EA044757B1 - MONOCLONAL ANTIBODY THAT SPECIFICALLY BINDS TO CD20 - Google Patents

Description

Field of technology

The present invention relates to the field of biotechnology, namely to antibodies to CD20 or antigen-binding fragments thereof, as well as their use for the treatment of diseases that are associated with B cells. More specifically, the present invention relates to monoclonal antibodies that specifically bind to CD20 (CD20 B lymphocyte antigen). The invention also relates to a nucleic acid encoding the antibody or an antigen-binding fragment thereof, an expression vector, a method for producing the antibody, and the use of the antibody for the treatment of diseases or disorders associated with B cells, namely the B lymphocyte antigen CD20.

State of the art

Lymphocytes are one of several populations of white blood cells; they specifically recognize and respond to foreign antigen. The three main classes of lymphocytes are B lymphocytes (B cells), T lymphocytes (T cells), and natural killer (NK) cells. B lymphocytes are the cells responsible for the production of antibodies and the humoral immune response. The cells mature in the bone marrow and leave it, expressing an antigen-binding antibody 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 rapidly divide and its progeny differentiate to form memory B cells and effector cells called plasmacytes. Memory B cells have a long lifespan and continue to express membrane-bound antibody with the same specificity as the original parent cell. Plasmocytes do not produce membrane-bound antibody, but instead produce a secreted form of the antibody. Secreted antibodies are the main effector molecules of the humoral immune response.

The CD20 antigen (also called the human B cell-specific antigen, BP35) is a hydrophobic transmembrane protein with a molecular weight of approximately 35 kDa localized on pre-B and mature B cells (Valentine et al., J Biol Chem 264(19), 1989, pp. 1282-11287, 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 lymphomas (HXJI) (Anderson et al., Blood 63(6), 1984, cc. 1424-1433), but it is not found on hematopoietic stem cells, pro- B cells, healthy plasma cells or other healthy tissues (Tedder et al., J. Immunol. 135(2), 1985, pp. 973-979). CD20 likely regulates the early stage(s) of the activation process of the cell initiation and differentiation cycle (Tedder et al., supra) and possibly functions as calcium ion channels (Tedder et al., J. Cell. Biochem. 14D, 1990, p. 195).

Given that CD20 expression occurs on B cells in lymphomas, this antigen may represent a valuable therapeutic target in the treatment of these lymphomas.

The antibody rituximab (RITUXAN, MabThera, Acellbia), which is a genetically engineered chimeric mouse/human monoclonal antibody to the human CD20 antigen, is used to treat patients who suffer from relapsed or mildly refractory or follicular CD20-positive B-cell disease. Hodgkin's lymphoma. Rituximab is an antibody designated C2B8 in US 5,736,137 and US 5,776,456. In vitro mechanism of action studies have demonstrated that rituximab binds human complement and lyses lymphoid B cell lines through complement-dependent cytotoxicity (CDC) (Reff et al., Blood 83 (2), 1994, pp. 435-445). In addition, this antibody has significant activity in the antibody-mediated cell-dependent cytotoxicity (ADCC) assay. In preclinical in vivo studies, rituximab reduced the number of B cells in the peripheral blood, lymph nodes, and bone marrow of cynomolgus cynomolgus monkeys (Macaca fascicularis), likely due to complement and cell-mediated processes (Reff et al., Blood 83 (2), 1994, pp. 435-445).

In addition, it was later discovered that antibodies to CD20, such as rituximab, are also an effective therapeutic agent in the treatment of various autoimmune diseases, for example, rheumatoid arthritis (patents RU 2358762, RU 2489166 and RU 2457860) or Wegener's granulomatosis (patent RU 2326127).

The main limitation associated with the use of mouse antibodies for human treatment is the formation of human anti-mouse antibodies (HAMA) (see, for example, Miller R.A. et al. Monoclonal antibody therapeutic trials in seven patients with T-cell lymphoma, Blood, 62, 1983, cc 988-995; and Schroff R.W. 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) regions of rodent antibodies are fused to human constant (C) regions still retain the ability to elicit a strong immune response (NACA, human chimeric antibody response) (Neuberger et al., Nature (London), 314 , 1985, pp. 268-270).

An effective approach to overcome these limitations in the use of monoclonal antibodies in clinical settings is the humanization of a murine antibody or an antibody derived from a non-human species (Jones et al., Nature (London), 321, 1986, pp. 522-525; Riechman et al.

- 1 044757 dr., Nature (London), 332, 1988, cc. 323-327).

Thus, it seems important to create new therapeutic antibodies to the CD20 antigen that, when administered to patients, have minimal or no antigenicity, intended primarily for long-term treatment. The present invention solves this problem.

The monoclonal antibody BCD-132 selectively and specifically binds to the CD20 antigen and is an effective inhibitor of the CD20 antigen, while the antibody of the invention is minimally antigenic when administered to patients.

Brief description of the invention

In one aspect, the present invention provides a monoclonal antibody or antigen binding fragment thereof that specifically binds CD20 and includes:

1) a heavy chain variable domain that has the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 6;

2) a light chain variable domain which has the amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 8.

In some embodiments, the monoclonal antibody or antigen binding fragment thereof includes:

1) a heavy chain variable domain, which has the amino acid sequence presented in SEQ ID NO: 2;

2) a light chain variable domain, which has the amino acid sequence set forth in SEQ ID NO: 4.

In some embodiments, the monoclonal antibody or antigen binding fragment thereof includes:

1) a heavy chain variable domain, which has the amino acid sequence presented in SEQ ID NO: 6;

2) a light chain variable domain, which has the amino acid sequence set forth in SEQ ID NO: 8.

In some embodiments, the monoclonal antibody includes:

1) a heavy chain that has the amino acid sequence presented in SEQ ID NO: 1 or SEQ ID NO: 5;

2) a light chain that has the amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 7.

In some embodiments, the monoclonal antibody includes:

1) a heavy chain that has the amino acid sequence presented in SEQ ID NO: 1;

2) a light chain that has the amino acid sequence set forth in SEQ ID NO: 3.

In some embodiments, the monoclonal antibody includes:

1) a heavy chain that has the amino acid sequence presented in SEQ ID NO: 5;

2) a light chain that has the amino acid sequence set forth in SEQ ID NO: 7.

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

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

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

In one aspect, the present invention relates to a nucleic acid that encodes the above antibody.

In some embodiments, the nucleic acid is DNA.

In one aspect, the present invention relates to an expression vector containing the above nucleic acid.

In one aspect, the present invention relates to a method of obtaining a host cell for producing the above antibody, which includes transforming the cell with the above vector.

In one aspect, the present invention relates to a host cell for producing the above antibody containing the above nucleic acid.

In one aspect, the present invention relates to a method for producing said antibody, which comprises culturing said host cell in a culture medium under conditions sufficient to produce said antibody, if necessary, and then isolating and purifying the resulting antibody.

In one aspect, the present invention relates to a pharmaceutical composition for the treatment of a disease or disorder mediated by CD20, containing the above antibody or antigen binding fragment thereof in a therapeutically effective amount, in combination with one or

- 2 044757 with several pharmaceutically acceptable excipients.

In some embodiments, the pharmaceutical composition is for treating a disease or disorder, wherein the disease or disorder is selected from:

a) cancer or disorder; or

b) an autoimmune disease or disorder.

In some embodiments, the pharmaceutical composition is for the treatment of a cancer or disorder that is selected from the group consisting of: B cell lymphoma or leukemia.

In some embodiments, the pharmaceutical composition is for treating a B cell lymphoma that is selected from: non-Hodgkin's lymphoma (NHL) or Hodgkin's disease (Hodgkin's lymphoma).

In some embodiments, the pharmaceutical composition is for the treatment of leukemia that is selected from: chronic lymphocytic leukemia (CLL) or small cell lymphocytic lymphoma (SLL).

In some embodiments, the pharmaceutical composition is for the treatment of an autoimmune disease or disorder that is 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 disease intestinal, idiopathic thrombocytopenic purpura (ITP), thrombotic thrombohemolytic purpura (TTP), autoimmune thrombocytopenia, multiple sclerosis, psoriasis, IgA-associated nephropathy, IgM-associated polyneuropathy, myasthenia gravis, vasculitis, ANCA vasculitis, solid organ transplant rejection, graft reaction against the host (GVHD), diabetes mellitus, Raynaud's syndrome, Sjögren's syndrome and glomerulonephritis.

In one aspect, the present invention relates to a pharmaceutical combination for the prevention or treatment of a disease or disorder mediated by CD20, which contains the above antibody or antigen binding fragment thereof and at least one other therapeutically active compound.

In some embodiments, the pharmaceutical combination includes another therapeutically active antitumor compound that is selected from a chemotherapeutic agent, an antibody, or an antihormonal agent.

In one aspect, the present invention provides a method of inhibiting the biological activity of CD20 in a subject in need of such inhibition, which comprises administering to the subject an effective amount of the above antibody or antigen binding fragment thereof.

In one aspect, the present invention provides a method of treating a disease or disorder mediated by CD20, which includes administering to a subject in need of such treatment the above antibody or antigen binding fragment thereof or the above pharmaceutical composition, in a therapeutically effective amount.

In some embodiments, the method of treating a disease or disorder relates to a disease or disorder that is selected from:

a) cancer or disorder; or

b) an autoimmune disease or disorder.

In some embodiments, the method of treating the disease or disorder relates to a cancer disease or disorder that is selected from the group consisting of: B cell lymphoma or leukemia.

In some embodiments, the method of treating the disease or disorder is a B cell lymphoma that is selected from: non-Hodgkin's lymphoma (NHL) or Hodgkin's disease (Hodgkin's lymphoma).

In some embodiments, the method of treating the disease or disorder relates to a leukemia that is selected from: chronic lymphocytic leukemia (CLL) or small cell lymphocytic lymphoma (SLL).

In some embodiments, the method of treating a disease or disorder relates to an autoimmune disease or disorder that is 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 thrombohemolytic purpura (TTP), autoimmune thrombocytopenia, multiple sclerosis, psoriasis, IgA-associated nephropathy, IgM-associated polyneuropathy, myasthenia gravis, vasculitis, ANCA vasculitis, solid organ transplant rejection, graft-versus-host disease (GVHD), diabetes mellitus, Raynaud's syndrome, Sjögren's syndrome and glomerulonephritis.

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

- 3 044757

In some embodiments, the above antibody or antigen binding fragment thereof is used to treat a disease or disorder, wherein the disease or disorder is selected from:

a) cancer or disorder; or

b) an autoimmune disease or disorder.

In some embodiments, the above antibody or antigen binding fragment thereof is used to treat a cancer or disorder that is selected from the group consisting of: B cell lymphoma or leukemia.

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

In some embodiments, the above antibody or antigen binding fragment thereof is used to treat a leukemia that is selected from: chronic lymphocytic leukemia (CLL) or small cell lymphocytic lymphoma (SLL).

In some embodiments, the above antibody or antigen binding fragment thereof is used to treat an autoimmune disease or disorder that is selected from the group consisting of: rheumatoid arthritis, juvenile rheumatoid arthritis (Still's disease), systemic lupus erythematosus (SLE), lupus nephritis, ulcerative colitis, disease Wegener's, inflammatory bowel disease, idiopathic thrombocytopenic purpura (ITP), thrombotic thrombohemolytic purpura (TTP), autoimmune thrombocytopenia, multiple sclerosis, psoriasis, IgA-associated nephropathy, IgM-associated polyneuropathy, myasthenia gravis, vasculitis, ANCA vasculitis, parenchymal graft rejection chilly organs, graft-versus-host disease (GVHD), diabetes mellitus, Raynaud's syndrome, Sjögren's syndrome and glomerulonephritis.

Brief description of drawings

Fig. 1. Map of the pEE CK expression vector.

Fig. 2. Map of the pEE NS expression vector.

Fig. 3. Schematic representation of the IgG1 format.

Fig. 4. Scheme for the synthesis of a combinatorial naive human library.

Fig. 5. Phagemid map for cloning Fab phage display libraries.

Fig. 6. Map of the pLL expression plasmid for Fab production.

Fig. 7. Analysis of the interaction of candidate BCD132L with biotinylated CD20 peptide on a Forte Bio Octet RED 386 instrument.

Fig. 8. Analysis of the interaction of candidate BCD132L-026 with biotinylated CD20 peptide on a Forte Bio Octet RED 386 instrument.

Fig. 9. Analysis of the interaction of candidate BCD132L-028 with biotinylated CD20 peptide on a Forte Bio Octet RED 386 instrument.

Fig. 10. Analysis of the interaction of candidate BCD132L-075 with biotinylated CD20 peptide on a Forte Bio Octet RED 386 instrument.

Fig. 11. Analysis of the interaction of candidate BCD132L-077 with biotinylated CD20 peptide on a Forte Bio Octet RED 386 instrument.

Fig. 12. Analysis of the interaction of candidate BCD132L-028 with biotinylated CD20 peptide on a Forte Bio Octet RED 386 instrument.

Fig. 13. Analysis of the interaction of candidates BCD132L-077 (1,2) with biotinylated CD20 peptide on a Forte Bio Octet RED 386 instrument.

Fig. 14. Analysis of the interaction of candidates BCD132L-077 (3,4) with biotinylated CD20 peptide on a Forte Bio Octet RED 386 instrument.

Fig. 15. Analysis of the interaction of candidates BCD132L-077 (5,6) with biotinylated CD20 peptide on a Forte Bio Octet RED 386 instrument.

Fig. 16. Analysis of the interaction of candidates BCD132L-028 with FcyRIIIa-158F on a Forte Bio Octet RED 386 instrument.

Fig. 17. Analysis of the interaction of candidates BCD132L-077 (1,2) with FcyRHIa-158F on a Forte Bio Octet RED 386 instrument.

Fig. 18. Analysis of the interaction of candidates BCD132L-077 (3,4) with FcyRIIIa-158F on a Forte Bio Octet RED 386 instrument.

Fig. 19. Analysis of the interaction of candidates BCD132L-077 (5,6) with FcyRIIIa-158F on a Forte Bio Octet RED 386 instrument.

Fig. 20. Analysis of the interaction of candidates BCD132L-028 with FcyRIIIa-158V on a Forte Bio Octet RED 386 instrument.

Fig. 21. Analysis of the interaction of candidates BCD132L-077 (1,2) with FcyRIIIa-158V on a Forte Bio Octet RED 386 instrument.

Fig. 22. Analysis of the interaction of candidates BCD132L-077 (3,4) with FcyRIIIa-158V on the Forte device

- 4 044757

Bio Octet RED 386.

Fig. 23. Analysis of the interaction of candidates BCD132L-077 (5,6) with FcyRIIIa-158V on the Forte device

Bio Octet RED 386.

Fig. 24. Map of the pSX expression vector.

Fig. 25. Evaluation of the specific binding of BCD-132-L-028 and BCD-132-L-077 to the CD20 receptor on the WIL2-S cell line by flow cytometry in comparison with MabThera.

Fig. 26. Assessment of complement-dependent cytotoxicity of BCD-132-L-028 and BCD-132-L-077 relative to MabThera.

BCD-132-L-028 • Mabthera BCD-132-L-028 ▼ BCD-132-L-077.

Fig. 27. Assessment of complement-dependent cytotoxicity of BCD-132-L-028 and BCD-132-L-077 relative to MabThera.

BCD-132-L-077.

• Mabthera BCD-132-L-028 ▼ BCD-132-L-077.

Fig. 28. Evaluation of antibody-dependent cellular cytotoxicity of BCD-132-L-028 and BCD-132-L-077 relative to MabThera. Low affinity CD16 reporter line, WIL2-S targeted cell line.

CD-132-L-028 • Mabthera BCD-132-L-028 ▼ BCD-132-L-077.

Fig. 29. Evaluation of antibody-dependent cellular cytotoxicity of BCD-132-L-028 and BCD-132-L-077 relative to MabThera. Low affinity CD16 reporter line, WIL2-S targeted cell line.

BCD-132-L-077 • Mabthera BCD-132-L-028 ▼ BCD-132-L-077.

Fig. 30. Evaluation of antibody-dependent cellular cytotoxicity of BCD-132-L-028 and BCD-132-L-077 relative to MabThera. High affinity CD16 reporter line, WIL2-S targeted cell line.

BCD-132-L-028 • Mabthera BCD-132-L-028 ▼ BCD-132-L-077.

Fig. 31. Evaluation of antibody-dependent cellular cytotoxicity of BCD-132-L-028 and BCD-132-L-077 relative to MabThera. High affinity CD16 reporter line, WIL2-S targeted cell line.

BCD-132-L-077 • Mabthera BCD-132-L-028 ▼ BCD-132-L-077.

Fig. 32. Evaluation of CD19+ B cell depletion caused by antibodies BCD-132-L-028 and BCD-132-L077 in comparison with MabThera, on whole blood of healthy donors with the FF (A) FV (B) and W (C) allotype.

Fig. 33. Evaluation of antibody-dependent cellular cytotoxicity (ADCC) of BCD-132-L-028 and BCD132-L-077 relative to the commercial antibody Rituximab.

Fig. 34. Severity of the inflammatory response (BCD132-L-077).

Fig. 35. Severity of demyelination (BCD132-L-077).

Fig. 36. Average curves of changes in concentration in the blood serum of primates after repeated intravenous administration of the drug BCD132-L-077 at doses of 22.0, 44.0, 88.0 mg/kg.

Description of the invention

Definitions and general methods.

Unless otherwise defined herein, scientific and technical terms used in connection with the present invention will have meanings that are commonly understood by those skilled in the art.

In addition, unless the context otherwise requires, terms in the singular include plural terms, and plural terms include singular terms. In general, the classification and methods of cell culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, organic synthesis chemistry, medicinal and pharmaceutical chemistry, and hybridization and protein and nucleic acid chemistry described herein are well known to those skilled in the art. widely used in this field. Enzymatic reactions and purification methods are carried out in accordance with the manufacturer's instructions, as is typically carried out in the art, or as described herein.

Antibody related definitions.

CD20, or B-lymphocyte antigen CD20 is a protein coreceptor located on the surface of Blymphocytes. Product of the human MS4A1 gene. The exact function of this protein has not yet been established, but it is assumed that it is involved in the activation and proliferation of B lymphocytes.

The MS4A1 gene belongs to the MS4A (membrane-spanning 4A) family, which includes at least 25 other genes. The genes of this family are grouped at the 11q12-13 locus. Presumably, the corresponding proteins have a similar spatial structure: they are membrane proteins that span the membrane 4 times and have N- and C-terminal cytoplasmic domains (JANAS

E. ET ALL., Functional role of lipid rafts in CD20 activity? Biochem Soc Symp. 2005;(72):165-75).

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

a) cancers or disorders from the group including: non-Hodgkin's lymphoma (NHL), Hodgkin's disease (Hodgkin's lymphoma), chronic lymphocytic leukemia (CLL) or small cell lymphocytic lymphoma (SLL);

b) autoimmune diseases or disorders from the group including: rheumatoid arthritis, juvenile rheumatoid arthritis (Still's disease), systemic lupus erythematosus (SLE), lupus nephritis, ulcerative colitis, Wegener's disease, inflammatory bowel disease, idiopathic thrombocytopenic purpura (ITP), thrombotic thrombohemolytic purpura (TTP), autoimmune thrombocytopenia, multiple sclerosis, psoriasis, IgA-associated nephropathy, IgM-associated polyneuropathy, myasthenia gravis, vasculitis, ANCA vasculitis, solid organ transplant rejection, graft-versus-host disease (GVHD), diabetes mellitus, Raynaud's syndrome, Sjogren's syndrome and glomerulonephritis.

The term binding molecule includes antibodies and immunoglobulins.

The term antibody or immunoglobulin (Ig) as used herein includes whole antibodies and any antigen-binding fragment (ie, antigen-binding portion) or individual chains thereof. The term antibody refers to a glycoprotein containing at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding portion thereof. Each heavy chain contains a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. There are five known types of mammalian antibody heavy chains, which are designated by Greek letters: α, ..., ε, γ and μ. The type of heavy chain present determines the class of the antibody; these chains are found in antibodies of the IgA, IgD, IgE, IgG and IgM types, respectively. Different heavy chains differ in size and composition; α and γ contain approximately 450 amino acids, and μ and ε consist of approximately 550 amino acids. Each heavy chain contains two regions, i.e. constant region and variable region. The constant region is identical in all antibodies of the same isotype, but differs in antibodies of different isotypes. The heavy chains γ, α and δ contain a constant region, which consists of three constant domains CH1, CH2 and CH3 (arranged in a row) and a hinge region, which provides flexibility (Woof J., Burton D., Nat Rev Immunol 4, 2004, cc.89-99); The μ and ε heavy chains contain a constant region, which consists of four constant domains CH1, CH2, CH3 and CH4. In mammals, only two types of light chains are known, which are designated lambda (λ) and kappa (k). Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The approximate length of the light chain is 211-217 amino acids. Preferably the light chain is a kappa (k) light chain and the CL constant domain is preferably a C-kappa (k) chain.

Antibodies of the invention may be antibodies of any class (eg, IgA, IgD, IgE, IgG and IgM, preferably IgG) or subclass (eg, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2, preferably IgG1).

The VH and VL regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs), interspersed between regions that are more conserved called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The heavy and light chain variable regions contain a binding domain that interacts with the antigen. Antibody constant regions can mediate the binding of immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells), and the first component (Clq) of the classical complement system.

The term antigen-binding portion of an antibody or antigen-binding fragment (or simply antibody portion or antibody fragment) as used herein refers to one or more antibody fragments that retain the ability to specifically bind 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 included in the term antigen-binding moiety of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH 1 domains; (ii) F(ab')2 fragment, a divalent fragment containing two Fab fragments linked by a disulfide bridge in the hinge region; (iii) Fd fragment consisting of VH and CH1 domains; (iv) an Fv fragment, consisting of VL and VH domains in a single antibody arm, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a VH/VHH domain; and (vi) a dedicated complementarity determining region (CDR). In addition, the two regions of the Fv fragment, VL and VH, are encoded by different genes; they can be connected using recombinant methods using a synthetic linker, which makes it possible to obtain them in the form of a single protein chain, in which the VL and VHs pair to form monovalent molecules (known as single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). It is intended that such single chain molecules are also included in the term antigen binding portion of an antibody. Such antibody fragments are prepared using conventional methods known to those skilled in the art, and these fragments are screened in the same manner as intact antibodies.

