A monoclonal antibody that specifically binds to CD20

Abstract

The present invention relates to the field of bioengineering and provides a monoclonal antibody that specifically binds to CD20. The present invention also relates to DNA encoding the antibody, a corresponding expression vector and a method for producing the same, and a therapeutic method using the antibody.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to anti-CD20 antibodies or antigen-binding fragments thereof, and their use for treating diseases associated with B cells. More specifically, the present invention relates to monoclonal antibodies that specifically bind to CD20 (B lymphocyte antigen CD20). The present invention also relates to nucleic acids encoding the antibodies or antigen-binding fragments thereof, expression vectors, methods for producing the antibodies, and the use of the antibodies for treating diseases or conditions associated with B cells, particularly the B lymphocyte antigen CD20. Background Art

[0002] Lymphocytes are one of the many types of white blood cells; they 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 responsible for 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 a membrane-bound antibody is specific, the cell begins to divide rapidly, and its descendants differentiate into memory B cells and effector cells called "plasma cells". Memory B cells have a longer lifespan and continue to express membrane-bound antibodies with the same specificity as the original parent cell. Plasma cells do not produce membrane-bound antibodies; instead, they produce antibodies in a secretable form. Secreted antibodies are the main effector molecules of humoral immunity.

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

[0004] Considering the expression of CD20 in B-cell lymphomas, this antigen may be a valuable therapeutic target in the treatment of said lymphomas.

[0005] The antibody rituximab (RITUXAN, MabThera, Acellbia), a genetically engineered chimeric mouse / human monoclonal antibody directed against the human antigen CD20, is indicated for the treatment of patients with low-grade relapsed or refractory or follicular CD20-positive B-cell non-Hodgkin's lymphoma. Rituximab is the antibody referred to as "C2B8" in US 5,736,137 and US 5,776,456. In vitro mode of action studies have shown that rituximab binds to human complement and lyses B-lymphoid cell lines via complement-dependent cytotoxicity (CDC) (Reff et al., Blood 83 (2), 1994, pp. 435-445). In addition, this antibody has significant activity in an antibody-dependent cellular cytotoxicity (ADCC) assay. 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, pp. 435-445).

[0006] Furthermore, it was later discovered that anti-CD20 antibodies such as rituximab are also 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).

[0007] 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, pp. 988-995; and Schroff RW et al. «Human anti-murine immunoglobulin response in patients receiving monoclonal antibody therapy», Cancer Res., 45, 1985, pp. 879-885). Even chimeric molecules in which the variable (V) domains of rodent antibodies are fused to human constant (C) regions are still able to elicit a significant immune response (HACA, human anti-chimeric antibody response) (Neuberger et al., Nature (London), 314, 1985, pp. 268-270).

[0008] A powerful approach to overcome these limitations in the clinical use of monoclonal antibodies is the "humanization" of murine antibodies or antibodies from non-human species (Jones et al., Nature (London), 321, 1986, pp. 522-525; Riechman et al., Nature (London), 332, 1988, pp. 323-327).

[0009] Therefore, it would be beneficial to generate therapeutic antibodies against the CD20 antigen that produce minimal or no antigenicity when administered to a patient and that are primarily intended for use in long-term treatment.The present invention addresses this problem.

[0010] Monoclonal antibody BCD-132 selectively and specifically binds to the CD20 antigen and is a potent inhibitor of the CD20 antigen; in addition, the antibodies of the present invention have minimal antigenicity when administered to a patient. Summary of the Invention

[0011] In one aspect, the present invention relates to a monoclonal antibody or antigen-binding fragment thereof that specifically binds to CD20 and comprises:

[0012] 1) a heavy chain variable domain comprising the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 6;

[0013] 2) a light chain variable domain comprising the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 8.

[0014] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises:

[0015] 1) a heavy chain variable domain comprising the amino acid sequence shown in SEQ ID NO: 2;

[0016] 2) a light chain variable domain comprising the amino acid sequence shown in SEQ ID NO: 4.

[0017] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises:

[0018] 1) a heavy chain variable domain comprising the amino acid sequence shown in SEQ ID NO: 6;

[0019] 2) a light chain variable domain comprising the amino acid sequence shown in SEQ ID NO: 8.

[0020] In some embodiments, the monoclonal antibody comprises:

[0021] 1) a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 5;

[0022] 2) a light chain comprising the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 7.

[0023] In some embodiments, the monoclonal antibody comprises:

[0024] 1) a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 1;

[0025] 2) A light chain comprising the amino acid sequence shown in SEQ ID NO: 3.

[0026] In some embodiments, the monoclonal antibody comprises:

[0027] 1) a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 5;

[0028] 2) A light chain comprising the amino acid sequence shown in SEQ ID NO: 7.

[0029] In some embodiments, the monoclonal antibody that specifically binds to CD20 is a full-length IgG antibody.

[0030] In some embodiments, the monoclonal antibody is of human IgG1, IgG2, IgG3, IgG4 isotype.

[0031] In some embodiments, the monoclonal antibody is of the human IgG1 isotype.

[0032] In one aspect, the present invention relates to nucleic acids encoding the above-mentioned antibodies.

[0033] In some embodiments, the nucleic acid is DNA.

[0034] In one aspect, the present invention relates to an expression vector comprising the above-mentioned nucleic acid.

[0035] In one aspect, the present invention relates to a method for producing a host cell for producing the above-mentioned antibody, the method comprising transforming the cell with the above-mentioned vector.

[0036] In one aspect, the present invention relates to a host cell for producing the above-mentioned antibody, wherein the host cell comprises the above-mentioned nucleic acid.

[0037] In one aspect, the present invention relates to a method for producing the above-mentioned antibody, which comprises culturing the above-mentioned host cell in a culture medium under conditions sufficient to produce the antibody, and then isolating and purifying the obtained antibody if necessary.

[0038] In one aspect, the present invention relates to a pharmaceutical composition for treating a disease or condition mediated by CD20, comprising a therapeutically effective amount of the antibody or antigen-binding fragment thereof in combination with one or more pharmaceutically acceptable excipients.

[0039] In some embodiments, the pharmaceutical composition is intended for use in treating a disease or condition, wherein the disease or condition is selected from:

[0040] a) a neoplastic disease or condition, or

[0041] b) Autoimmune diseases or disorders.

[0042] In some embodiments, the pharmaceutical composition is intended for use in treating a neoplastic disease or disorder selected from the group consisting of: B-cell lymphoma or leukemia.

[0043] In some embodiments, the pharmaceutical composition is intended for the treatment of a B-cell lymphoma selected from the group consisting of non-Hodgkin's lymphoma (NHL) or Hodgkin's disease (Hodgkin's lymphoma).

[0044] 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).

[0045] In some embodiments, the pharmaceutical composition is intended for use in treating an autoimmune disease or disorder selected from the group consisting of 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, solid organ transplant rejection, graft-versus-host disease (GvHD), diabetes, Raynaud's syndrome, Sjögren's syndrome, and glomerulonephritis.

[0046] In one aspect, the present invention relates to a pharmaceutical combination for preventing or treating a disease or condition mediated by CD20, comprising the antibody or antigen-binding fragment thereof and at least one therapeutically active compound.

[0047] In some embodiments, the drug combination comprises a different therapeutically active anti-tumor compound selected from a chemotherapeutic agent, an antibody, or an anti-hormonal agent.

[0048] 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 administering an effective amount of the antibody or antigen-binding fragment thereof.

[0049] In one aspect, the present invention relates to a method for treating a disease or condition mediated by CD20, comprising administering to a subject in need of such treatment a therapeutically effective amount of the antibody or antigen-binding fragment thereof or the pharmaceutical composition.

[0050] In some embodiments, the method for treating a disease or condition is directed to a disease or condition selected from:

[0051] a) a neoplastic disease or condition, or

[0052] b) Autoimmune diseases or disorders.

[0053] In some embodiments, the method for treating a disease or condition relates to a neoplastic disease or condition selected from the group consisting of: B-cell lymphoma or leukemia.

[0054] In some embodiments, the method for treating a disease or condition involving a B-cell lymphoma selected from: non-Hodgkin lymphoma (NHL) or Hodgkin's disease (Hodgkin's lymphoma).

[0055] In some embodiments, the method for treating a disease or condition involving a leukemia selected from chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL).

[0056] In some embodiments, the method for treating a disease or disorder is directed to an autoimmune disease or disorder selected from the group consisting of 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, solid organ transplant rejection, graft-versus-host disease (GvHD), diabetes, Raynaud's syndrome, Sjögren's syndrome, and glomerulonephritis.

[0057] In one aspect, the present invention relates to the use of the antibody or antigen-binding fragment thereof or the pharmaceutical composition for treating a disease or disorder mediated by CD20 in a subject in need of such treatment.

[0058] In some embodiments, the antibody or antigen-binding fragment thereof is used to treat a disease or condition, wherein the disease or condition is selected from:

[0059] a) a neoplastic disease or condition, or

[0060] b) Autoimmune diseases or disorders.

[0061] In some embodiments, the antibody or antigen-binding fragment thereof is used to treat a neoplastic disease or disorder selected from the group consisting of: B-cell lymphoma or leukemia.

[0062] In some embodiments, the antibody or antigen-binding fragment thereof is used to treat a B-cell lymphoma selected from non-Hodgkin lymphoma (NHL) or Hodgkin's disease (Hodgkin's lymphoma).

[0063] In some embodiments, the antibody or antigen-binding fragment thereof is used to treat a leukemia selected from chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL).

[0064] In some embodiments, the antibody or antigen-binding fragment thereof is used to treat an autoimmune disease or disorder selected from the group consisting of 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, solid organ transplant rejection, graft-versus-host disease (GvHD), diabetes, Raynaud's syndrome, Sjögren's syndrome, and glomerulonephritis. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 . Map of the expression vector pEE CK.

[0066] Figure 2 . Map of the expression vector pEE HC.

[0067] Figure 3 . Schematic diagram of the IgG1 format.

[0068] Figure 4 . A synthesis scheme for a combinatorial library used in human experiments for the first time.

[0069] Figure 5 . Map of the phagemid used for cloning the Fab phage display library.

[0070] Figure 6 . Diagram of the expression plasmid pLL used for Fab culture.

[0071] Figure 7 BCD132L candidate-biotinylated CD20 peptide interactions were analyzed on a Forte Bio Octet RED 386.

[0072] Figure 8 The interaction of the BCD132L-026 candidate with biotinylated CD20 peptide was analyzed on a Forte Bio Octet RED 386.

[0073] Figure 9 The interaction of the BCD132L-028 candidate with biotinylated CD20 peptide was analyzed on a Forte Bio Octet RED 386.

[0074] Figure 10 The interaction of the BCD132L-075 candidate with biotinylated CD20 peptide was analyzed on a Forte Bio Octet RED 386.

[0075] Figure 11 The interaction of the BCD132L-077 candidate with biotinylated CD20 peptide was analyzed on a Forte Bio Octet RED 386.

[0076] Figure 12 The interaction of the BCD132L-028 candidate with biotinylated CD20 peptide was analyzed on a Forte Bio Octet RED 386.

[0077] Figure 13The interaction of the BCD132L-077 (1,2) candidate with a biotinylated CD20 peptide was analyzed on a Forte Bio Octet RED 386.

[0078] Figure 14 The interaction of the BCD132L-077 (3,4) candidate with biotinylated CD20 peptide was analyzed on a Forte Bio Octet RED 386.

[0079] Figure 15 The interaction of the BCD132L-077 (5,6) candidate with biotinylated CD20 peptide was analyzed on a Forte Bio Octet RED 386.

[0080] Figure 16 The BCD132L-028 candidate-FcγRIIIa-158F interaction was analyzed on a Forte Bio Octet RED 386.

[0081] Figure 17 The BCD132L-077 (1,2) candidate-FcγRIIIa-158F interaction was analyzed on a Forte Bio Octet RED 386.

[0082] Figure 18 The BCD132L-077 (3,4) candidate-FcγRIIIa-158F interaction was analyzed on a Forte Bio Octet RED 386.

[0083] Figure 19 The BCD132L-077 (5,6) candidate-FcγRIIIa-158F interaction was analyzed on a Forte Bio Octet RED 386.

[0084] Figure 20 The BCD132L-028 candidate-FcγRIIIa-158V interaction was analyzed on a Forte Bio Octet RED 386.

[0085] Figure 21 The BCD132L-077 (1,2) candidate-FcγRIIIa-158V interaction was analyzed on a Forte Bio Octet RED 386.

[0086] Figure 22The BCD132L-077 (3,4) candidate-FcγRIIIa-158V interaction was analyzed on a Forte Bio Octet RED 386.

[0087] Figure 23 The BCD132L-077 (5,6) candidate-FcγRIIIa-158V interaction was analyzed on a Forte Bio Octet RED 386.

[0088] Figure 24 . Map of the expression vector pSX.

[0089] Figure 25 Specific binding of BCD-132-L-028 and BCD-132-L-077 to the CD20 receptor on the WIL2-S cell line was measured using flow cytometry compared to MabThera.

[0090] Figure 26 Measurement of complement-dependent cytotoxicity of BCD-132-L-028 and BCD-132-L-077 compared to MabThera.

[0091] BCD-132-L-028

[0092] ● MabThera ■ BCD-132-L-028 ▼ BCD-132-L-077.

[0093] Figure 27 Measurement of complement-dependent cytotoxicity of BCD-132-L-028 and BCD-132-L-077 compared to MabThera.

[0094] BCD-132-L-077

[0095] ● MabThera ■ BCD-132-L-028 ▼ BCD-132-L-077.

[0096] Figure 28 Measurement of antibody-dependent cellular cytotoxicity of BCD-132-L-028 and BCD-132-L-077 compared to MabThera, a low-affinity CD16 reporter cell line targeting the WIL2-S cell line.

[0097] BCD-132-L-028

[0098] ● MabThera ■ BCD-132-L-028 ▼ BCD-132-L-077.

[0099] Figure 29 Measurement of antibody-dependent cellular cytotoxicity of BCD-132-L-028 and BCD-132-L-077 compared to MabThera, a low-affinity CD16 reporter cell line targeting the WIL2-S cell line.

[0100] BCD-132-L-077

[0101] ● MabThera ■ BCD-132-L-028 ▼ BCD-132-L-077.

[0102] Figure 30 Measurement of antibody-dependent cellular cytotoxicity of BCD-132-L-028 and BCD-132-L-077 compared to MabThera, a low-affinity CD16 reporter cell line targeting the WIL2-S cell line.

[0103] BCD-132-L-028

[0104] ● MabThera ■ BCD-132-L-028 ▼ BCD-132-L-077.

[0105] Figure 31 Measurement of antibody-dependent cellular cytotoxicity of BCD-132-L-028 and BCD-132-L-077 compared to MabThera, a low-affinity CD16 reporter cell line targeting the WIL2-S cell line.

[0106] BCD-132-L-077

[0107] ● MabThera ■ BCD-132-L-028 ▼ BCD-132-L-077.

[0108] Figure 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 with FF (A), FV (B), and VV (C) allotypes.

[0109] Figure 33 Measurement of antibody-dependent cellular cytotoxicity (ADCC) of BCD-132-L-028 and BCD-132-L-077 relative to the commercially available antibody rituximab.

[0110] Figure 34 . Severity of inflammatory response (BCD132-L-077).

[0111] Figure 35 . Severity of demyelination (BCD132-L-077).

[0112] Figure 36 Smoothed concentration curves in primate serum following 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

[0113] Definition and general approach

[0114] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0115] Further, unless the context otherwise requires, singular terms shall include the plural, and plural terms shall include the singular. Generally, the classes and methods of cell culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, medical and pharmaceutical chemistry, and hybridization and chemistry of proteins and nucleic acids described herein are well known and widely used by those skilled in the art. Enzyme reactions and purification methods are performed according to the manufacturer's specifications, as is common in the art or as described herein.

[0116] Definitions related to antibodies

[0117] CD20, or B-lymphocyte antigen CD20, is a protein co-receptor found on the surface of B lymphocytes. CD20 is the product of the human MS4A1 gene. The exact function of this protein remains unknown; however, it is believed to play a role in the activation and proliferation of B lymphocytes.

[0118] 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 chromosome locus 11q12-13. The corresponding proteins are expected to have similar spatial structures: they have a four-transmembrane topology with N-terminal and C-terminal cytoplasmic domains (JANAS E. ET ALL., Functional role of lipid rafts in CD20 activity). , Biochem Soc Symp. 2005;(72):165-75).

[0119] Amplification of this gene and / or overexpression of its protein has been observed in many cancers or autoimmune diseases, including:

[0120] a) A neoplastic disease or condition from the group comprising: non-Hodgkin's lymphoma (NHL), Hodgkin's disease (Hodgkin's lymphoma), chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL).

[0121] b) an autoimmune disease or disorder 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, solid organ transplant rejection, graft-versus-host disease (GvHD), diabetes, Raynaud's syndrome, Sjögren's syndrome and glomerulonephritis.

[0122] The term "binding molecule" includes antibodies and immunoglobulins.

[0123] As used herein, the terms "antibody" or "immunoglobulin" (Ig) include whole 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 an 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, represented by the Greek letters: α, δ, ε, γ, and μ. The type of heavy chain present defines the class of antibody; 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, a constant region and a variable region. The constant region is identical in all antibodies of the same isotype, but differs in antibodies of different isotypes. Heavy chains γ, α and δ have a constant region consisting of three constant domains CH1, CH2 and CH3 (in a line), and a hinge region for increasing flexibility (Woof J., Burton D., Nat Rev Immunol 4, 2004, cc.89-99); heavy chains μ and ε have a constant region consisting of four constant domains CH1, CH2, CH3 and CH4. In mammals, only two types of light chains, represented by lambda (λ) and kappa (κ), are known. 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 to 217 amino acids. Preferably, the light chain is a kappa (κ) light chain, and the constant domain CL is preferably C kappa (κ).

[0124] 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).

[0125] The VL and VH regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs), which are interspersed between more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from amino terminus to carboxyl 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 the antigen. The constant region of an antibody can 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.

[0126] As used herein, the terms "antigen-binding portion" or "antigen-binding fragment" of an antibody (or simply "antibody portion" or "antibody fragment") refer 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 comprising 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 (Ward et al., (1989) Nature 341:544-546) consisting of the VH / VHH domains; and (vi) extracted complementarity determining regions (CDRs). In addition, the two regions of the Fv fragment, VL and VH, are encoded by separate genes and can be linked using recombinant methods using synthetic linkers that enable them to accept a single protein chain in which the VL and VH regions pair to form a monovalent molecule (called single-chain Fv (scFv); see, e.g., Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883). It is assumed that such single-chain molecules are also included in 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.

[0127] Preferably, the CDRs of the antigen-binding portion or whole antibody antigen-binding portion of the invention are derived from a mouse, llama or human donor library, or are substantially human, wherein certain amino acid residues are changed, for example, substituted with different amino acid residues, in order to optimize the properties of the specific antibody, such as KD, koff, IC50, EC50, ED50. Preferably, the framework regions of the antibodies of the invention are human or substantially human (at least 80, 85, 90, 95, 96, 97, 98 or 99% human).

