EA037973B1 - Gitr antibodies, methods and uses - Google Patents

Abstract

Provided herein are antibodies that specifically bind to GITR. Also described are related polynucleotides capable of encoding the provided GITR-specific antibodies or antigen-binding fragments, cells expressing the provided antibodies or antigen-binding fragments, as well as associated vectors and detectably labeled antibodies or antigen-binding fragments. In addition, methods of using the provided antibodies are described. For example, the provided antibodies may be used to enhance an immune response in a subject against cancer.

Description

Cross-references to related claims

This application claims the priority of Provisional U.S. Patent Application No. 62/305270, filed March 8, 2016, and Provisional U.S. Patent Application No. 62/407106, filed October 12.

2016 year. The entire content of the aforementioned applications is incorporated herein by reference in its entirety.

Sequence listing

The currently pending application contains a sequence listing electronically filed in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy of this list, created on February 21, 2017, is named JBI5082USNP_SL.txt and is 92,231 bytes in size.

The technical field to which the invention relates

As provided herein, the description relates to monoclonal antibodies that specifically bind to the glucocorticoid-induced tumor necrosis factor receptor (GITR), and to methods of making and using the disclosed antibodies.

State of the art

Glucocorticoid-induced TNFR-associated protein (GITR; also called AITR, TNFRSF18, or CD357), which belongs to the TNFR superfamily, is expressed in many components of the innate and acquired immune systems and stimulates the formation of acquired and innate immunity (Nocentini G et al., (1994 ) PNAS 94: 6216-6221; Hanabuchi S et al., (2006) Blood 107: 3617-3623; Nocentini G & Riccardi C (2005) Eur J Immunol 35: 1016-1022; Nocentini G et al., (2007) Eur J Immunol 37: 1165-1169). This protein is expressed in several cells and tissues, including T-, B-, dendritic (DC) natural killer (NK) cells, and is activated by its ligand, GITR-L, which in most cases is expressed on antigen-presenting cells (APC ), on endothelial cells, as well as on tumor cells.

The GITR-GITRL system is involved in the formation of an autoimmune / inflammatory response and enhances the response to infection and tumors. For example, the administration of the GITR-Fc fusion protein to animals facilitates the course of autoimmune / inflammatory diseases, and the GITR signal is effective in the treatment of viral, bacterial and parasitic infections, and also stimulates the immune response against tumors (Nocentini G et al., (2012) Br J Pharmacol 165 : 2089-99). These effects are associated with several concurrent mechanisms, including co-activation of effector T cells, inhibition of regulatory T cells, modulation of NK and dendritic cell function, activation of macrophages, and regulation of the extravasation process. Membrane expression of GITR increases after T cell activation (Hanabuchi S et al, (2006) supra; Nocentini G & Riccardi C supra). Its launch leads to co-activation of effector T-lymphocytes (McHugh RS et al, (2002) Immunity 16: 311-323; Shimizu J et al, (2002) Nat Immunol 3: 135-142; Roncheti S et al, (2004) Eur J Immunol 34: 613-622; Tone M et al, (2003) PNAS 100: 15059-15064). GITR activation increases resistance to tumors and viral infections, it is involved in autoimmune / inflammatory processes and regulates leukocyte extravasation (Nocentini G & Riccardi C (2005) supra; Cuzzocrea S et al, (2004) J Leukoc Biol 76: 933-940 ; Shevach EM & Stephens GL (2006) Nat Rev Immunol 6: 613-618; Cuzzocrea S et al, (2006) J Immunol 177: 631-641; Cuzzocrea S et al, (2007) FASEB J 21: 117-129) ...

The expression level of human GITR in peripheral (non-activated) T cells is very low. After activation of T cells, the level of GITR rises sharply in CD4 'and CD8 ⁺ cells for several days (Kwon B et al., (1999) J Biol Chem 274: 6056-6061; Gurney AL et al., (1999) Curr Biol 9: 215-218; Ronchetti S et al, (2004) supra; Shimizu J et al, (2002) supra; Ji HB et al, (2004) supra; Ronchetti S et al, (2002) Blood 100: 350-352 ; Li Z et al., (2003) J Autoimmun 21: 83-92), with a higher level of GITR expression in CD4 + cells than in CD8 ⁺ cells (Kober J et al., (2008) Eur J Immunol 38 (10): 267888; Bianchini R et al., (2011) Eur J Immunol 41 (8): 2269-78).

The role of human GITR in modulating immune responses indicates that it may be a suitable target for antibody-based therapy for diseases such as cancer. Anti-GITR antibodies are described (for example, in WO 200610502, WO 2011028683, WO 2015031667, WO 20150353637, WO 2015187835, WO 2015184099, US 9255151 and US 9255152), but there is an urgent need for new agents and methods for modulating GITR activity against diseases such as malignant neoplasm.

Disclosure of the essence of the invention

This document provides antibodies that specifically bind to GITR, as well as antigen-binding fragments thereof. In addition, corresponding polynucleotides capable of encoding the proposed GITR-specific antibodies and antigen-binding fragments, cells expressing the proposed antibodies and antigen-binding fragments, as well as corresponding vectors and detectably labeled antibodies and antigen-binding fragments are described. Moreover, methods of using the proposed antibodies and antigen-binding fragments are described. For example, due to the role that GITR plays in modulating the immune response, GITR specific antibodies can be used to treat a variety of diseases or disorders associated with GITR in which modulation of the immune response is desired. For example, GITR-specific antibodies can be used in a wide variety of immunotherapy treatments, such as in the treatment of various forms of malignant neoplasms.

GITR-specific antibodies

This document describes isolated antibodies and antigennegative fragments specific for GITR. In some embodiments, the GITR-specific antibodies and antigen-binding fragments bind to human GITR. In some embodiments, the GITR-specific antibodies and antigen-binding fragments bind to human GITR and cynomolgus GITR. In some embodiments, the GITR-specific antibodies and antigen-binding fragments bind to an epitope comprising one or more residues from the extracellular domain (ECD) of GITR as set forth in SEQ ID NO: 59. Such a GITR-specific antibody or antigen-binding fragment can bind to a GITR binding affinity of 30 nM or less, can induce an increase in luciferase expression in an NF-kB luciferase gene assay, and can induce in vitro ADCC with an EC ₅₀ of 67 ng / ml or less.

Table 1 provides a summary of examples of some of the GITR-specific antibodies described herein.

Table 1. CDR sequences of mAbs engineered against human GITR (SEQ ID NO :)

ID HC-CDR1 HC-CDR2 HC-CDR3 LC-CDR1 LC-CDR2 LC-CDR3 TRGB5 GFTFSGYW (1) ISGSGGST (5) AKDFYWDAFDY (12) QSVSSY (28) DAS (32) QQRSNWPLT (35) TRGB14 GFTFSSYA (2) ISGSGGST (5) AKPIRGLDY (13) QSVNNF (29) DAS (32) QQGFNAPLT (36) TRGB20 GFTFSGYW (1) ISSDGGSK (6) AKEWYDHYAALDY (14) QSVNSF (30) YAS (33) QQYIRWPLT (37) TRGB23 GGTFSSYA (3) IIPIFGTA (7) ARHGNWLHFNLDY (15) QSVSSY (28) DAS (32) QQRSNWPLT (35) TRGB25 TRGB190 GGTFSSYA (3) IIPIFGTA (7) ARHRRFWLDY (16) QSVSSY (28) DAS (32) QQRSNWPLT (35) TRGB31 GYSFTSYW (4) IDPSDSDT (8) ARVFPYYGLVLDY (17) QSVSSY (28) DAS (32) QQRSNWPLT (35) TRGB34 GYSFTSYW (4) IYPGDSDT (9) ARDYGWHDFDY (18) QSVSSY (28) DAS (32) QQRSNWPLT (35) TRGB35 GYSFTSYW (4) IDPGDSDT (10) ARHRWSTSLLLDY (19) QSVSSY (28) DAS (32) QQRSNWPLT (35) TRGB120 GGTFSSYA (3) IIPIFGTA (7) ARPRRNTNELDY (20) QSISSY (31) AAS (34) QQSYSTPLT (38) TRGB127 GGTFSSYA (3) IIPIFGNA (11) ARHVYKRGVLNY (21) QSISSY (31) AAS (34) QQSYSTPLT (38) TRGB134 GGTFSSYA (3) IIPIFGTA (7) ARHRWGSGNLDY (22) QSVSSY (28) DAS (32) QQRSNWPLT (35) TRGB144 GGTFSSYA (3) IIPIFGTA (7) ARHGFQRGYLDY (23) QSVSSY (28) DAS (32) QQRSNWPLT (35) TRGB153 GGTFSSYA (3) IIPIFGTA (7) ARHAWLGHLDY (24) QSVSSY (28) DAS (32) QQRSNWPLT (35) TRGB159 GGTFSSYA (3) IIPIFGTA (7) ARHGRNSGRLDY (25) QSVSSY (28) DAS (32) QQRSNWPLT (35) TRGB160 TRGB191 TRGB191. CLF GFTFSNYW (27) ISGSGGST (5) AKDFYWDSFDY (26) QSVSSY (28) DAS (32) QQRSNWPLT (35) TRGB162 GGTFSSYA (3) IIPIFGNA (11) ARHVYKRGVLNY (21) QSVSSY (28) DAS (32) QQRSNWPLT (35)

In some embodiments, a GITR-specific antibody or antigen-binding fragment thereof is provided, comprising a heavy chain containing CDR1, CDR2, and CDR3 of any of the antibodies described in table. 1, and a light chain containing CDR1, CDR2 and CDR3 of any of the antibodies described in table. one.

The IgG class in humans is divided into four isotypes: IgG1, IgG2, IgG3, and IgG4. These isotypes have more than 95% homology in amino acid sequence to the Fc regions, but have significant differences in amino acid composition and structure of the hinge region. The Fc region mediates effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). In ADCC and ADCP, the Fc region of an antibody binds to Fc receptors (FcgRs) on the surface of effector immune cells, such as natural killer cells and macrophages, resulting in lysis and phagocytosis of target cells. In CDC, antibodies destroy target cells by triggering a cascade of complement reactions on the cell surface. The antibodies described herein include antibodies with the described variable domain features in combination with any of the IgG isotypes, including modified versions in which the Fc sequence has been modified to affect various effector functions.

In some embodiments, the antibodies comprise the CDR regions of the antibodies shown in table. 1 above. In some embodiments, the antibodies described are capable of binding to GITR

- 2,037973 with a dissociation constant of 30 nM or less, measured by surface plasmon resonance (SPR). In some embodiments, the disclosed antibodies are capable of inducing an increase in luciferase expression when assayed for the NF-kB luciferase gene. In some embodiments, the disclosed antibodies are capable of inducing ADCC in vitro with an EC _{50 of} 67 ng / ml or less.

Along with the disclosed GITR-specific antibodies and antigen-binding fragments, polynucleotide sequences capable of encoding the disclosed antibodies and antigen-binding fragments are also provided. In addition, vectors are provided containing the disclosed polynucleotides, as well as cells expressing GITR-specific antibodies or antigen-binding fragments provided herein. In addition, cells are described that are capable of expressing the described vectors. These cells can be mammalian cells (eg, 293F cells, CHO cells), insect cells (eg, Sf7 cells), yeast cells, plant cells, or bacterial cells (eg E. coli). The process for the production of the disclosed antibodies or antigen-binding fragments is also provided.

Methods for Using GITR-Specific Antibodies In addition, methods are described in which the described GITR-specific antibodies or antigen-binding fragments are used. Specific antibodies used in the methods described in this section include antibodies with the set of CDRs described for antibodies in table. 1. For example, the key role that GITR plays in the immune response makes it an attractive target for immunotherapy, including inducing or enhancing an immune response against desired tumor antigens or pathogenic antigens (eg, viruses and other pathogenic organisms). Thus, GITR-specific antibodies find application in the treatment of various forms of malignant neoplasms and infectious diseases.

As noted above, GITR activation leads to the simultaneous transmission of an activating signal to CD4 + and CD8 + T cells and prevents the suppression of the immune response by regulatory T cells. Thus, in one embodiment, the GITR-specific antibody is administered to inhibit the suppression of effector T cell activity by regulatory T cells. The assay for such inhibition can be performed in a variety of ways known to those of skill in the art, including, for example, by monitoring T cell proliferation, expression of known activation markers, or cytokine secretion. In another embodiment, a GITR-specific antibody is administered to an individual to reduce the level of regulatory T cells, eg, tumor regulatory T cells. In yet another embodiment, effector T cell activity is induced or enhanced by administration of a GITR-specific antibody as described herein. Specific analysis methods for each of the listed methods are given in the examples section.

GITR Specific Antibody Kits

This document describes kits containing the described GITR-specific antibodies or antigen-binding fragments thereof. The described kits can be used to carry out methods that use the GITR-specific antibodies or antigennegative fragments of the present invention, or other methods known to those skilled in the art. In some embodiments, the described kits may include antibodies or antigen binding fragments described herein and reagents designed to detect the presence of GITR in a biological sample. Accordingly, the kits described may include one or more antibodies or antigen-binding fragments thereof described herein, a vessel for storing the antibody or fragment when not in use, instructions for using the antibody or fragment, an antibody or fragment immobilized on a solid support, and / or detectably labeled forms of an antibody or fragment as described herein.

Brief Description of Drawings

FIG. 1A-1C. Agonist activity exhibited by anti-GITR mAb as measured by a standard screening panel. The data presented is the Cell-Titer Glo signal from cells that were treated with the indicated reagent after transfection of either the NFkB luciferase reporter gene alone (Luc) or a GITR expression vector with the NF-kB luciferase reporter gene (GITR-Luc). Antibodies that produced a signal in excess of that observed for PBS were tentatively categorized as agonists.

FIG. 2A-2E. Agonist activity exhibited by anti-GITR mAb in next generation sequencing panel. The data shown is the Cell-Titer Glo signal from cells that have been treated with this reagent after transfection of either the NF-kB luciferase reporter gene alone or a GITR expression vector with the NF-kB luciferase reporter gene.

FIG. 3A-3D. Effect of anti-GITR antibody ligation on NF-kB activity. These results reflect SEAP activity as an indicator of NF-kB activation in HEK-Blue NF-kB cells stably transfected with GITR. Cells were treated with various concentrations of anti-GITR antibody in the absence (3A, 3B) or in the presence (3C, 3D) of 25 ng / ml soluble GITR ligand. Wednesday and

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CNTO3930 was used as a control without antibody and with isotypic antibody.

FIG. 4A and 4B. Effect of anti-GITR antibodies on memory T cell responses to CMV and TT.

Seroreactive PBMCs were washed with CMV and TT antigen in the absence or presence of anti-GITR antibodies, isotypic control antibody CNTO3930, or in the absence of antibodies. The supernatant was taken and analyzed for the presence of IFNy.

FIG. 5A and 5B. Antitumor activity of a single agent surrogate anti-GITR antibody (DTA-1) in the syngeneic MC38 colon carcinoma model. DTA-1 or isotypic rat IgG2b was administered 200 μg / mouse to animals on the days marked with black arrows (n = 10 per group), starting from the moment when tumor volumes reached 100 mm ³ . DTA-1 administration resulted in complete tumor regression in 5/10 animals.

FIG. 6A-6C. The combination of an anti-GITR surrogate antibody (DTA-1) and an anti-PD-1 antibody (RMP114) results in synergistic antitumor activity in the syngeneic MC38 colon carcinoma model. Isotypic rat IgG2b (6A), DTA-1 (6B) or DTA-1 + RMP1-14 (6C) were administered at 100 μg / antibody / mouse to animals on the days marked with black arrows (n = 10 per group), starting from the moment when the tumor volumes reached 200 mm ³ . The combined treatment led to tumor regression in 5/10 animals.

FIG. 7A-7C. The combination of an anti-GITR surrogate antibody (DTA-1) and an anti-CTLA-4 antibody (9D9) results in synergistic antitumor activity in the syngeneic MC38 colon carcinoma model. Isotypic rat IgG2b (7A), DTA-1 (7B) or DTA-1 + 9D9 (7C) were administered at 100 μg / antibody / mouse to the animals on the days marked with black arrows (n = 10 per group), starting from the moment when tumor volumes reached 200 mm ³ . The combined treatment led to tumor regression in 3/10 animals.

FIG. 8A-8D. The combination of an anti-GITR surrogate antibody (DTA-1) and an anti-OX-40 antibody (OX86) is more effective than administering OX-86 alone in the syngeneic MC38 colon carcinoma model. Three injections of isotypic rat IgG2b (8A) or DTA-1 (8B) were administered at 100 μg / antibody / mouse to the animals on the days marked with black arrows (n = 10 per group), starting from the moment when tumor volumes reached 200 mm ³ . DTA-1 (d1) sequencing followed by OX-86 (d5, d9, Fig. 7D) was more efficient than isotypic Ab followed by OX-86 (7C).

FIG. 9A-9F. Combination anti-GITR / anti-PD-1 therapy with vaccination stimulates the expansion, function and differentiation of Ag-specific CD8 + T cells. Intact B6 mice without tumor (n = 5 / group) were immunized once with peptide OVA _257-264 (day 0) along with mono or combination therapy: 200 μg anti-GITR or control rat IgG on days 0, 3 and 6 and 200 μg anti- PD-1 on days 3, 6, 9 and 12. Desired immune responses were monitored on day 7 (d7) and day 14 (d14) in blood and / or spleen. A, ELISpot assay of IFNY-secreting T cells from the spleen of mice stimulated with OVA _257-264 -specific peptide (d7). B, bar graph plots polyfunctional subpopulations of single, double and triple positive CD8 + T cells secreting effector cytokines IFNy, TNFa, and IL-2 in response to OVA _257-264 stimulation in the spleen (d7). C, cytolytic phenotype profile (d7). D, OVA-specific CD8 + T cells in peripheral blood at d7. Dot plots representing data for 5 mice given in D. E, OVA-specific CD8 + T cells in peripheral blood at d14. EF, OVA differentiation of tetramer-specific CD8 + memory T cells in blood taken from treated mice at d14 after immunization. Each of the above experiments was repeated at least twice with similar results. * P <0.05; ** P <0.01; *** P <0.001. Error bars indicate the standard error of the mean (STEM). EM: memory effector cells; CM: central memory cells.

FIG. 10A-10D. In vivo combination therapy in combination with vaccination promotes B16-OVA tumor rejection in mice. And, for the developed B16-OVA tumors (~ 30-40 mm ³ ), treatment with the indicated drugs was carried out. B, time control was performed for individual tumor response, tumor group measurements (mean +/- SEM, C) and survival (D). The graph reflects the average tumor volume for the groups of animals studied, and the graph shows the indicator without tumor / total (C). The graphs show the results of 1 of 3 independent experiments. * P <0.05; ** P <0.01; *** P <0.001.

FIG. 11A-11D. Vax / anti-GITR / anti-PD-1 combination therapy provides a synergistic effect of enhancing the frequency and function of vaccine-induced antigen-specific CD8 + TIL responses. Provides a summary of intracellular cytokine staining for IFNy, TNFa, IFNy / TNFa and CD107a / IFNy in CD8 + TIL after stimulation with the restricted peptide OVA _257-264 (CD8) (AB) or stimulation with PMA / ION (D) after 12-15 days after tumor implantation. C, bar graph represents the percentage of H2-K ^b -SIINFEKL-restricted OVA tetramer-specific CD8 + TIL from all CD45 + cells in the tumor. The experiments were repeated at least twice with similar results. * P <0.05; ** P <0.01; *** P <0.001. The error intervals reflect the standard error of the mean for n = 4-5 / group.

FIG. 12A-12D. The combination therapy increases the infiltration of CD8 + T cells and decreases the frequency of Treg in B16-OVA tumors. A, Treg percentages determined from spleen samples from non-tumor mice from FIG. 9B-E, cohorts of B16-OVA tumor mice received combinations of Vax, anti-GITR, or -PD-1 (as in FIG. 10). B, CD8 ⁺ TIL as a percentage of total CD45 + cells 15 days after tumor implantation. CD. Representative flow cytometric dot plots and pooled data represent the percentage of Tregs for CD45 + TIL and the ratio of CD8 'effector T cells to Tregs in tumors from treated mice 15 days after tumor implantation. Statistical analyzes were compared with Vax / anti-GITR / anti-PD-1. Results reflect data from 2-3 independent experiments with 4-5 mice in each group. * P <0.05; ** P <0.01; *** P <0.001. Error bars indicate st.osh.sr.

FIG. 13A-13D. The efficacy of Vax / anti-GITR / anti-PD-1 depends on CD8 'T cells and treatment induces long-term memory. A. Dosage regimen for the study of therapeutic exhaustion. B6 mice (n = 10 / group) were injected sc with 4x105 B16-OVA tumor cells, and when the diameter of the tumors reached ~ 40 mm ³ , the population of CD8 cells, CD4 cells or NK cells was reduced by injecting 200 μg / mouse mAbs on days 7 , 8, 9, 11, 14, 17; day 8 was the day of initiation of Vax / anti-GITR / anti-PD-1 or IgG treatment. The vaccine was administered on day 8; anti-GITR on days 8 and 14; anti-PD-1 on days 10, 13, 16 and 19 after tumor implantation. B, tumor volume was monitored twice a week (mean +/- SEM). CD, tumor-free mice (n = 6-9 in each group) after combination treatment were re-injected with B16OVA (2x105; C) or B16.F10 (1.5x105; D) cells in the same flank six months after primary tumor rejection. Mice of comparable age were used as a control for repeated administration. Results reflect data from 2-3 independent experiments.

FIG. 14A-14D. Vax / anti-GITR / anti-PD-1 combination therapy results in the expansion of tumor-specific CD8 ⁺ TILs and induces tumor elimination mediated by KLRG1 ⁺ CD8 ⁺ memory effector T cells. A, representative scatter plots represent percentages of H2-K ^b -SIINFEKL-restricted OVA-specific CD8 ⁺ T cells, (B) percentages of KLRG1 ⁺ CD8 ⁺ TIL, and (C) percentages of tetramer-binding KLRG1 ⁺ CD8 ⁺ TIL 15 days after tumor inoculation (4-5 mice / group). D, B6 mice (10 in each group) were injected SC with 4x105 B16-OVA tumor cells, and on day 8, after tumor diameters reached ~ 50 mm ³ , treatment was started as in FIG. 13.200 μg aKLRG1 mAb was administered on days 7, 8, 9, 11, 14, 17, 20; day 8 was the day of initiation of treatment. Tumor volume and survival were monitored twice a week. All graphs show the average +/- SOS. for at least two independent experiments. * P <0.05; ** P <0.01; *** P <0.001.

FIG. 15A and 15B. The dual anti-GITR / anti-PD-1 combination provides a synergistic effect when combined with a TRP2 peptide vaccine to induce regression of advanced B16-OVA tumors. B6 mice (10 in each group) sc injected with 4x10 ⁵ B16-OVA tumor cells; when the tumors reached ~ 50 mm ³ , treatment was started. A, exemplary regimen for TRP2-peptide vaccination alone or in combination with anti-GITR / anti-PD-1 therapy (matched isotypic IgG) for mice with B16-OVA tumors. B, tumor growth was monitored over time, in groups (mean +/- SEM), as well as in individual mice. Experiments were performed at least twice with similar results. ** P <0.01

FIG. 16. Depletion of the CD25 'cell population with Vax / anti-GITR / anti-PD-1 combination therapy did not suppress or enhance tumor efficacy. Combination therapy and 200 μg anti-CD25 dosing was performed as shown in FIG. 5A. Tumor volume was monitored twice a week (the graph shows the mean +/- SEM). Results reflect data from two independent experiments with 10 mice in each group.

FIG. 17A-17C. Anti-KLRGI antibody depletes the target population of KLRG1 ⁺ CD8 ⁺ . AB, intact tumor-free mice were injected with a combination of Vax / anti-GITR / anti-PD-1 and isotype as in FIG. 9. Anti-KLRG1 treated mice were injected with 100 μg of anti-KLRG1 mAb at 2, 4 and 6 days post vaccination. Mice were sacrificed on day 7 after vaccination and lymphocytes were harvested from blood and spleen to assess the expression of CD8, KLRG1 and CD44. A, Percentage of CD8 + T cells in the spleen after anti-KLRG-1 antibody treatment. B, Representative plots of flow cytometry data showing the percentages of KLRG1 ⁺ CD8 ⁺ in blood and spleen, and (C) summary data for the frequency and / or total number of KLRG1 ⁺ CD8 ⁺ CD44 ⁺ and KLRG1 ⁺ CD8 ⁺ Tet ⁺ cells (left and right panels, respectively) in the blood and spleen. Results reflect data from 2 independent experiments with 5 mice in each group. * P <0.05; ** P <0.01; *** P <0.001. Error bars indicate st.osh.sr.

FIG. 18A and 18B contain an example of tumor growth (FIG. 12A) and survival (FIG. 12B) of mice (n = 10 in each group) implanted with B16-0VA cells (400,000), followed by treatment with either the anti-CD122 mAb 5H4 (administered 5 times at intervals of 2-3 days), either with a peptide vaccine complex (1 dose), or a combination thereof, as shown, on the 7th day after implantation.

FIG. 19A-19D. Shows the frequency of TILs harvested on day 16 after B16-OVA implantation (400,000 cells) and the percentage of tumor infiltrating G-MDSCs (CD11b ⁺ Ly6G ⁺ Ly6C ^- ) (Fig.13A) tumor infiltrating cytokine secreting IFNY ⁺ TNFa ⁺ CD8 ⁺ T cells upon ex-vivo stimulation of 5 037973 OVA _257-264 peptide (Fig.13B), H2-K ^b -SIINFEKL OVA-specific CD8 'TIL (Fig.13C) and tumor-infiltrating Tregs (CD4 + CD44 + FOXP3 + CD25 +) (Fig.13D). aCD122: anti-CD122 monoclonal antibody. * P <0.05; ** P <0.01; *** P <0.001; **** P <0.0001.

FIG. 20A-20C. Demonstrates that the anti-CD122 therapy enhances vaccine-induced responses of antigen-specific CD8 + T cells in the periphery of mice without tumors: Percentage of H2-K ^b restricted SIINFEKL-OVA-specific CD8 + T cells (Figure 31A.); the percentage of the population of CD8 + CD122 + T cells after administration of anti-CD122 in the blood of mice without tumor (Fig. 31B), the percentage of the population of CD8 + CD122 + T cells after tumor treatment in mice with tumor (Fig. 31C). aCD122: anti-CD122 monoclonal antibody; Vax: vaccine. * P <0.05; ** P <0.01; *** P <0.001; **** P <0.0001. Error bars indicate st.osh.sr.

FIG. 21A and 21B. Demonstrates survival of mice implanted with B16OVA cells (400,000) followed by anti-CD122 in combination with peptide vaccination (3 doses) (Fig. 32A). The mice receiving the vaccine / anti-CD122 in FIG. 32A, which survived and without tumors, were reintroduced with B16-ova cells; the graph depicts the percentage of mice with rejection of the second tumor provocation (Fig. 32B).

FIG. 22A and 22B. Demonstrates tumor growth and survival in intact mice (n = 10 in each group) that received a B16-0VA cell implant (400,000), followed by treatment with either anti-CD122 or anti-GITR, or a combination of anti-CD122 and anti-GITR, as shown on day 4 after implantation. * P <0.05; ** P <0.01; *** P <0.001; **** P <0.0001.

FIG. 23. Difference in deuteration levels for each GITR-CED segment with or without TRGB191.CLF. Each block corresponds to peptides that can be mapped and the level of exchange compared to the control after 60, 600, 3600 and 14 400 sec. Gray blocks: no deuteration protection; dark gray blocks: high protection for mAb binding; light gray blocks: moderate protection with mAb binding.

FIG. 24. Structural model with spatial filling of GITR ECD monomer with epitope TRGB191 highlighted in black.

FIG. 25A and 25B. ADCC activity for TRGB191.CLF on primary CD4 ⁺ and CD8 ⁺ T cells at rest and in an activated state. TRGB191.CLF and CNTO3930 (isotypic control antibody) were assessed for ADCC activity on resting CD4 ⁺ (A, left panel) and resting CD8 ⁺ T cells (B, left panel) or activated CD4 ⁺ ( A, right panel) and CD8 ⁺ T cells (B, right panel). The percentage of specific lysis is given in the form of mean ± standard error of the mean (STEM). E: T ratios are shown in the legend. N = 3 replicates, n = 12 per data point. [Ab] = antibody concentration.

FIG. 26. ADCC activity for TRGB191.CLF on the JJN-3 cell line. TRGB191.CLF and CNTO3930 (isotypic control antibody) were evaluated for ADCC activity using JJN-3 target cells and NK-92 158 V / V effector cells. The percentage of specific lysis is given in the form of mean ± standard error of the mean (STEM). N = 6 replicates, n = 12 to 24 per data point. [Ab] = antibody concentration.

FIG. 27. TRGB191.CLF and isotypic control antibody CNTO3930 were evaluated for ADCC activity on JJN-3 target cells and in vitro differentiated T _reg as target cells using NK-92 158 V / V effector cells. The percentage of specific lysis is given in the form of mean ± standard error of the mean (STEM). N = 3 replicates, n = 12 per data point. [Ab] = antibody concentration, EC ₅₀ = half maximum effective concentration, B _max = maximum lysis.