Preferably, the CDR of the antigen binding region or the entire antigen binding region of the antibodies of the invention is of mouse, llama or human donor library origin, or is substantially human in origin with certain amino acid residues altered, for example, substituted with different amino acid residues, so as to optimize specific properties of the antibody, for example KD , koff, IC50, EC50, ED50. Preferably, the framework regions of the antibody of the invention are of human origin or substantially human origin (at least 80, 85, 90, 95, 96, 97, 98 or 99% human origin).

In other embodiments, the antigen binding region of an antibody 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 human species.

The term variable refers to the fact that certain segments of variable domains vary widely in sequence among antibodies. Domain V mediates antigen binding and determines the specificity of a particular antibody for its particular antigen. However, variability is not evenly distributed across the 110 amino acid variable domain region. In contrast, V regions are composed of invariant fragments called framework regions (FRs) of 15–30 amino acids separated by shorter regions of extreme variability called hypervariable regions or CDRs. Each variable domain of the native heavy and light chains contains four FRs, generally adopting a beta-sheet configuration, linked by three hypervariable regions that form binding loops and, in some cases, are part of a beta-sheet structure. The hypervariable regions on each chain are held together in close proximity by the FR and, with the hypervariable regions on the other chain, contribute to the formation of the antigen-binding site of antibodies. Constant domains are not directly involved in the binding of the antibody to the antigen, but exhibit various effector functions, such as the participation of the antibody in antibody-dependent cellular cytotoxicity (ADCC).

The term hypervariable region as used herein refers to the amino acid residues of an antibody that are responsible for antigen binding. Typically, the hypervariable region contains amino acid residues from the complementarity determining region or CDR and/or such residues from the hypervariable loop.

In some cases, it may also be preferable to modify one or more amino acid residues of the CDR regions to increase binding affinity to the target epitope. This is known as affinity maturation and in some cases may be performed in connection with humanization, for example in situations where humanization of an antibody results in a decrease in binding specificity or affinity and it is not possible to sufficiently improve binding specificity or affinity using back mutations alone. Various affinity maturation methods are known in the art, for example, the in vitro scanning saturation mutagenesis method described by Burks et al., Proc Natl Acad Sci USA, 94:412-417 (1997), and the in vitro stepwise affinity maturation method proposed by Wu et al., Proc Natl Acad Sci USA 95:6037 6042 (1998).

Framework regions (FR) are variable domain residues other than CDR residues. Typically, each variable domain has four FRs, defined as FR1, FR2, FR3 and FR4. If CDRs are defined according to Kabat, the light chain FR residues are located approximately in the region of residues 1-23 (LCFR1), 35-49 (LCFR2), 57-88 (LCFR3 and 98-107 (LCFR4), and the heavy chain FR residues are located approximately in the region of residues 1-30 (HCFR1), 36-49 (HCFR2), 66-94 (HCFR3) and 103-113 (HCFR4) in the heavy chain. Where CDR regions contain amino acid residues from hypervariable loops, FR light chain residues are located approximately at residues 1-25 (LCFR1), 33-49 (LCFR2), 53-90 (LCFR3) and 97-107 (LCFR4) in the light chain , and heavy chain FR residues are located at approximately residues 1-25 (HCFRI), 33-52 (HCFR2), 56-95 (HCFR3) and 102-113 (HCFR4) in the heavy chain residues. In some examples, when the CDR contains amino acids from both the Kabat CDR and amino acids from the hypervariable loop, the FRs are adjusted accordingly. For example, when CDRH1 includes amino acids H26-H35, heavy chain FR1 residues are at positions 1-25 and FR2 residues are at positions 36-49.

A crystallizable fragment of an immunoglobulin (fragment crystallizable region, Fc region, Fc) is the terminal part of an immunoglobulin molecule that interacts with the Fc receptor on the cell surface and with some proteins of the complement system. This property allows antibodies to activate the immune system. The Fc region of IgG, IgA and IgD isotypes consists of two identical protein fragments, respectively, the second and third constant domains of two heavy chains; in the case of the IgM and IgE isotypes, Fc contains three heavy chain constant domains (CH 2-4 domains) in each polypeptide chain.

An antibody of the present invention that binds a target antigen is an antibody that binds the antigen with sufficient affinity such that the antibody can be used as a diagnostic and/or therapeutic agent when targeting a protein or cell or tissue expressing the antigen and with little cross-reacts with other proteins to a degree. According to analytical methods: fluorescence-activated cell sorting (FACS), radioimmunoprecipitation assay (RIA) or ELISA, in such embodiments the degree of binding of the antibody to the non-target protein (non-target protein) is less than 10% of the binding of the antibody with a specific target protein. With respect to the binding of an antibody to a target molecule, the term specific binding or expression specifically binding to or specific to a particular polypeptide or epitope on a particular target polypeptide means binding that is markedly (measurably) different from a nonspecific interaction (for example, in the case of bH1-44 or bH1-81 non-specific interaction is binding to bovine serum albumin, casein, fetal bovine serum or neutravidin).

Specific binding can be quantified, for example, by measuring the binding of a molecule compared to the binding of a control molecule. For example, specific binding can be determined by a competitive reaction with another molecule similar to the target, for example, with an excess of unlabeled target. In this case, specific binding is indicated if the binding of the labeled target to the probe is competitively inhibited by an excess of unlabeled target. As used herein, the term specific binding or expression specifically binds to or is specific to a particular polypeptide or epitope on a particular target polypeptide can be characterized by a molecule having a Kd to the target of 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 higher. In one embodiment, the term specific binding refers to binding in which a molecule binds to a specific polypeptide or epitope on a specific polypeptide without substantially binding to any other polypeptide or epitope on the polypeptide.

The term Ka, as used herein, refers to the rate of association of a particular antibody-antigen interaction.

The term Kd, as used herein, refers to the rate of dissociation of a particular antibody-antigen interaction.

Binding affinity generally refers to the strength of the aggregate non-covalent interactions between a single binding site of a molecule (eg, antibody) and its binding partner (eg, antigen). Unless otherwise specified, binding affinity refers to the intrinsic binding affinity, which reflects the 1:1 interaction between members of a binding pair (eg, antibody and antigen). The affinity of a molecule X for its partner Y can usually be represented by the dissociation constant (Kd). It is desirable that the Kd value is about 200, 150, 100, 60, 50, 40, 30, 20, 10, 8, 6, 4, 2, 1 nM or less. Affinity can be measured by conventional methods known in the art, including methods described herein. Low-affinity antibodies typically bind antigen slowly and tend to dissociate easily, whereas high-affinity antibodies typically bind antigen more quickly and tend to remain bound longer. Various methods for measuring binding affinity are known in the art, any of these methods can be used for the purposes of the present invention.

In one embodiment of the invention, Kd or the Kd value of this invention is measured by surface plasmon resonance methods on a BIAcore™-2000 or BIAcore™-3000 instrument (BIAcore, Inc., Piscataway, NJ) at 25°C using chips with immobilized CM5 antigen at ~ 10 relative units (response units, RU). Briefly, carboxymethyldextran biosensor chips (CM5, BIAcore Inc.) were activated with N -ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N -hydroxysuccinimide (NHS) according to the manufacturer's instructions. The antigen is diluted with 10 mM sodium acetate, pH 4.8, to a concentration of 5 μg/ml (~0.2 μM), and then injected at a flow rate of 5 μl/min until approximately 10 relative units (RU) of bound protein are reached. After antigen administration, a 1 M ethanolamine solution is injected to block unreacted groups. For kinetic measurements, 2-fold serial dilutions of Fab (eg, 0.78 to 500 nM) are injected into PBS with 0.05% Tween 20 (PBST) at 25°C with a flow rate of approximately 25 μl/min. The values of the rate of association (kon) and rate of dissociation (koff) are calculated,

- 8 044757 using a simple Langmuir model for one-plus-one binding (BIAcore Evaluation Software version 3.2), by simultaneously obtaining an association and dissociation sensorogram. The equilibrium dissociation constant (Kd) is calculated as the ratio koff/kon. See, for example, Chen, Y., et al., (1999) J. Mol. Biol. 293:865-881. If, according to the above surface plasmon resonance method, the association rate exceeds 106 M ^-1 s ^-1 , then it can be determined by the fluorescence quenching method, which measures the increase or decrease in the intensity of fluorescent emission (excitation = 295 nm; emission (radiation) = 340 nm, band 16 nm) at 25°C of a 20 nM solution of antibody against the antigen (Fab form) in PBS, pH 7.2, in the presence of increasing concentrations of antigen measured using a spectrometer such as a stopped-flow spectrophotometer (Aviv Instruments) or an SLM-Aminco spectrophotometer (ThermoSpectronie) 8000 series with stirred cuvette.

The term koff refers to the dissociation rate constant of a particular binding molecule-antigen interaction. The dissociation rate constant koff can be measured by biolayer interferometry, such as the Octet™ system.

The association rate (on-rate) or kon of this invention can also be determined by the same surface plasmon resonance method described above on a BIAcore™-2000 or BIAcore™-3000 instrument (BIAcore, Inc., Piscataway, NJ) at 25°C. using chips with immobilized CM5 antigen at ~10 relative units (response units, RU). Briefly, carboxymethyldextran biosensor chips (CM5, BIAcore Inc.) were activated with N -ethyl-N'-(3dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N -hydroxysuccinimide (NHS) according to the manufacturer's instructions. The antigen is diluted with 10 mM sodium acetate, pH 4.8, to a concentration of 5 μg/ml (~0.2 μM), and then injected at a flow rate of 5 μl/min until approximately 10 relative units (RU) of bound protein are reached. After antigen administration, a 1 M ethanolamine solution is injected to block unreacted groups.

Unless specifically stated otherwise, the expressions biologically active, and biological activity, and biological characteristics, in relation to a polypeptide of this invention, mean having the ability to bind to a biological molecule.

The expression biological molecule refers to a nucleic acid, protein, carbohydrate, lipid, and combinations thereof. In one embodiment of the invention, the biological molecule exists in nature.

Antibody regions, such as Fab and F(ab') ₂ fragments, can be prepared from whole antibodies using traditional methods such as papain or pepsin digestion of whole antibodies. Moreover, antibodies, antibody portions, and immunoadhesion molecules can be produced using standard recombinant DNA techniques, for example, as described herein.

The term recombinant antibody means an antibody that is expressed in a cell or cell line containing nucleotide sequence(s) that encode antibodies, wherein said nucleotide sequence(s) are not naturally associated with the cell.

The term variant antibody as used herein refers to an antibody having an amino acid sequence that differs from the amino acid sequence of its parent antibody by adding, deleting and/or substituting one or more amino acid residues relative 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; and in some embodiments carrying out the invention from one to about ten) additions, deletions and/or substitutions of amino acids relative to the parent antibody. In some embodiments, additions, deletions and/or substitutions are made at CDR regions of the variant antibody. Identity or homology to a variant antibody sequence is defined herein as the percentage of amino acid residues in the variant antibody sequence that are identical to those of the parent antibody, after sequence alignment and the introduction of gaps, if necessary, to achieve the maximum percentage of sequence identity. The variant antibody retains the ability to bind to the same antigen, and preferably epitope, to which the parent antibody binds, and in some embodiments, at least one property or biological activity is superior to that of the parent antibody. For example, a variant antibody may have, for example, greater binding affinity, a longer half-life, a lower IC50 value, or an increased ability to inhibit the biological activity of an antigen compared to the parent antibody. Of particular interest herein is a variant antibody that exhibits biological activity that is at least 2-fold (preferably at least 5-, 10-, or 20-fold) greater than the biological activity of the parent antibody.

The term bispecific antibody means an antibody containing an antigen binding domain

- 9 044757 or antigen binding domains that are capable of specifically binding to two different epitopes on one biological molecule or are capable of specifically binding to epitopes on two different biological molecules. A bispecific antibody is also referred to herein as having dual specificity or being a dual-specific antibody.

The term chimeric antibody refers broadly to an antibody that contains one or more regions from one antibody and one or more regions from one or more other antibodies, typically an antibody that is partly of human origin and partly of non-human origin, that is, derived in part from a non-human animal such as a mouse, rat or other rodent, or a camelid such as a llama or alpaca. Chimeric antibodies are preferred over non-human antibodies in order to reduce the risk of an anti-antibody immune response in humans, such as an anti-mouse antibody response in humans in the case of a mouse antibody. An example of a typical chimeric antibody is one in which the variable region sequences are murine while the constant region sequences are human. In the case of a chimeric antibody, the non-human parts may be further modified to humanize the antibody.

The term humanization refers to the fact that when an antibody is wholly or partly of non-human origin, such as a mouse or llama antibody produced by immunizing mice or llamas, respectively, with an antigen of interest, or is a chimeric antibody based on such a mouse or llama antibody, can be replaced certain amino acids, for example in the framework regions and constant domains of the heavy and light chains, in order to avoid or minimize the immune response in humans. The specificity of the interaction of the antibody with the target antigen is inherent mainly in the amino acid residues located in the six CDR regions of the heavy and light chain. Therefore, amino acid sequences within the CDR regions are much more variable between individual antibodies compared to sequences outside the CDR regions. Since CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of a specific natural antibody, or more generally of any specific antibody of a given amino acid sequence, for example, by constructing expression vectors that express the CDR sequences -regions from a specific antibody and framework sequences of another antibody. As a result, it is possible to humanize a non-human antibody and largely retain the binding specificity and affinity of the original antibody. Although it is impossible to accurately predict immunogenicity and thereby the immune response directed against an antibody in a person to a particular antibody, non-human antibodies are generally more immunogenic than human antibodies. Chimeric antibodies in which foreign (eg, rodent or camel) constant regions have been replaced with sequences of human origin have shown generally lower immunogenicity than antibodies of completely foreign origin, and there is a tendency to use humanized or fully human antibodies in therapeutic antibodies. Chimeric antibodies or other non-human antibodies can thus be humanized to reduce the risk of an immune response directed against the antibody in humans.

For chimeric antibodies, humanization typically involves modification of framework regions of the variable region sequences. Amino acid residues that are part of complementarity determining regions (CDR regions) will most often not be changed due to humanization, although in some cases it may be desirable to change individual amino acid residues of the CDR region, for example, to remove a glycosylation site, a deamidation site, an aspartate isomerization site, or an undesired cysteine or methionine residue. N-linked glycosylation occurs by attaching an oligosaccharide chain to an asparagine residue in the tripeptide sequence Asn-X-Ser or Asn-X-Thr, where X can be any amino acid except Pro. Removal of the N-glycosylation site can be achieved by mutating the Asn or Ser/Thr residue with another residue, preferably by a conservative substitution. Deamidation of asparagine and glutamine residues can occur depending on factors such as pH and surface exposure. Asparagine residues are particularly susceptible to deamidation, primarily if they are present in the Asn-Gly sequence, and to a lesser extent in other dipeptide sequences such as Asn-Ala. In the presence of such a deamidated region, for example Asn-Gly in the CDR region sequence, it may be preferable to remove this region, usually by a conservative substitution to remove one of the residues involved.

Numerous methods for humanizing an antibody sequence are known in the art. One of the most commonly used methods is CDR transplantation. CDR site transplantation can be based on the Kabat CDR site definitions, although a more recent publication (Magdelaine-Beuzelin et al., Crit Rev. Oncol Hematol. 64:210 225 (2007)) suggests that the IMGT® definition (the international ImMunoGeneTics information system®, www.imgt.org)

- 10 044757 can improve the result of humanization (see Lefranc et al., Dev. Comp Immunol. 27:55-77 (2003)). In some cases, transplantation of a CDR region may reduce the specificity and binding affinity, and therefore the biological activity, of the CDR grafted non-human antibody compared to the parent antibody from which the CDR regions are derived. Back mutations (sometimes referred to as framework repairs) can be applied at selected CDR positions of the transplanted antibody, typically framework regions, to restore the specificity and binding affinity of the parent antibody. Determining positions for possible back mutations can be done using information available in the literature and antibody databases. Amino acid residues that are candidates for back mutations are typically located on the surface of the antibody molecule, while residues that are recessed or have a low degree of surface exposure will generally not be affected by changes. An alternative method of humanization to CDR region grafting and reverse mutation is surface modification in which non-surface-exposed non-human residues are retained while surface-exposed residues are modified into human residues.

There are two technologies for producing fully human antibodies: using in vitro assembled phage libraries or in vivo immunization of humanized animals (mice, rats, etc.).

The construction of combinatorial phage antibody libraries begins with the selection of the source of the gene repertoire, depending on which several types of antibody libraries can be distinguished: naive, immune or synthetic. Naïve and immune libraries are constructed using naturally rearranged genes encoding variable domains of immunoglobulins from healthy donors or donors immune to any antigen, respectively. To do this, mRNA of lymphoid cells that produce antibodies is isolated. Most often these are peripheral blood lymphocytes, but in some cases splenocytes are used [Sheets M.D., Amersdorfer P., Finnern R., Sargent P., Lindquist E., Schier R., et al. Efficient construction of a large nonimmune phage antibody library: the production of high affinity human single-chain antibodies to protein antigens. Proc Natl Acad Sci USA 1998,95:6157-6162 and de Haard H. J., van Neer N., Reurs A., Hufton S. E., Roovers R. C., Henderikx P., et al. A large non-immunized human Fab fragment phage library that allows rapid isolation and kinetic analysis of high affinity antibodies. J Biol Chem 1999,274:18218-18230], tonsil cells or bone marrow lymphocytes [Vaughan T.J., Williams A.J., Pritchard K., Osbourn J.K., Pope A.R., Earnshaw J.C., et al. Human antibodies with sub-nanomolar affinities isolated from a large non-immunized phage display library. Nat Biotechnol 1996,14:309-314]. Based on mRNA, cDNA is synthesized, while oligo-dT primers and statistical hexaoligonucleotides can be used to prime the reaction, which makes it possible to obtain cDNA copies of all possible variants of genes encoding variable domains of antibodies [Ulitin A.B., Kapralova M.B., Laman A.G. ., Shepelyakovstkaya A.O., Bulgakova E.V., Fursova K.K., et al. Human miniantibody library in phage display format. Creation and testing. DAN: Nauka publishing house; 2005].

One or more primers can be used immediately, limiting the set of amplified genes to one or several gene families of variable domains or antibody isotypes already at the cDNA level [Marks J.D., Hoogenboom H.R., Bonnert T.P., McCafferty J., Griffiths A.D., Winter G. Bypassing immunization. Human antibodies from V-gene libraries displayed on phage. J Mol Biol 1991,222:581-597]. Primers used to amplify genes encoding immunoglobulins are complementary to their most conserved regions. Their sequences are selected from gene collections that are organized in databases such as the Kabat or V BASE database. The design of the primers also provides for the presence of internal restriction sites in them, allowing the cloning of PCR products into the corresponding vectors.

The construction of synthetic libraries is based on replacing natural CDRs with a set of random sequences, which allows the creation of a huge variety of antigen-binding sites.

Phage display is the first and most widely used in vitro technology for antibody discovery. In 1985, Smith discovered that foreign DNA sequences could be cloned into the filamentous bacteriophage M13 in such a way that the cloned gene sequences were expressed on the surface of the phage particles as fusion proteins (Smith G.P.: Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 1985, 228:1315-1317). Thus, it is possible to select fusion proteins of interest based on their ability to bind other proteins. This discovery was combined with PCR amplification techniques to allow cDNA cloning of the immunoglobulin gene repertoire to create a variety of phage libraries containing variable domains that can be used to rapidly search for target-specific monoclonal antibodies. The repertoire of phage libraries reflects the antibody repertoire of B lymphocytes of each person or animal whose blood was used to create the library. In 1995, two papers reported the creation of genetically engineered mice that expressed fully human antibodies, the repertoire of which could be comparable to those obtained by hybridoma technology (Lonberg N., Taylor L.D., Harding F.A., Trounstine M., Higgins

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K.M., Schramm S.R., Kuo C.C., Mashayekh R., Wymore K., McCabe J.G. et al.: Antigen-specific human antibodies from mice comprising four distinct genetic modifications. Nature 1994, 368:856-859) In these animals, the genes of their own endogenous heavy and k light chains of immunoglobulins were purposefully destroyed and transgenes were introduced, which were segments of the human heavy and k light chain genes. It turns out that the human gene repertoire can be used by the mouse immune system to create highly specific and high-affinity antibodies to a wider variety of antigens. Although transgenic mice express B cell receptors that are essentially mouse-human hybrids (human immunoglobulin, mouse Iga, Ige and other signaling molecules), their B cells develop and mature normally.

The term monoclonal antibody or mAb refers to an antibody that is synthesized and isolated by a distinct clonal population of cells. The clonal population may be a clonal population of immortalized cells. In some embodiments, the immortalized cells in the clonal population are hybrid cells, hybridomas, which are typically produced by fusing individual B lymphocytes from immunized animals with individual lymphocytic tumor cells. Hybridomas are a type of engineered cell and do not occur in nature.

Native antibodies are typically heterotetrameric glycoproteins with a molecular weight of approximately 150,000 Daltons, consisting of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by a single covalent disulfide bond, while the number of disulfide bonds between heavy chains varies among immunoglobulin isotypes. Each heavy and light chain also has evenly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (VH) at one end, followed by several constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at the other end. The light chain constant domain is aligned with the first heavy chain constant domain, and the light chain variable domain is aligned with the heavy chain variable domain. It is believed that specific amino acid residues form the interface between the light chain and heavy chain variable domains.

