Anti-Gal9 immunosuppressive binding molecule

Abstract

This invention demonstrates an inhibitory anti-GAL9 binding molecule, an antibody construct, a pharmaceutical composition comprising the binding molecule and the antibody construct, and methods of using thereof.

Description

[0001] 1. Cross-reference of related applications

[0002] This application claims the benefit of previously co-pending U.S. Provisional Patent Application No. 62 / 900,105, filed September 13, 2019, and U.S. Provisional Patent Application No. 62 / 855,590, filed May 31, 2019, pursuant to 35U.SC119(e).

[0003] 2. Sequence List

[0004] This application contains a sequence listing, which is filed together with other application documents and is incorporated herein by reference in its entirety. The computer-readable vector txt file containing the nucleotide or amino acid sequence listing was created in November 2021 and is 486,128 bytes in size. 3. Background Technology

[0006] Autoimmune diseases are caused by an imbalance within the immune system, leading to an immune-mediated attack on the body's own cells and tissues. Currently, the "gold standard" for treating autoimmune diseases is systemic immunosuppression through immunosuppressants, including glucocorticoids, anti-cytokine antibodies such as anti-TNF-α, anti-IL-1, anti-IL-5, anti-IL-6, anti-IL-17, and anti-IL-23 antibodies, and small molecule drugs that reduce inflammatory cytokine signaling, such as JAK / STAT inhibitors. However, nonspecific systemic immunosuppression makes patients more susceptible to infectious diseases and may have other serious side effects.

[0007] Immunotherapy holds great promise for treating autoimmune diseases. Galectin-9 (GAL9) is an S-type lectin β-galactosidase-binding protein with its N-terminal and C-terminal carbohydrate-binding domains linked by a linker peptide. GAL9 is associated with the regulation of cell-cell and cell-matrix interactions. GAL9 has been shown to bind soluble PD-L2, and at least some immunological effects of PD-L2 have been proposed to be mediated by the binding of multimeric PD-L2 to GAL9 rather than by PD-1 (WO 2016 / 008005, incorporated herein by reference in its entirety). However, the mechanisms by which GAL9 and PD-L2 influence immune effector function are not fully understood.

[0008] More targeted treatments are still needed to restore balance to the immune system by modulating immune effector cells to establish a more clinically viable cytokine profile. Such therapies may be useful in improving the treatment of autoimmune and inflammatory diseases. 4. Overview of the Invention

[0010] The invention arose in part from the unexpected discovery that PD-L2 is overexpressed in autoimmune diseases and that inhibiting the Galectin-9 / PD-L2 pathway can modulate immune effector cells to produce a wider range of clinically beneficial cytokines.

[0011] Therefore, this paper discloses various GAL9 binding molecules, their antigen-binding moieties, and antibodies that specifically bind to and antagonize human GAL9 (Galectin-9). Inhibition of GAL9 using the disclosed anti-human GAL9 binding molecules reduces the secretion and production of pro-inflammatory cytokines, increases the secretion and production of anti-inflammatory cytokines, and reduces the surface expression of stimulating molecules.

[0012] Pharmaceutical compositions comprising GAL9-binding molecules are also disclosed. The anti-GAL9-binding molecules, their antigen-binding moieties, and antibodies disclosed herein can be used as pharmaceutical compositions on their own or in combination with other therapeutic agents or procedures to treat, prevent, and / or diagnose autoimmune diseases, inflammatory diseases, or diseases that elicit inflammatory responses, such as infections. Anti-GAL9-binding molecules are particularly useful for diseases or conditions in which the interaction between GAL9 and PD-L2 plays a significant role in the pathogenesis. Anti-GAL9-binding molecules are beneficial for treating, reducing inflammation, reducing autoimmune responses, prolonging remission, inducing remission, re-establishing immune tolerance, improving organ function, reducing disease progression, reducing the risk of developing a second disease, or increasing overall survival in subjects.

[0013] In a first aspect, the present invention discloses 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 from any one of the ABS clones selected from P9-01, P9-02A, P9-03, P9-06, P9-07, P9-11, P9-12, P9-14, P9-23, P9-24, P9-25, P9-29, P9-30, P9-34, P9-37, P9-38, P9-40, P9-41, P9-42, P9-43, P9-44, P9-45, P9-46, P9-50, P9-51, P9-52, P9-53, P9-56 and P9-57.

[0014] In a second aspect, the present invention 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 from any one of the ABS clones selected from P9-01, P9-02A, P9-03, P9-06, P9-07, P9-11, P9-12, P9-14, P9-23, P9-24, P9-25, P9-29, P9-30, P9-34, P9-37, P9-38, P9-40, P9-41, P9-42, P9-43, P9-44, P9-45, P9-46, P9-50, P9-51, P9-52, P9-53, P9-56, and P9-57.

[0015] In a third aspect, the present invention discloses 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 from any one of the ABS clones selected from P9-01, P9-02A, P9-03, P9-06, P9-07, P9-11, P9-12, P9-14, P9-23, P9-24, P9-25, P9-29, P9-30, P9-34, P9-37, P9-38, P9-40, P9-41, P9-42, P9-43, P9-44, P9-45, P9-46, P9-50, P9-51, P9-52, P9-53, P9-56 and P9-57.

[0016] In a fourth aspect, the present invention discloses a Galectin-9 (GAL9) antigen-binding molecule comprising a first antigen-binding site specific to a first epitope of a first GAL9 antigen, which comprises a VL sequence and a VH sequence from any ABS clone selected from P9-01, P9-02A, P9-03, P9-06, P9-07, P9-11, P9-12, P9-14, P9-23, P9-24, P9-25, P9-29, P9-30, P9-34, P9-37, P9-38, P9-40, P9-41, P9-42, P9-43, P9-44, P9-45, P9-46, P9-50, P9-51, P9-52, P9-53, P9-56 and P9-57.

[0017] In some embodiments, the GAL9 antigen-binding molecule comprises a whole immunoglobulin heavy chain "IgG1" sequence containing a VH sequence and a whole immunoglobulin light chain sequence containing a VL sequence, wherein the VH and VL sequences are derived from any one of the ABS clones selected from P9-01, P9-02A, P9-03, P9-06, P9-07, P9-11, P9-12, P9-14, P9-23, P9-24, P9-25, P9-29, P9-30, P9-34, P9-37, P9-38, P9-40, P9-41, P9-42, P9-43, P9-44, P9-45, P9-46, P9-50, P9-51, P9-52, P9-53, P9-56, and P9-57.

[0018] In some embodiments, the GAL9 antigen-binding molecule comprises a complete immunoglobulin heavy chain "IgG4" sequence containing the VH sequence and a complete immunoglobulin light chain sequence containing the VL sequence, wherein the VH and VL sequences are derived from any one of the ABS clones selected from P9-01, P9-02A, P9-03, P9-06, P9-07, P9-11, P9-12, P9-14, P9-23, P9-24, P9-25, P9-29, P9-30, P9-34, P9-37, P9-38, P9-40, P9-41, P9-42, P9-43, P9-44, P9-45, P9-46, P9-50, P9-51, P9-52, P9-53, P9-56, and P9-57.

[0019] In some implementations, the GAL9 antigen-binding molecule may contain a GAL9 antigen, which is a human GAL9 antigen.

[0020] In some implementations, the GAL9 antigen-binding molecule may further include a second antigen-binding site.

[0021] In some embodiments, the second antigen binding site is specific to the GAL9 antigen. In other embodiments, the second antigen binding site is the same as the first antigen binding site.

[0022] In other embodiments, the second antigen binding site is specific to the second epitope of the first GAL9 antigen.

[0023] 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 VLCDRs from another ABS clone selected from P9-01, P9-02A, P9-03, P9-06, P9-07, P9-11, P9-12, P9-14, P9-23, P9-24, P9-25, P9-29, P9-30, P9-34, P9-37, P9-38, P9-40, P9-41, P9-42, P9-43, P9-44, P9-45, P9-46, P9-50, P9-51, P9-52, P9-53, P9-56, and P9-57.

[0024] In some implementations, the second antigen binding site comprises the VL and VH sequences from another ABS clone.

[0025] In some embodiments, the second antigen binding site comprises a complete immunoglobulin heavy chain sequence including a VH sequence and a complete immunoglobulin light chain sequence including a VL sequence.

[0026] In some implementations, the second antigen binding site is specific to antigens that are different from the first GAL9 antigen.

[0027] In some embodiments, the first antigen binding site comprises all three VH CDRs, all three VL CDRs, or all three VH CDRs and all three VLCDRs from any ABS clone selected from P9-01, P9-02A, P9-03, P9-06, P9-07, P9-11, P9-12, P9-14, P9-23, P9-24, P9-25, P9-29, P9-30, P9-34, P9-37, P9-38, P9-40, P9-41, P9-42, P9-43, P9-44, P9-45, P9-46, P9-50, P9-51, P9-52, P9-53, P9-56, and P9-57.

[0028] In some embodiments, the first antigen binding site comprises all three VH CDRs, all three VL CDRs, or all three VH CDRs and all three VL CDRs from any ABS clone selected from P9-11, P9-24, P9-34, and P9-37.

[0029] In some implementations, the first antigen binding site comprises all three VH CDRs, all three VL CDRs, or all three VH CDRs and all three VL CDRs from any ABS clone selected from P9-11, P9-24, and P9-34.

[0030] In some implementations, the first antigen binding site comprises all three VHCDRs, all three VL CDRs, or all three VH CDRs and all three VL CDRs from ABS clone P9-11.

[0031] In some implementations, the first antigen binding site comprises all three VHCDRs, all three VL CDRs, or all three VH CDRs and all three VL CDRs from ABS clone P9-24.

[0032] In some implementations, the first antigen binding site comprises all three VHCDRs, all three VL CDRs, or all three VH CDRs and all three VL CDRs from ABS clone P9-34.

[0033] In some implementations, the first antigen binding site comprises all three VHCDRs, all three VL CDRs, or all three VH CDRs and all three VL CDRs from ABS clone P9-37.

[0034] In some embodiments, the GAL9 antigen-binding molecule includes antibody formats selected from the group consisting of: full-length antibodies, Fab fragments, F(ab)'2 fragments, Fvs, scFvs, tandem scFvs, diabody antibodies, scDiabodies, DARTs, single-chain VHH camel antibodies, tandAbs, minibody antibodies, and B-body. B-body is described in the pre-license publication US No. 2018 / 0118811, the entire contents of which are incorporated herein by reference.

[0035] In some embodiments, the GAL9 antigen-binding molecule reduces TNF-α secretion from activated immune cells upon contact, wherein the reduction is approximately 30%, 35%, 40%, 45%, 50%, 55%, or 60% relative to activated immune cells treated with a control agent.

[0036] In some embodiments, the GAL9 antigen-binding molecule reduces IFN-γ secretion from activated immune cells upon contact, wherein the reduction is approximately 20%, 25%, 30%, 35%, 40%, 45%, or 50% relative to activated immune cells treated with a control agent.

[0037] In some embodiments, the GAL9 antigen-binding molecule increases IL-10 secretion from activated immune cells upon contact, wherein the increase is approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40% relative to activated immune cells treated with a control.

[0038] In some implementations, the GAL9 antigen-binding molecule does not regulate PD-1 surface expression on activated immune cells, relative to activated immune cells treated with a control agent.

[0039] In some implementations, the GAL9 antigen-binding molecule does not modulate PD-L1 surface expression on activated immune cells, relative to activated immune cells treated with a control agent.

[0040] In some implementations, the GAL9 antigen-binding molecule does not regulate CTLA-4 surface expression on activated immune cells, relative to activated immune cells treated with a control agent.

[0041] In some implementations, the GAL9 antigen-binding molecule does not regulate TIM3 surface expression on activated immune cells, relative to activated immune cells treated with a control agent.

[0042] In some implementations, the GAL9 antigen-binding molecule does not modulate LAG3 surface expression on activated immune cells compared to activated immune cells treated with a control agent.

[0043] In some implementations, relative to activated CD8 treated with a control agent + T cells, GAL9 antigen-binding molecules reduce activated CD8 + 4-1BB is expressed on the surface of T cells.

[0044] In some implementations, relative to activated CD8 treated with a control agent + T cells, GAL9 antigen-binding molecule reduces CD40L activity in activated CD8 + Surface expression on T cells.

[0045] In some implementations, the GAL9 antigen-binding molecule reduces the expression of activated OX40 on CD8 T cells compared to activated CD8 T cells treated with a control agent.

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

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

[0048] In some implementations, the control antibody is selected from a group consisting of ECA42 clone anti-GAL9 antibody, RG9.1 clone anti-GAL9 antibody, RG9.35 clone anti-GAL9 antibody, anti-PD1 antibody, 108A2 clone anti-GAL9 antibody and isotype control antibody that does not bind to GAL9.

[0049] In some implementation schemes, activated immune cells, activated CD8 + T cells, or activated dendritic cells, are activated by stimulation with peptides, anti-CD3, or dendritic cells.

[0050] In a fifth aspect, this disclosure provides a GAL9 antigen-binding molecule that reduces TNF-α secretion by activated immune cells, wherein the reduction is approximately 30%, 35%, 40%, 45%, 50%, 55%, or 60% relative to activated immune cells treated with a control agent.

[0051] In a sixth aspect, this disclosure provides a GAL9 antigen-binding molecule that reduces IFN-γ secretion by activated immune cells, wherein the reduction is approximately 20%, 25%, 30%, 35%, 40%, 45%, or 50% relative to activated immune cells treated with a control agent.

[0052] In a seventh aspect, the present invention provides a GAL9 antigen-binding molecule that increases the secretion of IL-10 by activated immune cells, wherein the increase is approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40% relative to activated immune cells treated with a control agent.

[0053] In an eighth aspect, the GAL9 antigen-binding molecule provided by the present invention does not regulate PD-1 surface expression on activated immune cells, relative to activated immune cells treated with a control agent.

[0054] In a ninth aspect, the GAL9 antigen-binding molecule provided by the present invention does not regulate PD-L1 surface expression on activated immune cells, relative to activated immune cells treated with a control agent.

[0055] In a tenth aspect, the GAL9 antigen-binding molecule provided by the present invention does not regulate the surface expression of CTLA-4 on activated immune cells, relative to activated immune cells treated with a control agent.

[0056] In the eleventh aspect, compared with activated immune cells treated with a control agent, the GAL9 antigen-binding molecule provided by the present invention does not regulate the surface expression of TIM3 on activated immune cells.

[0057] In a twelfth aspect, the GAL9 antigen-binding molecule provided by the present invention does not regulate the surface expression of LAG3 on activated immune cells, relative to activated immune cells treated with a control agent.

[0058] In a thirteenth aspect, the present invention provides a GAL9 antigen-binding molecule that reduces the activation of CD8 T cells relative to activated CD8 T cells treated with a control agent. + 4-1BB is expressed on the surface of T cells.

[0059] In a fourteenth aspect, the present invention provides a GAL9 antigen-binding molecule that, relative to activated CD8 T cells treated with a control agent, reduces the activity of CD40L in activated CD8 T cells. + Surface expression on T cells.

[0060] In a fifteenth aspect, the present invention provides a GAL9 antigen-binding molecule that reduces the expression of activated OX40 on CD8 T cells relative to activated CD8 T cells treated with a control agent.

[0061] In a sixteenth aspect, the GAL9 antigen-binding molecule disclosed in this invention exhibits one or more of the following properties: A) reducing TNF-α secretion by activated immune cells, wherein the reduction is approximately 30%, 35%, 40%, 45%, 50%, 55%, or 60% relative to activated immune cells treated with a control agent; B) reducing IFN-γ secretion by activated immune cells, wherein the reduction is approximately 20%, 25%, 30%, 35%, 40%, 45%, or 50% relative to activated immune cells treated with a control agent; C) increasing IL-10 secretion by activated immune cells, wherein the increase is approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40% relative to activated immune cells treated with a control agent; D) not regulating PD-1 surface expression of activated immune cells relative to activated immune cells treated with a control agent; E) not regulating PD-L1 surface expression of activated immune cells relative to activated immune cells treated with a control agent. F) Compared to activated immune cells treated with the control, it does not regulate the surface expression of CTLA-4 on activated immune cells; G) Compared to activated immune cells treated with the control, it does not regulate the surface expression of TIM3 on activated immune cells; H) Compared to activated immune cells treated with the control, it does not regulate the surface expression of LAG3 on activated immune cells. I) Compared to activated CD8+ treated with the control... + For T cells, reducing CD8 activation + 4-1BB surface expression on T cells; J) relative to activated CD8 cells treated with a control agent. + For T cells, reducing CD8 activation + CD40L expression on the surface of T cells; or K) relative to activated CD8 cells treated with a control agent. + For T cells, reducing CD8 activation + OX40 surface expression on T cells.

[0062] In some implementations, the control agent is either a negative control or a positive control.

[0063] In some implementations, the control agent is a control antibody.

[0064] In some implementations, the control antibody is selected from the following: ECA42 clone anti-GAL9 antibody, RG9.1 clone anti-GAL9 antibody, RG9.35 clone anti-GAL9 antibody, anti-PD1 antibody, 108A2 clone anti-GAL9 antibody and isotype control antibody that does not bind to GAL9.

[0065] In some implementations, the activated immune cells are stimulated by peptides, anti-CD3, or dendritic cells.

[0066] In some embodiments, the GAL9 antigen-binding molecules of the fifth to fifteenth aspects provided herein comprise 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 from any one of the ABS clones selected from P9-01, P9-02A, P9-03, P9-06, P9-07, P9-11, P9-12, P9-14, P9-23, P9-24, P9-25, P9-29, P9-30, P9-34, P9-37, P9-38, P9-40, P9-41, P9-42, P9-43, P9-44, P9-45, P9-46, P9-50, P9-51, P9-52, P9-53, P9-56, and P9-57.

[0067] In some implementations, the VL and VH sequences are derived from any of the ABS clones selected from P9-01, P9-02A, P9-03, P9-06, P9-07, P9-11, P9-12, P9-14, P9-23, P9-24, P9-25, P9-29, P9-30, P9-34, P9-37, P9-38, P9-40, P9-41, P9-42, P9-43, P9-44, P9-45, P9-46, P9-50, P9-51, P9-52, P9-53, P9-56, and P9-57.

[0068] In certain specific embodiments, the GAL9 antigen-binding molecule comprises a complete immunoglobulin heavy chain sequence including the VH sequence and a complete immunoglobulin light chain sequence including the VL sequence, wherein the VH and VL sequences are derived from any one of the ABS clones selected from P9-01, P9-02A, P9-03, P9-06, P9-07, P9-11, P9-12, P9-14, P9-23, P9-24, P9-25, P9-29, P9-30, P9-34, P9-37, P9-38, P9-40, P9-41, P9-42, P9-43, P9-44, P9-45, P9-46, P9-50, P9-51, P9-52, P9-53, P9-56, and P9-57.

[0069] In some implementations, the GAL9 antigen is the human GAL9 antigen.

[0070] In some implementations, the GAL9 antigen-binding molecule further includes a second antigen-binding site.

[0071] In some implementations, the second antigen binding site is specific to the GAL9 antigen.

[0072] In some embodiments, the second antigen binding site is the same as the first antigen binding site.

[0073] In some implementations, the second antigen binding site is specific to the second epitope of the first GAL9 antigen.

[0074] In some embodiments, the second antigen binding site comprises all three VH CDRs and all three VL CDRs from another ABS clone selected from P9-01, P9-02A, P9-03, P9-06, P9-07, P9-11, P9-12, P9-14, P9-23, P9-24, P9-25, P9-29, P9-30, P9-34, P9-37, P9-38, P9-40, P9-41, P9-42, P9-43, P9-44, P9-45, P9-46, P9-50, P9-51, P9-52, P9-53, P9-56, and P9-57.

