Anti-glucocorticoid-induced TNFR-related (GITR) protein antibodies and uses thereof

Abstract

The present invention provides agonistic and non-agonistic antibodies that specifically bind to a glucocorticoid-induced TNFR-related (GITR) protein, comprising a modified human constant region (Fc) and optionally a reduced fucosylation content. Polynucleotide sequences encoding the antibodies, pharmaceutical compositions thereof and methods of treating cancer are also provided.

Description

[Technical field]

[0001] The present invention is in the field of immunotherapy and relates to engineered antibodies, polynucleotide sequences encoding them, as well as pharmaceutical compositions and therapeutic uses. [Background technology]

[0002] Cancer treatment has progressed significantly over the past decade, driven by immunomodulatory therapies that enhance host antitumor defenses. In particular, immune checkpoint inhibitors such as monoclonal antibodies (mAbs), which target the immune system rather than directly recognize tumors, have been shown to induce durable responses in a subset of patients treated across multiple tumor types (Ribas, A. & Wolchok, JD Cancer immunotherapy using checkpoint blockade. Science 80-, 359, 1350-1355, 2018). These antibodies can be divided into two groups based on the means by which they induce anti-tumor immune responses: (i) agonist mAbs, which stimulate activating coregulatory receptors in the immune synapse (Mayes et al., A. The promise and challenges of immune agonist antibody development in cancer. Nature Reviews Drug Discovery, 17, 509-527, 2018), and (ii) antagonist mAbs, which block inhibitory signaling pathways in the immune synapse, thereby attenuating regulatory (inhibitory) mechanisms (Sharma et al. Science 348, 56-61, 2015).

[0003] Antibodies consist of two structural regions: the variable fragment (Fab), which mediates antigen binding, and the constant fragment (Fc), which mediates downstream effector functions via interactions with Fc receptors on (innate) immune cells or proteins of the complement system.

[0004] Fcγ receptors (FcγR) belong to the immunoglobulin superfamily and share many structural and functional properties. FcγRs can be classified as activating or inhibitory based on the presence of intracellular activating (immunoreceptor tyrosine-based activation motifs, ITAM) or inhibitory (immunoreceptor tyrosine-based inhibitory motifs, ITIM) signaling motifs. Three activating FcγRs are expressed in mice (FcγRI, FcγRIII, and FcγRIV) and humans (FcγRI, FcγRIIA, and FcγRIIIA), and a single inhibitory FcγRIIB is expressed in both species. Each FcγR has a distinct cellular expression pattern. Engagement of activating FcγRs induces effector mechanisms such as antibody-dependent cellular cytotoxicity (ADCC), phagocytosis, and production of proinflammatory cytokines. Signaling through the inhibitory receptor, FcγRIIB, results in negative functions such as suppression of inflammatory immune responses. Engagement of activating or inhibitory FcγRs by the Fc domain of an antibody is determined by the IgG subclass and Fc glycan composition (Pincetic, A. et al., Type I and type II Fc receptors regulate innate and adaptive immunity. Nat. Immunol. 15, 707-716, 2014).

[0005] Dendritic cells are a heterogeneous population of antigen-presenting cells that infiltrate tumors. Although dendritic cells play a key role in priming and maintaining local immunity, dendritic cell function is often reduced or suppressed by factors encountered in the tumor microenvironment. In addition, dendritic cell populations with immunosuppressive activity are also recruited to tumors, limiting T cell infiltration and promoting tumor growth. Activation of dendritic cells plays a key role in priming antitumor T cell immunity, thereby representing a major therapeutic target for cancer immunotherapy (Whily et al., Dendritic Cells and Cancer: From Biology to Therapeutic Intervention. Cancer 11, 4, 521. 2019).

[0006] The main principle guiding the rational design and development of antibodies to date has been the need to engineer the Fab domain of an antibody to enhance its effect on specific receptor targets on immune cells. However, recent in vivo findings have highlighted the importance of the Fc domain for enhancing the efficacy of checkpoint antibody inhibitors by recruiting additional types of immune cells expressing either activating and / or inhibitory FcγRs (Dahan, R. et al., Therapeutic Activity of Agonistic, Human Anti-CD40 Monoclonal Antibodies Requires Selective FcγR Engagement. Cancer Cell 29, 820-831, 2016; Vargas, FA et al., Fc Effector Function Contributes to the Activity of Human Anti-CTLA-4 Antibodies. Cancer Cell 33, 649-663. e4, 2018).

[0007] Fc binding to activating FcγR is necessary to induce antibody effector functions such as intratumoral Treg depletion by anti-CTLA4 (cytotoxic T-lymphocyte-associated protein 4 / CD125), anti-OX-40 (CD134), or anti-4-1BB (Bulliard, Y. et al., OX40 engagement depletes intratumoral Tregs via activating FcγRs, leading to anti-tumor efficacy. Immunol. Cell Biol. 92, 475-480, 2014; Buchan, S Let al., Antibodies to Costimulatory Receptor 4-1BB Enhance Anti-tumor Immunity via T Regulatory Cell Depletion and Promotion of CD8 T Cell Effector Function. Immunity 49, 958-970. e7, 2018). Another Fc binding is via FcγR in the tumor microenvironment (TME), e.g., by anti-PD-L1 (programmed death ligand 1) and anti-CD73 mAb. +It can induce reprogramming of myeloid cells and natural killer cells (NK). (Bulliard, Y. et al., Activating Fc γ receptors contribute to the anti-tumor activities of immunoregulatory receptor-targeting antibodies. J. Exp. Med. 210, 1685-1693, 2013; Dahan, R. et al., FcγRs Modulate the Anti-tumor Activity of Antibodies Targeting the PD-1 / PD-L1 Axis. Cancer Cell 28, 285-295, 2015; and Dahan, R. & Ravetch, JV Co-targeting of Adenosine Signaling Pathways for Immunotherapy: Potentiation by Fc Receptor Engagement. Cancer Cell 30, 2016).

[0008] Conversely, binding of inhibitory FcγRIIB provides an inert scaffold for antibody cross-linking that enhances the activity of agonistic mAbs that primarily target tumor necrosis factor receptors (TNFRs), such as anti-CD40 antibodies (Li, F. & Ravetch, JV Anti-tumor activities of agonistic anti-TNFR antibodies require differential FcRγIIB co-engagement in vivo. Proc. Natl. Acad. Sci., 110(48), 19501-6, 2013). Thus, agonistic mAbs can act through multiple mechanisms in vivo (e.g., TNFR activation and Treg depletion), which can synergize their activity for optimal therapeutic efficacy. Therefore, the selection of an appropriate scaffold for each antibody (consisting of IgG isoforms or Fc region variants that selectively engage or avoid specific FcγR pathways) is crucial to achieve optimal anti-tumor activity.

[0009] One of the aforementioned immune checkpoint molecules is the glucocorticoid-induced TNFR-related (GITR, also called TNFRSF18 and CD357) protein. GITR is a member of the cell surface TNF receptor superfamily and is constitutively expressed at high levels on regulatory T cells (Treg) and at low levels on naive and memory T cells (G. Nocentini, C. Riccardi GITR: a modulator of immune response and inflammation. Adv Exp Med Biol, 647 156-173, 2009).

[0010] GITR induction coactivates effector T lymphocytes and modulates regulatory T cell (Treg) activity. GITR is activated by GITR ligand (GITRL), which is primarily expressed on antigen-presenting cells (APCs). GITR activation increases resistance to tumor and viral infections, participates in autoimmune / inflammatory processes, and modulates leukocyte extravasation. Thus, GITR is an attractive target for immunotherapy due to its ability to promote effector T cell function and prevent regulatory T cell suppression.

[0011] Activation of T cells by several different stimuli rapidly increases GITR expression within 24 hours in both regulatory T cells (Treg) and effector T cells (Teff). (LT Krausz, et al., GITR-GITRL system, a novel player in shock and inflammation. Scientific World Journal, 7 533-566, 2007). FoxP3, a key regulatory protein for Treg development and function, promotes high levels of GITR expression in mature T cells, whereas classical NFκB signaling induces GITR expression in activated T cells, suggesting cell type-specific regulation of expression (Y. Zhan, et al., Glucocorticoid-induced TNF receptor expression by T cells is reciprocally regulated by NF-kappaB and NFAT. J Immunol, 181 5405-5413, 2008).

[0012] Low to moderate levels of GITR are also detected on innate immune cells after activation (S.Hanabuchi,et al.,Human plasmacytoid predendritic cells activate NK cells through glucocorticoid-induced tumor necrosis factor receptor-ligand (GITRL).Blood,107 3617-3623,2006). Within innate cell types, the highest induction is observed on activated natural killer cells (NK), with levels comparable to GITR expression on activated T effector (Teff) cells. Only moderate levels are seen on activated macrophages and dendritic cells (DCs) (DLClouthier,THWatts Cell-specific and context-dependent effects of GITR in cancer,autoimmunity,and infection.Cytokine Growth Factor Rev,25 91-106,2014).

[0013] The high level of GITR expression in activated Tregs is an important difference that becomes more evident during in vivo assessment of GITR regulation. Numerous studies have reported that tumor-infiltrating Treg-expressing GITR cells are present in tumors such as non-small cell lung cancer (NSCLC), renal cell carcinoma, melanoma, glioblastoma, colorectal cancer, breast cancer and ovarian cancer (Curiel, T., et al., Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival. Nat Med 10, 942-949, 2004., Zhu et al., Evaluation of glucocorticoid-induced TNF receptor (GITR) expression in breast cancer and across multiple tumor types. Mod Pathol 33, 1753-1763, 2020; Vence L, et al. Characterization and Comparison of GITR Expression in Solid Tumors. Clin Cancer Res. 2019, 25(21), 6501-6510).

[0014] Anti-mouse GITR antibodies were found to depend on FcγR-mediated Treg depletion and CD8 T cell proliferation for anti-tumor activity (D. Coe, et al., Depletion of regulatory T cells by anti-GITR mAb as a novel mechanism for cancer immunotherapy. Cancer Immunol Immunotherapy, 59, 1367-1377, 2010).

[0015] Anti-GITR agonist antibodies, either as monotherapy or in combination with additional immunotherapy, demonstrate significant therapeutic potential by enhancing effector T cells in the tumor microenvironment (TME) and suppressing Treg responses, as demonstrated in preclinical models (A.D. Cohen et al., Agonist anti-GITR monoclonal antibody induces melanoma tumor immunity in mice by altering regulatory T cell stability and intra-tumor accumulation. PLoS One. 5, 2010; D. A. Chaer, J. et al., Modulation of GITR for cancer immunotherapy. Curr. Opin. Immunol. 24 217-224 2012; L. Lu, et al. Combined PD-1 blockade and GITR triggering induce a potent anti-tumor immunity in murine cancer models and synergizes with chemotherapeutic drugs. J Transl Med, 12, 36-47, 2014; Zappasodi R, et al. Rational design of anti-GITR-based combination immunotherapy.Nat Med.2019,25(5),759-766).However, significant barriers to its clinical translation have arisen: the promising preclinical activity has yet to be reproduced in clinical trials (Tran B.,et al.,Dose escalation results from a first-in-human,phase 1 study of glucocorticoid-induced TNF receptor-related protein agonist AMG 228 in patients with advanced solid tumors.J Immunother Cancer.2018,6(1),93;and Siu LL,et al.,Preliminary results of a phase I / II a study of BMS-986156,glucocorticoid-induced tumor necrosis factor receptor-related gene (GITR)agonist),alone and in combination with nivolumab in patients with advanced solid tumors.J Clin Oncol.2017,35(15_suppl),104).

[0016] The anti-mouse GITR (mGITR) agonist antibody, called DTA-1, is the most widely used IgG2b rat antibody in research (J. Shimizu, et al., Stimulation of CD25+ CD4+ regulatory T cells through GITR breaks immunological self-tolerance. Nat Immunol. 3, 135-142, 2002). Currently, there are few antibodies in preclinical or clinical (Phase I / II) trials, such as MK-4166 from Merck & Co Inc. (U.S. Patent No. 8,709,424), ragifilimab from Incyte Corp. (U.S. Patent No. 10,155,818), REGN-6569 from Regeneron Pharmaceuticals Inc. (U.S. Patent No. 10,738,126), PTZ-522 from Potenza Therapeutics Inc. (U.S. Patent Application Publication No. 2018208665), GWN-323 from Novartis AG (U.S. Patent No. 10,662,247), and BMS-986156 from Bristol-Myers Squibb Co. (U.S. Patent No. 9,228,016). WO 2015187835 discloses anti-human GITR (hGITR) antibodies with engineered Fc regions with increased binding to FcγRIIB and increased ADCC activity.

[0017] US Patent No. 7,812,135, US Patent No. 1,121,358 and US Patent No. 2019010241 disclose specific mAbs against human GITR.

[0018] US Patent No. 9,040,041 discloses a method for enhancing activation of antibody-dependent cellular phagocytosis (ADCP) by antibodies containing engineered human Fc. The protein targets of the engineered antibodies are Ep-CAM, CD19, CD20, CD22, CD30, CD33, CD40, CD40L, CD52, Her2 / neu, EGFR, IGF-1R, EpCAM, MUC1, GD3, CEA, CA125, HLA-DR, MUC18, and prostate-specific membrane antigen (PMSA).

[0019] WO 2019 / 125846 discloses human IgG Fc region variants specific for the tumor antigen sLeA that have improved effector function and higher binding affinity to hFcγRIIA, hFcγRIIIA, and hFcγRIIB. One of the antibodies, hIgG1-G236A / A330L / I332E (GAALIE), has enhanced binding affinity to activating hFcRIIA and hFcRIIIA, and lower binding to inhibitory FcγRIIB, and also exhibits enhanced ADCC activity.

[0020] Weitzenfeld P. et al. disclose carbohydrate-targeting antibodies that contain a combination of specific mutations in the Fc domain that result in enhanced affinity for both activating hFcγRs, hFcγRIIA and hFcγRIIIA, while reducing binding to the inhibitory receptor, hFcγRIIB (Antibodies targeting sialyl Lewis A mediate tumor clearance through distinct effector pathways. J. Clin. Invest, 129, 3952-3962, 2019).

[0021] US Pat. No. 1,0479,838 discloses a specific antibody that binds to CD40, which comprises an Fc region that has been modified to enhance the specificity of binding to FcγRIIB.

[0022] WO2003035835 discloses cell lines with reduced ability to attach fucose to N297-linked carbohydrates resulting in hypofucosylation of expressed antibodies, with approximately 80-100% of the glycoproteins containing mature core carbohydrate structures lacking fucose. The antibodies produced bind human FcγRIIIA and have improved ADCC activity compared to fucosylated antibodies. One of the antibody variants disclosed has increased binding to FcγRIIA and FcγRIIIB.

[0023] The consequences of human FcγR interactions with anti-human GITR antibodies have not been comprehensively addressed, and there remains an unmet need for suitable IgG scaffolds to improve the anti-tumor activity of therapeutic anti-GITR antibodies through selective binding to Fcγ receptors in the TME. Summary of the Invention

[0024] According to some embodiments, the present invention provides antibodies that bind to glucocorticoid-induced TNFR-related (GITR) and have improved therapeutic anti-tumor activity compared to known antibodies against this protein. The present invention provides anti-GITR antibodies that include Fc modifications that result in increased binding to at least one of the activating receptors FcγRIIA and FcγRIIIA, and optionally decreased binding to the inhibitory FcγRIIB receptor. It was unexpectedly found that the Fc modified antibodies of the present invention have a high binding ratio to FcγRIIA and / or FcγRIIIA compared to FcγRIIB, and therefore cannot participate in FcγRIIB-mediated antibody cross-linking, yet induce enhanced in vivo anti-tumor effects and long-term anti-tumor immunity. Antibodies with the same Fab region but with a lower binding ratio to FcγRIIA and / or FcγRIIIA compared to FcγRIIB could not induce anti-tumor effects and long-term anti-tumor immunity.

[0025] The present invention provides therapeutic antibodies that bind with high affinity and specificity via their Fab region to GITR, via their Fc region to the activating FcγRIIA and FcγRIIIA receptors, and with comparable lower binding affinity to the inhibitory receptor FcγRIIB. The antibodies of the present invention are characterized by having a modified human IgG1 Fc region, which comprises a substitution of the glycine residue at position 236 with an alanine residue (G236A) and at least one further modification selected from reduced glycosylation content (e.g. afucosylation) and substitution of alanine at position 330 with leucine (A330L) and substitution of isoleucine at position 332 with glutamic acid (I332E). The numbering of the amino acid residues in the Fc region is according to the EU index.

[0026] Thus, according to one aspect, the invention provides an antibody or a composition comprising at least one antibody that specifically binds to GITR, wherein the antibody comprises a variable region (Fab) and a modified human IgG1 constant region (Fc), and wherein the antibody has an increased binding ratio to (RIIA and / or RIIIA) / RIIB compared to an antibody having the same Fab and parent unmodified human IgG1 Fc.

[0027] According to some embodiments, the human IgG1 Fc region modification results in an increased binding ratio of the antibody to RIIA / RIIBRIIIA / RIIB; and (RIIA+RIIIA) / RIIB.

[0028] According to some embodiments of this aspect, the invention provides an antibody that specifically binds to GITR, the antibody comprising a variable region (Fab) and a modified human IgG1 Fc region, the modified human IgG1 Fc region comprising: i. a human IgG1 Fc region comprising an asparagine residue (N) at position 297, a substitution G236A of the amino acid residue glycine (G) at position 236 with an alanine residue (A), and a fucosylation content of up to about 40% of all glycan structures (herein Afuco-G236A); and ii. a human IgG1 Fc region comprising the amino acid substitutions: a substitution of the glycine residue at position 236 with an alanine residue (G236A), a substitution of the alanine residue at position 330 with a leucine residue (A330L), and a substitution of the isoleucine residue at position 332 with a glutamic acid residue (I332E) (herein referred to as GAALIE); The antibody is selected from the group consisting of:

[0029] The present invention therefore provides an isolated afucosylated antibody that specifically binds to a glucocorticoid-induced TNFR-related (GITR) protein, the antibody comprising a variable region (Fab) and a modified human IgG1 constant region (Fc) comprising an asparagine residue (N) at position 297, a substitution G236A for the amino acid residue glycine (G) at position 236 to an alanine residue (A) (herein afuco-G236A).

[0030] The present invention also provides an antibody that specifically binds to a glucocorticoid-induced TNFR-related (GITR) protein, wherein the antibody comprises a variable region (Fab) and a modified human IgG1 constant region (Fc), wherein the modified human IgG comprises amino acid substitutions (herein GAALIE) of the glycine residue at position 236 to an alanine residue (G236A), of the alanine residue at position 330 to a leucine residue (A330L), and of the isoleucine residue at position 332 to a glutamic acid residue (I332E).

[0031] According to some embodiments, the GITR is human GITR (hGITR).

[0032] According to some embodiments, the antibody is a humanized antibody.

[0033] According to some embodiments, the antibody is a chimeric antibody.

[0034] Any binding site, hypervariable region (HVR) or Fab of an antibody that specifically recognizes GITR, particularly hGITR, can be used to generate the IgG1-Fc region modified antibodies of the present invention. This includes agonist, non-agonist, and mixed agonist / non-agonist antibodies, as well as antibodies that do not elicit a response after binding to hGITR or mediate any of the activities associated with binding to hGITR. In some embodiments, binding to hGITR on T cells is used only to target the antibodies of the present invention to the required location of the immune synapse, while the modified Fc region mediates their activity on FcγR expressing cells.

[0035] According to some embodiments, the Fab of the anti-hGITR antibody is derived from an agonist antibody that induces GITR activation mediated by MAPK signaling.

[0036] According to some embodiments, the Fab of the anti-hGITR antibody is derived from a non-agonistic antibody.

[0037] According to some embodiments, the Fab of the anti-hGITR antibody comprises a set of six CDR sequences of an antibody specific for human GITR.

[0038] According to some embodiments, the antibody comprises a set of six CDR sequences, the set comprising: i. a heavy chain (HC) CDR1 comprising the sequence GFSLSTSGMG (SEQ ID NO:1), a HC CDR2 comprising the sequence IWWDDDK (SEQ ID NO:2), a HC CDR3 comprising the sequence ARTRRYFPFAY (SEQ ID NO:3), a light chain (LC) CDR1 comprising the sequence QNVGTN (SEQ ID NO:4), a LC CDR2 comprising the sequence SAS or the sequence SAST (SEQ ID NO:5), a LC CDR3 comprising the sequence QQYNTDPLT (SEQ ID NO:6); and ii. An HC CDR1 comprising the sequence SYGMH (SEQ ID NO:7), an HC CDR2 comprising the sequence VIWYEGSNKYYADSVKG (SEQ ID NO:8), an HC CDR3 comprising the sequence GGSMVRGDYYYGMDV (SEQ ID NO:9), an LC CDR1 comprising the sequence RASQGISSALA (SEQ ID NO:10), an LC CDR2 comprising the sequence DASSLES (SEQ ID NO:11), and an LC CDR3 comprising the sequence QQFNSYPYT (SEQ ID NO:12). iii. A HC CDR1 comprising the sequence GYTFTRYW (SEQ ID NO: 25), a HC CDR2 comprising the sequence IYPGDGDT (SEQ ID NO: 26), a HC CDR3 comprising the sequence ARNPLTTATAWFVY (SEQ ID NO: 27), a LC CDR1 comprising the sequence ENIYSN (SEQ ID NO: 28), a LC CDR2 comprising the sequence AAT, and a LC CDR3 comprising the sequence QHFWGPPWT (SEQ ID NO: 29). Each option represents a separate embodiment of the present invention.

[0039] According to some embodiments, the Afco-G236A antibody comprises a human IgG1 heavy chain constant region comprising the following sequence:

[0040] [Table 1]

[0041] According to some embodiments, the afucosylated-G236A antibody comprises a human kappa light chain constant region comprising the following sequence: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 14).

[0042] According to some embodiments, the afuco-G236A antibody comprises a heavy chain comprising the sequence set forth in SEQ ID NO:13 and a light chain comprising the sequence set forth in SEQ ID NO:14.

[0043] According to some embodiments, the GAALIE antibody (Fc region substitutions G236A, A330L and I332E) has the heavy chain constant region sequence:

[0044] [Table 2]

[0045] According to some embodiments, the GAALIE antibody comprises a heavy chain comprising the sequence set forth in SEQ ID NO:15, and a light chain comprising the sequence set forth in SEQ ID NO:14.

[0046] According to some embodiments, the Fc region modification results in enhanced dendritic cell (DC) activation, inflammatory cytokine production, or both.

[0047] According to some embodiments, the Fc region modifications do not result in enhanced ADCC activity.

[0048] According to some embodiments, the Fc region modifications result in dendritic cell activation leading to upregulation of DC activation markers without enhanced ADCC.

[0049] Conjugates comprising the above-mentioned anti-hGITR antibodies are also within the scope of the present invention. These conjugates may contain a moiety, such as a detectable probe or a toxin, attached to the anti-GITR antibody of the present invention. The moiety may be attached to any part of the antibody, so long as it does not interfere with its binding to hGITR and to human FcγRIIA and FcγRIIIA.

[0050] The present invention also provides, according to another aspect, a polynucleotide sequence encoding at least one chain of the above-mentioned antibody which specifically binds to hGITR.

[0051] According to some embodiments, the antibody encoded by the polynucleotide sequence of the present invention comprises a variable region (Fab) that recognizes hGITR, and a modified human IgG1 constant region (Fc), wherein the modified human IgG1 Fc region comprises: i. a human IgG1 Fc region comprising an N at position 279 and the substitution G236A; and ii. Amino acid substitutions: selected from human IgG1 Fc region, including G236A, A330L and I332E (herein GAALIE).

[0052] According to some embodiments, the polynucleotide sequence encodes at least one chain of an antibody with high affinity and specificity for hGITR, wherein the antibody comprises a modified IgG1 Fc region comprising a substitution of the amino acid residue at position 236, glycine, with an alanine residue (G236A), a substitution of the amino acid residue at position 330, alanine, with a leucine residue (A330L), and a substitution of the amino acid residue at position 332, isoleucine, with a glutamic acid residue (I332E).

[0053] According to some embodiments, the polynucleotide sequence encodes the amino acid heavy or light chain of the above-mentioned antibody.

[0054] According to some embodiments, the encoded heavy chain is human IgG1.

[0055] According to some embodiments, the polynucleotide sequence encodes at least one amino acid chain of an antibody selected from a humanized antibody and a chimeric antibody.

[0056] According to some embodiments, the polynucleotide sequence encodes an antibody chain comprising the amino acid sequence set forth in any one of SEQ ID NOs: 13, 14, or 15, or an analog or derivative thereof having at least 90% sequence identity to any of the amino acid sequences. Each possibility represents a separate embodiment of the present invention.

[0057] According to some embodiments, the polynucleotide sequence encodes an antibody heavy chain amino acid sequence comprising a sequence selected from SEQ ID NO: 13 and SEQ ID NO: 15, or a variant thereof having at least 90% sequence identity to this amino acid sequence.

[0058] According to some embodiments, the polynucleotide encoding the antibody heavy chain is set forth in (SEQ ID NO:22), and (SEQ ID NO:23); or a variant thereof having at least 80% sequence identity to this polynucleotide sequence.

[0059] According to some embodiments, the polynucleotide sequence encodes an antibody light chain amino acid sequence comprising the sequence set forth in SEQ ID NO: 14, or a variant thereof having at least 90% sequence identity to that amino acid sequence.

[0060] According to some embodiments, a polynucleotide sequence encoding an antibody light chain amino acid sequence comprising the sequence set forth in SEQ ID NO:24, or a variant thereof having at least 80% sequence identity to said amino acid sequence.

[0061] Vectors, plasmids and constructs carrying the polynucleotide sequences disclosed above are provided according to a further aspect of the present invention.

[0062] According to some embodiments, the vector, plasmid or construct comprises a polynucleotide sequence encoding a heavy chain, said polynucleotide sequence being selected from the group consisting of SEQ ID NO: 13 and SEQ ID NO: 15, or a variant thereof having at least 80% sequence identity to said polynucleotide sequence.

[0063] According to some embodiments, the vector, plasmid or construct comprises a polynucleotide sequence encoding a heavy chain selected from the group consisting of SEQ ID NO: 13 and 15, or a variant thereof having at least 80% sequence identity, and a polynucleotide sequence encoding a light chain of SEQ ID NO: 14, or a variant thereof having at least 80% sequence identity.

[0064] In yet another aspect, the invention provides a host cell or a population or culture of host cells comprising at least one of the above polynucleotide sequences, vectors or constructs, which cells are capable of producing at least one chain of the above-mentioned antibody.

[0065] The present invention also provides a composition comprising a plurality of antibody molecules that specifically bind to a glucocorticoid-inducible TNFR-related (GITR) protein, each antibody molecule comprising a variable region (Fab), and a modified human IgG1 constant region (Fc region), wherein the Fc region comprises an asparagine residue (N) at position 297, a substitution G236A for amino acid residue glycine (G) at position 236 to an alanine residue (A), and about 65-100% of the antibody molecules in the composition comprise a mature core carbohydrate structure lacking fucose attached to the asparagine residue (N) at position 297 of the Fc region.

[0066] According to some embodiments, about 80-100% of the antibody molecules in the composition comprise a mature core carbohydrate structure lacking fucose attached to the asparagine residue (N) at position 297 of the Fc region.

[0067] According to yet other embodiments, about 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 80%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the antibody molecules in the composition comprise a mature core carbohydrate structure lacking a fucose attached to the asparagine residue (N) at position 297 of the Fc region.

[0068] According to other embodiments, at least 70%, 75% or 80% of the IgG Fc regions of the antibody molecules are non-fucosylated. According to some specific embodiments, about 60-80% of the antibody molecules are afucosylated. Each option represents a separate embodiment of the present invention.

