Monoclonal antibodies that specifically bind to GITR.

Abstract

The present invention relates to the field of biotechnology, and particularly to antibodies or their antigen-binding fragments, and to the use thereof. More particularly, the present invention relates to monoclonal antibodies that bind specifically to GITR. The invention also relates to a nucleic acid that codes for said antibody or for an antigen-binding fragment thereof, an expression vector, a method for producing the antibody, and the use of said antibody for treating diseases or disorders associated with GITR.

Description

[0001] A monoclonal antibody that specifically binds to GITR

[0002] Field of technology

[0003] The present invention relates to the field of biotechnology, namely to antibodies or antigen-binding fragments thereof, and their use. More specifically, the present invention relates to monoclonal antibodies that specifically bind to GITR (Glucocorticoid-induced TNFR-related protein / tumor necrosis factor receptor superfamily, member 18 / membrane protein, receptor from the tumor necrosis factor receptor superfamily). The invention also relates to a nucleic acid encoding this antibody or 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 GITR.

[0004] State of the art

[0005] TNFRSF18, GITR (Glucocorticoid-induced TNFR-related protein / tumor necrosis factor receptor superfamily) is a membrane protein, a receptor from the tumor necrosis factor receptor superfamily.

[0006] GITR is a type I transmembrane protein consisting of 216 amino acids and a molecular mass of 26 kDa. The N-terminal extracellular domain contains three TNFR-Cys repeats and an N-glycosylation site. Three cysteine-rich domains and the cytoplasmic tail of GITR share significant homology with 4-1BB, OX40, and CD27 (Nocentini et al. (1997) Proc. Natl. Acad. Sci. 94: 6216–6221).

[0007] Human GITR is expressed at low levels by immunoreactive T cells, with CD4+ cells showing higher expression compared to CD8+ cells. GITR expression increases significantly for several days after T cell activation. GITR is constitutively expressed at high levels in regulatory T cells (Treg), such as CD4+CD25+ or CD8+CD25+ cells, and is further increased upon activation of these cells (Nocentini and Riccardi (2005) E. J. Immunol. 35:1016)

[0008] However, GITR expression is not limited to T cells alone. Some studies have also indicated that GITR is expressed by NK cells, macrophages, B cells, dendritic cells, mast cells, and monocytes (Nocentini and Riccardi (2005) E. J. Immunol. 35:1016–1022). GITR is expressed in lymph nodes, peripheral blood leukocytes, and to a lesser extent in the spleen; it is constitutively expressed in high amounts on Tregs and in low amounts on naive T cells and memory cells.

[0009] However, GITR is expressed not only on immune cells but also on tumor cells. RNA-Seq analysis of GITR expression data was performed in 33 tumor types, with high expression levels found in head and neck squamous cell carcinoma (HNSCC), non-small cell lung cancer (NSCLC), breast cancer, esophageal cancer, and bladder cancer.

[0010] Similar results were obtained during RNA-Seq analysis of samples from 24 tumor types. Thus, GITR is expressed not only on immune cells but also on the tumor cell membrane. In tumor samples, GITRL-Fc increased the expression of genes associated with T cells, CD8 T cells, cytotoxicity, Th1 cells, IFN-gamma, NK, Teff, and T-cell activation markers.

[0011] Expression of GITR and its ligand is not limited to hematopoietic cells. GITR is also expressed on keratinocytes, the precursors of osteoclasts, and GITRL is expressed on endothelial cells.

[0012] GITRL is a type II transmembrane protein, as is typical of most members of the TNF ligand family. Current studies indicate that human GITRL typically exists as a trimer, although it can also exist as a monomer or form other multimeric forms (Chattopadhyay, et al. (2007) Proc. Natl. Acad. Sci. 104:19452–19457; Zhou, et al. (2008) Proc. Natl. Acad. Sci. 105:635–640). There is evidence to suggest that a soluble form of GITRL is also produced (Baltz, et al . (2008) Blood 112:3735–3743; Mahesh, et al . (2006) Eur . J. Immunol. 36: 2128–2138). GITRL is expressed primarily on antigen-presenting cells (APCs), including macrophages, B cells, dendritic cells, and endothelial cells, which can function as APCs (Nocentini and Riccardi (2005) E. J. Immunol. 35:1016–1022; Agostini, et al . (2005) Infect. Immun. 73:7502–7508; and Nocentini, et al . (2007) EJImmunol. 37 : 11651169) .

[0013] Binding of GITRL on APCs to GITR on immunoreactive T cells triggers GITR signaling, which co-stimulates immunoreactive T cells and inhibits the suppressive activity of regulatory T cells. GITR signaling serves as a coactivation signal for both CD4+ and CD8+ naive T cells, thereby inducing or enhancing proliferation and effector function, particularly when T cell receptor (TCR) stimulation is near optimal (Schaer, et al. (2012) Curr. Opin. Immunol. 24:217224). More specifically, GITR can exert several actions on effector T cells and regulatory T cells, including: costimulation and activation of effector T cells such that they become more resistant to inhibition, inhibition of regulatory T cells, decreased sensitivity of effector T cells to suppression by regulatory T cells, and partial removal of regulatory T cells from the circulation (Nocentini, et al . (2007) Eur . J. Immunol. 37:1165–1169).

[0014] The primary functions of GITR, and therefore the primary effects of anti-GITR antibodies, are to enhance the proliferation and function of effector T cells and inhibit the suppressive action of Tregs. Teff cells are initially generated with an inability to withstand the inhibitory tumor microenvironment and suppression by Tregs. Stimulation of GITR during repeated priming and expansion stages, through an anti-GITR agonist, soluble GITR ligand, or a DC vaccine, modulates the tumor response of Teff and Tregs in favor of the former, leading to tumor regression. Thus, anti-GITR ensures Teff resistance to Treg suppression.

[0015] Taken together, these functions, particularly costimulation of immunoreactive T cells and neutralization of the suppressive activity of regulatory T cells, mean that GITR activation leads to an enhanced immune response. This activation could potentially restore immune responses to infections and tumors. Accordingly, molecules capable of activating GITR would be valuable as immunostimulatory agents in conditions where an enhanced immune response is needed.

[0016] The antibody should have GITR receptor agonist properties, with effector, cytotoxic properties against Treg lymphocytes.

[0017] Various antibodies to GITR are known from the prior art (e.g. WO2015187835, WO2015031667, WO2017068186, W02017096189, WO2017214548).

[0018] Currently, 23 GITR agonists (antibodies / recombinant GITRL) are in preclinical and clinical trials. Only two antibodies (TRX518 and INCAGN1876) are in Phase 2 clinical trials, while AMG228, MEDI1873, and MK-4166 are in Phase 1 clinical trials. Very little clinical data has been presented.

[0019] However, at present, there is not a single antibody in the world that specifically binds to GITR that has been approved for therapeutic use.

[0020] In connection with the above, it is relevant to create new agonistic antibodies that interact with GITR, activate the receptor, suppress the suppressive action of T-regulatory lymphocytes, inhibit / deplete the T-suppressor (regulatory) link of immunity due to ADCC effector properties.

[0021] BCD-166 is an agonistic monoclonal antibody that interacts with GITR, activates the receptor, suppresses the suppressive action of T-regulatory lymphocytes, inhibits / depletes the T-suppressor (regulatory) component of immunity due to ADCC effector properties, thereby increasing the number of CD8+ and CD4+ effector cells and activating the T-effector component of immunity in the tumor microenvironment.

[0022] Brief description of the invention

[0023] In one aspect, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to GITR, comprising:

[0024] (a) a heavy chain variable domain comprising:

[0025] (i) CDR1 with an amino acid sequence selected from the group:

[0026] NYGMH (SEQ ID NO: 1) or YYWMY (SEQ ID NO: 12);

[0027] (ii) CDR2 with an amino acid sequence selected from the group:

[0028] VIWFDGSNKFYTDSVKG (SEQ ID NO: 2) or AISWNGGRTYYAESMKG (SEQ ID NO: 13) ;

[0029] (iii) CDR3 with an amino acid sequence selected from the group:

[0030] ELGGYYYDSSGFRPYYYGMDV (SEQ ID NO: 3) or NRYYSDPNYGMNL (SEQ ID NO: 14), and

[0031] (b) a light chain variable domain comprising:

[0032] (i) CDR1 with an amino acid sequence selected from the group:

[0033] RASQSIGSWLA (SEQ ID NO: 7) or TGTSTDIGTYKYIS (SEQ ID NO:

[0034] 17) ;

[0035] (ii) CDR2 with an amino acid sequence selected from the group:

[0036] AASTLQR (SEQ ID NO: 8) or GVSHRPS (SEQ ID NO: 18);

[0037] (iii) CDR3 with an amino acid sequence selected from the group:

[0038] QQSHSHPLT (SEQ ID NO: 9) or SSYTSSGTW (SEQ ID NO: 19) .

[0039] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable domain that comprises CDRs 1, 2, and 3 with amino acid sequences represented by SEQ ID NOs: 1, 2, and 3, respectively.

[0040] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable domain that comprises CDRs 1, 2, and 3 with amino acid sequences represented by SEQ ID NOs: 12, 13, and 14, respectively.

[0041] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises a light chain variable domain that comprises CDRs 1, 2, and 3 with amino acid sequences represented by SEQ ID NOs: 7, 8, and 9, respectively.

[0042] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises a light chain variable domain that comprises CDRs 1, 2, and 3 with amino acid sequences represented by SEQ ID NOs: 17, 18, and 19, respectively.

[0043] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises:

[0044] - a heavy chain variable domain that contains CDRs 1, 2 and 3 with amino acid sequences represented by the sequences SEQ ID NO: 1, 2 and 3, respectively;

[0045] - a light chain variable domain that contains CDRs 1, 2 and 3 with amino acid sequences represented by SEQ ID NO: 7, 8 and 9, respectively.

[0046] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises:

[0047] - a heavy chain variable domain that contains CDRs 1, 2 and 3 with amino acid sequences represented by the sequences SEQ ID NO: 12, 13 and 14, respectively;

[0048] - a light chain variable domain that contains CDRs 1, 2 and 3 with amino acid sequences represented by SEQ ID NO: 17, 18 and 19, respectively.

[0049] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable domain that comprises an amino acid sequence that is at least 90% homologous to the amino acid sequence of SEQ ID NO: 4.

[0050] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 4.

[0051] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable domain that comprises an amino acid sequence that is at least 90% homologous to the amino acid sequence of SEQ ID NO: 15.

[0052] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 15.

[0053] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises a light chain variable domain that comprises an amino acid sequence that is at least 90% homologous to the amino acid sequence

[0054] SEQ ID NO: 10.

[0055] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 10.

[0056] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises a light chain variable domain that comprises an amino acid sequence that is at least 90% homologous to the amino acid sequence

[0057] SEQ ID NO: 20.

[0058] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 20.

[0059] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises:

[0060] a heavy chain variable domain that comprises an amino acid sequence that is at least 90% homologous to the amino acid sequence of SEQ ID NO: 4;

[0061] a light chain variable domain that comprises an amino acid sequence that is at least 90% homologous to the amino acid sequence of SEQ ID NO: 10.

[0062] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises:

[0063] a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 4;

[0064] a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 10.

[0065] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises:

[0066] a heavy chain variable domain that comprises an amino acid sequence that is at least 90% homologous to the amino acid sequence of SEQ ID NO: 15;

[0067] a light chain variable domain that comprises an amino acid sequence that is at least 90% homologous to the amino acid sequence of SEQ ID NO: 20.

[0068] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises:

[0069] a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 15;

[0070] a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 20.

[0071] In some embodiments, the monoclonal antibody that specifically binds to GITR is a full-length IgG antibody. In some embodiments, the monoclonal IgG antibody is of the human IgG1, IgG2, IgG3, or IgG4 isotype.

[0072] In some embodiments, the IgG monoclonal antibody is of the human IgG1 isotype.

[0073] In some embodiments, a monoclonal antibody that specifically binds to GITR includes an E345R mutation in the Fc region to increase agonist properties, antibody-dependent cellular cytotoxicity (ADCC), but not complement-dependent cytotoxicity (CDC).

[0074] In some embodiments, the monoclonal antibody comprises a heavy chain comprising an amino acid sequence that is at least 90% homologous to the sequence of SEQ ID NO: 5.

[0075] In some embodiments, the monoclonal antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 5.

[0076] In some embodiments, the monoclonal antibody comprises a heavy chain comprising an amino acid sequence that is at least 90% homologous to the sequence of SEQ ID NO: b.

[0077] In some embodiments, the monoclonal antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 6.

[0078] In some embodiments, the monoclonal antibody comprises a light chain comprising an amino acid sequence that is at least 90% homologous to the sequence of SEQ ID NO: 11.

[0079] In some embodiments, the monoclonal antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 11.

[0080] In some embodiments, the monoclonal antibody comprises:

[0081] - a heavy chain comprising an amino acid sequence at least 90% homologous to the sequence of SEQ ID NO: 5;

[0082] - a light chain comprising an amino acid sequence at least 90% homologous to the sequence of SEQ ID NO: 11.

[0083] In some embodiments, the monoclonal antibody comprises:

[0084] - a heavy chain containing the amino acid sequence SEQ ID NO: 5;

[0085] - a light chain comprising the amino acid sequence of SEQ ID NO: 11.

[0086] In some embodiments, the monoclonal antibody comprises:

[0087] - a heavy chain comprising an amino acid sequence at least 90% homologous to the sequence of SEQ ID NO: 6;

[0088] - a light chain comprising an amino acid sequence at least 90% homologous to the sequence of SEQ ID NO: 11.

[0089] In some embodiments, the monoclonal antibody comprises: - a heavy chain comprising the amino acid sequence of SEQ ID NO: 6;

[0090] - a light chain comprising the amino acid sequence of SEQ ID NO: 11.

[0091] In some embodiments, the monoclonal antibody comprises a heavy chain comprising an amino acid sequence that is at least 90% homologous to the sequence of SEQ ID NO: 16.

[0092] In some embodiments, the monoclonal antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 16.

[0093] In some embodiments, the monoclonal antibody comprises a light chain comprising an amino acid sequence that is at least 90% homologous to the sequence of SEQ ID NO: 21.

[0094] In some embodiments, the monoclonal antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 21.

[0095] In some embodiments, the monoclonal antibody comprises:

[0096] - a heavy chain comprising an amino acid sequence at least 90% homologous to the sequence of SEQ ID NO: 16;

[0097] - a light chain comprising an amino acid sequence at least 90% homologous to the sequence of SEQ ID NO: 21.

[0098] In some embodiments, the monoclonal antibody comprises:

[0099] - a heavy chain containing the amino acid sequence SEQ ID NO: 16;

[0100] - a light chain comprising the amino acid sequence of SEQ ID NO: 21.

[0101] In one aspect, the present invention relates to an isolated nucleic acid that encodes any of the above-mentioned antibodies or antigen-binding fragments thereof.

[0102] In some embodiments, the nucleic acid is

[0103] DNA.

[0104] In one aspect, the present invention relates to an expression vector comprising the above nucleic acid.

[0105] In one aspect, the present invention relates to a method for producing a host cell for producing the above antibody or antigen-binding fragment thereof, which comprises transforming the cell with the above vector.

[0106] In one aspect, the present invention relates to a host cell for producing the above-mentioned antibody or antigen-binding fragment thereof, comprising the above-mentioned nucleic acid.

[0107] In one aspect, the present invention relates to a method for producing the above-mentioned antibody or antigen-binding fragment thereof, which comprises culturing the above-mentioned host cell in a culture medium under conditions sufficient to produce the said antibody, if necessary, followed by isolating and purifying the resulting antibody.

[0108] In one aspect, the present invention relates to a pharmaceutical composition for the treatment of a disease or disorder mediated by GITR, comprising the above-mentioned antibody or antigen-binding fragment thereof in a therapeutically effective amount, in combination with one or more pharmaceutically acceptable excipients.

[0109] In some embodiments, the pharmaceutical composition is intended for the treatment of a disease or disorder mediated by GITR selected from the group: cervical cancer, head and neck cancer, gastric cancer, breast cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, and non-small cell lung cancer.

[0110] In one aspect, the present invention relates to a pharmaceutical composition for the treatment of a disease or disorder mediated by GITR, comprising the above-mentioned antibody or antigen-binding fragment thereof in a therapeutically effective amount and at least one therapeutically active antitumor compound in a therapeutically effective amount.

[0111] In some embodiments, the pharmaceutical composition is intended for the treatment of a disease or disorder mediated by GITR selected from the group of cervical cancer, head and neck cancer, gastric cancer, breast cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, and non-small cell lung cancer.

[0112] In some embodiments, the pharmaceutical composition includes a therapeutically active antitumor compound that is selected from a chemotherapeutic agent, an antibody, or an antihormonal agent.

[0113] In some embodiments, the pharmaceutical composition includes a therapeutically active antitumor compound that is an antibody selected from the group: antibodies to PD1, antibodies to PD-L1, antibodies to CTLA4, antibodies to anti 4-1BB, antibodies to 0X40, or combinations thereof.

[0114] In some embodiments, the pharmaceutical composition includes a therapeutically active antitumor compound that is a small molecule.

[0115] In some embodiments, the pharmaceutical composition comprises a therapeutically active antitumor compound selected from the group of activators of innate or acquired immunity. In some embodiments of the pharmaceutical composition, the above-mentioned antibody and at least one therapeutically active antitumor compound are administered sequentially.

[0116] In some embodiments of the pharmaceutical composition, the above antibody and at least one therapeutically active antitumor compound are administered simultaneously.

[0117] In one aspect, the present invention relates to a method for inhibiting the biological activity of GITR in a subject in need of such inhibition, comprising administering to the subject an effective amount of the above-mentioned antibody or antigen-binding fragment thereof.

[0118] In one aspect, the present invention relates to a method of treating a disease or disorder mediated by GITR, comprising administering to a subject in need of such treatment the above-mentioned antibody or antigen-binding fragment thereof or the above-mentioned pharmaceutical composition in a therapeutically effective amount.

[0119] In some embodiments, the method of treatment includes a disease or disorder that is selected from the group: cervical cancer, head and neck cancer, gastric cancer, breast cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, NSCLC

[0120] (non-small cell lung cancer).

[0121] 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 the treatment of a disease or disorder mediated by GITR in a subject in need of such treatment.

[0122] In some embodiments, the use includes a disease or disorder that is selected from the group: cervical cancer, head and neck cancer, gastric cancer, breast cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, NSCLC

[0123] (non-small cell lung cancer).

[0124] Brief description of the drawings

[0125] Fig. 1. Plasmid for the production of pEE-GITR-TEV-Fc lama protein.

[0126] Fig. 2. Plasmid for the production of pEE-GITR-Fc protein.

[0127] Fig. 3. Map of the pEE-humGITR-ligand-foldon-EPEA plasmid.

[0128] Fig. 4. SDS gel electrophoresis in 4-20% PAGE

[0129] 1. Actemra 2.5 mg

[0130] 2. Fermentas unstained PW marker

[0131] 3.

[0132] 4. GITR-TEV-Fc-lama +b-ME 10 ml

[0133] 5. Angiopoietin 2-H6F + b-ME 10 ml

[0134] 6.

[0135] 7. GITR-TEV-Fc-lama -b-ME 10 ml 8. Angiopoietin 2-H6F - b-ME 10 ml

[0136] Fig. 5. SDS gel electrophoresis

[0137] 1. Fermentas unstained PW marker

[0138] 2.

[0139] 3. hGITR ligand-EPEA medium before purification 5 ml

[0140] 4. hGITR ligand-EPEA medium after purification 5 ml

[0141] 5. hGITR ligand -EPEA

[0142] Fig. b. Scheme of synthesis of combinatorial naive human library

[0143] Fig. 7. Map of the pH5 phagemid for cloning Fab phage display libraries.

[0144] Fig. 8. Map of the pLL expression plasmid for the production of secreted Fabs.

[0145] Fig. 9. Map of the expression vector pEE-BCD1 66-01-001-VH- НС encoding the heavy chain of antibodies for transient production of antibodies in mammalian cells

[0146] Fig. 10. Map of the expression vector pEE-BCD166-01-001-VK-SK encoding the light chain of antibodies for transient production of antibodies in mammalian cells

[0147] Fig. 11. SDS gel electrophoresis in 12% PAGE + b-ME

[0148] 1. Actemra 5 ml

[0149] 2. Fermentas unstained PW marker

[0150] 3.

[0151] 4. Anti-GITR antibody lot #1161 medium before purification 5 ml

[0152] 5. Anti-GITR antibody lot #1161 medium after purification 5 ml

[0153] 6. anti-GITR antibody lot#1161 5 ml

[0154] 7. anti-GITR antibody lot#1162 5 ml

[0155] 8. anti-GITR antibody lot#1163 5 ml

[0156] 9. anti-GITR antibody lot#1164 5 ml

[0157] 10 anti-GITR antibody lot#1165 5 ml

[0158] 11 anti-GITR antibody lot#1166 medium before purification 5 ml 12 anti-GITR antibody lot#1166 medium after purification 5 ml

[0159] 13 anti-GITR antibody lot#1166 5 ml

[0160] 14 anti-GITR antibody lot#1167 5 ml

[0161] Fig. 12. SDS gel electrophoresis in 12% PAGE + b-ME

[0162] 1. Fermentas unstained PW marker

[0163] 2. Anti-GITR antibody lot #1208 medium before purification 10 ml

[0164] 3. Anti-GITR antibody lot #1208 medium after purification 10 ml

[0165] 4. anti-GITR antibody lot#1208 5 ml

[0166] 5. anti-GITR antibody lot#1209 5 ml

[0167] 6. anti-GITR antibody lot#1210 5 ml

[0168] 7. anti-GITR antibody lot#1211 5 ml

[0169] 8. anti-GITR antibody lot#1212 5 ml

[0170] 9. anti-GITR antibody lot#1213 5 ml

[0171] 10. Anti-GITR antibody lot #1214 medium before purification 10 ml

[0172] 11. Anti-GITR antibody lot#1214 medium after purification 10 ml 12. Anti-GITR antibody lot#1214 5 ml

[0173] Fig. 13. Graph with the results of testing the specific agonistic activity of anti-GITR monoclonal antibodies of lots 1161 (BCD166-01-01), 1210 BCD166-01-011 and 1213 BCD166-01-014 in a reporter cell line test.

[0174] Fig. 14. Graph showing the results of testing the specific binding of the BCD166-01-01 antibody to human GITR, mouse GITR, and cynomolgus monkey GITR.

[0175] Fig. 15. Graph showing the results of testing the specific binding of the BCD166-01-011 antibody to human GITR, mouse GITR, and cynomolgus monkey GITR.

[0176] Fig. 16. Graph showing the results of testing the specific binding of the BCD166-01-014 antibody to human GITR, mouse GITR, and cynomolgus monkey GITR.

[0177] Fig. 17. Graph with the results of the activation level in the cellular agonist test of the studied BCD166-01-01, BCD166-02-01, BCD166-01-014.

[0178] Fig. 18. Graph with the results of the EC50 value in the cellular ADCC test of the studied BCD166-02-01, BCD166-01-01, BCD166-01-014, BCD166-02-014.

[0179] Fig. 19. Graph with the results of the CDC level of the studied BCD166-01-01, BCD166-02-01, BCD166-01-014.

[0180] Fig. 20. Graph showing the results of the effect of the studied anti-GITR candidates on immunoreactive cells (NK) in comparison with the negative control.

[0181] Fig. 21. Graph showing the results of the effect of the studied anti-GITR candidates on immunoreactive cells (B) in comparison with the negative control.

[0182] Fig. 22. Graph showing the results of the effect of the studied anti-GITR candidates on immunoreactive cells (CD3) in comparison with the negative control.

