Activating anti-GAL9 binding molecule

Abstract

The present invention discloses an anti-GAL9 antibody construct, a pharmaceutical composition comprising the construct, and a method of using the same.

Description

[0001] 1. Cross-reference to related applications

[0002] This application claims the benefit of prior co-pending U.S. Provisional Patent Application No. 62 / 964,487, filed on January 22, 2020, U.S. Provisional Patent Application No. 62 / 900,105, filed on September 13, 2019, and U.S. Provisional Patent Application No. 62 / 855,590, filed on May 31, 2019, pursuant to U.S.C. §119(e).

[0003] 2. Sequence Listing

[0004] This application contains a sequence listing submitted via EFS-Web, which is incorporated herein by reference in its entirety. The ASCII copy created on April 9, 2020 is named 42700WO_CRF_sequencelisting.txt and is 389,339 bytes in size. 3. Background Technology

[0006] Immunotherapy holds enormous potential for treating cancer. However, tumors can become resistant to immunotherapy, for example by recruiting immunosuppressive cells or signaling molecules into the tumor microenvironment or by coercing immune checkpoint signaling pathways.

[0007] Galectin-9 (GAL9) is an S-type lectin β-galactoside binding protein whose N- and C-terminal carbohydrate binding domains are connected by a linker peptide. GAL9 is involved in regulating cell-cell and cell-matrix interactions. GAL9 has been shown to bind to soluble PD-L2, and it has been suggested that at least some of the immune effects of PD-L2 are mediated by the binding of multimeric PD-L2 to GAL9 rather than by PD-1 (WO 2016 / 008005, which is incorporated herein by reference in its entirety). However, the mechanism by which GAL9 and PD-L2 affect immune effector function has not been fully characterized.

[0008] There remains a need for therapeutic agents that can enhance immune effector function and reduce immunosuppressive or T cell exhaustion pathways. Such therapeutic agents could be used to improve cancer immunotherapy. 4. Summary of the Invention

[0010] In a first aspect, the present disclosure provides a Galectin-9 (GAL9) antigen binding molecule comprising a first antigen binding site (ABS) specific for a first epitope of a first GAL9 antigen, wherein the first antigen binding site comprises all three VH CDRs from any one ABS clone selected from P9-02B, P9-04, P9-05, P9-08, P9-09, P9-10, P9-15, P9-16, P9-18, P9-19, P9-20, P9-21, P9-22, P9-27, P9-28, P9-31, P9-32, P9-36, P9-39, P9-49, P9-54, and P9-58.

[0011] In a second aspect, the present disclosure provides a Galectin-9 (GAL9) antigen binding molecule comprising a first antigen binding site specific for a first epitope of a first GAL9 antigen, wherein the first antigen binding site comprises all three VL CDRs from any one ABS clone selected from P9-02B, P9-04, P9-05, P9-08, P9-09, P9-10, P9-15, P9-16, P9-18, P9-19, P9-20, P9-21, P9-22, P9-27, P9-28, P9-31, P9-32, P9-36, P9-39, P9-49, P9-54, and P9-58.

[0012] In a third aspect, the present disclosure provides a Galectin-9 (GAL9) antigen binding molecule comprising a first antigen binding site specific for a first epitope of a first GAL9 antigen, wherein the first antigen binding site comprises all three VH CDRs and all three VL CDRs from any one ABS clone selected from P9-02B, P9-04, P9-05, P9-08, P9-09, P9-10, P9-15, P9-16, P9-18, P9-19, P9-20, P9-21, P9-22, P9-27, P9-28, P9-31, P9-32, P9-36, P9-39, P9-49, P9-54, and P9-58.

[0013] In a fourth aspect, the present disclosure provides a galectin-9 (GAL9) antigen binding molecule comprising a first antigen binding site specific for a first epitope of a first GAL9 antigen, comprising a VL sequence and a VH sequence from any one ABS clone selected from P9-02B, P9-04, P9-05, P9-08, P9-09, P9-10, P9-15, P9-16, P9-18, P9-19, P9-20, P9-21, P9-22, P9-27, P9-28, P9-31, P9-32, P9-36, P9-39, P9-49, P9-54, and P9-58.

[0014] In some embodiments, the GAL9 antigen binding molecule comprises a complete immunoglobulin heavy chain "IgG1" sequence comprising a VH sequence and a complete immunoglobulin light chain sequence comprising a VL sequence, wherein the VH sequence and the VL sequence are from any one ABS clone selected from P9-02B, P9-04, P9-05, P9-08, P9-09, P9-10, P9-15, P9-16, P9-18, P9-19, P9-20, P9-21, P9-22, P9-27, P9-28, P9-31, P9-32, P9-36, P9-39, P9-49, P9-54 and P9-58.

[0015] In some embodiments, the GAL9 antigen binding molecule comprises a complete immunoglobulin heavy chain "IgG4" sequence comprising a VH sequence and a complete immunoglobulin light chain sequence comprising a VL sequence, wherein the VH sequence and the VL sequence are from any one ABS clone selected from P9-02B, P9-04, P9-05, P9-08, P9-09, P9-10, P9-15, P9-16, P9-18, P9-19, P9-20, P9-21, P9-22, P9-27, P9-28, P9-31, P9-32, P9-36, P9-39, P9-49, P9-54 and P9-58.

[0016] In some embodiments, the GAL9 antigen binding molecule comprises a complete immunoglobulin heavy chain "IgG3" sequence comprising a VH sequence and a complete immunoglobulin light chain sequence comprising a VL sequence, wherein the VH sequence and the VL sequence are from any one ABS clone selected from P9-02B, P9-04, P9-05, P9-08, P9-09, P9-10, P9-15, P9-16, P9-18, P9-19, P9-20, P9-21, P9-22, P9-27, P9-28, P9-31, P9-32, P9-36, P9-39, P9-49, P9-54 and P9-58.

[0017] In some embodiments, the GAL9 antigen binding molecule may comprise a GAL9 antigen that is a human GAL9 antigen.

[0018] In some embodiments, the GAL9 antigen binding molecule may further comprise a second antigen binding site.

[0019] In certain embodiments, the second antigen binding site is specific for the GAL9 antigen. In other embodiments, the second antigen binding site is identical to the first antigen binding site.

[0020] In other embodiments, the second antigen binding site is specific for a second epitope of the first GAL9 antigen.

[0021] In some embodiments, the second antigen binding site comprises all three VH CDRs, all three VL CDRs, or all three VH CDRs and all three VL CDRs from another ABS clone selected from P9-02B, P9-04, P9-05, P9-08, P9-09, P9-10, P9-15, P9-16, P9-18, P9-19, P9-20, P9-21, P9-22, P9-27, P9-28, P9-31, P9-32, P9-36, P9-39, P9-49, P9-54, and P9-58.

[0022] In some embodiments, the second antigen binding site comprises a VL sequence and a VH sequence from another ABS clone.

[0023] In some embodiments, the second antigen binding site comprises a complete immunoglobulin heavy chain sequence comprising a VH sequence and a complete immunoglobulin light chain sequence comprising a VL sequence, wherein the VH sequence and VL sequence are from another ABS clone.

[0024] In some embodiments, the second antigen binding site is specific for an antigen other than the first GAL9 antigen.

[0025] In some embodiments, the first antigen binding site comprises all three VH CDRs, all three VL CDRs, or all three VH CDRs and all three VL CDRs from any ABS selected from P9-02B, P9-04, P9-05, P9-08, P9-09, P9-10, P9-15, P9-16, P9-18, P9-19, P9-20, P9-21, P9-22, P9-27, P9-28, P9-31, P9-32, P9-36, P9-39, P9-49, P9-54, and P9-58.

[0026] In some embodiments, the first antigen binding site comprises all three VH CDRs, all three VL CDRs, or all three VH CDRs and all three VL CDRs from any one ABS clone selected from P9-18, P9-15, P9-21, and P9-28.

[0027] In some embodiments, the first antigen binding site comprises all three VH CDRs, all three VL CDRs, or all three VH CDRs and all three VL CDRs from ABS clone P9-15.

[0028] In some embodiments, the first antigen binding site comprises all three VHCDRs, all three VL CDRs, or all three VH CDRs and all three VL CDRs from ABS clone P9-18.

[0029] In some embodiments, the first antigen binding site comprises all three VHCDRs, all three VL CDRs, or all three VH CDRs and all three VL CDRs from ABS clone P9-21.

[0030] In some embodiments, the first antigen binding site comprises all three VH CDRs, all three VL CDRs, or all three VH CDRs and all three VL CDRs from ABS clone P9-22.

[0031] In some embodiments, the first antigen binding site comprises all three VHCDRs, all three VL CDRs, or all three VH CDRs and all three VL CDRs from ABS clone P9-28.

[0032] In some embodiments, the GAL9 antigen binding molecule comprises an antibody format selected from the group consisting of a full-length antibody, a Fab fragment, a F(ab)'2 fragment, a Fv, a scFv, a tandcFv, a Diabody, a scDiabody, a DART, a single-chain VHH derived antibody, a tandAb, a minibody, and a B-body. B-bodies are described in U.S. Pre-Grant Publication No. US 2018 / 0118811, which is incorporated herein by reference in its entirety.

[0033] In some embodiments, the GAL9 antigen binding molecule increases TNF-α secretion by activated immune cells, wherein the increase is greater than a 20, 30, 40, 50, 60, 70, or 80-fold increase relative to activated immune cells treated with a control agent.

[0034] In some embodiments, the GAL9 antigen binding molecule increases IFN-γ secretion by activated immune cells, wherein the increase is greater than a 1.2-fold increase relative to activated immune cells treated with a control agent.

[0035] In some embodiments, the GAL9 antigen binding molecule increases CD40L surface expression on activated CD8+ T cells, wherein the increase is greater than a 2-fold increase relative to activated CD8+ T cells treated with a control agent.

[0036] In some embodiments, the GAL9 antigen binding molecule increases OX40 surface expression on activated CD8+ T cells, wherein the increase is greater than a 2-fold increase relative to activated CD8+ T cells treated with a control agent.

[0037] In some embodiments, the GAL9 antigen binding molecule increases IL-12 production by activated dendritic cells (DCs), wherein the increase is greater than a 20-fold increase relative to activated DCs treated with a control agent.

[0038] In some embodiments, the GAL9 antigen binding molecule increases PD-L2 surface expression on activated dendritic cells (DCs), wherein the increase is greater than a 4-fold increase relative to activated DCs treated with a control agent.

[0039] In some embodiments, the control agent is a negative control agent or a positive control agent.

[0040] In some embodiments, the control agent is a control antibody.

[0041] In some embodiments, the control antibody is selected from the group consisting of: ECA 42 clone anti-GAL9 antibody, RG9.1 clone anti-GAL9 antibody, RG9.35 clone anti-GAL9 antibody, anti-PD1 antibody, and non-GAL9 binding isotype control antibody.

[0042] In some embodiments, the activated immune cells, activated CD8+ T cells, or activated DCs are activated by peptide stimulation (eg, by a peptide or peptides known to induce an immune response).

[0043] In a fifth aspect, the present disclosure provides that a GAL9 antigen binding molecule increases TNF-α secretion by activated immune cells, wherein the increase is greater than an 80-fold increase relative to activated immune cells treated with a control agent.

[0044] In a sixth aspect, the present disclosure provides that a GAL9 antigen binding molecule increases IFN-γ secretion by activated immune cells, wherein the increase is greater than a 1.2-fold increase relative to activated immune cells treated with a control agent.

[0045] In a seventh aspect, the present disclosure provides that a GAL9 antigen binding molecule increases CD40L surface expression on activated CD8+ T cells, wherein the increase is greater than a 2-fold increase relative to activated CD8+ T cells treated with a control agent.

[0046] In an eighth aspect, the disclosure provides that a GAL9 antigen binding molecule increases OX40 surface expression on activated CD8+ T cells, wherein the increase is greater than a 2-fold increase relative to activated CD8+ T cells treated with a control agent.

[0047] In a ninth aspect, the disclosure provides that a GAL9 antigen binding molecule increases IL-12 production by activated dendritic cells (DCs), wherein the increase is greater than a 20-fold increase relative to activated DCs treated with a control agent.

[0048] In a tenth aspect, the present disclosure provides that a GAL9 antigen binding molecule increases PD-L2 surface expression on activated dendritic cells (DCs), wherein the increase is greater than a 4-fold increase relative to activated DCs treated with a control agent.

[0049] In an eleventh aspect, the present disclosure provides GAL9 antigen binding molecules that exhibit one or more of the following properties: A) increase TNF-α secretion by activated immune cells, wherein the increase is greater than an 80-fold increase relative to activated immune cells treated with a control agent; B) increase IFN-γ secretion by activated immune cells, wherein the increase is greater than a 1.2-fold increase relative to activated immune cells treated with a control agent; C) increase CD40L surface expression by activated CD8+ T cells, wherein the increase is greater than an 1.2-fold increase relative to activated CD8+ T cells treated with a control agent; , wherein the increase is greater than a 2-fold increase; D) increasing OX40 surface expression on activated CD8+ T cells, wherein the increase is greater than a 2-fold increase relative to activated CD8+ T cells treated with a control agent; E) increasing IL-12 production by activated dendritic cells (DCs), wherein the increase is greater than a 20-fold increase relative to activated DCs treated with a control agent; F) increasing PD-L2 surface expression on activated dendritic cells (DCs), wherein the increase is greater than a 4-fold increase relative to activated DCs treated with a control agent.

[0050] In some embodiments, the control agent is a negative control agent or a positive control agent.

[0051] In some embodiments, the control agent is a control antibody.

[0052] In some embodiments, the control antibody is selected from the group consisting of: ECA 42 clone anti-GAL9 antibody, RG9.1 clone anti-GAL9 antibody, RG9.35 clone anti-GAL9 antibody, anti-PD1 antibody, and non-GAL9 binding isotype control antibody.

[0053] In some embodiments, the activated immune cells, activated CD8+ T cells, or activated DCs are activated by peptide stimulation (eg, by a peptide or peptides known to induce an immune response).

[0054] In some embodiments, the GAL9 antigen binding molecules of the fifth to eleventh aspects provided herein comprise a first antigen binding site specific for a first epitope of a first GAL9 antigen, wherein the first antigen binding site comprises all three VH CDRs and all three VL CDRs from any one ABS clone selected from P9-02B, P9-04, P9-05, P9-08, P9-09, P9-10, P9-15, P9-16, P9-18, P9-19, P9-20, P9-21, P9-22, P9-27, P9-28, P9-31, P9-32, P9-36, P9-39, P9-49, P9-54, and P9-58.

[0055] In some embodiments, the VL sequence and the VH sequence are from any one ABS clone selected from P9-02B, P9-04, P9-05, P9-08, P9-09, P9-10, P9-15, P9-16, P9-18, P9-19, P9-20, P9-21, P9-22, P9-27, P9-28, P9-31, P9-32, P9-36, P9-39, P9-49, P9-54, and P9-58.

[0056] In some embodiments, the GAL9 antigen binding molecule comprises a complete immunoglobulin heavy chain sequence comprising a VH sequence and a complete immunoglobulin heavy chain sequence comprising a VL sequence, wherein the VH sequence and the VL sequence are from any one ABS clone selected from P9-02B, P9-04, P9-05, P9-08, P9-09, P9-10, P9-15, P9-16, P9-18, P9-19, P9-20, P9-21, P9-22, P9-27, P9-28, P9-31, P9-32, P9-36, P9-39, P9-49, P9-54, and P9-58.

[0057] In some embodiments, the GAL9 antigen is a human GAL9 antigen.

[0058] In some embodiments, the GAL9 antigen binding molecule further comprises a second antigen binding site.

[0059] In some embodiments, the second antigen binding site is specific for the GAL9 antigen.

[0060] The GAL9 antigen binding molecule of claim 48, wherein the second antigen binding site is identical to the first antigen binding site.

[0061] In some embodiments, the second antigen binding site is specific for a second epitope of the first GAL9 antigen.

[0062] In some embodiments, the second antigen binding site comprises all three VH CDRs and all three VL CDRs from another ABS clone selected from P9-02B, P9-04, P9-05, P9-08, P9-09, P9-10, P9-15, P9-16, P9-18, P9-19, P9-20, P9-21, P9-22, P9-27, P9-28, P9-31, P9-32, P9-36, P9-39, P9-49, P9-54, and P9-58.

[0063] In some embodiments, the second antigen binding site comprises a VL sequence and a VH sequence from another ABS clone.

[0064] In some embodiments, the second antigen binding site comprises a complete immunoglobulin heavy chain sequence containing a VH sequence and a complete immunoglobulin light chain sequence containing a VL sequence from another ABS clone.

[0065] In some embodiments, the second antigen binding site is specific for an antigen other than the first GAL9 antigen.

[0066] In some embodiments, the first antigen binding site comprises all three VH CDRs and all three VL CDRs from any one ABS clone selected from P9-10, P9-15, P9-18, P9-21, P9-22, and P9-28.

[0067] In some embodiments, the first antigen binding site comprises all three VH CDRs and all three VL CDRs from any one ABS clone selected from P9-10, P9-15, P9-18, P9-21, P9-22, and P9-28.

[0068] In some embodiments, the first antigen binding site comprises all three VH CDRs and all three VL CDRs from ABS clone P9-15.

[0069] In some embodiments, the first antigen binding site comprises all three VH CDRs and all three VL CDRs from ABS clone P9-18.

[0070] In some embodiments, the first antigen binding site comprises all three VH CDRs and all three VL CDRs from ABS clone P9-21.

[0071] In some embodiments, the first antigen binding site comprises all three VH CDRs and all three VL CDRs from ABS clone P9-22.

[0072] In some embodiments, the first antigen binding site comprises all three VH CDRs and all three VL CDRs from ABS clone P9-28.

[0073] In some embodiments, the GAL9 antigen binding molecule comprises an antibody format selected from the group consisting of a full length antibody, a Fab fragment, an Fv, a scFv, a tandem scFv, a Diabody, a scDiabody, a DART, a tandAb, a minibody, and a B-body.

[0074] In a twelfth aspect, the present disclosure provides a GAL9 antigen binding molecule that binds to the same epitope as the GAL9 antigen binding molecule of any of the preceding claims.

[0075] In a thirteenth aspect, the present disclosure provides a GAL9 antigen binding molecule that competes for binding with the GAL9 antigen binding molecule of any preceding claim.

[0076] In some embodiments, the GAL9 antigen binding molecule is purified.

[0077] In a fourteenth aspect, the present disclosure provides a pharmaceutical composition comprising the GAL9 antigen binding molecule of any one of the preceding claims and a pharmaceutically acceptable diluent.

[0078] In a fifteenth aspect, the present disclosure provides a method for treating a subject having cancer, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition as provided herein.

[0079] In some embodiments, the cancer is selected from the group consisting of pancreatic cancer, ovarian cancer, breast cancer, lung cancer, gastric cancer, melanoma, Ewing sarcoma, chronic lymphocytic leukemia, mantle cell lymphoma, B-ALL, hematological cancers, head and neck squamous cell carcinoma, prostate cancer, colon cancer, kidney cancer, and uterine cancer.

[0080] In some embodiments, the cancer is selected from the group consisting of breast cancer, colon cancer, lung cancer, and prostate cancer, cancers of the blood and lymphatic system (including Hodgkin's disease, leukemia, lymphoma, multiple myeloma, and Waldenstrom's disease), skin cancer (including malignant melanoma), cancers of the digestive tract (including head and neck cancer, esophageal cancer, stomach cancer, pancreatic cancer, liver cancer, colon and rectal cancer, anal cancer), cancers of the reproductive and urinary systems (including kidney cancer, bladder cancer, testicular cancer, prostate cancer), cancers of women (including breast cancer, ovarian cancer, gynecological cancer, and choriocarcinoma), and brain, bone carcinoid, nasopharyngeal, retroperitoneal, thyroid, and soft tissue tumors.