[0069] According to yet other embodiments, the invention provides compositions comprising a plurality of afuco-G236A antibodies comprising less than 20%, less than 25%, or less than 30% fucosylation of the human IgG1 Fc region, with each option representing a separate embodiment of the invention.

[0070] According to some embodiments, the composition contains at least 50%, 60%, 70%, 80% or 90% less fucose moieties attached to N-297 of the human IgG1 Fc region of the afuco-G236A antibody molecule compared to a non-afucosylated antibody, with each option representing a separate embodiment of the present invention.

[0071] According to some embodiments, the GITR is human GITR (hGITR).

[0072] According to some embodiments, each of the antibody molecules comprises a set of six CDR sequences, the sets being: i. a heavy chain (HC) CDR1 comprising the sequence GFSLSTSGMG (SEQ ID NO:1), a HC CDR2 comprising the sequence IWWDDDK (SEQ ID NO:2), a HC CDR3 comprising the sequence ARTRRYFPFAY (SEQ ID NO:3), a light chain (LC) CDR1 comprising the sequence QNVGTN (SEQ ID NO:4), a LC CDR2 comprising the sequence SAS or the sequence SAST (SEQ ID NO:5), and a LC CDR3 comprising the sequence QQYNTDPLT (SEQ ID NO:6); ii. an HC CDR1 comprising the sequence SYGMH (SEQ ID NO:7), an HC CDR2 comprising the sequence VIWYEGSNKYYADSVKG (SEQ ID NO:8), an HC CDR3 comprising the sequence GGSMVRGDYYYGMDV (SEQ ID NO:9), an LC CDR1 comprising the sequence RASQGISSALA (SEQ ID NO:10), an LC CDR2 comprising the sequence DASSLES (SEQ ID NO:11), and an LC CDR3 comprising the sequence QQFNSYPYT (SEQ ID NO:12); and iii. A HC CDR1 comprising the sequence GYTFTRYW (SEQ ID NO: 25), a HC CDR2 comprising the sequence IYPGDGDT (SEQ ID NO: 26), a HC CDR3 comprising the sequence ARNPLTTATAWFVY (SEQ ID NO: 27), a LC CDR1 comprising the sequence ENIYSN (SEQ ID NO: 28), a LC CDR2 comprising the sequence AAT, and a LC CDR3 comprising the sequence QHFWGPPWT (SEQ ID NO: 29).

[0073] According to some embodiments, each of the antibody molecules comprises the heavy chain sequence set forth in SEQ ID NO: 13, the light chain sequence set forth in SEQ ID NO: 14, or both.

[0074] According to some embodiments, the antibody molecule is a chimeric antibody or a humanized antibody.

[0075] According to some embodiments, the composition is in the form of a pharmaceutical composition further comprising at least one carrier, excipient, or diluent.

[0076] There is provided according to yet another aspect of the invention a pharmaceutical composition comprising at least one antibody or antibody conjugate as described above which specifically binds to GITR and at least one carrier, excipient or diluent.

[0077] According to some embodiments, the pharmaceutical composition comprises as an active ingredient an antibody comprising a variable region (Fab) that binds to GITR and a modified human IgG1 constant region (Fc), wherein the modified human IgG1 Fc region comprises: i. a human IgG1 Fc region containing the substitution G236A and a fucosylation content of 20-40% of all glycan structures (herein afuco-G236A); and ii. Amino acid substitutions: selected from human IgG1 Fc region, including G236A, A330L and I332E (herein GAALIE).

[0078] According to some embodiments, the pharmaceutical composition is an isolated afucosylated antibody that specifically binds to a glucocorticoid-induced TNFR-related (GITR) protein, wherein the antibody comprises a variable region (Fab) and a modified human IgG1 constant region (Fc) comprising an asparagine residue (N) at position 297, a substitution of amino acid residue glycine (G) at position 236 with an alanine residue (A) at G236A (herein afuco-G236A); The present invention comprises as an active ingredient an antibody or multiple antibodies selected from an antibody that specifically binds to a TR protein, the antibody comprising a variable region (Fab) and a modified human IgG1 constant region (Fc), the modified human IgG comprising the following amino acid substitutions (referred to herein as GAALIE): a substitution of the glycine residue at position 236 with an alanine residue (G236A), a substitution of the alanine residue at position 330 with a leucine residue (A330L), and a substitution of the isoleucine residue at position 332 with a glutamic acid residue (I332E).

[0079] According to some embodiments, the pharmaceutical composition comprises as an active ingredient at least one antibody or conjugate thereof that specifically binds to GITR, the antibody comprising a modified IgG1 Fc region comprising a substitution of the amino acid residue glycine (G) at position 236 with alanine (A), and a fucosylation content of up to 40% of the total glycan structures.

[0080] According to some embodiments, the pharmaceutical composition comprises as an active ingredient at least one antibody that specifically binds to GITR, or a conjugate thereof, wherein the antibody comprises a modified IgG1 Fc region comprising a substitution of the amino acid residue glycine at position 236 with an alanine residue (G236A), a substitution of the amino acid residue alanine at position 330 with a leucine residue (A330L), and a substitution of the amino acid residue isoleucine at position 332 with a glutamic acid residue (I332E).

[0081] According to some embodiments, the pharmaceutical composition comprises an antibody comprising a set of six CDRs, wherein heavy chain CDR1 comprises SEQ ID NO:1; heavy chain CDR2 comprises SEQ ID NO:2; heavy chain CDR3 comprises SEQ ID NO:3; light chain CDR1 comprises SEQ ID NO:4; light chain CDR2 comprises SEQ ID NO:5; and light chain CDR3 comprises SEQ ID NO:6.

[0082] According to some embodiments, the pharmaceutical composition comprises an antibody comprising a set of six CDRs, wherein heavy chain CDR1 comprises SEQ ID NO:7; heavy chain CDR2 comprises SEQ ID NO:8; heavy chain CDR3 comprises SEQ ID NO:9; light chain CDR1 comprises SEQ ID NO:10; light chain CDR2 comprises SEQ ID NO:11; and light chain CDR3 comprises SEQ ID NO:12.

[0083] According to certain embodiments, the pharmaceutical composition comprises an antibody comprising a heavy chain having a sequence selected from SEQ ID NOs: 13 and 15, and a light chain having the sequence of SEQ ID NO: 14. Each possibility represents a separate embodiment of the present invention.

[0084] The pharmaceutical compositions of the present invention may be formulated for administration by any means suitable for administering antibodies. According to some embodiments, the pharmaceutical compositions are formulated for intravenous (iv) administration. In some embodiments, the pharmaceutical compositions are formulated for administration by injection or infusion.

[0085] Also provided is a pharmaceutical composition comprising at least one antibody or antibody conjugate as described above for use in enhancing the immune costimulatory activity of hGITR.

[0086] According to some embodiments, a pharmaceutical composition comprising at least one Fc modified antibody against hGITR as described above is for use in activating dendritic cells, activating phagocytosis and enhancing the production of pro-inflammatory cytokines.

[0087] According to some embodiments, the pharmaceutical composition is for use in treating cancer or tumors.

[0088] According to some embodiments, the tumor is a solid tumor. According to other embodiments, the cancer is a hematological cancer.

[0089] According to some embodiments, the cancer is a metastatic cancer.

[0090] According to some particular embodiments, the cancer is selected from the group consisting of non-small cell lung cancer (NSCLC), renal cell carcinoma, melanoma, glioblastoma, colorectal cancer, breast cancer and ovarian cancer.

[0091] According to some embodiments, the treatment of cancer or tumor comprises administering or administering at least one additional anti-cancer therapy. According to certain embodiments, the additional anti-cancer therapy is selected from surgery, chemotherapy, radiation therapy, immunotherapy and hormonal therapy.

[0092] According to some embodiments, the treatment of cancer or tumor comprises administration of an anti-hGITR antibody or conjugate thereof and an additional anti-cancer agent, according to some embodiments, the additional anti-cancer agent is selected from the group consisting of an immunomodulatory agent, an agent that binds to a tumor antigen or a receptor overexpressed on a tumor cell, and a chemotherapeutic agent.

[0093] According to some embodiments, treating a cancer or tumor includes delaying, slowing, or preventing tumor growth or recurrence.

[0094] According to some embodiments, treating cancer or tumors results in the prevention or reduction of the formation, growth or spread of metastases in a subject.

[0095] According to some embodiments, treating a cancer or tumor results in an enhanced anti-tumor effect.

[0096] According to some embodiments, treating a cancer or tumor results in enhanced long-term anti-tumor immunity.

[0097] According to some embodiments, cancer or tumor treatment results in modulation of the tumor microenvironment by targeting hGITR and through Fc region-mediated FcγRIIA and / or FcγRIIIA activation of effector cells.

[0098] According to some embodiments, the pharmaceutical composition comprising at least one Fc modified antibody against hGITR as described above is for use in enhancing anti-tumor effects by activating effector cells via hGITR and via FcγRIIA and / or FcγRIIIA.

[0099] According to some embodiments, the pharmaceutical composition comprising at least one Fc-modified antibody against hGITR as described above is for use in inducing long-term anti-tumor immunity.

[0100] According to some embodiments, a pharmaceutical composition comprising at least one Fc modified antibody against hGITR as described above is for use in depleting or inhibiting regulatory T cells (Tregs).

[0101] According to some embodiments, the pharmaceutical composition comprising at least one Fc modified antibody against hGITR as described above is for use in activating dendritic cells.

[0102] According to some embodiments, pharmaceutical compositions comprising at least one Fc modified antibody against hGITR as described above are for use in modulating the tumor microenvironment by targeting GITR and through Fc region-mediated FcγRIIA and / or FcγRIIA activation of effector cells.

[0103] According to some embodiments, pharmaceutical compositions comprising at least one Fc modified antibody against hGITR as described above are for use in delaying, slowing or preventing tumor growth and metastasis formation or spread.

[0104] According to yet another aspect and in some embodiments, the present invention provides a method of enhancing activity of hGITR in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an anti-GITR antibody or conjugate thereof as defined herein, or a pharmaceutical composition comprising same, wherein the anti-GITR antibody has agonistic activity.

[0105] According to yet another aspect, the present invention provides a method for treating cancer or tumor, comprising administering to a subject in need thereof a pharmaceutical composition comprising a therapeutically effective amount of an Fc region-modified anti-hGITR antibody or a conjugate thereof described herein.

[0106] According to some embodiments, the method of treating cancer or tumor in a subject in need thereof comprises administering or administering at least one additional anti-cancer therapy, according to certain embodiments, the additional anti-cancer therapy is selected from surgery, chemotherapy, radiation therapy, immunotherapy, and hormonal therapy.

[0107] According to some embodiments, the method of treating a cancer or tumor comprises administering an anti-hGITR antibody or conjugate thereof and an additional anti-cancer agent, according to some embodiments, the additional anti-cancer agent is selected from the group consisting of an immunomodulatory agent, an agent that binds to a tumor antigen or a receptor overexpressed on a tumor cell, and a chemotherapeutic agent.

[0108] According to some embodiments, the immunomodulatory agent is a checkpoint inhibitor.

[0109] According to some embodiments, the method of treating cancer or tumor in a subject in need thereof results in the prevention or reduction of formation, growth or recurrence in the subject.

[0110] According to some embodiments, the method of treating cancer or tumors includes delaying, slowing or preventing metastasis formation or spread.

[0111] According to some embodiments, the method of treating cancer or tumors comprises enhancing an anti-tumor effect.

[0112] According to some embodiments, the method of treating cancer or tumors comprises inducing long-term anti-tumor immunity.

[0113] According to some embodiments, a method of treating cancer or tumors comprises modulating the tumor microenvironment by targeting hGITR and by Fc region-mediated FcγRIIA and / or FcγRIIIA activation of effector cells.

[0114] According to some embodiments, the present invention provides methods for activating dendritic cells, activating phagocytosis, and enhancing the production of pro-inflammatory cytokines, the methods comprising administration of at least one Fc-modified antibody against hGITR as described above.

[0115] According to some embodiments, the present invention provides a method for preventing, delaying, depleting or inhibiting Tregs comprising administering at least one Fc-engineered antibody to hGITR as described above.

[0116] The invention also provides a kit comprising an antibody, antibody conjugate or composition as described above, packaged in packaging material and identified in or on the packaging material.

[0117] Methods for producing and purifying the anti-GITR antibodies or antibody conjugates of the invention are also included within the scope. Any method known in the art for producing, isolating and purifying recombinant antibodies may be used.

[0118] According to some embodiments, the antibodies are produced by transient transfection into cells of plasmids containing polynucleotide sequences encoding the heavy and light chain pairs.

[0119] According to some embodiments, a method for producing an Fc engineered antibody against GITR comprises: (i) transfecting a cell with a vector, plasmid, or construct containing polynucleotide sequences encoding the heavy and light chain pairs. (ii) culturing a cell containing at least one polynucleotide encoding the antibody heavy or light chain under conditions allowing expression of the heavy and / or light chain; and (iii) recovering the strand from the cell.

[0120] According to other embodiments, the antibodies are assembled intracellularly and secreted into the cell culture supernatant, then harvested, isolated and purified.

[0121] According to some embodiments, the separation and purification includes one or more of the following steps: collecting the culture medium, centrifuging to pellet the cells and any particulate matter, filtering, purifying using chromatography, and buffer exchanging.

[0122] According to some embodiments, the chromatography is protein G chromatography.

[0123] According to certain embodiments, the antibody fragment humanized antibody is purified to a level of at least 95%, 96%, 97%, 98%, 99% or even higher levels of purity (less than 5% host cell contaminants w / w or w / v).

[0124] Any method known in the art for producing afucosylated antibodies may be used to produce the Fc-afucosylated antibodies of the present invention.

[0125] According to some embodiments, the antibody is afucosylated without altering the amino acid glycosylation site, for example without substituting or deleting the asparagine (N) amino acid residue at position 297 of the Fc.

[0126] According to some embodiments, afucosylation of the antibody is performed in a post-translational transfection step.

[0127] According to some embodiments, the method of producing an afucosylated Fc modified antibody comprises adding 50-500 μM 2-deoxy-2-fluoro-L-fucose to the transfection medium. [Brief description of the drawings]

