Antibodies to Galectin-3 and methods of use thereof

Abstract

Provided herein are compositions, methods, and uses involving antibodies that specifically bind to the carbohydrate binding domain (CBD) of galectin-3 (LGALS3).Also provided herein are uses and methods for managing, treating, or preventing disorders, such as cancer.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 122,714, filed December 8, 2020, the entire contents of which are incorporated herein by reference.

[0002] The present technology relates generally to the preparation of immunoglobulin-related compositions (e.g., antibodies or antigen-binding fragments thereof) that specifically bind to Galectin-3 protein, and their uses. In particular, the present technology relates to the preparation of Galectin-3-binding antibodies and their use in the detection and treatment of Galectin-3-associated cancer, cardiovascular disease, fibrosis, and inflammation. [Background technology]

[0003] Galectins are a family of small, highly conserved eukaryotic proteins that recognize specific complex sugars on glycosylated cell surface proteins. Galectins are essential in linking cancer cells with the stromal microenvironment to regulate development, adhesion, signaling, invasion, and immune system interactions. Galectins can be found in the nucleus, cytoplasm, and pericellular space. Extracellular galectins are primarily released in exosomes, and do not appear to be classically secreted from the ER or Golgi. Humans possess at least 12 different galectins, which are variably expressed in different tissues and developmental stages. In the last decade, it has become clear that human galectins, especially galectin-3 (LGALS3), represent a key link between the microenvironment and tumor cells. Notably, the biological functions of glycoproteins and other surface glycans are primarily dependent on the specific glycans connected to the Golgi, resulting in unique galectin selectivity. Extracellularly, LGALS3 is involved in regulating cell membrane residence time, adhesion, migration, invasion, and angiogenic functions. Although LGALS3 binds to other natural ligands, its highest affinity ligand is the most proximal lactosamine disaccharide in the poly-lactosamine chain that decorates many O- and N-glycan species. By binding and polymerization, LGALS3 forms a lattice and regulates the location and residence time of growth factor receptors, including EGFR, PDGFR, integrins, and CTLA4, among others (Figures 1A-1B). Activation of downstream signaling molecules such as SRC, ERK, AKT, and FAK leads to the generation of key molecules involved in metastasis and invasion. On the surface of T cells, CTLA4 surface concentration is stabilized by LGALS3, causing immune suppression. Loss of the LGALS3 lattice inhibits the behavior of multiple cancer and immune cells. In addition to cancer, excess LGALS3 is also associated with kidney disease, liver fibrosis, pulmonary fibrosis, heart failure, and parasitic diseases (see Figure 2A). Summary of the Invention

[0004] Provided herein are compositions, methods, and uses involving antibodies that specifically bind to Galectin-3 (LGALS3) and modulate expression and / or activity of LGALS3 to manage or treat LGALS-mediated disorders, such as cancer. The present disclosure provides antibodies or antigen-binding fragments thereof that specifically bind to Galectin-3 (LGALS3) polypeptides. In some embodiments, the LGALS3 polypeptide comprises SEQ ID NO: 12.

[0005] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof, comprising a heavy chain immunoglobulin variable domain (V H ) and light chain immunoglobulin variable domains (V L ), (a) V H is selected from the group consisting of SEQ ID NOs: 7, 19, 29, 39, and 49 H - a CDR1 sequence and a V selected from the group consisting of SEQ ID NOs: 8, 20, 30, 40, and 50 H - a CDR2 sequence and a V selected from the group consisting of SEQ ID NOs: 9, 21, 31, 41, and 51 H and / or (b) the CDR3 sequence V L is selected from the group consisting of SEQ ID NOs: 2, 14, 24, 34, and 44 L CDR1 sequence and a V selected from the group consisting of SEQ ID NOs: 3, 15, 25, 35, and 45 L CDR2 sequence and a V sequence selected from the group consisting of SEQ ID NOs: 4, 16, 26, 36, and 46 L and CDR3 sequence.

[0006] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof that specifically binds to galectin-3 (LGALS3), the antibody or antigen-binding fragment thereof comprising a heavy chain variable region (V H ) and the light chain variable region (V L ), (a) V H V comprising SEQ ID NO:7 H Complementarity determining region (CDR) 1 sequence and V comprising SEQ ID NO:8 H CDR2 sequence and V comprising SEQ ID NO:9 H CDR3 sequence, and / or VL V containing SEQ ID NO:2 L CDR1 sequence and V comprising SEQ ID NO:3 L CDR2 sequence and V comprising SEQ ID NO:4 L and (b) a CDR3 sequence, H V comprising SEQ ID NO: 19 H Complementarity determining region (CDR) 1 sequence and V comprising SEQ ID NO: 20 H CDR2 sequence and V comprising SEQ ID NO:21 H CDR3 sequence, and / or V L V comprising SEQ ID NO: 14 L CDR1 sequence and V comprising SEQ ID NO:15 L CDR2 sequence and V comprising SEQ ID NO: 16 L CDR3 sequence, or (c) V H V comprising SEQ ID NO:29 H Complementarity determining region (CDR) 1 sequence and V comprising SEQ ID NO: 30 H CDR2 sequence and V comprising SEQ ID NO:31 H CDR3 sequence, and / or V L V comprising SEQ ID NO: 24 L CDR1 sequence and V comprising SEQ ID NO:25 L CDR2 sequence and V comprising SEQ ID NO:26 L CDR3 sequence, or (d) V H V comprising SEQ ID NO:39 H Complementarity determining region (CDR) 1 sequence and V comprising SEQ ID NO: 40 H CDR2 sequence and V comprising SEQ ID NO:41 H CDR3 sequence, and / or V L V comprising SEQ ID NO:34 L CDR1 sequence and V comprising SEQ ID NO:35 L CDR2 sequence and V comprising SEQ ID NO:36 L CDR3 sequence; or (e) V H V comprising SEQ ID NO:49 H Complementarity determining region (CDR) 1 sequence and V comprising SEQ ID NO: 50 H CDR2 sequence and V comprising SEQ ID NO:51 H CDR3 sequence, and / or V L V comprising SEQ ID NO: 44L CDR1 sequence and V comprising SEQ ID NO:45 L CDR2 sequence and V comprising SEQ ID NO:46 L and a CDR3 sequence. The antibody or antigen-binding fragment thereof comprises a human or humanized heavy chain variable domain (V H ) and / or a human or humanized light chain variable domain (V L In some embodiments, V H comprises an amino acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:18, SEQ ID NO:28, SEQ ID NO:38, and SEQ ID NO:48, and / or V L comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 13, SEQ ID NO: 23, SEQ ID NO: 33, and SEQ ID NO: 43. Additionally or alternatively, in some embodiments, the antigen-binding fragment is a Fab, F(ab')2, Fab', Fv, or scFv.

[0007] Additionally or alternatively, in certain embodiments, the antibody or antigen-binding fragment is selected from the group consisting of SEQ ID NO:6 and SEQ ID NO:1, SEQ ID NO:18 and SEQ ID NO:13, SEQ ID NO:28 and SEQ ID NO:23, SEQ ID NO:38 and SEQ ID NO:33, and SEQ ID NO:48 and SEQ ID NO:43, respectively. H Amino acid sequence and V L In some embodiments, the antibody or antigen-binding fragment is a humanized or fully human antibody, or an antigen-binding fragment thereof.

[0008] In any of the foregoing embodiments, the antibody comprises a heavy chain constant region and a light chain constant region of human origin. In certain embodiments of the antibodies of the present technology, the heavy chain constant region has an isotype selected from the group consisting of gamma 1, gamma 2, gamma 3, and gamma 4, and / or the light chain constant region has an isotype selected from the group consisting of kappa and lambda. Additionally or alternatively, in some embodiments, the antibody is an immunoglobulin comprising two identical heavy chains and two identical light chains. In certain embodiments, the immunoglobulin is an IgG. In some embodiments, the antibody is a monoclonal antibody, a single chain antibody, or any composition comprising an antigen-binding fragment thereof. Additionally or alternatively, in some embodiments, the antibody or antigen-binding fragment thereof is generated by immunizing a transgenic mouse.

[0009] In some embodiments, the antibody or antigen-binding fragment of the present technology inhibits Gal-3-PE binding to tumor cells. In certain embodiments, the tumor cells are ovarian tumor cells (e.g., OVCAR3 cells). Additionally or alternatively, in some embodiments, the antibody or antigen-binding fragment thereof inhibits LGALS3 binding to glycosylated cell surface proteins (e.g., glycosylated cell surface receptors). In certain embodiments, the antibody or antigen-binding fragment inhibits LGALS3 binding to one or more of glycosylated mucin-1 (MUC1), mucin-4 (MUC4), mucin-16 (MUC16), disialoganglioside, GD2, epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR), insulin-like growth factor receptor (IGFR), cMET / hepatocyte growth factor receptor (HGFR), integrins, or CTLA4. In some embodiments, glycosylated MUC16 is N-glycosylated at Asn1800 or Asn1806. In embodiments of any and all of the antibodies or antigen-binding fragments disclosed herein, the antibody or antigen-binding fragment inhibits growth of tumors expressing a glycosylated form of MUC16.

