Monoclonal antibody that specifically binds to GITR

Abstract

The present invention relates to biotechnology, particularly to antibodies or antigen-binding fragments thereof, and their uses. More particularly, the present invention relates to monoclonal antibodies that specifically bind to GITR. The present invention also relates to nucleic acids encoding the antibodies or antigen-binding fragments thereof, expression vectors, methods for preparing the antibodies, and uses of the antibodies in the treatment of diseases or disorders associated with GITR.

Description

Technical Field

[0001] The present invention relates to biotechnology, in particular to antibodies or antigen-binding fragments thereof, and further to their use. More specifically, the present invention relates to monoclonal antibodies that specifically bind to GITR (glucocorticoid-induced TNFR-related protein / TNFRSF18 / tumor necrosis factor receptor superfamily, member 18). The present invention also relates to nucleic acids encoding said antibodies or antigen-binding fragments thereof, expression vectors, methods for preparing said antibodies, and the use of said antibodies in the treatment of diseases or disorders associated with GITR.

Background Art

[0002] TNFRSF18, GITR (glucocorticoid-induced TNFR-related protein / TNFRSF18 / tumor necrosis factor receptor superfamily) is a membrane protein that is a receptor belonging to the tumor necrosis factor receptor superfamily.

[0003] GITR is a type I transmembrane protein consisting of 216 amino acids and having a molecular weight of 26 kDa. The N-terminal extracellular domain contains three TNFR-Cys repeats and an N-glycosylation site. The three cysteine-rich domains and the cytoplasmic tail of GITR have significant homology with 4-1BB, OX40 and CD27 (Nocentini et al. (1997) Proc. Natl. Acad. Sci. 94: 6216-6221).

[0004] Human GITR is expressed at low levels in responder T cells and shows increased expression in CD4+ cells compared to CD8+ cells. GITR expression is significantly upregulated over several days after T cell activation. GITR is constitutively expressed at high levels in regulatory T cells (Tregs), such as CD4+CD25+ or CD8+CD25+ cells, and is further upregulated when these cells are activated (Nocentini and Riccardi (2005) E. J. Immunol. 35: 1016).

[0005] However, GITR expression is not limited to T cells only. Numerous studies have shown that GITR is also expressed in NK cells, macrophages, B cells, dendritic cells, mast cells, and monocytes (Nocentini and Riccardi (2005) E. J. Immunol. 35: 1016 - 1022).

[0006] GITR is expressed in lymph nodes, peripheral blood leukocytes, and to a lesser extent in the spleen. It is constitutively expressed at high levels in Tregs and at low levels in naive T cells and memory cells.

[0007] However, GITR is expressed not only in immune cells but also in tumor cells. RNA - Seq data analysis of GITR expression in 33 tumor types has revealed that GITR is highly expressed in HNSCC (head and neck squamous cell carcinoma), NSCLC (non - small cell lung cancer), breast cancer, esophageal cancer, and bladder cancer.

[0008] RNA - Seq analysis of samples from 24 tumor types showed similar results. Thus, GITR is expressed not only in immune cells but also on the membrane of tumor cells. In tumor samples, GITRL - Fc increased gene expression related to T cells, CD8 T cells, cytotoxicity, Th1 cells, interferon - gamma, NK cells, Teff cells, and T - cell activation markers.

[0009] The expression of GITR and its ligand is not limited to hematopoietic cells. GITR is also expressed in keratinocytes and osteoclast precursors, while GITRL is expressed in endothelial cells.

[0010] GITRL is a type II transmembrane protein, as is typical for most TNF ligand family members. Current research indicates that typically, human GITRL exists as a trimer, but may also exist as a monomer or assemble into 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 some evidence suggesting 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) such as macrophages, B cells, dendritic cells, and endothelial cells that 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) E. J. Immunol. 37:1165 - 1169).

[0011] When GITRL on APC binds to GITR on responder T cells, GITR signaling is initiated, thereby co-stimulating the responder T cells and inhibiting the inhibitory activity of regulatory T cells. GITR signaling functions as a co-activation signal to both CD4+ and CD8+ naive T cells, thereby inducing or enhancing proliferation and effector functions, especially when T cell receptor (TCR) stimulation is in an optimal proximity (Schaer et al. (2012) Curr. Opin. Immunol. 24: 217 - 224). More specifically, GITR may have numerous effects on effector T cells and regulatory T cells, such effects including co-stimulating and activating effector T cells to make them more resistant to inhibition, inhibiting regulatory T cells, reducing the sensitivity of effector T cells to suppression by regulatory T cells, and partially depleting regulatory T cells from the circulation (Nocentini et al. (2007) Eur. J. Immunol. 37: 1165 - 1169).

[0012] The main function of GITR, and thus the main action of anti-GITR antibodies, is to enhance the proliferation and functionalization of effector T cells and inhibit the inhibitory action of Tregs. Teff cells are initially generated lacking the ability to withstand the inhibitory tumor microenvironment and suppression by Treg cells. Stimulation of GITR in the secondary stages of priming and expansion via agonist anti-GITR antibodies, soluble GITR ligands or DC vaccines modulates both Teff and Treg tumor responses, favoring the former and thus promoting tumor regression. Thus, anti-GITR antibodies provide Teff with resistance to Treg suppression.

[0013] Collectively, the aforementioned functions, particularly the co-stimulation of responder T cells and the elimination of the suppressor activity of regulatory T cells, mean that GITR activation results in an enhanced immune response. Such activation has the potential to restore the immune response against infections and tumors. Therefore, molecules capable of activating GITR are expected to be beneficial as immune stimulants under conditions where it is desirable to initiate an enhanced immune response.

[0014] The antibody must have the properties of a GITR agonist, along with effector and cytotoxic properties against Treg lymphocytes. Various antibodies against GITR are known in the art (for example, from WO2015187835, WO2015031667, WO2017068186, WO2017096189, WO2017214548).

[0015] Currently, 23 anti-GITR agonists (antibodies / recombinant GITRL) are in preclinical and clinical trials. Only two antibodies are in phase 2 clinical trials (TRX518, INCAGN1876), and AMG228, MEDI1873, MK-4166 are in phase 1 clinical trials. Little clinical data has been presented.

[0016] However, currently there is no antibody in the world that specifically binds to GITR and is approved for therapeutic use.

Summary of the Invention

Problems to be Solved by the Invention

[0017] In relation to the above, it is important to create a novel agonist antibody that interacts with GITR, activates the receptor, eliminates the suppressor action of regulatory T cells, and inhibits / depletes the T-suppressor (regulatory) component of the immune system via ADCC effector properties.

Means for Solving the Problems

[0018] BCD-166 is an agonistic monoclonal antibody that interacts with GITR, activates the receptor, eliminates the suppressor function of regulatory T cells, and inhibits / losses the T-suppressor (regulatory) component of the immune system through its ADCC effector properties, thereby increasing the number of CD8+ and CD4+ effector cells and activating the T-effector component of the immune system in the tumor microenvironment.

[0019] In one aspect, the invention provides an isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to GITR, (a)(i) the following groups: NYGMH (SEQ ID NO: 1) or YYWMY (SEQ ID NO: 12) and comprises a CDR1 containing an amino acid sequence selected from the group consisting of: (ii) the following groups: VIWFDGSNKFYTDSVKG (SEQ ID NO: 2) or AISWNGGRTYYAESMKG (SEQ ID NO: 13) and comprises a CDR2 containing an amino acid sequence selected from the group consisting of: (iii) the following groups: ELGGYYYDSSGFRPYYYGMDV (SEQ ID NO: 3) or NRYYSDPNYGMNL (SEQ ID NO: 14) and comprises a CDR3 containing an amino acid sequence selected from the group consisting of: and comprises a heavy chain variable domain, and (b)(i) the following groups: RASQSIGSWLA (SEQ ID NO: 7) or TGTSTDIGTYKYIS (SEQ ID NO: 17) and comprises a CDR1 containing an amino acid sequence selected from the group consisting of: (ii) the following groups: AASTLQR (SEQ ID NO: 8) or GVSHRPS (SEQ ID NO: 18) and comprises a CDR2 containing an amino acid sequence selected from the group consisting of: (iii) the following groups: QQSHSHPLT (SEQ ID NO: 9) or SSYTSSGTVV (SEQ ID NO: 19) and comprises a CDR3 containing an amino acid sequence selected from the group consisting of: and comprises a light chain variable domain Relates to a monoclonal antibody or an antigen-binding fragment thereof.

[0020] In some embodiments, the monoclonal antibody or an antigen-binding fragment thereof comprises a heavy chain variable domain comprising CDR1, 2, and 3 comprising amino acid sequences represented by SEQ ID NOs: 1, 2, and 3, respectively.

[0021] In some embodiments, the monoclonal antibody or an antigen-binding fragment thereof comprises a heavy chain variable domain comprising CDR1, 2, and 3 comprising amino acid sequences represented by SEQ ID NOs: 12, 13, and 14, respectively.

[0022] In some embodiments, the monoclonal antibody or an antigen-binding fragment thereof comprises a light chain variable domain comprising CDR1, 2, and 3 comprising amino acid sequences represented by SEQ ID NOs: 7, 8, and 9, respectively.

[0023] In some embodiments, the monoclonal antibody or an antigen-binding fragment thereof comprises a light chain variable domain comprising CDR1, 2, and 3 comprising amino acid sequences represented by SEQ ID NOs: 17, 18, and 19, respectively.

[0024] In some embodiments, the monoclonal antibody or an antigen-binding fragment thereof - a heavy chain variable domain comprising CDR1, 2, and 3 comprising amino acid sequences represented by SEQ ID NOs: 1, 2, and 3, respectively; - a light chain variable domain comprising CDR1, 2, and 3 comprising amino acid sequences represented by SEQ ID NOs: 7, 8, and 9, respectively comprises.

[0025] In some embodiments, the monoclonal antibody or an antigen-binding fragment thereof - a heavy chain variable domain comprising CDR1, 2, and 3 comprising amino acid sequences represented by SEQ ID NOs: 12, 13, and 14, respectively; - a light chain variable domain comprising CDR1, 2, and 3 comprising amino acid sequences represented by SEQ ID NOs: 17, 18, and 19, respectively comprises.

[0026] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 4.

[0027] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 15. In some embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 15.

[0028] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises a light chain variable domain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 10. In some embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 10.

[0029] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises a light chain variable domain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 20. In some embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 20.

[0030] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof - a heavy chain variable domain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 4; - a light chain variable domain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 10 comprises.

[0031] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof - comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 4; - comprises a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 10 and comprises.

[0032] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof - comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 15; - comprises a light chain variable domain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 20 and comprises.

[0033] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof - comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 15; - comprises a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 20 and comprises.

[0034] 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 a monoclonal IgG antibody of human IgG1, IgG2, IgG3 or IgG4 isotype.

[0035] In some embodiments, the monoclonal IgG antibody is a monoclonal IgG antibody of human IgG1 isotype. In some embodiments, the monoclonal antibody that specifically binds to GITR comprises an E345R mutation in the Fc fragment such that it has agonist properties and increases antibody-dependent cell-mediated cytotoxicity (ADCC), but does not increase complement-dependent cytotoxicity (CDC).

[0036] In some embodiments, the monoclonal antibody comprises a heavy chain comprising an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 5. In some embodiments, the monoclonal antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 5.

[0037] In some embodiments, the monoclonal antibody comprises a heavy chain comprising an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 6. In some embodiments, the monoclonal antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 6.

[0038] In some embodiments, the monoclonal antibody comprises a light chain comprising an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 11. In some embodiments, the monoclonal antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 11.

[0039] In some embodiments, the monoclonal antibody - a heavy chain comprising an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 5; - a light chain comprising an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 11 and comprises.

[0040] In some embodiments, the monoclonal antibody - a heavy chain comprising the amino acid sequence of SEQ ID NO: 5; - a light chain comprising the amino acid sequence of SEQ ID NO: 11 and comprises.

[0041] In some embodiments, the monoclonal antibody - a heavy chain comprising an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 6; - a light chain comprising an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 11 and comprises.

[0042] In some embodiments, the monoclonal antibody - 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 and comprises.

[0043] In some embodiments, the monoclonal antibody comprises a heavy chain comprising an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 16. In some embodiments, the monoclonal antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 16.

[0044] In some embodiments, the monoclonal antibody comprises a light chain comprising an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 21. In some embodiments, the monoclonal antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 21.

[0045] In some embodiments, the monoclonal antibody - a heavy chain comprising an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 16; - a light chain comprising an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 21 comprises.

[0046] In some embodiments, the monoclonal antibody - a heavy chain comprising the amino acid sequence of SEQ ID NO: 16; - a light chain comprising the amino acid sequence of SEQ ID NO: 21 comprises.

[0047] In one aspect, the present invention relates to an isolated nucleic acid encoding any of the above antibodies or antigen-binding fragments thereof. In some embodiments, the nucleic acid is DNA.

[0048] In one aspect, the present invention relates to an expression vector comprising the above nucleic acid. In one aspect, the present invention relates to a method for obtaining a host cell for producing the antibody or antigen-binding fragment thereof, the method comprising transforming the cell with the vector.

[0049] In one aspect, the present invention relates to a host cell comprising the nucleic acid for preparing the antibody or antigen-binding fragment thereof. In one aspect, the present invention relates to a method for obtaining the antibody or an antigen-binding fragment thereof, the method comprising culturing the host cell in a culture medium under conditions sufficient to produce the antibody, and, optionally, subsequently isolating and purifying the obtained antibody.

[0050] In one aspect, the present invention relates to a pharmaceutical composition for treating a GITR-mediated disease or disorder, comprising a therapeutically effective amount of the antibody or an antigen-binding fragment thereof in combination with one or more pharmaceutically acceptable excipients.

[0051] In some embodiments, the pharmaceutical composition is intended to be used for treating a GITR-mediated disease or disorder selected from the group consisting 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).

[0052] In one aspect, the present invention relates to a pharmaceutical composition for treating a GITR-mediated disease or disorder, comprising a therapeutically effective amount of the antibody or an antigen-binding fragment thereof, and a therapeutically effective amount of at least one anti-tumor compound having therapeutic activity.

[0053] In some embodiments, the pharmaceutical composition is intended to be used for treating a GITR-mediated disease or disorder selected from the group consisting 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).

[0054] In some embodiments, the pharmaceutical composition comprises an anti-tumor compound having therapeutic activity selected from a chemotherapeutic agent, an antibody or an anti-hormonal agent. In some embodiments, the pharmaceutical composition comprises an anti-tumor compound having therapeutic activity, which is an antibody selected from the group consisting of an anti-PD1 antibody, an anti-PD-L1 antibody, an anti-CTLA4 antibody, an anti-4-1BB antibody, an anti-OX40 antibody or a combination thereof.

[0055] In some embodiments, the pharmaceutical composition comprises an antitumor compound having therapeutic activity that is a small molecule. In some embodiments, the pharmaceutical composition comprises an antitumor compound having therapeutic activity selected from the group of activators of innate or adaptive immunity.

[0056] In some embodiments of the pharmaceutical composition, the antibody and at least one antitumor compound having therapeutic activity are administered sequentially. In some embodiments of the pharmaceutical composition, the antibody and at least one antitumor compound having therapeutic activity are administered simultaneously.

[0057] In one aspect, the present invention relates to a method for inhibiting the biological activity of GITR in a subject in need thereof, the method comprising administering an effective amount of the antibody or antigen-binding fragment thereof.

[0058] In one aspect, the present invention relates to a method for treating a disease or disorder mediated by GITR, the method comprising administering a therapeutically effective amount of the antibody or antigen-binding fragment thereof or the pharmaceutical composition to a subject in need of such treatment.

[0059] In some embodiments, the method for treatment includes a disease or disorder selected from the group including cervical cancer, head and neck cancer, gastric cancer, breast cancer, renal cell cancer, CRC (colorectal cancer), (OC) ovarian cancer, NSCLC (non-small cell lung cancer).

[0060] In one aspect, the present invention relates to the use of the antibody or antigen-binding fragment thereof or the pharmaceutical composition for treatment in a subject in need of such treatment of a disease or disorder mediated by GITR.

[0061] In some embodiments, the use includes a disease or disorder selected from the following group: cervical cancer, head and neck cancer, gastric cancer, breast cancer, renal cell cancer, CRC (colorectal cancer), (OC) ovarian cancer, NSCLC (non-small cell lung cancer).

Brief Description of the Drawings

[0062]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Figure 15

Figure 16

Figure 17

Figure 18

Figure 19

Figure 20

Figure 21

Figure 22

Figure 23

Figure 24

Figure 25

Figure 26

Figure 27

Figure 28

Figure 29

Figure 30

Figure 31

Figure 32

Figure 33

Mode for Carrying Out the Invention

[0063] Definitions and General Methods Unless otherwise specified, all technical terms and scientific terms used in this specification have the same meaning as commonly understood by those skilled in the art.

[0064] Furthermore, unless the context requires otherwise, singular terms shall include pluralities and plural terms shall include singulars. Typically, classifications and methods of cell culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, organic synthetic chemistry, medicinal chemistry and pharmaceutical chemistry, as well as the hybridization and chemical properties of the proteins and nucleic acids described herein are well known and widely used by those skilled in the art. Enzyme reactions and purification methods are carried out according to the manufacturer's instructions, as commonly practiced in the art or as described herein. Definitions related to antibodies TNFRSF18, GITR (glucocorticoid-induced TNFR-related protein / TNFRSF18 / tumor necrosis factor receptor superfamily) is a membrane protein that is a receptor belonging to the tumor necrosis factor receptor superfamily. GITR is a type I transmembrane protein consisting of 216 amino acids and having a molecular weight of 26 kDa. The N-terminal extracellular domain contains three TNFR-Cys repeats and an N-glycosylation site. The three cysteine-rich domains and the cytoplasmic tail of GITR have significant homology with 4-1BB, OX40 and CD27 (Nocentini et al. (1997) Proc. Natl. Acad. Sci. 94: 6216-6221).

[0065] Amplification of the GITR gene and / or overexpression of its protein have been found in many cancer diseases including cervical cancer, head and neck cancer, gastric cancer, breast cancer, renal cell cancer, CRC (colorectal cancer), (OC) ovarian cancer, NSCLC (non-small cell lung cancer).

[0066] The term "binding molecule" includes antibodies and immunoglobulins. The terms "antibody" or "immunoglobulin" (Ig), as used herein, include the entire antibody and any antigen-binding fragment (i.e., "antigen-binding portion") or single chains thereof. The term "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds or by the antigen-binding portions. Each heavy chain comprises a heavy-chain variable region (referred to herein by the abbreviation VH) and a heavy-chain constant region. Five types of mammalian Ig heavy chains, represented by the Greek letters: α, δ, ε, γ and μ are known. The type of heavy chain present defines the class of the antibody; these chains are found in IgA, IgD, IgE, IgG, and IgM antibodies, respectively. The distinct heavy chains differ in size and composition; α and γ contain approximately 450 amino acids, while μ and ε have approximately 550 amino acids. Each heavy chain has two regions, a constant region and a variable region. The constant region is identical in all antibodies of the same isotype, but differs in antibodies of different isotypes. Heavy chains γ, α and δ have a constant region composed of three constant domains CH1, CH2 and CH3 (in a row), as well as a hinge region to add flexibility (Woof J., Burton D., Nat Rev Immunol 4, 2004, cc.89-99); heavy chains μ and ε have a constant region composed of four constant domains CH1, CH2, CH3 and CH4. In mammals, only two types of light chains, represented by lambda (λ) and kappa (κ) are known. Each light chain consists of a light-chain variable region (referred to herein by the abbreviation VL) and a light-chain constant region. The approximate length of the light chain is 211-217 amino acids. Preferably, the light chain is a kappa (κ) light chain, and the constant domain CL is preferably C kappa (κ).

[0067] The "antibodies" according to the invention may belong to any class (e.g., IgA, IgD, IgE, IgG, and IgM, preferably IgG) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2, preferably IgG1).

[0068] The VL and VH regions can be further classified into hypervariable regions called complementarity-determining regions (CDRs) that are interspersed between more highly conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of the antibody can mediate the binding of immunoglobulins to various cells of the immune system (e.g., effector cells) and host tissues or factors such as the first component of the classical complement system (Clq).

[0069] The term "antigen-binding portion" or "antigen-binding fragment" (or simply "antibody portion" or "antibody fragment") of an antibody, 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 fragments of a full-length antibody can perform the function of binding to the antigen of the antibody. Examples of binding fragments included in the term "antigen-binding portion" of an antibody include: (i) Fab fragment, i.e., a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) F(ab’)2 fragment, i.e., a divalent fragment containing two Fab fragments linked by a disulfide bridge in the hinge region; (iii) Fd fragment consisting of the VH and CH1 domains; (iv) Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) dAb fragment consisting of the VH / VHH domain (Ward et al., (1989) Nature 341:544-546); and (vi) extracted complementarity-determining regions (CDRs). In addition, the two regions of the Fv fragment, VL and VH, are encoded by separate genes, and they can be joined using recombinant methods that use a synthetic linker that allows them to accept a single protein chain in which the VL region and the VH region pair to form a monovalent molecule (known as single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain molecules are also considered to be included in the term "antigen-binding portion" of an antibody. Such antibody fragments can be obtained using conventional techniques known to those skilled in the art, and the fragments are screened in the same manner as intact antibodies.

[0070] Preferably, the CDRs of the antigen-binding portion of the present invention or the antigen-binding portion of the whole antibody are derived from a mouse, llama or human donor library or are of substantially human origin, and specific amino acid residues have been altered, e.g., substituted with different amino acid residues, to optimize the properties of the specific antibody, such as KD, koff, IC50, EC50, ED50. Preferably, the framework regions of the antibodies of the present invention are of human origin or are of substantially human origin (at least 80, 85, 90, 95, 96, 97, 98 or 99% are of human origin).

[0071] In other embodiments, the antigen-binding portion of the present invention may be derived from other non-human species such as, but not limited to, mouse, llama, rabbit, rat or hamster. Alternatively, the antigen-binding region may be of human species origin.

[0072] The term "variable" refers to the fact that certain portions of the variable domains vary significantly in sequence among antibodies. The V domains mediate antigen binding and determine the specificity of each particular antibody for its particular antigen. However, the variability is not uniformly distributed over the 110 amino acids of the variable domain. Instead, the V region consists of 15-30 amino acid framework regions (FRs) separated by shorter regions of extreme variability called "hypervariable regions" or CDRs. Each variable domain of the native heavy and light chains contains four FRs, which generally assume a beta-sheet configuration connected by three hypervariable regions that form loops that connect, and in some cases form part of, the beta-sheet structure. The hypervariable regions in each chain are held together in close proximity to the FRs and, together with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of the antibody. The constant domains do not directly participate in the binding of the antibody to the antigen but exhibit various effector functions such as the participation of the antibody in antibody-dependent cell-mediated cytotoxicity (ADCC).