[0128] [Figure 1A] Schematic diagram of the FcγR pathway for an agonistic anti-GITR antibody, showing both activation of the FcγR pathway (FIG. 1A) and involvement of FcγR as a scaffold for antibody cross-linking (FIG. 1B). [Figure 1B] Schematic diagram of the FcγR pathway for an agonistic anti-GITR antibody, showing both activation of the FcγR pathway (FIG. 1A) and involvement of FcγR as a scaffold for antibody cross-linking (FIG. 1B). [Diagram 2] Schematic diagram of an exemplary cloning strategy used to produce some of the antibodies of the present invention. Human IgG1 was cloned and introduced into a mammalian expression vector. hIgG1-labeled bands of heavy (HC) and light (LC) chain constant regions were extracted from a 1% agarose gel, linked to the heavy and light chain variable regions (VH and VL) of anti-mouse GITR (mGITR), and used for expression and production of Fc region variant antibodies. [Figure 3A]Non-reduced (Fig. 3A) and reduced (Fig. 3B) SDS-PAGE of chimeric rat anti-mGITR hIgG1 Fc region mutant based on rat anti-mGITR antibody DTA-1. In non-reduced SDS-PAGE, the disulfide bond between the heavy and light chains is intact and therefore only one band is evident at the size of the intact antibody (Fig. 3A), whereas in reduced SDS-PAGE (Fig. 3B) two distinct bands appear, one representing the heavy chain approx. 50 kDa and the other the light chain approx. 25 kDa. Lane 1 - molecular weight marker, lane 2 - anti-mGITR V11, lane 3 - afucosylated anti-mGITR, lane 4 - N279A, lane 5 - GAALIE, lane 6 - GASDALIE, lane 7 - G236A, lane 8 - anti-mGITR IgG1. [Figure 3B] Non-reduced (Fig. 3A) and reduced (Fig. 3B) SDS-PAGE of chimeric rat anti-mGITR hIgG1 Fc region mutant based on rat anti-mGITR antibody DTA-1. In non-reduced SDS-PAGE, the disulfide bond between the heavy and light chains is intact and therefore only one band is evident at the size of the intact antibody (Fig. 3A), whereas in reduced SDS-PAGE (Fig. 3B) two distinct bands appear, one representing the heavy chain approx. 50 kDa and the other the light chain approx. 25 kDa. Lane 1 - molecular weight marker, lane 2 - anti-mGITR V11, lane 3 - afucosylated anti-mGITR, lane 4 - N279A, lane 5 - GAALIE, lane 6 - GASDALIE, lane 7 - G236A, lane 8 - anti-mGITR IgG1. [Figure 4] 1 is a graphical representation showing similar binding affinity of several chimeric anti-mGITR hIgG1 Fc engineered variants (including the Fab of anti-mGITR DTA-1) to plate-bound mouse GITR. ELISA OD450 values ​​were plotted against increasing concentrations of tested antibody. Data are shown as mean + SEM. [Figure 5A]Graphs showing differential binding affinities of chimeric anti-mGITR hIgG1 Fc region mutants (including the Fab of DTA-1) to the indicated hFcγRs: hFcγRIa (FIG. 5A), hFcγRIIA (FIG. 5B), hFcγRIIB (FIG. 5C), and hFcγRIIIA (FIG. 5D). Data are presented as mean + SEM. [Figure 5B] Graphs showing differential binding affinities of chimeric anti-mGITR hIgG1 Fc region mutants (including the Fab of DTA-1) to the indicated hFcγRs: hFcγRIa (FIG. 5A), hFcγRIIA (FIG. 5B), hFcγRIIB (FIG. 5C), and hFcγRIIIA (FIG. 5D). Data are presented as mean + SEM. [Figure 5C] Graphs showing differential binding affinities of chimeric anti-mGITR hIgG1 Fc region mutants (including the Fab of DTA-1) to the indicated hFcγRs: hFcγRIa (FIG. 5A), hFcγRIIA (FIG. 5B), hFcγRIIB (FIG. 5C), and hFcγRIIIA (FIG. 5D). Data are presented as mean + SEM. [Figure 5D] Graphs showing differential binding affinities of chimeric anti-mGITR hIgG1 Fc region mutants (including the Fab of DTA-1) to the indicated hFcγRs: hFcγRIa (FIG. 5A), hFcγRIIA (FIG. 5B), hFcγRIIB (FIG. 5C), and hFcγRIIIA (FIG. 5D). Data are presented as mean + SEM. [Figure 6] Pharmacokinetic assay results for several chimeric anti-mGITR IgG1 Fc region variants containing the Fab of DTA-1, measured as in vivo half-life of the antibody. Data are presented as mean±SEM. n=3. [Figure 7A]Differential antitumor activity of chimeric anti-mGITR hIgG Fc region variants. FcγR humanized (hFcγR) mice were inoculated with MC38 cells and treated with chimeric human Fc region variants of the DTA-1 antibody. Tumor progression and overall survival of mice were followed. Untreated mice (n=10) (Figure 7A), IgG1-treated (n=8) (Figure 7B), IgG1-N297A-treated (n=10) (Figure 7C), IgG1-V11-treated (n=10) (Figure 7D), IgG1-G236A-treated (n=9) (Figure 7E), and Afuco-IgG1-treated (n=10) (Figure 7F). An unpaired two-tailed t-test was used to compare the Fc region variants with the IgG1-N297A group (Figure 7G) and to compare survival probability between the Fc region variants (Figure 7H). Asterisks indicate statistical comparison with IgG1-N297A. Data are presented as mean + SEM. [Figure 7B] Differential antitumor activity of chimeric anti-mGITR hIgG Fc region variants. FcγR humanized (hFcγR) mice were inoculated with MC38 cells and treated with chimeric human Fc region variants of the DTA-1 antibody. Tumor progression and overall survival of mice were followed. Untreated mice (n=10) (Figure 7A), IgG1-treated (n=8) (Figure 7B), IgG1-N297A-treated (n=10) (Figure 7C), IgG1-V11-treated (n=10) (Figure 7D), IgG1-G236A-treated (n=9) (Figure 7E), and Afuco-IgG1-treated (n=10) (Figure 7F). An unpaired two-tailed t-test was used to compare the Fc region variants with the IgG1-N297A group (Figure 7G) and to compare survival probability between the Fc region variants (Figure 7H). Asterisks indicate statistical comparison with IgG1-N297A. Data are presented as mean + SEM. [Figure 7C]Differential antitumor activity of chimeric anti-mGITR hIgG Fc region variants. FcγR humanized (hFcγR) mice were inoculated with MC38 cells and treated with chimeric human Fc region variants of the DTA-1 antibody. Tumor progression and overall survival of mice were followed. Untreated mice (n=10) (Figure 7A), IgG1-treated (n=8) (Figure 7B), IgG1-N297A-treated (n=10) (Figure 7C), IgG1-V11-treated (n=10) (Figure 7D), IgG1-G236A-treated (n=9) (Figure 7E), and Afuco-IgG1-treated (n=10) (Figure 7F). An unpaired two-tailed t-test was used to compare the Fc region variants with the IgG1-N297A group (Figure 7G) and to compare survival probability between the Fc region variants (Figure 7H). Asterisks indicate statistical comparison with IgG1-N297A. Data are presented as mean + SEM. [Figure 7D] Differential antitumor activity of chimeric anti-mGITR hIgG Fc region variants. FcγR humanized (hFcγR) mice were inoculated with MC38 cells and treated with chimeric human Fc region variants of the DTA-1 antibody. Tumor progression and overall survival of mice were followed. Untreated mice (n=10) (Figure 7A), IgG1-treated (n=8) (Figure 7B), IgG1-N297A-treated (n=10) (Figure 7C), IgG1-V11-treated (n=10) (Figure 7D), IgG1-G236A-treated (n=9) (Figure 7E), and Afuco-IgG1-treated (n=10) (Figure 7F). An unpaired two-tailed t-test was used to compare the Fc region variants with the IgG1-N297A group (Figure 7G) and to compare survival probability between the Fc region variants (Figure 7H). Asterisks indicate statistical comparison with IgG1-N297A. Data are presented as mean + SEM. [Figure 7E]Differential antitumor activity of chimeric anti-mGITR hIgG Fc region variants. FcγR humanized (hFcγR) mice were inoculated with MC38 cells and treated with chimeric human Fc region variants of the DTA-1 antibody. Tumor progression and overall survival of mice were followed. Untreated mice (n=10) (Figure 7A), IgG1-treated (n=8) (Figure 7B), IgG1-N297A-treated (n=10) (Figure 7C), IgG1-V11-treated (n=10) (Figure 7D), IgG1-G236A-treated (n=9) (Figure 7E), and Afuco-IgG1-treated (n=10) (Figure 7F). An unpaired two-tailed t-test was used to compare the Fc region variants with the IgG1-N297A group (Figure 7G) and to compare survival probability between the Fc region variants (Figure 7H). Asterisks indicate statistical comparison with IgG1-N297A. Data are presented as mean + SEM. [Figure 7F] Differential antitumor activity of chimeric anti-mGITR hIgG Fc region variants. FcγR humanized (hFcγR) mice were inoculated with MC38 cells and treated with chimeric human Fc region variants of the DTA-1 antibody. Tumor progression and overall survival of mice were followed. Untreated mice (n=10) (Figure 7A), IgG1-treated (n=8) (Figure 7B), IgG1-N297A-treated (n=10) (Figure 7C), IgG1-V11-treated (n=10) (Figure 7D), IgG1-G236A-treated (n=9) (Figure 7E), and Afuco-IgG1-treated (n=10) (Figure 7F). An unpaired two-tailed t-test was used to compare the Fc region variants with the IgG1-N297A group (Figure 7G) and to compare survival probability between the Fc region variants (Figure 7H). Asterisks indicate statistical comparison with IgG1-N297A. Data are presented as mean + SEM. [Figure 7G]Differential antitumor activity of chimeric anti-mGITR hIgG Fc region variants. FcγR humanized (hFcγR) mice were inoculated with MC38 cells and treated with chimeric human Fc region variants of the DTA-1 antibody. Tumor progression and overall survival of mice were followed. Untreated mice (n=10) (Figure 7A), IgG1-treated (n=8) (Figure 7B), IgG1-N297A-treated (n=10) (Figure 7C), IgG1-V11-treated (n=10) (Figure 7D), IgG1-G236A-treated (n=9) (Figure 7E), and Afuco-IgG1-treated (n=10) (Figure 7F). An unpaired two-tailed t-test was used to compare the Fc region variants with the IgG1-N297A group (Figure 7G) and to compare survival probability between the Fc region variants (Figure 7H). Asterisks indicate statistical comparison with IgG1-N297A. Data are presented as mean + SEM. [Figure 7H] Differential antitumor activity of chimeric anti-mGITR hIgG Fc region variants. FcγR humanized (hFcγR) mice were inoculated with MC38 cells and treated with chimeric human Fc region variants of the DTA-1 antibody. Tumor progression and overall survival of mice were followed. Untreated mice (n=10) (Figure 7A), IgG1-treated (n=8) (Figure 7B), IgG1-N297A-treated (n=10) (Figure 7C), IgG1-V11-treated (n=10) (Figure 7D), IgG1-G236A-treated (n=9) (Figure 7E), and Afuco-IgG1-treated (n=10) (Figure 7F). An unpaired two-tailed t-test was used to compare the Fc region variants with the IgG1-N297A group (Figure 7G) and to compare survival probability between the Fc region variants (Figure 7H). Asterisks indicate statistical comparison with IgG1-N297A. Data are presented as mean + SEM. [Figure 8A]Graphical representation of the protection provided by anti-GITR treatment in a tumor rechallenge model as an indicator of long-term immunity. Naive and MC38-bearing hFcγR mice treated with anti-mGITR Fc region mutants that fully responded to previous treatment and were tumor-free; IgG1 (n=4), IgG1-N297A (n=1), IgG1-G236A (n=6), Afuco-IgG1 (n=7) were rechallenged and tumor growth (FIG. 8A) and survival probability (FIG. 8B) were followed. Asterisks indicate statistical comparison with naive mice. Data are presented as mean + SEM. [Figure 8B] Graphical representation of the protection provided by anti-GITR treatment in a tumor rechallenge model as an indicator of long-term immunity. Naive and MC38-bearing hFcγR mice treated with anti-mGITR Fc region mutants that fully responded to previous treatment and were tumor-free; IgG1 (n=4), IgG1-N297A (n=1), IgG1-G236A (n=6), Afuco-IgG1 (n=7) were rechallenged and tumor growth (FIG. 8A) and survival probability (FIG. 8B) were followed. Asterisks indicate statistical comparison with naive mice. Data are presented as mean + SEM. [Figure 9A] Long-term protection against tumor reappearance and memory responses by anti-mGITR mAb. MC38-bearing hFcγR mice treated with anti-GITR Fc region variants that fully responded to previous treatment and were tumor-free were compared to the same type of tumor-free mice injected with antibodies that deplete CD4 and CD8 cells. Comparison of tumor growth in T cell-depleted versus non-T cell-depleted mice for each Fc region variant treatment: untreated (n=6, FIG. 9A), IgG1-G236A (T cell-depleted n=4, non-T cell-depleted n=2, FIG. 9B), Afuco-IgG1 (T cell-depleted n=3, non-T cell-depleted n=2, FIG. 9C), and Afuco-IgG1-G236A (T cell-depleted n=2, non-T cell-depleted n=4, FIG. 9D). Comparison of tumor growth over time (FIG. 9E) and survival probability (FIG. 9F) was performed. Asterisks indicate statistical comparison between T cell depleted and non-T cell depleted mice treated with the same Fc region variants. Data are presented as mean + SEM. [Figure 9B]Long-term protection against tumor reappearance and memory responses by anti-mGITR mAb. MC38-bearing hFcγR mice treated with anti-GITR Fc region variants that fully responded to previous treatment and were tumor-free were compared to the same type of tumor-free mice injected with antibodies that deplete CD4 and CD8 cells. Comparison of tumor growth in T cell-depleted versus non-T cell-depleted mice for each Fc region variant treatment: untreated (n=6, FIG. 9A), IgG1-G236A (T cell-depleted n=4, non-T cell-depleted n=2, FIG. 9B), Afuco-IgG1 (T cell-depleted n=3, non-T cell-depleted n=2, FIG. 9C), and Afuco-IgG1-G236A (T cell-depleted n=2, non-T cell-depleted n=4, FIG. 9D). Comparison of tumor growth over time (FIG. 9E) and survival probability (FIG. 9F) was performed. Asterisks indicate statistical comparison between T cell depleted and non-T cell depleted mice treated with the same Fc region variants. Data are presented as mean + SEM. [Figure 9C] Long-term protection against tumor reappearance and memory responses by anti-mGITR mAb. MC38-bearing hFcγR mice treated with anti-GITR Fc region variants that fully responded to previous treatment and were tumor-free were compared to the same type of tumor-free mice injected with antibodies that deplete CD4 and CD8 cells. Comparison of tumor growth in T cell-depleted versus non-T cell-depleted mice for each Fc region variant treatment: untreated (n=6, FIG. 9A), IgG1-G236A (T cell-depleted n=4, non-T cell-depleted n=2, FIG. 9B), Afuco-IgG1 (T cell-depleted n=3, non-T cell-depleted n=2, FIG. 9C), and Afuco-IgG1-G236A (T cell-depleted n=2, non-T cell-depleted n=4, FIG. 9D). Comparison of tumor growth over time (FIG. 9E) and survival probability (FIG. 9F) was performed. Asterisks indicate statistical comparison between T cell depleted and non-T cell depleted mice treated with the same Fc region variants. Data are presented as mean + SEM. [Figure 9D]Long-term protection against tumor reappearance and memory responses by anti-mGITR mAb. MC38-bearing hFcγR mice treated with anti-GITR Fc region variants that fully responded to previous treatment and were tumor-free were compared to the same type of tumor-free mice injected with antibodies that deplete CD4 and CD8 cells. Comparison of tumor growth in T cell-depleted versus non-T cell-depleted mice for each Fc region variant treatment: untreated (n=6, FIG. 9A), IgG1-G236A (T cell-depleted n=4, non-T cell-depleted n=2, FIG. 9B), Afuco-IgG1 (T cell-depleted n=3, non-T cell-depleted n=2, FIG. 9C), and Afuco-IgG1-G236A (T cell-depleted n=2, non-T cell-depleted n=4, FIG. 9D). Comparison of tumor growth over time (FIG. 9E) and survival probability (FIG. 9F) was performed. Asterisks indicate statistical comparison between T cell depleted and non-T cell depleted mice treated with the same Fc region variants. Data are presented as mean + SEM. [Figure 9E] Long-term protection against tumor reappearance and memory responses by anti-mGITR mAb. MC38-bearing hFcγR mice treated with anti-GITR Fc region variants that fully responded to previous treatment and were tumor-free were compared to the same type of tumor-free mice injected with antibodies that deplete CD4 and CD8 cells. Comparison of tumor growth in T cell-depleted versus non-T cell-depleted mice for each Fc region variant treatment: untreated (n=6, FIG. 9A), IgG1-G236A (T cell-depleted n=4, non-T cell-depleted n=2, FIG. 9B), Afuco-IgG1 (T cell-depleted n=3, non-T cell-depleted n=2, FIG. 9C), and Afuco-IgG1-G236A (T cell-depleted n=2, non-T cell-depleted n=4, FIG. 9D). Comparison of tumor growth over time (FIG. 9E) and survival probability (FIG. 9F) was performed. Asterisks indicate statistical comparison between T cell depleted and non-T cell depleted mice treated with the same Fc region variants. Data are presented as mean + SEM. [Figure 9F]Long-term protection against tumor reappearance and memory responses by anti-mGITR mAb. MC38-bearing hFcγR mice treated with anti-GITR Fc region variants that fully responded to previous treatment and were tumor-free were compared to the same type of tumor-free mice injected with antibodies that deplete CD4 and CD8 cells. Comparison of tumor growth in T cell-depleted versus non-T cell-depleted mice for each Fc region variant treatment: untreated (n=6, FIG. 9A), IgG1-G236A (T cell-depleted n=4, non-T cell-depleted n=2, FIG. 9B), Afuco-IgG1 (T cell-depleted n=3, non-T cell-depleted n=2, FIG. 9C), and Afuco-IgG1-G236A (T cell-depleted n=2, non-T cell-depleted n=4, FIG. 9D). Comparison of tumor growth over time (FIG. 9E) and survival probability (FIG. 9F) was performed. Asterisks indicate statistical comparison between T cell depleted and non-T cell depleted mice treated with the same Fc region variants. Data are presented as mean + SEM. [Figure 10A] 10A-C are graphical representations of binding of chimeric Fc region-modified anti-mGITR (DTA-1 Fab) variants to hFcγRIIA (FIG. 10A), hFcγRIIIA (FIG. 10B), and hFcγRIIB (FIG. 10C) as measured by ELISA. Data are shown as mean + SEM. [Figure 10B] 10A-C are graphical representations of binding of chimeric Fc region-modified anti-mGITR (DTA-1 Fab) variants to hFcγRIIA (FIG. 10A), hFcγRIIIA (FIG. 10B), and hFcγRIIB (FIG. 10C) as measured by ELISA. Data are shown as mean + SEM. [Figure 10C] 10A-C are graphical representations of binding of chimeric Fc region-modified anti-mGITR (DTA-1 Fab) variants to hFcγRIIA (FIG. 10A), hFcγRIIIA (FIG. 10B), and hFcγRIIB (FIG. 10C) as measured by ELISA. Data are shown as mean + SEM. [Figure 11A]11A is a graphical representation of enhanced FcγRIIA and FcγRIIIA activity mediated by Fc-region-modified chimeric anti-mouse GITR antibodies. Humanized FcγR mice were inoculated with MC38 cells, and when tumors reached an average volume of 115 mm3, they were treated with the indicated human Fc-region variants and monitored for tumor progression: untreated mice (FIG. 11A), IgG1-G236A (FIG. 11B), Afuco-IgG1 (FIG. 11C), Afuco-IgG1-G236A (FIG. 11D), comparing the average tumor growth of all Fc-region variants; an unpaired two-tailed t-test was used to compare survival probability (FIG. 11F) between the Afuco-IgG1-G236A and Afuco-IgG1 groups (FIG. 11E). Data are shown as mean + SEM. [Figure 11B] 11A is a graphical representation of enhanced FcγRIIA and FcγRIIIA activity mediated by Fc-region-modified chimeric anti-mouse GITR antibodies. Humanized FcγR mice were inoculated with MC38 cells, and when tumors reached an average volume of 115 mm3, they were treated with the indicated human Fc-region variants and monitored for tumor progression: untreated mice (FIG. 11A), IgG1-G236A (FIG. 11B), Afuco-IgG1 (FIG. 11C), Afuco-IgG1-G236A (FIG. 11D), comparing the average tumor growth of all Fc-region variants; an unpaired two-tailed t-test was used to compare survival probability (FIG. 11F) between the Afuco-IgG1-G236A and Afuco-IgG1 groups (FIG. 11E). Data are shown as mean + SEM. [Figure 11C] 11A is a graphical representation of enhanced FcγRIIA and FcγRIIIA activity mediated by Fc-region-modified chimeric anti-mouse GITR antibodies. Humanized FcγR mice were inoculated with MC38 cells, and when tumors reached an average volume of 115 mm3, they were treated with the indicated human Fc-region variants and monitored for tumor progression: untreated mice (FIG. 11A), IgG1-G236A (FIG. 11B), Afuco-IgG1 (FIG. 11C), Afuco-IgG1-G236A (FIG. 11D), comparing the average tumor growth of all Fc-region variants; an unpaired two-tailed t-test was used to compare survival probability (FIG. 11F) between the Afuco-IgG1-G236A and Afuco-IgG1 groups (FIG. 11E). Data are shown as mean + SEM. [Figure 11D] 11A is a graphical representation of enhanced FcγRIIA and FcγRIIIA activity mediated by Fc-region-modified chimeric anti-mouse GITR antibodies. Humanized FcγR mice were inoculated with MC38 cells, and when tumors reached an average volume of 115 mm3, they were treated with the indicated human Fc-region variants and monitored for tumor progression: untreated mice (FIG. 11A), IgG1-G236A (FIG. 11B), Afuco-IgG1 (FIG. 11C), Afuco-IgG1-G236A (FIG. 11D), comparing the average tumor growth of all Fc-region variants; an unpaired two-tailed t-test was used to compare survival probability (FIG. 11F) between the Afuco-IgG1-G236A and Afuco-IgG1 groups (FIG. 11E). Data are shown as mean + SEM. [Figure 11E] 11A is a graphical representation of enhanced FcγRIIA and FcγRIIIA activity mediated by Fc-region-modified chimeric anti-mouse GITR antibodies. Humanized FcγR mice were inoculated with MC38 cells, and when tumors reached an average volume of 115 mm3, they were treated with the indicated human Fc-region variants and monitored for tumor progression: untreated mice (FIG. 11A), IgG1-G236A (FIG. 11B), Afuco-IgG1 (FIG. 11C), Afuco-IgG1-G236A (FIG. 11D), comparing the average tumor growth of all Fc-region variants; an unpaired two-tailed t-test was used to compare survival probability (FIG. 11F) between the Afuco-IgG1-G236A and Afuco-IgG1 groups (FIG. 11E). Data are shown as mean + SEM. [Figure 11F] 11A is a graphical representation of enhanced FcγRIIA and FcγRIIIA activity mediated by Fc-region-modified chimeric anti-mouse GITR antibodies. Humanized FcγR mice were inoculated with MC38 cells, and when tumors reached an average volume of 115 mm3, they were treated with the indicated human Fc-region variants and monitored for tumor progression: untreated mice (FIG. 11A), IgG1-G236A (FIG. 11B), Afuco-IgG1 (FIG. 11C), Afuco-IgG1-G236A (FIG. 11D), comparing the average tumor growth of all Fc-region variants; an unpaired two-tailed t-test was used to compare survival probability (FIG. 11F) between the Afuco-IgG1-G236A and Afuco-IgG1 groups (FIG. 11E). Data are shown as mean + SEM. [Figure 12A] Graphical representation of enhanced antitumor activity of chimeric anti-mGITR hIgG Fc region variants. When tumors reached a mean volume of 115 mm3, humanized FcγR mice were inoculated with MC38 cells and treated with the indicated chimeric antibody variants (based on DTA-1). Tumor progression in mice was followed: untreated mice (Figure 12A), IgG1 (Figure 12B), IgG1-N297A (Figure 12C), Afuco-IgG1-G236A (Figure 12D), comparison of mean tumor growth for all Fc region variants, Afuco-IgG1-G236A group compared to IgG1 group using unpaired two-tailed t-test (Figure 12E) and survival probability (Figure 12F). Data are presented as mean + SEM. [Figure 12B] Graphical representation of enhanced antitumor activity of chimeric anti-mGITR hIgG Fc region variants. When tumors reached a mean volume of 115 mm3, humanized FcγR mice were inoculated with MC38 cells and treated with the indicated chimeric antibody variants (based on DTA-1). Tumor progression in mice was followed: untreated mice (Figure 12A), IgG1 (Figure 12B), IgG1-N297A (Figure 12C), Afuco-IgG1-G236A (Figure 12D), comparison of mean tumor growth for all Fc region variants, Afuco-IgG1-G236A group compared to IgG1 group using unpaired two-tailed t-test (Figure 12E) and survival probability (Figure 12F). Data are presented as mean + SEM. [Figure 12C] Graphical representation of enhanced antitumor activity of chimeric anti-mGITR hIgG Fc region variants. When tumors reached a mean volume of 115 mm3, humanized FcγR mice were inoculated with MC38 cells and treated with the indicated chimeric antibody variants (based on DTA-1). Tumor progression in mice was followed: untreated mice (Figure 12A), IgG1 (Figure 12B), IgG1-N297A (Figure 12C), Afuco-IgG1-G236A (Figure 12D), comparison of mean tumor growth for all Fc region variants, Afuco-IgG1-G236A group compared to IgG1 group using unpaired two-tailed t-test (Figure 12E) and survival probability (Figure 12F). Data are presented as mean + SEM. [Figure 12D]Graphical representation of enhanced antitumor activity of chimeric anti-mGITR hIgG Fc region variants. When tumors reached a mean volume of 115 mm3, humanized FcγR mice were inoculated with MC38 cells and treated with the indicated chimeric antibody variants (based on DTA-1). Tumor progression in mice was followed: untreated mice (Figure 12A), IgG1 (Figure 12B), IgG1-N297A (Figure 12C), Afuco-IgG1-G236A (Figure 12D), comparison of mean tumor growth for all Fc region variants, Afuco-IgG1-G236A group compared to IgG1 group using unpaired two-tailed t-test (Figure 12E) and survival probability (Figure 12F). Data are presented as mean + SEM. [Figure 12E] Graphical representation of enhanced antitumor activity of chimeric anti-mGITR hIgG Fc region variants. When tumors reached a mean volume of 115 mm3, humanized FcγR mice were inoculated with MC38 cells and treated with the indicated chimeric antibody variants (based on DTA-1). Tumor progression in mice was followed: untreated mice (Figure 12A), IgG1 (Figure 12B), IgG1-N297A (Figure 12C), Afuco-IgG1-G236A (Figure 12D), comparison of mean tumor growth for all Fc region variants, Afuco-IgG1-G236A group compared to IgG1 group using unpaired two-tailed t-test (Figure 12E) and survival probability (Figure 12F). Data are presented as mean + SEM. [Figure 12F] Graphical representation of enhanced antitumor activity of chimeric anti-mGITR hIgG Fc region variants. When tumors reached a mean volume of 115 mm3, humanized FcγR mice were inoculated with MC38 cells and treated with the indicated chimeric antibody variants (based on DTA-1). Tumor progression in mice was followed: untreated mice (Figure 12A), IgG1 (Figure 12B), IgG1-N297A (Figure 12C), Afuco-IgG1-G236A (Figure 12D), comparison of mean tumor growth for all Fc region variants, Afuco-IgG1-G236A group compared to IgG1 group using unpaired two-tailed t-test (Figure 12E) and survival probability (Figure 12F). Data are presented as mean + SEM. [Figure 13A]Figure 13 shows T cell frequency in the TME 1, 4, and 8 days after the initiation of anti-GITR Fc region variant treatment. Humanized FcγR mice bearing refractory MC38 tumors were treated with chimeric anti-mGITR Fc region variants IgG1-N297A (NA), Afuco-IgG1-G236A (GA-aFuc), and IgG1. Mice were sacrificed and tumors were harvested at three time points after treatment: 1, 4 (total of 2 mAb doses), and 8 (total of 3 mAb doses), and untreated mice were used as controls. Figures 13A-13C, Flow cytometry analysis of Treg frequency detected for CD45+CD3+CD11b-CD8-CD4+FoxP3+: 1 day after the initiation of treatment (Figure 13A), 4 days after the initiation of treatment (Figure 13B), and 8 days after the initiation of treatment (Figure 13C). Figures 13D-183F, Flow cytometry analysis of CD8+ frequencies detected in CD45+CD3+CD11b-CD8+CD4-: 1 day after initiation of treatment (Figure 13D), 4 days after initiation of treatment (Figure 13E), and 8 days after initiation of treatment (Figure 13F). [Figure 13B] Figure 13 shows T cell frequency in the TME 1, 4, and 8 days after the initiation of anti-GITR Fc region variant treatment. Humanized FcγR mice bearing refractory MC38 tumors were treated with chimeric anti-mGITR Fc region variants IgG1-N297A (NA), Afuco-IgG1-G236A (GA-aFuc), and IgG1. Mice were sacrificed and tumors were harvested at three time points after treatment: 1, 4 (total of 2 mAb doses), and 8 (total of 3 mAb doses), and untreated mice were used as controls. Figures 13A-13C, Flow cytometry analysis of Treg frequency detected for CD45+CD3+CD11b-CD8-CD4+FoxP3+: 1 day after the initiation of treatment (Figure 13A), 4 days after the initiation of treatment (Figure 13B), and 8 days after the initiation of treatment (Figure 13C). Figures 13D-183F, Flow cytometry analysis of CD8+ frequencies detected in CD45+CD3+CD11b-CD8+CD4-: 1 day after initiation of treatment (Figure 13D), 4 days after initiation of treatment (Figure 13E), and 8 days after initiation of treatment (Figure 13F). [Figure 13C]Figure 13 shows T cell frequency in the TME 1, 4, and 8 days after the initiation of anti-GITR Fc region variant treatment. Humanized FcγR mice bearing refractory MC38 tumors were treated with chimeric anti-mGITR Fc region variants IgG1-N297A (NA), Afuco-IgG1-G236A (GA-aFuc), and IgG1. Mice were sacrificed and tumors were harvested at three time points after treatment: 1, 4 (total of 2 mAb doses), and 8 (total of 3 mAb doses), and untreated mice were used as controls. Figures 13A-13C, Flow cytometry analysis of Treg frequency detected for CD45+CD3+CD11b-CD8-CD4+FoxP3+: 1 day after the initiation of treatment (Figure 13A), 4 days after the initiation of treatment (Figure 13B), and 8 days after the initiation of treatment (Figure 13C). Figures 13D-183F, Flow cytometry analysis of CD8+ frequencies detected in CD45+CD3+CD11b-CD8+CD4-: 1 day after initiation of treatment (Figure 13D), 4 days after initiation of treatment (Figure 13E), and 8 days after initiation of treatment (Figure 13F). [Figure 13D] Figure 13 shows T cell frequency in the TME 1, 4, and 8 days after the initiation of anti-GITR Fc region variant treatment. Humanized FcγR mice bearing refractory MC38 tumors were treated with chimeric anti-mGITR Fc region variants IgG1-N297A (NA), Afuco-IgG1-G236A (GA-aFuc), and IgG1. Mice were sacrificed and tumors were harvested at three time points after treatment: 1, 4 (total of 2 mAb doses), and 8 (total of 3 mAb doses), and untreated mice were used as controls. Figures 13A-13C, Flow cytometry analysis of Treg frequency detected for CD45+CD3+CD11b-CD8-CD4+FoxP3+: 1 day after the initiation of treatment (Figure 13A), 4 days after the initiation of treatment (Figure 13B), and 8 days after the initiation of treatment (Figure 13C). Figures 13D-183F, Flow cytometry analysis of CD8+ frequencies detected in CD45+CD3+CD11b-CD8+CD4-: 1 day after initiation of treatment (Figure 13D), 4 days after initiation of treatment (Figure 13E), and 8 days after initiation of treatment (Figure 13F). [Figure 13E]Figure 13 shows T cell frequency in the TME 1, 4, and 8 days after the initiation of anti-GITR Fc region variant treatment. Humanized FcγR mice bearing refractory MC38 tumors were treated with chimeric anti-mGITR Fc region variants IgG1-N297A (NA), Afuco-IgG1-G236A (GA-aFuc), and IgG1. Mice were sacrificed and tumors were harvested at three time points after treatment: 1, 4 (total of 2 mAb doses), and 8 (total of 3 mAb doses), and untreated mice were used as controls. Figures 13A-13C, Flow cytometry analysis of Treg frequency detected for CD45+CD3+CD11b-CD8-CD4+FoxP3+: 1 day after the initiation of treatment (Figure 13A), 4 days after the initiation of treatment (Figure 13B), and 8 days after the initiation of treatment (Figure 13C). Figures 13D-183F, Flow cytometry analysis of CD8+ frequencies detected in CD45+CD3+CD11b-CD8+CD4-: 1 day after initiation of treatment (Figure 13D), 4 days after initiation of treatment (Figure 13E), and 8 days after initiation of treatment (Figure 13F). [Figure 13F] Figure 13 shows T cell frequency in the TME 1, 4, and 8 days after the initiation of anti-GITR Fc region variant treatment. Humanized FcγR mice bearing refractory MC38 tumors were treated with chimeric anti-mGITR Fc region variants IgG1-N297A (NA), Afuco-IgG1-G236A (GA-aFuc), and IgG1. Mice were sacrificed and tumors were harvested at three time points after treatment: 1, 4 (total of 2 mAb doses), and 8 (total of 3 mAb doses), and untreated mice were used as controls. Figures 13A-13C, Flow cytometry analysis of Treg frequency detected for CD45+CD3+CD11b-CD8-CD4+FoxP3+: 1 day after the initiation of treatment (Figure 13A), 4 days after the initiation of treatment (Figure 13B), and 8 days after the initiation of treatment (Figure 13C). Figures 13D-183F, Flow cytometry analysis of CD8+ frequencies detected in CD45+CD3+CD11b-CD8+CD4-: 1 day after initiation of treatment (Figure 13D), 4 days after initiation of treatment (Figure 13E), and 8 days after initiation of treatment (Figure 13F). [Figure 13G]Figures 13G-13I, CD8 / Treg ratio in the TME of mice: 1 day after the start of treatment (Figure 13G), 4 days after the start of treatment (Figure 13H), and 8 days after the start of treatment (Figure 13I). [Figure 13H] Figures 13G-13I, CD8 / Treg ratio in the TME of mice: 1 day after the start of treatment (Figure 13G), 4 days after the start of treatment (Figure 13H), and 8 days after the start of treatment (Figure 13I). [Figure 13I] Figures 13G-13I, CD8 / Treg ratio in the TME of mice: 1 day after the start of treatment (Figure 13G), 4 days after the start of treatment (Figure 13H), and 8 days after the start of treatment (Figure 13I). [Figure 14A] Flow cytometric analysis of DC activation status within TME and dLN from humanized FcγR mice bearing refractory MC38 tumors treated with either Afuco-IgG1-G236A (GA-aFuc), IgG1 Fc-silent N297A (NA), and IgG1. TME and dLN were harvested 4 and 8 days after treatment initiation, and untreated mice served as controls (untreated). DC were detected for CD45+CD11b-NK1.1-F4 / 80-CD11c+MHCII+; activated CD80 CD was detected for CD45+CD11b-NK1.1-F4 / 80-CD11c+MHCII+CD80+CD86-; activated CD86 DC were detected for CD45+CD11b-NK1.1-F4 / 80-CD11c+MHCII+CD80-CD86+. Figure 14A shows the percentage of DCs in the TME 4 days after the start of treatment. Figure 14B shows the geometric mean fluorescence intensity (gMFI) of activated CD80 DCs in the TME 4 days after the start of treatment. Figure 14C shows the gMFI of activated CD86 DCs in the TME 4 days after the start of treatment. Figure 14D shows the percentage of DCs in the dLN 4 days after the start of treatment. Figure 14E shows the geometric mean fluorescence intensity (gMFI) of activated CD80 DCs in the dLN 4 days after the start of treatment. Figure 14F shows the gMFI of activated CD86 DCs in the dLN 4 days after the start of treatment. Figure 14G shows the percentage of DCs in the TME 8 days after the start of treatment. [Figure 14B]Flow cytometric analysis of DC activation status within TME and dLN from humanized FcγR mice bearing refractory MC38 tumors treated with either Afuco-IgG1-G236A (GA-aFuc), IgG1 Fc-silent N297A (NA), and IgG1. TME and dLN were harvested 4 and 8 days after treatment initiation, and untreated mice served as controls (untreated). DC were detected for CD45+CD11b-NK1.1-F4 / 80-CD11c+MHCII+; activated CD80 CD was detected for CD45+CD11b-NK1.1-F4 / 80-CD11c+MHCII+CD80+CD86-; activated CD86 DC were detected for CD45+CD11b-NK1.1-F4 / 80-CD11c+MHCII+CD80-CD86+. Figure 14A shows the percentage of DCs in the TME 4 days after the start of treatment. Figure 14B shows the geometric mean fluorescence intensity (gMFI) of activated CD80 DCs in the TME 4 days after the start of treatment. Figure 14C shows the gMFI of activated CD86 DCs in the TME 4 days after the start of treatment. Figure 14D shows the percentage of DCs in the dLN 4 days after the start of treatment. Figure 14E shows the geometric mean fluorescence intensity (gMFI) of activated CD80 DCs in the dLN 4 days after the start of treatment. Figure 14F shows the gMFI of activated CD86 DCs in the dLN 4 days after the start of treatment. Figure 14G shows the percentage of DCs in the TME 8 days after the start of treatment. [Figure 14C]Flow cytometric analysis of DC activation status within TME and dLN from humanized FcγR mice bearing refractory MC38 tumors treated with either Afuco-IgG1-G236A (GA-aFuc), IgG1 Fc-silent N297A (NA), and IgG1. TME and dLN were harvested 4 and 8 days after treatment initiation, and untreated mice served as controls (untreated). DC were detected for CD45+CD11b-NK1.1-F4 / 80-CD11c+MHCII+; activated CD80 CD was detected for CD45+CD11b-NK1.1-F4 / 80-CD11c+MHCII+CD80+CD86-; activated CD86 DC were detected for CD45+CD11b-NK1.1-F4 / 80-CD11c+MHCII+CD80-CD86+. Figure 14A shows the percentage of DCs in the TME 4 days after the start of treatment. Figure 14B shows the geometric mean fluorescence intensity (gMFI) of activated CD80 DCs in the TME 4 days after the start of treatment. Figure 14C shows the gMFI of activated CD86 DCs in the TME 4 days after the start of treatment. Figure 14D shows the percentage of DCs in the dLN 4 days after the start of treatment. Figure 14E shows the geometric mean fluorescence intensity (gMFI) of activated CD80 DCs in the dLN 4 days after the start of treatment. Figure 14F shows the gMFI of activated CD86 DCs in the dLN 4 days after the start of treatment. Figure 14G shows the percentage of DCs in the TME 8 days after the start of treatment. [Figure 14D]Flow cytometric analysis of DC activation status within TME and dLN from humanized FcγR mice bearing refractory MC38 tumors treated with either Afuco-IgG1-G236A (GA-aFuc), IgG1 Fc-silent N297A (NA), and IgG1. TME and dLN were harvested 4 and 8 days after treatment initiation, and untreated mice served as controls (untreated). DC were detected for CD45+CD11b-NK1.1-F4 / 80-CD11c+MHCII+; activated CD80 CD was detected for CD45+CD11b-NK1.1-F4 / 80-CD11c+MHCII+CD80+CD86-; activated CD86 DC were detected for CD45+CD11b-NK1.1-F4 / 80-CD11c+MHCII+CD80-CD86+. Figure 14A shows the percentage of DCs in the TME 4 days after the start of treatment. Figure 14B shows the geometric mean fluorescence intensity (gMFI) of activated CD80 DCs in the TME 4 days after the start of treatment. Figure 14C shows the gMFI of activated CD86 DCs in the TME 4 days after the start of treatment. Figure 14D shows the percentage of DCs in the dLN 4 days after the start of treatment. Figure 14E shows the geometric mean fluorescence intensity (gMFI) of activated CD80 DCs in the dLN 4 days after the start of treatment. Figure 14F shows the gMFI of activated CD86 DCs in the dLN 4 days after the start of treatment. Figure 14G shows the percentage of DCs in the TME 8 days after the start of treatment. [Figure 14E]Flow cytometric analysis of DC activation status within TME and dLN from humanized FcγR mice bearing refractory MC38 tumors treated with either Afuco-IgG1-G236A (GA-aFuc), IgG1 Fc-silent N297A (NA), and IgG1. TME and dLN were harvested 4 and 8 days after treatment initiation, and untreated mice served as controls (untreated). DC were detected for CD45+CD11b-NK1.1-F4 / 80-CD11c+MHCII+; activated CD80 CD was detected for CD45+CD11b-NK1.1-F4 / 80-CD11c+MHCII+CD80+CD86-; activated CD86 DC were detected for CD45+CD11b-NK1.1-F4 / 80-CD11c+MHCII+CD80-CD86+. Figure 14A shows the percentage of DCs in the TME 4 days after the start of treatment. Figure 14B shows the geometric mean fluorescence intensity (gMFI) of activated CD80 DCs in the TME 4 days after the start of treatment. Figure 14C shows the gMFI of activated CD86 DCs in the TME 4 days after the start of treatment. Figure 14D shows the percentage of DCs in the dLN 4 days after the start of treatment. Figure 14E shows the geometric mean fluorescence intensity (gMFI) of activated CD80 DCs in the dLN 4 days after the start of treatment. Figure 14F shows the gMFI of activated CD86 DCs in the dLN 4 days after the start of treatment. Figure 14G shows the percentage of DCs in the TME 8 days after the start of treatment. [Figure 14F]Flow cytometric analysis of DC activation status within TME and dLN from humanized FcγR mice bearing refractory MC38 tumors treated with either Afuco-IgG1-G236A (GA-aFuc), IgG1 Fc-silent N297A (NA), and IgG1. TME and dLN were harvested 4 and 8 days after treatment initiation, and untreated mice served as controls (untreated). DC were detected for CD45+CD11b-NK1.1-F4 / 80-CD11c+MHCII+; activated CD80 CD was detected for CD45+CD11b-NK1.1-F4 / 80-CD11c+MHCII+CD80+CD86-; activated CD86 DC were detected for CD45+CD11b-NK1.1-F4 / 80-CD11c+MHCII+CD80-CD86+. Figure 14A shows the percentage of DCs in the TME 4 days after the start of treatment. Figure 14B shows the geometric mean fluorescence intensity (gMFI) of activated CD80 DCs in the TME 4 days after the start of treatment. Figure 14C shows the gMFI of activated CD86 DCs in the TME 4 days after the start of treatment. Figure 14D shows the percentage of DCs in the dLN 4 days after the start of treatment. Figure 14E shows the geometric mean fluorescence intensity (gMFI) of activated CD80 DCs in the dLN 4 days after the start of treatment. Figure 14F shows the gMFI of activated CD86 DCs in the dLN 4 days after the start of treatment. Figure 14G shows the percentage of DCs in the TME 8 days after the start of treatment. [Figure 14G]Flow cytometric analysis of DC activation status within TME and dLN from humanized FcγR mice bearing refractory MC38 tumors treated with either Afuco-IgG1-G236A (GA-aFuc), IgG1 Fc-silent N297A (NA), and IgG1. TME and dLN were harvested 4 and 8 days after treatment initiation, and untreated mice served as controls (untreated). DC were detected for CD45+CD11b-NK1.1-F4 / 80-CD11c+MHCII+; activated CD80 CD was detected for CD45+CD11b-NK1.1-F4 / 80-CD11c+MHCII+CD80+CD86-; activated CD86 DC were detected for CD45+CD11b-NK1.1-F4 / 80-CD11c+MHCII+CD80-CD86+. Figure 14A shows the percentage of DCs in the TME 4 days after the start of treatment. Figure 14B shows the geometric mean fluorescence intensity (gMFI) of activated CD80 DCs in the TME 4 days after the start of treatment. Figure 14C shows the gMFI of activated CD86 DCs in the TME 4 days after the start of treatment. Figure 14D shows the percentage of DCs in the dLN 4 days after the start of treatment. Figure 14E shows the geometric mean fluorescence intensity (gMFI) of activated CD80 DCs in the dLN 4 days after the start of treatment. Figure 14F shows the gMFI of activated CD86 DCs in the dLN 4 days after the start of treatment. Figure 14G shows the percentage of DCs in the TME 8 days after the start of treatment. [Figure 15A] 15A is a graphical representation of the differential binding affinity of humanized anti-hGITR (TRX518) IgG1 Fc region mutants to hGITR and the indicated hFcγRs. The binding affinity of anti-human IgG1 Fc region mutants to hGITR (FIG. 15A) and human FcγRIIA (FIG. 15B) and FcγRIIIA (FIG. 15C) was assessed using a comparative ELISA. OD450 values ​​were plotted against increasing concentrations of test antibody to assess binding to plate-bound proteins. Data are shown as mean + SEM. [Figure 15B]15A is a graphical representation of the differential binding affinity of humanized anti-hGITR (TRX518) IgG1 Fc region mutants to hGITR and the indicated hFcγRs. The binding affinity of anti-human IgG1 Fc region mutants to hGITR (FIG. 15A) and human FcγRIIA (FIG. 15B) and FcγRIIIA (FIG. 15C) was assessed using a comparative ELISA. OD450 values ​​were plotted against increasing concentrations of test antibody to assess binding to plate-bound proteins. Data are shown as mean + SEM. [Figure 15C] 15A is a graphical representation of the differential binding affinity of humanized anti-hGITR (TRX518) IgG1 Fc region mutants to hGITR and the indicated hFcγRs. The binding affinity of anti-human IgG1 Fc region mutants to hGITR (FIG. 15A) and human FcγRIIA (FIG. 15B) and FcγRIIIA (FIG. 15C) was assessed using a comparative ELISA. OD450 values ​​were plotted against increasing concentrations of test antibody to assess binding to plate-bound proteins. Data are shown as mean + SEM. [Figure 16] Graphical representation of the effect of blood T cell frequency after treatment with anti-hGITR antibodies. Humanized FcγR mice and hGITR mice inoculated with MC38 tumors were treated with humanized Fc region modified variants of anti-hGITR antibodies IgG1-N297A and Afuco-IgG1-G236A, both containing the Fab of TRX518. After a single dose of mAb, blood was collected at the following time points: 2 days before treatment, and 2, 22 and 96 hours after treatment. Data are presented as mean ± SEM and ordinary one-way ANOVA with Tukey's multiple comparison statistical test. n=5 for all groups. [Figure 17A]Figure 17 depicts T cell frequency in the TME after anti-hGITR treatment. Humanized FcγR / GITR mice bearing refractory MC38 tumors were treated with anti-hGITR Fc region variant IgG1-N297A (TRX518) and Afuco-IgG1-G236A. Tumors were harvested 4 days after the start of treatment (a total of 2 mAb doses), single cell suspensions were generated, and modulation of the immune compartments of the TME of CD4 FoxP3- (Figure 17A), CD8 (Figure 17B), and Treg (Figure 17C) was analyzed by flow cytometry. Data are presented as mean ± SEM and Kruskal-Wallis statistical test was performed. [Figure 17B] Figure 17 depicts T cell frequency in the TME after anti-hGITR treatment. Humanized FcγR / GITR mice bearing refractory MC38 tumors were treated with anti-hGITR Fc region variant IgG1-N297A (TRX518) and Afuco-IgG1-G236A. Tumors were harvested 4 days after the start of treatment (a total of 2 mAb doses), single cell suspensions were generated, and modulation of the immune compartments of the TME of CD4 FoxP3- (Figure 17A), CD8 (Figure 17B), and Treg (Figure 17C) was analyzed by flow cytometry. Data are presented as mean ± SEM and Kruskal-Wallis statistical test was performed. [Figure 17C] Figure 17 depicts T cell frequency in the TME after anti-hGITR treatment. Humanized FcγR / GITR mice bearing refractory MC38 tumors were treated with anti-hGITR Fc region variant IgG1-N297A (TRX518) and Afuco-IgG1-G236A. Tumors were harvested 4 days after the start of treatment (a total of 2 mAb doses), single cell suspensions were generated, and modulation of the immune compartments of the TME of CD4 FoxP3- (Figure 17A), CD8 (Figure 17B), and Treg (Figure 17C) was analyzed by flow cytometry. Data are presented as mean ± SEM and Kruskal-Wallis statistical test was performed. [Figure 18A]Timeline of in vivo NK depletion assay from inoculation of refractory MC38 tumor cells into mice, blood was drawn and tumors harvested, to day 4 from initiation of anti-mGITR hIgG1 Fc region mutant treatment, including timeline for NK depletion with αNK1.1. Depletion of Tregs with Afuco-IgG1-G236A requires evaluation of NK cells. [Figure 18B] Flow cytometry of NK cells in blood samples of mice on day 0 (FIG. 18B) and day 4 (FIG. 18C) from the start of Afuco-IgG1-G236A or IgG1 Fc-silent N297A (NA) treatment and after NK cell depletion. NK cells were detected for CD45+CD3-NKp46+, blood from mice treated with PBS only was used as negative control. [Figure 18C] Flow cytometry of NK cells in blood samples of mice on day 0 (FIG. 18B) and day 4 (FIG. 18C) from the start of Afuco-IgG1-G236A or IgG1 Fc-silent N297A (NA) treatment and after NK cell depletion. NK cells were detected for CD45+CD3-NKp46+, blood from mice treated with PBS only was used as negative control. [Figure 18D] The cell numbers per mg of tumor are shown for NK cells (Figure 18D), Treg cells (detected for CD45+CD3+CD11b-CD8-CD4+FoxP3+, Figure 18E), and CD8+ cells (detected for CD45+CD3+CD11b-CD8+CD4-, Figure 18F). [Figure 18E] The cell numbers per mg of tumor are shown for NK cells (Figure 18D), Treg cells (detected for CD45+CD3+CD11b-CD8-CD4+FoxP3+, Figure 18E), and CD8+ cells (detected for CD45+CD3+CD11b-CD8+CD4-, Figure 18F). [Figure 18F]The cell numbers per mg of tumor are shown for NK cells (Figure 18D), Treg cells (detected for CD45+CD3+CD11b-CD8-CD4+FoxP3+, Figure 18E), and CD8+ cells (detected for CD45+CD3+CD11b-CD8+CD4-, Figure 18F). [Figure 19A] ELISA analysis measuring binding of novel anti-mGITR bispecific variants to different hFcγRs compared to the monospecific Afuco-IgG1-G236A (DTA1 GA-aFuc, circles) that served as a control. Nonagonistic bispecific antibodies were generated based on the Fc scaffolds of (i) Afuco-IgG1-G236A and Synagsis antibodies (GITR / Afuco-IgG1-G236A Syn, DTA1 / Syn GA-aFuc, triangles) and (ii) IgG1-N297A and Synagsis (GITR / NA Syn DTA1 / NA Syn, upside-down triangles). Abs were assessed by ELISA for their binding to FcγRIIA (Figure 19A), FcγRIIB (Figure 19B) and FcγRIIIA (Figure 19C). Figure 19D HPLC characterization of trypsin-digested bispecific anti-GITR GITR / Afuco-IgG1-G236A Synagis non-agonist antibody. [Figure 19B]ELISA analysis measuring binding of novel anti-mGITR bispecific variants to different hFcγRs compared to the monospecific Afuco-IgG1-G236A (DTA1 GA-aFuc, circles) that served as a control. Nonagonistic bispecific antibodies were generated based on the Fc scaffolds of (i) Afuco-IgG1-G236A and Synagsis antibodies (GITR / Afuco-IgG1-G236A Syn, DTA1 / Syn GA-aFuc, triangles) and (ii) IgG1-N297A and Synagsis (GITR / NA Syn DTA1 / NA Syn, upside-down triangles). Abs were assessed by ELISA for their binding to FcγRIIA (Figure 19A), FcγRIIB (Figure 19B) and FcγRIIIA (Figure 19C). Figure 19D HPLC characterization of trypsin-digested bispecific anti-GITR GITR / Afuco-IgG1-G236A Synagis non-agonist antibody. [Figure 19C] ELISA analysis measuring binding of novel anti-mGITR bispecific variants to different hFcγRs compared to the monospecific Afuco-IgG1-G236A (DTA1 GA-aFuc, circles) that served as a control. Nonagonistic bispecific antibodies were generated based on the Fc scaffolds of (i) Afuco-IgG1-G236A and Synagsis antibodies (GITR / Afuco-IgG1-G236A Syn, DTA1 / Syn GA-aFuc, triangles) and (ii) IgG1-N297A and Synagsis (GITR / NA Syn DTA1 / NA Syn, upside-down triangles). Abs were assessed by ELISA for their binding to FcγRIIA (Figure 19A), FcγRIIB (Figure 19B) and FcγRIIIA (Figure 19C). [Figure 19D] HPLC characterization of trypsin-digested bispecific anti-GITR GITR / Afuco-IgG1-G236A Synagis non-agonist antibody. [Figure 20A]Figure 1 shows the binding capacity of a novel non-agonistic bispecific anti-mGITR mAb with an N297A substitution in the Fc, denoted "mGITR / NA Synagsis," compared to monospecific mIgG1-N297A (DTA-1 / Synagsis NA, squares). A non-GITR binding antibody with an N297A substitution in the Fc region as an isotype control (isotype control NA, triangles) was assayed for binding capacity by ELISA and OD450 values ​​were plotted against increasing concentrations of the indicated antibodies. [Figure 20B] IL-2 secretion by isolated T cells from wild-type (WT) mice (activated with anti-CD3 Ab) after incubation with increasing concentrations of anti-mGITR / NA Synagsis (DTA-1 / Synagsis NA, squares), monospecific mIgG1-N297A (DTA-1 NA filled circles), or a non-GITR binding antibody with a N297A substitution in the Fc region (isotype control NA) as an isotype control (triangles). ELISA was performed on supernatants of activated T cells incubated with Ab. [Figure 21] Flow cytometry analysis of the fraction of Tregs in the TME of hFcγR mice bearing MC38 tumors after different treatments. Mice were treated (IP) with 100 μg, 200 μg, or 400 μg of the humanized GA-aFuc Fc region variant of the non-agonistic bispecific Ab (GITR / syn GA-aFuc) or 100 μg of mouse anti-DTA-1 (anti-GITR) GA-aFuc Ab. Tumors from untreated mice were analyzed similarly as negative controls. [Figure 22A] Tumor volume (mm3) progression in MC38 tumor-bearing hFcγR mice after treatment with 100 μg GA-aFuc Ab (black triangles) or 200 μg nonagonistic GITR / syn GA-aFuc Ab (white triangles). Untreated mice served as controls (black circles). Figure 22A shows tumor volume measurements on day 20. Figure 22B shows tumor volumes for mice in all groups on day 13 from the start of treatment. Figure 22C shows tumor volumes for mice in GA-aFuc Ab and GITR / syn GA-aFuc Ab test groups on day 15 from the start of treatment. [Figure 22B]Tumor volume (mm3) progression in MC38 tumor-bearing hFcγR mice after treatment with 100 μg GA-aFuc Ab (black triangles) or 200 μg nonagonistic GITR / syn GA-aFuc Ab (white triangles). Untreated mice served as controls (black circles). Figure 22A shows tumor volume measurements on day 20. Figure 22B shows tumor volumes for mice in all groups on day 13 from the start of treatment. Figure 22C shows tumor volumes for mice in GA-aFuc Ab and GITR / syn GA-aFuc Ab test groups on day 15 from the start of treatment. [Figure 22C] Tumor volume (mm3) progression in MC38 tumor-bearing hFcγR mice after treatment with 100 μg GA-aFuc Ab (black triangles) or 200 μg nonagonistic GITR / syn GA-aFuc Ab (white triangles). Untreated mice served as controls (black circles). Figure 22A shows tumor volume measurements on day 20. Figure 22B shows tumor volumes for mice in all groups on day 13 from the start of treatment. Figure 22C shows tumor volumes for mice in GA-aFuc Ab and GITR / syn GA-aFuc Ab test groups on day 15 from the start of treatment. [Figure 23A] Flow cytometry analysis of sections of Tregs and dendritic cells (DCs) in the TME of hFcγR mice bearing MC38 tumors after treatment with 200 μg GITR / syn GA-aFuc (triangles) and 100 μg GA-aFuc (squares), respectively. Tumors from untreated mice were analyzed similarly to negative controls (circles). Tregs were detected by CD45+CD3+CD11b-CD8-CD4+FoxP3+ and DCs by CD45+CD11b-NK1.1-F4 / 80-CD11c+MHCII+. [Figure 23B]Flow cytometry analysis of sections of Tregs and dendritic cells (DCs) in the TME of hFcγR mice bearing MC38 tumors after treatment with 200 μg GITR / syn GA-aFuc (triangles) and 100 μg GA-aFuc (squares), respectively. Tumors from untreated mice were analyzed similarly to negative controls (circles). Tregs were detected by CD45+CD3+CD11b-CD8-CD4+FoxP3+ and DCs by CD45+CD11b-NK1.1-F4 / 80-CD11c+MHCII+. [Figure 23C] Flow cytometric analysis of DC activation by detecting peaks of CD80+ or ​​CD86+ expressing DC, respectively. Tumors from untreated mice were analyzed similarly to negative controls. DC were detected by CD45+CD11b-NK1.1-F4 / 80-CD11c+MHCII+, CD80+DC were detected by addition of anti-CD80, and CD86+DC were detected by addition of anti-CD86. [Figure 23D] Flow cytometric analysis of DC activation by detecting peaks of CD80+ or ​​CD86+ expressing DC, respectively. Tumors from untreated mice were analyzed similarly to negative controls. DC were detected by CD45+CD11b-NK1.1-F4 / 80-CD11c+MHCII+, CD80+DC were detected by addition of anti-CD80, and CD86+DC were detected by addition of anti-CD86. [Figure 24A] Figure 17 shows tumor volume progression monitoring over 17 days in wild type WT C57BL / 6J mice bearing MC38 tumors after treatment with 100 μg of DTA-1 mIgG2a (squares) or 100 μg of anti-GITR-mIgG2a N279A Ab (DTA-1 mIgG2a NA, open triangles). Untreated mice served as controls (filled circles). [Figure 24B]Figures 24B-24E, Flow cytometry analysis of modulation of immune compartments in the TME of MC38 tumor-bearing WT mice treated with 100 μg DTA-1 mIgG2a Ab (squares) and 100 μg DTA-1 mIgG2a NA (triangles) or untreated mice (circles) used as control. Tumors were harvested and analyzed 4 days after treatment. Number of Tregs / mg tumor detected using anti-FoxP3 in all test groups. [Figure 24C] Figures 24B-24E, Flow cytometry analysis of modulation of immune compartments in the TME of MC38 tumor-bearing WT mice treated with 100 μg DTA-1 mIgG2a Ab (squares) and 100 μg DTA-1 mIgG2a NA (triangles) or untreated mice (circles) used as control. Tumors were harvested and analyzed 4 days after treatment. Percentage of DCs from total lymphocytes (detected using anti-CD11c / anti-CD45) in all study groups. [Figure 24D] Figures 24B-24E, Flow cytometry analysis of modulation of immune compartments in the TME of MC38 tumor-bearing WT mice treated with 100 μg DTA-1 mIgG2a Ab (squares) and 100 μg DTA-1 mIgG2a NA (triangles) or untreated mice (circles) used as control. Tumors were harvested and analyzed 4 days after treatment. Delta gMFI of CD80+ activated DCs in all test groups. [Figure 24E] Figures 24B-24E, Flow cytometry analysis of modulation of immune compartments in the TME of MC38 tumor-bearing WT mice treated with 100 μg DTA-1 mIgG2a Ab (squares) and 100 μg DTA-1 mIgG2a NA (triangles) or untreated mice (circles) used as control. Tumors were harvested and analyzed 4 days after treatment. Delta gMFI of CD86+ activated DCs in all test groups. [Diagram 25] Graphical representation of tumor volume (mm3) progression over 13 days in MC38 tumor-bearing BATF3- / - mice treated with 100 μg of DTA-1 mIgG2a (squares) Ab and 100 μg of DTA-1 mIgG2a-NA Ab (triangles), or untreated mice used as controls (circles). [Figure 26A] Graphical representation of tumor volume (mm3) progression in XCR1-iDTR and ZBTB46-iDTR NC38 tumor-bearing mice treated with 100 μg DTA-1 mIgG2a Ab, 20 ng / g body weight diphtheria toxin (DT), or a combination of 100 μg DTA-1 mIgG2a Ab and 20 ng / g body weight DT, respectively. Untreated mice were used as controls. DCs were depleted by injection of 20 ng / g DT every other day. Figure 26A: XCR1-iDTR, a model of DT-mediated inducible cDC1 cell depletion. Tumor volume (mm3) progression at day 13 in XCR1-iDTR mice. Figure 26B: Tumor volume (mm3) progression at day 20 in ZBTB46-iDTR mice, a model of DT-mediated inducible cDC1 and cDC2 cell depletion. [Figure 26B] Graphical representation of tumor volume (mm3) progression in XCR1-iDTR and ZBTB46-iDTR NC38 tumor-bearing mice treated with 100 μg DTA-1 mIgG2a Ab, 20 ng / g body weight diphtheria toxin (DT), or a combination of 100 μg DTA-1 mIgG2a Ab and 20 ng / g body weight DT, respectively. Untreated mice were used as controls. DCs were depleted by injection of 20 ng / g DT every other day. Figure 26A: XCR1-iDTR, a model of DT-mediated inducible cDC1 cell depletion. Tumor volume (mm3) progression at day 13 in XCR1-iDTR mice. Figure 26B: Tumor volume (mm3) progression at day 20 in ZBTB46-iDTR mice, a model of DT-mediated inducible cDC1 and cDC2 cell depletion. [Figure 27A] The percentage of Tregs in the TME and DCs in the dLNs from MC38 tumor-bearing mice treated with 100 μg of mouse anti-GITR Ab, 100 μg of anti-CD25 Ab, or untreated mice are shown. Organs were harvested 4 days after treatment. [Figure 27B] The percentage of Tregs in the TME and DCs in the dLNs from MC38 tumor-bearing mice treated with 100 μg of mouse anti-GITR Ab, 100 μg of anti-CD25 Ab, or untreated mice are shown. Organs were harvested 4 days after treatment. [Figure 27C] Figure 27 shows the delta gMFI of CD80+ and CD86+ activated DCs in dLNs from mice treated with 100 μg mouse anti-GITR Ab, 100 μg mouse anti-CD25 Ab, or untreated mice, respectively. dLNs were harvested 4 days after treatment. Figure 27C shows the delta gMFI of CD80+ activated DCs. Figure 27D shows the delta gMFI of CD86+ activated DCs. [Figure 27D] Figure 27 shows the delta gMFI of CD80+ and CD86+ activated DCs in dLNs from mice treated with 100 μg mouse anti-GITR Ab, 100 μg mouse anti-CD25 Ab, or untreated mice, respectively. dLNs were harvested 4 days after treatment. Figure 27C shows the delta gMFI of CD80+ activated DCs. Figure 27D shows the delta gMFI of CD86+ activated DCs. [Figure 28A] Figure 1 shows the progression of tumor volume in MC38 tumor-bearing mice treated three times with 100 μg / mouse of IgG1-N297A (N297A) or IgG1-GAALIE (GAALIE). Untreated mice were used as controls. Each line represents one animal. [Figure 28B] Figure 1 shows the progression of tumor volume in MC38 tumor-bearing mice treated three times with 100 μg / mouse of IgG1-N297A (N297A) or IgG1-GAALIE (GAALIE). Untreated mice were used as controls. Each line represents one animal. [Figure 28C] Figure 1 shows the progression of tumor volume in MC38 tumor-bearing mice treated three times with 100 μg / mouse of IgG1-N297A (N297A) or IgG1-GAALIE (GAALIE). Untreated mice were used as controls. Each line represents one animal. [Figure 28D] Graphical representation of the mean tumor volume progression for each treatment group IgG1-N297A (N297A, n=8), IgG1-GAALIE (GAALIE, n=9), control, untreated mice (untreated n=8). An unpaired two-tailed t-test was used to compare the IgG1-N297A and IgG1-GAALIE groups. Data are presented as mean + SEM.

