Anti-gal3 antibodies and uses thereof

Abstract

To provide antibodies that specifically bind to Gal3 and methods of use thereof.SOLUTION: In some embodiments, provided are methods of inducing immune activation or promoting T cell or Natural Killer cell proliferation with an antibody that specifically binds to Gal3; methods and compositions of reducing fibrosis or propensity thereof in a tissue with antibodies that specifically bind to Gal3; or an anti-Gal3 antibody that disrupts the interaction between Gal3 and TIM-3.SELECTED DRAWING: Figure 7B

Description

[Technical field]

[0001] Related Technology This application claims the benefit of U.S. Provisional Patent Application No. 62 / 798,945, filed January 30, 2019, and U.S. Provisional Patent Application No. 62 / 798,949, filed January 30, 2019, each of which is incorporated by reference in its entirety.

[0002] Array list reference This application has been filed with a sequence listing in electronic format. The sequence listing has been submitted as a sequence listing file named IMMUT003.TXT, created and last saved on January 29, 2020, which is 146,561 bytes in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.

[0003] Technical Field In some embodiments, disclosed herein are antibodies (or "anti-Gal3 antibodies") that specifically bind Gal3, disrupt the interaction of Gal3 with TIM-3, and promote T cell or natural killer (NK) cell proliferation. Also disclosed herein are methods of using antibodies to induce an immune response and methods of treatment. Disclosed herein are methods of reducing fibrosis or a propensity for fibrosis in a tissue by contacting the tissue with an antibody that specifically binds Gal3. Also described herein are methods of disrupting Gal3-TIM-3 interaction with an antibody that specifically binds Gal3 under conditions that reduce the expression of one or more fibrosis biomarkers in the tissue. [Background technology]

[0004] Galectin 3 (Gal3) is a lectin, or carbohydrate-binding protein, with specificity for β-galactosides. In human cells, Gal3 is expressed and can be found in the nucleus, cytoplasm, cell surface, and extracellular space. T-cell immunoglobulin and mucin-domain-containing-3 (TIM-3) is a protein expressed on immune cells such as T cells, dendritic cells, NK cells, and monocytes. Summary of the Invention

[0005] In some embodiments, disclosed herein are antibodies that specifically bind to Gal3 and disrupt the interaction of Gal3 with TIM-3 (or "anti-Gal3 antibodies"). In some embodiments, disclosed herein are antibodies that specifically bind to Gal3 and promote T cell or natural killer cell proliferation. Also disclosed herein in some embodiments are methods of using antibodies to elicit an immune response and methods of treatment.

[0006] The embodiments of the invention provided herein are described as the following numbered options: 1. A method for inducing immune activation, the method comprising: contacting a plurality of cells, including Gal3-expressing cells and TIM-3-expressing cells, with an antibody under conditions that disrupt the interaction between Gal3 and TIM-3; The antibody specifically binds to Gal3, and upon binding to the antibody, the Gal3-expressing cells express cytokines that induce immune activation, and the antibody is not IMT001. 2. The method of option 1, wherein the cytokine is an interferon. 3. The method of option 2, wherein the interferon is IFNγ. 4. The method of option 3, wherein the IFNγ production is 150%, 160%, 170%, 180%, 190%, 200% or more IFNγ production by isotype antibody. 5. The method of option 1, wherein the cytokine is an interleukin. 6. The method of option 5, wherein the interleukin is IL-2. 7. The method of any one of options 1-6, wherein the immune activation comprises proliferation of CD3+ T lymphocytes, CD4+ T helper cells, CD8+ cytotoxic T cells, natural killer cells, or a combination thereof. 8. The method of any one of options 1 to 7, wherein the immune activation comprises an increase in the M1 macrophage population within a plurality of cells. 9. The method of any one of options 1 to 8, wherein the immune activation comprises a reduction in the M2 macrophage population within a plurality of cells. 10. A method for promoting T cell or natural killer (NK) cell proliferation, the method comprising: 16. A method comprising contacting a plurality of cells, the plurality of cells comprising T cells, NK cells, and Gal3-expressing cells, with an antibody under conditions that affect proliferation of T cells and / or NK cells within the plurality of cells, wherein the antibody specifically binds to Gal3, and the antibody is not IMT001. 11. The method of claim 10, wherein the plurality of cells further comprises TIM-3 expressing cells. 12. The method of option 11, wherein the antibody further disrupts the interaction of Gal3 with TIM-3. 13. A method for inducing immune activation, the method comprising: A method comprising contacting a plurality of cells, including Gal3-expressing cells and TIM-3-expressing cells, with an antibody under conditions that disrupt the interaction between Gal3 and TIM-3, wherein the antibody specifically binds to Gal3, and the Gal3-TIM-3 interaction is reduced to less than 70%, less than 60%, less than 59%, less than 50%, less than 40%, less than 34%, less than 30%, less than 20%, less than 14%, less than 10%, less than 7%, less than 5%, less than 4%, or less than 1%. 14. The method of option 13, wherein the interaction occurs at one or more residues of Gal3 selected from regions 145-168, 160-177, or 165-184, and the residue positions correspond to positions 145-168, 160-177, or 165-184 of SEQ ID NO:1. 15. The method of option 13, wherein the interaction occurs at one or more residues of Gal3 selected from regions 149-156, 152-168, 163-169, 163-177, or 163-171, and wherein the residue positions correspond to positions 149-156, 152-168, 163-169, 163-177, or 163-171 of SEQ ID NO:1. 16. The method of any one of options 13-15, wherein the interaction occurs at one or more residues of TIM-3 selected from regions 91-111 or 82-111, and the residue positions correspond to positions 91-111 or 82-111 of SEQ ID NO:2. 17. The method of any one of options 13-15, wherein the interaction occurs at one or more residues of TIM-3 selected from regions 91-111, 107-117, 96-102, 100-106, or 92-119, and wherein the residue positions correspond to positions 91-111, 107-117, 96-102, 100-106, or 92-119 of SEQ ID NO:2. 18. The method of any one of options 13 to 17, wherein the TIM-3 is human TIM-3. 19. The method of any one of options 1 to 18, wherein the Gal3-expressing cell is a tumor cell. 20. The method of any one of options 1 to 19, wherein the plurality of cells are present within the tumor microenvironment (TME). 21. Any of options 1 to 20, wherein the antibody induces a reduction in tumor cells in the TME. The method according to any one of claims 1 to 5. 22. The method of any one of options 1 to 21, wherein the plurality of cells further comprises tumor infiltrating lymphocytes (TILs). 23. The method of any one of options 1-22, wherein the plurality of cells further comprises CD3+ T lymphocytes, CD4+ T helper cells, CD8+ cytotoxic T cells, or a combination thereof. 24. The method of any one of options 1, 10, 13, or 22, wherein said contacting further induces TIL proliferation. 25. The method of any one of options 1, 10, 13, or 23, wherein the contacting further induces proliferation of CD3+ T lymphocytes, CD4+ T helper cells, CD8+ cytotoxic T cells, or a combination thereof. 26. The method of any one of options 1, 10, 13, or 22-25, wherein the contacting further comprises increasing proliferation of M1 macrophages. 27. The method of any one of options 1, 10, 13, or 22-26, wherein the contacting further comprises reducing the M2 macrophage population within the TME. 28. The method of any one of options 1-27, wherein the antibody binds to at least one amino acid residue in the Gal3 region corresponding to residues 1-20 of SEQ ID NO:1. 29. The method of any one of options 1-27, wherein the antibody binds to at least one amino acid residue within the Gal3 region corresponding to residues 41-91 of SEQ ID NO:1. 30. The method of any one of options 1-27 or 29, wherein the antibody binds to at least one amino acid residue within the Gal3 region corresponding to residues 41-71 of SEQ ID NO:1. 31. The method of any one of options 1-27 or 29, wherein the antibody binds to at least one amino acid residue within the Gal3 region corresponding to residues 71-91 of SEQ ID NO:1. 32. The method of any one of options 1-31, wherein the antibody binds to at least one amino acid residue in peptide_1, peptide_4, peptide_5, peptide_6, peptide_7, or peptide_8. 33. The antibody has a K of less than 1 nM, 1.2 nM, 2 nM, 5 nM, 10 nM, 13.5 nM, 15 nM, 20 nM, 25 nM, or 30 nM. D The method according to any one of options 1 to 32, comprising: 34. The method of any one of options 1-33, wherein the antibody comprises a humanized antibody. 35. The method of any one of options 1-34, wherein the antibody comprises a full-length antibody or a binding fragment thereof. 36. The method of any one of options 1-35, wherein the antibody comprises a bispecific antibody or a binding fragment thereof. 37. The method of any one of options 1-36, wherein the antibody comprises a monovalent Fab', a bivalent Fab2, a single chain variable fragment (scFv), a diabody, a minibody, a nanobody, a single domain antibody (sdAb), or a camelid antibody or binding fragment thereof. 38. The method of any one of options 1-37, wherein the antibody comprises an IgG framework. 39. The method of any one of options 1-38, wherein the antibody comprises an IgG1, IgG2, or IgG4 framework. 40. The method of any one of options 1-39, wherein the antibody further comprises an Fc mutation. 41. The method of any one of options 1-33 or 35-40, wherein the antibody comprises a chimeric antibody. 42. The method of any one of options 1, 10, or 13, wherein the method further comprises administering the antibody to a subject prior to the contacting step. 43. The method of option 42, wherein the subject has been diagnosed with cancer. 44. The method of option 43, wherein the cancer is a solid tumor. 45. The method of option 44, wherein the cancer is breast cancer, colorectal cancer, renal cancer, liver cancer, or lung cancer. 46. ​​The method of option 43, wherein the cancer is a hematological tumor. 47. The method of any one of options 43 to 46, wherein the cancer is a metastatic cancer. 48. The method of any one of options 43 to 46, wherein the cancer is a recurrent or refractory cancer. 49. The method of any one of options 42-48, wherein the antibody is formulated for systemic administration. 50. The method of any one of options 42 to 49, wherein the antibody is formulated for parenteral administration. 51. The method of any one of options 42 to 50, wherein the antibody is administered in combination with an additional therapeutic agent. 52. The method of option 51, wherein the antibody and the additional therapeutic agent are administered simultaneously. 53. The method of option 51, wherein the antibody and the additional therapeutic agent are administered sequentially. 54. The method of option 53, wherein the antibody is administered prior to administration of the additional therapeutic agent. 55. The method of option 53, wherein the antibody is administered after the additional therapeutic agent is administered. 56. The method of any one of options 51 to 55, wherein the additional therapeutic agent comprises an immune checkpoint modulator. 57. The method of any one of options 51-55, wherein the additional therapeutic agent comprises a chemotherapeutic agent, a targeted therapeutic agent, a hormonal therapeutic agent, or a stem cell-based therapeutic agent. 58. The method of any one of options 1 to 57, wherein the subject is a human. 59. The method of option 58, wherein the antibody is administered either before or after surgery. 60. The method of option 58, wherein the antibody is administered concomitantly with, prior to, or after radiation therapy. 61. The antibody is the K of antibody IMT001. D Higher K D Option 1 to option 60, comprising the method of any one of options 1 to 60. 62. A method for reducing fibrosis or a tendency thereof in a tissue, the method comprising: contacting the tissue with an antibody that specifically binds to the Gal3 antibody under conditions such that the expression level of a fibrosis biomarker is reduced in the tissue. 63. The method of option 62, wherein the tissue further comprises TIM-3-expressing cells. 64. The method of option 63, wherein the antibody further disrupts the interaction between Gal3 and TIM-3. 65. The method of option 63, wherein the antibody does not interfere with the interaction between Gal3 and TIM-3. 66. The method of any one of options 62 to 65, wherein the at least one fibrosis biomarker includes alpha-smooth muscle actin (alpha-SMA). 67. The method of any one of options 62 to 65, wherein the at least one fibrosis biomarker comprises fibronectin. 68. The method of any one of options 62 to 65, wherein the at least one fibrosis biomarker includes alpha-smooth muscle actin (alpha-SMA) and fibronectin. 69. The method of any one of options 62 to 68, wherein the tissue is renal tissue or liver tissue. 70. The tissue is selected from the group consisting of liver tissue, kidney tissue, skin tissue, lung tissue, heart tissue, brain tissue, intestinal tissue, The method of any one of options 62-68, wherein the tissue is selected from the group consisting of bone marrow tissue, and soft tissue. 71. The method of any one of options 62-70, wherein expression of the at least one fibrosis biomarker in the tissue treated with the antibody is less than expression of the at least one fibrosis biomarker in a control tissue treated with an mIgG2b antibody. 72. The method of any one of options 62-71, wherein the antibody results in a reduction in accumulation of extracellular matrix proteins in the tissue. 73. The method of option 72, wherein the extracellular matrix protein comprises collagen. 74. The method of option 73, wherein the tissue comprises collagen-producing cells. 75. The method of option 74, wherein the collagen-producing cells are fibroblasts. 76. The method of option 75, wherein the fibroblasts are activated with a fibrogenic cytokine. 77. The method of option 76, wherein the fibrogenic cytokine is TGF-β1. 78. The method of any one of options 62 to 77, wherein the tissue has high expression of TGF-β1. 79. The method of any one of options 62-78, wherein the antibody comprises a humanized antibody. 80. The method of any one of options 62-79, wherein the antibody comprises a full-length antibody or a binding fragment thereof. 81. The method of any one of options 62-79, wherein the antibody comprises a bispecific antibody or a binding fragment thereof. 82. The method of any one of options 62-79, wherein the antibody comprises a chimeric antibody. 83. The method of any one of options 62-82, wherein the antibody binds to at least one amino acid residue in the Gal3 region corresponding to residues 1-20 of SEQ ID NO:1. 84. The method of any one of options 62-82, wherein the antibody binds to at least one amino acid residue in the Gal3 region corresponding to residues 41-91 of SEQ ID NO:1. 85. The method of any one of options 62-82 or 84, wherein the antibody binds to at least one amino acid residue in the Gal3 region corresponding to residues 41-71 of SEQ ID NO:1. 86. The method of any one of options 62-82 or 84, wherein the antibody binds to at least one amino acid residue in the Gal3 region corresponding to residues 71-91 of SEQ ID NO:1. 87. The method of any one of options 62 to 86, wherein the antibody binds to at least one amino acid residue in peptide_1, peptide_4, peptide_5, peptide_6, peptide_7, or peptide_8. 88. The antibody has a K of less than 1 nM, 1.2 nM, 2 nM, 5 nM, 10 nM, 13.5 nM, 15 nM, 20 nM, 25 nM, or 30 nM. D The method according to any one of options 62 to 87, comprising: 89. The method of any one of options 62-88, wherein the antibody comprises a monovalent Fab', a bivalent Fab2, a single chain variable fragment (scFv), a diabody, a minibody, a nanobody, a single domain antibody (sdAb), or a camelid antibody or binding fragment thereof. 90. The method of any one of options 62-89, wherein the antibody comprises an IgG framework. 91. The method of any one of options 62-90, wherein the antibody comprises an IgG1, IgG2, or IgG4 framework. 92. The antibody of any one of options 62 to 91, further comprising an Fc mutation. method. 93. The method of any one of options 62-92, wherein the method further comprises administering the antibody to a subject prior to the contacting step. 94. The method of option 93, wherein the subject has been diagnosed with a fibrotic disease. 95. The method of option 94, wherein the fibrotic disease is renal fibrosis. 96. The method of option 94, wherein the fibrotic disease is liver fibrosis. 97. The method of any one of options 93 to 96, wherein the antibody is formulated for systemic administration. 98. The method of any one of options 93 to 96, wherein the antibody is formulated for parenteral administration. 99. The method of any one of options 93 to 98, wherein the subject is a mammal. 100. The method of any one of options 64 and 66-99, wherein the Gal3-TIM-3 interaction is reduced to less than 70%, less than 60%, less than 59%, less than 50%, less than 40%, less than 34%, less than 30%, less than 20%, less than 14%, less than 10%, less than 7%, less than 5%, less than 4%, or less than 1%. 101. The method of option 100, wherein the interaction occurs at one or more residues of Gal3 selected from regions 145-168, 160-177, or 165-184, and the residue positions correspond to positions 145-168, 160-177, or 165-184 of SEQ ID NO:1. 102. The method of option 100, wherein the interaction occurs at one or more residues of Gal3 selected from regions 149-156, 152-168, 163-169, or 163-171, and the residue positions correspond to positions 149-156, 152-168, 163-169, or 163-171 of SEQ ID NO:1. 103. A method according to any one of options 100 to 102, wherein the interaction occurs at one or more residues of TIM-3 selected from regions 90 to 122 or 82 to 111, and the residue positions correspond to positions 90 to 122 or 82 to 111 of SEQ ID NO:2. 104. The method of any one of options 100-102, wherein the interaction occurs at one or more residues of TIM-3 selected from regions 91-111, 107-117, 96-102, 100-106, or 92-119, and wherein the residue positions correspond to positions 91-111, 107-117, 96-102, 100-106, or 92-119 of SEQ ID NO:2. 105. An anti-Gal3 antibody for use in treating an immune-related disease in a subject, wherein said anti-Gal3 antibody induces activation of the immune system. 106. An anti-Gal3 antibody for use in the treatment of an immune-related disease according to option 105, wherein the anti-Gal3 antibody inhibits the interaction between Gal3 and TIM-3. 107. An anti-Gal3 antibody for use in the treatment of an immune-related disease according to option 105 or 106, wherein the activation of the immune system comprises proliferation of CD3+ T lymphocytes, CD4+ T helper cells, CD8+ cytotoxic T cells, NK cells, M1 macrophages, or a combination thereof. 108. An anti-Gal3 antibody for use in the treatment of an immune-related disease according to any one of options 105 to 107, wherein the activation of the immune system comprises a reduction in M2 macrophages. 109. An anti-Gal3 antibody for use in the treatment of an immune-related disease according to any one of options 105 to 108, wherein the immune-related disease is cancer. 110. An anti-Gal3 antibody for use in the treatment of an immune-related disease according to option 109, wherein the cancer is breast cancer, colorectal cancer, renal cancer, liver cancer, lung cancer, or a hematological tumor. 111. An anti-Gal3 antibody for use in the treatment of an immune-related disease according to option 109 or 110, wherein the cancer is a metastatic cancer, a recurrent cancer, or a refractory cancer. 112. An anti-Gal3 antibody for use in treating an immune-related disease according to any one of options 109 to 111, wherein the anti-Gal3 antibody is administered in combination with an additional therapeutic agent, such as an immune checkpoint modulator, a chemotherapeutic agent, a targeted therapeutic agent, a hormonal therapeutic agent, a stem cell-based therapeutic agent, surgery, or radiation therapy. 113. An anti-Gal3 antibody for use in the treatment of an immune-related disease according to any one of options 105 to 108, wherein the immune-related disease is fibrosis and the anti-Gal3 antibody results in a reduction in the accumulation of extracellular matrix proteins in tissues. 114. An anti-Gal3 antibody for use in the treatment of an immune-related disease according to option 113, wherein the extracellular matrix protein comprises collagen. 115. An anti-Gal3 antibody for use in treating an immune-related disease according to option 113 or 114, wherein the expression level of the at least one fibrosis biomarker in the subject is reduced, and the at least one fibrosis biomarker comprises α-SMA, fibronectin, or both. 116. An anti-Gal3 antibody for use in treating an immune-related disease according to any one of options 113 to 115, wherein the tissue is selected from the group consisting of liver tissue, kidney tissue, skin tissue, lung tissue, heart tissue, brain tissue, intestinal tissue, bone marrow tissue, and soft tissue. 117. An anti-Gal3 antibody for use in treating an immune-related disease according to any one of options 113 to 116, wherein the fibrosis is renal fibrosis, hepatic fibrosis, pulmonary fibrosis, cardiac fibrosis, or vascular fibrosis. In some embodiments, this may be administered intravenously or subcutaneously. 118. An anti-Gal3 antibody for use in treating an immune-related disease according to any one of options 105 to 117, wherein the anti-Gal3 antibody is formulated for systemic, parenteral, intravenous or subcutaneous administration. 119. An anti-Gal3 antibody for use in treating an immune-related disease according to any one of options 105 to 118, wherein the subject is a human. 120. The anti-Gal3 antibody is selected from the group consisting of 2D10.2B2, 3B11.2G2, 4A11.2B5, 4G2.2G6, 6H6.2D6, 7D8.2D8, 12G5.D7, 13A12.2E5, 13G4.2F8, 13H12.2F8, 14H10.2C9, 15F10.2D6, 15G7.2A7, 19B5.2E6, 19D9.2E5, and 20D11. The method of any one of options 1 to 104, wherein the nucleic acid is selected from the group consisting of one or more of: .2C6, 20H5.A3, 23H9.2E4, 24D12.2H9, 846.1F5, 846.2H3, 846T.1H2, 9H2.2H10, IMT001-4, IMT006-1, IMT006-5, IMT006-8, and mIMT001. 121. The anti-Gal3 antibody is selected from the group consisting of 2D10.2B2, 3B11.2G2, 4A11.2B5, 4G2.2G6, 6H6.2D6, 7D8.2D8, 12G5.D7, 13A12.2E5, 13G4.2F8, 13H12.2F8, 14H10.2C9, 15F10.2D6, 15G7.2A7, 19B5.2E6, 19D9.2E5, 20D11.2C6, and 20H5.A. 3, 23H9.2E4, 24D12.2H9, 846.1F5, 846.2H3, 846T.1H2, 9H2.2H10, IMT001-4, IMT006-1, IMT006-5, IMT006-8, and mIMT001. 121. The method of any one of options 1 to 104, wherein the anti-Gal3 antibody is IMT001-4, IMT006-1, IMT006-5, or IMT006-8. 122. The anti-Gal3 antibody is selected from the group consisting of 2D10.2B2, 3B11.2G2, 4A11.2B5, 4G2.2G6, 6H6.2D6, 7D8.2D8, 12G5.D7, 13A12.2E5, 13G4.2F8, 13H12.2F8, 14H10.2C9, 15F10.2D6, 15G7.2A7, 19B5.2E6, 19D9.2E5, 20D11.2C6, 20H5.A3, 23H9.2E4, 24D12.2H9, 846.1F5, 846.2H3, 846T.1H2, 9H2.2H10, IMT001-4, IMT006-1, An anti-Gal3 antibody for use in treating an immune-related disease according to any one of options 105 to 119, selected from the group consisting of IMT006-5, IMT006-8, and mIMT001. 123. An anti-Gal3 antibody for use in treating an immune-related disease according to any one of options 105 to 119, wherein the anti-Gal3 antibody is IMT001-4, IMT006-1, IMT006-5, or IMT006-8. 124. An anti-GAL3 antibody, the anti-GAL3 antibody comprising at least an HCDR3 in any one of the antibodies shown in Figures 35A to 36B. 125. The anti-GAL3 antibody of option 124, further comprising all three HCDRs of any one of the antibodies of Figures 35A-36B. 126. The anti-GAL3 antibody of option 125, further comprising all three LCDRs of any one of the antibodies of Figures 35A-36B. 127. An anti-Gal3 antibody, wherein the anti-Gal3 antibody comprises any one of the heavy chain sequences in Figure 36A, or a sequence having at least 80% identity thereto. 128. An anti-Gal3 antibody, wherein the anti-Gal3 antibody comprises any one of the light chain sequences in Figure 36B, or a sequence having at least 80% identity thereto. 129. The anti-GAL3 antibody of claim 128, further comprising any one of the heavy chain sequences in Figure 36A, or a sequence having at least 80% identity thereto. 130. An anti-Gal3 antibody comprising 6 CDRs, said 6 CDRs having at least 80% identity with any set of 6 CDRs in Figures 35A and 35B across their combined sequences. 131. An anti-GAL3 antibody, said anti-GAL3 antibody comprising at least one of the CDRs from FIG. 38. 132. An anti-GAL3 antibody, said anti-GAL3 antibody comprising at least two of the CDRs from FIG. 38. 133. An anti-GAL3 antibody, said anti-GAL3 antibody comprising at least three of the CDRs from FIG. 38. 134. An anti-GAL3 antibody, said anti-GAL3 antibody comprising at least four of the CDRs from FIG. 38. 135. An anti-GAL3 antibody, said anti-GAL3 antibody comprising at least five of the CDRs from FIG. 38. 136. An anti-GAL3 antibody, said anti-GAL3 antibody comprising six of the CDRs from FIG. 38. 137. An anti-GAL3 antibody, wherein the anti-GAL3 antibody comprises six of the CDRs from Figure 38, and all six are from a single bin. 138. An anti-GAL3 antibody, said anti-GAL3 antibody comprising six of the CDRs from Figure 38, or a set of six CDRs having at least 80% identity thereto over their entire sequences.

[0007] In addition to the above figures, further views and variations will be readily apparent from the following description of the drawings and illustrative embodiments. It should be understood that these drawings depict typical embodiments and are not intended to be limiting in scope. [Brief description of the drawings]

[0008] [Figure 1A] Figures 1A-C illustrate the results of a co-immunoprecipitation assay showing that human Gal3 (hGal3) specifically pulled down human TIM-3 (hTIM-3). Figure 1A shows TIM-3 expression in 293T cells co-transfected with a plasmid encoding HA-tagged hTIM-3 and plasmids encoding hGal3, hGal9, or hCEACAM1. [Figure 1B] FIG. 1B shows the expression of hGal9, hGal3, or hCEACAM1. [Figure 1C]Figure 1C shows that hGal3, but not CEACAM1, pulled down HA-tagged hTIM-3 in cotransfected 293T cells. The results show that human Gal9 (hGal9) pulled down hTIM-3, but did so together with protein aggregates (Figure 1B), indicating that the binding of hGal9 to hTIM-3 was likely nonspecific. [Diagram 2] The results of a pull-down assay using a fusion protein consisting of the hTIM-3 extracellular domain fused to the Fc portion of hIgG (hTIM-3 Fc) are shown. The results indicate that the binding of Gal3 to TIM-3 was specific. As shown in this figure, hTIM-3 Fc, but not hFc or hPD1 Fc, pulled down overexpressed Flag-tagged hGal3 protein from 293T cells. [Diagram 3] Figure 1 shows the results of a cell adhesion assay demonstrating the specific interaction of hGal3 with hTIM-3. As shown in the figure, significantly more A20 cells expressing hGal3 (A20 Gal3 cells) were able to adhere to plates coated with hTIM-3 Fc than to plates coated with hVISTA Fc or hPD1 Fc. The results also show that more A20 PDL1 cells were able to adhere to plates coated with hPD1 Fc than to plates coated with human VISTA Fc (hVISTA Fc) or hTIM-3 Fc. [Figure 4A] FIG. 4A shows live (left peak) and dead (right peak) A20 cells by flow cytometry analysis. [Figure 4B]Figure 4B-C shows the results of flow cytometry analysis of live (Figure 4B) and dead (Figure 4C) cells stained with anti-hFc APC antibody. In group 1, A20 Gal3 cells were incubated without mTIM-3 Fc protein as a control; in group 2, A20 Gal3 cells were incubated with mTIM-3 Fc protein; in groups 3, 4, and 5, in addition to mTIM-3 Fc protein, anti-mouse TIM-3 polyclonal antibody (R&D System, Minneapolis, MN) (group 3), monoclonal antibody RMT3-23 (Bio X Cell, West Lebanon, NH) (group 4), and monoclonal antibody 215015 (R&D System) (group 5) were added to test whether these antibodies could block the binding of Gal3 to Tim3. [Figure 4C] Figure 4B-C shows the results of flow cytometry analysis of live (Figure 4B) and dead (Figure 4C) cells stained with anti-hFc APC antibody. In group 1, A20 Gal3 cells were incubated without mTIM-3 Fc protein as a control; in group 2, A20 Gal3 cells were incubated with mTIM-3 Fc protein; in groups 3, 4, and 5, in addition to mTIM-3 Fc protein, anti-mouse TIM-3 polyclonal antibody (R&D System, Minneapolis, MN) (group 3), monoclonal antibody RMT3-23 (Bio X Cell, West Lebanon, NH) (group 4), and monoclonal antibody 215015 (R&D System) (group 5) were added to test whether these antibodies could block the binding of Gal3 to Tim3. [Figure 5A] Figures 5A-C show ELISA results demonstrating the specific binding of Gal3 to TIM-3. In Figure 5A, plates were coated with mGal3 at 10 μg / ml, and it was found that the mGal3 polyclonal antibody (mGal3 pAb) and monoclonal antibody IMT001, but not monoclonal antibody M3 / 38, blocked the interaction of Gal3 with Tim3. [Figure 5B]FIG. 5B shows that lactose blocked Gal9 but not Gal3 binding to TIM-3, indicating that the binding between Gal3 and Tim3 is a sugar-independent binding. [Figure 5C] Figure 5C shows that antibody RMT3-23 blocked phosphatidylserine (PS) but did not block Gal3 binding to TIM-3, indicating that the epitope on TIM-3 that binds Gal3 is distinct from that which binds PS. [Figure 6A] Figures 6A-B show that overexpressed Gal3 suppressed T cell activation. Figure 6A shows that mouse A20 cell clones #41, #31, and #15 overexpress Gal3. [Figure 6B] FIG. 6B shows that when these cells were mixed with murine DO11.10T cells, much less IL-2 was produced compared to the parental A20 cells. [Figure 7A] Figures 7A-E show that Gal3 antibody has anti-tumor activity in a lung metastasis model: Figure 7A shows high expression of Gal3 in B16F10 tumor cells. [Figure 7B] FIG. 7B shows representative images of whole lungs from the three treatment groups. [Figure 7C] FIG. 7C shows the number of metastatic colonies (mean±standard deviation of the mean) on the surface of the left lung lobe. [Figure 7D] Figures 7D and 7E show lung and body weights (mean ± standard error of the mean) of the different treatment groups. Compared to animals treated with isotope control, animals treated with monoclonal anti-human Gal3 antibody showed a significant reduction in tumor number (p<0.01) (Figure 7B) and much less tumor burden as indicated by lung weight (p<0.05) (Figure 7D). However, animals treated with PD1 antibody showed no significant reduction in tumor number or tumor burden in this lung metastasis model (p>0.05). [Figure 7E] FIG. 7E shows that animals treated with either PD1 or Gal3 antibodies had similar body weights to the control group, and there were no adverse effects associated with administration of either antibody. [Figure 8A] Figures 8A-C show anti-tumor activity of Gal3 antibody in 4T1 orthotopic tumor induced lung metastasis. Figure 8A shows images of metastatic tumor colonies in the lungs of mice implanted with 4T1 cells and then treated with either control antibody ("isotype") or IMT001. Antibodies were administered intraperitoneally on days 0, 3, 7, 10 and 14 for a period of 30 days. Images were taken on day 30, when the mice were sacrificed. [Figure 8B] FIG. 8B shows the body weight measurements of these mice over the same period. [Figure 8C] FIG. 8C shows the number of metastatic tumor colonies on the surface of the left lung lobe in these mice on day 30. [Figure 9] Figure 1 shows tumor growth in mice implanted with Renca tumor cells and treated with Gal3 antibody. Compared to mice implanted with Renca tumor cells and treated with an isotype control antibody ("Iso"), mice treated with Gal3 antibody ("IMT001") showed a significant reduction in tumor size (p<0.05), whereas the anti-mouse PD-1 antibody 29F had no effect (p>0.05). [Figure 10] Figure 1 shows tumor growth in mice implanted with MC38 colon cancer cells and treated with anti-Gal3 antibody. Mice treated with Gal3 antibody ("IMT001") showed a significant reduction in tumor size (p<0.05) compared to mice implanted with MC38 colon cancer cells and treated with an isotype control antibody ("Iso"). [Figure 11A] 11A-D show the results of epitope mapping. A peptide array derived from the hGal3 protein sequence was synthesized (FIG. 11A) and dot-blotted with the anti-Gal3 antibody IMT001 (FIG. 11B). [Figure 11B] 11A-D show the results of epitope mapping. A peptide array derived from the hGal3 protein sequence was synthesized (FIG. 11A) and dot-blotted with the anti-Gal3 antibody IMT001 (FIG. 11B). [Figure 11C]Peptides_5 and 6 showed good signals indicating that anti-Gal3 monoclonal antibody, IMT001, could bind to these peptides. To further map the binding epitopes of IMT001 to these peptides, several shorter peptides derived from these peptide sequences were synthesized (FIG. 11C) and their binding to IMT001 was measured by ELISA (FIG. 11D). The peptide with the sequence GQAPPGAYPG (SEQ ID NO:28) produced the highest signal. [Figure 11D] Peptides_5 and 6 showed good signals indicating that anti-Gal3 monoclonal antibody, IMT001, could bind to these peptides. To further map the binding epitopes of IMT001 to these peptides, several shorter peptides derived from these peptide sequences were synthesized (FIG. 11C) and their binding to IMT001 was measured by ELISA (FIG. 11D). The peptide with the sequence GQAPPGAYPG (SEQ ID NO:28) produced the highest signal. [Figure 12] Immune cell counts are summarized from mice engrafted with B16F10 cells expressing various lymphocyte markers: CD3, CD4, CD8, CD19, or DX5. These mice were treated with isotype control antibody or IMT001. [Figure 13A] Figures 13A-B show Gal3 expression on tumor-associated macrophages of human lung cancer in an immunohistochemistry (IHC) assay. IMT001 was used to stain human lung cancer frozen slides to detect Gal3 expression on tumor-associated macrophages. Figure 13A shows the results of staining squamous cell carcinoma. [Figure 13B] FIG. 13B shows the results of staining adenocarcinoma. [Figure 14A] 14A-C show that Gal3 expression was detected in human M2 macrophages (FIG. 14C), but not in dendritic cells (DCs) (FIG. 14A) or M1 macrophages (FIG. 14B). [Figure 14B]14A-C show that Gal3 expression was detected in human M2 macrophages (FIG. 14C), but not in dendritic cells (DCs) (FIG. 14A) or M1 macrophages (FIG. 14B). [Figure 14C] 14A-C show that Gal3 expression was detected in human M2 macrophages (FIG. 14C), but not in dendritic cells (DCs) (FIG. 14A) or M1 macrophages (FIG. 14B). [Figure 15A] 15A-D show the immunoreactivity of Gal3 antibody ("IMT001") in mouse macrophage / T cell responses. [Figure 15B] FIG. 15B shows detection of Gal3 expression by IHC in the mouse macrophage cell line RAW264.7 compared to the control (FIG. 15A). [Figure 15C] FIG. 15C shows expression of Gal3 in mouse macrophage cell lines by flow cytometry using cells stained with IMT001. [Figure 15D] The anti-Gal3 antibody IMT001, but not the anti-mouse PD-1 antibody 29F, enhanced IL-2 production in RAW macrophage / DO11.10 T cell mixed reactions (FIG. 15D). [Figure 16] Figure 1 illustrates ELISA assessment of GAL3-TIM3 interaction blocking by GAL3-binding antibodies. Results demonstrate that Gal3-targeting antibodies show differential blocking of Gal3-TIM3 binding. Percentage of TIM3-GAL3 binding in the absence of antibody is shown. [Figure 17A] Figures 17A-17B illustrate the ELISA evaluation of anti-GAL3 antibodies that bind to peptide fragments of GAL3. Figure 17A: Antibodies mab1, mab3, mab4, and mab5. [Figure 17B] Figure 17B: Antibodies mab2, mab3, mab6, and mab7. The results demonstrate that Gal3-targeted antibodies show differential blocking of Gal3-TIM3 binding. [Figure 18]1 illustrates ELISA competitive binding assessment of anti-GAL3 antibodies that bind to GAL3. The results show that Gal3-targeted antibodies mab1 (801) and mab4 (804), but not mab5 (805), compete bidirectionally for binding to Gal3. [Figure 19A] Figures 19A-C illustrate biolayer interferometry assessment of the association and dissociation kinetics of anti-Gal3 antibodies with Gal3 (Gal3 binding antibody affinity). Figure 19A: mab1; kD = 13.5 nM. [Figure 19B] Figure 19B: mab4; kD=1.2 nM. [Figure 19C] Figure 19C: mab5; kD=32 nM. [Figure 20] 1 illustrates a CMV antigen recall assay evaluation of GAL3 enhancement of T cell antigen-specific responsiveness. The results demonstrate that Gal3-targeted antibodies display differential activation of T cells upon CMV-induced antigen recall. [Figure 21A] Figures 21A-C illustrate MALDI-MS identification of GAL3 and TIM3 regions that mediate the interaction of TIM3 with GAL3. Note that the amino acid enumeration is based on the mature protein after processing of the signal peptide. See also Table 2. Figure 21A illustrates sequences that may be involved in the binding interface. [Figure 21B] FIG. 21B illustrates residues that may be involved in the interaction. [Figure 21C] Figure 21C illustrates the sequence positions mapped to TIM-3 and Gal3, respectively, with amino acid numbering based on the mature protein after processing of the signal peptide. [Figure 22] Western blot analysis of fibrosis markers, α-smooth muscle actin (α-SMA) and fibronectin in kidney tissue lysates from male unilateral urethral obstruction (UUO) mice treated with IMT001 and mIgG2b (control) for 14 days after ureteral ligation or sham-treated without antibody treatment. GAPDH was used as a loading control. [Figure 23]Western blot analysis of fibrosis markers, α-SMA and fibronectin in liver tissue lysates from non-obese diabetic and infected (N-IF) mice. Animals were treated with IMT001, anti-Gal3 and mIgG2b (control) antibodies for 40 days. GAPDH was used as a loading control. [Figure 24] Galectin-3 targeted antibodies were assessed for their ability to block binding of GAL3 to TIM3 by ELISA at 3 μg / ml. Bars represent the mean ± standard deviation. [Diagram 25] Alignment of GAL3 peptides with the ability to bind GAL3-TIM-3 blocking GAL3-targeting antibodies. [Figure 26] Identification of Galectin-3 binding antibody bins by antibody competition. Values ​​represent inhibition as assessed by biolayer interferometry. [Figure 27] Humanized anti-GAL3 antibodies were evaluated for blocking GAL3-TIM-3 by ELISA in a titration series. Values ​​plotted represent the mean ± standard deviation. [Figure 28A] Figure 28A-D. Tumor burden in mice implanted with subcutaneous MBT2 tumors and treated with control, IMT001, anti-PD-L1 antibody, or a combination thereof (Figure 28A), or treated with control, IMT001, anti-PD-1 antibody, or a combination thereof (Figure 28C). Tumor burden plots for anti-PD-L1 antibody (Figure 28B) or anti-PD-1 (Figure 28D) represent daily measurements from individual animals. [Figure 28B] Figure 28A-D. Tumor burden in mice implanted with subcutaneous MBT2 tumors and treated with control, IMT001, anti-PD-L1 antibody, or a combination thereof (Figure 28A), or treated with control, IMT001, anti-PD-1 antibody, or a combination thereof (Figure 28C). Tumor burden plots for anti-PD-L1 antibody (Figure 28B) or anti-PD-1 (Figure 28D) represent daily measurements from individual animals. [Figure 28C]Figure 28A-D. Tumor burden in mice implanted with subcutaneous MBT2 tumors and treated with control, IMT001, anti-PD-L1 antibody, or a combination thereof (Figure 28A), or treated with control, IMT001, anti-PD-1 antibody, or a combination thereof (Figure 28C). Tumor burden plots for anti-PD-L1 antibody (Figure 28B) or anti-PD-1 (Figure 28D) represent daily measurements from individual animals. [Figure 28D] Figure 28A-D. Tumor burden in mice implanted with subcutaneous MBT2 tumors and treated with control, IMT001, anti-PD-L1 antibody, or a combination thereof (Figure 28A), or treated with control, IMT001, anti-PD-1 antibody, or a combination thereof (Figure 28C). Tumor burden plots for anti-PD-L1 antibody (Figure 28B) or anti-PD-1 (Figure 28D) represent daily measurements from individual animals. [Figure 29A] Figure 29A-D. Assessment of hepatocellular carcinoma formation in normal and human IgG4 (huIgG4) or IMT001-4 treated STAM-CDAA mice by gross histology (Figure 29A) and counting (Figure 29B). Arrows highlight tumor-bearing areas. [Figure 29B] Assessment of hepatocellular carcinoma formation in normal and human IgG4 (huIgG4) or IMT001-4 treated STAM-CDAA mice by gross histology (FIG. 29A) and counting (FIG. 29B). Arrows highlight tumor-bearing areas. [Figure 29C] Hematoxylin and eosin stained sections of liver samples assessed for tumor formation (FIG. 29C). Arrows highlight areas with tumors. [Figure 29D] Quantification of α-fetoprotein in serum of STAM-CDAA mice treated with huIgG4 or IMT001-4 (FIG. 29D). Circles indicate the mean per animal, lines indicate the mean per group. [Figure 30A] Figure 30A-D. Hematoxylin and eosin stained sections of liver from the MCD mouse model of NASH liver fibrosis treated with isotype control or mIMT001 (Figure 30A). [Figure 30B]Histological measurements of hepatic steatosis, hepatocyte ballooning, intralobular inflammation, or image-based quantification of NAFLD Activity Score (NAS) (FIG. 30B). [Figure 30C] Picosirius red staining of liver specimens from mice treated as in (A) (FIG. 30C). [Figure 30D] Quantification based on images of Sirius red staining (FIG. 30D). Bars represent the mean of 7 animals±standard error of the mean. [Figure 31A] Figures 31A-B. Picosirius red staining of liver specimens from the choline-deficient, L-amino acid restricted, high fat diet (CDAA-HFD) STAM model of liver fibrosis treated with isotype control or IMT001-4 (Figure 31A). [Figure 31B] Quantitation based on images of Sirius red staining (FIG. 31B). Bars represent the mean value of 5 fields from each of 7 animals±standard error of the mean. [Figure 32A] Figures 32A-C. Assessment of serum markers of renal fibrosis KIM-1 and NGAL (Figure 32A) and picosirius red staining (Figure 32B) of kidney specimens from mice treated with isotype control, IMT001-4, IMT001-6, or metformin in a mouse unilateral urethral obstruction (UUO) model. [Figure 32B] Assessment of serum markers of renal fibrosis KIM-1 and NGAL (FIG. 32A) and picosirius red staining (FIG. 32B) of kidney specimens from mice treated with isotype control, IMT001-4, IMT001-6, or metformin in a mouse unilateral urethral obstruction (UUO) model. [Figure 32C] Image-based Picosirius Red staining quantification (FIG. 32C). Bars represent the mean ± standard error of the mean of triplicate assessments from each of 7 animals per group. Points represent individual animal mean Picosirius Red staining, bars indicate group mean values. [Figure 33A]Figures 33A-B. IHC assessment of collagen 1a1 (Col1a1) deposition in kidney specimens treated with isotype control or mIMT001 on day 1 after UUO (Figure 33A). [Figure 33B] Black arrows correspond to areas of fibrillar collagen deposition. Image-based quantification (FIG. 33B). Bars represent the mean ± standard error of the mean of 10 fields from each of 7 animals per group. [Figure 34A] Figure 34A-B. Masson's trichrome staining assessment of pulmonary fibrosis in a bleomycin-induced pulmonary fibrosis mouse model treated with isotype control or mIMT001 (Figure 34A). [Figure 34B] Ashcroft scores of tissue sections (FIG. 34B). Bars represent the mean±standard error of the mean of 10 fields from each of 8 animals per group. [Figure 35A] Figure 35A shows some embodiments of the VH CDR regions of various embodiments of anti-GAL3 antibodies. In some embodiments, any of the methods or compositions provided herein can include one or more of the CDRs provided herein, including one, two, or three CDRs. [Figure 35B] Figure 35B shows some embodiments of the VL CDR regions of various embodiments of anti-GAL3 antibodies. In some embodiments, any of the methods or compositions provided herein can include one or more of the CDRs provided herein, including one, two, or three CDRs. [Figure 36A-1] Figure 36A shows several embodiments of the complete VH regions of various embodiments of anti-GAL3 antibodies. In some embodiments, any of the methods or compositions provided herein can include any one of these VH regions. [Figure 36A-2] Continued from Figure 36A. [Figure 36B-1] Figure 36B shows several embodiments of the complete VL regions of various embodiments of anti-GAL3 antibodies. In some embodiments, any of the methods or compositions provided herein can include any one of these VL regions. [Figure 36B-2] Continued from Figure 36B. [Figure 37-1] Figure 37 shows several embodiments of various GAL3 antibodies (including complete heavy or kappa chain sequences). In some embodiments, any one or more of the VH / VL and / or CDRs provided in other figures can be paired with one or more of the relevant sequences in Figure 37 (e.g., the IgG4 section or kappa sequence). [Figure 37-2] Continued from Figure 37. [Figure 38-1] Figure 38 shows an alignment of some embodiments of the VH or VL CDR regions of various embodiments of anti-Gal3 antibodies. In some embodiments, any of the methods or compositions provided herein may use any one, two, three, four, five, or six of the consensus CDRs provided in Figure 38. [Figure 38-2] Continued from Figure 38. [Figure 38-3] Continued from Figure 38. [Figure 38-4] Continued from Figure 38. [Figure 38-5] Continued from Figure 38. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] Galectin 3 (Gal3, GAL3, or Gal-3) is expressed in several cell types and is associated with a wide range of physiological and pathological processes, including cell adhesion, cell activation and chemoattraction, cell cycle, apoptosis, cell growth and division, and tumor progression and metastasis. Gal3 is expressed in tumor cells and cells in the tumor microenvironment, such as tumor-associated macrophages, especially M2 macrophages. In addition, it is involved in the activation of various profibrotic factors that promote fibroblast proliferation and transformation and mediate collagen production. Furthermore, Gal3 is thought to play an important role in fibrosis in various tissues, including the liver, kidney, lung, and myocardium.

