Activated anti-GAL9 binding molecules

Abstract

Anti-GAL9 antibody constructs, pharmaceutical compositions containing the constructs, and methods for their use are presented.

Description

Background Art

[0001] [3. Background]

[0003] Immunotherapy has great potential in the treatment of cancer. However, tumors can be resistant to immunotherapy, for example, by mobilizing immunosuppressive cells or signaling molecules into the tumor microenvironment or by utilizing immune checkpoint signaling pathways.

[0002]

[0004] Galectin-9 (GAL9) is an S-type lectin beta-galactoside-binding protein in which the N-terminal carbohydrate-binding domain and the C-terminal carbohydrate-binding domain are connected by a linker peptide. GAL9 has been suggested to be involved in the regulation of cell-cell interactions and cell-matrix interactions. GAL9 has been shown to bind to soluble PD-L2, and it has been suggested that at least part of the immunological effect of PD-L2 is mediated not by PD-1 but by the binding of multimeric PD-L2 to GAL9 (International Publication No. 2016 / 008005. This document is incorporated herein by reference in its entirety). However, the mechanism by which GAL9 and PD-L2 affect immune effector functions has not yet been fully characterized.

[0003]

[0005] There is still a need for therapeutic agents that can enhance immune effector functions and reduce immunosuppressive or T cell exhaustion pathways. Such therapeutic agents may be useful for improving cancer immunotherapy.

Summary of the Invention

[0004] [4. Summary]

[0006] In a first aspect, the Disclosure provides a galectin-9 (GAL9) antigen-binding molecule comprising a first antigen-binding site (ABS) specific to a first epitope of a first GAL9 antigen, wherein the first antigen-binding site comprises all three VH CDRs derived 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.

[0005]

[0007] In a second aspect, the present disclosure provides a galectin-9 (GAL9) antigen-binding molecule comprising a first antigen-binding site specific to a first epitope of a first GAL9 antigen, wherein the first antigen-binding site comprises all three VL CDRs derived 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.

[0006]

[0008] In a third aspect, the present disclosure provides a galectin-9 (GAL9) antigen-binding molecule comprising a first antigen-binding site specific to a first epitope of a first GAL9 antigen, wherein the first antigen-binding site comprises all three VH CDRs and all three VL CDRs derived 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.

[0007]

[0009] In a fourth aspect, the present disclosure provides a galectin-9 (GAL9) antigen-binding molecule comprising a first antigen-binding site specific to a first epitope of a first GAL9 antigen, and comprising a VL sequence and a VH sequence derived 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.

[0008]

[0010] In some embodiments, the GAL9 antigen-binding molecule comprises a fully immunoglobulin heavy chain "IgG1" sequence containing a VH sequence, and a fully immunoglobulin light chain sequence containing a VL sequence, wherein the VH and VL sequences are derived 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.

[0009]

[0011] In some embodiments, the GAL9 antigen-binding molecule comprises a fully immunoglobulin heavy chain "IgG4" sequence containing a VH sequence, and a fully immunoglobulin light chain sequence containing a VL sequence, wherein the VH and VL sequences are derived 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.

[0010]

[0012] In some embodiments, the GAL9 antigen-binding molecule comprises a fully immunoglobulin heavy chain "IgG3" sequence containing a VH sequence, and a fully immunoglobulin light chain sequence containing a VL sequence, wherein the VH and VL sequences are derived 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.

[0011]

[0013] In some embodiments, the GAL9 antigen-binding molecule may contain the human GAL9 antigen, which is the GAL9 antigen.

[0012]

[0014] In some embodiments, the GAL9 antigen-binding molecule may further include a second antigen-binding site.

[0013]

[0015] In certain embodiments, the second antigen-binding site is specific to the GAL9 antigen. In other embodiments, the second antigen-binding site is identical to the first antigen-binding site.

[0014]

[0016] In other embodiments, the second antigen-binding site is specific to the second epitope of the first GAL9 antigen.

[0015]

[0017] 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 derived 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.

[0016]

[0018] In some embodiments, the second antigen-binding site includes VL and VH sequences derived from another ABS clone.

[0017]

[0019] In some embodiments, the second antigen-binding site includes a full immunoglobulin heavy chain sequence containing a VH sequence and a full immunoglobulin light chain sequence containing a VL sequence, derived from another ABS clone.

[0018]

[0020] In some embodiments, the second antigen-binding site is specific to antigens other than the first GAL9 antigen.

[0019]

[0021] 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 derived 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.

[0020]

[0022] 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 derived from any one of the ABS clones selected from P9-18, P9-15, P9-21, and P9-28.

[0021]

[0023] In some embodiments, the first antigen-binding site comprises all three VH CDRs and all three VL CDRs derived from ABS clone P9-15, or all three VH CDRs and all three VL CDRs.

[0022]

[0024] In some embodiments, the first antigen-binding site comprises all three VH CDRs and all three VL CDRs derived from ABS clone P9-18, or all three VH CDRs and all three VL CDRs.

[0023]

[0025] In some embodiments, the first antigen-binding site comprises all three VH CDRs and all three VL CDRs derived from ABS clone P9-21, or all three VH CDRs and all three VL CDRs.

[0024]

[0026] In some embodiments, the first antigen-binding site comprises all three VH CDRs and all three VL CDRs derived from ABS clone P9-22, or all three VH CDRs and all three VL CDRs.

[0025]

[0027] In some embodiments, the first antigen-binding site comprises all three VH CDRs and all three VL CDRs derived from ABS clone P9-28, or all three VH CDRs and all three VL CDRs.

[0026]

[0028] In some embodiments, the GAL9 antigen-binding molecule includes an antibody format selected from the group consisting of full-length antibody, Fab fragment, F(ab)'2 fragment, Fv, scFv, tandcFv, diabody, scDiabody, DART, single-chain VHH camel antibody, tandAb, minibody, and B-body. The B-body is described in U.S. Patent Application Publication 2018 / 0118811, which is incorporated herein by reference in its entirety.

[0027]

[0029] In some embodiments, the GAL9 antigen-binding molecule increases TNF-α secretion by activated immune cells, with increases greater than 20, 30, 40, 50, 60, 70, or 80 times compared to activated immune cells treated with a control agent.

[0028]

[0030] In some embodiments, the GAL9 antigen-binding molecule increases IFN-γ secretion by activated immune cells, with the increase being greater than 1.2 times compared to activated immune cells treated with the control agent.

[0029]

[0031] In some embodiments, the GAL9 antigen-binding molecule increases CD40L surface expression on activated CD8+ T cells, with the increase being greater than 2-fold compared to activated CD8+ T cells treated with the control agent.

[0030]

[0032] In some embodiments, the GAL9 antigen-binding molecule increases OX40 surface expression on activated CD8+ T cells, with the increase being greater than 2-fold compared to activated CD8+ T cells treated with the control agent.

[0031]

[0033] In some embodiments, the GAL9 antigen-binding molecule increases IL-12 production in activated dendritic cells (DCs), with the increase being greater than 20-fold compared to activated DCs treated with the control agent.

[0032]

[0034] In some embodiments, the GAL9 antigen-binding molecule increased PD-L2 surface expression on activated dendritic cells (DCs), with the increase being greater than four-fold compared to activated DCs treated with the control agent.

[0033]

[0035] In some embodiments, the control agent is either a negative control agent or a positive control agent.

[0034]

[0036] In some embodiments, the control agent is a control antibody.

[0035]

[0037] In some embodiments, the control antibody is selected from the group consisting of 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.

[0036]

[0038] In some embodiments, activated immune cells, activated CD8+ T cells, or activated DCs were activated by peptide stimulation, for example, by a peptide or a combination of peptides known to induce an immune response.

[0037]

[0039] In a fifth aspect, the disclosure provides a GAL9 antigen-binding molecule that increases TNF-α secretion by activated immune cells, wherein the increase is greater than 80-fold compared to activated immune cells treated with a control agent.

[0038]

[0040] In a sixth aspect, the disclosure provides a GAL9 antigen-binding molecule that increases IFN-γ secretion by activated immune cells, wherein the increase is greater than 1.2 times compared to activated immune cells treated with a control agent.

[0039]

[0041] In a seventh aspect, the disclosure provides a GAL9 antigen-binding molecule that increases CD40L surface expression of activated CD8+ T cells, wherein the increase is greater than 2-fold compared to activated CD8+ T cells treated with a control agent.

[0040]

[0042] In an eighth aspect, the disclosure provides a GAL9 antigen-binding molecule that increases OX40 surface expression on activated CD8+ T cells, wherein the increase is greater than twofold compared to activated CD8+ T cells treated with a control agent.

[0041]

[0043] In a ninth aspect, the disclosure provides a GAL9 antigen-binding molecule that increases IL-12 production in activated dendritic cells (DCs), wherein the increase is greater than 20-fold compared to activated DCs treated with a control agent.

[0042]

[0044] In a tenth aspect, the disclosure provides a GAL9 antigen-binding molecule that increases PD-L2 surface expression on activated dendritic cells (DCs), wherein the increase is greater than four-fold compared to activated DCs treated with a control agent.

[0043]

[0045] In the eleventh aspect, the present disclosure provides a GAL9 antigen-binding molecule exhibiting one or more of the following characteristics: A) increasing TNF-α secretion by activated immune cells, the increase being greater than 80-fold compared to activated immune cells treated with a control agent; B) increasing IFN-γ secretion by activated immune cells, the increase being greater than 1.2-fold compared to activated immune cells treated with a control agent; C) increasing CD40L surface expression of activated CD8+ T cells, the increase being greater than 2-fold compared to activated CD8+ T cells treated with a control agent; D) increasing OX40 surface expression of activated CD8+ T cells, the increase being greater than 1.2-fold compared to activated CD8+ T cells treated with a control agent. E) An increase greater than 2 times compared to T cells; F) Increased IL-12 production in activated dendritic cells (DCs) with an increase greater than 20 times compared to activated DCs treated with the control agent; and G) Increased PD-L2 surface expression on activated dendritic cells (DCs) with an increase greater than 4 times compared to activated DCs treated with the control agent.

[0044]

[0046] In some embodiments, the control agent is either a negative control agent or a positive control agent.

[0045]

[0047] In some embodiments, the control agent is a control antibody.

[0046]

[0048] In some embodiments, the control antibody is selected from the group consisting of 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.

[0047]

[0049] In some embodiments, activated immune cells, activated CD8+ T cells, or activated DCs were activated by peptide stimulation, for example, by a peptide or a combination of peptides known to induce an immune response.

[0048]

[0050] In some embodiments, the GAL9 antigen-binding molecules described in the fifth to eleventh embodiments provided herein include a first antigen-binding site specific to a first epitope of a first GAL9 antigen, the first antigen-binding site including all three VH CDRs and all three VL CDRs derived 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.

[0049]

[0051] In some embodiments, the VL and VH sequences are derived 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.

[0050]

[0052] In some specific embodiments, the GAL9 antigen-binding molecule comprises a full immunoglobulin heavy chain sequence containing a VH sequence and a full immunoglobulin light chain sequence containing a VL sequence, wherein the VH and VL sequences are derived 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.

[0051]

[0053] In some embodiments, the GAL9 antigen is human GAL9 antigen.

[0052]

[0054] In some embodiments, the GAL9 antigen-binding molecule further comprises a second antigen-binding site.

[0053]

[0055] In some embodiments, the second antigen-binding site is specific to the GAL9 antigen.

[0054]

[0056] The GAL9 antigen-binding molecule according to claim 48, wherein the second antigen-binding site is identical to the first antigen-binding site.

[0055]

[0057] In some embodiments, the second antigen-binding site is specific to the second epitope of the first GAL9 antigen.

[0056]

[0058] In some embodiments, the second antigen-binding site includes all three VH CDRs and all three VL CDRs derived 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.

[0057]

[0059] In some embodiments, the second antigen-binding site includes VL and VH sequences derived from another ABS clone.

[0058]

[0060] In some embodiments, the second antigen-binding site includes a full immunoglobulin heavy chain sequence containing a VH sequence and a full immunoglobulin light chain sequence containing a VL sequence, derived from another ABS clone.

[0059]

[0061] In some embodiments, the second antigen-binding site is specific to antigens other than the first GAL9 antigen.

[0060]

[0062] In some embodiments, the first antigen-binding site comprises all three VH CDRs and all three VL CDRs derived from any one of the ABS clones selected from P9-10, P9-15, P9-18, P9-21, P9-22, and P9-28.

[0061]

[0063] In some embodiments, the first antigen-binding site comprises all three VH CDRs and all three VL CDRs derived from any one of the ABS clones selected from P9-10, P9-15, P9-18, P9-21, P9-22, and P9-28.

[0062]

[0064] In some embodiments, the first antigen-binding site includes all three VH CDRs and all three VL CDRs derived from ABS clone P9-15.

[0063]

[0065] In some embodiments, the first antigen-binding site includes all three VH CDRs and all three VL CDRs derived from ABS clone P9-18.

[0064]

[0066] In some embodiments, the first antigen-binding site includes all three VH CDRs and all three VL CDRs derived from ABS clone P9-21.

[0065]

[0067] In some embodiments, the first antigen-binding site includes all three VH CDRs and all three VL CDRs derived from ABS clone P9-22.

[0066]

[0068] In some embodiments, the first antigen-binding site includes all three VH CDRs and all three VL CDRs derived from ABS clone P9-28.

[0067]

[0069] In some embodiments, the GAL9 antigen-binding molecule includes an antibody format selected from the group consisting of full-length antibody, Fab fragment, Fv, scFv, tandem scFv, diabody, scdiabody, DART, tandAb, minibody, and Bbody.

[0068]

[0070] In a twelfth aspect, the disclosure provides a GAL9 antigen-binding molecule that binds to the same epitope as the GAL9 antigen-binding molecule described in any one of the above claims.

[0069]

[0071] In a thirteenth aspect, the disclosure provides a GAL9 antigen-binding molecule that competes for binding with the GAL9 antigen-binding molecule described in any one of the above claims.

[0070]

[0072] In some embodiments, the GAL9 antigen-binding molecule is purified.

[0071]

[0073] In a fourteenth aspect, the disclosure provides a pharmaceutical composition comprising a GAL9 antigen-binding molecule according to any one of the above claims and a pharmaceutically acceptable excipient.

[0072]

[0074] In a 15th aspect, the present disclosure provides a method for treating a subject having cancer, comprising the step of administering a therapeutically effective amount of a pharmaceutical composition as provided herein to the subject.

[0073]

[0075] In some embodiments, the cancer is selected from the group consisting of pancreatic cancer, ovarian cancer, breast cancer, lung cancer, stomach cancer, melanoma, Ewing's sarcoma, chronic lymphocytic leukemia, mantle cell lymphoma, B-ALL, hematological cancer, head and neck squamous cell carcinoma, prostate cancer, colon cancer, kidney cancer, and uterine cancer.

[0074]

[0076] In some embodiments, 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 Waldenström's disease), skin cancer (including malignant melanoma), cancers of the gastrointestinal tract (including head and neck cancer, esophageal cancer, stomach cancer, pancreatic cancer, liver cancer, colon and rectal cancer, and anal cancer), cancers of the reproductive and urinary system (including kidney cancer, bladder cancer, testicular cancer, and prostate cancer), cancers of women (including breast cancer, ovarian cancer, gynecological cancer, and choriocarcinoma), and tumors of the brain, carcinoid bone, nasopharynx, retroperitoneum, thyroid, and soft tissues.