[0075] In some implementations, the second antigen binding site comprises VL and VH sequences from other ABS clones.

[0076] In some embodiments, the second antigen binding site comprises a complete immunoglobulin light chain sequence containing the VL sequence and a complete immunoglobulin heavy chain sequence containing the VH sequence from another ABS clone.

[0077] In some implementations, the second antigen binding site is specific to antigens that are different from the first GAL9 antigen.

[0078] In some implementations, the first antigen binding site comprises all three VH CDRs and all three VL CDRs from any one of the ABS clones selected from P9-11, P9-24, P9-34, and P9-37.

[0079] In some implementations, the first antigen binding site comprises all three VH CDRs and all three VL CDRs from any ABS clone selected from P9-11, P9-24, and P9-34.

[0080] In some implementations, the first antigen binding site comprises all three VH CDRs and all three VL CDRs from ABS clone P9-11.

[0081] In some implementations, the first antigen binding site comprises all three VH CDRs and all three VL CDRs from ABS clone P9-24.

[0082] In some implementations, the first antigen binding site comprises all three VH CDRs and all three VL CDRs from ABS clone P9-34.

[0083] In some implementations, the first antigen binding site comprises all three VH CDRs and all three VL CDRs from ABS clone P9-37.

[0084] In some implementations, the GAL9 antigen-binding molecule includes antibody formats selected from the group consisting of: full-length antibody, Fab fragment, variable region fragment (Fv), single-chain variable region fragment (scFv), tandem scFv, diabody, scDiabody, DART, tandAb, minibody, and B-body.

[0085] In a seventeenth aspect, this disclosure provides a GAL9 antigen-binding molecule that binds to the same epitope as the GAL9 antigen-binding molecule of any of the preceding claims.

[0086] In an eighteenth aspect, this disclosure provides a GAL9 antigen-binding molecule that competes with the GAL9 antigen-binding molecule of any of the preceding claims.

[0087] In some implementations, the GAL9 antigen-binding molecule is purified.

[0088] In a nineteenth aspect, this disclosure provides a pharmaceutical composition comprising the GAL9 antigen-binding molecule of any of the preceding claims and a pharmaceutically acceptable diluent.

[0089] In a twentieth aspect, this disclosure provides a method for treating a subject suffering from an autoimmune disease, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition provided herein.

[0090] In some implementations, subjects with autoimmune diseases showed increased PD-L2 expression on dendritic cells compared to those from healthy controls.

[0091] In some implementation schemes, autoimmune diseases are selected from the group consisting of inflammatory bowel disease, Crohn's disease, ulcerative colitis, colitis, celiac disease, rheumatoid arthritis, Behcet's disease, amyloidosis, psoriasis, psoriatic arthritis, systemic lupus erythematosus nephritis, graft-versus-host disease (GVHD), non-alcoholic steatohepatitis (NASH), and ankylosing spondylitis.

[0092] In some implementations, administering a therapeutically effective amount of the GAL-binding molecule itself or the pharmaceutical composition results in reduced inflammation, reduced autoimmune response, prolonged remission, induced remission, reconstitution of immune tolerance, improved organ function, reduced disease progression, reduced risk of progression or occurrence of a second disease, or increased overall survival. 5. Brief description of the attached diagram

[0094] Figure 1A and 1B This demonstrates various applications, such as those used in the P9-01 anti-human Gal9 candidate antibody presented herein.

[0095] CDR and Frame Numbering Systems – Illustrative Examples of Chothia, Martin (ABA), and Kabat.

[0096] Figure 2 CD11c from Crohn's disease patients was shown. + Density contour plot of the percentage of blood dendritic cells detected as positive for PD-L1 or PD-L2 expression, compared with labeled isotype IgG controls.

[0097] Figure 3A and 3B A scatter plot showing the percentage of blood dendritic cells expressing PD-L1 or PD-L2 in healthy controls or patients with Crohn's disease. Figure 3C and 3D A scatter plot showing geometric mean fluorescence (GMI) of PD-L1 or PD-L2 expression on the surface of blood dendritic cells in healthy controls or Crohn's disease patients.

[0098] Figure 4A and 4B Representative confocal images showing the expression of DNA (DAPI; blue), PD-L1 (green), and PD-L2 (red) on dendritic cells from two healthy controls (4A) and three Crohn's disease patients (4B); presented in grayscale in the accompanying figures.

[0099] Figures 5A-5C The figures show the mean concentrations of cytokines secreted by PMBCs in patients with Crohn's disease (CD) after treatment with anti-CD3 mimicking TCR activation and either anti-PD-L2 (αPD-L2) or IgG controls. Figures 5A-5B show the mean concentrations of TNF-α and IFN-γ after treatment of PMBCs in CD patients with either anti-PD-L2 or IgG controls. Figure 5C The mean ratio of IL-10:TNF-α secretion is shown after PMBC in CD patients treated with anti-PD-L2 and IgG controls.

[0100] Figure 6 The image shows anti-CD3 activated mouse CD4 after treatment with sPD-L2 or co-treatment with sPD-L2 and inhibitory anti-mouse anti-GAL9 (108A2). + TNF-α secreted by T cells.

[0101] Figure 7 The study demonstrated the reduction of CD4+ in mice infected with malaria after treatment with mouse inhibitory anti-mouse GAL9 (108A2) and activating anti-mouse GAL9 (RG9.1) antibodies. + Representative confocal images of DNA (DAPI; blue), PD-L1 (green), PD-1 (red), and OX40 (yellow) expression in T cells; presented in grayscale in the accompanying figures.

[0102] Figure 8A and 8B The image shows mouse CD4 counts that survived treatment with sPD-L2 or with sPD-L2 and mouse inhibitory anti-GAL9 (108A2) antibody. + and CD8 + A bar chart showing the percentage of T cells.

[0103] Figure 9A and 9B The image shows mouse CD4 cells co-cultured with dendritic cells (stimulated) and treated with either blocking anti-PD-L2 (clone Ty25) or inhibitory anti-GAL9 (108A2) mouse antibodies. + A bar chart of INF-γ (9A) and TNF-α (9B) secreted by T cells, compared with unstimulated CD4+ in the control group. + Compared to T cells.

[0104] Figure 10A and 10B The secretion of INF-γ (10A) and TNF-α (10B) from PBMCs stimulated in vitro with treated HCMV peptides using various anti-human GAL9 candidates, known tool mAbs, anti-PD-1 antibodies, IgG control antibodies (IgG Ctrl), and vehicle controls (PBS control). Black diamonds indicate the secretion of activated PBMCs stimulated by the tool mAb and anti-PD-1 antibody.

[0105] Figure 11A-11C The study showed that, compared with the IgG control antibody (IgG), IFN-γ and TNF-α secreted from HCMV peptides in vitro stimulated by PBMCs were reduced after treatment with anti-human GAL9 P9-11, P9-37, or P9-57.

[0106] Figures 12A-12C The study showed that, compared with the IgG control antibody (IgG), TNF-α (12A), IFN-γ (12B), and IL-10 (12C) secreted from HCMV peptides, in vitro stimulated PBMCs, after treatment with anti-human GAL9 candidates P9-11, P9-24, or P9-34 were secreted.

[0107] Figure 13A and 13B The study demonstrated that treatment with inhibitory anti-mouse GAL9 (108A2) and anti-human GAL9 P9-11, P9-24, or P9-34 reduced the activity of anti-CD3 activated mouse CD3. + A bar chart showing the ratio of TNF-α:IL-10 secretion by T cells (13A) and the ratio of IFN-γ:IL-10 secretion (13B). 6. Detailed Description of the Invention

[0109] 6.1. Definition

[0110] Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. As used herein, the following terms have the meanings assigned to them hereinafter.

[0111] "Antigen binding site" or "ABS" refers to the region in the GAL9 binding molecule that specifically recognizes or binds to a given antigen or epitope.

[0112] As used herein, the terms "treat" or "treatment" are used in their most widely accepted clinical sense. These terms include, but are not limited to, alleviating the signs or symptoms of a disease; improving the signs or symptoms of a disease; reducing symptoms; reducing the extent of a disease; stabilizing (i.e., not worsening) the state of a disease; delaying or slowing the progression of a disease; improving or mitigating the state of a disease; achieving remission (whether partial or complete), whether detectable or undetectable; curing; and prolonging survival compared to expected survival without treatment. Unless otherwise expressly stated, "treat" or "treatment" does not imply prevention or avoidance of disease.

[0113] "Subject" or "individual" or "animal" or "patient" or "mammal" means any subject on whom diagnosis, prognosis, or treatment is desired, particularly a mammalian subject. Mammal subjects include humans, livestock, farm animals and zoo, sporting, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cows, dairy cows, and so on. Unless otherwise stated, "patient" refers to a human "subject".

[0114] The term "sufficient amount" refers to an amount that is sufficient to produce the desired effect, such as an amount sufficient to regulate the aggregation of proteins in cells.

[0115] The term "therapeutic effective dose" refers to the amount that can effectively improve the symptoms of a disease.

[0116] The term "preventive effective dose" refers to the amount that can effectively prevent the symptoms of a disease.

[0117] 6.2. Other Interpretive Conventions

[0118] Unless otherwise specified, all sequences mentioned in this article refer to amino acid sequences.

[0119] Unless otherwise specified, the residue numbering of the antibody constant region is based on 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. The residue numbering is determined by the position of the residue in the endogenous constant region sequence, regardless of the physical position of the residue in the GAL9 binding molecular chain described herein.

[0120] Unless otherwise specified as "Kabat CDR", "Chothia CDR", "Contact CDR" or "IMGT CDR", all references to "CDR" refer to the CDR defined using the Martin (ABA) definition.

[0121] "Endogenous sequence" or "native sequence" refers to any sequence, including nucleic acid and amino acid sequences, that originates from an organism, tissue, or cell that has not been artificially modified or mutated.

[0122] Polypeptide chain numbers (e.g., “first” polypeptide chain, “second” polypeptide chain, etc., or polypeptide “chain 1”, “chain 2”, etc.) are used herein as unique identifiers for specific polypeptide chains that form the bound molecule and are not intended to imply the order or number of different polypeptide chains within the bound molecule.

[0123] In this disclosure, "comprise", "comprising", "containing", "having", "include", "including" and their linguistic variations have the meanings given to them under U.S. patent law, allowing for the presence of additional components beyond those explicitly stated.

[0124] As used herein, unless the context clearly indicates otherwise, the singular forms "a," "an," and "the" include plural references. The terms "include," "such as," etc., are intended to express unrestricted inclusion unless otherwise specified.

[0125] The ranges provided in this document are understood as a simplified representation of all values ​​within the range, including the endpoints. For example, the range 1 to 50 is understood to include values ​​from 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, ...

[0126] Any number, combination of numbers, or subrange consisting of the group consisting of 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.

[0127] Unless otherwise specified or apparent from the context, the term "approximately" as used herein is understood to mean within the normal tolerance range in the field, such as within 2 standard deviations of the mean. "Approximately" can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the listed values.

[0128] 6.3. Overview

[0129] This disclosure provides Galectin-9 (GAL9) antigen-binding molecules, such as anti-GAL9 antibodies and their antigen-binding fragments; compositions comprising GAL9 binding molecules; pharmaceutical compositions comprising GAL9 binding molecules; and methods of treating subjects with diseases or conditions using GAL9 binding molecules. Specifically, the invention provides a variety of GAL9 antigen-binding molecules that are inhibitory, acting as inhibitors of the immune system, reducing the secretion and production of pro-inflammatory cytokines in various immune cells, increasing the secretion and production of anti-inflammatory cytokines, and reducing the surface expression of stimulating molecules.

[0130] GAL9 antigen-binding molecules are particularly useful for treating autoimmune or inflammatory diseases in subjects. In some embodiments, the composition and method are used to treat infections that cause an inflammatory response in the subject. Anti-GAL9 binding molecules are particularly suitable for treating diseases or conditions in which the GAL9 / PD-L2 interaction significantly promotes the pathogenesis. In some embodiments, the anti-GAL9 binding molecule is administered to the subject as a pharmaceutical composition, or in combination with other therapeutic agents or procedures.

[0131] 6.4. GAL9 antigen-binding molecule

[0132] In a first aspect, an antigen-binding molecule is provided. In each embodiment, the antigen-binding molecule contains at least a first antigen-binding site specific to the GAL9 antigen; therefore, the binding molecule is referred to as...

[0133] GAL9 antigen-binding molecule or GAL9 binding molecule.

[0134] The GAL9 antigen-binding molecule described in this article binds specifically to the GAL9 antigen.

[0135] As used herein, "GAL9 antigen" refers to members and homologs of the Galectin-9 family. GAL9 is also known as LGALS9, HUAT, LGALS9A, tumor antigen HOM-HD-21, and ecalectin. In certain embodiments, the GAL9 binding molecule has an antigen-binding site that specifically binds to at least a portion of one or more GAL9 domains, such as the junction between the first and second GAL9 domains.

[0136] In the specific implementation plan, the GAL9 antigen is human. (GenBank login number #)

[0137] NP_033665.1 describes a typical human GAL9 protein, including its sequence and domain features, the entire contents of which are incorporated herein by reference. SEQ ID NO:6 provides the full-length sequence of the GAL9 protein.

[0138] MAFSGSQAPYLSPAVPFSGTIQGGLQDGLQITVNGTVLSSSGTRFAVNFQTGFSGNDIAF

[0139] HFNPRFEDGGYVVCNTRQNGSWGPEERKTHMPFQKGMPFDLCFLVQSSDFKVMVNGI

[0140] LFVQYFHRVPFHRVDTISVNGSVQLSYISFQNPRTVPVQPAFSTVPFSQPVCFPPRPRGR

[0141] RQKPPGVWPANPAPITQTVIHTVQSAPGQMFSTPAIPPMMYPHPAYPMPFITTILGGLY

[0142] PSKSILLSGTVLPSAQRFHINLCSGNHIAFHLNPRFDENAVVRNTQIDNSWGSEERSLPR

[0143] KMPFVRGQSFSVWILCEAHCLKVAVDGQHLFEYYHRLRNLPTINRLEVGGDIQLTHVQT[SEQ IDNO:6]

[0144] In various embodiments, the GAL9 binding molecule additionally binds specifically to at least one antigen other than the GAL9 antigen.

[0145] 6.4.1. Functional characteristics of GAL9 antigen-binding molecules

[0146] In a typical implementation, upon contact, the GAL9 antigen-binding molecule regulates cytokine secretion from immune cells or activated immune cells (e.g., increasing or decreasing cytokine secretion). 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+ cells. + T cells. In practice, T cells are CD4+ cells. + T cells. In some implementations, T cells are CD3+ cells. + T cells.

[0147] The effect of GAL9 antigen-binding molecules on the secretion of cytokines by immune cells can be determined by any suitable method. For example, the effect of GAL9 antigen-binding molecules on the secretion of cytokines by immune cells can be determined in vivo, in vitro, or ex vivo. In some embodiments, cytokine secretion is determined in activated immune cells in contact with GAL9 antigen-binding molecules, compared to activated immune cells controlled by a control agent, such as a control antigen-binding molecule or a mediator. Immune cells can be activated by stimulation with peptides. For example, immune cells can be activated by peptides or multiple peptides known to induce an immune response. Multiple peptides known to induce an immune response can originate from infection by pathogens, such as viral or bacterial infections.

[0148] The control agent can be a negative control or a positive control. In some embodiments, the GAL9 antigen-binding molecule increases cytokine secretion in immune cells relative to the negative control agent or 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 can be a Gal9 antibody clone RG9.1 (Cat. No. BE0218, InVivoMabAntibodies) or...

[0149] RG9.35. RG9.1 and RG9.35 are both in Fukushima A, Sumi T, Fukuda K, Kumagai N,

[0150] Nishida T et al. (2008) described in Roles of galectin-9 in the development of experimental allergic conjunctivitis in mice. IntArch Allergy Immunol 146:36-43, the full text of which is incorporated herein by reference. The GAL9 control antibody can be the GAL9 antibody clone ECA42.

[0151] (Cat. No. LS-C 179449, LifeSpan BioScience). The GAL9 control antibody can be GAL9 antibody clone 108A2 ( (San Diego, CA). In some embodiments, the GAL9 antigen-binding molecule reduces the secretion of pro-inflammatory cytokines in immune cells compared to a control antibody. In some embodiments, the GAL9 antigen-binding molecule increases the secretion of inhibitory cytokines in immune cells compared to a control antibody.

[0152] Cytokine secretion by immune cells can be assessed using any suitable method. For example, cytokine secretion in in vitro or ex vivo immune cell culture models can be assessed by analyzing the cytokine content in the cultured cell supernatant, for instance, using a cytokine bead array.

[0153] In some embodiments, the cytokine is TNF-α. In some embodiments, compared with the control agent described herein, the GAL9 antigen-binding molecule reduces the secretion of TNF-α in activated immune cells by at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%. In some embodiments, compared with the control agent described herein, the GAL9 antigen-binding molecule reduces TNF-α secretion in activated immune cells by at least 1%-5%, 5-10%, 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%, or 85-90%. In some embodiments, compared with the control agent described herein, the GAL9 antigen-binding molecule reduces TNF-α secretion in activated immune cells by approximately 30%-50%.

[0154] In some embodiments, the cytokine is IFN-γ. In some embodiments, compared with the control agent described herein, the GAL9 antigen-binding molecule reduces IFN-γ secretion in activated immune cells by at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%. In some embodiments, compared with the control agent described herein, the GAL9 antigen-binding molecule reduces IFN-γ secretion in activated immune cells by at least 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, 35-40%, 40%-45%, 45%-50%, 50%-55%, 55%-60%, 60%-65%, or 70%-75%. In some implementations, compared with the control agent described herein, the GAL9 antigen-binding molecule reduces the secretion of IFN-γ in activated immune cells by approximately 20%–40%.

[0155] In some embodiments, the cytokine is IL-10. In some embodiments, compared with the control agent described herein, the GAL9 antigen-binding molecule increases IL-10 secretion in activated immune cells by at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%. In some embodiments, compared with the control agent described herein, the GAL9 antigen-binding molecule increases IL-10 secretion in activated immune cells by at least 1%-5%, 5%-10%, 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, 35-40%, 40-45%, or 45%-50%. In some embodiments, compared with the control agent described herein, the GAL9 antigen-binding molecule increases IL-10 secretion in activated immune cells by about 5%-30%.

[0156] In some embodiments, the GAL9 antigen-binding molecule does not modulate the surface expression of immune checkpoint molecules (e.g., stimulatory or inhibitory checkpoint molecules) upon contact with activated immune cells treated with a control. The term "non-modulation" means that treatment with the GAL9 binding molecule provided herein results in no substantial increase or decrease in the expression of immune checkpoint molecules compared to a control. In some embodiments, no substantial increase in surface expression (e.g., non-modulated expression) means an increase in cell surface expression not exceeding a 1.01X, 1.02X, 1.03X, 1.04X, 1.05X, 1.06X, 1.07X, 1.08X, 1.09X, 1.1X, 1.2X, or 1.3X fold change relative to activated immune cells treated with a control. In some implementations, no substantial reduction in surface expression (e.g., no regulation of expression) means that the reduction in cell surface expression relative to activated immune cells treated with a control agent does not exceed a change of 0.01X, 0.02X, 0.03X, 0.04X, 0.05X, 0.06X, 0.07X, 0.08X, 0.09X, 0.1X, or 0.2X fold.

[0157] In some embodiments, no substantial increase in surface expression (e.g., unregulated expression) means an increase of approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% in surface expression relative to activated immune cells treated with a control. In some embodiments, no substantial decrease in surface expression (e.g., unregulated expression) relative to activated immune cells treated with a control means a decrease of approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% in surface expression.

