Anti-gitr antibodies and uses thereof

Abstract

This article provides antibodies that specifically bind to glucocorticoid-induced tumor necrosis factor receptor (GITR) and antigen-binding fragments thereof, compositions comprising said antibodies or antigen-binding fragments thereof, and methods of using said antibodies or antigen-binding fragments thereof, including, for example, therapeutic methods using said antibodies or antigen-binding fragments thereof.

Description

Technical Field

[0001] This disclosure relates to antibodies that specifically bind to glucocorticoid-induced tumor necrosis factor receptor (GITR), their antigen-binding fragments, and methods of using them.

[0002] sequence list

[0003] An official copy of the sequence list, in ASCII format, was submitted electronically via EFS-Web along with the instruction manual. The file name is 10671WO01_Sequence_Listing_ST25.TXT, created on March 5, 2021, and is approximately 60 kilobytes in size. The sequence list contained in this ASCII format document is part of the instruction manual and is incorporated herein by reference in its entirety. Background Technology

[0004] Glucocorticoid-induced tumor necrosis factor receptor (GITR) is a member of the tumor necrosis factor receptor superfamily (TNFRSF). GITR expression is constitutively high on regulatory T cells, and low / moderate on naive T cells, NK cells, and granulocytes, and can be induced upon activation. GITR interacts with its ligand GITRL, which is primarily expressed on antigen-presenting cells. GITR receptor activation can both enhance the proliferation and function of effector T cells and attenuate the repression induced by regulatory T cells. Therefore, the regulation of GITR activity can serve as a basis for cancer immunotherapy and immune disorders. Consequently, agents that modulate GITR activity, such as antibodies, are needed. Summary of the Invention

[0005] This disclosure provides antibodies that bind to the glucocorticoid-induced tumor necrosis factor receptor (GITR) and their antigen-binding fragments. The antibodies provided herein are particularly useful for targeting GITR-expressing immune cells, such as effector T cells, regulatory T cells, and natural killer (NK) cells. The antibodies are especially useful because in vivo truncation is minimized.

[0006] The antibodies provided in this article may be full-length (e.g., IgG1 or IgG4 antibodies) or may contain only the antigen-binding portion (e.g., Fab, F(ab')2 or scFv fragments), and may be modified to affect function, such as eliminating residual effector functions (Reddy et al., 2000, J. Immunol. 164: 1925-1933).

[0007] The exemplary anti-GITR antibodies provided herein are listed in Tables 7, 8, and 9. Table 7 lists the amino acid sequence identifiers of the heavy chain variable region (HCVR), light chain variable region (LCVR), heavy chain complementarity-determining region (HCDR1, HCDR2, and HCDR3), and light chain complementarity-determining region (LCDR1, LCDR2, and LCDR3) of the exemplary anti-GITR antibodies. Table 8 lists the nucleic acid sequence identifiers of the HCVR, LCVR, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the exemplary anti-GITR antibodies. Table 9 provides the sequence identifiers of the full-length heavy and light chain sequences of the exemplary anti-GITR antibodies.

[0008] This disclosure provides antibodies or antigen-binding fragments thereof that specifically bind to GITR, wherein the antibodies or antigen-binding fragments thereof comprise three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) within the heavy chain variable region (HCVR) amino acid sequence selected from the group consisting of SEQ ID NO: 22, 28, 34, and 40; and three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) within the light chain variable region (LCVR) amino acid sequence of SEQ ID NO: 10.

[0009] This disclosure provides antibodies or antigen-binding fragments thereof that specifically bind to GITR, wherein the antibodies or antigen-binding fragments thereof comprise an HCVR amino acid sequence selected from the group consisting of SEQ ID NO:22, 28, 34 and 40; and an LCVR amino acid sequence of SEQ ID NO:10.

[0010] This disclosure provides antibodies that specifically bind to GITR or antigen-binding fragments thereof, wherein the antibodies comprise a heavy chain amino acid sequence selected from the group consisting of SEQ ID NO:26, 32, 38 and 44; and a light chain amino acid sequence of SEQ ID NO:20.

[0011] This disclosure provides antibodies or antigen-binding fragments thereof that specifically bind to GITR, wherein the antibodies or antigen-binding fragments thereof comprise three heavy chain CDRs within the HCVR amino acid sequence of SEQ ID NO:2 modified with N101D, N101E, N101S or N101T mutations; and three light chain CDRs within the LCVR amino acid sequence of SEQ ID NO:10.

[0012] This disclosure provides antibodies or antigen-binding fragments thereof that specifically bind to GITR, wherein the antibodies or antigen-binding fragments thereof comprise three heavy chain CDRs within the HCVR amino acid sequence of SEQ ID NO:2 modified with mutations of N101A, N101F, N101G, N101H, N101I, N101K, N101L, N101M, N101P, N101Q, N101R, N101V, N101W, or N101Y; and three light chain CDRs within the LCVR amino acid sequence of SEQ ID NO:10.

[0013] This disclosure provides antibodies or antigen-binding fragments thereof that specifically bind to GITR, wherein the antibodies or antigen-binding fragments thereof comprise three heavy chain CDRs within the HCVR amino acid sequence of SEQ ID NO:2 modified with mutations of S103A, S103D, S103E, S103F, S103G, S103H, S103I, S103K, S103L, S103M, S103N, S103P, S103Q, S103R, S103T, S103V, S103W, or S103Y; and three light chain CDRs within the LCVR amino acid sequence of SEQ ID NO:10.

[0014] Antibodies provided in Table 1 of this paper and disclosed in U.S. 2017 / 0355774A1 have been explicitly excluded, as they lack N101 or S103 modification or mutation in HCVR.

[0015] This disclosure provides antibodies or antigen-binding fragments thereof that specifically bind to GITR, the antibodies or antigen-binding fragments comprising HCVR and LCVR, wherein the HCVR comprises an HCDR3 amino acid sequence selected from any of the HCDR3 amino acid sequences listed in Table 7. The HCVR may also comprise an amino acid sequence selected from any of the HCVR amino acid sequences listed in Table 7, or a substantially similar sequence to them having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. The LCVR may comprise an LCVR amino acid sequence listed in Table 7, or a substantially similar sequence to them having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0016] This disclosure also provides antibodies or antigen-binding fragments thereof that specifically bind to GITR, said antibodies or antigen-binding fragments thereof comprising HCVR and LCVR amino acid sequence pairs (HCVR / LCVR), said HCVR and LCVR amino acid sequence pairs comprising HCVR amino acid sequences listed in Table 7 that pair with LCVR amino acid sequences listed in Table 7. According to certain embodiments, this disclosure provides antibodies or antigen-binding fragments thereof comprising the HCVR / LCVR amino acid sequence pairs contained in any of the exemplary anti-GITR antibodies listed in Table 7. In some embodiments, said HCVR / LCVR amino acid sequence pairs are selected from the group consisting of: SEQ ID NO: 22 / 10, 28 / 10, 34 / 10, and 40 / 10.

[0017] This disclosure also provides antibodies or antigen-binding fragments thereof that specifically bind to GITR, said antibodies or antigen-binding fragments comprising: a heavy chain CDR3 (HCDR3) comprising an amino acid sequence selected from any of the HCDR3 amino acid sequences listed in Table 7.

[0018] This disclosure also provides antibodies or antigen-binding fragments thereof that specifically bind to GITR, said antibodies or antigen-binding fragments thereof comprising an HCDR3 and LCDR3 amino acid sequence pair (HCDR3 / LCDR3), said HCDR3 and LCDR3 amino acid sequence pair comprising any of the HCDR3 amino acid sequences listed in Table 7 that pair with the LCDR3 amino acid sequences provided in Table 7. According to certain embodiments, this disclosure provides antibodies or antigen-binding fragments thereof comprising the HCDR3 / LCDR3 amino acid sequence pair contained in any of the exemplary anti-GITR antibodies listed in Table 7. In one embodiment, said HCDR3 / LCDR3 amino acid sequence pair is selected from the group consisting of: SEQ ID NO:24 / 16, 30 / 16, 36 / 16, and 42 / 16.

[0019] This disclosure also provides antibodies or antigen-binding fragments thereof that specifically bind to GITR, said antibodies or antigen-binding fragments comprising a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained in any of the exemplary anti-GITR antibodies listed in Table 7. In some embodiments, the amino acid sequence set of said HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 is selected from the group consisting of: SEQ ID NO: 4-6-24-12-14-16, 4-6-30-12-14-16, 4-6-36-12-14-16, and 4-6-42-12-14-16.

[0020] In one related embodiment, this disclosure provides an antibody or antigen-binding fragment thereof that specifically binds to GITR, said antibody or antigen-binding fragment comprising a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained in the HCVR / LCVR amino acid sequence pairs defined by any of the exemplary anti-GITR antibodies listed in Table 7. For example, this disclosure includes an antibody or antigen-binding fragment thereof that specifically binds to GITR, said antibody or antigen-binding fragment comprising the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence set contained in the HCVR / LCVR amino acid sequence pairs selected from the group consisting of SEQ ID NO: 24 / 16, 30 / 16, 36 / 16, and 42 / 16.

[0021] This disclosure also provides nucleic acid molecules encoding anti-GITR antibodies or portions thereof. For example, this disclosure provides nucleic acid molecules encoding any of the HCVR amino acid sequences listed in Table 7; in some embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCVR nucleic acid sequences listed in Table 8, or a substantially similar sequence to them having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0022] This disclosure also provides nucleic acid molecules encoding the LCVR amino acid sequences listed in Table 7; in some embodiments, the nucleic acid molecules comprise polynucleotide sequences of the LCVR nucleic acid sequences listed in Table 8, or sequences substantially similar to them having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0023] This disclosure also provides nucleic acid molecules encoding any of the HCDR3 amino acid sequences listed in Table 7; in some embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR3 nucleic acid sequences listed in Table 8, or a sequence substantially similar to the polynucleotide sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0024] This disclosure also provides a nucleic acid molecule encoding HCVR, wherein the HCVR comprises a set of three CDRs (i.e., HCDR1-HCDR2-HCDR3), wherein the amino acid sequence set of HCDR1-HCDR2-HCDR3 is defined as any of the exemplary anti-GITR antibodies listed in Table 7.

[0025] This disclosure also provides nucleic acid molecules encoding both HCVR and LCVR, wherein the HCVR comprises the amino acid sequence of any one of the HCVR amino acid sequences listed in Table 7, and wherein the LCVR comprises the amino acid sequence of any one of the LCVR amino acid sequences listed in Table 7. In some embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any one of the HCVR nucleic acid sequences listed in Table 8, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with it, and a polynucleotide sequence selected from any one of the LCVR nucleic acid sequences listed in Table 8, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with it. In some embodiments according to this aspect provided herein, the nucleic acid molecule encodes HCVR and LCVR, wherein both HCVR and LCVR are derived from the same anti-GITR antibody listed in Table 7.

[0026] This disclosure also provides recombinant expression vectors capable of expressing polypeptides containing heavy chain variable regions or light chain variable regions of anti-GITR antibodies. For example, this disclosure includes recombinant expression vectors comprising any of the aforementioned nucleic acid molecules, namely nucleic acid molecules encoding any of the HCVR, LCVR, and / or CDR sequences shown in Table 7. The scope of this disclosure also includes host cells, such as eukaryotic host cells, such as mammalian host cells, in which such vectors have been introduced. Exemplary eukaryotic host cells include yeast and mammalian cells, such as vertebrate cells, such as mouse, rat, monkey, or human cell lines, such as HKB11 cells, PER.C6 cells, HEK cells, or CHO cells. This document also provides methods for producing antibodies or portions thereof by culturing host cells under conditions that allow for the production of antibodies or antibody fragments, and recovering the antibodies and antibody fragments thus produced.

[0027] This disclosure includes anti-GITR antibodies having a modified glycosylation pattern. In some embodiments, modifications to remove unwanted glycosylation sites may be useful, or antibodies lacking the fucose moiety may be present on the oligosaccharide chain, for example, to enhance antibody-dependent cell cytotoxicity (ADCC) function (see Shield et al. (2002) JBC 277:26733). In other applications, galactosylation modifications may be made to alter complement-dependent cytotoxicity (CDC).

[0028] In another aspect, this disclosure provides a pharmaceutical composition comprising a recombinant human antibody or a fragment thereof that specifically binds to GITR and a pharmaceutically acceptable carrier. In a related aspect, this disclosure features a composition that is a combination of an anti-GITR antibody and a second therapeutic agent. In one embodiment, the second therapeutic agent is any pharmaceutical agent advantageously combined with the anti-GITR antibody. This disclosure also provides antibody-drug conjugates (ADCs) comprising an anti-GITR antibody conjugated to a cytotoxic agent. Exemplary combination therapies, combination formulations, and ADCs relating to the anti-GITR antibodies of this disclosure are disclosed elsewhere herein.

[0029] In another aspect, this disclosure provides pharmaceutical compositions comprising a recombinant human antibody or a fragment thereof that specifically binds to GITR, for use in manufacturing medicaments for killing tumor cells or for inhibiting or attenuating tumor cell growth or otherwise treating patients with cancer. In some aspects, the pharmaceutical composition is therapeutically combined with a second therapeutic agent. In some aspects, the second therapeutic agent is a PD-1 inhibitor. In some aspects, the PD-1 inhibitor is cimiprilmab.

[0030] In another aspect, this disclosure provides a treatment method for killing tumor cells or inhibiting or attenuating tumor cell growth or otherwise treating a patient with cancer using the anti-GITR antibody or antigen-binding fragment of an antibody provided herein. The treatment method according to this aspect includes administering a therapeutically effective amount of a pharmaceutical composition comprising the antibody or antigen-binding fragment of an antibody provided herein to a subject in need. The treated condition is any disease or condition that is improved, alleviated, inhibited, or prevented by targeting GITR and / or by increasing T cell proliferation or function and / or inhibiting regulatory T cell-induced repressive activity. In some aspects, the method further includes administering a second therapeutic agent to the patient or subject in need. In some aspects, the second therapeutic agent is a PD-1 inhibitor. In some aspects, the PD-1 inhibitor is cimipril malabsorption.

[0031] In another aspect, this disclosure provides the use of a recombinant human antibody or fragment thereof that specifically binds to GITR, or a pharmaceutical composition comprising said antibody or fragment thereof, for the manufacture of a medicament for killing tumor cells or for inhibiting or attenuating tumor cell growth or otherwise treating a patient with cancer.

[0032] In another aspect, this disclosure provides a treatment method for killing tumor cells or inhibiting or attenuating tumor cell growth, or otherwise treating a patient with cancer, using an anti-GITR antibody or a combination of the antigen-binding portion of an anti-GITR antibody and an anti-PD1 antibody or a combination of the antigen-binding portions of an anti-PD1 antibody. In some aspects, the anti-PD1 antibody is cimipril. The treatment method according to this aspect comprises administering a therapeutically effective amount of a pharmaceutical composition to a subject in need, said pharmaceutical composition comprising a combination of an anti-GITR and an anti-PD1 antibody or antigen-binding fragment composition. The treated condition is any disease or condition that is improved, alleviated, inhibited, or prevented by targeting both GITR and PD1.

[0033] In another aspect, this disclosure provides a method for treating cancer, the method comprising administering to a subject in need of (i) an anti-GITR antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising: HCDR1 comprising the amino acid sequence of SEQ ID NO:4, HCDR2 comprising the amino acid sequence of SEQ ID NO:6, HCDR3 comprising the amino acid sequence of SEQ ID NO:24, LCDR1 comprising the amino acid sequence of SEQ ID NO:12, LCDR2 comprising the amino acid sequence of SEQ ID NO:14, and LCDR3 comprising the amino acid sequence of SEQ ID NO:16; and (ii) cimiprimab. In some aspects, the cancers are selected from the group consisting of: squamous cell carcinoma of the skin, cutaneous squamous cell carcinoma (CSCC), myeloma, lung cancer, melanoma, head and neck squamous cell carcinoma (SCCHN), small cell lung cancer, non-small cell lung cancer (NSCLC), cervical cancer such as cervical squamous cell carcinoma (cervical SCC), breast cancer and renal cell carcinoma (RCC), adenocarcinoma, colorectal cancer (CRC), pancreatic cancer, head and neck cancer, prostate cancer, glioblastoma multiforme, malignant glioma, osteosarcoma, colorectal cancer, gastric cancer (e.g., gastric cancer with MET amplification), malignant mesothelioma, multiple myeloma, ovarian cancer, synovial sarcoma, thyroid cancer, breast cancer (including triple-negative breast cancer), testicular cancer, esophageal cancer, uterine cancer, endometrial cancer, liver cancer, immune checkpoint blockade (ICB) initiating cancer and ICB-experienced cancer.

[0034] In another aspect, this disclosure provides the use of a medicament for a method of manufacturing a drug using an anti-GITR antibody or an antigen-binding fragment thereof for treating cancer in a subject with such need, said antibody or antigen-binding fragment comprising: HCDR1 comprising the amino acid sequence of SEQ ID NO:4, HCDR2 comprising the amino acid sequence of SEQ ID NO:6, HCDR3 comprising the amino acid sequence of SEQ ID NO:24, LCDR1 comprising the amino acid sequence of SEQ ID NO:12, LCDR2 comprising the amino acid sequence of SEQ ID NO:14, and LCDR3 comprising the amino acid sequence of SEQ ID NO:16. The method comprises administering an anti-GITR antibody or an antigen-binding fragment thereof and cimipril to a subject. In some aspects, the cancers are selected from the group consisting of: squamous cell carcinoma of the skin, cutaneous squamous cell carcinoma (CSCC), myeloma, lung cancer, melanoma, head and neck squamous cell carcinoma (SCCHN), small cell lung cancer, non-small cell lung cancer (NSCLC), cervical cancer such as cervical squamous cell carcinoma (cervical SCC), breast cancer and renal cell carcinoma (RCC), adenocarcinoma, colorectal cancer (CRC), pancreatic cancer, head and neck cancer, prostate cancer, glioblastoma multiforme, malignant glioma, osteosarcoma, colorectal cancer, gastric cancer (e.g., gastric cancer with MET amplification), malignant mesothelioma, multiple myeloma, ovarian cancer, synovial sarcoma, thyroid cancer, breast cancer (including triple-negative breast cancer), testicular cancer, esophageal cancer, uterine cancer, endometrial cancer, liver cancer, immune checkpoint blockade (ICB) initiating cancer and ICB-experienced cancer.

[0035] In another embodiment, this disclosure provides an anti-GITR antibody or an antigen-binding fragment thereof in a method for treating cancer in a subject with such need, the method comprising administering to a subject with such need (i) an anti-GITR antibody or an antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof comprising: HCDR1 comprising the amino acid sequence of SEQ ID NO:4, HCDR2 comprising the amino acid sequence of SEQ ID NO:6, HCDR3 comprising the amino acid sequence of SEQ ID NO:24, LCDR1 comprising the amino acid sequence of SEQ ID NO:12, LCDR2 comprising the amino acid sequence of SEQ ID NO:14, and LCDR3 comprising the amino acid sequence of SEQ ID NO:16; and (ii) cimiprimab. In some aspects, the cancers are selected from the group consisting of: squamous cell carcinoma of the skin, cutaneous squamous cell carcinoma (CSCC), myeloma, lung cancer, melanoma, head and neck squamous cell carcinoma (SCCHN), small cell lung cancer, non-small cell lung cancer (NSCLC), cervical cancer such as cervical squamous cell carcinoma (cervical SCC), breast cancer and renal cell carcinoma (RCC), adenocarcinoma, colorectal cancer (CRC), pancreatic cancer, head and neck cancer, prostate cancer, glioblastoma multiforme, malignant glioma, osteosarcoma, colorectal cancer, gastric cancer (e.g., gastric cancer with MET amplification), malignant mesothelioma, multiple myeloma, ovarian cancer, synovial sarcoma, thyroid cancer, breast cancer (including triple-negative breast cancer), testicular cancer, esophageal cancer, uterine cancer, endometrial cancer, liver cancer, immune checkpoint blockade (ICB) initiating cancer and ICB-experienced cancer.

[0036] Other implementation methods will become apparent upon referring to the following detailed description. Attached Figure Description

[0037] Figure 1 The truncation increase observed in CompAb1 during incubation in monkey serum or PBS was depicted. The estimated truncation rate in monkey serum was approximately 1.5% / day, and the estimated truncation rate in PBS was approximately 0.5% / day.

[0038] Figure 2 The truncation increase observed in CompAb1 when administered at 10 mg / kg in mice was depicted. CompAb1 was incorporated into mouse serum and PBS as a day 0 control prior to pull-down (affinity purification).

[0039] Figure 3 The truncation increase in CompAb1 antibody following incubation in IgG-depleted human serum was depicted, while no truncation increase was observed in variants mAb1 or mAb2. CompAb1 antibody showed a truncation rate of 1.9% / day at the C-terminus and 0.7% / day at the N-terminus.

[0040] Figure 4 The study depicted the truncation increase observed in CompAb1 (also known as CompAb1) antibody after in vitro incubation in mouse serum, while no truncation increase was observed in variants mAb1 or mAb2. The estimated truncation rate of CompAb1 antibody in mouse serum was at least 1.7% / day.

[0041] Figure 5A , Figure 5B and Figure 5C The image depicts the state of Jurkat / hCD20 cells ( Figure 5A Jurkat / hCD20 / hGITR cells ( Figure 5B ) and Jurkat / hCD20 / MfGITR cells ( Figure 5C On the cell surface of mAb1 and IgG1 isotype controls at concentrations ranging from 18 pM to 300 nM, binding was detected by flow cytometry using Alexa647-labeled anti-human IgG. Fluorescence intensity was plotted as a geometric MFI. h, human; Mf, cynomolgus macaque (Macaca fascicularis).

[0042] Figure 6 The binding of antibodies to activated (CD25+) human primary T cells was depicted. Primary human T cells from four individual donors were stimulated in vitro with anti-CD2 / anti-CD3 / anti-CD28-coated beads. Flow cytometry was used to detect the binding of antibodies to activated (CD25+) human primary T cells. + and CD25 - Original CD4 + and CD8 + On T cells, AF647-conjugated mAb1 and AF647-conjugated IgG1 isotype controls bound at concentrations ranging from 8 pM to 200 nM. Fluorescence intensity was plotted as a geometric MFI. (Hu, Human)

[0043] Figure 7 The binding of antibodies to CD69+ cynomolgus monkey primary T cells was depicted. Primary cynomolgus monkey T cells from four individual donors were stimulated in vitro with anti-CD2 / anti-CD3 / anti-CD28-coated beads. Flow cytometry was used to detect the binding of antibodies to CD69+ cynomolgus monkey primary T cells. + and CD69 - Crab-eating macaque original CD4 + and CD8 + On T cells, AF647-conjugated mAb1 and AF647-conjugated IgG1 isotype controls bound to the cell surface at concentrations ranging from 8 pM to 200 nM. Fluorescence intensity was plotted as a geometric MFI. Mf, cynomolgus macaque (cynomolgus monkey)

[0044] Figure 8A , Figure 8B , Figure 8C and Figure 8D This study depicted Fc-mediated NFAT activity in Jurkat / NFAT-Luc / hFcγR3a and Jurkat / NFAT-Luc / MfFcγR3a effector cells. Jurkat / NFAT-Luc / hFcγR3a and Jurkat / NFAT-Luc / MfFcγR3a were compared with 916 fM to 60 nM mAb1 or IgG1 isotype controls or 45.8 fM to 3 nM anti-CD20 IgG1 (including antibody-free controls) and Jurkat / hCD20 cells (…). Figure 8A , Figure 8C ) or Jurkat / hCD20 / hGITR cells ( Figure 8B ) or Jurkat / hCD20 / MfGITR cells ( Figure 8D The cells were incubated together (effect cell to target cell ratio of 1:2). Signal transduction in Jurkat / NFAT-Luc / hFcγR3a and Jurkat / NFAT-Luc / MfFcγR3a cells was detected as luciferase activity and measured by quantification of the luminescent signal, reported in relative optical units (RLU). Data were obtained from measurements performed in duplicate wells and plotted as mean ± SD.

[0045] Figure 9A , Figure 9B , Figure 9C , Figure 9D , Figure 9E and Figure 9F The antibody-mediated ADCC targeting Jurkat T cells engineered to express human or cynomolgus monkey GITR was depicted. Jurkat / hCD20 ( Figure 9A , Figure 9D ), Jurkat / hCD20 / hGITR ( Figure 9B , Figure 9E ) or Jurkat / hCD20 / MfGITR ( Figure 9C , Figure 9FTarget cells were incubated with human NK cells (effective cell to target cell ratio of 5:1) and mAb1, IgG1 isotype control, and anti-CD20 IgG1 at concentrations ranging from 9.5 fM to 10 nM for 3.5 h. Cytotoxicity was determined using the commercially available CytoTox-Glo assay, a luminescent cytotoxicity assay that measures the relative number of dead cells in a cell population using a luminescent readout. The dashed lines in each graph represent the level of nonspecific cytotoxicity observed after the addition of NK cells in the absence of antibodies. Data from assays performed in triplicate are plotted as mean ± SD.

[0046] Figure 10A , Figure 10B , Figure 10C and Figure 10D Antibody-mediated ADCC targeting human primary T cells was described. Human primary Treg cells were isolated from PBMCs (3 donors). Figure 10A , Figure 10C ) and CD8 + T cells ( Figure 10B , Figure 10D (Target cells) were stimulated and expanded in culture and incubated with human primary NK cells (effective cells) isolated from whole blood (two donors) at a ratio of 5:1 (effective cells to target cells). mAb1, IgG1 isotype control, or anti-CD3 IgG1 at concentrations ranging from 169 fM to 10 nM were incubated with effector and target cells for 3.5 hours. Cytotoxicity was determined using the commercially available CytoTox-Glo assay, a luminescent cytotoxicity assay that measures the relative number of dead cells in a cell population. Dashed lines in each graph represent the level of nonspecific cytotoxicity observed after the addition of NK cells in the absence of antibodies. Data from assays performed in triplicate are plotted as mean ± SD.