[0075]

[0077] In some embodiments, cancer is a virus-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. [Brief explanation of the drawing]

[0076] [5. Brief description of the drawing] [Figure 1] This figure shows the results of administering immunoactivated anti-GAL9 (α-GAL9) antibody to a colon cancer tumor model. BALB / c mice were subcutaneously transplanted with CT26 tumor cell line cells and treated with control rat IgG or anti-GAL9 antibody P9-18 or P9-21. All treatments were administered intraperitoneally (IP) at 200 μg on days 7, 11, 15, and 19. n=10 mice / group. Tumor growth was evaluated by measuring tumor volume. Mice treated with P9-18 and P9-21 showed reduced growth of transplanted CT26 tumors compared to those treated with control IgG. [Figure 2] This figure shows the results of administering an immunoactivated anti-GAL9 antibody to a melanoma tumor model. C57BL / 6 mice were subcutaneously transplanted with B16.F0 tumor cell line cells and treated with control IgG or α-GAL9 antibody P9-18 or P9-21. All treatments were administered intraperitoneally (IP) at 200 μg on days 7, 11, 15, and 19. n=10 mice / group. Tumor growth was evaluated by measuring tumor volume. Mice treated with P9-18 and P9-21 showed reduced growth of transplanted B16.F0 tumors compared to those treated with control IgG. [Figure 3A] This figure shows INF-γ secreted from activated PBMCs stimulated in vitro with various GAL9 antibody candidates, known comparative tool antibodies (Tool mAbs), anti-PD-1 antibodies, control antibodies (IgG Ctrl), and vehicle controls (PBS Ctrl). The black diamonds indicate secretion from activated PBMCs stimulated with the positive controls, comparative tool mAbs and anti-PD-1 antibodies. [Figure 3B] This figure shows TNF-α secreted from activated PBMCs stimulated in vitro with various GAL9 antibody candidates, known comparative tool antibodies (tool mAbs), anti-PD-1 antibodies, control antibodies (IgG Ctrl), and vehicle controls (PBS Ctrl). The black diamonds indicate secretion from activated PBMCs stimulated with the positive controls, comparative tool mAbs and anti-PD-1 antibodies. [Figure 4-1] This figure shows the levels of immunostimulatory markers CD27, CD40L, ICOS, 4-1BB, and OX40 on the surface of activated CD8+ T cells stimulated in vitro with various GAL9 antibody candidates or IgG control antibodies. [Figure 4-2] This is a continuation of Figure 4-1. [Figure 5] This figure shows representative flow cytometry plots quantifying IL-12 production by DCs stimulated in vitro with control IgG or α-GAL9 candidate P9-18, as well as those stimulated with a stained control. [Figure 6A] This figure shows representative flow cytometry plots of TNF-α secretion by CD56+ NK cells after 72 hours of stimulation with a 5 μg dose of control antibody P9-55 (clone 55), anti-GAL9 candidate antibody P9-15 (clone 15), or α-GAL9 candidate antibody P9-18 (clone 18). [Figure 6B] This figure shows representative flow cytometry plots of TNF-α secretion by CD56+ NK cells after 72 hours of stimulation with a 20 μg dose of control antibody P9-55 (clone 55), anti-GAL9 candidate antibody P9-15 (clone 15), or α-GAL9 candidate antibody P9-18 (clone 18). [Figure 7A-1] This figure illustrates an example of the Martin numbering scheme, along with various CDR definitions (Chothia, AbM, Kabat, Contact, IMGT) applicable to the P9-28 anti-GAL9 candidate antibodies provided herein. Figures 7A-1 to 7E-2 disclose sequence numbers 187 and 188, respectively, in order of appearance. [Figure 7A-2] This is a continuation of Figure 7A-1. [Figure 7B-1] This figure illustrates an example of the Martin numbering scheme, along with various CDR definitions (Chosia, AbM, Kabat, Contact, IMGT) applicable to the P9-28 anti-GAL9 candidate antibodies provided herein. Figures 7A-1 to 7E-2 disclose sequence numbers 187 and 188, respectively, in order of appearance. [Figure 7B-2] This is a continuation of Figure 7B-1. [Figure 7C-1] This figure illustrates an example of the Martin numbering scheme, along with various CDR definitions (Chosia, AbM, Kabat, Contact, IMGT) applicable to the P9-28 anti-GAL9 candidate antibodies provided herein. Figures 7A-1 to 7E-2 disclose sequence numbers 187 and 188, respectively, in order of appearance. [Figure 7C-2] This is a continuation of Figure 7C-1. [Figure 7D-1] This figure illustrates an example of the Martin numbering scheme, along with various CDR definitions (Chosia, AbM, Kabat, Contact, IMGT) applicable to the P9-28 anti-GAL9 candidate antibodies provided herein. Figures 7A-1 to 7E-2 disclose sequence numbers 187 and 188, respectively, in order of appearance. [Figure 7D-2] This is a continuation of Figure 7D-1. [Figure 7E-1] This figure illustrates an example of the Martin numbering scheme, along with various CDR definitions (Chosia, AbM, Kabat, Contact, IMGT) applicable to the P9-28 anti-GAL9 candidate antibodies provided herein. Figures 7A-1 to 7E-2 disclose sequence numbers 187 and 188, respectively, in order of appearance. [Figure 7E-2] This is a continuation of Figure 7E-1. [Figure 8A]This figure shows representative confocal microscope images demonstrating the co-localization and clustering of GAL9 and PD-L2 on DCs after treatment with IgG control. Blue staining indicates DNA (DAPI), red staining indicates PD-L2, green staining indicates CD11c, and yellow staining indicates GAL9. The unlabeled microscope image is bright-field. The attached figure is rendered in grayscale. [Figure 8B] This figure shows representative confocal microscope images demonstrating the colocalization and clustering of GAL9 and PD-L2 on DCs after treatment with P9-18. Blue staining indicates DNA (DAPI), red staining indicates PD-L2, green staining indicates CD11c, and yellow staining indicates GAL9. The unlabeled microscope image is bright-field. The attached figure is rendered in grayscale. [Figure 8C] This figure shows representative confocal microscope images demonstrating the colocalization and clustering of GAL9 and PD-L2 on DCs after treatment with P9-21. Blue staining indicates DNA (DAPI), red staining indicates PD-L2, green staining indicates CD11c, and yellow staining indicates GAL9. The unlabeled microscope image is bright-field. The attached figure is rendered in grayscale. [Figure 9A] This figure shows a representative confocal image demonstrating the retention of PD-L2 and PD-L1 on the surface of CT26 tumor cells after treatment with anti-GAL9 P9-18 (Figure 9B) compared to an IgG control (Figure 9A). The spots in the image highlight the increased expression of PD-L2 and PD-L1 ligands. Blue staining indicates DNA (DAPI), red staining indicates PD-L2, and green staining indicates PD-L1. The attached figure is rendered in grayscale. [Figure 9B]This figure shows a representative confocal image demonstrating the retention of PD-L2 and PD-L1 on the surface of CT26 tumor cells after treatment with anti-GAL9 P9-18 (Figure 9B) compared to an IgG control (Figure 9A). The spots in the image highlight the increased expression of PD-L2 and PD-L1 ligands. Blue staining indicates DNA (DAPI), red staining indicates PD-L2, and green staining indicates PD-L1. The attached figure is rendered in grayscale. [Figure 10A] This figure shows representative data from an EBV-infected humanized mouse model treated with anti-GAL9 P9-15. Figure 10A shows an overview of the protocol along with the treatment timeline. [Figure 10B] This figure shows representative data from EBV-infected humanized mouse models treated with anti-GAL9 P9-15. Figure 10B shows images of spleens from mice treated with IgG control and P9-15. Arrows indicate undue tumor growth in IgG-controlled mice. [Figure 10C] This figure shows representative data from EBV-infected humanized mouse models treated with anti-GAL9 P9-15. Figure 10C shows a bar graph of spleen weight. [Figure 10D] This figure shows representative data from an EBV-infected humanized mouse model treated with anti-GAL9 P9-15. Figure 10D shows a bar graph of the number of cells per spleen. [Figure 10E] This figure shows representative data from a humanized mouse model infected with EBV treated with anti-GAL9 P9-15. Figure 10E shows a bar graph of splenic viral load. [Figure 11] This figure shows representative data from EBV-infected humanized mouse models treated with anti-GAL9 P9-28. Figure 10A shows an overview of the protocol along with the treatment timeline. Figure 11 shows images of spleens from IgG-controlled and anti-GAL9 P9-28 treated mice. Arrows indicate uncontrolled tumor growth in IgG-controlled treated mice. [Figure 12] Figure 12A is P9-18-IgG1 (diamond shape) [ka] ), sFc-P9-18-IgG2a (inverted triangle [ka] ), P9-18-IgG2a (round shape) [ka] ), and IgG (IgG2a) control #1 (black square) [ka] This figure shows the in vivo evaluation of tumor growth in the CT26 tumor model by sFc-P9-18 IgG2a (inverted triangle). Figure 12B shows sFc-P9-18 IgG2a (inverted triangle). [ka] ), P9-18-IgG2a (round shape) [ka] ), and IgG (IgG2a) control #2 (black diamond shape) [ka] This figure shows the in vivo evaluation of immunological memory in CT26 tumors previously treated with ). [Figure 13] This figure shows bar graphs of the mean percentage of PD-L1+ or PD-L2+ tumor-associated dendritic cells (CD11c+) and the mean cell surface expression (GMI) of PD-L1 or PD-L2 on tumor-associated dendritic cells (CD11c+) after treatment with anti-GAL9 P9-18 or a control. [Figure 14]This figure shows bar graphs of the mean percentage of PD-L1+ or PD-L2+ tumor cells and the mean cell surface expression level (GMI) of PD-L1 or PD-L2 on tumor cells after treatment with anti-GAL9 P9-18 or IgG control. [Modes for carrying out the invention]

[0077] [6. Detailed explanation] 6.1.Definition

[0093] Unless otherwise defined, all technical and scientific terms used herein have the meanings generally understood by those skilled in the art in which this invention pertains. Where used herein, the following terms have the meanings assigned to them below.

[0078]

[0094] The term "antigen-binding site" or "ABS" refers to a region of a GAL9-binding molecule that specifically recognizes or binds to a given antigen or epitope.

[0079]

[0095] As used herein, the terms “to treat” or “to cure” are used in their most broadly accepted clinical sense. This sense includes, but is not limited to, reducing the signs or symptoms of a disease; improving the signs or symptoms of a disease; alleviating symptoms; reducing the severity of the disease; achieving a stable (i.e., non-worsening) state of the disease; delaying or slowing the progression of the disease; improving or easing the disease state; achieving remission, whether detectable or undetectable (whether partial or total); curing; and prolonging survival compared to expected survival without treatment. Unless otherwise specified, “to treat” or “to cure” is not intended to prevent or prevent a disease.

[0080]

[0096] "Subject," "individual," "animal," "patient," or "mammal" refers to any subject, particularly mammalian subjects, for whom diagnosis, prognosis, or therapy is desired. Mammalian subjects include humans, domesticated animals, livestock, and zoo animals, sporting animals, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, and cows. Unless otherwise stated, "patient" is intended to refer to a human "subject."

[0081]

[0097] The term "sufficient amount" means an amount sufficient to produce the desired effect, for example, an amount sufficient to regulate protein aggregation in cells.

[0082]

[0098] The term "therapeutic dose" refers to the amount of a substance that is effective in improving the symptoms of a disease.

[0083]

[0099] The term "preventive effective dose" refers to the amount of a substance that is effective in preventing the symptoms of a disease.

[0084] 6.2. Other Interpretive Rules

[0100] Unless otherwise specified, all references to sequences in this specification refer to amino acid sequences.

[0085]

[0101] Unless otherwise specified, antibody constant region residue numbering conforms to the Eu index as described in www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html#refs (accessed August 22, 2017), which is incorporated herein by reference in its entirety, and residue numbers identify 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 molecule described herein.

[0086]

[0102] Unless otherwise specified as "Kabatto CDR," "Chothia CDR," "Contact CDR," or "IMGT CDR," all references to "CDR" refer to CDRs as defined using the Martin (AbM) definition.

[0087]

[0103] "Endogenous sequence" or "natural sequence" means any sequence, including both nucleic acid and amino acid sequences, that originates from an organism, tissue, or cell and has not been artificially modified or mutated.

[0088]

[0104] 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 forming a binding molecule and are not intended to imply any order or quantity of different polypeptide chains within the binding molecule.

[0089]

[0105] In this disclosure, “comprises,” “comprising,” “containing,” “having,” “includes,” “including,” and their linguistic variations have the meanings assigned to them under U.S. patent law and permit the presence of additional components other than those expressly described.

[0090]

[0106] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. Terms such as "include" and "such as" are intended to convey inclusion, not limitation, unless otherwise specifically indicated.

[0091]

[0107] The ranges provided herein are understood to be a concise representation of all values ​​within the range, including the endpoints described. For example, the range 1 to 50 is understood to include any number, combination of numbers, or subrange of the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50.

[0092]

[0108] Unless otherwise stated or it is otherwise obvious from the context, the term “about” as used herein is understood to mean within the normal tolerance range in the art, for example, within two standard deviations of the mean. “About” can be understood to mean within about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value.

[0093] 6.3. General overview

[0109] This disclosure provides galectin-9 (GAL9) antigen-binding molecules such as anti-GAL9 antibodies and their antigen-binding fragments, compositions containing GAL9-binding molecules, and pharmaceutical compositions containing GAL9-binding molecules. In particular, this disclosure provides various GAL9 antigen-binding molecules that are irritating, act as activators of the immune system, increase the secretion and production of various cytokines in various immune cells, and increase the surface expression of irritating molecules.

[0094]

[0110] Methods for treating a target disease or condition by administering an immunostimulatory galectin-9 antibody-binding molecule are also provided in this disclosure. The methods provided in this disclosure are particularly useful for treating proliferative disorders or cancer. In some embodiments, the cancer is a virus-induced cancer, such as a cancer caused by infection with an oncovirus or tumor virus. In some embodiments, the compositions and methods provided in this disclosure can be used for treating immunosuppressive diseases or conditions, such as malaria, HIV, or AIDs.

[0095] 6.4.GAL9 antigen binding molecule

[0111] In the first embodiment, an antigen-binding molecule is provided. In all embodiments, the antigen-binding molecule comprises at least a first antigen-binding site specific to the GAL9 antigen, and therefore the binding molecule is referred to as a GAL9 antigen-binding molecule or a GAL9-binding molecule.

[0096]

[0112] The GAL9 antigen-binding molecules described herein specifically bind to the GAL9 antigen.

[0097]

[0113] As used herein, “GAL9 antigen” refers to galectin-9 family members and homologs. GAL9 is also known as LGALS9, HUAT, LGALS9A, tumor antigen HOM-HD-21, and ekalectin. In certain embodiments, a GAL9-binding molecule has an antigen-binding site that specifically binds to at least one portion of more than one GAL9 domain, such as the junction between a first GAL9 domain and a second GAL9 domain.

[0098]

[0114] In certain embodiments, the GAL9 antigen is human. A standard human GAL9 protein, including its sequence and domain features, is described in GenBank acceptance number NP_033665.1, which is incorporated herein by reference in its entirety. Sequence ID 6 provides the full-length GAL9 protein sequence. MAFSGSQAPYLSPAVPFSGTIQGGLQDGLQITVNGTVLSSSGTRFAVNFQTGFSGNDIAFHFNPRFEDGGYVVCNTRQNGSWGPEERKTHMPFQKGMPFDLCFLVQSSDFKVMVNGILFVQYFHRVPFHRVDTISVNGSVQLSYISFQNPRTVPVQPAFSTVPFSQPVCFPPRPRGRRQKPPGVWPANPAPITQTVIHTVQSAPGQMFSTPAIPPMMYPHPAYPMPFITTILGGLYPSKSILLSGTVLPSAQRFHINLCSGNHIAFHLNPRFDENAVVRNTQIDNSWGSEERSLPRKMPFVRGQSFSVWILCEAHCLKVAVDGQHLFEYYHRLRNLPTINRLEVGGDIQLTHVQT[Sequence ID 6]

[0099]

[0115] In various embodiments, the GAL9-binding molecule binds more specifically to at least one antigen in addition to the GAL9 antigen.

[0100] 6.4.1. Functional characteristics of the GAL9 antigen-binding molecule

[0116] In some embodiments, upon contact, the GAL9 antigen-binding molecule increases cytokine secretion by activated immune cells, such as 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 + These are T cells. In some embodiments, T cells are CD4 + These are T cells. In some embodiments, the immune cells are natural killer (NK) cells. In some embodiments, the immune cells are dendritic cells (DCs).

[0101]

[0117] The effect of the GAL9 antigen-binding molecule on immune cell cytokine secretion can be determined by any preferred means. For example, the effect of the GAL9 antigen-binding molecule on immune cell cytokine secretion can be determined in vivo, ex vivo, or in vitro. In some embodiments, cytokine secretion is determined in activated immune cells contacted with the GAL9 antigen-binding molecule compared to activated immune cells contacted with a control agent, such as a control antigen-binding molecule or vehicle control. Immune cells can be activated by peptide stimulation. For example, immune cells can be activated by a peptide or a set of peptides known to induce an immune response. The control agent may be a negative control or a positive control. In some embodiments, the GAL9 antigen-binding molecule increases cytokine secretion from immune cells compared to a negative control agent or a negative control antigen-binding molecule. In some embodiments, the negative control antigen-binding molecule is an isotype control-binding molecule that does not bind to 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 may be GAL9 antibody clone RG9.1 (catalog number BE0218, InVivoMab antibody) 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," IntArch Allergy Immunol 146: pp. 36-43. This document is incorporated herein by reference in its entirety. The GAL9 control antibody may also be GAL9 antibody clone ECA42 (catalog number LS-C179449, LifeSpan BioScience). In some embodiments, the GAL9 antigen-binding molecule increases cytokine secretion by immune cells compared to the positive control antibody.

[0102]

[0118] Cytokine secretion by immune cells can be evaluated by any appropriate means. For example, cytokine secretion in in vitro or ex vivo immune cell culture models can be evaluated by analyzing the cytokine content of the cultured cell supernatant, for instance, using a cytokine bead array.

[0103]

[0119] In some embodiments, the cytokine is IFN-γ. In some embodiments, the GAL9 antigen-binding molecule increases IFN-γ secretion from 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 from 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%.

[0104]

[0120] In some embodiments, the cytokine is TNF-α. In some embodiments, the GAL9 antigen-binding molecule increases TNF-α secretion from activated immune cells by at least 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950%, 10,000%, 10,500%, 11,000%, 11,500%, 12,000%, 12,500%, 13,000%, 13,500%, and 14,000% compared to the negative control agents described herein. Increase by 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 from activated immune cells by at least 100% to 150%, 150% to 200%, 200% to 250%, 250% to 300%, 300% to 350%, 350% to 400%, 400% to 450%, 500% to 550%, 550% to 600%, 600% to 650%, 650% to 700%, 700% to 750%, and 750% compared to the negative control agents described herein. ~800%, 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 0%, 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 Increase by 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%.

[0105]

[0121] In various embodiments, activated immune cells include T cells, CD8 cells, and other similar cells. + T cells, NK cells, CD4 + These are T cells or dendritic cells (DCs).

[0106]

[0122] In some embodiments, the GAL9 antigen-binding molecule increases the surface expression of one or more costimulatory molecules on immune cells, such as human immune cells. In certain embodiments, the GAL9 antigen-binding molecule increases the surface expression of one or more costimulatory molecules on activated immune cells. In certain embodiments, the immune cells are T cells. In certain embodiments, the activated immune cells are CD8+ T cells. In certain embodiments, the activated immune cells are NK cells. In certain embodiments, the activated immune cells are dendritic cells.