The term isolated as used to describe the various antibodies herein means an antibody that has been identified and isolated and/or recovered from the cell or cell culture in which it is expressed. Impurities (contaminants) from the natural environment are materials that typically interfere with the diagnostic or therapeutic use of the polypeptide, and may include enzymes, hormones, and other protein or non-protein solutes. In preferred embodiments, the antibody is purified (1) to an extent sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence using a spin glass sequencer (Edman sequencer), or (2) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie Brilliant Blue or preferably Silver staining. The isolated antibody includes antibodies in situ within the recombinant cells, since at least one component of the natural environment of the polypeptide is missing. Typically, the isolated polypeptide is obtained through at least one purification step.

An isolated nucleic acid molecule is a nucleic acid molecule that has been identified and separated from at least one contaminant nucleic acid molecule with which it is typically associated in a natural source of antibody nucleic acid. The isolated nucleic acid molecule differs from the form or set in which it occurs naturally. Thus, the isolated nucleic acid molecule is different from the nucleic acid molecule that naturally exists in cells. However, an isolated nucleic acid molecule includes a nucleic acid molecule found in cells in which the antibody is normally expressed, for example, if the nucleic acid molecule has a chromosomal location that is different from its location in cells in vivo.

The term epitope as used herein refers to the portion (determinant) of an antigen that specifically binds to a binding molecule (eg, an antibody or related molecule, such as a bispecific binding molecule). Epitope determinants typically consist of reactive surface groups of molecules, such as amino acids or carbohydrates, or sugar side chains, and typically have specific three-dimensional structural characteristics as well as specific charge characteristics. The epitope can be linear or conformational. In a linear epitope, all points of interaction between the protein (such as an antigen) and the interacting molecule (such as an antibody) occur linearly along the primary amino acid sequence of the protein. In a conformational epitope, the interaction points occur through amino acid residues on the protein that are separated from each other in the primary amino acid sequence. Once the desired epitope of an antigen has been identified, antibodies to that epitope can be generated using techniques well known in the art. In addition, generation and characterization

- 12 044757 antibodies or other binding molecules can shed light on information about desired epitopes.

Based on this information, binding molecules can then be competitively screened for binding to the same or similar epitopes, for example by performing competition studies to find binding molecules that compete for binding to the antigen.

The term peptide linker as used herein means any peptide capable of connecting domains of length depending on the domains it links, containing any amino acid sequence. Preferably, the peptide linker is greater than 5 amino acids in length and consists of any set of amino acids selected from G, A, S, P, E, T, D, K.

The term in vitro refers to a biological entity, biological process, or biological reaction outside the body, simulated under artificial conditions. For example, in vitro cell growth should be understood as the growth of cells in an environment outside the body, such as a test tube, culture flask, or microplate.

The term IC ₅₀ (50% inhibitory concentration) refers to the drug concentration at which a measurable activity or response, such as growth or proliferation of cells such as tumor cells, is inhibited by 50%. The IC ₅₀ value can be estimated using appropriate log dose response curves using special statistical curve processing programs.

The term IC ₅₀ (50% inhibitory concentration or half-maximal inhibition concentration) refers to drug concentrations and indicates how much inhibitory substance is needed to inhibit a biological process by 50%. The IC ₅₀ value can be estimated using appropriate log dose response curves using special statistical curve processing programs.

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

The term ED50 (EC50) (50% effective dose/concentration) refers to the drug concentrations at which 50% of the measured biological effect (may include cytotoxicity) is achieved.

The term antiproliferative action implies stopping or inhibiting the growth of proliferating cells, such as cancer cells.

The term antibody effector function refers to the biological activities associated with the Fc region (native Fc region sequence or Fc region amino acid sequence variants) of an antibody and varies depending on the isotype of the antibody. Examples of antibody effector functions are: Cl _q - binding; complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down-regulation of cell surface receptors (eg, B cell receptor, BCR) and B cell activation.

Antibody-dependent cellular cytotoxicity or ADCC refers to a cell-mediated response in which nonspecific cytotoxic cells that express Fc receptors (FcRs) (eg, natural killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on the target cell and then cause lysis or phagocytosis of the target cell. The primary cells for mediating ADCC, NK cells, express only FcyRIII while monocytes express FcyRI, FcyRII and FcyRIII. FcR expression on hematopoietic cells is summarized in Table. 3 on page 464 in Ravetch and Kinet, Annu. Rev. Immunol 9: 457-92 (1991). To evaluate the ADCC activity of a molecule of interest, in vitro ADCC assays can be performed, such as those described in US Pat. Nos. 5,500,362 or 5,821,337. Suitable effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of a molecule of interest can be assessed in vivo, for example, in an animal model such as the model described in Clynes et al. PNAS (USA) 95: 652–656 (1998).

Human effector cells are leukocytes that express one or more FcRs and perform effector functions. Preferably the cells express at least FcyRIII and exert ADCC effector function. 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 source, for example from blood or PBMC, as described herein.

The terms Fc receptor and FcR are used to describe a receptor that binds to the Fc region of an antibody. A preferred FcR is a human FcR with a native sequence. In addition, a preferred FcR is an FcR that binds an IgG antibody (gamma receptor), and preferred receptors include receptors of the FcyRI, FcyRII and FcyRIII subclasses including allelic variants and alternatively spliced forms of these receptors. FcyRII receptors include FcyRIIA (activating receptor) and FcyRIIB (inhibitory receptor), which are similar

- 13 044757 amino acid sequences that differ mainly in their cytoplasmic domains. The activating receptor FcyRIIA contains a tyrosine-based immunoreceptor activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcyRIIB contains a tyrosine-based immunoreceptor inhibitory motif (ITIM) in its cytoplasmic domain (reviewed in Daeron, Annu. Rev. Immunol. 15: 203-234 (1997)). A review on FcR is presented in Ravetch and Kinet, Annu. Rev. Immunol 9: 457-92 (1991). Other FcRs, including FcRs that will be identified in the future, are included herein under the term FcRs. The term also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgG into the fetus.

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 (C1q) to a molecule (such as an antibody) complexed with its antigen. To assess complement activation, a CDC assay can be performed, for example, as described in Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996).

The term identity or homology should be interpreted to mean the percentage of amino acid residues in a candidate sequence that are identical to those of the corresponding sequence to which it is compared, after sequence comparison and the introduction of gaps, if necessary to achieve the maximum percentage of identity for the complete sequence and without taking into account any conservative substitutions as part of sequence identity. Neither the N- or C-terminal extension nor the insertion segments should be interpreted as reducing identity or homology. Methods and computer programs for comparison are well known. Sequence identity can be determined using sequence analysis software (eg, Sequence Analysis Software Package, Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Ave., Madison, WI 53705). This software approaches such sequences by determining the degree of homology for a variety of substitutions, deletions (eliminations) and other modifications.

The phrase homologous, with respect to an antibody polypeptide sequence, should be interpreted as an antibody exhibiting at least 70%, preferably 80%, more preferably 90%, and most preferably 95% sequence identity to the polypeptide sequence. The term nucleic acid sequence should be construed as a sequence of nucleotides exhibiting at least 85%, preferably 90%, more preferably 95%, and most preferably 97% sequence identity to the nucleic acid sequence.

Modification(s) of the amino acid sequences of the antibodies described in this publication are proposed. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody. Antibody amino acid sequence variants are produced 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 in the amino acid sequences of the antibody. Any combination of deletion, insertion and substitution is performed to produce the final construct, provided that the final construct has the required characteristics. Amino acid changes can also alter post-translational processes in the antibody, such as changes in the number or position of glycosylation sites.

A variant of modifying the amino acid sequences of antibodies using amino acid substitutions. This option is the replacement of at least one amino acid residue in the antibody molecule with another residue. The sites of greatest interest for substitution mutagenesis include the hypervariable regions or CDRs, but changes in the FR or Fc region have also been proposed. Conservative substitutions are shown in table. And under the heading are preferred replacements. If such substitutions result in a change in biological activity, then additional significant changes, identified as exemplified substitutions in Table A, or changes further described below in the description of amino acid classes, can be introduced and product screening can be performed.

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Table A Original balance Replacement examples Preferred Substitutions Ala (A) Val; Leu; lie Val Arg(R) Lys; Gin; Asn Lys Asn(Ν) Gin; His; Asp, Lys; Arg Gin Asp (D) Glu; Asn Glu Cys(C) Ser; Ala Ser Gln(Q) Asn; Glu Asn Glu(E) Asp; Gin Asp Gly(G) Ala Ala His(H) Asn; Gin; Lys; Arg Arg He(I) Leu; Val; Met; Ala; Ph; Norleucine Leu Leu (L) Norleucine; Ile; Val; Met; Ala; Phe lie Lys (K) Arg; Gin; Asn Arg Met(M) Leu; Ph; lie Leu Phe(F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser(S) Thr Thr Thr (T) Val; Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Ph; Thr; Ser Phe Val(V) lie; Leu; Met; Ph; Ala; Norleucine Leu

The terms nucleic acid, nucleic sequence or nucleic acid sequence, polynucleotide, oligonucleotide, polynucleotide sequence and nucleotide sequence, as used interchangeably herein, refer to a distinct sequence of nucleotides, modified or unmodified, defining a fragment or region of nucleic acid, whether or not containing unnatural nucleotides. and being either double-stranded DNA or RNA, or single-stranded DNA or RNA, or transcription products of these DNAs.

It should also be mentioned here that this invention does not relate to nucleotide sequences in their natural chromosomal environment, i.e. in its natural state. The sequences of the present invention have been isolated and/or purified, i.e. were taken directly or indirectly, for example by copying, and their environment was at least partially modified. Thus, it should also be understood here as isolated nucleic acids obtained by genetic recombination, for example by host cells, or obtained by chemical synthesis.

Nucleotide sequence reference covers its complement unless otherwise noted. Thus, reference to a nucleic acid having a specific sequence should be understood to include its complementary strand with its complementary sequence.

The expression control sequences refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. Suitable control sequences for prokaryotes are, for example, a promoter, optionally an operator and a ribosome binding site. As is known, promoters, polyadenylation signals and enhancers are present in eukaryotic cells.

A nucleic acid is operably linked if it is in a functional relationship with another nucleotide sequence. For example, the DNA of a presequence or secretory leader sequence is operably linked to the DNA of a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects transcription of the sequence; a ribosome binding site is operably linked to a coding sequence if it is located in a manner that can facilitate translation. Generally, operably linked means that the linked DNA sequences are contiguous, and in the case of a secretory leader sequence, contiguous and in reading phase. However, enhancers do not have to be contiguous.

The term vector as used herein means a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. Somehow

- 15 044757 In some embodiments of the invention, the vector is a plasmid, i.e. a circular, double-stranded portion of DNA into which additional DNA segments can be ligated. In some embodiments, the vector is a viral vector in which additional DNA segments can be ligated into the viral genome. In some embodiments, the vectors are capable of autonomous replication in the host cell into which they are introduced (eg, bacterial vectors having a bacterial origin of replication and mammalian episomal vectors). In other embodiments, vectors (eg, non-episomal mammalian vectors) can be integrated into the host cell genome upon introduction into the host cell, and thus replicate along with the host gene. Moreover, some vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as recombinant expression vectors (or simply expression vectors).

The term recombinant host cell (or simply host cell) as used herein means a cell into which a recombinant expression vector has been introduced. The present invention relates to host cells, which may include, for example, a vector in accordance with the present invention described above. The present invention also relates to host cells that include, for example, a nucleotide sequence encoding a heavy chain or antigen binding portions thereof, a nucleotide sequence encoding a light chain or antigen binding portions thereof, or both, a first binding domain and/or a second binding domain of a binding molecule according to this invention. It should be understood that recombinant host cell and host cell mean not only the specific cell claimed, but also the progeny of such a cell. Since modifications may occur in subsequent generations due to mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but such cells are still included within the scope of the term host cell as used herein.

The term excipient is used herein to describe any ingredient other than the compound(s) of this invention.

The term CD20-mediated disease or disorder includes all diseases or disorders that are either directly or indirectly associated with CD20, including the etiology, development, progression, persistence or pathology of the disease or disorder. Treat, cure, and therapy refer to a method of alleviating or eliminating a biological disorder and/or at least one of its associated symptoms. As used herein, the term ameliorate a disease, disorder or condition means reducing the severity and/or frequency of symptoms of the disease, disorder or condition. In addition, references to treatment contained herein include references to curative, palliative, and preventive therapies.

In one aspect, the subject of treatment or patient is a mammal, preferably a human subject.

The above subject may be male or female of any age.

The term disorder means any condition that can be improved by treatment according to the present invention. The definition of this term includes chronic and acute disorders or diseases, including pathological conditions that predispose a mammal to the occurrence of a given disorder.

The terms cancer or cancerous refer to or describe a physiological condition in mammals that is typically characterized by unregulated cell growth/proliferation. This definition covers benign and malignant cancers. Examples of cancers include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma and leukemia. More specific examples of such cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, squamous cell lung carcinoma, peritoneal cancer, hepatic cell carcinoma, gastric cancer, including gastrointestinal cancer, pancreatic cancer, glioblastoma, glioma, cervical cancer, cancer ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial and uterine carcinoma, salivary gland carcinoma, kidney cancer (renal cell carcinoma), prostate cancer (prostate cancer), cancer of the external female genitalia, thyroid cancer, liver carcinoma, anal carcinoma, penile carcinoma, melanoma and various types of head and neck cancer.

The terms immune response, autoimmune reaction, autoimmune inflammation refer, for example, to the action of lymphocytes, antigen-presenting cells, phagocytic cells, granulocytes and soluble macromolecules produced by these cells or liver cells (including antibodies, cytokines and complement resulting from selective damage, destruction or elimination from the human body of invasive pathogens, pathogen-infected cells or tissues, cancer cells or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues).

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The terms immune response, autoimmune reaction, autoimmune inflammation refer, for example, to the action of lymphocytes, antigen-presenting cells, phagocytic cells, granulocytes and soluble macromolecules produced by these cells or liver cells (including antibodies, cytokines and complement resulting from selective damage, destruction or elimination from the human body of invasive pathogens, pathogen-infected cells or tissues, cancer cells or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues).

The term autoantibodies as used herein refers to antibodies directed against autoantigens. 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), nuclear proteins (e.g., SS-A(Ro), SS-B(La), Scl-70, centromeres, Jo- 1, histadyl-tRNA synthetase, threonyl-tRNA synthetase, PM-1, Mi-2, histones or chromatin), cellular receptors (eg acetylcholine receptor, thyretropin receptor), cellular proteins (eg cardiolipin, e2GPl), cell membrane proteins (eg aquaporin, desmoglein) RNA-protein complexes (eg ribonucleoproteins and Sm), red blood cell and platelet receptor glycoproteins.

The term autoimmune disease as used herein refers to a non-malignant disease or disorder arising from and directed against an individual's own (auto) antigens and/or tissues.

This definition includes, but is not limited to, rheumatoid arthritis, juvenile chronic arthritis, septic arthritis, Lyme arthrosis, psoriatic arthritis, reactive arthritis, spondyloarthropathy, systemic lupus erythematosus, Crohn's disease, ulcerative colitis, inflammatory bowel disease, diabetes mellitus, thyroiditis, asthma, allergic diseases, psoriasis, atopic dermatitis, scleroderma, graft-versus-host disease, graft rejection, acute or chronic immune diseases associated with transplantation, sarcoidosis, Kawasaki disease, Graves' disease, nephrotic syndrome, chronic fatigue syndrome, Wegener's granulomatosis, Henoch-Schönlein purpura , microscopic renal vasculitis, chronic active hepatitis, uvenita, septic shock, toxic shock syndrome, septic syndrome, cachexia, acquired immunodeficiency syndrome, acute transverse myelitis, Huntington's chorea, Parkinson's disease, Alzheimer's disease, stroke, primary biliary cirrhosis, hemolytic anemia, adults (acute) respiratory distress syndrome, alopecia, alopecia areata, seronegative arthropathy, arthropathy, Reiter's disease, psoriatic arthropathy, ulcerative colitis-associated arthropathy, atopic allergies, autoimmune bullous diseases, pemphigus vulgaris, pemphigus foliaceus, pemphigoid disease, linear IgA, IgG -associated diseases, autoimmune hemolytic anemia, Coombs-positive hemolytic anemia, pernicious anemia, juvenile pernicious anemia, giant cell arteritis, arthritis, primary sclerosing hepatitis A, cryptogenic autoimmune hepatitis, fibrosing lung diseases, cryptogenic fibrous alveolitis, post-inflammatory interstitial lung diseases, interstitial pneumonitis, chronic eosinophilic pneumonia, post-infectious interstitial lung diseases, gouty arthritis, autoimmune hepatitis, autoimmune hepatitis type I (classical autoimmune hepatitis or lipoid), autoimmune hepatitis type II, osteoarthritis, primary sclerosing cholangitis, psoriasis, idiopathic leukopenia, autoimmune neutropenia, renal NOS diseases [renal NOS-disease], glomerulonephritis, microscopic renal vasculitis, discoid lupus erythematosis, idiopathic or male NOS fertility [autoimmunity to sperm], all subtypes of multiple sclerosis, sympathetic ophthalmia, secondary pulmonary hypertension in connective tissue diseases, Goodpasture's syndrome, pulmonary manifestation of nodular polyarthritis, acute rheumatic fever, rheumatoid spondylitis, ankylosing spondylitis, Still's disease, systemic scleroderma, focal scleroderma, Sjögren's syndrome, Sjögren's disease, Behçet's disease, ankylosing spondylitis, spondyloarthritis, axial enthesitis, relapsing polychondritis, Takayasu syndrome, autoimmune thrombocytopenia, idiopathic thrombocytopenia, autoimmune thyroiditis , autoimmune gastritis, autoimmune polyglandular syndrome, hyperthyroidism, Hoshimoto's disease, autoimmune atrophic hypothyroidism, primary mexidema, phacogenic uveitis, primary vasculitis, vitiligo, acute liver diseases, chronic liver diseases, allergies, asthma, mental illnesses (including depression and schizophrenia), diseases mediated by Th2 type and Th1 type, conjunctivitis, allergic contact dermatitis, allergic rhinitis, alpha-1-antitrypsin deficiency, amyotrophic lateral sclerosis, anemia, dietary fibrosis, diseases associated with cytokine therapy, demyelinating diseases, dermatitis, iridocyclitis/uveitis/optic neuritis , ischemic reperfusion injury, ischemic stroke, juvenile rheumatoid arthritis, autoimmune enteropathy, autoimmune hearing loss, autoimmune lymphoproliferative syndrome, autoimmune myocarditis, autoimmune cardiomyopathy, Coxsackie myocarditis, Dressler's syndrome, lupus nephritis, angioedema, including hereditary, urticaria, superficial hydradenitis, lichen planus, lichen sclerosus, lichenoid pityriasis, vitiligo, Addison's disease, autoimmune polyendocrine syndrome, autoimmune pancreatitis, celiac disease, microscopic colitis, anti-17 044757 phospholipid syndrome, autoimmune lymphoproliferative syndrome, cold agglutinin disease, essential cryoglobulinemia , Evans syndrome, pernicious anemia, red cell aplasia, CREST syndrome, eosinophilic fasciitis, Felty syndrome, overlap syndrome, chronic Lyme disease, Parry-Romberg syndrome, palindromic rheumatism, rheumatism, acute rheumatic fever, retroperitoneal syndrome, sarcoidosis, Schnitzler syndrome, undifferentiated connective tissue disease, dermatomyositis , polymyositis, fibromyalgia, myasthenia gravis, neuromyotonia, acute disseminated encephalomyelitis, Guillain-Barré syndrome, Deivik's opticomyelitis, Hashimoto's encephalopathy, Lambert-Eaton myasthenic syndrome, eosinophilic gruanulomatosis with polyangiitis, leukocytoclastic vasculitis, lupus vasculitis, rheumatoid vasculitis, nodular polyangiitis, autoimmune premature ovarian failure and blepharitis. The antibody may also treat any combination of the disorders listed above.

A therapeutically effective amount is the amount of therapeutic agent administered during treatment that will relieve, to a certain extent, one or more symptoms of the disease being treated.

The term chronic administration refers to the continuous (long-term) use of the agent(s), as opposed to the acute (short-term) route of administration, so as to maintain the initial therapeutic effect (activity) for an extended period of time.

Intermittent use refers to treatment that is not administered consistently without interruption, but rather is intermittent in nature.

In the present specification and in the following claims, unless the context otherwise requires, the words have, include and contain, or variations thereof such as has, having, includes, including, contains or containing, are to be understood to include the specified whole or group of wholes, but not to the exclusion of any other whole or group of wholes.

Detailed Description of the Invention

Antibody.

The present invention relates to a monoclonal antibody that specifically binds to CD20.

In one aspect, the present invention provides a monoclonal antibody or antigen binding fragment thereof that specifically binds CD20 and includes:

1) a heavy chain variable domain, which has the amino acid sequence EVQLVQPGAEVVKPGASVKVSCKASGYTFTSYNMHWVRQAPGRGLEWMGAIYPGNGDTSYNQKFKG RVTMTRDKSTSTVYMELSSLRSEDTAVYYCARSTYYGGDWYFNVWGQGTLVTVSS (SEQ ID NO: 2);

or

QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGRGLEWMGAIYPGNGDTSYN QKFKGRATLTRDTSTSTVYMELSSLRSEDTAVYYCARSTYYGGDWYFNVWGQGTLVTVSS (SEQ ID NO: 6);

2) a light chain variable domain, which has the amino acid sequence QIVLSQSPAILSASPGERVTLTCRASSSVSYIHWFQQKPGKAPKPLIYATSNLASGVPSRFSGSGS IGTDFSLTISRVEPEDFAVYYCQQWTSNPPTFGGGTKVEIK (SEQ ID NO: 4) or

QIVLSQSPATLSASPGERATMTCRASSSVSYIHWFQQKPGKAPKPLIYATSNLASGVPSRF SGSGSGTDFTLTISRLEPEDFATYYCQQWTSNPPTFGGGTKVEIK (SEQ ID NO: 8).