[0183] Fig. 23. Graph showing the results of the effect of the studied anti-GITR candidates on immunoreactive cells (CD8+ T cells) in comparison with the negative control.

[0184] Fig. 24. Graph showing the results of the effect of the studied anti-GITR candidates on immunoreactive cells (CD4+ T cells) in comparison with the negative control.

[0185] Fig. 25. Analysis of antibody-dependent depletion of nTreg cells under the influence of the studied anti-GITR candidates for the cellular material of donor 1.

[0186] Fig. 26. Analysis of antibody-dependent nTreg depletion under the influence of the studied anti-GITR candidates for the cellular material of donor 2.

[0187] Fig. 27. Analysis of antibody-dependent depletion of iTreg under the influence of the studied anti-GITR candidates for the cellular material of donor 1 and donor 2. Fig. 28. Results of the analysis of the effect of anti-GITR antibodies on the level of secretion of pro-inflammatory cytokines IL-2 and IFN-γ, which determine the anti-cancer effect.

[0188] Fig. 29. Table of data on the binding affinity constants of the candidate BCD166-02-01 to gamma receptors.

[0189] Fig. 30. Stability graph of BCD166-02-01 after 72-hour incubation at 50°C.

[0190] Fig. 31. Stability graph of BCD166-01-014 after 72-hour incubation at 50°C.

[0191] Fig. 32. Average values ​​of tumor volumes in groups during the experiment.

[0192] Fig. 33. Tumor growth inhibition index on day 33.

[0193] Description of the invention

[0194] Definitions and general methods

[0195] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those skilled in the art.

[0196] Furthermore, unless the context otherwise requires, singular terms include plural terms, and plural terms include singular terms. The generally used classification and methods of cell culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, organic synthetic chemistry, medicinal and pharmaceutical chemistry, as well as hybridization and protein and nucleic acid chemistry, described herein are well known to those skilled in the art and are widely used in the art. Enzymatic reactions and purification methods are carried out in accordance with the manufacturer's instructions, as commonly practiced in the art, or as described herein.

[0197] Antibody-related definitions

[0198] TNFRSF18, GITR (Glucocorticoid-induced TNFR-related protein / tumor necrosis factor receptor superfamily) is a membrane protein, a receptor of the tumor necrosis factor receptor superfamily. GITR is a type I transmembrane protein consisting of 216 amino acids, its molecular mass is 26 kDa. The N-terminal extracellular domain contains three TNFR-Cys repeats and an N-glycosylation site. Three cysteine-rich domains and the cytoplasmic tail of GITR have significant homology to 4-1BB, OX40, and CD27 (Nocentini et al. (1997) Proc.

[0199] Natl. Acad. Sci. 94:6216–6221). GITR gene amplification and / or overexpression of its protein have been detected in many cancers, including cervical cancer, head and neck cancer, gastric cancer, breast cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, and non-small cell lung cancer.

[0200] The term "binding molecule" includes antibodies and immunoglobulins.

[0201] The term "antibody" or "immunoglobulin" (Ig), as used herein, includes whole antibodies and any antigen-binding fragment (i.e., "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 linked 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. Five types of mammalian antibody heavy chains are known, designated by the Greek letters a, d, e, g, and m. The type of heavy chain present determines the class of the antibody; these chains are found in IgA, IgD, IgE, IgG, and IgM antibodies, respectively. Different heavy chains vary in size and composition; a and g contain approximately 450 amino acids, while m and e consist of approximately 550 amino acids. Each heavy chain contains two regions, i.e.,A constant region and a variable region. The constant region is identical in all antibodies of the same isotype but differs between antibodies of different isotypes. Heavy chains g and d contain a constant region, which consists of three constant domains CHI, CH2, and CH3 (arranged in a row) and a hinge region that confers flexibility (Woof J., Burton D., Nat Rev Immunol 4, 2004, pp. 89-99); heavy chains m and e contain a constant region, which consists of four constant domains CHI, CH2, CH3, and CH4. Only two types of light chains are known in mammals, designated lambda (l) 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 a light chain is 211-217 amino acids. Preferably, the light chain is a kappa (k) light chain and the constant domain CL is preferably a C-kappa (k).

[0202] "Antibodies" according to the invention may be antibodies of any class (e.g., IgA, IgD, IgE, IgG and IgM, preferably IgG) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgAl and IgA2, preferably IgG1).

[0203] The VH and VL regions can be further subdivided into regions of hypervariability, called complementarity-determining regions (CDRs), interspersed with regions that are more conserved, called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain the binding domain that interacts with the antigen. The constant regions of antibodies can mediate the binding of immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0204] 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 fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments included within the term "antigen-binding portion" of an antibody include (i) the Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) the F (ab f) the 2 fragment, a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region; (iii) the Fd fragment, consisting of the VH and CH1 domains; (iv) the Fv fragment, consisting of the VL and VH domains in a single arm of the antibody, (v) the dAb fragment (Ward et al ., (1989) Nature 341:544–546), which consists of the VH / VHH domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, the two regions of the Fv fragment, VL and VH, are encoded by different genes and can be joined by recombinant techniques using a synthetic linker that allows them to be produced as a single protein chain in which the VL and VH regions are paired to form monovalent molecules (known as single-chain Fv (scFv); see, e.g., Bird et al . (1988) Science 242:423–426; and Huston et al . (1988) Proc. Natl. Acad. Sci. USA 85:5879–5883). Such single-chain molecules are also intended to be included within the term "antigen-binding portion" of an antibody.Such antibody fragments are obtained using conventional methods known to those skilled in the art, and these fragments are screened in the same manner as intact antibodies.

[0205] Preferably, the CDRs of the antigen-binding region or the entire antigen-binding region of the antibodies of the invention are of mouse, llama, or donor human library origin, or are of substantially human origin with certain amino acid residues altered, for example, substituted with different amino acid residues, in order to optimize specific properties of the antibody, for example, KD, koff, IC50, EC50, ED50. Preferably, the framework regions of the antibodies of the invention are of human origin or are of substantially human origin (at least 80, 85, 90, 95, 96, 97, 98, or 99% human origin).

[0206] In other embodiments, the antigen-binding region of the antibody of the invention may be from other non-human species, including, but not limited to, mouse, llama, rabbit, rat, or hamster. Alternatively, the antigen-binding region may be from a human species.

[0207] The term "variable" refers to the fact that certain segments of the variable domains vary widely in sequence among antibodies. The V domain mediates antigen binding and determines the specificity of a particular antibody to its specific antigen. However, variability is unevenly distributed across the 110-amino acid stretch of the variable domains. In contrast, V regions consist 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 native heavy and light chains contains four FRs, generally adopting a beta-sheet configuration, linked by three hypervariable regions that form loops that link and, in some cases, are part of a beta-sheet structure.The hypervariable regions in each chain are held together in close proximity by FRs and, together with the hypervariable regions of the other chain, contribute to the formation of the antigen-binding site of antibodies. The constant domains are not directly involved in antibody-antigen binding but exhibit various effector functions, such as participation in antibody-dependent cellular cytotoxicity (ADCC).

[0208] 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 those from the "hypervariable loop."

[0209] In some cases, it may also be advantageous to alter one or more amino acid residues of the CDR regions in order to increase the binding affinity for the target epitope. This is known as "affinity maturation" and may in some cases be performed in conjunction 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 the binding specificity or affinity using backmutations alone. Various methods for affinity maturation are known in the art, such as the in vitro scanning saturation mutagenesis method described by Burks et al ., Proc Natl Acad Sci USA, 94:412-417 (1997), and the stepwise in vitro affinity maturation method proposed by Wu et al ., Proc Natl Acad Sci USA 95:6037-6042 (1998).

[0210] Framework regions (FRs) are the residues of a variable domain other than the CDR residues. Typically, each variable domain has four FRs, defined as FR1, FR2, FR3, and FR4. When CDRs are defined according to Rabat, 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. When the CDR regions contain amino acid residues from the hypervariable loops, the light chain FR residues are located at approximately residues 1-25 (LCFR1), 33-49 (LCFR2), 53-90 (LCFR3), and 97-107 (LCFR4) in the light chain, and the heavy chain FR residues are located at approximately residues 1-25 (HCFR1), 33-52 (HCFR2), 56-95 (HCFR3), and 102-113 (HCFR4) in the heavy chain.In some examples, when a CDR contains amino acids from both the Rabat CDR and the hypervariable loop, the FRs are adjusted accordingly. For example, when CDRH1 includes amino acids H26-H35, the heavy chain's FR1 residues are at positions 1-25, and the FR2 residues are at positions 36-49.

[0211] The crystallizable fragment of immunoglobulin (Fc region, Fc) is the terminal portion of the immunoglobulin molecule that interacts with the Fc receptor on the cell surface and with certain 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, the second and third constant domains of the two heavy chains, respectively; in the case of IgM and IgE isotypes, Fc contains three heavy chain constant domains (CH2-4 domains) in each polypeptide chain.

[0212] An antibody of the present invention that "binds" a target antigen is one that binds the antigen with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent when targeting a protein or a cell or tissue expressing the antigen, and cross-reacts to a low degree with other proteins. In such embodiments, the degree of binding of the antibody to a non-target protein (an "off-target protein") is less than 10% of the binding of the antibody to the specific target protein, as determined by fluorescence-activated cell sorting (FACS), radioimmunoprecipitation assay (RIA), or ELISA.With respect to the binding of an antibody to a target molecule, the term "specific binding" or the expressions "specifically binds to" or "specific for" a particular polypeptide or epitope on a particular target polypeptide means binding that is measurably different from non-specific interaction (e.g., in the case of LB1-44 or LB1-81, non-specific interaction is binding to bovine serum albumin, casein, fetal bovine serum, or neutravidin).

[0213] Specific binding can be determined quantitatively, for example, by determining the binding of a molecule relative to the binding of a control molecule. Specific binding can also be determined by a competitive reaction with another molecule similar to the target, such as an excess of unlabeled target. In this case, specific binding is indicated if the binding of the labeled target to the probe is competitively inhibited by the excess of unlabeled target.As used herein, the term "specific binding" or the expressions "specifically binds to" or "specific for" a particular polypeptide or epitope on a particular target polypeptide may be characterized by an example of 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 of the invention, the term "specific binding" refers to binding in which a molecule binds to a particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptide or epitope on the polypeptide.

[0214] The term "Ka" as used herein refers to the rate of association of a particular antibody-antigen interaction.

[0215] The term "Kd" as used herein refers to the dissociation rate of a particular antibody-antigen interaction.

[0216] "Binding affinity" generally refers to the strength of the combined non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, "binding affinity" refers to the intrinsic (characteristic, true) binding affinity, which reflects the 1:1 interaction between the members of the binding pair (e.g., an antibody and an antigen). The affinity of molecule X for its partner Y can typically be represented by the dissociation constant (Kd). Desirably, the Kd value is approximately 200 nM, 150 nM, 100 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 8 nM, 6 nM, 4 nM, 2 nM, 1 nM, or less. Affinity can be measured by conventional methods known in the art, including the methods described herein.Low-affinity antibodies typically bind to antigen slowly and tend to dissociate easily, whereas high-affinity antibodies typically bind to antigen more quickly and tend to remain bound longer. Various methods for measuring binding affinity are known in the art, and any of these methods can be used for the purposes of the present invention.

[0217] In one embodiment, the "Kb" or "Kb value" of the present invention is measured by surface plasmon resonance methods on a BIAcore™-2000 or BIAcore™-3000 instrument (BIAcore, Inc., Piscataway, NJ) at 25°C using CM5 antigen-immobilized chips at ~10 relative units (RU). Briefly, carboxymethyldextran (CM5, BIAcore Inc.) biosensor chips are 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 achieved. Following antigen injection, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (e.g., from 0.78 nM to 500 nM) are injected into PBS containing 0.05% Tween 20 (PBST) at 25°C at a flow rate of approximately 25 μl / min. The association rate (koff) and dissociation rate (koff) values ​​are calculated using the simple Langmuir model for one-plus-one binding (BIAcore Evaluation Software version 3.2) by simultaneously acquiring the association and dissociation sensorgram. The equilibrium dissociation constant (koff) is calculated as the ratio koff / kon. See, e.g., Chen, Y., et al . , (1999) J. Mol. Biol. 293: 865–881. If the association rate exceeds 10 according to the above surface plasmon resonance method, 6 M -1 sec -1, then it can be determined by the fluorescence quenching method, which measures the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, band 16 nm) at 25 °C of a 20 nM solution of antibody to antigen (Fab form) in PBS, pH 7.2, in the presence of increasing concentrations of antigen measured with a spectrometer such as a stopped-flow spectrophotometer (Aviv Instruments) or an SLM-Aminco (ThermoSpectronie) 8000 series spectrophotometer with a stirred cuvette.

[0218] The term "Koff" refers to the dissociation rate constant of a specific interaction between a binding molecule and an antigen. The dissociation rate constant, koff, can be measured using biolayer interferometry, such as the Octet™ system.

[0219] The "oh-rate" or "cop" of the present 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 CM5 antigen-immobilized chips at ~10 relative units (RU). Briefly, carboxymethyldextran (CM5, BIAcore Inc.) biosensor chips are activated with -ethyl-'-(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 achieved. Following antigen injection, a 1 M ethanolamine solution is added to block unreacted groups.

[0220] Unless otherwise specifically stated, the terms "biologically active," "biological activity," and "biological characteristics," when used with respect to a polypeptide of the present invention, mean having the ability to bind to a biological molecule.

[0221] The term "biological molecule" refers to a nucleic acid, protein, carbohydrate, lipid, or combination thereof. In one embodiment, the biological molecule exists in nature.

[0222] Antibody fragments, such as Fab and F(ab')2 fragments, can be obtained from whole antibodies using traditional methods such as papain or pepsin digestion of whole antibodies. Furthermore, antibodies, antibody portions, and immunoadhesion molecules can be produced using standard recombinant DNA techniques, such as those described herein.

[0223] The term "recombinant antibody" means an antibody that is expressed in a cell or cell line containing nucleotide sequence(s) that encodes antibodies, wherein said nucleotide sequence(s) is(are) not associated with the cell in nature.

[0224] 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 the addition, deletion, and / or substitution of one or more amino acid residues relative to the sequence of the parent antibody. In a preferred embodiment, a variant antibody comprises at least one or more (e.g., one to twelve, such as two, three, four, five, six, seven, eight or nine, ten, eleven, or twelve; and in some embodiments, one to about ten) amino acid additions, deletions, and / or substitutions relative to the parent antibody. In some embodiments, the additions, deletions, and / or substitutions are made in the CDR regions of the variant antibody.Identity or homology with respect to the sequence of a variant antibody is defined herein as the percentage of amino acid residues in the variant antibody sequence that are identical to residues of the parent antibody, after alignment of the sequences and introduction of gaps, if necessary, to achieve the maximum percentage of sequence identity. A variant antibody retains the ability to bind to the same antigen, and preferably the epitope, to which the parent antibody binds, and in some embodiments, has at least one property or biological activity superior to that of the parent antibody. For example, a variant antibody may have, for example, a more pronounced 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 a biological activity that is at least 2-fold (preferably at least 5-fold, 10-fold, or 20-fold) greater than the biological activity of the parent antibody.

[0225] The term "bispecific antibody" means an antibody comprising an antigen-binding domain or domains that are capable of specifically binding to two different epitopes on a single biological molecule or capable of specifically binding to epitopes on two different biological molecules. A bispecific antibody is also referred to herein as having "dual specificity" or as being an antibody with "dual specificity."

[0226] 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 human and partly non-human, that is, derived partly from a non-human animal, such as a mouse, rat, or other rodent, or camelids such as a llama or alpaca. Chimeric antibodies are preferred over non-human antibodies to reduce the risk of an immune response directed against human antibodies, such as a response directed against mouse antibodies 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 can be further modified to humanize the antibody.

[0227] The term "humanization" refers to the fact that when an antibody is of completely or partially non-human origin, such as a mouse or llama antibody obtained by immunizing mice or llamas, respectively, with an antigen of interest, or is a chimeric antibody based on such a mouse or llama antibody, certain amino acids can be substituted, for example, in the framework regions and constant domains of the heavy and light chains, to avoid or minimize the immune response in humans. The specificity of antibody interaction with the target antigen is primarily determined by the amino acid residues located within the six CDR regions of the heavy and light chains. Therefore, amino acid sequences within the CDR regions are much more variable between individual antibodies than sequences outside the CDR regions.Because 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, any specific antibody with a given amino acid sequence, for example, by constructing expression vectors that express CDR sequences from a specific antibody and framework sequences from another antibody. As a result, it is possible to "humanize" a non-human antibody while largely preserving the binding specificity and affinity of the original antibody. Although it is impossible to accurately predict immunogenicity and thus the immune response directed against an antibody in humans to a specific antibody, non-human antibodies are generally more immunogenic than human antibodies.Chimeric antibodies, in which foreign (e.g., rodent or camel) constant regions have been replaced with human-derived sequences, have generally shown lower immunogenicity than antibodies of completely foreign origin, and there is a trend toward using 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 anti-antibody immune response in humans.

[0228] For chimeric antibodies, humanization typically involves modification of the framework regions of the variable region sequences. The amino acid residues that are part of the complementarity-determining regions (CDRs) will most often not be altered by humanization, although in some cases it may be desirable to alter individual amino acid residues of a CDR, for example, to remove a glycosylation site, a deamidation site, an aspartate isomerization site, or an unwanted 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-linked glycosylation site can be achieved by mutating the Asn or Ser / Thr residue with another residue, preferably by 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. When such a deamidated site is present, for example, Asn-Gly in a CDR sequence, it may be preferable to remove this site, typically by a conservative substitution to remove one of the involved residues.

[0229] Numerous methods for humanizing an antibody sequence are known in the art. One of the most commonly used methods is CDR grafting. CDR grafting can be based on the Rabat CDR definitions, although a more recent publication (Magdelaine-Beuzelin et al., Crit Rev. Oncol Hematol. 64:210–225 (2007)) suggests that definition of IMGT® (the international ImMunoGeneTics information system®, www.imgt.org) may improve the humanization outcome (see Lefranc et al., Dev. Comp Immunol. 27:55–77 (2003)). In some cases, CDR grafting may reduce the binding specificity and affinity, and hence biological activity, of the CDR-grafted non-human antibody compared to the parent antibody from which the CDRs are derived.Back mutations (sometimes referred to as "framework repairs") can be applied to selected CDR positions of the grafted antibody, typically in the framework regions, to restore the specificity and binding affinity of the parent antibody. Positions for potential back mutations can be determined 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 are generally unaffected. Humanization, an alternative to CDR grafting and back mutation, is a surface modification in which non-human residues not exposed on the surface are retained, while surface-exposed residues are modified to resemble human residues.

[0230] 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.).

[0231] Construction of combinatorial phage antibody libraries begins with the selection of a gene repertoire source, which can be used to distinguish several types of antibody libraries: naive, immune, or synthetic. Naive and immune libraries are constructed using naturally rearranged genes encoding the variable domains of immunoglobulins from healthy or immune donors, respectively. For this purpose, mRNA from antibody-producing lymphoid cells is isolated. Most often, these are peripheral blood lymphocytes, but in some cases, splenocytes are used [Sheets MD, Amersdorfer P, Finnern R, Sargent P, Lindquist E, Schier R, et al. Efficient construction of a large nonimmune phage antibody library: the production of high-affinity human single-chain antibodies to protein antigens. Proc Natl Acad Sci USA 1998,95:6157-6162 and de Haard HJ, van Neer N, Reurs A, Hufton SE, Roovers RC, Henderikx P, et al .A large non-immunized human Fab fragment phage library that allows rapid isolation and kinetic analysis of high affinity antibodies. J Biol Chem 1999,274:18218-18230.], tonsil cells or bone marrow lymphocytes [Vaughan TJ, Williams AJ, Pritchard K, Osbourn JK, Pope AR, Earnshaw JC, et al. Human antibodies with sub-nanomolar affinities isolated from a large non-immunized phage display library. Nat Biotechnol 1996,14:309-314]. cDNA is synthesized on the basis of mRNA, and oligo-dT primers and random hexaoligonucleotides can be used for priming the reaction, which makes it possible to obtain cDNA copies of all possible variants of the genes encoding the variable domains of antibodies [Ulitin AB, Kapralova MV, Daman AG, Shepelyakovskaya AO, Bulgakova EV, Fursova KK, et al. Library of human miniantibodies in phage display format. Creation and testing. DAN: Nauka Publishing House; 2005.].

[0232] One or more primers can be used immediately, limiting the set of amplified genes to one or more variable domain gene families or antibody isotypes already at the cDNA level [Marks JD, Hoogenboom HR, Bonnert TP, McCafferty J, Griffiths AD, Winter G. Bypassing immunization. Human antibodies from V-gene libraries displayed on phage. J Mol Biol 1991, 222: 581–597]. Primers used to amplify genes encoding immunoglobulins are complementary to their most conserved regions. Their sequences are selected from gene collections organized into databases, such as the Rabat or V BASE databases. Primer design also includes internal restriction sites that allow cloning of PCR products into the corresponding vectors.

[0233] The construction of synthetic libraries is based on the replacement of natural CDRs with a set of random sequences, which allows the creation of a huge variety of antigen-binding sites.

[0234] 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 such that the cloned gene sequences were expressed on the surface of the phage particles as fusion proteins (Smith GP: Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 1985, 228:1315–1317). Thus, fusion proteins of interest could be selected based on their ability to bind other proteins. This discovery was combined with PCR amplification techniques to clone cDNA repertoires of immunoglobulin genes to generate diverse phage libraries containing variable domains that could be used to rapidly screen for target-specific monoclonal antibodies.The repertoire of phage libraries reflects the antibody repertoire of the B lymphocytes of each individual 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 whose repertoires could be compared with those obtained by hybridoma technology (Lonberg N, Taylor LD, Harding FA, Trounstine M, Higgins KM, Schramm SR, Kuo CC, Mashayekh R, Wymore K, McCabe JG et al.: Antigen-specific human antibodies from mice comprising four distinct genetic modifications. Nature 1994, 368:856–859). These animals had their own endogenous heavy and K light chain genes deliberately disrupted and were given transgenes representing 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 generate highly specific and high-affinity antibodies to a wider variety of antigens. Although transgenic mice express B-cell receptors that are essentially hybrids of mouse and human receptors (human immunoglobulin, mouse Ig, Irb, and other signaling molecules), their B cells develop and mature normally. In some cases, it may also be advantageous to alter one or more amino acid residues of the CDR regions to increase binding affinity for the target epitope. This is known as "affinity maturation" and may in some cases be performed in conjunction with humanization, for example in situations where humanization of an antibody results in decreased specificity or binding affinity, and it is not possible to sufficiently improve specificity or binding affinity using backmutations alone.Various affinity maturation methods are known in the art, such as the in vitro scanning saturation mutagenesis method described by Burks et al., Proc Natl Acad Sci USA, 94:412-417 (1997), and the stepwise in vitro affinity maturation method proposed by Wu et al., Proc Natl Acad Sci USA 95:6037-6042 (1998).

[0235] The term "monoclonal antibody" or "mAb" refers to an antibody synthesized and secreted 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, or 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 naturally.

[0236] 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 one covalent disulfide bond, while the number of disulfide bonds between heavy chains varies among immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (VH) at one end, followed by several constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at the other end. The constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the variable domain of the light chain is aligned with the variable domain of the heavy chain.Specific amino acid residues are thought to form the interface between the variable domains of the light chain and heavy chain.