[0158] In some embodiments, the absence of a substantial increase or decrease in surface expression is determined by comparing the surface expression level to the noise level in the detection (e.g., in vivo, ex vivo, or in vitro). In some embodiments, the absence of a substantial increase or decrease in surface expression is determined by comparing the surface expression level to the standard deviation in the detection (e.g., in vivo, ex vivo, or in vitro).

[0159] The effect of GAL9 antigen-binding molecules on the surface expression of one or more immune checkpoint molecules can be determined by any suitable 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, in vitro, or ex vivo.

[0160] In some embodiments, one or more immune checkpoint molecules are selected from PD-1, PD-L1, CTLA-4, TIM3, LAG3, TIGIT, and PVRIG. In some embodiments, one or more checkpoint molecules are selected from PD-1, PD-L1, TIM3, and LAG3. In some embodiments, the immune checkpoint molecule is PD-1 or PD-L1. In many embodiments, the activated (e.g., stimulated) immune cells are T cells, CD8 cells, etc. + T cells, CD4 + T cells, CD3 + T cells, or PBMCs.

[0161] In some embodiments, the immune checkpoint molecule is PD-1. In some embodiments, it is relative to activated CD4 treated with a control agent. + or CD8 + T cells, activated CD8 cells treated with GAL9 antigen-binding molecules + or CD4 +T cell expression of PD-1 on the surface showed an increase not exceeding 1.01X, 1.02X, 1.03X, 1.04X, 1.05X, 1.06X, 1.07X, 1.08X, 1.09X, 1.1X, 1.2X, or 1.3X fold. In some embodiments, this is relative to activated CD4 cells treated with a control agent. + or CD8 + T cells, activated CD8 cells treated with GAL9 antigen-binding molecules + or CD4 + T cell expression on the PD-1 surface showed a decrease of no more than 0.01X, 0.02X, 0.03X, 0.04X, 0.05X, 0.06X, 0.07X, 0.08X, 0.09X, 0.1X, or 0.2X times.

[0162] In some implementations, activated CD8 cells treated with GAL9 antigen-binding molecules + or CD4 + T cells exhibited relative activation of CD4 cells compared to those treated with a control agent. + or CD8 + The expression of PD-1 on the surface of T cells increased by no more than approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15%. In some embodiments, activated CD8 cells treated with GAL9 antigen-binding molecules... + or CD4 + T cells exhibited relative activation of CD4 cells compared to those treated with a control agent. + Or a reduction of no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% in CD8 T cell expression on PD-1 surface.

[0163] In some embodiments, the immune checkpoint molecule is PD-L1. In some embodiments, it is relative to activated CD4 treated with a control agent. + or CD8 + T cells, activated CD8 cells treated with GAL9 antigen-binding molecules + or CD4 + T cells showed no more than a fold change in PD-L1 surface expression. In some embodiments, relative to activated CD4 cells treated with a control agent... + or CD8 +T cells, activated CD8 cells treated with GAL9 antigen-binding molecules + or CD4 + T cells showed an increase in PD-L1 surface expression not exceeding 1.01X, 1.02X, 1.03X, 1.04X, 1.05X, 1.06X, 1.07X, 1.08X, 1.09X, 1.1X, 1.2X, or 1.3X fold. In some embodiments, this is relative to activated CD4 cells treated with a control agent. + or CD8 + T cells, activated CD8 cells treated with GAL9 antigen-binding molecules + or CD4 + T cell expression on the PD-L1 surface showed a decrease of no more than 0.01X, 0.02X, 0.03X, 0.04X, 0.05X, 0.06X, 0.07X, 0.08X, 0.09X, 0.1X, or 0.2X times.

[0164] In some implementations, activated CD8 cells treated with GAL9 antigen-binding molecules + or CD4 + T cells exhibited relative activation of CD4 cells compared to those treated with a control agent. + or CD8 + The expression of PD-L1 on the surface of T cells increased by no more than approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15%. In some embodiments, activated CD8 cells treated with GAL9 antigen-binding molecules... + or CD4 + T cells exhibited relative activation of CD4 cells compared to those treated with a control agent. + Or a reduction of no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% in the expression of CD8 T cells on the PD-L1 surface.

[0165] In some embodiments, the immune checkpoint molecule is CTLA-4. In some embodiments, it is relative to activated CD4 treated with a control agent. + or CD8 + T cells, activated CD8 cells treated with GAL9 antigen-binding molecules + or CD4 +T cell expression on the CTLA-4 surface showed an increase not exceeding 1.01X, 1.02X, 1.03X, 1.04X, 1.05X, 1.06X, 1.07X, 1.08X, 1.09X, 1.1X, 1.2X, or 1.3X fold. In some embodiments, this is relative to activated CD4 treated with a control agent. + or CD8 + T cells, activated CD8 cells treated with GAL9 antigen-binding molecules + or CD4 + T cell expression on the CTLA-4 surface showed a decrease of no more than 0.01X, 0.02X, 0.03X, 0.04X, 0.05X, 0.06X, 0.07X, 0.08X, 0.09X, 0.1X, or 0.2X times.

[0166] In some implementations, activated CD8 cells treated with GAL9 antigen-binding molecules + or CD4 + T cells exhibited relative activation of CD4 cells compared to those treated with a control agent. + or CD8 + The expression of CTLA-4 on the surface of T cells showed an increase of no more than approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15%. In some embodiments, activated CD8 cells were treated with GAL9 antigen-binding molecules. + or CD4 + T cells exhibited relative activation of CD4 cells compared to those treated with a control agent. + Or a reduction of no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% in CD8 T cell expression on CTLA-4 surface.

[0167] In some embodiments, the immune checkpoint molecule is TIM3. In some embodiments, it is relative to activated CD4 treated with a control agent. + or CD8 + T cells, activated CD8 cells treated with GAL9 antigen-binding molecules + or CD4 +T cell expression on the TIM3 surface showed an increase not exceeding 1.01X, 1.02X, 1.03X, 1.04X, 1.05X, 1.06X, 1.07X, 1.08X, 1.09X, 1.1X, 1.2X, or 1.3X fold. In some embodiments, relative to activated CD4 cells treated with a control agent... + or CD8 + T cells, activated CD8 cells treated with GAL9 antigen-binding molecules + or CD4 + T cell expression on the TIM3 surface showed a decrease of no more than 0.01X, 0.02X, 0.03X, 0.04X, 0.05X, 0.06X, 0.07X, 0.08X, 0.09X, 0.1X, or 0.2X times.

[0168] In some implementations, activated CD8 cells treated with GAL9 antigen-binding molecules + or CD4 + T cells exhibited relative activation of CD4 cells compared to those treated with a control agent. + or CD8 + The expression of TIM3 on the surface of T cells showed an increase of no more than approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15%. In some embodiments, activated CD8 cells were treated with GAL9 antigen-binding molecules. + or CD4 + T cells exhibited relative activation of CD4 cells compared to those treated with a control agent. + Or a reduction of no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% in TIM3 surface expression of CD8 T cells.

[0169] In some embodiments, the immune checkpoint molecule is LAG3. In some embodiments, it is relative to activated CD4 treated with a control agent. + or CD8 + T cells, activated CD8 cells treated with GAL9 antigen-binding molecules + or CD4 +T cell expression on the LAG3 surface showed an increase not exceeding 1.01X, 1.02X, 1.03X, 1.04X, 1.05X, 1.06X, 1.07X, 1.08X, 1.09X, 1.1X, 1.2X, or 1.3X fold. In some embodiments, relative to activated CD4 cells treated with a control agent... + or CD8 + T cells, activated CD8 cells treated with GAL9 antigen-binding molecules + or CD4 + T cell expression on the LAG3 surface showed a reduction of no more than 0.01X, 0.02X, 0.03X, 0.04X, 0.05X, 0.06X, 0.07X, 0.08X, 0.09X, 0.1X, or 0.2X fold. In some embodiments, activated CD8 cells were treated with GAL9 antigen-binding molecules. + or CD4 + T cells exhibited relative activation of CD4 cells compared to those treated with a control agent. + or CD8 + The expression of LAG3 on the surface of T cells increased by no more than approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15%. In some embodiments, activated CD8 cells treated with GAL9 antigen-binding molecules... + or CD4 + T cells exhibited relative activation of CD4 cells compared to those treated with a control agent. + Or a reduction of no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% in the expression of CD8 T cells on the LAG3 surface.

[0170] In some embodiments, the GAL9 antigen-binding molecule reduces the surface expression of one or more co-stimulatory molecules on immune cells (e.g., human immune cells). In some embodiments, the GAL9 antigen-binding molecule reduces the surface expression of one or more co-stimulatory molecules on activated immune cells. In a particular embodiment, the activated immune cells are T cells. In a specific embodiment, the activated immune cells are CD8 cells. + T cells. In some embodiments, one or more co-stimulatory molecules are selected from 4-1BB, CD40L, and OX40. In some embodiments, one or more co-stimulatory molecules are selected from 4-1BB and

[0171] CD40L. In some implementations, the co-stimulatory molecule is OX40.

[0172] The effect of GAL9 antigen-binding molecules on the surface expression of one or more co-stimulatory molecules can be determined by any suitable 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, in vitro, or ex vivo.

[0173] In some embodiments, the GAL9 antigen-binding molecule reduces the surface expression of one or more co-stimulatory molecules on activated immune cells compared to activated immune cells treated with a control agent. Exemplary control agents are described herein. In a particular embodiment, the control agent is an isotype control binding molecule that does not bind to GAL9.

[0174] In some implementations, relative to activated CD8 treated with a control agent + T cells, GAL9 antigen-binding molecules reduce 4-1BB in activated CD8 + Surface expression on T cells. In some implementations, this is relative to activated CD8 cells treated with a control agent. + T cells, activated CD8 cells treated with GAL9 antigen-binding molecules + T cell 4-1BB surface expression is reduced by at least approximately 0.1X, 0.2X, 0.3X, 0.4X, 0.5X, or 0.6X. In some embodiments, activated CD8 cells are treated with GAL9 antigen-binding molecules. + T cells, relative to activated CD8 cells treated with a control agent + T cells showed a decrease in 4-1BB surface expression of approximately 0.1X-0.2X, 0.2X-0.3X, 0.3X-0.4X, 0.4X-0.5X, or 0.5X-0.6X.

[0175] In some implementations, relative to activated CD8 treated with a control agent + T cells, GAL9 antigen-binding molecules reduce activated CD8 + CD40L is expressed on the surface of T cells. In some embodiments, activated CD8T cells treated with GAL9 antigen-binding molecules are compared with activated CD8T cells treated with a control agent. + CD40L expression on T cells is reduced by at least approximately 0.1X, 0.2X, 0.3X, 0.4X, 0.5X, or 0.6X. In some embodiments, activated CD8 cells are treated with GAL9 antigen-binding molecules. +Compared to activated CD8 T cells treated with the control, CD40L surface expression was reduced by approximately 0.1X-0.2X, 0.2X-0.3X, 0.3X-0.4X, 0.4X-0.5X, or 0.5X-0.6X.

[0176] In some implementations, relative to activated CD8 treated with a control agent + GAL9 antigen-binding molecules reduce OX40 surface expression in activated CD8 T cells. + In some implementations, activated CD8 cells are treated with GAL9 antigen-binding molecules. + T cells, compared to activated CD8 T cells treated with a control agent, showed at least a 0.1X reduction, a 0.2X reduction, a 0.3X reduction, a 0.4X reduction, a 0.5X reduction, or a 0.6X reduction in OX40 surface expression. In some embodiments, activated CD8 T cells treated with GAL9 antigen-binding molecules... + Compared to activated CD8 T cells treated with the control, T cells showed a reduction in OX40 surface expression of approximately 0.1X-0.2X, 0.2X-0.3X, 0.3X-0.4X, 0.4X-0.5X, or 0.5X-0.6X.

[0177] This disclosure also provides GAL9 antigen-binding molecules with various clinical benefits that improve the health of subjects suffering from autoimmune or inflammatory diseases. The subject may be a mammal. The mammal may be a mouse. In some embodiments, the mammal is a human.

[0178] In some embodiments, the GAL9 antigen-binding molecule reduces autoimmune responses in the subject. In some embodiments, the GAL9 antigen-binding molecule reduces inflammation in the subject. Inflammation can be systemic or localized in an organ or tissue. In some embodiments, the GAL9 antigen-binding molecule prolongs remission of the subject's disease or condition. In some embodiments, the GAL9 antigen-binding molecule induces remission in the subject. In some embodiments, the GAL9 antigen-binding molecule re-establishes immune tolerance in the subject (e.g., by improving the cytokine profile or environment). Re-establishment of immune tolerance can be a reduction in pro-inflammatory cytokines, an increase in inhibitory cytokines, or a combination thereof. In some embodiments, the GAL9 antigen-binding molecule improves organ function in the subject. In some embodiments, the GAL9 antigen-binding molecule reduces the risk / likelihood of disease progression or the development of a second disease such as cancer or infection. In some embodiments, the GAL9 antigen-binding molecule improves overall survival in the subject.

[0179] 6.4.2. Variable Region

[0180] In a typical implementation, the GAL9 binding molecule has a variable region amino acid sequence of the antibody, 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 below, respectively.

[0181] 6.4.2.1. VH region

[0182] In typical embodiments, the GAL9 binding molecule described herein includes a variable domain sequence of the antibody heavy chain. In typical antibody arrangements in nature and in the GAL9 binding molecule described herein, a specific VH amino acid sequence binds to a specific VL amino acid sequence to form an antigen-binding site. In various embodiments, the VH amino acid sequence is a mammalian sequence, including human sequences, synthetic sequences, or combinations of non-human mammalian, mammalian, and / or synthetic sequences, as further described in detail in Sections 6.4.2.3 and 6.4.2.4 above. In various embodiments, the VH amino acid sequence is a mutant sequence of a naturally occurring sequence.

[0183] 6.4.2.2. VL region

[0184] The useful VL amino acid sequence in the GAL9 binding molecule described herein is the variable domain sequence of the antibody light chain. In the typical arrangement of natural antibodies and the antibody constructs described herein, a specific VL amino acid sequence binds to a specific VH amino acid sequence to form an antigen-binding site. In different embodiments, the VL amino acid sequence is a mammalian sequence, including human sequences, synthetic sequences, or combinations of human, non-human mammalian, mammalian, and / or synthetic sequences, as further described in detail in Sections 6.4.2.3 and 6.4.2.4 below.

[0185] In various embodiments, the VL amino acid sequence is a variant of a naturally occurring sequence. In some embodiments, the VL amino acid sequence is a λ (lambda) light chain variable domain sequence. In some embodiments, the VL amino acid sequence is a κ (kappa) light chain variable domain sequence. In a preferred embodiment, the VL amino acid sequence is a κ (kappa) light chain variable domain sequence.

[0186] 6.4.2.3. Complementarity Determining Zone

[0187] The VH and VL amino acid sequences contain highly variable sequences called "complementarity-determining regions" (CDRs), which are typically three CDRs (CDR1, CDR2, and CDR3). In various embodiments, the CDRs are mammalian sequences, including but not limited to sequences from mice, rats, hamsters, rabbits, camels, donkeys, goats, and humans. In a preferred embodiment, the CDR is a human sequence. In various embodiments, the CDR is a naturally occurring sequence. In different embodiments, the CDR is a naturally occurring sequence that has been mutated to alter the binding affinity of the antigen-binding site for a specific antigen or epitope. In some embodiments, the naturally occurring CDR is mutated in vivo through affinity maturation and high-frequency somatic mutagenesis. In some embodiments, the CDR is mutated in vitro by methods including but not limited to PCR mutagenesis and chemical mutagenesis. In many embodiments, the CDR is a synthetic sequence, including but not limited to CDRs obtained from random sequence CDR libraries and rationally designed CDR libraries. The Martin numbering scheme is used to determine the boundaries of the CDRs. See [link to documentation]. Figure 1A-1B This is applicable to the P9-01 anti-human GAL9 candidate provided in this paper.

[0188] In various implementations, CDRs identified as binding to the target antigen are further mutated (i.e., "affinity maturation") to achieve desired binding properties, such as increased affinity for the target antigen relative to the original CDR. For example, degenerate oligonucleotides can be used to selectively introduce diversity into CDRs, including those identified as binding to the target antigen. Various randomization schemes can be employed. For instance, "soft-randomization" can be used to provide a highly wild-type sequence bias at a given amino acid position, allowing variation at a given position in the CDR across all 20 amino acids while biasing towards the wild-type sequence through non-equivalent levels of incorporation at four bases per codon position. As an illustrative example of soft randomization, if an approximately 50% wild-type sequence is desired, each base per codon maintains 70% wild-type and 10% wild-type for each of the other nucleotides, and degenerate oligonucleotides are used to create a focused phage library around the selected CDRs. The resulting phage particles are then used for phage panning as needed under various stringent selection conditions.

[0189] 6.4.2.4. Grafting of the Frame Region and CDR

[0190] The amino acid sequences of VH and VL contain "framework region" (FR) sequences. FRs are typically conserved sequence regions that act as a scaffold for intercalating CDRs (see Section 6.4.2.3), and are generally arranged in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (from N-terminus to C-terminus). In various embodiments, FRs are mammalian sequences, including but not limited to sequences from mice, rats, hamsters, rabbits, camels, donkeys, goats, and humans. In a preferred embodiment, FRs are human sequences. In various embodiments, FRs are naturally occurring sequences. In different embodiments, FRs are synthetic sequences, including but not limited to rationally designed sequences.

[0191] In various embodiments, FRs and CDRs are derived from the same naturally occurring variable domain sequence. In various embodiments, FRs and CDRs are derived from different variable domain sequences, wherein the CDR is grafted onto the FR scaffold, and the CDR provides specificity for a specific antigen. In some embodiments, the grafted CDRs are all derived from the same naturally occurring variable domain sequence. In some embodiments, the grafted CDRs are derived from different variable domain sequences. In some embodiments, the grafted CDR is a synthetic sequence, including but not limited to CDRs obtained from random sequence CDR libraries and rationally designed CDR libraries. In some embodiments, the grafted CDR and FR are from the same species. In some embodiments, the grafted CDR and FR are from different species. In a preferred embodiment of the grafted CDR, the antibody is "humanized," wherein the grafted CDR is a non-human mammalian sequence, 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 6,407,213, the entire contents of which are incorporated herein by reference. In various implementations, a portion or specific sequence of the FR of one species is used to replace a portion or specific sequence of the FR of another species.

[0192] 6.4.3. Exemplary amino acid sequence of GAL9-binding molecule

[0193] In various implementations, the GAL9 binding molecule contains a specific VH CDR3 (CDR-H3) sequence and a specific VLCDR3 (CDR-L3) sequence.

[0194] In some embodiments, the GAL9 binding molecule comprises CDR-H3 and CDR-L3 from any of the ABS clones selected from P9-01, P9-02A, P9-03, P9-06, P9-07, P9-11, P9-12, P9-14, P9-23, P9-24, P9-25, P9-29, P9-30, P9-34, P9-37, P9-38, P9-40, P9-41, P9-42, P9-43, P9-44, P9-45, P9-46, P9-50, P9-51, P9-52, P9-53, P9-56, and P9-57. 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, which is used throughout this disclosure.

[0195] In a currently preferred embodiment, the GAL9 binding molecule comprises CDR-H3 and CDR-L3 of ABS clone P9-11.

[0196] In some embodiments, the GAL9 binding molecule comprises all three VH CDRs from any one of the ABS clones selected from P9-01, P9-02A, P9-03, P9-06, P9-07, P9-11, P9-12, P9-14, P9-23, P9-24, P9-25, P9-29, P9-30, P9-34, P9-37, P9-38, P9-40, P9-41, P9-42, P9-43, P9-44, P9-45, P9-46, P9-50, P9-51, P9-52, P9-53, P9-56, and P9-57. In a currently preferred embodiment, the GAL9 binding molecule comprises all three VH CDRs of the ABS clone P9-11.