In some embodiments, the monoclonal antibody or antigen binding fragment thereof includes:

1) heavy chain variable domain, which has an amino acid sequence

EVQLVQPGAEWKPGASVKVSCKASGYTFTSYNMHWVRQAPGRGLEWMGAIYPGNGDTSYN QKFKGRVTMTRDKSTSTVYMELSSLRSEDTAVYYCARSTYYGGDWYFNVWGQGTLVTVSS (SEQ ID NO: 2);

2) a light chain variable domain, which has the amino acid sequence QIVLSQSPAILSASPGERVTLTCRASSSVSYIHWFQQKPGKAPKPLIYATSNLASGVPSRFSGSGS GTDFSLTISRVEPEDFAVYYCQQWTSNPPTFGGGTKVEIK (SEQ ID NO: 4).

In some embodiments, the monoclonal antibody or antigen binding fragment thereof includes:

1) a heavy chain variable domain, which has the amino acid sequence QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGRGLEWMGAIYPGNGDTSYNQKFKG RATLTRDTSTSTVYMELSSLRSEDTAVYYCARSTYYGGDWYFNVWGQGTLVTVSS (SEQ ID NO: 6);

2) a light chain variable domain, which has the amino acid sequence QIVLSQSPATLSASPGERATMTCRASSSVSYIHWFQQKPGKAPKPLIYATSNLASGVPSRFSGSGS GTDFTLTISRLEPEDFATYYCQQWTSNPPTFGGGTKVEIK (SEQ ID NO: 8).

In some embodiments, the monoclonal antibody includes:

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1) a heavy chain that has an amino acid sequence

EVQLVQPGAEWKPGASVKVSCKASGYTFTSYNMHWVRQAPGRGLEWMGAIYPGNGDTSYNQKFKG RVTMTRDKSTSTVYMELSSLRSEDTAVYYCARSTYYGGDWYFNVWGQGTLVTVSSASTKGPSVFPL APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLG TQ TYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 1) or

SSSL GTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR TPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKC KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI AVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 5);

2) a light chain, which has an amino acid sequence

QIVLSQSPAILSASPGERVTLTCRASSSVSYIHWFQQKPGKAPKPLIYATSNLASGVPSRF SGSGSGTDFSLTISRVEPEDFAVYYCQQWTSNPPTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSG TASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC EVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 3) or

QIVLSQSPATLSASPGERATMTCRASSSVSYIHWFQQKPGKAPKPLIYATSNLASGVPSRF SGSGSGTDFTLTISRLEPEDFATYYCQQWTSNPPTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSG TASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC EV THQGLSSPVTKSFNRGEC (SEQ ID NO: 7).

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

Monoclonal antibody BCD132-077 includes:

1) a heavy chain that has an amino acid sequence

EVQLVQPGAEWKPGASVKVSCKASGYTFTSYNMHWVRQAPGRGLEWMGAIYPGNGDTSYNQKFKG RVTMTRDKSTSTVYMELSSLRSEDTAVYYCARSTYYGGDWYFNVWGQGTLVTVSSASTKGPSVFPL APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLG TQ TYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT CVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 1) ;

2) a light chain, which has an amino acid sequence

QIVLSQSPAILSASPGERVTLTCRASSSVSYIHWFQQKPGKAPKPLIYATSNLASGVPSRF SGSGSGTDFSLTISRVEPEDFAVYYCQQWTSNPPTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSG TASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC EVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 3).

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

Monoclonal antibody BCD132-028 includes:

1) a heavy chain that has an amino acid sequence

QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGRGLEWMGAIYPGNGDTSYNQKFKG RATLTRDTSTSTVYMELSSLRSEDTAVYYCARSTYYGGDWYFNVWGQGTLVTVSSASTKGPSVFPL APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLG TQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT CVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 5) ;

2) a light chain, which has an amino acid sequence

QIVLSQSPATLSASPGERATMTCRASSSVSYIHWFQQKPGKAPKPLIYATSNLASGVPSRFSGS GTDFTLTISRLEPEDFATYYCQQWTSNPPTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASW CLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH Q GLSSPVTKSFNRGEC (SEQ ID NO: 7).

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

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

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

Nucleic acid molecules.

The present invention also relates to nucleic acid molecules, namely sequences encoding a monoclonal antibody that specifically binds to CD20, according to this invention, which are described herein, optionally including any peptide linker sequence connecting them.

A reference to a nucleotide sequence covers its complement unless otherwise noted. Thus, reference to a nucleic acid having a specific sequence should be understood to include its complementary strand with its complementary sequence. The term polynucleotide as used herein means a polymeric form of nucleotides at least 10 bases in length, either ribonucleotides or deoxynucleotides, or a modified form of any type of nucleotide. The term includes single- and double-stranded forms.

In one aspect, the present invention provides a nucleic acid molecule comprising a nucleotide sequence that encodes an amino acid sequence selected from SEQ ID NOs: 1-8. The nucleic acid molecule may also contain any combination of these nucleotide sequences.

In one aspect, the present invention provides a nucleic acid molecule comprising a nucleotide sequence that encodes a monoclonal antibody or antigen binding fragment thereof that specifically binds CD20, and includes:

1) a heavy chain variable domain that has the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 6;

2) a light chain variable domain which has the amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 8.

In some embodiments, the nucleic acid molecule contains a nucleotide sequence that encodes a monoclonal antibody or antigen binding fragment thereof includes:

1) a heavy chain variable domain, which has the amino acid sequence presented in SEQ ID NO: 2;

2) a light chain variable domain, which has the amino acid sequence set forth in SEQ ID NO: 4.

In some embodiments, the nucleic acid molecule contains a nucleotide sequence that encodes a monoclonal antibody or antigen binding fragment thereof includes:

1) a heavy chain variable domain, which has the amino acid sequence presented in SEQ ID NO: 6;

2) a light chain variable domain, which has the amino acid sequence set forth in SEQ ID NO: 8.

In some embodiments, the nucleic acid molecule contains a nucleotide sequence that encodes a monoclonal antibody that includes:

1) a heavy chain that has the amino acid sequence presented in SEQ ID NO: 1 or SEQ ID NO: 5;

2) a light chain that has the amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 7.

In some embodiments, the nucleic acid molecule contains a nucleotide sequence that encodes a monoclonal antibody that includes:

1) a heavy chain that has the amino acid sequence presented in SEQ ID NO: 1;

2) a light chain that has the amino acid sequence set forth in SEQ ID NO: 3.

In some embodiments, the nucleic acid molecule contains a nucleotide sequence that encodes a monoclonal antibody that includes:

1) a heavy chain that has the amino acid sequence presented in SEQ ID NO: 5;

2) a light chain that has the amino acid sequence set forth in SEQ ID NO: 7.

In any of the above embodiments, the nucleic acid molecules may be isolated.

The nucleic acid molecule of this invention can be isolated from any source that produces a monoclonal antibody that specifically binds to CD20. In certain embodiments of the invention, the nucleic acid molecule of this invention may be synthesized rather than isolated.

In one embodiment of the invention, nucleic acid molecules encoding domains

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VH (SEQ ID NO: 2 or SEQ ID NO: 6) or VL (SEQ ID NO: 4 or SEQ ID NO: 8) are converted into full-length antibody genes by insertion into an expression vector already encoding the heavy chain constant domains (CH ) or light chain (CL), respectively, such that the VH segment is operably connected to the CH segment(s) in the vector and/or the VL segment is operatively connected to the CL segment in the vector. In another embodiment of the invention, nucleic acid molecules encoding VH and/or VL domains are converted into genes along the entire length of the antibody by joining, for example, ligation, a nucleic acid molecule encoding VH and/or VL domains to a nucleic acid molecule encoding CH and /or CL domains using standard molecular biology techniques. Nucleic acid molecules encoding the entire length of the heavy and/or light chains can then be expressed from the cell into which they have been introduced.

Nucleic acid molecules can be used to express large amounts of recombinant monoclonal antibody that specifically binds to CD20.

Vector.

In yet another aspect, the present invention provides a vector suitable for expressing any of the nucleotide sequences described herein.

The present invention relates to vectors containing nucleic acid molecules that encode any of the amino acid sequences of a monoclonal antibody that specifically binds to CD20, or parts thereof (for example, the heavy chain sequences of the first and/or the heavy and/or light chain of the second binding domains) as described in this document. The present invention further relates to vectors containing nucleic acid molecules encoding fusion proteins, modified antibodies, antibody fragments.

In some embodiments, a monoclonal antibody that specifically binds CD20 of the invention is expressed by inserting DNA encoding part or all of the first and second binding domain sequences (e.g., heavy and light chain sequences, wherein the binding domain comprises heavy and light chain sequences). chains) prepared as described above in expression vectors such that the genes are operably linked to the necessary expression control sequences, such as transcriptional and translational control sequences. Expression vectors include plasmids, retroviruses, adenoviruses, adeno-associated viruses (AAV), plant viruses such as cauliflower mosaic virus, tobacco mosaic virus, cosmid, YAC, EBV-derived episomes and the like. DNA molecules can be ligated into a vector such that the transcription and translation control sequences in the vector perform the 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 used. DNA molecules encoding partial or entire sequences of the first and second binding domains (eg, heavy and light chain sequences, where the binding domain contains a heavy and light chain sequence) can be introduced into separate vectors. In one embodiment of the invention, any combination of the above DNA molecules is introduced into the same expression vector. DNA molecules can be introduced into the expression vector by standard methods (eg, by ligating complementary restriction sites on the antibody gene fragment and the vector, or blunt-end ligation if restriction sites are not present).

A suitable vector is one that encodes functionally complete human immunoglobulin CH or CL sequences with an appropriate restriction site engineered so that any VH or VL sequence can be readily incorporated and expressed as described above. The NS and LC coding of genes in such vectors may contain intronic sequences, which leads to an overall increase in antibody protein products by stabilizing the corresponding mRNA. Intronic sequences are flanked by splice donor and splice acceptor sites, which determine where RNA splicing will occur. The location of the intronic sequences can be either in the variable or constant regions of the antibody chains, or in both the variable and constant regions when multiple introns are used. Termination of polyadenylation and transcription can occur downstream of the native chromosome site of the encoded regions. The recombinant expression vector may also encode a signal peptide that facilitates production of an antibody chain by the host cell. The antibody chain gene can be cloned into a vector such that the signal peptide is linked to the amino terminus of the immunoglobulin chain. The signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (ie, a non-immunoglobulin protein signal peptide).

In addition to the antibody gene chain, the recombinant expression vectors of the present invention may carry regulatory sequences that control the expression of the antibody chain genes in the host cell. Those skilled in the art will appreciate that the design of an expression vector, including the choice of regulatory sequences, may depend on factors such as selection

- 21 044757 host cell for transformation, level of expression of the desired protein, etc. Preferred regulatory sequences for a mammalian host expression cell include viral elements that provide high level expression of proteins in mammalian cells, such as promoters and/or enhancers derived from a retroviral LTR, cytomegalovirus (CMV) (eg, CMV promoter/enhancer), simian virus 40 (SV40) (eg, SV40 promoter/enhancer), adenovirus (eg, adenovirus large late promoter (AdMLP)), polyoma virus, as well as strong mammalian promoters such as the native immunoglobulin promoter or the actin promoter. For further description of viral regulatory elements and their sequences, see, for example, US patents 5168062, 4510245 and 4968615. Methods for expressing binding molecules such as plant antibodies, including the description of promoters and vectors, as well as transformation of plants, are known in the art . See, for example, US Pat. No. 6,517,529. Methods for expressing polypeptides in bacterial or fungal cells, such as yeast cells, are also well known in the art.

In addition to antibody chain genes and regulatory sequences, the recombinant expression vectors of the invention may carry additional sequences, such as sequences that regulate replication of the vector in host cells (eg, origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, for example, US Pat. Nos. 4,399,216, 4,634,665, and 5,179,017). For example, typically a selectable marker gene confers resistance to drugs, such as G418, hygromycin, or methotrexate, to the host cell into which the vector is introduced. For example, selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr host cells for methotrexate selection/amplification), the neo gene (for G418 selection) and the glutamate synthetase gene.

The term expression control sequence as used herein refers to polynucleotide sequences that are necessary to influence the expression and processing of the coding sequences to which they are ligated. Expression control sequences include the corresponding transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that increase translation efficiency (ie, the Kozak consensus sequence); sequences that increase protein stability; and, if desired, sequences that enhance protein secretion. The nature of such control sequences varies depending on the host organism; in prokaryotes, such control sequences typically include a promoter, a ribosome binding site, and transcription termination sequences; in eukaryotes, such control sequences typically include promoters and transcription termination sequences. The term control sequences includes, at a minimum, all components whose presence is essential for expression and processing, and may also include additional components whose presence is beneficial, such as leader sequences and fusion sequences.

Host cells.

An additional aspect of the present invention relates to methods for producing a monoclonal antibody that specifically binds to CD20 according to the present invention. One embodiment of the invention relates to a method of producing a monoclonal antibody that specifically binds to CD20, as defined herein, comprising obtaining a recombinant host cell capable of expressing a monoclonal antibody that specifically binds to CD20, culturing said host cell under conditions suitable for expressing the production of a monoclonal antibody that specifically binds to CD20, and isolating the resulting monoclonal antibody that specifically binds to CD20. A monoclonal antibody that specifically binds to CD20 produced by such expression in such recombinant host cells is referred to herein as a recombinant monoclonal antibody that specifically binds to CD20. The invention also relates to progeny cells of such host cells and a monoclonal antibody that specifically binds to CD20 prepared in a similar manner.

Nucleic acid molecules encoding a monoclonal antibody that specifically binds to CD20 of the invention and vectors containing these nucleic acid molecules can be used to transfect a suitable mammal or cell thereof, a plant or cell thereof, or a bacterial or yeast host cell. Conversion can occur by any known method for introducing polynucleotides into a host cell. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, transfection with a complex of nucleic acid and a positively charged polymer, transfection with a nucleic acid and calcium phosphate precipitate, polybrene-mediated transfection, protoplast fusion, transfection with liposome-encapsulated polynucleotides, and direct microinjection of DNA into nuclei. In addition, nucleic acid molecules

- 22 044757 can be introduced into mammalian cells by viral vectors. Methods for transfecting cells are well known in the art. See, for example, US Pat. 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, including, for example, Agrobacterium-mediated transformation, biolistic transformation, direct injection, electroporation, and viral transformation. Methods for transforming bacterial and yeast cells are also well known in the art.

Mammalian cell lines used as hosts for transformation are well known in the art and include many immortalized cell lines available. These include, for example, Chinese hamster ovary (CHO) cells, NS0 cells, SP2 cells, HEK-293T cells, 293 Freestyle cells (Invitrogen), NIH-3T3 cells, HeLa cells, hamster kidney (HK) cells, African kidney cells vervet monkeys (COS), human hepatocellular carcinoma cells (eg Hep G2), A549 cells and a number of other cell lines. Cell lines are selected by determining which cell lines have high levels of expression and provide the desired characteristics of the protein produced. Other cell lines that can be used are insect cell lines such as Sf9 or Sf21 cells. When recombinant expression vectors encoding a monoclonal antibody that specifically binds CD20 are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a time sufficient to express the antibodies in the host cells or, preferably, releasing the antibodies into the culture medium , in which host cells are grown. A monoclonal antibody that specifically binds to CD20 can be isolated from the culture medium using standard protein purification methods. Plant host cells, for example, include Nicotiana, Arabidopsis, duckweed, corn, wheat, potato, etc. Host bacterial cells include Escherichia and Streptomyces species. Yeast host cells include Schizosaccharomyces pombe, Saccharomyces cerevisiae and Pichia pastoris.

In addition, the level of production of a monoclonal antibody that specifically binds to CD20 of the present invention from a producing cell line can be enhanced by a number of known methods. For example, the glutamine synthetase gene expression system (GS system) is common enough to enhance expression under certain conditions. The GS system is discussed in whole or in part in connection with patents EP 0216846, 0256055, 0323997 and 0338841.

It is likely that a monoclonal antibody that specifically binds CD20 derived from different cell lines or transgenic animals will have different glycosylation profiles. However, a monoclonal antibody that specifically binds to CD20, encoded by the nucleic acid molecules described herein, or containing the amino acid sequences described herein, is part of the present invention, regardless of the glycosylation state of the binding molecules and in general, regardless of the presence or absence of post-translational modifications.

Obtaining antibodies.

The invention also relates to methods and processes for producing a monoclonal antibody that specifically binds to CD20, and antigen-binding fragments thereof.

Monoclonal antibodies.

Monoclonal antibodies can be created, for example, using the hybridoma-based method, which was first described by Kohler et al., Nature, 256, 1975, p. 495, or using recombinant DNA techniques (US 4816567).

When using the hybridoma method, a mouse or other suitable host animal, such as a hamster, is immunized according to the procedure described above to induce the production of lymphocytes that produce or can produce antibodies having the ability to specifically bind to the protein used for immunization . In another embodiment, lymphocytes can be obtained by in vitro immunization. Following immunization, the lymphocytes are isolated and then fused with a myeloma cell line using a suitable coupling agent such as polyethylene glycol to produce a hybridoma cell.

The hybridoma cells thus obtained are seeded and grown in a suitable culture medium which preferably contains one or more substances that inhibit the growth or survival of the unfused parent myeloma cells. For example, if the parent myeloma cells do not contain the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), then the culture medium for hybridomas, as a rule, should include hypoxanthine, aminopterin and thymidine (HAT medium), i.e. substances that inhibit the growth of HGPRT-deficient cells.

Preferred myeloma cell fusion components are cells that are readily fusionable, maintain consistently high levels of antibody production by the selected antibody-producing cells, and are sensitive to a selective environment in which unrelated parent cells are selected for. Preferred myeloma cell lines are murine myeloma lines, such as the murine tumor cell lines MOPC-21 and MPC-11, which are available from the Salk Institute Cell Distribution Center, San 23 044757

Diego, pc. California, USA, and lines SP-2 or X63-Ag8-653, which are available from the American Type Culture Collection, Rockville, CA. Maryland, USA. The use of human myeloma and mouse-human heteromyeloma cell lines for the production of monoclonal antibodies has also been described (Kozbor, J. Immunol., 133, 1984, p. 3001).

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

The binding affinity of a monoclonal antibody can, for example, be determined using the Scatchard assay described in Munson et al., Anal. Biochem., 107, 1980, p. 220.

Once hybridoma cells that produce antibodies of the required specificity, affinity, and/or activity have been identified, clones can be subcloned using the limiting dilution method and grown using standard methods. Suitable media for this purpose include, for example, DMEM or RPMI-1640 media. In addition, hydriboma cells can be grown in vivo as ascites tumors in animals, for example by intraperitoneal (i.p.) injection of cells into mice.

Monoclonal antibodies secreted by subclones can be separated from culture medium, ascites fluid or serum using conventional antibody purification methods, for example, affinity chromatography (for example, using protein A- or protein Q-Sepharose), or ion exchange chromatography, hydroxylapatite chromatography, gel electrophoresis, dialysis, etc.

DNA encoding monoclonal antibodies can be easily isolated and sequenced using conventional procedures (eg, using oligonucleotide probes that have the ability to specifically bind to the genes encoding the heavy and light chains of murine antibodies). The preferred source of such DNA is hybridoma cells. Once isolated, the DNA can be incorporated into expression vectors that are then transfected into host cells, such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells, which do not produce antibody protein without transfection, resulting in synthesis of monoclonal antibodies in recombinant host cells.

In yet another embodiment, monoclonal antibodies or antibody fragments can be isolated from phage antibody libraries generated using methods described in McCafferty et al., Nature, 348, 1990, cc. 552-554. In Clackson et al., Nature, 352, 1991, cc. 624-628 and Marks et al., J. Mol. Biol., 222, 1991, pp. 581-597 describe the isolation of mouse and human antibodies, respectively, using phage libraries. Subsequent publications described the production of high-affinity (nM-range) human antibodies by chain shuffling (Marks et al., Bio/Technology, 10, 1992, pp. 779-783), and combinatorial infection and in vivo recombination as a strategy construction of very large phage libraries (Waterhouse et al., Nucl. Acids. Res., 21, 1991, cc. 2265-2266). Thus, these methods represent a viable alternative to traditional methods for isolating monoclonal antibodies based on monoclonal antibody hybridomas.

The DNA encoding an antibody can also be modified, for example, to produce chimeric or fusion antibody polypeptides, for example by replacing the heavy and light chain constant region (CH and CL) sequences with homologous murine sequences (US 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA: 81, 1984, p. 6851) or by covalently linking the coding sequence of an immunoglobulin to all or part of the coding sequence of a non-immunoglobulin polypeptide (heterologous polypeptide). Non-immunoglobulin polypeptide sequences can be replaced with constant regions of an antibody or replaced with variable regions of an antibody's antigen binding site, creating a chimeric bivalent antibody that contains one antigen binding site having specificity for an antigen and another antigen binding site having specificity for a different antigen.

Human antibodies and a technique based on the use of a phage display library.

It is now possible to produce transgenic animals (eg mice) which, following immunization, can produce the full spectrum of human antibodies without the production of endogenous immunoglobulin. For example, homozygous deletion of the antibody heavy chain ( _JH ) gene junction regions in chimeric and germline mutant mice has been described to result in complete inhibition of endogenous antibody production. Transfer of a germline human immunoglobulin gene repertoire into such mutant germline mice results in the production of human antibodies upon antigen challenge (US 5545806, 5569825, 5591669 (all to GenPharm); 5545807; and WO 97/17852).

Alternatively, phage display technology can be used to produce human antibodies and antibody fragments in vitro from immunoglobulin variable region (V) genes from immunized donors (McCafferty et al., Nature, 348, 1990, pp. 552-i553). In this technique, antibody V region genes are cloned in frame with either the major or minor envelope protein gene of a filamentous bacteriophage, such as M13 or fd, and presented

- 24 044757 in the form of functional antibody fragments on the surface of the phage particle. Since the filamentous particle contains a single-stranded DNA copy of the phage genome, selection for the functional properties of the antibody also results in the selection of the gene encoding the antibody that has these properties. Thus, the phage mimics some of the properties of a B cell. Phage presentation can be carried out in various formats. Various sources of V gene segments can be used for phage presentation. Clackson et al., Nature, 352, 1991, cc. 624-628 isolated different sets of anti-oxazolone antibodies from a small random combinatorial library of V genes obtained from the spleens of immunized mice. A spectrum of V genes obtained from immunized human donors can be constructed, and antibodies to a different set of antigens (including autoantigens) can be isolated generally according to the methods described in Marks et al., J. Mol. Biol., 222, 1991, cc. 581-597.