[0107]

[0123] In some embodiments, one or more co-stimulatory molecules are selected from 4-1BB, CD27, CD40L, ICOS, and OX40. In some embodiments, one or more co-stimulatory molecules are selected from 4-1BB, CD27, CD40L, and OX40. In some embodiments, one or more co-stimulatory molecules are selected from 4-1BB, CD40L, and OX40.

[0108]

[0124] The effect of GAL9 antigen-binding molecules on the surface expression of one or more co-stimulatory molecules can be determined by any preferred means. For example, the effect of GAL9 antigen-binding molecules on the surface expression of one or more co-stimulatory molecules can be determined in vivo, ex vivo, or in vitro.

[0109]

[0125] In some embodiments, the GAL9 antigen-binding molecule increases the surface expression of one or more costimulatory molecules on 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.

[0110]

[0126] In some embodiments, the GAL9 antigen-binding molecule increases CD40L surface expression in activated CD8+ T cells compared 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 0.1×, 0.2×, 0.3×, 0.4×, 0.5×, 0.6×, 0.7×, 0.8×, 0.9×, 1×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, or greater than 10× increase in CD40L surface expression compared to activated CD8+ T cells treated with a control agent. In some embodiments, activated CD8+ T cells treated with a GAL9 antigen-binding molecule exhibit approximately 0.1 × to 10 ×, 0.5 × to 5 ×, 1 × to 4 ×, or approximately 1.5 × to 2.5 × increases in CD40L surface expression compared to activated CD8+ T cells treated with a control agent.

[0111]

[0127] In some embodiments, the GAL9 antigen-binding molecule increases OX40 surface expression in activated CD8+ T cells compared 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 a 0.1× increase, 0.2× increase, 0.3× increase, 0.4× increase, 0.5× increase, 0.6× increase, 0.7× increase, 0.8× increase, 0.9× increase, 1× increase, 2× increase, 3× increase, 4× increase, 5× increase, 6× increase, 7× increase, 8× increase, 9× increase, 10× increase, or greater than 10× increase in OX40 surface expression compared to activated CD8+ T cells treated with a control agent. In some embodiments, activated CD8+ T cells treated with a GAL9 antigen-binding molecule exhibit approximately 0.1 × to 10 ×, 0.5 × to 5 ×, or approximately 1.0 × to 2.0 × increases in OX40 surface expression compared to activated CD8+ T cells treated with a control agent.

[0112]

[0128] In some embodiments, the GAL9 antigen-binding molecule increases 4-1BB surface expression in activated CD8+ T cells compared 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 a 0.1× increase, 0.2× increase, 0.3× increase, 0.4× increase, 0.5× increase, 0.6× increase, 0.7× increase, 0.8× increase, 0.9× increase, 1× increase, 2× increase, 3× increase, 4× increase, 5× increase, 6× increase, 7× increase, 8× increase, 9× increase, 10× increase, or greater than 10× increase in 4-1BB surface expression compared to activated CD8+ T cells treated with a control agent. In some embodiments, activated CD8+ T cells treated with a GAL9 antigen-binding molecule exhibit approximately 0.1 × to 10 ×, 0.2 × to 2 ×, or approximately 0.5 × to 1 × increases in 4-1BB surface expression compared to activated CD8+ T cells treated with a control agent.

[0113]

[0129] In some embodiments, the GAL9 antigen-binding molecule increases CD27 surface expression in activated CD8+ T cells compared to activated CD8+ T cells treated with a control agent. In some embodiments, activated CD8+ T cells treated with the GAL9 antigen-binding molecule show at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, and 24% increased CD27 surface expression compared to activated CD8+ T cells treated with a control agent. Increase, 25% increase, 26% increase, 27% increase, 28% increase, 29% increase, 30% increase, 31% increase, 32% increase, 33% increase, 34% increase, 35% increase, 36% increase, 37% increase, 38% increase, 39% increase, 40% increase, 41% increase, 42% increase, 43% increase, 44% increase, 45% increase, 46% increase, 47% increase, 48% increase, 49% increase, 50 % increase, 51% increase, 52% increase, 53% increase, 54% increase, 55% increase, 56% increase, 57% increase, 58% increase, 59% increase, 60% increase, 61% increase, 62% increase, 63% increase, 64% increase, 65% increase, 66% increase, 67% increase, 68% increase, 69% increase, 70% increase, 71% increase, 72% increase, 73% increase, 74% increase, 75% increase, 7 The increases are 6%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, activated CD8+ T cells treated with the GAL9 antigen-binding molecule show at least about 1% to 100%, 5% to 50%, 10% to 40%, or about 20% to 30% of CD27 surface expression compared to activated CD8+ T cells treated with the control agent.

[0114]

[0130] In some embodiments, the GAL9 antigen-binding molecule increases ICOS surface expression in activated CD8+ T cells compared to activated CD8+ T cells treated with a control agent. In some embodiments, activated CD8+ T cells treated with the GAL9 antigen-binding molecule show at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, and 24% increased ICOS surface expression compared to activated CD8+ T cells treated with a control agent. Increase, 25% increase, 26% increase, 27% increase, 28% increase, 29% increase, 30% increase, 31% increase, 32% increase, 33% increase, 34% increase, 35% increase, 36% increase, 37% increase, 38% increase, 39% increase, 40% increase, 41% increase, 42% increase, 43% increase, 44% increase, 45% increase, 46% increase, 47% increase, 48% increase, 49% increase, 50 % increase, 51% increase, 52% increase, 53% increase, 54% increase, 55% increase, 56% increase, 57% increase, 58% increase, 59% increase, 60% increase, 61% increase, 62% increase, 63% increase, 64% increase, 65% increase, 66% increase, 67% increase, 68% increase, 69% increase, 70% increase, 71% increase, 72% increase, 73% increase, 74% increase, 75% increase, 7 The increase is 6%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, activated CD8+ T cells treated with the GAL9 antigen-binding molecule show an increase of at least approximately 1% to 100%, 5% to 50%, 10% to 40%, or approximately 20% to 30% in ICOS surface expression compared to activated CD8+ T cells treated with the control agent.

[0115]

[0131] 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 microscopic techniques, such as confocal microscopy.

[0116]

[0132] In some embodiments, the GAL9 antigen-binding molecule increases PD-L2 expression on the surface of dendritic cells (DCs). In some embodiments, the GAL9 antigen-binding molecule decreases PD-L1 expression on the surface of dendritic cells (DCs). In some embodiments, the DCs are activated DCs. This specification describes the activation of immune cells, including DCs. Surface expression of proteins, including PD-L1 and PD-L2, on DCs can be evaluated by any suitable means. For example, the percentage of DCs exhibiting detectable surface PD-L1 and / or PD-L2 can be measured, for example, by flow cytometry. In some embodiments, a population of dendritic cells treated with the GAL9 antigen-binding molecule exhibits a larger percentage of surface PD-L2-positive cells compared to a control population of dendritic cells 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, a population of dendritic cells treated with a GAL9 antigen-binding molecule shows an increase of approximately 0.1 × to 100 ×, 0.5 × to 20 ×, 1 × to 10 ×, or approximately 5 × to 6 × in the percentage of DCs exhibiting detectable surface PD-L2 expression compared to a control population of dendritic cells treated with a control agent, such as an isotype-controlled antigen-binding molecule. In some embodiments, a population of dendritic cells treated with a GAL9 antigen-binding molecule shows a decrease of approximately 1% to 50%, 5% to 30%, or approximately 10% to 20% in the percentage of DCs exhibiting detectable surface PD-L1 expression compared to a control population of dendritic cells treated with a control agent, such as an isotype-controlled antigen-binding molecule.

[0117]

[0133] In some embodiments, the GAL9 antigen-binding molecule increases the cell surface aggregation of PD-L2 in dendritic cells (DCs). In some embodiments, the DCs are activated DCs. Activation of immune cells, including DCs, is described herein. In some embodiments, the increase in PD-L2 cell surface aggregation is compared to DCs treated with a control agent. The control agent is described herein. In some embodiments, the control agent is an isotype antigen-binding molecule that does not bind to GAL9. The cell surface aggregation of PD-L2 in DCs can be evaluated by any preferred means, for example, confocal microscopy.

[0118]

[0134] In some embodiments, the GAL9 antigen-binding molecule increases IL-12 production by DCs. The DCs may be activated DCs. In some embodiments, the GAL9 antigen-binding molecule increases IL-12 production in DCs compared 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, a population of DCs treated with the GAL9 antigen-binding molecule shows an increase of approximately 0.1 × to 100 ×, 10 × to 75 ×, 20 × to 40 ×, 25 × to 35 ×, or approximately 28 × in the percentage of IL-12-positive DCs compared to a population of DCs treated with a control agent.

[0119]

[0135] 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 DC cells. In some embodiments, the immune cells may be NK cells.

[0120]

[0136] In some embodiments, the GAL9 antigen-binding molecule reduces the tumor burden of the subject. The subject may be a mammal. The mammal may be a mouse. In some embodiments, the mammal is a human. In some embodiments, the GAL9 antigen-binding molecule prevents tumor growth of the subject. The tumor may 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 compared to a subject 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.

[0121] 6.4.2. Variable Region

[0137] The GAL9-binding molecules described herein have an antibody variable region domain amino acid sequence, including VH and VL antibody domain sequences. The VH and VL sequences are described in more detail in sections 6.4.2.1 and 6.4.2.2, respectively.

[0122] 6.4.2.1.VH area

[0138] The VH amino acid sequence of the GAL9-binding molecules described herein is the antibody heavy chain variable domain sequence. In typical antibody configurations in both naturally occurring and the GAL9-binding molecules described herein, a specific VH amino acid sequence, in association with a specific VL amino acid sequence, forms an antigen-binding site. In various embodiments, the VH amino acid sequence is a human sequence, a synthetic sequence, or a mammalian sequence including a combination of a non-human mammalian sequence, a mammalian sequence, and / or a synthetic sequence, 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 variant sequence of a naturally occurring sequence.

[0123] 6.4.2.2.VL area

[0139] The VL amino acid sequences useful for GAL9-binding molecules described herein are antibody light chain variable domain sequences. In typical configurations in both natural antibodies and antibody constructs described herein, a specific VL amino acid sequence, in association with a specific VH amino acid sequence, forms an antigen-binding site. In various embodiments, the VL amino acid sequence is a human sequence, a synthetic sequence, or a mammalian sequence comprising a combination of a human sequence, a non-human mammalian sequence, a mammalian sequence, and / or a synthetic sequence, as described in further detail in sections 6.4.2.3 and 6.4.2.4 below.

[0124]

[0140] In various embodiments, the VL amino acid sequence is a variant 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.

[0125] 6.4.2.3. Complementarity Determination Region

[0141] The VH and VL amino acid sequences include highly variable sequences referred to as "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 alter the binding affinity of an antigen-binding site to a particular antigen or epitope. In certain embodiments, naturally occurring CDRs are mutated in vivo in a host by affinity maturation and somatic hypermutation. In certain embodiments, the CDRs are 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, random sequence CDR libraries and CDRs obtained from reasonably designed CDR libraries. CDR boundaries were determined using the Martin numbering scheme. Please refer to Figures 7A-1 to 7E-2.

[0126]

[0142] In various embodiments, a CDR identified to bind to a target antigen is further mutated to achieve desired binding characteristics, such as increased affinity for the target antigen compared to the original CDR (i.e., "affinity maturation"). For example, the introduction of targeted diversity into a CDR, including one identified to bind to a target antigen, can be done using degenerate oligonucleotides. Various randomization schemes can be used. For example, "soft randomization" can be used, which provides a high bias against the wild-type sequence identity of a given amino acid position, such as doping each codon position with four bases at non-equivalent levels, thereby biasing the wild-type sequence. As an example of soft randomization, if it is desired to achieve approximately 50% wild-type sequence, each base in each codon is maintained at 70% wild-type, and the other nucleotides are maintained at 10% each. A phage library focused around a selected CDR can be constructed using degenerate oligonucleotides, and the resulting phage particles can be used for phage panning under various stringent selective conditions as needed.

[0127] 6.4.2.4. Framework Area and CDR Porting

[0143] The VH and VL amino acid sequences include a “framework region” (FR) sequence. The FR acts as a scaffold for scattered CDRs (see Section 6.4.2.3) and is a generally conserved sequence region, typically in the FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 configuration (N-terminus to C-terminus). In various embodiments, the FR is a mammalian sequence, including, but not limited to, mouse, rat, hamster, rabbit, camel, donkey, goat, and human sequences. In preferred embodiments, the FR is a human sequence. In various embodiments, the FR is a naturally occurring sequence. In various embodiments, the FR is a synthetic sequence, including, but not limited to, a reasonably designed sequence.

[0128]

[0144] In various embodiments, both FR and CDR originate from the same naturally occurring variable domain sequence. In various embodiments, FR and CDR originate from different variable domain sequences, with the CDR being implanted in the FR scaffold and the CDR providing specificity for a particular antigen. In certain embodiments, all implanted CDRs originate from the same naturally occurring variable domain sequence. In certain embodiments, the implanted CDRs originate from different variable domain sequences. In certain embodiments, the implanted CDRs are synthetic sequences, including, but not limited to, random sequence CDR libraries and CDRs obtained from rationally designed CDR libraries. In certain embodiments, the implanted CDRs and FR originate from the same species. In certain embodiments, the implanted CDRs and FR originate from different species. In preferred embodiments of implanted CDRs, the antibody is “humanized,” and the implanted CDRs are non-human mammalian sequences, including, but not limited to, mouse, rat, hamster, rabbit, camel, donkey, and goat sequences, while the FR is a human sequence. Humanized antibodies are discussed in more detail in U.S. Patent No. 6,407,213. The entirety of this document is incorporated herein by reference with respect to all that it teaches. In various embodiments, a portion or specific sequence of FR derived from one species is used to replace a portion or specific sequence of FR from another species.

[0129] 6.4.3. Exemplary amino acid sequences of GAL9-binding molecules

[0145] In various embodiments, the GAL9-binding molecule includes a specific VH CDR3 (CDR-H3) sequence and a specific VL CDR3 (CDR-L3) sequence.

[0130]

[0146] In some embodiments, the GAL9-binding molecule includes CDR-H3 and CDR-L3 derived 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. The VH CDR amino acid sequences of the ABS clones are disclosed in Table 3. The VL CDR amino acid sequences of the ABS clones are disclosed in Table 4. For clarity, each GAL9 ABS clone is assigned a unique ABS clone number used throughout this disclosure.

[0131]

[0147] In one current preferred embodiment, the GAL9-binding molecule comprises CDR-H3 and CDR-L3 of ABS clone P9-28.

[0132]

[0148] In some embodiments, the GAL9-binding molecule comprises all three VH CDRs derived 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 one current preferred embodiment, the GAL9-binding molecule comprises all three VH CDRs derived from ABS clone P9-28.

[0133]

[0149] In some embodiments, the GAL9-binding molecule comprises all three VL CDRs derived 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 one current preferred embodiment, the GAL9-binding molecule comprises all three VL CDRs derived from ABS clone P9-28.

[0134]

[0150] In some embodiments, the GAL9-binding molecule comprises all six CDRs derived 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 one current preferred embodiment, the GAL9-binding molecule comprises all six CDRs derived from ABS clone P9-28.

[0135]

[0151] In some embodiments, the GAL9-binding molecule includes a VH amino acid sequence, a VL amino acid sequence, or both VH and VL amino acid sequences derived 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 complete immunoglobulin heavy chain and immunoglobulin light chain sequences, as well as the VH and VL amino acid sequences, are provided in Table 6. In certain current preferred embodiments, the GAL9-binding molecule comprises a VH amino acid sequence, a VL amino acid sequence, or both VH and VL amino acid sequences derived from ABS clone P9-28.

[0136]

[0152] In some embodiments, the GAL9-binding molecule comprises a complete IgG heavy chain sequence and a complete IgG light chain sequence derived 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 one current preferred embodiment, the GAL9-binding molecule comprises a complete IgG heavy chain sequence and a complete IgG light chain sequence derived from the ABS clone P9-28.

[0137] 6.4.4. Steady-state region

[0153] In a GAL9-binding molecule, the GAL9-binding molecule may 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 the antibody. The constant region can refer to the CH1, CH2, CH3, CH4, or CL constant domain.

[0138]

[0154] 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 preferred embodiments, the constant region sequence is a human sequence. In certain embodiments, the constant region sequence is derived from an antibody light chain. In certain embodiments, the constant region sequence is derived from a lambda or kappa light chain. In certain embodiments, the constant region sequence is derived from an antibody heavy chain. In certain embodiments, the constant region sequence is an antibody heavy chain sequence that is an IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM isotype. In certain embodiments, the constant region sequence is derived from an IgG isotype. In preferred embodiments, the constant region sequence is derived from an IgG1 isotype.

[0139]

[0155] Exemplary steady-state regions and their modifications are described in International Publication No. 2018075692, which is incorporated herein by reference in its entirety.

[0140] 6.4.4.1. CH1 and CL regions

[0156] The CH1 amino acid sequence is the sequence of the second domain of the antibody heavy chain, relative to the N-terminus to the C-terminus of the innate antibody heavy chain architecture, as described herein. 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 preferred embodiments, the CH1 sequence is a human sequence. In certain embodiments, the CH1 sequence is derived from IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM isotypes. In preferred embodiments, the CH1 sequence is derived from the IgG1 isotype. In preferred embodiments, the CH1 sequence is UniProt acceptance number P01857 amino acids 1-98.