[0237] The term "isolated" as used to describe the various antibodies described herein means an antibody that has been identified and separated and / or recovered from the cell or cell culture in which it is expressed. 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 proteinaceous or non-proteinaceous solutes. In preferred embodiments, the antibody is purified (1) to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence using a spinning glass cup sequencer (Edman sequencer), or (2) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie Brilliant Blue or, preferably, silver staining.An isolated antibody includes antibodies produced in situ within recombinant cells, as at least one component of the polypeptide's natural environment is absent. Typically, an isolated polypeptide is obtained through at least one purification step.

[0238] 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 normally associated in the natural source of the antibody nucleic acid. An isolated nucleic acid molecule is distinct from the form or composition in which it is naturally found. Thus, an isolated nucleic acid molecule is distinct from a nucleic acid molecule naturally present in cells. However, an isolated nucleic acid molecule includes a nucleic acid molecule present in cells that normally express the antibody, for example, if the nucleic acid molecule has a chromosomal location different from its natural cellular location.

[0239] The term "epitope," as used herein, refers to the portion (determinant) of an antigen that specifically binds to a binding molecule (e.g., an antibody or related molecule, such as a bispecific binding molecule). Epitope determinants typically consist of chemically active 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. An epitope can be "linear" or "conformational." In a linear epitope, all points of interaction between a protein (e.g., an antigen) and an interacting molecule (such as an antibody) occur linearly along the primary amino acid sequence of the protein. In a conformational epitope, the points of interaction occur across amino acid residues on the protein that are separated from each other in the primary amino acid sequence.Once a desired epitope of an antigen is identified, antibodies to that epitope can be generated using techniques well known in the art. Furthermore, the generation and characterization of antibodies or other binding molecules can provide insight into the desired epitopes. Based on this information, binding molecules can then be competitively screened for binding to the same or similar epitopes, for example, by conducting competition studies to find binding molecules that compete for binding to the antigen.

[0240] The term "peptide linker" as used herein means any peptide capable of connecting domains of a length depending on the domains it links together, comprising 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, and K.

[0241] The term "in vitro" refers to a biological object, biological process, or biological reaction outside the body, simulated under artificial conditions. For example, in vitro cell growth should be understood as cell growth in an environment outside the body, such as a test tube, culture flask, or microplate.

[0242] The term "IC50" (50% inhibitory concentration) refers to the drug concentration at which the measured activity or response, such as the growth or proliferation of cells such as tumor cells, is inhibited by 50%. The IC50 value can be estimated using the corresponding log dose response curves, using specialized statistical software for curve processing.

[0243] The term GI50 (50% growth inhibition) refers to the concentrations of a drug at which the proliferation of cells, such as tumor cells, is inhibited by 50%.

[0244] The term ED50 (EC50) (50% effective dose / concentration) refers to the drug concentration at which 50% of the measurable biological effect is achieved (may include cytotoxicity).

[0245] The term "antiproliferative action" refers to stopping or inhibiting the growth of proliferating cells, such as cancer cells.

[0246] The term "effector function" of an antibody refers to the types of biological activity associated with the Fc region (either the native Fc region sequence or Fc region amino acid sequence variants) of an antibody, and varies depending on the antibody isotype. Examples of antibody effector functions include: C1 q — binding; complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor, BCR) and B cell activation.

[0247] “Antibody-dependent cellular cytotoxicity” or “ADCC” refers to a cell-mediated response in which nonspecific cytotoxic cells that express Fc receptors (FcRs) (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on a target cell and then cause lysis or phagocytosis of the target cell. The primary cells for mediating ADCC, NK cells, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcγR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9: 457–92 (1991). To assess the ADCC activity of a molecule of interest, in vitro ADCC assays can be performed, such as those described in U.S. Patents W 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, ADCC activity of the molecule of interest can be assessed in vivo, for example, in an animal model such as that described in Clynes et al. PNAS (USA) 95: 652–656 (1998).

[0248] "Human effector cells" are leukocytes that express one or more FcRs and perform effector functions. Preferably, the cells express at least FcγRIII and perform ADCC effector 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, such as blood or PBMCs, as described herein.

[0249] The terms "Fc receptor" and "FcR" are used to describe a receptor that binds the Fc region of an antibody. A preferred FcR is the native-sequence human FcR. Additionally, a preferred FcR is the FcR that binds IgG antibody (the gamma receptor), and preferred receptors include receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. FcγRII receptors include FcγRI IA (the "activating receptor") and FcγRI IB (the "inhibitory receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. The activating receptor FcγRI IA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRI IB contains a tyrosine-based immunoreceptor inhibitory motif (ITIM) in its cytoplasmic domain (see review in Baeron, Annu. Rev. Immunol.15: 203–234 (1997). A review of FcRs 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 "FcR". The term also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgG to the fetus.

[0250] "Complement-dependent cytotoxicity" and "CBC" refer to the ability of a molecule to lyse a target in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (Clq) to a molecule (e.g., an antibody) complexed with its antigen. To assess complement activation, a CBC assay can be performed, such as that described by Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996).

[0251] 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 as necessary to achieve the maximum percentage identity for the complete sequence, and without considering any conservative substitutions as part of the sequence identity. Neither N- or C-terminal extensions nor insertion segments should be construed as reducing identity or homology. Methods and computer programs for comparison are well known. Sequence identity can be determined using sequence analysis software (e.g., Sequence Analysis Software Package, Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Ave., Madison, WI 53705).This software is suitable for such sequences by determining the degree of homology for various substitutions, deletions (eliminations) and other modifications.

[0252] The phrase "homologous" when used with respect to the polypeptide sequence of an antibody should be interpreted as an antibody exhibiting at least 70%, preferably 80%, more preferably 90%, and most preferably 95% sequence identity with respect to the polypeptide sequence. The term when used with respect to a nucleic acid sequence should be interpreted as a sequence of nucleotides exhibiting at least 85%, preferably 90%, more preferably 95%, and most preferably 97% sequence identity with respect to the nucleic acid sequence.

[0253] 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. Variants of the amino acid sequence of the antibody are obtained 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 deletions, insertions, and substitutions is carried out to obtain the final construct, provided that the final construct has the desired characteristics. Amino acid changes can also alter post-translational processes in the antibody, such as changing the number or position of glycosylation sites.

[0254] A variant of modifying the amino acid sequences of antibodies using amino acid substitutions. This variant involves replacing at least one amino acid residue in the antibody molecule with another residue. Locations of greatest interest for substitution mutagenesis include the hypervariable regions or CDRs, but changes in the FR or Fc region are also contemplated. Conservative substitutions are shown in Table A under the heading "preferred substitutions." If such substitutions result in altered biological activity, additional significant changes, termed "example substitutions" in Table A, or changes further described below under the amino acid classes, can be introduced, and product screening can be performed.

[0255]

[0256] The terms "nucleic acid", "nucleic sequence" or "nucleic acid sequence", "polynucleotide", "oligonucleotide", "polynucleotide sequence" and "nucleotide sequence", which are used interchangeably in this description, denote a distinct sequence of nucleotides, modified or unmodified, defining a fragment or section of nucleic acid, containing or not containing non-natural nucleotides and being either double-stranded DNA or RNA, or single-stranded DNA or RNA, or transcription products of said DNA.

[0257] It should also be noted here that this invention does not relate to nucleotide sequences in their natural chromosomal environment, i.e., in their natural state. The sequences of this invention have been isolated and / or purified, i.e., taken directly or indirectly, for example, by copying, while their environment has been at least partially modified. Thus, this also includes isolated nucleic acids obtained by genetic recombination, for example, using host cells, or obtained by chemical synthesis.

[0258] A reference to a nucleotide sequence includes its complement unless otherwise stated. Thus, a reference to a nucleic acid having a particular sequence should be understood to include its complementary strand with its complementary sequence.

[0259] The term "control sequences" refers to DNA sequences required for the expression of a functionally linked coding sequence in a given host organism. Control sequences suitable for prokaryotes include, for example, a promoter, optionally an operator, and a ribosome binding site. Eukaryotic cells are known to contain promoters, polyadenylation signals, and enhancers.

[0260] 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 the transcription of the sequence; a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Typically, "operably linked" means that the linked DNA sequences are contiguous, and in the case of a secretory leader sequence, they are contiguous and in the reading phase. However, enhancers do not necessarily have to be contiguous.

[0261] The term "vector," as used herein, refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. In some embodiments, the vector is a plasmid, i.e., a circular double-stranded piece 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, vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and mammalian episomal vectors).In other embodiments of the invention, vectors (e.g., non-episomal mammalian vectors) can be integrated into the host cell genome upon introduction into the host cell, thereby replicating along with the host gene. Furthermore, 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").

[0262] 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 that may comprise, for example, a vector according to the present invention, as described above. The present invention also relates to host cells that comprise, for example, a nucleotide sequence encoding the heavy chain or its antigen-binding portions, a nucleotide sequence encoding the light chain or its antigen-binding portions, or both, of the first binding domain and / or the second binding domain of the binding molecule of the present 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.Because 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" when used herein.

[0263] The term "excipient" is used herein to describe any ingredient other than the compound(s) of the present invention.

[0264] The term "GITR-mediated disease or disorder" means all diseases or disorders that are either directly or indirectly related to GITR, including the etiology, development, progression, persistence, or pathology of the disease or disorder.

[0265] "Treat," "treatment," 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 "alleviate" a disease, disorder, or condition means reducing the severity and / or frequency of symptoms of the disease, disorder, or condition. Furthermore, references herein to "treatment" include references to curative, palliative, and prophylactic therapy.

[0266] In one aspect, the subject of treatment, or patient, is a mammal, preferably a human subject. Said subject may be male or female of any age.

[0267] The term "disorder" means any condition that can be ameliorated by treatment according to the present invention. This term is defined to include chronic and acute disorders or diseases, including pathological conditions that predispose a mammal to developing the disorder.

[0268] The terms "cancer" or "cancerous" refer to or describe a physiological condition in mammals typically characterized by unregulated (i) cell growth / proliferation. This definition encompasses both 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, hepatocellular carcinoma, gastric cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial 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.

[0269] 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 the selective damage, destruction or elimination from the human body of invasive pathogens, cells or tissues infected with pathogens, cancer cells or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues).

[0270] A "therapeutically effective amount" is defined as the amount of a therapeutic agent administered during treatment that will relieve, to a specified degree, one or more symptoms of the disease being treated.

[0271] The term "chronic" administration refers to continuous (long-term) administration of the agent(s), as opposed to acute (short-term) administration, so as to maintain the initial therapeutic effect (activity) over an extended period of time.

[0272] "Intermittent" use refers to treatment that is not administered consecutively without interruption, but is rather periodic in nature.

[0273] In the present description 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 as including the stated whole or group of wholes, but not excluding any other whole or group of wholes.

[0274] Detailed description of the invention

[0275] Antibody

[0276] The present invention relates to a monoclonal antibody that specifically binds to GITR.

[0277] In one aspect, the present invention relates to a monoclonal antibody or antigen-binding fragment thereof that specifically binds to GITR, comprising:

[0278] (a) a heavy chain variable domain comprising:

[0279] (i) CDR1 with an amino acid sequence selected from the group:

[0280] NYGMH (SEQ ID NO: 1) or YYWMY (SEQ ID NO: 12);

[0281] (ii) CDR2 with an amino acid sequence selected from the group:

[0282] VIWFDGSNKFYTDSVKG (SEQ ID NO: 2) or AISWNGGRTYYAESMKG (SEQ ID NO: 13) ;

[0283] (iii) CDR3 with an amino acid sequence selected from the group:

[0284] ELGGYYYDSSGFRPYYYGMDV (SEQ ID NO: 3) or NRYYSDPNYGMNL (SEQ ID NO: 14), and

[0285] (b) a light chain variable domain comprising:

[0286] (i) CDR1 with an amino acid sequence selected from the group:

[0287] RASQSIGSWLA (SEQ ID NO: 7) or TGTSTDIGTYKYIS (SEQ ID NO:

[0288] 17) ;

[0289] (ii) CDR2 with an amino acid sequence selected from the group:

[0290] AASTLQR (SEQ ID NO: 8) or GVSHRPS (SEQ ID NO: 18); (iii) CDR3 with an amino acid sequence selected from the group:

[0291] QQSHSHPLT (SEQ ID NO: 9) or SSYTSSGTW (SEQ ID NO: 19) .

[0292] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable domain that comprises CDRs 1, 2, and 3 with amino acid sequences represented by SEQ ID NOs: 1, 2, and 3, respectively.

[0293] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable domain that comprises CDRs 1, 2, and 3 with amino acid sequences represented by SEQ ID NOs: 12, 13, and 14, respectively.

[0294] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises a light chain variable domain that comprises CDRs 1, 2, and 3 with amino acid sequences represented by SEQ ID NOs: 7, 8, and 9, respectively.

[0295] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises a light chain variable domain that comprises CDRs 1, 2, and 3 with amino acid sequences represented by SEQ ID NOs: 17, 18, and 19, respectively.

[0296] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises:

[0297] - a heavy chain variable domain that contains CDRs 1, 2 and 3 with amino acid sequences represented by the sequences SEQ ID NO: 1, 2 and 3, respectively;

[0298] - a light chain variable domain that contains CDRs 1, 2 and 3 with amino acid sequences represented by SEQ ID NO: 7, 8 and 9, respectively.

[0299] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises:

[0300] - a heavy chain variable domain that contains CDRs 1, 2 and 3 with amino acid sequences represented by the sequences SEQ ID NO: 12, 13 and 14, respectively;

[0301] - a light chain variable domain that contains CDRs 1, 2 and 3 with amino acid sequences represented by SEQ ID NO: 17, 18 and 19, respectively.

[0302] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable domain that comprises an amino acid sequence that is at least 90% homologous to the amino acid sequence EVQLVQSGGGWQPGKSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWVAVIWFDGSNKFYTDSV KGRFTISRDNSKDTLSLQMNSLRAEDTAVYYCARELGGYYYDSSGFRPYYYGMDVWGQGTMVTV SS ( SEQ ID NO: 4). In some embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable domain that comprises an amino acid sequence

[0303] EVQLVQSGGGWQPGKSLRLSCAASGTFFSNYGMHWVRQAPGKGLEWVAVIWFDGSNKFYTDSV KGRFTISRDNSKDTLSLQMNSLRAEDTAVYYCARELGGYYYDSSGFRPYYYGMDVWGQGTMVTV SS ( SEQ ID NO: 4) .

[0304] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable domain that comprises an amino acid sequence that is at least 90% homologous to the amino acid sequence

[0305] QVQLVQSGGGLVQPGGSLRLSCAASGFFSYWMYWVRQAPGKGLEWVSAISWNGGRTY YAESMKGRFTISRDNAQNTLYLQMNSLKSEDTAVYYCAKNRYYSDPNYGMNLWGKGTTVTVSS

[0306] ( SEQ ID NO: 15) .

[0307] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable domain that comprises the amino acid sequence

[0308] QVQLVQSGGGLVQPGGSLRLSCAASGFTFSYYWMYWVRQAPGKGLEWVSAISWNGGRTYYAESM KGRFTISRDNAQNTLYLQMNSLKSEDTAVYYCAKNRYYSDPNYGMNLWGKGTTVTVSS ( SEQ

[0309] ID NO: 15) .

[0310] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises a light chain variable domain that comprises an amino acid sequence that is at least 90% homologous to the amino acid sequence

[0311] DVVMTQSPSSVSASVGDRVTITCRASQS IGSWLAWYQQKPGEAPKLLIYAASTLQRGVP SRFSGGGYGTEFTLTISSLQPEDFATYFCQQSHSHPLTFGGGTKVEIK ( SEQ ID NO: 10) .

[0312] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises a light chain variable domain that comprises the amino acid sequence DVVMTQSPSSVSASVGDRVTITCRASQS IGSWLAWYQQKPGEAPKLLIYAASTLQRGVPSRFSG GGYGTEFTLTISSLQPEDFATYFCQQSHSHPLTFGGGTKVEIK (SEQ ID NO: 10).

[0313] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises a light chain variable domain that comprises an amino acid sequence that is at least 90% homologous to the amino acid sequence QSALTQPASVSGSPGQS ITISCTGTSTDIGTYKYISWYQQHPGKAPKLI IYGVSHRPSGVSDRF SGSKSDNTASLTISGLQAEDEADYYCSSYTSSGTWFGGGTKVTVL (SEQ ID NO: 20).

[0314] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises a light chain variable domain that comprises the amino acid sequence QSALTQPASVSGSPGQS ITISCTGTSTDIGTYKYISWYQQHPGKAPKLI IYGVSHRPSGVSDRF SGSKSDNTASLTISGLQAEDEADYYCSSYTSSGTWFGGGTKVTVL (SEQ ID NO: 20).

[0315] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises:

[0316] a heavy chain variable domain that comprises an amino acid sequence at least 90% homologous to the amino acid sequence EVQLVQSGGGWQPGKSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWVAVIWFDGSNKFYTDSV KGRFTISRDNSKDTLSLQMNSLRAEDTAVYYCARELGGYYYDSSGFRPYYYGMDVWGQGTMVTV SS (SEQ ID NO: 4);

[0317] a light chain variable domain that contains an amino acid sequence that is at least 90% homologous to the amino acid sequence of

[0318] DVVMTQSPSSVSASVGDRVTITCRASQSIGSWLAWYQQKPGEAPKLLIYAASTLQRGVPSRFSG GGYGTEFTLTISSLQPEDFATYFCQQSHSHPLTFGGGTKVEIK ( SEQ ID NO: 10) .

[0319] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises:

[0320] the variable domain of the heavy chain, which contains the amino acid sequence

[0321] EVQLVQSGGGWQPGKSLRLSCAASGTFFSNYGMHWVRQAPGKGLEWVAVIWFDGSNKFYTDSV KGRFTISRDNSKDTLSLQMNSLRAEDTAVYYCARELGGYYYDSSGFRPYYYGMDVWGQGTMVTV SS ( SEQ ID NO: 4) ;

[0322] the variable domain of the light chain, which contains the amino acid sequence

[0323] DVVMTQSPSSVSASVGDRVTITCRASQSIGSWLAWYQQKPGEAPKLLIYAASTLQRGVPSRFSG GGYGTEFTLTISSLQPEDFATYFCQQSHSHPLTFGGGTKVEIK (SEQ ID NO: 10) .

[0324] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises:

[0325] a heavy chain variable domain that contains an amino acid sequence that is at least 90% homologous to the amino acid sequence

[0326] QVQLVQSGGGLVQPGGSLRLSCAASGFTFSYYWMYWVRQAPGKGLEWVSAISWNGGRTYYAESM KGRFTISRDNAQNTLYLQMNSLKSEDTAVYYCAKNRYYSDPNYGMNLWGKGTTVTVSS ( SEQ ID NO: 15) ;

[0327] a light chain variable domain that contains an amino acid sequence that is at least 90% homologous to the amino acid sequence of

[0328] QSALTQPASVSGSPGQSITISCTGTSTDIGTYKYISWYQQHPGKAPKLIIYGVSHRPSGVSDRF SGSKSDNTASLTISGLQAEDEADYYCSSYTSSGTWFGGGTKVTVL ( SEQ ID NO: 20) .

[0329] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises:

[0330] the variable domain of the heavy chain, which contains the amino acid sequence

[0331] QVQLVQSGGGLVQPGGSLRLSCAASGFTFSYYWMYWVRQAPGKGLEWVSAISWNGGRTYYAESM KGRFTISRDNAQNTLYLQMNSLKSEDTAVYYCAKNRYYSDPNYGMNLWGKGTTVTVSS ( SEQ ID NO: 15) ;

[0332] the variable domain of the light chain, which contains the amino acid sequence

[0333] QSALTQPASVSGSPGQSITISCTGTSTDIGTYKYISWYQQHPGKAPKLIIYGVSHRPSGVSDRF SGSKSDNTASLTISGLQAEDEADYYCSSYTSSGTWFGGGTKVTVL (SEQ ID NO: 20) .

[0334] In some embodiments, the GITR-specific monoclonal antibody is a full-length IgG antibody. In some embodiments, the IgG monoclonal antibody is of the human IgG1, IgG2, IgG3, or IgG4 isotype.

[0335] In some embodiments, the IgG monoclonal antibody is of the human IgG1 isotype.

[0336] In some embodiments, a monoclonal antibody specific to GITR comprises an E345R mutation in the Fc region to increase agonist properties, antibody-dependent cellular cytotoxicity (ADCC), but not complement-dependent cytotoxicity (CDC).

[0337] In some embodiments, the monoclonal antibody comprises a heavy chain comprising an amino acid sequence that is at least 90% homologous to the sequence

[0338] EVQLVQSGGGWQPGKSLRLSCAASGTFFSNYGMHWVRQAPGKGLEWVAVIWFDGSNKFYTDSV KGRFTISRDNSKDTLSLQMNSLRAEDTAVYYCARELGGYYYDSSGFRPYYYGMDVWGQGTMVTV SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLF PPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK ( SEQ ID NO: 5) .

[0339] In some embodiments, the monoclonal antibody comprises a heavy chain comprising an amino acid sequence

[0340] EVQLVQSGGGWQPGKSLRLSCAASGTFFSNYGMHWVRQAPGKGLEWVAVIWFDGSNKFYTDSV KGRFTISRDNSKDTLSLQMNSLRAEDTAVYYCARELGGYYYDSSGFRPYYYGMDVWGQGTMVTV SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLF PPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK (SEQ ID NO: 5) .

[0341] In some embodiments, the monoclonal antibody comprises a heavy chain comprising an amino acid sequence that is at least 90% homologous to the sequence

[0342] EVQLVQSGGGWQPGKSLRLSCAASGTFFSNYGMHWVRQAPGKGLEWVAVIWFDGSNKFYTDSV KGRFTISRDNSKDTLSLQMNSLRAEDTAVYYCARELGGYYYDSSGFRPYYYGMDVWGQGTMVTV SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLF PPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRRPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK (SEQ ID NO: 6) .

[0343] In some embodiments, the monoclonal antibody comprises a heavy chain comprising an amino acid sequence

[0344] EVQLVQSGGGWQPGKSLRLSCAASGTFFSNYGMHWVRQAPGKGLEWVAVIWFDGSNKFYTDSV KGRFTISRDNSKDTLSLQMNSLRAEDTAVYYCARELGGYYYDSSGFRPYYYGMDVWGQGTMVTV SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLF PPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRRPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK (SEQ ID NO: 6) .

[0345] In some embodiments, the monoclonal antibody comprises a light chain comprising an amino acid sequence that is at least 90% homologous to the sequence

[0346] DVVMTQSPSSVSASVGDRVTITCRASQS IGSWLAWYQQKPGEAPKLLIYAASTLQRGVPSRFSG GGYGTEFTLTISSLQPEDFATYFCQQSHSHPLTFGGGTKVEIKRTVAAPSVFI FPPSDEQLKSG TASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC ( SEQ ID NO: 11) .

[0347] In some embodiments, the monoclonal antibody comprises a light chain comprising an amino acid sequence

[0348] DVVMTQSPSSVSASVGDRVTITCRASQS IGSWLAWYQQKPGEAPKLLIYAASTLQRGVPSRFSG GGYGTEFTLTISSLQPEDFATYFCQQSHSHPLTFGGGTKVEIKRTVAAPSVFI FPPSDEQLKSG TASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 11) .

[0349] In some embodiments, the monoclonal antibody comprises:

[0350] - a heavy chain containing an amino acid sequence at least 90% homologous to the sequence

[0351] EVQLVQSGGGWQPGKSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWVAVIWFDGSNKFYTDSV KGRFTISRDNSKDTLSLQMNSLRAEDTAVYYCARELGGYYYDSSSGFRPYYYGMDVWGQGTMVTV SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPPVTVSWNSGALTCGVHTFPAVLQSSGL YSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPPAPELLGGPSVFLF PPKPKDTLMISRTPEVTCVWDVSHEDPEWKFNWYVDGVEVHNACTKPREEQYNSTYRWSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK (SEQ ID NO: 5) ;

[0352] is a light chain containing an amino acid sequence at least 90% homologous to the sequence

[0353] DVVMTQSPSSVSASVGDRVTITCRASQS IGSWLAWYQQKPGEAPKLLIYAASTLQRGVPSRFSG GGYGTEFTLTISSLQPEDFATYFCQSHSHPLTFGGGTKVEIKRTVAAPSVFI FPPSDEQLKSG TASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 11) .

