Anti-GAL3 antibodies and uses thereof

Abstract

Disclosed herein are antibodies that specifically bind to Gal3 and methods of use thereof. In some embodiments, also described herein are methods of inducing immune activation or promoting proliferation of T cells or natural killer cells with antibodies that specifically bind to Gal3. Also disclosed herein are methods and compositions for reducing fibrosis, or a predisposition thereof, in tissue with antibodies that specifically bind to Gal3. In some cases, the anti-Gal3 antibodies also disrupt the interaction between Gal3 and TIM-3.

Description

[0001] This application is a divisional application of a Chinese patent application with application number 202080025814.4, filing date January 29, 2020, and invention title "Anti-Gal3 Antibodies and Their Uses".

[0002] Cross-reference to related applications

[0003] This application claims the benefit of U.S. Provisional Application Serial No. 62 / 798,945, filed January 30, 2019, and U.S. Provisional Application Serial No. 62 / 798,949, filed January 30, 2019, each of which is incorporated herein by reference in its entirety.

[0004] Reference to the Sequence Listing

[0005] This application is filed with a sequence listing in electronic format. The sequence listing is provided as a file entitled Seq List IMMUT003.TXT, which was created and last saved on January 29, 2020, and is 146,561 bytes in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety. Technical Field

[0006] In some embodiments, antibodies (or "anti-Gal3 antibodies") that specifically bind to Gal3 and disrupt the interaction between Gal3 and TIM-3 and promote the proliferation of T cells or natural killer (NK) cells are disclosed herein. Also disclosed herein are methods of using the antibodies to elicit an immune response and therapeutic methods. Also disclosed herein is a method of reducing fibrosis or its propensity in a tissue by contacting the tissue with an antibody that specifically binds to Gal3. Also described herein is a method of disrupting the Gal3-TIM-3 interaction with an antibody that specifically binds to Gal3 under conditions that reduce the expression of one or more fibrosis biomarkers in a tissue. Background Art

[0007] Galectin-3 (Gal3) is a lectin or carbohydrate-binding protein that is specific 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 protein-3 (TIM-3) is a protein expressed on immune cells such as T cells, dendritic cells, NK cells, and monocytes. Summary of the Invention

[0008] In some embodiments, antibodies (or "anti-Gal3 antibodies") that specifically bind to Gal3 and disrupt the interaction between Gal3 and TIM-3 are disclosed herein. In some embodiments, antibodies that specifically bind to Gal3 and promote the proliferation of T cells or natural killer cells are disclosed herein. In some embodiments, methods of eliciting an immune response and methods of treatment using the antibodies are also disclosed herein.

[0009] Embodiments of the invention provided herein are described by the following numbered alternatives:

[0010] 1. A method of inducing immune activation, comprising:

[0011] contacting a plurality of cells comprising 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, wherein the Gal3-expressing cells express cytokines that induce immune activation upon binding to the antibody, and wherein the antibody is not IMT001.

[0012] 2. The method according to alternative 1, wherein the cytokine is interferon.

[0013] 3. The method according to alternative 2, wherein the interferon is IFNγ.

[0014] 4. The method according to alternative 3, wherein IFNγ production is 150%, 160%, 170%, 180%, 190%, 200% or more of IFNγ production by an isotype antibody.

[0015] 5. The method according to alternative 1, wherein the cytokine is interleukin.

[0016] 6. The method according to alternative 5, wherein the interleukin is IL-2.

[0017] 7. The method according to any one of alternatives 1-6, wherein immune activation comprises the proliferation of CD3+ T lymphocytes, CD4+ T helper cells, CD8+ cytotoxic T cells, natural killer cells, or a combination thereof.

[0018] 8. The method according to any one of alternatives 1-7, wherein immune activation comprises an increase in the population of intracellular M1 macrophages.

[0019] 9. The method according to any one of alternatives 1-8, wherein immune activation comprises a decrease in the population of intracellular M2 macrophages.

[0020] 10. A method of promoting the proliferation of T cells or natural killer (NK) cells, comprising:

[0021] Under conditions that enable the proliferation of T cells and / or NK cells in multiple cell types, contacting multiple cell types including T cells, NK cells, and Gal3-expressing cells with an antibody, wherein the antibody specifically binds to Gal3, and wherein the antibody is not IMT001.

[0022] 11. The method according to alternative 10, wherein the multiple cell types further comprise TIM-3-expressing cells.

[0023] 12. The method according to alternative 11, wherein the antibody further disrupts the interaction between Gal3 and TIM-3.

[0024] 13. A method of inducing immune activation, comprising:

[0025] Contacting multiple cell types 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 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%.

[0026] 14. The method according to alternative 13, wherein the interaction occurs at one or more residues of Gal3 selected from regions 145-168, 160-177, or 165-184, wherein the residue positions correspond to positions 145-168, 160-177, or 165-184 of SEQ ID NO:1.

[0027] 15. The method according to alternative 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, wherein the residue positions correspond to positions 149-156, 152-168, 163-169, 163-177, or 163-171 of SEQ ID NO:1.

[0028] 16. The method according to any one of alternatives 13-15, wherein the interaction occurs at one or more residues of TIM-3 selected from regions 91-111 or 82-111, wherein the residue positions correspond to positions 91-111 or 82-111 of SEQ ID NO:2.

[0029] 17. The method according to any one of alternatives 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 the residue positions herein correspond to positions 91 - 111, 107 - 117, 96 - 102, 100 - 106, or 92 - 119 of SEQ ID NO:2.

[0030] 18. The method according to any one of alternatives 13 - 17, wherein TIM - 3 is human TIM - 3.

[0031] 19. The method according to any one of alternatives 1 - 18, wherein the Gal3 - expressing cells are tumor cells.

[0032] 20. The method according to any one of alternatives 1 - 19, wherein the plurality of cells are located within the tumor microenvironment (TME).

[0033] 21. The method according to any one of alternatives 1 - 20, wherein the antibody induces a reduction of tumor cells within the TME.

[0034] 22. The method according to any one of alternatives 1 - 21, wherein the plurality of cells further comprises tumor - infiltrating lymphocytes (TIL).

[0035] 23. The method according to any one of alternatives 1 - 22, wherein the plurality of cells further comprises CD3+ T lymphocytes, CD4+ T helper cells, CD8+ cytotoxic T cells, or a combination thereof.

[0036] 24. The method according to any one of alternatives 1, 10, 13, or 22, wherein the contact further induces TIL proliferation.

[0037] 25. The method according to any one of alternatives 1, 10, 13, or 23, wherein the contact further induces the proliferation of CD3+ T lymphocytes, CD4+ T helper cells, CD8+ cytotoxic T cells, or a combination thereof.

[0038] 26. The method according to any one of alternatives 1, 10, 13, or 22 - 25, wherein the contact further comprises an increase in M1 macrophage proliferation.

[0039] 27. The method according to any one of alternatives 1, 10, 13, or 22 - 26, wherein the contact further comprises a reduction of the M2 macrophage population within the TME.

[0040] 28. The method according to any one of alternative modes 1-27, wherein the antibody binds to at least one amino acid residue within the Gal3 region corresponding to residues 1-20 of SEQ ID NO:1.

[0041] 29. The method according to any one of alternative modes 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.

[0042] 30. The method according to any one of alternative modes 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.

[0043] 31. The method according to any one of alternative modes 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.

[0044] 32. The method according to any one of alternative modes 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.

[0045] 33. The method according to any one of alternative modes 1-32, wherein the antibody comprises 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 。

[0046] 34. The method according to any one of alternative modes 1-33, wherein the antibody comprises a humanized antibody.

[0047] 35. The method according to any one of alternative modes 1-34, wherein the antibody comprises a full-length antibody or its binding fragment.

[0048] 36. The method according to any one of alternative modes 1-35, wherein the antibody comprises a bispecific antibody or its binding fragment.

[0049] 37. The method according to any one of alternative modes 1-36, wherein the antibody comprises a monovalent Fab’, bivalent Fab2, single-chain variable fragment (scFv), diabody, minibody, nanobody, single-domain antibody (sdAb) or camelized antibody or its binding fragment.

[0050] 38. The method according to any one of alternative modes 1-37, wherein the antibody comprises an IgG framework.

[0051] 39. The method according to any one of alternative modes 1 - 38, wherein the antibody comprises an IgG1, IgG2 or IgG4 framework.

[0052] 40. The method according to any one of alternative modes 1 - 39, wherein the antibody further comprises an Fc mutation.

[0053] 41. The method according to any one of alternative modes 1 - 33 or 35 - 40, wherein the antibody comprises a chimeric antibody.

[0054] 42. The method according to any one of alternative modes 1, 10 or 13, further comprising administering the antibody to a subject prior to the contacting step.

[0055] 43. The method according to alternative mode 42, wherein the subject is diagnosed with cancer.

[0056] 44. The method according to alternative mode 43, wherein the cancer is a solid tumor.

[0057] 45. The method according to alternative mode 44, wherein the cancer is breast cancer, colorectal cancer, renal cancer, liver cancer or lung cancer.

[0058] 46. The method according to alternative mode 43, wherein the cancer is a hematological malignancy.

[0059] 47. The method according to any one of alternative modes 43 - 46, wherein the cancer is metastatic cancer.

[0060] 48. The method according to any one of alternative modes 43 - 46, wherein the cancer is recurrent or refractory cancer.

[0061] 49. The method according to any one of alternative modes 42 - 48, wherein the antibody is formulated for systemic administration.

[0062] 50. The method according to any one of alternative modes 42 - 49, wherein the antibody is formulated for parenteral administration.

[0063] 51. The method according to any one of alternative modes 42 - 50, wherein the antibody is administered in combination with another therapeutic agent.

[0064] 52. The method according to alternative mode 51, wherein the antibody and the another therapeutic agent are administered simultaneously.

[0065] 53. The method according to alternative mode 51, wherein the antibody and the another therapeutic agent are administered sequentially.

[0066] 54. The method according to alternative mode 53, wherein the antibody is administered before the another therapeutic agent.

[0067] 55. The method according to alternative 53, wherein the antibody is administered after administration of an additional therapeutic agent.

[0068] 56. The method according to any one of alternatives 51 - 55, wherein the additional therapeutic agent comprises an immune checkpoint modulator.

[0069] 57. The method according to any one of alternatives 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.

[0070] 58. The method according to any one of the previous alternatives, wherein the subject is a human.

[0071] 59. The method according to alternative 58, wherein the antibody is administered before or after surgery.

[0072] 60. The method according to alternative 58, wherein the antibody is administered in combination with radiotherapy, before radiotherapy, or after radiotherapy.

[0073] 61. The method according to any one of the previous alternatives, wherein the antibody has a K D higher than the K of antibody IMT001 D .

[0074] 62. A method of reducing fibrosis or its propensity in a tissue, comprising:

[0075] contacting the tissue with an antibody that specifically binds to an antibody against Gal3 under conditions that result in a reduced level of expression of a fibrosis biomarker in the tissue.

[0076] 63. The method according to alternative 62, wherein the tissue further comprises TIM - 3 expressing cells.

[0077] 64. The method according to alternative 63, wherein the antibody further disrupts the interaction between Gal3 and TIM - 3.

[0078] 65. The method according to alternative 63, wherein the antibody does not disrupt the interaction between Gal3 and TIM - 3.

[0079] 66. The method according to any one of alternatives 62 - 65, wherein at least one fibrosis biomarker comprises α - smooth muscle actin (α - SMA).

[0080] 67. The method according to any one of alternatives 62 - 65, wherein at least one fibrosis biomarker comprises fibronectin.

[0081] 68. The method according to any one of alternatives 62 - 65, wherein at least one fibrotic biomarker comprises α - smooth muscle actin (α - SMA) and fibronectin.

[0082] 69. The method according to any one of alternatives 62 - 68, wherein the tissue is kidney tissue or liver tissue.

[0083] 70. The method according to any one of alternatives 62 - 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.

[0084] 71. The method according to any one of alternatives 62 - 70, wherein the expression of at least one fibrotic biomarker in the tissue treated with the antibody is less than the expression of at least one fibrotic biomarker in the control tissue treated with the mIgG2b antibody.

[0085] 72. The method according to any one of alternatives 62 - 71, wherein the antibody causes a reduction in the accumulation of extracellular matrix proteins in the tissue.

[0086] 73. The method according to alternative 72, wherein the extracellular matrix protein comprises collagen.

[0087] 74. The method according to alternative 73, wherein the tissue comprises cells that produce collagen.

[0088] 75. The method according to alternative 74, wherein the cells that produce collagen are fibroblasts.

[0089] 76. The method according to alternative 75, wherein the fibroblasts are activated by fibrogenic cytokines.

[0090] 77. The method according to alternative 76, wherein the fibrogenic cytokine is TGF - β1.

[0091] 78. The method according to any one of alternatives 62 - 77, wherein the tissue has elevated TGF - β1 expression.

[0092] 79. The method according to any one of alternatives 62 - 78, wherein the antibody comprises a humanized antibody.

[0093] 80. The method according to any one of alternatives 62 - 79, wherein the antibody comprises a full - length antibody or a binding fragment thereof.

[0094] 81. The method according to any one of alternatives 62 - 79, wherein the antibody comprises a bispecific antibody or a binding fragment thereof.

[0095] 82. The method according to any one of alternatives 62 - 79, wherein the antibody comprises a chimeric antibody.

[0096] 83. The method according to any one of alternatives 62 - 82, wherein the antibody binds to at least one amino acid residue within the Gal3 region corresponding to residues 1 - 20 of SEQ ID NO:1.

[0097] 84. The method according to any one of alternatives 62 - 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.

[0098] 85. The method according to any one of alternatives 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.

[0099] 86. The method according to any one of alternatives 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.

[0100] 87. The method according to any one of alternatives 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.

[0101] 88. The method according to any one of alternatives 62 - 87, wherein the antibody comprises a KD 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.

[0102] 89. The method according to any one of alternatives 62 - 88, wherein the antibody comprises a monovalent Fab’, bivalent Fab2, single-chain variable fragment (scFv), diabody, minibody, nanobody, single-domain antibody (sdAb) or camelized antibody or a binding fragment thereof.

[0103] 90. The method according to any one of alternatives 62 - 89, wherein the antibody comprises an IgG framework.

[0104] 91. The method according to any one of alternatives 62 - 90, wherein the antibody comprises an IgG1, IgG2 or IgG4 framework.

[0105] 92. The method according to any one of alternatives 62 - 91, wherein the antibody further comprises an Fc mutation.

[0106] 93. The method according to any one of alternatives 62 - 92, further comprising administering an antibody to a subject prior to the contacting step.

[0107] 94. The method according to alternative 93, wherein the subject is diagnosed with a fibrotic disease.

[0108] 95. The method according to alternative 94, wherein the fibrotic disease is renal fibrosis.

[0109] 96. The method according to alternative 94, wherein the fibrotic disease is hepatic fibrosis.

[0110] 97. The method according to any one of alternatives 93 - 96, wherein the antibody is formulated for systemic administration.

[0111] 98. The method according to any one of alternatives 93 - 96, wherein the antibody is formulated for parenteral administration.

[0112] 99. The method according to any one of alternatives 93 - 98, wherein the subject is a mammal.

[0113] 100. The method according to any one of alternatives 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%.

[0114] 101. The method according to alternative 100, wherein the interaction occurs at one or more residues of Gal3 selected from regions 145 - 168, 160 - 177 or 165 - 184, wherein the residue positions correspond to positions 145 - 168, 160 - 177 or 165 - 184 of SEQ ID NO:1.

[0115] 102. The method according to alternative 100, wherein the interaction occurs at one or more residues of Gal3 selected from regions 149 - 156, 152 - 168, 163 - 169 or 163 - 171, wherein the residue positions correspond to positions 149 - 156, 152 - 168, 163 - 169 or 163 - 171 of SEQ ID NO:1.

[0116] 103. The method according to any one of alternatives 100 - 102, wherein the interaction occurs at one or more residues of TIM - 3 selected from regions 90 - 122 or 82 - 111, wherein the residue positions correspond to positions 90 - 122 or 82 - 111 of SEQ ID NO:2.

[0117] 104. The method according to any one of alternatives 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 the residue positions herein correspond to positions 91 - 111, 107 - 117, 96 - 102, 100 - 106, or 92 - 119 of SEQ ID NO:2.

[0118] 105. An anti - Gal3 antibody for treating immune - related diseases in a subject, wherein the anti - Gal3 antibody induces activation of the immune system.

[0119] 106. The anti - Gal3 antibody for treating immune - related diseases according to alternative 105, wherein the anti - Gal3 antibody inhibits the interaction between Gal3 and TIM - 3.

[0120] 107. The anti - Gal3 antibody for treating immune - related diseases according to alternative 105 or 106, wherein the activation of the immune system includes the proliferation of CD3+ T lymphocytes, CD4+ T helper cells, CD8+ cytotoxic T cells, NK cells, M1 macrophages, or a combination thereof.

[0121] 108. The anti - Gal3 antibody for treating immune - related diseases according to any one of alternatives 105 - 107, wherein the activation of the immune system includes a reduction in M2 macrophages.

[0122] 109. The anti - Gal3 antibody for treating immune - related diseases according to any one of alternatives 105 - 108, wherein the immune - related disease is cancer.

[0123] 110. The anti - Gal3 antibody for treating immune - related diseases according to alternative 109, wherein the cancer is breast cancer, colorectal cancer, kidney cancer, liver cancer, lung cancer, or hematological malignancy.

[0124] 111. The anti - Gal3 antibody for treating immune - related diseases according to alternative 109 or 110, wherein the cancer is metastatic cancer, recurrent cancer, or refractory cancer.

[0125] 112. The anti - Gal3 antibody for treating immune - related diseases according to any one of alternatives 109 - 111, wherein the anti - Gal3 antibody is administered in combination with another 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.

[0126] 113. An anti-Gal3 antibody for treating an immune-related disease according to any one of alternative modes 105-108, wherein the immune-related disease is fibrosis, and the anti-Gal3 antibody results in a decrease in the accumulation of extracellular matrix proteins in tissues.

[0127] 114. An anti-Gal3 antibody for treating an immune-related disease according to alternative mode 113, wherein the extracellular matrix protein comprises collagen.

[0128] 115. An anti-Gal3 antibody for treating an immune-related disease according to alternative mode 113 or 114, wherein the expression level of at least one fibrosis biomarker in a subject is decreased, and wherein the at least one fibrosis biomarker comprises α-SMA, fibronectin, or both.

[0129] 116. An anti-Gal3 antibody for treating an immune-related disease according to any one of alternative modes 113-115, 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.

[0130] 117. An anti-Gal3 antibody for treating an immune-related disease according to any one of alternative modes 113-116, wherein the fibrosis is renal fibrosis, hepatic fibrosis, pulmonary fibrosis, cardiac fibrosis, or vascular fibrosis. In some embodiments, this can be administered IV or subcutaneously.

[0131] 118. An anti-Gal3 antibody for treating an immune-related disease according to any one of alternative modes 105-117, wherein the anti-Gal3 antibody is formulated for systemic administration, parenteral administration, intravenous administration, or subcutaneous administration.

[0132] 119. An anti-Gal3 antibody for treating an immune-related disease according to any one of alternative modes 105-118, wherein the subject is a human.

[0133] 120. The method according to any one of alternative modes 1 - 104, wherein the anti-Gal3 antibody is one or more 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.

[0134] 121. The method according to any one of alternative modes 1 - 104, wherein the anti-Gal3 antibody is an antibody having 1, 2, 3, 4, 5, or 6 CDRs within one or more of the CDRs from 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.

[0135] 121. The method according to any one of alternative modes 1 - 104, wherein the anti-Gal3 antibody is IMT001-4, IMT006-1, IMT006-5, or IMT006-8.

[0136] 122. An anti-Gal3 antibody for treating immune-related diseases according to any one of alternative modes 105-119, wherein 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.

[0137] 123. An anti-Gal3 antibody for treating immune-related diseases according to any one of alternative modes 105-119, wherein the anti-Gal3 antibody is IMT001-4, IMT006-1, IMT006-5 or IMT006-8.

[0138] 124. An anti-GAL3 antibody comprising at least Figures 35A - 36B the HCDR3 within any one of the antibodies.

[0139] 125. The anti-GAL3 antibody according to alternative mode 124, which further comprises Figures 35A - 36B all 3 HCDRs within any one of the antibodies.

[0140] 126. The anti-GAL3 antibody according to alternative mode 125, which further comprises Figures 35A - 36B all 3 LCDRs within any one of the antibodies.

[0141] 127. An anti-GAL3 antibody comprising Figure 36A any one of the inner heavy chain sequences, or a sequence having at least 80% identity therewith.

[0142] 128. An anti-GAL3 antibody comprising Figure 36B any one of the inner light chain sequences, or a sequence having at least 80% identity therewith.

[0143] 129. The anti-GAL3 antibody according to alternative mode 128, which further comprises Figure 36A any one of the inner heavy chain sequences, or a sequence having at least 80% identity therewith.

[0144] 130. An anti-GAL3 antibody comprising 6 CDRs, wherein the 6 CDRs, in their combined sequence, are identical toFigure 35A and 35B the six CDRs of any group within are at least 80% identical.

[0145] 131. An anti-GAL3 antibody comprising at least one CDR from Figure 38 ...

[0146] 132. An anti-GAL3 antibody comprising at least two CDRs from Figure 38 ...

[0147] 133. An anti-GAL3 antibody comprising at least three CDRs from Figure 38 ...

[0148] 134. An anti-GAL3 antibody comprising at least four CDRs from Figure 38 ...

[0149] 135. An anti-GAL3 antibody comprising at least five CDRs from Figure 38 ...

[0150] 136. An anti-GAL3 antibody comprising at least five CDRs from Figure 38 ...

[0151] 137. An anti-GAL3 antibody comprising six CDRs from Figure 38 ... and all six of which are from a single bin.

[0152] 138. An anti-GAL3 antibody comprising six CDRs from Figure 38 ... or a set of six CDRs that are at least 80% identical to them in their entire sequence. BRIEF DESCRIPTION OF THE DRAWINGS

[0153] In addition to the features described above, additional features and variations will become apparent from the following drawings and description of exemplary embodiments. It should be understood that these drawings depict typical embodiments and are not intended to limit the scope.

[0154] Figure 1A -Panel C illustrates the results of a co-immunoprecipitation assay indicating that human Gal3 (hGal3) specifically pulls down hTIM-3. Figure 1A Shows TIM-3 expression in 293T cells co-transfected with a plasmid encoding HA-tagged hTIM-3 and a plasmid encoding hGal3, hGal9, or hCEACAM1. Figure 1B Shows the expression of hGal9, hGal3, or hCEACAM1. Figure 1Cshowed that hGal3, rather than CEACAM1, pulled down HA-tagged hTIM-3 in co-transfected 293T cells. The results also showed that human Gal9 (hGal9) pulled down hTIM-3, but the pull-down was accompanied by protein aggregation ( Figure 1B ), indicating that the binding between hGal9 and hTIM-3 can be non-specific.

[0155] Figure 2 showed the results of pull-down assays using fusion proteins including the extracellular domain of hTIM-3 (hTIM-3Fc) fused to the Fc portion of hIgG. The results showed that the binding between Gal3 and TIM-3 was specific. As shown in the figure, hTIM-3Fc, rather than hFc or hPD1Fc, pulled down overexpressed, Flag-tagged hGal3 protein from 293T cells.

[0156] Figure 3 showed the results of cell adhesion assays, indicating specific interaction between hGal3 and hTIM-3. As shown in the figure, a significantly higher number of A20 cells expressing hGal3 (A20 Gal3 cells) were able to adhere to plates coated with hTIM-3Fc compared to plates coated with hVISTA Fc or hPD1 Fc. The results also indicated that a higher number of A20 PDL1 cells were able to adhere to plates coated with hPD1Fc compared to plates coated with human VISTA Fc (hVISTAFc) or plates coated with hTIM-3Fc.

[0157] Figure 4A showed live A20 cells (left peak) and dead A20 cells (right peak) analyzed by flow cytometry.

[0158] Figure 4B -C showed the results of flow cytometry analysis of live cells ( Figure 4B ) and dead cells ( Figure 4C ) stained with anti-hFc APC antibody. In group 1, A20 Gal3 cells were not incubated with mTIM-3Fc protein as a control; in group 2, A20 Gal3 cells were incubated with mTIM-3Fc protein; in groups 3, 4, and 5, in addition to mTIM-3Fc protein, anti-mouse TIM-3 polyclonal antibody (R&D System, Minneapolis, MN) (group 3), monoclonal antibody RMT3-23 (Bio Xcell, West Lebanon, NH) (group 4), monoclonal antibody 215015 (R&DSystems) (group 5) were added to test whether these antibodies could block the binding of Gal3 and Tim3.

[0159] Figure 5A -C shows the ELISA results indicating the specific binding of Gal3 to TIM-3. In Figure 5A , it shows that the plate was coated with 10 μg / ml of mGal3, and the interaction between Gal3 and Tim3 was blocked by mGal3 polyclonal antibody (mGal3pAb) and monoclonal antibody IMT001, rather than monoclonal antibody M3 / 38. Figure 5B It shows that lactose blocked Gal9, rather than Gal3, from binding to TIM-3, indicating that the binding between Gal3 and Tim3 is sugar-independent. Figure 5C It shows that the antibody RMT3-23 blocked phosphatidylserine (PS), rather than Gal3, from binding to TIM-3, indicating that the epitopes on TIM-3 that bind to Gal3 are different from those that bind to PS.

[0160] Figure 6A -B shows that overexpressed Gal3 inhibits T cell activation. Figure 6A It shows that mouse A20 cell clones #41, #31, and #15 overexpress Gal3. Figure 6B It shows that when these cells were mixed with mouse DO11.10 T cells, much less IL-2 was produced compared to parental A20 cells.

[0161] Figure 7A -E shows that the Gal3 antibody has anti-tumor activity in the lung metastasis model. Figure 7A It shows the high expression of Gal3 on B16F10 tumor cells. Figure 7B It shows representative images of the whole lungs of three treatment groups. Figure 7C It shows the number (mean ± SEM) of metastatic colonies on the surface of the left lung lobe. Figure 7D And Figure 7E It shows the lung weights and body weights (mean ± SEM) of different treatment groups. Animals treated with the monoclonal anti-human Gal3 antibody showed a significant reduction in the number of tumors (p < 0.01) ( Figure 7B ) and much less tumor burden as indicated by lung weight (p < 0.05) ( Figure 7D ) compared to animals treated with the isotype control. However, animals treated with the PD1 antibody did not show a significant reduction in the number of tumors or tumor burden in this lung metastasis model (p > 0.05). Figure 7E It shows that animals treated with the PD1 antibody or Gal3 antibody had similar body weights to the control group, indicating no side effects associated with the administration of either antibody.

[0162] Figure 8A -C shows the anti-tumor activity of the Gal3 antibody in 4T1 orthotopic tumor-induced lung metastasis. Figure 8AImages of pulmonary metastatic tumor colonies in mice that had been implanted with 4T1 cells and then treated with a control antibody (“isotype”) or IMT001 are shown. The antibodies were administered intraperitoneally on days 0, 3, 7, 10, and 14 during a 30-day period. Images were taken on day 30 when the mice were sacrificed. Figure 8B Shows the body weight measurements of these mice during the same period. Figure 8C Shows the number of metastatic tumor colonies on the surface of the left lung lobe of these mice on day 30.

[0163] Figure 9 Shows tumor growth in mice implanted with Renca tumor cells and treated with Gal3 antibody. Mice treated with Gal3 antibody (“IMT001”) showed much reduced tumor size (p < 0.05) compared to mice implanted with Renca tumor cells and treated with an isotype control antibody (“iso”), while anti-mouse PD-1 antibody 29F had no effect (p > 0.05).

[0164] Figure 10 Shows tumor growth in mice implanted with MC38 colon cancer cells and treated with anti-Gal3 antibody. Mice treated with Gal3 antibody (“IMT001”) showed much reduced tumor size (p < 0.05) compared to mice implanted with MC38 tumor cells and treated with an isotype control antibody (“iso”).

[0165] Figure 11A -D shows the results of epitope mapping. Peptide arrays were synthesized from the hGal3 protein sequence ( Figure 11A ) and dot blotted with anti-Gal3 antibody IMT001 ( Figure 11B ). Peptides 5 and 6 showed good signals, indicating that the anti-Gal3 monoclonal antibody, IMT001, could bind to these peptides. To further map the binding epitope of IMT001 on these peptides, several shorter peptides were synthesized from these peptide sequences ( Figure 11C ) and their binding to IMT001 was measured by ELISA ( Figure 11D ). The peptide with the sequence GQAPPGAYPG (SEQ ID NO:28) produced the highest signal.

[0166] Figure 12 Summarizes the numbers of immune cells from mice implanted with B16F10 cells expressing various lymphocyte markers: CD3, CD4, CD8, CD19, or DX5. These mice had been treated with an isotype control antibody or IMT001.