[0141]

[0157] The CL amino acid sequences useful for GAL9-binding molecules described herein are antibody light chain constant domain sequences based on the innate antibody light chain architecture. 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.

[0142]

[0158] In certain embodiments, the CL amino acid sequence is a lambda (λ) light chain constant domain sequence. In certain embodiments, the CL amino acid sequence is a human lambda light chain constant domain sequence. In preferred embodiments, the lambda (λ) light chain sequence is UniProt acceptance number P0CG04.

[0143]

[0159] In certain embodiments, the CL amino acid sequence is the kappa(κ) light chain constant domain sequence. In preferred embodiments, the CL amino acid sequence is the human kappa(κ) light chain constant domain sequence. In preferred embodiments, the kappa light chain sequence is UniProt acceptance number P01834.

[0144]

[0160] In certain embodiments, both the CH1 sequence and the CL sequence are endogenous sequences. In certain embodiments, the CH1 sequence and the CL sequence each include orthogonal modifications to the endogenous CH1 and CL sequences, respectively, as discussed in more detail in Section 6.4.4.1 below. The CH1 and CL sequences may also be parts thereof, and may be either endogenous sequences or modified sequences, so that a domain having the CH1 sequence or a portion thereof can be associated with a domain having the CL sequence or a portion thereof.

[0145] 6.4.4.2. Modification of CH1 and CL orthogonality

[0161] In certain embodiments, the CH1 and CL sequences contain orthogonal modifications to the endogenous CH1 and CL sequences, respectively. These orthogonal mutations are generally described in more detail in sections 6.4.6.1 to 6.4.6.3 below.

[0146]

[0162] In certain embodiments, the orthogonal modification in the endogenous CH1 and CL sequences is a genetically engineered disulfide bridge selected from genetically engineered cysteine ​​at position 138 of the CH1 sequence and position 116 of the CL sequence, position 128 of the CH1 sequence and position 119 of the CL sequence, or position 129 of the CH1 sequence and position 210 of the CL sequence, with numbering as discussed in more detail in U.S. Patent Nos. 8,053,562 and 9,527,927, each of which is incorporated herein by reference in its entirety. In preferred embodiments, the genetically engineered cysteine ​​is located at position 128 of the CH1 sequence and position 118 of the CL kappa sequence, with numbering as per the Eu index.

[0147]

[0163] In a series of preferred embodiments, the mutations providing non-endogenous cysteine ​​amino acids are either the F118C mutation of the CL sequence having the corresponding A141C in the CH1 sequence, or the F118C mutation of the CL sequence having the corresponding L128C in the CH1 sequence, or the S162C mutation of the CL sequence having the corresponding P171C mutation in the CH1 sequence, numbered according to the Eu index.

[0148]

[0164] In various embodiments, the orthogonal mutations in the CL and CH1 sequences are charge-pair mutations. In certain embodiments, the charge-pair mutation is an F118S, F118A, or F118V mutation in the CL sequence having the corresponding A141L in the CH1 sequence, or a T129R mutation in the CL sequence having the corresponding K147D in the CH1 sequence, numbered according to the Eu index, as described in more detail in Bonisch et al. (Protein Engineering, Design & Selection, 2017, pp. 1-12). This document is incorporated herein by reference in all that it teaches. In a series of preferred embodiments, the charge-pair mutation is an N138K mutation in the CL sequence having the corresponding G166D in the CH1 sequence, or an N138D mutation in the CL sequence having the corresponding G166K in the CH1 sequence, numbered according to the Eu index.

[0149] 6.4.4.3.CH2 area

[0165] In the GAL9-binding molecules described herein, the GAL9-binding molecule may have a CH2 amino acid sequence. The CH2 amino acid sequence is the CH2 amino acid sequence of the third domain of the antibody heavy chain, relative to the N-terminus to the C-terminus of the innate antibody heavy chain architecture, as described herein. 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 preferred embodiments, the CH2 sequence is a human sequence. In certain embodiments, the CH2 sequence is derived from an IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM isotype. In preferred embodiments, the CH2 sequence is derived from an IgG1 isotype.

[0150]

[0166] In certain embodiments, the CH2 sequence is an endogenous sequence. In preferred embodiments, the sequence is UniProt acceptance number P01857 amino acids 111-223.

[0151]

[0167] In a series of embodiments, the GAL9-binding molecule has more than one set of pairs of CH2 domains having a CH2 sequence, and the first set has one or more orthologous sets of CH2 amino acid sequences derived from a first isotype and CH2 amino acid sequences derived from another isotype. The orthologous CH2 amino acid sequences can interact with CH2 amino acid sequences derived from a common isotype as described herein, but do not interact significantly with CH2 amino acid sequences derived from another isotype present in the GAL9-binding molecule. In certain embodiments, the entire set of CH2 amino acid sequences is derived from the same species. In preferred embodiments, the entire set of CH2 amino acid sequences is a human CH2 amino acid sequence. In other embodiments, the sets of CH2 amino acid sequences are derived from different species. In certain embodiments, the first set of CH2 amino acid sequences is derived from the same isotype as other non-CH2 domains in the GAL9-binding molecule. In certain embodiments, the first set has CH2 amino acid sequences derived from an IgG isotype, and one or more orthologous sets have CH2 amino acid sequences derived from an IgM or IgE isotype. In certain embodiments, one or more of the set of CH2 amino acid sequences are endogenous CH2 sequences. In other embodiments, one or more of the set of CH2 amino acid sequences are endogenous CH2 sequences with one or more mutations. In certain embodiments, one or more mutations are orthogonal knob-hole mutations, orthogonal charge-pair mutations, or orthogonal hydrophobic mutations. Orthologous CH2 amino acid sequences useful for GAL9-binding molecules are described in more detail in International Publication Nos. 2017 / 011342 and International Publication Nos. 2017 / 106462. These documents are incorporated herein by reference in their entirety.

[0152] 6.4.4.4.CH3 area

[0168] The CH3 amino acid sequence is the sequence of the C-terminal domain of the antibody heavy chain, relative to the N-terminus to the C-terminus of the natural antibody heavy chain architecture, as described herein.

[0153]

[0169] 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 preferred embodiments, the CH3 sequence is a human sequence. In certain embodiments, the CH3 sequence is derived from IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, or IgG4 isotypes, or the CH4 sequence is derived from IgE or IgM. In certain embodiments, the CH3 sequence is derived from an IgG isotype. In preferred embodiments, the CH3 sequence is derived from an IgG1 isotype.

[0154]

[0170] In certain embodiments, the CH3 sequence is an endogenous sequence. In certain embodiments, the CH3 sequence is UniProt acceptance number P01857 amino acids 224-330. In various embodiments, the CH3 sequence is a segment of the endogenous CH3 sequence. In certain embodiments, the CH3 sequence has an endogenous CH3 sequence lacking N-terminal amino acids G224 and Q225. In certain embodiments, the CH3 sequence has an endogenous CH3 sequence lacking C-terminal amino acids P328, G329, and K330. In certain embodiments, the CH3 sequence has an endogenous CH3 sequence lacking both N-terminal amino acids G224 and Q225, as well as C-terminal amino acids P328, G329, and K330. In preferred embodiments, the GAL9-binding molecule has multiple domains having a CH3 sequence, where the CH3 sequence can refer to both a fully endogenous CH3 sequence and a CH3 sequence lacking an N-terminal amino acid, a C-terminal amino acid, or both.

[0155]

[0171] In certain embodiments, the CH3 sequence is an endogenous sequence having one or more mutations. In certain embodiments, the mutations are one or more orthogonal mutations introduced into the endogenous CH3 sequence to lead to a specific pair of a particular CH3 sequence, as described in more detail in sections 6.4.6.1 to 6.4.6.3 below.

[0156]

[0172] In certain embodiments, the CH3 sequence is genetically engineered to reduce the immunogenicity of the antibody by replacing a specific amino acid of one allotype with an amino acid of another allotype, as described in more detail in Stickler et al. (Genes Immun. April 2011; Vol. 12 (No. 3): pp. 213-221), which is referred to herein as isoallotypic mutation. This document is incorporated herein by reference with respect to all that it teaches. In certain embodiments, a specific amino acid of the G1m1 allotype is replaced. In preferred embodiments, the CH3 sequence has isoallotypic mutations D356E and L358M.

[0157]

[0173] In some embodiments, the IgG1 CH3 amino acid sequence includes the following mutations: P343V; Y349C; and tripeptide insertions, 445P, 446G, 447K. In other preferred embodiments, domain B has a human IgG1 CH3 sequence having the following mutations: T366K; and tripeptide insertions, 445K, 446S, 447C. In yet another preferred embodiment, domain B has a human IgG1 CH3 sequence having the following mutations: Y349C and tripeptide insertions, 445P, 446G, 447K.

[0158]

[0174] In some embodiments, the IgG1 CH3 amino acid sequence includes a 447C mutation that would otherwise be incorporated into the endogenous CH3 sequence.

[0159] 6.4.5. Antigen binding site

[0175] In some embodiments, a VL or VH amino acid sequence and an identical VL or VH amino acid sequence together form a first antigen-binding site (ABS). The antigen-binding site (ABS) is capable of specifically binding to an antigen epitope. Antigen binding by the ABS is described in more detail in Section 6.4.5.1 below.

[0160]

[0176] In an alternative embodiment, for example, if the GAL9-binding molecule is a single-domain antibody, the VH or VL amino acid sequence forms a first ABS.

[0161]

[0177] In some embodiments, the GAL9 antigen-binding molecule includes a second ABS. In some embodiments, the second ABS is specific to 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.

[0162]

[0178] In some embodiments, the second ABS is specific to a different epitope of the first GAL9 antigen. For example, the first ABS includes a CDR or variable domain derived 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 this case, the second ABS may include a CDR or variable domain derived 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.

[0163]

[0179] In some embodiments, the GAL9 antigen-binding molecule is multispecific; for example, the second ABS of the GAL9 antigen-binding molecule specifically binds to a different antigen than the GAL9 antigen to which the first ABS specifically binds.

[0164] 6.4.5.1. Antigen binding by ABS

[0180] ABS and GAL9-binding molecules containing such ABS are said to "recognize" an epitope (or more generally an antigen) to which the ABS specifically binds, and the epitope (or more generally the antigen) is said to be the "recognition specificity" or "binding specificity" of the ABS.

[0165]

[0181] ABS is said to bind to its specific antigen or epitope with a particular affinity. As described herein, "affinity" refers to the strength of the non-covalent intermolecular force interaction between one molecule and another. The affinity, i.e., the strength of the interaction, can be expressed as the dissociation equilibrium constant (K D ), and the lower the K D value, the stronger the intermolecular interaction. The K D value of an antibody construct is measured by methods well known in the art, including but not limited to biolayer interferometry (e.g., Octet / FORTEBIO®), surface plasmon resonance (SPR) technology (e.g., Biacore®), and cell binding assays. For the purposes of this specification, affinity is the dissociation equilibrium constant measured by biolayer interferometry using Octet / FORTEBIO®.

[0166]

[0182] "Specific binding" as used herein refers to the affinity between an ABS and its cognate antigen or epitope, and the K D value is less than 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, or 10 -10 M.

[0167]

[0183] The number of ABS in a GAL9-binding molecule as described herein defines the "valence" of the GAL9-binding molecule. A GAL9-binding molecule with a single ABS is said to be "monovalent". A GAL9-binding molecule with multiple ABS is said to be "polyvalent". A polyvalent GAL9-binding molecule with two ABS is "divalent". A polyvalent GAL9-binding molecule with three ABS is "trivalent". A polyvalent GAL9-binding molecule with four ABS is "tetravalent".

[0168]

[0184] In various polyvalent embodiments, all of the ABS have the same recognition specificity. Such a GAL9-binding molecule is a "single-specificity," "polyvalent" binding construct. In other polyvalent embodiments, at least two of the ABS have different recognition specificities. Such a GAL9-binding molecule is polyvalent and "multi-specific." In a polyvalent embodiment where the ABS collectively have two recognition specificities, the GAL9-binding molecule is "bi-specific." In a polyvalent embodiment where the ABS collectively have three recognition specificities, the GAL9-binding molecule is "triple-specific."

[0169]

[0185] In a multivalent embodiment where ABS collectively has multiple recognition specificities for different epitopes present on the same antigen, the GAL9-binding molecule is "multiparatopic." In a multivalent embodiment where ABS collectively recognizes two epitopes of the same antigen, it is "biparatopic."

[0170]

[0186] In various polyvalent embodiments, the polyvalentity of the GAL9-binding molecule enhances the avidity of the GAL9-binding molecule to a specific target. As described herein, “avidity” refers to the overall strength of the interaction between two or more molecules, for example, a polyvalent GAL9-binding molecule to a specific target, and avidity is the cumulative strength of the interaction provided by the affinity of multiple ABSs. Avidity can be measured in the same way as that used to determine affinity, as described above. In a particular embodiment, the avidity of the GAL9-binding molecule to a specific target is such that the interaction is a specific binding interaction, and the avidity between the two molecules is 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, or 10 -10 Less than M K D It has a value. In a particular embodiment, the avidity of a GAL9-binding molecule to a particular target is such that the interaction is a specific binding interaction. D The affinity of one or more individual ABSs is such that they themselves are eligible for specific binding to their respective antigens or epitopes. D It does not have a value. In certain embodiments, avidity is the cumulative strength of the interaction provided by the affinities of multiple ABS to a common specific target or to separate antigens on a complex, such as separate antigens found on individual cells. In certain embodiments, avidity is the cumulative strength of the interaction provided by the affinities of multiple ABS to separate epitopes on a common individual antigen.

[0171] 6.4.6. Modification of Orthogonality

[0187] In the GAL9-binding molecules described herein, the GAL9-binding molecule may have a constant region domain including orthogonal modifications. The amino acid sequence of the constant region domain is described in more detail in Section 6.4.4 above.

[0172]

[0188] As described herein, “orthogonal modification” or synonymous “orthogonal mutation” is one or more genetically 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, an orthogonal modification decreases the affinity of a domain having an orthogonal modification to a domain lacking a complementary orthogonal modification. In certain embodiments, an orthogonal modification is a mutation in the endogenous antibody domain sequence. In various embodiments, an orthogonal modification is a modification of the N-terminus or C-terminus of the endogenous antibody domain sequence, including, but not limited to, amino acid additions or deletions. In certain embodiments, examples of orthogonal modifications include, but are not limited to, genetically engineered disulfide crosslinks, knob-in-hole mutations, and charge-pair mutations, as described in more detail in sections 6.4.6.1 to 6.4.6.3 below. In certain embodiments, orthogonal modifications include, but are not limited to, combinations of orthogonal modifications selected from genetically engineered disulfide crosslinks, knob-in-hole mutations, and charge-pair mutations. In certain embodiments, orthogonal modifications can be combined with immunogenicity-reducing amino acid substitutions, such as isoallotype mutations, as described in more detail in Section 6.4.4.4 above.

[0173] 6.4.6.1. Orthogonal Gene Manipulation Disulfide Crosslinking

[0189] In various embodiments, orthogonal modifications include mutations that generate a genetically engineered disulfide bridge between a first domain and a second domain. As described herein, a “genetically engineered disulfide bridge” is a mutation that provides two or more domains with non-endogenous cysteine ​​amino acids such that a non-natural disulfide bond is formed when two or more domains are associated. Genetically engineered disulfide bridges are described in more detail in Merchant et al. (Nature Biotech (1998) Vol. 16: pp. 677-681). The entirety of that document is incorporated herein by reference with respect to all that it teaches. In certain embodiments, the genetically engineered disulfide bridge improves orthogonal association between specific domains. In certain embodiments, the mutations that generate a genetically engineered disulfide bridge are the K392C mutation in one of the first or second CH3 domains and the D399C mutation in the other CH3 domain. In a preferred embodiment, the mutations that generate the genetically engineered disulfide crosslink are the S354C mutation in one of the first or second CH3 domains and the Y349C mutation in the other CH3 domain. In another preferred embodiment, the mutations that generate the genetically engineered disulfide crosslink are the 447C mutation in both the first and second CH3 domains, provided by the elongation of the C-terminus of the CH3 domain into which the KSC tripeptide sequence is incorporated.

[0174] 6.4.6.2. Orthogonal Knob-Hole Mutations

[0190] In various embodiments, orthogonal modifications include knob-hole (synonymously, knob-in-hole) mutations. As described herein, a knob-hole mutation is a mutation that alters the steric features of the surface of a first domain such that the first domain preferentially associates with a second domain having a complementary steric mutation over association with a domain that does not have a complementary steric mutation. Knob-hole mutations are described in more detail in U.S. Patents 5,821,333 and 8,216,805, each of which is incorporated herein by reference in whole. In various embodiments, knob-hole mutations are combined with genetically engineered disulfide crosslinks, as described in more detail in Merchant et al. (Nature Biotech (1998) Vol. 16: pp. 677-681). In various embodiments, knob-hole mutations, isoallotyping mutations, and genetically engineered disulfide mutations are combined.

[0175]

[0191] In certain embodiments, the knob-in-hole mutations are the T366Y mutation in the first domain and the Y407T mutation in the second domain. In certain embodiments, the knob-in-hole mutations are the F405A mutation in the first domain and the T394W mutation in the second domain. In certain embodiments, the knob-in-hole mutations are the T366Y mutation and the F405A mutation in the first domain, and the T394W mutation and the Y407T mutation in the second domain. In certain embodiments, the knob-in-hole mutations are the T366W mutation in the first domain and the Y407A mutation in the second domain. In certain embodiments, the combination of knob-in-hole mutations and genetically modified disulfide mutations is the S354C and T366W mutations in the first domain, and the Y349C, T366S, L368A, and Y407V mutations in the second domain. In preferred embodiments, combinations of knob-in-hole mutations, isoallotype mutations, and genetically modified disulfide mutations include the S354C and T366W mutations in the first domain, and the Y349C, D356E, L358M, T366S, L368A, and Y407V mutations in the second domain.