[0047] Figure 11A , Figure 11B and Figure 11C Antibody-mediated ADCP was depicted in Jurkat T cells engineered to express human or cynomolgus monkey GITR. Jurkat / hCD20 cells labeled with CellTrace CFSE dye were used. Figure 11A ), Jurkat / hCD20 / hGITR ( Figure 11B ) or Jurkat / hCD20 / MfGITR ( Figure 11CTarget cells were incubated for 1 to 2 hours with phagocytes derived from human primary monocytes labeled with CellTrace far-red dye and mAb1, IgG1 isotype controls, or anti-CD20 IgG1 (including antibody-free controls) at concentrations ranging from 381 fM to 100 nM. Phagocytosis was analyzed by fluorescence imaging on the Opera Phoenix high-content screening system and measured as relative fluorescence intensity (RFU) within the far-red labeled cell population (phagocytes labeled with far-red dye, detected in the 647 nm emission channel) of target cells (target cells labeled with CFSE, detected in the 488 nm emission channel). Data from measurements performed in duplicate wells were plotted as mean ± SD.

[0048] Figure 12A , Figure 12B , Figure 12C , Figure 12D , Figure 12E and Figure 12F The effect of anti-GITR antibody on anti-CD3-mediated primary CD4+ T cell proliferation was depicted. Enriched human primary T cells (6 donors) were co-incubated with serially diluted mAb1 or IgG1 isotype controls at concentrations ranging from 32 fM to 133 nM, including an antibody-free control, in the presence of a fixed concentration (2 nM) of stimulating anti-CD3 and HEK293 / FcγR2b helper cells. Data were obtained from assays performed in triplicate and plotted as mean ± SD. T cell proliferation was measured by detecting tritium decay (from tritized thymidine incorporated into dividing cells) and reported as CPM. Dashed lines in each plot represent the level of proliferation observed after the addition of stimulating anti-CD3 in the absence of titrated antibody.

[0049] Figure 13 The effect of anti-GITR antibody on the binding of human GITR to human GITR-L was described. The binding of human GITR protein hGITR.mmH to immobilized human GITR-L at concentrations ranging from 3.4 pM to 200 nM was evaluated by ELISA, and the EC50 of the binding was determined. 50 Value. EC calculated based on the combination with hGITR-L. 50 To determine the ability of an antibody to block the binding of immobilized human GITR-L, a fixed concentration of recombinant hGITR.mmH (1.5 nM) was pre-incubated with mAb1 or IgG1 isotype controls at concentrations ranging from 51 pM to 3 μm and added to wells containing immobilized human GITR-L. The binding of hGITR.mmH was detected using a TMB substrate with an HRP-conjugated cMyc antibody, and the OD was measured spectrophotometrically. 450 .

[0050] Figure 14 The effect of anti-GITR antibody on ADCs is illustrated. Target cells of Jurkat / hCD20, Jurkat / hCD20 / hGITR, or Jurkat / hCD20 / MfGITR were incubated for 3.5 h with or without 5% NHS, along with mAb1, an IgG1 isotype control ranging from 477 fM to 500 nM, and anti-CD20 IgG1 (including an antibody-free control set at 119 fM). Cytotoxicity was determined using the commercially available CytoTox-Glo assay, a luminescent cytotoxicity assay that measures the relative number of dead cells in a cell population. Dashed lines in each graph represent the level of nonspecific cytotoxicity observed after the addition of 5% NHS in the absence of antibody. Data from assays performed in triplicate are plotted as mean ± SD.

[0051] Figure 15A and Figure 15B This demonstrates that when incubated with soluble GITR, anti-GITR antibodies cannot form immune complexes capable of binding C1q. The study compared 30 nM of GITR mAb1 or IgG1 isotype controls with recombinant human GITR extracellular domain protein monomer hGITR.mmH. Figure 15A ) or dimer hGITR.mFc( Figure 15B Incubate together at a 1:1 molar ratio. Control wells containing only the antibody hGITR.mmH or hGITR.mFc were also evaluated. All samples were diluted 50-fold to the kit's detection solution prior to analysis. High (38 μg Eq / mL) and low (15 μg Eq / mL) HAGG positive controls were analyzed in parallel, and C1q binding for each control sample was observed to be within the expected range of 11 to 26 μg Eq / mL and less than 4 μg Eq / mL, respectively. The dashed line represents 4 μg Eq / mL. Data from samples tested in triplicate are plotted as mean ± SD.

[0052] Figure 16A and Figure 16B The study depicted the effects of donor 555105 in the presence of human PD-L1. Figure 16A ) and donor 555130 ( Figure 16B Enhanced IL-2 release from primary CD3+ T cells stimulated with anti-CD3. This was achieved with a fixed concentration (20 nM) of cimipril or a range of concentrations (76 fM to 200 nM) of IgG4. PIn the presence of an isotype control, enriched human primary T cells were incubated with serially diluted mAb1 or IgG1 isotype controls at a 1:2 antigen-presenting cell to T cell ratio in the presence of RBL-2H3 / αCD3 or RBL-2H3 / αCD3 / hPD-L1. Data were obtained from assays performed in triplicate and plotted as mean ± SD. IL-2 release was measured using the PerkinElmer Human IL-2 Kit according to the manufacturer's protocol.

[0053] Figure 17 This study describes the depletion of intratumoral T regulatory cells and the increased CD8+ T cell / T regulatory cell ratio caused by anti-GITR antibody in GITR / GITR-L humanized mice subcutaneously challenged with MC38 mouse colon tumor cells.

[0054] Figure 18 In GITR / GITR-L-PD-1 humanized mice, the combination of 1 mg / kg cimiprimab and 10 mg / kg mAb1 resulted in a greater reduction in MC38 tumor growth compared to cimiprimab alone.

[0055] Figure 19A , Figure 19B , Figure 19C , Figure 19D and Figure 19E The results showed that cimiprilmab at 1 mg / kg was used in combination with 0.1 mg / kg ( Figure 19E ), 1.0 mg / kg ( Figure 19D ) and 10 mg / kg mAb1 ( Figure 19C In mice treated with combination therapy with cimipril alone, the efficacy was lower than that of cimipril alone. Figure 19B MC38 tumors showed a higher frequency of clearance. Treatment with isotype control, such as... Figure 19A As shown.

[0056] Figure 20 The study demonstrated that mice with MC38 tumors treated with a combination of 1 mg / kg cimiprimab and 10 mg / kg mAb1 had higher survival rates compared to cimiprimab alone. Detailed Implementation

[0057] Modulating the tumor microenvironment by targeting intratumoral immune cells is a therapeutic approach for cancer treatment. Specifically, targeting co-stimulatory and co-inhibitory immune checkpoint receptors expressed on the cell surface of intratumoral T cells can enhance the endogenous antitumor response by increasing cytotoxicity against tumor cells and downregulating local immune suppression.

[0058] Tumor necrosis factor receptor superfamily member 18 (TNFRSF18), also known as glucocorticoid-induced tumor necrosis factor receptor-associated protein (GITR), is a co-stimulatory receptor expressed on regulatory T cells (Tregs) and non-Tregs (called conventional T cells), as well as other cells of the immune system. GITR is expressed at low levels on resting T cells and is upregulated upon T cell activation, with higher expression on activated Tregs than on activated conventional T cells (Shimizu et al., Nature Immunology, 23(2):135-142, 2002; Krausz et al., The Scientific World Journal, 7:533-66, 2007; Knee et al., European Journal of Cancer, 67:1-10, 2016). The differential expression profile of GITR on activated Tregs makes it an attractive target for preferentially depleting activated intratumoral Tregs to promote antitumor immunity.

[0059] mAb1 and mAb2 are human IgG1 GITR agonist antibodies that preferentially deplete activated Tregs. GITR agonist antibodies can induce antitumor immunity by preferentially depleting intratumoral Tregs in an FcγR-dependent manner (e.g., antibody-dependent cytotoxicity / phagocytosis [ADCC / ADCP]), which is associated with GITR cell surface expression. These GITR antibodies can synergize with programmed cell death-1 (PD-1) checkpoint blockade to generate long-term antitumor responses in preclinical models. In some respects, anti-GITR antibodies are combined with PD-1 inhibitors, such as cimipril. While not wishing to be limited to theory, PD-1 inhibitors can restore the ability of anti-GITR antibodies to enhance anti-CD3-stimulated T cell activation. In some respects, the combination of the anti-GITR antibodies disclosed herein with PD-1 inhibitors therapeutically resulted in a greater reduction in tumor growth compared to PD-1 inhibitors alone. In some respects, the combination of the anti-GITR antibodies disclosed herein with PD-1 inhibitors therapeutically resulted in a higher frequency of tumor clearance compared to PD-1 inhibitors alone. In some respects, the combination of the anti-GITR antibody disclosed in this article with a PD-1 inhibitor resulted in higher survival rates in tumor-bearing subjects compared to PD-1 inhibitors alone.

[0060] Before describing this disclosure, it should be understood that this disclosure is not limited to the specific methods and experimental conditions described, as such methods and conditions can vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, as the scope of this disclosure will be defined only by the appended claims.

[0061] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. As used herein, when referring to a specifically enumerated numerical value, the term “about” means that the value can vary from the enumerated value by no more than 1%. For example, as used herein, the expression “about 100” includes 99 and 101 and all values ​​between them (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0062] Although any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of this disclosure, preferred methods and materials are described hereafter. All patents, applications, and non-patent publications referenced in this specification are incorporated herein by reference in their entirety.

[0063] Glucocorticoid-induced tumor necrosis factor receptor

[0064] As used herein, the term "glucocorticoid-induced tumor necrosis factor receptor," "GITR," etc., refers to the human glucocorticoid-induced tumor necrosis factor receptor, which contains the amino acid sequence shown in SEQ ID NO:49 (NCBI accession number #NP_004186.1). The term "GITR" includes both monomeric and multimeric GITR molecules. As used herein, the term "monomer human GITR" means a GITR protein or a portion thereof that does not contain or have any multimerizing domains and exists as a single GITR molecule (not directly physically linked to another GITR molecule) under normal conditions. An exemplary monomeric GITR molecule is a molecule referred to herein as "hGITR.mmh" containing the amino acid sequence of SEQ ID NO:45 (see, for example, Example 3 herein). The term "dimeric human GITR," as used herein, refers to a construct containing two GITR molecules linked together by a linker, covalent bond, non-covalent bond, or multimerizing domain such as an antibody Fc domain. Exemplary dimerized GITR molecules include those referred to herein as “hGITR.mFc” and “hGITR.hFc”, which contain the amino acid sequences of SEQ ID NO:46 and SEQ ID NO:47, respectively.

[0065] All references to proteins, peptides, and protein fragments in this document are intended to refer to the human form of the various proteins, peptides, or protein fragments, unless explicitly stated to be derived from a non-human species. Therefore, unless it is specified to be derived from a non-human species, such as "mouse GITR," "monkey GITR," etc., the expression "GITR" refers to the human GITR.

[0066] As used herein, the term "cell surface expressed GITR" means one or more GITR proteins or their extracellular domains expressed on the surface of cells, either in vitro or in vivo, such that at least a portion of the GITR protein is exposed to the extracellular space of the cell membrane and is accessible to the antigen-binding portion of an antibody. "Cell surface expressed GITR" may include or consist of GITR proteins expressed on the surface of cells that normally express GITR proteins. Alternatively, "cell surface expressed GITR" may include or consist of GITR proteins expressed on the surface of cells that normally do not express human GITRs but have been engineered to express GITRs on their surface.

[0067] Antibodies and antigen-binding fragments of antibodies

[0068] As used herein, the term "antibody" means any antigen-binding molecule or molecular complex containing at least one complementarity-determining region (CDR) that specifically binds to or interacts with a particular antigen (e.g., GITR). The term "antibody" includes immunoglobulin molecules comprising four polypeptide chains (i.e., two heavy (H) chains and two light (L) chains interconnected by disulfide bonds), and their multimers (e.g., IgM). Each heavy chain contains a heavy chain variable region (abbreviated herein as HCVR or V). H ) and the heavy-chain constant region. The heavy-chain constant region contains three structural domains: C H 1. C H 2 and C H 3. Each light chain contains a light chain variable region (abbreviated as LCVR or V in this document). L The light chain constant region contains a structural domain (C) and a light chain constant region. L 1). V H District and V L The region can be further subdivided into highly variable regions, called complementarity-determining regions (CDRs), interspersed with more conservative regions, called framing regions (FRs). Each V H and V L It consists of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In the different embodiments provided herein, the FRs of the anti-GITR antibody (or its antigen-binding portion) may be identical to the human germline sequence, or may be naturally or artificially modified. The common amino acid sequence can be defined based on the side-by-side analysis of two or more CDRs.

[0069] In some embodiments provided herein, the anti-GITR antibodies provided herein are human antibodies. As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies provided herein may include, for example, amino acid residues in the CDR and particularly in CDR3 that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro or by somatic mutations in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which a germline CDR sequence derived from another mammalian species (such as a mouse) has been inserted into a human frame sequence. The term "human antibody" does not include naturally occurring molecules that exist normally in a living, unmodified organism without modification or human intervention / manipulation.

[0070] As used herein, the term "anti-GITR antibody" includes monovalent antibodies and monospecific bivalent antibodies, as well as bispecific antibodies comprising a first arm that binds to GITR and a second arm that binds to a second (target) antigen, wherein the anti-GITR arm comprises any of the HCVR / LCVR or CDR sequences listed in Table 7 herein. The term "anti-GITR antibody" also includes antibody-drug conjugates (ADCs) comprising an anti-GITR antibody or its antigen-binding moiety conjugated to a drug or toxin (i.e., a cytotoxic agent). The term "anti-GITR antibody" also includes antibody-radionium conjugates (ADCs) comprising an anti-GITR antibody or its antigen-binding moiety conjugated to a radionuclide.

[0071] In some embodiments, the antibodies provided herein may be recombinant human antibodies. The term "recombinant human antibody" as used herein is intended to include all human antibodies prepared, expressed, constructed, or isolated in a recombinant manner, such as antibodies expressed using a recombinant expression vector (further described below) transfected into host cells, antibodies isolated from a recombinant combined human antibody library (further described below), antibodies isolated from animals transgenic with human immunoglobulin genes (e.g., mice) (see, for example, Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295), or antibodies prepared, expressed, constructed, or isolated by any other means involving splicing a human immunoglobulin gene sequence into another DNA sequence. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in some embodiments, such recombinant human antibodies undergo in vitro mutagenesis (or, when using animals transgenic with respect to human Ig sequences, in vivo somatic cell mutagenesis) and thus the V of the recombinant antibody… H and V L The amino acid sequence of the region is, although it originates from human lineage V H and V LSequences and related sequences, but which may not naturally exist within the human antibody germline library in vivo.

[0072] The terms "specific binding" or "specific binding to" refer to the formation of a relatively stable complex between an antibody or its antigen-binding fragment and the antigen under physiological conditions. Specific binding is characterized by a minimum of approximately 1 x 10-1 -8 M or lower equilibrium dissociation constant (, smaller K) D (Indicating a tighter binding). Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, etc. As described herein, methods have been used... Real-time label-free biolayer interferometry analysis on the HTX biosensor identified antibodies that specifically bind to GITR. Furthermore, multispecific antibodies binding to one domain of GITR and one or more other antigens, or bispecific antibodies binding to two different regions of GITR, are still considered "specifically binding" antibodies as used herein.

[0073] The term "high affinity" antibody refers to antibodies obtained through methods such as real-time label-free biolayer interferometry, for example... HTX biosensors, or through surface plasmon resonance, such as BIACORE. TM Or, as measured by solution affinity ELISA, those with at least 10 Ω to hGITR. -8 M, at least about 10 -9 M, at least about 10 -10 M or at least about 10 -11 Binding affinity of M (denoted as K) D Monoclonal antibodies.

[0074] The terms "slow dissociation rate," "Koff," or "kd" refer to the rate at which the antibody dissociates at a rate of 1 × 10⁻⁶. -3 s -1 or smaller or 1×10 -4 s -1 Or a smaller rate constant dissociates from the GITR, such as through real-time label-free biolayer interferometry analysis, for example... HTX biosensors, or through surface plasmon resonance, such as BIACORE. TM It has been determined.

[0075] As used in this article, the term "K" D "Intended to refer to the equilibrium dissociation constant of a specific antibody-antigen interaction."

[0076] The antibodies provided herein may be isolated antibodies. As used herein, "isolated antibody" means an antibody that has been identified and isolated and / or recovered from at least one component of the natural environment. For example, for the purposes of this disclosure, an antibody that has been isolated or removed from at least one component of an organism, or from tissues or cells where the antibody is naturally present or produced, is an "isolated antibody." Isolated antibodies also include in situ antibodies within recombinant cells. Isolated antibodies are antibodies that have undergone at least one purification or isolation step. According to certain embodiments, isolated antibodies may be substantially free of other cellular material and / or chemicals.

[0077] As used herein, the terms “antigen-binding portion” and “antigen-binding fragment” of an antibody include any naturally occurring, enzymatically available, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. As used herein, the terms “antigen-binding fragment” or “antibody fragment” of an antibody refer to one or more fragments of an antibody that retain the ability to bind.

[0078] As used herein, the term "antibody" also includes the antigen-binding fragment of a whole antibody molecule. As used herein, the terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, etc., include any naturally occurring, enzymatically available, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen-binding fragments of antibodies can be derived from whole antibody molecules, for example, using any suitable standard technique, such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding variable and optionally constant antibody domains. Such DNA is known and / or readily available from, for example, commercial sources, DNA libraries (including, for example, phage-antibody libraries), or can be synthesized. DNA can be sequenced and manipulated chemically or using molecular biology techniques, for example, to align one or more variable and / or constant domains into suitable conformations, or to introduce codons, generate cysteine ​​residues, modify, add, or delete amino acids, etc.

[0079] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) the smallest recognition unit consisting of amino acid residues mimicking the hypervariable region of an antibody (e.g., a separated complementarity-determining region (CDR) such as a CDR3 peptide) or a restricted FR3-CDR3-FR4 peptide. As used herein, other engineered molecules (such as domain-specific antibodies, single-domain antibodies, domain-deficient antibodies, chimeric antibodies, CDR-transplanted antibodies, dimers, triplets, tetramers, microbodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains) are also included in the description “antigen-binding fragments”.

[0080] Antigen-binding fragments of antibodies typically include at least one variable domain. Variable domains can have any size or amino acid composition and will generally contain at least one CDR adjacent to or conforming to one or more frame sequences. In the presence of V... H Domain and V L In the antigen-binding fragment associated with the structural domain, V H and V L The domains can be positioned relative to each other in any suitable arrangement. For example, the variable region can be a dimer and contain V. H -V H V H -V L or V L -V L Dimer. Alternatively, the antigen-binding fragment of the antibody may contain monomer V. H or V L Structural domain.

[0081] In some embodiments, the antigen-binding fragment of the antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of the variable and constant domains that may be found within the antigen-binding fragment of the antibody of this disclosure include: (i) V H -C H 1; (ii)V H -C H 2; (iii)V H -C H 3; (iv)V H -C H 1-C H 2; (v)V H -C H 1-C H 2-C H 3;(vi)VH -C H 2-C H 3;(vii)V H -C L (viii)V L -C H 1; (ix)V L -C H 2; (x)V L -C H 3;(xi)V L -C H 1-C H 2;(xii)V L -C H 1-C H 2-C H 3; (xiii)V L -C H 2-C H 3; and (xiv)V L -C L In any configuration of the variable and constant domains (including any of the exemplary configurations listed above), the variable and constant domains may be directly connected to each other or may be connected via full-length or partial hinge or linker regions. The hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60, or more) amino acids, resulting in a flexible or semi-flexible connection between adjacent variable and / or constant domains in a single polypeptide molecule. Furthermore, the antigen-binding fragment of the antibody of this disclosure may comprise each other and / or one or more monomers V having any of the variable and constant domain configurations listed above. H or V L The structural domains (e.g., via disulfide bonds) are non-covalently associated homodimers or heterodimers (or other polymers).

[0082] Just like whole antibody molecules, antigen-binding fragments can be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies typically contain at least two distinct variable domains, each capable of specifically binding to a single antigen or a different epitope on the same antigen. Using conventional techniques available in the art, any form of multispecific antibody, including the exemplary bispecific antibody forms disclosed herein, can be adapted to the context of the antigen-binding fragments of the antibodies disclosed herein.

[0083] Human antibodies can exist in two forms associated with hinge heterogeneity. In one form, the immunoglobulin molecule comprises a stable four-chain construct of approximately 150–160 kDa, where the dimers are held together by interchain heavy chain disulfide bonds. In the second form, the dimers are not linked by interchain disulfide bonds, and the approximately 75–80 kDa molecule consists of covalently coupled light and heavy chains (half-antibodies). These forms are extremely difficult to separate even after affinity purification.

[0084] The frequency of the second form in various intact IgG isotypes is due to, but not limited to, structural differences associated with the hinge region isotype of the antibody. A single amino acid substitution in the hinge region of the human IgG4 hinge can significantly reduce the occurrence of the second form (Angal et al. (1993) Molecular Immunology 30:105) to levels typically observed with the human IgG1 hinge. This invention covers the occurrence of the second form in the hinge, C... H 2 or C H Antibodies with one or more mutations in region 3 may be desired, for example, in production, to increase the yield of the desired antibody form.

[0085] Modification of antibodies and their antigen-binding fragments

[0086] The anti-GITR antibodies disclosed herein may contain one or more amino acid substitutions, insertions, and / or deletions in the frame and / or CDR regions of the heavy and light chain variable domains compared to the sequence of the antibodies disclosed herein, or may be naturally or artificially modified. In some embodiments, the frame regions of the antibody (or its antigen-binding fragment) may be identical to human germline sequences, such as those of the antibodies provided herein, or may be naturally or artificially modified. One or more amino acids in a given frame region (or one or more frame regions) may be substituted, and the substitution may be conserved or non-conserved. Substitution of one or more CDR residues or omission of one or more CDRs is also possible. Antibodies have been described in the scientific literature, in which one or two CDRs may be assigned for binding. Padlan et al. (1995 FASEB J.9:133-139) analyzed the contact region between the antibody and its antigen based on published crystal structures and concluded that only about one-fifth to one-third of the CDR residues actually contact the antigen. Padlan also discovered numerous antibodies in which one or two CDRs lack amino acids that contact the antigen (see also Vajdos et al. 2002 J Mol Biol 320:415-428). Therefore, the antibodies presented herein can be effectively modified in the CDR region and / or frame region, provided that the modified antibody retains one or more desired characteristics, such as the antibody or its antigen-binding fragment having a density of less than about 10. -9 M's EC 50It binds to hGITR; and / or exhibits a reduced truncation rate compared to antibodies lacking N101 or S103 modification.

[0087] A given CDR can be modified relative to the CDR sequence of an antibody provided herein, and such modifications can include conserved or non-conserved substitutions. Desired substitutions can be determined through molecular modeling and / or empirically. For example, one or more CDR residues can be substituted by amino acids occupying corresponding positions in the sequence of another human antibody or by a common sequence of such sequences.

[0088] In addition, the antigen-binding fragment may be an antibody disclosed herein, but modified to omit one or more CDRs and / or one or more frame regions, as long as the modified antibody (also known as the antigen-binding fragment) remains bound to the hGITR.

[0089] Based on previous research, non-antigen-contacting CDR residues (e.g., residues H60-H65 in HCDR2 are typically undesirable) can be identified from the Kabat CDR region located outside the Chothia CDR through molecular modeling and / or empirical evidence. The antibodies or their antigen-binding fragments presented herein can be modified to remove or replace a given CDR, particularly a non-antigen-contacting CDR. Light chain CDRs can be replaced with, for example, universal light chain CDRs.

[0090] The fully human anti-GITR monoclonal antibodies disclosed herein may contain one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR regions of the heavy and light chain variable domains compared to the corresponding germline sequence or the sequence disclosed herein. Such modifications or mutations can be readily identified by comparing the amino acid sequence disclosed herein with a germline sequence available from, for example, a public antibody sequence database, or by comparing the amino acid sequence with the amino acid sequence of an antibody provided herein, such as any of the antibody sequences provided in Table 7.

[0091] This disclosure includes antibodies and antigen-binding fragments thereof derived from any of the amino acid sequences provided herein, wherein one or more amino acids within one or more frame regions and / or CDRs are modified, provided that the modified antibody retains one or more desired characteristics, such as the antibody or its antigen-binding fragment having a ratio of less than about 10. -9 M's EC 50Binding to hGITR; and / or exhibiting a reduced truncation rate in vivo or in vitro. After obtaining an antibody-antigen binding fragment modified with one or more frame regions and / or CDRs, one or more desired properties of the antibody-antigen binding fragment can be readily tested, such as improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as applicable), reduced immunogenicity, etc. Antibody-antigen binding fragments obtained in this general manner are covered within this disclosure.

[0092] This disclosure includes antibodies and their antigen-binding fragments derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids within one or more frames and / or CDR regions are mutated to corresponding residues of the germline sequence yielding the antibody, or corresponding residues of another human germline sequence, or conserved amino acid substitutions of corresponding germline residues (such sequence changes are collectively referred to herein as "germline mutations"). Those skilled in the art can readily generate a variety of antibodies and antigen-binding fragments comprising one or more individual germline mutations or combinations thereof, starting from the heavy and light chain variable region sequences disclosed herein. In some embodiments, V H and / 或VL All frame and / or CDR residues within the domain are mutated back to residues in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are mutated back to the original germline sequence, for example, mutated residues present only in the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or mutated residues present only in CDR1, CDR2, or CDR3. In other embodiments, one or more of the frame and / or one or more CDR residues are mutated to one or more corresponding residues of a different germline sequence (i.e., a germline sequence different from the one from which the antibody was originally derived). Furthermore, the antibody of this disclosure may contain any combination of two or more germline mutations within the frame and / or CDR region, for example, where certain individual residues are mutated to corresponding residues of a specific germline sequence, while certain other residues different from the original germline sequence are retained or mutated to corresponding residues in a different germline sequence. After obtaining an antibody-antigen binding fragment containing one or more germline mutations, one or more desired properties of the antibody-antigen binding fragment can be readily tested, such as improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, etc. Antibody-antigen binding fragments obtained in this general manner are covered within this disclosure.

[0093] This disclosure also includes anti-GITR antibodies comprising variants of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein having one or more conserved substitutions. For example, this disclosure includes anti-GITR antibodies having HCVR, LCVR, and / or CDR amino acid sequences having, for example, 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc., conserved amino acid substitutions relative to any of the HCVR, LCVR, and / or CDR amino acid sequences shown in Table 7 herein.

