Anti-gal3 antibodies and compositions

Abstract

The present invention relates to anti-Gal3 antibodies and methods of using them to treat diseases and conditions that benefit from modulating Gal3 activity, such as fibrotic diseases, inflammatory diseases, autoimmune diseases, immune-mediated disorders, cancer, neurodegenerative diseases, metabolic diseases, or infectious diseases.

Description

Background of the Invention

[0002] Galectins are multifunctional proteins that specifically bind to β-galactoside-containing ligands through their carbohydrate recognition domain (CRD).

[0003] Galectin 3 (Gal3) is a member of this family, which includes 15 galectins that are highly evolutionarily conserved in mammals. Most galectins contain two carbohydrate recognition domains (CRDs) that are either homodimers (galectins 1, 2, 5, 7, 10, 11, 13, 14, 15) or heterodimers (galectins 5, 6, 8, 9, 12). Gal3 is the only monomeric galectin that contains a single CRD linked to a collagen-like N-terminal domain, and has also been described to oligomerize through this N-terminal domain depending on environmental conditions.

[0004] Gal3 can bind to several sugar-containing ligands, preferentially recognizing N-acetyl-lactosamine structures present in intracellular, membrane-associated or extracellular proteins. The asialofetuin (ASF) glycoprotein is one of the prototypical ligands for Gal3, and it can also bind to galectin 1 (Gal1) or galectin 7 (Gal7).

[0005] Gal3 is a multi-compartment protein, present in cells, outside cells or in combination with cell membranes. Intracellular Gal3 has been described as regulating several processes related to cell growth or apoptosis pathways or cell signaling events. When combined with the ligand of the plasma membrane, it was observed that Gal3 affects the expression, location and activity of several cell surface receptors, thus regulating various biological functions such as cell migration or cell adhesion. In the extracellular compartment, Gal3 is mainly involved in maintaining the integrity and cell adhesion process of the extracellular matrix. In some cases, the expression destruction of Gal3 and / or its extracellular interactome have been shown to cause several pathological processes related to inflammation, fibrotic immune system disorders, angiogenesis, tumor metastasis and progression and cell homeostasis.

[0006] Gal3 is widely expressed in cells of the innate immune system, among others, and has been shown to stimulate inflammatory responses by increasing chemoattraction and stimulating myeloid cells (including macrophages) to produce proinflammatory cytokines. Gal3 is also expressed by fibroblasts and endothelial cells, and in combination with activated macrophages, promotes tissue fibrosis through proliferation of fibroblasts and increased collagen production.

[0007] Increased Gal3 expression is associated with many fibrotic diseases, including heart failure, renal failure, systemic sclerosis, pulmonary fibrosis, liver fibrosis, atherosclerosis, as well as inflammatory diseases, autoimmune diseases, immune-mediated disorders, neurodegenerative diseases, metabolic diseases, infectious diseases and different types of cancer. Therefore, inhibition of Gal3 interactions is an attractive mechanism for treating these conditions.

[0008] The main forms used to target Gal3 are small molecules and large complex carbohydrates such as pectin derivatives. They are generally described as being able to target Gal3, but sometimes also other galectins, thus lacking selectivity.

[0009] For example, TD139 (GB-0139) developed by Galecto and Bristol-Myers Squibb or Belapectin (GR-MD-02) developed by Galectin Therapeutics are considered to be dual inhibitors of Gal3 and Gal1. Therefore, they show poor selectivity to Gal3, respectively. In addition, TD139 has poor bioavailability and is formulated to be inhaled twice a day, can mainly reach the lungs, and is developed for the treatment of idiopathic pulmonary fibrosis. This route of administration may also be disadvantageous for the treatment of other conditions.

[0010] This dual selectivity is also associated with the complex carbohydrate Davanat (GM-CT-01, also developed by Galectin Therapeutics) or GCS-100, developed by La Jolla Pharmaceuticals.

[0011] Another small molecule in development is GB1211 from Galecto, an oral inhibitor described as targeting Gal3, including intracellular Gal3, as it can cross the plasma membrane, which is different from our antibodies that target extracellular Gal3.

[0012] The identification of small molecule or complex carbohydrate Gal3 inhibitors has encountered many obstacles due to the fact that sugar chemistry design is complex and difficult and may result in compounds with low selectivity and / or potency and / or bioavailability.

[0013] Antibody therapeutic approaches are suitable for reaching targets with low druggability and can show higher selectivity, potency, and bioavailability compared to chemical entities and pectin derivatives.

[0014] True Binding recently described an anti-Gal3 antibody with its TB001 monoclonal antibody in preclinical stage and TB006 monoclonal antibody in phase 1 for Alzheimer's disease and acute ischemic stroke indications. These antibodies are described as inhibitors of the interaction of Gal3 and TIM3. They are not described as specifically targeting Gal3-CRD.

[0015] Therefore, there is a need to develop new therapeutic entities that specifically target Gal3, especially Gal3 in its CRD, to reach the site of interaction with its ligand, avoiding the selectivity, bioavailability or efficacy problems encountered in the development of small and medium molecule compounds or pectin derivatives. Summary of the invention

[0016] The present invention relates to novel monoclonal antibodies that bind to extracellular galectin 3 (Gal3), in particular to the carbohydrate recognition domain of Gal3 (Gal3-CRD), and do not bind to either galectin 1 (Gal1) or galectin 7 (Gal7).

[0017] The invention also relates to pharmaceutical compositions comprising one or more of these antibodies, and the use of the antibodies and pharmaceutical compositions for inhibiting disease progression in patients or for ameliorating, preventing and / or treating patients suffering from Gal3-related diseases or Gal3-related disorders, including fibrotic diseases such as heart failure, renal failure, systemic sclerosis, pulmonary fibrosis and liver fibrosis, and including inflammatory diseases, autoimmune diseases, neurodegenerative diseases, metabolic diseases, infectious diseases and various cancers. It is expected that the antibodies of the invention, whether alone or in combination with another therapeutic molecule, exhibit specific properties compared to molecules currently being developed for Gal3-related diseases or Gal3-related disorders.

[0018] In one embodiment, the present invention provides an anti-Gal3 antibody or antigen-binding fragment thereof, wherein the anti-Gal3 antibody or antigen-binding fragment thereof is any antibody referred to herein as antibody D06, D11, E01, E02, E07, G03, H07, H10, E12 or B12, or a variant thereof, wherein the variant may comprise certain minimal amino acid changes relative to antibody D06, D11, E01, E02, E07, G03, H07, H10, E12 or B12 without losing the antigen-binding specificity of the parent antibody.

[0019] In one embodiment, the anti-Gal3 antibody or antigen-binding fragment thereof competes for binding to human Gal3 with an antibody whose heavy (H) CDR1-3 and light (L) CDR1-3 are identical to or derived from the H-CDR1-3 and L-CDR1-3 of antibody D06, D11, E01, E02, E07, G03, H07, H10, E12 or B12.

[0020] In one embodiment, the anti-Gal3 antibody or antigen-binding fragment thereof comprises H-CDR1-3 comprising the H-CDR1-3 sequence of antibody D06, D11, E01, E02, E07, G03, H07, H10, E12 or B12, respectively.

[0021] In one embodiment, the anti-Gal3 antibody or antigen-binding fragment thereof comprises L-CDR1-3, which comprises the L-CDR1-3 sequence of antibody D06, D11, E01, E02, E07, G03, H07, H10, E12 or B12, respectively.

[0022] In one embodiment, the anti-Gal3 antibody or antigen-binding fragment thereof comprises the H-CDR1-3 and L-CDR1-3 amino acid sequences of antibody D06, D11, E01, E02, E07, G03, H07, H10, E12 or B12.

[0023] In one embodiment, the anti-Gal3 antibody or antigen-binding fragment thereof comprises a heavy chain complementarity determining region (H-CDR) 1 comprising the amino acid sequence of any one of SEQ ID NOs: 1-5.

[0024] In one embodiment, the anti-Gal3 antibody or antigen-binding fragment thereof comprises a heavy chain complementarity determining region (H-CDR) 2 comprising the amino acid sequence of any one of SEQ ID NOs: 6-11.

[0025] In one embodiment, the anti-Gal3 antibody or antigen-binding fragment thereof comprises a heavy chain complementarity determining region (H-CDR) 3 comprising the amino acid sequence of any one of SEQ ID NOs: 12-19.

[0026] In one embodiment, the anti-Gal3 antibody or antigen-binding fragment thereof comprises a heavy chain complementarity determining region (L-CDR) 1 comprising the amino acid sequence of any one of SEQ ID NOs: 20-26.

[0027] In one embodiment, the anti-Gal3 antibody or antigen-binding fragment thereof comprises a heavy chain complementarity determining region (L-CDR) 2 comprising the amino acid sequence of any one of SEQ ID NOs: 27-29.

[0028] In one embodiment, the anti-Gal3 antibody or antigen-binding fragment thereof comprises a heavy chain complementarity determining region (L-CDR) 3 comprising the amino acid sequence of any one of SEQ ID NOs: 30-37.

[0029] In one embodiment, the anti-Gal3 antibody or antigen-binding fragment thereof has a heavy chain variable domain (VH) that is at least 90% (e.g., at least 92%, at least 95%, at least 98% or at least 99%) identical to the VH amino acid sequence of SEQ ID NO: 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 81, 82, 84, 85, 87, 88, 90, 92, 94, 95, 97, 99, 101, 103 or 104.

[0030] In one embodiment, the anti-Gal3 antibody or antigen-binding fragment thereof has a VH comprising SEQ ID NO: 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 81, 82, 84, 85, 87, 88, 90, 92, 94, 95, 97, 99, 101, 103, or 104.

[0031] In one embodiment, the anti-Gal3 antibody or antigen-binding fragment thereof has a heavy chain (HC) comprising a VH amino acid sequence of SEQ ID NO: 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 81, 82, 84, 85, 87, 88, 90, 92, 94, 95, 97, 99, 101, 103 or 104 and a heavy chain constant region amino acid sequence of SEQ ID NO: 58 or 59.

[0032] In one embodiment, the anti-Gal3 antibody or antigen-binding fragment thereof has a light chain variable domain (VL) that is at least 90% (e.g., at least 92%, at least 95%, at least 98% or at least 99%) identical to the VL amino acid sequence of SEQ ID NO: 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 83, 86, 89, 91, 93, 96, 98, 100, 102 or 105.

[0033] In one embodiment, the anti-Gal3 antibody or antigen-binding fragment thereof has a VL comprising SEQ ID NO: 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 83, 86, 89, 91, 93, 96, 98, 100, 102, or 105.

[0034] In one embodiment, the anti-Gal3 antibody or antigen-binding fragment thereof has a light chain (LC) comprising the VL amino acid sequence of SEQ ID NO: 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 83, 86, 89, 91, 93, 96, 98, 100, 102 or 105 and the light chain constant region amino acid sequence of SEQ ID NO: 60.

[0035] In certain embodiments, the anti-Gal3 antibody or antigen-binding fragment thereof of the present invention comprises the following H-CDR 1-3 and L-CDR 1-3 amino acid sequences:

[0036] a) SEQ ID NOs: 1, 6, 12, 20, 27 and 30, respectively;

[0037] b) SEQ ID NO: 1, 6, 13, 21, 27 and 31, respectively;

[0038] c) SEQ ID NO: 1, 6, 14, 22, 27 and 31, respectively;

[0039] d) SEQ ID NOs: 5, 7, 3, 21, 28 and 32, respectively;

[0040] e) SEQ ID NOs: 3, 8, 15, 23, 27 and 33, respectively;

[0041] f) SEQ ID NO: 2, 6, 16, 24, 27 and 34, respectively;

[0042] g) SEQ ID NO: 1, 9, 14, 24, 27 and 34, respectively;

[0043] h) SEQ ID NO: 2, 10, 17, 25, 27 and 35, respectively;

[0044] i) SEQ ID NO: 4, 11, 18, 21, 29 and 36, respectively; or

[0045] j) SEQ ID NO: 2, 10, 19, 26, 27 and 37, respectively.

[0046] In certain embodiments, the anti-Gal3 antibodies or antigen-binding fragments thereof of the invention:

[0047] a) a VH having at least 90% (e.g., at least 92%, at least 95%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 38, 81 or 82; and a VL having at least 90% (e.g., at least 92%, at least 95%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 39 or 83;

[0048] b) a VH having at least 90% (e.g., at least 92%, at least 95%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 40, 84 or 85; and a VL having at least 90% (e.g., at least 92%, at least 95%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 41 or 86;

[0049] c) a VH having at least 90% (e.g., at least 92%, at least 95%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 42, 87 or 88; and a VL having at least 90% (e.g., at least 92%, at least 95%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 43 or 89;

[0050] d) a VH having at least 90% (e.g., at least 92%, at least 95%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 44 or 90; and a VL having at least 90% (e.g., at least 92%, at least 95%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 45 or 91;

[0051] e) a VH having at least 90% (e.g., at least 92%, at least 95%, at least 98%, or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 46 or 92; and a VL having at least 90% (e.g., at least 92%, at least 95%, at least 98%, or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 47 or 93;

[0052] f) a VH having at least 90% (e.g., at least 92%, at least 95%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 48, 94 or 95; and a VL having at least 90% (e.g., at least 92%, at least 95%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 49 or 96;

[0053] g) a VH having at least 90% (e.g., at least 92%, at least 95%, at least 98%, or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 50 or 97; and a VL having at least 90% (e.g., at least 92%, at least 95%, at least 98%, or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 51 or 98;

[0054] h) a VH having at least 90% (e.g., at least 92%, at least 95%, at least 98%, or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 52 or 99; and a VL having at least 90% (e.g., at least 92%, at least 95%, at least 98%, or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 53 or 100;

[0055] i) a VH having at least 90% (e.g., at least 92%, at least 95%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 54 or 101; and a VL having at least 90% (e.g., at least 92%, at least 95%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 55 or 102; or

[0056] j) a VH having at least 90% (e.g., at least 92%, at least 95%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:56, 103 or 104; and a VL having at least 90% (e.g., at least 92%, at least 95%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:57 or 105.

[0057] In certain embodiments, the anti-Gal3 antibodies or antigen-binding fragments thereof of the invention:

[0058] a) a VH comprising, or consisting of, an amino acid sequence of SEQ ID NO: 38, 81 or 82; and a VL comprising, or consisting of, an amino acid sequence of SEQ ID NO: 39 or 83;

[0059] b) a VH comprising, or consisting of, an amino acid sequence of SEQ ID NO: 40, 84 or 85; and a VL comprising, or consisting of, an amino acid sequence of SEQ ID NO: 41 or 86;

[0060] c) a VH comprising, or consisting of, an amino acid sequence of SEQ ID NO: 42, 87 or 88; and a VL comprising, or consisting of, an amino acid sequence of SEQ ID NO: 43 or 89;

[0061] d) a VH comprising, or consisting of, an amino acid sequence of SEQ ID NO: 44 or 90; and a VL comprising, or consisting of, an amino acid sequence of SEQ ID NO: 45 or 91;

[0062] e) a VH comprising, or consisting of, an amino acid sequence of SEQ ID NO: 46 or 92; and a VL comprising, or consisting of, an amino acid sequence of SEQ ID NO: 47 or 93;

[0063] f) a VH comprising, or consisting of, an amino acid sequence of SEQ ID NO: 48, 94 or 95; and a VL comprising, or consisting of, an amino acid sequence of SEQ ID NO: 49 or 96;

[0064] g) a VH comprising, or consisting of, an amino acid sequence of SEQ ID NO: 50 or 97; and a VL comprising, or consisting of, an amino acid sequence of SEQ ID NO: 51 or 98;

[0065] h) a VH comprising, or consisting of, an amino acid sequence of SEQ ID NO: 52 or 99; and a VL comprising, or consisting of, an amino acid sequence of SEQ ID NO: 53 or 100;

[0066] j) a VH comprising, or consisting of, an amino acid sequence of SEQ ID NO: 54 or 101; and a VL comprising, or consisting of, an amino acid sequence of SEQ ID NO: 55 or 102; or

[0067] i) a VH comprising, or consisting of, an amino acid sequence of SEQ ID NO: 56, 103 or 104; and a VL comprising, or consisting of, an amino acid sequence of SEQ ID NO: 57 or 105.

[0068] In certain embodiments, the anti-Gal3 antibody:

[0069] a) a LC having an amino acid sequence comprising or consisting of SEQ ID NOs: 39 and 60; and a HC having an amino acid sequence comprising or consisting of SEQ ID NOs: 38 and 58;

[0070] b) a LC having an amino acid sequence comprising or consisting of SEQ ID NOs: 41 and 60; and a HC having an amino acid sequence comprising or consisting of SEQ ID NOs: 40 and 58;

[0071] c) a LC having an amino acid sequence comprising or consisting of SEQ ID NOs: 43 and 60; and a HC having an amino acid sequence comprising or consisting of SEQ ID NOs: 42 and 58;

[0072] d) a LC having an amino acid sequence comprising or consisting of SEQ ID NOs: 45 and 60; and a HC having an amino acid sequence comprising or consisting of SEQ ID NOs: 44 and 58;

[0073] e) a LC having an amino acid sequence comprising or consisting of SEQ ID NOs: 47 and 60; and a HC having an amino acid sequence comprising or consisting of SEQ ID NOs: 46 and 58;

[0074] f) LC having an amino acid sequence comprising or consisting of SEQ ID NOs: 49 and 60; and HC having an amino acid sequence comprising or consisting of SEQ ID NOs: 48 and 58;

[0075] g) a LC having an amino acid sequence comprising or consisting of SEQ ID NOs: 51 and 60; and a HC having an amino acid sequence comprising or consisting of SEQ ID NOs: 50 and 58;

[0076] h) a LC having an amino acid sequence comprising or consisting of SEQ ID NOs: 53 and 60; and a HC having an amino acid sequence comprising or consisting of SEQ ID NOs: 52 and 58;

[0077] i) a LC having an amino acid sequence comprising or consisting of SEQ ID NOs: 55 and 60; and a HC having an amino acid sequence comprising or consisting of SEQ ID NOs: 54 and 58;

[0078] j) a LC having an amino acid sequence comprising or consisting of SEQ ID NOs: 57 and 60; and a HC having an amino acid sequence comprising or consisting of SEQ ID NOs: 56 and 58.

[0079] In certain embodiments, the anti-Gal3 antibody:

[0080] a) a LC having an amino acid sequence comprising or consisting of SEQ ID NOs: 39 and 60; and a HC having an amino acid sequence comprising or consisting of SEQ ID NOs: 38 and 59;

[0081] b) a LC having an amino acid sequence comprising or consisting of SEQ ID NOs: 41 and 60; and a HC having an amino acid sequence comprising or consisting of SEQ ID NOs: 40 and 59;

[0082] c) a LC having an amino acid sequence comprising or consisting of SEQ ID NOs: 43 and 60; and a HC having an amino acid sequence comprising or consisting of SEQ ID NOs: 42 and 59;

[0083] d) a LC having an amino acid sequence comprising or consisting of SEQ ID NOs: 45 and 60; and a HC having an amino acid sequence comprising or consisting of SEQ ID NOs: 44 and 59;

[0084] e) a LC having an amino acid sequence comprising or consisting of SEQ ID NOs: 47 and 60; and a HC having an amino acid sequence comprising or consisting of SEQ ID NOs: 46 and 59;

[0085] f) LC having an amino acid sequence comprising or consisting of SEQ ID NOs: 49 and 60; and HC having an amino acid sequence comprising or consisting of SEQ ID NOs: 48 and 59;

[0086] g) a LC having an amino acid sequence comprising or consisting of SEQ ID NOs: 51 and 60; and a HC having an amino acid sequence comprising or consisting of SEQ ID NOs: 50 and 59;

[0087] h) a LC having an amino acid sequence comprising or consisting of SEQ ID NOs: 53 and 60; and a HC having an amino acid sequence comprising or consisting of SEQ ID NOs: 52 and 59;

[0088] i) a LC having an amino acid sequence comprising or consisting of SEQ ID NOs: 55 and 60; and a HC having an amino acid sequence comprising or consisting of SEQ ID NOs: 54 and 59;

[0089] j) a LC having an amino acid sequence comprising or consisting of SEQ ID NOs: 57 and 60; and a HC having an amino acid sequence comprising or consisting of SEQ ID NOs: 56 and 59.