As described above, human antibodies can also be produced in vitro by activated B cells (see US 5567610 and 5229275).

Antibody fragments.

In certain circumstances, it may be appropriate to use antibody fragments rather than full antibodies. The smaller size of the fragments facilitates their rapid clearance and may allow for better penetration into solid tumors.

Various methods have been developed to obtain antibody fragments. Traditionally, these fragments were obtained by proteolytic cleavage of intact antibodies. However, these fragments can now be produced directly from recombinant host cells. Fab, Fv, and scFv antibody fragments can be expressed and secreted from E. coli, allowing the production of large quantities of these fragments to be facilitated. Antibody fragments can be isolated from the phage antibody libraries described above. In another embodiment, Fab'-SH fragments can be directly isolated from E. coli and chemically cross-linked to produce F(ab')2 fragments (Carter et al., Bio/Technology, 10, 1992, pp. 163-167). According to another approach, F(ab')2 fragments can be isolated directly from recombinant host cell cultures. Fab and F(ab') ₂ fragments with increased in vivo half-lives that retain epitope-binding receptor residues are described in US Pat. No. 5,869,046. Other techniques for producing antibody fragments will be apparent to those skilled in the art. In other embodiments, the antibody selected is a single chain Fv fragment (scFv) (see WO 93/16185; US 5,571,894 and US US 5,587,458). Fv and scFv are the only species with intact binding sites lacking constant regions; as a result, they can be used for reduced nonspecific binding when used in vivo. ScFv-carrying fusion proteins can be engineered to produce an effector protein fusion at either the N- or C-terminus of the scFv. The antibody fragment may also be a linear antibody, for example, as described in US Pat. No. 5,641,870. Such linear antibody fragments may be monospecific or bispecific.

Pharmaceutical compositions.

Another aspect of the invention is a pharmaceutical composition containing as an active ingredient (or as the only active ingredient) a monoclonal antibody that specifically binds to CD20.

The pharmaceutical composition may include at least one monoclonal antibody that specifically binds to CD20 and at least one component selected from the group consisting of pharmaceutically acceptable and pharmacologically compatible excipients.

The pharmaceutical composition may include at least one monoclonal antibody that specifically binds to CD20 and one or more additional binding molecules (eg, antibodies) that target one or more corresponding surface receptors. In some embodiments, the compositions are intended to improve, prevent, or treat disorders that may be associated with CD20.

Pharmaceutical composition means a composition comprising a monoclonal antibody that specifically binds to CD20 according to the invention and at least one of the components selected from the group consisting of pharmaceutically acceptable and pharmacologically compatible excipients such as excipients, diluents, diluents, carriers, auxiliary, distributing, delivery vehicles, preservatives, stabilizers, emulsifiers, suspending agents, thickeners, prolonged delivery regulators, the choice and ratio of which depends on the nature and method of administration and dosage. The pharmaceutical compositions of the present invention and methods for their manufacture will be readily apparent to those skilled in the art. The production of pharmaceutical compositions should preferably comply with GMP (Good Manufacturing Practices) requirements. The composition may include a buffer composition, tonic agents, stabilizers and solubilizers. Prolonged action of the composition can be achieved by using agents that slow down the absorption of the active pharmaceutical ingredient, for example, aluminum monostearate and gelatin. Examples of suitable carriers, solvents, diluents and delivery vehicles are water, ethanol, polyalcohols, as well as mixtures thereof, oils and injectable organic esters.

- 25 044757

Medicine (drug) - a substance or mixture of substances in the form of a pharmaceutical composition in the form of tablets, capsules, powders, lyophilisates, injections, infusions, ointments and other finished forms, intended to restore, correct or change physiological functions in humans and animals, and also for the treatment and prevention of diseases, diagnostics, anesthesia, contraception, cosmetology and other things. Any method of administering peptides, proteins or antibodies accepted in the art can suitably be used for the monoclonal antibody that specifically binds to CD20 of the present invention.

The term pharmaceutically acceptable means one or more compatible liquid or solid components that are suitable for administration to a mammal, preferably a human.

The term excipient or excipient is used herein to describe any component other than those previously described in this invention. These are substances of inorganic or organic origin, used in the production process of medicines to give them the necessary physical and chemical properties.

The term buffer, buffer composition, buffering agent refers to a solution capable of maintaining a pH value due to the interaction of the acidic and alkaline components included in its composition, which allows the monoclonal antibody preparation that specifically binds to CD20 to exhibit resistance to changes in pH. In general, pH values of the pharmaceutical composition from 4.0 to 8.0 are preferred. As buffering agents, for example, acetate, phosphate, citrate, histidine, succinate, etc. can be used. buffer solutions, but not limited to them.

The terms tonic agent, osmolytic or osmotic agent as used herein refer to an excipient that can add osmotic pressure to a liquid antibody preparation. An isotonic drug is a drug that has an osmotic pressure equivalent to that of human blood. Isotonic drugs typically have an osmotic pressure of approximately 250 to 350 mOsm/kg. Polyols, mono- and disaccharides, amino acids, metal salts such as sodium chloride, etc. can be used as isotonic agents, but are not limited to them.

By stabilizer is meant an excipient or a mixture of two or more excipients that provide physical and/or chemical stability of the active agent. Amino acids can be used as stabilizers, for example, but not limited to, arginine, histidine, glycine, lysine, glutamine, proline; surfactants, e.g. polysorbate 20 (trade name Tween 20), polysorbate 80 (trade name Tween 80), polyethylene-polypropylene glycol and its copolymers (trade names Poloxaner, Pluronic), sodium dodecyl sulfate (SDS) ), but not limited to them; antioxidants, for example, but not limited to, methionine, acetylcysteine, ascorbic acid, monothioglycerol, sulfuric acid salts, etc.; chelating agents, for example, but not limited to, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), sodium citrate and the like.

A pharmaceutical composition is stable if the active agent maintains its physical stability and/or chemical stability and/or biological activity through its stated shelf life at storage temperature, for example, 2-8°C. It is preferred that the active agent retains both physical and chemical stability as well as biological activity. The storage period is selected based on the results of stability studies during accelerated and natural storage.

The pharmaceutical composition of this invention may be manufactured, packaged or widely sold as a single unit dose or a plurality of unit dose units in a finished dosage form. As used herein, the term unit dose means a discrete amount of a pharmaceutical composition containing a predetermined amount of the active ingredient. The amount of active ingredient is typically equal to the dosage of the active ingredient to be administered to the subject, or a convenient portion of such dosage, such as one-half or one-third of such dosage.

The pharmaceutical compositions of the present invention are generally suitable for parenteral administration in the form of sterile medicinal products intended for introduction into the human body without breaking the integrity of the skin or mucous membranes, bypassing the gastrointestinal tract by injection, infusion or implantation. For example, parenteral administration is intended to include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intravenous, intra-arterial, intrathecal, intraventricular, intraurethral, intracranial, intra-articular, transdermal injection or infusion; and renal dialysis infusion techniques. Intratumoral delivery, such as intratumoral injection, may also be applicable. Regional perfusion is also provided. Preferred embodiments of the invention include intravenous and subcutaneous routes. Any method of administering peptides or proteins accepted in the art can suitably be used for the monoclonal antibody that specifically binds to CD20 of the present invention.

- 26 044757

Injectable drugs may be manufactured, packaged or sold in unit dosage form, such as, but not limited to, ampoules, vials, polymer containers, pre-filled syringes, auto-injection devices. Drugs for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous bases, pastes and the like.

Another embodiment of the invention is a medicament for parenteral administration, wherein the pharmaceutical composition is provided in dry form, i.e. powder or granules for dissolution in a suitable solvent (eg, sterile pyrogen-free water) before administration. Such a medicinal product can be obtained, for example, by lyophilization, i.e. a process known in the art as freeze drying, which involves freezing the preparation and then removing the solvent from the frozen contents.

The monoclonal antibody that specifically binds to CD20 of the present invention may also be administered intranasally or by inhalation, alone, in the form of a mixture with a suitable pharmaceutically acceptable excipient from an inhaler, such as a pressurized aerosol container, pump, spray, nebulizer) or nebulizer, in which whether or not a suitable propellant is used, or in the form of nasal drops or spray.

The drug for parenteral administration may be immediate or modified release. Modified release drugs include delayed, sustained, pulsatile, controlled, targeted and programmable release.

Therapeutic use of a monoclonal antibody that specifically binds to CD20 according to the present invention.

In one aspect, a monoclonal antibody that specifically binds to CD20 of the present invention is used in the treatment of disorders that are associated with CD20 activity.

In one aspect, the subject of treatment or patient is a mammal, preferably a human subject.

The above subject may be male or female and of any age.

In the case of a tumor (eg, a cancerous tumor), a therapeutically effective amount of an antibody or antibody fragment (eg, an antibody or antibody fragment that specifically binds CD20) can reduce the number of cancer cells; reduce the initial size of the tumor; inhibit (ie, slow down to some extent and preferably stop) the infiltration of cancer cells into peripheral organs; inhibit (ie, slow down to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or alleviate, to some extent, one or more symptoms associated with the disorder. The antibody or antibody fragment may, to some extent, prevent the growth and/or kill existing cancer cells, and may cause a cytostatic and/or cytotoxic effect. In cancer therapy, in vivo efficacy can be determined, for example, by assessing survival, time to disease progression (TTP), tumor response rate (RR), duration of response, and/or quality of life.

As used herein, the terms co-administration, co-administered and in combination with refer to a monoclonal antibody that specifically binds to CD20, one or more other therapeutic agents and is intended to mean, refer to or include:

1) simultaneous administration of such a combination of a monoclonal antibody that specifically binds to CD20 according to this invention and a therapeutic agent to a patient in need of treatment, when such components are formulated together in a single dosage form from which said components are released substantially simultaneously to said patient,

2) simultaneous administration of such a combination of a monoclonal antibody that specifically binds to CD20 according to this invention and a therapeutic agent to a patient who needs treatment, when such components are formulated separately in different dosage forms, the administration of which occurs at substantially the same time to the specified patient , after which the specified components are released almost simultaneously to the specified patient,

3) sequential administration of such a combination of a monoclonal antibody that specifically binds to CD20 according to this invention and a therapeutic agent to a patient who needs treatment, when such components are formulated separately from each other in separate dosage forms that are taken in sequential time by the specified patient with a significant time interval between each administration, after which the specified components are released at practically different times to the specified patient; and

4) sequential administration of such a combination of a monoclonal antibody that specifically binds to CD20, according to this invention and a therapeutic agent to a patient who needs treatment, when such components are formulated together in a single dosage form from which the release of these components occurs in a controlled manner, after which they at the same time

- 27 044757 are released separately, sequentially or jointly at the same time and/or at different times to a specified patient, where each part can be administered by one or different routes.

The monoclonal antibody that specifically binds to CD20 of the present invention can be administered without additional therapeutic treatment, i.e. as an independent therapy. In addition, treatment with a monoclonal antibody that specifically binds to CD20 according to the present invention may include at least one additional therapeutic treatment (combination therapy). In some embodiments, a monoclonal antibody that specifically binds to CD20 may be co-administered or formulated with another drug/drug for the treatment of cancer or an autoimmune disease.

The term cytotoxic agent as used herein refers to a substance that inhibits or prevents the functioning 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 Lu radioactive isotopes), chemotherapeutic agents and toxins such as low molecular weight toxins or enzymatically active toxins originating from bacteria, fungi, plants or animals, including fragments and/or variants thereof.

A chemotherapy agent is a chemical compound used to treat a malignant tumor. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN®); alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquinone, meturedopa and uredopa; ethyleneimines and methylmelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylmelamine; acetogenins (eg bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol; colchicines; betulinic acid; camptothecin (including synthetic analogue of topotecan (HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin and 9-aminocamptothecin); bryostatin; kallistatin; CC-1065 (including its synthetic analogues adozelesin, carzelesin and bizelesin); podophyllotoxin; podophyllic acid; teniposide; cryptophycins (eg, cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogues KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphasine, holophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembiquine, phenesterine, prednimustine, trofosfamide, uramustine; nitrosoureas such as carmustine, chlorosotocin, fotemustine, lomustine, nimustine and ranimustine; antibiotics such as enediyne antibiotics (eg calicheamicin, e.g. calicheamicin gamma II and calicheamicin omega II (see, for example, Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994)); dinemycin, including dinemycin A; esperamycin; as well as neocarcinostatin chromophore and related chromophores of enediin antibiotics, aclacinomysins, actinomycin, outramycin, azaserin, bleomycins, cactinomycin, carabicin, carminomycin, carzinophyllin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6di azo-5-oxo-norleucine, doxorubicin (including ADRIAMICIN®, morpholinodoxorubicin, cyanomorpholinodoxorubicin, 2-pyrrolinodoxorubicin, doxorubicinoNO in injectable liposomes (DOXOL®), liposomal doxorubicin TLC D-99 (MYOCET®), pegylated liposomal doxorubicin (CAELYX®) and deoxydoxorubicin), epi rubicin, ezorubicin, idarubicin , marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rhodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate, gemcitabine (GEMZAR®), tegafur (UFTORAL®), capecitabine (XELODA®), epothilone and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprin, thioguanine; pyrimidine analogues such as ancytabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocytabine, floxuridine; drugs that suppress adrenal function, such as aminoglutethimide, mitotane, trilostane; a folic acid compensator such as folinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diazichon; elfornitine; elliptinium acetate; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerin; pentostatin; phenomet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sisofiran; spirogermanium; tenuazonic acid; triaziquon; 2,2',2-trichlorotriethylamine; trichothecenes (eg, T-2 toxin, verracurine A, roridin A and anguidine); urethane; dacarbazine; mannomustin; mitobronitol; mitolactol; pipobromance; gacytosine; arabinoside (ara-C); thiotepa; a taxoid, such as paclitaxel (TAXOL®), an engineered albumin-associated nanoparticle formulation of paclitaxel (ABRAXANETM), and docetaxel (TAXOTERE®); chlorambucil; 6-thioguanine; mercaptopurine; methotrexate; platinum-based agents such as cisplatin, oxaliplatin and carboplatin; vinca alkaloids, which prevent the polymerization of tubulin from nascent microtubules, including vinb-28 044757 lastine (VELBAN®), vincristine (ONCOVIN®), vindesine (ELDISINE®, FILDESIN®) and vinorelbine (NAVELBINE®); etoposide (VP-16); ifosfamide; mitoxantrone; leucovorin; Novantrone; edatrexate; daunomycin; aminopterin; ibandronate; 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/zoledronate (ZOMETA®), alendronate (FOSAMAX®), pamidronate (AREDIA®), tiludronate (SKELID® ) or risendronate (ACTONEL®); troxacitabine (1,3-dioxolane nucleoside analogue of cytosine); antisense oligonucleotides, for example oligonucleotides that inhibit gene expression in signaling pathways involved in aberrant cell proliferation, such as, for example, PKC-alpha, Raf, H-Ras and epidermal growth factor receptor (EGF-R); vaccines such as THERATOPE® vaccine and gene therapy vaccines such as ALLOVECTIN® vaccine, LEUVECTIN® vaccine and VAXID® vaccine; topoisomerase 1 inhibitor (eg LURTOTECAN®); rmRH (eg ABARELIX®); BAY439006 (sorafenib; Bayer); SU-11248 (Pfizer); perifosine, COX-2 inhibitor (eg, celecoxib or etoricoxib), proteasome inhibitor (eg, PS341); bortezomib (VELCADE®); CCI-779; tipifarnib (811577); orafenib, ABT510; a Bcl-2 inhibitor such as oblimersen sodium (GENASENSE®); pixantron; EGFR inhibitors (see definition below); tyrosine kinase inhibitors (see definition below); and pharmaceutically acceptable salts, acids or derivatives of any of the above; and combinations of two or more of the above, such as CHOP, short for combination therapy of cyclophosphamide, doxorubicin, vincristine and prednisolone, and FOLFOX, short for oxaliplatin treatment regimen (ELOXATINTM) in combination with 5-FU and leucovorin.

Also included in this definition are antihormonal agents that act by regulating or inhibiting the action of hormones on tumors, such as antiestrogens with a mixed agonist/antagonist profile, including tamoxifen (NOLVADEX®), 4-hydroxytamoxifen, trioxifene, toremifene (FARESTON®); idoxifene, droloxifene, raloxifene (EVISTA®), trioxifene, keoxifene and selective estrogen receptor modulators (SERMs) such as SERM3; pure antiestrogens without agonist properties, such as fulvestrant (FASLODEX®) and EM800 (such agents may block estrogen receptor (ER) dimerization, inhibit DNA binding, increase ER metabolism, and/or decrease ER levels); aromatase inhibitors, including steroidal aromatase inhibitors such as formestane and exemestane (AROMAS IN®), and non-steroidal aromatase inhibitors such as anastrazole (ARIMIDEX®), letrozole (FEMARA®) and aminoglutethimide and other aromatase inhibitors, including vorozole (RIVISOR®) , megestrol acetate (MEGASE®), fadrozole, imidazole; luteinizing hormone releasing hormone agonists including leuprolide (LUPRON® and ELIGARD®), goserelin, buserelin and tripterelin; 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; onapristone; antiprogesterones; estrogen receptor down-regulators (ERDs); antiandrogens such as flutamide, nilutamide and bicalutamide; testolactone; and pharmaceutically acceptable salts, acids or derivatives of any of the above; as well as a combination of two or more of the above means.

To treat the above autoimmune diseases or related autoimmune conditions, a monoclonal antibody that specifically binds to CD20 of the present invention may be administered to a patient in combination with another therapeutic agent such as an immunosuppressant, an anti-inflammatory drug, a systemic hormonal drug, an antineoplastic agent, and an immunomodulatory agent. drug or others using a multi-drug regimen. A monoclonal antibody that specifically binds to CD20 may be administered concomitantly, sequentially, or alternately with another therapeutic agent or following resistance to another therapy. The other therapeutic agent may be administered at the same or lower doses than those used in the art. The choice of the preferred other therapeutic agent should depend on many factors, including the type of disease being treated as well as the patient's medical history.

As used herein, the term immunosuppressant used for adjunctive therapy refers to substances that act to suppress or mask the patient's immune system. Such agents may be substances that inhibit the production of cytokines, down-regulate or suppress the expression of autoantigens, or mask major histocompatibility complex (MHC) antigens. Examples of such agents are steroids, such as glucocorticosteroids, such as prednisone, methylprednisolone and dexamethasone; 2-amino-6-aryl-5-substituted pyrimidines (see US 4665077), azathioprine (or cyclophosphamide if there is a negative reaction to azathioprine); bromocriptine; glutaraldehyde (which masks MHC antigens, as described in US 4120649); anti-idiotypic antibodies to MHC antigens and MHC fragments; cyclosporine A; cytokine or cytokine receptor antagonists, including antibody to

- 29 044757 interferon-γ -β or -α; antibodies to tumor necrosis factor-α, antibodies to tumor necrosis factor-β; antibodies to interleukin-2 and antibodies to IL-2 receptor; antibodies to L3T4; heterologous antilymphocyte globulin; rap-T antibodies, preferably anti-CD3 antibodies or anti-CD4/CD4a antibodies; a soluble peptide containing an LFA-3 binding domain (WO 90/08187, published July 26, 1990); streptokinase; TGF-p; streptodornase; Host RNA or DNA; FK506; RS-61443; deoxyspergualin; rapamycin; T cell receptor (US 5114721); T cell receptor fragments (Offner et al., Science 251, 1991, pp. 430-432; WO 90/11294; and WO 91/01133); and anti-T cell receptor antibodies (EP 340109), such as T10B9.

For the treatment of rheumatoid arthritis, a monoclonal antibody of the invention that specifically binds to CD20 can be administered to a patient, alone or in combination with one or more of the following drugs: DMARDs (DMARDs (eg, methotrexate, leflunomide, sulfasalazine) , NSAIDs or (non-steroidal anti-inflammatory drugs, e.g. cyclooxygenase inhibitors), corticosteroids (e.g. prednisolone, budesonide).DMARDs commonly used to treat RA are hydroxycloroquine, sulfasalazine, methorexate, leflunomide, azathioprine, D-penicillamine, drugs based on gold (oral), gold-based drugs (intramuscular), minocycline, cyclosporine, Staphylococcal obtained by immunoadsorption on protein A. Common methods of treating RA are described, for example, in J.A. Singh et al., 2015 American College of Rheumatology Guideline for the Treatment of Rheumatoid Arthritis. Arthritis Care Res (Hoboken) 68, 1-25 (2016).

It is intended that the monoclonal antibody that specifically binds to CD20 of the present invention can be used in methods of treatment as described above, can be used in treatment as described above, and/or can be used in the manufacture of medicaments for treatment as described above.

Doses and routes of administration.

A monoclonal antibody that specifically binds to CD20 of the present invention will be administered in an amount effective to treat the condition in question, i.e. in doses and for periods of time necessary to achieve the desired result. The therapeutically effective amount may vary depending on factors such as the specific condition being treated, the age, sex and weight of the patient, and whether administration of a monoclonal antibody that specifically binds to CD20 is a stand-alone treatment or is given in combination with one or more additional medications or treatments.

Drug regimens can be adjusted to provide the optimal desired response. For example, a single bolus may be administered, multiple divided doses may be administered over time, or the dose may be proportionally decreased or increased depending on the acuity of the therapeutic situation. It is particularly advantageous to formulate parenteral compositions in unit dosage form for ease of administration and uniformity of dosage. Unit dosage form as used herein refers to physically discrete units suitable as unitary dosages for patients/subjects 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 specification for unit dosage forms of the present invention is generally dictated by and directly depends on (a) the unique characteristics of the chemotherapeutic agent and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the technique of compounding such active compound for treating sensitivity in subjects.