[0094] When referring to nucleic acids or fragments thereof, the terms "substantially identical" or "substantially the same" indicate that, when optimally aligned with another nucleic acid (or its complementary strand) by appropriate nucleotide insertions or deletions, at least about 95%, and more preferably at least about 96%, 97%, 98%, or 99% of the nucleotide bases exhibit nucleotide sequence identity, as measured by any well-known sequence identity algorithm discussed below, such as FASTA, BLAST, or GAP. In some cases, a nucleic acid molecule substantially identical to a reference nucleic acid molecule may encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0095] When applied to peptides, the term "substantially similar" or "substantially analogous" means that, when optimally aligned, such as using the procedures GAP or BESTFIT with default gap weights, two peptide sequences share at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence similarity. In some respects, dissimilar residue positions differ due to conserved amino acid substitutions. A "conserved amino acid substitution" is an amino acid substitution in which an amino acid residue is replaced by another amino acid residue with a side chain (R group) having similar chemical properties (e.g., charge or hydrophobicity). Generally, conserved amino acid substitutions do not substantially alter the functional properties of a protein. In cases where the conserved substitutions of two or more amino acid sequences differ from each other, the percentage of sequence identity or degree of similarity can be adjusted upwards to correct for the conserved nature of the substitutions. The means for making such adjustments are well known to those skilled in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24:307-331, which is incorporated herein by reference. Examples of amino acid groups containing side chains with similar chemical properties include: (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; (2) aliphatic-hydroxy side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartic acid and glutamic acid; and (7) sulfur-containing side chains, namely cysteine ​​and methionine. Preferred conserved amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conserved substitution is any change that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443-1445, which is incorporated herein by reference. A “moderately conservative” substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0096] Sequence identity and / or similarity of peptides are typically measured using sequence analysis software. Protein analysis software uses similarity measures to match similar sequences, specifying various substitutions, deletions, and other modifications, including conserved amino acid substitutions. For example, the GCG software contains programs such as Gap and Bestfit, which can be used with default parameters to determine sequence homology or sequence identity between closely related peptides (such as homologous peptides from organisms of different species) or between wild-type proteins and their mutant counterparts. See, for example, GCG version 6.1. Peptide sequences can also be compared using FASTA (a program in GCG version 6.1) with default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides alignment of the best overlapping regions between the query and search sequences and the percentage of sequence identity (Pearson (2000) ibid.). Sequences can also be compared using the Smith-Waterman homology search algorithm, which uses an affine vacancy search with a vacancy opening penalty of 12, a vacancy extension penalty of 2, and a BLOSUM matrix of 62. When comparing the sequences provided in this paper with databases containing a large number of sequences from different organisms, another preferred algorithm is the computer program BLAST using default parameters, especially BLASTP or TBLASTN. See, for example, Altschul. et al. (1990) J.Mol.Biol.215:403-410 and Altschul. et al. (1997) Nucleic Acids Res.25:3389-402, each of which is incorporated herein by reference.

[0097] Anti-GITR antibodies containing N101 or S103 modification

[0098] According to certain embodiments of this disclosure, anti-GITR antibodies or antigen-binding fragments thereof modified with N101 or S103 in HCVR are provided. Such modifications provide the disclosed antibodies with desirable characteristics in terms of in vitro and in vivo stability.

[0099] The antibodies provided in Table 1 of this paper and disclosed in U.S. 2017 / 0355774 were explicitly excluded because they lack N101 or S103 modification or mutation in HCVR.

[0100] In some respects, this article provides isolated antibodies or antigen-binding fragments thereof with N101 or S103 modifications in HCVR. Anti-GITR antibodies or antigen-binding fragments thereof with such modifications may further exhibit one or more properties selected from the following:

[0101] (a) A truncation rate of less than 0.5% per day in human, monkey, or mouse serum; and

[0102] (b) The in vivo truncation rate in mice was approximately 0%.

[0103] The truncation rate can be determined by, for example, the determination methods described in Examples 2, 5 and 6.

[0104] In some cases, an anti-GITR antibody or its antigen-binding fragment may include three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) within the heavy chain variable region (HCVR) amino acid sequence selected from the group consisting of SEQ ID NO:22, 28, 34, and 40; and three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) within the light chain variable region (LCVR) amino acid sequence of SEQ ID NO:10.

[0105] In some respects, an anti-GITR antibody or its antigen-binding fragment may comprise an HCVR amino acid sequence selected from the group consisting of SEQ ID NO:22, 28, 34 and 40; and an LCVR amino acid sequence of SEQ ID NO:10.

[0106] In some respects, the anti-GITR antibody may comprise a heavy chain amino acid sequence selected from the group consisting of SEQ ID NO:26, 32, 38 and 44; and a light chain amino acid sequence of SEQ ID NO:20.

[0107] In some aspects, an anti-GITR antibody or its antigen-binding fragment may comprise three heavy chain CDRs within the HCVR amino acid sequence of SEQ ID NO:2 modified with N101A, N101F, N101G, N101H, N101I, N101K, N101L, N101M, N101P, N101Q, N101R, N101V, N101W, or N101Y mutations; and three light chain CDRs within the LCVR amino acid sequence of SEQ ID NO:10. For example, an anti-GITR antibody or its antigen-binding fragment may comprise three heavy chain CDRs within the HCVR amino acid sequence of SEQ ID NO:2 modified with N101D, N101E, N101S, or N101T mutations; and three light chain CDRs within the LCVR amino acid sequence of SEQ ID NO:10.

[0108] In some respects, the anti-GITR antibody or its antigen-binding fragment may include three heavy chain CDRs within the HCVR amino acid sequence of SEQ ID NO:2 modified by mutations of S103A, S103D, S103E, S103F, S103G, S103H, S103I, S103K, S103L, S103M, S103N, S103P, S103Q, S103R, S103T, S103V, S103W or S103Y; and three light chain CDRs within the LCVR amino acid sequence of SEQ ID NO:10.

[0109] The antibodies listed in Table 1 of this paper and disclosed in US 2017 / 0355774 were explicitly excluded. These antibodies lack N101 or S103 modifications or mutations in HCVR.

[0110] Anti-GITR antibodies containing Fc variants

[0111] According to certain embodiments of this disclosure, an anti-GITR antibody is provided, the anti-GITR antibody comprising an Fc domain, the Fc domain containing one or more mutations that, for example, enhance or reduce the binding of the antibody to the FcRn receptor at an acidic pH compared to a neutral pH. For example, this disclosure includes an anti-GITR antibody at the C0 of the Fc domain. H 2 or C HRegion 3 contains mutations, one or more of which increase the affinity of the Fc domain for FcRn under acidic conditions (e.g., in endosomes with a pH range of about 5.5 to about 6.0). When administered to animals, such mutations can lead to an increase in the serum half-life of the antibody. Non-limiting examples of such Fc modifications include, for example, modifications at positions 250 (e.g., E or Q); 250 and 428 (e.g., L or F); 252 (e.g., L / Y / F / W or T), 254 (e.g., S or T), and 256 (e.g., S / R / Q / E / D or T); or modifications at positions 428 and / or 433 (e.g., H / L / R / S / P / Q or K) and / or 434 (e.g., H / F or Y); or modifications at positions 250 and / or 428; or modifications at positions 307 or 308 (e.g., 308F, V308F) and 434. In one embodiment, the modifications include 428L (e.g., M428L) and 434S (e.g., N434S) modifications; 428L, 259I (e.g., V259I) and 308F (e.g., V308F) modifications; 433K (e.g., H433K) and 434 (e.g., 434Y) modifications; 252, 254 and 256 (e.g., 252Y, 254T and 256E) modifications; 250Q and 428L modifications (e.g., T250Q and M428L); and 307 and / or 308 modifications (e.g., 308F or 308P).

[0112] For example, this disclosure includes anti-GITR antibodies comprising an Fc domain containing one or more pairs or groups of mutations selected from the group consisting of: 250Q and 248L (e.g., T250Q and M248L); 252Y, 254T, and 256E (e.g., M252Y, S254T, and T256E); 428L and 434S (e.g., M428L and N434S); and 433K and 434F (e.g., H433K and N434F). All possible combinations of the aforementioned Fc domain mutations and other mutations within the variable domains of antibodies disclosed herein are covered within the scope of this disclosure.

[0113] Biological characteristics of anti-GITR antibodies

[0114] This disclosure includes antibodies that bind to monomeric human GITR with high affinity and antigen-binding fragments thereof. For example, this disclosure includes K+ with a binding affinity of less than about 12.0 nM. DAnti-GITR antibodies conjugated to monomeric human GITR (e.g., hGITR.mmh) are measured, for example, by surface plasmon resonance at 25°C, using the assay form defined in Example 3 herein, or a substantially similar assay. In some embodiments, K is provided at 25°C at concentrations less than about 12 nM, less than about 10 nM, less than about 5 nM, or less than about 2.0 nM. D Anti-GITR antibodies conjugated to monomeric human GITR can be measured, for example, by surface plasmon resonance, using the assay form defined in Example 3 herein, or a substantially similar assay. This disclosure also provides Ki at concentrations less than about 110 nM, less than about 90 nM, less than about 70 nm, less than about 50 nM, less than about 25 nM, or less than about 15 nM. D Anti-GITR antibodies conjugated to monomeric human GITR were measured, for example, by surface plasmon resonance at 25°C, using the assay form provided in Example 3 of this document, wherein the antibody was secreted in conditioned culture.

[0115] This disclosure also includes antibodies and antigen-binding fragments thereof that bind to monomeric human GITR (e.g., hGITR.mmh) with a dissociation half-life (t1 / 2) greater than about 2 minutes, as measured by surface plasmon resonance at 25°C, for example using the assay form defined in Example 3 herein, or a substantially similar assay. According to some embodiments, anti-GITR antibodies are provided that bind to monomeric human GITR at 25°C with a t1 / 2 greater than about 2 minutes, greater than about 4 minutes, greater than about 6 minutes, greater than about 7 minutes, or longer, as measured by surface plasmon resonance, for example using the assay form defined in Example 3 herein, or a substantially similar assay.

[0116] This disclosure also includes antibodies that bind to dimer human GITRs (e.g., hGITR.mFc) with high affinity and their antigen-binding fragments. For example, this disclosure includes K+ with a concentration of less than about 1 nM. D Anti-GITR antibodies binding to the human GITR dimer are measured, for example, by surface plasmon resonance at 25°C, using the assay form defined in Example 3 herein, or a substantially similar assay. According to certain embodiments, anti-GITR antibodies are provided that, at 25°C, have a Kc of less than about 1 nM, less than about 900 pM, less than about 800 pM, less than about 700 pM, less than about 600 pM, less than about 500 pM, less than about 400 pM, less than about 300 pM, less than about 200 pM, or less than about 100 pM. DCombined with human GITR dimers, measured, for example, by surface plasmon resonance, using the assay form defined in Example 3 herein, or a substantially similar assay. This disclosure also includes Ki at concentrations less than about 5 nM, less than about 1 nM, less than about 300 pM, or less than about 100 pM. D Anti-GITR antibodies binding to human GITR dimers were measured, for example, by surface plasmon resonance at 25°C, using the assay form provided in Example 3 of this document, wherein the antibody was secreted in conditioned culture.

[0117] This disclosure also includes antibodies and antigen-binding fragments thereof that bind to dimer human GITR (e.g., hGITR.mFc) with a dissociation half-life (t1 / 2) greater than about 4 minutes, as measured by surface plasmon resonance at 25°C, for example using the assay form defined in Example 3 herein, or a substantially similar assay. According to some embodiments, anti-GITR antibodies are provided that bind to dimer human GITR at 25°C with a t1 / 2 greater than about 4 minutes, greater than about 9 minutes, greater than about 10 minutes, greater than about 35 minutes, or greater than about 70 minutes or longer, as measured by surface plasmon resonance, for example using the assay form defined in Example 3 herein, or a substantially similar assay.

[0118] This disclosure also includes antibodies that bind to Jurkat / hCD20 target cells expressing human or monkey GITR and antigen-binding fragments thereof. For example, antibodies are provided herein that bind with high affinity to Jurkat / hCD20 cells expressing human or monkey GITR. For example, this disclosure includes EC50 fragments with an affinity of less than about 4 nM. 50 Anti-GITR antibodies against Jurkat / hCD20 target cells expressing human GITR are measured, for example, by mean fluorescence intensity (MFI), using the assay form defined in Example 7 herein, or a substantially similar assay. In some embodiments, EC50 at concentrations less than about 4 nM, less than about 3 nM, or less than about 2 nM is provided. 50 Anti-GITR antibodies against Jurkat / hCD20 target cells expressing human or monkey GITR are measured, for example, by MFI, using the assay form defined in Example 7 of this document, or a substantially similar assay.

[0119] This disclosure also includes antibodies and antigen-binding fragments thereof that bind to anti-CD3 / anti-CD28 stimulated human and cynomolgus monkey primary T cells. CD25 and CD69 are alternative biomarkers for activation of human and cynomolgus monkey primary T cells, respectively. For example, antibodies that bind with high affinity to anti-CD3 / anti-CD28 stimulated human and cynomolgus monkey primary T cells are provided herein using the assay form defined in Example 8 herein, or a substantially similar assay. In some aspects, anti-GITR antibodies or antigen-binding fragments thereof may be combined with PD-1 inhibitors such as cimiprilmab. While not wishing to limit oneself to theory, cimiprilmab can restore the ability of anti-GITR antibodies to enhance anti-CD3 stimulated T cell activation in the presence of inhibitory PD-L1 / PD1 signaling.

[0120] This disclosure also includes antibodies and antigen-binding fragments thereof that mediate NFAT activation via human or cynomolgus monkey FcγR3a (an Fc receptor that mediates ADCC and is primarily expressed on NK cells). For example, this document provides antibodies that mediate NFAT activation via human or cynomolgus monkey FcγR3a, as evaluated in ADCC reporter factor bioassays. For example, this disclosure includes, for instance, antibodies that mediate NFAT activation via human or cynomolgus monkey FcγR3a in ADCC reporter factor bioassays at EC50 values ​​less than about 32 pM. 50 Anti-GITR antibodies activated by NFAT mediated by human or cynomolgus monkey FcγR3a are measured, for example by luminescence, using the assay form defined in Example 9 of this document, or a substantially similar assay. In some embodiments, EC50 at less than about 32 pM or less than about 16 pM is provided. 50 Anti-GITR antibodies activated by human or cynomolgus monkey FcγR3a-mediated NFAT, as measured by luminescence, for example using the assay form defined in Example 9 of this document, or substantially similar assays.

[0121] The antibodies disclosed herein can function via complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC). “Complement-dependent cytotoxicity” (CDC) refers to the lysis of cells expressing antigens by the antibodies provided herein in the presence of complement. “Antibody-dependent cell-mediated cytotoxicity” (ADCC) refers to a cell-mediated response in which nonspecific cytotoxic cells expressing Fc receptors (FcRs) (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize antibodies bound to target cells, thereby causing lysis of the target cells. CDC and ADCC can be measured using assays well known and available in the art. (See, for example, U.S. Patent Nos. 5,500,362 and 5,821,337, and Clynes et al. (1998) Proc. Natl. Acad. Sci. (USA) 95:652-656). The constant region of an antibody is important for its ability to fix complement and mediate cell-dependent cytotoxicity. Therefore, antibody isotypes can be selected based on whether antibody-mediated cytotoxicity requires the antibody.

[0122] This disclosure also includes antibodies that mediate ADCC and their antigen-binding fragments. For example, this document provides antibodies that mediate ADCC against Jurkat / hCD20 / hGITR and Jurkat / hCD20 / MfGITR (target cells) in the presence of human primary NK cells (effector cells). For example, this disclosure includes EC50 at less than about 14 pM in the presence of human primary NK cells. 50 Anti-GITR antibodies mediating ADCC against Jurkat / hCD20 / hGITR and Jurkat / hCD20 / MfGITR are measured, for example, by luminescence, using the assay form defined in Example 10 herein, or a substantially similar assay. In some embodiments, ECGs are measured in the presence of human primary NK cells at concentrations of less than about 14 pM, or less than about 13 pM, or less than about 12 pM, or less than about 11 pM, or less than about 10 pM. 50 Mediating ADCC for Jurkat / hCD20 / hGITR and Jurkat / hCD20 / MfGITR, such as by luminescence, measured, for example, using the assay form defined in Example 10 of this document, or a substantially similar assay.

[0123] In another example, this document provides antibodies that mediate ADCC against human primary T cells (target cells) in the presence of human primary NK cells (effector cells). For example, this disclosure includes sub-nanomolar EC in the presence of human primary NK cells. 50Anti-GITR antibodies mediating ADCC against human primary T cells, such as those measured by luminescence, for example using the assay form defined in Example 11 of this document, or substantially similar assays.

[0124] This disclosure also includes antibodies that mediate ADCPs and their antigen-binding fragments. For example, this document provides antibodies that mediate concentration-dependent ADCPs against Jurkat / hCD20 / hGITR and Jurkat / hCD20 / MfGITR (target cells) in the presence of phagocytes (effector cells) derived from human primary monocytes. For example, this disclosure includes antibodies that mediate ADCPs at sub-nanomolar levels in the presence of phagocytes derived from human primary monocytes. 50 Anti-GITR antibodies mediating concentration-dependent ADCPs of Jurkat / hCD20 / hGITR and Jurkat / hCD20 / MfGITR, as measured by green fluorescence intensity, for example using the assay form defined in Example 12 of this document, or substantially similar assays.

[0125] This disclosure also includes antibodies that enhance anti-CD3-mediated T cell proliferation and their antigen-binding fragments. For example, this document provides enhancement of primary CD4 cell proliferation mediated by stimulating CD3 antibodies in the presence of HEK293 / FcγR2b helper cells. + Antibodies that promote T cell proliferation. For example, this disclosure includes antibodies that enhance primary CD4 cell proliferation mediated by stimulating CD3 antibodies in the presence of HEK293 / FcγR2b helper cells. + Anti-GITR antibodies against T cell proliferation, EC 50 The values ​​were in the sub-nanomolar range and the maximum T cell proliferation (measured in CPM of tritium decay) was 2.3 to 3.1 times the background, for example, measured using a assay form as defined in Example 13 herein, or a substantially similar assay.

[0126] In some respects, this article provides isolated antibodies or antigen-binding fragments thereof with N101 or S103 modifications in the HCVR amino acid sequence. Anti-GITR antibodies or antigen-binding fragments thereof with such modifications may further exhibit one or more properties selected from the following:

[0127] (a) As measured by surface plasmon resonance, KD binds to human GITR monomers at 25 °C at a concentration of less than about 12 nM;

[0128] (b) Binding of human GITR monomers at 25°C for more than 2 minutes at t1 / 2;

[0129] (c) KD-binding dimer human GITR at 25 °C with less than about 1 nM, as measured by surface plasmon resonance; and

[0130] (d) Binding of human GITR dimer at 25°C for more than 5 minutes at t1 / 2.

[0131] In some respects, this article provides isolated antibodies or antigen-binding fragments thereof with N101 or S103 modifications in the HCVR amino acid sequence. Anti-GITR antibodies or antigen-binding fragments thereof with such modifications may further exhibit one or more properties selected from the following:

[0132] (a) with EC less than about 4 nM 50 Binding to GITR on the surface of human and cynomolgus monkey cells;

[0133] (b) with EC less than approximately 40 pM 50 Enhance Fc-mediated NFAT activity in Jurkat / NFAT-Luc / hFcγR3a and / or Jurkat / NFAT-Luc / MfFcγR3a effector cells;

[0134] (c) EC at less than approximately 20 pM in the presence of primary human NK cells 50 Mediates ADCC targeting human primary T cells;

[0135] (d) Compared to Jurkat T cells that do not express GITR, Jurkat T cells engineered to express human or cynomolgus monkey GITR mediate at least about 5-fold ADCP;

[0136] (e) Enhances the proliferation of anti-CD3-mediated primary CD4+ T cells;

[0137] (f) By preferentially consuming intratumoral T cells in an FcγR-dependent manner reg To induce anti-tumor immunity; and

[0138] (g) with an IC of less than about 7 nM 50 It blocks the binding of human monomeric GITR to human GITR ligand.

[0139] The antibodies of the present invention may have one or more of the foregoing biological characteristics, or any combination thereof. The foregoing list of biological characteristics of the antibodies provided herein is not intended to be exhaustive. Other biological characteristics of the antibodies of this disclosure will be apparent to those skilled in the art from a review of this disclosure, including the examples of work herein.

[0140] Antibodies that block the binding of GITR to GITR ligands

[0141] This disclosure includes antibodies that block the binding of human GITR ligand (hGITRL) to human GITR, for example, as determined in the assay form described in Example 14 herein.

[0142] In some embodiments, the antibodies provided herein block human GITR ligand (hGITRL). In some embodiments, the antibody or its antibody-binding fragment blocks human GITR ligand with a blocking percentage greater than about 90%, IC50. 50 Less than about 7.0 nM, as described in Example 14 or a substantially similar assay. In some embodiments, the antibody or its antibody-binding fragment blocks the human GITR ligand by a blocking percentage greater than about 90%, greater than about 95%, or greater than about 98%, IC50. 50 Less than about 10 nM, less than about 7.0 nM, less than about 5.0 nM, or less than about 3.0 nM, as described in Example 14 or a substantially similar assay.

[0143] Epitope plotting and related techniques

[0144] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site (called a complementary site) in the variable region of an antibody molecule. A single antigen can have more than one epitope. Therefore, different antibodies can bind to different regions on the antigen and can have different biological effects. Epitopes can be conformational or linear. Conformational epitopes are generated by the spatial juxtaposition of amino acids from different segments of a linear polypeptide chain. Linear epitopes are generated from neighboring amino acid residues in the polypeptide chain. In some cases, epitopes may include sugar, phosphoryl, or sulfonyl groups on the antigen.

[0145] The antibody-binding epitope of this disclosure may consist of a single adjacent sequence of 3 or more amino acids (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) of the GITR protein. Alternatively, the epitope may consist of multiple non-adjacent amino acids (or amino acid sequences) of the GITR. In some embodiments, the epitope is located on or near the GITRL-binding domain of the GITR. In other embodiments, the epitope is located outside the GITR-binding domain of the GITR, for example, on the surface of the GITR, without interfering with the binding site of GITRL to the GITR when the antibody binds to such an epitope.

[0146] Various techniques known to those skilled in the art can be used to determine whether an antibody "interacts with one or more amino acids" within a peptide or protein. Exemplary techniques include, for example, conventional cross-blocking assays (such as...) Antibodies(As described in Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY)), alanine scanning mutation analysis, peptide blotting analysis (Reineke, 2004, Methods Mol Biol 248:443-463), and peptide cleavage analysis. Additionally, methods such as epitope excision, epitope extraction, and chemical modification of the antigen can be employed (Tomer, 2000, Protein Science 9:487-496). Another method for identifying amino acids within peptides that interact with antibodies is hydrogen / deuterium exchange detected by mass spectrometry. Generally, the hydrogen / deuterium exchange method involves deuterating the protein of interest and then binding an antibody to the deuterated protein. The protein / antibody complex is then transferred to water to allow hydrogen-deuterium exchange to occur at all residues except those protected by the antibody (which remain deuterated). After antibody dissociation, the target protein is cleaved by protease and analyzed by mass spectrometry to reveal the deuterated residues corresponding to the specific amino acids that interact with the antibody. See, for example, Ehring (1999) Analytical Biochemistry 267(2):252-259; Engen and Smith (2001) Anal. Chem. 73:256A-265A.

[0147] This disclosure also includes anti-GITR antibodies that bind to the same epitope as any of the specific exemplary antibodies described herein (e.g., antibodies containing any of the amino acid sequences listed in Table 7 herein). Similarly, this disclosure also includes anti-GITR antibodies that compete with any of the specific exemplary antibodies described herein (e.g., antibodies containing any of the amino acid sequences listed in Table 7 herein) for binding to GITR.

[0148] Using conventional methods known in the art and exemplified herein, it can be readily determined whether an antibody binds to the same epitope as a reference anti-GITR antibody or competes for binding with a reference anti-GITR antibody. For example, to determine whether an experimental antibody binds to the same epitope as a reference anti-GITR antibody provided herein, the reference antibody is bound to the GITR protein. The ability of the experimental antibody to bind to the GITR molecule is then evaluated. If the experimental antibody binds to the GITR after saturation binding with the reference anti-GITR antibody, it can be concluded that the experimental antibody binds to a different epitope than the reference anti-GITR antibody. On the other hand, if the experimental antibody does not bind to the GITR molecule after saturation binding with the reference anti-GITR antibody, the experimental antibody may bind to the same epitope as the reference anti-GITR antibody provided herein. Additional routine experiments (e.g., peptide mutation and binding assays) can then be performed to confirm whether the observed lack of binding by the experimental antibody is actually due to binding to the same epitope as the reference antibody or whether steric hindrance (or another phenomenon) causes the observed lack of binding. Such experiments can be performed using ELISA, RIA, Biacore, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art. According to certain embodiments of this disclosure, if, for example, an excess of 1, 5, 10, 20, or 100 times that of one antibody inhibits the binding of another antibody by at least 50%, but preferably 75%, 90%, or even 99%, as measured in a competitive binding assay, then the two antibodies bind to the same (or overlapping) epitope (see, for example, Junghans et al., Cancer Res. 1990:50:1495-1502). Alternatively, if substantially all amino acid mutations in the antigen that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of the other antibody, then the two antibodies are considered to bind to the same epitope. If only a subset of amino acid mutations that reduce or eliminate the binding of one antibody reduces or eliminates the binding of the other antibody, then the two antibodies are considered to have “overlapping epitopes.”

[0149] To determine whether an antibody competes with (or cross-competes with) a reference anti-GITR antibody for binding, the binding method described above is performed in two directions: In the first direction, the reference antibody is bound to the GITR protein under saturation conditions, and then the binding of the test antibody to the GITR molecule is evaluated. In the second direction, the test antibody is bound to the GITR molecule under saturation conditions, and then the binding of the reference antibody to the GITR molecule is evaluated. If only the first (saturated) antibody can bind to the GITR molecule in both directions, it can be concluded that the test antibody and the reference antibody compete for binding to the GITR. As will be understood by those skilled in the art, the antibody competing for binding with the reference antibody may not necessarily bind to the same epitope as the reference antibody, but may spatially block the binding of the reference antibody by binding to overlapping or adjacent epitopes.

[0150] Preparation of human antibodies

[0151] The anti-GITR antibody disclosed herein may be a fully human antibody. Methods for generating monoclonal antibodies (including fully human monoclonal antibodies) are known in the art. Any such known methods may be used in the context of this disclosure to prepare human antibodies that specifically bind to human GITR.