[0090] In certain embodiments, the anti-Gal3 antibody:

[0091] a) a LC having an amino acid sequence comprising or consisting of SEQ ID NOs: 83 and 60; and a HC having an amino acid sequence comprising or consisting of SEQ ID NOs: 81 or 82 and SEQ ID NOs: 58 or 59;

[0092] b) a LC having an amino acid sequence comprising or consisting of SEQ ID NOs: 86 and 60; and a HC having an amino acid sequence comprising or consisting of SEQ ID NOs: 84 or 85 and SEQ ID NOs: 58 or 59;

[0093] c) a LC having an amino acid sequence comprising or consisting of SEQ ID NOs: 89 and 60; and a HC having an amino acid sequence comprising or consisting of SEQ ID NOs: 87 or 88 and SEQ ID NOs: 58 or 59;

[0094] d) a LC having an amino acid sequence comprising or consisting of SEQ ID NOs: 91 and 60; and a HC having an amino acid sequence comprising or consisting of SEQ ID NO: 90 and SEQ ID NOs: 58 or 59;

[0095] e) a LC having an amino acid sequence comprising or consisting of SEQ ID NOs: 93 and 60; and a HC having an amino acid sequence comprising or consisting of SEQ ID NO: 92 and SEQ ID NOs: 58 or 59;

[0096] f) a LC having an amino acid sequence comprising or consisting of SEQ ID NOs: 96 and 60; and a HC having an amino acid sequence comprising or consisting of SEQ ID NOs: 94 or 95 and SEQ ID NOs: 58 or 59;

[0097] g) a LC having an amino acid sequence comprising or consisting of SEQ ID NOs: 98 and 60; and a HC having an amino acid sequence comprising or consisting of SEQ ID NO: 97 and SEQ ID NOs: 58 or 59;

[0098] h) a LC having an amino acid sequence comprising or consisting of SEQ ID NOs: 100 and 60; and a HC having an amino acid sequence comprising or consisting of SEQ ID NO: 99 and SEQ ID NOs: 58 or 59;

[0099] i) a LC having an amino acid sequence comprising or consisting of SEQ ID NOs: 102 and 60; and a HC having an amino acid sequence comprising or consisting of SEQ ID NO: 101 and SEQ ID NOs: 58 or 59;

[0100] j) a LC having an amino acid sequence comprising or consisting of SEQ ID NOs: 105 and 60; and a HC having an amino acid sequence comprising or consisting of SEQ ID NOs: 103 or 104 and SEQ ID NOs: 58 or 59.

[0101] In one embodiment, the anti-Gal3 antibody competes for binding to human Gal3 with antibody D06, D11, E01, E02, E07, G03, H07, H10, B12, or E12.

[0102] In one embodiment, the anti-Gal3 antibody competes for binding to human Gal3 with antibody D06-G1, D06-G2, D11-G1, D11-G2, E01-G, E02-G, E07-G1, E07-G2, G03-G1, G03-G2, H07-G, H10-G, B12-G, E12-G1, or E12-G2.

[0103] In some embodiments, the antibodies of the invention are of the IgG isotype, such as the IgG isotype subclass IgG1 or IgG2. The IgG1 constant chain sequence is shown in SEQ ID NO 58. In certain embodiments, the antibodies comprise at least one mutation in the Fc region. In a specific embodiment, the antibodies comprise one or more mutations at heavy chain amino acid positions 228, 234, and 235, the positions being numbered according to EU or Numbering scheme number. For example, one or both of the amino acid residues at positions 234 and 235 can be mutated from Leu to Ala, and / or the amino acid residue at position 228 can be mutated from Ser to Pro. The IgG1 constant chain sequence with positions 234 and 235 mutated from Leu to Ala is shown in SEQ ID NO 59.

[0104] In another aspect, the present invention provides a pharmaceutical composition comprising at least one anti-Gal3 antibody or antigen-binding fragment thereof as described herein and a pharmaceutically acceptable excipient, optionally comprising an additional therapeutic agent as described herein.

[0105] The present invention also provides an isolated nucleic acid molecule, which comprises a nucleotide sequence encoding a heavy chain or VH of an anti-Gal3 antibody or antigen-binding fragment, a nucleotide sequence encoding a light chain or VL of an anti-Gal3 antibody or antigen-binding fragment, or a nucleotide sequence encoding a heavy chain or VH or a light chain or VL of an anti-Gal3 antibody or antigen-binding fragment thereof. The present invention also provides a combination of isolated nucleic acid molecules encoding anti-Gal3 antibodies or antigen-binding fragments thereof, in particular a first isolated nucleic acid molecule and a second isolated nucleic acid molecule, wherein the first isolated nucleic acid molecule comprises or consists of a sequence encoding a heavy chain sequence or a VH sequence, and the second isolated nucleic acid molecule comprises or consists of a sequence encoding a light chain sequence or a VL sequence.

[0106] The present invention also provides a vector comprising such an isolated nucleic acid molecule or a combination of isolated nucleic acid molecules, wherein the vector may further comprise an expression control sequence.

[0107] The present invention also provides a host cell comprising a nucleotide sequence encoding the heavy chain or antigen-binding fragment thereof of an anti-Gal3 antibody or antigen-binding fragment thereof as described herein, a nucleotide sequence encoding the light chain or antigen-binding fragment thereof of an anti-Gal3 antibody or antigen-binding fragment thereof as described herein, or a nucleotide sequence encoding both.

[0108] The present invention also provides a method for producing an antibody or an antigen-binding fragment thereof as described herein, the method comprising: providing a host cell, the host cell comprising a nucleotide sequence encoding a heavy chain or VH of an anti-Gal3 antibody or an antigen-binding fragment thereof as described herein and a nucleotide sequence encoding a light chain or VL of an anti-Gal3 antibody or an antigen-binding fragment thereof as described herein, or a nucleotide sequence encoding both, or the host cell comprising a combination of isolated nucleic acid molecules encoding anti-Gal3 antibodies or antigen-binding fragments thereof, the isolated nucleic acid molecules being particularly a first isolated nucleic acid molecule and a second isolated nucleic acid molecule, the first isolated nucleic acid molecule comprising or consisting of a sequence encoding a heavy chain sequence or a VH sequence, the second isolated nucleic acid molecule comprising or consisting of a sequence encoding a light chain sequence or a VL sequence; culturing the host cell under conditions suitable for expressing the antibody or fragment thereof; and isolating the resulting recombinant antibody or fragment.

[0109] The present invention also provides a multispecific (eg, bispecific) binding molecule comprising an antigen-binding fragment of an anti-Gal3 antibody described herein and an antigen-binding fragment of a different antibody, such as another anti-Gal3 antibody (eg, described herein) or an antibody targeting a different protein.

[0110] The present invention also provides a method for improving a patient's condition, preventing and / or treating a patient with a Gal3-related disorder or disease, including fibrotic diseases, inflammatory diseases, autoimmune diseases, immune-mediated disorders, neurodegenerative diseases, metabolic diseases, infectious diseases, and various cancers, the method comprising administering to the patient an anti-Gal3 antibody or antigen-binding fragment thereof, a pharmaceutical composition, or a bispecific binding molecule as described herein. Unless otherwise indicated, a patient herein refers to a human patient.

[0111] The present invention also provides a method of inhibiting extracellular Gal3 interaction in a patient, comprising administering to the patient an anti-Gal3 antibody or antigen-binding fragment thereof, pharmaceutical composition or bispecific binding molecule described herein.

[0112] The present invention also provides a method for improving a patient's condition, preventing and / or treating a patient's fibrotic disease, inflammatory disease, autoimmune disease, immune-mediated disorder, neurodegenerative disease, metabolic disease, infectious disease, cancer, the method comprising administering to the patient an anti-Gal3 antibody or antigen-binding fragment thereof, a pharmaceutical composition or a bispecific binding molecule as described herein. In some embodiments, the fibrotic disease originates from a tissue selected from the group consisting of skin, lung, liver, heart, kidney and blood vessels.

[0113] The present invention also provides the use of an antibody composition comprising an anti-Gal3 antibody or an antigen-binding fragment thereof as described herein in the preparation of a medicament for improving the patient's condition in a patient in need thereof, preventing and / or treating a patient suffering from a Gal3-related disease, including fibrotic diseases such as heart failure, renal failure, systemic sclerosis, pulmonary fibrosis and liver fibrosis, and including inflammatory diseases, autoimmune diseases, immune-mediated disorders, neurodegenerative diseases, metabolic diseases, infectious diseases and various cancers, and / or inhibiting Gal3 interactions.

[0114] The present invention also provides an anti-Gal3 antibody or antigen-binding fragment as described herein, for improving the patient's condition in a patient in need thereof, preventing and / or treating a patient suffering from a Gal3-related disease or Gal3-related disorder, including fibrotic diseases, autoimmune diseases, immune-mediated disorders, neurodegenerative diseases, metabolic diseases, infectious diseases, and various cancers, and / or inhibiting Gal3 interactions.

[0115] The present invention also provides a preparation comprising an anti-Gal3 antibody or antigen-binding fragment as described herein, wherein the preparation is suitable for detecting human Gal3 in a patient in need thereof, or for improving a patient's condition, preventing and / or treating a patient suffering from a Gal3-related disease, including fibrotic diseases, inflammatory diseases, autoimmune diseases, immune-mediated disorders, neurodegenerative diseases, metabolic diseases, infectious diseases, and various cancers, and / or for inhibiting Gal3 interactions. BRIEF DESCRIPTION OF THE DRAWINGS

[0116] Figure 1The binding specificity of scFvs to human Gal3 full length and CRD domain (Hu-Gal3-FL, black bars, and Hu-Gal3-CRD, white bars, respectively), human Gal1 (Hu-Gal1, gray bars), and human Gal7 (Hu-Gal7, black bars), as well as mouse Gal3 (m-Gal3-FL, gray bars) detected by ELISA is shown. The control without antigen is represented by a dotted bar graph (the last bar on the right side of each group). The first two bars are positive and negative controls, respectively: anti-Gal3, anti-Gal1 and anti-Gal7 commercial antibodies, confirming the coating of the five antigens tested, as well as unrelated scFvs tested on the five antigens.

[0117] Figure 2 Shown is the inhibition of Gal3-ASF complex formation by scFv expressed in the supernatant detected by competitive ELISA. Absorbance results are expressed as a percentage, where ASF with an irrelevant scFv corresponds to 100% signal (upper dashed line) and the lower dashed line corresponds to the signal without binding, i.e. 100% inhibition (secondary antibody alone). Data represent three independent experiments and values ​​are expressed as mean ± SEM.

[0118] Figure 3 Human and mouse cross-reactivity to Hu-Gal3-FL (1st white bar), Hu-Gal3-CRD (2nd grey bar) and m-Gal3-FL (3rd grey bar) detected by ELISA is shown. The first 2 groups are controls: a commercial anti-Gal3 antibody to confirm coating of Hu-Gal3 antigen, and an irrelevant IgG tested on the 3 antigens, respectively.

[0119] Figure 4 Shown is the lack of binding of anti-Gal3 antibodies to Hu-Gal1 (2nd black bar) and Hu-Gal7 (3rd black bar) compared to the Hu-Gal3-CRD positive control (1st white bar). The first 3 groups of bars are positive and negative controls, anti-Gal1 and anti-Gal7 antibodies, respectively, to confirm coating of the two antigens tested under the 3 conditions and an irrelevant IgG tested.

[0120] Figure 5 Binding of antibodies to Hu-Gal3-FL and m-Gal3-FL is shown as circles and triangles, respectively. EC50 is the best fit value (Hill equation).

[0121] Figure 6Dose response ELISA of anti-Gal3 antibody inhibition of ASF binding to Hu-Gal3-CRD is shown. The measured (triangles) and fitted (curve) OD450 nm for each anti-Gal3 antibody concentration are black. The measured (circles) OD450 nm for an irrelevant antibody is grey.

[0122] Figure 7 shows the evolution of skin thickness changes during two independent studies, one of which was compared with a control group of mice treated with TD139, E07 and D11 antibodies ( Fig. 7A ), and in another study compared with control mice treated with TD139, E07, and H10 antibodies ( Figure 7B ). Data are presented as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001 compared with HOCl group. ###p<0.001 compared with control group. Repeated measures ANOVA was performed daily with Dunnett post hoc test.

[0123] Figure 8 shows a depiction of the Sirius red stained lung ( Fig. 8A ) and skin( Figure 8B ) Histogram of Image J software-assisted quantification of collagen deposition in sections. Data are expressed as mean ± SEM. *p<0.05, **p<0.01 compared with HOCl group. ###p<0.001 compared with control group. One-way ANOVA (mouse as random effect) was mixed with Dunnett's post hoc test.

[0124] Fig. 9 Shown are histograms depicting IL-5 and IL-6 levels in plasma at day 42 in the control group, HOCl group, and HOCl antibody or TD139 treated groups. Data are expressed as mean ± SEM. **p<0.01 compared to the HOCl group. ##p<0.01 compared to the control group. Repeated measures ANCOVA (day -1 measurements as covariates) were performed on log-transformed data every day with Dunnett post hoc tests.

[0125] Fig.10 Shown are histograms depicting the quantification of Gal3 in mouse plasma in the control group, HOCl group, and HOCl antibody or TD139 treated groups before test item administration (day 0) and at day 42. Data are expressed as mean ± SEM. ***p<0.001 compared to the HOCL group. ##p<0.01 compared to the control group. ANCOVA (day -1 measurements as covariates and mice as random effects) was mixed with Dunnett post hoc tests.

[0126] Fig.11A heat map representation of genes differentially expressed in whole blood cells on day 42 in the control group, HOCl group, HOCl E07 antibody-treated group, and TD139-treated group using RNA sequencing method is shown.

[0127] Fig.12 Shown are the changes in body weight from day -1 to day 21 of bleomycin-treated mice with or without treatment with D06, D11, E07, or TD139.

[0128] Fig.13 Shown are the time to weight gain (TWG) and time to baseline (TTB) of bleomycin-treated mice with or without D06, D11, E07, or TD139 treatment. Data are expressed as medians of individual values ​​and quartiles predicted by nonlinear models. *p<0.05, **p<0.01 compared to the bleo group. Logrank test followed by Bonferroni-Holm multiplicity adjustment.

[0129] Fig.14 Shown are pictures representing sections made on the lungs before staining with picrosirius red and scored using the modified Ashcroft fibrosis score.

[0130] Fig.15 The frequencies of the median Ashcroft scores for lung fibrosis in bleomycin-treated mice without any treatment or treated with D06 antibody, D11 antibody, E07 antibody, or TD139 are shown.

[0131] Fig.16 A scatter plot of neutrophil counts in bronchoalveolar lavage fluid from rats in an acute respiratory distress syndrome (ARDS) model is shown. Black circles indicate controls that did not receive LPS stimulation and were not treated, black squares indicate positive controls that received LPS stimulation but were not treated, and white squares indicate groups that received LPS stimulation and were treated with dexamethasone (Dexa) as a reference. Groups that received anti-Gal3 antibody treatment before LPS stimulation are indicated by black triangles for D11 antibody, inverted black triangles for D06 antibody, black diamonds for E07 antibody, and white circles for H10 antibody. Data are expressed as mean + / - SEM. ####p<0.0001 between LPS and control (CTRL) groups; **p<0.01; ****p<0.0001 compared to the positive control LPS group. One-way ANOVA was performed on log-transformed data using the Dunnett adjustment method.

[0132] Fig.17A scatter plot of protein quantification in bronchoalveolar lavage fluid from rats in an acute respiratory distress syndrome (ARDS) model is shown. Black circles represent controls that did not receive LPS stimulation and were not treated, black squares represent positive controls that received LPS stimulation but were not treated, and white squares represent groups that received LPS stimulation and were treated with dexamethasone (Dexa) as a reference. The groups that received anti-Gal3 antibody treatment before LPS stimulation were represented by black triangles for D11 antibody, inverted black triangles for D06 antibody, black diamonds for E07 antibody, and white circles for H10 antibody. Data are expressed as mean + / - SEM. #p<0.05 between LPS and control groups; **p<0.051 compared with the positive control LPS group. One-way ANOVA was performed on log-transformed data using the Dunnett adjustment method.

[0133] Fig.18 A scatter plot of TNFα quantified by MSD in rat plasma from an acute respiratory distress syndrome (ARDS) model is shown. Black circles represent controls that did not receive LPS stimulation and were not treated, black squares represent positive controls that received LPS stimulation but were not treated, and white squares represent groups that received LPS stimulation and were treated with dexamethasone (Dexa) as a reference. The groups that received anti-Gal3 antibody treatment before LPS stimulation were represented by black triangles for D11 antibody, inverted black triangles for D06 antibody, black diamonds for E07 antibody, and white circles for H10 antibody. Data are expressed as mean + / - SEM. ####p<0.0001 between LPS and control groups; **p<0.01; ****p<0.0001 compared to the positive control LPS group. One-way ANOVA was performed on log-transformed data using the Dunnett adjustment method.

[0134] Fig.19 A scatter plot of KC-GRO quantified by MSD in rat plasma from an acute respiratory distress syndrome (ARDS) model is shown. Black circles indicate controls that did not receive LPS stimulation and were not treated, black squares indicate positive controls that received LPS stimulation but were not treated, and white squares indicate groups that received LPS stimulation and were treated with dexamethasone (Dexa) as a reference. Groups that received anti-Gal3 antibody treatment before LPS stimulation are indicated by black triangles for D11 antibody, inverted black triangles for D06 antibody, black diamonds for E07 antibody, and white circles for H10 antibody. Data are expressed as mean + / - SEM. ####p<0.0001 between LPS and control groups; **** other p<0.0001 values ​​are indicated compared to the positive control LPS group. One-way ANOVA was performed on log-transformed data using the Dunnett adjustment method. DETAILED DESCRIPTION

[0135] The present invention provides a novel anti-human galectin 3 (Gal3) antibody. The human Gal3 polypeptide sequence can be obtained under UniProt accession number P17931 (LEG3_HUMAN).

[0136] The present invention relates to novel monoclonal antibodies that target extracellular galectin 3 (Gal3) and bind neither to galectin 1 (Gal1) nor to galectin 7 (Gal7), and also to pharmaceutical compositions comprising one or more of these antibodies.

[0137] The present invention also relates to the use of the anti-Gal3 antibodies and pharmaceutical compositions for inhibiting Gal3 interactions in patients in need thereof or for improving, preventing and / or treating patients suffering from Gal3-related diseases or Gal3-related disorders, including fibrotic diseases, inflammatory diseases, autoimmune diseases, immune-mediated disorders, neurodegenerative diseases, metabolic diseases, infectious diseases and various cancers.

[0138] Tissue fibrosis is a progressive debilitating disease characterized by the massive accumulation of extracellular matrix 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 kidneys, liver, lungs, heart, skin, pancreas, intestines, eyes, nervous system, joints, tendons, mediastinum or retroperitoneum. Characteristics of tissue fibrosis include epithelial and endothelial damage and dysfunction, abnormal proliferation of myofibroblasts, smooth muscle cells and stellate cells, and deposition of extracellular matrix proteins. The presence of cytokines, chemokines, growth factors and angiogenic factors further regulates the activation of cells that produce extracellular matrix proteins in the profibrotic process.

[0139] The terms "antibody" (Ab) or "immunoglobulin" (Ig) as used herein refer to a tetramer comprising two heavy (H) chains (about 50-70 kDa) and two light (L) chains (about 25 kDa) interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable domain (VH) and a heavy chain constant region (CH). Each light chain consists of a light chain variable domain (VL) and a light chain constant region (CL). The VH and VL domains can be further subdivided into highly variable regions, called "complementarity determining regions" (CDRs), interspersed with more conserved regions, called "framework regions" (FRs). Each VH and VL consists of three CDRs (H-CDR herein refers to CDRs from the heavy chain; L-CDR herein refers to CDRs from the light chain) and four FRs, arranged in the following order from amino terminus to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The designation of amino acid numbers in the heavy or light chain can be based on the EU numbering or Definition (Lefranc et al., Dev Comp Immunol 27(1):55-77 (2003)); or the definition of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD (1987 and 1991)); Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987); or Chothia et al., Nature 342:878-883 (1989).

[0140] The term "recombinant antibody" refers to an antibody expressed by a cell or cell line that contains a nucleotide sequence encoding the antibody, wherein the nucleotide sequence is not naturally associated with the cell.

[0141] The terms "isolated protein," "isolated polypeptide," or "isolated antibody" refer to a protein, polypeptide, or antibody that, by virtue of its source or derivation from which it is derived, (1) is not associated with naturally associated components that accompany it in its native state, (2) is free of other proteins from the same species, (3) is expressed by cells from a different species, and / or (4) does not occur in nature. Thus, a polypeptide that is chemically synthesized or synthesized in a cellular system different from the cells from which it naturally originates will be "isolated" from its naturally associated components. A protein may also be rendered substantially free of naturally associated components by isolation, using protein purification techniques known in the art.

[0142] As used herein, the term "germline" refers to the nucleotide and amino acid sequences of antibody genes and gene fragments when they are passed from parents to offspring through reproductive cells. Germline sequences are different from the nucleotide sequences encoding antibodies in mature B cells, which have been changed by recombination and hypermutation events during B cell maturation. Antibodies that "utilize" specific germline sequences have nucleotide or amino acid sequences that are more closely aligned with the germline nucleotide sequence or its specified amino acid sequence than with any other germline nucleotide or amino acid sequence.