[0010] In one aspect, the disclosure provides an antibody conjugate comprising an agent conjugated to any of the antibodies or antigen-binding fragments described herein. The agent can be an imaging agent or a cytotoxic agent. In some embodiments, the antibody or antigen-binding fragment is a bispecific antibody. In certain embodiments, the bispecific antibody specifically binds to CD3. Additionally or alternatively, in some embodiments, the bispecific antibody comprises an immunoglobulin that specifically binds to LGALS3, and the light chain of the immunoglobulin is conjugated via a peptide linker to a single chain variable fragment (scFv) that specifically binds to CD3.

[0011] In another aspect, the disclosure provides a bispecific antibody conjugate comprising an agent conjugated to any of the bispecific antibodies or antigen-binding fragments described herein. The agent can be an imaging agent or a cytotoxic agent.

[0012] Also disclosed herein are scFv conjugates, including an agent conjugated to any of the scFvs described herein. The agent can be an imaging agent or a cytotoxic agent. Also provided herein are chimeric antigen receptors (CARs) including any of the antibodies or antigen-binding fragments (e.g., scFvs) described herein, and T cells recombinantly expressing the above-mentioned CARs.

[0013] In one aspect, the disclosure provides a recombinant nucleic acid sequence encoding any of the antibodies or antigen-binding fragments or CARs described herein. In some embodiments, the recombinant nucleic acid sequence comprises any one of the polynucleotides of SEQ ID NO:5, SEQ ID NO:17, SEQ ID NO:27, SEQ ID NO:37, SEQ ID NO:47, SEQ ID NO:10, SEQ ID NO:22, SEQ ID NO:32, SEQ ID NO:42, or SEQ ID NO:52, or a portion thereof encoding at least one CDR sequence.

[0014] In another aspect, the disclosure provides a vector comprising any of the recombinant nucleic acid sequences disclosed herein. In some embodiments, the recombinant nucleic acid sequence is operably linked to a promoter. In yet another aspect, the disclosure provides an isolated cell comprising any of the recombinant nucleic acid sequences or vectors disclosed herein.

[0015] In one aspect, the disclosure provides a pharmaceutical composition comprising a therapeutically effective amount of any of the antibodies, antigen-binding fragments, antibody conjugates, bispecific antibodies or antibody conjugates, scFvs, scFv conjugates, CARs, T cells, recombinant nucleic acid sequences, vectors, or isolated cells described herein and a pharma- ceutically acceptable carrier.

[0016] In another aspect, the disclosure provides a method of treating cancer in a patient in need thereof, comprising administering to said patient an effective amount of any of the pharmaceutical compositions disclosed herein. The cancer may be ovarian, lung, pancreatic, breast, uterine, fallopian tube, or primary peritoneal cancer, and / or metastatic cancer. In some embodiments, the pharmaceutical composition inhibits metastasis in the patient. Additionally or alternatively, in some embodiments, the method further comprises administering to the patient a therapeutically effective amount of an additional therapeutic agent. In certain embodiments, the patient is a human patient.

[0017] In yet another aspect, the present disclosure provides a method for detecting LGALS3 protein expression levels in a biological sample, comprising contacting the biological sample with any of the antibodies or antigen-binding fragments disclosed herein and detecting binding to LGALS3 protein in the biological sample.

[0018] Also disclosed herein are kits for the detection and / or treatment of galectin-3-associated diseases or conditions (e.g., galectin-3-associated cancers, cardiovascular diseases, fibrosis, and inflammation), comprising at least one immunoglobulin-related composition of the present technology (e.g., any of the antibodies, antigen-binding fragments, antibody conjugates, bispecific antibodies or antibody conjugates, scFvs, scFv conjugates, CARs, T cells, recombinant nucleic acid sequences, vectors, or isolated cells described herein) and instructions for use. In certain embodiments, the immunoglobulin-related composition is linked to one or more detectable labels. In one embodiment, the one or more detectable labels comprise a radioactive label, a fluorescent label, or a chromogenic label. Additionally or alternatively, in some embodiments, the kit further comprises a secondary antibody that specifically binds to the immunoglobulin-related composition described herein. In some embodiments, the secondary antibody is linked to at least one detectable label selected from the group consisting of a radioactive label, a fluorescent label, or a chromogenic label. [Brief description of the drawings]

[0019] [Figure 1A] The structure and activity of extracellular galectin-3 (LGALS3) is shown, which forms a pentamer to link cell surface cancer molecules, e.g., MUC16 in ovarian cancer, to signaling molecules through contact with signaling receptors, e.g., EGFR and integrins. Activation of downstream signaling molecules, such as SRC, ERK, AKT, and FAK, then leads to the production of molecules important for metastasis and invasion. [Figure 1B] We show how antibody inhibitors that bind to the LGALS3 carbohydrate-binding domain disrupt the LGALS3 surface complex and block activation of 'outside-in' signalling by destabilising the cell surface receptor. [Figure 2A] The experimental in vitro and in vivo function of Galectin-3 and the effect of potential blocking antibodies on treatment with the provided antibodies are shown. [Figure 2B]Figure 1 shows the primary structure of the LGALS3 carbohydrate-binding domain (CBD). The LGALS3 CBD is divided into five subdomains, each of which binds a single sugar residue, conventionally labeled ABCD and E. In this diagram, highly conserved amino acids are underlined, while domains C, D and E are shown in boxed regions. In particular, R186 (*) is important for the function of LGALS3. The LGALS3 CBD sequence is highly homologous to mouse galectin-3, but distinct from GAL1, GAL7 and GAL9. Each of these family members has a similar sugar-binding domain, but LGALS3 is the only lectin with a polymerization domain. [Figure 3A-3B] 3 shows the development and optimization of an assay for Gal-3-PE binding to OVCAR3 cells. FIG. 3A shows the binding of the indicated concentrations of PE-labeled Gal-3 (Gal-3-PE) to the indicated numbers of OVCAR3 cells. The number of OVCAR3 cells bound to Gal-3-PE as detected by a Mirrorball® flow cytometer is plotted. FIG. 3B shows the binding of the indicated concentrations of PE-labeled Gal-3 protein (Gal-3-PE) to the indicated numbers of MOLT-4 cells. The number of MOLT-4 cells bound to Gal-3-PE as detected by a Mirrorball® flow cytometer is plotted. [Figure 4]Figure 1 shows exemplary results of a secondary functional screen to select antibodies that inhibit Gal-3-PE binding to OVCAR3 cells. Reactions to measure Gal-3-PE binding to OVCAR3 cells were set up in the presence of PBS (negative control) or the indicated antibodies obtained by enrichment (crude purification of antibodies) from the supernatants of hybridoma clones selected in the primary binding screen. The extent of binding as measured by Mirrorball® laser scanning flow cytometer was plotted. The secondary screen showed that five antibodies subsequently blocked Gal-3-PE binding to OVCAR3 cells. Clone 14D11 did not block Gal-3-PE binding to OVCAR3 cells in this assay. [Figure 5A-5B] Species cross-reactivity of the indicated antibodies is shown. ELISA was used to detect binding to mouse Gal-3 (FIG. 5A) or human Gal-3 (FIG. 5B). OD450 values ​​for each clone are shown. As shown, clones 39F02, 38E05, and 46H02 bound to mouse Gal-3, whereas clones 20F08 and 12H07 did not bind to mouse Gal-3. [Figures 6A-6D] Figure 6 shows the dose response of blocking Gal-3-PE binding to OVCAR3 cells by purified anti-Gal-3 antibody hits. Reactions to measure binding of Gal-3-PE to OVCAR3 cells were set up in the presence of PBS (negative control) or increasing concentrations of the indicated antibodies. Purified antibodies were used in the binding reactions. The effect of antibody concentration on the extent of inhibition of Gal-3-PE binding to OVCAR3 by antibody clones 20F08 and 12H07 (Figure 6A), 46H02 (Figure 6B), clones 38E05 and 39F02 (Figure 6C), also with identical sequences, and 14D11.2D3 (Figure 6D) are shown. These data showed that the three antibodies (46H02, 20F08, 12H07) showed dose-dependent blocking of Gal-3 binding OVCAR3 cells. [Figure 7A-7B]Figure 7A shows the binding kinetics of monoclonal antibodies (mAbs) 46H02 (Figure 7A) and 20F08 (Figure 7B) to human Gal-3 as determined by surface plasmon resonance (SPR). Line graphs show the change in resonance units (RU, reflecting the change in analyte binding capacity of the surface) as a function of time (seconds) upon addition of mAb at the indicated concentrations. These data showed that mAbs 46H02 and 20F08 bind human Gal-3 with KDs of 10 nM and 44 nM, respectively. [Figure 8] Figure 1 shows the light chain species determination of the indicated antibodies. The light chain species of the indicated mAbs was determined by ELISA using anti-mouse kappa, anti-mouse lambda, anti-human kappa, and anti-human lambda antibodies. The data showed that 46H02 has a fully human F(ab)2, while the other hits have a human HC and a mouse LC. [Figure 9A-9C]9A shows the results of an epitope binning assay for mAb46H02 and 14D11.2D2 by surface plasmon resonance (SPR). FIG. 9A shows the premix epitope binning assay strategy used herein. In this strategy, the test antibody (analyte) is immobilized on a surface, the ligand is flowed over the analyte, and binding is detected by SPR. The ligand was either antigen alone or a premixed solution of antigen complexed with a second antibody. A premixed solution of antigen complexed with a test antibody (analyte) was used as a positive control to indicate a blocking reaction. As shown, antigen alone generates a characteristic SPR profile (left panel) indicating binding of the analyte to the antigen. When the ligand is a premixed solution of antigen complexed with a second antibody, an SPR profile indicating binding of the analyte to the antigen is obtained if the second antibody binds to a distinct epitope on the antigen (middle panel). The SPR profile of the positive control shows no binding, indicating that the antigen complexed with the test antibody (analyte) blocks the binding of the analyte (right panel). Figure 9B shows a premix epitope binning assay using mAb2D3 as the analyte and Gal-3, Gal-3+2D3 complex, or Gal-3+46H02 complex as the ligand. Figure 9C shows a premix epitope binning assay using mAb46H02 as the analyte and Gal-3, Gal-3+2D3 complex, or Gal-3+46H02 complex as the ligand. These data indicate that mAb46H02 and 14D11 do not compete with each other and therefore bind distinct epitopes. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] The present disclosure is not limited in terms of the specific embodiments described in this application, but are intended as single illustrations of individual aspects of the present disclosure. Not all of the various embodiments of the present disclosure are described herein. As will be apparent to those skilled in the art, many modifications and variations of the present disclosure can be made without departing from its spirit and scope. In addition to those enumerated herein, functionally equivalent methods and apparatuses within the scope of the present disclosure will be apparent to those skilled in the art from the foregoing description. Such modifications and variations are intended to fall within the scope of the appended claims. The present disclosure should be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled.