FIG. 28A and 28B. ADCC activity for TRGB191.CLF using high and low affinity effector cells of FcyRIIIA polymorphisms. TRGB191.CLF and CNTO3930 were evaluated for ADCC activity on JJN-3 target cells using NK-92 effector cells expressing (A) high affinity 158V / V variant or (B) low affinity 158F / F variant. The percentage of specific lysis is given in the form of mean ± standard error of the mean (STEM). N = 3 replicates, n = 12 per data point. [Ab] = antibody concentration, EC ₅₀ = half maximum effective concentration, B _max = maximum lysis.

FIG. 29. Treatment with DTA-1 + FGK4.5 leads to more complete tumor regression in the MC38 model, starting from tumor volumes of 100 mm ³ .

FIG. 30. Treatment with DTA-1 + FGK4.5 leads to more complete tumor regression in the MC38 model, starting with tumor volumes of 230 mm ³ .

FIG. 31. Treatment with DTA-1 + OX86 leads to more complete tumor regression in the MC38 model, starting from tumor volumes of 100 mm ³ .

FIG. 32. Treatment with DTA-1 + RMP1-14 leads to more complete tumor regression in the MC38 model, starting from tumor volumes of 100 mm ³ .

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Implementation of the invention Definitions

Various terms are used throughout the specification and claims to relate to aspects of the specification. Such terms shall have their usual meaning in the art, unless otherwise indicated. Other specifically defined terms are to be interpreted according to the definitions given in this document.

When used in this description and in the appended claims, the singular includes the plural, unless the text clearly indicates otherwise. For example, the designation “cell” includes a combination of two or more cells and the like.

In the context of this document, the term about when indicating a measurable quantity, such as quantity, duration in time, and the like, is considered to encompass deviations up to ± 10% of the indicated value, since such deviations are acceptable for the implementation of the described methods. Unless otherwise indicated, all numbers expressing quantities of ingredients, characteristics such as molecular weight, reaction conditions, and the like used in the specification and claims are to be understood as modified in all instances by the term about. Accordingly, unless otherwise indicated, the numerical parameters specified in the following description and the accompanying claims are approximate values that may vary depending on the desired properties that you want to obtain by means of the present invention. As a last resort, but not as an attempt to limit the application of the theory of equivalents to the scope of the claims, each numerical parameter should at least be considered in light of the number of significant digits presented and using standard rounding techniques.

Although the numerical ranges and parameters setting the broad scope of the subject matter are approximate, the numerical values set forth in the specific examples are presented as precisely as possible. However, any numerical value by its nature contains certain errors that inevitably follow from the standard deviation found in the corresponding test measurements.

The term isolated means that a biological component (eg, a nucleic acid, peptide, or protein) has been substantially separated, obtained separately from, or purified from other biological components of an organism in which the component occurs naturally, i.e. from other chromosomal and extrachromosomal DNA, RNA and proteins. Thus, nucleic acids, peptides and proteins that have been isolated include nucleic acids and proteins purified by standard purification methods. Isolated nucleic acids, peptides and proteins can be part of a composition and still be considered isolated if such composition is not part of the original nucleic acid, peptide or protein environment. The term also includes nucleic acids, peptides and proteins obtained by recombinant expression in a host cell, as well as chemically synthesized nucleic acids. As used herein, the term isolated antibody or antigen binding fragment means an antibody or antigen binding fragment that is substantially free of other antibodies or antigen binding fragments having other antigenic specificities (e.g., an isolated antibody that specifically binds to GITR is substantially free of antibodies, which specifically bind to antigens other than GITR). However, an isolated antibody that specifically binds to an epitope, isoform, or variant of GITR may have cross-reactivity with other related antigens, for example, from other species (such as GITR species homologues).

As used herein, the terms glucocorticoid-induced TNFR-linked protein and GITR specifically include human GITR protein, for example, as described in GenBank accession # AF241229, NCBI reference sequence: NP_004186.1 and UniProtKB / Swiss-Prot accession number Q9Y5U5 (cf. also Kwon et al 1999, J. Biol Chem 274, 6056-6061). GITR is also known in the scientific literature as AITR, CD357, TNFRSF18, and GITR-D.

As used herein, the terms GITR ligand, GITRL and GITR-L refer to a glucocorticoid-induced TNFR-linked protein ligand. GITRL is also known as activation-induced TNF-linked ligand (AITRL) and is a member of the tumor necrosis factor 18 (TNFSF18) ligand superfamily. GenBank accession # AF125303 contains an example human GITRL nucleic acid sequence. GenBank ™ accession # NP 005083 and SwissProt accession # Q9UNG2 provide examples of human GITRL amino acid sequences. In a specific embodiment, the GITRL is a human GITRL with SEQ ID NO: 65.

Antibody refers to all isotypes of immunoglobulins (IgG, IgA, IgE, IgM, IgD and IgY), including various monomeric, polymeric and chimeric forms, unless otherwise indicated. In particular, the term antibody encompasses polyclonal antibodies, monoclonal antibodies (mAbs), and antibody-like polypeptides such as chimeric antibodies and humanized antibodies.

Antigen binding fragments are any protein structure capable of exhibiting binding affinity for a particular antigen. Antigen binding fragments include fragments obtained by any known method, for example, enzymatic digestion,

- 7 037973 peptide synthesis and recombinant methods. Some antigen-binding fragments are composed of parts of intact antibodies that retain the antigen-binding specificity of the original antibody molecule. For example, antigen binding fragments can contain at least one variable region (heavy chain or light chain variable region) or one or more CDRs of an antibody known to bind to a particular antigen. Examples of suitable antigen binding fragments include, but are not limited to, diabodies and single chain molecules, as well as Fab, F (ab ') 2, Fc, Fabc and Fv molecules, single chain (Sc) antibodies, individual light chains of antibodies, individual heavy chains of antibodies, chimeric fusions of chains of antibodies or CDRs with other proteins, protein scaffolds, monomers or dimers of heavy chains, monomers or dimers of light chains, dimers consisting of one heavy and one light chain, a monovalent fragment consisting of the VL, VH, CL and CH1 domains, or a monovalent antibody described in WO 2007059782, bivalent fragments containing two Fab fragments connected by a disulfide bridge in the hinge region, an Fd fragment consisting essentially of the V.sub.H and C.sub.H1 domains; An Fv fragment consisting essentially of the VL and VH domains of a single arm of an antibody, a dAb fragment (Ward et al., Nature 341, 544-546 (1989)), which consists essentially of a VH domain and is also called a domain antibody (Holt et al; Trends Biotechnol 2003 Nov .; 21 (11): 484-90); a camel-type antibody (camelid) or nanobody (Revets et al; Expert Opin Biol Ther. 2005 Jan .; 5 (1): 111-24); highlighted complementarity determining regions (CDRs); and the like. All antibody isotypes can be used to obtain antigen-binding fragments. In addition, antigen-binding fragments can include non-antigenic protein scaffolds into which polypeptide segments can be successfully inserted in an orientation that confers affinity for a given antigen of interest, such as protein scaffolds. Antigen-binding fragments can be obtained recombinantly or by enzymatic or chemical cleavage of intact antibodies. The phrase "antibody" or antigennegative fragment thereof can be used to indicate that the given antigennegative fragment includes one or more amino acid segments of the antibody related to the specified phrase.

The terms CDR and the plural for CDR refer to the complementarity determining region (CDR), with three such regions defining the binding nature of the light chain variable region (CDRL1, CDRL2 and CDRL3), and three defining the binding nature of the heavy chain variable region (CDRH1, CDRH2 and CDRH3). CDRs contribute to the functional activity of an antibody molecule and are separated by amino acid sequences that include skeletal or framework regions. The precise definition of the boundaries and extent of CDRs depends on various classification and numbering systems. Therefore, CDRs can be distinguished using Kabat, Chothia, contact, or any other boundary definitions. Despite the different boundaries, each of these systems is characterized by a certain degree of overlap in the part that constitutes the so-called hypervariable region within the variable sequences. Thus, CDR definitions in accordance with such systems may differ in length and border regions relative to the adjacent wireframe region. See, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. NIH Publication No. 91-3242 (1991); Chothia et al., Canonical Structures For the Hypervariable Regions of Immunoglobulins, J. Mol. Biol. 196: 901 (1987); and MacCallum et al., Antibody-Antigen Interactions: Contact Analysis and Binding Site Topography, J. Mol. Biol. 262: 732 (1996)), each of which is incorporated herein by reference in its entirety.

Typically, CDRs form a loop structure that can be classified as a canonical structure. The term canonical structure refers to the backbone conformation that antigen binding loops (CDRs) adopt. As a result of comparative structural studies, it was found that five of the six antigen-binding loops have only a limited set of available conformations. Each canonical structure can be characterized by the torsion angles of the polypeptide backbone. Therefore, the corresponding loops between antibodies can have very similar three-dimensional structures, despite the high level of variability of the amino acid sequence in most regions of the loops (Chothia et al., Canonical Structures For the Hypervariable Regions of Immunoglobulins, J. Mol. Biol. 196: 901 (1987) ; Chothia et al., Conformations of Immunoglobulin Hypervariable Regions, I 342: 877 (1989); Martin and Thornton, Structural Families in Loops of Homologous Proteins: Automatic Classification, Modeling and Application to Antibodies, J. Mol. Biol. 263: 800 (1996)), each of which is incorporated by reference in its entirety. Moreover, there is a certain relationship between the formed loop structure and the surrounding amino acid sequences. The conformation of a particular canonical class is determined by the chain length and amino acid residues located at key positions in the loop, as well as within the conserved framework (i.e. outside the loop). Therefore, assignment to a specific canonical class can be made based on the presence of such key amino acid residues.

The term polypeptide is used interchangeably with the term protein and in its broadest sense refers to a compound with two or more subunit amino acids, amino acid analogs, or peptidomimetics. Subunits can be linked by peptide bonds. In another embodiment, the subunit can be linked by other bonds, for example, a complex

- 8 037973 ester, ester, etc. When used in this document, the term amino acid refers to natural and / or artificial, or synthetic amino acids, including glycine, as well as D and L optical isomers, amino acid analogs and peptidomimetics. A peptide of three or more amino acids is usually called an oligopeptide if the peptide chain is short enough. In the case of a long peptide chain, the peptide is usually referred to as a polypeptide or protein. The term specifically binds or binds in a specific manner, or derivatives thereof, when used in the context of antibodies or antibody fragments, refers to binding, via the domains encoded by immunoglobulin genes, to one or more epitopes of a protein of interest without preferentially binding other molecules in a sample containing a mixed population of molecules. Typically, an antibody binds to a related antigen with a Kd of less than about 1x10 ^-8 M as measured in a surface plasmon resonance assay or cell binding assay. Phrases such as [antigen] -specific antibody (eg, GITR-specific antibody) mean that said antibody specifically binds to said antigen.

The term polynucleotide, which is synonymous with the term nucleic acid molecule, nucleotides, or nucleic acids, refers to any polyribonucleotide or polydeoxyribonucleotide, which can be unmodified RNA or DNA or modified RNA or DNA. Polynucleotides include, but are not limited to, single and double stranded DNA, DNA that is a mixture of single and double stranded regions, single and double stranded RNA, RNA that is a mixture of single and double stranded regions, hybrid molecules containing DNA and RNA that may be single stranded or, more typically, double stranded, or a mixture of single and double stranded regions. In addition, a polynucleotide refers to three-stranded regions containing RNA or DNA, or both RNA and DNA. The term polynucleotide also includes DNA or RNA containing one or more modified bases and DNA or RNA with backbones modified for stability or other purposes. Modified bases include, for example, tritylated bases and atypical bases such as inosine. Various modifications can be made to DNA and RNA; therefore, the term polynucleotide encompasses chemically, enzymatically or metabolically modified forms of polynucleotides commonly found in nature, as well as chemical forms of DNA and RNA that are characteristic of viruses and cells. The term polynucleotide also encompasses relatively short nucleic acid chains, often referred to as oligonucleotides.

A vector is a replicon, such as a plasmid, phage, cosmid or virus, into which another nucleotide segment can be operatively inserted so that replication or expression of the segment occurs.

As used herein, the term “host cell” can refer to any type of cell, for example, a primary cell, a cell in culture, or a cell from a cell line. In specific embodiments, the term “host cell” refers to a cell transfected with a nucleic acid molecule and to the descendants or potential descendants of such a cell. The descendants of such a cell may not be identical to the parent cell transfected with the nucleic acid molecule, for example, due to mutations or environmental influences that may occur in subsequent generations, or the integration of the nucleic acid molecule into the genome of the host cell. The terms expression and production are used synonymously herein and refer to the biosynthesis of a gene product. These terms encompass the transcription of a gene into RNA. These terms also encompass the translation of RNA into one or more polypeptides and further encompass all post-transcriptional and post-translational modifications of natural origin.

Expression or production of an antibody or antigen-binding fragment thereof can occur within the cytoplasm of a cell or in an extracellular medium, for example, in a growth medium for cell culture. The meaning of a term is essentially the same and may differ depending on the context in which it is used. Due to the potential for variation in the natural sequence of light and heavy chains and genes encoding them, it is expected that a certain level of variation can be found within the amino acid sequences or genes encoding antibodies or antigen-binding fragments described herein, which practically do not affect them. unique binding properties (eg specificity and affinity). This expectation is partly due to the degeneracy of the genetic code, as well as the evolutionary success of conservative variants of amino acid sequences, which do not appreciably change the nature of the encoded protein. Accordingly, in the context of nucleotide sequences, substantially the same means at least 65% identity between two or more sequences. Preferably, the term refers to at least 70% identity between two or more sequences, more preferably at least 75% identity, more preferably at least 80% identity, more preferably at least 85% identity, more preferably at least 90% identity identity, more preferably at least 91% identity, more preferably at least 92% identity, more preferably at least 93% identity, more preferably at least 94% identity, more preferably by

9 037973 at least 95% identity, more preferably at least 96% identity, more preferably at least 97% identity, more preferably at least 98% identity, and more preferably at least 99% or more identity. The percentage identity between two sequences is a function of the number of identical positions common to the sequences (i.e.% homology = number of identical positions / total number of positions / 100), taking into account the number of gaps and the length of each gap introduced for optimal aligning the two sequences. The percentage identity between two nucleotide or amino acid sequences can be determined, for example, using the algorithm described in E. Meyers and W. Miller, Comput. Appl. Biosci 4, 11-17 (1988), which is embedded in the ALIGN program (version 2.0) using the PAM120 residue weighting table with a gap length penalty of 12 and a gap penalty of 4. In addition, the percent identity of two amino acid sequences can be determined using the algorithm described in Needleman and Wunsch, J. Mol. Biol. 48, 444-453 (1970).

The degree of variation that is possible within the amino acid sequence of a protein without significantly affecting the function of the protein is much lower than in the nucleotide sequence, since the principles of degeneracy are not applicable to amino acid sequences. Accordingly, in the context of an antibody or antigen binding fragment, substantially the same refers to an antibody or antigen binding fragments having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. described antibodies or antigennegative fragments. Other options for implementation include GITR-specific antibodies or antigennegative fragments that have a framework, backbone, or other non-binding regions that do not have significant identity with the antibodies and antigennegative fragments described herein, but necessarily include one or more CDRs or other sequences required for linking that have 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the sequences described herein.

Binding affinity generally refers to the strength of the total sum of non-covalent interactions between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen). Unless otherwise indicated, as used herein, the term binding affinity refers to an internal binding affinity that reflects a 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 determined by the dissociation constant (KD). Affinity can be measured and / or expressed in a variety of ways known to those skilled in the art, including, but not limited to, equilibrium dissociation constant (KD) and equilibrium association (binding) constant (K _A ). KD is calculated from the ratio k _off / k _on , while KA is calculated from the ratio k _on / k _off . kon refers to the rate constant of the association of, for example, an antibody to an antigen, and koff refers to the rate constant of the dissociation of, for example, an antibody to an antigen. kon and koff can be determined by methods known to those skilled in the art, such as surface plasmon resonance.

The term “individual” means human and non-human animals, including all vertebrates, eg mammals and non-mammals, eg non-human primates, mice, rabbits, sheep, dogs, cats, horses, cows, chickens, amphibians and reptiles. In many embodiments of the described methods, the individual is a human.

GITR-specific antibodies and antigen-binding fragments

This document describes isolated monoclonal antibodies or antigennegative fragments that specifically bind to GITR. The general structure of an antibody molecule contains an antigen-binding domain, which includes heavy and light chains, and an Fc domain, which performs various functions, including complement fixation and binding to antibody receptors.

Described GITR-specific antibodies or antigennegative fragments include all isotypes, IgA, IgD, IgE, IgG and IgM, and synthetic multimers of the four-chain structure of immunoglobulins. The disclosed antibodies or antigen binding fragments also include the IgY isotype essentially found in chicken or turkey serum and in the yolk of a chicken or turkey egg.

GITR-specific antibodies and antigen-binding fragments can be obtained from any species by recombinant methods. For example, antibodies or antigen-binding fragments can be murine, rat, goat, equine, porcine, bovine, chicken, rabbit, camel, donkey, human, or chimeric variants. For the purpose of administering to a human, non-human antibodies or antigen binding fragments can be genetically or structurally altered so that they are less antigenic when administered to a human patient.

In some embodiments, the antibodies or antigen binding fragments are chimeric. As used herein, the term chimeric means an antibody or antigen-binding fragment thereof containing at least some part of at least one variable domain derived from the amino acid sequence of an antibody of a non-human mammalian, rodent or reptile, while the remaining portions of the antibody or antigen-binding fragment thereof fragments are of human origin.

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In some embodiments, the antibodies are humanized antibodies. Humanized antibodies can be chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (eg, Fv, Fab, Fab ', F (ab') 2, or other antigen-binding antibody subsequences) containing a minimal sequence derived from a non-human animal immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from the complementarity determining region (CDR) of the recipient are replaced by residues from the complementarity determining region of a non-human species (donor antibody), for example, a mouse, rat or rabbit having the desired specificity, affinity and ability. Typically, a humanized antibody will contain substantially all of at least one, and usually two, variable domains in which all or substantially all of the CDR regions correspond to those in an immunoglobulin from a non-human animal, and all or substantially all the framework regions correspond to those of the human immunoglobulin sequence. A humanized antibody may contain at least a portion of an immunoglobulin constant region (Fc), typically a human immunoglobulin.

Antibodies or antigennegative fragments described herein can take various forms, but will include one or more CDRs of antibodies, as shown in table. 1. This document describes isolated antibodies and antigennegative fragments that specifically bind to GITR. In some embodiments, the GITR-specific antibodies or antigen-binding fragments are human IgG or derivatives thereof. Although the GITR specific antibodies or antigen binding fragments exemplified herein are human, the exemplified antibodies or antigen binding fragments can be chimerized.

In some embodiments, a GITR-specific antibody or antigen-binding fragment thereof is provided, comprising a heavy chain containing CDR1, CDR2, and CDR3 of any of the antibodies described in table. 1, and a light chain containing CDR1, CDR2 and CDR3 of any of the antibodies described in table. one.

In some embodiments, the GITR-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 1, a heavy chain CDR2 comprising SEQ ID NO: 5, a heavy chain CDR3 comprising SEQ ID NO: 12, a light chain CDR1 comprising SEQ ID NO: 28, the light chain CDR2 containing SEQ ID NO: 32, and the light chain CDR3 containing SEQ ID NO: 35. This GITR-specific antibody or antigen-binding fragment may contain human framework sequences. Such a GITR-specific antibody or antigen-binding fragment can bind to a GITR affinity of 30 nM or less, can induce an increase in luciferase expression in an NF-kB luciferase gene assay, and can induce in vitro ADCC with an EC ₅₀ of 67 ng / ml or less. In some embodiments, the GITR-specific antibodies and antigen-binding fragments comprise a heavy chain substantially the same or identical to SEQ ID NO: 39 and a light chain substantially the same or identical to SEQ ID NO: 55. The heavy chain and light chain of the antibodies described in this paragraph are suitable for introduction into bispecific constructs in which one arm is an anti-GITR antibody arm.

In some embodiments, the GITR-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 2, a heavy chain CDR2 comprising SEQ ID NO: 5, a heavy chain CDR3 comprising SEQ ID NO: 13, a light chain CDR1 comprising SEQ ID NO: 29, the light chain CDR2 containing SEQ ID NO: 32, and the light chain CDR3 containing SEQ ID NO: 36. This GITR-specific antibody or antigen-binding fragment may contain human framework sequences. Such a GITR-specific antibody or antigen-binding fragment can bind to a GITR affinity of 30 nM or less, can induce an increase in luciferase expression in an NF-kB luciferase gene assay, and can induce in vitro ADCC with an EC ₅₀ of 67 ng / ml or less. In some embodiments, the GITR-specific antibodies and antigen-binding fragments comprise a heavy chain substantially the same or identical to SEQ ID NO: 40 and a light chain substantially the same or identical to SEQ ID NO: 56 The heavy chain and light chain of antibodies described in this paragraph are suitable for introduction into bispecific constructs in which one arm is an anti-GITR antibody arm.

In some embodiments, the GITR-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 1, a heavy chain CDR2 comprising SEQ ID NO: 6, a heavy chain CDR3 comprising SEQ ID NO: 14, a light chain CDR1 comprising SEQ ID NO: 30, the light chain CDR2 containing SEQ ID NO: 33 and the light chain CDR3 containing SEQ ID NO: 37. This GITR-specific antibody or antigen-binding fragment may contain human framework sequences. Such a GITR-specific antibody or antigen-binding fragment can bind to a GITR affinity of 30 nM or less, can induce an increase in luciferase expression in an NF-kB luciferase gene assay, and can induce in vitro ADCC with an EC ₅₀ .

- 11 037973 equal to 67 ng / ml or less. In some embodiments, the GITR-specific antibodies and antigen-binding fragments comprise a heavy chain substantially the same or identical to SEQ

ID NO: 41, and a light chain substantially the same or identical to SEQ ID NO: 57. The heavy chain and light chain of the antibodies described in this paragraph are suitable for introduction into bispecific constructs in which one arm is an anti-GITR antibody arm.

In some embodiments, the GITR-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 3, a heavy chain CDR2 comprising SEQ ID NO: 7, a heavy chain CDR3 comprising SEQ ID NO: 15, a light chain CDR1 comprising SEQ ID NO: 28, the light chain CDR2 containing SEQ ID NO: 32, and the light chain CDR3 containing SEQ ID NO: 35. This GITR-specific antibody or antigen-binding fragment may contain human framework sequences. Such a GITR-specific antibody or antigen-binding fragment can bind to a GITR affinity of 30 nM or less, can induce an increase in luciferase expression in an NF-kB luciferase gene assay, and can induce in vitro ADCC with an EC ₅₀ of 67 ng / ml or less. In some embodiments, the GITR-specific antibodies and antigen-binding fragments comprise a heavy chain substantially the same or identical to SEQ ID NO: 42 and a light chain substantially the same or identical to SEQ ID NO: 55. The heavy chain and light chain of the antibodies described in this paragraph are suitable for introduction into bispecific constructs in which one arm is an anti-GITR antibody arm.

In some embodiments, the GITR-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 3, a heavy chain CDR2 comprising SEQ ID NO: 7, a heavy chain CDR3 comprising SEQ ID NO: 16, a light chain CDR1 comprising SEQ ID NO: 28, the light chain CDR2 containing SEQ ID NO: 32, and the light chain CDR3 containing SEQ ID NO: 35. This GITR-specific antibody or antigen-binding fragment may contain human framework sequences. Such a GITR-specific antibody or antigen-binding fragment can bind to a GITR affinity of 30 nM or less, can induce an increase in luciferase expression in an NF-kB luciferase gene assay, and can induce in vitro ADCC with an EC ₅₀ of 67 ng / ml or less. In some embodiments, the GITR-specific antibodies and antigen-binding fragments comprise a heavy chain substantially the same or identical to SEQ ID NO: 43 and a light chain substantially the same or identical to SEQ ID NO: 55. The heavy chain and light chain of the antibodies described in this paragraph are suitable for introduction into bispecific constructs in which one arm is an anti-GITR antibody arm.

In some embodiments, the GITR-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 4, a heavy chain CDR2 comprising SEQ ID NO: 8, a heavy chain CDR3 comprising SEQ ID NO: 17, a light chain CDR1 comprising SEQ ID NO: 28, the light chain CDR2 containing SEQ ID NO: 32, and the light chain CDR3 containing SEQ ID NO: 35. This GITR-specific antibody or antigen-binding fragment may contain human framework sequences. Such a GITR-specific antibody or antigen-binding fragment can bind to a GITR affinity of 30 nM or less, can induce an increase in luciferase expression in an NF-kB luciferase gene assay, and can induce in vitro ADCC with an EC ₅₀ of 67 ng / ml or less. In some embodiments, the GITR-specific antibodies and antigen-binding fragments comprise a heavy chain substantially the same or identical to SEQ ID NO: 44 and a light chain substantially the same or identical to SEQ ID NO: 55. The heavy chain and light chain of the antibodies described in this paragraph are suitable for introduction into bispecific constructs in which one arm is an anti-GITR antibody arm.

In some embodiments, the GITR-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 4, a heavy chain CDR2 comprising SEQ ID NO: 9, a heavy chain CDR3 comprising SEQ ID NO: 18, a light chain CDR1 comprising SEQ ID NO: 28, the light chain CDR2 containing SEQ ID NO: 32, and the light chain CDR3 containing SEQ ID NO: 35. This GITR-specific antibody or antigen-binding fragment may contain human framework sequences. Such a GITR-specific antibody or antigen-binding fragment can bind to a GITR affinity of 30 nM or less, can induce an increase in luciferase expression in an NF-kB luciferase gene assay, and can induce in vitro ADCC with an EC ₅₀ of 67 ng / ml or less. In some embodiments, the GITR-specific antibodies and antigen-binding fragments comprise a heavy chain substantially the same or identical to SEQ ID NO: 45 and a light chain substantially the same or identical to SEQ ID NO: 55. The heavy chain and light chain of the antibodies described in this paragraph are suitable for introduction into bispecific constructs in which one arm is an anti-GITR antibody arm.

In some embodiments, the GITR-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 4, a heavy chain CDR2 comprising SEQ ID NO: 10, a heavy chain CDR3 comprising SEQ ID NO: 19, a light chain CDR1 comprising SEQ ID NO: 28, light chain CDR2 containing SEQ ID NO: 32, and light chain CDR3 containing SEQ ID

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NO: 35. This GITR-specific antibody or antigen-binding fragment may contain human framework sequences. Such a GITR-specific antibody or antigen-binding fragment can bind to a GITR affinity of 30 nM or less, can induce an increase in luciferase expression in an NF-kB luciferase gene assay, and can induce in vitro ADCC with an EC50 of 67 ng / ml or less. In some embodiments, the GITR-specific antibodies and antigen-binding fragments comprise a heavy chain substantially the same or identical to SEQ ID NO: 46 and a light chain substantially the same or identical to SEQ ID NO: 55. The heavy chain and light chain of the antibodies described in this paragraph are suitable for introduction into bispecific constructs in which one arm is an anti-GITR antibody arm.

In some embodiments, the GITR-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 3, a heavy chain CDR2 comprising SEQ ID NO: 7, a heavy chain CDR3 comprising SEQ ID NO: 20, a light chain CDR1 comprising SEQ ID NO: 31, the light chain CDR2 containing SEQ ID NO: 34, and the light chain CDR3 containing SEQ ID NO: 38. This GITR-specific antibody or antigen-binding fragment may contain human framework sequences. Such a GITR-specific antibody or antigen-binding fragment can bind to a GITR affinity of 30 nM or less, can induce an increase in luciferase expression in an NF-kB luciferase gene assay, and can induce in vitro ADCC with an EC50 of 67 ng / ml or less. In some embodiments, the GITR-specific antibodies and antigen-binding fragments comprise a heavy chain substantially the same or identical to SEQ ID NO: 47 and a light chain substantially the same or identical to SEQ ID NO: 58. The heavy chain and light chain of the antibodies described in this paragraph are suitable for introduction into bispecific constructs in which one arm is an anti-GITR antibody arm.

In some embodiments, the GITR-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 3, a heavy chain CDR2 comprising SeQ ID NO: 11, a heavy chain CDR3 comprising SEQ ID NO: 21, a light chain CDR1 comprising SEQ ID NO: 31, the light chain CDR2 containing SEQ ID NO: 34, and the light chain CDR3 containing SEQ ID NO: 38. This GITR-specific antibody or antigen-binding fragment may contain human framework sequences. Such a GITR-specific antibody or antigen-binding fragment can bind to a GITR affinity of 30 nM or less, can induce an increase in luciferase expression in an NF-kB luciferase gene assay, and can induce in vitro ADCC with an EC ₅₀ of 67 ng / ml or less. In some embodiments, the GITR-specific antibodies and antigen-binding fragments comprise a heavy chain substantially the same or identical to SEQ ID NO: 48 and a light chain substantially the same or identical to SEQ ID NO: 58. The heavy chain and light chain of the antibodies described in this paragraph are suitable for introduction into bispecific constructs in which one arm is an anti-GITR antibody arm.