[0073] As used herein, the term "hypervariable region" refers to the amino acid residues of an antibody involved in antigen binding. Typically, hypervariable regions generally include amino acid residues from "complementary determining regions" or "CDRs" and / or amino acid residues from "hypervariable loops".

[0074] In certain cases, it may be desirable to alter one or more CDR amino acid residues in order to improve the binding affinity to a target epitope. This is known as "affinity maturation" and may optionally be carried out in combination with humanization, for example, in situations where humanization of an antibody causes a reduction in binding specificity or affinity and reversion mutations alone are not sufficient to fully improve binding specificity or affinity. A variety of affinity maturation methods are known in the art, such as the in vitro scanning saturation mutagenesis method described by Burks et al., Proc Natl Acad Sci USA, 94:412-417 (1997), and the stepwise in vitro affinity maturation method by Wu et al., Proc Natl Acad Sci USA 95:6037-6042 (1998).

[0075] The "framework region" (FR) consists of variable domain residues other than CDR residues. Each variable domain typically has four FRs, identified as FR1, FR2, FR3, and FR4. When CDRs are defined according to Kabat, the light chain FR residues are located approximately at residues 1-23 (LCFR1), 35-49 (LCFR2), 57-88 (LCFR3), and 98-107 (LCFR4), and the heavy chain FR residues are located approximately at residues 1-30 (HCFR1), 36-49 (HCFR2), 66-94 (HCFR3), and 103-113 (HCFR4) in the heavy chain. When CDRs include amino acid residues from hypervariable loops, the light chain FR residues are located approximately at 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 approximately at residues 1-25 (HCFR1), 33-52 (HCFR2), 56-95 (HCFR3), and 102-113 (HCFR4) in the heavy chain residues. In some cases, when CDRs include amino acids from both Kabat-defined CDRs and hypervariable loop CDRs, the FR residues are expected to be adjusted accordingly. For example, when CDRH1 includes amino acids H26-H35, the heavy chain FR1 residues are at positions 1-25 and the FR2 residues are at positions 36-49.

[0076] The crystallizable region of an immunoglobulin fragment ("Fc region, Fc") is the "tail" region of an immunoglobulin molecule that interacts with cell surface Fc receptors and is also a protein of part of the complement system. This property enables antibodies to activate the immune system. In IgG, IgA, and IgD antibody isotypes, the Fc region is composed of two identical protein fragments from the second and third constant domains of the two heavy chains, and in IgM and IgE isotypes, the Fc region contains three heavy chain constant domains (CH domains 2-4) in each polypeptide chain.

[0077] The antibody of the present invention that "binds" to the target antigen binds to the antigen with sufficient affinity such that the antibody can be used as a diagnostic agent and / or a therapeutic agent, can target the protein or cell expressing the antigen, and refers to an antibody with little cross-reactivity with other proteins. Analytical methods: According to fluorescence-activated cell sorting (FACS), radioimmunoassay (RIA) or ELISA, in such embodiments, the degree of antibody binding to non-target proteins is less than 10% of the antibody binding to specific target proteins. With respect to the binding of an antibody to a target molecule, the terms "specific binding" to a specific polypeptide or epitope on a specific polypeptide target, or "specifically binds" thereto or "is specific" thereto mean binding that is distinguishable (to a measurable degree) from non-specific interactions (for example, in the case of bH1-44 or bH1-81, non-specific interactions are binding to bovine serum albumin, casein, fetal bovine serum or neutravidin).

[0078] Specific binding can be measured, for example, by determining the binding of a molecule as compared to the binding of a control molecule. For example, specific binding can be determined by competition with a control molecule similar to the target, such as an excess of unlabeled target. In this case, specific binding is indicated when the binding of the labeled target to the probe is competitively inhibited by the excess unlabeled target. As used herein, the terms "specific binding" or "binds specifically" or "is specific for" to a particular polypeptide or an epitope on a particular polypeptide target can be described by a molecule having a Kd 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 greater, to the target. In one aspect, the term "specific binding" refers to binding when a molecule binds to a particular polypeptide or an epitope on a particular polypeptide without substantially binding to any other polypeptide or polypeptide epitope.

[0079] As used herein, the term "Ka" refers to the association (on) rate of a particular antibody-antigen interaction. As used herein, the term "Kd" refers to the dissociation (off) rate of a particular antibody-antigen interaction.

[0080] "Binding affinity" generally refers to the overall strength of non-covalent interaction between a single binding site of a molecule (such as an antibody) and its binding partner (such as an antigen). Unless otherwise specified, "binding affinity" refers to the intrinsic (characteristic, true) binding affinity that reflects the 1:1 interaction between the members of the binding pair (such as an antibody and an antigen). The affinity of molecule X for its binding partner Y can generally be represented by the dissociation constant (Kd). Preferred Kd values are about 200 nM, 150 nM, 100 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 8 nM, 6 nM, 4 nM, 2 nM, 1 nM, or less. Affinity can be measured by common methods known in the art, including those described herein. Antibodies with low affinity usually bind to antigens slowly and tend to dissociate easily, while antibodies with high affinity usually bind to antigens more quickly and tend to remain bound for a longer time. Various methods for measuring binding affinity are known in the art, and any of them can be used for the purposes of the present invention.

[0081] In one aspect, the "Kd" or "Kd value" is measured at 25°C using a surface plasmon resonance assay with an immobilized antigen CM5 chip and approximately 10 response units (RU) using a BIAcore™-2000 or BIAcore®-3000 (BIAcore, Inc., Piscataway, N.J.). Briefly, a carboxymethylated dextran biosensor chip (CM5, BIAcore Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the manufacturer's instructions. The antigen is diluted to 5 μg / ml (about 0.2 μM) in 10 mM sodium acetate, pH 4.8 and then injected at a flow rate of 5 μl / min to achieve protein coupling of approximately 10 response units (RU). After injection of the antigen, a 1 M ethanolamine solution is injected to block unreacted groups. For measurement of the reaction rate, two-fold serial dilutions of the Fab in PBS containing 0.05% Tween 20 (PBST) (e.g., from 0.78 nM to 500 nM) are injected at 25°C at a flow rate of approximately 25 μl / min. The on-rate (kon) and off-rate (koff) are calculated by fitting the association and dissociation sensorgrams simultaneously using a simple 1:1 Langmuir binding model (BIAcore evaluation software, version 3.2). The equilibrium dissociation constant (Kd) is calculated as the ratio of koff / kon. See, for example, Chen, Y. et al., (1999) J. Mol. Biol. 293:865-881. The on-rate is 10 6 M -1 s -1When it exceeds, the on-rate can be determined by measuring the increase or decrease in the fluorescence emission intensity at 25°C of a 20 nM anti-antigen antibody solution (Fab form) in PBS, pH 7.2 in the presence of increasing concentrations of antigen, using a fluorescence quenching technique (excitation = 295 nm; emission = 340 nm, 16 nm bandpass) where the on-rate is measured with a spectrometer, for example, a spectrophotometer equipped with a stop-flow (Aviv Instruments) or an 8000 series SLM-Aminco spectrophotometer (ThermoSpectronic) having a stirred cuvette.

[0082] The term "koff" refers to the off-rate constant of a specific interaction between a binding molecule and an antigen. The off-rate constant koff can be measured using biolayer interferometry, for example, using an Octet™ system.

[0083] The "on-rate" or "kon" according to the present invention can also be measured at 25°C using the above-described surface plasmon resonance assay with a BIAcore™-2000 or BIAcore®-3000 (BIAcore, Inc., Piscataway, N.J.) using an immobilized antigen CM5 chip at approximately 10 relative units (response units, RU). Briefly, a carboxymethylated dextran biosensor chip (CM5, BIAcore Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the manufacturer's instructions. The antigen is diluted to 5 μg / ml (about 0.2 μM) in 10 mM sodium acetate, pH 4.8 and then injected at a flow rate of 5 μl / min to achieve coupling of approximately 10 response units (RU) of protein. After injection of the antigen, a 1 M ethanolamine solution is injected to block unreacted groups.

[0084] Unless otherwise specified, the terms "biologically active", "biological activity", and "biological characteristics" with respect to the polypeptides of the present invention mean having the ability to bind to a biomolecule.

[0085] The term "biomolecule" refers to nucleic acids, proteins, carbohydrates, lipids, and combinations thereof. In one aspect, the biomolecule is naturally occurring. Antibody fragments, such as Fab and F(ab')2 fragments, can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion of whole antibodies. Additionally, antibodies, portions thereof, and immunoadhesion molecules can be prepared using standard recombinant DNA techniques, such as as described herein.

[0086] The term "recombinant antibody" is intended to refer to an antibody expressed in a cell or cell line containing a nucleotide sequence encoding the antibody, where the nucleotide sequence is not naturally associated with the cell.

[0087] As used herein, the term "variant antibody" is intended to refer to an antibody having an amino acid sequence that differs from that of its "parent" antibody based on the addition, deletion, and / or substitution of one or more amino acid residues as compared to the sequence of the parent antibody. In preferred embodiments, the variant antibody comprises at least one or more (e.g., 1 to 12, such as 2, 3, 4, 5, 6, 7, 8, or 9, 10, 11, or 12; in some embodiments, 1 to about 10) amino acid additions, deletions, and / or substitutions as compared to the parent antibody. In some embodiments, such additions, deletions, and / or substitutions are made within the CDRs of the variant antibody. Identity or homology with respect to the sequence of a variant antibody is defined herein as the percentage of amino acid residues in the variant antibody sequence that are identical to the residues of the parent antibody after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percentage of sequence identity. A variant antibody retains the ability to bind to the same antigen, preferably an epitope, to which the parent antibody binds, and in some embodiments, has at least one property or biological activity that is superior to that of the parent antibody. For example, the variant antibody may have, compared to the parent antibody, a stronger binding affinity, a longer half-life, a lower IC50 value, or an enhanced ability to inhibit the biological activity of the antigen. Variant antibodies of particular interest herein are those that exhibit an enhancement of biological activity of at least 2-fold (preferably at least 5-fold, 10-fold, or 20-fold) as compared to the parent antibody.

[0088] The term "bispecific antibody" refers to an antibody having antigen-binding domains capable of specific binding to two distinct epitopes on a single biomolecule or to epitopes on two distinct biomolecules. Bispecific antibodies are also referred to herein as having "bispecificity" or as "bispecific" antibodies.

[0089] The term "chimeric antibody" is intended to broadly refer to an antibody that includes one or more regions of one antibody and one or more regions of one or several other antibodies, typically partially human and partially non-human antibodies, i.e., antibodies derived from partially non-human animals such as mice, rats, or other rodents, or camelids such as llamas and alpacas. Chimeric antibodies are generally preferred over non-human antibodies to reduce the risk of a human anti-antibody immune response, e.g., a human anti-mouse antibody immune response in the case of a mouse antibody. An example of a typical chimeric antibody is one in which the variable region sequence is a mouse sequence while the constant region sequence is human. In the case of chimeric antibodies, the non-human portion may be further modified to humanize the antibody.

[0090] The term "humanization" is intended to mean that an antibody, which has a fully or partially non-human origin, e.g., a mouse or llama antibody obtained by immunizing a mouse or llama with the antigen of interest respectively, or a chimeric antibody based on such a mouse or llama antibody, can have specific amino acids, particularly specific amino acids in the framework regions and constant domains of the heavy and light chains, replaced in order to avoid or minimize an immune response in humans. The specificity of the interaction of an antibody with its target antigen resides mainly in the amino acid residues located in the six CDR regions of the heavy and light chains. For this reason, the amino acid sequences within the CDRs have much higher variability between individual antibodies than the sequences outside the CDRs. Since the CDR sequences are involved in most antibody-antigen interactions, it is possible to express a specific naturally occurring antibody, or more generally, a recombinant antibody that mimics the properties of any specific antibody having said amino acid sequences, by constructing an expression vector that expresses the CDR sequences from a specific antibody and the framework sequences from a different antibody. As a result, it is possible to "humanize" a non-human antibody and, in many cases, retain the binding specificity and affinity of the original antibody. Although it is impossible to accurately predict immunogenicity and thereby accurately predict the human anti-antibody response to a specific antibody, non-human antibodies typically have higher immunogenicity than human antibodies. Chimeric antibodies have generally been shown to have lower immunogenicity than those of completely foreign origin when the foreign (e.g., rodent or camelid) constant regions are replaced with sequences of human origin, and the trend in therapeutic antibodies is towards humanized or fully human antibodies. Therefore, chimeric antibodies or other antibodies of non-human origin may be humanized to reduce the risk of a human anti-antibody response.

[0091] In the case of chimeric antibodies, humanization typically involves modification of the framework regions of the variable region sequences. Amino acid residues that are part of the complementarity-determining regions (CDRs) are, in most cases, not expected to be modified by humanization, although in some cases it may be desirable to modify individual amino acid residues of the CDRs, for example, to remove glycosylation sites, deamidation sites, aspartic acid isomerization sites, or undesirable cysteine or methionine residues. N-linked glycosylation is created by attaching an oligosaccharide chain to an asparagine residue at the tripeptide sequence Asn-X-Ser or Asn-X-Thr, where X can be any amino acid except Pro. Removal of N-glycosylation sites can be achieved by mutating either the Asn or Ser / Thr residue to a different residue, preferably by a method of 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 when present mainly in the sequence Asn-Gly, and also, to a lesser extent, when present in other dipeptide sequences such as Asn-Ala. When the CDR sequence contains such deamidation sites, particularly Asn-Gly, it may be desirable to remove this site by conservative substitution to delete one of the relevant residues.

[0092] Numerous methods for humanizing antibody sequences are known in the art. One commonly used method is CDR grafting. CDR grafting may be based on the CDR definitions by Kabat, although the final version (Magdelaine-Beuzelin et al., Crit Rev. Oncol Hematol. 64:210 - 225 (2007)) suggests that the definitions of IMGT® (international ImMunoGeneTics information system®; www.imgt.org) can improve the results of humanization (see Lefranc et al., Dev. Comp Immunol. 27:55 - 77 (2003)). In some cases, CDR grafting can reduce the binding specificity and affinity, and thus the biological activity, of the CDR-grafted non-human antibody compared to the parental antibody from which the CDRs were obtained. Back mutations (which are sometimes referred to as "framework region repair") may typically be introduced at selected positions of the CDR-grafted antibody in the framework region to restore the binding specificity and affinity of the parental antibody. Identification of positions for potential back mutations can be performed using information available in the literature and antibody databases. Amino acid residues that are candidates for back mutations are typically those located on the surface of the antibody molecule, whereas buried residues or residues with a low degree of surface exposure are usually not expected to be altered. An alternative humanization technique to CDR grafting and back mutations is resurfacing, in which non-human-origin residues that are not surface-exposed are retained, whereas surface residues are changed to human residues.

[0093] Fully human antibodies can be generated using two techniques: the use of phage libraries collected in vitro or the immunization of humanized animals (such as mice, rats, etc.) in vivo.

[0094] The construction of combinatorial phage antibody libraries starts with the selection of a gene repertoire source, and accordingly, several types of antibody libraries can be distinguished as naive, immune, and synthetic. Naive and immune libraries are constructed using genes that are naturally rearranged and encode the variable immunoglobulin domains of healthy donors or donors immunized with a specific antigen. For this purpose, mRNA is isolated from antibody-producing lymphoid cell lines. Peripheral blood lymphocytes are mainly used, but in some cases, splenocytes [Sheets MD, Amersdorfer P, Finnern R, Sargent P, Lindquist E, Schier R et al., Efficient construction of a large nonimmune phage antibody library: the production of high-affinity human single-chain antibodies to protein antigens. Proc Natl Acad Sci USA 1998, 95:6157~6162 and de Haard HJ, van Neer N, Reurs A, Hufton SE, Roovers RC, Henderikx P et al., A large non-immunized human Fab fragment phage library that permits rapid isolation and kinetic analysis of high affinity antibodies. J Biol Chem 1999, 274:18218~18230.], tonsil cells, or bone marrow lymphocytes [Vaughan TJ, Williams AJ, Pritchard K, Osbourn JK, Pope AR, Earnshaw JC et al., Human antibodies with sub-nanomolar affinities isolated from a large non-immunized phage display library. Nat Biotechnol 1996, 14:309~314.] have also been used.Subsequently, cDNA is synthesized based on mRNA, and both an oligo-dT primer and statistically designed hexanucleotides that generate cDNA copies of all possible variants of the gene encoding the variable domain of the antibody can be used [Ulitin AB, Kapralova MV, Laman AG, Shepelyakovskaya AO, Bulgakova EB, Fursova KK et al., The library of human miniantibodies in the phage display format: Designing and testing DAN: Izd-vo “Nauka”; 2005.].

[0095] At the cDNA level, one or several primers can be used simultaneously to limit the range of amplified genes to one or several gene families of variable domains or antibody isotypes [Marks JD, Hoogenboom HR, Bonnert TP, McCafferty J, Griffiths AD, Winter G. Bypassing immunization. Human antibodies from V-gene libraries displayed on phage. J Mol Biol 1991, 222: 581~597]. Primers used for amplification of genes encoding immunoglobulins are complementary to their most conserved regions. Their sequences are selected from collections of genes systematically constructed in databases such as the Kabat or V BASE databases. Primer design also provides internal restriction sites for cloning PCR products into appropriate vectors.

[0096] The construction of synthetic libraries is based on the replacement of sets of random sequences of natural CDRs. In this case, it is possible to generate a huge number of antigen-binding sites. Phage display is one of the most powerful and widely used in vitro technologies for antibody screening. In 1985, Smith discovered that foreign DNA sequences can be cloned into the filamentous bacteriophage M13 and that such cloned sequences can be expressed as fusion proteins on the surface of phage particles (Smith GP: Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 1985, 228: 1315 - 1317.). Thus, it is possible to select the fusion protein of interest based on its ability to bind to other proteins. By combining this discovery with PCR amplification methods, it became possible to clone the cDNA repertoire of immunoglobulin genes and create various phage libraries containing variable domains that can be used to rapidly screen for target - specific monoclonal antibodies. The phage library repertoire reflects that of all human or animal B - cell antibodies using blood to create the library. In 1995, two papers described the production of genetically engineered mice capable of expressing fully human antibodies, and their repertoire is comparable to that 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). In these animals, their endogenous immunoglobulin heavy - chain and k - light - chain genes were deliberately disrupted and subsequently transgenes, which are segments of human heavy - chain and k - light - chain genes, were introduced. It was found that the human gene repertoire can be used by the mouse immune system to produce antibodies with high specificity and high affinity for a greater variety of antigens.Despite the fact that transgenic mice express a B cell receptor that is essentially a hybrid of mouse and human components (human immunoglobulins, mouse Igα, Igβ, and other signaling molecules), their B cells develop and mature normally.

[0097] In certain cases, it may be desirable to alter one or more CDR amino acid residues to improve the binding affinity to a target epitope. This is known as "affinity maturation" and may optionally be carried out in combination with humanization, for example, in situations where humanization of an antibody causes a reduction in binding specificity or affinity and reversion mutations alone are not sufficient to fully improve binding specificity or affinity. 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 by Wu et al., Proc Natl Acad Sci USA 95:6037 - 6042 (1998).

[0098] The term "monoclonal antibody" or "mAb" refers to an antibody that is synthesized and isolated by a distinct clone population of cells. The clone population may be a clone population of immortalized cells. In some embodiments, the immortalized cells in the clone population are hybrid cells, i.e., typically hybridomas produced by fusing individual B lymphocytes from an immunized animal with individual cells from a lymphocyte tumor. Hybridomas are a type of constructed cell and do not occur naturally.

[0099] A "natural antibody" is typically a glycoprotein heterotetramer of approximately 150,000 daltons composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to the heavy chain by a single covalent disulfide bond, although the number of disulfide bonds varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intra-chain disulfide cross-links. Each heavy chain has a variable domain (VH) at one end, followed by a number of constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at the other end. The constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Certain amino acid residues are thought to form the boundary between the light chain variable domain and the heavy chain variable domain.

[0100] The term "isolated" as used herein to describe various antibodies refers to an antibody that has been identified and separated from, and / or regenerated from, the cells or cell culture in which it is expressed. Impurities (contaminating components) from its natural environment are substances that are expected to interfere with the diagnostic or therapeutic use of the polypeptide, examples of which can include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In a preferred embodiment, the antibody is purified to homogeneity by (1) at least to an extent sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence by use of a spinning cup sequenator (Edman sequenator), or (2) SDS-PAGE under non-reducing or reducing conditions using Coomassie Brilliant Blue, or preferably silver staining. An isolated antibody includes an antibody present in the location within a recombinant cell after at least one component of the polypeptide's natural environment has been eliminated. An isolated polypeptide is typically prepared by at least one purification step.

[0101] An "isolated" nucleic acid molecule is a nucleic acid molecule that has been identified and separated from at least one nucleic acid molecule impurity to which it is bound in the natural source of the antibody nucleic acid. An isolated nucleic acid molecule is different from the form or population in which it is found in nature. Thus, an isolated nucleic acid molecule is different from the nucleic acid molecules that exist in cells in their natural state. However, for example, if a nucleic acid molecule is located at a chromosomal location different from the location where it is localized in cells in its natural state, the isolated nucleic acid molecule includes the nucleic acid molecules that exist in cells in which the antibody is normally expressed.

[0102] The term "epitope," as used herein, is intended to refer to the portion (determinant) of an antigen that specifically binds to a binding molecule (e.g., an antibody or a related molecule, such as a bispecific binding molecule). Epitope determinants usually consist of chemically active surface groups of a molecule, such as amino acids or carbohydrates or sugar side chains, and typically include the characteristics of a specific three-dimensional structure, in addition to the characteristics of a specific charge. An epitope may be either "linear" or "conformational." In a linear epitope, all of the points of interaction between a molecule (e.g., an antibody) that interacts with a protein (e.g., an antigen) are linearly present along the primary amino acid sequence of the protein. In a conformational epitope, the points of interaction are present across amino acid residues on the protein that are separated from each other in the primary amino acid sequence. Once the desired epitope of an antigen has been determined, it is possible to generate an antibody against that epitope using techniques well known in the art. In addition, the generation and characterization of an antibody or other binding molecule may reveal information about the desired epitope. Based on this information, it is possible to competitively screen antibodies for binding to the same or identical epitopes, for example, by performing competition studies to find binding molecules that compete with each other for binding to the antigen.

[0103] As used herein, the term "peptide linker" is intended to mean any peptide having the ability to combine domains, the length of which depends on the domains to be joined to each other and includes any amino acid sequence. Preferably, the peptide linker has a length of more than 5 amino acids and consists of any set of amino acids selected from G, A, S, P, E, T, D, K.

