EA044786B1 - MONOCLONAL ANTIBODY THAT SPECIFICALLY BINDS TO GITR - Google Patents

Description

Field of technology

The invention relates to the field of biotechnology, namely to antibodies or their antigen-binding fragments, as well as 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/membrane protein, receptor from the factor receptor superfamily tumor necrosis). The invention also relates to a nucleic acid encoding the antibody or an antigen binding fragment thereof, an expression vector, a method for producing the antibody, and the use of the antibody for the treatment of diseases or disorders associated with GITR.

State of the art

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

GITR is a type I transmembrane protein that consists of 216 amino acids and has a molecular weight of 26 kDa. The N-terminal extracellular domain contains 3 TNFR-Cys repeats and an N-glycosylation site. The 3 cysteine-rich domains and cytoplasmic tail of GITR share significant homology with 4-1BB, OX40, and CD27 (Nocentini, et al. (1997) Proc. Natl. Acad. Sci. 94:6216-6221).

Human GITR is expressed at low levels by immunoreactive T cells, with CD4+ cells showing higher expression compared to CD8+ cells. GITR expression is significantly increased for 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 increased upon activation of these cells (Nocentini and Riccardi (2005) E. J. Immunol. 35:1016)

However, GITR expression is not limited exclusively to T cells. Some studies have also indicated that GITR is expressed by NK cells, macrophages, B cells, dendritic cells, mast cells and monocytes (Nocentini and Riccardi (2005) E. J. Immunol. 35:1016-1022).

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

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

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

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

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

Binding of GITRL on APCs to GITR on immunoreactive T cells triggers GITR signaling, which co-stimulates immunoreactive T cells and inhibits the suppressive activity of regulatory T cells. GITR signaling serves as a coactivation signal for both CD4+ and CD8+ naïve T cells, thereby inducing or enhancing proliferation and effector function, particularly when T cell receptor (TCR) stimulation is close to optimal (Schaer, et al. (2012) Curr Opin Immunol 24:217224). More specifically, GITR may have several effects on effector T cells and regulatory T cells, including: co-stimulating and activating effector T cells so that they become more resistant to inhibition, inhibiting regulatory T cells, reducing the sensitivity of effector T cells to suppression by regulatory T cells and partial removal of regulatory T cells from the circulation (Nocentini, et al. (2007) Eur. J. Immunol. 37:1165-1169).

The main functions of GITR, and therefore the main effects of anti-GITR antibodies, are

- 1 044786 enhancing the proliferation and functioning of effector T cells, as well as an inhibitory effect on the suppressive effect of Tregs. Teff cells are generated initially with an inability to resist the inhibitory tumor microenvironment and Treg cell suppression. Stimulation of GITR during repeated stages of priming and expansion, through an anti-GITR agonist, a soluble GITR ligand, or a DC vaccine, modulates Teff and Treg tumor responses in favor of the former, leading to tumor regression. Thus, anti-GITR confers Teff resistance to Treg suppression.

Together, the above functions, particularly co-stimulation of immunoreactive T cells and neutralization of suppressive activity of regulatory T cells, mean that activation of GITR leads to an enhanced immune response. Such activation could potentially lead to restoration of immune responses to infections and tumors.

Accordingly, molecules capable of activating GITR would be valuable as immunostimulating agents in conditions where it is necessary to induce an enhanced immune response.

The antibody must have GITR receptor agonist properties, with effector, cytotoxic properties against Treg lymphocytes.

Various antibodies to GITR are known from the prior art (for example, WO 2015187835, WO 2015031667, WO 2017068186, WO 2017096189, WO 2017214548).

There are currently 23 GITR agonists (antibodies/recombinant GITRLs) in preclinical and clinical studies. Only 2 antibodies are in phase 2 of the clinical trial (TRX518, INCAGN1876), in phase 1 of the clinical trial - AMG228, MEDI1873, MK-4166. Very little clinical data is presented.

However, at the moment, there is no antibody in the world that specifically binds to GITR approved for therapeutic use.

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

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

Brief description of the invention

In one aspect, the present invention provides an isolated monoclonal antibody or antigen binding fragment thereof that specifically binds GITR, comprising:

(a) a heavy chain variable domain containing:

(i) CDR1 with an amino acid sequence selected from the group: NYGMH (SEQ ID NO: 1) or YYWMY (SEQ ID NO: 12);

(ii) CDR2 with an amino acid sequence selected from the group: VIWFDGSNKFYTDSVKG (SEQ ID NO: 2) or AISWNGGRTYYAESMKG (SEQ ID NO: 13);

(iii) a CDR3 with an amino acid sequence selected from the group: ELGGYYYDSSGFRPYYYGMDV (SEQ ID NO: 3) or NRYYSDPNYGMNL (SEQ ID NO: 14), and (b) a light chain variable domain comprising:

(i) CDR1 with an amino acid sequence selected from the group: RASQSIGSWLA (SEQ ID NO: 7) or TGTSTDIGTYKYIS (SEQ ID NO: 17);

(ii) CDR2 with an amino acid sequence selected from the group: AASTLQR (SEQ ID NO: 8) or GVSHRPS (SEQ ID NO: 18);

(iii) CDR3 with an amino acid sequence selected from the group: QQSHSHPLT (SEQ ID NO: 9) or SSYTSSGTVV (SEQ ID NO: 19).

In some embodiments, the monoclonal antibody or antigen binding fragment thereof includes a heavy chain variable domain that contains CDRs 1, 2, and 3 with the amino acid sequences represented by SEQ ID NO: 1, 2, and 3, respectively.

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

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

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

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

- 2044786 heavy chain variable domain, which contains CDRs 1, 2 and 3 with the amino acid sequences represented by the sequences SEQ ID NO: 1, 2 and 3, respectively;

a light chain variable domain which contains CDRs 1, 2 and 3 with the amino acid sequences represented by SEQ ID NOs: 7, 8 and 9, respectively.

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

a heavy chain variable domain which contains CDRs 1, 2 and 3 with the amino acid sequences represented by SEQ ID NOs: 12, 13 and 14, respectively;

a light chain variable domain which contains CDRs 1, 2 and 3 with the amino acid sequences represented by SEQ ID NOs: 17, 18 and 19, respectively.

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

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

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

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

In some embodiments, the monoclonal antibody or antigen binding fragment thereof includes a light chain variable domain that contains an amino acid sequence that is at least 90% homologous to the amino acid sequence of SEQ ID NO: 10.

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

In some embodiments, the monoclonal antibody or antigen binding fragment thereof includes a light chain variable domain that contains an amino acid sequence that is at least 90% homologous to the amino acid sequence of SEQ ID NO: 20.

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

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

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

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

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

a heavy chain variable domain which contains the amino acid sequence of SEQ ID NO: 4;

a light chain variable domain which contains the amino acid sequence of SEQ ID NO: 10.

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

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

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

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

a heavy chain variable domain which contains the amino acid sequence of SEQ ID NO: 15;

a light chain variable domain which contains the amino acid sequence of SEQ ID NO: 20.

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

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

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

In some embodiments, a monoclonal antibody that specifically binds to GITR includes an E345R mutation in the Fc fragment to enhance agonistic properties, antibody-dependent cellular cytotoxicity (ADCC), but not complement-dependent cytotoxicity (CDC).

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

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

In some embodiments, the monoclonal antibody includes a heavy chain containing an amino acid sequence that is at least 90% homologous to the sequence of SEQ ID NO: 6.

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

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

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

In some embodiments, the monoclonal antibody includes:

a heavy chain containing an amino acid sequence at least 90% homologous to the sequence of SEQ ID NO: 5;

a light chain containing an amino acid sequence that is at least 90% homologous to the sequence of SEQ ID NO: 11.

In some embodiments, the monoclonal antibody includes:

a heavy chain containing the amino acid sequence of SEQ ID NO: 5;

light chain containing the amino acid sequence SEQ ID NO: 11.

In some embodiments, the monoclonal antibody includes:

a heavy chain containing an amino acid sequence at least 90% homologous to the sequence of SEQ ID NO: 6;

a light chain containing an amino acid sequence that is at least 90% homologous to the sequence of SEQ ID NO: 11.

In some embodiments, the monoclonal antibody includes:

a heavy chain containing the amino acid sequence of SEQ ID NO: 6;

light chain containing the amino acid sequence SEQ ID NO: 11.

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

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

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

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

In some embodiments, the monoclonal antibody includes:

a heavy chain containing an amino acid sequence at least 90% homologous to the sequence of SEQ ID NO: 16;

a light chain containing an amino acid sequence that is at least 90% homologous to the sequence of SEQ ID NO: 21.

In some embodiments, the monoclonal antibody includes:

a heavy chain containing the amino acid sequence of SEQ ID NO: 16;

light chain containing the amino acid sequence SEQ ID NO: 21.

In one aspect, the present invention provides an isolated nucleic acid that encodes any of the above antibodies or an antigen binding fragment thereof.

In some embodiments, the nucleic acid is DNA.

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

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

In one aspect, the present invention relates to a host cell for producing the above antibody or an antigen binding fragment thereof containing the above nucleic acid.

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In one aspect, the present invention relates to a method for producing said antibody or an antigen binding fragment thereof, comprising culturing said host cell in a culture medium under conditions sufficient to produce said antibody, if necessary, and then isolating and purifying the resulting antibody.

In one aspect, the present invention provides a pharmaceutical composition for treating a disease or disorder mediated by GITR, comprising the above antibody or antigen binding fragment thereof in a therapeutically effective amount, in combination with one or more pharmaceutically acceptable excipients.

In some embodiments, the pharmaceutical composition is for treating a disease or disorder mediated by GITR selected from the group: cervical cancer, head and neck cancer, gastric cancer, breast cancer, renal cell cancer, colorectal cancer ), OC (ovarian cancer), NSCLC (non-small cell lung cancer).

In one aspect, the present invention provides a pharmaceutical composition for treating a disease or disorder mediated by GITR, comprising the above antibody or antigen binding fragment thereof in a therapeutically effective amount and at least one therapeutically active antitumor compound in a therapeutically effective amount.

In some embodiments, the pharmaceutical composition is for treating a disease or disorder mediated by GITR selected from the group of cervical cancer, head and neck cancer, gastric cancer, breast cancer, renal cell cancer, colorectal cancer. , OC (ovarian cancer), NSCLC (non-small cell lung cancer).

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

In some embodiments, the pharmaceutical composition includes a therapeutically active antitumor compound, an antibody selected from the group: anti-PD1 antibodies, anti-PD-L1 antibodies, anti-CTLA4 antibodies, anti-4-1BB antibodies, anti-OX40 antibodies, or combinations thereof.

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

In some embodiments, the pharmaceutical composition includes a therapeutically active antitumor compound that is selected from the group of innate or adaptive immune activators.

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

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

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

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

In some embodiments, the method of treatment includes a disease or disorder that is selected from the group: cervical cancer, head and neck cancer, gastric cancer, breast cancer, renal cell cancer, colorectal cancer, ovarian cancer ( ovarian cancer), NSCLC (non-small cell lung cancer).

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

In some embodiments, the use includes a disease or disorder that is selected from the group: cervical cancer, head and neck cancer, gastric cancer, breast cancer, renal cell cancer, colorectal cancer, ovarian cancer ovary), NSCLC (non-small cell lung cancer).

Brief description of drawings

Fig. 1. Plasmid for protein production pEE-GITR-TEV-Fc lama.

Fig. 2. Plasmid for protein production pEE-GITR-Fc.

Fig. 3. Map of plasmid pEE-humGITR-ligand-foldon-EPEA.

Fig. 4. SDS gel electrophoresis in 4-20% PAGE

1. Actemra 2.5 mg;

2. marker Fermentas unstained PW;

3. -;

4. GITR-TEV-Fc-lama + β-ME 10 ml;

5. Angiopoietin 2-H6F + β-ME 10 ml;

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6. -;

7. GITR-TEV-Fc-lama - β-ME 10 ml;

8. Angiopoietin 2-H6F - β-ME 10 ml;

Fig. 5. SDS gel electrophoresis

1. marker Fermentas unstained PW;

2. -;

3. hGITR ligand -EPEA medium before purification 5 ml;

4. hGITR ligand -EPEA medium after purification 5 ml;

5. hGITR ligand -EPEA.

Fig. 6. Scheme for the synthesis of a combinatorial naive human library.

Fig. 7. pH5 phagemid map for cloning Fab phage display libraries.

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

Fig. 9. Map of the expression vector pEE-BCD166-01-001-VH-HC, encoding the antibody heavy chain for transient antibody production in mammalian cells.

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

Fig. 11. SDS gel electrophoresis in 12% PAGE + β-ME

1. Actemra 5 ml;

2. marker Fermentas unstained PW;

3. -;

4. anti-GITR antibody lot#1161 pre-purification medium 5 ml;

5. anti-GITR antibody lot#1161 medium after purification 5 ml;

6. anti-GITR antibody lot#1161 5 ml;

7. anti-GITR antibody lot#1162 5 ml;

8. anti-GITR antibody lot#1163 5 ml;

9. anti-GITR antibody lot#1164 5 ml;

10. anti-GITR antibody lot#1165 5 ml4

11. anti-GITR antibody lot#1166 medium before purification 5 ml;

12. anti-GITR antibody lot#1166 medium after purification 5 ml;

13. anti-GITR antibody lot#1166 5 ml;

14. anti-GITR antibody lot#1167 5 ml.

Fig. 12. SDS gel electrophoresis in 12% PAGE + β-ME

1. marker Fermentas unstained PW;

2. anti-GITR antibody lot#1208 pre-purification medium 10 ml;

3. anti-GITR antibody lot#1208 medium after purification 10 ml;

4. anti-GITR antibody lot#1208 5 ml;

5. anti-GITR antibody lot#1209 5 ml;

6. anti-GITR antibody lot#1210 5 ml;

7. anti-GITR antibody lot#1211 5 ml;

8. anti-GITR antibody lot#1212 5 ml;

9. anti-GITR antibody lot#1213 5 ml;

10. anti-GITR antibody lot#1214 medium before purification 10 ml;

11. anti-GITR antibody lot#1214 medium after purification 10 ml;

12. anti-GITR antibody lot#1214 5 ml.

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

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

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

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

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

Fig. 18. Graph with the results of the EC50 value in the cell ADCC test of the studied BCD16602-01, BCD166-01-01, BCD166-01-014, BCD166-02-014.

Fig. 19. Graph with the results of the CDC level of the studied BCD166-01-01, BCD166-02-01, BCD16601-014.

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

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

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

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

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

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

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

Fig. 27. Analysis of antibody-dependent iTreg depletion under the influence of the studied anti-GITR candidates for cellular material of donor 1 and donor 2

Fig. 28. Results of an analysis of the effect of anti-GITR antibodies on the level of secretion of pro-inflammatory cytokines IL-2 and IFN-γ, which determine the anti-cancer effect.

Fig. 29. Data table of binding affinity constants for candidate BCD166-02-01 gamma receptors.

Fig. 30. Stability graph of BCD166-02-01 during 72-hour incubation at 50°C.