In some embodiments, the GITR-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 3, a heavy chain CDR2 comprising SEQ ID NO: 7, a heavy chain CDR3 comprising SEQ ID NO: 22, a light chain CDR1 comprising SEQ ID NO: 28, the light chain CDR2 containing SEQ ID NO: 32, and the light chain CDR3 containing SEQ ID NO: 35. This GITR-specific antibody or antigen-binding fragment may contain human framework sequences. Such a GITR-specific antibody or antigen-binding fragment can bind to a GITR affinity of 30 nM or less, can induce an increase in luciferase expression in an NF-kB luciferase gene assay, and can induce in vitro ADCC with an EC ₅₀ of 67 ng / ml or less. In some embodiments, the GITR-specific antibodies and antigen-binding fragments comprise a heavy chain substantially the same or identical to SEQ ID NO: 49 and a light chain substantially the same or identical to SEQ ID NO: 55. The heavy chain and light chain of the antibodies described in this paragraph are suitable for introduction into bispecific constructs in which one arm is an anti-GITR antibody arm.

In some embodiments, the GITR-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 3, a heavy chain CDR2 comprising SEQ ID NO: 7, a heavy chain CDR3 comprising SEQ ID NO: 23, a light chain CDR1 comprising SEQ ID NO: 28, the light chain CDR2 containing SEQ ID NO: 32, and the light chain CDR3 containing SEQ ID NO: 35. This GITR-specific antibody or antigen-binding fragment may contain human framework sequences. Such a GITR-specific antibody or antigen-binding fragment can bind to a GITR affinity of 30 nM or less, can induce an increase in luciferase expression in an NF-kB luciferase gene assay, and can induce in vitro ADCC with an EC ₅₀ of 67 ng / ml or less. In some embodiments, the GITR-specific antibodies and antigen-binding fragments comprise a heavy chain substantially the same or identical to SEQ ID NO: 50 and a light chain substantially the same or identical to SEQ ID NO: 55. The heavy chain and light chain of the antibodies described in this paragraph are suitable for introduction into bispecific constructs in which one arm is an anti-GITR antibody arm.

- 13 037973

In some embodiments, the GITR-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 3, a heavy chain CDR2 comprising SeQ ID NO: 7, a heavy chain CDR3 comprising SEQ ID NO: 24, a light chain CDR1 comprising SEQ ID NO: 28, the light chain CDR2 containing SEQ ID NO: 32, and the light chain CDR3 containing SEQ ID NO: 35. This GITR-specific antibody or antigen-binding fragment may contain human framework sequences. Such GITR-specific antibody or antigen-binding fragment can bind GITR affinity of 30 nM or less, can induce an increase luciferase expression in the analysis of the luciferase gene and the NF-kB could induce ADCC in vitro with EC5 ₀ equal to 67 ng / ml or less. In some embodiments, the GITR-specific antibodies and antigen-binding fragments comprise a heavy chain substantially the same or identical to SEQ ID NO: 51 and a light chain substantially the same or identical to SEQ ID NO: 55. The heavy chain and light chain of the antibodies described in this paragraph are suitable for introduction into bispecific constructs in which one arm is an anti-GITR antibody arm.

In some embodiments, the GITR-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 3, a heavy chain CDR2 comprising SeQ ID NO: 7, a heavy chain CDR3 comprising SEQ ID NO: 25, a light chain CDR1 comprising SEQ ID NO: 28, the light chain CDR2 containing SEQ ID NO: 32, and the light chain CDR3 containing SEQ ID NO: 35. This GITR-specific antibody or antigen-binding fragment may contain human framework sequences. Such GITR-specific antibody or antigen-binding fragment can bind GITR affinity of 30 nM or less, can induce an increase luciferase expression in the analysis of the luciferase gene and the NF-kB could induce ADCC in vitro with EC5 ₀ equal to 67 ng / ml or less.

In some embodiments, the GITR-specific antibodies and antigen-binding fragments comprise a heavy chain substantially the same or identical to SEQ ID NO: 52 and a light chain substantially the same or identical to SEQ ID NO: 55. The heavy chain and light chain of the antibodies described in this paragraph are suitable for introduction into bispecific constructs in which one arm is an anti-GITR antibody arm.

In some embodiments, the GITR-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 27, a heavy chain CDR2 comprising SEQ ID NO: 5, a heavy chain CDR3 comprising SEQ ID NO: 26, a light chain CDR1 comprising SEQ ID NO: 28, the light chain CDR2 containing SEQ ID NO: 32, and the light chain CDR3 containing SEQ ID NO: 35. This GITR-specific antibody or antigen-binding fragment may contain human framework sequences. Such a GITR-specific antibody or antigen-binding fragment can bind to a GITR affinity of 30 nM or less, can induce an increase in luciferase expression in an NF-kB luciferase gene assay, and can induce in vitro ADCC with an EC ₅₀ of 67 ng / ml or less. In some embodiments, the GITR-specific antibodies and antigen-binding fragments comprise a heavy chain substantially the same or identical to SEQ ID NO: 53 and a light chain substantially the same or identical to SEQ ID NO: 55. The heavy chain and light chain of the antibodies described in this paragraph are suitable for introduction into bispecific constructs in which one arm is an anti-GITR antibody arm.

In some embodiments, the GITR-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 3, a heavy chain CDR2 comprising SeQ ID NO: 11, a heavy chain CDR3 comprising SEQ ID NO: 21, a light chain CDR1 comprising SEQ ID NO: 28, the light chain CDR2 containing SEQ ID NO: 32, and the light chain CDR3 containing SEQ ID NO: 35. This GITR-specific antibody or antigen-binding fragment may contain human framework sequences. Such a GITR-specific antibody or antigen-binding fragment can bind to a GITR affinity of 30 nM or less, can induce an increase in luciferase expression in an NF-kB luciferase gene assay, and can induce in vitro ADCC with an EC ₅₀ of 67 ng / ml or less. In some embodiments, the GITR-specific antibodies and antigen-binding fragments comprise a heavy chain substantially the same or identical to SEQ ID NO: 54 and a light chain substantially the same or identical to SEQ ID NO: 55. The heavy chain and light chain of the antibodies described in this paragraph are suitable for introduction into bispecific constructs in which one arm is an anti-GITR antibody arm.

In some embodiments, the GITR-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 3, a heavy chain CDR2 comprising SEQ ID NO: 7, a heavy chain CDR3 comprising SEQ ID NO: 16, a light chain CDR1 comprising SEQ ID NO: 28, the light chain CDR2 containing SEQ ID NO: 32, and the light chain CDR3 containing SEQ ID NO: 35. This GITR-specific antibody or antigen-binding fragment may contain human framework sequences. Such a GITR-specific antibody or antigen-binding fragment can bind to a GITR affinity of 30 nM or less, can induce an increase in luciferase expression in an NF-kB luciferase gene assay, and can induce in vitro ADCC with an EC ₅₀ .

- 14 037973 equal to 67 ng / ml or less. In some embodiments, the GITR-specific antibodies and antigen-binding fragments comprise a heavy chain substantially the same or identical to SEQ

ID NO: 63, and a light chain substantially the same or identical to SEQ ID NO: 55. The heavy chain and light chain of the antibodies described in this paragraph are suitable for introduction into bispecific constructs in which one arm is an anti-GITR antibody arm.

In some embodiments, the GITR-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 27, a heavy chain CDR2 comprising SEQ ID NO: 5, a heavy chain CDR3 comprising SEQ ID NO: 26, a light chain CDR1 comprising SEQ ID NO: 28, the light chain CDR2 containing SEQ ID NO: 32, and the light chain CDR3 containing SEQ ID NO: 35. This GITR-specific antibody or antigen-binding fragment may contain human framework sequences. Such a GITR-specific antibody or antigen-binding fragment can bind to a GITR affinity of 30 nM or less, can induce an increase in luciferase expression in an NF-kB luciferase gene assay, and can induce in vitro ADCC with an EC ₅₀ of 67 ng / ml or less. In some embodiments, the GITR-specific antibodies and antigen-binding fragments comprise a heavy chain substantially the same or identical to SEQ ID NO: 64 and a light chain substantially the same or identical to SEQ ID NO: 55. The heavy chain and light chain of the antibodies described in this paragraph are suitable for introduction into bispecific constructs in which one arm is an anti-GITR antibody arm.

Also described are isolated polynucleotides encoding antibodies or antigen-binding fragments that specifically bind to GITR. Isolated polynucleotides capable of encoding variable domain segments provided herein can be incorporated into the same or different vectors to produce antibodies or antigen binding fragments.

Polynucleotides encoding recombinant antigen binding proteins are also within the scope of the disclosure. In some embodiments, the disclosed polynucleotides (and the peptides they encode) include a leader sequence. Any leader sequence known in the art can be used. The leader sequence can include, without limitation, a restriction site or a translation initiation site.

GITR-specific antibodies or antigen-binding fragments described herein include variants having single or multiple amino acid substitutions, deletions, or additions that retain biological properties (e.g., binding affinity or immune effector activity) of the described GITR-specific antibodies or antigen-binding fragments ... Such variants may include: (a) variants in which one or more amino acid residues are replaced by conservative or non-conservative amino acids, (b) variants in which one or more amino acids are attached to or removed from the polypeptide, (c) variants, c which one or more amino acids include a substituent group, and (d) variants in which the polypeptide is fused to another peptide or polypeptide, such as a fusion partner, protein tag, or other chemical functional group capable of conferring beneficial properties on the polypeptide, for example, an epitope for an antibody, a polyhistidine sequence, a biotin residue, and the like. Antibodies or antigennegative fragments described herein can include variants in which amino acid residues of one kind are replaced by corresponding residues of another kind (in conservative or non-conservative positions). In other embodiments, the implementation of amino acid residues in non-conservative positions are replaced by conservative or non-conservative residues. Techniques for producing such variants, including genetic (deletions, mutations, and the like), chemical and enzymatic, are known to those skilled in the art.

The GITR-specific antibodies or antigen-binding fragments described herein can refer to several antibody isotypes such as IgM, IgD, IgG, IgA, and IgE. In some embodiments, the antibody isotype is an IgG1, IgG2, IgG3, or IgG4 isotype, preferably an IgG1 isotype. The specificity of an antibody or antigen-binding fragment thereof is largely determined by the amino acid sequence and location of the CDRs. Therefore, CDRs of one isotype can be transferred to another isotype without altering the antigenic specificity. In an alternative embodiment, techniques have been established that cause hybridomas to switch from one antibody isotype to another (isotype switching) without altering antigenic specificity. Accordingly, such antibody isotypes are included within the scope of the disclosed antibodies or antigen binding fragments.

GITR-specific antibodies or antigen-binding fragments described herein have binding affinities for GITR that contain a dissociation constant (K _D ) of less than about 30 nM. The affinity of the disclosed GITR-specific antibodies or antigen-binding fragments can be determined by a variety of methods known in the art, such as surface plasmon resonance or solid phase ELISA methods. SPR affinity assays include assays performed using a BIAcore 3000 instrument and assays are performed at room temperature (e.g., 15 037973 at or near 25 ° C), with antibodies capable of binding to GITR are captured on a BIAcore sensor chip using an anti-Fc antibody (for example, goat anti-human IgG Fc antibody, manufactured by Jackson ImmunoResearch laboratories, item code 109-005-098) in an amount of about 75 OU, followed by collection of association and dissociation data upon flow rate μL / min.

In addition, vectors are provided containing the polynucleotides described herein. The vectors can be expression vectors. Therefore, recombinant expression vectors containing a sequence encoding a polypeptide of interest are considered to be within the scope of this disclosure. The expression vector may contain one or more additional sequences, for example, but not limited to, regulatory sequences (eg, promoter, enhancer), a selection marker, and a polyadenylation signal. Vectors for transformation of a wide variety of host cells are widely known and include, but are not limited to, plasmids, phagemids, cosmids, baculoviruses, bacmids, artificial bacterial chromosomes (BAC), artificial yeast chromosomes (YAC), and other bacterial, yeast and viral vectors.

Recombinant expression vectors, within the scope of the disclosure, include synthetic, genomic, or cDNA-derived nucleic acid fragments that encode at least one recombinant protein that can be operably linked to suitable regulatory elements. Such regulatory elements can include a transcriptional promoter, sequences encoding suitable binding sites for mRNA ribosome binding, and sequences that control the termination of transcription and translation. Expression vectors, especially mammalian expression vectors, can also include one or more non-transcribed elements, for example, an origin of replication, an acceptable promoter and enhancer associated with the expressed gene, other 5 'or 3' flanking non-transcribed sequences, 5 'or 3' untranslated sequences (eg, required ribosome binding sites), a polyadenylation site, splice donor and acceptor sites, or transcription termination sequences. In addition, a replication origin can be built in to provide replication capability in the host cell.

Transcription and translation control sequences in expression vectors for transforming vertebrate cells can be obtained from viral sources.

Examples of vectors that can be constructed are described in Okayama and Berg, 3 Mol. Cell. Biol. 280 (1983).

In some embodiments, the sequence encoding the antibody or antigen-binding fragment is placed under the control of an effective constitutive promoter, such as, for example, promoters of the following genes: hypoxanthine phosphoribosyl transferase (HPRT), adenosine deaminase, pyruvate kinase, beta-actin, human myosin, human hemoglobin and others. In addition, many viral promoters function constitutively in eukaryotic cells and are suitable for use in the described embodiments. Such viral promoters include, but are not limited to, cytomegalovirus immediate-early promoter (CMV), SV40 early and late promoters, mouse mammary tumor virus (MMTV) promoter, Maloney leukemia virus (LTR) virus, human immunodeficiency virus (HIV) long terminal repeats (LTR) , Epstein-Barr virus (EBV), Rous sarcoma virus (RSV) and other retroviruses and the herpes simplex virus thymidine kinase promoter. In one embodiment, the coding sequence of a GITR-specific antibody or antigen-binding fragment thereof is placed under the control of an inducible promoter, for example, a metallothionein promoter, a tetracycline-inducible promoter, a doxycycline-inducible promoter, promoters containing one or more IS-stimulated interferon reactive elements (ISREs) reacting elements ( R 2 ', 5'oligoadenylate synthases, Mx genes, ADAR1 and the like.

The vectors described herein can contain one or more internal ribosome entry regions (IRES). The inclusion of the IRES sequence in fusion vectors can be useful for enhancing the expression of certain proteins. In some embodiments, the vector system may include one or more polyadenylation regions (eg, SV40) that may be located above or below any of the aforementioned nucleic acid sequences. The vector components can be stitched together or arranged to provide optimal spacing for expression of gene products (i.e., by introducing spacer nucleotides between open reading frames (ORF)), or otherwise arranged. Regulatory elements such as the IRES motif can also be positioned to provide optimal spacing for expression.

Vectors may contain selectable markers well known in the art. Selectable markers include positive and negative selection markers, antibiotic resistance genes (e.g., neomycin resistance gene, hygromycin resistance gene, kanamycin resistance gene, tetracycline resistance gene, penicillin resistance gene), genes

16 037973 glutamate synthase, HSV-TK, HSV-TK derivatives for ganciclovir selection or bacterial purine nucleoside phosphorylase gene for 6-methylpurine selection (Gadi et al., 7 Gene Ther. 1738-1743 (2000)).

The nucleotide sequence encoding the selectable marker or cloning site may be located above or below the nucleotide sequence encoding the polypeptide or cloning site of interest.

The vectors described herein can be used to transform various cells with genes encoding the disclosed antibodies or antigen binding fragments. For example, vectors can be used to generate cells that produce a GITR-specific antibody or antigen-binding fragment. Therefore, in another aspect of the invention, host cells are described transformed with vectors containing a nucleotide sequence encoding an antibody or antigen-binding fragment thereof that specifically binds to GITR, for example, antibodies or antigen-binding fragments described and exemplified herein.

Numerous methods of introducing foreign genes into cells are known in the art and can be used to construct recombinant cells to carry out the described methods in accordance with the various embodiments described and exemplified herein. The technique used should ensure stable transfer of the heterologous gene sequence into the host cell so that the heterologous gene sequence can be inherited and expressed by the progeny of the cell, and in such a way that the necessary development and physiological functions of the recipient cells are not disturbed. Techniques that can be used include, but are not limited to, chromosome transfer (e.g., cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer), physical methods (e.g., transfection, spheroplast fusion, microinjection, electroporation, liposomal carrier), vector viral transfer (eg, recombinant DNA viruses, recombinant RNA viruses) and the like (described in Cline, 29 Pharmac. Ther. 69-92 (1985)). Calcium phosphate precipitation and polyethylene glycol (PEG) -induced fusion of bacterial protoplasts with mammalian cells can also be used to transform cells.

Cells suitable for use in the expression of the GITR-specific antibodies or antigen-binding fragments described herein preferably include eukaryotic cells, more preferably plant, rodent or human derived cells, for example, without limitation, NSO, CHO, CHOK1 cell lines , perC.6, Tk-ts13, BHK, HEK293 cells, cOs7, T98G, CV-1 / EBNA, L cells, C127, 3T3, HeLa, NS1, Sp2 / 0 myeloma cells, BHK and the like. In addition, the expression of antibodies can be carried out using hybridoma cells. Methods for producing hybridomas are well known in the art.

Cells transformed with the expression vectors described herein can be selected or screened for recombinant expression of the antibodies or antigen-binding fragments described herein. Recombination-positive cells are propagated and subclones displaying the desired phenotype, for example, high level of expression, enhanced growth characteristics, or the ability to produce proteins with the desired biochemical properties, for example, due to protein modification or altered post-translational modifications, are screened. These phenotypes may be due to the intrinsic properties of a given subclone or mutation. Mutations can be made by the use of chemical agents, UV light, radiation, viruses, insertional mutagens, inhibition of DNA mating error repair, or a combination of such methods.

Methods that use GITR-specific antibodies for treatment

Provided herein are GITR-specific antibodies or antigen-binding fragments thereof for therapeutic use. In particular, these antibodies or antigen-binding fragments can be used in the treatment of malignant neoplasms. Accordingly, the invention provides a method for treating cancer, comprising administering an antibody as described herein, for example, GITR-specific antibodies or antigen-binding fragments. Several aspects of the biology of GITR make it a potential target for the treatment of various forms of malignant neoplasms. First of all, activation of GITR, as detailed above, activates the immune system. In addition, GITR-expressing effector T cells and regulatory T cells infiltrate many types of tumors, with little or no GITR expression on non-hematopoietic cells. This distribution profile means that GITR-expressing cells can concentrate in tumors. This combination of activity and distribution together determines the attractiveness of the approach of using GITR as a target for the treatment of various forms of malignant neoplasms. Antigen binding proteins can be used to treat both solid tumors and hematological malignant neoplasms, including leukemia.

Antibodies used in these methods include the antibodies described herein above, such as a GITR-specific antibody or antigen-binding fragment with the properties described in table. 1, for example with CDR or variable domain sequences which are also described in the following description of these antibodies.

In some embodiments, the implementation described herein, the immunoeffective properties of GITR-specific antibodies can be modulated by Fc modifications using techniques known to those of skill in the art. For example, Fc fragment effector functions such as Clq binding, complement dependent cytotoxicity (CDC), antibody dependent cell mediated cytotoxicity (ADCC), antibody dependent cell mediated phagocytosis (ADCP), downregulation of cell surface receptors (e.g., B cell receptor; BCR) and the like, can be provided and / or controlled by modifying residues in the Fc fragment responsible for these actions.

Antibody-dependent cell-mediated cytotoxicity, or ADCC, is a cell-mediated response in which nonspecific cytotoxic cells expressing Fc (FcRc) receptors (eg, natural killer (EK) cells, neutrophils, and macrophages) recognize the bound antibody on the target cell and subsequently induce lysis of the target cell.

The ability of monoclonal antibodies to induce ADCC can be enhanced by designing their oligosaccharide component. Human IgG1 or IgG3 undergo N-glycosylation at Asn297 by most glycans in the well-known 2-antenna forms G0, G0F, G1, G1F, G2 or G2F. Antibodies produced by unconstructed CHO cells typically have a glycan fucose content of at least about 85%. Removal of central fucose from oligosaccharides of the 2-antenna complex type attached to Fc regions enhances ADCC of antibodies through improved Fc gamma.RIIIa binding without altering antigen binding or CDC activity. Such mAbs can be produced by a variety of methods that have been reported to result in the successful expression of relatively high defucosylation antibodies carrying Fs-oligosaccharides of the 2-antenna complex type, such as by controlling the osmolality of the culture medium (Konno et al., Cytotechnology 64 : 249-65, 2012), the use of the variant CHO Lec13 cell line as a host cell line (Shields et al., J Biol Chem 277: 26733-26740, 2002), the use of the variant CHO cell line EB66 ( Olivier et al., MAbs; 2 (4), 2010; electronic edition before print; PMID: 20562582), the use of the rat hybridoma cell line YB2 / 0 as a host cell line (Shinkawa et al., J Biol Chem 278: 3466-3473, 2003), introduction of small interfering RNAs, especially to the gene of alpha-1,6-fucosyltransferrase (FUT8) (Mori et al., Biotechnol Bioeng 88: 901-908, 2004) or co-expression of beta-1,4-N -acetylglucosaminyltransferase III and Golgi alpha-mannosidase II or an effective inhibitor of alpha-mannosidase I, kifunensin (Ferrara et al., J Biol Chem 281: 5032-5036, 2006, Ferrara et al., Biotechnol Bioeng 93: 851-861, 2006; Xhou et al., Biotechnol Bioeng 99: 652-65, 2008).

In some embodiments, the implementation described herein, ADCC caused by antibodies to GITR, can also be enhanced by introducing certain substitutions in the Fc region of the antibody. Examples of substitutions are, for example, substitutions at amino acid positions 256, 290, 298, 312, 356, 330, 333, 334, 360, 378 or 430 (residue numbering according to the EC index), as described in US patent No. 6,737,056.

Pharmaceutical compositions and administration

The pharmaceutical compositions of the present invention comprise: a) an effective amount of a GITR-specific antibody or antibody fragment of the present invention, and b) a pharmaceutically acceptable carrier that may be inert or physiologically active. In preferred embodiments, the GITR-specific antibody is a GITR-specific antibody as described above, or an antigen-binding fragment thereof. As used herein, the term pharmaceutically acceptable carriers includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, and the like that are physiologically compatible. Examples of suitable carriers, diluents and / or excipients include one or more of water, saline, phosphate buffered saline (PBS), dextrose, glycerol, ethanol, and the like, or any combination thereof. In many cases, it will be preferable to include in the composition isotonic agents such as sugars, polyhydric alcohols, or sodium chloride. In particular, suitable examples of an acceptable carrier include: (1) Dulbecco's phosphate buffered saline, pH about 7.4, containing or not containing from about 1 mg / ml to 25 mg / ml human serum albumin, (2) 0.9% saline (0.9% w / v sodium chloride (NaCl)) and (3) 5% (w / v) dextrose; and it may also contain an antioxidant such as tryptamine and a stabilizing agent such as Tween 20®.

The compositions described herein can also contain additional therapeutic agent as required for the particular disorder to be treated. It is preferred that the GITR-specific antibody or antibody fragment and the additional active compound have complementary activities that do not adversely affect each other. In a preferred embodiment, the additional therapeutic agent is cytarabine, anthracycline, histamine dihydrochloride, or interleukin 2. In a preferred embodiment, the additional therapeutic agent is a chemotherapeutic agent.

The compositions of the invention can be presented in various forms. These forms include, for example, liquid, semi-solid and solid dosage forms, but the preferred form depends on the intended route of administration and the therapeutic use. Typical preferred compositions are in the form of solutions suitable for injection or infusion. The preferred route of administration is parenteral (eg, intravenous, intramuscular, intraperitoneal, subcutaneous). In a preferred embodiment, the compositions of the invention are administered by intravenous bolus, or they are administered as a continuous infusion over a period of time. In another preferred embodiment, they are administered intramuscularly, subcutaneously, intraarticularly, intratumorally, near a tumor, into diseased tissue, or near diseased tissue so that they have local and systemic therapeutic effects.

Sterile compositions for parenteral administration can be prepared by introducing an antibody, an antibody fragment or an antibody conjugate of the present invention in the desired amount in a suitable solvent, followed by sterilization by microfiltration. As a solvent or carrier, water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, and a combination thereof can be used. In many cases, it will be preferable to include in the composition isotonic agents such as sugars, polyhydric alcohols, or sodium chloride. These compositions can also contain auxiliary substances, in particular, wetting, isotonizing, emulsifying, dispersing and stabilizing agents. Sterile compositions for parenteral administration may also be formulated as sterile solid compositions, which dissolve during use in sterile water or any other injectable sterile medium.

The GITR-specific antibody or antibody fragment can also be administered orally. As solid compositions for oral administration, tablets, pills, powders (gelatin capsules, sachets) or granules can be used. In these compositions, the active ingredient according to the invention is mixed under a stream of argon with one or more inert diluents such as starch, cellulose, sucrose, lactose or silicon dioxide. These compositions may also contain substances other than diluents, for example, one or more lubricants such as magnesium stearate or talc, a colorant, a coating (sugar-coated tablet), or a glaze.

As liquid compositions for oral administration, pharmaceutically acceptable solutions, suspensions, emulsions, syrups and elixirs containing inert diluents such as water, ethanol, glycerin, vegetable oils or paraffin oil can be used. These compositions may contain substances other than diluents, for example, wetting agents, sweetening agents, thickening agents, flavoring or stabilizing products.

Doses depend on the desired effect, the duration of treatment and the route of administration used; they essentially range from 5 to 1000 mg per day orally for adults, in single doses ranging from 1 mg to 250 mg of active ingredient. In general, the appropriate dosage is determined by the physician depending on age, body weight and other factors specific to the individual to be treated.

Also provided herein are methods of killing Tregs by administering to a patient in need of a GITR-specific antibody with ADCC activity and which is capable of causing immune cells to kill GITR-expressing Tregs. For therapeutic purposes, you can use any of the GITR-specific antibodies or antibody fragments of the invention. In preferred embodiments, the GITR-specific antibody is a GITR-specific antibody as described above, or an antigen-binding fragment thereof.

In a preferred embodiment, GITR-specific antibodies or antibody fragments of the invention are used to treat a hyperproliferative disorder in mammals. In a more preferred embodiment, one of the above-described pharmaceutical compositions comprising a GITR-specific antibody or antibody fragment of the invention is used to treat a hyperproliferative disorder in mammals. In one embodiment, the disorder is cancer. A wide variety of cancers have been shown to contain GITR-positive immune cells. Accordingly, such tumors are particularly attractive targets. Such tumors include, for example, melanoma (including stage III and stage IV malignant melanoma), lung cancer (eg, non-small cell lung cancer - NSCLC), head and neck cancer, prostate cancer, renal cell carcinoma, and colorectal cancer. In preferred embodiments, the GITR-specific antibody is a GITR-specific antibody as described above, or an antigen-binding fragment thereof.

Other cancers that can be treated with antigen binding proteins include but are not limited to breast cancer, prostate cancer, endometrial cancer, bladder cancer, kidney cancer, esophageal cancer, testicular cancer, ovarian cancer, bladder cancer, squamous cell carcinoma (eg, squamous cell carcinoma of the head and neck - SCCHN), uveal melanoma, follicular lymphoma, cervical cancer, brain cancer, cancer

- 19 037973 pancreas, liver cancer, lymphoma, Hodgkin's disease, multiple myeloma, gastrointestinal cancer and astrocytic cancer.

When treating any of the above forms of malignant neoplasms, the proposed methods of treatment can be used to further suppress tumor growth, induce tumor regression, increase progression-free survival and / or prolong the overall survival of an individual suffering from a tumor. In some embodiments, the implementation of the GITR specific antibodies can also delay or prevent the onset of metastasis. Treatment progress can be tracked in a variety of ways. For example, inhibition can lead to a decrease in tumor size and / or decrease metabolic activity within the tumor. Both of these parameters can be measured through, for example, an MRI scan or a PET scan. Inhibition can also be monitored by biopsy to confirm the level of necrosis, tumor cell death, and the level of vascularization within the tumor. The extent of metastasis can be monitored by known methods.

Accordingly, the pharmaceutical compositions of the invention are used to treat or prevent metastasis in various forms of cancer, including but not limited to breast cancer, prostate cancer, endometrial cancer, bladder cancer, kidney cancer, esophageal cancer, testicular cancer, ovarian cancer, squamous cell carcinoma (e.g. squamous cell carcinoma of the head and neck SCCHN), uveal melanoma, follicular lymphoma, cervical cancer, brain cancer, pancreatic cancer, liver cancer, lymphoma, Hodgkin's disease, multiple myeloma, gastrointestinal cancer, and astrocytic cancer.

