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 would benefit from modulating Gal3 activity, such as fibrotic diseases, inflammatory diseases, autoimmune diseases, five immune-mediated disorders, cancer, neurodegenerative diseases, metabolic diseases, or infectious diseases.

Description

[Technical field]

[0001] 2. Background of the Invention Galectins are multifunctional proteins that specifically bind β-galactoside-containing ligands through their carbohydrate recognition domains (CRDs).

[0002] Galectin-3 (Gal3) is a member of this family, which is evolutionarily highly conserved and includes 15 galectins in mammals. Most galectins contain two carbohydrate recognition domains (CRDs) that are either homodimers (galectin-1, -2, -5, -7, -10, -11, -13, -14, -15) or heterodimers (galectin-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 it has also been described to undergo oligomerization through this N-terminal domain depending on environmental conditions.

[0003] Gal3 preferentially recognizes N-acetyl-lactosamine structures present in intracellular, membrane-bound, or extracellular proteins and can bind to several carbohydrate-containing ligands. Asialofetuin (ASF) glycoprotein is one of the prototype ligands of Gal3, and it can also associate with galectin-1 (Gal1) or galectin-7 (Gal7).

[0004] Gal3 is a multicompartmental protein that is present intracellularly, extracellularly, or associated with the cell membrane. Intracellularly, Gal3 has been described to regulate several processes related to cell proliferation or apoptosis pathways or cell signaling events. Upon association with ligands at the plasma membrane, Gal3 has been observed to affect the expression, localization, 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 of the extracellular matrix and cell adhesion processes. In several conditions, disruption of Gal3 expression and / or its extracellular interactome has been shown to trigger several pathological processes related to inflammation, fibrosis, immune system dysregulation, angiogenesis, tumor metastasis and progression, and cellular homeostasis.

[0005] Gal3 is widely expressed, inter alia, in cells of the innate immune system and has been shown to stimulate inflammatory responses by increasing chemoattraction and stimulating the production of proinflammatory cytokines by cells of the myeloid lineage, including macrophages. Gal3 is also expressed by fibroblasts and endothelial cells and, in combination with activated macrophages, promotes tissue fibrosis through increased fibroblast proliferation and collagen production.

[0006] Increased expression of Gal3 is associated with many fibrotic diseases, including heart failure, kidney 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 various types of cancer. Therefore, inhibition of Gal3 interactions is an attractive mechanism for treating the listed conditions.

[0007] The primary therapeutic approaches used to target Gal3 are small molecules and large complex carbohydrates, such as pectin derivatives, which are often described as being able to target Gal3, but sometimes also target other galectins and thus lack selectivity.

[0008] For example, TD139 (GB-0139) developed by Galect and Bristol-Myers Squibb or Verapectin (GR-MD-02) developed by Galectin Therapeutics, Inc. are said to be dual inhibitors of Gal3 and Gal1. Thus, they each show low selectivity for Gal3. Moreover, TD139 has low bioavailability, is formulated to be inhaled twice a day to reach mainly the lungs, and is developed for the treatment of idiopathic pulmonary fibrosis. Such an administration route may also be disadvantageous for the treatment of other conditions.

[0009] This dual selectivity is also associated with the complex carbohydrate Davanat (GM-CT-01, also developed by Galectin Therapeutics, Inc.) or GCS-100, developed by La Jolla Pharmaceuticals.

[0010] Another small molecule in development is GB1211 from Galect, which is an oral inhibitor that is described as targeting Gal3, including intracellular Gal3, due to its ability to cross the cell plasma membrane, unlike our antibody which targets extracellular Gal3.

[0011] The identification of Gal3 inhibitors that are small molecules or complex carbohydrates encounters many challenges, which arise from the complex and difficult carbohydrate chemistry design and can lead to compounds with low selectivity and / or potency and / or bioavailability.

[0012] Antibody-based therapeutic approaches are suitable for reaching targets with low druggability and could show higher selectivity, potency, and bioavailability compared to chemical entities and pectin derivatives.

[0013] Anti-Gal3 antibodies have recently been described by True Binding, whose TB001 monoclonal antibody is in preclinical trials and whose TB006 monoclonal antibody is in Phase 1 clinical trials for the indications of Alzheimer's disease and acute ischemic stroke. These antibodies are described as inhibitors of the interaction of Gal3 with TIM-3. They have not been described as specifically targeting the Gal3-CRD.

[0014] There is therefore a need to develop novel therapeutic entities that specifically target Gal3, particularly within its glycan recognition domain, to reach the site of interaction with its ligands, avoiding the concerns of selectivity, bioavailability or efficacy encountered with small molecule compounds or pectin derivatives under development.

[0015] Summary of the Invention The present invention relates to a novel monoclonal antibody that binds to extracellular galectin-3 (Gal3), in particular to the carbohydrate recognition domain of Gal3 (Gal3-CRD), but does not bind to galectin-1 (Gal1) or galectin-7 (Gal7).

[0016] The invention also relates to pharmaceutical compositions comprising one or more of these antibodies, and to uses of the antibodies and pharmaceutical compositions for inhibiting disease progression in patients, or for ameliorating, preventing and / or treating patients suffering from Gal3-associated diseases or disorders, including fibrotic diseases such as heart failure, renal failure, systemic sclerosis, pulmonary fibrosis, and liver fibrosis, as well as inflammatory diseases, autoimmune diseases, neurodegenerative diseases, metabolic diseases, infectious diseases, and some cancers.Compared to currently available molecules under development that target Gal3-associated diseases or disorders, the antibodies of the invention are believed to exhibit unique properties, either alone or in combination with another therapeutic molecule.

[0017] In one embodiment, the invention provides an anti-Gal3 antibody or antigen-binding fragment thereof, wherein the anti-Gal3 antibody or antigen-binding fragment thereof is any of the antibodies referred to herein as antibodies D06, D11, E01, E02, E07, G03, H07, H10, E12 or B12, or a variant thereof, wherein the variant may contain some minimal amino acid changes compared to antibody D06, D11, E01, E02, E07, G03, H07, H10, E12 or B12 without losing the antigen-binding specificity of the parent antibody.

[0018] In one embodiment, the anti-Gal3 antibody or antigen-binding fragment thereof competes for binding to human Gal3 with an antibody whose heavy chain (H) CDRs1-3 and light chain (L) CDRs1-3 are the same as or derived from the H-CDRs1-3 and L-CDRs1-3 of antibody D06, D11, E01, E02, E07, G03, H07, H10, E12, or B12.

[0019] In one embodiment, the anti-Gal3 antibody or antigen-binding fragment thereof comprises H-CDR1-3, which contain the H-CDR1-3 sequences of antibodies D06, D11, E01, E02, E07, G03, H07, H10, E12, or B12, respectively.

[0020] In one embodiment, the anti-Gal3 antibody or antigen-binding fragment thereof comprises L-CDR1-3, which comprises the L-CDR1-3 sequences of antibodies 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 the amino acid sequences of H-CDR1-3 and L-CDR1-3 of antibody D06, D11, E01, E02, E07, G03, H07, H10, E12, or B12.

[0022] In one embodiment, the anti-Gal3 antibody or antigen-binding fragment thereof comprises heavy chain complementarity determining region (H-CDR) 1 comprising any one of the amino acid sequences of SEQ ID NOs: 1-5.

[0023] In one embodiment, the anti-Gal3 antibody or antigen-binding fragment thereof comprises heavy chain complementarity determining region (H-CDR) 2 comprising the amino acid sequence of any one of SEQ ID NOs:6-11.

[0024] In one embodiment, the anti-Gal3 antibody or antigen-binding fragment thereof comprises a heavy chain complementarity determining region (H-CDR) 3 comprising any one of the amino acid sequences of SEQ ID NOs: 12-19.

[0025] In one embodiment, the anti-Gal3 antibody or antigen-binding fragment thereof comprises a heavy chain complementarity determining region (L-CDR) 1 comprising any one of the amino acid sequences of SEQ ID NOs: 20 to 26.

[0026] In one embodiment, the anti-Gal3 antibody or antigen-binding fragment thereof comprises heavy chain complementarity determining region (L-CDR) 2 comprising the amino acid sequence of any one of SEQ ID NOs:27-29.

[0027] In one embodiment, the anti-Gal3 antibody or antigen-binding fragment thereof comprises a heavy chain complementarity determining region (L-CDR) 3 comprising any one of the amino acid sequences of SEQ ID NOs: 30-37.

[0028] 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 in sequence 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.

[0029] 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.

[0030] 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 the amino acid sequence of the heavy chain constant region of SEQ ID NO: 58 or 59.

[0031] 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 in sequence 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.

[0032] 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.

[0033] In one embodiment, the anti-Gal3 antibody or antigen-binding fragment thereof has a light chain (LC) comprising a 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 a light chain constant region amino acid sequence of SEQ ID NO: 60.

[0034] In certain embodiments, the anti-Gal3 antibody or antigen-binding fragment thereof of the invention comprises: a) SEQ ID NOs: 1, 6, 12, 20, 27 and 30, respectively; b) SEQ ID NOs: 1, 6, 13, 21, 27 and 31, respectively; c) SEQ ID NOs: 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 NOs: 2, 6, 16, 24, 27 and 34, respectively; g) SEQ ID NOs: 1, 9, 14, 24, 27 and 34, respectively; h) SEQ ID NOs: 2, 10, 17, 25, 27 and 35, respectively; i) SEQ ID NOs: 4, 11, 18, 21, 29 and 36, respectively; or j) SEQ ID NOs: 2, 10, 19, 26, 27 and 37, respectively It includes the amino acid sequences of H-CDR1 to 3 and L-CDR1 to 3.

[0035] In certain embodiments, the anti-Gal3 antibody or antigen-binding fragment thereof of the invention comprises: a) a VH having at least 90% (e.g., at least 92%, at least 95%, at least 98% or at least 99%) identical in sequence 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%) identical in sequence to the amino acid sequence of SEQ ID NO: 39 or 83; b) a VH having at least 90% (e.g., at least 92%, at least 95%, at least 98% or at least 99%) identical in sequence 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%) identical in sequence to the amino acid sequence of SEQ ID NO: 41 or 86; c) a VH having at least 90% (e.g., at least 92%, at least 95%, at least 98% or at least 99%) identical in sequence 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%) identical in sequence to the amino acid sequence of SEQ ID NO: 43 or 89; d) a VH having at least 90% (e.g., at least 92%, at least 95%, at least 98% or at least 99%) identical in sequence 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%) identical in sequence to the amino acid sequence of SEQ ID NO: 45 or 91; e) a VH having at least 90% (e.g., at least 92%, at least 95%, at least 98% or at least 99%) identical in sequence 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%) identical in sequence to the amino acid sequence of SEQ ID NO: 47 or 93; f) a VH having at least 90% (e.g., at least 92%, at least 95%, at least 98% or at least 99%) identical in sequence 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%) identical in sequence to the amino acid sequence of SEQ ID NO: 49 or 96; g) a VH having at least 90% (e.g., at least 92%, at least 95%, at least 98% or at least 99%) identical in sequence 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%) identical in sequence to the amino acid sequence of SEQ ID NO: 51 or 98; h) a VH having at least 90% (e.g., at least 92%, at least 95%, at least 98% or at least 99%) identical in sequence 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%) identical in sequence to the amino acid sequence of SEQ ID NO: 53 or 100; i) a VH having at least 90% (e.g., at least 92%, at least 95%, at least 98% or at least 99%) identical in sequence 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%) identical in sequence to the amino acid sequence of SEQ ID NO: 55 or 102; or j) having a VH whose sequence is at least 90% (e.g., at least 92%, at least 95%, at least 98% or at least 99%) identical to the amino acid sequence of SEQ ID NO: 56, 103 or 104, and a VL whose sequence is at least 90% (e.g., at least 92%, at least 95%, at least 98% or at least 99%) identical to the amino acid sequence of SEQ ID NO: 57 or 105.

[0036] In certain embodiments, the anti-Gal3 antibody or antigen-binding fragment thereof of the invention comprises: a) having a VH comprising or consisting of the amino acid sequence of SEQ ID NO: 38, 81 or 82 and a VL comprising or consisting of the amino acid sequence of SEQ ID NO: 39 or 83; b) a VH comprising or consisting of the amino acid sequence of SEQ ID NO: 40, 84 or 85 and a VL comprising or consisting of the amino acid sequence of SEQ ID NO: 41 or 86; c) a VH comprising or consisting of the amino acid sequence of SEQ ID NO: 42, 87 or 88 and a VL comprising or consisting of the amino acid sequence of SEQ ID NO: 43 or 89; d) having a VH comprising or consisting of the amino acid sequence of SEQ ID NO: 44 or 90 and a VL comprising or consisting of the amino acid sequence of SEQ ID NO: 45 or 91; e) a VH comprising or consisting of the amino acid sequence of SEQ ID NO: 46 or 92 and a VL comprising or consisting of the amino acid sequence of SEQ ID NO: 47 or 93; f) having a VH comprising or consisting of the amino acid sequence of SEQ ID NO: 48, 94 or 95 and a VL comprising or consisting of the amino acid sequence of SEQ ID NO: 49 or 96; g) having a VH comprising or consisting of the amino acid sequence of SEQ ID NO: 50 or 97 and a VL comprising or consisting of the amino acid sequence of SEQ ID NO: 51 or 98; h) having a VH comprising or consisting of the amino acid sequence of SEQ ID NO: 52 or 99 and a VL comprising or consisting of the amino acid sequence of SEQ ID NO: 53 or 100; j) a VH comprising or consisting of the amino acid sequence of SEQ ID NO: 54 or 101 and a VL comprising or consisting of the amino acid sequence of SEQ ID NO: 55 or 102; or i) having a VH comprising or consisting of the amino acid sequence of SEQ ID NO: 56, 103 or 104, and a VL comprising or consisting of the amino acid sequence of SEQ ID NO: 57 or 105.

[0037] In certain embodiments, the anti-Gal3 antibody is a) having a LC comprising or consisting of the amino acid sequences of SEQ ID NOs: 39 and 60 and a HC comprising or consisting of the amino acid sequences of SEQ ID NOs: 38 and 58; b) having a LC comprising or consisting of the amino acid sequences of SEQ ID NOs: 41 and 60 and a HC comprising or consisting of the amino acid sequences of SEQ ID NOs: 40 and 58; c) having a LC comprising or consisting of the amino acid sequences of SEQ ID NOs: 43 and 60 and a HC comprising or consisting of the amino acid sequences of SEQ ID NOs: 42 and 58; d) having a LC comprising or consisting of the amino acid sequence of SEQ ID NO: 45 and 60 and a HC comprising or consisting of the amino acid sequence of SEQ ID NO: 44 and 58; e) having a LC comprising or consisting of the amino acid sequences of SEQ ID NOs: 47 and 60 and a HC comprising or consisting of the amino acid sequences of SEQ ID NOs: 46 and 58; f) having a LC comprising or consisting of the amino acid sequences of SEQ ID NOs: 49 and 60 and a HC comprising or consisting of the amino acid sequences of SEQ ID NOs: 48 and 58; g) having a LC comprising or consisting of the amino acid sequence of SEQ ID NOs: 51 and 60 and a HC comprising or consisting of the amino acid sequence of SEQ ID NOs: 50 and 58; h) having a LC comprising or consisting of the amino acid sequences of SEQ ID NOs: 53 and 60 and a HC comprising or consisting of the amino acid sequences of SEQ ID NOs: 52 and 58; i) having a LC comprising or consisting of the amino acid sequence of SEQ ID NOs: 55 and 60 and a HC comprising or consisting of the amino acid sequence of SEQ ID NOs: 54 and 58; j) having a LC comprising or consisting of the amino acid sequence of SEQ ID NO:57 and 60 and a HC comprising or consisting of the amino acid sequence of SEQ ID NO:56 and 58.

[0038] In certain embodiments, the anti-Gal3 antibody is a) having a LC comprising or consisting of the amino acid sequences of SEQ ID NOs: 39 and 60 and a HC comprising or consisting of the amino acid sequences of SEQ ID NOs: 38 and 59; b) having a LC comprising or consisting of the amino acid sequences of SEQ ID NOs: 41 and 60 and a HC comprising or consisting of the amino acid sequences of SEQ ID NOs: 40 and 59; c) having a LC comprising or consisting of the amino acid sequences of SEQ ID NOs: 43 and 60 and a HC comprising or consisting of the amino acid sequences of SEQ ID NOs: 42 and 59; d) having a LC comprising or consisting of the amino acid sequences of SEQ ID NOs: 45 and 60 and a HC comprising or consisting of the amino acid sequences of SEQ ID NOs: 44 and 59; e) having a LC comprising or consisting of the amino acid sequences of SEQ ID NOs: 47 and 60 and a HC comprising or consisting of the amino acid sequences of SEQ ID NOs: 46 and 59; f) having a LC comprising or consisting of the amino acid sequences of SEQ ID NOs: 49 and 60 and a HC comprising or consisting of the amino acid sequences of SEQ ID NOs: 48 and 59; g) having a LC comprising or consisting of the amino acid sequences of SEQ ID NOs: 51 and 60 and a HC comprising or consisting of the amino acid sequences of SEQ ID NOs: 50 and 59; h) having a LC comprising or consisting of the amino acid sequences of SEQ ID NOs: 53 and 60 and a HC comprising or consisting of the amino acid sequences of SEQ ID NOs: 52 and 59; i) having a LC comprising or consisting of the amino acid sequence of SEQ ID NOs: 55 and 60 and a HC comprising or consisting of the amino acid sequence of SEQ ID NOs: 54 and 59; j) having a LC comprising or consisting of the amino acid sequence of SEQ ID NO: 57 and 60 and a HC comprising or consisting of the amino acid sequence of SEQ ID NO: 56 and 59.

[0039] In certain embodiments, the anti-Gal3 antibody is a) having a LC comprising or consisting of the amino acid sequences of SEQ ID NOs: 83 and 60, and a HC comprising or consisting of the amino acid sequences of SEQ ID NOs: 81 or 82 and SEQ ID NOs: 58 or 59; b) having a LC comprising or consisting of the amino acid sequences of SEQ ID NOs: 86 and 60 and a HC comprising or consisting of the amino acid sequences of SEQ ID NOs: 84 or 85 and SEQ ID NOs: 58 or 59; c) having a LC comprising or consisting of the amino acid sequences of SEQ ID NOs: 89 and 60 and a HC comprising or consisting of the amino acid sequences of SEQ ID NOs: 87 or 88 and SEQ ID NOs: 58 or 59; d) having a LC comprising or consisting of the amino acid sequences of SEQ ID NO: 91 and 60 and a HC comprising or consisting of the amino acid sequences of SEQ ID NO: 90 and SEQ ID NO: 58 or 59; e) having a LC comprising or consisting of the amino acid sequences of SEQ ID NO: 93 and 60, and a HC comprising or consisting of the amino acid sequences of SEQ ID NO: 92 and SEQ ID NO: 58 or 59; f) having a LC comprising or consisting of the amino acid sequences of SEQ ID NOs: 96 and 60 and a HC comprising or consisting of the amino acid sequences of SEQ ID NOs: 94 or 95 and SEQ ID NOs: 58 or 59; g) having a LC comprising or consisting of the amino acid sequences of SEQ ID NO: 98 and 60, and a HC comprising or consisting of the amino acid sequences of SEQ ID NO: 97 and SEQ ID NO: 58 or 59; h) having a LC comprising or consisting of the amino acid sequences of SEQ ID NO: 100 and 60 and a HC comprising or consisting of the amino acid sequences of SEQ ID NO: 99 and SEQ ID NO: 58 or 59; i) having a LC comprising or consisting of the amino acid sequences of SEQ ID NO: 102 and 60, and a HC comprising or consisting of the amino acid sequences of SEQ ID NO: 101 and SEQ ID NO: 58 or 59; j) having a LC comprising or consisting of the amino acid sequences of SEQ ID NO: 105 and 60 and a HC comprising or consisting of the amino acid sequences of SEQ ID NO: 103 or 104 and SEQ ID NO: 58 or 59.