Thus, those skilled in the art will appreciate from the disclosure provided herein that dosages and dosage regimens will be adjusted in accordance with methods well known in the therapeutic field. This means that the maximum tolerated dose can be easily determined and the effective amount to provide a detectable therapeutic effect for the patient can also be determined, as well as the timing requirements for each agent to achieve a detectable therapeutic effect for the patient. Thus, although certain dosages and dosage regimens are given as examples herein, these examples are not intended to limit in any way the dosages and dosage regimens that may be needed for a patient to practice the present invention.

It should be noted that dosage levels may vary depending on the type and severity of the condition to be alleviated and may include one or more doses. In addition, it should be understood that for any particular patient, specific dosage regimens must be adjusted over time according to individual need and at the discretion of the healthcare professional administering or supervising the administration of the compositions, and that the concentration ranges given herein are provided only by way of example and are not intended to limit the scope or practice of the claimed compositions. In addition, the dosage regimen of the compositions of this invention may be based on various factors, including the type of disease

- 30 044757 information, age, weight, gender, patient's health conditions, severity of condition, route of administration and the specific monoclonal antibody used that specifically binds to CD20. Thus, the dosage regimen may vary widely, but can be determined regularly using standard methods. For example, dosages may be adjusted based on pharmacokinetic and pharmacodynamic parameters, which may include clinical effects such as toxic effects or laboratory values. Thus, the present invention covers individual dose escalation, which is determined by a qualified specialist. Determination of the required dosage and regimens are well known in the relevant art and will be apparent to one skilled in the art upon familiarization with the concepts disclosed herein.

Examples of suitable routes of administration are provided above.

It is expected that a suitable dose of a monoclonal antibody that specifically binds to CD20 of 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, for example about 1-20 mg/kg. A monoclonal antibody that specifically binds to CD20 may be administered, for example, at a dose of at least 0.25 mg/kg, such as at least 0.5 mg/kg, including at least 1 mg/kg, e.g. at least 1.5 mg/kg, for example also at least 2 mg/kg, for example at least 3 mg/kg, including at least 4 mg/kg, for example at least 5 mg/kg; and, for example, up to a maximum of 50 mg/kg, including up to a maximum of 30 mg/kg, for example, up to a maximum of 20 mg/kg, including up to a maximum of 15 mg/kg. Administration will be repeated usually at suitable intervals, for example once a week, once every two weeks, once every three weeks or once every four weeks, and for as long as is considered appropriate by the responsible physician, which may in some cases increase or reduce dose if necessary.

Diagnostic uses and compositions.

The monoclonal antibody that specifically binds to CD20 of the present invention is also used for diagnostic purposes (eg, in vitro, ex vivo). For example, a given monoclonal antibody that specifically binds to CD20 of the present invention can be used to detect or measure the level of CD20 in samples obtained from a patient, for example, a tissue sample or a body fluid sample such as inflammatory exudate, blood, serum, fluid intestines, saliva or urine. Suitable detection and measurement methods include immunological methods such as flow cytometry, enzyme-linked immunosorbent assay (ELISA), chemiluminescence assay, radioimmunoassay and immunohistology. The invention further includes kits, for example diagnostic kits, containing a monoclonal antibody that specifically binds to CD20, according to the present invention, described herein.

For a better understanding of the invention, the following examples are provided. These examples are provided for illustrative purposes only and should not be construed as limiting the scope of the invention in any form.

All publications, patents and patent applications referenced in this specification are incorporated herein by reference. Although the foregoing invention has been described in some detail by way of illustration and example in order to avoid ambiguity, those skilled in the art will appreciate from the teachings disclosed herein that certain changes and modifications may be made without departing from the spirit and scope of the appended embodiments. implementation of the invention.

Examples.

For a better understanding of the invention, the following examples are provided. These examples are provided for illustrative purposes only and should not be construed as limiting the scope of the invention in any form.

All publications, patents and patent applications referenced in this specification are incorporated herein by reference. Although the foregoing invention has been described in some detail by way of illustration and example in order to avoid ambiguity, those skilled in the art will appreciate from the teachings disclosed herein that certain changes and modifications may be made without departing from the spirit and scope of the appended embodiments. implementation of the invention.

Materials and general methods.

General information regarding the nucleotide sequences of the light and heavy chains of human immunoglobulin is presented in: Kabat E.A. et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service Publishing, National Institutes of Health, Bethesda, MD, 1991. Amino acid chains of antibodies are numbered according to EU numbering (Edelman G.M. et al., Proc Natl Acad Sci USA 63, 1969, pp. 78-85 Kabat E. A. et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda , MD, 1991).

Recombinant DNA methods.

For DNA manipulation, standard methods were used as described in Sambrook J. et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York,

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1989. Molecular biology reagents were used according to the manufacturers' instructions.

Gene synthesis.

The required gene segments were obtained from oligonucleotides created by chemical synthesis. Gene segments ranging from 300 to 4000 kb in length, which are flanked by unique restriction sites, were assembled by annealing and ligation of oligonucleotides, including PCR amplification and subsequent cloning through the specified restriction sites. The DNA sequences of the subcloned gene fragments were confirmed by DNA sequencing.

Determination of DNA sequences.

DNA sequences were determined by Sanger sequencing.

Analysis of DNA and protein sequences and processing of sequence data.

Infomax's Vector NT1 Advance suite, version 8.0, was used to generate, map, analyze, annotate, and illustrate sequences.

Expression vectors.

To express the described antibodies and antigens, variants of expression plasmids intended for expression in prokaryotic cells (E. coli) and short-term expression in eukaryotic cells (for example, in CHO cells) were used. In addition to the antibody expression cassette, the vectors contained: an origin of replication that ensures replication of the specified plasmid in E. coli, genes that confer resistance in E. coli to various antibiotics (for example, ampicillin and kanamycin).

Fusion of genes containing the antibody chains described, as described below, was accomplished by PCR and/or gene synthesis and assembly using known recombination methods and procedures by joining appropriate nucleic acid segments, for example, using unique restriction sites in appropriate vectors. Subcloned nucleotide sequences were confirmed by DNA sequencing. For short-term transfections, large quantities of plasmids were generated by obtaining plasmids from transformed E. coli cultures.

Example 1. Production of recombinant control antibodies in a suspension culture of mammalian cells.

An antibody with the published Rituximab sequence was used as a control. The genes for the variable domains of the heavy and light chains of the antibody were synthesized and cloned into the vectors pEE-HC, pEE-CK (Fig. 1, 2), intended for protein production in mammalian cells, at the restriction sites Sall/NheI and SalI/BstWI, respectively.

Plasmids were produced in the required quantities in E. coli cells and purified using a Qiagen kit.

Control antibodies were produced in cells of a permanent cell line derived from Chinese hamster ovary cells (CHO-T line). Suspension cultivation was carried out in flasks on an orbital shaker-incubator, using serum-free media produced by HyCell TransFx-C with the addition of 8 mM L-glutamine and 1 g/l Pluronic 68. For transient cell expression at a concentration of 2-2.2x106 cells/ml transfected with linear polyethylenimine (PEI MAX, Polysciences). The DNA/PEI ratio was 1:3/1:10. 9 days after transfections, the culture liquid was separated from the cells by filtration through a depth filter with a pore size of 0.5/0.22 μm. Target proteins were isolated from the culture liquid using affinity chromatography on the bacterial protein Protein A.

The purity of the resulting protein solution was assessed using SDS gel electrophoresis under reducing and non-reducing conditions.

Example 2. Creation of a naive FAB library of human antibodies MeganLibTM.

Total B cell RNA from individual blood samples from over a thousand human donors was isolated using the RNeasy Mini Kit according to the proposed protocol (from QIAGEN). RNA concentration was determined using a Nanovue kit (from GE Healthcare) and the quality of the isolated RNA was checked using 1.5% agarose gel electrophoresis.

The reverse transcription reaction was performed using the MMLV RT kit (from Evrogen) according to the recommended protocol using MMuLV reverse transcriptase and random hexamer oligonucleotides as primers.

Reverse transcription products were used as a template in a two-step polymerase chain reaction to obtain variable domain genes flanked by restriction sites using a set of oligonucleotides according to the authors' protocols [J Biol Chem. 1999 Jun 25; 274(26): 18218-30].

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

Example 3. Selection of FAB library by phage display method.

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Specific human phage Fab antibodies against CD20 were obtained from the combinatorial phage Fab display library MeganLibTM. Selection was carried out on eukaryotic cells expressing human CD20 using the phage display method [Nat Biotechnol. 1996 Mar;14(3):309-14; J Mol Biol.1991 Dec 5;222(3): 581-97], but using magnetic particles and the KingFisher Flex device, since the use of this technique allows up to 96 different biopanning schemes and options to be carried out in parallel.

In biopanning selection, biotinylated eukaryotic cells were immobilized on the surface of streptavidin magnetic particles by incubating the cells with the particles for 20 min on a rotator. Next, the particles were washed with PBS (pH 7.4), then the particles were blocked with a solution of 2% skim milk in PBS (pH 7.4) for 1 hour. Then a solution of phages pre-incubated with antigen-negative cells was added to the magnetic particles with bound cells in PBS (pH 7.4) with 2% skim milk. This mixture was incubated for 40 min with stirring. Unbound phages were removed during several washes of magnetic particles with a PBS solution (pH 7.4) with 0.1% Tween 20. The number of washes was increased from round to round (10 washes in the 1st round, 20 washes in the 2nd round, and 20 washes in the 2nd round). m - 30). Phages bound to the antigen on the surface of magnetic particles were eluted from the particles with a 100 mM Gly-HCl solution (pH 2.2) for 15 min with stirring; after elution, a 1 M TRIS-HCl solution (pH 7.6) was neutralized. Bacteria of the E. coli TGI strain were infected with the obtained phages, phages were produced in them, they were isolated and used in the next round of selection. After three to four rounds, their DNA (phagemids) was isolated from the phages, and the genes for the variable domains of the antibodies were cloned into expression vectors (Fig. 6) to produce Fabs in E. coli cells.

Example 4. Library screening.

Primary screening.

Fab production was carried out according to a standard procedure: bacterial cells are transformed with expression vectors containing Fab genes, and the subsequent addition of an inducer that triggers transcription of the lac operon to the medium during cultivation of the resulting transformants induces Fab expression.

Next, an ELISA was performed for the binding of Fab to the CD20 peptide immobilized on the substrate. Detection of antigen-bound Fabs was performed using an anti human Fab HRP-conjugated secondary antibody (from Pierce-ThermoScientific).

The Rituximab Fab sequence inserted into the expression plasmid pLL was used as a positive control (Fig. 6).

As a result of primary screening, clones with the ability to bind to the target CD20 peptide were selected. The material was transferred for secondary screening.

Secondary screening.

The purpose of the secondary screening was to select clones producing Fabs that interact with the CD20 peptide and do not interact with other antigens IL6R-Fc, PCSK9-VG-FE, PD-1-Fc.

Fab production was carried out according to standard methods. Next, an ELISA of Fab binding to various antigens immobilized on a substrate was carried out according to the standard procedure.

As a result of secondary screening, Fab-producing clones that specifically bind only the target CD20 peptide were selected.

Example 5. Optimization of leading candidates.

The selected candidates were optimized to enhance humanization. To select replacement points, we used the Humanizer tool, which is part of the YLab software package (developed by Biocad). Germline functional V, D, J segments of various biological species were taken as a source of data from the IMGT database. Human segments were used as positive references, rats and mice were used as negative references. Using this data, the tool suggested replacement positions.

For further selection, 1000 candidates were generated with different subsets of the selected substitution set. The resulting candidates were modeled based on the crystal structure of the complex of the original candidate with the CD20 target. The models were obtained using the BENDER programs (developed by Biocad) and BioLuminate (part of the Schrodinger Suite software package, developed by Schrodinger). The resulting models were evaluated by calculating the average MM-GBSA using the OPLS 2005 force field on 100 ns molecular dynamics trajectories. Molecular dynamics trajectories were obtained using the Desmond program (part of the Schrodinger Suite software package, developed by Schrodinger). Among the results obtained, a cluster of 133 candidates clearly stood out, which were proposed for further synthesis along with the control candidate.

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

For the resulting 133 candidates, codon optimization for CHO was carried out using the OligoDesigner tool of the YLab software package (Biocad). The optimized sequences of the variable domains of the heavy and light chains were synthesized de novo and cloned into the vectors pEENS, pEE-CK (IgG1 format) at the Sall/Nhe1 and Sall/BsiW1 restriction sites, respectively (Fig. 1, 2). Sche

- 33 044757 mathematical image of the IgGl format in FIG. 3.

The resulting genetic constructs were used to transform the CHO-T cell line. Proteins were isolated and purified according to standard methods by affinity chromatography on the bacterial protein Protein A as described in example 1. Electrophoresis was carried out under denaturing conditions in 7.5% PAGE. The productivity of 22 candidates was below the threshold level (50 mg/l), and accordingly they were not taken for isolation and purification.

Example 7: Sequencing of high-affinity clones.

Variable domain genes of positive clones were sequenced according to standard protocols on an Applied Biosystems 3130 Genetic Analyzer (Applied Biosystems) and analyzed.

Example 8. Determination of the affinity of full-length antibodies using Forte Bio Octert RED 384.

The kD values of the resulting full-length candidates were assessed on a Forte Bio Octet RED 384 instrument.

SAX biosensors and CD20 peptide modified with biotin (Sigma Aldrich) were used for this work. Rituximab antibody was used as a control. SAX biosensors were immersed in a solution with biotinylated CD20 peptide at a concentration of 20 μg/ml, where the peptide was immobilized. Further analysis was carried out at 30°C using PBS containing 0.1% Tween-20 and 0.1% BSA as a running buffer.

After a baseline was prescribed in the buffer solution, the sensors were immersed in wells with an antibody solution at a concentration of 10 μg/ml for 150 s, where association of the complex occurs. Then the dissociation of the complex in the buffer solution was detected for 300 s.

Baseline-subtracted binding curves were analyzed using Octet Data Analysis (version 9.0) according to standard procedures using a 1:1 interaction model.

116 candidates were submitted for analysis. Of these, 67 candidates showed no binding to the peptide. The remaining 49 candidates interacted with the peptide with nanomolar and micromolar affinities (Figure 7 and Table 1).

Table 1

Candidate Response (nm) kD (M) kon(1/Ms) kdis(1/s) Full R ^A 2 BCD132 No No No No No L-001 binding binding binding binding binding BCD132 No No No No No L-002 binding binding binding binding binding BCD132 L-003 0.2576 1.87E-07 3.06E+05 5.73E-02 0.9909 BCD132 No No No No No L-004 binding binding binding binding binding BCD132 No No No No No L-007 binding binding binding binding binding BCD132 L-008 0.1189 7.51E-07 1.85E+05 1.39E-01 0.9899 BCD132 No No No No No L-009 binding binding binding binding binding BCD132 No No No No No L-010 binding binding binding binding binding

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BCD132 L-011 0.2545 4.35E-07 3.41E+05 1.48E-01 0.996 BCD132 L-012 No binding No binding No binding No binding No binding BCD132 L-013 No binding No binding No binding No binding No binding BCD132 L-015 0.7312 1.51E-07 3.00E+05 4.54E-02 0.9981 BCD132 L-016 No binding No binding No binding No binding No binding BCD132 L-017 No binding No binding No binding No binding No binding BCD132 L-018 No binding No binding No binding No binding No binding BCD132 L-019 No binding No binding No binding No binding No binding BCD132 L-020 No binding No binding No binding No binding No binding BCD132 L-021 No binding No binding No binding No binding No binding BCD132 L-022 No binding No binding No binding No binding No binding BCD132 L-023 No binding No binding No binding No binding No binding BCD132 L-024 0.2377 2.72E-07 5.8 6E+05 1.59E-01 0.9969 BCD132 L-025 No binding No binding No binding No binding No binding BCD132 L-026 0.189 9.74E-08 8.72E+05 8.50E-02 0.9761 BCD132 L-028 0.2631 1.35E-07 9.30E+05 1.26E-01 0.9849 BCD132 L-029 No binding No binding No binding No binding No binding BCD132 L-030 0.7685 1.11E-05 2.39E+03 2.65E-02 0.8223 BCD132 L-031 No binding No binding No binding No binding No binding BCD132 L-033 No binding No binding No binding No binding No binding BCD132 L-034 0.8465 8.45E-06 3.73E+03 3.15E-02 0.9308 BCD132 L-035 1.3668 6.5 6E-0 6 3.35E+03 2.20E-02 0.9829 BCD132 L-037 No binding No binding No binding No binding No binding BCD132 L-038 No binding No binding No binding No binding No binding BCD132 L-039 1,493 6.91E-06 2.44E+03 1.69E-02 0.9924 BCD132 L-040 1,099 9.14E-06 3.11E+03 2.84E-02 0.9675

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BCD132 L-041 No binding No binding No binding No binding No binding BCD132 L-042 0.745 8.51E-06 4.46E+03 3.80E-02 0.8425 BCD132 L-043 No binding No binding No binding No binding No binding BCD132 L-044 0.638 8.90E-06 3.55E+03 3.16E-02 0.8215 BCD132 L-045 0.7287 1.04E-05 3.70E+03 3.83E-02 0.9562 BCD132 L-046 0.4142 1.38E-05 4.68E+03 6.44E-02 0.9478 BCD132 L-047 No binding No binding No binding No binding No binding BCD132 L-048 No binding No binding No binding No binding No binding BCD132 L-049 No binding No binding No binding No binding No binding BCD132 L-050 No binding No binding No binding No binding No binding BCD132 L-051 No binding No binding No binding No binding No binding BCD132 L-052 No binding No binding No binding No binding No binding BCD132 L-053 0.1125 1.36E-07 4.64E+05 6.31E-02 0.9252 BCD132 L-054 0.083 1.59E-07 3.10E+05 4.92E-02 0.8279 BCD132 L-055 No binding No binding No binding No binding No binding BCD132 L-056 No binding No binding No binding No binding No binding BCD132 L-057 No binding No binding No binding No binding No binding BCD132 L-058 0.1308 1,ZOE-07 2.08E+06 2.70E-01 0.9744 BCD132 L-059 No binding No binding No binding No binding No binding BCD132 L-060 No binding No binding No binding No binding No binding BCD132 L-061 No binding No binding No binding No binding No binding BCD132 L-062 No binding No binding No binding No binding No binding BCD132 L-063 No binding No binding No binding No binding No binding BCD132 L-064 No binding No binding No binding No binding No binding BCD132 L-065 0.0551 6.48E-08 1.01E+06 6.53E-02 0.8386 BCD132 L- 0 6 6 0.0398 1.14E-05 4.10E+03 4.66E-02 0.8193

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BCD132 L-068 0.0984 9.34E-08 9.50E+05 8.87E-02 0.876 BCD132 L-069 0.1078 1.29E-07 6.96E+05 8.96E-02 0.8737 BCD132 L-070 No binding No binding No binding No binding No binding BCD132 L-071 0.3793 1.36E-07 7.59E+05 1.03E-01 0.9755 BCD132 L-072 0.2878 9.15E-08 7.82E+05 7.16E-02 0.8842 BCD132 L-073 No binding No binding No binding No binding No binding BCD132 L-074 No binding No binding No binding No binding No binding BCD132 L-075 0.4394 1.01E-07 8.20E+05 8.29E-02 0.9752 BCD132 L-076 0.2166 8.14E-08 8.45E+05 6.88E-02 0.7702 BCD132 L-077 0.065 7.48E-08 9.24E+05 6.91E-02 0.8382 BCD132 L-079 0.1921 1.88E-07 3.16E+05 5.96E-02 0.9433 BCD132 L-080 No binding No binding No binding No binding No binding BCD132 L-081 No binding No binding No binding No binding No binding BCD132 L-082 0.0695 7.99E-08 1.05E+06 8.41E-02 0.9063 BCD132 L-083 No binding No binding No binding No binding No binding BCD132 L-084 0.3092 1.86E-07 3.41E+05 6.35E-02 0.9697 BCD132 L-085 No binding No binding No binding No binding No binding BCD132 L-087 No binding No binding No binding No binding No binding BCD132 L-088 No binding No binding No binding No binding No binding BCD132 L-089 0.1279 5.94E-08 1.75E+06 1.04E-01 0.879 BCD132 L-092 No binding No binding No binding No binding No binding BCD132 L-093 No binding No binding No binding No binding No binding BCD132 L-094 No binding No binding No binding No binding No binding BCD132 L-095 0.5518 7.72E-08 1.04E+06 7.99E-02 0.9778 BCD132 L-097 0.2544 7.32E-08 1.59E+06 1.17E-01 0.9603 BCD132 L-098 No binding No binding No binding No binding No binding

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BCD132 L-099 No binding No binding No binding No binding No binding BCD132 L-100 0.639 8.07E-08 7.36E+05 5.95E-02 0.98 BCD132 L-101 No binding No binding No binding No binding No binding BCD132 L-102 No binding No binding No binding No binding No binding BCD132 L-103 No binding No binding No binding No binding No binding BCD132 L-104 No binding No binding No binding No binding No binding BCD132 L-105 0.207 6.28E-08 1.08E+06 6.75E-02 0.8085 BCD132 L-106 0.2847 4.65E-08 2.30E+06 1.07E-01 0.8997 BCD132 L-107 No binding No binding No binding No binding No binding BCD132 L-109 No binding No binding No binding No binding No binding BCD132 L-110 No binding No binding No binding No binding No binding BCD132 L-111 No binding No binding No binding No binding No binding BCD132 L-112 0.3355 6,ZOE-08 1.86E+06 1.17E-01 0.9556 BCD132 L-113 No binding No binding No binding No binding No binding BCD132 L-114 0.3495 6.03E-08 1.22E+06 7.36E-02 0.877 BCD132 L-115 No binding No binding No binding No binding No binding BCD132 L-116 No binding No binding No binding No binding No binding BCD132 L-117 No binding No binding No binding No binding No binding BCD132 L-118 0.5571 9.04E-08 9.57E+05 8.66E-02 0.973 BCD132 L-119 0.2468 4.91E-08 2.3 6E+0 6 1.16E-01 0.9109 BCD132 L-120 0.3083 6.95E-08 1.69E+06 1.18E-01 0.9443 BCD132 L-121 0.1834 5.97E-08 2.52E+06 1.50E-01 0.9316 BCD132 L-123 No binding No binding No binding No binding No binding BCD132 L-124 No binding No binding No binding No binding No binding BCD132 L-126 0.2006 4.24E-08 2.19E+06 9.29E-02 0.8596 BCD132 L-129 0.4355 9.00E-08 1.11E+06 9.98E-02 0.9724 BCD132 L-130 0.4193 8.67E-08 1.08E+06 9.31E-02 0.9485 BCD132 L-131 0.4494 9.39E-08 9.77E+05 9.17E-02 0.9612 BCD132 L-132 0.7019 1.07E-07 8.38E+05 8.97E-02 0.9888 BCD132 L-133 0.2948 5.66E-08 2.12E+06 1.20E-01 0.9316

Candidates BCD132L-026, BCD132L-028, BCD132L-075 and BCD132L-077 were selected based on the results of the above analysis.