[0129] Any embodiment disclosed hereinabove can be optionally combined with one or any combination of the subject matter of another embodiment disclosed herein. Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given below. However, it should be understood that the detailed description and specific examples, while showing preferred embodiments of the present invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art from this detailed description. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0130] The present invention provides an antibody specific for human protein glucocorticoid-induced TNFR-related (hGITR, also called CD357 or TNFRSF18), which comprises a modified human IgG1 Fc and shows an enhanced binding ratio to activating FcγRIIA and / or FcγRIIIA compared to binding to FcγRIIB, enhancing the control of tumor growth and overall survival. The Fc modified anti-hGITR antibody of the present invention was designed by combining two strategies - glycoengineering, i.e., altering the content of sugar moieties (e.g., afucosylation), and protein engineering by substitution of one or more amino acids (e.g., G236A). In the process of developing the anti-GITR antibody of the present invention, initial experiments were performed in human FcγR mice to test chimeric anti-mouse GITR antibodies comprising human IgG1 Fc region and its variants. Following promising anti-tumor activity, an anti-hGITR antibody comprising a modified hIgG1 Fc region was generated and tested in a human FcγR-human GITR mouse model.

[0131] The enhanced affinity of the antibodies of the present invention to FcγRIIA and / or FcγRIIIA, compared to the reduced or unchanged binding affinity to FcγRIIB, results in a significant increase in the activation / inhibition ratio. This ratio directly correlates with the ability of the antibodies to stimulate the immune system through dendritic cell activation, phagocytosis, and the production of pro-inflammatory cytokines, and subsequently induce anti-tumor effects and long-term anti-tumor immunity. Surprisingly, the Fc-modified antibodies of the present invention enhance anti-tumor immune activity without the need to activate the GITR receptor.

[0132] Without wishing to be bound by any theory of mechanism of action, it is suggested that the reduced binding of the anti-hGITR antibody of the present invention to FcγRIIB of Fc-modified IgG1 impairs the cross-linking activity of the antibody compared to binding to activating receptors.Antibody cross-linking mechanism via binding to FcγRIIB is known for TNFR family members due to their role in enhancing agonist activity.Surprisingly, the Fc-modified anti-hGITR antibody of the present invention shows significant enhancement of anti-tumor activity even in the absence of such cross-linking.

[0133] Compared to antibodies comprising substantially the same antigen-binding site (e.g., the antibodies referred to herein as "IgG1", "IgG1-N297A" or "V11"), the antibodies of the present invention (e.g., the antibodies referred to as "afucosylated IgG1-G236A" or "afuco-G236A" and "GAALIE") showed enhanced binding to both FcγRIIA and FcγRIIIA activating receptors compared to binding to the inhibitory receptor FcγRIIB. The "V11" Fc variant, which shows high affinity for FcγRIIB, failed to induce antitumor effects and long-term antitumor immunity.

[0134] The anti-hGITR antibody of the present invention reduces Treg frequency in the TME by an Fc-mediated depletion mechanism. The reduction of CD4 Treg occurs at an early time point with the Fc-modified Afuco-G236A antibody. Anti-hGITR-mediated Treg depletion is FcγR dependent. The Afuco-G236A anti-hGITR antibody of the present invention demonstrated FcγR-mediated Treg depletion, whereas the antibody TRX518, currently in clinical trials, which is an Fc region silent antibody, does not mediate Treg depletion. Furthermore, the Afuco-G236A Fc-modified variant of the present invention showed the highest increase in both CD80 and CD86 DC activation marker density in tumors and draining lymph nodes (dLNs) among all Fc region variants tested. IgG1 Fc-silentN297A showed no increase in CD80 or CD86 markers.

[0135] Again, unexpectedly, the Fc modified antibodies of the invention bind primarily to activating receptors, do not enhance cross-linking, and exhibit increased anti-cancer activity compared to similar antibodies with different Fc scaffolds.

[0136] definition As used herein, the term "glucocorticoid-inducible TNFR family-related receptor (abbreviated herein as "GITR"), also known as TNF receptor superfamily 18 (TNFRSF18), AITR, CD357, ENERGEN, or GITR-D, refers to a member of the tumor necrosis factor / nerve growth factor receptor family. Human members are type I transmembrane proteins characterized by three cysteine ​​pseudorepeats in the extracellular domain (Nocentini, G. et al. (1997) Proc. Natl. Acad. Sci., USA 94:6216-622). The Fc modified antibodies of the invention may be directed against any human GITR protein variant expressed on human cancer cells and / or human immune cells. Non-limiting examples of human protein variants include the 241 amino acid protein with accession numbers NM_004195.3, accession number NP_004186.1 and GI:4759246, the 255 amino acid protein with accession numbers NP_683699.1 and GI:23238194, and the 234 amino acid protein with accession numbers NP_683700.1 and GI:23238197. Non-limiting exemplary mouse GITR protein variant accession numbers are NM_021985.3, the 132 amino acid protein with accession number NP_068820.1 and the 228 amino acid protein with accession number NM_009400.3, accession number NP_033426.1.