[0104] The term "in vitro" refers to an ex vivo biological entity, biological process, or biological reaction under artificial conditions. For example, cells grown in vitro are understood to be cells that have grown in an ex vivo environment, such as in a test tube, culture vial, or microtiter plate.

[0105] The term "IC 50 " (50% inhibitory concentration), as used herein, refers to the concentration of a drug at which a measurable activity or response, such as the growth / proliferation of cells such as tumor cells, is inhibited by 50%. The IC 50 value can be calculated using an appropriate dose-response curve using special statistical software for curve fitting.

[0106] The term GI50 (50% growth inhibition) refers to the concentration of a drug at which the growth of cells such as tumor cells is inhibited by 50%. The term "ED50" (EC50) (50% effective dose / concentration) refers to the concentration of a drug that produces 50% of a biological effect (such as, for example, cytotoxicity).

[0107] The term "antiproliferative effect" is intended to mean the arrest or inhibition of the growth of cells such as cancer cells. The term "effector function" of an antibody refers to the biological activity resulting from the Fc region of the antibody (the sequence of the native Fc region or an amino acid variant of the Fc region), and such activities vary depending on the antibody isotype. Examples of antibody effector functions include Cl qBinding and complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor, BCR), and B cell activation are included.

[0108] "Antibody-dependent cell cytotoxicity" or "ADCC" refers to a cell-mediated response in which non-specific cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) expressing Fc receptors (FcR) recognize bound antibodies on target cells, subsequently causing lysis or phagocytosis of the target cells. NK cells, which are the main cells mediating ADCC, express only FcγRJII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression in hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To evaluate the ADCC activity of the molecule of interest, an in vitro ADCC assay, such as those described in U.S. Patent Nos. 5,500,362 or 5,821,337, may be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or in addition, the ADCC activity of the molecule of interest can be evaluated in vivo, for example, in an animal model, such as those disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).

[0109] "Human effector cells" are leukocytes that express one or more FcRs and exhibit effector functions. Preferably, such cells express at least FcγRIII and exhibit ADCC effector functions. Examples of human leukocytes mediating ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils, with PBMC and NK cells being preferred. Effector cells can be isolated from their natural sources, for example, from blood or PBMC as described herein.

[0110] The term "Fc receptor" or "FcR" is used to describe receptors that bind to the Fc region of an antibody. Preferred FcRs are native sequence human FcRs. Further, preferred FcRs are those that bind to IgG antibodies (gamma receptors), examples of which include receptors of the FcγRI, FcγRII, and FcγRIII subclasses, as well as allelic variants and alternatively spliced forms of these receptors. As FcγRII receptors, there are mainly FcγRIIA ("activating receptor") and FcγRIIB ("inhibitory receptor") which have similar amino acid sequences that differ mainly in their cytoplasmic domains. The activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain (see the review in Daeron, Annu.Rev.Immunol. 15:203-234 (1997)). FcRs are reviewed in Ravetch and Kinet, Annu.Rev.Immunol 9:457-92 (1991). Other FcRs, including those that may be identified in the future, are encompassed by the term "FcR" herein. This term also includes the neonatal receptor, FcRn, which is involved in the transfer of maternal IgG to the fetus.

[0111] "Complement-dependent cytotoxicity" and "CDC" refer to the ability of a molecule to lyse a target in the presence of complement. The complement activation pathway begins with the binding of the first component of the complement system (C1q) to a molecule (e.g., an antibody) complexed with an alloantigen. To assess complement activation, a CDC assay, such as the assay described in Gazzano-Santoro et al., J.Immunol.Methods 202:163 (1996).

[0112] The terms "identity" or "homology" are to be interpreted as meaning the percentage of amino acid residues in a candidate sequence that are identical to the residues of the corresponding sequence being compared, after introducing gaps as necessary to achieve the maximum percent identity over the entire sequence, and no conservative substitutions are to be considered as part of sequence identity. Neither N- nor C-terminal extensions or insertions are to be interpreted as reducing identity or homology. Methods and computer programs for alignment are well known in the art. Sequence identity can be measured using sequence analysis software (e.g., Sequence Analysis Software Package, Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Ave., Madison, WI 53705). This software matches similar sequences by assigning degrees of homology to various substitutions, deletions (exclusions), and other modifications.

[0113] The term "homologous" is to be interpreted, with respect to the polypeptide sequence of an antibody, as an antibody having at least 70%, preferably 80%, more preferably 90%, most preferably 95% sequence identity with respect to the polypeptide sequence. With respect to a nucleic acid sequence, this term is to be interpreted as a nucleotide sequence having at least 85%, preferably 90%, more preferably 95%, most preferably 97% sequence identity with respect to the nucleic acid sequence.

[0114] Modifications of the amino acid sequences of the antibodies described herein are provided. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibody are prepared by introducing appropriate nucleotide changes into the antibody nucleic acid or by peptide synthesis. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions is made to arrive at the final construct, provided that the final construct has the desired characteristics. Also, changing the amino acid, for example, the number or position of glycosylation sites, can also alter the post-translational processes in the antibody.

[0115] Variants of the modification of the amino acid sequence of the antibody using amino acid substitution. Such variants are those in which at least one amino acid residue in the antibody molecule is replaced with a different residue. The most interesting sites for substitution mutagenesis include the hypervariable regions or CDRs, although FR or Fc modifications are also expected. Conservative substitutions are shown under "Preferred Substitutions" in Table A. If such substitutions cause a change in biological activity, further substantial changes may be made, which are represented as "Exemplary Substitutions" described in Table A or, when describing classes of amino acids, the changes described in more detail below, and the product may also be screened.

[0116] [Table 1]

[0117] The terms "nucleic acid", "nucleic acid sequence", "nucleic acid sequence", "polynucleotide", "oligonucleotide", "polynucleotide sequence" and "nucleotide sequence" are used interchangeably herein and mean the exact sequence of nucleotides, such sequences being modified or unmodified, determining a fragment or region of a nucleic acid, containing or not containing unnatural nucleotides, and being either double-stranded DNA or RNA, single-stranded DNA or RNA, or a transcript of said DNA.

[0118] It is also herein included that the present invention is not related to its natural chromosomal environment, i.e., the nucleotide sequence in its natural state. The sequences of the present invention are isolated and / or purified, i.e., they are sampled directly or indirectly, for example by copying, and their environment is at least partially modified. Thus, it is to be considered that isolated nucleic acids obtained by recombinant genetics by means of, for example, host cells, or obtained by chemical synthesis, are also described herein.

[0119] References to nucleotide sequences include, unless otherwise specified, their complements. Thus, references to nucleic acids having a particular sequence are to be understood as including the complementary strand having the complementary sequence thereto.

[0120] The term "control sequence" refers to a DNA sequence necessary for the expression of an operably linked coding sequence in a particular host organism. Control sequences suitable for prokaryotes include, for example, a promoter, optionally an operator sequence, and a ribosome binding site. It is known that eukaryotic cells utilize a promoter, a polyadenylation signal, and an enhancer.

[0121] Nucleic acids are "operably linked" when they are arranged so as to be in a functional relationship with another nucleic acid sequence. For example, DNA regarding a precursor sequence or a secretory leader sequence is operably linked to DNA regarding a polypeptide when it is expressed as a precursor protein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence when it affects the transcription of the sequence; a ribosome binding site is operably linked to a coding sequence when it is arranged so as to facilitate translation. Generally, "operably linked" means that the linked DNA sequences are continuous, and in the case of a secretory leader, continuous in the reading frame. However, an enhancer may not be continuous.

[0122] As used herein, the term "vector" means 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 fragment of DNA to which additional DNA segments can be ligated. In some embodiments, the vector is a viral vector to which additional DNA segments can be ligated to a viral genome. In some embodiments, the vector is capable of autonomous replication in a host cell into which it is introduced (e.g., a bacterial vector having a bacterial origin of replication and an episomal mammalian vector). In a further embodiment, the vector (e.g., a non-episomal mammalian vector) may be integrated into the genome of the host cell when introduced into the host cell and thereby replicated with the host gene. Further, a particular vector is capable of directing the expression of a gene to which it is operably linked. Such a vector is referred to herein as a "recombinant expression vector" (or simply an "expression vector").

[0123] The term "recombinant host cell" (or simply "host cell"), as used herein, is intended to refer to a cell into which a recombinant expression vector has been introduced. The present invention relates to host cells, which may, for example, contain a vector according to the present invention as described above. The present invention also relates, for example, to host cells containing a nucleotide sequence encoding a heavy chain or an antigen-binding portion thereof, a nucleotide sequence encoding a light chain or an antigen-binding portion thereof, or both, of the first binding domain and / or the second binding domain of a binding molecule of the present invention. It is understood that the terms "recombinant host cell" and "host cell" are intended to refer to not only a particular target cell but also the progeny of such a cell. Such progeny may not actually be identical to the parent cell due to, for example, mutations or the influence of the environment, but such cells are still included within the scope of the term "host cell" as used herein.

[0124] The term "excipient" is used herein to describe any component other than the compounds of the present invention. The term "disease or disorder mediated by GITR" refers to any disease or disorder that is directly or indirectly associated with GITR, including the etiology, onset, progression, persistence, or pathology of the disease or disorder.

[0125] "Treat", "treating", and "treatment" refer to a method of reducing or preventing at least one of a biological disorder and / or its attendant symptoms. As used herein, "reducing" a disease, disorder, or condition means reducing the severity and / or frequency of the symptoms of the disease, disorder, or condition. Further, references to "treatment" herein include references to curative, palliative, and prophylactic treatment.

[0126] In one aspect, the subject to be treated, or patient, is a mammal, preferably a human subject. The subject may be either male or female and of any age.

[0127] The term "disorder" means any condition that is expected to benefit from treatment with the compounds of the present invention. This definition includes chronic and acute disorders or diseases, including pathological conditions that predispose mammals to the disorder in question.

[0128] The terms "cancer" and "cancerous" refer to, or describe, a physiological condition typically characterized by unregulated cell growth / proliferation in a mammal. This definition includes both benign and malignant cancerous diseases. Examples of cancerous diseases include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias. More specific examples of such cancerous diseases include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma and squamous cell carcinoma of the lung, peritoneal cancer, hepatocellular cancer, gastric cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, liver carcinoma, anal cancer, penile cancer, melanoma, and various head and neck cancers.

[0129] The terms "immune response", "autoimmune response" and "autoimmune inflammation" refer to, for example, the action of lymphocytes, antigen-presenting cells, phagocytes, granulocytes, and soluble macromolecules produced by said cells or liver cells (such as antibodies, cytokines and complements produced as a result of selective damage, destruction or elimination of normal cells or tissues from the human body in the case of invasive pathogens, pathogen-infected cells or tissues, cancer cells, or autoimmune or pathological inflammation).

[0130] "Therapeutically effective amount" is intended to mean an amount of a therapeutic agent administered that is expected to alleviate to some extent one or more of the symptoms of the disorder being treated. The use of the term "chronic" refers to the continuous (unceasing) use of a drug such that its initial therapeutic action (activity) is maintained over a long period, as opposed to acute (temporary) routes of administration.

[0131] "Intermittent" use refers to a procedure that is not carried out consistently without interruption, but in fact the procedure is rather intermittent. As used herein, the words "comprise", "have", "include", or variations such as "comprises", "comprising", "has", "having", "includes", or "including", and all of their grammatical variations, are understood to include the stated integer or group of integers, but not to exclude any other integer or group of integers. Detailed Description of the Invention Antibody The present invention relates to monoclonal antibodies that specifically bind to GITR.

[0132] In one aspect, the present invention is a monoclonal antibody or an antigen-binding fragment thereof that specifically binds to GITR, (a)(i) The following group: NYGMH (SEQ ID NO: 1) or YYWMY (SEQ ID NO: 12) comprising a CDR1 having an amino acid sequence selected from the group consisting of: (ii) The following group: VIWFDGSNKFYTDSVKG (SEQ ID NO: 2) or AISWNGGRTYYAESMKG (SEQ ID NO: 13) comprising a CDR2 having an amino acid sequence selected from the group consisting of: (iii) The following group: ELGGYYYDSSGFRPYYYGMDV (SEQ ID NO: 3) or NRYYSDPNYGMNL (SEQ ID NO: 14) comprising a CDR3 having an amino acid sequence selected from the group consisting of: comprising a heavy chain variable domain, and (b)(i) The following group: RASQSIGSWLA (SEQ ID NO: 7) or TGTSTDIGTYKYIS (SEQ ID NO: 17) comprising a CDR1 having an amino acid sequence selected from the group consisting of: (ii) The following group: CDR2 comprising an amino acid sequence selected from AASTLQR (SEQ ID NO: 8) or GVSHRPS (SEQ ID NO: 18); (iii) The following group: CDR3 comprising an amino acid sequence selected from QQSHSHPLT (SEQ ID NO: 9) or SSYTSSGTVV (SEQ ID NO: 19) A light chain variable domain comprising Relates to a monoclonal antibody or an antigen-binding fragment thereof comprising

[0133] In some embodiments, the monoclonal antibody or an antigen-binding fragment thereof comprises a heavy chain variable domain comprising CDR1, 2, and 3 comprising amino acid sequences represented by the sequences of SEQ ID NOs: 1, 2, and 3, respectively.

[0134] In some embodiments, the monoclonal antibody or an antigen-binding fragment thereof comprises a heavy chain variable domain comprising CDR1, 2, and 3 comprising amino acid sequences represented by the sequences of SEQ ID NOs: 12, 13, and 14, respectively.

[0135] In some embodiments, the monoclonal antibody or an antigen-binding fragment thereof comprises a light chain variable domain comprising CDR1, 2, and 3 comprising amino acid sequences represented by the sequences of SEQ ID NOs: 7, 8, and 9, respectively.

[0136] In some embodiments, the monoclonal antibody or an antigen-binding fragment thereof comprises a light chain variable domain comprising CDR1, 2, and 3 comprising amino acid sequences represented by the sequences of SEQ ID NOs: 17, 18, and 19, respectively.

[0137] In some embodiments, the monoclonal antibody or an antigen-binding fragment thereof - A heavy chain variable domain comprising CDR1, 2, and 3 comprising amino acid sequences represented by the sequences of SEQ ID NOs: 1, 2, and 3, respectively; - A light chain variable domain comprising CDR1, 2, and 3 comprising amino acid sequences represented by the sequences of SEQ ID NOs: 7, 8, and 9, respectively ​​​

[0138] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof - comprises a heavy-chain variable domain comprising CDR1, 2, and 3, which comprise amino acid sequences represented by SEQ ID NOs: 12, 13, and 14, respectively; - comprises a light-chain variable domain comprising CDR1, 2, and 3, which comprise amino acid sequences represented by SEQ ID NOs: 17, 18, and 19, respectively .

[0139] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises a heavy-chain variable domain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence EVQLVQSGGGVVQPGKSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWVAVIWFDGSNKFYTDSVKGRFTISRDNSKDTLSLQMNSLRAEDTAVYYCARELGGYYYDSSGFRPYYYGMDVWGQGTMVTVSS (SEQ ID NO: 4).

[0140] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises a heavy-chain variable domain comprising the amino acid sequence EVQLVQSGGGVVQPGKSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWVAVIWFDGSNKFYTDSVKGRFTISRDNSKDTLSLQMNSLRAEDTAVYYCARELGGYYYDSSGFRPYYYGMDVWGQGTMVTVSS (SEQ ID NO: 4).

[0141] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises a heavy-chain variable domain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence QVQLVQSGGGLVQPGGSLRLSCAASGFTFSYYWMYWVRQAPGKGLEWVSAISWNGGRTYYAESMKGRFTISRDNAQNTLYLQMNSLKSEDTAVYYCAKNRYYSDPNYGMNLWGKGTTVTVSS (SEQ ID NO: 15).

[0142] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising the amino acid sequence QVQLVQSGGGLVQPGGSLRLSCAASGFTFSYYWMYWVRQAPGKGLEWVSAISWNGGRTYYAESMKGRFTISRDNAQNTLYLQMNSLKSEDTAVYYCAKNRYYSDPNYGMNLWGKGTTVTVSS (SEQ ID NO: 15).

[0143] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises a light chain variable domain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence DVVMTQSPSSVSASVGDRVTITCRASQSIGSWLAWYQQKPGEAPKLLIYAASTLQRGVPSRFSGGGYGTEFTLTISSLQPEDFATYFCQQSHSHPLTFGGGTKVEIK (SEQ ID NO: 10).

[0144] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises a light chain variable domain comprising the amino acid sequence DVVMTQSPSSVSASVGDRVTITCRASQSIGSWLAWYQQKPGEAPKLLIYAASTLQRGVPSRFSGGGYGTEFTLTISSLQPEDFATYFCQQSHSHPLTFGGGTKVEIK (SEQ ID NO: 10).

[0145] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises a light chain variable domain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence QSALTQPASVSGSPGQSITISCTGTSTDIGTYKYISWYQQHPGKAPKLIIYGVSHRPSGVSDRFSGSKSDNTASLTISGLQAEDEADYYCSSYTSSGTVVFGGGTKVTVL (SEQ ID NO: 20).

[0146] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises a light chain variable domain comprising the amino acid sequence QSALTQPASVSGSPGQSITISCTGTSTDIGTYKYISWYQQHPGKAPKLIIYGVSHRPSGVSDRFSGSKSDNTASLTISGLQAEDEADYYCSSYTSSGTVVFGGGTKVTVL (SEQ ID NO: 20).

[0147] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof, - a heavy chain variable domain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence EVQLVQSGGGVVQPGKSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWVAVIWFDGSNKFYTDSVKGRFTISRDNSKDTLSLQMNSLRAEDTAVYYCARELGGYYYDSSGFRPYYYGMDVWGQGTMVTVSS (SEQ ID NO: 4); - a light chain variable domain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence DVVMTQSPSSVSASVGDRVTITCRASQSIGSWLAWYQQKPGEAPKLLIYAASTLQRGVPSRFSGGGYGTEFTLTISSLQPEDFATYFCQQSHSHPLTFGGGTKVEIK (SEQ ID NO: 10) comprises.

[0148] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof, - a heavy chain variable domain comprising the amino acid sequence EVQLVQSGGGVVQPGKSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWVAVIWFDGSNKFYTDSVKGRFTISRDNSKDTLSLQMNSLRAEDTAVYYCARELGGYYYDSSGFRPYYYGMDVWGQGTMVTVSS (SEQ ID NO: 4); - a light chain variable domain comprising the amino acid sequence DVVMTQSPSSVSASVGDRVTITCRASQSIGSWLAWYQQKPGEAPKLLIYAASTLQRGVPSRFSGGGYGTEFTLTISSLQPEDFATYFCQQSHSHPLTFGGGTKVEIK (SEQ ID NO: 10) comprises.

[0149] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof - a heavy chain variable domain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 15: QVQLVQSGGGLVQPGGSLRLSCAASGFTFSYYWMYWVRQAPGKGLEWVSAISWNGGRTYYAESMKGRFTISRDNAQNTLYLQMNSLKSEDTAVYYCAKNRYYSDPNYGMNLWGKGTTVTVSS; - a light chain variable domain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 20: QSALTQPASVSGSPGQSITISCTGTSTDIGTYKYISWYQQHPGKAPKLIIYGVSHRPSGVSDRFSGSKSDNTASLTISGLQAEDEADYYCSSYTSSGTVVFGGGTKVTVL comprises.

[0150] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof - a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 15: QVQLVQSGGGLVQPGGSLRLSCAASGFTFSYYWMYWVRQAPGKGLEWVSAISWNGGRTYYAESMKGRFTISRDNAQNTLYLQMNSLKSEDTAVYYCAKNRYYSDPNYGMNLWGKGTTVTVSS; - a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 20: QSALTQPASVSGSPGQSITISCTGTSTDIGTYKYISWYQQHPGKAPKLIIYGVSHRPSGVSDRFSGSKSDNTASLTISGLQAEDEADYYCSSYTSSGTVVFGGGTKVTVL comprises.

[0151] In some embodiments, the monoclonal antibody specific for GITR is a full-length IgG antibody. In some embodiments, the monoclonal IgG antibody is a monoclonal IgG antibody of the human IgG1, IgG2, IgG3 or IgG4 isotype.

[0152] In some embodiments, the monoclonal IgG antibody is a monoclonal IgG antibody of the human IgG1 isotype. In some embodiments, the monoclonal antibody specific for GITR contains an E345R mutation in the Fc fragment such that it has agonist properties and increases antibody-dependent cell cytotoxicity (ADCC), but does not increase complement-dependent cell cytotoxicity (CDC).

[0153] In some embodiments, the monoclonal antibody has an amino acid sequence

[0154]

Chemical formula

[0155] and comprises a heavy chain containing an amino acid sequence that is at least 90% identical to the sequence In some embodiments, the monoclonal antibody has an amino acid sequence

[0156]

Chemical formula

[0157] and comprises a heavy chain containing the sequence In some embodiments, the monoclonal antibody has an amino acid sequence

[0158]

Chemical formula

[0159] and comprises a heavy chain containing an amino acid sequence that is at least 90% identical to the sequence In some embodiments, the monoclonal antibody has an amino acid sequence

[0160]

Chemical formula

[0161] comprises a heavy chain comprising In some embodiments, the monoclonal antibody comprises a light chain comprising an amino acid sequence that is at least 90% identical to the sequence DVVMTQSPSSVSASVGDRVTITCRASQSIGSWLAWYQQKPGEAPKLLIYAASTLQRGVPSRFSGGGYGTEFTLTISSLQPEDFATYFCQQSHSHPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 11).

[0162] In some embodiments, the monoclonal antibody comprises a light chain comprising the amino acid sequence DVVMTQSPSSVSASVGDRVTITCRASQSIGSWLAWYQQKPGEAPKLLIYAASTLQRGVPSRFSGGGYGTEFTLTISSLQPEDFATYFCQQSHSHPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 11).

[0163] In some embodiments, the monoclonal antibody - a heavy chain comprising an amino acid sequence that is at least 90% identical to the sequence

[0164]

Chemical formula

[0165] ; - A light chain comprising an amino acid sequence having at least 90% homology to the sequence DVVMTQSPSSVSASVGDRVTITCRASQSIGSWLAWYQQKPGEAPKLLIYAASTLQRGVPSRFSGGGYGTEFTLTISSLQPEDFATYFCQQSHSHPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 11) comprising.

[0166] In some embodiments, the monoclonal antibody that specifically binds to GITR is BCD166-01-001. The monoclonal antibody BCD166-01-001 - amino acid sequence

[0167]

Chemical formula

[0168] comprising a heavy chain; - A light chain comprising the amino acid sequence DVVMTQSPSSVSASVGDRVTITCRASQSIGSWLAWYQQKPGEAPKLLIYAASTLQRGVPSRFSGGGYGTEFTLTISSLQPEDFATYFCQQSHSHPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 11) comprising.

