for the treatment of a disease or disorder according to embodiment 26, wherein the autoimmune disease or disorder is selected from the group comprising: rheumatoid arthritis, juvenile rheumatoid arthritis (Still's disease), systemic lupus erythematosus (SLE), lupus nephritis, ulcerative colitis, Wegener's disease, inflammatory bowel disease, idiopathic thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura (TTP), autoimmune thrombocytopenia, multiple sclerosis, psoriasis, IgA nephropathy, IgM polyneuropathy, myasthenia gravis, vasculitis, ANCA vasculitis, parenchymal organ graft rejection, graft versus host disease (GvHD), diabetes, Raynaud's syndrome, Sjogren's syndrome and glomerulonephritis. [Aspect 31] Use of the antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 9, or the pharmaceutical composition according to embodiment 16, for treatment of a disease or disorder mediated by CD20 in a subject in need of such treatment. [Aspect 32] If the disease or disorder is: a) a neoplastic disease or disorder; or b) an autoimmune disease or disorder 28. Use of an antibody or antigen-binding fragment thereof according to embodiment 27, selected from the group consisting of: [Aspect 33] 33. Use of the antibody or antigen-binding fragment thereof according to embodiment 32, wherein the neoplastic disease or disorder is selected from the group comprising: B-cell lymphoma or leukemia. [Aspect 34] 34. The use of the antibody or antigen-binding fragment thereof according to embodiment 33, wherein the B-cell lymphoma is selected from: non-Hodgkin's lymphoma (NHL) or Hodgkin's disease (Hodgkin's lymphoma). [Aspect 35] 34. The use of the antibody or antigen-binding fragment thereof according to embodiment 33, wherein the leukemia is selected from: chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL). [Aspect 36] 33. The use of the antibody or antigen-binding fragment thereof according to embodiment 32, wherein the autoimmune disease or disorder is selected from the group comprising: rheumatoid arthritis, juvenile rheumatoid arthritis (Still's disease), systemic lupus erythematosus (SLE), lupus nephritis, ulcerative colitis, Wegener's disease, inflammatory bowel disease, idiopathic thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura (TTP), autoimmune thrombocytopenia, multiple sclerosis, psoriasis, IgA nephropathy, IgM polyneuropathy, myasthenia gravis, vasculitis, ANCA vasculitis, parenchymal organ graft rejection, graft versus host disease (GvHD), diabetes, Raynaud's syndrome, Sjogren's syndrome, and glomerulonephritis.< / biocad> < / biocad>

Claims

1. A monoclonal antibody or antigen-binding fragment thereof that specifically binds to CD20: 1) a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:2; and 2) A light chain variable domain comprising the amino acid sequence shown in SEQ ID NO:

4. The monoclonal antibody or antigen-binding fragment thereof comprising:

2. A monoclonal antibody or antigen-binding fragment thereof that specifically binds to CD20: 1) a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:6; and 2) A light chain variable domain comprising the amino acid sequence shown in SEQ ID NO:

8. The monoclonal antibody or antigen-binding fragment thereof comprising:

3. 1) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:1; and 2) A light chain comprising the amino acid sequence shown in SEQ ID NO:

3. The monoclonal antibody of claim 1 , comprising:

4. 1) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:5; and 2) A light chain comprising the amino acid sequence shown in SEQ ID NO:

7. The monoclonal antibody of claim 2, comprising:

5. The monoclonal antibody according to any one of claims 1 to 4, wherein the antibody that specifically binds to CD20 is a full-length IgG antibody.

6. The monoclonal antibody of claim 5 , wherein the full-length IgG antibody is of human IgG1, IgG2, IgG3, or IgG4 isotype.

7. The monoclonal antibody of claim 6, wherein the full-length IgG antibody is of the human IgG1 isotype.

8. A nucleic acid encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 7.

9. The nucleic acid of claim 8, wherein the nucleic acid is DNA.

10. An expression vector comprising the nucleic acid according to any one of claims 8 to 9.

11. A method for the production of a host cell for producing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, comprising the step of transforming the cell with the vector according to claim 10.