[0167] Figure 13A-Panel B shows Gal3 expression on tumor-associated macrophages in human lung cancer in an immunohistochemistry (IHC) assay. IMT001 was used to stain frozen sections of human lung cancer to detect Gal3 expression on tumor-associated macrophages. Figure 13A Results from staining squamous cell carcinoma are shown and Figure 13B results from staining adenocarcinoma are shown.

[0168] Figure 14A -Panel C shows Gal3 expression detected on human M2 macrophages ( Figure 14C ) but not on dendritic cells (DC) ( Figure 14A ) or M1 macrophages ( Figure 14B ).

[0169] Figure 15A -Panel D shows the immunological activity of the Gal3 antibody (“IMT001”) in a mouse macrophage / T cell response. Figure 15B Gal3 expression detected by IHC on the mouse macrophage cell line RAW264.7 is shown compared to a control ( Figure 15A ). Figure 15C Gal3 expression on the mouse macrophage cell line by flow cytometry using cells stained with IMT001 is shown. The anti-Gal3 antibody IMT001, but not the anti-mouse PD-1 antibody 29F, enhanced IL-2 production in the RAW macrophage / DO11.10 T cell mixed response ( Figure 15D ).

[0170] Figure 16 Illustrated is the ELISA assessment of GAL3-TIM3 interaction blockade by a GAL3-binding antibody. Results illustrate that Gal3-targeted antibodies exhibit different blockade of Gal3-TIM3 binding. The percentage of TIM3-GAL3 binding in the absence of antibody is shown.

[0171] Figures 17A - 17B Illustrated is the ELISA assessment of anti-GAL3 antibodies with peptide fragments that bind to GAL3. Figure 17A : antibodies mab1, mab3, mab4, and mab5; Figure 17B : antibodies mab2, mab3, mab6, and mab7. Results illustrate that Gal3-targeted antibodies exhibit different blockade of Gal3-TIM3 binding.

[0172] Figure 18 Illustrated is the ELISA competitive binding assessment of anti-GAL3 antibodies that bind to GAL3. Results illustrate that the Gal3-targeted antibodies mab1 (801) and mab4 (804), but not mab5 (805), competitively bind to Gal3 bidirectionally.

[0173] Figure 19A -C illustrates the binding and dissociation kinetics of anti-Gal3 antibodies and the biolayer interferometry assessment of Gal3 (Gal3-binding antibody affinity). Figure 19A : mab1; kD = 13.5 nM. Figure 19B : mab4; kD = 1.2 nM. Figure 19C : mab5; kD = 32 nM.

[0174] Figure 20 Illustrates the assessment of the GAL3-enhanced CMV antigen recall test for T-cell antigen-specific responsiveness. The results illustrate that Gal-3-targeted antibodies exhibit different T-cell activation through CMV-induced antigen recall.

[0175] Figure 21A -C illustrates the MALDI-MS identification of the GAL3 and TIM3 regions that mediate the interaction between TIM3 and GAL3. Note that the amino acid numbering is based on the mature protein after processing of the signal peptide. See also Table 2. Figure 21A Illustrates the potential sequences involved in the binding interface. Figure 21B Illustrates the potential residues involved in the interaction. Figure 21C Illustrates the sequence positions mapped on the respective TIM-3 and Gal3. Note that the amino acid numbering is based on the mature protein after processing of the signal peptide.

[0176] Figure 22 Shows the Western blot analysis of the fibrosis markers, α-smooth muscle actin (α-SMA) and fibronectin, in kidney tissue lysates from male unilateral ureteral obstruction (UUO) mice treated with IMT001 and mIgG2b (control) antibodies for 14 days after ureteral ligation or sham-treated without antibodies. GAPDH was used as a loading control.

[0177] Figure 23 Shows the Western blot analysis of the fibrosis markers, α-SMA and fibronectin, in liver tissue lysates from non-obese diabetic and inflamed (N-IF) mice. Animals were treated with IMT001, anti-Gal3 antibody, and mIgG2b (control) antibody for 40 days. GAPDH was used as a loading control.

[0178] Figure 24 . The ability of galectin-3-targeted antibodies to block the binding of GAL3 and TIM3 was evaluated by ELISA at 3 μg / mL. Bars represent mean + / - standard deviation.

[0179] Figure 25.Comparison of the ability of GAL3 peptide and GAL3-targeted antibodies that bind and block GAL3-TIM3.

[0180] Figure 26 .Identification of galectin-3 binding antibodies by antibody competition. Values represent inhibition as assessed by biolayer interferometry.

[0181] Figure 27 .Blocking of GAL3-TIM3 by humanized anti-GAL3 antibodies was evaluated by ELISA in a titration series. The plotted values represent mean + / − standard deviation.

[0182] Figure 28A -D. Tumor volumes of mice with subcutaneous MBT2 tumor implants and treated with control, IMT001, anti-PD-L1 antibody or combinations thereof ( Figure 28A ), or with control, IMT001, anti-PD-1 antibody or combinations thereof ( Figure 28C ). Graphs of tumor volumes of anti-PD-L1 ( Figure 28B ) or anti-PD-1 ( Figure 28D ) represent measurements of individual animals taken daily.

[0183] Figure 29A -D. Hepatocellular carcinoma formation in normal and STAM-CDAA mice treated with human IgG4 (huIgG4) or IMT001-4 was evaluated by gross histology ( Figure 29A ) and enumerated ( Figure 29B ). Arrows highlight areas with tumors. Tumor formation was evaluated in hematoxylin and eosin-stained sections of liver samples ( Figure 29C ). Arrows highlight areas with tumors. Alpha-fetoprotein ( Figure 29D ) in the sera of STAM-CDAA mice treated with huIgG4 or IMT001-4 was quantified. Circles indicate the mean for each animal and the lines indicate the mean for each group.

[0184] Figure 30A -D. Hematoxylin and eosin-stained sections of livers from MCD mouse models of NASH liver fibrosis treated with isotype control or mIMT001 ( Figure 30A ). Histological findings of steatosis, hepatocyte ballooning, lobular inflammation or NAFLD activity score (NAS) were measured based on image quantification ( Figure 30B ). Picosirius red-stained liver samples from mice treated as in (A) ( Figure 30C ). Sirius red staining was quantified based on images ( Figure 30D)。The bars represent the mean of 7 animals + / - the standard error of the mean.

[0185] Figure 31A -B. Sirius red-stained liver samples from a choline-deficient L-amino acid-defined high-fat diet (CDAA-HFD) STAM model of liver fibrosis treated with isotype control or IMT001-4 ( Figure 31A ). Sirius red staining was quantified based on images ( Figure 31B ). The bars represent the mean + / - the standard error of the mean of 5 fields from each of 7 animals.

[0186] Figure 32A -C. Serum markers of renal fibrosis, KIM-1 and NGAL, were evaluated in mice treated with isotype control, IMT001-4, IMT001-6, or metformin in a mouse unilateral ureteral obstruction (UUO) model ( Figure 32A ) and Sirius red-stained kidney samples ( Figure 32B ). Sirius red staining was quantified based on images ( Figure 32C ). The bars represent the mean + / - the standard error of the mean of three evaluations from each of seven animals in each group. The dots represent the mean Sirius red staining of individual animals, and the bars indicate the mean group values.

[0187] Figure 33A -B. IHC evaluation of collagen 1a1 (Col1a1) deposition in kidney samples treated with isotype control or mIMT001 on the first day after treatment in a UUO model ( Figure 33A ). The black arrows correspond to areas of fibrotic collagen deposition. Quantification was based on images ( Figure 33B ). The bars represent the mean + / - the standard error of the mean of 10 fields from each of 7 animals in each group.

[0188] Figure 34A -B. Masson's trichrome staining evaluation of lung fibrosis in a bleomycin-induced mouse model of lung fibrosis treated with isotype control or mIMT001 ( Figure 34A ). Ashcroft scoring of tissue sections ( Figure 34B ). The bars represent the mean + / - the standard error of the mean of 10 fields from each of 8 animals in each group.

[0189] Figure 35A Depicted are some embodiments of the VH CDR regions of various embodiments of anti-GAL3 antibodies. In some embodiments, any method or composition provided herein may include one or more of the CDRs provided herein, including 1, 2, or 3 of them.

[0190] Figure 35BDepicts some embodiments of the VL CDR regions of various embodiments of anti-GAL3 antibodies. In some embodiments, any method or composition provided herein may include one or more of the CDRs provided herein, including 1, 2, or 3 of them.

[0191] Figure 36A Depicts some embodiments of the complete VH regions of various embodiments of anti-GAL3 antibodies. In some embodiments, any method or composition provided herein may include any one of these VH regions.

[0192] Figure 36B Depicts some embodiments of the complete VL regions of various embodiments of anti-GAL3 antibodies. In some embodiments, any method or composition provided herein may include any one of these VL regions.

[0193] Figure 37 Depicts some embodiments of various GAL3 antibodies (including complete heavy chain or κ chain sequences). In some embodiments, any one or more of the VH / VL and / or CDRs provided in other figures may be paired with Figure 37 any one or more of the relevant sequences in

[0194] Figure 38 Depicts an alignment of some embodiments of the VH CDR or VL CDR regions of various embodiments of anti-Gal3 antibodies. In some embodiments, any method or composition provided herein may use any 1, 2, 3, 4, 5, or 6 of the consensus CDRs provided in Figure 38 Detailed description

[0196] Galectin-3 (Gal3, GAL3, or Gal-3) is expressed in a variety of cell types and is involved in a wide range of physiological and pathological processes, which include cell adhesion, cell activation and chemotaxis, cell cycle, apoptosis, cell growth and differentiation, and tumor progression and metastasis. Gal3 is expressed on tumor cells and in the tumor microenvironment, such as tumor-associated macrophages, especially M2 macrophages. Further, it is also involved in promoting fibroblast proliferation and transformation and mediating the activation of various profibrotic factors that produce collagen. In addition, Gal3 is thought to play a key role in fibrogenesis in various tissues including the liver, kidney, lung, and myocardium.

[0197] TIM-3 is a molecule expressed on immune cells, especially on T cells, and can inhibit immune responses, such as T cell signaling, through interaction with Gal3. Anti-Gal3 antibodies interfere with the interaction between Gal3 and TIM-3 and activate immune responses. ​

[0198] Tumors are generally associated with immune infiltration as part of a reactive stroma that is enriched with macrophages. Tumor-associated macrophages (TAMs) play an important role in promoting tumor growth by facilitating neovascularization and matrix degradation. When associated with tumors, macrophages demonstrate functional polarization into one of two phenotypically distinct macrophage subsets: M1 macrophages or M2 macrophages. M1 macrophages are known to produce pro-inflammatory cytokines and play an active role in cell destruction, while M2 macrophages primarily clear debris and promote angiogenesis and wound repair. Thus, many tumors with a large population of TAMs have increased tumor growth rates, local proliferation, and distant metastasis. The M2 macrophage population phenotypically resembles the TAM population that promotes tumor growth and development. In some cases, in addition to expressing Gal3, 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 scavenger 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.

[0199] Tissue fibrosis is a progressive debilitating disease characterized by the massive accumulation of extracellular matrix (ECM) proteins such as collagen and fibronectin, leading to tissue scarring, organ damage, organ dysfunction, and subsequent organ failure. Tissue fibrosis can be located in the kidney, liver, lung, heart, skin, pancreas, intestine, eye, nervous system, joints, tendons, mediastinum, or retroperitoneal space. Features of tissue fibrosis include epithelial and endothelial injury and dysfunction; abnormal proliferation of myofibroblasts (MFb), smooth muscle cells, and stellate cells; and ECM deposition. The presence of cytokines, chemokines, growth factors, and angiogenic factors further regulates the activation of cells that produce ECM during the pro-fibrotic process.

[0200] Galectin-3 (Gal3) is known to play important roles in cell proliferation, adhesion, differentiation, angiogenesis, and apoptosis. In addition, it is involved in promoting fibroblast proliferation and transformation and mediating the activation of various pro-fibrotic factors that mediate collagen production. Furthermore, Gal3 is thought to play a key role in fibrogenesis in various tissues including the liver, kidney, lung, and myocardium.

[0201] In some embodiments, methods for reducing fibrosis or its propensity in a tissue are disclosed herein. In some embodiments, reducing fibrosis or its propensity in a tissue includes preventing fibrosis from occurring in normal tissue. In some embodiments, reducing fibrosis or its propensity in a tissue includes slowing or arresting the progression of fibrosis in fibrotic tissue. In some embodiments, reducing fibrosis or its propensity in a tissue includes reducing the amount of the degree of fibrosis in fibrotic tissue. In some embodiments, reducing fibrosis or its propensity in a tissue includes eliminating fibrosis in fibrotic tissue.

[0202] In some embodiments, methods for monitoring the progression of tissue fibrosis by monitoring one or more fibrosis biomarkers are also described herein. In additional cases, methods for treating tissue fibrosis with an anti-Gal3 antibody are disclosed herein, wherein the anti-Gal3 antibody disrupts the interaction between Gal3 and TIM-3.

[0203] 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 meant to be 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. It will be readily understood that the aspects of the present disclosure, as generally described herein and illustrated in the drawings, can be arranged, substituted, combined, separated, and designed in a variety of different configurations, all of which are explicitly contemplated herein.

[0204] Unless otherwise defined, the 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 the purposes of this disclosure, the following terms are defined as follows.

[0205] As used herein, the articles "a" and "an" refer to one or 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.

[0206] "About" means a variation of up to 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% in mass, level, value, quantity, frequency, percentage, dimension, size, amount, weight, or length.

[0207] Throughout this specification, unless the context requires otherwise, the words "comprise", "comprising" and "include" will be understood to imply the inclusion of the stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. "Consisting of" means including and limited to whatever follows the phrase "consisting of". Thus, the phrase "consisting of" indicates that the listed elements are essential or mandatory and that no other elements may be present. "Consisting essentially of" means including any elements listed with the accompanying phrase and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure of the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are essential or mandatory, but other elements are optional and may or may not be present depending on whether they materially affect the activity or action of the listed elements.

[0208] 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 of blocking or disrupting the interaction between Gal3 and the TGF-β receptor in vitro or in vivo using 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 of blocking or disrupting the interaction between Gal3 and TIM-3 using an anti-Gal3 antibody or a binding fragment thereof or a composition comprising an anti-Gal3 antibody or a binding fragment thereof is used for treating, curing or preventing a disease or disorder in a subject. In some embodiments, the disease or disorder is cancer, breast cancer, colorectal cancer, kidney cancer, liver cancer, lung cancer or hematological malignancy. In some embodiments, in some embodiments, the cancer is metastatic cancer, recurrent cancer or refractory cancer. In some embodiments, the antibody is administered in combination with another 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.

[0209] As used herein, the terms "individual", "subject" and "patient" mean any mammal or bird. In some embodiments, the mammal is a human. In some embodiments, the mammal is non-human and includes, but is not limited to, farm animals (e.g., cows, pigs, horses, chickens, etc.), sport animals, pets, primates, dogs, cats, mice and rats. None of these terms require or are limited to a condition characterized by the supervision (e.g., continuous or intermittent) of a health care worker (e.g., a doctor, a registered nurse, a licensed practical nurse, a physician assistant, a health care provider or a hospice worker).

[0210] As used herein, the terms "polypeptide", "peptide" and "protein" are used interchangeably to refer to a polymer of amino acids of any length. The polymer may be linear, cyclic or branched, it may include modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass amino acid polymers that have been modified, e.g., by sulfation, glycosylation, lipidation, acetylation, phosphorylation, iodination, methylation, oxidation, proteolytic processing, phosphorylation, isoprenylation, racemization, selenylation, transfer-RNA mediated addition of amino acids to proteins, such as arginylation, ubiquitination or any other manipulation, such as conjugation to a label component.

[0211] As used herein, the term "amino acid" refers to natural and / or non-natural or synthetic amino acids, including both glycine and D or L optical isomers, as well as amino acid analogs and peptidomimetics.

[0212] A polypeptide or amino acid sequence "derived from" a designated protein refers to the source of the polypeptide. Preferably, the polypeptide has an amino acid sequence that is substantially the same as the amino acid sequence of the 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 with the polypeptide encoded in the sequence. The term also includes polypeptides expressed from the designated nucleic acid sequence.

[0213] As used herein, the term "antibody" is intended to include any polypeptide chain-containing molecular structure having a particular shape that is suitable for and recognizes an epitope, where one or more non-covalent binding interactions stabilize the complex between the molecular structure and the epitope. The antibodies utilized in the present invention may be polyclonal antibodies, although monoclonal antibodies are preferred because they can be replicated by cell culture or recombinantly and can be modified to reduce their antigenicity.

[0214] In addition to intact immunoglobulins (or their recombinant counterparts), immunoglobulin fragments or "binding fragments" that include the epitope-binding site (e.g., Fab', F(ab')2, single-chain variable fragment (scFv), diabody, minibody, nanobody, single-domain antibody (sdAb), or other fragments) can be used as the antibody portion in the present invention. Such antibody fragments can be generated from whole immunoglobulins by cleavage with ricin, pepsin, papain, or other proteases. Minimal immunoglobulins can be designed using recombinant immunoglobulin technology. For example, an "Fv" immunoglobulin for use in the present invention can be generated 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 form an α-helical or β-sheet motif). Nanobodies or single-domain antibodies can also be derived from alternative organisms such as camels, dromedaries, llamas, alpacas, or sharks. In some embodiments, the antibody can be a conjugate, such as a polyethylene glycolated antibody, a drug, a radioisotope, or a toxin conjugate. Monoclonal antibodies against a specific epitope or combination of epitopes will allow targeting and / or depletion of cell populations that express the marker. A variety of techniques can be used to screen cell populations that express the marker using monoclonal antibodies and include magnetic separation using antibody-coated magnetic beads, "panning" using antibodies attached to a solid matrix (i.e., a plate), and flow cytometry (e.g., U.S. Patent No. 5,985,660, which is hereby expressly incorporated by reference in its entirety).

[0215] As used herein, the term "humanized" in reference to a non-human (e.g., rodent or primate) antibody is a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof that contains the minimal sequence derived from the non-human immunoglobulin.

[0216] 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 is known in the art, the variable regions of the heavy and light chains each consist of four framework regions (FRs) connected by three complementarity-determining regions (CDRs), also known as hypervariable regions, and contribute to the formation of the antigen-binding site of the antibody. If variants of the subject variable region are desired, particularly substitutions in amino acid residues outside the CDR regions (i.e., in the framework regions), appropriate amino acid substitutions can be identified by comparing the subject variable region with the variable regions of other antibodies that contain CDR1 and CDR2 sequences in the same canonical class as the subject variable region, preferably conservative amino acid substitutions (Chothia and Lesk, J Mol Biol 196(4):901-917, 1987).

[0217] In some embodiments, the unambiguous delineation of the CDRs and the identification of the residues comprising the antibody binding site are accomplished by resolving the structure of the antibody and / or the structure of the antibody-ligand complex. In some embodiments, this can be accomplished by any of a variety of techniques known to those of skill in the art, such as X-ray crystallography. In some embodiments, a variety of analytical methods can be employed to identify or approximate the CDR regions. In some embodiments, a variety of analytical methods can be employed to identify or approximate the CDR regions. Examples of such methods include, but are not limited to, the Kabat definition, the Chothia definition, the IMGT method (Lefranc et al., 2003, Dev Comp Immunol. 27:55-77), computational programs such as Paratome (Kunik et al., 2012, Nucl Acids Res. W521-4), the AbM definition, and the conformational definition.

[0218] The Kabat definition is the 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 the Chothia definition takes into account the positions 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 set of integrated computer programs produced by the Oxford Molecular Group of the model antibody structure. 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 a knowledge database and ab initio methods to model the tertiary structure of antibodies from the primary sequence, such as those described by Samudrala et al., 1999, "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach", PROTEINS, Structure, Function and Genetics Suppl., 3:194-198. 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 method, herein called the "conformational definition" of CDR, the positions of CDRs can be identified as the residues that make an enthalpic contribution to antigen binding. See, e.g., Makabe et al., 2008, Journal of Biological Chemistry, 283:1156-1166. There are other CDR boundary definitions that may not strictly follow one of the above methods but will still overlap with at least a portion of the Kabat CDRs and can be shortened or lengthened although they do not significantly affect the prediction or experimental findings of antigen binding according to a particular residue or group of residues. As used herein, CDR can refer to CDRs defined by any method known in the art, including combinations of methods. The methods used herein can utilize CDRs defined according to any of these methods.In some embodiments containing more than one CDR, the CDRs can be defined according to any one of Kabat, Chothia, extended, IMGT, Paratome, AbM, and / or conformational definitions, or any combination of the foregoing. In some embodiments, the residue numbering of the variable region is 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).

[0219] As is known in the art, the "constant region" of an antibody refers to the constant region of an antibody light chain or the constant region of an antibody heavy chain, either alone or in combination.

[0220] As used herein, the term "compete" with respect to an antibody means that a first antibody or an antigen-binding portion thereof binds an epitope in a manner sufficiently similar to a second antibody or an antigen-binding portion thereof such that the binding of the first antibody to its cognate epitope in the presence of the second antibody is detectably reduced compared to the binding of the first antibody in the absence of the second antibody. An alternative scenario where the binding of the second antibody to its epitope is also detectably reduced in the presence of the first antibody may or may not be the case. That is, the first antibody can inhibit the binding of the second antibody to its epitope, while the second antibody does not inhibit the binding of the first antibody to its corresponding epitope. However, in the case where each antibody detectably inhibits the binding of the other antibody to its cognate epitope or ligand, whether to the same, greater, or lesser extent, the antibodies are said to "cross-compete" with each other for binding to their respective epitopes. The present invention encompasses competing and cross-competing antibodies. Regardless of the mechanism by which such competition or cross-competition occurs (e.g., steric hindrance, conformational change, or binding to a common epitope or a portion thereof), based on the teachings provided herein, one of ordinary skill in the art will recognize that such competing and cross-competing antibodies are encompassed and can be used in the methods disclosed herein.

[0221] An antibody that "specifically binds" or "preferentially binds" (used interchangeably herein) to an epitope is a well-understood term in the art, and methods for determining such specific or preferential binding are also well known in the art. A molecule is said to exhibit "specific binding" or "preferential binding" if it reacts or associates more frequently and / or more rapidly, and / or for a longer duration and / or with greater affinity with a particular cell or substance compared to the molecule with an alternative cell or substance. An antibody "specifically binds" or "preferentially binds" to a target if the antibody binding has greater affinity and / or avidity, and / or is more facile, and / or has a longer duration compared to the antibody binding to other substances. For example, an antibody that specifically or preferentially binds to a CFD epitope is an antibody that binds to that epitope with greater affinity and / or avidity, and / or more facilely, and / or for a longer duration compared to the antibody binding to other CFD epitopes or non-CFD epitopes. It can also be understood from reading this definition that, for example, an antibody (or portion or epitope) that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. Thus, "specific binding" or "preferential binding" does not necessarily require (although it can include) exclusive binding. Usually, but not necessarily, reference to binding means preferential binding.

[0222] As used herein, "substantially pure" refers to a material that is at least 50% pure (i.e., free of contaminants), more preferably at least 90% pure, more preferably at least 95% pure, still more preferably at least 98% pure, and most preferably at least 99% pure.

[0223] "Host cell" includes an individual cell or a cell culture that can be or has been a recipient of a vector for incorporation of a polynucleotide insert. Host cells include progeny of a single host cell, and due to natural, accidental or deliberate mutations, the progeny may not necessarily be identical to the original parental cell (in morphology or genomic DNA complement). Host cells include cells transfected in vivo with the polynucleotides of the present invention.

[0224] As is known in the art, the term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain. The "Fc region" can 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 generally defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxyl terminus. The numbering of residues in the Fc region is according to the EU index numbering in Kabat. 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 contains two constant domains, CH2 and CH3. As is known in the art, the Fc region can exist in dimer or monomer form.

[0225] As used herein, "vector" means a construct capable of delivering and preferably expressing one or more genes or sequences of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as producer cells.

[0226] As used herein, "expression control sequence" means a nucleic acid sequence that directs the transcription of a nucleic acid. The expression control sequence can be a promoter, such as a constitutive or inducible promoter, or an enhancer. The expression control sequence is operably linked to the nucleic acid sequence to be transcribed.

[0227] As used herein, "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" includes any material which, when combined with an active ingredient, permits the ingredient to retain its biological activity and which does not react with the immune system of the subject. Examples include, but are not limited to, any standard pharmaceutical carrier, such as phosphate buffered saline, water, emulsions such as oil / water emulsions, various types of wetting agents, detergents such as polysorbate 20 to prevent aggregation, and sugars such as sucrose as cryoprotectants. A preferred diluent for aerosol or parenteral administration is phosphate buffered saline (PBS) or normal (0.9%) saline. Compositions comprising such carriers are formulated by well-known conventional methods (see, e.g., Remington's Pharmaceutical Sciences, 18th Edition, A. Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990 and Remington, The Science and Practice of Pharmacy, 20th Edition, Mack Publishing, 2000).

[0228] As used herein, the term "k 结合 " refers to the rate constant for the binding of an antibody (or bioconjugate) to an antigen. Specifically, the rate constants (k 结合 and k 解离 ) and the equilibrium dissociation constant are measured using full-length antibodies and / or Fab antibody fragments (i.e., monovalent) and CFD.

[0229] As used herein, the term "k 解离 " refers to the rate constant for the dissociation of an antibody (or bioconjugate) from an antibody / antigen complex.

[0230] As used herein, the term "K D " refers to the equilibrium dissociation constant for an antibody-antigen (or bioconjugate-antigen) interaction.

[0231] As used herein, the term "treatment" (and well understood in the art) means a method for obtaining a beneficial or desired result, including a clinical result, in the condition of a subject. Beneficial or desired clinical results can include, but are not limited to, alleviating or improving one or more symptoms or conditions, reducing the severity of a disease, stabilizing (i.e., not worsening) the disease state, preventing the transmission or spread of a disease, delaying or slowing the progression of a disease, improving or alleviating the disease state, reducing disease recurrence and remission, whether partial or complete, whether detectable or undetectable. "Treatment" as used herein also includes prophylactic treatment. Treatment methods include administering to a subject a therapeutically effective amount of an active agent. The administration step can consist of a single administration or can include a series of administrations. The composition is administered to the subject in an amount and for a duration sufficient to treat the patient. The length of the treatment cycle depends on a variety of factors, such as the severity of the condition, the age and genetic characteristics of the patient, the concentration of the active agent, the activity of the composition used in the treatment or a combination thereof. It should also be understood that the effective dose of a reagent for treatment or prevention can be increased or decreased during the course of a particular treatment or prevention regimen. Dose changes can be effected and become apparent by standard diagnostic assays known in the art. In some embodiments, long-term administration may be required.

[0232] The term "administer" 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 micro-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, intra-arterial, intradermal, subcutaneous, intraperitoneal, intraventricular and intracranial. Other delivery modes include, but are not limited to, the use of liposomal formulations, intravenous infusions, transdermal patches, etc. By "co-administer" is meant administering the first compound described herein either immediately before or immediately after administering the second compound described herein.

[0233] As used herein, the term "therapeutic target" refers to a gene or gene product that, upon modulation of its activity, can provide modulation (e.g., by modulating expression, bioactivity, etc.) of a disease phenotype (e.g., fibrosis or cancer). As used throughout, "modulation" means an increase or decrease in the indicated phenomenon (e.g., modulation of bioactivity refers to an increase or a decrease in bioactivity).

[0234] As used interchangeably herein, the terms “cancer,” “tumor,” “neoplasm,” and “carcinoma” refer to cells that exhibit relatively autonomous growth such that they display an abnormal growth phenotype characterized by a significant loss of control of cell proliferation. In general, the cells of interest for detection or treatment in the present application include pre-cancerous (e.g., benign), malignant, pre-metastatic, metastatic, and non-metastatic cells. Detection of cancer cells is of particular interest. The term “normal,” as used in the context of “normal cells,” means cells that have an untransformed phenotype or display the morphology of non-transformed cells of the tissue type being examined. “Carcinoma phenotype” generally refers to any of the various biological phenomena that are characteristic of cancer cells and that can vary with cancer type. The carcinoma phenotype is typically identified by abnormalities in, for example, cell growth or proliferation (e.g., uncontrolled growth or proliferation), cell cycle regulation, cell motility, cell-cell interactions, or metastasis.

[0235] The term “tumor microenvironment” refers to the cellular environment in which a tumor exists, including tumor cells and surrounding blood vessels, immune cells, fibroblasts, bone marrow-derived inflammatory cells, lymphocytes, signaling molecules, and extracellular matrix.

[0236] The term “immune cell” refers to hematopoietic-derived cells that are 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 monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.

[0237] As used herein, 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 cytotoxicity), and activation of cytokine-responsive cells, such as macrophages. The term “activate an immune response” refers to enhancing the level of a T cell-mediated and / or B cell-mediated immune response using methods known to those of skill in the art. In some embodiments, the enhanced level 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%.