[0128] In other embodiments, the antigen binding portion 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.

[0129] The term "variable domain" refers to the fact that certain parts of the variable domain are very different in sequence between antibodies. The V domain mediates antigen binding and determines the specificity of each particular antibody for its specific antigen. However, variability is not evenly distributed across the 110 amino acid span of the variable domain. Instead, the V region consists of an invariant segment called a framework region (FR) of 15-30 amino acids, which is separated by a shorter region of extreme variability called a "hypervariable region" or CDR. The variable domains of native heavy and light chains each contain four FRs, which largely adopt a β-sheet configuration and are connected by three hypervariable regions that form loops connecting the β-sheet structure and, in some cases, forming parts of the β-sheet structure. The hypervariable regions in each chain are tightly bound together by the FRs and, together with the hypervariable regions in the other chain, contribute to the formation of the antigen binding site of the antibody. The constant domain is not directly involved in the binding of the antibody to the antigen, but rather displays various effector functions, such as antibody participation in antibody-dependent cellular cytotoxicity (ADCC).

[0130] As used herein, the term "hypervariable region" refers to the amino acid residues of an antibody that are responsible for antigen binding. A hypervariable region generally comprises amino acid residues from a "complementarity determining region" or "CDR" and / or those residues from a "hypervariable loop."

[0131] In some cases, it may also be desirable to change one or more CDR amino acid residues to improve binding affinity to the target epitope. This is referred to as "affinity maturation," and can optionally be performed in conjunction with humanization, such as where the humanization of an antibody results in reduced binding specificity or affinity, and where binding specificity or affinity can not be fully improved by a single back mutation. Various affinity maturation methods are known in the art, such as by Burks et al., Proc Natl Acad Sci USA, 94:412–417 (1997) in vitro scanning saturation mutagenesis methods described, and by Wu et al., Proc Natl Acad Sci USA 95:6037–6042 (1998) stepwise in vitro affinity maturation methods.

[0132] " Framework region " (FR) is those variable domain residues except CDR residues.Each variable domain generally has four FRs identified as FR1, FR2, FR3 and FR4.If CDR is defined according to Kabat, then light chain FR residues are approximately located at residues 1-23 (LCFR1), 35-49 (LCFR2), 57-88 (LCFR3) and 98-107 (LCFR4), while heavy chain FR residues are approximately located at residues 1-30 (HCFR1), 36-49 (HCFR2), 66-94 (HCFR3) and 103-113 (HCFR4) in the heavy chain. If the CDRs comprise amino acid residues from a hypervariable loop, the light chain FR residues are positioned approximately at residues 1-25 (LCFR1), 33-49 (LCFR2), 53-90 (LCFR3), and 97-107 (LCFR4) in the light chain, while the heavy chain FR residues are positioned approximately at residues 1-25 (HCFR1), 33-52 (HCFR2), 56-95 (HCFR3), and 102-113 (HCFR4) in the heavy chain. In some cases, when the CDRs comprise amino acids from both CDRs as defined by Kabat and amino acids from a hypervariable loop, the FR residues will be adjusted accordingly. For example, when CDRH1 includes amino acids H26-H35, the heavy chain FR1 residues are at positions 1-25, and the FR2 residues are at positions 36-49.

[0133] The crystallizable fragment region of an immunoglobulin ("Fc region, Fc") is the "tail" region of the immunoglobulin molecule that interacts with cell surface Fc receptors and several proteins of the complement system. This property allows antibodies to activate the immune system. In IgG, IgA, and IgD antibody isotypes, the Fc region consists of two identical protein fragments, one from the second constant domain and the other from the third constant domain of each heavy chain; in IgM and IgE isotypes, the Fc region contains three heavy chain constant domains (CH domains 2-4) in each polypeptide chain.

[0134] An antibody of the present invention that "binds" to a target antigen is one that binds to the antigen with sufficient affinity to allow the antibody to be used as a diagnostic and / or therapeutic agent targeting proteins or cells expressing the antigen, and that cross-reacts slightly with other proteins. In such embodiments, the extent of binding of the antibody to non-target proteins is less than 10% of the binding of the antibody to the specific target protein, depending on the analytical method: fluorescence activated cell sorting (FACS), radioimmunoassay (RIA), or ELISA. With respect to binding of an antibody to a target molecule, the term "specifically binds" or "specifically binds to" or "specifically for" a particular polypeptide or epitope on a particular polypeptide target means binding that is significantly (measurably) different from nonspecific interactions (e.g., in the case of bH1-44 or bH1-81, the nonspecific interaction is binding to bovine serum albumin, casein, fetal calf serum, or neutravidin).

[0135] Specific binding can be measured by, for example, comparing the combination of a molecule with that of a control molecule.For example, specific binding can be measured by competing with a control molecule similar to a target (such as an excessive unlabeled target). In this case, if the combination of the target of labeling and the probe is competitively inhibited by an excessive unlabeled target, specific binding is indicated. As used herein, the term "specific binding" or "specifically binds to" or "is specific for" the epi-position on a specific polypeptide or a specific polypeptide target can be described by having a molecule with the following Kd for target: at least about 200 nM, or at least about 150 nM, or at least about 100 nM, or at least about 60 nM, or at least about 50 nM, or at least about 40 nM, or at least about 30 nM, or at least about 20 nM, or at least about 10 nM, or at least about 8 nM, or at least about 6 nM, or at least about 4 nM, or at least about 2 nM, or at least about 1 nM or larger. In one embodiment, the term "specific binding" refers to binding wherein a molecule binds to a specific polypeptide or epitope on a specific polypeptide while not substantially binding to any other polypeptide or polypeptide epitope.

[0136] As used herein, the term "Ka" refers to the on rate of a specific antibody-antigen interaction.

[0137] As used herein, the term "Kd" refers to the off rate of a particular antibody-antigen interaction.

[0138] "Binding affinity" generally refers to the intensity of the sum 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 inherent (property, true) binding affinity, which reflects a 1:1 interaction between a pair of members (e.g., an antibody and an antigen). The affinity of a molecule X for its binding partner Y can generally be represented by a dissociation constant (Kd). Preferred Kd values are approximately 200 nM, 150 nM, 100 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 8 nM, 6 nM, 4 nM, 2 nM, 1 nM or less. Affinity can be measured by common methods known in the art, including methods described herein. Low-affinity antibodies generally bind antigen slowly and tend to dissociate easily, while high-affinity antibodies generally bind antigen faster and tend to remain in conjunction for longer. Various methods of measuring binding affinity are known in the art, any of which can be used for the purposes of the present invention.

[0139] In one embodiment, "Kd" or "Kd value" is measured by using a surface plasmon resonance assay using a BIAcore™-2000 or BIAcore®-3000 (BIAcore, Inc., Piscataway, NJ) at 25°C using an immobilized antigen CM5 chip with ~10 response units (RU). 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) with 10 mM sodium acetate pH 4.8 and then injected at a flow rate of 5 μl / minute to achieve approximately 10 response units (RU) of coupled protein. After antigen administration, a 1 M ethanolamine solution is applied to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (e.g., 0.78 nM to 500 nM) were injected into PBS with 0.05% Tween 20 (PBST) at 25°C at a flow rate of approximately 25 μl / min. Association rates (kon) and dissociation rates (koff) were calculated by simultaneously fitting the association and dissociation sensorgrams using a simple one-to-one Langmuir binding model (BIAcore Evaluation Software version 3.2). The equilibrium dissociation constant (Kd) was calculated as the ratio koff / kon. See, e.g., Chen, Y. et al., (1999) J. Mol. Biol. 293:865-881. If the association rate exceeds 10 by the surface plasmon resonance assay above, the association rate is 10. 6 M −1 s −1 , the on-rate can be determined using a fluorescence quenching technique that measures 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 spectrophotometer equipped with stopped-flow (Aviv Instruments) or a 8000 series SLM-Aminco spectrophotometer with a stirred cuvette (ThermoSpectronic).

[0140] The term "koff" refers to the dissociation rate constant for a specific interaction between a binding molecule and an antigen. The dissociation rate constant, koff, can be measured using biolayer interferometry, for example, using an Octet™ system.

[0141] Can also be by using above surface plasmon resonance determination method, use BIAcore -2000 or BIAcore -3000 (BIAcore, Inc., Piscataway, NJ), at 25 ℃, with the immobilized antigen CM5 chip of ~ 10 relative units (response unit, RU), measure " binding rate " or " kon " according to the present invention.In brief, according to the manufacturer's instructions, with N- ethyl -N '- (3- dimethylaminopropyl) -carbodiimide hydrochloride (EDC) and N- hydroxysuccinimide (NHS), activate carboxymethylated dextran biosensor chip (CM5, BIAcore Inc.).Antigen is diluted to 5 μ g / ml (~ 0.2 μ M) with 10 mM sodium acetate pH 4.8, then injected with the flow rate of 5 μ l / minute, to reach the coupled protein of about 10 response units (RU).After antigen is applied, 1M ethanolamine solution is used to block unreacted groups.

[0142] Unless otherwise indicated, the terms "biologically active" and "biological activity" and "biological property" with respect to the polypeptides of the present invention mean the ability to bind to a biological molecule.

[0143] The term "biomolecule" refers to nucleic acids, proteins, carbohydrates, lipids, and combinations thereof. In one embodiment, the biomolecule exists in nature.

[0144] Antibody fragments, such as Fab and F(ab')2 fragments, can be prepared from intact antibodies using conventional techniques, such as papain or pepsin digestion of intact antibodies. In addition, antibodies, portions thereof and immunoadhesion molecules can be prepared using standard recombinant DNA techniques, such as those described herein.

[0145] The term "recombinant antibody" is intended to refer to an antibody expressed in a cell or cell line that contains a nucleotide sequence encoding the antibody, where the nucleotide sequence is not naturally associated with the cell.

[0146] As used herein, the term "variant antibody" is intended to refer to an antibody that has an amino acid sequence that is different from the amino acid sequence of its "parent" antibody by adding, deleting and / or replacing one or more amino acid residues compared to the sequence of the parent antibody. In a preferred embodiment, the variant antibody comprises at least one or more (e.g., one to twelve, e.g., two, three, four, five, six, seven, eight or nine, ten, eleven or twelve) amino acids compared to the parent antibody; in some embodiments, the variant antibody comprises one to about ten) amino acid additions, deletions and / or replacements. In some embodiments, such additions, deletions and / or replacements are made in the CDRs of the variant antibody. With respect to the identity or homology of the sequence of the variant antibody, it is defined herein as the percentage of amino acid residues equivalent to the parent antibody residues in the variant antibody sequence after aligning the sequences and, if necessary, introducing gaps to achieve the maximum percentage of sequence identity. The variant antibody retains the ability to bind to the same antigen to which the parent antibody binds, and preferably an epitope; and in some embodiments, at least one property or biological activity is superior to those of the parent antibody. For example, compared to the parent antibody, the variant antibody can have, for example, stronger binding affinity, longer half-life, lower IC50 or enhanced ability to inhibit the biological activity of the antigen. The variant antibody of particular concern herein is an antibody that exhibits at least 2-fold (preferably at least 5-fold, 10-fold or 20-fold) enhanced biological activity compared to the parent antibody.

[0147] The term "bispecific antibody" refers to an antibody having an antigen-binding domain that can specifically bind to two different epitopes on a single biomolecule, or can specifically bind to epitopes on two different biomolecules. Bispecific antibodies are also referred to herein as having "dual specificity" or "dual-specific" antibodies.

[0148] In a broad sense, the term "chimeric antibody" is intended to refer to an antibody that comprises one or more regions of an antibody and one or more regions of one or more other antibodies, wherein the other antibodies are typically part human and part non-human antibodies, i.e., partly derived from non-human animals, such as pests such as mice and rats, or camelids, such as llamas and alpacas. Chimeric antibodies are generally preferred relative to non-human antibodies to reduce the risk of human anti-antibody immune responses, such as in the case of murine antibodies, of human anti-mouse antibody immune responses. Common examples of chimeric antibodies are antibodies in which the variable region sequence is a murine sequence and the constant region sequence is a human antibody. In the case of chimeric antibodies, the non-human portion can be subjected to further changes to humanize the antibody.

[0149] The term "humanization" is intended to refer to the fact that when an antibody is of fully or partially non-human origin, for example, a mouse or llama antibody obtained by immunizing a mouse or llama with a target antigen, or a chimeric antibody based on such an antibody from a mouse or llama, certain amino acids, particularly in the framework regions and constant domains of the heavy and light chains, can be substituted to avoid or minimize an immune response in humans. Antibodies interact with the target antigen predominantly 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 variable between individual antibodies than the amino acid sequences outside the CDRs. Because the CDR sequences are responsible for most of the antibody-antigen interactions, recombinant antibodies can be expressed, for example, by constructing an expression vector that expresses the CDR sequences from a specific antibody and the framework sequences from a different antibody, which mimics the properties of a specific naturally occurring antibody, or more generally, any specific antibody having the amino acid sequence. As a result, a non-human antibody can be "humanized" and, to a large extent, the binding specificity and affinity of the original antibody are preserved. Although it is impossible to accurately predict the immunogenicity of a particular antibody and, therefore, the human anti-antibody response, non-human antibodies are generally more immunogenic than human antibodies. Chimeric antibodies, in which a foreign (e.g., pest or camelid) constant region has been replaced by a sequence of human origin, have been shown to be generally less immunogenic than those of completely foreign origin, and the trend in therapeutic antibodies is toward humanized or fully human antibodies. Therefore, chimeric antibodies or other antibodies of non-human origin can be humanized to reduce the risk of human anti-antibody responses.

[0150] For chimeric antibodies, humanization generally involves the modification of the framework region of the variable region sequence. The amino acid residues of the part of the complementary determining region (CDR) are most often not modified by means of humanization, although in some cases, it may be desirable to modify each amino acid residue of the CDR, for example, to delete glycosylation sites, deamidation sites, aspartic acid isomerization sites or unwanted cysteine or methionine residues. N-linked glycosylation is prepared by attaching oligosaccharide chains to the asparagine residues in the tripeptide sequence Asn-X-Ser or Asn-X-Thr, where X can be any amino acid except Pro. The removal of N-glycosylation sites can be achieved by using different residue mutations Asn or Ser / Thr residues, preferably by conservative substitution. The deamidation of asparagine and glutamine residues can occur depending on such factors as pH and surface exposure. Asparagine residues are particularly susceptible to deamidation, primarily when present in the sequence Asn-Gly, and less so in other dipeptide sequences such as Asn-Ala. If a CDR sequence contains such a deamidation site, particularly Asn-Gly, it may be desirable to remove this site, typically by deleting one of the implicated residues, by conservative substitution.

[0151] Numerous methods for the humanization of antibody sequences are known in the art. A commonly used method is CDR transplantation. CDR transplantation can be based on the CDR definition by Kabat, although the latest version (Magdelaine-Beuzelin et al., Crit Rev. Oncol Hematol. 64: 210 225 (2007)) prompts IMGT® (International ImMunoGeneTics Information System®, www.imgt.org) definition can improve humanization results (see Lefranc et al., Dev. Comp Immunol. 27: 55-77 (2003)). In some cases, compared with the parent antibody from which the CDR is obtained, CDR transplantation can reduce the binding specificity and affinity of the non-human antibody from which the CDR is transplanted, and therefore reduce biological activity. Back mutations (which are sometimes referred to as "framework region repairs") can be introduced at the selected position (usually in the framework region) of the CDR-transplanted antibody to restore the binding specificity and affinity of the parent antibody. The information available in the literature and antibody databases can be used to perform the identification of the position for possible back mutations. The amino acid residues that are candidates for back mutations are typically those located on the surface of the antibody molecule, while buried residues or residues with a lower degree of surface exposure are typically not altered. Another humanization technique for CDR grafting and back mutation is resurfacing, in which non-surface exposed residues of non-human origin are retained, while surface residues are changed to human residues.

[0152] Fully human antibodies can be generated using two techniques: using in vitro collected phage libraries or in vivo immunization of humanized animals (mice, rats, etc.).

[0153] The construction of a combinatorial phage antibody library begins with the selection of the source of the gene reservoir. This is determined by the different types of antibody libraries that can be distinguished: naive, immune, and synthetic. Naive and immune libraries are constructed using naturally reconstructed genes encoding variable immunoglobulin domains from healthy donors or donors immunized with a certain antigen, respectively. For this purpose, mRNA is isolated from antibody-producing lymphoid cell lines. Peripheral blood lymphocytes are primarily 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 proteinantigens. 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 Fabfragment 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 J et al. Efficient construction of a large nonimmune phage antibody library: the production of high-affinity human single-chain antibodies to proteinantigens. 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 Fabfragment phage library that permits rapid isolation and kinetic analysis of high affinity antibodies. J Biol Chem 1999, 274: 18218-18230.] have also been used. 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.].cDNA is then synthesized based on the mRNA, and both oligo-dT primers and statistically designed hexanucleotides can be used, which generate cDNA copies of all possible variants of the gene encoding the antibody variable domain [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.].

[0154] One or more primers can be used simultaneously to restrict the scope of amplified genes to one or more variable domain gene families or antibody isotypes, now available at the cDNA level [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]. Primers used to amplify immunoglobulin-encoding genes are complementary to their most conserved regions. Their sequences are selected from gene collections organized into databases, such as the Kabat or V BASE databases. The primer design also provides internal restriction sites for cloning the PCR product into an appropriate vector.

[0155] The construction of synthetic libraries is based on replacing the natural CDRs with a set of random sequences. In this way, a wide variety of antigen-binding sites can be generated.

[0156] Phage display is one of the most powerful and widely used in vitro techniques for searching antibodies. In 1985, Smith discovered that foreign DNA sequences can be cloned into filamentous phage M13, and such cloned sequences can be expressed on the surface of phage particles as fusion proteins (Smith GP: Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 1985, 228: 1315-1317.). Therefore, target fusion proteins can be selected based on their ability to bind to other proteins. This discovery is combined with PCR amplification methods, which allows the cDNA reservoir of immunoglobulin genes to be cloned to produce various phage libraries containing variable domains, which can be used to quickly search for target-specific monoclonal antibodies. The phage library reservoir reflects the B cell antibody reservoir of each person or animal whose blood is used to generate the library. In 1995, two papers reported the generation of genetically modified mice expressing a repertoire of fully human antibodies comparable to those 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 chain and kappa light chain immunoglobulin genes were intentionally disrupted, followed by the introduction of transgenes that were segments of human heavy chain and kappa light chain genes. This demonstrated that the human gene repertoire could be used by the mouse immune system to produce highly specific and high-affinity antibodies against a wider variety of antigens. Although the transgenic mice express B cell receptors that are actually a mixture of mouse and human components (human immunoglobulins, mouse Igα, Igβ, and other signaling molecules), their B cells develop and mature normally.