Fig. 31. Stability graph of BCD166-01-014 during 72-hour incubation at 50°C.

Fig. 32. Average values of tumor volumes in groups during the experiment.

Fig. 33. Tumor growth inhibition rate on day 33.

Description of the invention

Definitions and general methods.

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

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

Antibody related definitions.

TNFRSF18, GITR (eng. Glucocorticoid-induced TNFR-related protein / glucocorticoid-induced tumor necrosis factor receptor / TNFRSF18 / tumor necrosis factor receptor superfamily) is a membrane protein, a receptor from the tumor necrosis factor receptor superfamily. GITR is a type I transmembrane protein that consists of 216 amino acids and has a molecular weight of 26 kDa. The N-terminal extracellular domain contains 3 TNFR-Cys repeats and an Nglycosylation site. The 3 cysteine-rich domains and cytoplasmic tail of GITR share significant homology with 4-1BB, OX40, and CD27 (Nocentini, et al. (1997) Proc. Natl. Acad. Sci. 94:6216-6221).

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

The term binding molecule includes antibodies and immunoglobulins.

The term antibody or immunoglobulin (Ig) as used herein includes whole antibodies and any antigen-binding fragment (ie, antigen-binding portion) or individual chains thereof. The term antibody refers to a glycoprotein containing at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding portion thereof. Each heavy chain contains a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. There are five known types of mammalian antibody heavy chains, which are designated by Greek letters: α, δ, ε, γ and μ. The type of heavy chain present determines the class of the antibody; these chains are found in antibodies of the IgA, IgD, IgE, IgG and IgM types, respectively. Different heavy chains differ in size and composition; α and γ contain approximately 450 amino acids, and μ and ε consist of approximately 550 amino acids. Each heavy chain contains two regions, i.e. constant region and variable region. The constant region is identical in all antibodies of the same isotype, but differs in antibodies of different

- 7 044786 isotype. The heavy chains γ, α and δ contain a constant region, which consists of three constant domains CH1, CH2 and CH3 (arranged in a row) and a hinge region, which provides flexibility (Woof J., Burton D., Nat Rev Immunol. 4, 2004 , pp. 89-99); The μ and ε heavy chains contain a constant region, which consists of four constant domains CH1, CH2, CH3 and CH4. In mammals, only two types of light chains are known, which are designated lambda (λ) and kappa (k). Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The approximate length of the light chain is 211-217 amino acids. Preferably the light chain is a kappa (k) light chain and the CL constant domain is preferably a C-kappa (k) chain.

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

The VH and VL regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs), interspersed between regions that are more conserved called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The heavy and light chain variable regions contain a binding domain that interacts with the antigen. Antibody constant regions can mediate the binding of immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells), and the first component (C1q) of the classical complement system.

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

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

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

The term variable refers to the fact that certain segments of variable domains vary widely in sequence among antibodies. Domain V mediates antigen binding and determines the specificity of a particular antibody for its particular antigen. However, variability is not evenly distributed across the 110 amino acid variable domain region. In contrast, V regions are composed of invariant fragments called framework regions (FRs) of 15–30 amino acids separated by shorter regions of extreme variability called hypervariable regions or CDRs. Each variable domain of the native heavy and light chains contains four FRs, generally adopting a beta-sheet configuration, linked by three hypervariable regions that form binding loops and, in some cases, are part of a beta-sheet structure. The hypervariable regions in each chain are held together in

- 8 044786 close proximity with FR and with hypervariable regions of another chain contribute to the formation of the antigen-binding site of antibodies. Constant domains are not directly involved in the binding of the antibody to the antigen, but exhibit various effector functions, such as the participation of the antibody in antibody-dependent cellular cytotoxicity (ADCC).

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

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

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

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

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

Specific binding can be quantified, for example, by measuring the binding of a molecule compared to the binding of a control molecule. For example, specific binding can be determined by a competitive reaction with another molecule similar to the target, for example, with an excess of unlabeled target. In this case, specific binding is indicated if the binding of the labeled target to the probe is competitively inhibited by an excess of unlabeled target. As used herein, the term specific binding or expression specifically binds to or is specific to a particular polypeptide or epitope on a particular target polypeptide can be characterized by a molecule having a Kd to the target of at least about 200 nM, or at least about 150 nM, or at least about 100 nM, or at least about 60 nM, or at least about 50 nM, or at least about 40 nM, or at least about 30 nM, or at least about 20 nM, or at least about 10 nM, or at least about 8 nM, or at least about 6 nM, or at least about 4 nM, or

- 9 044786 at least about 2 nM, or at least about 1 nM or higher. In one embodiment, the term specific binding refers to binding in which a molecule binds to a specific polypeptide or epitope on a specific polypeptide without substantially binding to any other polypeptide or epitope on the polypeptide.

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

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

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

In one embodiment of the invention, Kd or the Kd value of this invention is measured by surface plasmon resonance methods on a BIAcore™-2000 or BIAcore™-3000 instrument (BIAcore, Inc., Piscataway, NJ) at 25°C using chips with immobilized CM5 antigen at ~ 10 relative units (response units, RU). Briefly, carboxymethyldextran biosensor chips (CM5, BIAcore Inc.) were activated with N -ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N -hydroxysuccinimide (NHS) according to the manufacturer's instructions. The antigen is diluted with 10 mM sodium acetate, pH 4.8, to a concentration of 5 μg/ml (~0.2 μM), and then injected at a flow rate of 5 μl/min until approximately 10 relative units (RU) of bound protein are reached. After antigen administration, 1 M ethanolamine solution is injected to block unreacted groups. For kinetic measurements, 2-fold serial dilutions of Fab (eg, 0.78 nM to 500 nM) are injected into PBS with 0.05% Tween 20 (PBST) at 25°C with a flow rate of approximately 25 μl/min. Association rate (kon) and dissociation rate (koff) values are calculated using a simple Langmuir one-plus-one binding model (BIAcore Evaluation Software version 3.2) by simultaneously obtaining association and dissociation sensorgrams. The equilibrium dissociation constant (Kd) is calculated as the ratio koff/kon. See, for example, Chen, Y., et al., (1999) J. Mol. Biol. 293:865-881. If, according to the above surface plasmon resonance method, the association rate exceeds 106 M- ¹ s- ¹ , then it can be determined by the fluorescence quenching method, which measures the increase or decrease in the intensity of fluorescent emission (excitation = 295 nm; emission (radiation) = 340 nm, band 16 nm) at 25°C of a 20 nM solution of antibody against the antigen (Fab form) in PBS, pH 7.2, in the presence of increasing concentrations of antigen measured using a spectrometer such as a stopped-flow spectrophotometer (Aviv Instruments) or an SLM-Aminco spectrophotometer (ThermoSpectronie) 8000 series with stirred cuvette.

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

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

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

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

- 10 044786

Antibody regions, such as Fab and F(ab') ₂ fragments, can be prepared from whole antibodies using traditional methods such as papain or pepsin digestion of whole antibodies.

Moreover, antibodies, antibody portions, and immunoadhesion molecules can be produced using standard recombinant DNA techniques, for example, as described herein.

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

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

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

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

The term humanization refers to the fact that when an antibody is of wholly or partly non-human origin, such as a mouse or llama antibody produced by immunizing mice or llamas, respectively, with an antigen of interest, or is a chimeric antibody based on such a mouse or llama antibody, it may replace certain amino acids, for example, in the framework regions and constant domains of the heavy and light chains, in order to avoid or minimize the immune response in humans. The specificity of the interaction of the antibody with the target antigen is inherent mainly in the amino acid residues located in the six CDR regions of the heavy and light chain. Therefore, amino acid sequences within the CDR regions are much more variable between individual antibodies compared to sequences outside the CDR regions. Since CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of a specific natural antibody, or more generally of any specific antibody of a given amino acid sequence, for example, by constructing expression vectors that express the CDR sequences -areas of specific antibodies and framework

- 11 044786 sequence of another antibody. As a result, it is possible to humanize a non-human antibody and largely retain the binding specificity and affinity of the original antibody. Although it is impossible to accurately predict immunogenicity and thus the immune response directed against an antibody in a person to a particular antibody, non-human antibodies are generally more immunogenic than human antibodies. Chimeric antibodies in which foreign (eg, rodent or camel) constant regions have been replaced with sequences of human origin have shown generally lower immunogenicity than antibodies of completely foreign origin, and there is a tendency to use humanized or fully human antibodies in therapeutic antibodies. Chimeric antibodies or other non-human antibodies can thus be humanized to reduce the risk of an immune response directed against the antibody in humans.

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

Numerous methods for humanizing an antibody sequence are known in the art. One of the most commonly used methods is CDR transplantation. CDR site transplantation can be based on the Kabat CDR site definitions, although a more recent publication (Magdelaine-Beuzelin et al., Crit Rev. Oncol Hematol. 64:210 225 (2007)) suggests that the IMGT® definition (the international ImMunoGeneTics information system®, www.imgt.org) can improve the outcome of humanization (see Lefranc et al., Dev. Comp Immunol. 27:55-77 (2003)). In some cases, transplantation of a CDR region may reduce the specificity and binding affinity, and therefore the biological activity, of the CDR grafted non-human antibody compared to the parent antibody from which the CDR regions are derived. Back mutations (sometimes referred to as framework repairs) can be applied at selected CDR positions of the transplanted antibody, typically framework regions, to restore the specificity and binding affinity of the parent antibody. Determining positions for possible back mutations can be done using information available in the literature and antibody databases. Amino acid residues that are candidates for back mutations are typically located on the surface of the antibody molecule, while residues that are recessed or have a low degree of surface exposure will generally not be affected by changes. An alternative method of humanization to CDR region grafting and reverse mutation is surface modification in which non-surface-exposed non-human residues are retained while surface-exposed residues are modified into human residues.

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

The construction of combinatorial phage antibody libraries begins with the selection of the source of the gene repertoire, depending on which several types of antibody libraries can be distinguished: naive, immune or synthetic. Naïve and immune libraries are constructed using naturally reorganized genes encoding variable domains of immunoglobulins from healthy donors or donors immune to any antigen, respectively. To do this, mRNA of lymphoid cells that produce antibodies is isolated. Most often these are peripheral blood lymphocytes, but in some cases splenocytes are used [Sheets 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 U S A 1998,95:6157-6162 and de Haard HJ, van Neer N, Reurs A, Hufton SE, Roovers RC, Henderikx P, et al. A large non-immunized human Fab fragment phage library that allows rapid isolation and kinetic analysis of high affinity antibodies. J Biol Chem 1999,274:18218-18230.], tonsil cells or bone marrow lymphocytes [Vaughan TJ, Williams

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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]. Based on mRNA, cDNA is synthesized, while oligo-dT primers and statistical hexaoligonucleotides can be used to prime the reaction, which allows one to obtain cDNA copies of all possible variants of genes encoding variable domains of antibodies [Ulitin A.B., Kapralova M.B., Laman A.G. ., Shepelyakovstkaya A.O., Bulgakova E.V., Fursova K.K., et al. Human miniantibody library in phage display format. Creation and testing. DAN: Nauka publishing house; 2005].

One or more primers can be used immediately, limiting the set of amplified genes to one or several gene families of variable domains or antibody isotypes already at the cDNA level [Marks 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 1991,222:581597]. Primers used to amplify genes encoding immunoglobulins are complementary to their most conserved regions. Their sequences are selected from gene collections that are organized in databases such as the Kabat or V BASE database. The design of the primers also provides for the presence of internal restriction sites in them, allowing the cloning of PCR products into the corresponding vectors.

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

Phage display is the first and most widely used in vitro technology for antibody discovery. In 1985, Smith discovered that foreign DNA sequences could be cloned into the filamentous bacteriophage M13 such that the cloned gene sequences were expressed on the surface of the phage particles as fusion proteins (Smith GP: Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 1985, 228:1315-1317). Thus, it is possible to select fusion proteins of interest based on their ability to bind other proteins. This discovery was combined with PCR amplification techniques to allow cDNA cloning of the immunoglobulin gene repertoire to create a variety of phage libraries containing variable domains that can be used to rapidly search for target-specific monoclonal antibodies. The repertoire of phage libraries reflects the antibody repertoire of B lymphocytes of each person or animal whose blood was used to create the library. In 1995, two papers reported the creation of genetically engineered mice that expressed fully human antibodies, the repertoire of which could be comparable to the resulting 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, the genes of their own endogenous heavy and k light chains of immunoglobulins were purposefully destroyed and transgenes were introduced, which are segments of the human heavy and k light chain genes. It turns out that the human gene repertoire can be used by the mouse immune system to create highly specific and high-affinity antibodies to a wider variety of antigens. Although transgenic mice express B cell receptors that are essentially mouse-human hybrids (human immunoglobulin, mouse Iga, Ige, and other signaling molecules), their B cells develop and mature normally.

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

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

Native antibodies are typically heterotetrameric glycoproteins with a molecular weight of approximately 150,000 daltons, consisting of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by a single covalent disulfide bond, while the number of disulfide bonds between heavy chains varies among immunoglobulin isotypes. Each heavy and light chain also has evenly spaced intrachain chains.

- 13 044786 point disulfide bridges. Each heavy chain has a variable domain (VH) at one end, followed by several constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at the other end. The light chain constant domain is aligned with the first heavy chain constant domain, and the light chain variable domain is aligned with the heavy chain variable domain. It is believed that specific amino acid residues form the interface between the light chain and heavy chain variable domains.

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

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

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

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

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

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

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

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

The term antiproliferative action implies stopping or inhibiting the growth of

- 14 044786 proliferating cells, such as cancer cells.

The term antibody effector function refers to the biological activities associated with the Fc region (native Fc region sequence or Fc region amino acid sequence variants) of an antibody and varies depending on the isotype of the antibody. Examples of antibody effector functions are: C1q binding; complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down-regulation of cell surface receptors (eg, B cell receptor, BCR) and B cell activation.

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

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

The terms Fc receptor and FcR are used to describe a receptor that binds to the Fc region of an antibody. A preferred FcR is a human FcR with a native sequence. In addition, a preferred FcR is an FcR that binds an IgG antibody (gamma receptor), and preferred receptors include receptors of the FcyRI, FcyRII and FcyRIII subclasses, including allelic variants and alternatively spliced forms of these receptors. FcyRII receptors include FcyRIIA (activating receptor) and FcyRIIB (inhibitory receptor), which have similar amino acid sequences that differ mainly in their cytoplasmic domains. The activating receptor FcyRIIA contains a tyrosine-based immunoreceptor activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcyRIIB contains a tyrosine-based immunoreceptor inhibitory motif (ITIM) in its cytoplasmic domain (reviewed in Daeron, Annu. Rev. Immunol. 15: 203-234 (1997)). A review on FcR is presented in Ravetch and Kinet, Annu. Rev. Immunol 9: 457-92 (1991). Other FcRs, including FcRs that will be identified in the future, are included herein under the term FcRs. The term also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgG into the fetus.