[0169] In some embodiments, the monoclonal antibody - sequence

[0170]

Chemical formula

[0171] A heavy chain comprising an amino acid sequence that is at least 90% homologous; - A light chain comprising an amino acid sequence that is at least 90% homologous to the sequence DVVMTQSPSSVSASVGDRVTITCRASQSIGSWLAWYQQKPGEAPKLLIYAASTLQRGVPSRFSGGGYGTEFTLTISSLQPEDFATYFCQQSHSHPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 11) Comprising.

[0172] In some embodiments, the monoclonal antibody that specifically binds to GITR is BCD166-02-001. The difference between BCD166-02-001 and BCD166-01-001 is the E345R mutation in the Fc fragment to increase agonist properties and antibody-dependent cell cytotoxicity (ADCC) but not complement-dependent cell cytotoxicity (CDC).

[0173] The monoclonal antibody BCD166-02-001 - Amino acid sequence

[0174]

Chemical formula

[0175] A heavy chain comprising - A light chain comprising the amino acid sequence DVVMTQSPSSVSASVGDRVTITCRASQSIGSWLAWYQQKPGEAPKLLIYAASTLQRGVPSRFSGGGYGTEFTLTISSLQPEDFATYFCQQSHSHPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 11) comprising

[0176] In some embodiments, the monoclonal antibody comprises a heavy chain comprising an amino acid sequence

[0177]

Chem.

[0178] that is at least 90% identical to the sequence In some embodiments, the monoclonal antibody comprises a heavy chain comprising the amino acid sequence

[0179]

Chem.

[0180] comprising In some embodiments, the monoclonal antibody comprises a light chain comprising an amino acid sequence that is at least 90% identical to the sequence QSALTQPASVSGSPGQSITISCTGTSTDIGTYKYISWYQQHPGKAPKLIIYGVSHRPSGVSDRFSGSKSDNTASLTISGLQAEDEADYYCSSYTSSGTVVFGGGTKVTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 21).

[0181] In some embodiments, the monoclonal antibody comprises a light chain having the amino acid sequence QSALTQPASVSGSPGQSITISCTGTSTDIGTYKYISWYQQHPGKAPKLIIYGVSHRPSGVSDRFSGSKSDNTASLTISGLQAEDEADYYCSSYTSSGTVVFGGGTKVTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 21).

[0182] In some embodiments, the monoclonal antibody - a heavy chain comprising an amino acid sequence that is at least 90% identical to the sequence

[0183]

Chemical formula

[0184] ; - a light chain comprising an amino acid sequence that is at least 90% identical to the sequence QSALTQPASVSGSPGQSITISCTGTSTDIGTYKYISWYQQHPGKAPKLIIYGVSHRPSGVSDRFSGSKSDNTASLTISGLQAEDEADYYCSSYTSSGTVVFGGGTKVTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 21). comprises

[0185] In some embodiments, the monoclonal antibody that specifically binds to GITR is BCD166-01-014. The monoclonal antibody BCD166-01-014 - an amino acid sequence

[0186]

Chemical formula

[0187] comprising a heavy chain comprising - a light chain comprising the amino acid sequence QSALTQPASVSGSPGQSITISCTGTSTDIGTYKYISWYQQHPGKAPKLIIYGVSHRPSGVSDRFSGSKSDNTASLTISGLQAEDEADYYCSSYTSSGTVVFGGGTKVTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 21). Nucleic acid molecule The present invention also relates to a nucleic acid molecule as described herein, optionally comprising any peptide linker sequence connected thereto, in particular to a sequence encoding a monoclonal antibody that specifically binds to GITR according to the present invention.

[0188] References to nucleotide sequences include their complements, unless otherwise specified. Thus, references to a nucleic acid having a particular sequence are to be understood to include its complementary strand having the complementary sequence. The term "polynucleotide" as used herein means a polymeric form of nucleotides, ribonucleotides, or deoxyribonucleotides, or modified forms of any type of nucleotide, having a length of at least 10 bases. This term includes single-stranded and double-stranded forms.

[0189] In one aspect, the present invention relates to a nucleic acid molecule comprising a nucleotide sequence encoding an amino acid sequence selected from SEQ ID NOs: 1 to 21. The nucleic acid molecule may also include any combination of said nucleotide sequences.

[0190] In one aspect, the present invention specifically binds to GITR and (a)(i) the following group: NYGMH (SEQ ID NO: 1) or YYWMY (SEQ ID NO: 12) A CDR1 comprising an amino acid sequence selected from: (ii) The following group: VIWFDGSNKFYTDSVKG (SEQ ID NO: 2) or AISWNGGRTYYAESMKG (SEQ ID NO: 13) A CDR2 comprising an amino acid sequence selected from: (iii) The following group: ELGGYYYDSSGFRPYYYGMDV (SEQ ID NO: 3) or NRYYSDPNYGMNL (SEQ ID NO: 14) A CDR3 comprising an amino acid sequence selected from: A heavy chain variable domain comprising, and (b) (i) The following group: RASQSIGSWLA (SEQ ID NO: 7) or TGTSTDIGTYKYIS (SEQ ID NO: 17) A CDR1 comprising an amino acid sequence selected from: (ii) The following group: AASTLQR (SEQ ID NO: 8) or GVSHRPS (SEQ ID NO: 18) A CDR2 comprising an amino acid sequence selected from: (iii) The following group: QQSHSHPLT (SEQ ID NO: 9) or SSYTSSGTVV (SEQ ID NO: 19) A CDR3 comprising an amino acid sequence selected from: A light chain variable domain comprising, and Relates to a nucleic acid comprising a nucleotide sequence encoding a monoclonal antibody or an antigen-binding fragment thereof comprising the same.

[0191] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a monoclonal antibody or an antigen-binding fragment thereof comprising a heavy chain variable domain comprising CDR1, 2, and 3 comprising the amino acid sequences represented by the sequences of SEQ ID NOs: 1, 2, and 3, respectively.

[0192] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a monoclonal antibody or an antigen-binding fragment thereof that comprises a heavy chain variable domain comprising CDR1, 2, and 3, each comprising the amino acid sequence set forth in SEQ ID NOs: 12, 13, and 14, respectively.

[0193] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a monoclonal antibody or an antigen-binding fragment thereof that comprises a light chain variable domain comprising CDR1, 2, and 3, each comprising the amino acid sequence set forth in SEQ ID NOs: 7, 8, and 9, respectively.

[0194] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a monoclonal antibody or an antigen-binding fragment thereof that comprises a light chain variable domain comprising CDR1, 2, and 3, each comprising the amino acid sequence set forth in SEQ ID NOs: 17, 18, and 19, respectively.

[0195] In some embodiments, the nucleic acid molecule - a heavy chain variable domain comprising CDR1, 2, and 3, each comprising the amino acid sequence set forth in SEQ ID NOs: 1, 2, and 3, respectively; - a light chain variable domain comprising CDR1, 2, and 3, each comprising the amino acid sequence set forth in SEQ ID NOs: 7, 8, and 9, respectively comprises a nucleotide sequence encoding a monoclonal antibody or an antigen-binding fragment thereof.

[0196] In some embodiments, the nucleic acid molecule - a heavy chain variable domain comprising CDR1, 2, and 3, each comprising the amino acid sequence set forth in SEQ ID NOs: 12, 13, and 14, respectively; - a light chain variable domain comprising CDR1, 2, and 3, each comprising the amino acid sequence set forth in SEQ ID NOs: 17, 18, and 19, respectively comprises a nucleotide sequence encoding a monoclonal antibody or an antigen-binding fragment thereof.

[0197] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a monoclonal antibody or an antigen-binding fragment thereof that comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 4.

[0198] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a monoclonal antibody or an antigen-binding fragment thereof that comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 4.

[0199] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a monoclonal antibody or an antigen-binding fragment thereof that comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 15.

[0200] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a monoclonal antibody or an antigen-binding fragment thereof that comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 15.

[0201] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a monoclonal antibody or an antigen-binding fragment thereof that comprises a light chain variable domain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 10.

[0202] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a monoclonal antibody or an antigen-binding fragment thereof that comprises a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 10.

[0203] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a monoclonal antibody or an antigen-binding fragment thereof that comprises a light chain variable domain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 20.

[0204] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a monoclonal antibody or an antigen-binding fragment thereof that comprises a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 20.

[0205] In some embodiments, the nucleic acid molecule - comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 4; - comprises a light chain variable domain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 10 and comprises a nucleotide sequence encoding a monoclonal antibody or an antigen-binding fragment thereof.

[0206] In some embodiments, the nucleic acid molecule - comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 4; - comprises a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 10 and comprises a nucleotide sequence encoding a monoclonal antibody or an antigen-binding fragment thereof.

[0207] In some embodiments, the nucleic acid molecule - comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 15; - comprises a light chain variable domain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 20 and comprises a nucleotide sequence encoding a monoclonal antibody or an antigen-binding fragment thereof.

[0208] In some embodiments, the nucleic acid molecule - comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 15; - comprises a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 20 and comprises a nucleotide sequence encoding a monoclonal antibody or an antigen-binding fragment thereof.

[0209] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a monoclonal antibody that specifically binds to GITR and is a full-length IgG antibody. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a monoclonal IgG antibody of the human IgG1, IgG2, IgG3, or IgG4 isotype.

[0210] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a monoclonal IgG antibody of the human IgG1 isotype. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a monoclonal antibody that specifically binds to GITR and contains an E345R mutation in the Fc fragment, such that it has agonist properties and increases antibody-dependent cell-mediated cytotoxicity (ADCC) but does not increase complement-dependent cytotoxicity (CDC).

[0211] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a monoclonal antibody comprising a heavy chain having an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 5. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a monoclonal antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 5.

[0212] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a monoclonal antibody comprising a heavy chain having an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 6. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a monoclonal antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 6.

[0213] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a monoclonal antibody comprising a light chain having an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 11. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a monoclonal antibody comprising a light chain having the amino acid sequence of SEQ ID NO: 11.

[0214] In some embodiments, the nucleic acid molecule - a heavy chain having an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 5; - A light chain comprising an amino acid sequence that is at least 90% homologous to the sequence of SEQ ID NO: 11 It comprises a nucleotide sequence encoding a monoclonal antibody comprising

[0215] In some embodiments, the nucleic acid molecule - A heavy chain comprising the amino acid sequence of SEQ ID NO: 5; - A light chain comprising the amino acid sequence of SEQ ID NO: 11 It comprises a nucleotide sequence encoding a monoclonal antibody comprising

[0216] In some embodiments, the nucleic acid molecule - A heavy chain comprising an amino acid sequence that is at least 90% homologous to the sequence of SEQ ID NO: 6; - A light chain comprising an amino acid sequence that is at least 90% homologous to the sequence of SEQ ID NO: 11 It comprises a nucleotide sequence encoding a monoclonal antibody comprising

[0217] In some embodiments, the nucleic acid molecule - 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 It comprises a nucleotide sequence encoding a monoclonal antibody comprising

[0218] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a monoclonal antibody comprising a heavy chain comprising an amino acid sequence that is at least 90% homologous to the sequence of SEQ ID NO: 16. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a monoclonal antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 16.

[0219] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a monoclonal antibody comprising a light chain comprising an amino acid sequence that is at least 90% homologous to the sequence of SEQ ID NO: 21. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a monoclonal antibody comprising a light chain comprising the amino acid sequence of SEQ ID NO: 21.

[0220] In some embodiments, the nucleic acid molecule - a heavy chain comprising an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 16; - a light chain comprising an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 21 and comprises a nucleotide sequence encoding a monoclonal antibody.

[0221] In some embodiments, the nucleic acid molecule - a heavy chain comprising the amino acid sequence of SEQ ID NO: 16; - a light chain comprising the amino acid sequence of SEQ ID NO: 21 and comprises a nucleotide sequence encoding a monoclonal antibody.

[0222] In some embodiments, the nucleic acid is DNA. In any of the above embodiments, the nucleic acid molecule may be isolated. The nucleic acid molecule of the present invention may 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 may not be isolated but may be synthetic.

[0223] In one aspect of the present invention, a nucleic acid molecule encoding a VH (SEQ ID NO: 4 or SEQ ID NO: 15) or VL (SEQ ID NO: 10 or SEQ ID NO: 20) domain is already inserted into an expression vector that already encodes a heavy chain constant (CH) or light chain constant (CL) domain, respectively, such that the VH segment is operably linked to the CH segment within the vector and / or the VL segment is operably linked to the CL segment within the vector, and is transformed into an antibody gene over its full length. In another aspect of the present invention, a nucleic acid molecule encoding a VH and / or VL domain is transformed into a gene over the full length of an antibody by ligating, for example, a nucleic acid molecule encoding a VH and / or VL domain to a nucleic acid molecule encoding a CH and / or CL domain using standard molecular biological techniques. Subsequently, the nucleic acid molecule encoding the full length of the heavy chain and / or light chain can be expressed from the cell into which it has been introduced.

[0224] The nucleic acid molecule can be used to express a monoclonal antibody that specifically binds to a large amount of recombinant GITR. Vector In another aspect, the present invention relates to a vector suitable for the expression of any of the nucleotide sequences described herein.

[0225] The present invention relates to a vector comprising a nucleic acid molecule encoding any of the amino acid sequences of a monoclonal antibody or a portion thereof (e.g., the heavy chain sequence of the first binding domain and / or the heavy chain and / or light chain sequence of the second binding domain) that specifically binds to GITR described herein. The present invention further relates to a vector comprising a nucleic acid molecule encoding a fusion protein, a modified antibody, an antibody fragment.

[0226] In some embodiments, monoclonal antibodies that specifically bind to GITR according to the present invention are expressed by inserting DNA (e.g., light and heavy chain sequences, where the binding domain comprises light and heavy chain sequences) that partially or fully encodes the sequence of the first or second binding domain obtained as described above into an expression vector such that the gene is operably linked to the required expression control sequences, e.g., transcription and translation control sequences. Examples of expression vectors include plasmids, retroviruses, adenoviruses, adeno-associated viruses (AAV), plant viruses such as cauliflower mosaic virus, tobacco mosaic virus, cosmids, YACs, EBV-derived episomes, and the like. The DNA molecule can be ligated into the vector such that the transcription and translation control sequences within the vector function to regulate the transcription and translation of the intended DNA. The expression vector and expression control sequences can be selected to be compatible with the expression host cell used. DNA molecules that partially or fully encode the sequences of the first and second binding domains (e.g., heavy and light chain sequences, where the binding domain comprises heavy and light chain sequences) can be introduced into individual vectors. In one embodiment, any combination of said DNA molecules is introduced into the same expression vector. The DNA molecules can be introduced into the expression vector by standard methods (e.g., ligation of antibody gene fragments and complementary restriction sites into the vector, or blunt-end ligation if no restriction sites are present).

[0227] Suitable vectors encode a functionally complete human CH or CL immunoglobulin sequence with appropriate restriction sites engineered so that any VH or VL sequence, as described above, can be readily inserted and expressed. The HC and LC coding portions of the genes in such vectors may contain intron sequences, which as a result can enhance the overall antibody protein yield by stabilizing the corresponding mRNA. The intron sequences have splice donor and splice acceptor sites at their ends that determine where RNA splicing occurs. The position of the intron sequences may be in either the variable or constant region of the antibody chain, or in both the variable and constant regions if multiple introns are used. Polyadenylation and transcription termination may occur at natural chromosomal sites downstream of the coding region. The recombinant expression vector may also encode a signal peptide that facilitates secretion of the antibody chain from the host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the immunoglobulin chain. The signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide derived from a non-immunoglobulin protein).

[0228] In addition to the antibody chain gene, the recombinant vector expression of the present invention may have regulatory sequences that control the expression of the antibody chain gene in a host cell. It will be understood by those skilled in the art that the design of the expression vector may depend on factors such as the choice of host cell to be transformed, including the selection of regulatory sequences, and the level of expression of the desired protein. Preferred regulatory sequences for expression host cells in mammals include viral elements that ensure high-level protein expression in mammalian cells, such as promoters and / or enhancers derived from retroviral LTRs, promoters and / or enhancers derived from cytomegalovirus (CMV) (e.g., CMV promoter / enhancer), promoters and / or enhancers derived from simian virus 40 (SV40) (e.g., SV40 promoter / enhancer), promoters and / or enhancers derived from adenovirus (e.g., major late promoter adenovirus (AdMLP)), polyomavirus, and strong mammalian promoters such as natural immunoglobulin promoters or actin promoters. For further description of viral control elements and their sequences, see, for example, U.S. Patent Nos. 5,168,062, 4,510,245, and 4,968,615. Methods for expressing binding molecules, such as antibodies, in plants are known in the art, including descriptions of promoters and vectors, as well as plant transformation. See, for example, U.S. Patent No. 6,517,529. Methods for expressing polypeptides in bacterial cells or fungal cells, such as yeast cells, are also well known in the art.

[0229] In addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors of the present invention may have additional sequences, such as sequences that regulate the replication of the vector in the host cell (e.g., origin of replication) and selectable marker genes. The selectable marker gene facilitates the selection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665, and 5,179,017). For example, typically, the selectable marker gene confers resistance to pharmaceutical substances, such as G418, hygromycin, or methotrexate, on the host cells into which the vector has been introduced. For example, examples of selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr host cells during methotrexate selection / amplification), the neo gene (for G418 selection), and the glutamate synthase gene.

[0230] As used herein, the term "expression control sequence" is intended to refer to polynucleotide sequences necessary to effect the expression and processing of the 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., Kozak consensus sequences); sequences that enhance protein stability; and, if desired, sequences that enhance protein secretion. The nature of such control sequences differs depending on the host organism, and in prokaryotes, such control sequences generally include the promoter of the ribosome binding site and transcription termination sequences; in eukaryotes, typically, such control sequences include a promoter and transcription termination sequences. The term "control sequence" is intended to include at least all components whose presence is essential for expression and processing, and may include additional components, such as leader sequences and fusion partner sequences, whose presence is advantageous. Host cell A further aspect of the present invention relates to a method for obtaining a monoclonal antibody that specifically binds to GITR according to the present invention. One embodiment of the present invention is a method for obtaining a monoclonal antibody that specifically binds to GITR as defined herein, comprising the production of a recombinant host cell capable of expressing a monoclonal antibody that specifically binds to GITR, culturing the host cell under conditions suitable for the expression / production of a monoclonal antibody that specifically binds to GITR, and isolating the obtained monoclonal antibody that specifically binds to GITR. A monoclonal antibody that specifically binds to GITR produced by such expression in such a recombinant host cell is referred to herein as a "monoclonal antibody that specifically binds to recombinant GITR". The present invention also relates to the progeny of cells from such host cells, and to monoclonal antibodies that specifically bind to GITR produced in the same way.

[0231] Nucleic acid molecules encoding monoclonal antibodies that specifically bind to GITR according to the present invention and vectors containing these nucleic acid molecules can be used for transfection of suitable mammalian or their cells, plants or their cells, bacterial or yeast host cells. To introduce the polynucleotide into the host cell, transformation can be done by any known technique. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, cationic polymer-nucleic acid complex transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, encapsulation of the polynucleotide into liposomes, and direct microinjection of DNA into the nucleus. In addition, the nucleic acid molecule may be introduced into mammalian cells by a viral vector. Methods for transfecting cells are well known in the art. See, for example, U.S. Patent Nos. 4,399,216, 4,912,040, 4,740,461 and 4,959,455. Methods for transforming plant cells are well known in the art and include, for example, Agrobacterium-mediated transformation, gene gun transformation, direct injection, electroporation and viral transformation. Methods for transforming bacterial and yeast cells are also well known in the art.

[0232] Mammalian cell lines used as hosts for transformation are well known in the art, and examples thereof include a plurality of immortalized cell lines available. Examples of these include, for example, Chinese hamster ovary (CHO) cells, NS0 cells, SP2 cells, HEK-293T cells, FreeStyle 293 cells (Invitrogen), NIH-3T3 cells, HeLa cells, baby hamster kidney (BHK) cells, African green monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, and numerous other cell lines. The cell line is selected by determining which cell line has a high expression level and provides the necessary characteristics of the protein to be produced. Other cell lines that can be used are insect cell lines, such as Sf9 or Sf21 cells. When a recombinant expression vector encoding a monoclonal antibody that specifically binds to GITR is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a period sufficient to enable expression of the antibody in the host cell or, more preferably, secretion of the antibody into the culture medium in which the host cell is grown. The monoclonal antibody that specifically binds to GITR can be reconstituted from the culture medium using standard protein purification techniques. Examples of plant host cells include, for example, the genus Nicotiana, Arabidopsis, duckweed, corn, wheat, potato, etc. Examples of bacterial host cells include species of the genus Escherichia and Streptomyces. Examples of yeast host cells include Schizosaccharomyces pombe, Saccharomyces cerevisiae, and Pichia pastoris.

[0233] Furthermore, the production level of monoclonal antibodies that specifically bind to GITR according to the present invention from production cell lines can be enhanced using a number of known techniques. For example, the glutamine synthetase gene expression system (GS system) is a common approach for enhancing expression under certain conditions. The GS system has been discussed in whole or in part in relation to EP0216846, 0256055, 0323997, and 0338841.

[0234] Monoclonal antibodies that specifically bind to GITR of various cell lines or transgenic animals may be expected to have different glycosylation profiles when compared to each other. 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 of the binding molecule, and more generally, regardless of the presence or absence of post-translational modifications. Preparation of Antibodies The present invention also relates to methods and processes for obtaining monoclonal antibodies that specifically bind to GITR and antigen-binding fragments thereof. Monoclonal antibody Monoclonal antibodies may be prepared using the hybridoma method first described by Kohler et al., Nature 256, 1975, p. 495, or using recombinant DNA methods (US4816567).

[0235] In the hybridoma method, a mouse, or other suitable host animal, such as a hamster, is immunized according to the methods described above to produce lymphocytes that produce or are capable of producing antibodies that are expected to specifically bind to the protein used for immunization. According to another aspect, lymphocytes can be obtained as a result of in vitro immunization. After immunization, the lymphocytes are fused with a myeloma cell line using a suitable fusing agent, such as polyethylene glycol, to produce hybridoma cells.

[0236] The hybridoma cells obtained by the above-described method may be cultured in a suitable culture medium, preferably a medium containing one or more substances that inhibit the growth or survival of the unfused parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine-guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridoma will typically be hypoxanthine, aminopterin, and thymidine (HAT medium), i.e., a medium expected to contain substances that prevent the growth of HGPRT-deficient cells.

[0237] Preferred cells used as components for myeloma cell fusion are those that fuse efficiently, support stable high-level antibody production by the selected antibody-producing cells, and are sensitive to the medium in which unfused parental cells are selected. Preferred myeloma cell lines are mouse myeloma lines, such as the MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, California, USA, and those derived from SP-2 or X63-Ag8-653 cells available from the American Type Culture Collection, Rockville, Maryland, USA. Also, human myelomas and mouse-human heteromyeloma cell lines have been described with respect to the production of monoclonal antibodies (Kozbor, J. Immunol., 133, 1984, p. 3001).