[0081] In some embodiments, the cancer is a virally induced tumor caused by an oncovirus. In some embodiments, the oncovirus is Epstein-Barr virus (EBV), hepatitis B virus, hepatitis C virus, human papillomavirus, human T-lymphotropic virus 1 (HTLV-1), Kaposi's sarcoma-associated herpesvirus (KHSV), Merkel cell polyomavirus, or cytomegalovirus. 5. Brief Description of the Figures

[0083] Figure 1 Results are shown for the administration of an immune-activating anti-GAL9 (α-GAL9) antibody in a colon cancer tumor model. BALB / c mice were subcutaneously implanted with CT26 tumor line cells and treated with control rat IgG or the anti-GAL9 antibodies P9-18 or P9-21. All treatments were 200 μg administered intraperitoneally (IP) on days 7, 11, 15, and 19. n=10 / group. Tumor growth was assessed by measuring tumor volume. Mice treated with P9-18 and P9-21 showed reduced growth of implanted CT26 tumors compared to those treated with control IgG.

[0084] Figure 2 Results are shown for administration of immune-activating anti-GAL9 antibodies in a melanoma tumor model. C57BL / 6 mice were subcutaneously implanted with B16.F0 tumor line cells and treated with control IgG or α-GAL9 antibodies P9-18 or P9-21. All treatments were administered 200 μg intraperitoneally (IP) on days 7, 11, 15, and 19. n=10 mice / group. Tumor growth was assessed by measuring tumor volume. Mice treated with P9-18 and P9-21 showed reduced growth of implanted B16.F0 tumors compared to mice treated with control IgG.

[0085] Figure 3A and 3BShown are INF-γ (3A) and TNF-α (3B) secretion by activated PBMCs stimulated in vitro with various GAL9 antibody candidates, known comparative tool antibodies (Tool mAb), anti-PD-1 antibodies, control antibodies (IgG Ctrl), and vehicle controls (PBS Ctrl). Black diamond shapes show secretion by activated PBMCs stimulated with comparative tool mAbs and anti-PD-1 antibodies (positive control).

[0086] Figure 4 The results show that activated CD8 + T cells, and the levels of the immunostimulatory markers CD27, CD40L, ICOS, 4-1BB, and OX40 on their surface.

[0087] Figure 5 Shown are representative flow cytometric plots quantifying IL-12 production by DCs stimulated in vitro with control IgG or α-GAL9 candidate P9-18, along with staining controls.

[0088] Figure 6A and 6B The results show that the control antibody P9-55 (clone 55), the anti-GAL9 candidate antibody P9-15 (clone 15) or the α-GAL9 candidate antibody P9-18 (clone 18) were used at 5 μg ( Figure 6A ) or 20 μg ( Figure 6B ) after 72 hours of stimulation, CD56 + Representative flow cytometric plots of TNF-α secretion by NK cells.

[0089] Figures 7A-7E Shown are exemplified by the Martin numbering scheme and various CDR definitions - Chothia, AbM, Kabat, Contact, IMGT - as applied to the P9-28 anti-GAL9 candidate antibody provided herein. Figures 7A-7E SEQ ID NOs: 187 and 188 are disclosed in order of appearance, respectively.

[0090] Figures 8A-8C Representative confocal microscopy images are shown, demonstrating the effect of IgG control ( Figure 8A )、P9-18( Figure 8B ) and P9-21( Figure 8C Colocalization and clustering of GAL9 and PD-L2 on DCs after treatment with PD-L2. Blue staining indicates DNA (DAPI), red staining indicates PD-L2, green staining indicates CD11c, and yellow staining indicates GAL9. Unlabeled micrographs are bright field; images are presented in grayscale in the accompanying figures.

[0091] Figure 9A and 9B Representative confocal images are shown, demonstrating the effect of IgG control ( Figure 9A ) compared with anti-GAL9P9-18 ( Figure 9B Retention of PD-L2 and PD-L1 on the surface of CT26 tumor cells after treatment with PD-L1. The spots in the image highlight increased expression of PD-L2 and PD-L1 ligands. Blue staining shows DNA (DAPI), red staining shows PD-L2, and green staining shows PD-L1; all are presented in grayscale in the accompanying figures.

[0092] Figure 10A -E shows representative data from a humanized mouse model of EBV infection treated with anti-GAL9 P9-15. Figure 10A A schematic diagram of the protocol with treatment timeline is shown. Figure 10B Images of spleens from mice treated with IgG control and P9-15 are shown. Arrows point to uncontrolled tumor growth in IgG control mice. Figure 10C A bar graph of spleen weights is shown. Figure 10D Histograms of the number of cells per spleen are shown. Figure 10E A histogram of spleen viral load is shown.

[0093] Figure 11 Shown are representative data from a humanized mouse model of EBV infection treated with anti-GAL9 P9-28. Figure 10A A schematic diagram of the protocol with treatment timeline is shown. Figure 11 Images of spleens from IgG control and anti-GAL9 P9-28 treated mice are shown. Arrows point to uncontrolled tumor growth in IgG control treated mice.

[0094] Figure 12 A shows in vivo evaluation of tumor growth in the CT26 tumor model using P9-18-IgG1 (diamonds - ◇-), sFc-P9-18-IgG2a (inverted triangles - ▽-), P9-18 IgG2a (circles - ○-), and IgG (IgG2a) control #1 (black squares - ■-).

[0095] Figure 12 B shows in vivo assessment of immune memory in previously treated CT 26 tumors using sFc-P9-18 IgG2a (inverted triangles -▽-), P9-18 IgG2a (circles -○-), and IgG (IgG2a) control #2 (black diamonds -◆-).

[0096] Figure 13 showed that after treatment with anti-GAL9 P9-18 or control, PD-L1+ or PD-L2 + Tumor-associated dendritic cells (CD11c + ) and the average percentage of tumor-associated dendritic cells (CD11c + ) are bar graphs of the mean cell surface expression (GMI) of PD-L1 or PD-L2 on the PD-L1 or PD-L2 cells.

[0097] Figure 14 It was shown that after treatment with anti-GAL9 P9-18 or IgG control, PD-L1 + or PD-L2 + Bar graphs of the mean percentage of tumor cells and the mean cell surface expression level (GMI) of PD-L1 or PD-L2 on tumor cells. 6. Detailed Description of the Invention

[0099] 6.1. Definitions

[0100] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, the following terms have the following meanings.

[0101] "Antigen binding site" or "ABS" refers to the region of a GAL9 binding molecule that specifically recognizes or binds a given antigen or epitope.

[0102] As used herein, the term "treat" or "treatment" is used in its most widely accepted clinical sense. The term includes, but is not limited to, alleviating the signs or symptoms of a disease; improving the signs or symptoms of a disease; relieving symptoms; lessening the extent of a disease; stabilizing (i.e., not worsening) the disease state; delaying or slowing the progression of a disease; improving or palliating the disease state; remission (whether partial or complete), whether detectable or undetectable; curing; prolonging survival compared to expected survival if not receiving treatment. Unless expressly stated otherwise, "treat" or "treatment" does not mean prophylaxis or prevention of a disease.

[0103] "Subject" or "individual" or "animal" or "patient" or "mammal" refers to any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or treatment is desired. Mammalian subjects include humans, livestock, farm animals, and zoo, sport, or pet animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, and the like. Unless otherwise indicated, "patient" means a human "subject."

[0104] The term "sufficient amount" refers to an amount sufficient to produce a desired effect, such as an amount sufficient to modulate protein aggregation in a cell.

[0105] The term "therapeutically effective amount" is an amount effective to ameliorate symptoms of a disease.

[0106] The term "prophylactically effective amount" is an amount effective to prevent symptoms of disease.

[0107] 6.2. Other Explanations

[0108] Unless otherwise indicated, all references to sequences herein are to amino acid sequences.

[0109] Unless otherwise indicated, antibody constant region residue numbering is according to the Eu index as described at www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html#refs (accessed August 22, 2017), which is incorporated herein by reference in its entirety, and residue numbering identifies residues according to their position in the endogenous constant region sequence, regardless of the physical position of the residue within the chain of the GAL9-binding molecules described herein.

[0110] Unless otherwise indicated as a "Kabat CDR," "Chothia CDR," "Contact CDR," or "IMGT CDR," all references to "CDRs" refer to CDRs defined using the Martin (AbM) definition.

[0111] "Endogenous sequence" or "native sequence" refers to any sequence, including nucleic acid and amino acid sequences, that originates from an organism, tissue or cell and has not been modified or mutated by man.

[0112] Polypeptide chain numbers (e.g., "first" polypeptide chain, "second" polypeptide chain, etc., or polypeptide "chain 1," "chain 2," etc.) are used herein as unique identifiers for specific polypeptide chains that form a binding molecule, but are not intended to refer to the order or number of these different polypeptide chains within the binding molecule.

[0113] In this disclosure, the words "comprises," "comprising," "containing," "having," "includes," "including," and variations thereof, have the meanings ascribed to them in U.S. patent law, allowing for the presence of additional components in addition to those explicitly listed.

[0114] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Unless specifically stated otherwise, the terms "including," "such as," and the like are intended to mean inclusive rather than limiting.

[0115] 38,39,40,41,42,43,44,45,46,47,48,49, and 50.

[0116] Unless otherwise specified or apparent from the context, as used herein, the term "about" should be understood as within the normal tolerance in the art, for example, within 2 standard deviations of the mean. "About" can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05% or 0.01% of the stated value.

[0117] 6.3. General Overview

[0118] The present invention provides galectin-9 (GAL9) antigen-binding molecules, such as anti-GAL9 antibodies and antigen-binding fragments thereof; compositions comprising the GAL9 binding molecules; and pharmaceutical compositions comprising the GAL9 binding molecules. The present disclosure particularly provides various GAL9 antigen-binding molecules that are stimulatory, acting as activators of the immune system, increasing the secretion and production of various cytokines in various immune cells, and increasing the surface expression of stimulatory molecules.

[0119] The present disclosure also provides methods for treating a disease or condition in a subject by administering an immunostimulatory Galectin-9 antibody binding molecule. The methods provided herein are particularly suitable for treating proliferative diseases or cancer. In some embodiments, the cancer is a virally induced cancer, such as a cancer caused by infection with an oncovirus or tumor virus. In some embodiments, the compositions and methods provided herein can be used to treat immunosuppressive diseases or conditions, such as malaria, HIV, or AIDs.

[0120] 6.4. GAL9 Antigen Binding Molecules

[0121] In a first aspect, an antigen binding molecule is provided. In each embodiment, the antigen binding molecule comprises at least a first antigen binding site specific for the GAL9 antigen; therefore, the binding molecule is referred to as a GAL9 antigen binding molecule or a GAL9 binding molecule.

[0122] The GAL9 antigen binding molecules described herein specifically bind to the GAL9 antigen.

[0123] As used herein, "GAL9 antigen" refers to members and homologs of the galectin-9 family. GAL9 is also known as LGALS9, HUAT, LGALS9A, tumor antigen HOM-HD-21, and ecalectin. In certain embodiments, a GAL9 binding molecule has an antigen binding site that specifically binds to at least a portion of more than one GAL9 domain (e.g., the junction between a first and a second GAL9 domain).

[0124] In a specific embodiment, the GAL9 antigen is human. GenBank Accession No. NP_033665.1 describes a representative human GAL9 protein, including its sequence and domain characteristics, and is incorporated herein by reference in its entirety. SEQ ID NO: 6 provides the full-length GAL9 protein sequence.

[0125] MAFSGSQAPYLSPAVPFSGTIQGGLQDGLQITVNGTVLSSSGTRFAVNFQTGFSGNDIAFHFNPRFEDGGYVVCNTRQNGSWGPEERKTHMPFQKGMPFDLCFLVQSSDFKVMVNGILFVQYFHRVPFHRVDTISVNGSVQLSYISFQNPRTVPVQPAFSTVPFSQPVCFPPRPRGRRQ KPPGVWPANPAPITQTVIHTVQSAPGQMFSTPAIPPMMYPHPAYPMPFITTILGGLYPSKSILLSGTVLPSAQRFHINLCSGNHIAFHLNPRFDENAVVRNTQIDNSWGSEERSLPRKMPFVRGQSFSVWILCEAHCLKVAVDGQHLFEYYHRLRNLPTINRLEVGGDIQLTHVQT[SEQ ID NO:6]

[0126] In various embodiments, the GAL9 binding molecule also specifically binds to at least one antigen other than the GAL9 antigen.

[0127] 6.4.1. Functional Characteristics of GAL9 Antigen Binding Molecules

[0128] In some embodiments, upon contact therewith, the GAL9 antigen binding molecule increases cytokine secretion of activated immune cells (e.g., activated human immune cells). In some embodiments, the immune cells are peripheral blood mononuclear cells (PBMCs). In some embodiments, the immune cells are T cells. In some embodiments, the T cells are effector T cells. In some embodiments, the T cells are CD8 + In some embodiments, the T cells are CD4 +In some embodiments, the immune cell is a natural killer (NK) cell. In some embodiments, the immune cell is a dendritic cell (DC).

[0129] The influence of GAL9 antigen binding molecules on the cytokine secretion of immune cells can be measured by any suitable method. For example, the influence of GAL9 antigen binding molecules on the cytokine secretion of immune cells can be measured in vivo, in vitro or in vitro. In some embodiments, compared with the activated immune cells of contact control agents (such as control antigen binding molecules or solvent controls), the cytokine secretion in the activated immune cells of contact GAL9 antigen binding molecules is measured. Immune cells can be activated by peptide stimulation. For example, immune cells can be activated by a peptide or multiple peptides known to induce an immune response. The control agent can be a negative control or a positive control. In some embodiments, relative to the negative control agent or negative control antigen binding molecule, the GAL9 antigen binding molecules increase the cytokine secretion in the immune cells. In some embodiments, the negative control antigen binding molecule is an isotype control binding molecule that is not in conjunction with GAL9. In some embodiments, the positive control antibody is an anti-PD1 antibody, such as nivolumab. In some embodiments, the positive control antibody is a GAL9 control antibody. The GAL9 control antibody can be a Gal9 antibody clone RG9.1 (Catalog No. BE0218, InVivoMab Antibodies) or RG9.35. Both RG9.1 and RG9.35 are described in Fukushima A, Sumi T, Fukuda K, Kumagai N, Nishida T, et al. (2008), "Roles of GALectin-9 in the development of experimental allergic conjunctivitis in mice," Int Arch Allergy Immunol 146:36-43, which is incorporated herein by reference in its entirety. The GAL9 control antibody can be a Gal9 antibody clone ECA42 (Catalog No. LS-C179449, LifeSpan BioScience). In some embodiments, the GAL9 antigen binding molecule increases cytokine secretion in immune cells relative to the positive control antibody.

[0130] Cytokine secretion by immune cells can be assessed by any suitable method. By way of example only, cytokine secretion by in vitro or ex vivo immune cell culture models can be assessed by analyzing the cytokine content of cultured cell supernatants, such as by cytokine bead arrays.

[0131] In some embodiments, the cytokine is IFN-γ. In some embodiments, the GAL9 antigen binding molecule increases IFN-γ secretion in activated immune cells by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, or 120%. In some embodiments, the GAL9 antigen binding molecule increases IFN-γ secretion in activated immune cells by at least 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, 35%-40%, 40%-45%, 45%-50%, 50%-55%, 55%-60%, 60%-65%, 70%-75%, 75%-80%, 80%-85%, 85%-90%, 90%-95%, 95%-100%, 100%-105%, 105%-110%, 110%-115%, or 115%-120%.

[0132] In some embodiments, the cytokine is TNF-α. In some embodiments, the GAL9 antigen binding molecule increases TNF-α secretion in activated immune cells by at least 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950%, 10,00%, 10,500%, 11,000%, 11,500%, 12,000%, 12,500%, 13,000%, 13,500%, 14,000%, 15,000%, 16,000%, 17,000%, 18,000%, 19,000%, 20,000%, 21,000%, 22,000%, 23,000%, 24,000%, 25,000%, 26,000%, 27,000%, 28,000%, 29,000%, 30,000%, 31,000%, 32,000%, 33,000%, 34,000%, 35,000%, 36,000%, 37,000%, 38,000%, 39,000%, 40,000%, 41,000%, 42,000%, 43,000%, 44,000%, 45,000%, 46,000%, 47,000%, 48,000%, 49,000%, 50,000%, 51,000%, 52,000%, 53, %, 14,500%, 15,000%, 15,500%, 16,000%, 16,500%, 17,000%, 17,500%, 18,000%, 18,500%, 19,000%, 19,500%, 20,000%, 20,500%, 30,000%, 30,500%, 40,000%, 40,500%, 50,000%, 50,500%, 60,000%, 60,500%, 70,000%, 70,500%, 80,000%, 80,500%, 90,000% or 90,500%.In some embodiments, the GAL9 antigen binding molecule increases TNF-α secretion in activated immune cells by at least 100%-150%, 150%-200%, 200%-250%, 250%-300%, 300%-350%, 350%-400%, 400%-450%, 500%-550%, 550%-600%, 600%-650%, 650%-700%, 700%-750%, 750%-800%, 800%-800%, 800%-900%, 800%-950%, 800%-900%, 800%-10 ... 800%-850%, 850%-900%, 900%-950%, 950%-10,000%, 10,000%-10,500%, 10,500%-11,000%, 11,000-11,500%, 11,500-12,000%, 12,000%-12,500%, 13,000%-13,500%, 13,500%-14,000%, 14,000%-14,500%, 14,500%-15,000% 、15,000-15,500%、15,550%-16,000%、16,000%-16,500%、17,000%-17,500%、17,500%-18,000%、17,500%-18,500%、18,500%-19,000%、19,000%-19,500%、19,500%-20,000%、20,000%-20,500%、20,500%-30,000%、30,000%- 30,500%, 30,500%-40,000%, 40,000%-40,500%, 45,500%-50,000%, 50,000%-50,500%, 55,500%-60,000%, 60,000%-60,500%, 70,000%-70,500%, 70,500%-80,000%, 80,000%-80,500%, 85,000%-90,000% or 90,000%-90,500%.

[0133] In various embodiments, the activated immune cells are T cells, CD8 + T cells, NK cells, CD4 + T cells or dendritic cells (DCs).

[0134] In some embodiments, the GAL9 antigen binding molecules increase the surface expression of one or more costimulatory molecules on immune cells (e.g., human immune cells). In certain embodiments, the GAL9 antigen binding molecules increase the surface expression of one or more costimulatory molecules in activated immune cells. In specific embodiments, the immune cells are T cells. In specific embodiments, the activated immune cells are CD8+ In some embodiments, the activated immune cells are NK cells. In some embodiments, the activated immune cells are dendritic cells.

[0135] In some embodiments, one or more costimulatory molecules are selected from 4-1BB, CD 27, CD40L, ICOS and OX40. In some embodiments, one or more costimulatory molecules are selected from 4-1BB, CD27, CD40L and OX40. In some embodiments, one or more costimulatory molecules are selected from 4-1BB, CD40L and OX40.

[0136] The effect of a GAL9 antigen binding molecule on the surface expression of one or more costimulatory molecules can be determined by any suitable method. For example, the effect of a GAL9 antigen binding molecule on the surface expression of one or more costimulatory molecules can be determined in vivo, ex vivo, or in vitro.