[0010] TIM-3 is a molecule expressed in immune cells, particularly T cells, and can suppress immune responses, such as T cell signaling, by interacting with Gal3. Anti-Gal3 antibodies interfere with the interaction between Gal3 and TIM-3, activating immune responses.

[0011] Tumors are often associated with immune infiltrates as part of a macrophage-enriched reactive stroma. Tumor-associated macrophages (TAMs) play a key role in promoting tumor growth by promoting neovascularization and matrix degradation. When associated with tumors, macrophages exhibit functional polarization into one of two phenotypically distinct subsets of macrophages: M1 macrophages or M2 macrophages. M1 macrophages produce proinflammatory cytokines and play an active role in cell destruction, whereas M2 macrophages It has been found that TAMs primarily remove debris and promote angiogenesis and wound healing. As a result, many tumors with a large number of TAMs have increased tumor growth rates, local growth, and distant metastasis. M2 macrophage populations are phenotypically similar to TAM populations that promote tumor growth and tumor development. In addition to expressing Gal3, in some cases, M2 macrophages also express one or more cell surface markers selected from the group consisting of CD206, IL-4r, IL-1ra, decoy IL-1rll, IL-10r, CD23, macrophage removing receptors A and B, Ym-1, Ym-2, low density receptor-related protein 1 (LRP1), IL-6r, CXCR1 / 2, CD136, CD14, CD1a, CD1b, CD93, CD226, (FcyR), and PD-L1.

[0012] Tissue fibrosis is a progressive debilitating disease characterized by abundant accumulation of extracellular matrix (ECM) proteins such as collagen and fibronectin, leading to tissue scarring, organ damage, organ function decline, and then organ failure. Tissue fibrosis can be present in the kidney, liver, heart, skin, pancreas, intestine, eye, nervous system, joints, tendons, mediastinum, or retroperitoneum. Features of tissue fibrosis include epithelial and endothelial injury and dysfunction, abnormal proliferation of myofibroblasts (MFb), smooth muscle cells, and astrocytes, and ECM deposition. The presence of cytokines, chemokines, growth factors, and angiogenic factors further regulate the activation of ECM-producing cells during the proliferation process.

[0013] Galectin 3 (Gal3) has been shown to play important roles in cell proliferation, adhesion, differentiation, angiogenesis, and apoptosis. In addition, it promotes fibroblast proliferation and transformation and is involved in the activation of various profibrotic factors that mediate collagen production. Furthermore, Gal3 appears to play an important role in fibrosis in various tissues, including the liver, kidney, lung, and myocardium.

[0014] In some embodiments, a method for reducing fibrosis or its tendency in tissue is disclosed herein using an anti-Gal3 antibody. In some embodiments, reducing fibrosis or its tendency in tissue comprises preventing fibrosis from occurring in normal tissue. In some embodiments, reducing fibrosis or its tendency in tissue comprises slowing or stopping the progression of fibrosis in fibrotic tissue. In some embodiments, reducing fibrosis or its tendency in tissue comprises reducing the amount of fibrosis in fibrotic tissue. In some embodiments, reducing fibrosis or its tendency in tissue comprises eliminating fibrosis in fibrotic tissue.

[0015] Also described herein, in some embodiments, are methods for monitoring the progression of tissue fibrosis by monitoring one or more fibrosis biomarkers.Furthermore, disclosed herein are methods for treating tissue fibrosis with anti-Gal3 antibodies, where the anti-Gal3 antibody disrupts the interaction of Gal3 with TIM-3.

[0016] In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, like symbols generally identify like components unless the context dictates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. As generally described herein and illustrated in the figures, aspects of the present disclosure can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are expressly contemplated herein.

[0017] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. For purposes of this disclosure, the following terms are defined below.

[0018] The articles "a" and "an" refer to one or to more than one (e.g., at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0019] "About" means an amount, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% of the reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight or length.

[0020] Throughout this specification, unless the context requires otherwise, the terms "comprise", "comprises" and "comprising" will be understood to mean the inclusion of the specified step or element or group of steps or elements, but not the exclusion of other steps or elements or group of steps or elements. "Consisting of" means including, but is limited to, whatever follows the phrase "consisting of". Thus, the phrase "consisting of" indicates that the recited elements are necessary or mandatory, and no other elements may be present. "Consisting essentially of" means including any elements recited after the phrase, and is limited to other elements that do not interfere with or contribute to the activity or action specified in this disclosure for the recited elements. Thus, the phrase "consisting essentially of" means that the recited elements are necessary or mandatory, but other elements are optional and may or may not be present depending on whether they substantially affect the activity or action of the recited elements.

[0021] In some embodiments, an anti-Gal3 antibody or a binding fragment thereof or a composition comprising an anti-Gal3 antibody or a binding fragment thereof is provided. In some embodiments, a method is provided using an anti-Gal3 antibody or a binding fragment thereof or a composition comprising an anti-Gal3 antibody or a binding fragment thereof to block or disrupt the interaction between Gal3 and TGF-β receptor either in vitro or in vivo. In some embodiments, a method is used using an anti-Gal3 antibody or a binding fragment thereof or a composition comprising an anti-Gal3 antibody or a binding fragment thereof to treat, cure, or prevent a disease or disorder in a subject. In some embodiments, the disease or disorder is cancer, breast cancer, colorectal cancer, renal cancer, liver cancer, lung cancer, or a hematological tumor. In some embodiments, the cancer is metastatic cancer, recurrent cancer, or refractory cancer. In some embodiments, the antibody is administered in combination with an additional therapeutic agent, such as an immune checkpoint inhibitor, a chemotherapeutic agent, a targeted therapeutic agent, a hormonal therapeutic agent, or a stem cell-based therapeutic agent. In some embodiments, the disease or disorder is fibrosis in a tissue, such as liver tissue, kidney tissue, skin tissue, lung tissue, heart tissue, brain tissue, intestinal tissue, bone marrow tissue, or soft tissue.

[0022] As used herein, the terms "individual," "subject," and "patient" refer to any mammal or bird. In some embodiments, the mammal is a human. In some embodiments, the mammal is non-human, including, but not limited to, farm animals (e.g., cows, pigs, horses, chickens, etc.), sport animals, pets, primates, dogs, cats, mice, and rats. None of the terms require or are limited to conditions characterized by the supervision (e.g., constant or intermittent) of a medical professional (e.g., a physician, registered nurse, nurse practitioner, physician assistant, handyman, or hospice worker).

[0023] As used herein, the terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear, cyclic, or branched, may contain modified amino acids, and may be interrupted by non-amino acids. The term also encompasses amino acid polymers modified by, for example, sulfation, glycolation, lipidation, acetylation, phosphorylation, iodination, methylation, oxidation, protein processing, phosphorylation, prenylation, racemization, selenization, transfer RNA-mediated addition of amino acids to proteins such as arginylation, ubiquitination, or other manipulation such as conjugation with a labeling moiety.

[0024] As used herein, the term "amino acid" refers to either natural and / or unnatural or synthetic amino acids, including glycine and both the D or L optical isomers, as well as amino acid analogs and peptidomimetics.

[0025] A polypeptide or amino acid sequence "derived from" a specified protein refers to the origin of the polypeptide. Preferably, the polypeptide has an amino acid sequence that has substantial identity to a polypeptide encoded in the sequence, or a portion thereof, where the portion consists of at least 10-20 amino acids, or at least 20-30 amino acids, or at least 30-50 amino acids, or is immunologically identifiable as the polypeptide encoded in the sequence. The term also includes polypeptides expressed from a specified nucleic acid sequence.

[0026] As used herein, the term "antibody" is intended to include any polypeptide chain-containing molecular structure having a specific shape that fits and recognizes an epitope, where one or more non-covalent interactions stabilize the complex between the molecular structure and the epitope. Although monoclonal antibodies are preferred, since they may be reproduced in cell culture or recombinantly, and can be modified to reduce their antigenicity, the antibodies utilized in the present invention may be polyclonal.

[0027] In addition to whole immunoglobulins (or their recombinant counterparts), immunoglobulin fragments or "binding fragments" that contain epitope-binding sites (e.g., Fab', F(ab')2, single chain variable fragment (scFv), or other fragments) are useful as antibody moieties in the present invention. Such antibody fragments may be generated from whole immunoglobulins by lysine, pepsin, or other protease cleavage. Minimal immunoglobulins may be engineered using recombinant immunoglobulin technology. For example, "Fv" immunoglobulins for use in the present invention may be produced by linking the variable light chain region to the variable heavy chain region via a peptide linker (e.g., polyglycine or another sequence that does not generate an alpha helix or beta sheet motif). Nanobodies or single domain antibodies may also be derived from alternative organisms such as dromedaries, camels, llamas, alpacas, or sharks. In some embodiments, the antibody may be a conjugate, e.g., a pegylated antibody, a drug, a radioisotope, or a toxin conjugate. Monoclonal antibodies against a specific epitope, or combination of epitopes, would allow for targeting and / or depletion of cell populations expressing the marker. Monoclonal antibodies can be used to screen for cell populations expressing the marker using a variety of techniques, including magnetic separation using antibody-coated magnetic bead "panning" with antibody bound to a solid matrix (i.e., plate) and flow cytometry (see, e.g., U.S. Patent No. 5,985,660, expressly incorporated herein by reference in its entirety).

[0028] As used herein, the term "humanized" when applied to non-human (e.g., rodent or primate) antibodies is a hybrid immunoglobulin, immunoglobulin chain or fragment thereof which contains minimal sequence derived from non-human immunoglobulin.

[0029] The "variable region" of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, either alone or in combination. As known in the art, each of the variable regions of the heavy and light chains consists of four framework regions (FRs) linked by three complementarity determining regions (CDRs), also known as hypervariable regions, which contribute to the formation of the antigen-binding site of the antibody. In particular, when a variant of the subject variable region is desired that includes the substitution of amino acid residues outside the CDR regions (i.e., within the framework regions), suitable amino acid substitutions, preferably conservative amino acid substitutions, can be identified by comparing the subject variable region with the variable regions of other antibodies that include CDR1 and CDR2 sequences in the same canonical class as the subject variable region (Chothia and Lesk, J Mol Biol 196(4):901-917,1987).

[0030] In some embodiments, the final delineation of the CDRs and the identification of the residues that comprise the binding site of the antibody is performed by analysis of the structure of the antibody and / or analysis of the structure of the antibody-ligand complex. In some embodiments, this can be performed by any of a variety of techniques known to those of skill in the art, such as X-ray crystallography. In some embodiments, various analytical methods can be used to identify or approximate the CDR regions. In some embodiments, various analytical methods can be used to identify or approximate the CDR regions. Examples of such methods include the Kabat definition, the Chothia definition, the IMGT approach (Lefranc et al., 2003 Dev Comp Immunol. 27:55-77), computer programs such as Paratome (Kunik et al., 2012, Nucl Acids Res.W521-4), the AbM definition, and the conformational definition.

[0031] The Kabat definition is a standard for numbering residues in antibodies and is commonly used to identify CDR regions. See, e.g., Johnson & Wu, 2000, Nucleic Acids Res., 28:214-8. The Chothia definition is similar to the Kabat definition, but it takes into account the location of certain structural loop regions. See, e.g., Chothia et al., 1986, J. Mol. Biol., 196:901-17; Chothia et al., 1989, Nature, 342:877-83. The AbM definition uses a comprehensive suite of computer programs created by the Oxford Molecular Group to model antibody structures. See, e.g., Martin et al., 1989, Proc Natl Acad Sci (USA), 86:9268-9272; "AbM.TM., A Computer Program for Modeling Variable Regions of Antibodies," Oxford, UK; Oxford Molecular, Ltd. The AbM definition uses a combination of knowledge databases and ab initio methods, such as those described in Samudrala et al., 1999, "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach," in PROTEINS, Structure, Function and Genetics Suppl., 3:194-198, to model the tertiary structure of an antibody from the primary sequence. The contact definition is based on the analysis of available complex crystal structures. See, e.g., MacCallum et al., 1996, J. Mol. Biol., 5:732-45. In another approach, referred to herein as the "conformational definition" of the CDRs, the positions of the CDRs may be identified as residues that contribute enthalpy to antigen binding. See, e.g., Makabe et al., 2008, Journal of Biological Chemistry, 283:1156-1166.Still other CDR association definitions, which may be shortened or extended in light of predicted or experimental findings that certain residues or groups of residues do not significantly affect antigen binding, may not strictly follow one of the above approaches but will nevertheless overlap with at least a portion of the Kabat CDRs as used herein. In the present specification, the CDRs may represent CDRs defined by any approach known in the art, including a combination of approaches. The methods used herein may utilize CDRs defined according to any of these approaches. In some embodiments that include more than one CDR, the CDRs may be defined according to Kabat, Chothia, extended, IMGT, Paratome, AbM, and / or conformational definitions, or a combination of any of the foregoing. In some embodiments, the residue numbers of the variable regions are numbered using the IMGT numbering system. In the sequences provided herein, the CDRs are mapped according to IMGT (https: / / world wide web.Ebi.ac.uk / ipd / imgt / hla / align.html).

[0032] As known in the art, a "constant region" of an antibody refers to the constant region of the antibody light chain or the constant region of the antibody heavy chain, either alone or in combination.

[0033] The term "compete" as used herein in connection with antibodies means that a first antigen, or antigen-binding portion thereof, binds to an epitope in a manner sufficiently similar to the binding of a second antibody, or antigen-binding portion thereof, such that the result of binding of the first antibody to its cognate epitope is detectably reduced in the presence of the second antibody, compared to the binding of the first antibody in the absence of the second antibody. If the binding of the second antibody to its epitope is detectably reduced in the presence of the first antibody, the alternative need not be. That is, a first antibody can inhibit a second antibody from binding to its epitope without the second antibody inhibiting the first antibody from binding to its respective epitope. However, if each antibody detectably inhibits the other antibody from binding to its cognate epitope or ligand, whether to the same extent, a greater extent, or a lesser extent, the antibodies are considered to "cross-compete" with each other for binding of their respective epitopes. Both competing and cross-competing antibodies are encompassed by the present invention. Regardless of the mechanism by which such competition or cross-competition occurs (e.g., steric hindrance, conformational changes, or binding to a common epitope, or portion thereof), one of skill in the art will recognize, based on the teachings provided herein, that such competing and / or cross-competing antibodies are encompassed and may be useful in the methods disclosed herein.

[0034] Antibodies that "preferentially bind" or "specifically bind" (used interchangeably herein) to an epitope are terms well understood in the art, and methods for determining such specific or preferential binding are well known in the art. A molecule is considered to exhibit "specific binding" or "preferential binding" if it reacts or associates with another cell or substance more frequently and / or more rapidly and / or for a longer duration and / or with greater affinity than it reacts or associates with another cell or substance. An antibody "specifically binds" or "preferentially binds" to a target if it binds with greater affinity and / or avidity and / or more rapidly and / or for a longer duration than it binds to other substances. For example, an antibody that specifically or preferentially binds to a CFD epitope is an antibody that binds with greater affinity and / or avidity and / or more readily and / or for a longer duration than it binds to other CFD epitopes or non-CFD epitopes. For example, it is understood by reading this definition that an antibody (or moiety or epitope) that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. As such, "specific binding" or "preferential binding" does not necessarily require (although it can include) exclusive binding. Generally, but not necessarily, references to binding refer to preferential binding.

[0035] As used herein, "substantially pure" refers to a material that is at least 50% pure (i.e., free from contaminants), more preferably at least 90% pure, more preferably at least 95% pure, even more preferably at least 98% pure, and most preferably at least 99% pure.

[0036] A "host cell" includes an individual cell or cell culture that can be or has been the recipient of a vector for incorporating a polynucleotide insert. A host cell includes the progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or genomic DNA complement) to the original parent due to natural, accidental, or deliberate mutation. A host cell includes cells transfected in vivo with a polynucleotide of the invention.

[0037] As known in the art, the term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain. The "Fc region" may be a native sequence Fc region or a variant Fc region. Although the boundaries of the Fc region of an immunoglobulin heavy chain may vary, the human IgG heavy chain Fc region is usually defined as extending from an amino acid residue at Cys226, or Pro230, to its carboxylic acid terminus. The numbering of residues in the Fc region is that of the EU index as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991. The Fc region of an immunoglobulin generally comprises two constant domains, CH2 and CH3. As known in the art, the Fc region may exist in a dimer or monomer.

[0038] As used herein, "vector" refers to a construct that can deliver and preferably express one or more genes or sequences of interest in host cell.Examples of vectors include, but are not limited to, virus vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells such as production cells.

[0039] As used herein, "expression control sequence" refers to a nucleic acid sequence that directs the transcription of a nucleic acid. An expression control sequence can be a promoter, such as a constitutive or inducible promoter, or an enhancer. An expression control sequence is operably linked to the nucleic acid sequence to be transcribed.

[0040] As used herein, a "pharmaceutical acceptable carrier" or "pharmaceutical acceptable excipient" includes any material that, when combined with an active ingredient, allows the ingredient to retain biological activity and is non-reactive with the subject's immune system. Examples include, but are not limited to, any of the standard pharmaceutical carriers, such as phosphate buffered saline, water, emulsions such as oil / water emulsions, various types of wetting agents, surfactants such as polysorbate 20 to prevent clumping, and sugars such as sucrose as cryoprotectants. A preferred diluent for aerosol or parenteral administration is phosphate buffered saline (PBS) or physiological (0.9%) saline. Compositions containing such carriers are formulated by well-known conventional methods (see, for example, Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990; and Remington, The Science and Practice of Pharmacy 20th Ed. Mack 1990). (See Publishing, 2000).

[0041] As used herein, the term "k on " represents the rate constant for the association of an antibody (or bioconjugate) to an antigen. In particular, the rate constant (k on and k off ) and equilibrium dissociation constants for full-length antibodies and / or Fab antibody fragments (i.e., monovalent) and and CFD to measure.

[0042] As used herein, the term "k off " represents the rate constant for dissociation of the antibody (or bioconjugate) from the antibody / antigen complex.

[0043] As used herein, the term "k D " represents the equilibrium dissociation constant of an antibody-antigen (or bioconjugate-antigen) interaction.

[0044] As used herein, the term "treating" or "treatment" (as well understood in the art) refers to an approach to obtain a beneficial or desired outcome of a condition in a subject, including clinical outcomes. Beneficial or desired outcomes may include, but are not limited to, reduction or amelioration of one or more symptoms or conditions, minimization of the extent of the disease, stabilization (i.e., not worsening) of the disease state, prevention of disease infection or spread, delay or slowing of disease progression, remission or alleviation of the disease state, reduction of disease recurrence, and remission, whether partial or complete, and whether detectable or undetectable. As used herein, "treating" and "treatment" also include prophylactic treatment. The treatment method includes administering a therapeutically effective amount of an active ingredient to the subject. The administration step may consist of a single administration or may include a series of administrations. The composition is administered to the subject in an amount and for a period sufficient to treat the patient. The length of the treatment period depends on various factors, such as the severity of the condition, the age and genetic profile of the patient, the concentration of the active agent, the activity of the composition used for treatment, or a combination thereof. It will be appreciated that the effective dosage of an agent used for treatment or prevention may increase or decrease over the course of a particular treatment or prevention regime. Modifications in dosage may be effected and accounted for by standard diagnostic assays known in the art. In some embodiments, chronic administration may be required.

[0045] The term "administering" includes oral administration, topical contact, administration as a suppository, intravenous, intraperitoneal, intramuscular, intralesional, intrathecal, intranasal, or subcutaneous administration, or implantation of a sustained release device, such as a mini-osmotic pump, into a subject. Administration is by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, for example, intravenous, intramuscular, intraarteriolar, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial. Other methods of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, and the like. "Concurrent administration" means administration of a first compound described herein simultaneously with, immediately before, or immediately after administration of a second compound described herein.

[0046] As used herein, the term "therapeutic target" refers to a gene or gene product that, upon modulation of its activity (e.g., by modulation of expression, biological activity, and the like), can provide modulation of a disease phenotype (e.g., fibrosis or cancer). As used throughout, "modulation" is intended to refer to an increase or decrease in the indicated phenomenon (e.g., modulation of biological activity refers to an increase in biological activity or a decrease in biological activity).

[0047] The terms "cancer", "neoplasm", "tumor" and "tumor" are used interchangeably herein to refer to cells that exhibit relatively autonomous growth and, as a result, an abnormal growth phenotype characterized by a significant loss of cell growth control. Generally, cells of interest for detection or treatment in this application include precancerous (e.g., benign), malignant, premetastatic, metastatic, and nonmetastatic cells. Detection of cancerous cells is of particular interest. When used in the context of "normal cells", the term "normal" is intended to refer to cells of a non-transformed phenotype or cells that exhibit the morphology of non-transformed cells of the tissue type being examined. "Cancerous phenotype" generally refers to any of a variety of biological phenomena that have the characteristics of cancerous cells, phenomena that may vary with the type of cancer. A cancerous phenotype may also be, for example, a cell growth disorder. The tumor may be characterized by abnormalities such as proliferation or proliferation (eg, uncontrolled growth or proliferation), cell cycle regularity, cell motility, cell-cell interactions, or metastasis.

[0048] The term "tumor microenvironment" refers to the cellular environment in which a tumor resides, including tumor cells and surrounding blood vessels, immune cells, fibroblasts, bone marrow-derived inflammatory cells, lymphocytes, signaling molecules, and extracellular matrix.

[0049] The term "immune cells" refers to cells of hematopoietic origin involved in the specific recognition of antigens. Immune cells include antigen-presenting cells (APCs), such as dendritic cells or macrophages, B cells, T cells, natural killer cells, and myeloid cells, such as macrophages, eosinophils, mast cells, basophils, and granulocytes.

[0050] The term "immune response" refers to T cell-mediated, NK cell-mediated, macrophage-mediated, and / or B cell-mediated immune responses. Exemplary immune responses include B cell responses (e.g., antibody production), NK cell responses, or T cell responses (e.g., cytokine production, and cytotoxic activity) and activation of cytokine-responsive cells, e.g., macrophages. The term "activation of immune response" refers to enhancement of the level of T cell-mediated and / or B cell-mediated immune response using methods known to those of skill in the art. In some embodiments, the level of enhancement is at least 20-50%, alternatively at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 120%, at least 150%, or at least 200%.

[0051] As used herein, the term "transforming growth factor beta receptor" (TGF-b receptor) refers to a family of serine / threonine kinase receptors expressed on the cell surface that are specific for the protein transforming growth factor beta (TGF-b). Interaction of TGF-b with the receptor activates signaling pathways that are responsible for many functions, including, but not limited to, cell proliferation, differentiation (e.g., stem cells, immune cells), apoptosis, homeostasis, chemotaxis, inflammation, and immune cell activation.

[0052] As used herein, the term "fibrosis" refers to a condition in which tissues or organs harden or scar as a result of the irregular production of extracellular matrix, such as collagen proteins. Fibrosis is associated with chronic inflammation, where immune cells, such as macrophages, signal fibroblasts to respond by expressing extracellular matrix proteins. This signaling occurs through pathways such as the TGF-b pathway, although there are other profibrotic pathways as well. Fibrosis includes, but is not limited to, liver fibrosis, bridging fibrosis, cirrhosis, renal fibrosis, pulmonary fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, cardiovascular fibrosis, arterial fibrosis, venous thrombosis, cardiac fibrosis, pulmonary artery fibrosis, arthrofibrosis, Crohn's disease, Dupuytren's contracture, keloids, mediastinal fibrosis, myelofibrosis, Peyronie's disease, nephrogenic systemic fibrosis, progressive massive fibrosis, retroperitoneal fibrosis, or systemic sclerosis.

[0053] The terms "w / w% (weight / weight%)" or "wt / wt% (weight / weight%)" refer to the percentage expressed by weight of a component or agent relative to the total weight of the composition multiplied by 100.

[0054] In some embodiments, disclosed herein is a method of inducing immune activation comprising contacting an anti-Gal3 antibody with a plurality of cells comprising Gal3-expressing cells and TIM-3-expressing cells. In some embodiments, disclosed is a method of reducing fibrosis comprising contacting a tissue comprising Gal3-expressing cells and at least one fibrosis biomarker with an anti-Gal3 antibody for a time sufficient to reduce expression of at least one fibrosis biomarker in the tissue. Disclosed herein, in some embodiments, anti-Gal3 antibodies result in a reduction in the accumulation of one or more extracellular matrix proteins in tissues, including, but not limited to, collagen.

[0055] In some cases, upon binding with the anti-Gal3 antibody, the Gal3 expressing cells express a cytokine that induces immune activation. In some cases, the cytokine is an interferon. In some cases, the interferon is IFNγ. In some cases, the IFNγ production is 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 300%, 400%, 500%, 600% or more of the IFNγ production by the isotype antibody. In some cases, the IFNγ production is 150% of the IFNγ production by the isotype antibody. In some cases, the IFNγ production is 160% of the IFNγ production by the isotype antibody. In some cases, the IFNγ production is 170% of the IFNγ production by the isotype antibody. In some cases, the IFNγ production is 180% of the IFNγ production by the isotype antibody. In some cases, IFNγ production is 190% of IFNγ production by the isotype antibody. In some cases, IFNγ production is 200% of IFNγ production by the isotype antibody. In some cases, IFNγ production is more than 200% of IFNγ production by the isotype antibody. In some cases, IFNγ production is more than 300% of IFNγ production by the isotype antibody. In some cases, IFNγ production is more than 400% of IFNγ production by the isotype antibody. In some cases, IFNγ production is more than 500% of IFNγ production by the isotype antibody. In some cases, the cytokine is an interleukin. In some cases, the interleukin is IL-2.

[0056] In some cases, immune activation includes proliferation of CD3+ T lymphocytes, CD4+ T helper cells, CD8+ cytotoxic T cells, natural killer (NK) cells, or a combination thereof. In some cases, immune activation includes proliferation of CD3+ T lymphocytes. In some cases, immune activation includes proliferation of CD4+ T helper cells. In some cases, immune activation includes proliferation of CD8+ cytotoxic T cells. In some cases, immune activation includes proliferation of NK cells. In some cases, immune activation includes proliferation of T cells and NK cells.

[0057] In some cases, the immune activation includes an increase in the M1 macrophage population in the plurality of cells. In some cases, the immune activation includes a decrease in the M2 macrophage population in the plurality of cells. In some cases, the immune activation includes an increase in the M1 macrophage population in the plurality of cells and a decrease in the M2 macrophage population in the plurality of cells.

[0058] In some cases, the anti-Gal3 antibody binds to Gal3 and disrupts the interaction of Gal3 with TIM-3. In some cases, disrupting the interaction of Gal3 with TIM-3 includes partial inhibition of the interaction of Gal3 with TIM-3. In some cases, disrupting the interaction of Gal3 with TIM-3 includes complete inhibition of the interaction of Gal3 with TIM-3. In some cases, the Gal3-TIM-3 interaction is reduced by less than 99%, less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 59%, less than 55%, less than 50%, less than 45%, less than 40%, less than 34%, less than 30%, less than 25%, less than 20%, less than 14%, less than 10%, less than 7%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%. In some cases, the Gal3-TIM-3 interaction is reduced by less than 70%. In some cases, the Gal3-TIM-3 interaction is reduced by less than 60%. In some cases, the Gal3-TIM-3 interaction is reduced by less than 59%. In some cases, the Gal3-TIM-3 interaction is reduced by less than 50%. In some cases, the Gal3-TIM-3 interaction is reduced by less than 40%. In some cases, the Gal3-TIM- In some cases, the Gal3-TIM-3 interaction is reduced to less than 34%. In some cases, the Gal3-TIM-3 interaction is reduced to less than 30%. In some cases, the Gal3-TIM-3 interaction is reduced to less than 20%. In some cases, the Gal3-TIM-3 interaction is reduced to less than 14%. In some cases, the Gal3-TIM-3 interaction is reduced to less than 10%. In some cases, the Gal3-TIM-3 interaction is reduced to less than 7%. In some cases, the Gal3-TIM-3 interaction is reduced to less than 5%. In some cases, the Gal3-TIM-3 interaction is reduced to less than 4%. In some cases, the Gal3-TIM-3 interaction is reduced to less than 1%.

[0059] In some cases, the interaction between Gal3 and TIM-3 occurs at one or more residues of Gal3 selected from regions 145-168, 160-177, or 165-184, the residue positions corresponding to positions 145-168, 160-177, or 165-184 of SEQ ID NO:1. In some cases, the interaction between Gal3 and TIM-3 occurs at one or more residues of Gal3 from region 145-168, the residue positions corresponding to positions 145-168 of SEQ ID NO:1. In some cases, the interaction between Gal3 and TIM-3 occurs at one or more residues of Gal3 from region 160-177, the residue positions corresponding to positions 160-177 of SEQ ID NO:1. In some cases, the interaction between Gal3 and TIM-3 occurs at one or more residues of Gal3 from region 165-184, the residue positions corresponding to positions 165-184 of SEQ ID NO:1. In some cases, the interaction between Gal3 and TIM-3 occurs at one or more residues of Gal3 selected from regions 149-156, 152-168, 163-169, or 163-171, where the residue positions correspond to positions 149-156, 152-168, 163-169, or 163-171 of SEQ ID NO:1. In some cases, the interaction between Gal3 and TIM-3 occurs at one or more residues of Gal3 from region 149-156, where the residue positions correspond to positions 149-156 of SEQ ID NO:1. In some cases, the interaction between Gal3 and TIM-3 occurs at one or more residues of Gal3 from region 152-168, where the residue positions correspond to positions 152-168 of SEQ ID NO:1. In some cases, the interaction between Gal3 and TIM-3 occurs at one or more residues of Gal3 from region 163-169, which residue positions correspond to positions 163-169 of SEQ ID NO: 1. In some cases, the interaction between Gal3 and TIM-3 occurs at one or more residues of Gal3 from region 163-171, which residue positions correspond to positions 163-171 of SEQ ID NO: 1.