[0238] Preferably, the binding specificity of the monoclonal antibody produced by the 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).

[0239] The binding affinity of the monoclonal antibody can be determined, for example, by the Scatchard analysis described by Munson et al., Anal. Biochem., 107:220 (1980).

[0240] Once hybridoma cells that produce antibodies with desirable specificity, affinity, and / or activity have been identified, the clones can be subcloned by limiting dilution and grown by standard methods. Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, hybridoma cells can be grown in vivo as ascites tumors in animals, for example, by injecting the cells intraperitoneally (i.p.) into mice.

[0241] Monoclonal antibodies secreted by subclones can be separated from the culture medium, ascites, or serum by conventional antibody purification techniques, such as affinity chromatography (e.g., using protein A or protein G-sepharose), ion exchange chromatography, hydroxylapatite chromatography, gel electrophoresis, dialysis, etc.

[0242] DNA encoding the monoclonal antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specific binding to the genes encoding the heavy and light chains of the mouse antibody). Hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA can be placed in an expression vector and then transfected into host cells that do not produce the antibody protein unless transfected, such as Escherichia coli (E. coli) cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells, to achieve the synthesis of the monoclonal antibody in the recombinant host cell.

[0243] In a further aspect of the invention, the monoclonal antibody or antibody fragment may be isolated from an antibody phage library generated using the techniques described in McCafferty et al., Nature, 348:552-554 (1990). Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991) describe the isolation of mouse and human antibodies using phage libraries, respectively. Subsequent publications describe the production of high affinity (in the nM range) human antibodies by chain shuffling (Marks et al., Bio / Technology, 10:779-783 (1992), as well as combinatorial infection and in vivo recombination as strategies for constructing very large phage libraries (Waterhouse et al., Nucl. Acids Res. 21:2265-2266 (1993). Thus, these techniques are a practical alternative to the conventional monoclonal antibody hybridoma technology for the isolation of monoclonal antibodies.

[0244] DNA encoding the antibody can be modified to produce, for example, chimeric or fusion antibody polypeptides, for example, by substituting the heavy and light chain (CH and CL) constant region sequences with homologous mouse sequences (US 4816567 and Morrison et al., Proc. Natl. Acad. Sci. USA: 81:6851 (1984), or by covalently fusing the immunoglobulin coding sequences with all or part of the coding sequences of a non-immunoglobulin polypeptide (heterologous polypeptide) so as to produce them. The non-immunoglobulin polypeptide sequences may be substituted for the constant regions of the antibody, or they may be substituted for the variable domains of the antigen-binding center of the antibody such that a chimeric bivalent antibody is created that contains one antigen-binding site having specificity for one antigen and another antigen-binding site having specificity for a different antigen. Human antibodies and methods based on phage display libraries It is now possible to produce transgenic animals (e.g., mice) that can produce the full range of human antibodies without endogenous immunoglobulin production after immunization. For example, it has been described that homozygous deletion of the antibody heavy chain joining region (JH) gene in chimeric and germline mutant mice results in complete inhibition of endogenous antibody production. Transfer of a human germline immunoglobulin gene array into such germline mutant mice results in the production of human antibodies after antigen challenge (US5545806, 5569825, 5591669 (all GenPharm); 5545807; and WO97 / 17852).

[0245] Alternatively, phage display technology (McCafferty et al., Nature, 348:552-553 (1990)) can be used to obtain human antibodies and antibody fragments from the immunoglobulin variable (V) region gene repertoire derived from the body of an in vitro immunized donor. According to this technology, antibody V region genes are cloned in-frame with either the major or minor coat protein gene of filamentous bacteriophages, such as M13 or fd, and presented as functional antibody fragments on the surface of phage particles. Since the filamentous particles contain a single-stranded DNA copy of the phage genome, selection based on the functional properties of the antibody also results in the selection of the gene encoding the antibody exhibiting said properties. Thus, phage mimics some of the properties of B cells. Phage display can be carried out in various ways. A number of sources of V gene segments can be used for phage display. Clackson et al., Nature, 352:624-628 (1991) isolated various arrays of anti-oxazolone antibodies from a small random combinatorial library of V genes derived from the spleens of immunized mice. A repertoire of V genes from non-immunized human donors can be constructed, and antibodies against various arrays of antigens (including autoantigens) can be isolated essentially according to the techniques described by Marks et al., J. Mol. Biol. 222:581-597 (1991).

[0246] As described above, human antibodies can also be produced by activated B cells in vitro (see US5567610 and 5229275). Antibody fragment In certain circumstances, it may be desirable to use antibody fragments rather than whole antibodies. Smaller sized fragments may contribute to their rapid clearance and may also contribute to better penetration into dense tumors.

[0247] A variety of techniques for the production of antibody fragments have been developed. Conventionally, these fragments were obtained via proteolytic digestion of intact antibodies. However, these fragments can now be obtained directly from recombinant host cells. Fab, Fv and ScFv antibody fragments can be expressed and secreted from E. coli and thus enable the easy production of large amounts of these fragments. Antibody fragments may also be isolated from the antibody phage libraries described above. According to another aspect, Fab'-SH fragments can be isolated directly from E. coli and chemically coupled to form F(ab')2 fragments (Carter et al., Bio / Technology 10:163-167 (1992)). According to another approach, F(ab')2 fragments may be isolated directly from recombinant host cell cultures. Fab and F(ab')2 with increased in vivo half-lives that retain receptor residues that bind to the epitope are described in US5869046. Other techniques for the production of antibody fragments will be apparent to those skilled in the art. In another aspect of the invention, the optimal antibody is a single-chain Fv fragment (scFv) (see WO93 / 16185; US5571894 and US5587458). Fv and scFv are the only species with intact binding sites lacking constant regions; as a result they are suitable for reducing non-specific binding during in vivo use. An scFv fusion protein can be constructed to result in the fusion of an effector protein at either the N or C terminus of the scFv. Antibody fragments may also be "linear antibodies" as described, for example, in U.S.5641870. Such linear antibody fragments may be monospecific or bispecific. Pharmaceutical composition In another aspect, the present invention provides a pharmaceutical composition comprising a monoclonal antibody that specifically binds to GITR as an active ingredient (or as the only active ingredient).

[0248] The pharmaceutical composition may comprise at least one monoclonal antibody that specifically binds to at least one GITR, and at least one component selected from the group consisting of pharmaceutically acceptable and pharmacologically compatible excipients.

[0249] The pharmaceutical composition may comprise at least one monoclonal antibody that specifically binds to at least one GITR, and one or more additional binding molecules (such as antibodies) that target one or more of the corresponding surface receptors. In some embodiments of the present invention, the composition is intended to improve, prevent, or treat disorders that may be associated with GITR.

[0250] "Pharmaceutical composition" means a composition comprising a monoclonal antibody that specifically binds to GITR according to the present invention, and at least one component selected from the group consisting of pharmaceutically acceptable and pharmacologically compatible excipients, such as bulking agents, solvents, diluents, carriers, adjuvants, dispersants, delivery agents, preservatives, stabilizers, emulsifiers, suspending agents, thickening agents, prolonged delivery controllers, and the selection and ratio thereof depend on the type and route of administration and the dosage. The pharmaceutical compositions of the present invention and methods for their preparation will be readily apparent to those skilled in the art. The pharmaceutical composition should preferably be manufactured in accordance with the requirements of GMP (Good Manufacturing Practice; standards for the manufacture and quality control of pharmaceuticals). The composition may contain a buffer composition, an isotonic agent, a stabilizer, and a solubilizing agent. The long-term action of the composition may be achieved by agents that delay the absorption of the pharmaceutical active ingredient, such as aluminum monostearate and gelatin. Examples of suitable carriers, solvents, diluents, and delivery agents include water, ethanol, polyhydric alcohols and mixtures thereof, oils, and organic esters for injection.

[0251] "Medicine (drug)" refers to compounds, or mixtures of compounds as pharmaceutical compositions, in the form of tablets, capsules, powders, lyophilized agents, injections, drip infusions, ointments, and other immediately usable forms, intended for restoring, improving or modifying physiological functions in humans and animals, as well as treating and preventing diseases, diagnosis, anesthesia, contraception, cosmetology, etc. Any method for administering peptides, proteins or antibodies recognized in the art can be suitably employed for the monoclonal antibody that specifically binds to GITR according to the present invention.

[0252] The term "pharmaceutically acceptable" refers to one or more compatible liquid or solid components suitable for administration in mammals, preferably humans. The term "excipient" is used herein to describe any component other than the above components of the present invention. These are substances having inorganic or organic properties used in pharmaceutical manufacturing to impart the physicochemical properties required for drug products.

[0253] The terms "buffer solution", "buffer composition", "buffer agent" refer to a solution that enables resistance to pH changes by the action of its acid-base conjugate components and allows the drug of the monoclonal antibody that specifically binds to CD20 to withstand pH changes. Generally, a pharmaceutical composition preferably has a pH in the range of 4.0 to 8.0. Examples of buffer solutions used include, but are not limited to, buffer solutions such as acetic acid buffer solution, phosphate buffer solution, citrate buffer solution, histidine buffer solution, succinate buffer solution, etc.

[0254] The terms "tonicity agent", "osmotic pressure regulator", or "osmotic agent", as used herein, refer to excipients that can increase the osmotic pressure of a liquid antibody formulation. A "isotonic" drug is a drug that has an osmotic pressure equivalent to that of human blood. Isotonic drugs typically have an osmotic pressure of about 250 to 350 mOsm / kg. Examples of tonicity agents used include, but are not limited to, polyols, sugars and sucrose, amino acids, metal salts such as sodium chloride, and the like.

[0255] "Stabilizer" refers to an excipient or a mixture of two or more excipients that provide physical and / or chemical stability to the active pharmaceutical agent. Stabilizers include amino acids such as, but not limited to, arginine, histidine, glycine, lysine, glutamine, proline; surfactants such as, but not limited to, polysorbate 20 (trade name: Tween 20), polysorbate 80 (trade name: Tween 80), polyethylene - polypropylene glycol and their copolymers (trade name: Poloxamer, Pluronic, sodium dodecyl sulfate (SDS); antioxidants such as, but not limited to, methionine, acetylcysteine, ascorbic acid, monothioglycerol, sulfite, etc.; chelating agents such as, but not limited to, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), sodium citrate, and the like.

[0256] A pharmaceutical composition is "stable" if the active pharmaceutical agent retains its physical stability and / or chemical stability and / or biological activity at the storage temperature, for example, at a storage temperature of 2 - 8°C, during a specified shelf life. Preferably, the active pharmaceutical agent retains both physical and chemical stability, in addition to biological activity. The storage period is adjusted based on the results of stability tests under accelerated or natural aging conditions.

[0257] The pharmaceutical composition of the present invention can be manufactured, packaged, or widely sold in the form of a ready-to-use formulation, in the form of a single unit dose or multiple single unit doses. The term "single unit dose" as used herein refers to a separate quantity of the pharmaceutical composition containing a predetermined amount of the active ingredient. The amount of the active ingredient is typically equal to the dose of the active ingredient to be administered to the subject or a convenient fraction of such a dose, such as half or one-third of such a dose.

[0258] The pharmaceutical composition according to the present invention is typically suitable for parenteral administration as a sterile preparation intended for administration to the human body by breaking through the skin or mucosal barrier or bypassing the gastrointestinal tract by utilizing injection, infusion, and implantation. For example, parenteral administration includes, inter alia, subcutaneous, intraperitoneal, intramuscular, intrasternal, intravenous, intraarterial, intrathecal, intracardiac, intraurethral, intracranial, intra-articular, transdermal injection or infusion; and infusion techniques for renal dialysis. Intratumoral delivery, such as intratumoral injection, can also be employed. Local perfusion is also provided. Preferred embodiments of the present invention include intravenous and subcutaneous routes. Any method recognized in the art for administering peptides or proteins can be suitably employed for monoclonal antibodies that specifically bind to GITR according to the present invention.

[0259] Injectable preparations can be prepared, packaged, or sold in unit dosage forms, for example, in the form of ampoules, vials, plastic containers, prefilled syringes, self-injecting devices, etc., without being limited thereto. Preparations for parenteral administration include, inter alia, suspensions, solutions, emulsions in oily or aqueous bases, pastes, and the like.

[0260] In another aspect, the present invention provides a composition for parenteral administration comprising a pharmaceutical composition provided in a dried (i.e., powdered or granular) form for reconstitution using a suitable vehicle (e.g., sterile pyrogen-free water) prior to administration. Such formulations may be prepared, for example, by a lyophilization process known as freeze-drying in the art, which process involves freezing the product and subsequently removing the solvent from the frozen material.

[0261] The monoclonal antibody that specifically binds to GITR according to the present invention may also be administered intranasally or by inhalation, either alone or as a mixture with a suitable pharmaceutically acceptable excipient, from an inhaler such as a pressurized aerosol container, pump, spray, atomizer, or nebulizer, with or without the use of a suitable propellant, or as a nasal spray or spray.

[0262] Dosage forms for parenteral administration may be formulated for immediate release or modified release. Modified release formulations include delayed, sustained, pulsed, controlled, targeted, and programmed release. Therapeutic use of monoclonal antibodies that specifically bind to GITR according to the present invention In one aspect, the monoclonal antibody that specifically binds to GITR according to the present invention is useful in the treatment of disorders associated with (and thereby mediated by) GITR activity.

[0263] In one aspect, the subject or patient to be treated is a mammal, preferably a human subject. The subject may be either male or female and of any age. In some aspects, the monoclonal antibody or antigen-binding fragment thereof that specifically binds to GITR is used to treat a disease or disorder mediated by GITR, where the disease or disorder is selected from the group including cervical cancer, head and neck cancer, gastric cancer, breast cancer, renal cell cancer, CRC (colorectal cancer), (OC) ovarian cancer, NSCLC (non-small cell lung cancer).

[0264] In the case of a tumor (e.g., cancer), a therapeutically effective amount of an antibody or a fragment thereof (e.g., an antibody or a fragment thereof that specifically binds to GITR) reduces the number of cancer cells; reduces the initial tumor size; inhibits the invasion of cancer cells into peripheral organs (i.e., slows it down to some extent, preferably stops it); suppresses tumor metastasis (i.e., slows it down to some extent, preferably stops it); inhibits tumor growth to some extent; and / or alleviates one or more of the symptoms associated with the disorder to some extent. The antibody or a fragment thereof can, to some extent, prevent the growth of existing cancer cells and / or kill it, which may be cytostatic and / or cytotoxic. In the case of cancer therapy, the in vivo efficacy can be measured, for example, by evaluating survival, the time to tumor progression (TTP), the tumor response rate (RR) to treatment, the duration of the response, and / or the quality of life.

[0265] As used herein, the terms "co-administered", "co-administering", and "in combination with" are intended to mean, refer to, or include the following when referring to a monoclonal antibody that specifically binds to GITR and one or more different therapeutic agents according to the present invention: 1) The simultaneous administration of 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, and if such components are formulated together in a single dosage form, it releases the components to the patient at substantially the same time, 2) The simultaneous administration of 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, and if such components are formulated separately in separate dosage forms, it is taken by the patient at substantially the same time, and then immediately thereafter the components are released to the patient at substantially the same time, 3) The sequential administration of 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, where such components are formulated into separate dosage forms apart from each other, is taken by the patient at successive times with a significant time interval between each administration, and then immediately thereafter, the components are released to the patient at substantially different times; 4) The sequential administration of 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, where such components are formulated together into a single dosage form, releases the components in a controlled manner, and then immediately thereafter, it is released to the patient simultaneously, continuously, or concomitantly at the same and / or different times, in which case each portion may be administered by either the same or different routes.

[0266] The monoclonal antibody that specifically binds to GITR according to the present invention can be administered without additional therapeutic treatment, i.e., as an independent therapy. Further, treatment with the monoclonal antibody that specifically binds to GITR according to the present invention may include at least one additional therapeutic treatment (combination therapy). In some aspects of the present invention, the monoclonal antibody that specifically binds to CD20 may be administered concomitantly or formulated together with another drug therapy / preparation for the treatment of cancer.

[0267] The term "cytotoxic substance" as used herein refers to a substance that inhibits or prevents the function of cells and / or causes the destruction of cells. This term includes radioisotopes (e.g., At 211 、I 131 、I 125 、Y 90 、Re 186 、Re 188 、Sm 153 、Bi 212 、P 32and radioisotopes of Lu), chemotherapeutic agents, and toxins, such as small molecule toxins, or enzymatically active toxins of bacterial, fungal, plant or animal origin, and further intended to include fragments and / or variants thereof.

[0268] "Chemotherapeutic agent" is a compound used to treat malignant tumors. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN (registered trademark)); alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carbocone, meturedopa, and uredopa; ethyleneimines and methylamelamines such as altretamine, triethylenemelamine, triethylenethiophosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; acetogenins (such as bratasin and bratasinone); delta-9-tetrahydrocannabinol (MARINOL (registered trademark) which is dronabinol); beta-lapachone; lapachol; colchicine; betulinic acid; camptothecin (such as synthetic analogs topotecan (HYCAMTIN (registered trademark)), CPT-11 (irinotecan, CAMPTOSAR (registered trademark)), acetylcamptothecin, scopoletin, and 9-aminocamptothecin); bryostatin; calistatin; CC-1065 (such as its adozelesin, carzelesin, and bizelesin synthetic analogs); podophyllotoxin; podophyllinic acid; teniposide; cryptophycin (such as cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (such as synthetic analogs KW-2189 and CB1-TM1); erythrobicin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustard such as chlorambucil, chloronaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosourea such as carmustine, chloroozotocin, 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. Intl. Ed. Engl., 33:183-186 (1994)); dynemicin, e.g., dynemicin A, etc.; esperamicin; in addition, neocarzinostatin chromophore and related pigment proteins, enediyne antibiotic chromophores), aclacinomycin, actinomycin, authramycin, azaserine, bleomycin, cactinomycin, carabicin, calminomycin, cardinophilin, chromomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (e.g., ADRIAMYCIN®, morpholinodoxorubicin, cyanomorpholinodoxorubicin, 2-pyrrolinodoxorubicin, doxorubicin HCl liposome injection (DOXOL®), liposomal doxorubicin TLC D-99 (MYOCET®), pegylated liposomal doxorubicin (CAELYX®), and deoxydoxorubicin, etc.), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, e.g., mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptozocin, streptozocin, tubercidin, ubenimex, dinostatin, zorubicin; antimetabolites, such as methotrexate, gemcitabine (GEMZAR®), tegafur (UFTORAL®), capecitabine (XELODA®), epothilone, and 5-fluorouracil (5-FU); folic acid analogs, such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs, such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine;Pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, didoxuridine, doxifluridine, enocitabine, floxuridine; anti-adrenal agents, such as aminoglutethimide, mitotane, trilostane; folic acid supplements, such as frolinic acid; aceglutone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demeclocycline; diaziquone; elfornithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids, such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rizoxin; 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; gacytosine; arabinoside (“Ara-C”); thiotepa; taxoids, such as paclitaxel (TAXOL®), albumin-engineered nanoparticle formulation of paclitaxel (ABRAXANE™), and docetaxel (TAXOTERE®); chlorambucil; 6-thioguanine; mercaptopurine; methotrexate; platinum agents, such as cisplatin, oxaliplatin, and carboplatin;Vinca that prevents microtubule formation by tubulin polymerization, such as vinblastine (VELBAN®), vincristine (ONCOVIN®), vindesine (ELDISINE®), FILDESIN®, and vinorelbine (NAVELBINE®); etoposide (VP16); ifosfamide; mitoxantrone; leucovorin; novantrone; edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid, such as bexarotene (TARGRETIN®); bisphosphonates, such as clodronate (e.g., BONEFOS® or OSTAC®), etidronate (DIDROCAL®), NE-58095, zoledronic acid / zoledronate (ZOMETA®), alendronate (FOSAMAJX®), pamidronate (AREDIA®), tiludronate (SKELID®), or risedronate (ACTONEL®); troxacitabine (1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those that inhibit the expression of genes in signal transduction pathways involved in abnormal cell proliferation, such as PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines, such as the THERATOPE® vaccine and gene therapy vaccines, such as the ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; topoisomerase 1 inhibitors (e.g., LURTOTECAN®); rmRH (e.g., ABARELIX®); BAY439006 (sorafenib; Bayer); SU-11248 (Pfizer); perifosine, COX-2 inhibitors (e.g., celecoxib or etoricoxib), proteasome inhibitors (e.g., PS341); bortezomib (VELCADE®); CCI-779; tipifarnib (Rl 1577);or afenib, ABT510; Bcl-2 inhibitors such as oblimersen sodium (GENASENSE®); pixantrone; EGFR inhibitors (see the following definitions); tyrosine kinase inhibitors (see the following definitions); and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing; in addition, combinations of two or more of the foregoing, for example CHOP, which is an abbreviation for a therapy combining cyclophosphamide, doxorubicin, vincristine, and prednisone, and FOLFOX, which is an abbreviation for a treatment regimen using oxaliplatin (ELOXATINTM) in combination with 5-FU and leucovovin.;

[0269] This definition includes antihormonal agents that act to modulate or inhibit the hormonal action on tumors, such as antiestrogen agents having a mixed agonist / antagonist profile, such as tamoxifen (NOLVADEX®), 4-hydroxytamoxifen, toremifene (FARESTON®), idoxifene, droloxifene, raloxifene (EVTSTA®), trioxifene, keoxifene, and selective estrogen receptor modulators (SERMs), such as SERM3, etc.; pure antiestrogen agents having no agonist properties, such as fulvestrant (FASLODEX®), and EM800 (such agents can block estrogen receptor (ER) dimerization, inhibit DNA binding, increase ER turnover, and / or suppress ER levels); aromatase inhibitors, such as steroidal aromatase inhibitors, such as formestane and exemestane (AROMASIN®), and non-steroidal aromatase inhibitors, such as anastrozole (AREVIIDEX®), letrozole (FEMARA®) and aminoglutethimide, etc., and other aromatase inhibitors, such as vorozole (RIVISOR®), megestrol acetate (MEGASE®), fadrozole, imidazole, etc.; luteinizing hormone-releasing hormone agonists, such as leuprolide (LUPRON® and ELIGARD®), goserelin, buserelin, and tripterelin, etc.; sex steroids, such as progestins, such as megestrol acetate and medroxyprogesterone acetate, estrogens, such as diethylstilbestrol and Premarin, and androgen / retinoids, such as fluoxymesterone, all-trans retinoic acid and fenretinide, etc.; onapristone; antiprogesterone agents; estrogen receptor downregulators (ERD); antiandrogen agents, such as flutamide, nilutamide and bicalutamide; testolactone; and pharmaceutically acceptable salts, acids or derivatives of any of the above;In addition, combinations of two or more of the above are also included.;

[0270] Other therapeutic agents that can be used in combination with an antibody that specifically binds to GITR according to the present invention may be inhibitors of growth factor function. For example, such inhibitors may include growth factor antibodies and growth factor receptor antibodies (e.g., anti-erbB2 antibody trastuzumab [Herceptin], anti-EGFR antibody panitumumab, anti-erbB1 antibody cetuximab [Erbitux, C225] and any growth factor or growth factor receptor antibody disclosed by Stern et al., Critical reviews in oncology / haematology, 2005, Vol. 54, pp. 11-29); angiogenesis inhibitors, e.g., those that inhibit the action of vascular endothelial growth factor [e.g., bevacizumab (Avastin), an anti-vascular endothelial growth factor antibody], anti-vascular endothelial growth factor receptor antibodies, e.g., anti-KDR antibody and anti-flt1 antibody; antisense therapeutic agents, e.g., those directed against the targets listed above, e.g., ISIS2503, anti-ras antisense or G3139 (Genasense), anti-bcl2 antisense; gene therapy approaches, e.g., approaches for replacing abnormal genes, e.g., abnormal p53 or abnormal BRCA1 or BRCA2, approaches using GDEPT (gene-directed enzyme prodrug therapy), cytosine deaminase, thymidine kinase or bacterial nitroreductase enzymes, and approaches for increasing the patient's tolerance to chemotherapy or radiotherapy, e.g., multidrug resistance gene therapy, etc.;Ex vivo and in vivo approaches for increasing the immunogenicity of a patient's tumor cells, such as treatment with immunotherapy approaches, for example, alemtuzumab (campath-1H), a monoclonal antibody directed against CD52, or treatment with an antibody directed against CD22, cytokines, for example transfection with interleukin 2, interleukin 4 or granulocyte macrophage colony stimulating factor, approaches for reducing T cell anergy, for example, treatment with a monoclonal antibody that inhibits CTLA-4 function, approaches using transfected immune cells, for example dendritic cells transfected with cytokines, approaches using tumor cell lines transfected with cytokines, and approaches using anti-idiotype antibodies, adoptive T cell transfer using non-specifically activated or ex vivo targeted T cells to the specific antigen of interest; inhibitors of proteolysis, for example proteasome inhibitors, for example bortezomib; therapeutic approaches by biotherapy, for example those using peptides or proteins (for example antibodies or soluble extracellular receptor domain constructs) that either block receptor ligands, block ligands that bind to receptors, or reduce receptor signaling (for example, by enhancing receptor degradation or reducing expression levels).;

[0271] Other therapeutic agents that can be used in combination with the antibody specifically binding to GITR according to the invention may be antibodies selected from the group comprising anti-PD1 antibodies, anti-PD-L1 antibodies, anti-CTLA4 antibodies, anti-4-1BB antibodies, anti-OX40 antibodies or combinations thereof.