[0040] In one embodiment, the anti-Gal3 antibody competes with antibody D06, D11, E01, E02, E07, G03, H07, H10, B12, or E12 for binding to human Gal3.

[0041] In one embodiment, the anti-Gal3 antibody competes 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 for binding to human Gal3.

[0042] In some embodiments, the antibody of the invention is an antibody of isotype IgG, such as an antibody of isotype IgG subclass IgG1 or IgG2. The sequence of the IgG1 constant chain is provided in SEQ ID NO: 58. In certain embodiments, the antibody comprises at least one mutation in the Fc region. In certain embodiments, the antibody comprises a mutation in one or more of amino acid positions 228, 234, and 235 of the heavy chain, which are numbered according to the EU numbering or IMGT® numbering system. For example, one or both of the amino acid residues at positions 234 and 235 may be mutated from leucine to alanine, and / or the amino acid residue at position 228 may be mutated from serine to proline. The sequence of the IgG1 constant chain mutated from leucine to alanine at positions 234 and 235 is provided in SEQ ID NO: 59.

[0043] In another aspect, the present invention provides pharmaceutical compositions comprising at least one anti-Gal3 antibody or antigen-binding fragment thereof as described herein and a pharma- ceutically acceptable excipient, optionally together with an additional therapeutic agent as described herein.

[0044] The present invention further provides isolated nucleic acid molecules comprising a nucleotide sequence encoding the heavy chain or VH of an anti-Gal3 antibody or antigen-binding fragment thereof, comprising a nucleotide sequence encoding the light chain or VL of an anti-Gal3 antibody or antigen-binding fragment thereof, or comprising a nucleotide sequence encoding both the heavy chain or VH or the light chain or VL of an anti-Gal3 antibody or antigen-binding fragment thereof. The present invention further provides combinations of isolated nucleic acid molecules encoding anti-Gal3 antibodies or antigen-binding fragments thereof, in particular combinations of a first isolated nucleic acid molecule comprising or consisting of a sequence encoding the heavy chain or VH sequence and a second isolated nucleic acid molecule comprising or consisting of a sequence encoding the light chain or VL sequence.

[0045] The present invention also provides vectors comprising such isolated nucleic acid molecules or combinations of isolated nucleic acid molecules, wherein the vectors may further comprise expression control sequences.

[0046] The present invention also provides a host cell containing a nucleotide sequence encoding the heavy chain or antigen-binding fragment thereof, a nucleotide sequence encoding the light chain or antigen-binding fragment thereof, or both, of an anti-Gal3 antibody or antigen-binding fragment thereof described herein.

[0047] The present invention also provides a host cell comprising a nucleotide sequence encoding the heavy chain or VH and / or the light chain or VL of an anti-Gal3 antibody or antigen-binding fragment thereof as described herein, or a combination of isolated nucleic acid molecules encoding an anti-Gal3 antibody or antigen-binding fragment thereof, in particular a combination of a first isolated nucleic acid molecule comprising or consisting of a sequence encoding a heavy chain or VH sequence and a second isolated nucleic acid molecule comprising or consisting of a sequence encoding a light chain or VL sequence, culturing the host cell under conditions suitable for expression of the antibody or fragment thereof, and isolating the resulting recombinant antibody or fragment.

[0048] The present invention also provides multispecific (e.g., bispecific) binding molecules that include an antigen-binding fragment of an anti-Gal3 antibody described herein and an antigen-binding fragment of another, distinct antibody, e.g., another anti-Gal3 antibody (e.g., as described herein) or an antibody that targets a different protein.

[0049] The present invention also provides methods for preventing and / or treating a patient having a Gal3-associated disorder or disease, including fibrotic diseases, inflammatory diseases, autoimmune diseases, immune-mediated disorders, neurodegenerative diseases, metabolic diseases, infectious diseases, and some cancers, comprising administering to the patient an anti-Gal3 antibody or antigen-binding fragment thereof, pharmaceutical composition, or bispecific binding molecule, as described herein, to ameliorate the patient's condition. Unless otherwise specified, patient refers to a human patient herein.

[0050] The present invention also provides a method for inhibiting extracellular Gal3 interactions in a patient, comprising administering to the patient an anti-Gal3 antibody or antigen-binding fragment thereof, pharmaceutical composition, or bispecific binding molecule as described herein.

[0051] The invention further provides a method for preventing and / or treating a fibrotic disease, an inflammatory disease, an autoimmune disease, an immune-mediated disorder, a neurodegenerative disease, a metabolic disease, an infectious disease, a cancer in a patient, comprising administering to the patient an anti-Gal3 antibody or antigen-binding fragment thereof, pharmaceutical composition, or bispecific binding molecule, as described herein, to ameliorate the condition of the patient. In some embodiments, the fibrotic disease originates in a tissue selected from the skin, lung, liver, heart, kidney, and blood vessels.

[0052] The present invention further provides use of an antibody composition comprising an anti-Gal3 antibody or antigen-binding fragment thereof as described herein for the manufacture of a medicament for the amelioration of a patient's condition, for preventing and / or treating a patient with a Gal3-associated 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 some cancers, and / or for inhibiting Gal3 interactions in a patient in need thereof.

[0053] The present invention further provides anti-Gal3 antibodies or antigen-binding fragments as described herein for ameliorating a patient's condition, for preventing and / or treating a patient having a Gal3-associated disease or a Gal3-associated disorder, including fibrotic diseases, autoimmune diseases, immune-mediated disorders, neurodegenerative diseases, metabolic diseases, infectious diseases, and some cancers, and / or for inhibiting Gal3 interactions in a patient in need thereof.

[0054] The present invention further provides articles of manufacture comprising an anti-Gal3 antibody or antigen-binding fragment as described herein, wherein the articles of manufacture are suitable for detecting human Gal3 or ameliorating a patient's condition, preventing and / or treating patients with Gal3-associated diseases, including fibrotic diseases, inflammatory diseases, autoimmune diseases, immune-mediated disorders, neurodegenerative diseases, metabolic diseases, infectious diseases, and some cancers, and / or inhibiting Gal3 interactions in patients in need thereof. [Brief description of the drawings]

[0055] [Figure 1] FIG. 1 shows the binding specificity of scFvs against human Gal3 full-length and CRD domain (Hu-Gal3-FL, black bars, and Hu-Gal3-CRD, white bars, respectively), against human Gal1 (Hu-Gal1, grey striped bars) and human Gal7 (Hu-Gal7, black striped bars), and against mouse Gal3 (m-Gal3-FL, grey bars) by ELISA. Antigen-free controls are shown by polka dot bars (last bars on the right for each group). The first two groups of bars are positive and negative controls, respectively: commercial anti-Gal3, anti-Gal1, and anti-Gal7 antibodies to confirm coverage of the five antigens tested, and an irrelevant scFv tested against the five antigens. [Diagram 2] Figure 2 shows the inhibition of Gal3-ASF (asialofetuin) complex formation by scFv expressed in the supernatant by competitive ELISA. Absorbance results are expressed as percentages, where irrelevant scFv and asialofetuin (ASF) correspond to 100% signal (upper dotted line) and the lower dotted line corresponds to no binding, i.e., 100% inhibited signal (secondary antibody only). Data are representative of three independent experiments and values ​​are expressed as mean ± standard error. [Diagram 3] Figure 3 shows human and mouse cross-reactivity of Hu-Gal3-FL (first white bar), Hu-Gal3-CRD (second grey bar), and m-Gal3-FL (third striped bar) by ELISA. The first two groups are controls, respectively: a commercial anti-Gal3 antibody to confirm coverage of the Hu-Gal3 antigen, and an irrelevant IgG tested against the three antigens. [Figure 4]Figure 4 shows that there is no binding of anti-Gal3 antibodies to Hu-Gal1 (second black bar) and Hu-Gal7 (third striped bar) compared to the positive control to Hu-Gal3-CRD (first white bar). The first three groups of bars are positive and negative controls, respectively: anti-Gal1 and anti-Gal7 antibodies to confirm coverage of the two antigens tested, and an irrelevant IgG tested in three conditions. [Diagram 5] Figure 5 shows the antibody binding to Hu-Gal3-FL and m-Gal3-FL as circles and triangles, respectively. EC50 is the best fit value (Hill equation). [Figure 6] Figure 6 shows dose-response inhibition of asialofetuin (ASF) binding to Hu-Gal3-CRD by anti-Gal3 antibodies by ELISA. The measured (triangles) and fitted (curve) OD450nm for each anti-Gal3 antibody concentration is in black. The measured (circles) OD450nm for irrelevant antibodies is in grey. [Figure 7] Figure 7 shows the evolution of changes in skin thickness during two independent studies compared to a control group of mice treated with TD139, E07 and D11 antibodies in one study (Figure 7A) and with TD139, E07 and H10 antibodies in the other study (Figure 7B). Data are quoted as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001 compared to the HOCl group. ###p<0.001 compared to the control group. Repeated measures ANOVA with Dunnett's post-hoc test per day. [Figure 8] Figure 8 shows histograms illustrating quantification of collagen deposition in lung (Figure 8A) and skin (Figure 8B) sections stained with picrosirius red, assisted by ImageJ software. Data are expressed as mean ± SEM. *p<0.05, **p<0.01 compared to HOCl group. ###p<0.001 compared to control group. Mixed one-way ANOVA with Dunnett's post-hoc test (mice as random effect). [Figure 9]Figure 9 shows histograms depicting plasma IL-5 and IL-6 levels on day 42 in control, HOCl, and HOCl antibody or TD139 treated groups. Data are expressed as mean ± SEM. **p<0.01 compared to HOCl group. ##p<0.01 compared to control group. Repeated measures analysis of covariance on log transformed data (previous day measurements as covariates) with Dunnett's post hoc test per day. [Figure 10] Figure 10 shows histograms showing quantification of Gal3 in mouse plasma in control, HOCl, and HOCl antibody or TD139 treated groups before administration of test article (day 0) and on day 42. Data are expressed as mean ± SEM. ***p<0.001 compared to HOCL group. ##p<0.01 compared to control group. Dunnett's post-hoc test and mixed analysis of covariance (previous day measurement as covariate and mouse as random effect). [Figure 11] FIG. 11 shows a heat map of differentially expressed genes in whole blood at day 42 using RNA sequencing in control, HOCl, HOCl E07 antibody-treated, and TD139-treated groups. [Figure 12] FIG. 12 shows the change in body weight of bleomycin-treated mice with or without treatment with D06, D11, E07 or TD139 from day 1 to day 21. [Figure 13] Figure 13 shows time to weight gain (TWG) and time to baseline (TTB) in bleomycin-treated mice with or without treatment with D06, D11, E07 or TD139. Data are expressed as individual values ​​and median with interquartile range predicted by nonlinear model. *p<0.05, **p<0.01 compared to bleomycin group. Log-rank test followed by Bonforney-Holm correction for multiplicity. [Figure 14] FIG. 14 shows photographs showing sections made of the lungs prior to staining with picrosirius red and scoring using the modified Ashcroft fibrosis score. [Figure 15]FIG. 15 shows the incidence of median Ashcroft scores of pulmonary fibrosis in bleomycin-treated mice with no treatment or with treatment with D06, D11, E07 antibodies or TD139. [Figure 16] FIG. 16 shows a scatter plot dot blot of neutrophil counts in bronchoalveolar lavage fluid of acute respiratory distress syndrome (ARDS) model rats. The controls without any lipopolysaccharide challenge and without any treatment are shown by black circles, the positive controls challenged with lipopolysaccharide but without any treatment are shown by black squares, and the groups challenged with lipopolysaccharide and dexamethasone (Dexa) as a reference treatment are shown by white squares. The groups treated with anti-Gal3 antibodies before lipopolysaccharide challenge are shown by black triangles for D11 antibody, black inverted triangles for D06 antibody, black diamonds for E07 antibody, and white circles for H10 antibody. Data are expressed as mean ± standard error. ####p<0.0001 between lipopolysaccharide and control groups; **p<0.01; ****p<0.0001 compared to the positive control lipopolysaccharide group. One-way ANOVA on log-transformed data using Dunnett's correction method. [Figure 17] FIG. 17 shows scatter plot dot blots of protein quantification in bronchoalveolar lavage fluid of acute respiratory distress syndrome (ARDS) model rats. Controls without any lipopolysaccharide challenge and no treatment are indicated by black circles, positive controls with lipopolysaccharide challenge but no treatment are indicated by black squares, and groups with lipopolysaccharide challenge and dexamethasone (Dexa) as reference treatment are indicated by white squares. Groups treated with anti-Gal3 antibody before lipopolysaccharide challenge are indicated by black triangles for D11 antibody, black inverted triangles for D06 antibody, black diamonds for E07 antibody, and white circles for H10 antibody. Data are expressed as mean ± standard error. #p<0.05 between lipopolysaccharide group and control group; **p<0.051 compared to positive control lipopolysaccharide group. One-way ANOVA on log-transformed data with Dunnett correction. [Figure 18] Figure 18 shows the scatter plot dot blot of quantification of tumor necrosis factor alpha in plasma of acute respiratory distress syndrome (ARDS) model rats by MSD method. The control without any treatment without any lipopolysaccharide loading is shown by black circle, the positive control without any treatment but with lipopolysaccharide loading is shown by black square, and the group with lipopolysaccharide loading and dexamethasone (Dexa) as reference treatment is shown by white square. The groups treated with anti-Gal3 antibody before lipopolysaccharide loading are shown by black triangle for D11 antibody, black inverted triangle for D06 antibody, black diamond for E07 antibody, and white circle for H10 antibody. Data are expressed as mean ± standard error. ####p<0.0001 between lipopolysaccharide group and control group; **p<0.01; ****p<0.0001 compared to the positive control lipopolysaccharide group. One-way ANOVA on log-transformed data with Dunnett's correction method. [Figure 19] FIG. 19 shows a scatter plot dot blot of the quantification of KC (keratinocyte-derived cytokine)-GRO (proliferation-related oncogene) in plasma of acute respiratory distress syndrome (ARDS) model rats by MSD method. The control without any treatment without any lipopolysaccharide loading is shown by a black circle, the positive control without any treatment but with lipopolysaccharide loading is shown by a black square, and the group with lipopolysaccharide loading and dexamethasone (Dexa) as a reference treatment is shown by an open square. The group treated with anti-Gal3 antibody before lipopolysaccharide loading is shown by a black triangle for D11 antibody, a black inverted triangle for D06 antibody, a black diamond for E07 antibody, and an open circle for H10 antibody. Data are expressed as the mean ± standard error. ####p<0.0001 between lipopolysaccharide and control groups; ****other values ​​p<0.0001 as indicated compared to the positive control lipopolysaccharide group. One-way ANOVA on log-transformed data with Dunnett's correction method.

[0056] Detailed Description of the Invention The present invention provides novel anti-human Galectin-3 (Gal3) antibodies. The human Gal3 polypeptide sequence is available under UniProt accession number P17931 (LEG3_HUMAN).

[0057] The present invention relates to novel monoclonal antibodies that target extracellular galectin-3 (Gal3) and do not bind to galectin-1 (Gal1) or galectin-7 (Gal7), and pharmaceutical compositions containing one or more of these antibodies.

[0058] The present invention also relates to the use of said anti-Gal3 antibodies and pharmaceutical compositions to inhibit Gal3 interactions in patients in need thereof or to ameliorate, prevent and / or treat patients having Gal3-associated diseases or Gal3-associated disorders, including fibrotic diseases, inflammatory diseases, autoimmune diseases, immune-mediated disorders, neurodegenerative diseases, metabolic diseases, infectious diseases, and some cancers.

[0059] Tissue fibrosis is a progressive debilitating disease characterized by abundant accumulation of extracellular matrix proteins such as collagen and fibronectin, which leads to tissue scarring, organ damage, reduced organ function, and subsequent organ failure.Tissue fibrosis can be localized in the kidney, liver, lung, heart, skin, pancreas, intestine, eye, nervous system, joints, tendons, diaphragm, or retroperitoneum.Characteristics of tissue fibrosis include epithelial and endothelial damage and dysfunction, abnormal proliferation of myofibroblasts, smooth muscle cells, and astrocytes, and deposition of extracellular matrix proteins.The presence of cytokines, chemokines, growth factors, and angiogenic factors further regulates the activation of extracellular matrix protein-producing cells during the profibrotic process.

[0060] As used herein, the term "antibody" (Ab) or "immunoglobulin" (Ig) refers to a tetramer comprising two heavy (H) chains (about 50-70 kDa) and two light (L) chains (about 25 kDa) connected together by disulfide bonds. Each heavy chain comprises a heavy chain variable domain (VH) and a heavy chain constant domain (CH). Each light chain comprises a light chain variable domain (VL) and a light chain constant domain (CL). The VH and VL domains can be further subdivided into regions of hypervariability, termed "complementarity determining regions" (CDRs), interspersed with more conserved regions, termed "framework regions" (FRs). Each VH and VL is composed of three CDRs (H-CDR herein refers to the CDRs of the heavy chain; L-CDR herein refers to the CDRs of the light chain) and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Number assignment of amino acids within the heavy or light chain may follow EU numbering or IMGT® definitions (Lefranc et al., Dev Comp Immunol 27(1):55-77 (2003)); or 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).

[0061] The term "recombinant antibody" refers to an antibody expressed from a cell or cell line that contains a nucleotide sequence(s) encoding the antibody, where the nucleotide sequence(s) is not naturally associated with the cell.

[0062] The term "isolated protein," "isolated polypeptide," or "isolated antibody" refers to a protein, polypeptide, or antibody that, by reason of its source or origin, (1) is not associated with naturally associated components that accompany it in its natural state, (2) is free of other proteins from the same species, (3) is expressed by cells from a different species, and / or (4) is not naturally occurring. Thus, a polypeptide that is chemically synthesized or synthesized in a cellular system other than the cell of natural origin 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 that are well known in the art.

[0063] The term "germline" as used herein refers to the nucleotide and amino acid sequences of antibody genes and gene segments as they are passed from parents to offspring through germ cells. Germline sequences are distinct from the nucleotide sequences that encode antibodies in mature B cells, which have been altered by recombination and hypermutation events during the course of B cell maturation. An antibody that "utilizes" a particular germline sequence has a nucleotide or amino acid sequence that aligns with a specific nucleotide or amino acid sequence more precisely than any other germline nucleotide or amino acid sequence.