[0152] Use, for example, VELOCIMMUNE TM This technique, or any other similar known method for generating fully human monoclonal antibodies, initially isolates a high-affinity chimeric antibody against GITR having a human variable region and a mouse constant region. As described in the Experimental Section below, the desired characteristics of the antibody are characterized and selected, including affinity, ligand blocking activity, selectivity, epitopes, etc. If desired, the mouse constant region is replaced with the desired human constant region (e.g., wild-type or modified IgG1 or IgG4) to generate a fully human anti-GITR antibody. While the selected constant region can vary depending on the specific application, high-affinity antigen binding and target-specific characteristics are present in the variable region. In some cases, fully human anti-GITR antibodies are isolated directly from antigen-positive B cells.

[0153] bioequivalent

[0154] The anti-GITR antibodies and antibody fragments disclosed herein encompass proteins having an amino acid sequence different from that of the antibodies but retaining the ability to bind human GITR. When compared with a parental sequence, such variant antibodies and antibody fragments contain one or more additions, deletions, or substitutions of amino acids but exhibit biological activity substantially equivalent to that of the antibodies. Similarly, the DNA sequences encoding anti-GITR antibodies disclosed herein encompass sequences containing one or more additions, deletions, or substitutions of nucleotides compared to the disclosed sequences, but encoding anti-GITR antibodies or antibody fragments substantially bioequivalent to the anti-GITR antibodies or antibody fragments of the present invention. Examples of such variant amino acid and DNA sequences have been discussed above.

[0155] Two antigen-binding proteins or antibodies are considered bioequivalent if, for example, they are drug equivalents or drug substitutes that do not show significant differences in absorption rate and extent when administered at the same molar dose in a single or multiple doses under similar experimental conditions. If some antibodies are identical in extent of absorption but differ in absorption rate, they may be considered equivalents or drug substitutes, but still bioequivalent because this difference in absorption rate is intentional and reflected in the product information, is not necessary for achieving effective in vivo drug concentrations (e.g., in long-term use), and is considered medically irrelevant to the specific drug product under investigation.

[0156] In one implementation, two antigen-binding proteins are bioequivalent if they do not differ clinically in terms of their safety, purity, and potency.

[0157] In one implementation, the two antigen-binding proteins are bioequivalent if a patient can make one or more such switches without an expected increase in the risk of adverse reactions, including clinically significant changes in immunogenicity or reduced effectiveness, compared to continuous therapy without switching between the reference product and the biological product.

[0158] In one implementation, the two antigen-binding proteins are bioequivalent if they function through one or more co-operating mechanisms targeting one or more conditions of use to the extent that such mechanisms are known.

[0159] Bioequivalence can be demonstrated through in vivo and in vitro methods. Bioequivalence measures include, for example, (a) in vivo testing in humans or other mammals, in which the concentration of an antibody or its metabolites in blood, plasma, serum, or other biological fluids over time is measured; (b) in vitro testing that is correlated with and can reasonably predict in vivo bioavailability data; (c) in vivo testing in humans or other mammals, in which appropriate acute pharmacological effects of the antibody (or its target) over time are measured; and (d) clinical trials that establish well-controlled safety, efficacy, or bioavailability or bioequivalence of the antibody.

[0160] Bioequivalent variants of the anti-GITR antibodies of the present invention can be constructed, for example, by various substitutions of residues or sequences, or by deletion of terminal or internal residues or sequences not required for biological activity. For example, cysteine ​​residues that are not essential for biological activity can be deleted or replaced with other amino acids to prevent the formation of unwanted or incorrect intramolecular disulfide bridges during renaturation. In other cases, the bioequivalent antibody may comprise an anti-GITR antibody variant comprising amino acid alterations that modify the glycosylation characteristics of the antibody, such as mutations that eliminate or remove glycosylation.

[0161] Species selectivity and species cross-reactivity

[0162] According to certain embodiments, this disclosure provides anti-GITR antibodies that bind to human GITR but not to GITRs from other species. The invention also includes anti-GITR antibodies that bind to human GITRs and GITRs from one or more non-human species. For example, the anti-GITR antibodies provided herein may bind to human GITRs and, depending on the circumstances, may bind to or not bind to one or more of the GITRs of mice, rats, guinea pigs, hamsters, gerbils, pigs, cats, dogs, rabbits, goats, sheep, cows, horses, camels, cynomolgus monkeys, marmosets, rhesus monkeys, or chimpanzees. According to certain exemplary embodiments of this disclosure, anti-GITR antibodies are provided that specifically bind to human GITRs and cynomolgus monkey (e.g., cynomolgus macaque) GITRs. Other anti-GITR antibodies provided herein bind to human GITRs but do not bind to or only weakly bind to cynomolgus monkey GITRs.

[0163] Multispecific antibodies

[0164] The antibodies disclosed herein can be monospecific or multispecific (e.g., bispecific). Multispecific antibodies can be specific to different epitopes of a single target polypeptide, or can contain antigen-binding domains specific to more than one target polypeptide. See, for example, Tutt et al., 1991, J. Immunol. 147:60-69; Kufer et al., 2004, Trends Biotechnol. 22:238-244. The anti-GITR antibodies of this disclosure can be linked to another functional molecule (e.g., another peptide or protein) or co-expressed with another functional molecule. For example, an antibody or fragment thereof can be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent association, or other means) to one or more other molecular entities (such as another antibody or antibody fragment) to produce a bispecific or multispecific antibody with a second binding specificity.

[0165] This disclosure includes a bispecific antibody wherein one arm of an immunoglobulin binds to human GITR, while the other arm of the immunoglobulin is specific for a second antigen. The GITR-binding arm may comprise any of the HCVR / LCVR or CDR amino acid sequences listed in Table 7 herein. In some embodiments, the GITR-binding arm binds to human GITR and blocks the binding of GITRL to GITR. In other embodiments, the GITR-binding arm binds to human GITR but does not block the binding of GITRL to GITR. In some embodiments, the GITR-binding arm binds to human GITR and activates GITR signaling. In other embodiments, the GITR-binding arm blocks GITRL-mediated receptor stimulation. This disclosure also includes a bispecific antibody wherein one arm of the antibody binds to a first epitope of human GITR, while the other arm of the antibody binds to a second, different epitope of human GITR.

[0166] Exemplary bispecific antibody forms that may be used in the context of this disclosure involve the use of a first immunoglobulin (Ig) C H 3 structural domains and second Ig C H 3 structural domains, where the first and second IgC H The three domains differ from each other by at least one amino acid, and wherein at least one amino acid difference reduces the binding of the bispecific antibody to protein A compared to a bispecific antibody without amino acid differences. In one embodiment, the first IgC H 3-domain binding protein A and second IgC H Domain 3 contains mutations that reduce or eliminate protein A binding, such as H95R modification (based on IMGT exon numbering; H435R based on EU numbering). Second C H 3 may also include Y96F modifications (according to IMGT; according to EU Y436F). In the second CH Other modifications that may be found within 3 include: in the case of IgG1 antibodies, D16E, L18M, N44S, K52N, V57M, and V82I (according to IMGT; D356E, L358M, N384S, K392N, V397M, and V422I according to EU); in the case of IgG2 antibodies, N44S, K52N, and V82I (IMGT; N384S, K392N, and V422I according to EU); and in the case of IgG4 antibodies, Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (according to IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I according to EU). These variations in the form of bispecific antibodies are covered within the scope of this disclosure.

[0167] Other exemplary bispecific forms that may be used in the context of this disclosure include, but are not limited to, scFv-based or bispecific forms, IgG-scFv fusions, dual variable domain (DVD)-Ig, tetrageneous hybridomas, buttonholes, common light chains (e.g., common light chains with buttonholes), CrossMab, CrossFab, (SEED) bodies, leucine zippers, Duobody, IgG1 / IgG2, dual-action Fab (DAF)-IgG and Mab 2 Bispecific forms (for a review of the aforementioned forms, see, for example, Klein et al., 2012, mAbs 4:6, 1-11, and the references cited therein). Bispecific antibodies can also be constructed using peptide / nucleic acid conjugations, for example, where non-natural amino acids with orthogonal chemical reactivity are used to generate site-specific antibody-oligonucleotide conjugations, which are then self-assembled into multimeric complexes with defined composition, valence, and geometry. (See, for example, Kazane et al., J. Am. Chem. Soc. [Epub: December 4, 2012]).

[0168] Therapeutic preparations and administration

[0169] This disclosure provides pharmaceutical compositions comprising the anti-GITR antibody or its antigen-binding fragment thereof. The pharmaceutical compositions provided herein are formulated with suitable carriers, excipients, and other agents that provide improved transfer, delivery, tolerability, etc. Numerous suitable formulations can be found in all formularies known to pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, and lipid-containing (cationic or anionic) vesicles (such as LIPOFECTIN). TM Life Technologies, Carlsbad, CA), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsion carbon waxes (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbon waxes. See also Powell et al., “Compendium of excipients for parenteral formulations”, PDA (1998), J Pharm Sci Technol 52:238-311.

[0170] The dosage of the antibody administered to a patient can vary depending on the patient's age and size, the target disease, the condition, the route of administration, etc. Preferred dosages are typically calculated based on body weight or body surface area. In adult patients, it may be advantageous to administer the disclosed antibody intravenously in a single dose of about 0.01 to about 20 mg / kg body weight, more preferably about 0.02 to about 7, about 0.03 to about 5, or about 0.05 to about 3 mg / kg body weight. The frequency and duration of treatment can be adjusted depending on the severity of the condition. Effective dosages and schedules for anti-GITR antibody administration can be determined empirically; for example, patient progression can be monitored through periodic assessments, and the dosage adjusted accordingly. Furthermore, interspecies scaling of dosages can be performed using methods well known in the art (e.g., Mordenti et al., 1991, Pharmaceut. Res. 8:1351).

[0171] Various delivery systems are known and can be used to administer the pharmaceutical compositions provided herein, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, and receptor-mediated endosomes (see, for example, Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Methods of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compositions can be administered via any convenient route, such as by infusion or bolus injection, by absorption through the epithelial or mucosal inner layer of the skin (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and can be administered together with other bioactive agents. Administration can be systemic or local.

[0172] The pharmaceutical compositions of this disclosure can be delivered subcutaneously or intravenously using standard needles and syringes. Furthermore, for subcutaneous delivery, pen-type delivery devices are readily applicable for delivering the pharmaceutical compositions of this disclosure. Such pen-type delivery devices can be reusable or disposable. Reusable pen-type delivery devices generally utilize a replaceable cartridge containing the pharmaceutical composition. Once all the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen-type delivery device can then be reused. In disposable pen-type delivery devices, there is no replaceable cartridge. In fact, disposable pen-type delivery devices have a reservoir pre-filled with the pharmaceutical composition within the device. Once the pharmaceutical composition in the reservoir is emptied, the entire device is discarded.

[0173] Various reusable pen-type delivery devices and auto-injector delivery devices are used for subcutaneous delivery of the pharmaceutical compositions disclosed herein. Examples include, but are not limited to, AUTOPEN. TM (Owen Mumford, Inc., Woodstock, UK), DISSETRONIC TM Pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX75 / 25 TM Pen, HUMALOG TM Pen, HUMALIN 70 / 30 TM Pen (Eli Lilly and Co., Indianapolis, IN), NOVOPEN TM I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR TM (NovoNordisk,Copenhagen,Denmark)、BD TMPen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN TM OPTIPEN PRO TM OPTIPEN STARLET TM and OPTICLIK TM (sanofi-aventis, Frankfurt, Germany), etc. Examples of disposable pen delivery devices that can be used for subcutaneous delivery of the pharmaceutical compositions disclosed herein include, but are not limited to, SOLOSTAR. TM Pen (sanofi-aventis), FLEXPEN TM (Novo Nordisk) and KWIKPEN TM (EliLilly), SURECLICK TM Automatic injectors (Amgen, Thousand Oaks, CA), PENLET TM (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP), and HUMIRA TM Pens (Abbott Labs, Abbott Park IL), etc.

[0174] In some cases, the drug composition can be delivered in a controlled-release system. In one embodiment, a pump can be used (see Langer, ibid.; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201). In another embodiment, polymeric materials can be used; see Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida. In yet another embodiment, the controlled-release system can be placed near the target of the composition, thus requiring only a fraction of the systemic dose (see, for example, Goodson, 1984, in Medical Applications of Controlled Release, ibid., Vol. 2, pp. 115-138). Other controlled-release systems are discussed in the review in Langer, 1990, Science 249:1527-1533.

[0175] Injectable formulations can include dosage forms for intravenous, subcutaneous, intradermal, and intramuscular injection, infusion, etc. These injectable formulations can be prepared using well-known methods. For example, injectable formulations can be prepared by dissolving, suspending, or emulsifying, for instance, the antibodies or their salts described above in a sterile aqueous or oily medium conventionally used for injection. Aqueous media for injection include, for example, physiological saline, isotonic solutions containing glucose and other adjuvants, which can be used in combination with suitable solubilizers (such as alcohols (e.g., ethanol), polyols (e.g., propylene glycol, polyethylene glycol), nonionic surfactants [e.g., polysorbate 80, HCO-50 (a polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc.]. Oily media include, for example, sesame oil, soybean oil, which can be used in combination with solubilizers such as methyl benzoate, benzyl alcohol, etc. The resulting injection is preferably filled in a suitable ampoule.

[0176] Advantageously, the pharmaceutical compositions described above for oral or oral administration are prepared into dosage forms suitable for unit doses corresponding to the dosage of the active ingredient. Such unit-dose dosage forms include, for example, tablets, pills, capsules, ampoules, suppositories, etc. The amount of the aforementioned antibody contained is typically about 5 to about 500 mg per dosage form in a unit dose; particularly in injectable forms, for other dosage forms, the amount of the aforementioned antibody contained is preferably about 5 to about 100 mg and about 10 to about 250 mg.

[0177] Therapeutic uses of antibodies

[0178] This disclosure includes methods comprising administering a therapeutic composition comprising an anti-GITR antibody (e.g., an anti-GITR antibody comprising any of the HCVR / LCVR or CDR sequences listed in Table 7 herein) to a subject in need. The therapeutic composition may comprise any of the anti-GITR antibodies disclosed herein, their antigen-binding fragments, or ADCs, and a pharmaceutically acceptable carrier or diluent.

[0179] The antibodies provided herein are particularly useful for treating, preventing, and / or improving any disease or condition associated with or mediated by GITR expression or activity, or any disease or condition that can be treated by blocking the interaction between GITR and GITRL, and / or stimulating GITR activity and / or signaling. For example, the antibodies and antigen-binding fragments disclosed herein can be used to treat immune and proliferative diseases or conditions, such as cancer, by modulating immune responses, i.e., antitumor responses, through, for example, GITR activation.

[0180] In some embodiments, the antibodies described herein do not rely on their ligand blocking ability (e.g., via ADCC) to consume cells expressing high levels of GITR. The antibodies and antigen-binding fragments of this disclosure can be used to treat diseases or conditions by enhancing immune responses. This disclosure includes methods for modulating an anti-tumor immune response in a subject, the method comprising administering the anti-GITR antibody or antigen-binding fragment described herein to the subject. In some embodiments, the antibodies or antigen-binding fragments of this disclosure enhance the ratio of intratumoral T effector cells to T regulatory cells that is beneficial for treatment. In some embodiments, the antibodies or antigen-binding fragments of this disclosure enhance the consumption of intratumoral T regulatory cells that is beneficial for treatment. In some embodiments, the antibodies or antigen-binding fragments of this disclosure increase the ratio of CD8+ T cells to T regulatory cells that is beneficial for treatment. In some embodiments, the antibodies or antigen-binding fragments of this disclosure promote T cell survival.

[0181] Example diseases or conditions that can be treated with antibody-antigen binding fragments include immune and proliferative diseases or conditions, such as cancer. The antibody-antigen binding fragments disclosed herein can be used to treat primary and / or metastatic tumors occurring in the brain and meninges, oropharynx, lungs and bronchial tree, gastrointestinal tract, male and female reproductive tracts, muscles, bones, skin and appendages, connective tissue, spleen, immune system, hematopoietic cells and bone marrow, liver and urinary tract, and special sensory organs such as the eyes. In some embodiments, the antibody-antigen binding fragments disclosed herein are used to treat solid tumors or hematogenous tumors. In some embodiments, the antibodies provided herein are used to treat one or more of the following: squamous cell carcinoma of the skin, cutaneous squamous cell carcinoma (CSCC), myeloma, lung cancer, melanoma, head and neck squamous cell carcinoma (SCCHN), small cell lung cancer, non-small cell lung cancer (NSCLC), cervical cancer such as cervical squamous cell carcinoma (cervical SCC), breast cancer and renal cell carcinoma (RCC), adenocarcinoma, colorectal cancer (CRC), pancreatic cancer, head and neck cancer, prostate cancer, malignant glioma, osteosarcoma, colorectal cancer, gastric cancer (e.g., gastric cancer with MET amplification), malignant mesothelioma, multiple myeloma, ovarian cancer, synovial sarcoma, thyroid cancer, breast cancer (including triple-negative breast cancer), testicular cancer, esophageal cancer, uterine cancer, endometrial cancer, or liver cancer. In some aspects, the antibodies of this disclosure are used to treat immune checkpoint blockade (ICB)-initiated cancers. In some aspects, the antibodies of this disclosure are used to treat ICB-experienced cancers.

[0182] In some aspects, the antibodies disclosed herein are used to treat colorectal cancer. In some aspects, the antibodies disclosed herein are used to treat head and neck squamous cell carcinoma (HNSCC or SCCHN). In some aspects, the antibodies disclosed herein are used to treat cutaneous squamous cell carcinoma (CSCC). In some aspects, the antibodies disclosed herein are used to treat gastric cancer. In some aspects, the antibodies disclosed herein are used to treat non-small cell lung cancer (NSCLC). In some aspects, the antibodies disclosed herein are used to treat cervical cancer. In some aspects, the antibodies disclosed herein are used to treat esophageal cancer. In some aspects, the antibodies disclosed herein are used to treat melanoma. In some aspects, the antibodies disclosed herein are used to treat triple-negative breast cancer. In some aspects, the antibodies disclosed herein are used to treat renal cell carcinoma. In some aspects, the antibodies disclosed herein are used to treat breast cancer.

[0183] In some implementations, the antibodies provided herein can be used to treat autoimmune diseases, including but not limited to alopecia areata, autoimmune hepatitis, celiac disease, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic anemia, inflammatory bowel disease, inflammatory myopathy, multiple sclerosis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma, and Sjögren's syndrome. (Syndrome), systemic lupus erythematosus, vitiligo, autoimmune pancreatitis, autoimmune urticaria, autoimmune thrombocytopenic purpura, Crohn's disease, type I diabetes, eosinophilic fasciitis, eosinophilic gastroenteritis, Goodpasture's syndrome, myasthenia gravis, psoriatic arthritis, rheumatic fever, ulcerative colitis, vasculitis, and Wegener's granulomatosis.

[0184] In the context of the treatment methods described herein, anti-GITR antibodies can be administered as monotherapy (i.e., as the sole treatment agent) or in combination with one or more other treatment agents (examples of which are described elsewhere herein).

[0185] Combination therapies and formulations

[0186] This document also provides combination therapies utilizing the anti-GITR antibody of this disclosure and any other therapeutic agents that can be advantageously combined with the antibody or its antigen-binding fragment of this disclosure.

[0187] This disclosure includes compositions and therapeutic formulations comprising any of the anti-GITR antibodies described herein combined with one or more additional therapeutically active components, and treatment methods comprising administering such combinations to subjects in need of them.

[0188] The antibodies disclosed herein can be synergistically combined with one or more anticancer drugs or therapies for treating cancer, including, for example, in some embodiments, the antibodies provided herein for treating one or more of the following: squamous cell carcinoma of the skin, cutaneous squamous cell carcinoma (CSCC), myeloma, lung cancer, melanoma, head and neck squamous cell carcinoma (SCCHN), small cell lung cancer, non-small cell lung cancer (NSCLC), cervical cancer such as cervical squamous cell carcinoma (cervical SCC), breast cancer and renal cell carcinoma (RCC), adenocarcinoma, colorectal cancer (CRC), pancreatic cancer, head and neck cancer, prostate cancer, glioblastoma multiforme, malignant glioma, osteosarcoma, colorectal cancer, gastric cancer (e.g., gastric cancer with MET amplification), malignant mesothelioma, multiple myeloma, ovarian cancer, synovial sarcoma, thyroid cancer, breast cancer (including triple-negative breast cancer), testicular cancer, esophageal cancer, uterine cancer, endometrial cancer, or liver cancer. In some aspects, the antibodies disclosed herein are used to treat immune checkpoint blockade (ICB)-initiated cancers. In some respects, the antibodies disclosed herein are used to treat ICB-induced cancer.

[0189] In some aspects, the antibodies disclosed herein are used to treat colorectal cancer. In some aspects, the antibodies disclosed herein are used to treat head and neck squamous cell carcinoma (HNSCC or SCCHN). In some aspects, the antibodies disclosed herein are used to treat cutaneous squamous cell carcinoma (CSCC). In some aspects, the antibodies disclosed herein are used to treat gastric cancer. In some aspects, the antibodies disclosed herein are used to treat non-small cell lung cancer (NSCLC). In some aspects, the antibodies disclosed herein are used to treat cervical cancer. In some aspects, the antibodies disclosed herein are used to treat esophageal cancer. In some aspects, the antibodies disclosed herein are used to treat melanoma. In some aspects, the antibodies disclosed herein are used to treat triple-negative breast cancer. In some aspects, the antibodies disclosed herein are used to treat renal cell carcinoma. In some aspects, the antibodies disclosed herein are used to treat breast cancer.

[0190] This article considers using the anti-GITR antibodies presented herein in combination with immunostimulatory therapy and / or immunosupportive therapy to suppress tumor growth and / or enhance cancer patient survival. Immunostimulatory therapy includes direct immunostimulation therapy, which increases immune cell activity by “releasing the brakes” or “stepping on the gas” to activate the immune response on suppressed immune cells. Examples include targeting other checkpoint receptors, vaccination, and adjuvants. Forms of immunosupportive therapy can increase tumor antigenicity by promoting immunogenic cell death, inflammation, or have other indirect effects that promote antitumor immune responses. Examples include radiation, chemotherapy, anti-angiogenic agents, and surgery.

[0191] This disclosure includes a method for modulating an antitumor immune response in a subject, the method comprising administering to the subject an anti-GITR antibody in combination with one or more agonist antibodies targeting activating receptors and one or more blocking antibodies targeting inhibitory receptors, the blocking antibodies enhancing T cell stimulation to promote tumor destruction.

[0192] This disclosure includes a method for modulating an antitumor immune response in a subject, the method comprising administering to the subject a combination of the anti-GITR antibody or antigen-binding fragment described herein and one or more isolated antibodies or antigen-binding fragments thereof, said isolated antibody or antigen-binding fragment binding to a second T-cell activation receptor (i.e., other than GITR). In some embodiments, the second T-cell activation receptor is CD28, OX40, CD137, CD27, or VEM. This disclosure also includes formulations comprising the anti-GITR antibody or antigen-binding fragment provided herein and an antibody or antigen-binding fragment binding to the second T-cell activation receptor.

[0193] In various embodiments, one or more antibodies of this disclosure may be combined with: antibodies against PD-L1, antibodies against PD-1 (e.g., nivolumab, cimiprimab), LAG-3 inhibitors, CTLA-4 inhibitors (e.g., ipilimumab), TIM3 inhibitors, BTLA inhibitors, TIGIT inhibitors, CD47 inhibitors, another T-cell co-inhibitor or ligand antagonist (e.g., antibodies against CD-28, 2B4, LY108, LAIR1, ICOS, CD160, or VISTA), indoleamine-2,3-dioxygenase (IDO) inhibitors, vascular endothelial growth factor (VEGF) antagonists [e.g., "VEGF traps", such as aflibercept or as in US 7,087,Other VEGF inhibitory fusion proteins shown in 411, or anti-VEGF antibodies or their antigen-binding fragments (e.g., bevacizumab or ranibizumab) or small molecule kinase inhibitors of the VEGF receptor (e.g., sunitinib, sorafenib, or pazopanib), Ang2 inhibitors (e.g., nesvacumab), transforming growth factor β (TGFβ) inhibitors, epidermal growth factor receptor (EGFR) inhibitors (e.g., erlotinib, cetuximab), co-stimulatory receptor agonists (e.g., glucocorticoid-induced TNFR-related protein agonists), tumor-specific antigens (e.g., CA9, CA125, melanoma-associated antigen 3 (MAGE3), carcinoembryonic antigen (CEA), vimentin, tumor-M2-PK, prostate-specific antigen (P... Antibodies against SA, mucin-1, MART-1, and CA19-9; vaccines (e.g., BCG, cancer vaccines); adjuvants that enhance antigen presentation (e.g., granulocyte-macrophage colony-stimulating factor); bispecific antibodies (e.g., CD3xCD20 bispecific antibodies, PSMAxCD3 bispecific antibodies); cytotoxins; and chemotherapy agents (e.g., dacarbazine, temozolomide, cyclophosphamide, docetaxel, doxorubicin, daunorubicin, cisplatin, carboplatin, gemcitabine, and methotrexate). The treatment options include pterin, mitoxantrone, oxaliplatin, paclitaxel, and vincristine; cyclophosphamide; radiotherapy; IL-6R inhibitors (e.g., sarilumab); IL-4R inhibitors (e.g., dupilumab); IL-10 inhibitors; cytokines (such as IL-2, IL-7, IL-21, and IL-15); antibody-drug conjugates (ADCs) (e.g., anti-CD19-DM4 ADCs and anti-DS6-DM4 ADCs); anti-inflammatory drugs (e.g., corticosteroids and nonsteroidal anti-inflammatory drugs); dietary supplements (such as antioxidants); or any palliative treatment for cancer. In some embodiments, the anti-GITR antibody of this disclosure can be used in combination with a cancer vaccine to enhance the antitumor response, said cancer vaccine including dendritic cell vaccines, oncolytic viruses, tumor cell vaccines, etc. Examples of cancer vaccines that can be used in combination with the anti-GITR antibodies disclosed herein include the MAGE3 vaccine for melanoma and bladder cancer, the MUC1 vaccine for breast cancer, and EGFRv3 (e.g., Rindopepimut) or ALVAC-CEA (for CEA+ cancers) for brain cancers (including glioblastoma multiforme).