[0176] 6.4.6.3. Orthogonal Charge Pair Displacement

[0192] In various embodiments, the orthogonal modification is a charge-pair mutation. As used herein, a charge-pair mutation is a mutation that affects the charge of an amino acid on the surface of a domain such that the domain preferentially associates with a second domain having a complementary charge-pair mutation compared to association with a domain that does not have a complementary charge-pair mutation. In certain embodiments, the charge-pair mutation improves the orthogonal association between specific domains. Charge-pair mutations are described in more detail in U.S. Patents 8,592,562, 9,248,182, and 9,358,286, each of which is incorporated herein by reference with respect to all that they teach. In certain embodiments, the charge-pair mutation improves the stability between specific domains. In a preferred embodiment, the charge-pair mutation is a T366K mutation in a first domain and an L351D mutation in the other domain.

[0177]

[0193] In certain 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 of VH and the corresponding Q38K of VL, or Q39K of VH and the corresponding Q38E of VL, as described in more detail in Igawa et al. (Protein Eng. Des. Sel, 2010, Vol. 23, pp. 667-677). This document is incorporated herein by reference with respect to all that it teaches.

[0178] 6.4.7. Trivalent and tetravalent GAL9-binding molecules

[0194] 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.”

[0179] 6.5. GAL9-binding molecular architecture

[0195] The antigen-binding sites described herein, including specific CDR subsets, can be formatted into any binding molecular architecture, including, but not limited to, full-length antibodies, Fab fragments, Fv, scFv, tandem scFv, diabodies, scdiabodies, DART, tandAb, minibodies, camel 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, pp. 182-212). This document is incorporated herein by reference in all its teachings. The antigen-binding sites described herein, including specific CDR subsets, can also be formatted into a “B-body” format, as described in more detail in U.S. Patent Application Publication No. 2018 / 0118811 and International Publication No. 2018 / 075692. Each of these documents is incorporated herein by reference in its entirety.

[0180] 6.6. Further Modification

[0196] In a further series of embodiments, the GAL9-binding molecule has additional modifications.

[0181] 6.6.1. Antibody-drug conjugates

[0197] In various embodiments, a GAL9-binding molecule is conjugated to a therapeutic agent (i.e., a drug) to form a GAL9-binding molecule-drug conjugate. Examples of therapeutic agents include, but are not limited to, chemotherapeutic agents, contrast 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 attached to the GAL9-binding molecule via a linker peptide, as discussed in more detail in Section 6.6.3 below.

[0182]

[0198] Methods for preparing antibody-drug conjugates (ADCs) that can be configured to conjugate a drug to the GAL9-binding molecule disclosed herein are described in the following literature: for example, U.S. Patent No. 8,624,003 (pot method), U.S. Patent No. 8,163,888 (one-step method), U.S. Patent No. 5,208,020 (two-step method), U.S. Patent No. 8,337,856, U.S. Patent No. 5,773, Publication No. 001, U.S. Publication No. 7,829,531, U.S. Publication No. 5,208,020, U.S. Publication No. 7,745,394, International Publication No. 2017 / 136623, International Publication No. 2017 / 015502, International Publication No. 2017 / 015496, International Publication No. 2017 / 015495, International Publication No. 2004 / 010957, International Publication No. 2005 / 077090, International Publication No. 2005 / 082023, International Publication International Publication No. 2006 / 065533, International Publication No. 2007 / 030642, International Publication No. 2007 / 103288, International Publication No. 2013 / 173337, International Publication No. 2015 / 057699, International Publication No. 2015 / 095755, International Publication No. 2015 / 123679, International Publication No. 2015 / 157286, International Publication No. 2017 / 165851, International Publication No. 2009 / 073445, International Publication No. 20 International Publication No. 10 / 068759, International Publication No. 2010 / 138719, International Publication No. 2012 / 171020, International Publication No. 2014 / 008375, International Publication No. 2014 / 093394, International Publication No. 2014 / 093640, International Publication No. 2014 / 160360, International Publication No. 2015 / 054659, International Publication No. 2015 / 195925, International Publication No. 2017 / 160754, Storz (MAbs. (November-December 2015; Vol. 7 (No. 6): pp. 989-1009), Lambert et al. (Adv Ther, 2017, Vol. 34: p. 1015), Diamantis et al. (British Journal of Cancer, 2016, Vol. 114, pp. 362-367), Carrico et al. (Nat Chem Biol, 2007, Vol. 3: pp. 321-322), We et al. (Proc Natl Acad Sci USA, 2009, Vol. 106: pp. 3000-3005), Rabuka et al. (Curr Opin Chem Biol.), 2011, Vol. 14: pp. 790-796), Hudak et al. (Angew Chem Int Ed Engl., 2012: pp. 4161-4165), Rabuka et al. (Nat Protoc., 2012, Vol. 7: pp. 1052-1067), Agarwal et al. (Proc Natl Acad Sci USA., 2013, Vol. 110: pp. 46-51), Agarwal et al. (Bioconjugate Chem., 2013, Vol. 24: pp. 846-851), Barfield et al. (Drug Dev. and D., 2014, Vol. 14: pp. 34-41), Drake et al. (Bioconjugate Chem., 2014, Vol. 25: pp. 1331-1341), Liang et al. (J Am Chem Soc., 2014, Vol. 136: (pp. 10850-1083), Drake et al. (Curr Opin Chem Biol., 2015, Vol. 28: pp. 174-1780), and York et al. (BMC Biotechnology, 2016, Vol. 16 (No. 1): p. 23). Each of these publications is incorporated herein by reference in its entirety for all that it teaches.

[0183] 6.6.2. Additional coupling parts

[0199] In various embodiments, the GAL9-binding molecule has modifications including one or more additional binding moieties. In certain embodiments, the binding moieties are antibody fragments or antibody formats, including, but not limited to, full-length antibodies, Fab fragments, Fv, scFv, tandem scFv, diabodies, scdiabodies, DART, tandAb, minibodies, camel 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, pp. 182-212). This document is incorporated herein by reference with respect to all that it teaches.

[0184]

[0200] In certain embodiments, one or more additional binding moieties are attached to the C-terminus of the first or third polypeptide chain. In certain embodiments, one or more additional binding moieties are attached to the C-terminuses of both the first and third polypeptide chains. In certain embodiments, one or more additional binding moieties are attached to the C-terminuses of both the first and third polypeptide chains. In certain embodiments, the individual portions of one or more additional binding moieties are attached separately to the C-terminuses of the first and third polypeptide chains so that such portions form a functional binding moiety.

[0185]

[0201] In certain embodiments, one or more additional binding sites are attached to the N-terminus of any of the polypeptide chains (e.g., the first, second, third, fourth, fifth, or sixth polypeptide chains). In certain embodiments, the individual parts of the additional binding sites are attached separately to the N-terminuses of different polypeptide chains so that these parts form a functional binding site.

[0186]

[0202] In certain embodiments, one or more additional binding sites are specific to different antigens or epitopes of ABS within the GAL9 binding molecule. In certain embodiments, one or more additional binding sites are specific to the same antigen or epitope of ABS within the GAL9 binding molecule. In certain embodiments, if the modification consists of two or more additional binding sites, the additional binding sites are specific to the same antigen or epitope. In certain embodiments, if the modification consists of two or more additional binding sites, the additional binding sites are specific to different antigens or epitopes.

[0187]

[0203] In certain embodiments, one or more additional binding moieties are attached to the GAL9-binding molecule using in vitro methods including reactive chemistry and affinity tagging systems, as discussed in more detail in Section 6.6.3 below. In certain embodiments, one or more additional binding moieties are attached to the GAL9-binding molecule via Fc-mediated binding (e.g., protein A / G). In certain embodiments, one or more additional binding moieties are attached to the GAL9-binding molecule using recombinant DNA techniques, such as encoding the nucleotide sequence of the fusion product of the GAL9-binding molecule and the additional binding moieties in the same expression vector (e.g., plasmid).

[0188] 6.6.3. Functional Groups / Reactive Groups

[0204] In various embodiments, the GAL9-binding molecule has modifications including functional groups or chemical reaction groups that can be used in downstream processes such as linkage with additional parts (e.g., drug conjugates and additional binding parts as discussed in more detail in sections 6.6.1 and 6.6.2 above) and downstream purification processes.

[0189]

[0205] In certain embodiments, modifications are chemical reaction groups including, but are not limited to, reactive thiols (e.g., maleimide-based reactive groups), reactive amines (e.g., N-hydroxysuccinimide-based reactive groups), "click chemical" groups (e.g., reactive alkyne groups), and aldehydes having formylglycine (FGly). In certain embodiments, modifications are functional groups including affinity peptide sequences (e.g., HA, HIS, FLAG, GST, MBP, and Strep systems, etc.). In certain embodiments, the functional group or chemical reaction group has a cleavable peptide sequence. In certain embodiments, the cleavable peptide is cleaved by means including, but are not limited to, photocleavage, chemical cleavage, protease cleavage, reducing conditions, and pH conditions. In certain embodiments, protease cleavage is carried out by an intracellular protease. In certain embodiments, protease cleavage is carried out by an extracellular or membrane-bound protease. ADC therapy utilizing protease cleavage is described in more detail in Choi et al. (Theranosties, 2012; Vol. 2 (No. 2): pp. 156-178). This document is incorporated herein by reference to all that it teaches.

[0190] 6.6.4. Reduction of Effector Functions

[0206] In certain embodiments, the GAL9-binding molecule has one or more genetically modified mutations in the amino acid sequence of the antibody domain that reduce the effector function naturally associated with antibody binding. Effector functions include, but are not limited to, cellular functions resulting from Fc receptors that bind to the Fc portion of the antibody, such as antibody-dependent cell-mediated cytotoxicity (ADCC), complement binding (e.g., Clq binding), antibody-dependent cell-mediated phagocytosis (ADCP), and opsonization. Exemplary genetically engineered mutations that reduce effector function are described in more detail in U.S. Patent Application Publication 2017 / 0137530, Armour et al. (Eur. J. Immunol. Vol. 29 (No. 8) (1999), pp. 2613-2624), Shields et al. (J. Biol. Chem. Vol. 276 (No. 9) (2001), pp. 6591-6604), and Oganesyan et al. (Acta Cristallographica D64 (2008), pp. 700-704). Each of these documents is incorporated herein by reference in its entirety.

[0191] 6.7. Purification method

[0207] This specification provides methods for purifying GAL9-binding molecules. Purification steps include, but are not limited to, purifying 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, including the use of protein A, protein G, or protein A / G reagents, may be performed. Multiple repetitions of a single purification method may be performed. Combinations of purification methods may be performed.

[0192] 6.7.1. Assembly and Purity of the Composite

[0208] In embodiments of the present invention, at least four distinct polypeptide chains are associated together to form a complete complex, i.e., a GAL9-binding molecule. However, incomplete complexes may also be formed that do not contain at least four distinct polypeptide chains. For example, an incomplete complex may be formed that has only one, two, or three of the polypeptide chains. In other examples, an incomplete complex may contain more than three polypeptide chains but not at least four distinct polypeptide chains. For example, an incomplete complex may be improperly associated with one or more copies of the distinct polypeptide chains. In the method of the present invention, the complex, i.e., the fully assembled GAL9-binding molecule, is purified from the incomplete complex.

[0193]

[0209] Methods for evaluating the effectiveness and efficiency of the 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 evaluated according to various criteria. Examples of criteria, but are not limited to, include: 1) evaluating the percentage of total protein in the eluate provided by the fully assembled GAL9-binding molecule; 2) evaluating the concentration ratio or increase percentage of the method for purifying the desired product, e.g., comparing the total protein in the eluate provided by the fully assembled GAL9-binding molecule with the total protein of the starting sample; and 3) evaluating the percentage of total protein, or the decrease percentage of undesirable products, e.g., the incomplete complexes described above, including determining the percentage or decrease percentage of specific undesirable products (e.g., unaccompanied single polypeptide chains, dimers of any combination of polypeptide chains, or trimers of any combination of polypeptide chains). Purity can be evaluated after any combination of the methods described herein.

[0194] 6.8. Manufacturing method

[0210] The GAL9-binding molecules described herein can be readily produced by expression using standard cell-free translation, transient transfection, and stable transfection methods currently used in antibody production. In certain embodiments, Expi293 cells (ThermoFisher) can be used to produce GAL9-binding molecules using ThermoFisher protocols or reagents 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, Vol. 19: p. 11). This document is incorporated herein by reference in all respects it provides.

[0195]

[0211] The expressed proteins, though not limited to these, can be readily separated from undesirable proteins and protein complexes using various purification strategies, including the use of protein A, protein G, or protein A / G reagents. Further purification can be achieved using ion exchange chromatography, as routinely used in the art.

[0196] 6.9. Pharmaceutical Compositions

[0212] In another embodiment, a pharmaceutical composition is provided comprising a GAL9-binding molecule as described herein and a pharmaceutically acceptable carrier or excipient. In a typical embodiment, the pharmaceutical composition is sterile.

[0197]

[0213] In various embodiments, the pharmaceutical composition contains GAL9-binding molecules at concentrations ranging from 0.1 mg / ml to 100 mg / ml. In specific embodiments, the pharmaceutical composition contains GAL9-binding molecules at concentrations 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 contains GAL9-binding molecules at concentrations exceeding 10 mg / ml. In certain embodiments, the GAL9-binding molecules are present at concentrations 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 higher. In certain embodiments, the GAL9-binding molecules are present at concentrations exceeding 50 mg / ml.

[0198]

[0214] In various embodiments, pharmaceutical compositions are described in more detail in U.S. Patent Nos. 8,961,964, 8,945,865, 8,420,081, 6,685,940, 6,171,586, 8,821,865, 9,216,219, U.S. Patent Application No. 10 / 813,483, International Publication No. 2014 / 066468, International Publication No. 2011 / 104381, and International Publication No. 2016 / 180941. Each of these documents is incorporated herein in its entirety.

[0199] 6.10. Treatment

[0215] In another embodiment, a treatment method is provided which includes the step of administering a GAL9-binding molecule as described herein to a patient having a disease or condition in an amount effective for the treatment of the patient.

[0200] 6.10.1. Target

[0216] In some embodiments, the subject is a mammal. In some embodiments, the mammal is a mouse. In preferred embodiments, the mammal is a human.

[0201] 6.10.2. Combination Therapy

[0217] GAL9-binding molecules can be used alone or in combination with other therapeutic agents or procedures to treat or prevent a disease or condition. Depending on the disease to be treated, the GAL9-binding molecule may be administered either simultaneously with or sequentially with a second therapeutic agent.

[0202]

[0218] In some embodiments, anti-GAL9 binding molecules are used in clinical settings or in combination with agents or procedures within the scope of current standard treatment to treat or prevent diseases or conditions such as proliferative disorders or cancer. In some embodiments, 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.

[0203] 6.10.3. Proliferative Disorders

[0219] In some embodiments, the treatment involves administering one or more GAL9-binding molecules as described herein to a subject with a proliferative disorder in an amount effective for treating the subject.

[0204]

[0220] In some embodiments, the treatment involves administering one or more GAL9-binding molecules in an effective amount as described herein to treat cancer and / or precancerous conditions. In some embodiments, the treatment involves administering one or more GAL9-binding molecules in an effective amount as described herein in combination with another cancer therapy and / or treatment regimen (such as radiation or surgery).

[0205]

[0221] In various embodiments, cancer is cancer of the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal tract, gums, head, kidney, liver, lung, nasopharynx, neck, head and neck, ovaries, prostate, pancreas, skin, stomach, testes, tongue, or uterus.