[0021] It is to be understood that this disclosure is not limited to particular applications, methods, reagents, compounds, compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0022] In addition, where features or aspects of the disclosure are described in terms of a Markush group, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual members or subgroups of members of the Markush group.

[0023] As would be understood by one of ordinary skill in the art, for any or all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of those subranges. Any recited range can be readily recognized as fully descriptive and allowing for the same range to be broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third, and upper third, etc. As would also be understood by one of ordinary skill in the art, all language such as "up to," "at least," "greater than," "less than," etc., includes the recited numbers and refers to a range that can be subsequently broken down into subranges as discussed above. Finally, as would be understood by one of ordinary skill in the art, a range includes each individual member. Thus, for example, a group having 1-3 cells refers to a group having 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers to a group having 1, 2, 3, 4, or 5 cells, and so on.

[0024] definition Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which this disclosure belongs. The following references provide those skilled in the art with general definitions of many of the terms used in the technology of the present invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed.1994), The Cambridge Dictionary of Science and Technology (Walker ed.,1988), The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991), and Hale&Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless otherwise specified. The terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the disclosure.

[0025] As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise.

[0026] As used herein, when used to modify a numerical value or numerical range, the term "about" indicates a variation of 5%-10% above and 5%-10% below that value or range while remaining within the intended meaning of the cited value or range.

[0027] As used herein, the term "administration" of an agent to a subject includes any route of introducing or delivering an agent to a subject to perform its intended function. Administration may be by any suitable route, including, but not limited to, intravenous, intramuscular, intraperitoneal, subcutaneous, and other suitable routes described herein. Administration includes self-administration and administration by another.

[0028] The term "amino acid" refers to naturally occurring and non-naturally occurring amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally encoded amino acids are the 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine), as well as pyrrolysine and selenocysteine. Amino acid analogs refer to agents that have the same basic chemical structure as naturally occurring amino acids (i.e., an alpha carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium). Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. In some embodiments, the amino acids that form the polypeptides are in the D form. In some embodiments, the amino acids forming the polypeptide are in the L-form. In some embodiments, a first plurality of amino acids forming the polypeptide are in the D-form and a second plurality of amino acids are in the L-form.

[0029] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted one-letter codes.

[0030] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to naturally occurring amino acid polymers as well as to amino acid polymers in which one or more amino acid residues are non-naturally occurring amino acids, e.g., amino acid analogs. The terms encompass amino acid chains of any length, including full-length proteins, in which the amino acid residues are linked by covalent peptide bonds.

[0031] As used herein, a "control" is a substitute sample used in an experiment for comparison purposes. A control can be "positive" or "negative". For example, if the purpose of an experiment is to determine the correlation of the effectiveness of a therapeutic agent for treatment of a particular type of disease, a positive control (a composition known to exhibit the desired therapeutic effect) and a negative control (a subject or sample that does not receive therapy or receives a placebo) are typically used.

[0032] As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount of an agent sufficient to achieve a desired therapeutic effect. In the context of therapeutic applications, the amount of therapeutic peptide administered to a subject may depend on the type and severity of infection, as well as individual characteristics such as general health, age, sex, weight, and tolerance to drugs. It may also depend on the extent, severity, and type of disease. Those skilled in the art will be able to determine the appropriate dosage depending on these and other factors.

[0033] As used herein, the term "expression" refers to the process by which a polynucleotide is transcribed into mRNA and / or the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in eukaryotic cells. The expression level of a gene can be determined by measuring the amount of mRNA or protein in a cell or tissue sample. In one embodiment, the expression level of a gene from a sample can be directly compared to the expression level of that gene from a control or reference sample. In another embodiment, the expression level of a gene from a sample can be directly compared to the expression level of that gene from the same sample after administration of a composition disclosed herein. The term "expression" also refers to one or more of the following events: (1) production of an RNA template from a DNA sequence within a cell (e.g., by transcription), (2) processing of the RNA transcript within the cell (e.g., by splicing, editing, 5' capping, and / or 3' end formation), (3) translation of the RNA sequence into a polypeptide or protein within the cell, (4) post-translational modification of the polypeptide or protein within the cell, (5) presentation of the polypeptide or protein on the cell surface, and (6) secretion or presentation or release of the polypeptide or protein from the cell.

[0034] The term "linker" refers to a synthetic sequence (e.g., an amino acid sequence) that connects or links two sequences, e.g., links two polypeptide domains. In some embodiments, the linker contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids.

[0035] As used herein, the term "antibody" refers not only to intact antibody molecules, but also to fragments of antibody molecules that retain immunogen-binding ability. Such fragments are also well known in the art and are commonly used both in vitro and in vivo. Thus, as used herein, the term "antibody" refers not only to intact immunoglobulin molecules, but also to the well-known active fragments F(ab')2 and Fab. F(ab')2 and Fab fragments, which lack the Fc fragment of intact antibodies, may be cleared more rapidly from the circulation and have less nonspecific tissue binding than intact antibodies (Wahl et al., J. Nucl. Med. 24:316-325 (1983)). The antibodies of the present technology include whole natural antibodies, monoclonal antibodies, human antibodies, humanized antibodies, camelized antibodies, multispecific antibodies, bispecific antibodies, chimeric antibodies, Fab, Fab', single chain V region fragments (scFv), single domain antibodies (e.g., nanobodies and single domain camelid antibodies), V NAR Antibodies and antigen-binding fragments include non-conventional antibodies and antigen-binding fragments, bispecific T cell engager (BiTE) antibodies, minibodies, disulfide-linked Fvs (dsFvs), and anti-idiotypic (anti-Id) antibodies, intrabodies, fusion polypeptides, any of the above. In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an antigen-binding site. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgAl, and IgA2), or subclass.