[0354] In some embodiments, the monoclonal antibody that specifically binds to GITR is BCD166-01-001.

[0355] Monoclonal antibody BCD166-01-001 includes:

[0356] - a heavy chain containing the amino acid sequence EVQLVQSGGGWQPGKSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWVAVIWFDGSNKFYTDSV KGRFTISRDNSKDTLSLQMNSLRAEDTAVYYCARELGGYYYDSSGFRPYYYGMDVWGQGTMVTV SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLF PPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK (SEQ ID NO: 5) ;

[0357] - a light chain containing the amino acid sequence DVVMTQSPSSVSASVGDRVTITCRASQSIGSWLAWYQQKPGEAPKLLIYAASTLQRGVPSRFSG GGYGTEFTLTISSLQPEDFATYFCQQSHSHPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSG TASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 11).

[0358] In some embodiments, the monoclonal antibody comprises:

[0359] - a heavy chain containing an amino acid sequence at least 90% homologous to the sequence

[0360] EVQLVQSGGGWQPGKSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWVAVIWFDGSNKFYTDSV KGRFTISRDNSKDTLSLQMNSLRAEDTAVYYCARELGGYYYDSSSGFRPYYYGMDVWGQGTMVTV SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPPVTVSWNSGALTCGVHTFPAVLQSSGL YSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPPAPELLGGPSVFLF PPKPKDTLMISRTPEVTCVWDVSHEDPEWKFNWYVDGVEVHNACTKPREEQYNSTYRWSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRRPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYCTTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK (SEQ ID NO: b) ;

[0361] is a light chain containing an amino acid sequence at least 90% homologous to the sequence

[0362] DVVMTQSPSSVSASVGDRVTITCRASQSIGSWLAWYQQKPGEAPKLLIYAASTLQRGVPSRFSG GGYGTEFTLTISSLQPEDFATYFCQSHSHPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSG TASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 11) .

[0363] In some embodiments, the monoclonal antibody that specifically binds to GITR is BCD166-02-001.

[0364] BCD166-02-001 differs from BCD166-01-001 by one mutation E345R in the Fc fragment to increase agonist properties, antibody-dependent cellular cytotoxicity (ADCC) but not complement-dependent cytotoxicity (CDC).

[0365] Monoclonal antibody BCD166-02-001 includes:

[0366] - heavy chain containing the amino acid sequence EVQLVQSGGGWQPGKSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWVAVIWFDGSNKFYTDSV KGRFTISRDNSKDTLSLQMNSLRAEDTAVYYCARELGGYYDSSGFRPYYYGMDVWGQGTMVTV SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLF PPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRRPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK (SEQ ID NO: b);

[0367] - a light chain comprising the amino acid sequence DVVMTQSPSSVSASVGDRVTITCRASQSIGSWLAWYQQKPGEAPKLLIYAASTLQRGVPSRFSG GGYGTEFTLTISSLQPEDFATYFCQQSHSHPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSG TASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 11). In some embodiments, the monoclonal antibody comprises a heavy chain comprising an amino acid sequence at least 90% homologous to the sequence

[0368] QVQLVQSGGGLVQPGGSLRLSCAASGFTFSYYWMYWVRQAPGKGLEWVSAISWNGGRTYYAESM KGRFTISRDNAQNTLYLQMNSLKSEDTAVYYCAKNRYYSDPNYGMNLWGKGTTVTVSSASTKGP SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWT VPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLN GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK ( SEQ ID NO: 16) .

[0369] In some embodiments, the monoclonal antibody comprises a heavy chain comprising an amino acid sequence

[0370] QVQLVQSGGGLVQPGGSLRLSCAASGFTFSYYWMYWVRQAPGKGLEWVSAISWNGGRTYYAESM KGRFTISRDNAQNTLYLQMNSLKSEDTAVYYCAKNRYYSDPNYGMNLWGKGTTVTVSSASTKGP SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWT VPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLN GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK (SEQ ID NO: 16) .

[0371] In some embodiments, the monoclonal antibody comprises a light chain comprising an amino acid sequence that is at least 90% homologous to the sequence

[0372] QSALTQPASVSGSPGQS ITISCTGTSTDIGTYKYISWYQQHPGKAPKLI IYGVSHRPSGVSDRF SGSKSDNTASLTISGLQAEDEADYYCSSYTSSGTWFGGGTKVTVLGQPKAAPSVTLFPPSSEE LQANKATLVCLISDFYРGAVTVAWKADSSРVKAGVETTТРSKQSNNKYAASSYLSLTREQWKSН RSYSCQVTHEGSTVEKTVAPTECS ( SEQ ID NO: 21) .

[0373] In some embodiments, the monoclonal antibody comprises a light chain comprising an amino acid sequence

[0374] QSALTQPASVSGSPGQS ITISCTGTSTDIGTYKYISWYQQHPGKAPKLIIYGVSHRPSGVSDRF SGSKSDNTASLTISGLQAEDEADYYCSSYTSSGTWFGGGTKVTVLGQPKAAPSVTLFPPSSEE LQANKATLVCLISDFYРGAVTVAWKADSSРVKAGVETTТРSKQSNNKYAASSYLSLTREQWKSН RSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 21) .

[0375] In some embodiments, the monoclonal antibody comprises:

[0376] - a heavy chain comprising an amino acid sequence at least 90% homologous to the sequence QVQLVQSGGGLVQPGGSLRLSCAASGFTFSYYWMYWVRQAPGKGLEWVSAISWNGGRTYYAESM KGRFTISRDNAQNTLYLQMNSLKSEDTAVYYCAKNRYYSDPNYGMNLWGKGTTVTVSSASTKGP SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWT VPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLN GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK (SEQ ID NO: 16) . - a light chain comprising an amino acid sequence at least 90% homologous to the sequence QSALTQPASVSGSPGQSITISCTGTSTDIGTYKYISWYQQHPGKAPKLIIYGVSHRPSGVSDRF SGSKSDNTASLTISGLQAEDEADYYCSSYTSSGTWFGGGTKVTVLGQPKAAPSVTLFPPSSEE LQANKATLVCLISDFYРGAVTVAWKADSSРVKAGVEТТТРSKQSNNKYAASSYLSLТРЕQWKSН RSYSCQVTHEGSTVEKTVAPTECS ( SEQ ID NO: 21).

[0377] In some embodiments, the monoclonal antibody that specifically binds to GITR is BCD166-01-014.

[0378] Monoclonal antibody BCD166-01-014 includes:

[0379] - heavy chain containing the amino acid sequence QVQLVQSGGGLVQPGGSLRLSCAASGFTFSYYWMYWVRQAPGKGLEWVSAISWNGGRTYYAESM KGRFTISRDNAQNTLYLQMNSLKSEDTAVYYCAKNRYYSDPNYGMNLWGKGTTVTVSSASTKGP SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLN GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK ( SEQ ID NO: 16) .

[0380] - a light chain containing the amino acid sequence QSALTQPASVSGSPGQSITISCTGTSTDIGTYKYISWYQQHPGKAPKLIIYGVSHRPSGVSDRF SGSKSDNTASLTISGLQAEDEADYYCSSYTSSGTWFGGGTKVTVLGQPKAAPSVTLFPPSSEE LQANKATLVCLISDFYРGAVTVAWKADSSРVKAGVEТТТРSKQSNNKYAASSYLSLТРЕQWKSН RSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 21).

[0381] Nucleic acid molecules

[0382] The present invention also relates to nucleic acid molecules, namely, sequences encoding a monoclonal antibody that specifically binds to GITR, according to the invention, as described herein, optionally including any peptide linker sequence connecting them.

[0383] A reference to a nucleotide sequence includes its complement, unless otherwise indicated. Thus, a reference to a nucleic acid having a certain sequence should be understood as including its complementary strand with its complementary sequence. The term "polynucleotide" as referred to 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 relates to a nucleic acid molecule comprising a nucleotide sequence that encodes an amino acid sequence selected from SEQ ID NOs: 1-21. The nucleic acid molecule may also comprise any combination of said nucleotide sequences.

[0384] In one aspect, the present invention relates to a nucleic acid that comprises a nucleotide sequence that encodes a monoclonal antibody or antigen-binding fragment thereof that specifically binds to GITR, and comprises:

[0385] (a) a heavy chain variable domain comprising:

[0386] (i) CDR1 with an amino acid sequence selected from the group:

[0387] NYGMH (SEQ ID NO: 1) or YYWMY (SEQ ID NO: 12);

[0388] (ii) CDR2 with an amino acid sequence selected from the group:

[0389] VIWFDGSNKFYTDSVKG (SEQ ID NO: 2) or AISWNGGRTYYAESMKG (SEQ ID NO: 13) ;

[0390] (iii) CDR3 with an amino acid sequence selected from the group:

[0391] ELGGYYYDSSGFRPYYYGMDV (SEQ ID NO: 3) or NRYYSDPNYGMNL (SEQ ID NO: 14), and

[0392] (b) a light chain variable domain comprising:

[0393] (i) CDR1 with an amino acid sequence selected from the group:

[0394] RASQSIGSWLA (SEQ ID NO: 7) or TGTSTDIGTYKYIS (SEQ ID NO:

[0395] 17) ;

[0396] (ii) CDR2 with an amino acid sequence selected from the group:

[0397] AASTLQR (SEQ ID NO: 8) or GVSHRPS (SEQ ID NO: 18);

[0398] (iii) CDR3 with an amino acid sequence selected from the group:

[0399] QQSHSHPLT (SEQ ID NO: 9) or SSYTSSGTW (SEQ ID NO: 19) .

[0400] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a monoclonal antibody or antigen-binding fragment thereof that includes a heavy chain variable domain that contains CDRs 1, 2, and 3 with amino acid sequences represented by SEQ ID NOs: 1, 2, and 3, respectively.

[0401] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a monoclonal antibody or antigen-binding fragment thereof, which comprises a heavy chain variable domain that comprises CDRs 1, 2, and 3 with amino acid sequences represented by the sequences of SEQ ID NOs: 12, 13, and 14, respectively. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a monoclonal antibody or antigen-binding fragment thereof, which comprises a light chain variable domain that comprises CDRs 1, 2, and 3 with amino acid sequences represented by the sequences of SEQ ID NOs: 7, 8, and 9, respectively.

[0402] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a variant monoclonal antibody or antigen-binding fragment thereof that includes a light chain variable domain that comprises CDRs 1, 2, and 3 with amino acid sequences represented by SEQ ID NOs: 17, 18, and 19, respectively.

[0403] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a monoclonal antibody or antigen-binding fragment thereof that includes:

[0404] - a heavy chain variable domain that contains CDRs 1, 2 and 3 with amino acid sequences represented by the sequences SEQ ID NO: 1, 2 and 3, respectively;

[0405] - a light chain variable domain that contains CDRs 1, 2 and 3 with amino acid sequences represented by SEQ ID NO: 7, 8 and 9, respectively.

[0406] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a monoclonal antibody or antigen-binding fragment thereof that includes:

[0407] - a heavy chain variable domain that contains CDRs 1, 2 and 3 with amino acid sequences represented by the sequences SEQ ID NO: 12, 13 and 14, respectively;

[0408] - a light chain variable domain that contains CDRs 1, 2 and 3 with amino acid sequences represented by SEQ ID NO: 17, 18 and 19, respectively.

[0409] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a monoclonal antibody or antigen-binding fragment thereof that includes a heavy chain variable domain that comprises an amino acid sequence that is at least 90% homologous to the amino acid sequence of SEQ ID NO: 4.

[0410] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a monoclonal antibody or antigen-binding fragment thereof that includes a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 4.

[0411] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a monoclonal antibody or antigen-binding fragment thereof that includes a heavy chain variable domain that comprises an amino acid sequence that is at least 90% homologous to the amino acid sequence of SEQ ID NO: 15.

[0412] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a monoclonal antibody or antigen-binding fragment thereof that includes a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 15.

[0413] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a monoclonal antibody or antigen-binding fragment thereof that includes a light chain variable domain that comprises an amino acid sequence that is at least 90% homologous to the amino acid sequence of SEQ ID NO: 10.

[0414] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a monoclonal antibody or antigen-binding fragment thereof that includes a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 10.

[0415] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a monoclonal antibody or antigen-binding fragment thereof that includes a light chain variable domain that comprises an amino acid sequence that is at least 90% homologous to the amino acid sequence of SEQ ID NO: 20.

[0416] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a monoclonal antibody or antigen-binding fragment thereof that includes a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 20.

[0417] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a monoclonal antibody or antigen-binding fragment thereof that includes:

[0418] a heavy chain variable domain that comprises an amino acid sequence that is at least 90% homologous to the amino acid sequence of SEQ ID NO: 4;

[0419] a light chain variable domain that comprises an amino acid sequence that is at least 90% homologous to the amino acid sequence of SEQ ID NO: 10.

[0420] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a monoclonal antibody or antigen-binding fragment thereof that includes:

[0421] a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 4;

[0422] a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 10. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a monoclonal antibody or antigen-binding fragment thereof that includes:

[0423] a heavy chain variable domain that comprises an amino acid sequence that is at least 90% homologous to the amino acid sequence of SEQ ID NO: 15;

[0424] a light chain variable domain that comprises an amino acid sequence that is at least 90% homologous to the amino acid sequence of SEQ ID NO: 20.

[0425] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a monoclonal antibody or antigen-binding fragment thereof that includes:

[0426] a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 15;

[0427] a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 20.

[0428] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a monoclonal antibody that specifically binds to GITR, which is a full-length IgG antibody.

[0429] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes an IgG monoclonal antibody that is of the human IgG1, IgG2, IgG3, or IgG4 isotype.

[0430] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes an IgG monoclonal antibody that is of the human IgG1 isotype.

[0431] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a monoclonal antibody that specifically binds to GITR, which includes an E345R mutation in the Fc region to increase agonist properties, antibody-dependent cellular cytotoxicity (ADCC), but not complement-dependent cytotoxicity (CDC).

[0432] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a monoclonal antibody that includes a heavy chain comprising an amino acid sequence that is at least 90% homologous to the sequence of SEQ ID NO: 5.

[0433] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a monoclonal antibody that includes a heavy chain comprising the amino acid sequence of SEQ ID NO: 5.

[0434] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a monoclonal antibody that includes a heavy chain comprising an amino acid sequence that is at least 90% homologous to the sequence of SEQ ID NO: b.

[0435] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a monoclonal antibody that includes a heavy chain comprising the amino acid sequence of SEQ ID NO: b.

[0436] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a monoclonal antibody that includes a light chain that comprises an amino acid sequence that is at least 90% homologous to the sequence of SEQ ID NO: 11.

[0437] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a monoclonal antibody that includes a light chain comprising the amino acid sequence of SEQ ID NO: 11.

[0438] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a monoclonal antibody that includes:

[0439] - a heavy chain comprising an amino acid sequence at least 90% homologous to the sequence of SEQ ID NO:

[0440] 5;

[0441] - a light chain comprising an amino acid sequence at least 90% homologous to the sequence of SEQ ID NO: 11.

[0442] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a monoclonal antibody that includes:

[0443] - a heavy chain containing the amino acid sequence SEQ ID NO: 5;

[0444] - a light chain comprising the amino acid sequence of SEQ ID NO: 11.

[0445] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a monoclonal antibody that includes:

[0446] - a heavy chain comprising an amino acid sequence at least 90% homologous to the sequence of SEQ ID NO: 6;

[0447] - a light chain comprising an amino acid sequence at least 90% homologous to the sequence of SEQ ID NO: 11.

[0448] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a monoclonal antibody that includes:

[0449] - a heavy chain comprising the amino acid sequence of SEQ ID NO: 6; - a light chain comprising the amino acid sequence of SEQ ID NO: 11.

[0450] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a monoclonal antibody that includes a heavy chain comprising an amino acid sequence that is at least 90% homologous to the sequence of SEQ ID NO: 16.

[0451] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a monoclonal antibody that includes a heavy chain comprising the amino acid sequence of SEQ ID NO: 16.

[0452] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a monoclonal antibody that includes a light chain that comprises an amino acid sequence that is at least 90% homologous to the sequence of SEQ ID NO: 21.

[0453] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a monoclonal antibody that includes a light chain comprising the amino acid sequence of SEQ ID NO: 21.

[0454] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a monoclonal antibody that includes:

[0455] - a heavy chain comprising an amino acid sequence at least 90% homologous to the sequence of SEQ ID NO: 16;

[0456] - a light chain comprising an amino acid sequence at least 90% homologous to the sequence of SEQ ID NO: 21.

[0457] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a monoclonal antibody that includes:

[0458] - a heavy chain containing the amino acid sequence SEQ ID NO: 16;

[0459] - a light chain comprising the amino acid sequence of SEQ ID NO: 21.

[0460] In some embodiments, the nucleic acid is

[0461] DNA.

[0462] In any of the above embodiments of the invention, the nucleic acid molecules may be isolated.

[0463] The nucleic acid molecule of the present invention can be isolated from any source that produces a monoclonal antibody that specifically binds to GITR. In certain embodiments, the nucleic acid molecule of the present invention can be synthesized rather than isolated. In one embodiment, nucleic acid molecules encoding the VH (SEQ ID NO: 4 or SEQ ID NO: 15) or VL (SEQ ID NO: 10 or SEQ ID NO: 20) domains are converted into full-length antibody genes by insertion into an expression vector already encoding the constant domains of the heavy chain (CH) or light chain (CL), respectively, such that the VH segment is operably linked to the CH segment(s) in the vector and / or the VL segment is operably linked to the CL segment in the vector.In another embodiment of the invention, nucleic acid molecules encoding the VH and / or VL domains are converted into genes along the entire length of the antibody by joining, for example, by ligation, a nucleic acid molecule encoding the VH and / or VL domains to a nucleic acid molecule encoding the 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.

[0464] Nucleic acid molecules can be used to express large amounts of a recombinant monoclonal antibody that specifically binds to GITR.

[0465] Vector

[0466] In another aspect, the present invention relates to a vector suitable for expressing any of the nucleotide sequences described herein.

[0467] The present invention relates to vectors containing nucleic acid molecules encoding any of the amino acid sequences of a monoclonal antibody that specifically binds to GITR, or portions thereof (e.g., the heavy chain sequences of the first and / or heavy and / or light chain of the second binding domains) as described herein. The present invention further relates to vectors containing nucleic acid molecules encoding fusion proteins, modified antibodies, and antibody fragments.

[0468] In some embodiments, a monoclonal antibody that specifically binds to GITR according to 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), obtained as described above, into 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 viruses, cosmids, YACs, EBV-derived episomes, and the like.DNA molecules can be ligated into a vector such that 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 part or all of the first and second binding domain sequences (e.g., heavy and light chain sequences, where the binding domain comprises a heavy and light chain sequence) can be introduced into separate vectors. In one embodiment of the invention, any combination of the aforementioned DNA molecules is introduced into the same expression vector.DNA molecules can be introduced into the expression vector by standard methods (e.g., by ligation of complementary restriction sites on the antibody gene fragment and the vector, or by blunt-end ligation if restriction sites are not present).

[0469] A suitable vector is one that encodes functionally complete CH or CL sequences of human immunoglobulin with an appropriate restriction site engineered so that any VH or VL sequence can be easily incorporated and expressed as described above. The HC and LC-encoding 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 intronic sequences can be either in the variable or constant regions of the antibody chains, or in both variable and constant regions when multiple introns are used. Termination of polyadenylation and transcription can occur downstream of the native chromosomal site of the encoded regions.The recombinant expression vector may also encode a signal peptide that facilitates production of the antibody chain by the host cell. The antibody chain gene may be cloned into the vector such that the signal peptide is linked to the reading frame of the amino terminus of the immunoglobulin chain. The signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide of a non-immunoglobulin protein). 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 the expression vector, including the choice of regulatory sequences, may depend on factors such as the selection of the host cell for transformation, the expression level of the desired protein, etc.Preferred regulatory sequences for a mammalian expression host 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) (e.g., the CMV promoter / enhancer), simian virus 40 (SV40) (e.g., the SV40 promoter / enhancer), adenovirus (e.g., the 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 descriptions of viral regulatory elements and sequences thereof, see, for example, U.S. Patents 5,168,062, 4,510,245, and 4,968,615. Methods for expressing binding molecules such as plant antibodies, including the description of promoters and vectors, as well as plant transformation, are known in the art. See, for example, U.S. Patent 6,517,529.Methods for expressing polypeptides in bacterial cells or fungal cells, such as yeast cells, are also well known in the art.

[0470] In addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors of the invention may carry additional sequences, such as sequences that regulate vector replication in host cells (e.g., origins of replication) and selectable marker genes. A selectable marker gene facilitates the selection of host cells into which the vector has been introduced (see, e.g., U.S. Patents 4,399,216, 4,634,665, and 5,179,017). For example, a selectable marker gene typically confers resistance to drugs such as G418, hygromycin, or methotrexate on the host cell into which the vector has been introduced. For example, selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr host cells for methotrexate selection / amplification), the neo gene (for G418 selection), and the glutamate synthetase gene.

[0471] The term "expression control sequence" as used herein refers to polynucleotide sequences that are necessary to influence the expression and processing of coding sequences to which they are ligated. Expression control sequences include appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., the Kozak consensus sequence); sequences that enhance protein stability; and, optionally, 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.

[0472] Host cells

[0473] An additional aspect of the present invention relates to methods for producing a monoclonal antibody that specifically binds to GITR according to the invention. One embodiment of the invention relates to a method for producing a monoclonal antibody that specifically binds to GITR, as defined herein, comprising producing a recombinant host cell capable of expressing a monoclonal antibody that specifically binds to GITR, culturing said host cell under conditions suitable for the expression of a monoclonal antibody that specifically binds to GITR, and isolating the resulting monoclonal antibody that specifically binds to GITR. A monoclonal antibody that specifically binds to GITR, obtained by such expression in such recombinant host cells, is referred to herein as a "recombinant monoclonal antibody that specifically binds to GITR."The invention also relates to progeny cells of such host cells and a monoclonal antibody that specifically binds to GITR, obtained similarly.

[0474] Nucleic acid molecules encoding a monoclonal antibody that specifically binds to GITR, according to the invention, and vectors containing these nucleic acid molecules can be used to transfect a suitable mammal or its cell, a plant or its cell, or a bacterial or yeast host cell. The transformation 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 a nucleic acid and a positively charged polymer, transfection with a nucleic acid-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 can be introduced into mammalian cells by viral vectors.Methods for transfecting cells are well known in the art. See, for example, U.S. Patents 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.

[0475] Mammalian cell lines used as transformation hosts are well known in the art and include a variety of immortalized cell lines readily 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 (BHK) cells, African green monkey kidney (COS) cells, human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, and a number of other cell lines. Cell lines are selected by determining which cell lines have high expression levels and provide the desired characteristics of the protein produced. Other cell lines that can be used include insect cell lines such as Sf9 or Sf21 cells.When recombinant expression vectors encoding a monoclonal antibody that specifically binds to GITR 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, by isolating the antibodies into the growth medium in which the host cells are grown. The monoclonal antibody that specifically binds to GITR can be isolated from the growth medium using standard protein purification methods. Plant host cells, for example, include Nicotiana, Arabidopsis, duckweed, corn, wheat, potato, etc. Bacterial host cells include Escherichia and Streptomyces species. Yeast host cells include Schizosaccharomyces pombe, Saccharomyces cerevisiae, and Pichia pastoris.Furthermore, the production level of the monoclonal antibody that specifically binds to GITR according to the present invention from a producing cell line can be enhanced using a number of known methods. For example, the glutamine synthetase gene expression system (GS system) is widely used 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.