[0060] In some cases, the interaction between Gal3 and TIM-3 occurs at one or more residues of TIM-3 selected from regions 91-111, or 82-111, where the residue positions correspond to positions 91-111, or 82-111 of SEQ ID NO:2. In some cases, the interaction between Gal3 and TIM-3 occurs at one or more residues of TIM-3 from region 91-111, where the residue positions correspond to positions 91-111 of SEQ ID NO:2. In some cases, the interaction between Gal3 and TIM-3 occurs at one or more residues of TIM-3 from region 82-111, where the residue positions correspond to positions 82-111 of SEQ ID NO:2. In some cases, the interaction between Gal3 and TIM-3 occurs at one or more residues of TIM-3 selected from regions 91-111, 107-117, 96-102, 100-106, or 92-119, where residue positions herein correspond to positions 91-111, 107-117, 96-102, 100-106, or 92-119 of SEQ ID NO:2. In some cases, the interaction between Gal3 and TIM-3 occurs at one or more residues of TIM-3 from region 91-111, where residue positions correspond to positions 91-111 of SEQ ID NO:2. In some cases, the interaction between Gal3 and TIM-3 occurs at one or more residues of TIM-3 from region 107-117, where residue positions correspond to positions 107-117 of SEQ ID NO:2. In some cases, the interaction between Gal3 and TIM-3 occurs at one or more residues of TIM-3 from the region 96-102, the residue positions corresponding to positions 96-102 of SEQ ID NO: 2. In some cases, the interaction between Gal3 and TIM-3 occurs at one or more residues of TIM-3 from the region 96-102, the residue positions corresponding to positions 96-102 of SEQ ID NO: 2. Optionally, the interaction between Gal3 and TIM-3 occurs at one or more residues of TIM-3 from region 100-106, where the residue positions correspond to positions 100-106 of SEQ ID NO: 2. Optionally, the interaction between Gal3 and TIM-3 occurs at one or more residues of TIM-3 from region 92-119, where the residue positions correspond to positions 92-119 of SEQ ID NO: 2. Optionally, the TIM-3 is human TIM-3.

[0061] In some embodiments, disclosed herein is a method of promoting T cell or natural killer (NK) cell proliferation, comprising contacting a plurality of cells, including T cells, NK cells, and Gal3-expressing cells, with an anti-Gal3 antibody for a time sufficient to promote proliferation of T cells or NK cells in the plurality of cells. In some embodiments, disclosed herein is a method of promoting T cell and natural killer (NK) cell proliferation, comprising contacting a plurality of cells, including T cells, NK cells, and Gal3-expressing cells, with an anti-Gal3 antibody for a time sufficient to promote proliferation of T cells and NK cells in the plurality of cells. In some embodiments, the plurality of cells further comprises TIM-3-expressing cells. In some embodiments, the anti-Gal3 antibody binds to Gal3 and disrupts the interaction of Gal3 with TIM-3. In some embodiments, the anti-Gal3 antibody binds to Gal3 and disrupts the interaction of Gal3 with TIM-3. In some embodiments, the anti-Gal3 antibody binds Gal3 and disrupts greater than 25%, greater than 50%, greater than 100%, or more of the interaction of Gal3 with TIM-3.

[0062] In some embodiments, the plurality of cells further comprises tumor infiltrating lymphocytes (TILs). Optionally, the plurality of cells further comprises CD3+ T lymphocytes, CD4+ T helper cells, CD8+ cytotoxic T cells, or a combination thereof. Optionally, the plurality of cells further comprises CD3+ T lymphocytes. Optionally, the plurality of cells further comprises CD4+ T helper cells. Optionally, the plurality of cells further comprises CD8+ cytotoxic T cells. Optionally, the plurality of cells further comprises CD3+ T lymphocytes and CD4+ T helper cells. Optionally, the plurality of cells further comprises CD3+ T lymphocytes and CD8+ cytotoxic T cells. Optionally, the plurality of cells further comprises CD4+ T helper cells and CD8+ cytotoxic T cells. Optionally, the plurality of cells further comprises CD3+ T lymphocytes, CD4+ T helper cells, and CD8+ cytotoxic T cells.

[0063] In some embodiments, the contacting further induces TIL proliferation. Optionally, the contacting further induces proliferation of CD3+ T lymphocytes, CD4+ T helper cells, CD8+ cytotoxic T cells, or a combination thereof. Optionally, the contacting further induces proliferation of CD3+ T lymphocytes. Optionally, the contacting further induces proliferation of CD4+ T helper cells. Optionally, the contacting further induces proliferation of CD8+ cytotoxic T cells. Optionally, the contacting further induces proliferation of CD3+ T lymphocytes and CD4+ T helper cells. Optionally, the contacting further induces proliferation of CD3+ T lymphocytes and CD8+ cytotoxic T cells. Optionally, the contacting further induces proliferation of CD4+ T helper cells and CD8+ cytotoxic T cells. Optionally, the contacting further induces proliferation of CD3+ T lymphocytes, CD4+ T helper cells, and CD8+ cytotoxic T cells.

[0064] In some embodiments, the contacting further comprises increasing proliferation of M1 macrophages. In some embodiments, the contacting further comprises decreasing the population of M2 macrophages in the TME. In some embodiments, the contacting further comprises increasing proliferation of M1 macrophages and decreasing the population of M2 macrophages in the TME.

[0065] In some embodiments, the anti-Gal3 antibody comprises at least one residue 1-6 of SEQ ID NO:1. 20. Optionally, the anti-Gal3 antibody binds to at least one amino acid residue in a Gal3 region corresponding to at least one residue 41-91 of SEQ ID NO:1. Optionally, the anti-Gal3 antibody binds to at least one amino acid residue in a Gal3 region corresponding to at least one residue 41-71 of SEQ ID NO:1. Optionally, the anti-Gal3 antibody binds to at least one amino acid residue in a Gal3 region corresponding to at least one residue 71-91 of SEQ ID NO:1. Optionally, the anti-Gal3 antibody binds to at least one amino acid residue in peptide_1, peptide_4, peptide_5, peptide_6, peptide_7, or peptide_8. Optionally, the anti-Gal3 antibody binds to at least one amino acid residue in peptide_1. Optionally, the anti-Gal3 antibody binds to at least one amino acid residue in peptide_4. Optionally, the anti-Gal3 antibody binds to at least one amino acid residue in peptide_5. In some cases, the anti-Gal3 antibody binds to at least one amino acid residue in peptide_6. In some cases, the anti-Gal3 antibody binds to at least one amino acid residue in peptide_7. In some cases, the anti-Gal3 antibody binds to at least one amino acid residue in peptide_8.

[0066] In some embodiments, the anti-Gal3 antibody has a binding affinity (K D In some embodiments, the anti-Gal3 antibody has a K of less than 1 nM. D In some embodiments, the anti-Gal3 antibody has a K of less than 1.2 nM. D In some embodiments, the anti-Gal3 antibody has a K of less than 2 nM. D In some embodiments, the anti-Gal3 antibody has a K of less than 5 nM. D In some embodiments, the anti-Gal3 antibody has a K of less than 10 nM. DIn some embodiments, the anti-Gal3 antibody has a K of less than 13.5 nM. D In some embodiments, the anti-Gal3 antibody has a K of less than 15 nM. D In some embodiments, the anti-Gal3 antibody has a K of less than 20 nM. D In some embodiments, the anti-Gal3 antibody has a K of less than 25 nM. D In some embodiments, the anti-Gal3 antibody has a K of less than 30 nM. D Includes.

[0067] In some embodiments, the anti-Gal3 antibody comprises a humanized antibody. In other embodiments, the anti-Gal3 antibody comprises a chimeric antibody. In some embodiments, the anti-Gal3 antibody comprises a full-length antibody or a binding fragment thereof. In some embodiments, the anti-Gal3 antibody comprises a bispecific antibody or a binding fragment thereof. In some embodiments, the anti-Gal3 antibody comprises a monovalent Fab', a bivalent Fab2, a single chain variable fragment (scFv), a diabody, a minibody, a nanobody, a single domain antibody (sdAb), or a camelid antibody or a binding fragment thereof.

[0068] In some embodiments, the anti-Gal3 antibody comprises a bispecific antibody or a binding fragment thereof. Exemplary bispecific antibody formats include, but are not limited to, Knobs-into-Holes (KiH), Asymmetric Re-engineering Technology-Immunoglobulin (ART-Ig), Triomab, Quadroma, Bispecific Monoclonal Antibody (BiMAb, BsmAb, BsAb, bsMab, BS-Mab, or Bi-MAb), Asymmetric, Bispecific Engagement by T Cell Receptor-based Antibodies (BEAT), Bispecific T Cell Engager (BiTE), Biclonics, Fab-scFv-Fc, Two-in-One / Dual-Action Fab (DNL), Adaptir (pre-SCORPION), Tandem diabody (TandAb), Dual Affinity Re-Targeting (DART), Nanobody, Triplebody, Tandem scFv (taFv), Triple Head, Tandem dAb / VHH, Trispecific dAb / VHH, or Quadraspecific dAb / VHH. In some cases, the anti-Gal3 antibody is a bispecific antibody or a binding fragment thereof that comprises the bispecific antibody format shown in FIG. 2 of Brinkmann and Kontermann, “The making of bispecific antibod ies,” MABS 9(2):182-212(2017).

[0069] In some embodiments, the anti-Gal3 antibody comprises a framework region selected from IgM, IgG (e.g., IgG1, IgG2, IgG3, or IgG4), IgA, or IgE. In some cases, the anti-Gal3 antibody comprises an IgM framework. In some cases, the anti-Gal3 antibody comprises an IgG (e.g., IgG1, IgG2, IgG3, or IgG4) framework. In some cases, the anti-Gal3 antibody comprises an IgG1 framework. In some cases, the anti-Gal3 antibody comprises an IgG2 framework. In some cases, the anti-Gal3 antibody comprises an IgG4 framework. In some embodiments, the anti-Gal3 antibody can further comprise an Fc mutation. In some embodiments, any one or more of the Fc or kappa regions of FIG. 37 can be paired with any of the CDR, VH / VL sequences herein, including FIGs. 35A-36B.

[0070] In some embodiments, the anti-Gal3 antibody comprises one or more mutations in the framework region, such as the CH1 domain, the CH2 domain, the CH3 domain, the hinge region, or a combination thereof. In some cases, the one or more mutations modulate Fc receptor interaction to enhance Fc effector function, such as, for example, ADCC and / or complement-dependent cytotoxicity (CDC). In some cases, the one or more mutations stabilize the antibody and / or increase the half-life of the antibody. In further cases, the one or more mutations modulate glycosylation.

[0071] In some embodiments, the Fc region comprises one or more mutations that modulate Fc receptor interaction to enhance effector function, such as, for example, ADCC and / or CDC. In such embodiments, exemplary residues that modulate effector function when mutated include S228, S239, K326, A330, I332, or E333, where the residue positions correspond to IgG1 and the residue numbering is according to Kabat numbering (EU index of Kabat et al 1991 Sequences of Proteins of Immunological Interest). In some embodiments, the one or more mutations comprise S228P, S239D, K326W, A330L, I332E, E333A, E333S, or a combination thereof. In some cases, the one or more mutations comprise S228P, S239D, I332E, or a combination thereof. In some embodiments, the one or more mutations include S228P, S239D, A330L, I332E, or a combination thereof. In some embodiments, the one or more mutations include K326W, E333S, or a combination thereof. In some embodiments, the mutation includes E333A. In some embodiments, the Fc region is an IgG4 Fc region. In some embodiments, the S228P mutation is in the hinge region of IgG4. In some embodiments, the S228P mutation enhances the stability of IgG4 by preventing Fab arm exchange.

[0072] In some embodiments, the anti-Gal3 antibody comprises a humanization score quantified as the overall sequence similarity of the humanized antibody compared to IMGT curated human germline antibodies. In some embodiments, the anti-Gal3 antibody comprises a humanization score of about 70, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, or about 95. In some embodiments, the anti-Gal3 antibody comprises a humanization score of about 80. In some embodiments, the anti-Gal3 antibody comprises a humanization score of about 83. In some embodiments, the anti-Gal3 antibody comprises a humanization score of about 85. In some embodiments, the anti-Gal3 antibody comprises a humanization score of about 87. In some embodiments, the anti-Gal3 antibody comprises a humanization score of about 90. In some cases, the anti-Gal3 antibody is about 70, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, or about 95, optionally about 80, about 85, or about 87. Optionally, the anti-Gal3 antibody comprises a light chain humanization score of about 70, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, or about 95, optionally about 80, about 83, or about 85.

[0073] In some embodiments, the anti-Gal3 antibody comprises a complementarity determining region (CDR) as defined herein. In some embodiments, the CDR is a portion of the heavy chain (VH) of the antibody. In some embodiments, the CDR is a portion of the light chain (VL) of the antibody. In some embodiments, the VH comprises a VH CDR1, a VH CDR2, and / or a VH CDR3. In some embodiments, the VH CDR1 comprises one of the sequences of SEQ ID NOs: 37-64. In some embodiments, the VH CDR2 comprises one of the sequences of SEQ ID NOs: 65-92. In some embodiments, the VH CDR3 comprises one of the sequences of SEQ ID NOs: 93-120. In some embodiments, the VL comprises a VL CDR1, a VL CDR2, and / or a VL CDR3. In some embodiments, the VL CDR1 comprises one of the sequences of SEQ ID NOs: 121-148. In some embodiments, the VL CDR2 comprises one of the sequences of SEQ ID NOs: 149-176. In some embodiments, the VL CDR3 comprises one of the sequences in SEQ ID NOs: 177-204. In some embodiments, the VH comprises one of the sequences in SEQ ID NOs: 205-232. In some embodiments, the VL comprises one of the sequences in SEQ ID NOs: 233-260. In some embodiments, the anti-Gal3 antibody comprises a hIgG4 constant region. In some embodiments, the hIgG4 constant region comprises a hIgG4 constant region within SEQ ID NOs: 261, 263, 265, or 267. In some embodiments, the anti-Gal3 antibody comprises a hκ constant region. In some embodiments, the hκ constant region comprises a hκ constant region within SEQ ID NOs: 262, 264, 266, or 268.

[0074] In some embodiments, the anti-Gal3 comprises a sequence shown in Figures 35A-B, 36A-B, or 37. In some embodiments, the anti-Gal3 antibody is selected from the group consisting of 2D10.2B2, 3B11.2G2, 4A11.2B5, 4G2.2G6, 6H6.2D6, 7D8.2D8, 12G5.D7, 13A12.2E5, 13G4.2F8, 13H12.2F8, 14H10.2C9, 15F10.2D6, 15G7.2A7, 19B5.2E6, 19D9.2E5, 20D11.2C6, 20H5.A3, 23H9.2E4, 24D12.2H9, 846.1F5, 846.2H3, 846T.1H2, 9H2.2H10, IMT001-4, IMT006-1, IMT006-5, IMT006-8, and mIMT001 (IMT001). In some embodiments, the anti-Gal3 antibody is selected from the group consisting of 2D10.2B2, 3B11.2G2, 4A11.2B5, 4G2.2G6, 6H6.2D6, 7D8.2D8, 12G5.D7, 13A12.2E5, 13G4.2F8, 13H12.2F8, 14H10.2C9, 15F10.2D6, 15G7.2A7, 19B5.2E6 , 19D9.2E5, 20D11.2C6, 20H5.A3, 23H9.2E4, 24D12.2H9, 846.1F5, 846.2H3, 846T.1H2, 9H2.2H10, IMT001-4, IMT006-1, IMT006-5, IMT006-8, or mIMT001, or any combination thereof. In some embodiments, the anti-Gal3 antibody is mIMT001 (IMT001). In some embodiments, the anti-Gal3 antibody is not mIMT001 (IMT001). In some embodiments, the anti-Gal3 antibody is 4A11.2B5. In some embodiments, the anti-Gal3 antibody is mIMT001 and / or 4A11.2B5. In some embodiments, the anti-Gal3 antibody comprises one, two, or three H CDRs from mIMT001 and / or 4A11.2B5. In some embodiments, the anti-Gal3 antibody comprises one, two, or three LCDRs from mIMT001 and / or 4A11.2B5.In some embodiments, the anti-Gal3 antibody comprises one, two, or three H CDRs from mIMT001 and / or 4A11.2B5 and one, two, or three LCDRs from mIMT001 and / or 4A11.2B5. In some embodiments, the anti-Gal3 antibody comprises one, two, or three H CDRs from mIMT001 and / or 4A11.2B5. The antibodies comprise one, two, or three HCDRs from mIMT001 and / or 4A11.2B5 and one, two, or three LCDRs from mIMT001 and / or 4A11.2B5, or which are substituted with one, two, three, four, five, six, seven, or eight amino acids. In some embodiments, the anti-Gal3 antibodies comprise one, two, or three HCDRs from mIMT001 and / or 4A11.2B5 and one, two, or three LCDRs from mIMT001 and / or 4A11.2B5, and further comprise mIMT001 and / or 4A11.2B5 VH and VL sequences (shown in Figures 36A and 36B) or sequences having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to the VH and VL sequences.

[0075] In some embodiments, the anti-Gal3 antibody is any one of IMT001-4, IMT006-1, IMT006-5, or IMT006-8. In some embodiments, the anti-Gal3 antibody is any one of IMT001-4, IMT006-1, IMT006-5, and / or IMT006-8. In some embodiments, the anti-Gal3 antibody comprises one, two, or three H CDRs from any one of IMT001-4, IMT006-1, IMT006-5, and / or IMT006-8. In some embodiments, the anti-Gal3 antibody comprises one, two, or three L CDRs from any one of IMT001-4, IMT006-1, IMT006-5, and / or IMT006-8. In some embodiments, the anti-Gal3 antibody comprises one, two or three LCDRs from any one of IMT001-4, IMT006-1, IMT006-5, and / or IMT006-8 and one, two or three LCDRs from any one of IMT001-4, IMT006-1, IMT006-5, and / or IMT006-8. In some embodiments, the anti-Gal3 antibody comprises one, two or three LCDRs from any one of IMT001-4, IMT006-1, IMT006-5, and / or IMT006-8 and one, two or three LCDRs from any one of IMT001-4, IMT006-1, IMT006-5, and / or IMT006-8, or which are substituted with one, two, three, four, five, six, seven or eight amino acids. In some embodiments, the anti-Gal3 antibody comprises one, two, or three LCDRs from any one of IMT001-4, IMT006-1, IMT006-5, and / or IMT006-8 and one, two, or three LCDRs from any one of IMT001-4, IMT006-1, IMT006-5, and / or IMT006-8, and further comprises IMT001-4, IMT006-1, IMT006-5, and / or IMT006-8 VH and VL sequences (shown in Figures 36A and 36B) or sequences with at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to the VH and VL sequences.

[0076] In some embodiments, the anti-GAL3 antibody is selected from the group consisting of 2D10.2B2, 3B11.2G2, 4A11.2B5, 4G2.2G6, 6H6.2D6, 7D8.2D8, 12G5.D7, 13A12.2E5, 13G4.2F8, 13H12.2F8, 14H10.2C9, 15F10.2D6, 15G7.2A7, 19B5.2E6, 19 D9.2E5, 20D11.2C6, 20H5.A3, 23H9.2E4, 24D12.2H9, 846.1F5, 846.2H3, 846T.1H2, 9H2.2H10, IMT001-4, IMT006-1, IMT006-5, IMT006-8, and mIMT001 (IMT001) also compete for binding with one or more of the following:

[0077] In some embodiments, the anti-GAL3 antibody comprises at least the HCDR3 of any one of the antibodies depicted in Figures 35A-36B. In some embodiments, the anti-GAL3 antibody further comprises all three HCDRs of any one of the antibodies depicted in Figures 35A-36B. In some embodiments, the anti-GAL3 antibody further comprises all three HCDRs of any one of the antibodies depicted in Figures 35A-36B. The LCDR further includes all three LCDRs in any one of the above.

[0078] In some embodiments, the anti-GAL3 antibody comprises any one of the heavy chain sequences in FIG. 36A or a sequence having at least 80% identity thereto, such as 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity thereto.

[0079] In some embodiments, the anti-GAL3 antibody comprises any one of the light chain sequences in Figure 36B or a sequence having at least 80% identity thereto, such as 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity thereto. In some embodiments, the anti-GAL3 antibody further comprises any one of the heavy chain sequences in Figure 36A or a sequence having at least 80% identity thereto, such as 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity thereto.

[0080] In some embodiments, the anti-GAL3 antibody comprises six CDRs, which have at least 80% identity, such as 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity, to any set of six CDRs in Figures 35A and 35B across their combined sequences.

[0081] In some embodiments, the anti-GAL3 antibody comprises at least one of the CDRs from FIG. 38 (with 1, 2, or 3 amino acid conservative substitutions). The anti-GAL3 antibody comprises at least two of the CDRs from FIG. 38 (with 1, 2, or 3 amino acid conservative substitutions). The anti-GAL3 antibody comprises at least three of the CDRs from FIG. 38 (with 1, 2, or 3 amino acid conservative substitutions). The anti-GAL3 antibody comprises at least four of the CDRs from FIG. 38 (with 1, 2, or 3 amino acid conservative substitutions). The anti-GAL3 antibody comprises at least five of the CDRs from FIG. 38 (with 1, 2, or 3 amino acid conservative substitutions). The anti-GAL3 antibody comprises six of the CDRs from FIG. 38 (with 1, 2, or 3 amino acid conservative substitutions). In some embodiments, the anti-GAL3 antibody comprises six of the CDRs from FIG. 38, all six being from a single bin. In some embodiments, the anti-GAL3 antibody comprises six of the CDRs from FIG. 38, or a set of six CDRs that have at least 80% identity thereto over their entire sequences.

[0082] In some embodiments, the method of inducing immune activation comprises, consists essentially of, or consists of contacting a plurality of cells, including Gal3-expressing cells and TIM-3-expressing cells, with an antibody under conditions that disrupt interaction between Gal3 and TIM-3, where the antibody specifically binds Gal3, and where upon binding of the antibody, the Gal3-expressing cells express a cytokine that induces immune activation. In some embodiments, the cytokine is an interferon or an interleukin. In some embodiments, the cytokine is IFNγ or IL-2. In some embodiments, immune activation comprises proliferation of CD3+ T lymphocytes, CD4+ T helper cells, CD8+ cytotoxic T cells, natural killer cells, or combinations thereof. In some embodiments, the method of promoting T cell or NK cell proliferation comprises, consists essentially of, or consists of contacting a plurality of cells, including T cells, NK cells, and Gal3-expressing cells, with an antibody under conditions that affect proliferation of T cells and / or NK cells in the plurality of cells, where the antibody specifically binds Gal3. In some embodiments, a method of inducing immune activation comprises, consists essentially of, or consists of contacting a plurality of cells, including Gal3-expressing cells and TIM-3-expressing cells, with an antibody under conditions that disrupt interaction between Gal3 and TIM-3, wherein the antibody specifically binds to Gal3, and Gal3-TIM-3 interaction is reduced to less than 70%, less than 60%, less than 59%, less than 50%, less than 40%, less than 34%, less than 30%, less than 20%, less than 14%, less than 10%, less than 7%, less than 5%, less than 4%, or less than 1%. The method of reducing fibrosis or propensity for fibrosis in a tissue comprises, consists essentially of, or consists of contacting the tissue with an antibody that specifically binds to the anti-Gal3 antibody under conditions such that the expression level of a fibrosis biomarker is reduced in the tissue. Some embodiments are anti-Gal3 antibodies for use in treating an immune-related disease in a subject, wherein the anti-Gal3 antibody induces activation of the immune system. In some embodiments or any of the foregoing embodiments, the anti-Gal3 antibody is 2D10.2B2, 3B11.2G2, 4A11.2B5, 4G2.2G6, 6H6.2D6, 7D8.2D8, 12G5.D7, 13A12.2E5, 13G4.2F8, 13H12.2F8, 14H10.2C9, 15F10.2D6, 15G7.2A7, 15G7.2A8, 15G7.2B9 ... Selected from the group consisting of 9B5.2E6, 19D9.2E5, 20D11.2C6, 20H5.A3, 23H9.2E4, 24D12.2H9, 846.1F5, 846.2H3, 846T.1H2, 9H2.2H10, IMT001-4, IMT006-1, IMT006-5, IMT006-8, and mIMT001 (IMT001). In some embodiments, the anti-Gal3 antibody is selected from the group consisting of 2D10.2B2, 3B11.2G2, 4A11.2B5, 4G2.2G6, 6H6.2D6, 7D8.2D8, 12G5.D7, 13A12.2E5, 13G4.2F8, 13H12.2F8, 14H10.2C9, 15F10.2D6, 15G7.2A7, 19B5.2E6 , 19D9.2E5, 20D11.2C6, 20H5.A3, 23H9.2E4, 24D12.2H9, 846.1F5, 846.2H3, 846T.1H2, 9H2.2H10, IMT001-4, IMT006-1, IMT006-5, IMT006-8, or mIMT001, or any combination thereof. In some embodiments or any of the foregoing embodiments, the anti-Gal3 antibody is mIMT001 (IMT001). In some embodiments or any of the foregoing embodiments, the anti-Gal3 antibody is mIMT001 (IMT001). In some embodiments or any of the foregoing embodiments, the anti-Gal3 antibody is 4A11.2B5, IMT001-4, IMT006-1, IMT006-5, and / or IMT006-8.In some embodiments or any of the foregoing embodiments, the anti-Gal3 antibody is mIMT001, 4A11.2B5, IMT001-4, IMT006-1, IMT006-5, and / or IMT006-8. In some embodiments or any of the foregoing embodiments, the anti-Gal3 antibody is one or more of IMT001-4, IMT006-1, IMT006-5, or IMT006-8. In some embodiments or any of the foregoing embodiments, the anti-Gal3 antibody is mIMT001 (IMT001). In some embodiments or any of the foregoing embodiments, the anti-Gal3 antibody is IMT001-4, IMT006-1, IMT006-5, and / or IMT006-8.

[0083] Regarding the properties of the various antibodies, IMT001-4, IMT006-1, and IMT006-5 are humanized antibodies. mIMT001 is the mouse antibody from which IMT001 is derived. 4A11.2B5 is the original mouse antibody from which IMT006-1 and IMT006-5 are derived. mIMT001, 2D10.2B2, 3B11.2G2, 4A11.2B5, 4G2.2G6, 6H6.2D6, 7D8.2D8, 12G5.D7, 13A12.2E5, 13G4.2F8, 13H12.2F8, 14H10.2C9, 15F10.2D6, 15G7.2A7, 19B5.2E6, 19D9.2E5, 20D11.2C6, 20H5.A3, 23H9.2E4, 24D12.2H9, 846.1F5, 846.2H3, 846T.1H2, and 9H2.2H10 are all mouse antibodies. IMT001-4, IMT006-1, IMT006-5, and IMT006-8 are all humanized antibodies.

[0084] Treatment method In some embodiments, a method of inducing immune activation is provided, comprising contacting a plurality of cells comprising, consisting essentially of, or consisting of Gal3-expressing cells and TIM-3-expressing cells with an antibody under conditions that disrupt the interaction of Gal3 with TIM-3. Disclosed herein are methods that consist of, or consist of: In some embodiments, the antibody is an anti-Gal3 antibody.

[0085] In some embodiments, disclosed herein are methods of reducing fibrosis, comprising contacting a tissue containing Gal3-expressing cells and at least one fibrosis biomarker with an anti-Gal3 antibody for a time sufficient to reduce the expression of at least one fibrosis biomarker in the tissue. In some cases, the anti-Gal3 antibody results in a reduction in the accumulation of one or more extracellular matrix proteins in tissues, including, but not limited to, collagen.

[0086] In some embodiments, the anti-Gal3 antibody is not IMT001. In some embodiments, the antibody is IMT001. In some embodiments, the anti-Gal3 antibody is 4A11.2B5. In some embodiments, the anti-Gal3 antibody is IMT001-4, IMT006-1, IMT006-5, or IMT006-8.

[0087] In some embodiments, the anti-Gal3 antibody inhibits or disrupts the interaction of Gal3 with TIM-3. In some embodiments, the Gal3-TIM-3 interaction is at least 99%, 95%, 90%, 80%, 78%, 70%, 66%, 60%, 56%, 52%, 50%, 40%, 30%, 29%, 27%, 20%, 19%, 17%, 10%, 5%, 4%, 3%, 2%, 1%, 0%, about 99%, about 95%, about 90%, about 80%, about 78%, about 70%, about 66%, about 60%, about 56%, about 52%, about 50%, about 40%, about 30%, about 29%, about 28%, about 36%, about 38%, about 39%, about 40%, about 45%, about 45%, about 40%, about 5 ... 7%, about 20%, about 19%, about 17%, about 10%, about 5%, about 4%, about 3%, about 2%, about 1%, about 0%, less than 99%, less than 95%, less than 90%, less than 80%, less than 78%, less than 70%, less than 66%, less than 60%, less than 56%, less than 52%, less than 50%, less than 40%, less than 30%, less than 29%, less than 27%, less than 20%, less than 19%, less than 17%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%.

[0088] In some embodiments, the anti-Gal3 antibody does not inhibit or disrupt the interaction of Gal3 with TIM-3.

[0089] In some embodiments, the interaction occurs at one or more residues of GAL3 selected from regions 145-168, 160-177, or 165-184, where the residue positions correspond to positions 145-168, 160-177, or 165-184 of SEQ ID NO:1. In some embodiments, the interaction occurs at one or more residues of GAL3 selected from regions 149-156, 152-168, 163-169, or 163-171, where the residue positions correspond to positions 149-156, 152-168, 163-169, or 163-171 of SEQ ID NO:1. In some embodiments, the interaction occurs at one or more residues of TIM-3 selected from regions 90-122, or 82-111, where the residue positions correspond to positions 90-122, or 82-111 of SEQ ID NO:2. In some embodiments, the interaction occurs at one or more residues of TIM-3 selected from regions 91-111, 107-117, 96-102, 100-106, or 92-119, where residue positions herein correspond to positions 91-111, 107-117, 96-102, 100-106, or 92-119 of SEQ ID NO:2.

[0090] In some embodiments, upon binding with the antibody, Gal3-expressing cells express cytokines that induce immune activation. As used herein, the term "cytokine" refers to small proteins, polypeptides, or peptides involved in cell signaling. Cytokines include chemokines, interferons, interleukins, lymphokines, tumor necrosis factors, CCL1, CCl2, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL29, CCL30, CCL31, CCL32, CCL33, CCL34, CCL35, CCL36, CCL37, CCL38, CCL39, CCL40, CCL41, CCL42, CCL43, CCL44, CCL45, CCL46, CCL47, CCL48, CCL49, CCL50, CCL51, CCL52, CCL53, CCL54, CCL55, CCL56, CCL57, CCL58, CCL59, CCL60, CCL61, CCL62, CCL63, CCL64, CCL65, CCL66, CCL67, CCL68, CCL69, CCL70, CCL71, CCL72, CCL73, CCL74, CCL75, CCL76, CCL77, CCL78, CCL79, CCL80, CCL81, CCL82, CCL83, CCL84, CCL85, CCL86, CCL87, CCL88, CCL CL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, C XCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, CX3CL1, XCL1, XCL2, INFα, INFβ, INFγ, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL- 7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, Examples of such antibodies include, but are not limited to, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, IL-37, IL-38, GM-CSF, TNFα, TNFβ, TNFγ, TNFSF4, TNFSF5, TNFSF6, TNFSF7, TNFSF8, TNFSF9, TNFSF10, TNFSF11, TNFSF12, TNFSF13, TNFSF13B, TNFSF14, TNFSF15, TNFSF18, or TNFSF19, or any combination thereof.

[0091] In some embodiments, the cytokine is an interferon. In some embodiments, the interferon is IFNγ. In some embodiments, the antibody provides IFNγ production that is 100%, 150%, 160%, 170%, 180%, 190%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more of the IFNγ production from an isotype antibody. In some embodiments, the cytokine is an interleukin. In some embodiments, the interleukin is IL-2.

[0092] In some embodiments, the immune activation or immune system activation comprises, consists essentially of, or consists of proliferation of CD3+ T lymphocytes, CD4+ T helper cells, CD8+ cytotoxic T cells, THF cells, Th3 cells, Th17 cells, natural killer T (NKT) cells, or natural killer (NK) cells, or a combination thereof. In some embodiments, the immune activation or immune system activation comprises, consists essentially of, or consists of promotion of T cell or NK cell proliferation. In some embodiments, the immune activation or immune system activation comprises, consists essentially of, or consists of an increase in M1 macrophage, neutrophil, mast cell, eosinophil, basophil, or dendritic cell populations in a plurality of cells. In some embodiments, the immune activation or immune system activation comprises, consists essentially of, or consists of a decrease in M2 macrophage populations in a plurality of cells.

[0093] In some embodiments, the TIM-3 is human TIM-3.

[0094] In some embodiments, the plurality of cells comprises, consists essentially of, or consists of tumor cells. In some embodiments, the plurality of cells resides within a tumor microenvironment (TME) and comprises, consists essentially of, or consists of tumor cells and immune cells. In some embodiments, the TME comprises tumor cells, immune cells, cancer-associated fibroblasts, myeloid-derived suppressor cells, neutrophils, tumor-infiltrating lymphocytes (TILs), or any combination thereof. In some embodiments, the plurality of cells comprises, consists essentially of, or consists of CD3+ T lymphocytes, CD4+ T helper cells, CD8+ cytotoxic T cells, THF cells, Th3 cells, Th17 cells, natural killer T (NKT) cells, natural killer (NK) cells, M1 macrophages, neutrophils, mast cells, eosinophils, basophils, or dendritic cells. In some embodiments, the anti-TIM-3 antibody induces a reduction of tumor cells within the TME.

[0095] In some embodiments, the antibody comprises a nucleic acid sequence corresponding to at least one of residues 1-20 of SEQ ID NO:1. In some embodiments, the antibody binds to at least one amino acid residue in a Gal3 region corresponding to at least one residue 41-91 of SEQ ID NO:1. In some embodiments, the antibody binds to at least one amino acid residue in a Gal3 region corresponding to at least one residue 41-71 of SEQ ID NO:1. In some embodiments, the antibody binds to at least one amino acid residue in a Gal3 region corresponding to at least one residue 71-91 of SEQ ID NO:1.

[0096] In some embodiments, the antibody binds to at least one amino acid residue of peptide_1, peptide_2, peptide_3, peptide_4, peptide_5, peptide_6, peptide_7, peptide_8, peptide_9, peptide_10, peptide_11, peptide_12, peptide_13, peptide_14, peptide_15, peptide_16, peptide_17, peptide_18, peptide_19, peptide_20, peptide_21, peptide_22, peptide_23, or peptide_24, or any combination thereof.

[0097] In some embodiments, the antibody is 1 fM, 10 fM, 100 fM, 1 pM, 10 pM, 100 pM, 1 nM, 1.2 nM, 2 nM, 5 nM, 10 nM, 13.5 nM, 15 nM, 20 nM, 25 nM, 30 nM, 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 1 μM, 10 μM, 100 μM, about 1 fM, about 10 fM, about 100 fM, about 1 pM, about 10 pM, about 100 pM, about 1 nM, about 1.2 nM, about 2 nM, about 5 nM, about 10 nM, about 13.5 nM, about 15 nM, about 20 nM, about 25 nM, about 30 nM M, about 100 nM, about 200 nM, about 300 nM, about 400 nM, about 500 nM, about 1 μM, about 10 μM, about 100 μM, less than 1 fM, less than 10 fM, less than 100 fM, less than 1 pM, less than 10 pM, less than 100 pM, less than 1 nM, less than 1.2 nM, less than 2 nM, less than 5 nM, less than 10 nM, less than 13.5 nM, less than 15 nM, less than 20 nM, less than 25 nM, less than 30 nM, less than 100 nM, less than 200 nM, less than 300 nM, less than 400 nM, less than 500 nM, less than 1 μM, 10 μM, or less than 100 μM.

[0098] In some embodiments, the antibody comprises a humanized antibody. In some embodiments, the antibody comprises a full-length antibody or binding fragment thereof. In some embodiments, the antibody comprises a bispecific antibody or binding fragment thereof. In some embodiments, the antibody comprises a monovalent Fab', a bivalent Fab2, a single chain variable fragment (scFv), a diabody, a minibody, a nanobody, a single domain antibody (sdAb), or a camelid antibody or binding fragment thereof. In some embodiments, the antibody comprises an IgG framework. In some embodiments, the antibody comprises an IgG1, IgG2, or IgG framework. In some embodiments, the antibody further comprises an Fc mutation. In some embodiments, the antibody comprises a chimeric antibody.

[0099] In some embodiments, the anti-Gal3 antibody is selected from the group consisting of 2D10.2B2, 3B11.2G2, 4A11.2B5, 4G2.2G6, 6H6.2D6, 7D8.2D8, 12G5.D7, 13A12.2E5, 13G4.2F8, 13H12.2F8, 14H10.2C9, 15F10.2D6, 15G7.2A7, 19B5.2E6, In some embodiments, the anti-Gal3 antibody is selected from the group consisting of 19D9.2E5, 20D11.2C6, 20H5.A3, 23H9.2E4, 24D12.2H9, 846.1F5, 846.2H3, 846T.1H2, 9H2.2H10, IMT001-4, IMT006-1, IMT006-5, IMT006-8, and mIMT001 (IMT001). In some embodiments, the anti-Gal3 antibody is mIMT001 (IMT001). In some embodiments, the anti-Gal3 antibody is not mIMT001 (IMT001). In some embodiments, the anti-Gal3 antibody is 4A11.2B5. In some embodiments, the anti-Gal3 antibody is mIMT001 and / or 4A11.2B5. In some embodiments, the antibody is selected from the group consisting of these antibodies (IM The antibody competes for binding to Gal3 with one or more of IMT001-4, IMT006-1, IMT006-5, and / or IMT006-8. In some embodiments, the antibody is one or more of IMT001-4, IMT006-1, IMT006-5, or IMT006-8. In some embodiments, the antibody for the method comprises one or more of the CDRs from one or more of IMT001-4, IMT006-1, IMT006-5, or IMT006-8. In some embodiments, the antibody for the method comprises one or more of the VH, VL, or VH and VL from one or more of IMT001-4, IMT006-1, IMT006-5, or IMT006-8.

[0100] In some embodiments, the method further comprises administering an anti-Gal3 antibody to the subject prior to the contacting step.

[0101] In some embodiments, the subject has been diagnosed with cancer.

[0102] In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is breast cancer, colorectal cancer, renal cancer, liver cancer, lung cancer, prostate cancer, melanoma, bladder cancer, uterine cancer, pancreatic cancer, thyroid cancer, brain cancer, bone cancer, sarcoma, or gastric cancer. In some embodiments, the lung cancer is non-small cell lung cancer (NSCLC), lung adenocarcinoma, squamous cell carcinoma, large cell carcinoma, or small cell lung cancer (SCLC).

[0103] In some embodiments, the cancer is a hematological tumor, and is leukemia, non-Hodgkin's lymphoma, Hodgkin's lymphoma, multiple myeloma, acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, acute monocytic leukemia, or any combination thereof.