[0238] As used herein, the term “transforming growth factor β receptor” (TGF-β receptor) refers to a family of serine / threonine kinase receptors that are expressed on the cell surface and are specific for the protein transforming growth factor β (TGF-β). The interaction between TGF-β and the receptor triggers signaling pathways that are responsible for many functions, including but not limited to cell growth, differentiation (e.g., stem cells, immune cells), apoptosis, homeostasis, chemotaxis, inflammation, and immune cell activation.

[0239] As used herein, the term "fibrosis" refers to a medical condition in which hardening or scarring of a tissue or organ results from unregulated production of extracellular matrix such as collagen proteins. Fibrosis is associated with chronic inflammation, in which immune cells such as macrophages signal to fibroblasts to express extracellular matrix proteins in response. This signaling is achieved through pathways such as the TGF-β pathway, although there are also other profibrotic pathways. 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 arterial fibrosis, joint fibrosis, Crohn's disease, Dupuytren's contracture, keloid, mediastinal fibrosis, myelofibrosis, Peyronie's disease, nephrogenic systemic fibrosis, progressive massive fibrosis, retroperitoneal fibrosis, or systemic sclerosis.

[0240] The term " % w / w" or " % wt / wt" means a percentage expressed as the weight of a component or reagent multiplied by 100 and divided by the total weight of the composition.

[0241] In some embodiments, methods of inducing immune activation are disclosed herein, including contacting an anti-Gal3 antibody with a plurality of cells including Gal3-expressing cells and TIM-3-expressing cells. In some embodiments, methods of reducing fibrosis are disclosed herein, including contacting a tissue including Gal3-expressing cells and at least one fibrosis biomarker with an anti-Gal3 antibody for a sufficient time to reduce the expression of at least one fibrosis biomarker in the tissue. In some embodiments, the anti-Gal3 antibody results in a reduction in the accumulation of one or more extracellular matrix proteins in the tissue, including but not limited to collagen.

[0242] In some cases, after binding to an anti-Gal3 antibody, Gal3-expressing cells express cytokines that induce immune activation. In some cases, the cytokine is 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 of an isotype antibody. In some cases, the IFNγ production is 150% of the IFNγ production of an isotype antibody. In some cases, the IFNγ production is 160% of the IFNγ production of an isotype antibody. In some cases, the IFNγ production is 170% of the IFNγ production of an isotype antibody. In some cases, the IFNγ production is 180% of the IFNγ production of an isotype antibody. In some cases, the IFNγ production is 190% of the IFNγ production of an isotype antibody. In some cases, the IFNγ production is 200% of the IFNγ production of an isotype antibody. In some cases, the IFNγ production is greater than 200% of the IFNγ production of an isotype antibody. In some cases, the IFNγ production is greater than 300% of the IFNγ production of an isotype antibody. In some cases, the IFNγ production is greater than 400% of the IFNγ production of an isotype antibody. In some cases, the IFNγ production is greater than 500% of the IFNγ production of an isotype antibody. In some cases, the cytokine is interleukin. In some cases, the interleukin is IL-2.

[0243] In some cases, immune activation includes the 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 the proliferation of CD3+ T lymphocytes. In certain cases, immune activation includes the proliferation of CD4+ T helper cells. In certain cases, immune activation includes the proliferation of CD8+ cytotoxic T cells. In certain cases, immune activation includes the proliferation of NK cells. In certain cases, immune activation includes the proliferation of T cells and NK cells.

[0244] In some cases, immune activation includes an increase in multiple intracellular M1 macrophage populations. In some cases, immune activation includes a decrease in multiple intracellular M2 macrophage populations. In some cases, immune activation includes an increase in multiple intracellular M1 macrophage populations and a decrease in multiple intracellular M2 macrophage populations.

[0245] In some cases, an anti-Gal3 antibody binds to Gal3 and disrupts the interaction between Gal3 and TIM-3. In some cases, disruption of the interaction between Gal3 and TIM-3 includes partial inhibition of the interaction between Gal3 and TIM-3. In some cases, disruption of the interaction between Gal3 and TIM-3 includes complete inhibition of the interaction between Gal3 and TIM-3. In some cases, the Gal3-TIM-3 interaction is reduced to 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 to less than 70%. In some cases, the Gal3-TIM-3 interaction is reduced to less than 60%. In some cases, the Gal3-TIM-3 interaction is reduced to less than 59%. In some cases, the Gal3-TIM-3 interaction is reduced to less than 50%. In some cases, the Gal3-TIM-3 interaction is reduced to less than 40%. 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%.

[0246] 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, wherein the residue positions correspond 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, wherein the residue positions correspond 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, wherein the residue positions correspond 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, wherein the residue positions correspond 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, wherein 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, wherein 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, wherein 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, wherein the 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, wherein the residue positions correspond to positions 163-171 of SEQ ID NO:1.

[0247] 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, wherein 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, wherein 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, wherein 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, wherein the residue positions 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, wherein 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 107-117, wherein the 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 region 96-102, wherein the residue positions correspond 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 region 100-106, wherein the residue positions correspond to positions 100-106 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 92-119, wherein the residue positions correspond to positions 92-119 of SEQ ID NO:2. In some cases, TIM-3 is human TIM-3.

[0248] In some embodiments, methods for promoting the proliferation of T cells or natural killer (NK) cells are disclosed herein, including contacting a plurality of cells including T cells, NK cells, and Gal3-expressing cells with an anti-Gal3 antibody for a sufficient time to promote the proliferation of T cells or NK cells in the plurality of cells. In some embodiments, methods for promoting the proliferation of T cells and natural killer (NK) cells are disclosed herein, including contacting a plurality of cells including T cells, NK cells, and Gal3-expressing cells with an anti-Gal3 antibody for a sufficient time to promote the proliferation of T cells and NK cells in the plurality of cells. In some embodiments, the plurality of cells further includes TIM-3-expressing cells. In some embodiments, the anti-Gal3 antibody binds to Gal3 and disrupts the interaction between Gal3 and TIM-3. In some embodiments, the anti-Gal3 antibody binds to Gal3 and disrupts the interaction between Gal3 and TIM-3. In some embodiments, the anti-Gal3 antibody binds to Gal3 and disrupts the interaction between Gal3 and TIM-3 by greater than 25%, greater than 50%, greater than 100%, or greater than 200%.

[0249] In some embodiments, the plurality of cells further includes tumor infiltrating lymphocytes (TIL). In some cases, the plurality of cells further includes CD3+ T lymphocytes, CD4+ T helper cells, CD8+ cytotoxic T cells, or a combination thereof. In some cases, the plurality of cells further includes CD3+ T lymphocytes. In some cases, the plurality of cells further includes CD4+ T helper cells. In some cases, the plurality of cells further includes CD8+ cytotoxic T cells. In some cases, the plurality of cells further includes CD3+ T lymphocytes and CD4+ T helper cells. In some cases, the plurality of cells further includes CD3+ T lymphocytes and CD8+ cytotoxic T cells. In some cases, the plurality of cells further includes CD4+ T helper cells and CD8+ cytotoxic T cells. In some cases, the plurality of cells further includes CD3+ T lymphocytes, CD4+ T helper cells, and CD8+ cytotoxic T cells.

[0250] In some embodiments, the contact further induces TIL proliferation. In some cases, the contact further induces the proliferation of CD3+ T lymphocytes, CD4+ T helper cells, CD8+ cytotoxic T cells, or a combination thereof. In some cases, the contact further induces the proliferation of CD3+ T lymphocytes. In some cases, the contact further induces the proliferation of CD4+ T helper cells. In some cases, the contact further induces the proliferation of CD8+ cytotoxic T cells. In some cases, the contact further induces the proliferation of CD3+ T lymphocytes and CD4+ T helper cells. In some cases, the contact further induces the proliferation of CD3+ T lymphocytes and CD8+ cytotoxic T cells. In some cases, the contact further induces the proliferation of CD4+ T helper cells and CD8+ cytotoxic T cells. In some cases, the contact further induces the proliferation of CD3+ T lymphocytes, CD4+ T helper cells, and CD8+ cytotoxic T cells.

[0251] In some embodiments, the contact further includes an increase in M1 macrophage proliferation. In some embodiments, the contact further includes a decrease in the population of M2 macrophages in the TME. In some embodiments, the contact further includes an increase in M1 macrophage proliferation and a decrease in the population of M2 macrophages in the TME.

[0252] In some embodiments, the anti-Gal3 antibody binds to at least one amino acid residue within the Gal3 region corresponding to residues 1-20 of SEQ ID NO:1. In some cases, the anti-Gal3 antibody binds to at least one amino acid residue within the Gal3 region corresponding to residues 41-91 of SEQ ID NO:1. In some cases, the anti-Gal3 antibody binds to at least one amino acid residue within the Gal3 region corresponding to residues 41-71 of SEQ ID NO:1. In some cases, the anti-Gal3 antibody binds to at least one amino acid residue within the Gal3 region corresponding to residues 71-91 of SEQ ID NO:1. In some cases, the anti-Gal3 antibody binds to at least one amino acid residue within peptide_1, peptide_4, peptide_5, peptide_6, peptide_7, or peptide_8. In some cases, the anti-Gal3 antibody binds to at least one amino acid residue within peptide_1. In some cases, the anti-Gal3 antibody binds to at least one amino acid residue within peptide_4. In some cases, the anti-Gal3 antibody binds to at least one amino acid residue within peptide_5. In some cases, the anti-Gal3 antibody binds to at least one amino acid residue within peptide_6. In some cases, the anti-Gal3 antibody binds to at least one amino acid residue within peptide_7. In some cases, the anti-Gal3 antibody binds to at least one amino acid residue within peptide_8.

[0253] In some embodiments, the anti-Gal3 antibody comprises a binding affinity (K) of 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, or less than 30 nM D) In some embodiments, the anti-Gal3 antibody comprises a K of less than 1 nM D In some embodiments, the anti-Gal3 antibody comprises a K of less than 1.2 nM D In some embodiments, the anti-Gal3 antibody comprises a K of less than 2 nM D In some embodiments, the anti-Gal3 antibody comprises a K of less than 5 nM D In some embodiments, the anti-Gal3 antibody comprises a K of less than 10 nM D In some embodiments, the anti-Gal3 antibody comprises a K of less than 13.5 nM D In some embodiments, the anti-Gal3 antibody comprises a K of less than 15 nM D In some embodiments, the anti-Gal3 antibody comprises a K of less than 20 nM D In some embodiments, the anti-Gal3 antibody comprises a K of less than 25 nM D In some embodiments, the anti-Gal3 antibody comprises a K of less than 30 nM D

[0254] In some embodiments, the anti-Gal3 antibody comprises a humanized antibody. In other embodiments, the anti-Gal3 antibody comprises a chimeric antibody. In some cases, the anti-Gal3 antibody comprises a full-length antibody or a binding fragment thereof. In some cases, the anti-Gal3 antibody comprises a bispecific antibody or a binding fragment thereof. In some cases, the anti-Gal3 antibody comprises a monovalent Fab’, bivalent Fab2, single-chain variable fragment (scFv), diabody, minibody, nanobody, single-domain antibody (sdAb), or camelized antibody or a binding fragment thereof.

[0255] ​In some embodiments, the anti-Gal3 antibody is 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), enzyme-linked immunosorbent assay, bispecific engagement of T cell receptor-based antibodies (BEAT), bispecific T cell engager (BiTE), biclone, Fab-scFv-Fc, two-in-one / dual action Fab (DAF), FinomAb, scFv-Fc-(Fab)-fusion, docking and locking (DNL), Adaptir (previously SCORPION), tandem diabody (TandAb), bispecific affinity retargeting (DART), nanobody, trispecific, tandem scFv (taFv), trispecific, tandem dAb / VHH, trispecific dAb / VHH or tetravalent dAb / VHH. In some cases, the anti-Gal3 antibody is a bispecific antibody or a binding fragment thereof, which includes the bispecific antibody formats illustrated in Brinkmann and Kontermann, “The making of bispecific antibodies,” MABS 9(2):182-212(2017) Figure 2 of the bispecific antibody formats.

[0256] In some embodiments, the anti-Gal3 antibody includes a framework region selected from IgM, IgG (e.g., IgG1, IgG2, IgG3 or IgG4), IgA or IgE. In some cases, the anti-Gal3 antibody includes an IgM framework. In some cases, the anti-Gal3 antibody includes an IgG (e.g., IgG1, IgG2, IgG3 or IgG4) framework. In some cases, the anti-Gal3 antibody includes an IgG1 framework. In some cases, the anti-Gal3 antibody includes an IgG2 framework. In some cases, the anti-Gal3 antibody includes an IgG4 framework. In some embodiments, the anti-Gal3 antibody may further include an Fc mutation. In some embodiments, Figure 37 any one or more of the Fc region or κ region in Figures 35A - 36B can be paired with any CDR, VH / VL sequence herein, including

[0257] In some embodiments, the anti-Gal3 antibody comprises one or more mutations in the framework region, e.g., in the CH1 domain, CH2 domain, CH3 domain, hinge region, or a combination thereof. In some cases, one or more mutations modulate Fc receptor interaction, e.g., to increase Fc effector functions such as ADCC and / or complement-dependent cytotoxicity (CDC). In some cases, one or more mutations stabilize the antibody and / or increase the half-life of the antibody. In other cases, one or more mutations modulate glycosylation.

[0258] In some embodiments, the Fc region comprises one or more mutations that modulate Fc receptor interaction, e.g., to enhance effector functions such as 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 the Kabat numbering (EU index of Kabat et al. 1991 Sequences of Proteins of Immunological Interest). In some embodiments, one or more mutations comprise S228P, S239D, K326W, A330L, I332E, E333A, E333S, or a combination thereof. In some cases, one or more mutations comprise S228P, S239D, I332E, or a combination thereof. In some cases, one or more mutations comprise S228P, S239D, A330L, I332E, or a combination thereof. In some cases, one or more mutations comprise K326W, E333S, or a combination thereof. In some cases, the mutation comprises 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.

[0259] In some embodiments, the anti-Gal3 antibody comprises a humanization score that quantifies the overall sequence similarity of the humanized antibody compared to an IMGT-selected germline antibody. In some embodiments, the anti-Gal3 antibody comprises a humanization score that quantifies the overall sequence similarity of the humanized antibody compared to an IMGT-curated germline antibody. In some embodiments, the anti-Gal3 antibody comprises a humanization score of 70 or greater, 80 or greater, 81 or greater, 82 or greater, 83 or greater, 84 or greater, 85 or greater, 86 or greater, 87 or greater, 88 or greater, 89 or greater, 90 or greater, or 95 or greater. In some embodiments, the anti-Gal3 antibody comprises a humanization score of 80 or greater. In some embodiments, the anti-Gal3 antibody comprises a humanization score of 83 or greater. In some embodiments, the anti-Gal3 antibody comprises a humanization score of 85 or greater. In some embodiments, the anti-Gal3 antibody comprises a humanization score of 87 or greater. In some embodiments, the anti-Gal3 antibody comprises a humanization score of 90 or greater. In some cases, the anti-Gal3 antibody comprises a humanization score of 70 or greater, 80 or greater, 81 or greater, 82 or greater, 83 or greater, 84 or greater, 85 or greater, 86 or greater, 87 or greater, 88 or greater, 89 or greater, 90 or greater, or 95 or greater, optionally 80 or greater, 85 or greater, or 87 or greater for the heavy chain. In some cases, the anti-Gal3 antibody comprises a humanization score of 70 or greater, 80 or greater, 81 or greater, 82 or greater, 83 or greater, 84 or greater, 85 or greater, 86 or greater, 87 or greater, 88 or greater, 89 or greater, 90 or greater, or 95 or greater, optionally 80 or greater, 83 or greater, or 85 or greater for the light chain.

[0260] In some embodiments, the anti-Gal3 antibody comprises complementarity determining regions (CDRs) as provided herein. In some embodiments, the CDR is part of the heavy chain (VH) of the antibody. In some embodiments, the CDR is part of the light chain (VL) of the antibody. In some embodiments, VH comprises VH CDR1, VH CDR2, and / or VH CDR3. In some embodiments, VH CDR1 comprises one of the sequences of SEQ ID NO: 37-64. In some embodiments, VH CDR2 comprises one of the sequences of SEQ ID NO: 65-92. In some embodiments, VH CDR3 comprises one of the sequences of SEQ ID NO: 93-120. In some embodiments, VL comprises VL CDR1, VL CDR2, and / or VL CDR3. In some embodiments, VL CDR1 comprises one of the sequences of SEQ ID NO: 121-148. In some embodiments, VL CDR2 comprises one of the sequences of SEQ ID NO: 149-176. In some embodiments, VL CDR3 comprises one of the sequences of SEQ ID NO: 177-204. In some embodiments, VH comprises one of the sequences of SEQ ID NO: 205-232. In some embodiments, VL comprises one of the sequences of SEQ ID NO: 233-260. In some embodiments, the anti-Gal3 antibody comprises an hIgG4 constant region. In some embodiments, the hIgG4 constant region comprises an hIgG4 constant region sequence within SEQ ID NO: 261, 263, 265, or 267. In some embodiments, the anti-Gal3 antibody comprises an hκ constant region. In some embodiments, the hκ constant region comprises an hκ constant region sequence within SEQ ID NO: 262, 264, 266, or 268.

[0261] In some embodiments, the anti-Gal3 comprises Figure 35A-B, the sequences depicted in 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 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 1, 2, or 3 HCDRs from mIMT001 and / or 4A11.2B5. In some embodiments, the anti-Gal3 antibody comprises 1, 2, or 3 LCDRs from mIMT001 and / or 4A11.2B5. In some embodiments, the anti-Gal3 antibody comprises 1, 2, or 3 HCDRs from mIMT001 and / or 4A11.2B5 and 1, 2, or 3 LCDRs from mIMT001 and / or 4A11.2B5. In some embodiments, the anti-Gal3 antibody comprises 1, 2, or 3 HCDRs from mIMT001 and / or 4A11.2B5 and 1, 2, or 3 LCDRs from mIMT001 and / or 4A11.2B5, optionally having 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions therein.In some embodiments, the anti-Gal3 antibody comprises 1, 2, or 3 HCDRs from mIMT001 and / or 4A11.2B5 and 1, 2, or 3 LCDRs from mIMT001 and / or 4A11.2B5, and further comprises the mIMT001 and / or 4A11.2B5 VH and VL sequences (such as. Figure 36A and 36B shown therein) or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH and VL sequences.

[0262] 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 1, 2, or 3 HCDRs from any one of IMT001-4, IMT006-1, IMT006-5, and / or IMT006-8. In some embodiments, the anti-Gal3 antibody comprises 1, 2, or 3 LCDRs from any one of IMT001-4, IMT006-1, IMT006-5, and / or IMT006-8. In some embodiments, the anti-Gal3 antibody comprises 1, 2, or 3 HCDRs from any one of IMT001-4, IMT006-1, IMT006-5, and / or IMT006-8 and 1, 2, or 3 LCDRs from any one of IMT001-4, IMT006-1, IMT006-5, and / or IMT006-8. In some embodiments, the anti-Gal3 antibody comprises 1, 2, or 3 HCDRs from any one of IMT001-4, IMT006-1, IMT006-5, and / or IMT006-8 and 1, 2, or 3 LCDRs from any one of IMT001-4, IMT006-1, IMT006-5, and / or IMT006-8, alternatively having 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions therein. In some embodiments, the anti-Gal3 antibody comprises 1, 2, or 3 HCDRs from any one of IMT001-4, IMT006-1, IMT006-5, and / or IMT006-8 and 1, 2, or 3 LCDRs from any one of IMT001-4, IMT006-1, IMT006-5, and / or IMT006-8, and further comprises any one of the IMT001-4, IMT006-1, IMT006-5, and / or IMT006-8 VH and VL sequences (such as Figure 36A and36B sequences that are shown therein or sequences that are at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to the VH and VL sequences.

[0263] In some embodiments, the anti-GAL3 antibody competes for binding with one or more of the following: 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).

[0264] In some embodiments, the anti-Gal3 antibody comprises at least Figures 35A - 36B the HCDR3 within any of the antibodies of. In some embodiments, the anti-GAL3 antibody further comprises Figures 35A - 36B all 3 HCDRs within any of the antibodies of. In some embodiments, the anti-GAL3 antibody further comprises Figures 35A - 36B all 3 LCDRs within any of the antibodies of.

[0265] In some embodiments, the anti-Gal3 antibody comprises Figure 36A any one of the inner heavy chain sequences, or a sequence that is at least 80% identical thereto, such as a sequence that is 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical.

[0266] In some embodiments, the anti-Gal3 antibody comprises Figure 36B any one of the inner light chain sequences, or a sequence that is at least 80% identical thereto, such as a sequence that is 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical. In some embodiments, the anti-GAL3 antibody further comprises Figure 36A any one of the inner heavy chain sequences, or a sequence that is at least 80% identical thereto, such as a sequence that is 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical.

[0267] In some embodiments, the anti-Gal3 antibody comprises 6 CDRs, and in their combined sequences, the 6 CDRs have at least 80% identity with any 6 CDR set within Figure 35A and 35B , such as 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity.

[0268] In some embodiments, the anti-Gal3 antibody comprises at least one CDR from Figure 38 (with 1, 2 or 3 conservative amino acid substitutions). The anti-GAL3 antibody comprises at least two CDRs from Figure 38 (with 1, 2 or 3 conservative amino acid substitutions). The anti-GAL3 antibody comprises at least three CDRs from Figure 38 (with 1, 2 or 3 conservative amino acid substitutions) (with 1, 2 or 3 conservative amino acid substitutions). The anti-GAL3 antibody comprises at least four CDRs from Figure 38 (with 1, 2 or 3 conservative amino acid substitutions). The anti-GAL3 antibody comprises at least five CDRs from Figure 38 (with 1, 2 or 3 conservative amino acid substitutions). The anti-GAL3 antibody comprises six CDRs from Figure 38 (with 1, 2 or 3 conservative amino acid substitutions). In some embodiments, the anti-Gal3 antibody comprises six CDRs from Figure 38 , and wherein said six are from a single bin. In some embodiments, the anti-Gal3 antibody comprises six CDRs from Figure 38 , or in their entire sequences, a set of 6 CDRs having at least 80% identity therewith.

[0269] In some embodiments, it is a method of inducing immune activation, which comprises, consists essentially of, or consists of: contacting a plurality of cells comprising 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 wherein the Gal3-expressing cells express cytokines that induce immune activation upon binding to the antibody. In some embodiments, the cytokine is interferon or interleukin. In some embodiments, the cytokine is IFNγ or IL-2. In some embodiments, immune activation comprises the proliferation of CD3+ T lymphocytes, CD4+ T helper cells, CD8+ cytotoxic T cells, natural killer cells, or a combination thereof. In some embodiments, it is a method of promoting T cell or NK cell proliferation, which comprises, consists essentially of, or consists of: contacting a plurality of cells comprising T cells, NK cells, and Gal3-expressing cells with an antibody under conditions that achieve the proliferation of T cells and / or NK cells in the plurality of cells, wherein the antibody specifically binds to Gal3. In some embodiments, it is a method of inducing immune activation, which comprises, consists essentially of, or consists of: contacting a plurality of cells comprising 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 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%. In some embodiments, it is a method of reducing fibrosis or its tendency in a tissue, which comprises, consists essentially of, or consists of: contacting the tissue with an antibody that specifically binds to an anti-Gal3 antibody under conditions that reduce the expression level of a fibrosis biomarker in the tissue. In some embodiments, it is an anti-Gal3 antibody for 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 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 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 not 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 not 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.

[0270] Regarding the properties of various antibodies, note that IMT001-4, IMT006-1, and IMT006-5 are humanized antibodies. mIMT001 is a murine antibody derived from IMT001. 4A11.2B5 is an original murine antibody derived from IMT006-1 and IMT006-5. 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, 9H2.2H10 are all murine antibodies. IMT001-4, IMT006-1, IMT006-5, and IMT006-8 are all humanized antibodies.

[0271] Treatment methods

[0272] In some embodiments, methods of inducing immune activation are disclosed herein, comprising, consisting essentially of, or consisting of: contacting a plurality of cells comprising Gal3-expressing cells and TIM-3 expressing cells, consisting essentially of Gal3-expressing cells and TIM-3 expressing cells, or consisting of Gal3-expressing cells and TIM-3 expressing cells with an antibody under conditions that disrupt the interaction between Gal3 and TIM-3. In some embodiments, the antibody is an anti-Gal3 antibody.

[0273] In some embodiments, methods of reducing fibrosis are disclosed herein, which comprise contacting a tissue comprising Gal3-expressing cells and at least one fibrosis biomarker with an anti-Gal3 antibody for a sufficient time to reduce the expression of at least one fibrosis biomarker in the tissue. In some cases, the anti-Gal3 antibody results in a decrease in the accumulation of one or more extracellular matrix proteins in the tissue, including but not limited to collagen.

[0274] 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.

[0275] In some embodiments, the anti-Gal3 antibody inhibits or disrupts the interaction between Gal3 and TIM-3. In some embodiments, the Gal3-TIM-3 interaction is reduced to 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 27%, 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%.

[0276] In some embodiments, the anti-Gal3 antibody does not inhibit or disrupt the interaction between Gal3 and TIM-3.

[0277] In some embodiments, the interaction occurs at one or more residues of Gal3 selected from regions 145-168, 160-177 or 165-184, wherein 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, wherein 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, wherein 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, wherein the residue positions herein correspond to positions 91-111, 107-117, 96-102, 100-106 or 92-119 of SEQ ID NO:2.

[0278] In some embodiments, Gal3-expressing cells express cytokines that induce immune activation upon binding to an antibody. As used herein, the term "cytokine" refers to small proteins, polypeptides, or peptides that participate in cell signaling. Cytokines include, but are not limited to, 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, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, 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, 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.

[0279] In some embodiments, the cytokine is an interferon. In some embodiments, the interferon is IFNγ. In some embodiments, the IFNγ production induced by the antibody 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.

[0280] In some embodiments, immune activation or activation of the immune system comprises, consists essentially of, or consists of: proliferation of CD3+ T lymphocytes, CD4+ T helper cells, CD8+ cytotoxic T cells, TFH cells, Th3 cells, Th17 cells, natural killer T (NKT) cells, or natural killer (NK) cells, or a combination thereof. In some embodiments, immune activation or activation of the immune system comprises, consists essentially of, or consists of: promoting T cell or NK cell proliferation. In some embodiments, immune activation or activation of the immune system comprises, consists essentially of, or consists of: an increase in populations of multiple intracellular M1 macrophages, neutrophils, mast cells, eosinophils, basophils, or dendritic cells. In some embodiments, immune activation or activation of the immune system comprises, consists essentially of, or consists of: a decrease in populations of multiple intracellular M2 macrophages.

[0281] In some embodiments, TIM-3 is human TIM-3.

[0282] In some embodiments, the multiple cells comprise, consist essentially of, or consist of: tumor cells. In some embodiments, the multiple cells are within the tumor microenvironment (TME) and comprise, consist essentially of, or consist of: tumor cells and immune cells. In some embodiments, the TME comprises tumor cells, immune cells, cancer-associated fibroblasts, myeloid-derived suppressor factor cells, neutrophils, tumor-infiltrating lymphocytes (TILs), or any combination thereof. In some embodiments, the multiple cells comprise, consist essentially of, or consist of: CD3+ T lymphocytes, CD4+ T helper cells, CD8+ cytotoxic T cells, TFH 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 in tumor cells within the TME.

[0283] In some embodiments, the antibody binds to at least one amino acid residue within the Gal3 region corresponding to residues 1-20 of SEQ ID NO:1. In some embodiments, the antibody binds to at least one amino acid residue within the Gal3 region corresponding to residues 41-91 of SEQ ID NO:1. In some embodiments, the antibody binds to at least one amino acid residue within the Gal3 region corresponding to residues 41-71 of SEQ ID NO:1. In some embodiments, the antibody binds to at least one amino acid residue within the Gal3 region corresponding to residues 71-91 of SEQ ID NO:1.

[0284] In some embodiments, the antibody binds to at least one amino acid residue within 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.

[0285] In some embodiments, the antibody comprises a K of 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, 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, less than 10 μM, or less than 100 μM D .

[0286] In some embodiments, the antibody comprises a humanized antibody. In some embodiments, the antibody comprises a full-length antibody or a binding fragment thereof. In some embodiments, the antibody comprises a bispecific antibody or a binding fragment thereof. In some embodiments, the antibody comprises a monovalent Fab’, bivalent Fab2, single-chain variable fragment (scFv), diabody, minibody, nanobody, single-domain antibody (sdAb), or camelized antibody, or a binding fragment thereof. In some embodiments, the antibody comprises an IgG framework. In some embodiments, the antibody comprises an IgG1, IgG2, or IgG4 framework. In some embodiments, the antibody further comprises an Fc mutation. In some embodiments, the antibody comprises a chimeric antibody.

[0287] 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 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 competes for binding to Gal3 with one or more of these antibodies, including any one of IMT001-4, IMT006-1, IMT006-5, and / or IMT006-8. In some embodiments, the antibody is one or more of the following: IMT001-4, IMT006-1, IMT006-5, or IMT006-8. In some embodiments, the antibody used in the method comprises one or more CDRs from one or more of the following: IMT001-4, IMT006-1, IMT006-5, or IMT006-8. In some embodiments, the antibody used in the method comprises one or more VH, VL, or VH and VL from one or more of the following: IMT001-4, IMT006-1, IMT006-5, or IMT006-8.