[0064] The term "affinity" refers to a measure of attraction between an antigen and an antibody. The intrinsic attractiveness of an antibody to an antigen is typically expressed as the binding affinity equilibrium constant (KD) of a particular antibody-antigen interaction. An antibody is said to specifically bind to an antigen when the KD is 1 mM or less, preferably 100 nM or less. The KD binding affinity constant can be measured, for example, by surface plasmon resonance (Biacore™) or biolayer interferometry, for example using the IBIS MX96SPR system from IBIS Technologies or the Octet™ system from ForteBio.

[0065] The term "humanized" refers to the fact that when an antibody is of fully or partially non-human origin (e.g. mouse or chicken antibodies obtained from immunization of mice or chickens, respectively, with an antigen of interest, or chimeric antibodies based on such mouse or chicken antibodies), it is possible to exchange certain amino acids, especially in the framework and constant regions of the heavy and light chains, to avoid or minimize immune responses in humans. Although it is not possible to accurately predict the immunogenicity of a particular antibody, and thus the human response to the antibody, 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 are replaced with sequences of human origin, have been shown to be generally less immunogenic than antibodies of completely foreign origin, and the trend for therapeutic antibodies is towards humanized or fully human antibodies. Chimeric or other antibodies of non-human origin may therefore be humanized to reduce the risk of a human response to the antibody.

[0066] Numerous methods for humanizing antibody sequences are known in the art, see, for example, the review by Almagro & Fransson, Front Biosci. 13:1619-1633 (2008). One commonly used method is CDR grafting, which, for example, for mouse-derived chimeric antibodies, involves identifying the human germline gene counterparts for the mouse variable domain genes and grafting the mouse CDR sequences into this framework. The specificity of the antibody's interaction with the target antigen resides primarily in the amino acid residues located within the six CDRs of the heavy and light chains. Therefore, the amino acid sequences within the CDRs are much more variable between individual antibodies than the sequences outside the CDRs. Since the CDR sequences are involved in the majority of antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of a particular naturally occurring antibody, or more generally any particular antibody with a given amino acid sequence, for example, by constructing an expression vector that expresses the CDR sequences from a particular antibody grafted into framework sequences from a different antibody. As a result, it is possible to "humanize" a non-human antibody and still substantially maintain the binding specificity and affinity of the original antibody. CDR grafting may be based on the Kabat CDR definition, although recent publications (Magdelaine-Beuzelin et al., Crit Rev. Oncol Hematol. 64:210-225(2007)) suggest that the IMGT® definition (International ImMunoGeneTics Information System®, www.imgt.org) (or EU numbering) may improve humanization results (see Lefranc et al., Dev. Comp Immunol. 27:55-77 (2003)).

[0067] In some cases, CDR grafting may reduce the binding specificity and affinity of the CDR-grafted antibody compared to the parent antibody from which the CDRs were obtained, and thus may reduce the biological activity. Back mutations (sometimes referred to as "framework repair") may be introduced at selected positions of the CDR-grafted antibody, typically within the framework regions, to re-establish the binding specificity and affinity of the parent antibody. Possible positions for back mutations can be identified using information available in the literature and antibody databases. Candidate amino acid residues for back mutations are typically residues located on the surface of the antibody molecule, although buried or low surface-exposed residues will usually not be altered. An alternative humanization technique to CDR grafting and back mutation is resurfacing, in which non-surface-exposed residues of the non-human source are maintained, but surface residues are altered to human residues.

[0068] In certain cases, it may also be desirable to modify one or more CDR amino acid residues to improve binding affinity to the target epitope. This is known as "affinity maturation" and may be performed, optionally in conjunction with humanization, for example, in situations where humanization of an antibody reduces binding specificity or affinity and back mutation alone is not sufficient to improve binding specificity or affinity. Various affinity maturation methods are known in the art, such as the in vitro scanning saturation mutagenesis method described by Burks et al., Proc Natl Acad Sci USA, 94:412-417 (1997), and the stepwise in vitro affinity maturation method of Wu et al., Proc Natl Acad Sci USA 95:6037-6042 (1998).

[0069] The term "antigen-binding fragment" of an antibody (or simply "antibody fragment") as used herein 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 the antibody. Examples of binding fragments encompassed within the term "antigen-binding fragment" include: (i) Fab fragment: a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragment: a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region; (iii) Fd fragment consisting of the VH and CH1 domains; (iv) Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) dAb fragment consisting of the VH domain; and (vi) an isolated complementarity determining region (CDR) capable of specifically binding to an antigen. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they may be connected by a synthetic linker that allows them to be produced using recombinant techniques as a single protein chain in which the VL and VH domains pair to form a monovalent molecule, known as 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 encompassed within the term "antigen-binding fragment" of an antibody. Other forms of single chain antibodies, such as diabodies, are also encompassed. Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow pairing between the two domains on the same chain, which forces the domains to pair with the complementary domains of another chain and creates two antigen-binding sites.

[0070] Antibody fragments, such as Fab and F(ab')2 fragments, can be prepared from whole antibodies using conventional techniques, such as digestion of whole antibodies with papain or pepsin. Furthermore, antibodies, antibody fragments, and immunoadhesin molecules can be obtained using standard recombinant DNA techniques, for example, as described herein.

[0071] The class (isotype) and subclass of an anti-Gal3 antibody can be determined by any method known in the art. Generally, the class and subclass of an antibody can be determined using antibodies that are specific for a particular class and subclass of antibody. Such antibodies are commercially available. The class and subclass can be determined by ELISA, Western blot, and other techniques. Alternatively, the class and subclass can be determined by sequencing all or a fragment of the constant region of the heavy and / or light chain of the antibody, comparing their amino acid sequences with known amino acid sequences of various classes and subclasses of immunoglobulins, and determining the class and subclass of the antibody.

[0072] Unless otherwise specified, the numbering of amino acid residues of all antibodies referred to in this disclosure is under the EU numbering or IMGT® numbering system.

[0073] Anti-Gal3 antibody The present invention provides antibodies directed against Gal3, and antigen-binding fragments thereof. One advantage of the novel anti-Gal3 antibodies of the present invention is that they can selectively bind to Gal3, in particular to the CRD of Gal3, but not to Gal1 or Gal7 (see Example 4). They can inhibit the binding of Gal3 to its ligands (see Example 6). Another possible advantage of the anti-Gal3 antibodies of the present invention is the low level of secondary effector function in antibodies carrying the "LALA" mutation (L234A / L235A), which prevents significant binding of the antibody to human FcγR (Fcγ receptor), thus preventing the elimination of effector T cells. In another embodiment, the present invention provides antibodies with germline framework mutations within the variable domain of the antibody outside the CDR regions, which allows for potentially reduced immunogenicity when administered to humans, while substantially maintaining the specificity and affinity of the parent antibody.

[0074] 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 in sequence to any one of SEQ ID NOs: 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 in sequence, e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical in sequence to the VH amino acid sequence of SEQ ID NOs: 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.

[0075] In one embodiment, the anti-Gal3 antibody has at least 90% sequence identity to any one of SEQ ID NOs: 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., a VH antigen 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 at least 90% identical in sequence to SEQ ID NO:58 or 59, e.g., at least 92% identical, e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:58 or 59.

[0076] In one embodiment, the anti-Gal3 antibody has a heavy chain (HC) comprising a VH amino acid sequence of any one of SEQ ID NOs: 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.

[0077] 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 in sequence to the VL amino acid sequence of any one of SEQ ID NOs: 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, e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 83, 86, 89, 91, 93, 96, 98, 100, 102 or 105.

[0078] In one embodiment, the anti-Gal3 antibody has at least 90% sequence identity to the VL amino acid sequence of any one of SEQ ID NOs: 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 90% sequence identity to any one of SEQ ID NOs: 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 83, 86, 89, 91, 93, 96, 98, 100, 102, or 105. a light chain variable domain (VL) that is at least 92% identical, e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical; and a light chain constant region amino acid sequence that is at least 90% identical in sequence to SEQ ID NO:60, e.g., at least 92% identical, e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:60.

[0079] In another embodiment, the anti-Gal3 antibody has a light chain comprising any one of SEQ ID NOs: 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.

[0080] In certain embodiments, the anti-Gal3 antibody or antigen-binding fragment of the invention comprises: a) SEQ ID NOs: 1, 6, 12, 20, 27 and 30, respectively; b) SEQ ID NOs: 1, 6, 13, 21, 27 and 31, respectively; c) SEQ ID NOs: 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 NOs: 2, 6, 16, 24, 27 and 34, respectively; g) SEQ ID NOs: 1, 9, 14, 24, 27 and 34, respectively; h) SEQ ID NOs: 2, 10, 17, 25, 27 and 35, respectively; i) SEQ ID NOs: 4, 11, 18, 21, 29 and 36, respectively; or j) SEQ ID NOs: 2, 10, 19, 26, 27 and 37, respectively It includes the amino acid sequences of H-CDR1 to 3 and L-CDR1 to 3.

[0081] In some embodiments, the anti-Gal3 antibody or antigen-binding fragment of the invention comprises: a) SEQ ID NOs: 38 and 39, respectively; b) SEQ ID NOs: 40 and 41, respectively; c) SEQ ID NOs: 42 and 43, respectively; d) SEQ ID NOs: 44 and 45, respectively; e) SEQ ID NOs: 46 and 47, respectively; f) SEQ ID NOs: 48 and 49, respectively; g) SEQ ID NOs: 50 and 51, respectively; h) SEQ ID NOs: 52 and 53, respectively; i) SEQ ID NOs: 54 and 55, respectively; j) SEQ ID NOs: 56 and 57, respectively; k) SEQ ID NOs: 81 and 83, respectively; l) SEQ ID NOs: 82 and 83, respectively; m) SEQ ID NOs: 84 and 86, respectively; n) SEQ ID NOs: 85 and 86, respectively; o) SEQ ID NOs: 87 and 89, respectively; p) SEQ ID NOs: 88 and 89, respectively; q) SEQ ID NOs: 90 and 91, respectively; r) SEQ ID NOs: 92 and 93, respectively; s) SEQ ID NOs: 94 and 96, respectively; t) SEQ ID NOs: 95 and 96, respectively; u) SEQ ID NOs: 97 and 98, respectively; v) SEQ ID NOs: 99 and 100, respectively; w) SEQ ID NOs: 101 and 102, respectively; x) SEQ ID NOs: 103 and 105, respectively; or y) SEQ ID NOs: 104 and 105, respectively The present 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 amino acid sequence of

[0082] In some embodiments, the anti-Gal3 antibody or antigen-binding fragment of the invention comprises: a) SEQ ID NOs: 38 and 39, respectively; b) SEQ ID NOs: 40 and 41, respectively; c) SEQ ID NOs: 42 and 43, respectively; d) SEQ ID NOs: 44 and 45, respectively; e) SEQ ID NOs: 46 and 47, respectively; f) SEQ ID NOs: 48 and 49, respectively; g) SEQ ID NOs: 50 and 51, respectively; h) SEQ ID NOs: 52 and 53, respectively; i) SEQ ID NOs: 54 and 55, respectively; j) SEQ ID NOs: 56 and 57, respectively; k) SEQ ID NOs: 81 and 83, respectively; l) SEQ ID NOs: 82 and 83, respectively; m) SEQ ID NOs: 84 and 86, respectively; n) SEQ ID NOs: 85 and 86, respectively; o) SEQ ID NOs: 87 and 89, respectively; p) SEQ ID NOs: 88 and 89, respectively; q) SEQ ID NOs: 90 and 91, respectively; r) SEQ ID NOs: 92 and 93, respectively; s) SEQ ID NOs: 94 and 96, respectively; t) SEQ ID NOs: 95 and 96, respectively; u) SEQ ID NOs: 97 and 98, respectively; v) SEQ ID NOs: 99 and 100, respectively; w) SEQ ID NOs: 101 and 102, respectively; x) SEQ ID NOs: 103 and 105, respectively; or y) SEQ ID NOs: 104 and 105, respectively The VH and VL have the amino acid sequence:

[0083] In certain embodiments, the anti-Gal3 antibody is a) having a LC comprising or consisting of the amino acid sequences of SEQ ID NO: 39 and 60; and a HC comprising or consisting of the amino acid sequences of SEQ ID NO: 38 and SEQ ID NO: 58 or 59; b) having a LC comprising or consisting of the amino acid sequences of SEQ ID NO: 41 and 60; and a HC comprising or consisting of the amino acid sequences of SEQ ID NO: 40 and SEQ ID NO: 58 or 59; c) having a LC comprising or consisting of the amino acid sequences of SEQ ID NO: 43 and 60; and a HC comprising or consisting of the amino acid sequences of SEQ ID NO: 42 and SEQ ID NO: 58 or 59; d) having a LC comprising or consisting of the amino acid sequences of SEQ ID NO: 45 and 60; and a HC comprising or consisting of the amino acid sequences of SEQ ID NO: 44 and SEQ ID NO: 58 or 59; e) having a LC comprising or consisting of the amino acid sequences of SEQ ID NO: 47 and 60; and a HC comprising or consisting of the amino acid sequences of SEQ ID NO: 46 and SEQ ID NO: 58 or 59; f) having a LC comprising or consisting of the amino acid sequences of SEQ ID NO: 49 and 60; and a HC comprising or consisting of the amino acid sequences of SEQ ID NO: 48 and SEQ ID NO: 58 or 59; g) having a LC comprising or consisting of the amino acid sequences of SEQ ID NO: 51 and 60; and a HC comprising or consisting of the amino acid sequences of SEQ ID NO: 50 and SEQ ID NO: 58 or 59; h) having a LC comprising or consisting of the amino acid sequences of SEQ ID NO: 53 and 60; and a HC comprising or consisting of the amino acid sequences of SEQ ID NO: 52 and SEQ ID NO: 58 or 59; i) having a LC comprising or consisting of the amino acid sequences of SEQ ID NO: 55 and 60; and a HC comprising or consisting of the amino acid sequences of SEQ ID NO: 54 and SEQ ID NO: 58 or 59; j) having a LC comprising or consisting of the amino acid sequences of SEQ ID NO: 57 and 60; and a HC comprising or consisting of the amino acid sequences of SEQ ID NO: 56 and SEQ ID NO: 58 or 59.

[0084] In certain embodiments, the anti-Gal3 antibody is a) having a LC comprising or consisting of the amino acid sequences of SEQ ID NO: 83 and 60; and a HC comprising or consisting of the amino acid sequences of SEQ ID NO: 81 or 82 and SEQ ID NO: 58 or 59; b) having a LC comprising or consisting of the amino acid sequences of SEQ ID NO: 86 and 60; and a HC comprising or consisting of the amino acid sequences of SEQ ID NO: 84 or 85 and SEQ ID NO: 58 or 59; c) having a LC comprising or consisting of the amino acid sequences of SEQ ID NOs: 89 and 60; and a HC comprising or consisting of the amino acid sequences of SEQ ID NOs: 87 or 88 and SEQ ID NOs: 58 or 59; d) having a LC comprising or consisting of the amino acid sequences of SEQ ID NO: 91 and 60; and a HC comprising or consisting of the amino acid sequences of SEQ ID NO: 90 and SEQ ID NO: 58 or 59; e) having a LC comprising or consisting of the amino acid sequences of SEQ ID NO: 93 and 60; and a HC comprising or consisting of the amino acid sequences of SEQ ID NO: 92 and SEQ ID NO: 58 or 59; f) having a LC comprising or consisting of the amino acid sequences of SEQ ID NOs: 96 and 60; and a HC comprising or consisting of the amino acid sequences of SEQ ID NOs: 94 or 95 and SEQ ID NOs: 58 or 59; g) having a LC comprising or consisting of the amino acid sequences of SEQ ID NO: 98 and 60; and a HC comprising or consisting of the amino acid sequences of SEQ ID NO: 97 and SEQ ID NO: 58 or 59; h) having a LC comprising or consisting of the amino acid sequences of SEQ ID NO: 100 and 60; and a HC comprising or consisting of the amino acid sequences of SEQ ID NO: 99 and SEQ ID NO: 58 or 59; i) having a LC comprising or consisting of the amino acid sequences of SEQ ID NO: 102 and 60; and a HC comprising or consisting of the amino acid sequences of SEQ ID NO: 101 and SEQ ID NO: 58 or 59; j) having a LC comprising or consisting of the amino acid sequences of SEQ ID NO: 105 and 60; and a HC comprising or consisting of the amino acid sequences of SEQ ID NO: 103 or 104 and SEQ ID NO: 58 or 59.

[0085] In some embodiments, the anti-Gal3 antibody or antigen-binding fragment of the invention comprises the amino acid sequence of H-CDR1-3 and L-CDR1-3 of antibody D06, D11, E01, E02, E07, G03, H07, H10, B12, or E12.

[0086] In some embodiments, an anti-Gal3 antibody or antigen-binding fragment of the invention comprises a VH and VL that are at least 90% identical in amino acid sequence to the VH and VL, respectively, of antibody D06, D11, E01, E02, E07, G03, H07, H10, B12, or E12.

[0087] In some embodiments, an anti-Gal3 antibody or antigen-binding fragment of the invention comprises a VH and a VL that are the VH and VL, respectively, of antibody D06, D11, E01, E02, E07, G03, H07, H10, B12, or E12.

[0088] In some embodiments, an anti-Gal3 antibody or antigen-binding fragment of the invention comprises a VH and VL that are at least 90% identical in amino acid sequence to the VH and VL, respectively, of 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.

[0089] In some embodiments, an anti-Gal3 antibody or antigen-binding fragment of the invention comprises a VH and VL that are the VH and VL, respectively, 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.

[0090] In some embodiments, the anti-Gal3 antibody or antigen-binding fragment thereof of the 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 foregoing antibodies or antibody-binding fragments thereof.

[0091] In some embodiments, none of the anti-Gal3 antibodies or antigen-binding fragments thereof binds to Gal1 or Gal7.

[0092] In some embodiments, any of the anti-Gal3 antibodies or antigen-binding fragments thereof may inhibit and / or reduce the expression of Gal3 and / or the interaction of Gal3 with its ligands, thereby enabling the amelioration, prevention and / or treatment of a Gal3-associated disease or a Gal3-associated disorder.

[0093] In some embodiments, any of the anti-Gal3 antibodies or antigen-binding fragments thereof can suppress plasma levels of IL-6 and / or IL-5.

[0094] In some embodiments, the anti-Gal3 antibodies or antigen-binding fragments thereof may reduce skin thickness or collagen deposition in patients with a fibrotic disease.

[0095] The class of the anti-Gal3 antibody obtained by the methods described herein may be exchanged or switched to another class or subclass. In one embodiment of the present invention, nucleic acid molecules encoding VL or VH are isolated using methods well known in the art so that they are free of nucleic acid sequences encoding CL or CH, respectively. The nucleic acid molecules encoding VL or VH are then operably linked to nucleic acid sequences encoding CL or CH, respectively, from a different class of immunoglobulin molecule. This may be accomplished using vectors or nucleic acid molecules that contain CL or CH chains, as described above. For example, an anti-Gal3 antibody that was originally IgM may be class switched to IgG. Furthermore, class switching may be used to convert one IgG subclass to another, for example, from IgG1 to IgG2 or from IgG1 to IgG4. The kappa light chain constant region may be changed, for example, to a gamma light chain constant region. A preferred method for producing an antibody of the present invention having a desired Ig isotype includes the steps of isolating a nucleic acid molecule encoding the heavy chain of an anti-Gal3 antibody and a nucleic acid molecule encoding the light chain of an anti-Gal3 antibody, obtaining the variable domain of the heavy chain, ligating the variable domain of the heavy chain to a constant region of a heavy chain of the desired isotype, expressing the light chain and the ligated heavy chain in a cell, and collecting an anti-Gal3 antibody having the desired isotype.