Complement-dependent cytotoxicity and CDC refer to the ability of a molecule to lyse a target in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (C1q) to a molecule (such as an antibody) complexed with its antigen. To assess complement activation, a CDC assay can be performed, for example, as described in Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996).

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

The phrase homologous, with regard to the polypeptide sequence of the antibody, should only be used

- 15 044786 act as an antibody exhibiting at least 70%, preferably 80%, more preferably 90%, and most preferably 95% sequence identity to the polypeptide sequence. The term nucleic acid sequence should be construed as a sequence of nucleotides exhibiting at least 85%, preferably 90%, more preferably 95%, and most preferably 97% sequence identity to the nucleic acid sequence.

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

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

Table A

Original balance Replacement examples Preferred replacements Ala (A) Val; Leu; Ile Val Arg(K) Lys; Gin; Asn Lys Asn(N) Gin; His; Asp, Lys; Arg Gin Asp (D) Glu; Asn Glu Suz (C) Ser; Ala Ser Gln[Q} Asn; Glu Asn Glu(E) Asp; Gin Asp Gly [G] Ala Ala His(H) Asn; Gin; Lys; Arg Arg He(I) Leu; Val; Met; Ala; Ph; Horleucine Leu Leu (L) Norleucine; Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gin; Asn Arg Met(M) Leu; Ph; Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Val; Ser Ser Trp[W} Tyr; Phe Tyr Tyr[Y} Trp; Ph; Thr; Ser Phe Val(V) Ile; Leu; Met; Ph; Ala; Horleucine Leu

The terms nucleic acid, nucleic sequence or nucleic acid

- 16 044786 sequence, polynucleotide, oligonucleotide, polynucleotide sequence and nucleotide sequence, as used interchangeably herein, mean a distinct sequence of nucleotides, modified or unmodified, defining a fragment or region of nucleic acid, containing or not containing unnatural nucleotides and being either double-stranded DNA or RNA, or single-stranded DNA or RNA, or transcription products of said DNA.

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

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

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

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

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

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

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

The term GITR-mediated disease or disorder includes all diseases or disorders that are either directly or indirectly associated with GITR, including etiology, development, progression,

- 17 044786 persistence or pathology of a disease or disorder.

Treat, cure, and therapy refer to a method of alleviating or eliminating a biological disorder and/or at least one of its associated symptoms. As used herein, the term ameliorate a disease, disorder or condition means reducing the severity and/or frequency of symptoms of the disease, disorder or condition. In addition, references to treatment contained herein include references to curative, palliative, and preventive therapies.

In one aspect, the subject of treatment or patient is a mammal, preferably a human subject. The above subject may be male or female of any age.

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

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

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

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

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

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

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

Detailed Description of the Invention

Antibody.

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

In one aspect, the present invention provides a monoclonal antibody or antigen binding fragment thereof that specifically binds to GITR, comprising:

(a) a heavy chain variable domain containing:

(i) CDR1 with an amino acid sequence selected from the group: NYGMH (SEQ ID NO: 1) or YYWMY (SEQ ID NO: 12);

(ii) CDR2 with an amino acid sequence selected from the group: VIWFDGSNKFYTDSVKG (SEQ ID NO: 2) or AISWNGGRTYYAESMKG (SEQ ID NO: 13);

(iii) a CDR3 with an amino acid sequence selected from the group: ELGGYYYDSSGFRPYYYGMDV (SEQ ID NO: 3) or NRYYSDPNYGMNL (SEQ ID NO: 14), and (b) a light chain variable domain comprising:

(i) CDR1 with an amino acid sequence selected from the group: RASQSIGSWLA (SEQ ID NO: 7) or TGTSTDIGTYKYIS (SEQ ID NO: 17);

(ii) CDR2 with an amino acid sequence selected from the group: AASTLQR (SEQ ID NO:

- 18 044786

8) or GVSHRPS (SEQ ID NO: 18);

(iii) CDR3 with an amino acid sequence selected from the group: QQSHSHPLT (SEQ ID

NO: 9) or SSYTSSGTVV (SEQ ID NO: 19).

In some embodiments, the monoclonal antibody or antigen binding fragment thereof includes a heavy chain variable domain that contains CDRs 1, 2, and 3 with the amino acid sequences represented by SEQ ID NO: 1, 2, and 3, respectively.

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

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

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

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

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

a light chain variable domain which contains CDRs 1, 2 and 3 with the amino acid sequences represented by SEQ ID NOs: 7, 8 and 9, respectively.

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

a heavy chain variable domain which contains CDRs 1, 2 and 3 with the amino acid sequences represented by SEQ ID NOs: 12, 13 and 14, respectively;

a light chain variable domain which contains CDRs 1, 2 and 3 with the amino acid sequences represented by SEQ ID NOs: 17, 18 and 19, respectively.

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

EVQLVQSGGGVVQPGKSLRLSCAASGTFFSNYGMHWVRQAPGKGLEWVAVIWFDGSN KFYTDSVKGRFTISRDNSKDTLSLQMNSLRAEDTAVYYCARELGGYYYDSSGFRPYYY GMDVWGQGTMVTVSS (SEQ Ш NO: 4).

In some embodiments, the monoclonal antibody or antigen binding fragment thereof includes a heavy chain variable domain that contains the amino acid sequence

EVQLVQSGGGVVQPGKSLRLSCAASGTFFSNYGMHWVRQAPGKGLEWVAVIWFDGSN KFYTDSVKGRFTISRDNSKDTLSLQMNSLRAEDTAVYYCARELGGYYYDSSGFRPYYY GMDVWGQGTMVTVSS (SEQ Ш NO: 4).

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

QVQLVQSGGGLVQPGGSLRLSCAASGFTFSYYWMYWVRQAPGKGLEWVSAIS WNGGRTYYAESMKGRFTISRDNAQNTLYLQMNSLKSEDTAVYYCAKNRYYSDPNYG MNLWGKGTTVTVSS (SEQ ID NO: 15).

In some embodiments, the monoclonal antibody or antigen binding fragment thereof includes a heavy chain variable domain that contains the amino acid sequence

QVQLVQSGGGLVQPGGSLRLSCAASGFTFSYYWMYWVRQAPGKGLEWVSAISWNGG RTYYAESMKGRFTISRDNAQNTLYLQMNSLKSEDTAVYYCAKNRYYSDPNYGMNLWG KGTTVTVSS (SEQ Ш NO: 15).

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

DVVMTQSPSSVSASVGDRVTITCRASQSIGSWLAWYQQKPGEAPKLLIYAASTL QRGVPSRFSGGGYGTEFTLTISSLQPEDFATYFCQQSHSHPLTFGGGTKVEIK (SEQ ID NO: 10).

In some embodiments, the monoclonal antibody or antigen binding fragment thereof includes a light chain variable domain that contains the amino acid sequence

DVVMTQSPSSVSASVGDRVTITCRASQSIGSWLAWYQQKPGEAPKLLIYAASTLQRGVP SRFSGGGYGTEFTLTISSLQPEDFATYFCQQSHSHPLTFGGGTKVEIK (SEQ ID NO: 10).

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

- 19 044786

QSALTQPASVSGSPGQSITISCTGTSTDIGTYKYISWYQQHPGKAPKLIIYGVSHRPSGVS DRFSGSKSDNTASLTISGLQAEDEADYYCSSYTSSGTVVFGGGTKVTVL (SEQ ID NO: 20).

In some embodiments, the monoclonal antibody or antigen binding fragment thereof includes a light chain variable domain that contains the amino acid sequence

QSALTQPASVSGSPGQSITISCTGTSTDIGTYKYISWYQQHPGKAPKLIIYGVSHRPSGVS DRFSGSKSDNTASLTISGLQAEDEADYYCSSYTSSGTVVFGGGTKVTVL (SEQ ID NO: 20).

In some embodiments, the monoclonal antibody or antigen binding fragment thereof includes: a heavy chain variable domain that contains an amino acid sequence that is at least 90% homologous to the amino acid sequence

EVQLVQSGGGVVQPGKSLRLSCAASGTFFSNYGMHWVRQAPGKGLEWVAVIWFDGSN KFYTDSVKGRFTISRDNSKDTLSLQMNSLRAEDTAVYYCARELGGYYYDSSGFRPYYY GMDVWGQGTMVTVSS (SEQ Ш NO: 4);

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

DVVMTQSPSSVSASVGDRVTITCRASQSIGSWLAWYQQKPGEAPKLLIYAASTLQRGVP SRFSGGGYGTEFTLTISSLQPEDFATYFCQQSHSHPLTFGGGTKVEIK (SEQ ID NO: 10).

In some embodiments, the monoclonal antibody or antigen binding fragment thereof includes: a heavy chain variable domain that contains an amino acid sequence

EVQLVQSGGGVVQPGKSLRLSCAASGTFFSNYGMHWVRQAPGKGLEWVAVIWFDGSN KFYTDSVKGRFTISRDNSKDTLSLQMNSLRAEDTAVYYCARELGGYYYDSSGFRPYYY GMDVWGQGTMVTVSS (SEQ Ш NO: 4);

a light chain variable domain that contains the amino acid sequence DVVMTQSPSSVSASVGDRVTITCRASQSIGSWLAWYQQKPGEAPKLLIYAASTLQRGVP SRFSGGGYGTEFTLTISSLQPEDFATYFCQQSHSHPLTFGGGTKVEIK (SEQ Ш NO: 10).

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

QVQLVQSGGGLVQPGGSLRLSCAASGFTFSYYWMYWVRQAPGKGLEWVSAISWNGG RTYYAESMKGRFTISRDNAQNTLYLQMNSLKSEDTAVYYCAKNRYYSDPNYGMNLWG KGTTVTVSS (SEQ Ш NO: 15);

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

QSALTQPASVSGSPGQSITISCTGTSTDIGTYKYISWYQQHPGKAPKLIIYGVSHRPSGVS DRFSGSKSDNTASLTISGLQAEDEADYYCSSYTSSGTVVFGGGTKVTVL (SEQ ID NO: 20).

In some embodiments, the monoclonal antibody or antigen binding fragment thereof includes: a heavy chain variable domain that contains an amino acid sequence

QVQLVQSGGGLVQPGGSLRLSCAASGFTFSYYWMYWVRQAPGKGLEWVSAISWNGG RTYYAESMKGRFTISRDNAQNTLYLQMNSLKSEDTAVYYCAKNRYYSDPNYGMNLWG KGTTVTVSS (SEQ ID NO: 15);

a light chain variable domain that contains the amino acid sequence QSALTQPASVSGSPGQSITISCTGTSTDIGTYKYISWYQQHPGKAPKLIIYGVSHRPSGVS DRFSGSKSDNTASLTISGLQAEDEADYYCSSYTSSGTVVFGGGTKVTVL (SEQ ID NO: 20).

In some embodiments, the GITR-specific monoclonal antibody is a full-length IgG antibody.

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

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

In some embodiments, the GITR-specific monoclonal antibody includes an E345R mutation in the Fc fragment to enhance agonistic properties, antibody-dependent cellular cytotoxicity (ADCC), but not complement-dependent cytotoxicity (CDC).

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

- 20 044786

EVQLVQSGGGVVQPGKSLRLSCAASGTFFSNYGMHWVRQAPGKGLEWVAVIWFDGSN KFYTDSVKGRFTISRDNSKDTLSLQMNSLRAEDTAVYYCARELGGYYYDSSGFRPYYY GMDVWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLS SVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 5).

In some embodiments, the monoclonal antibody includes a heavy chain comprising the amino acid sequence

EVQLVQSGGGVVQPGKSLRLSCAASGTFFSNYGMHWVRQAPGKGLEWVAVIWFDGSN KFYTDSVKGRFTISRDNSKDTLSLQMNSLRAEDTAVYYCARELGGYYYDSSGFRPYYY GMDVWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLS SVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ Ш NO: 5).

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

EVQLVQSGGGVVQPGKSLRLSCAASGTFFSNYGMHWVRQAPGKGLEWVAVIWFDGSN KFYTDSVKGRFTISRDNSKDTLSLQMNSLRAEDTAVYYCARELGGYYYDSSGFRPYYY GMDVWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLS SVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPRRPQVYTLPPSRDELTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ Ш NO: 6).

In some embodiments, the monoclonal antibody includes a heavy chain comprising the amino acid sequence

EVQLVQSGGGVVQPGKSLRLSCAASGTFFSNYGMHWVRQAPGKGLEWVAVIWFDGSN KFYTDSVKGRFTISRDNSKDTLSLQMNSLRAEDTAVYYCARELGGYYYDSSGFRPYYY GMDVWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLS SVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPRRPQVYTLPPSRDELTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 6).

In some embodiments, the monoclonal antibody includes a light chain containing an amino acid sequence that is at least 90% homologous to the sequence

DVVMTQSPSSVSASVGDRVTITCRASQSIGSWLAWYQQKPGEAPKLLIYAASTLQRGVP SRFSGGGYGTEFTLTISSLQPEDFATYFCQQSHSHPLTFGGGTKVEIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL TLSKADYEKHK VYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 11).

In some embodiments, the monoclonal antibody includes a light chain comprising the amino acid sequence

DVVMTQSPSSVSASVGDRVTITCRASQSIGSWLAWYQQKPGEAPKLLIYAASTLQRGVP SRFSGGGYGTEFTLTISSLQPEDFATYFCQQSHSHPLTFGGGTKVEIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL TLSKADYEKHK VYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 11).

In some embodiments, the monoclonal antibody includes:

a heavy chain containing an amino acid sequence that is at least 90% homologous to the sequence

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EVQLVQSGGGVVQPGKSLRLSCAASGTFFSNYGMHWVRQAPGKGLEWVAVIWFDGSN KFYTDSVKGRFTISRDNSKDTLSLQMNSLRAEDTAVYYCARELGGYYYDSSGFRPYYY GMDVWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLS SVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 5);

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

DVVMTQSPSSVSASVGDRVTITCRASQSIGSWLAWYQQKPGEAPKLLIYAASTLQRGVP SRFSGGGYGTEFTLTISSLQPEDFATYFCQQSHSHPLTFGGGTKVEIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL TLSKADYEKHK VYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 11).

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

Monoclonal antibody BCD166-01-001 includes:

heavy chain containing an amino acid sequence

EVQLVQSGGGVVQPGKSLRLSCAASGTFFSNYGMHWVRQAPGKGLEWVAVIWFDGSN KFYTDSVKGRFTISRDNSKDTLSLQMNSLRAEDTAVYYCARELGGYYYDSSGFRPYYY GMDVWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLS SVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ Ш NO: 5);

light chain containing the amino acid sequence

DVVMTQSPSSVSASVGDRVTITCRASQSIGSWLAWYQQKPGEAPKLLIYAASTLQRGVP SRFSGGGYGTEFTLTISSLQPEDFATYFCQQSHSHPLTFGGGTKVEIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL TLSKADYEKHK VYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 11).