Similarly, this document further provides a method of inhibiting the growth of selected populations of cells comprising contacting GITR-expressing immune cells with an effective amount of a GITR-specific antibody or antibody fragment of the present invention, alone or in combination with other therapeutic agents. In preferred embodiments, the GITR-specific antibody is a GITR-specific antibody as described above, or an antigen-binding fragment thereof. In a preferred embodiment, the additional therapeutic agent is immunotherapy, that is, an immunostimulating agent that induces or enhances an immune response. Such agents may include, for example: 1) dendritic cell activators, 2) vaccine adjuvants, 3) T cell stimulants, 4) immunological checkpoint inhibitors, and 5) inhibitors of suppressor cells, cytokines and / or enzymes.

In one embodiment, the immunostimulatory agent is a cancer vaccine. In addition to cancer vaccines that contain cancer-associated antigens, vaccines that are used in combination with a GITR-specific antibody include, but are not limited to, GM-CSF, DNA and cell-based vaccines, dendritic cell vaccines, recombinant viral vaccines (eg vaccinia virus) and heat shock protein (HSP) vaccines. Vaccines used also include tumor vaccines such as those derived from melanoma cells; and they can be autologous or allogeneic. Vaccines can be based on, for example, peptides, DNA or cells. In one embodiment, the GITR-specific antibody is administered in combination with a CD8 / CD4 Ag-specific peptide vaccine.

In clinical use, a therapeutically effective amount of a GITR-specific antibody or antigen-binding fragment is administered to an individual in need of such treatment. For example, GITR-specific antibodies and antigen-binding fragments thereof can be used to treat cancers that contain GITR-positive immune cells. In preferred embodiments, the GITR-specific antibody is a GITR-specific antibody as described above, or an antigen-binding fragment thereof. In some embodiments, the implementation of the individual is a mammal, preferably a human. In some embodiments, the GITR-specific antibody or antigen-binding fragment is administered as a sterility tested solution.

Dosage regimens for the above methods of treatment and use are adjusted to provide the optimal desired response (eg, therapeutic response). For example, you can perform a single bolus administration, administer several divided doses over a period of time, or the dose can be proportionally reduced or increased according to the indications of the therapeutic situation. Parenteral compositions can be prepared in standard dosage units for ease of administration and dosage uniformity.

Effective dosages and dosage regimens for GITR-specific antibodies and fragments depend on the disease or condition being treated and can be determined by one of ordinary skill in the art. A non-limiting example of a range of a therapeutically effective amount of a compound of the present invention is the range of about 0.001-10 mg / kg, for example about 0.001-5 mg / kg, for example about 0.001-2 mg / kg, for example about 0.001-1 mg / kg, for example about 0.001, about 0.01, about 0.1, about 1, or about 10 mg / kg.

- 20 037973

A physician or veterinarian skilled in the art can easily determine and prescribe the required effective amount of the pharmaceutical composition. For example, a physician or veterinarian may start with lower doses of the GITR-specific antibody or fragment included in the pharmaceutical composition than is necessary to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. In general, an acceptable daily dose of a GITR-specific antibody of the present invention will be that amount of the compound that is the lowest dose that produces a therapeutic effect. Administration can be, for example, parenteral, such as intravenous, intramuscular, or subcutaneous. In one embodiment, the implementation of the GITR-specific antibody or fragment can be entered by infusion at a weekly dose, calculated in mg / m ² . Such doses, for example, can be calculated based on the mg / kg doses described above, according to the following formula: dose (mg / kg) x 70. This introduction can be repeated, for example, from 1 to 8 times, for example from 3 to 5 times. The introduction can be carried out by continuous infusion over a period of time from 2 to 24 hours, for example from 2 to 12 hours. side toxic effects.

In one embodiment, the GITR-specific antibody or fragment can be administered at a weekly dose, calculated as a fixed dose, administered up to eight times, eg, four to six times, once a week. This pattern can be repeated one or more times as needed, for example, after six months or twelve months. Such fixed doses, for example, may be based on the above doses in mg / kg based on an assumed body weight of 70 kg. The dose can be determined or adjusted by measuring the amount of the GITR-specific antibody of the present invention in the blood after administration, for example, by obtaining a biological sample and using anti-idiotypic antibodies targeting the GITR-binding region of the GITR-specific antibodies of the present invention.

In one embodiment, the implementation of the GITR-specific antibody or fragment can be administered in the form of maintenance therapy, for example, once a week for a period of six or more months.

The GITR-specific antibody or fragment can also be administered prophylactically to reduce the risk of developing malignant neoplasms, slow the onset of events in the progression of malignant neoplasms, and / or reduce the risk of recurrence in the event of remission of malignant neoplasms.

GITR-specific antibodies and fragments thereof described herein can also be administered as part of a combination therapy, i. E. in combination with other therapeutic agents relevant to the treatment of a disease or condition. Accordingly, in one embodiment, the antibody-containing medicament is intended to be combined with one or more additional therapeutic agents, such as a chemotherapeutic agent. In some embodiments, other therapeutic agents, without limitation, include antineoplastic agents, including alkylating agents, including: nitrogen mustards such as mechlorethamine, cyclophosphamide, ifosfamide, melphalan, and chlorambucil; nitrosoureas such as carmustine (BCNU), lomustine (CCNU) and semustine (methyl-CCNU); Temodal ™ (temozolamide), ethyleneimines / methylmelamine, eg triethylene melamine (TEM), triethylene, thiophosphoramide (thiotepa), hexamethylmelamine (HMM, altretamine); alkyl sulfonates such as busulfan; triazines such as dacarbazine (DTIC); antimetabolites, including folic acid analogs, for example methotrexate and trimetrexate, pyrimidine analogs, for example 5-fluorouracil (5FU), fluorodeoxyuridine, gemcitabine, cytosine arabinoside (AraC, cytarabine), 5-azacytidine, 2,2'-difluorodeoxyurate analogs , 6-thioguanine, azathioprine, 2'-deoxycoformycin (pentostatin), erythrohydroxynonyladenine (EHNA), fludarabine phosphate, and 2-chlorodeoxyadenosine (cladribine, 2-CdA); natural products including antimitotic drugs such as paclitaxel, vinca alkaloids including vinblastine (VLB), vincristine and vinorelbine, taxotere, estramustine and estramustine phosphate; pipodphilotoxins such as etoposide and teniposide; antibiotics such as actimomycin D, daunomycin (rubidomycin), doxorubicin, mitoxantrone, idarubicin, bleomycin, plikamycin (mithramycin), mitomycin C, and actinomycin; enzymes such as L-asparaginase; biological response modulators such as interferon alpha, IL-2, G-CSF, and GM-CSF; various agents, including platinum coordination complexes such as cisplatin and carboplatin, anthraceneodions such as mitoxantrone, substituted urea such as hydroxyurea, methylhydrazine derivatives including N-methylhydrazine (MIH) and procarbazine, adrenocorticoid suppressants such as mitotane (o, p- DDD) and aminoglutethimide; hormones and antagonists, including adrenocorticosteroid antagonists such as prednisone and equivalents, dexamethasone and aminoglutethimide; Gemzar ™ (gemcitabine), a progestin such as hydroxyprogesterone caproate, medroxyprogesterone acetate, and megestrol acetate; estrogens such as diethylstilbestrol and ethinyl estradiol equivalents; antiestrogens such as tamoxifen; androgens, including testosterone propionate and fluoxymesterone / equivalents; antiandrogens, e.g. flutamide, analogs

- 21 037973 gonadotropin-releasing hormone and leuprolide; and non-steroidal antiandrogens such as flutamide. Therapies targeting an epigenetic mechanism, including but not limited to histone deacetylase inhibitors, demethylating agents (eg, Vidaza), and transcriptional repression factor releasing therapies (ATRA), may also be combined with GITR antibodies.

Additional specific examples of chemotherapeutic agents include taxol, taxenes (eg, docetaxel and taxotere), modified paclitaxel (eg, Abraxane and Opaxio), doxorubicin, Avastin®, Sutent, Nexavar, and other multi-kinase inhibitors, cisplatin and carboplatin, gemtabosin ... Specific inhibitors of other kinases can also be used in combination with GITR antibodies, including, but not limited to, MAPK pathway inhibitors (eg, ERK, JNK, and p38 inhibitors), PI3 kinase / AKT inhibitors, and Pim inhibitors. Other inhibitors include Hsp90 inhibitors, proteasome inhibitors (eg Velcade) and inhibitors of various mechanisms of action, such as Trisenox.

Such combined administration can be simultaneous, separate, or sequential, in any order. When administered simultaneously, agents can be administered as a single composition or as separate compositions, as appropriate.

In one embodiment, a GITR-specific antibody or fragment thereof is administered in combination with another immunomodulatory agent, and such an immunomodulatory agent may be selected, for example, from the group consisting of a dendritic cell activator such as CD40 ligand and anti-CD40 agonist antibodies, and antigen presentation enhancing agents, T cell tropism enhancing agents, inhibitors of tumor associated immunosuppressive factors such as TGF-β (transforming growth factor beta) and IL-10.

Some methods include administering a GITR-specific antibody or fragment thereof with a vaccine adjuvant. Such adjuvants include, for example, IL-12 and various Tol-like receptor (TLR) agonists, including CpG (a TLR 9 agonist), monophosphoryl lipid A (MPL is a TLR4 agonist), PolyI: C or PolyICLC (a TLR3 agonist), and resikimod and 852A (TLR 7/8 agonists).

In other therapeutic approaches, the GITR-specific antibody is administered in combination with T cell growth factors such as IL-15 and / or IL-17, or activators of similar molecules. In similar methods, a T cell stimulant is coupled with a GITR antibody. Such stimulants include 4-1BB agonists, for example, an anti-4-1BB antibody agonist and 4-1BBL.

In one embodiment, a GITR-specific antibody or fragment thereof is administered with an inhibitor of T cell checkpoints, eg, molecules that send an inhibitory signal to the immune system. Examples of such agents include PD-1 or PD-L1 (B7-H1) inhibitors, such as anti-PD-1 antibodies, including nivolumab (Bristol-Myers Squibb) and pembrolizumab, also known as MK-3475 (Merck), pidilizumab (Curetech), AMP-224 (Amplimmune) and anti-PD-L1 antibodies including MPDL3280A (Roche), MDX-1105 (Bristol Myer Squibb), MEDI-4736 (AstraZeneca) and MSB0010718C (Merck). Other checkpoint inhibitors include CTLA-4 antagonists such as anti-CTLA-4 antibodies. An example of an anti-CTLA4 antibody is Yervoy® (ipilimumab) available from Bristol-Myers Squibb. Other examples of CTLA-4 antibodies include tremelimumab (Pfizer), tisilimumab (AstraZeneca), and AMGP-224 (Glaxo Smith Kline).

In other methods, a GITR-specific antibody or fragment thereof is administered in combination with an inhibitor of an enzyme that has an immunosuppressive effect. An example is 1methyltryptophan (1MT), which is a small molecule inhibitor of indoleamine 2,3 dioxygenase.

A GITR-specific antibody or fragment thereof can also be used in combination with TVEC (talimogen lagerparepvec) developed by Amgen.

In some embodiments, a GITR-specific antibody or fragment thereof is administered in combination with a bispecific antibody. The bispecific antibody can target the host's immune system, in particular the cytotoxic activity of T cells, against cancer cells.

The GITR-specific antibody or fragment thereof can also be used in combination with various forms of targeted therapy. Examples of targeted therapy include, without limitation, the use of therapeutic antibodies. Examples of antibodies without limitation include antibodies that bind to cell surface proteins Her2, CDC20, CDC33, mucin-like glycoprotein and epidermal growth factor receptor (EGFR) present on tumor cells, OX40, PD-1, CD122, CD40, CTLA-4, and if necessary, inducing a cytostatic and / or cytotoxic effect on tumor cells exhibiting such proteins. Examples of such antibodies also include HERCEPTIN® (trastuzumab), which can be used to treat breast cancer and other forms of malignancy, and RITUXAN® (rituximab), ZEVALIN ™ (ibritumomab tuxetan), and LYMPHOCIDE ™ (epratuzumab), which can be used to treatment of non-Hodgkin's lymphoma and other forms of malignant neoplasms. A number of other examples of antibodies also include panitumumab (VECTIBIX®), ERBITUX® (IMC-C225) ;; BEXXAR ™ (iodine-131-22 037973 tositumomab); KDR (kinase domain receptor) inhibitors; anti-VEGF antibodies and antagonists (eg Avastin® and VEGAF-TRAP); antibodies to the VEGF receptor and antigen binding regions; anti-Ang-1 and -Ang-2 antibodies and antigen binding regions; antibodies to Tie-2 and other Ang-1 and Ang-2 receptors; Tie-2 ligands; antibodies to Tie-2 kinase inhibitors; Hif-1a and Campath ™ inhibitors (alemtuzumab). In some embodiments, anti-cancer therapy agents are polypeptides that selectively induce apoptosis in tumor cells, including, but not limited to, TNF-linked TRAIL polypeptide.

In one embodiment, a GITR-specific antibody or fragment thereof as described herein is used in combination with one or more anti-angiogenic agents that reduce angiogenesis. Some such agents include, but are not limited to, IL-8 antagonists; Campath, B-FGF; FGF antagonists; Tek antagonists (Cerretti et al., US publication No. 2003/0162712; Cerretti et al., US patent No. 6413932, and Cerretti et al., US patent No. 6521424); anti-TWEAK agents (which include, but are not limited to, antibodies and antigen binding regions); soluble TWEAK receptor antagonists (Wiley, US Pat. No. 6,727,225); the disintegrin domain ADAM as an antagonist of the binding of integrin to its ligands (Fanslow et al., US publication No. 2002/0042368); anti-ephrine receptor and anti-ephrin antibodies; antigen binding regions or antagonists (US patent No. 5981245; 5728813; 5969110; 6596852; 6232447; 6057124); anti-VEGF agents (for example, antibodies or antigen binding regions that specifically bind VEGF, or soluble VEGF receptors or their ligand binding regions), for example Avastin® or VEGF-TRAP ™, and anti-VEGF receptor agents (for example, antibodies or antigen binding regions, which specifically bind to them), EGFR inhibiting agents (for example, antibodies or antigen binding regions that specifically bind to them), for example panitumumab, IRESSA ™ (gefitinib), TARCEVA ™ (erlotinib), anti-Ang-1 and anti-Ang- 2 agents (for example, antibodies or antigen binding regions that specifically bind to them or their receptors, for example, Tie-2 / TEK), and anti-Tie-2 kinase inhibitory agents (for example, antibodies or antigen binding regions that specifically bind and inhibit activity of growth factors, for example antagonists of hepatocyte growth factor (HGF, also known as scattering factor), and antibodies or antigen-binding regions that are specific eski bind its c-met receptor (eg rilotumumab and AMG 337, Amgen); antagonists to PDGF-BB; antibodies and antigen binding regions to PDGFBB ligands; and PDGFR kinase inhibitors.

Other anti-angiogenic agents that can be used in combination with a GITR-specific antibody or fragment thereof include agents such as MMP-2 (matrix metalloproteinase 2) inhibitors, MMP-9 (matrix metalloproteinase 9) inhibitors, and COXII (cyclooxygenase II) inhibitors. Examples of suitable COX-II inhibitors include CELEBREX ™ (celecoxib), valdecoxib, and rofecoxib.

A GITR-specific antibody or fragment thereof as described can also be used in combination with a growth factor inhibitor. Examples of such agents, without limitation, include agents that can inhibit the EGF-R (epidermal growth factor receptor) response, for example, EGF-R antibodies (eg, panitumumab (VECTIBIX®)), EGF antibodies, and molecules that inhibit EGF-R; VEGF (Vascular Endothelial Growth Factor) inhibitors such as VEGF receptors and molecules capable of inhibiting VEGF; and inhibitors of erbB2 receptors, eg organic molecules or antibodies that bind to the erbB2 receptor, eg HERCEPTIN® (Genentech, Inc.). EGF-R inhibitors, which are described, for example, in US Pat. No. 5,747,498, WO 98/14451, WO 95/19970 and WO 98/02434.

In some therapeutic applications, in particular, in the case of metastasis of a malignant neoplasm to bone with a negative effect on bone, it may be advisable to administer a GITR-specific antibody or a fragment thereof with a therapeutic agent that prevents further bone loss or promotes the restoration of lost bone mass. Accordingly, a GITR-specific antibody or fragment thereof can be administered together with a therapeutically effective amount of an agent that promotes the growth of bone mass (anabolic) or an agent that prevents bone resorption, including but not limited to: bone morphogenetic factors designated BMP-1 to BMP -12; transforming growth factor-β and members of the TGF-β family; fibroblast growth factors from FGF-1 to FGF-10; inhibitors of interleukin-1 (including IL-1ra, antibodies to IL-1 and antibodies to IL-1 receptors); TNFa inhibitors (including etanercept, adalimumab, and infliximab); RANK ligand inhibitors (including soluble RANK, osteoprotegerin, and antagonist antibodies that specifically bind RANK or RANK ligand, such as denosumab (XGEVA®)), Dkk-1 inhibitors (such as Dkk-1 antibodies), parathyroid hormone, group E prostaglandins , bisphosphonates and bone-strengthening minerals such as fluoride and calcium. Anabolic agents that can be used in combination with GITR antibodies and their functional fragments include parathyroid hormone and insulin-like growth factor (IGF), the latter agent being preferably used in combination with IGF-binding

- 23 037973 protein. An IL-1 receptor antagonist suitable for such combination therapy is described in WO

89/11540, and a suitable soluble TNF receptor-1 is described in WO 98/01555. Examples of RANK ligand antagonists are disclosed, for example, in WO 03/086289, WO 03/002713, US Pat. Nos. 6,740,511 and

6479635.

In one embodiment, a method for treating cancer comprises administering to an individual in need of such treatment a therapeutically effective amount of a GITR-specific antibody, as described herein, in combination with radiation therapy. Radiation therapy can include radiation or appropriate administration of radiopharmaceuticals to a patient. The radiation source can be external or internal to the patient being treated (radiation therapy can, for example, be external beam radiation therapy (EBRT) or brachytherapy (BT)). Radioactive elements that can be used in practice for these methods include, for example, radium, cesium-137, iridium-192, americium-241, gold-198, cobalt-57, copper-67, technetium-99, iodine- 12 3, iodine-131 and indium-111.

GITR detection methods

Provided herein are methods for detecting GITR in a biological sample by contacting the sample with an antibody or antigen-binding fragment thereof described herein. As described herein, a sample can be obtained from urine, blood, serum, plasma, saliva, ascites fluid, circulating cells, circulating tumor cells, cells not associated with tissues (i.e., free cells), tissues (e.g., surgically excised tumor tissue, biopsy materials, including those obtained using fine needle aspiration biopsy), histological preparations, and the like. In some embodiments, the methods described comprise detecting GITR in a biological sample by contacting the sample with any of the GITR-specific antibodies or antigen-binding fragments thereof described herein.

In some embodiments, the implementation of the sample can be contacted with more than one of the GITR-specific antibodies or antigennegative fragments described herein. For example, the sample can be contacted with a first GITR-specific antibody or antigen-binding fragment thereof and then contacted with a second GITR-specific antibody or antigen-binding fragment thereof, wherein the first antibody or antigen-binding fragment and the second antibody or antigen-binding fragment are not one and the same antibody or antigen-binding fragment. In some embodiments, prior to contacting the sample, the first antibody or antigen-binding fragment thereof may be attached to a surface, such as the surface of a multi-well plate, a chip, or a similar substrate. In other embodiments, prior to contacting with the sample, the first antibody or antigen-binding fragment thereof may not be attached or fixed to anything.

Described GITR-specific antibodies and antigen-binding fragments can be labeled for detection. In some embodiments, labeled antibodies and antigen-binding fragments can facilitate the detection of GITR by the methods described herein. Many such labels are well known to those skilled in the art. For example, acceptable labels include, but are not limited to, radioactive labels, fluorescent labels, epitope labels, biotin, chromophore labels, ECL labels, or enzymes. More specifically, the disclosed labels include ruthenium, ^1U In-DOTA, ^1U In-diethylenetriaminepentaacetic acid (DTPA), horseradish peroxidase, alkaline phosphatase and beta-galactosidase, polyhistidine (HIS-tag), acridine dyes, cyanine dyes, fluorine dyes oxazine dyes, phenanthridine dyes, rhodamine dyes, Alexa Fluor® dyes, and the like.

The described GITR-specific antibodies and antigen-binding fragments can be used in various assays to detect GITR in a biological sample. Some suitable assays include, but are not limited to, Western blot, radioimmunoassay, surface plasmon resonance, immunofluorimetry, immunoprecipitation, equilibrium dialysis, immunodiffusion, electrochemiluminescence (ECL) immunoassay, immunohistochemistry, fluorescence assay cell sorting cytometry (FACS).

GITR Detection Kits

This document offers kits for detecting GITR in a biological sample. These kits include one or more of the GITR-specific antibodies described herein, or an antigen-binding fragment thereof, and instructions for using the kit.

The proposed GITR-specific antibody or antigennegative fragment can be in solution; be lyophilized; attached to a substrate, carrier, or tablet; or can be labeled and detectable.

The kits described may also include additional components used to implement the methods described herein. By way of example, the kits may contain means for obtaining a sample from an individual, a control sample, or a reference sample, for example, a sample from an individual with slowly progressing cancer and / or an individual without malignant neoplasm,

- 24 037973 one or more sample compartments and / or instruction materials describing the implementation of the methods of the invention and tissue specific controls or standards.

The means for determining the GITR level may, for example, additionally include buffers and other reagents for use in an assay to determine the GITR level. The instructions can be, for example, printed instructions for performing the assay and / or instructions for assessing the level of GITR expression.

The kits described may also include means for isolating a sample from an individual. These means may contain one or more items of equipment or reagents that can be used to obtain a fluid or tissue from an individual. Means for obtaining a sample from an individual can also be means for isolating blood components, such as serum, from a blood sample. Preferably, the kit is for human use.

Examples of embodiments of the invention

For a better and more complete description of the subject matter of the invention, this section in the form of a numbered list provides examples of the implementation of the presented subject matter of the invention.

Below are the options for implementation in the form of a numbered list.

Embodiments

1. An isolated antibody or antigen-binding fragment thereof that specifically binds to human GITR and contains:

a) Heavy chain CDR1 with amino acid sequence SEQ ID NO: 1, heavy chain CDR2 with amino acid sequence SEQ ID NO: 5, and heavy chain CDR3 with amino acid sequence SEQ ID NO: 12, light chain CDR1 with amino acid sequence SEQ ID NO: 28 , Light chain CDR2 with the amino acid sequence of SEQ ID NO: 32, and light chain CDR3 with the amino acid sequence of SEQ ID NO: 35;

b) Heavy chain CDR1 with amino acid sequence SEQ ID NO: 2, heavy chain CDR2 with amino acid sequence SEQ ID NO: 5, and heavy chain CDR3 with amino acid sequence SEQ ID NO: 13, light chain CDR1 with amino acid sequence SEQ ID NO: 29 , Light chain CDR2 with the amino acid sequence of SEQ ID NO: 32, and light chain CDR3 with the amino acid sequence of SEQ ID NO: 36;

c) Heavy chain CDR1 with amino acid sequence SEQ ID NO: 1, heavy chain CDR2 with amino acid sequence SEQ ID NO: 6, and heavy chain CDR3 with amino acid sequence SEQ ID NO: 14, light chain CDR1 with amino acid sequence SEQ ID NO: 30 , Light chain CDR2 with the amino acid sequence of SEQ ID NO: 33, and light chain CDR3 with the amino acid sequence of SEQ ID NO: 37;

d) Heavy chain CDR1 with amino acid sequence SEQ ID NO: 3, heavy chain CDR2 with amino acid sequence SEQ ID NO: 7, and heavy chain CDR3 with amino acid sequence SEQ ID NO: 15, light chain CDR1 with amino acid sequence SEQ ID NO: 28 , Light chain CDR2 with the amino acid sequence of SEQ ID NO: 32, and light chain CDR3 with the amino acid sequence of SEQ ID NO: 35;

e) Heavy chain CDR1 with amino acid sequence SEQ ID NO: 3, heavy chain CDR2 with amino acid sequence SEQ ID NO: 7, and heavy chain CDR3 with amino acid sequence SEQ ID NO: 16, light chain CDR1 with amino acid sequence SEQ ID NO: 28 , Light chain CDR2 with the amino acid sequence of SEQ ID NO: 32, and light chain CDR3 with the amino acid sequence of SEQ ID NO: 35;

f) Heavy chain CDR1 with amino acid sequence SEQ ID NO: 4, heavy chain CDR2 with amino acid sequence SEQ ID NO: 8, and heavy chain CDR3 with amino acid sequence SEQ ID NO: 17, light chain CDR1 with amino acid sequence SEQ ID NO: 28 , Light chain CDR2 with the amino acid sequence of SEQ ID NO: 32, and light chain CDR3 with the amino acid sequence of SEQ ID NO: 35;

g) Heavy chain CDR1 with amino acid sequence SEQ ID NO: 4, heavy chain CDR2 with amino acid sequence SEQ ID NO: 9, and heavy chain CDR3 with amino acid sequence SEQ ID NO: 18, light chain CDR1 with amino acid sequence SEQ ID NO: 28 , Light chain CDR2 with the amino acid sequence of SEQ ID NO: 32, and light chain CDR3 with the amino acid sequence of SEQ ID NO: 35;

h) Heavy chain CDR1 with amino acid sequence SEQ ID NO: 4, heavy chain CDR2 with amino acid sequence SEQ ID NO: 10, and heavy chain CDR3 with amino acid sequence SEQ ID NO: 19, light chain CDR1 with amino acid sequence SEQ ID NO: 28 , Light chain CDR2 with the amino acid sequence of SEQ ID NO: 32, and light chain CDR3 with the amino acid sequence of SEQ ID NO: 35;

i) Heavy chain CDR1 with amino acid sequence SEQ ID NO: 3, heavy chain CDR2 with amino acid sequence SEQ ID NO: 7, and heavy chain CDR3 with amino acid sequence SEQ ID NO: 20, light chain CDR1 with amino acid sequence SEQ ID NO: 31 , Light chain CDR2 with amino acid sequence SEQ ID NO: 34, and light chain CDR3 with amino

- 25,037973 buto acid sequence SEQ ID NO: 38;

j) Heavy chain CDR1 with amino acid sequence SEQ ID NO: 3, heavy chain CDR2 with amino acid sequence SEQ ID NO: 11, and heavy chain CDR3 with amino acid sequence SEQ ID NO: 21, light chain CDR1 with amino acid sequence SEQ ID NO: 31 , Light chain CDR2 with the amino acid sequence of SEQ ID NO: 34, and light chain CDR3 with the amino acid sequence of SEQ ID NO: 38;

k) Heavy chain CDR1 with amino acid sequence SEQ ID NO: 3, heavy chain CDR2 with amino acid sequence SEQ ID NO: 7, and heavy chain CDR3 with amino acid sequence SEQ ID NO: 22, light chain CDR1 with amino acid sequence SEQ ID NO: 28 , Light chain CDR2 with the amino acid sequence of SEQ ID NO: 32, and light chain CDR3 with the amino acid sequence of SEQ ID NO: 35;

l) Heavy chain CDR1 with amino acid sequence SEQ ID NO: 3, heavy chain CDR2 with amino acid sequence SEQ ID NO: 7, and heavy chain CDR3 with amino acid sequence SEQ ID NO: 23, light chain CDR1 with amino acid sequence SEQ ID NO: 28 , Light chain CDR2 with the amino acid sequence of SEQ ID NO: 32, and light chain CDR3 with the amino acid sequence of SEQ ID NO: 35;

m) Heavy chain CDR1 with amino acid sequence SEQ ID NO: 3, heavy chain CDR2 with amino acid sequence SEQ ID NO: 7, and heavy chain CDR3 with amino acid sequence SEQ ID NO: 24, light chain CDR1 with amino acid sequence SEQ ID NO: 28 , Light chain CDR2 with the amino acid sequence of SEQ ID NO: 32, and light chain CDR3 with the amino acid sequence of SEQ ID NO: 35;

n) Heavy chain CDR1 with amino acid sequence SEQ ID NO: 3, heavy chain CDR2 with amino acid sequence SEQ ID NO: 7, and heavy chain CDR3 with amino acid sequence SEQ ID NO: 25, light chain CDR1 with amino acid sequence SEQ ID NO: 28 , Light chain CDR2 with the amino acid sequence of SEQ ID NO: 32, and light chain CDR3 with the amino acid sequence of SEQ ID NO: 35;

o) Heavy chain CDR1 with amino acid sequence SEQ ID NO: 27, heavy chain CDR2 with amino acid sequence SEQ ID NO: 5, and heavy chain CDR3 with amino acid sequence SEQ ID NO: 26, light chain CDR1 with amino acid sequence SEQ ID NO: 28 , Light chain CDR2 with the amino acid sequence of SEQ ID NO: 32, and light chain CDR3 with the amino acid sequence of SEQ ID NO: 35; or

p) Heavy chain CDR1 with amino acid sequence SEQ ID NO: 3, heavy chain CDR2 with amino acid sequence SEQ ID NO: 11, and heavy chain CDR3 with amino acid sequence SEQ ID NO: 21, light chain CDR1 with amino acid sequence SEQ ID NO: 28 , Light chain CDR2 with the amino acid sequence of SEQ ID NO: 32, and light chain CDR3 with the amino acid sequence of SEQ ID NO: 35.