[0143] The term "affinity" refers to the measure of the attraction between an antigen and an antibody. The intrinsic attraction of an antibody to an antigen is usually expressed as the binding affinity equilibrium constant (KD) of a specific antibody-antigen interaction. When KD≤1 mM, preferably ≤100 nM, the antibody is considered to specifically bind to the antigen. The KD binding affinity constant can be measured, for example, by surface plasmon resonance (BIAcoreTM) or biolayer interferometry, for example using an IBIS MX96 SPR system from IBIS Technologies or an OctetTM system from ForteBio.

[0144] The term "humanization" refers to the fact that when an antibody is wholly or partially of non-human origin (e.g., a mouse or chicken antibody obtained by immunizing a mouse or chicken, respectively, with an antigen of interest, or a chimeric antibody based on such a mouse or chicken antibody), certain amino acids may be substituted, particularly in the framework and constant regions of the heavy and light chains, to avoid or minimize an immune response in humans. Although it is not possible to accurately predict the immunogenicity of a particular antibody, and thus human anti-antibody responses, non-human antibodies tend to be more immunogenic in humans than human antibodies. Chimeric antibodies, in which foreign (e.g., rodent or avian) constant regions have been replaced with human sequences, have been shown to be generally less immunogenic than completely foreign antibodies, and the trend for therapeutic antibodies is towards humanized or fully human antibodies. Thus, chimeric antibodies or other antibodies of non-human origin may be humanized to reduce the risk of human anti-antibody responses.

[0145] Many methods for humanization of antibody sequences are known in the art; see, for example, Almagro & Fransson, Front Biosci. 13: 1619-1633 (2008) review. A commonly used method is CDR transplantation, for example, for mouse chimeric antibodies, the method includes identifying human germline genes corresponding to mouse variable region genes and transplanting mouse CDR sequences into the framework. The specificity of antibody-target antigen interaction lies mainly in the amino acid residues located in the six CDRs of the heavy and light chains. Therefore, the variability of the amino acid sequence within the CDR between each antibody is much greater than the sequence outside the CDR. Since the CDR sequence is responsible for most antibody-antigen interactions, it is possible to express a recombinant antibody that simulates a specific naturally occurring antibody or more generally has any specific antibody properties of a given amino acid sequence, such as by constructing an expression vector that expresses the CDR sequence from a specific antibody and transplants it into the framework sequence from a different antibody. Therefore, non-human antibodies can be "humanized" and the binding specificity and affinity of the original antibody are still substantially maintained. CDR grafting can be based on the Kabat CDR definition, although a recent publication (Magdelaine-Beuzelin et al., Crit Rev. Oncol Hematol. 64:210-225 (2007)) has suggested that Definition (International ImMunoGeneTics Information System www.imgt.org) (or EU numbering) may improve the results of humanization (see, LeFranc et al., Dev. Comp Immunol. 27:55-77 (2003)).

[0146] In some cases, compared with the parent antibody from which the CDR is obtained, CDR transplantation can reduce the binding specificity and affinity of the CDR transplanted antibody, thereby reducing its biological activity. Back mutations (sometimes referred to as "framework repair") can be introduced at selected positions of CDR transplanted antibodies, usually in the framework region, to re-establish the binding specificity and affinity of the parent antibody. The position of possible back mutations can be determined using the information available in the literature and antibody databases. The amino acid residues that are candidates for back mutations are usually those located on the surface of the antibody molecule, and the residues that are buried or have low surface exposure are usually not changed. The alternative humanization technology of CDR transplantation and back mutation is surface reconstruction, in which the non-surface exposed residues of non-human origin are retained, and the surface residues are changed to human residues.

[0147] In some cases, in order to improve the binding affinity to the target epitope, it may also be necessary to change one or more CDR amino acid residues. This is referred to as "affinity maturation" and can optionally be carried out in conjunction with humanization, such as when the humanization of an antibody causes a reduction in binding specificity or affinity and cannot fully improve binding specificity or affinity only by back mutation. A variety of affinity maturation methods are known in the art, such as Burks et al., Proc Natl Acad Sci USA, 94:412-417 (1997) described in vitro scanning saturation mutagenesis method, and Wu et al., Proc Natl Acad Sci USA 95:6037-6042 (1998) described in stepwise in vitro affinity maturation method.

[0148] As used herein, the term "antigen-binding fragment" of an antibody (or simply "antibody fragment") refers to one or more fragments or fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., human Gal3 or a fragment thereof). It has been shown that certain fragments of a full-length antibody can perform the antigen-binding function of an antibody. Examples of binding fragments encompassed by the term "antigen-binding fragment" include (i) Fab fragments: monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragments: bivalent fragments comprising two Fab fragments linked by a disulfide bond at the hinge region; (iii) Fd fragments consisting of VH and CH1 domains; (iv) Fv fragments consisting of the VL and VH domains of a single arm of an antibody, (v) dAb fragments consisting of a VH domain; and (vi) isolated complementary determining regions (CDRs) capable of specifically binding to an antigen. In addition, although the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be connected by a synthetic linker using recombinant methods, which enables them to be made into a single protein chain, in which the VL and VH domains are paired to form a monovalent molecule (called single-chain Fv (scFv or scFvs)). The present invention also includes antigen-binding molecules comprising VH and / or VL. In the case of VH, the molecule may also include one or more of the CH1, hinge, CH2 or CH3 regions. Such single-chain antibodies are also intended to be included in the term "antigen-binding fragment" of an antibody. Other forms of single-chain antibodies, such as double-chain antibodies (diabodies) are also included. Double-chain antibodies are bivalent bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but a too short linker is used so that the two domains on the same chain cannot be paired, thereby forcing the domains to pair with the complementary domains of another chain and produce two antigen-binding sites.

[0149] Antibody fragments, such as Fab and F(ab')2 fragments, can be prepared from intact antibodies using conventional techniques (e.g., papain or pepsin digestion of intact antibodies). In addition, antibodies, antibody fragments and immunoadhesion molecules can be obtained using standard recombinant DNA techniques (e.g., as described herein).

[0150] The class (isotype) and subclass of anti-Gal3 antibodies can be determined by any method known in the art. Generally, the class and subclass of antibodies can be determined using antibodies that are specific to antibodies of a particular class and subclass. These antibodies are commercially available. The class and subclass can be determined by ELISA, Western blotting and other techniques. Alternatively, the class and subclass can be determined by sequencing all or a fragment of the constant region of the antibody heavy chain and / or light chain, comparing its amino acid sequence with the known amino acid sequence of immunoglobulins of various classes and subclasses, and determining the class and subclass of the antibody.

[0151] Unless otherwise indicated, all antibody amino acid residue numbers referred to in this disclosure are based on EU numbering or Those with numbering schemes.

[0152] Anti-Gal3 antibody

[0153] The present invention provides antibodies and antigen-binding fragments thereof against Gal3. One advantage of the novel anti-Gal3 antibodies of the present invention is that they can selectively bind to Gal3, in particular to Gal3 CRD, but neither bind to Gal1 nor Gal7 (see Example 4). They can inhibit Gal3 from binding to its ligand (see Example 6). Another potential advantage of the anti-Gal3 antibodies of the present invention is the low level of secondary effector function in antibodies with a "LALA" mutation (L234A / L235A), which hinders the significant binding of the antibody to human FcgR (Fcγ receptor), thereby hindering the exhaustion of effector T cells. In another embodiment, the present invention provides antibodies with germline framework mutations in the variable domains of antibodies outside the CDR region, thereby allowing for potential reduction of immunogenicity when administered to humans while substantially retaining the specificity and affinity of the parent antibody.

[0154] In one embodiment, the anti-Gal3 antibody or antigen-binding fragment thereof has a heavy chain variable domain (VH) that is at least 90% identical to any one of SEQ ID NO:38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 81, 82, 84, 85, 87, 88, 90, 92, 94, 95, 97, 99, 101, 103 or 104, for example, at least 92% identical to the VH amino acid sequence of SEQ ID NO:38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 81, 82, 84, 85, 87, 88, 90, 92, 94, 95, 97, 99, 101, 103 or 104, for example, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the VH amino acid sequence of SEQ ID NO:38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 81, 82, 84, 85, 87, 88, 90, 92, 94, 95, 97, 99, 101,

[0155] In one embodiment, the anti-Gal3 antibody has a heavy chain variable domain (VH) that is at least 90% identical to any one of SEQ ID NO:38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 81, 82, 84, 85, 87, 88, 90, 92, 94, 95, 97, 99, 101, 103 or 104, e.g., at least 92% identical to the VH amino acid sequence of SEQ ID NO:38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 81, 82, 84, 85, 87, 88, 90, 92, 94, 95, 97, 99, 101, 103 or 104, e.g., at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the VH amino acid sequence of SEQ ID NO:38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 81, 82, 84, 85, 87, 88, 90, 92, 94, 95, 97, 99, 101, 103 or 104, and a heavy chain constant region that is identical to any one of SEQ ID NO: ID NO:58 or 59 is at least 90% identical, such as at least 92% identical, such as at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:58 or 59.

[0156] In one embodiment, the anti-Gal3 antibody has a heavy chain (HC) comprising the VH amino acid sequence of any one of SEQ ID NO: 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 81, 82, 84, 85, 87, 88, 90, 92, 94, 95, 97, 99, 101, 103 or 104 and the heavy chain constant region amino acid sequence of SEQ ID NO: 58 or 59.

[0157] In one embodiment, the anti-Gal3 antibody or antigen-binding fragment thereof has a light chain variable domain (VL) that is at least 90% identical to the VL amino acid sequence of any one of SEQ ID NO: 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 83, 86, 89, 91, 93, 96, 98, 100, 102, or 105, e.g., at least 92% identical to any one of SEQ ID NO: 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 83, 86, 89, 91, 93, 96, 98, 100, 102, or 105, e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the VL amino acid sequence of any one of SEQ ID NO: 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 83, 86, 89, 91, 93, 96, 98, 100, 102, or 105.

[0158] In one embodiment, the anti-Gal3 antibody has a light chain variable domain (VL) that is at least 90% identical to the VL amino acid sequence of any one of SEQ ID NO:39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 83, 86, 89, 91, 93, 96, 98, 100, 102, or 105, for example, at least 92% identical to any one of SEQ ID NO:39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 83, 86, 89, 91, 93, 96, 98, 100, 102, or 105, for example, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NO:60; and a light chain constant region amino acid sequence that is at least 90% identical to the VL amino acid sequence of any one of SEQ ID NO:60, for example, at least 92% identical to any one of SEQ ID NO:60 NO:60 is at least 92% identical to the sequence, such as at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the sequence.

[0159] In another embodiment, the anti-Gal3 antibody has a light chain comprising any one of SEQ ID NO:39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 83, 86, 89, 91, 93, 96, 98, 100, 102 or 105 and SEQ ID NO:60.

[0160] In certain embodiments, the anti-Gal3 antibody or antigen-binding fragment of the invention comprises the following H-CDR 1-3 and L-CDR 1-3 amino acid sequences:

[0161] a) SEQ ID NOs: 1, 6, 12, 20, 27 and 30, respectively;

[0162] b) SEQ ID NO: 1, 6, 13, 21, 27 and 31, respectively;

[0163] c) SEQ ID NO: 1, 6, 14, 22, 27 and 31, respectively;

[0164] d) SEQ ID NOs: 5, 7, 3, 21, 28 and 32, respectively;

[0165] e) SEQ ID NOs: 3, 8, 15, 23, 27 and 33, respectively;

[0166] f) SEQ ID NO: 2, 6, 16, 24, 27 and 34, respectively;

[0167] g) SEQ ID NO: 1, 9, 14, 24, 27 and 34, respectively;

[0168] h) SEQ ID NO: 2, 10, 17, 25, 27 and 35, respectively;

[0169] i) SEQ ID NO: 4, 11, 18, 21, 29 and 36, respectively; or

[0170] j) SEQ ID NO: 2, 10, 19, 26, 27 and 37, respectively.

[0171] In some embodiments, the anti-Gal3 antibody or antigen-binding fragment of the invention comprises a VH and a VL that are 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the following amino acid sequence:

[0172] a) SEQ ID NOs: 38 and 39, respectively;

[0173] b) SEQ ID NOs: 40 and 41, respectively;

[0174] c) SEQ ID NOs: 42 and 43, respectively;

[0175] d) SEQ ID NOs: 44 and 45, respectively;

[0176] e) SEQ ID NOs: 46 and 47, respectively;

[0177] f) SEQ ID NOs: 48 and 49, respectively;

[0178] g) SEQ ID NOs: 50 and 51, respectively;

[0179] h) SEQ ID NOs: 52 and 53, respectively;

[0180] i) SEQ ID NOs: 54 and 55, respectively;

[0181] j) SEQ ID NOs: 56 and 57, respectively;

[0182] k) SEQ ID NOs: 81 and 83, respectively;

[0183] 1) SEQ ID NO: 82 and 83, respectively;

[0184] m) SEQ ID NOs: 84 and 86, respectively;

[0185] n) SEQ ID NOs: 85 and 86, respectively;

[0186] o) SEQ ID NOs: 87 and 89, respectively;

[0187] p) SEQ ID NOs: 88 and 89, respectively;

[0188] q) SEQ ID NOs: 90 and 91, respectively;

[0189] r) SEQ ID NOs: 92 and 93, respectively;

[0190] s) SEQ ID NOs: 94 and 96, respectively;

[0191] t) SEQ ID NOs: 95 and 96, respectively;

[0192] u) SEQ ID NOs: 97 and 98, respectively;

[0193] v) SEQ ID NOs: 99 and 100, respectively;

[0194] w) SEQ ID NOs: 101 and 102, respectively;

[0195] x) SEQ ID NO: 103 and 105, respectively; or

[0196] y) SEQ ID NO: 104 and 105, respectively.

[0197] In some embodiments, the anti-Gal3 antibody or antigen-binding fragment of the invention comprises VH and VL, wherein VH and VL have the following amino acid sequences:

[0198] a) SEQ ID NOs: 38 and 39, respectively;

[0199] b) SEQ ID NOs: 40 and 41, respectively;

[0200] c) SEQ ID NOs: 42 and 43, respectively;

[0201] d) SEQ ID NOs: 44 and 45, respectively;

[0202] e) SEQ ID NOs: 46 and 47, respectively;

[0203] f) SEQ ID NOs: 48 and 49, respectively;

[0204] g) SEQ ID NOs: 50 and 51, respectively;

[0205] h) SEQ ID NOs: 52 and 53, respectively;

[0206] i) SEQ ID NOs: 54 and 55, respectively;

[0207] j) SEQ ID NOs: 56 and 57, respectively;

[0208] k) SEQ ID NOs: 81 and 83, respectively;

[0209] 1) SEQ ID NO: 82 and 83, respectively;

[0210] m) SEQ ID NOs: 84 and 86, respectively;

[0211] n) SEQ ID NOs: 85 and 86, respectively;

[0212] o) SEQ ID NOs: 87 and 89, respectively;

[0213] p) SEQ ID NOs: 88 and 89, respectively;

[0214] q) SEQ ID NOs: 90 and 91, respectively;

[0215] r) SEQ ID NOs: 92 and 93, respectively;

[0216] s) SEQ ID NOs: 94 and 96, respectively;

[0217] t) SEQ ID NOs: 95 and 96, respectively;

[0218] u) SEQ ID NOs: 97 and 98, respectively;

[0219] v) SEQ ID NOs: 99 and 100, respectively;

[0220] w) SEQ ID NOs: 101 and 102, respectively;

[0221] x) SEQ ID NO: 103 and 105, respectively; or

[0222] y) SEQ ID NO: 104 and 105, respectively.

[0223] In certain embodiments, the anti-Gal3 antibody:

[0224] a) a LC having an amino acid sequence comprising or consisting of SEQ ID NOs: 39 and 60; and a HC having an amino acid sequence comprising or consisting of SEQ ID NO: 38 and SEQ ID NOs: 58 or 59;

[0225] b) a LC having an amino acid sequence comprising or consisting of SEQ ID NOs: 41 and 60; and a HC having an amino acid sequence comprising or consisting of SEQ ID NO: 40 and SEQ ID NOs: 58 or 59;

[0226] c) a LC having an amino acid sequence comprising or consisting of SEQ ID NOs: 43 and 60; and a HC having an amino acid sequence comprising or consisting of SEQ ID NO: 42 and SEQ ID NOs: 58 or 59;

[0227] d) a LC having an amino acid sequence comprising or consisting of SEQ ID NOs: 45 and 60; and a HC having an amino acid sequence comprising or consisting of SEQ ID NO: 44 and SEQ ID NOs: 58 or 59;

[0228] e) a LC having an amino acid sequence comprising or consisting of SEQ ID NOs: 47 and 60; and a HC having an amino acid sequence comprising or consisting of SEQ ID NO: 46 and SEQ ID NOs: 58 or 59;

[0229] f) a LC having an amino acid sequence comprising or consisting of SEQ ID NOs: 49 and 60; and a HC having an amino acid sequence comprising or consisting of SEQ ID NO: 48 and SEQ ID NOs: 58 or 59;

[0230] g) a LC having an amino acid sequence comprising or consisting of SEQ ID NOs: 51 and 60; and a HC having an amino acid sequence comprising or consisting of SEQ ID NO: 50 and SEQ ID NOs: 58 or 59;

[0231] h) a LC having an amino acid sequence comprising or consisting of SEQ ID NOs: 53 and 60; and a HC having an amino acid sequence comprising or consisting of SEQ ID NO: 52 and SEQ ID NOs: 58 or 59;

[0232] i) a LC having an amino acid sequence comprising or consisting of SEQ ID NOs: 55 and 60; and a HC having an amino acid sequence comprising or consisting of SEQ ID NO: 54 and SEQ ID NOs: 58 or 59;

[0233] j) a LC having an amino acid sequence comprising or consisting of SEQ ID NOs: 57 and 60; and a HC having an amino acid sequence comprising or consisting of SEQ ID NO: 56 and SEQ ID NOs: 58 or 59.

[0234] In certain embodiments, the anti-Gal3 antibody:

[0235] a) a LC having an amino acid sequence comprising or consisting of SEQ ID NOs: 83 and 60; and a HC having an amino acid sequence comprising or consisting of SEQ ID NOs: 81 or 82 and SEQ ID NOs: 58 or 59;

[0236] b) a LC having an amino acid sequence comprising or consisting of SEQ ID NOs: 86 and 60; and a HC having an amino acid sequence comprising or consisting of SEQ ID NOs: 84 or 85 and SEQ ID NOs: 58 or 59;

[0237] c) a LC having an amino acid sequence comprising or consisting of SEQ ID NOs: 89 and 60; and a HC having an amino acid sequence comprising or consisting of SEQ ID NOs: 87 or 88 and SEQ ID NOs: 58 or 59;

[0238] d) a LC having an amino acid sequence comprising or consisting of SEQ ID NOs: 91 and 60; and a HC having an amino acid sequence comprising or consisting of SEQ ID NO: 90 and SEQ ID NOs: 58 or 59;

[0239] e) a LC having an amino acid sequence comprising or consisting of SEQ ID NOs: 93 and 60; and a HC having an amino acid sequence comprising or consisting of SEQ ID NO: 92 and SEQ ID NOs: 58 or 59;

[0240] f) a LC having an amino acid sequence comprising or consisting of SEQ ID NOs: 96 and 60; and a HC having an amino acid sequence comprising or consisting of SEQ ID NOs: 94 or 95 and SEQ ID NOs: 58 or 59;

[0241] g) a LC having an amino acid sequence comprising or consisting of SEQ ID NOs: 98 and 60; and a HC having an amino acid sequence comprising or consisting of SEQ ID NO: 97 and SEQ ID NOs: 58 or 59;

[0242] h) a LC having an amino acid sequence comprising or consisting of SEQ ID NOs: 100 and 60; and a HC having an amino acid sequence comprising or consisting of SEQ ID NO: 99 and SEQ ID NOs: 58 or 59;

[0243] i) a LC having an amino acid sequence comprising or consisting of SEQ ID NOs: 102 and 60; and a HC having an amino acid sequence comprising or consisting of SEQ ID NO: 101 and SEQ ID NOs: 58 or 59;

[0244] j) a LC having an amino acid sequence comprising or consisting of SEQ ID NOs: 105 and 60; and a HC having an amino acid sequence comprising or consisting of SEQ ID NOs: 103 or 104 and SEQ ID NOs: 58 or 59.

[0245] In some embodiments, the anti-Gal3 antibody or antigen-binding fragment of the invention comprises the H-CDR 1-3 and L-CDR 1-3 amino acid sequences of antibody D06, D11, E01, E02, E07, G03, H07, H10, B12, or E12.

[0246] In some embodiments, the anti-Gal3 antibody or antigen-binding fragment of the invention comprises a VH and a VL, wherein the VH and VL are at least 90% identical in amino acid sequence to the VH and VL of antibody D06, D11, E01, E02, E07, G03, H07, H10, B12 or E12, respectively.

[0247] In some embodiments, the anti-Gal3 antibody or antigen-binding fragment of the invention comprises a VH and a VL, which are the VH and VL of antibody D06, D11, E01, E02, E07, G03, H07, H10, B12, or E12, respectively.