In some embodiments, the monoclonal antibody includes:

a heavy chain containing an amino acid sequence that is at least 90% homologous to the sequence

EVQLVQSGGGVVQPGKSLRLSCAASGTFFSNYGMHWVRQAPGKGLEWVAVIWFDGSN KFYTDSVKGRFTISRDNSKDTLSLQMNSLRAEDTAVYYCARELGGYYYDSSGFRPYYY GMDVWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLS SVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPRRPQVYTLPPSRDELTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ Ш NO: 6);

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

D VVMTQ SP S S VS AS VGDRVTITCRASQ SIGS WL AW YQQKPGEAPKLLIYAASTLQRGVP SRFSGGGYGTEFTLTISSLQPEDFATYFCQQSHSHPLTFGGGTKVEIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL TLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 11).

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

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

Monoclonal antibody BCD166-02-001 includes:

heavy chain containing an amino acid sequence

EVQLVQSGGGVVQPGKSLRLSCAASGTFFSNYGMHWVRQAPGKGLEWVAVIWFDGSN

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KFYTDSVKGRFTISRDNSKDTLSLQMNSLRAEDTAVYYCARELGGYYYDSSGFRPYYY GMDVWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC DKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPRRPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 6);

light chain containing the amino acid sequence

DVVMTQSPSSVSASVGDRVTITCRASQSIGSWLAWYQQKPGEAPKLLIYAASTLQRGVP SRFSGGGYGTEFTLTISSLQPEDFATYFCQQSHSHPLTFGGGTKVEIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL TLSKADYEKHK VYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 11).

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

QVQLVQSGGGLVQPGGSLRLSCAASGFTFSYYWMYWVRQAPGKGLEWVSAISWNGG RTYYAESMKGRFTISRDNAQNTLYLQMNSLKSEDTAVYYCAKNRYYSDPNYGMNLWG KGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSL GTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSRDELTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 16).

In some embodiments, the monoclonal antibody includes a heavy chain comprising the amino acid sequence

QVQLVQSGGGLVQPGGSLRLSCAASGFTFSYYWMYWVRQAPGKGLEWVSAISWNGG RTYYAESMKGRFTISRDNAQNTLYLQMNSLKSEDTAVYYCAKNRYYSDPNYGMNLWG KGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSL GTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSRDELTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ Ш NO: 16).

In some embodiments, the monoclonal antibody includes a light chain containing an amino acid sequence that is at least 90% homologous to the sequence

QSALTQPASVSGSPGQSITISCTGTSTDIGTYKYISWYQQHPGKAPKLIIYGVSHRPSGVS DRFSGSKSDNTASLTISGLQAEDEADYYCSSYTSSGTVVFGGGTKVTVLGQPKAAPSVT LFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAAS SYLSLTPEQWKS HRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 21).

In some embodiments, the monoclonal antibody includes a light chain comprising the amino acid sequence

QSALTQPASVSGSPGQSITISCTGTSTDIGTYKYISWYQQHPGKAPKLIIYGVSHRPSGVS DRFSGSKSDNTASLTISGLQAEDEADYYCSSYTSSGTVVFGGGTKVTVLGQPKAAPSVT LFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAAS SYLSLTPEQWKS HRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 21).

In some embodiments, the monoclonal antibody includes:

a heavy chain containing an amino acid sequence that is at least 90% homologous to the sequence

QVQLVQSGGGLVQPGGSLRLSCAASGFTFSYYWMYWVRQAPGKGLEWVSAISWNGG RTYYAESMKGRFTISRDNAQNTLYLQMNSLKSEDTAVYYCAKNRYYSDPNYGMNLWG KGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSL GTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSRDELTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ Ш NO: 16).

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

- 23 044786

QSALTQPASVSGSPGQSITISCTGTSTDIGTYKYISWYQQHPGKAPKLIIYGVSHRPSGVS DRFSGSKSDNTASLTISGLQAEDEADYYCSSYTSSGTVVFGGGTKVTVLGQPKAAPSVT LFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAAS SYLSLTPEQWKS HRSYSCQVTHEGSTVEKTVAPTECS (SEQ Ш NO: 21).

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

Monoclonal antibody BCD166-01-014 includes:

heavy chain containing an amino acid sequence

QVQLVQSGGGLVQPGGSLRLSCAASGFTFSYYWMYWVRQAPGKGLEWVSAISWNGG RTYYAESMKGRFTISRDNAQNTLYLQMNSLKSEDTAVYYCAKNRYYSDPNYGMNLWG KGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSL GTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSRDELTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ Ш NO: 16).

light chain containing the amino acid sequence

QSALTQPASVSGSPGQSITISCTGTSTDIGTYKYISWYQQHPGKAPKLIIYGVSHRPSGVS DRFSGSKSDNTASLTISGLQAEDEADYYCSSYTSSGTVVFGGGTKVTVLGQPKAAPSVT LFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAAS SYLSLTPEQWKS HRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 21).

Nucleic acid molecules.

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

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

In one aspect, the present invention provides a nucleic acid molecule comprising a nucleotide sequence that encodes an amino acid sequence selected from SEQ ID NO: 1-21. The nucleic acid molecule may also contain any combination of these nucleotide sequences.

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

(a) a heavy chain variable domain containing:

(i) CDR1 with an amino acid sequence selected from the group: NYGMH (SEQ ID NO: 1) or YYWMY (SEQ ID NO: 12); (ii) CDR2 with an amino acid sequence selected from the group: VIWFDGSNKFYTDSVKG (SEQ ID NO: 2) or AISWNGGRTYYAESMKG (SEQ ID NO: 13);

(iii) a CDR3 with an amino acid sequence selected from the group: ELGGYYYDSSGFRPYYYGMDV (SEQ ID NO: 3) or NRYYSDPNYGMNL (SEQ ID NO: 14), and (b) a light chain variable domain containing:

(i) CDR1 with an amino acid sequence selected from the group: RASQSIGSWLA (SEQ ID NO: 7) or TGTSTDIGTYKYIS (SEQ ID NO: 17);

(ii) CDR2 with an amino acid sequence selected from the group: AASTLQR (SEQ ID NO: 8) or GVSHRPS (SEQ ID NO: 18);

(iii) CDR3 with an amino acid sequence selected from the group: QQSHSHPLT (SEQ ID NO: 9) or SSYTSSGTVV (SEQ ID NO: 19).

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

In some embodiments, the nucleic acid molecule contains a nucleotide sequence that encodes a monoclonal antibody or an antigen binding fragment thereof, which includes a heavy chain variable domain that contains CDRs 1, 2 and 3 with the amino acid sequences represented by SEQ ID NOs: 12, 13 and 14, respectively .

In some embodiments, the nucleic acid molecule contains a nucleotide sequence that encodes a monoclonal antibody or an antigen binding fragment thereof, which includes a light chain variable domain that contains CDRs 1, 2, and 3 with the amino acid sequences represented by SEQ ID NO: 7, 8 and 9 respectively.

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

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

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

a light chain variable domain which contains CDRs 1, 2 and 3 with the amino acid sequences represented by SEQ ID NOs: 7, 8 and 9, respectively.

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

a heavy chain variable domain which contains CDRs 1, 2 and 3 with the amino acid sequences represented by SEQ ID NOs: 12, 13 and 14, respectively;

a light chain variable domain which contains CDRs 1, 2 and 3 with the amino acid sequences represented by SEQ ID NOs: 17, 18 and 19, respectively.

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

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

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

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

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

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

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

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

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

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

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

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

- 25 044786 includes:

a heavy chain variable domain which contains the amino acid sequence of SEQ ID NO: 4;

a light chain variable domain which contains the amino acid sequence of SEQ ID NO: 10.

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

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

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

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

a heavy chain variable domain which contains the amino acid sequence of SEQ ID NO: 15;

a light chain variable domain that contains the amino acid sequence of SEQ ID NO: 20.

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

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

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

In some embodiments, the nucleic acid molecule contains a nucleotide sequence that encodes a monoclonal antibody that specifically binds to GITR, which includes an E345R mutation in the Fc fragment to enhance agonistic properties, antibody-dependent cellular cytotoxicity (ADCC), but not complement-dependent cytotoxicity (CDC).

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

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

In some embodiments, the nucleic acid molecule contains a nucleotide sequence that encodes a monoclonal antibody that includes a heavy chain containing an amino acid sequence that is at least 90% homologous to the sequence of SEQ ID NO: 6.

In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a monoclonal antibody that includes a heavy chain comprising the amino acid sequence of SEQ ID NO: 6.

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

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

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

a heavy chain containing an amino acid sequence at least 90% homologous to the sequence of SEQ ID NO: 5;

a light chain containing an amino acid sequence that is at least 90% homologous to the sequence of SEQ ID NO: 11.

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

a heavy chain containing the amino acid sequence of SEQ ID NO: 5;

light chain containing the amino acid sequence SEQ ID NO: 11.

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

a heavy chain containing an amino acid sequence at least 90% homologous to the sequence of SEQ ID NO: 6;

a light chain containing an amino acid sequence that is at least 90% homologous to the sequence of SEQ ID NO: 11.

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

a heavy chain containing the amino acid sequence of SEQ ID NO: 6;

light chain containing the amino acid sequence SEQ ID NO: 11.

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

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

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

a heavy chain containing an amino acid sequence at least 90% homologous to the sequence of SEQ ID NO: 16;

a light chain containing 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 that encodes a monoclonal antibody that includes:

a heavy chain containing the amino acid sequence of SEQ ID NO: 16;

light chain containing the amino acid sequence SEQ ID NO: 21.

In some embodiments, the nucleic acid is DNA.

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

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

In one embodiment, nucleic acid molecules encoding the VH (SEQ ID NO: 4 or SEQ ID NO: 15) or VL (SEQ ID NO: 10 or SEQ ID NO: 20) domains are converted into full-length antibody genes by insertion into an expression vector already encoding heavy chain (CH) or light chain (CL) constant domains, respectively, such that the VH segment is operably linked to the CH segment(s) in the vector and/or the VL segment is operably linked to the CL segment in the vector. In another embodiment of the invention, nucleic acid molecules encoding VH and/or VL domains are converted into genes along the entire length of the antibody by joining, for example, ligation, a nucleic acid molecule encoding VH and/or VL domains to a nucleic acid molecule encoding CH and /or CL domains using standard molecular biological techniques. Nucleic acid molecules encoding the entire length of the heavy and/or light chains can then be expressed from the cell into which they have been introduced.

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

Vector.

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

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

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

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

In addition to the antibody gene chain, the recombinant expression vectors of the present invention may carry regulatory sequences that control the expression of the antibody chain genes in the host cell. Those skilled in the art will appreciate that the design of an expression vector, including the choice of regulatory sequences, may depend on factors such as the selection of the host cell for transformation, the level of expression of the desired protein, etc. Preferred regulatory sequences for a mammalian host expression cell include viral elements that provide high level expression of proteins in mammalian cells, such as promoters and/or enhancers derived from a retroviral LTR, cytomegalovirus (CMV) (eg, CMV promoter/enhancer), simian virus 40 (SV40) (eg, SV40 promoter/enhancer), adenovirus (eg, adenovirus large late promoter (AdMLP)), polyoma virus, as well as strong mammalian promoters such as the native immunoglobulin promoter or the actin promoter. For further description of viral regulatory elements and their sequences, see, for example, US patents 5,168,062, 4,510,245 and 4,968,615. Methods for expressing binding molecules such as plant antibodies, including the description of promoters and vectors, as well as transformation of plants, are known in the art. See, for example, US Patent 6,517,529. Methods for expressing polypeptides in bacterial or fungal cells, such as yeast cells, are also well known in the art.

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

- 28 044786

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

Host cells.

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

Nucleic acid molecules encoding a monoclonal antibody that specifically binds to GITR of the invention and vectors containing these nucleic acid molecules can be used to transfect a suitable mammal or cell thereof, a plant or cell thereof, or a bacterial or yeast host cell. Conversion can occur by any known method for introducing polynucleotides into a host cell. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, transfection with a complex of nucleic acid and a positively charged polymer, transfection with a nucleic acid-calcium phosphate precipitate, polybrene-mediated transfection, protoplast fusion, transfection with liposome-encapsulated polynucleotides, and direct microinjection of DNA into kernels. In addition, nucleic acid molecules can be introduced into mammalian cells by viral vectors. Methods for transfecting cells are well known in the art. See, for example, US Pat. Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455. Methods for transforming plant cells are well known in the art, including, for example, Agrobacterium-mediated transformation, biolistic transformation, direct injection, electroporation, and viral transformation. Methods for transforming bacterial and yeast cells are also well known in the art.

Mammalian cell lines used as hosts for transformation are well known in the art and include many immortalized cell lines available. These include, for example, Chinese hamster ovary (CHO) cells, NS0 cells, SP2 cells, HEK293T cells, 293 Freestyle cells (Invitrogen), NIH-3T3 cells, HeLa cells, hamster kidney (HK) cells, African green monkey kidney cells (COS), human hepatocellular carcinoma cells (eg Hep G2), A549 cells and a number of other cell lines. Cell lines are selected by determining which cell lines have high levels of expression and provide the desired characteristics of the protein produced. Other cell lines that can be used are insect cell lines such as Sf9 or Sf21 cells. When recombinant expression vectors encoding a monoclonal antibody that specifically binds to GITR are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a time sufficient to express the antibodies in the host cells or, preferably, releasing the antibodies into a culture medium , in which host cells are grown. A monoclonal antibody that specifically binds to GITR can be isolated from the culture medium using standard protein purification methods. Plant host cells, for example, include Nicotiana, Arabidopsis, duckweed, corn, wheat, potato, etc. Host bacterial cells include Escherichia and Streptomyces species. Yeast host cells include Schizosaccharomyces pombe, Sac

- 29 044786 charomyces cerevisiae and Pichia pastoris.

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

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

Obtaining antibodies.

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

Monoclonal antibodies.

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

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

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

Preferred myeloma cell fusion components are cells that are readily fusionable, maintain consistently high levels of antibody production by the selected antibody-producing cells, and are sensitive to a selective environment in which unrelated parent cells are selected for. Preferred myeloma cell lines are murine myeloma lines, such as the murine tumor cell lines MOPC-21 and MPC-11, which are available from the Salk Institite Cell Distribution Center, San Diego, NY. California, USA, and lines SP-2 or X63-Ag8-653, which are available from the American Type Culture Collection, Rockville, CA. Maryland, USA. The use of human myeloma and mouse-human heteromyeloma cell lines for the production of monoclonal antibodies has also been described (Kozbor, J. Immunol., 133, 1984, p. 3001).

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

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

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

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

DNA encoding monoclonal antibodies can be easily isolated and sequenced using conventional procedures (eg, using oligonucleotide probes that have the ability to specifically bind to the genes encoding the heavy and light chains of murine antibodies). The preferred source of such DNA is hybridoma cells. After selection

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The DNA can be incorporated into expression vectors that are then transfected into host cells, such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells, which without transfection do not produce antibody protein, resulting in the synthesis of monoclonal antibodies in recombinant host cells.