[0194] In some embodiments, one or more anti-GITR antibodies described herein are administered in combination with one or more anti-PD1 antibodies, including but not limited to those described in U.S. Patent Publication No. 2015 / 0203579 and U.S. Patent No. 9,987,500, each of which is incorporated herein by reference in its entirety. In some embodiments, the anti-GITR antibody is an antibody derived from U.S. Patent No. 2017 / 0355774A1 and modified as described herein. In some embodiments, the anti-PD1 antibody is cimiprizumab, pembrolizumab, or nivolumab.

[0195] In some embodiments, the anti-GITR antibody provided herein may be administered in combination with radiotherapy to produce a long-lasting antitumor response and / or enhance the survival of cancer patients. In some embodiments, the anti-GITR antibody provided herein may be administered before, simultaneously with, or after radiotherapy to a cancer patient. For example, radiotherapy may be administered to the tumor lesion at one or more doses, followed by one or more doses of the anti-GITR antibody provided herein. In some embodiments, radiotherapy may be administered locally to the tumor lesion to enhance the local immunogenicity of the patient's tumor (adjuvant radiation) and / or kill tumor cells (ablative radiation), followed by systemic administration of the anti-GITR antibody provided herein. For example, intracranial radiation may be administered to a patient with brain cancer (e.g., glioblastoma multiforme) in combination with systemic administration of the anti-GITR antibody provided herein. In some embodiments, the anti-GITR antibody provided herein may be administered in combination with radiotherapy and a chemotherapy agent (e.g., temozolomide) or a VEGF antagonist (e.g., aflibercept).

[0196] In some embodiments, the anti-GITR antibody provided herein may be administered in combination with one or more antiviral drugs to treat chronic viral infections caused by LCMV, HIV, HPV, HBV, or HCV. Examples of antiviral drugs include, but are not limited to, zidovudine, lamivudine, abacavir, ribavirin, lopinavir, efavirenz, cobicistat, tenofovir, rilpivirine, and corticosteroids. In some embodiments, the anti-GITR antibody provided herein may be administered in combination with any antagonist of a LAG3 inhibitor, a CTLA-4 inhibitor, or another T-cell co-inhibitor to treat chronic viral infections.

[0197] In some embodiments, the anti-GITR antibody provided herein can be combined with an antibody targeting an Fc receptor on immune cells for the treatment of autoimmune diseases. In one embodiment, the antibody provided herein or a fragment thereof is administered in combination with an antibody or antigen-binding protein targeting a specific antigen to an autoimmune tissue. In some embodiments, the antibody provided herein or an antigen-binding fragment thereof is administered in combination with an antibody or antigen-binding protein targeting a T-cell receptor or B-cell receptor, said T-cell receptor or B-cell receptor including, but not limited to, Fcα (e.g., CD89), Fcγ (e.g., CD64, CD32, CD16a, and CD16b), CD19, etc. The antibodies or fragments thereof provided herein can be combined with any drugs or therapies known in the art (e.g., corticosteroids and other immunosuppressants) for the treatment of autoimmune diseases or conditions, including but not limited to alopecia areata, autoimmune hepatitis, celiac disease, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic anemia, inflammatory bowel disease, inflammatory myopathy, multiple sclerosis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma, Sjögren's syndrome, systemic lupus erythematosus, vitiligo, autoimmune pancreatitis, autoimmune urticaria, autoimmune thrombocytopenic purpura, Crohn's disease, type I diabetes, eosinophilic fasciitis, eosinophilic gastroenteritis, Goodpasseur syndrome, myasthenia gravis, psoriatic arthritis, rheumatic fever, ulcerative colitis, vasculitis, and Wegener's granulomatosis.

[0198] This disclosure also includes methods for modulating antitumor immune responses in subjects, the methods comprising administering to a subject a combination of the anti-GITR antibody or antigen-binding fragment described herein with one or more isolated antibodies or antigen-binding fragments thereof, said isolated antibodies or antigen-binding fragments thereof binding to a T-cell inhibitory receptor. In some embodiments, the T-cell inhibitory receptor is CTLA-4, PD-1, TIM-3, BTLA, VISTA, or LAG-3. This disclosure also includes formulations comprising the anti-GITR antibody or antigen-binding fragment thereof provided herein and an antibody or antigen-binding fragment binding to said T-cell inhibitory receptor. In some aspects, for example, in the presence of PD1 signaling, cimiprilmab restores the ability of anti-GITR antibodies to enhance anti-CD3-stimulated T-cell activation.

[0199] This disclosure also includes methods for treating cancer by administering the antibodies described herein or their antigen-binding fragments or preparations to a subject in conjunction with radiation or chemotherapy.

[0200] In some embodiments, the anti-GITR antibody of this disclosure is formulated and / or administered in combination with one or more additional therapeutically active ingredients selected from the group consisting of: EGFR antagonists (e.g., anti-EGFR antibodies [e.g., cetuximab or panitumumab] or EGFR small molecule inhibitors [e.g., gefitinib or erlotinib]), antagonists of another EGFR family member such as Her2 / ErbB2, ErbB3, or ErbB4 (e.g., anti-ErbB2 [e.g., trastuzumab or T-DM1]). [The following are listed as examples of inhibitors:] anti-ErbB3 or anti-ErbB4 antibodies or small molecule inhibitors of ErbB2, ErbB3 or ErbB4 activity; EGFRvIII antagonists (e.g., antibodies that specifically bind to EGFRvIII); cMET antagonists (e.g., anti-cMET antibodies); IGF1R antagonists (e.g., anti-IGF1R antibodies); B-raf inhibitors (e.g., vemurafenib, sorafenib, GDC-0879, PLX-4720); PDGFR-α inhibitors (e.g., anti-PDGFR-α antibodies); PDGFR-β inhibitors (e.g., anti-PDGFR-β antibodies or small molecule kinase inhibitors, such as imatinib mesylate or sunitinib malate); PDGF ligand inhibitors (e.g., anti-PDGF-A, PDGF-B, PDGF-C or PDGF-D antibodies, aptamers, siRNA, etc.); VEGF antagonists (e.g., VEGF traps such as aflibercept, see, for example, US...] 7,087,411 (also referred to herein as “VEGF inhibitory fusion protein”), anti-VEGF antibodies (e.g., bevacizumab), small molecule kinase inhibitors of the VEGF receptor (e.g., sunitinib, sorafenib, or perazapani)), DLL4 antagonists (e.g., anti-DLL4 antibodies disclosed in US2009 / 0142354, such as REGN421), Ang2 antagonists (e.g., anti-Ang2 antibodies disclosed in US 2011 / 0027286, such as H1H685P), FOLH1 antagonists (e.g., anti-FOLH1 antibodies), STEAP1 or STEAP2 antagonists (e.g., anti-STEAP1 or anti-STEAP2 antibodies), TMPRSS2 antagonists (e.g., anti-TMPRSS2 antibodies), MSLN antagonists (e.g., anti-MSLN antibodies), CA9 antagonists (e.g., anti-CA9 antibodies), uroplakin antagonists (e.g., anti-u Examples of agents that can be beneficially administered in combination with the antibodies provided herein include, for example, tamoxifen, aromatase inhibitors, and cytokine inhibitors, including small molecule cytokine inhibitors and antibodies that bind to cytokines such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-11, IL-12, IL-13, IL-17, IL-18, or their respective receptors.

[0201] This disclosure includes compositions and therapeutic formulations comprising any of the anti-GITR antibodies described herein in combination with one or more chemotherapeutic agents. Examples of chemotherapeutic agents include: alkylating agents, such as thiotepa and cyclophosphamide (Cytoxan™); alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylmelamines, including altretamine, triethylenemelamine, and triethylenephosphamide. Hydroxylenol, triethylenethiophosphoramide, and trimethylomelamine; nitrogen mustards, such as chlorambucil, chlomaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, and mechlorethamine hydrochloride. Oxygen hydrochloride, melphalan, novombhichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitroureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimnustine;Antibiotics, such as aclacinomysin, actinomycin, autramycin, azaserine, bleomycin, cactinomycin C, calicheamicin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin D, daunorubicin, detorubicin, 6-diazo-5-oxo-L-leucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, and mycophenolic acid. Folic acid, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites, such as methotrexate and fluorouracil (5-FU); folic acid analogues. Examples of analogues include: denopterin, methotrexate, pteropterin, and trimetrexate; purine analogues include: fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; and pyrimidine analogues include: ancitabine, azacitidine, 6-azouridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and fluxuridine.Androgens, such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, and testolactone; anti-adrenergics, such as aminoglutethimide, mitotane, and trilostane; folic acid supplements, such as frolinic acid; aceglatone; aldophosphamide glycoside; and aminolevulinic acid. acid); amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfomithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; mitoguazone; mitoxantrone; mopidamnol; nitraerine; pentostatin; phenamet; pirarubicin; podophyllinic acid acid); 2-ethylhydrazine; procarbazine; PSKTM; razoxane; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2”-trichlorotriethylamine; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactalol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa;Taxanes, such as paclitaxel (Taxol™, Bristol-Myers Squibb Oncology, Princeton, NJ) and docetaxel (Taxotere™; Aventis Antony, France); chloranmbucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C. C); mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; esperamicins; capecitabine; and pharmaceutically acceptable salts, acids, or derivatives of any of the above. This definition also includes anti-hormonal agents (which act to regulate or inhibit the effects of hormones on tumors), such as anti-estrogens including, for example, tamoxifen, raloxifene, 4(5)-imidazole (an aromatase inhibitor), 4-hydroxytamoxifen, trioxifene, raloxifene hydrochloride, LY117018, onapristone, and toremifene (Fareston), and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0202] The anti-GITR antibodies described herein can also be used in combination with and / or formulated in combination with antiviral agents, antibiotics, analgesics, corticosteroids, steroids, oxygen, antioxidants, COX inhibitors, cardioprotective agents, metal chelators, IFN-γ and / or NSAIDs.

[0203] One or more additional therapeutically active ingredients, such as any of the pharmaceutical agents listed above or their derivatives, may be administered before, concurrently with, or immediately after the administration of the anti-GITR antibody of the present invention; (for the purposes of this disclosure, such administration regimens are considered as administration of the anti-GITR antibody in combination with additional therapeutically active ingredients). This disclosure includes pharmaceutical compositions wherein the anti-GITR antibody of the present disclosure is formulated in combination with one or more additional therapeutically active ingredients as described elsewhere herein.

[0204] The additional therapeutically active component may be administered to the subject prior to administration of the anti-GITR antibody of this disclosure. For example, if the first component is administered 1 week, 72 hours, 60 hours, 48 ​​hours, 36 hours, 24 hours, 12 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, 30 minutes, 15 minutes, 10 minutes, 5 minutes, or less than 1 minute before administration of the second component, the first component may be considered to have been administered "before" the second component. In other embodiments, the additional therapeutically active component may be administered to the subject after administration of the anti-GITR antibody of this disclosure. For example, if the first component is administered 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, or 72 hours after the administration of the second component, then the first component can be considered to be administered "after" the second component. In other embodiments, one or more additional therapeutically active components may be administered to the subject concurrently with the administration of the anti-GITR antibody of this disclosure. For the purposes of this disclosure, "concurrent" administration includes, for example, administration to the subject in a single dosage form (e.g., co-formulated) or administration to the subject in separate dosage forms at approximately 30 minutes or less between each other of the anti-GITR antibody and the additional therapeutically active component. If administered in separate dosage forms, each dosage form may be administered via the same route (e.g., both the anti-GITR antibody and the other therapeutically active component may be administered intravenously, subcutaneously, etc.); alternatively, each dosage form may be administered via different routes (e.g., the anti-GITR antibody may be administered intravenously, while the other therapeutically active component may be administered subcutaneously). In any case, for the purposes of this disclosure, administration of the components in a single dosage form, in a single dosage form via the same route, or in a single dosage form via different routes is considered "simultaneous administration." For the purposes of this disclosure, administration of the anti-GITR antibody "before," "simultaneously with," or "after" administration of the other therapeutically active component (those terms as defined above) is considered administration of the anti-GITR antibody in "combination" with the other therapeutically active component.

[0205] This disclosure includes pharmaceutical compositions wherein the anti-GITR antibody of this disclosure is formulated in combination with one or more of other therapeutically active components as described elsewhere herein in a variety of dosage combinations.

[0206] In the exemplary embodiments provided herein, including administration of anti-GITR antibodies in combination with VEGF antagonists (e.g., VEGF traps such as aflibercept), including administration of a combination formulation comprising anti-GITR antibodies and VEGF antagonists, a single component may be administered to a subject in multiple dose combinations and / or in combination formulations. For example, anti-GITR antibodies may be administered to a subject and / or contained in a combination formulation in amounts selected from the group consisting of: 0.01 mg, 0.02 mg, 0.03 mg, 0.04 mg, 0.05 mg, 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1.0 mg, 1.5 mg, 2.0 mg, 2.5 mg, 3.0 mg, 3.5 mg, 4.0 mg, 4.5 mg, 5.0 mg, 6.0 mg, 7.0 mg, 8.0 mg, 9.0 mg, and 10.0 mg; and VEGF antagonists (e.g., aflibercept) may be administered in multiple dose combinations. For example, VEGF traps such as aflibercept can be administered to subjects in doses selected from the following groups and / or contained in combination formulations: 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1.0 mg, 1.1 mg, 1.2 mg, 1.3 mg, 1.4 mg, 1.5 mg, 1.6 mg, 1.7 mg, 1.8 mg, 1.9 mg, 2.0 mg, 2.1 mg, 2.2 mg, 2.3 mg, 2.4 mg, 2.5 mg, 2.6 mg, 2.7 mg, 2.8 mg, 2.9 mg, and 3.0 mg. The combination / combination formulation may be administered to the subject according to any of the administration regimens disclosed elsewhere herein, including, for example, twice a week, once a week, once every two weeks, once every three weeks, once a month, once every two months, once every three months, once every four months, once every five months, once every six months, etc.

[0207] Application plan

[0208] According to certain embodiments of this disclosure, multiple doses of an anti-GITR antibody (or a pharmaceutical composition comprising an anti-GITR antibody and any of the other therapeutically active agents mentioned herein) can be administered to a subject within a defined timeframe. Methods according to this aspect include sequentially administering multiple doses of the anti-GITR antibody provided herein to a subject. As used herein, “sequentially administered” means that each dose of the anti-GITR antibody is administered to the subject at different time points, for example, on different days separated by predetermined intervals (e.g., hours, days, weeks, or months). This disclosure includes methods that sequentially administer a single initial dose of the anti-GITR antibody, followed by one or more second doses of the anti-GITR antibody, and optionally subsequently one or more third doses of the anti-GITR antibody to a patient.

[0209] The terms “initial dose,” “second dose,” and “third dose” refer to the temporal sequence of administration of the anti-GITR antibody as described herein. Thus, “initial dose” is the dose administered at the start of a treatment regimen (also known as the “basal dose”); “secondary dose” is the dose administered after the initial dose; and “tertiary dose” is the dose administered after the secondary dose. The initial, second, and third doses may contain the same amount of anti-GITR antibody, but typically may differ from each other in terms of administration frequency. However, in some embodiments, the amount of anti-GITR antibody contained in the initial, second, and / or third doses may differ from each other during treatment (e.g., appropriately up- or down-regulated). In some embodiments, two or more (e.g., 2, 3, 4, or 5) doses are administered at the start of a treatment regimen as a “loading dose,” followed by subsequent doses (e.g., “maintenance doses”) administered at a lower frequency.

[0210] In some exemplary embodiments of this disclosure, each second and / or third dose is administered 1 to 26 (e.g., 1, 1) immediately following the previous dose. 1 / 2、2、2 1 / 2, 3, 3 1 / 2、4、4 1 / 2, 5, 5 1 / 2, 6, 6 1 / 2、7、7 1 / 2、8、8 1 / 2, 9, 9 1 / 2, 10, 10 1 / 2, 11, 11 1 / 2, 12, 12 1 / 2, 13, 13 1 / 2, 14, 14 1 / 2, 15, 15 1 / 2, 16, 16 1 / 2, 17, 17 1 / 2, 18, 18 1 / 2, 19, 19 1 / 2, 20, 20 1 / 2、21、21 1 / 2、22、22 1 / 2、23、23 1 / 2、24、24 1 / 2、25、25 1 / 2, 26, 26 1 (2 or more) weeks of administration. As used herein, the phrase “immediately following the previous dose” means that in a sequence of multiple administrations, the dose of the anti-GITR antibody is administered to the patient before the next dose in the sequence, without intermediate doses.

[0211] The methods provided in this article may include administering any number of second and / or third doses of the anti-GITR antibody to a patient. For example, in some embodiments, only a single secondary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8 or more) secondary doses are administered to the patient. Similarly, in some embodiments, only a single tertiary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8 or more) tertiary doses are administered to the patient. The administration regimen may be continued indefinitely throughout the lifetime of a particular subject, or until such treatment is no longer therapeutically necessary or beneficial.

[0212] In embodiments involving multiple second doses, each second dose may be administered at the same frequency as the other second doses. For example, each second dose may be administered to the patient 1 to 2 weeks or 1 to 2 months immediately following the previous dose. Similarly, in embodiments involving multiple third doses, each third dose may be administered at the same frequency as the other third doses. For example, each third dose may be administered to the patient 2 to 12 weeks immediately following the previous dose. In some embodiments provided herein, the frequency of administration of the second and / or third doses to the patient may vary during the treatment regimen. During treatment, the physician may also adjust the administration frequency based on the individual patient's needs following clinical examination.

[0213] This disclosure includes administration regimens in which a loading dose of 2 to 6 times is administered to a patient at a first frequency (e.g., once a week, once every two weeks, once every three weeks, once a month, once every two months, etc.), followed by two or more maintenance doses administered to the patient at a lower frequency. For example, according to this aspect provided herein, if the loading dose is administered once a month, the maintenance dose may be administered to the patient once every six weeks, once every two months, once every three months, etc.

[0214] Diagnostic uses of antibodies

[0215] The anti-GITR antibody disclosed herein can also be used to detect and / or measure GITR or GITR-expressing cells in a sample, for example, for diagnostic purposes. For instance, the anti-GITR antibody or a fragment thereof can be used to diagnose symptoms or diseases characterized by abnormal GITR expression (e.g., overexpression, underexpression, lack of expression, etc.). Exemplary diagnostic assays for GITR may include, for example, exposing a sample obtained from a patient to the anti-GITR antibody provided herein, wherein the anti-GITR antibody is labeled with a detectable marker or reporter molecule. Alternatively, an unlabeled anti-GITR antibody may be combined with a secondary antibody that is itself detectably labeled for diagnostic applications. The detectable marker or reporter molecule may be a radioisotope, such as... 3 H, 14 C 32 P, 35 S or 125 I; fluorescent or chemiluminescent components such as luciferin or rhodamine; or enzymes such as alkaline phosphatase, β-galactosidase, horseradish peroxidase, or luciferase. Specific exemplary assays that can be used to detect or measure GITR in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and immuno-PET (e.g., 89 Zr、 64 Cu, etc.) and fluorescence activated cell sorting (FACS).

[0216] Samples that can be used in the GITR diagnostic assay according to this disclosure include any tissue or fluid sample available from a patient under normal or pathological conditions, containing a detectable amount of GITR protein or fragments thereof. Typically, GITR levels are measured in a specific sample obtained from a healthy patient (e.g., a patient without a disease or symptom associated with abnormal GITR levels or activity) to initially establish a baseline or standard GITR level. This baseline GITR level can then be compared to GITR levels measured in samples obtained from individuals suspected of having GITR-related diseases or symptom.

[0217] Example

[0218] The following examples are provided to give those skilled in the art a complete disclosure and description of how to prepare and use the methods and compositions provided herein, and are not intended to limit the scope of what the inventors consider their invention. Efforts have been made to ensure the accuracy of the figures used (e.g., amounts, temperatures, etc.), but some experimental errors and biases should be taken into account. Unless otherwise stated, parts are parts by weight, molecular weights are average molecular weights, temperatures are in degrees Celsius, and pressures are at or near atmospheric pressure.

[0219] Example 1. Generation of anti-GITR antibodies

[0220] Immunization with an immunogen containing the soluble extracellular domain of human GITR Anti-GITR antibodies were obtained from mice (i.e., engineered mice containing DNA encoding the variable regions of the heavy and κ light chains of human immunoglobulins). Antibody immune responses were monitored using a GITR-specific immunoassay. Several fully human anti-GITR antibodies were isolated directly from antigen-positive B cells without fusing with myeloma cells, as described in US2007 / 0280945A1.

[0221] Table 1 lists the amino acid sequence identifiers of the heavy and light chain variable regions and CDRs of the selected anti-GITR antibody (CompAb1 antibody), as previously disclosed in US2017 / 0355774A1. The corresponding nucleic acid sequence identifiers are listed in Table 2. Table 3 provides the full-length heavy and light chain sequences of the CompAb1 antibody.

[0222] Table 1: Amino Acid Sequence Identifiers

[0223]

[0224] Table 2: Nucleic Acid Sequence Identifiers

[0225]

[0226]

[0227] Table 3: Full-length heavy chain and light chain sequences

[0228]

[0229] Example 2. Evaluation and quantification of truncated antibody CompAb1 against GITR

[0230] Characterization of the CompAb1 antibody revealed that a portion of it was cleaved during protein expression. The following analyses were performed to identify the point of truncation and determine the amount of antibody that underwent truncation.

[0231] CompAb1 antibody samples were analyzed using reversed-phase liquid chromatography with a UV detector and an online Waters Vion IMS QTof mass spectrometer to determine the molecular weight of the antibody and its variants. Each antibody sample was injected into a Waters ACQUITY UPLC BEH300 C4 column (1.7 μm, 2.1 mm × 50 mm), which was equilibrated before injection with 99% mobile phase A (0.1% formic acid in Milli-Q aqueous solution) and 1% mobile phase B (0.1% formic acid in acetonitrile solution). The column temperature was maintained at 80 °C. Proteins were eluted using the Waters ACQUITY UPLC system at a flow rate of 0.25 mL / min. After sample injection, the total mobile phase gradient was maintained at 1% mobile phase B for the first 3 minutes, then linearly increased from 1% to 20% over 2 minutes, followed by a second linear increase from 20% to 35% over 15 minutes, a third linear increase from 35% to 70% over 5 minutes, and finally increased to 90% over the next 2 minutes. The elution profile of the eluted proteins was monitored at 215 nm using a photodiode array detector, and the mass of the eluted proteins was measured using a Waters Vion IMS QTof mass spectrometer.

[0232] Using this method, truncated variants of the full-length GITR antibody CompAb1 were identified. In UV chromatograms, the truncated form of the antibody eluted earlier than the full-length antibody. The mass of the truncated form was correlated with the N residues in the CDR3 region of the CompAb1 heavy chain. 101 and P 102 The fragmentation corresponds to the cleavage between the samples. Based on the UV chromatographic peak areas of multiple batches of CompAb1, the abundance of the truncated form was estimated to be approximately 5% of the total antibody. The locations of these fragments were verified by simplified peptide mapping analysis, in which CompAb1 was digested with trypsin into peptides ending in lysine or arginine, separated and analyzed by reversed-phase LC, followed by mass spectrometry on a Q Exactive mass spectrometer. The trypsin peptides corresponding to the full-length antibody and those ending in N... 101 and P 102 Shorter peptides (corresponding to truncated sites in CDR3) are identified based on their precise mass and fragment spectra.

[0233] The truncated levels of CompAb1 in serum increased over time, both in vitro and in vivo.

[0234] CompAb1 was incubated in phosphate-buffered saline (PBS) and monkey serum at 37°C for up to 28 days to investigate its stability under in vitro physiological conditions. Additionally, mice were administered 10 mg / kg of CompAb1, and serum was collected on days 1 and 8 to investigate its stability in vivo.

[0235] CompAb1 was purified from serum samples using magnetic beads coated with anti-human Fc antibodies. The purified CompAb1 was eluted from the beads under acidic conditions and then digested with the enzyme Lys-C, which cleaves the full-length antibody into lysine-terminated peptides. Samples were separated using reversed-phase LC gradient separation and analyzed by Q Exactive HF mass spectrometry. Based on their precise mass and fragment spectra, peptides corresponding to the full-length antibody and those in N were identified in all samples. 101 / P 102 Lys-C-derived peptides from truncated antibodies. The percentage of truncation in each sample was calculated using the peak area of ​​the extracted ion chromatography corresponding to the mass of the intact and truncated antibodies.

[0236] The results showed that incubation in PBS led to an increase in CompAb1 truncation from 10.3% to 24.5% over 28 days, while incubation in monkey serum led to the same increase in CompAb1 truncation from 7.4% to 48.7% over 28 days. Figure 1 The truncated level of CompAb1 in the serum of treated mice increased from 7.7% to 54% after 8 days. Figure 2 ).

[0237] Example 3. Generation of heavy chain variants of the anti-GITR antibody CompAb1 that maintains GITR binding.

[0238] Screening was performed to identify amino acids that prevent the formation of CDR3. 101 NP 102 or 102 PS 103 CompAb1 heavy chain (HC) variants were truncated while retaining anti-GITR function. Double-stranded DNA fragments representing variants of the CompAb1 heavy chain (HC) variable domain were ordered and synthesized from IDT (gBlock gene fragments), subsequently cloned into expression vectors containing the human IgG1 heavy chain constant region. A total of 36 variants were generated by replacing N101 or S103 of CompAb1 HC with all amino acids except cysteine. See Table 4.

[0239] Antibodies are generated after transient expression of each HC variant and the CompAb1 light chain (1-39 lineage universal light chains) in CHO cells.

[0240] Table 4: CompAb1 Heavy Chain Variants

[0241]

[0242]

[0243] The antibody-containing supernatant was collected and sent for screening by Biacore to measure GITR binding.

[0244] The binding affinity and kinetic constants of human anti-GITR antibodies were determined by surface plasmon resonance.

[0245] Biacore experiments targeting antibodies secreted in conditioned medium were performed as follows. Antibodies were captured for 80 seconds on a CM5 anti-human Fc conjugated surface using a Biacore 2000 instrument at 25°C at a flow rate of 8 μL / min. Approximately 1000-2000 RU of each antibody were captured. 100 nM hGITR.mmH (soluble monomer hGITR, SEQ ID NO:45) or 50 nM hGITR.mFc (dimeric cynomolgus macaque GITR, SEQ ID NO:46) was injected onto the surface at a flow rate of 50 μL / min for 2 minutes. Dissociation was measured for 2 minutes. K was calculated by fitting a dual-reference sensor map to a 1:1 binding model. D and t 1 / 2 .