[0272] Other therapeutic agents that can be used in combination with the antibody specifically binding to GITR according to the invention may be antitumor compounds having therapeutic activity selected from the group of activators of innate or adaptive immunity.

[0273] The monoclonal antibodies that specifically bind to GITR according to the present invention are intended to be usable in the methods of treatment described above, in the treatments described above, and / or in the manufacture of pharmaceutical agents for the treatments described above. Dosage and Route of Administration The monoclonal antibodies that specifically bind to GITR according to the present invention are expected to be administered in an effective amount in the treatment of the condition in question, i.e., in the dosage and for the period necessary to achieve the desired result. The therapeutically effective amount may vary depending on factors such as the particular condition being treated, the age, sex and weight of the patient, and further, whether the monoclonal antibody that specifically binds to GITR is being administered as a sole treatment or in combination with one or more additional drugs or treatments.

[0274] The dosing regimen may be adjusted to provide an optimal response. For example, a single bolus may be administered, or divided doses may be administered over a long period of time, or the dosage may be proportionally decreased or increased depending on the exigency of the treatment situation. For ease of administration and uniformity of dosage, it is particularly advantageous to formulate parenteral compositions in unit dosage form. Unit dosage form, as used herein, is intended to refer to physically discrete units suitable as a unitary dosage for the patient / subject to be treated, each unit containing a predetermined quantity of the active compound calculated to produce the desired therapeutic effect in association with the desired pharmaceutical carrier. Details regarding the unit dosage forms of the present invention are typically determined 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 compounds for the treatment of sensitivity in such subjects.

[0275] Accordingly, one of ordinary skill in the art will recognize that, based on the disclosure provided herein, the dosages and dosing regimens will be adjusted according to methods well known in the therapeutic arts. That is, the maximum tolerated volume can be readily established, the effective amount that will produce a detectable therapeutic effect in a patient can be determined, and likewise, the transient requirements for administration of each agent to produce a detectable therapeutic effect in a patient can be determined. Accordingly, while specific dosages and dosing regimens are exemplified herein, these examples in no way limit the dosages and dosing regimens that can be provided to a patient in the practice of aspects of the invention.

[0276] It should be noted that the dosage values may vary depending on the type and severity of the condition to be alleviated and may include single or multiple dosages. Further, for any particular subject, the specific dosing regimen should be adjusted over time according to the individual needs and the judgment of the medical professional administering or managing the administration of the composition. Further, it will be understood that the dosage ranges described herein are merely exemplary and are not intended to limit the scope or practice of the claimed composition. Further, the dosing regimen using the compositions of the invention may be based on various factors such as the type of disease, the age, weight, sex, medical condition, severity of the condition, route of administration, and the specific monoclonal antibody that specifically binds to the GITR employed. Accordingly, the dosing regimen can be widely varied but can be routinely determined using standard methods. For example, the dosage may be adjusted based on pharmacokinetic or pharmacodynamic parameters, such as clinical effects such as toxicity and / or experimental values. Accordingly, the invention encompasses dose titration within a patient determined by one of ordinary skill in the art. Methods for determining appropriate dosages and regimens are well known in the art and will be understood by one of ordinary skill in the art given the concepts disclosed herein.

[0277] Examples of suitable methods of administration are provided above. The preferred dosage of the monoclonal antibody that specifically binds to GITR according to the present invention is expected to be in the range of 0.1 to 200 mg / kg, preferably 0.1 to 100 mg / kg, such as about 0.5 to 50 mg / kg, for example about 1 to 20 mg / kg. The monoclonal antibody that specifically binds to GITR may be administered, for example, at a dosage of at least 0.25 mg / kg, for example, at least 0.5 mg / kg, for example at least 1 mg / kg, etc., for example, at least 1.5 mg / kg, for example, at least 2 mg / kg, for example, at least 3 mg / kg, for example at least 4 mg / kg, etc., for example, at least 5 mg / kg; up to a maximum value of 50 mg / kg, for example up to a maximum value of 30 mg / kg, etc., for example, up to a maximum value of 20 mg / kg, for example up to a maximum value of 15 mg / kg. Administration is typically repeated over a length of time considered appropriate by the attending physician, at appropriate time intervals, such as once a week, once every two weeks, once every three weeks, or once every four weeks, and the dosage can be increased or decreased as needed in some cases. Diagnostic uses and compositions The monoclonal antibody that specifically binds to GITR according to the present invention is also used in diagnostic processes (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 a sample obtained from a patient (e.g., a tissue sample or a sample of body fluid, such as an inflammatory exudate, blood, serum, intestinal fluid, saliva, or urine). Suitable methods for detection and measurement include immunoassays, such as flow cytometry, enzyme-linked immunosorbent assay (ELISA), chemiluminescence assay, radioimmunoassay, and immunohistology. The present invention further includes kits, such as diagnostic kits, containing the monoclonal antibody that specifically binds to GITR described herein.

Examples

[0278] The following examples are provided for a better understanding of the present invention. These examples are for illustrative purposes only and are not to be construed as limiting the scope of the present invention in any way. All publications, patents, and patent applications cited herein are hereby incorporated by reference into this specification. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of the present invention that some changes and modifications may be made thereto without departing from the essence and scope of the appended claims. Materials and General Methods General information regarding the nucleotide sequences of human immunoglobulin light and heavy chains is shown in Kabat, 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) 78-85; Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991). Recombinant DNA Technology Standard methods were used to manipulate DNA as described in Sambrook, J. et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. Molecular biology reagents were used according to the manufacturer's protocols. Gene Synthesis Desired gene segments were prepared from oligonucleotides synthesized chemically. Gene segments with lengths of 300 - 4000 kb having specific restriction sites at their ends were assembled by annealing and ligation of oligonucleotides including PCR amplification, and then cloned via the indicated restriction sites. The DNA sequences of the subcloned gene fragments were confirmed by DNA sequencing. DNA sequencing The DNA sequences were determined by Sanger sequencing. Sequence analysis of DNA and proteins and management of sequence data Infomax's Vector NT1 Advance suite version 8.0 was used for sequence creation, mapping, analysis, annotation, and illustration. Expression vector For the expression of the described antibodies and antigens, variants of expression plasmids intended for expression in prokaryotic cells (E. coli) and transient expression in eukaryotic cells (e.g., in CHO cells) were applied. The vectors, in addition to the antibody expression cassette, contained an origin of replication enabling the replication of the plasmid in E. coli and genes conferring resistance to various antibiotics in E. coli (e.g., resistance to ampicillin and kanamycin).

[0279] Fusion genes containing the described antibody chains, as explained below, were generated by PCR and / or gene synthesis and assembled using known recombinant methods and techniques by connection of the corresponding nucleic acid segments, for example, using unique restriction sites in the corresponding vectors. The subcloned nucleic acid sequences were verified by DNA sequencing. For transient transfection, larger amounts of plasmids were prepared by plasmid preparation from transformed E. coli cultures.

[0280] Example 1 Production of recombinant antigens and antibodies in suspension culture of mammalian cells To prepare recombinant antigens based on the sequences of the extracellular portions of human GITR and orthologs, the inventors generated a number of constructs containing the antigen extracellular domains of human GITR (https: / / www.uniprot.org / uniprot / Q9Y5U5), cynomolgus macaque (Macaca mulatta) mcyGITR (https: / / www.uniprot.org / uniprot / Q1PBC4), and murine musGITR (https: / / www.uniprot.org / uniprot / O35714). The gene sequences were synthesized by PCR from a matrix encoding the full-length human GITR antigen (https: / / www.rndsystems.com / products / human-gitr-tnfrsf18-np_004186-versaclone-cdna_rdc0359) from plasmid vector RDC0359 and assembled from synthetic oligonucleotides for the GITR ortholog genes. The GITR gene sequences were cloned into the SalI / NotI restriction sites of a plasmid (Figure 1) for protein production in mammalian cells with an Fc IgG1 llama (Lama glama) tag. Furthermore, the antigen sequence is separated from the Fc by the protease-labile site of TEV protease; this site was further used to cleave the Fc fragment by treatment with TEV protease to also obtain a tag-free version of the antigen. This sequence was further cloned into a plasmid (Figure 2) containing a FLAG-EPEA-tag (C-tag, GE Healthcare) at the C-terminus of the protein also at the SalI / NotI restriction sites. Additionally, recombinant human GITR-ligand (GITRL) (https: / / www.uniprot.org / uniprot / Q9UNG2) was similarly cloned into a plasmid (Figure 3) containing a homotrimeric foldon [1] domain and an EPEA-tag at the C-terminus of GITRL. The required amounts of plasmid were produced in E. coli cells and purified using the Maxiprep Qiagen kit.

[0281] Antibodies and antigens were generated in an established cell line obtained from Chinese hamster ovary cells (CHO-K1) according to published protocols [Biotechnol Bioeng. September 20, 2005; 91(6):670 - 677, Liao Metal., 2004; Biotechnol Lett. June 2006; 28(11):843 - 848; Biotechnol Bioeng. November 5, 2003; 84(3):332 - 342]. Cells constitutively expressing the EBNA1 (Epstein - Barr virus nuclear antigen 1) protein gene were used. Suspension cultures were carried out in flasks on an orbital shaker using a serum - free medium from Life Technologies according to the manufacturer's guidelines. For transient expression, cells at a concentration of 2×10 6 / ml were transfected with linear polyethyleneimine (PEI MAX, Polysciences). The DNA / PEI ratio was 1:3 - 1:10. Five to seven days after transfection, the cell culture was centrifuged at 2000g for 20 minutes and filtered through a 0.22 μm filter. The target protein was isolated from the culture supernatant by affinity HPLC.

[0282] Recombinant GITR and GITRL proteins containing an EPEA - tag (glutamic acid - proline - glutamic acid - alanine) at the C - terminus of the protein were isolated and purified from the culture supernatant using a CaptureSelect C - tag affinity matrix adsorbent. The culture supernatant was passed through a chromatography column pre - packed with 5 ml of the C - tag adsorbent, and then the column was washed with 25 ml of PBS to remove non - specifically bound components. The bound antigen was eluted under gentle conditions using 20 mM Tris, 2 M MgCl2 at pH 7.0 - 7.4. The protein was then dialyzed against PBS (pH 7.4) using a semi - permeable dialysis membrane, filtered (0.22 μm), transferred to tubes, and stored at - 70°C.

[0283] The recombinant GITR-TEV-Fc antigen was isolated and purified from the culture supernatant using a Protein A column for affinity HPLC. The clarified culture supernatant was passed through a 5 ml HiTrap rProteinA 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 remove nonspecifically binding components. The bound antigen was eluted using 0.1 M glycine buffer (pH 3). The major protein elution peak was collected and brought to neutral pH with 1 M Tris buffer (pH 8). All steps were carried out at a flow rate of 110 cm / hour. The protein was then dialyzed against PBS (pH 7.4) using SnakeSkin Dialysis Tubing technology, filtered (0.22 μm), transferred to tubes, and stored at -70 °C.

[0284] All recombinant IgG antibodies were purified on a 1 ml HiTrap rProteinA FF column (GE Healthcare) according to the aforementioned procedure for the antigen. The purity of the resulting protein solution was evaluated by SDS gel electrophoresis (examples are shown in Figures 4 and 5).

[0285] Example 2 Construction of the naive human antibody Fab-library MeganLib™ Total RNA of B lymphocytes from blood samples from more than a thousand individual human donors was isolated using the RNeasy Mini Kit (QIAGEN) according to the suggested protocol. RNA concentration assays were performed using the Nanovue kit (GE Healthcare), and the quality of the isolated RNA was tested by 1.5% agarose gel electrophoresis.

[0286] The reverse transcription reaction was carried out using the MMLV RT kit (Evrogen) according to the recommended protocol, using MMuLV reverse transcriptase and random hexamer oligonucleotides as primers.

[0287] The reverse transcription product was used as a matrix in a two-step polymerase chain reaction to obtain a variable domain gene having a restriction site at the end, and the reaction was carried out using an oligonucleotide kit according to the protocol by [J Biol Chem. 1999 Jun 25;274(26):18218~30].

[0288] The obtained DNA preparation VL-CK-VH (Figure 6) was treated with NheI / Eco91I restriction endonucleases and ligated to the original phagemid pH5 (Figure 7). The ligation product was transformed into SS320 strain electrocompetent cells prepared according to the protocol [Methods Enzymol. 2000;328:333~63.]. The repertoire of the combinatorial phage Fab display library MeganLib (trademark) was 10 11 transformants. The Fab library phage products were prepared according to the procedure described initially [J Mol Biol. December 5, 1991;222(3):581~97].

[0289] Example 3 Construction of an immune Fab library of hybrid llama-human antibodies (CHARM) Total RNA of B lymphocytes from individual blood samples of llamas immunized with recombinant GITR antigen, which exhibited the highest specific serum titer, was isolated using an RNeasy mini kit according to the suggested protocol (QIAGEN). The RNA concentration assay was performed using a Nanovue kit (GE Healthcare), and the quality of the isolated RNA was tested by 1.5% agarose gel electrophoresis.

[0290] The reverse transcription reaction was carried out using an MMLV RT kit (Evrogen) according to the recommended protocol, using MMuLV reverse transcriptase and random hexamer oligonucleotides as primers.

[0291] The reverse transcription product was used as a matrix in a two-step polymerase chain reaction to obtain a variable domain gene having a restriction site at the end, and the reaction was carried out using an oligonucleotide kit according to the protocol by [J Biol Chem. 1999 Jun 25;274(26):18218~30].

[0292] The obtained DNA preparation VL-CK-VH (Figure 6) was treated with NheI / Eco91I restriction endonuclease and ligated to the original phagemid pH5 (Figure 7). The ligation product was transformed into SS320 strain electrocompetent cells prepared according to the protocol [Methods Enzymol. 2000;328:333~63.]. The repertoire of the combinatorial phage Fab display library MeganLib™ was 10 11 transformants. The Fab library phage products were prepared according to the procedure described initially [J Mol Biol. Dec 5, 1991;222(3):581~97]. Table 1 shows a summary table of the immune library products.

[0293]

Table 2

[0294] Example 4 Selection of Phage Antibody Fab-Library Specific anti-GITR human phage Fab antibodies were obtained from the combinatorial phage Fab display library MeganLib™ and the immune CHARM library described in Examples 2 and 3. The selection was performed with recombinant human GITR antigen by phage display [Nat Biotechnol. Mar 1996;14(3):309~14; J Mol Biol. Dec 5, 1991;222(3):581~97].

[0295] The selection of the immune phage library was carried out with EIA / RIA Tube High Binding 5 ml immunotubes. The antigen (0.5 ml, 2 μg / ml) was adsorbed overnight at +4 °C in carbonate buffer (0.1 M NaHCO3, pH 9.5). The tubes 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 0.5% powdered milk in PBST with a total volume (5 ml) was added for blocking. The blocking was changed for each round; for example, if 0.5% milk in PBST was used in the first round, 1% BSA in PBST was used in the second round. The tubes were incubated on a shaker at room temperature for 1 hour. The tubes were washed with PBST, and 2 ml / tube of blocking buffer and phage library were added to a concentration of about 2×10 12 phage particles per ml. The inventors incubated it with the phage at room temperature for 1 - 2 hours. In further rounds, the inventors used 4 ml of phage supernatant from the previous round, which was pre-clarified in a centrifuge at 17,000 g for 10 - 15 minutes. The tubes were washed 20 times with PBST. Phage was eluted from the surface of the tubes using 0.5 ml of 0.1 M glycine buffer pH 2.2: the glycine buffer was added to the tubes and the solution was gently stirred at room temperature for 15 minutes. The phage solution was then transferred to a clean non-sorbing tube containing 100 μl of 1 M TrisHCl pH 8.0 for neutralization. The tubes were kept on ice until the cells were infected.

[0296] After selection, the M13 bacteriophage is cultured using the Escherichia coli TG1 strain as the host. Amplification is carried out by infecting the host strain culture with the phage and subsequently growing it for 12 - 15 hours.

[0297] After selection, 0.5 - 1 ml of phage was added to the cell culture (OD 600= 0.3 to 0.4), incubated at 37 °C for 1.5 hours. Then the cells were centrifuged at 3,000 - 4,000 rpm for 10 - 15 minutes, resuspended in 1 ml of medium, and plated on Petri dishes containing the antibiotic for selection (ampicillin). The colonies were grown at 30 °C in a thermostat. After 12 - 15 hours, the inventors counted the number of colonies and washed the cells off the culture dishes with 5 - 10 ml of LB medium. 100 μl of the cell suspension was added to 20 ml of antibiotic medium (ampicillin). The inventors increased the density to OD600 = 0.35 - 0.5 by 37. К07 phage helper was added at 1 μl / 10 ml of culture (particles 10 10 per ml), incubated at 37 °C for 1.5 hours without shaking. Then an equal volume of medium containing a single dose of ampicillin (100 μg / ml) and a double dose of kanamycin (40 μg / ml) and a double dose of IPTG (0.2 mM) was added to the cell culture. The cell culture was loaded onto a shaker and the phage was cultured at 30 °C for 4 - 5 hours. The cell culture was centrifuged at 17,000 g for 25 - 30 minutes and the supernatant was collected in a tube. Then the obtained supernatant was used for the next round of selection or the phage was isolated by precipitation for further storage.

[0298] The phage was isolated by the following method: 1 / 6 of the volume of a solution containing 20% polyethylene glycol and 2.5 M sodium chloride was added to the supernatant and stirred strongly. The solution was incubated in ice for at least 3 hours. Then the solution was centrifuged at 8,000 g for 10 minutes and the precipitate containing the obtained phage was diluted with 1 ml of TBS buffer (Tris-borate buffer).

[0299] After 2 - 3 rounds, the bacteriophage was isolated and the inventors analyzed the specific binding of the polyclonal phage to human GITR and non-specific antigens. Example 5 Analysis of polyclonal phage from the second and third rounds of selection After three rounds of selection of two naive MeganLib libraries and six immune libraries against the target antigen, the inventors investigated the specific and non-specific binding of polyclonal phages from the second and third rounds using ELISA.

[0300] The target GITR antigen was adsorbed overnight onto the plastic surface of the ELISA plate in carbonate buffer (0.1 M NaHCO3, pH 9.5) to analyze specifically binding phages (2 μg / ml), or the presence of GCSF, hIL6R-Fc, interferon alpha-2b, rituximab (2 μg / ml) to analyze non-specifically binding phages. The plate was 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 then 0.5% powdered milk in PBST at 300 μl / well was added for blocking. The plate was incubated on a shaker for 1 hour at room temperature. The plate was washed with PBST, and 50 μl / well of phage solution from the second and third rounds of selection diluted 1:2 to 1:256 in blocking buffer was added to the plate. The plate was incubated on a shaker for 1 hour at room temperature. The plate was washed with PBST and coated with anti-M13 horseradish peroxidase conjugate antibody in blocking buffer. After 1 hour of incubation, the plate was washed and 50 μl / well of reaction substrate (H2О2 - 0.02% and TMB) in acetate buffer pH 5.0 was added. The plate was incubated in the dark at room temperature until a slight background appeared in the negative control (but for no more than 20 minutes). The reaction was quenched by adding 25 μl / well of 10% H2SO4, and the OD of the wells was measured at 450 nm.

[0301] From the assay, specific (5-fold background signal) binding of one human naive Fab library and six immune Fab CHARM libraries after the third round of selection against the target antigen, as well as the absence of significant non-specific binding (less than 2-fold background signal) was revealed.

[0302] The variable domain genes of the antibodies from these libraries were recloned into the expression vector pLL-Fab (Figure 8) to produce a secretion-capable soluble form of Fab in E. coli cells.