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

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

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

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

Alternatively, phage display technology can be used to obtain human antibodies and antibody fragments in vitro from immunoglobulin variable region (V) genes from immunized donors (McCafferty et al., Nature, 348, 1990, pp. 552-553). In this technique, antibody V region genes are cloned in frame with either the major or minor envelope protein gene of a filamentous bacteriophage, such as M13 or fd, and presented as functional antibody fragments on the surface of the phage particle. Since the filamentous particle contains a single-stranded DNA copy of the phage genome, selection for the functional properties of the antibody also results in the selection of the gene encoding the antibody that has these properties. Thus, the phage mimics some of the properties of a B cell. Phage presentation can be carried out in various formats. Various sources of V gene segments can be used for phage presentation. Clackson et al., Nature, 352, 1991, p. 624-628 isolated different sets of anti-oxazolone antibodies from a small random combinatorial library of V genes obtained from the spleen of immunized mice. A spectrum of V genes obtained from immunized human donors can be constructed, and antibodies to a different set of antigens (including autoantigens) can be isolated generally according to the methods described in Marks et al., J. Mol. Biol., 222, 1991, p. 581-597.

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

Antibody fragments.

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

Various methods have been developed to obtain antibody fragments. Traditionally, these fragments were obtained by proteolytic cleavage of intact antibodies. However, these fragments can now be produced directly from recombinant host cells. Fab, Fv and scFv fragments of antibodies can be expressed and secreted from E. coli, which allows for easy production of large quantities of these fragments. Antibody fragments can be isolated from the phage antibody libraries described above. In another embodiment, Fab'-SH fragments can be directly isolated from E. coli and chemically cross-linked to produce F(ab')2 fragments (Carter et al.,

- 31 044786

Bio/Technology, 10, 1992, p. 163-167). According to another approach, F(ab') ₂ fragments can be isolated directly from a culture of recombinant host cells. Fab and F(ab') ₂ fragments with increased in vivo half-lives that retain epitope-binding receptor residues are described in US Pat. No. 5,869,046. Other techniques for producing antibody fragments will be apparent to those skilled in the art. In other embodiments, the antibody selected is a single chain Fv fragment (scFv) (see WO 93/16185; US Pat. No. 5,571,894 and US Pat. No. 5,587,458). Fv and scFv are the only species with intact binding sites lacking constant regions; as a result, they can be used for reduced nonspecific binding when used in vivo. ScFv-carrying fusion proteins can be engineered to produce an effector protein fusion at either the N- or C-terminus of the scFv. The antibody fragment may also be a linear antibody, for example, as described in US Pat. No. 5,641,870. Such linear antibody fragments may be monospecific or bispecific.

Pharmaceutical compositions.

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

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

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

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

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

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

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

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

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

By stabilizer is meant an excipient or a mixture of two or more excipients that provide physical and/or chemical stability of the active agent. Amino acids can be used as stabilizers, for example, but not limited to, arginine, histidine, glycine, lysine, glutamine, proline; surfactants, e.g. polysorbate 20 (trade name Tween 20), polysorbate 80 (trade name Tween 80), polyethylene-polypropylene glycol and its copolymers (trade name Poloxaner),

Pluronic), sodium dodecyl sulfate (SDS), but not limited to; antioxidants, for example, but not limited to, methionine, acetylcysteine, ascorbic acid, monothioglycerol, sulfuric acid salts, etc.; chelating agents, for example, but not limited to, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), sodium citrate and the like.

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

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

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

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

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

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

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

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

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

In one aspect, the subject of treatment or patient is a mammal, preferably a human subject. The above subject may be male or female and of any age.

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

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

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

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

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

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

4) sequential administration of such a combination of a monoclonal antibody that specifically binds to GITR, according to this invention and a therapeutic agent to a patient who needs treatment, when such components are formulated together in a single dosage form from which the release of these components occurs in a controlled manner, after which they simultaneously, sequentially or jointly released at the same time and/or different times to a specified patient, where each part can be administered by one or different routes.

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. In addition, treatment with a monoclonal antibody that specifically binds to GITR according to the present invention may include at least one additional therapeutic treatment (combination therapy). In some embodiments, a monoclonal antibody that specifically binds to GITR may be co-administered or formulated with another drug/drug for the treatment of cancer.

The term cytotoxic agent as used herein refers to a substance that inhibits or prevents the functioning of cells and/or causes cell destruction. The term is intended to include radioactive isotopes (e.g. At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² and Lu radioactive isotopes), chemotherapeutic agents and toxins such as low molecular weight toxins or enzymatically active toxins originating from bacteria, fungi, plants or animals, including fragments and/or variants thereof.

A chemotherapy agent is a chemical compound used to treat a malignant tumor. Examples of chemotherapeutic agents include alkylating agents,

- 34 044786 such as thiotepa and cyclophosphamide (CYTOXAN®); alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquinone, meturedopa and uredopa; ethyleneimines and methylmelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylmelamine; acetogenins (eg bullatacin and bullatacinone); delta-9tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol; colchicines; betulinic acid; camptothecin (including synthetic analogue of topotecan (HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin and 9-aminocamptothecin); bryostatin; kallistatin; CC-1065 (including its synthetic analogues adozelesin, carzelesin and bizelesin); podophyllotoxin; podophyllic acid; teniposide; cryptophycins (eg, cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogues KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphasine, holophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembiquine, phenesterine, prednimustine, trofosfamide, uramustine; nitrosoureas such as carmustine, chlorosotocin, fotemustine, lomustine, nimustine and ranimustine; antibiotics such as enediyne antibiotics (eg calicheamicin, e.g. calicheamicin gamma II and calicheamicin omega II (see, for example, Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994)); dinemycin, including dinemycin A; esperamycin; as well as the neocarcinostatin chromophore and related chromophores of chromoproteins - enediin antibiotics, aclacinomysins, actinomycin, outramycin, azaserin, bleomycins, cactinomycin, carabicin, carminomycin, carzinophyllin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6 diazo-5-oxo-E -norleucine, doxorubicin (including ADRIAMICIN®, morpholinodoxorubicin, cyanomorpholinodoxorubicin, 2-pyrrolinodoxorubicin, doxorubicino HCl injectable liposomes (DOXOL®), liposomal doxorubicin TLC D-99 (MYOCET®), pegylated liposomal doxorubicin (CAELYX®) and deoxygen oxorubicin), epirubicin , ezorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rhodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate, gemcitabine (GEMZAR®), tegafur (UFTORAL®), capecitabine (XELODA®), epothilone and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprin, thioguanine; pyrimidine analogues such as ancytabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocytabine, floxuridine; drugs that suppress adrenal function, such as aminoglutethimide, mitotane, trilostane; a folic acid compensator such as folinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diazichon; elfornitine; elliptinium acetate; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerin; pentostatin; phenomet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sisofiran; spirogermanium; tenuazonic acid; triaziquon; 2,2',2''-trichlorotriethylamine; trichothecenes (eg, T-2 toxin, verracurine A, roridin A and anguidine); urethane; dacarbazine; mannomustin; mitobronitol; mitolactol; pipobromance; gacytosine; arabinoside (ara-C); thiotepa; a taxoid, such as paclitaxel (TAXOL®), an engineered albumin-associated nanoparticle formulation of paclitaxel (ABRAXANETM), and docetaxel (TAXOTERE®); chlorambucil; 6-thioguanine; mercaptopurine; methotrexate; platinum-based agents such as cisplatin, oxaliplatin and carboplatin; Vinca alkaloids, which prevent the polymerization of tubulin from nascent microtubules, including vinblastine (VELBAN®), vincristine (ONCOVIN®), vindesine (ELDISINE®, FILDESIN®) and vinorelbine (NAVELBINE®); etoposide (VP-16); ifosfamide; mitoxantrone; leucovorin; Novantrone; edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid, including bexarotene (TARGRETIN®); bisphosphonates such as clodronate (eg, BONEFOS® or OSTAC®), etidronate (DIDROCAL®), NE-58095, zoledronic acid/zoledronate (ZOMETA®), alendronate (FOSAMAX®), pamidronate (AREDIA®), tiludronate (SKELID® ) or risendronate (ACTONEL®); troxacitabine (1,3-dioxolane nucleoside analogue of cytosine); antisense oligonucleotides, for example oligonucleotides that inhibit gene expression in signaling pathways involved in aberrant cell proliferation, such as, for example, PKC-alpha, Raf, H-Ras and epidermal growth factor receptor (EGF-R); vaccines such as THERATOPE® vaccine and gene therapy vaccines such as ALLOVECTIN® vaccine, LEUVECTIN® vaccine and VAXID® vaccine; topoisomerase 1 inhibitor (eg LURTOTECAN®); rmRH (eg ABARELIX®); BAY439006 (sorafenib; Bayer); SU-11248 (Pfizer); perifosine, COX-2 inhibitor (eg, celecoxib or etoricoxib), proteasome inhibitor (eg, PS341); bortezomib (VELCADE®); CCI-779; tipifarnib (811577); orafenib, ABT510; a Bcl-2 inhibitor such as oblimersen sodium (GENASENSE®); pixantron; EGFR inhibitors (see definition below); inhibitors

- 35 044786 tyrosine kinases (see definition below); and pharmaceutically acceptable salts, acids or derivatives of any of the above; and combinations of two or more of the above, such as CHOP, short for combination therapy of cyclophosphamide, doxorubicin, vincristine and prednisolone, and FOLFOX, short for oxaliplatin treatment regimen (ELOXATINTM) in combination with 5-FU and leucovorin.

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

Other therapeutic agents that can be used in combination with an antibody that specifically binds to GITR of the present invention may be inhibitors of growth factor function, for example, such inhibitors include growth factor antibodies and growth factor receptor antibodies (for example, the anti-erbB2 antibody trastuzumab [ Herceptin], anti-EGFR antibody panitumumab, anti-erbB1 antibody cetuximab [Erbitux, C225] and any growth factor or growth factor receptor antibodies disclosed by Stern et al.Critical reviews in oncology/haematology, 2005, Vol.54, pp11-29 ); antiangiogenic agents such as inhibitory effects of vascular endothelial growth factor [eg, anti-vascular endothelial growth factor antibody bevacizumab (Avastin)], anti-vascular endothelial growth factor receptor antibodies such as anti-KDR antibodies and anti-flt1 antibodies; antisense therapies, for example, directed to the above targets, such as ISIS 2503, an anti-ras antisense agent or G3139 (Genasense), an anti-bc12 antisense agent; gene therapy approaches, including, for example, approaches that replace aberrant genes such as aberrant p53 or aberrant BRCA1 or BRCA2, GDEPT (gene-directed enzyme prodrug therapy), approaches using cytosine deaminase, thymidine kinase or the bacterial nitroreductase enzyme, and approaches aimed at increasing patient tolerance to chemotherapy or radiation therapy, such as multidrug resistance gene therapy; immunotherapeutic approaches including, for example, treatment with Alemtuzumab (campath-1H), a monoclonal antibody targeting CD52, or treatment with antibodies targeting CD22, ex vivo and in vivo approaches to enhance the immunogenicity of the patient's tumor cells, transfection with cytokines such as interleukin 2 , interleukin 4 or granulocyte-macrophage colony-stimulating factor, approaches aimed at reducing T cell anergy, such as treatment with monoclonal antibodies that inhibit CTLA-4 function, approaches using transfected immune cells, such as cytokine-transfected dendritic cells, approaches using cytokine-transfected tumor cell lines and approaches using anti-idiotypic antibodies, adoptive T cell transfer using T cells subjected to nonspecific activation or targeting a specific antigen of interest ex vivo; protein degradation inhibitors such as a proteasome inhibitor such as Velcade (bortezomide); biotherapeutic therapeutic approaches, for example, using peptides or proteins (such as antibodies or soluble external receptor domain constructs) that sequester ligand receptors, block ligand binding to the receptor, or attenuate receptor signaling (eg, due to increased receptor degradation or decreased expression levels).

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

Another therapeutic agent that may be used in combination with an antibody that specifically binds to GITR of the present invention may be a therapeutically active antitumor compound that is selected from the group of innate or adaptive immune activators.

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

Doses and routes of administration.

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

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

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

It should be noted that dosage values may vary depending on the type and severity of the condition to be alleviated, and may include one or more doses. In addition, it should be understood that for any particular patient, specific dosage regimens must be adjusted over time according to individual need and at the discretion of the healthcare professional administering or supervising the administration of the compositions, and that the concentration ranges given herein are provided only by way of example and are not intended to limit the scope or practice of the claimed compositions. In addition, the dosage regimen of the compositions of this invention may be based on various factors, including the type of disease, age, weight, gender, medical conditions of the patient, severity of the condition, route of administration, and the particular monoclonal antibody used that specifically binds to GITR. Thus, the dosage regimen may vary widely, but can be determined regularly using standard methods. For example, dosages may be adjusted based on pharmacokinetic and pharmacodynamic parameters, which may include clinical effects such as toxic effects or laboratory values. Thus, the present invention covers individual dose escalation, which is determined by a qualified specialist. Determination of the required dosage and regimens are well known in the relevant art and will be apparent to one skilled in the art upon familiarization with the concepts disclosed herein.

Examples of suitable routes of administration are provided above.

It is expected that a suitable dose of a monoclonal antibody that specifically binds to GITR of the present invention will be in the range of 0.1-200 mg/kg, preferably 0.1-100 mg/kg, including about 0.5-50 mg /kg, for example about 1-20 mg/kg. A monoclonal antibody that specifically binds to GITR can be administered, for example, at a dose of at least 0.25 mg/kg, for example, at least 0.5 mg/kg, including at least 1 mg/kg, for example , at least 1.5 mg/kg, for example as well as at least 2 mg/kg, for example at least 3 mg/kg, including at least 4 mg/kg, for example at least 5 mg /kg; and for example up to a maximum of 50 mg/kg, including up to a maximum of 30 mg/kg, for example, up to a maximum of 20 mg/kg, including up to a maximum of 15 mg/kg. Administration will be repeated usually at suitable intervals, for example once a week, once every two weeks, once every three weeks or once every four weeks, and for as long as is considered appropriate by the responsible physician, which may in some cases increase or reduce dose if necessary.

Diagnostic uses and compositions.

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

Examples

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

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

Materials and general methods.

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

Recombinant DNA methods.

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

Gene synthesis.

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

Determination of DNA sequences.

DNA sequences were determined by Sanger sequencing.

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

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

Expression vectors.

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

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

- 38 044786

Example 1.

Production of recombinant antigens and antibodies in suspension culture of mammalian cells.

To prepare recombinant antigens based on the sequence of the extracellular part of human GITR and orthologs, a number of constructs were created containing the extracellular domain of the human GITR antigen (https://www.uniprot.org/uniprot/Q9Y5U5), mcyGITR Macaca mulatta (https://www. uniprot.org/uniprot/Q1PBC4) and musGITR Mus musculus (https://www.uniprot.org/uniprot/O35714). Gene sequences were synthesized using PCR from a template encoding the full-length human GITR antigen from the plasmid vector RDC0359 (https://www.rndsystems.com/products/human-gitr-tnfrsf18-np_004186-versaclone-cdna_rdc0359) and assembled from synthetic oligonucleotides for GITR ortholog genes. The GITR gene sequence was cloned into a plasmid for protein production in mammalian cells with the Lama glama IgG1 Fc tag (Fig. 1) at the SalI/NotI restriction sites. In addition, the antigen sequence is separated from the Fc protease-labile site of TEV protease, which was also used to cleave the Fc fragment by treatment with TEV protease and produce a tag-free version of the antigen.