[0096] The anti-Gal3 antibody of the present invention can be an IgG, IgM, IgE, IgA, or IgD molecule, but is typically an IgG isotype, such as an IgG subclass IgG1, IgG2a, or IgG2b, IgG3, or IgG4. In one embodiment, the antibody is an IgG1. In another embodiment, the antibody is an IgG2 or IgG4.

[0097] In one embodiment, the anti-Gal3 antibody may contain at least one mutation in the Fc region. Many different Fc mutations are known, but these mutations herein confer altered effector functions. For example, in many cases, such as when ligand / receptor interactions are undesirable or in the case of antibody-drug conjugates, it may be desirable to reduce or eliminate effector functions.

[0098] In one embodiment, the anti-Gal3 antibody comprises at least one mutation in the Fc region that reduces effector function. Amino acid positions in the Fc region that may be advantageously mutated to reduce effector function include one or more of 228, 233, 234, and 235, where the amino acid positions are numbered according to the EU numbering or IMGT® numbering system.

[0099] In one embodiment, one or both of the amino acid residues at positions 234 and 235 may be mutated, e.g., from leucine to alanine (L234A / L235A). These mutations reduce the effector function of the Fc region of an IgG1 antibody. The sequence of such a mutated IgG1 constant chain is provided in SEQ ID NO: 59. Additionally or alternatively, the amino acid residue at position 228 may be mutated, e.g., to proline. In some embodiments, the amino acid residue at position 233 may be mutated, e.g., to proline, the amino acid residue at position 234 may be mutated, e.g., to valine, and / or the amino acid residue at position 235 may be mutated, e.g., to alanine. Amino acid positions are numbered according to the EU numbering or the IMGT® numbering system.

[0100] In some embodiments, if the antibody is of the IgG4 subclass, it may contain a S228P mutation, i.e., a proline at position 228, where amino acid positions are numbered according to the EU numbering or IMGT® numbering system. This mutation is known to reduce undesired Fab arm exchange.

[0101] In another embodiment, a fusion antibody can be made that includes all or a fragment of the anti-Gal3 antibody of the present invention linked to another polypeptide. In a particular embodiment, only the variable domain of the anti-Gal3 antibody is linked to the polypeptide. In a particular embodiment, the VH domain of the anti-Gal3 antibody is linked to a first polypeptide, while the VL domain of the anti-Gal3 antibody is linked to a second polypeptide that is associated with the first polypeptide, such that the VH and VL domains can interact with each other to form an antigen-binding site. In another preferred embodiment, the VH domain is separated from the VL domain by a linker (e.g., a single-chain antibody) such that the VH and VL domains can interact with each other. The VH-linker-VL antibody is then linked to a polypeptide of interest. In addition, a fusion antibody can be made in which two (or more) single-chain antibodies are linked to each other. This is useful when one wishes to create a bivalent or polyvalent antibody on a single polypeptide chain, or when one wishes to create a bispecific antibody.

[0102] In other embodiments, other modified antibodies can be prepared using nucleic acid molecules encoding anti-Gal3 antibodies, such as "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)), which can be prepared following the teachings of the specification using standard molecular biology techniques.

[0103] The anti-Gal3 antibodies or antigen-binding fragments of the invention may be derivatized or linked to another molecule (e.g., another peptide or protein). Generally, the antibody or fragment thereof is derivatized such that Gal3 binding is not adversely affected by the derivatization or labeling. Thus, the antibodies and antibody fragments of the invention are intended to include both intact and modified forms of the human anti-Gal3 antibodies described herein. For example, the antibodies or antibody fragments of the invention may be functionally linked (by chemical conjugation, genetic fusion, non-covalent binding, or other methods) to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or diabody), a detection agent, a pharmaceutical agent, and / or a protein or peptide that can mediate the association of the antibody or antibody fragment with another molecule (e.g., a streptavidin core region or a polyhistidine tag).

[0104] One type of derivatized antibody is produced by crosslinking two or more antibodies (of the same or different species, e.g., to make bispecific antibodies). Suitable crosslinkers include those that are heterobifunctional or homobifunctional (e.g., disuccinimidyl suberate), which have two separate reactive groups separated by a suitable spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester). Anti-Gal3 antibodies or antigen-binding fragments thereof may also be derivatized with chemical groups, such as polyethylene glycol (PEG) groups, methyl or ethyl groups, or carbohydrate groups. These groups may be useful to improve the biological characteristics of the antibody, e.g., to increase serum half-life.

[0105] The antibody according to the present invention may also be labeled. The term "label" or "labeled" as used herein refers to the incorporation of another molecule into the antibody. In one embodiment, the label is a detectable marker, such as the incorporation of a radiolabeled amino acid or the attachment of a biotinyl moiety to the polypeptide, which can be detected by a marked avidin (e.g., streptavidin containing a fluorescent marker or an enzymatic activity that can be detected by optical or colorimetric methods). In another embodiment, the label or marker is a therapeutic agent, such as a drug conjugate or a toxin. Various methods of labeling polypeptides and glycoproteins are known in the art and may be used. Examples of labels for polypeptides include the following: radioisotopes or radionuclides (e.g., 3H, 14C, 15N, 35S, 90Y, 99Tc, 111In, 125I, 131I), fluorescent labels (e.g., FITC (fluorescein isothiocyanate), rhodamine, lanthanide fluorophores), enzyme labels (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, predetermined polypeptide epitopes recognized by secondary reporters (e.g., sequences of leucine zipper pairs, binding sites for secondary antibodies, metal binding domains, enzymes, etc.). Examples of suitable labeling agents include, but are not limited to, pitope tags), magnetic agents, e.g., gadolinium chelators, toxins, e.g., pertussis toxin, taxol, cytochalasin B, gramicin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracenedione, mitoxantrone, mithramycin, actinomycin D, dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, and analogs or homologs thereof. In some embodiments, labels are attached with spacer arms of various lengths to reduce potential steric hindrance.

[0106] In certain embodiments, the antibodies of the invention may exist in their neutral form (including zwitterionic forms) or as positively or negatively charged species. In some embodiments, the antibodies may be complexed with counterions to form pharma- ceutically acceptable salts.

[0107] The term "pharmaceutically acceptable salts" refers to complexes comprising one or more antibodies and one or more counterions, where the counterions are derived from pharmaceutically acceptable inorganic and organic acids and bases.

[0108] Bispecific binding molecules In a further aspect, the invention provides bispecific binding molecules having the binding specificity of an anti-Gal3 antibody or antigen-binding fragment thereof described herein (e.g., including an antigen-binding fragment) and the binding specificity of another anti-Gal3 antibody or antigen-binding fragment thereof (e.g., another anti-Gal3 antibody described herein), or an antibody targeting the same or a different protein, e.g., another immune checkpoint protein, a cancer antigen, or another cell surface molecule whose activity mediates a disease condition, such as cancer. Such bispecific binding molecules are known in the art, and examples of various types of bispecific binding molecules are provided elsewhere herein.

[0109] Nucleic Acid Molecules and Vectors The present invention also provides nucleic acid molecules and nucleic acid 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 and the light chain amino acid sequence or VL sequence of the anti-Gal3 antibody or antigen-binding fragment thereof. In some embodiments, a combination of isolated nucleic acid molecules encodes an anti-Gal3 antibody or antigen-binding fragment thereof, in particular a first isolated nucleic acid molecule comprising or consisting of a sequence encoding the heavy chain sequence or VH sequence, and a second isolated nucleic acid molecule comprising or consisting of a sequence encoding the 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 antigen-binding fragment thereof.

[0110] Reference to a nucleotide sequence also includes its complement unless otherwise specified. Thus, a reference to a nucleic acid having a particular sequence should be understood to include its complementary strand with its complementary sequence. The term "polynucleotide" as referred to herein means a polymeric form of nucleotides of at least 10 bases in length, either ribonucleotides or deoxynucleotides, or any type of nucleotide in modified form. The term includes single-stranded and double-stranded forms.

[0111] 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 of the nucleotide sequences listed herein, for example to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 61-80, or to a nucleotide sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NOs: 38-57. The term "sequence identity" in the context of nucleic acid sequences refers to the residues in two sequences that are the same when aligned for maximum matching. The length of sequence identity comparison can be over a stretch of 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. Many different algorithms that can be used to measure nucleotide sequence identity are known in the art. For example, polynucleotide sequences can be compared using FASTA, Gap, or Bestfit, which are programs of the Wisconsin Package version 10.0, Genetics Computer Group (GCG), Madison, Wis. FASTA includes, for example, the FASTA2 and FASTA3 programs, which provide alignments of the regions of best overlap between the query and search sequences, and percent sequence identity (see, e.g., 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 specified, default parameters for 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 (word size of 6, NOPAM factor for the scoring matrix) or using Gap with its default parameters as provided in GCG version 6.1.

[0112] In one aspect, the invention provides a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 61 to 80. In certain embodiments, the nucleic acid molecule comprises the 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.

[0113] In any of the above embodiments, the nucleic acid molecule may be isolated.

[0114] In a further aspect, 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 to which it is linked. In some embodiments, the vector is a plasmid, i.e., a circular double-stranded piece of DNA, into which additional DNA segments may be ligated. In some embodiments, the vector is a viral vector, in which additional DNA segments may be ligated into the viral genome. In some embodiments, the vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). In other embodiments, the vectors (e.g., non-episomal mammalian vectors) may be integrated into the genome of the host cell upon introduction into the host cell, thereby replicating along with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors").

[0115] The present invention provides vectors containing nucleic acid molecules encoding the heavy chain or VH of an anti-Gal3 antibody or antigen-binding fragment thereof of the present invention, the light chain or VL of an anti-Gal3 antibody or antigen-binding fragment thereof of the present invention, or both the heavy and light chains or both the VH and VL of an anti-Gal3 antibody or antigen-binding fragment thereof of the present invention. The present invention further provides vectors containing nucleic acid molecules encoding fusion proteins, modified antibodies, antibody fragments, and probes thereof.

[0116] Nucleic acid molecules encoding the heavy and / or light chains of the anti-Gal3 antibody or antigen-binding fragment 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 isolated from animals immunized with human Gal3 antigen, or from immortalized cells produced from such B cells. Methods for isolating nucleic acids encoding antibodies are well known in the art. mRNA can be isolated and used to generate cDNA for use in 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.

[0117] In some embodiments, the nucleic acid molecules of the invention may comprise a nucleotide sequence encoding a VH domain from an anti-Gal3 antibody or antigen-binding fragment of the invention joined in frame to a nucleotide sequence encoding a heavy chain constant region from any source. Similarly, the nucleic acid molecules of the invention may comprise a nucleotide sequence encoding a VL domain from an anti-Gal3 antibody or antigen-binding fragment of the invention joined in frame to a nucleotide sequence encoding a light chain constant region from any source.

[0118] In a further aspect of the invention, nucleic acid molecules encoding the variable domains of the heavy (VH) and / or light (VL) chains can be "converted" into full-length antibody genes. In one embodiment, nucleic acid molecules encoding the VH or VL domains are converted into full-length antibody genes by insertion into an expression vector already encoding a heavy chain constant region (CH) or a light chain constant region (CL), respectively, such that the VH segment is operably linked to the CH segment(s) in the vector and / or the VL segment is operably linked to the CL segment in the vector. In another embodiment, nucleic acid molecules encoding the VH and / or VL domains are converted into full-length antibody genes, e.g., by linking, e.g., ligating, the nucleic acid molecules encoding the VH and / or VL domains to the nucleic acid molecules encoding the CH and / or CL regions using standard molecular biology techniques. Nucleic acid molecules encoding the full-length heavy and / or light chains can then be expressed from a cell into which they have been introduced and the anti-Gal3 antibody can be isolated.

[0119] The nucleic acid molecules can be used to recombinantly express large amounts of anti-Gal3 antibodies. The nucleic acid molecules can be used to generate chimeric antibodies, bispecific antibodies, single chain antibodies, immunoadhesins, diabodies, mutated antibodies, and antibody derivatives, as described herein.

[0120] 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 acids can be used as probes in diagnostic methods or as PCR primers to amplify DNA regions that can be used to isolate additional nucleic acid molecules that encode, inter alia, variable domains of anti-Gal3 antibodies. In some embodiments, the nucleic acid molecules are oligonucleotides. In some embodiments, the oligonucleotides are derived from the highly variable domains of the heavy and light chains of the antibody of interest. In some embodiments, the oligonucleotides encode all or part of one or more CDRs of an anti-Gal3 antibody or antigen-binding fragment thereof as described herein.

[0121] In another embodiment, the nucleic acid molecules and vectors can be used to generate mutated anti-Gal3 antibodies or antigen-binding fragments thereof. The antibodies can be mutated in the heavy and / or light chain variable domains, for example, to modify the binding properties of the antibodies. For example, mutations can be made in one or more of the CDRs to increase or decrease the KD of the anti-Gal3 antibody or antigen-binding fragment thereof, or to modify the binding specificity of the antibody or antigen-binding fragment thereof. In another embodiment, one or more mutations are made in amino acid residues that are known to be altered compared to germline in the monoclonal antibody of the present invention. The mutations can be made in the CDR or framework regions of the variable domain, or in the constant region. In a preferred embodiment, the mutations are made in the variable domain. In some embodiments, one or more mutations are made in amino acid residues that are known to be altered compared to germline in the CDR or framework regions of the variable domain of the antibody or antigen-binding fragment thereof of the present invention.

[0122] In another embodiment, the framework region(s) are mutated such that the resulting framework region(s) have the amino acid sequence of the corresponding germline gene. Mutations can be made in the framework regions or constant regions to extend the half-life of the anti-Gal3 antibody. See, for example, PCT Publication No. WO00 / 09560. Mutations in the framework regions or constant regions can also be made to alter the immunogenicity of the antibody and / or to provide a site for covalent or non-covalent attachment to another molecule. According to the present invention, a single antibody can have mutations in any one or more of the CDR regions or framework regions of the variable domain or in the constant region.

[0123] In some embodiments, the anti-Gal3 antibody or antigen-binding fragment thereof of the present invention is expressed by inserting DNA encoding the partial or full-length light and heavy chains obtained as described above into an expression vector, whereby the genes are operably linked to necessary expression control sequences, such as transcriptional and translational control sequences. Expression vectors include plasmids, retroviruses, adenoviruses, adeno-associated viruses (AAV), episomes derived from plant viruses, such as cauliflower mosaic virus, tobacco mosaic virus, cosmids, YACs (yeast artificial chromosomes), and Epstein-Barr virus. The antibody coding sequence may be ligated into the vector such that the transcriptional and translational control sequences in the vector perform their intended function of regulating the transcription and translation of the antibody coding sequence. The expression vector and expression control sequences may be selected to be compatible with the expression host cell used. The antibody light chain coding sequence and the antibody heavy chain coding sequence may be inserted into separate vectors and 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 may be operably linked to the same expression control sequence (e.g., a common promoter), to separate identical expression control sequences (e.g., promoters), or to different expression control sequences (e.g., promoters). The antibody coding sequence may be inserted into the expression vector by standard methods (e.g., ligation of complementary restriction enzyme sites on the antibody gene fragment and vector, or blunt end ligation if no restriction enzyme sites are present).

[0124] A convenient vector is one that encodes a functionally complete human CH or CL immunoglobulin sequence, with appropriate restriction enzyme sites engineered to allow easy insertion and expression of any VH or VL sequence, as described above. The genes encoding the HC and LC in such vectors may contain intron sequences, which will enhance the yield of the total antibody protein by stabilizing the associated mRNA. The intron sequences are flanked by splice donor and splice acceptor sites, which determine where RNA splicing will occur. The location of the intron sequence may be either within the variable or constant region of the antibody chain, or both within the variable and constant regions if multiple introns are used. Polyadenylation and transcription termination may occur at native chromosomal sites downstream of the coding region. The recombinant expression vector may also encode a signal peptide that facilitates secretion of the antibody chain from the host cell. The antibody chain gene may be cloned into the vector such that the signal peptide is linked in frame to the amino terminus of the immunoglobulin chain. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (ie, a signal peptide from a non-immunoglobulin protein).

[0125] In addition to the antibody chain genes, the recombinant expression vectors of the invention can carry regulatory sequences that control the expression of the antibody chain genes in a host cell. It will be understood by those skilled in the art that the design of the expression vector, including the selection of the regulatory sequence, can depend on factors such as the choice of the host cell to be transformed, the level of expression of protein desired, etc. Preferred regulatory sequences for expression in mammalian host cells include viral elements that direct high level expression of proteins in mammalian cells, such as retroviral LTRs (long terminal repeats), cytomegalovirus (CMV) (e.g., the CMV promoter / enhancer), simian virus 40 (SV40) (e.g., the SV40 promoter / enhancer), adenovirus (e.g., the adenovirus major late promoter (AdMLP), promoters and / or enhancers derived from polyoma, and strong mammalian promoters such as the native immunoglobulin and actin promoters. For further description of viral regulatory elements, and sequences thereof, see, e.g., U.S. Pat. Nos. 5,168,062, 4,510,245, and 4,968,615. Methods for expressing antibodies in plants, including descriptions of promoters and vectors, as well as plant transformation, are known in the art. See, e.g., U.S. Pat. No. 6,517,529. Methods for expressing polypeptides in bacterial or fungal cells, such as yeast cells, are also well known in the art.

[0126] In addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors of the invention may carry additional sequences, such as sequences that regulate replication of the vector in a host cell (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of a host cell into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665, and 5,179,017). For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin, or methotrexate, on the host cell into which the vector has been introduced. For example, selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr host cells with methotrexate selection / amplification), the neo gene (for G418 selection), and the glutamate synthetase gene.

[0127] The term "expression control sequences" as used herein refers to polynucleotide sequences necessary for causing expression and processing of coding sequences to which they are ligated. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals, such as splicing and polyadenylation signals; sequences that stabilize mRNA in the cytoplasm; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and sequences that enhance protein secretion, if desired. The nature of such control sequences varies depending on the host organism; in prokaryotes, such control sequences generally include promoters, ribosomal binding sites, and transcription termination sequences; in eukaryotes, such control sequences generally include promoters and transcription termination sequences. The term "control sequences" is intended to include, at a minimum, all components whose presence is essential for expression and processing, and may also include additional components whose presence is advantageous, such as leader sequences and fusion partner sequences.

[0128] Host Cells and Methods for Producing Antibodies and Antibody Compositions An additional aspect of the invention relates to antibody compositions of the invention and methods for producing antibodies and antigen-binding fragments thereof. One embodiment of this aspect of the invention relates to a method for producing an antibody as defined herein, comprising the steps of providing a recombinant host cell capable of expressing the antibody, culturing the host cell under conditions suitable for expression of the antibody, and isolating the resulting antibody. An antibody produced by such expression in such a recombinant host cell is referred to herein as a "recombinant antibody". The invention also provides progeny of such host cells, and antibodies produced thereby.