12. A host cell for producing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, comprising the nucleic acid according to any one of claims 8 to 9.

13. 17. A method for the production of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, comprising culturing a host cell according to claim 12 in a medium under conditions sufficient to produce said antibody or antigen-binding fragment thereof.

14. The method of claim 13, further comprising the step of isolating and purifying the produced antibody or antigen-binding fragment thereof.

15. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 7 in combination with one or more pharma- ceutically acceptable excipients.

16. A pharmaceutical combination comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 7 and at least one different therapeutically active compound selected from the group consisting of immunosuppressants, anti-inflammatory drugs, systemic hormonal drugs, anti-neoplastic and immunomodulatory drugs.

17. 17. The pharmaceutical combination according to claim 16, wherein the different therapeutically active compounds are different therapeutically active anti-tumor compounds selected from chemotherapeutic agents, antibodies or anti-hormonal agents.

18. 17. A pharmaceutical combination according to claim 16 for the prevention or treatment of a disease mediated by CD20.

19. If the disease is: a) a neoplastic disease, or b) Autoimmune disease 19. The pharmaceutical combination according to claim 18, selected from the group consisting of

20. 20. The pharmaceutical combination according to claim 19, wherein the neoplastic disease is selected from the group comprising: B-cell lymphoma or leukemia.

21. 21. The pharmaceutical combination of claim 20, wherein the B-cell lymphoma is selected from: non-Hodgkin's lymphoma (NHL) or Hodgkin's disease (Hodgkin's lymphoma).

22. 21. The pharmaceutical combination of claim 20, wherein the leukemia is selected from: chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL).

23. 20. The pharmaceutical combination of claim 19, wherein the autoimmune disease is selected from the group comprising: rheumatoid arthritis, juvenile rheumatoid arthritis (Still's disease), systemic lupus erythematosus (SLE), lupus nephritis, ulcerative colitis, Wegener's disease, inflammatory bowel disease, idiopathic thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura (TTP), autoimmune thrombocytopenia, multiple sclerosis, psoriasis, IgA nephropathy, IgM polyneuropathy, myasthenia gravis, vasculitis, ANCA vasculitis, parenchymal organ graft rejection, graft versus host disease (GvHD), diabetes mellitus, Raynaud's syndrome, Sjogren's syndrome and glomerulonephritis.

24. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, a pharmaceutical composition according to claim 15, or a pharmaceutical combination according to claim 16, for the manufacture of a medicament for the treatment of a disease mediated by CD20.

25. If the disease is: a) a neoplastic disease, or b) Autoimmune disease 25. The use according to claim 24, wherein the

26. 26. The use according to claim 25, wherein the neoplastic disease is selected from the group comprising: B-cell lymphoma or leukemia.

27. 27. The use according to claim 26, wherein the B cell lymphoma is selected from: non-Hodgkin's lymphoma (NHL) or Hodgkin's disease (Hodgkin's lymphoma).

28. 27. The use according to claim 26, wherein the leukemia is selected from: chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL).

29. 26. The use according to claim 25, wherein the autoimmune disease is selected from the group comprising: rheumatoid arthritis, juvenile rheumatoid arthritis (Still's disease), systemic lupus erythematosus (SLE), lupus nephritis, ulcerative colitis, Wegener's disease, inflammatory bowel disease, idiopathic thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura (TTP), autoimmune thrombocytopenia, multiple sclerosis, psoriasis, IgA nephropathy, IgM polyneuropathy, myasthenia gravis, vasculitis, ANCA vasculitis, parenchymal organ graft rejection, graft versus host disease (GvHD), diabetes mellitus, Raynaud's syndrome, Sjogren's syndrome and glomerulonephritis.

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