The sequence was also cloned into a plasmid containing the FLAG-EPEA-tag (C-tag GE Healthcare) at the C-terminus of the protein (Fig. 2) at the SalI/NotI restriction sites. In addition, a similar cloning of the human recombinant GITR ligand (GITRL) https://www.uniprot.org/uniprot/Q9UNG2) was carried out into a plasmid containing the homotrimerizing foldon domain [1] and EPEA-tag on the C-terminus GITRL (Fig. 3). Plasmids were produced in the required quantities in E. coli cells and purified using the Maxiprep Qiagen kit.

Antibodies and antigens were produced in cells of a permanent cell line derived from Chinese hamster ovary cells (line CHO-K1), according to published protocols [Biotechnol Bioeng. 2005 Sep 20; 91(6):670-677, Liao Metal., 2004; Biotechnol Lett. 2006 Jun; 28(11):843-848; Biotechnol Bioeng. 2003 Nov 5; 84(3):332-342]. Cells constitutively expressing the EBNA1 protein gene (Epstein-Barrvirus nuclear antigen 1) were used. Suspension cultivation was carried out in flasks on an orbital shaker, using serum-free media from Life Technologies Corporation and according to the manufacturer's instructions. For transient expression, cells were transfected at a concentration of 2x10 ⁶ /ml using linear polyethylenimine (PEI MAX, Polysciences). The DNA/PEI ratio was 1:3/1:10. 5-7 days after transfection, the culture medium was centrifuged at 2000 g for 20 min and filtered through a filter with a pore size of 0.22 μm. Target proteins were isolated from the culture liquid using affinity chromatography.

Recombinant proteins GITR and GITRL, containing an EPEA-tag (glutamic acid-proline-glutamic acidalanine) at the C-terminus of the protein, were isolated and purified from the culture liquid using the CaptureSelect C-tag Affinity Matrix sorbent. The culture liquid was passed through a chromatographic column pre-filled with 5 ml of C-tag sorbent, then the column was washed with 25 ml of PBS to wash out nonspecifically binding components. Bound antigen was eluted under mild conditions using 20 mM Tris, 2M MgCl ₂ pH7.0-7.4. The protein was then transferred to PBS (pH 7.4) by dialysis using a semi-permeable dialysis membrane, filtered (0.22 μm), transferred to tubes and stored at -70°C.

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

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

Example 2.

Creation of a naive Fab library of human antibodies MeganLibTM.

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

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

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

18218-30].

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

Example 3.

Generation of llama-human hybrid antibody Fab libraries (CHARMs).

Total B cell RNA from individual blood samples immunized with recombinant Lama Glama GITR antigens that demonstrated the highest specific serum titer was isolated using the RNeasy Mini Kit according to the proposed protocol (from QIAGEN). RNA concentration was determined using a Nanovue kit (from GE Healthcare) and the quality of the isolated RNA was checked using 1.5% agarose gel electrophoresis.

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

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

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

Table 1

Order number Cipher Name Type of libraries/type of affiliation Repertoire, CFU Concentration, units/ml 1 Libl-60 BCD166 IL KalifVHH.b Ph immune single-domain library from llama Kalif according to the GITR project (plasmid pSCK) 5x1 OE ⁸ 2x10E ¹³ 2 Libl-61 BCD166 IL BlackVHH.b Ph immune single-domain library from llama Black according to the GITR project (plasmid pSCK) 5x1 OE ⁸ 2x10E ¹³ 3 Libl-62 BCD166 Ch_L_VH ILB lack.bph Fab Charm Lib lambda from llama Black according to the GITR project (plasmid pH6) 5x1 OE ⁷ 4x10E ¹³ 4 Libl-63 BCD166 Ch K VH ILB lack.bph Fab Charm Lib kappa from Black llama according to the GITR project (plasmid pH6) 5x1 OE ⁷ 4x10E ¹³ 5 Libl-64 BCD166 Ch_L_VH ILK alif.bph Fab Charm Lib lambda from llama Kalif according to the GITR project (plasmid pHb) 5x1 OE ⁷ ZxYUE ¹³ 6 Libl-65 BCD166 Ch K VH IL Kalif.bph Fab Charm Lib kappa from llama Kalif according to the GITR project (plasmid pHb) 5x1 OE ⁷ 4.7x10E ¹³

- 40 044786

Example 4.

Selection of Fab libraries of phage antibodies.

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

Selection of immune phage libraries was carried out on an EIA/RIA Tube High Binding 5 ml immunotube. Antigen (0.5 ml 2 μg/ml) was adsorbed in carbonate buffer (0.1 M NaHCO ₃ , pH 9.5) overnight at + 4C. Then the tubes were 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 added the full volume (5 ml) of 0.5% milk powder to PBST for driving. The filling alternates from round to round, for example, if in the 1st round it was 0.5% milk in PBST, then in the 2nd round it was 1% BSA in PBST. Incubated for an hour at room temperature on a rocking chair. We washed the tubes with PBST and added 2 ml of filling buffer and phage libraries to a concentration of approximately 2x10 ¹² phage particles per ml. They were incubated with phages for 1-2 hours at room temperature. In subsequent rounds, 4 ml of phage supernatant from the previous round, pre-clarified in a centrifuge at 17,000 g for 10-15 min, was used. The tubes were washed 20 times with PBST. Phages were eluted from the surface of the tubes with 0.5 ml of 0.1 M glycine buffer pH 2.2: glycine buffer was added to the tubes and gently stirred for 15 min at room temperature. Then the phage solution was transferred into clean non-sorbing tubes with 100 μl of 1 M Tris HCl pH8.0 for neutralization. The tubes were kept on ice until the cells were infected.

Bacteriophage M13 after selection is cultivated using E. coli strain TG1 as a host. Amplification is carried out by infecting a culture of the host strain with phages and subsequent growth for 12-15 hours.

After selection, 0.5-1 ml of phages were mixed with cell culture (OD ₆₀₀ = 0.3-0.4) and incubated for 1.5 hours at 37C. Then the cells were pelleted at 3000-4000 rpm for 10-15 min, resuspended in 1 ml of medium and ground into Petri dishes with a selection antibiotic (ampicillin). The colonies were grown in a thermostat at 30C. After 12-15 hours, the number of colonies was calculated and the cells were washed off the dishes with 5-10 ml of LB medium. 100 μl of cell suspension was added to 20 ml of medium with antibiotic (ampicillin). Increased to OD ₆₀₀ =0.35-0.5 per 37. Helper phage K07 was added at the rate of 1 µl per 10 ml of culture (10 ¹⁰ particles per ml) and incubated at 37C for 1.5 hours without shaking. Then an equal volume of medium with one-time ampicillin (100 μg/ml) and two-time kanamycin (40 μg/ml) and two-time IPTG (0.2mM) was added to the cell culture. We moved it to a rocking chair and produced the phage for 4-5 hours at 30C. The cell culture was centrifuged for 25-30 min at 17,000 g and the supernatant was collected into test tubes. The resulting supernatant was then used in the next round of selection or the phage was isolated by sedimentation for subsequent storage.

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

After two to three rounds, bacteriophages were isolated and the specific binding of polyclonal phage to human GITR and nonspecific antigens was analyzed.

Example 5.

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

After 3 rounds of selection of 2 naive MeganLib libraries and 6 immune libraries on the target antigen, specific and nonspecific binding of polyclonal phages obtained after 2 and 3 rounds was checked using ELISA.

The target antigen GITR to analyze the presence of specifically binding phages (2 μg _/ ml) or GCSF, hIL6R-Fc, interferon alpha 2b, Rituximab (2 µg/ml) for the analysis of non-specifically binding phages. Then the plates were 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 added 300 μl/well of 0.5% dry milk in PBST for driving. Incubated for an hour at room temperature on a rocking chair. We washed the PBST plates and added 50 μl of phage solutions obtained after rounds 2 and 3 of selection, diluted with filling buffer from 2 to 256 times. Incubated for an hour at room temperature on a rocking chair. We washed the plates with PBST and applied horseradish peroxidase-conjugated anti-M13 antibody in loading buffer. After incubation for an hour, the plates were washed and 50 μl of the reaction substrate (H ₂ O ₂ 0.02% and TMB) in acetate buffer pH-5.0 was added. Incubate at room temperature in the dark until a slight background appears in negative controls (but no more than 20 min). The reaction was stopped by adding 25 µl per well of 10% H ₂ SO ₄ and the OD in the wells was measured at 450 nm.

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

Antibody variable domain genes from these libraries were recloned into expression

- 41 044786 vector pLL-Fab (Fig. 8) for producing a secreted soluble form of Fab in E. coli cells.

Example 6.

Screening for Fabs that specifically bind human GITR.

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

Example 7.

Analysis of nonspecific binding of selected Fabs to other antigens.

ELISA was also used to analyze the nonspecific binding of the studied Fab fragments to other antigens. The study was carried out as described above, but IL6R-Fc, INFa2b, PCSK9-VG-FE, PD-1-Fc (2.5 μg/ml in 1x carbonate buffer) were used as immobilization antigens. GITR-TEV-Fc (0.2 μg/ml in 1x carbonate buffer) was used as a control for specific binding. All subsequent steps were carried out according to a standard ELISA protocol using a high-throughput automated platform based on the Genetix Qpix2xt (from Molecular Device) and Tecan Freedom EVO 200 (from Tecan) robotic systems. Clones in which the color signal of nonspecific binding did not exceed the background signal and was 5X less than the value of specific binding were considered positive and their genes were sequenced to determine the sequence of the antibody variable domain genes and determine uniqueness. As a result of sequencing, 30 unique sequences were selected for conversion into the full-length IgG1 antibody format.

Example 8.

Production of recombinant antibodies in suspension culture of mammalian cells.

Cloning of the genes for the variable domains of antibodies from example 7 was carried out according to standard methods. For this purpose, PCR products were developed containing genes for the variable domains of the heavy and light chains of antibodies. The heavy chain variable domain was cloned into the pEE-Hc IgG1 vector at the SalI/NheI restriction sites, a schematic map of which is shown in Fig. 9). The light chain variable domain was ligated into the pEE-SK vector using the SalI/BsiWI restriction sites, a schematic map of which is shown in (Fig. 10).

Antibodies were produced in cells of a permanent cell line derived from Chinese hamster ovary cells (line CHO-K1), according to published protocols [Biotechnol Bioeng. 2005 Sep 20; 91(6):670-677, Liao Metal., 2004; Biotechnol Lett. 2006 Jun; 28(11):843-848; Biotechnol Bioeng. 2003 Nov 5;84(3):332-342]. Cells constitutively expressing the EBNA1 protein gene (EpsteinBarrvirus nuclear antigen 1) were used.

To produce antibodies in a transient expression system, CHO-K1-S cells were used. Cells were cultured in flasks with baffles (125, 250, 500, 1000 and 3000 ml) in a mixture of CHO-S-SFM II and FreeStyle CHO media (1:1) with 4 mM glutamine, 0.05 mg/ml gentamicin and 10 μg /ml ciprofloxacin in orbital shaker-incubators at +37C in the presence of 5% CO ₂ , at 110 or 150 rpm, depending on the size of the flask. Cells were subcultured 3 times a week, seeding density 0.2x10 ⁶ cells/ml.

For transfection, cells were seeded the day before at a density of 0.8x10 ⁶ cells/ml; transfection was carried out one day later, in a cell culture with a density of 2x10 ⁶ cells/ml. To prepare the transfection mixture, RPMI-1640 medium with 2 mM glutamine and 0.05 mg/ml gentamicin was used. The vectors were diluted separately in the medium at a rate of 0.75 μg/ml, and the transfecting agent polyethylenimine was diluted separately.

- 42 044786 (PEI), based on PEI:DNA 7:1 by weight. Dilutions of vectors and PEI were mixed and incubated for 10 min at room temperature, after which the transfection mixture was added to the cells and the cells were cultured under standard conditions.

The next day after transfection, a mixture of CHO-S-SFM II and FreeStyle CHO media (1:1) with 0.05 mg/ml gentamicin and 10 μg/ml ciprofloxacin, 1 mM sodium valproate and 10% feeding F12.7 was added to the cells. and the cells were cultured in orbital shaker-incubators at +34C in the presence of 5% CO ₂ at 110 or 150 rpm, depending on the size of the flask. On days 3-4 after transfection, 10% F12.7 feeder was added to the cells.

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

The purity of the resulting protein solution was assessed using SDS gel electrophoresis Fig. 11 and 12. All antibodies meet the purity required for the study of physicochemical properties and activity for cell tests.

table 2

Lot number Protein name Antibody concentration determined by OCTETRED96 device (mg/l) 1161 BCD166-NHVKITSel3-5-H3-25 147.0 1162 BCD 166-NHVKSel3 -1 -С 1 -9 127.9 1163 BCD166-NHVKSel3-l-Al-l 119.5 1164 BCD 166-SPF2Sel3 -4-F1 -3 8 148.0 1165 1208 1209 1210 BCD 166-SPF2Sel3-4-G8-43 BCD 166- 0 l_VH_ChLVHILB Sel2_MP 1_A9_3HC(DEL)_VL_ChLVHILB Sel2_MP 1_A9_3 CL BCD 166- 0 l_VH_ChLVHILB Sel2_MP IB 1120HC(DEL)_VL_ChLVHILB Sel2_MP IB 1120 -CL BCD 166- 0 l_VH_ChLVHILB Sel2_MP IF 1016HC(DEL)_VL_ChLVHILB Sel2_MP IF 1016 -CL 188.0 145.4 163.0 52.0 1211 BCD 166- 0 l_VH_ChKVHILKSel3_MP2_B9_57- HC(DEL)_VK_ChKVHILKSel3_MP2_B9_57 -CK 125.2 1212 BCD 166- 0 l_VH_ChKVHILKSel3_MP2_D2_5HC(DEL)_VL_ChKVHILKSel3_MP2_D2_5 - C.L. 136.0 1213 BCD 166- 01_VH_ChLVHILKSel2_MP2_Gl_5- HC(DEL)_VL_ChLVHILKSel2_MP2_Gl_5CL 109.0 1214 BCD 166- 01_VH_ChKVHILKSel3_MP2_H8_55HC(DEL)_VK_ChKVHILKSel3_MP2_H8_5 5 -CK 187.4

Example 9.

Kinetic analysis of the interaction of anti-GITR IgG1 antibodies with human GITR.

Binding affinity constants of anti-GITR antibodies to human, rhesus and cynomolgus GITR were obtained using an OctetRed 96 instrument according to ForteBio instructions. Antigen 25 μg/ml was nonspecifically immobilized on the surface of second generation amino-reactive sensors (from AR2G) (ForteBio using a standard protocol according to the manufacturer's instructions for preparation

- 43 044786 and immobilization of AR2G sensors).

anti-GITR antibodies.