[0476] It is likely that monoclonal antibodies that specifically bind to GITR from different cell lines or transgenic animals will differ from each other in their glycosylation profiles. However, monoclonal antibodies that specifically bind to GITR, encoded by the nucleic acid molecules described herein or comprising the amino acid sequences provided herein, are part of the present invention, regardless of the glycosylation state of the binding molecules and, in general, regardless of the presence or absence of post-translational modifications.

[0477] Obtaining antibodies

[0478] The invention also relates to methods and processes for producing a monoclonal antibody that specifically binds to GITR, and antigen-binding fragments thereof.

[0479] Monoclonal antibodies

[0480] Monoclonal antibodies can be generated, for example, by the hybridoma method first described by Kohler et al., Nature, 256, 1975, p. 495, or by recombinant DNA techniques (US 4816567).

[0481] Using the hybridoma method, a mouse or other suitable host animal, such as a hamster, is immunized using the above-described method to induce the formation of lymphocytes that produce or can produce antibodies capable of specifically binding to the protein used for immunization. Alternatively, 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 hybridoma cells.

[0482] The resulting hybridoma cells 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 parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT or HPRT), the hybridoma culture medium typically contains hypoxanthine, aminopterin, and thymidine (HAT medium), substances that inhibit the growth of HGPRT-deficient cells.

[0483] Preferred myeloma cell fusion components are those that readily fuse, maintain stable, high levels of antibody production by the selected antibody-producing cells, and are sensitive to a selective medium for selecting unrelated parental cells. Preferred myeloma cell lines are murine myeloma lines, such as the murine tumor cell lines MOPC-21 and MPC-11, available from the Salk Institute Cell Distribution Center, San Diego, CA, USA, and the SP-2 or X63-Ad8-653 lines, available from the American Type Culture Collection, Rockville, MD, 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).

[0484] Preferably, the binding specificity of monoclonal antibodies produced using hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay such as a radioimmunoassay (RIA) or an enzyme-linked immunosorbent assay (ELISA).

[0485] The binding affinity of a monoclonal antibody can, for example, be determined using the Scatchard analysis described by Munson et al., Anal. Biochem., 107, 1980, p. 220.

[0486] Once hybridoma cells producing antibodies of the desired specificity, affinity, and / or activity are identified, the clones can be subcloned using limiting dilutions and grown using standard methods. Suitable media for this purpose include, for example, DMEM or RPMI-1640. Furthermore, hybridoma cells can be grown in vivo as ascites tumors in animals, for example, by intraperitoneal (ip) injection of the cells into mice.

[0487] Monoclonal antibodies secreted by subclones can be separated from the culture medium, ascites fluid, or serum using conventional antibody purification methods, such as affinity chromatography (e.g., using protein A or protein Q-Sepharose), or ion exchange chromatography, hydroxyapatite chromatography, gel electrophoresis, dialysis, etc.

[0488] DNA encoding monoclonal antibodies can be readily isolated and sequenced using conventional procedures (e.g., using oligonucleotide probes that specifically bind to genes encoding the heavy and light chains of mouse antibodies). Hybridoma cells are a preferred source of such DNA. Once isolated, the DNA can be incorporated into expression vectors, which are then transfected into host cells such as E. coli, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells, which do not produce antibody protein without transfection, resulting in the synthesis of monoclonal antibodies in recombinant host cells.

[0489] According to another embodiment of the invention, monoclonal antibodies or antibody fragments can be isolated from antibody phage libraries generated using the methods described in McCafferty et al., Nature, 348 (1990), pp. 552-554. Clackson et al., Nature, 352 (1991), pp. 624-628 and Marks et al., J. Mol. Biol., 222 (1991), pp. 581-597 describe the isolation of murine 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 for constructing very large phage libraries (Waterhouse et al., Nucl. Acids. Res., 21, 1991, pp. 2265–2266). These methods thus represent a viable alternative to traditional monoclonal antibody hybridoma-based isolation methods.

[0490] The DNA encoding the antibody may also be modified, for example, to produce chimeric or fusion antibody polypeptides, for example by replacing the heavy and light chain constant region sequences (CH and CL) with homologous murine sequences (US 4816567 and Morrison et al., Proc. Natl. Acad. Sci. USA: 81, 1984, p. 6851) or by covalently linking the immunoglobulin coding sequence 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 the antigen-binding site of an antibody, creating a chimeric bivalent antibody that contains one antigen-binding site specific for an antigen and another antigen-binding site specific for a different antigen.

[0491] Human antibodies and a phage display library-based approach

[0492] It is now possible to produce transgenic animals (e.g., mice) that, upon immunization, can produce a full spectrum of human antibodies without producing endogenous immunoglobulin. For example, it has been described that homozygous deletion of the antibody heavy chain gene junction regions (Dn segment) in chimeric and germline mutant mice results in complete inhibition of endogenous antibody production. Transfer of a human immunoglobulin germline gene complement into such germline mutant mice results in the production of human antibodies upon antigen challenge (US 5,545,806, 5,569,825, 5,591,669 (all to GenPharm); 5,545,807; and WO 97 / 17852).

[0493] Alternatively, phage display technology can be used to produce human antibodies and antibody fragments in vitro from the immunoglobulin variable region (V) gene repertoire from immunized donors (McCafferty et al., Nature, 348 (1990), pp. 552–553). In this technique, antibody V region genes are cloned in frame with either the major or minor coat protein gene of a filamentous bacteriophage, such as M13 or fd, and presented as functional antibody fragments on the surface of a phage particle. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selection for functional antibody properties also selects for a gene encoding an antibody that exhibits these properties. The phage thus mimics some of the properties of a B cell. Phage presentation can be accomplished in a variety of formats. Various sources of V gene segments can be used for phage presentation.Clackson et al., Nature, 352 (1991), pp. 624–628, isolated diverse repertoires of antibodies to oxazolone from a small, random combinatorial library of V genes obtained from the spleens of immunized mice. A repertoire of V genes derived from immunized human donors can be constructed, and antibodies to a diverse repertoire of antigens (including autoantigens) can be isolated generally according to the methods described by Marks et al., J. Mol. Biol., 222 (1991), pp. 581–597.

[0494] As described above, human antibodies can also be produced in vitro by activated B cells (see US 5,567,610 and 5,229,275).

[0495] Antibody fragments

[0496] In certain circumstances, it may be appropriate to use antibody fragments rather than whole antibodies. The smaller size of fragments facilitates their rapid clearance and may improve penetration into solid tumors.

[0497] Various methods have been developed for producing antibody fragments. Traditionally, these fragments were obtained by proteolytic cleavage of intact antibodies. However, these fragments can now be produced directly using recombinant host cells. Fab, Fv, and scFv antibody fragments can be expressed and secreted from E. coli, which facilitates the production of large quantities of these fragments. Antibody fragments can be isolated from the antibody phage libraries described above. According to another variant, 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-life that retain epitope-binding receptor residues are described in US 5,869,046.Other techniques for producing antibody fragments will be apparent to those skilled in the art. In other embodiments, the antibody of choice is a single-chain Fv fragment (scFv) (see WO 93 / 16185; US 5,571,894 and 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 non-specific binding in vivo. Fusion proteins bearing scFv can be designed to fusion an effector protein to either the N- or C-terminus of the scFv. The antibody fragment can also be a "linear antibody", such as described in US 5,641,870. Such linear antibody fragments can be monospecific or bispecific.

[0498] Pharmaceutical compositions

[0499] Another aspect of the invention is a pharmaceutical composition comprising as an active ingredient (or as the only active ingredient) a monoclonal antibody that specifically binds to GITR.

[0500] The pharmaceutical composition may include at least one monoclonal antibody that specifically binds to GITR and at least one component selected from the group consisting of pharmaceutically acceptable and pharmacologically compatible excipients.

[0501] A pharmaceutical composition may include at least one monoclonal antibody that specifically binds to GITR and one or more additional binding molecules (e.g., antibodies) that target one or more corresponding surface receptors. In some embodiments, the compositions are intended to ameliorate, prevent, or treat disorders that may be associated with GITR."Pharmaceutical composition" means a composition comprising a monoclonal antibody that specifically binds to GITR according to the invention and at least one component selected from the group consisting of pharmaceutically acceptable and pharmacologically compatible excipients, such as fillers, solvents, diluents, carriers, auxiliary, distributing, delivery vehicles, preservatives, stabilizers, emulsifiers, suspending agents, thickeners, prolonged delivery regulators, the selection and ratio of which depends on the nature and route of administration and dosage. Pharmaceutical compositions of the present invention and methods for their manufacture will be readily apparent to those skilled in the art. The manufacture of pharmaceutical compositions should preferably comply with GMP (good manufacturing practice) requirements. The composition may include a buffer composition, tonicity agents, stabilizers and solubilizers.Prolonged action of the composition can be achieved by using agents that delay the absorption of the active pharmaceutical ingredient, such as aluminum monostearate and gelatin. Examples of suitable carriers, solvents, diluents, and delivery vehicles include water, ethanol, polyols, and mixtures thereof, oils, and injectable organic esters.

[0502] "Medicine (preparation)" means 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 modify physiological functions in humans and animals, as well as for the treatment and prevention of diseases, diagnostics, anesthesia, contraception, cosmetology, and other applications. Any method of administering peptides, proteins, or antibodies accepted in the art can be appropriately used for the monoclonal antibody that specifically binds to GITR according to the present invention.

[0503] The term "pharmaceutically acceptable" means one or more compatible liquid or solid components that are suitable for administration to a mammal, preferably a human.

[0504] The term "excipient" or "auxiliary substance" 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 manufacturing process of medicinal products to impart the necessary physicochemical properties.

[0505] The terms "buffer," "buffer composition," and "buffering agent" refer to a solution capable of maintaining a pH value through the interaction of its acidic and alkaline components, which allows the monoclonal antibody preparation that specifically binds to GITR to exhibit resistance to changes in pH. Generally, the preferred pH of the pharmaceutical composition is between 4.0 and 8.0. Buffer solutions such as acetate, phosphate, citrate, histidine, succinate, and similar solutions, but are not limited to them, can be used as buffering agents.

[0506] The terms "tonicity agent," "osmolytic," or "osmotic agent," as used herein, refer to an excipient that can adjust the osmotic pressure of a liquid antibody formulation. An "isotonic" formulation is one that has an osmotic pressure equivalent to that of human blood. Isotonic formulations typically have an osmotic pressure of approximately 250 to 350 mOsm / kg. Polyols, mono- and disaccharides, amino acids, metal salts such as sodium chloride, and the like can be used as isotonic agents, but are not limited to them.

[0507] By "stabilizer" is meant an auxiliary substance or a mixture of two or more auxiliary substances that ensure physical and / or chemical stability of the active agent. Amino acids such as, but not limited to, arginine, histidine, glycine, lysine, glutamine, proline; surfactants such as, but not limited to, polysorbate 20 (trade name Tween 20), polysorbate 80 (trade name Tween 80), polyethylene-polypropylene glycol and its copolymers (trade names Poloxamer (Poloxaner), Pluronic (Pluronic) ), sodium dodecyl sulfate (SDS); antioxidants such as, but not limited to, methionine, acetylcysteine, ascorbic acid, monothioglycerol, salts of sulfur acids, etc.; chelating agents such as, but not limited to, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), sodium citrate, etc.

[0508] A pharmaceutical composition is considered "stable" if the active ingredient maintains its physical stability and / or chemical stability and / or biological activity throughout the stated shelf life at a storage temperature of, for example, 2-8°C. Preferably, the active ingredient should maintain both physical and chemical stability, as well as biological activity. The shelf life is determined based on the results of stability studies under accelerated and natural storage.

[0509] The pharmaceutical composition of this invention may be manufactured, packaged, or commercially sold as a single unit dose or multiple unit doses in a finished dosage form. As used herein, the term "unit dose" refers to a discrete quantity of the pharmaceutical composition containing a predetermined amount of the active ingredient. The quantity of active ingredient typically equals the dosage of the active ingredient to be administered to the subject, or a convenient fraction of such a dosage, such as one-half or one-third of such a dosage.

[0510] The pharmaceutical compositions of the present invention are generally suitable for parenteral administration as sterile medicaments intended for administration to the human body through the skin or mucous membranes, bypassing the gastrointestinal tract, by injection, infusion, or implantation. For example, parenteral administration is contemplated to include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intravenous, intraarterial, intrathecal, intraventricular, intraurethral, ​​intracranial, intra-articular, transdermal injection or infusion; and renal dialysis infusion techniques. Intratumoral delivery, such as intratumoral injection, may also be useful. Regional perfusion is also contemplated. Preferred embodiments of the invention include intravenous and subcutaneous routes.Any method of administering peptides or proteins accepted in the art can be suitably used for the monoclonal antibody that specifically binds to GITR of the present invention.

[0511] Injectable medicinal products may be manufactured, packaged, or sold in unit dosage forms, such as, but not limited to, ampoules, vials, polymer containers, prefilled syringes, and autoinjector devices. Medicinal products for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous bases, pastes, and the like.

[0512] Another embodiment of the invention is a medicinal product for parenteral administration, wherein the pharmaceutical composition is provided in dry form, i.e., a powder or granules for reconstitution in a suitable solvent (e.g., sterile, pyrogen-free water) prior to 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 drug and then removing the solvent from the frozen contents.

[0513] The monoclonal antibody that specifically binds to GITR according to the present invention can also be administered intranasally or by inhalation, either alone, as a mixture with a suitable pharmaceutically acceptable excipient from an inhaler (such as a pressurized aerosol container, pump, spray, nebulizer) or a nebulizer, with or without a suitable propellant, or as nasal drops or spray. The parenteral drug can be immediate-release or modified-release. Modified-release drugs include delayed-release, sustained-release, pulsatile, controlled-release, targeted-release, and programmed-release.

[0514] Therapeutic use of a monoclonal antibody that specifically binds to GITR according to the present invention

[0515] In one aspect, a monoclonal antibody that specifically binds to GITR according to the present invention is used in the treatment of disorders that are associated (mediated) by GITR activity.

[0516] In one aspect, the treatment subject or patient is a mammal, preferably a human subject. Said subject may be male or female and of any age.

[0517] In some embodiments, a monoclonal antibody or antigen-binding fragment thereof that specifically binds to GITR is used to treat a disease or disorder mediated by GITR, wherein the disease or disorder is selected from the group: cervical cancer, head and neck cancer, gastric cancer, breast cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, and non-small cell lung cancer.

[0518] In the case of a tumor (e.g., a cancer tumor), a therapeutically effective amount of an antibody or antibody fragment (e.g., an antibody or antibody fragment that specifically binds GITR) can reduce the number of cancer cells; reduce the initial size of the tumor; inhibit (i.e., slow to some extent and, preferably, stop) the infiltration of peripheral organs by cancer cells; inhibit (i.e., slow to some extent and, preferably, stop) tumor metastasis; inhibit, to some extent, tumor growth; and / or alleviate, to some extent, one or more symptoms caused by the disorder. The antibody or antibody fragment can, to some extent, prevent the growth of and / or kill existing cancer cells, it can cause a cytostatic and / or cytotoxic effect.In cancer therapy, in vivo efficacy can be determined, for example, by assessing survival time, time to disease progression (TTP), tumor response rate (RR), duration of response, and / or quality of life.

[0519] As used herein, the terms "co-administration," "co-administered," and "in combination with" refer to a monoclonal antibody that specifically binds to GITR with one or more other therapeutic agents and are intended to mean, refer to, or include: 1) the simultaneous administration of such a combination of a monoclonal antibody that specifically binds to GITR according to the 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,

[0520] 2) the simultaneous administration of such a combination of a monoclonal antibody that specifically binds to GITR according to the present invention and a therapeutic agent to a patient in need of treatment, when such components are formulated separately in different dosage forms, the administration of which occurs at substantially the same time to said patient, after which said components are released substantially simultaneously to said patient,

[0521] 3) sequentially administering such a combination of a monoclonal antibody that specifically binds to GITR according to the present invention and a therapeutic agent to a patient in need of treatment, wherein such components are formulated separately from each other in separate dosage forms that are administered in sequential order of time to said patient with a significant time interval between each administration, whereupon said components are released at substantially different times to said patient; and

[0522] 4) sequential administration of such a combination of a monoclonal antibody that specifically binds to GITR, according to the present 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 the release of said components occurs in a controlled manner, after which they are simultaneously, sequentially or jointly released at the same time and / or different times to said patient, where each part can be administered by the same or different routes.

[0523] The monoclonal antibody that specifically binds to GITR according to the present invention can be administered without additional therapeutic treatment, i.e., as a standalone therapy. Furthermore, treatment with the monoclonal antibody that specifically binds to GITR according to the present invention can include at least one additional therapeutic treatment (combination therapy). In some embodiments, the monoclonal antibody that specifically binds to GITR can be co-administered or formulated with another drug / therapeutic agent for the treatment of cancer.

[0524] The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents cell function 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 , Re188 , Sm 153 , Bi 212 , P 32 and radioactive isotopes (Lu), chemotherapeutic agents and toxins, such as small molecular weight toxins or enzymatically active toxins, originating from bacteria, fungi, plants or animals, including fragments and / or variants thereof.

[0525] A "chemotherapeutic agent" is a chemical compound used to treat a malignant tumor. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN ® ) ; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquinone, meturedopa, and uredopa; ethyleneimines and methylmelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylmelamine; acetogenins (e.g., bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL ®) ; beta-lapachone; lapachol; colchicines; betulinic acid; camptothecin (including the synthetic analogue topotecan (HYCAMTIN ® ), SRT-11 (irinotecan, CAMPTOSAR ®) , acetyl camptothecin, scopolectin and 9-aminocamptothecin); bryostatin; callistatin; CC-1065 (including its synthetic analogs adozelesin, carzelesin and bizelesin); podophyllotoxin; podophyllinic acid; teniposide; cryptophycins (eg, cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogs KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uramustine; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as enediyne antibiotics (e.g., calicheamicin, e.g., calicheamicin gamma II and calicheamicin omega II (see, e.g., Agnew, Chem. Inti. Ed. Engl., 33: 183-186 (1994)); dynemicin, including dynemicin A; esperamicin; as well as the neocarzinostatin chromophore and related chromoprotein chromophores enediyne antibiotics, aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including ADRIAMICIN. ® , morpholinodoxorubicin, cyanomorpholinodoxorubicin, 2-pyrrolinodoxorubicin, doxorubicin HCl in injectable liposomes (DOXOL ® ), liposomal doxorubicin TLC D-99 (MYOCET ® ), pegylated liposomal doxorubicin (CAELYX ®) and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate, gemcitabine (GEMZAR ® ) , tegafur (UFTORAL ® ), capecitabine (XELODA ®) , epothilone and 5-fluorouracil (5-FU); Folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate ; Purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; Pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; Adrenal suppressants such as aminoglutethimide, mitotane, trilostane; Folic acid compensator such as folinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerin; pentostatin; fenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; polysaccharide complex PSK ®(JHS Natural Products, Eugene, OR) ; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2>>-trichlorotriethylamine; trichothecenes (e.g., T-2 toxin, verracurin A, roridin A, and anguidine) ; urethane; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosin; arabinoside ("aga-C") ; thiotepa; a taxoid such as paclitaxel (TAXOL ® ), a paclitaxel drug based on engineered albumin-linked nanoparticles (ABRAXANETM) and docetaxel (TAXOTERE ® ) ; chlorambucil; 6-thioguanine; mercaptopurine; methotrexate; platinum-based agents such as cisplatin, oxaliplatin, and carboplatin; vinca alkaloids that prevent tubulin polymerization from nascent microtubules, including vinblastine (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, such as oligonucleotides that inhibit the expression of genes in signal transduction pathways involved in aberrant cell proliferation, such as PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such as THERATOPE vaccine ®and gene therapy vaccines, such as the ALLOVECTIN vaccine ® , LEUVECTIN vaccine ® and the 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; Bcl-2 inhibitor such as oblimersen sodium (GENASENSE ®) ; pixantrone; EGFR inhibitors (see definition below); tyrosine kinase inhibitors (see definition below); and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing; and combinations of two or more of the foregoing such as CHOP, an abbreviation for combination therapy with cyclophosphamide, doxorubicin, vincristine, and prednisolone, and FOLFOX, an abbreviation for the treatment regimen of oxaliplatin (ELOXATINTM) in combination with 5-FU and leucovorin.

[0526] Also included in the 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, trioxifen, 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 turnover, and / or decrease ER levels); aromatase inhibitors, including steroidal aromatase inhibitors such as formestane and exemestane (AROMAS IN ® ), and nonsteroidal aromatase inhibitors such as anastrozole (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 foregoing; and combinations of two or more of the foregoing.

[0527] Other therapeutic agents that can be used in combination with an antibody that specifically binds to GITR according to the present invention can be inhibitors of growth factor function, for example, such inhibitors include growth factor antibodies and growth factor receptor antibodies (e.g., the anti-erbB2 antibody trastuzumab [Herceptin], the anti-EGFR antibody panitumumab, the anti-erbB1 antibody cetuximab [Erbitux, C225], and any of the growth factor or growth factor receptor antibodies disclosed by Stern et al. Critical reviews in oncology / haematology, 2005, Vol. 54, pp. 11-29); antiangiogenic agents such as those that inhibit the effects of vascular endothelial growth factor [eg, the anti-vascular endothelial cell growth factor antibody bevacizumab (Avastin)], antibodies against vascular endothelial growth factor receptors such as anti-KDR antibodies and anti-fltl antibodies;Antisense therapies, such as those directed at the above targets, such as ISIS 2503, anti-ras antisense agents or G3139 (Genasense), anti-bc12 antisense agents; Gene therapy approaches, including, for example, approaches to replace aberrant genes such as aberrant p53 or aberrant BRCA1 or BRCA2, GDEPT (gene-directed enzyme prodrug therapy) approaches using cytosine deaminase, thymidine kinase or bacterial nitroreductase enzyme, and approaches aimed at increasing patient tolerance to chemotherapy or radiotherapy, such as multidrug resistance gene therapy;Immunotherapeutic approaches, including, for example, treatment with Alemtuzumab (campath-lH), a monoclonal antibody directed to CD52, or treatment with antibodies directed to CD22, ex vivo and in vivo approaches to enhance the immunogenicity of a patient's tumor cells, transfection with cytokines such as interleukin 2, interleukin 4, or granulocyte-macrophage colony-stimulating factor, approaches aimed at reducing T cell anergy such as treatment with monoclonal antibodies that inhibit CTLA-4 function, approaches using transfected immune cells such as cytokine-transfected dendritic cells, approaches using cytokine-transfected tumor cell lines, and approaches using anti-idiotypic antibodies, adoptive T cell transfer using T cells subjected to non-specific activation or targeted to a specific antigen of interest ex vivo;protein degradation inhibitors, such as a proteasome inhibitor such as Velcade (bortezamide); biotherapeutic therapeutic approaches, such as those using peptides or proteins (such as antibodies or soluble receptor external domain constructs) that sequester ligand receptors, block ligand binding to the receptor, or attenuate receptor signaling (e.g., due to increased receptor degradation or decreased expression levels).

[0528] Another therapeutic agent that can be used in combination with an antibody that specifically binds to GITR according to the present invention can be an antibody that is selected from the group: antibodies to PD1, antibodies to PD-L1, antibodies to CTLA4, antibodies to anti-4-1BB, antibodies to OX40, or combinations thereof.