[0246] Table 5: Biacore binding affinity: CHOt supernatant

[0247]

[0248]

[0249] NB: No binding was detected under the conditions of use.

[0250] IC: Uncertain data

[0251] Biacore results from the CHOt supernatant showed that, compared to CompAb1, at least four HC variants (N101D, N101E, N101S, and N101T) maintained similar binding to both the monomer (hGITR.mmh) and dimer (hGITR.mFc) GITR. See Table 5.

[0252] Notably, many variants lost GITR binding relative to the parent antibody. Four N101 variant candidates that maintained similar GITR binding to CompAb1 were selected for further characterization. These variants were generated in CHO stable (CHO) cell lines and subsequently purified.

[0253] For the purified antibodies, Biacore experiments were performed as follows. Using a Biacore T-200 instrument, antibodies were captured on a CM5 anti-human Fc conjugated surface at a flow rate of 8 μL / min for 30 seconds. Approximately 120 RU of each antibody was captured. 10 nM hGITR.mmH (soluble monomer hGITR, SEQ ID NO:45) or 5 nM hGITR.mFc (dimeric cynomolgus macaque GITR, SEQ ID NO:46) was injected onto the surface at a flow rate of 50 μL / min for 5 minutes. Dissociation was measured after 10 minutes. K was calculated by fitting a dual-reference sensor map to a 1:1 binding model. D and t 1 / 2 .

[0254] Compared to CompAb1, the four purified N101 variants showed Ki values ​​for monomeric (hGITR.mmh) and dimeric (hGITR.mFc) GITRs. D and t 1 / 2 As shown in Table 6.

[0255] Table 6: Biacore binding affinity of variants produced by CHO cells

[0256]

[0257] Example 4. Amino acid and nucleic acid sequences of the variable regions of the heavy and light chains.

[0258] Table 7 lists the amino acid sequence identifiers of the heavy and light chain variable regions and CDRs of the anti-GITR antibody variants of CompAb1. The corresponding nucleic acid sequence identifiers are listed in Table 8.

[0259] Table 7: Amino Acid Sequence Identifiers

[0260]

[0261] Table 8: Nucleic Acid Sequence Identifiers

[0262]

[0263] As will be understood by those skilled in the art, antibodies with a specific Fc isotype can be converted into antibodies with different Fc isotypes, but in any case, the variable domains (including CDRs) (represented by the numerical identifiers shown in Tables 7 and 8) will remain the same and are expected to have the same or substantially similar binding properties, regardless of the nature of the Fc domains.

[0264] Table 9 provides exemplary full-length heavy chain (HC) and light chain (LC) nucleic acid and amino acid sequence identifiers for selected anti-GITR antibodies.

[0265] Table 9: Sequence identifiers of the full-length heavy and light chain sequences of exemplary anti-GITR antibodies

[0266]

[0267] Example 5. Evaluation and quantification of truncated variants of the anti-GITR antibody CompAb1

[0268] As described above, truncated variants of the full-length anti-GITR antibody CompAb1 were identified. The quality of the truncated form is related to residue N in the CDR3 region of the CompAb1 heavy chain. 101 and P 102 The cleavage between them corresponds to the subsequent N in CompAb1. 101 / P 102 N at the truncated site 101 Novel antibody variants with a single amino acid substitution at a specific position were identified. These novel antibody variants were analyzed to confirm their identity and determine whether the novel variants exhibited truncation.

[0269] Variant truncation was analyzed using denaturing SEC (run at low temperature and mild pH). CompAb1 and mAb1 were diluted to 1 mg / mL and 5 μg of each sample was injected into a Waters ACQUITY UPLC BEH200 SEC column (1.7 μm, 4.6 mm x 300 mm). The column was equilibrated with 100% mobile phase A (Milli-Q aqueous solution of 30% acetonitrile, 0.1% formic acid, and 0.1% trifluoroacetic acid) before injection. Column temperature was off. Proteins were isocratically eluted over 5 minutes at a flow rate of 0.10 mL / min using the Waters ACQUITY UPLC system with 100% mobile phase A.

[0270] Analysis of the intact antibodies by denaturing SEC-MS without heating the column showed no truncation in mAb1 and mAb2 (Table 10). These results were confirmed by simplified peptide mapping and LC-MS analysis of the variants (data not shown).

[0271] Table 10: In CompAb1 and N 101 The percentage of truncation measured in the variant.

[0272]

[0273] NA: Unanalyzed

[0274] Example 6: No truncation was observed in the N101D and N101E variants of CompAb1 in serum stability analysis.

[0275] CompAb1 and variant candidates mAb1 (N101D) and mAb2 (N101E), identified by Biacore as the most likely original candidates in terms of binding to human GITR protein (see previous example), were incubated at 37°C in IgG-depleted human and mouse serum and HEPES buffer to test their stability under physiological conditions. Additionally, CompAb1, mAb1, and mAb2 (10 mg / kg) were administered to mice, and serum was collected after 4 hours, 1 day, 2 days, 4 days, and 8 days. Antibodies were purified by anti-Fc pull-down affinity and digested using Lys-C. The Lys-C digestion and LC-MS methods used only low temperatures for antibody denaturation and low column temperatures to prevent artificial truncation of the N101D variant. Following serum incubation, the truncation level in the CompAb1 molecule increased, but no truncation was detected in mAb1 or mAb2 throughout the time period. Figure 3 and Figure 4 Similarly, variable levels of truncation were detected in CompAb1 administered to mice, but no truncation was detected in variants mAb1 or mAb2. See Table 11.

[0276] Table 11: In vitro and in vivo stability of mAb1 and mAb2

[0277]

[0278] Example 7. Binding of anti-GITR antibody to GITR on cell surface

[0279] The binding of mAb1 to Jurkat / hCD20 / hGITR and Jurkat / hCD20 / MfGITR cells was evaluated using flow cytometry. Jurkat / hCD20 cells were used as a negative control cell line for GITR binding. Cells were added to 96-well V-plates (2 x 10⁻⁶ cells / wells). 5 Up to 4x10 5 Cells were incubated at 100 cells / well in Fc blocking solution, followed by sequential incubation on ice with primary antibody (mAb1 or IgG1 isotype control) at final concentrations ranging from 18 pM to 300 nM and secondary antibody (AF647 conjugated anti-human IgG) at 5 μg / mL. Cells were then stained with a live / dead fixable green dead cell stain according to the manufacturer's instructions. Cells were fixed with BDCytofix and filtered through an AcroPrep Advance plate, followed by collection on an Intellicyt iQue Screener PLUS flow cytometer. Data were analyzed using FlowJo software. For EC 50The effective concentration (at 50% activity) was determined by analyzing the measured geometric MFI value using a 4-parameter logistic equation on a 9-point response curve with a GraphPad Prism. The binding fold was determined by taking the ratio of the highest MFI on the curve to the MFI of the well containing only the secondary antibody.

[0280] mAb1 showed concentration-dependent binding with Jurkat / hCD20 cells engineered to express hGITR or MfGITR, and its EC50... 50 The values ​​were in the nanomolar range; no binding was detected with Jurkat / hCD20 cells (Figure 5). The IgG1 isotype control did not show binding with any of the three cell lines tested. EC50 values ​​for mAb1 and IgG1 isotype controls were reported. 50 Values ​​(if applicable), maximum MFI (highest average MFI within the tested dose range), and binding fold calculated as the maximum MFI above background (secondary antibody only). Results are summarized in Table 12.

[0281] Table 12: Binding of antibodies to Jurkat T cells engineered to express human or cynomolgus monkey GITR

[0282]

[0283] a Maximum MFI is the highest average MFI value within the tested concentration range (18 pM to 300 nM).

[0284] b The fold increase was used as the maximum MFI (secondary antibody only) above the background level for calculation.

[0285] h, human; Mf, cynomolgus monkey; ND, undetermined because concentration-dependent binding was not observed.

[0286] In summary, mAb1 showed concentration-dependent binding to Jurkat / hCD20 target cells expressing human or monkey GITR, with similar potency (ECG). 50 ) and maximum binding multiple.

[0287] Example 8: Binding of anti-GITR antibody to activated human and cynomolgus monkey primary T cells

[0288] In this experiment, the binding of mAb1 to anti-CD3 / anti-CD28 stimulated human and cynomolgus monkey primary T cells was evaluated. CD25 and CD69 were used as surrogate markers for activation of human and cynomolgus monkey primary T cells, respectively.

[0289] Peripheral blood mononuclear cells (PBMCs) were isolated from human or cynomolgus monkey whole blood (4 donors each) using density centrifugation via Ficoll-PaquePLUS in SepMate tubes (for human PBMCs) or 50 mL conical tubes (for cynomolgus monkey PBMCs) and transferred to fresh conical tubes for incubation in erythrocyte (RBC) lysis buffer. The PBMCs were then washed and transferred to T75 culture flasks for T-cell activation and expansion using a T-cell activation / expansion kit for human or non-human primates. After 4 days, activation beads were removed from the culture using a MACSiMAG separator, and cells were collected and counted.

[0290] The binding of mAb1 to human or cynomolgus monkey T cells in activated PBMCs was evaluated using flow cytometry. Cells were added to 96-well V-shaped plates (3 x 10⁶ cells / wells). 5 Cells were incubated in Fc blocking / monocyte blocking solution (cells / well) and then on ice with a mixture of fluorophore-conjugated antibodies (anti-CD3, anti-CD4, anti-CD8, and anti-CD25 [activation markers of human T cells] or anti-CD69 [activation markers of cynomolgus monkey T cells]), AF647-conjugated mAb1, or AF647-conjugated IgG1 isotype control (final concentration range 8 pM to 200 nM) for T cell phenotypic analysis and live / dead fixable blue dead cell stain (according to manufacturer's instructions). Cells were fixed with BD stabilizing fixative and filtered through AcroPrep Advance plates, followed by collection on a BDLSLRFortessa X-20 flow cytometer. Data were analyzed using FlowJo software. For EC 50 The geometric MFI value was determined by analyzing the measured values ​​using a 4-parameter logistic equation on a 12-point response curve with GraphPad Prism.

[0291] mAb1 showed concentration-dependent binding to activated human and cynomolgus monkey primary T cells. Compared to the IgG1 isotype control, mAb1 showed minimal binding to inactivated human and cynomolgus monkey primary T cells. Figure 6 and Figure 7 Tables 13 and 14 summarize the maximum MFI values ​​(detected at the highest tested concentration of 200 nM) for the binding of mAb1 and IgG1 isotype controls.

[0292] Table 13: Anti-GITR antibodies and activated (CD25) + ) and inactive (CD25) - Binding of human primary T cells

[0293]

[0294]

[0295] a The maximum MFI was determined at an unsaturated concentration of 200 nM (the highest concentration tested).

[0296] Table 14: Related to activated (CD69) + ) and inactive (CD69) - Binding of primary T cells from cynomolgus monkeys

[0297]

[0298] a The maximum MFI was determined at an unsaturated concentration of 200 nM (the highest concentration tested).

[0299] Example 9. Activation of NFAT in engineered Jurkat T cells via FcγR3a

[0300] An ADCC reporter factor bioassay was developed (Parekh et al., mAbs, 4(3):310-318, 2012) to evaluate the ability of mAb1 to activate nuclear factor (NFAT) in T cells via human or cynomolgus monkey FcγR3a (an Fc receptor that mediates ADCC and is primarily expressed on NK cells). Jurkat / NFAT-Luc / hFcγR3a or Jurkat / NFAT-Luc / MfFcγR3a effector cells and Jurkat / hCD20 / hGITR or Jurkat / hCD20 / MfGITR target cells were used in these assays. Jurkat / hCD20 cells were used as control target cells for evaluation.

[0301] Two replicates of mAb1 or IgG1 isotype control (final concentration range 916 fM to 60 nM) or anti-CD20 IgG1 (final concentration range 45.8 fM to 3 nM) were prepared and administered to effector cells (2.5 x 10⁻⁶ cells / mL). 4 (5 x 10 cells / well) together in target cells (5 x 10) 4 In the presence of 10 cells / well, the cells were incubated in Jurkat complete medium (RPMI supplemented with 10% fetal bovine serum (FBS), 100 U / mL penicillin, 100 μg / mL streptomycin, and 292 μg / mL L-glutamine). Wells without antibodies were included as a control for background signal transduction. Plates were incubated at 37°C and 5% CO2 for 5 hours. The plates were then equilibrated to room temperature for 10 minutes, followed by the addition of One-Glo luciferase substrate to the wells and incubation for 3 minutes. Luciferase activity was captured as a luminescent signal and expressed in relative optical units (RLU) using an ENVISION microplate reader. For EC... 50The RLU values ​​were determined using a 4-parameter logistic equation on a 9-point response curve using GraphPad Prism. The fold change in activity was determined by comparing the highest RLU on the curve with the RLU of the well without antibody.

[0302] The results are summarized in Table 15. In the presence of Jurkat / hCD20 / hGITR or Jurkat / hCD20 / MfGITR, mAb1 mediated a concentration-dependent increase in NFAT signaling in Jurkat / NFAT-Luc / hFcγR3a and Jurkat / NFAT-Luc / MfFcγR3a, respectively, and its EC50... 50 The values ​​were in the sub-nanomolar range (Figure 8). In the presence of Jurkat / hCD20 and Jurkat / hCD20 / hGITR or Jurkat / hCD20 / MfGITR, the positive control, anti-CD20 IgG1, mediated a concentration-dependent increase in NFAT signaling in Jurkat / NFAT-Luc / hFcγR3a and Jurkat / NFAT-Luc / MfFcγR3a, respectively, with EC50 values ​​in the sub-nanomolar range. 50 The values ​​were in the sub-nanomolar range. In the absence of target cells (i.e., the culture medium contained no cells other than effector cells), no increase in baseline NFAT activation was observed under any test conditions with mAb1 or anti-CD20 IgG1, or with an IgG1 isotype control.

[0303] Table 15: Summary of mAb1-mediated NFAT activation in Jurkat / NFAT-Luc / hFcγR3a and Jurkat / NFAT-Luc / MfFcγR3a effector cells

[0304]

[0305] a The maximum RLU was defined as the highest average RLU value within the tested concentration range (916 fM to 60 nM for mAb1 and IgG1 isotype controls, and 45.8 fM to 3 nM for anti-CD20 IgG1).

[0306] b The fold change in activity was calculated as the maximum RLU above the background (antibody-free).

[0307] h, human; Mf, cynomolgus monkey; ND, undetermined because concentration-dependent luciferase activity was not observed.

[0308] Example 10: Anti-GITR antibody-mediated ADCC against Jurkat T cells engineered to express human or cynomolgus monkey GITR

[0309] ADCC assays were performed to evaluate the ability of mAb1 to induce ADCC against T cells expressing human or cynomolgus monkey GITR. In the first assay, target cells included Jurkat / hCD20 / hGITR cells or Jurkat / hCD20 / MfGITR cells; Jurkat / hCD20 cells were included as a control target cell line. In the second assay, target cells included stimulated / expanded Tregs (regulatory T cells) and CD8+ cells from human PBMCs (from the same three donors). + T cells. Human NK cells isolated from leukocyte-rich whole blood (from two donors used in the assay with engineered Jurkat T cells and two donors used in the assay with primary T cells) were used as effector cells in the ADCC assay. IgG1 isotype controls were evaluated in parallel with mAb1. Anti-CD20 IgG1 or anti-CD3 IgG1 was used as a positive control to induce ADCC against engineered Jurkat T cells and human primary T cells, respectively, in the presence of NK cells.

[0310] In the presence of primary human NK cells (effector cells), mAb1 mediates concentration-dependent ADCC against Jurkat / hCD20 / hGITR and Jurkat / hCD20 / MfGITR (target cells); cytotoxic ECGs. 50 Values ​​were in the sub-nanomolar range. In contrast, the IgG1 isotype control did not mediate ADCC against any target cell line at concentrations ranging from 9.5 fM to 10 nM. Addition of NK cells in the absence of antibody treatment resulted in a low percentage of nonspecific cytotoxicity against the target cell line.

[0311] The ability of NK cells to induce ADCC against the same target cell lines was evaluated using a positive control, anti-CD20 IgG1. Anti-CD20 IgG1 mediated ADCC against Jurkat / hCD20, Jurkat / hCD20 / hGITR, and Jurkat / hCD20 / MfGITR target cells in a concentration-dependent manner, with its cytotoxic EC50 values... 50 The values ​​are in the sub-nanomolar range. Representative data from two human NK donors are shown in Figure 9 and summarized in Table 16.

[0312] Table 16: Antibody-mediated ADCC against engineered Jurkat T cells

[0313]

[0314]

[0315] aMaximum cytotoxicity % was determined as the highest average percentage of cytotoxicity within the tested concentration range (9.5 fM to 10 nM). h, human; Mf, cynomolgus monkey (cynomolgus macaque); ND, undetermined because no concentration-dependent cytotoxicity was observed.

[0316] Example 11: Anti-GITR antibody-mediated ADCC targeting human primary T cells

[0317] Following the manufacturer's recommended protocol, PBMCs were isolated from human whole blood (from 3 donors) by density centrifugation using Ficoll-Paque PLUS density gradient medium and SepMate tubes. Subsequently, erythrocyte lysis buffer was added to the isolated PBMCs to remove unwanted RBCs. The PBMCs were then washed, and CD8+ was separated using CD8 microbeads according to the manufacturer's instructions. + T cells. Precipitate the remaining CD8-negative cells and use CD4+. + CD25 + Regulatory T cell isolation kit isolates CD4 cells according to the manufacturer's protocol. + CD25 + T cells.

[0318] CD8 was confirmed using flow cytometry. + and CD4 + / CD25 + Isolation of T cells (i.e., Tregs). CD8... + T cells were seeded into T75 flasks containing IL-2-supplemented medium and expanded using CD3 / CD28 Dynabead according to the manufacturer's instructions (1 bead: 1 cell). Tregs were seeded into T75 flasks containing IL-2 and rapamycin-supplemented medium and expanded using CD3 / CD28 MACSiBead particles according to the manufacturer's instructions (4 beads: 1 cell). After 5 days of culture, the beads were removed, and the cells were incubated for 24 hours in medium without IL-2 or rapamycin before use in the ADCC assay.

[0319] Prior to the cytotoxicity assay, CD8 was subjected to treatment as described in Example 8. + T cells and Tregs were stained, and antibody binding capacity (ABC) was assessed by flow cytometry. Antibody binding capacity is defined as the number of antibody molecules bound to the cell surface under saturation conditions. The ABC of GITR antibodies from Miltenyi Biotec was determined using the Quantum Simply Cellular AF647 MESF (Soluble Fluorescent Dye Molecule) Bead Kit according to the manufacturer's instructions.

[0320] target cells (5x10) 3 Three copies of each cell / well were added to opaque white 96-well plates, followed by the addition of mAb1, IgG1 isotype control or anti-CD20 (for engineered Jurkat T cells) or anti-CD3 (for human primary T cells), and IgG1 (final concentration range 9.5 fM to 10 nM for engineered Jurkat T cells, and 169 fM to 10 nM for human primary T cells). Human NK cells (2.5 x 10⁻⁶ cells / well) were then added to assay medium (RPMI supplemented with 1% bovine serum albumin (BSA), 100 U / mL penicillin, 100 μg / mL streptomycin, and 292 μg / mL L-glutamine). 4 (cells / well). A control sample containing all components except the antibody was incorporated into each experiment to determine the background signal of the assay (i.e., nonspecific lysis of target cells in the presence of NK cells). To assess spontaneous lysis, separate untreated target cells (target cells) and separate effector cells (effector cells) were incubated in separate wells.

[0321] The plate was incubated at 37°C and 5% CO2 for 3.5 hours. The plate was then equilibrated to room temperature for 10 minutes, followed by the addition of CytoTox Glo reagent to the wells while shaking, and held for 15 minutes. The luminescence signal was measured using an ENVISION microplate reader as a reading of cytotoxicity.

[0322] mAb1-mediated concentration-dependent ADCC against human primary T cells (target cells) from three of three donors tested in the presence of human primary NK cells (effective cells), with cytotoxic EC50. 50 The values ​​were in the sub-nanomolar range. In 4 out of 6 test conditions (2 effector cell donors × 3 target cell donors), the values ​​were consistent with CD8. + Compared to T cells, mAb1-mediated cytotoxicity against Tregs was significantly greater. See Figure 10 and Table 17.

[0323] Conversely, the IgG1 isotype control did not mediate ADCC against any primary T cell donor. Adding NK cells in the absence of antibody treatment resulted in a low percentage of nonspecific cytotoxicity against primary T cells.

[0324] The ability of NK cells to induce ADCC was also evaluated in an assay using the same NK and T cell donors with a positive control anti-CD3 IgG1. Anti-CD3 IgG1 mediated ADCC against primary T cells in a concentration-dependent manner, with its cytotoxic EC50... 50Values ​​are in the sub-nanomolar range. Representative data from two primary NK donors tested alongside three primary T cell donors are shown in Figure 10 and summarized in Table 17.

[0325] Table 17: Antibody-mediated ADCC against human primary T cells

[0326]

[0327] a Maximum cytotoxicity % was determined as the highest average percentage of cytotoxicity within the tested concentration range (169 fM to 10 nM).

[0328] b mAb1 and CD8 from donor 0400N + Compared to T cells, it mediated significantly stronger ADCC against Tregs (p<0.0001) and compared to Tregs from donor 0200R, it mediated significantly stronger ADCC against CD8. + T cells showed significantly stronger ADCC (p<0.0001).

[0329] c Anti-CD3 IgG1 mediates CD8 compared to Treg from donor 5100X. + T cells showed significantly stronger ADCC (p<0.0001).

[0330] d mAb1 and CD8 from all 3 donors + Compared to T cells, it mediates significantly stronger ADCC against Tregs (p<0.0001).

[0331] The statistical significance of ADCC for each NK cell donor under all experimental conditions was determined by two-way ANOVA and Tukey's post-hoc test for multiple comparisons (α = 0.05).

[0332] ABC, antibody binding capacity (antibody bound per cell based on fluorescence intensity measured by flow cytometry); ND, undetermined because no concentration-dependent cytotoxicity was observed.

[0333] Example 12: Anti-GITR antibody-mediated ADCP against Jurkat T cells engineered to express human or cynomolgus monkey GITR

[0334] An ADCP (antibody-dependent phagocytosis) assay was performed to assess the ability of mAb1 to induce ADCP on T cells expressing human or cynomolgus monkey GITR. Target cells included Jurkat / hCD20 / hGITR cells or Jurkat / hCD20 / MfGITR cells; Jurkat / hCD20 cells were included as a control target cell line. ADCP was induced from CD14 cells in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF). + Phagocytes differentiated from monocytes were used as effector cells in the ADCP assay. IgG1 isotype controls were evaluated in parallel with mAb1. Anti-CD20 IgG1 was used as a positive control to induce ADCP in engineered Jurkat T cells.

[0335] Frozen CD14 obtained from Lonza + Cells were thawed and resuspended in cell culture medium supplemented with 50 ng / mL GM-CSF (supplemented with 10% FBS, 100 U / mL penicillin, 100 μg / mL streptomycin, 292 μg / mL L-glutamine, 1% human AB serum, NaPyr (sodium pyruvate), HEPES, NEAA (non-essential amino acids), and 0.01 mM β-mercaptoethanol in RPMI), and sputtered at 5 x 10⁻⁶ ppm. 4 Cells / well plates were seeded into clear-bottomed, collagen-coated 96-well black plates to differentiate into phagocytes within 13 days, with fresh GM-CSF (50 ng / ml) added on days 6 and 12.

[0336] Prior to ADCP assay, target cells and phagocytes derived from monocytes were labeled by incubating with CellTrace CFSE (carboxyfluorescein succinimide) dye or CellTrace far-red dye at 37°C and 5% CO2 for 15 minutes.

[0337] target cells (5x10) 5 Two copies of each cell / well were added to 96-well U-shaped plates, followed by the addition of mAb1, IgG1 isotype control, or anti-CD20 IgG1 (final concentration range 381 fM to 100 nM) to assay medium (RPMI supplemented with 10% FBS, 100 U / mL penicillin, 100 μg / mL streptomycin, and 292 μg / mL L-glutamine) and incubated on ice for at least 15 minutes. Antibody-free target cells were included as a background phagocytic control. The target cell mixture, with or without titrated antibodies, was then transferred to a plate containing adherent far-red labeled phagocytes and incubated at 37°C, 5% CO2 for 1 to 2 hours.

[0338] After incubation, remove the culture medium containing unattached cells from the wells and rinse the wells with PBS. Add a solution of 3.7% formaldehyde in PBS to the wells to fix the cells for 20 minutes. Wash the wells with PBS and store the plate at 4°C until analysis.

[0339] Phagocytosis was analyzed by fluorescence imaging using Harmony software on the Opera Phoenix high-content screening system, acquiring images in the 488 nm (CFSE-labeled target cells) and 647 nm (far-infrared-labeled phagocytes) emission channels. Image analysis was performed in Columbus software, and phagocyte populations were selected by image segmentation in the 647 nm emission channel, and phagosis was quantified by calculating the 488 nm fluorescence intensity (from CFSE-labeled target cells) within each phagocyte as relative fluorescence units (RFU). For EC 50 To determine the activity, for engineered Jurkat T cells or human primary T cells, the RFU values ​​were analyzed using a 4-parameter logistic equation on 10-point or 9-point response curves with GraphPad Prism. The fold change in activity was determined by the ratio of the highest RFU on the curve to the RFU of the well without antibody.

[0340] mAb1 mediates concentration-dependent ADCP of Jurkat / hCD20 / hGITR and Jurkat / hCD20 / MfGITR (target cells) in the presence of phagocytic cells derived from human primary monocytes (effector cells). EC50 of the phagocytic effect of Jurkat / hCD20 / hGITR could not be calculated. 50 The value, because 4-parameter logistic regression does not converge to a single value; the ECG of the phagocytic effect of Jurkat / hCD20 / MfGITR. 50 Values ​​were in the sub-nanomolar range. The maximum levels of phagocytosis mediated by mAb1 in Jurkat / hCD20 / hGITR and Jurkat / hCD20 / MfGITR cells were comparable, with activities 5.92-fold and 6.98-fold higher than background (antibody-free), respectively. Conversely, the IgG1 isotype control did not mediate ADCP in any target cell line. Data from a single human primary monocyte-derived phagocytic cell donor are shown in Figure 11 and summarized in Table 18.