[0157] The term "monoclonal antibody" or "mAb" refers to an antibody that is synthesized and isolated from a separate clonal population of cells. The clonal population can be a clonal population of immortalized cells. In some embodiments, the immortalized cells in the clonal population are hybrid cells—hybridomas—typically produced by fusing individual B lymphocytes from an immunized animal with individual somatic cells from a lymphoma. Hybridomas are a type of constructed cell and do not occur in nature.

[0158] "Natural antibodies" are typically heterotetrameric glycoproteins of approximately 150,000 daltons, consisting of two identical light (L) chains and two identical heavy (H) chains. Each light chain is connected to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds varies in the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (VH) at one end, followed by multiple constant domains. Each light chain has a variable domain (VL) at one end, and a constant domain at its other end. The constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the variable domain of the light chain is aligned with the variable domain of the heavy chain. It is believed that specific amino acid residues form an interface between the light chain and the heavy chain variable domains.

[0159] The term "isolated" as used in this specification to describe various antibodies refers to antibodies that have been identified and separated and / or regenerated from the cells or cell cultures in which the antibody is expressed. Impurities (contaminant components) from its natural environment are materials that will interfere with the diagnostic or therapeutic use of the polypeptide and may include enzymes, hormones, and other proteins or non-protein solutes. In preferred embodiments, (1) the antibody is purified to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence using a spinning cup sequencer (Edman sequenator), or (2) the antibody is purified to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie Brilliant Blue or preferably silver staining. Isolated antibodies include in situ antibodies within recombinant cells because at least one component of the natural environment of the polypeptide is absent. Isolated polypeptides are typically prepared by at least one purification step.

[0160] An "isolated" nucleic acid molecule is one that is identified and separated from at least one nucleic acid molecule-impurity with which it is associated in the natural source of the antibody nucleic acid. An isolated nucleic acid molecule is different from the form or collection in which it is found under natural conditions. Thus, an isolated nucleic acid molecule is different from a nucleic acid molecule that exists in a cell under natural conditions. However, an isolated nucleic acid molecule includes a nucleic acid molecule that is located in a cell in which the antibody is normally expressed, for example, if the nucleic acid molecule has a chromosomal location that is different from its natural location in the cell.

[0161] As used herein, the term "epitope" is intended to refer to a portion (determinant) of an antigen that specifically binds to a binding molecule (e.g., an antibody or related molecule, such as a bispecific binding molecule). Epitope determinants are typically grouped by the chemically active surface of the molecule, such as amino acids or carbohydrates or sugar side chains, and typically comprise specific three-dimensional structural characteristics and specific charge characteristics. An epitope can be "linear" or "conformational." In a linear epitope, all interaction points 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, interaction points occur across amino acid residues on the protein that are separated from each other in the primary amino acid sequence. Once the desired epitope has been determined, antibodies against the epitope can be generated using techniques well known in the art. In addition, the generation and characterization of antibodies or other binding molecules can illustrate information about the desired epitope. Based on this information, antibodies that are combined with the same or equivalent epitopes can then be competitively screened, for example, by conducting competition studies to find binding molecules that compete with each other for binding to the antigen.

[0162] As used herein, the term "peptide linker" is intended to mean any peptide capable of combining domains, whose length depends on the domains to which it is bound, and comprising any amino acid sequence. Preferably, the peptide linker has a length of more than 5 amino acids and consists of any amino acid set selected from G, A, S, P, E, T, D, K.

[0163] The term "in vitro" refers to a biological entity, biological process, or biological reaction that is grown under artificial conditions outside the body. For example, cells grown in vitro are understood to be cells grown in an environment outside the body, such as in a test tube, culture vial, or microtiter plate.

[0164] As used herein, the term "IC 50 " (Inhibitory concentration 50%) refers to the concentration of drug at which a measurable activity or response, such as the growth / proliferation of cells, such as tumor cells, is inhibited by 50%. 50 Values can be calculated using appropriate dose-response curves using specialized statistical software for curve fitting.

[0165] 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 the biological process by 50%. IC 50 Values can be calculated using appropriate dose-response curves using specialized statistical software for curve fitting.

[0166] The term GI50 (growth inhibition 50%) refers to the concentration of drug at which the proliferation of cells, such as tumor cells, is inhibited by 50%.

[0167] The term "ED50" (EC50) (50% effective dose / concentration) refers to the concentration of a drug that produces 50% of the biological effect, which may include cytotoxicity.

[0168] The term "anti-proliferative activity" is intended to mean the arrest or inhibition of the growth of cells, such as cancer cells.

[0169] The term antibody "effector function" refers to the biological activity attributable to the Fc region (native Fc region sequence or Fc region amino acid variant) of an antibody, which varies with the antibody isotype. Examples of antibody effector functions include: q 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.

[0170] "Antibody-dependent cellular cytotoxicity" or "ADCC" refers to a cell-mediated response in which nonspecific cytotoxic cells expressing Fc receptors (FcRs), 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. NK cells, the primary cells for mediating ADCC, express only FcγRJII, while 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. Nos. 5,500,362 or 5,821,337, can be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, ADCC activity of the molecule of interest can be assessed in vivo, for example, in a animal model such as that disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).

[0171] "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 can be isolated from their natural sources, such as blood or PBMCs as described herein.

[0172] 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. In addition, a preferred FcR is an FcR that binds to 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 ("inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. The activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain (see review in Daëron, 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 to be identified in the future, are encompassed by the term "FcR" herein. The term also includes the neonatal receptor FcRn, which is responsible for the transfer of maternal IgG to the fetus.

[0173] "Complement-dependent cytotoxicity" and "CDC" refer 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 (Clq) to a molecule (e.g., an antibody) complexed with a cognate antigen. To assess complement activation, a CDC assay is used, for example, as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996).

[0174] The term "identity" or "homology" should be interpreted as meaning the percentage of amino acid residues in a candidate sequence that are equivalent to the residues in the corresponding sequence to which it is compared, after aligning the sequences and introducing gaps where necessary to achieve the maximum percent identity for the entire sequence, and without considering any conservative substitutions as part of sequence identity. Extensions or insertions at the N-terminus or the C-terminus should not be interpreted as reducing identity or homology. Methods and computer programs for comparison are well known in the art. Sequence identity can 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 (eliminations) and other modifications.

[0175] The term "homologous" with respect to polypeptide sequences of antibodies should be interpreted as antibodies that exhibit at least 70%, preferably 80%, more preferably 90%, and most preferably 95% sequence identity with respect to the polypeptide sequence. The term with respect to nucleic acid sequences should be interpreted as nucleotide sequences that exhibit at least 85%, preferably 90%, more preferably 95%, and most preferably 97% sequence identity with respect to the nucleic acid sequence.

[0176] Modifications of the amino acid sequences of the antibodies 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 may be prepared by introducing appropriate nucleotide changes into the antibody nucleic acid, or by peptide synthesis. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions may be prepared to obtain the final construct, provided that the final construct has the desired properties. Amino acid changes may also alter post-translational processes in the antibody, such as changing the number or position of glycosylation sites.

[0177] Amino acid substitutions are used to modify variants of the amino acid sequence of an antibody. Such variants are those that replace at least one amino acid residue in the antibody molecule with a different residue. The most favorable sites for substitution mutagenesis include hypervariable regions or CDRs, but FR or Fc changes are also contemplated. Conservative substitutions are shown under "preferred substitutions" in Table 1. If such substitutions result in changes in biological activity, further substantial changes may be made, which are indicated as "exemplary substitutions" in Table A, or, when describing amino acid classes, the changes described in more detail below, and product screening may also be performed.

[0178]

[0179] The terms "nucleic acid," "nucleic acid sequence," "nucleic acid sequence," "polynucleotide," "oligonucleotide," "polynucleotide sequence," and "nucleotide sequence" are used interchangeably in this specification to refer to the precise sequence of nucleotides, modified or unmodified, defining segments or regions of nucleic acid, with or without non-natural nucleotides, and whether double-stranded DNA or RNA, single-stranded DNA or RNA, or the transcription products of said DNA.

[0180] This also includes that the present invention does not relate to nucleotide sequences in their natural chromosomal environment, i.e. in the native state. The sequences of the present invention have been isolated and / or purified, i.e., they have been sampled directly or indirectly, for example by copying, and their environment has been at least partially modified. Thus, isolated nucleic acids obtained by recombinant genetics, for example with the aid of host cells, or isolated nucleic acids obtained by chemical synthesis should also be mentioned.

[0181] Unless otherwise indicated, reference to a nucleotide sequence encompasses its complement. Thus, reference to a nucleic acid having a particular sequence should be understood to encompass its complementary strand and its complementary sequence.

[0182] The term "control sequence" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. Suitable control sequences for prokaryotes include, for example, a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0183] A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, if the DNA for a presequence or secretory leader is expressed as a preprotein that participates in the secretion of the polypeptide, it is operably linked to the DNA for the polypeptide; if a promoter or enhancer affects the transcription of the sequence, it is operably linked to a coding sequence; if a ribosome binding site is positioned so as to promote translation, it is operably linked to a coding sequence. Generally, "operably linked" means that the DNA sequences to be linked are contiguous, and in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not need to be contiguous.

[0184] As used herein, the term "vector" refers to a nucleic acid molecule that can transport another nucleic acid that it is connected to. In some embodiments, the vector is a plasmid, i.e., other DNA segments can be connected to the circular double-stranded DNA sheet therein. In some embodiments, the vector is a viral vector, in which other DNA segments can be connected to the viral genome. In some embodiments, the vector can be autonomously replicated in the host cell they are introduced into (for example, bacterial vectors and additional mammalian vectors with bacterial replication initiation sites). In further embodiments, the vector (for example, non-additional mammalian vectors) can be integrated into the genome of the host cell after being introduced into the host cell, and thus replicated together with the host gene. In addition, some vectors can instruct the expression of the gene that they are operably connected to. This type of vector is referred to as "recombinant expression vector" (or simply "expression vector") in this article.

[0185] As used herein, the term "recombinant host cell" (or simply "host cell") is intended to refer to a cell into which a recombinant expression vector has been introduced. The present invention relates to a host cell, which may include, for example, the vector according to the present invention described above. The present invention also relates to a host cell, which comprises, for example, a nucleotide sequence encoding the heavy chain or antigen-binding portion thereof of the first binding domain and / or the second binding domain of a binding molecule of the present invention, a nucleotide sequence encoding a light chain or its antigen-binding portion thereof, or both. It should be understood that "recombinant host cell" and "host cell" are intended to refer not only to a specific subject cell, but also to the offspring of such cells. Because modification can occur in subsequent generations due to mutations or environmental influences, in fact, such offspring may not be equivalent to the parental cell, however, such cells are still included within the scope of the term "host cell" as used herein.

[0186] The term "excipient" is used herein to describe any ingredient other than a compound of the invention.

[0187] The term "a disease or condition mediated by CD20" refers to any disease or condition that is directly or indirectly associated with CD20, including the etiology, development, progression, persistence, or pathology of the disease or condition. "Treat," "treating," and "treatment" refer to a method of alleviating or eliminating at least one of a biological condition and / or its attendant symptoms. As used herein, "alleviating" a disease, disorder, or condition means reducing the severity and / or frequency of the symptoms of the disease, disorder, or condition. Further, references herein to "treating" include references to curative, palliative, and preventative treatments.

[0188] In one aspect, the subject or patient being treated is a mammal, preferably a human subject. The subject can be male or female of any age.

[0189] The term "disorder" means any condition that would benefit from treatment with a compound of the invention. This is intended to include both chronic and acute disorders or diseases, including those pathological conditions which predispose a mammal to the disorder in question.

[0190] The terms "cancer" and "cancerous" refer to a physiological condition or describe a physiological condition in a mammal that is generally characterized by unregulated cell growth / proliferation. This definition encompasses both benign and malignant cancerous diseases. Examples of cancerous diseases include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias. More specific examples of such cancerous diseases include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, adenocarcinoma and squamous cell carcinoma of the lung, 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 cancer or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, melanoma, and various head and neck cancers.

[0191] The terms "immune response," "autoimmune response," and "autoimmune inflammation" refer to the actions of, for example, lymphocytes, antigen-presenting cells, phagocytes, granulocytes, and soluble macromolecules produced by such cells or hepatocytes, including antibodies, cytokines, and complement, produced as a result of the selective damage, destruction, or elimination of invading pathogens, cells or tissues infected by pathogens, cancer cells, or, in the case of autoimmunity or pathological inflammation, normal cells or tissues from the human body.

[0192] The terms "immune response," "autoimmune response," and "autoimmune inflammation" refer to the actions of, for example, lymphocytes, antigen-presenting cells, phagocytes, granulocytes, and soluble macromolecules produced by such cells or hepatocytes, including antibodies, cytokines, and complement, produced as a result of the selective damage, destruction, or elimination of invading pathogens, cells or tissues infected by pathogens, cancer cells, or, in the case of autoimmunity or pathological inflammation, normal cells or tissues from the human body.

[0193] As used herein, the term "autoantibody" refers to an antibody directed against an autoantigen. Autoantigens include, but are not limited to, nucleic acids (e.g., double-stranded DNA or RNA, single-stranded DNA or RNA, or a combination thereof), nucleoproteins (e.g., SS-A (Ro), SS-B (La), Scl-70, centromeres, Jo-1, histidyl-tRNA synthetase, threonyl-tRNA synthetase, PM-1, Mi-2, histones, and chromatin), cell receptors (e.g., acetylcholine receptor, thyroid stimulating hormone receptor), cell proteins (e.g., cardiolipin, β2GP1), cell membrane proteins (e.g., aquaporins, desmoglein), RNA protein complexes (e.g., RNP and Sm), erythrocyte and platelet receptor glycoproteins.

[0194] As used herein, the term "autoimmune disease" refers to a non-malignant disease or disorder arising from and directed against an individual's own (self) antigens and / or tissues.

[0195] The term encompasses, 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 mellitus, thyroiditis, asthma, allergic disorders, psoriasis, atopic dermatitis, scleroderma, "graft-versus-host" reactions, 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, and microscopic renal vasculitis. , chronic active hepatitis, uvenita, septic shock, toxic shock syndrome, sepsis syndrome, cachexia, acquired immunodeficiency syndrome, acute transverse myelitis, Huntington's disease, Parkinson's disease, Alzheimer's disease, stroke, primary biliary cirrhosis, hemolytic anemia, adult (acute) respiratory distress syndrome, alopecia, alopecia areata, seronegative arthropathy, arthropathy, Reiter's disease, psoriatic arthropathy associated with ulcerative colitis arthropathy, atopic allergy, autoimmune bullous diseases, pemphigus vulgaris, pemphigus flakes, pemphigoid disease, linear IgA , IgG-related disease, autoimmune hemolytic anemia, Coombs-positive hemolytic anemia, pernicious anemia, juvenile pernicious anemia, giant cranial arteritis, arthritis, primary sclerosing hepatitis A, cryptogenic autoimmune hepatitis, fibrosing lung disease, cryptogenic fibrosing alveolitis, postinflammatory interstitial lung disease, interstitial pneumonia, chronic eosinophilic pneumonia, postinfectious interstitial lung disease, gouty arthritis, autoimmune hepatitis, autoimmune hepatitis type I (classic autoimmune hepatitis or lupus-like), autoimmune hepatitis type II, osteoarthritis, primary sclerosing cholangitis, psoriasis, idiopathic leukocytopenia neutropenia, autoimmune neutropenia, renal disease (NOS), glomerulonephritis, microscopic renal vasculitis, discoid lupus erythematosus, idiopathic or NOS male infertility (autoimmunity to sperm), multiple sclerosis (all subtypes), sympathetic ophthalmia, pulmonary hypertension secondary to connective tissue disease, Goodpasture's syndrome, pulmonary manifestations of nodular polyarthritis, acute rheumatic fever, rheumatoid spondylitis, ankylosing spondylitis, Still's disease, systemic scleroderma, localized scleroderma, Sjögren's syndrome, Sjögren's disease, Behçet's disease, ankylosing spondylitis, spondyloarthritis, axial enthesitis enthesitis), relapsing polychondritis, Takayasu's disease, autoimmune thrombocytopenia, idiopathic thrombocytopenia, autoimmune thyroid disease, autoimmune gastritis, polyglandular autoimmune syndrome, hyperthyroidism, Hashimoto's disease, autoimmune atrophic hypothyroidism, primary myxedema, phacogenic uveitisuveitis), 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-1 antitrypsin deficiency, amyotrophic lateral sclerosis, anemia, cystic fibrosis, conditions associated with cytokine therapy, demyelinating diseases, dermatitis, iridocyclitis / uveitis / optic neuritis, ischemia-reperfusion injury, ischemic stroke, juvenile rheumatoid arthritis, autoimmune bowel disease, autoimmune hearing loss, autoimmune lymphoproliferative syndrome, autoimmune myocarditis, autoimmune cardiomyopathy, coxsackievirus myocarditis, Dressler syndrome, lupus nephritis, angioedema including hereditary angioedema, urticaria, hidradenitis suppurativa, lichen planus, lichen sclerosus, pityriasis lichenoides, vitiligo, Addison's disease disease, autoimmune polyendocrine syndrome, autoimmune pancreatitis, celiac disease, microscopic colitis, antiphospholipid syndrome, autoimmune lymphoproliferative syndrome, cold agglutinin disease, essential cryoglobulinemia, Evan's syndrome, pernicious anemia, red cell aplasia, CREST syndrome, eosinophilic fasciitis, Felty's syndrome, overlap syndrome, chronic Lyme disease, Parrot II syndrome, relapsing rheumatic disease, rheumatic disease, acute rheumatic disease Fever, retroperitoneal syndrome, sarcoidosis, Schnitzler syndrome, undifferentiated connective tissue disease, dermatomyositis, polymyositis, fibromyalgia, myasthenia gravis, neuromyotonia, acute disseminated encephalomyelitis, Guillain-Barre syndrome, Devic's disease, Hashimoto's encephalopathy, Lanyon myasthenic syndrome, eosinophilic granulomatosis with polyangiitis, leukocytoclastic vasculitis, lupus vasculitis, rheumatoid vasculitis, polyangiitis nodosa, autoimmune premature ovarian failure and blepharitis. The antibody can also treat any combination of the above-mentioned conditions.

[0196] A "therapeutically effective amount" is intended to mean an amount of the therapeutic agent being administered which relieves to some extent one or more of the symptoms of the condition being treated.

[0197] The term "chronic" use refers to the continuous (uninterrupted) use of an agent, as opposed to an acute (temporary) route of administration, in order to maintain the initial therapeutic effect (activity) over an extended period of time.

[0198] "Intermittent" use refers to treatment that is not continued without interruption but is cyclical in nature.

[0199] As used herein, the words “comprise,” “have,” “include,” or variations such as “comprises,” “comprising,” “has,” “having,” “includes,” or “including,” and all grammatical variations thereof, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

[0200] Disclosure of the Invention

[0201] Antibody

[0202] The present invention relates to monoclonal antibodies that specifically bind to CD20.