2. An isolated antibody or antigen-binding fragment thereof that specifically binds to human GITR and contains a heavy chain region selected from the group consisting of SEQ ID NOs: 39-54, 63 and 64.

3. The antibody of embodiment 2, wherein the antibody or antigen-binding fragment thereof comprises a light chain region selected from the group consisting of SEQ ID NO: 55-58.

4. The antibody of embodiment 2, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain region selected from the group consisting of SEQ ID NOs: 39-54, 63 and 64 and a light chain region selected from the group consisting of SEQ ID NO: 55-58.

5. The antibody of embodiment 4, wherein:

a) the region of the heavy chain contains SEQ ID NO: 39, paired with the region of the light chain containing SEQ ID NO: 55;

b) the region of the heavy chain contains SEQ ID NO: 40, paired with the region of the light chain containing SEQ ID NO: 56;

c) the region of the heavy chain contains SEQ ID NO: 41, paired with the region of the light chain containing SEQ ID NO: 57;

d) the region of the heavy chain contains SEQ ID NO: 42, paired with the region of the light chain containing SEQ ID NO: 55;

e) the region of the heavy chain contains SEQ ID NO: 43, paired with the region of the light chain containing SEQ ID NO: 55;

f) the region of the heavy chain contains SEQ ID NO: 44, paired with the region of the light chain containing SEQ ID NO: 55;

g) the region of the heavy chain contains SEQ ID NO: 45, paired with the region of the light chain containing SEQ ID NO: 55;

h) the region of the heavy chain contains SEQ ID NO: 46, paired with the region of the light chain containing SEQ ID NO: 55;

i) the region of the heavy chain contains SEQ ID NO: 47 paired with the region of the light chain containing

- 26 037973

SEQ ID NO: 58;

j) the region of the heavy chain contains SEQ ID NO: 48, paired with the region of the light chain containing

SEQ ID NO: 58;

k) the region of the heavy chain contains SEQ ID NO: 49, paired with the region of the light chain containing

SEQ ID NO: 55;

l) the region of the heavy chain contains SEQ ID NO: 50, paired with the region of the light chain containing SEQ ID NO: 55;

m) the region of the heavy chain contains SEQ ID NO: 51, paired with the region of the light chain containing SEQ ID NO: 55;

n) the region of the heavy chain contains SEQ ID NO: 52, paired with the region of the light chain containing SEQ ID NO: 55;

o) the region of the heavy chain contains SEQ ID NO: 53, paired with the region of the light chain containing SEQ ID NO: 55;

p) the region of the heavy chain contains SEQ ID NO: 54, paired with the region of the light chain containing SEQ ID NO: 55;

q) the region of the heavy chain contains SEQ ID NO: 63, paired with the region of the light chain containing SEQ ID NO: 55; or

r) the region of the heavy chain contains SEQ ID NO: 64, paired with the region of the light chain containing SEQ ID NO: 55.

6. An antibody or antigen-binding fragment of embodiment 5, wherein the antibody specifically binds to human GITR through interaction with GITR (SEQ ID NO: 62 amino acid residues:

a) 40-45 and

b) 75-79.

7. An antibody or antigen-binding fragment of embodiment 1, which binds to a polypeptide having the amino acid sequence of SEQ ID NO: 59.

8. An antibody or antigen-binding fragment of embodiment 1, wherein such antibody or antigen-binding fragment specifically binds to human GITR with a binding affinity of at least 30 nM as indicated by surface plasmon resonance measurements using the experimental conditions described in Example 9.

9. An antibody or antigen-binding fragment according to embodiment 1, wherein such antibody or antigen-binding fragment induces an increase in luciferase expression when assayed for the NF-kB luciferase gene.

10. An antibody or antigen binding fragment of embodiment 1, wherein such antibody or antigen binding fragment induces in vitro ADCC with an EC _{50 of} less than about 67 ng / ml.

11. The antibody or antigen-binding fragment of embodiment 1, which is a human antibody or antigen-binding fragment.

12. The antigen binding fragment of embodiment 1, which is a Fab fragment, Fab2 fragment, or a single chain antibody.

13. The antibody or antigen binding fragment of embodiment 1, which is recombinant.

14. The antibody or antigen-binding fragment of embodiment 1, which is of the IgG1, IgG2, IgG3, or IgG4 isotype.

15. The antibody or antigen binding fragment of embodiment 1 is of the IgG1 isotype.

16. An antibody or antigen-binding fragment according to any one of embodiments 1, wherein the antibody or antigen-binding fragment thereof specifically binds to human GITR or cynomolgus GITR.

17. A polynucleotide encoding an antibody or antigen binding fragment according to any one of Embodiments 1.

18. A vector containing the polynucleotide of embodiment 17.

19. A host cell containing the vector of embodiment 18.

20. A method of producing an antibody or antigen-binding fragment, comprising:

culturing the host cell as described in Embodiment 19 under conditions such that the antibody or antigen binding fragment is expressed; and recovering the antibody or antigen binding molecule from the culture.

21. A method of ameliorating a symptom of a malignant neoplasm or other neoplastic condition, comprising administering an antibody or antigen-binding fragment thereof according to embodiment 1 to an individual in an amount sufficient to alleviate a symptom of a malignant neoplasm or other neoplastic condition in the individual.

22. The method of embodiment 21, wherein the individual is a human.

23. The method of embodiment 21, further comprising one or more of the following:

- 27 037973

a) chemotherapy

b) conducting radiation therapy; or

c) the introduction of one or more therapeutic agents.

24. The method of embodiment 23, wherein the additional therapeutic agent is an immunostimulating agent.

25. The method of embodiment 24, wherein the immunostimulatory agent is selected from the group consisting of an anti-PD-1 antibody, an anti-CTLA-4 antibody, an anti-CD122 antibody, an anti-CD40 antibody, an anti-OX40 antibody, and a CD8 Ag-specific OVA peptide vaccine ...

26. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to embodiment 1 and a pharmaceutically acceptable carrier.

27. A kit comprising the antibody or antigen-binding fragment thereof according to embodiment 1 and packaging therefor.

Examples of

The examples below are provided in addition to the above description and for the purpose of a better understanding of the subject matter of the invention described herein. These examples should not be construed as limiting the described subject matter of the invention. It should be understood that the examples and embodiments described herein are for illustrative purposes only, and in light of this, various modifications and variations will be apparent to those skilled in the art, which should be included within the scope of the invention and that may be made without departing from the scope. inventions.

Example 1. Materials.

GITR ECD molecules.

Recombinant (h) human GITR-Fc fusion protein (R&D Systems cat # 689-GR) corresponding to amino acids 26 to 161 of hGITR (SEQ ID NO: 59). The protein was biotinylated for phage panning studies. This protein was also used for binding and affinity measurements.

GITR cell lines.

GITR was expressed in HEK293F cells by transfection or lentiviral transduction to confirm anti-GITR antibody reactivity, to test phage panels and next generation sequencing panels, and to test for cross-reactivity of GITR mAbs against cynomolgus GITR.

The transfected cells presented the following GITR sequences:

Human GITR (SEQ ID NO: 60)

QRPTGGPGCGPGRLLLGTGTDARCCRVHTTRCCRDYPGEECCSEWDCMCVQPEFHCGDP

CCTTCRHHPCPPGQGVQSQGKFSFGFQCIDCASGTFSGGHEGHCKPWTDCTQFGFLTVFPGNKT

HNAVCVPGSPPAEPLGWLTWLLAVAACVLLLTSAQLGLHIWQLRSQCMWPRETQLLLEVPPST

EDARSCQFPEEERGERSAEEKGRLGDLWV

Javanese macaque GITR (SEQ ID NO: 61)

QRPTGGPGCGPGRLLLGTGKDARCCRVHPTRCCRDYQSEECCSEWDCVCVQPEFHCGNP

CCTTCQHHPCPSGQGVQPQGKFSFGFRCVDCALGTFSRGHDGHCKPWTDCTQFGFLTVFPGNKT

HNAVCVPGSPPAEPPGWLTIVLLAVAACVLLLTSAQLGLHIWQLGSQPTGPRETQLLLEVPPST

EDASSCQFPEEERGERLAEEKGRLGDLWV

Cells after lentiviral transduction presented the following GITR sequences: Human GITR (SEQ ID NO: 62)

MAQHGAMGAFRALCGLALLCALSLGQRPTGGPGCGPGRLLLGTGTDARCCRVHTTRCCR

DYPGEECCSEWDCMCVQPEFHCGDPCCTTCRHHPCPPGQGVQSQGKFSFGFQCIDCASGTFSGG

HEGHCKPWTDCTQFGFLTVFPGNKTHNAVCVPGSPPAEPLGWLTWLLAVAACVLLLTSAQLGL

HIWQLRSQCMWPRETQLLLEVPPSTEDARSCQFPEEERGERSAEEKGRLGDLWV

Javanese macaque GITR (SEQ ID NO: 61)

QRPTGGPGCGPGRLLLGTGKDARCCRVHPTRCCRDYQSEECCSEWDCVCVQPEFHCGNP

CCTTCQHHPCPSGQGVQPQGKFSFGFRCVDCALGTFSRGHDGHCKPWTDCTQFGFLTVFPGNKT

HNAVCVPGSPPAEPPGWLTIVLLAVAACVLLLTSAQLGLHIWQLGSQPTGPRETQLLLEVPPST

EDASSCQFPEEERGERLAEEKGRLGDLWV

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Transient expression of HEK293F cells was performed by plating the cells in Freestyle ™ 293 medium (Gibco No. 12338) at a density of 1e ⁶ cells / ml in a volume of 30 ml in 125 ml ventilated vials with shaking at 130 rpm for 24 h before transfection. Transfection was performed using Freestyle max reagent (Invitrogen # 16447). For a single transfection, 30 ml in a separate tube, 37.5 μl of Freestyle Max reagent was diluted in 1 ml of OptiMEM medium (Gibco # 31985). In a separate tube, 37.5 μg of DNA (300 nanograms of target and 37.2 μN of foreign carrier plasmid) was mixed in 1 ml of OptiMEM. The contents of the two tubes were then mixed together, incubated in a biosafety cabinet for 1 minute, and then the mixture was added directly to the vial of HEK293F cells. After 48 hours of growth, the cells were ready for use in the assays indicated.

Lentiviral particles representing full length GITR were prepared in Genecopoeia (Genecopoeia catalog number LPP-U0202-LV105-200-S for human GITR and Genecopoeia catalog number LPP-U0202-LV105-200-S for cynomolgus macaque GITR), transduced into cells according to the manufacturer's protocol. The transduced cells were selected for stable integration of the plasmid, and then individual cells were sorted using a BD Biosciences FACS Jazz cell sorter. GITR surface expression was quantified by flow cytometry staining with R&D Systems FAB689P anti-huGITR antibody and analyzed on BD Biosciences Accuri C6.

Example 2. Detection of GITR antibodies using phage display technology.

De novo pIX Fab libraries were panned (Shi, L., et al J Mol Biol, 2010.397 (2): pp. 385-396. WO 2009/0854 62) containing the heavy chain libraries VH1-69, 3-23 5-51 and paired with the light chain libraries V _K VLK3-20, VLK4-1, VLK3-11 and VLK1-39, regarding biotinylated fusion GITR-ECD Fc human at a concentration of 100 nM (cycles 1-3) or 10 nM ( cycle 4) in a selection process similar to that described in Rothe et al, J Mol Biol 376: 1182-1200, 2008 and Steidl et al, Mol Immunol. 46: 135-144, 2008.

The pIX gene was excised from phagemid DNA after the fourth round of panning and soluble his-tagged Fab coding regions were obtained. Fab fragments were expressed in E. coli and screened for GITR binding by ELISA. Briefly, 96 well Nunc Maxisorp Plates (Nunc # 437111) were coated with either streptavidin (Promega) or anti-human ovine Fd (binding site # PC075) in PBS at 5 μg / ml at 4 ° C overnight. Bacterial cultures containing the Fab expression vector were grown in 1 μl of 2xYT (carbenicillin) in deep-well culture plates until turbidity (OD600 * 0.6). The Fab expression was then induced by the addition of 1 mM IPTG. The cultures were grown overnight at 30 ° C and then the next day they were purified by centrifugation. Streptavidin-coated plates were washed three times with TBS, 0.5% Tween-20 (Sigma # 79039-10PAK), loaded with biotinylated GITR-Fc at 5 μg / ml and kept at room temperature for 30 minutes. Using a Biomek liquid handling robot (Beckman Coulter), Maxisorp anti-Fd coated plates and streptavidin coated plates were washed three times with TBS, 0.5% Tween-20 (Sigma # 79039-10PAK) and blocked with 200 μl PBS-Tween (0, 5%) + skim milk powder (3%) per well for one hour at room temperature. At this and all subsequent steps, the plates were washed three times with TBS with 0.5% Tween-20 (Sigma # 79039-10PAK). Each well was injected with 50 μl of Fab supernatant, and then incubated for one hour at room temperature. After washing, 50 μl of goat anti-human kappa-HRP (Southern Biotech) diluted 1: 5000 in TBST with 0.3% milk was added and the plates were incubated for one hour at room temperature. The plates were washed and 50 μl of PoD chemiluminescent substrate (Roche # 121-5829500001) was added according to the manufacturer's instructions. Then the plates were scanned for luminescence on an Envision flatbed scanner (Perkin Elmer). For DNA sequencing, clones were selected that were positive for expression by ELISA and GITR binding by ELISA. As part of this phage panning process, only 50 unique Fab sequences were found. The unique heavy chain V regions were cloned into human IgG1_G1m (17.1) expression vectors, the unique light chains were cloned into human kappa expression vectors and the resulting antibodies were tested for binding activity by ELISA.

Example 3. Preliminary description of the properties of GITR antibodies obtained using phage display technology.

Human GITR binding assay.

The binding of GITR antibodies to engineered cells was assessed using FACS. The purpose of this screening assay was to identify antibodies that bind to cells expressing hGITR.

Briefly, 300,000 cells per well were seeded in a 96-well plate (Greiner bio one cat # 651261) and the cells were pelleted. Cells were washed with 100 μl FACS staining buffer (BD Pharmingen Stain Buffer (BSA) cat # 554657), incubated at 4 ° C for 30 min with a mixture of 50 μl FACS staining buffer and 20 μl per well of crude antibody supernatants, after

- 29 037973 which was washed once with 200 μl of FACS staining buffer. For registration, cells were incubated for 30 minutes at 4 ° C with 50 μl / well Alexa Fluor 488 goat anti-human IgG (H + L) antibody (Molecular Probes cat # A11013) at 2 μg / ml in FACS staining buffer. Cells were washed once with 200 μl per well of FACS staining buffer, resuspended in 150 μl per well of FACS staining buffer, and then transferred to FACS tubes that contained 200 μl per well of FACS staining buffer. FACS analysis was then performed. The analysis was repeated for data consistency and the best binding molecules were selected for further development.

Analysis of the luciferase gene NF-kB.

To assess the agonist activity of GITR antibodies, a panel was screened as part of the NF-kB luciferase gene assay. Briefly, HEK293 cells were transiently transfected with a reporter plasmid encoding the luciferase gene under the control of the NF-kB promoter together with the human GITR expression plasmid. The cells were allowed to recover from transfection and express human GITR for four hours at 37 ° C, after which the analysis could be performed. Recombinant human GITR ligand (R&D Systems 6987-GL / CF) was added to the positive control wells at a final concentration of 2.5 μg / ml to confirm that the assay results were as expected. The test anti-GITR antibodies were added to the experimental wells at a final concentration of 5 micrograms per ml. The plates were then incubated at 37 ° C for four hours. Efficient GITR signaling was expected to activate the NF-kB pathway followed by luciferase expression, which could be recorded by adding Steady Glo as indicated by the manufacturer (Promega cat # E2550), with the final luminescence measured on an Envision plate reader (Perkin Elmer) ...

Fifteen antibodies induced an increase in luciferase expression compared to treatment with PBS alone (FIG. 1). Antibodies that led to an increase in luciferase expression compared to treatment with PBS alone were preliminarily classified as agonists, while the rest of the antibodies could be antagonists or simply bind to GITR without affecting signal transduction.

Example 4 Detection of GITR Antibodies in Next Generation Sequencing.

Following the discovery of the first set of anti-GITR antibodies, DNA samples from the last round of phage selection were submitted to Beckman Coulter Genomics for next generation sequencing for heavy chain V regions using the Roche 454 sequencing platform. at IMGT and then more extensively analyzed in-house using Janssen's 3DX computer program. The idea of using next-generation sequencing for more detailed analysis of phage selection results has recently been developed into a method to increase the number and quality of antibodies detected (Ravn et al, Methods 60 (2013) pg 99-110).

The sequences provided by IMGT were filtered to exclude low quality samples, or samples containing stop codons, and then the remaining sequences were sorted by heavy chain CDR3. This approach was chosen because it was assumed that CDR3 determines the most significant part of the binding energy for the antigen, and the greatest diversity in phage libraries is located in the CDR3 of the heavy chain. Eighty-seven V regions were selected for DNA synthesis and cloning into human IgG1 G1m (17) vector based on the frequency of observation and the absence of cysteines, methionines or highly hydrophobic sequences.

After synthesis, putative anti-GITR heavy chains were tested for binding to GITR as described above. Since the next generation sequencing dataset did not contain data on the corresponding light chain partners for these heavy chain V regions, the heavy chains were co-transfected with each of the 4 germline light chain genes found in the phage libraries: Vk3-20, Vk4-1, Vk3 -11 and Vk1-39. Crude antibody supernatants from these four standard transfections were tested by ELISA for the ability to bind to the recombinant human ECD-Fc GITR fusion protein. The most strongly binding compounds were selected for further development.

Example 5. Preliminary description of the properties of GITR antibodies obtained by sequencing next generation.

After the binding of the anti-GITR mAb from the next generation sequencing kit to the GITR ECD-Fc fusion protein in ELISA was demonstrated, a portion of the kit was tested for binding to cell surface GITR as described in Example 3. Those showing binding were tested for agonist activity using the assay the luciferase gene NF-kB, as described in Example 3. At a concentration of 40 micrograms per ml, antibodies that induced an increase in luciferase expression equal to at least a 20% increase over the background observed with natural ligand treatment were considered to have agonist activity (Fig. . 2).

Example 6. Cross-reactivity for the Javanese macaque.

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Purified antibodies after phage display and next generation sequencing were tested for binding to cynomolgus GITR using flow cytometry. Transiently transfected cells were incubated at 2-8 ° C for 30 min with 0.1 mg / ml of test antibodies and incubated at 2-8 ° C for 30 min with PE-labeled goat anti-human IgG antibody. The cells were then washed and analyzed on a MACSQuant flow cytometer. Antibodies classified as positively binding showed a shift of 1.5 to 2 log units in the mean fluorescence intensity of cells transfected with human or cynomolgus GITR compared to the mean fluorescence intensity of cells transfected with the blank vector. The binding results are summarized in table. 2.

Table 2. Binding of anti-GITR antibodies to HEK293f cells transiently transfected with human or cynomolgus GITR. Note that this table only shows antibodies that showed cell binding to cynomolgus GITR — tested antibodies, which are not shown, did not bind to cynomolgus GITR. Antibodies were scored ++ for a strong shift in the binding curve and + for a moderate shift in binding.

Thus, in the end, it was demonstrated that a panel of 16 GITR antibodies - all of them are shown in table. 3 binds to GITR in humans and Javanese macaques.

Table 3. CDR sequences of 16 candidate GITR mAbs that showed binding to human and cynomolgus GITR (SEQ ID NO :)

ID HC-CDR1 HC-CDR2 HC-CDR3 LC-CDR1 LC-CDR2 LC-CDR3 TRGB5 GFTFSGYW (1) ISGSGGST (5) AKDFYWDAFDY (12) QSVSSY (28) DAS (32) QQRSNWPLT (35) TRGB14 GFTFSSYA (2) ISGSGGST (5) AKPIRGLDY (13) QSVNNF (29) DAS (32) QQGFNAPLT (36) TRGB20 GFTFSGYW (1) ISSDGGSK (6) AKEWYDHYAALDY (14) QSVNSF (30) YAS (33) QQYIRWPLT (37) TRGB23 GGTFSSYA (3) IIPIFGTA (7) ARHGNWLHFNLDY (15) QSVSSY (28) DAS (32) QQRSNWPLT (35) TRGB25 GGTFSSYA (3) IIPIFGTA (7) ARHRRFWLDY (16) QSVSSY (28) DAS (32) QQRSNWPLT (35) TRGB31 GYSFTSYW (4) IDPSDSDT (8) ARVFPYYGLVLDY (17) QSVSSY (28) DAS (32) QQRSNWPLT (35) TRGB34 GYSFTSYW (4) IYPGDSDT (9) ARDYGWHDFDY (18) QSVSSY (28) DAS (32) QQRSNWPLT (35) TRGB35 GYSFTSYW (4) IDPGDSDT (10) ARHRWSTSLLLDY (19) QSVSSY (28) DAS (32) QQRSNWPLT (35) TRGB120 GGTFSSYA (3) IIPIFGTA (7) ARPRRNTNELDY (20) QSISSY (31) AAS (34) QQSYSTPLT (38) TRGB127 GGTFSSYA (3) IIPIFGNA (ID ARHVYKRGVLNY (21) QSISSY (31) AAS (34) QQSYSTPLT (38) TRGB134 GGTFSSYA (3) IIPIFGTA (7) ARHRWGSGNLDY (22) QSVSSY (28) DAS (32) QQRSNWPLT (35) TRGB144 GGTFSSYA (3) IIPIFGTA (7) ARHGFQRGYLDY (23) QSVSSY (28) DAS (32) QQRSNWPLT (35) TRGB153 GGTFSSYA (3) IIPIFGTA (7) ARHAWLGHLDY (24) QSVSSY (28) DAS (32) QQRSNWPLT (35) TRGB159 GGTFSSYA (3) IIPIFGTA (7) ARHGRNSGRLDY (25) QSVSSY (28) DAS (32) QQRSNWPLT (35) TRGB160 GFTFSNYW (27) ISGSGGST (5) AKDFYWDSFDY (26) QSVSSY (28) DAS (32) QQRSNWPLT (35) TRGB162 GGTFSSYA (3) IIPIFGNA (ID ARHVYKRGVLNY (21) QSVSSY (28) DAS (32) QQRSNWPLT (35)

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The VH and VL regions for the 16 GITR mAbs are shown in Table 1 below. 4.

Table 4. Heavy and light chain sequences for 16 GITR mAb candidates that showed binding to human and cynomolgus GITR

MAb identifier Heavy chain amino acid sequence SEQ ID NO: Light chain amino acid sequence SEQ ID NO: TRGB5 EVQLLESGGGLVQPGGSLRLSC AASGFTFSGYWMSWVRQAPGKG LEWVSAISGSGGSTYYADSVKG RETISRDNSKNTLYLQMNSLRA EDTAVYYCAKDFYWDAFDYWGQ GTLVTVSSASTKGPSVFPLAPS SKSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKS CDKTHTCPPCPAPELLGGPSVF LFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQK SLSLSPGK 39 EIVLTQSPATLSLSPGERAT LSCRASQSVSSYLAWYQQKP GQAPRLLIYDASNRATGIPA RFSGSGSGTDFTLTISSLEP EDFAVYYCQQRSNWPLTFGQ GTKVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFY PREAKVQWKVDNALQSGNSQ ESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC 55 TRGB14 EVQLLESGGGLVQPGGSLRLSC AASGFTFSSYAMSWVRQAPGKG LEWVSAISGSGGSTYYADSVKG RETISRDNSKNTLYLQMNSLRA EDTAVYYCAKPIRGLDYWGQGT LVTVSSASTKGPSVFPLAPSSK STSGGTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLF PPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNA KTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPP SRDELTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSL SLSPGK 40 EIVLTQSPATLSLSPGERAT LSCRASQSVNNFLAWYQQKP GQAPRLLIYDASNRATGIPA RFSGSGSGTDFTLTISSLEP EDFAVYYCQQGFNAPLTFGQ GTKVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFY PREAKVQWKVDNALQSGNSQ ESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC 56 TRGB20 EVQLLESGGGLVQPGGSLRLSC AASGFTFSGYWMNWVRQAPGKG LEWVSGISSDGGSKYYADSVKG RETISRDNSKNTLYLQMNSLRA EDTAVYYCAKEVVYDHYAALDY WGQGTLVTVSSASTKGPSVFPL APSSKSTSGGTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLG TQTYICNVNHKPSNTKVDKKVE PKSCDKTHTCPPCPAPELLGGP SVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQV YTLPPSRDELTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK 41 EIVLTQSPATLSLSPGERAT LSCRASQSVNSFLAWYQQKP GQAPRLLIYYASNRATGIPA RFSGSGSGTDFTLTISSLEP EDFAVYYCQQYIRWPLTFGQ GTKVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFY PREAKVQWKVDNALQSGNSQ ESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC 57 TRGB23 QVQLVQSGAEVKKPGSSVKVSC KASGGT FSSYAISWVRQAPGQG LEWMGGIIPIFGTANYAQKFQG RVTITADESTSTAYMELSSLRS 42 EIVLTQSPATLSLSPGERAT LSCRASQSVSSYLAWYQQKP GQAPRLLIYDASNRATGIPA RFSGSGSGTDFTLTISSLEP 55

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EDTAVYYCARHGNWLHFNLDYW GQGTLVTVSSASTKGPSVFPLA PSSKSTSGGTAALGCLVKDYFP EPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVY TLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK EDFAVYYCQQRSNWPLTFGQ GTKVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFY PREAKVQWKVDNALQSGNSQ ESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQF LSSPECVT TRGB25 QVQLVQSGAEVKKPGSSVKVSC KASGGT FSSYAISWVRQAPGQG LEWMGGIIPIFGTANYAQKFQG RVTITADESTSTAYMELSSLRS EDTAVYYCARHRRFWLDYWGQG TLVTVSSASTKGPSVFPLAPSS KST SGGTAALGCLVKDY FPE PV TVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPELLGGPSVFL FPPKPKDTLMISRTPEVTCVVV DVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLP PSRDELTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKS LSLSPGK 43 EIVLTQSPATLSLSPGERAT LSCRASQSVSSYLAWYQQKP GQAPRLLIYDASNRATGIPA RFSGSGSGTDFTLTISSLEP EDFAVYYCQQRSNWPLTFGQ GTKVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFY PREAKVQWKVDNALQSGNSQ ESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC 55 TRGB31 EVQLVQSGAEVKKPGESLKISC KGSGYSFTSYWIGWVRQMPGKG LEWMGIIDPSDSDTRYSPSFQG QVTISADKSISTAYLQWSSLKA SDTAMYYCARVFPYYGLVLDYW GQGTLVTVSSASTKGPSVFPLA PSSKSTSGGTAALGCLVKDYFP EPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVY TLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYT 44 EIVLTQSPATLSLSPGERAT LSCRASQSVSSYLAWYQQKP GQAPRLLIYDASNRATGIPA RFSGSGSGTDFTLTISSLEP EDFAVYYCQQRSNWPLTFGQ GTKVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFY PREAKVQWKVDNALQSGNSQ ESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC 55

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QKSLSLSPGK TRGB34 EVQLVQSGAEVKKPGESLKISC KGSGYSFTSYWIGWVRQMPGKG LEWMGIIYPGDSDTRYSPSFQG QVTISADKSISTAYLQWSSLKA SDTAMYYCARDYGWHDFDYWGQ GTLVTVSSASTKGPSVFPLAPS SKSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKS CDKTHTCPPCPAPELLGGPSVF LFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQK SLSLSPGK 45 EIVLTQSPATLSLSPGERAT LSCRASQSVSSYLAWYQQKP GQAPRLLIYDASNRATGIPA RFSGSGSGTDFTLTISSLEP EDFAVYYCQQRSNWPLTFGQ GTKVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFY PREAKVQWKVDNALQSGNSQ ESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC 55 TRGB35 EVQLVQSGAEVKKPGESLKISC KGSGYSFTSYWISWVRQMPGKG LEWMGIIDPGDSDTRYSPSFQG QVTISADKSISTAYLQWSSLKA SDTAMYYCARHRWSTSLLLDYW GQGTLVTVSSASTKGPSVFPLA PSSKSTSGGTAALGCLVKDYFP EPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVY TLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK 46 EIVLTQSPATLSLSPGERAT LSCRASQSVSSYLAWYQQKP GQAPRLLIYDASNRATGIPA RFSGSGSGTDFTLTISSLEP EDFAVYYCQQRSNWPLTFGQ GTKVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFY PREAKVQWKVDNALQSGNSQ ESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC 55 TRGB120 QVQLVQSGAEVKKPGSSVKVSC KASGGT FSSYAISWVRQAPGQG LEWMGGIIPIFGTANYAQKFQG RVTITADESTSTAYMELSSLRS EDTAVYYCARPRRNTNELDYWG QGTLVTVSSASTKGPSVFPLAP SSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPK SCDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEV 47 DIQMTQSPSSLSASVGDRVT ITCRASQSISSYLNWYQQKP GKAPKLLIYAASSLQSGVPS RFSGSGSGTDFTLTISSLQP EDFATYYCQQSYSTPLTFGQ GTKVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFY PREAKVQWKVDNALQSGNSQ ESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC 58