[0137] In some embodiments, the GAL9 antigen binding molecule increases the surface expression of one or more co-stimulatory molecules in activated immune cells compared to activated immune cells treated with a control agent. Exemplary control agents are described herein. In certain embodiments, the control agent is an isotype control binding molecule that does not bind to GAL9.

[0138] In some embodiments, the GAL9 antigen binding molecules increase the surface expression of CD40L on activated CD8+ T cells relative to activated CD8+ T cells treated with a placebo. In some embodiments, the activated CD8+ T cells treated with the GAL9 antigen binding molecules exhibit an increase in CD40L surface expression of at least about 0.1-fold, 0.2-fold, 0.3-fold, 0.4-fold, 0.5-fold, 0.6-fold, 0.7-fold, 0.8-fold, 0.9-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or greater than 10-fold relative to activated CD8+ T cells treated with a placebo. In some embodiments, the activated CD8+ T cells treated with the GAL9 antigen binding molecules exhibit an increase in CD40L surface expression of about 0.1-fold to 10-fold, about 0.5-fold to 5-fold, about 1-fold to 4-fold, or about 1.5-fold to 2.5-fold relative to activated CD8+ T cells treated with a placebo.

[0139] In some embodiments, relative to activated CD8+T cells treated with a placebo, the GAL9 antigen binding molecules increase the OX40 surface expression of activated CD8+T cells. In some embodiments, relative to activated CD8+T cells treated with a placebo, activated CD8+T cells treated with GAL9 antigen binding molecules exhibit an increase in OX40 surface expression of at least about 0.1 times, 0.2 times, 0.3 times, 0.4 times, 0.5 times, 0.6 times, 0.7 times, 0.8 times, 0.9 times, 1 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times or more than 10 times. In some embodiments, relative to activated CD8+T cells treated with a placebo, activated CD8+T cells treated with GAL9 antigen binding molecules exhibit an increase in OX40 surface expression of about 0.1 times-10 times, an increase of about 0.5 times-5 times or an increase of about 1.0 times-2.0 times.

[0140] In some embodiments, relative to the activated CD8+T cells treated with a control agent, the GAL9 antigen binding molecules increase the 4-1BB surface expression of the activated CD8+T cells. In some embodiments, relative to the activated CD8+T cells treated with a control agent, the activated CD8+T cells treated with the GAL9 antigen binding molecules show that 4-1BB surface expression increases by at least about 0.1 times, 0.2 times, 0.3 times, 0.4 times, 0.5 times, 0.6 times, 0.7 times, 0.8 times, 0.9 times, 1 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times or more than 10 times. In some embodiments, relative to the activated CD8+T cells treated with a control agent, the activated CD8+T cells treated with the GAL9 antigen binding molecules show that 4-1BB surface expression increases by about 0.1 times -10 times, about 0.2 times -2 times or about 0.5 times -1 times.

[0141] In some embodiments, the GAL9 antigen binding molecule increases CD27 surface expression on activated CD8+ T cells relative to activated CD8+ T cells treated with a control agent. In some embodiments, activated CD8+ T cells treated with the GAL9 antigen binding molecule exhibit at least about an increase in CD27 surface expression of 1%, an increase of 2%, an increase of 3%, an increase of 4%, an increase of 5%, an increase of 6%, an increase of 7%, an increase of 8%, an increase of 9%, an increase of 10%, an increase of 11%, an increase of 12%, an increase of 13%, an increase of 14%, an increase of 15%, an increase of 16%, an increase of 17%, an increase of 18%, an increase of 19%, an increase of 20%, an increase of 21%, an increase of 22%, an increase of 23%, an increase of 24%, an increase of 25%, an increase of 26%, an increase of 27%, an increase of 28%, an increase of 29%, an increase of 30%, an increase of 31%, an increase of 32%, an increase of 33%, an increase of 34%, an increase of 35%, an increase of 36%, an increase of 37%, an increase of 38%, an increase of 39%, an increase of 40%, an increase of 41%, an increase of 42%, an increase of 43%, an increase of 44%, an increase of 45%, an increase of 46%, an increase of 47%, an increase of 48%, an increase of 49%, an increase of 50%, an increase of 51%, an increase of 52%, an increase of 53%, an increase of 54%, an increase of 55%, an increase of 56%, an increase of 57%, an increase of 58%, an increase of 59%, an increase of 60%, an increase of 61%, an increase of 62%, an increase of 63%, an increase of 64%, an increase of Increase by 19%, increase by 20%, increase by 21%, increase by 22%, increase by 23%, increase by 24%, increase by 25%, increase by 26%, increase by 27%, increase by 28%, increase by 29%, increase by 30%, increase by 31%, increase by 32%, increase by 33%, increase by 34%, increase by 35%, increase by 36%, increase by 37%, increase by 38%, increase by 39%, increase by 40%, increase by 41%, increase by 42%, increase by 43%, increase by 44%, increase by 45%, increase by 46%, increase by 47%, increase by 48%, increase by 49%, increase by 50%, increase by 51%, increase by 52%, increase by 53%, increase by 54%, increase by 55%, increase by 56%, increase by 57%, increase by 58%, increase by 59%, increase by 60%, increase by 61%, increase by 62%, increase by 63%, increase by 64%, increase by 65%, increase by 66%, increase by 67%, increase by 68%, increase by 69%, increase by 70%, increase by 71%, increase by 72%, increase by 73 In some embodiments, the activated CD8+ T cells treated with the GAL9 antigen binding molecule exhibit an increase in CD27 surface expression of at least about 1%-100%, an increase of 5%-50%, an increase of 10%-40%, or an increase of about 20%-30% relative to activated CD8+ T cells treated with a control agent.

[0142] In some embodiments, the GAL9 antigen binding molecule increases ICOS surface expression on activated CD8+ T cells relative to activated CD8+ T cells treated with a control agent. In some embodiments, activated CD8+ T cells treated with the GAL9 antigen binding molecule exhibit at least about an increase in ICOS surface expression of 1%, an increase of 2%, an increase of 3%, an increase of 4%, an increase of 5%, an increase of 6%, an increase of 7%, an increase of 8%, an increase of 9%, an increase of 10%, an increase of 11%, an increase of 12%, an increase of 13%, an increase of 14%, an increase of 15%, an increase of 16%, an increase of 17%, an increase of 18%, an increase of 19%, an increase of 20%, an increase of 21%, an increase of 22%, an increase of 23%, an increase of 24%, an increase of 25%, an increase of 26%, an increase of 27%, an increase of 28%, an increase of 29%, an increase of 30%, an increase of 31%, an increase of 32%, an increase of 33%, an increase of 34%, an increase of 35%, an increase of 36%, an increase of 37%, an increase of 38%, an increase of 39%, an increase of 40%, an increase of 41%, an increase of 42%, an increase of 43%, an increase of 44%, an increase of 45%, an increase of 46%, an increase of 47%, an increase of 48%, an increase of 49%, an increase of 50%, an increase of 51%, an increase of 52%, an increase of 53%, an increase of 54%, an increase of 55%, an increase of 56%, an increase of 57%, an increase of 58%, an increase of 59%, an increase of 60%, an increase of 61%, an increase of 62%, an increase of 63%, an increase of 64%, an increase of 6 %, increase by 19%, increase by 20%, increase by 21%, increase by 22%, increase by 23%, increase by 24%, increase by 25%, increase by 26%, increase by 27%, increase by 28%, increase by 29%, increase by 30%, increase by 31%, increase by 32%, increase by 33%, increase by 34%, increase by 35%, increase by 36%, increase by 37%, increase by 38%, increase by 39%, increase by 40%, increase by 41%, increase by 42%, increase by 43%, increase by 44%, increase by 45%, increase by Increase by 46%, increase by 47%, increase by 48%, increase by 49%, increase by 50%, increase by 51%, increase by 52%, increase by 53%, increase by 54%, increase by 55%, increase by 56%, increase by 57%, increase by 58%, increase by 59%, increase by 60%, increase by 61%, increase by 62%, increase by 63%, increase by 64%, increase by 65%, increase by 66%, increase by 67%, increase by 68%, increase by 69%, increase by 70%, increase by 71%, increase by 72%, increase by 73 In some embodiments, the activated CD8+ T cells treated with the GAL9 antigen binding molecule exhibit an increase in ICOS surface expression of at least about 1%-100%, an increase of 5%-50%, an increase of 10%-40%, or an increase of about 20%-30% relative to activated CD8+ T cells treated with a control agent.

[0143] In some embodiments, the GAL9 antigen binding molecule increases the retention of PD-L1, PD-L2, or both PD-L1 and PD-L2 on the surface of tumor cells. In some embodiments, the increased retention of PD-L1, PD-L2, or both PD-L1 and PD-L2 on the surface of tumor cells is demonstrated by microscopy techniques (e.g., confocal microscopy).

[0144] In some embodiments, the GAL9 antigen binding molecules increase the expression of PD-L2 on the surface of dendritic cells (DC). In some embodiments, the GAL9 antigen binding molecules reduce the expression of PD-L1 on the surface of dendritic cells (DC). In some embodiments, DC is an activated DC. The activation of immune cells (including DC) is described herein. The surface expression of proteins (including PD-L1 and PD-L2) on DC can be assessed by any suitable method. For example, the percentage of DCs showing detectable surface PD-L1 and / or PD-L2 can be measured by, for example, flow cytometry. In some embodiments, compared with a control dendritic cell population treated with a control agent, a dendritic cell population treated with a GAL9 antigen binding molecule shows a higher percentage of cells being surface PD-L2 positive. Exemplary control agents are described herein. In some embodiments, the control agent is an isotype antigen binding molecule that does not bind GAL9. In some embodiments, relative to a control dendritic cell population treated with a control agent (e.g., an isotype control antigen binding molecule), a dendritic cell population treated with a GAL9 antigen binding molecule exhibits an increase in the percentage of DCs that present detectable surface PD-L2 expression by about 0.1-100 fold, 0.5-20 fold, 1-10 fold, or about 5-6 fold. In some embodiments, relative to a control dendritic cell population treated with a control agent (e.g., an isotype control antigen binding molecule), a dendritic cell population treated with a GAL9 antigen binding molecule exhibits a decrease in the percentage of DCs that present detectable surface PD-L1 expression by about 1%-50%, about 5%-30%, or about 10%-20%.

[0145] In some embodiments, the GAL9 antigen binding molecules increase the cell surface aggregation of PD-L2 in dendritic cells (DCs). In some embodiments, DCs are activated DCs. Activation of immune cells (including DCs) is described herein. In some embodiments, the increase in cell surface aggregation of PD-L2 is relative to DCs treated with a control agent. Control agents are described herein. In some embodiments, the control agent is an isotype antigen binding molecule that does not bind to GAL9. Cell surface aggregation of PD-L2 in DCs can be assessed by any suitable means, such as confocal microscopy.

[0146] In some embodiments, the GAL9 antigen binding molecules increase IL-12 production in DCs. The DCs may be activated DCs. In some embodiments, the GAL9 antigen binding molecules increase IL-12 production in DCs relative to DCs treated with a control agent. Exemplary control agents are described herein. In some embodiments, the control agent is an isotype antigen binding molecule that does not bind to GAL9. In some embodiments, the DC population treated with the GAL9 antigen binding molecules exhibits an increase in the percentage of IL-12-positive DCs by approximately 0.1-fold to 100-fold, 10-fold to 75-fold, 20-fold to 40-fold, 25-fold to 35-fold, or approximately 28-fold compared to the DC population treated with the control agent.

[0147] In some embodiments, the GAL9 antigen binding molecule induces clustering of GAL9 and PD-L2 on the surface of immune cells. In some embodiments, the immune cells may be DCs. In some embodiments, the immune cells may be NK cells.

[0148] In some embodiments, the GAL9 antigen binding molecule reduces tumor burden in a subject. The subject can be a mammal. The mammal can be a mouse. In some embodiments, the mammal is a human. In some embodiments, the GAL9 antigen binding molecule prevents the growth of a tumor in a subject. The tumor can be, for example, a colon tumor. In some embodiments, the GAL9 antigen binding molecule reduces tumor growth. In some embodiments, the GAL9 antigen binding molecule reduces tumor growth by about 25%, 50%, or more than 50%. In some embodiments, the tumor is a melanoma tumor. In some embodiments, the reduction in tumor growth is relative to a subject treated with a control agent. Exemplary control agents are described herein. In some embodiments, the control agent is an isotype of the antigen binding molecule that does not bind to GAL9.

[0149] 6.4.2. Variable Region

[0150] The GAL9 binding molecules described herein have the variable region domain amino acid sequences of antibodies, including VH and VL antibody domain sequences. The VH and VL sequences are described in more detail below in Sections 6.4.2.1 and 6.4.2.2, respectively.

[0151] 6.4.2.1.VH region

[0152] The VH amino acid sequence in the GAL9 binding molecules described herein is an antibody heavy chain variable domain sequence. In a typical natural antibody arrangement and in the GAL9 binding molecules described herein, a specific VH amino acid sequence associates with a specific VL amino acid sequence to form an antigen binding site. In various embodiments, the VH amino acid sequence is a mammalian sequence, including a human sequence, a synthetic sequence, or a combination of non-human mammalian, mammalian, and / or synthetic sequences, as described in further detail in Sections 6.4.2.3 and 6.4.2.4 above. In various embodiments, the VH amino acid sequence is a mutant sequence of a naturally occurring sequence.

[0153] 6.4.2.2.VL Zone

[0154] The VL amino acid sequences that can be used in the GAL9 binding molecules described herein are antibody light chain variable domain sequences. In the typical arrangement of natural antibodies and the antibody constructs described herein, a particular VL amino acid sequence associates with a particular VH amino acid sequence to form an antigen binding site. In various embodiments, the VL amino acid sequence is a mammalian sequence, including a human sequence, a synthetic sequence, or a combination of human, non-human mammalian, mammalian, and / or synthetic sequences, as described in further detail in Sections 6.4.2.3 and 6.4.2.4 below.

[0155] In various embodiments, the VL amino acid sequence is a mutant sequence of a naturally occurring sequence. In certain embodiments, the VL amino acid sequence is a lambda (λ) light chain variable domain sequence. In certain embodiments, the VL amino acid sequence is a kappa (κ) light chain variable domain sequence. In preferred embodiments, the VL amino acid sequence is a kappa (κ) light chain variable domain sequence.

[0156] 6.4.2.3. Complementarity-Determining Regions

[0157] The VH and VL amino acid sequences contain highly variable sequences called "complementarity determining regions" (CDRs), typically three CDRs (CDR1, CDR2, and CDR3). In various embodiments, the CDRs are mammalian sequences, including but not limited to mouse, rat, hamster, rabbit, camel, donkey, goat, and human sequences. In preferred embodiments, the CDRs are human sequences. In various embodiments, the CDRs are naturally occurring sequences. In various embodiments, the CDRs are naturally occurring sequences that have been mutated to change the binding affinity of the antigen binding site to a specific antigen or epitope. In certain embodiments, the naturally occurring CDRs have been mutated in a host in vivo by affinity maturation and somatic hypermutation. In certain embodiments, the CDRs have been mutated in vitro by methods including but not limited to PCR mutagenesis and chemical mutagenesis. In various embodiments, the CDRs are synthetic sequences, including but not limited to CDRs obtained from random sequence CDR libraries and rationally designed CDR libraries. The Martin numbering scheme is used to determine the CDR boundaries. See Figures 7A-7E .

[0158] In various embodiments, the CDR identified as binding to the target antigen is further mutated (i.e., "affinity maturation") to achieve desired binding characteristics, such as an increase in affinity for the target antigen relative to the original CDR. For example, degenerate oligonucleotides can be used to introduce diversity targeting into CDRs (including those identified as binding to the target antigen). Various randomization schemes can be adopted. For example, "soft randomization" can be used, which provides a high bias towards the wild-type sequence at a given amino acid position, such as allowing a given position in a CDR to change in all twenty kinds of amino acids, while biasing towards the wild-type sequence by mixing four bases at each codon position with unequal levels. As an illustrative example of soft randomization, if it is desired to achieve approximately 50% wild-type sequence, each base of each codon maintains 70% wild-type and the other nucleotides are each 10%, and degenerate oligonucleotides are used to prepare a focused phage library around the selected CDR, wherein the resulting phage particles are used for phage panning, which can be performed under various stringent selection conditions as needed.

[0159] 6.4.2.4. Framework Region and CDR Grafting

[0160] The VH and VL amino acid sequences comprise "framework region" (FR) sequences. FRs are typically conserved sequence regions that serve as a scaffold for interspersed CDRs (see Section 6.4.2.3), typically arranged in FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (from N-terminus to C-terminus). In various embodiments, FRs are mammalian sequences, including but not limited to mouse, rat, hamster, rabbit, camel, donkey, goat, and human sequences. In a preferred embodiment, FRs are human sequences. In various embodiments, FRs are naturally occurring sequences. In various embodiments, FRs are synthetic sequences, including but not limited to rationally designed sequences.

[0161] In a plurality of embodiments, FR and CDR are all from the same naturally occurring variable domain sequence. In a plurality of embodiments, FR and CDR are from different variable domain sequences, wherein CDR is transplanted onto the FR support, wherein CDR provides specificity to a particular antigen. In certain embodiments, the CDRs of the transplant are all derived from the same naturally occurring variable domain sequence. In certain embodiments, the CDRs of the transplant are derived from different variable domain sequences. In certain embodiments, the CDRs of the transplant are synthetic sequences, including but not limited to the CDRs obtained from a random sequence CDR library and a rationally designed CDR library. In certain embodiments, the CDRs of the transplant and FR are from the same species. In certain embodiments, the CDRs of the transplant and FR are from different species. In a preferred transplant CDR embodiment, the antibody is "humanized", wherein the CDRs of the transplant are non-human mammalian sequences, including but not limited to mouse, rat, hamster, rabbit, camel, donkey and goat sequences, and FR are human sequences. Humanized antibodies are discussed in more detail in U.S. Patent No. 6,407,213, the full content of which is incorporated herein by reference. In various embodiments, portions or specific sequences of FRs from one species are used to replace portions or specific sequences of FRs from another species.

[0162] 6.4.3. Exemplary Amino Acid Sequences of GAL9 Binding Molecules

[0163] In various embodiments, the GAL9 binding molecule comprises a specific VH CDR3 (CDR-H3) sequence and a specific VLCDR3 (CDR-L3) sequence.

[0164] In some embodiments, the GAL9 binding molecule comprises a CDR-H3 and a CDR-L3 from any one of the ABS clones selected from P9-02B, P9-04, P9-05, P9-08, P9-09, P9-10, P9-15, P9-16, P9-18, P9-19, P9-20, P9-21, P9-22, P9-27, P9-28, P9-31, P9-32, P9-36, P9-39, P9-49, P9-54, and P9-58. The VH CDR amino acid sequences of these ABS clones are disclosed in Table 3. The VL CDR amino acid sequences of these ABS clones are disclosed in Table 4. For clarity, each GAL9 ABS clone is assigned a unique ABS clone number, which is used throughout this disclosure.

[0165] In a presently preferred embodiment, the GAL9 binding molecule comprises CDR-H3 and CDR-L3 of ABS clone P9-28.

[0166] In some embodiments, the GAL9 binding molecule comprises all three VH CDRs from one of the ABS clones selected from P9-02B, P9-04, P9-05, P9-08, P9-09, P9-10, P9-15, P9-16, P9-18, P9-19, P9-20, P9-21, P9-22, P9-27, P9-28, P9-31, P9-32, P9-36, P9-39, P9-49, P9-54, and P9-58. In a preferred embodiment, the GAL9 binding molecule comprises all three VH CDRs from the ABS clone P9-28.