[0203] In one aspect, the present invention relates to a monoclonal antibody or antigen-binding fragment thereof that specifically binds to CD20 and comprises:

[0204] 1) a heavy chain variable domain comprising the amino acid sequence EVQLVQPGAEVVKPGASVKVSCKASGYTFTSYNMHWVRQAPGRGLEWMGAIYPGNGDTSYNQKFKGRVTMTRDKSTSTVYMELSSLRSEDTAVYYCARSTYYGGDWYFNVWGQGTLVTVSS (SEQ ID NO: 2);

[0205] or

[0206] QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGRGLEWMGAIYPGNGDTSYNQKFKGRATLTRDTSTSTVYMELSSLRSEDTAVYYCARSTYYGGDWYFNVWGQGTLVTVSS (SEQ ID NO: 6);

[0207] 2) a light chain variable domain comprising the amino acid sequence QIVLSQSPAILSASPGERVTLTCRASSSVSYIHWFQQKPGKAPKPLIYATSNLASGVPSRFSGSGSGTDFSLTISRVEPEDFAVYYCQQWTSNPPTFGGGTKVEIK (SEQ ID NO: 4)

[0208] or

[0209] QIVLSQSPATLSASPGERATMTCRASSSVSYIHWFQQKPGKAPKPLIYATSNLASGVPSRFSGSGSGTDFTLTISRLEPEDFATYYCQQWTSNPPTFGGGTKVEIK (SEQ ID NO: 8).

[0210] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises:

[0211] 1) a heavy chain variable domain comprising the amino acid sequence EVQLVQPGAEVVKPGASVKVSCKASGYTFTSYNMHWVRQAPGRGLEWMGAIYPGNGDTSYNQKFKGRVTMTRDKSTSTVYMELSSLRSEDTAVYYCARSTYYGGDWYFNVWGQGTLVTVSS (SEQ ID NO: 2);

[0212] 2) a light chain variable domain comprising the amino acid sequence QIVLSQSPAILSASPGERVTLTCRASSSVSYIHWFQQKPGKAPKPLIYATSNLASGVPSRFSGSGSGTDFSLTISRVEPEDFAVYYCQQWTSNPPTFGGGTKVEIK (SEQ ID NO: 4).

[0213] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises:

[0214] 1) a heavy chain variable domain comprising the amino acid sequence QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGRGLEWMGAIYPGNGDTSYNQKFKGRATLTRDTSTSTVYMELSSLRSEDTAVYYCARSTYYGGDWYFNVWGQGTLVTVSS (SEQ ID NO: 6);

[0215] 2) a light chain variable domain comprising the amino acid sequence QIVLSQSPATLSASPGERATMTCRASSSVSYIHWFQQKPGKAPKPLIYATSNLASGVPSRFSGSGSGTDFTLTISRLEPEDFATYYCQQWTSNPPTFGGGTKVEIK (SEQ ID NO: 8).

[0216] In some embodiments, the monoclonal antibody comprises:

[0217] 1) A heavy chain comprising the amino acid sequence EVQLVQPGAEVVKPGASVKVSCKASGYTFTSYNMHWVRQAPGRGLEWMGAIYPGNGDTSYNQKFKGRVTMTRDKSTSTVYMELSSLRSEDTAVYYCARSTYYGGDWYFNVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 1)

[0218] or

[0219] QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGRGLEWMGAIYPGNGDTSYNQKFKGRATLTRDTSTSTVYMELSSLRSEDTAVYYCARSTYYGGDWYFNVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 5);

[0220] 2) A light chain comprising the amino acid sequence QIVLSQSPAILSASPGERVTLTCRASSSVSYIHWFQQKPGKAPKPLIYATSNLASGVPSRFSGSGSGTDFSLTISRVEPEDFAVYYCQQWTSNPPTFGGG TKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 3)

[0221] or

[0222] QIVLSQSPATLSASPGERATMTCRASSSVSYIHWFQQKPGKAPKPLIYATSNLASGVPSRFSGSGSGTDFTLTISRLEPEDFATYYCQQWTSNPPTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 7).

[0223] In some embodiments, the monoclonal antibody that specifically binds to CD20 is BCD132-077.

[0224] Monoclonal antibody BCD132-077 contains:

[0225] 1) A heavy chain comprising the amino acid sequence EVQLVQPGAEVVKPGASVKVSCKASGYTFTSYNMHWVRQAPGRGLEWMGAIYPGNGDTSYNQKFKGRVTMTRDKSTSTVYMELSSLRSEDTAVYYCARSTYYGGDWYFNVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 1);

[0226] 2) A light chain comprising the amino acid sequence QIVLSQSPAILSASPGERVTLTCRASSSVSYIHWFQQKPGKAPKPLIYATSNLASGVPSRFSGSGSGTDFSLTISRVEPEDFAVYYCQQWTSNPPTFGGG TKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 3).

[0227] In some embodiments, the monoclonal antibody that specifically binds to CD20 is BCD132-L-028.

[0228] Monoclonal antibody BCD132-L-028 contains:

[0229] 1) A heavy chain comprising the amino acid sequence QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGRGLEWMGAIYPGNGDTSYNQKFKGRATLTRDTSTSTVYMELSSLRSEDTAVYYCARSTYYGGDWYFNVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 5);

[0230] 2) A light chain comprising the amino acid sequence QIVLSQSPATLSASPGERATMTCRASSSVSYIHWFQQKPGKAPKPLIYATSNLASGVPSRFSGSGSGTDFTLTISRLEPEDFATYYCQQWTSNPPTFGGG TKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 7).

[0231] In some embodiments, the monoclonal antibody that specifically binds to CD20 is a full-length IgG antibody.

[0232] In some embodiments, the monoclonal antibody is of human IgG1, IgG2, IgG3, IgG4 isotype.

[0233] In some embodiments, the monoclonal antibody is of the human IgG1 isotype.

[0234] Nucleic acid molecules

[0235] The present invention also relates to nucleic acid molecules, in particular sequences encoding monoclonal antibodies that specifically bind to CD20 according to the present invention, as described herein, optionally including any peptide linker sequences linked thereto.

[0236] Unless otherwise indicated, reference to a nucleotide sequence encompasses its complement. Therefore, reference to a nucleic acid having a specific sequence should be understood to encompass the nucleic acid of its complementary strand and its complementary sequence. As used herein, the term "polynucleotide" means a polymeric form of nucleotides, or ribonucleotides, or deoxyribonucleotides, or a modified form of any type of nucleotide, of at least 10 bases in length. The term includes single-stranded and double-stranded forms.

[0237] 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 NO: 1 to 8. The nucleic acid molecule may also comprise any combination of said nucleotide sequences.

[0238] In one aspect, the present invention relates to a nucleic acid molecule comprising a nucleotide sequence encoding a monoclonal antibody or antigen-binding fragment thereof that specifically binds to CD20 and comprising:

[0239] 1) a heavy chain variable domain comprising the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 6;

[0240] 2) a light chain variable domain comprising the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 8.

[0241] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a monoclonal antibody or antigen-binding fragment thereof comprising:

[0242] 1) a heavy chain variable domain comprising the amino acid sequence shown in SEQ ID NO: 2;

[0243] 2) a light chain variable domain comprising the amino acid sequence shown in SEQ ID NO: 4.

[0244] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a monoclonal antibody or antigen-binding fragment thereof comprising:

[0245] 1) a heavy chain variable domain comprising the amino acid sequence shown in SEQ ID NO: 6;

[0246] 2) a light chain variable domain comprising the amino acid sequence shown in SEQ ID NO: 8.

[0247] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a monoclonal antibody comprising:

[0248] 1) a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 5;

[0249] 2) a light chain comprising the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 7.

[0250] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a monoclonal antibody comprising:

[0251] 1) a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 1;

[0252] 2) A light chain comprising the amino acid sequence shown in SEQ ID NO: 3.

[0253] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a monoclonal antibody comprising:

[0254] 1) a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 5;

[0255] 2) A light chain comprising the amino acid sequence shown in SEQ ID NO: 7.

[0256] In any of the above embodiments, the nucleic acid molecule can be isolated.

[0257] The nucleic acid molecules of the present invention can be isolated from any source that produces monoclonal antibodies that specifically bind to CD20. In certain embodiments, the nucleic acid molecules of the present invention can be synthesized rather than isolated.

[0258] In one embodiment, nucleic acid molecules encoding VH (SEQ ID NO: 2 or SEQ ID NO: 6) or VL (SEQ ID NO: 4 or SEQ ID NO: 8) domains are transformed into antibody genes along their entire length by insertion into expression vectors that already encode heavy chain constant (CH) or light chain constant (CL) domains, respectively, such that the VH segment is operably linked to the CH segment within the vector, and / or the VL segment is operably linked to the CL segment within the vector. In another embodiment, nucleic acid molecules encoding VH and / or VL domains are transformed into genes along the entire length of the antibody by linking (e.g., conjugating) nucleic acid molecules encoding VH and / or VL domains to nucleic acid molecules encoding CH and / or CL domains using standard molecular biology techniques. Nucleic acid molecules encoding heavy and / or light chains along their entire length can then be expressed by the cells into which they have been introduced.

[0259] Nucleic acid molecules can be used to express large amounts of recombinant monoclonal antibodies that specifically bind to CD20.

[0260] carrier

[0261] In another aspect, the present invention relates to vectors suitable for expressing any of the nucleotide sequences described herein.

[0262] As described herein, the present invention relates to vectors comprising nucleic acid molecules encoding any amino acid sequence of a monoclonal antibody or a portion thereof that specifically binds to CD20 (e.g., the heavy chain sequence of the first binding domain, and / or the heavy and / or light chain sequences of the second binding domain). The present invention further provides vectors comprising nucleic acid molecules encoding fusion proteins, modified antibodies, and antibody fragments.

[0263] In some embodiments, the monoclonal antibody that specifically binds to CD20 according to the present invention is expressed by inserting DNA obtained as described above into an expression vector, wherein the DNA partially or completely encodes the sequence of the first binding domain or the second binding domain (e.g., light chain and heavy chain sequences, wherein the binding domain comprises light chain and heavy chain sequences), such that the gene is operably linked to necessary expression control sequences, such as transcription and translation control sequences. Expression vectors include plasmids, retroviruses, adenoviruses, adeno-associated viruses (AAVs), plant viruses, such as cauliflower mosaic virus, tobacco mosaic virus, cosmids, YACs, EBV-derived episomes, and the like. The DNA molecule can be ligated into the vector so that the transcription and translation control sequences within the vector perform their intended function of regulating DNA transcription and translation. The expression vector and expression control sequences can be selected to be compatible with the expression host cell being used. DNA molecules that partially or completely encode the sequence of the first binding domain and the second binding domain (e.g., heavy chain and light chain sequences, wherein the binding domain comprises heavy chain and light chain sequences) can be introduced into each vector. In one embodiment, any combination of the DNA molecules is introduced into the same expression vector. The DNA molecule can be introduced into the expression vector by standard methods (eg, ligation of complementary restriction sites on the antibody gene fragment and the vector, or blunt-end ligation if no restriction sites are present).

[0264] Suitable vectors are vectors encoding functionally complete human CH or CL immunoglobulin sequences, with suitable restriction sites for transformation so that, as described above, any VH or VL sequence can be easily inserted and expressed. The genes encoding HC and LC in such vectors can include intron sequences, which can lead to enhanced total antibody protein yields by stabilizing the corresponding mRNA. The flanks of the intron sequences are splice donor and splice acceptor sites, which determine where RNA splicing occurs. When using multiple introns, the position of the intron sequence can be in the variable region or constant region of the antibody chain or in both the variable region and the constant region. Polyadenylation and transcription termination can occur at the natural chromosomal site downstream of the coding region. Recombinant expression vectors can also encode signal peptides, which promote the secretion of antibody chains from host cells. The antibody chain gene can be cloned into a vector so that the signal peptide is connected to the amino terminus of the immunoglobulin chain in frame. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin).

[0265] In addition to the antibody chain genes, the recombinant vector expression of the present invention can also carry regulatory sequences that control the expression of the antibody chain genes in the host cell. It will be understood by those skilled in the art that the design of the expression vector, including the selection of regulatory sequences, can depend on such factors as the selection of the host cell to be transformed, the expression level of the desired protein, etc. The preferred control sequences for the expression host cells in mammals include viral elements that ensure high-level protein expression in mammalian cells, such as promoters and / or enhancers derived from retrovirus LTR, 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 natural immunoglobulin promoters or actin promoters. For further description of viral control elements and their sequences, see, for example, U.S. Patent Nos. 5,168,062, 4,510,245, and 4,968,615. Methods for expressing binding molecules such as antibodies in plants, including descriptions of promoters and vectors and plant transformation, are known in the art. See, for example, U.S. Patent No. 6,517,529. Methods for expressing polypeptides in bacterial cells or fungal cells, such as yeast cells, are also well known in the art.

[0266] In addition to antibody chain genes and regulatory sequences, the recombinant expression vector of the present invention can also carry other sequences, such as sequences (such as replication origins) and selectable marker genes regulating the replication of the vector in the host cell. Selectable marker genes promote the selection of the host cell that the vector has been introduced therein (see, for example, U.S. Patent numbers 4,399,216, 4,634,665 and 5,179,017). For example, usually, selectable marker genes give resistance to medicaments, such as G418, hygromycin or methotrexate to the host cell that the vector has been introduced therein. For example, selectable marker genes include dihydrofolate reductase (DHFR) gene (for use in dhfr host cells during methotrexate selection / amplification), neo gene (selected for G418) and glutamate synthetase gene.

[0267] As used herein, the term "expression control sequence" is intended to refer to polynucleotide sequences that are necessary to achieve expression and processing of the coding sequences to which they are 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 (i.e., Kozak consensus sequences); sequences that enhance protein stability; and, where necessary, sequences that enhance protein secretion. The nature of such control sequences varies depending on the host organism; in prokaryotes, such control sequences generally include promoters and transcription termination sequences for ribosome binding sites; in eukaryotes, typically, such control sequences include promoters and transcription termination sequences. The term "control sequence" is intended to include at least all components whose presence is necessary for expression and processing, and may also include additional components whose presence is advantageous, such as leader sequences and fusion partner sequences.

[0268] host cells

[0269] 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 generating a recombinant host cell capable of expressing a monoclonal antibody that specifically binds to CD20, culturing the host cell under conditions suitable for expression / production of the 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 progeny of cells derived from such a host cell, and similarly produced monoclonal antibodies that specifically bind to CD20.

[0270] Nucleic acid molecules encoding monoclonal antibodies that specifically bind to CD20 according to the present invention and vectors containing these nucleic acid molecules can be used to transfect suitable mammals or cells thereof, plants or cells thereof, bacteria or yeast host cells. Transformation can be performed 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, polynucleotide encapsulation in liposomes, and direct microinjection of DNA into the nucleus. In addition, nucleic acid molecules can be introduced into mammalian cells via 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, bioballistic transformation, direct injection, electroporation, and viral transformation. Methods for transforming bacterial and yeast cells are also well known in the art.

[0271] Mammalian cell lines used as hosts for transformation are well known in the art and include a number of available immortalized cell lines. These include, for example, Chinese hamster ovary (CHO) cells, NSO 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., Hep G2), A549 cells, and many other cell lines. Cell lines are selected by determining which cell lines have high expression levels and provide the necessary properties of the protein 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 culture medium in which the host cell is grown. Monoclonal antibodies that specifically bind to CD20 can 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.

[0272] In addition, various known techniques can be used to enhance production levels of monoclonal antibodies that specifically bind to CD20 according to the present invention from production cell lines. For example, the glutamine synthetase gene expression system (GS system) is a common method used to enhance expression under certain conditions. The GS system, in whole or in part, is discussed in conjunction with ER Nos. 0216846, 0256055, 0323997, and 0338841.

[0273] Monoclonal antibodies that specifically bind to CD20 expressed by different cell lines or in transgenic animals are likely to have different glycosylation profiles 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 the glycosylation of the binding molecules and, generally, the presence or absence of post-translational modifications.

[0274] Antibody preparation

[0275] The present invention also relates to methods and processes for producing monoclonal antibodies and antigen-binding fragments thereof that specifically bind to CD20.

[0276] Monoclonal antibodies

[0277] Monoclonal antibodies may be prepared using the hybridoma method first described by Kohler et al., Nature 256, 1975, p. 495, or may be prepared using recombinant DNA methods (US 4816567).

[0278] In the hybridoma method, mice or other appropriate host animals, such as hamsters, are immunized according to the above methods to elicit lymphocytes that produce antibodies or are capable of producing antibodies that specifically bind to the protein used for immunization. According to another embodiment, lymphocytes can be generated by in vitro immunization. Following immunization, the lymphocytes are fused with a myeloma cell line using a suitable fusing agent, such as polyethylene glycol, to produce hybridoma cells.

[0279] The hybridoma cells produced in the above manner can be cultured in a suitable culture medium that preferably contains 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 culture medium for the hybridomas will typically include hypoxanthine, aminopterin, and thymidine (HAT medium), substances that prevent the growth of HGPRT-deficient cells.

[0280] Preferred cells for use as components for myeloma cell fusion are cells that fuse efficiently, support stable high-level production of antibodies by selected antibody-producing cells, and are sensitive to culture media in which unfused parental cells are selected. Preferred myeloma cell lines are murine myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, California, USA, and SP-2 or X63-Ag8-653 cells available from the American Type Culture Collection, Rockville, Maryland, USA. Human myeloma and mouse-human heteromyeloma cell lines for the production of monoclonal antibodies have also been described (Kozbor, J. Immunol., 133, 1984, p. 3001).

[0281] 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).

[0282] The binding affinity of the monoclonal antibody can be determined, for example, by Scatchard analysis as described in Munson et al., Anal. Biochem., 107:220 (1980).

[0283] In case the hybridoma cell of producing required specificity, affinity and / or active antibody is identified, clone just can be carried out subclone by limiting dilution procedure, and by standard method growth.Suitable culture medium for this purpose comprises for example D-MEM or RPMI-1640 culture medium.In addition, hybridoma cell can be grown in vivo as ascites tumor in animal, for example, by being injected into mouse by cell intraperitoneal (ip) injection.

[0284] The monoclonal antibodies secreted by the subclones can be separated from the culture medium, ascites fluid or serum by conventional antibody purification techniques, such as affinity chromatography (e.g., using protein A- or protein G-Sepharose) or ion exchange chromatography, hydroxyapatite chromatography, gel electrophoresis, dialysis, and the like.

[0285] The DNA encoding the monoclonal antibody is easily separated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can bind specifically to the genes encoding the heavy and light chains of mouse antibodies). Hybridoma cells serve as a preferred source of this type of DNA. After separation, the DNA can be placed in an expression vector, which is then transfected into a host cell to obtain the synthesis of the monoclonal antibody in a recombinant host cell, such as an Escherichia coli cell, a monkey COS cell, a Chinese hamster ovary (CHO) cell, or a myeloma cell, which does not produce an antibody protein if not transfected.