[0129] The term "recombinant host cell" (or simply "host cell") as used herein means a cell into which a recombinant expression vector has been introduced. The present invention provides host cells, which may, for example, comprise a vector as described in the present invention above. The present invention also provides host cells, which may, for example, comprise a nucleotide sequence encoding the heavy chain or an antigen-binding fragment thereof, a nucleotide sequence encoding the light chain or an antigen-binding fragment thereof, or both, of the anti-Gal3 antibody or antigen-binding fragment thereof of the present invention. It should be understood that "recombinant host cell" and "host cell" refer not only to the particular subject cell, but also to the progeny of such a cell. Because certain modifications may occur in successive generations due to mutations or environmental influences, such progeny may not in fact be identical to the parent cell, but are still within the scope of the term "host cell" as used herein.

[0130] The nucleic acid molecules encoding anti-Gal3 antibodies and vectors containing these nucleic acid molecules can be used for transfection of suitable mammalian, plant, bacterial, or yeast host cells. Transformation can be by any known method for introducing polynucleotides into host cells. 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 polynucleotide(s) into liposomes, and direct microinjection of DNA into the nucleus. Additionally, nucleic acid molecules can be introduced into mammalian cells by viral vectors. Methods for transforming cells are well known in the art. See, for example, U.S. Patent Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455. Methods for transforming plant cells are well known in the art and include, for example, Agrobacterium-mediated transformation, biolistic transformation, direct injection, electroporation, and viral transformation. Methods for transforming bacterial and yeast cells are also well known in the art.

[0131] Mammalian cell lines available as hosts for expression are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC). These include, among others, Chinese hamster ovary (CHO) cells, NS0 cells, SP2 cells, HEK-293T cells, 293 freestyle cells (Invitrogen), NIH-3T3 cells, HeLa cells, baby hamster kidney (BHK) cells, African green monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., HepG2), A549 cells, and many other cell lines. Cell lines of particular preference are selected by determining which cell lines have high expression levels. Other cell lines that may be used are insect cell lines, such as Sf9 cells or Sf21 cells. When a recombinant expression vector encoding an antibody gene is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a time sufficient to allow expression of the antibody in the host cell, or more preferably, secretion of the antibody into the culture medium in which the host cell is growing. Antibodies may be recovered from the culture medium using standard protein purification methods. Plant host cells include, for example, Nicotiana, Arabidopsis, duckweed, corn, wheat, potato, etc. Bacterial host cells include E. coli and Streptomyces species. Yeast host cells include Schizosaccharomyces pombe, Saccharomyces cerevisiae, and Pichia pastoris.

[0132] Furthermore, expression of the antibodies of the invention or antigen-binding fragments thereof from production cell lines can be enhanced using a number of known techniques. For example, the glutamine synthetase gene expression system (GS system) is a common approach for enhancing expression under certain conditions. The GS system is discussed in whole or in part in connection with European Patent Nos. 0216846, 0256055, 0323997, and 0338841.

[0133] Antibodies expressed by different cell lines or in transgenic animals will likely have different glycosylation patterns from each other. However, all antibodies encoded by the nucleic acid molecules provided herein or comprising the amino acid sequences provided herein are part of the invention, regardless of the glycosylation state of the antibody, and more generally, regardless of the presence or absence of post-translational modification(s).

[0134] Pharmaceutical Compositions Another aspect of the present invention is a pharmaceutical composition comprising as an active ingredient (or as the only active ingredient) an anti-Gal3 antibody or antigen-binding fragment thereof, bispecific binding molecule, or antibody composition of the present invention. The pharmaceutical composition may comprise any anti-Gal3 antibody or antigen-binding fragment thereof, bispecific binding molecule, or antibody composition as described herein. In some embodiments, the pharmaceutical composition is intended for the amelioration, prevention, and / or treatment of a Gal3-associated disorder or disease. As used herein, a Gal3-associated disorder or disease refers to a condition whose progression is improved or slowed by modulating Gal3 expression, interaction, and / or activity. In certain embodiments, the composition is intended for the amelioration, prevention, and / or treatment of fibrotic diseases, inflammatory diseases, autoimmune diseases, immune-mediated disorders, neurodegenerative diseases, metabolic diseases, infectious diseases, and some cancers.

[0135] Generally, the antibodies, antigen-binding fragments, and bispecific binding molecules of the invention are suitable for administration as a formulation in association with one or more pharma- ceutically acceptable excipient(s), e.g., as described below.

[0136] A pharmaceutical composition of the invention will comprise one or more anti-Gal3 antibodies or antigen-binding fragments thereof, or bispecific binding molecules of the invention, such as one or two anti-Gal3 antibodies or antigen-binding fragments thereof, or bispecific binding molecules. In one embodiment, the composition comprises one anti-Gal3 antibody or antigen-binding fragment thereof of the invention. In one embodiment, the composition comprises more than one anti-Gal3 antibody or antigen-binding fragment thereof of the invention.

[0137] In another embodiment, the pharmaceutical composition may comprise at least one anti-Gal3 antibody or antigen-binding fragment thereof, e.g., one anti-Gal3 antibody or antigen-binding fragment thereof, as well as one or more additional antibodies that target one or more associated cell surface receptors, e.g., one or more cancer-associated receptors.

[0138] The term "excipient" is used herein to describe any ingredient other than the compound(s) of the invention. The choice of excipient(s) will depend largely on factors such as the specific mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form. As used herein, "pharmaceutically acceptable excipient" includes any and all solvents, dispersion media, coating agents, antibacterial and antifungal agents, isotonicity agents, and absorption delaying agents, which are physiologically compatible. Some examples of pharmaceutically acceptable excipients are water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, as well as combinations thereof. In many cases, it will be preferable to include an isotonicity agent, such as a sugar, a polyalcohol, such as mannitol, sorbitol, or sodium chloride, in the composition. Additional examples of pharmaceutically acceptable substances are wetting agents or small amounts of auxiliary substances, such as wetting agents or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the antibody.

[0139] The pharmaceutical compositions of the present invention and methods for their preparation will be readily apparent to those skilled in the art. Such compositions and methods for their preparation can be found, for example, in Remington's Pharmaceutical Sciences, 19th Edition (Mack Publishing, 1995). The pharmaceutical compositions are preferably manufactured under GMP (Good Manufacturing Practice) conditions.

[0140] The pharmaceutical composition of the present invention can be prepared, packaged, or sold in bulk, 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 that contains a predetermined amount of active ingredient.The amount of active ingredient is generally equivalent to the dose of active ingredient that would be administered to a subject, or a convenient fraction of such a dose, such as half or one third of such a dose.

[0141] Any method accepted in the art for administering peptides, proteins, or antibodies may be suitably used for the antibodies and antigen-binding fragments of the present invention.

[0142] The pharmaceutical composition of the present invention is typically suitable for parenteral administration. As used herein, "parenteral administration" of a pharmaceutical composition includes any administration route characterized by physically creating a break in the tissue of a subject and administering the pharmaceutical composition through the break in the tissue, and thus generally administers directly into the bloodstream, intramuscularly, or into an internal organ. Thus, parenteral administration includes, but is not limited to, administering the pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a non-surgical wound that penetrates the tissue, and the like. In particular, parenteral administration is considered to include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrathoracic, intravenous, intraarterial, intrathecal, intraventricular, intraurethral, ​​intracranial, intratumoral, and intrasynovial injection or infusion; and kidney dialysis fluid replacement techniques. Localized perfusion is also considered. Particular embodiments include intravenous and subcutaneous routes.

[0143] A formulation of a pharmaceutical composition suitable for parenteral administration typically includes the active ingredient in combination with a pharma- ceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for administration in a bolus or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, for example, in ampoules, or in multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and the like. Such formulations may further include one or more additional components, including, but not limited to, suspending agents, stabilizing agents, or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in a dry form (i.e., powder or granules) for reconstitution with a suitable vehicle (e.g., sterile, pyrogen-free water) prior to parenteral administration of the reconstituted composition. Parenteral formulations also include aqueous solutions, which may contain excipients such as salts, carbohydrates, and buffers (preferably pH 3-9), although in some applications they may be more suitably formulated as sterile non-aqueous solutions or as dry forms for use in conjunction with a suitable vehicle such as sterile pyrogen-free water. Exemplary parenteral dosage forms include solutions or suspensions in sterile aqueous solutions, for example, aqueous propylene glycol or dextrose solutions. Such dosage forms may be suitably buffered, if desired. Other parenterally administrable formulations that are useful include those that contain the active ingredient in microcrystalline form or in a liposomal formulation. Formulations for parenteral administration may be formulated to be immediate and / or modified release. Modified release formulations include delayed, sustained, pulsed, controlled, targeted, and programmed release.

[0144] For example, in one embodiment, a sterile injectable solution can be prepared by incorporating an anti-Gal3 antibody, its antigen-binding fragment, bispecific binding molecule, or antibody composition in the required amount in a suitable solvent with one or a combination of ingredients listed above, if necessary, followed by sterile filtration. In general, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other ingredients as required from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred preparation methods are vacuum drying and freeze-drying, which produce a powder of the active ingredient and any additional desired ingredients from a previously sterile-filtered solution thereof. The proper fluidity of the solution can be maintained, for example, by the use of a coating agent such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Prolonged absorption of the injectable composition can be achieved by including a substance that delays absorption, such as monostearate salts and gelatin, in the composition, and / or by using a modified release coating (e.g., a slow release coating).

[0145] The antibodies of the invention may also be administered intranasally or by inhalation, typically in the form of a dry powder from a dry powder inhaler (either alone, in a mixture, or as particles of the components mixed, e.g., with suitable pharma- ceutical excipients), as an aerosol spray from pressurized containers, pumps, sprays, atomizers (preferably those using electrohydrodynamics to produce a fine mist) or nebulizers, with or without the use of a suitable propellant, or as intranasal drops.

[0146] The pressurized container, pump, spray, atomizer, or nebulizer will generally contain a solution or suspension of the antibody of the invention, which may include, for example, a material suitable for dispersing, solubilizing, or extending the release of the active propellant(s) as a solvent.

[0147] Prior to use in a dry powder or suspension formulation, the drug is generally micronized to a size suitable for delivery by inhalation (typically less than 5 μm). This can be achieved by any suitable comminuting method, such as spiral jet milling, fluid bed jet milling, supercritical fluid processing to form nanoparticles, high pressure homogenization, or spray drying.

[0148] 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.

[0149] A solution formulation suitable for generating a fine mist using electrohydrodynamics in an atomizer may contain a suitable dose of an antibody of the invention per actuation, and actuation volumes may vary, for example, from 1 μL to 100 μL.

[0150] Formulations for inhaled / intranasal administration may be formulated to be immediate and / or modified release, including delayed-, sustained-, pulsed-, controlled-, targeted-, and programmed-release.

[0151] In the case of dry powder inhalers and aerosols, the dosage unit is determined by means of a valve, which delivers a metered amount. Units according to the invention are typically adapted to administer a metered dose or "puff" of the antibody of the invention. The total daily dose will typically be administered in a single dose or, more usually, in divided doses throughout the day.

[0152] The antibodies and antibody fragments of the invention may also be formulated for the oral route of administration, which may involve swallowing, so that the compound enters the digestive tract, and / or buccal, lingual, or sublingual administration, whereby the compound enters the bloodstream directly from the mouth.

[0153] Formulations suitable for oral administration include solid, semi-solid, and liquid systems, such as tablets; soft or hard capsules, liquids, or powders containing multiparticulates or nanoparticles; lozenges (including those filled with liquid); chewables; gels; rapidly disintegrating dosage forms; films; vaginal suppositories; sprays; and buccal / mucoadhesive patches.

[0154] Liquid formulations include suspensions, solutions, syrups, and elixirs. Such formulations can be used as fillers in soft or hard capsules (e.g., made from gelatin or hydroxypropylmethylcellulose), typically containing a carrier such as water, ethanol, polyethylene glycol, propylene glycol, methylcellulose, or a suitable oil, and one or more emulsifiers and / or suspending agents. Liquid formulations can also be prepared by the reconstitution of a solid, for example, from a sachet.

[0155] Therapeutic Uses of the Antibodies and Compositions of the Invention In one embodiment, the anti-Gal3 antibodies and antigen-binding fragments thereof, anti-Gal3 antibody compositions, and bispecific binding molecules of the invention are used to inhibit Gal3 expression, interaction, and / or activity in a human in need thereof. The anti-Gal3 antibodies or antigen-binding fragments thereof of the invention may be administered alone or in combination (sequentially or simultaneously) with other therapeutic agents.

[0156] In certain embodiments, the antibodies or antigen-binding fragments thereof, compositions, or bispecific binding molecules are for use in the amelioration, prevention and / or treatment of many fibrotic diseases, inflammatory diseases, autoimmune diseases, immune-mediated disorders, neurodegenerative diseases, metabolic diseases, infectious diseases, and some cancers.

[0157] The term "fibrosis" refers to a medical condition in which tissues or organs harden or scar as a result of dysregulated production of extracellular matrix, such as collagen proteins. Fibrosis is associated with chronic inflammation, in which immune cells, such as macrophages, signal fibroblasts to respond by expressing extracellular matrix proteins. This signaling is accomplished through pathways such as the IL-6 pathway, the IL-5 pathway, and other pro-fibrotic pathways as well. Fibrosis includes, but is not limited to, hepatic fibrosis, bridging fibrosis, liver cirrhosis, renal fibrosis, pulmonary fibrosis, asbestosis, silicosis, coal worker's pneumoconiosis, and diffuse dust fibrosis, chemotherapy-induced pulmonary fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, polycystic kidney disease, cardiovascular fibrosis, Chagas' disease, arterial fibrosis, venous thrombosis, cardiac fibrosis, pulmonary arterial fibrosis, arthritic fibrosis, Dupuytren's contracture, keloids, 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.

[0158] 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 invention include cancers, e.g., carcinomas, sarcomas, leukemias, and lymphomas, e.g., T-cell lymphomas; metastatic cancers, which may further include malignant melanoma (e.g., advanced or metastatic malignant melanoma), non-small cell lung cancer, head and neck squamous cell carcinoma, renal cell carcinoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, glioblastoma, glioma, squamous cell lung cancer, small cell lung cancer, hepatocellular carcinoma, bladder cancer, upper urinary tract cancer, esophageal cancer, esophagogastric junction cancer, gastric cancer, liver cancer, colon cancer, colorectal cancer, multiple myeloma, sarcoma, acute myeloid leukemia, chronic myelogenous leukemia, myelodysplastic syndrome, nasopharyngeal carcinoma, chronic lymphocytic leukemia, acute The cancer may include lymphoblastic leukemia, small lymphocytic lymphoma, ovarian cancer, gastrointestinal cancer, primary peritoneal cancer, fallopian tube cancer, urothelial cancer, HTLV (human T-cell leukemia virus)-associated T-cell leukemia / lymphoma, prostate cancer, genitourinary cancer, meningioma, adrenal cortical carcinoma, gliosarcoma, fibrosarcoma, kidney cancer, breast cancer, pancreatic cancer, endometrial cancer, basal cell carcinoma of the skin, appendix cancer, bile duct cancer, salivary gland cancer, advanced Merkel cell carcinoma, diffuse large B-cell lymphoma, follicular lymphoma, intermediate splenomegaly, or solid tumors. The cancer may be, for example, early stage, intermediate stage, late stage, or metastatic stage.

[0159] The terms "cancer", "tumor" and "cellular cancer" are used synonymously herein and refer to cells that exhibit relatively autonomous growth and thus exhibit an abnormal growth phenotype characterized by a significant loss of control of cell growth. Generally, cells of interest for detection or treatment in this application include precancerous (e.g., benign), malignant, premetastatic, metastatic, and non-metastatic cells. Detection of cancerous cells is of particular interest. The term "normal" as used in the context of "normal cells" is meant to refer to cells that have an unaltered phenotype or exhibit unaltered cell morphology of the tissue type being examined. "Cancerous phenotype" generally refers to any of a variety of biological phenomena characteristic of cancerous cells, which may vary depending on the type of cancer. Cancerous phenotypes are generally identified by abnormalities, such as abnormalities in cell growth or proliferation (e.g., uncontrolled growth or proliferation), cell cycle regulation, cell migration, cell-cell interactions, or metastasis.

[0160] In one embodiment, the anti-Gal3 antibodies and antigen-binding fragments thereof, antibody compositions, or bispecific binding molecules of the invention may be used to prevent or treat an inflammatory disease or an autoimmune disease or disorder or an immune-mediated disease or disorder, such as psoriasis, rheumatoid arthritis, juvenile idiopathic arthritis, osteoarthritis, ulcerative colitis, ankylosing spondylitis, autoimmune uveitis, primary biliary cholangitis, autoimmune and inflammatory nephropathy, sepsis, type 1 diabetes, inflammatory myopathies, myocarditis, pericarditis, allergies, asthma, gastric ulcers, gastritis, systemic lupus erythematosus, Sjogren's syndrome, inflammatory bowel disease (IBD), Crohn's disease, atopic dermatitis, systemic sclerosis, or scleroderma.

[0161] In one embodiment, 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 metabolic disorders, heart disease, heart failure, myocardial infarction, pathological angiogenesis, atherosclerosis, hypertension, pulmonary hypertension, venous embolism, 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, and liver disorders such as non-alcoholic fatty liver disease.

[0162] In one aspect, the anti-Gal3 antibodies and antigen-binding fragments thereof, antibody compositions, or bispecific binding molecules of the present invention may 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 and binding fragments thereof disclosed herein disrupt the interaction between Gal3 and a protein associated with a neurodegenerative disease. In some embodiments, the protein associated with a neurodegenerative disease causes disease due to misfolding or aggregation of the protein in a subject. These diseases may include the death of neurons or other cell types associated with the nervous system. Non-limiting examples of neurological 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, neurogenesis, Tourette's syndrome, neuroinfections, meningitis, encephalitis, bovine spongiform encephalopathy, West Nile virus encephalitis, neurological complications of AIDS, Fragile X syndrome, Guillain-Barre syndrome, brain metastases, or brain cancer, or others known to those of skill in the art.

[0163] 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 coronaviruses, papillomaviruses, influenza viruses, encephalomyocarditis viruses, and Trypanosoma cruzi.

[0164] "Treat," "treating," and "treatment" refer to a method of reducing or eliminating at least one of a biological disease or disorder and / or its attendant symptoms. As used herein, "relieving" a disease, disorder, or condition means reducing the severity and / or frequency of occurrence of the symptoms of the disease, disorder, or condition. Furthermore, references herein to "treatment" include references to curative treatment, palliative treatment, and prophylactic treatment.

[0165] "Therapeutically effective amount" refers to that amount of the therapeutic agent being 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 may result, for example, in tumor regression, extended survival time, elimination of cancer cells, diminished disease progression, reversal of metastasis, or other clinical endpoint desired by the medical practitioner.

[0166] Also described herein, in some embodiments, are methods of monitoring the progression of tissue fibrosis by monitoring one or more fibrosis biomarkers, including or consisting of smooth muscle actin, fibronectin, collagen, elastin, laminin, hyaluronic acid, or proteoglycans, or any combination thereof.