[0036] In certain embodiments, an antibody is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (herein referred to as V H ) and heavy chain constant (C H The heavy chain constant region is composed of three domains, CH1, CH2 and CH3. Each light chain comprises a light chain variable region (herein referred to as V L (abbreviated as ) and light chain constant C LThe light chain constant region consists of one domain, C L It consists of V H and V L The regions can be further subdivided into regions of hypervariability, called complementarity determining regions (CDRs), interspersed with regions that are more conserved, called framework regions (FRs). H and V L is composed of three CDRs and four FRs arranged in the following order from amino terminus to carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (Cl q). As used interchangeably herein, the terms "antigen-binding portion," "antigen-binding fragment," or "antigen-binding region" of an antibody refer to the region or portion of an antibody that binds to an antigen and confers antigen specificity to the antibody, and fragments of antigen-binding proteins, e.g., antibodies, include one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., a peptide / HLA complex). It has been shown that the antigen-binding function of an antibody can be exerted by fragments of a full-length antibody. Examples of antigen-binding portions encompassed by the term "antibody fragment" of an antibody include the V L , V H , C L and a Fab fragment, which is a monovalent fragment consisting of a CHI domain; a F(ab)2 fragment, which is a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region; H and an Fd fragment consisting of a CHI domain, a V L and V H Fv fragment consisting of domains, V H These include dAb fragments consisting of domains (Ward et al., Nature 341:544-546 (1989)), as well as isolated complementarity determining regions (CDRs).

[0037] Antibodies and antibody fragments may be derived wholly or partially from mammals (e.g., humans, non-human primates, goats, guinea pigs, hamsters, horses, mice, rats, rabbits, and sheep) or from antibody-producing animals that are non-mammals (e.g., chickens, ducks, geese, snakes, caudal amphibians). Antibodies and antibody fragments may be produced in animals or produced outside animals, for example from yeast or phage (e.g., as single antibodies or antibody fragments or as part of an antibody library).

[0038] In addition, the two domains V of the Fv fragment L and V H are encoded by separate genes, but these are referred to as V L and V H The antibody fragments can be joined using recombinant methods by synthetic linkers that allow the domains to be produced as a single protein chain that pairs to form a monovalent molecule. These are known as single-chain Fvs (scFvs), see, e.g., Bird et al., Science 242:423-426 (1988), and Huston et al., Proc. Natl. Acad. Sci. 85:5879-5883 (1988). These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as intact antibodies.

[0039] An "isolated antibody" or "isolated antigen binding protein" is one that has been identified and separated and / or recovered from a component of its natural environment. A "synthetic antibody" or "recombinant antibody" is generally produced using recombinant techniques, or using peptide synthesis techniques known to those of skill in the art.

[0040] As used herein, the term "single chain variable fragment" or "scFv" refers to a V H :V L Heavy chains (V) of immunoglobulins (e.g., mouse or human) covalently linked to form heterodimers H ) and light chain (V L) is a fusion protein of the variable regions of the heavy chain (V H ) and light chain (V L ) are either directly connected or connected by a peptide-encoding linker (e.g., about 10, 15, 20, 25 amino acids), H N-terminus of V L or V H The C-terminus of V L The linker is usually rich in glycine for flexibility and rich in serine or threonine for solubility. The linker may link the heavy and light chain variable regions of the extracellular antigen-binding domain.

[0041] Despite the removal of the constant region and the introduction of the linker, the scFv protein retains the specificity of the original immunoglobulin. Single-chain Fv polypeptide antibodies can be synthesized by the VFv polypeptide synthesis method, as described by Huston et al. (Proc. Nat. Acad. Sci. USA, 85:5879-5883 (1988)). H and V LThe polypeptide can be expressed from a nucleic acid containing a sequence encoding the polypeptide. See also U.S. Patent Nos. 5,091,513, 5,132,405, and 4,956,778, and U.S. Patent Publication Nos. 2005 / 0196754 and 2005 / 0196754. Antagonistic scFvs with inhibitory activity have been described (e.g., Zhao et al., Hybridoma (Larchmt) 27(6):455-51 (2008); Peter et al., J Cachexia Sarcopenia Muscle (2012); Shieh et al., J Imunol 183(4):2277-85 (2009); Giomarelli et al., Thromb Haemost 97(6):955-63 (2007); Fife et al., J Clin Invst 116(8):2252-61 (2006); Brocks et al., Immunotechnology 3(3):173-84 (1997); Moosmayer et al., Ther See Immunol 2(10):31-40 (1995). Agonistic scFvs with stimulatory activity have been described (see, e.g., Peter et al., J Biol Chem 25278(38):36740-7 (2003); Xie et al., Nat Biotech 15(8):768-71 (1997); Ledbetter et al., Crit Rev Immunol 17(5-6):427-55 (1997); Ho et al., Bio Chim Biophys Acta 1638(3):257-66 (2003)).

[0042] As used herein, "F(ab)" refers to the fragment of an antibody structure that binds an antigen but is monovalent and does not have the Fc portion; for example, an antibody digested with the enzyme papain produces two F(ab) fragments and an Fc fragment (the heavy (H) chain constant region, the Fc region that does not bind antigen).

[0043] As used herein, "F(ab)2" refers to an antibody fragment produced by pepsin digestion of a whole IgG antibody, which fragment has two antigen-binding (ab') (bivalent) regions, each of which has a distinct antigen-binding domain (ab'). 1 The "F(ab')2" fragment contains two separate amino acid chains of the light (L) chain and part of the heavy chain that are linked together by an S-S bond to bind the antigen, and the remaining parts of the heavy chain are linked together. The "F(ab')2" fragment can be divided into two individual Fab' fragments.

[0044] As used herein, "CDR" is defined as the complementarity determining region amino acid sequence of an antibody, which is the hypervariable region of the immunoglobulin heavy and light chains. See, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 4th USDepartment of Health and Human Services, National Institutes of Health (1987). Generally, an antibody contains three heavy and three light chain CDRs or CDR regions in the variable region. CDRs provide the majority of contact residues for the binding of the antibody to the antigen or epitope. In certain embodiments, the CDR regions are delineated using the Kabat system (Kabat, EA, et al. Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242 (1991)).

[0045] As used herein, the terms "constant region" or "constant domain" are interchangeable and have their common meaning in the art. The constant region is the portion of an antibody, e.g., the carboxyl-terminal portion of the light and / or heavy chain, that is not directly involved in binding the antibody to an antigen but can exhibit various effector functions, e.g., interaction with Fc receptors. The constant region of an immunoglobulin molecule generally has a more conserved amino acid sequence compared to the immunoglobulin variable domain.

[0046] As used herein, "epitope" is a term in the art and can refer to a localized region of an antigen to which an antibody can specifically bind. An epitope can be, for example, contiguous amino acids of a polypeptide (linear or branched epitope), or an epitope can be, for example, the combination of two or more discontinuous regions of a polypeptide or polypeptides (conformational, nonlinear, discontinuous, or noncontinuous epitope).

[0047] As used herein, the term "ligand" refers to a molecule that binds to a receptor. In some embodiments, a ligand binds to a receptor on another cell, allowing recognition and / or interaction between the cells.

[0048] As used herein, the term "affinity" refers to a measure of binding strength. Without being bound by theory, affinity depends on the closeness of stereochemical compatibility between the antibody combination site and the antigenic determinant, the size of the contact area between them, and the distribution of charged and hydrophobic groups. Affinity also includes the term "avidity", which refers to the strength of antigen-antibody binding after the formation of a reversible complex (e.g., either monovalent or multivalent). Methods for calculating the affinity of an antibody to an antigen are known in the art, including the use of binding experiments to calculate affinity. Antibody activity in functional assays (e.g., flow cytometry assays) also reflects antibody affinity. Antibodies and affinities can be characterized by phenotype and compared using functional assays (e.g., flow cytometry assays). Nucleic acid molecules useful in the subject matter disclosed herein include any nucleic acid molecule encoding a polypeptide or a fragment thereof. In certain embodiments, nucleic acid molecules useful in the subject matter disclosed herein include nucleic acid molecules encoding an antibody or an antigen-binding portion thereof. Such nucleic acid molecules do not need to be 100% identical to endogenous nucleic acid sequences, but typically exhibit substantial identity. A polynucleotide having "substantial homology" or "substantial identity" to an endogenous sequence can typically hybridize with at least one strand of a double-stranded nucleic acid molecule. "Hybridize" refers to a pair that forms a double-stranded molecule between complementary polynucleotide sequences (e.g., genes described herein), or portions thereof, under various stringency conditions. (See, e.g., Wahl, GM and SL Berger, Methods Enzymol. 152:399 (1987); Kimmel, AR Methods Enzymol. 152:507 (1987)).

[0049] As used herein, the terms "specifically bind" and "specifically recognize" are similar terms in the context of antibodies and refer to an antibody or antigen-binding fragment thereof that binds to an antigen (e.g., an epitope or immune complex) via its antigen-binding site and does not exclude cross-reactivity of the antibody or antigen-binding fragment with other antigens, as would be understood by one of skill in the art.