[0248] In some embodiments, the anti-Gal3 antibody or antigen-binding fragment of the invention comprises a VH and a VL, wherein the VH and VL are at least 90% identical in amino acid sequence to the VH and VL of antibodies D06-G1, D06-G2, D11-G1, D11-G2, E01-G, E02-G, E07-G1, E07-G2, G03-G1, G03-G2, H07-G, H10-G, B12-G, E12-G1 or E12-G2, respectively.

[0249] In some embodiments, the anti-Gal3 antibody or antigen-binding fragment of the invention comprises a VH and a VL, wherein the VH and VL are respectively the VH and VL of antibody D06, D11, E01, E02, E07, G03, H07, H10, B12, E12, D06-G1, D06-G2, D11-G1, D11-G2, E01-G, E02-G, E07-G1, E07-G2, G03-G1, G03-G2, H07-G, H10-G, B12-G, E12-G1, or E12-G2.

[0250] In some embodiments, the anti-Gal3 antibody or antigen-binding fragment thereof of the present invention is antibody D06, D11, E01, E02, E07, G03, H07, H10, B12, E12, D06-G1, D06-G2, D11-G1, D11-G2, E01-G, E02-G, E07-G1, E07-G2, G03-G1, G03-G2, H07-G, H10-G, B12-G, E12-G1 or E12-G2, or an antigen-binding fragment thereof, or an antibody or antigen-binding fragment thereof having the same amino acid sequence as the antibody or antigen-binding fragment thereof.

[0251] In some embodiments, any of the anti-Gal3 antibodies or antigen-binding fragments thereof do not bind Gal1 or Gal7.

[0252] In some embodiments, any of the anti-Gal3 antibodies or antigen-binding fragments thereof can inhibit and / or reduce Gal3 expression and / or the interaction of Gal3 with its ligand, thereby allowing for the improvement, prevention and / or treatment of Gal3-related diseases or Gal3-related disorders.

[0253] In some embodiments, any of the anti-Gal3 antibodies or antigen-binding fragments thereof can inhibit IL-6 and / or IL-5 levels in plasma.

[0254] In some embodiments, any of the anti-Gal3 antibodies or antigen-binding fragments thereof can reduce skin thickness or collagen deposition in a patient suffering from a fibrotic disease.

[0255] The class of the anti-Gal3 antibody obtained by the method described herein can be changed or converted to another class or subclass. In one aspect of the invention, a nucleic acid molecule encoding VL or VH is separated using methods well known in the art so that it does not include a nucleic acid sequence encoding CL or CH, respectively. The nucleic acid molecule encoding VL or VH is then operably linked to a nucleic acid sequence encoding CL or CH from a different class of immunoglobulin molecules, respectively. This can be achieved using a vector or nucleic acid molecule comprising a CL or CH chain, as described above. For example, an anti-Gal3 antibody that was originally IgM can be converted to IgG by class conversion. In addition, class conversion can be used to convert one IgG subclass to another, such as from IgG1 to IgG2 or from IgG1 to IgG4. The κ light chain constant region can be changed to, for example, a λ light chain constant region. A preferred method for producing an antibody of the present invention having a desired Ig isotype comprises the steps of isolating a nucleic acid molecule encoding an anti-Gal3 antibody heavy chain and a nucleic acid molecule encoding an anti-Gal3 antibody light chain, obtaining the variable region of the heavy chain, linking the variable region of the heavy chain to a heavy chain constant region of the desired isotype, expressing the light chain and the linked heavy chain in a cell, and collecting an anti-Gal3 antibody having the desired isotype.

[0256] The anti-Gal3 antibodies of the present invention can be IgG, IgM, IgE, IgA or IgD molecules, but are typically IgG isotypes, such as IgG subclasses IgG1, IgG2a or IgG2b, IgG3 or IgG4. In one embodiment, the antibody is IgG1. In another embodiment, the antibody is IgG2 or IgG4.

[0257] In one embodiment, the anti-Gal3 antibody may comprise at least one mutation in the Fc region. Many different Fc mutations are known, wherein these mutations provide altered effector functions. For example, in many cases, it is desirable to reduce or eliminate effector functions, such as when ligand / receptor interactions are undesirable, or in the case of antibody-drug conjugates.

[0258] In one embodiment, the anti-Gal3 antibody comprises at least one mutation in the Fc region that reduces effector function. Fc region amino acid positions that may be advantageous for mutation to reduce effector function include one or more of positions 228, 233, 234, and 235, wherein amino acid positions are numbered according to EU or Numbering scheme number.

[0259] In one embodiment, one or both amino acid residues at positions 234 and 235 may be mutated, for example, from Leu to Ala (L234A / L235A). These mutations reduce the effector function of the Fc region of the IgG1 antibody. The sequence of such a mutated IgG1 constant chain is shown in SEQ ID NO 59. In addition, or alternatively, the amino acid residue at position 228 may be mutated, for example, to Pro. In some embodiments, the amino acid residue at position 233 may be mutated, for example, to Pro, the amino acid residue at position 234 may be mutated, for example, to Val, and / or the amino acid residue at position 235 may be mutated, for example, to Ala. The amino acid positions are numbered according to EU or Numbering scheme number.

[0260] In some embodiments, when the antibody is of IgG4 subclass, it may comprise the mutation S228P, i.e. having a proline at position 228, wherein the amino acid positions are numbered according to EU or Numbering scheme No. This mutation is known to reduce undesired Fab arm exchange.

[0261] In another embodiment, it comprises all or a fragment of an anti-Gal3 antibody of the present invention connected to another polypeptide. In certain embodiments, only the variable domain of the anti-Gal3 antibody is connected to the polypeptide. In certain embodiments, the VH domain of the anti-Gal3 antibody is connected to the first polypeptide, and the VL domain of the anti-Gal3 antibody is connected to the second polypeptide, and the second polypeptide is combined with the first polypeptide in such a way that the VH and VL domains can interact to form an antigen binding site. In another preferred embodiment, the VH domain is separated from the VL domain by a linker, so that the VH and VL domains can interact with each other (e.g., a single-chain antibody). The VH-linker-VL antibody is then connected to the polypeptide of interest. In addition, a fusion antibody can be produced in which two (or more) single-chain antibodies are connected to each other. This is useful if you want to produce a bivalent or multivalent antibody on a single polypeptide chain, or if you want to produce a bispecific antibody.

[0262] In other embodiments, other modified antibodies can be prepared using nucleic acid molecules encoding anti-Gal3 antibodies. For example, "kappa bodies" (Ill et al., Protein Eng. 10: 949-57 (1997)); "minibodies" (Martin et al., EMBO J. 13: 5303-9 (1994)); "diabodies" (Holliger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993)) or "Janusins" (Traunecker et al., EMBO J. 10: 3655-3659 (1991) and Traunecker et al., Int. J. Cancer (Suppl.) 7: 51-52 (1992)) can be prepared using standard molecular biology techniques according to the teachings of this specification.

[0263] The anti-Gal3 antibody or antigen-binding fragment of the present invention may be derivatized or linked to another molecule (e.g., another peptide or protein). Typically, the antibody or fragment thereof is derivatized so that Gal3 binding is not adversely affected by derivatization or labeling. Therefore, the antibodies and antibody fragments of the present invention are intended to include intact and modified forms of the human anti-Gal3 antibodies described herein. For example, the antibodies or antibody fragments of the present invention may be functionally linked (by chemical coupling, genetic fusion, non-covalent binding or other means) to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or a diabody), a detection agent, an agent, and / or a protein or peptide that can mediate binding of the antibody or antibody fragment to another molecule (e.g., a streptavidin core region or a polyhistidine tag).

[0264] One type of derivatized antibody is produced by cross-linking two or more antibodies (of the same type or of different types, e.g., to produce bispecific antibodies). Suitable cross-linking agents include heterobifunctional cross-linking agents having two differently reactive groups separated by a suitable spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester); or homobifunctional cross-linking agents (e.g., disuccinimidyl suberate). Anti-Gal3 antibodies or antigen-binding fragments thereof can also be derivatized with chemical groups such as polyethylene glycol (PEG), methyl or ethyl groups, or carbohydrate groups. These groups can be used to improve the biological properties of antibodies, such as increasing serum half-life.

[0265] The antibodies of the present invention may also be labeled. As used herein, the term "label" or "labeled" refers to the incorporation of another molecule into an antibody. In one embodiment, the label is a detectable marker, such as a radiolabeled amino acid or a polypeptide attached to a biotin moiety that can be detected by a labeled avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods). In another embodiment, the label or marker can be therapeutic, such as a drug conjugate or toxin. Various methods of labeling polypeptides and glycoproteins are known in the art and can be used. Examples of polypeptide labels include, but are not limited to, the following: radioisotopes or radionuclides (e.g., 3H, 14C, 15N, 35S, 90Y, 99Tc, 111In, 125I, 131I), fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzyme labels (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotin groups, predetermined polypeptide epitopes recognized by secondary reporter molecules (e.g., leucine zipper pair sequences, binding sites of secondary antibodies, metal binding domains, epitope tags), magnetic agents (e.g., gadolinium chelates), toxins (e.g., pertussis toxin, paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and their analogs or homologs). In some embodiments, the labels are attached via spacer arms of various lengths to reduce potential steric hindrance.

[0266] In certain embodiments, the antibodies of the invention may exist in a neutral form (including zwitterionic form) or as a positively or negatively charged species. In some embodiments, the antibodies may be complexed with a counterion to form a pharmaceutically acceptable salt.

[0267] The term "pharmaceutically acceptable salt" refers to a complex comprising one or more antibodies and one or more counterions, wherein the counterions are derived from pharmaceutically acceptable inorganic and organic acids and bases.

[0268] Bispecific Binding Molecules

[0269] In other aspects, the present invention provides bispecific binding molecules having the binding specificity of an anti-Gal3 antibody or its antigen binding fragment as described herein (e.g., comprising an antigen binding fragment) and another anti-Gal3 antibody or its antigen binding fragment (e.g., another anti-Gal3 antibody as described herein) or an antibody targeting the same or different proteins, such as another immune checkpoint protein, a cancer antigen, or another cell surface molecule whose activity mediates a disease state such as cancer. Such bispecific binding molecules are known in the art, and examples of different types of bispecific binding molecules are given elsewhere herein.

[0270] Nucleic acid molecules and vectors

[0271] The present invention also provides nucleic acid molecules and sequences encoding the anti-Gal3 antibodies or antigen-binding fragments thereof described herein. In some embodiments, different nucleic acid molecules encode the heavy chain or VH sequence of the anti-Gal3 antibody or its antigen-binding fragment and encode the light chain amino acid sequence or VL sequence. In some embodiments, the combination of isolated nucleic acid molecules encodes the anti-Gal3 antibody or its antigen-binding fragment, in particular the first isolated nucleic acid molecule and the second isolated nucleic acid molecule, the first isolated nucleic acid molecule comprising or consisting of a sequence encoding a heavy chain sequence or VH sequence, and the second isolated nucleic acid molecule comprising or consisting of a sequence encoding a light chain sequence or VL sequence. In other embodiments, the same nucleic acid molecule encodes the heavy chain or VH sequence and the light chain or VL sequence of the anti-Gal3 antibody or its antigen-binding fragment.

[0272] Unless otherwise indicated, reference to a nucleotide sequence encompasses its complementary sequence. Thus, reference to a nucleic acid having a particular sequence should be understood to encompass its complementary strand and its complementary sequence. The term "polynucleotide" as used herein refers to a polymeric form of nucleotides of at least 10 bases in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide. The term includes single-stranded and double-stranded forms.

[0273] The present invention also provides nucleotide sequences that are at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to one or more nucleotide sequences described herein (e.g., to a nucleotide sequence selected from SEQ ID NO: 61-80, or to a nucleotide sequence encoding an amino acid sequence selected from SEQ ID NO: 38-57). In the context of nucleic acid sequences, the term "percentage of sequence identity" refers to the residues that are identical in two sequences when aligned for maximum correspondence. The length of the sequence identity comparison can exceed at least about 9 nucleotides, usually at least about 18 nucleotides, more usually at least about 24 nucleotides, typically at least about 28 nucleotides, more typically at least about 32 nucleotides, and preferably at least about 36, 48 or more nucleotides. There are many different algorithms known in the art that can be used to measure nucleotide sequence identity. For example, the programs FASTA, GAP or Bestfit in the Wisconsin Package version 10.0 of the Genetics Computer Group (GCG), Madison, Wisconsin, USA, can be used to compare polynucleotide sequences. FASTA (including, for example, FASTA2 and FASTA3) provides the comparison and sequence identity percentage of the best overlapping region between the query and search sequences (see, for example, Pearson, Methods Enzymol. 183: 63-98 (1990); Pearson, Methods Mol. Biol. 132: 185-219 (2000); Pearson, Methods Enzymol. 266: 227-258 (1996); and Pearson, J. Mol. Biol. 276: 71-84 (1998)). Unless otherwise stated, the default parameters of a particular program or algorithm are used. For example, percent sequence identity between nucleic acid sequences can be determined using FASTA with its default parameters (wordlength 6 and NOPAM coefficients for the scoring matrix) or using GAP provided in GCG version 6.1 with its default parameters.

[0274] In one aspect, the present invention provides a nucleic acid molecule comprising a nucleotide sequence selected from SEQ ID NOs: 61 to 80. In certain embodiments, the nucleic acid molecule comprises a nucleotide sequence of SEQ ID NOs: 61 and 62, SEQ ID NOs: 63 and 64, SEQ ID NOs: 65 and 66, SEQ ID NOs: 67 and 68, SEQ ID NOs: 69 and 70, SEQ ID NOs: 71 and 72, SEQ ID NOs: 73 and 74, SEQ ID NOs: 75 and 76, SEQ ID NOs: 77 and 78, or SEQ ID NOs: 79 and 80.

[0275] In any of the above embodiments, the nucleic acid molecule may be isolated.

[0276] On the other hand, the present invention provides a vector suitable for expressing one of the chains of an antibody or antigen-binding fragment thereof as described herein. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid connected thereto. In some embodiments, the vector is a plasmid, i.e., a circular double-stranded DNA fragment to which another DNA segment can be connected. In some embodiments, the vector is a viral vector, wherein the other DNA segment can be connected to the viral genome. In some embodiments, the vector can replicate autonomously in the host cell into which they are introduced (e.g., bacterial vectors and episomal mammalian vectors having a bacterial origin of replication). In other embodiments, the vector (e.g., a non-episomal mammalian vector) can be integrated into the genome of the host cell after being introduced into the host cell, thereby replicating with the host genome. In addition, certain vectors can direct the expression of genes operably connected thereto. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors").

[0277] The present invention provides a vector comprising a nucleic acid molecule encoding the heavy chain or VH of the anti-Gal3 antibody or its antigen-binding fragment of the present invention, the light chain or VL of the anti-Gal3 antibody or its antigen-binding fragment of the present invention, or the heavy chain and light chain or VH and VL of the anti-Gal3 antibody or its antigen-binding fragment of the present invention. The present invention also provides a vector comprising a nucleic acid molecule encoding a fusion protein, a modified antibody, an antibody fragment, and a probe.

[0278] Nucleic acid molecules encoding the heavy and / or light chains of the anti-Gal3 antibodies or antigen-binding fragments thereof of the present invention can be isolated from any source that produces such antibodies or fragments. In various embodiments, the nucleic acid molecules are isolated from B cells expressing anti-Gal3 antibodies, or from immortalized cells produced by such B cells, and the anti-Gal3 antibodies are isolated from animals immunized with human Gal3 antigen. Methods for isolating nucleic acids encoding antibodies are well known in the art. mRNA can be isolated and used to produce cDNA for polymerase chain reaction (PCR) or cDNA cloning of antibody genes. In certain embodiments, the nucleic acid molecules of the present invention can be synthesized rather than isolated.

[0279] In some embodiments, the nucleic acid molecules of the present invention may comprise a nucleotide sequence encoding a VH domain from an anti-Gal3 antibody or antigen-binding fragment of the present invention, which is linked in frame to a nucleotide sequence encoding a heavy chain constant region from any source. Similarly, the nucleic acid molecules of the present invention may comprise a nucleotide sequence encoding a VL domain from an anti-Gal3 antibody or antigen-binding fragment of the present invention, which is linked in frame to a nucleotide sequence encoding a light chain constant region from any source.

[0280] In another aspect of the present invention, nucleic acid molecules encoding the variable domains of heavy chains (VH) and / or light chains (VL) can be "converted" into full-length antibody genes. In one embodiment, nucleic acid molecules encoding VH or VL domains are converted into full-length antibody genes by inserting into expression vectors that have encoded heavy chain constant (CH) regions or light chain constant (CL) regions, respectively, so that the VH segment is effectively connected to the CH segment in the vector, and / or the VL segment is effectively connected to the CL segment in the vector. In another embodiment, nucleic acid molecules encoding VH and / or VL domains are linked (link) (e.g., ligated) to nucleic acid molecules encoding CH and / or CL regions using standard molecular biology techniques, and nucleic acid molecules encoding VH and / or VL domains are converted into full-length antibody genes. Nucleic acid molecules encoding full-length heavy chains and / or light chains can then be expressed from cells into which nucleic acid molecules encoding full-length heavy chains and / or light chains are introduced and anti-Gal3 antibodies can be isolated.

[0281] The nucleic acid molecules can be used to recombinantly express large amounts of anti-Gal3 antibodies. As described herein, the nucleic acid molecules can also be used to produce chimeric antibodies, bispecific antibodies, single chain antibodies, immunoadhesins, diabodies, mutant antibodies, and antibody derivatives.

[0282] In another embodiment, the nucleic acid molecules of the present invention are used as probes or PCR primers for specific antibody sequences. For example, the nucleic acid can be used as a probe in a diagnostic method or as a PCR primer to amplify a DNA region, which can be used to separate other nucleic acid molecules encoding anti-Gal3 antibody variable regions. In some embodiments, the nucleic acid molecule is an oligonucleotide. In some embodiments, the oligonucleotide is derived from the highly variable domains of the heavy and light chains of the antibody of interest. In some embodiments, the oligonucleotide encodes all or part of one or more CDRs of an anti-Gal3 antibody or its antigen-binding fragment as described herein.

[0283] In another embodiment, nucleic acid molecules and vectors can be used to prepare mutated anti-Gal3 antibodies or antigen-binding fragments thereof. Antibodies can mutate in the variable domains of the heavy chain and / or light chain, for example to change the binding properties of the antibody. For example, mutations can be made in one or more CDRs, or the KD of the anti-Gal3 antibody or its antigen-binding fragment can be increased or decreased, or the binding specificity of the antibody or its antigen-binding fragment can be changed. In another embodiment, in the monoclonal antibodies of the present invention, one or more mutations are made at amino acid residues known to have changed compared to the germline. Mutations can be made in the CDRs or framework regions of the variable regions or in the constant regions. In a preferred embodiment, mutations are produced in the variable domains. In some embodiments, one or more mutations are made in the CDRs or framework regions of the variable domains of the antibodies of the present invention or their antigen-binding fragments at amino acid residues known to have changed compared to the germline.

[0284] In another embodiment, the framework region is mutated so that the resulting framework region has the amino acid sequence of the corresponding germline gene. Mutations can be made in the framework region or the constant region to increase the half-life of the anti-Gal3 antibody. See, for example, PCT Publication WO 00 / 09560. Mutations can also be made in the framework region or the constant region to change the immunogenicity of the antibody, and / or to provide a site for covalent or non-covalent binding to another molecule. According to the present invention, a single antibody can have a mutation in any one or more CDRs or framework regions of the variable region or in the constant region.

[0285] In some embodiments, the anti-Gal3 antibody or antigen-binding fragment thereof of the present invention is expressed by inserting the DNA encoding partial or full-length light and heavy chains obtained as described above into an expression vector so that the gene is operably linked to necessary expression control sequences such as transcription and translation control sequences. Expression vectors include plasmids, retroviruses, adenoviruses, adeno-associated viruses (AAV), plant viruses such as cauliflower mosaic virus, tobacco mosaic virus, cosmids, YACs, EBV-derived episomes, etc. The antibody coding sequence can be linked to a vector so that the transcription and translation control sequences in the vector play their intended functions of regulating the transcription and translation of the antibody coding sequence. Expression vectors and expression control sequences compatible with the expression host cells used can be selected. The antibody light chain coding sequence and the antibody heavy chain coding sequence can be inserted into separate vectors and can be operably linked to the same or different expression control sequences (e.g., promoters). In one embodiment, both coding sequences are inserted into the same expression vector and can be operably linked to the same expression control sequence (e.g., a common promoter) to separate the same expression control sequence (e.g., a promoter), or to different expression control sequences (e.g., promoters). The antibody coding sequence can be inserted into the expression vector by standard methods (e.g., joining complementary restriction sites on the antibody gene fragment and the vector, or, if no restriction sites are present, blunt-end ligation).