[0137] An antibody, or immunoglobulin, comprises two heavy chains and two light chains linked together by disulfide bonds, with each light chain joined to a respective heavy chain by disulfide bonds in a "Y" configuration. Each heavy chain contains at one end a variable domain (V H ) and has several constant domains (C H Each light chain has at one end a variable domain (V L ), and a constant domain (C L), where the light chain variable domain is aligned with the variable domain of the heavy chain and the light chain constant domain is aligned with the first constant domain (CH1) of the heavy chain. The variable domains of each pair of light and heavy chains form the antigen-binding site (Fab). The light and heavy chain domains have the same overall structure, each domain containing four framework regions whose sequences are relatively conserved and connected by three hypervariable domains known as complementarity determining regions (CDRs). These domains contribute to the specificity and affinity of the antigen-binding site.

[0138] "Framework region" and "FR" are known in the art to refer to the non-CDR portions of the heavy and light chain variable regions. Generally, there are four FRs (FR-H1, FR-H2, FR-H3, and FR-H4) in each full-length heavy chain variable region, and four FRs (FR-L1, FR-L2, FR-L3, and FR-L4) in each full-length light chain variable region.The precise amino acid sequence boundaries of a given CDR or FR can be determined by Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme); Al-Lazikani et al. (1997) JMB273927-948 ("Chothia" numbering scheme); MacCallum et al., J. Mol. Biol. 262:732-745 (1996), "Antibody-antigen interactions: Contact analysis and binding site topography," J. Mol. Biol. 262, 732-745. ("Contact" numbering scheme); Lefranc MP et al., "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains," Dev Comp Immunol, 2003 Jan., 27(1):55-77 ("IMGT" numbering scheme); Honegger A and Pluckthun A, "Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool," J Mol Biol, 2001 Jun 8;309(3):657-70 ("Aho" numbering scheme); and Whitelegg NR and Rees AR "WAM: an improved algorithm for modelling antibodies on the WEB" Protein Eng. 2000 Dec;13(12):819-24 ("AbM" numbering scheme).

[0139] There are several methods known in the art for determining the CDR sequences of a given antibody molecule, but no standard, defined method. The determination of CDR sequences from the heavy and light chain variable regions of an antibody can be performed according to any method known in the art, including but not limited to the Kabat, Chothia, and IMGT methods. The set of selected CDRs according to the present invention may comprise sequences identified by more than one method, i.e., for example, some CDR sequences may be determined using Kabat and some using IMGT. According to some embodiments, the CDR sequences of the antibody variable regions are determined using the Kabat and / or Chothia methods. In certain embodiments, the CDRs of the antibodies described herein may be defined by a method selected from Kabat, Chothia, IMGT, Aho, AbM, or a combination thereof. In some embodiments, the CDRs are defined using Kabat.

[0140] Alterations (e.g., substitutions) may be made in the CDRs, for example, to improve antibody affinity. Such alterations may be made in codons encoding CDRs that have a high mutation rate during somatic maturation (e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and the resulting mutants may be tested for binding affinity. Affinity maturation (e.g., using error-prone PCR, chain shuffling, randomization of CDRs, or site / oligonucleotide-directed mutagenesis) may be used to improve antibody affinity (e.g., Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (2001)). CDR residues involved in antigen binding may be specifically identified, for example, using alanine scanning mutagenesis or modeling (e.g., Cunningham and Wells Science, 244:1081-1085 (1989)). In particular, CDR-H3 and CDR-L3 are often targeted.

[0141] Further included within the scope of the present invention are chimeric antibodies, human and humanized antibodies, recombinant and engineered antibodies, and conjugates thereof. Furthermore, DNA encoding the variable regions of an antibody can be inserted into DNA encoding another antibody to produce a chimeric antibody.

[0142] Antibodies herein specifically include "chimeric" antibodies. Chimeric antibodies are molecules whose different portions are derived from different animal species, such as those with variable regions derived from a murine mAb and human immunoglobulin constant regions. Antibodies with variable region framework residues substantially derived from a human antibody and complementarity determining regions substantially derived from a mouse antibody are also called humanized antibodies. Chimeric antibodies are primarily used to reduce immunogenicity in applications and increase yields in production, for example, murine mAbs have high yields from hybridomas but are highly immunogenic in humans, so human / murine chimeric mAbs are used. Chimeric antibodies and methods for their production are known in the art (e.g., WO 86 / 01533, WO 97 / 02671, WO 90 / 07861, WO 92 / 22653, and U.S. Pat. Nos. 5,693,762, 5,693,761, 5,585,089, 5,530,101, and 5,225,539).

[0143] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In most cases, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate having the desired specificity, affinity, and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, humanized antibodies will comprise substantially all of at least one, and typically two, variable domains, with all or substantially all of the hypervariable loops corresponding to those of a non-human immunoglobulin and all or substantially all of the FRs being those of human immunoglobulin sequences. Humanized antibodies typically comprise a modified human immunoglobulin constant region (Fc).

[0144] Non-human antibodies may be humanized by any method known in the art. In one method, non-human complementarity determining regions (CDRs) are inserted (grafted) into a human antibody or consensus antibody framework sequence. Further changes can then be introduced into the antibody framework to adjust affinity or immunogenicity.

[0145] For example, U.S. Patent No. 5,585,089 to Queen et al. discloses humanized immunoglobulins and methods for their preparation, which contain complementarity determining regions (CDRs) from a donor immunoglobulin and heavy and light chain variable region frameworks from human acceptor immunoglobulin heavy and light chains, and which contain amino acids from the donor immunoglobulin framework outside of the Kabat and Chothia CDRs, and which donor amino acids substitute for corresponding amino acids in the acceptor immunoglobulin heavy or light chain framework. U.S. Patent No. 5,225,539 to Winter also discloses modified antibodies or antigen-binding fragments thereof and methods for preparing them, wherein the variable domains of the antibodies or antigen-binding fragments have framework regions of a first immunoglobulin heavy or light chain variable domain and complementarity determining regions of a second immunoglobulin heavy or light chain variable domain, the second immunoglobulin heavy or light chain variable domain differing from the first immunoglobulin heavy or light chain variable domain in antigen-binding specificity, antigen-binding affinity, species, class or subclass.

[0146] A "human antibody" is one that has an amino acid sequence that corresponds to that of an antibody produced by a human and / or that has been made using any of the techniques for making human antibodies disclosed herein. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues. Human antibodies can be produced using a variety of techniques known in the art. In one embodiment, human antibodies are selected from a phage library, which expresses human antibodies (Vaughan et al. Nature Biotechnology 1996 14309-314; Sheets et al. PNAS (USA), 1998, 95, 6157-6162); Hoogenboom and Winter, J. Mol. Biol., 1991, 227, 381; Marks et al., J. Mol. Biol., 1991, 222, 581). Human antibodies can also be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Patent Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, and 5,661,016, as well as the following chemical publications: Marks et al, Bio / Technology 10:779-783 (1992); Lonberg et al., Nature 368:856-859 (1994); Morrison, Nature 368:812-13 (1994); Fishwild et al., Nature Biotechnology 14:845-51 (1996); Neuberger, Nature Biotechnology 14:826 (1996); Lonberg and Huszar, Intern. Rev. Immunol. 13:65-93 (1997); (1995).Alternatively, human antibodies may be prepared through immortalization of human B lymphocytes that produce antibodies against a target antigen (such B lymphocytes may be harvested from an individual or immunized in vitro). See, e.g., Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol., 147 (1):86-95 (1991); and U.S. Patent No. 5,750,373.

[0147] As used herein, the term "site-directed mutagenesis" refers to an in vitro procedure in which custom-designed oligonucleotide primers are used to impart desired mutations to double-stranded DNA plasmids. The most widely used method incorporates mutations into the plasmid by inverse PCR using standard primers. For example, one of these methods utilizes special overlapping affinity primers designed to introduce specific desired substitutions.

[0148] Affinity is the strength of binding of a single molecule to its ligand. Affinity is typically measured using the equilibrium dissociation constant (K D ), which is used to assess and rank the order strength of bimolecular interactions. The binding of an antibody to its antigen is a reversible process, and the rate of the binding reaction is proportional to the concentration of the reactants.

[0149] K D is the equilibrium dissociation constant between an antibody and its antigen, k off / k on It is the ratio of K D and affinity are inversely proportional. D The K value is related to the concentration of the antibody (the amount of antibody needed for a particular experiment). D The lower the value (lower the concentration) and therefore the higher the affinity of the antibody.

[0150] Ki refers to the inhibition constant used to describe the binding affinity of a molecule to its ligand (e.g., antibody and antigen, enzyme and ligand). Ki is also known as K D Ki represents the dissociation constant as K, or more narrowly, for the binding of a ligand whose binding reduces the activity of the binding molecule. The binding equilibrium described by the Ki value depends on the kinetic mechanism of inhibition.

[0151] Ki is measured via inhibition kinetics, in a non-limiting example by competitive ELISA, but when binding is measured more directly, for example by surface plasmon resonance (SPR), it is referred to as "K D " is preferred (Scarano S, Mascini M, Turner AP, Minunni M. Surface plasmon resonance imaging for affinity-based biosensors. Biosens Bioelectron. 2010, 25: 957-66). Lower K D and K i Values ​​represent higher affinity and inhibition.

[0152] The Fc region of monoclonal antibodies acts as an important bridge between adaptive and innate immune responses. When antigens expressed on the surface of cancer cells, virus-infected cells or invading pathogens are recognized by specific antibodies, the cells or pathogens are coated with antibodies. The Fc region of these surface-bound antibodies assists in the elimination of targets by different mechanisms. First, the Fc region can interact with the C1 molecule of the complement system, leading to activation of the classical pathway of the complement system. The Fc region can also recruit phagocytes via Fc receptors and activate the phagocytosis (ADCP) pathway, as well as ADCC mediated by NK cells macrophages and further effector cells. Among these mechanisms, studies on rituximab (anti-CD20) and trastuzumab (anti-HER2 / neu, also known as Herceptin) suggest that ADCC / ADCP is an important mechanism of action for the elimination of cancer cells.

[0153] As used below, the term "dendritic cell (DC) activation" refers to a process triggered by pathogens, inflammatory stimuli, or by T helper lymphocytes. DC activation exhibits multiple changes that allow the activated DC to transform into the most efficient antigen-presenting cells. One of these changes is a significant increase in the surface expression of T cell costimulatory molecules, such as CD80 and CD86, which serve as markers for activated DC.

[0154] The Fc region modifications of the present invention may be performed by site / oligonucleotide-directed mutagenesis resulting in the substitution, addition or deletion of at least one amino acid residue of the polypeptide, or may be carried out at the glycosylation level of the Fc region, for example, by the removal or addition of at least one oligosaccharide (N-glycan) moiety, or more specifically, by the reduction of fucose units (afucosylation).

[0155] As used herein, the term "afucosylated antibody" refers to an antibody whose Fc region has a significantly reduced glycan structure, i.e., more than 50% reduction or no fucose, compared to a parent antibody, including an unmodified IgG1.

[0156] Significantly reduced fucosylation according to some embodiments of the present invention refers to a fucose content or level of up to about 40% of all glycan structures. According to some embodiments, the antibody comprises 20-40% fucose of all glycan structures on its Fc region.

[0157] Afucosylation, i.e., reduction of the fucose content in an antibody Fc, may be performed by any method known in the art, including cell lines in which the fucosylation machinery has either been genetically knocked out, blocked or modified (e.g., CHO cells). According to some embodiments, afucosylation is performed post-translationally.

[0158] Afucosylation may also be performed according to any one of the following non-limiting approaches: (i) Fucosyltransferase (FUT) inhibitors, such as the L-fucose analog, 2-fluoroperacetylated fucose (2FF), which interferes with the normal synthesis of GDP-fucose and inhibits core fucosylation (Mishra N et al., "Comparison of two glycoengineering strategies to control the fucosylation of a monoclonal antibody". J Biotechnol. 2020; Zhou, Y., et al., "Inhibition of fucosylation by 2-fluorofucose suppresses human liver cancer HepG2 cell proliferation and migration as well as tumor formation". Sci Rep 7, 11563 (2017). (ii) Lec13 cells as a host cell line for the production of afucosylated antibodies (Shields RL, et al., Lack of fucose on human IgG1 N-linked oligosaccharide improves binding to human Fcgamma RIII and antibody-dependent cellular toxicity. J Biol Chem. 2002, 277:26733-40). (iii) GDP-keto-6-deoxymannose 3,5-epimerase / 4-reductase (FX)-knockout CHO cell lines for producing antibodies with fully afucosylated N-glycans (Louie S., et al., FX knockout CHO hosts can express desired ratios of fucosylated or afucosylated antibodies with high titer and comparable product quality. Biotechnol Bioeng. 2017, 114: 632-44) (iv) FUT8 (fucosyltransferase 8)- / - cell lines that have been shown to express fully defucosylated antibodies compared to the same antibodies produced in the parent cell line (Yamane-Ohnuki, N. et al., Establishment of FUT8 knockout Chinese hamster ovary cells: An ideal host cell line for producing completely defucosylated antibodies with enhanced antibody-dependent cellular cytotoxicity. Biotechnology and bioengineering, 87, 614-622). (v) Rat hybridoma YB2 / 0 cells, which have lower levels of Fut8 (fucosyltransferase 8) mRNA than CHO cells (Shinkawa T. et al., The absence of fucose but not the presence of galactose or bisecting N-acetylglucosamine of human IgG1 complex-type oligosaccharides shows the critical role of enhancing antibody-dependent cellular cytotoxicity. J Biol Chem. 2003 Jan 31, 278(5):3466-73). (vi) FUT8 siRNA CHO cells (Mori K. et al., Engineering Chinese hamster ovary cells to maximize effector function of produced antibodies using.Biotechnol Bioeng,2004,88:901-8). (vii) CHO-gmt3 (CHO-glycosylation mutant 3) cells, which showed a complete lack of core fucose on the N-glycans attached to EPO-Fc fusion proteins and IgG1 antibodies produced in the cells (Chan KF et al., Inactivation of GDP-fucose transporter gene (Slc35c1) in CHO cells by ZFNs, TALENs and CRISPR-Cas9 for production of fucose-free antibodies. Biotechnol J. 2016, 11: 399-414), (viii) CHO cells overexpressing both GnT-III and αManII yielded the highest levels of bisected and afucosylated glycans on IgG antibodies (Ferrara C. et al., Modulation of therapeutic antibody effector functions by glycosylation engineering: influence of Golgi enzyme localization domain and co-expression of heterologous beta1,4-N-acetylglucosaminyltransferase III and Golgi alpha-mannosidase II. Biotechnol Bioeng, 2006, 93:851-61). (ix) Small molecules that inhibit antibody fucosylation to complement existing platforms that involve genetic engineering of cell lines for the production of afucosylated antibodies (Okeley et al., Development of orally active inhibitors of protein and cellular fucosylation. Proc Natl Acad Sci USA, 2013, 110:5404-9). (x) Alternative expression platforms such as plants (Loos A. IgG-Fc glycoengineering in non-mammalian expression hosts. Arch Biochem Biophys, 2012, 526:167-73).

[0159] According to some embodiments, afucosylation is performed by adding 20-600 μM 2-deoxy-2-fluoro-L-fucose to the transfection medium.

[0160] Antibody fucose content and percent afucosylation may be determined by any method known in the art, including, but not limited to, mass spectrometry.

[0161] Sequence identity is the percentage of amino acids or nucleotides that are exactly the same between two different sequences. Sequence similarity allows conservative substitution of amino acids to be determined as identical amino acids. The polynucleotide sequences described herein can be codon-optimized for expression in a particular cell, such as a human cell. Codon optimization does not change the encoded amino acid sequence of the antibody chain, but may, for example, increase expression in the cell.

[0162] Mutants, analogs and derivatives of the antibody sequence are also within the scope of this application. These include, but are not limited to, conservative and non-conservative substitutions, insertions and deletions of amino acids within the sequence. Such modifications and the resulting antibody analogs or variants are within the scope of the present invention as long as they confer or even improve the binding profile to hGITR and human FcγR.

[0163] The term "antibody variant" as used herein refers to an antibody derived from another antibody by one or more conservative amino acid substitutions and / or by altering the glycosylation content of its Fc region. Variants according to the invention may be generated that preserve the overall molecular structure of the encoded protein. Given the properties of the individual amino acids that make up the disclosed protein products, some rational substitutions will be recognized by those skilled in the art.

[0164] Conservative substitutions of amino acids known to those skilled in the art are within the scope of the present invention. Conservative amino acid substitutions include replacing one amino acid with another having the same type of functional group or side chain, e.g., aliphatic, aromatic, positively charged, negatively charged. These substitutions may enhance oral bioavailability, penetration, and targeting to specific cell populations, immunogenicity, and the like. Those skilled in the art will recognize that individual substitutions, deletions, or additions to peptide, polypeptide, or protein sequences that change, add, or delete a single amino acid or a small percentage of amino acids in the encoded sequence are "conservatively modified variants," where the changes result in the replacement of an amino acid with a chemically similar amino acid. Conservative substitution lists that result in functionally similar amino acids are well known in the art. For example, according to one list known in the art, the following six groups each contain amino acids that are conservative substitutions for each other: 1) Alanine (A), Serine (S), Threonine (T), 2) Aspartic acid (D), glutamic acid (E), 3) Asparagine (N), Glutamine (Q), 4) Arginine (R), Lysine (K), 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V), and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0165] The term "antibody conjugate" as used herein refers to any molecule comprising an antibody of the present invention. For example, a fusion protein in which an antibody is linked to another entity, such as an anti-cancer drug or an identifiable moiety, is considered an antibody conjugate.

[0166] The term "nucleic acid" refers to a single- or double-stranded sequence (polymer) of deoxyribonucleotides or ribonucleotides. Furthermore, polynucleotides include variants of naturally occurring polynucleotides, unless otherwise specified. According to one embodiment, the nucleic acid may be selected from the group consisting of, but is not limited to, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), peptide nucleic acid (PNA), locked nucleic acid (LNA), and analogs thereof. The term encompasses DNA, RNA, single- or double-stranded, and chemical modifications thereof.

[0167] As used herein, the term "polynucleotide" refers to a long nucleic acid containing more than 150 nucleotides.

[0168] The terms "nucleic acid" and "polynucleotide" are used interchangeably herein.

[0169] The antibodies described herein are encoded by nucleic acids. In certain embodiments, the nucleic acid is a component of a vector that can be used to transfer a polypeptide encoding a polynucleotide into a cell. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a genomic integration vector, or "integration vector," which can be integrated into the chromosomal DNA of a host cell. Another type of vector is an "episomal" vector, e.g., a nucleic acid capable of extrachromosomal replication. A vector capable of directing the expression of an operably linked gene is referred to herein as an "expression vector." Suitable vectors include plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, viral vectors, and the like. In expression vectors, regulatory elements such as promoters, enhancers, polyadenylation signals, and the like, for use in controlling transcription can be derived from mammalian, microbial, viral, or insect genes. The ability to replicate in a host, usually conferred by an origin of replication, and a selection gene that facilitates recognition of transformants may also be incorporated. Vectors derived from viruses, such as lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses, may also be used. The plasmid vector can be linearized for integration into a chromosomal location. The vector can contain sequences that direct site-specific integration (e.g., AttP-AttB recombination) into a defined location or set of restriction sites in the genome. Additionally, the vector can contain sequences from transposable elements.

[0170] Nucleic acids encoding the antibodies described herein can be used to infect, transfect, transform, or transfect suitable cells, thus allowing the production of antibodies for commercial or therapeutic use. Standard cell lines and methods for producing antibodies from large scale cell culture are known in the art. In some embodiments, the cells are eukaryotic cells. In some embodiments, the eukaryotic cells are mammalian cells. In certain embodiments, the mammalian cells are cell lines useful for producing antibodies and are Chinese Hamster Ovary (CHO) cells, NS0 mouse myeloma cells, or PER.C6® cells. In certain embodiments, the nucleic acid encoding the antibody is integrated into a genomic locus of a cell useful for producing the antibody. In certain embodiments, methods of making an antibody are described herein, comprising culturing a cell comprising a nucleic acid encoding the antibody under in vitro conditions sufficient to allow the production and secretion of the antibody.

[0171] Any method known for the production of recombinant antibodies may be used to produce the antibodies of the invention. According to one method, cells transformed with a nucleotide sequence encoding an antibody polypeptide are cultured under effective conditions that allow for the expression of large amounts of the recombinant polypeptide or polypeptides. Effective culture conditions include, but are not limited to, effective media, bioreactors, temperature, pH and oxygen conditions that allow for protein production. Effective media refers to any medium in which cells are cultured to produce the recombinant polypeptide of the invention. Such media typically include aqueous solutions having assimilable carbon, nitrogen and phosphate sources, as well as other nutrients such as appropriate salts, minerals, metals and vitamins. The cells of the invention can be cultured in conventional fermentation bioreactors, shake flasks, test tubes, microtiter dishes and petri dishes. The culture can be carried out at temperatures, pH and oxygen content suitable for the recombinant cells. Such culture conditions are within the expertise of one of ordinary skill in the art.

[0172] Depending on the vector and host system used for production, the resulting polypeptide of the invention may either remain within the recombinant cell, be secreted into the fermentation medium, be secreted into the space between two cell membranes, or be retained on the outer surface of a cellular or viral membrane.

[0173] After a given time in culture, recovery of the recombinant antibody is effected, for example, by collecting the whole fermentation medium containing the antibody polypeptide, with or without further steps of separation or purification.

[0174] If the antibody polypeptide is expressed in cells, the cell membrane is preferably disrupted to release the polypeptide, using methods known in the art, including homogenization.

[0175] Any method known in the art for expressing and purifying antibodies may be used to produce the antibodies of the invention, including, but not limited to, those described in Vazquez-Lombardi et al., 2018, Nature Protocols, 13, 1, 99-117.

[0176] It should be emphasized that different sequencing methods used on the same protein or nucleotide sequence may result in slightly different sequences due to technical issues and different primers, especially at the ends of the sequence.

[0177] Notwithstanding the above, antibodies of some embodiments of the present invention can be purified using a variety of standard protein purification techniques, including, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalin A chromatography, protein A / G / L separation, mixed mode chromatography, metal affinity chromatography, lectin affinity chromatography, chromatofocusing, and differential solubilization.

[0178] Pharmacology and Treatment Methods In pharmaceutical and drug formulations, the active agent is preferably utilized together with one or more pharma- ceutically acceptable carrier(s) and, optionally, any other therapeutic ingredients. The carrier(s) must be pharma- ceutically acceptable in the sense that it is compatible with the other ingredients of the formulation and is not unduly deleterious to the recipient thereof. The active agent, as described above, is provided in an amount effective to achieve the desired pharmacological effect and in an amount appropriate to achieve the desired exposure.

[0179] The antibody of the present invention as an active ingredient is dissolved, dispersed or mixed in an excipient that is pharma- ceutically acceptable and compatible with the active ingredient, as is well known. Suitable excipients are, for example, water, saline, phosphate buffered saline (PBS), dextrose, glycerol, ethanol, and the like, and combinations thereof. Other suitable carriers are well known to those skilled in the art. In addition, if necessary, the composition may contain minor amounts of auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, and the like.

[0180] According to some embodiments, the pharmaceutical composition comprises 1-50 mg / mL of an anti-hGITR antibody. According to some embodiments, the pharmaceutical composition comprises a basic amino acid. According to some embodiments, the pharmaceutical composition comprises a sugar. According to some embodiments, the pharmaceutical composition comprises a surfactant. According to some embodiments, the pharmaceutical composition comprises a basic amino acid, a sugar, and a surfactant. According to some embodiments, the pharmaceutical composition comprises (i) 1-10 mg / mL of a basic amino acid; (ii) 10-200 mg / mL of a sugar; (iii) 0.01-1 mg / mL of a surfactant; (iv) 1-50 mg / mL of an anti-hGITR antibody.

[0181] According to some embodiments, the basic amino acid is selected from the group consisting of histidine, arginine, lysine and ornithine, each possibility representing a separate embodiment of the present invention.

[0182] The term "sugar" refers to monosaccharides, disaccharides, and polysaccharides, examples of sugars include, but are not limited to, sucrose, trehalose, dextrose, and the like. According to some embodiments, the sugar is selected from the group consisting of sucrose, trehalose, glucose, dextrose, and maltose. Each possibility represents a separate embodiment of the present invention. According to some embodiments, the composition comprises 10-200, 10-100, 50-150, or 70-100 mg / mL of sugar. Each possibility represents a separate embodiment of the present invention.

[0183] According to yet other embodiments, the composition comprises a polyol, including but not limited to mannitol and sorbitol.

[0184] According to some embodiments, the surfactant is non-anionic. According to some embodiments, the surfactant is selected from the group consisting of polysorbates, sorbitan esters, and poloxamers. Each possibility represents a separate embodiment of the present invention. According to some embodiments, the composition comprises 0.01-10, 0.01-1, 0.05-5, or 0.1-1 mg / mL of the surfactant. Each possibility represents a separate embodiment of the present invention.

[0185] Typically, the antibodies and conjugates thereof of the present invention are suspended in sterile saline for therapeutic use. Alternatively, pharmaceutical compositions may be formulated to control the release of the active ingredient or to extend its presence in the patient's system. Many suitable drug delivery systems are known, including, for example, implantable drug release systems, hydrogels, hydroxymethylcellulose, microcapsules, liposomes, microemulsions, microspheres, and the like. Controlled release preparations can be prepared through the use of polymers to complex or adsorb the molecules according to the present invention. For example, biocompatible polymers include matrices of poly(ethylene-co-vinyl acetate) and matrices of polyanhydride copolymers of stearic acid dimer and sebacic acid. The release rate of the molecules, i.e., antibodies, according to the present invention from such matrices depends on the molecular weight of the molecule, the amount of the molecule in the matrix, and the size of the particles dispersed.

[0186] The pharmaceutical compositions of the invention are formulated for administration by any suitable means, such as intravenous, subcutaneous, intramuscular, oral, topical, intranasal, intraarterial, intraarticular, intralesional, intratumoral or parenteral. Typically, intravenous (iv) administration is used to deliver antibodies. In some embodiments, the antibody or antibody conjugate is administered by injection.

[0187] According to one aspect, the invention provides a method of treating cancer comprising administering to a subject in need thereof a pharmaceutical composition comprising a therapeutically effective amount of an antibody or antibody conjugate described herein.

[0188] As used herein, the terms "subject," "individual," or "patient" refer to an individual who has been diagnosed with, is suspected of suffering from, or is at risk of developing at least one disease for which the described compositions and methods are useful for treating. In some embodiments, the individual is a mammal. In some embodiments, the individual is a human.

[0189] It will be apparent to those skilled in the art that a therapeutically effective amount of a molecule according to the invention will depend on, among other things, the administration schedule, the unit dose of the molecule administered, whether the molecule is administered in combination with other therapeutic agents, the immune status and health of the patient, the therapeutic activity of the administered molecule, its persistence in the circulation, and the judgment of the treating physician.