[0050] The term "substantially homologous" or "substantially identical" refers to a polypeptide or nucleic acid molecule that exhibits at least 50% or more homology or identity to a reference amino acid sequence (e.g., any one of the amino acid sequences described herein) or nucleic acid sequence (e.g., any one of the nucleic acid sequences described herein). For example, such a sequence is at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% homologous or identical at the amino acid level or nucleic acid to a sequence (e.g., a wild-type or naturally occurring sequence) used for comparison. In some embodiments, a substantially homologous or substantially identical polypeptide contains one or more amino acid substitutions, insertions, or deletions compared to the sequence used for comparison. In some embodiments, a substantially homologous or substantially identical polypeptide contains one or more unnatural amino acids or amino acid analogs, including D-amino acids and retro-inverso amino acids, to replace the homologous sequence.

[0051] Sequence homology or sequence identity is typically measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. In an exemplary approach to determining the degree of identity, the BLAST program may be used, e.g. -3 ~e -100 A probability score of 0.05 indicates closely related sequences.

[0052] As used herein, the term "analog" refers to a structurally related polypeptide or nucleic acid molecule that has the function of a reference polypeptide or nucleic acid molecule.

[0053] As used herein, the term "conservative sequence modifications" refers to amino acid modifications that do not significantly affect or change the binding characteristics of the anti-LGALS3 antibody or antigen-binding fragment thereof disclosed herein that comprises the amino acid sequence. Conservative modifications can include amino acid substitutions, additions, and deletions. Modifications can be introduced into the human scFv of the anti-LGALS3 antibody or antigen-binding fragment thereof disclosed herein by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Amino acids can be classified into groups according to physicochemical properties, such as charge and polarity. Conservative amino acid substitutions are those in which an amino acid residue is replaced with an amino acid within the same group. For example, amino acids can be classified by charge, with positively charged amino acids including lysine, arginine, and histidine, negatively charged amino acids including aspartic acid and glutamic acid, and neutrally charged amino acids including alanine, asparagine, cysteine, glutamine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. Additionally, amino acids can be classified by polarity, with polar amino acids including arginine (basic polar), asparagine, aspartic acid (acidic polar), glutamic acid (acidic polar), glutamine, histidine (basic polar), lysine (basic polar), serine, threonine, and tyrosine, and non-polar amino acids including alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine. Thus, one or more amino acid residues in a CDR region can be replaced with other amino acid residues from the same group, and the altered antibodies can be tested for retained function (i.e., the functions shown in (c)-(l) above) using the functional assays described herein. In certain embodiments, no more than one, no more than two, no more than three, no more than four, no more than five residues in a specified sequence or CDR region are altered.

[0054] As used herein, the term "heterologous nucleic acid molecule or polypeptide" refers to a nucleic acid molecule (e.g., a cDNA, DNA, or RNA molecule) or a polypeptide that is not normally present in a cell or a sample obtained from a cell. The nucleic acid may be from another organism, or may be, for example, an mRNA molecule that is not normally expressed in the cell or sample.

[0055] As used herein, the term "modulate" refers to a positive or negative change. Exemplary modulation includes a change of about 1%, about 2%, about 5%, about 10%, about 25%, about 50%, about 75%, or about 100%.

[0056] As used herein, the term "increase" refers to a positive change of at least about 5%, including but not limited to a positive change of about 5%, about 10%, about 25%, about 30%, about 50%, about 75%, or about 100%.

[0057] As used herein, the term "reduce" refers to a negative change of at least about 5%, including but not limited to a negative change of about 5%, about 10%, about 25%, about 30%, about 50%, about 75%, or about 100%.

[0058] As used herein, an "isolated" polynucleotide or nucleic acid molecule is one that is separated from other nucleic acid molecules that are present in the natural source of the nucleic acid molecule (e.g., mouse or human). Furthermore, an "isolated" nucleic acid molecule, e.g., a cDNA molecule, may be substantially free of other cellular material, or culture medium if produced by recombinant techniques, or substantially free of chemical precursors or other chemicals if chemically synthesized. For example, the language "substantially free" includes preparations of polynucleotides or nucleic acid molecules that have less than about 15%, 10%, 5%, 2%), 1%), 0.5%), or 0.1%) of other materials, e.g., cellular material, culture medium, other nucleic acid molecules, chemical precursors, and / or other chemicals.

[0059] As used herein, the term "isolated cell" refers to a cell that is separated from molecules and / or cellular components that naturally accompany the cell.

[0060] An "effective amount" (or "therapeutically effective amount") is an amount sufficient to produce a beneficial or desired clinical result upon treatment. An effective amount may be administered to a subject in one or more doses. In terms of treatment, an effective amount is an amount sufficient to palliate, ameliorate, stabilize, reverse or slow the progression of a disease (e.g., a neoplasm) or otherwise reduce the pathological consequences of a disease (e.g., a neoplasm). An effective amount is generally determined by a physician on a case-by-case basis and is within the skill of one of ordinary skill in the art. Several factors are typically considered in determining the appropriate dosage to achieve an effective amount. These factors include the age, sex, and weight of the subject, the condition being treated, the severity of the condition, and the form and effective concentration of the engineered immune cells being administered.

[0061] As used herein, the term "neoplasm" refers to a disease characterized by pathological proliferation of cells or tissues and subsequent migration or invasion into other tissues or organs. Neoplastic growth is typically uncontrolled and progressive, occurring under conditions that do not induce proliferation of normal cells or cause their cessation. Neoplasms can affect a variety of cell types, tissues, or organs, including, but not limited to, bladder, colon, bone, brain, breast, cartilage, glia, esophagus, fallopian tube, gallbladder, heart, intestine, kidney, liver, lung, lymph node, nervous tissue, ovary, pleura, pancreas, prostate, skeletal muscle, skin, spinal cord, spleen, stomach, testes, thymus, thyroid, trachea, urinary tract, ureter, urethra, uterus, and vagina, or organs selected from the group consisting of tissues or cell types thereof. Neoplasms include cancers, such as sarcomas, carcinomas, or plasmacytomas (malignant tumors of plasma cells).

[0062] As used herein, the term "treat" or "treatment" refers to clinical intervention in an attempt to change the disease course of the individual or cell being treated, and can be performed either for prophylaxis or during the course of clinical pathology. The therapeutic effect of treatment includes, but is not limited to, preventing the onset or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and remission or improved prognosis. By preventing the progression of a disease or disorder, treatment can not only prevent deterioration due to the disorder in a subject who has been affected or diagnosed, or a subject who is suspected of having the disorder, but also prevent the onset of the disorder or symptoms of the disorder in a subject who is at risk of the disorder or a subject who is suspected of having the disorder.

[0063] As used herein, the term "subject" refers to any animal (e.g., a mammal), including but not limited to a human, non-human primate, rodent, etc. (e.g., the recipient of a particular treatment or from which cells are obtained).

[0064] overview Galectin-3 (LGALS3) is a β-galactoside-binding protein secreted by many cells, but the protein lacks signal sequences for translocation to the endoplasmic reticulum and Golgi compartments and entry into the classical secretory pathway. LGALS3 is found in intracellular and extracellular locations of cells and has pleiotropic biological functions, such as cell growth, cell adhesion, and cell-cell interactions. Depending on its localization and post-translational modifications, such as cleavage and phosphorylation, it can exhibit anti- or pro-apoptotic activity. Cleavage of galectin-3 has been reported to be involved in angiogenic ability and apoptosis resistance. Phosphorylation of galectin-3 regulates its carbohydrate-binding ability.

[0065] Preliminary studies by the inventors in ovarian cancer using artificially synthesized chimeric LGALS3 have established that LGALS3 plays a key role in the growth, spreading and invasive properties of ovarian cancer (Rao, et al. (2017) ACS Chem. Biol. 12(8):2085-2096, incorporated by reference in its entirety). This work utilized both knockdown shRNA suppression of LGALS3 and a chimeric antibody in which the native low affinity LGALS3 carbohydrate binding domain was replaced with an antibody variable region. Knockdown experiments showed that tumor expression of LGALS3 is required for the oncogenic effect of ovarian cancer mucin MUC16 / CA125. When LGALS3 knockdown cells were implanted into nude mice, growth was highly restricted, confirming the essential role of LGALS3 for tumor growth in vivo. Furthermore, the enhancement of growth / invasion by LGALS3 was shown to be extracellular. Invasion studies showed that Matrigel invasion of ovarian cancer cells was dependent on LGALS3. A low-affinity chimeric antibody containing a fusion protein of the unmodified carbohydrate-binding domain of LGALS3 linked to an Fc backbone can block LGALS3 function and prevent Matrigel gel invasion, whereas a chimeric blocking antibody for galectin-1 was not effective. In addition, the introduction of low-affinity chimeric anti-LGALS3 blocking antibodies reduces the in vivo growth of xenografts in mice. These chimeric antibodies had no significant adverse effects on the host mice, consistent with the reported minimal effects of LGALS3 knockout (Wright et al., (2017) J Leukoc Biol 101 (3): 717-726).