[0529] Another therapeutic agent that can be used in combination with an antibody that specifically binds to GITR according to the present invention may be a therapeutically active antitumor compound that is selected from the group of activators of innate or acquired immunity.

[0530] It is contemplated that the monoclonal antibody that specifically binds to GITR of the present invention may be used in the methods of treatment as described above, may be used in the treatment as described above, and / or may be used in the manufacture of medicaments for the treatment as described above.

[0531] Dosage and administration routes

[0532] The monoclonal antibody that specifically binds to GITR according to the present invention will be administered in an amount effective for the treatment of the condition in question, i.e., at 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 patient's age, gender, and weight, and whether the monoclonal antibody that specifically binds to GITR is administered as a stand-alone treatment or in combination with one or more additional drugs or therapies.

[0533] Dosage regimens can be adjusted to provide the optimal desired response. For example, a single bolus may be administered, several divided doses may be administered over a period of time, or the dose may be proportionally decreased or increased depending on the exigencies 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 the patients / subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.The specification for the unit dosage forms of the present invention is generally dictated by and directly dependent on (a) the unique characteristics of the chemotherapeutic agent and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of compounding such active compound for the treatment of sensitivity in subjects.

[0534] Thus, those skilled in the art will appreciate, based on the disclosure provided herein, that doses and dosage regimens are adjusted according to methods well known in the therapeutic art. This means that the maximum tolerated dose can be readily established, and an effective amount providing a detectable therapeutic effect for a patient can also be determined, as can the timing of administration of each agent to achieve a detectable therapeutic effect for the patient. Thus, although certain doses and administration regimens are provided as examples herein, these examples in no way limit the doses and administration regimens that may be required for a patient practicing the present invention.

[0535] It should be noted that dosage values ​​may vary depending on the type and severity of the condition to be alleviated and may include one or more doses. Furthermore, it should be understood that for any particular patient, specific administration regimens should be adjusted over time according to the individual need and at the discretion of the healthcare professional administering or supervising the administration of the compositions, and that the concentration ranges provided herein are provided only as an example and are not intended to limit the scope or practice of the claimed compositions. Furthermore, the dosage regimen with the compositions of the present invention can be based on various factors, including the type of disease, age, weight, gender, health condition of the patient, severity of the condition, route of administration, and the particular monoclonal antibody used that specifically binds to GITR.Thus, the dosage regimen may vary widely but can be determined regularly using standard methods. For example, doses can be adjusted based on pharmacokinetic and pharmacodynamic parameters, which may include clinical effects, such as toxic effects, or laboratory values. Thus, the present invention encompasses individualized dose escalation, as determined by a skilled artisan. Determining the required dose and regimens are well known in the art and will be readily apparent to those skilled in the art upon familiarity with the teachings disclosed herein.

[0536] Examples of suitable routes of administration are provided above.

[0537] It is contemplated that a suitable dose of a monoclonal antibody that specifically binds to GITR according to the invention will be in the range of 0.1-200 mg / kg, preferably 0.1-100 mg / kg, including about 0.5-50 mg / kg, such as about 1-20 mg / kg. The monoclonal antibody that specifically binds to GITR can be administered, for example, at a dose of at least 0.25 mg / kg, such as at least 0.5 mg / kg, including at least 1 mg / kg, such as at least 1.5 mg / kg, such as at least 2 mg / kg, such as at least 3 mg / kg, including at least 4 mg / kg, such as at least 5 mg / kg; and for example up to 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.The administration will usually be repeated at suitable intervals, such as once a week, once every two weeks, once every three weeks, or once every four weeks, and for as long as deemed appropriate by the responsible physician, who may in some cases increase or decrease the dose if necessary.

[0538] Diagnostic uses and compositions

[0539] The monoclonal antibody that specifically binds to GITR according to the present invention is also used for diagnostic purposes (e.g., in vitro, ex vivo). For example, the monoclonal antibody that specifically binds to GITR according to the present invention can be used to detect or measure the level of GITR in samples obtained from a patient, for example, a tissue sample or a body fluid sample, such as inflammatory exudate, blood, blood serum, intestinal fluid, 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 the monoclonal antibody that specifically binds to GITR according to the present invention, as described herein.

[0540] Examples

[0541] For a better understanding of the invention, the following examples are provided. These examples are given for illustrative purposes only and should not be construed as limiting the scope of the invention in any way.

[0542] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference. Although the above invention has been described in some detail by way of illustration and example to avoid ambiguity, those skilled in the art, based on the teachings disclosed in this invention, will readily understand that certain changes and modifications can be made without departing from the spirit and scope of the accompanying embodiments of the invention.

[0543] Materials and general methods

[0544] General information on the nucleotide sequences of the light and heavy chains of human immunoglobulin is provided by Rabat E. A. et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991. The amino acids of the antibody chains are numbered according to the EU numbering (Edelman G. M. et al., Proc. Natl. Acad. Sci. USA 63, 1969, pp. 78–85; Rabat E. A. et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991).

[0545] Recombinant DNA methods

[0546] DNA manipulations were performed using standard methods described in Sambrook J. et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. Molecular biology reagents were used according to the manufacturers' instructions.

[0547] Gene synthesis

[0548] The desired gene segments were obtained from oligonucleotides created by chemical synthesis. Gene segments ranging from 300 to 4000 kb in length, flanked by unique restriction sites, were assembled by annealing and ligation of the 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.

[0549] DNA sequencing

[0550] DNA sequences were determined by Sanger sequencing.

[0551] DNA and protein sequence analysis and sequence data processing Infomax's Vector NT1 Advance suite, version 8.0, was used to create, map, analyze, annotate, and illustrate sequences.

[0552] Expression vectors

[0553] Expression plasmids designed for expression in prokaryotic cells (E. coli) and short-term expression in eukaryotic cells (e.g., CHO cells) were used to express the described antibodies and antigens. In addition to the antibody expression cassette, the vectors contained a replication initiation site, which ensures replication of the plasmid in E. coli, and genes conferring resistance to various antibiotics (e.g., ampicillin and kanamycin) in E. coli.

[0554] Fusion of genes containing the described antibody chains, as described below, was accomplished by PCR and / or gene synthesis and assembly using known recombination methods and procedures by joining corresponding nucleic acid segments, for example, by using unique restriction sites in the corresponding 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.

[0555] Example 1

[0556] Production of recombinant antigens and antibodies in suspension culture of mammalian cells

[0557] To prepare recombinant antigens based on the sequence of the extracellular portion of human GITR and orthologs, a series of constructs were created containing the extracellular domain of the human GITR antigen (https: / / www . uniprot . org / uniprot / Q9Y5U5 ), mcyGITR Macaca mulatta (https: / / www . uniprot . org / uniprot / Q1PBC4 ) and musGITR Mus musculus (https: / / www . uniprot . org / uniprot / 035714 ).

[0558] Gene sequences were synthesized by PCR from a template encoding the full-length human GITR antigen from the plasmid vector RDC0359.

[0559] (https : / / www. rndsystems . com / products / human-gitr-tnfrsf18- np 004186-versaclone-cdna rdc0359) and assembled from synthetic oligonucleotides for GITR ortholog genes. The GITR gene sequence was cloned into a plasmid for protein production in mammalian cells with the Lama glama IgG1 Fc tag (Fig. 1) using the Sall / Notl restriction sites. In addition, the antigen sequence is separated from the Fc by a TEV protease-labile site, which was also used to cleave the Fc fragment by treatment with TEV protease and obtain a tag-free version of the antigen. The sequence was also cloned into a plasmid containing a FLAG-EPEA-tag (C-tag GE Healthcare) at the C-terminus of the protein (Fig. 2) using the Sall / Notl restriction sites. In addition, a similar cloning of the recombinant human GITR ligand (GITRL) was performed into a plasmid containing the foldon homotrimerizing domain [1] and the EPEA-tag at the C-terminus of GITRL (Fig. 3).Plasmids were produced in the required quantities in E. coli cells and purified using the Qiagen Maxiprep kit.

[0560] Antibodies and antigens were produced in a constant cell line derived from Chinese hamster ovary cells (CHO-K1 line) according to published protocols [Biotechnol Bioeng. 2005 Sep 20; 91(6): 670–677, Liao Metal., 2004; Biotechnol Lett. 2006 Jun; 28 (11): 843–848; Biotechnol Bioeng. 2003 Nov 5; 84 (3): 332–342]. Cells constitutively expressing the EBNA1 (Epstein-Barrvirus nuclear antigen 1) protein gene were used. Suspension cultivation was performed in flasks on an orbital shaker using serum-free media from Life Technologies Corporation and according to the manufacturer's instructions. For transient expression, cells at a concentration of 2*10 6 / ml were transfected using linear polyethyleneimine (PEI MAX, Polysciences). The DNA / PEI ratio was 1:3 / 1:10. Five to seven days after transfection, the culture medium was centrifuged at 2000 g for 20 minutes and filtered through a 0.22 μm filter. Target proteins were isolated from the culture fluid using affinity chromatography.

[0561] Recombinant GITR and GITRL proteins, containing the EPEA tag (glutamic acid-proline-glutamic acid-alanine) at the C-terminus, were isolated and purified from culture fluid using the CaptureSelect C-tag Affinity Matrix sorbent. The culture fluid was passed through a chromatography column pre-packed with 5 ml of C-tag sorbent, then washed with 25 ml of PBS to remove non-specifically bound components. Bound antigen was eluted under mild conditions using 20 mM Tris, 2 M MgCl2, pH 7.0-7.4. The protein was then converted to PBS (pH 7.4) by dialysis using a semipermeable dialysis membrane, filtered (0.22 μm), transferred to tubes, and stored at -70°C.

[0562] Recombinant GITR-TEV-Fc antigen was isolated and purified from the culture fluid using a protein A affinity chromatography column. The clarified culture fluid was passed through a 5 ml HiTrap rProtein A Sepharose FF column (GE Healthcare) equilibrated with phosphate-buffered saline (PBS, pH 7.4). The column was then washed with 5 column volumes of PBS to wash out non-specific binding components. Bound antigen was eluted using 0.1 M glycine buffer pH 3. The main protein elution peak was collected and its pH was adjusted to neutrality with 1 M Tris buffer (pH 8). All steps were performed at a flow rate of 110 cm / h. The protein was then converted to PBS (pH 7.4) by dialysis using SnakeSkin Dialysis Tubing technology, filtered (0.22 μm), transferred into test tubes and stored at -70°C.

[0563] All recombinant IgG antibodies were purified using a 1 ml Hi Trap rProteinA FF column (GE Healthcare) according to the procedure described above for antigens. The purity of the resulting protein solution was assessed by SDS gel electrophoresis (see examples in Figs. 4 and 5).

[0564] Example 2

[0565] Creation of a naive Fab library of human antibodies MeganLibTM

[0566] Total B-lymphocyte RNA was isolated from individual blood samples from over a thousand human donors using the RNeasy Mini Kit according to the suggested protocol (from QIAGEN). RNA concentration was determined using the Nanovue kit (from GE Healthcare), and the quality of the isolated RNA was verified by electrophoresis in a 1.5% agarose gel.

[0567] The reverse transcription reaction was performed using the MMLV RT kit (Evrogen) according to the recommended protocol using MMuLV reverse transcriptase and random hexameric oligonucleotides as primers.

[0568] The 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].

[0569] The resulting VL-CK-VH DNA preparation (Fig. 6) was treated with restriction endonucleases NheI / Eco91I and ligated into the original pH5 phagemid (Fig. 7). The ligation products were transformed into electrocompetent cells of the SS320 strain, prepared according to the protocols [Methods Enzymol. 2000;328: 333-63]. The repertoire of the combinatorial phage Fab-display library MeganLibTM was 10 11 transformants. Phage preparations from Fab libraries were prepared according to the procedure described previously [J Mol Biol. 1991 Dec 5; 222 (3): 581–97].

[0570] Example 3

[0571] Generation of immune Fab libraries of hybrid llama-human antibodies (CHARM).

[0572] Total B-lymphocyte RNA from individual blood samples immunized with recombinant Lama Glama GITR antigens that demonstrated the highest serum specific titer was isolated using the RNeasy Mini Kit according to the suggested protocol (from QIAGEN). RNA concentration was determined using the Nanovue kit (from GE Healthcare), and the quality of the isolated RNA was verified by electrophoresis in a 1.5% agarose gel.

[0573] The reverse transcription reaction was performed using the MMLV RT kit (Evrogen) according to the recommended protocol using MMuLV reverse transcriptase and random hexameric oligonucleotides as primers.

[0574] The 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].

[0575] The resulting VL-CK-VH DNA preparation (Fig. 6) was treated with restriction endonucleases NheI / Eco91I and ligated into the original pH5 phagemid (Fig. 7). The ligation products were transformed into electrocompetent cells of the SS320 strain, prepared according to the protocols [Methods Enzymol. 2000;328: 333-63]. The repertoire of the combinatorial phage Fab-display library MeganLibTM was 10 11 transformants. Phage preparations from Fab libraries were prepared according to the procedure described previously [J Mol Biol. 1991 Dec 5;222(3): 581–97]. A summary table of immune library preparations is given in Table 1.

[0576] Table 1.

[0577]

[0578] Example 4

[0579] Selection of Fab libraries of phage antibodies

[0580] Specific human phage Fab antibodies against GITR were obtained from the combinatorial phage Fab display library MeganLibTM and immune CHARM libraries described in Examples 2 and 3. Selection was performed on recombinant human GITR antigens by the phage display method [Nat Biotechnol. 1996 Mar ; 14 ( 3 ): 309–14 ; J Mol Biol. 1991 Dec 5 ; 222 ( 3 ): 581–97].

[0581] Selection of immune phage libraries was performed on an EIA / RIA Tube High Binding 5 ml immunotube. Antigen (0.5 ml, 2 μg / ml) was adsorbed in carbonate buffer (0.1 M NaHC03, pH 9.5) overnight at + 4C. The tubes were then washed with PBST (10 tM phosphate buffered saline pH 7.3-7.5, 137 mM NaCl and 2.7 tM KCl, 0.1% Polysorbate 20) and the full volume (5 ml) of 0.5% dry milk in PBST was added for loading. Loading was alternated from round to round, for example, if in round 1 it was 0.5% milk in PBST, then in round 2 it was 1% BSA in PBST. Incubated for an hour at room temperature on a shaker. The tubes were washed with PBST and 2 ml of loading buffer and phage libraries were added to them to a concentration of approximately 2*10 12phage particles per ml. Incubated with phages for 1-2 hours at room temperature. For subsequent rounds, 4 ml of phage supernatant from the previous round, pre-clarified by centrifugation at 17:00 Od for 10-15 minutes, was used. The tubes were washed 20 times with PBST. Phages were eluted from the surface of the tubes with 0.5 ml of 0.1 M glycine buffer pH 2.2: glycine buffer was added to the tubes and gently stirred for 15 minutes at room temperature. Then the phage solution was transferred to clean non-sorbent tubes with 100 μl of 1 M TrisHCl pH 8.0 for neutralization. The tubes were kept on ice until infection of the cells. After selection, bacteriophage M13 was cultured using the E. coli strain TG1 as a host. Amplification is carried out by infecting the host strain culture with phages and subsequent growth for 12-15 hours.

[0582] 0.5-1 ml of phages after selection were mixed with cell culture (OP боо=0.3-0.4) and incubated for 1.5 hours at 37C. Then the cells were pelleted at 3000-4000 rpm for 10-15 minutes, resuspended in 1 ml of medium and ground onto Petri dishes with an antibiotic for selection (ampicillin). Colonies were grown in a thermostat at ZOS. After 12-15 hours, the number of colonies was counted and the cells were washed from the dishes with 5-10 ml of LB medium. 100 μl of the cell suspension was added to 20 ml of medium with an antibiotic (ampicillin). They grew to ODboo = 0.35-0.5 per 37. Helper phage K07 was added at a rate of 1 μl per 10 ml of culture (10 10particles per ml) and incubated at 37C for 1.5 hours without shaking. Then, an equal volume of medium with single ampicillin (100 μg / ml), double kanamycin (40 μg / ml), and double IPTG (0.2 mm) was added to the cell culture. The culture was placed on a shaker and the phage was grown for 4-5 hours at 30°C. The cell culture was centrifuged for 25-30 minutes at 17,000 g, and the supernatant was collected in test tubes. The resulting supernatant was then used in the next round of selection, or the phage was isolated by precipitation for subsequent storage.

[0583] Phages were isolated as follows: 1 / 6 of the volume of a solution containing 20% ​​polyethylene glycol and 2.5 M sodium chloride was added to the supernatant and mixed vigorously. The solution was incubated on ice for at least 3 hours. The solution was then centrifuged for 10 minutes at 8000 g, and the resulting precipitate containing the phages was dissolved in 1 ml of TBS (Tris-Borate Buffer).

[0584] After two or three rounds, bacteriophages were isolated and an analysis of the specific binding of the polyclonal phage to human GITR and non-specific antigens was performed.

[0585] Example 5

[0586] Analysis of polyclonal phage obtained after 2 and 3 rounds of selection.

[0587] After 3 rounds of selection of 2 naive MeganLib libraries and 6 immune libraries on the target antigen, specific and non-specific binding of polyclonal phages obtained after the 2nd and 3rd rounds was tested using ELISA.

[0588] The target antigen GITR was adsorbed onto plastic ELISA plates overnight in carbonate buffer (0.1 M IaHCO3, pH 9.5) to analyze the presence of specifically binding phages (2 μg / ml) or G-CSF, hIL6R-Fc, interferon alpha 2b, Rituximab (2 μg / ml) to analyze non-specifically binding phages. The plates were then washed with PBST (10 mM phosphate-buffered saline pH 7.3-7.5, 137 mM NaCl and 2.7 mM KCl, 0.1% Polysorbate 20) and 300 μl / well of 0.5% dry milk in PBST were added for loading. The plates were incubated for an hour at room temperature on a shaker. The plates were washed with PBST and added with 50 µl of phage solutions obtained after rounds 2 and 3 of selection, diluted 2 to 256 times with loading buffer. The mixture was incubated for 1 hour at room temperature on a shaker. The plates were washed with PBST and coated with horseradish peroxidase-conjugated anti-M13 antibody in loading buffer. After incubation for 1 hour, the plates were washed and 50 µl of reaction substrate (0.02% H2O2 and TMB) in acetate buffer pH 5.0 were added.The reaction was incubated at room temperature in the dark until a slight background appeared in the negative controls (but no more than 20 min). The reaction was stopped by adding 25 µl of 10% H2SO4 per well, and the OD in the wells was measured at 450 nm.

[0589] The analysis revealed specific (5X excess over background signal) binding of 1 naive human Fab and 6 immune Fab CHARM libraries after the 3rd round of selection on the target antigen and the absence of significant (less than 2X excess) non-specific binding.

[0590] The variable domain genes of antibodies from these libraries were recloned into the pLL-Fab expression vector (Fig. 8) to produce a secreted soluble form of Fab in E. coli cells.

[0591] Example 6

[0592] Screening of Fabs that specifically bind human GITR

[0593] ELISA was used to detect Fab secreted into the medium from the E. coli producer monoclones obtained from Example 5 that specifically bind human GITR. Fab with the published sequence Fab-gitr3215 (patent pending) was used as a positive control. To analyze specific binding, wells of ELISA plates (medium binding from Greiner bio one) were coated with 50 μl of GITR (0.2 μg / ml in 1* carbonate buffer), sealed, and incubated overnight at 4°C. All subsequent steps were performed according to the standard ELISA protocol using a high-throughput automated platform based on the Genetix Qpix2xt robotic systems (from Molecular Device) and Tecan Freedom EVO 200 (from Tecan). To block nonspecific binding, blocking buffer BB (200 µl of 0.5% nonfat milk in PBS) was added. The plates were incubated on a shaker for 1 hour at room temperature.After washing with PBS-Tween, 50 µl of the test cell supernatant containing the Fab under study, mixed with an equal volume of BB, was added per well. The plates were again incubated with shaking for one hour at room temperature, after which each well of the plates was washed three times with PBS-Tween buffer. After washing, 50 µl / well of anti-human Fab HRP-conjugated secondary antibody (Pierce-ThermoScientific) was added at a ratio of 1:5000 in PBS-Tween. The plates were shaken on a rotary shaker (50 min, room temperature) and washed three times with PBS-Tween buffer, as described above. The colorimetric signal was developed by adding TMB (50 µl / well) until saturation (3-5 min on average), then further development was stopped by adding a stop solution (30 µl / well, 10% sulfuric acid). The color signal was measured at a wavelength of 450 nm using a suitable Tesap-Sunrise plate reader (Tesap).The degree of antibody binding was proportional to the color signal produced. Clones with a color signal 5X greater than the background signal and comparable to the signal of the control antibody Fab-gitr3215 were tested for nonspecific binding in an ELISA.

[0594] Example 7

[0595] Analysis of non-specific binding of selected Fabs to other antigens

[0596] ELISA was also used to analyze the non-specific binding of the studied Fab fragments to other antigens. The study was carried out as described above, but IL6R-Fc, INF 2b, PCSK9-VG-FE, PD-1-Fc (2.5 μg / ml in 1 c carbonate buffer). GITR-TEV-Fc (0.2 μg / ml in 1 c(carbonate buffer). All subsequent steps were performed according to the standard ELISA protocol using a high-throughput automated platform based on the Genetix Qpix2xt robotic systems (Molecular Device) and Tesap Freedom EVO 200 (Tesap). Clones in which the color signal of nonspecific binding did not exceed the background signal and was 5X lower than the value of specific binding were considered positive, and their genes were sequenced to determine the sequence of the variable domain genes of antibodies and determine uniqueness. As a result of sequencing, 30 unique sequences were selected for conversion to the full-length IgG1 antibody format.

[0597] Example 8

[0598] Production of recombinant antibodies in suspension culture of mammalian cells

[0599] Cloning of the genes of the variable domains of the antibodies from Example 7 was performed according to the standard technique. For this, PCR products containing the genes of the variable domains of the heavy and light chains of the antibodies were generated. The variable domain of the heavy chain was cloned into the pEE-Hc IgG1 vector at the Sall / Nhel restriction sites, the schematic map of which is shown in Fig. 9). The variable domain of the light chain was ligated into the pEE-CK vector at the Sall / BsiWI restriction sites, the schematic map of which is shown in (Fig. 10). Antibodies were produced in cells of a constant cell line obtained from Chinese hamster ovary cells (line CH0-K1), according to published protocols [Biotechnol Bioeng. 2005 Sep 20; 91 (b): 670-677, Liao Metal., 2004; Biotechnol Lett. 2006 Jun; 28 ( 11 ): 843–848; Biotechnol Bioeng. 2003 Nov 5; 84 (3): 332–342. Cells constitutively expressing the EBNA1 (Epstein-Barrvirus nuclear antigen 1) protein gene were used.

[0600] To produce antibodies in the transient expression system, CHO-K1-S cells were used. The cells were cultured in baffled flasks (125, 250, 500, 1000, and 3000 ml) in a mixture of CHO-S-SEM II and Freestyle CHO media (1:1) with 4 mM glutamine, 0.05 mg / ml gentamicin, and 10 μg / ml ciprofloxacin in orbital shaker-incubators at +37°C in the presence of 5% CO2, at 110 or 150 rpm depending on the flask size. The cells were passaged 3 times a week at a seeding density of 0.2*106 cells / ml.

[0601] For transfection, cells were seeded the day before at a density of 0.8*106 cells / ml, transfection was performed 24 hours later, on a cell culture at a density of 2*106 cells / ml. RPMI-1640 medium with 2 mM glutamine and 0.05 mg / ml gentamicin was used to prepare the transfection mixture. Vectors were diluted separately in the medium at a rate of 0.75 μg / ml, and the transfection agent polyethyleneimine (PEI) was diluted separately at a PEI:DNA ratio of 7:1 by weight. The vector and PEI dilutions were mixed and incubated for 10 minutes at room temperature, after which the transfection mixture was added to the cells and the cells were cultured under standard conditions.