Antibodies were added at concentrations. The assay was performed at 30°C using PBS containing 0.1% Tween-20 and 0.1% BSA as running buffer.

The resulting baseline-subtracted sensorograms were analyzed using the Octet Data Analysis program (version 8.0) according to the standard procedure using a 1:1 interaction model. The resulting affinity constants are given in Table. 3. All antibodies tested are highly affinity and specific for human GITR.

Table 3

Affinity constants of anti-GITR antibodies when interacting with human GITR

N^mer^ Name Lot number KD(M) 1 BCD166-NHVKITSel3-5-H3-25 1161 6.38E' ¹¹ 2 BCD166-NHVKSel3-l-Cl-9 1162 6.81E' ¹⁰ 3 BCD166-NHVKSel3-l-Al-l 1163 1.96E' ¹⁰ 4 BCD166-SPF2Sel3-4-Fl-38 1164 5.76E- ¹⁰ 5 BCD166-SPF2Sel3-4-G8-43 1165 3.30E' ¹¹ 7 BCD 166-0 IVHChL VHILB Sel2_MP 1_A9_3 HC (DEL)VLChL VHILB Sel2_MP 1 _A9_3 -CL 1208 7.37E' ¹¹ 8 BCD 166-0 IVHChL VHILB Sel2_MP IB 11_20- HC(DEL)_VL_ChLVHILBSel2_MP IB 1120-CL 1209 6.54E' ¹⁰ 9 B CD 166-0 I VH ChL VHILB Sel2_MP I F 10_ 16HC (DEL)VLChL VHILB S el2_MP 1 _F 10_ 16 -CL 1210 1.84E' ¹¹ 10 BCD 166-0 l_VH_ChKVHILKSel3_MP2_B9_57- HC(DEL)_VK_ChKVHILKSel3_MP2_B9_57-CK 1211 3.13E' ¹⁰ AND BCD 166-01_VH_ChKVHILKSel3_MP2_D2_5- HC(DEL)_VL_ChKVHILKSel3_MP2_D2_5-CL 1212 <1.0E' ¹² 12 BCD 166-0 l_VH_ChLVHILKSel2_MP2_Gl_5- HC(DEL)_VL_ChLVHILKSel2_MP2_Gl_5-CL 1213 ^8.50E'09 13 BCD 166-0 l_VH_ChKVHILKSel3_MP2_H8_5 5- HC(DEL)_VK_ChKVHILKSel3_MP2_H8_55-CK 1214 1.98E' ¹⁰

Example 10.

Test to determine anti-GITR specific agonistic activity.

For the test, we used the HEK293-GITR-NFkB-Luc cell line, created on the basis of the Hek-293 cell line, stably expressing GITR on the surface and containing the gene encoding firefly luciferase under the control of the NFkB promoter.

The test was carried out in a 96-well culture plate. The suspension in each well contained HEK293-GITR-NFkB-Luc cells and the test antibody at the concentration indicated on the graph. All components of the suspension were prepared in a nutrient medium for cell cultivation. After adding all components, the plate was incubated at 37°C, 5% CO ₂ and then, using a luminescence assessment kit and a plate reader, the luciferase intensity in the wells was measured.

The results of testing (Fig. 13) the specific activity of anti-GITR monoclonal antibodies revealed that antibodies of lots 1161, 1210 and 1213 are functionally active and exhibit significant agonistic activity with an upper plateau level comparable to both the control antibody anti-GITR3215 antibody and GITRL. These antibodies were renamed according to table. 4.

Table 4

'K^mer^ Old name Lot number New name 1 BCD16 6-NHVKITSel3-S-H3-2 5 1161 BCD166-01-01 3 BCD166- 01_VH_ChLVHILBSel2_MPl_F10 _16- HC(DEL)_VL_ChLVHILBSel2_MP l_F10_16- CL 1210 BCD166-01—Oil 2 BCD166- 01_VH_ChLVHILKSel2_MP2_Gl_ 5- HC(DEL)_VL_ChLVHILKSel2_MP 2_G1_5-CL 1213 BCD166-01-014

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Example 11.

Enzyme-linked immunosorbent assay for interactions of anti-GITR antibodies with GITR from different organisms.

ELISA was used to measure the comparative affinities of antibodies to GITR-Fc from different organisms. For binding assay, wells of ELISA (medium binding from Greiner bio one) plates were coated with 50 μl of human, cynomolgus, mouse GITR-Fc (0.5 μg/ml in 1x carbonate buffer), sealed and incubated overnight at 4°C . All subsequent 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 monkey GITR and did not bind to mouse GITR (Fig. 14). Antibody BCD166-01-011 specifically bound to human GITR, but did not show binding to cynomolgus monkey and mouse GITR (Fig. 15). Antibody BCD166-01-014 specifically bound to human GITR, had much weaker binding to cynomolgus monkey GITR, and was completely absent for mouse GITR (Fig. 16). Therefore, candidate BCD166-01-011 was excluded from further work due to lack of apparent specificity for macaque GITR. Candidates BCD166-01-01 and BCD166-01-014 have been finalized for further development.

Example 12.

Kinetic analysis of the interaction of anti-GITR IgG1 antibodies with macaca mulatta GITR and macaca Cynomolgus GITR.

Binding affinity constants of anti-GITR antibodies to GITR of rhesus and cynomolgus monkeys were obtained using an OctetRed 96 instrument according to ForteBio instructions. Antigen 25 μg/ml was nonspecifically immobilized on the surface of second generation amino-reactive sensors (from AR2G) (ForteBio using a standard protocol according to the manufacturer's instructions for the preparation and immobilization of AR2G sensors).

anti-GITR antibodies.

Antibodies were added at concentrations. The assay was performed at 30°C using PBS containing 0.1% Tween-20 and 0.1% BSA as running buffer.

The resulting baseline-subtracted sensorograms were analyzed using the Octet Data Analysis program (version 8.0) according to the standard procedure using a 1:1 interaction model. The resulting affinity constants are given in Table. 5 and 6. All antibodies tested are highly affinity and specific for macaque GITR, which warrants further study in this relevant in vivo animal model.

Table 5 Affinity constants of anti-GITR antibodies when interacting with macaca mulatta GITR

Our anti-GITR antibody Lot# KD(M) 1 BCD166-01-O1 1285 9.34E ^-11 2 BCD166-01-O14 1284 1.92E- ¹⁰

Table 6

Affinity constants of anti-GITR antibodies for interaction with macaca Cynomolgus GITR

Kame of anti-GITR antibody Lot# KD(M) 1 ECD166-01-01 1285 5.42E- ¹⁰ 2 BCD166-01—014 1284 5.53E-° ^a

Example 13.

Cell-based in vitro assay to determine anti-GITR specific agonist activity of wild-type and mutant candidates with enhanced agonist activity.

For a possible enhancement of the agonistic activity of the anti-GITR antibody BCD166-01-01, a human derivative, based on studies of such substitutions leading to oligomerization of the antibody in WO 2005047327, WO 2006104989 (A2), WO 2007005612 (A2) and Science. 2014 Mar 14; 343(6176):1260-3, the E345R mutation was selected and introduced into the IgG1 Fc region of the antibody. It has been shown that such a mutation can lead to oligomerization of the antibody on the cell surface after binding to antigen, and enhancement of various effector abilities of ADCC, ADCP, CDC, as well as pharmacokinetics (PK). The goal was to obtain an antibody with enhanced agonistic capacity, ADCC, but not CDC. Mutagenesis was carried out according to standard genetic engineering protocols and the recombinant antibody was synthesized according to Example 8. The antibody was named and is used hereinafter as BCD16602-01. Two parent antibodies BCD166-01-01, BCD166-01-014 were also taken into the test.

The test was carried out similarly to the test in example 10.

The results are shown in Fig. 17 indicate a 5-fold increase in the level of activation in the cellular agonistic test of the studied BCD166-02-01 in comparison with the predecessor BCD166-01-01, into which the E345R mutation was introduced, which is caused by antigen-dependent oligomerization of anti-GITR BCD166-02-01 antibodies on the target cell. Moreover, not only the level of the upper activation plateau naturally increased, but also the EC50 value changed more than 20 times in the direction of increase.

- 45 044786

Example 14.

In vitro cell-based antibody-dependent cellular cytotoxicity (ADCC) test for anti-GITR antibodies.

The test examined ADCC activity for candidates BCD166-02-01. Two antibodies BCD166-01-01, BCD166-01-014 were also taken into the test. Additionally, similar to candidate BCD166-01-01, the E345R mutation was introduced into candidate BCD166-01-014 (named BCD166-02-014) to enhance ADCC activity compared to the wild-type form.

The test used the Jurkat-NFAT-Luc-CD16 cell line, created on the basis of the Jurkat cell line, stably expressing CD16 on the surface and containing the gene encoding firefly luciferase under the control of the NFAT promoter; as well as the Hek-293-GITR cell line, created on the basis of the Hek-293 cell line, stably expressing GITR on the surface.

The test was carried out in a 96-well culture plate. The suspension in each well contained a mixture of Jurkat-NFAT-Luc-CD16 effector cells and Hek-293-GITR target cells, as well as the analyzed antibodies at the concentration indicated in the graph. All components of the suspension were prepared in a nutrient medium for cell cultivation. After adding all components, the plates were incubated at 37°C, 5% CO ₂ and then, using a Lucifera assessment kit and a plate reader, the luminescence intensity in the wells was measured.

The results are shown in Fig. 18 indicate a 40-fold increase in the EC50 value in the cellular ADCC test of the studied BCD166-02-01 in comparison with the predecessor BCD166-01-01, into which the E345R mutation was introduced, which is caused by antigen-dependent oligomerization of anti-GITR BCD166-02-01 antibodies on the target cell. A 5-fold increase in the EC50 value in the cellular ADCC test of the studied BCD166-02-14 was also reliably determined in comparison with the predecessor BCD166-01-14, into which the E345R mutation was introduced. Together, the combined data clearly demonstrate a significant positive effect of substituting E345R in the Fc portion of IgG1 antibodies on the various effector characteristics required to create a highly active GITR agonist.

Example 15.

Cellular in vitro test to determine complement dependent cytotoxicity (CDC) for antiGITR antibodies.

The antibodies tested in this test were BCD166-01-01, BCD166-01-014, BCD166-02-01 and the control antibody anti-GITR-3215.

The test was carried out in a 96-well culture plate. The suspension in each well contained HEK-293-GITR cells, as well as the analyzed antibodies at the specified concentration and human serum complement. The plate with the described suspension was incubated for 4 hours at 37°C, 5% CO ₂ . Next, a solution of Alamar blue was added to each well and then incubated at 37°C, 5% CO ₂ . Next, using a plate reader, the fluorescence value in the wells was measured (excitation wavelength 544 nm, emission wavelength 590 nm).

The results are shown in Fig. 19 indicate insignificant CDC activity, no more than 10% up to a concentration of 1 μg/ml, which is the limit for the estimated therapeutic dose expected for humans, for antibodies BCD166-01-01, BCD166-02-01 and the control antibody anti-GITR- 3215. For the candidate BCD166-01-14, the activity at 1 μg/mL CDC was about 30% lysis, indicating a visible effect but possibly little effect in vivo. Together, the combined data clearly indicate little effect of the E345R substitution in the Fc portion of the IgG1 antiGITR candidates.

Example 16.

Cellular in vitro test to determine the cytotoxicity of anti-GITR candidates.

To assess the negative or positive effect of the studied anti-GITR candidates on changes in the number of different populations of immunoreactive cells, this test was performed. This test tested antibodies BCD166-01-01, BCD166-01-014, BCD166-02-01 and the control antibody antiCD20 (rituximab).

PBMCs were isolated from whole blood of healthy donors by Ficoll density gradient centrifugation. The test was carried out in a 96-well culture plate. The suspension in each well contained PBMCs and the antibody indicated on the graph at concentrations of 25, 1, and 0 μg/ml. After mixing PBMCs and antibodies, the plate was incubated for 16 hours at 37°C, 5% CO ₂ . Next, the proportion of CD56+, CD19+, CD3+, CD4+ and CD8+ subpopulations of PBMCs in the suspensions was assessed using direct staining of the suspensions with fluorescently labeled antibodies against the corresponding CDs and subsequent analysis of the cells on a flow cytometer. For CD56+, CD19+, CD3+ cells, the graphs show the proportion of all cells in the analyzed suspension; for CD4+, CD8+ - the proportion of CD3+ cells.

The results presented in Fig. 20-24 indicate an insignificant negative effect of the studied anti-GITR candidates, no more than a 10% decrease in the number of immunoreactive cells compared to the negative control. In the case of the NK cell population, the inventors observed an approximately 30-70% increase in cell number depending on the dose. At the same time, the control anti-46 044786 anti-CD20 body also showed an increase in the percentage of NK cells, but almost naturally a complete depletion of the CD20+ B cell population. Thus, all candidates considered do not exhibit nonspecific cytotoxicity in vitro on human blood cells.

Example 17.

Cellular in vitro test for determining antibody-dependent depletion of nTreg cell population of a human.

One of the main proposed mechanisms for the therapeutic anti-oncogenic action of antiGITR antibodies is believed to be ADCC-dependent depletion of GITR+ cells of the nTreg population. This test tested antibodies BCD166-01-01, BCD166-01-014, BCD166-02-01 and the control antibody antiCTLA4 (ipilimumab).

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

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

Antibody solutions, a suspension of NK cells, and a suspension of nTreg were added to the culture plate. The plate was incubated at 37°C with 5% _CO2 for 16 hours.

Cell suspension samples were stained using fluorescently labeled antibodies against CD3, CD4, CD25, FoxP3 (Biolegend, USA). Samples of stained cell suspensions were analyzed using flow cytometry.

The results of the analysis of antibody-dependent nTreg depletion under the influence of the studied antibodies are presented in Fig. 25 for cell material from donor 1 and FIG. 26 donor 2 indicate a significant 70-100% decrease in the cell population, i.e. depleting activity of the studied anti-GITR candidates especially for donor material 2. In addition, comparative analysis with the control antiCTLA4 antibody ipilimumab demonstrates greater activity in favor of anti-GITR antibodies especially for the final candidate BCD166-02-01.

Example 18.

Cellular in vitro test for the analysis of antibody-dependent depletion of iTreg cell population of a human.

One of the main proposed mechanisms for the therapeutic anti-oncogenic action of antiGITR antibodies is believed to be ADCC-dependent depletion of GITR+ cells in the iTreg population. In this test, antibodies BCD166-01-01, BCD166-01-014, BCD166-02-01 were tested.

PBMCs were isolated from whole blood of healthy donors by Ficoll density gradient centrifugation. Monocytes were isolated from PBMC as a population of cells that bind to culture plastic. To obtain dendritic cells, monocytes were incubated in the presence of 1000 U/ml GM-CSF (Peprotech, USA) and 500 U/ml IL-4 (Thermo Scientific, USA) at 37°C 5% CO ₂ for 120 hours. Added to the culture medium LPS solution (Sigma, USA) to a concentration of 0.5 μg/ml, cells were incubated at 37°C 5% CO ₂ for 48 hours.