[0104] In some embodiments, the cancer is a metastatic cancer. In some embodiments, the cancer is a recurrent or refractory cancer. The staging of the cancer or tumor is used to determine the progression of the spread of the cancer or tumor within the patient. The commonly recognized criteria for the classification of solid tumors are the TNM classification criteria, which differentiate tumors based on the size of the tumor (T), the extent of spread to lymph nodes (N), and metastatic potential (M). These classifications are further grouped into stages, with stage 0 growth being non-malignant, stage I and II tumors being locally contained, stage III tumors having spread to nearby lymph nodes, and stage IV tumors having metastasized. Although the TNM criteria are a widely used classification method, alternative or modified criteria may be used that represent the behavior of a particular cancer type. Thus, although these criteria are useful in determining progression, the prognosis of early or late stages of a cancer or tumor is independent of the particular classification.

[0105] Disclosed herein, in some embodiments, is a method of reducing fibrosis or its tendency in a tissue of a subject by contacting the tissue with an antibody. In some embodiments, the antibody specifically binds to Gal3 or is an anti-Gal3 antibody. In some embodiments, the contacting induces an expression level of at least one fibrosis biomarker to be reduced in the tissue. In some embodiments, the blocking solution comprises at least one TIM-3 expressing cell. In some embodiments, the anti-Gal3 antibody interferes with the interaction of Gal3 with TIM-3. In some embodiments, the anti-Gal3 antibody does not interfere with the interaction of Gal3 with TIM-3.

[0106] In some embodiments, reducing fibrosis or its propensity in a tissue comprises preventing fibrosis from occurring in normal tissue. In some embodiments, reducing fibrosis or its propensity in a tissue comprises slowing or stopping the progression of fibrosis in fibrotic tissue. In some embodiments, reducing fibrosis or its propensity in a tissue comprises preventing fibrosis in fibrotic tissue. In some embodiments, reducing fibrosis or the propensity for fibrosis in a tissue comprises eliminating fibrosis in a fibrotic tissue.

[0107] Also described herein, in some embodiments, are methods for monitoring the progression of tissue fibrosis by monitoring one or more fibrosis biomarkers.Disclosed herein, in some embodiments, are methods for treating tissue fibrosis with anti-Gal3 antibodies, wherein the anti-Gal3 antibody disrupts the interaction of Gal3 with TIM-3.

[0108] In some embodiments, at least one fibrosis biomarker comprises, consists essentially of, or consists of alpha-smooth muscle actin (alpha-SMA), fibronectin, collagen, collagen I, collagen III, collagen IV, elastin, laminin, hyaluronic acid, or proteoglycan, or any combination thereof. In some embodiments, at least one fibrosis biomarker comprises, consists essentially of, or consists of alpha-smooth muscle actin (alpha-SMA). In some embodiments, at least one fibrosis biomarker comprises, consists essentially of, or consists of fibronectin. In some embodiments, at least one fibrosis biomarker comprises, consists essentially of, or consists of alpha-smooth muscle actin (alpha-SMA) and fibronectin.

[0109] In some embodiments, the tissue is selected from the group consisting of liver tissue, kidney tissue, skin tissue, lung tissue, heart tissue, brain tissue, colorectal tissue, intestinal tissue, bone marrow tissue, breast tissue, prostate tissue, bladder tissue, uterine tissue, pancreatic tissue, thyroid tissue, muscle tissue, stomach tissue, and soft tissue. In some embodiments, the tissue is kidney tissue or liver tissue.

[0110] In some embodiments, expression of at least one fibrosis biomarker in tissue treated with an anti-Gal3 antibody is less than expression of at least one fibrosis biomarker in control tissue treated with an mIgG2b antibody.

[0111] In some embodiments, anti-Gal3 antibodies result in a decrease in the accumulation of extracellular matrix (ECM) proteins in tissues. In some embodiments, the extracellular matrix is ​​a protein that is involved in the formation of a number of proteins, including agrin, nidogen, cadherin, clathrin, collagen, defensin, elastin, entactin, fibrillin, fibronectin, keratin, laminin, microtubule-actin crosslinking factor 1, SPARC-like protein, nesprin (Nesprin 1, Nesprin 2, Nesprin 3, Nesprin 4, Nesprin 5, Nesprin 6, Nesprin 7, Nesprin 8, Nesprin 9, Nesprin 10, Nesprin 11, Nesprin 12, Nesprin 13, Nesprin 14, Nesprin 15, Nesprin 16, Nesprin 17, Nesprin 18, Nesprin 19, Nesprin 20, Nesprin 21, Nesprin 22, Nesprin 23, Nesprin 24, Nesprin 25, Nesprin 26, Nesprin 27, Nesprin 28, Nesprin 29, Nesprin 30, Nesprin 31, Nesprin 32, Nesprin 33, Nesprin 34, Nesprin 35, Nesprin 36, Nesprin 37, Nesprin 38, Nesprin 39 ... In some embodiments, the tissue comprises, consists essentially of, or consists of a protein selected from the group consisting of fibrogenic cytokines TGF-β, TGF-β1, IL-1β, TNF-α, or GM-CSF. In some embodiments, the tissue has high expression of a fibrogenic cytokine.

[0112] In some embodiments, the antibody binds to at least one amino acid residue in a Gal3 region corresponding to at least one residue 1-20 of SEQ ID NO:1. In some embodiments, the antibody binds to at least one amino acid residue in a Gal3 region corresponding to at least one residue 41-91 of SEQ ID NO:1. In some embodiments, the antibody binds to at least one amino acid residue in a Gal3 region corresponding to at least one residue 41-91 of SEQ ID NO:1. In some embodiments, the antibody binds to at least one amino acid residue in the Gal3 region corresponding to at least one of residues 41-71 of SEQ ID NO:1. In some embodiments, the antibody binds to at least one amino acid residue in the Gal3 region corresponding to at least one of residues 71-91 of SEQ ID NO:1.

[0113] In some embodiments, the subject has been diagnosed with a fibrotic disease or fibrosis. In some embodiments, the subject has been diagnosed with a fibrotic disease. In some embodiments, the fibrotic disease is renal fibrosis. In some embodiments, the fibrotic disease is liver fibrosis. In some embodiments, the antibody is formulated for systemic administration. In some embodiments, the antibody is formulated for parenteral administration. In some embodiments, the subject is a mammal.

[0114] In some embodiments, the fibrotic disease or fibrosis is liver fibrosis, bridging fibrosis, cirrhosis, renal (kidney) fibrosis, pulmonary fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, cardiovascular fibrosis, arterial fibrosis, venous thrombosis, arthrofibrosis, Crohn's disease, Dupuytren's contracture, keloid, mediastinal fibrosis, myelofibrosis, Peyronie's disease, nephrogenic systemic fibrosis, progressive massive fibrosis, retroperitoneal fibrosis, or systemic sclerosis.In some embodiments, the fibrotic disease is renal (kidney) fibrosis.In some embodiments, the fibrotic disease is liver fibrosis.

[0115] In some embodiments, the method includes an antibody that binds Gal3 but disrupts the interaction of Gal3 with TIM-3. This can be a direct disruption of the interaction zone of Gal3 with TIM-3, or an indirect change such as binding that results in a conformational change in Gal3 such that it is no longer bound or active with TIM-3. This can also be achieved by binding to the first section of Gal3, some other portion of the antibody disrupting or altering the interaction of Gal3 with TIM-3.

[0116] In some embodiments, the use of anti-Gal3 antibodies in the manufacture of a medicament or composition is disclosed. In some embodiments, the medicament or composition is used to treat immune-related diseases. In some embodiments, the medicament or composition is used to treat cancer. In some embodiments, the medicament or composition is used to treat fibrotic diseases or fibrosis.

[0117] In some embodiments, the anti-Gal3 antibody is for use in treating a disease in a subject. In some embodiments, the anti-Gal3 antibody inhibits the interaction of Gal3 with TIM-3. In some embodiments, the anti-Gal3 antibody does not inhibit the interaction of Gal3 with TIM-3.

[0118] In some embodiments, the anti-Gal3 antibody is for use in the treatment of a disease, the disease is immune-related, and the anti-Gal3 antibody induces activation of the immune system of the subject. In some embodiments, the immune-related disease is an autoimmune disease. In some embodiments, the immune-related disease is an immunodeficiency. In some embodiments, the immune deficiency is immunosenescence, humoral immune deficiency, B cell deficiency, T cell deficiency, neutropenia, asplenia, or complement deficiency. In some embodiments, the activation of the immune system is a reduction in CD3+ T lymphocytes, CD4+ T helper cells, CD8+ cytotoxic T cells, T FH cell, T h 3 cells, T h In some embodiments, activation of the immune system comprises the expansion of M17 cells, natural killer T (NKT) cells, NK cells, or M1 macrophages, or a combination thereof. In some embodiments, activation of the immune system comprises the reduction of M2 macrophages.

[0119] In some embodiments, the anti-Gal3 antibody is for use in the treatment of a disease, the disease is cancer, and the anti-TIM-3 antibody is for use in the treatment of the cancer.

[0120] In some embodiments, the anti-Gal3 antibody is for use in the treatment of a disease. and the disease is a fibrotic disease or fibrosis. In some embodiments, the anti-Gal3 antibody for use in treating the disease results in a decrease in accumulation of extracellular matrix proteins in a tissue.

[0121] In some embodiments, the anti-Gal3 antibody for use in treating a disease is administered in combination with an additional therapeutic agent, such as an immune checkpoint modulator, a chemotherapeutic agent, a targeted therapeutic agent, a hormonal therapeutic agent, a stem cell-based therapeutic agent, surgery, or radiation therapy.

[0122] In some embodiments, the antibody is formulated for systemic administration, hi some embodiments, the antibody is formulated for parenteral, subcutaneous, intramuscular, intradermal, or intravenous administration, or any combination thereof.

[0123] In some embodiments, the anti-Gal3 antibody is administered to the subject in combination with an additional therapeutic agent. In some embodiments, the additional therapeutic agent comprises an immunotherapeutic agent. In some embodiments, the additional therapeutic agent comprises an immune checkpoint modulator. In some embodiments, the additional therapeutic agent comprises a chemotherapeutic agent, a targeted therapeutic agent, a hormonal therapeutic agent, or a stem cell-based therapeutic agent.

[0124] In some embodiments, the additional therapeutic agent comprises an immunotherapy agent. In some embodiments, the immunotherapy is adoptive cellular therapy. Exemplary cell therapies include Adaptimmune's AFP TCR, MAGE-A10 TCR, or NY-ESO-TCR; Unum Therapeutics' ACTR087 / rituximab; Juno Therapeutics' anti-BCMA CAR-T cell therapy, anti-CD19 "armored" CAR-T cell therapy, JCAR014, JCAR018, JCAR020, JCAR023, JCAR024, or JTCR016; Celgene / Juno Therapeutics' JCAR017; Intrexon's anti-CD19 CAR-T cell therapy; Kite Pharma's anti-CD19 CAR-T cell therapy, axicabtagene ciloleucel, KITE-718, KITE-439, or NY-ESO-1 T cell receptor therapy; Sorrento Therapeutics' anti-CEA CAR-T therapy; TNK Therapeutics / Sorrento anti-PSMA CAR-T cell therapy from Atara Biotherapeutics; ATA520 from Atara Biotherapeutics; AU101 and AU105 from Aurora BioPharma; valtalucel-T (CMD-003) from Cell Medica; bb2121 from bluebird bio; BPX-501, BPX-601, or BPX-701 from Bellicum Pharmaceuticals; BSK01 from Kiromic; IMCgp100 from Immunocore; JTX-2011 from Jounce Therapeutics; LN-144 or LN-145 from Lion Biotechnologies; MB-101 or MB-102 from Mustang Bio; NKR-2 from Celyad; PNK-007 from Celgene; tisagenlecleucel-T from Novartis Pharmaceuticals; or TT12 from Tessa Therapeutics.

[0125] In some embodiments, the immunotherapy is a dendritic cell-based therapy.

[0126] In some embodiments, the immunotherapy comprises a cytokine-based therapy including, for example, an interleukin (IL) such as IL-2, IL-15, or IL-21, interferon (IFN)-α, or granulocyte-macrophage colony-stimulating factor (GM-CSF).

[0127] In some embodiments, the immunotherapy comprises an immune checkpoint modulator. Exemplary immune checkpoint modulators include nivolumab (Opdivo) from Bristol-Myers Squibb, pembrolizumab (Keytruda) from Merck, AGEN 2034 from Agenus, BGB-A317 from BeiGene, Bl-754091 from Boehringer-Ingelheim Pharmaceuticals, CBT-501 (genolimuzumab) from CBT Pharmaceuticals, INCSHR1210 from Incyte, JNJ-63723283 from Janssen Research & Development, MEDI0680 from MedImmune, MGA012 from MacroGenics, PDR001 from Novartis Pharmaceuticals, PF-06801591 from Pfizer, and REGN2810 from Regeneron Pharmaceuticals / Sanofi. PD-1 modulators such as (SAR439684), or TSR-042 from TESARO; CTLA-4 modulators such as ipilimumab (Yervoy), or AGEN1884 from Agenus; PD-L1 modulators such as durvalumab (Imfinzi) from AstraZeneca, atezolizumab (MPDL3280A) from Genentech, avelumab from EMD Serono / Pfizer, CX-072 from CytomX Therapeutics, FAZ053 from Novartis Pharmaceuticals, KN035 from 3D Medicine / Alphamab, LY3300054 from Eli Lilly, or M7824 (anti-PD-L1 / TGFβ trap) from EMD Serono; BMS-986016 from Bristol-Myers Squibb, IMP701 from Novartis Pharmaceuticals, or M7824 (anti-PD-L1 / TGFβ trap) from Novartis LAG3 modulators such as LAG525 from Pharmaceuticals, or REGN3767 from Regeneron Pharmaceuticals;OX40 modulators such as BMS-986178 from Bristol-Myers Squibb, GSK3174998 from GlaxoSmithKline, INCAGN1949 from Agenus / Incyte, MEDI0562 from MedImmune, PF-04518600 from Pfizer, or RG7888 from Genentech; GITR modulators such as GWN323 from Novartis Pharmaceuticals, INCAGN1876 from Agenus / Incyte, MEDI1873 from MedImmune, MK-4166 from Merck, or TRX518 from Leap Therapeutics; KIR modulators such as lirilumab from Bristol-Myers Squibb; or TIM modulators such as MBG453 from Novartis Pharmaceuticals or TSR-022 from Tesaro;

[0128] In some embodiments, the additional therapeutic agent comprises a chemotherapeutic agent. Exemplary chemotherapeutic agents include alkylating agents such as cyclophosphamide, mechlorethamine, chlorambucil, melphalan, dacarbazine, or nitrosoureas; anthracyclines such as daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, or valrubicin; cytoskeletal disruptors such as paclitaxel, docetaxel, abraxane, or taxotere; epothilones; histone deacetylase inhibitors such as vorinostat or romidepsin; Topoisomerase I inhibitors, such as irinotecan or topotecan; Topoisomerase II inhibitors, such as etoposide, teniposide, or tafluposide; Kinase inhibitors, such as bortezomib, erlotinib, gefitinib, imatinib, vemurafenib, or vismodegib; Azacitidine, azathioprine, capecitabine, cytarabine, doxifluridine, fluorouracil, gemcitabine, hydrozyurea, mercaptopurine, methotrexate, or thioguanidine. nucleotide and precursor analogs such as rifampin; platinum-based agents such as carboplatin, cisplatin, or oxaliplatin; retinoid agents such as tretinoin, alitretinoin, or bexarotene; or vinca alkaloid agents such as vinblastine, vincristine, vindesine, or vinorelbine. Not determined.

[0129] In some embodiments, the additional therapeutic agent comprises a hormonal therapeutic agent.Exemplary hormonal therapeutic agents include, but are not limited to, aromatase inhibitors such as letrozole, anastrozole, exemestane or aminoglutethimide; gonadotropin-releasing hormone (GnRH) analogs such as leuprorelin or goserelin; selective estrogen receptor modulators (SERMs) such as tamoxifen, raloxifene, toremifene or fulvestrant; antiandrogens such as flutamide or bicalutamide; progestogens such as megestrol acetate or medroxyprogesterone acetate; androgens such as fluoxymesterone; estrogens such as estrogen diethylstilbestrol (DES), estrace or polyestradiol phosphate; or somatostatin analogs such as octreotide.

[0130] In some embodiments, the additional therapeutic agent is a first line therapeutic agent.

[0131] In some embodiments, the anti-Gal3 antibody and the additional therapeutic agent are administered simultaneously. In some embodiments, the anti-Gal3 antibody and the additional therapeutic agent are administered sequentially. In some embodiments, the anti-Gal3 antibody is administered to the subject prior to administration of the additional therapeutic agent. In some embodiments, the anti-Gal3 antibody is administered to the subject after administration of the additional therapeutic agent.

[0132] In some embodiments, the additional therapeutic agent and the anti-Gal3 antibody are formulated as separate dosages.

[0133] In some embodiments, the subject undergoes surgery. In some cases, the anti-Gal3 antibody and optionally additional therapeutic agent are administered to the subject before surgery. In some embodiments, the anti-Gal3 antibody and optionally additional therapeutic agent are administered to the subject after surgery.

[0134] In some embodiments, the subject is receiving radiation. In some embodiments, the anti-Gal3 antibody and optionally additional therapeutic agent are administered to the subject before or after radiation therapy. In some cases, the anti-Gal3 antibody and optionally additional therapeutic agent are administered to the subject before receiving radiation.

[0135] In some embodiments, the subject is a mammal, hi some embodiments, the subject is a human.

[0136] In some embodiments, disclosed herein are methods of reducing fibrosis or propensity for fibrosis in a tissue by contacting the tissue with an antibody that specifically binds Gal3. Also described herein, in some embodiments, are methods of disrupting Gal3-TIM-3 interaction with an antibody that specifically binds Gal3 under conditions that reduce expression of one or more fibrosis biomarkers in the tissue.

[0137] In certain embodiments, disclosed herein are methods of reducing fibrosis or a propensity for fibrosis in a tissue, comprising contacting the tissue with an antibody that specifically binds Gal3 under conditions such that the expression level of a fibrosis biomarker is reduced in the tissue. In some embodiments, the tissue further comprises TIM-3 expressing cells. In some embodiments, the antibody further interferes with the interaction of Gal3 with TIM-3. In some embodiments, the antibody does not interfere with the interaction of Gal3 with TIM-3. In some embodiments, the at least one fibrosis biomarker comprises alpha-smooth muscle actin (alpha-SMA). In some embodiments, In some embodiments, the at least one fibrosis biomarker comprises fibronectin. In some embodiments, the at least one fibrosis biomarker comprises α-smooth muscle actin (α-SMA) and fibronectin. In some embodiments, the tissue is kidney or liver tissue. In some embodiments, the tissue is selected from the group consisting of liver tissue, kidney tissue, skin tissue, lung tissue, heart tissue, brain tissue, intestine tissue, bone marrow tissue, and soft tissue. In some embodiments, the expression of the at least one fibrosis biomarker in the tissue treated with the antibody is less than the expression of said at least one fibrosis biomarker in a control tissue treated with an mIgG2b antibody. In some embodiments, the antibody results in a reduced accumulation of an extracellular matrix protein in the tissue. In some embodiments, the extracellular matrix protein comprises collagen. In some embodiments, the tissue comprises collagen-producing cells. In some embodiments, the collagen-producing cells are fibroblasts. In some embodiments, the fibroblasts are activated by a fibrogenic cytokine. In some embodiments, the fibrogenic cytokine is TGF-β1. In some embodiments, the tissue has high TGF-β1 expression.

[0138] Antibody production In some embodiments, anti-Gal3 antibodies are raised by standard protocols by injecting the production animal with an antigen composition. See, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. When using the whole protein or a larger portion of the protein, the antibodies may be raised by immunizing the production animal with the protein and a suitable adjuvant (e.g., Freund's, Freund's complete, oil-in-water emulsion, etc.). When using smaller peptides, it is advantageous to conjugate the peptide with a larger molecule to produce an immunostimulatory conjugate. Commonly used conjugate proteins that are commercially available for such use include bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH). To raise antibodies against a specific epitope, peptides derived from the complete sequence may be used. Alternatively, to generate antibodies against a relatively short peptide portion of the protein target, a good immune response may be elicited when the polypeptide is conjugated to a carrier protein such as ovalbumin, BSA, or KLH.

[0139] Polyclonal or monoclonal anti-Gal3 antibodies can be produced from animals genetically engineered to produce human immunoglobulins. Transgenic animals can be produced by first creating a "knockout" animal that does not produce the animal's natural antibodies, and then stably transforming the animal with human antibody loci (e.g., by using human artificial chromosomes). In such cases, only human antibodies are then produced by the animal. Techniques for producing such animals and deriving antibodies therefrom are described in U.S. Patent Nos. 6,162,963 and 6,150,584, which are incorporated herein by reference in their entirety. Such antibodies can also be referred to as human xenogeneic antibodies.

[0140] Alternatively, anti-Gal3 antibodies can be produced from phage libraries containing human variable regions, see U.S. Patent No. 6,174,708, which is incorporated herein by reference in its entirety.

[0141] In some aspects of some embodiments disclosed herein, the anti-Gal3 antibodies are produced by hybridomas.

[0142] For monoclonal anti-Gal3 antibodies, hybridomas may be produced by isolation of stimulated immune cells, such as from the spleen of the inoculated animal. These cells are then cultured in a microbial cell culture medium. The fusion cells can be fused to immortalized cells, such as myeloma cells or transformed cells, which can replicate indefinitely in cell culture, thereby producing an immortal immunoglobulin-secreting cell line. The immortal cell line utilized can be selected to be deficient in enzymes required for utilization of a particular nutrient. Many such cell lines (such as myelomas) are known to those skilled in the art, for example, thymidine kinase (TK) or hypoxanthine guanine phosphoribosyltransferase (HGPRT). These deficiencies allow for selection against the fused cells by their ability to grow, for example, on hypoxanthine aminopterin thymidine medium (HAT).

[0143] In addition, anti-Gal3 antibodies may be produced by genetic engineering.

[0144] The anti-Gal3 antibodies disclosed herein can have a reduced tendency to induce undesired immune responses in humans, such as anaphylactic shock, and can also exhibit a reduced tendency to prime immune responses that prevent repeated dosing of antibody therapeutics or imaging agents (e.g., human anti-mouse antibody "HAMA" reactions). Such anti-Gal3 antibodies include, but are not limited to, humanized, chimeric, or xenogeneic human anti-Gal3 antibodies.

[0145] Chimeric anti-Gal3 antibodies can be produced by recombinant means, for example by combining mouse variable light and heavy chain regions (VK and VH) obtained from a mouse (or other animal derived) hybridoma clone with human constant light and heavy chain regions to produce an antibody that contains primarily human domains. The production of such chimeric antibodies is well known in the art and may be performed by standard means (e.g., as described in U.S. Patent No. 5,624,659, which is incorporated herein by reference in its entirety).

[0146] The term "humanized" when applied to non-human (e.g., rodent or primate) antibodies, is a hybrid immunoglobulin, immunoglobulin chain or fragment thereof that contains minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementarity determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, rabbit or primate having the desired specificity, affinity and capacity. In some embodiments, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and optimize antibody performance and to minimize immunogenicity when introduced into the human body. In some embodiments, a humanized antibody will comprise substantially all of at least one, and usually two, variable domains, with all or substantially all of the CDR regions corresponding to those of a non-human immunoglobulin and all or substantially all of the FR regions being those of a human immunoglobulin sequence. The humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), usually that of a human immunoglobulin.

[0147] Humanized antibodies can be engineered to contain human-like immunoglobulin domains and incorporate only the complementarity determining regions of an animal-derived antibody. This can be done by carefully examining the hypervariable loop sequences of the monoclonal antigen binding units or variable regions of a monoclonal antibody and matching them to the structure of human antigen binding units or human antibody chains. See, e.g., U.S. Patent No. 6,187,287, which is incorporated herein by reference in its entirety.

[0148] Methods for humanizing non-human antibodies are well known in the art. A "humanized" antibody is an antibody in which at least part of the sequence has been altered from its original form to make it more similar to a human immunoglobulin. In some variations, the heavy (H) and light (L) chain constant (C) sequences are In some embodiments, the V region is replaced with a human sequence. This can be a fusion polypeptide comprising a variable (V) region and a heterologous immunoglobulin C region. In some embodiments, the complementarity determining regions (CDRs) comprise non-human antibody sequences, while the V framework regions are converted to human sequences. See, for example, EP 0329400. In some embodiments, the V region is humanized by designing consensus sequences of human and mouse V regions and converting residues outside the CDRs that differ between the consensus sequences.

[0149] In principle, the framework sequences of humanized antibodies can serve as templates for CDR grafting; however, it has been shown that direct replacement of CDRs with such frameworks can result in significant loss of binding affinity with the antigen. Glaser et al. (1992) J. Immunol. 149:2606; Tempest et al. (1992) Biotechnology 9:266; and Shalaby et al. (1992) J. Exp. Med. 17:217. Human antibodies (HuAbs) The more homologous to the original murine antibody (muAb), the less likely the human framework will introduce distortions in the murine CDRs that could reduce affinity. Based on sequence homology searches against antibody sequence databases, HuAb IC4 provides good framework homology with muM4TS.22, although other highly homologous HuAbs, particularly kappa L chains from human subgroup I or H chains from human subgroup III, are suitable as well. Kabat et al. (1987). Various computer programs, such as ENCAD (Levitt et al. (1983) J. Mol. Biol. 168:595), can be utilized to predict the ideal sequence for the V region. Thus, the present invention encompasses HuAbs with different variable (V) regions. It is within the skill of the art to determine suitable V region sequences and optimize these sequences. Methods for obtaining antibodies with reduced immunogenicity are also described in U.S. Pat. No. 5,270,202 and European Patent No. 699,755.

[0150] Using three-dimensional models of parental and humanized sequences, humanized antibodies can be produced by a process of analysis of the parental sequences and various conceptual humanized products. Three-dimensional immunoglobulin models are familiar to those skilled in the art. Computer programs are available that illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays allows analysis of the possible role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the consensus and import sequences to achieve desired antibody characteristics, such as increased affinity for the target antigen.

[0151] The method of humanizing the subject antigen-binding unit may be as follows: The best-matching germline acceptor heavy and light chain variable regions are selected based on the homology, canonical structure and physical properties of the human antibody germline for grafting. Computer modeling of the mVH / VL to the hVH / VL to be grafted is performed to generate a prototype humanized antibody sequence. If the modeling indicates the need for framework backmutation, a second variant with the indicated FW changes is generated. The selected germline framework and DNA framework encoding the mouse CDRs are synthesized. The synthetic DNA fragment is subcloned into an IgG expression vector and the sequence is confirmed by DNA sequencing. The humanized antibody is expressed in cells such as 293F and the protein is tested, for example, in MDM phagocytosis assays and antigen binding assays. The humanized antigen-binding unit is compared to the parent antigen-binding unit in antigen-binding affinity, for example, by FACS on cells expressing the target antigen. If the affinity is more than half that of the parent antigen-binding unit, a second round of humanized variants can be generated and tested as described above.

[0152] As noted above, anti-Gal3 antibodies can be either "monovalent" or "multivalent." The former have one binding site per antigen-binding unit, while the latter contain multiple binding sites capable of binding more than one antigen of the same or different kinds. Depending on the number of binding sites, an antigen-binding unit can be bivalent (having two antigen-binding sites), trivalent (having three antigen-binding sites), tetravalent (having four antigen-binding sites), etc.

[0153] Multivalent anti-Gal3 antibodies can be further classified based on their binding specificity. A "monospecific" anti-Gal3 antibody is a molecule that can bind to one or more antigens of the same kind. A "multispecific" anti-Gal3 antibody is a molecule that has binding specificity for at least two different antigens. Although such molecules will usually only bind to two different antigens (i.e., bispecific anti-Gal3 antibodies), antibodies with additional specificities, such as trispecific antibodies, are encompassed by this expression as used herein. The present disclosure further provides multispecific anti-Gal3 antibodies. Multispecific anti-Gal3 antibodies are multivalent molecules that can bind to at least two different antigens, for example, bispecific and trispecific molecules that exhibit binding specificity for two and three different antigens, respectively.

[0154] Monoclonal antibodies can be obtained by injecting a mouse with an antigen, e.g., a composition containing Gal3 or an epitope thereof, removing the spleen to obtain B lymphocytes, fusing the B lymphocytes with myeloma cells to produce hybridomas, cloning the hybridomas, culturing clones that produce antibodies against the antigen, and isolating the antibodies from the hybridoma culture medium.

[0155] The monoclonal antibodies produced can be isolated and purified from the hybridoma culture by a variety of established techniques. Such isolation techniques include affinity chromatography with protein A sepharose, size exclusion chromatography, and ion exchange chromatography. See, for example, Coligan, pp. 2.7.1-2.7.12 and pp. 2.9.1-2.9.3. See, for example, Baines et al., "Purification of Immunoglobulin G (IgG)," in METHODS IN MOLECULAR BIOLOGY, VOL. 10, pp. 79-104 (The Humana Press, Inc. 1992). After initial production of antibodies against the target protein, the antibodies can be sequenced and then produced by recombinant techniques. Humanization and chimerization of mouse antibodies and antibody fragments are well known to those skilled in the art. See, e.g., Leung et al. Hybridoma 13:469 (1994); U.S. Patent Application Publication No. 2014 / 0099254(A1), each of which is incorporated by reference in its entirety.

[0156] Human antibodies can be produced using transgenic mice that have been genetically engineered to produce specific human antibodies in response to challenge with a target protein. See, e.g., McCafferty et al., Nature Genet. 7:13 (1994); Lonberg et al., Nature 368:856 (1994). Human antibodies against target proteins can also be constructed by genetic or chromosomal transfection methods, phage display technology, or in vitro activated B cells. See, e.g., McCafferty et al., 1990, Nature 348:552-553; U.S. Patent Nos. 5,567,610 and 5,229,275.

[0157] In some embodiments, Gal3-TIM-3 interaction is reduced to less than 70%, less than 60%, less than 59%, less than 50%, less than 40%, less than 34%, less than 30%, less than 20%, less than 14%, less than 10%, less than 7%, less than 5%, less than 4%, or less than 1%.

[0158] Polynucleotides and Vectors In some embodiments, the disclosure provides any of the anti-Gal3 antibodies disclosed herein. In some embodiments, the present disclosure provides an isolated nucleic acid encoding any of the anti-Gal3 antibodies disclosed herein. In some embodiments, the present disclosure provides a vector comprising a nucleic acid sequence encoding any of the anti-Gal3 antibodies disclosed herein. In some embodiments, the present disclosure provides an isolated nucleic acid encoding the light chain CDRs and the heavy chain CDRs of the anti-Gal3 antibodies disclosed herein.

[0159] The anti-Gal3 antibody of interest can be produced by recombinant DNA technology, synthetic chemical technology, or a combination thereof. For example, the sequences encoding the desired components of the anti-Gal3 antibody, which usually include light chain CDR and heavy chain CDR, are assembled and cloned into an expression vector using standard molecular techniques known in the art. These sequences may be assembled from PCR-generated fragments using respective template nucleic acids, or from other vectors encoding the desired protein sequences by assembly of synthetic oligonucleotides encoding the desired sequences. An expression system can be produced by transfecting appropriate cells with an expression vector containing the anti-Gal3 antibody of interest.

[0160] Nucleotide sequences corresponding to the variable regions of the light or heavy chains of existing antibodies can be readily obtained and sequenced using conventional techniques, including but not limited to hybridization, PCR, and DNA sequencing. Hybridoma cells producing monoclonal antibodies serve as the preferred source of antibody nucleotide sequences. A vast number of hybridoma cells producing monoclonal antibody arrays may be obtained from public or private repositories. The largest depository is the American Type Culture Collection (atcc.org), which provides a diverse collection of well-characterized hybridoma cell lines. Alternatively, antibody nucleotides can be obtained from immunized or non-immunized rodents or humans, and from organs such as spleen and peripheral blood lymphocytes. Specific techniques applicable to the extraction and synthesis of antibody nucleotides are described in Orlandi et al. (1989) Proc. Natl. Acad. Sci. USA 86:3833-3837; Larrick et al. (1989) Biochem. Biophys. Res. Commun. 160:1250-1255; Sastry et al. (1989) Proc. Natl. Acad. Sci., USA 86:5728-5732; and U.S. Patent No. 5,969,108.

[0161] Polynucleotides encoding anti-Gal3 antibodies can also be modified, for example, by substituting coding sequences for human heavy and light chain constant regions in place of the homologous non-human sequences, thus producing chimeric antibodies that retain the binding specificity of the original anti-Gal3 antibody.

[0162] Host cells for antibody production In some embodiments, the present disclosure provides a host cell expressing any one of the anti-Gal3 antibodies disclosed herein. The subject host cell typically comprises a nucleic acid encoding any one of the anti-Gal3 antibodies disclosed herein. In some embodiments, the host cell is a Chinese Hamster Ovary (CHO) cell. In some embodiments, the host cell is an NSO cell.

[0163] The present invention provides a host cell transfected with a polynucleotide, vector, or library of said vectors. The vector can be introduced into a suitable prokaryotic or eukaryotic cell by any of several suitable means, including electroporation, microprojectile bombardment; lipofection (combining the vector with an infectious agent), transfection using calcium chloride, rubidium chloride, calcium phosphate, DEAE dextran, or other substances. The choice of means for vector introduction often depends on the characteristics of the host cell.

[0164] For most animal cells, either of the above methods is suitable for vector delivery. Preferred animal cells are vertebrate cells, preferably mammalian cells, capable of expressing exogenously introduced gene products in large amounts, e.g., milligram levels. Non-limiting examples of preferred cells are NIH3T3 cells, COS, HeLa, and CHO cells.

[0165] Once introduced into suitable host cells, the expression of anti-Gal3 antibody can be determined using any nucleic acid or protein assay known in the art.For example, the presence of transcribed mRNA of light chain CDR or heavy chain CDR or anti-Gal3 antibody can be detected and / or quantified by conventional hybridization assay (e.g., Northern blot analysis), amplification method (e.g., RT-PCR), SAGE (U.S. Pat. No. 5,695,937), and array-based technology using probes that are complementary to any region of the polynucleotide encoding anti-Gal3 antibody (see, e.g., U.S. Pat. Nos. 5,405,783, 5,412,087 and 5,445,934).

[0166] Vector expression can also be determined by testing for expressed anti-Gal3 antibodies. A variety of techniques are available in the art for protein analysis. These techniques include, but are not limited to, radioimmunoassays, ELISA (enzyme linked immunoradiometric assays), "sandwich" immunoassays, immunoradiometric assays, in situ immunoassays (e.g., using colloidal gold, enzyme or radioisotope labels), Western blot analysis, immunoprecipitation assays, immunofluorescence assays, and SDS-PAGE.

[0167] payload In some embodiments, the anti-Gal3 antibody comprises a payload. In some cases, the payload comprises a small molecule, a protein or functional fragment thereof, a peptide, or a nucleic acid polymer.

[0168] In some cases, the number of payloads attached to the anti-Gal3 antibody (e.g., drug / antibody ratio or DAR) is about 1:1, i.e., one payload to one anti-Gal3 antibody. In some cases, the ratio of payload to anti-Gal3 antibody is about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, or 20:1. In some cases, the ratio of payload to anti-Gal3 antibody is about 2:1. In some cases, the ratio of payload to anti-Gal3 antibody is about 3:1. In some cases, the ratio of payload to anti-Gal3 antibody is about 4:1. In some cases, the ratio of payload to anti-Gal3 antibody is about 6:1. In some cases, the ratio of payload to anti-Gal3 antibody is about 8: 1. In some cases, the ratio of payload to anti-Gal3 antibody is about 12:1.

[0169] In some embodiments, the payload is a small molecule. In some embodiments, the small molecule is a cytotoxic payload. Exemplary cytotoxic payloads include, but are not limited to, microtubule disrupting agents, DNA modifying agents, or Akt inhibitors.

[0170] In some embodiments, the payload comprises a microtubule disrupting agent. Exemplary microtubule disrupting agents include, but are not limited to, 2-methoxyestradiol, auristatin, chalcone, colchicine, combretastatin, cryptophycin, dictyostatin, discodermolide, dolastatin, erytherobin, epothilone, halichondrin, laulimalide, maytansine, noscapinoid, paclitaxel, peloruside, phomopsin, podophyllotoxin, rhizoxin, spongistatin, taxane, tubulysin, vinca alkaloid, vinorelbine, or derivatives or analogs thereof.

[0171] In some embodiments, the maytansine is a maytansinoid. In some embodiments, the maytansinoid is DM1, DM4, or ansamitocin. In some embodiments, the maytansinoid is DM1. In some embodiments, the maytansinoid is DM4. In some embodiments, the maytansinoid is ansamitocin. In some embodiments, the maytansinoid is a maytansinoid derivative or analog, such as those described in U.S. Patent Nos. 5,208,020, 5,416,064, 7,276,497, and 6,716,821, or U.S. Patent Application Publication Nos. 2013 / 029900 and 2013 / 0323268.

[0172] In some embodiments, the payload is a dolastatin, or a derivative or analog thereof. In some embodiments, the dolastatin is dolastatin 10 or dolastatin 15, or a derivative or analog thereof. In some embodiments, the dolastatin 10 analog is auristatin, sobridotin, simprostatin 1, or simprostatin 3. In some embodiments, the dolastatin 15 analog is cemadotin or tacidotin.

[0173] In some embodiments, the dolastatin 10 analog is an auristatin or an auristatin derivative. In some embodiments, the auristatin or auristatin derivative is auristatin E (AE), auristatin F (AF), auristatin E5-benzoylvalerate (AEVB), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), or monomethyl auristatin D (MMAD), auristatin PE, or auristatin PYE. In some embodiments, the auristatin derivative is monomethyl auristatin E (MMAE). In some embodiments, the auristatin derivative is monomethyl auristatin F (MMAF). In some embodiments, auristatin E is an auristatin derivative or analog, such as those described in U.S. Patent Nos. 6,884,869, 7,659,241, 7,498,298, 7,964,566, 7,750,116, 8,288,352, 8,703,714, and 8,871,720.