[0602] The next day after transfection, a mixture of CHO-S-SEM II and Freestyle CHO media (1:1) with 0.05 mg / ml gentamicin and 10 μg / ml ciprofloxacin, 1 mM sodium valproate and 10% F12.7 feeding agent was added to the cells and the cells were cultured in orbital shaker-incubators at +34°C in the presence of 5% CO2, at 110 or 150 rpm depending on the flask size. On days 3-4 after transfection, 10% F12.7 feeding agent was added to the cells.

[0603] On day 7 post-transfection, cell fluid samples were collected to measure the concentration of the produced protein on Protein-A Biosense chips using an OctetRed 96 instrument according to the ForteBio instructions (Table 2). Recombinant antibodies were isolated from the culture fluid and purified using a protein A affinity chromatography column. The clarified culture fluid was passed through a 1 ml Hi Trap rProteinA FF column (GE Healthcare), which was equilibrated with phosphate-buffered saline (PBS, pH 7.4). The column was then washed with 5 column volumes of PBS to wash out non-specifically binding components. Bound antibody was eluted using 0.1 M glycine buffer pH 3. The main protein elution peak was collected and its pH was adjusted to neutrality using 1 M Tris buffer (pH 8). All stages were carried out at a flow rate of 110 cm / h.The protein was then dialyzed into PBS (pH 7.4) using SnakeSkin Dialysis Tubing technology, filtered (0.22 μm), transferred into tubes and stored at -70°C.

[0604] The purity of the resulting protein solution was assessed using SDS gel electrophoresis (Fig. 11 and Fig. 12). All antibodies meet the purity required for studying the physicochemical properties and activity for cellular tests.

[0605] Table 2.

[0606] Example 9

[0607] Kinetic analysis of the interaction of anti-GITR IgGl antibodies with human GITR

[0608] Binding affinity constants of anti-GITR antibodies to human, rhesus macaque, and cynomolgus macaque GITR were obtained using an OctetRed 96 instrument according to the ForteBio instructions. Antigen (25 μg / ml) was non-specifically immobilized on the surface of second-generation amine-reactive sensors (from AR2G) (ForteBio according to the standard protocol according to the manufacturer's instructions for the preparation and immobilization of AR2G sensors). Anti-GITR antibodies were added at a concentration of 0.5 mg / ml. The assay was performed at 30°C using PBS containing 0.1% Tween-20 and 0.1% BSA as a running buffer.

[0609] The resulting baseline-subtracted sensorgrams were analyzed using Octet Data Analysis (version 8.0) according to the standard procedure using a 1:1 interaction model. The resulting affinity constants are presented in Table 3. All antibodies studied are high-affinity and specific to human GITR.

[0610] Table 3. Affinity constants of anti-GITR antibodies when interacting with human GITR.

[0611] Example 1 O

[0612] Test for determination of anti-GITR specific agonist activity.

[0613] For the test, we used the HEK293-GITR-NFkB-Luc cell line, created on the basis of the HEK-293 cell line, which stably expresses GITR on the surface and contains the gene encoding firefly luciferase under the control of the NFkB promoter.

[0614] The assay was performed in a 96-well culture plate. The suspension in each well contained HEK293-GITR-NFkB-Luc cells and the antibody under study at the concentration indicated on the graph. All suspension components were prepared in cell culture medium. After adding all components, the plate was incubated at 37°C, 5% CO2, and then the luciferase intensity in the wells was measured using a luminescence assay kit and a plate reader.

[0615] Results of testing the specific activity of anti-GITR monoclonal antibodies (Fig. 13) revealed that antibodies from lots 1161, 1210, and 1213 are functionally active and exhibit significant agonist activity with an upper plateau level comparable to both the control antibody aHTM-GITR3215 and GITRL. These antibodies were renamed according to Table 4.

[0616] Table 4.

[0617]

[0618] Example 11

[0619] Enzyme-linked immunosorbent assay of the interactions of anti-GITR antibodies with GITR from different organisms

[0620] ELISA was used to measure the comparative affinity of antibodies to GITR-Fc from different organisms. For the binding assay, wells of ELISA plates (medium binding from Greiner bio one) were coated with 50 µl of GITR-Fc from human, cynomolgus, and mouse (0.5 µg / ml in 1 cThe plates were then washed with water (carbonate buffer), sealed, and incubated overnight at 4°C. All subsequent steps were performed using the standard ELISA protocol described in Example b. Anti-GITR antibody BCD166-01-01 bound specifically to human and cynomolgus monkey GITR and did not bind to mouse GITR (Fig. 14). Antibody BCD166-01-011 bound specifically to human GITR but showed no binding to cynomolgus monkey or mouse GITR (Fig. 15). Antibody BCD166-01-014 bound specifically to human GITR, showed much weaker binding to cynomolgus monkey GITR, and showed no binding to mouse GITR (Fig. 16). Thus, candidate BCD166-01-011 was excluded from further work due to a lack of apparent specificity for macaque GITR. Candidates BCD166-01-01 and BCD166-01-014 were finalized for further development.

[0621] Example 12

[0622] Kinetic analysis of the interaction of anti-GITR IgGl antibodies with macaca mulatta GITR and macaca cynomolgus GITR

[0623] Binding affinity constants of anti-GITR antibodies to rhesus macaque and cynomolgus macaque GITR were obtained using an OctetRed 96 instrument according to the ForteBio instructions. Antigen (25 μg / ml) was non-specifically immobilized on the surface of second-generation amine-reactive sensors (from AR2G) (ForteBio according to the standard protocol according to the manufacturer's instructions for the preparation and immobilization of AR2G sensors). Anti-GITR antibodies were added at a concentration of 0.1%. The assay was performed at 30°C using PBS containing 0.1% Tween-20 and 0.1% BSA as a running buffer.

[0624] The resulting baseline-subtracted sensorgrams were analyzed using Octet Data Analysis (version 8.0) according to the standard procedure using a 1:1 interaction model. The resulting affinity constants are presented in Tables 5 and 6. All antibodies studied were high-affinity and specific for macaque GITR, which warrants further study in this relevant in vivo animal model.

[0625] Table 5.

[0626] Affinity constants of anti-GITR antibodies when interacting with tacaca mulatta GITR.

[0627] Table 6.

[0628] Affinity constants of anti-GITR antibodies in interactions with the cynomolgus macaca GITR.

[0629]

[0630] Example 13

[0631] A cell-based in vitro assay for the determination of anti-GITR specific agonist activity of wild-type and mutant candidates with enhanced agonist activity.

[0632] To potentially enhance the agonist activity of the human-derived anti-GITR antibody BCD166-01-01, a mutation E345R was selected and introduced into the IgG1 Fc region of the antibody based on studies of such substitutions leading to antibody oligomerization in W02005047327, W02006104989 (A2), W02007005612 (A2) and Science. 2014 Mag 14;343 (6176):1260-3. It was shown that such a mutation can lead to antibody oligomerization on the cell surface after binding to the antigen, and to an increase in various effector abilities of ADCC, ADCP, CDC, as well as pharmacokinetics (PK). The goal was to obtain an antibody with enhanced agonist activity, ADCC, but not CDC. Mutagenesis was performed according to standard genetic engineering protocols, and the recombinant antibody was synthesized according to Example 8. The antibody was named and is used hereinafter as BCD166-02-01. The two parental antibodies, BCD166-01-01 and BCD166-01-014, were also tested.

[0633] The test was carried out similarly to the test in Example 10.

[0634] The results shown in Fig. 17 demonstrate a 5-fold increase in the activation level in the cellular agonist assay of the studied BCD166-02-01 compared to its predecessor, BCD166-01-01, which contained the E345R mutation. This increase is due to antigen-dependent oligomerization of the anti-GITR BCD166-02-01 antibody on the target cell. Moreover, not only did the upper plateau of activation increase, but the EC50 value also increased more than 20-fold.

[0635] Example 14

[0636] Cell-based in vitro antibody-dependent cellular cytotoxicity (ADCC) assay for anti-GITR antibodies.

[0637] The assay examined the ADCC activity of candidate BCD166-02-01. Two antibodies, BCD166-01-01 and BCD166-01-014, were also included in the assay. Furthermore, similar to candidate BCD166-01-01, the E345R mutation was introduced into candidate BCD166-01-014 (which was named BCD166-02-014) to increase ADCC activity compared to the wild-type form.

[0638] The test used the Jurkat-NFAT-Luc-CD1 6 cell line, created on the basis of the Jurkat cell line, which stably expresses CD16 on the surface and contains the gene encoding firefly luciferase under the control of the NFAT promoter; as well as the Hek-293-GITR cell line, created on the basis of the Hek-293 cell line, which stably expresses GITR on the surface.

[0639] The test was performed in a 96-well culture plate.

[0640] The suspension in each well contained a mixture of effector cells.

[0641] Jurkat-NFAT-Luc-CD1 6 and Hek-293-GITR target cells, as well as the analyzed antibodies at the concentrations indicated in the graph, were added. All suspension components were prepared in cell culture medium. After adding all components, the plates were incubated at 37°C, 5% CO2, and then the luminescence intensity in the wells was measured using a Lucifer assay kit and a plate reader.

[0642] The results shown in Fig. 18 indicate a 40-fold increase in the EC50 value in the cellular ADCC assay of the studied BCD166-02-01 compared to the precursor BCD166-01-01, into which the E345R mutation was introduced, which is due to the antigen-dependent oligomerization of the anti-GITR BCD166-02-01 antibody on the target cell. A 5-fold increase in the EC50 value in the cellular ADCC assay of the studied BCD166-02-14 was also reliably determined compared to the precursor BCD166-01-14, into which the E345R mutation was introduced. Together, the combined data clearly indicate a significant positive effect of the E345R substitution in the Fc portion of IgG1 antibodies on various effector characteristics necessary for the creation of a highly active GITR agonist.

[0643] Example 15

[0644] Cell-based in vitro complement-dependent cytotoxicity (CDC) assay for anti-GITR antibodies.

[0645] In this test, antibodies BCD166-01-01, BCD166-01-014, BCD166-02-01 and the control antibody anti-GITR-3215 were examined.

[0646] The test was performed in a 96-well culture plate. The suspension in each well contained HEK-293-GITR cells, as well as the analyzed antibodies at the specified concentration and human serum complement. The plate with the described suspension was incubated for 4 hours at 37 °C, 5% CO2. Next, a solution of Alamar blue was added to each well, and further incubation was carried out at 37 °C, 5% CO2. Then, using a plate reader, the fluorescence value in the wells was measured (excitation wavelength 544 nm, emission wavelength 590 nm).

[0647] The results shown in Fig. 19 indicate insignificant CDC activity, i.e., no more than 10% at a concentration of 1 μg / mL, which is the limit for the estimated therapeutic dose intended for humans, for the BCD166-01-01, BCD166-02-01 antibodies, and the control antibody anti-GITR-3215. For candidate BCD166-01-14, the CDC activity at a concentration of 1 μg / mL was approximately 30% lysis, indicating a visible effect, but possibly weak in vivo. Taken together, the combined data clearly indicate an insignificant effect of the E345R substitution in the Fc portion of the IgG1 anti-GITR candidates.

[0648] Example 16

[0649] In vitro cellular assay for determination of cytotoxicity of anti-GITR candidates.

[0650] To assess the negative or positive impact of the anti-GITR candidates being studied on changes in the numbers of various immunoreactive cell populations, this assay was conducted. The antibodies BCD166-01-01, BCD166-01-014, BCD166-02-01, and the control antibody anti-CD20 (rituximab) were tested.

[0651] PBMCs were isolated from whole blood of healthy donors by Ficoll density gradient centrifugation. The assay was performed in a 96-well culture plate. The suspension in each well contained PBMCs and the antibody indicated on the graph at concentrations of 25, 1, and 0 μg / ml. After mixing the PBMCs and antibodies, the plate was incubated for 16 h at 37°C, 5% CO2. Next, the proportion of CD56+, CD19+, CD3+, CD4+, and CD8+ PBMC subsets in the suspensions was assessed by direct staining of the suspensions with fluorescently labeled antibodies against the corresponding CDs and subsequent cell analysis on a flow cytometer. For CD56+, CD19+, CD3+ cells, the graphs show the proportion of all cells in the analyzed suspension; for CD4+, CD8+, the proportion of CD3+ cells.

[0652] The results presented in Figs. 20–24 indicate an insignificant negative effect of the studied anti-GITR candidates, i.e., no more than a 10% decrease in the number of immunoreactive cells compared to the negative control. For the NK cell population, we observed an approximately 30–70% increase in cell number depending on the dose. The control anti-CD20 antibody also showed an increase in the percentage of NK cells, but a nearly complete depletion of the CD20+ B cell population. Thus, all the candidates under consideration exhibit no nonspecific cytotoxicity in vitro on human blood cells.

[0653] Example 17

[0654] A cellular in vitro test for the determination of antibody-dependent depletion of the human nTreg cell population.

[0655] One of the main proposed mechanisms of the therapeutic anti-oncogenic action of anti-GITR antibodies is ADCC-dependent depletion of GITR. +nTreg cell population. This test examined the antibodies BCD166-01-01, BCD166-01-014, BCD166-02-01 and the control antibody anti-CTLA4 (ipilimumab).

[0656] Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood of healthy donors by Ficoll density gradient centrifugation. PBMCs were enriched for nTreg using the CD4+ CD25+ Regulatory T Cell Isolation Kit, human (Miltenyi Biotec, Germany). The resulting cells were activated using magnetic particles immobilized with anti-CD3 and anti-CD28 antibodies.

[0657] NK cells were isolated from PBMCs using the NK Cell Isolation Kit, human (Miltenyi Biotec, Germany).

[0658] Antibody solutions, NK cell suspension, and nTreg suspension were added to a culture plate. The plate was incubated at 37°C with 5% CO2 for 16 hours.

[0659] Cell suspension samples were stained using fluorescently labeled antibodies against SV3, CB4, CB25, and FoxP3 (Biolegend, USA). The stained cell suspension samples were analyzed using flow cytofluorometry.

[0660] The results of the analysis of antibody-dependent depletion of nTreg under the action of the studied antibodies presented in Fig. 25 for the cellular material of donor 1 and in Fig. 26 for donor 2 indicate a significant 70-100% decrease in the cell population, i.e., the depleting activity of the studied anti-GITR candidates, especially for the material of donor 2. In addition, a comparative analysis with the control anti-CTLA4 antibody ipilimumab demonstrates greater activity in favor of anti-GITR antibodies, especially for the final candidate BCV166-02-01.

[0661] Example 18

[0662] A cellular in vitro assay for the analysis of antibody-dependent depletion of human iTreg cell populations.

[0663] One of the main proposed mechanisms of the therapeutic anti-oncogenic action of anti-GITR antibodies is ADCC-dependent depletion of GITR+ iTreg cells. The antibodies BCD166-01-01, BCD166-01-014, and BCD166-02-01 were tested in this assay.

[0664] PBMCs were isolated from whole blood of healthy donors by Ficoll density gradient centrifugation. Monocytes were isolated from PBMCs as a population of cells that bind to the culture plastic. To obtain dendritic cells, monocytes were incubated in the presence of 1000 U / ml GM-CSF (Peprotech, USA) and 500 U / ml IL-4 (Thermo Scientific, USA) at 37°C, 5% CO2 for 120 hours. LPS solution (Sigma, USA) was added to the culture medium to a concentration of 0.5 μg / ml, and the cells were incubated at 37°C, 5% CO2 for 48 hours. Antibody dilution solutions, dendritic cell suspension, and PBMCs were added to the culture plate. The plate was incubated at 37°C, 5% CO2 for 120 hours.

[0665] Cell suspension samples were stained with fluorescently labeled antibodies against CD3, CD4, CD25, and FoxP3 (Biolegend, USA). The stained cell suspension samples were analyzed by flow cytometry. Anti-GITR antibodies BCD166-01-01, BCD166-01-14, and BCD166-02-01 induce iTreg depletion in vitro.

[0666] The results of the analysis of antibody-dependent iTreg depletion under the action of the studied antibodies, presented in Fig. 27 for the cellular material of donor 1 and donor 2, indicate a significant 2-5X depleting activity of the studied anti-GITR candidates, especially for the material of donor 1. The final candidate BCD166-02-01 demonstrates sufficient iTreg depleting activity.

[0667] Example 19

[0668] In vitro cellular assay for the determination of secretion of proinflammatory cytokines IL-2 and IFN-γ under the influence of anti-GITR antibody in a human cell population.

[0669] PBMCs were isolated from whole blood of healthy donors by Ficoll density gradient centrifugation.

[0670] Anti-C3 and aHTHGITR antibodies were immobilized in the wells of a 96-well plate. To do this, 100 µl of DPBS solution containing the aHTHGITR antibody BCD166-02-01 at the concentration indicated on the graph and the anti-C3 antibody at a suboptimal concentration were added to the corresponding wells of the plate and the plate was incubated for 16 hours at room temperature.

[0671] The test was performed in a 96-well plate with anti-SVZ and anti-GITR antibodies pre-immobilized in the wells. The suspension in each well contained 40,000 PBMCs, as well as anti-CD28 antibody at a suboptimal concentration. All suspension components were prepared in RPMI-1640 medium containing 10% FBS. After adding the cell suspension, the plate was incubated for 6 days at 37°C, 5% CO2. On the 4th day of incubation, aliquots of the culture fluid were removed from the wells. The concentrations of IL-2 and IFN-γ in the culture fluid on the 4th and 6th days of incubation were then determined using ELISA.

[0672] Results from the analysis of the effect of the anti-GITR antibody on the secretion of proinflammatory cytokines IL-2 and IFN-γ, which mediate the anticancer effect, are presented in Fig. 28. They demonstrate a significant increase in the concentration of these substances in the culture fluid. Thus, candidate BCD166-02-01 exhibits high activity in activating cytokine secretion, which may indicate a significant anti-oncogenic effect.

[0673] Example 2O

[0674] Analysis of binding of antibody BCD166-02-01 to human FcRn, FcgRIIIal58V, FcgRIIIal58F, FcgRIIal31H, FcgRIIal31R, FcgRIIb and FcgRIa receptors.

[0675] Binding affinity constants of the BCD166-02-01 antibody to human FcRn, FcgRIIIal58V, FcgRIIIal58F, FcgRIIal31H, FcgRIIal31R, FcgRIIb, and FcgRIa were obtained using an OctetRed 96 instrument (ForteBio). Biotinylated receptors were immobilized on the surface of streptavidin sensors (SA). Association and dissociation of receptors and antibodies were performed and analyzed in a working buffer (PBS containing 0.1% Tween-20 and 0.1% BSA: with the FcRn receptor at pH 6, with the other receptors - in a buffer with pH 7.4.) at 30 °C. The resulting sensorgrams were analyzed according to 1:1 or 2:1 models and affinity constants were calculated using Octet Data Analysis Software 8.0 User Guide Copyright 2011©.

[0676] The results of the analysis of the affinity of BCD166-02-01 to human FcRn, FcgRIIIal58V, FcgRIIIal58F, FcgRIIal31H, FcgRIIal31R, FcgRIIb and FcgRIa are presented in Fig. 29. They demonstrate values ​​that are several times higher than the literature data and the data repeatedly measured in our company for IgGl isotype antibodies, which indicates a noticeable but not qualitative effect of the E345R mutation in the Fc fragment on the kinetics of monomeric antibodies adsorbed on these receptors from solution.

[0677] Example 21

[0678] Determination of colloidal and thermal stability by the protein aggregation point using dynamic light scattering

[0679] To determine the aggregation temperature of the studied samples, the dependence of particle size in the medium on temperature was obtained using the dynamic light scattering method using a DynaPro® Plate Reader II (Wyatt Technology Corp.) with gradual heating from 40 to 85°C. The results are presented in Table 7.

[0680] Table 7. BCD166-02-01 aggregation point

[0681] It can be concluded that the BCD166-02-01 molecule has high thermocolloidal stability (the aggregation point in 20mM Acetate, pH=5.0 and 20mM His, pH=5.5 buffer solutions is > 65°).

[0682] Similar data were obtained for BCD166-01-014.

[0683] Example 22

[0684] Determination of thermal stability under thermal stress of 50 °C

[0685] The test samples were placed in an air-heated thermostat and maintained at 50°C for 72 hours. After heating, intact and stressed samples were analyzed by size-exclusion HPLC (SEC HPLC) with a UV detector and capillary isoelectric focusing. Chromatography was performed on an Agilent 1100 HPLC system using a Tosoh TSK-Gel G3000SWXL column, with detection at 220 nm. Charge heterogeneity was determined by capillary isoelectric focusing on a Labchip GX II Caliper instrument. Working solutions were prepared and the chip was prepared according to standard procedures using the NT Protein Charge Variant Labeling Kit and Protein Charge Variant Buffer Kit, PerkinElmer.

[0686] The resulting stability data for BCD-166 (for BCD166-02-01 and BCD166-01-014) during incubation at 50 °C are presented in Table 8, and Fig. 30 (for BCD166-02-01) and Fig. 31 (for BCD166-01-014) show the combined chromatograms for 72-hour incubation at 50 °C.

[0687] General conclusion: the sample has high colloidal and thermal stability.

[0688] * D is the difference between the purity of the stressed sample and the purity of the intact sample (incoming control), %.

[0689] Table 8. Dependence of monomer content according to exclusive HPLC and EF for BCD-166.

[0690] Example 23

[0691] Evaluation of the antitumor activity of BCD-166 drugs in a subcutaneous xenograft model

[0692] To evaluate the antitumor activity of BCD-166 drugs, a subcutaneous tumor xenograft model was used. For this, 3x10 6A2058 cells mixed with Matrigel®. On day 7 after cell inoculation, animals were divided into groups as shown in Table 9.

[0693] Table 9. Distribution of animals into groups in the experiment to determine the antitumor activity of BCD-166 drugs

[0694]

[0695] The drugs were administered intraperitoneally at a dose of 20 mg / kg on days 1, 5, 8, and 12 after animals were divided into groups. The negative control group was administered a histidine buffer. The body weight of the mice and the linear dimensions of the tumor were measured throughout the experiment. The tumor volume was calculated using the formula V = L x W x H x n / 6. The tumor growth inhibition index (TGI) was used as a criterion for the effectiveness of the test drug, which is calculated taking into account the average tumor volume in the negative control group (V c ) and the drug group (V t ) according to the formula:

[0696] Based on the experimental results, BCD-166-01-014 and BCD-166-02-01 demonstrated significant antitumor activity, while BCD-166-01-01 did not. The results are shown in Figures 32 and 33.

Claims

DEPCT6519 / 01 / 25651. Monoclonal antibody isolated or sub-fragment that binds specifically to the GITR composed of: (a) heavy chain variant domain composed of: (i) CDR1 composed of amino acid sequences selected from the groups: NYGMH(SEQIDNO:1) or YYWMY(SEQIDNO:12); (ii) CDR2 composed of amino acid sequences selected from the groups: VIWFDGSNKFYTDSVKG(SEQIDNO:2) or AISWNGGRTYYAESMKG(SEQIDNO:13); (iii) CDR3 composed of amino acid sequences selected from the groups: (a) light chain variant domains composed of: (i) CDR1 composed of amino acid sequences selected from the groups: RASQSIGSWLA(SEQIDNO:3) or TGTSTDIGTYKYIS(SEQIDNO:17); (ii) CDR2 composed of amino acid sequences selected from the groups: AASTLQR(SEQIDNO:8) or GVSHRPS(SEQIDNO:18); (iii) CDR3 composed of amino acid sequences selected from the groups: QQSHSHPLT(SEQIDNO:9) or SSYTSSGTVV(SEQIDNO:19).Monoclonal antibody or sub-fragments that bind to the antigen of that thing according to claim 1, in which the heavy chain variable domain comprises: (i) CDR1, CDR2 and CDR3 acid sequences which are represented by the sequences SEQIDNO:1, SEQIDNO:2 and SEQIDNO:3, respectively; (ii) CDR1, CDR2 and CDR3 acid sequences which are represented by the sequences SEQIDNO:12, SEQIDNO:13 and SEQIDNO:14, respectively.