[0286] Convenient vectors are vectors encoding functionally intact human CH or CL immunoglobulin sequences, which have suitable restriction sites of engineering approaches so that any VH or VL sequence can be easily inserted and expressed, as described above. The genes encoding HC and LC in such vectors may contain intron sequences, which can increase the total yield of antibody protein by stabilizing the relevant mRNA. The two sides of the intron sequence are splicing donor and splicing acceptor sites, which determine the position where RNA splicing occurs. The position of the intron sequence can be located in the variable region or constant region of the antibody chain, or in both the variable region and the constant region when multiple introns are used. Polyadenylation and transcription termination can occur at the natural chromosome site downstream of the coding region. Recombinant expression vectors can also encode signal peptides that promote antibody chain secretion from host cells. The antibody chain gene can be cloned into a vector so that the signal peptide is connected to the amino terminus of the immunoglobulin chain in frame. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin).

[0287] In addition to the antibody chain genes, the recombinant expression vectors of the present invention can carry regulatory sequences that control the expression of the antibody chain genes in host cells. It will be appreciated by those skilled in the art that the design of the expression vector, including the selection of regulatory sequences, may depend on factors such as the selection of the host cell to be transformed, the expression level of the desired protein, and the like. Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high-level protein expression in mammalian cells, such as promoters and / or enhancers derived from: retroviral LTR, cytomegalovirus (CMV) (e.g., CMV promoter / enhancer), simian virus 40 (SV40) (e.g., SV40 promoter / enhancer), adenovirus (e.g., adenovirus major late promoter (AdMLP)), polyoma virus, and strong mammalian promoters such as natural immunoglobulins and actin promoters. For further description of viral regulatory elements and sequences thereof, see, for example, U.S. Patents 5,168,062, 4,510,245, and 4,968,615. Methods for expressing antibodies in plants, including descriptions of promoters and vectors, and plant transformation, are known in the art. See, e.g., U.S. Patent No. 6,517,529. Methods for expressing polypeptides in bacterial cells or fungal cells such as yeast cells are also well known in the art.

[0288] In addition to the antibody chain gene and the regulatory sequence, the recombinant expression vector of the present invention can carry other sequences, such as sequences (e.g., replication origins) and selectable marker genes that the regulatory vector replicates in the host cell. The selectable marker gene helps to select the host cell that has been introduced into the vector (see, e.g., U.S. Patents 4,399,216, 4,634,665 and 5,179,017). For example, usually the selectable marker gene gives the host cell that has been introduced into the vector resistance to drugs such as G418, hygromycin or methotrexate. For example, selectable marker genes include dihydrofolate reductase (DHFR) gene (for carrying out methotrexate selection / amplification in DHFR-host cells), neo gene (selected for G418) and glutamate synthetase gene.

[0289] The term "expression control sequence" as used herein refers to a polynucleotide sequence necessary for achieving expression and processing of a coding sequence connected thereto. Expression control sequences include suitable transcription initiation, termination, promoter and enhancer sequences; effective RNA processing signals, such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and sequences that enhance protein secretion when necessary. The nature of these control sequences varies depending on the host organism; in prokaryotes, such control sequences typically include promoters, ribosome binding sites, and transcription termination sequences; in eukaryotes, such control sequences typically include promoters and transcription termination sequences. The term "control sequence" is intended to include at least all components whose presence is necessary for expression and processing, and may also include other components whose presence is advantageous, such as leader sequences and fusion partner sequences.

[0290] Host cells and methods for producing antibodies and antibody compositions

[0291] Another aspect of the present invention relates to a method for producing the antibody compositions and antibodies and antigen-binding fragments thereof of the present invention. An embodiment of this aspect of the present invention relates to a method for producing an antibody as defined herein, comprising providing a recombinant host cell capable of expressing the antibody, culturing the host cell under conditions suitable for expressing the antibody, and isolating the resulting antibody. Antibodies produced by such expression in such recombinant host cells are referred to herein as "recombinant antibodies". The present invention also provides progeny cells of such host cells, and antibodies produced therefrom.

[0292] As used herein, the term "recombinant host cell" (or simply "host cell") refers to a cell into which a recombinant expression vector has been introduced. The present invention provides a host cell, which may include, for example, the above-mentioned vector according to the present invention. The present invention also provides a host cell, which includes, for example, a nucleotide sequence encoding the heavy chain or antigen-binding fragment of the anti-Gal3 antibody or its antigen-binding fragment of the present invention, a nucleotide sequence encoding the light chain or antigen-binding fragment of the anti-Gal3 antibody or its antigen-binding fragment of the present invention, or a nucleotide sequence encoding both. It should be understood that "recombinant host cell" and "host cell" refer not only to a specific subject cell, but also to the progeny of such a cell. Due to mutations or environmental influences, certain modifications may occur in subsequent generations, so such progeny may actually be different from the parent cell, but are still included in the scope of the term "host cell" as used herein.

[0293] Nucleic acid molecules encoding anti-Gal3 antibodies and vectors containing these nucleic acid molecules can be used to transfect suitable mammalian, plant, bacterial or yeast host cells. Transformation can introduce polynucleotides into host cells by any known method. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art, including dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of polynucleotides in liposomes, and direct microinjection of DNA into the nucleus. In addition, nucleic acid molecules can be introduced into mammalian cells through viral vectors. Transformation methods are well known in the art. See, for example, U.S. Patents 4,399,216, 4,912,040, 4,740,461 and 4,959,455. Methods for transforming plant cells are well known in the art, including, for example, Agrobacterium-mediated transformation, gene gun transformation, direct injection, electroporation and viral transformation. Methods for transforming bacterial and yeast cells are also well known in the art.

[0294] Mammalian cell lines that can be used as expression hosts are well known in the art, including many immortalized cell lines that can be obtained from the American Type Culture Collection (ATCC). These cells especially include Chinese hamster ovary (CHO) cells, NSO cells, SP2 cells, HEK-293T cells, 293Freestyle cells (Invitrogen), NIH-3T3 cells, HeLa cells, baby hamster kidney (BHK) cells, African green monkey kidney cells (COS), human hepatocellular carcinoma cells (such as Hep G2), A549 cells and many other cell lines. It is determined which cell lines have high expression levels to select a particularly preferred cell line. Other cell lines that can be used are insect cell lines, such as Sf9 or Sf21 cells. When the recombinant expression vector encoding the antibody gene is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a period of time sufficient to allow the antibody to be expressed in the host cell, or more preferably, a period of time sufficient to allow the antibody to be secreted into the culture medium for the growth of the host cell. Standard protein purification methods can be used to reclaim the antibody from the culture medium. Plant host cells include, for example, Nicotiana, Arabidopsis, duckweed, corn, wheat, potato, etc. Bacterial host cells include Escherichia coli and Streptomyces species. Yeast host cells include Schizosaccharomyces pombe, Saccharomyces pombe, and Pichia pastoris.

[0295] In addition, many known techniques can be used to enhance the expression of the antibodies or antigen-binding fragments thereof of the present invention in production cell lines. For example, the glutamine synthetase gene expression system (GS system) is a common method for enhancing expression under certain conditions. The GS system is discussed in whole or in part in European Patents 0 216 846, 0 256 055, 0 323 997 and 0 338841.

[0296] Antibodies expressed by different cell lines or antibodies expressed in transgenic animals may have different glycosylation patterns from each other. However, all antibodies encoded by the nucleic acid molecules provided herein or all antibodies comprising the amino acid sequences provided herein are part of the present invention, regardless of the glycosylation state of the antibody, and more generally, regardless of the presence or absence of post-translational modifications.

[0297] Pharmaceutical composition

[0298] Another aspect of the present invention is a pharmaceutical composition comprising an anti-Gal3 antibody or antigen-binding fragment thereof of the present invention as an active ingredient (or as the sole active ingredient), a bispecific binding molecule or an antibody composition. The pharmaceutical composition may comprise any anti-Gal3 antibody or antigen-binding fragment thereof, a bispecific binding molecule or an antibody composition as described herein. In some embodiments, the pharmaceutical composition is intended to improve, prevent and / or treat a Gal3-related disorder or disease. As used herein, a Gal3-related disorder or Gal3-related disease refers to a condition that improves or slows its progression by regulating Gal3 expression, interaction and / or activity. In certain embodiments, the composition is intended to improve, prevent and / or treat fibrotic diseases, inflammatory diseases, autoimmune diseases, immune-mediated disorders, neurodegenerative diseases, metabolic diseases, infectious diseases, and a variety of cancers.

[0299] Typically, the antibodies, antigen-binding fragments, and bispecific binding molecules of the invention are suitably administered as a formulation in association with one or more pharmaceutically acceptable excipients, such as described below.

[0300] The pharmaceutical composition of the present invention will contain one or more anti-Gal3 antibodies or antigen-binding fragments thereof or bispecific binding molecules of the present invention, such as one or two anti-Gal3 antibodies or antigen-binding fragments thereof or bispecific binding molecules. In one embodiment, the composition comprises a single anti-Gal3 antibody or antigen-binding fragment thereof of the present invention. In one embodiment, the composition comprises a plurality of anti-Gal3 antibodies or antigen-binding fragments thereof of the present invention.

[0301] In another embodiment, the pharmaceutical composition may comprise at least one anti-Gal3 antibody or antigen-binding fragment thereof, such as one anti-Gal3 antibody or antigen-binding fragment thereof, and one or more other antibodies targeting one or more relevant cell surface receptors, such as one or more cancer-associated receptors.

[0302] The term "excipient" is used herein to describe any ingredient other than the compounds of the present invention. The choice of excipient depends to a large extent on factors such as the specific mode of administration, the effect of the excipient on solubility and stability, and the properties of the dosage form. As used herein, "pharmaceutically acceptable excipients" include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delay agents that are physiologically compatible. Some examples of pharmaceutically acceptable excipients are water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In many cases, isotonic agents, such as sugars, polyols (such as mannitol, sorbitol), or sodium chloride are preferably included in the composition. Another example of a pharmaceutically acceptable substance is a wetting agent or a small amount of auxiliary substance, such as a wetting agent or emulsifier, a preservative, or a buffer, which increases the shelf life of the antibody or enhances effectiveness.

[0303] The pharmaceutical compositions of the present invention and methods for preparing the same are obvious to those skilled in the art. Such compositions and methods for preparing the same can be found, for example, in Remington's Pharmaceutical Sciences, 19th edition (Mack Publishing Company, 1995). The pharmaceutical compositions are preferably produced under GMP (Good Manufacturing Practice) conditions.

[0304] The pharmaceutical compositions of the present invention can be prepared, packaged or sold in batches as a single unit dose or as a plurality of single unit doses. As used herein, a "unit dose" is a discrete amount of a pharmaceutical composition containing a predetermined amount of an active ingredient. The amount of the active ingredient is generally equal to the dose of the active ingredient to be administered to a subject or a convenient fraction of such a dose, such as half or one-third of the dose.

[0305] Any art-accepted method for administering peptides, proteins, or antibodies can be applied to the antibodies and antigen-binding fragments of the invention.

[0306] The pharmaceutical composition of the present invention is generally suitable for parenteral administration. As used herein, "parenteral administration" of a pharmaceutical composition includes any route of administration characterized by physically destroying the tissue of a subject and administering the pharmaceutical composition through a breach in the tissue, thus generally resulting in direct administration in the bloodstream, in a muscle, or in an internal organ. Therefore, parenteral administration includes, but is not limited to, administering the pharmaceutical composition by injecting the composition, administering the composition through a surgical incision, administering the composition through a tissue penetration non-surgical wound, etc. In particular, it is contemplated that parenteral administration includes, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrasternal, intravenous, intraarterial, intrathecal, intraventricular, intraurethral, ​​intracranial, intratumoral, and intrasynovial injection or infusion; and renal dialysis infusion techniques. Regional perfusion is also contemplated. Specific embodiments include intravenous and subcutaneous approaches.

[0307] The preparation of the pharmaceutical composition suitable for parenteral administration generally comprises an active ingredient combined with a pharmaceutically acceptable carrier such as sterile water or sterile isotonic saline. Such preparations can be prepared, packaged or sold in a form suitable for push injection or continuous administration. Injectable preparations can be prepared, packaged or sold in unit dosage form (e.g., in an ampoule or a multi-dose container containing a preservative). Preparations for parenteral administration include, but are not limited to, emulsions, pastes, etc. in suspensions, solutions, oily or aqueous vehicles. Such preparations can also include one or more additional ingredients, including but not limited to suspending agents, stabilizers or dispersants. In one embodiment, the active ingredient for the preparation for parenteral administration is provided in a dry (i.e., powder or granule) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) before parenteral administration of the reconstructed composition. Parenteral preparations also include aqueous solutions, which may contain excipients such as salts, carbohydrates and buffers (preferably to a pH of 3 to 9), but for some applications, they may be more suitably formulated as sterile non-aqueous solutions or formulated as dry forms to be used in combination with suitable vehicles such as sterile pyrogen-free water. Exemplary parenteral administration forms include solutions or suspensions in sterile aqueous solutions, such as propylene glycol or glucose aqueous solutions. If necessary, such dosage forms may be suitably buffered. Other useful parenteral administration preparations include those comprising the active ingredient of microcrystalline form or liposome preparations. Preparations for parenteral administration may be formulated into quick-release and / or sustained-release preparations (modified release). Sustained-release preparations include delayed release, sustained release, pulse release, controlled release, targeted release and programmed release.

[0308] For example, in one aspect, a sterile injectable solution can be prepared by incorporating an anti-Gal3 antibody, an antigen-binding fragment thereof, a bispecific binding molecule or an antibody composition in a desired amount into a suitable solvent, incorporating one or a combination of the ingredients listed above as needed, and then filtering and sterilizing. Typically, a dispersion is prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other ingredients required from those listed above. In the case of sterile powders for preparing sterile injectable solutions, preferred preparation methods are vacuum drying and freeze drying, which produce a powder of the active ingredient plus any other desired ingredients from a previously sterile filtered solution thereof. The appropriate fluidity of the solution can be maintained, for example, by using a coating such as lecithin, by maintaining the desired particle size in the case of a dispersion, and by using a surfactant. Extended absorption of the injectable composition can be achieved by including an agent that delays absorption (e.g., monostearate and gelatin) in the composition and / or by using a modified-release coating (e.g., a slow-release coating).

[0309] The antibodies of the invention can also be administered intranasally or by inhalation, usually in the form of a dry powder (alone, as a mixture or as mixed component particles, e.g., mixed with a suitable pharmaceutically acceptable excipient) from a dry powder inhaler, as an aerosol spray (with or without a suitable propellant) from a pressurized container, pump, spray, atomizer (preferably an atomizer using electrohydrodynamics to produce a fine mist) or nebuliser or as nasal drops.

[0310] The pressurized container, pump, spray, atomizer or nebulizer typically contains a solution or suspension of the antibody of the invention containing, for example, a suitable agent for dispersing, dissolving or prolonging the release of the active substance, a propellant as a solvent.

[0311] Prior to use in dry powder or suspension formulations, the drug product is typically micronized to a size suitable for delivery by inhalation (usually less than 5 microns). This can be achieved by any suitable comminution method, such as spiral jet milling, fluidized bed jet milling, supercritical fluid processing to form nanoparticles, high pressure homogenization or spray drying.

[0312] Capsules, blisters and cartridges for use in an inhaler or insufflator may be formulated containing a powder mix of a compound of the invention, a suitable powder base and a performance modifier.

[0313] Suitable solution formulations for nebulizers that use electrohydrodynamics to generate a fine mist may contain a suitable dose of an antibody of the invention per actuation, and the actuation volume may vary, for example, from 1 μL to 100 μL.

[0314] Preparations for inhaled / intranasal administration can be formulated as immediate and / or sustained release preparations. Sustained release preparations include delayed release, sustained release, pulsed release, controlled release, targeted release, and programmed release.

[0315] In the case of dry powder inhalers and aerosols, the dosage unit is determined by a valve that delivers a metered dose. The device of the present invention is generally arranged to administer a metered dose or "spray" the antibody of the present invention. The total daily dose is generally administered in a single dose, or more generally, as divided doses throughout the day.

[0316] The antibodies and antibody fragments of the invention can also be formulated for oral administration. Oral administration can include swallowing, so that the compound enters the gastrointestinal tract, and / or buccal, lingual or sublingual administration, through which the compound enters the bloodstream directly from the mouth.

[0317] Formulations suitable for oral administration include solid, semisolid and liquid systems, such as tablets; soft or hard capsules containing multi- or nanoparticles, liquids or powders; lozenges (including liquid-filled); chewables; gels; fast-dispersing dosage forms; films; ovules; sprays; and buccal / mucosal adhesive patches.

[0318] Liquid preparations include suspensions, solutions, syrups and elixirs. Such preparations can be used as fillers in soft or hard capsules (e.g., made of gelatin or hydroxypropyl methylcellulose) and typically contain carriers such as water, ethanol, polyethylene glycol, propylene glycol, methylcellulose or suitable oils, and one or more emulsifiers and / or suspending agents. Liquid preparations can also be prepared by reconstitution of solids, such as reconstitution from a pouch.

[0319] Therapeutic uses of the antibodies and compositions of the invention

[0320] In one aspect, the anti-Gal3 antibodies and antigen-binding fragments thereof, anti-Gal3 compositions and bispecific binding molecules of the present invention are used to inhibit Gal3 expression, interaction and / or activity in a person in need thereof. The anti-Gal3 antibodies or antigen-binding fragments thereof of the present invention can be administered alone or in combination with other therapeutic agents (sequentially or simultaneously).

[0321] In certain embodiments, the antibodies or antigen-binding fragments thereof, compositions or bispecific binding molecules are used to improve, prevent and / or treat a number of fibrotic diseases, inflammatory diseases, autoimmune diseases, immune-mediated disorders, neurodegenerative diseases, metabolic diseases, infectious diseases and various cancers.

[0322] The term "fibrosis" refers to a medical condition in which tissues or organs become hardened or scarred due to unregulated production of an extracellular matrix such as collagen. Fibrosis is associated with chronic inflammation, in which immune cells such as macrophages signal fibroblasts to express extracellular matrix proteins in response. This signaling is achieved through pathways such as the IL-6 pathway, the IL-5 pathway, although there are other profibrotic pathways. Fibrosis includes, but is not limited to, hepatic fibrosis, bridging fibrosis, cirrhosis, renal fibrosis, pulmonary fibrosis, asbestosis, silicosis, coal workers' pneumoconiosis and diffuse dust fibrosis, chemotherapeutic agent-induced pulmonary fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, polycystic kidney disease, cardiovascular fibrosis, Chagas disease, arterial fibrosis, venous thrombosis, cardiac fibrosis, pulmonary artery fibrosis, arthrofibrosis, Dupuytren's contracture, keloid, mediastinal fibrosis, myelofibrosis, Peyronie's disease, nephrogenic systemic fibrosis, tubulointerstitial fibrosis, glomerulosclerosis progressive massive fibrosis, aristolochic acid and Balkan endemic nephropathy, progressive massive fibrosis, retroperitoneal fibrosis, or systemic sclerosis.

[0323] In some embodiments, the cancers prevented or treated by the anti-Gal3 antibodies, antigen-binding fragments, bispecific binding molecules and / or antibody compositions of the present invention may include cancers such as carcinomas, sarcomas, leukemias and lymphomas, such as T-cell lymphomas; metastatic cancers. It may also include melanoma (e.g., advanced or metastatic melanoma), non-small cell lung cancer, head and neck squamous cell carcinoma, renal cell carcinoma, Hodgkin lymphoma, non-Hodgkin lymphoma, glioblastoma, glioma, squamous cell lung cancer, small cell lung cancer, hepatocellular carcinoma, bladder cancer, upper urinary tract cancer, esophageal cancer, gastroesophageal junction cancer, gastric cancer, liver cancer, colon cancer, colorectal cancer, multiple myeloma, sarcoma, acute myeloid leukemia, chronic myeloid leukemia, myelodysplastic syndrome, Nasopharyngeal carcinoma, chronic lymphocytic leukemia, acute lymphoblastic leukemia, small lymphocytic lymphoma, ovarian cancer, gastrointestinal cancer, primary peritoneal cancer, fallopian tube cancer, urothelial carcinoma, HTLV-related T-cell leukemia / lymphoma, prostate cancer, genitourinary cancer, meningioma, adrenocortical carcinoma, gliosarcoma, fibrosarcoma, renal cancer, breast cancer, pancreatic cancer, endometrial cancer, basal cell carcinoma of the skin, appendix cancer, biliary tract cancer, salivary gland cancer, advanced Merkel cell carcinoma, diffuse large B-cell lymphoma, follicular lymphoma, mesothelioma or solid tumors. Cancer can be, for example, early, intermediate, advanced or metastatic stage.