Solutions of antibody dilutions, a suspension of dendritic cells, and PBMCs were added to the culture plate. The plate was incubated at 37°C with 5% _CO2 for 120 hours.

Cell suspension samples were stained using fluorescently labeled antibodies against CD3, CD4, CD25, FoxP3 (Biolegend, USA). Samples of stained cell suspensions were analyzed using flow cytometry. Antibodies against GITR BCD166-01-01, BCD166-01-14 and BCD166-02-01 cause iTreg depletion in vitro.

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

Example 19.

Cellular in vitro test to determine the secretion of proinflammatory cytokines IL-2 and IFN-γ under the influence of anti-GITR antibodies in a human cell population.

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

Anti-CD3 and anti-GITR antibodies were immobilized in the wells of a 96-well plate. To do this, add 100 μl of a DPBS solution containing the anti-GITR antibody BCD166-02-01 at the concentration indicated in the graph and the anti-CD3 antibody at a suboptimal concentration into the corresponding wells of the plate, and incubate the plate for 16 hours at room temperature.

The test was performed in a 96-well plate with anti-CD3 and anti-GITR antibodies pre-immobilized in the wells. The suspension in each well contained 40,000 PBMCs as well as anti-CD28 antibody at a suboptimal concentration. All suspension components were prepared in RPMI-1640 medium containing 10% FBS. After adding the cell suspension, the plate was incubated for 6 days at 37°C, 5% CO2. On the 4th day of incubation, aliquots of the culture liquid were taken from the wells. Next, the concentration of IL-2 and IFN-γ was determined in the culture liquid on the 4th and 6th days of incubation using

ELISA.

The results of the analysis of the effect of anti-GITR antibodies on the level of secretion of pro-inflammatory cytokines IL-2 and IFN-γ, which determine the anti-cancer effect, are presented in Fig. 28 demonstrate a significant increase in the concentration of these substances in the culture fluid. Thus, candidate BCD166-02-01 demonstrates high activity in activating cytokine secretion, which may indicate a significant anti-oncogenic effect.

Example 20.

Analysis of the binding of antibody BCD166-02-01 to human FcRn, FcgRIIIal58V, FcgRIIIa158F, FcgRIIa131H, FcgRIIa131R, FcgRIIb and FcgRIa receptors.

The binding affinity constants of the antibody BCD166-02-01 to human FcRn, FcgRIIIa158V, FcgRIIIa158F, FcgRIIa131H, FcgRIIa131R, FcgRIIb and FcgRIa were obtained using an OctetRed 96 instrument (from ForteBio). Biotinylated receptors were immobilized on the surface of streptavidin sensors (SA). The association and dissociation of receptors and antibodies were carried out and analyzed in a working buffer (PBS containing 0.1% Tween-20 and 0.1% BSA: with the FcRn receptor with pH6, with the remaining receptors in a buffer with pH7.4.) at 30 °C. The resulting sensorgrams were analyzed according to 1:1 or 2:1 models and affinity constants were calculated using Octet Data Analysis Software 8.0 User Guide Copyright 2011©.

The results of the affinity analysis of BCD166-02-01 for human FcRn, FcgRIIIa158V, FcgRIIIa158F, FcgRIIa131H, FcgRIIa131R, FcgRIIb and FcgRIa are presented in FIG. 29. They demonstrate values that are multiply enhanced in comparison with the literature data and data repeatedly measured in our company for antibodies of the IgG1 isotype, which indicates a noticeable, but not qualitative, effect of the E345R mutation in the Fc fragment on the kinetics of monomeric antibodies adsorbed on these receptors from solution.

Example 21.

Determination of colloidal and thermal stability based on the protein aggregation point using dynamic light scattering.

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

Table 7

Aggregation point BCD166-02-01

Samples studied Aggregation point BCD166-02-01 20 shM Acetate, pH=5.0 73.9°С ±0.1°С 20 mM His, pH=5.5 73.9°С ±0.1°С

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

Similar data were obtained for BCD166-01-014.

Example 22.

Determination of thermal stability at thermal stress of 50°C.

The test samples were placed in an air thermostat and thermostated at 50°C for 72 hours. After heating, intact and stressed samples were transferred for analysis by size exclusion HPLC (SEC HPLC) with a UV detector and the capillary isoelectric focusing method. Chromatography was performed on an Agilent 1100 HPLC system on a Tosoh TSK-Gel G3000SWXL column, detection was carried out at a wavelength of 220 nm. Determination of charge heterogeneity by capillary isoelectric focusing was carried out on a Labchip GX II, Caliper device. The preparation of working solutions and preparation of the chip were carried out according to standard methods using the HT Protein Charge Variant Labeling Kit and Protein Charge Variant Buffer Kit, PerkinElmer.

The resulting stability data for BCD-166 (for BCD166-02-01 and BCD166-01-014) when incubated at 50°C are presented in table. 8 and FIG. 30 (for BCD166-02-01) and 31 (for BCD166-01-014) show combined chromatograms for a 72-hour incubation at 50°C.

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

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Claims (37)

Table 8 Dependence of monomer content according to exclusive HPLC and EF for BCD-166 Buffer solution Conditions for sample under study E HPLC Form profile Aggregate content, % Monomer content, % Fragment content, % Alkaline fraction, % Main fraction, % Acid fraction, % 20 shM Acetate, pH=5.0 Intact 0.396 93.332 1.272 4.57 64.43 31.00 72 h at 50°С 0.432 93,664 0.905 4.01 53.90 37.09 Δ* 0.036 0.332 -0.367 -0.56 -5.53 6.09 20 mM His, pH=5.5 Intact 0.416 99.110 0.474 4.43 66.93 23.59 72 h at 50°С 0.393 99.193 0.410 4.39 60.93 34.69 D* -0.023 0.033 -0.064 -0.09 -6.00 6.10 *Δ - difference between the purity of the stressed sample and the purity of the intact sample (input control), %. Example 23. Evaluation of the antitumor activity of BCD-166 drugs on the model of subcutaneous xenografts. To evaluate the antitumor activity of BCD-166 drugs, a model of subcutaneous tumor xenografts was used. To do this, humanized huNOG-EXL mice were subcutaneously transplanted with 3x10 ⁶ cells of the A2058 line mixed with Matrigel®. On day 7 after cell inoculation, animals were divided into groups, as presented in table. 9. Table 9 Distribution of animals into groups in the experiment to determine the antitumor activity of BCD-166 drugs Group Number of animals Dose BCD-166-01-01 10 20 mg/kg BCD-166-02-01 10 20 mg/kg BCD-166-01-014 9 20 mg/kg Negative control 10 - The drugs were administered intraperitoneally at a dose of 20 mg/kg on days 1, 5, 8, 12 after the animals were divided into groups. The negative control group was injected with histidine buffer. During the experiment, the body weight of the mice and the linear dimensions of the tumor were measured. To calculate the tumor volume, the formula V=LxWxHxn/6 was used. As a criterion for the effectiveness of the study drug, we used the tumor growth inhibition index (TGR), which is calculated taking into account the average tumor volume in the negative control group ( _Vc ) and the drug group ( _Vt ) using the formula: XX I · - K TRO(%)= -^^*100 According to the results of the experiment, the drugs BCD-166-01-014 and BCD-166-02-01 showed significant antitumor activity, the drug BCD-166-01-01 did not show significant antitumor activity. The results are shown in Fig. 32 and 33. CLAIM 1. An isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to GITR, comprising: (a) a heavy chain variable domain comprising: (i) CDR1 with amino acid sequence SEQ ID NO: 1; (ii) CDR2 with amino acid sequence SEQ ID NO: 2; (iii) a CDR3 with amino acid sequence SEQ ID NO: 3, and (b) a light chain variable domain comprising: (i) CDR1 with amino acid sequence SEQ ID NO: 7; (ii) CDR2 with amino acid sequence SEQ ID NO: 8; (iii) CDR3 with amino acid sequence SEQ ID NO: 9. - 49 044786 2. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable domain contains an amino acid sequence that is at least 90% homologous to the amino acid sequence of SEQ ID NO: 4. 3. A monoclonal antibody or an antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable domain contains the amino acid sequence of SEQ ID NO: 4. 4. The monoclonal antibody or antigen binding fragment thereof according to claim 1, wherein the light chain variable domain contains an amino acid sequence that is at least 90% homologous to the amino acid sequence of SEQ ID NO: 10. 5. A monoclonal antibody or an antigen-binding fragment thereof according to claim 1, wherein the light chain variable domain contains the amino acid sequence of SEQ ID NO: 10. 6. Monoclonal antibody or its antigen-binding fragment according to claim 1, where: the heavy chain variable domain contains an amino acid sequence that is at least 90% homologous to the amino acid sequence of SEQ ID NO: 4; the light chain variable domain contains an amino acid sequence that is at least 90% homologous to the amino acid sequence of SEQ ID NO: 10. 7. Monoclonal antibody or antigen-binding fragment thereof according to claim 6, where: the heavy chain variable domain contains the amino acid sequence of SEQ ID NO: 4; the light chain variable domain contains the amino acid sequence of SEQ ID NO: 10. 8. The monoclonal antibody of claim 1, wherein the antibody that specifically binds to GITR is a full-length IgG antibody. 9. The monoclonal antibody of claim 8, wherein the full-length IgG antibody is of the human IgG1, IgG2, IgG3 or IgG4 isotype. 10. The monoclonal antibody according to claim 9, wherein the full-length IgG antibody is of the human IgG1 isotype. 11. The monoclonal antibody of claim 1, wherein the antibody that specifically binds to GITR includes an E345R mutation in the Fc fragment to enhance agonistic properties, antibody-dependent cellular cytotoxicity (ADCC), but not complement-dependent cytotoxicity (CDC). 12. Monoclonal antibody according to claim 1, including a heavy chain containing: (i) an amino acid sequence that is at least 90% homologous to the sequence of SEQ ID NO: 5; or (ii) an amino acid sequence that is at least 90% homologous to the sequence of SEQ ID NO: 6. 13. Monoclonal antibody according to claim 1, including: (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. 14. A monoclonal antibody according to claim 1, comprising a light chain containing an amino acid sequence that is at least 90% homologous to the sequence of SEQ ID NO: 11. 15. A monoclonal antibody according to claim 1, comprising a light chain containing the amino acid sequence of SEQ ID NO: 11. 16. Monoclonal antibody according to claim 1, including: (i) a heavy chain containing an amino acid sequence that is at least 90% homologous to the sequence of SEQ ID NO: 5; a light chain containing an amino acid sequence at least 90% homologous to the sequence of SEQ ID NO: 11; or (ii) a heavy chain containing an amino acid sequence that is at least 90% homologous to the sequence of SEQ ID NO: 6; a light chain containing an amino acid sequence that is at least 90% homologous to the sequence of SEQ ID NO: 11. 17. Monoclonal antibody according to claim 1, including: a heavy chain containing the amino acid sequence of SEQ ID NO: 5; light chain containing the amino acid sequence SEQ ID NO: 11. 18. Monoclonal antibody according to claim 1, including: a heavy chain containing the amino acid sequence of SEQ ID NO: 6; light chain containing the amino acid sequence SEQ ID NO: 11. 19. An isolated nucleic acid that encodes an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 18. 20. The nucleic acid according to claim 19, wherein the nucleic acid is DNA. 21. An expression vector containing the nucleic acid according to any one of claims 19 and 20. 22. A method for obtaining a host cell for producing an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 18, including transforming the cell with a vector according to claim 21. 23. A host cell for producing an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 18, containing a nucleic acid according to any of claims 19 and 20. - 50 044786 24. A method for producing an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 18, consisting in cultivating the host cell according to claim 23 in a culture medium under conditions sufficient to obtain said antibody, followed by isolating and purifying the resulting antibody. 25. A pharmaceutical composition for the treatment of a disease or disorder mediated by GITR, comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 18 in a therapeutically effective amount, in combination with one or more pharmaceutically acceptable excipients. 26. The pharmaceutical composition according to claim 25, intended for the treatment of a disease or disorder mediated by GITR, selected from the group: cervical cancer (cervical cancer), head and neck cancer, gastric cancer, breast cancer (breast cancer), renal cell cancer, CRC (colorectal cancer), OC (ovarian cancer), NSCLC (non-small cell lung cancer). 27. A pharmaceutical composition for treating a disease or disorder mediated by GITR, comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 18 in a therapeutically effective amount and at least one therapeutically active antitumor compound in a therapeutically effective amount. 28. The pharmaceutical composition according to claim 27, intended for the treatment of a disease or disorder mediated by GITR, selected from the group of cervical cancer (cervical cancer), head and neck cancer, gastric cancer, breast cancer (breast cancer), renal cell cancer, colorectal cancer (colorectal cancer), OC (ovarian cancer), NSCLC (non-small cell lung cancer). 29. The pharmaceutical composition according to claim 27, wherein the therapeutically active antitumor compound is selected from a chemotherapeutic agent, an antibody, or an antihormonal agent. 30. Pharmaceutical composition according to claim 29, where the therapeutically active antitumor compound is: (i) is an antibody selected from the group: anti-PD1 antibodies, anti-PD-L1 antibodies, anti-CTLA4 antibodies, anti-4-1BB antibodies, anti-OX40 antibodies, or combinations thereof, or (ii) is a small molecule, or (iii) selected from the group of activators of innate or acquired immunity. 31. The pharmaceutical composition according to claim 30, wherein the antibody according to claims 1 to 18 and at least one therapeutically active antitumor compound are administered sequentially. 32. The pharmaceutical composition according to claim 30, wherein the antibody according to claims 1 to 18 and at least one therapeutically active antitumor compound are administered simultaneously. 33. A method of inhibiting the biological activity of GITR in a subject in need of such inhibition, comprising administering to the subject an effective amount of an antibody or antigen binding fragment thereof according to any one of claims 1 to 18. 34. A method of treating a disease or disorder mediated by GITR, comprising administering to a subject in need of such treatment an antibody or antigen binding fragment thereof according to any one of claims 1 to 18 or a pharmaceutical composition according to claims 25 or 27 in a therapeutically effective amount. 35. A method for treating a disease or disorder according to claim 34, where the disease or disorder is selected from the group: cervical cancer (cervical cancer), head and neck cancer, stomach cancer, breast cancer (breast cancer), renal cell cancer, colorectal cancer cancer), OC (ovarian cancer), NSCLC (non-small cell lung cancer). 36. Use of an antibody or antigen binding fragment thereof according to any one of claims 1 to 18 or a pharmaceutical composition according to claim 25 or 27 for treating, in a subject in need of such treatment, a disease or disorder mediated by GITR. 37. Use according to claim 36, where the disease or disorder is selected from the group: cervical cancer (cervical cancer), head and neck cancer, stomach cancer, breast cancer (breast cancer), renal cell cancer, colorectal cancer, ovarian cancer (ovarian cancer), NSCLC (non-small cell lung cancer). -