[0190] As used herein, the term "therapeutically effective amount" refers to an amount of a drug that is effective to treat a disease or disorder in a mammal. In the case of cancer, a therapeutically effective amount of a drug may reduce the number of cancer cells; reduce tumor size; inhibit (i.e., slow to some extent, and preferably stop) cancer cell invasion into peripheral organs; inhibit (i.e., slow to some extent, and preferably stop) tumor metastasis; inhibit tumor growth to some extent; and / or alleviate to some extent one or more symptoms associated with the disorder. To the extent that a drug may prevent growth and / or kill existing cancer cells, it may be cytostatic and / or cytotoxic. For cancer treatment, efficacy in vivo may be determined, for example, by assessing survival time, time to disease progression (TTP), response rate (RR), duration of response, and / or quality of life.

[0191] The term "cancer" generally refers to or describes a physiological condition in mammals characterized by uncontrolled cell proliferation. Cancers amenable to treatment by the present invention include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include squamous cell carcinoma, lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung), cancer of the peritoneum, hepatocellular carcinoma, gastric or stomach cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney or renal cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatic cancer, and ovarian cancer. carcinoma), and various types of head and neck cancer, as well as B-cell lymphomas (including low-grade / follicular non-Hodgkin's lymphoma (NHL)); small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small noncleaved cell NHL; giant mass disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal blood vessel growth associated with nevus syndrome, edema (such as that associated with brain tumors), and Meigs' syndrome. According to some embodiments, the cancer is selected from the group consisting of non-small cell lung cancer (NSCLC), renal cell carcinoma, melanoma, glioblastoma, colorectal cancer, breast cancer and ovarian cancer.Cancerous conditions modifiable for treatment according to the invention include metastatic cancer.

[0192] The pharmaceutical composition according to the invention may be administered together or in combination with an anti-cancer composition.

[0193] As used herein, the term "combination" or "combination therapy" can refer to either simultaneous administration of the combined items or sequential administration of the combined items. As described herein, when combination refers to sequential administration of the items, the items can be administered in any temporal order.

[0194] As used herein, the term "treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented.

[0195] According to some embodiments, the methods of treating cancer include administering the pharmaceutical composition as part of a treatment regimen that includes administration of at least one additional anti-cancer agent or treatment.

[0196] According to some embodiments, the anti-cancer agent is selected from the group consisting of antimetabolites, antimitotics, taxanes, topoisomerase inhibitors, topoisomerase II inhibitors, asparaginases, alkylating agents, antitumor antibiotics, immune-modulators, checkpoint inhibitors, antibodies targeting tumor antigens, and combinations thereof, with each possibility representing a separate embodiment of the present invention.

[0197] According to some embodiments, the antimetabolite is selected from the group consisting of cytarabine, fludarabine, fluorouracil, mercaptopurine, methotrexate, thioguanine, gemcitabine, and hydroxyurea. According to some embodiments, the mitotic inhibitor is selected from the group consisting of vincristine, vinblastine, and vinorelbine. According to some embodiments, the topoisomerase inhibitor is selected from the group consisting of topotecan and irenotecan. According to some embodiments, the alkylating agent is selected from the group consisting of busulfan, carmustine, lomustine, chlorambucil, cyclophosphamide, cisplatin, carboplatin, ifosfamide, mechlorethamine, melphalan, thiotepa, dacarbazine, and procarbazine. According to some embodiments, the antitumor antibiotic is selected from the group consisting of bleomycin, dactinomycin, daunorubicin, doxorubicin, idarubicin, mitomycin, mitoxantrone, and plicamycin. According to some embodiments, the topoisomerase II is selected from the group consisting of etoposide and teniposide.Each possibility represents a separate embodiment of the present invention.

[0198] According to some specific embodiments, the additional anticancer agent is selected from the group consisting of bevacizumab, carboplatin, cyclophosphamide, doxorubicin hydrochloride, gemcitabine hydrochloride, topotecan hydrochloride, thiotepa, and combinations thereof, each possibility representing a separate embodiment of the present invention.

[0199] The antibodies according to the invention may also be used as part of a combination therapy with at least an immunomodulatory agent, activated lymphocyte cells, a kinase inhibitor or a chemotherapeutic agent.

[0200] In some embodiments, the anti-cancer agent is an immunomodulatory agent, such as an antibody against an immune checkpoint inhibitor, whether an agonist or antagonist.

[0201] Checkpoint immunotherapeutic blockade has proven to be an exciting new arena for cancer treatment. Immune checkpoint pathways consist of a set of costimulatory and inhibitory molecules that work in concert to maintain self-tolerance and protect tissues from damage by the immune system under physiological conditions. Tumors exploit specific checkpoint pathways to evade the immune system. Thus, inhibition of such pathways has emerged as a promising anticancer therapeutic strategy.

[0202] The anti-cytotoxic T-lymphocyte 4 (CTLA-4) antibody ipilimumab (approved in 2011) was the first immunotherapy agent to show benefit in treating cancer patients. The antibody interferes with inhibitory signals during antigen presentation to T cells. The anti-programmed cell death 1 (PD-1) antibody pembrolizumab (approved in 2014) blocks the negative immunoregulatory signaling of the PD-1 receptor expressed by T cells. Additional anti-PD-1 agents were submitted for regulatory approval in 2014 for the treatment of non-small cell lung cancer (NSCLC). Currently, active research is exploring many other immune checkpoints, notably CEACAM1, NKG2A, B7-H3, B7-H4, VISTA, lymphocyte activation gene 3 (LAG3), CD137, OX40 (also called CD134), and killer cell immunoglobulin-like receptors (KIRs).

[0203] According to another embodiment, the additional anti-cancer agent is a chemotherapeutic agent, which may be administered together with the antibody according to the invention or separately, includes mitoxantrone, topoisomerase inhibitors, vinca-derived spindle poisons: vinblastine, vincristine, vinorelbine (taxol), paclitaxel, docetaxel; alkylating agents: mechlorethamine, chlorambucil, cyclophosphamide, melphalan, ifosfamide; methotrexate; 6-mercaptopurine; 5-fluorouracil, cytarabine, gemcitabine; podophyllotoxins: etoposide, irinotecan. , topotecan, dacarbazine; antibiotics: doxorubicin (adriamycin), bleomycin, mitomycin; nitrosoureas: carmustine (BCNU), lomustine, epirubicin, idarubicin, daunorubicin; inorganic ions: cisplatin, carboplatin; interferons, asparaginase; hormones: tamoxifen, leuprolide, flutamide, and megestrol acetate.

[0204] According to some embodiments, the chemotherapeutic agent is selected from alkylating agents, antimetabolites, folic acid analogs, pyrimidine analogs, purine analogs and related inhibitors, vinca alkaloids, epipodophyllotoxins, antibiotics, L-asparaginase, topoisomerase inhibitors, interferons, platinum coordination complexes, anthracenedione-substituted ureas, methylhydrazine derivatives, adrenal cortical suppressants, corticosteroids, progestins, estrogens, antiestrogens, androgens, antiandrogens, and gonadotropin-releasing hormone analogs. According to another embodiment, the chemotherapeutic agent is selected from the group consisting of 5-fluorouracil (5-FU), leucovorin (LV), irinotecan, oxaliplatin, capecitabine, paclitaxel, and docetaxel. One or more chemotherapeutic agents can be used.

[0205] According to yet another aspect, the invention provides a method of treating cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of an antibody or antibody conjugate according to the invention.

[0206] Toxicity and therapeutic efficacy of the compositions described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, by determining the IC50 (concentration producing 50% inhibition) and maximum tolerated dose for the compound of interest. The data obtained from these cell culture assays, and animal studies can be used to formulate a range of dosages for use in humans. The dosage may vary depending upon, among other relevant factors, the dosage form used, the dosing regimen selected, the composition of the agent used for treatment, and the route of administration utilized. The exact formulation, route of administration, and dosage can be selected by the individual physician in view of the patient's condition. Depending on the severity and responsiveness of the condition being treated, administration can also be a single dose of a sustained release composition, with the course of treatment continuing for several days to several weeks, or until a cure is effected or a diminution of the disease state is achieved. The amount of composition administered will, of course, depend on the subject being treated, the severity of the affliction, the means of administration, the judgment of the prescribing physician, and all other relevant factors.

[0207] The term "administering" or "administration of" a substance, compound, or agent to a subject can be performed using one of a variety of methods known to those of skill in the art. For example, the compound or agent can be administered enterally or parenterally. Enteral refers to administration via the gastrointestinal tract, including orally, sublingually, or rectally. Parenteral administration includes administration intravenously, intradermally, intramuscularly, intraperitoneally, subcutaneously, ocularly, sublingually, intranasally, by inhalation, intraspinal, intracerebral, and transdermally (e.g., by absorption through the skin duct). The compound or agent can also be suitably introduced by rechargeable or biodegradable polymeric devices or other devices, such as patches and pumps, or formulations, which provide sustained, sustained, or controlled release of the compound or agent. Administration can also be performed, for example, once, multiple times, and / or for one or more extended periods of time. In some embodiments, administration includes both direct administration, including self-administration, and indirect administration, including the act of prescribing a drug. For example, as used herein, a physician who instructs a patient to self-administer a drug or have another person administer the drug and / or provides the patient with a prescription for the drug administers the drug to a patient.

[0208] Antibodies are generally administered in the range of about 0.1 to about 50 mg / kg patient weight, generally about 0.5 to about 20 mg / kg, and often about 1 to about 10 mg / kg. In this regard, it is preferred to use antibodies that have a circulating half-life of at least 12 hours, preferably at least 4 days, more preferably up to 21 days. In some instances, it may be effective to administer a large loading dose followed by intermittent (e.g., weekly) maintenance doses over the course of the treatment. Antibodies can also be delivered by sustained release delivery systems, pumps, and other known delivery systems for continuous infusion.

[0209] The term "about" means that an acceptable range of error for a particular value, for example, up to 5% or 10%, should be assumed.

[0210] The terms "a," "an," and "the" are used interchangeably herein to mean one or more.

[0211] The term "and / or" is used to indicate that either or both of the stated cases may occur; for example, A and / or B includes (A and B) as well as (A or B).

[0212] The term "or" is used herein to indicate alternatives that may be combined where appropriate, i.e., the term "or" includes each listed alternative separately as well as combinations thereof where combinations are not mutually exclusive.

[0213] The terms "comprising", "comprise(s)", "including, include(s)", "having", "has" and "contain(s)" are used interchangeably herein and mean "consisting at least in part of". When interpreting each description herein containing the term "comprising", other features or features preceded by this term may also be present. Related terms such as "comprise" and "comprises" are to be interpreted in a similar manner. The terms "have", "has", "having" and "comprising" may also encompass and be replaced by the terms "consisting of" and "consisting essentially of". The term "consisting of" excludes any element, step, or procedure not specifically described or listed. The term "consisting essentially of" means that a composition or component may contain additional ingredients, but only if the additional ingredients do not materially alter the basic and novel characteristics of the claimed composition or method.

[0214] The following methods and examples are presented to more fully illustrate some embodiments of the invention, and should not be construed as limiting the scope of the invention in any way.

[0215] Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological, immunological, and recombinant DNA techniques. Such techniques are well known in the art. Other general references referring to well-known procedures are provided throughout this document for the convenience of the reader.

[0216] Materials and Methods ELISA assay The binding specificity and affinity of the antibody variants were determined by ELISA using recombinant mouse or human GITR (SinoBiological). ELISA plates (Nunc) were coated with the indicated recombinant GITR (1 μg / mL / well) overnight at 4 °C. All successive steps were performed at room temperature in phosphate-buffered saline PBS supplemented with 2% bovine serum albumin (BSA). After washing, the plates were blocked for 1 h and subsequently incubated with serially diluted antibody samples for 1 h. After washing, the plates were incubated with HRP-conjugated anti-mouse IgG (Jackson ImmunoResearch) for 1 h. Detection was performed with TMB-soluble reagent (3,3',5,5'-tetramethylbenzidine, Scy Tek Laboratories), and the reaction was stopped by adding 0.18 M sulfuric acid. The absorbance at 450 nm was immediately recorded using a SpectraMax Plus spectrophotometer (Molecular Devices), and background absorbance from negative control samples was subtracted.

[0217] For FcγR binding ELISA, the following modifications of the above protocol were performed: human FcγR soluble ectodomain (2 μg / mL / well) was immobilized on the plate. After washing, the plate was blocked with 10% BSA in PBS for 40 min, followed by incubation with serially diluted antibodies for 2 h.

[0218] In vivo studies in mice C57BL / 6J mice were purchased from Harlan Laboratories. FcγR humanized mice (mFcγRα - / - , Fcgr1 - / - , hFcγRI + , hFcγRIIAR 131+ , hFcγRIIB + , hFcγRIIIAF 158+、 and hFcγRIIIB +) was generated on a C57Bl / 6J background as described in Rankin CT et al. (CD32B, the human inhibitory Fc-gamma receptor IIB, as a target for monoclonal antibody therapy of B-cell lymphoma. Blood, 2006, 108, 2384-91).

[0219] To evaluate the regulation of immune cells after administration of anti-hGITR Fc region mutants, mice in which FcγR and hGITR were humanized (huFcγR / hGITR) were generated.

[0220] For pharmacokinetic assays to evaluate the effect of Fc region modifications on the in vivo half-life of antibodies, MC38 (murine colon adenocarcinoma) tumor-bearing human FcγR mice were injected with the hIgG1 Fc region variant of DTA-1 anti-murine GITR (200 μg / mouse) and bled at the indicated time points.

[0221] To assess antitumor activity (either by single challenge or by initial challenge), FcγR-humanized mice inoculated with MC38 cells were treated with a chimeric human Fc region variant based on the Fab of the anti-mGITR antibody DTA-1. Mice aged 8–10 weeks were anesthetized and implanted subcutaneously (sc) with MC38 cells (2 × 10 6 The tumor volume was measured every 2 to 3 days using an electronic caliper and calculated using the formula (L2 2 After tumor inoculation, mice were quantified for tumor size (day 0), with an average of 55 mm 3 Or 115mm 3 (unless otherwise indicated) and received intraperitoneal (ip) injections of Ab or PBS. Mice were treated with antibody variants at 100 μg / mouse, including hIgG1, as indicated, with additional antibody treatments on days 3 and 6.

[0222] For rechallenge experiments evaluating long-term immune and memory responses, mice with complete response and rejection of the primary tumor (tumor-free mice) were followed for 62 days after treatment initiation and then injected with MC38 tumor cells (2×10 6 ) were reinoculated.

[0223] For CD4 / CD8 depletion experiments, some tumor-free mice were injected with antibodies against CD4 and / or CD8 before rechallenge.

[0224] Tissue processing and flow cytometry For functional experiments, mice were challenged and treated as above and sacrificed on day 8 unless otherwise indicated. Spleens were dissected through a 70 μm nylon cell strainer, incubated with red blood cell lysis buffer (Sigma) and washed. Tumors were mechanically cut into small pieces and transferred to GentleMACS™ C tubes (Milteny) containing 0.33 mg / mL DNase (Sigma-Aldrich) and 0.27 mg / mL Liberase TL (Roche). GentleMACS™ Octo Dissociator program "m_impTumor_02_01" (Milteny) was run twice, followed by incubation at 37°C for 40 min with continuous rotation at 25 rpm. Program "m_impTumor_03_01" (Milteny) was run twice before dissociation through a 70 μm nylon cell strainer and washing. Lymph nodes were dissected through a 70 μm nylon cell strainer and washed.

[0225] Different cell populations were identified after exclusion of dead cells using the live / dead fixable blue dead cell satin kit (Invitrogen). For intracellular staining, cells were fixed and permeabilized with Foxp3 Fix / Perm buffer kit (BioLeagend). CountBright™ Absolute Counting Beads (Life Technologies) were added before acquisition. Cell populations were identified using the following markers: BioLegened Dendritic Cell (CD11b +CD11c + MHCII + F4 / 80 - ), NK cells (NKp46 + CD3 - CD8 T cells (CD3 + CD8 + CD4 - CD4 T cells (CD3 + CD4 + CD8 - ), CD4 effector T cells (CD3 + CD4 + CD8 - Foxp3 - CD44 + ), and CD4 regulatory T cells (CD3 + CD4 + CD8 - Foxp3 + ).

[0226] mass spectrometry Samples were digested with trypsin using the S-trap method, followed by HILIC enrichment of glycopeptides. The resulting peptides were analyzed using nanoflow liquid chromatography (nanoAcquity) coupled to high-resolution high-mass accuracy mass spectrometry (Fusion Lumos). The resulting data were searched against human IgG1 glycosylated peptide and human Fc glycan databases using Byonic. IDs were manually verified and quantified using Skyline (v19.1.0.193). Experiments were performed and analyzed by the INCPM unit at the Weizmann Institute of Science.

[0227] Determination of dissociation constants (KD) by surface plasmon resonance (SPR). SPR experiments were performed using a Biacore™ T200 (Cytiva) instrument. Antibodies were captured on a Protein G chip. All measurements were performed using PBS-Tween 0.05%. Antibodies at a concentration of 5 μg / mL were immobilized for 20 seconds at a flow rate of 10 μL / min. FcγRs were prepared in different concentration ranges: 0.78 nM-200 nM FcγRIIA, 2.34-600 nM FcγRIIIA and 2-500 nM FcγRIIB. Each concentration was injected for 180 seconds at a flow rate of 30 μL / min and allowed to dissociate for 340 seconds. After each cycle, the surface was regenerated with glycine buffer pH 1.5. Background binding to a blank immobilization pathway was subtracted from each binding event. Data were fitted to a two-state 1:1 binding model using the T200 Evaluation Software to obtain the K D The activation to inhibition affinity ratio for each mutant was calculated as follows: K D Activation / K D Inhibition.

[0228] statistical analysis When comparing two groups, and to compare groups in experiments assessing the proportion of cell types, an unpaired two-tailed t test was used. Data were analyzed with GraphPad Prism software (GraphPad) and a p value of <0.05 was considered statistically significant and is shown in figures. * ≦0.05, ** p ≦ 0.01 and *** p≦0.001 is indicated. Asterisks indicate statistical comparisons shown on the graphs. EXAMPLES

[0229] Reference is now made to the following examples which, together with the above descriptions, illustrate the invention in a non-limiting manner.

[0230] Example 1. Generation and characterization of anti-GITR hIgG1 Fc region mutants To generate chimeric anti-mouse GITR antibody with human IgG1 from rat anti-mouse DTA-1 clone, heavy and light chain variable regions were sequenced from DTA-1 hybridoma clone and synthesized (by BioBasic). To generate humanized anti-human GITR antibody, anti-hGITR antibody heavy chain (HC) and light chain (LC) sequences were used and synthesized (by BioBasic). The sequences were PCR amplified and cloned into mammalian expression vector with human IgG1 constant region. Figure 2 shows gel electrophoresis results of HC and LC expression vector after insertion of anti-GITR VH and VL. Figure 2 shows gel electrophoresis results of HC and LC expression vector after insertion of anti-GITR VH and VL from TRX518 (this gel is DTA-1). Lane 1 - 1 Kb molecular weight marker (Mw), lane 2 - 100 bp Mw, lane 3 - uncut expression vector, lane 4 and 5 - vector with anti-GITR VH insert, lane 6 - vector with anti-GITR VL and LC constant regions (Fc), lane 7 - HC IgG1 expression vector with constant region (Fc), lane 8 - HC IgG1 expression vector with constant region (Fc) with V11 mutation in the constant heavy chain region. Lane 9 - LC IgG1 expression vector. For the generation of Fc-modified variants of human IgG1, the site-directed mutagenesis technique was applied to generate predefined point mutations by PCR using specific primers (Agilent Technologies) listed in Table 1 according to the manufacturer's instructions.

[0231] [Table 3]

[0232] Mutant plasmid sequences were verified by direct sequencing (Life Science Core Facility, Weizmann Institute of Science). To produce antibodies, heavy and light chain expression vectors were transiently transfected into Expi293 cells (ThermoFisher). Antibodies secreted into the supernatant were purified by Protein G Sepharose 4 Fast Flow (GE Healthcare). Purified antibodies were dialyzed in PBS, sterile filtered (0.22 μm), and purity was assessed by SDS-PAGE followed by Imperial brand blue staining (ThermoFisher), demonstrating that all antibodies were expressed in the expected form. For the generation of afucosylated Fc region variants, 200 μM 2-deoxy-2-fluoro-L-fucose (Carbosynth) was added to the transfection medium as an inhibitor of fucosyltransferase and to the supernatant 1 day after transfection. Figures 3A-B show SDS-pages stained with Imperial brand blue of the expressed anti-GITR variants: lane 1 - molecular weight marker, lane 2 - anti-mGITR V11, lane 3 - afucosylated anti-mGITR, lane 4 - N279A, lane 5 - GAALIE, lane 6 - GASDALIE, lane 7 - G236A, lane 8 anti-mGITR IgG1. Figure 3A shows the results of non-reducing SDS-PAGE. Figure 3B shows the results of reducing SDS-PAGE.

[0233] To test whether the activity of anti-mouse GITR (mGITR) chimeric antibodies with human IgG1 requires interaction with hFcγR and to determine whether such interactions can be further engineered to optimize the activity of the parent antibody, the variable regions of anti-mouse GITR (DTA-1) antibody were cloned into human IgG1. Different mutations were introduced into the CH2 domain of the Fc region by site-directed mutagenesis to generate a series of mutant hIgG1 anti-mGITR antibodies. Binding affinities were measured using SPR analysis with immobilized FcγR and soluble antibodies, and the results are shown in Table 2.

[0234] [Table 4] * Magnification=K D (IgG1) / K D Fc region variants). nb=no binding.

[0235] After production, the binding affinity of the Fc region mutants to mouse GITR was measured and verified by comparative ELISA. Figure 4 shows the results of ELISA assay of the Fc region mutants to mouse GITR, including IgG1 (closed circle), IgG1-V11 (closed square), IgG1-N297A (inverted triangle), IgG1-G236A (diamond), Afuco-IgG1 (triangle), IgG-GAALIE (asterisk), IgG1-GASDALIE (open square), and Afuco-IgG1-G236A. As shown in Figure 4, the chimeric anti-mGITR (containing Fab of DTA-1) hIgG1 Fc region mutants showed similar binding affinity to the parent antibody, indicating that the Fc region modification does not impair binding to mouse GITR.

[0236] Furthermore, the binding affinity of anti-mGITR antibodies with modified human IgG1 to each FcγR was compared and evaluated using comparative ELISA. Figures 5A-5D show comparative EISA results for binding of anti-mGITR Fc region mutants: IgG1 (closed circle), IgG1-V11 (closed square), Afuco-IgG1 (triangle), IgG1-N297A (inverted triangle), IgG-GAALIE (open circle), IgG1-GASDALIE (open square), and IgG1-G236A (open diamond). Anti-mouse GITR antibodies with DTA-1 binding site and modified human IgG1 show different binding affinities to the indicated hFcγR. A human IgG1 mutant with multiple Fc region mutations G236A / S239D / A330L / I332E (referred to as "GASDALIE") mainly enhances hFcRγIIIA binding, but also binds to hFcγRIIA and hFcγRIIB. A similar mutant without the S239D mutation (G236A / A330L / I332E, termed "GAALIE") loses binding to hFcγRIIB but preserves enhanced binding to hFcRγIIIA and hFcRγIIA. The mutant "IgG1-G236A" shows selective enhancement of hFcγRIIA binding, and the "IgG1-V11" mutant (G237D / P238D / H268D / P271G / A330R, GDPDHDPGAR) selectively enhances hFcγRIIB and shows no binding to human FcγRIIIA. The aglycosylated FcγR-null mutant "IgG1-N297A" lacks the ability to engage the hFcγR pathway due to modifications at the glycosylation site. A post-translationally glycoengineered IgG1 mutant containing unmodified Fc-N297, "Afuco-IgG1," enhances binding to hFcγRIIIA.

[0237] Two non-fucosylated forms of the anti-GITR variants, IgG1 and IgG1-G236A, were produced using a fucosyltransferase inhibitor (2FF), which was added to the transfection medium of IgG1 and IgG1-G236A producing cells one day after transfection. Fucosylation was determined by mass spectrometry and the percentage of non-fucosylated forms is shown in Table 3. Antibody concentrations were measured using a NanoDrop instrument.

[0238] [Table 5]

[0239] Example 2. Pharmacokinetics of anti-mGITR IgG1 Fc region mutants The effect of Fc region modification on the in vivo half-life of the antibody was tested. MC38 tumor-bearing human FcγR mice were injected with hIgG1 Fc region variant of anti-mGITR containing Fab of DTA-1 (200 μg / mouse) and bled at the indicated time points. Serum was stored at -80°C until collection at all time points. Antibody concentration in serum was determined using a standard colorimetric ELISA assay. Briefly, assay plates were coated with recombinant mouse GITR (1 μg / mL, Sino Biological, #13643-H08H) and incubated overnight at 4°C. Plates were then blocked for 2 hours with PBS containing 10% FCS. Serial dilutions of serum were added to the plates and incubated for 2 hours. After washing, plates were incubated with horseradish peroxidase-conjugated anti-human IgG (#109-035-088, Jackson IummunoResearch) for 1 hour. The absorbance at 450 nm was immediately recorded using a SpectraMax Plus spectrophotometer (Molecular Devices) and background absorbance from negative control samples was subtracted. Figure 6 shows the ELISA results of the in vivo half-life (PK assay) of the Fc-engineered variants, IgG1 (closed circle), IgG1-V11 (closed square), IgG1-N297A (inverted triangle), IgG1-G236A (open diamond), Afuco-IgG1 (triangle), and Afuco-IgG1-G236A (open circle).

[0240] As shown in FIG. 6, the in vivo half-lives of the Fc-engineered variants are similar to that of the parent IgG1-bearing antibody, except for "IgG1-V11," which shows reduced blood levels 4 days after injection.

[0241] Example 3. In vivo antitumor activity of anti-GITR hIgG1 Fc region mutants The antitumor activity of anti-mGITR hIgG Fc region mutants was evaluated in vivo in FcγR humanized mice inoculated with MC38 (murine colon adenocarcinoma) cells and treated with the human Fc region mutant of DTA-1. - / - , Fcgr1 - / - , hFcγRI + , hFcγRIIAR 131+ , hFcγRIIB + , hFcγRIIIAF 158+、 and hFcγRIIIB + ) were generated on a C57Bl / 6J background as described in Rankin CT, et al., Blood, 2006. Eight to ten week old mice were anesthetized and injected with MC38 cells (2 × 10 6 ) was implanted subcutaneously (sc). The tumor volume was measured every 2 to 3 days using an electronic caliper and calculated using the formula (L2 2 After tumor inoculation, mice were quantified for tumor size (day 0), with an average of 55 mm 3 (unless otherwise indicated) and received intraperitoneal (ip) injections of Ab or PBS. For antitumor activity, mice were treated with 100 μg / mouse anti-mGITR (DTA-1 line) containing hIgG and received additional antibody treatments on days 3 and 6. For rechallenge experiments, tumor-free mice from all treatment groups were inoculated with MC38 tumor cells (2 × 10 6 The mice were re-challenged with 100% IgG4-immunoglobulin (IgG4-immunoglobulin) and 100% IgG4-immunoglobulin (IgG4-immunoglobulin) in the presence of ...

[0242] Tumor volume and survival were assessed for each test animal for each human Fc region variant antibody and compared between the Fc variant and IgG1-N297A groups using an unpaired two-tailed t-test. Each line represents one animal. Results were as follows: untreated mice (n=10), no tumor-free mice as expected (Figure 7A), IgG1-treated (n=8), 4 / 8 tumor-free mice (Figure 7B), IgG1-N297A-treated (n=10), 1 / 10 tumor-free mice (Figure 7C), IgG1-V11-treated (n=10), 0 / 10 tumor-free mice (Figure 7D), IgG1-G236A-treated (n=9), 6 / 9 tumor-free mice (Figure 7E), Afuco-IgG1-treated (n=10), 8 / 10 tumor-free mice (Figure 7F). The average tumor volumes of all tested animals per each Fc region variant are shown in Figure 7G for naive (untreated, black diamonds), IgG1 (black circles), IgG1-N297A (inverted triangles), IgG1-G236A (open diamonds), and Afuco-IgG1 (triangles). The survival probability over time (up to 60 days after the start of treatment) was also followed, and the results are shown in Figure 7H for untreated mice (untreated, black line), IgG1 (dashed line), IgG1-N297A (dotted line), IgG1-V11 (dashed-dotted line), IgG1-G236A (dashed-dotted line), and Afuco-IgG1 (line ending in vertical segment). As shown in Figure 7H, several treatments showed an enhanced survival probability, 80% for Afuco-IgG1, 66.6% for IgG1-G236A, and 50% for IgG1. Treatment with the Fc-engineered or glycoengineered variants IgG1-G236A and Afuco-IgG1 results in reduced tumor growth and increased overall survival compared to other Fc region variants.