[0167] In some embodiments, the anti-Gal3 antibody or antigen-binding fragment thereof results in a reduction in the accumulation of extracellular matrix proteins in tissues. In some embodiments, the anti-Gal3 antibody or antigen-binding fragment thereof is for use in the amelioration, prevention, and / or treatment of a disease, wherein the disease is a fibrotic disease or fibrosis. In some embodiments, the anti-Gal3 antibody or antigen-binding fragment thereof for use in the amelioration, prevention, and / or treatment of a disease results in a reduction in the accumulation of extracellular matrix proteins in tissues. In some embodiments, the extracellular matrix comprises or consists of agrin, nidogen, cadherin, clathrin, collagen, defensin, elastin, entactin, fibrillin, fibronectin, keratin, laminin, microtubule-actin crosslinking factor 1, SPARC-like protein, nesprin, fibrous sheath interacting protein, myomesin, nebulin, plakophilin, integrin, talin, exportin, transportin, tenascin, perlecan, sortilin-related receptor, tensin, or titin, or any combination thereof. 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 profibrogenic cytokine is TGF (transforming growth factor)-β, IL-1β, TNF-α, IL-5, or IL-6. In some embodiments, the tissue has elevated profibrogenic cytokine expression.

[0168] The anti-Gal3 antibodies or antigen-binding fragments thereof, antibody compositions, or bispecific binding molecules of the invention can be administered alone or in combination with one or more other drugs or antibodies (or in any combination thereof). Thus, the pharmaceutical compositions, methods, and uses of the invention also encompass embodiments of combination (co-administration) with other active agents, as detailed below.

[0169] As used herein, the terms “co-administration,” “co-administered,” and “in combination with,” referring to anti-Gal3 antibodies and antigen-binding fragments thereof, antibody compositions, and bispecific binding molecules of the invention together with one or more other therapeutic agents, are intended to mean, refer to, and include the following: a) simultaneous administration to a patient in need of treatment of such combinations of an antibody / antigen-binding fragment / antibody composition / bispecific binding molecule of the invention and therapeutic agent(s), when such components are formulated together into a single dosage form, which releases the components substantially simultaneously to the patient; b) substantially simultaneous administration to a patient in need of treatment of such combinations of antibodies / antigen-binding fragments / antibody compositions / bispecific binding molecules of the invention and therapeutic agent(s), where such components are formulated separately from one another into separate dosage forms which are ingested by the patient at substantially the same time, at which time the components are released to the patient at substantially the same time; c) sequential administration to a patient in need of treatment of such combinations of antibodies / antigen-binding fragments / antibody compositions / bispecific binding molecules of the invention and therapeutic agent(s), where such components are formulated separately from one another into separate dosage forms which are ingested in successive doses by the patient with significant dosage intervals between them, wherein the components are released to the patient at substantially different times; d) Sequential administration to said patient of such combinations of antibodies / antigen-binding fragments / antibody compositions / bispecific binding molecules of the invention and therapeutic agent(s), where such components are formulated together into a single dosage form which releases the components in a controlled manner, where they are released simultaneously, sequentially, and / or overlappingly at the same and / or different times to said patient, where each portion may be administered either by the same route or by different routes.

[0170] 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 a sole 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 additional further therapeutic treatment (combination therapy). The additional therapeutic treatment may further include administration of other additional therapeutic agents to the patient, such as immune checkpoint modulators, chemotherapeutic agents, anti-neoplastic agents, anti-angiogenic agents, hormonal therapeutic agents, stem cell-based therapeutic agents, surgery, and / or radiation therapy. The additional therapeutic treatment may further include administration of other additional therapeutic agents to the patient, such as PD1 / PDL1 blockade therapy and / or CTLA blockade therapy. In some embodiments, the PD1 / PDL1 blockade therapy comprises administration of 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, to the patient. In some embodiments, the CTLA4 blockade therapy comprises administration of a therapeutic agent, such as ipilimumab and / or tremelimumab, to the patient.

[0171] It is also contemplated that the anti-Gal3 antibodies or antigen-binding fragments thereof, antibody compositions, or bispecific binding molecules of the invention may be used in adjunctive therapy with yet other additional therapeutic agents that are tyrosine kinase inhibitors. These are synthetic, primarily quinazoline-derived, low molecular weight molecules that inhibit ligand-induced receptor phosphorylation by interacting with the intracellular tyrosine kinase domain of the receptor and competing for the intracellular Mg-ATP binding site.

[0172] In certain embodiments, the antibodies and antigen-binding fragments, antibody compositions, or bispecific binding molecules of the invention may be administered in combination with yet another additional therapeutic agent that is another Gal3 pathway inhibitor that may target Gal3 or one or more of its ligands. Examples of such inhibitors include other anti-Gal3 antibodies or Gal3-targeting small molecules.

[0173] It is understood that the antibodies and antigen-binding fragments thereof, antibody compositions, and bispecific binding molecules of the invention can be used in methods of amelioration, prevention, and / or treatment as described herein, can be for use in amelioration, prevention, and / or treatment as described herein, and / or can be for use in the manufacture of a medicament for amelioration, prevention, and / or treatment as described herein. The invention also provides kits and articles of manufacture comprising the antibodies and antigen-binding fragments thereof, antibody compositions, and bispecific binding molecules described herein.

[0174] Dose and Route of Administration The antibodies or antigen-binding fragments thereof, antibody compositions, or bispecific binding molecules of the invention will be administered in an amount effective to ameliorate, prevent, and / or treat the condition in question, i.e., at dosages and for periods of time necessary to achieve the desired result. The therapeutically effective amount may vary depending on factors such as the particular condition being treated, the age, sex, and weight of the patient, and whether the antibody is administered as the sole treatment or in combination with one or more additional anti-cancer treatments.

[0175] The dosage regimen may be adjusted to provide the optimum desired response. For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be programmatically reduced or increased as indicated by the exigencies of the therapeutic situation. For ease of administration and uniformity of dose, it is particularly advantageous to formulate parenteral compositions in dosage unit form. As used herein, dosage unit form refers to a physically discrete unit suitable as a unitary dose for the patient / subject to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect, together with the required pharmaceutical carrier. The specifications of the dosage unit form of the present invention are generally dictated by and directly dependent on (a) the unique characteristics of the chemotherapeutic agent and the specific therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the technology of compounding such active compound for the treatment of hypersensitivity in an individual.

[0176] Thus, one skilled in the art will understand, based on the disclosure provided herein, that the dose and dosage regimen will be adjusted according to methods well known in the therapeutic arts. That is, the maximum tolerated dose can be readily established, and the effective amount that provides a detectable therapeutic benefit to the patient can be determined, as can the time requirements for administering each agent to provide a detectable therapeutic benefit to the patient. Thus, although specific doses and dosage regimens are exemplified herein, these examples in no way limit the doses and dosage regimens that can be provided to a patient in practicing the present invention.

[0177] It should be noted that the dosage values ​​may vary with the type and severity of the condition to be alleviated, and may include single or multiple doses. For any particular subject, the specific dosage regimen should be adjusted over time according to the individual needs and the professional judgment of the person administering or supervising the administration of the composition, and it should be further understood that the dosage ranges set forth herein are merely exemplary and are not intended to limit the scope or practice of the embodied compositions. Furthermore, dosage regimens using the compositions of the present invention may be based on a variety of factors, including the type of disease, the patient's age, weight, sex, medical condition, the severity of the condition, the route of administration, and the specific antibody used. Thus, dosage regimens may vary widely, but can be routinely determined using standard methods. For example, the dose may be adjusted based on pharmacokinetic or pharmacodynamic parameters, which may include clinical effects such as toxic effects and / or clinical laboratory values. Thus, the present invention encompasses intrapatient dose escalation, as determined by those skilled in the art. Determination of appropriate dosages and regimens is well known in the relevant art and will be understood to be within the scope of those of ordinary skill in the art once provided with the teachings disclosed herein.

[0178] It is contemplated that a suitable dose of the antibody, antigen-binding fragment, antibody composition, or bispecific binding molecule of the invention would be 0.1-100 mg / kg, such as about 0.5-50 mg / kg, such as about 1-20 mg / kg. The antibody, antigen-binding fragment, antibody composition, or bispecific binding molecule may be administered, for example, at a dose of at least 0.25 mg / kg, such as at least 0.5 mg / kg, such as at least 1 mg / kg, such as at least 1.5 mg / kg, such as at least 2 mg / kg, such as at least 3 mg / kg, such as at least 4 mg / kg, such as at least 5 mg / kg; and for example at a dose of up to 50 mg / kg, such as at a maximum of 30 mg / kg, such as at a maximum of 20 mg / kg, such as at a maximum of 15 mg / kg. Administration is usually repeated at appropriate intervals, such as once per week, once per two weeks, once per three weeks, or once per four weeks, as long as deemed appropriate by the attending physician, who may optionally increase or decrease the dose if necessary.

[0179] An effective amount for tumor therapy may be measured by its ability to reverse disease progression, by stabilizing disease progression and / or ameliorating symptoms in a patient, e.g., by causing regression of tumor size. The ability of an antibody, antigen-binding fragment, antibody composition, or bispecific binding molecule of the invention to inhibit cancer may be evaluated by in vitro assays, e.g., as described in the Examples, as well as in appropriate animal models predictive of efficacy in human tumors. An appropriate dosage regimen will be selected to provide the optimal therapeutic response in each particular situation, and will be administered, e.g., as a single bolus or as a continuous infusion, with the possibility of adjusting the dose as dictated by the exigencies of each case.

[0180] Diagnostic Uses and Compositions The antibodies of the present invention are also useful in diagnostic processes (e.g., in vitro, ex vivo). For example, the antibodies can be used to detect and / or measure the level of Gal3 in a sample from a patient (e.g., a tissue sample, or a body fluid sample, such as inflammatory exudate, blood, serum, intestinal fluid, saliva, or urine). Suitable detection and measurement methods include immunological methods, such as flow cytometry, enzyme-linked immunosorbent assay (ELISA), chemiluminescence assay, radioimmunoassay, and immunohistochemistry. The present invention further encompasses kits (e.g., diagnostic kits) comprising the antibodies described herein.

[0181] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those skilled in the art. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. In case of conflict, the present specification (including definitions) will control.

[0182] Generally, the nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, medicinal and pharmaceutical chemistry, and protein and nucleic acid chemistry, and hybridization described herein are those well known and commonly used in the art. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein.

[0183] Further, unless otherwise required by context, singular terms include plurals and plural terms include the singular. Throughout this specification and the embodiments, the terms "have" and "comprise", or variations such as "has", "having", "comprises" or "comprises" 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.

[0184] In order that this invention may be better understood, the following examples are set forth. These examples are merely illustrative and should not be construed as limiting the scope of the invention in any way.

[0185] Working Example Example 1: Generation of a novel anti-Gal3scFv A novel galectin-3 inhibitor monoclonal antibody was obtained using the Mabqi human phage display platform.

[0186] Several constructs were generated for the 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 were previously controlled and did not show any protein degradation.

[0187] Recombinant proteins were immobilized on Nunc xisoap plates via the use of their histidine tag: streptavidin was coated at 10 μg / mL in TBS, 100 μL / well and incubated for 1 h at room temperature. Afterwards, the plates were blocked with 200 μL of TBS-BSA 4% for 2 h. A solution containing 5 mM NiSO4 and 1 μg / mL Tris-NTA-biotin in TBS was incubated for 30 min at room temperature. The plates were washed three times with TBST 0.1% and 100 μL of NiSO4 / Tris-NTA-biotin solution was added to the wells for 1 h at room temperature. The plates were washed again and recombinant proteins were added at 20 μg / mL in TBS, 100 μL / well and incubated overnight at 4°C.

[0188] Plates were washed three times with TBST 0.1% and the phage library was removed onto wells coated with streptavidin and NiSO4 / Tris-NTA-biotin only: 50 μL of library was incubated with 50 μL of TBS-BSA (bovine serum albumin) 4% for 1 h at room temperature.

[0189] The depleted library was incubated on the antigen-coated wells for 2 hours at room temperature. The wells were washed 10 times with TBST 0.1% and 3 times with TBS to remove non-specific phages.

[0190] Bound phages were eluted by adding 100 μL of trypsin-EDTA (Gibco, 25300-054) and incubated for 15 min at room temperature.

[0191] 200 μL of TG1 (absorbance = 0.3-0.5) was infected with 100 μL of eluted phages for 1 h at 37°C.

[0192] The infected TG1 was centrifuged at 4000 rpm for 10 min, the pellet was resuspended in 2× TY, ampicillin, and 2% glucose, and the culture was incubated overnight at 30°C and 200 rpm.

[0193] 20 μL of infected TG1 was added to 2 mL of 2×TY, ampicillin, glucose 2% and grown at 200 rpm at 37° C. When the optical density reached 0.5, TG1 was infected with helper phage for 1 h at 37° C., 200 rpm.

[0194] The TG1 culture was centrifuged at 4000 rpm for 10 min, the pellet was resuspended in 1 mL of 2xTY, ampicillin, kanamycin, and incubated overnight at 30°C, 200 rpm.

[0195] The culture was centrifuged at 4000 rpm for 15 min at 4° C., and the phages were precipitated using PEG-NaCl for the supernatant. These phages were used for the next round of panning.

[0196] Several rounds of panning allowed successful enrichment of multiple antigen-binding substances.

[0197] The monoclonal scFv were then sequenced and selected for unique clones.

[0198] Monoclonal scFvs were then produced in E. coli. HB2151 bacteria containing the pHEN phagemid expressing the scFvs were grown overnight at 37° C. and 220 rpm in 150 μL of 2TY-2% glucose-ampicillin in 96-well plates. This starter was used to inoculate more cultures in deep-well plates containing 2TY-IPTG 1 mM-ampicillin for induction of scFv expression. The induction culture was grown overnight at 30° C. and 220 rpm. The culture was centrifuged and polymyxin B sulfate 1 mg / mL in Tris 20 mM pH 8 was used for extraction of the periplasm of the scFvs from the pellet.

[0199] A summary of the SEQ ID NOs for the CDR domains, VH domains and VL domains is provided in Tables 1 and 2 below, and the DNA sequences corresponding to the VH and VL sequences are provided in Table 2.

[0200] [Table 1]

[0201] [Table 2]

[0202] Example 2: Human / mouse cross-reactive anti-Gal3scFv that does not bind to Gal1 or Gal7 The binding cross-reactivity and specificity of the scFvs against human Gal3 full-length and CRD domain (Hu-Gal3-FL and Hu-Gal3-CRD, respectively), against 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 (human-Gal7 as shown in SEQ ID NO: 109 and available under UniProt accession number P47929-LEG7_HUMAN), and against mouse full-length Gal3 (m-Gal3-FL) were tested by ELISA (Figure 1).

[0203] Hu-Gal3-FL, Hu-Gal3-CRD, m-Gal3-FL, Hu-Gal1 and Hu-Gal7 were immobilized at a final concentration of 3 μg / mL through their histidine tags on functionalized microplates after internalization. Periplasmic extracts containing scFvs were added to the wells and detected with an anti-scFv tag HRP (horseradish peroxidase) antibody. Rabbit antibodies directed against Gal3 (ab53082-Abcam reference), Gal1 (ab25138-Abcam reference) and Gal7 (ab108623-Abcam reference) were used at 5 μg / mL as positive controls to coat the antigen and detected with an anti-rabbit HRP secondary antibody (7074S-Cell Signaling reference).

[0204] All scFvs of the present invention show binding to both human and mouse Gal3 proteins, but no binding to human Gal1 or Gal7.

[0205] Example 3: Competitive inhibition assay of Gal3 binding to asialofetuin Asialofetuin (ASF) glycoprotein is often studied as a model ligand for galectins. In this assay, binding of ASF to immobilized human Gal3 (Hu-Gal3) is measured in the presence or absence of scFv (controls with secondary antibody only and with irrelevant scFv). Human galectin-3 (Hu-Gal3-FL) was immobilized at 3 μg / mL through its histidine tag on functionalized microplates as before. ASF (A1908-Sigma reference) was added at 30 μg / mL in each well with or without periplasmic extract containing scFv. Binding of ASF to Hu-Gal3 was detected with sheep anti-ASF antibody (secondary antibody, ab35184-Abcam reference) and donkey anti-sheep HRP antibody (tertiary antibody, ab6900-Abcam reference).

[0206] All scFvs of the invention, except for the irrelevant scFv, demonstrated inhibition of the ASF-Gal3 interaction (Figure 2).

[0207] Example 4: Generation of novel anti-Gal3 antibodies and design of anti-Gal3 antibodies with reduced immunogenicity Anti-Gal3 antibodies were then produced in IgG1 format using transient transfection in the CHO-K1 cell line, and the supernatant was collected by centrifugation, filtered, and subsequently purified using MabSelect SuRe™ (Cytiva).

[0208] Subsequently, anti-Gal3 antibodies with reduced immunogenicity were designed to obtain antibody molecules that have minimal immunogenicity when administered to humans while substantially retaining the specificity and affinity of the parent antibody.

[0209] The VH and VL regions of the antibody were BLASTed against a human IgG database to find the closest human germline sequence. Furthermore, the sequence was run through the EpiAnalyzer tool, which highlighted immunogenic regions. No immunogenicity was observed beyond tolerable levels.

[0210] After alignment of the VH or VL sequences with the germline sequences, a mutant antibody "-G" is proposed to have framework mutations corrected to germline, while two mutant antibodies -G1 and -G2 are proposed to be in borderline cases where the mutations are close to the CDR regions, where the G1 form has framework germline mutations that are not close to the CDR regions, and G2 contains framework germline mutations close to the CDR regions. The sequence numbers of the mutant antibodies VH and VL are provided in Table 3.

[0211] [Table 3]

[0212] Example 5: Human / mouse cross-reactive anti-Gal3 antibodies The human / mouse cross-reactivity of anti-Gal3 antibodies against human Gal3 full-length (Hu-Gal3-FL) and CRD domain (Hu-Gal3-CRD), and mouse Gal3 (m-Gal3-FL) was tested by ELISA.

[0213] Hu-Gal3-FL, Hu-Gal3-CRD and m-Gal3-FL were immobilized via their histidine tags on functionalized microplates at 3 μg / mL. Purified IgG was added to the wells (final concentration 15-20 μg / mL) and detected with an anti-Fab HRP antibody. A control rabbit antibody directed against Hu-Gal3 (reference ab53082 from Abcam) was used at 5 μg / mL as a positive control to coat the antigen and detected with an anti-rabbit HRP secondary antibody (reference 7074S from Cell Signaling).

[0214] The results are presented in Figure 3. The antibodies of the present invention show binding to both human and mouse Gal3 proteins. However, lower binding signals to human and mouse Gal3 were observed for the B12 antibody. Also, the signal of the E12 antibody was much lower to m-Gal3 than to Hu-Gal3-FL and Hu-Gal3-CRD.

[0215] Example 6: Antibody that specifically binds to Gal3 but not to Gal1 or Gal7 The specificity of the IgG against Gal3 was tested by ELISA to confirm the absence of binding to human Gal1 and Gal7.

[0216] Hu-Gal1 and Hu-Gal7 were immobilized at 3 μg / mL. Purified IgG was added to the wells (final concentration 15-20 μg / mL) and detected with an anti-Fab.HRP antibody. Rabbit antibodies directed against Gal1 (reference Abcam ab25138) and Gal7 (reference Abcam ab108623) were used at 5 μg / mL as positive controls to coat the antigen and detected with an anti-rabbit HRP secondary antibody (7074S - reference Cell Signaling).

[0217] The results of the ELISA are presented in Figure 4. All anti-Gal3 antibodies of the present invention specifically bind to Gal3 and do not bind to human Gal1 and Gal7.