[0167] In some embodiments, the GAL9 binding molecule comprises all three VL CDRs from one of the ABS clones selected from P9-02B, P9-04, P9-05, P9-08, P9-09, P9-10, P9-15, P9-16, P9-18, P9-19, P9-20, P9-21, P9-22, P9-27, P9-28, P9-31, P9-32, P9-36, P9-39, P9-49, P9-54, and P9-58. In a currently preferred embodiment, the GAL9 binding molecule comprises all three VL CDRs from the ABS clone P9-28.

[0168] In some embodiments, the GAL9 binding molecule comprises all six CDRs from any one of the ABS clones selected from P9-02B, P9-04, P9-05, P9-08, P9-09, P9-10, P9-15, P9-16, P9-18, P9-19, P9-20, P9-21, P9-22, P9-27, P9-28, P9-31, P9-32, P9-36, P9-39, P9-49, P9-54, and P9-58. In a currently preferred embodiment, the GAL9 binding molecule comprises all six CDRs from the ABS clone P9-28.

[0169] In some embodiments, the GAL9 binding molecule comprises a VH amino acid sequence, a VL amino acid sequence, or both a VH and a VL amino acid sequence from any one of the ABS clones selected from P9-02B, P9-04, P9-05, P9-08, P9-09, P9-10, P9-15, P9-16, P9-18, P9-19, P9-20, P9-21, P9-22, P9-27, P9-28, P9-31, P9-32, P9-36, P9-39, P9-49, P9-54, and P9-58. Complete immunoglobulin heavy chain and immunoglobulin light chain sequences, as well as VH and VL amino acid sequences, are provided in Table 6. In certain presently preferred embodiments, the GAL9 binding molecule comprises a VH amino acid sequence, a VL amino acid sequence, or both a VH and a VL amino acid sequence from ABS clone P9-28.

[0170] In some embodiments, the GAL9 binding molecule comprises a complete IgG heavy chain sequence and a complete IgG light chain sequence from any ABS clone selected from P9-02B, P9-04, P9-05, P9-08, P9-09, P9-10, P9-15, P9-16, P9-18, P9-19, P9-20, P9-21, P9-22, P9-27, P9-28, P9-31, P9-32, P9-36, P9-39, P9-49, P9-54, and P9-58. In a currently preferred embodiment, the GAL9 binding molecule comprises a complete IgG heavy chain sequence and a complete IgG light chain sequence from ABS clone P9-28.

[0171] 6.4.4. Constant Region

[0172] In the GAL9 binding molecule, the GAL9 binding molecule can have a constant region domain sequence. As described herein, the constant region domain amino acid sequence is the sequence of the constant region domain of an antibody. The constant region can refer to a CH1, CH2, CH3, CH4, or CL constant domain.

[0173] In various embodiments, the constant region sequence is a mammalian sequence, including but not limited to mouse, rat, hamster, rabbit, camel, donkey, goat, and human sequences. In a preferred embodiment, the constant region sequence is a human sequence. In certain embodiments, the constant region sequence is from an antibody light chain. In specific embodiments, the constant region sequence is from a lambda or kappa light chain. In certain embodiments, the constant region sequence is from an antibody heavy chain. In specific embodiments, the constant region sequence is an antibody heavy chain sequence of an IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM isotype. In a specific embodiment, the constant region sequence is from an IgG isotype. In a preferred embodiment, the constant region sequence is from an IgG1 isotype.

[0174] Exemplary constant regions and modifications thereof are described in WO2018075692, which is herein incorporated by reference in its entirety.

[0175] 6.4.4.1.CH1 and CL areas

[0176] As described herein, the CH1 amino acid sequence is the sequence of the second domain of the antibody heavy chain, with reference to the N-terminus to the C-terminus of the natural antibody heavy chain structure. In certain embodiments, the CH1 sequence is an endogenous sequence. In various embodiments, the CH1 sequence is a mammalian sequence, including but not limited to mouse, rat, hamster, rabbit, camel, donkey, goat, and human sequences. In a preferred embodiment, the CH1 sequence is a human sequence. In certain embodiments, the CH1 sequence is from the IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM isotype. In a preferred embodiment, the CH1 sequence is from the IgG1 isotype. In some preferred embodiments, the CH1 sequence is amino acids 1-98 of UniProt Accession No. P01857.

[0177] With reference to the structure of a natural antibody light chain, the CL amino acid sequence useful in the GAL9 binding molecules described herein is an antibody light chain constant domain sequence. In certain embodiments, the CL sequence is an endogenous sequence. In various embodiments, the CL sequence is a mammalian sequence, including but not limited to mouse, rat, hamster, rabbit, camel, donkey, goat, and human sequences. In preferred embodiments, the CL sequence is a human sequence.

[0178] In certain embodiments, the CL amino acid sequence is a lambda (λ) light chain constant domain sequence. In some specific embodiments, the CL amino acid sequence is a human λ light chain constant domain sequence. In a preferred embodiment, the λ light chain sequence is the sequence of UniProt accession number P0CG04.

[0179] In certain embodiments, the CL amino acid sequence is a kappa (κ) light chain constant domain sequence. In a preferred embodiment, the CL amino acid sequence is a human κ light chain constant domain sequence. In a preferred embodiment, the κ light chain sequence is the sequence of UniProt accession number P01834.

[0180] In certain embodiments, both the CH1 sequence and the CL sequence are endogenous sequences. In certain embodiments, the CH1 sequence and the CL sequence separately comprise orthogonal modifications in the endogenous CH1 and CL sequences, respectively, as discussed in more detail in Section 6.4.4.1 below. The CH1 and CL sequences can also be portions thereof, having endogenous or modified sequences, such that a domain having a CH1 sequence or a portion thereof can associate with a domain having a CL sequence or a portion thereof.

[0181] 6.4.4.2. Orthogonal modification of CH1 and CL

[0182] In certain embodiments, the CH1 sequence and the CL sequence separately comprise orthogonal modifications in the endogenous CH1 and CL sequences, respectively. Orthogonal mutations, in general, are described in more detail below in Sections 6.4.6.1-6.4.6.3.

[0183] In some specific embodiments, the orthogonal modification in the endogenous CH1 and CL sequences is an engineered disulfide bridge selected from the group consisting of an engineered cysteine at position 138 of the CH1 sequence and position 116 of the CL sequence, an engineered cysteine at position 128 of the CH1 sequence and position 119 of the CL sequence, or an engineered cysteine at position 129 of the CH1 sequence and position 210 of the CL sequence, numbered and discussed in greater detail in U.S. Patent No. 8,053,562 and U.S. Patent No. 9,527,927, each of which is incorporated herein by reference in its entirety. In a preferred embodiment, the engineered cysteine is at position 128 of the CH1 sequence and position 118 of the CLκ sequence, numbered by Eu index.

[0184] In a series of preferred embodiments, the mutation providing a non-endogenous cysteine amino acid is an F118C mutation in the CL sequence with a corresponding A141C in the CH1 sequence, or an F118C mutation in the CL sequence with a corresponding L128C in the CH1 sequence, or an S162C mutation in the CL sequence with a corresponding P171C mutation in the CH1 sequence, as numbered by the Eu index.

[0185] In various embodiments, the orthogonal mutations in the CL sequence and the CH1 sequence are charge pair mutations. In specific embodiments, the charge pair mutations are F118S, F118A, or F118V mutations in the CL sequence and the corresponding A141L in the CH1 sequence, or T129R mutations in the CL sequence and the corresponding K147D in the CH1 sequence, as numbered by the Eu index and described in more detail in Bonisch et al. (Protein Engineering, Design & Selection, 2017, pp. 1-12), the entire contents of which are incorporated herein by reference. In a series of preferred embodiments, the charge pair mutations are N138K mutations in the CL sequence and the corresponding G166D in the CH1 sequence, or N138D mutations in the CL sequence and the corresponding G166K in the CH1 sequence, as numbered by the Eu index.

[0186] 6.4.4.3.CH2 Area

[0187] In the GAL9 binding molecules described herein, the GAL9 binding molecules may have a CH2 amino acid sequence. As described herein, the CH2 amino acid sequence is the CH2 amino acid sequence of the third domain of the antibody heavy chain, with reference to the N-terminus to the C-terminus of the natural antibody heavy chain structure. In various embodiments, the CH2 sequence is a mammalian sequence, including but not limited to mouse, rat, hamster, rabbit, camel, donkey, goat, and human sequences. In a preferred embodiment, the CH2 sequence is a human sequence. In certain embodiments, the CH2 sequence is from the IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM isotype. In a preferred embodiment, the CH2 sequence is from the IgG1 isotype.

[0188] In certain embodiments, the CH2 sequence is an endogenous sequence. In a specific embodiment, the sequence is amino acids 111-223 of UniProt Accession No. P01857.

[0189] In one series of embodiments, a GAL9 binding molecule comprises one or more paired CH2 domains having CH2 sequences, wherein a first group comprises CH2 amino acid sequences from a first isotype and one or more orthologous groups of CH2 amino acid sequences from another isotype. As described herein, orthologous CH2 amino acid sequences are capable of interacting with CH2 amino acid sequences from a shared isotype but do not significantly interact with CH2 amino acid sequences from another isotype present in the GAL9 binding molecule. In specific embodiments, all groups of CH2 amino acid sequences are from the same species. In preferred embodiments, all groups of CH2 amino acid sequences are human CH2 amino acid sequences. In other embodiments, the groups of CH2 amino acid sequences are from different species. In a specific embodiment, the first group of CH2 amino acid sequences is from the same isotype as the other non-CH2 domains in the GAL9 binding molecule. In a specific embodiment, the first group comprises CH2 amino acid sequences from the IgG isotype, and one or more orthologous groups comprise CH2 amino acid sequences from the IgM or IgE isotype. In certain embodiments, one or more sets of CH2 amino acid sequences are endogenous CH2 sequences. In other embodiments, one or more sets of CH2 amino acid sequences are endogenous CH2 sequences with one or more mutations. In specific embodiments, the one or more mutations are orthogonal knob-in-hole mutations, orthogonal charge pair mutations, or orthogonal hydrophobic mutations. Orthologous CH2 amino acid sequences that can be used in GAL9 binding molecules are described in more detail in International PCT Applications WO2017 / 011342 and WO2017 / 106462, which are incorporated herein by reference in their entirety.

[0190] 6.4.4.4.CH3 Area

[0191] With reference to the structure from N-terminus to C-terminus of a native antibody heavy chain, the CH3 amino acid sequence described herein is the sequence of the C-terminal domain of the antibody heavy chain.

[0192] In various embodiments, the CH3 sequence is a mammalian sequence, including but not limited to mouse, rat, hamster, rabbit, camel, donkey, goat, and human sequences. In a preferred embodiment, the CH3 sequence is a human sequence. In certain embodiments, the CH3 sequence is from an IgA1, IgA2, IgD, IgE, IgM, IgG1, IgG2, IgG3, or IgG4 isotype, or the CH4 sequence is from an IgE or IgM isotype. In a specific embodiment, the CH3 sequence is from an IgG isotype. In a preferred embodiment, the CH3 sequence is from an IgG1 isotype.

[0193] In certain embodiments, the CH3 sequence is an endogenous sequence. In specific embodiments, the CH3 sequence is amino acids 224-330 of UniProt accession number P01857. In multiple embodiments, the CH3 sequence is a segment of an endogenous CH3 sequence. In specific embodiments, the CH3 sequence comprises an endogenous CH3 sequence lacking the N-terminal amino acids G224 and Q225. In specific embodiments, the CH3 sequence comprises an endogenous CH3 sequence lacking the C-terminal amino acids P328, G329, and K330. In specific embodiments, the CH3 sequence comprises an endogenous CH3 sequence lacking the N-terminal amino acids G224 and Q225 and the C-terminal amino acids P328, G329, and K330. In preferred embodiments, the GAL9 binding molecule comprises multiple domains having a CH3 sequence, wherein the CH3 sequence can refer to a complete endogenous CH3 sequence as well as a CH3 sequence lacking the N-terminal amino acid, the C-terminal amino acid, or both.

[0194] In certain embodiments, the CH3 sequence is an endogenous sequence having one or more mutations. In specific embodiments, the mutations are one or more orthogonal mutations that are introduced into the endogenous CH3 sequence to direct specific pairing of a particular CH3 sequence, as described in more detail below in Sections 6.4.6.1-6.4.6.3.

[0195] In certain embodiments, the CH3 sequence is engineered to reduce the immunogenicity of the antibody by replacing specific amino acids of one allotype with specific amino acids of another allotype (referred to herein as isoallotype mutations), as described in more detail in Stickler et al. (Genes Immun. 2011 Apr; 12(3): 213-221), the entire teachings of which are incorporated herein by reference. In a specific embodiment, specific amino acids of the G1m1 allotype are replaced. In a preferred embodiment, the isoallotype mutations D356E and L358M are formed in the CH3 sequence.

[0196] In some embodiments, the IgG1 CH3 amino acid sequence comprises the following mutational changes: P343V; Y349C; and a tripeptide insertion, 445P, 446G, 447K. In other preferred embodiments, domain B has a human IgG1 CH3 sequence with the following mutational changes: T366K; and a tripeptide insertion, 445K, 446S, 447C. In other preferred embodiments, domain B has a human IgG1 CH3 sequence with the following mutational changes: Y349C; and a tripeptide insertion, 445P, 446G, 447K.

[0197] In some embodiments, the IgG1 CH3 amino acid sequence comprises a 447C mutation incorporated into the endogenous CH3 sequence.

[0198] 6.4.5. Antigen Binding Site

[0199] In some embodiments, the VL or VH amino acid sequence and the cognate VL or VH amino acid sequence associate and form a first antigen binding site (ABS). The antigen binding site (ABS) is capable of specifically binding to an epitope of an antigen. Antigen binding of the ABS is described in more detail in Section 6.4.5.1 below.

[0200] In alternative embodiments, for example where the GAL9 binding molecule is a single domain antibody, the VH or VL amino acid sequence forms the first ABS.

[0201] In some embodiments, the GAL9 antigen-binding molecule comprises a second ABS. In some embodiments, the second ABS is specific for the same GAL9 antigen as the first ABS. In some embodiments, the second ABS specifically binds to the same epitope of the same GAL9 antigen as the first ABS. In some embodiments, the second ABS is identical to the first ABS.

[0202] In some embodiments, the second ABS is specific for a different epitope from the first GAL9 antigen. For example, if the first ABS comprises CDRs from any ABS clone selected from P9-02B, P9-04, P9-05, P9-08, P9-09, P9-10, P9-15, P9-16, P9-18, P9-19, P9-20, P9-21, P9-22, P9-27, P9-28, P9-31, P9-32, P9-36, P9-39, P9-49, P9-54, and P9-58, or variable domains, the second ABS may comprise CDRs or variable domains from another ABS clone selected from P9-02B, P9-04, P9-05, P9-08, P9-09, P9-10, P9-15, P9-16, P9-18, P9-20, P9-21, P9-22, P9-27, P9-28, P9-31, P9-32, P9-36, P9-39, P9-49, P9-54, and P9-58.

[0203] In some embodiments, the GAL9 antigen binding molecule is multispecific, eg, the second ABS of the GAL9 antigen binding molecule specifically binds to an antigen that is different from the GAL9 antigen specifically bound by the first ABS.

[0204] 6.4.5.1. Antigen Binding via ABS

[0205] An ABS and GAL9-binding molecules comprising such an ABS are said to "recognize" an epitope (or more generally, an antigen) to which the ABS specifically binds, and this epitope (or more generally, antigen) is said to be the "recognition specificity" or "binding specificity" of the ABS.

[0206] ABS is considered to bind to its specific antigen or epitope with a specific affinity. As used herein, "affinity" refers to the strength of the non-covalent intermolecular interaction between one molecule and another. Affinity, i.e., the strength of the interaction, can be expressed as the dissociation equilibrium constant (K D ), where the lower K D A value of 0 indicates a stronger interaction between the molecules. D The values are measured by methods well known in the art, including but not limited to biolayer interferometry (e.g., ), surface plasmon resonance (SPR) technology (e.g., ) and cell binding assays. For the purposes of this article, affinity is measured using Dissociation equilibrium constants measured by biolayer interferometry.

[0207] As used herein, "specific binding" refers to the affinity between an ABS and its cognate antigen or epitope, where K D Values below 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M.

[0208] The number of ABSs in the GAL9 binding molecules described herein defines the "valency" of the GAL9 binding molecule. A GAL9 binding molecule with a single ABS is "monovalent." A GAL9 binding molecule with multiple ABSs is referred to as "multivalent." A multivalent GAL9 binding molecule with two ABSs is "bivalent," a multivalent GAL9 binding molecule with three ABSs is "trivalent," and a multivalent GAL9 binding molecule with four ABSs is "tetravalent."

[0209] In various multivalent embodiments, the multiple ABSs all have the same recognition specificity. Such GAL9 binding molecules are "monospecific," "multivalent" binding constructs. In other multivalent embodiments, at least two of the multiple ABSs have different recognition specificities. Such GAL9 binding molecules are multivalent and "multispecific." In multivalent embodiments in which the ABSs have a total of two recognition specificities, the GAL9 binding molecule is "bispecific." In multivalent embodiments in which the ABSs have a total of three recognition specificities, the GAL9 binding molecule is "trispecific."

[0210] In multivalent embodiments where the ABS collectively has multiple recognition specificities for multiple different epitopes present on the same antigen, the GAL9 binding molecule is "multiparatopic." Multivalent embodiments where the ABS collectively recognizes two epitopes on the same antigen are "biparatopic."

[0211] In multiple multivalent embodiments, the multivalency of the GAL9 binding molecule increases the avidity of the GAL9 binding molecule for a specific target. As used herein, "avidity" refers to the total strength of an interaction between two or more molecules, for example, a multivalent GAL9 binding molecule for a specific target, where avidity is the cumulative strength of the interaction provided by the affinities of multiple ABS. Avidity can be measured by the same methods as those used to determine affinity, as described above. In certain embodiments, the avidity of the GAL9 binding molecule for a specific target is such that the interaction is a specific binding interaction, where the avidity between the two molecules has a value less than 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M's K D In certain embodiments, the affinity of a GAL9 binding molecule for a particular target has a K D A value that makes the interaction a specific binding interaction, where the affinity of the individual ABS does not have a K that makes it bind specifically to the corresponding antigen or epitope D In certain embodiments, avidity is the cumulative strength of the interactions provided by the affinities of multiple ABS for separate antigens on a shared specific target or complex (e.g., multiple separate antigens present on a single cell). In certain embodiments, avidity is the cumulative strength of the interactions provided by the affinities of multiple ABS for multiple separate epitopes on a shared single antigen.

[0212] 6.4.6. Orthogonal modification

[0213] In the GAL9 binding molecules described herein, the GAL9 binding molecules may have a constant region domain comprising orthogonal modifications. The constant region domain amino acid sequences are described in more detail above in Section 6.4.4.

[0214] As described herein, an "orthogonal modification" or its synonym "orthogonal mutation" is one or more engineered mutations in the amino acid sequence of an antibody domain that increase the binding affinity of a first domain having an orthogonal modification to a second domain having a complementary orthogonal modification. In certain embodiments, the orthogonal modification reduces the affinity of a domain having an orthogonal modification to a domain lacking a complementary orthogonal modification. In certain embodiments, the orthogonal modification is a mutation in an endogenous antibody domain sequence. In various embodiments, the orthogonal modification is a modification of the N-terminus or C-terminus of an endogenous antibody domain sequence, including but not limited to amino acid additions or deletions. In specific embodiments, orthogonal modifications include but are not limited to engineered disulfide bridges, knob-in-hole mutations, and charge pair mutations, as described in more detail in Sections 6.4.6.1-6.4.6.3 below. In specific embodiments, orthogonal modifications include, but are not limited to, a combination of orthogonal modifications selected from, but not limited to, engineered disulfide bridges, knob-in-hole mutations, and charge pair mutations. In certain embodiments, orthogonal modifications can be combined with amino acid substitutions that reduce immunogenicity, such as allotypic mutations, as described in more detail above in Section 6.4.4.4.