[0324] The terms "cancer", "tumor" and "cancer" are used interchangeably herein and refer to cells that exhibit relatively autonomous growth, such that they exhibit an abnormal growth phenotype characterized by a significant loss of cell proliferation control. In general, the target cells for detection or treatment in the present application include precancerous (e.g., benign), malignant, pre-metastatic, metastatic and non-metastatic cells. The detection of cancer cells is of particular interest. The term "normal" used in the context of "normal cells" refers to cells of untransformed phenotypes or cells that exhibit the morphology of untransformed cells of the tissue type being examined. "Cancerous phenotype" generally refers to any of a variety of biological phenomena characteristic of cancerous cells, which can vary with the type of cancer. Cancerous phenotypes are typically identified by, for example, abnormalities in cell growth or proliferation (e.g., uncontrolled growth or proliferation), cell cycle regulation, cell migration, cell-cell interactions or metastasis, etc.

[0325] In one aspect, the anti-Gal3 antibodies and antigen-binding fragments thereof, antibody compositions or bispecific binding molecules of the invention can be used to prevent or treat inflammatory diseases or autoimmune disorders or immune-mediated diseases or immune-mediated disorders, such as psoriasis, rheumatoid arthritis, juvenile idiopathic arthritis, osteoarthritis, ulcerative colitis, ankylosing spondylitis, autoimmune uveitis, primary cholangitis, autoimmune and inflammatory kidney disease, sepsis, type 1 diabetes, inflammatory myopathy, myocarditis, pericarditis, allergic reactions, asthma, gastric ulcer, gastritis, systemic lupus erythematosus, Sjögren's syndrome ( syndrome, inflammatory bowel disease (IBD), Crohn's disease, atopic dermatitis, systemic sclerosis, or sclerosis cutis.

[0326] In one aspect, the anti-Gal3 antibodies and antigen-binding fragments thereof, antibody compositions or bispecific binding molecules of the present invention can be used to prevent or treat metabolic disorders, heart disease, heart failure, myocardial infarction, pathological angiogenesis, atherosclerosis, hypertension, pulmonary hypertension, venous thrombosis, metabolic diseases such as diabetes, type 2 diabetes, type 1 diabetes, insulin resistance, obesity, diastolic heart failure, asthma and other interstitial lung diseases, including Hermansky-Pudlak syndrome, mesothelioma, chronic obstructive pulmonary disease, endometriosis, liver diseases such as non-alcoholic steatohepatitis.

[0327] In one aspect, the anti-Gal3 antibodies and antigen-binding fragments thereof, antibody compositions or bispecific binding molecules of the present invention can be used to prevent or treat neurodegenerative diseases or diseases affecting the central and / or peripheral nervous system of a patient. The anti-Gal3 antibodies or binding fragments thereof disclosed herein disrupt the interaction between Gal3 and proteins associated with neurodegenerative diseases. In some embodiments, proteins associated with neurodegenerative diseases cause diseases due to misfolding or aggregation of proteins in a subject. These diseases may involve the death of neurons or other cell types associated with the nervous system. Non-limiting examples of nervous system disorders include encephalitis, Alzheimer's disease, Parkinson's disease, Huntington's disease, traumatic brain injury, spinal cord injury, multiple sclerosis, amyotrophic lateral sclerosis, olfactory dysfunction, aphasia, Bell's palsy, transmissible spongiform encephalopathy, Creutzfeldt-Jakob disease, fatal familial insomnia, epilepsy, seizures, neurodevelopment, Tourette's syndrome, neuroinfectious disorders, meningitis, encephalitis, bovine spongiform encephalopathy, West Nile virus encephalitis, neuro-AIDS, fragile X syndrome, Guillain-Barre syndrome, brain metastasis or brain cancer, or other diseases known to those skilled in the art.

[0328] In one aspect, the anti-Gal3 antibodies and antigen-binding fragments thereof, antibody compositions or bispecific binding molecules of the invention can be used to prevent or treat infectious diseases, such as viral or parasitic or pathogenic infections and their consequences, including those induced by coronavirus, papilloma virus, influenza virus, encephalomyocarditis virus, trypanosoma cruzi.

[0329] "Treatment" and its variants refer to a method of alleviating or eliminating at least one of a biological disease or disorder and / or its associated symptoms. As used herein, "alleviating" a disease, disorder or condition means reducing the severity and / or frequency of the symptoms of the disease, disorder or condition. In addition, "treatment" as used herein includes curative, palliative and preventive treatment.

[0330] A "therapeutically effective amount" refers to an amount of a therapeutic agent administered that will relieve to some extent one or more of the symptoms of the disease or disorder being treated. A therapeutically effective amount of an anti-cancer therapeutic agent can, for example, result in tumor shrinkage, increased survival, elimination of cancer cells, reduction in disease progression, reversal of metastasis, or other clinical endpoints desired by a health care professional.

[0331] In some embodiments, the present invention also describes a method of monitoring the progression of tissue fibrosis by monitoring one or more fibrosis biomarkers, wherein the fibrosis biomarkers comprise or consist of α-smooth muscle actin, fibronectin, collagen, elastin, laminin, hyaluronic acid, or proteoglycans, or any combination thereof.

[0332] In some embodiments, the anti-Gal3 antibody or its antigen-binding fragment results in a decrease in the accumulation of extracellular matrix proteins in a tissue. In some embodiments, the anti-Gal3 antibody or its antigen-binding fragment is used to improve, prevent and / or treat a disease, wherein the disease is a fibrotic disease or fibrosis. In some embodiments, the anti-Gal3 antibody or its antigen-binding fragment is used to improve, prevent and / or treat a disease that results in a decrease in the accumulation of extracellular matrix proteins in a tissue. In some embodiments, the extracellular matrix comprises agrin, nidogen, cadherin, clathrin, collagen, defensin, elastin, entactin, fibrillin, fibronectin, keratin, laminin, microtubule-actin cross-linking factor 1, SPARC-like protein, nesprin, fibrous sheath interacting protein, myosin, nebulin, plakophilin, integrin, talin, exportin, transporter, tenascin, perlecan, sortin-associated receptor, tensin or titin or any combination thereof or consists of the above. In some embodiments, the extracellular matrix protein comprises or consists of collagen. In some embodiments, the tissue comprises or consists of collagen-producing cells. 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-b, IL-1b, TNF-a, IL-5, or IL-6. In some embodiments, the tissue has elevated expression of a fibrogenic cytokine.

[0333] The anti-Gal3 antibody or antigen-binding fragment thereof, antibody composition or bispecific binding molecule of the present invention can be administered alone or in combination with one or more other drugs or antibodies (or as any combination thereof). Therefore, the pharmaceutical compositions, methods and uses of the present invention also include embodiments of combination (co-administration) with other active agents, as described in detail below.

[0334] As used herein, the terms "co-administration", "co-administered" and "combination" when referring to the anti-Gal3 antibodies and antigen-binding fragments thereof, antibody compositions and bispecific binding molecules of the present invention and one or more other therapeutic agents are intended to mean and do refer to and include the following:

[0335] a) when such combination of an antibody / antigen-binding fragment / antibody composition / bispecific binding molecule of the invention and a therapeutic agent are formulated together into a single dosage form, such combination is administered simultaneously to a patient in need of treatment, said dosage form releasing said components to said patient substantially simultaneously;

[0336] b) when such combination of the antibody / antigen-binding fragment / antibody composition / bispecific binding molecule of the invention and the therapeutic agent are formulated separately from each other into separate dosage forms, such combination is administered to a patient in need of treatment substantially simultaneously, said dosage forms are taken by said patient substantially simultaneously, and said components are subsequently released to said patient substantially simultaneously;

[0337] c) when such combinations of the antibodies / antigen-binding fragments / antibody compositions / bispecific binding molecules of the invention and therapeutic agents are formulated separately from one another into separate dosage forms, such combinations are administered sequentially to a patient in need of treatment, said dosage forms being taken by said patient at consecutive times with significant time intervals between each administration, whereupon said components are released to said patient at substantially different times; and

[0338] d) When such combinations of antibodies / antigen-binding fragments / antibody compositions / bispecific binding molecules of the invention and therapeutic agents are formulated together into a single dosage form that releases the components in a controlled manner, such combinations are administered sequentially to a patient in need of treatment, whereupon the components are released to the patient simultaneously, sequentially and / or overlappingly at the same and / or different times, wherein the various parts may be administered by the same or different routes.

[0339] The anti-Gal3 antibodies and antigen-binding fragments thereof, antibody compositions or bispecific binding molecules of the present invention may be administered without additional therapeutic treatment, i.e., as independent therapy (monotherapy). Alternatively, treatment with the anti-Gal3 antibodies and antigen-binding fragments thereof, antibody compositions or bispecific binding molecules of the present invention may include at least one other additional therapeutic treatment (combination therapy). The additional therapeutic treatment may include administering other additional therapeutic agents to the patient, such as immune checkpoint regulators, chemotherapeutic agents, anti-tumor agents, anti-angiogenic agents, hormone therapeutic agents, stem cell-based therapeutic agents, surgery and / or radiotherapy. The additional therapeutic treatment may include PD1 / PDL1 blocking therapy and / or CTLA4 blocking therapy, including administering other additional therapeutic agents to the patient. In some embodiments, PD1 / PDL1 blockade therapy comprises administering to the patient a therapeutic agent, such as pembrolizumab, nivolumab, cemiplimab, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, AMP-224, AMP-514, atezolizumab, avelumab, durvalumab, KN035, CK-301, AUNP12, CA-170, and / or BMS-986189. In some embodiments, CTLA4 blockade therapy comprises administering to the patient a therapeutic agent, such as ipilimumab and / or tremilimumab.

[0340] It is also contemplated that the anti-Gal3 antibodies or antigen-binding fragments thereof, antibody compositions or bispecific binding molecules of the invention can be used in combination with other additional therapeutic agents for adjuvant therapy, which are tyrosine kinase inhibitors. These are synthetic, low molecular weight molecules derived primarily from quinazolines that interact with the intracellular tyrosine kinase domain of the receptor and inhibit ligand-induced receptor phosphorylation by competing for intracellular Mg-ATP binding sites.

[0341] In certain aspects, the antibodies and antigen-binding fragments, antibody compositions or bispecific binding molecules of the invention can be administered in combination with other additional therapeutic agents, which are another inhibitor of the Gal3 pathway that can target Gal3 or one or more of its ligands. Examples of such inhibitors include other anti-Gal3 antibodies or small molecules targeting Gal3.

[0342] It should be understood that the antibodies and antigen-binding fragments thereof, antibody compositions and bispecific binding molecules of the present invention can be used in the improvement, prevention and / or treatment methods described herein, can be used in the improvement, prevention and / or treatment described herein, and / or can be used to manufacture the improvement, prevention and / or treatment drugs described herein. The present invention also provides kits and products, which contain antibodies and antigen-binding fragments thereof, antibody compositions and bispecific binding molecules described herein.

[0343] Dosage and route of administration

[0344] The antibodies or antigen-binding fragments thereof, antibody compositions or bispecific binding molecules of the invention will be administered in an effective amount to ameliorate, prevent and / or treat the condition in question, i.e., at a dosage and for a period of time necessary to achieve the desired result. The therapeutically effective amount may vary according to a variety of factors, such as the specific condition being treated, the age, sex and weight of the patient, and whether the antibody is administered as a stand-alone therapy or in combination with one or more additional anti-cancer treatments.

[0345] The dosage regimen may be adjusted to provide the optimal desired response. For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be gradually reduced or increased as the therapeutic situation requires. For ease of administration and uniformity of dosage, it is particularly advantageous to formulate parenteral compositions in dosage unit form. As used herein, dosage unit form refers to physically discrete units suitable as unit doses for the patient / subject to be treated; each unit contains a predetermined amount of active compound, calculated to produce the desired therapeutic effect in combination with the required pharmaceutical carrier. The specifications for the dosage unit forms of the present invention are generally determined by, and directly depend on, (a) the unique characteristics of the chemotherapeutic agent and the specific therapeutic or preventive effect to be achieved and (b) the inherent limitations of the technology for formulating such active compounds for treating individual sensitivities.

[0346] Therefore, it will be appreciated by those skilled in the art based on the disclosure provided herein that dosages and dosing regimens can be adjusted according to well-known methods in the therapeutic field. That is, the maximum tolerated dose can be easily established, and the effective amount that provides a detectable therapeutic benefit to the patient can also be determined, as well as the time requirements for administering each agent that provides a detectable therapeutic benefit to the patient can be determined. Therefore, although certain dosages and administration regimens are illustrated herein, these examples are by no means limiting the dosages and administration regimens that can be provided to patients when practicing the present invention.

[0347] It should be noted that dosage values ​​may vary with the type and severity of the condition to be alleviated, and may include a single dose or multiple doses. It should also be understood that for any particular subject, the specific dosage regimen should be adjusted over time according to individual needs and the professional judgment of the person administering or supervising the administration of the composition, and the dosage ranges described herein are exemplary only and are not intended to limit the scope or practice of a specific composition. In addition, the dosage regimen using the composition of the present invention can be based on a variety of factors, including disease type, age, weight, sex, the patient's medical condition, the severity of the condition, the route of administration, and the specific antibody used. Therefore, the dosage regimen can vary widely, but can be routinely determined using standard methods. For example, the dosage can be adjusted based on pharmacokinetic or pharmacodynamic parameters, which can include clinical effects, such as toxic effects and / or laboratory values. Therefore, the present invention includes intra-patient dose escalation as determined by a technician. Determining appropriate dosages and regimens is well known in the relevant art, and once the teachings disclosed herein are provided, those skilled in the art will understand that it is covered.

[0348] It is expected that the suitable dosage of the antibody, antigen binding fragment, antibody composition or bispecific binding molecule of the present invention will be in the range of 0.1-100mg / kg, such as about 0.5-50mg / kg, for example, about 1-20mg / kg.Antibody, antigen binding fragment, antibody composition or bispecific binding molecule can be administered, for example, at the following dosage: at least 0.25mg / kg, for example, at least 0.5mg / kg, for example, at least 1mg / kg, for example, at least 1.5mg / kg, for example, at least 2mg / kg, for example, at least 3mg / kg, for example, at least 4mg / kg, for example, at least 5mg / kg; and for example, up to 50mg / kg, for example, up to 30mg / kg, for example, up to 20mg / kg, for example, up to 15mg / kg.Usually, it is repeatedly administered at suitable intervals, for example, once a week, once every two weeks, once every three weeks or once every four weeks, and as long as the responsible doctor deems it appropriate, it can optionally increase or decrease the dosage as needed.

[0349] The effective amount for tumor treatment can be measured by its ability to stabilize disease progression and / or improve patient symptoms, and preferably reverses disease progression, for example, by reducing tumor size. The ability of the antibodies, antigen-binding fragments, antibody compositions or bispecific binding molecules of the present invention to inhibit cancer can be evaluated by in vitro assays, for example, as described in the Examples and in suitable animal models that predict the efficacy of human tumors. Select a suitable dosage regimen to provide the best therapeutic response in each specific case, for example, as a single bolus or as a continuous infusion, and adjust the dosage as needed for each case.

[0350] Diagnostic uses and compositions

[0351] The antibodies of the present invention can also be used in diagnostic procedures (e.g., in vitro, ex vivo). For example, the antibodies can be used to detect and / or measure the Gal3 level in a sample (e.g., a tissue sample, or a body fluid sample such as an inflammatory exudate, blood, serum, intestinal fluid, saliva, or urine) from a patient. Suitable detection and measurement methods include immunological methods, such as flow cytometry, enzyme-linked immunosorbent assay (ELISA), chemiluminescence assay, radioimmunoassay, and immunohistology. The present invention also includes a kit (e.g., a diagnostic kit) comprising the antibodies described herein.

[0352] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. In the event of a conflict, the present specification (including definitions) shall prevail.

[0353] Generally, the nomenclature used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, medicinal chemistry, and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art. Enzyme reactions and purification techniques are performed according to manufacturer's instructions, as commonly accomplished in the art or as described herein.

[0354] In addition, unless the context requires otherwise, singular terms shall include pluralities and plural terms shall include the singular. In the present specification and embodiments, the words "have" and "comprise" or variations thereof, such as "has, having", "comprises, comprising" will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

[0355] In order to better understand the present invention, the following examples are given. These examples are for illustrative purposes only and should not be understood as limiting the scope of the present invention in any way.

[0356] Example

[0357] Example 1: Production of novel anti-Gal3 scFv

[0358] Novel galectin-3 inhibitor monoclonal antibodies were obtained using the Mabqi human phage display platform.

[0359] Several constructs have been generated for panning experiments: hu-Gal3-FL (full length human Galectin 3, as shown in SEQ ID NO 106 and available under UniProt accession number P17931-LEG3_HUMAN), hu-Gal3-CRD (CRD domain of human Galectin 3, contained in SEQ ID NO 107) and m-Gal3-FL (full length mouse Galectin 3, as shown in SEQ ID NO 110 and available under UniProt accession number P16110-LEG3_MOUSE), which have been previously controlled and showed no protein degradation.

[0360] Recombinant proteins were immobilized via their histidine tags on Nunc Maxisorp plates: coated with 10 μg / mL of streptavidin in TBS, 100 μL / well, and incubated for 1 hour at room temperature. The plates were then blocked with 200 μL of TBS-BSA 4% for 2 hours. A TBS solution containing 5 mM NiSO4 and 1 μg / mL of Tris-NTA-biotin was incubated for 30 minutes at room temperature. The plates were washed 3 times with 0.1% TBST, and 100 μL of NiSO4 / Tris-NTA-biotin solution was added to the wells for 1 hour at room temperature. The plates were washed again, and 20 μg / mL of the recombinant protein in TBS was added, 100 μL / well, and incubated overnight at 4°C.

[0361] The plates were washed 3 times with 0.1% TBST and the phage library was depleted on wells coated with streptavidin only and NiSO4 / Tris-NTA-biotin: 50 μL of library was incubated with 50 μL of TBS-BSA 4% for 1 hour at room temperature.

[0362] The depleted library was incubated on the antigen-coated wells for 2 hours at room temperature. The wells were washed 10 times with 0.1% TBST and 3 times with TBS to remove nonspecific phage.

[0363] Bound phages were eluted by adding 100 μL of trypsin-EDTA (Gibco, 25300-054) and incubated at room temperature for 15 minutes.

[0364] 200 μL of TG1 (OD=0.3-0.5) was infected with 100 μL of the eluted phage at 37°C for 1 hour.

[0365] The infected TG1 were centrifuged at 4000 rpm for 10 min, the pellet was resuspended in 2xTY, ampicillin, Glc 2% and the culture was incubated overnight at 30°C, 200 rpm.

[0366] 20 μL of infected TG1 was added to 2 mL 2xTY, ampicillin, Glc 2% and grown at 37°C, 200 rpm. When OD reached 0.5, TG1 was infected with helper phage at 37°C, 200 rpm for 1 hour.

[0367] The TG1 culture was centrifuged at 4000 rpm for 10 minutes and the pellet was resuspended in 1 mL 2xTY, ampicillin, kanamycin and incubated at 30°C, 200 rpm overnight.

[0368] The culture was centrifuged at 4000 rpm for 15 minutes at 4°C, and the phages were precipitated with PEG-NaCl to obtain the supernatant. These phages were used for the next round of panning.

[0369] After several rounds of panning, a variety of antigen binders were successfully enriched.

[0370] Monoclonal scFvs were then sequenced and unique clones were selected.

[0371] Monoclonal scFvs were then produced in E. coli. HB2151 bacteria containing pHEN phagemids expressing scFvs were grown overnight at 37°C, 220rpm in 150 μL 2TY-2% glucose-ampicillin in 96-well plates. Larger cultures were inoculated into deep well plates containing 2TY-IPTG 1mM-ampicillin using this starter to induce scFv expression. Induced cultures were grown overnight at 30°C, 220rpm. The cultures were centrifuged and periplasmic scFv extraction was performed from the precipitate using 1mg / mL polymyxin B sulfate dissolved in 20mM Tris pH 8.

[0372] A summary of the SEQ ID numbers for the CDR, VH and VL domains is provided below in Tables 1 and 2, and the DNA sequences corresponding to the VH and VL sequences are provided in Table 2.

[0373] Table 1. SEQ ID NOs: H-CDR1-3 and L-CDR1-3 amino acid sequences of anti-Gal3 antibodies

[0374]

[0375]

[0376] Table 2. SEQ ID NOs of VH and VL proteins and DNA sequences of anti-Gal3 antibodies

[0377] Antibody VH(protein) VL(protein) VH(DNA) VL(DNA) D06 38 39 61 62 G03 48 49 71 72 H07 50 51 73 74 H10 44 45 67 68 B12 54 55 77 78 D11 40 41 63 64 E02 52 53 75 76 E07 42 43 65 66 E12 56 57 79 80 E01 46 47 69 70

[0378] Example 2: Human / mouse cross-reactive anti-Gal3 scFv that binds neither Gal1 nor Gal7

[0379] The scFvs were tested by ELISA for binding cross-reactivity and specificity to human Gal3 full length and CRD domain (Hu-Gal3-FL and Hu-Gal3-CRD, respectively), human full length Gal1 (Hu-Gal1, as shown in SEQ ID NO 108 and available under UniProt accession number P09382-LEG1_HUMAN) and human full length Gal7 (Hu-Gal7, as shown in SEQ ID NO 109 and available under UniProt accession number P47929-LEG7_HUMAN), as well as mouse full length Gal3 (m-Gal3-FL) ( Figure 1 ).