[0174] In some embodiments, the payload comprises a DNA denaturing agent. In some embodiments, the DNA denaturing agent comprises a DNA cleaving agent, a DNA intercalating agent, a DNA transcription inhibitor, or a DNA cross-linking agent. In some embodiments, the DNA cleaving agent comprises bleomycin A2, calicheamicin, or a derivative or analogue thereof. In some embodiments, the DNA emulsifying agent comprises doxorubicin, epirubicin, PNU-159682, duocarmycin, pyrrolobenzodiazepine, oligomycin C, daunorubicin, valrubicin, topotecan, or a derivative or analogue thereof. In some embodiments, the DNA transcription inhibitor comprises dactinomycin. In some embodiments, the DNA cross-linking agent comprises mitomycin C.

[0175] In some embodiments, the DNA modifying agent comprises amsacrine, anthracycline, camptothecin, doxorubicin, duocarmycin, enediyne, etoposide, indolinobenzodiadiazepine, netropsin, teniposide, or derivatives or analogs thereof.

[0176] In some embodiments, the anthracycline is doxorubicin, daunorubicin, epirubicin, idarubicin, mitomycin C, dactinomycin, mithramycin, nemorubicin, pixantrone, sabarubicin, or valrubicin.

[0177] In some embodiments, the analog of camptothecin is topotecan, irinotecan, ciratecan, cositecan, exatecan, lurtotecan, gimatecan, belotecan, rubitecan, Tecan, or SN-38.

[0178] In some embodiments, the duocarmycin is duocarmycin A, duocarmycin B1, duocarmycin B2, duocarmycin C1, duocarmycin C2, duocarmycin D, duocarmycin SA, or CC-1065. In some embodiments, the enediyne is calicheamicin, esperamicin, or dynemicin A.

[0179] In some embodiments, the pyrrolobenzodiazepine is anthramycin, abbeymycin, ticamycin, DC-81, mazethramycin, neothramycin A, neothramycin B, polothramycin, sivanomycin (DC-102), sibiromycin, or tomaymycin. In some embodiments, the pyrrolobenzodiazepine is a tomaymycin derivative, such as those described in U.S. Patent Nos. 8,404,678 and 8,163,736. In some embodiments, the pyrrolobenzodiazepines are such as those described in U.S. Pat. Nos. 8,426,402, 8,802,667, 8,809,320, 6,562,806, 6,608,192, 7,704,924, 7,067,511, 7,612,062, 7,244,724, 7,528,126, 7,049,311, 8,633,185, 8,501,934, and 8,697,688 and U.S. Patent Application Publication No. 2014 / 0294868.

[0180] In some embodiments, the pyrrolobenzodiazepine is a pyrrolobenzodiazepine dimer. In some embodiments, the PBD dimer is a symmetric dimer. Examples of target PBD dimers include, but are not limited to, SJG-136 (SG-2000), ZC-423 (SG2285), SJG-720, SJG-738, ZC-207 (SG2202), and DSB-120. In some embodiments, the PBD dimer is an asymmetric dimer. Examples of asymmetric PBD dimers include, but are not limited to, SJG-136 derivatives such as those described in U.S. Pat. Nos. 8,697,688 and 9,242,013 and U.S. Patent Application Publication No. 2014 / 0286970.

[0181] In some embodiments, the payload comprises an Akt inhibitor. In some cases, the Akt inhibitor comprises ipatasertib (GDC-0068) or a derivative thereof.

[0182] In some embodiments, the payload comprises a polymerase inhibitor, including, but not limited to, a polymerase II inhibitor, such as alpha-amantin, and a poly(ADP-ribose) polymerase (PARP) inhibitor. Exemplary PARP inhibitors include, but are not limited to, iniparib (BSI201), talazoparib (BMN-673), olaparib (AZD-2281), olaparib, rucaparib (AG014699, PF-01367338), veliparib (ABT-888), CEP9722, MK4827, BGB-290, or 3-aminobenzamide.

[0183] In some embodiments, the payload comprises a detectable moiety. Exemplary detectable moieties include a fluorescent dye, an enzyme, a substrate, a chemiluminescent moiety, a specific binding moiety such as streptavidin, avidin, or biotin, or a radioisotope.

[0184] In some embodiments, the payload comprises an immunomodulatory agent. Useful immunomodulatory agents include anti-hormonal agents that block hormone action on tumors and immunosuppressants that suppress cytokine production, downregulate self-antigen expression, or mask MHC antigens. Representative anti-hormonal agents include, for example, tamoxifen, raloxifene, aromatase-inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene. Antiestrogens; and antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and antiadrenal drugs. Illustrative immunosuppressants include, but are not limited to, 2-amino-6-aryl-5-substituted pyrimidines, azathioprine, cyclophosphamide, bromocriptine, danazol, dapsone, glutaraldehyde, anti-idiotypic antibodies to MHC antibodies and MHC fragments, cyclosporin A, steroids, such as glucocorticosteroids, streptokinase, or rapamycin.

[0185] In some embodiments, the payload comprises an immune modulator. Exemplary immune modulators include ganciclovir, etanercept, tacrolimus, sirolimus, voclosporin, cyclosporin, rapamycin, cyclophosphamide, azathioprine, mycophenolate mofetil, methotrexate, glucocorticoids and analogs thereof, xanthines, stem cell growth factors, lymphotoxins, hematopoietic factors, tumor necrosis factors (TNF), (e.g., TNFα), interleukins (e.g., interleukin-1 (IL-1)), and the like. , IL-2, IL-3, IL-6, IL-10, IL-12, IL-18, and IL-21), colony stimulating factors (e.g., granulocyte colony stimulating factor (G-CSF) and granulocyte macrophage colony stimulating factor (GM-CSF)), interferons (e.g., interferon-α, interferon-β, interferon-γ), stem cell growth factors designated "S1 factors," erythropoietin, and thrombopoietin, or combinations thereof.

[0186] In some embodiments, the payload comprises an immunotoxin, including, but not limited to, ricin, radionuclides, pokeweed antiviral protein, Pseudomonas exotoxin A, diphtheria toxin, ricin A chain, fungal toxins such as restrictocin, and phospholipase enzymes. See generally, "Chimeric Toxins," Olsnes and Pihl, Pharmac. Ther. 15:355-381 (1981); and "Monoclonal Antibodies for Cancer Detection and Therapy," eds. Baldwin and Byers, pp. 159-179, 224-266, Academic Press (1985).

[0187] In some embodiments, the payload comprises a nucleic acid polymer. In some embodiments, the nucleic acid polymer comprises a small interfering nucleic acid (siNA), a small interfering RNA (siRNA), a double-stranded RNA (dsRNA), a microRNA (miRNA), a small hairpin RNA (shRNA), an antisense oligonucleotide. In some embodiments, the nucleic acid polymer comprises, for example, a cytotoxic protein or peptide or an apoptosis-inducing protein or peptide. Exemplary cytotoxic proteins or peptides include bacterial cytotoxins such as alpha pore-forming toxins (e.g., cytolysin A from Escherichia coli (E. coli)), beta pore-forming toxins (e.g., alpha-hemolysin, PVL-Panton-Valentine leukocidin, aerolysin, Clostridium epsilon toxin, Clostridium perfringens enterotoxin), binary toxins (anthrax toxin, edema toxin, C. botulinum C2 toxin, C spirofome toxin, Clostridium perfringens iota toxin, C. perfringens cytotoxin, C. botulinum C2 toxin, C spirofome toxin, C. perfringens iota to ... iota toxins, C. difficile cytolethal toxins (A and B), prions, parasporins, cholesterol-dependent cytolysins (e.g., pneumolysin), small pore-forming toxins (e.g., gramicidin A), cyanotoxins (e.g., microcystin, nodularin), hematotoxins, neurotoxins (e.g., botulinum neurotoxins), cytotoxins, cholera toxins, diphtheria toxins, Pseudomonas exotoxin A, tetanus toxins, or immunotoxins (idarubicin, ricin A, CRM9, pokeweed antiviral protein, DT). Exemplary apoptosis-inducing proteins or peptides include apoptotic protease activating factor-1 (Apaf-1), cytochrome-c, caspase initiator protein (CAS), P2, CASP8, CASP9, CASP10), apoptosis-inducing factor (AIF), p53, p73, p63, Bcl-2, Bax, granzyme B, poly-ADP ribose polymerase (PARP), and 21-activated kinase 2 (PAK2). In some embodiments, the nucleic acid polymer comprises a nucleic acid decoy. In some embodiments, the nucleic acid decoy is a mimic of a protein-binding nucleic acid, such as an RNA-based protein-binding mimic. Exemplary nucleic acid decoys include transcription-activating region (TAR) decoys and Rev-responsive element (RRE) decoys.

[0188] In some cases, the payload is an aptamer. Aptamers are small oligonucleotide or peptide molecules that bind to specific target molecules. Exemplary nucleic acid aptamers include DNA aptamers, RNA aptamers, or XNA aptamers, which are RNA and / or DNA aptamers that contain one or more non-natural nucleotides. Exemplary nucleic acid aptamers include ARC19499 (Archemix Corp.), REG1 (Regado Biosciences), and ARC1905 (Ophthotech).

[0189] Nucleic acids according to some embodiments described herein optionally include natural nucleic acids, or one or more nucleotide analogs, or have a structure that is otherwise different from that of natural nucleic acids. For example, 2'-modifications include halo, alkoxy, and allyloxy groups. In some embodiments, the 2'-OH group is replaced with a group selected from H, OR, R, halo, SH, SR, NH2, NHR, NR2, or CN, where R is C1-C6 alkyl, alkenyl, or alkynyl, and halo is F, Cl, Br, or I. Examples of modified linkages include phosphorothioate and 5'-N-phosphoramidite linkages.

[0190] Nucleic acids having a variety of different nucleotide analogs, modified backbones, or non-natural internucleotide linkages are utilized according to some embodiments described herein. In some cases, the nucleic acids include natural nucleosides (i.e., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine) or modified nucleosides. Examples of modified nucleotides include base-modified nucleosides (e.g., aracytidine, inosine, isoguanosine, nebularine, pseudouridine, 2,6-diaminopurine, 2-aminopurine, 2-thiothymidine, 3-deaza-5-azacytidine, 2'-deoxyuridine, 3-nitropyrrole, 4-methylindole, 4-thiouridine, 4-thiothymidine, 2-aminoadenosine, 2-thiothymidine, 2-thiouridine, 5-bromocytidine, 5-iodouridine, inosine, 6-azauridine, 6-chloropurine, 7-deazaadenosine, 7-deazaguanosine, 8-azaadenosine, 8-azidoadenosine, benzimidazole, M1-methyladenosine, pyrrolo-pyrimidine, These include 2-amino-6-chloropurine, 3-methyladenosine, 5-propynylcytidine, 5-propynyluridine, 5-bromouridine, 5-fluorouridine, 5-methylcytidine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)-methylguanine, and 2-thiocytidine), chemically or biologically modified bases (e.g., methylated bases), modified sugars (e.g., 2'-fluororibose, 2'-aminoribose, 2'-azidoribose, 2'-O-methylribose, L-enantiomeric nucleosides arabinose, and hexose), modified phosphate groups (e.g., phosphorothioate and 5'-N-phosphoramidite linkages), and combinations thereof. Natural and modified nucleotide monomers for chemical synthesis of nucleic acids are readily available. In some cases, nucleic acids containing such modifications exhibit improved properties compared to nucleic acids consisting only of natural nucleotides.In some embodiments, the nucleic acid modifications described herein are utilized to reduce digestion by nucleases (e.g., exonucleases, endonucleases, etc.). For example, the structure of the nucleic acid may be stabilized by including nucleotide analogues at the 3' end of one or both strands to reduce digestion.

[0191] Different nucleotide modifications and / or backbone structures can be present at various positions in the nucleic acid. Such modifications include morpholinos, peptide nucleic acids (PNAs), methylphosphonate nucleotides, thiophosphonate nucleotides, 2'-fluoro N3-P5'-phosphoramidites, 1',5'-anhydrohexitol nucleic acids (HNAs), or combinations thereof.

[0192] Conjugation Chemistry In some embodiments, the payload is attached to the anti-Gal3 antibody described herein by native ligation. In some embodiments, the conjugation is as described in Dawson, et al. "Synthesis of proteins by native chemical ligation," Science 1994, 266, 776-779; Dawson, et al. "Modulation of Reactivity in Native Chemical Ligation through the Use of Thiol Additives," J. Am. Chem. Soc. 1997, 119, 4325-4329; Hackeng, et al. "Protein synthesis by native chemical ligation: Expanded scope by using straightforward methodology.," Proc. Natl. Acad. Sci. USA 1999, 96, 10068-10073; or Wu, et al. "Building complex glycopeptides: Development of a cysteine-free native chemical ligation protocol," Angew. Chem. Int. Ed. 2006, 45, 4116-4125. In some embodiments, the linkages are as described in US Pat. No. 8,936,910.

[0193] In some embodiments, the payload is coupled to the nuclease via a site-directed method utilizing "traceless" coupling technology (Philochem), as described herein. In some embodiments, the "traceless" coupling technique utilizes the N-terminal 1,2-aminothiol group of the coupling moiety, which is complexed with a polynucleic acid molecule containing an aldehyde group. (Casi et al., "Site-specific traceless coupling of potent cytotoxic drugs to recombinant antibodies for pharmacodelivery,” JACS 134(13):5887-5892 (2012)).

[0194] In some embodiments, payloads are conjugated to anti-Gal3 antibodies described herein by a site-directed method that utilizes an unnatural amino acid incorporated into the conjugation moiety. In some embodiments, the unnatural amino acid comprises p-acetylphenylalanine (pAcPhe). In some embodiments, the keto group of pAcPhe is selectively coupled to an alkoxy-amine derived conjugation moiety to generate an oxime bond. (Axup et al., J. Am. Chem. Soc. 1999, 143:1311-1322) al., “Synthesis of site-specific antibody-drug conjugates using unnatural amino acids,” PNAS 109(40):16101-16106 (2012)).

[0195] In some embodiments, the payload is attached to the anti-Gal3 antibody described herein by a site-directed method that utilizes enzyme catalysis. A typical method utilizes SMARTag™ technology (Redwood). In some embodiments, SMARTag™ technology generates formylglycine (FGly) residues from cysteine ​​by formylglycine generating enzyme (FGE) through oxidative treatment in the presence of an aldehyde tag, and then couples FGly to an alkylhydraine-functionalized polynucleic acid molecule by hydrazino-Pictet-Spengler (HIPS) ligation. (Wu et al., “Site-specific chemical modification of recombinant proteins produced in mammalian cells by using the genetically encoded aldehyde tag,”PNAS 106(9):3000-3005 (2009);Agarwal, et al.,“A Pictet-Spengler ligation for protein chemical modification,” PNAS 110(1):46-51 (2013)).

[0196] In some embodiments, the enzyme catalysis method includes microbial transglutaminase (mTG). In some cases, the payload is conjugated to the anti-Gal3 antibody using the microbial transglutaminase catalysis method. In some embodiments, mTG catalyzes the formation of a covalent bond between the amide side chain of glutamine in the recognition sequence and the primary amine of the functionalized polynucleic acid molecule. In some embodiments, mTG is produced from Streptomyces mobarensis. (See Strop et al., “Location matters: site of conjugation modulates stability and pharmacokinetics of antibody drug conjugates,” Chemistry and Biology 20(2)161-167(2013)).

[0197] In some embodiments, the payload is conjugated to the anti-Gal3 antibody by the methods described in WO 2014 / 140317, which utilize a sequence-specific transpeptidase.

[0198] In some embodiments, the payload is conjugated to the anti-Gal3 antibodies described herein by the methods described in U.S. Patent Application Publication Nos. 2015 / 0105539 and 2015 / 0105540.

[0199] Linker In some embodiments, the linker comprises a natural or synthetic polymer consisting of long chains of branched or unbranched monomers and / or crosslinked networks of monomers in two or three dimensions. In some embodiments, the linker includes a polysaccharide, lignin, rubber, or polyalkylene oxide (e.g., polyethylene glycol).

[0200] In some embodiments, the linker includes α-, ω-dihydroxypolyethylene glycol, biodegradable lactone-based polymers such as polyacrylic acid, polylactic acid (PLA), poly(glycolic acid) (PGA), polypropylene, polystyrene, polyolefins, polyamides, polycyanoacrylates, polyimides, polyethylene terephthalate (PET, PETG), polyethylene terephthalate (PE Examples of suitable block copolymers include, but are not limited to, poly(ethylene glycol), polytetramethylene glycol (PTG), or polyurethanes, as well as mixtures thereof. As used herein, a mixture refers to the use of different polymers within the same compound, as well as with respect to block copolymers. In some cases, a block copolymer is a polymer in which at least one polymer portion is built up from monomers of another polymer. In some embodiments, the linker comprises a polyalkylene oxide. In some embodiments, the linker comprises a PEG. In some embodiments, the linker comprises polyethyleneimide (PEI) or hydroxyethyl starch (HES).

[0201] In some embodiments, the polyalkylene oxide (e.g., PEG) is a polydisperse or monodisperse compound. In some embodiments, a polydisperse material comprises a polydisperse distribution of materials of different molecular weights, characterized by average weight (weight average) size and dispersity. In some embodiments, a monodisperse PEG comprises molecules of one size. In some embodiments, the linker is a polydisperse or monodisperse polyalkylene oxide (e.g., PEG), and the specified molecular weight is the average of the molecular weights of the polyalkylene oxide, e.g., PEG, molecules.

[0202] In some embodiments, the linker is a polyalkylene oxide (e.g., PEG), and the molecular weight of the polyalkylene oxide (e.g., PEG) is about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 5100, 5200, 5300, 5400, 5500, 5600, 5700, 5800, 5900, 6000, 6100, 6200, 6300, 6400, 6500, 6600, 6700, 6800, 6900, 7000, 7100, 7200, 7300, 7400, 7500, 7600, 7700, 7800, 7900, 8 00, 2500, 2600, 2700, 2800, 2900, 3000, 3250, 3350, 3500, 3750, 4000, 4250, 4500, 4600, 4750, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 10,000, 12,000, 20,000, 35,000, 40,000, 50,000, 60,000, or 100,000 Da.

[0203] In some embodiments, the polyalkylene oxide (e.g., PEG) is a dispersed PEG, which is a polymeric PEG that contains more than one repeating ethylene oxide unit. In some embodiments, the dispersed PEG (dPEG) contains 2-60, 2-50, or 2-48 repeating ethylene oxide units. In some embodiments, the dPEG contains about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30, 35, 40, 42, 48, 50 or more repeating ethylene oxide units. In some embodiments, the dPEG contains about 2 or more repeating ethylene oxide units. In some embodiments, the dPEG is synthesized stepwise from pure (e.g., about 95%, 98%, 99%, or 99.5%) starting materials as a single molecular weight compound. In some embodiments, the dPEG has a specific molecular weight, rather than an average molecular weight. In some instances, the dPEG described herein is dPEG from Quanta Biodesign, LMD.

[0204] In some embodiments, the linker is a dispersed PEG containing 2-60, 2-50, or 2-48 repeating ethylene oxide units, as appropriate. In some cases, the linker comprises a dPEG containing about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30, 35, 40, 42, 48, 50 or more repeating ethylene oxide units. In some cases, the linker is a dPEG from Quanta Biodesign, LMD.

[0205] In some embodiments, the linker is a polypeptide linker. In some embodiments, the polypeptide linker comprises at least 2, 3, 4, 5, 6, 7, 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, or more amino acid residues. In some embodiments, the polypeptide linker comprises at least 2, 3, 4, 5, 6, 7, 8, or more amino acid residues. In some embodiments, the polypeptide linker comprises at most 2, 3, 4, 5, 6, 7, 8, or fewer amino acid residues. In some embodiments, the polypeptide linker is a cleavable polypeptide linker (e.g., either enzymatically or chemically). In some embodiments, the polypeptide linker is a non-cleavable polypeptide linker. In some embodiments, the polypeptide linker comprises Val-Cit (valine-citrulline), Gly-Gly-Phe-Gly, Phe-Lys, Val-Lys, Gly-Phe-Lys, Phe-Phe-Lys, Ala-Lys, Val-Arg, Phe-Cit, Phe-Arg, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Ala, Ala-Leu-Ala-Leu, or Gly-Phe-Leu-Gly. In some embodiments, the polypeptide linker comprises a peptide such as Val-Cit (valine-citrulline), Gly-Gly-Phe-Gly, Phe-Lys, Val-Lys, Gly-Phe-Lys, Phe-Phe-Lys, Ala-Lys, Val-Arg, Phe-Cit, Phe-Arg, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Ala, Ala-Leu-Ala-Leu, or Gly-Phe-Leu-Gly. In some cases, the polypeptide linker comprises L-amino acids, D-amino acids, or a mixture of both L- and D-amino acids.

[0206] In some embodiments, the linker comprises a homobifunctional linker. Exemplary homobifunctional linkers include Lomant's reagents. Dithiobis(succinimidyl propionate) DSP, 3',3'-dithiobis(sulfosuccinimidyl propionate) (DTSSP), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl)suberate (BS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), ethylene glycobis(succinimidyl succinate) (EGS), disuccinimidyl glutarate (DSG), N,N'-disuccinimidyl carbonate (DSC), dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS), dimethyl-3,3'-dithiobispropionimidate (DTBP), 1,4-di-3'-(2'-pyridyldithio)propionamidate difluoro-2,4-dinitrobenzene (DPDPB), bismaleimidohexane (BMH), halogenated aryl-containing compounds (DFDNB) such as 1,5-difluoro-2,4-dinitrobenzene or 1,3-difluoro-4,6-dinitrobenzene, 4,4'-difluoro-3,3'-dinitrophenyl sulfone (DFDNPS), bis-[β-4-azidosalicylamido)ethyl] disulfide (BASED), formaldehyde, glutaraldehyde, 1,4-butanediol diglycidyl ether, adipic acid dihydrazide, carbohydrazide, o-toluidine, 3,3'-dimethylbenzidine, benzidine, α,α'-ethylene-bis(iodoacetamide), or N,N'-hexamethylene-bis(iodoacetamide).

[0207] In some embodiments, the linker comprises a heterobifunctional linker. Exemplary heterobifunctional linkers include N-succinimidyl 3-(2-pyridyldithio)propionate (sPDP), long-chain N-succinimidyl 3-(2-pyridyldithio)propionate (LC-sPDP), water-soluble long-chain N-succinimidyl 3-(2-pyridyldithio)propionate (sulfo-LC-sPDP), succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene (sMPT), sulfosuccinimidyl-6-[α-methyl-α-(2-pyridyldithio)toluamide]hexanoate (sulfo-LC-sMPT), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC), sulfosuccini ... )cyclohexane-1-carboxylate (sulfo-sMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBs), m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBs), N-succinimidyl (4-iodoacetyl)aminobenzoate (sIAB), sulfosuccinimidyl (4-iodoacetyl)aminobenzoate (sulfo-sIAB), succinimidyl-4-(p-maleimidophenyl)butyrate (sMPB), sulfosuccinimidyl-4-(p-maleimidophenyl)butyrate (sulfo-sMPB), N-(γ-maleimidobutyryloxy)succinimide ester (GMBs), N-(γ-maleimidobutyryloxy)sulfosuccinimide Mido esters (sulfo-GMBs), succinimidyl 6-((iodoacetyl)amino)hexanoate (sIAX), succinimidyl 6-[6-(((iodoacetyl)amino)hexanoyl)amino]hexanoate (sIAXX), succinimidyl Amine-reactive and sulfhydryl-reactive crosslinkers such as 4-(((iodoacetyl)amino)methyl)cyclohexane-1-carboxylate (sIAC), succinimidyl 6-((((4-iodoacetyl)amino)methyl)cyclohexane-1-carbonyl)amino)hexanoate (sIACX), p-nitrophenyl iodoacetate (NPIA), carbonyl-reactive and sulfhydryl-reactive crosslinkers such as 4-(4-N-maleimidophenyl)butyric acid hydrazide (MPBH), 4-(N-maleimidomethyl)cyclohexane-1-carboxyl-hydrazide-8 (M2C2H), 3-(2-pyridyldithio)propionyl hydrazide (PDPH) Crosslinking agents, N-hydroxysuccinimidyl-4-azidosalicylate (NHs-AsA), N-hydroxysulfosuccinimidyl-4-azidosalicylate (sulfo-NHs-AsA), sulfosuccinimidyl-(4-azidosalicylamido)hexanoate (sulfo-NHs-LC-AsA), sulfosuccinimidyl-2-(ρ-azidosalicylamido)ethyl-1,3′-dithiopropionate (sAsD), N-hydroxysuccinimidyl-4-azidobenzoate (HsAB), N-hydroxysulfosuccinimidyl-4-azidobenzoate (sulfo-HsAB), N-succinimidyl Sulfosuccinimidyl-6-(4′-azido-2′-nitrophenylamino)hexanoate (sANPAH), sulfosuccinimidyl-6-(4′-azido-2′-nitrophenylamino)hexanoate (sulfo-sANPAH), N-5-azido-2-nitrobenzoyloxysuccinimide (ANB-NOs), sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido)-ethyl-1,3′-dithiopropionate (sAND), N-succinimidyl-4(4-azidophenyl)1,3′-dithiopropionate (sADP), N-sulfosuccinimidyl(4-azidophenyl)-1,3′-dithiopropionate (sADP),3′-dithiopropionate (sulfo-sADP), sulfosuccinimidyl 4-(ρ-azidophenyl)butyrate (sulfo-sAPB), sulfosuccinimidyl 2-(7-azido-4-methylcoumarin-3-acetamido)ethyl-1,3′-dithiopropionate (sAED), sulfosuccinimidyl 7-azido-4-methylcoumarin-3-acetate (sulfo-sAMCA), ρ-nitrophenyl These include, but are not limited to, amine-reactive and photoreactive crosslinkers such as diazopyruvate (ρNPDP), ρ-nitrophenyl-2-diazo-3,3,3-trifluoropropionate (PNP-DTP), sulfhydryl-reactive and photoreactive crosslinkers such as 1-(ρ-azidosalicylamido)-4-(iodoacetamido)butane (AsIB), N-[4-(ρ-azidosalicylamido)butyl]-3′-(2′-pyridyldithio)propionamide (APDP), benzophenone-4-iodoacetamide, benzophenone-4-maleimidocarbonyl-reactive and photoreactive crosslinkers such as ρ-azidobenzoylhydrazide (ABH), carboxylate-reactive and photoreactive crosslinkers such as 4-(ρ-azidosalicylamido)butylamine (AsBA), and arginine-reactive and photoreactive crosslinkers such as ρ-azidophenyl glyoxal (APG).

[0208] In some embodiments, the linker comprises a benzoic acid group or a derivative thereof. In some embodiments, the benzoic acid group or a derivative thereof comprises para-aminobenzoic acid (PABA). In some embodiments, the benzoic acid group or a derivative thereof comprises gamma-aminobutyric acid (GABA).

[0209] In some embodiments, the linker comprises one or more of a maleimide group, a peptide moiety, and / or a benzoic acid group, in any combination. In some embodiments, the linker comprises a combination of a maleimide group, a peptide moiety, and / or a benzoic acid group. In some embodiments, the maleimide group is maleimidocaproyl (mc). In some embodiments, the peptide group is val-cit. In some embodiments, the benzoic acid group is PABA. In some embodiments, the linker is mc-val-cit In some cases, the linker comprises a val-cit-PABA group. In further cases, the linker comprises a mc-val-cit-PABA group.

[0210] In some embodiments, the linker is a self-disintegrating linker or a self-eliminating linker. In some cases, the linker is a self-disintegrating linker. In other cases, the linker is a self-eliminating linker (e.g., a cyclized self-eliminating linker). In some embodiments, the linker comprises the linker described in U.S. Pat. No. 9,089,614 or WO 2015 / 038426.

[0211] In some embodiments, the linker is a dendritic linker. In some embodiments, the dendritic linker comprises a branched multifunctional linker moiety. In some embodiments, the dendritic linker comprises a PAMAM dendrimer.

[0212] In some embodiments, the linker is a traceless linker or a linker that does not leave a linker moiety (e.g., an atom or a linker group) on the antibody or payload after cleavage. Exemplary traceless linkers include, but are not limited to, germanium linkers, silicon linkers, sulfur linkers, selenium linkers, nitrogen linkers, phosphorus linkers, boron linkers, chromium linkers, or phenylhydrazide linkers. In some cases, the linker is a traceless aryl-triazene linker as described in Hejesen, et al., "A traceless aryl-triazene linker for DNA-directed chemistry," Org Biomol Chem 11(15):2493-2497 (2013). In some embodiments, the linker is a traceless linker as described in Blaney, et al., "Traceless solid-phase organic synthesis," Chem.Rev.102:2607-2024 (2002). In some embodiments, the linker is a traceless linker as described in US Pat. No. 6,821,783.

[0213] Pharmaceutical Compositions In some embodiments, the pharmaceutical formulation for reducing tissue fibrosis can include the above-mentioned anti-Gal3 antibody. The anti-Gal3 antibody can be formulated for systemic administration. Alternatively, the anti-Gal3 antibody can be formulated for parenteral administration.

[0214] In some embodiments, the anti-Gal3 antibody is further formulated as a pharmaceutical composition. In some embodiments, the pharmaceutical composition is formulated for administration to a subject by multiple routes of administration, including, but not limited to, parenteral (e.g., intravenous, subcutaneous, intramuscular, intraarterial, intradermal, intraperitoneal, intravitreal, intracerebral, or intraventricular), oral, intranasal, buccal, rectal, or transdermal administration routes. In some embodiments, the pharmaceutical composition described herein is formulated for parenteral (e.g., intravenous, subcutaneous, intramuscular, intraarterial, intradermal, intraperitoneal, intravitreal, intracerebral, or intraventricular) administration. In some embodiments, the pharmaceutical composition described herein is formulated for oral administration. In still other embodiments, the pharmaceutical composition described herein is formulated for intranasal administration.

[0215] In some embodiments, pharmaceutical formulations include, but are not limited to, aqueous liquid dispersions, self-emulsifying dispersions, solid solutions, liposomal dispersions, aerosols, solid dosage forms, powders, immediate release formulations, controlled release formulations, fast dissolving formulations, tablets, capsules, pills, delayed release formulations, sustained release formulations, pulsed release formulations, multiparticulate formulations (e.g., nanoparticle formulations), and immediate and controlled release combined formulations.

[0216] In some embodiments, the pharmaceutical composition comprises acetic acid, boric acid, citric acid, lactic acid, phosphoric acid, and and pH adjusters or buffers, including acids such as hydrochloric acid, bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate, and trishydroxymethylaminomethane, and buffers such as citrate / dextrose, sodium bicarbonate, and ammonium chloride. Such acids, bases, and buffers are included in amounts necessary to maintain the pH of the composition within an acceptable range.

[0217] In some embodiments, the pharmaceutical composition comprises one or more salts in an amount necessary to bring the osmolality of the composition into an acceptable range.Such salts include those having sodium, potassium or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate or bisulfite anions; suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite and ammonium sulfate.

[0218] In some embodiments, the pharmaceutical composition further comprises a diluent that is used to stabilize the compound, since the diluent can provide a more stable environment.Salts dissolved in buffers (which can be pH controlled or pH maintained) are utilized as diluents in the art, including but not limited to phosphate buffered saline.In certain embodiments, the diluent increases the bulk of the composition, making it easier to compress and creating sufficient bulk for a homogeneous blend for capsule filling. Such compounds may include, for example, lactose, starch, mannitol, sorbitol, dextrose, microcrystalline cellulose such as Avicel®; dibasic calcium phosphate, dicalcium phosphate dihydrate; tricalcium phosphate, calcium phosphate; anhydrous lactose, spray-dried lactose; pregelatinized starch, compressible sugars such as Di-Pac® (Amstar); mannitol, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose acetate stearate, sucrose-based diluents, confectioners' sugar; monobasic calcium sulfate monohydrate, calcium sulfate dihydrate; calcium lactate trihydrate, dextrate; hydrolyzed cereal solids, amylose; powdered cellulose, calcium carbonate; glycine, kaolin; mannitol, sodium chloride; inositol, bentonite, and the like.

[0219] In some embodiments, the pharmaceutical preparation may further comprise an additional therapeutic agent. The additional therapeutic agent may have an anti-fibrotic effect. The additional therapeutic agent may be an inhibitor of growth factors, cytokines and ligands such as matrix metalloproteinases (MMPs). The additional therapeutic agent may be an inhibitor of TGF-β, ALK5, BMP-7, PDGF, platelet-derived growth factor, VEGF, TNF, HGF, IL-13, chemokine (CC motif) ligand 2; CCR5, MMPs and TIMPs. Additional therapeutic agents include SHP-627 (FT011), hydronidone (F351), PXS-25, diciteltide (P-144), fresolimumab (GC-1008), LY2382770, STX-100, CWHM-12, SB-431542, THR-184, PF-06473871, RXI-109, FG-3019, imatinib, BOT-191, nilotinib (AMN-107), dasatinib, nintedanib (BIBF-1120), sorafenib (BAY 43-9006), thalidomide, pomalidomide, etanercept, belimumab, refanalin (BB-3), dextrose (QAX-576), tralokinumab, anakinra, rilonacept, SAR156597, carlumab (CNTO-888), bindarit, maraviroc, RS-504393, Actimune, interferon, alpha oral lozenge, batimastat (BB-49), marimastat, mastectomy It can be tentan, bosentan, ambrisentan, sparsentan (RE-021), atrasentan, losartan, BMS-986020, SAR-100842, PAR1 antagonism, curcumin, silymarin, beta-caryophyllene, beraprost, iloprost, treprostinil, aviptadil, sivelestat, UK-396082, serelaxin, PRM-151, or dioscin, NTU281.

[0220] Treatment regimen In some embodiments, the anti-Gal3 antibody disclosed herein is administered for therapeutic use. In some embodiments, the anti-Gal3 antibody is administered once a day, twice a day, three times a day or more. The anti-Gal3 antibody is administered daily, daily, every other day, five days a week, once a week, every other week, every second week of the month, every third week of the month, once a month, twice a month, three times a month or more. The anti-Gal3 antibody is administered for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 18 months, 2 years, 3 years or more.

[0221] If the patient's condition improves, administration of anti-Gal3 antibody may be continued, at the discretion of the physician; alternatively, the dose of anti-Gal3 antibody administered may be temporarily reduced or temporarily suspended for a certain period of time (i.e., a "drug holiday"). In some embodiments, the length of the drug holiday may vary from 2 days to 1 year, including, by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or 365 days. Dose reductions during drug holidays can be, by way of example only, 10% to 100%, including 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0222] Once improvement of the patient's condition has occurred, a maintenance dose is administered if necessary, after which the dosage or frequency of administration, or both, can be symptomatically reduced to a level at which the improved disease, disorder, or condition is maintained.

[0223] In some embodiments, a given amount of drug that corresponds to such an amount will vary depending on factors such as the particular compound, the severity of the disease, the identity (e.g., weight) of the subject or host requiring treatment, but is nevertheless routinely determined in a manner known in the art depending on the particular circumstances surrounding the case, including, for example, the particular drug being administered, the route of administration, and the subject or host being treated. In some embodiments, the desired dose is conveniently provided in a single dose or divided doses administered simultaneously (or over a short period of time) or subdoses at appropriate intervals, for example, twice a day, three times a day, four times a day or more.

[0224] Because of the large number of variables involved in an individual treatment regimen, the foregoing ranges are merely suggestive, and significant deviations from these recommendations are not uncommon. Such dosages will vary depending on a number of variables, including but not limited to the activity of the compound being used, the disease or condition being treated, the manner of administration, the requirements of the individual subject, the severity of the disease or condition being treated, and the judgment of the physician.

[0225] In some embodiments, the toxicity and therapeutic efficacy of such treatment regimens are determined by standard pharmaceutical procedures in cell cultures or experimental animals, including, but not limited to, determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which is expressed as the ratio between LD50 and ED50. Compounds that exhibit high therapeutic indices are preferred. The data obtained from cell culture assays and animal studies are used to formulate a dosage range for use in humans. The dosage of such compounds is preferably within a range of circulating concentrations that includes the ED50 with minimal toxicity. Dosages vary within this range depending on the dosage form used and the route of administration utilized.

[0226] Kits / Manufacturing Supplies In some embodiments, the present invention provides a method for the treatment of a cancer using one or more of the compositions and methods described herein. Disclosed herein are kits and articles of manufacture for use in the methods described herein. Such kits include carriers, packages, or containers partitioned to receive one or more containers, such as vials, tubes, and the like, each of which contains one of the separate elements to be used in the methods described herein. Suitable containers include, for example, bottles, vials, syringes, and test tubes. In some embodiments, the containers are manufactured from a variety of materials, such as glass or plastic.

[0227] The articles of manufacture provided herein include packaging materials. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, bags, containers, bottles, and any packaging material suitable for the selected formulation and the desired method of administration and treatment.

[0228] For example, a container may comprise an anti-Gal3 antibody disclosed herein, a host cell for producing one or more of the antibodies described herein, and / or a vector comprising a nucleic acid molecule encoding an antibody described herein. Such a kit may optionally comprise an identification or label or instructions for its use in the methods described herein.

[0229] The kit will typically include a label and / or instructions indicating the contents and a package insert with instructions. An instruction set will also typically be included.

[0230] In some embodiments, the label is on or associated with the container. In some embodiments, the label is on the container when letters, numbers, or other characters that form the label are attached, molded, or etched on the container; the label is associated with the container when, for example, the label is in the container or carrier that holds the container as a package insert. In some embodiments, the label is used to indicate that the contents are to be used for a particular therapeutic purpose. The label also indicates instructions for using the contents, such as in the methods described herein.

[0231] In some embodiments, the pharmaceutical composition is provided in a pack or dispenser device containing one or more unit dosage forms comprising a compound provided herein. The pack comprises, for example, metal or plastic foil, such as a blister pack. In some embodiments, the pack or dispenser device is accompanied by instructions for administration. In some embodiments, the pack or dispenser device is also accompanied by a notice associated with the container in a format prescribed by a government agency regulating the manufacture, use, or sale of pharmaceuticals, the notice reflecting approval by the government agency of the form of drug for human or veterinary administration. Such notice is, for example, a label approved by the U.S. Food and Drug Administration for medical drugs, or an approved product insert. In some embodiments, a composition comprising a compound provided herein formulated in a compatible pharmaceutical carrier is prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.