[0286] In a further embodiment, monoclonal antibodies or antibody fragments can 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 using phage libraries, respectively. Subsequent publications described the generation of high-affinity (nM range) human antibodies by chain shuffling (Marks et al., Bio / Technology, 10:779-783 (1992), and combinatorial infection and in vivo recombination as a strategy for the construction of very large phage libraries (Waterhouse et al., Nucl. Acids. Res. 21:2265-2266 (1993). Therefore, these techniques are viable alternatives to conventional monoclonal antibody hybridoma technology for isolation of monoclonal antibodies.

[0287] The DNA encoding the antibody can be modified, for example, by substituting heavy and light chain (CH and CL) constant region sequences for the homologous murine sequences (US 4816567 and Morrison et al., Proc. Natl. Acad. Sci. USA: 81: 6851 (1984)), or by covalently fusing the immunoglobulin coding sequence to all or part of the coding sequence for a non-immunoglobulin polypeptide (heterologous polypeptide), e.g., to produce chimeric or fusion antibody polypeptides. The non-immunoglobulin polypeptide sequences can replace the constant regions of the antibody, or they can replace the variable domains of the antigen-binding center of the antibody to produce a chimeric bivalent antibody comprising one antigen-binding site with specificity for an antigen and another antigen-binding site with specificity for a different antigen.

[0288] Human antibodies based on phage display libraries and methods

[0289] Transgenic animals (e.g., mice) can be produced at present, which can all produce various human antibodies after immunization without endogenous immunoglobulin production. For example, homozygous deletions of the antibody heavy chain joining region (JH) gene in chimeric and germline mutant mice have been described, resulting in the complete suppression of endogenous antibody production. The human germline immunoglobulin gene array is transferred into such germline mutant mice, resulting in the production of human antibodies after antigen attack (US 5545806, 5569825, 5591669 (all GenPharm); 5545807; and WO 97 / 17852).

[0290] Alternatively, phage display technology (McCafferty et al., Nature, 348: 552-553 (1990), can be used to produce human antibodies and antibody fragments in vitro from a reservoir of immunoglobulin variable (V) region genes from an immunized donor organism. According to this technology, antibody V region genes are cloned in frame with the major or minor coat protein genes of a filamentous phage (e.g., M13 or fd) and displayed as functional antibody fragments on the surface of the phage particle. Because the filamentous particles contain a single-stranded DNA copy of the phage genome, selection based on the functional properties of the antibody also results in selection of genes encoding antibodies that exhibit said properties, and thus, the phage mimics some of the properties of B cells. Phage display can be performed in various formats. Several sources of V gene segments can be used for phage display. Clackson et al., Nature, 352: 624-628 (1991), isolated a variety of anti-oxazolone antibodies from a small random combinatorial library of V genes derived from the spleens of immunized mice. V gene repositories from unimmunized human donors can be constructed and can essentially follow the method described by Marks et al., J. Mol. Biol. 222:581-597 (1991) describes a technique for isolating antibodies directed against a variety of antigens, including self-antigens.

[0291] As described above, human antibodies can also be generated by in vitro activated B cells (see US Pat. Nos. 5,567,610 and 5,229,275).

[0292] Antibody fragments

[0293] In some cases, it may be advisable to use antibody fragments rather than whole antibodies. The small size of the fragments facilitates their rapid clearance and may allow for better penetration into dense tumors.

[0294] Developed the various technologies for producing antibody fragments. Routinely, these fragments are derived via the proteolytic digestion of intact antibodies. However, these fragments can be directly produced by recombinant host cells at present. Fab, Fv and ScFv antibody fragments can be expressed in E. coli and secreted therefrom, thereby allowing the generation of a large number of these fragments to be promoted. Antibody fragments can be separated from the antibody phage library described above. According to another embodiment, Fab'-SH fragments can 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 can be directly isolated from recombinant host cell culture. Fab and F(ab')2 with increased in vivo half-life, retaining epitope binding receptor residues, are described in US5869046. 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 with complete binding sites that lack constant regions; as a result, they are suitable for reducing nonspecific binding during in vivo use. scFv fusion proteins can be constructed to produce an effector protein fused to the N-terminus or C-terminus of the scFv. Antibody fragments can also be "linear antibodies," as described, for example, in U.S. Patent 5641870. Such linear antibody fragments can be monospecific or bispecific.

[0295] Pharmaceutical composition

[0296] 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.

[0297] The pharmaceutical composition may include at least one monoclonal antibody that specifically binds to CD20, and at least one component selected from pharmaceutically acceptable and pharmacologically compatible excipients.

[0298] 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 improve, prevent, or treat conditions that may be associated with CD20.

[0299] "Pharmaceutical composition" means a composition comprising a monoclonal antibody that specifically binds to CD20 according to the present invention, and at least one component selected from pharmaceutically acceptable and pharmacologically compatible excipients, such as fillers, solvents, diluents, carriers, adjuvants, dispensing agents, delivery agents, preservatives, stabilizers, emulsifiers, suspending agents, thickeners, and delivery control agents, the selection and proportion of which depend on the type and route of administration and the dosage. The pharmaceutical compositions of the present invention and methods for their preparation will be readily apparent to those skilled in the art. Pharmaceutical compositions should preferably be manufactured in accordance with GMP (Good Manufacturing Practice) requirements. The composition may include a buffer component, a tonicity agent, a stabilizer, and a solubilizer. Prolonged action of the composition may be achieved by agents that slow the absorption of the active pharmaceutical ingredient, such as aluminum monostearate and gelatin. Examples of suitable carriers, solvents, diluents, and delivery agents include water, ethanol, polyols, and mixtures thereof, oils, and organic esters for injection.

[0300] "Drugs" are compounds (or mixtures of compounds as pharmaceutical compositions) in tablets, capsules, powders, lyophilized preparations, injections, infusions, ointments and other ready-to-use forms that are intended for use in restoring, improving or modifying physiological functions in humans and animals, as well as for the treatment and prevention of diseases, for diagnosis, anesthesia, contraception, cosmetics and other purposes. Any method recognized in the art for administering peptides, proteins or antibodies can be suitably used with the monoclonal antibodies that specifically bind to CD20 according to the present invention.

[0301] The term "pharmaceutically acceptable" refers to one or more compatible liquid or solid components that are suitable for administration to a mammal, preferably a human.

[0302] The term "excipient" is used herein to describe any ingredient other than the above-mentioned ingredients of the present invention. These are substances of inorganic or organic nature that are used in the manufacture of pharmaceuticals in order to give the necessary physicochemical properties to the pharmaceutical product.

[0303] The terms "buffer," "buffer component," and "buffer" refer to a solution that is capable of resisting pH changes through the action of its acid-base conjugate components and allows the monoclonal antibody drug that specifically binds to CD20 to resist pH changes. Generally, the pharmaceutical composition preferably has a pH in the range of 4.0 to 8.0. Examples of buffers include, but are not limited to, acetate, phosphate, citrate, histidine, succinate, and the like.

[0304] As used herein, the terms "tonic agent," "osmotic agent," or "osmotic agent" refer to excipients that increase the osmotic pressure of a liquid antibody formulation. An "isotonic" drug is one that has an osmotic pressure comparable to that of human blood. Isotonic drugs typically have an osmotic pressure of about 250 to 350 mOsm / kg. Examples of isotonic agents include, but are not limited to, polyols, sugars and sucrose, amino acids, metal salts such as sodium chloride, and the like.

[0305] "Stabilizer" refers to an excipient or a mixture of two or more excipients that provides physical and / or chemical stability to the active agent. Stabilizers include amino acids such as, but not limited to, arginine, histidine, glycine, lysine, glutamine, and 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 names: poloxamer, pluronic, sodium dodecyl sulfate (SDS); antioxidants such as, but not limited to, methionine, acetylcysteine, ascorbic acid, monothioglycerol, and sulfites; and chelating agents such as, but not limited to, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), and sodium citrate.

[0306] A pharmaceutical composition is "stable" if the active agent maintains its physical stability and / or chemical stability and / or biological activity during a specified shelf life at a specified storage temperature, 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 testing under accelerated or natural aging conditions.

[0307] The pharmaceutical compositions of the present invention can be manufactured, packaged, or widely marketed as a single unit dose or multiple single unit doses in the form of ready-to-use preparations. As used herein, the term "single unit dose" refers to a discrete amount of a pharmaceutical composition containing a predetermined amount of an active ingredient. The amount of the active ingredient is generally equal to the dose of the active ingredient to be administered to a subject, or a convenient fraction of such a dose, such as half or one-third of such a dose.

[0308] The pharmaceutical compositions according to the present invention are generally suitable for parenteral administration as sterile formulations, which are intended to be administered to humans by injection, infusion, and implantation, bypassing the gastrointestinal tract and through breaks in the skin and mucosal barriers. For example, parenteral administration includes, inter alia, subcutaneous, intraperitoneal, intramuscular, intrasternal, intravenous, intraarterial, intrathecal, intraventricular, intraurethral, intracranial, intrasynovial, and transcutaneous injection or infusion; and renal dialysis infusion techniques. Intratumoral delivery, such as intratumoral injection, can also be employed. Regional perfusion is also provided. Preferred embodiments include intravenous and subcutaneous routes. Any method recognized in the art for administering peptides or proteins can be suitably used with the monoclonal antibodies that specifically bind to CD20 according to the present invention.

[0309] Injectable formulations can be prepared, packaged or sold in unit dosage form (without limitation), for example, in ampoules, vials, plastic containers, prefilled syringes, automatic injection devices. Formulations for parenteral administration include suspensions, solutions, emulsions in oily or aqueous bases, pastes, and the like.

[0310] In another embodiment, the present invention provides a composition for parenteral administration, comprising a pharmaceutical composition provided in dry (i.e., powder or granular) form for reconstitution with a suitable matrix (e.g., sterile, pyrogen-free water) prior to administration. Such formulations can be prepared, for example, by a lyophilization process, which is known in the art as freeze drying and involves freezing the product, followed by removal of the solvent from the frozen material.

[0311] The monoclonal antibodies that specifically bind to CD20 according to the present invention can also be administered intranasally, alone or as a mixture with a suitable pharmaceutically acceptable excipient, or by inhalation from an inhaler (e.g., a pressurized aerosol container, pump, spray, nebulizer, or atomizer), with or without a suitable propellant, or as nasal drops or spray.

[0312] Dosage forms for parenteral administration can be formulated to be immediate release or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted-, and programmed-release.

[0313] Therapeutic uses of monoclonal antibodies that specifically bind to CD20 according to the present invention

[0314] In one aspect, monoclonal antibodies that specifically bind to CD20 according to the present invention can be used to treat disorders associated with (mediated by) CD20 activity.

[0315] In one aspect, the subject is a mammal, preferably a human subject. The subject can be male or female of any age.

[0316] In the case of tumors (e.g., cancer), a therapeutically effective amount of an antibody or fragment thereof (e.g., an antibody or fragment thereof that specifically binds to CD20) can reduce the number of cancer cells; reduce the size of the initial tumor; inhibit (i.e., slow down to some extent and preferably stop) the infiltration of cancer cells into surrounding organs; inhibit (i.e., slow down 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 condition. The antibody or fragment thereof can, to some extent, prevent growth and / or kill existing cancer cells, and can be cytostatic and / or cytotoxic. For cancer treatment, in vivo efficacy can be measured, for example, by evaluating survival, time to tumor progression (TTP), tumor response rate (RR) to treatment, duration of response, and / or quality of life.

[0317] As used herein, the terms "co-administered," "co-administered," and "in combination with," in reference to a monoclonal antibody that specifically binds to CD20 and one or more different therapeutic agents, are intended to mean, refer to, or include:

[0318] 1) such combination of a monoclonal antibody that specifically binds to CD20 according to the present invention and a therapeutic agent is administered simultaneously 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 at substantially the same time,

[0319] 2) such combination of a monoclonal antibody that specifically binds to CD20 according to the present invention and a therapeutic agent is administered simultaneously to a patient in need of treatment, where such components are formulated separately from one another into separate dosage forms, said separate dosage forms are taken by said patient at substantially the same time, whereupon said components are released to said patient at substantially the same time,

[0320] 3) sequential administration of such combinations 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 into separate dosage forms, which are taken by said patient at consecutive times with a substantial time interval between each administration, whereupon said components are released to said patient at substantially different times; and

[0321] 4) Such combinations of a monoclonal antibody that specifically binds to CD20 according to the present invention and a therapeutic agent are administered sequentially to a patient in need of treatment, where such components are formulated together into a single dosage form that releases the components in a controlled manner, whereupon they are released to the patient simultaneously, sequentially, or in combination at the same and / or different times, wherein each part can be administered by the same or different routes.

[0322] The monoclonal antibodies that specifically bind to CD20 according to the present invention can be administered without further therapeutic treatment, i.e., as a stand-alone therapy. Furthermore, treatment with the monoclonal antibodies that specifically bind to CD20 according to the present invention can include at least one additional therapeutic treatment (combination therapy). In some embodiments, the monoclonal antibodies that specifically bind to CD20 can be administered or formulated in combination with another pharmaceutical product / agent for the treatment of cancer or autoimmune disease.

[0323] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents the function of cells and / or causes cell destruction. The term is intended to include 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, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and / or variants thereof.

[0324] A "chemotherapeutic agent" is a chemical compound that can be used 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, carboquinone, meturedopa, and uredopa; ethyleneimines and methylmelamines, including hexamethylmelamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylmelamine; acetogenins (e.g., bratasin and bratasinone); delta-9-tetrahydrocannabinol (dronabinol MARINOL); ® ); β-lapachone; lapachol; colchicine; betulinic acid; camptothecin (including the synthetic analogue topotecan (HYCAMTIN ® ), CPT-11 (irinotecan, CAMPTOSAR ®), acetylcamptothecin, scopolectin, and 9-aminocamptothecin); bryostatin; callystatin; CC-1065 (including its synthetic analogs adolesin, carzelesin, and biszelesin); podophyllotoxin; podophyllic acid; teniposide; candidins (e.g., candidin 1 and candidin 8); dolastatin; duocarmycin (including its synthetic analogs KW-2189 and CB1-TM1); eleutherobin; pan cratistatin; sarcodictyin; spongestatin; nitrogen mustards, such as chlorambucil, naphthiazolin, clofosamide, estramustine, ifosfamide, methylchloroethylamine, nitrogen oxide mustard hydrochloride, melphalan, new nitrogen mustard, phenylephrine, prednimustine, trofosamide, uracil mustard; nitrosoureas, such as carmustine, chlorozolin, fotemustine, lomustine, nimustine and ranimustine; antibiotics, such as enediyne antibiotics (e.g., calicheamicins, such as calicheamicin gamma II and calicheamicin omega II (see, e.g., Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994)); danemycins, including danemycin A; epothilones; and the neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclarubicin, actinomycin D, anthramycin, azaserine, bleomycin, actinomycin C, carabicin, carminomycin, carmomycin, chromomycin, dactinomycin, daunorubicin, detoximcin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including ADRIAMYCIN ® Morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolido-doxorubicin, doxorubicin HCl liposome injection (DOXOL ® ), Doxorubicin Liposomal TLC D-99 (MYOCETCA ® ) and pegylated doxorubicin liposomes (CAELYX ® ) and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, mexilomycin, mitomycins such as mitomycin C, mycophenolic acid, noramycin, olivomycin, peplomycin, porfibrinocin, puromycin, triferon-adriamycin, rhodorubicin, streptozocin, streptozocin, tuberculin, ubenimex, zoloft, daunorubicin; antimetabolites such as methotrexate, gemcitabine (GEMZAR ® ), Tegafur (UFTORAL ® ), Capecitabine (XELODA ®), epothilones, and 5-fluorouracil (5-FU); folic acid analogs such as folinic acid, methotrexate, pteropterin, and trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiopurine, and thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxiflurane, enocitabine, and floxuridine; antiadreners such as aminoglutethimide, mitotane, and trilostane; folic acid supplements such as folinic acid; acetyl glucosamine Aldehyde esters; aldophosphamide glycosides; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatrexate; defosfamide; demeclocycline; diacridone; eflornithine; eliplironium acetate; etoglucagon; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansine alkaloids, such as maytansine and ansamitocin; mitoguanidine; mitoxantrone; mopidarol; nitropropane; pentostatin; methambucil; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK ® Polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; cytochrome; siroxifen; spirogermanamine; tricholomanic acid; triazoline; 2,2',2"-trichlorotriethylamine; trichothecenes (e.g., T-2 toxin, verrucosporin A, baculozolin A, and serpentin); urethanes; dacarbazine; mannomustine; dibromomannitol; dibromodulcitol; pipobroman; gacytosine; cytarabine ("Ara-C"); thiotepa; taxanes, such as paclitaxel (TAXOL ® ), albumin-modified paclitaxel nanoformulation (ABRAXANETM) and docetaxel (TAXOTERE ® ); chlorambucil; 6-thioguanine; mercaptopurine; methotrexate; platinum agents such as cisplatin, oxaliplatin, and carboplatin; vinblastines, which prevent tubulin from polymerizing to form microtubules, including vinblastine (VELBAN ® ), vincristine (ONCOVIN ® ), ELDISINE ® ), FILDESIN ® ) and vinorelbine ® ); etoposide (VP16); ifosfamide; mitoxantrone; leucovorin; noxolin; edatrexate; daunomycin; methotrexate; ibandronate; the topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid, including bexarotene (TARGRETIN ® ); bisphosphonates, such as clodronate (eg, BONEFOS ® or OSTAC ® ), etidronate (DIDROCAL® ), NE-58095, zoledronic acid / zoledronic acid salt (ZOMETA ® ), alendronate (FOSAMAJX ® ), Pamidronate (AREDIA ® ), tiludronate (SKELID ® ) or risedronate (ACTONEL ® ); troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those that inhibit the expression of genes in signaling pathways involved in abnormal cell proliferation, such as PKC-α, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines, such as THERATOPE ® Vaccines and gene therapy vaccines, such as ALLOVECTIN ® LEUVECTIN ® Vaccines and VAXIDs ® vaccines; topoisomerase 1 inhibitors (e.g., LURTOTECAN®); rmRH (e.g., ABARELIX ® ); BAY439006 (sorafenib; Bayer); SU-11248 (Pfizer); perifosine, COX-2 inhibitors (such as celecoxib or etoricoxib), proteosome inhibitors (such as PS341); bortezomib (VELCADE ® ); CCI-779; tipifarnib (R1 1577); orafenib, ABT510; Bcl-2 inhibitors, such as oblimersen sodium (GENASENSE ® ); pixanol; EGFR inhibitors (see definition below); tyrosine kinase inhibitors (see definition below); and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing; and combinations of two or more of the foregoing, such as CHOP, an abbreviation for combination therapy with cyclophosphamide, doxorubicin, vincristine, and prednisolone, and FOLFOX, an abbreviation for treatment with oxaliplatin in combination with 5-FU and leucovorin (ELOXATIN™).