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Example 9. Determination of the affinity of final candidates from transient production to CD20 on

Forte Bio Octert RED 384.

SAX biosensors and CD20 peptide modified with biotin (Sigma Aldrich) were used for this work. Rituximab antibody was used as a control. SAX biosensors were immersed in a solution with biotinylated CD20 peptide at a concentration of 20 μg/ml, where the peptide was immobilized. Further analysis was carried out at 30°C using PBS containing 0.1% Tween-20 and 0.1% BSA as a running buffer.

After a baseline was prescribed in the buffer solution, the sensors were immersed in wells with an antibody solution at a concentration of 10 μg/ml for 210 s, where association of the complex occurs. Then the dissociation of the complex in the buffer solution was detected for 100 s.

Baseline-subtracted binding curves were analyzed using Octet Data Analysis (version 9.0) according to standard procedures using a 1:1 interaction model (see Figures 8-11).

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

Candidates BCD132L-028 and BCD132L-077 are selected based on the results of the above analysis.

Example 10. Obtaining a cell line that stably produces antibodies in the IgG1 format.

Based on the results of all the above tests, candidates BCD132-L028 and BCD132-L-077 performed best.

The sequences of their heavy and light chains were cloned into the pSX vector at the HindIII, XbaI sites (Fig. 24). The resulting plasmids were produced in E. soy cells and isolated using a BenchPro device in an amount of 600-700 μg. Plasmids were linearized overnight using Pvul endonuclease, then reprecipitated with ethanol and adjusted to a final concentration of 900–1100 ng/μl.

The CHO-K1-S cell line was cultured in S.3.87 MM medium (synthetic medium developed at BIOCAD without FBS) + 6 mM Glutamine. Transfection with genetic constructs containing the coding sequences of candidate chains BCD132-L-028, BCD132-L-077 was carried out using electroporation on a Nucleofector™ device (Lonza) according to the manufacturer's protocol.

The next day after transfection, selection of the transfected culture was carried out for 24 days by adding puromycin (final concentration 7.2 μg/ml) and hygromycin B (final concentration 640 μg/ml) to the medium. The cell population obtained after selection was cloned. Cell clones expressing BCD132-L-028, BCD132-L-077, respectively, were selected based on the results of the analysis of the level of the target protein and the homogeneity of its structure, taking into account the growth rate, homogeneity of the population and the absence of morphological changes, while candidates BCD132L-026 and BCD-132-L-075 were eliminated.

Example 11. Determination of the affinity of the final candidates developed in stable lines to CD20 on Forte Bio Octert RED 384.

SAX biosensors and CD20 peptide modified with biotin (Sigma Aldrich) were used for this work. Rituximab antibody was used as a control. SAX biosensors were immersed in a solution with biotinylated CD20 peptide at a concentration of 20 μg/ml, where the peptide was immobilized. Further analysis was carried out at 30°C using PBS containing 0.1% Tween-20 and 0.1% BSA as a running buffer.

After a baseline was prescribed in the buffer solution, the sensors were immersed in wells with an antibody solution at a concentration of 10 μg/ml for 150 s, where association of the complex occurs. Then the dissociation of the complex in the buffer solution was detected for 300 s.

Baseline-subtracted binding curves were analyzed using Octet Data Analysis (version 9.0) according to standard procedures using a 1:1 interaction model (FIGS. 12-15).

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

Example 12. Determination of the affinity of the final candidates developed in stable lines to FcyRIIIa-158F on Forte Bio Octert RED 384.

SAX biosensors and FcyRIIIa-158F proteins modified with biotin (Sigma Aldrich) were used for this work. SAX biosensors were immersed in a solution with biotinylated protein at a concentration of 5 μg/ml, where the protein was immobilized to a signal level of 0.5 nm. Further analysis was carried out at 30°C using PBS containing 0.1% Tween-20 and 0.1% BSA as a working buffer.

After a baseline was prescribed in the buffer solution, the sensors were immersed in wells with antibody solutions at different concentrations for 90 s, where association of the complex occurs. Then the dissociation of the complex in the buffer solution was detected for 150 s.

Baseline-subtracted binding curves were analyzed using Octet Data Analysis (version 9.0) according to standard procedures using a 1:1 interaction model (FIGS. 16-19). ______________________________________________________________________________________________

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

Example 13. Determination of the affinity of the final candidates developed in stable lines to FcYRIIIa-158V on Forte Bio Octert RED 384.

SAX biosensors and FcYRIIIa-158V proteins modified with biotin (Sigma Aldrich) were used for this work. SAX biosensors were immersed in a solution with biotinylated protein at a concentration of 5 μg/ml, where the protein was immobilized to a signal level of 0.5 nm. Further analysis was carried out at 30°C using PBS containing 0.1% Tween-20 and 0.1% BSA as a running buffer.

After a baseline was prescribed in the buffer solution, the sensors were immersed in wells with antibody solutions at different concentrations for 90 s, where association of the complex occurs. Then the dissociation of the complex in the buffer solution was detected for 150 s.

Baseline-subtracted binding curves were analyzed using Octet Data Analysis (version 9.0) according to standard procedures using a 1:1 interaction model (FIGS. 20-23). ______________________________________________________________________________________________

BCD132L028 1.78E-08 6.385E05 1.139E-02 0.9963 BCD132L077 (1.2) 2.38E-08 6.563E05 1.564E-02 0.9942 BCD132L077 (3.4) 1.66E-08 6.90 6E05 1.149E-02 0.9961 BCD132L077 (5, 6) 2.27E-08 7.311E05 1.659E-02 0.9952

Example 14. Evaluation of the specific binding of BCD-132-L-028 and BCD-132-L-077 to the CD20 receptor on the WIL2-S cell line by flow cytometry.

MabThera (Rituximab) was used as a control antibody. The analyzed samples and the control antibody were diluted to a concentration of 200 μg/ml and titrated in steps of 4 in Stain Buffer (PBS, 0.5% BSA, 0.1% _NaN3 ). A cell suspension of WIL2-S (ATCC® CRL8885) with a concentration of 1x10 ⁶ cells/ml was incubated with a titer of solutions of the standard and test samples. Mixed and incubated for 30 min on ice. After the incubation time, the plate was centrifuged, the supernatant was collected, 100 μl of Stain Buffer was added, resuspended and centrifuged. The supernatant was collected, and the pellet was resuspended in a solution of conjugated fluorescent anti-human Fc-PE antibodies (Jackson Immunoresearch, 109-115-098) in Stain Buffer. The plate was incubated for 30 min on ice in the dark. After the incubation time, the plate was centrifuged, the supernatant was collected, 100 μl of Stain Buffer was added, resuspended and centrifuged. The supernatant was collected, and the pellet was resuspended in 150 μl of Stain Buffer and analyzed using a Guava12HT flow cytometer (Merck Millipore). Data analysis was performed using the InCyte module of guavaSoft 3.1.1 software.

The level of specific binding to the CD20 receptor on the WIL2-S cell line of the studied antibodies BCD-132-L-028 and BCD-132-L-077 is similar to the level of specific binding of MabThera (rituximab). The results are shown in Fig. 25.

Example 15. Evaluation of complement-dependent cytotoxicity of BCD-132-L-028 and BCD-132-L-077.

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

The analysis was carried out in RPMI-1640 medium, 2 mM glutamine, 0.1% bovine serum albumin, 50 μg/ml gentamicin. A series of dilutions of the analyzed antibodies BCD-132-L-028, BCD-132L-077 and MabThera (Rituximab) was prepared from a concentration of 50 μg/ml. The prepared solutions were added to 96-well culture plates at a dose of 50 μl per well. A WIL2-S cell suspension of 1 x 10 ⁶ cells/ml was prepared and 50 µl was added per well of the plate. Complement working solution was prepared

- 40 044757 (Quidel, A113), added to culture plates at the rate of 50 µl/well.

The plates were incubated for 2 hours at 37°C, 5% CO ₂ . At the end of the incubation time, 15 μl of Alamar blue dye was added to the wells of the plate, and the plate was incubated at 37°C, 5% CO ₂ until a gradient color developed. The fluorescence level was assessed using a plate detector

Infinite M200Pro at excitation/emission wavelength 544/590 nm.

BCD-132-L-028 and BCD-132-L-077 show similar levels of complement-dependent cytotoxicity compared to MabThera (rituximab). The results are shown in Fig. 26, 27.

Example 16. Evaluation of antibody-dependent cellular cytotoxicity of BCD-132-L-028 and BCD-132-L077 using Jurkat-NFAT-CD16 reporter lines.

To evaluate antibody-dependent cellular cytotoxicity, WIL-2S (ATCC® CRL-8885™) was used as a target line. The reporter lines Jurkat-NFAT-CD16 High (CD16 allotype with high affinity, V158) and Jurkat-NFAT-CD16 Low (CD16 allotype with low affinity, F158), stably expressing the FcyRIIIa (CD16a) receptor and carrying the gene encoding luciferase under the control of NFAT-responsive elements.

A WIL-2S cell suspension of 0.5 x 10 ⁶ cells/ml was prepared in RPMI1640 with 2mM L-Gln, 4% (v/v) FBS with low IgG content and 5 μg/ml gentamicin. 25 μl/well of the target cell suspension was added to culture plates with white walls.

A titer of BCD-132-L-028 or BCD-132-L-077 and MabThera (rituximab) was added from 5 μg/ml in increments of 5 (25 μl/well), and a suspension of 3x10 ⁶ cells/ml of the reporter line Jurkat-NFAT- CD16 High or Low (25 µl/well). The plates were mixed and incubated at 37°C, 5% CO ₂ for 4-8 hours.

At the end of the incubation time, 75 μl/well of Bio-Glo luciferase activity detection reagent (Promega) was added and luminescence was measured using Infinite M200Pro at an integration time of 100 ms.

BCD-132-L-028 and BCD-132-L-077 are significantly superior in ADCC activity to MabThera: activity is 3-4 times higher when using a reporter line with a high affinity CD16 allotype, and 12-16 times higher when using a reporter line lines with a low affinity CD16 allotype. The results are shown in Fig. 28, 29 for a reporter line with a low affinity CD16 allotype and FIG. 30 , 31 for a reporter line with a high affinity CD16 allotype.

Example 17: Evaluation of CD19+ B cell depletion caused by BCD-132-L-028 and BCD-132-L-077 in whole blood from healthy donors.

The activity of the studied samples was assessed ex vivo using whole blood of healthy donors with allotypes of the CD16a receptor: FF (low-affinity receptor), FV (heterozygote), VV (high-affinity receptor).

MabThera (Rituximab) was used as a control antibody. Control and test antibodies were titrated in triplicates in a 96-well plate. The blood of a healthy donor was collected into vacuum tubes with Li-heparin. Incubated for 30 min at room temperature. 10 μl of prepared antibody solutions and 190 μl of whole blood were added to a 96-well plate with a marginal groove (Eppendorf). 5 ml of DPBS was added to the edge groove of a 96-well plate. The plate was mixed on an orbital shaker (2 mm) for 2 minutes at 600 rpm. Incubated in a CO2 incubator for 22 hours.

After the incubation time, the samples were stained with fluorescently labeled antibodies against CD45, CD3/CD19 (BD Pharmingen) for 1 hour. The cells were fixed and erythrocytes were lysed using BD Pharmingen lysis buffer. The lysis buffer was washed twice in Stain Buffer (DPBS, 0.1% NaN3, 0.5% BSA) and the number of CD45+CD3+ and CD45+CD19+ events was assessed (at least 10,000 events in the CD45+ gate). Data analysis was performed using the InCyte module of guavaSoft 3.1.1 software.

Relative B-cell depletion was assessed using the B-/T-cell ratio point without antibodies (the number of B-cells is taken as 100% = 0% B-cell depletion). B-/T-ce11 ratio was calculated using the formula _π η—, „ number of B cells

Β-ΛΓ-cell ratio =---------------- number of T cells

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

V-cell depletion, % = 100 - ( ----------------- * V/T —

B/T—cell ratio witnont antibody cell ratio with antibody)

Four-parameter curves were constructed using the R statistical data processing environment using the drc package.

The studied antibodies BCD-132-L-028 and BCD-132-L-077 are significantly more active than MabThera (rituximab). Thus, when using blood from donors with the FF allotype, BCD-132-L-028 and BCD132-L-077 cause depletion of about 50% of CD19+ cells, while MabThera (rituximab) - about 41 044757 about 20%. The ED50 values for MabThera (rituximab) are an order of magnitude higher than those for

BCD-132-L-028 and BCD-132-L-077 when using blood from donors with the CD16 FV and W allotype. Level

B-cell depletion of BCD-132-L-028 and BCD-132-L-077 is independent of the donor CD16 allotype. The results are shown in Fig. 32.

Example 18 Antibody-dependent cellular cytotoxicity (ADCC) activity assay of candidate anti-CD20 antibodies on the Ramos cell line using human peripheral blood mononuclear cells (PBMCs).

For ADCC analysis, the CD20-expressing Ramos cell line and PBMCs from healthy donors were used. Ramos cells were cultured in RPMI-1640 medium with 10% FBS (fetal bovine serum) at 37°C 5% CO ₂ and stained with the fluorescent dye Calcein AM, which only comes from cells with damaged cell walls. A cell suspension with a density of 10 ⁵ cells/ml was prepared in RPMI-1640 medium with 10% FBS.

PBMC were isolated from venous blood of healthy donors using Ficoll density gradient separation (1.077 g/cm ³ ). A cell suspension with a density of 5*10 ⁶ cells/ml was prepared in RPMI-1640 medium with 10% FBS.

For ADCC analysis, a series of 50 μl antibody dilutions were added to the wells of a 96-well plate. To these were added 100 μl of Ramos suspension and 50 μl of PBMC suspension. The plate was incubated for 4 hours at 37°C 5% CO ₂ . Half an hour before the end of incubation, 10 μl of 10% Tryton X-100 was added to the wells that determined maximum cell lysis. After the end of incubation, 100 μl of cell fluid was transferred without capturing the cells into a new plate. The fluorescence intensity of the samples was determined at an excitation/emission wavelength of 485/538 nm.

To calculate the efficiency of ADCC, we used the formula l Experimental data - background llp/

AULC =-------------------X

Full lysis - background

Based on the dependence of ADCC on antibody concentration, the dependence described by a 4-parameter equation was determined using the GraphPad Prism 6.0 package and the half-maximal effective concentration (EC50) was calculated.

According to the ADCC activity experiment, the anti-CD20 antibody candidates BCD132-L-028 and BCD-132-L-077 are superior to the commercial antibody Rituximab. The results are shown in Fig. 33.

Example 19. Study of the activity of the monoclonal antibody drug BCD132-L-077 upon repeated intravenous administration to cynomolgus monkeys (Macaca fascicularis) in a model of experimental autoimmune encephalomyelitis (EAE).

The study was conducted on male cynomolgus macaques (Macaca fascicularis). The total number of animals in the experiment was 12 animals, each group of animals included 4 monkeys. Two doses of the drug were used in the experiment: 5 mg/kg; 22 mg/kg, animals in the control group received a placebo drug. Data on experimental groups of animals are presented in table. 2.

Table 2. Distribution of animals by groups

Group number Number of animals A drug Method of administration Dose 1 4(^) BCD132-L-077 IV 5 mg/kg 2 4 (^) 22 mg/kg 3 4 I) Placebo -

To induce experimental autoimmune encephalomyelitis in M. fascicularis, a modified technique described in a number of publications was used. To sensitize primates, a recombinant protein produced by JSC BIOCAD was used, which is the extracellular domain of the human oligodendrocyte myelin protein (rhMOG, amino acids 1-125).

Each animal was injected three times with an emulsion containing 400 μg of protein in 400 μl of phosphate buffer solution mixed with 400 μl of Freund's complete adjuvant. Immediately after the first administration of rhMOG, monkeys were injected with a heat-inactivated B. pertussis vaccine.

First administration of rhMOG.

To prepare the emulsion, 10 mg of rhMOG was dissolved in 10 ml of phosphate buffer solution. 10 ml of Freund's complete adjuvant was added to the resulting solution. The resulting emulsion was administered by intradermal injection into 8 points of 100 μl (the total injection volume for each monkey will be 800 μl):

in the back area (between the shoulder blades, 2 on the right and 2 on the left of the spine);

in the groin area (1 on the right and 1 on the left);

in the axillary areas (1 on the right and 1 on the left).

After the first administration of rhMOG, 10 ¹⁰ inactivated B. pertussis particles were administered intravenously.

The interval between the first and second immunization was 28 days.

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Second administration of rhMOG.

To prepare the emulsion, 10 mg of rhMOG was dissolved in 10 ml of phosphate buffer solution. 10 ml of Freund's complete adjuvant was added to the resulting solution. The resulting emulsion was administered by intradermal injections into 8 points of 100 μl (the total injection volume for each monkey will be

800 µl):

in the back area (between the shoulder blades, 2 on the right and 2 on the left of the spine);

in the groin area (1 on the right and 1 on the left);

in the axillary areas (1 on the right and 1 on the left).

The interval between the second and third immunization was 14 days.

Third administration of rhMOG.

To prepare the emulsion, 10 mg of rhMOG was dissolved in 10 ml of phosphate buffer solution. 10 ml of Freund's complete adjuvant was added to the resulting solution. The resulting emulsion was administered by intradermal injection into 8 points of 100 μl (the total injection volume for each monkey will be 800 μl):

in the back area (between the shoulder blades, 2 on the right and 2 on the left of the spine);

in the groin area (1 on the right and 1 on the left);

in the axillary areas (1 on the right and 1 on the left).

Evaluation of the effectiveness of the drug BCD132-L-077 in the model of EAE (experimental allergic encephalomyelitis).

To assess the activity of the drug, a histological examination of the tissues of the brain and spinal cord was carried out. The severity of the inflammatory reaction in the tissues of the spinal cord and brain was assessed on a three-point scale according to Table. 3.

Table 3. Scale for assessing the severity of the inflammatory response

Points Severity of inflammation 0 no signs of inflammation; 1 rare (1-3 per section) foci of perivascular infiltration; 2 moderate frequency (4-10 per section) of foci of perivascular infiltration; possibly inflammation of the meninges; 3 widespread foci of perivascular infiltration and infiltration of nervous tissue with inflammatory cells.

To assess the severity of degenerative changes in the tissues of the spinal cord and brain of primates, we used the scale presented in Table. 4.

Table 4. Scale for assessing the severity of demyelination in the tissues of the spinal cord and brain

Points Severity of demyelination 0 no signs of demyelination; 1 rare (1-3 per section) foci of demyelination; 2 moderate frequency (4-10 per section) of demyelination foci; 3 extensive demyelination with large confluent lesions.

The results of assessing the severity of inflammation are presented in Fig. 34. It was shown that when the drug doses of 5.0 and 22.0 mg/kg used in the study were administered, there was a decrease in the overall score of the group in comparison with the value of the control group (control-placebo). The identified changes were not reliable. There was also no significant difference between the experimental groups.

Thus, we can talk about a comparable level of anti-inflammatory effect for the two tested doses of the drug.

The results of assessing the severity of degenerative changes in the nervous tissue are shown in Fig. 35. When using the study drug BCD132-L-077 at a minimum dose of 5.0 mg/kg, a significant decrease in the score characterizing demyelination was observed, compared to the control.

In the group of animals receiving the drug at a dose of 22.0 mg/kg, a decrease in the value of the assessed parameter was also shown, but it was not significant. There were no significant differences in values between the experimental groups, which allows us to determine the intensity of activity for the two doses as comparable.

The study showed that the drug in doses of 5.0 and 22.0 mg/kg has a comparable anti-inflammatory effect and to a similar extent reduces the level of demyelination of the nervous tissue of experimental primates. Based on these data, a dose of 5.0 mg/kg can be determined as the pharmacologically active dose (PDD).

Example 20. Study of the toxicity and main pharmacokinetic parameters (toxicokinetics) of the drug BCD132-L-077 after a single intravenous administration to cynomolgus monkeys (Macaca fascicularis).

The study was conducted on male cynomolgus macaques (Macaca fascicularis). After passing quarantine, the animals were divided into four experimental groups, three males in each

- 43 044757 group, in accordance with the doses of the administered drug; Body weight was used as a criterion for the distribution of monkeys into groups. Three doses of the drug were used in the experiment: 44 mg/kg; 88 mg/kg; 176 mg/kg, animals in the control group received a placebo drug. The evaluation criteria were the condition of the animals, the number of dead animals and the timing of their death. The animals were observed for 8 hours after injection, and then daily for 42 days.

Data on experimental groups of animals and doses of the study drug are presented in Table 5.