[0380] Hu-Gal3-FL, Hu-Gal3-CRD, m-Gal3-FL, Hu-Gal1 and Hu-Gal7 were immobilized on functionalized microplates via their histidine tags at a final concentration of 3 μg / mL after internal processing. Periplasmic extracts containing scFvs were added to the wells and detected with anti-scFv tag.HRP (horseradish peroxidase) antibodies. Rabbit antibodies against Gal3 (ab53082 - abcam reference), Gal1 (ab25138 - abcam reference) and Gal7 (ab108623 - abcam reference) were used as antigen coating positive controls at 5 μg / mL and detected with anti-rabbit.HRP secondary antibodies (7074S - Cell Signaling reference).

[0381] All scFvs of the present invention showed binding to human and mouse Gal3 proteins, but not to human Gal1 or Gal7.

[0382] Example 3: Competitive inhibition assay of Gal3 binding to asialofetuin

[0383] The asialofetuin (ASF) glycoprotein is often studied as a model ligand for galectins. In this assay, the binding of ASF to immobilized human Gal3 (Hu-Gal3) was measured in the presence or absence of scFv (secondary antibody and irrelevant scFv alone were used as controls). Human galectin 3 (Hu-Gal3-FL) was immobilized on functionalized microplates at 3 μg / mL via its histidine tag as described previously. 30 μg / mL of ASF (A1908 - Sigma reference) was added to each well of periplasmic extracts with or without scFv. The binding of ASF to Hu-Gal3 was detected using sheep anti-ASF antibody (secondary antibody, ab35184 - abcam reference) and donkey anti-sheep .HRP antibody (tertiary antibody, ab6900 - abcam reference).

[0384] All scFvs of the present invention, except for an unrelated scFv, showed inhibition of ASF-Gal3 interaction ( Figure 2 ).

[0385] Example 4: Preparation of novel anti-Gal3 antibodies and design of anti-Gal3 antibodies with reduced immunogenicity

[0386] Anti-Gal3 antibody was then produced in IgG1 format using transient transfection in the CHO-K1 cell line. The supernatant was collected by centrifugation, filtered, and then purified by MabSelect SuRe TM Purified by Cytiva.

[0387] Then, anti-Gal3 antibodies with reduced immunogenicity are designed in order to obtain antibody molecules with minimal immunogenicity when administered to humans while substantially retaining the specificity and affinity of the parent antibody.

[0388] The VH and VL regions of the antibodies were aligned to the human IgG database to find the closest human germline sequence. In addition, the sequences were run through the EpiAnalyzer tool, which highlights the immunogenic regions. It was observed that the immunogenicity did not exceed tolerable levels.

[0389] After aligning the VH or VL sequence with the germline sequence, a variant antibody "-G" was proposed to correct the germline framework mutation, while two variant antibodies -G1 and -G2 were proposed for critical cases where the mutation was close to the CDR region, the germline framework mutation of the G1 version was not close to the CDR region, and the germline framework mutation of the G2 version was close to the CDR region. The SEQ ID numbers of the variant antibodies VH and VL are provided in Table 3.

[0390] Table 3. SEQ ID NOs of anti-Gal3 variant antibody VH and VL protein sequences

[0391] Antibody VH V L D06-G1 81 83 D06-G2 82 83 D11-G1 84 86 D11-G2 85 86 E07-G1 87 89 E07-G2 88 89 H10-G 90 91 E01-G 92 93 G03-G1 94 96 G03-G2 95 96 H07-G 97 98 E02-G 99 100 B12-G 101 102 E12-G1 103 105 E12-G2 104 105

[0392] Example 5: Human / mouse cross-reactive anti-Gal3 antibodies

[0393] Anti-Gal3 antibodies were tested for human / mouse cross-reactivity on human Gal3 full length (Hu-Gal3-FL) and CRD domain (Hu-Gal3-CRD) and murine Gal3 (m-Gal3-FL) by ELISA.

[0394] Hu-Gal3-FL, Hu-Gal3-CRD and m-Gal3-FL were immobilized on functionalized microplates via their histidine tags at 3 μg / mL. Purified IgG was added to the wells (15-20 μg / mL final concentration) and detected with anti-Fab.HRP antibody. A control rabbit antibody against Hu-Gal3 (Abcam ab53082 reference) at 5 μg / mL was used as an antigen coating positive control and detected with an anti-rabbit.HRP secondary antibody (7074S - Cell Signaling reference).

[0395] The results are as follows Figure 3 As shown. The antibodies of the present invention were shown to bind both human and mouse Gal3 proteins. However, for the B12 antibody, lower binding signals were observed on human and mouse Gal3. Moreover, the signal of the E12 antibody was much lower on m-Gal3 than on Hu-Gal3-FL and Hu-Gal3-CRD.

[0396] Example 6: The antibody specifically binds to Gal3 and neither to Gal1 nor to Gal7.

[0397] The specificity of the IgG for Gal3 was tested by ELISA to examine the presence of binding to human Gal1 and Gal7.

[0398] Hu-Gal1 and Hu-Gal7 were immobilized at 3 μg / mL. Purified IgG was added to the wells (15-20 μg / mL final concentration) and detected with anti-Fab.HRP antibody. Rabbit antibodies against Gal1 (Abcam ab25138 reference) and Gal7 (Abcam ab108623 reference) at 5 μg / mL were used as antigen coating positive controls and detected with anti-rabbit.HRP secondary antibodies (7074S-CellSignaling reference).

[0399] ELISA results Figure 4 All anti-Gal3 antibodies of the present invention specifically bind to Gal3, but do not bind to human Gal1 and Gal7.

[0400] Example 7: Affinity of antibodies for Gal3 from several species

[0401] The interaction of anti-Gal3 antibodies with recombinant human Gal3 full length (Hu-Gal3-FL) and murine Gal3 (m-Gal3-FL) (from Novalix) was monitored by surface plasmon resonance (SPR) detection using a Biacore T-200 instrument.

[0402] Binding studies were performed at 25° C. in freshly prepared, filtered and degassed running buffer containing 10 mM HEPES, 150 mM NaCl and 0.05% P20 (HBS-P, Cytiva Life Sciences). Prior to binding studies, the CM5 sensor surface with immobilized anti-human Fc mAb was equilibrated with HBS-P by priming the instrument at least three to four times.

[0403] First, the anti-human Fc mAb was immobilized on the CM5 sensor surface using a standard protocol. The entire sensor surface was first activated by injecting a 1:1 (v / v) mixture of 400mM EDC and 100mM NHS at a flow rate of 10μL / min for 7 minutes. A sodium acetate pH 5 solution of 25μg / mL monoclonal anti-human Fc antibody (Millipore AP113) flowed at 10μL / min for 360s to reach 10000Ru in both flow cells, followed by an injection of 1M ethanolamine, pH 8.5, at a flow rate of 10μL / min for 7 minutes. HBS-P was used as the running buffer throughout the immobilization process.

[0404] Antibody samples were prepared at 5 μg / ml and injected at 10 μL / min for 30 sec. Typically, 400 to 680 resonance units of antibody were captured on the chip.

[0405] The detailed Kd values ​​for each tested antibody are shown in Table 4 below.

[0406] Table 4. Determination of Kd values ​​of human and mouse full-length Gal3

[0407]

[0408]

[0409] On human Gal3, the native antibodies of the invention showed Kd values ​​between 4nM and 15nM, and the variant antibodies showed Kd values ​​between 4nM and 18nM. On mouse Gal3, only the native antibodies were tested, and they showed lower Kd values ​​between 1nM and 8nM. There was no significant difference in the Kd values ​​between human and mouse antigens.

[0410] The Kd values ​​of the variant antibodies were similar to those of the native antibody. These similar binding characteristics indicate that mutations in the VH or VL domain frameworks had no effect on the interaction of the antibody with human Gal3.

[0411] Kd value is also determined by SPR using single cell kinetic analysis. A total of 5 concentrations of Gal3 CRD domains from people, dogs, rats and cynomolgus monkeys (Novalix) [0.5-50] nM are injected into the antibody capture surface for 210 seconds, followed by a dissociation phase of 30 μL / min for 600 seconds. At the end of each cycle, the capture surface is regenerated by injecting 2 pH 1.5 glycine at a speed of 10 μL / min for 30 seconds, and then additional cleaning is performed with pH 1.5 glycine. Fresh antibodies are captured at the beginning of each cycle.

[0412] The Kd values ​​for each antibody tested are shown in Table 5 below.

[0413] Table 5. Determination of Kd values ​​of Gal3-CRD in humans, rats, cynomolgus monkeys and dogs

[0414]

[0415] Example 8: EC50 determination for human and mouse Gal3 by ELISA

[0416] To determine the half maximal effective concentration (EC50) of the antibodies, ELISA dose-response experiments were performed on Hu-Gal3-FL and m-Gal3-FL.

[0417] Hu-Gal3-FL and m-Gal3-FL were immobilized via their His tags at 3 μg / mL on functionalized microplates as described previously. Serial dilutions of 3.3-fold purified IgG were added to wells containing immobilized Hu-Gal3-FL or m-Gal3-FL and detected with anti-Fab.HRP antibody.

[0418] Human / mouse Gal3 EC50 ratios were estimated from EC50 values ​​(sigmoidal dose-response equation: Y = bottom + X*(top - bottom) / (EC50 + X)), with low ratios indicating high cross-reactivity.

[0419] Of the ten antibodies tested, eight had measurable EC50s. The EC50s for the B12 and E12 antibodies were too high to be determined. Of the remaining eight antibodies, six had EC50 values ​​below 10 nM for human Gal3 and four had EC50 values ​​below 10 nM for mouse Gal3. The human / mouse Gal3 EC50 ratios for the D06, D11, E02, E07, and E01 antibodies were below four, confirming high cross-reactivity.

[0420] The results are shown in Figure 5 and Table 6 below.

[0421] Table 6. EC50 calculations for human and mouse Gal3

[0422] Antibody hGal3 EC50 (nM) mGal3 EC50 (nM) ratio D06 0.26 0.16 2 G03 0.52 13.6 26 H07 2.4 88 37 H10 0.20 5.9 30 D11 0.37 0.99 3 E02 34 76 2 E07 0.07 0.30 4 E01 14.3 25.9 2

[0423] Example 9: Competitive inhibition assay of Gal3 binding to asialo-fetuin and IC50 determination for human and mouse Gal3

[0424] The inhibitory properties and IC50 of anti-Gal3 antibodies were determined in an ELISA competition assay by measuring the binding of ASF to the CRD domain of immobilized human Gal3 in the presence or absence of different concentrations of antibodies.

[0425] Inhibition assays were performed on Hu-Gal3-CRD, which was immobilized via its His tag at 1 μg / mL on a functionalized microplate. Serial dilutions of 2-fold or 3-fold were made with purified IgG and added to the wells with immobilized Hu-Gal3-CRD. 8 μg / mL ASF (A1908 - Sigma reference) was added to each well with or without IgG. Binding of ASF to Hu-Gal3-CRD was detected using an HRP-labeled anti-ASF sheep antibody. The IC50 was estimated according to the model equation Y = bottom + (top - bottom) / (1 + (X / IC50)). The indicative maximum inhibition percentage was obtained using the following formula: 100% - ((average of the bottom plateau values ​​of IgG) × 100%) / average of the irrelevant IgG values)

[0426] All except the negative control (irrelevant IgG) showed inhibition of ASF binding to Hu-Gal3-CRD ( Figure 6 The IC50 values ​​of the antibodies ranged from 1.5 to 6 nM, and the estimated relative maximum inhibition percentages ranged from 55 to 97%, as shown in Table 7 below.

[0427] Therefore, these eight antibodies are antagonists of Gal3 / ASF binding.

[0428] Table 7. IC50 calculations for human Gal3 CRD

[0429] Antibody hGal3-CRD IC50(nM) Maximum inhibition % D06 5.1 82 G03 1.8 83 H07 6.0 74 H10 2.7 89 D11 2.2 99 E02 5.2 65 E07 1.5 89 E01 3.2 63

[0430] Example 10: Antibody Inhibitory Activity in Hypochlorous Acid (HOCl)-Induced Fibrosis Mouse Model

[0431] In a murine model of HOCl-induced fibrosis, the therapeutic effect of anti-Gal3 antibodies on lung and skin fibrosis was evaluated in two independent experiments.

[0432] In the first experiment, control mice (n = 12) were injected intradermally with 300 μL sterile phosphate buffered saline (PBS) for 5 consecutive days per week for 42 days. Mice treated with HOCl alone and mice treated with HOCl and antibody (HOCl-antibody) (n = 12 per group) were injected intradermally with 300 μL HOCl for 5 consecutive days per week for 42 days. Antibody-treated mice also received subcutaneous injections of 20 mg / kg antibody, starting from day -1, every 5 days between day 5 and day 40. Before injection, each antibody was dissolved in sterile, endotoxin-free PBS. In the second experiment, the same protocol was applied, except that the duration of the study was 63 days, and antibodies were given every 7 days from day -1 until day 62.

[0433] Mice injected with HOCl daily were also treated with 0.5 mg / kg of TD139 compound (HOCl-TD139), a small molecule Gal3 inhibitor, by intratracheal instillation. In the first experiment, administration began on day 21, and the compound was given every 3 days until day 39. In the second experiment, administration began on day 20, and the compound was given every 3 days until day 62. TD139 was initially dissolved in 100% DMSO and then diluted 50-fold with endotoxin-free PBS before administration.

[0434] A- Longitudinal assessment of skin thickness

[0435] The evolution of skin thickness was monitored every seven days by using an external caliper until sacrifice.

[0436] In the first experiment, Fig. 7A As shown, daily HOCl injections were associated with a rapid, sustained, and statistically significant increase in skin thickness compared with control mice, starting on day 7 and reaching a maximum of 3.9-fold on day 42. The D11 and E07 monoclonal antibodies significantly reduced the HOCl-induced increase in skin thickness starting on day 21, and this effect persisted until day 42. The reduction in skin thickness in mice receiving the reference product TD139 was less than that in mice receiving either antibody, reaching statistical significance only on days 28 and 42.

[0437] In the second experiment, the skin thickness of HOCL-treated mice continued to increase significantly from day 7, reaching a maximum of 5.9-fold on day 63 compared with control mice ( Figure 7B E07 from day 7 and H10 from day 14 significantly reduced the increase in skin thickness induced by HOCl, and this effect lasted until day 63.

[0438] Thus, the antibodies of the present invention have the ability to significantly reduce the fibrosis index of longitudinally measured skin thickness to a similar or greater extent than TD139 ( Fig. 7A and 7B ), using a once-weekly dosing regimen for at least 9 weeks ( Figure 7B ).

[0439] B- Quantification of skin and lung collagen

[0440] On the day of the end of the study of the first experiment (day 42), skin and lung samples were collected, fixed with formalin and processed into separate paraffin blocks. Skin and lung sections were cut from the paraffin blocks and, after rehydration, stained with Picorisrius Red according to the manufacturer's protocol. Subsequently, each slide was examined and remotely analyzed using the web-based Image J software.

[0441] Compared with vehicle mice, daily intradermal injection of HOCl and lung ( Fig. 8A ) and skin( Figure 8B ) is associated with increased collagen deposition in the

[0442] Subcutaneous injection of D06, D11, and E07 antibodies, as well as intratracheal instillation of the reference compound TD139, restored lung collagen levels to those of control mice, reaching statistical significance for E07.

[0443] Subcutaneous injection of D11 antibody and reference compound TD139 significantly reduced the level of skin collagen compared with the HOCl group. A non-significant downward trend was also observed after treatment with E07 and D06 antibodies.

[0444] Thus, the antibodies of the invention have the ability to significantly reduce the fibrotic markers of collagen deposition in the lung and skin as measured histologically after 6 weeks of treatment.

[0445] Effects of C- on the levels of interleukin-6 (IL-6) and interleukin-5 (IL-5) in mouse plasma

[0446] In the first experiment, a mixed effects model with repeated measures was performed on the control, HOCl-treated, HOCl-antibody-treated, and HOCl-TD139-treated groups. The effect of HOCl administration on plasma IL-6 and IL-5 levels compared to the control group was evaluated. The effects of antibody and TD139 treatment were compared to the HOCl group ( Fig. 9 ). IL-6 and IL-5 were quantified by using MSD technology from Mesoscale.

[0447] After administration of HOCl, the plasma levels of IL-6 increased significantly. Treatment with E07 or D11 antibodies significantly reduced plasma IL-6 levels to values ​​similar to those of control mice, while TD139 had no activity. Therefore, the antibodies of the present invention have a better ability to reduce IL-6 levels in plasma after HOCl treatment than TD139.

[0448] E07 and D11 antibodies as well as TD139 also significantly reduced plasma IL-5 levels compared with the HOCl group.

[0449] D-antibody treatment resets Gal3 plasma levels to those of healthy mice

[0450] In the first experiment, Gal3 plasma levels were measured on day -1 (before antibody administration) and on the termination day (day 42) using the mouse Galectin-3 DuoSet ELISA kit (R&D Systems, DY1197) according to the manufacturer's instructions. Plasma samples were diluted 1:200 to fit the dynamic range of the assay.

[0451] On day -1, as expected, no significant differences were observed between all groups ( Fig.10 ). A significant increase in Gal3 was observed in the plasma of HOCl-treated mice on day 42. Treatment with E07 and D11 antibodies and TD139 compound restored plasma Gal3 levels to values ​​similar to those of the control group.

[0452] E-Genes that are dysregulated under pathological conditions are almost completely restored to normal after treatment with anti-Gal3 antibodies

[0453] In the first experiment, global gene expression in whole blood cells was examined by RNA sequencing on day 42.

[0454] Whole blood was collected in RNA protection tubes (Qiagen) and RNA was extracted using the RNeasy Protect Animal Blood Kit and optional on-column DNAse I digestion. TM) and analyzed on a Bioanalyzer (Agilent) using an RNA 6000 nanochip to determine RNA quality (RIN). Total RNA that met the QC criteria was included in the study. Purified RNA samples were used as input for TruSeq strand-specific RNA-seq (poly A selection and globin removal) according to the manufacturer's protocol (Illumina). After library preparation, samples that met the required criteria were sequenced on a NovaSeq 6000 sequencer (Illumina).

[0455] Raw reads were processed using the in-house RNAExp Expression pipeline 2.1 and quality was assessed using the FastQC tool. Reads were then trimmed for adapters using Cutadapt and the trimmed reads were aligned to the mouse mm39 reference genome using the STAR alignment tool. The quality of the aligned data was assessed using the Samtools and Picard tools. FeatureCount was then used to quantify the aligned reads for each gene. The sample filtering step identified 2 outliers, one in the control group and one in the TD139 treatment group, which were discarded for further analysis. Read counts were normalized using the variance stabilizing transformation vst function from the DESeq2 R package.

[0456] 55,416 genes were detected in the data. 29,947 non-protein coding genes were removed. Samples with a count of 0 or more than 95% expression level below 1 in all samples were filtered out. Finally, the RNA-seq data included 10,508 genes. The genes differentially expressed between groups were determined using a linear model of the vst transformed gene expression dataset (lmFit function from the limma R package). The resulting p-values ​​were adjusted for multiple hypothesis testing and filtered to retain differentially expressed genes, where the false discovery rate (FDR) adjusted p-value was <0.05 and the difference fold (FC) value was ≥1.3.

[0457] 510 genes were found to be differentially regulated between the pathological HOCl group and the control group (484 genes were upregulated and 26 genes were downregulated). E07 antibody treatment counteracted the pathological pattern of 445 genes, accounting for 87.2% of these genes, resulting in an overall gene expression profile very close to that of the control group ( Fig.11 ). In contrast, only 7 genes (accounting for 1.37% of the deregulated genes between HOCl and the control group) were found to be counteracted after treatment with the reference compound TD139. Therefore, the E07 antibody of the present invention showed a clear advantage over TD139.

[0458] Example 11: Antibody activity in a bleomycin-induced pulmonary fibrosis mouse model

[0459] The therapeutic effects of anti-Gal3 antibodies on lung gene expression and pulmonary fibrosis were evaluated in a murine model of bleomycin-induced fibrosis.

[0460] Control bleomycin mice, designated "Bleo", received 1.65 mg / kg bleomycin via the oropharyngeal route on day 1. Control unstimulated mice, designated "CTRL" or "control," did not receive bleomycin on day 1, but received the saline vehicle of bleomycin. Antibody-treated mice also received 1.65 mg / kg bleomycin via the oropharyngeal route on day 1, and subcutaneous injections of 20 mg / kg of the antibody starting on day -1 and days 4, 8, 12, 16, and 20. Each antibody was dissolved in sterile, endotoxin-free PBS prior to injection.