[0341] The ability of human primary monocyte-derived phagocytes to induce ADCP in the same target cell lines was evaluated using a positive control, anti-CD20 IgG1. Anti-CD20 IgG1 mediated ADCP in Jurkat / hCD20, Jurkat / hCD20 / hGITR, and Jurkat / hCD20 / MfGITR target cells in a concentration-dependent manner, and its phagocytic EC50 was [not specified in the original text]. 50 The value is in the sub-nanomolar range.

[0342] Table 18: Antibody-mediated ADCP of engineered Jurkat T cells

[0343]

[0344] a The maximum RFU was determined as the highest average RFU value within the tested concentration range (381 fM to 100 nM).

[0345] b The fold change in activity was calculated as the maximum RFU above the background (antibody-free).

[0346] h, human; Mf, cynomolgus monkey (crab-eating macaque); NC, not calculated because no unique fit could be found for the dataset; ND, undetermined because no concentration-dependent phagocytosis was observed.

[0347] Example 13: Effect of anti-GITR antibody on anti-CD3-mediated primary CD4+ T cell proliferation

[0348] The effect of mAb1 on the proliferation of primary CD4+ T cells mediated by a stimulating CD3 antibody was evaluated in the presence of HEK293 / FcγR2b helper cells. Human primary CD4+ T cells from six donors were evaluated.

[0349] HEK293 / FcγR2b cells were incubated at 37°C and 5% CO2 in 50 μg / mL mitomycin C for 30 min, and then incubated at 1 x 10⁻⁶ cells / mL in 37°C and 5% CO2. 5 10 cells / well were seeded into 96-well flat-bottomed tissue culture plates and incubated overnight.

[0350] CD4 was isolated from PBMCs from 6 donors using density gradient centrifugation with the Ficoll-Paque PLUS gradient. + T cells were then used, followed by the consumption of memory T cells using human CD45RO microbeads according to the manufacturer's protocol; initial CD45RO microbeads were then used. + T cells were resuspended in assay medium (RPMI supplemented with 5% human AB serum, 100 U / mL penicillin, 100 μg / mL streptomycin and 292 μg / mL L-glutamine).

[0351] On the day of the assay, HEK293 / FcγR2b cells seeded on plates were pre-incubated with 1.2 μg / mL OKT3 at room temperature for 20 minutes, and then 5 x 10⁻⁶ cells were added. 5 An initial CD4 + T cells / well and mAb1 or IgG1 isotype controls with final concentrations ranging from 32 fM to 133 nM.

[0352] The plate was incubated at 37°C and 5% CO2 for 4 days, and 0.5 uCi of trityl thymidine was added to the cells. The plate was then incubated for another 16 hours. Thymidine, and therefore tritium, will be incorporated into the dividing cells in higher amounts.

[0353] After incubation, cells were harvested onto 96-well UniFilter plates and 35 μL of scintillation buffer was added to each well. Tritium incorporation was measured in counts per minute (CPM) using a TopCount NXT microplate scintillation and luminescence counter. All serial dilutions were tested in duplicate.

[0354] Using GraphPad Prism TM The software determines the antibody's EC50 based on a 4-parameter logistic equation on a 12-point dose-response curve. 50 The maximum proliferation was determined as the average maximum CPM detected within the tested concentration range. The fold change in activity was determined by taking the ratio of the highest CPM on the curve to the CPM of the well without antibody.

[0355] mAb1 enhanced anti-CD3-mediated T cell proliferation in a concentration-dependent manner at sub-nanomolar EC50 values ​​(Figure 12) and was associated with a 2.3–3.1-fold increase in maximum T cell proliferation above background (measured in CPM of tritium decay). In contrast, the IgG1 isotype control did not promote a concentration-dependent enhancement of T cell proliferation. The results are summarized in Table 19.

[0356] Table 19: Anti-GITR antibodies against CD3-mediated primary CD4+ + Effects of T cell proliferation

[0357]

[0358] a The maximum CPM was defined as the highest average CPM value within the tested concentration range (32 fM to 133 nM).

[0359] b The fold change in activity was calculated as the maximum CPM above the background (antibody-free).

[0360] NC, not calculated because the 4-parameter logistic regression does not converge to a single value; ND, undetermined because no concentration-dependent proliferation was observed.

[0361] Example 14: Effect of anti-GITR antibody on the binding of GITR to GITR-L in blocking ELISA

[0362] The binding of recombinant hGITR to its ligand hGITR-L was determined using an enzyme-linked immunosorbent assay (ELISA). Recombinant hGITR-L diluted in PBS at a concentration of 2 μg / mL was passively adsorbed onto a microtiter plate and incubated overnight at 4°C, followed by blocking with PBS containing 0.5% BSA. Then, monomeric recombinant human GITR extracellular domain protein hGITR.mmH at concentrations ranging from 3.4 pM to 200 nM was added in duplicate to the plate. The plate was incubated at room temperature for 1 hour and then washed four times with PBS (PBST) containing 0.05% Tween-20. The binding of hGITR.mmH to the plate was detected at 0.33 μg / mL using a horseradish peroxidase (HRP)-conjugated goat anti-cMyc antibody, and visualization was performed using a colorimetric HRP substrate 3-3',5-5'-tetramethylbenzidine (TMB) according to the manufacturer's recommended procedure. Absorbance data (OD) at 450 nm were analyzed. 450 Plotting was performed based on the concentration of recombinant hGITR.mmH. Separate buffer samples were included to determine the background signal; however, their OD... 450 The data was not plotted on the response curve. The combined data was analyzed using a 4-parameter logistic equation on the 11-point response curve using GraphPad Prism software, and the EC was calculated. 50 Value. EC 50 The value was defined as the concentration of hGITR.mmH at which 50% of maximum binding was observed. Select values ​​close to EC. 50 The concentration of the value (within the linear range) is used as the fixed hGITR.mmH concentration for the blocking assay.

[0363] An ELISA-based blocking assay was developed to determine the ability of mAb1 to block the binding of recombinant human GITR (hGITR.mmH) to hGITR-L. A fixed concentration of recombinant human hGITR.mmH (1.5 nM) was pre-incubated for 1 hour with either mAb1 or an IgG1 isotype control (51 pM to 3 μM). The pre-incubated solution was transferred to duplicate wells of a microtiter plate previously coated with GITR-L as described above. The plate was incubated at room temperature for 1 hour, then washed four times with PBST, and the bound hGITR.mmH was detected as described above. OD 450 The absorbance data at each point were plotted based on antibody concentration. Binding data were analyzed using a 4-parameter logistic equation on the 11-point response curve using GraphPad Prism software, and the IC50 was calculated. 50 Value. IC 50 The value is defined as the antibody concentration required to block the binding of 50% hGITR.mmH to the plate-coated hGITR-L.

[0364] The effect of mAb1 or IgG1 isotype controls on the binding of monomeric recombinant human GITR extracellular domain protein (hGITR.mmH) to immobilized human GITR-L (6His.GCN4.G4Sx3.hGITR-L) is shown in the figure. Figure 13 Table 20 summarizes the IC50 values ​​of the antibodies. 50 Value and maximum inhibition percentage.

[0365] Table 20: Blocking of binding between hGITR.mmH and immobilized human GITR-L

[0366] mAb1 6.66E-09 98 IgG1 isotype control ND -6

[0367] Maximum blocking percentage = the percentage of blockage at the maximum antibody concentration

[0368] ND, undetermined, as no concentration-dependent inhibition of binding was observed.

[0369] Recombinant hGITR.mmH binds to immobilized human GITR-L, EC in a concentration-dependent manner. 50 A value of 2.05 nM was chosen to be close to EC. 50 A concentration of 1.5 nM (within the linear range) was used as the fixed recombinant hGITR.mmH concentration for the blocking assay.

[0370] mAb1 blocks the binding of hGITR.mmH to hGITR-L in a concentration-dependent manner, with a maximum inhibition rate of 98% and an IC50 value of 1.5%. 50 The value was 6.66 nM. At any concentration tested, the IgG1 isotype control did not inhibit the binding of hGITR.mmH to human GITR-L.

[0371] Example 15: The effect of anti-GITR antibodies on CDC

[0372] The ability of mAb1 to mediate CDC against engineered Jurkat T cells expressing human or cynomolgus monkey GITR in the presence of serum complement was evaluated using a cytotoxicity assay. Maximum cytotoxicity percentage and EC50 were reported. 50 value.

[0373] CDC assays were performed to evaluate the ability of mAb1 to induce CDC against T cells expressing human or cynomolgus monkey GITR. Target cells included Jurkat / hCD20 / hGITR cells or Jurkat / hCD20 / MfGITR cells; Jurkat / hCD20 cells were included as a control target cell line. IgG1 isotype controls were evaluated in parallel with mAb1. Anti-CD20 IgG1 was used as a positive control to induce CDC against engineered Jurkat T cells in the presence of serum complement factors.

[0374] Target cells (5 x 10³ cells / well) were added in triplicate to opaque white 96-well plates. Then, mAb1, an IgG1 isotype control, or anti-CD20 IgG1 (final concentration range 477 fM to 500 nM) was added to assay medium (RPMI supplemented with 1% BSA, 100 U / mL penicillin, 100 μg / mL streptomycin, and 292 μg / mL L-glutamine). Serum (final volume 5%) was then added. Separate assay medium was added in parallel to assess lysis in the absence of complement factors. A buffer control sample containing all components except the antibody was incorporated into each experiment to determine the background signal of the assay (i.e., nonspecific lysis of target cells).

[0375] The plate was incubated at 37°C and 5% CO2 for 3.5 hours. The plate was then equilibrated to room temperature for 30 minutes, followed by the addition of CytoTox Glo reagent to the wells while shaking, and held for 15 minutes. The luminescence signal was measured using an ENVISION microplate reader as a reading of cytotoxicity.

[0376] The cytotoxic response is calculated as follows:

[0377]

[0378] a SBS, Spontaneous Background Signal

[0379] For EC50 determination, the percentage of cytotoxicity was analyzed using a 4-parameter logistic equation on a 12-point response curve with GraphPad Prism.

[0380] In the presence of 5% NHS, mAb1 did not mediate CDC against Jurkat / hCD20, Jurkat / hCD20 / hGITR, or Jurkat / hCD20 / MfGITR target cells in the concentration range of 477 fM to 500 nM. Similarly, no CDC was observed in target cells co-incubated with the IgG1 isotype control antibody. Figure 14 ).

[0381] The ability of NHS to induce CDC in the same target cell lines was evaluated using a positive control, anti-CD20 IgG1. In the presence of NHS, anti-CD20 IgG1 mediated CDC in a concentration-dependent manner against all three target cell lines. In the absence of NHS, anti-CD20 IgG1 did not mediate lysis against any of the target cell lines tested.

[0382] Example 16: The ability of anti-GITR antibodies to form immune complexes that bind to C1q

[0383] The ability of mAb1 to form immune complexes that bind C1q was evaluated using recombinant soluble GITR extracellular domain protein in monomeric (hGITR.mmH) or dimeric (hGITR.mFc) form and the MicroVue CIC-C1q EIA kit. As a positive control, the binding of thermoagglutinin (HAGG) to C1q was also evaluated.

[0384] A 30 nM mAb1 or IgG1 isotype control was incubated with hGITR.mmH or hGITR.mFc at a 1:1 molar ratio. Controls containing only 30 nM mAb1, IgG1 isotype control, hGITR.mmH, or hGITR.mFc were also evaluated. Samples were incubated in 0.1% (w / v) bovine serum albumin (BSA) (pH 7.4) in DPBS at 37°C for 30 minutes. Each sample was evaluated in triplicate.

[0385] To examine the binding of the complex to C1q, the sample was diluted 50-fold to the kit assay buffer in an ELISA plate coated with C1q protein and incubated at room temperature for 60 minutes. Wells were washed to remove unbound protein, and horseradish peroxidase (HRP)-conjugated goat anti-human IgG (MicroVue CIC-C1q EIA kit) was added to each well to detect C1q-bound immune complexes. After washing away unbound HRP conjugates, the bound complex was detected by adding enzyme substrate. Absorbance was read at 405 nm using a Perkin Elmer VICTOR X5 multilabel microplate reader.

[0386] Linear reference curves were generated using the CIC-C1q assay to determine the HAGG standard, based on absorbance values ​​of 0, 15, and 38 HAGG μg equivalents / mL (μg Eq / mL). The μg Eq / mL concentration for each test sample was determined with reference to this standard curve. High and low HAGG positive controls were also performed. The high HAGG control was expected to produce 11 to 26 μg Eq / mL in this assay, and the low HAGG control was expected to be below 4 μg Eq / mL. The kit manufacturer defines values ​​less than 4 μg Eq / mL as negative for significant levels of C1q binding in CIC.

[0387] No C1q binding was observed in samples containing mAb1 and either hGITR.mmH or hGITR.mFc (Figure 15). Similarly, no C1q binding was observed in samples containing IgG1 isotype control and either hGITR.mmH or hGITR.mFc. No C1q binding was observed in samples containing only mAb1, IgG1 isotype control, hGITR.mmH, or hGITR.mFc. High and low HAGG positive controls were analyzed in parallel, and C1q binding was observed in each sample within the expected corresponding range.

[0388] Example 17: Effect of anti-GITR antibody combined with cimipril on IL-2 release from human primary T cells stimulated with engineered RBL-2H3 cells

[0389] This embodiment examines mAb1 against primary human CD3 from two donors. + The agonistic effect of T cells on anti-CD3-stimulated IL-2 release. CD3 + T cells were isolated from peripheral blood mononuclear cells (PBMCs) from two donors. For one donor, PBMCs were isolated from peripheral blood using density gradient centrifugation with a Ficoll-Paque PLUS gradient. For the other donor, PBMCs were isolated from peripheral blood from a healthy donor using the EasySep Direct Human PBMC Isolation Kit from Stem Cell Technologies, following the manufacturer's protocol. The PBMCs isolated from both donors were individually frozen in FBS containing 10% DMSO.

[0390] For CD3 + For T cell isolation, frozen PBMCs in vials were thawed in a 37°C water bath and diluted in stimulation medium (X-VIVO 15 cell culture medium supplemented with 10% FBS, HEPES, NaPyr, NEAA, and 0.01 mM BME) containing 50 U / ml benzonase nuclease. Cells were centrifuged at 1200 rpm for 10 minutes, resuspended in EasySep buffer, and isolated using the StemCell Technologies EasySep T cell isolation kit according to the manufacturer's protocol.

[0391] CD3 + T cells were resuspended in stimulation medium and 1x10⁻⁶ cells were added. 5 Cells per well were seeded onto 96-well round-bottom plates at a concentration of [number] cells / well. RBL-2H3 / αCD3 or RBL-2H3 / αCD3 / hPD-L1 cells were initiated in primary stimulation medium with 10 μg / mL mitomycin C at a concentration of 10 × 10⁻⁶ cells / well. 6Cells were treated at a concentration of 10 cells / mL. After incubation at 37°C and 5% CO2 for 1 hour, the mitomycin C-treated cells were washed three times with D-PBS containing 2% FBS and added to a final concentration of 5 × 10⁻⁶ cells / mL. 4 T cells per well were then placed in the wells. Subsequently, mAb1 or IgG1 isotype controls at final concentrations ranging from 3 pM to 200 nM and cimipril or IgG4 at a fixed concentration of 20 nM were added. P Antibody combinations for isotype control were added to the wells. The last spot of the 10-spot dilution contained no titrating antibody. The plate was incubated at 37°C with 5% CO2 for 3 days, followed by centrifugation to pellet the cells. For IL-2 release, 5 μL of supernatant was tested using the PerkinElmer Human IL-2 Kit according to the manufacturer's protocol. IL-2 measurements were obtained using a PerkinElmer Envision multilabel microplate reader. All serial dilutions were tested in triplicate.

[0392] The EC50 of the antibody was determined using a 4-parameter logistic equation on a 10-point dose-response curve using GraphPad Prism software. 50 The maximum IL-2 release was determined as the average maximum response detected within the tested concentration range.

[0393] In the absence of PD-L1 on RBL-2H3 / aCD3 target cells, mAb1 increased IL-2 release in a dose-dependent manner, independent of a fixed concentration of cimipril. Both T cell donors exhibited similar ECGs. 50 And the maximum IL-2 value, without depending on cimiprilmab (Figure 16).

[0394] In the presence of PD-L1 on RBL-2H3 / αCD3 cells, baseline IL-2 levels were decreased for both T cell donors compared to target cells lacking PD-L1. However, the addition of a fixed concentration of cimiprimab increased baseline IL-2 levels. For both T cell donors, mAb1 was independent of cimiprimab, resulting in a dose-dependent increase in IL-2, and the addition of cimiprimab further increased the maximum IL-2 level (Figure 16). The results are summarized in Table 21.

[0395] Table 21: Summary of the effects of mAb1 combined with cimiprimab on IL-2 release from anti-CD3 stimulated primary T cells

[0396]

[0397] aThe maximum IL-2 concentration is the highest average IL-2 concentration value recorded within the tested antibody concentration range (76 fM to 200 nM).

[0398] b Cimipril or IgG4 P The isotype control was tested at a fixed concentration of 20 nM.

[0399] NC, not calculated because the 4-parameter logistic regression does not converge to a single value; ND, undetermined because concentration-dependent IL-2 release was not observed.

[0400] Overview

[0401] The above examples demonstrate that mAb1 can bind to human and cynomolgus monkey GITR expressed on the cell surface of T cells and enhance Fcγ receptor-mediated NFAT activation in surrogate ADCC reporter factor assays. Therefore, compared to CD8 + T cells: mAb1 induces ADCP in engineered T cells and mediates preferential ADCC against Tregs. On the other hand, mAb1 cannot mediate CDC in engineered GITR-expressing T cells or form immune complexes capable of binding C1q. mAb1 also blocks the binding of human GITR to human GITR-L. mAb1 leads to a dose-dependent increase in IL-2 independently of cimiprimab, and the addition of cimiprimab further increases the maximum IL-2 level.

[0402] Example 18: Anti-GITR antibodies deplete T regulatory cells within tumors and increase the ratio of CD8+ T cells to T regulatory cells.

[0403] In this experiment, the ability of the parental anti-GITR antibody CompAb1 and its related variants mAb1 and mAb2 to deplete regulatory T cells within tumors was evaluated. MC38 mouse colon tumor cells (3.0 x 10⁻⁶) were used. 5 Twelve-week-old female GITR / GITR-L humanized mice were subcutaneously attacked with 1 cell / mouse. CompAb1, mAb1, or mAb2 was administered via intraperitoneal (ip) injection on day 6. Mice were euthanized on day 11, and tumor tissue was collected for FACS analysis. FACS samples were acquired using a BDFortessa X20 and analyzed using Flowjo software.

[0404] Data shown Figure 17In summary, single-dose antibody therapy with anti-GITR antibodies mediated a significant reduction in intratumoral Treg cells and an increase in the CD8+ T cell / T-reg ratio. Compared to the N101E variant mAb2, CompAb1 and the N101D variant mAb1 more persistently depleted Tregs and increased the CD8 / Treg ratio.

[0405] Example 19: Antitumor efficacy of mAb1 alone in GITR / GITR-L humanized mice and in combination with mAb1 and cimipril in GITR / GITR-L / PD-1 humanized mice carrying mouse colorectal cancer tumors.

[0406] On day 0, MC38 colorectal cancer cells (in 3 x 10⁻⁶ mcgs in 100 μL PBS) were subcutaneously implanted on the right side of female GITR / GITR-L / PD-1 humanized mice aged 9 to 14 weeks. 5 (cells). Six days post-implantation (day 6), when the tumor averaged 43 mm. 3 Mice were randomly divided into 5 groups and administered the first dose of either 10 mg / kg IgG1 isotype control + 1 mg / kg IgG4P isotype control (n=7), 10 mg / kg IgG1 isotype control + 1 mg / kg cimiprimab (n=7), 10 mg / kg mAb1 + 1 mg / kg cimiprimab (n=7), 1 mg / kg mAb1 + 1 mg / kg cimiprimab (n=6), or 0.1 mg / kg mAb1 + 1 mg / kg cimiprimab (n=7). All mice received a second dose on day 13, for a total of 2 doses. Mice were bled on days 7, 12, 14, and 20 to monitor serum antibody concentrations. Tumor growth was monitored by measuring tumor volume on days 5, 10, 13, 17, 20, 26, 28, 31, 34, 38, 41, 45, 48, 52, 60, 68, and 75. All mice were monitored until day 75 unless euthanized prematurely due to tumor burden.

[0407] Compared with isotype control antibody (1 mg / kg IgG4) P Compared with mice treated with a combination of 1 mg / kg IgG1 and isotype control, mice treated with 1 mg / kg cimiprimab in combination with 0.1, 1, or 10 mg / kg mAb1 showed a statistically significant reduction in tumor growth. Figure 18Compared with mice treated with cimiprimab alone (1 mg / kg cimiprimab combined with 10 mg / kg IgG1 isotype control), mice treated with 1 mg / kg cimiprimab combined with 10 mg / kg mAb1 (but not 0.1 or 1 mg / kg mAb1) showed a numerically greater reduction in tumor growth; however, the difference did not reach statistical significance (adjusted p = 0.0525).

[0408] Multiple doses of 1 mg / kg cimipril alone induced tumor clearance in 2 out of 7 mice. Figure 19B Adding mAb1 to cimiprimab increased tumor clearance frequency in 5 out of 7 mice at the highest tested dose of 10 mg / kg mAb1. Figure 19C However, lower doses of 1 mg / kg and 0.1 mg / kg mAb1 did not increase the frequency of tumor clearance (2 out of 6 mice and 2 out of 7 mice, respectively). Figure 19D and Figure 19E All mice that achieved tumor clearance remained tumor-free until the end of the study (day 75, approximately 9 weeks after the last dose). As expected, no tumor clearance was observed in mice administered the isotype control antibody. Figure 19A ).

[0409] The combined Gehan-Breslow-Wilcoxon test detected a statistically significant difference in survival rates between groups (p = 0.0105); a separate Gehan-Breslow-Wilcoxon test was performed for intergroup comparisons. Mice treated with the isotype control antibody (no mice survived) showed a significant increase in survival rate (33% and 71%, respectively) in mice treated with 1 mg / kg cimiprimab in combination with 1 mg / kg or 10 mg / kg mAb1. Figure 20 Furthermore, compared with mice treated with cimiprimab alone, mice treated with a combination of 1 mg / kg cimiprimab and 10 mg / kg mAb1 (but not 0.1 mg / kg or 1 mg / kg mAb1) showed a significant increase in survival (29% survival).