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HNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYT LPPSREEMTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTT PPVLDSNHGSHKSR TRGB127 QVQLVQSGAEVKKPGSSVKVSC KASGGT FSSYAISWVRQAPGQG LEWMGGIIPIFGNANYAQKFQG RVTITADESTSTAYMELSSLRS EDTAVYYCARHVYKRGVLNYWG QGTLVTVSSASTKGPSVFPLAP SSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPK SCDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYT LPPSREEMTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK 48 DIQMTQSPSSLSASVGDRVT ITCRASQSISSYLNWYQQKP GKAPKLLIYAASSLQSGVPS RFSGSGSGTDFTLTISSLQP EDFATYYCQQSYSTPLTFGQ GTKVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFY PREAKVQWKVDNALQSGNSQ ESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC 58 TRGB134 QVQLVQSGAEVKKPGSSVKVSC KASGGT FSSYAISWVRQAPGQG LEWMGGIIPIFGTANYAQKFQG RVTITADESTSTAYMELSSLRS EDTAVYYCARHRWGSGNLDYWG QGTLVTVSSASTKGPSVFPLAP SSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPK SCDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYT LPPSREEMTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK 49 EIVLTQSPATLSLSPGERAT LSCRASQSVSSYLAWYQQKP GQAPRLLIYDASNRATGIPA RFSGSGSGTDFTLTISSLEP EDFAVYYCQQRSNWPLTFGQ GTKVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFY PREAKVQWKVDNALQSGNSQ ESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC 55 TRGB144 QVQLVQSGAEVKKPGSSVKVSC KASGGT FSSYAISWVRQAPGQG LEWMGGIIPIFGTANYAQKFQG RVTITADESTSTAYMELSSLRS EDTAVYYCARHGFQRGYLDYWG QGTLVTVPLTAGASTKGPS fifty EIVLTQSPATLSLSPGERAT LSCRASQSVSSYLAWYQQKP GQAPRLLIYDASNRATGIPA RFSGSGSGTDFTLTISSLEP EDFAVYYCQQRSNWPLTFGQ GTKVEIKRTVAAPSVFIFPP SDENQLYKSGFNQDN 55

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QSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPK SCDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYT LPPSREEMTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK ESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC TRGB153 QVQLVQSGAEVKKPGSSVKVSC KASGGT FSSYAISWVRQAPGQG LEWMGGIIPIFGTANYAQKFQG RVTITADESTSTAYMELSSLRS EDTAVYYCARHAWLGHLDYWGQ GTLVTVSSASTKGPSVFPLAPS SKSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKS CDKTHTCPPCPAPELLGGPSVF LFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTL PPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQK SLSLSPGK 51 EIVLTQSPATLSLSPGERAT LSCRASQSVSSYLAWYQQKP GQAPRLLIYDASNRATGIPA RFSGSGSGTDFTLTISSLEP EDFAVYYCQQRSNWPLTFGQ GTKVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFY PREAKVQWKVDNALQSGNSQ ESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC 55 TRGB159 QVQLVQSGAEVKKPGSSVKVSC KASGGT FSSYAISWVRQAPGQG LEWMGGIIPIFGTANYAQKFQG RVTITADESTSTAYMELSSLRS EDTAVYYCARHGRNSGRLDYWG QGTLVTVSSASTKGPSVFPLAP SSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPK SCDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYT LPPSREEMTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK 52 EIVLTQSPATLSLSPGERAT LSCRASQSVSSYLAWYQQKP GQAPRLLIYDASNRATGIPA RFSGSGSGTDFTLTISSLEP EDFAVYYCQQRSNWPLTFGQ GTKVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFY PREAKVQWKVDNALQSGNSQ ESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC 55 TRGB160 QVQLLESGGGLVQPGGSLRLSCA ASGFTFSNYWMSWVRQAPGKGLE WVSAISGSGGSTYYADSVKGRFT 53 EIVLTQSPATLSLSPGERAT LSCRASQSVSSYLAWYQQKP GQAPRLLIYDASNRATGIPA 55

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ISRDNSKNTLYLQMNSLRAEDTA VYYCAKDFYWDSFDYWGQGTLVT VSSASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLS SWTVPSSSLGTQTYICNVNHKP SNTKVDKKVEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLM ISRTPEVTCVWDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKC KVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK RFSGSGSGTDFTLTISSLEP EDFAVYYCQQRSNWPLTFGQ GTKVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFY PREAKVQWKVDNALQSGNSQ ESVTEQDSKDSTYSLSSTLT LSKFADYEKTHACE TRGB162 QVQLVQSGAEVKKPGSSVKVSC KASGGT FSSYAISWVRQAPGQG LEWMGGIIPIFGNANYAQKFQG RVTITADESTSTAYMELSSLRS EDTAVYYCARHVYKRGVLNYWG QGTLVTVSSASTKGPSVFPLAP SSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPK SCDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYT LPPSREEMTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK 54 EIVLTQSPATLSLSPGERAT LSCRASQSVSSYLAWYQQKP GQAPRLLIYDASNRATGIPA RFSGSGSGTDFTLTISSLEP EDFAVYYCQQRSNWPLTFGQ GTKVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFY PREAKVQWKVDNALQSGNSQ ESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC 55

Example 7 Evaluation of anti-GITR antibodies in a functional cell killing assay.

Antibodies that bind to human and cynomolgus GITR were tested for activity in the ADCC and CDC assays. For comparison, the isotypic control antibody huIgG1 was used in such assays. ADCC assays were used to study cell killing performed by NK-92 cells genetically modified to express the high affinity FcYRIIIa 176V / V polymorphism. Three kinds of target cells were used: HuT102 cells that endogenously express human GITR, pooled HT1080-huGITR stable transfectants, and HEK293 cells that transiently transfected with human GITR or cynomolgus GITR. For ADCC assays, target cells were labeled with Calcein AM, washed, resuspended in assay medium, and plated at 50,000 cells / 50 μl / well in 96-well V-bottom plates. Anti-hGITR or control antibodies (100 μl / well) were added to the wells at various concentrations. NK-92 176V effector cells were washed, resuspended in assay medium and plated at 50,000 cells / 50 μl / well or 100,000 cells / 50 μl / well along with target cells and antibodies. As a control, we used pure medium (background signal), only target cells (spontaneous lysis signal), cells that will ultimately be treated with Triton X-100 (maximum lysis signal), and an isotypic control antibody at a final concentration of 1 μg / ml. After 1 h incubation at 37 ° C, complete cell lysis was induced in the wells with max. signal by adding 20 μl of 2% Triton X100, after which the plates were centrifuged. 100 μl of supernatants were removed and added to black clear-bottomed plates. Fluorescence intensity units (FI) were measured using a Molecular Devices SpectraMax5. The percentage of specific lysis was calculated after subtracting the average FI observed for pure media from all wells. To determine the percentage of specific lysis, the formula (Sample - Spontaneous lysis) / (Max lysis - Spontaneous lysis) x100 was used. Half-maximum effective concentration (EC ₅₀ ) analysis was performed for each antibody in Prism.

Table 5 shows the activity of GITR antibodies in various cell lines tested.

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Table 5. Activity of GITR antibodies in ADCC assay

TRGB23 ^A eu ₅₀ (ng / ml) TRGB25 ^A EC ₅ o (ng / ml) TRGB31 EC ₅₀ (ng / ml) TRGB34 EC ₅₀ (ng / ml) TRGB153 eu ₅₀ (ng / ml) TRGB159 eu ₅₀ (ng / ml) TRGB160 eu ₅₀ (ng / ml) NiT102 From 1.6 up to 1, 9 6.8 to 9.4 2.9 to 3.7 1.3 to 1.7 From 0.6 BEFORE 18 From 32.7 UP TO 61.1 From 11.2 up to 21.3 HT1080- hGITR From 0.9 up to 4.5 15.0 to 67.4 2.0 to 4.2 2.4 to 9, 8 From 0.01 UP TO 1.2 6,7 4.2 to 26, 6 HEK293- hGITR 4.4 52.0 7.6 5, 8 nineteen 2.9 30.2 HEK293- GITR Javanese macaque 10.2 465.9 No activity No activity 2.5 25, 8 56.7

^A TRGB23 is TRGB25, TRGB25 is TRGB19.

Example 8. Construction of a Dual Gene and Production of Low Fucose Molecules.

In preparation for the preparation of the cell line for TRGB25, TRGB153, TRGB159 and TRGB160, a double gene was constructed. During this procedure, it was found that the TRGB25 heavy chain belonged to the human IgG1_G1m allotype (17,1), and not to the preferred human IgG1_G1m allotype (17). During the construction of the double gene, the V region of the TRGB25 heavy chain was transformed into the structure of the human IgG1_G1m allotype (17), resulting in the creation of a new protein TRGB190. At this point, the TRGB160 framework mutation at the amine end of the heavy chain was also detected. During the construction of the double gene, the terminal amine residue in the heavy chain of TRGB160 was changed from Q to E, thereby generating a new protein TRGB191. In addition, it was decided to prepare a low fucose version of TRGB191, that is, TRGB191.CLF. Table 6 shows the sequences of such modified anti-GITR antibodies.

Table 6. VH and V _L sequences for 2 GITR mAb candidates with framework modification. CDRs remained unchanged and. identical to parent

MAb identifier Amino Acid Sequence VH SEQ ID NO: Amino acid sequence VL SEQ ID NO: TRGB190 TRGB191. CLF QVQLVQSGAEVKKPGSSVKVSC KASGGT FSSYAISWVRQAPGQG LEWMGGIIPIFGTANYAQKFQG RVTITADESTSTAYMELSSLRS EDTAVYYCARHRRFWLDYWGQG TLVTVSSASTKGPSVFPLAPSS KST SGGTAALGCLVKDY FPE PV TVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPELLGGPSVFL FPPKPKDTLMISRTPEVTCVVV DVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLP PSREEMTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKS LSLSPGK EVQLLESGGGLVQPGGSLRLSC AASGFTFSNYWMSWVRQAPGKG LEWVSAISGSGGSTYYADSVKG RFTISRDNSKNTLYLQMNSLRA EDTAVYYCAKDFYWDSFDYWGQ GTLVTVSSASTKGPSVFPLAPS SKSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKS CDKTHTCPPCPAPELLGGPSVF LFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTL PPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQK SLSLSPGK 63 64 EIVLTQSPATLSLSPGERAT LSCRASQSVSSYLAWYQQKP GQAPRLLIYDASNRATGIPA RFSGSGSGTDFTLTISSLEP EDFAVYYCQQRSNWPLTFGQ GTKVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFY PREAKVQWKVDNALQSGNSQ ESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC EIVLTQSPATLSLSPGERAT LSCRASQSVSSYLAWYQQKP GQAPRLLIYDASNRATGIPA RFSGSGSGTDFTLTISSLEP EDFAVYYCQQRSNWPLTFGQ GTKVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFY PREAKVQWKVDNALQSGNSQ ESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC 55 55

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Example 9. Measurement of affinity by the SPR method.

The affinities of GITR antibodies to recombinant human GITR ECD were measured by surface plasmon resonance (SPR) using the ProteOn XPR36 protein interaction analysis chip system (BioRad).

For each variant, the rates of association and dissociation with the GITR ECD were measured. The biosensor surface was prepared by the method of covalent binding of goat antibodies to human IgG (Fc) to the surface of the GLC chip (BioRad) according to the manufacturer's instructions for the chemical reaction of amine binding. 8800 RU (response units) of goat anti-human IgG (Fc) antibodies (Jackson ImmunoResearch laboratories product code 109-005-098) were immobilized. The immobilized OEs also included a goat anti-mouse Fc antibody that was added to capture other antibodies not included in the description. Since a 1: 1 mixture was used, it is estimated that about 50% of these immobilized OEs were goat anti-human Fc antibodies. Binding kinetics experiments were performed at 25 ° C in rolling buffer (PBS pH 7.4, 0.005% P20, 3 mM EDTA). Four-fold (1: 3) serial dilutions of human GITR ECD or cynomolgus GITR ECD were prepared in rolling buffer, starting at 100 nM. On average, 300 OU mAbs (174-600) were captured for each channel of the sensor chip. As a reference surface, reference spots (a surface modified with goat anti-human IgG (Fc) antibodies) were used that did not contain the captured potential antibody. After the capture of the mAb, a 3-minute injection (association phase) of an antigen solution at a concentration of 40 μl / min was performed, followed by a 10-minute injection of a running buffer (dissociation phase). The chip surface was regenerated by injecting 0.85% phosphoric acid at a rate of 100 μl / min. The data were processed using the software of the device. A double reference subtraction of the data was carried out by subtracting the graphs obtained from the injection of the buffer from the reference graphs obtained from the injections of the analytes. Kinetic data analysis was performed using a 1: 1 Langmuir binding model with group fit. The result for each mAb is presented in the format kon or association rate, koff or dissociation rate and KD (equilibrium dissociation constant) (Table 7).

Table 7. RPR affinity results for anti-GITR mAb binding to human and cynomolgus GITR ECD protein

shah GITR AVERAGE to _op (1 / Ms) AVERAGE k _off (1 / s) Ko (pm) ΓΠϋΓΌΟ R Man 7.93E + 05 4.31E-05 54.3 1KbbZэ Javan macaque 2.37E + 05 9.31E-04 3936 Man 1.40Е + 05 1.50Е-04 1077 TRGB160 Javanese macaques 8.80Е + 04 4.19E-05 476 Man 9.96E + 05 2.02E-05 20.2 TRGB153 Javanese macaques 2.99E + 05 2.57E-03 8597 Man 5.32E + 05 1.62E-03 3045 TRGB159 Javanese macaques 1.85E + 05 1.97E-03 10629 Negative Human / Javanese No binding in tested the control toque conditions

The results indicated that several antibodies met the target of binding to the cynomolgus GITR ECD with an affinity within five-fold compared to binding to the human GITR ECD. Obviously, these results contradict the data on cell killing discussed in example 7, where most of the antibodies killed cells expressing cynomolgus macaque GITR with only marginally less efficacy than against cells expressing human GITR protein. There is a potential for misfolding of truncated extracellular GITR domains that are overexpressed in insect cells, leading to a discrepancy between cell killing experiments and affinity assays. Correct folding is also likely for full-length GITR expressed in human cells, and therefore binding affinity measurements to GITR-expressing cells are more likely to match observed cell killing activity. To test these possibilities, an assessment of the affinity of these antibodies for cells that express human or cynomolgus GITR should be carried out.

Example 10. Measurement of affinity by the MSD method.

Cell affinity experiments were performed to assess the binding of anti-GITR antibodies using human and cynomolgus GITR transfected HEK293 cell lines using MSD-based cell affinity measurement technology (MSD-CAT). MSD-CAT was developed as a proprietary label-free method for determining affinity on intact cells in a high-throughput format. The maternal HEK293 cell line without any GITR expression was used as a negative control.

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To measure the affinity of the interaction using this technique, a series of solutions were prepared with a fixed concentration of anti-GITR antibody (300, 60, 12, 2.4 pM) and a varying concentration of cells expressing human or cynomolgus monkey GITR (2.0x107-1.0x103 cells / ml), and equilibrated by rotating the plates for 18 hours at 4 ° C. These samples were prepared in DMEM Glutamax medium (Invitrogen, item no. 10569-044) with 0.05% azide, 1% BSA, 3 mM EDTA. After equilibration, the plates were centrifuged for 5 min at 2000 rpm and free anti-GITR mAbs were recorded in the supernatant. Free anti-GITR mAbs in the mixture were monitored by electroluminescence (ECL) using an MSD reader. For registration, MSD-streptavidin plates (MesoScale Discovery, product code L11SA-1) were coated with 0.1 μg / ml biotinylated anti-human GITR antigen in assay buffer at 50 μl / well and equilibrated overnight (~ 16 h at 4 ° C). C). After equilibration, the plates were blocked by adding 150 μl / well of assay buffer without removing the coating antigen, incubated for ~ 1 h at room temperature, and washed three times with wash buffer. Supernatants from centrifuged plates were transferred to antigen-coated plates (50 μl / well), incubated for 60 min, and then washed three times with wash buffer. Then 50 μl / well 0.7 μg / ml ruthenium conjugate F (ab ') 2 donkey anti-human IgG antibody (H + L) (Jackson ImmunoResearch; item code 709-006-149) was added and incubated for 1 hour. For 1 h, plates were washed three times with wash buffer and 150 μl MSD read buffer (MesoScale Discovery cat # R92TC-1; prepared by diluting stock 1: 3 in deion. H2O) was added to each well. Thereafter, the plates were immediately scanned on an MSD Sector Imager 6000 scanner to determine the luminescence levels. The ECL signal determined by the MSD scanner was expressed as% free antibody in the mixture and the data analyzed for affinity determination using a custom equation (derived from mass law of mass action) entered into the Prism software. Concentration of free mAb versus receptor concentration was analyzed by non-linear least squares in a 1: 1 binding model to determine binding affinities. Table 8 shows the values of the cell binding affinity for all tested molecules. Table 8. MSD-CAT results for assessing the affinity of anti-GITR mAb for HEK293 cell lines expressing human and cynomolgus GITR

Try Study Human GITR (K _D , pM) Javanese macaque GITR (K _D , nM) 2¾ fold difference (Macaque / Human) TRGB190 2 3 41 ± 22 17 ± 4 63 ± 40 * (91 ± 97) 27 ± 8 1.5 1.6 one 29.2 194 6, 6 TRGB25 2 3 26 ± 11 19 ± 4 47 ± N / O 45 ± 29 1.8 2.4 4 23 ± 1 29 ± 7 1,3 2 287 ± 182 424 ± 155 1.5 TRGB191.CLF 3 83 ± 18 262 ± 159 3.2 4 158 ± 86 321 ± 63 2.0 one 112 711 6, 3 TRGB160 2 273 ± 192 464 ± 244 * (598 ± 481) 1.7 3 83 ± 43 210 ± 78 2.5 TRGB153 one -ίο ** 115 11.4

Affinity values for cell surface expressed GITR were measured by MSD-CAT for TRGB25, TRGB190, TRGB160, TRGB191.CLF and TRGB153. Four studies were conducted, with the results of the first study being considered preliminary and included only one measurement. Subsequent studies with a large number of repeated measurements were carried out (studies 2 and 3). The results of studies 2 and 3 show that in the case of TRGB190, the affinity of the mAb for GITR of cynomolgus macaque is 1.5-1.6 times lower than for human GITR. The results of studies 2 and 3 also show that in the case of TRGB191.CLF, the affinity of the mAb for Cynomolgus GITR is 1.5-3.2 times lower than for human GITR. Study 4 was conducted at a later time to confirm the data for TRGB191.CLF from previous studies. The results of study 4 show that in the case of TRGB191.CLF, the affinity of the mAb for Cynomolgus macaque GITR is 2.0 times lower than for human GITR.

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Example 11. GITR signal via NF-kB and the effect of GITRL blocking on signal transmission.

To stimulate adaptive immunity, it is necessary to multiply and preserve antigen-primed T-lymphocytes. Signals necessary for growth and survival are largely transmitted through the NF-kB channel in activated T cells. Interferon gamma (IFNy), a well-known target gene for the transcription factor NF-kB, is an essential cytokine for immunity to foreign pathogens, and is produced by the Th1 effector T cells of CD4 and CD8 cytotoxic T lymphocytes (CTL) after antigen-specific immunity has developed (Schoenborn JR, Wilson CB Adv Immunol 2007; 96: 41-101).

Members of the tumor necrosis factor receptor (TNFR) superfamily, which includes GITR, can be a source of costimulatory signal for T cells. It is initiated by binding to its respective ligands and by recruiting adapter proteins known as TRAF (TNF Receptor Associated Factor), which can signal the NF-kB pathway. In addition, the intensity of co-stimulated signaling depends on receptor oligomerization, which can be achieved in the presence of trimeric or hexameric soluble ligands or by antibody-mediated cross-linking.

To record the effect of anti-GITR antibody ligation on NF-kB activity, a modified version of the HEK-Blue NF-kB system (Invivogen) was used. These cells express the SEAP (secreted embryonic alkaline phosphatase) reporter gene under the control of a minimal promoter fused to five NF-kB and AP-1 binding sites. They were stably transfected to express human GITR. Cross-linking of the GITR receptor in this system determines the activity of NF-kB, which can be detected by the secretion of SEAP in the supernatant. Trimeric soluble chimeric protein ligand GITR (R&D Systems) was used as a positive control.

For agonist testing, 25,000 HEK-Blue-NF-KB-GITR cells were treated with serially diluted 1: 2 portions of soluble GITRL (starting at 100 ng / ml) or anti-GITR antibody (starting at 1 μg / ml) in the presence of 5-fold excess antibody cross-linking (antiHA or anti-Fc, respectively) for 16-20 hours. The supernatant (40 μl) was removed and mixed with 160 μl of Quanti-Blue ™ reagent. The colorimetric reaction was incubated at 37 ° C for up to 1 hour before being analyzed on a spectrophotometer at OD-650 nm.

To test antibodies in the antagonistic mode, cells were treated with serially diluted 1: 2 portions of anti-GITR antibody (starting at 2 μg / ml) in the presence of 25 ng / ml constant concentration of soluble GITRL. Antagonists included antibodies that blocked the binding and activity against NF-kB for soluble GITRL by more than 50%. The following are representative graphs that serve as examples of experimental variability.

In agonist mode, the anti-GITR antibodies were able to cross-link GITR on HEK-Blue-NF-KB-GITR cells, resulting in a dose-dependent increase in NF-kB activity compared to the isotypic control antibody, CNTO3930 (FIG. 3). In the presence of sGITRL, it was noted that some anti-GITR antibodies reduce the level of sGITR-dependent activation of NF-kB, while other antibodies do not show a similar effect, even when administered at 400-fold concentration. It was found that TRGB191.CLF, GTRB45, and GTRB49 do not block the GITRL: GITR interaction by more than 30%, which may be due to the variability of the assay. GTRB45 and GTRB49 are uncharacterized antibodies that also bind to GITR.

Example 12 Antibodies against GITR can enhance the response of memory T cells to CMV and TT antigen.

A distinctive feature of immunity is the generation of memory T cells against foreign antigens, so that in the case of repeated exposure, the immune response will form much faster.

Cytomegalovirus (CMV), a herpevirus, is a common infection that usually occurs without symptoms in healthy adults and children. It is estimated that 50-80% of adults are infected with CMV by the time they reach 40 years of age. Tetanus toxin (TT) is produced by the bacteria Clostridium tetani. Most adults in the United States are vaccinated against tetanus 5 times before age 6, and are boosted every 10 years thereafter.

By exposing seropositive individuals to appropriate antigens, memory T cells can be reactivated to generate a secondary immune response. An increase in the level of expression of GITR on T cells and expression of GITRL on antigen presenting cells has been demonstrated. An agonist GITR antibody can enhance T cell activation through signaling through GITR, thereby further enhancing the antigen-specific immune response.

Interferon gamma (IFNy) is an essential cytokine for immunity to foreign pathogens and is produced by Th1 effector T cells of CD4 and CD8 cytotoxic T lymphocytes (CTL) after antigen-specific immunity has developed (Schoenborn JR, Wilson CB. Adv Immunol. 2007; 96 : 41-101).

In this example, we have developed a secondary response analysis method for CMV and TT to characterize our anti-GITR antibodies in terms of their ability to enhance T cell activation by

- 41 037973 Tory was assessed by the secretion of IFNy. In short, 150,000 PBMCs obtained from seropositive donors to CMV and TT were incubated in the presence of 0.1 μg / ml CMV antigen or TT antigen (complete CMV antigen, Astarte # 1004; TT antigen, Astarte # 10 02) in wells c precoated with a series of 1: 2 serial dilutions of the test antibodies, ranging from 5 μg / ml to 156 ng / ml (from left to right on the x-axis). The supernatant was harvested after 4-6 days and IFNy levels were quantified using MSD. An antigen-only control was used to assess donor reactivity, and CNTO3930 was administered as an isotypic control antibody. For each antibody concentration, the assay was repeated several times (n = 6).

TRGB191.CLF enhances CMV-dependent activation of memory T cells in a dose-dependent manner, as shown by measurements of IFNy secretion, the maximum of which was observed at [Ab] = 5 μg / ml (Fig. 4A). When analyzing the secondary response for TT, a peak of T cell coactivation was observed at [Ab] = 625 ng / ml (Fig. 4B).

Example 13 Monoimmunotherapy using anti-GITR induces robust anti-tumor immunity.

The effectiveness of anti-GITR in the formation of antitumor immunity can only be investigated in tumor models in which the host has an intact immune system. For this reason, the murine surrogate GITR antibody, DTA-1, was investigated in validated syngeneic colon carcinoma models CT26 and MC38 in Balb / C or C57 / BL6 mice, respectively.

Mice were implanted subcutaneously (sc) into the right flank with 5x10 ⁵ CT26 or MC38 tumor cells. On day 7 after implantation of tumor cells, mice were randomized to experimental groups with an average tumor size of approximately 85 mm ³ or 120 mm ³ , respectively.

Mice were injected with DTA-1 (BioXcell # BE00b3) or isotypic control rat IgG2b (clone LTF-2, BioXcell # BE0090) intraperitoneally in an amount of 200 μg / animal once every 3-4 days up to a total of 3 doses on days 7, 11 and 14 ( n = 10 / group). Tumor size was measured with a caliper twice a week until study completion. Tumor volume was calculated using the formula: Tumor volume (mm ³ ) = (dxsh2 / 2); where d is the length and w is the width of the tumor as measured with a measuring instrument and was monitored weekly throughout the study. The percentage of tumor growth inhibition (% TGI) was defined as the difference between the mean tumor volumes in the treatment group and the control group, calculated as TGI = [((TVc-TVt) / TVc) x100], where TVc is the mean tumor volume in this control group. , and TVt is the mean tumor volume in the treatment group. According to NCI criteria, a TGI> 60% is considered biologically significant.

In the MC38 model, statistically significant inhibition of tumor growth was achieved with the introduction of DTA-1 (80% TGI on day 21 compared to isotypic control, p <0.0001) with tumor regression, which was noted already on day 14 after administration of DTA-1, and on day 28, complete responses (CR) were achieved in 5/10 animals. CRs appeared to be stable, with no regrowth observed up to 35 days after the last dose of treatment.

In the CT26 model, statistically significant inhibition of tumor growth was achieved with the introduction of DTA-1 compared to isotype-treated control animals (> 65% TGI on day 27, p <0.0001) with tumor regression, which was observed in half of the group (5/10 animals ) as early as day 14, and with complete responses (CR) observed on day 31 (FIG. 5). CRs appeared to be stable, with no regrowth observed up to 42 days after the last dose of treatment.

Example 14 Combination anti-GITR therapy with immunological checkpoint antibodies and a T-cell agonist antibody enhances anti-tumor immunity.

Syngeneic colon carcinoma model MC38 was used to evaluate combination therapy of anti-GITR in combination with anti-PD-1, anti-CTLA-4 checkpoint blockade, or anti-OX-40 antibody.

Mice were implanted subcutaneously (sc) into the right flank with 5x10 ⁵ MC38 tumor cells. On days 14-21 after implantation of tumor cells, mice were randomized to experimental groups with an average tumor size of about 200 mm ³ . Mice were injected with surrogate anti-GITR (DTA-1, BioXcell # BE0063), anti-PD-1 (RMP1-14, BioXcell # BE0146), anti-CTLA-4 (9D9, BioXcell # BP0164), anti-OX40 (OX- 86, BioXcell No. BE0031) or isotypic control of rat IgG2b (LTF-2, BioXcell No. BE0090) intraperitoneally in an amount of 100 μg / animal once every 4 days for a total of 3 doses on days 1, 5 and 9 after randomization (n = 10 / group ). Tumor size was measured with a caliper twice a week until study completion. Tumor volume was calculated using the formula: Tumor volume (mm ³ ) = (dxsh2 / 2); where d is the length and w is the width of the tumor as measured with a measuring instrument and was monitored weekly throughout the study. The percentage of tumor growth inhibition (% TGI) was defined as the difference between the mean tumor volumes in the treatment group and the control group, calculated as TGI = [((TVc-TVt) / TVc) x100], where TVc is the mean tumor volume in this control group. , and TVt is the mean tumor volume in the treatment group. According to NCI criteria, a TGI> 60% is considered biologically significant.

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Statistically significant inhibition of tumor growth was achieved with the administration of anti-GITR compared to the isotypic control cohort, even if the administration was started when the tumors were larger and the dosage was reduced from 200 μg / mouse to 100 μg / mouse. In the anti-GITR + anti-PD-1 combination group, tumor regression was observed in 5/10 animals on day 26 after randomization (FIG. 6). In the anti-GITR + anti-CTLA-4 combination group, tumor regression was observed in 3/10 animals, and delayed tumor progression was noted in 2/10 animals (Fig. 7). Finally, the combination of anti-GITR (d1) + anti-OX40 (d5, d9) was more effective than anti-GITR monotherapy (d1, d5, d9) and anti-OX40 monotherapy (d5, d9) (Fig. 8).