[0243] To assess the ability of anti-GITR treatment to mediate long-term immune and memory responses, tumor-free mice from all treatment groups were rechallenged. Mice with complete response and rejection of the primary tumor were followed for 62 days after treatment initiation and then challenged with 2X10 6The mice were re-inoculated with MC38 tumor cells. Tumor progression and overall survival of the mice were followed, and the results are shown as follows: tumor volume over time (Figure 8A) (days after rechallenge) and survival probability (Figure 8B), calculated for each previous Fc region variant treatment (tumor-free animals). Figure 8A Naive (untreated, black diamonds, n=9), IgG1 (black circles, n=4), IgG1-N297A (inverted triangles, n=1), IgG1-G236A (white diamonds, n=6), and Afuco-IgG1 (triangles, n=7). Figure 8B Naive (untreated, black line), IgG1 (dashed line), IgG1-N297A (dotted line), IgG1-G236A (dashed-dotted line), and Afuco-IgG1 (dashed-dotted line). Although tumors developed rapidly in naive control mice, all animals previously treated with anti-GITR Ab exhibited long-term immunity, i.e., rejected tumors and fully survived rechallenge, indicating that GITR Abs may mediate long-term antitumor T cell memory protection against tumor reappearance.

[0244] To further evaluate the mechanism of long-term protection and memory response by GITR antibodies, experiments were performed in a similar setting as above, but in addition, some tumor-free mice were injected with antibodies against CD4 or CD8. MC38-bearing hFcγR mice treated with anti-GITR Fc region variants that fully responded to previous treatment and became tumor-free, and control naive mice were inoculated with MC38 cells, injected with antibodies against CD4 and CD8, and tumor growth was followed. PBS was used as a control treatment. The tumor volume of each animal tested was followed over time. Figures 9A-9D show the results for tumor growth progression in mice injected with αCD4 and αCD8 (dotted lines) and mice injected with PBS as a control (lines) in addition to treatment with anti-GITR Fc region variants. Figure 9A, control, untreated mice. Figure 9B, IgG1-G236A. Figure 9C, Afuco-IgG1. Figure 9D, Afuco-IgG1-G236A. FIG. 9E shows the mean tumor volumes of patients naive to each previous Fc region mutant treatment (n=6, black diamonds), IgG1-G236A with PBS (G236APBS, n=2, open diamonds and black line), IgG1-G236A with aCD4 and aCD8 (G236AαCD4+αCD8, n=4, open diamonds and dashed line), Afuco-IgG1 with PBS (afucosylated PBS, n=2, triangles and black line), Afuco-IgG1 with aCD4 and aCD8 (afucosylated αCD4+αCD8, n=3, triangles and dashed line), Afuco-IgG1-G236A with PBS (n=3, open squares and black line), and Afuco-IgG1-G236A with aCD4 and aCD8 (n=4, open squares and dashed line). Survival probability was calculated for each Fc region variant treatment prior to treatment as shown in Figure 9F: Naive (double dots), IgG1-G236A (line), IgG1-G236AαCD4+αCD8 (dashed line), Afuco-IgG1 (line), Afuco-IgG1 with αCD4 and αCD8 (dotted line), Afuco-IgG1-G236A (line), and Afuco-IgG1-G236A with αCD4 and αCD8 (dash-dash line). CD8+ and CD4+ T cell abundance in peripheral blood was determined by flow cytometry at various time points after antibody administration.For flow cytometric analysis, a fluorescently conjugated monoclonal antibody targeting the β subunit of mouse CD8 (clone YTS156.7.7; Biolegend) or an epitope that does not overlap with the anti-CD4 GK1.5 epitope (clone RM4-4; Thermofisher) was used to avoid competition with the depleting antibodies.

[0245] Taken together, the tumor growth and overall survival results argue for a T cell-dependent mechanism of long-term tumor immunity mediated by GITR antibodies. Tumor-free mice were rechallenged with a second injection of tumor cells and then depleted of their T cells to test the T cell dependency on tumor recurrence. T cells were found to be important for long-term immunity, as tumors grew and were not rejected in the absence of these cells.

[0246] Example 4. The afucosylated G236A mutant improves the activating / inhibitory affinity ratio of an antibody. The activating receptor FcγRIIA is 90% homologous to the inhibitory receptor FcγRIIB (Rankin CT et al., Blood, 2006, ibid.), therefore, binding to FcγRIIA and not to FcγRIIB is a difficult task. So far, several modifications have been introduced, most of which result in enhanced binding to FcγRIIB (DAE, variant 18 and GASDALIE disclosed in Smith P, et al., Mouse model recapitulating human Fcγ receptor structural and functional diversity. Proc Natl Acad Sci USA, 2012, 109, 6181-6). In the present invention, new Fc region mutants were generated by combining two strategies on the Fc scaffold: glycoengineering (afucosylation) and protein engineering by amino acid substitution (G236A) to generate afucosylated IgG1-G236A mutants. This dual engineered antibody has enhanced binding to both FcγRIIA and FcγRIIIA activating receptors and the ability to induce subsequent antibody-mediated effector functions, such as dendritic cell activation and phagocytosis, while minimizing engagement with inhibitory FcγRIIB. Figures 10A-C show the results of ELISA assays for binding of aGITRIgG1 (circles), Afuco-IgG1 (diamonds), Afuco-IgG1-G236A (triangles) and IgG1-G236A (squares) to FcγRIIA (Figure 10A), FcγRIIIA (Figure 10B) and FcγRIIB (Figure 10c).

[0247] The novel Afuco-IgG1-G236A mutant targeting GITR has improved affinity for both activating Fc receptors compared to other Abs tested, but apparently lower affinity for the inhibitory FcγRIIB when compared to hIgG1 (Figures 10A-10C).

[0248] Example 5. Requirement of FcγRIIA and FcγRIIIA for optimal anti-mGITR antibody-mediated anti-tumor immunity As both afucosylated IgG1 and IgG1-G236A resulted in similar enhancement of antitumor activity, further evaluation was performed to establish whether one of these Fc variants was superior to the other and whether they involved different mechanisms (FcγRIIA vs. FcγRIIIA pathways). Antitumor activity was compared in vivo. Humanized FcγR mice were inoculated with MC38 cells and tumors grew to 115 mm 3 Once the mean tumor volume reached 100% in the 100-mL mice, mice were treated with the indicated chimeric human Fc region variants (containing the anti-mGITR binding domain of the DTA-1 antibody), IgG1-G236A, Afuco-IgG1 and Afuco-IgG1-G236A. Tumor progression was monitored for each animal (Figures 11A-11D) as the mean tumor volume for each treatment group (Figure 11E) and as survival probability (Figure 11F). An unpaired two-tailed t-test was used to compare the Afuco-IgG1-G236A group with the IgG1 group (n=8 in all groups). The treatment groups were: untreated, no tumor-free mice (Figure 11A), IgG1-G236A-treated, 1 / 8 tumor-free mice (Figure 11B), Afuco-IgG1-treated, 0 / 8 tumor-free mice (Figure 11C), and Afuco-IgG1-G236A-treated, 2 / 8 tumor-free mice (Figure 11D). Figure 11E shows the mean tumor volumes for each treatment group, control, untreated mice (untreated, black diamonds), IgG-G236A (white diamonds), Afuco-IgG1 (triangles), and Afuco-IgG1-G236A (squares). Figure 11F shows the survival probability of mice in each of the test groups, untreated mice (untreated, lines), IgG-G236A (dashed lines), Afuco-IgG1 (dotted lines), and Afuco-IgG1-G236A (dash-dotted lines).

[0249] As shown in the results, all three Fc region variants still provide significant antitumor activity: mice treated with the Fc region variant Afuco-IgG1-G236A, which exhibits enhanced FcγRIIA and FcγRIIIA, provide a statistically significant reduction in tumor growth and complete response when compared to Afuco-IgG1, but not when compared to the IgG1-G236A variant.

[0250] When compared to IgG1 or IgG1-N297A, Fc scaffolds that are part of antibodies currently in clinical trials, the novel Afuco-IgG1-G236A shows superior antitumor activity and overall survival in advanced refractory tumor models. Humanized FcγR mice were inoculated with MC38 cells and tumors grew to an average volume of 115 mm. 3 Once the tumor reached 100% CI, mice were treated with the indicated human Fc region variants, IgG1, IgG1-N297A, or Afuco-IgG1-G236A. Mice were followed for tumor progression (Figures 12A-12D), mean tumor volume for each treatment group, and overall survival probability (Figures 12E-12F). Figure 12E shows the mean tumor volume for each treatment group, control, untreated mice (untreated, black diamonds), IgG1 (circles), IgG1-N297A (inverted triangles), and Afuco-IgG1-G236A (squares). Figure 12F shows the survival probability of mice in each of the test groups, untreated mice (untreated, line), IgG1 (dashed line), IgG1-N297A (dotted line), and Afuco-IgG1-G236A (dash-dotted line).

[0251] As demonstrated in Figures 12E and 12F, the double engineered-Fc region variant Afuco-IgG1-G236A reduces tumor growth and survival compared to other Fc region variants. An unpaired two-tailed t-test was used to compare the Afuco-IgG1-G236A group with the IgG1 group. The Fc region variant treatment groups were: untreated mice, no tumor-free mice (Figure 12A), IgG1-treated, 1 / 9 tumor-free mice (Figure 12B), IgG1-N297A-treated, 0 / 9 tumor-free mice (Figure 12C), Afuco-IgG1-G236A-treated, 5 / 9 tumor-free mice (Figure 12D).

[0252] Example 6. Impact of anti-mGITR hIgGs on the tumor microenvironment (TME) and draining lymph node (dLN) immune landscape After characterization of the Fc-engineered antibody mutants, Afuco-IgG1-G236A was found to be a remarkably efficient and suitable human IgG scaffold for GITR mAb, resulting in enhanced antitumor activity, the effect of anti-GITR Fc region mutants on the TME was evaluated. Humanized FcγR mice bearing refractory MC38 tumors were treated with 100 μg / mouse / intraperitoneal (IP) injection of anti-mGITR hIgG1 Fc region mutants, Afuco-IgG1-G236A (GA-aFuc), IgG1 Fc-silent N297A (NA), IgG1, and untreated mice were used as controls. Mice were sacrificed and tumors were harvested at three time points after treatment: day 1, day 4 (total of two mAb doses) and day 8 (total of three mAb doses). For evaluation of TME composition, single cell suspensions were generated and lymphocyte percentages were analyzed by flow cytometry. Flow cytometry results for the frequency of Treg in the TME of mice 1 day, 4 days, and 8 days after the start of anti-GITR Fc region mutant treatment are shown in Figures 13A to 13C, respectively, and CD45+CD3+CD11b-CD8-CD4+FoxP3+ of Treg was detected. Flow cytometry results for the frequency of CD8+ cells in the TME of mice 1 day, 4 days, and 8 days after the start of anti-GITR Fc region mutant treatment are shown in Figures 13D to 13F, respectively, and CD45+CD3+CD11b-CD8+CD4- of CD8+ cells was detected. CD8 / Treg ratios in the TME of mice 1 day, 4 days, and 8 days after the start of anti-GITR Fc region mutant treatment are shown in Figures 13G to 13I, respectively. 13A-13I, Naive mice (naive, diamonds), IgG1 (circles), IgG1-N297A (inverted triangles), and Afuco-IgG1-G236A (squares). An unpaired two-tailed t-test was used. Data are presented as mean + SEM.

[0253] As can be seen from Figures 13A-13I, a decrease in Treg frequency after treatment with all Fc region variants except IgG1 Fc-silent N297A (NA) is evident, indicating Fc-mediated depletion of intratumoral Tregs. A decrease in Treg frequency occurs at early time points (1 day) with the Fc-enhanced variant Afuco-IgG1-G236A (GA-aFuc), while CD8+ T cell frequency remains the same at this early time point. Nevertheless, at later time points (8 days), an increase in CD8+ T cell frequency was observed with the Fc-enhanced variants, but not with IgG1. GA-aFuc showed the highest CD8 / Treg ratio at early time points (4 days), whereas the other variants showed at later time points. CD8 / Treg ratio showed a significant increase at early time points only for GA-aFuc treatment compared to IgG1-N297A, whereas at later time points, an increase is also observed between the IgG1 Fc region variants compared to IgG1-N297A.

[0254] The DC activation status within the TME and dLN was assessed by measuring DC and DC activation markers in the TME and dLN of humanized FcγR treated with MC38 cells and humanized anti-GITR Fc region variants Afuco-IgG1-G236A (GA-aFuc), IgG1 Fc-silent N297A (NA), and IgG1. Tumors and dLN from untreated mice served as controls. Tumors and dLN were harvested 4 and 8 days after the start of mAb treatment, and single cell suspensions were then generated from the harvested organs and analyzed by flow cytometry. Figures 14A-14F are flow cytometry for detection of DCs and activated DCs in the TME (14A-14C) and dLN (14D-14F) 4 days after the start of treatment with either Afuco-IgG1-G236A (GA-aFuc, inverted triangles), IgG1 Fc-silent N297A (IgG1-N297A, triangles), and IgG1 (squares). Untreated mice served as control (untreated, circles). Figure 14A shows the percentage of DCs in the TME detected for CD45+CD11b-NK1.1-F4 / 80-CD11c+MHCII+. Figure 14B shows the geometric mean fluorescence intensity (gMFI) of activated CD80 DCs in the TME detected for CD45+CD11b-NK1.1-F4 / 80-CD11c+MHCII+. Figure 14C shows gMFI of activated CD86 DC in TME detected for CD45+CD11b-NK1.1-F4 / 80-CD11c+MHCII+. Figure 14D shows the percentage of DC in dLN detected as described in Figure 14A. Figure 14E shows the geometric mean fluorescence intensity (gMFI) of activated CD80 in dLN DC detected as described in Figure 14B. Figure 14F shows the gMFI of activated CD86 DC in dLN detected as described in Figure 14C. Figure 14G shows the percentage of DC in TME of treated mice described in Figure 14 8 days after the start of treatment. Geometric mean fluorescence intensity (gMFI) was calculated using FLOWJO software. Ordinary one-way ANOVA with Tukey's multiple comparison statistical test was used. Data are presented as mean + SEM.

[0255] As can be seen from Figures 14A-14G, the percentage of DCs in the TME decreased between days 4 and 8 after the start of treatment (Figures 14A and 14G). Compared to the DC percentage observed with the IgG1 mutant, an increase in the DC percentage was observed in the dLN 4 days after the start of treatment (Figure 14D). All Fc region mutants except IgG1 Fc-silent N297A (NA) showed an increase in the density of CD80 or CD86 activation markers in the TME and dLN (14B-14C and 14E-14F), while the Afuco-IgG1-G236A (GA-aFuc) mutant showed the highest increase for both markers.

[0256] Example 7. Impact of anti-human GITR hIgGs on the tumor microenvironment (TME) and draining lymph node (dLN) immune landscape To test whether the optimized Fc region variant (Afuco-IgG1-G236A) maintains its enhanced anti-tumor activity while targeting hGITR, the heavy and light chains of anti-hGITR humanized antibody TRX518 were synthesized and cloned into a mammalian expression vector to form an anti-hGITR humanized antibody. G236A and N297A mutations were introduced into the CH2 domain by site-directed mutagenesis to generate TRX518(N297A) and Afuco-IgG1-G236A Fc region variants of the same GITR mAb clone to enhance binding to activated hFcγR. The amino acid sequence of the heavy chain constant region of the Afuco-IgG1-G236A variant, in which alanine at position 236 is replaced by glycine, is set forth in SEQ ID NO: 13. The light chain constant region of this variant, like TRX518, follows SEQ ID NO: 14.

[0257] After production, the binding affinity of the anti-hGITR (anti-hGITR) Fc region variants was measured and validated by comparative ELISA, where OD450 values ​​were plotted against increasing concentrations of test antibody to assess binding to plate-bound proteins. Figure 15A shows the ELISA results for binding to hGITR of the humanized test antibodies, IgG1 (circles), hGITR TRX518 (TRX518(IgG1-N297A), inverted triangles), and Afuco-IgG1-G236A (squares). The binding affinity of the Fc region variants to the hFcγR members, RIIA and RIIIA, was also measured by ELISA. Figures 15B-15C show the ELISA results for binding to FcγRIIA and FcγRIIIA, respectively, of the tested antibodies, IgG1 (circles), hGITR TRX518 (TRX518(IgG1-N297A), inverted triangles), and Afuco-IgG1-G236A (squares).

[0258] The results shown in Figure 15A show that the different Fc scaffolds of anti-hGITR TRX518 preserved the binding affinity to hGITR with comparable binding affinity. The lack of binding to the two human FcγRs by TRX518 (IgG1-N297A, (triangles)) is evident, whereas "Afuco-IgG1-G236A" (squares) demonstrated increased binding to both human FcγRIIA and FcγRIIIA.

[0259] Example 8. FcγR-dependent tumor infiltrating lymphocyte (TIL) regulation after anti-GITR treatment To assess the impact of humanized anti-hGITR Fc region variants on regulating immune cells in the blood and TME, mice humanized for FcγR and GITR (hFcγR / hGITR) were generated.

[0260] Humanized FcγR / hGITR mice bearing refractory MC38 tumors were treated with anti-hGITR antibody TRX518 (IgG1-N297A) and its Fc region variant Afuco-IgG1-G236A. Blood Treg levels were assessed over time after a single dose of anti-hGITR Fc region variant. Figure 16 shows the results of blood Treg frequency after treatment with anti-hGITR antibody TRX518 (IgG1-N297A, inverted triangles), Afuco-IgG1-G236A (squares), and untreated mice as control (untreated, diamonds). The blood Treg frequency reduction occurred similarly after 22 h of treatment with both the Fc-silent IgG1-N297A mutant (triangles) and the Afuco-IgG1-G236A mutant (squares), but the Treg frequency reduction occurred at an earlier time point (2 h) after treatment with Afuco-IgG1-G236A than after treatment with IgG1 Fc-silent N297A. Four days after the start of treatment, Treg levels were restored in the blood.

[0261] Next, TME composition was evaluated, and tumors were harvested 4 days after treatment initiation (after two antibody doses), single cell suspensions were generated, and analyzed by flow cytometry for modulation of immune compartments in the TME. Figures 17A-17C show flow cytometry of the effect of different Ab variants on the percentage of CD4+FoxP3, CD8+, and Tregs in the TME, respectively. TRX518-IgG1-N297A (TRX518 (IgG1-N297A, inverted triangles), or Afuco-IgG1-G236A (squares), or untreated mice as control (untreated, diamonds). Unlike the Treg depletion in blood and in the huFcγR / GITR in vivo model described above for TRX518-IgG1-N297A, Treg depletion in the TME was exclusive to the Afuco-IgG1-G236A-treated group (Figure 17C), whereas no changes were observed in other TILs (Figures 17A-B). Anti-hGITR-mediated Treg depletion was found to be FcγR-dependent, in contrast to the Fc-silenced antibody TRX518 currently in clinical trials.

[0262] Example 9. Treg depletion by anti-GITR Afuco-IgG1-G236A mutant is NK-independent. For in vivo NK depletion assay, humanized FcγR mice were challenged with refractory MC38 tumors and treated twice (days 0 and 3) with 100 μg / mouse / injection IP, anti-mGITR hIgG1 Fc region variants, Afuco-IgG1-G236A (GA-aFuc) and IgG1 Fc-silent N297A (NA) while depleting NK cells. Humanized FcγR mice were challenged with refractory MC38 tumor cells 14 days (day -14) before the first treatment with anti-mGITR hIgG1 Fc region variants (day 0). NK cell depletion with αNK1.1 (250ug, #BE0036, BioXCell) was performed 1 and 2 days before and after the first treatment with anti-mGITR hIgG1 Fc region variants (days -2, -1, 1, and 2). Absence of NK cells was confirmed on days 0 and 4. Mice treated with PBS or PBS and αNK1.1 were used as controls. Blood was collected from test animals on days 0 and 4 after initiation of anti-mGITR hIgG1 Fc region mutant treatment and analyzed for the presence of NK cells by flow cytometry. Additionally, single cell suspensions generated from tumors harvested on day 4 after initiation of treatment were analyzed for modulation in the immune compartment in the TME by flow cytometry.

[0263] FIG. 18A shows a timeline from inoculation of mice with refractory MC38 tumor cells to day 4 from initiation of anti-mGITR hIgG1 Fc Region mutant treatment, when blood was taken and tumors harvested. FIG. 18B-FIG. 18C show flow cytometry of NK cells in blood samples from mice on days 0 and 4 from initiation of anti-mGITR hIgG1 Fc Region mutant treatment and after NK cell depletion, respectively. NK cells were detected for CD45+CD3-NKp46+, and blood from mice treated with PBS only was used as a negative control. FIG. 18D-18F show the cell counts per mg of tumor for NK cells (detected for CD45+CD3-NKp46+), Treg cells (detected for CD45+CD3+CD11b-CD8-CD4+FoxP3+), and CD8+ cells (detected for CD45+CD3+CD11b-CD8+CD4-), respectively. Mice treated with PBS (PBS, closed circles), PBS and αNK1.1 (PBS+αNK1.1, open circles), IgG1-N297A (closed squares), IgG1-N297A and αNK1.1 (IgG1-N297A+αNK1.1, open squares), Afuco-IgG1-G236A (closed triangles), Afuco-IgG1-G236A and αNK1.1 (Afuco-IgG1-G236A+αNK1.1, open triangles). An unpaired two-tailed t-test was used. Data are presented as mean + SEM.

[0264] As can be seen from Figures 18B-18C, significant depletion of NK cells was achieved in αNK1.1-injected mice as early as day 0 of the initiation of anti-mGITR hIgG1 Fc region mutant treatment compared to negative control mice injected with PBS only. Between day 0 and day 4 of the initiation of anti-mGITR hIgG1 Fc region mutant treatment, NK cell depletion was even significantly greater in αNK1.1-injected mice. As can be seen from Figures 18D-18F, the absolute number of intratumoral NK cells shows depletion in all αNK1.1-injected groups compared to PBS-injected control mice. In the afuco-IgG1-G236A (GA-aFuc)-treated groups, Treg cells were depleted in a manner independent of NK cells, whereas a much milder depletion of Tregs was observed in the IgG1 Fc-silent N297A (NA)-treated groups. CD8+ cell levels were preserved 4 days after treatment initiation in mice treated with either afuco-IgG1-G236A (GA-aFuc) or IgG1 Fc-silent N297A (NA). In vivo NK depletion assays demonstrated that Treg depletion was independent of the presence of NK.

[0265] Example 10. Generation and characterization of non-agonistic bispecific Afuco-IgG1-G236A anti-GITR antibodies To evaluate the need for bivalent GITR agonism in conjunction with enhanced FcγR targeting, nonagonistic bispecific antibodies were generated based on the Afuco-IgG1-G236A and IgG1-N297A Fc scaffolds and the Fab of Synagsis (palivizumab), a humanized anti-RSV (respiratory syncytial virus). The Afuco-IgG1-G236A Fc scaffold and Synagsis Fab were used to generate the GITR / Synagsis Afuco-IgG1-G236A bispecific antibody, and the IgG1-N297A and Synagis Fab were used to generate the GITR / NA Synagsis bispecific antibody. The novel bispecific antibodies were purified and characterized by ELISA, high performance liquid chromatography (HPLC) and mass spectrometry. For HPLC, samples of the two bispecific nonagonistic Abs were digested with trypsin using the S-trap method. The resulting peptides were analyzed using nanoflow liquid chromatography (nanoAcquity) coupled to high resolution, high mass accuracy mass spectrometry (Exploris). Each sample was analyzed separately on the instrument in random order in discovery mode. The data were processed using the Byonic search engine against IgG1 peptide and Fc glycan libraries. The data were quantified using Skyline and manually verified. Binding of the bispecific antibodies to FcγR, FcγRIIA, FcγRIIB and FcγRIIIA was measured. OD450 values ​​obtained in ELISA tests were plotted against increasing concentrations of the indicated antibodies to evaluate binding to plate-bound proteins. Monospecific afuco-IgG1-G236A was used as a control. Figures 19A-19C show ELISA results for binding of bispecific non-agonist variants, Afuco-IgG1-G236A (circles), GITR / Afuco-IgG1-G236A Syn (DTA1 / Syn GA-aFuc, triangles) and GITR / NA Syn (DTA1 / Syn, inverted triangles) to different hFcγRs, FcγRIIA, FcγRIIB and FcγRIIIA. Monospecific Afuco-IgG1-G236A (DTA1 GA-aFuc) was used as a control (circles). Figure 19D shows HPLC analysis of trypsin digested GITR / Afuco-IgG1-G236A Synagis non-agonist antibody.Data are presented as mean + SEM.

[0266] As can be seen from Figures 19A-19D, GITR / Afuco-IgG1-G236A SYNAGIS humanized bivalent (bispecific) and monovalent Afuco-IgG1-G236A (monospecific) antibodies have similar binding abilities to different hFcRs. Nevertheless, GITR / Afuco-IgG1-G236A SYNAGIS showed preferential binding of the Fc domain to activating Fc receptors. HPLC analysis revealed a protein purity of 80%. Furthermore, mass spectrometry revealed a yield of about 73.13% of the non-fucosylated form of GITR / Afuco-IgG1-G236A SYNAGIS. Bispecific anti-mouse GITR / Synagis was produced with high purity and with the desired binding properties due to both the Fab and Fc domains.

[0267] Example 11. In vitro activity assay of anti-mouse-GITR / Synagis bispecific mAb An anti-mouse GITR (mGITR) nonagonistic bispecific mAb with an N297A substitution in the Fc, termed mGITR / NA SYNAGIS, was generated. The ability of the novel mGITR / NA SYNAGIS to bind mGITR was assessed by ELISA in comparison to the monospecific mIgG1-N297A. Optical density 450 (OD450) values ​​were plotted against increasing concentrations of mGITR / NA SYNAGIS and mIgG1-N297A to assess binding to plate-bound proteins. T cells isolated from WT mice were then incubated with anti-CD3 and increasing concentrations of anti-mGITR / NA SYNAGIS or monospecific mIgG1-N297A. A non-GITR binding antibody with an N297A substitution in the Fc region served as an isotype control. After 24 hours of incubation, ELISA was performed on the supernatants to detect mouse IL-2 secreted by T cells. IL-2 ELISA was performed using Biolegened ELISA MAX™ Deluxe Set Mouse IL-2 (BLG-431004) according to the manufacturer's instructions. Data are presented as mean + SEM. Figure 20A shows the binding ability of mGITR to the novel mGITR / NA SYNAGIS (squares) compared to monospecific mIgG1-N297A (filled circles). Figure 20B shows mouse IL-2 (mIL-2) secretion (pg / mL) by T cells (activated with anti-CD3 antibody) after incubation with increasing concentrations of anti-mGITR / NA SYNAGIS (squares), monospecific mIgG1-N297A (filled circles), or a non-GITR binding antibody with a N297A substitution in the Fc region as an isotype control (isotype control NA, triangles). The dotted line represents the natural threshold of IL-2 concentration after activation with anti-CD3 and without Ab treatment.