[0218] Example 7: Affinity of antibodies to Gal3 from several species The interaction of recombinant full-length human Gal3 (Hu-Gal3-FL) and mouse Gal3 (m-Gal3-FL) (Novarix) with anti-Gal3 antibodies was monitored by surface plasmon resonance (SPR) detection using a Biacore T-200 instrument.

[0219] Binding studies were performed in freshly prepared, filtered, and degassed running buffer (HBS-P, Cytiva Life Sciences) containing 10 mM HEPES, 150 mM NaCl, and 0.05% P20 at 25° C. Before performing binding studies, the CM5 sensor surface immobilized with anti-human Fc monoclonal antibody was equilibrated with HBS-P by priming the instrument at least three to four times.

[0220] The CM5 sensor surface was first immobilized with anti-human Fc monoclonal antibody using standard protocols. All sensor surfaces were first activated by injecting a 1:1 (v / v) mixture of 400 mM EDC and 100 mM NHS at a flow rate of 10 μL / min for 7 min. Monoclonal anti-human Fc antibody (Millipore AP113) at 25 μg / mL in sodium acetate pH 5 was flowed at 10 μL / min for 360 s to reach 10000 RU on both flow cells, followed by injection of 1 M ethanolamine (pH 8.5) at a flow rate of 10 μL / min for 7 min. HBS-P was used as the running buffer during the entire immobilization procedure.

[0221] Antibody samples were prepared at 5 μg / ml and injected at 10 μL / min for 30 seconds. Typically, 400 to 680 resonance units of antibody were captured on the chip.

[0222] Detailed Kd values ​​for each antibody tested are shown in Table 4 below.

[0223] [Table 4]

[0224] Against human Gal3, the native antibodies of the present invention show Kd values ​​of 4 nM-15 nM, and the mutant antibodies show values ​​of 4 nM-18 nM. Against mouse Gal3, only the native antibodies have been tested, and they show lower Kd values ​​of 1 nM-8 nM. There is no significant difference in Kd values ​​between the human and mouse antigens.

[0225] The Kd values ​​of the mutant antibodies are also similar compared to the native antibody. These similar binding characteristics indicate that mutations within the framework of the VH or VL domains do not affect the interaction of the antibody with human Gal3.

[0226] Kd values ​​were also determined by surface plasmon resonance using single-cell kinetic analysis. A total of five concentrations of Gal3 CRD domain (Novarix) [0.5-50] nM from human, dog, rat, and cynomolgus monkey were injected over the antibody capture surface for 210 s, followed by a dissociation phase of 600 s at 30 μL / min. At the end of each cycle, the capture surface was regenerated by two injections of glycine (pH 1.5) for 30 s at 10 μL / min, followed by an additional wash with glycine (pH 1.5). New antibody was captured at the beginning of each cycle.

[0227] The Kd values ​​for each antibody tested are shown in Table 5 below.

[0228] [Table 5]

[0229] Example 8: ELISA EC50 Determination for Human and Mouse Gal3 To determine the 50% effective concentration (EC50) of the antibodies, dose-response experiments in ELISA were performed for Hu-Gal3-FL and m-Gal3-FL.

[0230] Hu-Gal3-FL and m-Gal3-FL were immobilized at 3 μg / mL via their histidine tags on functionalized microplates as before. 3.3-fold serial dilutions were performed with purified IgG, added to wells containing immobilized Hu-Gal3-FL or m-Gal3-FL, and detected with an anti-Fab HRP antibody.

[0231] The EC50 ratio of human / mouse Gal3 was estimated according to the EC50 values ​​(sigmoidal dose-response equation: Y = minimum + X * (Maximum-minimum) / (EC50+X), a low ratio indicates high cross-reactivity.

[0232] EC50 was measurable for 8 out of 10 antibodies tested. EC50 for B12 and E12 antibodies was too high to be determined. Of the 8 remaining antibodies, 6 had EC50 values ​​for human Gal3 and 4 for mouse Gal3 below 10 nM. Human / mouse Gal3 EC50 ratios were below 4 for D06, D11, E02, E07 and E01 antibodies, confirming high cross-reactivity.

[0233] The results are presented in Figures 5 and 6 below.

[0234] [Table 6]

[0235] Example 9: Competitive inhibition assay of Gal3 binding to asialofetuin and determination of IC50 for human and mouse Gal3 The inhibitory properties and IC50 of anti-Gal3 antibodies were determined by measuring the binding of asialofetuin to the immobilized CRD domain of human Gal3 in the absence or presence of various concentrations of antibodies in an ELISA competition assay.

[0236] Inhibition assays were performed on Hu-Gal3-CRD immobilized at 1 μg / mL through its histidine tag on functionalized microplates. 2- or 3-fold serial dilutions were performed with purified IgG, which were added to wells with immobilized Hu-Gal3-CRD. Asialofetuin (A1908-Sigma reference) was added at 8 μg / mL to each well with or without IgG. Binding of asialofetuin to Hu-Gal3-CRD was detected using an anti-asialofetuin sheep antibody labeled with HRP. IC50 was estimated according to the model formula Y=lowest value+(highest value-lowest value) / (1+(X / IC50)). The index maximum inhibition percentage was obtained using the formula: 100%-(((mean value of IgG plateau low values)×100%) / mean value of irrelevant IgG values).

[0237] All except the negative control (irrelevant IgG) showed inhibition of asialofetuin binding to Hu-Gal3-CRD (Figure 6). The IC50 of the antibodies ranged from 1.5 to 6 nM, and the estimated relative maximum inhibition ranged from 55 to 97%, as shown in Table 7 below.

[0238] Therefore, these eight antibodies are antagonists of Gal3 / asialofetuin binding.

[0239] [Table 7]

[0240] Example 10: Inhibitory activity of antibodies in a hypochlorous acid (HOCl)-induced fibrosis mouse model The therapeutic effect of anti-Gal3 antibody on pulmonary and cutaneous pulmonary fibrosis was evaluated in a HOCl-induced fibrosis mouse model in two independent experiments.

[0241] In the first experiment, control mice (n=12) received intradermal injections of 300 μL of sterile phosphate-buffered saline (PBS) once a day for 5 consecutive days per week for a period of 42 days. Mice treated with HOCl only and antibody plus HOCl (HOCl-antibody) (n=12 per group) received intradermal injections of 300 μL of HOCl once a day for 5 consecutive days per week for a period of 42 days. Mice treated with antibody also received subcutaneous injections of 20 mg / kg of antibody every 5 days from day 5 to day 40, starting 1 day prior. Each antibody was dissolved in sterile, endotoxin-free PBS and then injected. In the second experiment, the same protocol was applied, except that the study period was 63 days and the antibody was administered every 7 days starting 1 day prior until day 62.

[0242] Mice that received a daily injection of HOCl were also treated by intratracheal instillation of 0.5 mg / kg of TD139 compound, a small molecule Gal3 inhibitor (HOCl-TD139). In the first experiment, administration began on day 21, and the compound was administered every 3 days until day 39. In the second experiment, administration began on day 20, and the compound was administered every 3 days until day 62. TD139 was first dissolved in 100% DMSO, and then diluted 50-fold with endotoxin-free PBS before administration.

[0243] A- Longitudinal assessment of skin thickness The evolution of skin thickness was monitored every 7 days until sacrifice by using an explant.

[0244] In the first experiment, as shown in Figure 7A, once-daily injections of HOCl were associated with a rapid, sustained and statistically significant increase in skin thickness compared to control mice, beginning on day 7 and reaching a maximum of 3.9-fold on day 42. The HOCl-induced increase in skin thickness was significantly reduced by both D11 and E07 monoclonal antibodies, an effect that began on day 21 and persisted through day 42. Mice receiving the reference article TD139 showed a lower decrease in skin thickness than either antibody, reaching statistical significance only on days 28 and 42.

[0245] In the second experiment, a sustained significant increase in skin thickness in mice treated with HOCL compared to control mice began on day 7 and reached a maximum of 5.9-fold on day 63 (FIG. 7B). The HOCl-induced increase in skin thickness was significantly reduced by E07 from day 7 and by H10 from day 14, showing a sustained effect until day 63.

[0246] As a result, the antibodies of the invention have a significant ability to reduce an indicator of fibrosis, skin thickness, measured over time, to the same or greater extent than TD139 (Figures 7A and 7B) and for at least 9 weeks (Figure 7B) using a weekly dosing scheme.

[0247] B- Quantification of collagen in skin and lung At the end of the first study (day 42), skin and lung samples were collected, formalin fixed, and processed into individual paraffin wax blocks. Skin and lung sections were cut from the blocks, rehydrated, and stained with picrosirius red stain according to the manufacturer's protocol. Each slide was then examined and analyzed remotely using ImageJ software online.

[0248] Daily intradermal injection of HOCl was associated with increased collagen deposition in the lungs (FIG. 8A) and skin (FIG. 8B) compared to vehicle mice.

[0249] Subcutaneous injection of D06, D11 and E07 antibodies, as well as intratracheal instillation of the reference compound TD139, restored intrapulmonary collagen levels to those of control mice, reaching statistical significance for E07.

[0250] Subcutaneous injection of D11 antibody and reference compound TD139 significantly reduced skin collagen levels compared to the HOCl group. A non-significant downward trend was also observed after treatment with E07 and D06 antibodies.

[0251] As a result, the antibodies of the invention have a demonstrable ability to reduce collagen deposition, an indicator of fibrosis, in both lung and skin, as measured histologically after 6 weeks of treatment.

[0252] C-Effects on plasma interleukin-6 (IL-6) and interleukin-5 (IL-5) levels in mice In the first experiment, a mixed effects model for repeated measures data was performed for the control group, the HOCl-treated group, the HOCl-antibody treated group, and the HOCl-TD139 treated group. The effect of HOCl administration on plasma IL-6 and IL-5 levels was evaluated compared to the control group. The effect of antibody and TD139 treatment was compared to the HOCl group (Figure 9). IL-6 and IL-5 were quantified by using Mesoscale's MSD technology.

[0253] Plasma IL-6 levels increased significantly after HOCl administration. Treatment with E07 or D11 antibody significantly reduced plasma IL-6 levels to values ​​similar to those of control mice, while TD139 had no activity. As a result, the antibodies of the present invention have a better ability than TD139 to reduce plasma IL-6 levels after HOCl treatment.

[0254] The E07 and D11 antibodies, as well as TD139, significantly reduced the plasma IL-5 level compared to the HOCl group.

[0255] Treatment with D-antibody resets plasma Gal3 levels to those of healthy mice In the first experiment, plasma Gal3 levels were measured 1 day before (before antibody administration) and on the end 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 fall within the dynamic range of the assay.

[0256] As expected, no significant differences were observed among all groups one day prior (FIG. 10). On day 42, a significant increase in Gal3 was observed in the plasma of mice treated with HOCl. Treatment with E07 and D11 antibodies, as well as with the TD139 compound, restored plasma Gal3 levels to values ​​similar to those of the control group.

[0257] E - Genes dysregulated in pathological conditions are virtually completely normalized after treatment with anti-Gal3 antibodies In the first experiment, expression of all genes in whole blood cells was examined by RNA sequencing at day 42.

[0258] Whole blood was collected in RNA Protect tubes (Qiagen) and processed for RNA extraction using the RNeasy Protect Animal Blood Kit with optional on-column deoxynuclease I digestion. Total RNA samples were quantified with a Nanodrop 2000 (Thermo Fisher Scientific™) and analyzed with an RNA6000 Nano chip on a Bioanalyzer (Agilent) to determine RNA quality (RNA Integrity Number, RIN). Total RNA that met quality control standards was included in the study. Purified RNA samples were used as input for TruSeq strand-specific RNA sequencing (polyA selection and globin removal) according to the manufacturer's protocol (Illumina). After library preparation, samples that met the required criteria were sequenced on a NovaSeq6000 sequencer (Illumina).

[0259] Raw reads were processed using an in-house RNAExp Expression pipeline2.1 and quality was assessed using the FastQC tool. Reads were then trimmed for adapters using Cutadapt and trimmed reads were aligned to the mouse mm39 reference genome using the STAR aligner. Aligned data were assessed for quality using Samtool and Picard tools. Aligned reads per gene were then quantified using FeatureCount. A sample filter step identified two outliers, one in the control group and one in the TD139-treated group, which were removed from further analysis. Read counts were normalized by the variance-stabilizing transformation (vst) function in the DESeq2R package.

[0260] 55,416 genes were detected in the data. 29,947 non-protein coding genes were removed. Those with a count of 0 in all samples or expression levels less than 1 in more than 95% were filtered. At the end, the RNA-seq data contained 10,508 genes. Differentially expressed genes between groups were determined using a linear model (ImFit function in the limma R package) on the vst-transformed gene expression dataset. The resulting p-values ​​were adjusted for multiple hypothesis testing and filtered to retain differentially expressed genes with a false discovery rate (FDR) corrected p-value < 0.05 and a fold change (FC) value > 1.3.

[0261] 510 genes were found to be differentially regulated between pathological HOCl group and control group (484 up and 26 down). Treatment with E07 antibody counteracted this pathological pattern in 445 of these genes, accounting for 87.2%, resulting in a total gene expression profile very close to that of the control group (Figure 11). In contrast, only 7 genes, accounting for 1.37% of the dysregulated genes between HOCl group and control group, were found to be counteracted after treatment with reference compound TD139. As a result, the E07 antibody of the present invention shows a clear superiority over TD139.

[0262] Example 11: Activity of antibodies in a mouse model of bleomycin-induced pulmonary fibrosis The therapeutic effect of anti-Gal3 antibody on pulmonary gene expression and pulmonary fibrosis was evaluated in a bleomycin-induced fibrosis mouse model.

[0263] Control bleomycin mice, designated "BLEO", received 1.65 mg / kg bleomycin by the oropharyngeal route on day 1. Control unchallenged mice, designated "CTRL" or "Control", did not receive bleomycin but received the bleomycin saline vehicle on day 1. Antibody-treated mice also received 1.65 mg / kg bleomycin by the oropharyngeal route on day 1, as well as subcutaneous injections of 20 mg / kg antibody on days 4, 8, 12, 16, and 20, beginning one day prior. Prior to injection, each antibody was dissolved in sterile, endotoxin-free PBS.

[0264] Mice that received bleomycin were also treated with 0.5 mg / kg of the small molecule Gal3 inhibitor TD139 compound (bleo-TD139) administered oropharynx starting on day 12 and every 2 days until day 20. TD139 was first dissolved in 100% DMSO.

[0265] A-Weight Assessment The mice were weighed once a day. The weight gain curves are shown in FIG. 12. The control mice showed normal weight gain, with an average weight gain of 6.4% compared to their initial weight measured one day before. The mice treated with bleomycin showed a rapid mouse weight loss, reaching a maximum weight loss of 8.7% on day 8, followed by a weight gain phase from days 9 to 21. Subcutaneous injection of the antibodies D06 and E07 allowed a more rapid weight recovery, reaching values ​​close to those of the control mice from days 12 and 18, respectively, and completely regaining weight by day 21. The D11 antibody showed an average weight gain of 4.4% on day 21 compared to day 1 before. The TD139 compound showed a more modest efficacy on this parameter, with an average weight gain of 2.9% on day 21 compared to day 1 before, slightly higher than the untreated bleomycin group (+1.9%).

[0266] These results demonstrate that the antibodies of the invention acted by improving overall animal health as reflected by improved body weight of the mice.

[0267] B-Weight measurement Body weight measurements were evaluated by using two parameters, called time to weight gain (TWG) and time to baseline (TTB), shown in Figure 13. Time to weight gain corresponds to the inflection point when mice show an ascending gain curve during the course of the experiment and start to gain weight again. Time to baseline corresponds to the number of days required for mice to regain their initial weight (1 day prior).

[0268] The median TWG was 10 days in the untreated bleomycin group. It was significantly shortened by 2.8 days after treatment with D06 antibody, and by 1.3 and 2 days after treatment with D11 and E07 antibodies, respectively, with p-values ​​just above the statistical significance limit. The TD139 compound showed no effect on this parameter (p-value 0.53).

[0269] The median TTB was 20 days in the untreated bleomycin group. It was significantly shortened by 5.5 days, 2.7 days, and 4 days after treatment with D06, D11, and E07 antibodies, respectively, and by 2.7 days and 4 days after treatment with D11 and E07 antibodies, respectively, with p-values ​​just above the statistical significance limit. The TD139 compound showed no effect on this parameter (p-value 0.29).

[0270] These results demonstrate that the antibodies of the invention acted by improving overall animal health as reflected by improved body weight metrics in the mice.

[0271] C- Measurement of the extent of pulmonary fibrosis Mice were terminally euthanized on day 21. Lungs were collected for histopathological examination and gravity-fixed with 10% neutral buffered formalin (NBF) followed by immersion fixation in NBF.

[0272] Sections were taken and then placed onto three slides as indicated by the code numbers in FIG.

[0273] Three longitudinal sections parallel to and bisecting the main bronchus were trimmed from the left and right posterior lobes and mounted in cassettes 01 and 02, respectively. The right anterior, middle, and accessory lobes were sectioned longitudinally and mounted in cassette 03. All sections were stained with picrosirius red and blindly scored by a board-certified veterinary pathologist using a modified Ashcroft fibrosis score (0-8), as described by Huebner et al. (2008), where "0" corresponds to "normal" ("no fibrosis") and "8" corresponds to the highest fibrosis score possible, based on evaluation across all lung sections.

[0274] In mice that received bleomycin without any treatment, 87.6% of the animals had a median fibrosis score of 3 or 4, and only 7.4% had a score of 2 (Figure 15).

[0275] In mice treated with D06 antibody, the frequency of animals with a median fibrosis score of 3 or 4 was reduced to 64.5%, while 30% of the animals had a fibrosis score as low as 2.

[0276] Mice treated with D11 antibody also showed an improvement in the frequency of a score of 2 (17.8%), with the proportion of mice scoring 4 or 5 shifting significantly toward lower scores 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 bleomycin group, and the frequency of animals with a score of 3 increased to 61.1%, compared to 43.2% in the bleomycin group.

[0277] The TD139 compound did not show any signs of efficacy against fibrosis, with 49.4% of mice having a lung fibrosis score of 4 and 5 compared to 49.3% in the untreated bleomycin group, and 50.6% of mice having a lung fibrosis score of 2 and 3, which was similar to the untreated bleomycin group.

[0278] In conclusion, there was clear evidence of attenuated pulmonary fibrosis in mice treated with the D06 antibody, and to a lesser extent in mice treated with the D11 antibody.

[0279] Example 12: Antibody suppresses inflammatory responses in a rat model of ARDS (acute respiratory distress syndrome) The therapeutic effect of anti-Gal3 antibody on inflammation was evaluated in a rat model of ARDS (acute respiratory distress syndrome).

[0280] Positive control rats received three doses of sterile phosphate buffer 11, 6, and 1 day before challenge with lipopolysaccharide (0.25 mg / kg by subcutaneous route on day 1). Negative control rats received one dose of 10 mg / kg of dexamethasone (dissolved in 0.5% carboxymethylcellulose in deionized water) administered orally 2 hours before challenge with lipopolysaccharide (0.25 mg / kg by subcutaneous route on day 1). LPS (lipopolysaccharide) causes an acute inflammatory response, whereas dexamethasone has an anti-inflammatory effect and is therefore the reference treatment in this model.