[0325] Also included within this definition are antihormonal agents that act to modulate or inhibit the effects of hormones on tumors, such as antiestrogens with mixed agonist / antagonist profiles, including tamoxifen (NOLVADEX ® ), 4-hydroxytamoxifen, toremifene (FARESTON ® ), idoxifene, droloxifene, raloxifene (EVTSTA ®), troloxifene, raloxifene, and selective estrogen receptor modulators (SERMs), such as SERM3; pure antiestrogens without agonist properties, such as fulvestrant (FASLODEX ® ) and EM800 (these agents can block estrogen receptor (ER) dimerization, inhibit DNA binding, increase ER turnover, and / or suppress ER levels); aromatase inhibitors, including steroidal aromatase inhibitors such as formestane and exemestane (AROMASIN ® ), and nonsteroidal aromatase inhibitors such as anastrozole (AREVIIDEX ® ), letrozole (FEMARA ® ) and aminoglutethimide), and other aromatase inhibitors including vorazole (RIVISOR ® ), megestrol acetate (MEGASE ® ), fadrozole, imidazole; luteinizing hormone-releasing hormone agonists, including leuprorelin (LUPRON ® and Eligard ® ), goserelin, buserelin and triptorelin; sex steroids, including progestins, such as megestrol acetate and medroxyprogesterone acetate; estrogens, such as diethylstilbestrol and premarin; and androgens / retinoids, such as fluoxymesterone; all-trans retinoic acid and fenretinide; onasterone; antiprogestins; estrogen receptor downregulators (ERDs); antiandrogens, such as flutamide, nilutamide and bicalutamide; testolactone; and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing; and combinations of two or more of the foregoing.

[0326] In the treatment of the above-mentioned autoimmune diseases or related autoimmune conditions, monoclonal antibodies that specifically bind to CD20, such as those provided herein, can be administered to patients using a variety of drug regimens in combination with different therapeutic agents (e.g., immunosuppressants, anti-inflammatory drugs, systemic hormonal drugs, anti-tumor drugs, and immunomodulatory drugs, or others). Monoclonal antibodies that specifically bind to CD20 can be administered simultaneously, sequentially, or alternatingly with different therapeutic agents, or after resistance to different therapies is demonstrated. Different therapeutic agents can be administered at the same or lower doses than those used in the art. When selecting a preferred different therapeutic agent, many factors should be considered, including the type of disease to be treated and the patient's medical history.

[0327] As used herein, the term "immunosuppressive agent" used in adjunctive therapy refers to a substance that directly suppresses or masks the patient's immune system. Such agents can be substances that inhibit cytokine production, downregulate or suppress self-antigen expression, or mask major histocompatibility complex (MHC) antigens. Examples of such agents include steroids, such as glucocorticoids, e.g., prednisone, methylprednisolone, and dexamethasone; 2-amino-6-aryl-5-substituted pyrimidines (see US 4665077), azathioprine (or, in the case of adverse reactions to azathioprine, cyclophosphamide); bromocriptine; glutaraldehyde (which masks MHC antigens, as described in US 4120649); anti-idiotypic antibodies to MHC antigens and MHC fragments; cyclosporine A; cytokine and cytokine receptor antagonists, including interferon-γ, -β, or -α antibodies; anti-tumor necrosis factor antibodies; anti-interleukin-2 antibodies and anti-IL-2 receptor antibodies; anti-L3T4 antibodies, heterologous antilymphocyte globulins, pan-T antibodies, preferably anti-CD3 or anti-CD4 / CD4α antibodies; soluble peptides containing the LFA-3 binding domain (WO 90 / 08187, published June 26, 1990); streptokinase; TGF-β; streptodornase; host DNA / RNA; FK506; RS-61443; deoxyspergualin; rapamycin; T cell receptor (US Pat. No. 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 (ER340109), such as T10B9.

[0328] In the treatment of rheumatoid arthritis, the monoclonal antibody that specifically binds to CD20 according to the present invention can be administered to the patient 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, such as cyclooxygenase inhibitors), corticosteroids (e.g., prednisolone, budesonide). Common DMARDs used in the treatment of RA are hydroxychloroquine, sulfasalazine, methotrexate, leflunomide, azathioprine, D-penicillamine, gold-based preparations (oral), gold-based preparations (intramuscular), minocycline, cyclosporin, and staphylococci obtained by protein A immunoadsorption. Conventional methods for the treatment of 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).

[0329] The monoclonal antibodies that specifically bind to CD20 according to the present invention are intended to be used in the methods of treatment described above, to be used in the treatment described above, and / or to manufacture a medicament for use in the treatment described above.

[0330] Dosage and route of administration

[0331] 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 dosages and for durations necessary to achieve the desired result. The therapeutically effective amount can vary depending on factors such as the specific condition being treated, the age, sex, and weight of the patient, and whether the monoclonal antibody that specifically binds to CD20 is administered as a stand-alone therapy or in combination with one or more additional drugs or therapies.

[0332] The dosage regimen can be adjusted to provide the optimal response. For example, a single bolus can be administered, several divided doses can be administered over time, or the dose can be proportionally reduced or increased as indicated by the urgency of the therapeutic situation. It is particularly advantageous to formulate parenteral compositions in unit dosage form for ease of administration and uniformity of dosage. As used herein, unit dosage form is intended to refer to physically discrete units suitable as unit doses for the patient / subject to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect in combination with the required pharmaceutical carrier. The specifications of the unit dosage forms of the present invention are generally determined by the following factors and are directly dependent on the following: (a) the unique characteristics of the chemotherapeutic agent and the specific therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of formulating such active compounds for treating sensitivity in subjects.

[0333] Therefore, based on the disclosure provided herein, the skilled artisan will understand that dosages and dosage regimens can be adjusted according to methods well known in the therapeutic field. That is, the maximum tolerable dose can be easily determined, and the effective amount that provides a detectable therapeutic effect to the patient can also be determined, as well as the time requirement for administering each agent to provide a detectable therapeutic effect to the patient. Therefore, although certain dosages and administration regimens are exemplified herein, these examples are in no way limiting of the dosages and administration regimens that can be provided to patients in practicing the embodiments of the present invention.

[0334] It should be noted that dosage values may vary depending on the type and severity of the condition to be alleviated and may include a single dose or multiple doses. Furthermore, it should be understood that for any particular subject, the specific dosage regimen should be adjusted over time according to the individual need and the judgment of the medical professional administering or supervising 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, the administration regimen of the compositions of the present invention may be based on various 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 specific monoclonal antibody employed that specifically binds to CD20. Thus, dosage regimens may vary widely but can be routinely determined 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 encompasses intra-patient dose escalation determined by one skilled in the art. Methods for determining appropriate dosages and regimens are well known in the art and will be understood by one skilled in the art once provided with the concepts disclosed herein.

[0335] Examples of suitable methods of administration are provided above.

[0336] It is believed that suitable dosages of monoclonal antibodies that specifically bind to CD20 according to the present invention will be in the range of 0.1-200 mg / kg, preferably 0.1-100 mg / kg, including about 0.5-50 mg / kg, such as about 1-20 mg / kg. Monoclonal antibodies that specifically bind to CD20 can be administered, for example, at a dose of at least 0.25 mg / kg, such as at least 0.5 mg / kg, including at least 1 mg / kg, such as at least 1.5 mg / kg, such as at least 2 mg / kg, such as 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, such as up to 20 mg / kg, including up to 15 mg / kg. Administration is typically repeated at appropriate intervals, for example, once a week, once every two weeks, once every three weeks, or once every four weeks, and for a period of time deemed appropriate by the attending physician, who may, in some cases, increase or decrease the dose as necessary.

[0337] Diagnostic uses and compositions

[0338] The monoclonal antibodies that specifically bind to CD20 according to the present invention are also used in diagnostic procedures (e.g., in vitro, ex vivo). For example, the monoclonal antibodies herein that specifically bind to CD20 according to the present invention can be used to detect or measure CD20 levels in a sample obtained from a patient (e.g., a tissue sample or a body fluid sample, such as inflammatory exudate, blood, serum, intestinal 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, e.g., diagnostic kits comprising the monoclonal antibodies that specifically bind to CD20 as described herein.

[0339] The following examples are provided for a better understanding of the present invention. These examples are for illustrative purposes only and should not be construed as limiting the scope of the present invention in any way.

[0340] All publications, patents, and patent applications cited in this specification are incorporated herein 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 apparent to those skilled in the art, based on the teachings of this invention, that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended embodiments. Example

[0341] The following examples are provided for a better understanding of the present invention. These examples are for illustrative purposes only and should not be construed as limiting the scope of the present invention in any way.

[0342] All publications, patents, and patent applications cited in this specification are incorporated herein 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 apparent to those skilled in the art, based on the teachings of this invention, that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended embodiments.

[0343] Materials and general methods

[0344] General information about the nucleotide sequences of human immunoglobulin light and heavy chains is given 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 the antibody chains are numbered according to EU numbering and are referred to (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).

[0345] Recombinant DNA technology

[0346] 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.

[0347] gene synthesis

[0348] The desired gene segments were prepared from oligonucleotides prepared by chemical synthesis. Gene segments 300-4000 kb long, flanked by individual restriction sites, were assembled by annealing and ligation of oligonucleotides, including PCR amplification, and subsequently cloned via the indicated restriction sites. The DNA sequence of the subcloned gene fragments was confirmed by DNA sequencing.

[0349] DNA sequencing

[0350] DNA sequences were determined by Sanger sequencing.

[0351] DNA and protein sequence analysis and sequence data management

[0352] Infomax's Vector NT1 Advance suite version 8.0 was used for sequence creation, plotting, analysis, annotation, and interpretation.

[0353] expression vector

[0354] For expression of the antibodies and antigens, variants of expression plasmids intended for expression in prokaryotic cells (E. coli) and transient expression in eukaryotic cells (e.g., CHO cells) are used. In addition to the antibody expression cassette, the vector also contains an origin of replication that allows the plasmid to replicate in E. coli and genes that confer resistance to various antibiotics (e.g., ampicillin and kanamycin) in E. coli.

[0355] Fusion genes comprising the antibody chains described below are generated by PCR and / or gene synthesis and assembled using known recombinant methods and techniques by ligating the corresponding nucleic acid segments, e.g., using unique restriction sites in the corresponding vectors. The nucleic acid sequences of the subclones are verified by DNA sequencing. For transient transfection, larger quantities of plasmid are prepared from plasmid preparations of transformed E. coli cultures.

[0356] Example 1. Production of recombinant control antibodies in mammalian cell suspension culture

[0357] Antibodies with public sequences and rituximab were used as controls. The genes for the heavy and light chain variable domains of the antibodies were synthesized and cloned into the SalI / NheI and SalI / BstWI restriction sites of the pEE-HC and pEE-CK vectors, respectively ( Figure 1 、 2 ), which vector is intended for protein production in mammalian cells.

[0358] The required amount of plasmid was grown in E. coli cells and purified using a Qiagen kit.

[0359] Control antibodies were produced in cells of an established cell line derived from Chinese hamster ovary cells (CHO-T line). 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 × 10 6 Cells were isolated from the culture medium by filtration through a 0.5 / 0.22 µm depth filter within 9 days of transfection. Target protein was isolated from the culture medium by affinity HPLC using Protein A (a bacterial protein).

[0360] The purity of the obtained protein solution was assessed by reducing and non-reducing SDS gel electrophoresis.

[0361] Example 2. Construction of the Human Antibody FAB Library MeganLib™ for Initial Experimentation

[0362] Total RNA from B lymphocytes of blood samples from more than one thousand individual human donors was isolated using the RNeasy Mini Kit according to the recommended protocol (QIAGEN). RNA concentration was determined using the Nanovue kit (GE Healthcare) and the quality of the isolated RNA was checked by electrophoresis on 1.5% agarose gels.

[0363] Reverse transcription reactions were performed using the MMLV RT kit (Evrogen) according to the recommended protocol using MMuLV reverse transcriptase and random hexamer oligonucleotides as primers.

[0364] The reverse transcription product was used as a substrate in a two-stage polymerase chain reaction to generate the variable domain gene flanked by restriction sites; the reaction was performed according to the protocol by [J Biol Chem. 1999 Jun 25;274(26):18218-30] using an oligonucleotide kit.

[0365] The resulting DNA preparation VL-CK-VH ( Figure 4 ) and ligated into the original phagemid pH5 ( Figure 5 The ligation product was transformed into SS320 electrocompetent cells prepared according to the protocol [Methods Enzymol. 2000;328:333-63]. The reservoir of the combinatorial phage Fab display library MeganLib™ was 1011 transformants. Fab library phage products were prepared according to a previously described procedure [J Mol Biol. 1991 Dec 5;222(3):581-97].

[0366] Example 3. Selection of FAB library by phage display

[0367] Specific anti-CD20 human phage Fab antibodies were obtained from the combinatorial phage Fab display library MeganLib™. Biopanning was performed on eukaryotic cells expressing human CD20 by phage display [Nat Biotechnol. 1996 Mar; 14(3): 309-14; J Mol Biol. 1991 Dec 5; 222(3): 581-97], but using magnetic beads and a KingFisher Flex device because this technology allows up to 96 different biopanning schemes and variations to be performed simultaneously.

[0368] In the bio-panning process, by making cell and pearl incubate 20 minutes on rotator, biotinylated eukaryotic cells are fixed on the surface of streptavidin magnetic beads.Then pearl is washed with PBS (pH 7.4), then pearl is sealed with PBS (pH 7.4) solution of 2% skim milk for 1 hour.Then, the phage solution of pre-incubation in PBS (pH 7.4) supplemented with 2% skim milk together with antigen-negative cells is added in the magnetic beads with binding cells.The mixture is incubated 40 minutes under agitation.By using several circulations of PBS (pH 7.4) solution washing magnetic beads supplemented with 0.1% Tween-20, unbound phage is removed.The number of times of washing cycles is increased by wheel (10 washing cycles in the first round, and 30 washing cycles in the second and third rounds). Under stirring, 100 mM Gly-HCl solution (pH 2.2) was used to elute the phage bound to the antigen on the surface of the magnetic beads from the beads for 15 minutes, and then the solution was neutralized with 1M Tris-HCl (pH 7.6). The resulting phage was used to infect E. coli TG1 bacteria, which were cultured in the bacteria, isolated and used for the next round of selection. After three to four rounds, DNA (phagemid) was isolated from the phage, and the antibody variable domain genes were cloned into expression vectors ( Figure 6 ), for Fab production in E. coli cells.

[0369] Example 4. Library Screening

[0370] Preliminary screening

[0371] Fab is produced according to standard techniques: bacterial cells are transformed with an expression vector containing the Fab gene, and during cultivation of the resulting transformants, an inducer that triggers transcription of the lac operon is subsequently added to the culture medium to induce Fab expression.

[0372] We then performed an ELISA for Fab binding to substrate-immobilized CD20 peptide using an anti-human Fab HRP-conjugated secondary antibody (Pierce-ThermoScientific) to detect antigen-bound Fab.

[0373] The rituximab Fab sequence inserted into the expression plasmid pLL was used as a positive control ( Figure 6 ).

[0374] As a result of the primary screening, we selected clones that were able to bind to the target CD20 peptide. This material was transferred for secondary screening.

[0375] Secondary screening

[0376] The secondary screening aimed to select Fab-producing clones that interacted with CD20 peptide but not with other antigens IL6R-Fc, PCSK9-VG-FE, and PD-1-Fc.

[0377] Fabs were generated according to standard techniques. We then performed ELISAs for Fab binding to various substrate-immobilized antigens according to standard procedures.

[0378] As a result of the secondary screening, Fab-producing clones that only specifically bound to the target CD20 peptide were selected.

[0379] Example 5. Optimization of lead candidates

[0380] The candidate for selection is optimized to increase humanization. The Humanizer tool from the YLab software package (developed by Biocad) is used to select substitution points. Germline functional V, D, J segments from various biological species are obtained from the IMGT database and used as data sources. Human segments are used as positive references, while segments from rats and mice are used as negative references. Based on this data, the tool proposes positions to be substituted.

[0381] For further selection, we generated 1000 candidates with multiple subsets of the selected substitution sets. Based on the crystal structure of the initial candidate-target CD20 complex, the obtained candidates were modeled. Models were generated using BENDER (developed by Biocad) and BioLuminate (from Schrodinger Suite software, developed by Schrodinger). The obtained model was evaluated by calculating the average MM-GBSA along a 100 ns molecular dynamics trajectory using OPLS 2005 force field. Molecular dynamics trajectories were obtained using Desmond (Schrodinger Suite, developed by Schrodinger). In the results obtained, we clearly distinguished 133 candidate clusters, which were proposed for further synthesis together with control candidates.

[0382] Example 6. Preparation of full-length antibodies in IgG1 format

[0383] The 133 candidates prepared were codon-optimized in CHO cells using the OligoDesigner tool from the Ylab software package (BIOCAD). The optimized sequences of the heavy and light chain variable domains were synthesized de novo and cloned into the vectors pEE-HC and pEE-CK (IgG1 format) at the Sal1 / Nhe1 and Sal1 / BsiW1 restriction sites, respectively. Figure 1 、 2). A schematic diagram of the IgG1 form is shown in Figure 3 Given in.

[0384] The resulting genetic constructs were used to transform a CHO-T cell line. As described in Example 1, proteins were isolated and purified by affinity chromatography on bacterial protein A according to standard methods. 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.

[0385] Example 7. Sequencing of high-affinity clones

[0386] The variable domain genes of positive clones were sequenced and analyzed on an Applied Biosystems 3130 Genetic Analyzer (Applied Biosystems) according to standard protocols.

[0387] Example 8. Determination of affinity of full-length antibodies on the Forte Bio Octert RED 384

[0388] The resulting KD values for the full-length candidates were determined on a Forte Bio Octet RED 384.

[0389] SAX biosensors and biotinylated CD20 peptide (Sigma Aldrich) were used in this study. The antibody rituximab was used as a control. The SAX biosensors were immersed in a solution containing 20 μg / ml of biotinylated CD20 peptide, where the peptide was immobilized. PBS containing 0.1% Tween 20 and 0.1% BSA was used as the working buffer, and further analysis was performed at 30°C.

[0390] After baseline recording in buffer solution, the sensor was immersed in a well containing a 10 μg / ml antibody solution for 150 seconds, where the complex bound, and then the dissociation of the complex was monitored in buffer solution for 300 seconds.

[0391] Binding curves were analyzed using a 1:1 interaction model after subtraction of the reference signal, according to standard procedures using Octet Data Analysis software (version 9.0).

[0392] 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 ).

[0393] Table 1

[0394]

[0395]

[0396]

[0397]

[0398]

[0399]

[0400]

[0401] Based on the results of the above analysis, candidates BCD132L-026, BCD132L-028, BCD132L-075, and BCD132L-077 were selected.

[0402] Example 9. Determination of affinity of final candidates for CD20 after transient production using Forte Bio Octert RED 384

[0403] SAX biosensors and biotinylated CD20 peptide (Sigma Aldrich) were used in this study. The antibody rituximab was used as a control. The SAX biosensors were immersed in a solution containing 20 μg / ml of biotinylated CD20 peptide, where the peptide was immobilized. PBS containing 0.1% Tween 20 and 0.1% BSA was used as the working buffer, and further analysis was performed at 30°C.