[0303] Example 6 Screening for Fab that specifically binds to human GITR Using ELISA, Fabs specifically binding to human GITR secreted into the medium from monoclonal clones produced in E. coli according to Example 5 were detected. Fab with the published sequence, Fab-gitr3215 (patent application), was used as a positive control. For the specific binding assay, ELISA well plates (medium binding, Greiner bio one) were coated with 50 μl / well of GITR (0.2 μg / ml in 1× carbonate buffer), sealed, and incubated overnight at 4 °C. All further steps were performed using a high-performance automated platform based on robotic systems such as Genetix Qpix2xt (Molecular Devices) and Tecan Freedom EVO200 (Tecan) according to the standard ELISA protocol. Nonspecific binding was blocked by adding blocking buffer BB (0.5% fat-free milk in 200 μl of PBS). The plates were incubated on a shaker at room temperature for 1 hour. After washing with PBS-Tween, each cell was coated with 50 μl of cell supernatant containing the test Fab mixed with an equal volume of BB. The plates were incubated on a shaker at room temperature for 1 hour; further, the wells of each plate were washed three times with PBS-Tween buffer. After washing, each well was coated with an anti-human Fab HRP conjugate secondary antibody (Pierce-ThermoScientific) (1:5000) in PBS-Tween (50 μl / well). The plates were shaken on a rotary shaker (50 minutes at room temperature) and then washed three times with PBS-Tween buffer as described above. The colorimetric signal was developed by adding ТMВ (50 μl / well) until saturation (on average 3 - 5 minutes); further color development was blocked by adding a stop solution (30 μl / well, 10% sulfuric acid). The color signal was measured at 450 nm using an appropriate Tecan-Sunrise plate reader (Tecan). The antibody binding level was proportional to the production of the color signal. Clones with a color signal exceeding five times the background signal and comparable to the color signal of the control antibody Fab-gitr3215 were investigated by ELISA to detect nonspecific binding.

[0304] Example 7 Assay for non-specific binding of selected Fab to other antigens Also, ELISA was employed to assay for non-specific binding of the Fab fragments in question to other antigens. The study was carried out as described above, using IL6R-Fc, INFα2b, PCSK9-VG-FE, PD-1-Fc (2.5 μg / ml in 1× carbonate buffer) as the immobilized antigens. GITR-TEV-Fc (0.2 μg / ml in 1× carbonate buffer) was used as a control for specific binding. All further steps were carried out according to a standard ELISA protocol using a high-performance automated platform based on robotic systems such as Genetix Qpix2xt (Molecular Devices) and Tecan Freedom EVO200 (Tecan). Clones with a non-specific binding color signal not exceeding the background signal and having a value one-fifth that of the specific binding color signal were considered positive, and their genes were sequenced for determination of the antibody variable domain gene sequences and uniqueness. As a result of the sequencing, the inventors selected 30 unique sequences for conversion to the full-length IgG1 antibody format.

[0305] Example 8 Production of recombinant antibodies in suspension culture of mammalian cells The antibody variable domain genes from Example 7 were cloned according to standard methods. To achieve this, the inventors generated PCR products containing the antibody heavy and light chain variable domain genes. The heavy chain variable domain was cloned into the SalI / NheI restriction sites of the vector pEE-Hc IgG1. Its schematic map is shown in Figure 9. The light chain variable domain was ligated into the SalI / BsiWI restriction sites of the vector pEE-CK. Its schematic map is shown in Figure 10.

[0306] The antibody was generated in an established cell line obtained from Chinese hamster ovary cells (CHO-K1) according to a published protocol [Biotechnol Bioeng. September 20, 2005; 91(6):670-677, Liao Metal., 2004; Biotechnol Lett. June 2006; 28(11):843-848; Biotechnol Bioeng. November 5, 2003; 84(3):332-342]. Cells constitutively expressing the EBNA1 (Epstein-Barr virus nuclear antigen 1) protein gene were used.

[0307] Cells of the CHO-K1-S cell line were used to generate the antibody in a transient expression system. In baffled flasks (125, 250, 500, 1000 and 3000 ml), in a mixture of CHO-S-SFM II and FreeStyle CHO (1:1) medium supplemented with 4 mM glutamine, 0.05 mg / ml gentamicin and 10 μg / ml ciprofloxacin, in an orbital shaker-incubator, at +37 °C, 5% CO2, at 110 or 150 rpm depending on the flask size, the cells were cultured. The cells were subcultured three times a week at a plating density of 0.2×106 cells / ml.

[0308] For transfection, the cells were seeded at a density of 0.8×106 cells / ml the day before transfection, and transfection was performed using cell culture at a density of 2×106 cells / ml one day later. RPMI-1640 medium supplemented with 2 mM glutamine and 0.05 mg / ml gentamicin was used to prepare the transfection mixture. The vector was diluted separately in the medium at 0.75 μg / ml, and the transfection reagent polyethyleneimine (PEI) was diluted separately by weight at a PEI:DNA of 7:1. The dilutions of the vector and PEI were mixed, incubated at room temperature for 10 minutes, then this transfection mixture was introduced into the cells, and the cells were cultured under standard conditions.

[0309] On the day following transfection, a mixture of CHO-S-SFM II and FreeStyle CHO (1:1) medium supplemented with 0.05 mg / ml gentamicin, 10 μg / ml ciprofloxacin, 1 mM sodium valproate, and 10% supplied F12.7 was added to the cells, and the cells were cultured at +34 °C, 5% CO2 in an orbital shaker-incubator at 110 or 150 rpm depending on the flask size. On days 3 - 4 after transfection, 10% supplied F12.7 was added to the cells.

[0310] On day 7 after transfection, cell fluid samples were selected and the concentration of the protein produced on a Protein-A Biosense chip was measured using an OctetRed 96 according to the ForteBio protocol (Table 2). The recombinant antibody was isolated and purified from the culture using a protein A affinity HPLC column. The clarified culture was passed through a 1 ml HiTrap rProteinA 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 remove all non-specifically binding components. The bound antibody was eluted using 0.1 M glycine buffer (pH 3). The major protein elution peak was collected and brought to neutral pH with 1 M Tris buffer (pH 8). All steps were carried out at a flow rate of 110 cm / hour. The protein was then dialyzed against PBS (pH 7.4) using SnakeSkin Dialysis Tubing technology, filtered (0.22 μm), transferred to tubes, and stored at -70 °C.

[0311] The purity of the obtained protein solution was evaluated by SDS gel electrophoresis (Figures 11 and 12). All antibodies meet the purity levels required to investigate their physicochemical properties and activities in cell assays.

[0312]

Table 3

[0313] Example 9 Kinetic assay of anti-GITR IgG1 antibody-human GITR interaction The binding affinity constants of anti-GITR antibody-human / rhesus macaque / cynomolgus macaque GITR interactions were determined using Octet Red 96 according to the manufacturer's (ForteBio) protocol. 25 μg / ml of antigen was nonspecifically immobilized on the surface of the second-generation amine-reactive sensor (AR2G) of the anti-GITR antibody (using a standard protocol according to the manufacturer's instructions for the preparation and immobilization of the AR2G sensor, ForteBio). Antibodies were added at predetermined concentrations. The assay was performed at 30 °C using PBS containing 0.1% Tween-20 and 0.1% BSA as the assay buffer.

[0314] The sensogram obtained after subtracting the reference signal was analyzed using the 1:1 interaction model using Octet data analysis software (version 8.0) according to the standard procedure. The obtained affinity constants are shown in Table 3. All the tested antibodies showed high affinity and specificity for human GITR.

[0315] [Table 4]

[0316] Example 10 Determination of anti-GITR specific agonist activity For the assay, the inventors used a HEK293-GITR-NFkB-Luc cell line generated based on the Hek-293 cell line, which stably expresses GITR on the surface and contains a gene encoding firefly luciferase under the control of the NFkB promoter.

[0317] The assay was performed in a 96-well culture plate. The suspension in each well contained HEK293-GITR-NFkB-Luc cells and the test antibody at the concentrations as shown in the graph. All suspension components were prepared in nutrient cell culture medium. After adding all components, the plate was incubated at 37 °C and 5% CO2, and then the luciferase intensity in the wells was measured using a luminescence assay kit and a plate reader.

[0318] From the investigation of the specific activity of the anti-GITR monoclonal antibodies, it was elucidated that the antibodies of lots 1161, 1210, and 1213 were functionally active and exhibited significant agonist activity with an upper plateau equivalent to the agonist activity of both the control antibody, anti-GITR3215 antibody, and GITRL (Figure 13). These antibodies were renamed again according to Table 4.

[0319]

Table 5

[0320] Example 11 Enzyme-linked immunosorbent assay of the interaction between anti-GITR antibodies from different organisms and GITR The relative affinity of antibodies against GITR-Fc from different organisms was measured using ELISA. For the binding assay, wells of an ELISA plate (medium binding from Greiner bio one) were coated with 50 μl of human / cynomolgus / mouse GITR-Fc (0.5 μg / ml in 1× carbonate buffer), sealed, and incubated overnight at 4°C. All further steps were performed according to the standard ELISA protocol described in Example 6. Anti-GITR antibody BCD166-01-01 specifically bound to human and cynomolgus GITR and did not bind to mouse GITR (Figure 14). Antibody BCD166-01-011 specifically bound to human GITR but did not show binding to cynomolgus and mouse GITR (Figure 15). Antibody BCD166-01-014 specifically bound to human GITR, but its binding to cynomolgus GITR was weaker and no binding to mouse GITR was observed (Figure 16). Therefore, candidate BCD166-01-011 was excluded from further studies due to the lack of apparent specificity for cynomolgus GITR. For further development, candidates BCD166-01-01 and BCD166-01-014 were finally selected.

[0321] Example 12 Anti-GITR IgG1 antibody - Kinetic assay of cynomolgus macaque GITR / macaca Cynomolgus GITR interaction The binding affinity constants of anti-GITR antibodies and cynomolgus / rabettus GITR were obtained using an OctetRed 96 according to the manufacturer's (ForteBio) protocol. 25 μg / ml of the antigen was non-specifically immobilized on the surface of the second-generation amine-reactive sensor (AR2G) of the anti-GITR antibody (using a standard protocol according to the manufacturer's instructions for the preparation and immobilization of the AR2G sensor). Antibodies were added at a predetermined concentration. The assay was performed at 30°C using PBS containing 0.1% Tween-20 and 0.1% BSA as the working buffer.

[0322] The sensogram obtained after subtracting the reference signal was analyzed using a 1:1 interaction model using Octet data analysis software (version 8.0) according to the standard procedure. The affinity constants obtained are shown in Tables 5 and 6. All of the antibodies tested exhibited high affinity and specificity for monkey GITR, thus providing a basis for further research into this relevant in vivo animal model.

[0323]

Table 6

[0324]

Table 7

[0325] Example 13 An in vitro cell assay for determining the anti-GITR specific agonist activity of wild-type and mutant candidates with enhanced agonist activity.

[0326] Regarding the possible enhancement of the agonist activity of the anti-GITR antibody BCD166-01-01, a human derivative, and further based on the study of such substitutions leading to antibody oligomerization in WO2005047327, WO2006104989 (A2), WO2007005612 (A2) and Science. March 14, 2014; 343(6176):1260 - 3, the E345R mutation was selected and introduced into the Fc region of the IgG1 of the antibody. Such mutations have been shown to result in antibody oligomerization on the cell surface after binding to the antigen and can enhance various effector functions such as ADCC, ADCP, CDC, as well as pharmacokinetics (PK). The aim was to obtain an antibody with enhanced agonist ability and ADCC rather than CDC. Mutagenesis was performed according to standard genetic engineering protocols and recombinant antibodies were synthesized according to Example 8. The antibody was named, which is referred to as BCD166-02-01 herein. Also, two parental antibodies BCD166-01-01, BCD166-01-014 were also assayed.

[0327] The assay was performed in the same manner as in Example 10. The results shown in Figure 17 indicate that in the cell agonist assay of test BCD166-02-01, the activation level increased 5-fold 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 antibody BCD166-02-01 on the target cells. Furthermore, not only did the level of the upper plateau of activation increase spontaneously, but the EC50 value also increased by more than 20-fold in the upward direction.

[0328] Example 14 In vitro cell assay for antibody-dependent cell-mediated cytotoxicity (ADCC) of anti-GITR antibody In the assay, the inventors studied the ADCC activity of candidate BCD166-02-01. They also assayed two antibodies, BCD166-01-01 and BCD166-01-014. Furthermore, by analogy from candidate BCD166-01-01, an E345R mutation was introduced into candidate BCD166-01-014 (designated as BCD166-02-014), which increased the ADCC activity compared to the wild-type.

[0329] In the assay, the inventors used the Jurkat-NFAT-Luc-CD16 cell line, which was generated based on the Jurkat cell line and stably expressed CD16 on the surface and contained a gene encoding firefly luciferase under the control of the NFAT promoter; and the HEK-293-GITR cell line, which was generated based on the Hek-293 cell line and stably expressed GITR on the surface.

[0330] The assay was performed in a 96-well culture plate. The suspension in each well contained Jurkat-NFAT-Luc-CD16 effector cells and Hek-293-GITR target cells, in addition to the test antibody at the concentrations shown in the graph. All suspension components were prepared in nutrient cell culture medium. After adding all the components, the plate was incubated at 37°C and 5% CO2, and then the inventors measured the luminescence intensity in the wells using a luciferase assay kit and a plate reader.

[0331] The results shown in Figure 18 show a 40-fold increase in the EC50 value in the ADCC assay of test BCD166-02-01 compared to the EC50 value of the precursor BCD166-01-01 into which the E345R mutation was introduced, which is due to the antigen-dependent oligomerization of the anti-GITR antibody BCD166-02-01 in target cells. Also, a 5-fold increase in the EC50 value was reliably detected in the ADCC assay of test BCD166-02-14 compared to the EC50 value of the precursor BCD166-01-14 into which the E345R mutation was introduced. Clearly from the aggregated data, a significant positive effect of the E345R substitution in the Fc portion of the IgG1 antibody on different effector characteristics required for the production of highly active GITR agonists is shown.

[0332] Example 15 In Vitro Cellular Assay for Complement-Dependent Cytotoxicity (CDC) of Anti-GITR Antibodies In the assay, the inventors studied the antibodies BCD166-01-01, BCD166-01-014, BCD166-02-01 and the control anti-GITR-3215 antibody.

[0333] The assay was performed in a 96-well culture plate. The suspension in each well contained HEK-293-GITR cells, as well as the test antibody and human serum complement at the indicated concentrations. The plates containing the described suspension were incubated at 37 °C for 4 hours in 5% CO2. Further, alamar blue solution was added to each well and then the plates were incubated at 37 °C, 5% CO2. Further, the fluorescence intensity in the wells was measured using a plate reader (excitation wavelength of 544 nm, emission wavelength of 590 nm).

[0334] The results shown in Figure 19 indicate less than 10% CDC activity against a concentration of 1 μg / ml, which is a marginal concentration for inferring the therapeutic dose expected for human use with respect to the slight CDC activity, i.e., antibodies BCD166-01-01, BCD166-02-01 and the control anti-GITR-3215 antibody. In the case of candidate BCD166-01-14, the CDC activity at a concentration of 1 μg / ml is about 30% lysis, thus showing a clear effect, although not so much in vivo in some cases. It is clearly shown from the aggregated data that there is a non-significant effect of the E345R substitution in the Fc portion of the anti-GITR IgG1 antibody candidates.

[0335] Example 16 In Vitro Cytotoxicity Assay of Anti-GITR Candidates The assay was performed to evaluate the negative or positive effect of the test anti-GITR candidates on the change in the number of different populations of responder cells. In the assay, the inventors studied antibodies BCD166-01-01, BCD166-01-014, BCD166-02-01 and the control anti-CD20 antibody (rituximab).

[0336] PBMCs were isolated from whole blood from healthy donors by Ficoll density gradient centrifugation. The assay was performed in 96-well culture plates. The suspension in each well contained PBMCs and antibodies at concentrations of 25, 1 and 0 μg / ml as shown in the graph. After mixing the PBMCs and the antibodies, the plates were incubated at 37 °C for 16 h in 5% CO2. Subsequently, the ratios of the CD56+, CD19+, CD3+, CD4+ and CD8+ subpopulations of PBMCs in the suspension were measured by direct staining of the suspension with fluorescently labeled antibodies against the corresponding CDs and subsequent cell analysis using a flow cytometer. For CD56+, CD19+, CD3+ cells, the graph shows their ratios to all cells in the test suspension, whereas for CD4+, CD8+ the graph shows their ratios to CD3+ cells.

[0337] The results shown in FIGS. 20-24 indicate a not large negative effect of the test anti-GITR candidates, i.e., a reduction of 10% or less in the number of responder cells compared to the negative control. In the case of the NK cell population, the inventors observed an increase of about 30-70% in cell number depending on the dose. However, although the control anti-CD20 antibody also showed an increase in the percentage of NK cells, it also showed a substantial and almost complete depletion of the CD20+ B cell population. Therefore, all candidate problems do not exhibit non-specific in vitro cytotoxicity against human blood cells.

[0338] Example 17 In vitro cell assay regarding antibody-dependent depletion of human nTreg cell population GITR + ADCC-dependent depletion of the nTreg cell population is expected to be one of the major expected mechanisms for the therapeutic anti-tumorigenic action of anti-GITR antibodies. In the assay, the inventors studied the antibodies BCD166-01-01, BCD166-01-014, BCD166-02-01 and the control anti-CTLA4 antibody (ipilimumab).

[0339] Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood from healthy donors by Ficoll density gradient centrifugation. PBMCs were enriched for nTregs using the "CD4+CD25+ Regulatory T Cell Isolation Kit, human" (Miltenyi Biotec, Germany). The obtained cells were activated using magnetic beads coated with anti-CD3 and anti-CD28 antibodies.

[0340] NK cells were isolated from PBMCs using the "NK Cell Isolation Kit, human" (Miltenyi Biotec, Germany).

[0341] An antibody solution, an NK cell suspension, and an nTreg suspension were added to a culture plate. The plate was incubated at 37 °C and 5% CO2 for 16 hours. Samples of the cell suspension were stained using fluorescently labeled antibodies (Biolegend, USA) against CD3, CD4, CD25, and FoxP3. Samples of the stained cell suspension were analyzed by flow cytometry.

[0342] From the results of the analysis of antibody-dependent nTreg depletion under the action of the test antibodies shown in Figure 25 for donor 1 cell material and Figure 26 for donor 2, a significant 70 - 100% decrease in the cell population is shown, that is, depletion of the activity of the test anti-GITR candidates, especially in the case of donor 2 material. Furthermore, from the comparative analysis with the control anti-CTLA4 antibody ipilimumab, the anti-GITR antibodies, especially the final candidate BCD166-02-01, are shown to exhibit greater activity.

[0343] Example 18 In vitro cell assay for antibody-dependent depletion of human iTreg cell populations ADCC-dependent depletion of the GITR+iTreg cell population is expected to be one of the major expected mechanisms for the therapeutic anti-tumorigenic action of anti-GITR antibodies. In the assay, the inventors studied the antibodies BCD166-01-01, BCD166-01-014, and BCD166-02-01.

[0344] PBMCs were isolated from whole blood from healthy donors by Ficoll density gradient centrifugation. Monocytes were isolated from PBMCs as the population of cells that bind to culture plastic. To obtain dendritic cells, monocytes were incubated at 37 °C and 5% CO2 for 120 hours in the presence of 1000 units / ml of GM-CSF (Peprotech, USA) and 500 units / ml of IL-4 (Thermo Scientific, USA). 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 and 5% CO2 for 48 hours.

[0345] An antibody dilution solution, a dendritic cell suspension, and PBMC were added to a culture plate. The plate was incubated at 37 °C and 5% CO2 for 120 hours. Samples of the cell suspension were stained using fluorescently labeled antibodies (Biolegend, USA) against CD3, CD4, CD25, and FoxP3. Samples of the stained cell suspension were analyzed by flow cytometry. The anti-GITR antibodies BCD166-01-01, BCD166-01-14, and BCD166-02-01 induce iTreg depletion in vitro.

[0346] The results of the assay for antibody-dependent iTreg depletion under the action of the test antibodies shown in Figure 27 for donor 1 / donor 2 cell materials show a marked 2- to 5-fold depletion activity of the test anti-GITR candidates, especially for the donor 1 material. The final candidate, BCD166-02-01, shows sufficient iTreg depletion activity.

[0347] Example 19 In vitro cell assay regarding the secretion of pro-inflammatory cytokines IL-2 and IFN-γ under the action of anti-GITR antibodies in a human cell population PBMC were isolated from whole blood from healthy donors by Ficoll density gradient centrifugation.

[0348] Anti-CD3 and anti-GITR antibodies were immobilized in the wells of a 96-well plate. To achieve this, 100 μl / well of a DPBS solution containing the anti-GITR antibody BCD166-02-01 at the concentrations shown in the graph and the optimal lower limit concentration of the anti-CD3 antibody was added to the corresponding wells of the plate, and the plate was incubated at room temperature for 16 hours.

[0349] The assay was performed using a 96-well plate pre-coated with anti-CD3 and anti-GITR antibodies in the wells. The suspension in each well contained 40,000 PBMCs and the optimal lower limit concentration of anti-CD28 antibody. All suspension components were prepared in RPMI-1640 medium containing 10% FBS. After adding the cell suspension, the plate was incubated at 37 °C, 5% CO2 for 6 days. On the 4th day of incubation, an aliquot of the culture medium was collected from the wells. Subsequently, the concentrations of IL-2 and IFN-γ were measured using ELISA in the culture medium on the 4th and 6th days of incubation.

[0350] Analysis of the effect of anti-GITR antibody on the secretion levels of the pro-inflammatory cytokines IL-2 and IFN-γ, which contribute to the anti-cancer effect, showed a significant increase in the concentrations of these substances in the culture medium (results are shown in Figure 28). Therefore, candidate BCD166-02-01 exhibits high activity in cytokine secretion activation, which may indicate a significant anti-tumorigenic effect.

[0351] Example 20 Binding analysis of the interaction of antibody BCD166-02-01 with human FcRn, FcgRIIIa158V, FcgRIIIa158F, FcgRIIa131H, FcgRIIa131R, FcgRIIb and FcgRIa receptors The binding affinity constants of the antibody BCD166-02-01 with human FcRn, FcgRIIIa158V, FcgRIIIa158F, FcgRIIa131H, FcgRIIa131R, FcgRIIb and FcgRIa were determined using an OctetRed 96 (ForteBio). Biotinylated receptors were immobilized on the surface of streptavidin sensors (SA). The inventors performed and analyzed the association and dissociation of the receptor and the antibody in the use buffer at 30 °C (PBS containing 0.1% Tween-20 and 0.1% BSA: for the FcRn receptor, the inventors used a buffer at pH 6, and for the other receptors, the inventors used a buffer at pH 7.4). The obtained sensograms were analyzed according to a 1:1 or 2:1 model, and the affinity constants were calculated using the user guide of Octet data analysis software 8.0, copyright 2011 (C).