[0288] In some embodiments, the method further comprises administering an anti-Gal3 antibody to the subject prior to the contacting step.

[0289] In some embodiments, the subject is diagnosed with cancer.

[0290] In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is breast cancer, colorectal cancer, kidney 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).

[0291] In some embodiments, the cancer is a hematological malignancy, including but not limited to leukemia, non-Hodgkin lymphoma, Hodgkin lymphoma, multiple myeloma, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, acute monocytic leukemia, or any combination thereof.

[0292] In some embodiments, the cancer is a metastatic cancer. In some embodiments, the cancer is a recurrent cancer or a refractory cancer. The staging of a cancer or tumor is used to determine the progression of the spread of the cancer or tumor within a patient. The accepted standard for solid tumor classification is the TNM classification standard, which differentiates tumors based on the size of the tumor (T), the extent of spread to lymph nodes (N), and metastasis (M). These classifications are further divided into stages, where stage 0 growth is non-malignant, locally contained stage I and II tumors, stage III tumors have spread to nearby lymph nodes, and stage IV tumors have metastasized. Although the TNM standard is a widely used classification method, alternative or improved standards that represent the behavior of a specific cancer type can also be employed. Accordingly, although these standards can be used to determine progression, the prognosis of early or late stage cancer or tumor is independent of a specific classification.

[0293] In some embodiments, methods are disclosed herein for reducing fibrosis or the propensity thereof in a subject's tissue by contacting the tissue with an antibody. In some embodiments, the antibody specifically binds Gal3 or is an anti-Gal3 antibody. In some embodiments, the contacting induces the expression level of at least one fibrosis biomarker to be reduced in the tissue. In some embodiments, the tissue includes at least one TIM-3 expressing cell. In some embodiments, the anti-Gal3 antibody disrupts the interaction between Gal3 and TIM-3. In some embodiments, the anti-Gal3 antibody does not disrupt the interaction between Gal3 and TIM-3.

[0294] In some embodiments, reducing fibrosis or the propensity thereof in a tissue includes preventing fibrosis from occurring in normal tissue. In some embodiments, reducing fibrosis or the propensity thereof in a tissue includes slowing or halting the progression of fibrosis in fibrotic tissue. In some embodiments, reducing fibrosis or the propensity thereof in a tissue includes reducing the amount of the degree of fibrosis in fibrotic tissue. In some embodiments, reducing fibrosis or the propensity thereof in a tissue includes eliminating fibrosis in fibrotic tissue.

[0295] In some embodiments, methods are also described herein for monitoring the progression of tissue fibrosis by monitoring one or more fibrosis biomarkers. In some embodiments, methods for treating tissue fibrosis with an anti-Gal3 antibody are disclosed, wherein the anti-Gal3 antibody disrupts the interaction between Gal3 and TIM-3.

[0296] In some embodiments, at least one fibrosis biomarker comprises, consists essentially of, or consists of: alpha-smooth muscle actin (α-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 (α-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 (α-SMA) and fibronectin.

[0297] 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.

[0298] In some embodiments, the expression of at least one fibrosis biomarker in the tissue treated with the anti-Gal3 antibody is less than the expression of at least one fibrosis biomarker in the control tissue treated with the mIgG2b antibody.

[0299] In some embodiments, the anti-Gal3 antibody results in a decrease in the accumulation of extracellular matrix (ECM) proteins in tissues. In some embodiments, the extracellular matrix comprises, consists essentially of, or consists of: aggrecan, nestin, cadherin, clathrin, collagen, defensin, elastin, entactin, fibrillin, fibronectin, keratin, laminin, microtubule-actin cross-linking factor 1, SPARC-like protein, nesprin (nesprin-1, nesprin-2, nesprin-3), fibrous sheath interacting protein, intercalatedin, parietal actin, thrombospondin, integrin, talin, exportin, transporter, tenascin, orosin, sorting protein-related receptor, tensin, or titin, or any combination thereof. In some embodiments, the extracellular matrix protein comprises, consists essentially of, or consists of: collagen. In some embodiments, the tissue comprises, consists essentially of, or consists of: cells that produce collagen. In some embodiments, the collagen-producing cells are fibroblasts. In some embodiments, the fibroblasts are activated by fibrogenic cytokines. In some embodiments, the fibrogenic cytokine is TGF-β, TGF-β1, IL-1β, TNF-α, or GM-CSF. In some embodiments, the tissue has elevated fibrogenic cytokine expression.

[0300] In some embodiments, the antibody binds to at least one amino acid residue within the Gal3 region corresponding to residues 1-20 of SEQ ID NO:1. In some embodiments, the antibody binds to at least one amino acid residue within the Gal3 region corresponding to residues 41-91 of SEQ ID NO:1. In some embodiments, the antibody binds to at least one amino acid residue within the Gal3 region corresponding to residues 41-71 of SEQ ID NO:1. In some embodiments, the antibody binds to at least one amino acid residue within the Gal3 region corresponding to residues 71-91 of SEQ ID NO:1.

[0301] In some embodiments, the subject is diagnosed with a fibrotic disease or fibrosis. In some embodiments, the subject is 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.

[0302] In some embodiments, the fibrotic disease or fibrosis is hepatic fibrosis, bridging fibrosis, cirrhosis, renal (kidney) fibrosis, pulmonary fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, cardiovascular fibrosis, arterial fibrosis, venous thrombosis, joint fibrosis, 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 hepatic fibrosis.

[0303] In some embodiments, the method involves an antibody that binds to Gal3 but disrupts the interaction between Gal3 and TIM-3. This may be a direct obstruction of the interaction region between Gal3 and TIM-3, or an indirect alteration, such as binding that causes a conformational change in Gal3 such that it no longer binds to TIM-3 or is active with TIM-3. It can also be generated by binding to a first portion of Gal3, where some other portion of the antibody hinders or alters the interaction of Gal3 with TIM-3.

[0304] 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 for treating an immune-related disease. In some embodiments, the medicament or composition is for treating cancer. In some embodiments, the medicament or composition is for treating a fibrotic disease or fibrosis.

[0305] In some embodiments, it is an anti-Gal3 antibody for treating a disease in a subject. In some embodiments, the anti-Gal3 antibody inhibits the interaction between Gal3 and TIM-3. In some embodiments, the anti-Gal3 antibody does not inhibit the interaction between Gal3 and TIM-3.

[0306] In some embodiments, the anti-Gal3 antibody is for treating a disease, where the disease is immune-related, and where the anti-Gal3 antibody induces activation of the subject's immune system. In some embodiments, the immune-related disease is an autoimmune disease. In some embodiments, the immune-related disease is an immune deficiency. 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 includes the proliferation of CD3+ T lymphocytes, CD4+ T helper cells, CD8+ cytotoxic T cells, TFH cells, Th3 cells, Th17 cells, natural killer T (NKT) cells, NK cells, or M1 macrophages, or a combination thereof. In some embodiments, the activation of the immune system includes a decrease in M2 macrophages.

[0307] In some embodiments, an anti-Gal3 antibody is used to treat a disease, where the disease is cancer and an anti-TIM-3 antibody is used to treat cancer.

[0308] In some embodiments, an anti-Gal3 antibody is used to treat a disease, where the disease is a fibrotic disease or fibrosis. In some embodiments, an anti-Gal3 antibody is used to treat a disease that results in a decrease in the accumulation of extracellular matrix proteins in tissues.

[0309] In some embodiments, an anti-Gal3 antibody for treating a disease is administered in combination with another 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.

[0310] In some embodiments, the antibody is formulated for systemic administration. In some embodiments, the antibody is formulated for parenteral, subcutaneous, intramuscular, intradermal, or intravenous administration, or any combination thereof.

[0311] In some embodiments, an anti-Gal3 antibody is administered to a subject in combination with another therapeutic agent. In some embodiments, the other therapeutic agent includes an immunotherapeutic agent. In some embodiments, the other therapeutic agent includes an immune checkpoint modulator. In some embodiments, the other therapeutic agent includes a chemotherapeutic agent, a targeted therapeutic agent, a hormonal therapeutic agent, or a stem cell-based therapeutic agent.

[0312] In some embodiments, the additional therapeutic agent includes an immunotherapeutic agent. In some embodiments, the immunotherapy is adoptive cell therapy. Exemplary adoptive cell therapies include AFP TCR, MAGE-A10 TCR, or NY-ESO-TCR from Adaptimmune; ACTR087 / rituximab from Unum Therapeutics; anti-BCMA CAR-T cell therapy, anti-CD19 “armored” CAR-T cell therapy, JCAR014, JCAR018, JCAR020, JCAR023, JCAR024, or JTCR016 from Juno Therapeutics; JCAR017 from Celgene / Juno Therapeutics; anti-CD19 CAR-T cell therapy from Intrexon; anti-CD19 CAR-T cell therapy, axicabtagene ciloleucel, KITE-718, KITE-439, or NY-ESO-1 T-cell receptor therapy from Kite Pharma; anti-CEA CAR-T therapy from Sorrento Therapeutics; anti-PSMA CAR-T cell therapy from TNK Therapeutics / Sorrento Therapeutics; ATA520 from Atara Biotherapeutics; AU101 and AU105 from Aurora BioPharma; baltaleucel-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.

[0313] In some embodiments, the immunotherapy is dendritic cell-based therapy.

[0314] In some embodiments, immunotherapy includes cytokine-based therapies, including, for example, interleukins (IL) such as IL-2, IL-15, or IL-21; interferon (IFN)-α, or granulocyte macrophage colony-stimulating factor (GM-CSF).

[0315] In some embodiments, the immunotherapy includes immune checkpoint modulators. Exemplary immune checkpoint modulators include PD-1 modulators such as 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 (genolimzumab) from CBT Pharmaceuticals, INCSHR1210 from Incyte, JNJ-63723283 from Janssen Research & Development, MEDI0680 from MedImmune, MGA 012 from MacroGenics, PDR001 from Novartis Pharmaceuticals, PF-06801591 from Pfizer, REGN2810 (SAR439684) from Regeneron Pharmaceuticals / Sanofi, or TSR-042 from TESARO; CTLA-4 modulators such as ipilimumab (Yervoy) or AGEN 1884 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 capture) from EMD Serono; LAG3 modulators such as BMS-986016 from Bristol-Myers Squibb, IMP701 from Novartis Pharmaceuticals, LAG525 from Novartis 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 lerotilumab from Bristol-Myers Squibb; or TIM modulators such as MBG453 from Novartis Pharmaceuticals or TSR-022 from Tesaro.;

[0316] In some embodiments, the additional therapeutic agent includes a chemotherapeutic agent. Exemplary chemotherapeutic agents include, but are not limited to, alkylating agents such as cyclophosphamide, mechlorethamine, chlorambucil, melphalan, dacarbazine, or nitrosourea; anthracyclines such as daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, or valrubicin; cytoskeleton disruptors such as paclitaxel, docetaxel, Taxol, 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 talaporfin; kinase inhibitors such as bortezomib, erlotinib, gefitinib, imatinib, vemurafenib, or vismodegib; nucleotide analogs and precursor analogs such as azacitidine, azathioprine, capecitabine, cytarabine, capecitabine, fluorouracil, gemcitabine, hydroxyurea, mercaptopurine, methotrexate, or thioguanine; platinum-based agents such as carboplatin, cisplatin, or oxaliplatin; retinoids such as tretinoin, alitretinoin, or bexarotene; or vinca alkaloids and derivatives such as vinblastine, vincristine, vindesine, or vinorelbine.

[0317] In some embodiments, the additional therapeutic agent includes a hormone-based therapeutic agent. Exemplary hormone-based therapeutic agents include, but are not limited to, aromatase inhibitors such as letrozole, anastrozole, exemestane, or aminoglutethimide; gonadotropin-releasing hormone (GnRH) analogs such as leuprolide or goserelin; selective estrogen receptor modulators (SERMs) such as tamoxifen, raloxifene, toremifene, or fulvestrant; antiandrogens such as flutamide or bicalutamide; progesterones such as megestrol acetate or medroxyprogesterone acetate; androgens such as fluoxymesterone; estrogens such as diethylstilbestrol (DES), estradiol formulations, or estradiol valerate; or somatostatin analogs such as octreotide.

[0318] In some embodiments, the additional therapeutic agent is a first-line therapeutic agent.

[0319] 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 before the additional therapeutic agent. In some embodiments, the anti-Gal3 antibody is administered to the subject after the additional therapeutic agent.

[0320] In some embodiments, the additional therapeutic agent and the anti-Gal3 antibody are formulated as separate doses.

[0321] In some embodiments, the subject has undergone surgery. In some cases, the anti-Gal3 antibody and optionally the additional therapeutic agent are administered to the subject before surgery. In some embodiments, the anti-Gal3 antibody and optionally the additional therapeutic agent are administered to the subject after surgery.

[0322] In some embodiments, the subject has undergone radiation. In some embodiments, the anti-Gal3 antibody and optionally the additional therapeutic agent are administered to the subject during or after radiation therapy. In some cases, the anti-Gal3 antibody and optionally the additional therapeutic agent are administered to the subject before undergoing radiation.

[0323] In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0324] In some embodiments, methods are disclosed herein for reducing fibrosis or the propensity thereto in a tissue by contacting the tissue with an antibody that specifically binds to Gal3. In some embodiments, methods are also described herein for disrupting Gal3-TIM-3 interactions by an antibody that specifically binds to Gal3 under conditions that reduce the expression of one or more fibrosis biomarkers in the tissue.

[0325] In certain embodiments, methods of reducing fibrosis or its propensity in a tissue are disclosed herein, including: contacting the tissue with an antibody that specifically binds to an antibody against Gal3 under conditions that result in a reduced expression level of a fibrosis biomarker in the tissue. In some embodiments, the tissue further comprises TIM-3 expressing cells. In some embodiments, the antibody further disrupts the interaction between Gal3 and TIM-3. In some embodiments, the antibody does not disrupt the interaction between Gal3 and TIM-3. In some embodiments, at least one fibrosis biomarker comprises alpha-smooth muscle actin (α-SMA). In some embodiments, at least one fibrosis biomarker comprises fibronectin. In some embodiments, at least one fibrosis biomarker comprises alpha-smooth muscle actin (α-SMA) and fibronectin. In some embodiments, the tissue is renal tissue or hepatic tissue. In some embodiments, the tissue is selected from the group consisting of: hepatic tissue, renal tissue, skin tissue, lung tissue, heart tissue, brain tissue, intestinal tissue, bone marrow tissue, and soft tissue. In some embodiments, the expression of at least one fibrosis biomarker in the tissue treated with the antibody is less than the expression of 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 extracellular matrix proteins in the tissue. In some embodiments, the extracellular matrix protein comprises collagen. In some embodiments, the tissue comprises cells that produce collagen. In some embodiments, the cells that produce collagen 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 elevated TGF-β1 expression.

[0326] Antibody production

[0327] In some embodiments, anti-Gal3 antibodies are produced by injecting production animals with an antigen composition by standard protocols. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. When using an entire protein or a larger portion of a protein, antibodies can be produced by immunizing production animals with the protein and a suitable adjuvant (e.g., Freund's, Freund's complete, oil-in-water emulsion, etc.). When using a smaller peptide, it is advantageous to conjugate the peptide to a larger molecule to prepare an immunostimulatory conjugate. Commonly available conjugate proteins for this purpose include bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH). To produce antibodies against a specific epitope, peptides derived from the full sequence can be utilized. Alternatively, to produce antibodies against a relatively short peptide portion of a protein target, excellent immune responses can be elicited if the polypeptide is linked to a carrier protein, such as ovalbumin, BSA, or KLH.

[0328] Polyclonal or monoclonal anti-Gal3 antibodies can be produced from animals that have been genetically altered to produce human immunoglobulins. Transgenic animals can be produced by initially generating "knockout" animals that do not produce the animal's native antibodies and stably transforming the animals with human antibody loci (e.g., by using human artificial chromosomes). In such cases, only human antibodies are then produced by the animals. 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 be referred to as human xenogeneic antibodies.

[0329] Alternatively, anti-Gal3 antibodies can be produced from a phage library containing human variable regions. See U.S. Patent No. 6,174,708, which is incorporated herein by reference in its entirety.

[0330] In some aspects of some embodiments disclosed herein, anti-Gal3 antibodies are produced by hybridomas.

[0331] For monoclonal anti-Gal3 antibodies, hybridomas can be formed by isolating stimulated immune cells, such as those from the spleen of immunized animals. These cells can then be fused to immortal cells, such as myeloma cells or transformed cells, which are capable of replicating indefinitely in cell culture, thereby generating an immortalized immunoglobulin-secreting cell line. The immortal cell line utilized can be selected to be deficient in an enzyme necessary for the utilization of certain nutrients. Many such cell lines (such as myelomas) are known to those of skill in the art and include, for example: thymidine kinase (TK) or hypoxanthine-guanine phosphoribosyltransferase (HGPRT). These deficiencies allow selection of the fused cells based on their ability to grow, for example, on hypoxanthine aminopterin thymidine medium (HAT).

[0332] Alternatively, anti-Gal3 antibodies can be produced by genetic engineering.

[0333] The anti-Gal3 antibodies disclosed herein may have a reduced tendency to induce an undesired immune response in humans, such as, for example, anaphylactic shock, and may also exhibit a reduced tendency to elicit an immune response (such as, for example, a human anti-mouse antibody “HAMA” response) that would prevent repeated dosing with an antibody therapeutic or imaging agent. Such anti-Gal3 antibodies include, but are not limited to, humanized, chimeric, or xenogeneic human anti-Gal3 antibodies.

[0334] Chimeric anti-Gal3 antibodies can be prepared, for example, by recombinant means of combining murine variable light and heavy chain regions (VK and VH) obtained from a murine (or other animal source) hybridoma clone with human constant light and heavy chain regions so as to produce an antibody that predominantly has human domains. The production of such chimeric antibodies is well known in the art and can be achieved by standard means (as, for example, described in U.S. Patent No. 5,624,659, which is incorporated herein by reference in its entirety).

[0335] The term "humanized" as applied to non-human (e.g., rodent or primate) antibodies is a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof that contains minimal sequence derived from a non-human immunoglobulin. In most cases, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the complementarity-determining regions (CDRs) of the recipient are replaced by residues from the CDRs of a non-human species (donor antibody) such as mouse, rat, rabbit, or primate having the desired specificity, affinity, and capacity. In some embodiments, residues of the Fv framework regions (FRs) of the human immunoglobulin are replaced by the corresponding non-human residues. Additionally, humanized antibodies may include residues that are neither present in the recipient antibody nor in the input CDR or framework sequences. These modifications are made to further improve and optimize antibody performance and to minimize immunogenicity when introduced into humans. In some examples, a humanized antibody will include substantially all of at least one, and usually two, variable regions in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. A humanized antibody may also include at least a portion of the immunoglobulin constant region (Fc), usually those of a human immunoglobulin.

[0336] 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 accomplished by carefully examining the sequences of the hypervariable loops of the monoclonal antigen-binding unit or the variable regions of a monoclonal antibody and matching them to the structure of a human antigen-binding unit or a human antibody chain. See, e.g., U.S. Patent No. 6,187,287, which is incorporated herein by reference in its entirety.

[0337] Methods for humanizing non-human antibodies are well known in the art. A "humanized" antibody is one in which at least a portion of the sequence has been altered from its original form to make it more like a human immunoglobulin. In some versions, the heavy (H) and light (L) chain constant (C) regions are replaced with human sequences. This can be a fusion polypeptide that includes variable (V) regions and a heterologous immunoglobulin C region. In some versions, the complementarity-determining regions (CDRs) include non-human antibody sequences while the V framework regions have also been converted to human sequences. See, e.g., EP 0329400. In some versions, the V regions are humanized by designing a consensus sequence of human and mouse V regions and converting residues outside the different CDRs between the consensus sequences.

[0338] In principle, framework sequences from humanized antibodies can be used as templates for CDR grafting; however, it has been shown that directly substituting CDRs into such frameworks results in a significant loss of binding affinity for 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. The higher the homology of the human antibody (HuAb) to the original murine antibody (muAb), the less likely the human framework will introduce distortions that can reduce affinity into the murine CDRs. Based on sequence homology searches of antibody sequence databases, HuAb IC4 provides good framework homology for muM4TS.22, although other highly homologous HuAbs will also be suitable, particularly the κL chain from human subgroup I or the H chain from human subgroup III. Kabat et al. (1987). Various computer programs such as ENCAD (Levitt et al. (1983) J. Mol. Biol. 168:595) can be used to predict the optimal sequences for the V regions. The present invention thus encompasses HuAbs having different variable (V) regions. Determining suitable V region sequences and optimizing these sequences are within the skill of the art. Methods for obtaining antibodies with reduced immunogenicity are also described in U.S. Patent No. 5,270,202 and EP 699,755.

[0339] Humanized antibodies can be prepared by a process of analyzing the parental sequence and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are familiar to those skilled in the art. Computer programs are available to interpret and display the possible three-dimensional conformational structures of selected candidate immunoglobulin sequences. Examination of these displays allows analysis of the possible role of residues in the function of the candidate immunoglobulin sequence, i.e., analysis of the ability of the residues to affect the candidate immunoglobulin's ability to bind its antigen. In this way, FR residues can be selected and combined from consensus and input sequences to achieve desired antibody characteristics, such as increased affinity for the target antigen.

[0340] The process for humanizing a subject antigen-binding unit can be as follows. The most suitable germline receptor heavy and light chain variable regions are selected based on the homology, canonical structure, and physical properties of human antibody germlines for transplantation. In silico modeling of mVH / VL versus transplanted hVH / VL is performed, and a prototype humanized antibody sequence is generated. If the modeling indicates that framework back mutations are required, a second variant with the indicated FW changes is generated. DNA fragments encoding the selected germline frameworks and murine CDRs are synthesized. The synthesized DNA fragments are 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 antigen-binding affinity of the humanized antigen-binding unit is compared to that of the parental antigen-binding unit, for example, by FACS of cells expressing the target antigen. If the affinity is more than 2-fold lower than the parental antigen-binding unit, a second round of humanized variants can be generated and tested as described above.

[0341] As described above, an anti-Gal3 antibody can be "monovalent" or "multivalent". The former has one binding site per antigen-binding unit, while the latter contains multiple binding sites capable of binding more than one of the same or different types of antigens. Depending on the number of binding sites, the antigen-binding unit can be bivalent (having two antigen-binding sites), trivalent (having three antigen-binding sites), tetravalent (having four antigen-binding sites), etc.

[0342] Multivalent anti-Gal3 antibodies can be further classified based on their binding specificities. A "monospecific" anti-Gal3 antibody is a molecule capable of binding to one or more antigens of the same type. A "multispecific" anti-Gal3 antibody is a molecule having binding specificities for at least two different antigens. While such a molecule will typically only bind two different antigens (i.e., a bispecific anti-Gal3 antibody), as used herein, antibodies having additional specificities, such as trispecific antibodies, are also encompassed by this expression. The present disclosure further provides multispecific anti-Gal3 antibodies. A multispecific anti-Gal3 antibody is a multivalent molecule capable of binding at least two different antigens, for example, bispecific and trispecific molecules respectively exhibiting binding specificities for two and three different antigens.

[0343] Monoclonal antibodies can be obtained by injecting a mouse with a composition comprising an antigen, such as 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, selecting the positive clones that produce antibodies against the antigen, culturing the clones that produce antibodies against the antigen, and isolating the antibodies from the hybridoma culture.

[0344] The monoclonal antibodies generated can be isolated and purified from the hybridoma cultures by various well-established techniques. Such isolation techniques include affinity chromatography with protein-A agarose, size-exclusion chromatography, and ion-exchange chromatography. See, e.g., Coligan, at pages 2.7.1-2.7.12 and 2.9.1-2.9.3. See also Baines et al., "Purification of Immunoglobulin G (IgG)", in METHODS IN MOLECULAR BIOLOGY, Vol. 10, pages 79-104 (The Humana Press, Inc. 1992). After initially generating an antibody against a target protein, the antibody can be sequenced and subsequently prepared by recombinant techniques. Humanization and chimerization of murine antibodies and antibody fragments are well known to those skilled in the art. See, e.g., Leung et al., Hybridoma 13:469 (1994); US20140099254A1, each of which is hereby expressly incorporated by reference in its entirety.

[0345] Human antibodies can be generated using transgenic mice that have been genetically engineered to produce specific human antibodies in response to antigen challenge with the target protein. See Green et al., Nature Genet. 7:13 (1994), Lonberg et al., Nature 368:856 (1994). Human antibodies against the target protein can also be constructed by genetic or chromosomal transfection methods, phage display techniques, or in vitro activation of B cells. See, e.g., McCafferty et al., 1990, Nature 348:552-553; U.S. Patent Nos. 5,567,610 and 5,229,275, each of which is hereby expressly incorporated by reference in its entirety.

[0346] In some embodiments, the Gal3-TIM-3 interaction can be 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%.

[0347] Polynucleotides and vectors

[0348] In some embodiments, the present disclosure provides an isolated nucleic acid encoding any anti-Gal3 antibody disclosed herein. In some embodiments, the present disclosure provides a vector comprising a nucleic acid sequence encoding any anti-Gal3 antibody disclosed herein. In some embodiments, the present invention provides an isolated nucleic acid encoding the light chain CDR and heavy chain CDR of the anti-Gal3 antibody disclosed herein.

[0349] Subject anti-Gal3 antibodies can be prepared by recombinant DNA technology, synthetic chemical techniques, or combinations thereof. For example, using standard molecular techniques known in the art, the sequences encoding the desired components of the anti-Gal3 antibody, including the light chain CDR and the heavy chain CDR, are typically assembled and cloned into an expression vector. These sequences can be assembled from other vectors encoding the desired protein sequences, from fragments generated by PCR using the respective template nucleic acids, or by the assembly of synthetic oligonucleotides encoding the desired sequences. An expression system can be created by transfecting a suitable cell with an expression vector comprising the anti-Gal3 antibody of interest.

[0350] Using conventional techniques including, but not limited to, hybridization, PCR, and DNA sequencing, nucleotide sequences corresponding to various regions of the light or heavy chain of an existing antibody can be readily obtained and sequenced. Hybridoma cells that produce monoclonal antibodies are used as a preferred source of antibody nucleotide sequences. Hybridoma cells that produce a series of monoclonal antibodies in large quantities can 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 organs such as the spleen and peripheral blood lymphocytes are formed. Specific techniques applicable to the extraction and synthesis of antibody nucleotides are described in Orlandi et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86:3833-3837; Larrick et al. (1989) Biochem. Biophys. Res. Commun. 160:1250-1255; Sastry et al. (1989) Proc. Natl. Acad. Sci., U.S.A. 86:5728-5732, and U.S. Patent No. 5,969,108.

[0351] The polynucleotide encoding the anti-Gal3 antibody can also be modified, for example, by substituting the coding sequences of the human heavy and light chain constant regions for the homologous non-human sequences. In this way, a chimeric antibody is prepared that retains the binding specificity of the original anti-Gal3 antibody.

[0352] Host cells for antibody production

[0353] In some embodiments, the present disclosure provides host cells expressing any one of the anti-Gal3 antibodies disclosed herein. The subject host cells generally comprise 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 NS0 cell.

[0354] The present invention provides host cells transfected with the polynucleotides, vectors or vector libraries described above. The vector can be introduced into a suitable prokaryotic or eukaryotic cell by any of a number of suitable means, including electroporation, biolistic bombardment; lipofection, infection (wherein the vector is conjugated to a pathogen), transfection with calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran or other substances. The choice of method for introducing the vector will generally depend on the characteristics of the host cell.

[0355] For most animal cells, any of the above-mentioned methods are suitable for vector delivery. Preferred animal cells are vertebrate cells, preferably mammalian cells, which are capable of expressing an exogenously introduced gene product in large amounts, e.g., at the milligram level. Non-limiting examples of preferred cells are NIH3T3 cells, COS, HeLa and CHO cells.

[0356] Once introduced into a suitable host cell, the expression of the anti-Gal3 antibody can be determined using any nucleic acid or protein assay known in the art. For example, the presence of the transcription mRNA of the light chain CDR or heavy chain CDR or the anti-Gal3 antibody can be detected and / or quantified by conventional hybridization assays (e.g., Northern blot analysis), amplification procedures (e.g., RT-PCR), SAGE (U.S. Patent No. 5,695,937), and array-based techniques (see, e.g., U.S. Patent Nos. 5,405,783, 5,412,087, and 5,445,934), using a probe complementary to any region of the polynucleotide encoding the anti-Gal3 antibody.

[0357] The expression of the vector can also be determined by examining the expressed anti-Gal3 antibody. A variety of techniques for protein analysis are available in the art. They include, but are not limited to, radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), "sandwich" immunoassay, immunoradiometric assay, in situ immunoassay (using, e.g., colloidal gold, enzyme or radioisotope labeling), Western blot analysis, immunoprecipitation assay, immunofluorescence assay, and SDS-PAGE.