[0410] This disclosure is not limited in scope to the specific embodiments described herein. In fact, various modifications provided herein will be readily apparent to those skilled in the art from the foregoing description and accompanying drawings, in addition to those described herein. Such modifications are intended to fall within the scope of the appended claims. sequence list <110> Regeneron Pharmaceuticals, Inc. R. BABB (BABB, Robert) D. DUDGEON (DUDGEON, Drew) Y. HUANG (HUANG, Yu) R. MOLDEN (MOLDEN, Rosalynn) W. OLSON (OLSON, William) M. SLEEMAN (SLEEMAN, Matthew) D. SKOKOS (SKOKOS, Dimitris) B. WANG (WANG, Bei) <120> Anti-GITR Antibodies and Uses Thereof <130> 10671WO01 <140> TBA <141> 2021-03-05 <150> 62 / 986,494 <151> 2020-03-06 <160> 49 <170> PatentIn Version 3.5 <210> 1 <211> 360 <212> DNA <213> Artificial Sequence <220> <223> Synthesized <400> 1 caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60 acctgcattg tctctggtgg ctccatcagt ggttacttct ggaactggat ccggcagccc 120 ccagggaagg gacttgaatg gattggttat atctattaca gtgggaccac catctacaac 180 ccctccctca agagtcgatt caccatatca ctagacacgt ccaagaacca gttctcccta 240 aagctgacct ctgtgaccgc tgcggacacg gccgtatatt actgtgcgag agagtcgtat 300 aatccctcgc cgcgatattt tgaccactgg ggccagggaa ccctggtcac cgtctcctca 360 <210> 2 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 2 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ile Val Ser Gly Gly Ser Ile Ser Gly Tyr 20 25 30 Phe Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Tyr Tyr Ser Gly Thr Thr Ile Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Phe Thr Ile Ser Leu Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Thr Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Glu Ser Tyr Asn Pro Ser Pro Arg Tyr Phe Asp His Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 3 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> synthesis <400> 3 ggtggctcca tcagtggtta cttc 24 <210> 4 <211> 8 <212> PRT <213> Artificial sequence <220> <223> synthesis <400> 4 Gly Gly Ser Ile Ser Gly Tyr Phe 1 5 <210> 5 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> synthesis <400> 5 atctattaca gtgggaccac c 21 <210> 6 <211> 7 <212> PRT <213> Artificial sequence <220> <223> synthesis <400> 6 Ile Tyr Tyr Ser Gly Thr Thr 1 5 <210> 7 <211> 42 <212> DNA <213> Artificial sequence <220> <223> synthesis <400> 7 gcgagagagt cgtataatcc ctcgccgcga tattttgacc ac 42 <210> 8 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> synthesized <400> 8 Ala Arg Glu Ser Tyr Asn Pro Ser Pro Arg Tyr Phe Asp His 1 5 10 <210> 9 <211> 327 <212> DNA <213> Artificial Sequence <220> <223> synthesized <400> 9 gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgcc gggcaagtca gagcattagc agctatttaa attggtatca gcagaaacca 120 gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccgtca 180 aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240 gaagattttg caacttacta ctgtcaacag agttacagta cccctccgat caccttcggc 300 caagggacac gactggagat taaacga 327 <210> 10 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 10 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Pro 85 90 95 Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg 100 105 <210> 11 <211> 18 <212> DNA <213> artificial sequence <220> <223> synthesis <400> 11 cagagcatta gcagctat 18 <210> 12 <211> 6 <212> PRT <213> artificial sequence <220> <223> synthesis <400> 12 Gln Ser Ile Ser Ser Tyr 1 5 <210> 13 <211> 9 <212> DNA <213> Artificial sequence <220> <223> synthesis <400> 13 gctgcatcc 9 <210> 14 <211> 3 <212> PRT <213> Artificial sequence <220> <223> synthesis <400> 14 Ala Ala Ser 1 <210> 15 <211> 30 <212> DNA <213> Artificial sequence <220> <223> synthesis <400> 15 caacagagtt acagtacccc tccgatcacc 30 <210> 16 <211> 10 <212> PRT <213> Artificial sequence <220> <223> synthesis <400> 16 Gln Gln Ser Tyr Ser Thr Pro Pro Ile Thr 1 5 10 <210> 17 <211> 1353 <212> DNA <213> Artificial sequence <220> <223> synthesis <400> 17 caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60 acctgcattg tctctggtgg ctccatcagt ggttacttct ggaactggat ccggcagccc 120 ccagggaagg gacttgaatg gattggttat atctattaca gtgggaccac catctacaac 180 ccctccctca agagtcgatt caccatatca ctagacacgt ccaagaacca gttctcccta 240 aagctgacct ctgtgaccgc tgcggacacg gccgtatatt actgtgcgag agagtcgtat 300 aatccctcgc cgcgatattt tgaccactgg ggccagggaa ccctggtcac cgtctcctca 360 gcctccacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 420 ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 480 tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 540 ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 600 tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 660 aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 720 ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 780 gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 840 tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 900 agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 960 gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 1020 aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag 1080 ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 1140 gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 1200 ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 1260 cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1320 cagaagtccc tctccctgtc tccgggtaaa tga 1353 <210> 18 <211> 450 <212> PRT <213> Artificial Sequence <220> <223> synthesized <400> 18 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ile Val Ser Gly Gly Ser Ile Ser Gly Tyr 20 25 30 Phe Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Tyr Tyr Ser Gly Thr Thr Ile Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Phe Thr Ile Ser Leu Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Thr Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Glu Ser Tyr Asn Pro Ser Pro Arg Tyr Phe Asp His Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Light 450 <210> 19 <211> 648 <212> DNA <213> artificial sequence <220> <223> synthesis <400> 19 gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgcc gggcaagtca gagcattagc agctatttaa attggtatca gcagaaacca 120 gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccgtca 180 aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240 gaagattttg caacttacta ctgtcaacag agttacagta cccctccgat caccttcggc 300 caagggacac gactggagat taaacgaact gtggctgcac catctgtctt catcttcccg 360 ccatctgatg agcagttgaa atctggaact gcctctgttg tgtgcctgct gaataacttc 420 tatcccagag aggccaaagt acagtggaag gtggataacg ccctccaatc gggtaactcc 480 caggagagtg tcacagagca ggacagcaag gacagcacct acagcctcag cagcaccctg 540 acgctgagca aagcagacta cgagaaacac aaagtctacg cctgcgaagt cacccatcag 600 ggcctgagct cgcccgtcac aaagagcttc aacaggggag agtgttag 648 <210> 20 <211> 215 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 20 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Pro 85 90 95 Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg Thr Val Ala 100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140 Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180 185 190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195 200 205 Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 21 <211> 360 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 21 caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60 acctgcattg tctctggtgg ctccatcagt ggttacttct ggaactggat ccggcagccc 120 ccagggaagg gacttgaatg gattggttat atctattaca gtgggaccac catctacaac 180 ccctccctca agagtcgatt caccatatca ctagacacgt ccaagaacca gttctcccta 240 aagctgacct ctgtgaccgc tgcggacacg gccgtatatt actgtgcgag agagtcgtat 300 gacccctccc cgcgatattt tgaccactgg ggccagggaa ccctggtcac cgtctcctca 360 <210> 22 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> synthesized <400> 22 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ile Val Ser Gly Gly Ser Ile Ser Gly Tyr 20 25 30 Phe Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Tyr Tyr Ser Gly Thr Thr Ile Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Phe Thr Ile Ser Leu Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Thr Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Glu Ser Tyr Asp Pro Ser Pro Arg Tyr Phe Asp His Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 23 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> synthesized <400> 23 gcgagagagt cgtatgaccc ctccccgcga tattttgacc ac 42 <210> 24 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> synthesized <400> 24 Ala Arg Glu Ser Tyr Asp Pro Ser Pro Arg Tyr Phe Asp His 1 5 10 <210> 25 <211> 1353 <212> DNA <213> Artificial Sequence <220> <223> synthesized <400> 25 caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60 acctgcattg tctctggtgg ctccatcagt ggttacttct ggaactggat ccggcagccc 120 ccagggaagg gacttgaatg gattggttat atctattaca gtgggaccac catctacaac 180 ccctccctca agagtcgatt caccatatca ctagacacgt ccaagaacca gttctcccta 240 aagctgacct ctgtgaccgc tgcggacacg gccgtatatt actgtgcgag agagtcgtat 300 gacccctccc cgcgatattt tgaccactgg ggccagggaa ccctggtcac cgtctcctca 360 gcctccacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 420 ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 480 tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 540 ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 600 tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 660 aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 720 ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 780 gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 840 tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 900 agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 960 gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 1020 aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag 1080 aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag 1140 gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 1200 ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 1260 cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1320 cagaagtccc tctccctgtc tccgggtaaa tga 1353 <210> 26 <211> 450 <212> PRT <213> Artificial Sequence <220> <223> Synthesis <400> 26 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ile Val Ser Gly Gly Ser Ile Ser Gly Tyr 20 25 30 Phe Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Tyr Tyr Ser Gly Thr Thr Ile Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Phe Thr Ile Ser Leu Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Thr Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Glu Ser Tyr Asp Pro Ser Pro Arg Tyr Phe Asp His Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys 450 <210> 27 <211> 360 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 27 caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60 acctgcattg tctctggtgg ctccatcagt ggttacttct ggaactggat ccggcagccc 120 ccagggaagg gacttgaatg gattggttat atctattaca gtgggaccac catctacaac 180 ccctccctca agagtcgatt caccatatca ctagacacgt ccaagaacca gttctcccta 240 aagctgacct ctgtgaccgc tgcggacacg gccgtatatt actgtgcgag agagtcgtat 300 gagccctccc cgcgatattt tgaccactgg ggccagggaa ccctggtcac cgtctcctca 360 <210> 28 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 28 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ile Val Ser Gly Gly Ser Ile Ser Gly Tyr 20 25 30 Phe Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Tyr Tyr Ser Gly Thr Thr Ile Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Phe Thr Ile Ser Leu Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Thr Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Glu Ser Tyr Glu Pro Ser Pro Arg Tyr Phe Asp His Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 29 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 29 gcgagagagt cgtatgagcc ctccccgcga tattttgacc ac 42 <210> 30 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 30 Ala Arg Glu Ser Tyr Glu Pro Ser Pro Arg Tyr Phe Asp His 1 5 10 <210> 31 <211> 1353 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 31 caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60 acctgcattg tctctggtgg ctccatcagt ggttacttct ggaactggat ccggcagccc 120 ccagggaagg gacttgaatg gattggttat atctattaca gtgggaccac catctacaac 180 ccctccctca agagtcgatt caccatatca ctagacacgt ccaagaacca gttctcccta 240 aagctgacct ctgtgaccgc tgcggacacg gccgtatatt actgtgcgag agagtcgtat 300 gagccctccc cgcgatattt tgaccactgg ggccagggaa ccctggtcac cgtctcctca 360 gcctccacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 420 ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 480 tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 540 ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 600 tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 660 aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 720 ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 780 gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 840 tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 900 agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 960 gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 1020 aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag 1080 ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 1140 gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 1200 ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 1260 cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1320 cagaagtccc tctccctgtc tccgggtaaa tga 1353 <210> 32 <211> 450 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 32 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ile Val Ser Gly Gly Ser Ile Ser Gly Tyr 20 25 30 Phe Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Tyr Tyr Ser Gly Thr Thr Ile Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Phe Thr Ile Ser Leu Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Thr Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Glu Ser Tyr Glu Pro Ser Pro Arg Tyr Phe Asp His Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys 450 <210> 33 <211> 360 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 33 caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60 acctgcattg tctctggtgg ctccatcagt ggttacttct ggaactggat ccggcagccc 120 ccagggaagg gacttgaatg gattggttat atctattaca gtgggaccac catctacaac 180 ccctccctca agagtcgatt caccatatca ctagacacgt ccaagaacca gttctcccta 240 aagctgacct ctgtgaccgc tgcggacacg gccgtatatt actgtgcgag agagtcgtat 300 tccccctccc cgcgatattt tgaccactgg ggccagggaa ccctggtcac cgtctcctca 360 <210> 34 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 34 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ile Val Ser Gly Gly Ser Ile Ser Gly Tyr 20 25 30 Phe Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Tyr Tyr Ser Gly Thr Thr Ile Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Phe Thr Ile Ser Leu Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Thr Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Glu Ser Tyr Ser Pro Ser Pro Arg Tyr Phe Asp His Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 35 <211> 42 <212> DNA <213> Artificial sequence <220> <223> synthesis <400> 35 gcgagagagt cgtattcccc ctccccgcga tattttgacc ac 42 <210> 36 <211> 14 <212> PRT <213> Artificial sequence <220> <223> synthesis <400> 36 Ala Arg Glu Ser Tyr Ser Pro Ser Pro Arg Tyr Phe Asp His 1 5 10 <210> 37 <211> 1353 <212> DNA <213> Artificial sequence <220> <223> synthesis <400> 37 caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60 acctgcattg tctctggtgg ctccatcagt ggttacttct ggaactggat ccggcagccc 120 ccagggaagg gacttgaatg gattggttat atctattaca gtgggaccac catctacaac 180 ccctccctca agagtcgatt caccatatca ctagacacgt ccaagaacca gttctcccta 240 aagctgacct ctgtgaccgc tgcggacacg gccgtatatt actgtgcgag agagtcgtat 300 tccccctccc cgcgatattt tgaccactgg ggccagggaa ccctggtcac cgtctcctca 360 gcctccacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 420 ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 480 tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 540 ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 600 tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 660 aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 720 ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 780 gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 840 tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 900 agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 960 gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 1020 aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag 1080 ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 1140 gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 1200 ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 1260 cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1320 cagaagtccc tctccctgtc tccgggtaaa tga 1353 <210> 38 <211> 450 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 38 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ile Val Ser Gly Gly Ser Ile Ser Gly Tyr 20 25 30 Phe Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Tyr Tyr Ser Gly Thr Thr Ile Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Phe Thr Ile Ser Leu Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Thr Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Glu Ser Tyr Ser Pro Ser Pro Arg Tyr Phe Asp His Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Light 450 <210> 39 <211> 360 <212> DNA <213> artificial sequence <220> <223> synthesis <400> 39 caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60 acctgcattg tctctggtgg ctccatcagt ggttacttct ggaactggat ccggcagccc 120 ccagggaagg gacttgaatg gattggttat atctattaca gtgggaccac catctacaac 180 ccctccctca agagtcgatt caccatatca ctagacacgt ccaagaacca gttctcccta 240 aagctgacct ctgtgaccgc tgcggacacg gccgtatatt actgtgcgag agagtcgtat 300 accccctccc cgcgatattt tgaccactgg ggccagggaa ccctggtcac cgtctcctca 360 <210> 40 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Synthesis <400> 40 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ile Val Ser Gly Gly Ser Ile Ser Gly Tyr 20 25 30 Phe Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Tyr Tyr Ser Gly Thr Thr Ile Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Phe Thr Ile Ser Leu Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Thr Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Glu Ser Tyr Thr Pro Ser Pro Arg Tyr Phe Asp His Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 41 <211> 42 <212> DNA <213> Artificial sequence <220> <223> synthesis <400> 41 gcgagagagt cgtatacccc ctccccgcga tattttgacc ac 42 <210> 42 <211> 14 <212> PRT <213> Artificial sequence <220> <223> synthesis <400> 42 Ala Arg Glu Ser Tyr Thr Pro Ser Pro Arg Tyr Phe Asp His 1 5 10 <210> 43 <211> 1353 <212> DNA <213> Artificial sequence <220> <223> synthesis <400> 43 caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60 acctgcattg tctctggtgg ctccatcagt ggttacttct ggaactggat ccggcagccc 120 ccagggaagg gacttgaatg gattggttat atctattaca gtgggaccac catctacaac 180 ccctccctca agagtcgatt caccatatca ctagacacgt ccaagaacca gttctcccta 240 aagctgacct ctgtgaccgc tgcggacacg gccgtatatt actgtgcgag agagtcgtat 300 accccctccc cgcgatattt tgaccactgg ggccagggaa ccctggtcac cgtctcctca 360 gcctccacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 420 ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 480 tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 540 ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 600 tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 660 aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 720 ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 780 gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 840 tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 900 agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 960 gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 1020 aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag 1080 ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 1140 gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 1200 ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 1260 cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1320 cagaagtccc tctccctgtc tccgggtaaa tga 1353 <210> 44 <211> 450 <212> PRT <213> Artificial Sequence <220> <223> synthesized <400> 44 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ile Val Ser Gly Gly Ser Ile Ser Gly Tyr 20 25 30 Phe Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Tyr Tyr Ser Gly Thr Thr Ile Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Phe Thr Ile Ser Leu Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Thr Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Glu Ser Tyr Thr Pro Ser Pro Arg Tyr Phe Asp His Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Light 450 <210> 45 <211> 194 <212> PRT <213> artificial sequence <220> <223> synthesis <400> 45 Met His Arg Pro Arg Arg Arg Gly Thr Arg Pro Pro Pro Leu Ala Leu 1 5 10 15 Leu Ala Ala Leu Leu Leu Ala Ala Arg Gly Ala Asp Ala Gln Arg Pro 20 25 30 Thr Gly Gly Pro Gly Cys Gly Pro Gly Arg Leu Leu Leu Gly Thr Gly 35 40 45 Thr Asp Ala Arg Cys Cys Arg Val His Thr Thr Arg Cys Cys Arg Asp 50 55 60 Tyr Pro Gly Glu Glu Cys Cys Ser Glu Trp Asp Cys Met Cys Val Gln 65 70 75 80 Pro Glu Phe His Cys Gly Asp Pro Cys Cys Thr Thr Cys Arg His His 85 90 95 Pro Cys Pro Pro Gly Gln Gly Val Gln Ser Gln Gly Lys Phe Ser Phe 100 105 110 Gly Phe Gln Cys Ile Asp Cys Ala Ser Gly Thr Phe Ser Gly Gly His 115 120 125 Glu Gly His Cys Lys Pro Trp Thr Asp Cys Thr Gln Phe Gly Phe Leu 130 135 140 Thr Val Phe Pro Gly Asn Lys Thr His Asn Ala Val Cys Val Pro Gly 145 150 155 160 Ser Pro Pro Ala Glu Pro Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu 165 170 175 Gly Gly Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu His His His His 180 185 190 His His <210> 46 <211> 399 <212> PRT <213> Artificial Sequence <220> <223> Synthesized <400> 46 Met His Arg Pro Arg Arg Arg Gly Thr Arg Pro Pro Pro Leu Ala Leu 1 5 10 15 Leu Ala Ala Leu Leu Leu Ala Ala Arg Gly Ala Asp Ala Gln Arg Pro 20 25 30 Thr Gly Gly Pro Gly Cys Gly Pro Gly Arg Leu Leu Leu Gly Thr Gly 35 40 45 Thr Asp Ala Arg Cys Cys Arg Val His Thr Thr Arg Cys Cys Arg Asp 50 55 60 Tyr Pro Gly Glu Glu Cys Cys Ser Glu Trp Asp Cys Met Cys Val Gln 65 70 75 80 Pro Glu Phe His Cys Gly Asp Pro Cys Cys Thr Thr Cys Arg His His 85 90 95 Pro Cys Pro Pro Gly Gln Gly Val Gln Ser Gln Gly Lys Phe Ser Phe 100 105 110 Gly Phe Gln Cys Ile Asp Cys Ala Ser Gly Thr Phe Ser Gly Gly His 115 120 125 Glu Gly His Cys Lys Pro Trp Thr Asp Cys Thr Gln Phe Gly Phe Leu 130 135 140 Thr Val Phe Pro Gly Asn Lys Thr His Asn Ala Val Cys Val Pro Gly 145 150 155 160 Ser Pro Pro Ala Glu Pro Glu Pro Arg Gly Pro Thr Ile Lys Pro Cys 165 170 175 Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser Val 180 185 190 Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser 195 200 205 Pro Ile Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro Asp 210 215 220 Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala Gln 225 230 235 240 Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser 245 250 255 Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys 260 265 270 Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr Ile 275 280 285 Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln Val Tyr Val Leu Pro 290 295 300 Pro Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr Leu Thr Cys Met 305 310 315 320 Val Thr Asp Phe Met Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn 325 330 335 Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser 340 345 350 Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn 355 360 365 Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His Glu Gly Leu 370 375 380 His Asn His His Thr Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys 385 390 395 <210> 47 <211> 393 <212> PRT Artificial Sequence <220> <223> Synthesis <400> 47 Met His Arg Pro Arg Arg Arg Gly Thr Arg Pro Pro Pro Leu Ala Leu 1 5 10 15 Leu Ala Ala Leu Leu Leu Ala Ala Arg Gly Ala Asp Ala Gln Arg Pro 20 25 30 Thr Gly Gly Pro Gly Cys Gly Pro Gly Arg Leu Leu Leu Gly Thr Gly 35 40 45 Thr Asp Ala Arg Cys Cys Arg Val His Thr Thr Arg Cys Cys Arg Asp 50 55 60 Tyr Pro Gly Glu Glu Cys Cys Ser Glu Trp Asp Cys Met Cys Val Gln 65 70 75 80 Pro Glu Phe His Cys Gly Asp Pro Cys Cys Thr Thr Cys Arg His His 85 90 95 Pro Cys Pro Pro Gly Gln Gly Val Gln Ser Gln Gly Lys Phe Ser Phe 100 105 110 Gly Phe Gln Cys Ile Asp Cys Ala Ser Gly Thr Phe Ser Gly Gly His 115 120 125 Glu Gly His Cys Lys Pro Trp Thr Asp Cys Thr Gln Phe Gly Phe Leu 130 135 140 Thr Val Phe Pro Gly Asn Lys Thr His Asn Ala Val Cys Val Pro Gly 145 150 155 160 Ser Pro Pro Ala Glu Pro Asp Lys Thr His Thr Cys Pro Pro Cys Pro 165 170 175 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 180 185 190 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 195 200 205 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 210 215 220 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 225 230 235 240 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 245 250 255 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 260 265 270 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 275 280 285 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu 290 295 300 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 305 310 315 320 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 325 330 335 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 340 345 350 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 355 360 365 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 370 375 380 Lys Ser Leu Ser Leu Ser Pro Gly Lys 385 390 <210> 48 <211> 194 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 48 Met His Arg Pro Arg Arg Arg Gly Thr Arg Pro Pro Pro Leu Ala Leu 1 5 10 15 Leu Ala Ala Leu Leu Leu Ala Ala Arg Gly Ala Asp Ala Gln Arg Pro 20 25 30 Thr Gly Gly Pro Gly Cys Gly Pro Gly Arg Leu Leu Leu Gly Thr Gly 35 40 45 Lys Asp Ala Arg Cys Cys Arg Val His Pro Thr Arg Cys Cys Arg Asp 50 55 60 Tyr Gln Gly Glu Glu Cys Cys Ser Glu Trp Asp Cys Val Cys Val Gln 65 70 75 80 Pro Glu Phe His Cys Gly Asn Pro Cys Cys Thr Thr Cys Gln His His 85 90 95 Pro Cys Pro Ser Gly Gln Gly Val Gln Pro Gln Gly Lys Phe Ser Phe 100 105 110 Gly Phe Arg Cys Val Asp Cys Ala Leu Gly Thr Phe Ser Arg Gly His 115 120 125 Asp Gly His Cys Lys Pro Trp Thr Asp Cys Thr Gln Phe Gly Phe Leu 130 135 140 Thr Val Phe Pro Gly Asn Lys Thr His Asn Ala Val Cys Val Pro Gly 145 150 155 160 Ser Pro Pro Ala Glu Pro Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu 165 170 175 Gly Gly Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu His His His His 180 185 190 His His <210> 49 <211> 241 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 49 Met Ala Gln His Gly Ala Met Gly Ala Phe Arg Ala Leu Cys Gly Leu 1 5 10 15 Ala Leu Leu Cys Ala Leu Ser Leu Gly Gln Arg Pro Thr Gly Gly Pro 20 25 30 Gly Cys Gly Pro Gly Arg Leu Leu Leu Gly Thr Gly Thr Asp Ala Arg 35 40 45 Cys Cys Arg Val His Thr Thr Arg Cys Cys Arg Asp Tyr Pro Gly Glu 50 55 60 Glu Cys Cys Ser Glu Trp Asp Cys Met Cys Val Gln Pro Glu Phe His 65 70 75 80 Cys Gly Asp Pro Cys Cys Thr Thr Cys Arg His His Pro Cys Pro Pro 85 90 95 Gly Gln Gly Val Gln Ser Gln Gly Lys Phe Ser Phe Gly Phe Gln Cys 100 105 110 Ile Asp Cys Ala Ser Gly Thr Phe Ser Gly Gly His Glu Gly His Cys 115 120 125 Lys Pro Trp Thr Asp Cys Thr Gln Phe Gly Phe Leu Thr Val Phe Pro 130 135 140 Gly Asn Lys Thr His Asn Ala Val Cys Val Pro Gly Ser Pro Pro Ala 145 150 155 160 Glu Pro Leu Gly Trp Leu Thr Val Val Leu Leu Ala Val Ala Ala Cys 165 170 175 Val Leu Leu Leu Thr Ser Ala Gln Leu Gly Leu His Ile Trp Gln Leu 180 185 190 Arg Ser Gln Cys Met Trp Pro Arg Glu Thr Gln Leu Leu Leu Glu Val 195 200 205 Pro Pro Ser Thr Glu Asp Ala Arg Ser Cys Gln Phe Pro Glu Glu Glu 210 215 220 Arg Gly Glu Arg Ser Ala Glu Glu Lys Gly Arg Leu Gly Asp Leu Trp 225 230 235 240 Wave

Claims

1. An isolated antibody or antigen-binding fragment thereof that binds to a glucocorticoid-induced tumor necrosis factor receptor (GITR), wherein the antibody or antigen-binding fragment comprises (i) three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3), wherein the amino acid sequence of HCDR1 is SEQ ID NO: 4, the amino acid sequence of HCDR2 is SEQ ID NO: 6, and the amino acid sequence of HCDR3 is SEQ ID NO: 24 or SEQ ID NO: 30; and (ii) three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3), wherein the amino acid sequence of LCDR1 is SEQ ID NO: 12, the amino acid sequence of LCDR2 is SEQ ID NO: 14, and the amino acid sequence of LCDR3 is SEQ ID NO:

16.

2. The isolated antibody or its antigen-binding fragment as described in claim 1, wherein the amino acid sequence of HCDR3 is SEQ ID NO:

24.

3. The isolated antibody or antigen-binding fragment thereof as claimed in claim 2, wherein the isolated antibody or antigen-binding fragment thereof comprises a heavy chain variable region (HCVR) having the amino acid sequence of SEQ ID NO:

22.

4. The isolated antibody or its antigen-binding fragment as described in claim 1, wherein the amino acid sequence of HCDR3 is SEQ ID NO:

30.

5. The isolated antibody or antigen-binding fragment thereof as claimed in claim 4, wherein the isolated antibody or antigen-binding fragment thereof comprises HCVR having the amino acid sequence of SEQ ID NO:

28.

6. The isolated antibody or antigen-binding fragment thereof as described in any one of claims 1-5, wherein the isolated antibody or antigen-binding fragment thereof comprises an LCVR having the amino acid sequence of SEQ ID NO:

10.

7. The isolated antibody or antigen-binding fragment thereof as claimed in claim 1, wherein the isolated antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO:

26.

8. The isolated antibody or antigen-binding fragment thereof as claimed in claim 1, wherein the isolated antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO:

32.

9. The isolated antibody or antigen-binding fragment thereof as claimed in claim 7 or 8, wherein the isolated antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, wherein the light chain comprises the amino acid sequence of SEQ ID NO:

20.

10. A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof as described in any one of claims 1-9 and a pharmaceutically acceptable carrier.

11. A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof as described in any one of claims 1-9 and a pharmaceutically acceptable diluent.

12. Use of the antibody or antigen-binding fragment thereof as described in any one of claims 1-9 in the preparation of a medicament for treating cancer in a subject.

13. The use as described in claim 12, wherein the drug is used in combination with an antibody or antigen-binding fragment thereof that binds to a T-cell activation receptor, wherein the T-cell activation receptor is CD28, OX40, CD137, CD27, or HVEM.

14. The use as described in claim 12, wherein the drug is used in combination with an antibody or antigen-binding fragment thereof that binds to a T-cell inhibitory receptor, wherein the T-cell inhibitory receptor is CTLA-4, PD-1, TIM-3, BTLA, VISTA, or LAG-3.

15. The use as claimed in claim 14, wherein the T-cell inhibitory receptor is PD-1, and the antibody binding to the T-cell inhibitory receptor is cimipril.

16. The use as described in claim 12, wherein the drug is used in combination with radiotherapy.

17. The use as described in claim 12, wherein the drug is used in combination with a chemotherapy agent.

18. The use as described in any one of claims 12-17, wherein the cancer is selected from the group consisting of: squamous cell carcinoma of the skin (CSCC), myeloma, lung cancer, melanoma, cervical cancer, renal cell carcinoma (RCC), adenocarcinoma, colorectal cancer (CRC), head and neck cancer, malignant glioma, osteosarcoma, gastric cancer, malignant mesothelioma, ovarian cancer, synovial sarcoma, testicular cancer, esophageal cancer, uterine cancer, and liver cancer.

19. The use as claimed in any one of claims 12-17, wherein the cancer is selected from the group consisting of breast cancer, pancreatic cancer, prostate cancer, and thyroid cancer.

20. The use as described in claim 18, wherein the lung cancer is small cell lung cancer.

21. The use as described in claim 18, wherein the lung cancer is non-small cell lung cancer (NSCLC).

22. The use as claimed in claim 18, wherein the cervical cancer is cervical squamous cell carcinoma (cervical SCC).

23. The use as claimed in claim 19, wherein the breast cancer is triple-negative breast cancer.

24. The use as claimed in claim 18, wherein the head and neck cancer is head and neck squamous cell carcinoma (SCCHN).

25. The use as claimed in claim 18, wherein the malignant glioma is glioblastoma multiforme.

26. The use as claimed in claim 18, wherein the gastric cancer is a gastric cancer with MET amplification.

27. The use as claimed in claim 18, wherein the uterine cancer is endometrial cancer.

28. The use as claimed in claim 18, wherein the myeloma is multiple myeloma.

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