[0066] Described herein is an immunization strategy for isolating inhibitory high affinity antibodies against LGALS3. The primary sequence of the LGALS3 carbohydrate binding domain (CBD) is shown in FIG. 2B. Boxes indicate the C, D, and E domains of CBD, which are important for lactosamine binding. Highly conserved amino acids are underlined. R186 (*) is important for GAL3 function. The GAL3CBD sequence is highly homologous to mouse galectin-3, but is distinct from GAL1, GAL7, and GAL9. Each of these family members has a similar sugar-binding domain, but LGALS3 is the only lectin with a polymerization domain. As described in the examples herein, to generate antibodies against LGALS3CBD, mice were immunized with peptides and / or fusion constructs containing LGALS3, or portions thereof including the amino acid sequence of SEQ ID NO:53. Anti-LGALS3 antibodies were screened using peptides and / or fusion constructs containing LGALS3, or portions thereof. The isolated antibodies bind to native human LGALS3 and to the mouse homolog Lgal3.

[0067] In exemplary embodiments, anti-Gal3 antibodies are generated using genetically modified animals expressing chimeric antibody genes. For example, in exemplary embodiments, anti-Gal3 antibodies provided herein are generated using AlivaMab® Mouse Kappa mice and / or AlivaMab® Mouse Lambda mice (also referred to herein, interchangeably, as AlivaMab® Kappa Mice and AlivaMab® Lambda Mice, respectively). Antibodies generated by AlivaMab® Kappa Mice comprise a chimeric immunoglobulin heavy (IgH) chain and a human immunoglobulin kappa (κ) light chain. Antibodies generated by AlivaMab® Lambda Mice comprise a chimeric IgH chain and a human immunoglobulin lambda (λ) light chain. The chimeric IgH chain of the AlivaMab® Mouse antibody is a human variable heavy chain (VH) chain. H) domain, a human diversity heavy chain (DH) domain and a human joining heavy chain (JH) domain, a human constant heavy chain 1 (CH1) domain, a human upper hinge region (except for Cμ, which naturally lacks an upper hinge region), a mouse middle hinge region, a mouse CH2 domain, and a mouse CH3 domain. In an exemplary embodiment, as shown in Figure 8, AlivaMab® Mouse comprises: (1) a fully human variable heavy chain domain (V H ) and the human light chain domain (V L ) and (2) an antibody comprising a human variable heavy domain (V H ) and mouse light chain domain (V L In an exemplary embodiment, hit (e.g., anti-Gal3 antibodies) identification is performed using ELISA. In an exemplary embodiment, enriched clones are identified using an assay to measure inhibition of Gal-3-PE binding to OVCAR3 (FIG. 4). In an exemplary embodiment, a mixture of antibodies containing a fully human variable heavy domain (V H ) and the human light chain domain (V L Identification of antibodies containing the V ) is performed using ELISA. In a further exemplary embodiment, the V ) of antibodies identified from AlivaMab® Mouse is H and / or V L The regions are used to generate additional constructs described herein. In a further exemplary embodiment, the V H and / or V L The CDRs of the regions are used to generate the additional constructs described herein.

[0068] Various studies have demonstrated the role of LGALS3 in tumor biology and inflammation. A partial list of the effects and predicted outcomes of targeting LGALS3 is shown in FIG. 2A. Many of the predicted effects are expected to be beneficial in cancer treatment. Although the effects of LGALS3 are multifaceted, even the complete loss of LGALS3 in knockout mouse models is consistent with near-normal development, and some immunosuppressive effects are not substantially different from TNF inhibitors. Based on these studies, the antibodies provided herein are expected to be useful therapeutic agents for the treatment of LGALS3-mediated diseases and conditions, including but not limited to cancer and inflammation.

[0069] Antibodies and antigen-binding fragments thereof, as well as polypeptides, such as fusion proteins, conjugates, and / or chimeric antigen receptors, that comprise such antibodies or fragments, and cells expressing them, are provided. Some of the antibodies and fragments specifically bind to epitopes of the LGALS3 protein. Such antibodies are referred to herein as "anti-LGALS3 antibodies."

[0070] In some embodiments, the anti-LGALS3 antibodies and antigen-binding fragments thereof described herein inhibit or treat cancer. In some embodiments, the anti-LGALS3 antibodies and antigen-binding fragments thereof described herein inhibit tumor cell invasion and growth. In some embodiments, the anti-LGALS3 antibodies and antigen-binding fragments thereof described herein inhibit tumor angiogenesis. In some embodiments, the anti-LGALS3 antibodies and antigen-binding fragments thereof described herein inhibit metastasis. In some embodiments, the anti-LGALS3 antibodies and antigen-binding fragments thereof described herein inhibit metastatic tumor growth. In some embodiments, the anti-LGALS3 antibodies and antigen-binding fragments thereof described herein inhibit activation of cancer-associated signaling molecules, e.g., AKT or ERK.

[0071] In some embodiments, the anti-LGALS3 antibodies and antigen-binding fragments thereof described herein inhibit the induction of T cell apoptosis by LGALS3.

[0072] In some embodiments, the anti-LGALS3 antibodies and antigen-binding fragments thereof described herein inhibit immunosuppression of T cells.

[0073] In some embodiments, the anti-LGALS3 antibodies and antigen-binding fragments thereof described herein inhibit or treat cardiovascular disease, hi some embodiments, the anti-LGALS3 antibodies and antigen-binding fragments thereof described herein inhibit or treat heart failure, coronary heart disease, or myocardial infarction.

[0074] In some embodiments, the anti-LGALS3 antibodies and antigen-binding fragments thereof described herein inhibit or treat pulmonary fibrosis.

[0075] In some embodiments, the anti-LGALS3 antibodies and antigen-binding fragments thereof described herein inhibit or treat renal disease. In some embodiments, the anti-LGALS3 antibodies and antigen-binding fragments thereof described herein inhibit or treat glomerulonephritis. In some embodiments, the anti-LGALS3 antibodies and antigen-binding fragments thereof described herein inhibit or treat renal cell carcinoma.

[0076] Also provided herein are polynucleotides (e.g., isolated polynucleotides) that include nucleic acid sequences (e.g., complementary DNA (cDNA)) and antigen-binding fragments, heavy chains, or light chains thereof, encoding such antibodies. Also provided are vectors (e.g., expression vectors) and cells (e.g., isolated cells or ex vivo cells) that include nucleic acid sequences (e.g., complementary DNA (cDNA)) and polynucleotides (e.g., isolated polynucleotides) that include antigen-binding fragments, heavy chains, or light chains thereof, encoding such antibodies. Also provided are methods of making such antibodies, antigen-binding fragments, heavy chains, light chains, vectors, and cells. In other aspects, provided herein are methods and uses for anti-LGALS3 antibodies for treating or managing certain conditions or disorders described herein, such as treating or managing cancer. Related compositions (e.g., pharmaceutical compositions), kits, and diagnostic methods are also provided.

[0077] As used herein, the terms "LGALS3," "LGALS3 polypeptide," "LGALS3 peptide," "Gal-3," "Gal-3 polypeptide," or "Gal-3 peptide" are used interchangeably herein to refer to galectin-3 that binds β-galactosides, as well as functional homologs and fragments thereof. GenBank™ Accession No. NM_002306.4 (SEQ ID NO: 11) provides an exemplary human LGALS3 nucleic acid sequence. GenBank™ Accession No. NP_002297.2 (SEQ ID NO: 12) provides an exemplary human LGALS3 amino acid sequence.

[0078] Anti-LGALS3 antibody Anti-LGALS3 antibodies or antigen-binding fragments thereof can include, for example, monoclonal antibodies, polyclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy chain and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-antibody heavy chain pairs, intrabodies, single domain antibodies, monovalent antibodies, single chain antibodies, or single chain variable fragments (scFv), camelid antibodies, affibodies, and disulfide-linked Fvs (dsFv), or fragments thereof. Such antibodies can be produced by methods known in the art.