[0281] Antibody-treated rats received three doses of antibody (14 mg / kg) 11, 6, and 1 days before LPS challenge (0.25 mg / kg by subcutaneous route on day 1). Each antibody was dissolved in sterile PBS before injection.

[0282] Experimental control rats received three doses of sterile phosphate buffer on days 11, 6, and 1 without LPS challenge on day 1. For all rats, plasma sampling was performed 4 hours after LPS challenge.

[0283] A-Effect on neutrophil count in bronchoalveolar lavage fluid (BALF) The effect of treatment with anti-Gal3 antibodies in rats challenged with LPS was evaluated by quantification of neutrophils in bronchoalveolar lavage fluid and compared with the positive control group (LPS challenge with PBS treatment) (Figure 16). In the control rats, the neutrophil count was significantly increased after LPS challenge, indicating that the rats developed an inflammatory response in response to LPS challenge. The neutrophil count level was decreased after treatment with the anti-inflammatory drug dexamethasone. The neutrophil count was also decreased in rats treated with anti-Gal3 antibodies D11, D06, E07, and H10.

[0284] Effect on blood-air barrier damage (protein concentration in bronchoalveolar lavage fluid) The effect of treatment with anti-Gal3 antibody in LPS-challenged rats was evaluated by quantification of protein in bronchoalveolar lavage fluid (by BCA (bicinchoninic acid) assay) and compared with the positive control group (LPS challenge with PBS treatment) (Figure 17). The presence of protein in bronchoalveolar lavage fluid reflects damage to the blood-air barrier. In the control group of rats, the total protein in bronchoalveolar lavage fluid was significantly increased after LPS challenge, indicating that there is rapid damage to the blood-air barrier in this group. The total protein count level in bronchoalveolar lavage fluid was decreased after treatment with the anti-inflammatory drug dexamethasone. In rats treated with anti-Gal3 antibodies D11, D06, E07 and H10, the total protein in bronchoalveolar lavage fluid was also decreased with statistical significance in the E07 group.

[0285] B-Effects on plasma tumor necrosis factor alpha (TNFα) and KC (keratinocyte-derived cytokine)-GRO (proliferation-related oncogene) levels in rats The effect of anti-Gal3 antibody treatment in LPS-challenged rats was evaluated on plasma cytokine levels, namely TNFα and KC-GRO cytokines, and compared with the positive control group (LPS-challenged with PBS treatment) (Figures 18A and 18B, respectively). TNFα and KC-GRO were quantified by using Mesoscale's MSD technology.

[0286] In control rats, plasma TNFα levels were significantly increased after LPS challenge, indicating that the rats developed an inflammatory response in response to LPS challenge. TNFα levels were reduced after treatment with the anti-inflammatory drug dexamethasone. Plasma tumor necrosis factor α levels were also reduced in rats treated with anti-Gal3 antibodies D11, D06, E07, and H10, showing an intermediate profile between LPS-challenged rats and dexamethasone-treated LPS-challenged rats.

[0287] Plasma KC-GRO levels were also reduced after treatment with the four antibodies to a similar extent as with dexamethasone.

[0288] As a result, the antibodies of the invention exert an anti-inflammatory effect by reducing neutrophil counts, reversing blood-air barrier damage, and reducing plasma cytokine levels of TNFα and KC-CRO.

Claims

1. An anti-galectin-3 (anti-Gal3) antibody or its antigen-binding fragment, wherein the anti-Gal3 antibody or its antigen-binding fragment specifically binds to galectin-3 but does not bind to galectin-1 or galectin-7.

2. The anti-Gal3 antibody or antigen-binding fragment thereof according to claim 1, wherein the anti-Gal3 antibody or antigen-binding fragment thereof binds to extracellular galectin 3.

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

4. The anti-Gal3 antibody or its antigen-binding fragment, - Heavy chain complementarity determination region (H-CDR): a) H-CDR1 containing or consisting of one of the amino acid sequences of SEQ ID NOs. 1 to 5, b) H-CDR2 containing or consisting of one of the amino acid sequences of SEQ ID NOs: 6 to 11, and c) H-CDR3 containing or consisting of one of the amino acid sequences of SEQ ID NOs: 3, 12-19, and - Light chain complementarity determination region (L-CDR): a) L-CDR1 containing or consisting of one of the amino acid sequences of SEQ ID NOs. 20 to 26, b) L-CDR2 contained in the amino acid sequence of SEQ ID NOs. 39, 43, 45, 55, 89, or 96 as defined according to Kabat or IMGT, and c) L-CDR3 containing or consisting of one of the amino acid sequences of SEQ ID NOs. 30 to 37 The anti-Gal3 antibody or antigen-binding fragment thereof according to claim 1, comprising:

5. The anti-Gal3 antibody or its antigen-binding fragment, a) H-CDR1-3, each containing or consisting of the amino acid sequences of SEQ ID NOs: 1, 6, and 12, respectively. L-CDR1, which contains or consists of the amino acid sequence of SEQ ID NO: 20 L-CDR2, and the amino acid sequence of Sequence ID No. 39 as defined by Kabat or IMGT, L-CDR3 containing or consisting of the amino acid sequence of SEQ ID NO: 30; b) H-CDR1-3, each containing or consisting of the amino acid sequences of SEQ ID NOs: 1, 6, and 13, respectively. L-CDR1, which contains or consists of the amino acid sequence of SEQ ID NO: 21 L-CDR2, and the amino acid sequence of Sequence ID No. 39 as defined by Kabat or IMGT, L-CDR3 containing or consisting of the amino acid sequence of SEQ ID NO: 31; c) H-CDR1-3, each containing or consisting of the amino acid sequences of SEQ ID NOs: 1, 6, and 14, respectively. L-CDR1, which contains or consists of the amino acid sequence of SEQ ID NO: 22 L-CDR2, and included in the amino acid sequence of Sequence ID No. 43 or 89 as defined by Kabat or IMGT, L-CDR3 containing or consisting of the amino acid sequence of SEQ ID NO: 31; d) H-CDR1-3, each containing or consisting of the amino acid sequences of SEQ ID NOs. 5, 7, and 3, respectively. L-CDR1, which contains or consists of the amino acid sequence of SEQ ID NO: 21 L-CDR2, and the amino acid sequence of Sequence ID No. 45 as defined by Kabat or IMGT, L-CDR3 containing or consisting of the amino acid sequence of SEQ ID NO: 32; e) H-CDR1-3, each containing or consisting of the amino acid sequences of SEQ ID NOs: 3, 8, and 15, respectively. L-CDR1, which contains or consists of the amino acid sequence of SEQ ID NO: 23 L-CDR2, and the amino acid sequence of Sequence ID No. 39 as defined by Kabat or IMGT, L-CDR3 containing or consisting of the amino acid sequence of SEQ ID NO: 33; f) H-CDR1-3, each containing or consisting of the amino acid sequences of SEQ ID NOs: 2, 6, and 16, respectively. L-CDR1, which contains or consists of the amino acid sequence of SEQ ID NO: 24 L-CDR2, and included in the amino acid sequence of Sequence ID No. 39 or 96 as defined by Kabat or IMGT, L-CDR3 containing or consisting of the amino acid sequence of SEQ ID NO: 34; g) H-CDR1-3, each containing or consisting of the amino acid sequences of SEQ ID NOs: 1, 9, and 14, respectively. L-CDR1, which contains or consists of the amino acid sequence of SEQ ID NO: 24 L-CDR2, and included in the amino acid sequence of Sequence ID No. 39 or 96 as defined by Kabat or IMGT, L-CDR3 containing or consisting of the amino acid sequence of SEQ ID NO: 34; h) H-CDR1-3, each containing or consisting of the amino acid sequences of SEQ ID NOs: 2, 10, and 17, respectively. L-CDR1, which contains or consists of the amino acid sequence of SEQ ID NO: 25 L-CDR2, and the amino acid sequence of Sequence ID No. 39 as defined by Kabat or IMGT, L-CDR3 containing or consisting of the amino acid sequence of SEQ ID NO: 35; i) H-CDR1-3, each containing or consisting of the amino acid sequences of SEQ ID NOs: 4, 11, and 18, L-CDR1, which contains or consists of the amino acid sequence of SEQ ID NO: 21 L-CDR2, and the amino acid sequence of Sequence ID No. 55 as defined by Kabat or IMGT, L-CDR3 containing or consisting of the amino acid sequence of SEQ ID NO: 36; or j) H-CDR1-3, each containing or consisting of the amino acid sequences of SEQ ID NOs: 2, 10, and 19, respectively. L-CDR1, which contains or consists of the amino acid sequence of SEQ ID NO: 26 L-CDR2, and the amino acid sequence of Sequence ID No. 39 as defined by Kabat or IMGT, L-CDR3 containing or consisting of the amino acid sequence of SEQ ID NO: 37 The anti-Gal3 antibody or antigen-binding fragment thereof according to claim 1, comprising:

6. The anti-Gal3 antibody or its antigen-binding fragment, a) A heavy chain variable domain (VH) containing an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NOs: 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) Light chain variable domains (VLs) containing an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NOs: 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 83, 86, 89, 91, 93, 96, 98, 100, 102, or 105. The anti-Gal3 antibody or antigen-binding fragment thereof according to claim 1, comprising:

7. The anti-Gal3 antibody or its antigen-binding fragment is a) Heavy chain variable domains (VH) comprising or consisting of the amino acid sequences of SEQ ID NOs: 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) comprising or consisting of the amino acid sequence of SEQ ID NOs: 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 83, 86, 89, 91, 93, 96, 98, 100, 102, or 105. The anti-Gal3 antibody or antigen-binding fragment thereof according to claim 1, comprising:

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

105.

9. Regarding the binding of the anti-Gal3 antibody or its antigen-binding fragment to human Gal3, a) Antibody D06 having VH containing or consisting of the amino acid sequence of SEQ ID NO: 38, and VL containing or consisting of the amino acid sequence of SEQ ID NO: 39; b) Antibody D11 having VH containing or consisting of the amino acid sequence of SEQ ID NO: 40, and VL containing or consisting of the amino acid sequence of SEQ ID NO: 41; c) Antibody E01 having VH containing or consisting of the amino acid sequence of SEQ ID NO: 46, and VL containing or consisting of the amino acid sequence of SEQ ID NO: 47; d) Antibody E02 having VH containing or consisting of the amino acid sequence of SEQ ID NO: 52, and VL containing or consisting of the amino acid sequence of SEQ ID NO: 53; e) Antibody E07 having VH containing or consisting of the amino acid sequence of SEQ ID NO: 42, and VL containing or consisting of the amino acid sequence of SEQ ID NO: 43; f) Antibody G03 having VH containing or consisting of the amino acid sequence of SEQ ID NO: 48, and VL containing or consisting of the amino acid sequence of SEQ ID NO: 49; g) Antibody H07 having VH containing or consisting of the amino acid sequence of SEQ ID NO: 50, and VL containing or consisting of the amino acid sequence of SEQ ID NO: 51; h) Antibody H10 having VH containing or consisting of the amino acid sequence of SEQ ID NO: 44, and VL containing or consisting of the amino acid sequence of SEQ ID NO: 45; i) Antibody B12 having VH containing or consisting of the amino acid sequence of SEQ ID NO: 54, and VL containing or consisting of the amino acid sequence of SEQ ID NO: 55; j) Antibody E12 having VH containing or consisting of the amino acid sequence of SEQ ID NO: 56, and VL containing or consisting of the amino acid sequence of SEQ ID NO: 57; k) Antibody D06-G1 having VH containing or consisting of the amino acid sequence of SEQ ID NO: 81, and VL containing or consisting of the amino acid sequence of SEQ ID NO: 83; l) Antibody D06-G2 having VH containing or consisting of the amino acid sequence of SEQ ID NO: 82, and VL containing or consisting of the amino acid sequence of SEQ ID NO: 83; m) Antibody D11-G1 having VH containing or consisting of the amino acid sequence of SEQ ID NO: 84, and VL containing or consisting of the amino acid sequence of SEQ ID NO: 86; n) Antibody D11-G2 having VH containing or consisting of the amino acid sequence of SEQ ID NO: 85, and VL containing or consisting of the amino acid sequence of SEQ ID NO: 86; o) Antibody E01-G having VH containing or consisting of the amino acid sequence of SEQ ID NO: 92, and VL containing or consisting of the amino acid sequence of SEQ ID NO: 93; p) Antibody E02-G having VH containing or consisting of the amino acid sequence of SEQ ID NO: 99, and VL containing or consisting of the amino acid sequence of SEQ ID NO: 100; q) Antibody E07-G1 having VH containing or consisting of the amino acid sequence of SEQ ID NO: 87, and VL containing or consisting of the amino acid sequence of SEQ ID NO: 89; r) Antibody E07-G2 having VH containing or consisting of the amino acid sequence of SEQ ID NO: 88, and VL containing or consisting of the amino acid sequence of SEQ ID NO: 89; s) Antibody G03-G1 having VH containing or consisting of the amino acid sequence of SEQ ID NO: 94, and VL containing or consisting of the amino acid sequence of SEQ ID NO: 96; t) Antibody G03-G2 having VH containing or consisting of the amino acid sequence of SEQ ID NO: 95, and VL containing or consisting of the amino acid sequence of SEQ ID NO: 96; u) Antibody H07-G having VH containing or consisting of the amino acid sequence of SEQ ID NO: 97, and VL containing or consisting of the amino acid sequence of SEQ ID NO: 98; v) Antibody H10-G having VH containing or consisting of the amino acid sequence of SEQ ID NO: 90, and VL containing or consisting of the amino acid sequence of SEQ ID NO: 91; w) Antibody B12-G having VH containing or consisting of the amino acid sequence of SEQ ID NO: 101, and VL containing or consisting of the amino acid sequence of SEQ ID NO: 102; x) Antibody E12-G1 having VH containing or consisting of the amino acid sequence of SEQ ID NO: 103, and VL containing or consisting of the amino acid sequence of SEQ ID NO: 105; or y) Antibody E12-G2 having VH containing or consisting of the amino acid sequence of SEQ ID NO: 104, and VL containing or consisting of the amino acid sequence of SEQ ID NO:

105. The anti-Gal3 antibody or antigen-binding fragment thereof according to claim 1, which competes with the above.

10. The anti-Gal3 antibody according to claim 1, wherein the anti-Gal3 antibody is an isotype IgG antibody.

11. The anti-Gal3 antibody according to claim 10, wherein the anti-Gal3 antibody is an antibody of isotype IgG subclass IgG1 or IgG4.

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

13. The anti-Gal3 antibody according to claim 12, wherein the anti-Gal3 antibody contains a mutation at one or more positions 228, 234, and 235 of the heavy chain amino acids, which are numbered according to the EU numbering system.

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 leucine to alanine, and / or the amino acid residue at position 228 is mutated from serine to proline.

15. A pharmaceutical composition comprising an anti-Gal3 antibody or an antigen-binding fragment thereof according to any one of claims 1 to 14.

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

17. A combination of isolated nucleic acid molecules encoding an anti-Gal3 antibody or an antigen-binding fragment thereof, as described in any one of claims 1 to 14.

18. The combination of isolated nucleic acid molecules according to claim 17, which is a combination of a first isolated nucleic acid molecule containing or consisting of a sequence encoding a VH sequence, and a second isolated nucleic acid molecule containing or consisting of a sequence encoding a VL sequence.

19. A vector comprising an isolated nucleic acid molecule according to claim 16, wherein the vector further comprises an expression control sequence.

20. A vector comprising the combination of isolated nucleic acid molecules described in Claim 17, wherein the vector further comprises an expression control sequence.

21. A host cell comprising the isolated nucleic acid molecule described in claim 16.

22. A host cell comprising the combination of isolated nucleic acid molecules described in Claim 17.

23. A host cell comprising the vector according to claim 19.

24. A host cell comprising the vector according to claim 20.

25. A method for producing an anti-Gal3 antibody or an antigen-binding fragment thereof, comprising the steps of: preparing host cells according to claim 21; culturing the host cells under conditions suitable for the expression of the anti-Gal3 antibody or its antigen-binding fragment; and isolating the resulting anti-Gal3 antibody or its antigen-binding fragment.

26. A method for producing an anti-Gal3 antibody or an antigen-binding fragment thereof, comprising the steps of: preparing the host cells described in Claim 22; culturing the host cells under conditions suitable for the expression of the anti-Gal3 antibody or its antigen-binding fragment; and isolating the anti-Gal3 antibody or its antigen-binding fragment obtained as a result.

27. ​​A method for producing an anti-Gal3 antibody or an antigen-binding fragment thereof, comprising the steps of: preparing the host cells described in Claim 23; culturing the host cells under conditions suitable for the expression of the anti-Gal3 antibody or its antigen-binding fragment; and isolating the anti-Gal3 antibody or its antigen-binding fragment obtained as a result.

28. A method for producing an anti-Gal3 antibody or an antigen-binding fragment thereof, comprising the steps of: preparing the host cells described in Claim 24; culturing the host cells under conditions suitable for the expression of the anti-Gal3 antibody or its antigen-binding fragment; and isolating the anti-Gal3 antibody or its antigen-binding fragment obtained as a result.

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

30. A pharmaceutical composition according to claim 15 for treating Gal3-related diseases.

31. The pharmaceutical composition according to claim 30, wherein the Gal3-related disease is a fibrous disease, an inflammatory disease, an autoimmune disease, an immune-mediated disorder, a neurodegenerative disease, a metabolic disease, an infectious disease, or cancer.

32. The pharmaceutical composition of claim 31, wherein the fibrous disease originates from a tissue selected from the skin, lungs, liver, heart, kidneys, and blood vessels.

33. The pharmaceutical composition according to claim 30, for use in combination with an additional therapeutic agent or treatment.

34. The pharmaceutical composition of claim 33, wherein the additional therapeutic agent or treatment is an immune checkpoint modulator, a chemotherapeutic agent, an antineoplastic agent, an anti-angiogenic agent, a hormonal therapy agent, a stem cell-based therapeutic agent, surgery, radiotherapy, another inhibitor of the Gal3 pathway, or PD1 / PDL1 blockade therapy and / or CTLA4 blockade therapy.

35. a) for treating Gal3-related diseases; or b) for treating fibrous diseases, inflammatory diseases, autoimmune diseases, immune-mediated disorders, neurodegenerative diseases, metabolic diseases, infections, or cancer; or c) To inhibit Gal3 interaction Use of an anti-Gal3 antibody or its antigen-binding fragment according to any one of claims 1 to 14 for the manufacture of a pharmaceutical product.

36. a) When treating Gal3-related diseases; or b) when treating fibrous diseases, inflammatory diseases, autoimmune diseases, immune-mediated disorders, neurodegenerative diseases, metabolic diseases, infections, or cancer; or c) When inhibiting Gal3 interaction An anti-Gal3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 14, for use.

37. a) for treating Gal3-related diseases; or b) for treating fibrous diseases, inflammatory diseases, autoimmune diseases, immune-mediated disorders, neurodegenerative diseases, metabolic diseases, infections, or cancer; or c) To inhibit Gal3 interaction A product comprising an anti-Gal3 antibody or an antigen-binding fragment thereof, as described in any one of claims 1 to 14, which is suitable.

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