3. Monoclonal antibody or sub-fragments The subunits that bind to the antigen of that thing according to claim1, where the light chain variant domains consist of: (i) CDR1, CDR2 and CDR3 consisting of amino acid sequences represented by the sequences of SEQIDNO:7, SEQIDNO:8 and SEQIDNO:9, respectively; (ii) CDR1, CDR2 and CDR3 consisting of amino acid sequences represented by the sequences of SEQIDNO:17, SEQIDNO:18 and SEQIDNO:19, respectively.4.Monoclonal antibodies or sub-fragments that bind to the antigen of that thing according to claim 1, where - heavy chain variant domains comprised of CDR1, CDR2 and CDR3 comprised of amino acid sequences denoted by the sequences of SEQIDNO:1, SEQIDNO:2 and SEQIDNO:3, respectively; - light chain variant domains comprised of CDR1, CDR2 and CDR3 comprised of amino acid sequences denoted by the sequences of SEQIDNO:7, SEQIDNO:8 and SEQIDNO:9, respectively.

5. Monoclonal antibodies or sub-fragments that bind to the antigen of that thing according to claim 1, where - heavy chain variant domains comprised of CDR1, CDR2 and CDR3 comprised of amino acid sequences denoted by the sequences of SEQIDNO:7, SEQIDNO:8 and SEQIDNO:9, respectively. The clonal antibody or sub-fragment that binds to the antigen of that thing according to claim 1, where - the heavy chain variant domains comprised of CDR1, CDR2 and CDR3, which are composed of amino acid sequences denoted by the sequences of SEQIDNO:12, SEQIDNO:13 and SEQIDNO:14, respectively; - the light chain variant domains comprised of CDR1, CDR2 and CDR3, which are composed of amino acid sequences denoted by the sequences of SEQIDNO:17, SEQIDNO:18 and SEQIDNO:19, respectively.6.Monoclonal antibody or antigen-binding subfragment of that thing under claim 1, in which the heavy chain variant domain contains: (i) an amino acid sequence that is at least 90% similar to the amino acid sequence of SEQIDNO:4; or (ii) an amino acid sequence that is at least 90% similar to the amino acid sequence of SEQIDNO:

157. Monoclonal antibody or antigen-binding subfragment of that thing under claim 1, in which (i) the heavy chain variant domain contains the amino acid sequence of SEQIDNO:4; (ii) the heavy chain variant domain contains the amino acid sequence of SEQIDNO:

158. Monoclonal antibody or antigen-binding subfragment of that thing under claim 1, where the light chain variant domain contains: (i) an amino acid sequence that is at least 90% similar to the amino acid sequence of SEQIDNO:10; or (ii) an amino acid sequence that is at least 90% similar to the amino acid sequence of SEQIDNO:2010. Monoclonal antibody or antigen-binding subfragment of that thing under claim 1, where (i) the light chain variant domain contains the amino acid sequence of SEQIDNO:10; or (ii) the light chain variant domain contains the amino acid sequence of SEQIDNO:2010.Monoclonal antibody or sub-fragment binding to the antigen of that thing under claim 1, where (i) - heavy chain variant domain contains an amino acid sequence that is at least 90% similar to the amino acid sequence of SEQIDNO:4; - light chain variant domain contains an amino acid sequence that is at least 90% similar to the amino acid sequence of SEQIDNO:10; or (ii) - heavy chain variant domain contains an amino acid sequence that is at least 90% similar to the amino acid sequence of SEQIDNO:15; - light chain variant domain contains an amino acid sequence that is at least 90% similar to the amino acid sequence of SEQIDNO:

15. At least 90% with the amino acid sequence of SEQIDNO:2011. Monoclonal antibody or subfragment binding to the antigen of that thing according to claim 10, where - the heavy chain variant domain contains the amino acid sequence of SEQIDNO:4; - the light chain variant domain contains the amino acid sequence of SEQIDNO:1012. Monoclonal antibody or subfragment binding to the antigen of that thing according to claim 10, where - the heavy chain variant domain contains the amino acid sequence of SEQIDNO:15; - the light chain variant domain contains the amino acid sequence of SEQIDNO:2013.Monoclonal antibody according to claim 1, where the antibody specifically binding to GITR is a full-length IgG antibody14. Monoclonal antibody according to claim 13, where the full-length IgG antibody is associated with human IgG1, IgG2, IgG3, IgG4 isotypes15. Monoclonal antibody according to claim 14, where the full-length IgG antibody is associated with human IgG1 isotype16. Monoclonal antibody according to claim 1, where the antibody specifically binding to GITR incorporates the E345R mutation in the Fc subfract to enhance the antibody properties.

17. Monoclonal antibody under claim 1 comprising a heavy chain containing: (i) an amino acid sequence that is at least 90% similar to the sequence of SEQIDNO:5; or (ii) an amino acid sequence that is at least 90% similar to the sequence of SEQIDNO:

6.

18. Monoclonal antibody under claim 1 comprising: (i) a heavy chain containing the amino acid sequence of SEQIDNO:5; or (ii) a heavy chain containing the amino acid sequence of SEQIDNO:

6. 19.Monoclonal antibody under claim 1 composed of a light chain containing an amino acid sequence that is at least 90% similar to the sequence of SEQIDNO:1120. Monoclonal antibody under claim 1 composed of a light chain containing the amino acid sequence of SEQIDNO:1121. Monoclonal antibody under claim 1 composed of: (i)- a heavy chain containing an amino acid sequence that is at least 90% similar to the sequence of SEQIDNO:5;- a light chain containing an amino acid sequence that is at least 90% similar to the sequence of SEQIDNO:11; or (ii)- a chain 1. Monoclonal antibody according to claim 1 consisting of: - Heavy chain containing the amino acid sequence of SEQIDNO:6; - Light chain containing the amino acid sequence of SEQIDNO:1122.

2. Monoclonal antibody according to claim 1 consisting of: - Heavy chain containing the amino acid sequence of SEQIDNO:5; - Light chain containing the amino acid sequence of SEQIDNO:1123.

3. Monoclonal antibody according to claim 1 consisting of: - Heavy chain containing the amino acid sequence of SEQIDNO:6; - Light chain containing the amino acid sequence of SEQIDNO:1124.Monoclonal antibody under claim 1, composed of a heavy chain containing an amino acid sequence that is at least 90% similar to the sequence of SEQIDNO:1625. Monoclonal antibody under claim 1, composed of a heavy chain containing the amino acid sequence of SEQIDNO:1626. Monoclonal antibody under claim 1, composed of a light chain containing an amino acid sequence that is at least 90% similar to the sequence of SEQIDNO:2127. Monoclonal antibody under claim 1, composed of... A light chain incorporating the amino acid sequence of SEQIDNO:2128. A monoclonal antibody according to claim 1 incorporating: - a heavy chain incorporating the amino acid sequence with at least 90% similarity to the sequence of SEQIDNO:16; - a light chain incorporating the amino acid sequence with at least 90% similarity to the sequence of SEQIDNO:2129. A monoclonal antibody according to claim 1 incorporating: - a heavy chain incorporating the amino acid sequence of SEQIDNO:16; - a light chain incorporating the amino acid sequence of SEQIDNO:2130.

31. A nucleic acid isolated that encodes an antibody or antigen-binding fragment according to one of the claims in 1-29.

32. A nucleic acid according to claim 30, where the nucleic acid is DNA.

33. An expression vector composed of a nucleic acid according to one of the claims in 30-31.

34. A method of obtaining host cells for the acquisition of an antibody or antigen-binding fragment according to one of the claims in 1-29, composed of a vector-based cell transfection according to claim 32.

35. A host cell.

35. The method of obtaining antibodies or antigen-binding fragments of the substance as specified in any of the claims 1-29, which includes the culturing of host cells as specified in claim 34 in a culture medium under conditions sufficient to obtain such antibodies, if necessary, followed by isolation and purification of the obtained antibodies. 36.

37. A pharmaceutical component under claim 36 focused on therapeutic use for a GITR-mediated disease or disorder, selected from the following groups: cervical cancer, head and neck cancer, gastric cancer, breast cancer, renal cell carcinoma, CRC (colorectal cancer), (OC) ovarian cancer, NSCLC (non-small cell lung cancer).

38. A pharmaceutical component for therapeutic use for a GITR-mediated disease or disorder, selected from the following groups: cervical cancer, head and neck cancer, gastric cancer, breast cancer, renal cell carcinoma, CRC (colorectal cancer), (OC) ovarian cancer, NSCLC (non-small cell lung cancer).

39. A pharmaceutical component for therapeutic use for a GITR-mediated disease or disorder, containing an antibody or antigen-binding fraction of any of the above-mentioned claims in claims 1-29 in an effective therapeutic dose and at least one therapeutic antitumor compound in an effective therapeutic dose.

40. Pharmaceutical composition under claim 38, where the therapeutic focus is on the treatment of a disease or disorder mediated by GITR, selected from the group of cervical cancer, head and neck cancer, gastric cancer, breast cancer, renal cell carcinoma, CRC (colorectal cancer), (OC) ovarian cancer, NSCLC (non-small cell lung cancer).

41. Pharmaceutical composition under claim 40, where the therapeutic antitumor compound is selected from chemotherapy, antibodies, or antihormonal agents.

42. Pharmaceutical composition under claim 41, where the therapeutic antitumor compound is (i) an antibody selected from the group: anti-PD1 antibody, anti-PD-L1 antibody, anti-CTLA4 antibody, anti-4-1BB antibody, anti-OX40 antibody, or a combination thereof, or (ii) a small molecule, or (iii) selected from the group of innate or acquired immunogenic stimulators.

43. Pharmaceutical composition under claim 41, where the antibody under claim 29 and at least one therapeutic antitumor compound are given in sequence.

44. A method for the inhibition of GITR biological activity in the test sample requiring inhibition, consisting of the administration of an effective dose of an antibody or antigen-binding subunit of that substance as specified in claims 1-29.

45. A method for the treatment of a GITR-mediated disease or disorder consisting of the administration in the test sample requiring the treatment of an antibody or antigen-binding subunit of that substance as specified in claims 1-29 or a pharmaceutical component as specified in claims 36 or 38 in an effective dose.

46. A method for the treatment of a disease or disorder as specified in claim 45, where the disease or disorder is selected from the following groups: cervical cancer, head and neck cancer, gastric cancer, breast cancer, renal cell carcinoma, CRC (colorectal cancer), (OC) ovarian cancer, NSCLC (non-small cell lung cancer). 47.The use of antibodies or antigen-binding subunits of any of the claims in 1-29 or pharmaceutical components in claims 36 or 38 for therapeutic purposes in the test subjects for the disease or disorder mediated by GITR48. The use in claim 47, where the disease or disorder is selected from a group that includes: cervical cancer, head and neck cancer, gastric cancer, breast cancer, renal cell carcinoma, CRC (colorectal cancer), (OC) ovarian cancer, NSCLC (non-small cell lung cancer).-----------------------------------------------------------DEPCT651.Monoclonal antibodies, isolated or fragmented, that bind their antigens specifically to GITR, comprise: (a) variable heavy chain domains comprise: (i) CDR1, comprised of an amino acid sequence selected from the group:NYGMH(SEQIDNO:1) or YYWMY(SEQIDNO:12); (ii) CDR2, comprised of an amino acid sequence selected from the group:VIWFDGSNKFYTDSVKG(SEQIDNO:2) or AISWNGGRTYYAESMKG(SEQIDNO:13); (iii) CDR3, comprised of an amino acid sequence selected from the group: (a) the variable domain of the light chain which comprises: (i) CDR1 which comprises amino acid sequences selected from the group: RASQSIGSWLA(SEQIDNO:7) or TGTTSTDIGTYKYIS(SEQIDNO:17); (ii) CDR2 which comprises amino acid sequences selected from the group: AASTLQR(SEQIDNO:8) or GVSHRPS(SEQIDNO:18); (iii) CDR3 which comprises amino acid sequences selected from the group: QQSHSHPLT(SEQIDNO:9) or SSYTSSGTW(SEQIDNO:19)2.Monoclonal antibodies or fragments which bind their antigens according to claim I, in which the variable domains of the heavy chain comprise: (i) the acid sequences CDR1, CDR2 and CDR3 represented by the sequences SEQIDNO:1, SEQIDNO:2 and SEQIDNO:3, respectively; (ii) the acid sequences CDR1, CDR2 and CDR3 represented by the sequences SEQIDNO:12, SEQIDNO:13 and SEQIDNO:14, respectively.

3. Monoclonal antibodies or fragments which bind their antigens according to claim I, in which the variable domains of the heavy chain comprise: (i) the acid sequences CDR1, CDR2 and CDR3 represented by the sequences SEQIDNO:12, SEQIDNO:13 and SEQIDNO:14, respectively. The fragments which bind their antigens according to claim1, in which the light chain variant domains consist of: (i) CDR1, CDR2 and CDR3, which consist of amino acid sequences represented by the sequences SEQIDNO:7, SEQIDNO:8 and SEQIDNO:9, respectively; (ii) CDR1, CDR2 and CDR3, which consist of amino acid sequences represented by the sequences SEQIDNO:17, SEQIDNO:18 and SEQIDNO:19, respectively.4.Monoclonal antibodies or fragments that bind to their antigens according to claim1, where - the variable domain of the heavy chain consists of CDR1, CDR2 and CDR3, which are comprised of amino acid sequences represented by the sequences SEQIDNO:1, SEQIDNO:2 and SEQIDNO:3, respectively; - the variable domain of the light chain consists of CDR1, CDR2 and CDR3, which are comprised of amino acid sequences represented by the sequences SEQIDNO:7, SEQIDNO:8 and SEQIDNO:9, respectively.

5. Monoclonal antibodies or fragments that bind to their antigens according to claim1, where - the variable domain of the heavy chain consists of CDR1, CDR2 and CDR3, which are comprised of amino acid sequences represented by the sequences SEQIDNO:7, SEQIDNO:8 and SEQIDNO:9, respectively. Clonal antibodies or fragments that bind to their antigens according to claim1, where - the variable domain of the heavy chain consists of CDR1, CDR2 and CDR3, which are comprised of amino acid sequences represented by the sequences SEQIDNO:12, SEQIDNO:13 and SEQIDNO:14, respectively; - the variable domain of the light chain consists of CDR1, CDR2 and CDR3, which are comprised of amino acid sequences represented by the sequences SEQIDNO:17, SEQIDNO:18 and SEQIDNO:19, respectively.6.Monoclonal antibodies or fragments that bind their antigens according to claim 1, in which the variable heavy chain domain contains: (i) an amino acid sequence that is at least 90% similar to the amino acid sequence of SEQIDNO:4; or (ii) an amino acid sequence that is at least 90% similar to the amino acid sequence of SEQIDNO:

157. Monoclonal antibodies or fragments that bind their antigens according to claim 1, in which (i) the variable heavy chain domain contains the amino acid sequence of SEQIDNO:4; (ii) the variable heavy chain domain contains the amino acid sequence of SEQIDNO:

158. Monoclonal antibodies or fragments that bind their antigens according to claim 1, in which the variable light chain domain contains: (i) an amino acid sequence that is at least 90% similar to the amino acid sequence of SEQIDNO:10; or (ii) an amino acid sequence that is at least 90% similar to the amino acid sequence of SEQIDNO:209.Monoclonal antibodies or fragments that bind their antigens according to claim1 where (i) the variable domain of the light chain contains the amino acid sequence of SEQIDNO:10; or (ii) the variable domain of the light chain contains the amino acid sequence of SEQIDNO:2010. Monoclonal antibodies or fragments that bind their antigens according to claim1 where (i) - the variable domain of the heavy chain contains the amino acid sequence that is at least 90% similar to the amino acid sequence of SEQIDNO:4; - the variable domain of the light chain contains the amino acid sequence that is at least 90% similar to the amino acid sequence of SEQIDNO:2010. (ii) - a variable domain of the heavy chain consisting of an amino acid sequence that is at least 90% similar to the amino acid sequence of SEQIDNO:15; - a variable domain of the light chain consisting of an amino acid sequence that is at least 90% similar to the amino acid sequence of SEQIDNO:2011. Monoclonal antibodies or fragments that bind their antigens according to claim10, where - a variable domain of the heavy chain consisting of the amino acid sequence of SEQIDNO:4; - a variable domain of the light chain consisting of the amino acid sequence of SEQIDNO:1012.Monoclonal antibodies or fragments that bind to their antigens according to claim10, where - the variable domain of the heavy chain incorporates the amino acid sequence SEQIDNO:15; - the variable domain of the light chain incorporates the amino acid sequence SEQIDNO:2013. Monoclonal antibodies according to claim1, where the antibody that specifically binds to GITR is a full-length IgG antibody14. Monoclonal antibodies according to claim13, where the full-length IgG antibody is associated with human IgG1, IgG2, IgG3, IgG4 isotypes15. Monoclonal antibodies according to claim14, where the full-length IgG antibody is associated with human IgG1 isotype16. Monoclonal antibodies according to claim1, where the antibody that specifically binds to GITR incorporates the E345R mutation in the Fc fragment to enhance antibody-dependent cell cytotoxicity (ADCC) but not complement-dependent cell cytotoxicity (CDC) properties17.Monoclonal antibody under claim 1, consisting of a heavy chain incorporating: (i) an amino acid sequence with at least 90% similarity to the sequence of SEQIDNO:5; or (ii) an amino acid sequence with at least 90% similarity to the sequence of SEQIDNO:

618. Monoclonal antibody under claim 1, consisting of: (i) a heavy chain incorporating the amino acid sequence of SEQIDNO:5; or (ii) a heavy chain incorporating the amino acid sequence of SEQIDNO:

619. Monoclonal antibody under claim 1, consisting of a light chain incorporating an amino acid sequence with at least 90% similarity to the sequence of SEQIDNO:1120. Monoclonal antibody under claim 1, consisting of a light chain incorporating the amino acid sequence of SEQIDNO:1121.Monoclonal antibodies under claim 1, comprising: (i)-heavy chain containing an amino acid sequence that is at least 90% similar to the sequence of SEQIDNO:5;-light chain containing an amino acid sequence that is at least 90% similar to the sequence of SEQIDNO:11; or (ii)-heavy chain containing an amino acid sequence that is at least 90% similar to the sequence of SEQIDNO:6;-light chain containing an amino acid sequence that is at least 90% similar to the sequence of SEQIDNO:1122. Monoclonal Antibody according to claim 1, comprising: - heavy chain containing the amino acid sequence of SEQIDNO:5; - light chain containing the amino acid sequence of SEQIDNO:1123. Monoclonal antibody according to claim 1, comprising: - heavy chain containing the amino acid sequence of SEQIDNO:6; - light chain containing the amino acid sequence of SEQIDNO:1124. Monoclonal antibody according to claim 1, comprising: - heavy chain containing the amino acid sequence of SEQIDNO:1625.Monoclonal antibody under claim 1, consisting of: - a heavy chain incorporating the amino acid sequence of SEQIDNO:1626. Monoclonal antibody under claim 1, consisting of: - a light chain incorporating the amino acid sequence that is at least 90% homologous to the sequence of SEQIDNO:2127. Monoclonal antibody under claim 1, consisting of: - a light chain incorporating the amino acid sequence of SEQIDNO:2128. Monoclonal antibody under claim 1, consisting of: - a heavy chain incorporating the amino acid sequence that is at least 90% similar to the sequence of SEQIDNO:16; - a light chain incorporating the amino acid sequence that is at least 90% similar to the sequence of SEQIDNO:16; A monoclonal antibody according to claim 1, consisting of: - a heavy chain containing the amino acid sequence of SEQIDNO:16; - a light chain containing the amino acid sequence of SEQIDNO:2130. A single nucleic acid encoding an antibody or a fragment binding to its antigen according to one of claims 1-2931. A nucleic acid according to claim 30, where the nucleic acid is DNA32. An expression vector consisting of a nucleic acid according to one of claims 30-3133.Methods for obtaining host cells to obtain antibodies or antigen-binding fragments under any of the claims 1-29, incorporating vector transformation of the cells under claim 3234. Host cells to obtain antibodies or antigen-binding fragments under any of the claims 1-29, incorporating nucleic acids under any of the claims 30-3135. Methods for obtaining antibodies or antigen-binding fragments under any of the claims 1-29, incorporating the culture of host cells under claim 34 in culture medium under conditions sufficient to obtain such antibodies, if necessary, followed by isolation and purification of the obtained antibodies.

36. Pharmaceutical components for the treatment of diseases or disorders mediated by GITRs, incorporating antibodies or antigen-binding fragments under any of the claims 1-29 in therapeuticly effective doses in combination with one or more pharmaceutically acceptable drug additives.37.

38. A pharmaceutical compound under claim 36 intended for the treatment of a disease or disorder mediated by GITR, selected from the following groups: cervical cancer, head and neck cancer, gastric cancer, breast cancer, renal cell carcinoma, CRC (colon cancer) (OC), ovarian cancer, NSCLC (non-small cell lung cancer).

39. A pharmaceutical compound under claim 38 intended for the treatment of a disease or disorder mediated by GITR, which includes antibodies or fragments binding to their antigens under any of claims 1-29 in a therapeutic dose and at least one therapeuticly active antitumor compound in a therapeutic dose.

30. A pharmaceutical compound under claim 38 intended for the treatment of a disease or disorder mediated by GITR, selected from the following groups: cervical cancer, head and neck cancer, gastric cancer, breast cancer, renal cell carcinoma, CRC (colon cancer) (OC), ovarian cancer, NSCLC (non-small cell lung cancer).

41. A pharmaceutical compound under claim 38 in which the therapeuticly active antitumor compound is selected from chemotherapy, antibodies, or antihormonal agents.

42. Pharmaceutical composition under claim 41 in which the antitumor compound of therapeutic activity under claim 29 and at least one therapeutic antitumor compound are administered, respectively.

43. Pharmaceutical composition under claim 41 in which the antibody under claim 1-29 and at least one therapeutic antitumor compound are administered concomitantly.

44. Method of inhibition of the biological activity of GITR in the desired trial participants which includes the administration of an effective dose of the antibody or its antigen-binding fragment under one of the claims 1-29. 45.GITR-mediated therapeutic methods for diseases or disorders comprising the administration of antibodies or their antigen-binding fragments as per claims 1-29 or pharmaceutical components as per claims 36 or 38 in therapeutic doses to trial patients requiring such therapy.

46. Therapeutic methods for diseases or disorders as per claim 45, selecting diseases or disorders from the following groups: cervical cancer, head and neck cancer, gastric cancer, breast cancer, renal cell carcinoma, CRC (colon cancer), (OC) ovarian cancer, NSCLC (non-small cell lung cancer).

47. The use of antibodies or their antigen-binding fragments as per claims 1-29 or pharmaceutical components as per claims 36 or 38 for therapeutic purposes in trial patients requiring such therapy for diseases or disorders mediated by GITR.48.Under claim 47, select a disease or disorder from a group that includes: cervical cancer, head and neck cancer, stomach cancer, breast cancer, renal cell carcinoma, CRC (colon cancer) (OC), ovarian cancer, NSCLC (non-small cell lung cancer).

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