[0268] As can be seen in Figures 20A-20B, the bispecific mGITR / NA Synergis has a lower binding capacity for mGITR compared to the monospecific mIgG1-N297A. ~3Already at an antibody concentration of 10 μg / mL, the OD450 of mGITR / NA SYNAGIS is significantly lower (0.18) compared to mIgG1-N297A (0.6). The saturation point is reached by mIgG1-N297A at about 10-1.5 μg / mL, plateauing at an OD450 value of about 0.9, while mGITR / NA SYNAGIS reaches the same OD value and plateaus at an antibody concentration of about 100 μg / mL. This difference is consistent with the valency of the antibodies, with each bispecific mGITR / NA SYNAGIS antibody being able to bind only one mGITR molecule, while each monospecific mIgG1-N297A antibody is able to bind two mGITR molecules.

[0269] mGITR / NA SYNAGIS did not induce mIL-2 secretion by anti-CD3 activated murine T cells at increasing concentrations. The levels of mIL-2 secreted after incubation of T cells with mGITR / NA SYNAGIS were even lower than the non-GITR binding isotype control antibody. The lack of ability of mGITR / NA SYNAGIS to induce IL-2 secretion by T cells indicates a lack of agonist activity. However, incubation with mIgG1-N297A reduced IL-2 secretion by approximately 10 -3 ~about 10 -1.5 Antibody concentrations between 10 μg / mL induced mIL-2 secretion by anti-CD3-activated mouse T cells in a dose-dependent manner, and the secretion level remained the same with increasing concentrations of mIgG1-N297A. -3 ~about 10 1.5 At concentrations in the μg / mL range, mIgG1-N297A demonstrated agonistic activity by binding to mGITR in a dose-dependent manner and IL-2 secretion by activated T cells. Bispecific technology enabled the generation of a non-agonistic anti-GITR.

[0270] Example 12. Anti-GITR mutant, GA-aFuc and bispecific non-agonist humanized afuco-IgG1 show in vivo anti-tumor activity. Humanized FcγR mice were challenged with refractory MC38 tumors. 14 days after challenge, mice were treated with 100 μg of mouse anti-DTA-1 (anti-GITR) GA-aFuc Ab or 100 μg, 200 μg, or 400 μg of humanized GA-aFuc Fc region variant of nonagonistic bispecific antibody (GITR / syn GA-aFuc) Ab. Untreated mice served as negative controls. 24 hours after treatment, tumors were harvested, single cell suspensions were generated, and Treg regulation in the TME was analyzed by flow cytometry. Figure 21 shows flow cytometry analysis of the percentage of Treg in the TME after treatment with 100 μg (squares), 200 μg (triangles), or 400 μg (diamonds) of GITR / syn GA-aFuc, and 100 μg of GA-aFuc (inverted triangles). Untreated mice served as control (untreated, circles). Tumors from untreated mice were analyzed similarly to negative controls. Tregs were detected by CD45+CD3+CD11b-CD8-CD4+FoxP3+. Data are presented as mean+SEM. (n=5 mice / group, one-way ANOVA).

[0271] As can be seen in Figure 21, non-agonistic and agonistic αGITR antibodies demonstrated significant ability to immunomodulate the TME by in vivo Treg depletion. The reduction in Treg percentage in the TME correlated with the concentration of the novel non-agonistic GITR / syn GA-aFuc.

[0272] Example 13. Agonistic and non-agonistic humanized afuco-IgG1 GA variants show in vivo anti-tumor activity. To further evaluate the in vivo anti-cancer immunity of GITR / syn GA-aFuc and GA-aFuc, hFcγR mice bearing refractory MC38 tumors were treated with GA-aFuc Ab or nonagonistic GITR / syn GA-aFuc Ab 14 days after tumor cell inoculation, and tumor progression (tumor volume) was monitored for 20 days. Untreated mice served as controls and were sacrificed on day 13 to prevent further animal suffering. Figures 22A-22C show the tumor volume (mm ) in hFcγR mice bearing MC38 tumors after treatment with 100 μg of GA-aFuc Ab (black triangles) or 200 μg of nonagonistic GITR / syn GA-aFuc Ab (white triangles). 3 ) progression results are shown. Untreated mice served as controls (filled circles) with n=9 per test group. Figure 22A shows tumor volume measurements on day 20. Figure 22B shows tumor volumes for mice in all groups on day 13 from the start of treatment. Figure 22C shows tumor volumes for mice in the GA-aFuc Ab and GITR / syn GA-aFuc Ab test groups on day 15 from the start of treatment. Untreated mice were sacrificed on day 13 but are not shown in this figure.

[0273] From Figures 22A-22C, it is clear that both non-agonistic aGITR Abs, GITR / syn GA-aFuc and GA-aFuc, demonstrated successful anti-tumor efficacy in vivo. However, non-agonistic GITR / syn GA-aFuc demonstrated significantly greater cancer immunity compared to the agonistic aGITR antibody GA-aFuc. Thus, non-agonistic aGITR variants demonstrated increased ability to suppress tumor growth in vivo compared to the corresponding Fc agonistic aGITR mAb.

[0274] Example 14. In vivo dendritic cell activation by agonistic and non-agonistic humanized afuco-IgG1 GA variants Humanized FcγR mice were inoculated with refractory MC38 tumors. Mice were treated twice with 100 μg GA-aFuc Ab or 200 μg GITR / syn GA-aFuc Ab, 0 and 3 days after treatment initiation. Untreated mice served as negative controls. Tumors were harvested 24 hours after the second treatment, single cell suspensions were generated, and the TME was analyzed by flow cytometry for Treg depletion and dendritic cell (DC) activation. Figures 23A-23B show flow cytometry analysis of Treg and DC fractions in the TME after treatment with 200 μg GITR / syn GA-aFuc (GITR / syn Afuco-IgG1-G236A inverted open triangles) and 100 μg GA-aFuc (Afuco-IgG1-G236A inverted black triangles), respectively. Tumors from untreated mice were analyzed similarly to the negative controls (circles). Tregs were detected by CD45+CD3+CD11b-CD8-CD4+FoxP3+ and DCs by CD45+CD11b-NK1.1-F4 / 80-CD11c+MHCII+. Data are shown as mean+SEM. (n=5 mice / group, one-way ANOVA). Figures 23C-23D show flow cytometric analysis of DC activation by detecting the percentage of CD80+ or ​​CD86+ expressing DCs, respectively. GITR / syn Afuco-IgG1-G236A (inverted open triangles), Afuco-IgG1-G236A inverted filled triangles, tumors from untreated mice, as well as negative controls (circles) were analyzed. Geometric mean fluorescence intensity (gMFI) was calculated using FLOWJO software. DCs were detected by CD45+CD11b-NK1.1-F4 / 80-CD11c+MHCII+, CD80+DCs were detected by addition of anti-CD80, and CD86+DCs were detected by addition of anti-CD86. Data are presented as mean ± SEM, and each dot represents an individual mouse (n=5 mice / group, one-way ANOVA).

[0275] 23A-23B show that Tregs were significantly depleted after treatment with either aGITR Ab, GITR / syn GA-aFuc, or GA-aFuc, whereas DC levels were stable, compared to control mice, indicating that the agonistic and / or non-agonistic activity of aGITR Ab does not affect or modulate the levels of DCs.

[0276] Figures 23C-23D show that treatment with either GITR / syn GA-aFuc or GA-aFuc Ab significantly increased CD80+DC. Treatment with GITR / syn GA-aFuc Ab significantly increased the level of CD86+DC, and there was less increase in CD86+DC after treatment with GA-aFuc Ab. Fc region-mediated DC activation was demonstrated by both GITR / syn GA-aFuc or GA-aFuc Ab.

[0277] Example 15. In vivo anticancer activity of murine αGITR-mIgG2a mutants in wild-type mice. The anti-tumor activity of anti-GITR-mIgG2a Abs with preferential binding to activating mouse receptors FcγRI and FcγRIV was evaluated. Wild-type (WT) C57BL / 6J mice bearing MC38 tumors were treated 8 days after tumor cell inoculation with 100 μg of mouse anti-GITR (DTA-1) Ab (DTA-1 mIgG2a) or 100 μg of mIgG2a-N297A Ab (DTA-1 mIgG2a NA), the corresponding mouse Fc with enhanced activating Fc receptors. Tumor progression was monitored by measuring tumor volume for 17 days. Untreated mice were used as controls.

[0278] Figure 24A shows the results of monitoring tumor volume progression over 17 days in WT mice bearing MC38 tumors after treatment with 100 μg DTA-1 mIgG2a (squares) or 100 μg DTA-1 mIgG2a NAAb (triangles). Untreated mice served as controls (filled circles). Data are presented as mean ± SEM, n=9 mice / group.

[0279] As can be seen in FIG. 24A, mice treated with 100 μg of Fc-bound DTA-1 mIgG2a Ab showed a statistically significant reduction in tumor growth and a complete response when compared to mIgG2a-N297A.

[0280] MC38 tumor-bearing WT mice were then treated with 100 μg DTA-1 mIgG2a Ab and 100 μg DTA-1 mIgG2a NA, with untreated mice serving as negative controls. Four days after treatment, tumors were harvested, single cell suspensions were generated, and modulation in the immune compartment in the TME was analyzed by flow cytometry. Immunomodulatory parameters assayed were Treg numbers / mg tumor (detected using anti-FoxP3) and DC percentage from total lymphocytes (detected using anti-CD11c / anti-CD45), DC activation status (detected using anti-CD80 or anti-CD86). Figures 24B-24E show flow cytometry analysis of the above mentioned parameters in harvested tumors of WT mice treated with 100 μg DTA-1 mIgG2a Ab (mIgG2a, squares) and 100 μg DTA-1 mIgG2a NA (mIgG2a-N297A, triangles) or untreated mice (PBS, circles) used as control. Each dot represents an individual mouse n=6 mice / group and data are presented as mean ± SEM and analyzed using one-way ANOVA. Delta gMFI was calculated using FLOWJO software. Unpaired t-tests were used. Figure 24B shows the number of Tregs / mg tumor detected using anti-FoxP3 in all test groups. Figure 24C shows the percentage of DCs of total lymphocytes (detected using anti-CD11c / anti-CD45) in all test groups. Figure 24D shows the delta gMFI of CD80+ activated DCs in all test groups. FIG. 24E shows the delta gMFI of CD86+ activated DCs in all test groups.

[0281] As can be seen from Figures 24B-24E, both DTA-1 mIgG2a and DTA-1 mIgG2a NA induced a pronounced and significant Treg depletion in vivo compared to untreated mice. Furthermore, DTA-1 mIgG2a Ab induced a significantly greater Treg depletion compared to the Fc-null antibody mIgG2a NA, indicating that Ab specificity and Fc interactions contribute to Ab immunomodulatory capacity. In vivo treatment with DTA-1 mIgG2a Ab modulated TME immune cell populations and significantly increased DC percentage compared to DTA-1 mIgG2a NA, suggesting that Fc interactions are important for increasing DC in the TME. Furthermore, in vivo treatment with DTA-1 mIgG2a Ab significantly increased DC activation as reflected by the number of CD80+ and CD86+ activated DC in the TME compared to both untreated mice and DTA-1 mIgG2a NA. Notably, in the case of CD80+ activated DCs, the increase in this DC population with DTA-1 mIgG2a Ab compared to DTA-1 mIgG2a NA was even greater than the increase compared to untreated mice. In summary, DTA-1 mIgG2a was shown to enhance antitumor responses, enhance Treg depletion, increase DC percentage in the TME, and elevate DC activation markers CD86 and CD80 compared to Fc null antibody.

[0282] This Fc-binding antibody, DTA-1mIgG2a, was further evaluated in various DC knockout (KO) mouse models to determine the dependence of the anti-GITR anti-tumor effect on the presence of DC.

[0283] Example 16. In vivo anti-cancer activity of murine αGITR-mIgG2a mutants in the BATF3- / - mouse model cDC1 cells are a subset of DCs that are highly efficient at presenting cell-associated antigens in association with class I MHC molecules (MHC-I). This property of cDC1 cells is particularly evident in the CD8+ / CD9+ expression of CD8+ / CD9+ cells against infected or tumor cells. + Contributing to their key role in T cell priming. Only the DC subset of cDC1 cells, bearing MC38 tumors, lacks BATF3- / - Mice were treated with DTA-1 mIgG2a Ab or DTA-1 mIgG2a-NA Ab 9 days after inoculation. Tumor volume ( mm3 ) progression was monitored for 13 days. Untreated mice were used as negative controls and were sacrificed 10 days after treatment to avoid unnecessary suffering of the animals. Figure 25 shows the progression of BATF3 mice bearing MC38 tumors treated with 100 μg of DTA-1 mIgG2a (mIgG2a, squares) Ab and 100 μg of DTA-1 mIgG2a-NA Ab (mIgG2a NA triangles). - / - Tumor volume (mm) over 13 days in mice or untreated mice (PBS, circles) used as control 3 ) Progression results are shown. Data are presented as mean ± SEM, n=8, unpaired t-test.

[0284] Figure 25 shows that tumor progression in all groups is essentially the same for the first 6 days after treatment. From day 6 to day 9 after treatment, the tumor growth rate of mice treated with Fc-binding Ab, DTA-1 mIgG2a, begins to decrease, while the tumor growth rate of the other groups remains essentially the same. From day 10 after treatment, mice treated with DTA-1 mIgG2a Ab showed a statistically significant decrease in tumor growth compared to mIgG2a-N297A and untreated mice. Without wishing to be bound by any theory or mechanism of action, the results indicate that the lack of cDC1 cells prevents the efficacy of Fc-null aGITR Ab, DTA-1 mIgG2a-NA Ab, rendering it ineffective, likely due to defective T cell priming and subsequent lack of agonistic effect. The delayed effect of DTA-1 mIgG2a Ab (detected only from day 10 after treatment) is due to preserved Treg depletion activity, but likely due to lack of agonism on effector cells.

[0285] Example 17. In vivo anti-cancer activity of murine αGITR-mIgG2a mutants in XCR1-iDTR and ZBTB46-iDTR mouse models XCR1-iDTR and ZBTB46-iDTR mouse models are models in which DCs can be transiently depleted via the DT receptor (DTR) upon injection of diphtheria toxin (DT). XCR1-iDTR is an inducible cDC1 cell depletion model, and ZBTB46-iDTR is an inducible cDC1 and cDC2 cell depletion model. cDC2 cells are particularly efficient in priming CD4+ T cells and cDC1 cells, and promote CD8+ T cell priming, as previously described. XCR1-iDTR and ZBTB46-iDTR mice bearing MC38 tumors were treated with 100 μg DTA-1 mIgG2a Ab, 20 ng / g body weight DT, or a combination of 100 μg DTA-1 mIgG2a Ab and DT 20 ng / g body weight. DT was injected at 20 ng / g every other day to deplete DCs. Tumor volume progression was monitored for 13 days (XCR1-iDTR mice) or 20 days (ZBTB46-iDTR mice). Figures 26A-26B show the progression of tumor volume (mm) in XCR1-iDTR and ZBTB46-iDTR mice treated with 100 μg DTA-1 mIgG2a Ab (mIgG2a, closed squares), 20 ng / g body weight of DT (DTx, circles), or a combination of 100 μg DTA-1 mIgG2a Ab and 20 ng / g body weight of DT (mIgG2a+DTx, open squares), respectively. 3 ) Progression results are shown. Untreated mice served as controls (untreated, circles), n = 5–10 mice / group for XCR1-iDTR assay and n = 8 mice / group for ZBTB46-iDTR assay. Data are presented as mean ± SEM, unpaired t-test.

[0286] As seen in Figure 26A, a similar trend to the BATF3- / - mouse model assay was observed in the XCR1-iDTR mouse model, with a delayed effect of DTA-1 mIgG2a Ab on reducing tumor growth in mice treated with DTA-1 mIgG2a Ab compared to mice treated with a combination of DTA-1 mIgG2a Ab and DT. In the XCR1-iDTR assay, the effect of the mIgG2a antibody was abolished upon DT administration, further suggesting that DCs, particularly cDC1s, are required for mediating successful anti-tumor activity of Fc-bound DTA-1 antibodies.

[0287] As seen in Figure 26B, in the ZBTB46-iDTR model, in which both cDC1 and cDC2 cell populations were depleted, the efficacy of Fc-binding DTA-1 mIgG2a Ab disappeared upon cDC depletion with DT. However, treatment with DTA-1 mIgG2a Ab alone showed a marked and significant lower progression of tumor volume compared to treatment with the combination of DTA-1 mIgG2a Ab and DT. Treatment with DTA-1 mIgG2a Ab alone stopped tumor growth from the early stage at day 3 after treatment.

[0288] Without wishing to be bound by any theory or mechanism of action, the Fc-engaged DTA-1 antibody requires engagement with DCs to mediate the enhanced antitumor effect observed. In the absence of these cells in the system, the antibody loses efficacy and even has a neutralized effect relative to that of the untreated population, possibly due to deleterious binding to other effector cells in the environment.

[0289] Example 18. In vivo aGITR effect on Treg depletion-mediated DC activation The in vivo effect of anti-GITR Ab on Treg depletion-mediated DC activation was compared with that of anti-CD25, a Treg depleting antibody known in the art. For that purpose, tumors and dLNs from mice C57BL / 6J with refractory MC38 treated with 100 μg of anti-mouse GITR mIgG2a or 100 μg of anti-CD25 were harvested 4 days after treatment. Single cell suspensions were generated from harvested organs and Treg regulation in immune compartments in the TME and dLNs or DC activation in the dLNs were analyzed by flow cytometry. Untreated mice served as control. Figures 27A-27B show the Treg percentage in the TME and DC percentage in the dLNs, respectively, from mice treated with 100 μg of mouse anti-GITR Ab (aGITR(DTA-1), squares), 100 μg of anti-CD25 Ab (aCD25, triangles), or untreated mice (untreated, circles). Tregs were detected by CD45+CD3+CD11b-CD8-CD4+FoxP3+, and DCs were detected by CD45+CD11b-NK1.1-F4 / 80-CD11c+MHCII+. Each dot represents an individual mouse, n=5 / 6 mice / group. Data are shown as mean±SEM and analyzed using one-way ANOVA. Figures 27C-27D show CD80+ and CD86+ activated DCs, respectively, in dLNs from mice treated with 100 μg mouse anti-GITR Ab (aGITR(DTA-1), squares), 100 μg mouse anti-CD25 Ab (aCD25, triangles), or untreated mice (untreated, circles). Activated CD80+DCs were detected for CD45+CD11b-NK1.1-F4 / 80-CD11c+MHCII+CD80+, and activated CD86 DCs were detected for CD45+CD11b-NK1.1-F4 / 80-CD11c+MHCII+CD86+. Each dot represents an individual mouse, n=5 / 6 mice / group. Delta gMFI was calculated using FLOWJO software. Data are shown as mean ± SEM and analyzed using one-way ANOVA.

[0290] Figures 27A-27B show significant and almost complete depletion of Tregs in the TME in both anti-GITR AB and anti-CD25 treated mice compared to untreated mice. Anti-GITR AB showed a significant increase in the proportion of DCs in the dLN compared to anti-CD25 and untreated mice. The proportion of DCs in untreated and anti-CD25 treated mice was similar.

[0291] 27C-27D show that anti-GITR Ab induced significant activation of CD80+ compared to untreated mice and a marked increase compared to anti-CD25. Furthermore, anti-GITR Ab induced significant activation of CD86+ DC compared to anti-CD25 and an even greater increase compared to untreated mice.

[0292] Although anti-GITR and anti-CD25 treatment mediated almost complete depletion of Tregs, only anti-GITR increased DCs and induced their activation, indicating that in vivo Treg depletion-mediated DC activation is GITR dependent.

[0293] Example 19. Requirement of FcγRIIA and FcγRIIIA for optimal anti-mGITR antibody-mediated anti-tumor immunity Humanized FcγR mice were inoculated with MC38 cells, and tumors grew to 55 mm 3Once the mean tumor volume of 100 μg / mouse was reached, the mice were treated with the chimeric human Fc region variants. Tumor-bearing mice were treated with IgG1-N297A or IgG1-GAALIE every 3 days for a total of 3 treatments (100 μg / mouse of each treatment). Tumor progression was monitored for each animal as the mean tumor volume for each treatment group. Figures 28A-28C show the progression of tumor volume in mice treated with IgG1-N297A (N297A) or IgG1-GAALIE (GAALIE), with untreated mice used as controls (untreated). Each line represents one animal. Figure 28D shows the results of the mean tumor volume for each treatment group IgG1-N297A (N297A, n=8), IgG1-GAALIE (GAALIE, n=9), control, untreated mice (untreated n=8). An unpaired two-tailed t-test was used to compare the IgG1-N297A group with the IgG1-GAALIE group. Data are presented as mean + SEM.

[0294] As can be seen from Figures 28A-28D, by the 8th day after treatment, there was a clear reduction in tumor volume in mice treated with IgG1-GAALIE, while mice treated with IgG1-N297A show an increase in tumor volume. As demonstrated in Example 5, GAALIE shows enhanced FcγRIIa and FcγRIIIa, which reflects a statistically significant reduction in tumor growth compared to IgG1-N297A herein. IgG1-GAALIE shows superior antitumor activity.

[0295] Example 20. In vivo comparison of antitumor immunity of aGITR mutants in MC38 and B16F10 cancer-bearing mice hFcγR mice inoculated with MC38 or B16F10 cells are treated with human Fc region variants of DTA-1 (anti-mouse GITR): IgG1-N297A, GAALIE, IgG1 and GA-aFuc. Tumor volume progression and overall survival of mice are monitored. Tumor immune profiles after treatment are then mapped using single-cell multi-omics, which allows parallel single-cell genome and transcriptome co-amplification and multi-level analysis based on single-cell data.

[0296] Example 21. Creation of an animal model for evaluation of anti-hGITR binding and activity Humanized GITR / FcγR, GITR-Tg mice are generated using two different approaches, a genetic engineering approach and a more traditional breeding approach. For the genetic engineering approach, human bacterial artificial chromosome clones (BACs) containing the GITR gene and its associated gene expression genomic regulatory elements are evaluated. For the breeding approach, the humanized FcγR mice described above are crossed with hGITR knock-in mice, in which mouse GITR is replaced with human sequences. Anti-hGITR binding and activity are evaluated in vivo, for example, using two commercially available humanized GITR mice, Biocytogen and GenOway.

[0297] Example 22. In vitro anti-hGITR-mediated peripheral blood mononuclear cell (PBMC) activation To test the activation potential of anti-human GITR mAb, fresh or frozen human PBMC from donors are evenly distributed into tubes and incubated with anti-hGITR Ab and anti-human CD3. The activation status of PBMC is assessed by T cell proliferation (CellTraceViolet dye) and expression of CD25, CD44 by flow cytometry. The presence of IL-2 is detected using ELISA.

Claims

1. An isolated afucosylated antibody that specifically binds to glucocorticoid-inducible TNFR-related (GITR) proteins, wherein the antibody comprises a variable region (Fab) and a modified human IgG1 constant region (Fc), The modified human IgG1 includes a substitution of the asparagine residue (N) at position 297 and the amino acid residue glycine (G) at position 236 for an alanine residue (A), G236A (referred to herein as afuco-G236A), or The modified human IgG1 includes amino acid substitutions (GAALIE as specified herein) of the substitution of the glycine residue at position 236 to an alanine residue (G236A), the alanine residue at position 330 to a leucine residue (A330L), and the isoleucine residue at position 332 to a glutamic acid residue (I332E). Isolated afucosylated antibody.

2. The antibody according to claim 1, wherein the GITR is human GITR (hGITR).

3. The Fab of the antibody comprises a set of six CDR sequences, and the set comprises i. Heavy chain (HC) CDR1 containing the sequence GFSLSTSGMG (SEQ ID NO: 1), HC CDR2 containing the sequence IWWDDDK (SEQ ID NO: 2), HC CDR3 containing the sequence ARTRRYFPFAY (SEQ ID NO: 3), light chain (LC) CDR1 containing the sequence QNVGTN (SEQ ID NO: 4), LC CDR2 containing the sequence SAS or SAST (SEQ ID NO: 5), LC CDR3 containing the sequence QQYNTDPLT (SEQ ID NO: 6); ii. HC CDR1 containing the sequence SYGMH (SEQ ID NO: 7), HC CDR2 containing the sequence VIWYEGSNKYYADSVKG (SEQ ID NO: 8), HC CDR3 containing the sequence GGSMVRGDYYYGMDV (SEQ ID NO: 9), LC CDR1 containing the sequence RASQGISSALA (SEQ ID NO: 10), LC CDR2 containing the sequence DASSLES (SEQ ID NO: 11), and LC CDR3 containing the sequence QQFNSYPYT (SEQ ID NO: 12); and iii. The antibody according to claim 2, selected from the group consisting of HC CDR1 containing the sequence GYTFTRYW (SEQ ID NO: 25), HC CDR2 containing the sequence IYPGDGDT (SEQ ID NO: 26), HC CDR3 containing the sequence ARNPLTTATAWFVY (SEQ ID NO: 27), LC CDR1 containing the sequence ENIYSN (SEQ ID NO: 28), LC CDR2 containing the sequence AAT, and LC CDR3 containing the sequence QHFWGPPWT (SEQ ID NO: 29).

4. A heavy chain sequence described in SEQ ID NO: 13, a light chain sequence described in SEQ ID NO: 14, or both thereof, The heavy chain sequence described in SEQ ID NO: 15, the light chain sequence described in SEQ ID NO: 14, or both, The antibody according to claim 1.

5. The antibody according to claim 1, wherein the antibody is a chimeric antibody or a humanized antibody.

6. A conjugate comprising the antibody described in claim 1.

7. A polynucleotide sequence encoding at least one chain of an antibody according to any one of claims 1 to 5 or an antibody conjugate according to claim 6, wherein the polynucleotide sequence preferably comprises a sequence selected from SEQ ID NOs: 22 and SEQ ID NOs: 23 encoding an antibody heavy chain, or a variant thereof having at least 80% sequence identity with the polynucleotide sequence.

8. A vector, plasmid, or construct comprising at least one polynucleotide sequence as described in claim 7, or a host cell comprising the vector, plasmid, or construct.

9. A vector, plasmid, or construct according to claim 8, comprising: a polynucleotide sequence selected from SEQ ID NOs. 22 and SEQ ID NOs. 23 encoding an antibody heavy chain; or a variant thereof having at least 80% sequence identity; and the polynucleotide sequence of SEQ ID NOs. 24 or a variant thereof having at least 80% sequence identity.

10. A composition comprising a plurality of antibodies according to Claim 1, wherein the Fc region comprises a substitution G236A where the asparagine residue (N) at position 297 and the amino acid residue glycine (G) at position 236 are replaced with an alanine residue (A), and about 65 to 100% of the antibodies in the composition comprises a mature core carbohydrate structure lacking fucose bound to the asparagine residue (N) at position 297 of the Fc region.

11. A pharmaceutical composition comprising, as an active ingredient, at least one antibody as described in claim 1, and at least one carrier, excipient, or diluent.

12. The pharmaceutical composition according to claim 11 for use in enhancing the immunocostimulatory activity of hGITR, enhancing the production of pro-inflammatory cytokines, depleting or inhibiting regulatory T cells (Treg), or activating dendritic cells.

13. A pharmaceutical composition according to claim 11 for use in the treatment of cancer or tumor, wherein the treatment optionally comprises administering or performing at least one additional anticancer therapy.

14. A pharmaceutical composition for use according to claim 12 or 13, wherein the treatment results in delaying, slowing or preventing tumor growth or recurrence, enhancing antitumor effects, modifying the tumor microenvironment, inducing long-term antitumor immunity, or preventing or reducing the formation, growth or spread of metastases.

15. A kit comprising an antibody according to any one of claims 1 to 5, a conjugate according to claim 6, or a composition according to claim 10, the kit being packaged in a packaging material and identified by printing in or on the packaging material.

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