[0232] Fibrosis Biomarkers In some embodiments, the puncture specimen is characterized by one or more biomarkers, such as collagen, extracellular matrix (ECM) molecules and enzymes, cytokines, proteomic markers, or genetic markers. In some cases, fibrosis biomarkers include collagen (I, III, and IV), procollagen N-terminal peptides, fibronectin, elastin, laminin, α-smooth muscle actin (α-SMA), hyaluronic acid (HA), proteoglycans, YKL-40, TIMP-1, TIMP-2, MMP-2, MMP-9, TGFβ, TNFα, angiotensin-II, microfibril-associated protein 4 (MFAP-4), tropomyosin, AZIN1, TLR4, TRPM5, AQP2, Or SNP of STXBP5L, but not limited thereto. The expression or absence of certain biomarkers is associated with one or more fibrotic diseases. When treated with anti-Gal3 antibody, the increase or decrease of such biomarkers can indicate the reduction of tissue fibrosis.

[0233] In some embodiments, the fibrosis biomarker is α-smooth muscle actin (α-SMA). α-SMA is the predominant 42 kDa actin isoform in vascular smooth muscle cells. Myofibroblasts are a form of fibroblasts that are partially differentiated into a smooth muscle phenotype. In particular, myofibroblasts can contract by using cytoskeletal proteins, including α-SMA. In some fibrotic diseases, it has been observed that there is an accumulation of myofibroblasts that leads to an increase in extracellular matrix. Thus, altered expression (e.g., high expression) of α-SMA correlates with myofibroblast activation and further serves as a fibrosis biomarker.

[0234] In some embodiments, the fibrosis biomarker is fibronectin. Fibronectin is a high molecular weight (about 440 kDa) glycoprotein in the extracellular matrix, and further binds to integrins, collagens, fibrins, and heparan sulfate proteoglycans. Fibronectin plays a major role in cell adhesion, growth, migration, and differentiation, and is further involved in wound healing among other functions. Fibronectin can be soluble plasma fibronectin or insoluble cellular fibronectin, and can be type I, type II, or type III. Altered expression (e.g., decreased expression) of fibronectin is associated with fibrosis.

[0235] In some embodiments, the fibrosis biomarker is transforming growth factor (TGF)-β1. TGF-β1 is a polypeptide member of the TGF-β superfamily of cytokines, and TGF-β1 is involved in cell growth, cell proliferation, cell differentiation, and apoptosis. Furthermore, collagen-producing cells, such as fibroblasts, are activated by fibrogenic cytokines such as TGF-β1. In the context of fibrosis, TGF-β1 has been proposed to be a master regulator and potent inducer of ECM synthesis. Furthermore, TGF-β1 is produced by various cells, such as macrophages, neutrophils, activated alveolar epithelial cells, endothelial cells, fibroblasts, and myofibroblasts. Activation of TGF-β1 leads to increased expression of proinflammatory and fibrogenic cytokines, such as TNF-α, PDGF, IL-1β, and / or IL-13, further enhancing and perpetuating the fibrotic response.

[0236] In some embodiments, administering an anti-Gal3 antibody to a tissue site of interest modulates the presence and / or expression of one or more fibrosis biomarkers. In some embodiments, the anti-Gal3 antibody alters the presence or absence or expression of one or more fibrosis biomarkers selected from collagen (I, III and IV), procollagen N-terminal peptide, fibronectin, elastin, laminin, α-smooth muscle actin (α-SMA), hyaluronic acid (HA), proteoglycan, YKL-40, TIMP-1, TIMP-2, MMP-2, MMP-9, TGFβ, TNFα, angiotensin-II, microfibril associated protein 4 (MFAP-4), tropomyosin, AZIN1, TLR4, TRPM5, AQP2, and STXBP5L SNPs. In some embodiments, the anti-Gal3 antibody alters the presence or absence or expression of α-SMA, fibronectin, TGF-β1, or a combination thereof. In some embodiments, administration of an anti-Gal3 antibody at a tissue site of interest results in a decrease in the expression of α-SMA. In some embodiments, administration of an anti-Gal3 antibody at a tissue site of interest results in an increase in the expression of fibronectin. In some embodiments, administration of an anti-Gal3 antibody at a tissue site of interest results in a decrease in the expression of TGF-β1.

[0237] In some embodiments, one or more of the fibrosis biomarkers are utilized to monitor the presence or absence of fibrosis or the progression of fibrosis.

[0238] In some cases, decreased expression of the fibrosis biomarkers disclosed herein may indicate decreased tissue fibrosis.

[0239] In some cases, the expression of at least one fibrosis biomarker in tissue treated with anti-Gal3 antibody is different from the expression of said at least one fibrosis biomarker in control tissue treated with control antibody. In some cases, the control antibody is an anti-Gal3 antibody that does not bind to the one or more epitopes and / or does not interfere with the interaction of Gal3 with TIM-3. In some cases, the control antibody is an IgG2b antibody, such as a mouse IgG2b (mIgG2b) antibody. In some cases, the expression of at least one fibrosis biomarker in tissue treated with anti-Gal3 antibody is less than the expression of at least one fibrosis biomarker in control tissue treated with mIgG2b antibody.

[0240] Fibrotic disorders In some embodiments, anti-Gal3 antibody can be administered to treat one or more fibrotic diseases.Fibrotic disease can be liver fibrosis.Fibrotic disease can be pulmonary fibrosis.Fibrotic disease can be cystic fibrosis, idiopathic pulmonary fibrosis, myelofibrosis, interstitial lung disease, liver fibrosis, progressive massive fibrosis, cirrhosis, renal fibrosis, cardiac fibrosis, interstitial pneumonia, pulmonary fibrosis, pancreatic fibrosis, myelofibrosis, intestinal fibrosis, arthrofibrosis, retinal fibrosis, hepatitis C-related fibrosis, or nephrogenic systemic fibrosis.

[0241] In some cases, anti-Gal3 antibody can be administered to fibrotic diseases associated with α-SMA or fibronectin expression. Fibrotic diseases associated with α-SMA increase can be renal fibrosis, liver fibrosis, cirrhosis, hepatitis C-related fibrosis, cardiac fibrosis, pulmonary fibrosis, interstitial lung disease, idiopathic pulmonary fibrosis, interstitial pneumonia, bone marrow fibrosis, arthrofibrosis, retinal fibrosis, or nephrogenic systemic fibrosis. Fibrotic diseases associated with fibronectin expression can be cystic fibrosis, pulmonary fibrosis, idiopathic pulmonary fibrosis, bone marrow fibrosis, interstitial lung disease, liver fibrosis, progressive massive fibrosis, cirrhosis, renal fibrosis, cardiac fibrosis, interstitial pneumonia, pulmonary fibrosis, pancreatic fibrosis, bone marrow fibrosis, intestinal fibrosis, arthrofibrosis, retinal fibronectin, hepatitis C-related fibrosis, or nephrogenic systemic fibrosis.

[0242] The subject of treatment may be diagnosed with a fibrotic disease. In some embodiments, the subject of treatment may be a human, rat, mouse, or other animal. In some embodiments, the subject of treatment may be a mammal. In some embodiments, the mammal may be a human. The mammal may be a primate. The primate may be a chimpanzee or a gorilla.

[0243] In some embodiments, the anti-Gal3 antibody binds to a specific epitope within the Gal3 protein. In some cases, the anti-Gal3 antibody can bind to at least 1, 2, 3, 4, 5, 6, 10, 15, or 20 amino acid residues within the Gal3 region corresponding to residues 2-21 of SEQ ID NO:1 (hGal3). In some embodiments, the anti-Gal3 antibody can bind to at least 1, 2, 3, 4, 5, 6, 10, 15, or 20 amino acid residues corresponding to residues 42-71 of SEQ ID NO:1. In other embodiments, the anti-Gal3 antibody can bind to at least 1, 2, 3, 4, 5, 6, 10, 15, 20, 30, 40, or 50 amino acid residues corresponding to residues 42-91 of SEQ ID NO:1. Alternatively, the anti-Gal3 antibody can bind to at least 1, 2, 3, 4, 5, 6, 10, 15, or 20 amino acid residues corresponding to residues 72-91 of SEQ ID NO:1. In some cases, the anti-Gal3 antibody is a nucleotide sequence that binds residues 2-21 and 42-71; 42-91; 2-21 and and 72-91; or 2-21 and 42-91. Gal3 and TIM-3 sequences are listed in Table 1.

[0244] [Table 1-1] [Table 1-2]

[0245] In some embodiments, the anti-Gal3 antibody may bind to at least 1, 2, 3, 4, 5, 6, 10, 15, or 20 amino acid residues within a peptide shown in Table 2 (and shown in FIG. 11A).

[0246] [Table 2]

[0247] In some embodiments, the anti-Gal3 antibody may bind to at least 1, 2, 3, 4, 5, 6, 10, 15, or 20 amino acid residues in peptide_1 (SEQ ID NO:3), peptide_5 (SEQ ID NO:4), peptide_6 (SEQ ID NO:5), or peptide_8 (SEQ ID NO:6). In some embodiments, the anti-Gal3 antibody may bind to at least 1, 2, 3, 4, 5, 6, 10, 15, or 20 amino acid residues in peptide_1 (SEQ ID NO:3). In some embodiments, the anti-Gal3 antibody may bind to at least 1, 2, 3, 4, 5, 6, 10, 15, or 20 amino acid residues in peptide_5 (SEQ ID NO:4). In some embodiments, the anti-Gal3 antibody may bind to at least 1, 2, 3, 4, 5, 6, 10, 15, or 20 amino acid residues in peptide_6 (SEQ ID NO:5). In some embodiments, the anti-Gal3 antibody may bind to at least 1, 2, 3, 4, 5, 6, 10, 15, or 20 amino acid residues within peptide_8 (SEQ ID NO:6).

[0248] In some embodiments, the anti-Gal3 antibody further disrupts the interaction of Gal3 with TIM-3, a molecule expressed in immune cells, particularly T cells, that can inhibit immune responses, e.g., T cell signaling, by interacting with Gal3.

[0249] In some embodiments, Gal3-TIM-3 antibodies are designed based on the interface where the interaction between Gal3 and TIM-3 occurs. The interaction for Gal3 can occur at one or more residues selected from regions 145-168, 149-168, 160-177, and / or 165-184, where the regions correspond to positions 145-168, 149-168, 160-177, and 165-184 of SEQ ID NO:1. In some embodiments, the interaction for Gal3 can occur at one or more residues within region 145-177, where region 145-177 corresponds to positions 145-177 of SEQ ID NO:1. The interaction can occur at one or more residues within region 160-184, where region 160-184 corresponds to positions 160-184 of SEQ ID NO:1. In some embodiments, the interaction may occur at one or more residues within the region 145-184, which corresponds to positions 145-168 of SEQ ID NO:1.

[0250] In some embodiments, the Gal3-TIM-3 antibody disrupts the interaction of Gal3 with TIM-3, and the interaction for Gal3 involves one or more residues selected from regions 145-168, 149-168, 160-177, and / or 165-184 of SEQ ID NO:1. The interaction for Gal3 can occur at one or more residues within region 145-177 of SEQ ID NO:1. The interaction can occur at one or more residues within region 160-184 of SEQ ID NO:1. The interaction can occur at one or more residues within region 145-184 of SEQ ID NO:1.

[0251] In some embodiments, the interaction may occur at one or more residues of Gal3 selected from regions 149-156, 152-171, 152-169, 152-168, 163-169, or 163-171, which regions correspond to positions 149-156, 152-171, 152-169, 152-168, 163-169, and 163-171 of SEQ ID NO:1. In some embodiments, the Gal3-TIM-3 antibody disrupts the interaction of Gal3 with TIM-3, the interaction for Gal3 involving one or more residues selected from regions 149-156, 152-171, 152-169, 152-168, 163-169, or 163-171, the regions corresponding to positions 149-156, 152-171, 152-169, 152-168, 163-169, and 163-171 of SEQ ID NO:1. The interaction can occur at one or more residues of Gal3 selected from region 149-156, the region corresponding to positions 149-156 of SEQ ID NO:1. The interaction can occur at one or more residues of Gal3 within region 163-169, the region corresponding to positions 163-169 of SEQ ID NO:1. The interaction may occur at one or more residues of Gal3 within region 163-171, which region corresponds to positions 163-171 of SEQ ID NO:1. The interaction may occur at one or more residues of Gal3 within region 152-169, which region corresponds to positions 152-169 of SEQ ID NO:1. The interaction may occur at one or more residues of Gal3 within region 152-171, which region corresponds to positions 152-171 of SEQ ID NO:1. The interaction may occur at one or more residues of Gal3 within region 163-171, which region corresponds to positions 163-171 of SEQ ID NO:1.

[0252] In some embodiments, the Gal3-TIM-3 antibody disrupts the interaction of Gal3 with TIM-3, and the interaction for Gal3 involves one or more residues selected from regions 149-156, 152-171, 152-169, 152-168, 163-169, or 163-171 of SEQ ID NO:1. The interaction can occur at one or more residues of Gal3 within region 149-156 of SEQ ID NO:1. The interaction can occur at one or more residues of Gal3 within region 163-169 of SEQ ID NO:1. The interaction can occur at one or more residues of Gal3 within region 163-171 of SEQ ID NO:1. The interaction can occur at one or more residues of Gal3 within region 152-169 of SEQ ID NO:1. The interaction can occur at one or more residues of Gal3 within region 152-171 of SEQ ID NO:1. The interaction may occur at one or more residues of Gal3 within the region 163-171 of SEQ ID NO:1.

[0253] A Gal3-TIM-3 antibody can interact with at least 1, 2, 3, 4, 5, 6, 10, 15, 20, 30, or 40 amino acid residues within a Gal3 region that interferes with TIM-3 at the positions described herein.

[0254] The interaction with TIM-3 may occur at one or more residues corresponding to positions 72-104 and / or 64-93, which correspond to positions 90-122 and 82-111 of SEQ ID NO:2. Alternatively, the interaction with TIM-3 may occur at one or more residues at positions 91-111, 107-117, 96-102, 100-106, and / or 92-119, which correspond to positions 91-111, 107-117, 96-102, 100-106, and 92-119 of SEQ ID NO:2. The interaction can occur at one or more residues at positions 91-117, 91-119, 96-117, 100-117, or 96-106. Gal3-TIM-3 interfering antibodies can be designed to interact with at least 1, 2, 3, 4, 5, 6, 10, 15, 20, 30, or 40 amino acid residues within the TIM-3 region that interferes with Gal3 at the positions described herein.

[0255] In some cases, the interaction may occur at one or more residues of Gal3 selected from regions 149-156, 152-168, 163-169, and / or 163-171 of SEQ ID NO:1; as well as one or more residues corresponding to positions 90-122 and / or 82-111 of SEQ ID NO:2. The interaction may occur at one or more residues of Gal3 selected from regions 149-156, 152-168, 163-169, and / or 163-171 of SEQ ID NO:1; as well as one or more residues at positions 91-111, 107-117, 96-102, 100-106, and / or 92-119 of SEQ ID NO:2. Interactions with Gal3 can occur at one or more residues selected from regions 145-168, 160-177, and / or 165-184 of SEQ ID NO: 1; and one or more residues corresponding to positions 90-122 and / or 82-111 of SEQ ID NO: 2. Interactions with Gal3 can occur at one or more residues selected from regions 145-168, 160-177, and / or 165-184 of SEQ ID NO: 1; and one or more residues at positions 91-111, 107-117, 96-102, 100-106, and / or 92-119 of SEQ ID NO: 2. Gal3-TIM-3 interfering antibodies can be designed to interact with at least 1, 2, 3, 4, 5, 6, 10, 15, 20, 30, or 40 amino acid residues on the Gal3 region and on TIM-3 that interfere with each other at the positions described herein.

[0256] For any of the embodiments provided herein, the anti-Gal3 antibody used can be replaced with another anti-Gal3 antibody, including but not limited to 2D10.2B2, 3B11.2G2, 4A11.2B5, 4G2.2G6, 6H6.2D6, 7D8.2D8, 12G5.D7, 13A12.2E5, 13G4.2F8, 13H12.2F8, 14H10.2C9, 15F10.2D6, 15G7.2A7, 19B5.2E6, 19D9. 2E5, 20D11.2C6, 20H5.A3, 23H9.2E4, 24D12.2H9, 846.1F5, 846.2H3, 846T.1H2, 9H2.2H10, IMT001-4, IMT006-1, IMT006-5, IMT006-8, and mIMT001 (IMT001). The anti-Gal3 antibodies were: 2D10.2B2, 3B11.2G2, 4A11.2B5, 4G2.2G6, 6H6.2D6, 7D8.2D8, 12G5.D7, 13A12.2E5, 13G4.2F8, 13H12.2F8, 14H10.2C9, 15F10.2D6, 15G7.2A7, 19B5.2E6, 19D9.2 The anti-Gal3 antibody may be mIMT001-4, IMT006-1, IMT006-5, IMT006-8, or mIMT001, or any combination thereof. The anti-Gal3 antibody may be mIMT001. The anti-Gal3 antibody may be an antibody other than mIMT001. The anti-Gal3 antibody may be 4A11.2B5. The anti-Gal3 antibody may be one or more of IMT001-4, IMT006-1, IMT006-5, or IMT006-8. The anti-Gal3 antibody may be 4A11.2B5. The anti-Gal3 antibody can be IMT001-4. The anti-Gal3 antibody can be IMT006-1. The anti-Gal3 antibody can be IMT006-5. The anti-Gal3 antibody can be IMT006-8. In some embodiments, the antibody comprises one or more of the CDRS, VH, and / or VL of any one or more of these antibodies. EXAMPLES

[0257] Certain aspects of the above embodiments are disclosed in further detail in the following examples, which in no way limit the scope of the disclosure. Those skilled in the art will recognize that many other embodiments are also within the scope of the invention, as described hereinabove and in the claims.

[0258] Example 1: Generation of Gal3 overexpressing cell lines American Tissue and Cell Culture Collection (AT A20, a murine B lymphocyte cell line obtained from Cell Signaling Technology, Inc. (Cell Signaling, Inc., Manassas, VA), was transfected with a nucleic acid construct encoding Flag-tagged human Gal3 protein or Flag-tagged human PDL1 protein. The construct further contains an antibiotic resistance marker. Transduced cells were selected based on antibiotic resistance to generate A20 cells stably expressing Flag-tagged human Gal3 protein (A20 hPDL1 cells) or A20 cells stably expressing Flag-tagged human PDL1 protein (A20 hPDL1 cells).

[0259] Example 2. Gal3 specifically binds to TIM-3 This example describes various assays that were performed to assess the interaction between Gal3 and TIM-3.

[0260] Binding assay - co-immunoprecipitation Co-immunoprecipitation experiments were performed to test whether TIM-3 specifically interacts with Gal3. 293T cells were co-transfected with a plasmid encoding HA-tagged TIM-3 and a plasmid encoding Flag-tagged Gal3, Flag-tagged Gal9, or Flag-tagged CEACAM1. Transfection was performed using Lipofectamine 3000 (Waltham, MA) according to the manufacturer's protocol. Transfected cells were grown overnight, then washed and lysed in 1 ml of lysis buffer. Lysed cells were centrifuged and the supernatant (lysate) was collected. Lysates were prepared, separated by SDS PAGE, and probed with anti-HA (Figure 1A) and anti-Flag antibodies (Figure 1B), respectively. Both anti-Flag and anti-HA antibodies were purchased from Sigma. Arrows in Figure 1A and Figure 1B indicate the presence of various proteins.

[0261] For immunoprecipitation, anti-Flag agarose beads (Abcam, Cambridge, MA) were added to the supernatant (lysate) obtained above. The beads and lysate were incubated at 4°C overnight with rotation to allow the Flag-tagged proteins to bind. The beads were then washed in triplicate with lysis buffer, mixed with 1x SDS PAGE sample buffer, boiled and resolved by SDS-PAGE. The SDS-PAGE gel was transferred to a membrane that was probed with an anti-HA antibody (Figure 1C). In Figure 1A-C, columns 1-3 are results from lysates obtained from cells co-transfected with a plasmid encoding HA-tagged TIM-3 and a plasmid encoding Flag-tagged Gal3; cells co-transfected with a plasmid encoding HA-tagged TIM-3 and a plasmid encoding Flag-tagged Gal9, or cells co-transfected with a plasmid encoding HA-tagged TIM-3 and a plasmid encoding Flag-tagged CEACAM1, respectively.

[0262] As shown in Figure 1A-C, the results show that human Gal3 specifically pulls down human TIM-3, whereas human CEACAM1 was unable to pull down HA-tagged human TIM-3. Human Gal9 also appeared to pull down human TIM-3 (Figure 1C, column 2), but this was likely nonspecific due to Gal9 protein aggregation - the molecular weight of Gal9 is likely much larger than its actual size of 40 kDa. The conclusion that the interaction of Gal9 with TIM-3 is nonspecific in nature is also supported by the evidence shown below in Figure 5B.

[0263] Further co-immunoprecipitation experiments were performed to test whether Gal3 specifically interacts with TIM-3. Flag-human Gal3 plasmid (OriGene, Rockville, MD) was transfected into 293T cells at 80% confluence. Transfection was performed in 10 cm plates using Lipofectamine 3000 as described above. After overnight transfection, cells were replaced on 10 cm plates coated with human Fc, human PD1-Fc, or human TIM-3 Fc for 3 h. Cells were washed once in 1×PBS and then lysed in 1 ml lysis buffer. Cell lysates were collected and centrifuged. After centrifugation, protein G beads were added to the resulting supernatant and incubated with rotation at 4°C for 4 h. The beads were then washed three times with lysis buffer and then 1×SDS PAGE sample buffer was added. Samples containing beads were boiled, separated by SDS-PAGE, and transferred onto membranes. The membrane was then probed with anti-Flag antibody. As shown in Figure 2, human TIM-3 specifically pulled down Flag-tagged Gal3. In contrast, neither human Fc nor human PD1 Fc was able to pull down TIM-3. This indicates that Gal3 does not bind to Fc or PD1 and that the binding of Gal3 to TIM-3 is specific.

[0264] Binding assay - cell adhesion assay Next, a cell adhesion assay was performed to confirm the binding of Gal3 to TIM-3. In this experiment, a 96-well plate was coated with human Fc, human PD1 Fc, human VISTA-Fc, and human TIM-3-Fc overnight at 4°C, and then blocked with 2% BSA in PBS at 37°C for 2 hours. A20, A20 overexpressing human Gal3 (A20 Gal3) cells, or A20 overexpressing human PDL1 (A20 PDL1) cells were seeded into the wells coated with the various Fc proteins. The plate was then centrifuged at 720 rpm before stopping. The plate was incubated at 37°C for 30 minutes and then submerged in PBS. The plate was gently inverted 180° and kept in the inverted position for 30 minutes. After the plate was replaced and removed from the PBS, 200 μl of solution was removed from each well and discarded, and the remaining solution, approximately 100 μl in volume, was transferred to the 96-well plate. Cells were counted by flow cytometric analysis.

[0265] The results (Figure 3) show that the number of A20 expressing human Gal3 (A20 Gal3) cells that adhered to human TIM-3 Fc-coated plates was significantly greater than the number of cells that adhered to plates coated with human VISTA-Fc or human PD1 Fc. As expected, since PDL1 is a known ligand for PD1, the number of A20 PDL1 cells found to adhere to hPD1 Fc was significantly greater than those that adhered to plates coated with human VISTA-Fc or human TIM-3 Fc. These results further confirmed that the interaction between Gal3 and TIM-3 is specific.

[0266] Blocking assay - flow cytometry Flow cytometry analysis was performed to evaluate the binding of Gal3 to TIM-3 using A20 cells. A20 Gal3 cells were incubated with 10% FBS HBSS solution with or without mouse TIM-3 Fc for 20 min on ice. There were five experimental groups: in group 1, A20 Gal3 cells were incubated without mTIM-3 Fc protein as a control; in group 2, A20 Gal3 cells were incubated with mTIM-3 Fc protein; in groups 3, 4, and 5, in addition to mTIM-3 Fc protein, anti-mouse TIM-3 polyclonal antibody (R&D System, Minneapolis, MN) (group 3), monoclonal antibody RMT3-23 (Bio X Cell, West Lebanon, NH) (group 4), and monoclonal antibody 215015 (R&D System) (group 5) were added to evaluate whether these antibodies could bind Gal3 to TIM-3. For blocking, the cells were treated with the mentioned antibodies. The cells were incubated with 10% FBS HBSS solution containing mTiM-3 Fc for 20 min, then added with 10% FBS HBSS containing mTiM-3 Fc for 20 min. The samples were centrifuged and the pellet was added to 10% FBS HBSS containing APC-conjugated anti-hFc antibody (Jackson ImmunoResearch, West Grove, PA) for 20 min. After spinning, live / dead cells were stained with Violet Dead Cell Dye Kit (Life Technologies). The stained cells were subjected to flow analysis.

[0267] Figure 4 shows that mTIM-3 can bind to Gal3 protein on dead and live cells and that Gal3 and dead cells bind to different epitopes on TIM-3. Figure 4A shows live A20 cells (peak on the left) and dead A20 cells (peak on the right) by flow cytometry analysis. In this assay, TIM-3 Fc binds to both dead cells (Figure 4C, column 2) and Gal3 expressed on live cells (Figure 4B, column 2). However, mTIM-3 monoclonal antibody RMT3-23 blocked TIM-3 binding to dead cells (Figure 4C, column 4) but did not block binding to Gal3 expressed on live cells (Figure 4B, column 4). This indicates that Gal3 and dead cells bind to different epitopes on TIM-3. As a control, neither the mTIM-3 polyclonal antibody nor the monoclonal antibody 215015 (R&D System, Minneapolis, MN) had any effect on TIM-3 binding to Gal3 (Fig. 4B , columns 3 and 5) or to dead cells (Fig. 4C , columns 3 and 5), respectively.

[0268] Blocking assay - ELISA ELISA assays were also performed to test the interaction of Gal3 with TIM-3. 96-well ELISA plates (ThermoFisher Scientific) were coated with mouse Gal3 protein (BioLegend, San Diego, CA) in PBS or human Gal9 protein (R&D systems) in PBS or phosphatidylserine (PS) (Sigma) in ethanol overnight at 4°C. The plates were washed three times with TBST and then blocked with 2% BSA-containing PBS buffer at room temperature for 1 hour. In Figure 5A, different anti-Gal3 antibodies, namely mGal3 polyclonal antibody (R&D systems), mAb IMT001 (described in WO 2019 / 023247, expressly incorporated herein by reference in its entirety), mAb M3 / 38 (ThermoFisher Scientific) (Figure 5A), were added to the wells coated with Gal3. The antibodies were incubated for 10 minutes, then mouse TIM-3 Fc was added to the plate and incubated for an additional hour. The plate was then washed three times and then incubated with anti-human IgG-HRP (Jackson ImmunoResearch). After washing three times with TBST, the plate was developed with TMB substrate (TMB subtract) (GeneTex, Irvine, CA) and the reaction was stopped with 1N HCl. Optical density (OD) was read at 450 nm. Results are expressed as the mean OD ± standard deviation of duplicates. The results in Figure 5A showed that, among all antibodies tested, mouse Gal3 polyclonal antibody and monoclonal antibody IMT001 blocked the interaction of Gal3 with TIM-3 (Figure 5A).

[0269] In Figure 5B, mouse Gal3 protein (BioLegend) in PBS (groups 1 and 2) or PS (Sigma-Aldrich, St. Louis, MO) in ethanol (groups 3 and 4) were coated onto plates and incubated overnight at 4°C. Anti-mTIM-3 mouse antibody, mAb RMT3-23 (Bio X cell), was added to the coated plates only for groups 2 and 4. Secondary anti-human IgG-HRP antibody and substrate were added as above to detect binding of mMTIM-3 to mGal3 or PS. The results showed a dramatic reduction in signal in group 4 compared to group 3, indicating that RMT3-23 blocks PS from binding to TIM-3; whereas the results showed a dramatic reduction in signal compared to group 1. There was no dramatic decrease in signal in group 2, indicating that RMT3-23 did not block Gal3 from binding to TIM-3. Because TIM-3 binds to dead cells through interaction with PS externalized and exposed on the dead cell surface, these experiments confirmed the observations in Figures 4A-C that Gal3 and PS bind to different epitopes on TIM-3.

[0270] For sugar-dependent assays, ELISA plates were coated with either mGal3 (groups 1 and 2) or hGal9 (groups 3 and 4). Mouse TIM-3 Fc protein (R&D systems) was added to ELISA plates coated (groups 2 and 4) or not coated (groups 1 and 3) with 25 mM α-lactose (Sigma-Aldrich) for 1 h at room temperature. Secondary anti-human IgG-HRP antibody and substrate were added as above to detect binding of mMTIM-3-Fc to mGal3 or hGal9. Figure 5C shows that lactose blocks Gal9 binding to TIM-3, as shown by a dramatic 10-fold decrease in the signal in group 4 (with lactose present) compared to group 3 (without lactose present), indicating sugar-dependent binding of Gal9 to TIM-3. In contrast, although the blocking effect of lactose on Gal3 binding to TIM-3 was minimal, there was no significant difference between group 2 (with the presence of lactose) and group 1 (without the presence of lactose) in the signal resulting from the binding of TIM-3 to Gal3, indicating that the interaction of Gal3 with TIM-3 was not affected by the presence of sugar, i.e., the interaction was sugar-independent.

[0271] Example 3. Overexpression of Gal3 suppresses T cell activation This example describes experiments performed to evaluate the functional properties of Gal3 overexpression in A20 cells.

[0272] A20 clones, #41, #31, and #15, stably overexpressing hGal3, were generated as described above. Figure 6A shows the results of flow cytometry analysis showing the hGal3 expression levels in these clones. A20 cells or A20 Gal3 clones were mixed with mouse D011.10 T cells. The mixture was placed into each well of a flat 96-well plate, and then OVA323-339 peptide (Invivogen, San Diego, CA) was added to the plate. After overnight incubation, the supernatant was used to measure IL-2 production of T cells by ELISA (Thermo Fisher Scientific). As shown in Figure 6B, IL-2 production by mouse D011.10 T cells was significantly reduced when mixed with any of the three mouse A20 cell clones compared to when T cells were mixed with parental A20 cells.

[0273] Example 4. Anti-Gal3 antibody exhibits anti-tumor activity in a mouse lung metastasis model In this example, experiments were performed to evaluate the antitumor efficacy of Gal3:TIM-3 inhibitors in vivo. Animal experiments were performed in accordance with protocols approved by the Molecular Medicine Research Institute Institutional Animal Care and Use Committee. C57BL / 6 mice were housed upon arrival in a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care. Thirty-six 7-week-old female mice were randomly assigned into three groups (n=12). On day 0, B16F10 cells (2×10 in 0.1 ml PBS) were inoculated into the 30-well plate. 5 100 pieces) were washed, resuspended in PBS, and injected into the tail vein of mice using a syringe with a 27-gauge needle. After injection of B16F10 cells, mouse IgG2b (Bio X Cell, West Lebanon, NH) was injected on days 0, 3, 7, and 10, mPD1 antibody (Bio X Cell, West Lebanon, NH) was injected on days 0, 3, 7, 10, and 15. Animals were intraperitoneally administered 10 mg / Kg of Gal3 antibody IMT001. The Gal3 antibody clone IMT001 used in this experiment recognizes an epitope corresponding to peptide_5 (PGAYPGQAPPGAYPGQAPPG, SEQ ID NO: 7) of Gal3. On day 21, animals were humanely sacrificed and lung tissue was removed and fixed in 10% buffered formaldehyde solution. The number of black metastatic colonies on one side of the left lobe of the lung was counted (Figure 7B). Results were expressed as mean ± standard error of the mean. Statistical analysis was performed in comparison with the IgG control group using one-way ANOVA.

[0274] Figure 7A shows that the mean fluorescence intensity (MFI) of B16F10 cells stained with anti-mGal3 antibody is almost 10-fold higher than that of cells stained with isotype control antibody. In detail, B16F10 cells were incubated with 10% FBS HBSS solution containing control rat IgG PE or rat anti-mouse Gal3 PE antibody (Thermo Fisher Scientific, Waltham, MA) for 20 min on ice. After spinning, live / dead cells were stained with Violet Dead Cell Dye Kit (Thermo Fisher Scientific, Waltham, MA). The stained cells were subjected to flow analysis. Figure 7B shows representative images of whole lungs from the three treatment groups. Figure 7C shows the number of metastatic colonies (mean ± standard deviation of the mean) on the left lung lobe surface. Figure 7D and Figure 7E show the lung weight and body weight (mean ± standard error of the mean) of the different treatment groups. Compared with the isotype control group, the Gal3 antibody treatment group showed a significant (about 46%) reduction in tumor numbers as indicated by the number of black metastatic colonies (p<0.01). However, compared with the isotype control group, the anti-mouse PD1 antibody 29F did not show a significant antitumor effect in this lung metastatic model (p>0.05).

[0275] Example 5. Anti-Gal3 antibodies show anti-tumor activity in a 4T1 orthotopic tumor-induced lung metastasis model Animal experiments followed protocols approved by the Molecular Medicine Research Institute Institutional Animal Care and Use Committee. Seven-week-old female Balb / c mice were housed upon arrival in a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care. On the day of tumor implantation, 4T1 cells were harvested, washed, and resuspended in PBS. Mice were anesthetized with inhalation anesthetic (3-5% isoflurane in medical air). 2 × 10 5 Cells were injected subcutaneously into the mammary gland using a syringe with a 25-ga needle. Mice were randomly assigned to two groups (n=10). After injection of 4T1 cells, animals were intraperitoneally administered 10mg / Kg of mouse IgG2b (Bio X Cell) on days 0, 3 and 7 or Gal3 antibody IMT001 on days 0, 3, 7, 10 and 14. Tumor burden and body weight were monitored twice weekly. On day 30, mice were humanely sacrificed and lung tissue was inflated with 30% sucrose, removed and fixed in Bouin's fixative (Sigma-Aldrich). The number of metastatic colonies on one side of the left lobe of the lung was counted. Results were expressed as mean ± standard error of the mean. Statistical analysis was performed using an unpaired T-test in comparison with the IgG control group.

[0276] Figure 8A shows representative images of whole lungs from treatment groups. Figure 8B shows the body weights (mean ± standard error of the mean) of the different treatment groups. Figure 8C shows the number of metastatic colonies (mean ± standard deviation of the mean) in one side of the left lung lobe. Compared to mice treated with isotype control antibody, animals treated with monoclonal anti-Gal3 antibody showed a significant reduction in the number of lung metastases (p<0.05).

[0277] Example 6. Anti-Gal3 antibodies show anti-tumor activity in primary murine RENCA kidney tumor model To evaluate the antitumor efficacy of Gal3:TIM-3 inhibitors in primary tumor models Experiments were performed on the 10-well plate (Figure 9). Animal experiments were performed according to protocols approved by the Molecular Medicine Research Institute Institutional Animal Care and Use Committee. Balb / c mice were housed in an Association for Assessment and Accreditation of Laboratory Animal Care-accredited facility upon arrival. Seven-week-old female mice were randomly assigned to three groups (n=15). On the day of tumor implantation, mice were anesthetized with inhalation anesthetic (3-5% isoflurane in medical air), Renca cells were washed and resuspended in PBS, and then 2 × 10 cells were cultured in 0.1 mL of PBS. 5 Cells were injected subcutaneously using a syringe equipped with a 25-ga needle. After Renca cell injection, animals were intraperitoneally administered either mouse IgG2b (Bio X Cell) on days 0, 3 and 7, mPD1 antibody (BioX Cell) on days 0, 3 and 7, or 10mg / Kg of Gal3 antibody IMT001 on days 0, 3, 7, 10 and 14. Tumor volumes in the control group were between 2000 and 2500mm. 3 Animals were humanely sacrificed when the IgG2b concentration reached 0.01 mg / kg / day. Results were expressed as mean ± standard error of the mean. Statistical analysis was performed in comparison with the IgG2b control group using an unpaired t-test.

[0278] The results show the antitumor activity of Gal3 antibody (IMT001) in a renal cancer model. When compared with the isotype control group, the anti-Gal3 antibody treatment group showed a significant (about 35%) reduction in tumor growth (p<0.05), whereas the anti-PD-1 antibody had no effect (Figure 9).

[0279] Example 7. Anti-Gal3 antibodies show anti-tumor activity in primary murine MC38 COLON tumor model Animal experiments followed protocols approved by the Molecular Medicine Research Institute Institutional Animal Care and Use Committee. Seven-week-old female C57BL / 6 mice were housed upon arrival in an Association for Assessment and Accreditation of Laboratory Animal Care-accredited facility. On the day of tumor implantation, MC38 mouse colon adenocarcinoma cells were harvested, washed, and resuspended in PBS. Mice were anesthetized with inhalation anesthetic (3-5% isoflurane in medical air). 5 × 10 5 Cells were injected subcutaneously into the right flank of mice using a syringe with a 25-ga needle. On day 7, tumor volume was measured and mice were randomly assigned to two groups (n=8). Animals were administered 10mg / Kg of mouse IgG2b (BioXCell) or Gal3 antibody IMT001 intraperitoneally on days 7, 10, 14, 17 and 22. Tumor volume and body weight were monitored twice weekly. Tumor volume was 3000mm 3 Animals were humanely sacrificed when the IgG antibody titer reached 1.0. Results were expressed as mean ± standard error of the mean. Statistical analysis was performed using unpaired T-tests in comparison with the IgG control group.

[0280] The results in Figure 10 show that the IMT001 antibody has anti-tumor activity in the MC38 colon cancer model. Compared to mice treated with an isotype control antibody, IMT001 antibody-treated mice showed a significant reduction in tumor burden (about 33%) at day 24 (p<0.05).

[0281] Example 8. Epitope Binding of Gal3 Antibody Clone IMT001 A peptide array containing 24 20-amino acid peptides overlapping by 10 amino acids and covering the entire human Gal3 protein sequence was synthesized (Genscript, Piscataway, NJ) (Figure 11A). 20 μg of each peptide was dot blotted onto the membrane. After blocking with 5% milk in PBS, the membrane was incubated with 1 μg / ml IMT001 antibody overnight at 4°C. After three washes, the membrane was incubated with 1:8000 diluted anti-mIgG HRP antibody (Southern Biotech, Birmingham, Alabama) for 1 hour. After three washes, the membrane was incubated with Western ECL blotting substrate (Bio-Rad, Hercules, CA) and developed (FIG. 11b). Peptides 5 (SEQ ID NO:7) and 6 (SEQ ID NO:8) showed good signals, indicating that the epitope on hGal3 to which IMT001 binds is PGAYPGQAPPGAYPGQAPPGAYPGAPGAYP.