[0206]

[0222] In some embodiments, cancerous or precancerous tumors include neoplasms, malignant tumors, carcinomas, undifferentiated tumors, giant cell and spindle cell carcinomas, small cell carcinoma, papillary carcinoma, squamous cell carcinoma, head and neck squamous cell carcinoma, lymphoepithelial carcinoma, basal cell carcinoma, follicular carcinoma, transitional cell carcinoma, papillary transitional cell carcinoma, adenocarcinoma, gastrinoma, malignant, cholangiocarcinoma, hepatocellular carcinoma, combined hepatocellular carcinoma and cholangiocarcinoma, trabecular adenocarcinoma, adenoid cystic carcinoma, adenocarcinoma of adenomatous polyps, adenocarcinoma, familial adenomatous polyposis, solid carcinoma, carcinoid tumors, malignant, bronchioloalveolar adenocarcinoma, papillary adenocarcinoma, chromophobe carcinoma, eosinophilic carcinoma, eosinophilic adenocarcinoma, basophilic carcinoma, clear cell adenocarcinoma, granular cell carcinoma, follicular adenocarcinoma, papillary and follicular adenocarcinoma, and nonencapsulating sclerosing carcinoma. Carcinoma, adrenocortical carcinoma, endometrioid carcinoma, adnexal carcinoma, apocrine gland carcinoma, sebaceous gland carcinoma, ceruminous gland carcinoma, mucoepidermoid carcinoma, cystadenocarcinoma, pancreatic adenocarcinoma, pancreatic ductal adenocarcinoma, cystadenocarcinoma, pancreatic neuroendocrine tumor (PanNET), pancreatic adenosquamous carcinoma, pancreatic signet ring cell carcinoma, pancreatic hepatoid carcinoma, pancreatic glial carcinoma, anaplastic carcinoma, and anaplastic carcinoma with osteoclast-like giant cells, pancreatic acinar cell carcinoma, solid pseudopapillary neoplasm, pancreatic blastoma, rare exocrine cancers of thePancreas), serous cystadenoma of the pancreas, mucinous cystic neoplasm of the pancreas, papillary cystadenocarcinoma, papillary serous cystadenocarcinoma, mucinous cystadenocarcinoma, mucinous adenocarcinoma, signet ring cell carcinoma, invasive ductal carcinoma, medullary carcinoma, lobular carcinoma, inflammatory carcinoma, Paget's disease, mammary, acinar cell carcinoma, adenosquamous carcinoma, adenoma with squamous metaplasia, thymoma, malignant, ovarian stromal tumor, malignant theca, malignant granulosa cell tumor, malignant androblastoma, malignant Sertoli cell carcinoma, Leydig cell tumor, malignant lipid cell tumor, malignant paraganglioma, malignant extramammary paraganglioma, malignant pheochromocytoma, angioglobulin angiosarcoma (glomang iosarcoma), malignant melanoma, achromatic melanoma, superficial spreading melanoma, melanoma of giant pigmented nevus, epithelioid cell melanoma, blue nevus, malignant sarcoma, fibrosarcoma, fibrous histiocytoma, malignant myxosarcoma, liposarcoma, leiomyosarcoma, rhabdomyosarcoma, embryonic rhabdomyosarcoma, alveolar rhabdomyosarcoma, stromal sarcoma, mixed tumor, malignant Müllerian mixed tumor, nephroblastoma, hepatoblastoma, carcinosarcoma, mesenchymal tumor, malignant Brenner tumor, malignant phyllodes tumor, malignant synovial sarcoma, mesothelioma, malignant undifferentiated germ cell tumor, embryonic carcinoma, teratoma, malignant ovarian goiter, malignant choriocarcinoma, mesonephroma, malignant angiosarcoma, hemangioendothelioma, malignant carcinoma Posi sarcoma, periangiocarcinoma, malignant, lymphangiosarcoma, osteosarcoma, paraosteal osteosarcoma, chondrosarcoma, chondroblastoma, malignant mesenchymal chondrosarcoma, giant cell tumor of bone, Ewing's sarcoma, odontogenic tumor, malignant epiamelosarcoma, ameloblastoma, malignant ameloblastoma, pineal glandoma, malignant chordoma, glioma, malignant ependymoma, astrocytoma, protoplasmic astrocytoma, fibrous astrocytoma, astrocytoma, glioblastoma, oligodendronoma, oligodendronoma, primitive neuroectodermal, cerebellar sarcoma, ganglioblastoma, neuroblastoma, retinoblastoma, olfactory retinoblastoma, meningioma, malignant, nerve fiber Sarcoma, schwannoma, malignant granular cell tumor, malignant lymphoma, Hodgkin's disease, Hodgkin's granuloma, malignant lymphoma, small lymphocytic lymphoma, malignant lymphoma, large cell lymphoma, diffuse lymphoma, malignant lymphoma, follicular lymphoma, mycosis fungoides, other certain non-Hodgkin lymphomas, malignant histiocytosis, multiple myeloma, mast cell sarcoma, immunoproliferative bowel disease, leukemia, lymphocytic leukemia, plasma cell leukemia, erythroleukemia, lymphosarcoma, myeloid leukemia, basophilic leukemia, eosinophilic leukemia, monocytic leukemia, mast cell leukemia, megakaryoblastic leukemia, myelosarcoma, or hairy cell leukemia.

[0207]

[0223] In some embodiments, the cancer is a virus-induced cancer, such as a cancer caused by infection with an oncovirus or tumor virus (also known as a "cancer virus"). In some embodiments, the cancer virus is a DNA virus. In some embodiments, the cancer virus is an RNA virus.

[0208]

[0224] In some embodiments, cancerous or precancerous tumors are associated with or caused by oncoviruses. Non-exclusive examples of oncoviruses include 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.

[0209]

[0225] In some embodiments, cancerous or precancerous tumors are associated with or caused by cancer viruses that directly induce transformation of infected host cells, thereby regulating host cell proliferation and survival, or conversely increasing genetic instability, initiating a DNA damage response that accelerates cancer acquisition, and causing mutations in the host cell genome.

[0210]

[0226] In some embodiments, cancerous or precancerous tumors are associated with or caused by cancer viruses that induce chronic inflammation in the host. For example, infection with HBV and HCV can induce chronic hepatitis associated with oxidative DNA damage, followed by cirrhosis, and in some cases, lead to the development of hepatocellular carcinoma.

[0211]

[0227] In some embodiments, cancerous or precancerous tumors are associated with or caused by cancer viruses that are not carcinogenic but inhibit the host's immune system, thereby interfering with immune surveillance and enabling the emergence of mutated malignant cells, such as those in HIV-infected patients.

[0212]

[0228] In some embodiments, treatment comprises administering to a subject having an (one or more) infectious disease, such as an infectious disease caused by HIV, HCV, HBV, EBV, or HPV, one or more GAL9-binding molecules as described herein.

[0213]

[0229] In some embodiments, treatment comprises administering to a subject having HIV or AIDS one or more GAL9-binding molecules as described herein in an amount effective for treating the subject.

[0214] 6.10.4. Administration

[0230] The GAL9-binding molecule can be administered to a subject by any route known in the art. For example, the GAL9-binding molecule can be administered to a human subject via, 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 molecule can be administered to a subject by itself or as a pharmaceutical composition. Exemplary pharmaceutical compositions are described herein.

Example

[0215] 6.11. Example

[0231] The following examples are provided by way of illustration and not limitation. In particular, the methods for expressing and purifying various antigen-binding proteins and their use in various assays as described in more detail below are non-limiting and exemplary.

[0216] 6.11.1. Methods 6.11.1.1. Expi293 Expression

[0232] The various antigen-binding proteins tested were expressed using the Expi293 transient transfection system according to the manufacturer's instructions (Thermo Fisher Scientific). In short, unless otherwise stated, plasmids encoding individual strands were mixed in a 1:1 mass ratio and transfected into Expi293 cells using the ExpiFectamine293 transfection kit. Cells were cultured at 37°C, 8% CO2, and 100% humidity with shaking at 125 rpm. Transfected cells were given nutrients 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.

[0217] 6.11.1.2. ExpiCHO expression

[0233] Various GAL9 antigen-binding proteins are tested and expressed using the ExpiCHO transient transfection system according to the manufacturer's instructions. In short, plasmids encoding individual strands are mixed, for example, in a 1:1 mass ratio and transfected with ExpiCHO using the ExpiFectamine CHO transfection kit.

[0218]

[0234] Cells are cultured at 37°C, 8% CO2, and 100% humidity with shaking at 125 rpm. Transfected cells are generally given nutrients once, 16-18 hours after transfection. Cells are harvested on day 5 by centrifugation at 2000 g for 10 minutes. The supernatant is then collected for affinity chromatography purification.

[0219] 6.11.1.3. Purification of Protein A

[0235] Clarified supernatants containing various antigen-binding proteins were separated using a gravity flow purifier with either protein A (ProtA) resin or anti-CH1 resin. In cases where direct comparison was performed, the supernatants containing various antigen-binding proteins were divided into two equal samples. For ProtA purification, a 1 mL protein A column (GE Healthcare) was equilibrated with PBS (5 mM potassium sodium phosphate, pH 7.4, 150 mM sodium chloride). The sample was loaded onto the column at 5 mL / min. The sample was eluted using 0.1 M sodium acetate, pH 3.5. The eluate was monitored by absorbance at 280 nm, and the elution peaks were pooled for analysis.

[0220] 6.11.1.4.SDS-Page Analysis

[0236] Samples containing various isolated antigen-binding proteins were analyzed by reducing and non-reducing SDS-PAGE for the presence of complete products, incomplete products, and overall purity. 2 μg of each sample was added to 15 μL of SDS loading buffer. Reducing samples were incubated at 75°C for 10 minutes in the presence of a 10 mM reducing agent. Non-reducing samples were incubated at 70°C for 5 minutes without a reducing agent. Both reducing and non-reducing samples were loaded onto 4-15% gradient TGX gels (BioRad) with electrophoresis buffer and run at 220 volts for 30 minutes. After electrophoresis, the gels were washed with deionized (DI) water and stained using GelCode Blue Safe Protein Stain (ThermoFisher). Before analysis, the gels were destained with DI water. Densitometry analysis of scanned images of the destained gels was performed using standard image analysis software, and the relative abundance of bands in each sample was calculated.

[0221] 6.11.1.5. IEX chromatography

[0237] Samples containing various isolated antigen-binding proteins were analyzed by cation exchange chromatography for the ratio of complete products to incomplete products and impurities. The clarified supernatant was analyzed using 5 ml of MonoS (GE Lifesciences) on an AKTA Purifier FPLC. The MonoS column was equilibrated with buffer A (10 mM MES pH 6.0). The sample was loaded onto the column at 2 ml / min. The sample was eluted using a 0-30% gradient of buffer B (10 mM MES pH 6.0, 1 M sodium chloride) over 6 column bed volumes (CV). The eluate was monitored by absorbance at 280 nm, and the purity of the sample was calculated by peak integration to identify the abundance of monomer peaks and impurity peaks. The monomer peaks and impurity peaks were pooled separately for analysis by SDS-PAGE as described above.

[0222]

[0238] For analytical SEC chromatography, each sample at a 1 mg / mL concentration was loaded onto the column at a flow rate of 1 ml / min. The samples were eluted at 1.5 CV using a homogeneous PBS solvent flow. The eluted material was monitored by absorbance at 280 nm, and the elution peaks were analyzed by peak integration.

[0223] 6.11.1.6.Mass spectrometry

[0239] Samples containing various isolated antigen-binding proteins were analyzed by mass spectrometry to confirm their correct species based on molecular weight. All analyses were performed by a third-party research institution. In short, the samples were treated with an enzyme cocktail to remove glycosylation. The samples were tested under reducing conditions to specifically identify each chain by molecular weight, and under non-reducing conditions to determine the molecular weight of all complexes in the sample. Mass spectrometry was used to determine the number of endogenous products based on molecular weight.

[0224] 6.11.1.7. Antibody discovery by phage display

[0240] Phage display of human Fab libraries was performed using a standard protocol. Human GAL9 protein was purchased from Acro Biosystems (Human Gal9 His-tag catalog number LG9-H5244), and EZ-Link NHS-PEG was used with a standard protocol. 12 -Biotinization was performed using Biotin (Thermo Scientific, catalog number 21312). The phage clones were screened for their ability to bind to the GAL9 protein by phage ELISA using a standard protocol.

[0225]

[0241] In short, we constructed a phage library in Fab format using expression vectors (also called phagemids) that could be replicated and expressed in phages. Both the heavy and light chains were encoded in the same expression vector, with the heavy chain fused to a truncated variant of the phage coat protein pIII. The light and heavy chain-pIII fusions were expressed as separate polypeptides and assembled in bacterial periplasms where the redox potential allowed for disulfide bond formation, forming phage display antibodies containing candidate ABSs.

[0226]

[0242] Libraries were created using sequences derived from specific human heavy chain variable domains (VH3-23) and specific human light chain variable domains (Vκ-1). In the screening libraries, all three CDRs of the VH domain were diversified to match the positional amino acid frequencies by CDR length found in the human antibody repertoire. In the generated light chain variable domains within the screening libraries, diversification was introduced only in VL CDR3 (L3). Light chain VL CDR1 (L1) and CDR2 (L2) retained human germline sequences.

[0227]

[0243] The heavy chain scaffold (SEQ ID NO: 2), light chain scaffold (SEQ ID NO: 4), complete heavy chain Fab polypeptide (SEQ ID NO: 1), and complete light chain Fab polypeptide (SEQ ID NO: 3) used in the phage display library are shown below, where the lowercase "x" indicates a modified CDR amino acid used to create the library. Phage display VH scaffold [SEQ ID NO: 2]: EVQLVESGGGLVQPGGSLRLSCAASGFTFxxxxIHWVRQAPGKGLEWVAxxxxxxxxxxxYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARxxxxxxxxxxxxxDYWGQGTLVTVSSAS Phage display VL scaffold [SEQ ID NO: 4]: DIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYSASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQxxxxxxTFGQGTKVEIKRT Phage display heavy chain Fab polypeptide [SEQ ID NO: 1]: EVQLVESGGGLVQPGGSLRLSCAASGFTFxxxxIHWVRQAPGKGLEWVAxxxxxxxxxxxYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARxxxxxxxxxxxxxDYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC Phage display light chain Fab polypeptide [SEQ ID NO: 3]: DIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYSASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQxxxxxxTFGQGTKVEIK RTVAAPSVFIFPPSDSQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0228]

[0244] The diversity was created by Kunkel mutagenesis using primers that introduce diversity into VH CDR1(H1), VH CDR2(H2), VH CDR3(H3), and VL CDR3(L3), mimicking the diversity found in the natural antibody repertoire, as described in more detail in Kunkel, TA (PNAS January 1, 1985, Vol. 82(2), pp. 488-492). This literature is incorporated herein by reference in its entirety. Briefly, single-stranded DNA was prepared from isolated phages using standard procedures, and Kunkel mutagenesis was performed. Chemically synthesized DNA was then electroporated into MC1061F- cells. The phagemides obtained after overnight culture were digested with restriction enzymes (BamHI and XbaI) to remove wild-type sequences. The digested samples were electroporated into TG1 cells and then harvested. The collected cells were subcultured and infected with the M13K07 helper phage to produce a phage library.

[0229]

[0245] Phage panning was performed using standard procedures. Briefly, the first round of phage panning was performed using targets immobilized on streptavidin magnetic beads, which were then used to pan approximately 5 × 10⁶ of a prepared library in a 1 mL volume in PBST-2% BSA. 12 The phages were subjected to phages. After 1 hour of incubation, bead-bound phages were separated from the supernatant using a magnetic stand. The beads were washed three times to remove nonspecifically bound phages, and then OD (Oral Processing) was performed. 600Approximately 0.6 ER2738 cells (5 mL) were added. After 20 minutes, the infected cells were treated with 25 mL of 2×YT+ ampicillin and M13K07 helper phage (final concentration, approximately 10 10 Phages were subcultured in pfu / ml and allowed to grow overnight at 37°C with vigorous shaking. The following day, phages were prepared by PEG precipitation using standard procedures. Prior to panning, phages specific to the SAV-coated beads were removed. A second round of panning was performed using a KingFisher magnetic bead handler with 100 nM bead-immobilized antigen using standard procedures. In total, 3-4 rounds of phage panning were performed to enrich phages presenting Fab specific to the target antigen. Target-specific enrichment was confirmed using polyclonal and monoclonal phage ELISA. Isolated Fab clones containing candidate ABS were determined using DNA sequencing.

[0230]

[0246] The VL and VH domains identified by the above phage screening were reformatted to a bivalent, monospecific, natural human full-length IgG1 architecture. Natural human full-length IgG single heavy-chain architecture [SEQ ID NO: 5]: EVQLVESGGGLVQPGGSLRLSCAASGFTFxxxxIHWVRQAPGKGLEWVAxxxxxxxxxxxYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARxxxxxxxxxxxxxDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK [SEQ ID NO:5] Native human full-length IgG1 light chain architecture: Corresponds to phage display light chain Fab. See [SEQ ID NO:3].

[0231] 6.11.1.8. Octet determination of binding kinetics

[0247] To measure the qualitative binding affinity in the GAL9 conjugate discovery experiment, the IgG1 reformat conjugate was immobilized on the biosensor of an Octet (Pall ForteBio) biolayer interferometer.

[0232]

[0248] Next, soluble GAL9 antigen was added to this system and binding was measured. By visualizing the slope of the dissociation phase of the octet sensogram, the qualitative binding affinity was evaluated from the weakest ( + ) to the strongest ( +++ ). A slow off-rate is represented by the negligible drop in the dissociation phase of the sensogram, indicating tight antibody binding ( +++). To obtain the precise rate constant of monovalent affinity, at least five concentrations (approximately 10-20 × K) are used during the binding step. D From 0.1 × K D Dilution series containing GAL9 analytes (within a range of values, 2-fold dilution) were measured. During the dissociation phase, the sensor was immersed in a buffer solution without GAL9 analytes to allow dissociation of the complex bound to the sensor surface. Using Octet dynamics analysis software, the rate constant and equilibrium binding constant were calculated based on the binding rate and dissociation curve. The analysis was performed globally (global fitting), and the rate constant was simultaneously derived from all analyte concentrations included in the experiment.

[0233] 6.11.1.9. Epitope Binning

[0249] Anti-GAL9 candidates formatted as described above as bivalent, monospecific, natural human full-length IgG1 were tested for GAL9 binding in a pairwise manner using an octet-based "tandem" assay. Briefly, biotinylated GAL9 was immobilized on a streptavidin sensor, and two anti-GAL9 candidates were conjugated in tandem. A competitive blockade profile was generated to determine whether a given anti-GAL9 candidate blocked the binding of the other anti-GAL9 candidates to GAL9. Anti-GAL9 candidates that competed for the same or non-overlapping binding regions were grouped together and placed in the same bin.