[0215] 6.4.6.1. Orthogonal engineered disulfide bridges

[0216] In various embodiments, orthogonal modifications include mutations that create engineered disulfide bridges between the first and second domains. As described herein, an "engineered disulfide bridge" is an amino acid mutation that provides non-endogenous cysteine in two or more domains, such that a non-natural disulfide bond is formed when the two or more domains associate. Engineered disulfide bridges are described in more detail in Merchant et al. (Nature Biotech (1998) 16: 677-681), the entire contents of which are incorporated herein by reference. In certain embodiments, engineered disulfide bridges improve the orthogonal association between specific domains. In a specific embodiment, the mutation that creates an engineered disulfide bridge is a K392C mutation in one of the first or second CH3 domains and a D399C in the other CH3 domain. In a preferred embodiment, the mutation that creates an engineered disulfide bridge is an S354C mutation in one of the first or second CH3 domains and a Y349C in the other CH3 domain. In another preferred embodiment, the mutation that creates the engineered disulfide bridge is a 447C mutation in both the first and second CH3 domains, which is provided by extending the C-terminus of the CH3 domains to incorporate a KSC tripeptide sequence.

[0217] 6.4.6.2. Orthogonal Knob-in-Holes Mutation

[0218] In various embodiments, orthogonal modifications include knob-hole (synonymously, knob-into-hole) mutations. As described herein, knob-hole mutations are mutations that change the spatial characteristics of the surface of a first domain, whereby the first domain will preferentially associate with a second domain having a complementary spatial mutation relative to association with a domain without a complementary spatial mutation. Knob-hole mutations are described in more detail in U.S. Patent No. 5,821,333 and U.S. Patent No. 8,216,805, each of which is incorporated herein in its entirety. In various embodiments, knob-hole mutations are combined with engineered disulfide bridges, as described in more detail in Merchant et al. (Nature Biotech (1998) 16: 677-681), the entire contents of which are incorporated herein by reference. In various embodiments, knob-hole mutations, allotypic mutations, and engineered disulfide bridge mutations are combined.

[0219] In certain embodiments, the knob-in-hole mutation is a T366Y mutation in the first domain and a Y407T mutation in the second domain. In certain embodiments, the knob-in-hole mutation is a F405A in the first domain and a T394W in the second domain. In certain embodiments, the knob-in-hole mutation is a T366Y mutation and F405A in the first domain, and T394W and Y407T in the second domain. In certain embodiments, the knob-in-hole mutation is a T366W mutation in the first domain and a Y407A in the second domain. In certain embodiments, the combined knob-in-hole mutation and engineered disulfide mutation is an S354C and T366W mutation in the first domain, and a Y349C, T366S, L368A, and Y407V mutation in the second domain. In a preferred embodiment, the combined knob-to-hole, allotypic and engineered disulfide mutations are S354C and T366W mutations in the first domain, and Y349C, D356E, L358M, T366S, L368A and Y407V mutations in the second domain.

[0220] 6.4.6.3. Orthogonal Charge Pair Mutation

[0221] In a plurality of embodiments, orthogonal modification is charge pair mutation.As used herein, charge pair mutation is the mutation of the amino acid charge that affects the surface of the domain, thus relative to the association with the domain without complementary charge pair mutation, the domain will preferentially associate with the second domain with complementary charge pair mutation.In certain embodiments, charge pair mutation improves the orthogonal association between specific domains.Charge pair mutation is described in more detail in U.S. Patent No. 8,592,562, U.S. Patent No. 9,248,182 and U.S. Patent No. 9,358,286, all of which are incorporated herein by reference.In certain embodiments, charge pair mutation improves the stability between specific domains.In preferred embodiments, charge pair mutation is T366K mutation in the first domain and L351D mutation in another domain.

[0222] In some embodiments, the orthogonal mutation is a charge pair mutation at the VH / VL interface. In preferred embodiments, the charge pair mutation at the VH / VL interface is Q39E in VH with the corresponding Q38K in VL, or Q39K in VH with the corresponding Q38E in VL, as described in more detail in Igawa et al. (Protein Eng. Des. Sel., 2010, Vol. 23, 667-677), the entire contents of which are incorporated herein by reference.

[0223] 6.4.7. Trivalent and Tetravalent GAL9 Binding Molecules

[0224] In another series of embodiments, the GAL9 binding molecule has three antigen binding sites and is therefore referred to as "trivalent." In various embodiments, the GAL9 binding molecule has four antigen binding sites and is therefore referred to as "tetravalent."

[0225] 6.5. GAL9 Binding Molecular Structure

[0226] Antigen binding sites as described herein, including specific CDR subgroups, can form any binding molecular structure, including but not limited to full-length antibodies, Fab fragments, Fv, scFv, tandem scFv, Diabody, scDiabody, DART, tandAb, miniantibodies, Camelidae VHH and other antibody fragments or forms known to those skilled in the art. Exemplary antibody and antibody fragment forms are described in detail in Brinkmann et al. (MABS, 2017, Vol. 9, No. 2, 182-212), the full contents of which are incorporated herein by reference. Antigen binding sites as described herein, including specific CDR subgroups, can also be formatted as "B-body" forms, as described in more detail in U.S. Publication No. US 2018 / 0118811 and International Application Publication No. WO2018 / 075692 before authorization, each of which is incorporated herein by reference in its entirety.

[0227] 6.6. Other modifications

[0228] In another series of embodiments, the GAL9 binding molecules have additional modifications.

[0229] 6.6.1. Antibody-drug conjugates

[0230] In a number of embodiments, the GAL9 binding molecule is conjugated to a therapeutic agent (i.e., a drug) to form a GAL9 binding molecule-drug conjugate. Therapeutic agents include, but are not limited to, chemotherapeutic agents, imaging agents (e.g., radioisotopes), immunomodulators (e.g., cytokines, chemokines, or checkpoint inhibitors), and toxins (e.g., cytotoxic agents). In certain embodiments, the therapeutic agent is connected to the GAL9 binding molecule via a linker peptide, as discussed in more detail in Section 6.6.3 below.

[0231] Methods for preparing antibody-drug conjugates (ADCs) suitable for conjugating drugs to the GAL9 binding molecules disclosed herein are described, for example, in U.S. Pat. No. 8,624,003 (pot method), U.S. Pat. No. 8,163,888 (one-step method), U.S. Pat. No. 5,208,020 (two-step method), U.S. Pat. No. 8,337,856, U.S. Pat. No. 5,773,001, U.S. Pat. No. 7,829,531, U.S. Pat. No. 5,208,020, U.S. Pat. No. 7,745,394, WO 2017 / 136623, WO 2017 / 015502, WO 2017 / 015496, WO 2017 / 015495, WO 2004 / 010957, WO 2005 / 077090, WO 2005 / 082023、WO2006 / 065533、WO 2007 / 030642、WO 2007 / 103288、WO 2013 / 173337、WO 2015 / 057699、WO2015 / 095755、WO 2015 / 123679、WO 2015 / 157286、WO 2017 / 165851、WO 2009 / 073445、WO2010 / 068759、WO 2010 / 138719、WO 2012 / 171020、WO 2014 / 008375、WO 2014 / 093394、WO2014 / 093640、WO 2014 / 160360, WO 2015 / 054659, WO 2015 / 195925, WO 2017 / 160754, Storz (MAbs. 2015 Nov-Dec; 7(6): 989-1009), Lambert et al. (Adv Ther, 201734: 1015), Diamantis et al. (British Journal of Cancer, 2016, 114, 362-367), Carrico et al. (Nat Chem Biol, 2007.3: 321-2), We et al. (Proc Natl Acad Sci USA, 2009.106: 3000-5), Rabuka et al. (Curr Opin Chem Biol., 201114: 790-6), Hudak et al. (Angew Chem Int Ed Engl., 2012: 4161-5), Rabuka et al. (Nat Protoc., 20127: 1052-67), Agarwal et al (Proc Natl Acad Sci USA, 2013, 110: 46-51), Agarwal et al (Proc Natl Acad 846-851), Barfield et al (Drug Dev. and D., 2014, 14: 34-41), Drake et al (Bioconjugate Chem., 2014, 25: 1331-41), Liang et al (J Am Chem Soc., 2014, 136: 10850-3), Drake et al (Curr Opin Chem Biol., 2015, 28: 174-80), and York et al (BMC Biotechnology, 2016, 16(1): 23), the entire contents of which are each incorporated herein by reference in their entirety.

[0232] 6.6.2. Other combined parts

[0233] In various embodiments, the GAL9 binding molecule has modifications comprising one or more other binding moieties. In certain embodiments, the binding moiety is an antibody fragment or antibody format, including but not limited to full-length antibodies, Fab fragments, Fv, scFv, tandem scFv, Diabody, scDiabody, DART, tandAb, miniantibodies, camelid VHH, and other antibody fragments or formats known to those skilled in the art. Exemplary antibody and antibody fragment formats are described in detail in Brinkmann et al. (MABS, 2017, Vol. 9, No. 2, 182-212), the entire contents of which are incorporated herein by reference.

[0234] In certain embodiments, the one or more additional binding moieties are attached to the C-termini of the first or third polypeptide chain. In certain embodiments, the one or more additional binding moieties are attached to the C-termini of both the first and third polypeptide chains. In certain embodiments, the one or more additional binding moieties are attached to the C-termini of both the first and third polypeptide chains. In certain embodiments, individual portions of the one or more additional binding moieties are separately attached to the C-termini of the first and third polypeptide chains such that the portions form a functional binding moiety.

[0235] In certain embodiments, one or more additional binding moieties are attached to the N-terminus of any polypeptide chain (e.g., the first, second, third, fourth, fifth, or sixth polypeptide chain). In certain embodiments, each individual portion of the additional binding moiety is separately attached to the N-terminus of a different polypeptide chain such that the portions form a functional binding moiety.

[0236] In certain embodiments, one or more additional binding moieties are specific for different antigens or epitopes of the ABS within the GAL9 binding molecule. In certain embodiments, one or more additional binding moieties are specific for the same antigen or epitope of the ABS within the GAL9 binding molecule. In certain embodiments, wherein the modification is two or more additional binding moieties, the additional binding moieties are specific for the same antigen or epitope. In certain embodiments, wherein the modification is two or more additional binding moieties, the additional binding moieties are specific for different antigens or epitopes.

[0237] In certain embodiments, one or more additional binding moieties are linked to the GAL9 binding molecule using in vitro methods, including but not limited to reactive chemistry and affinity tag systems, as discussed in more detail in Section 6.6.3 below. In certain embodiments, one or more additional binding moieties are linked to the GAL9 binding molecule via Fc-mediated binding (e.g., Protein A / G). In certain embodiments, one or more additional binding moieties are linked to the GAL9 binding molecule using recombinant DNA technology, such as a nucleotide sequence encoding a fusion product between the GAL9 binding molecule and the additional binding moiety on the same expression vector (e.g., plasmid).

[0238] 6.6.3. Functional groups / reactive groups

[0239] In various embodiments, the GAL9 binding molecules have modifications comprising functional groups or chemically reactive groups that can be used in downstream processes, such as attachment of other moieties (e.g., drug conjugates and other binding moieties, as discussed in more detail in Sections 6.6.1 and 6.6.2, above) and downstream purification processes.

[0240] In certain embodiments, the modification is a chemically reactive group, including but not limited to reactive thiols (e.g., maleimide-based reactive groups), reactive amines (e.g., N-hydroxysuccinimide-based reactive groups), "click chemistry" groups (e.g., reactive alkynes), and aldehydes with formylglycine (FGly). In certain embodiments, the modification is a functional group, including but not limited to affinity peptide sequences (e.g., HA, HIS, FLAG, GST, MBP, and Strep systems, etc.). In certain embodiments, the functional group or chemically reactive group has a cleavable peptide sequence. In certain embodiments, the cleavable peptide is cleaved by means including but not limited to: photocleavage, chemical cleavage, protease cleavage, reducing conditions, and pH conditions. In specific embodiments, protease cleavage is performed by intracellular proteases. In specific embodiments, protease cleavage is performed by extracellular or membrane-associated proteases. ADC therapy using protease cleavage is described in more detail in Choi et al. (Theranostics, 2012; 2(2): 156-178.), the entire contents of which are incorporated herein by reference.

[0241] 6.6.4. Reduced effector function

[0242] In certain embodiments, the GAL9 binding molecule has one or more engineered mutations in the amino acid sequence of the antibody domain that reduce effector functions naturally associated with antibody binding. Effector functions include, but are not limited to, cellular functions resulting from binding of Fc receptors to the Fc portion of an antibody, such as antibody-dependent cellular cytotoxicity (ADCC, also known as antibody-dependent cell-mediated cytotoxicity), complement fixation (e.g., C1q binding), antibody-dependent cell-mediated phagocytosis (ADCP), and opsonization. Exemplary engineered mutations that reduce effector function are described in more detail in U.S. Pub. No. 2017 / 0137530, Armour et al. (Eur. J. Immunol. 29(8)(1999)2613-2624), Shields et al. (J. Biol. Chem. 276(9)(2001)6591-6604), and Oganesyan et al. (Acta Cristallographica D64(2008)700-704), each of which is incorporated herein by reference in its entirety.

[0243] 6.7. Purification Methods

[0244] Provided herein are methods for purifying GAL9 binding molecules. Purification steps include, but are not limited to, purification of GAL9 binding molecules based on protein characteristics such as size (e.g., size exclusion chromatography), charge (e.g., ion exchange chromatography), or hydrophobicity (e.g., hydrophobic interaction chromatography). In one embodiment, cation exchange chromatography is performed. Other purification methods known to those skilled in the art may be performed, including but not limited to the use of Protein A, Protein G, or Protein A / G reagents. Multiple iterations of a single purification method may be performed. Combinations of purification methods may be performed.

[0245] 6.7.1. Assembly and purity of the complex

[0246] In some embodiments of the present invention, at least four different polypeptide chains associate together to form a complete complex, i.e., a GAL9-binding molecule. However, incomplete complexes that do not contain the at least four different polypeptide chains can also form. For example, incomplete complexes can form that have only one, two, or three of the polypeptide chains. In other examples, an incomplete complex can contain more than three polypeptide chains but not the at least four different polypeptide chains, e.g., an incomplete complex that is inappropriately associated with more than one copy of a different polypeptide chain. The methods of the present invention purify the complex, i.e., a fully assembled GAL9-binding molecule, from the incomplete complex.

[0247] Methods for evaluating the efficacy and efficiency of purification steps are well known to those skilled in the art and include, but are not limited to, SDS-PAGE analysis, ion exchange chromatography, size exclusion chromatography, and mass spectrometry. Purity can also be assessed according to various criteria. Examples of criteria include, but are not limited to: 1) assessing the percentage of total protein provided by the fully assembled GAL9 binding molecules in the eluate, 2) assessing the enrichment factor or percentage increase of the method for purifying the desired product, for example, comparing the total protein provided by the fully assembled GAL9 binding molecules in the eluate to the total protein in the starting sample, 3) assessing the percentage of total protein or the percentage reduction of undesirable products (e.g., the incomplete complex described above), including determining the percentage or percentage reduction of specific undesirable products (e.g., unassociated single polypeptide chains, dimers of any combination of polypeptide chains, or trimers of any combination of polypeptide chains). Purity can be assessed following any combination of the methods described herein.

[0248] 6.8. Preparation method

[0249] The GAL9 binding molecules described herein can be readily prepared by expression using standard cell-free translation, transient transfection, and stable transfection methods currently used for antibody preparation. In some specific embodiments, the production of GAL9 binding molecules can be performed using Expi293 cells (ThermoFisher) and protocols and reagents from ThermoFisher, such as ExpiFectamine, or other reagents known to those skilled in the art, such as polyethyleneimine, as described in detail in Fang et al. (Biological Procedures Online, 2017, 19:11), the entire contents of which are incorporated herein by reference.

[0250] Various purification strategies can be used, including but not limited to the use of Protein A, Protein G or Protein A / G reagents, to readily separate the expressed protein from unwanted proteins and protein complexes. Further purification can be performed using ion exchange chromatography routinely used in the art.

[0251] 6.9. Pharmaceutical Compositions

[0252] In another aspect, a pharmaceutical composition is provided, comprising a GAL9 binding molecule as described herein and a pharmaceutically acceptable carrier or diluent. In typical embodiments, the pharmaceutical composition is sterile.

[0253] In various embodiments, the pharmaceutical composition comprises the GAL9 binding molecule at a concentration of 0.1 mg / ml to 100 mg / ml. In specific embodiments, the pharmaceutical composition comprises the GAL9 binding molecule at a concentration of 0.5 mg / ml, 1 mg / ml, 1.5 mg / ml, 2 mg / ml, 2.5 mg / ml, 5 mg / ml, 7.5 mg / ml, or 10 mg / ml. In some embodiments, the pharmaceutical composition comprises the GAL9 binding molecule at a concentration greater than 10 mg / ml. In certain embodiments, the GAL9 binding molecule is present at a concentration of 20 mg / ml, 25 mg / ml, 30 mg / ml, 35 mg / ml, 40 mg / ml, 45 mg / ml, or even 50 mg / ml or more. In a specific embodiment, the GAL9 binding molecule is present at a concentration greater than 50 mg / ml.

[0254] In various embodiments, the pharmaceutical compositions are described in more detail in U.S. Patent No. 8,961,964, U.S. Patent No. 8,945,865, U.S. Patent No. 8,420,081, U.S. Patent No. 6,685,940, U.S. Patent No. 6,171,586, U.S. Patent No. 8,821,865, U.S. Patent No. 9,216,219, U.S. Application No. 10 / 813,483, WO 2014 / 066468, WO 2011 / 104381, and WO 2016 / 180941, each of which is herein incorporated in its entirety.

[0255] 6.10. Treatment methods

[0256] In another aspect, methods of treatment are provided, comprising administering to a patient suffering from a disease or disorder a GAL9 binding molecule as described herein in an amount effective to treat the patient.

[0257] 6.10.1. Subjects

[0258] In some embodiments, the subject can be a mammal. In some embodiments, the mammal is a mouse. In preferred embodiments, the mammal is a human.

[0259] 6.10.2. Combination therapy

[0260] GAL9 binding molecules can be used alone or in combination with other therapeutic agents or methods to treat or prevent diseases or conditions. The GAL9 binding molecules can be administered simultaneously or sequentially with the second therapeutic agent, depending on the disease to be treated.

[0261] In some embodiments, the anti-GAL9 binding molecules are used in combination with agents or procedures used in the clinic or within the current standard of care to treat or prevent a disease or condition (such as a proliferative disease or cancer). In some embodiments, the GAL9 binding molecules are administered in combination with immune checkpoint inhibitors such as anti-PD-L1 antibodies, anti-PD-1 antibodies, anti-CTLA4 antibodies, anti-LAB3 antibodies, anti-TIM1 antibodies, anti-TIGIT antibodies, and anti-PVRIG antibodies.

[0262] 6.10.3. Proliferative diseases

[0263] In some embodiments, treating comprises administering to a subject having a proliferative disease one or more GAL9 binding molecules as described herein in an amount effective to treat the subject.