[0197] In some embodiments, the GAL9 binding molecule comprises all three VL CDRs from any one of the ABS clones selected from P9-01, P9-02A, P9-03, P9-06, P9-07, P9-11, P9-12, P9-14, P9-23, P9-24, P9-25, P9-29, P9-30, P9-34, P9-37, P9-38, P9-40, P9-41, P9-42, P9-43, P9-44, P9-45, P9-46, P9-50, P9-51, P9-52, P9-53, P9-56, and P9-57. In a currently preferred embodiment, the GAL9 binding molecule comprises all three VL CDRs of the ABS clone P9-11.

[0198] In some embodiments, the GAL9 binding molecule comprises all six CDRs from any one of the ABS clones selected from P9-01, P9-02A, P9-03, P9-06, P9-07, P9-11, P9-12, P9-14, P9-23, P9-24, P9-25, P9-29, P9-30, P9-34, P9-37, P9-38, P9-40, P9-41, P9-42, P9-43, P9-44, P9-45, P9-46, P9-50, P9-51, P9-52, P9-53, P9-56, and P9-57. In a currently preferred embodiment, the GAL9 binding molecule comprises all six CDRs of the ABS clone P9-11.

[0199] In some embodiments, the GAL9-binding molecule comprises a VH amino acid sequence, a VL amino acid sequence, or a VH and VL amino acid sequence from any of the ABS clones selected from P9-01, P9-02A, P9-03, P9-06, P9-07, P9-11, P9-12, P9-14, P9-23, P9-24, P9-25, P9-29, P9-30, P9-34, P9-37, P9-38, P9-40, P9-41, P9-42, P9-43, P9-44, P9-45, P9-46, P9-50, P9-51, P9-52, P9-53, P9-56, and P9-57. Table 6 provides the complete immunoglobulin heavy chain and immunoglobulin light chain sequences, as well as the VH and VL amino acid sequences. In a currently preferred embodiment, the GAL9 binding molecule comprises a VH amino acid sequence, a VL amino acid sequence, or a VH and VL amino acid sequence from ABS clone P9-11.

[0200] In some embodiments, the GAL9 binding molecule comprises a complete ABS clone selected from any one of P9-01, P9-02A, P9-03, P9-06, P9-07, P9-11, P9-12, P9-14, P9-23, P9-24, P9-25, P9-29, P9-30, P9-34, P9-37, P9-38, P9-40, P9-41, P9-42, P9-43, P9-44, P9-45, P9-46, P9-50, P9-51, P9-52, P9-53, P9-56, and P9-57.

[0201] The IgG heavy chain sequence and the complete IgG light chain sequence. In a currently preferred embodiment, the GAL9 binding molecule comprises the complete IgG heavy chain sequence and the complete IgG light chain sequence from ABS clone P9-11.

[0202] 6.4.4. Constant Region

[0203] In some implementations, the GAL9 binding molecule contains an antibody constant region domain sequence. As described herein, the constant region domain amino acid sequence is the sequence of the antibody's constant region domain. The constant region may refer to the CH1, CH2, CH3, CH4, or CL constant domain.

[0204] In various embodiments, the constant region sequence is a mammalian sequence, including but not limited to sequences from mice, rats, hamsters, rabbits, camels, donkeys, goats, and humans. In a preferred embodiment, the constant region sequence is a human sequence. In some embodiments, the constant region sequence is derived from the antibody light chain. In specific embodiments, the constant region sequence is derived from the λ or κ light chain. In some embodiments, the constant region sequence is derived from the antibody heavy chain. In specific embodiments, the constant region sequence is an antibody heavy chain sequence that is an isotype of IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM. In a particular embodiment, the constant region sequence is derived from the IgG isotype. In a preferred embodiment, the constant region sequence is derived from the IgG1 isotype.

[0205] An exemplary constant region and its modifications are described in WO2018075692, the entire contents of which are incorporated herein by reference.

[0206] 6.4.4.1. CH1 and CL regions

[0207] The CH1 amino acid sequence, as described herein, refers to the native antibody heavy chain architecture from the N-terminus to the C-terminus and is the sequence of the second domain of the antibody heavy chain. In some embodiments, the CH1 sequence is an endogenous sequence. In many embodiments, the CH1 sequence is a mammalian sequence, including but not limited to sequences from mice, rats, hamsters, rabbits, camels, donkeys, goats, and humans. In a preferred embodiment, the CH1 sequence is a human sequence. In some embodiments, the CH1 sequence is derived from an IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM isotype. In a preferred embodiment, the CH1 sequence is derived from an IgG1 isotype. In a preferred embodiment, the CH1 sequence is amino acids 1-98 of UniProt accession number P01857.

[0208] The useful CL amino acid sequence in the GAL9 binding molecule described herein is the antibody light chain constant domain sequence, referencing the native antibody light chain structure. In some embodiments, the CL sequence is an endogenous sequence. In various embodiments, the CL sequence is a mammalian sequence, including but not limited to sequences from mice, rats, hamsters, rabbits, camels, donkeys, goats, and humans. In a preferred embodiment, the CL sequence is a human sequence.

[0209] In some embodiments, the CL amino acid sequence is the lambda(λ) light chain constant domain sequence. In a particular embodiment, the CL amino acid sequence is the human λ light chain constant domain sequence. In a preferred embodiment, the lambda(λ) light chain sequence is UniProt accession number P0CG04.

[0210] In some embodiments, the CL amino acid sequence is a kappa(κ) light chain constant domain sequence. In a preferred embodiment, the CL amino acid sequence is a human kappa(κ) light chain constant domain sequence. In a preferred embodiment, the κ light chain sequence is UniProt accession number P01834.

[0211] In some embodiments, both the CH1 and CL sequences are endogenous sequences. In some embodiments, the CH1 and CL sequences include orthogonal modifications of 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 portions thereof, whether endogenous or modified sequences, such that a domain having the CH1 sequence or a portion thereof may be associated with a domain having the CL sequence or a portion thereof.

[0212] 6.4.4.2. Orthogonal Modification of CH1 and CL

[0213] In some implementations, the CH1 and CL sequences contain orthogonal modifications of the endogenous CH1 and CL sequences, respectively. Generally, orthogonal variations are described in more detail in Sections 6.4.6.1–6.4.6.3 below.

[0214] In a particular embodiment, the orthogonal modification in the endogenous CH1 and CL sequences is an engineered disulfide bridge of an engineered cysteine ​​residue selected from positions 138 and 116 of the CH1 sequence, positions 128 and 119 of the CH1 sequence and the CL sequence, or positions 129 and 210 of the CH1 sequence, as numbered and discussed in more detail in U.S. Patent Nos. 8,053,562 and 9,527,927 (each incorporated herein by reference in its entirety). In a preferred embodiment, the engineered cysteine ​​residue is located at position 128 of the CH1 sequence and position 118 of the CL Kappa sequence, numbered according to the Eu index.

[0215] In a series of preferred embodiments, the mutations provided for non-endogenous cysteine ​​amino acids are the F118C mutation in the CL sequence numbered by the Eu index and the corresponding A141C mutation in the CH1 sequence, or the F118C mutation in the CL sequence and the corresponding L128C mutation in the CH1 sequence, or the S162C mutation in the CL sequence and the corresponding P171C mutation in the CH1 sequence.

[0216] In various embodiments, the orthogonal mutations in the CL and CH1 sequences are charge-pair mutations. In specific embodiments, the charge-pair mutations are F118S, F118A, or F118V mutations in the CL sequence with the corresponding A141L mutation in the CH1 sequence, or T129R mutations in the CL sequence with the corresponding K147D mutation in the CH1 sequence, as numbered by the Eu index and described in more detail in Bonisch et al. (Protein Engineering, Design & Selection, 2017, pp. 1-12), all of which are incorporated herein by reference. In a range of preferred embodiments, the charge-pair mutations are N138K mutations in the CL sequence with the corresponding G166D mutation in the CH1 sequence, or N138D mutations in the CL sequence with the corresponding G166K mutation in the CH1 sequence, as numbered by the Eu index.

[0217] 6.4.4.3. CH2 region

[0218] In the GAL9-binding molecule described herein, the GAL9-binding molecule may have a CH2 amino acid sequence. The CH2 amino acid sequence, as described herein, is the CH2 amino acid sequence of the third domain of the antibody heavy chain, referring to the native antibody heavy chain architecture from the N-terminus to the C-terminus. In various embodiments, the CH2 sequence is a mammalian sequence, including but not limited to sequences from mice, rats, hamsters, rabbits, camels, donkeys, goats, and humans. In a preferred embodiment, the CH2 sequence is a human sequence. In some embodiments, the CH2 sequence is derived from an isotype of IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM. In a preferred embodiment, the CH2 sequence is derived from an isotype of IgG1.

[0219] In some embodiments, the CH2 sequence is an endogenous sequence. In a particular embodiment, this sequence is UniProt accession number P01857, amino acid 111-223.

[0220] In a series of embodiments, the GAL9-binding molecule has one or more paired sets of CH2 domains with CH2 sequences, wherein the first set has a CH2 amino acid sequence from a first isotype and a CH2 amino acid sequence from one or more orthologous sets from another isotype. As described herein, the orthologous CH2 amino acid sequences can interact with CH2 amino acid sequences from a common isotype, but cannot significantly interact with CH2 amino acid sequences from another isotype present in the GAL9-binding molecule. In a particular embodiment, all sets of CH2 amino acid sequences are from the same species. In a preferred embodiment, all sets of CH2 amino acid sequences are human CH2 amino acid sequences. In other embodiments, the sets of CH2 amino acid sequences are from different species. In a specific embodiment, the first set of CH2 amino acid sequences is from the same isotype as other non-CH2 domains in the GAL9-binding molecule. In one specific embodiment, the first set of CH2 amino acid sequences is from the IgG isotype, while one or more orthologous sets have CH2 amino acid sequences from the IgM or IgE isotype. In some embodiments, one or more sets of CH2 amino acid sequences are endogenous CH2 sequences. In other embodiments, one or more sets of CH2 amino acid sequences are endogenous CH2 sequences having one or more mutations. In specific embodiments, the one or more mutations are orthogonal knockhole mutations, orthogonal charge-pair mutations, orthogonal hydrophobic mutations. Orthogonal CH2 amino acid sequences useful for GAL9 binding molecules are described in more detail in international PCT applications WO2017 / 011342 and WO2017 / 106462, the entire contents of which are incorporated herein by reference.

[0221] 6.4.4.4.CH3 region

[0222] The CH3 amino acid sequence described in this article is the sequence of the C-terminal domain of the antibody heavy chain, referring to the native antibody heavy chain structure from the N-terminus to the C-terminus.

[0223] In various embodiments, the CH3 sequence is a mammalian sequence, including but not limited to sequences from mice, rats, hamsters, rabbits, camels, donkeys, goats, and humans. In a preferred embodiment, the CH3 sequence is a human sequence. In some embodiments, the CH3 sequence is derived from IgA1, IgA2, IgD, IgE, IgM, IgG1, IgG2, IgG3, IgG4 isotypes or from the CH4 sequence of an IgE or IgM isotype. In a specific embodiment, the CH3 sequence is derived from an IgG isotype. In a preferred embodiment, the CH3 sequence is derived from an IgG1 isotype.

[0224] In some embodiments, the CH3 sequence is an endogenous sequence. In a particular embodiment, the CH3 sequence is amino acids 224-330 of UniProt accession number P01857. In many embodiments, the CH3 sequence is a segment of an endogenous CH3 sequence. In a particular embodiment, the CH3 sequence is an endogenous CH3 sequence lacking N-terminal amino acids G224 and Q225. In a particular embodiment, the CH3 sequence is an endogenous CH3 sequence lacking C-terminal amino acids P328, G329, and K330. In a special embodiment, the CH3 sequence is an endogenous CH3 sequence lacking both N-terminal amino acids G224 and Q225 and C-terminal amino acids P328, G329, and K330. In a preferred embodiment, a GAL9 binding molecule has multiple domains having a CH3 sequence, wherein the CH3 sequence can refer to the complete endogenous CH3 sequence or a CH3 sequence lacking N-terminal amino acids, C-terminal amino acids, or both.

[0225] In some embodiments, the CH3 sequence is an endogenous sequence with one or more mutations. In specific embodiments, the mutations are one or more orthogonal mutations introduced into the endogenous sequence.

[0226] The specific pairing of CH3 sequences that guides a particular CH3 sequence is described in more detail in Sections 6.4.6.1-6.4.6.3 below.

[0227] In some embodiments, the CH3 sequence is engineered to reduce the immunogenicity of the antibody by replacing a specific amino acid of one allotype with an amino acid of another allotype, referred to herein as an isoallotype mutation, as described in more detail in Stickler et al. (Genes Immun. 2011 Apr; 12(3):213-221), all of which are incorporated herein by reference. In a particular embodiment, a specific amino acid of the G1m1 allotype is substituted. In a preferred embodiment, isoallotype mutations D356E and L358M are made in the CH3 sequence.

[0228] In some embodiments, the IgG1 CH3 amino acid sequence includes the following mutational changes: P343V; Y349C; and a tripeptide insertion, 445P, 446G, 447K. In other preferred embodiments, domain B has the human IgG1 CH3 sequence with the following mutational changes: T366K; and a tripeptide insertion, 445K, 446S, 447C. In other preferred embodiments, domain B has the human IgG1 CH3 sequence with the following mutational changes: Y349C and a tripeptide insertion, 445P, 446G, 447K.

[0229] In some implementations, the IgG1 CH3 amino acid sequence includes the 447C mutation incorporated into other endogenous CH3 sequences.

[0230] 6.4.5. Antigen binding site

[0231] In some embodiments, a VL or VH amino acid sequence is associated with a homologous VL or VH amino acid sequence to form a first antigen-binding site (ABS). The antigen-binding site (ABS) is capable of specifically binding to an epitope of the antigen. The antigen binding at the antigen-binding site is described in more detail in Section 6.4.5.1 below.

[0232] In another embodiment, for example, where the GAL9 binding molecule is a single-domain antibody, the VH or VL amino acid sequence forms the first ABS.

[0233] In some embodiments, the GAL9 antigen-binding molecule comprises 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.

[0234] In some implementations, the second ABS is specific to different epitopes of the first GAL9 antigen. For example, if the first ABS contains a CDR or variable domain from any of the ABS clones selected from P9-01, P9-02A, P9-03, P9-06, P9-07, P9-11, P9-12, P9-14, P9-23, P9-24, P9-25, P9-29, P9-30, P9-34, P9-37, P9-38, P9-40, P9-41, P9-42, P9-43, P9-44, P9-45, P9-46, P9-50, P9-51, P9-52, P9-53, P9-56, and P9-57. The second ABS may contain a CDR or variable structural domain from another ABS clone selected from P9-01, P9-02A, P9-03, P9-06, P9-07, P9-11, P9-12, P9-14, P9-23, P9-24, P9-25, P9-29, P9-30, P9-34, P9-37, P9-38, P9-40, P9-41, P9-42, P9-43, P9-44, P9-45, P9-46, P9-50, P9-51, P9-52, P9-53, P9-56, and P9-57.

[0235] In some implementations, the GAL9 antigen-binding molecule is multispecific; for example, the second ABS of the GAL9 antigen-binding molecule specifically binds to an antigen different from the GAL9 antigen specifically bound by the first ABS.

[0236] 6.4.5.1. Binding of antigen to ABS

[0237] ABS, and GAL9-binding molecules including the ABS, are referred to as the epitope (or more generally, the antigen) that "recognizes" the ABS and binds specifically to it, and the epitope (or more generally, the antigen) is referred to as the "recognition specificity" or "binding specificity" of the ABS.

[0238] It is said that ABS binds to its specific antigens or epitopes with a specific affinity. As discussed in this article, "affinity" refers to the strength of the non-covalent intermolecular force between one molecule and another. Affinity, i.e., the strength of the interaction, can be expressed as the dissociation equilibrium constant (K0). D ), where the lower K D The value refers to the stronger interaction between molecules. The K value of the antibody construct... D The values ​​are measured using methods well-known in the art, including but not limited to biological layer interferometers (such as...). ), surface plasmon resonance (SPR) technology (e.g. (and cell binding assays. For the purposes of this article, affinity refers to the ability to bind to cells using...) The dissociation equilibrium constant of the biolayer interferometry method.

[0239] The term "specific binding" used in this article refers to the affinity between ABS and its homologous antigens or epitopes, where K... D Value below 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M.

[0240] The number of ABS in a GAL9-binding molecule, as described in this article, defines the "valence" of the GAL9-binding molecule. A GAL9-binding molecule with a single ABS is "monovalent". A GAL9-binding molecule with multiple ABS is called "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".

[0241] In various multivalent embodiments, all the multiple ABS have the same recognition specificity. Such GAL9 binding molecules are "single-specific" and "multivalent" binding constructs. In other multivalent embodiments, at least two of the multiple ABS have different recognition specificities. Such GAL9 binding molecules are multivalent and "multispecific". In multivalent embodiments where the ABS collective has two recognition specificities, the GAL9 binding molecule is "bispecific". In multivalent embodiments where the ABS collective has three recognition specificities, the GAL9 binding molecule is "trispecific".

[0242] In a multivalent implementation of ABS that collectively has multiple recognition specificities for different epitopes present on the same antigen, the GAL9 binding molecule is "multiparatopic". In a multivalent implementation of ABS that jointly recognizes two epitopes on the same antigen, it is "biparatopic".

[0243] In various multivalent embodiments, the multivalence of the GAL9-binding molecule enhances its affinity for a specific target. As described herein, "affinity" refers to the overall strength of the interaction between two or more molecules, such as the effect of a multivalent GAL9-binding molecule on a specific target, where the affinity is the cumulative strength of the interaction provided by the affinity of multiple ABS molecules. Affinity can be measured using the same methods described above for determining affinity. In some embodiments, the affinity of the GAL9-binding molecule for a specific target is such that the interaction is a specific binding interaction, where the K of the affinity between the two molecules is... D Value below 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M. In some implementations, the affinity of the GAL9 binding molecule for a specific target has a K... D Values ​​that make the interaction a specific binding interaction, wherein the affinity of one or more individual ABS does not have a K value that conforms to the individual binding of their respective antigens or epitopes. D Value. In some embodiments, affinity is the cumulative strength of the interaction provided by the affinity of multiple ABS for individual antigens on a shared specific target or complex (such as a single antigen found on a single cell). In some embodiments, affinity is the cumulative strength of the interaction provided by the affinity of multiple ABS for individual epitopes on a common individual antigen.

[0244] 6.4.6. Orthogonal Modification

[0245] In the GAL9-binding molecules described herein, the GAL9-binding molecules may have constant region structural domains containing orthogonal modifications. The amino acid sequences of the constant region domains are described in more detail in Section 6.4.4 above.

[0246] The "orthogonal modification" or synonymous "orthogonal mutation" described herein refers to one or more engineered mutations in the amino acid sequence of an antibody domain that increase the binding affinity of a first domain with an orthogonal modification to a second domain with a complementary orthogonal modification. In some embodiments, orthogonal modification reduces the affinity of an orthogonally modified domain for a domain lacking a complementary orthogonal modification. In some embodiments, orthogonal modification is a mutation in the sequence of an endogenous antibody domain. In various embodiments, orthogonal modification is an N-terminal or C-terminal modification of the endogenous antibody domain sequence, including but not limited to the addition or deletion of amino acids. In specific embodiments, orthogonal modification includes, but is not limited to, engineered disulfide bridges, knock-in-hole mutations, and charge-pair mutations, as described in more detail in Sections 6.4.6.1–6.4.6.3 below. In specific embodiments, orthogonal modification includes combinations of orthogonal modifications selected from, but not limited to, engineered disulfide bridges, knock-in-hole mutations, and charge-pair mutations. In certain implementations, orthogonal modifications can be combined with amino acid substitutions that reduce immunogenicity, such as isoallotype mutations, as described in more detail in Section 6.4.4.4 above.