[0358] Payload

[0359] In some embodiments, the anti-Gal3 antibody further comprises a payload. In some cases, the payload comprises a small molecule, a protein or a functional fragment thereof, a peptide or a nucleic acid polymer.

[0360] In some cases, the number of payloads conjugated to an anti-Gal3 antibody (e.g., drug-to-antibody ratio or DAR) is about 1:1, one payload per 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.

[0361] In some embodiments, the payload is a small molecule. In some embodiments, the small molecule is a cytotoxin payload. Exemplary cytotoxin payloads include, but are not limited to, microtubule disruptors, DNA modifiers, or Akt inhibitors.

[0362] In some embodiments, the payload includes a microtubule disruptor. Exemplary microtubule disrupting agents include, but are not limited to, 2-methoxyestradiol, auristatin, chalcone, colchicine, combretastatin, cryptophycin, dictyostatin, discodermolide, dolastatin, halichondrin, epothilone, spongistatin, laulimalide, maytansine, noscapaxin, paclitaxel, peloruside, phomopsin, podophyllotoxin, rhizoxin, spongistatin, taxane, tubulysin, vinca alkaloids, vinorelbine, or derivatives or analogs thereof.

[0363] In some embodiments, maytansine is a maytansine alkaloid. In some embodiments, the maytansine alkaloid is DM1, DM4, or ansamitocin. In some embodiments, the maytansine alkaloid is DM1. In some embodiments, the maytansine alkaloid is DM4. In some embodiments, the maytansine alkaloid is ansamitocin. In some embodiments, the maytansine alkaloid is a maytansine alkaloid 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. Publication Nos. 2013029900 and US20130323268.

[0364] In some embodiments, the payload is dolastatin, or a derivative or analogue thereof. In some embodiments, the dolastatin is dolastatin 10 or dolastatin 15, or a derivative or analogue thereof. In some embodiments, the dolastatin 10 analogue is auristatin, sobralidotin, symplostatin 1 or symplostatin 3. In some embodiments, the dolastatin 15 analogue is cemadotin or tesetaxel.

[0365] In some embodiments, the dolastatin 10 analogue is auristatin or an auristatin derivative. In some embodiments, the auristatin or auristatin derivative is auristatin E (AE), auristatin F (AF), auristatin E 5-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, the auristatin is an auristatin derivative or analogue, 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.

[0366] In some embodiments, the payload comprises a DNA modifier. In some embodiments, the DNA modifier comprises a DNA cleavage agent, a DNA intercalator, a DNA transcription inhibitor or a DNA crosslinking agent. In some embodiments, the DNA cleavage agent comprises bleomycin A2, calicheamicin or a derivative or analogue thereof. In some embodiments, the DNA intercalator comprises doxorubicin, epirubicin, PNU-159682, idarubicin, 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 crosslinking agent comprises mitomycin C.

[0367] In some embodiments, the DNA modifier comprises amsacrine, anthracycline, camptothecin, doxorubicin, idarubicin, enediyne, etoposide, indolobenzodiazepine, netropsin, teniposide or a derivative or analogue thereof.

[0368] In some embodiments, the anthracycline is doxorubicin, daunorubicin, epirubicin, idarubicin, mitomycin-C, dactinomycin, mithramycin, nemorubicin, pixantrone, zabicipurine or valrubicin.

[0369] In some embodiments, analogs of camptothecin are topotecan, irinotecan, silatecan, cositecan, elsatecan, letozotecan, gemitecan, belotecan, rubitecan, or SN-38.

[0370] In some embodiments, duocarmycin is duocarmycin A, duocarmycin B1, duocarmycin B2, duocarmycin C1, duocarmycin C2, duocarmycin D, duocarmycin SA, or CC-1065. In some embodiments, enediyne is calicheamicin, esperamicin, or dynemicin A.

[0371] In some embodiments, pyrrolobenzodiazepine is anthramycin, streptonigrin, calicheamicin, DC-81, mazethramycin, neothramycins A, neothramycin B, porothramycin, prothracarcin, sibanomicin (DC-102), sibiromycin, or turimycin. In some embodiments, pyrrolobenzodiazepine is a turimycin derivative, such as those described in U.S. Patent Nos. 8,404,678 and 8,163,736. In some embodiments, pyrrolobenzodiazepine is such as those described in U.S. Patent Nos. 8,426,402, 8,802,667, 8,809,320, 6,562,806, 6,608,192, 7,704,924, 7,067,511, US 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. Publication No. US20140294868.

[0372] In some embodiments, pyrrolobenzodiazepine is a pyrrolobenzodiazepine dimer. In some embodiments, the PBD dimer is a symmetric dimer. Examples of symmetric 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. Patent Nos. 8,697,688 and 9,242,013 and U.S. Publication No. 20140286970.

[0373] In some embodiments, the payload includes an Akt inhibitor. In some cases, the Akt inhibitor includes ipatasertib (GDC-0068) or a derivative thereof.

[0374] In some embodiments, the payload comprises a polymerase inhibitor, including but not limited to polymerase II inhibitors such as α-amanitin and poly(ADP-ribose) polymerase (PARP) inhibitors. Exemplary PARP inhibitors include but are not limited to Iniparib (BSI 201), talazoparib (BMN-673), olaparib (AZD-2281), olaparib, rucaparib (AG014699, PF-01367338), veliparib (ABT-888), CEP 9722, MK 4827, BGB-290 or 3-aminobenzamide.

[0375] In some embodiments, the payload comprises a detectable moiety. Exemplary detectable moieties include fluorescent dyes; enzymes; substrates; chemiluminescent moieties; specific binding moieties such as streptavidin, avidin or biotin; or radioisotopes.

[0376] In some embodiments, the payload comprises an immunomodulator. Useful immunomodulators include antihormones that block the action of hormones on tumors and immunosuppressive agents that inhibit cytokine production, downregulate the expression of autoantigens or mask MHC antigens. Representative antihormones include antiestrogens, including for example tamoxifen, raloxifene, aromatase that inhibits 4(5)-imidazole, 4-hydroxytamoxifen, trioxifene, raloxifene, LY 117018, onapnstone and toremifene; and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide, goserelin; and antiadrenal agents. Illustrative immunosuppressive agents include but are not limited to 2-amino-6-aryl-5-substituted pyrimidines, azathioprine, cyclophosphamide, bromocriptine, danazol, dapsone, glutaraldehyde, anti-idiotypic antibodies against MHC antigens and MHC fragments, cyclosporin A, steroids such as glucocorticoids, streptokinase or rapamycin.

[0377] In some embodiments, the payload comprises an immunomodulator. Exemplary immunomodulators include, but are not limited to, gancyclovier, etanercept, tacrolimus, sirolimus, voclosporin, cyclosporine, rapamycin, cyclophosphamide, azathioprine, mycophenolate mofetil, methotrexate, glucocorticoids and their analogs, xanthine, stem cell growth factor, lymphotoxin, hematopoietic factors, tumor necrosis factor (TNF) (e.g., TNFα), interleukins (e.g., interleukin-1 (IL-1), 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 factor known as "S1 factor", erythropoietin, and thrombopoietin, or combinations thereof.

[0378] In some embodiments, the payload comprises an immunotoxin. Immunotoxins include, but are not limited to, ricin, radionuclides, pokeweed antiviral protein, Pseudomonas exotoxin A, diphtheria toxin, ricin A-chain, mycotoxins such as restrictocin and phospholipase. Generally, see "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).

[0379] In some embodiments, the payload comprises a nucleic acid polymer. In some embodiments, the nucleic acid polymer comprises short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), antisense oligonucleotides. In some embodiments, the nucleic acid polymer comprises mRNA encoding, for example, a cytotoxin protein or peptide or an apoptosis-triggering protein or peptide. Exemplary cytotoxin proteins or peptides include bacterial cytotoxins such as α-pore-forming toxins (e.g., hemolysin A from Escherichia coli), β-pore-forming toxins (e.g., α-hemolysin, PVL-leukocidin, aerolysin, Clostridium perfringens epsilon toxin, Clostridium perfringens enterotoxin), binary toxins (anthrax toxin, edema toxin, Clostridium botulinum C2 toxin, Clostridium spiroforme toxin, Clostridium perfringens iota toxin, Clostridium difficile lethal toxin (A and B)), prions, paracoccin, cholesterol-dependent cytolysins (e.g., pneumolysin), small pore-forming toxins (e.g., gramicidin A), cyanotoxins (e.g., microcystin, nodularin), hemotoxins, neurotoxins (e.g., botulinum neurotoxin), cytotoxins, cholera toxin, diphtheria toxin, Pseudomonas aeruginosa exotoxin A, tetanus toxin or immunotoxins (idarubicin, ricin A, CRM9, pokeweed antiviral protein, DT). Exemplary apoptosis-triggering proteins or peptides include apoptotic protease-activating factor-1 (Apaf-1), cytochrome-c, caspase initiator proteins (CASP2, CASP8, CASP9, CASP10), apoptosis-inducing factor (AIF), p53, p73, p63, Bcl-2, Bax, granzyme B, poly(ADP-ribose) polymerase (PARP) and P21-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 trans-activation region (TAR) decoys and Rev response element (RRE) decoys.

[0380] In some cases, the payload is an aptamer. An aptamer is a small oligonucleotide or peptide molecule that binds to a specific target molecule. Exemplary nucleic acid aptamers include DNA aptamers, RNA aptamers or XNA aptamers, which are RNA and / or DNA aptamers comprising one or more non-natural nucleotides. Exemplary nucleic acid aptamers include ARC19499 (Archemix Corp.), REG1 (Regado Biosciences) and ARC1905 (Ophthotech).

[0381] Nucleic acids according to some embodiments described herein optionally include naturally occurring nucleic acids, or one or more nucleotide analogs or have a structure different from that of naturally occurring nucleic acids. For example, 2'-modifications include halo, alkoxy, and allyloxy. In some embodiments, the 2'-OH group is replaced by a group selected from H, OR, R, halo, SH, SR, NH 2 , NHR, NR 2 or CN, where R is C 1 -C 6 alkyl, alkenyl, or alkynyl, and halo is F, Cl, Br, or I. Examples of modified linkages include phosphorothioate and 5'-N-phosphoramidite linkages.

[0382] Nucleic acids with various different nucleotide analogs, modified backbones, or non-naturally occurring internucleoside linkages are utilized according to some embodiments described herein. In some cases, the nucleic acid includes 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., cytarabine, inosine, isoguanosine, tubercidin, 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-deazaguanosine, 7-deazaguanosine, 8-azaguanosine, 8-azidoadenosine, benzimidazole, M1-methyladenosine, pyrrolopyrimidine, 2-amino-6-chloropurine, 3-methyladenosine, 5-propynylcytidine, 5-propynyluridine, 5-bromouridine, 5-fluorouridine, 5-methylcytidine, 7-deazaguanosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(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 nucleoside arabinose, and hexose), modified phosphate groups (e.g., phosphorothioate and 5'-N-phosphoramidite linkages), and combinations thereof. Natural and modified nucleotide monomers for nucleic acid chemical synthesis are readily available. In some cases, nucleic acids containing such modifications exhibit improved properties relative to nucleic acids composed only of naturally occurring nucleotides. In some embodiments, the nucleic acid modifications described herein are utilized to reduce and / or prevent digestion by nucleases (e.g., exonucleases, endonucleases, etc.). For example, the structure of the nucleic acid can be stabilized by including nucleotide analogs at the 3' end of one or both strands to reduce digestion.

[0383] Different nucleotide modifications and / or backbone structures may be present at different positions of the nucleic acid. Such modifications include morpholine, peptide nucleic acid (PNA), methylphosphonate nucleotides, thiolphosphonate nucleotides, 2'-fluoro N3P5'-amidite, 1',5'-anhydrohexitol nucleic acid (HNA), or combinations thereof.

[0384] Conjugation chemistry

[0385] In some embodiments, the payload is conjugated 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 conjugation is as described in U.S. Patent No. 8,936,910.

[0386] In some embodiments, the payload is conjugated to the anti-Gal3 antibodies described herein by a directed-site approach utilizing the “traceless” coupling technology (Philochem). In some embodiments, the “traceless” coupling technology utilizes the N-terminal 1,2-aminothiol group on the binding moiety, which is then conjugated to a poly-nucleic acid molecule containing an aldehyde group. (See Casi et al., “Site-specific traceless coupling of potent cytotoxic drugs to recombinant antibodies for pharmacodelivery,” JACS 134(13):5887-5892 (2012)).

[0387] In some embodiments, the payload is conjugated to the anti-Gal3 antibodies described herein by a directed-site approach utilizing unnatural amino acids incorporated into the binding moiety. In some embodiments, the unnatural amino acid includes p-acetylphenylalanine (pAcPhe). In some embodiments, the keto group of pAcPhe is selectively conjugated to an alkoxy-amine-derived conjugation moiety to form an oxime bond. (See Axup et al., “Synthesis of site-specific antibody-drug conjugates using unnatural amino acids,” PNAS 109(40):16101-16106 (2012)).

[0388] In some embodiments, the payload is conjugated to the anti-Gal3 antibodies described herein by a directed-site approach utilizing an enzyme-catalyzed process. In some embodiments, the directed-site approach utilizes the SMARTag TM technology (Redwood). In some embodiments, the SMARTag TMThe technology involves generating formylglycine (FGly) residues from cysteine via an oxidation process by formylglycine-generating enzyme (FGE) in the presence of an aldehyde tag, followed by conjugating FGly to an alkylhydrain-functionalized polynucleic acid molecule via a hydrazino-Pictet-Spengler (HIPS) ligation. (See 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)).

[0389] In some embodiments, the enzyme-catalyzed process involves microbial transglutaminase (mTG). In some cases, a payload is conjugated to an anti-Gal3 antibody using a process catalyzed by microbial transglutaminase. In some embodiments, mTG catalyzes the formation of a covalent bond between the amide side chain of glutamine within a recognition sequence and the primary amine of a 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)).

[0390] In some embodiments, the payload is conjugated to the anti-Gal3 antibody by the method described in PCT Publication No. WO2014 / 140317, which utilizes a sequence-specific transpeptidase.

[0391] In some embodiments, the payload is conjugated to the anti-Gal3 antibody described herein by the methods described in U.S. Patent Publication Nos. 2015 / 0105539 and 2015 / 0105540.

[0392] Linker

[0393] In some embodiments, the linker described above comprises a natural or synthetic polymer, which consists of a long chain of branched or unbranched monomers and / or a two- or three-dimensional monomer cross-linked network. In some embodiments, the linker comprises a polysaccharide, lignin, rubber, or polyalkylen oxide (e.g., polyethylene glycol).

[0394] In some embodiments, the linker includes, but is not limited to, α-, ω-dihydroxy polyethylene glycol, biodegradable lactone-based polymers such as polyacrylic acid, polylactic acid (PLA), poly(glycolic acid) (PGA), polypropylene, polystyrene, polyolefin, polyamide, polycyanoacrylate, polyimide, polyethylene terephthalate (PET, PETG), polyethylene terephthalate (PETE), polytetramethylene glycol (PTG), or polyurethane, and mixtures thereof. As used herein, mixtures refer to the use of different polymers in the same compound and to block copolymers. In some cases, a block copolymer is a polymer in which at least one portion of the polymer is composed of monomers of another polymer. In some embodiments, the linker comprises a polyalkylene oxide. In some embodiments, the linker comprises PEG. In some embodiments, the linker comprises polyvinyl imide (PEI) or hydroxyethyl starch (HES).

[0395] In some cases, the polyalkylene oxide (e.g., PEG) is a polydisperse or monodisperse compound. In some embodiments, a polydisperse material comprises a dispersed distribution of materials of different molecular weights, characterized by an average weight (weight average) size and dispersity. In some embodiments, monodisperse PEG comprises molecules of one size. In some embodiments, the linker is a polydisperse or monodisperse polyalkylene oxide (e.g., PEG) and the indicated molecular weight represents the average molecular weight of the polyalkylene oxide, e.g., PEG molecules.

[0396] In some embodiments, the linker comprises 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, 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.

[0397] In some embodiments, the polyalkylene oxide (e.g., PEG) is discrete PEG, where the discrete PEG is polymeric PEG that includes more than one repeating ethylene oxide unit. In some embodiments, the discrete PEG (dPEG) includes 2 to 60, 2 to 50 or 2 to 48 repeating ethylene oxide units. In some embodiments, the dPEG includes 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 includes about 2 or more repeating ethylene oxide units. In some cases, the dPEG is synthesized in a stepwise manner from pure (e.g., about 95%, 98%, 99% or 99.5%) starting materials into a single molecular weight compound. In some cases, the dPEG has a specific molecular weight rather than an average molecular weight. In some cases, the dPEG described herein is from Quanta Biodesign, LMD.

[0398] In some embodiments, the linker is discrete PEG, optionally including 2 to 60, 2 to 50 or 2 to 48 repeating ethylene oxide units. In some cases, the linker includes a dPEG comprising 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 dPEG from Quanta Biodesign, LMD.

[0399] 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 cases, the polypeptide linker is a cleavable polypeptide linker (e.g., enzymatically or chemically). In some cases, 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.

[0400] In some embodiments, the linker comprises a homobifunctional linker. Exemplary homobifunctional linkers include, but are not limited to, Lomant's reagent dithiobis(succinimidyl propionate) DSP, 3’3’-dithiobis(sulfosuccinimidyl propionate) (DTSSP), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl) suberate (BS), disuccinimidyl tartrate (DST), bissulfosuccinimidyl tartrate (sulfosuccinimidyl DST), ethylene glycol bis(succinimidyl succinate) (EGS), disuccinimidyl glutarate (DSG), N,N’-disuccinimidyl carbonate (DSC), dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP), dimethylsuberimidate (DMS), dimethyl-3,3’-dithiobispropionimidate (DTBP), 1,4-di-3’-(2’'-pyridyldithio)propionamido)butane (DPDPB), bismaleimidohexane (BMH), aryl halide-containing compounds (DFDNB), such as for example 1,5-difluoro-2,4-dinitrobenzene or 1,3-difluoro-4,6-dinitrobenzene, 4,4’-difluoro-3,3’-dinitrophenylsulfone (DFDNPS), bis-[β-(4-azidosalicylamido)ethyl] disulfide (BASED), formaldehyde, glutaraldehyde, 1,4-butanediol diglycidyl ether, adipic dihydrazide, carbohydrazide, o-toluidine, 3,3’-dimethylbenzidine, benzidine, α,α’-p-diaminodiphenyl, diiodo-p-xylene sulfonic acid, N,N'-ethylene-bis(iodoacetamide) or N,N’-hexamethylene-bis(iodoacetamide).

[0401] In some embodiments, the linker includes a heterobifunctional linker. Exemplary heterobifunctional linkers include, but are not limited to, amine-reactive and thiol crosslinkers such as 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 (sulfonyl-LC-sPDP), succinimidyl oxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene (sMPT), sulfosuccinimidyl-6-[α-methyl-α-(2-pyridyldithio)toluidine]hexanoate (sulfonyl-LC-sMPT), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC), sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfonyl-sMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBs), m-maleimidobenzoyl-N-hydroxy sulfosuccinimide ester (sulfonyl-MBs), N-succinimidyl (4-iodoacetyl)aminobenzoate (sIAB), sulfosuccinimidyl (4-iodoacetyl)aminobenzoate (sulfonyl-sIAB), succinimidyl-4-(p-maleimidophenyl)butyrate (sMPB), sulfosuccinimidyl-4-(p-maleimidophenyl)butyrate (sulfonyl-sMPB), N-(γ-maleimidobutyryloxy)succinimide ester (GMB), N-(γ-maleimidobutoxy)sulfosuccinimide ester (sulfonyl-GMB), succinimidyl 6-((iodoacetyl)amino)hexanoate (sIAX), succinimidyl 6-[6-(((iodoacetyl)amino)hexanoyl)amino]hexanoate (sIAXX), succinimidyl 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 thiol-reactive crosslinkers such as 4-(4-N-maleimidophenyl)butyric acid hydrazide (MPBH), 4-(N-maleimidomethyl)cyclohexane-1-carboxy-hydrazide-8(M 2 C 2(H), 3-(2-pyridyldithio)propionyl hydrazide (PDPH), amine-reactive and photoreactive cross-linking agents such as N-hydroxysuccinimidyl-4-azidosalicylic acid (NHs-AsA), N-hydroxysulfosuccinimidyl-4-azidosalicylic acid (sulfonyl-NHs-AsA), sulfosuccinimidyl-(4-azidosalicylamido)hexanoate (sulfonyl-NHs-LC-AsA), sulfosuccinimidyl-2-(p-azidosalicylamido)ethyl-1,3'-dithiopropionate (sAsD), N-hydroxysuccinimidyl-4-azidobenzoate (HsAB), N-hydroxysulfosuccinimidyl-4-azidobenzoate (sulfonyl-HsAB), N-succinimidyl-6-(4'-azido-2'-nitroanilino)hexanoate (sANPAH), sulfosuccinimidyl-6-(4'-azido-2'-nitroanilino)hexanoate (sulfonyl-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 (sulfonyl-sADP), sulfosuccinimidyl 4-(p-azidophenyl)butyrate (sulfonyl-sAPB), sulfosuccinimidyl 2-(7-azido-4-methylcoumarin-3-acetamido)ethyl-1,3'-dithiopropionate (sAED), sulfosuccinimidyl 7-azido-4-methylcoumarin-3-acetate (sulfonyl-sAMCA), p-nitrophenyl diazopyruvate (ρNPDP), p-nitrophenyl-2-diazo-3,3,3-trifluoropropionate (PNP-DTP), thiol-reactive and photoreactive cross-linking agents such as 1-(p-azidosalicylamido)-4-(iodoacetamido)butane (AsIB), N-[4-(p-azidosalicylamido)butyl]-3'-(2'-pyridyldithio)propanamide (APDP), benzophenone-4-iodoacetamide, benzophenone-4-maleimide carbonyl-reactive and photoreactive cross-linking agents such as p-azidobenzoyl hydrazide (ABH), carboxylate-reactive and photoreactive cross-linking agents such as 4-(p-azidosalicylamido)butylamine (AsBA) and arginine-reactive and photoreactive cross-linking agents such as p-azidophenylglyoxal (APG).

[0402] 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 p-aminobenzoic acid (PABA). In some embodiments, the benzoic acid group or a derivative thereof comprises γ-aminobutyric acid (GABA).

[0403] 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 moiety is val-cit. In some embodiments, the benzoic acid group is PABA. In some embodiments, the linker comprises an mc-val-cit group. In some cases, the linker comprises a val-cit-PABA group. In other cases, the linker comprises an mc-val-cit-PABA group.

[0404] In some embodiments, the linker is a self-immolative linker or a self-eliminating linker. In some cases, the linker is a self-immolative linker. In other cases, the linker is a self-eliminating linker (e.g., a cyclized self-eliminating linker). In some embodiments, the linker comprises a linker described in U.S. Patent No. 9,089,614 or PCT Publication No. WO2015038426.

[0405] 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.

[0406] In some embodiments, the linker is a traceless linker or a linker that leaves no linker moiety (e.g., an atom or a linker group) of the antibody or payload upon 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 benzohydrazide linkers. In some cases, the linker is a traceless aryl-triazene linker as described by 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 by Blaney et al., “Traceless solid-phase organicsynthesis,” Chem.Rev.102:2607-2024(2002). In some embodiments, the linker is a traceless linker as described in U.S. Patent No. 6,821,783.

[0407] Pharmaceutical compositions

[0408] In some embodiments, a pharmaceutical formulation for reducing tissue fibrosis may include the anti-Gal3 antibody described above. The anti-Gal3 antibody may be formulated for systemic administration. Alternatively, the anti-Gal3 antibody may be formulated for parenteral administration.

[0409] In some embodiments, the anti-Gal3 antibody is further formulated into a pharmaceutical composition. In some embodiments, the pharmaceutical composition is formulated for administration to a subject by a variety of routes of administration, including but not limited to parenteral (e.g., intravenous, subcutaneous, intramuscular, intra-arterial, intradermal, intraperitoneal, intravitreal, intracerebral, or intraventricular), oral, intranasal, buccal, rectal, or transdermal routes of administration. In some embodiments, the pharmaceutical composition described herein is formulated for parenteral (e.g., intravenous, subcutaneous, intramuscular, intra-arterial, intradermal, intraperitoneal, intravitreal, intracerebral, or intraventricular) administration. In some embodiments, the pharmaceutical composition described herein is formulated for oral administration. In other embodiments, the pharmaceutical composition described herein is formulated for intranasal administration.

[0410] In some embodiments, the pharmaceutical formulation includes but is 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, sustained-release formulations, extended-release formulations, pulsatile-release formulations, multi-particle formulations (e.g., nanoparticle formulations), and mixed immediate-release and controlled-release formulations.

[0411] In some embodiments, the pharmaceutical composition further includes a pH regulator or buffer, which includes acids such as acetic acid, boric acid, citric acid, lactic acid, phosphoric acid, and hydrochloric acid; bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate, and tris(hydroxymethyl)aminomethane; and buffers such as citrate / dextrose, sodium bicarbonate, and ammonium chloride. Such acids, bases, and buffers are included in the amounts required to maintain the pH of the composition within an acceptable range.

[0412] In some embodiments, the pharmaceutical composition includes one or more salts in the amounts required to make the osmotic pressure of the composition within an acceptable range. Such salts include salts 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.

[0413] In some embodiments, the pharmaceutical composition further comprises a diluent for stabilizing the compound as they can provide a more stable environment. Salts dissolved in buffer solutions (which can also provide pH control or maintenance) are used as diluents in the art, including but not limited to phosphate buffered saline solutions. In certain embodiments, the diluent increases the volume of the composition to facilitate compression or to produce a sufficient volume for a homogeneous blend for capsule filling. Such compounds include, for example, lactose, starch, mannitol, sorbitol, dextrose, microcrystalline cellulose such as calcium hydrogen phosphate, dicalcium phosphate dihydrate; tricalcium phosphate, calcium phosphate; anhydrous lactose, spray-dried lactose; pregelatinized starch, compressible sugar, such as (Amstar); mannitol, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose acetate stearate, sucrose-based diluents, confectioner's sugar; calcium sulfate hemihydrate, calcium sulfate dihydrate; calcium lactate trihydrate, glucose binder; hydrolyzed cereal solids, amylose; powdered cellulose, calcium carbonate; glycine, kaolin; mannitol, sodium chloride; inositol, bentonite, etc.

[0414] In some embodiments, the pharmaceutical formulation may further comprise an additional therapeutic agent. The additional therapeutic agent may have an anti-fibrotic effect. The additional therapeutic agent may be a ligand, such as an inhibitor of growth factors, cytokines, and 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 (C-C motif) ligand 2; CCR5, MMP, and TIMP. The additional therapeutic agent may be SHP-627 (FT011), oxanimone (F351), PXS-25, Disitertide (P-144), nonsulmonab (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), deucravacitinib (QAX-576), tralokinumab, anakinra, rilonacept, SAR156597, carlumab (CNTO-888), bindarit, maraviroc, RS-504393, Actimmune, alpha oral interferon, batimastat (BB-49), marimastat, macitentan, bosentan, ambrisentan, Sparsentan (RE-021), atrasentan, losartan, BMS-986020, SAR-100842, PAR1 antagonist, curcumin, silymarin, beta-caryophyllene, beraprost, travoprost, altudil, sivelestat sodium, UK-396082, Serelaxin, PRM-151 or diosgenin, NTU281.

[0415] Therapeutic regimens

[0416] In some embodiments, the anti-Gal3 antibodies disclosed herein are administered for therapeutic applications. In some embodiments, the anti-Gal3 antibodies are administered once a day, twice a day, three times a day or more. The anti-Gal3 antibodies are administered daily, every day, every other day, five days a week, once a week, every other week, two weeks a month, three weeks a month, once a month, twice a month, three times a month or more. The anti-Gal3 antibodies are 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 longer.

[0417] In the event that the patient's condition does improve, at the discretion of the physician, administration of the anti-Gal3 antibody is continued; alternatively, the dose of the anti-Gal3 antibody being administered is temporarily reduced or temporarily suspended for a length of time (i.e., a "drug holiday"). In some embodiments, the length of the drug holiday varies between 2 days and 1 year and includes, 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. The dose reduction during the drug holiday is from 10% - 100% and includes, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%.

[0418] Once the patient's condition shows improvement, a maintenance dose is administered as necessary. Subsequently, due to the effect of the symptoms, the dose or frequency or both of the administered drug is reduced to a level that maintains the improved disease, disorder or condition.

[0419] In some embodiments, the amount of a given agent corresponding to such amount depends on factors such as the specific compound, the severity of the disease, the characteristics of the subject or host to be treated (e.g., body weight), however, it is routinely determined in a manner known in the art according to the specific circumstances surrounding the case, including for example the specific agent being administered, the route of administration and the subject or host being treated. In some embodiments, the required dose is conveniently provided as a single dose or as separate doses administered simultaneously (or within a short period of time) or at appropriate intervals, such as two, three, four or more sub-doses per day.