Example 15 Combined anti-GITR and anti-PD1 vaccination therapy induces sustained antigen-specific expansion, function and differentiation of CD8 + T cells in tumor-free mice.

The mechanisms by which combination therapy targeting PD-1 blockade GITR enhances the Ag-specific CD8 + T cell response under vaccination conditions has been investigated. To study this problem, tumor-free mice were immunized once with the OVA peptide vaccine with the immunodominant CTL epitope OVA257-264 (hereinafter referred to as Vax) and injected with 200 μg of anti-GITR on days 0, 3 and 6 and 200 μg of anti-PD-1 on days 3 , 6, 9 and 12. Vax / anti-GITR / anti-PD-1 combination therapy enhanced CD8 + effector function compared to control, as evidenced by increased levels of splenic Ag-specific IFNy ELISpot responses, polyfunctional CD8 + T cell responses, and increased levels of CD107a / IFNY CD8 + T cells exhibiting cytolytic activity (Fig. 9A, B and C, respectively). The triple therapy induced a significantly higher frequency of polyfunctional effector CD8 + T cells expressing single IFNy, dual IFNY / TNFa, and triple ZFNy / TNFa / IL-2 compared to other treatment regimens and control groups (Fig. 9B). Direct staining with the tetramer OVA267-264 H-2K ^b- SIINFEKL Vax / anti-GITR / anti-PD-1 significantly increased the response rate of OVA tetramer-specific CD8 + T cells in peripheral blood on days 7 and 14 (Fig. 9D and 9E ), indicating directed migration of target-specific CD8 + T cells. The high frequencies of Th1 inflammatory cytokine secreting effector cells suggest that the in vivo anti-GITR / anti-PD-1 combination can enhance vaccine-induced responses of Ag-specific CD8 + T cells.

The degree of the combination therapy-induced shift in the differentiation of Ag-specific CD8 + T cells towards the effector phenotype as opposed to the memory phenotype was determined by the results of surface expression of CD44 and CD62L 14 days after priming with the vaccine. The phenotypic profile of central memory cells (CM) usually corresponds to CD44 + and CD62L +, and the profile of effector memory cells (EM) is CD44 ⁺ and CD62L. There was a significant increase in the population of OVA-tetramer-specific EM and CM CD8 + T cells in mice receiving triple combination therapy compared with other groups (Fig. 9E). In addition, it was noted that the predominant population of KLRG1 + CD8 + T cells represents the optimal effector subpopulation for protective immunity (25-27) and, apparently, a vital subpopulation that correlates with efficacy with anti-cancer immunotherapy regimens (23, 28- 29). Therefore, as an analog, the phenotype of a population of Ag-specific CD8 + T cells was characterized for expression on the cell surface of killer cells of member 1 (KLRG1) of the lectin-like receptor G subfamily. As shown in FIG. 9F, the percentages of tetramer-specific KLRG1 + effector CD8 + memory T cells were significantly higher in the triple combination therapy group compared to control groups. The presented results collectively demonstrate that the combination of anti-GITR / anti-PD-1 with vaccination can enhance the expansion and function of active Ag-specific CD8 + memory T cells in vivo.

Example 16. Combination therapy with vaccination induces tumor regression and increases survival in mice with tumors.

After confirming the enhancement of the response of Ag-specific effector CD8 + T cells induced by triple combination therapy in the absence of a tumor, the question arises whether such a combination can induce an antitumor response within the weakly immunogenic model of B16-OVA melanoma. B16-OVA tumor cells were implanted in cohorts of intact recipient B6 mice (n = 10 / group). Several days after implantation, after tumors reached an average size of ~ 30-40 mm ³ , mice were randomized and treated as described in FIG. 10A. With the scheme of administration of antibodies without a vaccine, tumor growth was moderately slowed down, but did not lead to the disappearance of the tumor, apparently, due to the weak induction of Ag-specific T cells. Likewise, neither Vax monotherapy nor combination with anti-GITR or anti-PD-1 mAb provided more than 10-20% survival. However, tumors in mice treated with Vax / anti-GITR / anti-PD-1 grew significantly slower than in all other groups (Fig. 10B-10C). Interestingly, Vax / anti-GITR / anti-PD-1 combination therapy significantly increased tumor regression and survival in approximately 50% of mice compared to other combination therapy options or vaccine monotherapy (FIGS. 10C-10D). Taken together, these data indicate that the combination of anti-GITR antibody targeting and anti-PD-1 antibody blockade can provide a synergistic effect when combined with vaccine use to improve overall survival.

Example 17 Combined Vαx / anti-GITR / anti-PD-1 immunotherapy induces AG-specific polyfunctional CD8 + T cells and reduces the TREG population in tumors.

To understand the mechanism of action of the combination therapy, we analyzed the Ag-specific phenotype and the functional response of CD8 + effector and CD4 ⁺ Tregs isolated from tumors after various types of immunotherapy. Given the important role of immunity of polyfunctional effector CD8 + T cells in anti-tumor immunity (Villarreal DO, et al. Cancer Res 2014; 74: 1789-800; Slaney CY, et al. Cancer Res 2014; 74: 7168-7174), the Ag population -specific CD8 ⁺ T cells and its expression of IFNy and TNFa in response to ex vivo stimulation with the OVA257-264 SIINFEKL peptide was studied 15 days after tumor implantation (Fig. 11A). Vax / anti-GITR / anti-PD1 combination therapy significantly increased IFNy and TNFa production from effector CD8 ⁺ T cells in tumors compared to all other groups (Fig. 11A). Moreover, Vax / anti-GITR / anti-PD-1 therapy showed a synergistic effect, as evidenced by the higher frequency of OVA-specific IFNY / TNFa double positive CD8 ⁺ T cells within the tumor (Fig. 11A). Since cytolytic CD8 ⁺ CTLs are essential components of tumor defense (Villarreal DO, et al. Cancer Res 2014; 74: 1789-800; Slaney CY, et al. Cancer Res 2014; 74: 7168-7174), by the expression marker CD107a determined the cytolytic potential of cells undergoing degranulation. The results show that tumor infiltrating CD8 ⁺ lymphocytes (TIL) isolated from tumor mice treated with Vax / anti-GITR / anti-PD-1 had a significantly higher frequency of CD8 ⁺ T cells with lytic activity relative to OVA257- 264 compared to the control, which means that such T cells had a higher potential for targeting tumor cells (Fig. 11B). The triple combination also induced a higher frequency of OVA-tetramer-specific CD8 ⁺ T cells targeting tumor migration (Fig. 11C). Moreover, a similar trend was observed for the frequency of CD8 ⁺ T cells secreting IFNy, TNFa and / or expressing CD107a when stimulated with PMA / ION, indicating that the Vax / anti-GITR / anti-PD-1 combination overall, more functional CD8 ⁺ T cell responses (Fig. 11D). Vαx / anti-GITR / α anti-PD-1 TIL stimulated with PMA / ION had a higher frequency of cytolytic CD8 ⁺ T cells co-expressing CD107a ⁺ IFNY ⁺ . This correlates with a significant increase in cytolytic activity with significant growth control and / or regression of developed tumors in mice.

Since one of the mechanisms of action of the anti-GITR mAb leads to a decrease in the level of CD4 ⁺ Treg in tumors (Cohen AD, et al. PloS one 2010; 5: e10436; Schaer DA, et al. Curr Opin Immunol 2012; 24: 217-224; Schaer DA, et al. Immunother Cancer 2014: 15: 2-7), evaluated the effects of Vax / anti-GITR / anti-PD-1 combination immunotherapy on such cells in tumors. At the same time, before evaluating the Treg population in tumors, the Treg population in the spleen was monitored on day 14 in tumor-free mice using the scheme in FIG. 10, but for intact mice. There was a significant decrease in Treg in the Vax / anti-GITR / anti-PD-1 treated group compared to the other immunotherapy groups (Fig. 12A). Thus, based on these results, it was assumed that triple combination therapy would lead to a decrease in Treg levels in tumors. Monitoring the population on day 15 after tumor implantation showed that both anti-GITR / anti-PD-1 and Vax / anti-GITR / anti-PD-1 immunotherapy options in a similar manner and dramatically reduced Treg infiltration into the tumor (Fig. 12C-12D). indicating that the combination of anti-GITR in both variants can reduce the number of Tregs infiltrating the tumor. The triple combination generally showed a more effective reduction in Treg in tumors compared to all treated groups. The overall decline in Treg populations in most vaccinated groups may have been associated with a favorable Th1 response in TME, converting TME from suppressive to inflammatory (Tatsumi T, et al. J Exp Med 2002; 196: 619-628; Fridman WH, et al. Nat Rev Cancer 2012; 12: 298-306). All immunotherapy options except anti-GITR / anti-PD1 significantly increased CD8 ⁺ T cell infiltration into the tumor (FIG. 12B), which is likely due to the induction of Ag-specific CTL responses elicited by the peptide vaccine, as shown in FIG. 9 and FIG. 11A. As a result, intra-tumor CD8 / Treg ratios dramatically increased, with the triple combination therapy being statistically more effective than any other Ab combination therapy (Fig.12D), a response that has been described as correlating with therapeutic efficacy in the melanoma model ( Quezada SA, et al. J Clin Invest 2006; 116: 1935-1945). Taken together, the synergistic effects of the Vax / anti-GITR / anti-PD-1 combination, enhancing tumor-responsive CTL responses, decreasing Treg numbers, and leading to increased effector T-cell to Treg ratios in tumors, may form a more Ag-specific inflammatory microenvironment. , which is more capable of mediating the removal of the tumor.

Example 18 Vαx / anti-GITR / anti-PD-1 combination therapy induced CD8 ⁺ T cell mediated B16-OVA tumor rejection and generated longer-term memory.

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Tumor-infiltrating CD8 'T cells showed synergistic enhancement with peptide immunization in Vax / anti-GITR / anti-PD-1 combination therapy, suggesting that increased induction of active CTL responses was most likely to be critical to the efficacy of combination therapy. For this reason, the effect of effector cell populations on tumor rejection induced by combination therapy was investigated. As shown in FIG. 13A, tumor-bearing mice were depleted of CD8 + T cells, CD4 + T cells, and NK cells during therapy. The results show that CD8 depletion completely destroyed the beneficial effects provided by Vax / anti-GITR / anti-PD-1, as none of the mice survived 22 days after implantation (Fig. 13B). In contrast, depletion of CD4 and NK cells did not affect the anti-tumor activity of Vax / anti-GITR / anti-PD-1 therapy (Fig. 13B), suggesting that these cells did not play any role in the observed efficacy. Overall, there were no statistical differences in tumors between control mice or those treated with anti-CD8 alone or αnu-NK1.1 alone. Consistent with previous research (Fujiwara S, et al. J Invest Dermatol 2014; 134: 1884-92), we observed tumor growth retardation and a significant difference in observed survival (p = 0.0037; CD4-depleted versus isotype) with the anti-CD4-only group (FIG. 13B). However, there was no additional benefit of administering aCD4 (FIG. 13B) or aCD25 (FIG. 16) with Vax / anti-GITR / anti-PD-1 combination therapy, indicating that the combination may act independently of helper T- cells or depletion of regulatory CD4 + T cells. Overall, the results indicate that CD8 'T cells are the main effector population responsible for tumor survival and inducing tumor rejection.

The ultimate goal of vaccination and active immunotherapy against malignant neoplasm is the generation of T cells with long-term memory that can respond quickly to subsequent Ag exposure. To assess memory responses, experiments were performed to re-challenge surviving animals without tumors 6 months after completion of treatment. All mice that survived the first tumor challenge with Vax / anti-GITR / anti-PD-1 treatment survived the second tumor challenge for the same tumor 6 months later (Fig.13C), indicating prolonged anti-tumor immunity and inducing long-term responses. memory. In addition, if mice that recovered from Vax / anti-GITR / anti-PD-1 treatment were re-challenged with a B16.F10 maternal tumor line that did not express OVA, ~ 80% of mice did not have a tumor that rejected when re-challenge ( Fig.13D). Overall, these data show that Vax / anti-GITR / anti-PD-1 combination therapy can induce long-term memory responses as well as epitope spread against other antigens expressed by tumor cells.

Example 19 Vax / anti-GITR / anti-PD-1 combination induces active AG-specific tumor infiltrating KLRG1 + effector CD8 + T cells important for tumor control and resection.

In-depth research in this area has shown that CTLs play a major role in tumor rejection, and the number of tumor-infiltrating effector CD8 + T cells often correlates with a favorable prognosis (Blohm, U., et al. Eur. J. Immunol. 2006; 36: 468 -477; Boissonnas, A. et al. J. Exp. Med. 2007; 204: 345-356; Steer, HJ, et al. Oncogene 2010; 29: 6301-6313). Recently, a number of studies have supported the hypothesis that a subpopulation of KLRG1 ⁺ effector CD8 ⁺ memory T cells can predict therapeutic efficacy against pathogens and tumors (Villarreal DO, et al. Molecular Therapy 2015; 10: 1653-1662; Olson JA, et al Immunity 2013; 38: 1250-60; Cush SS, Flano E. J Immunol 2011; 186: 4051-8; Ye F, et al J Immunol 2012; 189: 5206-11; van Duikeren S, et al. J Immunol 2012; 189: 3397-403; Villarreal DO, et al. Cancer Res 2014; 74: 1789-800; Slaney CY, et al. Cancer Res 2014; 74: 7168-7174; Brunner SM, et al. Hepatology 2015 ; 61: 1957-67). The increase in the number of KLRG1 ⁺ CD8 ⁺ T cells in the peripheral blood of tumor-free mice in FIG. 9F shows that such cells can be an immune correlate for complete tumor regression induced by triple combination therapy (FIG. 10). Thus, we studied the possible relationship of tumor regression with the ability of such therapy to induce stable responses of tumor-infiltrating KLRG1 ⁺ effector Ag-specific CD8 ⁺ memory T cells. Twelve days after tumor implantation (5 days after initiation of therapy; Fig.10A), it was noted that Vax / antiGITR / anti-PD-1 combination therapy resulted in the greatest increase in tetramer-specific CD8 ⁺ T cell responses in tumors (Fig. 14A). Then, a subpopulation of effector ^{memory CD8 +} T cells was assessed using the expression marker KLRG1. Interestingly, Vax / anti-GITR / anti-PD-1 therapy resulted in a ~ 2-fold increase in the incidence of tumor-infiltrating KLRG1 ⁺ CD8 ⁺ effector cells and KLRG1 ⁺ CD8 ⁺ Tet ⁺ cells compared to all other groups (Fig.14B -14C), indicating that Ag-specific KLRG1 ⁺ CD8 ⁺ effector cells can be targeted to the tumor site to activate rapid effector function. Overall, we showed that the generation of more KLRG1 ⁺ CD8 ⁺ effector T cells correlated with the regression of advanced tumors observed with Vax / anti-GITR / anti-PD-1 combination therapy.

If the expansion of the KLRG1 + CD8 + subpopulation is one of the mechanisms that contributed to the provision of more effective tumor control / regression in combination therapy Vax / anti

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GITR / anti-PD-1, it was important to determine whether depletion of the KLRG1'CD8'CD44 'subpopulation of effector T cells would result in loss of control over tumor growth. First, the ability of the anti-KLRG1 (aKLRG1) antibody to deplete the target population was determined. For this analysis, two groups of tumor-free mice were vaccinated with Vax / anti-GITR / anti-PD-1 combination therapy, and one group received 200 μg anti-KLRGl mAb (200 μg) on days 0, 2, 4 and 6 post-vaccination. and on day 7 after initiation of treatment, the expression of KLRG1 on CD8 'T cells from blood and spleen was monitored (Fig. 17). It was noted that the anti-KLRG1 mAb reduced the percentage of CD8 'T cells (Fig. 17A) and reduced the target population of KLRG1 + CD8 + CD44 + (Fig. 17B-17C). Mice treated with Vax / antiGITR / anti-PD-1 anti-KLRG1 showed a significant decrease in the frequency and / or absolute total number of KLRG1 + CD8 + CD44 + and KLRG1 ⁺ CD8 ⁺ Tet ⁺ populations in the blood and spleen compared to the control group that did not receive KLRG1 ( Fig.17B-17C). The contribution of the KLRG1 ⁺ CD8 ⁺ CD44 ⁺ population to the stimulation of triple combination therapy-induced tumor rejection by depleting KLRG1 ⁺ CD8 ⁺ CD44 ⁺ cells in tumor-bearing mice was then assessed. The results show that depletion of KLRG1 significantly reduced the level of protection, since mice with reduced KLRG1 Ab levels showed faster tumor growth than those receiving combination therapy without decreasing KLRG1 levels (Fig. 14D). More surprising was the fact that combination therapy with aKLRG1 reduction did not result in advanced tumor regression and long-term survival without aKLRG1 administration (0% versus 40% tumor rejection). Taken together, these results indicate that the increase in the number of Ag-specific KLRG1 + effector CD8 ⁺ T cells induced by triple combination therapy was the mechanism by which such dynamics contributed to the control of tumor growth, regression and long-term survival in this therapeutic model of melanoma. Thus, the expansion of such a subpopulation of effector CD8 ⁺ T cells may be a major advantage for future immunotherapy strategies.

Example 20 An anti-GITR / anti-PD-1 combination therapy demonstrates a synergistic effect with an autoantigenic tumor-associated antigen vaccine to enhance anti-tumor efficacy.

The main problem for the development of effective anti-cancer immunotherapy regimens is the generation of active anti-tumor responses against weakly immunogenic tumor-associated antigens (TAA), for example, autoantigens. Shown in FIG. The 13D results indicate that Vax / anti-GITR / anti-PD-1 combination therapy likely induced epitope extension to other TAA melanomas outside the OVA. Thus, this raised the question of whether anti-GITR / anti-PD-1 combination therapy could enhance the efficacy of a vaccine encoding a tumor-associated autoantigen. The tyrosinase-associated melanoma tumor antigen protein-2 (TRP-2) was chosen because it is one of the most extensively studied weak immunogenic melanoma tumor antigens. A single dose of TRP2 monovaccination has been shown to have a potent antitumor effect in prophylaxis in mice with B16 melanoma (Avogadri F, et al. Cancer Immuno Res 2014; 2: 448-458; Pedersen SR, et al. J Immunol 2013; 191: 3955 -3967). At the same time, this activity of tumor control is significantly reduced and limited during therapy. Likewise, anti-GITR / anti-PD-1 antibody monotherapy was of limited efficacy (FIG. 10). Therefore, for 8-day-old tumors (with an average tumor diameter of ~ 50 mm ³ ), severe therapy was performed using anti-GITR / anti-PD-1 combination therapy with simultaneous triple immunization of mice with the TRP2 peptide vaccine, as shown in FIG. 15A.

The combined treatment of advanced tumors showed significant suppression of tumor growth compared to control groups (Fig. 15B), indicating that such treatment is capable of impairing autoantigen tolerance. More importantly, 3x TRP2 / anti-GITR6 / anti-PD-1 combination therapy resulted in complete and sustained regression in ˜20% of mice, whereas monotherapy did not induce complete regression (FIG. 15B). This observation further confirms that anti-GITR / anti-PD-1 can exhibit a synergistic effect in the presence of vaccines, enhancing anti-tumor immunity. Overall, these studies support the notion that the anti-GITR / anti-PD-1 combination can be used as an immunotherapy to enhance vaccine-induced responses against tumor autoantigens and non-tumor antigens.

Example 21 Administration of anti-CD122 demonstrates a synergistic effect in the presence of tumor vaccine and anti-GITR MAB for optimal therapeutic efficacy.

Despite the fact that anti-CD122 monotherapy delayed tumor progression, it did not lead to a cure with more severe therapeutic intervention for 7-day tumors (with an average tumor diameter of ~ 30 mm ³ ) under the conditions tested (Fig. 18A-B). Therefore, in order to increase the intensity of the tumor-specific immune response, peptide anti-cancer vaccine against neoplastic antigen OVA (SIINFEKL) was used in combination with anti-CD122 therapy. Therapeutic intervention for 7-day developed tumors

- 46,037973 cholah using aHTu-CD122 and a single dose of peptide vaccine showed significant suppression of tumor growth, leading to ~ 10% long-term survival, while monotherapy with any one of the agents resulted in only little or no effect (Fig. 18A-B ). TIL analysis showed that when anti-CD122 was combined with a vaccine, there was a significant reduction in the frequency of G-MDSC compared to monotherapy with each agent (Fig. 19A-E). In addition, the combination therapy also significantly increased the production of dual IFNY / TNFa from effector CD8 ⁺ TILs and synergistically increased the level of OVA-tetramer-specific CD44 + CD8 + memory T cells in tumors (Fig. 19A-D). An increase in the level of OVA-tetramer-specific CD8 + T cells was also noted in the periphery in tumor-free mice receiving combination therapy with the vaccine and anti-CD122 (Fig. 20A-C). The combination therapy markedly reduced the proportion of CD4 ⁺ Tregs compared to monotherapy with each agent (Fig.19D), indicating that the increase in the overall level of protection observed in the combination group was associated with (1) an increase in the response of Ag-specific CD8 + T cells. (2) a decrease in G-MDSC, and (3) a decrease in the population of CD4 ⁺ Treg in the tumor. Such changes can create more favorable conditions for tumor rejection.

The primer-stimulation strategy of vaccination was used on days 7, 10 and 14 to treat 7-day advanced tumors and showed a longer-term survival (30%) compared to a single dose of vaccine under these treatment conditions (Fig. 21A). Since there has been information in the literature that CD8 + CD122 + T cells have the properties of CD8 + memory T cells (Li S et al., Cell Mol Immunol 2014; 11: 326-31; Liu J et al., Front Immunol 2015; 6: 494), we investigated whether the selection of such a population as a target could influence the generation of T cells with long-term memory. A second challenge with tumor survivors after priming-stimulation on day 80 after treatment did not result in tumor growth, indicating the formation and maintenance of certain levels of memory T cells during the combination therapy (Fig. 21B).

Finally, in light of the increased efficacy due to the additional benefits of lowering Treg levels in studies of combination Vax / anti-CD122 and anti-CD4 / anti-CD122 regimens, the possibility of using only anti-CD122 to achieve a synergistic effect with anti-GITR mAb immunotherapy was investigated. the number of CD4 + Tregs in the tumor (Schaer DA et al., Curr Opin Immunol 2012; 24: 217-224).

Therapeutic intervention for 4-day tumors using anti-CD122 mAb and anti-GITR combination therapy showed synergistic effects with significant tumor growth suppression resulting in ~ 40% long-term survival compared to anti-CD122 monotherapy (Figs.22A-22B ). These studies have opened up additional opportunities for the development of GITR-targeted approaches in combination with complementary anti-cancer immunotherapy regimens.

Example 22. Mapping of the TRGB191 binding epitope.

To identify the TRGB191 binding epitopes on the extracellular domain of human GITR, a mass spectrometric study of isotopic hydrogen-deuterium exchange in solution (HDX-MS) was performed.

Pepsin / protease XIII digestion and LC-MS.

For digestion with pepsin / protease XIII, 3.2 μg human GITR in 133 μl control buffer (50 mM phosphate, 100 mM sodium chloride at pH 7.4) was denatured by adding 135 μl 4 M guanidine hydrochloride, 0.85 M TCEP buffer (final pH 2 , 5) with incubation of the mixture for 3 min at 25 ° C. The mixture was then digested with pepsin / protease XIII on a column and the resulting peptides were analyzed using an UPLC-MS system including a Waters Acquity HPLC coupled to a Q Exactive ™ Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo). The peptides were separated on a 50 mm x 1 mm C8 column with a 19 min gradient from 2-28% Solvent B (0.2% formic acid in acetonitrile) for samples containing human GITR. Solvent A - 0.2% formic acid in water. The injector valve and pepsin / protease XIII column and their respective connecting tubes are located inside the refrigerated housing, where the temperature is maintained at 11 ° C. And the second changeover valve, column C8, and their corresponding stainless steel connecting pipes are located inside another refrigerated circulation housing, where the temperature is maintained at 0 ° C. Peptide identification is performed by searching MS / MS data compared to human GITR sequence using Mascot. The mass tolerance for precursor and product ions is 10 ppm and 0.05 Da, respectively.

Determination of glycan mass.

μg of human GITR was deglycosylated by incubation with 1 μl of PNGase F at 37 ° C overnight. The sample was then dried and the glycan was redissolved, after which it was incubated with 5 μl of 400 mM procainamide (prepared in a 3: 7 ratio of acetic acid: DMSO (v / v) and 1 M sodium cyanoborohydride) at 65 ° C for 3 h. To remove excess labeling reagents, the sample was redissolved in 90% ACN to a total solution volume of 500 μl. After conditioning the HILICSPE plate with 200 μl water and 200 μl 90% ACN, the sample was loaded onto the HILIC-SPE plates, washed with 200 μl

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90% ACN and eluted with 50 μl of 20% ACN. 75 μl of ACN was added before further analysis.

Glycan masses were measured using UPLC-MS, which included UPLC ACQUITY and

Bruker MicroTOF QII.

HDX.

A mixture of 8 μl human GITR (3.2 μg) or 8 μl human GITR and mAb (3.2 μg: 24 μg) was incubated with 125 μl deuterated water injection buffer (50 mM sodium phosphate, 100 mM sodium chloride at pH 7.4) for 0, 60, 300, 1800, 7200 and 14400 s at 25 ° C. The exchange of hydrogen-deuterium was stopped by adding 135 μl of 4 M guanidine hydrochloride, 0.85 M TCEP buffer (final pH 2.5). The samples, after stopping the exchange, were then loaded onto a pepsin / protease XIII digestion column and analyzed by LC-MS as described above. Mass spectra were recorded only in MS mode.

MS raw data is processed using HDX Workbench software for analyzing MS H / D exchange data (J. Am. Soc. Mass Spectrom. 2012, 23 (9), 15121521). Deuterium levels are calculated using the average mass difference between the deuterated peptide and its native form (to).

Results.

Deuterium levels in the identified peptides were monitored by mass shift on LC-MS. Native human GITRECD showed a significant reduction in the level of deuteration when binding to mAb TRGB191.CLF at residues 28-50 and 70-79 in SEQ ID NO: 62. Therefore, such regions with a significant decrease in the level of deuteration upon binding to mAbs were assigned to the epitope peptides, which are highlighted in dark or light gray in FIG. 23.

Modeling an epitope in the GITR structure.

HDX-MC experiments on the binding of mAb TRGB191 to human GITR ECD indicate an epitope discontinuity that is localized within two peptide regions in GITR:

region 1 (residues 28-50 in SEQ ID NO: 62), region 2 (residues 70-79 in SEQ ID NO: 62).

The binding epitope structure of mAb TRGB191 was further refined by mapping HDX data within a 3D GITR model derived from the crystal structure of GITR ECD complexed with TRGB159 Fab. According to the above structure, a significant part of the two peptides is inaccessible to the solvent. The available peptide fragments are spatially adjacent and include residues 40-45 and 75-79 in SEQ ID NO: 62 (highlighted in FIG. 24).

The structure was determined as follows.

The GITR: TRGB159 complex was prepared by mixing Fab with a 25% molar excess of GITR ECD in 20 mM Tris, pH 8.5, 250 mM NaCl and incubated at 4 ° C overnight. The formation of the complex was monitored on a Superdex 200 column. The crystallization of the complex was carried out by vapor diffusion in a sessile drop at 20 ° C. Crystals suitable for X-ray diffraction analysis were obtained from 14% PEG 3350 and 0.2 M Na formate in 0.1 M HEPES buffer, pH 7.5. To collect X-ray data, one crystal was soaked for a few seconds in a mother liquor containing a cryoprotectant solution supplemented with 24% glycerol and instantly cooled in liquid nitrogen. X-ray diffraction data were acquired on Advanced Photon Source (Argonne, IL) using a Mar225 detector. Diffraction intensities were recorded with a resolution of 2.8 Angstroms and processed using the XDS program (Kabsch, W. (2010). XDS. Acta Cryst. D66, 125-132.). The structure was established by molecular substitution using the Phaser software (McCoy, AJ, Grosse-Kunstleve, RW, Adams, PD, Winn, MD, Storoni, LC & Read, RJ (2007). J. Appl. Crystallinity 40, 658-674) using structure 5116 from the Protein Data Bank as a search model. When placing the Fab inside the unit cell, the GITR molecule was injected manually based on electron density using the Coot program (Emsley, P., Lohkamp, B., Scott, WG & Cowtan, K. (2010) Acta Cryst. D66, 486- 501).

Example 23 ADCC Activity for TRGB191.CLF on Primary Activated T Cells and JJN-3 Cell Line.