[0404] After baseline recording in buffer solution, the sensor was immersed in a well containing an antibody solution at a concentration of 10 μg / ml for 210 seconds, where the complex binds. The dissociation of the complex was then monitored in buffer for 100 seconds.

[0405] Binding curves were analyzed using a 1:1 interaction model after subtraction of the reference signal using Octet Data Analysis software (version 9.0) according to standard procedures (see Figure 8-11 ).

[0406] BCD132L-026 0.5411 3.99E-08 6.98E+05 2.79E-02 0.9795 BCD132L-028 0.4703 6.28E-08 4.17E+05 2.62E-02 0.9924 BCD132L-075 0.8719 6.23E-08 3.30E+05 2.06E-02 0.9969 BCD132L-077 0.4774 2.76E-08 6.36E+05 1.75E-02 0.9413

[0407] Based on the results of the above analysis, candidates BCD132L-028 and BCD132L-077 were selected.

[0408] Example 10. Preparation of a cell line that stably produces antibodies in the IgG1 format

[0409] Based on the results of the above assays, BCD132-L-028 and BCD132-L-077 showed the best performance. Their heavy chain sequences and light chain sequences were cloned into the HindIII and XbaI sites of the vector pSX ( Figure 24 The resulting plasmid was cultured in E. coli cells, and 600-700 μg was isolated using BenchPro. The plasmid was linearized overnight with PvuI endonuclease and then reprecipitated with ethanol to a final concentration of 900-1100 ng / μl.

[0410] CHO-K1-S cell line was cultured in S.3.87 MM medium (a synthetic medium without FBS developed by BIOCAD) + 6 mM glutamine. Transfection with a gene construct containing the coding sequence of the BCD132-L-028 and BCD132-L-077 candidate chains was performed by electroporation using a Nucleofector™ (Lonza) according to the manufacturer's protocol.

[0411] On the second day after transfection, the transfected cultures were placed under selection for 24 days by adding puromycin (final concentration of 7.2 μg / ml) and hygromycin B (final concentration of 640 μg / ml) to the culture medium. The selected cell populations were cloned. Based on the analysis results of target protein levels / structural homogeneity, cell clones expressing BCD132-L-028 and BCD132-L-077 were selected, respectively, considering the growth rate, population homogeneity, and the absence of morphological changes, while candidates BCD132-L-026 and BCD-132-L-075 were excluded.

[0412] Example 11. Determination of the affinity of final candidates cultured in stable cell lines for CD20 on a Forte Bio Octert RED 384

[0413] SAX biosensors and biotinylated CD20 peptide (Sigma Aldrich) were used in this study. The antibody rituximab was used as a control. The SAX biosensors were immersed in a solution containing 20 μg / ml of biotinylated CD20 peptide, where the peptide was immobilized. PBS containing 0.1% Tween 20 and 0.1% BSA was used as the working buffer, and further analysis was performed at 30°C.

[0414] After baseline recording in buffer solution, the sensor was immersed in a well containing a 10 μg / ml antibody solution for 150 seconds, where the complex bound, and then the dissociation of the complex was monitored in buffer solution for 300 seconds.

[0415] Binding curves were analyzed using a 1:1 interaction model after subtraction of the reference signal using Octet Data Analysis software (version 9.0) according to standard procedures ( Figure 12-15 ).

[0416] BCD132L-028 0.2717 7.57E-08 9.05E+05 6.85E-02 0.9754 BCD132L-077 (1,2) 0.4031 7.39E-08 6.41E+05 4.74E-02 0.9846 BCD132L-077 (3,4) 0.2913 7.49E-08 8.47E+05 6.34E-02 0.9776 BCD132L-077 (5,6) 0.5857 6.71E-08 9.52E+05 6.39E-02 0.9682

[0417] Example 12. Determination of the affinity of final candidates cultured in stable cell lines for FcγRIIIa-158F on a Forte Bio Octert RED 384

[0418] SAX biosensors and biotinylated FcγRIIIa-158F protein (Sigma Aldrich) were used in this study. The SAX biosensors were immersed in a solution containing 5 μg / ml of biotinylated protein, where the protein was immobilized to a signal level of 0.5 nM. PBS containing 0.1% Tween 20 and 0.1% BSA was used as the working buffer, and further analysis was performed at 30°C.

[0419] After baseline recording, the sensor was immersed in a well containing antibody solution at various concentrations for 90 seconds, where the complex bound. The dissociation of the complex was then monitored in buffer for 150 seconds.

[0420] Binding curves were analyzed using a 1:1 interaction model after subtraction of the reference signal using Octet Data Analysis software (version 9.0) according to standard procedures ( Figure 16-19 ).

[0421] BCD132L-028 5.93E-08 7.273E05 4.316E-02 0.9946 BCD132L-077 (1,2) 4.47E-08 7.658E05 3.423E-02 0.9976 BCD132L-077 (3,4) 5.41E-08 8.084E05 4.375E-02 0.991 BCD132L-077 (5,6) 4.17E-08 9.231E05 3.845E-02 0.9954

[0422] Example 13. Determination of the affinity of the final candidates cultured in stable cell lines for FcγRIIIa-158V on a Forte Bio Octert RED 384

[0423] SAX biosensors and biotinylated FcγRIIIa-158V protein (Sigma Aldrich) were used in this study. The SAX biosensors were immersed in a solution containing 5 μg / ml of biotinylated protein, where the protein was immobilized to a signal level of 0.5 nM. PBS containing 0.1% Tween 20 and 0.1% BSA was used as the working buffer, and further analysis was performed at 30°C.

[0424] After baseline recording, the sensor was immersed in a well containing antibody solution at various concentrations for 90 seconds, where the complex bound. The dissociation of the complex was then monitored in buffer for 150 seconds.

[0425] Binding curves were analyzed using a 1:1 interaction model after subtraction of the reference signal using Octet Data Analysis software (version 9.0) according to standard procedures ( Figure 20-23 ).

[0426] BCD132L-028 1.78E-08 6.385E05 1.139E-02 0.9963 BCD132L-077 (1,2) 2.38E-08 6.563E05 1.564E-02 0.9942 BCD132L-077 (3,4) 1.66E-08 6.906E05 1.149E-02 0.9961 BCD132L-077 (5,6) 2.27E-08 7.311E05 1.659E-02 0.9952

[0427] Example 14. Specific binding of BCD-132-L-028 and BCD-132-L-077 to the CD20 receptor on the WIL2-S cell line was measured using flow cytometry.

[0428] MabThera (rituximab) was used as a control antibody. Samples and control antibodies were diluted to a concentration of 200 μg / ml and titrated in 4 increments in staining buffer (PBS, 0.5% BSA, 0.1% NaN3). The concentration was 1×10 6 The WIL2-S (ATCC® CRL8885) cell suspension of individual cells / ml was incubated with the standard solution of titration and the test sample. The suspension was stirred and incubated on ice for 30 minutes. After the incubation time, the plate was centrifuged, the supernatant was collected, 100 μl of staining buffer was added, and the mixture was resuspended and centrifuged. The supernatant was collected and the precipitate was resuspended in the fluorescent anti-human Fc-PE antibody (Jackson Immunoresearch, 109-115-098) solution of the conjugate in the staining buffer. The plate was incubated on ice for 30 minutes in the dark. After the incubation time, the plate was centrifuged, the supernatant was collected, 100 μl of staining buffer was added, and the mixture was resuspended and centrifuged. The supernatant was collected and the precipitate was resuspended in the staining buffer of 150 μl and measured by flow cytometer Guava12HT (Merck Millipore). The InCyte module of guavaSoft 3.1.1 software was used to analyze the data.

[0429] The test antibodies BCD-132-L-028 and BCD-132-L-077 bound to the CD20 receptor on the WIL2-S cell line at levels equivalent to MabThera (rituximab). Figure 25 middle.

[0430] Example 15. Measurement of complement-dependent cytotoxicity of BCD-132-L-028 and BCD-132-L-077.

[0431] The WIL2-S (ATCC® CRL-8885™) cell line was used for complement-dependent cytotoxicity assays.

[0432] The assay was performed in RPMI-1640 supplemented with 2 mM glutamine, 0.1% bovine serum albumin, and 50 μg / ml gentamicin. The test antibodies BCD-132-L-028, BCD-132-L-077, and MabThera (rituximab) were serially diluted starting at a concentration of 50 μg / ml. The resulting solution was added to a 96-well plate at 50 μl / well. A 1 × 10 6 A WIL2-S cell suspension at 10 cells / ml was prepared and added to the plate at 50 μl / well. A working solution of complement (Quidel, A113) was prepared and added to the plate at 50 μl / well.

[0433] The plate was incubated for 2 hours at 37°C, 5% CO2. After the incubation time, 15 μl / well of Alamar blue dye was added to the wells, and the plate was incubated at 37°C, 5% CO2 until a gradient staining was observed. Fluorescence was measured at excitation / emission wavelengths of 544 / 590 nm using an Infinite M200Pro plate reader.

[0434] The complement-dependent cytotoxicity levels of BCD-132-L-028 and BCD-132-L-077 were equivalent to those of MabThera (rituximab). Figure 26 、 Figure 27 middle.

[0435] Example 16. Measurement of antibody-dependent cellular cytotoxicity of BCD-132-L-028 and BCD-132-L-077 using the reporter line Jurkat-NFAT-CD16.

[0436] WIL-2S (ATCC® CRL-8885™) was used as a target line for measuring antibody-dependent cellular cytotoxicity. As effector cells, we employed the reporter lines Jurkat-NFAT-CD16 High (a high-affinity allotype of CD16, V158) and Jurkat-NFAT-CD16 Low (a 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 an NFAT response element.

[0437] We prepared 0.5 × 10 6 25 μl / well of the target cell suspension was added to a culture plate with white walls.

[0438] We added titrated concentrations of BCD-132-L-028 or BCD-132-L-077 and MabThera (rituximab) starting from 5 μg / ml in increments of 5 (25 μl / well), and 6 The plate was incubated at 37°C, 5% CO2 for 4-8 hours with a suspension of 100 cells / ml of the reporter line Jurkat-NFAT-CD16 High or Low (25 μl / well).

[0439] After the incubation time, we added 75 μΐ / well of Bio-Glo Luciferase Assay Reagent (Promega) and measured luminescence using an Infinite M200Pro at 100 ms integration time.

[0440] Compared to MabThera, BCD-132-L-028 and BCD-132-L-077 showed significantly higher ADCC activity: 3-4 times higher when using a reporter line with a high-affinity allotype of CD16, and 12-16 times higher when using a reporter line with a low-affinity allotype of CD16. Figure 28 middle; Figure 29 Results are shown for a reporter line with a low affinity allotype of CD16, whereas Figure 30 、 Figure 31 Results are shown for a reporter line with a high affinity allotype of CD16.

[0441] Example 17. Measurement of CD19+ B cell depletion induced by BCD-132-L-028 and BCD-132-L-077 using whole blood from healthy donors.

[0442] The activity of the test samples was measured ex vivo using whole blood from healthy donors with CD16a receptor allotypes: FF (low affinity receptor), FV (heterozygous), VV (high affinity receptor).

[0443] MabThera (rituximab) was used as a control antibody. The control antibody and the test antibody were titrated in triplicate in 96-well plates. Blood from healthy donors was collected into vacuum tubes with Li-heparin. The tubes were incubated at room temperature for 30 minutes. 10 μl of the 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 plate was stirred at 600 rpm for 2 minutes on an orbital shaker (2 mm) and then cultured in a CO2 incubator for 22 hours.

[0444] After the incubation period, the samples were stained for 1 hour with fluorescently labeled antibodies against CD45, CD3 / CD19 (BD Pharmingen). The cells were fixed and lysed with BD Pharmingen lysis buffer, followed by two washes in staining buffer (DPBS, 0.1% NaN3, 0.5% BSA) from the lysis buffer. The number of CD45+CD3+ and CD45+CD19+ events was measured (at least 10,000 events within the CD45+ gate). Data were analyzed using the InCyte module of guavaSoft 3.1.1 software.

[0445] Relative B cell depletion was measured using the B / T cell ratio of the spot without antibody (the number of B cells was considered 100% = 0% B cell depletion). The B / T cell ratio was calculated using the following formula:

[0446]

[0447] The percentage of B cell depletion was calculated using the following formula:

[0448]

[0449] The statistical environment R with the extension package drc is used to plot four-parameter curves.

[0450] Compared to MabThera (rituximab), the test antibodies BCD-132-L-028 and BCD-132-L-077 showed significantly higher activity. Thus, when using blood donations of the FF allotype, BCD-132-L-028 and BCD-132-L-077 induced approximately 50% depletion of CD19+ cells, while MabThera (rituximab) induced approximately 20% depletion. When using blood donations of the CD16 allotypes FV and VV, the ED50 values of MabThera (rituximab) were significantly higher than those of BCD-132-L-028 and BCD-132-L-077. The level of B cell depletion by BCD-132-L-028 and BCD-132-L-077 was not dependent on the donor CD16 allotype. The results are shown in Figure 32 middle.

[0451] Example 18. Antibody-dependent cellular cytotoxicity (ADCC) activity assay of anti-CD20 antibody candidates on Ramos cell line using human peripheral blood mononuclear cells (PBMCs).

[0452] The CD-20 expressing Ramos cell line and PBMC from healthy donors were used for ADCC assay. Ramos cells were cultured in RPMI-1640 medium supplemented with 10% FBS (fetal bovine serum) at 37°C under 5% CO2. The cells were stained with the fluorescent dye calcein AM, which can only escape from cells with damaged cell walls. 5 cells / ml of cell suspension.

[0453] Separation was performed by Ficoll density gradient (1.077 g / cm 3 PBMCs were isolated from venous blood of healthy donors. The cells were cultured at a density of 5 × 10 6 cells / ml of cell suspension.

[0454] A series of antibody dilutions were added to the wells of a 96-well plate at 50 μl / well for ADCC assays. 100 μl / well of Ramos suspension and 50 μl / well of PBMC suspension were added. The plate was incubated at 37°C with 5% CO2 for 4 hours. 30 minutes before the end of the incubation, 10 μl / well of 10% Tryton X-100 was added to the largest lysis well. After incubation, 100 μl / well of cell fluid was transferred without transferring the cells to a new plate. Fluorescence was measured at excitation / emission wavelengths of 485 / 538 nm.

[0455] ADCC efficacy was calculated using the following formula:

[0456]

[0457] Based on ADCC as a function of antibody concentration, we determined the dependence described by a 4-parameter equation and calculated the half-maximal effective concentration (EC50) using the GraphPad Prism 6.0 software package.

[0458] According to the ADCC assay, the anti-CD20 antibody candidates BCD-132-L-028 and BCD-132-L-077 showed better performance than rituximab. Figure 33 middle.

[0459] Example 19. The activity of the BCD132-L-077 monoclonal antibody product was investigated in the experimental autoimmune encephalomyelitis (EAE) model following repeated intravenous administration in cynomolgus monkeys (Macaca fascicularis).

[0460] This study was conducted in male cynomolgus macaques (Macaca fascicularis). A total of 12 animals participated in the experiment, with each group consisting of four monkeys. Two product doses were used: 5 mg / kg and 22 mg / kg. Animals in the control group received a placebo. Data regarding the animal experimental groups are presented in Table 2.

[0461] Table 2. Animal groups.

[0462]

[0463] To induce experimental autoimmune encephalomyelitis in cynomolgus macaques, a modified technique described in numerous publications was used. To sensitize the primates, a recombinant protein from JSC BIOKAD, the extracellular domain of human myelin oligodendrocyte protein (rhMOG, amino acids 1-125), was used.

[0464] Each animal was injected three times with an emulsion containing 400 μg of protein mixed with 400 μl of complete Freund's adjuvant in 400 μl of phosphate-buffered saline.Immediately after the first administration of rhMOG, the monkeys were injected with a heat-killed Bordetella pertussis (B. pertussis) vaccine.

[0465] First administration of rhMOG

[0466] To prepare the emulsion, 10 mg of rhMOG was dissolved in 10 ml of phosphate-buffered saline. 10 ml of Freund's complete adjuvant was added to the resulting solution. The resulting emulsion was administered subcutaneously via 100 μl injections at eight sites (total administration volume per monkey: 800 μl):

[0467] 4 injections in the spinal area (between the shoulder blades, 2 on the right side of the spinal cord and 2 on the left side);

[0468] 2 injections in the inguinal area (1 on the right side and 1 on the left side);

[0469] 2 injections in the armpit (1 on the right side and 1 on the left side);

[0470] After the first administration of rhMOG, 10 10 particles of inactivated Bordetella pertussis.

[0471] The interval between the first and second immunizations was 28 days.

[0472] Second administration of rhMOG

[0473] To prepare the emulsion, 10 mg of rhMOG was dissolved in 10 ml of phosphate-buffered saline. 10 ml of Freund's complete adjuvant was added to the resulting solution. The resulting emulsion was administered subcutaneously via 100 μl injections at eight sites (total administration volume per monkey: 800 μl):

[0474] 4 injections in the spinal area (between the shoulder blades, 2 on the right side of the spinal cord and 2 on the left side);

[0475] 2 injections in the inguinal area (1 on the right side and 1 on the left side);

[0476] 2 injections in the armpit (1 on the right side and 1 on the left side);

[0477] The interval between the second and third immunizations is 14 days.

[0478] The third administration of rhMOG

[0479] To prepare the emulsion, 10 mg of rhMOG was dissolved in 10 ml of phosphate-buffered saline. 10 ml of Freund's complete adjuvant was added to the resulting solution. The resulting emulsion was administered subcutaneously via 100 μl injections at eight sites (total administration volume per monkey: 800 μl):

[0480] 4 injections in the spinal area (between the shoulder blades, 2 on the right side of the spinal cord and 2 on the left side);

[0481] 2 injections in the inguinal area (1 on the right side and 1 on the left side);

[0482] 2 injections in the armpit (1 on the right side and 1 on the left side);

[0483] Evaluation of the efficacy of BCD132-L-077 in the experimental autoimmune encephalomyelitis (EAE) model

[0484] To measure product activity, histological examination of brain and spinal cord tissue was performed. The severity of the inflammatory response in spinal cord and brain tissue was scored on a three-point scale according to Table 3.

[0485] Table 3. Inflammatory Response Severity Scale

[0486] score Severity of inflammation 0 No inflammatory signs; 1 Rare (1-3 per section) perivascular infiltrates; 2 Moderate frequency (4-10 per section) of perivascular infiltrates; possible inflammation of the meninges; 3 Ubiquitous perivascular infiltrates and inflammatory cell infiltration of neural tissue.

[0487] As shown in Table 4, the severity of degenerative changes in the spinal cord and brain tissues of primates was scored.

[0488] Table 4. Spinal cord / brain demyelination severity scale

[0489] score Severity of demyelination 0 No signs of demyelination; 1 Rare (1-3 per section) demyelination foci; 2 moderate frequency (4-10 per section) of demyelinated foci; 3 There is widespread demyelination with large fusion foci.