[0420] The foregoing ranges are merely suggestive, since there are a large number of variables regarding an individual's treatment regimen and quite large deviations from these recommended values are not uncommon. Such dosage is altered depending upon many variables, including but not limited to the activity of the compound being used, the disease or disorder to be treated, the mode of administration, the requirements of the individual subject, the severity of the disease or disorder being treated, and the judgment of the practitioner.

[0421] 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 LD50 (the dose lethal to 50% of the population) and ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between the toxic and therapeutic effects is the therapeutic index, and is expressed as the ratio between LD50 and ED50. Compounds are preferably displayed with a high therapeutic index. Data obtained from cell culture assays and animal studies are used to formulate a range of dosage for humans. The dosage of such compounds preferably lies within a cyclic concentration range that includes ED50 with minimal toxicity. The dosage varies within this range depending upon the dosage form employed and the route of administration utilized.

[0422] Kits / articles

[0423] In some embodiments, kits and articles of manufacture are disclosed for use with one or more of the compositions and methods described herein. Such kits include a carrier, package, or container that is partitioned to receive one or more containers, such as vials, tubes, etc., each container including one of the separate elements for the methods described herein. Suitable containers include, for example, bottles, vials, syringes, and test tubes. In some embodiments, the containers are formed from a variety of materials such as glass or plastic.

[0424] The articles provided herein contain a packaging material. 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 intended mode of administration and treatment.

[0425] For example, the container includes 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 the antibody described herein. Such kits optionally include identifying instructions, labels, or directions related to their use in the methods described herein.

[0426] Kits generally include a label listing the contents and / or instructions for use, as well as a package insert with instructions for use. A set of instructions will generally also be included.

[0427] In some embodiments, the label is on or associated with the container. In some embodiments, the label is on the container when the letters, numbers, or other characters forming the label are attached, molded, or etched into the container itself; the label is associated with the container when the label is present in a reservoir or carrier that also houses the container, such as a packaging insert. In some embodiments, the label is used to indicate that the contents are for a specific therapeutic application. The label also indicates instructions for using the contents, such as in the methods described herein.

[0428] In some embodiments, the pharmaceutical composition is present in a packaging or dispenser device containing one or more unit dosage forms comprising the compounds provided herein. For example, the packaging contains a metal or plastic foil, such as a blister pack. In some embodiments, the packaging or dispenser device is accompanied by instructions for administration. In some embodiments, the packaging or dispenser is also accompanied by a notice associated with the container, in a form prescribed by a government agency that regulates the manufacture, use, or sale of pharmaceutical formulations, the notice reflecting the agency's approval of the form of the pharmaceutical for human or veterinary administration. For example, such notice is a prescription drug label or an approved product insert approved by the U.S. Food and Drug Administration. In some embodiments, a composition containing the compounds provided herein formulated in a compatible pharmaceutical carrier is also prepared, placed in a suitable container, and labeled for the treatment of the designated condition.

[0429] Fibrosis biomarkers

[0430] In some embodiments, fibrosis 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, but are not limited to, collagen (I, III, and IV), procollagen N-terminal propeptide, 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, SNPs of AZIN1, TLR4, TRPM5, AQP2, or STXBP5L. The expression or absence of certain biomarkers is associated with one or more fibrosis diseases. An increase or decrease in such biomarkers may indicate a reduction in tissue fibrosis when treated with anti-Gal3 antibody.

[0431] In some embodiments, the fibrosis biomarker is α - smooth muscle actin (α - SMA). α - SMA is a 42 kDa actin isoform that predominates in vascular smooth muscle cells and is involved in fibrogenesis. Myofibroblasts are a form of fibroblasts that have partially differentiated into a smooth muscle phenotype. In particular, myofibroblasts can contract by using cytoskeletal proteins including α - SMA. In several fibrotic diseases, the accumulation of myofibroblasts has been observed, leading to the expansion of the extracellular matrix. Thus, altered expression of α - SMA (e.g., elevated expression) is associated with the activation of myofibroblasts and, in other cases, can be used as a fibrosis biomarker.

[0432] In some embodiments, the fibrosis biomarker is fibronectin. Fibronectin is a high - molecular - weight (∼440 kDa) glycoprotein within the extracellular matrix and further binds to integrins, collagen, fibrin, and heparan sulfate proteoglycans. Fibronectin plays a major role in cell adhesion, growth, migration, and differentiation and is further involved in wound healing among numerous functions. Fibronectin can be soluble plasma fibronectin or insoluble cellular fibronectin and can be type I, type II, or type III. Altered expression of fibronectin (e.g., decreased expression) is associated with fibrosis.

[0433] 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. TGF - β1 is involved in cell growth, cell proliferation, cell differentiation, and apoptosis. In addition, collagen - producing cells, such as fibroblasts, are activated by fibrogenic cytokines such as TGF - β1. In a fibrotic context, TGF - β1 is considered a major regulator and potent inducer of ECM synthesis. Moreover, 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 an enhanced expression of pro - inflammatory responses and fibrogenic cytokines such as TNF - α, PDGF, IL - 1β, and / or IL - 13, further enhancing and maintaining the fibrotic response.

[0434] In some embodiments, administration of an anti-Gal3 antibody to an interested tissue site 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, alpha-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, SNPs of AZIN1, TLR4, TRPM5, AQP2, and STXBP5L. 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 to an interested tissue site results in a decrease in α-SMA expression. In some embodiments, administration of an anti-Gal3 antibody to an interested tissue site results in an increase in fibronectin expression. In some embodiments, administration of an anti-Gal3 antibody to an interested tissue site results in a decrease in TGF-β1 expression.

[0435] In some embodiments, one or more fibrosis biomarkers are utilized to monitor the presence or absence of fibrosis, or the progression of fibrosis.

[0436] In some cases, a decrease in the expression of the fibrosis biomarkers disclosed herein may indicate a decrease in tissue fibrosis.

[0437] In some cases, the expression of at least one fibrosis biomarker in a tissue treated with an anti-Gal3 antibody is different from the expression of at least one fibrosis biomarker in a control tissue treated with a control antibody. In some cases, the control antibody is an anti-Gal3 antibody that does not bind to one or more of the epitopes described above and / or does not disrupt the interaction between Gal3 and TIM-3. In some cases, the control antibody is an IgG2b antibody, such as a murine IgG2b (mIgG2b) antibody. In some cases, the expression of at least one fibrosis biomarker in a tissue treated with an anti-Gal3 antibody is lower than the expression of at least one fibrosis biomarker in a control tissue treated with an mIgG2b antibody.

[0438] Fibrotic diseases

[0439] In some embodiments, anti-Gal3 antibodies can be administered to treat one or more fibrotic diseases. The fibrotic disease can be liver fibrosis. The fibrotic disease can be pulmonary fibrosis. The fibrotic disease can be cystic fibrosis, idiopathic pulmonary fibrosis, myelofibrosis, interstitial lung disease, liver fibrosis, progressive massive fibrosis, cirrhosis, renal fibrosis, cardiac fibrosis, pneumonia, pulmonary fibrosis, pancreatic fibrosis, myelofibrosis, intestinal fibrosis, joint fibrosis, retinal fibrosis, hepatitis C-related fibrosis, or nephrogenic systemic fibrosis.

[0440] In some cases, anti-Gal3 antibodies can be administered to fibrotic diseases associated with the expression of α-SMA or fibronectin. Fibrotic diseases associated with increased α-SMA can be renal fibrosis, liver fibrosis, cirrhosis, hepatitis C-related fibrosis, cardiac fibrosis, pulmonary fibrosis, interstitial lung disease, idiopathic pulmonary fibrosis, pneumonia, myelofibrosis, joint fibrosis, retinal fibrosis, or nephrogenic systemic fibrosis. Fibrotic diseases associated with fibronectin expression can be cystic fibrosis, pulmonary fibrosis, idiopathic pulmonary fibrosis, myelofibrosis, interstitial lung disease, liver fibrosis, progressive massive fibrosis, cirrhosis, renal fibrosis, cardiac fibrosis, pneumonia, pulmonary fibrosis, pancreatic fibrosis, myelofibrosis, intestinal fibrosis, joint fibrosis, retinal fibronectin, hepatitis C-related fibrosis, or nephrogenic systemic fibrosis.

[0441] The subject to be treated can be diagnosed with a fibrotic disease. In some embodiments, the subject to be treated can be a human, a rat, a mouse, or other animal. In some embodiments, the subject to be treated can be a mammal. In some embodiments, the mammal can be a human. The mammal can be a primate. The primate can be a chimpanzee or a gorilla.

[0442] 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 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 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 can bind to Gal3 at one or more residues corresponding to residues 2-21 and 42-71; 42-91; 2-21 and 72-91; or 2-21 and 42-91. The Gal3 and TIM-3 sequences are listed in Table 1.

[0443]

[0444]

[0445] 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 within the peptide illustrated in Table 2 (and Figure 11A shown in).

[0446]

[0447] 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 within 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 can bind to at least 1, 2, 3, 4, 5, 6, 10, 15, or 20 amino acid residues within peptide_1 (SEQ ID NO:3). 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 within peptide_5 (SEQ ID NO:4). 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 within peptide_6 (SEQ ID NO:5). 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 within peptide_8 (SEQ ID NO:6).

[0448] In some embodiments, the anti-Gal3 antibody further disrupts the interaction between Gal3 and TIM-3. TIM-3 is a molecule expressed on immune cells, particularly T cells, and can inhibit immune responses, such as T cell signaling, through interaction with Gal3.

[0449] In some embodiments, Gal3-TIM-3 antibodies are designed based on the interface where the interaction between Gal3 and TIM-3 occurs. The interaction on 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 on 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 can occur at one or more residues within region 145-184, where region 145-184 corresponds to positions 145-168 of SEQ IDNO:1.

[0450] In some embodiments, the Gal3-TIM-3 antibody disrupts the interaction between Gal3 and TIM-3, wherein the interaction on 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 on 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.

[0451] In some embodiments, the interaction can occur at one or more residues of Gal3 selected from regions 149-156, 152-171, 152-169, 152-168, 163-169, or 163-171, wherein the 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 between Gal3 and TIM-3, wherein the interaction on Gal3 involves one or more residues selected from regions 149-156, 152-171, 152-169, 152-168, 163-169, or 163-171, wherein the regions correspond 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, wherein the region corresponds to position 149-156 of SEQ ID NO:1. The interaction can occur at one or more residues of Gal3 within region 163-169, wherein the region corresponds to position 163-169 of SEQ ID NO:1. The interaction can occur at one or more residues of Gal3 within region 163-171, wherein the region corresponds to 163-171 of SEQ ID NO:1. The interaction can occur at one or more residues of Gal3 within region 152-169, wherein the region corresponding to SEQ ID NO:1 is position 152-169. The interaction can occur at one or more residues of Gal3 within region 152-171, wherein the region corresponds to position 152-171 of SEQ ID NO:1. The interaction can occur at one or more residues of Gal3 within region 163-171, wherein the region corresponds to position 163-171 of SEQ ID NO:1.

[0452] In some embodiments, the Gal3-TIM-3 antibody disrupts the interaction between Gal3 and TIM-3, wherein the interaction on 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 can occur at one or more residues of Gal3 within region 163-171 of SEQ ID NO:1.

[0453] The 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 the Gal3 region that interfaces with TIM-3 at the positions described herein.

[0454] The interaction on TIM-3 can occur at one or more residues corresponding to positions 72-104 and / or 64-93, wherein the residues correspond to positions 90-122 and 82-111 of SEQ ID NO:2. Alternatively, the interaction on TIM-3 can occur at one or more residues at positions 91-111, 107-117, 96-102, 100-106 and / or 92-119, wherein the residues correspond to positions 91-111, 107-117, 96-102, 100-106 and 92-119 of SEQ ID NO:2. The interaction on TIM-3 can occur at one or more residues at positions 91-117, 91-119, 96-117, 100-117 or 96-106. The Gal3-TIM-3 disrupting antibody 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 interfaces with Gal3 at the positions described herein.

[0455] 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; and the one or more residues correspond 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; and at one or more residues at positions 91-111, 107-117, 96-102, 100-106, and / or 92-119 of SEQ ID NO:2. The interaction on Gal3 may occur at one or more residues selected from regions 145-168, 160-177, and / or 165-184 of SEQ ID NO:1; and the one or more residues correspond to positions 90-122 and / or 82-111 of SEQ ID NO:2. The interaction on Gal3 may occur at one or more residues selected from regions 145-168, 160-177, and / or 165-184 of SEQ ID NO:1; and at one or more residues at positions 91-111, 107-117, 96-102, 100-106, and / or 92-119 of SEQ ID NO:2. The Gal3-TIM-3 disrupting antibody may 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 face each other at the positions described herein.

[0456] For any embodiment provided herein, the anti-Gal3 antibody used may be replaced with another anti-Gal3 antibody. The anti-Gal3 antibody is optionally 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). The anti-Gal3 antibody can be 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. The anti-Gal3 antibody can be mIMT001. The anti-Gal3 antibody can be an antibody other than mIMT001. The anti-Gal3 antibody can be 4A11.2B5. The anti-Gal3 antibody can be one or more of IMT001-4, IMT006-1, IMT006-5, or IMT006 8. The anti-Gal3 antibody can be 4A11.2B5. The anti-Gal3 antibody can be an 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 CDRs, VH, and / or VL of any one or more of these antibodies. Example

[0457] Some aspects of the embodiments discussed above are disclosed in more detail in the following examples, which are not intended to limit the scope of the present disclosure in any way. Those skilled in the art will understand that many other embodiments also fall within the scope of the present invention, as described above herein and in the claims.

[0458] Example 1: Generation of Gal3 - overexpressing cell lines

[0459] A20, a murine B lymphoma cell line, was obtained from the American Type Culture Collection (ATCC, Manassas, VA) and transfected with a nucleic acid construct encoding Flag-tagged human Gal3 protein or Flag-tagged human PDL1 protein. The construct additionally contained an antibiotic resistance marker. Transformed cells were selected based on antibiotic resistance to generate A20 cells stably expressing Flag-tagged human Gal3 protein (A20 Gal3 cells) or A20 cells stably expressing Flag-tagged human PDL1 protein (A20 hPDL1 cells).

[0460] Example 2: Specific binding of Gal3 to TIM - 3

[0461] This example describes various assays performed to evaluate the interaction between Gal3 and TIM-3.

[0462] Binding assay - Immunoprecipitation

[0463] Co-immunoprecipitation experiments were performed to test whether TIM-3 interacts specifically 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. The transfected cells were grown overnight and then washed and lysed in 1 ml of lysis buffer. The lysed cells were centrifuged and the supernatant (lysate) was collected. The lysate was prepared and separated on SDS PAGE and probed with anti-HA ( Figure 1A ) and anti-Flag antibodies ( Figure 1B ). Both anti-Flag and anti-HA antibodies were purchased from Sigma. Figure 1A and Figure 1B The arrows in

[0464] indicate the presence of various proteins. Figure 1C ) and Figure 1A-C, lanes 1-3 represent the results of lysates from cells co-transfected with a plasmid encoding TIM-3 tagged with HA and a plasmid encoding Gal3 tagged with Flag, cells co-transfected with a plasmid encoding TIM-3 tagged with HA and a plasmid encoding Gal9 tagged with Flag, or cells co-transfected with a plasmid encoding TIM-3 tagged with HA and a plasmid encoding CEACAM1 tagged with Flag, respectively.

[0465] The results, as Figure 1A shown in -C, indicate that human Gal3 specifically pulls down human TIM-3, while human CEACAM1 cannot pull down HA-tagged human TIM-3. Although it seems that human Gal9 also pulls down human TIM-3 ( Figure 1C lane 2), this seems to be non-specific due to Gal9 protein aggregation – the molecular weight of Gal9 seems to be much larger than its actual size of 40 kDa. The conclusion that the interaction between Gal9 and TIM-3 is non-specific in nature is also supported by the evidence shown in the following Figure 5B as well.

[0466] Additional co-immunoprecipitation experiments were performed to test whether Gal3 specifically interacts with TIM-3. The Flag-human Gal3 plasmid (OriGene, Rockville, MD) was transfected into 293T cells, which were at 80% confluency. As described above, transfection was performed in a 10 cm plate using lipofectamine 3000. After transfection overnight, the cells were replaced on a 10 cm plate that had been coated with human Fc, human PD1-Fc, or human TIM-3 Fc for 3 hours. The cells were washed once in 1xPBS and then lysed in 1 ml of lysis buffer. The cell lysates were collected and centrifuged. Protein G beads were added to the supernatant formed after centrifugation and incubated with rotation at 4°C for 4 hours. Then the beads were washed 3x with lysis buffer, followed by the addition of 1x SDS PAGE sample buffer. The samples containing the beads were boiled and separated on SDS-PAGE and transferred to a membrane. Then the membrane was probed with an anti-Flag antibody. As Figure 2 shown, human TIM-3 specifically pulls down Flag-tagged Gal3. In contrast, neither human Fc nor human PD1 Fc can pull down TIM-3. This shows that Gal3 does not bind to Fc or PD1 Fc, and the binding between Gal3 and TIM-3 is specific.

[0467] Binding assay – Cell adhesion assay

[0468] Next, a cell adhesion assay was performed to confirm the binding of Gal3 and TIM-3. In this experiment, 96-well plates were coated overnight at 4 °C with human Fc, human PD1-Fc, human VISTA-Fc, and human TIM-3-Fc, and then blocked with 2% BSA in PBS for 2 hours at 37 °C. A20, A20 cells overexpressing human Gal3 (A20 Gal3), or A20 cells overexpressing human PDL1 (A20 PDL1) were seeded into the wells coated with the various Fc proteins described above. The plates were then centrifuged at 720 rpm and then stopped. The plates were incubated at 37 °C for 30 minutes and then immersed in PBS. The plates were slowly flipped 180 degrees and held in the flipped position for 30 minutes. After flipping the plates back and removing them from the PBS, 200 μl of the solution from each well was taken out and discarded, and the remaining solution (volume approximately 100 μl) was transferred into a 96-well plate. Cells were counted by flow cytometry analysis.

[0469] Results ( Figure 3 ) showed that the number of A20 (A20 Gal3) cells expressing human Gal3 attached to the plate coated with human TIM-3 Fc was significantly greater than those attached to the plates coated with human VISTA Fc or human PD1 Fc. As expected, since PDL1 is a known ligand of PD1, the number of A20 PDL1 cells attached to hPD1 Fc was shown to be significantly greater than those attached to the 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.

[0470] Blocking assay – Flow cytometry

[0471] Flow cytometry analysis was performed using A20 cells to evaluate the binding between TIM-3 and Gal3. A20 Gal3 cells were incubated on ice for 20 minutes with 10% FBS HBSS solution with or without murine TIM-3Fc. There were five experimental groups: In group 1, A20 Gal3 cells were incubated without mTIM-3Fc protein as a control; in group 2, A20 Gal3 cells were incubated with mTIM-3Fc protein; in groups 3, 4, and 5, in addition to mTIM-3Fc 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), monoclonal antibody 215015 (R&D Systems) (group 5) were added to test whether these antibodies could block the binding of Gal3 and TIM-3. For blocking, the cells were incubated with 10% FBS HBSS containing the mentioned antibodies, and then 10% FBS HBSS containing mTIM-3Fc was added for 20 minutes. The samples were centrifuged and the pellets were added with 10% FBS HBSS containing APC-conjugated anti-hFc antibody (Jackson ImmunoResearch, West Grove, PA) for 20 minutes. After spinning, live / dead cells were stained with Violet Dead Cell Staining Kit (Life Technologies). Flow analysis was performed on the stained cells.

[0472] Figure 4 shows that mTIM-3 is capable of binding to Gal 3 protein on both dead and live cells, and Gal3 binds to different epitopes on TIM-3 on dead cells. Figure 4A Flow cytometry analysis of live A20 cells (left peak) and dead A20 cells (right peak) is shown. In this assay, TIM-3Fc binds to dead cells ( Figure 4C , row 2) and Gal3 expressed on live cells ( Figure 4B , row 2). However, the mTIM-3 monoclonal antibody RMT3-23 blocks the binding of TIM-3 to dead cells ( Figure 4C , row 4), but does not block the binding of TIM-3 to Gal3 expressed on live cells ( Figure 4B , row 4). This shows that Gal3 and dead cells bind to different epitopes on TIM-3. As a control, the mTIM-3 polyclonal antibody and monoclonal antibody 215015 (R&D System, Minneapolis, MN) did not affect the binding of TIM-3 to Gal3 ( Figure 4B , row 3 and row 5) or dead cells ( Figure 4C, the 3rd and 5th rows) have no effect.

[0473] Blocking assay – ELISA

[0474] ELISA assays were also performed to test the interaction between Gal3 and TIM-3. A 96-well ELISA plate (ThermoFisher Scientific) was 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, and incubated overnight at 4°C. The plates were washed three times with TBST and then blocked with PBS buffer containing 2% BSA for 1 h at room temperature. In Figure 5A , different anti-Gal3 antibodies, namely mGal3 polyclonal antibody (R&D systems), mAb IMT001 (also described in WO 2019 / 023247, which is hereby expressly incorporated by reference in its entirety), mAb M3 / 38 (ThermofisherScientific)( Figure 5A ), were added to the wells that had been coated with Gal3. The antibodies were incubated for 10 min, and then mouse TIM-3Fc was added to the plate and incubated for an additional hour. The plates were then washed three times and subsequently incubated with anti-human-IgG-HRP (Jackson ImmunoResearch) for 1 h at room temperature. After washing three times with TBST, the color was developed with TMB subtract (GeneTex, Irvine, CA), and the reaction was terminated with 1 N HCl. The optical density (OD) was read at 450 nm. The results were expressed as the mean OD ± SD of replicates. Figure 5A The results in Figure 5A ) showed that among all the antibodies tested, the mouse Gal3 polyclonal antibody and the monoclonal antibody IMT001 blocked the interaction between Gal3 and TIM-3(

[0475] In Figure 5BIn this experiment, mouse Gal3 protein (BioLegend) in PBS (Groups 1 and 2) or PS in ethanol (Sigma-Aldrich, St. Louis, MO) (Groups 3 and 4) was 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. The secondary anti-human-IgG-HRP antibody and substrate were added as described above to detect the binding of mTIM-3 to mGal3 or PS. The results showed that the signal in Group 4 was significantly reduced compared to Group 3, indicating that RMT3-23 blocked the binding of PS to TIM-3; meanwhile, the results showed that the signal in Group 2 was not significantly reduced compared to Group 1, indicating that RMT3-23 did not block the binding of Gal3 to TIM-3. Since TIM-3 binds to dead cells through its interaction with externalized and exposed PS on the surface of dead cells, these experiments confirmed Figures 4A - 4C the observations in

[0476] that Gal3 and PS bind to different epitopes on TIM-3. For the sugar-dependence assay, ELISA plates were coated with mGal3 (Groups 1 and 2) or hGal9 (Groups 3 and 4). Mouse TIM-3Fc protein (R&D systems) was added to the ELISA plates coated with (Groups 2 and 4) or without (Groups 1 and 3) 25 mM α-lactose (Sigma-Aldrich) at room temperature for 1 h. The secondary anti-human-IgG-HRP antibody and substrate were added as described above to detect the binding of mTIM-3-Fc to mGal3 or hGal9. Figure 5C It was shown that lactose blocked the binding of Gal9 to TIM-3, as indicated by a more than 10-fold significant reduction in the signal in Group 4 (with lactose) compared to Group 3 (without lactose), indicating sugar-dependent binding between Gal9 and TIM-3. Conversely, although the blocking effect of lactose on the binding of Gal3 to TIM-3 was minimal, there was no significant difference in the signal generated from the binding of TIM-3 and Gal3 between Group 2 (with lactose) and Group 1 (without lactose). This shows that the interaction between Gal3 and TIM-3 is affected by the presence of sugar, i.e., the interaction is sugar-independent.

[0477] Example 3: Overexpressed Gal3 inhibits T cell activation

[0478] This example describes experiments conducted to evaluate the functional properties of overexpression of Gal3 in A20 cells.

[0479] A20 clones #41, #31, and #15 stably overexpressing hGal3 were generated as described above. Figure 6AShows the results of flow cytometry analysis, showing the hGal3 expression levels in these clones. Cells of A20 or A20Gal3 clones were mixed with murine DO11.10 T cells. The mixture was placed in each well of a flat-bottom 96-well plate, and then the 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 (ThermoFisher Scientific). As Figure 6B shown, compared to when T cells were mixed with parental A20 cells, when mixed with any of the three murine A20 cell clones, the IL-2 production by murine DO11.10 T cells was significantly reduced ( Figure 6B ).

[0480] Example 4: Anti - Gal3 antibody shows anti - tumor activity in a murine lung metastasis model

[0481] The experiments in this example were conducted to evaluate the anti-tumor efficacy of Gal3:TIM-3 inhibitors in vivo. Animal experiments were conducted according to a protocol approved by the Institutional Animal Care and Use Committee of the Institute of Molecular Medicine. C57BL / 6 mice were placed in a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care after arrival. Thirty-six 7-week-old female mice were randomly divided into three groups (n = 12). On day 0, B16F10 cells (2 x 10 5 cells in 0.1 mL PBS) were washed and resuspended in PBS, and then injected into the tail vein of mice using a syringe with a 27-ga needle. After injection of B16F10 cells, murine IgG2b at 10 mg / Kg (Bio X Cell, West Lebanon, NH) was administered intraperitoneally to the animals on days 0, 3, 7, and 10, mPD1 antibody at 10 mg / Kg (Bio X Cell, West Lebanon, NH) on days 0, 3, and 7, or Gal3 antibody IMT001 at 10 mg / Kg on days 0, 3, 7, 10, and 15. The Gal3 antibody clone IMT001 used in this experiment recognizes the epitope corresponding to peptide_5 (PGAYPGQAPPGAYPGQAPPG, SEQ ID NO: 7) on Gal3. On day 21, the animals were humanely sacrificed, and the lung tissues were removed and fixed in 10% buffered formaldehyde solution. The number of black metastatic colonies on one surface of the left lobe in the lung was counted ( Figure 7B ). Results are expressed as mean ± SEM. Statistical analysis was performed using one-way ANOVA compared to the IgG control group.

[0482] Figure 7AThe mean fluorescence intensity (MFI) of B16F10 cells stained with anti-mGal3 antibody was nearly ten-fold higher than that of cells stained with isotype control antibody. Specifically, B16F10 cells were incubated on ice for 20 minutes with 10% FBS HBSS solution containing control rat IgG PE or rat anti-mouse Gal3 PE antibody (Thermo Fisher Scientific, Waltham, MA). After spinning, live / dead cells were stained with Violet Dead Cell Staining Kit (Thermo Fisher Scientific, Waltham, MA). Flow analysis was performed on the stained cells. Figure 7B Representative images of the whole lungs from three treatment groups are shown. Figure 7C The number of metastatic colonies (mean ± SEM) on the surface of the left lung lobe is shown. Figure 7D and Figure 7E The lung weight and body weight (mean ± SEM) of different treatment groups are shown. Compared with the isotype control group, the Gal3 antibody treatment group showed a significant (about 46%) reduction in the number of tumors (p < 0.01), as indicated by the number of black metastatic colonies. However, compared with the isotype control group, anti-mouse PD1 antibody 29F did not show a significant anti-tumor effect in this lung metastasis model (p > 0.05).

[0483] Example 5: Anti - Gal3 antibody shows anti - tumor activity in a 4T1 orthotopic tumor - induced lung metastasis model

[0484] Animal experiments were conducted in accordance with the protocol approved by the Institutional Animal Care and Use Committee of the Institute of Molecular Medicine. Female Balb / c mice at 7 weeks of age were placed in a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International upon arrival. On the day of tumor implantation, 4T1 cells were collected, washed and resuspended in PBS. Mice were anesthetized by inhalation anesthetic (3% to 5% isoflurane in medical grade air). 2 x 10 5 cells in 0.1 mL PBS were subcutaneously injected into the mammary gland using a syringe with a 25-ga needle. Mice were randomly divided into two groups (n = 10). After injection of 4T1 cells, mice were intraperitoneally injected with 10 mg / Kg of mouse IgG2b (Bio X cell) on days 0, 3 and 7 or with 10 mg / Kg of Gal3 antibody IMT001 on days 0, 3, 7, 10 and 14. Tumor volume and body weight were monitored twice a week. On day 30, mice were humanely sacrificed and lung tissues were inflated with 30% sucrose, removed and fixed in Bouin's solution (Sigma-Aldrich). The number of metastatic colonies on one surface of the left lung lobe was counted. Results are expressed as mean ± SEM. Statistical analysis was performed using unpaired T-test compared with the IgG control group.

[0485] Figure 8A Representative images of the whole lungs from the treatment groups are shown.Figure 8B Shows the body weights (mean ± SEM) of different treatment groups. Figure 8C Shows the number of metastatic colonies on one surface of the left lung lobe (mean ± SEM). Animals treated with monoclonal anti-human Gal3 antibody showed a significant reduction in the number of lung metastases compared to mice treated with isotype control antibody (p<0.05).