Table 5. Distribution of animals into groups in the experiment to determine the effectiveness of the drug BCD132-L-077

Group number Number of animals A drug Method of administration Dose 1 h R) BCD132-L-077 IV 44 mg/kg 2 3 R) 88 mg/kg 3 3 R) 176 mg/kg 4 3 R) Placebo -

The evaluation criteria were the condition of the animals, the number of dead animals and the timing of their death.

As part of the study, a clinical examination was carried out for 8 hours after injection, and then daily, in addition to assessing:

animal weight;

body temperature;

general urine analysis;

general blood test according to indicators: red blood cell count, white blood cell count, hemoglobin concentration;

biochemical analysis of blood serum for the following indicators: lactate dehydrogenase, total bilirubin, total protein, glucose, aspartate aminotransferase, alanine aminotransferase;

concentration of the drug in the blood serum.

The research results showed that the drug under study did not cause death of M. fascicularis when administered once intravenously, and the administration process was well tolerated by experimental animals. There was no effect of the drug on integral toxicity indicators, as well as on the functional state of organs according to the parameters assessed. In the selected dose range, the drug has linear pharmacokinetics.

Example 21 Pharmacokinetics and immunogenicity study of repeated intravenous administration of BCD132-L-077 to cynomolgus monkeys (Macaca fascicularis) for four weeks followed by a two-week dosage-free period.

Pharmacokinetic parameters and immunogenicity of the drug with repeated intravenous administration were studied in doses of 22.0; 44.0 and 88.0 mg/kg. The total number of animals was 18 mature monkeys (Macaca fascicularis), aged 4-7 years, 9 females and 9 males. The animals were divided into 3 groups according to the dose of the administered drug.

Table 6. Distribution of experimental animals by groups

Group number Number of animals A drug Method of administration Dose 1 3 R) 3 (?) BCD132-L-077 IV 22 mg/kg 2 h R) 3 (5) 44 mg/kg 3 3 3 (?) 88 mg/kg

The level of BCD132-L-077 in primate serum was assessed using an enzyme-linked immunosorbent assay. During the study, in order to establish the possible influence of the formation of drug-binding antibodies on pharmacokinetic parameters, the immunogenicity of the drug BCD132-L-077 was assessed. The value of the accumulation coefficient was also calculated as the ratio of AUC _ss168 (336-504) to AUC ₀ - ₁₆₈ .

To calculate the AUC value ₀ . ₁₆₈ collected serum immediately before the first administration of the drug, and then 0.25, 24, 72 and 168 hours after administration; To calculate _the AUC value of _ss168 ( _336.504 ), serum was collected immediately before the fourth administration of the drug, and then 0.25, 24, 72 and 168 hours after it. When calculating pharmacokinetic parameters, only data from those animals in which CAT was not detected were used. That is data from animals that had an immune reaction to the drug were excluded from the calculation of pharmacokinetic parameters. The cumulation of the drug was assessed using the cumulation index, for which AUC values of _0-168 and

AUC _ss168 ( _336-504 ) and calculated the value using the formula (336-504) ^^^0—168 where AUC _ss168 ( _336-504 ) is the equilibrium value of the area under the drug concentration curve for the period corresponding to the dosing interval (168 hours) at repeated administration;

AUC _0-168 is the area under the concentration curve of the drug from the moment it enters the body until 168 hours upon first administration.

Primate blood serum was used to analyze the level of antibodies binding to the drug BCD13-2L-077. To conduct the study, samples were taken before the first administration, and then at 4 and 7 weeks of the experiment.

Example 22. Comparison of pharmacokinetic parameters with repeated intravenous administration of increasing doses (22.0, 44.0, 88.0 mg/kg) of the drug BCD132-L-077.

In fig. Figure 36 shows average curves of changes in the concentration of the drug BCD132-L-077 over time in the blood serum of primates. In table Table 7 shows the group average values of the main pharmacokinetic parameters.

Table 7. Comparative data of the main pharmacokinetic parameters when administering increasing doses of the drug BCD132-L-077 (To calculate steady-state (ss) values, a period of 336-504 hours was used, corresponding to the dosing interval (168 hours)

FC parameter UNITS measured Dose (mg/kg) 22 µg/ml 44 µg/ml 80 µg/ml Dick <7 Dick <7 Dick <7 AUCss (0-168) meg/mlh: 13435.59 11150.8 13301.04 8719.1 34145.90 16765.5 AUC S3 _{ 33ΰ-504) mcg/ml"h 26227.47 16819.4 22366.93 10370.6 9708S.33 94675.6 Css [min) µg/ml 82.64 75_2 84.5S 63_9 162.SS 74_7 Szv (check) µg/ml 326.S9 241.7 427.03 266_5 1212.72 830.9 Szz µg/ml 156.12 1001 133.14 617 577.91 563.5 T1/2 h 94.76 50_3 155.3S 111-2 471.00 782_5 C1 l/h 0.00924 0.004 0.01S73 0.015 0.01228 0.005 CLss ml/h 4.550 2.37 10.323 6.33 7.352 5.70 Vss MP 521.65 186.9 1639.68 1170-5 2384.08 2213.3 MRTiaat h 66.37 136 68.30 175 71.51 9.2

Pharmacokinetic parameters of BCD132-L-077 were calculated using steady-state AUC values during the dosing period 336-504 hours (168 hours) after drug administration. The initial AUC calculated in the first dosing interval (1-168 hours) was directly related to the dose used. In the minimum dose group, this parameter was 13435.59±11150.80 (mcg/ml)h, in the average dose group 13301.04±8719.10 (mcg/ml)h and 34145.90±16765.50 (mcg/ml) )h in the maximum dose group. AUC calculated at steady state (336-504 h) also depended on the dose used. In the group of animals receiving the drug at a dose of 22 mg/kg, AUCss168(336-504) was 26227.47±16819.40 (µg/ml)h, in the group of the average dose (44 mg/kg) the figure was 22366.93± 10370.60 (µg/ml)h and 97088.33±94675.60 (µg/ml)h in the maximum dose group. The half-life (T1/2) in the group of animals receiving the drug at a dose of 22 mg/kg was 94.76±50.30 hours, in the average dose group 155.38±111.20 hours and 471.00±782. 50 hours in the maximum dose group. Clearance in the minimum dose group was 0.00924±0.004 l/h, in the medium dose group 0.01873±0.015 l/h and 0.01228±0.005 l/h in the maximum dose group. The mean retention time of the drug in the body (MRT) in the minimum dose group was 66.37 ± 13.60 hours, in the average dose group 68.30 ± 17.50 hours and 71.51 ± 9.20 hours. Volume of distribution calculated in steady state (V _dss ) was 521.65±186.90 ml/kg, 1639.68±1170.50 ml/kg, 2384.08±2213.30 ml/kg in the minimum, average and maximum dose groups, respectively. The average concentration calculated in the steady state (C _ss ) was 156.12±100.10 µg/ml, 133.14±61.70 µg/ml, 577.91±563.50 µg/ml in the minimal and moderate groups and maximum doses, respectively. The maximum concentration calculated at steady state (C _ssmax ) was 326.89±241.70 µg/ml, 427.03±266.50 µg/ml, 1212.72±830.90 µg/ml. The minimum concentration calculated at steady state ( _Cssmin ) was 82.64±75.20 µg/ml, 84.58±63.90 µg/ml,

- 45 044757

162.88±74.70 µg/ml. The data obtained indicate that the concentration of the drug in the blood serum of primates is directly dependent on the dose of BCD132L-077 used in the experiment.

For the group of animals that received the drug at a minimum dose of 22.0 mg/kg, the value of the accumulation index (R) was 1.95. For the group of animals receiving BCD-132 at an average dose of 44.0 mg/kg, the accumulation index R was equal to 1.68. For the group of animals that received the drug at the maximum dose (88.0 mg/kg), the R index value was 2.84. The experimental data obtained indicate that the value of the cumulation index does not depend on the dose of the drug used.

During the study, in order to establish the possible influence of the formation of drug-binding antibodies on pharmacokinetic parameters, the immunogenicity of the drug BCD132-L-077 was assessed. Experimental data indicate the presence of CAT in 11.11% of all animals in the study. No sex dependence was found; CATs were recorded in one male and one female. Drug-binding antibodies were detected in the medium and maximum dose groups, in only one animal in both groups. Assessment of pharmacokinetic parameters is possible in all groups of animals, provided that experimental data for animals with the established presence of binding antibodies are excluded from processing.

Example 23. Study of the toxicity and local irritant effect of repeated intravenous administration of the monoclonal antibody preparation BCD132-L-077 to cynomolgus monkeys (Macaca fascicularis) for four weeks followed by a two-week period free from administration.

The total number of animals in the experiment was 30 heads of Java poppies (Masaca fascicularis), aged 4-7 years, 15 females and 15 males. Each group of animals included 6 monkeys. The toxicity of the drug after repeated administration was studied at doses of 22, 44 and 88 mg/kg. The animals were divided into 5 groups according to the dose of the administered drug and the period of euthanasia:

drug BCD132-L-077 at a minimum dose of 22 mg/kg (3 females and 3 males);

drug BCD132-L-077 at an average dose of 44 mg/kg (3 females and 3 males);

drug BCD132-L-077 at a maximum dose of 88 mg/kg, euthanasia after completion of drug administration (3 females and 3 males);

drug BCD132-L-077 at a maximum dose of 88 mg/kg, euthanasia after the end of the recovery period (3 females and 3 males);

placebo control (3 females and 3 males).

Data on experimental groups of animals are presented in table. 8.

Table 8. Distribution of experimental animals by groups

Number groups Number of animals A drug Method of administration Dova 1 Z(^) BCD132-L-077 IV 22 mt/kg 3 (P 2 3 44 mt/kg 3 {*) 3 з {<0 38 mt/kg з m 3* 3(J) 38mg/kg z {*) 4 3 (<Ό Placebo - 3 m

3* - satellite group, 3 - main group

As part of the study, clinical examination was carried out daily, and the weight of the animals was also assessed;

body temperature;

general urine analysis;

ECG;

hemostasis indicators: activated partial thromboplastin time, fibrinogen concentration, prothrombin time;

general blood test according to indicators: number of red blood cells, number of leukocytes, hemoglobin concentration, lymphocytes, monocytes, neutrophils, eosinophils, basophils;

- 46 044757 biochemical analysis of blood serum for the following indicators: lactate dehydrogenase, total bilirubin, total protein, glucose, aspartate aminotransferase, alanine aminotransferase, cholesterol, triglycerides, urea, kratenin, sodium, potassium, alkaline phosphatase;

pathomorphological and histological studies.

The local irritant effect was assessed based on examination data, as well as the results of histological examination. Tissues at the injection site and draining lymph nodes were collected for histological examination.

According to histological studies, the drug under study does not have a local irritating effect.

Data obtained in a toxicity study of the therapeutic monoclonal antibody drug BCD132-L-077 produced by BIOCAD JSC indicate that the drug under study does not have a toxic effect on the main organs and organ systems of experimental animals when administered multiple times weekly intravenously for 4 weeks. The dose without observed adverse effect (NOE) established in this study corresponds to the maximum dose of the study drug BCD132-L-077 and is 88 mg/kg.

Example 24. Study of the antitumor activity of the drug BCD132-L-028 upon repeated intraperitoneal administration of humanized human NK cells to immunodeficient mice of the hIL15-NOG line on a model of subcutaneous xenografts using the human lymphoma cell line Raji.

The study plans to use 7 groups of immunodeficient transgenic mice of the hIL15-NOG line, constitutively expressing human IL-15, weighing 15.0-25.0 g. The total number of animals participating in the experiment is 84 females. Data by groups are presented in table. 9.

Table 9. Distribution of experimental animals by groups

Number □troupes Total diI Ι-Ιι o ^C PNT|TX b group A drug Cell line quantities □ inoculi- roved MK cells Way One day I Dova, mg/kg 1 12 (?) BCD132-Ii—028 Raji IV - 6*10 ^£ cl w/b 10 mg/kg 2 12 {*) BCD132-L—028 thirty mt/kg 3 12 (*) BCD132-L-028 90 mg/kg 4 12 (?) Obinutuvumab thirty mg/kg 5 12 (?) Acellbil thirty mg/kg e 12 Placebo substance - - 7 12 Placebo substance -

Animals are weighed before the introduction of the tumor line, and then 2 times a week throughout the experiment.

The tumor node is measured after tumor injection 2 times a week throughout the experiment.

The volume of the tumor node is determined by the formula

V = n/6xLxWxH, where L, W, H are the linear dimensions of the tumor.

The criterion for the effectiveness of the study drug is the tumor growth inhibition index (TGI) and the tumor growth index (I), which are calculated using the formulas

TPO (%) = AND X 100, where V _k and Vo are the average tumor volume (mm ³ ) in the control and experimental groups, respectively.

k = where I is the tumor growth index, i is the day of the experiment, V _o is the tumor volume per day.

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

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The study plans to use four experimental groups of cynomolgus macaques, with three males in each group.

Monkeys will be kept in individual cages with the number of the animal in the experiment indicated.

Animals are randomly assigned to groups according to the doses of the substance administered, using body weight as a criterion.

Table 10. Distribution of experimental animals by groups

The drugs are planned to be administered intravenously into the cubital vein in the form of solutions in a sterile isotonic solution. Animals in the control group will be administered (placebo) isotonic solution using the same volumes and method as in the experimental groups.

As part of the study, a clinical examination is carried out daily, in addition, the following indicators are assessed:

1. weight of animals;

2. body temperature;

3. general urine test;

4. general blood test according to indicators: number of red blood cells, number of leukocytes, hemoglobin concentration;

5. biochemical analysis of blood serum according to indicators: lactate dehydrogenase, total bilirubin, total protein, glucose, aspartate aminotransferase, alanine aminotransferase;

6. study of the concentration of the drug in blood serum, calculation of the main pharmacokinetic parameters and assessment of the linearity of pharmacokinetics.

Example 26. Pharmacokinetics and immunogenicity study of repeated intravenous administration of the monoclonal antibody preparation BCD132-L-028 to cynomolgus monkeys (Macaca fascicularis) for 13 weeks followed by a 30-day dosage-free period.

Eighteen cynomolgus monkeys (9 males and 9 females) are planned to be used in a pharmacokinetics and immunogenicity study of repeated intravenous dosing for 13 weeks followed by a 30-day dosing-free period.

Animals will be divided into 3 groups (Table 11) in accordance with the doses of the administered substance, 3 females and 3 males in each: minimum dose group (6 mg/kg), intermediate dose group (20 mg/kg), maximum dose group (60 mg/kg).

Table 11. Distribution of experimental animals by groups

Group number Total animals in the group A drug Method of administration Dose, mg/kg 1 3 R) BCD132-L-028 IV 6 3 (?) 2 3 R) 20 3 (?) 3 3 R) 60 3 (?)

During the research it is planned:

1. Assess the concentration of the test drug in the blood serum of experimental animals.

2. Assess the accumulation of the study drug upon administration of increasing doses.

3. Assess the level of binding antibodies during repeated intravenous administration of the study drug.

Example 27. Toxicity and local irritation study of repeated intravenous administration of the monoclonal antibody preparation BCD132-L-028 to cynomolgus monkeys (Macaca fascicularis) for 13 weeks followed by a 30-day dosage-free period.

The study plans to use 5 experimental groups of cynomolgus macaques of 3 males and 3 females each: a minimum dose group (6 mg/kg, no necropsy), an intermediate dose group (20 mg/kg, no necropsy), a satellite maximum dose group (60 mg/kg, necropsy after completion of pe-

Claims (26)

period of drug administration), the main group of the maximum dose (60 mg/kg, necropsy after the end of the recovery period) and the placebo control group (necropsy after the end of the recovery period). Table 12. Distribution of experimental animals by groups (* - animals of the satellite group) Group number Number of animals Drug Method of administration Dose, mg/kg 1) a heavy chain that has the amino acid sequence presented in SEQ ID NO: 5; And 1) a heavy chain that has the amino acid sequence presented in SEQ ID NO: 1; And 1) a heavy chain that has the amino acid sequence presented in SEQ ID NO: 1 or SEQ ID NO: 5; And 1) the heavy chain variable domain has the amino acid sequence set forth in SEQ ID NO: 6; And 1) the heavy chain variable domain has the amino acid sequence set forth in SEQ ID NO: 2; And 1) a heavy chain variable domain, which has an amino acid sequence selected from the group: SEQ ID NO: 2, SEQ ID NO: 6; And 1. A monoclonal antibody or antigen-binding fragment thereof that specifically binds to CD20, including: 1 3 R) BCD132-L-028 IV 6 2) a light chain that has the amino acid sequence set forth in SEQ ID NO: 7. 2) a light chain that has the amino acid sequence set forth in SEQ ID NO: 3. 2) a light chain that has the amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 7. 2) the light chain variable domain has the amino acid sequence set forth in SEQ ID NO: 8. 2) the light chain variable domain has the amino acid sequence set forth in SEQ ID NO: 4. 2. Monoclonal antibody or its antigen-binding fragment according to claim 1, where: 2) a light chain variable domain, which has an amino acid sequence selected from the group: SEQ ID NO: 4, SEQ ID NO: 8. 2 3 R) 20 3. Monoclonal antibody or its antigen-binding fragment according to claim 1, where: 3 (?) A clinical examination of each animal will be carried out daily, in addition, the following will be assessed: the weight of the animals; body temperature (before administration, then weekly until the end of the experiment); influence on the cardiovascular system using the Poly-Spectrum cardiograph; general urine analysis; complete blood count according to the following indicators: number of erythrocytes, number of leukocytes, hemoglobin concentration, number of lymphocytes, number of monocytes, number of neutrophils, number of eosinophils, number of basophils; assessment of the effect on the blood coagulation system according to the following indicators: activated partial thromboplastin time, fibrinogen concentration, prothrombin time; biochemical analysis of blood serum for the following indicators: sodium, potassium, creatinine, urea, alkaline phosphatase, lactate dehydrogenase, total bilirubin, total protein, glucose, triglycerides, aspartate aminotransferase, alanine aminotransferase, total cholesterol. At the end of the drug administration period (week 14), it is planned to euthanize three males and three females from each group of animals. The remaining two males and two females in each group will be euthanized at the end of the recovery period (week 18). The local irritant effect will be assessed on the basis of necropsy data, as well as the results of histological examination of apnea. A section of the ulnar vein, tissue at the injection site and draining lymph nodes will be selected for histological examination. CLAIM 3 (?) 3 R) 3 (?) 3 3 R) 60 3 (?) 3 (?) 4. Monoclonal antibody according to claim 1, including: 4 3 R) Placebo - 5. Monoclonal antibody according to claim 4, including: - 49 044757 6. Monoclonal antibody according to claim 4, including: 7. The monoclonal antibody of claim 1, wherein the antibody that specifically binds to CD20 is a full-length IgG antibody. 8. Monoclonal antibody according to claim 7, where the full-length IgG antibody belongs to the human IgG1, IgG2, IgG3, IgG4 isotype. 9. The monoclonal antibody according to claim 8, wherein the full-length IgG antibody is of the human IgG1 isotype. 10. A nucleic acid that encodes an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 9. 11. Nucleic acid according to claim 10, wherein the nucleic acid is DNA. 12. An expression vector containing a nucleic acid according to any one of claims 10, 11. 13. A method for obtaining a host cell for producing an antibody or an antigen-binding fragment thereof according to any one of claims. 1 - 9, including transforming the cell with the vector according to claim 12. 14. A host cell for producing an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 9, containing a nucleic acid according to any one of claims. 10, 11. 15. A method for producing an antibody or an antigen-binding fragment thereof according to any one of claims 1-9, consisting in cultivating the host cell according to claim 14 in a culture medium under conditions sufficient to obtain said antibody or an antigen-binding fragment thereof, followed by isolation and purification of the resulting antibody or antigen-binding fragment thereof. 16. A pharmaceutical composition for the prevention or treatment of a disease or disorder mediated by CD20, comprising an antibody or antigen-binding fragment thereof according to any one of claims 19, in combination with one or more pharmaceutically acceptable excipients. 17. Pharmaceutical composition for prevention or treatment according to claim 16, where the disease or disorder is selected from: a) cancer or disorder; or b) an autoimmune disease or disorder. 18. The pharmaceutical composition according to claim 17, wherein the cancer or disorder is selected from the group consisting of B-cell lymphoma or leukemia. 19. The pharmaceutical composition according to claim 18, wherein the B-cell lymphoma is selected from non-Hodgkin's lymphoma (NHL) or Hodgkin's disease (Hodgkin's lymphoma). 20. The pharmaceutical composition according to claim 18, wherein the leukemia is selected from chronic lymphocytic leukemia (CLL) or small cell lymphocytic lymphoma (SLL). 21. The pharmaceutical composition according to claim 17, where the autoimmune disease or disorder is 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 disease intestinal, idiopathic thrombocytopenic purpura (ITP), thrombotic thrombohemolytic purpura (TTP), autoimmune thrombocytopenia, multiple sclerosis, psoriasis, IgA-associated nephropathy, IgM-associated polyneuropathy, myasthenia gravis, vasculitis, ANCA vasculitis, solid organ transplant rejection, graft reaction against the host (GVHD), diabetes mellitus, Raynaud's syndrome, Sjögren's syndrome and glomerulonephritis. 22. A pharmaceutical combination for the prevention or treatment of a disease or disorder mediated by CD20, comprising an antibody or antigen binding fragment thereof according to any one of claims 1 to 9 and at least one other therapeutically active compound selected from a chemotherapeutic agent, an antibody or an antihormonal agent. 23. A method of inhibiting the biological activity of CD20 in a subject in need of such inhibition, comprising administering to the subject an effective amount of an antibody or antigen binding fragment thereof according to any one of claims 1 to 9. 24. A method of treating a disease or disorder mediated by CD20, comprising administering to a subject in need of such treatment an antibody or antigen binding fragment thereof according to any one of claims 1 to 9 or a pharmaceutical composition according to claim 16 in a therapeutically effective amount. 25. The method of treating a disease or disorder according to claim 24, where the disease or disorder is selected from: a) cancer or disorder; or b) an autoimmune disease or disorder. 26. A method for treating a disease or disorder according to claim 25, where the oncological disease or -