[0490] The severity of inflammation was measured in Figure 34 Compared to the control group (sham control), a decrease in the group total score was shown for the 5.0 mg / kg and 22.0 mg / kg doses used in the study. The changes detected were not reliable. In addition, there were no significant differences between the experimental groups. Therefore, it can be said that the anti-inflammatory effect efficacy was comparable for the two product doses tested.

[0491] The severity of degenerative changes in neural tissue was measured in Figure 35 When the lowest dose of the test product, 5.0 mg / kg, BCD132-L-077, was used, a significant decrease in the demyelination score was observed compared to the control.

[0492] The group of animals that received the product at a dose of 22.0 mg / kg also showed a decrease in the value of the parameter in question, but it was not reliable. There were no significant differences in the values between the experimental groups; therefore, it can be concluded that the activity levels of the two doses are comparable.

[0493] The study showed that the product at doses of 5.0 mg / kg and 22.0 mg / kg showed comparable anti-inflammatory effects in terms of efficacy and reduced the level of demyelination in the neural tissue of experimental primates to a similar extent. Based on these data, the 5.0 mg / kg dose can be confirmed as the pharmacologically active dose (FAD).

[0494] Example 20. Toxicity and main pharmacokinetic parameters (toxicokinetics) of BCD132-L-077 were investigated following multiple subcutaneous administrations in cynomolgus monkeys (Macaca fascicularis).

[0495] The study was conducted in male cynomolgus macaques (Macaca fascicularis). After quarantine, the animals were divided into four experimental groups of three males each, depending on the product dose to be administered; body weight was used as the criterion for group assignment. Three product doses were used in the experiment: 44 mg / kg; 88 mg / kg; and 176 mg / kg. Animals in the control group received a placebo product. The animal's condition, the number of animals that died, and the time of their death were used as evaluation criteria. The animals were observed for 8 hours after injection and then once daily for 42 days.

[0496] The data on animal experimental groups and product dosages are given in Table 5:

[0497] Table 5. Animal groups used in the efficacy study of BCD132-L-077 product

[0498]

[0499] The condition of the animals, the number of dead animals and the time of their death were used as evaluation criteria.

[0500] Within the scope of the study, clinical examinations were performed 8 hours after injection and then daily; in addition, we assessed the following:

[0501] ∙ Animal weight;

[0502] ∙ Body temperature;

[0503] Urinalysis

[0504] ∙ Whole blood analysis for the following parameters: red blood cell count, white blood cell count, hemoglobin concentration;

[0505] Serum biochemistry analysis for the following parameters: lactate dehydrogenase, total bilirubin, total protein, glucose, aspartate aminotransferase, alanine aminotransferase;

[0506] ∙ Product concentration in serum.

[0507] According to the study, the test product did not induce mortality in cynomolgus macaques after a single intravenous administration, and the animals tolerated the administration procedure well. The product showed no effects on overall toxicity indicators or organ function across all parameters studied. The product exhibited linear pharmacokinetics within the selected dose range.

[0508] Example 21. Pharmacokinetics and immunogenicity of the BCD132-L-077 product were studied in cynomolgus monkeys (Macaca fascicularis) after four weeks of repeated intravenous administration followed by a two-week recovery period.

[0509] The pharmacokinetics and immunogenicity of the drug 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 monkeys and 9 male monkeys aged 4 to 7 years (cynomolgus macaques), participated in the study. The animals were divided into 3 groups based on the product dose to be administered.

[0510] Table 6. Animal groups.

[0511]

[0512] The levels of BCD132-L-077 in primate serum were measured by enzyme-linked immunosorbent assay. During the study, the immunogenicity of the BCD132-L-077 product was measured to confirm the possible effect of the formation of product-binding antibodies on the pharmacokinetic parameters. In addition, we measured the immunogenicity of the BCD132-L-077 product based on the ratio AUC ss168 (336-504) : AUC 0-168 Cumulative factors were calculated.

[0513] To calculate AUC 0-168 Values, serum was obtained immediately before the first product administration and then 0.25, 24, 72 and 168 hours after administration; to calculate AUCss 168 (336-504) Serum was obtained immediately before the fourth product administration and then at 0.25, 24, 72, and 168 hours after administration. Pharmacokinetic parameters were calculated based only on data from those animals in which no BAb was detected. Therefore, data from animals that showed an immune response to the product were excluded from the calculation of pharmacokinetic parameters. Product accumulation was measured by the accumulation index; for this purpose, AUC was determined. 0-168 and AUCss (336-504) value, and calculate the index by the following formula:

[0514]

[0515] Among them, AUCss 168 (336-504)It is the equilibrium value of the area under the product concentration-time curve over a period corresponding to the dosing interval (168 hours) under repeated administration;

[0516] AUC 0-168 It 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 administration.

[0517] To analyze the levels of antibodies binding to the BCD132-L-077 product, we used sera from primates. Samples were obtained for the study before the first administration and then at 4 and 7 weeks of the experiment.

[0518] 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.

[0519] Figure 36 Smoothed BCD132-L-077 product concentration-time curves for primate serum are shown. Table 7 shows the mean values of the main pharmacokinetic parameters for each group.

[0520] Table 7. Comparative data on key pharmacokinetic parameters under administration of escalating doses of BCD132-L-077 product (steady-state (ss) values were calculated using a period of 336-504 hours corresponding to the dosing interval (168 hours).

[0521]

[0522] The pharmacokinetic parameters of the BCD132-L-077 product were calculated using steady-state AUC values during the dosing period of 336-504 hours (168 hours) after product administration. The initial AUC calculated at the first dosing interval (1-168 hours) was directly related to the dose used. In the minimum dose group, this parameter was 13,435.59 ± 11,150.80 (μg / ml) hours; in the average dose group: this parameter was 13,301.04 ± 8,719.10 (μg / ml) hours; and in the maximum dose group: this parameter was 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 receiving a 22 mg / kg dose of the product, 22,366.93 ± 10,370.60 (μg / ml) hours in the mean dose group, and 97,088.33 ± 94,675.60 (μg / ml) hours in the maximum dose group. The half-life (T1 / 2) was 94.76 ± 50.30 hours in the group receiving a 22 mg / kg dose of the product, 155.38 ± 111.20 hours in the mean dose group, and 471.00 ± 782.50 hours in the maximum dose group. The clearance rate was 0.00924±0.004 l / hour in the minimum dose group, 0.01873±0.015 l / hour in the average dose group, and 0.01228±0.005 l / hour in the maximum dose group. The mean residence time (MRT) of the product was 66.37±13.60 hours in the minimum dose group, 68.30±17.50 hours in the average dose group, and 71.51±9.20 hours in the maximum dose group. The steady-state distribution volume (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, respectively. In the minimum dose group, average dose group, and maximum dose group, the average steady-state concentration (C ss ) were 156.12±100.10 μg / ml, 133.14±61.70 μg / ml, and 577.91±563.50 μg / ml, respectively. The maximum steady-state concentration (C ssmax ) were 326.89±241.70 μg / ml, 427.03±266.50 μg / ml, and 1,212.72±830.90 μg / ml. The minimum steady-state concentration (C ssmin) were 82.64±75.20 μg / ml, 84.58±63.90 μg / ml, 162.88±74.70 μg / ml. The data obtained indicate that the product concentration in the serum of primates is directly related to the BCD132-L-077 dose used in the experiments.

[0523] For the group of animals that received the product at the lowest dose of 22.0 mg / kg, the cumulative index (R) was 1.95. For the group of animals that received BCD-132 at an average dose of 44.0 mg / kg, the cumulative index R was 1.68. For the group of animals that received the product at the highest dose (88.0 mg / kg), the index R was 2.84. The experimental data obtained indicate that the cumulative index value does not depend on the dose used.

[0524] During the study, in order to confirm the possible effect of the formation of product-binding antibodies on pharmacokinetic parameters, the immunogenicity of the BCD132-L-077 product was measured. Experimental data indicated that BAbs were present in 11.11% of all animals participating in the study. No gender dependence was found; BAbs were observed in one male and one female. In only one animal from each group, product-binding antibodies were found in the average dose group and the maximum dose group. Pharmacokinetic parameters can be evaluated in all animal groups, provided that experimental data for animals for which the presence of binding antibodies has been confirmed are excluded from processing.

[0525] Example 23. Toxicity and local irritation effects were studied in cynomolgus monkeys (Macaca fascicularis) after four weeks of repeated intravenous administration of the BCD132-L-077 product followed by a two-week recovery period.

[0526] A total of 30 cynomolgus monkeys (Macaca fascicularis), aged 4 to 7 years, 15 males and 15 females, participated in the study. Each group consisted of 6 monkeys. The toxicity of the product under repeated administration was studied at doses of 22 mg / kg, 44 mg / kg, and 88 mg / kg. The animals were divided into 5 groups according to the dose of product to be administered and the time of euthanasia:

[0527] At the lowest dose of 22 mg / kg of the BCD132-L-077 product (3 females and 3 males);

[0528] At an average dose of 44 mg / kg of the BCD132-L-077 product (3 females and 3 males);

[0529] Euthanasia was performed after the administration of the BCD132-L-077 product at a maximum dose of 88 mg / kg (3 females and 3 males);

[0530] Euthanasia was performed after the recovery period (3 females and 3 males) at the maximum dose of 88 mg / kg of the BCD132-L-077 product;

[0531] Sham controls (3 females and 3 males).

[0532] The data on the animal experimental groups are shown in Table 8.

[0533] Table 8. Animal groups.

[0534]

[0535] Clinical examinations were performed daily within the scope of the study; in addition, we examined the following:

[0536] ∙ Animal weight;

[0537] ∙ Body temperature;

[0538] Urinalysis

[0539] ECG

[0540] ∙ Hemostasis parameters: activated partial thromboplastin time, fibrinogen concentration, prothrombin time;

[0541] ∙ Whole blood analysis for the following parameters: red blood cell count, white blood cell count, hemoglobin concentration, lymphocytes, monocytes, neutrophils, eosinophils, basophils;

[0542] Serum biochemistry analysis for the following parameters: lactate dehydrogenase, total bilirubin, total protein, glucose, aspartate aminotransferase, alanine aminotransferase, cholesterol, triglycerides, urea, creatinine, sodium, potassium, alkaline phosphatase;

[0543] ∙ Pathomorphological and histological studies.

[0544] The local irritation effect was evaluated based on the examination data and the results of histological examination.Tissues at the application site and draining lymph nodes were selected for histological examination.

[0545] Based on histological examination, the test product did not show local irritation effects.

[0546] Data obtained from a toxicity study of the BCD132-L-077 therapeutic monoclonal antibody product produced by JSC BIOKAD showed that after four weeks of repeated weekly intravenous administration, the test product did not exhibit toxic effects on major organs and organ systems in experimental animals. The no-observed-adverse-effect level (NOAEL) determined in this study corresponded to the maximum dose of the test product BCD132-L-077 and reached 88 mg / kg.

[0547] Example 24. The antitumor activity of the BCD132-L-028 product was studied after repeated intraperitoneal administration in immunodeficient hIL15-NOG mice humanized with human NK cells on a subcutaneous xenograft model using the Raji human lymphoma cell line.

[0548] In this study, we planned to use seven groups of immunodeficient transgenic hIL15-NOG mice, which constitutively express human IL-15 and weigh 15.0-25.0 g. A total of 84 female animals participated in the experiment. The data for each group are shown in Table 9.

[0549] Table 9. Animal groups.

[0550]

[0551] Animals were weighed prior to tumor cell line administration and then twice weekly throughout the experiment.

[0552] Following tumor cell administration, tumor nodules were measured twice weekly throughout the experiment.

[0553] The volume of tumor nodules was calculated using the following formula:

[0554] V = π / 6×L×W×H, where L, W, and H are the tumor linear dimensions.

[0555] The efficacy of the test product was evaluated by the tumor growth inhibition (TGI) index relative to the tumor growth index (I). The index was calculated by the following formula:

[0556]

[0557] Where V k and V o The median tumor volumes of the control and treatment groups were ( ).

[0558]

[0559] Where I is the tumor growth index, i is the experimental day, is the daily tumor volume.

[0560] Example 25. Study of the toxicity and main pharmacokinetic parameters (toxicokinetics) of the BCD132-L-028 monoclonal antibody product after a single intravenous administration in cynomolgus monkeys (Macaca fascicularis)

[0561] In this study, we planned to use four experimental groups of cynomolgus monkeys, each group consisting of three males. The monkeys were kept in individual cages with the number of animals according to the experimental instructions.

[0562] Using body weight as a criterion, animals are randomly assigned to groups according to the dose of the substance to be administered.

[0563] Table 10. Animal groups.

[0564]

[0565] The product is intended for intravenous administration as a sterile isotonic saline solution in the ulnar vein.Isotonic saline (placebo) is administered to animals in the control group using the same volume and method as used in the experimental groups.

[0566] Clinical examinations were performed daily within the scope of the study; in addition, we examined the following:

[0567] 1. Animal weight;

[0568] 2. Body temperature;

[0569] 3. Urinalysis;

[0570] 4. Whole blood analysis for the following parameters: red blood cell count, white blood cell count, hemoglobin concentration;

[0571] 5. Serum biochemistry analysis for the following parameters: lactate dehydrogenase, total bilirubin, total protein, glucose, aspartate aminotransferase, alanine aminotransferase;

[0572] 6. Study product concentrations in serum, calculate key pharmacokinetic parameters, and assess pharmacokinetic linearity.

[0573] Example 26. Study of the pharmacokinetics and immunogenicity of the BCD132-L-028 monoclonal antibody product in cynomolgus monkeys (Macaca fascicularis) after repeated intravenous administration for 13 weeks followed by a 30-day recovery period.

[0574] We plan to use 18 cynomolgus monkeys (9 males and 9 females) in the pharmacokinetic and immunogenicity studies after repeated intravenous administration for 13 weeks followed by a 30-day recovery period.

[0575] According to the dose of the substance to be administered, the animals were divided into 3 groups (Table 11), each group including 3 females and 3 males: minimum dose group (6 mg / kg), medium dose group (20 mg / kg), maximum dose group (60 mg / kg).

[0576] Table 11. Animal groups.

[0577]

[0578] In our research, we plan to:

[0579] 1. Evaluate the test product concentration in the serum of experimental animals;

[0580] 2. Evaluate the accumulation of the test product under administration of increasing doses;

[0581] 3. Evaluate the level of binding antibodies under repeated intravenous administration of the test product.

[0582] Example 27. Study of toxicity and local irritant effects after repeated intravenous administration of the BCD132-L-028 monoclonal antibody product in cynomolgus monkeys (Macaca fascicularis) for 13 weeks followed by a 30-day recovery period.

[0583] In this study, we planned to use five experimental groups of cynomolgus monkeys, each consisting of 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 after the end of the product administration period), a main maximum dose group (60 mg / kg, necropsy after the end of the recovery period), and a sham control group (necropsy after the end of the recovery period).

[0584] Table 12. Animal groups (*-animals from satellite group)

[0585]

[0586] A clinical examination of each animal was performed daily; in addition, we assessed the following:

[0587] ∙ Animal weight;

[0588] ∙ Body temperature (before administration and then weekly until termination of the experiment);

[0589] ∙ Use of Poly-Spectr electrocardiograph and its effects on the cardiovascular system;

[0590] Urinalysis

[0591] ∙ Whole 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;

[0592] ∙ Assessment of the effects on the coagulation system with respect to the following parameters: activated partial thromboplastin time, fibrinogen concentration, prothrombin time;

[0593] Serum biochemistry 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;

[0594] At the end of the product application period (week 14), we planned to euthanize three males and three females from each animal group. At the end of the recovery period (week 18), the remaining two males and two females in each group were euthanized. The local irritation effect was evaluated based on the results of autopsy data and histological examination. For histological examination, we selected the segment of the ulnar vein, the tissue at the application site, and the draining lymph nodes.

Claims

1. A monoclonal antibody that specifically binds to CD20, comprising: 1) a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 1 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 3; 2) a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 5 and a light chain comprising the amino acid sequence shown in SEQ ID NO:

7.

2. The monoclonal antibody according to claim 1, wherein the antibody that specifically binds to CD20 is a full-length IgG antibody.

3. The monoclonal antibody according to claim 2, wherein the intact IgG antibody is of the human IgG1 isotype.

4. A nucleic acid encoding the antibody according to any one of claims 1 to 3.

5. The nucleic acid according to claim 4, wherein the nucleic acid is DNA.

6. An expression vector comprising the nucleic acid according to any one of claims 4-5.

7. A method for producing a host cell for producing the antibody according to any one of claims 1 to 3, the method comprising transforming the cell with the vector according to claim 6.

8. A host cell for producing an antibody according to any one of claims 1 to 3, comprising a nucleic acid according to any one of claims 4 to 5.

9. A method for producing an antibody according to any one of claims 1 to 3, comprising culturing the host cell according to claim 8 in a culture medium under conditions sufficient to produce the antibody, and subsequently isolating and purifying the produced antibody, if necessary.

10. A pharmaceutical composition for preventing or treating a disease or condition mediated by CD20, comprising the antibody according to any one of claims 1 to 3 in combination with one or more pharmaceutically acceptable excipients.

11. The pharmaceutical composition for prevention or treatment according to claim 10, wherein the disease or condition is selected from: a) B-cell lymphoma or leukemia, or b) rheumatoid arthritis, systemic lupus erythematosus (SLE), lupus nephritis, 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, solid organ transplant rejection, graft-versus-host disease (GvHD), diabetes mellitus, Raynaud's syndrome, Sjögren's syndrome, and glomerulonephritis.

12. The pharmaceutical composition according to claim 11, wherein the B-cell lymphoma is selected from the group consisting of: non-Hodgkin's lymphoma (NHL) or Hodgkin's disease (Hodgkin's lymphoma).

13. The pharmaceutical composition according to claim 11, wherein the leukemia is selected from the group consisting of chronic lymphocytic leukemia (CLL) and small lymphocytic lymphoma (SLL).

14. A pharmaceutical combination for preventing or treating a disease or condition mediated by CD20, comprising an antibody according to any one of claims 1 to 3 and at least one different therapeutically active compound.

15. Pharmaceutical combination according to claim 14, wherein the different therapeutically active anti-tumor compounds are selected from chemotherapeutic agents, antibodies or anti-hormonal agents.

16. Use of a therapeutically effective amount of an antibody according to any one of claims 1 to 3 or a pharmaceutical composition according to claim 10 for the preparation of a medicament for treating a disease or condition mediated by CD20, wherein the disease or condition is selected from: a) B-cell lymphoma or leukemia, or b) rheumatoid arthritis, systemic lupus erythematosus (SLE), lupus nephritis, 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, solid organ transplant rejection, graft-versus-host disease (GvHD), diabetes mellitus, Raynaud's syndrome, Sjögren's syndrome, and glomerulonephritis.

17. Use according to claim 16, wherein the B-cell lymphoma is selected from the group consisting of: non-Hodgkin's lymphoma (NHL) or Hodgkin's disease (Hodgkin's lymphoma).

18. Use according to claim 16, wherein the leukemia is selected from the group consisting of chronic lymphocytic leukemia (CLL) and small lymphocytic lymphoma (SLL).

Images