[0264] In some embodiments, treatment comprises administering an effective amount of one or more GAL9 binding molecules described herein for treating cancer and / or precancerous lesions. In some embodiments, treatment comprises administering an effective amount of one or more GAL9 binding molecules as described herein in combination with another cancer therapeutic agent and / or treatment regimen (radiation, surgery, etc.).

[0265] In various embodiments, the cancer is bladder cancer, blood cancer, bone cancer, bone marrow cancer, brain cancer, breast cancer, colon cancer, esophageal cancer, gastrointestinal cancer, gum cancer, head cancer, kidney cancer, liver cancer, lung cancer, nasopharyngeal cancer, neck cancer, head and neck cancer, ovarian cancer, prostate cancer, pancreatic cancer, skin cancer, stomach cancer, testicular cancer, tongue cancer, or uterine cancer.

[0266] In some embodiments, the cancerous or precancerous tumor is a neoplasm, a malignant tumor, a carcinoma, an undifferentiated tumor, a giant cell and spindle cell carcinoma, a small cell carcinoma, a papillary carcinoma, a squamous cell carcinoma, a head and neck squamous cell carcinoma, a lymphoepithelial carcinoma, a basal cell carcinoma, a pilomatrix carcinoma, a transitional cell carcinoma, a papillary transitional cell carcinoma, adenocarcinoma, a gastrinoma, a malignant tumor, a bile duct carcinoma, a hepatocellular carcinoma, a combined hepatocellular and bile duct carcinoma, a trabecular adenocarcinoma, adenoid cystic carcinoma, adenocarcinoma in an adenomatous polyp, adenocarcinoma, a familial polyposis coli, a solid carcinoma, a carcinoid tumor, a malignant tumor, a bronchoalveolar adenocarcinoma, a papillary adenocarcinoma, a chromophobe carcinoma, an acidophil carcinoma, an oxyphilic adenocarcinoma, a basophil carcinoma carcinoma), clear cell adenocarcinoma, granular cell carcinoma, follicular adenocarcinoma, papillary and follicular adenocarcinoma, nonencapsulating sclerosing carcinoma, adrenocortical carcinoma, endometrioid carcinoma, skin appendage carcinoma, apocrine adenocarcinoma, sebaceous adenocarcinoma, ceraminous adenocarcinoma, mucoepidermoid carcinoma, cystadenocarcinoma, pancreatic adenocarcinoma, pancreatic ductal adenocarcinoma, cystadenocarcinoma, pancreatic neuroendocrine tumors (PanNET), adenosquamous carcinomas of the pancreas, signet ring cell carcinomas of the pancreas, hepatoid carcinomas of the pancreas, colloid carcinoma of the pancreas carcinomas of the pancreas, undifferentiated carcinomas of the pancreas, and undifferentiated carcinomas with osteoclast-like giant cellsOsteoclast-like giant cells of the pancreas, acinar cell carcinomas of the pancreas, solid pseudopapillary neoplasms of the pancreas, pancreatoblastoma, rare exocrine cancers of the pancreas, pancreatic serous cystadenomas, pancreatic mucinous cystic neoplasms, papillary cystadenocarcinoma, papillary serous cystadenocarcinoma, mucinous cystadenocarcinoma, mucinous adenocarcinoma, signet ring cell carcinoma, infiltrating ductal carcinoma carcinoma, medullary carcinoma, lobular carcinoma, inflammatory carcinoma, Paget's disease, mammary acinar cell carcinoma, adenosquamous carcinoma, adenocarcinoma with squamous metaplasia, thymoma, malignant, ovarian stromal tumor, malignant thecoma, malignant granulosa cell tumor, malignant androblastoma, malignant sertoli cell carcinoma, Leydig cell tumor, malignant lipid cell tumortumor), malignant paraganglioma, malignant extra-mammary paraganglioma, malignant pheochromocytoma, glomangiosarcoma, malignant melanoma, amelanotic melanoma, superficial spreading melanoma, melanoma in giant pigmented nevus, epithelioid cell melanoma, blue nevus, malignant sarcoma, fibrosarcoma, fibrous histiocytoma, malignant myxosarcoma myxosarcoma, liposarcoma, leiomyosarcoma, rhabdomyosarcoma, embryonal rhabdomyosarcoma, alveolarrhabdomyosarcoma, stromal sarcoma, mixed tumor, malignant mullerian mixed tumor, nephroblastoma, hepatoblastoma, carcinosarcoma, mesenchymoma, malignant Brenner tumor, malignant phyllodes tumor, malignant synovial sarcoma, mesothelioma, malignant dysgerminoma, embryonal carcinoma carcinoma), teratoma, malignant struma ovarii, malignant choriocarcinomachoriocarcinoma, mesonephroma, malignant hemangiosarcoma, hemangioendothelioma, malignant Kaposi's sarcoma, hemangiopericytoma, malignant lymphangiosarcoma, osteosarcoma, juxtacortical osteosarcoma, chondrosarcoma, chondroblastoma, malignant mesenchymal chondrosarcoma, giant cell tumor of bone, Ewing's sarcoma, odontogenic tumor, malignant ameloblastic odontosarcoma odontosarcoma), ameloblastoma, malignant ameloblastic fibrosarcoma, pinealoma, malignant chordoma, glioma, malignant ependymoma, astrocytoma, protoplasmic astrocytoma, fibrillary astrocytoma, astroblastoma, glioblastoma, oligodendroglioma, oligodendroblastoma, primitive neuroectodermal, cerebellar sarcoma sarcoma), ganglioneuroblastoma, neuroblastoma, retinoblastoma, olfactory neurogenic tumorstumor), meningioma, malignant, neurofibrosarcoma, neurilemmoma, malignant granular cell tumor, malignant lymphoma, Hodgkin's disease, Hodgkin's paragranuloma, malignant lymphoma, small lymphocytic malignant lymphoma, large cell diffuse malignant lymphoma, follicular mycosis fungoides, other specified non-Hodgkin's lymphomas, malignant histiocytosis, multiple myeloma, mast cell sarcoma, immunoproliferative small intestinal disease disease), leukemia, lymphoid leukemia, plasma cell leukemia, erythroleukemia, lymphosarcoma cell leukemia, myeloid leukemia, basophilic leukemia, eosinophilic leukemia, monocytic leukemia, mast cell leukemia, megakaryoblastic leukemia, myeloid sarcoma, or hairy cell leukemia.

[0267] In some embodiments, the cancer is a virally induced cancer, e.g., a cancer caused by infection with an oncogenic virus or tumor virus (also referred to as a "cancer virus"). In some embodiments, the cancer virus is a DNA virus. In some embodiments, the cancer virus is an RNA virus.

[0268] In some embodiments, the cancerous or precancerous tumor is associated with or caused by a cancer virus. Non-limiting examples of cancer viruses include: Epstein-Barr virus (EBV), hepatitis B virus, hepatitis C virus, human papillomavirus, human T-lymphotropic virus 1 (HTLV-1), Kaposi's sarcoma-associated herpes virus (KHSV), Merkel cell polyomavirus, or cytomegalovirus.

[0269] In some embodiments, cancerous or precancerous tumors are associated with or caused by cancer viruses that directly induce transformation of infected host cells, thereby modulating host cell growth and survival, or alternatively trigger a DNA damage response, which in turn increases genetic instability and accelerates the acquisition of oncogenic mutations in the host cell genome.

[0270] In some embodiments, the cancerous or precancerous tumor is associated with or caused by a cancer virus that induces chronic inflammation in the host. For example, HBV and HCV infection can induce chronic hepatitis associated with oxidative DNA damage, followed by cirrhosis and, in some cases, the development of hepatocellular carcinoma.

[0271] In some embodiments, cancerous or precancerous tumors are associated with or caused by cancer viruses that are not oncogenic but suppress the host's immune system, subverting immune surveillance, thereby allowing the emergence of mutated malignant cells, such as in HIV-infected patients.

[0272] In some embodiments, treatment comprises administering one or more GAL9 binding molecules as described herein to a subject having an infectious disease (eg, HIV, HCV, HBV, EBV, or HPV infection).

[0273] In some embodiments, treatment comprises administering one or more GAL9 binding molecules as described herein to a subject having HIV or AIDs in an amount effective to treat the subject.

[0274] 6.10.4. Application

[0275] The GAL9 binding molecules can be administered to a subject by any route known in the art. For example, the GAL9 binding molecules can be administered to a human subject by, for example, intravenous, subcutaneous, intramuscular, intradermal, intraarterial, intraperitoneal, intranasal, parenteral, pulmonary, topical, oral, sublingual, intratumoral, peritumoral, intralesional, intrasynovial, intrathecal, intracerebrospinal, or perilesional administration. The GAL9 binding molecules can be administered to a subject by themselves or as a pharmaceutical composition. Exemplary pharmaceutical compositions are described herein.

[0276] 6.11. Examples

[0277] The following examples are provided for illustration and not limitation. In particular, as described in more detail below, the methods for expressing and purifying the various antigen binding proteins and their use in various assays are non-limiting and illustrative.

[0278] 6.11.1. Methods

[0279] 6.11.1.1.Expi293 Expression

[0280] The various antigen-binding proteins tested were expressed using the Expi293 transient transfection system according to the manufacturer's instructions (Thermo Fisher Scientific). Briefly, unless otherwise specified, plasmids encoding each chain were mixed in a 1:1 mass ratio and transfected into Expi 293 cells using the ExpiFectamine 293 transfection kit. Cells were cultured at 37°C, 8% CO2, 100% humidity, and shaking at 125 rpm. Transfected cells were fed once 16-18 hours after transfection. Cells were harvested on day 5 by centrifugation at 2000 g for 10 minutes. The supernatant was collected for affinity chromatography purification.

[0281] 6.11.1.2. ExpiCHO Expression

[0282] Various GAL9 antigen binding proteins were tested and expressed using the ExpiCHO transient transfection system according to the manufacturer's instructions. Briefly, plasmids encoding each chain were mixed at a mass ratio of, for example, 1:1 and transfected into ExpiCHO using the ExpiFectamine CHO transfection kit.

[0283] Cells were cultured at 37°C, 8% CO2, 100% humidity, and shaking at 125 rpm. Transfected cells were typically fed once 16-18 hours after transfection. Cells were harvested on day 5 by centrifugation at 2000 g for 10 minutes. The supernatant was then collected for affinity chromatography purification.

[0284] 6.11.1.3. Protein A purification

[0285] Using protein A (ProtA) resin or anti-CH1 resin, on gravity flow purifier, separation contains the clear supernatant of various antigen-binding proteins.In the embodiment of carrying out head to head comparison, the supernatant containing various antigen-binding proteins is divided into two equal samples.For ProtA purification, 1mL protein A column (GE Healthcare) is balanced with PBS (5mM sodium potassium phosphate pH 7.4, 150mM sodium chloride).Sample is loaded onto the post with 5mL / min.Use 0.1M sodium acetate pH 3.5 elution sample.By the absorbance monitoring elution at 280nm, and merge elution peak for analysis.By the absorbance monitoring elution at 280nm, and merge elution peak for analysis.

[0286] 6.11.1.4. SDS-Page Analysis

[0287] By reducing and non-reducing SDS-PAGE, the presence of complete product, incomplete product and overall purity in the samples containing various separated antigen-binding proteins was analyzed. 2 μg of each sample was added to 15 μL SDS loading buffer. The reduced sample was incubated at 75°C for 10 minutes in the presence of 10mM reducing agent. The non-reduced sample was incubated at 70°C for 5 minutes without a reducing agent. The reduced and non-reduced samples were loaded into a 4%-15% gradient TGX gel (BioRad) containing running buffer and run at 220 volts for 30 minutes. After the run was completed, the gel was washed with deionized (DI) water and stained with GelCode Blue Safe Protein Stain (ThermoFisher). The gel was destained with DI water before analysis. Standard image analysis software was used to perform optical density analysis of the scanned images of the destained gel to calculate the relative abundance of the bands in each sample.

[0288] 6.11.1.5. IEX Chromatography

[0289] By cation exchange chromatography, the ratio of complete product to incomplete product and impurities in the samples containing various separated antigen-binding proteins was analyzed. The clarified supernatant was analyzed with 5mL MonoS (GE Lifesciences) on an AKTA purifier FPLC. The MonoS column was balanced with buffer A (10mM MES pH 6.0). The sample was loaded onto the column at 2mL / min. The sample was eluted over 6 bed volumes (CV) using a 0-30% gradient of buffer B (10mM MES pH 6.0, 1M sodium chloride). The elution was monitored by absorbance at 280nm, and the purity of the sample was calculated by peak integration to identify the abundance of the monomer peak and the contaminant peak. The monomer peak and the contaminant peak were merged respectively and analyzed by SDS-PAGE as described above.

[0290] For analytical SEC chromatography of each sample, 1 mg / ml was loaded onto the column at 1 ml / min. The sample was eluted using an isocratic flow of PBS over 1.5 CV. Elution was monitored by absorbance at 280 nm and the elution peak was analyzed by peak integration.

[0291] 6.11.1.6. Mass spectrometry

[0292] Samples containing various isolated antigen-binding proteins were analyzed by mass spectrometry to confirm the correct species by molecular weight. All analyses were performed by a third-party research organization. Briefly, samples were treated with an enzyme cocktail to remove glycosylation. Samples were tested in a reduced form to specifically identify each chain by molecular weight, and the molecular weights of all complexes in the sample were determined under non-reducing conditions. Mass spectrometry analysis was used to identify the number of unique products based on molecular weight.

[0293] 6.11.1.7. Antibody Discovery by Phage Display

[0294] Phage display of human Fab library was performed using standard protocol. Human GAL9 protein was purchased from Acro Biosystems (human Gal9 His-tag catalog number LG9-H5244) and stained with EZ-Link NHS-PEG using standard protocol. 12 - Biotin (ThermoScientific catalog number 21312) for biotinylation. Phage clones that can bind to GAL9 protein were screened by phage ELISA using standard protocols.

[0295] In brief, an expression vector capable of replication and expression in a phage (also referred to as a phagemid) is used to construct a phage library in the form of Fabs. The heavy and light chains are encoded in the same expression vector, wherein the heavy chain is fused to a truncated variant of the phage coat protein pill. The light chain and heavy chain-pIII fusions are expressed as separate polypeptides and assembled in the bacterial periplasm, where the redox potential allows the formation of disulfide bonds to form phage-displayed antibodies containing candidate ABS.

[0296] The library was formed using sequences derived from a specific human heavy chain variable domain (VH3-23) and a specific human light chain variable domain (Vk-1). For screening libraries, all three CDRs of the VH domain were diversified to match the positional amino acid frequency of the CDR lengths found in the human antibody repertoire. The light chain variable domain within the screening library was generated in which diversity was introduced only into the VL CDR3 (L3); the light chain VL CDR1 (L1) and CDR2 (L2) retained human germline sequences.

[0297] The heavy chain scaffold (SEQ ID NO: 2), light chain scaffold (SEQ ID NO: 4), full-length heavy chain Fab polypeptide (SEQ ID NO: 1), and full-length light chain Fab polypeptide (SEQ ID NO: 3) used in the phage display library are shown below, where lowercase "x" represents the CDR amino acid that was changed to form the library.

[0298] Phage display VH scaffold [SEQ ID NO:2]:

[0299] EVQLVESGGGLVQPGGSLRLSCAASGFTFxxxxIHWVRQAPGKGLEWVAxxxxxxxxxxxYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARxxxxxxxxxxxxxDYWGQGTLVTVSSAS

[0300] Phage display VL scaffold [SEQ ID NO:4]:

[0301] DIQMTQSPSSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYSASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQxxxxxxTFGQGTKVEIKRT

[0302] Phage displayed heavy chain Fab polypeptide [SEQ ID NO: 1]:

[0303] EVQLVESGGGLVQPGGSLRLSCAASGFTFxxxxIHWVRQAPGKGLEWVAxxxxxxxxxxxYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARxxxxxxxxxxxxxDYWGQ GTLVTVSSASTKGPSVFPLAPSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC

[0304] Phage displayed light chain Fab polypeptide [SEQ ID NO:3]:

[0305] DIQMTQSPSSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYSASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQxxxxxxTFGQGTKVEIK RTVAAPSVFIFPPSDSQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0306] Diversity was generated by Kunkel mutagenesis, where primers were used to introduce diversity into the VH CDR1 (H1), VH CDR2 (H2), VH CDR3 (H3), and VL CDR3 (L3) to mimic the diversity found in natural antibody repertoires, as described in more detail in Kunkel, TA (PNAS January 1, 1985, 82(2)488-492), the entire contents of which are incorporated herein by reference. Briefly, single-stranded DNA was prepared from isolated phage using standard procedures and subjected to Kunkel mutagenesis. The chemically synthesized DNA was then electroporated into MC1061F- cells. Phagemids obtained from overnight cultures were digested with restriction enzymes (Bam HI and Xba I) to remove wild-type sequences. The digested samples were electroporated into TG1 cells and then recovered. The recovered cells were subcultured and infected with M13K07 helper phage to generate a phage library.

[0307] Phage panning was performed using standard procedures. Briefly, the first round of phage panning was performed with targets immobilized on streptavidin magnetic beads with ~5 × 10 12 Each phage was treated in 1 mL of PBST-2% BSA. After one hour of incubation, the bead-bound phage was separated from the supernatant using a magnetic stand. The beads were washed three times to remove nonspecifically bound phage, and then the OD 600 After 20 minutes, the infected cells were inoculated in 25 mL of 2xYT+ampicillin and M13K07 helper phage (final concentration, 10 10pfu / ml) and grown overnight at 37°C with vigorous shaking. The next day, phage were prepared by PEG precipitation using standard procedures. Prior to panning, phage specific for SAV-coated beads were pre-cleared. A second round of panning was performed using a KingFisher magnetic bead processor using standard procedures with 100 nM bead-immobilized antigen. A total of 3-4 rounds of phage panning were performed to enrich for phage displaying Fabs specific for the target antigen. Target-specific enrichment was confirmed using polyclonal and monoclonal phage ELISAs. DNA sequencing was used to identify isolated Fab clones containing candidate Fabs.

[0308] The VL and VH domains identified in the above phage screening were reformed into a bivalent monospecific native human full-length IgG1 structure.

[0309] Native human full-length IgG1 heavy chain structure [SEQ ID NO: 5]:

[0310] EVQLVESGGGLVQPGGSLRLSCAASGFTFxxxxIHWVRQAPGKGLEWVAxxxxxxxxxxxYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARxxxxxxxxxxxxxDY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK[SEQ ID NO:5]

[0311] Natural human full-length IgG1 light chain structure:

[0312] Equivalent to phage displayed light chain Fab, see [SEQ ID NO: 3].

[0313] 6.11.1.8. Octet determination of binding kinetics

[0314] To measure the qualitative binding affinity of the discovered GAL9 binders, the reconstituted IgG1 form of the binders was immobilized on the biosensor of an Octet (Pall ForteBio) biolayer interferometer.

[0315] Soluble GAL9 antigen was then added to the system and binding was measured. Qualitative binding affinity was assessed by visualizing the slope of the dissociation phase of the octet sensorgram from weakest (+) to strongest (+++). A slow off-rate, indicated by a small drop in the dissociation phase of the sensorgram, indicated a tightly bound antibody (+++). To obtain accurate kinetic constants for monovalent affinity, a dilution series involving at least five concentrations of GAL9 analyte (ranging from approximately 10 to 20 × K D to 0.1×K D (value, 2-fold dilution). In the dissociation step, the sensor was immersed in a buffer solution without GAL9 analyte to dissociate the bound complex on the sensor surface. Kinetic and equilibrium binding constants were calculated based on the association and dissociation rate curves using Octet kinetic analysis software. A global analysis (global fitting) was performed in which kinetic constants were obtained simultaneously for all analyte concentrations included in the experiment.