[0079] A multispecific antibody or fragment thereof refers to an antibody or fragment thereof that can simultaneously bind to at least two targets with different structures, such as two different antigens, two different epitopes on the same antigen, or a hapten and an antigen or epitope. One specificity can be, for example, an antigen or epitope of B cells, T cells, bone marrow, plasma, or mast cells, such as CD3. Another specificity can be a different antigen on the same or different cell type, such as LGALS3. A multispecific, multivalent antibody has more than one binding site, where the binding sites have different specificities (e.g., bispecific diabodies), where one binding site reacts with one antigen and the other binding site reacts with another antigen.

[0080] Bispecific antibodies are antibodies that can bind simultaneously to two targets with different structures. Bispecific antibodies (bsAb) and bispecific antibody fragments (bsFab) have at least one arm that specifically binds to a first target, e.g., LGALS3, and at least one other arm that specifically binds to a second target, e.g., CD3. Various bispecific fusion proteins can be generated using molecular engineering. In one form, the bispecific fusion protein is bivalent, e.g., consisting of (i) an scFv with a single binding site for one antigen, and (ii) an antibody or Fab fragment with a single binding site for a second antigen. In another form, the bispecific fusion protein is tetravalent, e.g., consisting of an IgG with two binding sites for one antigen and two identical scFvs for the second antigen. See, e.g., WO 2011 / 1160119, which is incorporated herein by reference in its entirety.

[0081] Recent methods for generating bispecific monoclonal antibodies include the use of engineered recombinant monoclonal antibodies with additional cysteine ​​residues to cross-link more strongly than the more common immunoglobulin isotypes. See, e.g., FitzGerald et al., Protein Eng. 10(10):1221-1225, 1997. Another approach is to engineer recombinant fusion proteins linking two or more different single chain antibody or antibody fragment segments with the required bispecificity. See, e.g., Coloma et al., Nature Biotech. 15:159-163, 1997. A variety of bispecific fusion proteins can be generated using molecular engineering.

[0082] Bispecific fusion proteins linking two or more different single chain antibodies or antibody fragments can be produced in a similar manner. A variety of fusion proteins can be produced using recombinant methods. In certain embodiments, a flexible linker connects an scFv (e.g., an scFv targeting CD3) to the constant region of the light chain of a monoclonal antibody (e.g., an anti-LGALS3 antibody described herein). The appropriate linker sequence required for the in-frame connection of the heavy chain Fc to the scFv can be generated by PCR reaction using the V L The DNA fragment encoding the scFv is then ligated into a staging vector containing a DNA sequence encoding the CHI domain. The resulting construct is excised and the V kappa domain of the antibody (e.g., anti-LGALS3 antibody) is inserted into the V kappa domain. H The region is ligated into a vector containing a DNA sequence encoding the region. The resulting vector can be used to transfect a suitable host cell, such as a mammalian cell, for expression of the bispecific fusion protein.

[0083] In certain embodiments, the anti-LGALS3 antibodies and fragments thereof described herein can also be used to prepare functional bispecific single chain antibodies (bscAbs), also called diabodies, which can be produced in mammalian cells using recombinant methods. See, for example, Mack et al., Proc. Natl. Acad. Sci. USA, 92:7021-7025, 1995, which is incorporated herein by reference. For example, bscAbs can be produced using recombinant methods by connecting two single chain Fv fragments via a glycine-serine linker. The V of the two antibodies of interest can be expressed as L and V H The domains are isolated using standard PCR methods known in the art. Bispecific single chain antibodies and bispecific fusion proteins are included within the scope of the present technology.

[0084] In certain embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein refer to scFvs. scFv is an art-recognized term. An scFv comprises a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of an immunoglobulin, where the fusion protein retains the same antigen specificity as the whole immunoglobulin. H V via a peptide linker L In certain embodiments, the peptide linker is 5-25, 5-15, 10-20, 10-15, or 15-25 amino acid residues in length. In certain embodiments, the scFv peptide linker exhibits one or more characteristics suitable for a peptide linker known to those of skill in the art. In certain embodiments, the scFv peptide linker comprises amino acids that allow for solubility of the scFv peptide linker, e.g., serine and threonine. In certain embodiments, the scFv peptide linker comprises amino acids that allow for flexibility of the scFv peptide linker, e.g., glycine. In certain embodiments, the scFv peptide linker comprises a V H N-terminus of V L In certain embodiments, the scFv peptide linker is H The C-terminus of V L can be attached to the N-terminus of

[0085] In certain embodiments, the anti-LGALS3 antibody or antigen-binding fragment thereof described herein refers to a chimeric antigen receptor (CAR). CAR is an art-recognized term. CAR can target tumor-associated antigens (e.g., LGALS3). CARs provided herein are typically composed of scFv derived from LGALS3 glycosylation.

[0086] The CAR comprises an antibody, a transmembrane domain, which in some embodiments is a transmembrane domain from a T cell costimulatory molecule (e.g., a transmembrane domain from CD28, CD8, CD38, OX-40, or 4-1BB), and a primary signaling domain, e.g., a T cell receptor (TCR) zeta (ζ) chain cytoplasmic signaling domain. In some embodiments, the CAR further comprises one or more additional regions or domains, such as one or more spacers or linkers, including extracellular spacers, e.g., those derived from an antibody or other cell surface molecule, e.g., a spacer containing one or more of an antibody CH2, CH3, and / or hinge region, or a spacer derived from a CD28 molecule or a CD8 molecule, or other spacers. Also provided herein are cells engineered to express such CARs, e.g., T cells, e.g., those recombinantly expressing such CARs. The CAR-expressing T cells, upon recognition of a tumor expressing LGALS3, preferably induce T cell activation, proliferation, and / or lysis of cells of such tumors.

[0087] Anti-LGALS3 antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, or IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2), or any subclass (e.g., IgG2a or IgG2b) of immunoglobulin molecule. In certain embodiments, the antibodies described herein are IgG antibodies, or a class or subclass thereof. In certain embodiments, the antibodies described herein are IgG1 antibodies. In certain embodiments, the antibodies described herein are IgG2 antibodies. In certain embodiments, the antibodies described herein are IgG2a antibodies. In certain embodiments, the antibodies described herein are IgG2b antibodies. In certain embodiments, the antibodies described herein are IgG3 antibodies. In certain embodiments, the antibodies described herein are IgG4 antibodies. In certain embodiments, the antibodies described herein are a mixture of antibody types or subclasses. In certain embodiments, the antibodies described herein are a mixture of IgG2a and IgG2b antibodies. In a specific embodiment, the antibody is a humanized form of a rodent monoclonal antibody.

[0088] An antigen-binding fragment of an anti-LGALS3 antibody can be a Fab fragment, a F(ab')2 fragment, or a portion of an anti-LGALS3 antibody that contains amino acid residues (e.g., complementarity determining regions (CDRs)) that confer to the anti-LGALS3 antibody its specificity for the antigen. Anti-LGALS3 antibodies can be derived from any animal species, including rodents (e.g., mice, rats, or hamsters) and humans.

[0089] As used herein, the terms "variable region" or "variable domain" are used interchangeably and are common in the art. A variable region typically refers to a portion of an antibody, generally a portion of a light or heavy chain, typically about the amino-terminal 110-120 amino acids in a mature heavy chain and about the amino-terminal 90-100 amino acids in a mature light chain, that differs greatly in sequence between antibodies and is used in the binding and specificity of a particular antibody for its particular antigen. The variability in sequence is concentrated in these regions called complementarity determining regions (CDRs), while the more highly conserved regions in the variable domain are called framework regions (FRs). The CDRs are adjacent to the FRs. In general, the spatial orientation of the CDRs and FRs is as follows, from the N-terminus to the C-terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Without wishing to be bound by any particular mechanism or theory, it is believed that the CDRs of the light and heavy chains are primarily responsible for the interaction and specificity of the antibody with the antigen. In certain embodiments, the variable region is a rodent (e.g., mouse or rat) variable region. In certain embodiments, the variable region is a human variable region. In certain embodiments, the variable region comprises rodent (e.g., mouse or rat) CDRs and human framework regions (FRs). In certain embodiments, the variable region is a primate (e.g., non-human primate) variable region. In certain embodiments, the variable region comprises rodent or mouse CDRs and primate (e.g., non-human primate) framework regions (FRs).