[0486] Example 6: Anti - Gal3 antibody shows anti - tumor activity in a primary murine RENCA renal tumor model

[0487] Experiments were conducted to evaluate the anti-tumor efficacy of Gal3:TIM-3 inhibitor in a primary tumor model ( Figure 9 ). Animal experiments were performed according to a protocol approved by the Institutional Animal Care and Use Committee of the Institute of Molecular Medicine. Balb / c mice were placed in a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International upon arrival. Female mice at 7 weeks of age were randomly divided into three groups (n = 15). On the day of tumor implantation, mice were anesthetized by inhalation of anesthetic (3% to 5% isoflurane in medical-grade air). Renca cells were washed and resuspended in PBS, and then 2 x 10 5 cells in 0.1 mL PBS were subcutaneously injected using a syringe with a 25-ga needle. After injection of Renca cells, mice were intraperitoneally injected with 10 mg / Kg of mouse IgG2b (Bio X Cell) or mPD1 antibody (BioXCell) on days 0, 3, and 7, or with Gal3 antibody IMT001 on days 0, 3, 7, 10, and 14. When the tumor volume in the control group reached between 2000 - 2500 mm 3 , the animals were humanely euthanized. Results are expressed as mean ± SEM. Statistical analysis was performed using unpaired t-test compared to the IgG2b control group.

[0488] Results showed the anti-tumor activity of Gal3 antibody (IMT001) in a renal cancer model. The anti-Gal3 antibody treatment group showed a significant (about 35%) reduction in tumor growth compared to the isotype control group (p<0.05), while the anti-PD-1 antibody had no effect ( Figure 9 ).

[0489] Example 7: Anti - Gal3 antibody shows anti - tumor activity in a primary murine MC38 COLON tumor model

[0490] Animal experiments were conducted in accordance with protocols approved by the Institutional Animal Care and Use Committee of the Institute of Molecular Medicine. Female C57BL / 6 mice at 7 weeks of age were placed in a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care upon arrival. On the day of tumor implantation, MC38 murine colon adenocarcinoma cells were harvested, washed, and resuspended in PBS. Mice were anesthetized by inhalation of anesthetic (3% to 5% isoflurane in medical-grade air). 5 x 10 5 cells in 0.1 mL PBS were subcutaneously injected into the right side of the mice using a syringe with a 25-ga needle. On day 7, tumor volumes were measured and the mice were randomly divided into two groups (n = 8). On days 7, 10, 14, 17, and 22, mice were intraperitoneally administered 10 mg / Kg murine IgG2b (BioXCell) or the Gal3 antibody IMT001. Tumor volume and body weight were monitored twice a week. When the tumor volume reached 3000 mm 3 , the animals were humanely euthanized. Results are expressed as mean ± SEM. Statistical analysis was performed using unpaired T-tests compared to the IgG control group.

[0491] Figure 10 The results in

[0492] Example 8: Epitope Binding of Gal3 Antibody Clone IMT001

[0493] showed that the IMT001 antibody had anti-tumor activity in the MC38 colon cancer model. Mice treated with the IMT001 antibody showed a significant reduction in tumor burden (approx. 33%) on day 24 (p < 0.05) compared to mice treated with the isotype control antibody. Figure 11A ) A peptide array containing 2420 amino acid peptides that overlap by 10 amino acids and cover the entire human Gal3 protein sequence was synthesized (Genscript, Piscataway, NJ) ( Figure 11B ). 20 μg of each peptide was spot-blotted onto a membrane. After blocking with 5% milk in PBS, the membrane was incubated with 1 μg / ml IMT001 antibody overnight at 4°C. After washing three times, the membrane was incubated with a 1:8000 diluted anti-mIgG HRP antibody (Southern Biotech, Birmingham, AL) for one hour. After washing three times, the membrane was incubated with Western ECL blotting substrate (Bio-Rad, Hercules, CA) and developed (

[0494] To further define the binding epitope of IMT001 on the above peptide, eight shorter peptides derived from it were synthesized (Genscript, Piscataway, NJ)( Figure 11C ), and their binding to IMT001 was determined by ELISA( Figure 11D ). These peptides in PBS buffer were used to coat 96-well ELISA plates (Thermo Scientific) and incubated overnight at 4 °C. The plates were washed three times with TBST, followed by blocking with PBST buffer containing 2% BSA for 1 h at room temperature. IMT001 at 10 μg / mL was incubated in the coated ELISA plates for 1 h at room temperature. The plates were washed three times, and then incubated with anti-mouse-IgG-HRP diluted 1:8000 for 1 h at room temperature. After washing three times with TBST and terminating with 50 μL of 1 N HCl, the color was developed with 100 μL of TMB Substrate (GeneTex). The optical density (OD) was read at 450 nm. The results were expressed as the mean OD ± SD of replicates. Pep-2 showed a good signal, indicating that the binding epitope of IMT001 on human Gal3 is GQAPPGAYPG.

[0495] Example 9: Immunological Characteristics in Tumors of B16F10 Lung Metastasis Mice

[0496] Mice were implanted intravenously with 1 million B16F10 cells. Then, the mice were treated with IMT001 or isotype control (10 mg / kg I.P.) on days 0, 1, 3, and 7, and sacrificed on day 8 for lung immune cell isolation and phenotypic analysis. 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. Figure 12 The results in

[0497] Example 10: Gal3 Expression Detected on Macrophages Associated with Human Lung Cancer

[0498] showed that compared with isotype control antibody treatment, treatment with the anti-Gal3 antibody IMT001 increased the numbers of various immune effector cells in the tumor-bearing lungs, including CD3 T lymphocytes, CD4 T helper cells, CD8 cytotoxic T cells, CD19 B cells, and DX5 natural killer cells. This indicates that the anti-Gal3 antibody is capable of activating immune cells. 2 O 2Incubate the sections in the middle for 10 min to block endogenous peroxidase. After washing twice in PBS for 5 min each, incubate the sections in streptavidin reagent (Molecular Probes) for 15 min at room temperature, then rinse thoroughly with PBS, incubate in biotin reagent (Molecular Probes) for 15 min, and rinse again in PBS to block endogenous biotin background. Block the sections with 10% FBS, 200 μg / mL mIgG, and 200 μg / mL hIgG for 1 h, incubate overnight at 4 °C with the first antibody IMT001-biotin (5 μg / mL), wash three times, then incubate with the second antibody HRP-avidin (BioLegend) at 1:100 for 1 h and wash three times. Staining was visualized by incubation with DAB substrate (Vector Laboratories), and staining was stopped by immersing the sections in distilled water. Human lung cancer sections were finally counterstained with hematoxylin QS (Vector Laboratories), washed in distilled water, dehydrated in a series of graded ethanol and xylene solutions, and mounted in VectaMount TM mounting medium (Vector Laboratories).

[0499] Figure 13A -B showed that the crown-like tumor-associated macrophages in those human lung cancer sections (squamous cell carcinoma and adenocarcinoma) expressed Gal3, as demonstrated by their positive staining with IMT001.

[0500] Example 11: Gal3 Expression on Human M2 Macrophages

[0501] First, 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), or M1 macrophages, or M2 macrophages in the presence of GM-CSF plus IL-4, or GM-CSF, or M-CSF (Rocky Hill, NJ), respectively. Then, flow cytometry analysis was performed to detect Gal3 expression on human dendritic cells (DCs), M1, and M2 macrophages. Specifically, 100,000 DC, M1, or M2 cells were incubated on ice for 20 minutes with 100 μl of 10% FBS HBSS solution containing control mIgG-biotin (BioLegend) or IMT001-biotin at 10 μg / ml. The cells were then washed and incubated on ice with PE-streptavidin (BioLegend) at 1:1000 for 20 minutes. After spinning, live / dead cells were stained with the Violet Dead Cell Staining Kit (Life Technologies). Flow analysis was performed on the stained cells. Figure 14C The results in Figure 14C showed that the mean fluorescence intensity (MFI) of M2 cells stained with IMT001 was much higher than that of cells stained with the isotype control antibody, indicating specific binding of IMT001 to M2 cells, while dendritic cells ( Figure 14A ) and M1 macrophages ( Figure 14B ) were likely not stained.

[0502] Example 12: Anti-Gal3 Antibody Enhances Mouse T Cell Activity in Macrophage / T Cell Responses

[0503] The expression of Gal3 on mouse macrophages was detected by IHC and flow cytometry analysis. In the details of IHC, 100,000 cells per well were seeded overnight. On the next day, the cells were washed once with PBS, fixed with 3% formaldehyde for 10 min at room temperature, then washed twice with PBS and blocked for 1 h at room temperature in PBS containing 10% FBS and 200 μg / mL. After blocking, the cells were incubated with the first antibody mIgG-biotin (BioLegend) or IMT001-biotin at 10 μg / mL overnight at 4°C, washed three times with PBST, stained with avidin-HRP (1:1000) for 1 h at room temperature, and then washed three more times with PBST. The staining was developed using a peroxidase substrate and counterstained with hematoxylin QS (Vector Laboratories). The results showed that IMT001 clearly detected Gal3 expression on macrophages ( Figure 15A ) compared to the mIgG control ( Figure 15B ).

[0504] In the experiments of flow cytometry, 100,000 RAW cells were blocked with 10% FBS plus 200 μg / mL hIgG on ice for 20 min, and then incubated with 100 μl of 10% FBS HBSS solution containing control mIgG (BD Biosciences) or IMT001 at 10 μg / ml on ice for 20 min. Then the cells were washed and incubated with APC-conjugated anti-mFc antibody (Jackson ImmunoResearch) at 1:100 on ice for 20 min. After spinning, the live / dead cells were stained with Violet Dead Cell Staining Kit (Life Technologies). Flow analysis was performed on the stained cells. Figure 15C It was shown that the mean fluorescence intensity (MFI) of RAW cells stained with IMT001 was more than 10-fold higher than those of cells stained with the isotype control antibody.

[0505] The ability of IMT001 to activate T cells was demonstrated by the mixed lymphocyte reaction (MLR) assay. RAW mouse macrophages were mixed with DO11 mouse T cells at a 1:1 ratio, treated with OVA peptide, and cultured overnight at 37 °C in the presence of mIgG (BD Biosciences) at 10 μg / ml, anti-mPD1 antibody 29F (BioXCell) or IMT001. 50 μl of the culture medium was taken for mIL-2 measurement. mIL-2 production was measured according to the Mouse IL-2 ELISA Ready-SET-Go commercial kit from eBioscience.

[0506] Figure 15D It was shown that the IMT001 antibody, rather than the mouse PD-1 antibody 29F, enhanced IL-2 production compared to cells treated with mIgG or the mPD1 antibody, indicating reversal of macrophage-induced T cell inactivation.

[0507] Example 13: Identification of Antibodies Blocking Gal3-TIM-3 Interaction

[0508] To identify Gal3-targeting antibodies with the ability to block the interaction between Gal3 and TIM-3, purified Gal3 and TIM-3 proteins were incubated in the presence (or absence) of various Gal3-targeting antibodies or control antibodies or in the absence of antibodies, and the protein interaction was evaluated by ELISA.

[0509] The human Gal3 protein (Acro Biosystems, GA3-H5129) was diluted to a concentration of 0.5 μg / ml in phosphate-buffered saline (PBS) (Corning), and 100 μl of the diluted hGal3 was added to each well of a 96-well ELISA plate (ThermoFisher, 44-2404-21). After incubating the plate overnight at 4 °C, the plate was washed three times with 300 μl of PBS with 0.05% TWEEN (VWR) (PBST) per well. Then the plate was blocked for one hour at room temperature with gentle shaking with 200 μl of 2% bovine serum albumin (BSA) (Sigma) in PBST per well. Thereafter, the 2% BSA in PBST was removed, and 50 μl of 20 μg / ml of anti-Gal3 antibody in 2% BSA in PBST was added to the wells and incubated for 10 minutes with gentle shaking at room temperature. Antibodies mab1, mab2, mab3, mab4, mab5, mab6, and mab7 were used in the experiment. The antibodies used are listed in Table 3.

[0510] Then, 50 μl of 1 μg / ml of the extracellular domain protein of human TIM-3 (AcroBiosystems, TM3-H5229) in 2% BSA in PBST was added to the wells. The plate was incubated for one hour at room temperature with gentle shaking. Then the plate was washed three times with 300 μl of PBST per well, and 100 μl of 0.3 μg / ml of biotinylated anti-human TIM-3 antibody (R&D Systems, BAF2365) in 2% BSA in PBST was added to each well. The plate was incubated for one hour with gentle shaking and then washed three times with 300 μl of PBST per well. Then 100 μl of streptavidin-HRP (1:1000) (Jackson ImmunoResearch) was added to each well, and the plate was incubated for 30 minutes with gentle shaking at room temperature. Subsequently, the plate was washed three times with 300 μl of PBST per well, and 100 μl of TMB substrate (Fisher Scientific, 34029) was added to each well. The reaction was terminated with 50 μl of 1 M HCl (VWR) per well. The absorbance of the plate was read at 450 nm using a microplate reader (Molecular Devices). The percentage of inhibition of the Gal3-TIM-3 interaction was calculated as the fraction of the signal obtained in the absence of antibody minus the background signal.

[0511] As Figure 16As shown, anti-Gal3 antibodies exhibited different abilities to block the interaction between Gal3 and TIM-3. Each of the antibodies mab1, mab2, mab4, and IMT001 disrupted the 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. The antibodies mab3 and mab5 moderately disrupted the Gal3-TIM-3 binding, with the interaction reduced to 34% and 59% of the unblocked control, respectively. Finally, mab6 and mab7 did not affect the Gal3-TIM-3 binding. The results showed that the antibodies mab1, mab2, mab3, mab4, mab5, and IMT001 all blocked the Gal3-TIM-3 interaction to some extent. It also indicated that Gal3 binding alone was not sufficient to disrupt the interaction between Gal3 and TIM-3, and this disruptive activity required specific properties.

[0512]

[0513]

[0514] Example 14: Identification of Antibodies Binding to Different Epitopes of Gal3

[0515] To determine the epitope on Gal3 related to 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 Gal3 peptides.

[0516] A library of 20-amino acid peptides was generated, each representing certain regions of hGal3 (SEQ ID NO:1). At least 2 μg / ml of the generated hGal3 peptides: Peptide 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 was added to the wells of a 96-well ELISA plate (Thermo Fisher, 44-2404-21). As a positive control, 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. Then the plate was blocked with 2% BSA in 200 μl of PBST per well for one hour with gentle shaking at room temperature. Thereafter, the 2% BSA in PBST was removed and 0.1 μg / ml of the antibody in 2% BSA in 100 μl of PBST was added to the wells ( Figure 17A-B). As for the negative control group, the antibody was applied in the absence of hGal3 peptide or hGal3 protein.

[0517] Incubate the plate with gentle shaking at room temperature for one hour and then wash three times with 300 μl of PBST per well. Subsequently, add the HRP-conjugated secondary antibody to the wells and incubate with gentle shaking at room temperature for 30 minutes. After washing the plate three times with 300 μl of PBST per well, then add 100 ul of TMB substrate (Fisher Scientific, 34029) to each well. Terminate the reaction with 50 ul of 1M HCl (VWR) per well and read the plate at an absorbance of 450 nm using a microplate reader (Molecular Devices).

[0518] The 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 varying degrees ( Figure 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 binds to this region. Antibody mab2, an antibody with strong Gal3-TIM-3 blocking activity, binds to a different Gal3 peptide, peptide 1 (SEQ ID NO:3) ( Figure 17B ). In contrast, the anti-Gal3 antibody mab7, which does not have Gal3-TIM-3 blocking activity, showed binding activity to peptides 10 (SEQ ID NO:12) and 23 (SEQ ID NO:25), while mab6 failed to show substantial binding to any peptide but did show binding to hGal3 hGal3 protein, suggesting a non-linear binding epitope for this antibody. For clarity purposes, peptides that did not bind to any Gal3 antibody were not shown. Overall, these observations identified the sequences represented by peptides 1 (SEQID 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 IDNO:1).

[0519] Example 15: Binding Domain of Anti-Gal3 Antibody

[0520] To evaluate whether anti-Gal3 antibodies with Gal3-TIM-3 blocking activity bind to the same or overlapping regions of the Gal3 molecule, an epitope binning assay was performed to assess the ability of the antibodies to bind to Gal3 simultaneously.

[0521] 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, those that were "uncoated", after incubating the plate overnight at 4 °C, the plate was washed three times with 300 μl of PBST per well. The plate was blocked with 2% BSA in PBST at 200 μl per well for one hour with gentle shaking at room temperature, and the 2% BSA in PBST was removed. 50 μl of anti-hGal3 antibodies: mab1, mab4, or mab5 (4.2 μg / ml) in 2% BSA in PBST was added to the wells and pre-incubated for 10 minutes with gentle shaking at room temperature. No antibody was added to the wells of the second control group "no antibody" for pre-incubation.

[0522] After pre-incubation 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 one hour with gentle shaking at room temperature. No antibody was added to the wells of the third control group "blank" for incubation. Subsequently, the plate was washed three times with 300 μl of PBST per well, and then 100 μl of avidin-HRP (1:1000) (Jackson ImmunoResearch) was added to each well. The plate was incubated again for 30 minutes with gentle shaking at room temperature, and then washed three times with 300 μl of PBST per well. Then 100 μl of TMB substrate (Fisher Scientific, 34029) was added to each well. The reaction was terminated with 50 μl of 1 M HCl (VWR) per well, and the absorbance of the plate was read at 450 nm using a microplate reader (Molecular Devices).

[0523] As Figure 18Shown in the figure, the antibody binding is plotted as the percentage of the unblocked control. Pre-incubation with mab1 reduced the binding of mab1, mab4, and mab5 to hGal3 compared to pre-incubation with the isotype control, indicating that these antibodies share some overlapping binding domains. Similarly, pre-incubation with mab4 greatly reduced the subsequent binding of mab1, mab4, and mab5. Although pre-incubation with mab5 reduced the binding of mab5, it only minimally affected the binding of mab1 and mab4, indicating that the competition is asymmetric, which is usually the result of low-affinity antibodies.

[0524] Example 16: Gal3-TIM-3 Blocking Antibodies Show Different Biophysical Characteristics

[0525] To determine the biophysical characteristics of the Gal3-binding antibodies, biolayer interferometry evaluations were performed using purified Gal3 protein and various antibodies. The purified antibodies were loaded onto the anti-human Fc probe at 10 μg / mL for 180 seconds using a Gator (Probe Life, East Palo Alto, CA). After equilibration in the assay buffer for 30 seconds, the loaded probe was dipped into human Gal-3 in serial dilutions of 1:2 for association, starting from 500 nM. The association was observed for 300 seconds until equilibrium. Then the probe was dipped into the assay buffer for 300 seconds for dissociation.

[0526] Real-time plots of the association and dissociation of the Gal3-binding antibodies are depicted in Figure 19A -C. Antibody mab4 showed the strongest affinity with a K D of 1.2 nM, a k -1 of 1.05E+6 M-1 sec 结合 and a k -1 of 1.32E-3 sec 解离 ( Figure 19B ). Antibody mab1 exhibited the second-strongest affinity with a K D of 13.5 nM, a k -1 of 1.7E+6 M-1 sec 结合 and a k -1 of 2.29E-2 sec 解离 . Antibody mab5 showed the weakest affinity with a K D of 32.3 nM, a k -1 of 1.41E+6 M-1 sec 结合 and a k -1 of 4.57 sec 解离 . These binding affinities are qualitatively consistent with the predicted relative affinities from the antibody competition studies in Example 15.

[0527] Example 17: Gal3-Targeting Antibodies with Gal3-TIM-3 Blocking Activity Activate Antigen-Mediated T Cell Responses

[0528] To evaluate the ability of Gal3-targeting 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 rapidly thawed in a 37 °C water bath, resuspended in 20 ml of RPMI medium with 10% FBS, and centrifuged at 1500 RPM for 5 min. The medium was discarded, the pellet was resuspended in 20 ml of medium and counted by H&E exclusion, and diluted to a final concentration of 4 million / ml in serum-free medium (Lonza). 50 ul of medium plus cells (200,000 cells / well) were added to 60 inner wells of a 96-well round bottom plate and incubated at 37 °C for 30 min. Antibodies were added to the serum-free medium to a stock concentration of 4X the final concentration (40 ug / ml). 50 ul of the 4X concentration of the antibody was added directly to the PBMCs and incubated at 37 °C for 30 min. After 30 min of incubation of the PBMCs with the antibody, 100 ul of the 2X concentration (1 ug / ml) of CMV (Astarte Biologics, Cat.#1004) was added directly to the cells and incubated at 37 °C for 4 days. On day 4, 10 ul of cell supernatant was collected to measure the human IFN-γ concentration by ELISA using a human IFN-γ ELISA kit (Invitrogen).

[0529] As Figure 20 shown, samples treated with Gal3-targeting antibodies mab6 and mab7 that do not have TIM-3-Gal3 blocking activity induced similar levels of interferon-γ secretion as samples treated with an isotype control. In contrast, Gal3-targeting antibodies mab1, mab2, mab4 and the humanized antibody IMT001 that have TIM-3-Gal3 blocking activity demonstrated significantly increased levels of interferon-γ secretion. Notably, mab5, an antibody with partial Gal3-TIM-3 blocking activity but relatively low affinity for Gal3 failed to induce significant interferon-γ secretion, indicating that the immune activation property of Gal3-targeting antibodies requires an affinity threshold. Similarly, mab3, an antibody with partial Gal3-TIM-3 blocking activity produced ambiguous results in this T cell activation assay. Overall, these data suggest that Gal3-targeting antibodies can enhance antigen-specific T cell activation and only those antibodies with blocking TIM-3-Gal3 interaction possess this activity.

[0530] Example 18: Gal3-TIM-3 Binding Surface

[0531] To identify the amino acid residues mediating the interaction between Gal3 and TIM-3, cross-linking mass spectrometry was performed. 5 μl of purified Gal3 (4.62 μM) and TIM-3 (3.74 μM) were cross-linked using the K200 MALDI MS analysis kit (CovalX). 1 μl of the K200 stabilizer reagent (2 mg / ml) was added to 9 μl of the cross-linking mixture and incubated at room temperature for 3 hours. The incubated samples were analyzed by high-quality 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 cross-linked Gal3-TIM-3 products were identified to have MH+ = 26.886 kDa and MH+ = 34.397 kDa. The cross-linked proteins were digested with trypsin, chymotrypsin, ASPN-N, elastase, or thermolysin to form individual cross-linked peptides ( Figure 21A ). The sequences of the cross-linked peptides at the ligation sites were determined ( Figure 21A -C). Due to the intrinsically unstructured nature of this region, the Gal3-TIM-3 blocking epitope of Gal3 is not included in the crystal structure model of Gal3. Note that Figure 21A the amino acid numbering depicted in

[0532] reflects the amino acid numbering in the mature protein after signal peptide processing. See Table 4, which shows the amino acid numbering corresponding to SEQ ID NO:2. Figure 21A -C). Amino acid residues near TIM-3 amino acids at positions 73 - 101 were found to cross-link with residues near Gal3 amino acids at positions 145 - 184 (

[0533] -C). These amino acids are located in the exposed regions of each molecule, suggesting that these regions are involved in the protein-protein interaction between Gal3 and TIM-3. Importantly, the anti-Gal3 antibodies mab1, mab2, mab3, mab4, and mab5 appear to bind to different epitopes as identified in the peptide binding assay, corresponding to the first 2 - 21 N-terminal amino acids 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, where this region mediates the Gal-TIM-3 interface and binds to the Gal3-TIM-3 blocking antibody. Figure 21A and the representative amino acid coding from SEQ ID NO:2.

[0534] Table 4

[0535]

[0536] Example 19: Reduction of Murine Renal Fibrosis with Anti-Gal3 Antibody

[0537] To evaluate the effect of Gal3 inhibition on renal fibrosis, IMT001 was administered to a murine model of renal fibrosis disease. Since IMT001 also demonstrated Gal3-TIM-3 blocking activity, studies showed that disruption of Gal3-TIM-3 also had an impact on renal fibrosis.

[0538] A murine model of unilateral ureteral obstruction (UUO) was created using 8-week-old male C57BL / 6 mice. Animals were randomly divided into three groups (n = 5); sham, mouse IgG2b control, and IMT001. All animal studies were conducted in accordance with protocols approved by the Institutional Animal Care and Use Committee of the Institute of Molecular Medicine. On day 0, surgery was performed to ligate the left ureter of each animal. After surgery, 10 mg / kg of mIgG2b (BioXCell) or IMT001 was administered intraperitoneally to the animals on days 1, 5, and 10. Animals in the sham group were not treated. On days 4, 8, and 15, the animals were humanely sacrificed, and the left kidney tissues were surgically removed and snap-frozen for western blot analysis.

[0539] Thirty milligrams of snap-frozen kidney tissue from the 14-day treatment UUO group was homogenized in 500 μl of RIPA buffer (Thermo Scientific). The homogenate was placed on ice for 10 minutes and then centrifuged at 12,000 rpm for 10 min at 4 °C in a 1.5 mL Eppendorf tube. The supernatant containing the proteins was collected and quantified by A280 absorbance using a Nanodrop (Thermo Fisher). The protein samples were boiled for 10 min in 4X sample buffer containing β-mercaptoethanol (Bio-Rad). Equal amounts of protein lysates (20 μl / well; 10 μg / μl) were loaded onto a precast SDS-PAGE gel (Bio-Rad) and electrophoretically separated. The separated proteins were transferred onto a polyvinylidene difluoride membrane, which was subsequently blocked with 5% non-fat milk in phosphate buffer (Fisher Scientific MT21030CM) with 0.5% TWEEN (PBST). The membrane was 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). The Western blot data were normalized to GAPDH (1:5000 dilution) (Abcam ab181602). After washing three times with PBST, the membrane was incubated with the corresponding secondary antibody conjugated to horseradish peroxidase at a 1:5000 dilution for two hours at room temperature. The membrane was washed three times with PBST, and the protein bands were detected by enhanced chemiluminescence using the standard ECL detection method recommended and developed by the manufacturer (Bio-Rad). GAPDH was used as a loading control reference.

[0540] As Figure 22 illustrated, compared to animals treated with sham surgery, animals that underwent ureteral ligation and were treated with the non-specific isotype control antibody mIgG2b showed induction of the fibrosis markers smooth muscle actin (α-SMA) and fibronectin (lanes 1-3 versus lanes 7-9). Conversely, animals that underwent ureteral ligation and were treated with IMT001 showed reduced expression of the two fibrosis markers (lanes 4-6) relative to the IgG2b control (lanes 7-9), appearing more similar to sham-treated animals. These observations suggest that blocking Gal3 and disrupting the Gal3-TIM-3 interaction can reduce renal fibrosis.

[0541] Example 20: Reduction of Murine Hepatic Fibrosis with Anti-Gal3 Antibody

[0542] The Gal3-TIM-3 blocking antibody IMT001 was used in a genetic model of non-obese diabetic and inflamed (N-IF) mice with fibrosis to study the effect of Gal3 inhibition on liver fibrosis.

[0543] The N-IF mice were generated by crossing the 24αβNOD mice and the NOD.Rag2- / - mouse strain. The N-IF mice were backcrossed to the B6.Rag2- / - mouse strain for 10 generations. The mice (male and female) were divided into two groups: the IMT001 antibody treatment group and the mIgG2b antibody control group. The antibody was administered to the animals at 10 mg / kg body weight every four days for 40 days, and then the animals were sacrificed. All efforts were made to minimize suffering. Liver and kidney tissues were collected and rapidly 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 reference.

[0544] Animals treated with the Gal3-TIM-3 blocking antibody IMT001 had a significant reduction in the expression of the fibrosis markers a-SMA and fibronectin compared to animals treated with the mIgG2b isotype control ( Figure 23 ). These data suggest that Gal3-TIM-3 blockade by IMT001 reduces liver fibrosis in the N-IF model.

[0545] Example 21: Effects of Anti-Gal3 Antibodies with / without Gal3-TIM-3 Disruptive Properties on Fibrosis

[0546] To evaluate the effect of anti-Gal3 antibodies with and without Gal3-TIM-3 blocking activity on fibrosis, in vitro cell culture-based studies were conducted.

[0547] Normal rat kidney fibroblasts (NRK-49F) were grown to 80% confluence in RPMI medium containing 10% fetal bovine serum and penicillin / streptomycin antibiotics. The medium was removed and replaced with RPMI with penicillin / streptomycin but without fetal bovine serum to induce serum starvation for 24 hours, and then the resting cells were treated with control mIgG2b (10 mg / ml), TGF-β1 (1 ng / ml), or galectin-3 anti-IMT001 (10 mg / ml) and lysed in protein extraction buffer. The lysates were analyzed for fibroblast-to-myofibroblast markers, including α-SMA and fibronectin, which induce fibrotic disease, by Western blot analysis using GAPDH as a loading control reference. Similarly, normal human kidney proximal tubular cells (HK-2) (ATCC; Rockville, MD) were grown in keratinocyte medium in a humidified incubator at 37 °C under 5% CO 2 2. The cultured cells were treated with mIgG2b (10 mg / ml), TGF-β1 (1 ng / ml), or IMT001 (10 mg / ml) and evaluated by Western blot.