[0316] 6.11.1.9. Epitope Binning

[0317] Anti-GAL9 candidates formatted as bivalent monospecific native human full-length IgG1 as described above were tested for GAL9 binding in a paired fashion using an Octet-based "tandem" assay. Briefly, biotinylated GAL9 was immobilized on a streptavidin sensor and two anti-GAL9 candidates were bound in tandem. Competitive blocking profiles were generated to determine whether a given anti-GAL9 candidate blocked binding of a range of other anti-GAL9 candidates to GAL9. Anti-GAL9 candidates that competed for the same or non-overlapping binding regions were grouped together and referred to as belonging to the same bin.

[0318] 6.11.1.10. PBMC Activation and Galectin-9 Antibody Treatment

[0319] Aliquots of PepMix HCMVA (pp65) (>90%) Protein ID: P06725 (Cat. No. PM-PP 65-2, JPT Peptide Technologies) were prepared according to the manufacturer's instructions. TMHCMVA (pp65) is a mixture of overlapping 15mer peptides spanning the entire protein of the 65 kDa phosphorylated protein (pp65) of human cytomegalovirus (HHV-5) (Swiss-Prot ID: P06725). Aliquots of PepMix were used for immunostimulation of PBMCs to assess immune cell responses.

[0320] Frozen human peripheral blood mononuclear cells were thawed and resuspended in growth medium (10% FBS in RPMI) according to standard conditions.

[0321] The resuspended PBMCs were plated at 5×10 5 Cells were seeded in 96-well plates. Cells were mixed with 2 μg / mL PepMix TM HCMVA (pp65) was incubated with 40 μg / mL of candidate GAL9 antibodies or control antibodies in growth medium at 37° C. and 5% CO 2 for 24 hours.

[0322] 6.11.1.11. LEGENDplex Human Th Cytokine Assay

[0323] Following activation of PBMCs by PepMix HCMVA (pp65) and treatment with galectin-9 antibodies, cytokine secretion by PBMCs and specific immune cell subsets was assessed by cytokine bead array at 24 and 72 hours.

[0324] Collect 200 μl of cell culture supernatant and centrifuge to pellet cell debris. TM Human Th1 group (5-plex) (Cat. No. 740009, Biolegend), the resulting supernatant was analyzed. TM The human Th1 panel is a bead-based assay to allow for the simultaneous quantification of the human cytokines IL-2, IL-6, IL-10, IFN-γ, and TNF-α using flow cytometry.

[0325] Briefly, cytokine standards and capture bead mixtures were prepared according to the manufacturer's instructions.A 1:1:1 assay master mix of capture bead mixture: biotinylated detection antibody and assay buffer was prepared.

[0326] 12.5 μl supernatant samples or cytokine standards were incubated with 37.5 μl of the assay master mix. The plates were sealed, covered with foil, and shaken at 600 rpm for 2 hours at room temperature. The wells were then incubated with streptavidin-phycoerythrin (SA-PE) at room temperature for 30 minutes at 600 rpm. The beads were then washed twice and resuspended, and flow cytometry analysis was performed according to the manufacturer's instructions.

[0327] 6.11.1.12. Staining PBMCs with marker antibodies

[0328] After PBMC activation and galectin-9 antibody treatment as described above, PBMC immune cells were stained with marker antibodies according to the following procedure.

[0329] The cells were plated at 5 × 10 6 Resuspend cells in growth medium (10% FBS in RPMI) at 100 cells / mL. Aliquot 200 μL of the resuspended cells into 96-well plates and then incubate with a fixable viability dye. Incubate at 2-8° C. for 30 minutes to irreversibly label dead cells. Cells are then washed and incubated with human Fc blocking solution (Cat. No. 14-9161-73, eBiosciences) at room temperature for 10 minutes.

[0330] Prepare the antibody cocktail working solution according to the table below.

[0331]

[0332]

[0333] The wells were incubated with 10 μL of the diluted antibody mixture for 30 minutes at 2-8° C. The cells were then washed and resuspended and analyzed by flow cytometry.

[0334] For analysis of the immunostimulatory markers CD 27, CD40L, ICOS, 4-1BB, and OX 40, the same protocol provided above was followed, but cells were incubated with an alternative antibody cocktail as detailed in Table 2 below:

[0335]

[0336]

[0337] 6.11.2. Example 1: GAL9 binding arm discovery

[0338] As described above, a chemically synthesized Fab phage library with diversity introduced in the Fab CDRs was screened against the GAL9 antigen using a monoclonal phage ELISA format. Phage clones expressing Fabs that recognized GAL9 were sequenced.

[0339] This screen initially identified 52 GAL9 binding candidates (antigen-binding site clones). After reformatting the variable regions of these clones into a bivalent monospecific human IgG1 format, functional assays identified 22 antibodies with immunostimulatory properties.

[0340] Table 3 lists the VH CDR1 / 2 / 3 sequences from 22 activating ABS clones, showing only the residues of the CDRs that have been varied in constructing the library.

[0341] Table 4 lists the VL CDR1 / 2 / 3 sequences from the identified ABS clones; the light chain CDR1 and CDR2 sequences are invariant, and only the CDR3 residues that were varied in constructing the library are shown.

[0342]

[0343]

[0344]

[0345]

[0346] Table 5 presents the complete CDR sequences of 22 candidate anti-GAL9 immune-activating antibodies according to various definitions recognized in the art.

[0347]

[0348]

[0349]

[0350]

[0351]

[0352]

[0353]

[0354]

[0355]

[0356]

[0357]

[0358]

[0359]

[0360]

[0361]

[0362]

[0363]

[0364]

[0365]

[0366] Table 6 presents the complete immunoglobulin heavy chain and complete immunoglobulin light chain sequences, as well as the VH and VL sequences of various ABS candidates formatted into a bivalent monospecific human full-length IgG1 structure.

[0367]

[0368]

[0369]

[0370]

[0371]

[0372]

[0373]

[0374]

[0375]

[0376]

[0377]

[0378]

[0379]

[0380]

[0381]

[0382]

[0383]

[0384]

[0385]

[0386]

[0387]

[0388]

[0389]

[0390] Selected GAL9 binding candidates were analyzed for their binding properties: cross-reactive binding to murine GAL9, qualitative binding, epitope binning (bin 2 - candidates were binned with commercial antibody clone ECA8 from LS Bio [LS-C179448]; bin 3 - candidates were binned with commercial antibody clone ECA42 from LS Bio [LS-C179449], which is the "tool antibody" described in Figure 3), and monovalent affinity binding. The results of the analysis are shown in Table 7.

[0391]

[0392] Selected GAL9 binding candidates were further analyzed for sequence motifs that could adversely affect antibody properties relevant to clinical development, such as stability, mutagenesis, and immunogenicity. Computational analysis was performed according to Kumar and Singh (Developability of biotherapeutics: Computational approaches. Boca Raton: CRC Press, Taylor & Francis Group, 2016). The results of this analysis are presented in Table 8, demonstrating the presence of a limited number of unfavorable sequence motifs in the listed clones, indicating potential for further clinical development.

[0393]

[0394] 6.11.3. Example 2: Treatment with anti-GAL9 candidates increases cytokine production by human PBMCs

[0395] Candidate GAL9 ABS were formatted as bivalent monospecific native human full-length IgG1 heavy and light chain structures (SEQ ID NO: 5 and SEQ ID NO: 3, respectively) and tested for their effects on cytokine production by PBMCs following peptide stimulation. PBMCs were stimulated essentially as described above in Section 6.11.1. Briefly, PBMCs were harvested from human donors known to respond to human CMV virus (HCMV), placed in culture medium, stimulated with HCMV PepMix to elicit an antigen-specific response, and treated with one of the following: control IgG, comparator activating mAb (clone ECA42), α-PD1 (nivolumab), or a candidate anti-GAL9 antibody. Cytokine secretion was measured 24 and 72 hr after treatment by bead cytokine array. The results for IFN-γ and TNF-α are depicted in Figure 6. Figure 3A and 3B middle. Figures 3A-3B The data shown in are described in more detail in Tables 9 and 10 provided below.

[0396]

[0397]

[0398] It is noteworthy that PBMCs treated with candidates P9-15, P9-18, P9-21, and P9-28 exhibited increased IFN-γ and TNF-α secretion after stimulation relative to IgG controls and GAL9 comparison tool antibodies (clone ECA42). In addition, PBMCs treated with candidates P9-15, P9-18, P9-21, and P9-28 also significantly exhibited increased TNF-α production after stimulation relative to treatment with commercial α-PD1 antibodies. Thus, treatment of PBMCs with selected anti-GAL9 candidates can enhance cytokine secretion following peptide stimulation. Treatment with P9-54 resulted in a neutral response with no significant differences in TNF-α and IFN-γ secretion (data not shown).

[0399] 6.11.4. Example 3: Treatment with anti-GAL9 candidates increases TNF-α production by natural killer (NK) cells

[0400] The candidate GAL9 ABS were formatted as bivalent monospecific natural full-length human IgG1 heavy and light chain structures (SEQ ID NO: 5 and SEQ ID NO: 3, respectively) and tested for their effects on TNF-α secretion by NK cells (lineage, CD56+) after 72 hours of peptide stimulation. NK cells were treated with control antibody clone 55, GAL9 antibody candidate P9-15 (clone 15), or GAL9 antibody candidate P9-18 (clone 18) at a dose of 5 μg or 20 μg. After treatment, the TNF-α secretion levels of the cells were assessed by flow cytometry. NK cells (CD56+) secreting TNF-α + Representative data of the percentage of ) are presented in Figure 6.

[0401] Relative to clone P9-55 (negative control), treatment with GAL9 antibody candidate P9-18 or candidate P9-15 increased the percentage of NK cells that stained positive for TNF-α after stimulation. In the NK cell population treated with 5μg control antibody, 7.75% of the NK cells (CD56+) were TNF-α positive. In contrast, in the NK cell population treated with 5μg P9-18, 12.0% of the NK cells were TNF-α positive. And in the NK cells treated with 5μg P9-15, 22.5% of the NK cells were TNF-α positive. See Figure 6.

[0402] In the NK cell population treated with 20 μg of control antibody, 10.3% of the NK cells (CD56 + ) were TNF-α positive. In contrast, in the NK cell population treated with 20 μg of P9-18, 16.9% of the NK cells were TNF-α positive. And in the NK cells treated with 20 μg of P9-15, 28.5% of the NK cells were TNF-α positive. See Figure 6.

[0403] Thus, treatment with selected anti-GAL9 candidates was able to increase TNF-α production by NK cells following stimulation (see Figure 6).

[0404] 6.11.5. Example 4: Treatment with anti-GAL9 candidates increases IL-12 production in dendritic cells

[0405] Candidate GAL9 ABS were formatted as bivalent monospecific natural full-length human IgG1 heavy and light chain constructs (SEQ ID NO: 5 and SEQ ID NO: 3, respectively) and tested for their ability to express peptide-stimulated dendritic cells (lineage-negative, class II + , CD11c +PBMCs including dendritic cell (DC) populations were treated as described in Example 2 and IL-12 secretion levels were assessed using the IL-12 Secretion Assay-Detection Kit (PE) Human (Cat. No. 130-092-124, Miltenyi Biotec) according to the manufacturer's protocol. Representative data on the percentage of DCs secreting IL-12 are presented in Figure 5 middle.

[0406] Notably, treatment with the GAL9 antibody candidate, P9-18, increased the percentage of DCs that stained positive for IL-12 after stimulation relative to the IgG control. In the DC population treated with control IgG, 0.26% of these DCs were IL-12 positive. In contrast, in the DC population treated with P9-18, 7.74% of these DCs were IL-12 positive, a 28-fold increase in IL-12-positive DCs relative to the IgG control-treated population. Therefore, treatment of PBMCs with selected anti-GAL9 candidates was able to increase IL-12 production by DCs after stimulation.

[0407] 6.11.6. Example 5: Treatment with anti-GAL9 candidates increases surface expression of co-stimulatory molecules on CD8+ T cells

[0408] Candidate GAL9 ABS formatted as bivalent monospecific native full-length human IgG1 heavy and light chain constructs (SEQ ID NO: 5 and SEQ ID NO: 3, respectively) were tested for the expression of peptide-stimulated CD8 - Effect of T cell immunostimulatory surface marker expression. Treatment including CD8 + PBMCs of T cell populations were stained with marker antibodies as described herein and then harvested for flow cytometry. + The levels of immunostimulatory surface markers CD27, CD40L, ICOS, 4-1BB, and OX40 were assessed on T cells. Figure 4 "% value" indicates CD8 with detectable levels of the relevant markers + % of T cells Figure 4 showed that treatment with αGAL9 antibody candidates P9-15, P9-18, P9-21, and P9-28 increased CD8 + Immunostimulatory surface markers CD27, CD40L, ICOS, 4-1BB, and OX40 in T cells.

[0409] Immunostimulatory surface marker staining positive for CD8 + Representative data on the percentage of T cells are listed in Table 11 below.

[0410]

[0411] Notably, PBMCs treated with candidate P9-18 or P9-21 exhibited positive staining for various immunostimulatory surface markers of CD8 T cells upon stimulation, compared to IgG control, GAL9 comparator antibody (clone ECA42), and α-PD1. + Increased percentage of T cells, including CD8 T cells positive for CD40L and OX40 + The percentage of T cells increased more than 2-fold. Therefore, treatment of PBMCs with selected anti-GAL9 candidates was able to increase the number of CD8 + Expression of immunostimulatory surface markers on T cells. Similar immunostimulatory responses were observed with low-responder PBMC cells (donor 5) (data not shown).

[0412] 6.11.7. Example 6: Treatment with anti-GAL9 candidates alters PD-L1 and PD-L2 cell surface expression on dendritic cells (DCs)

[0413] Candidate GAL9 ABS were formatted as bivalent monospecific native full-length human IgG1 heavy and light chain constructs (SEQ ID NO: 5 and SEQ ID NO: 3, respectively) and tested for their effects on dendritic cells (lineage negative, class II, CD11c + The effect of PD-L1 and PD-L2 cell surface expression on PBMCs (cells) was investigated. PBMCs including a dendritic cell (DC) population were processed as described in Example 2 and then harvested for flow cytometry, and the levels of PD-L1 and PD-L2 were assessed on DCs. Representative data for the percentage of DCs that stained positive for PD-L1 and PD-L2 and the geometric mean fluorescence intensity (GMI) are presented in Table 12 below.

[0414]

[0415] Notably, PBMCs treated with candidate P9-18 showed an increase in the percentage of DCs that stained positive for PD-L2 after stimulation relative to the IgG control and the GAL9 comparison tool antibody (ECA42). Both P9-18 and P9-21 showed a decrease in the percentage of PD-L1 on DCs and a decrease in the geometric mean fluorescence (GMI) of PD-L1. Thus, treatment of PBMCs with selected anti-GAL9 candidates was able to alter the surface expression of PD-L1 and PD-L2 on DCs after stimulation.

[0416] 6.11.8. Example 7: Treatment with anti-GAL9 candidates results in clustering of GAL9 and PD-L2 on the cell surface of dendritic cells

[0417] Candidate GAL9 ABS were formatted as bivalent monospecific native human full-length IgG1 heavy and light chain constructs (SEQ ID NO: 5 and SEQ ID NO: 3, respectively) and tested for their effects on GAL9, PD-L1, and PD-L2 clustering on the dendritic cell ("DC") cell surface.

[0418] PBMCs including the dendritic cell (DC) population were processed as described in Example 2 and then fixed for confocal imaging analysis to assess the distribution of GAL9, CD11c, and PD-L2 on DCs.

[0419] Results / Conclusions

[0420] The IgG control ( Figure 8A )、P9-18( Figure 8B ) and P9-21( Figure 8C Confocal images of dendritic cells treated with ). Blue staining shows DNA (DAPI), red staining shows PD-L2, green staining shows CD11c, and yellow staining shows GAL9. Unlabeled images are bright field; images are presented in grayscale in the accompanying figures.

[0421] Treatment with candidate P9-18 or P9-21 showed colocalization and accumulation of GAL9 and PD-L2 on DCs compared to IgG control ( Figures 8B-8C ). Therefore, after stimulation, treatment with P9-18 or P9-21 can induce the colocalization and aggregation of GAL9 and PD-L2 on the cell surface of DCs.

[0422] 6.11.9. Example 8: Treatment with anti-GAL9 P9-18 preserves PD-L2 and PD-L1 expression on tumor cells

[0423] The effect of the anti-GAL9 candidate P9-18 on the cellular retention and distribution of PD-L2 and PD-L1 in tumor cells was tested.

[0424] Antibody

[0425] Candidate GAL9 ABS were formatted as bivalent monospecific native human full-length IgG1 heavy and light chain structures (SEQ ID NO: 5 and SEQ ID NO: 3, respectively). Anti-PD-L2 clone TY25 and anti-PD-L1 clone 10F.9G2 were obtained from BioXcell (Lebanon, NH).

[0426] Cell culture and immunostaining

[0427] CT26 tumor cells were cultured and treated with the anti-GAL9 candidate P9-18 or IgG control. Cells were fixed and stained with DAPI, anti-PD-L2, and anti-PD-L1 for confocal imaging analysis.

[0428] Results / Conclusions

[0429] Figure 9A and 9B Representative confocal images of CT26 tumor cells after treatment with P9-18 or IgG control are shown. Blue staining shows DNA (DAPI), red shows PD-L2, and green shows PD-L1; presented in grayscale in the accompanying figures. The images show that PD-L2 and PD-L1 remain on the surface of CT26 tumor cells after treatment with P9-18 compared to the IgG control. See Figure 9A and 9B . Figure 9B The spots in the figure highlight the increased PD-L2 and PD-L1 protein expression.

[0430] 6.11.10. Example 9: Treatment with anti-GAL9 P9-18 or P9-21 inhibits tumor growth in colon and melanoma tumor models

[0431] This study was conducted to determine whether the anti-GAL9 candidates P9-18 and P9-21 could inhibit tumor growth in colon and melanoma tumor models.

[0432] Antibody

[0433] Candidate GAL9 ABS were formatted as a bivalent monospecific format on a mouse IgG2a backbone.

[0434] Animals and treatment

[0435] BALB / c mice were subcutaneously implanted with the CT26 tumor line and treated with the anti-GAL9 candidates P9-18, P9-21, or IgG control. Treatment was given intraperitoneally (IP) at 200 μg on days 7, 11, 15, and 19, with 10 mice per treatment group. Tumor growth was assessed by measuring tumor volume. If tumors reached ~1000 mm 3 If the volume reaches , the mice were euthanized.

[0436] C57BL / 6 mice were implanted intradermally with the B16.F0 tumor line and treated with anti-GAL9 candidates P9-18, P9-21, or IgG control. Treatments were administered IP at 200 μg on days 3, 7, 11, and 15, with 10 mice per treatment group.

[0437] Results / Conclusions

[0438] Mice treated with P9-18 or P9-21 showed complete regression of CT26 tumors, whereas mice treated with IgG control showed continued tumor growth. Figure 1 Mice treated with P9-18 or P9-21 exhibited reduced B16.F0 tumor growth compared to mice treated with IgG control. Figure 2 Thus, P9-18 or P9-21 can inhibit tumor growth in colon and melanoma tumor models, including complete tumor regression in some cases.

[0439] 6.11.11. Example 10: Treatment with anti-GAL9 P9-15 results in fewer Epstein-Barr virus (EBV)-induced tumors and reduces viral load

[0440] This study was performed to determine the effects of the anti-GAL9 P9-15 candidate on Epsetin-Barr virus (EBV)-induced tumors in a humanized mouse model.