[0247] 6.4.6.1. Orthogonal engineered disulfide bridge

[0248] In various embodiments, orthogonal modification includes mutations that create engineered disulfide bridges between the first and second domains. As described herein, an "engineered disulfide bridge" is a mutation that provides a non-endogenous cysteine ​​amino acid in two or more domains to form a non-natural disulfide bond when the two or more domains bind. Engineered disulfide bridges are described in more detail by Merchant et al. (Nature Biotech (1998) 16:677-681), the entire contents of which are incorporated herein by reference. In some embodiments, the engineered disulfide bridge improves the orthogonal connection between specific domains. In one particular embodiment, the mutation that creates an engineered disulfide bridge is 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 mutation that creates an engineered disulfide bridge is 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 mutation that generates the engineered disulfide bridge is the 447C mutation in both the first and second CH3 domains, which is provided by the C-terminus extension of the CH3 domain of the KSC tripeptide sequence.

[0249] 6.4.6.2. Orthogonal knob-hole mutation

[0250] In various embodiments, orthogonal modification includes a knock-hole (synonymous, knock-in-hole) mutation. As described herein, a knock-hole mutation is a mutation that alters the stereotactic features of the surface of a first domain, thereby causing the first domain to preferentially bind to a second domain having a complementary stereotactic mutation, relative to binding to a domain without a complementary stereotactic mutation. Knob-hole mutations are described in more detail in U.S. Patent Nos. 5,821,333 and 8,216,805, each of which is incorporated herein in its entirety. In various embodiments, knock-hole mutations are combined with engineered disulfide bridges, as described in more detail in Merchant et al. (Nature Biotech (1998) 16:677-681), which is incorporated herein in its entirety by reference. In different embodiments, knock-hole mutations, isoallotype mutations, and engineered disulfide mutations are combined.

[0251] In some embodiments, the knock-in-hole mutation is the T366Y mutation in the first domain and the Y407T mutation in the second domain. In some embodiments, the knock-in-hole mutation is the F405A mutation in the first domain and the T394W mutation in the second domain. In some embodiments, the knock-in-hole mutation is the T366Y and F405A mutations in the first domain and the T394W and...

[0252] Y407T. In some embodiments, the knock-in-hole mutation is the T366W mutation in the first domain and the Y407A mutation in the second domain. In some embodiments, the combined knock-in-hole mutation and engineered disulfide mutation are the S354C and T366W mutations in the first domain and the Y407A mutation in the second domain.

[0253] Y349C, T366S, L368A, and Y407V mutations. In a preferred embodiment, the combined knock-in-hole mutation, isoallotype mutation, and engineered disulfide mutation are the S354C and T366W mutations in the first domain, and the Y349C, D356E, L358M, T366S, L368A, and Y407V mutations in the second domain.

[0254] 6.4.6.3. Abrupt change in orthogonal charge pairs

[0255] In various embodiments, orthogonal modification is a charge-pair mutation. As used herein, a charge-pair mutation is a mutation that affects the amino acid charge on the surface of a domain, such that the domain will preferentially bind to a second domain having a complementary charge-pair mutation, relative to binding to a domain without a complementary charge-pair mutation. In some embodiments, charge-pair mutations improve the orthogonal association between specific domains. Charge-pair mutations are described in U.S. Patent Nos. 8,592,562, 9,248,182, and 9,358,286, all teachings of which are incorporated herein by reference. In some embodiments, charge-pair mutations improve the stability between specific domains. In a preferred embodiment, the charge-pair mutation is a T366K mutation in the first domain and an L351D mutation in the other domain.

[0256] In a specific embodiment, the orthogonal mutation is a charge pair mutation at the VH / VL interface. In a preferred embodiment, the charge pair mutation at the VH / VL interface is Q39E in VH and the corresponding Q38K in VL, or Q39K in VH and the corresponding Q38E in VL, as described in more detail in Igawa et al. (Protein Eng. Des. Sel., 2010, vol. 23, 667-677), all of which are incorporated herein by reference.

[0257] 6.4.7. Trivalent and tetravalent GAL9-binding molecules

[0258] 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".

[0259] 6.5. GAL9 binding molecular structure

[0260] The antigen-binding sites described herein, including specific subsets of CDRs, can be formatted as any binding molecular architecture, including but not limited to full-length antibodies, Fab fragments, Fvs, scFvs, tandem scFvs, diabodies, scDiabodies, DARTs, tandAbs, 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, 182-212), all of which are incorporated herein by reference. The antigen-binding sites described herein, including specific subsets of CDRs, can also be formatted as "B-body" format, and a more detailed description can be found in U.S. Pre-Publication Publication No. US2018 / 0118811 and International Application Publication No. WO2018 / 075692, each of which is incorporated herein by reference in its entirety.

[0261] 6.6. Further Refinement

[0262] In a further series of embodiments, the GAL9-binding molecule has additional modifications.

[0263] 6.6.1. Antibody-Drug Conjugates

[0264] In various implementation schemes, the GAL9 binding molecule conjugates with the therapeutic agent (i.e., the drug) to form...

[0265] GAL9-binding molecule-drug conjugates. Therapeutic agents include, but are not limited to, chemotherapeutic agents, imaging agents (such as radioisotopes), immunomodulators (such as cytokines, chemokines, or checkpoint inhibitors), and toxins (such as cytotoxic agents). In some embodiments, the therapeutic agent is linked to the GAL9-binding molecule via a linker peptide, as discussed in more detail in Section 6.6.3 below.

[0266] Methods applicable to the preparation of antibody-drug conjugates (ADCs) that conjugate drugs with the GAL9 binding molecules disclosed herein are described in detail, for example, in the following: U.S. Patent No. 8,624,003 (canning 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. 5,773,001, U.S. Patent No. 7,829,531, U.S. Patent No. 5,208,020, U.S. Patent Nos. 7,745,394, WO 2017 / 136623, WO2017 / 015502, WO 2017 / 015496, WO 2017 / 015495, WO 2004 / 010957, WO 2005 / 077090, WO2005 / 082023, WO 2006 / 065533,WO 2007 / 030642,WO 2007 / 103288,WO2013 / 173337,WO2015 / 057699,WO 2015 / 095755,WO 2015 / 123679,WO 2015 / 157286,WO 2017 / 165851,WO2009 / 073445,WO 2010 / 068759,WO 2010 / 138719,WO2012 / 171020,WO 2014 / 008375,WO2014 / 093394,WO 2014 / 093640,WO 2014 / 160360,WO 2015 / 054659,WO 2015 / 195925,WO2017 / 160754,Storz(MAbs.2015Nov-Dec;7(6):989-1009),Lambert et al.(Adv Ther,2017 34:1015),Diamantis et al.(British Journal of Cancer, 2016, 114, 362-367), Carrico et al. (Nat Chem Biol, 2007.3:321-2), We et al. (Proc Natl Acad Sci USA, 2009.106:3000-5), Rabuka et al. (Curr Opin Chem Biol., 2011 14:790-6), Hudak et al.(Angew Chem Int Ed Engl., 2012: 4161-5), Rabuka et al. (Nat Protoc., 2012 7: 1052-67), Agarwal et al. (Proc Natl Acad Sci USA.,2013,110:46-51),Agarwal et al.(Bioconjugate Chem.,2013,24:846-851),Barfield et al.(Drug Dev.and D.,2014,14:34-41),Drake et al.(Bioconjugate Chem.,2014,25:1331-41),Liang et al.(J AmChem Soc.,2014,136:10850-3),Drake et al.(Curr Opin Chem Biol.,2015,28:174-80),andYork et al.(BMC Biotechnology,2016,16(1):23),(BMC Biotechnology,2016,16(1):23), each of which incorporates all of its teachings by citation. .

[0267] 6.6.2. Additional moiety

[0268] In various embodiments, the GAL9 binding molecule has modifications including one or more additional binding moieties. In some embodiments, the binding moieties are antibody fragments or antibody formats, including but not limited to full-length antibodies, Fab fragments, FVS, scFVS, tandem scFVS, diabetic antibodies, scDiabodies, DARTs, tandAbs, minibodies, camel VHH, and other antibody fragments or formats known to those skilled in the art. Exemplary antibodies and antibody fragment formats are described in [the document / reference].

[0269] The teachings of Brinkmann et al. (MABS, 2017, Vol. 9, No. 2, 182-212) are described in detail and all of their teachings are incorporated by reference.

[0270] In certain embodiments, the one or more additional binding moieties are attached to the C-terminus of the first or third polypeptide chain. In certain embodiments, the one or more additional binding groups are attached to the C-terminus of both the first and third polypeptide chains. In certain embodiments, the one or more additional binding moieties are attached to the C-terminus of the first and third polypeptide chains. In some embodiments, individual portions of the one or more additional binding moieties are attached to the C-terminus of the first and third polypeptide chains, respectively, thereby forming functional binding moieties.

[0271] In certain embodiments, one or more additional binding moieties are attached to the N-terminus of any polypeptide chain (e.g., a first, second, third, fourth, fifth, or sixth polypeptide chain). In some embodiments, the individual portions of the additional binding moieties are attached to the N-terminus of different polypeptide chains, thereby forming functional binding moieties.

[0272] In some embodiments, one or more additional binding moieties are specific for different antigens or epitopes of ABS within the GAL9-binding molecule. In some embodiments, one or more additional binding moieties are specific for the same antigen or epitope of ABS within the GAL9-binding molecule. In some embodiments where the modification consists of two or more additional binding moieties, the additional binding moieties are specific for the same antigen or epitope. In some embodiments where the modification consists of two or more additional binding moieties, the additional binding moieties are specific for different antigens or epitopes.

[0273] In some embodiments, the attachment of one or more additional binding moieties to the GAL9 binding molecule is performed using in vitro methods, including but not limited to reactive chemistry and affinity labeling systems, as discussed in more detail in Section 6.6.3 below. In some embodiments, one or more additional binding moieties are attached to the GAL9 binding molecule via Fc-mediated binding (e.g., protein A / G). In some embodiments, recombinant DNA technology is used to attach one or more additional binding moieties to the GAL9 binding molecule, for example, by encoding a nucleotide sequence of the fusion product between the GAL9 binding molecule and the additional binding moieties on the same expression vector (e.g., plasmid).

[0274] 6.6.3. Functional / Reactive Groups

[0275] In various embodiments, the GAL9 binding molecule has modifications including functional groups or chemically active groups that can be used in downstream processes, such as linkages with additional moieties (e.g., drug conjugates and additional binding moieties, as discussed in more detail in Sections 6.6.1 and 6.6.2 above) and downstream purification processes.

[0276] In some embodiments, the modification is a chemically reactive group, including but not limited to reactive thiols (e.g., maleimide-based reactive groups), reactive amines (e.g., N-hydroxysuccinimide-based reactive groups), "click chemistry" groups (e.g., reactive alkynyl groups), and aldehydes with formylglycine (FGly). In some embodiments, the modification is a functional group, including but not limited to affinity peptide sequences (e.g., HA, HIS, FLAG, GST, MBP, and Strep systems). In some embodiments, the functional group or chemically reactive group has a cleavable peptide sequence. In certain embodiments, the cleavable peptide is cleaved by means including but not limited to photocleavage, chemical cleavage, protease cleavage, reducing conditions, and pH conditions. In certain embodiments, protease cleavage is performed by an intracellular protease. In certain embodiments, protease cleavage is performed by an extracellular or membrane-associated protease. ADC therapies employing protease cleavage are described in more detail in Choi et al. (Theranosties, 2012; 2(2):156-178.), the entire teachings of which are incorporated herein by reference.

[0277] 6.6.4. Weakened effector function

[0278] In some embodiments, the GAL9 binding molecule contains one or more engineered mutations in the amino acid sequence of the antibody domain that attenuate effector functions naturally associated with antibody binding. Effector functions include, but are not limited to, cellular functions arising from the binding of the Fc receptor to the Fc portion of the antibody, such as antibody-dependent cell cytotoxicity (ADCC, also known as antibody-dependent cell-mediated cytotoxicity), complement fixation (e.g., Clq binding), antibody-dependent cell-mediated phagocytosis (ADCP), and opsonization. Exemplary engineered mutations that reduce effector function are cited in U.S. Publication No. 2017 / 0137530, Armour, et al. (Eur. J. Immunol. J. Immunol. 29(8)(1999) 2613-2624), Shields, et al. (J. Biol. Chem. 276(9)(2001) 6591-6604), and Oganesyan, et al. (Acta Cristalographica D64(2008) 700-704), each of which is incorporated herein by reference in its entirety.

[0279] 6.7. Purification methods

[0280] This document provides methods for purifying GAL9-bound molecules. Purification steps include, but are not limited to, purifying GAL9-bound 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 can be used, including but not limited to those using protein A, protein G, or protein A / G reagents. Multiple iterations of a single purification method can be performed. Combinations of various purification methods can be used.

[0281] 6.7.1. Assembly and Purity of the Complex

[0282] In embodiments of the invention, at least four distinct polypeptide chains bind together to form a complete complex, namely a GAL9-binding molecule. However, incomplete complexes can also be formed, not containing at least four distinct polypeptide chains. For example, incomplete complexes can form complexes with only one, two, or three polypeptide chains. In other examples, incomplete complexes may contain more than three polypeptide chains but not at least four distinct polypeptide chains; for example, an incomplete complex may inappropriately bind to copies of more than one distinct polypeptide chain. The method of the present invention purifies the complex, i.e., the fully assembled GAL9-binding molecule, from the incomplete complex.

[0283] Methods for evaluating the efficacy and efficiency of purification steps are well known to those skilled in the art, including but not limited to SDS-PAGE analysis, ion-exchange chromatography, size exclusion chromatography, and mass spectrometry. Purity can also be evaluated according to various standards. Examples of standards include, but are not limited to, 1) evaluating fully assembled...

[0284] 1) Assess the percentage of total protein in the eluent provided by the GAL9-binding molecule; 2) Evaluate the fold enrichment or percentage increase of the method for purifying the desired product, for example, by comparing the total protein in the eluent provided by the fully assembled GAL9-binding molecule with the protein in the starting sample; 3) Assess the percentage reduction of total protein or unwanted product, for example, 3) Assess the percentage of total protein or unwanted products, such as the incomplete complexes described above, including determining the percentage or reduction of specific unwanted products (such as unassociated single polypeptide chains, dimers of any combination of polypeptide chains, or trimers of any combination of polypeptide chains). Purity can be assessed after any combination of methods described herein.

[0285] 6.8. Manufacturing Method

[0286] The GAL9-binding molecule described herein can be readily manufactured using standard cell-free translation, transient transfection, and stable transfection methods currently used for antibody production. In a specific implementation, the GAL9-binding molecule can be produced using Expi293 cells (ThermoFisher).

[0287] ThermoFisher’s protocols and 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, 19:11), are incorporated herein by reference in their entirety.

[0288] Using various purification strategies, including but not limited to the use of protein A, protein G, or protein A / G reagents, the expressed protein can be readily separated from unwanted proteins and protein complexes. Further purification can be achieved using ion-exchange chromatography methods conventional in the art.

[0289] 6.9. Pharmaceutical Compositions

[0290] In another aspect, pharmaceutical compositions are provided that comprise the GAL9 binding molecule described herein and a pharmaceutically acceptable carrier or diluent. In a typical embodiment, the pharmaceutical composition is sterile.

[0291] In various embodiments, the pharmaceutical composition comprises a GAL9-binding molecule at a concentration of 0.1 mg / ml to 100 mg / ml. In specific embodiments, the pharmaceutical composition comprises a GAL9-binding molecule 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 comprises a GAL9-binding molecule at a concentration exceeding 10 mg / ml. In some embodiments, the concentration of the GAL9-binding molecule is 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 particular embodiments, the concentration of the GAL9-binding molecule present exceeds 50 mg / ml.

[0292] In various embodiments, the pharmaceutical composition is described 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. Application 10 / 813,483, WO 2014 / 066468, WO 2011 / 104381, and WO 2016 / 180941, each of which is incorporated herein by reference in its entirety.

[0293] 6.10. Treatment methods

[0294] In another aspect, a method of treatment is provided, which involves administering the GAL9 binding molecule described herein to a patient (e.g., a subject) suffering from a disease or condition in an effective amount (e.g., a therapeutically effective amount) that is effective for the patient.

[0295] 6.10.1. Subjects

[0296] In some embodiments, the subject is a mammal. In some embodiments, the mammal is a mouse. In a preferred embodiment, the mammal is a human. In some embodiments, the subject's immune cells, such as blood dendritic cells, have increased PD-L2 expression compared to immune cells from a healthy individual (e.g., a healthy control).

[0297] 6.10.2. Combined therapy

[0298] GAL9 binding molecules can be used alone or in combination with other therapeutic agents or procedures to treat or prevent diseases or conditions. GAL9 binding molecules can be administered simultaneously or sequentially, depending on the disease or condition to be treated.

[0299] Anti-GAL9 binding molecules can be used in combination with clinically used or current standard-of-care medications or procedures to treat or prevent a disease or condition.

[0300] In some implementations, the GAL9-binding molecule is administered in combination with a second immunosuppressant. In some implementations, the second immunosuppressant is a glucocorticoid (such as prednisone, dexamethasone, or hydrocortisone), a cell inhibitor, or an anti-cytokine antibody, including anti-TNFα, anti-IL1, anti-IL5, and anti-...

[0301] IL6, anti-IL-17 antibodies and anti-IL-23 antibodies, as well as small molecule drugs that reduce inflammatory cytokine signaling, such as JAK / STAT inhibitors, methotrexate, hydroxychloroquine, chloroquine, anti-CD25 or anti-CD52 antibodies, or drugs that act on immunoaffinity (e.g., cyclosporine or sirolimus, or any other drug known to inhibit or prevent the activity of the immune system).

[0302] In some implementations, the GAL9 binding molecule is administered in combination with one or more anti-inflammatory drugs.

[0303] 6.10.3. Autoimmune or inflammatory diseases

[0304] In some implementations, treatment includes administering the GAL9-binding molecule described herein to a subject suffering from an autoimmune or inflammatory disease at a dose effective in treating that subject.