Polymorphism in the FcyRIIIA gene (rs396991) leads to a change in amino acid substitution from valine to phenylalanine at position 158 (V158F), while the 158V allotype exhibits higher affinity for human IgG1 and increased ADCC; this polymorphism is sometimes referred to in the literature as V176F (Wu J, Edberg JC, Redecha PB, Bansal V, Guyre PM, Coleman K, Salmon JE, Kimberly RP. J Clin Invest; 1997; 100 (5): 1059-70). Cartron et al. it was confirmed that the homozygous genotype FcyRIIIA-158V is the only parameter associated with a positive clinical response to rituximab, which is an antibody whose mechanism of action includes ADCC of tumor cells (Cartron G, Dacheux L, Salles G, Solal-Celigny P, Bardos P, Colombat P, Watier H. Blood; 2002; 99 (3): 754-8).

TRGB191.CLF was prepared as a low fucosylation antibody, and as a result, it has about 10 times higher affinity for FcyRIIIA, the Fc receptor that is present on NK cells, compared to a conventional fucosylated variant (RFV) of the same mAb (K _D

- 48 037973 was ~ 37 nM versus ~ 370 nM in the high affinity variant of FcyRIIIA-158V, and ~ 180 nM versus 1750 nM in the low affinity variant of FcyRIIIA-158F, respectively).

Antibodies with increased affinity for FcyRIIIA have been shown to exhibit higher ADCC activity (Strohl W, Strohl L. Therapeutic Antibody Engineering - Current and Future Advances Driving the Strongest Growth Area in the Pharmaceutical Industry. 1st ed. Sawston: Woodhead Publishing; 2012) ... ADCC activity for TRGB191.CLF was evaluated against multiple target cells or cell lines expressing different levels of hGITR. For example, resting peripheral T cells express minimal levels of GITR, but expression of GITR on these cells increases when activated in vitro. The JJN-3 cell line is a human plasma leukemic cell line that expresses endogenous hGITR at more physiological levels than NiT102 cells, and more closely to those observed on activated T cells or in vitro differentiated T _regs .

ADCC activity for TRGB191.CLF was characterized for a wide range of E: T cell ratios on primary T cells at rest or on activated T cells (see FIG. 25) and on JJN-3 cells (see FIG. 26) with using effector cells NK-92 158V / V.

TRGB191.CLF exhibited minimal ADCC activity on resting non-activated CD4 + T cells (see FIG. 25A, left panel) and non-activated CD8 + T cells (see FIG. 25B, left panel). On activated primary T cells expressing GITR, JNJ64164711 elicited high ADCC activity with EC ₅₀ values ranging from 11 ng / ml to 32 ng / ml with the highest E: T ratio of 5: 1; the values varied depending on the E: T ratios (see Table 9). The B _max value also depended on the ratio of E: T cells and increased with the increase in the proportion of effector cells present in the system. The isotypic control antibody (CNTO3930) did not induce ADCC.

It was previously shown that JJN-3 cells express lower levels of GITR than HuT102 cells, and in a range that is more comparable to activated primary T cells and in vitroT _reg generated, despite the fact that the levels were several times higher. When testing JJN-3 cells using high affinity NK-92 158V effector cells over a wide range of E: T ratios (see Fig. 26), TRGB191.CLF induced ADCC activity in the range from 50 ng / ml to 130 ng / ml with B _max values, very close to those obtained in the ADCC assay with activated primary T cells (see Table 9). Isotypic control (CNTO3930) had no effect.

Table 9. Summary of JNJ-64164711-dependent NK-92-mediated ADCC activity as a function of E: T ratio

Primary Primary

activated CD4 ⁺ activated CD8 ⁺ T cells JJN-3 T cells ees ₅₀ (ng / ml) Lice ees ₅₀ (ng / ml) Lice ees ₅₀ (ng / ml) Lice E: T (5: 1) 11.32 95.48 32.18 93, 84 53.78 97.26 No No No No E: T (2: 1) data data data data 78.92 87.5 E: T (1: 1) 20.31 78.02 49, 02 62.71 83.35 76, 05 No No No No E: T (1: 2) data data data data 105 58.7 E: T (1: 5) 29, 61 51.11 47.5 37.97 126, 8 42.84

No data, conditions not investigated. Data for isotypic control of CNTO3930 and non-activated primary target cells are not provided as it was not possible to obtain a reliable curve fit using the software used. E: NK-92 158V / V effector cells; T: Target cells (CD4 + T cells, CD8 + T cells, or JJN-3 cells.

Example 24. ADCC activity for TRGB191.CLF on in vitro differentiated TREGs.

Recently published and our own data indicate that GITR is expressed on tumor-infiltrating lymphocytes present in the tumor microenvironment in mice and humans, with the highest expression level observed on CD4 + T _reg in solid tumors.

Peripheral CD4 + T cells were differentiated and expanded to transform into functionally suppressive T _regs , which were defined as CD4 + CD25 + FOXP3 +. These T _regs express similar levels of GITR compared to activated primary CD4 + and CD8 + T cells. TRGB191.CLF induced antibody-dependent Treg killing comparable to the activity observed with JJN-3 cells (see FIG. 27). Isotypic control (CNTO3930) not

- 49 037973 had no impact.

Example 25. Activity of ADCC TRGB191.CLF using high and low affinity effector cells of FCyRIIA polymorphisms.

TRGB191.CLF has about 10-fold increased affinity for FcyRIIIA-158V / V and FcyRIIIA-158F / F compared to RFV of the same mAb; its KD value for the low-affinity FcyRIIIA-158F / F variant was ~ 180 nM, which is about 2 times higher than the RFV affinity for the high-affinity receptor variant (370 nM).

The activity of ADCC TRGB191.CLF against JJN-3 cells was comparable with the use of NK-92 effector cells expressing either the high affinity variant of FcyRIIIA-158V / V or the low affinity variant of FcyRIIIA-158F / F, while the EC ₅₀ value varied ~ 2 times (40.01 ng / ml and 87.44 ng / ml, respectively) (see Fig. 28).

Example 26. The combination of anti-GITR with anti-CD40, anti-OX40, or anti-PDL-1 results in a more effective delay in tumor growth.

In animals receiving isotypic control antibodies with lower initial tumor volumes (~ 100 mm ³ ), the median time to endpoint (MTU) was approximately 19 days. Dosing of DTA-1 at a single dose of 10 mg / kg on day 1 resulted in 2 sustained complete regressions (CR) and MTU delay up to 29.3 days. FGK4.5, an anti-CD40 dosed at 2 mg / kg on days 1, 5 and 9, resulted in 6 CRs and a noticeable MTU delay of up to 60 days. The combination of a single injection of 10 mg / kg DTA-1 on day 1 along with FGK4.5 (three times once every 4 days) resulted in 8 CRs up to day 40, with 1 progression so far noted (FIG. 29).

In animals treated with isotypic control antibodies with large initial tumor volumes (~ 230 mm ³ ), the median time to endpoint (MTU) was approximately 10.5 days. FGK4.5, which was dosed at 10 mg / kg on days 1, 5 and 9, resulted in 1 complete response (CR) and a noticeable MTU delay of up to 33 days. The combination of a single injection of 10 mg / kg DTA-1 on day 1 along with FGK4.5 (three times once every 4 days) resulted in 4 sustained CRs (FIG. 30).

Combining DTA-1 (10 mg / kg, once daily, day 1) and OX86 antibodies (anti-OX40, 10 mg / kg, thrice once every 4 days starting from day 1) also resulted in more effective anti-growth responses. tumors with an increase from 2 CR with DTA-1 to 5 CR for the combination of DTA-1 with OX86. In the monotherapy variant, OX86 did not inhibit tumor progression (Fig. 31).

Combining DTA-1 (10 mg / kg, once daily, day 1) and RMP1-14 (αntu-PD-1, 10 mg / kg, three times once every 4 days) was also more effective than delaying one of the options therapy with its implementation two days after the first agent. Anti-PD-1 monotherapy resulted in 3 CRs, anti-GITR monotherapy resulted in 2 CRs, and anti-GITR and anti-PD-1 combination therapy, when administered simultaneously, resulted in 8 CRs. This efficacy was reduced to 4 or 5 CR if the dose of anti-PD-1 or anti-GITR was administered first in a sequential application, respectively (FIG. 32).

Brief description of the sequence listing

SEQ ID NO: A type View Description Subsequence one Protein Man TFGB5 and TRGB20-HCDR1 GFTFSGYW 2 Protein Man TFGB14-HCDR1 GFTFSSYA 3 PRT Man TFGB23, TFGB25, TFGB120, TFGB127, TFGB134, TFGB144, TFGB153, TFGB159, TRGB162 and GGTFSSYA

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TRGB190-HCDR1 4 Protein Man TRGB31, TRGB34 and TRGB35-HCDR1 GYSFTSYW five Protein Man TRGB5, TRGB14, TRGB160, TRGB191, TRGB191. CLFHCDR2 ISGSGGST 6 Protein Man TRGB20-HCDR2 ISSDGGSK 7 Protein Man TRGB23, TRGB25, TRGB120, TRGB134, TRGB144, TRGB153, TRGB159 and TRGB190-HCDR2 IIPIFGTA eight Protein Man TRGB31-HCDR2 IDPSDSDT nine Protein Man TRGB34-HCDR2 IYPGDSDT 10 Protein Man TRGB35-HCDR2 IDPGDSDT eleven Protein Man TRGB127. TRGB162-HCDR2 IIPIFGNA 12 Protein Man TRGB5-HCDR3 AKDFYWDAFDY 13 Protein Man TRGB14-HCDR3 AKPIRGLDY fourteen Protein Man TRGB20-HCDR3 AKEVVYDHYAALDY fifteen Protein Man TRGB23-HCDR3 ARHGNWLHFNLDY sixteen Protein Man TRGB25-HCDR3 ARHRRFWLDY 17 Protein Man TRGB31-HCDR3 ARVFPYYGLVLDY 18 Protein Man TRGB34-HCDR3 ARDYGWHDFDY nineteen Protein Man TRGB35-HCDR3 ARHRWSTSLLLDY twenty Protein Man TRGB120-HCDR3 ARPRRNTNELDY 21 Protein Man TRGB127 and TRGB162-HCDR3 ARHVYKRGVLNY 22 Protein Man TRGB134-HCDR3 ARHRWGSGNLDY 23 Protein Man TRGB144-HCDR3 ARHGFQRGYLDY 24 Protein Man TRGB153-HCDR3 ARHAWLGHLDY 25 Protein Man TRGB159-HCDR3 ARHGRNSGRLDY 26 Protein Man TRGB160, TRGB191, TRGB191. CLF-HCDR3 AKDFYWDSFDY 27 Protein Man TRGB160, TRGB191 and TRGB191. CLF-HCDR1 GFTFSNYW 28 Protein Man TRGB5, TRGB23, TRGB25, TRGB31, TRGB34, TRGB35, TRGB134, TRGB144, TRGB153, TRGB159, TRGB160, TRGB191, TRGB191. QSVSSY

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CLF and TRGB162- LCDR1 29 Protein Man TRGB14-LCDR1 QSVNNF thirty Protein Man TRGB20-LCDR1 QSVNSF 31 Protein Man TRGB120 and TRGB127-LCDR1 QSISSY 32 Protein Man TRGB5, TRGB14, TRGB23, TRGB25, TRGB31, TRGB34, TRGB35, TRGB134, TRGB144, TRGB153, TRGB159, TRGB160, TRGB191, TRGB191. CLF and TRGB162LCDR2 DAS 33 Protein Man TRGB20-LCDR2 YAS 34 Protein Man TRGB120 and TRGB127-LCDR2 AAS 35 Protein Man TRGB5, TRGB23, TRGB25, TRGB31, TRGB34, TRGB35, TRGB134, TRGB144, TRGB153, TRGB159, TRGB160, TRGB191, TRGB191. CLF and TRGB162LCDR3 QQRSNWPLT 36 Protein Man TRGB14-LCDR3 QQGFNAPLT 37 Protein Man TRGB20-LCDR3 QQYIRWPLT 38 Protein Man TRGB120 and TRGB127-LCDR3 QQSYSTPLT 39 Protein Man Heavy chain TRGB5 EVQLLESGGGLVQPGGSLRLSCAA SGFTFSGYWMSWVRQAPGKGLEWV SAISGSGGSTYYADSVKGRFTISR DNSKNTLYLQMNSLRAEDTAVYYC AKDFYWDAFDYWGQGTLVTVSSAS TKGPSVFPLAPSSKSTSGGTAALG CLVKDY FPE PVTVSWNSGALT SGV HTFPAVLQSSGLYSLSSVVTVPSS SLGTQTYICNVNHKPSNTKVDKKV EPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELT

- 52 037973

KNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK 40 Protein Man Heavy chain TRGB14 EVQLLESGGGLVQPGGSLRLSCAA SGFTFSSYAMSWVRQAPGKGLEWV SAISGSGGSTYYADSVKGRFTISR DNSKNTLYLQMNSLRAEDTAVYYC AKPIRGLDYWGQGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCL VKDY FPE PVTVSWNSGALT SGVHT FPAVLQSSGLYSLSSVVTVPSSSL GTQTYICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGGPSVF LFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDW LNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKN QVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK 41 Protein Man Heavy chain TRGB20 EVQLLESGGGLVQPGGSLRLSCAA SGFTFSGYWMNWVRQAPGKGLEWV SGISSDGGSKYYADSVKGRFTISR DNSKNTLYLQMNSLRAEDTAVYYC AKEVVYDHYAALDYWGQGTLVTVS SASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSVVTV PSSSLGTQTYICNVNHKPSNTKVD KKVEPKSCDKTHTCPPCPAPELLG GPSVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSRD ELTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 42 Protein Man Heavy chain TRGB23 QVQLVQSGAEVKKPGSSVKVSCKA SGGTFSSYAISWVRQAPGQGLEWM GGIIPIFGTANYAQKFQGRVTITA DESTSTAYMELSSLRSEDTAVYYC ARHGNWLHFNLDYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDY FPE PVTVSWNSGALT S GVHTFPAVLQSSGLYSLSSVVTVP SSSLGTQTYICNVNHKPSNTKVDK KVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDIAVEW

- 53 037973

ESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK 43 Protein Man Heavy chain TRGB25 QVQLVQSGAEVKKPGSSVKVSCKA SGGTFSSYAISWVRQAPGQGLEWM GGIIPIFGTANYAQKFQGRVTITA DESTSTAYMELSSLRSEDTAVYYC ARHRRFWLDYWGQGTLVTVSSAST KGPSVFPLAPSSKSTSGGTAALGC LVKDYFPEPVTVSWNSGALTSGVH TFPAVLQSSGLYSLSSVVTVPSSS LGTQTYICNVNHKPSNTKVDKKVE PKSCDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDELTK NQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK 44 Protein Man Heavy chain TRGB31 EVQLVQSGAEVKKPGESLKISCKG SGYSFTSYWIGWVRQMPGKGLEWM GIIDPSDSDTRYSPSFQGQVTISA DKSISTAYLQWSSLKASDTAMYYC ARVFPYYGLVLDYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDY FPE PVTVSWNSGALT S GVHTFPAVLQSSGLYSLSSVVTVP SSSLGTQTYICNVNHKPSNTKVDK KVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK 45 Protein Man Heavy chain TRGB34 EVQLVQSGAEVKKPGESLKISCKG SGYSFTSYWIGWVRQMPGKGLEWM GIIYPGDSDTRYSPSFQGQVTISA DKSISTAYLQWSSLKASDTAMYYC ARDYGWHDFDYWGQGTLVTVSSAS TKGPSVFPLAPSSKSTSGGTAALG CLVKDY FPE PVTVSWNSGALT SGV HTFPAVLQSSGLYSLSSVVTVPSS SLGTQTYICNVNHKPSNTKVDKKV EPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELT KNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLY

- 54 037973

SKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK 46 Protein Man Heavy chain TRGB35 EVQLVQSGAEVKKPGESLKISCKG SGYSFTSYWISWVRQMPGKGLEWM GIIDPGDSDTRYSPSFQGQVTISA DKSISTAYLQWSSLKASDTAMYYC ARHRWSTSLLLDYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDY FPE PVTVSWNSGALT S GVHTFPAVLQSSGLYSLSSVVTVP SSSLGTQTYICNVNHKPSNTKVDK KVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK 47 Protein Man Heavy chain TRGB120 QVQLVQSGAEVKKPGSSVKVSCKA SGGTFSSYAISWVRQAPGQGLEWM GGIIPIFGTANYAQKFQGRVTITA DESTSTAYMELSSLRSEDTAVYYC ARPRRNTNELDYWGQGTLVTVSSA STKGPSVFPLAPSSKSTSGGTAAL GCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPS SSLGTQTYICNVNHKPSNTKVDKK VEPKSCDKTHTCPPCPAPELLGGP SVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEM TKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK 48 Protein Man Heavy chain TRGB127 QVQLVQSGAEVKKPGSSVKVSCKA SGGTFSSYAISWVRQAPGQGLEWM GGIIPIFGNANYAQKFQGRVTITA DESTSTAYMELSSLRSEDTAVYYC ARHVY KRGVLNYWGQGTLVTVS SA STKGPSVFPLAPSSKSTSGGTAAL GCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPS SSLGTQTYICNVNHKPSNTKVDKK VEPKSCDKTHTCPPCPAPELLGGP SVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEM TKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHE

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ALHNHYTQKSLSLSPGK 49 Protein Man Heavy chain TRGB134 QVQLVQSGAEVKKPGSSVKVSCKA SGGTFSSYAISWVRQAPGQGLEWM GGIIPIFGTANYAQKFQGRVTITA DESTSTAYMELSSLRSEDTAVYYC ARHRWGSGNLDYWGQGTLVTVSSA STKGPSVFPLAPSSKSTSGGTAAL GCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPS SSLGTQTYICNVNHKPSNTKVDKK VEPKSCDKTHTCPPCPAPELLGGP SVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEM TKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK fifty Protein Man Heavy chain TRGB144 QVQLVQSGAEVKKPGSSVKVSCKA SGGTFSSYAISWVRQAPGQGLEWM GGIIPIFGTANYAQKFQGRVTITA DESTSTAYMELSSLRSEDTAVYYC ARHGFQRGYLDYWGQGTLVTVSSA STKGPSVFPLAPSSKSTSGGTAAL GCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPS SSLGTQTYICNVNHKPSNTKVDKK VEPKSCDKTHTCPPCPAPELLGGP SVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEM TKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK 51 Protein Man Heavy chain TRGB153 QVQLVQSGAEVKKPGSSVKVSCKA SGGTFSSYAISWVRQAPGQGLEWM GGIIPIFGTANYAQKFQGRVTITA DESTSTAYMELSSLRSEDTAVYYC ARHAWLGHLDYWGQGTLVTVSSAS TKGPSVFPLAPSSKSTSGGTAALG CLVKDY FPE PVTVSWNSGALT SGV HTFPAVLQSSGLYSLSSVVTVPSS SLGTQTYICNVNHKPSNTKVDKKV EPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK

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52 Protein Man Heavy chain TRGB159 QVQLVQSGAEVKKPGSSVKVSCKA SGGTFSSYAISWVRQAPGQGLEWM GGIIPIFGTANYAQKFQGRVTITA DESTSTAYMELSSLRSEDTAVYYC ARHGRNSGRLDYWGQGTLVTVSSA STKGPSVFPLAPSSKSTSGGTAAL GCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPS SSLGTQTYICNVNHKPSNTKVDKK VEPKSCDKTHTCPPCPAPELLGGP SVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEM TKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK 53 Protein Man Heavy chain TRGB160 QVQLLESGGGLVQPGGSLRLSCAA SGFTFSNYWMSWVRQAPGKGLEWV SAISGSGGSTYYADSVKGRFTISR DNSKNTLYLQMNSLRAEDTAVYYC AKD FYWDS FDYWGQGTLVTVS SAS TKGPSVFPLAPSSKSTSGGTAALG CLVKDY FPE PVTVSWNSGALT SGV HTFPAVLQSSGLYSLSSVVTVPSS SLGTQTYICNVNHKPSNTKVDKKV EPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK 54 Protein Man Heavy chain TRGB162 QVQLVQSGAEVKKPGSSVKVSCKA SGGTFSSYAISWVRQAPGQGLEWM GGIIPIFGNANYAQKFQGRVTITA DESTSTAYMELSSLRSEDTAVYYC ARHVY KRGVLNYWGQGTLVTVS SA STKGPSVFPLAPSSKSTSGGTAAL GCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPS SSLGTQTYICNVNHKPSNTKVDKK VEPKSCDKTHTCPPCPAPELLGGP SVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEM TKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK 55 Protein Man TRGB5, TRGB23, EIVLTQSPATLSLSPGERATLSCR

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TRGB25, TRGB31, TRGB34, TRGB35, TRGB134, TRGB144, TRGB153, TRGB159, TRGB160, TRGB162, TRGB190, TRGB191 and light chain TRGB191. CLF ASQSVSSYLAWYQQKPGQAPRLLI YDASNRATGIPARFSGSGSGTDFT LTISSLEPEDFAVYYCQQRSNWPL TFGQGTKVEIKRTVAAPSVFIFPP SDEQLKSGTASVVVCLLNNFYPREA KVQSGNKVDN 56 Protein Man Light chain TRGB14 EIVLTQSPATLSLSPGERATLSCR ASQSVNNFLAWYQQKPGQAPRLLI YDASNRATGIPARFSGSGSGTDFT LTISSLEPEDFAVYYCQQGFNAPL TFGQGTKVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFYPREA KVQWKVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC 57 Protein Man Light chain TRGB20 EIVLTQSPATLSLSPGERATLSCR ASQ SVNS FLAWYQQKPGQAPRLLI YYASNRATGIPARFSGSGSGTDFT LTISSLEPEDFAVYYCQQYIRWPL TFGQGTKVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFYPREA KVQWKVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC 58 Protein Man TRGB120 and TRGB127light chain DIQMTQSPSSLSASVGDRVTITCR ASQSISSYLNWYQQKPGKAPKLLI YAASSLQSGVPSRFSGSGSGTDFT LTISSLQPEDFATYYCQQSYSTPL TFGQGTKVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFYPREA KVQWKVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC 59 Protein Man STR 26-161 QRPTGGPGCGPGRLLLGTGTDARC CRVHTTRCCRDYPGEECCSEWDCM CVQPEFHCGDPCCTTCRHHPCPPG QGVQSQGKFSFGFQCIDCASGTFS GGHEGHCKPWTDCTQFGFLTVFPP NKT 60 Protein Man GITR 26-241 QRPTGGPGCGPGRLLLGTGTDARC CRVHTTRCCRDYPGEECCSEWDCM CVQPEFHCGDPCCTTCRHHPCPPG QGVQSQGKFSFGFQCIDCASGTFS GGHEGHCKPWTDCTQFGFLTVFPG NKT HNAVCVPGS P PAE PLGWLTVV LLAVAACVLLLTSAQLGLHIWQLR SQCMWPRETQLLLEVPPSTEDARS CQFPEEERGERSAEEKGRLGDLWV 61 Protein Javan macaque GITR QRPTGGPGCGPGRLLLGTGKDARC CRVHPTRCCRDYQSEECCSEWDCV

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CVQPEFHCGNPCCTTCQHHPCPSG QGVQPQGKFSFGFRCVDCALGTFS RGHDGHCKPWTDCTQFGFLTVFPG NKTHNAVCVPGSPPAEPPGWLTIV LLAVAACVLLLTSAQLLLGLHIWQLPRGET SQPTFP 62 Protein Man GITR full length MAQHGAMGAFRALCGLALLCALSL GQRPTGGPGCGPGRLLLGTGTDAR CCRVHTTRCCRDYPGEECCSEWDC MCVQPEFHCGDPCCTTCRHHPCPP GQGVQSQGKFSFGFQCIDCASGTF SGGHEGHCKPWTDCTQFGFLTVFP GNKTHNAVCVPGSPPAEPLGWLTV VLLAVAACVLLLT SAQLGL ΗIWQL RSQCMWPRETQLLLEVPPSTEDAR SCQFPEEERGERSAEEKGRLGDLW V 63 Protein Man TRGB190-VH QVQLVQSGAEVKKPGSSVKVSCKA SGGTFSSYAISWVRQAPGQGLEWM GGIIPIFGTANYAQKFQGRVTITA DESTSTAYMELSSLRSEDTAVYYC ARHRRFWLDYWGQGTLVTVSSAST KGPSVFPLAPSSKSTSGGTAALGC LVKDYFPEPVTVSWNSGALTSGVH TFPAVLQSSGLYSLSSVVTVPSSS LGTQTYICNVNHKPSNTKVDKKVE PKSCDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK 64 Protein Man TRGB191 and TRGB191. CLFVH EVQLLESGGGLVQPGGSLRLSCAA SGFTFSNYWMSWVRQAPGKGLEWV SAISGSGGSTYYADSVKGRFTISR DNSKNTLYLQMNSLRAEDTAVYYC AKDFYWDSFDYWGQGTLVTVSSAS TKGPSVFPLAPSSKSTSGGTAALG CLVKDY FPE PVTVSWNSGALT SGV HTFPAVLQSSGLYSLSSVVTVPSS SLGTQTYICNVNHKPSNTKVDKKV EPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK 65 Protein Man GITR-L MTLHPSPITCEFLFSTALISPKMC LSHLENMPLSHSRTQGAQRSSWKL WLFCSIVMLLFLCSFSWLIFIFLQ LETAKEPCMAKFGPLPSKWQMASS EPPCVNKVSDWKLEILQNGLYLIY GQVAPNANYNDVAPFEVRLYKNKD MIQTLTNGISGHKIHQNV

Claims (24)

CLAIM 1. An isolated antibody or antigen-binding fragment thereof that specifically binds to human GITR and contains the heavy chain CDR1 with the amino acid sequence SEQ ID NO: 27, the heavy chain CDR2 with the amino acid sequence SEQ ID NO: 5 and the heavy chain CDR3 with the amino acid sequence SEQ ID NO : 26, light chain CDR1 with amino acid sequence SEQ ID NO: 28, light chain CDR2 with amino acid sequence SEQ ID NO: 32 and light chain CDR3 with amino acid sequence SEQ ID NO: 35. 2. The antibody of claim 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain region comprising SEQ ID NO: 64 paired with a light chain region comprising SEQ ID NO: 55. 3. An antibody, or antigen-binding fragment, according to claim 1, wherein the antibody specifically binds to human GITR through interaction with amino acid residues of the GITR (SEQ ID NO: 62): a) 40-45 and b) 75-79. 4. An antibody or antigen-binding fragment according to claim 1, which binds to a polypeptide with the amino acid sequence of SEQ ID NO: 59. 5. An antibody or antigen-binding fragment according to claim 1, which specifically binds to human GITR with a binding affinity of at least 30 nM as measured by surface plasmon resonance. 6. An antibody or antigen-binding fragment according to claim 1, which induces an increase in luciferase expression in an NF-kB luciferase gene assay. 7. The antibody or antigen binding fragment of claim 1, which induces in vitro ADCC with an EC ₅₀ value of less than about 67 ng / ml. 8. An antibody or antigen-binding fragment according to claim 1, which is a human antibody or antigen-binding fragment. 9. An antibody or antigen binding fragment according to claim 1, wherein the antigen binding fragment is a Fab fragment, Fab2 fragment, or a single chain antibody. 10. The antibody or antigen binding fragment of claim 1, wherein the antibody or antigen binding fragment is recombinant. 11. An antibody or antigen-binding fragment according to claim 1, wherein the antibody or antigen-binding fragment thereof is of the IgG1, IgG2, IgG3 or IgG4 isotype. 12. An antibody or antigen-binding fragment according to claim 1, wherein the antibody or antigen-binding fragment thereof has an IgG1 isotype. 13. The antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen-binding fragment thereof specifically binds to human GITR or cynomolgus GITR. 14. A polynucleotide encoding an antibody or antigen-binding fragment according to claim 1. 15. An expression vector containing the polynucleotide of claim 14. 16. A host cell containing the vector of claim 15. 17. A method for producing an antibody or antigen-binding fragment, comprising culturing a host cell according to claim 16 under conditions allowing expression of the antibody or antigen-binding fragment, and isolating the antibody or antigen-binding molecule from the culture. 18. A method of alleviating a symptom of a malignant neoplasm or other neoplastic condition, comprising administering an antibody or antigen-binding fragment thereof according to claim 1 to an individual, if necessary, in an amount sufficient to alleviate a symptom of a malignant neoplasm or other neoplastic condition in an individual. 19. The method of claim 18, wherein the individual is a human. 20. The method of claim 18, further comprising one or more of the following: a) chemotherapy; b) performing radiation therapy, or c) the introduction of one or more therapeutic agents. 21. The method of claim 20, wherein the additional therapeutic agent is an immunostimulating agent. 22. The method of claim 21, wherein the immunostimulatory agent is selected from the group consisting of an anti-PD-1 antibody, an anti-CTLA-4 antibody, an anti-CD122 antibody, an anti-CD40 antibody, an anti-OX40 antibody, and a CD8 Ag-specific OVA peptide vaccine. 23. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to claim 1 and a pharmaceutically acceptable carrier. 24. A kit for alleviating a symptom of a malignant neoplasm or other neoplastic condition, characterized in that it contains an antibody or antigen-binding fragment thereof according to claim 1 and a package for it.