[0441] Epstein-Barr virus (EBV) is a gamma-herpes virus that infects human B cells. However, many human viruses do not infect mice. Therefore, to test the effect of anti-GAL9 P9-15 on EBV-induced tumors, we used human CD34 + Humanized mice with hematopoietic stem cells are used to generate mouse models reconstituted with human immune system cells.

[0442] Infection and treatment of humanized mice

[0443] Figure 10A A schematic diagram of the overall treatment regimen used in the study is shown. Briefly, immunodeficient mice were injected intravenously with CD34 + Humanized mice were then infected with EBV and incubated for three weeks to allow infection to develop. At the end of the infection period, mice were treated with two doses of anti-GAL9 P9-15 or IgG control on days 22 and 26. Ten days after treatment, live mice were euthanized and analyzed.

[0444] Generation of humanized NRG mice (hu-NRG)

[0445] Five female NRG (NOD-Rag1 null IL2rg null , NOD ragγ). Rag1 null The mutation results in mice lacking B and T cells and expressing IL2rg nullThe mutation blocks cytokine signaling through multiple receptors, resulting in a lack of functional NK cells. As a result, NRG mice are extremely immunodeficient, allowing engraftment of human CD34 + Hematopoietic stem cells.

[0446] Mice were irradiated twice with 275 cGy per dose (550 cGy in total) at 3-4 h intervals and injected intravenously with 5 × 10 4 CD34 + Human stem cells were then allowed to engraft for three weeks to generate humanized NRG ("hu-NRG") mice. The hu-NRG mice were weighed every two weeks for 12 weeks to assess their health. + Tail bleeding was performed at 4, 8, and 12 weeks after stem cell induction, and human CD45 was detected by flow cytometry analysis. + cells, including total monocytes (CD45 + ), T cells (CD3 + ) and B cells (CD19 + ), to monitor and confirm stable engraftment in mice.

[0447] Splenic tumors

[0448] Except in cases where mice died or were euthanized for ethical reasons, spleens were removed after euthanasia and examined to determine the number, cellularity, and weight of macroscopic tumors.

[0449] Assessment of EBV viral load

[0450] EBV load in spleen and blood was measured using real-time PCR.

[0451] Statistical analysis

[0452] Two-tailed Mann-Whitney U test was performed using GraphPad Prism 7 software (San Diego, CA).

[0453] Results / Conclusions

[0454] Spleens from mice treated with anti-GAL9 P9-15 showed fewer macroscopic tumors compared to spleens from mice treated with IgG control. Figure 10B Compared with IgG control-treated mice (mean 0.224 g / spleen, p-value < 0.0079), P9-15-treated mice had lighter spleen weight (mean 0.100 g / spleen) and significantly fewer splenocytes (22.14 × 10 6 10, compared with 51.04 × 10 in the IgG-treated control group. 6p-value < 0.0159). Figures 10C-10D Data are shown as mean values; error bars are ± SEM.

[0455] Furthermore, treatment with P9-15 suppressed viral load by 88%. P9-15-treated mice had an average viral load of 0.32 × 10 6 copies / μg of EBV, while IgG-treated mice had an average of 2×10 6 copies / μg of EBV (p value < 0.0079). Figure 10E Data are shown as mean values; error bars are ± SEM. These results suggest that treatment with P9-15 can reduce EBV-induced tumor development and control viral load.

[0456] 6.11.12. Example 11: Treatment with anti-GAL9 P9-28 results in fewer Epstein-Barr virus (EBV)-induced tumors

[0457] This study was conducted to determine the effects of the GAL9 P9-28 candidate on Epstein-Barr virus (EBV)-induced tumors in a humanized mouse model.

[0458] Animals, infection and treatment of humanized mice

[0459] This study was performed as described in Example 10 above.

[0460] Results / Conclusions

[0461] Anti-GAL9 P9-28 treated mice showed no macroscopic tumors in the spleen compared to IgG controls. Figure 11 Anti-GAL9 P9-23 treated mice were less likely to harbor tumors in the spleen, as inferred from their small spleen size and low cell number. These results suggest that treatment with P9-28 can reduce EBV-induced tumor development.

[0462] 6.11.13. Example 12: Anti-GAL9-silenced Fc P9-18 (sFcP9-18) has anti-tumor effects; sFcP9-18 and P9-18 can establish anti-tumor immune memory

[0463] This study was conducted to test the contribution of the Fc region to the antitumor effects of an immune-activating anti-GAL9 antibody. In addition, a rechallenge study was performed to determine whether P9-18 or sFcP9-18 could establish antitumor immune memory.

[0464] Antibody

[0465] The P9-18 antigen-binding site was formatted on a murine IgG1 backbone, a murine IgG2a backbone, or a murine IgG2a backbone with a mutation that nullifies Fc receptor binding (sFc). Silent Fc (sFc) P9-18 antibodies were generated by performing key point mutations that abolish Fc binding to Fc receptors.

[0466] CT26 cells

[0467] CT26 tumor cells were cultured in RPMI medium in a humidified incubator at 37°C in an atmosphere of 5% CO2 and 95% air.

[0468] Mice and treatment protocols

[0469] 7-10 mice were implanted subcutaneously with 1×10 5 CT 26 tumor cells were then treated with 200 μg IP of control IgG (mouse IgG2a), P9-18-IgG1 (murine IgG1 backbone), FcR-silenced sFcP9-18 (murine IgG2a backbone with Fc receptor binding null mutations), or P9-18 (murine IgG2a backbone) on days 7, 11, 15, and 19.

[0470] Tumor volume growth

[0471] Mice were monitored for up to 143 days, and tumors were measured with calipers every 1–3 days. Tumor volume (mm) was calculated according to the following formula: 3 ):tumor length × tumor width × 2 / 2.

[0472] Complete resolution reaction (CR)

[0473] Complete regression in this study was defined as a tumor volume of 0 mm2 for 20 consecutive measurements during the study period. 3 Animals were scored for complete resolution (CR) events every 1-3 days during the study.

[0474] Rechallenge of CT26 tumors

[0475] Following tumor clearance, tumor-free mice that survived the initial tumor clearance study were allowed to rest for 65–70 days. On day 107, animals were re-implanted with 1 × 10 5 CT26 tumor cells were added without other treatment. New control mice were given an IgG2a control treatment on day 113. Tumors were then allowed to grow for an additional 36 days. Tumor volume was determined as described above for days 107-143.

[0476] Results / Conclusions

[0477] The results of the tumor growth study showed that Figure 12 A. After administration of IgG (IgG2a) control antibody In mice treated with WT mice, tumors reached 900-1000 mm in size during the first 50 days. 3 In contrast, treatment with P9-18-IgG2a There was a 77% (7 / 9) CR, and treatment with sFcP9-18-IgG2a There was a 70% (7 / 10) CR.These results demonstrate that the Fc region of the P9-18 antibody is not required for anti-tumor effects.

[0478] P9-18 ABS reformatted in IgG1 backbone Tumor growth was not inhibited and showed similar tumor growth to the control.

[0479] The results of the rechallenge study were shown in Figure 12 In B, mice initially treated with P9-18-IgG2a without other treatment had 100% (7 / 7) CR to new tumors. Similarly, mice initially treated with sFcP9-18-IgG2a without other treatment had 100% (7 / 7) CR to new tumors. The results showed that the tumor growth was similar to that in the initial tumor clearance study. These data indicate that mice treated with P9-18 or sFc9-18 had established antitumor immune memory against CT26 tumor cells after initial treatment with P9-18-IgG2a or sFc9-18-IgG2a.

[0480] 6.11.14. Example 13: Treatment with anti-GAL9 P9-18 increases PD-L2 expression on tumor-associated dendritic cells and tumor cells

[0481] The effect of anti-GAL9 P9-18 on the cell surface expression of PD-L1 and PD-L2 on tumor-associated dendritic cells and tumor cells was tested.

[0482] Animals and treatment

[0483] 3-5 BALB / c mice were implanted subcutaneously with CT26 tumor cells and treated with P9-18 ABS formatted on a mouse IgG2a backbone or with a mouse IgG2a control. All treatments were administered at 200 μg (IP) on days 7 and 11.

[0484] Flow cytometry

[0485] On day 13, the tumors were dissected, digested, and isolated. CD45.1 was then isolated using anti-CD45.1 magnetic beads (Miltenyi Biotec, Germany). + A cell population that includes immune cells and tumor cells. Marked by CD45.1+ Cell populations were analyzed by flow cytometry for PD-L1 and PD-L2 cell surface expression on tumor-associated dendritic cells (CD11c+) and tumor cells. The reagents used are shown in Table 13 below.

[0486]

[0487]

[0488] *Labeled using the Lightning-Link Rapid DyLight 488 Labeling Kit.

[0489] Statistical analysis

[0490] Unpaired t-tests with Welch's correction were performed using GraphPad Prism 7 software (San Diego, CA).

[0491] Results / Conclusions

[0492] Figure 13 It was shown that after treatment with P9-18 (murine IgG2a backbone) or control, PD-L1 + or PD-L2 + Tumor-associated dendritic cells (CD11c + ) and the average percentage of PD-L1 or PD-L2 on tumor-associated dendritic cells (CD11c + Treatment with P9-18 significantly increased the mean cell surface expression level (GMI) of PDL2. + The percentage of tumor-associated dendritic cells. The expression of PD-L1 and PD-L2 on tumor-associated dendritic cells (GMI) was also significantly increased compared with controls. Figure 13 Data are shown as mean values; error bars are ± SEM.

[0493] Figure 14 Shown that after treatment with P9-18 (murine IgG2a backbone) or IgG control, PD-L1 + or PD-L2 + The mean percentage of tumor cells and the mean cell surface expression level (GMI) of PD-L1 or PD-L2 on tumor cells. Treatment with P9-18 significantly increased the amount of PD-L2 cell surface expression (GMI) on tumor cells, but did not increase PD-L1 cell surface expression. Figure 14 Data are shown as mean values; error bars are ± SEM. Without wishing to be bound by any theory, we speculate that PD-L2 + Tumor cells can inhibit PD-L1 from binding to PD-1 on the tumor.

[0494] 7. Equivalent Plan

[0495] Although various specific embodiments have been illustrated and described, the above is not restrictive. It should be understood that various changes can be made without departing from the spirit and scope of the present invention. After reading this specification, many changes will become apparent to those skilled in the art.

Claims

1. A galectin-9 (GAL9) antigen-binding molecule, comprising: a first antigen-binding site (ABS) that is specific for a first epitope of a first GAL9 antigen, wherein the first antigen-binding site comprises all three VH CDR sequences and all three VL CDR sequences from Ab clone P9-18, wherein: - According to Chothia, the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 are respectively composed of SEQ ID NOs: 559, 564, 569, 574, 579, and 584; - According to AbM, the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 are respectively composed of SEQ ID NOs: 560, 565, 570, 575, 580, and 585; - According to Kabat, the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 are respectively composed of SEQ ID NOs: 561, 566, 571, 576, 581, and 586; - According to Contact, the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 are respectively composed of SEQ ID NOs: 562, 567, 572, 577, 582, and 587; or - According to IMGT, the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 are respectively composed of SEQ ID NOs: 563, 568, 573, 578, 583, and 588.

2. The GAL9 antigen-binding molecule of claim 1, comprising a first antigen-binding site (ABS) that is specific for a first epitope of a first GAL9 antigen, wherein the first antigen-binding site comprises: a VL sequence shown in SEQ ID NO: 1064 and a VH sequence shown in SEQ ID NO: 1063.

3. The GAL9 antigen-binding molecule of claim 2, wherein the first antigen-binding site (ABS) further comprises a first IgG heavy chain polypeptide and a first IgG light chain polypeptide.

4. The GAL9 antigen-binding molecule of claim 1, wherein the GAL9 antigen is a human GAL9 antigen.

5. The GAL9 antigen-binding molecule of any one of claims 1-4, wherein the GAL9 antigen-binding molecule further comprises a second antigen-binding site (ABS).

6. The GAL9 antigen-binding molecule of claim 5, wherein the second ABS is specific for the GAL9 antigen.

7. The GAL9 antigen-binding molecule of claim 5, wherein the second ABS is specific for a second epitope of the first GAL9 antigen.

8. The GAL9 antigen-binding molecule of claim 5, wherein the second ABS is specific for the first epitope of the first GAL9 antigen and is identical to the first ABS.

9. The GAL9 antigen-binding molecule of claim 6, wherein the second ABS comprises all three VH CDRs and all three VL CDRs from another Ab clone selected from P9-02B, P9-04, P9-05, P9-08, P9-09, P9-10, P9-15, P9-16, P9-18, P9-19, P9-20, P9-21, P9-22, P9-27, P9-28, P9-31, P9-32, P9-36, P9-39, P9-49, P9-54, and P9-58.

10. The GAL9 antigen-binding molecule of claim 9, wherein the second antigen-binding site comprises the VL and VH sequences from said another Ab clone.

11. The GAL9 antigen-binding molecule of claim 10, wherein the second antigen-binding site comprises a full-length immunoglobulin heavy chain sequence containing the VH sequence from said another Ab clone and a full-length immunoglobulin light chain sequence containing the VL sequence from said another Ab clone.

12. The GAL9 antigen-binding molecule of claim 5, wherein the second antigen-binding site is specific for an antigen other than the first GAL9 antigen.

13. The GAL9 antigen-binding molecule of claim 5, wherein the second antigen-binding site comprises all three VH CDRs and all three VL CDRs from any one Ab clone selected from P9-18, P9-15, P9-21, P9-22, P9-28, and P9-32.

14. The GAL9 antigen-binding molecule of claim 5, wherein the second antigen-binding site comprises all three VH CDRs and all three VL CDRs from any one Ab clone selected from P9-18, P9-15, P9-21, and P9-28.

15. The GAL9 antigen-binding molecule of claim 5, wherein the second antigen-binding site comprises all three VH CDRs and all three VL CDRs from Ab clone P9-15.

16. The GAL9 antigen-binding molecule of claim 5, wherein the second antigen-binding site comprises all three VH CDRs and all three VL CDRs from Ab clone P9-18.

17. The GAL9 antigen-binding molecule of claim 5, wherein the second antigen-binding site comprises all three VH CDRs and all three VL CDRs from Ab clone P9-21.

18. The GAL9 antigen-binding molecule of claim 5, wherein the second antigen-binding site comprises all three VH CDRs and all three VL CDRs from Ab clone P9-28.

19. The GAL9 antigen-binding molecule of any one of claims 1-18, wherein the GAL9 antigen-binding molecule comprises an antibody form selected from full-length antibody, Fab fragment, Fv, scFv, tandem scFv, Diabody, scDiabody, DART, and tandAb.

20. The GAL9 antigen-binding molecule of claim 1, wherein the GAL9 antigen-binding molecule increases the TNF-α secretion of activated immune cells upon contact, and wherein the increase is greater than an 80-fold increase relative to the activated immune cells treated with a control agent.

21. The GAL9 antigen-binding molecule of claim 1, wherein the GAL9 antigen-binding molecule increases the IFN-γ secretion of activated immune cells upon contact, and wherein the increase is greater than a 1.2-fold increase relative to the activated immune cells treated with a control agent.

22. The GAL9 antigen-binding molecule of claim 1, wherein the GAL9 antigen-binding molecule increases the CD40L surface expression of activated CD8+ T cells upon contact, and wherein the increase is greater than a 2-fold increase relative to the activated CD8+ T cells treated with a control agent.

23. The GAL9 antigen-binding molecule of claim 1, wherein the GAL9 antigen-binding molecule increases the OX40 surface expression of activated CD8+ T cells upon contact, and wherein the increase is greater than a 2-fold increase relative to the activated CD8+ T cells treated with a control agent.

24. The GAL9 antigen-binding molecule of claim 1, wherein the GAL9 antigen-binding molecule increases the IL-12 production of activated dendritic cells (DCs) upon contact, and wherein the increase is greater than a 20-fold increase relative to the activated DCs treated with a control agent.

25. The GAL9 antigen-binding molecule of claim 1, wherein the GAL9 antigen-binding molecule increases the PD-L2 surface expression on activated dendritic cells (DCs) upon contact, and wherein the increase is greater than a 4-fold increase relative to the activated DCs treated with a control agent.

26. The GAL9 antigen-binding molecule of any one of claims 20-25, wherein the control agent is a negative control agent or a positive control agent.

27. The GAL9 antigen-binding molecule of claim 26, wherein the control agent is a control antibody.

28. The GAL9 antigen-binding molecule of claim 27, wherein the control antibody is selected from: ECA42 clone anti-GAL9 antibody, RG9.1 clone anti-GAL9 antibody, RG9.35 clone anti-GAL9 antibody, anti-PD1 antibody, and non-GAL9-binding isotype control antibody.

29. The GAL9 antigen-binding molecule of any one of claims 20-25, wherein the activated immune cells, activated CD8+ T cells, or activated DCs are activated by peptide stimulation.

30. The GAL9 antigen-binding molecule of claim 29, wherein the peptide stimulation is performed with one peptide or a plurality of peptides known to induce an immune response.

31. The GAL9 antigen-binding molecule according to claim 1, wherein the GAL9 antigen-binding molecule exhibits one or more of the following properties: A) increases the TNF-α secretion of activated immune cells, wherein the increase is greater than an 80-fold increase relative to the activated immune cells treated with a control agent, B) increases the IFN-γ secretion of activated immune cells, wherein the increase is greater than a 1.2-fold increase relative to the activated immune cells treated with a control agent, C) increasing the CD40L surface expression of activated CD8+ T cells, wherein the increase is greater than a 2-fold increase relative to activated CD8+ T cells treated with a control agent, D) increasing the OX40 surface expression of activated CD8+ T cells, wherein the increase is greater than a 2-fold increase relative to activated CD8+ T cells treated with a control agent, E) increasing the IL-12 production of activated dendritic cells (DCs), wherein the increase is greater than a 20-fold increase relative to activated DCs treated with a control agent, F) increasing the PD-L2 surface expression on activated dendritic cells (DCs), wherein the increase is greater than a 4-fold increase relative to activated DCs treated with a control agent.

32. The GAL9 antigen-binding molecule of claim 31, wherein the control agent is a negative control agent or a positive control agent.

33. The GAL9 antigen-binding molecule of claim 32, wherein the control agent is a control antibody.

34. The GAL9 antigen-binding molecule of claim 33, wherein the control antibody is selected from: ECA42 clone anti-GAL9 antibody, RG9.1 clone anti-GAL9 antibody, RG9.35 clone anti-GAL9 antibody, anti-PD1 antibody, and non-GAL9-binding isotype control antibody.

35. The GAL9 antigen-binding molecule of claim 31, wherein the activated immune cell, activated CD8+ T cell, or activated DC is stimulated by a peptide.

36. The GAL9 antigen-binding molecule of claim 35, wherein the activated immune cell, activated CD8+ T cell, or activated DC is activated by one peptide or a plurality of peptides known to induce an immune response.

37. The GAL9 antigen-binding molecule of claim 1, which is purified.

38. A pharmaceutical composition comprising the GAL9 antigen-binding molecule of any one of claims 1-37 and a pharmaceutically acceptable diluent.

39. Use of the pharmaceutical composition of claim 38 in the preparation of a medicament for treating cancer in a subject, wherein the cancer is selected from: pancreatic cancer, ovarian cancer, breast cancer, lung cancer, gastric cancer, melanoma, Ewing's sarcoma, mantle cell lymphoma, B-ALL, hematological cancer, head and neck squamous cell carcinoma, prostate cancer, colon cancer, kidney cancer, and uterine cancer.

40. The use of claim 39, wherein the cancer is chronic lymphocytic leukemia.

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