[0305] In some implementation schemes, autoimmune diseases include amyotrophic lateral sclerosis (ALS), achalasia, Addison's disease, adult-onset Still's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM / anti-TBM nephritis, antiphospholipid syndrome, autoimmune angioedema, autoimmune autonomic dysfunction, autoimmune encephalomyelitis, autoimmune hepatitis, autoimmune inner ear disease, autoimmune myocarditis, autoimmune oophoritis, autoimmune orchitis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune urticaria, axononeuropathy (AMAN), Balo's disease, Behcet's disease, benign mucosal herpes, cowpox, Castelman's disease, celiac disease, Chagas disease, chronic inflammatory demyelinating polyneuropathy, chronic relapsing multifocal osteomyelitis, Churg-Strauss syndrome, eosinophilic granulomatosis, and cicatricial pemphigoid. Cogan's syndrome, cold agglutinin disease, congenital heart block, Coxsackie myocarditis, CREST syndrome, Crohn's disease, herpes zoster, dermatomyositis, Devic disease (optic neuritis), discoid lupus, Dressier's syndrome, endometriosis, eosinophilic esophagitis (EoE), eosinophilic fasciitis, erythema nodosum, primary mixed cryoglobulinemia, Evans syndrome, fibromyalgia, fibrotic pneumonia, giant cell arteritis (temporal arteritis), giant cell myocarditis, glomerulonephritis, Goodpasture's syndrome, granulomatous polyangiitis, Graves' disease, Guillain-Barre syndrome, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schonleinpurpura (HSP), herpes gestationis or herpes gestationis (PG), purulent dermatitis (HS) (Acne Inversa, hypogammaglobulinemia, IgA nephropathy, IgG4-related sclerosis, immune thrombocytopenic purpura (ITP), inclusion body myositis, interstitial cystitis, juvenile arthritis, juvenile diabetes (type 1 diabetes), juvenile myositis, Kawasaki disease, Lambert-Eton syndrome, leukopenic vasculitis, lichen planus, woody conjunctivitis, linear IgA disease (LAD), lupus erythematosus, chronic Lyme disease, Meniere's disease, microscopic polyangiitis Inflammation, mixed connective tissue disease (MCTD), Mooren's ulcer, Mucha-Habermann disease, multifocal motor neuropathy (MMN) or MMNCB, multiple sclerosis, myasthenia gravis, myositis, somnolencephaly, neonatal lupus, optic neuritis, neutropenia, ocular catarrhal papules, optic neuritis, palmar rheumatoid disease (PR), PANDAS, paraneoplastic cerebellar degeneration (PCD), paroxysmal nocturnal hemoglobinuria (PNH), Parry Romberg syndrome, Pars planitis (peripheral uveitis)Parsonage-Turner syndrome, pemphigus, peripheral neuropathy, peripheral encephalomyelitis, pernicious anemia (PA), POEMS syndrome, polyarteritis nodosa, type I, II, or III polyglandular syndrome, polyrheumatoid arthritis, polymyositis, post-myocardial infarction syndrome, post-pericardectomy syndrome, primary biliary cirrhosis, primary sclerosing cholangitis, progesterone dermatitis, psoriasis, psoriatic arthritis, pure erythrocytosis, pyoderma, Raynaud's phenomenon, reactive arthritis, reflex sympathetic atrophy, relapsing polychondritis, restless legs syndrome, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcomatosis, Schmidt syndrome, dural dermatitis, scleroderma, Sjogren's syndrome, sperm and testicular autoimmunity, stiff-person syndrome, subacute bacterial endocarditis, Susac's syndrome, sympathetic ophthalmia, Takayasu's arteritis, testicular autoimmunity. Testicular autoimmune disease, stiff-person syndrome, subacute bacterial endocarditis, Sussac syndrome, sympathetic ophthalmitis, Caucasian arteritis, temporal arteritis, giant cell arteritis, thrombocytopenic purpura, Tolosa-Hunt syndrome, rhabdomyomyitis, type 1 diabetes mellitus, ulcerative colitis, undifferentiated connective tissue disease, uveitis, vasculitis, vitiligo, or Vogt-Koyanagi-Harada syndrome.

[0306] In some embodiments, the autoimmune disease is selected from the following: inflammatory bowel disease, Crohn's disease, ulcerative colitis, colitis, celiac disease, rheumatoid arthritis, Behcet's disease, amyloidosis, psoriasis, psoriatic arthritis, systemic lupus erythematosus nephritis, graft-versus-host disease (GVHD), non-alcoholic steatohepatitis (NASH), and ankylosing spondylitis. In a preferred embodiment, the disease is Crohn's disease.

[0307] In some embodiments, treatment includes administering a GAL9-binding molecule as described herein to a subject at risk of transplant rejection, in an amount that effectively reduces transplant rejection. In some embodiments, treatment includes administering a GAL9-binding molecule as described herein to a subject with graft-versus-host disease, in an amount that effectively reduces GvHD. In some embodiments, treatment includes administering a GAL9-binding molecule as described herein to a subject with a post-traumatic immune response, in an amount that effectively reduces inflammation. In some embodiments, treatment includes administering a GAL9-binding molecule as described herein to a subject with ischemia, in an amount that is effective in treating the subject. In some embodiments, treatment includes administering a GAL9-binding molecule as described herein to a subject who has experienced a stroke, in an amount that is effective in treating the subject.

[0308] In some implementations, treatment includes administering a GAL9-binding molecule to a subject with a viral infection in an amount that effectively reduces acute respiratory distress syndrome and / or acute cytokine release syndrome (cytokine storm). In a specific implementation, the viral infection is infection with the SARS-CoV-2 virus, and the disease is COVID-19.

[0309] 6.10.4. Administration

[0310] The GAL9 binding molecule can be administered to a subject via any route known in the art. For example, the GAL9 binding molecule can be administered to a human subject via, for example, intra-arterial, intramuscular, intradermal, intravenous, intraperitoneal, intranasal, parenteral, intrapulmonary, subcutaneous, local, oral, sublingual, intratumoral, intralesional, intrasynovial, intrathecal, intracerebrospinal fluid, or perivenous administration. The GAL9 binding molecule can be administered to the subject as a pharmaceutical composition or as a drug composition. Exemplary pharmaceutical compositions are described herein.

[0311] The anti-GAL9 binding molecules disclosed in this article can be administered alone or in combination with other therapeutic agents or procedures to treat or prevent diseases or conditions.

[0312] Depending on the condition or disease being treated, GAL9-binding molecular therapy can improve one or more clinical endpoints in subjects. Examples of improved clinical endpoints in subjects with a disease or condition include, but are not limited to: reduced inflammation, reduced autoimmune response, prolonged remission, induction of remission, re-establishment of immune tolerance, improved organ function, reduced risk of disease or condition progression, reduced risk of a second disease progression, increased overall survival, or combinations thereof.

[0313] 6.11. Example

[0314] The examples below are provided in an illustrative manner, not as limiting. In particular, the methods for expressing and purifying various antigen-binding proteins, and their use in the various assays described below, are non-limiting and illustrative.

[0315] 6.11.1. Method

[0316] 6.11.1.1.Expi293 expression

[0317] Following the manufacturer's instructions, various antigen-binding proteins were expressed using the Expi293 transient transfection system. In short, unless otherwise specified, plasmids encoding the individual strands were mixed at a 1:1 mass ratio and transfected into Expi 293 cells using the ExpiFectamine293 transfection kit. Cells were cultured at 37°C, 8% CO2, 100% humidity, and shaking at 125 rpm. 16–18 hours after transfection, the transfected cells were fed once. On day 5, cells were harvested by centrifugation at 2000g for 10 minutes. The supernatant was collected for affinity chromatography purification.

[0318] 6.11.1.2. ExpiCHO Expression

[0319] According to the manufacturer's instructions, various GAL9 antigen-binding proteins are expressed using the ExpiCHO transient transfection system. In short, plasmids encoding the respective strands are mixed at, for example, a 1:1 mass ratio and...

[0320] The ExpiFectamine CHO transfection kit was used to transfect ExpiCHO.

[0321] Cells were cultured at 37°C, 8% CO2, 100% humidity, and shaking at 125 rpm. Transfected cells were generally fed once 16-18 hours after transfection. On day 5, cells were harvested by centrifugation at 2000g for 10 minutes. The supernatant was then collected for affinity chromatography purification.

[0322] 6.11.1.3. Protein A Purification

[0323] Clear supernatants containing various antigen-binding proteins were separated using Protein A (ProtA) resin or anti-CH1 resin on a gravity flow purifier. In a head-to-head comparison example, the supernatants containing various antigen-binding proteins were split 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). Samples were loaded onto the column at a rate of 5 mL / min. The samples were eluted with 0.1 M sodium acetate, pH 3.5. Elution was monitored by absorbance at 280 nm, and the combined elution peaks were analyzed.

[0324] 6.11.1.4. SDS-Page Analysis

[0325] Samples containing various isolated antigen-binding proteins were analyzed by reducing and non-reducing SDS-PAGE to determine 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 min with 10 mM reducing agent. Non-reducing samples were incubated at 70°C for 5 min without reducing agent. Reducing and non-reducing samples were loaded into 4–15% gradient TGX gels (BioRad), run buffer was added, and the gels were run at 220 V for 30 min. After the run, the gels were washed with deionized water and stained with GelCode Blue Safe ProteinStain (ThermoFisher). The gels were destained with deionized water before analysis. Density analysis was performed on scanned images of the destained gels using standard image analysis software to calculate the relative abundance of bands in each sample.

[0326] 6.11.1.5. IEX Chromatography

[0327] Samples containing various isolated antigen-binding proteins were analyzed by cation exchange chromatography to determine the ratio of intact product to incomplete product and impurities. The clarified supernatant was analyzed using a 5-mL MonoS (GE Lifesciences) column on an AKTA Purifier FPLC. The MonoS column was equilibrated with buffer A containing 10 mM MES at pH 6.0. Samples were loaded onto the column at a rate of 2 mL / min. Elution was performed in 6 column volumes (CV) using a 0–30% gradient buffer B (10 mM MES pH 6.0, 1 M sodium chloride). Elution was monitored by absorbance at 280 nm, and sample purity was calculated by peak integration to determine the abundance of monomer and contaminant peaks. Monomer and contaminant peaks were pooled separately for SDS-PAGE analysis as described above.

[0328] Analytical SEC chromatography was performed by loading each sample at a concentration of 1 mg / mL onto the column at a rate of 1 mL / min. The samples were eluted using isocratic flow with 1.5 CV PBS. Elution was monitored using absorbance at 280 nm, and the elution peaks were analyzed by peak integration.

[0329] 6.11.1.6. Mass Spectrometry

[0330] Samples containing various isolated antigen-binding proteins were analyzed by mass spectrometry to confirm the correct molecular weight. All analyses were performed by third-party research institutions. In short, samples were treated with an enzyme cocktail to remove glycosylation. Samples were tested in a reducing format to specifically identify each chain by molecular weight. Samples were also tested under non-reducing conditions to determine the molecular weight of all complexes in the sample. Mass spectrometry was used to determine the quantity of unique products based on molecular weight.

[0331] 6.11.1.7. Discovering antibodies through phage display

[0332] Phage display of the human Fab library was performed using standard protocols. Human GAL9 protein was purchased from [source missing].

[0333] Aero Biosystems (Human Gal9 His-tag Cat#LG9-H5244), using EZ-Link NHS-PEG 12 - Biotin (ThermoScientific Cat#21312) was biotinylated according to standard protocol. The ability of phage clones to bind to the GAL9 protein was screened using a standard phage ELISA assay.

[0334] In short, a Fab-format phage library is constructed using expression vectors (also known as phage particles) capable of replicating and expressing within phages. Both the heavy and light chains are encoded in the same expression vector, with the heavy chain fused to a truncated variant of the phage capsid protein pIII. The light chain and heavy chain-pIII fusion are expressed as separate polypeptides and assembled in the bacterial periplasm, where redox potentials cause disulfide bonds to form, thereby creating a phage display antibody containing candidate ABS.

[0335] This library was created using sequences from specific human heavy chain variable domains (VH3-23) and specific human light chain variable domains (Vκ-1). For the selected library, all three CDRs of the VH domain were diversified to match the amino acid position frequencies calculated by CDR length found in the human antibody library. Light chain variable structures in the selected library introduced diversity only in VL CDR3 (L3); light chain VL CDR1 (LI) and CDR2 (L2) retained human germline sequences.

[0336] The heavy chain scaffold (SEQ ID NO:2), light chain scaffold (SEQ ID NO:4), complete heavy chain Fab peptide (SEQ ID NO:1), and complete light chain Fab peptide (SEQ ID NO:3) used in the phage display library are shown below, where lowercase "x" represents the CDR amino acid that changed during library creation.

[0337] Phage display of VH scaffold [SEQ ID NO:2]:

[0338] EVQLVESGGGLVQPGGSLRLSCAASGFTFxxxxIHWVRQAPGKGLEWVAxxxxxxxxxxxY

[0339] ADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARxxxxxxxxxxxxxDYWGQGTLVT

[0340] VSSAS

[0341] Phage display VL scaffold [SEQ ID NO:4]:

[0342] DIQMTQSPSSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYSASSLYSGVPS

[0343] RFSGSRSGTDFTLTISSLQPEDFATYYCQQxxxxxxTFGQGTKVEIKRT

[0344] Phage display of heavy chain Fab peptide [SEQ ID NO:1]:

[0345] EVQLVESGGGLVQPGGSLRLSCAASGFTFxxxxIHWVRQAPGKGLEWVAxxxxxxxxxxxY

[0346] ADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARxxxxxxxxxxxxxDYWGQGTLVT

[0347] VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL

[0348] QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC

[0349] Phage display of light chain Fab peptide [SEQ ID NO:3]:

[0350] DIQMTQSPSSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYSASSLYSGVPS

[0351] RFSGSRSGTDFTLTISSLQPEDFATYYCQQxxxxxxTFGQGTKVEIKRTVAAPSVFIFPPSDS

[0352] QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS

[0353] KADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0354] Diversity was generated by Kunkel mutagenesis, using primers to introduce diversity into VH CDR1(HI), CDR2(H2), and CDR3(H3), as well as VL CDR3, to mimic the diversity found in natural antibody libraries, as detailed in Kunkel, TA (PNAS Jan 1, 1985, 82(2) 488-492), which is incorporated herein by reference in its entirety. Briefly, single-stranded DNA was prepared from isolated phages using standard procedures and subjected to Kunkel mutagenesis. The chemically synthesized DNA was then electroporated into MC1061F-cells. Phage particles obtained from overnight cultures were digested with restriction enzymes (Bam HI and Xba I) to remove wild-type sequences. The digested samples were electroporated into TGI cells and then recovered. The recovered cells were passaged and then infected with M13K07 helper phages to generate a phage library.

[0355] Phage panning was performed using a standard procedure. In short, the first round of phage panning was conducted using targets immobilized on streptavidin magnetic beads, which were then placed in 1 mL of PBST-2% BSA with a prepared library of approximately 5 × 10⁶ phages. 12 Phage contact was performed. After one hour of incubation, the phage bound to the magnetic beads was separated from the supernatant using a magnetic rack. The beads were washed three times to remove non-specifically bound phage, and then added to OD. 600 In 0.6% ER2738 cells (5 mL). After 20 minutes, the infected cells were placed in 25 mL of 2xYT. + Ampicillin and M13K07 helper phage (final concentration, ~10) 10Passaged cultures were performed at pfu / ml and allowed to incubate overnight with vigorous shaking at 37°C. The following day, phages were prepared using a standard procedure via PEG precipitation. Prior to translation, phages specific to SAV-coated magnetic beads were pre-removed. A second round of panning was performed using a KingFisher magnetic bead processor and antigen fixed with 100 nM magnetic beads, following a standard procedure. A total of 3–4 rounds of phage panning were performed to enrich phages specific to the target antigen in the phage-displayed Fab. Target-specific enrichment was confirmed using polyclonal and monoclonal phage ELISA. DNA sequencing was used to identify isolated Fab clones containing candidate ABS.

[0356] The VL and VH domains identified in the above phage screening were reformulated into a bivalent, monospecific human full-length IgG1 architecture.

[0357] Natural human full-length IgG1 heavy chain structure [SEQ ID NO:5]:

[0358] EVQLVESGGGLVQPGGSLRLSCAASGFTFxxxxIHWVRQAPGKGLEWVAxxxxxxxxxxxYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARxxxxxxxxxxxxxDY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK[SEQ ID NO:5]

[0359] Natural human full-length IgG1 light chain structure:

[0360] Equivalent to bacteriophage display of the light chain Fab, see SEQ ID NO:3

[0361] 6.11.1.8. Octet determination in conjunction with kinetics

[0362] To measure qualitative binding affinity during the GAL9 binding discovery campaign, reformulated IgG1 conjugates were immobilized on the biosensor of an Octet (Pall ForteBio) biolayer interferometer.

[0363] The soluble GAL9 antigen was then added to the system and binding was measured. Qualitative binding affinity was determined by observing the dissociation phase of the octet induction plot, from the weakest ( + ) to the strongest ( +++ The slope of the induction graph is used for evaluation. A slow dissociation rate indicates that the decrease in the dissociation phase of the induction graph is negligible and indicates that the antibodies are tightly bound. +++ To obtain accurate kinetic constants for monovalent affinity, a series of dilutions involving at least five concentrations of the GAL9 analyte (ranging from approximately 10 to 20 X K) were measured during the binding step. D up to 0.1XK D (Value, 2-fold dilution). In the dissociation step, the sensor was immersed in a buffer solution free of GAL9 analyte, where the binding complex on the sensor surface dissociated. Kinetic and equilibrium binding constants were calculated using Octet kinetic analysis software based on the binding and dissociation rate curves. The analysis was performed globally (global fitting), where the kinetic constants were derived simultaneously from the concentrations of all analytes included in the experiment.

[0364] 6.11.1.9. Tabletop Sub-box

[0365] As described above, anti-GAL9 candidates are formatted as bivalent, monospecific, full-length human IgG1, and GAL9 binding is tested in pairs using an octet-based 'tandem' assay. In short, biotinylated GAL9 is immobilized on a streptavidin sensor, and two anti-GAL9 candidates are tandemly linked. A competitive blocking curve is generated to determine whether a given anti-GAL9 candidate blocks the binding of a set of other anti-GAL9 candidates to GAL9. Anti-GAL9 candidates competing for the same or non-overlapping binding regions are grouped and designated as belonging to the same bin.

[0366] 6.11.1.10. Activation of PBMCs and treatment with Galectin 9 antibody

[0367] Individual samples of PepMix HCMVA (pp65) (>90%) Protein ID: P06725 (Cat. No. PM-PP65-2, JPTPeptide Technologies) were prepared according to the manufacturer's instructions. TMHCMVA (pp65) is a 15-peptide-overlapping, complete protein span mixture obtained from the 65kDa phosphoprotein (pp65) of human cytomegalovirus (HHV-5) (Swiss-Prot ID: P06725) for stimulating immune cell responses.

[0368] Human peripheral blood mononuclear cells (PBMCs) were thawed under standard conditions and then resuspended in growth medium (10% FBS in RPMI).

[0369] The resuspended PBMC will be 5×10 5 Cells were seeded in 96-well plates. Cells were then inoculated with 2 μg / mL PepMix. TM HCMVA (pp65) was added to 40 μg / mL candidate GAL9 antibody or control antibody and cultured in growth medium for 24 hours at 37°C and 5% CO2.

[0370] 6.11.1.11. LEGENDplex Human Th Cytokine Detection Method

[0371] Following PBMC activation and treatment with the Galectin 9 antibody described herein, cytokine secretion from PBMCs and immune cell subsets was assessed using a cytokine bead array at 24 and 72 hours post-treatment, as shown below.

[0372] Collect 200 μl of cell culture supernatant and centrifuge to remove cell debris. Use LEGENDplex. TM The obtained supernatant was analyzed using a HumanThl Panel (5plex) (Cat. No. 740009, Biolegend). TM The Human Th1 Detection Kit is a bead-based detection method that allows for the simultaneous quantitative detection of human cytokines IL-2, IL-6, IL-10, IFN-γ, and TNF-α using flow cytometry.

[0373] In short, the cytokine standard and capture bead mixture were prepared according to the manufacturer's instructions. The capture bead mixture, biotinylated detection antibody, and detection buffer were mixed in a 1:1:1 ratio to prepare the detection stock solution.

[0374] Incubate 12.5 μL of supernatant sample or cytokine standard with 37.5 μL of stock solution. Seal the plate, cover with aluminum foil, and shake at 600 rpm for 2 hours at room temperature. Then incubate with streptavidin-phycoerythrin (SA-PE) at room temperature for 30 minutes with shaking at 600 rpm. Wash the beads twice and resuspend them, then perform flow cytometry analysis according to the manufacturer's instructions.

[0375] 6.11.1.12. Staining PBMCs with marker antibodies

[0376] Following the PBMC activation and Galectin 9 antibody treatment described in this article, the PBMC immune cells were stained with labeled antibodies according to the following procedure.