[0234] 6.11.1.10. PBMC activation and galectin-9 antibody treatment

[0250] Individual aliquots of PepMix HCMVA(pp65) (>90%) protein ID: P06725 (catalog number PM-PP65-2, JPT Peptide Technologies) were prepared according to the manufacturer's instructions. PepMix® HCMVA(pp65) is a mixture of duplicated 15-mer peptides encompassing the complete protein of the 65kDa phosphoprotein (pp65) (Swiss-Prot ID: P06725) of human cytomegalovirus (HHV-5). Aliquots of PepMix were used to immunostimulate PBMCs, and the immune cell response was evaluated.

[0235]

[0251] Frozen human peripheral blood mononuclear cells (PBMCs) were thawed according to standard conditions and then resuspended in growth medium (RPMI with 10% FBS).

[0236]

[0252] Resuspended PBMC in 5 × 10 5 Cells were seeded in 96-well plates. The cells were incubated in growth medium at 37°C and 5% CO2 for 24 hours with 2 μg / mL of PepMix® HCMVA (pp65) and 40 μg / mL of a candidate GAL9 antibody or a control antibody.

[0237] 6.11.1.11. LEGENDplex Human Th Cytokine Assay

[0253] Cytokine secretion by PBMCs and specific immune cell subpopulations was evaluated using a cytokine bead array 24 and 72 hours after PBMC activation induced by PepMix HCMVA (pp65) and galectin-9 antibody treatment, as follows.

[0238]

[0254] 200 μl of cell culture supernatant was collected and centrifuged to pellet the cell residue. The obtained supernatant was analyzed using the LEGENDplex® Human Thhl Panel (5-plex) (catalog number 740009, Biolegend). The LEGENDplex® Human Thhl Panel is a bead-based assay that enables simultaneous quantification of human cytokines IL-2, IL-6, IL-10, IFN-γ, and TNF-α using flow cytometry.

[0239]

[0255] In short, cytokine standards and capture bead mixtures were prepared according to the manufacturer's instructions. An assay master mix was prepared using a 1:1:1 ratio of capture bead mixture:biotinylation detection antibody:assay buffer.

[0240]

[0256] 12.5 μl of supernatant or cytokine standard was incubated with 37.5 μl of assay master mix. The plate was 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 600 rpm for 30 minutes at room temperature. The beads were then washed twice, resuspended, and flow cytometry analysis was performed according to the manufacturer's instructions.

[0241] 6.11.1.12. PBMC staining with marker antibodies

[0257] Following PBMC activation and galectin-9 antibody treatment as described herein, PBMC immunotherapies were stained with marker antibodies according to the following procedure.

[0242]

[0258] Cells 5 × 10 6 Cells were resuspended in growth medium (RPMI with 10% FBS) at a concentration of cells / mL. 200 μL of resuspended cells were aliquoted into 96-well plates and then incubated with Fixable Viability Dye eFluor® 780 at 2–8°C for 30 minutes to irreversibly label dead cells. The cells were then washed and incubated with human Fc barrier solution (catalog no. 14-9161-73, eBiosciences) at room temperature for 10 minutes.

[0243]

[0259] Antibody cocktail working solutions were prepared according to the following table. [Table 1]

[0244]

[0260] Wells were incubated with 10 μL of diluted antibody cocktail at 2–8°C for 30 minutes. Cells were then washed, resuspended, and analyzed by flow cytometry.

[0245]

[0261] Immunostimulatory markers CD27, CD40L, ICOS, 4-1BB, and OX40 were analyzed according to the same protocol provided above, but cells were incubated with alternative antibody cocktails as shown in Table 2 below.

[0246] [Table 2]

[0247] 6.11.2. Example 1: GAL9 coupling arm discovery experiment

[0262] Using the monoclonal phage ELISA format described above, chemically synthesized Fab phage libraries with introduced diversity in Fab CDRs were screened against the GAL9 antigen. Phage clones expressing Fab that recognizes GAL9 were sequenced.

[0248]

[0263] This experiment initially identified 52 GAL9 binding candidates (antigen-binding site clones). Functional assays performed after reformatting the variable regions of these clones to a bivalent monospecific human IgG1 format identified 22 antibodies with immunoactivating properties.

[0249]

[0264] Table 3 lists the VH CDR1 / 2 / 3 sequences from 22 activated ABS clones, showing only the CDR residues that were modified during library construction. Table 4 lists the VL CDR1 / 2 / 3 sequences from the identified ABS clones, showing that the light chain CDR1 and CDR2 sequences are unchanged, and only the CDR3 residues that were modified during library construction are shown. [Table 3] [Table 4]

[0250]

[0265] Table 5 shows the complete CDR sequences of 22 candidate anti-GAL9 immunoactivating antibodies, according to several definitions accepted in the art. [Table 5] TIFF0007870616000013.tif180149 TIFF0007870616000014.tif184149 TIFF0007870616000015.tif191149 TIFF0007870616000016.tif183149 TIFF0007870616000017.tif187149 TIFF0007870616000018.tif187149 TIFF0007870616000019.tif179149 TIFF0007870616000020.tif182149 TIFF0007870616000021.tif187149 TIFF0007870616000022.tif177149 TIFF0007870616000023.tif187149 TIFF0007870616000024.tif181149 TIFF0007870616000025.tif187149 TIFF0007870616000026.tif193149 TIFF0007870616000027.tif191149 TIFF0007870616000028.tif193149 TIFF0007870616000029.tif191149 TIFF0007870616000030.tif191149 TIFF0007870616000031.tif193149 TIFF0007870616000032.tif193149 TIFF0007870616000033.tif193149

[0251]

[0266] Table 6 shows the full immunoglobulin heavy chain and full immunoglobulin light chain sequences, as well as the VH and VL sequences, of various ABS candidates formatted in a bivalent monospecific human full-length IgG1 architecture. [Table 6] TIFF0007870616000035.tif175149 TIFF0007870616000036.tif175149 TIFF0007870616000037.tif175149 TIFF0007870616000038.tif175149 TIFF0007870616000039.tif175149 TIFF0007870616000040.tif179149 TIFF0007870616000041.tif175149 TIFF0007870616000042.tif179149 TIFF0007870616000043.tif182149 TIFF0007870616000044.tif174149 TIFF0007870616000045.tif88149

[0252]

[0267] The binding properties of the selected GAL9 binding candidates were analyzed: cross-reactivity with mouse GAL9; qualitative binding; epitope binning (bin 2 - candidate bin containing the commercially available antibody clone ECA8 [LS-C179448] from LSBio; bin 3 - candidate bin containing the commercially available antibody clone ECA42 [LS-C179449] from LSBio, which is the "tool antibody" referenced in Figures 3A and 3B); and monovalent affinity binding. The analysis results are shown in Table 7. [Table 7]

[0253]

[0268] The selected GAL9 binding candidates were further analyzed for sequence motifs that could adversely affect antibody properties relevant to clinical development, such as stability, mutability, and immunogenicity. Computer analysis was performed according to Kumar and Singh (Developability of biotherapeutics: computational approaches. Boca Raton: CRC Press, Taylor & Francis Group, 2016). The analysis results are shown in Table 8, indicating that the number of harmful sequence motifs present in the listed clones is limited, suggesting potential for further clinical development. [Table 8]

[0254] 6.11.3. Example 2: Treatment with an anti-GAL9 candidate increases cytokine production by human PBMCs.

[0269] Candidate GAL9 ABSs were formatted into bivalent, monospecific, natural human full-length IgG1 heavy and light chain architectures (SEQ ID NO: 5 and SEQ ID NO: 3, respectively), and their effects on cytokine production by PBMCs after peptide stimulation were tested. PBMCs were stimulated essentially as described in Section 6.11.1 above. Briefly, PBMCs were collected from human donors known to respond to human CMV virus (HCMV), cultured, stimulated with HCMV PepMix to pre-stimulate an antigen-specific response, and treated with either control IgG, a comparative tool-activated mAb (clonal ECA42), α-PD1 (nivolumab), or a candidate anti-GAL9 antibody. Cytokine secretion was measured 24 and 72 hours after treatment with a bead cytokine array. INF-γ and TNF-α results are illustrated in Figures 3A and 3B, respectively. The data shown in Figures 3A-3B are described in more detail in Tables 9 and 10 provided below. [Table 9] [Table 10]

[0255]

[0270] Notably, PBMCs treated with candidate P9-15, P9-18, P9-21, and P9-28 showed improved IFN-γ and TNF-α secretion after stimulation compared to both IgG control and GAL9 comparative tool antibody (clone ECA42). Furthermore, PBMCs treated with candidate P9-15, P9-18, P9-21, and P9-28 also showed improved TNF-α production after stimulation compared to treatment with a commercially available α-PD1 antibody. Therefore, treatment of PBMCs with selected anti-GAL9 candidates was able to improve cytokine secretion after peptide stimulation. Treatment with P9-54 resulted in a neutral response, with no significant difference in TNF-α or IFN-γ secretion (data not shown).

[0256] 6.11.4. Example 3: Treatment with an anti-GAL9 candidate increases TNF-α production by natural killer (NK) cells.

[0271] GAL9 ABS candidates were formatted into bivalent monospecific natural human full-length IgG1 heavy and light chain architectures (SEQ ID NO: 5 and SEQ ID NO: 3, respectively), and NK cells (strain, CD56) were stimulated 72 hours after peptide stimulation. + The effects of these antibodies on TNF-α production were investigated. NK cells were treated with 5 μg or 20 μg doses of control antibody clone 55, GAL9 antibody candidate P9-15 (clone 15), or GAL9 antibody candidate P9-18 (clone 18). After treatment, the level of TNF-α secretion from cells was evaluated by flow cytometry. NK cells that secreted TNF-α (CD56) + Representative percentage data for ) are shown in Figures 6A and 6B.

[0257]

[0272] Treatment with either candidate GAL9 antibody P9-18 or P9-15 increased the percentage of TNF-α-positive stained NK cells after stimulation compared to the negative control clone P9-55. In a population of NK cells treated with 5 μg of control antibody, such NK cells (CD56) increased. + 7.75% of the NK cells treated with 5 μg of P9-18 were TNF-α positive. In contrast, 12.0% of NK cells treated with 5 μg of P9-18 were TNF-α positive. Furthermore, 22.5% of NK cells treated with 5 μg of P9-15 were TNF-α positive. See Figures 6A and 6B.

[0258]

[0273] In a population of NK cells treated with 20 μg of control antibody, such NK cells (CD56 + ) 10.3% were TNF-α positive. In contrast, in a population of NK cells treated with 20 μg of P9-18, 16.9% of such NK cells were TNF-α positive. Furthermore, in NK cells treated with 20 μg of P9-15, 28.5% of such NK cells were TNF-α positive. See Figures 6A and 6B.

[0259]

[0274] Therefore, treatment with the selected anti-GAL9 candidate was able to increase TNF-α production by NK cells after stimulation. See Figures 6A and 6B.

[0260] 6.11.5. Example 4: Treatment with an anti-GAL9 candidate increases IL-12 production by dendritic cells.

[0275] GAL9 ABS candidates were formatted into bivalent monospecific natural human full-length IgG1 heavy and light chain architectures (SEQ ID NO: 5 and SEQ ID NO: 3, respectively), and after peptide stimulation, dendritic cells (lineage negative, class II) were tested. + CD11c +The effects of these methods on IL-12 secretion were tested. PBMCs containing a population of dendritic cells (DCs) were treated as described in Example 2, and then IL-12 secretion levels were evaluated using the IL-12 secretion assay-detection kit (PE), human (catalog number 130-092-124, Miltenyi Biotec) according to the manufacturer's protocol. Representative data for the percentage of DCs that secreted IL-12 are shown in Figure 5.

[0261]

[0276] Notably, treatment with the GAL9 antibody candidate P9-18 increased the percentage of IL-12-positive stained DCs after stimulation compared to the IgG control. In the DC population treated with control IgG, 0.26% of such DCs were IL-12 positive. In contrast, in the DC population treated with P9-18, 7.74% of such DCs were IL-12 positive, a 28-fold increase in IL-12-positive DCs compared to the IgG control-treated population. Therefore, treatment of PBMCs with the selected anti-GAL9 candidate was able to increase IL-12 production by DCs after stimulation.

[0262] 6.11.6. Example 5: Treatment with anti-GAL9 candidate increases surface expression of costimulatory molecules on CD8+ T cells.

[0277] GAL9 ABS candidates, formatted as bivalent monospecific natural human full-length IgG1 heavy and light chain architectures (SEQ ID NO: 5 and SEQ ID NO: 3, respectively), were stimulated with peptides, and then CD8 + The effects of these on the expression of T cell-mediated immunostimulatory surface markers were investigated. CD8 + PBMCs containing a population of T cells were treated as described in Example 2, stained with marker antibodies as described herein, and then recovered for flow cytometry. CD8 + The levels of immunostimulatory surface markers CD27, CD40L, ICOS, 4-1BB, and OX40 on T cells were evaluated. The data are shown in Figures 4-1 and 4-2. "% value" indicates CD8 with detectable levels of the relevant marker. +This represents the percentage of T cells. Figures 4-1 and 4-2 show that treatment with αGAL9 antibody candidates P9-15, P9-18, P9-21, and P9-28 compared to Ig control antibody clone ECA42 in CD8 + This study showed increased levels of the immunostimulatory surface markers CD27, CD40L, ICOS, 4-1BB, and OX40 in T cells.

[0263]

[0278] CD8 positively stained for immunostimulatory surface markers + Representative data for the percentage of T cells are shown in Table 11 below. [Table 11]

[0264]

[0279] Notably, PBMCs treated with candidate P9-18 or P9-21 showed positive staining of CD8 after stimulation for various immunostimulatory surface markers compared to IgG control, GAL9 comparative tool antibody (clone ECA42), and α-PD1. + This showed an increase in the percentage of T cells. This included CD8 cells positively stained for CD40L and OX40. + This includes an increase of more than twofold in the percentage of T cells. Therefore, treatment of PBMCs with the selected anti-GAL9 candidate leads to CD8 after stimulation. + We were able to improve the expression of surface markers stimulated by T cells. The same immune-stimulating response was observed in low-responsive PBMC cells and donor 5 (data not shown).

[0265] 6.11.7. Example 6: Treatment with an anti-GAL9 candidate alters PD-L1 and PD-L2 cell surface expression on dendritic cells (DCs).

[0280] GAL9 ABS candidates were formatted into bivalent monospecific natural human full-length IgG1 heavy and light chain architectures (SEQ ID NO: 5 and SEQ ID NO: 3, respectively), and after peptide stimulation, dendritic cells (lineage negative, class II, CD11c) were tested. +The effects of these methods on PD-L1 and PD-L2 cell surface expression were tested. PBMCs containing a population of dendritic cells (DCs) were treated as described in Example 2, then collected for flow cytometry, and the levels of PD-L1 and PD-L2 on the DCs were evaluated. Table 12 below shows representative data for the percentage of DCs positively stained for PD-L1 and PD-L2, as well as geometric mean fluorescence intensity (GMI). [Table 12]

[0266]

[0281] Notably, PBMCs treated with candidate P9-18 showed an increased percentage of dendritic cells (DCs) positively stained for PD-L2 after stimulation compared to IgG control and GAL9 comparative tool antibody (ECA42). Both P9-18 and P9-21 showed a decrease in the percentage of PD-L1 staining and a decrease in the geometric mean fluorescence (GMI) of PD-L1 on DCs. Therefore, treatment of PBMCs with selected anti-GAL9 candidates was able to alter PD-L1 and PD-L2 surface expression by DCs after stimulation.

[0267] 6.11.8. Example 7: Treatment with an anti-GAL9 candidate leads to clustering of GAL9 and PD-L2 on the cell surface of dendritic cells.

[0268]

[0282] GAL9 ABS candidates were formatted into bivalent, monospecific, natural human full-length IgG1 heavy and light chain architectures (SEQ ID NO: 5 and SEQ ID NO: 3, respectively), and their effects on the clustering of GAL9, PD-L1, and PD-L2 on the cell surface of dendritic cells ("DCs") were tested.

[0269]

[0283] PBMCs containing a population of dendritic cells (DCs) were processed as described in Example 2, then fixed for confocal imaging analysis, and the distribution of GAL9, CD11c, and PD-L2 on the dendritic cells was evaluated.

[0270]

[0284] Results / Conclusion

[0285] Confocal images of dendritic cells treated with IgG control (Figure 8A), P9-18 (Figure 8B), and P9-21 (Figure 8C) are shown. Blue staining indicates DNA (DAPI), red staining indicates PD-L2, green staining indicates CD11c, and yellow staining indicates GAL9. Unlabeled images are bright-field images. The attached figures are rendered in grayscale.

[0271]

[0286] Treatment with candidate P9-18 or P9-21 resulted in co-localization and clustering of GAL9 and PD-L2 on DCs compared to the IgG control (Figures 8B-8C). Therefore, treatment with P9-18 or P9-21 can induce co-localization and clustering of GAL9 and PD-L2 on the cell surface of DCs after stimulation.

[0272] 6.11.9. Example 8: Treatment with anti-GAL9 P9-18 preserves PD-L2 and PD-L1 expression on tumor cells.

[0287] The anti-GAL9 candidate P9-18 was tested for its effects on the cell retention and distribution of PD-L2 and PD-L1 in tumor cells.

[0273]

[0288] antibody

[0289] GAL9 ABS candidates were formatted into bivalent, monospecific, natural human full-length IgG1 heavy and light chain architectures (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, Inc. (New Hampshire, Lebanon).

[0274]

[0290] Cell culture and immunohistochemical staining

[0291] CT26 tumor cells were cultured and treated with either anti-GAL9 candidate P9-18 or an IgG control. The cells were fixed and stained with DAPI, anti-PD-L2, and anti-PD-L1 for confocal imaging analysis.