[0282] To further define the binding epitope of IMT001 on the peptide, eight short peptides derived from it were synthesized (Genscript, Piscataway, NJ) (Figure 11C) and their binding by IMT001 was determined by ELISA (Figure 11D). 96-well Elisa plates (Thermo Scientific) were coated with these peptides in PBS buffer and incubated overnight at 4°C. The plates were washed three times with TBST and then blocked with 2% BSA in PBST buffer for 1 hour at room temperature. 10 μg / mL of IMT001 was incubated on the coated Elisa plates for 1 hour at room temperature. The plates were washed three times and then incubated with a 1:8000 dilution of anti-mouse IgG-HRP for 1 hour at room temperature. After washing three times with TBST, the color was developed with 100 μL of TMB substrate (TMB subtract) (GeneTex) and stopped with 50 μL of 1N HCl. Optical density (OD) was read at 450 nm. Results were expressed as mean OD of duplicates ± standard deviation. Pep-2 showed good signal, indicating that the binding epitope of IMT001 on human Gal3 is GQAPPGAYPG.

[0283] Example 9. Immune profiling in B16F10 lung metastatic mouse tumors Mice were implanted intravenously with 1 million B16F10 cells. Mice were then treated with IMT001 or isotype control (intraperitoneally, 10 mg / kg) on ​​days 0, 1, 3, and 7, and sacrificed on day 8 to isolate and phenotype lung immune cells. Cells were isolated from the lungs and then stained with fluorescently labeled antibodies against lymphocyte markers CD3, CD4, CD8, CD19, DX5, and analyzed by flow cytometry. The results in Figure 12 show that, compared to isotype control antibody treatment, anti-Gal3 antibody increased the number of various immune effector cells, including CD3 T lymphocytes, CD4 T helper, CD8 cytotoxic T cells, CD19 B cells, and DX5 natural killer cells, in the lungs hosting the tumor. This indicates that anti-Gal3 antibody was able to activate immune cells.

[0284] Example 10. Ga detected in human lung cancer-associated macrophages l3 expression Immunohistochemistry (IHC) experiments were performed to detect Gal3 expression in human lung cancer. Human lung cancer frozen tissue slides (US Biomax Inc.) were fixed in 10% natural buffered formalin (Fisher Scientific) for 10 min at room temperature and washed twice for 5 min in PBS. Endogenous peroxidase was blocked by immersing the slides in 3% H2O2 for 10 min at room temperature. After washing twice for 5 min in PBS, the slides were incubated in streptavidin reagent (Molecular Probes) for 15 min at room temperature, then rinsed extensively in PBS, incubated in biotin reagent (Molecular Probes) for 15 min, and further rinsed in PBS to block endogenous biotin background. Slides were blocked with 10% FBS, 200 μg / mL mIgG and 200 μg / mL hIgG for 1 h, incubated with primary antibody IMT001-biotin (5 μg / mL) overnight at 4°C, washed three times, and then incubated with secondary antibody HRP Avidin (BioLegend) at 1:100 for 1 h and washed three times. Staining was performed by incubation with DAB substrate (Vector Laboratories) and stopped by immersing the slides in distilled water. Human lung cancer slides were finally counterstained in Hematoxylin QS (Vector Laboratories), washed in distilled water, and stained with graded ratios of ethanol / xylene. The sections were then dehydrated in ethyl acetate and mounted with VectaMount™ Mounting Medium (Vector Laboratories).

[0285] The results in Figures 13A-B show that canopy-shaped tumor-associated macrophages (squamous cell carcinoma and adenocarcinoma) in these human lung cancer slides express Gal3, as evidenced by their positive staining with IMT001.

[0286] Example 11. Gal3 expression in human M2 macrophages Primary human CD14 monocytes were isolated from peripheral blood mononuclear cells (PBMCs) using a CD14 cell positive selection kit (Miltenyi, Auburn, CA) and differentiated into dendritic cells (DCs), M1 macrophages, or M2 macrophages in the presence of GM-CSF+IL-4, or GM-CSF, or M-CSF (Rocky Hill, NJ), respectively. Flow cytometry analysis was performed to detect Gal3 expression in human dendritic cells (DCs), M1, and M2 macrophage cells. In detail, 100,000 DCs, M1, or M2 cells were incubated with 100 μl 10% FBS HBSS solution containing control mIgG-biotin (BioLegend) or IMT001-biotin at 10 μg / ml for 20 min on ice. The cells were then washed and incubated with PE-streptavidin (BioLegend) at 1:1000 for 20 min on ice. After spinning, live / dead cells were stained with Violet Dead Cell Dye Kit (Life Technologies). The stained cells were subjected to flow analysis. The results in Figure 14C show that the mean fluorescence intensity (MFI) of M2 cells stained with IMT001 was much higher than that of cells stained with isotype control antibody, indicating that IMT001 specifically binds to M2 cells but cannot stain dendritic cells (Figure 14A) and M1 macrophages (Figure 14B).

[0287] Example 12. Anti-Gal3 antibody enhances mouse T cell activation in macrophage / T cell reactions Gal3 expression on mouse macrophages was detected by both IHC and flow cytometry analysis. For IHC details, 100,000 cells were seeded per well overnight. On day 2, cells were washed once with PBS, fixed with 3% formaldehyde for 10 min at room temperature, then washed twice with PBS and blocked in 10% FBS and 200 μg / mL PBS at room temperature for 1 h. After blocking, cells were incubated with 10 μg / mL primary antibody mIgG-biotin (BioLegend) or IMT001-biotin overnight at 4°C, washed three times with PBST, stained with avidin-HRP (1:1000) for 1 h at room temperature, then washed three times again with PBST. Staining was performed with peroxidase substrate and counterstained with hematoxylin QS (Vector Laboratories). The results show that IMT001 clearly detected Gal3 expression on macrophages (FIG. 15B), compared to the mIgG control (FIG. 15A).

[0288] For flow cytometry experiments, 100,000 RAW cells were blocked with 10% FBS + 200 μg / ml hIgG for 20 min on ice, then incubated with 100 μl 10% FBS HBSS solution containing control mIgG (BD Biosciences) or IMT001 for 20 min on ice. The cells were then washed and incubated with APC-conjugated anti-mFc antibody (Jackson ImmunoResearch) at 1:100 for 20 min on ice. After spinning, live / dead cells were stained with Violet Dead Cell Dye Kit (Life Technologies). The stained cells were subjected to flow analysis. Figure 15C shows that the mean fluorescence intensity (MFI) of RAW cells stained with IMT001 is more than 10 times higher than that of cells stained with isotype control antibody.

[0289] The ability of IMT001 to activate T cells was demonstrated by mixed lymphocyte reaction (MLR) assay. RAW cells average mouse macrophage cells were mixed with D011 mouse T cells at a ratio of 1:1, treated with OVA peptide, and cultured in the presence of mIgG (BD Biosciences), anti-mPD1 antibody 29F (BioXCell) or IMT001 at 10 μg / ml at 37°C overnight. 50 μl of culture medium was taken for mIL-2 measurement. mIL-2 production was measured according to the commercial kit Mouse IL-2 Elisa Ready-SET-Go from eBioscience.

[0290] Figure 15D shows that IMT001 antibody, but not murine PD-1 antibody 29F, enhances IL-2 production compared to mIgG or mPD1 antibody treated cells, indicating reversal of macrophage-induced T cell inactivation.

[0291] Example 13: Identification of antibodies that block Gal3-TIM-3 interaction To identify Gal3-targeting antibodies capable of blocking Gal3-TIM-3 interaction, purified Gal3 and TIM-3 proteins were incubated in the presence (or absence) of various Gal3-targeting or control antibodies, or without antibody, and protein interaction was assessed by ELISA.

[0292] Human Gal3 protein (Acro Biosystems, GA3-H5129) was diluted in phosphate buffered saline (PDS) (Corning) to a concentration of 0.5 μg / ml, and 100 μl of diluted hGal3 was added to each well of a 96-well ELISA plate (Thermo Fisher, 44-2404-21). After incubating the plate overnight at 4°C, the plate was washed three times with 300 μl of PBS containing 0.05% TWEEN (VWR) (PBST) per well. The plate was then blocked with 200 μl of 2% bovine serum albumin (BSA) (Sigma) in PBST per well for 1 hour with gentle rocking at room temperature. The 2% BSA in PBST was then removed, and 50 μl of anti-Gal3 antibody at 20 μg / ml in 2% BSA in PBST was added to the wells and incubated for 10 minutes at room temperature with gentle rocking. The antibodies mab1, mab2, mab3, mab4, mab5, mab6, and mab7 were used in the experiments. The antibodies used are listed in Table 3.

[0293] Afterwards, 50 μl of 1 μg / ml human TIM-3 extracellular domain protein (Acro Biosystems, TM3-H5229) in 2% BSA in PBST was added to the wells. The plate was incubated at room temperature for 1 hour with gentle rocking. The plate was then washed three times with 300 μl of PBST per well, and 100 μl of 0.3 μg / ml anti-human TIM-3 biotinylated antibody (R&D Systems, BAF2365) in 2% BSA in PBST was added to each well. The plate was incubated for 1 hour with gentle rocking, and then washed three times with 300 μl of PBST per well. Then, 100 μl of avidin-HRP (1:1000) (Jackson ImmunoResearch) was added to each well, and the plate was incubated at room temperature for 30 minutes with gentle rocking. Plates were then washed three times with 300 μl PBST per well, and 100 μl TMB substrate (Fisher Scientific, 34029) was added to each well. The reaction was stopped with 50 μl 1 M HCl (VWR) per well. Plates were read at 450 nm absorbance using a plate reader (Molecular Devices). Percentage blockade of Gal3-TIM-3 interaction was calculated as a fraction of the signal obtained in the absence of antibody minus background signal.

[0294] As shown in Figure 16, anti-Gal3 antibodies showed differential ability to block the interaction of Gal3 with TIM-3. Antibodies mab1, mab2, mab3, mab4, and Each of IMT001 blocked Gal3-TIM-3 binding, resulting in a reduction of Gal3-TIM-3 binding to 14%, 4%, 10%, and 7% of the unblocked control (no antibody), respectively. Antibodies mab3 and mab5 blocked Gal3-TIM-3 binding moderately, reducing the interaction to 34% and 59% of the unblocked control, respectively. Finally, mab6 and mab7 had no effect on Gal3-TIM-3 binding. The results showed that antibodies mab1, mab2, mab4, mab5, and IMT001 all blocked Gal3-TIM-3 interaction to some degree. It was also demonstrated that Gal3 binding alone was not sufficient to block the interaction of Gal3 with TIM-3, and that specific properties were required for this blocking activity.

[0295] [Table 3]

[0296] Example 14: Identification of antibodies that bind to distinct epitopes of Gal3 To determine the epitope on Gal3 associated with the Gal3-TIM-3 antibody blocking site, an ELISA assay was performed by applying anti-Gal3 antibodies with and without Gal3-TIM-3 blocking activity to the Gal3 peptide.

[0297] A library of 20 amino acid peptides, each representing a specific region of hGal3 (SEQ ID NO:1), was generated. At least 2 μg / ml of the produced hGal3 peptides: peptides 1 (SEQ ID NO:3), 5 (SEQ ID NO:7), 6 (SEQ ID NO:8), 8 (SEQ ID NO:10), or 23 (SEQ ID NO:25) in 50 μl of PBS were added to the wells of a 96-well ELISA plate (Thermo Fisher, 44-2404-21). 0.1 μg / ml of full-length human galectin-3 protein (Acro Biosystems, GA3-H5129) in 100 μl of PBS was added to the wells of the ELISA plate. After incubating the plate overnight at 4° C., the plate was washed three times with 300 μl of PBST per well. The plate was then blocked with 200 μl of 2% BSA per well for 1 hour with gentle rocking at room temperature. Then, the 2% BSA in PBST was removed and 100 μl of 0.1 μg / ml antibody in 2% BSA in PBST was added to the wells (FIGS. 17A-B). As a negative control group, the antibody was applied without hGal3 peptide or hGal3 protein.

[0298] The plates were incubated at room temperature for 1 hour with gentle rocking and then washed three times with 300 μl PBST per well. HRP-conjugated secondary antibodies were then added to the wells and incubated at room temperature for 30 minutes with gentle rocking. After washing the plates three times with 300 μl PBST per well, 100 μl TMB substrate (Fisher Scientific, 34029) was added to each well. The reaction was stopped with 50 μl 1M HCl (VWR) per well and the plate was read on a plate reader (Molecular Devices). Plates were read at an absorbance of 450 nm using a 5'-dichloro-1,1'-dichloro-2,1'-triazolidinediaminetetraacetate (DTA) assay.

[0299] Anti-Gal3 antibodies, mab1, mab3, mab4, and IMT001, with known Gal3-TIM-3 blocking activity, bound to hGal3 peptides 5 (SEQ ID NO:7), 6 (SEQ ID NO:8), and 8 (SEQ ID NO:10) to various degrees (FIG. 17A), suggesting that these Gal3-TIM-3 blocking antibodies share some common epitopes on Gal3. Antibody mab5, an antibody with partial Gal3-TIM-3 blocking activity, also bound to this region. Antibody mab2, an antibody with potent Gal3-TIM-3 blocking activity, bound to a distinct Gal3 peptide, peptide 1 (SEQ ID NO:3) (FIG. 17B). In contrast, anti-Gal3 antibody mab7, which has no Gal3-TIM-3 blocking activity, showed binding activity with peptides 10 (SEQ ID NO:12) and 23 (SEQ ID NO:25), whereas mab6 showed no substantial binding to any of the peptides but did show binding to the hGal3 protein, suggesting a non-linear binding epitope for this antibody. Peptides that did not bind to any of the Gal3 antibodies are not shown for purposes of clarity. Overall, these observations confirmed the sequences represented by peptides 1 (SEQ ID NO:3), 5 (SEQ ID NO:7), 6 (SEQ ID NO:8), and 8 (SEQ ID NO:10) as features predictive of Gal3-TIM-3 blocking activity. These peptides correspond to the first 2-21 N-terminal amino acids of Gal3 and residues 52-71 and 72-91 of hGal3 (SEQ ID NO:1).

[0300] Example 15: Binding domains of anti-Gal3 antibodies To assess whether anti-Gal3 antibodies with Gal3-TIM-3 blocking activity bind to the same or overlapping regions of the Gal3 molecule, epitope binding assays were performed to assess the ability of the antibodies to co-bind with Gal3.

[0301] 100 μl of 0.1 μg / ml hGal3 (Acro Biosystems, GA3-H5129) was added to each well of a 96-well ELISA plate (Thermo Fisher, 44-2404-21) except for the control group "no coating". After incubating the plate overnight at 4°C, the plate was washed three times with 300 μl PBST per well. The plate was then blocked with 200 μl 2% BSA per well for 1 hour at room temperature with gentle rocking, and the 2% BSA in PBST was removed. 50 μl of anti-hGal3 antibody: mab1, mab4, or mab5 (4.2 μg / ml) in 2% BSA in PBST was added to the wells and preincubated for 10 minutes at room temperature with gentle rocking. Wells of the second control group, "no ab", were preincubated without the addition of antibody.

[0302] After preincubation with or without anti-Gal3 antibodies, 50 μl of biotinylated anti-Gal3 antibodies: mab1, mab4, and mab5 (0.2 μg / ml) in 2% BSA in PBST were added together to the wells and incubated for 1 h at room temperature with gentle rocking. A third control group, "blank" wells, was incubated without adding any antibody. The plates were then washed three times with 300 μl PBST per well, and then 100 μl of avidin-HRP (1:1000) (Jackson ImmunoReasearch) was added to each well. The plates were again incubated for 3 min at room temperature with gentle rocking, and then washed three times with 300 μl PBST per well. 100 μl of TMB substrate (Fisher Scientific, 34029) was then added to each well. The reaction was stopped with 50 μl per well of 1 M HCl (VWR) and the plates were read at an absorbance of 450 nm using a plate reader (Molecular Devices).

[0303] As shown in Figure 18, antibody binding plotted as a percentage of the unblocked control shows that preincubation with mab1 significantly increased antibody binding to the isotype control. We demonstrated that preincubation with hGal3 reduced the binding of mab1, mab4, and mab5 to hGal3 compared to preincubation with hGal3, indicating that these antibodies share some overlapping binding domains. Similarly, mab4 preincubation significantly reduced the latter binding of mab1, mab4, and mab5. Mab5 preincubation reduced the binding of mab5 but only minimally affected the binding of mab1 and mab4, indicating that the competition was asymmetric, which is often a consequence of low affinity antibodies.

[0304] Example 16. Gal3-TIM-3 blocking antibodies exhibit distinct biophysical properties To determine the biophysical properties of Gal3-binding antibodies, biolayer interference assays were performed with purified Gal3 protein and various antibodies. Anti-human Fc probes were loaded with purified antibodies at 10 μg / ml for 180 seconds using a Gator (Probe Life, East Palo Alto, CA). After balancing in assay buffer for 30 seconds, the loaded probes were immersed in serial 1:2 dilutions of human Gal3 starting at 500 nM for association. Association was observed for 300 seconds until equilibrium. The probes were then immersed in assay buffer for 300 seconds for association.

[0305] Real-time plots of Gal3-binding antibody association and dissociation are illustrated in Figures 19A-C. Antibody mab4 has a K D The strongest affinity is 1.05 × 10 6 M -1 seconds -1 k on and 1.32 × 10 -3 seconds -1 k off Antibody mab1 was found to have a K of 13.5 nM (Figure 19B). D The second strongest affinity was 1.7 × 10 6 M -1 seconds -1 k on and 2.29 × 10 -2 seconds -1 k offThe antibody mab5 showed a K D has the weakest affinity, 1.41 × 10 6 M -1 seconds -1 k on and 4.57 seconds -1 k off These binding affinities were qualitatively consistent with the relative affinities predicted from the antibody binding study in Example 15.

[0306] Example 17. Gal3-targeted antibodies with TIM-3 blocking activity activate antigen-mediated T cell responses To evaluate Gal3-targeted antibodies with Gal3-TIM-3 blocking activity to enhance T cell-mediated responses, a CMV antigen recall assay was used. Human peripheral blood mononuclear cells (PBMCs) (Astarte, donor ID 230) were quickly thawed in a 37°C water bath, resuspended in 20ml RPMI medium with 10% FBS, and centrifuged at 1500 RPM for 5 minutes. The medium was discarded and the pellet was resuspended in 20ml medium, counted by H&E exclusion, and diluted to a final concentration of 4 million cells / ml in serum-free medium (Lonza). 50μl of medium + cells (200,000 cells / well) were added to the 60 inner wells of a 96-well round-bottom plate and incubated for 30 minutes at 37°C. Antibodies were added to serum-free medium to a stock concentration of 4x (40μg / ml). 50 μl of antibody at 4× final concentration was added directly to the PBMCS and incubated for 30 min at 37° C. After incubating the PBMCs with antibody for 30 min, 100 μl of CMV (Astarte Biologics, Cat.#1004) at 2× concentration (1 μg / ml) was added directly to the cells and incubated for 4 days at 37° C. On the fourth day, 10 μl of cell supernatant was collected to measure human IFNγ concentration by ELISA with a human IFN-γ ELISA kit (Invitrogen).

[0307] As shown in Figure 20, samples treated with Gal3-targeting antibodies without TIM-3-Gal3 blocking activity, mab6 and mab7, induced similar levels of interferon-g secretion as isotype control-treated samples. In contrast, Gal3-targeting antibodies with TIM-3-Gal3 blocking activity, mab1, mab2, mab4, and humanized antibody IMT001, showed significantly increased levels of interferon-gamma secretion. Of note, mab5, an antibody with partial Gal3-TIM-3 blocking activity, although it has a relatively low affinity for Gal3, did not induce significant interferon-gamma secretion, indicating that an affinity threshold is required for the immune stimulatory properties of Gal3-targeting antibodies. Similarly, mab3, an antibody with partial Gal3-TIM-3 blocking activity, gives equivocal results in this T cell activation assay. Taken together, these data indicate that Gal3-targeting antibodies can enhance antigen-specific T cell activation, and that only those antibodies with the ability to block the TIM-3-Gal3 interaction possess this activity.

[0308] Example 18: Gal3-TIM-3 binding interface To identify the amino acid residues mediating the interaction between Gal3 and TIM-3, crosslinking mass spectroscopy was performed. 5 μl of purified Gal3 (4.62 μM) and TIM-3 (3.74 μM) were crosslinked using the K200 MALDI MS analysis kit (CovalX). 9 μl of the crosslinking mixture was added with 1 μl of K200 stabilizer (2 mg / ml) and incubated for 3 h at room temperature. The incubated samples were analyzed by high-mass MALDI analysis immediately after crystallization. For the analysis, the following parameters were applied: mass spectrometer: linear and positive mode, ion source 1: 20 kV, ion source 2: 17 kV, lens: 12 kV, pulsed ion extraction: 400 ns HM4, gain voltage: 3.14 kV, acceleration voltage: 20 kV. The crosslinked Gal3-TIM-3 products were identified with MH+=26.886 kDa and MH+=34.397 kDa. The cross-linked protein was digested with trypsin, chymotrypsin, ASPN-N, elastase, or thermolysin to generate separate cross-linked peptides (Figure 21A). The sequences of the cross-linked peptides at the binding site were determined (Figures 21A-C). The Gal3-TIM-3 blocking epitope of Gal3 was not included in the crystal structure model of Gal3 due to the inherent structurally undefined features of the region. Note that the amino acid counting depicted in Figure 21A reflects the amino acid numbering in the mature protein after signal peptide processing. See Table 4 showing the amino acid numbering corresponding to SEQ ID NO:2.

[0309] The amino acid residues near the TIM-3 amino acids at positions 73-101 were found to crosslink with the residues near the Gal3 amino acids at 145-184. These amino acids are in exposed regions of each molecule, suggesting that these regions are involved in protein-protein interactions of Gal3 and TIM-3. Importantly, the anti-Gal3 antibodies, mab1, mab2, mab3, mab4, and mab5, appear to bind to separate epitopes identified in peptide binding assays corresponding to the first 2-21 N-terminal amino acids of Gal3 and residues 52-71 and 72-91 of hGal3 (SEQ ID NO:1), as described in Example 14, suggesting that secondary or tertiary structure may be associated with the N-terminal region of Gal3, and that this region mediates the Gal-TIM-3 interface and binds to the Gal3-TIM-3 blocking antibodies.

[0310] Table 4 shows the respective amino acid numbering from FIG. 21A and SEQ ID NO:2.

[0311] [Table 4]

[0312] Example 19: Reduction of renal fibrosis in mice using anti-Gal3 antibody To evaluate the effect of Gal3 inhibition on renal fibrosis, IMT001 was administered to a mouse renal fibrotic disease model. Because IMT001 also exhibits Gal3-TIM-3 blocking activity, the study similarly demonstrated the effect of Gal3-TIM-3 blockade on renal fibrosis.

[0313] A unilateral urethral obstruction (UUO) mouse model was generated using 8-week-old male C57Bl·6 mice. Animals were randomly assigned to three groups (n=5); sham, mouse IgG2b control, and IMT001. All animal studies were performed in accordance with protocols approved by the Molecular Medicine Research Institute Institutional Animal Care and Use Committee. On day 0, surgery was performed and the left ureter was ligated in each animal. After surgery, animals were intraperitoneally administered either mIgG2b (BioXCell) or IMT001 at 10 mg / kg on days 1, 5, and 10. Animals in the sham group were left untreated. On days 4, 8, and 15, animals were humanely sacrificed and left kidney tissue was surgically removed and snap frozen for Western blot analysis.

[0314] Thirty mg of snap-frozen kidney tissue from the day 14 treatment UUO group was homogenized in 500 μl of RIPA buffer (Thermo Scientific). The homogenate was left on ice for 10 min and then centrifuged at 12000 rpm, 4°C for 10 min in a 1.5 mL Eppendorf tube. The supernatant containing protein was collected and quantified by A280 absorbance using a Nanodrop (ThermoFisher). Protein samples were boiled for 10 min in 4× sample buffer containing β-mercaptoethanol (Bio-Rad). Equal amounts of protein lysate (20 μl / well; 10 μg / μl) were loaded onto a precast SDS-PAGE gel (Bio-Rad) and separated by electrophoresis. The separated proteins were transferred to a polyvinylidene difluoride membrane and then eluted in phosphate-buffered saline containing 0.5% TWEEN (Fisher Scientific MT2103 The membranes were blocked with 5% nonfat dry milk in 0.0 cm (PBST). The membranes were incubated overnight at 4°C with primary antibodies targeting α-smooth muscle actin (SMA) (1:2000 dilution) (Sigma A5228) and fibronectin Fn-EIIIA (1:1000 dilution) (Abcam ab6328). Western blot data were normalized to GAPDH (1:5000 dilution) (Abcam ab181602). After washing three times with PBST, the membranes were incubated with the respective horseradish peroxidase-conjugated secondary antibodies at 1:5000 dilution for 2 h at room temperature. The membranes were washed three times with PBST and protein bands were detected by enhanced chemiluminescence using a standard ECL detection method recommended and developed by the manufacturer (Bio-Rad). GAPDH was used as a loading control standard.

[0315] As illustrated in Figure 22, animals subjected to ureteral ligation and treated with a nonspecific isotype control antibody, mIgG2b, showed induction of fibrosis markers smooth muscle actin (α-SMA) and fibronectin compared to sham-treated animals (rows 1-3 vs. rows 7-9). In contrast, animals subjected to ureteral ligation and treated with IMT001 showed decreased expression of both fibrosis markers (rows 4-6) compared to IgG2b controls (rows 7-9). These observations suggested that blocking Gal3 and disrupting Gal3-TIM-3 interactions can reduce renal fibrosis.

[0316] Example 20: Reduction of mouse liver fibrosis using anti-Gal3 antibody The Gal3-TIM-3 blocking antibody IMT001 was used in a non-obese diabetic and inflammatory (N-IF) mouse genetic model of fibrosis to test the effect of Gal3 inhibition on liver fibrosis.

[0317] N-IF mice were generated by crossing 24αβNOD and NOD.Rag2− / − mouse strains. N-IF mice were backcrossed with B6.Rag2− / − mouse strains for 10 generations. Mice (male and female) were divided into two groups: IMT001 antibody treatment group and mIgG2b antibody control group. Antibodies were administered to animals at 10 mg / kg body weight every 4 days for 40 days, after which the animals were sacrificed. Every effort was made to minimize suffering. Liver and kidney tissues were harvested and snap frozen in liquid nitrogen for Western blot analysis. Tissue processing and Western blot analysis were performed as in Example 19. GAPDH was used as a loading control standard.

[0318] Animals treated with the Gal3-TIM-3 blocking antibody, IMT001, had a significant decrease in expression of the fibrosis markers α-SMA and fibronectin compared to animals treated with the mIgG2b isotype control (Figure 23). These data suggest that Gal3-TIM-3 blockade with IMT001 reduces liver fibrosis in the N-IF model.

[0319] Example 21: Effect of anti-Gal3 antibodies with / without Gal3-TIM-3 blocking properties on fibrosis To evaluate the effect of anti-Gal3 antibodies with and without Gal3-TIM-3 blocking activity on fibrosis, in vitro cell culture-based studies are performed.

[0320] Normal rat kidney fibroblasts (NRK-49F) are grown to 80% confluence in RPMI medium containing 10% fetal bovine serum and penicillin / streptomycin antibiotics. Culture medium is removed and replaced with RPMI containing penicillin / streptomycin but no fetal bovine serum to induce serum starvation for 24 hours, and quiescent cells are treated with control mIgG2b (10 mg / ml), TGF-β1 (1 ng / ml) or galectin 3 antibody IMT001 (10 mg / ml) and dissolved in protein extraction buffer. Lysates were analyzed by Western blotting for induction of fibroblast-myoblast markers of fibrotic disease, including α-SMA and fibronectin, using GAPDH as a loading control standard. Similarly, normal human renal proximal tubule cells (HK-2) (ATCC; Rockville, MD) are grown in keratinocyte medium in a humidified incubator at 37°C with 5% CO2. Cultured cells are treated with either mIgG2b (10 mg / ml), TGF-β1 (1 ng / ml) or IMT001 (10 mg / ml) and assessed by Western blotting.

[0321] Example 22: Treatment of Patients with Fibrotic Disease A patient presenting with jaundice and fluid retention visits a physician. The physician diagnoses the patient with liver fibrosis and prescribes a treatment that includes an anti-Gal3 antibody. The treatment is administered orally to the patient at approximately 10 mg / kg of the patient's body weight daily for one month. In some cases, the anti-Gal3 antibody also has Gal3-TIM-3 blocking properties.

[0322] Example 23: Induction of immune system activation in human subjects using anti-Gal3 antibodies Human subjects or patients are selected according to criteria such as immune system disorders, autoimmune diseases, immunodeficiencies, immunosuppression, cancer or fibrosis, as required. Anti-Gal3 antibodies are administered systemically via parenteral, intravenous, intramuscular, intraarterial, intradermal, subcutaneous, intraperitoneal, intracerebroventricular, or intracranial routes. The subjects are monitored for effects on immune system disorders, autoimmune diseases, immunodeficiencies, immunosuppression, cancer or fibrosis. The subjects are also monitored by measuring blood, plasma or serum levels of cytokines such as IFNγ, TGF-β, TGF-β1, IL-1β, IL-2, TNF-α, or GM-CSF, using methods known in the art, such as gas chromatography, liquid chromatography, mass spectrometry, or enzyme-linked immunosorbent assay (ELISA).

[0323] Alternatively, leukocytes or TIM-3-rich leukocytes are isolated from subjects using techniques known in the art, such as centrifugation and fluorescent-activated cell sorting.Isolated leukocytes or TIM-3-rich leukocytes are contacted with anti-Gal3 antibody, which affects the production of at least one cytokine and induces immune activation.Contacted leukocytes or TIM-3-rich leukocytes are returned to subjects with syngeneic cells to treat immune-related diseases, such as cancer or fibrosis. The therapeutic benefit may be seen within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours, or 1, 2, 3, 4, 5, 6, 7 days, or 1, 2, 3, 4 weeks, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 years.

[0324] In some embodiments, administration of an anti-Gal3 antibody to a subject or contacting leukocytes or TIM-3-enriched leukocytes with an anti-Gal3 antibody reduces or eliminates less than 99%, less than 95%, 99%, less than 95%, less than 90%, less than 80%, less than 78%, less than 70%, less than 66%, less than normal. , the interaction between Gal3 and TIM-3 can be reduced to less than 60%, less than 56%, less than 52%, less than 50%, less than 40%, less than 30%, less than 29%, less than 27%, less than 20%, less than 19%, less than 17%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%.

[0325] Anti-Gal3 antibody can be produced and formulated under sterile conditions and regulated or controlled procedures.In this process, anti-Gal3 antibody is used in the manufacture of drugs or compositions.The formulated anti-Gal3 antibody is used in the treatment of immune-related diseases, such as cancer or fibrosis.

[0326] Methods for maintaining and guaranteeing sterility are based on the Good Manufacturing Practice (GMP) for drugs and quasi-drugs, the Basic Principles for the Handling and Use of Cell and Tissue-Based Pharmaceuticals (GTP), Good Laboratory Practice (GLP) and Good Distribution Practice (GDP) may be adhered to. Methods for maintaining and ensuring sterility include, but are not limited to, High Efficiency Particulate Air (HEPA) filtration, moist or dry heat, radiation, e.g., x-rays, gamma radiation, or UV light, sterilants or fumigants such as ethylene oxide, nitrogen dioxide, ozone, glutaraldehyde, formaldehyde, peracetic acid, chlorine dioxide, or hydrogen peroxide, aseptic filling and packaging of sterile containers, plastic film or wrap packaging, or vacuum packing.

[0327] Example 24: Discovery of antibodies with GAL3-TIM3 blocking activity To extend the findings made with the original antibody panel, an antibody discovery campaign was conducted to identify additional GAL3-binding antibodies with the ability to block GAL3 and TIM3 assembly. Balb / C, FVB, and CD-1F mice were inoculated three times at 7-day intervals with 50 μg of GAL3 protein fused to a 6-histidine tag separated by a linker, GAL3-ECD-His (Acro GA3-H5129; lot #819-43PS1-5E) in combination with a TLR agonist adjuvant mix (50 μg MPL, 20 μg CpG, 10 μg poly(I:C) and 10 μg R848), followed by 50 μg of GAL3-His alone subcutaneously at the groin, back of the neck, and base of the tail as well as at heel joints and intraperitoneal sites. Animals were sacrificed according to IACUC protocols and spleens, femurs, and lymph nodes (axillary, paraaxillary, mediastinal, superficial inguinal, iliac, sacral, and popliteal) were collected. Single cell suspensions of immunized lymph nodes (LNs), spleens, and bone marrow cells were obtained using two sterile snap-frozen glass slides in tissue culture Petri dishes containing 15 mL of DMEM. Bone marrow was extracted from femurs by end-cap flushing with a 5 mL syringe fitted with an 18-gauge needle. Cells from 3 animals were pelleted by centrifugation at 1200 RPM for 5 minutes, resuspended in 10 mL of DMEM (GIBCO 10564-011), and nucleated cells were counted by hemocytometer. Cells were pelleted at 1200 RPM, resuspended in SC buffer (PBS, 2% FBS and 1 mM EDTA) and plasma cells were isolated using the EasySep™ Mouse CD138 Positive Selection Kit (StemCell Technologies) following the manufacturer's recommended protocol. Highly enriched CD138 positive cells were pelleted by centrifugation at 1200 RPM for 5 minutes, resuspended in 50 mL electrofusion buffer (Eppendorf 940-00-220-6) and counted. Separately, SP2 / 0-mIL6 myeloma cells (ATCC CRL2016) were pelleted by centrifugation at 1200 RPM for 5 minutes, resuspended in 50 mL electrofusion buffer and counted.Myeloma cells and CD138 positive cells were mixed in a 1:1 ratio, the volume was increased to 50 mL with electrofusion buffer, the cells were pelleted by centrifugation at 1200 RPM for 5 minutes, and the supernatant was discarded. After repeating the washing step and pelleting in electrofusion buffer, the cells were diluted to 10 × 10. 6 Cells were resuspended in electrofusion buffer to a concentration of 1000 cells / ml, ∼9 mL of cell suspension was added to a BTX electrofusion chamber, and cells were fused using the 800V electrofusion protocol. Fused cells were rested for 5 min, transferred to tissue culture dishes containing 40 mL medium MM (DMEM, 15% FBS, 1% glutamax, and 1% penicillin / streptomycin), incubated for 1 h at 37°C under 8% CO2, resuspended with a pipette, pelleted by centrifugation at 1200 RPM for 5 min, resuspended in ClonaCell HY Liquid HAT selection medium (StemCell Technologies), and placed into 96-well tissue culture flat-bottom plates. After 10 days, supernatants were sampled and assessed for binding to isolated GAL3 by ELISA. 50 μl of 0.1 μg / mL GAL3-ECD-His (Acro GAL3-H5129; lot #819-43PS1-5E) resuspended in diluent (PBS containing 0.5% BSA) was added to each well for 45 min, the supernatant was discarded, and the plate was washed with phosphate-buffered saline (PBS) containing 0.05% Tween 20. A 1:5 dilution of hybridoma supernatant in 50 μl diluent was added to each well for 1 h, followed by five successive washes with 300 μl PBS / 0.05% Tween 20, followed by addition of 1:3000 dilution of goat anti-mammalian antibody conjugated to horseradish peroxidase in 50 μl diluent. Mouse Fc-specific antibody (Novex A16090) was added to each well for 1 h, then washed five consecutive times with 300 μl of PBS / 0.05% Tween 20. After washing, 50 μl of ABTS (Novex #00-202-4) was added to each well for 20-30 min before reading the absorbance at 405 nm using a spectrophotometer (Molecular Devices).

[0328] GAL3-binding antibodies were evaluated for their binding affinity by SPR. Kinetic experiments were performed at 25°C using BiacoreT200 in high performance mode. Ligand proteins, purified antibodies, were captured on a CM5 chip coupled with anti-human Fc or anti-mouse Fc antibodies, i.e., the three antibodies were captured simultaneously on flow cells #2, 3, and 4, respectively, while flow cell #1 was used as a reference. Analyte galectin 3 in HBS-EP buffer was injected across the four flow cells at concentrations of 100, 50, 25, 12.5, 6.25, 3.125, and 0 nM at a flow rate of 30 μL / min. The complexes were allowed to associate and dissociate for 240 and 300 s, respectively. The surface was regenerated by injecting 10 mM glycine pH 1.7 for 30 s (flow rate 30 μL / min). The data were fitted to a simple 1:1 interaction model using the global data analysis option available within the BiacoreT200 evaluation software V2.0. The affinity of Gal3 monoclonal antibodies was established to be greater than 30 nM for all antibodies tested (Table 24.1). Antibodies with affinities less than 2E-7 were selected for further characterization.