[0461] Mice receiving bleomycin were also treated with 0.5 mg / kg of TD139 compound (Bleo-TD139), a small molecule Gal3 inhibitor, orally every 2 days starting on day 12 until day 20. TD139 was initially dissolved in 100% DMSO.

[0462] A-Evolution of body weight

[0463] The mice were weighed daily. Their growth curves were as follows Fig.12 As shown in . Control mice grew normally, with an average percentage increase of 6.4% compared to their initial body weight measured on day -1. Mice treated with bleomycin showed a rapid decrease in their weight, reaching a maximum weight loss of 8.7% on day 8, followed by a phase of weight gain from day 9 to day 21. Subcutaneous injection of antibodies D06 and E07 resulted in faster weight recovery, reaching a weight close to that of control mice from day 12 and day 18, respectively, and full weight recovery on day 21. The D11 antibody showed an average weight percentage increase of 4.4% on day 21 compared to day -1. The TD139 compound showed a more modest efficacy on this parameter, with an average weight percentage increase of 2.9% on day 21 compared to day -1, slightly higher than the untreated bleomycin group (+1.9%).

[0464] These results suggest that the antibodies of the invention act by improving overall animal welfare, as reflected in improvements in their body weight.

[0465] B-Weight Index

[0466] Weight index was evaluated by using two parameters called time to weight gain (TWG) and time to baseline (TTB), e.g. Fig.13The weight gain time corresponds to the inflection point during the experiment when the mice show an ascending growth curve and begin to regain weight. The baseline time corresponds to the number of days required for the mice to regain their initial weight (day -1).

[0467] The median TWG for the untreated bleomycin group was 10 days. Treatment with the D06 antibody significantly reduced it by 2.8 days, and treatment with the D11 and E07 antibodies also reduced it by 1.3 and 2 days, respectively, with p values ​​slightly above the statistical significance limit. The TD139 compound had no effect on this parameter (p value of 0.53).

[0468] The median TTB of the untreated bleomycin group was 20 days. After treatment with D06, D11 and E07 antibodies, it was reduced by 5.5 days, 2.7 days and 4 days, respectively, and after treatment with D11 and E07 antibodies, it was also reduced by 2.7 days and 4 days, respectively, with p values ​​slightly above the statistical significance limit. TD139 compound had no effect on this parameter (p value of 0.29).

[0469] These results suggest that the antibodies of the invention act by improving overall animal welfare, as reflected in improvements in their body weight indicators.

[0470] C-Measurement of pulmonary fibrosis

[0471] Mice were terminally euthanized on day 21. Lungs were collected for histopathology and gravity inflation-fixed with 10% neutral buffered formalin (NBF) and then immersed in NBF.

[0472] Take the sections and place them on three slides, e.g. Fig.14 The code is shown above.

[0473] Three longitudinal sections parallel to and bisecting the main trunk bronchus were trimmed from the left and right caudal lobes and mounted in boxes 01 and 02, respectively. The right cranial, middle, and accessory lobes were longitudinally sectioned and mounted in box 03. All sections were stained with picrosirius red and scored in a blinded manner by a certified veterinary pathologist using a modified Ashcroft fibrosis score (0-8), where "0" corresponds to "normal" (no fibrosis) and "8" corresponds to the highest possible fibrosis score, as described by Hübner et al. (2008), but based on the evaluation of the entire lung section.

[0474] In mice that received bleomycin but no other treatment, 87.6% of the animals had a median fibrosis score of 3 or 4, and only 7.4% had a score of 2 ( Fig.15 ).

[0475] In mice treated with the D06 antibody, the frequency of animals with moderate fibrosis scores of 3 or 4 was reduced to 64.5%, while 30% of the animals had a low fibrosis score of 2.

[0476] Mice treated with the D11 antibody also showed an improvement in the frequency of score 2 (17.8%), and a significant shift toward lower scores in the percentage of mice with scores of 4 or 5 compared to the untreated bleomycin group. In particular, the frequency of animals with a score of 4 decreased to 20% compared to 44.4% in the bleo group, and the frequency of animals with a score of 3 increased to 61.1% compared to 43.2% in the bleo group.

[0477] The TD139 compound did not show any signs of antifibrotic efficacy, with 49.4% of mice having lung fibrosis scores of 4 and 5 compared to 49.3% in the untreated bleomycin group, and 50.6% of mice having lung fibrosis scores of 2 and 3, similar to the untreated bleomycin group.

[0478] In conclusion, there was clear evidence that lung fibrosis was reduced in mice that received the D06 antibody and, to a lesser extent, in mice that received the D11 antibody.

[0479] Example 12: Antibodies inhibit inflammatory responses in ARDS rat model

[0480] The therapeutic effect of anti-Gal3 antibody on inflammation was evaluated in an ARDS (acute respiratory distress syndrome) rat model.

[0481] Positive control rats received 3 doses of sterile phosphate buffered saline (0.25 mg / kg subcutaneously on day 1) on days -11, -6 and -1 before LPS challenge. Negative control rats received a single oral dose of 10 mg / kg dexamethasone (dissolved in 0.5% carboxymethylcellulose in deionized water) 2 hours before LPS challenge (0.25 mg / kg subcutaneously on day 1). LPS (lipopolysaccharide) causes an acute inflammatory response, while dexamethasone has anti-inflammatory effects and is therefore the reference treatment in this model.

[0482] Antibody-treated rats received 3 doses of antibody (14 mg / kg) on ​​days -11, -6 and -1 before LPS challenge (0.25 mg / kg subcutaneously on day 1). Prior to injection, each antibody was dissolved in sterile PBS.

[0483] Control rats in the experiment received 3 doses of sterile phosphate buffered saline on days -11, -6, and -1, but no LPS stimulation on day 1.

[0484] For all rats, plasma sampling was performed 4 h after LPS stimulation.

[0485] A-Effect on neutrophil count in bronchoalveolar lavage fluid (BALF)

[0486] The effect of anti-Gal3 antibody treatment in LPS-stimulated rats was evaluated by quantification of neutrophils in bronchoalveolar lavage fluid and compared with a positive control group (LPS stimulation, PBS treatment) ( Fig.16 ). In the control rats, neutrophil counts increased significantly after LPS stimulation, indicating that the rats produced an inflammatory response in response to LPS stimulation. After treatment with anti-inflammatory dexamethasone, neutrophil count levels decreased. Neutrophil counts were also reduced in rats treated with anti-Gal3 antibodies D11, D06, E07, and H10.

[0487] Effects on air-blood barrier damage (BAL protein concentration)

[0488] The effect of anti-Gal3 antibody treatment in LPS-stimulated rats was evaluated by quantification of protein in BALF (by BCA assay) and compared with the positive control group (LPS stimulation, PBS treatment) ( Fig.17 ). The presence of protein in BALF reflects damage to the air-blood barrier. In the control rats, total BALF protein increased significantly after LPS stimulation, indicating rapid damage to the air-blood barrier in this group. After treatment with the anti-inflammatory dexamethasone, the total BALF protein count level decreased. In rats treated with anti-Gal3 antibodies D11, D06, E07 and H10, total BALF protein also decreased, with statistical significance in the E07 group.

[0489] Effects of B- on the levels of TNFα and KC-GRO in rat plasma

[0490] The effects of anti-Gal3 antibody treatment on plasma cytokine levels (i.e., TNFα and KC-GRO cytokines) in LPS-stimulated rats were evaluated and compared with positive control groups (LPS stimulation, PBS treatment) ( Fig.18 A and Fig.18 B) TNFα and KC-GRO were quantified by using MSD technology from Mesoscale.

[0491] In the control rats, the plasma level of TNFα increased significantly after LPS stimulation, indicating that the rats had an inflammatory response to LPS stimulation. After treatment with anti-inflammatory dexamethasone, the TNFα level decreased. In rats treated with anti-Gal3 antibodies D11, D06, E07, and H10, the TNFα plasma level also decreased, presenting an intermediate state between LPS-stimulated rats and LPS-stimulated rats treated with dexamethasone.

[0492] KC-GRO plasma levels also decreased after treatment with the four antibodies, to a similar extent as after dexamethasone treatment.

[0493] Therefore, the antibodies of the present invention have anti-inflammatory effects by reducing neutrophil counts, reversing air-blood barrier damage, and lowering plasma TNFα and KC-GRO cytokine levels.

Claims

1. An anti-galectin-3 (anti-Gal3) antibody or an antigen-binding fragment thereof, wherein the antibody specifically binds to galectin-3 and does not bind to galectin-1 or galectin-7. 2 . The anti-Gal3 antibody or antigen-binding fragment according to claim 1 , wherein the anti-Gal3 antibody or antigen-binding fragment of the present invention binds to extracellular Gal3.

3. The anti-Gal3 antibody or antigen-binding fragment according to claim 1 or 2, wherein the anti-Gal3 antibody or antigen-binding fragment of the present invention inhibits the binding of Gal3 to its ligand, or is a Gal3 antagonist.

4. The anti-Gal3 antibody or antigen-binding fragment according to any one of claims 1 to 3, wherein the anti-Gal3 antibody comprises: - Heavy chain complementarity determining region (H-CDR): a) HCDR-1 comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 1-5; b) (H-CDR) 2, which comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 6-11; and c) (H-CDR) 3, which comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 12-19; ​​and -Light chain complementarity determining region (L-CDR): a) (L-CDR) 1, which comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 20-26; b) (L-CDR) 2, which comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 27-29; and c) (L-CDR) 3, which comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 30-37.

5. The anti-Gal3 antibody or antigen-binding fragment according to any one of claims 1 to 4, wherein the anti-Gal3 antibody comprises H-CDR 1-3 and L-CDR 1-3, and the H-CDR and L-CDR comprise or consist of the following amino acid sequence: a) SEQ ID NOs: 1, 6, 12, 20, 27 and 30, respectively; b) SEQ ID NO: 1, 6, 13, 21, 27 and 31, respectively; c) SEQ ID NO: 1, 6, 14, 22, 27 and 31, respectively; d) SEQ ID NOs: 5, 7, 3, 21, 28 and 32, respectively; e) SEQ ID NOs: 3, 8, 15, 23, 27 and 33, respectively; f) SEQ ID NO: 2, 6, 16, 24, 27 and 34, respectively; g) SEQ ID NO: 1, 9, 14, 24, 27 and 34, respectively; h) SEQ ID NO: 2, 10, 17, 25, 27 and 35, respectively; i) SEQ ID NO: 4, 11, 18, 21, 29 and 36, respectively; or j) SEQ ID NO: 2, 10, 19, 26, 27 and 37, respectively.

6. The anti-Gal3 antibody or antigen-binding fragment according to any one of claims 1 to 5, wherein the anti-Gal3 antibody comprises: a) a heavy chain variable domain VH which is at least 90% identical to the VH amino acid sequence of SEQ ID NO: 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 81, 82, 84, 85, 87, 88, 90, 92, 94, 95, 97, 99, 101, 103 or 104; and b) a light chain variable domain, VL, which is at least 90% identical to the VL amino acid sequence of SEQ ID NO: 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 83, 86, 89, 91, 93, 96, 98, 100, 102 or 105.

7. The anti-Gal3 antibody or antigen-binding fragment according to any one of claims 1 to 6, wherein a) the heavy chain variable domain VH comprises or consists of SEQ ID NO: 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 81, 82, 84, 85, 87, 88, 90, 92, 94, 95, 97, 99, 101, 103 or 104; and b) the light chain variable domain VL comprises or consists of SEQ ID NO: 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 83, 86, 89, 91, 93, 96, 98, 100, 102 or 105.

8. The anti-Gal3 antibody or antigen-binding fragment according to any one of claims 1 to 7, wherein the anti-Gal3 antibody or antigen-binding fragment of the present invention: a) a VH having an amino acid sequence comprising or consisting of SEQ ID NO: 38; and a VL having an amino acid sequence comprising or consisting of SEQ ID NO: 39; b) a VH comprising, or consisting of, an amino acid sequence of SEQ ID NO: 40; and a VL comprising, or consisting of, an amino acid sequence of SEQ ID NO: 41; c) a VH comprising, or consisting of, an amino acid sequence of SEQ ID NO: 42; and a VL comprising, or consisting of, an amino acid sequence of SEQ ID NO: 43; d) a VH comprising, or consisting of, an amino acid sequence of SEQ ID NO: 44; and a VL comprising, or consisting of, an amino acid sequence of SEQ ID NO: 45; e) a VH comprising, or consisting of, an amino acid sequence of SEQ ID NO: 46; and a VL comprising, or consisting of, an amino acid sequence of SEQ ID NO: 47; f) a VH comprising, or consisting of, an amino acid sequence of SEQ ID NO: 48; and a VL comprising, or consisting of, an amino acid sequence of SEQ ID NO: 49; g) a VH comprising, or consisting of, an amino acid sequence of SEQ ID NO: 50; and a VL comprising, or consisting of, an amino acid sequence of SEQ ID NO: 51; h) a VH comprising, or consisting of, an amino acid sequence of SEQ ID NO: 52; and a VL comprising, or consisting of, an amino acid sequence of SEQ ID NO: 53; i) a VH comprising, or consisting of, an amino acid sequence of SEQ ID NO: 54; and a VL comprising, or consisting of, an amino acid sequence of SEQ ID NO: 55; j) a VH comprising, or consisting of, an amino acid sequence of SEQ ID NO: 56; and a VL comprising, or consisting of, an amino acid sequence of SEQ ID NO: 57; k) a VH comprising, or consisting of, an amino acid sequence of SEQ ID NO: 81; and a VL comprising, or consisting of, an amino acid sequence of SEQ ID NO: 83; 1) a VH comprising, or consisting of, an amino acid sequence of SEQ ID NO: 82; and a VL comprising, or consisting of, an amino acid sequence of SEQ ID NO: 83; m) a VH comprising, or consisting of, an amino acid sequence of SEQ ID NO: 84; and a VL comprising, or consisting of, an amino acid sequence of SEQ ID NO: 86; n) a VH comprising, or consisting of, an amino acid sequence of SEQ ID NO: 85; and a VL comprising, or consisting of, an amino acid sequence of SEQ ID NO: 86; o) a VH comprising, or consisting of, an amino acid sequence of SEQ ID NO: 87; and a VL comprising, or consisting of, an amino acid sequence of SEQ ID NO: 89; p) a VH comprising, or consisting of, an amino acid sequence of SEQ ID NO: 88; and a VL comprising, or consisting of, an amino acid sequence of SEQ ID NO: 89; q) a VH comprising, or consisting of, an amino acid sequence of SEQ ID NO: 90; and a VL comprising, or consisting of, an amino acid sequence of SEQ ID NO: 91; r) a VH comprising, or consisting of, an amino acid sequence of SEQ ID NO: 92; and a VL comprising, or consisting of, an amino acid sequence of SEQ ID NO: 93; s) a VH comprising, or consisting of, an amino acid sequence of SEQ ID NO: 94; and a VL comprising, or consisting of, an amino acid sequence of SEQ ID NO: 96; t) a VH comprising, or consisting of, an amino acid sequence of SEQ ID NO: 95; and a VL comprising, or consisting of, an amino acid sequence of SEQ ID NO: 96; u) a VH comprising, or consisting of, an amino acid sequence of SEQ ID NO: 97; and a VL comprising, or consisting of, an amino acid sequence of SEQ ID NO: 98; v) a VH comprising, or consisting of, an amino acid sequence of SEQ ID NO: 99; and a VL comprising, or consisting of, an amino acid sequence of SEQ ID NO: 100; w) a VH comprising, or consisting of, an amino acid sequence of SEQ ID NO: 101; and a VL comprising, or consisting of, an amino acid sequence of SEQ ID NO: 102; x) a VH comprising, or consisting of, an amino acid sequence of SEQ ID NO: 103; and a VL comprising, or consisting of, an amino acid sequence of SEQ ID NO: 105; or y) a VH comprising, or consisting of, an amino acid sequence of SEQ ID NO: 104; and a VL comprising, or consisting of, an amino acid sequence of SEQ ID NO:

105.

9. The anti-Gal3 antibody or antigen-binding fragment of any one of claims 1-8, wherein the antibody competes for binding to human Gal3 with antibody D06, D11, E01, E02, E07, G03, H07, H10, B12, E12, D06-G1, D06-G2, D11-G1, D11-G2, E01-G, E02-G, E07-G1, E07-G2, G03-G1, G03-G2, H07-G, H10-G, B12-G, E12-G1 or E12-G2.

10. The anti-Gal3 antibody according to any one of claims 1 to 9, wherein the antibody is of isotype IgG. The anti-Gal3 antibody according to claim 10 , wherein the antibody is of the IgG subclass IgG1 or IgG4 isotype.

12. The anti-Gal3 antibody of claim 11, wherein the antibody comprises at least one mutation in the Fc region.

13. The anti-Gal3 antibody of claim 12, wherein the antibody comprises one or more mutations at heavy chain amino acid positions 228, 234 and 235, said positions being numbered according to the EU numbering scheme.

14. The anti-Gal3 antibody according to claim 13, wherein one or both of the amino acid residues at positions 234 and 235 are mutated from Leu to Ala, and / or wherein the amino acid residue at position 228 is mutated from Ser to Pro. 15 . A pharmaceutical composition comprising at least one anti-Gal3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 14 and a pharmaceutically acceptable excipient.

16. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the heavy chain or antigen-binding fragment thereof of the anti-Gal3 antibody according to any one of claims 1 to 14, or a nucleotide sequence encoding the light chain or antigen-binding fragment thereof of the anti-Gal3 antibody according to any one of claims 1 to 14, or a nucleotide sequence encoding both.

17. A combination of isolated nucleic acid molecules, wherein the isolated nucleic acid molecules encode the anti-Gal3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 14, in particular a first isolated nucleic acid molecule and a second isolated nucleic acid molecule, wherein the first isolated nucleic acid molecule comprises or consists of a sequence encoding a VH sequence, and the second isolated nucleic acid molecule comprises or consists of a sequence encoding a VL sequence.

18. A vector comprising the isolated nucleic acid molecule of claim 16 or a combination of isolated nucleic acid molecules of claim 17, wherein the vector further comprises an expression control sequence.

19. A host cell comprising the isolated nucleic acid molecule according to claim 16 or a combination of isolated nucleic acid molecules according to claim 17 or comprising the vector according to claim 18.

20. A method for producing an anti-Gal3 antibody or an antigen-binding fragment thereof, the method comprising providing the host cell according to claim 19, culturing the host cell under conditions suitable for expressing the antibody or fragment, and isolating the resulting antibody or fragment.

21. A bispecific binding molecule comprising the anti-Gal3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 14.

22. A method for treating a patient suffering from a Gal3-related disease, the method comprising administering to the patient an anti-Gal3 antibody or antigen-binding fragment according to any one of claims 1-14, a pharmaceutical composition according to claim 15, or a bispecific binding molecule according to claim 21.

23. The method of claim 22, wherein the Gal3-related disease is a fibrotic disease, an inflammatory disease, an autoimmune disease, an immune-mediated disorder, a neurodegenerative disease, a metabolic disease, an infectious disease, or cancer.

24. The method of claim 23, wherein the fibrotic disease originates from a tissue selected from the group consisting of skin, lung, liver, heart, kidney, and blood vessels.

25. The method of any one of claims 22-24, further comprising administering to the patient an additional therapeutic agent.

26. The method of claim 25, wherein the additional therapeutic agent is an immune checkpoint regulator, a chemotherapeutic agent, an anti-tumor agent, an anti-angiogenic agent, a hormonal therapy, a stem cell-based therapy, surgery, radiation therapy, another inhibitor of the Gal3 pathway, or a therapeutic agent administered during PD1 / PDL1 blockade therapy and / or CTLA4 blockade therapy.

27. Use of the anti-Gal3 antibody or antigen-binding fragment according to any one of claims 1 to 14 in the preparation of a medicament, wherein the medicament is used for: a) treating a patient suffering from a Gal3-related disease; or b) treating a patient for a fibrotic disease, inflammatory disease, autoimmune disease, immune-mediated disorder, neurodegenerative disease, metabolic disease, infectious disease, or cancer; or c) inhibiting Gal3 interaction in a patient in need thereof.

28. The antibody or antigen-binding fragment according to any one of claims 1 to 14, for use a) treating a patient suffering from a Gal3-related disease; or b) treating a patient for a fibrotic disease, inflammatory disease, autoimmune disease, immune-mediated disorder, neurodegenerative disease, metabolic disease, infectious disease, or cancer; or c) inhibiting Gal3 interaction in a patient in need thereof.

29. An article of manufacture comprising the antibody or antigen-binding fragment of any one of claims 1-14, wherein the article of manufacture is suitable for: a) treating a patient suffering from a Gal3-related disease; or b) treating a patient for a fibrotic disease, inflammatory disease, autoimmune disease, immune-mediated disorder, neurodegenerative disease, metabolic disease, infectious disease, or cancer; or c) inhibiting Gal3 interaction in a patient in need thereof.

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