[0275]

[0292] Results / Conclusion

[0293] Figures 9A and 9B show representative confocal images of CT26 tumor cells after treatment with P9-18 or IgG control. Blue staining indicates DNA (DAPI), red indicates PD-L2, and green indicates PD-L1. The attached figures are rendered in grayscale. Imaging shows that PD-L2 and PD-L1 are retained on the surface of CT26 tumor cells after treatment with P9-18 compared to the IgG control. See Figures 9A and 9B. The spots in Figure 9B highlight the increased expression of PD-L2 and PD-L1 proteins.

[0276] 6.11.10. Example 9: Treatment with anti-GAL9 P9-18 or P9-21 inhibits tumor growth in colon and melanoma tumor models.

[0294] This study was conducted to determine whether the anti-GAL9 candidates P9-18 and P9-21 can inhibit tumor growth in colon and melanoma tumor models.

[0277]

[0295] antibody

[0296] Candidate GAL9 ABS was formatted into a bivalent monospecific format of the mouse IgG2a skeleton.

[0278]

[0297] Animals and treatment

[0298] BALB / c mice were subcutaneously transplanted with CT26 tumor cells and treated with anti-GAL9 candidate P9-18, P9-21, or an IgG control. Treatment was administered intraperitoneally (IP) at 200 μg on days 7, 11, 15, and 19. There were 10 mice per treatment group. Tumor growth was evaluated by measuring tumor volume. Tumors were approximately 1000 mm². 3 The mice were euthanized once the volume reached a certain level.

[0279]

[0299] C57BL / 6 mice were subcutaneously transplanted with the B16.F0 tumor cell line and treated with anti-GAL9 candidate P9-18, P9-21, or an IgG control. Treatment consisted of 200 μg intracellular injections on days 3, 7, 11, and 15. There were 10 mice per treatment group.

[0280]

[0300] Results / Conclusion

[0301] Mice treated with P9-18 or P9-21 showed complete regression of CT26 tumors, while mice treated with an IgG control showed continued tumor growth. See Figure 1. Mice treated with P9-18 or P9-21 showed reduced B16.F0 tumor growth compared to mice treated with an IgG control. See Figure 2. Thus, P9-18 or P9-21 can inhibit tumor growth in colon and melanoma tumor models, including complete regression in some cases.

[0281] 6.11.11. Example 10: Treatment with anti-GAL9 P9-15 resulted in a reduction of Epstein-Barr virus (EBV)-induced tumors and decreased viral load.

[0302] This study was conducted to determine the efficacy of anti-GAL9 P9-15 candidates against Epstein-Barr virus (EBV)-induced tumors in a humanized mouse model.

[0282]

[0303] Epstein-Barr virus (EBV) is a gamma herpesvirus 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, human CD34 + Using humanized mice transplanted with hematopoietic stem cells, we created a mouse model reconstituted with human immune system cells.

[0283]

[0304] Infection and treatment of humanized mice

[0305] Figure 10A shows an overview of the overall treatment schedule used in the study. In short, immunodeficient mice were given CD34 + Human stem cells were injected intravenously, allowing for transplantation over the next 12 weeks. Humanized mice were then infected with EBV and incubated for 3 weeks to induce infection. After 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, surviving mice were euthanized and analyzed.

[0284]

[0306] Generation of humanized NRG mice (hu-NRG)

[0307] 5 female NRG (NOD-Rag1 null IL2rg null Rag1 (NODrag gamma) was used in each treatment group. null The mutation causes a loss of mouse B and T cells, and IL2rg null The mutation prevents cytokine signaling via multiple receptors, leading to a deficiency of functional NK cells. Therefore, NRG mice are extremely immunodeficient and human CD34 + This enables the engraftment of hematopoietic stem cells.

[0285]

[0308] Mice were irradiated with radiation twice at intervals of 3-4 hours, at a dose of 275 cGy per dose (550 cGy total), resulting in 5 × 10⁻⁶ 4 individual CD34 + Humanized NRG ("hu-NRG") mice were created by intravenously injecting human stem cells and then allowing them to engraft for three weeks. The body weight of the hu-NRG mice was measured every other week for 12 weeks to assess their health status. In addition, tail hemorrhage was induced at weeks 4, 8, and 12 after administration of human CD34+ stem cells to collect all mononuclear cells (CD45). + ), T cells (CD3 + ), and B cells (CD19 + ) including human CD45 + Stable engraftment in mice was monitored and confirmed using flow cytometry analysis to detect cells.

[0286]

[0309] Splenic tumor

[0310] Unless the mice died or were euthanized for ethical reasons, their spleens were removed and examined after euthanasia to determine the number of macroscopically visible tumors, cell count, and body weight.

[0287]

[0311] Evaluation of EBV viral load

[0312] EBV levels in the spleen and blood were measured using real-time PCR.

[0288]

[0313] statistical analysis

[0314] A two-tailed Mann-Whitney U test was performed using GraphPad Prism 7 software (San Diego, California).

[0289]

[0315] Results / Conclusion

[0316] Spleens from mice treated with anti-GAL9 P9-15 mice showed fewer macroscopically visible tumors than spleens from mice treated with IgG controls. See Figure 10B. P9-15 treated mice had lighter spleen weights (average 0.100 g per spleen) and significantly fewer spleen cells (51.04 × 10⁶ in IgG controls) compared to IgG control treated mice (average 0.224 g per spleen, p < 0.0079). 6 Compared to individual mice, P9-15 treated mice had a size of 22.14 × 10⁶. 6 (Number of cases, p-value < 0.0159). See Figures 10C-10D. Data are shown as means, and error bars are ±SEM.

[0290]

[0317] In addition, treatment with P9-15 controlled viral load by 88%. The mean EBV of P9-15-treated mice was 0.32 × 10⁶. 6 Although the count was copies / μg, the average EBV of IgG-treated mice was 2 × 10⁶. 6 The result was copies / μg (p-value < 0.0079). See Figure 10E. Data are shown as mean values, and error bars are ±SEM. These results indicate that treatment with P9-15 can reduce the incidence of EBV-induced tumors and control viral load.

[0291] 6.11.12. Example 11: Treatment with anti-GAL9 P9-28 results in fewer Epstein-Barr virus (EBV) induced tumors.

[0318] This study was conducted to determine the efficacy of anti-GAL9 P9-28 candidates against Epstein-Barr virus (EBV)-induced tumors in a humanized mouse model.

[0292]

[0319] Infection and treatment of animals and humanized mice

[0320] This study was conducted as described in Example 10 above.

[0293]

[0321] Results / Conclusion

[0322] Anti-GAL9 P9-28 treated mice did not show any visible macroscopic tumors in the spleen compared to IgG controls. See Figure 11. Anti-GAL9 P9-23 treated mice were less likely to have tumors in the spleen, as can be inferred from their lower cell counts and smaller spleen size. These results suggest that treatment with P9-28 can reduce the development of EBV-induced tumors.

[0294] 6.11.13. Example 12: Anti-GAL9 silent Fc P9-18 (sFcP9-18) has an antitumor effect, and sFcP9-18 and P9-18 can establish antitumor immunological memory.

[0323] This study was conducted to examine the contribution of the Fc region to the antitumor effect of immune-activating anti-Gal9 antibodies. In addition, a reload study was performed to determine whether P9-18 or sFcP9-18 can establish antitumor immunological memory.

[0295]

[0324] antibody

[0325] The P9-18 antigen-binding site was formatted to either the mouse IgG1 skeleton, the mouse IgG2a skeleton, or the mouse IgG2a skeleton with an Fc receptor-binding null mutation (sFc). Silent Fc(sFc)P9-18 antibodies were produced by creating key point mutations that inactivate the binding of Fc to the Fc receptor.

[0296]

[0326] CT26 cells

[0327] CT26 tumor cells were cultured in RPMI medium in a humidified incubator at 37°C, 5% CO2, and 95% air.

[0297]

[0328] Mouse and treatment schedule

[0329] 1 x 10 for 7-10 mice 5 CT26 tumor cells were subcutaneously transplanted and then treated with IP on days 7, 11, 15, and 19 with 200 μg of either control IgG (mouse IgG2a), P9-18-IgG1 (mouse IgG1 skeleton), FcR silent sFcP9-18 (mouse IgG2a skeleton with Fc receptor binding null mutation), or P9-18 (mouse IgG2a skeleton).

[0298]

[0330] Increased tumor volume

[0331] Mice were monitored for up to 143 days, and tumors were measured with calipers every 1-3 days. Tumor volume (mm²) 3 The value was calculated according to the formula: tumor length × tumor width × 2 / 2.

[0299]

[0332] Complete regression response (CR)

[0333] In this study, complete regression was defined as a tumor volume of 0 mm² over 20 consecutive measurements during the study. 3 This was defined as the condition being met. Animals were scored every 1-3 days during the study for complete regression (CR) events.

[0300]

[0334] CT26 Tumor Reloading

[0335] Tumor-free mice that survived the original initial tumor removal study were rested for 65-70 days after tumor removal. On day 107, without additional treatment, 1 × 10⁶ mice were subjected to a 1 × 10⁶ treatment. 5 Individual CT26 tumor cells were re-transplanted into animals. New control mice were treated with IgG2a control on day 113, after which tumor growth was allowed for a further 36 days. Tumor volume was determined as described above between days 107 and 143.

[0301]

[0336] Results / Conclusion

[0337] The results of the tumor growth study are shown in Figure 12A. IgG (IgG2a) control antibody ( [ka] In mice administered with ), tumors grew to 900-1000 mm in the first 50 days. 3 It reached that point. In contrast, P9-18-IgG2a( [ka] Treatment with ) showed a 77% (7 / 9) CR, and sFcP9-18-IgG2a ( [ka] Treatment with ) showed a 70% (7 / 10) complete response (CR). These results indicate that the Fc region of the P9-18 antibody is not necessary for its antitumor effect.

[0302]

[0338] P9-18 ABS reformatted to an IgG1 skeleton ( [ka] ) did not inhibit tumor growth and showed similar tumor growth to the control.

[0303]

[0339] The results of the reload study are shown in Figure 12B. Mice initially treated with P9-18-IgG2a showed a 100% (7 / 7) complete response (CR) to new tumors without additional treatment. Similarly, mice initially treated with sFcP9-18-IgG2a showed a 100% (7 / 7) CR to new tumors without additional treatment. Control IgG (IgG2a) antibody ( [ka] Treatment with ) resulted in tumor growth similar to that observed in initial tumor removal studies. These data indicate that mice treated with P9-18 or sFcP9-18 established antitumor immunological memory against CT26 tumor cells after initial treatment with P9-18-IgG2a or sFc9-18-IgG2a.

[0304] 6.11.14. Example 13: Treatment with anti-GAL9 P9-18 increases PD-L2 expression on tumor-associated dendritic cells and tumor cells.

[0340] The effects of anti-GAL9 P9-18 on PD-L1 and PD-L2 cell surface expression on tumor-associated dendritic cells and tumor cells were investigated.

[0305]

[0341] Animals and treatment

[0342] 3-5 BALB / c mice were subcutaneously transplanted with CT26 tumor cells and treated with P9-18 ABS formatted with a mouse IgG2a skeleton or a mouse IgG2a control. All treatments were administered via 200 μg (IP) on days 7 and 11.

[0306]

[0343] Flow cytometry

[0344] On the 13th day, the tumor was excised, digested, and dissected. Next, CD45.1 cells, including immune cells and tumor cells, were examined. + Cell populations were isolated using anti-CD45.1 magnetic beads (Miltenyi Biotec, Germany). + Labeling cell populations, tumor-associated dendritic cells (CD11c + ) and PD-L1 and PD-L2 cell surface expression on tumor cells were analyzed by flow cytometry. The reagents used are shown in Table 13 below. [Table 13]

[0307]

[0345] statistical analysis

[0346] We performed a Welch-adjusted independent t-test using GraphPad Prism 7 Software (San Diego, California).

[0308]

[0347] Results / Conclusion

[0348] Figure 13 shows PD-L1 after treatment with P9-18 (mouse IgG2a skeleton) or control. + Or PD-L2 + Tumor-associated dendritic cells (CD11c + The average percentage of ) and tumor-associated dendritic cells (CD11c + ) Indicates the mean cell surface expression level (GMI) of PD-L1 or PD-L2 on the above. Treatment with P9-18 is PDL2 + The percentage of tumor-associated dendritic cells was significantly increased. PD-L1 and PD-L2 expression (GMI) on tumor-associated dendritic cells was also significantly increased compared to the control. See Figure 13. Data are shown as means, and error bars represent ±SEM.

[0309]

[0349] Figure 14 shows PD-L1 after treatment with P9-18 (mouse IgG2a skeleton) or IgG control. + 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 are shown. 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. See Figure 14. Data are shown as means, and error bars are ±SEM. While we do not wish to be bound by theory, the inventors of PD-L2 + We assume that tumor cells can inhibit the binding of PD-L1 to PD-1 on the tumor.

[0310] [7. Equals]

[0350] While various specific embodiments are illustrated and described, the above specification is not limiting. It will be understood that various modifications can be made without departing from the spirit and scope of the invention. A number of variations will become apparent to those skilled in the art upon examination of this specification.

[0311] [1. Cross-reference of related applications]

[0001] This application asserts the interests under Section 119(e) of the prior concurrently pending U.S. Patent Provisional Application No. 62 / 964,487 filed on 22 January 2020, U.S. Patent Provisional Application No. 62 / 900,105 filed on 13 September 2019, and U.S. Patent Provisional Application No. 62 / 855,590 filed on 31 May 2019.

[0312] [2. Sequence Listing]

[0002] This application was filed via EFS-Web and includes a sequence listing which is incorporated herein in its entirety by reference. The above ASCII copy was created on 9 April 2020, named 42700WO_CRF_sequencelisting.txt, and has a size of 389,339 bytes.

Claims

1. A galectin-9 (GAL9) antigen-binding molecule comprising a first antigen-binding site (ABS) specific to a first epitope of a first GAL9 antigen, The first antigen-binding site is a GAL9 antigen-binding molecule containing all three VH CDRs and all three VL CDRs derived from any one of the ABS clones shown in the table below. Table 1 Table 2

2. The first ABS is specific to the first epitope of the first GAL9 antigen, and the first antigen-binding site is (a) The VL sequence of sequence number 1064 and the VH sequence of sequence number 1063, or (b) The VL sequence of sequence number 1056 and the VH sequence of sequence number 1055, or (c) The VL sequence of sequence number 1076 and the VH sequence of sequence number 1075, or (d) VL sequence of sequence number 1088 and VH sequence of sequence number 1087 A GAL9 antigen-binding molecule according to claim 1, comprising:

3. The GAL9 antigen-binding molecule according to claim 1 or 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 according to any one of claims 1 to 3, further comprising a second antigen-binding site (ABS).

5. The GAL9 antigen-binding molecule according to claim 4, wherein the second ABS is specific to the first or second epitope of the GAL9 antigen.

6. A GAL9 antigen-binding molecule according to any one of claims 1 to 5, comprising an antibody format selected from the group consisting of a full-length antibody, a Fab fragment, Fv, scFv, tandem scFv, a diabody, an sc diabody, a dual affinity retargeting (DART®) antibody, tandAb, a minibody, and a Bbody.

7. The aforementioned GAL9 antigen-binding molecule Upon contact, it increases TNF-α secretion by activated immune cells, and the increase is greater than 80 times compared to activated immune cells treated with the control agent, and / or Upon contact, it increases IFN-γ secretion by activated immune cells, and the increase is greater than 1.2 times compared to activated immune cells treated with the control agent, and / or Upon contact, it increases the surface expression of CD40L on activated CD8+ T cells, and the increase is greater than 2 times compared to activated CD8+ T cells treated with the control agent, and / or Upon contact, it increases OX40 surface expression on activated CD8+ T cells, and the increase is greater than 2-fold compared to activated CD8+ T cells treated with the control agent, and / or The GAL9 antigen-binding molecule, upon contact, increases IL-12 production in activated dendritic cells (DCs), and the increase is greater than 20 times compared to activated DCs treated with the control agent, and / or A GAL9 antigen-binding molecule according to any one of claims 1 to 6, wherein upon contact, it increases PD-L2 surface expression on activated dendritic cells (DCs), and the increase is greater than four times compared to activated DCs treated with a control agent.

8. The GAL9 antigen-binding molecule according to claim 7, wherein the control agent is a negative control agent or a positive control agent, and is optionally 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.

9. A GAL9 antigen-binding molecule that binds to the same epitope as the GAL9 antigen-binding molecule described in any one of claims 1 to 8.

10. A GAL9 antigen-binding molecule that competes for binding with the GAL9 antigen-binding molecule described in any one of claims 1 to 9.

11. A purified GAL9 antigen-binding molecule according to any one of claims 1 to 10.

12. A pharmaceutical composition comprising a GAL9 antigen-binding molecule according to any one of claims 1 to 11 and a pharmaceutically acceptable excipient.

13. A pharmaceutical composition according to claim 12 for treating cancer.

14. The pharmaceutical composition according to claim 13, wherein the cancer is selected from the group consisting of pancreatic cancer, ovarian cancer, breast cancer, lung cancer, gastric cancer, melanoma, Ewing's sarcoma, chronic lymphocytic leukemia, mantle cell lymphoma, B-ALL, hematological cancer, head and neck squamous cell carcinoma, prostate cancer, colon cancer, kidney cancer, uterine cancer, and virus-induced cancer.

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