[0352] Figure 29 shows the results of the affinity assay for the interaction of BCD166-02-01 with human FcRn, FcgRIIIa158V, FcgRIIIa158F, FcgRIIa131H, FcgRIIa131R, FcgRIIb and FcgRIa. These demonstrate multiple-fold values compared to literature data and data repeatedly measured in-house by the inventors for IgG1 isotype antibodies, and thus show a significant but not qualitative effect of the E345R mutation in the Fc fragment on the reaction rate of monomeric antibodies adsorbed onto these receptors from solution.

[0353] Example 21 Determination of colloidal and thermal stability by protein aggregation point using dynamic light scattering To determine the aggregation temperature of the sample under study by dynamic light scattering, a DynaPro® Plate Reader II (Wyatt Technology) was used to heat stepwise from 40 to 85 °C to obtain the dependence of the particle size in the medium on temperature. The results are shown in Table 7.

[0354] [Table 8]

[0355] It can be concluded that the molecule BCD166-02-01 has high thermal-colloidal stability (the aggregation points in 20 mM acetate, pH = 5.0 and 20 mM His, pH = 5.5 buffer solutions are >65°C).

[0356] Similar data were obtained for BCD166-01-014. Example 22 Determination of Thermal Stability under Thermal Stress at 50°C The test samples were placed in a thermostat-regulated air bath and adjusted to 50°C with a thermostat for 72 hours. After heating, the intact and stressed samples were analyzed by size exclusion HPLC (SEC HPLC) equipped with a UV detector and further by capillary isoelectric focusing. Chromatography was performed on an Agilent 1100 HPLC system on a TSK-Gel G3000SWXL column from Tosoh Corporation, and detection was performed at a wavelength of 220 nm. Charge heterogeneity was determined by capillary isoelectric focusing technology using a Labchip GX II, Caliper. The preparation of the solutions and chips used was carried out according to standard methods using the HT Protein Charge Variant Labeling Kit and Protein Charge Variant Buffer Kit from PerkinElmer.

[0357] The data obtained regarding the stability of BCD-166 (for BCD166-02-01 and BCD166-01-014) under incubation at 50°C are shown in Table 8; Figures 30 (for BCD166-02-01) and 31 (for BCD166-01-014) show the combined chromatograms under incubation at 50°C for 72 hours.

[0358] Overall conclusion: The samples have high colloidal and thermal stability.

[0359]

Table 9

[0360] Example 23 Evaluation of the antitumor activity of the BCD-166 product using a subcutaneous xenograft model The antitumor activity of the BCD-166 product was evaluated using a subcutaneous tumor xenograft model. To achieve this purpose, 3×10 6 cells of the A2058 cell line mixed with Matrigel® were transplanted subcutaneously into humanized huNOG-EXL mice. On the 7th day after cell implantation, the animals were grouped as shown in Table 9.

[0361]

Table 10

[0362] On days 1, 5, 8, and 12 after the animals were grouped, the product was administered intraperitoneally at a dose of 20 mg / kg. The negative control group was administered histidine buffer. The body weight of the mice and the length dimensions of the tumors were measured throughout the experiment. The tumor volume was calculated using the formula V = L×W×H×π / 6. The efficacy of the test product was evaluated by the tumor growth inhibition index (TGI) calculated taking into account the average tumor volume in the negative control group (V c ) and the group of the product (Vt):

[0363]

Number

[0364] From the experiment, the high antitumor activity of the BCD-166-01-014 and BCD-166-02-01 products was elucidated, whereas the BCD-166-01-01 product showed no significant antitumor activity at all. The results are shown in Figures 32 and 33. Non-limitingly, the present invention includes the following aspects. [Aspect 1] An isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to GITR, (a)(i) The following group: NYGMH (SEQ ID NO: 1) or YYWMY (SEQ ID NO: 12) and contains a CDR1 having an amino acid sequence selected therefrom; (ii) The following group: VIWFDGSNKFYTDSVKG (SEQ ID NO: 2) or AISWNGGRTYYAESMKG (SEQ ID NO: 13) and contains a CDR2 having an amino acid sequence selected therefrom; (iii) The following group: ELGGYYYDSSGFRPYYYGMDV (SEQ ID NO: 3) or NRYYSDPNYGMNL (SEQ ID NO: 14) and contains a CDR3 having an amino acid sequence selected therefrom and a heavy chain variable domain, and (b)(i) The following group: RASQSIGSWLA (SEQ ID NO: 7) or TGTSTDIGTYKYIS (SEQ ID NO: 17) and contains a CDR1 having an amino acid sequence selected therefrom; (ii) The following group: AASTLQR (SEQ ID NO: 8) or GVSHRPS (SEQ ID NO: 18) and contains a CDR2 having an amino acid sequence selected therefrom; (iii) The following group: QQSHSHPLT (SEQ ID NO: 9) or SSYTSSGTVV (SEQ ID NO: 19) and contains a CDR3 having an amino acid sequence selected therefrom and a light chain variable domain and a monoclonal antibody or antigen-binding fragment thereof containing the same. [Aspect 2] The heavy chain variable domain is (i) The amino acid sequences of CDR1, CDR2, and CDR3 represented by the sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, (ii) The CDR1, CDR2, and CDR3 amino acid sequences represented by the sequences of SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively The monoclonal antibody or antigen-binding fragment thereof according to embodiment 1, comprising [Embodiment 3] wherein the light chain variable domain (i) CDR1, CDR2 and CDR3 comprising amino acid sequences represented by the sequences of SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively; (ii) CDR1, CDR2 and CDR3 comprising amino acid sequences represented by the sequences of SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19, respectively comprising the monoclonal antibody or antigen-binding fragment thereof according to embodiment 1. [Embodiment 4] - The heavy chain variable domain comprises CDR1, CDR2 and CDR3 comprising amino acid sequences represented by the sequences of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively; - The light chain variable domain comprises CDR1, CDR2 and CDR3 comprising amino acid sequences represented by the sequences of SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively, the monoclonal antibody or antigen-binding fragment thereof according to embodiment 1. [Embodiment 5] - The heavy chain variable domain comprises CDR1, CDR2 and CDR3 comprising amino acid sequences represented by the sequences of SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, respectively; - The light chain variable domain comprises CDR1, CDR2 and CDR3 comprising amino acid sequences represented by the sequences of SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19, respectively, the monoclonal antibody or antigen-binding fragment thereof according to embodiment 1. [Embodiment 6] The heavy chain variable domain (i) an amino acid sequence having at least 90% homology to the amino acid sequence of SEQ ID NO: 4; or (ii) an amino acid sequence having at least 90% homology to the amino acid sequence of SEQ ID NO: 15 comprising the monoclonal antibody or antigen-binding fragment thereof according to embodiment 1. [Embodiment 7] (i) The heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 4, or (ii) The heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 15, The monoclonal antibody or antigen-binding fragment thereof according to embodiment 1. [Embodiment 8] The light chain variable domain is (i) An amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 10; or (ii) An amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 20 comprising The monoclonal antibody or antigen-binding fragment thereof according to embodiment 1. [Embodiment 9] (i) The light chain variable domain comprises the amino acid sequence of SEQ ID NO: 10; or (ii) The light chain variable domain comprises the amino acid sequence of SEQ ID NO: 20, The monoclonal antibody or antigen-binding fragment thereof according to embodiment 1. [Embodiment 10] (i) - The heavy chain variable domain comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 4; - The light chain variable domain comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 10; or (ii) - The heavy chain variable domain comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 15; - The light chain variable domain comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 20, The monoclonal antibody or antigen-binding fragment thereof according to embodiment 1. [Embodiment 11] - The heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 4; - The light chain variable domain comprises the amino acid sequence of SEQ ID NO: 10, The monoclonal antibody or antigen-binding fragment thereof according to embodiment 10. [Embodiment 12] - The heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 15; - The light chain variable domain comprises the amino acid sequence of SEQ ID NO: 20, The monoclonal antibody or antigen-binding fragment thereof according to embodiment 10. [Embodiment 13] The monoclonal antibody according to embodiment 1, wherein the antibody that specifically binds to GITR is a full-length IgG antibody. [Embodiment 14] The monoclonal antibody according to embodiment 13, wherein the full-length IgG antibody relates to human IgG1, IgG2, IgG3, IgG4 isotypes. [Embodiment 15] The monoclonal antibody according to embodiment 14, wherein the full-length IgG antibody relates to human IgG1 isotype. [Embodiment 16] The monoclonal antibody according to embodiment 1, wherein the antibody that specifically binds to GITR comprises an E345R mutation in the Fc fragment so as to have agonist properties and increase antibody-dependent cell-mediated cytotoxicity (ADCC), but not increase complement-dependent cytotoxicity (CDC). [Embodiment 17] (i) An amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 5; or (ii) An amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 6 The monoclonal antibody according to embodiment 1, comprising a heavy chain comprising the same. [Embodiment 18] (i) A heavy chain comprising the amino acid sequence of SEQ ID NO: 5; or (ii) A heavy chain comprising the amino acid sequence of SEQ ID NO: 6 The monoclonal antibody according to embodiment 1, comprising the same. [Embodiment 19] The monoclonal antibody according to embodiment 1, comprising a light chain comprising an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 11. [Embodiment 20] The monoclonal antibody according to embodiment 1, comprising a light chain comprising the amino acid sequence of SEQ ID NO: 11. [Embodiment 21] (i) - A heavy chain comprising an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 5; - A light chain comprising an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 11; or (ii) - A heavy chain comprising an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 6; - A light chain comprising an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 11 The monoclonal antibody according to embodiment 1, comprising the same. [Embodiment 22] - A heavy chain comprising the amino acid sequence of SEQ ID NO: 5; - A light chain comprising the amino acid sequence of SEQ ID NO: 11 The monoclonal antibody according to embodiment 1, comprising the same. [Embodiment 23] - 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 The monoclonal antibody according to embodiment 1, comprising the same. [Embodiment 24] The monoclonal antibody according to embodiment 1, comprising a heavy chain comprising an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 16. [Embodiment 25] The monoclonal antibody according to embodiment 1, comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 16. [Embodiment 26] The monoclonal antibody according to embodiment 1, comprising a light chain comprising an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 21. [Embodiment 27] The monoclonal antibody according to embodiment 1, comprising a light chain comprising the amino acid sequence of SEQ ID NO: 21. [Embodiment 28] - A heavy chain comprising an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 16; - A light chain comprising an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 21 The monoclonal antibody according to embodiment 1, comprising the same. [Embodiment 29] - A heavy chain comprising the amino acid sequence of SEQ ID NO: 16; - A light chain comprising the amino acid sequence of SEQ ID NO: 21 The monoclonal antibody according to embodiment 1, comprising the same. [Embodiment 30] An isolated nucleic acid encoding an antibody or an antigen-binding fragment thereof according to any one of embodiments 1 to 29. [Embodiment 31] The nucleic acid according to embodiment 30, which is DNA. [Embodiment 32] An expression vector comprising the nucleic acid according to embodiment 30 or 31. [Embodiment 33] A method for obtaining a host cell for obtaining an antibody or an antigen-binding fragment thereof according to any one of embodiments 1 to 29, the method comprising transforming a cell with the vector according to embodiment 32. [Embodiment 34] A host cell for obtaining an antibody or an antigen-binding fragment thereof according to any one of embodiments 1 to 29, comprising the nucleic acid according to embodiment 30 or 31. [Embodiment 35] A method for obtaining an antibody or an antigen-binding fragment thereof according to any one of embodiments 1 to 29, the method comprising culturing the host cell according to embodiment 34 in a culture medium under conditions sufficient to obtain the antibody, and optionally subsequently isolating and purifying the obtained antibody. [Embodiment 36] A pharmaceutical composition for treating a disease or disorder mediated by GITR, comprising a therapeutically effective amount of an antibody or an antigen-binding fragment thereof according to any one of embodiments 1 to 29, in combination with one or more pharmaceutically acceptable excipients. [Embodiment 37] The pharmaceutical composition according to embodiment 36, which is intended to treat a disease or disorder mediated by GITR selected from the following group: cervical cancer, head and neck cancer, gastric cancer, breast cancer, renal cell cancer, CRC (colorectal cancer), (OC) ovarian cancer, NSCLC (non-small cell lung cancer). [Embodiment 38] A pharmaceutical composition for treating a disease or disorder mediated by GITR, comprising an antibody or an antigen-binding fragment thereof according to any one of aspects 1 to 29 in a therapeutically effective amount and an anti-tumor compound having at least one therapeutically active amount. [Aspect 39] The pharmaceutical composition according to aspect 38, which is intended to treat a disease or disorder mediated by GITR selected from the following group: cervical cancer, head and neck cancer, gastric cancer, breast cancer, renal cell cancer, CRC (colorectal cancer), (OC) ovarian cancer, NSCLC (non-small cell lung cancer). [Aspect 40] The pharmaceutical composition according to aspect 38, wherein the anti-tumor compound having therapeutic activity is selected from chemotherapeutic agents, antibodies or anti-hormonal agents. [Aspect 41] The anti-tumor compound having therapeutic activity is (i) an antibody selected from the following 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 activators of natural or adaptive immunity, the pharmaceutical composition according to aspect 40. [Aspect 42] The pharmaceutical composition according to aspect 41, wherein the antibody according to aspect 29 and the anti-tumor compound having at least one therapeutic activity are administered sequentially. [Aspect 43] The pharmaceutical composition according to aspect 41, wherein the antibody according to any one of aspects 1 to 29 and the anti-tumor compound having at least one therapeutic activity are administered simultaneously. [Aspect 44] A method for inhibiting the biological activity of GITR in a subject in need of inhibition of the biological activity of GITR, the method comprising administering an effective amount of an antibody or an antigen-binding fragment thereof according to any one of aspects 1 to 29. [Aspect 45] A method for treating a disease or disorder mediated by GITR, the method comprising administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof according to any one of aspects 1 to 29 or a pharmaceutical composition according to aspect 36 or 38 to a subject in need of such treatment. [Aspect 46] A method for treating a disease or disorder according to aspect 45, wherein the disease or disorder is selected from the following group: cervical cancer, head and neck cancer, gastric cancer, breast cancer, renal cell cancer, CRC (colorectal cancer), (OC) ovarian cancer, NSCLC (non-small cell lung cancer). [Aspect 47] Use of an antibody or antigen-binding fragment thereof according to any one of aspects 1 to 29 or a pharmaceutical composition according to aspect 36 or 38 for the treatment of a subject in need of such treatment of a disease or disorder mediated by GITR. [Aspect 48] Use according to aspect 47, wherein the disease or disorder is selected from the group comprising cervical cancer, head and neck cancer, gastric cancer, breast cancer, renal cell cancer, CRC (colorectal cancer), (OC) ovarian cancer, NSCLC (non-small cell lung cancer).

Claims

**Claim 1** An isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to GITR, (a)(i) CDR1 comprising the amino acid sequence of NYGMH (SEQ ID NO: 1); (ii) CDR2 comprising the amino acid sequence of VIWFDGSNKFYTDSVKG (SEQ ID NO: 2); and, (iii) CDR3 comprising the amino acid sequence of ELGGYYYYDSSGFRPYYYGMDV (SEQ ID NO: 3) comprising a heavy chain variable domain, and, (b)(i) CDR1 comprising the amino acid sequence of RASQSIGSWLA (SEQ ID NO: 7); (ii) CDR2 comprising the amino acid sequence of AASTLQAR (SEQ ID NO: 8); and, (iii) CDR3 comprising the amino acid sequence of QQSHSHPLT (SEQ ID NO: 9) comprising a light chain variable domain, a monoclonal antibody or antigen-binding fragment thereof comprising the same. **Claim 2** An isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to GITR, (a)(i) CDR1 comprising the amino acid sequence of YYWMY (SEQ ID NO: 12); (ii) CDR2 comprising the amino acid sequence of AISWNGGRTYYAESMKG (SEQ ID NO: 13); and, (iii) CDR3 comprising the amino acid sequence of NRYYSDPNYGMNL (SEQ ID NO: 14) comprising a heavy chain variable domain, and, (b)(i) CDR1 comprising the amino acid sequence of TGTSSTDIGTYKYIS (SEQ ID NO: 17); (ii) CDR2 comprising the amino acid sequence of GVSHRPS (SEQ ID NO: 18); and, (iii) CDR3 comprising the amino acid sequence of SSYTSSGTVV (SEQ ID NO: 19) a light chain variable domain comprising A monoclonal antibody or an antigen-binding fragment thereof comprising:

3. The monoclonal antibody or antigen-binding fragment thereof of claim 1 , wherein the heavy chain variable domain comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:

4.

4. The monoclonal antibody or antigen-binding fragment thereof of claim 2, wherein the heavy chain variable domain comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:

15.

5. The monoclonal antibody or antigen-binding fragment thereof of claim 1 , wherein the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:

4.

6. The monoclonal antibody or antigen-binding fragment thereof of claim 2, wherein the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:

15.

7. The monoclonal antibody or antigen-binding fragment thereof of claim 1, wherein the light chain variable domain comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:

10.

8. The monoclonal antibody or antigen-binding fragment thereof of claim 2, wherein the light chain variable domain comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:

20.

9. The monoclonal antibody or antigen-binding fragment thereof of claim 1 , wherein the light chain variable domain comprises the amino acid sequence of SEQ ID NO:

10.

10. The monoclonal antibody or antigen-binding fragment thereof of claim 2, wherein the light chain variable domain comprises the amino acid sequence of SEQ ID NO:

20.

11. - The heavy chain variable domain comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 4; - The light chain variable domain comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 10, The monoclonal antibody or antigen-binding fragment thereof according to claim 1.

12. - The heavy chain variable domain comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 15; - The light chain variable domain comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 20, The monoclonal antibody or antigen-binding fragment thereof according to claim 2.

13. - The heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 4; - The light chain variable domain comprises the amino acid sequence of SEQ ID NO: 10, The monoclonal antibody or antigen-binding fragment thereof according to claim 11.

14. - The heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 15; - The light chain variable domain comprises the amino acid sequence of SEQ ID NO: 20, The monoclonal antibody or antigen-binding fragment thereof according to claim 12.

15. The monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the isolated monoclonal antibody that specifically binds to GITR is a full-length IgG antibody.

16. The monoclonal antibody or antigen-binding fragment thereof according to claim 15, wherein the full-length IgG antibody is a human IgG1, IgG2, IgG3, or IgG4 isotype.

17. The monoclonal antibody or antigen-binding fragment thereof according to claim 16, wherein the full-length IgG antibody is a human IgG1 isotype.

18. The isolated monoclonal antibody that specifically binds to GITR has agonist properties and increases antibody-dependent cell-mediated cytotoxicity (ADCC), but does not increase complement-dependent cytotoxicity (CDC), and contains the E345R mutation in the EU numbering in the Fc fragment. The monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2.

19. (i) an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 5; or (ii) an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 6 The monoclonal antibody or antigen-binding fragment thereof according to claim 1, comprising a heavy chain containing the same.

20. (i) a heavy chain containing the amino acid sequence of SEQ ID NO: 5; or (ii) a heavy chain containing the amino acid sequence of SEQ ID NO: 6 The monoclonal antibody or antigen-binding fragment thereof according to claim 1, comprising the same.

21. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, comprising a light chain containing an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO:

11.

22. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, comprising a light chain containing the amino acid sequence of SEQ ID NO:

11.

23. (i) - a heavy chain containing an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 5; and - a light chain containing an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 11; or (ii) - a heavy chain containing an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 6; and - a light chain containing an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 11 The monoclonal antibody or antigen-binding fragment thereof according to claim 1, comprising the same.

24. - A heavy chain comprising the amino acid sequence of SEQ ID NO: 5; and - A light chain comprising the amino acid sequence of SEQ ID NO: 11 The monoclonal antibody according to claim 1 or an antigen-binding fragment thereof, comprising:

25. - A heavy chain comprising the amino acid sequence of SEQ ID NO: 6; and - A light chain comprising the amino acid sequence of SEQ ID NO: 11 The monoclonal antibody according to claim 1 or an antigen-binding fragment thereof, comprising:

26. The monoclonal antibody according to claim 2 or an antigen-binding fragment thereof, comprising a heavy chain comprising an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO:

16.

27. The monoclonal antibody according to claim 2 or an antigen-binding fragment thereof, comprising a heavy chain comprising the amino acid sequence of SEQ ID NO:

16.

28. The monoclonal antibody according to claim 2 or an antigen-binding fragment thereof, comprising a light chain comprising an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO:

21.

29. The monoclonal antibody according to claim 2 or an antigen-binding fragment thereof, comprising a light chain comprising the amino acid sequence of SEQ ID NO:

21.

30. - A heavy chain comprising an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 16; and - A light chain comprising an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 21 The monoclonal antibody according to claim 2 or an antigen-binding fragment thereof, comprising:

31. - A heavy chain comprising the amino acid sequence of SEQ ID NO: 16; and - A light chain comprising the amino acid sequence of SEQ ID NO: 21 The monoclonal antibody according to claim 2 or an antigen-binding fragment thereof, comprising:

32. An isolated nucleic acid encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 31.

33. The nucleic acid according to claim 32, which is DNA.

34. An expression vector comprising the nucleic acid according to claim 32 or 33.

35. A method for obtaining a host cell for obtaining the antibody or antigen-binding fragment thereof according to any one of claims 1 to 31, comprising transforming cells with the vector according to claim 34 in vitro.

36. A host cell for obtaining the antibody or antigen-binding fragment thereof according to any one of claims 1 to 31, comprising the nucleic acid according to claim 32 or 33.

37. A method for obtaining the antibody or antigen-binding fragment thereof according to any one of claims 1 to 31, comprising culturing the host cell according to claim 36 in a culture medium.

38. The method according to claim 37, further comprising subsequently isolating and purifying the obtained antibody.

39. A pharmaceutical composition comprising a therapeutically effective amount of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 31 in combination with one or more pharmaceutically acceptable excipients.

40. A therapeutically effective amount of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 31, and a therapeutically effective amount of at least one antitumor compound having therapeutic activity, wherein the antitumor compound having therapeutic activity is selected from chemotherapeutic agents, antibodies or antihormonal agents The pharmaceutical composition according to claim 39.

41. The antitumor compound having therapeutic activity is (i) an antibody selected from the following group: an anti-PD1 antibody, an anti-PD-L1 antibody, an anti-CTLA4 antibody, an anti-4-1BB antibody, an anti-OX40 antibody, or a combination thereof, or (ii) selected from the group of activators of natural or adaptive immunity, The pharmaceutical composition according to claim 40.

42. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 31, and an antitumor compound having at least one therapeutic activity are administered sequentially, or, The antibody or antigen-binding fragment thereof according to any one of claims 1 to 31, and an antitumor compound having at least one therapeutic activity are administered simultaneously, The pharmaceutical composition according to claim 40.

43. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 31 or the pharmaceutical composition according to claim 39 or 40 for the manufacture of a drug therapy agent.

Images