[0548] Example 22: Treatment of Patients with Fibrotic Diseases

[0549] Patients presenting with jaundice and fluid retention are seen by a doctor. The doctor diagnoses the patient with liver fibrosis and prescribes a therapy comprising an anti-Gal3 antibody. The therapy is administered orally to the patient at about 10 mg / kg of patient body weight per day for one month. In some cases, the anti-Gal3 antibody also has Gal3-TIM-3 blocking properties.

[0550] Example 23: Induction of Immune System Activation in Human Subjects with Anti-Gal3 Antibody

[0551] Human subjects or patients are optionally selected according to criteria such as immune system irregularities, autoimmune diseases, immunodeficiencies, immunosuppression, cancer or fibrosis. The anti-Gal3 antibody is administered systemically by parenteral, intravenous, intramuscular, intra-arterial, intradermal, subcutaneous, intraperitoneal, intraventricular or intracranial routes. The effect of the subject on immune system irregularities, autoimmune diseases, immunodeficiencies, immunosuppression, cancer or fibrosis is monitored. The subject is also monitored by measuring the 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 analysis, liquid chromatography, mass spectrometry or enzyme-linked immunosorbent assay (ELISA).

[0552] Alternatively, leukocytes or TIM-3-rich leukocytes are isolated from the subject using techniques known in the art, such as centrifugation and fluorescence-activated cell sorting. The isolated leukocytes or TIM-3-rich leukocytes are contacted with the anti-Gal3 antibody to affect the production of at least one cytokine and induce immune activation. The contacted leukocytes or TIM-3-rich leukocytes can be autologously returned to the subject to treat immune-related diseases such as cancer or fibrosis. The therapeutic effect can be seen at 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 months, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 years.

[0553] In some embodiments, administering an anti-Gal3 antibody to a subject or contacting white blood cells or TIM-3-rich white blood cells with an anti-Gal3 antibody can reduce the interaction between Gal3 and TIM-3 to 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% of normal.

[0554] The anti-Gal3 antibody can be produced and prepared under aseptic conditions and in a regulated or controlled procedure. In this process, the anti-Gal3 antibody is used to manufacture a drug or composition. The prepared anti-Gal3 antibody is used to treat immune-related diseases such as cancer or fibrosis.

[0555] The methods for maintaining and ensuring sterility can comply with Good Manufacturing Practice (GMP), Good Tissue Practice (GTP), Good Laboratory Practice (GLP), and Good Distribution Practice (GDP) standards. The methods for maintaining and ensuring sterility include, but are not limited to, high-efficiency particulate air (HEPA) filtration, moist heat or dry heat, radiation such as X-rays, γ-rays, or UV light, bactericides or fumigants such as ethylene oxide, nitrogen dioxide, ozone, glutaraldehyde, formaldehyde, peracetic acid, chlorine dioxide, or hydrogen peroxide, preservative filling of sterile containers, packaging with plastic film or bags, or vacuum sealing.

[0556] Example 24: Discovery of Antibodies with GAL3-TIM3 Blocking Activity

[0557] To expand observations with antibodies from the original set, 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 at 7-day intervals with 50 μg of Gal3 protein fused to a linker spacer with a 6-histidine tag, GAL3-ECD-His (Acro GA3-H5129; lot number 819-43PS1-5E.), combined with a TLR agonist adjuvant mixture (50 μg MPL L, 20 μg CpG, 10 μg Poly(I:C), and 10 μg R848), repeated three times, followed by inoculation with 50 μg of GAL3-His, which was administered subcutaneously alone to the inguinal, posterior cervical, and tail base sites as well as the hock and intraperitoneal sites. Animals were sacrificed according to the IACUC protocol, and spleens, femurs, and lymph nodes (axillary, paraaxillary, mediastinal, superficial inguinal, iliac, sacral, and popliteal) were harvested. Single-cell suspensions of immune lymph nodes (LN), spleen, and bone marrow cells were obtained using two sterile frosted slides in a tissue culture dish with 15 mL of DMEM. Bone marrow was extracted from the femurs by washing through the end cap with a 5 mL syringe equipped with an 18-gauge needle. Cells from three animals were pelleted by centrifugation at 1200 RPM for 5 minutes, resuspended in 10 mL of DMEM (GIBCO 10564-011), and enumerated by counting the nucleated cells with a hemocytometer. The cells were pelleted at 1200 RPM and resuspended in SC buffer (PBS, 2% FBS, and 1 mM EDTA), and using the manufacturer's recommended protocol, with EasySep TMMouse CD138 Positive Selection Kit (StemCell Technologies) was used to isolate plasma cells. The enriched CD138-positive cells were centrifuged at 1200 RPM for 5 minutes to form a pellet, resuspended in 50 mL of electrofusion buffer (Eppendorf 940-00-220-6), and counted. Additionally, SP2 / 0-mIL6 myeloma cells (ATCC CRL2016) were centrifuged at 1200 RPM for 5 minutes to form a pellet, resuspended in 50 mL of electrofusion buffer, and counted. The myeloma cells and CD138-positive plasma cells were mixed at a 1:1 ratio, the volume was expanded to 50 mL with electrofusion buffer, the cells were centrifuged at 1200 RPM for 5 minutes to form a pellet, and the supernatant was discarded. After repeated washing and pelleting steps in electrofusion buffer, the cells were resuspended in electrofusion buffer to a concentration of 10 x 10^6 cells / mL. Up to 9 mL of the cell suspension was added to a BTX electrofusion chamber, and the cells were fused using an 800 V electrofusion protocol. The fused cells were allowed to stand for 5 minutes, transferred to a tissue culture dish containing 40 mL of medium MM (DMEM, 15% FBS, 1% glutamax, and 1% Pen / Strep), and incubated at 37 °C, 8% CO 2 for 1 hour, resuspended with a pipette, centrifuged at 1200 RPM for 5 minutes to form a pellet, resuspended in ClonaCell HY Liquid HAT Selection Medium (StemCell Technologies), and plated on a 96-well tissue culture flat bottom plate. Ten days later, the supernatant was sampled and the binding to isolated GAL3 was evaluated by ELISA. 50 μL of 0.1 μg / mL GAL3-ECD-His (Acro GA3-H5129; lot number 819-43PS1-5E) resuspended in diluent (PBS with 0.5% BSA) was added to each well for 45 minutes. The supernatant was discarded and the plate was washed with phosphate buffer (PBS) with 0.05% Tween 20. 50 μL of hybridoma supernatant diluted 1:5 in diluent was added to each well for 1 hour, followed by 5 consecutive 300 μL washes with PBS / 0.05% Tween 20. Then, 50 μL of goat anti-mouse Fc-specific antibody conjugated to horseradish peroxidase (Novex A16090) diluted 1:3000 in diluent was added to each well for 1 hour, followed by 5 consecutive 300 μL washes with PBS / 0.05% Tween 20. After washing, 50 μL of ABTS (Novex #00-202-4) was added to each well for 20 - 30 minutes, and then the absorbance was read at 405 nm on a spectrophotometer (Molecular Devices).

[0558] The binding affinity of Gal3-binding antibodies was evaluated by SPR. Kinetic experiments were performed at 25 °C in high performance mode on a Biacore T200. The ligand protein, the purified antibody, was captured onto a CM5 chip conjugated with anti-human Fc or anti-mouse Fc antibody. Three antibodies were captured onto 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 into all four flow cells at a flow rate of 30 μL / min at concentrations of 100, 50, 25, 12.5, 6.25, 3.125, and 0 nM. The complexes were allowed to associate and dissociate for 240 s and 300 s respectively. The surface was regenerated by injecting 10 mM glycine pH 1.7 (flow rate 30 μL / min) for 30 s. The data were fitted to a simple 1:1 interaction model using the global data analysis option obtained within the Biacore T200 evaluation software V2.0. It was confirmed that the affinity of the Gal3 monoclonal antibodies was greater than 30 nM for all the antibodies studied (Table 24.1). Antibodies with an affinity less than 2E-7 were selected for further characterization.

[0559] Table 24.1

[0560]

[0561]

[0562] The ability of the positive scoring wells to block the association of Gal3 and TIM3 was evaluated. To identify Gal3-targeting antibodies with the ability to block the interaction between Gal3 and TIM3, purified Gal3 and TIM3 proteins were incubated in the presence of the Gal3 immunohybridoma supernatant described above, or in the absence of antibody, and protein interaction was evaluated 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 added to the 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 with 0.05% Tween 20 [VWR, 0777]). The plate was then blocked with 2% BSA (EMD Millipore, 126609) in PBST for one hour with gentle shaking at room temperature. Thereafter, the 2% BSA in PBST was discarded, and antibodies or inhibitors (3-fold diluted starting from 20 μg / ml, 60 μg / ml, or 180 μM) in 2% BSA in PBST were added to the wells. Then, 2 μg / ml of human TIM3 (Aero Biosystems, TM3-H5229) in 2% BSA in PBST was added to the antibodies or inhibitors in the wells at a 1:1 ratio. The plate was incubated with gentle shaking for one hour at room temperature. Thereafter, the plate was 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 with gentle shaking for one hour and then washed three times with PBST. Then streptavidin-HRP (1:2000) was added to the wells. The plate was incubated with gentle shaking for one hour at room temperature and then washed three times with PBST. Then TMB substrate (Thermo Scientific, 34029) was added to each well. The reaction was terminated with 1 M HCl (JT Baker, 5620-02) and absorbance readings were taken at 450 nm using a microplate reader (Molecular Devices).

[0563] As Figure 24Depicted in , GAL3-binding antibodies exhibit variable ability to block the association of Gal3 and TIM3. Some antibodies are able to block the assembly of Gal3 and TIM3 to less than 5% of the level observed in the absence of Gal3-targeting antibodies including 846.2H3. Other Gal3-binding antibodies block the assembly of Gal3 and TIM3 to 5-20% of the level 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 block the assembly of Gal3 and TIM3 to 20-50% of the level observed in the absence of Gal3-targeting antibodies including 19B5.2E6, 13H12.2F8, and 23H9.2E4. Other Gal3-binding antibodies block the assembly of Gal3 and TIM3 to 50-75% of the level 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 show minimal blocking activity against the assembly of Gal3 and TIM3, reducing the binding of TIM3 and Gal3 by 25% or less in the absence of Gal3-targeting antibodies including 12G5.D7, 7D8.2D8, 9H2.2H1, 13G4.2F8, and 24D12.2H9.

[0564] Example 25: Different Epitopes of Gal3-Binding by Gal3-Targeting Antibodies with and without Gal3-TIM3 Blocking Activity Sites

[0565] To identify the 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, was generated and the ability to bind Gal3 antibodies was evaluated by ELISA.

[0566] hGal3 peptide at a concentration of at least 2 μg / ml or full-length human Gal3 protein (GenScript) at a concentration of 0.1 μg / ml in 50 μl of PBS, and human galectin-3 protein (Acro Biosystems, GA3-H5129) were each diluted to a concentration of at least 2 μg / ml or 0.1 μg / ml in PBS (Corning, 21-030-CM) and added to the 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 with 0.05% Tween 20 [VWR, 0777]). Then the plate was blocked with 2% BSA (EMD Millipore, 126609) in PBST for one hour with gentle shaking at room temperature. Thereafter, the 2% BSA in PBST was discarded, and the human galectin-3 hybridoma supernatant or antibody was diluted to a concentration of at least 0.1 μg / ml in 2% BSA in PBST and added to the wells. The plate was incubated for one hour with gentle shaking at room temperature and then washed three times with PBST. Then, 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 added to the wells. The plate was incubated for 30 minutes to 1 hour with gentle shaking at room temperature and then washed three times with PBST. Then the TMB substrate (Thermo Scientific, 34029) was added to each well. The reaction was terminated with 1 M HCl (JT Baker, 5620-02) and the absorbance was read at 450 nm using a microplate reader (Molecular Devices).

[0567] Gal3-binding antibodies were observed binding to the peptide array at multiple positions, and most binding was observed in peptides 1-8, summarized in Table 25.1. Significantly, all Gal3-binding antibodies with strong TIM3-Gal3 blocking activity demonstrated the ability to bind to peptide 4, 5, 6, or 7, corresponding to peptide sequences in the N-terminal domain of Gal. Specifically, six individual Gal3-binding antibodies with Gal3-TIM3 blocking activity (6H6.2D6, 20H5.A3, 20D11.2C6, 19B5.2E6, 15G7.2A7, 23H9.2E4) all bound to peptide 1 of Gal3, corresponding to amino acids 1-20 of Gal3, ADNFSLHDALSGSGNPNPQG (SEQ ID NO:3). In contrast, no Gal3-targeting antibodies with poor Gal3-TIM3 blocking activity were observed to bind peptide 1. Taken together, these data indicate that binding to Gal3 peptide 1 is predictive of the ability to block the interaction of Gal3 with TIM3. Similarly, three individual Gal3-binding antibodies with Gal3-TIM3 blocking activity (4G2.2G6, 3B11.2G2, and 13A12.2E5) bound to peptide 4 of Gal3, corresponding to amino acids 31-50 of Gal3, GAGGYPGASYPGAYPGQAPP (SEQ ID NO:6). In contrast, no Gal3-targeting antibodies with poor Gal3-TIM3 blocking activity were observed to bind peptide 4. Taken together, these data indicate that binding to Gal3 peptide 4 is predictive of the ability to block the interaction of Gal3 with TIM3. Further, thirteen Gal3-binding antibodies with Gal3-TIM3 blocking activity (mIMT001, 846T.1H2, 13H12.2F8, 19D9.2E5, 14H10.2C9, 2D10.2B2, 4A11.2B5, 846, 1B.32D.3B5, and 13A12.2E5) all bound to peptide 6 of Gal3, corresponding to amino acids 51-70 of Gal3, GAYPGQAPPGAYPGAPGAYP (SEQ ID NO:8). In contrast, no Gal3-targeting antibodies with poor Gal3-TIM3 blocking activity were observed to bind peptide 6. Taken together, these data indicate that binding to Gal3 peptide 6 is predictive of the ability to block the interaction of Gal3 with TIM3.In addition, eleven Gal3-binding antibodies with Gal3-TIM3 blocking activity (6H6.2D6, 20H5.A3, 20D11.2C6, 13H12.2F8, 19B5.2E6, 23H9.2E4, 15G7.2A7, 19D9.2C10, 27D8.2D8, 15F10.2D6, and 846.14A2) all bind to peptide 7 of Gal3, corresponding to amino acids 61-80 of Gal3, AYPGAPGAYPGAAPPGVYPG (SEQ ID NO:9). In contrast, no Gal3-targeting antibodies with poor Gal3-TIM3 blocking activity bind to peptide 7. Taken together, these data indicate that binding to Gal3 peptide 7 is predictive of the ability to block the interaction between Gal3 and TIM3. In summary, these data indicate that the binding of anti-Gal3 antibodies to Gal3 peptides 1, 4, 5, 6, and 7 serves as a prediction of the ability to block the interaction between Gal3 and TIM3.

[0568] As Figure 25 illustrated, peptides 4, 5, 6, and 7 share repetitive amino acid sequences including proline-glycine (PG) and tyrosine-proline-glycine (YPG), indicating a common feature that may explain the ability of Gal3-targeting antibodies to bind multiple Gal3 peptides. Further, 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 among peptides 4, 6, and 7, each having two such sequences separated by three amino acids. Accordingly, the presence of two closely juxtaposed GxYPG sequences may be predictive of the ability to bind Gal3-targeting antibodies with the ability to block Gal3 and TIM3. Additionally, the Grantham distances of alanine, glycine, and valine are Ala-Val: 64, Ala-Gly: 60, Val-Gly: 109, predicting that amino acids with similar low Grantham distances may similarly be able to substitute in the variable region, including proline and threonine.

[0569] Table 25.2. Galectin-3 Peptide Sequences

[0570]

[0571]

[0572] Example 26: Gal3-TIM3 Antibodies with Blocking Activity Compete for Binding to Gal3

[0573] To determine whether Gal3-binding antibodies with Gal3-TIM3 blocking activity bind to the same or overlapping regions of the Gal3 molecule, an antibody competition assay was performed to assess the ability of the antibodies to bind Gal3 simultaneously. An amine-reactive probe was loaded onto a Gator biosensor (Probe Life, Palo Alto, CA), equilibrated in dH20 for 60 seconds, immersed in 100 μl of 0.2 M EDC / 0.05 M NHS activation buffer for 30 seconds, and then immersed in a 20 μg / μl human Gal3-His solution in 10 mM NaOAc buffer, pH 5 until binding saturation, and quenched in 1 M ethanolamine, pH 8.5 for 300 seconds. After Gal3-Hi loading, the tip was immersed in 20 μg / mL saturating antibody and then sequentially in 5 μg / mL competing antibody. As Figure 26 shown, antibodies with competitive binding characteristics were assigned bins and correlated with blocking activity. After the initial bin assignment, subsequent competition experiments were performed with representative species from the bins as described to identify additional members of bins 1 and 3.

[0574] Twelve independent competitive antibody binding patterns against Gal3 were established. Significantly, a strong correlation was observed between the bins and the blocking of Gal3-TIM3 blocking activity. All antibodies from bins 1, 2, 3, 4, 5, and 6 significantly inhibited Gal3 binding to TIM3, summarized in Table 25.1. In contrast, the antibodies in bins 7 and 8 were slightly weaker blockers of Gal3 blockade of TIM3, although they had strong affinity for Gal3. The antibodies in bins 10, 11, and 12 did not have the ability to significantly inhibit the association of Gal3 and TIM3. Thus, the competitive binding bins of 1, 2, 3, 4, 5, and 6 were able to identify the ability of Gal3-binding antibodies to block the assembly of Gal3 and TIM3.

[0575] Example 27: Humanized GAL3-TIM3 Blocking Antibody Blocks GAL3-TIM3 Binding

[0576] Humanized variants of the GAL3-TIM3 blocking antibodies similarly demonstrated the ability to block the interaction of purified GAL3 and TIM3, as evaluated by ELISA, as Figure 27 illustrated. IMT001-4, IMT006-1, IMT006-5, and IMT006-8 demonstrated IC50 values of 5.6 nM, 26.5 nM, 4.1 nM, and 2.8 nM, respectively.

[0577] Example 28: GAL3-TIM3 Blocking Antibodies Demonstrate Combinatorial Antitumor Activity with Anti-PD1 or Anti-PD-L1 Antibodies

[0578] To evaluate the potential of GAL3-TIM3 blocking antibodies to affect tumor biology, in combination with antibodies targeting other immune regulatory checkpoint molecules, PD-1 and PD-L1, studies were conducted in mice bearing MBT-2 bladder tumor xenografts. Briefly, 7-week-old female C3H / HeJ mice (Jackson Laboratory) were anesthetized by inhalation of anesthetic (3% to 5% isoflurane in medical grade air), and 1x10 6 MBT-2 cells (Sekisui XenoTech, LLC) in 0.1 mL PBS were injected subcutaneously into the right flank. Seven days after tumor implantation, the mice were randomly divided into six groups (n = 9 - 10). Isotype control mIgG2b (BioXCell), anti-Gal3 (mIMT001), anti-PD1 (RMP1-14, BioXCell) plus mIgG2b, anti-PD1 (RMP1-14) plus mIMT001, anti-PDL1 (10F.9G2, BioXCell) plus mIgG2b, and anti-PDL1 (10F.9G2) plus mIMT001 were administered intraperitoneally to the mice. The 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 volume and body weight were monitored twice a week. Animals were humanely euthanized when tumor volume or animal health reached the endpoint defined by the IACUC. Results are expressed as mean ± SEM and were analyzed statistically by two-way ANOVA.

[0579] Animals treated with mIMT001 or huIgG did not show any significant reduction in tumor volume (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 volume after treatment ( Figure 28A -B). Significantly, 5 / 10 animals treated with the combination of mIMT001 and anti-PD-L1 antibody showed a strong anti-tumor response, representing a 66% increase in the response rate compared to animals treated with anti-PD-L1 antibody alone. These data indicate that the combination of antibodies blocking GAL3 and TIM3 with anti-PD-L1 antibody has significantly increased anti-tumor activity compared to anti-PD-L1 antibody alone.

[0580] Separate studies were conducted to evaluate the activity of the combination of mIMT001 with an anti-PD-1 antibody in mice with subcutaneous MBT-2 tumor implants. As in the PD-1 study, treatment with an isotype control or mIMT001 alone did not reduce tumor volume (data not shown). In contrast, treatment with the anti-PD-1 antibody resulted in an anti-tumor response in 3 / 10 animals, as demonstrated by a significant reduction in tumor volume ( Figure 28C -D). Significantly, 6 / 10 animals treated with the combination of mIMT001 and the anti-PD-1 antibody showed a strong anti-tumor response, representing a 100% increase in the response rate relative to animals treated with the anti-PD-1 antibody alone. These data indicate that the combination of an antibody blocking GAL3-TIM3 with an anti-PD-1 antibody has significantly increased anti-tumor activity compared to the anti-PD-1 antibody alone. Considering these data together with the PD-L1 combination study, these data indicate that GAL3-targeted antibodies that can block the GAL3-TIM3 interaction have the ability to more broadly enhance anti-tumor activity induced by PD-1-PD-L1 checkpoint interruption.

[0581] Example 29: GAL3-TIM3 Blocking Antibodies Demonstrate Single-Agent Antitumor Activity in HCC

[0582] Further studies evaluating the activity of GAL3-TIM3 blocking antibodies were conducted in the setting of a spontaneous hepatocellular carcinoma (HCC) model induced in STAM-CDAA mice. Briefly, two-day-old male C57Bl / 6 mice were induced to have pancreatic islet destruction by a single subcutaneous injection of 200 μg of streptozotocin and then were fed a CDAA high-fat diet (study diet #A06071302) starting at 4 weeks of age and continued throughout the duration of each study. At 8 weeks of age, the mice were divided into two groups (seven mice per group). Mice were treated by intraperitoneal injection twice weekly with a human anti-mIgG4 isotype control (hIgG4, 10 mg / kg) or a human anti-Gal3 antibody (IMT001-4, 10 mg / kg) for 4 weeks. All animal care and procedures were approved by the Immutics IACUC.

[0583] As Figure 29AAs described in -B, while tumors were not observed in animals maintained on a normal diet, STAM-CDAA animals treated with an isotype control antibody exhibited signs of multifocal tumorigenesis, as confirmed by gross observation, with severe ( > 5 tumors per liver) formation noted in 4 / 7 animals and moderate formation (3 - 5 tumors per liver) noted in 2 / 7 animals, while only 1 / 7 was noted to be grossly tumor-free. In contrast, in animals treated with IMT001-4, tumor formation was significantly reduced, with only 1 / 7 animals exhibiting severe tumor formation, indicating a 75% reduction in severe tumor formation, and 1 / 7 animals exhibiting moderate tumor formation, indicating a 50% reduction in moderate tumor formation. Correspondingly, animals treated with IMT001-4 did not exhibit signs of gross tumor formation in 5 / 7 animals, indicating a 400% increase in animals that were clearly tumor-free.

[0584] Microscopic examination of tumor samples stained with hematoxylin and eosin was performed to assess the histology of the observed tumors. Briefly, the liver was fixed in 4% paraformaldehyde (Electron Microscopy Sciences, Cat#15710S) for 24 hours, transferred to 70% EtOH for 72 hours, and then the samples were embedded in paraffin. 5 mM samples were sectioned and mounted on Apex advanced adhesive slides (Leica, Cat#3800080), then deparaffinized, rehydrated in sequential ethanol baths, stained in hematoxylin (Cat#HHS32-1L, MilliporeSigma) for 5 min, Define (Leica, Cat#3803590) for 1 min, bluing buffer (Leica, Cat#3802916) for 1 min and alcoholic eosin Y 515 (Leica, Cat#3801616) for 30 s, then dehydrated, cleared and coverslipped (Sakura Finetek, Cat#6500). Brightfield images were acquired under a Revolve microscope (DiscoverEcho, Inc.).

[0585] Consistent with the tumors observed grossly in the livers of animals treated with the isotype control, the tissue sections showed large multifocal areas of dysplastic hepatocytes surrounded by fatty change areas of fatty liver ( Figure 29C ). The fatty change was expected as a result of the diet administered. Liver sections from animals treated with IMT001-4 showed significantly fewer areas of plaques of dysplastic hepatocytes, Figure 29C with rare representative areas depicted in. Note that, in addition to the increased rarity of tumor plaques, the size of the tumor areas in animals treated with IMT001-4 was also significantly smaller than that of animals treated with the control.

[0586] To more systematically evaluate the abundance of HCC in STAM-CDAA mice, the serum levels of alpha-fetoprotein (AFP) (a human clinical biomarker of HCC emergence) were evaluated in isotype- and IMT001-4-treated animals. Serum AFP was measured by ELISA (R&D systems #MAFP00) according to the manufacturer's instructions.

[0587] Normal mice exhibited low levels of AFP in serum, however, in STAM-CDAA mice treated with isotype control, AFP was significantly elevated, with 3 / 6 animals showing >2000 ng AFP / mL, and 6 / 6 animals showing >1000 ng AFP / mL( Figure 29D ). In contrast, IMT...

Claims

1. An anti-GAL3 antibody, which comprises: HCDR1 consisting of the sequence of SEQ ID NO:39; HCDR2 consisting of the sequence of SEQ ID NO:67; HCDR3 consisting of the sequence of SEQ ID NO:95; LCDR1 consisting of the sequence of SEQ ID NO:123; LCDR2 consisting of the sequence of SEQ ID NO:151; and LCDR3 consisting of the sequence of SEQ ID NO:

179.

2. The anti-GAL3 antibody according to claim 1, which comprises: a heavy chain variable region (VH) comprising a sequence having at least 80% identity with SEQ ID NO:207; and a light chain variable region (VL) comprising a sequence having at least 80% identity with SEQ ID NO:

235.

3. The anti-GAL3 antibody according to claim 1, which comprises: a heavy chain variable region (VH) comprising SEQ ID NO:207; and a light chain variable region (VL) comprising SEQ ID NO:

235.

4. An anti-GAL3 antibody, which comprises: HCDR1 consisting of the sequence of SEQ ID NO:58; HCDR2 consisting of the sequence of SEQ ID NO:86; HCDR3 consisting of the sequence of SEQ ID NO:114; LCDR1 consisting of the sequence of SEQ ID NO:142; LCDR2 consisting of the sequence of SEQ ID NO:170; and LCDR3 consisting of the sequence of SEQ ID NO:

198.

5. The anti-GAL3 antibody according to claim 4, which comprises: a heavy chain variable region (VH) comprising a sequence having at least 80% identity with SEQ ID NO:226; and a light chain variable region (VL) comprising a sequence having at least 80% identity with SEQ ID NO:

254.

6. The anti-GAL3 antibody according to claim 4, which comprises: a heavy chain variable region (VH) comprising a sequence containing SEQ ID NO:226; and a light chain variable region (VL) comprising a sequence containing SEQ ID NO:

254.

7. The anti-GAL3 antibody according to any one of claims 1-6, wherein the antibody comprises a humanized antibody.

8. Use of the anti-GAL3 antibody according to any one of claims 1-7 in the preparation of a medicament for activating an immune response in a subject.

9. Use of the anti-GAL3 antibody according to any one of claims 1-7 in the preparation of a medicament for promoting the proliferation of T cells or natural killer (NK) cells.

10. Use of the anti-Gal3 antibody according to any one of claims 1-7 in the preparation of a medicament for reducing fibrosis or its tendency in a tissue.

11. Use according to any one of claims 8 - 10, wherein the use comprises contacting a plurality of cells comprising Gal3-expressing cells and TIM-3-expressing cells with the antibody under conditions that disrupt the interaction between Gal3 and TIM-3.

12. Use according to claim 11, 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%.

13. Use according to any one of claims 8 - 10, wherein the anti-Gal3 antibody binds to Gal3-expressing cells that express cytokines, and wherein binding of the antibody to Gal3 induces immune activation.

14. Use according to claim 13, wherein the cytokine is interferon or interleukin.

15. Use according to claim 14, wherein the immune activation comprises proliferation of CD3+ T lymphocytes, CD4+ T helper cells, CD8+ cytotoxic T cells, natural killer cells, M1 macrophages or a combination thereof.

16. Use according to any one of claims 11 - 15, wherein the contacting further comprises a reduction in the population of M2 macrophages within the TME.

17. Use according to any one of claims 11 - 16, wherein the antibody binds to at least one amino acid residue within the Gal3 region corresponding to residues 1 - 20 of SEQ ID NO:

1.

18. Use according to any one of claims 11 - 17, wherein the antibody comprises 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 .

19. Use according to any one of claims 11 - 18, wherein the antibody further comprises an Fc mutation and / or the antibody comprises a chimeric antibody.

20. Use according to any one of claims 11 - 19, wherein the use comprises administering the antibody to a subject prior to the contacting step.

Images