[0377] Cells were loaded at 5 × 10 6 Cells / mL were resuspended in growth medium (10% FBS in RPMI). 200 μL of the resuspended cells were distributed equally into 96-well plates and then treated with Fixable Viability Dye. 780 cells were incubated at 2–8°C for 30 minutes to irreversibly label dead cells. The cells were then washed and incubated with human Fc blocking solution (Cat. No. 14-9161-73, eBiosciences) at room temperature for 10 minutes.

[0378] Prepare the antibody cocktail working solution according to the table below.

[0379]

[0380]

[0381] Incubate the cells in a 10 μL diluted antibody cocktail at 2–8 °C for 30 minutes. Then wash and resuspend the cells, and analyze by flow cytometry.

[0382] To analyze the immunostimulatory markers CD27, CD40L, ICOS, 4-1BB, and OX40, the same protocol provided above was followed, but the cells were incubated with alternative antibody cocktails, as detailed in Table 2 below.

[0383]

[0384]

[0385] 6.11.2. Example 1: Increased PD-L2 expression in blood dendritic cells from Crohn's disease patients

[0386] Mice deficient in programmed death 1 (PD-1) develop various autoimmune-like diseases, suggesting that the PD-1 receptor plays an important role in both immunity and autoimmunity. PD-1 has two endogenous ligands, PD-L1 and PD-L2. The interaction between PD-1 and PD-L1 has been implicated in autoimmunity; however, the role of PD-L2 in autoimmunity remains unclear.

[0387] Crohn's disease (CD) is a chronic inflammatory disease of the gastrointestinal tract. While the exact cause of the disease is not fully understood, it is clear that an overactive immune system in CD patients leads to inflammation and damage to the gastrointestinal tract. This study aimed to determine the expression of PD-L2 and PD-L1 on blood dendritic cells from Crohn's disease patients.

[0388] Research participants

[0389] Peripheral blood was drawn from 29 adults diagnosed with Crohn's disease via colonoscopy. Patients at different stages of treatment were selected, but those who had received anti-TNF-α therapy were excluded. As a control, peripheral blood was drawn from 13 healthy adults who underwent screening for a family history of colorectal cancer.

[0390] Immunostaining

[0391] Single-cell suspensions obtained from 10 mL of whole blood were incubated with Fc receptor-binding antibodies to block non-specific Fc binding of specific antibodies. Fixable Viability Dye eFluor780 (ebioscience, San Diego, CA) was used to exclude dead cells from the analysis. The following anti-human monoclonal antibodies were used to evaluate cells: HLA-DRPerCP-Cy5.5 (clone G46-6; BD Bioscience, San Jose, CA); lineage cocktail BV510 [CD3 (clone OKT3) / CD14 (clone M5E2) / CD16 (clone 3G8) / CD19 (clone HIB19) / CD20 (clone 2H7) and CD56 (clone HCD56)]; CD1lc BV605 (clone 3.9; BioLegend, San Diego, CA).

[0392] Anti-human PD-L2 monoclonal antibody (clone MIH18; BioLegend, San Diego, CA) and anti-human PD-L1 monoclonal antibody (clone 29E.2A3; BioLegend, San Diego, CA) or control IgG were internally labeled using Lightning-Link Rapid DyLight 647 and Lightning-Link Rapid DyLight 488 (BioNovus Life Sciences, Cherrybrook, NSW, Australia), respectively. Cells were stained with anti-HLA-DR, anti-PD-L2, or anti-PD-L1 or IgG controls for 30 minutes at room temperature, then washed twice with PBS for 5 minutes each time, and then fixed with 1% paraformaldehyde-PBS, pH 7.25.

[0393] Flow cytometry

[0394] Cells were stained with Fixable Viability Dyes (FVD), and gating was performed on mononuclear cell regions in both side-scatter and front-scatter maps to capture only viable cells. Dendritic cells were defined as HLA-DR. + and Lin + Then, CD11c was performed in the total peripheral blood population. + Gating. At least 1×10⁻⁶ units must be collected from each donor. 4 One event.

[0395] Cells were analyzed using a BD LSR Fortessa flow cytometer, and data were analyzed using BD FACSDiva software (Becton & Dickinson, Franklin Lakes, NJ), FCS express (De Novo software, Glendale, CA), or FlowJo software (Tree Star; a subsidiary of Becton, Dickinson and Company, Ashland, OR).

[0396] Statistical analysis

[0397] The two-tailed nonparametric Mann-Whitney U test was performed using GraphPad Prism (GraphPad Software).

[0398] microscope

[0399] Microscopic samples are prepared by mounting stained, sorted cells onto a glass slide. Images are collected using a confocal microscope.

[0400] Results / Conclusions

[0401] Figure 2 It shows the CD11c of Crohn's disease patients + Isographs of dendritic cells (DCs) stained with IgG control, anti-PD-L1, or anti-PD-L2. We observed that 2.23% of the IgG control cells nonspecifically bound to DCs, while the anti-PD-L1 antibody stained 28.6% of the DCs with PD-L1. + Similarly, in the second experiment, the IgG control group had only 3.22% CD11c + DCs bind, and the anti-PD-L2 antibody detected that 62.7% of the DCs were PD-L2-bound. + .

[0402] Figures 3A-3BThe images show CD11c from healthy control donors and CD patients. + PD-L2 in blood dendritic cells + Percentage scatter plot of cells ( Figure 3B ) and in CD11c + PD-L1 in blood dendritic cells + Percentage scatter plot of cells ( Figure 3B The horizontal bars on the scatter plot represent the average value. Figure 3C-3D The CD11c values ​​of healthy control donors and Crohn's disease patients were displayed. + PD-L1 expression level on blood dendritic cells (GMI) Figure 3C Scatter plot of ) and PD-L2 expression level (GMI) Figure 3D The horizontal bars on the scatter plot represent the average value. A single asterisk "*" indicates a p-value of 0.0292. A double asterisk "**" indicates a p-value of 0.0032.

[0403] Figures 4A-4B Representative immunostaining of dendritic cells (DCs) from the blood of two healthy controls and three Crohn's disease patients is shown. DCs from healthy controls showed high PD-L1 (green) and PD-L2 (red) staining throughout the cells; presented in grayscale in the attached figure. In contrast, dendritic cells from Crohn's disease patients showed low PD-L1 expression and high levels of what appeared to be aggregated PD-L2. High staining of aggregated PD-L1 was observed in some cells.

[0404] The results showed that PD-L2 protein was expressed at higher levels in blood dendritic cells of Crohn's disease patients compared to healthy control donors (P = 0.0032), a statistically significant difference compared to PD-L1 (P = 0.0292). These results suggest that the PD-L2 pathway may play an important role in Crohn's disease and other autoimmune diseases.

[0405] 6.11.3. Example 2: Inhibition of PD-L2 in PBMCs of Crohn's disease patients leads to a clinically beneficial cytokine profile.

[0406] This study aimed to determine the effect of PD-L2 protein inhibition on the cytokine profile of PBMCs in patients with Crohn's disease (CD) compared to the IgG control group.

[0407] Research participants

[0408] Blood samples were obtained from 14 different Crohn's disease patients. Peripheral blood mononuclear cells (PBMCs) were isolated by density centrifugation on a Ficoll-Paque (Pharmacia, Freiburg, Germany) using heparinized blood. Isolated PBMCs from control groups and CD patients were added to wells pre-coated with anti-CD3 (2 × 10⁻⁶). 5 Cells / well). R10 medium supplemented with penicillin (100 IU / ml), streptomycin (0.1 mg / ml) and L-glutamine (0.29 g / ml). Control IgG or blocking anti-PD-L2 (MIH18) antibody was added to the culture at a dose of 20 μg / ml.

[0409] deal with

[0410] Matched PBMC samples were treated with IgG control or anti-human PD-L2 antibody clone MIH18 (BioLegend) for 36 hours before assay.

[0411] Cytokine assay

[0412] Use BD according to the manufacturer's instructions. TM Cytometric Bead Array (CBA) measures the concentrations of TNF-α, IFN-γ, and IL-10.

[0413] Statistical analysis

[0414] Wilcoxon paired signed-rank test was performed using GraphPad Prism (GraphPad Software).

[0415] Results / Conclusions

[0416] The average concentrations of TNF-α and IFN-γ in the matched samples were shown in Figures 5A-5B middle. Figure 5C The mean ratio of IL-10 to TNF-α is shown. These results indicate that inhibiting PD-L2 results in a clinically favorable cytokine profile in PMBCs of CD patients by reducing the levels of pro-inflammatory cytokines TNF-α and IFN-γ and increasing the levels of the inhibitory cytokine IL-10.

[0417] 6.11.4. Example 3: Regulation of mouse CD4 by stimulating or blocking the GAL9 / PD-L2 pathway + TNF-α secretion from T cells

[0418] Previously, we showed that GAL9 can bind soluble PD-L2, and that some immunological effects of PD-L2 are mediated through the binding of polymeric PD-L2 to GAL9, rather than through PD-1 / PD-L1 (WO 2016 / 008005, the full text of which is incorporated herein by reference). This study aims to determine whether stimulating or blocking the GAL9 / PD-L2 pathway can modulate mouse CD4. + TNF-α secretion in T cells.

[0419] animal

[0420] C57BL6 / J mice were used in the study. All animals used in the study were bred and cared for in accordance with the National Health and Medical Research Council (NHMRC) guidelines for animal use.

[0421] sPD-L2

[0422] Soluble mouse PD-L2 with human IgG1 Fc (sPD-L2) was custom-produced by Geneart (Germany).

[0423] Antibody

[0424] During treatment, an inhibitory anti-mouse GAL9 antibody clone 108A2 was used. San Diego, CA) or rat IgG2a control antibody. Anti-mouse GAL9 clone (108A2) binding to mouse Galectin-9 linker peptide (Oomizu, S. et al., PLoS One 7(1l):e48574(2012); Doi:10.1371 / journal.pone.0048574, incorporated herein by reference). Anti-CD3 (clone 145.2C11) (Aviva Systems Biology Corp. San Diego, CA) was used for stimulation. Cell isolation and CD4 + T cell stimulation

[0425] Mouse spleen cell suspensions were prepared from five mice. Unaffected CD4 cells were isolated using a kit from Miltenyi Biotec Inc. (Auburn, CA). + T cells. Mouse CD4 cells were stimulated with anti-CD3 clone 145.2C11 (Aviva Systems Biology Corp., San Diego, CA) at 5 μg / ml. + T cells were then treated with either IgG control or sPD-L2 (20 μg / ml), or with sPD-L2 and anti-GAL9 mAh clone 108A2 (both 20 μg / ml).+ T cells were then cultured for 36 hours.

[0426] Cytokine assay

[0427] After 36 hours of treatment, use BD according to the manufacturer's instructions. TM Cytometric Bead Array measures TNF-α concentration.

[0428] Statistical analysis

[0429] The nonparametric Mann-Whitney U test was performed using GraphPad Prism (GraphPad Software).

[0430] Results / Conclusions

[0431] Figure 6 A bar chart showing TNF-α concentration levels for each treatment group. Treatment of activated CD4+ with sPD-L2 alone. + T cells, compared to the IgG control, CD4 + T cell secretion of TNF-α was significantly increased (*P < 0.0001). The addition of inhibitory anti-mouse GAL9 antibody (108A2) resulted in a significant increase in TNF-α secretion compared to T cells treated with 108A2.

[0432] CD4 + Compared to T cells, and compared to IgG controls, activated CD4 + TNF-α secretion by T cells was significantly reduced, *P < 0.0001.

[0433] sPD-L2 binds to GAL9 on T cells, inducing TNF-α secretion, while inhibiting GAL9 blocks sPD-L2-mediated CD4+ secretion. + These results indicate that the GAL9 / PD-L2 pathway regulates stimulated CD4+ secretion. + TNF-α levels in T cells.

[0434] 6.11.5. Example 4: CD4 in malaria-infected mice + In T cells, inhibitory anti-mouse GAL9 (108A2) antibodies function independently of PD-1 / PD-L1, while activating anti-GAL9 antibodies do not.

[0435] This study aims to investigate the dependence of inhibitory and activating GAL9 antibodies on the PD-1 / PD-L1 pathway.

[0436] Mice infected with malaria can be used to study immune mechanisms and drug sensitivity. (Wykes, MN et al., Eur J Immunol. (2009) 39: 2004-7, the entire contents of which are incorporated herein by reference.) Furthermore, it has been demonstrated that the malaria parasite that causes malaria can utilize the PD-1 pathway to "disable" T cell function. The clear role of PD-1 in the pathogenesis of malaria was demonstrated when PD-1-deficient mice were shown to rapidly and completely clear P. chabaudi infection. Therefore, malaria infection models can be used to understand the relative roles of PD-1 and its ligands PD-L1 and PD-L2 in immunity.

[0437] Antibody

[0438] This study used an inhibitory anti-mouse GAL9 antibody (108A2) and an activating anti-mouse GAL9 antibody (RG9.1) (Cat.No.BE0218, InVivoMab Antibodies).

[0439] Mouse model infected with malaria

[0440] A cohort of C57BL / 6 mice was infected with nonfatal malaria (P. yoelii 17XNL). They were administered 10 mg of malaria intravenously. 5 Mice were cultured for 7 days after being infected with P. yoelii red blood cells to induce infection.

[0441] CD4 + Isolation and processing of T cells

[0442] Unaffected CD4 using Miltenyi Biotec + T-cell isolation kits were used to isolate CD4 cells from malaria-infected mice. + T cells. Next, the isolated T cells were cultured and treated overnight with control IgG antibody, inhibitory anti-mouse GAL9 antibody (108A2), or activating anti-mouse GAL9 antibody (RG9.1).

[0443] Immunostaining and microscopic examination

[0444] After processing, DAPI (for DNA detection) and Lightning-Link Rapid DyLight were used.

[0445] 647, 594, or 488 kit-labeled anti-OX40 (CD134), anti-PD-1, and anti-PD-L1

[0446] Cells were stained with (BioXCell, Lebanon, NH) antibodies. Immunostaining was observed using confocal imaging.

[0447] Results / Conclusions

[0448] Figure 7 The results show CD4 cells treated with IgG control, inhibitory anti-mouse GAL9 antibody (108A2), or activating anti-mouse GAL9 antibody (RG9.1). + Representative confocal images of T cells. Red staining shows the PD-1 receptor, green staining shows the PD-L1 ligand, yellow staining shows the OX40 receptor, and blue staining shows DNA (DAPI), presented in grayscale in the attached image.

[0449] We observed that treatment with activated anti-mouse GAL9 (RG9.1) antibody reduced the expression of PD-1 receptor (low staining levels) and PD-L1 ligand (significantly reduced staining levels). Conversely, we observed that treatment with inhibitory anti-GAL9 (108A2) had no effect on the expression of either PD-1 receptor (staining levels similar to IgG control levels) or PD-L1 ligand (staining levels similar to IgG control levels). Furthermore, we observed that treatment with inhibitory anti-GAL9 (108A2) resulted in a decrease in OX40 expression. These results suggest that inhibitory GAL9 antibody has a significant effect on CD40 expression. + It functions independently of the PD-1 / PD-L1 pathway in T cells.

[0450] 6.11.6. Example 5: Treatment of mice infected with malaria with inhibitory anti-mouse GAL9 (108A2) reduced PD-L2-mediated CD4 count. + and CD8 + T cell survival rate

[0451] This study aimed to determine the effect of inhibitory anti-mouse GAL9 (108A2) antibody on PD-L2-mediated CD4+ in mice infected with malaria. + and CD8 + The impact on T cell survival.

[0452] PD-L2 has been shown to increase parasite-specific CD4 levels. + and CD8 + The number of T cells is used to protect mice from fatal malaria infection, thereby mediating CD4+. + and CD8 + T cell survival in malaria-infected mice. See Karunarathne et al. Immunity (2016). Aug 16:45(2):333-45), the entire contents of which are incorporated herein by reference.

[0453] Mouse model infected with malaria

[0454] A group of 5 C57BL / 6 mice were infected with non-fatal malaria (P. yoelii 17XNL). 10 mg of the vaccine was administered intravenously. 5 Mice were cultured for 7 days after being infected with *P. yoelii* red blood cells to induce infection. All animals used in the study were bred and cared for in accordance with the National Health and Medical Research Council (NHMRC) guidelines for animal use.

[0455] sPD-L2

[0456] As a positive control, CD4 cells were treated with Geneart (Germany)'s custom-made soluble PD-L2 "sPD-L2". + and CD8 + T cells.

[0457] Cell isolation, processing and viability determination

[0458] By using CD4 that is unaffected + and CD8 + The FACS kit from Miltenyi Biotec Inc. (Auburn, CA) used to isolate CD4 from infected mice contained T cells. + and CD8 + T cells were isolated and then cultured at 37°C for 36 hours. Next, CD4 cells were treated with either 20 mg / ml sPD-L2 or 20 mg / ml anti-mouse GAL9 (108A2). + and CD8 + T cells. After treatment, cell viability was measured using viability dyes and flow cytometry.

[0459] Results / Conclusions

[0460] CD4 + T cells and CD8 + The results of the T cell survival assay were shown in the following... Figure 8A and Figure 8B In the middle. SPD-L2 processing increased CD4. + and CD8 + PD-L2-mediated survival in T cells. Conversely, treatment with sPD-L2 and anti-GAL9 (108A2) reduced CD4 cell survival. + and CD8 + PD-L2-mediated decreased survival rates in T cells. These results indicate that PD-L2 works in conjunction with GAL9 to mediate CD4+. + and CD8 + T cell survival.

[0461] 6.11.7. Example 6: Blocking the GAL9 / PD-L2 pathway reduces CD4 activation in malaria-infected mice. + Pro-inflammatory cytokines in T cells

[0462] This study aimed to determine whether blocking the GAL9 / PD-L2 pathway with either a blocking anti-PD-L2 antibody or an inhibitory anti-mouse GAL9 (108A2) antibody could reduce activated CD4+ in malaria-infected mice. + Secretion of pro-inflammatory cytokines in T cells.

[0463] Mouse model infected with malaria

[0464] A cohort of five C57BL / 6 mice was infected with the malaria strain P. yoelii 17XNL and cultured for 7 days to allow infection to occur. All animals used in the study were housed and cared for in accordance with the NHMRC's animal use guidelines.

[0465] Antibody

[0466] Using the blocking anti-mouse PD-L2 mAb clone TY25 (BioXCell, Lebanon, NH) or the inhibitory anti-mouse GAL9 clone 108A2 ( San Diego, CA.

[0467] Stimulation of cell isolation and co-culture

[0468] By using CD4 + T-cell isolation Miltenyi Biotec kit (Auburn, CA) and CD11c for DC isolation + Beads, CD4 isolated from mice infected with malaria + T cells and DC cells. Next, approximately 1 x 102 6 1 T cell and 2 x 10 5 The DCs were cultured in at least three copies of each well, and then incubated with 20 μg / ml of anti-PD-L2 mAb or 20 μg / ml of anti-Gal9 mAb for 36 hours.

[0469] Cytokine assay

[0470] After processing, use BD according to the manufacturer's instructions. TM Cytometric Bead Array (CBA) measures the concentration of IFN-γ or TNF-α.

[0471] Statistical analysis

[0472] Unpaired t-tests with Welch's correction were performed using GraphPad Prism (GraphPad Software).

[0473] Results / Conclusions

[0474] Figure 9A A bar chart showing the IFN-γ concentration detected in each treatment group is presented. Treatment with anti-PD-L2 or anti-GAL9 (108A2) resulted in a significant decrease in IFN-γ levels compared to the untreated co-culture control group.