[0329] [Table 5]

[0330] Wells were scored positive for their ability to block the association of GAL3 with TIM3. To identify GAL3-targeting antibodies capable of blocking the interaction of GAL3 with TIM3, purified GAL3 and TIM3 proteins were incubated in the presence of the GAL3-immunized hybridoma supernatants described above or without antibodies, and protein interaction was assessed by ELISA. Human galectin 3 protein (Acro Biosystems, GA3-H5129) was diluted to a concentration of 3 μg / ml in PBS (Corning, 21-030-CM) and plated in a 96-well ELISA plate (Thermo Fisher, 44-2404-21) was added to each well. After incubating the plate overnight at 4°C, the plate was washed three times with PBST (PBS containing 0.05% TWEEN20 [VWR, 0777]). The plate was then blocked with 2% BSA in PBST (EMD Millipore, 126609) for 1 hour with gentle rocking at room temperature. The 2% BSA in PBST was then discarded and the antibody or inhibitor (starting at 20 μg / ml, 60 μg / ml, or 180 μM and diluted 3-fold) in 2% BSA in PBST was added to the wells. Then, 2 μg / ml of human TIM3 (Aero Biosystems, TM3-H5229) in 2% BSA in PBST was added to the antibody or inhibitor in the wells at a 1:1 ratio. The plate was incubated at room temperature for 1 hour with gentle rocking. The plate was then washed three times with PBST, and 0.3 μg / ml of human TIM3 biotinylated antibody (R&D Systems, BAF2365) in 2% BSA in PBST was added to the wells. The plate was incubated for 1 hour with gentle rocking, then washed three times with PBST. Avidin-HRP (1:2000) was then added to the wells. The plate was incubated for 1 hour at room temperature with gentle rocking, then washed three times with PBST. TMB substrate (Thermo Scientific, 34029) was then added to each well. The reaction was stopped with 1M HCl (JT Baker, 5620-02) and read at 450 nm absorbance using a plate reader (Molecular Devices).

[0331] As illustrated in FIG. 24, Gal3-binding antibodies exhibited varying abilities to block the association of GAL3 with TIM3. Some antibodies were able to block assembly of GAL3 with TIM3 to less than 5% of the levels observed in the absence of GAL3-targeting antibodies, including 846.2H3. Other GAL3-binding antibodies blocked assembly of GAL3 with TIM3 to 5-20% of the levels observed in the absence of GAL3-targeting antibodies, including mIMT001, 846.1F5, 2D10.2B2, 6H6.2D6, 20H5.A3, and 846T.1H2. Other GAL3-binding antibodies blocked assembly of GAL3 with TIM3 to 20-50% of the levels observed in the absence of GAL3-targeting antibodies, including 19B5.2E6, 13H12.2F8, and 23H9.2E4. Other GAL3-binding antibodies blocked GAL3 and TIM3 assembly to 50-75% of the levels observed in the absence of GAL3-targeting antibodies, including 15G7.2A7, 4G2.2G6, 4A11.2B5, 14H10.2C9, 20D11.2C6, 19D9.2E5, 13A12.2E5, and 3B11.2G2. Other GAL3-binding antibodies showed minimal blocking activity against GAL3 and TIM3 assembly, reducing binding to 25% or less of TIM3 and GAL3 in the absence of GAL3-targeting antibodies, including 12G5.D7, 7D8.2D8, 9H2.2H1, 13G4.2F8, and 24D12.2H9.

[0332] Example 25. Gal3-targeting antibodies with and without GAL3-TIM3 blocking activity bind distinct epitopes on Gal3 To identify epitopes bound by Gal3 antibodies with and without GAL3-TIM3 blocking activity, a library of 20 amino acid peptides representing portions of Gal3, summarized in Table 24.1, were obtained and assessed by ELISA for their ability to bind to Gal3 antibodies.

[0333] At least 2 μg / ml of hGal3 peptide in 50 μl of PBS or 0.1 μg / ml of full-length human Gal3 protein (GenScript) and human galectin 3 protein (Acro Biosystems, GA3-H5129) were diluted in PBS (Corning, 21-030-CM) to a concentration of at least 2 μg / ml or 0.1 μg / ml, respectively, and added to wells of a 96-well ELISA plate (Thermo Fisher, 44-2404-21). After incubating the plate overnight at 4°C, the plate was washed three times with PBST (PBS containing 0.05% TWEEN20 [VWR, 0777]). The plate was then blocked with 2% BSA in PBST (EMD Millipore, 126609) for 1 h at room temperature with gentle rocking. Afterwards, the plate was washed three times with PBST (PBS containing 0.05% TWEEN20 [VWR, 0777]) for 1 h at room temperature with gentle rocking. The 2% BSA in ST was discarded, and human galectin-3 hybridoma supernatant or antibody was diluted in 2% BSA in PBST to a concentration of at least 0.1 μg / ml and added to the wells. The plate was incubated at room temperature for 1 h with gentle rocking, then washed three times with PBST. Goat anti-mouse IgG-HRP (Jackson ImmunoResearch, 115-036-1461) or goat anti-rat IgG-HRP (abcam, ab205720) diluted (1:4000) in 2% BSA in PBST was then added to the wells. The plate was incubated at room temperature for 30 min to 1 h with gentle rocking, then washed three times with PBST. TMB substrate (Thermo Scientific, 34029) was then added to each well. The reaction was stopped with 1M HCl (JT Baker, 5620-02) and read at an absorbance of 450 nm using a plate reader (Molecular Devices).

[0334] Binding of Gal3-binding antibodies to peptide arrays was observed at multiple locations, with the majority of binding observed with peptides 1-8 summarized in Table 25.1. Importantly, all Gal3-binding antibodies with potent TIM3-Gal3 blocking activity demonstrated the ability to bind peptides 4, 5, 6, or 7, which correspond to peptide sequences in the N-terminal domain of Gal3. Importantly, all six individual Gal3-binding antibodies with Gal3-TIM3 blocking activity (6H6.2D6, 20H5.A3, 20D11.2C6, 19B5.2E6, 15G7.2A7, 23H9.2E4) bound to peptide 1 of Gal3, which corresponds to amino acids 1-20 of Gal3, ADNFSLHDALSGSGNPNPQG (SEQ ID NO: 3). Conversely, no binding to peptide 1 was observed with Gal3-targeting antibodies with poor Gal3-TIM3 blocking activity. Taken together, these data indicate that binding to Gal3 peptide 1 predicts the ability to block the interaction of Gal3 with TIM3. Similarly, six individual Gal3-binding antibodies with Gal3-TIM3 blocking activity (4G2.2G6, 3B11.2G2, and 13A12.2E5) bound to peptide 4 of Gal3, which corresponds to amino acids 31-50 of Gal3, GAGGYPGASYPGAYPGQAPP (SEQ ID NO: 6). Conversely, no binding to peptide 4 was observed with Gal3-targeting antibodies with poor Gal3-TIM3 blocking activity. Taken together, these data indicate that binding to Gal3 peptide 4 predicts the ability to block the interaction of Gal3 with TIM3. Furthermore, all 13 Gal3-binding antibodies with Gal3-TIM3 blocking activity (mIMT001, 846T.1H2, 13H12.2F8, 19D9.2E5, 14H10.2C9, 2D10.2B2, 4A11.2B5, 846.2H3, 846.1F5, 3B11.2D2, and 13A12.2E5) bound peptide 6 of Gal3, which corresponds to amino acids 51-70 of Gal3, GAYPGQAPPGAYPGAPGAYP (SEQ ID NO: 8). Conversely, no binding to peptide 6 was observed with Gal3-targeting antibodies with insufficient Gal3-TIM3 blocking activity.Taken together, these data indicate that binding to Gal3 peptide 6 predicts the ability to block the interaction of Gal3 with TIM3. Furthermore, all 11 Gal3-binding antibodies with Gal3-TIM3 blocking activity (6H6.2D6, 20H5.A3, 20D11.2C6, 13H12.2F8, 19B5.2E6, 23H9.2E4, 15G7.2A7, 19D9.2E5, 14H10.2C9, 7D8.2D8, 15F10.2D6 and 846.14A2) bound to Gal3 peptide 7, which corresponds to amino acids 61-80 of Gal3, AYPGAPGAYPGAPAPGVYPG (SEQ ID NO: 9). Conversely, no binding to peptide 7 was observed with Gal3-targeting antibodies with insufficient Gal3-TIM3 blocking activity. Collectively, these data indicate that binding to Gal3 peptide 7 predicts the ability to block the interaction of Gal3 with TIM3. Overall, these data indicate that binding of anti-Gal3 antibodies to Gal3 peptides 1, 4, 5, 6, and 7 predicts the ability to block the interaction of Gal3 with TIM3.

[0335] As shown in FIG. 25, peptides 4, 5, 6, and 7 contain the proline-glycine Gal3-targeting antibodies share a repeating amino acid sequence consisting of tyrosine-proline (YPG) and tyrosine-proline (PG), indicating a common feature that may explain the ability of Gal3-targeting antibodies to bind multiple Gal3 peptides. Furthermore, the amino acid sequence glycine-x-tyrosine-proline-glycine (GxYPG), where x can be the amino acid alanine (A), glycine (G), or valine (V), is shared in peptides 4, 6, and 7, each of which has such a sequence separated by three amino acids. Thus, the presence of two GxYPG sequences in close juxtaposition may predict the ability to bind Gal3-targeting antibodies with the ability to block Gal3 and TIM3. In addition, the Grantham distances of alanine, glycine, and valine are Ala-Val: 64, Ala-Gly: 60, and Val-Gly: 109, which predicts that amino acids with similarly low Grantham distances may be similarly substituted in the variable region, including proline and threonine.

[0336] [Table 6]

[0337] Example 26. Gal3-TIM3 antibodies with blocking activity compete for binding to Gal3 To determine whether anti-Gal3 binding antibodies with Gal3-TIM3 blocking activity bind to the same or overlapping regions of the Gal3 molecule, antibody binning assays were performed. We performed amine-reactive probe assays to assess the ability of the antibodies to simultaneously bind Gal3. The buffer was loaded onto a Gator biosensor (Probe Life, Palo Alto, CA), equilibrated with dH20 for 60 seconds, and diluted with 100 μl of EDC 0.2 M / NHS immersion in 0.05M activation buffer for 30 seconds, followed by immersion in a solution of 20 μg / μl human Gal3-His in 10 mM NaOAc buffer, pH 5, until binding was saturated; The reaction was quenched in 1 M ethanolamine pH 8.5 for 300 seconds. After Gal3-His loading, the chip was immersed in 20 μg / mL saturating antibody, followed by immersion in 5 μg / mL competing antibody. Antibodies with competitive binding profiles were assigned to bins and correlated with blocking activity, as shown in FIG. 26. After initial bin assignment, subsequent competition experiments were performed using representative species from the bin set described to identify additional members of bins 1 and 3.

[0338] Twelve separate bins of competitive antibody binding patterns with Gal3 were established. Importantly, a strong association between blocking bins and Gal3-TIM3 blocking activity was observed. All antibodies from bins 1, 2, 3, 4, 5, and 6 significantly inhibited Gal3 binding to TIM3, as summarized in Table 25.1. In contrast, antibodies in bins 7 and 8 were somewhat weaker blockers of Gal3 blocking to TIM3, despite their strong affinity with Gal3. Antibodies in bins 10, 11, and 12 uniformly did not have the ability to significantly inhibit the association of Gal3 with TIM3. Thus, competitive binding bins 1, 2, 3, 4, 5, and 6 can identify the ability of Gal3-binding antibodies to block the assembly of Gal3 and TIM3.

[0339] Example 27: Humanized GAL3-TIM3 blocking antibodies block GAL3-TIM3 binding. Humanized variants of the GAL3-TIM3 blocking antibodies similarly demonstrated the ability to block the interaction of purified GAL3 with TIM3 as assessed by ELISA shown in Figure 27. IMT001-4, IMT006-1, IMT006-5, and IMT006-8 exhibited IC50s of 5.6 nM, 26.5 nM, 4.1 nM, and 2.8 nM, respectively.

[0340] Example 28: GAL3-TIM3 blocking antibodies show combined anti-tumor activity with anti-PD1 or anti-PD-L1 antibodies. To evaluate the potential of GAL3-TIM3 blocking antibodies to affect tumor biology, they were tested in mice bearing MBT-2 bladder tumor xenografts in combination with other antibodies targeting the immunoregulatory checkpoint molecules PD-1 and PD-L1. Briefly, 7-week-old female C3H / HeJ mice (Jackson Laboratory) were anesthetized with inhalation anesthetic (3-5% isoflurane in medical air) and then infused with 1 × 10 mAbs in 0.1 mL of PBS. 6 MBT-2 cells (Sekisui XenoTech, LLC) were injected into the right flank using a syringe with a 25-ga needle. Seven days after tumor implantation, mice were randomly assigned to six groups (n = 9–10). Isotype control mIgG2b (BioXCell), anti-Gal3 (mIMT001), anti-PD1 (RMP1-14, BioXCell) + mIgG2b, anti-PD1 (RMP1-14) + mIMT001, anti-PDL1 (10F.9G2, BioXCell) + mIgG2b, and anti-PDL1 (10F.9G2) + mIMT001 were administered intraperitoneally to mice. Isotype control and anti-Gal3 antibodies were administered at 20 mg / Kg on days 7, 9, 12, 14, and 16; anti-PD1 (RMP1-14, 10 mg / Kg) or anti-PDL1 (10F.9G2, 5 mg / Kg) were administered on days 8, 12, and 15. Tumor burden and body weight were monitored twice weekly. Animals were humanely sacrificed when tumor burden or animal health reached IACUC-defined endpoints. Results are expressed as mean ± standard error of the mean and statistical analysis was performed by two-way ANOVA.

[0341] Animals treated with mIMT001 or huIgG4 did not show a significant attenuation of tumor burden (data not shown). In contrast, as depicted in Figure 28, 3 / 10 animals treated with anti-PD-L1 antibody showed a strong anti-tumor response, as reflected by the reduction in tumor burden after treatment (Figure 28A-B). Importantly, 5 / 10 animals treated with a combination of mIMT001 and anti-PD-L1 antibody showed a strong anti-tumor response, with anti-PD-L The combination of GAL3 and TIM3 blocking antibodies with an anti-PD-L1 antibody significantly increased anti-tumor activity over the anti-PD-L1 antibody alone, demonstrating a 66% increase in response rate compared to animals treated with either antibody alone.

[0342] Another study was performed to evaluate the activity of mIMT001 in combination with anti-PD-1 antibody in mice implanted with subcutaneous MBT-2 tumors. As in the PD-1 study, treatment with isotype control or mIMT001 alone did not reduce tumor burden (data not shown). In contrast, treatment with anti-PD-1 antibody resulted in an anti-tumor response in 3 / 10 animals, as indicated by a significant reduction in tumor burden (Figure 28C-D). Importantly, 6 / 10 animals treated with the combination of mIMT001 and anti-PD-1 antibody showed potent Animals treated with anti-PD-1 antibody alone demonstrated robust anti-tumor responses, with a 100% increase in response rate compared to animals treated with anti-PD-1 antibody alone. These data indicate that the combination of GAL3- and TIM3-blocking antibodies with anti-PD-1 antibody significantly enhanced anti-tumor activity over anti-PD-1 antibody alone. Taken together with the PD-L1 combination studies, these data suggest that GAL3-targeting antibodies capable of blocking GAL3-TIM3 interactions have the potential to more broadly enhance anti-tumor activity induced by disruption of the PD-1-PD-L1 checkpoint.

[0343] Example 29: GAL3-TIM3 blocking antibodies show single-agent antitumor activity in HCC. Further studies evaluating the activity of GAL3-TIM3 blocking antibodies were evaluated in the setting of a spontaneous hepatocellular carcinoma (HCC) model induced in STAM-CDAA mice. Briefly, 2-day-old male C57B1 / 6 mice were injected with a single subcutaneous injection of 200 μg streptozotocin to cause island destruction and then fed a CDAA-high fat diet (Research Diet#A06071302) starting at 4 weeks of age and continuing for the entire duration of each study. At 8 weeks of age, mice were divided into 2 groups (7 mice each). Mice were treated with human anti-mIgG4 isotype control (hIgG4, 10 mg / kg) or human anti-Gal3 antibody (IMT001-4, 10 mg / kg) by intraperitoneal injection twice weekly for 4 weeks. All animal care and procedures were approved by Immutics IACUC.

[0344] As shown in Figures 29A-B, no tumors were observed in animals fed a normal diet, whereas STAM-CDAA animals treated with isotype control antibody showed signs of multifocal tumor formation as evident by macroscopic examination, with only 1 / 7 having no visible tumors, 4 / 7 animals showing severe (>5 tumors per liver) formation, and 2 / 7 animals showing moderate formation (3-5 tumors per liver). In contrast, tumor formation was significantly reduced in animals treated with IMT001-4, with only 1 / 7 animals showing severe tumor formation with a 75% reduction in severe tumor formation, and 1 / 7 animals showing moderate tumor formation with a 50% reduction in moderate tumor formation. Similarly, IMT001-4 treated animals showed visible signs of tumor formation in 5 / 7 animals, with a 400% increase in animals without visible tumors.

[0345] Microscopic examination of hematoxylin and eosin stained tumor specimens...

Claims

1. 1. A method for inducing immune activation, the method comprising: contacting a plurality of cells, including Gal3-expressing cells and TIM-3-expressing cells, with an antibody under conditions that disrupt the interaction between Gal3 and TIM-3; The antibody specifically binds to Gal3, and upon binding to the antibody, the Gal3-expressing cells express cytokines that induce immune activation, and the antibody is not IMT001.

2. The method of claim 1 , wherein the cytokine is an interferon.

3. 3. The method of claim 2, wherein the interferon is IFNγ.

4. 4. The method of claim 3, wherein the IFNγ production is 150%, 160%, 170%, 180%, 190%, 200% or more IFNγ production by isotype antibody.

5. The method of claim 1 , wherein the cytokine is an interleukin.

6. The method of claim 5, wherein the interleukin is IL-2.

7. The method of any one of claims 1 to 6, wherein the immune activation comprises proliferation of CD3+ T lymphocytes, CD4+ T helper cells, CD8+ cytotoxic T cells, natural killer cells, or a combination thereof.

8. The method of any one of claims 1 to 7, wherein the immune activation comprises an increase in the M1 macrophage population in a plurality of cells.

9. The method of any one of claims 1 to 8, wherein the immune activation comprises a reduction in the M2 macrophage population in a plurality of cells.

10. 1. A method for promoting T cell or natural killer (NK) cell proliferation, the method comprising: contacting a plurality of cells, including T cells, NK cells, and Gal3-expressing cells, with an antibody under conditions that affect proliferation of T cells and / or NK cells within the plurality of cells, wherein the antibody specifically binds Gal3, and the antibody is not IMT001. method.

11. The method of claim 10, wherein the plurality of cells further comprises TIM-3 expressing cells.

12. The method of claim 11, wherein the antibody further disrupts the interaction of Gal3 with TIM-3.

13. 1. A method for inducing immune activation, the method comprising: contacting a plurality of cells, including Gal3-expressing cells and TIM-3-expressing cells, with an antibody under conditions that disrupt the interaction between Gal3 and TIM-3; the antibody specifically binds Gal3, and the Gal3-TIM-3 interaction is reduced to less than 70%, less than 60%, less than 59%, less than 50%, less than 40%, less than 34%, less than 30%, less than 20%, less than 14%, less than 10%, less than 7%, less than 5%, less than 4%, or less than 1%. method.

14. 14. The method of claim 13, wherein the interaction occurs at one or more residues of Gal3 selected from regions 145-168, 160-177, or 165-184, said residue positions corresponding to positions 145-168, 160-177, or 165-184 of SEQ ID NO:

1.

15. 14. The method of claim 13, wherein the interaction occurs at one or more residues of Gal3 selected from regions 149-156, 152-168, 163-169, 163-177, or 163-171, said residue positions corresponding to positions 149-156, 152-168, 163-169, 163-177, or 163-171 of SEQ ID NO:

1.

16. 16. The method of any one of claims 13 to 15, wherein the interaction occurs at one or more residues of TIM-3 selected from regions 91-111 or 82-111, said residue positions corresponding to positions 91-111 or 82-111 of SEQ ID NO:

2.

17. 16. The method of any one of claims 13-15, wherein the interaction occurs at one or more residues of TIM-3 selected from regions 91-111, 107-117, 96-102, 100-106, or 92-119, wherein the residue positions correspond to positions 91-111, 107-117, 96-102, 100-106, or 92-119 of SEQ ID NO:

2.

18. The method of any one of claims 13 to 17, wherein the TIM-3 is human TIM-3.

19. The method of any one of claims 1 to 18, wherein the Gal3 expressing cell is a tumor cell.

20. The method of any one of claims 1 to 19, wherein the plurality of cells is present within a tumor microenvironment (TME).

21. The method of any one of claims 1 to 20, wherein the antibody induces a reduction of tumor cells within the TME.

22. The method of any one of claims 1 to 21, wherein the plurality of cells further comprises tumor infiltrating lymphocytes (TILs).

23. The method of any one of claims 1 to 22, wherein the plurality of cells further comprises CD3+ T lymphocytes, CD4+ T helper cells, CD8+ cytotoxic T cells, or a combination thereof.

24. 23. The method of any one of claims 1, 10, 13, or 22, wherein the contacting further induces TIL proliferation.

25. 24. The method of any one of claims 1, 10, 13, or 23, wherein the contacting further induces proliferation of CD3+ T lymphocytes, CD4+ T helper cells, CD8+ cytotoxic T cells, or a combination thereof.

26. 26. The method of any one of claims 1, 10, 13, or 22-25, wherein the contacting further comprises increasing proliferation of M1 macrophages.

27. 27. The method of any one of claims 1, 10, 13, or 22-26, wherein the contacting further comprises reducing the M2 macrophage population within the TME.

28. The method of any one of claims 1 to 27, wherein the antibody binds to at least one amino acid residue in the Gal3 region corresponding to residues 1 to 20 of SEQ ID NO:

1.

29. The method of any one of claims 1 to 27, wherein the antibody binds to at least one amino acid residue in the Gal3 region corresponding to residues 41 to 91 of SEQ ID NO:

1.

30. 30. The method of any one of claims 1 to 27 or 29, wherein the antibody binds to at least one amino acid residue in the Gal3 region corresponding to residues 41-71 of SEQ ID NO:

1.

31. 30. The method of any one of claims 1 to 27 or 29, wherein the antibody binds to at least one amino acid residue within the Gal3 region corresponding to residues 71-91 of SEQ ID NO:

1.

32. 32. The method of any one of claims 1-31, wherein the antibody binds to at least one amino acid residue within peptide_1, peptide_4, peptide_5, peptide_6, peptide_7, or peptide_8.

33. The antibody has a K of less than 1 nM, 1.2 nM, 2 nM, 5 nM, 10 nM, 13.5 nM, 15 nM, 20 nM, 25 nM, or 30 nM. Ｄ The method according to any one of claims 1 to 32, comprising:

34. The method of any one of claims 1 to 33, wherein the antibody comprises a humanized antibody.

35. The method of any one of claims 1 to 34, wherein the antibody comprises a full-length antibody or a binding fragment thereof.

36. The method of any one of claims 1 to 35, wherein the antibody comprises a bispecific antibody or a binding fragment thereof.

37. 37. The method of any one of claims 1 to 36, wherein the antibody comprises a monovalent Fab', a bivalent Fab2, a single chain variable fragment (scFv), a diabody, a minibody, a nanobody, a single domain antibody (sdAb), or a camelid antibody or binding fragment thereof.

38. The method of any one of claims 1 to 37, wherein the antibody comprises an IgG framework.

39. The method of any one of claims 1 to 38, wherein the antibody comprises an IgG1, IgG2, or IgG4 framework.

40. The method of any one of claims 1 to 39, wherein the antibody further comprises an Fc mutation.

41. The method of any one of claims 1 to 33 or 35 to 40, wherein the antibody comprises a chimeric antibody.

42. 14. The method of any one of claims 1, 10, or 13, wherein the method further comprises administering the antibody to a subject prior to the contacting step.

43. 43. The method of claim 42, wherein the subject has been diagnosed with cancer.

44. 44. The method of claim 43, wherein the cancer is a solid tumor.

45. 45. The method of claim 44, wherein the cancer is breast cancer, colorectal cancer, renal cancer, liver cancer, or lung cancer.

46. 44. The method of claim 43, wherein the cancer is a hematological tumor.

47. The method of any one of claims 43 to 46, wherein the cancer is a metastatic cancer.

48. The method according to any one of claims 43 to 46, wherein the cancer is a recurrent or refractory cancer.

49. The method of any one of claims 42-48, wherein the antibody is formulated for systemic administration.

50. The method of any one of claims 42-49, wherein the antibody is formulated for parenteral administration.

51. The method of any one of claims 42 to 50, wherein the antibody is administered in combination with an additional therapeutic agent.

52. 52. The method of claim 51, wherein the antibody and the additional therapeutic agent are administered simultaneously.

53. 52. The method of claim 51, wherein the antibody and the additional therapeutic agent are administered sequentially.

54. 54. The method of claim 53, wherein the antibody is administered prior to administration of the additional therapeutic agent.

55. 54. The method of claim 53, wherein the antibody is administered after the additional therapeutic agent is administered.

56. 56. The method of any one of claims 51-55, wherein the additional therapeutic agent comprises an immune checkpoint modulator.

57. 56. The method of any one of claims 51-55, wherein the additional therapeutic agent comprises a chemotherapeutic agent, a targeted therapeutic agent, a hormonal therapeutic agent, or a stem cell-based therapeutic agent.

58. The method of any one of claims 1 to 57, wherein the subject is a human.

59. 59. The method of claim 58, wherein the antibody is administered either pre-surgery or post-surgery.

60. 59. The method of claim 58, wherein the antibody is administered concomitantly with, prior to, or following radiation therapy.

61. The antibody is the K Ｄ Higher K Ｄ The method of any one of claims 1 to 60, comprising:

62. 1. A method for reducing fibrosis or a propensity for fibrosis in a tissue, the method comprising: contacting the tissue with an antibody that specifically binds to the Gal3 antibody under conditions such that the expression level of a fibrosis biomarker is reduced in the tissue. method.

63. The method of claim 62, wherein the tissue further comprises TIM-3 expressing cells.

64. The method of claim 63, wherein the antibody further disrupts the interaction of Gal3 with TIM-3.

65. The method of claim 63, wherein the antibody does not interfere with the interaction of Gal3 with TIM-3.

66. The method of any one of claims 62 to 65, wherein the at least one fibrosis biomarker comprises alpha-smooth muscle actin (alpha-SMA).

67. The method of any one of claims 62 to 65, wherein the at least one fibrosis biomarker comprises fibronectin.

68. The method of any one of claims 62 to 65, wherein the at least one fibrosis biomarker comprises alpha-smooth muscle actin (alpha-SMA) and fibronectin.

69. The method of any one of claims 62 to 68, wherein the tissue is kidney tissue or liver tissue.

70. 69. The method of any one of claims 62 to 68, wherein the tissue is selected from the group consisting of liver tissue, kidney tissue, skin tissue, lung tissue, heart tissue, brain tissue, intestinal tissue, bone marrow tissue, and soft tissue.

71. The method of any one of claims 62 to 70, wherein expression of the at least one fibrosis biomarker in the tissue treated with the antibody is less than expression of the at least one fibrosis biomarker in a control tissue treated with an mIgG2b antibody.

72. The method of any one of claims 62 to 71, wherein the antibody results in a reduction in accumulation of extracellular matrix proteins in the tissue.

73. 73. The method of claim 72, wherein the extracellular matrix protein comprises collagen.

74. 74. The method of claim 73, wherein the tissue comprises collagen-producing cells.

75. 75. The method of claim 74, wherein the collagen-producing cells are fibroblasts.

76. 76. The method of claim 75, wherein the fibroblasts are activated with a fibrogenic cytokine.

77. 77. The method of claim 76, wherein the fibrogenic cytokine is TGF-β1.

78. The method of any one of claims 62 to 77, wherein the tissue has high TGF-β1 expression.

79. The method of any one of claims 62 to 78, wherein the antibody comprises a humanized antibody.

80. The antibody according to any one of claims 62 to 79, wherein the antibody comprises a full-length antibody or a binding fragment thereof. The method according to any one of claims 1 to 5.

81. The method of any one of claims 62 to 79, wherein the antibody comprises a bispecific antibody or a binding fragment thereof.

82. The method of any one of claims 62 to 79, wherein the antibody comprises a chimeric antibody.

83. The method of any one of claims 62-82, wherein the antibody binds to at least one amino acid residue in the Gal3 region corresponding to residues 1-20 of SEQ ID NO:

1.

84. The method of any one of claims 62 to 82, wherein the antibody binds to at least one amino acid residue within the Gal3 region corresponding to residues 41-91 of SEQ ID NO:

1.

85. 85. The method of any one of claims 62-82 or 84, wherein the antibody binds to at least one amino acid residue within the Gal3 region corresponding to residues 41-71 of SEQ ID NO:

1.

86. 85. The method of any one of claims 62-82 or 84, wherein the antibody binds to at least one amino acid residue within the Gal3 region corresponding to residues 71-91 of SEQ ID NO:

1.

87. 87. The method of any one of claims 62-86, wherein the antibody binds to at least one amino acid residue within peptide_1, peptide_4, peptide_5, peptide_6, peptide_7, or peptide_8.

88. The antibody has a K of less than 1 nM, 1.2 nM, 2 nM, 5 nM, 10 nM, 13.5 nM, 15 nM, 20 nM, 25 nM, or 30 nM. Ｄ The method of any one of claims 62 to 87, comprising:

89. 89. The method of any one of claims 62-88, wherein the antibody comprises a monovalent Fab', a bivalent Fab2, a single chain variable fragment (scFv), a diabody, a minibody, a nanobody, a single domain antibody (sdAb), or a camelid antibody or binding fragment thereof.

90. The method of any one of claims 62 to 89, wherein the antibody comprises an IgG framework.

91. 91. The method of any one of claims 62-90, wherein the antibody comprises an IgG1, IgG2, or IgG4 framework.

92. The method of any one of claims 62 to 91, wherein the antibody further comprises an Fc mutation.

93. 93. The method of any one of claims 62-92, wherein the method further comprises administering the antibody to a subject prior to the contacting step.

94. 94. The method of claim 93, wherein the subject has been diagnosed with a fibrotic disease.

95. 95. The method of claim 94, wherein the fibrotic disease is renal fibrosis.

96. 95. The method of claim 94, wherein the fibrotic disease is liver fibrosis.

97. The method of any one of claims 93 to 96, wherein the antibody is formulated for systemic administration.

98. The method of any one of claims 93 to 96, wherein the antibody is formulated for parenteral administration.

99. The method of any one of claims 93 to 98, wherein the subject is a mammal.

100. 100. The method of any one of claims 64 and 66-99, wherein the Gal3-TIM-3 interaction is reduced to less than 70%, less than 60%, less than 59%, less than 50%, less than 40%, less than 34%, less than 30%, less than 20%, less than 14%, less than 10%, less than 7%, less than 5%, less than 4%, or less than 1%.

101. 101. The method of claim 100, wherein the interaction occurs at one or more residues of Gal3 selected from regions 145-168, 160-177, or 165-184, said residue positions corresponding to positions 145-168, 160-177, or 165-184 of SEQ ID NO:

1.

102. 101. The method of claim 100, wherein the interaction occurs at one or more residues of Gal3 selected from regions 149-156, 152-168, 163-169, or 163-171, said residue positions corresponding to positions 149-156, 152-168, 163-169, or 163-171 of SEQ ID NO:

1.

103. 13. The method of any one of claims 100-102, wherein the interaction occurs at one or more residues of TIM-3 selected from regions 90-122 or 82-111, said residue positions corresponding to positions 90-122 or 82-111 of SEQ ID NO:

2.

104. 13. The method of any one of claims 100-102, wherein the interaction occurs at one or more residues of TIM-3 selected from regions 91-111, 107-117, 96-102, 100-106, or 92-119, wherein the residue positions correspond to positions 91-111, 107-117, 96-102, 100-106, or 92-119 of SEQ ID NO:

2.

105. 1. An anti-Gal3 antibody for use in treating an immune-related disease in a subject, said anti-Gal3 antibody inducing activation of the immune system.

106. The anti-Gal3 antibody for use in treating an immune-related disease according to claim 105, wherein the anti-Gal3 antibody inhibits the interaction between Gal3 and TIM-3.

107. The activation of the immune system is characterized by the activation of CD3+ T lymphocytes, CD4+ T helper cells, CD8+ An anti-Gal3 antibody for use in treating an immune-related disease as described in claim 105 or 106, comprising proliferation of cytotoxic T cells, NK cells, M1 macrophages, or a combination thereof.

108. The anti-Gal3 antibody for use in treating an immune-related disease according to any one of claims 105 to 107, wherein the activation of the immune system comprises a reduction in M2 macrophages.

109. The anti-Gal3 antibody for use in treating an immune-related disease according to any one of claims 105 to 108, wherein the immune-related disease is cancer.

110. The cancer is breast cancer, colorectal cancer, renal cancer, liver cancer, lung cancer, or hematological malignancies. The anti-Gal3 antibody for use in treating an immune-related disease described in claim 109.

111. The anti-Gal3 antibody for use in treating an immune-related disease described in claim 109 or 110, wherein the cancer is a metastatic cancer, a recurrent cancer, or a refractory cancer.

112. The anti-Gal3 antibody for use in treating an immune-related disease according to any one of claims 109 to 111, wherein the anti-Gal3 antibody is administered in combination with an additional therapeutic agent, such as an immune checkpoint modulator, a chemotherapeutic agent, a targeted therapeutic agent, a hormonal therapeutic agent, a stem cell-based therapeutic agent, surgery, or radiation therapy.

113. The anti-Gal3 antibody for use in the treatment of an immune-related disease according to any one of claims 105 to 108, wherein the immune-related disease is fibrosis and the anti-Gal3 antibody results in a reduction in the accumulation of extracellular matrix proteins in tissues.

114. The anti-Gal3 antibody for use in treating an immune-related disease described in claim 113, wherein the extracellular matrix protein comprises collagen.

115. The anti-Gal3 antibody for use in treating an immune-related disease described in claim 113 or 114, wherein the expression level of the at least one fibrosis biomarker in the subject is decreased, and the at least one fibrosis biomarker comprises α-SMA, fibronectin, or both.

116. The anti-Gal3 antibody for use in treating an immune-related disease according to any one of claims 113 to 115, wherein the tissue is selected from the group consisting of liver tissue, kidney tissue, skin tissue, lung tissue, heart tissue, brain tissue, intestinal tissue, bone marrow tissue, and soft tissue.

117. The anti-Gal3 antibody for use in treating an immune-related disease according to any one of claims 113 to 116, wherein the fibrosis is renal fibrosis, hepatic fibrosis, pulmonary fibrosis, cardiac fibrosis, or vascular fibrosis.

118. The anti-Gal3 antibody for use in treating an immune-related disease according to any one of claims 105 to 117, wherein the anti-Gal3 antibody is formulated for systemic, parenteral, intravenous or subcutaneous administration.

119. The anti-Gal3 antibody for use in treating an immune-related disease according to any one of claims 105 to 118, wherein the subject is a human.

120. The anti-Gal3 antibodies include 2D10.2B2, 3B11.2G2, 4A11.2B5, 4G2.2G6, 6H6.2D6, 7D8.2D8, 12G5.D7, 13A12.2E5, 13G4.2F8, 13H12.2F8, 14H10.2C9, 15F10.2D6, 15G7.2A7, 19B5.2E6, 19D9.2E5, 20D11.2C6, 20H5.A3, 23H9.2E4, 24D12.2H9, 846.1F5, 846.2H3, 846T.

105. The method of any one of claims 1 to 104, wherein the antibody is selected from the group consisting of 1H2, 9H2.2H10, IMT001-4, IMT006-1, IMT006-5, IMT006-8, and mIMT001.

121. The method of any one of claims 1 to 104, wherein the anti-Gal3 antibody is one or more of IMT001-4, IMT006-1, IMT006-5, or IMT006-8.

122. The anti-Gal3 antibodies include 2D10.2B2, 3B11.2G2, 4A11.2B5, 4G2.2G6, 6H6.2D6, 7D8.2D8, 12G5.D7, 13A12.2E5, 13G4.2F8, 13H12.2F8, 14H10.2C9, 15F10.2D6, 15G7.2A7, 19B5.2E6, 19D9.2E5, 20D11.2C6, 20H5.A3, 23H9.2E4, 24D12.2H9, 846.1F5, 846.2H3, 846T.

120. An anti-Gal3 antibody for use in treating an immune related disease according to any one of claims 105 to 119, selected from the group consisting of 1H2, 9H2.2H10, IMT001-4, IMT006-1, IMT006-5, IMT006-8, and mIMT001.

123. The anti-Gal3 antibody for use in treating an immune-related disease according to any one of claims 105 to 119, wherein the anti-Gal3 antibody is IMT001-4, IMT006-1, IMT006-5, or IMT006-8.

124. An anti-Gal3 antibody, the anti-Gal3 antibody comprising at least an HCDR3 within any one of the antibodies depicted in Figures 35A-36B.

125. The anti-Gal3 antibody of claim 124, further comprising all three HCDRs of any one of the antibodies of Figures 35A-36B.

126. The anti-Gal3 antibody of claim 125, further comprising all three LCDRs of any one of the antibodies of Figures 35A-36B.

127. An anti-Gal3 antibody, the anti-Gal3 antibody comprising any one of the heavy chain sequences in FIG. 36A or a sequence having at least 80% identity thereto.

128. An anti-Gal3 antibody, the anti-Gal3 antibody comprising any one of the light chain sequences in FIG. 36B, or a sequence having at least 80% identity thereto.

129. The anti-Gal3 antibody of claim 128, further comprising any one of the heavy chain sequences in FIG. 36A, or a sequence having at least 80% identity thereto.

130. An anti-Gal3 antibody, said anti-Gal3 antibody comprising six CDRs, said six CDRs having at least 80% identity over their combined sequence with any set of six CDRs in Figures 35A and 35B.

131. An anti-Gal3 antibody, said anti-Gal3 antibody comprising at least one of the CDRs from FIG.

132. An anti-Gal3 antibody, said anti-Gal3 antibody comprising at least two of the CDRs from FIG.

133. An anti-Gal3 antibody, said anti-Gal3 antibody comprising at least three of the CDRs from FIG.

134. An anti-Gal3 antibody, said anti-Gal3 antibody comprising at least four of the CDRs from FIG.

135. An anti-Gal3 antibody, wherein the anti-Gal3 antibody comprises at least five of the CDRs from FIG.

136. An anti-Gal3 antibody, said anti-Gal3 antibody comprising six of the CDRs from FIG.

38.

137. An anti-Gal3 antibody, wherein the anti-Gal3 antibody comprises six of the CDRs from FIG. 38, and all six are from a single bin.

138. An anti-Gal3 antibody, said anti-Gal3 antibody comprising six of the CDRs from FIG. 38, or a set of six CDRs having at least 80% identity thereto over their entire sequences.

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