[0475] Figure 9B A bar chart showing the TNF-α concentration detected in each treatment group is presented. Treatment with anti-PD-L2 or inhibitory anti-mouse GAL9 antibody (108A2) significantly reduced TNF-α levels compared to the untreated co-culture control group. An asterisk "*" indicates statistical significance with a p-value < 0.05 compared to the control group. Notably, treatment with anti-PD-L2 and anti-GAL9 (108A2) reduced IFN-γ and TNF-α to approximately the same concentration levels.

[0476] 6.11.8. Example 7: Exploration of the binding arm of human GAL9 (anti-human GAL9)

[0477] As described above, using a monoclonal phage ELISA format, chemically synthesized Fab phage libraries containing diverse FabCDRs were screened for the GAL9 antigen. Phage clones expressing Fabs that recognize GAL9 were sequenced.

[0478] The activity initially identified 52 GAL9 binding candidates (antigen-binding site clones). After the variable regions of these clones were reformatted into a bivalent monospecific human IgG1 format, functional assays were performed, identifying 30 antibodies with immunosuppressive properties.

[0479] Table 3 lists the VH CDR1 / 2 / 3 sequences from 30 repressive ABS clones, showing only the CDR residues that were altered during library construction. Table 4 lists the VL CDR1 / 2 / 3 sequences from the identified ABS clones; the light chain CDR1 and CDR2 sequences are unchanged, and only the CDR3 residues are changed during library construction.

[0480]

[0481]

[0482]

[0483] Table 5 lists the complete CDR sequences of human candidate inhibitory anti-GAL9 antibodies, based on several definitions generally accepted in the field.

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[0485]

[0486]

[0487]

[0488]

[0489]

[0490]

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[0507]

[0508]

[0509]

[0510]

[0511]

[0512]

[0513]

[0514]

[0515]

[0516]

[0517]

[0518] Table 6 lists the complete immunoglobulin heavy and light chain sequences, as well as VH and VL sequences, of various ABS candidates formatted as a bivalent monospecific full-length human IgG1 architecture.

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[0520]

[0521]

[0522]

[0523]

[0524]

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[0542]

[0543]

[0544]

[0545]

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[0547]

[0548]

[0549]

[0550] Binding characteristics of the selected GAL9 binding candidates were analyzed: cross-reactivity with mouse GAL9; qualitative binding; epitope binning (bin 2 - candidate bin with LS Bio's commercial antibody clone ECA8 [LS-C179448]; bin 3 - candidate bin with LS Bio's commercial antibody clone ECA42 [LS-C179449] (the "tool antibody" mentioned in Figure 10)); and monovalent affinity binding. The results are listed in Table 7.

[0551]

[0552]

[0553]

[0554] Further analysis was conducted on the sequence motifs of the selected GAL9 binding candidates, which could adversely affect antibody properties relevant to clinical development, such as stability, variability, and immunogenicity. Computational analyses were performed according to Kumar and Singh (Developability of biotherapeutics: computational approaches. Boca Raton: CRC Press, Taylor & Francis Group, 2016). The results are shown in Table 8, demonstrating the presence of a limited number of unfavorable sequence motifs in the listed clones, indicating potential for further clinical development.

[0555]

[0556] 6.11.9. Example 8: Effects of anti-human GAL9 candidate on cytokine production from peripheral blood mononuclear cells (PBMCs)

[0557] Candidate anti-human GAL9 antigen binding sites (ABS) were formatted as bivalent, monospecific, native 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 human PBMCs after peptide stimulation were tested. This was essentially in accordance with the procedures described in Section 6.11.1 above.

[0558] PBMCs were stimulated. In brief, PBMCs were collected from human donors known to respond to human CMV (HCMV), placed in culture medium, stimulated with HCMV PepMix to elicit an antigen-specific response, and treated with one of the following: control IgG, a contrast-activated anti-human GAL9 instrument mAb (clone ECA42, mouse IgG2a), α-PD1 (Nivolumab), or a candidate anti-GAL9 antibody formatted as a bivalent monospecific full-length human IgG1 antibody. Cytokine secretion was measured at 24 and 72 hours post-treatment using a bead-based cytokine array. Results for INF-γ and TNF-α were obtained in… Figure 10A and 10B The data shown in Figure 10 is described below.

[0559] More detailed descriptions are available in Tables 9 and 10.

[0560]

[0561]

[0562] 6.11.10. Example 9 ：Treatment with anti-human GAL9 IgG1 antibodies P9-11, P9-37, or P9-57 reduced the production of TNF-α and IFN-γ in activated PBMCs.

[0563] The inhibitory anti-human GAL9 candidate selected from Example 7 was formatted as a bivalent monospecific human...

[0564] The IgG1 antibody was further tested on three other human donor PBMCs to demonstrate its ability to inhibit cytokine production in PBMCs.

[0565] Stimulating PBMC

[0566] Primary human PBMCs were collected from donors 19, RCB, and RG, which are known to strongly respond to human CMV virus (HCMV). The PBMCs were stimulated essentially as described in Section 6.11.1 above. In short, PBMCs were collected from donors known to respond to human CMV virus (HCMV), placed in culture medium, stimulated with HCMV PepMix to elicit an antigen-specific response, and treated with bivalent monospecific full-length human IgG1 antibodies in the format of P9-41, P9-42, P9-53, P9-11, P9-37, or P9-57, or a human IgG control.

[0567] Cytokine assay

[0568] According to the manufacturer's instructions, use BD 24 hours and 72 hours after treatment. TM Cytometric Bead Array (CBA) was used to measure the secretion of TNF-α and IFN-γ. Measurements were performed in quadruplicate.

[0569] Results / Conclusions

[0570] Figure 11A-11C The chart displays representative data processed over 72 hours. The mean is represented by horizontal bars on the scatter plot. The error bars represent the standard deviation.

[0571] Figure 11A-11B Scatter plots of TNF-α levels after treatment with human IgG control (hlgG) and inhibitory anti-human GAL9 candidate are shown. Figures 11A-11B show scatter plots of TNF-α levels after treatment with human IgG control (hlgG) and inhibitory anti-human GAL9 candidate. Compared with the IgG control, treatment with P9-11, P9-37, or P9-57 formatted with human IgG1 antibody reduced TNF-α levels in PBMCs from all three human donors. Figure 11CA scatter plot of IFN-γ levels after treatment with human control IgG (hlgG) or an anti-human GAL9 candidate is shown. Treatment with P9-11, P9-37, or P9-57 reduced IFN-γ levels in PBMCs compared to the control group.

[0572] Treatment with P9-41, P9-42, or P9-53 resulted in neutral or weak secretion of TNF-α and IFN-γ (data not shown).

[0573] 6.11.11. Example 10 ： Treatment with anti-human GAL9 P9-11, P9-24, or P9-34 can reduce the production of TNF-α and INF-γ in activated PBMCs and increase the production of IL-10.

[0574] This study aimed to determine the effect of the inhibitory anti-human GAL9 candidate selected in Example 7 on activated human...

[0575] Effects of TNF-α, IFN-γ and IL-10 secretion in PBMCs.

[0576] Stimulating PBMC

[0577] PBMCs were stimulated in essentially the manner described in Section 6.11.1 above. In short, PBMCs were collected from human donors known to be highly responsive to human CMV virus (HCMV), placed in cultures, stimulated with HCMV PepMix to elicit an antigen-specific response, and treated with one of P9-11, P9-24, and P9-34 formatted as bivalent monospecific human IgG1 antibodies, or a human IgG control.

[0578] Cytokine assay

[0579] Use BD according to the manufacturer's instructions. TM Cell counting bead array (CBA) was used to measure the secretion of cytokines TNF-α, IFN-γ, and IL-10 72 hours after treatment.

[0580] Results / Conclusions

[0581] Figure 12A The bar chart shows TNF-α levels after treatment with control IgG (hlgG) or an inhibitory anti-human GAL9 candidate. Treatment with anti-human IgG1 P9-11, P9-24, or P9-34 resulted in [a decrease in TNF-α levels compared to the IgG control group].

[0582] PBMCs secrete less TNF-α. Figure 12BThe bar chart shows IFN-γ levels after treatment with control IgG (hlgG) or an inhibitory anti-GAL9 candidate. Compared with the IgG control, treatment with anti-human GAL9 antibodies P9-11, P9-24, or P9-34 reduced IFN-γ secretion by PBMCs. Figure 12C The bar chart shows IL-10 levels after treatment with an inhibitory anti-human GAL9 candidate or IgG control. Treatment with P9-11, P9-24, or P9-34 antibodies increased IL-10 secretion in PBMCs compared to the control group.

[0583] 6.11.12. Example 11 ： Activated CD3 cells were treated with anti-human GAL9 antibodies P9-11, P9-24, or P9-34. + T cells can improve the cytokine profile, while anti-mouse GAL9 (108A2) results in complete blockade of cytokine secretion.

[0584] We measured the secretion of IFN-γ, TNF-α, or IL-10 cytokines to determine the effects of anti-mouse GAL9 (clone 108A2) and anti-human GAL9 antibodies P9-11, P9-24, or P9-34 (formatted as human IgG1 antibody) on mouse activated CD3. + The influence of cytokine profiles in T cells.

[0585] Animals and CD3 + T cell isolation

[0586] Five mice were used in each treatment group. All animals used in the study were bred and cared for in accordance with the NHMRC's animal use guidelines.

[0587] Antibody

[0588] Antibodies P9-11, P9-24, and P9-34, formatted as bivalent monospecific human IgG1 antibodies, and human IgG controls were used. Additionally, an inhibitory anti-mouse GAL9 clone 108A2"mGAL9" was used. San Diego, CA.

[0589] CD3 + T cell stimulation

[0590] CD3 + T cells (CD90.2) + CD3 + It was isolated from the spleen of juvenile mice. Mouse CD3 was stimulated with anti-CD3 clone 145.2C11 (Aviva Systems Biology Corp. San Diego, CA) at 5 μg / ml. +T cells, concentration of which is [value missing]. Next, stimulated CD3 [cells / cells / etc.]... + T cells were treated with either an IgG control group or an inhibitory antibody at 20 μg / ml and cultured for 72 hours.

[0591] Cytokine assay

[0592] After 72 hours of treatment, use BD according to the manufacturer's instructions. TM Cytometric Bead Array (CBA) measures the concentration of IFN-γ, TNF-α, or IL-10.

[0593] Statistical analysis

[0594] Nonparametric unpaired t-tests were performed using GraphPad Prism (GraphPad Software).

[0595] Results / Conclusions

[0596] The results show Figure 13A and 13B In this study, a decreased TNF-α:IL-10 or INF-γ:IL-10 ratio indicated a reduction in pro-inflammatory cytokines and an increase in the inhibitory cytokine IL-10. Treatment with anti-mouse GAL9 (108A2) antibody significantly reduced the secretion of TNF-α, IFN-γ, and IL-10. (See [link to relevant documentation]). Figure 13A Conversely, treatment with anti-human GAL9 antibodies P9-11, P9-24, or P9-34 (human IgG1 Fc) did not reduce TNF-α or IFN-γ secretion, while IL-10 secretion significantly increased. See Figure 13B An asterisk "*" indicates statistical significance (P < 0.05) compared to the control group.

[0597] Treatment with anti-human P9-11 and P9-24 antibodies (in human IgG1 format) resulted in an improved inflammatory environment, reduced TNF-α and IFN-γ secretion, and increased IL-10 secretion. Notably, treatment with anti-mouse GAL9 (108A2) led to complete blockade of cytokine responses, including IL-10 secretion. The difference in cytokine profiles produced by anti-human GAL9 and anti-mouse GAL9 (108A2) antibodies suggests that they have different mechanisms of action.

[0598] 6.11.13. Example 12: Treatment with anti-human GAL9 did not substantially alter the CD4+ and CD8+ of the stimulus. + The expression of immune checkpoint molecules in T cells and the reduction of CD8 + 4-1BB, CD40L, and OX40 co-stimulatory molecules in T cells

[0599] This study aimed to determine the effects of anti-human GAL9 candidates P9-11, P9-24, and P9-34 on stimulated CD8+. + and CD4 + The effect of anti-human GAL9 p9-11 on the expression of selected checkpoint molecules in T cells, and the effect of anti-human GAL9 p9-11 on stimulated CD8 + The effect of selected co-stimulatory molecules in T cells.

[0600] Stimulation and treatment

[0601] The stimuli mentioned above include CD8+ or CD4. + PBMCs of the T cell population were treated with anti-human GAL9 P9-11, P9-24, P9-34 or human IgG controls formatted as bivalent monospecific human IgG1 antibodies.

[0602] Immunomarkers

[0603] PMBCs in 5x10 6 Cells / mL were resuspended in RPMI containing 10% FBS. 200 μL of the resuspended cells were distributed equally into 96-well plates and then treated with Fixable Viability Dye. 780 was stained at 2–8 °C for 30 min to irreversibly label dead cells. Cells were then washed and incubated at room temperature with human Fc blocking solution (Cat. No. 14-9161-73, eBiosciences) for 10 min. Surface expression of PD-L1, PD-1, CTLA-4, TIM3, LAG3, 4-1BB, CD27, CD40L, ICOS, or OX40 was assessed by flow cytometry.

[0604] Flow cytometry

[0605] Flow cytometry analysis was performed using a BD LSR Fortessa flow cytometer and BD FACSDiva software (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). At least 5 × 10⁶ cells were collected for each sample. 5 One event.

[0606] CD4, which is positive for immune checkpoint molecules + or CD8 + Representative data on the percentage of T cells are listed in Tables 11 and 12 below. For co-stimulatory molecule-positive CD8... + The percentage of T cells is shown in Table 13 below.

[0607] "% value" represents the percentage of cells with a detectable level of the specified marker. "(x)" represents the fold change after treatment with the selected α-GAL9 candidate antibody compared to the human IgG control.

[0608]

[0609]

[0610]

[0611] Results / Conclusions

[0612] In stimulated CD8 + or CD4 + In T cells, the expression of any immune checkpoint molecules did not change substantially. However, we observed changes in the expression of stimulated CD8+ cells. + In T cells, the co-stimulatory molecules 4-1BB, CD40L, and OX40 were reduced. These results indicate that the effect of anti-human GAL9 candidates on cytokine responses is driven by GAL9 inhibition, and not through the PD-1 / PD-L1 immune checkpoint pathway or other checkpoint molecules such as CTLA-4, TIM3, or LAG3.

[0613] 7. Equivalent forms

[0614] While various specific embodiments have been described and illustrated, the foregoing specification is not restrictive. It is understood that various changes can be made without departing from the spirit and scope of the invention. Many variations will be apparent to those skilled in the art upon reading this specification.

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, wherein the first antigen-binding site comprises all three VH kabat CDRs and all three VL kabat CDRs from any of the Ab clones listed below:

2. The GAL9 antigen-binding molecule of claim 1, comprising a first ABS specific to a first epitope of a first GAL9 antigen, wherein the first antigen-binding site comprises: (a) The VL sequence as shown in SEQ ID NO: 1211 and the VH sequence as shown in SEQ ID NO: 1210; or (b) The VL sequence as shown in SEQ ID NO: 1227 and the VH sequence as shown in SEQ ID NO: 1226.

3. The GAL9 antigen-binding molecule of claim 1, wherein the first ABS further comprises a first IgG heavy chain polypeptide and a first light chain polypeptide.

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

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

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

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

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

9. The GAL9 antigen-binding molecule of claim 5, wherein the second ABS comprises all three VH kabat CDRs and all three VL kabat CDRs from another Ab clone selected from P9-01, P9-02A, P9-03, P9-06, P9-07, P9-12, P9-14, P9-23, P9-25, P9-29, P9-30, P9-37, P9-40, P9-41, P9-42, P9-43, P9-44, P9-45, P9-46, P9-50, P9-51, P9-52, P9-53, P9-56, and P9-57:

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

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

12. The GAL9 antigen-binding molecule of claim 5, wherein the second antigen-binding site is specific to an antigen different from the first GAL9 antigen.

13. The GAL9 antigen-binding molecule of any one of claims 1-4, 6-8 and 10-12, wherein the GAL9 antigen-binding molecule comprises an antibody format selected from the group consisting of: full-length antibody, Fab fragment, variable region fragment (Fv), single-chain variable region fragment (scFv), tandem scFv, diabody, scDiabody, DART, tandAb, minibody and B-body.

14. The GAL9 antigen-binding molecule of any one of claims 1-4, 6-8 and 10-12, wherein the GAL9 antigen-binding molecule reduces TNF-α secretion from activated immune cells upon contact, wherein the reduction is at least 30%, 35%, 40%, 45%, 50%, 55% or 60% relative to activated immune cells treated with a control agent.

15. The GAL9 antigen-binding molecule according to any one of claims 1-4, 6-8 and 10-12, wherein the GAL9 antigen-binding molecule reduces IFN-γ secretion of activated immune cells upon contact, wherein the reduction is at least 20%, 25%, 30%, 35%, 40%, 45% or 50% relative to activated immune cells treated with a control agent.

16. The GAL9 antigen-binding molecule of any one of claims 1-4, 6-8, and 10-12, wherein the GAL9 antigen-binding molecule increases the secretion of IL-10 by activated immune cells upon contact, wherein, The increase is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40% relative to activated immune cells treated with a control.

17. The GAL9 antigen-binding molecule according to any one of claims 1-4, 6-8 and 10-12, wherein the GAL9 antigen-binding molecule does not regulate PD-1 surface expression on activated immune cells relative to activated immune cells treated with a control agent.

18. The GAL9 antigen-binding molecule of any one of claims 1-4, 6-8 and 10-12, wherein the GAL9 antigen-binding molecule does not regulate PD-L1 surface expression on activated immune cells relative to activated immune cells treated with a control agent.

19. The GAL9 antigen-binding molecule of any one of claims 1-4, 6-8 and 10-12, wherein the GAL9 antigen-binding molecule does not regulate CTLA-4 surface expression on activated immune cells relative to activated immune cells treated with a control agent.

20. The GAL9 antigen-binding molecule of any one of claims 1-4, 6-8 and 10-12, wherein the GAL9 antigen-binding molecule does not regulate TIM-3 surface expression on activated immune cells relative to activated immune cells treated with a control agent.

21. The GAL9 antigen-binding molecule of any one of claims 1-4, 6-8 and 10-12, wherein the GAL9 antigen-binding molecule does not regulate LAG-3 surface expression on activated immune cells relative to activated immune cells treated with a control agent.

22. The GAL9 antigen-binding molecule of any one of claims 1-4, 6-8, and 10-12, wherein it is relative to CD8 treated with a control agent. + T cells, GAL9 antigen-binding molecule reduces CD8 + 4-1BB is expressed on the surface of T cells.

23. The GAL9 antigen-binding molecule of any one of claims 1-4, 6-8, and 10-12, wherein it is relative to CD8 treated with a control agent. + T cells, GAL9 antigen-binding molecule reduces CD8 + CD40L expression on the surface of T cells.

24. The GAL9 antigen-binding molecule of any one of claims 1-4, 6-8, and 10-12, wherein it is relative to CD8 treated with a control agent. + T cells, GAL9 antigen-binding molecule reduces CD8 + OX40 surface expression on T cells.

25. The GAL9 antigen-binding molecule of claim 14, wherein the control agent is a negative control or a positive control.

26. The GAL9 antigen-binding molecule of claim 25, wherein the control agent is a control antibody.

27. The GAL9 antigen-binding molecule of claim 26, wherein 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, 108A2 clone anti-GAL9 antibody and isotype control antibody without GAL9 binding.

28. The GAL9 antigen-binding molecule of claim 14, wherein the activated immune cells are activated by peptide stimulation, anti-CD3, or dendritic cell activation.

29. The GAL9 antigen-binding molecule of any one of claims 1-4, 6-8, 10-12 and 25-28, wherein it is purified.

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