[0090] CDRs are defined in various ways in the art, including Kabat, Chothia, and IMGT, as well as exemplary definitions. The Kabat definition is based on sequence variability (Kabat, Elvin A. et al., Sequences of Proteins of Immunological Interest. Bethesda: National Institutes of Health, 1983). With respect to the Kabat numbering system, (i) V HCDR1 is typically located at amino acid positions 31 to 35 of the heavy chain, and may optionally include one or two additional amino acids after amino acid position 35 (referred to as 35A and 35B in the Kabat numbering scheme); (ii) V H CDR2 is typically located at amino acid positions 50 to 65 of the heavy chain, and (iii) V H CDR2 is typically located at amino acid positions 95-102 of the heavy chain (Kabat, Elvin A. et al., Sequences of Proteins of Immunological Interest. Bethesda: National Institutes of Health, 1983). With respect to the Kabat numbering system, (i) V L CDR1 is typically located at amino acid positions 24 to 34 of the light chain, and (ii) V L CDR2 is typically located at amino acid positions 50 to 56 of the light chain, and (iii) V L CDR3 is typically located at amino acid positions 89-97 of the light chain (Kabat, Elvin A. et al., Sequences of Proteins of Immunological Interest. Bethesda: National Institutes of Health, 1983). As is well known to those skilled in the art, when using the Kabat numbering system, the actual linear amino acid sequence of an antibody variable domain may contain fewer or additional amino acids due to shortening or lengthening of FRs and / or CDRs, and therefore the Kabat number of an amino acid is not necessarily the same as the number of that linear amino acid.

[0091] The Chothia definition is based on the location of structural loop regions (Chothia et al. (1987) J. Mol. Biol. 196:901-917, and U.S. Patent No. 7,709,226). The term "Chothia CDR" and similar terms are art-recognized and refer to antibody CDR sequences as determined according to the method of Chothia and Lesk (1987) J. Mol. Biol. 196:901-917, and are referred to herein as "Chothia CDRs" (see also, e.g., U.S. Patent No. 7,709,226, and Martin, A., "Protein Sequence and Structure Analysis of Antibody Variable Domains," in Antibody Engineering, Kontermann and Diibel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001)). With respect to the Chothia numbering system, V H Using the Kabat numbering system to number the amino acid residues in a region, (i) V H CDR1 is typically located at amino acid positions 26-32 of the heavy chain; and (ii) V H CDR2 is typically located at amino acid positions 53-55 of the heavy chain, and (iii) V H CDR3 is typically located at amino acid positions 96 to 101 of the heavy chain. H Using the Kabat numbering system to number the amino acid residues in a region, (i) V H CDR1 is typically located at amino acid positions 26 to 32 or 34 of the heavy chain; and (ii) V H CDR2 is typically located at amino acid positions 52-56 of the heavy chain (in one embodiment, CDR2 is located at positions 52A-56, with 52A being after position 52); and (iii) V H CDR3 is typically located at amino acid positions 95-102 of the heavy chain (in one embodiment, there are no amino acids at positions numbered 96-100). LUsing the Kabat numbering system to number the amino acid residues in a region, (i) V L CDR1 is typically located at amino acid positions 26 to 33 of the light chain; and (ii) V L CDR2 is typically located at amino acid positions 50-52 of the light chain, and (iii) V L CDR3 is typically located at amino acid positions 91 to 96 of the light chain. L Using the Kabat numbering system to number the amino acid residues in a region, (i) V L CDR1 is typically located at amino acid positions 24 to 34 of the light chain, and (ii) V L CDR2 is typically located at amino acid positions 50 to 56 of the light chain, and (iii) V L CDR3 is typically located at amino acid positions 89-97 of the light chain (in one embodiment, there are no amino acids at positions numbered 96-100). These Chothia CDR positions may vary depending on the antibody and can be determined according to methods known in the art.

[0092] The definition of IMGT is from EVIGT ("IMGT®, the international ImMunoGeneTics information system® website imgt.org, founded and maintained by Marie-Paule Lefranc, Montpellier, France; see e.g., Lefranc, M.-P., 1999, The Immunologist, 7:132-136, and Lefranc, M.-P. et al., 1999, Nucleic Acids Res., 27:209-212, both of which are incorporated by reference in their entireties). With respect to the IMGT numbering system, (i) V H CDR1 is typically located at amino acid positions 25 to 35 of the heavy chain; and (ii) V H CDR2 is typically located at amino acid positions 51 to 57 of the heavy chain, and (iii) V H CDR2 is typically located at amino acid positions 93 to 102 of the heavy chain.L CDR1 is typically located at amino acid positions 27 to 32 of the light chain; and (ii) V L CDR2 is typically located at amino acid positions 50-52 of the light chain, and (iii) V L CDR3 is typically located at amino acid positions 89-97 of the light chain.

[0093] Sequence and structure of LGALS3 antibody In certain embodiments, the present disclosure provides a method for the preparation of a VGALS3 antibody comprising administering to a subject an antibody having the VGALS3 antibody a VGALS3 antibody sequence similar to that described herein, e.g., as shown in Table 6. H Anti-LGALS3 antibodies or antigen-binding fragments thereof are provided, which comprise the CDRs. In certain embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof provided herein comprise the V- or V-amino acids of the anti-LGALS3 antibodies shown in Table 6. H In certain embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof provided herein comprise CDR1 of the anti-LGALS3 antibodies shown in Table 6. H In certain embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof provided herein comprise CDR2 of the anti-LGALS3 antibodies shown in Table 6. H In certain embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof provided herein comprise the V CDR3 of the anti-LGALS3 antibodies shown in Table 6. H It may contain one, two, or all three of the CDRs.

[0094] In certain embodiments, the present disclosure provides a method for the preparation of a VGALS3 antibody comprising administering to a subject an antibody having the VGALS3 antibody a VGALS3 antibody sequence similar to that described herein, e.g., as shown in Table 6. L Anti-LGALS3 antibodies or antigen-binding fragments thereof are provided, which comprise the CDRs. In certain embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof provided herein comprise the V- or V-amino acids of the anti-LGALS3 antibodies shown in Table 6. L In certain embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof provided herein comprise CDR1 of the anti-LGALS3 antibodies shown in Table 6. LIn certain embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof provided herein comprise CDR2 of the anti-LGALS3 antibodies shown in Table 6. L In certain embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof provided herein comprise a V CDR3 of the anti-LGALS3 antibodies in Table 6. L It may contain one, two, or all three of the CDRs.

[0095] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof, comprising a heavy chain immunoglobulin variable domain (V H ) and light chain immunoglobulin variable domains (V L ), (a) V H is selected from the group consisting of SEQ ID NOs: 7, 19, 29, 39, and 49 H - a CDR1 sequence and a V selected from the group consisting of SEQ ID NOs: 8, 20, 30, 40, and 50 H - a CDR2 sequence and a V selected from the group consisting of SEQ ID NOs: 9, 21, 31, 41, and 51 H and / or (b) the CDR3 sequence V L is selected from the group consisting of SEQ ID NOs: 2, 14, 24, 34, and 44 L CDR1 sequence and a V selected from the group consisting of SEQ ID NOs: 3, 15, 25, 35, and 45 L CDR2 sequence and a V sequence selected from the group consisting of SEQ ID NOs: 4, 16, 26, 36, and 46 L and CDR3 sequence.

[0096] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof that specifically binds to galectin-3 (LGALS3), the antibody or antigen-binding fragment thereof comprising a heavy chain variable region (V H ) and the light chain variable region (V L ), (a) V H V comprising SEQ ID NO:7 H Complementarity determining region (CDR) 1 sequence and V comprising SEQ ID NO:8 H CDR2 sequence and V comprising SEQ ID NO:9 H CDR3 sequence, and / or V LV containing SEQ ID NO:2 L CDR1 sequence and V comprising SEQ ID NO:3 L CDR2 sequence and V comprising SEQ ID NO:4 L and (b) a CDR3 sequence, H V comprising SEQ ID NO: 19 H Complementarity determining region (CDR) 1 sequence and V comprising SEQ ID NO: 20 H CDR2 sequence and V comprising SEQ ID NO:21 H CDR3 sequence, and / or V L V comprising SEQ ID NO: 14 L CDR1 sequence and V comprising SEQ ID NO:15 L CDR2 sequence and V comprising SEQ ID NO: 16 L CDR3 sequence, or (c) V H V comprising SEQ ID NO:29 H Complementarity determining region (CDR) 1 sequence and V comprising SEQ ID NO: 30 H CDR2 sequence and V comprising SEQ ID NO:31 H CDR3 sequence, and / or V L V comprising SEQ ID NO: 24 L CDR1 sequence and V comprising SEQ ID NO:25 L CDR2 sequence and V comprising SEQ ID NO:26 L CDR3 sequence, or (d) V H V comprising SEQ ID NO:39 H Complementarity determining region (CDR) 1 sequence and V comprising SEQ ID NO: 40 H CDR2 sequence and V comprising SEQ ID NO:41 H CDR3 sequence, and / or V L V comprising SEQ ID NO:34 L CDR1 sequence and V comprising SEQ ID NO:35 L CDR2 sequence and V comprising SEQ ID NO:36 L CDR3 sequence; or (e) V H V comprising SEQ ID NO:49 H Complementarity determining region (CDR) 1 sequence and V comprising SEQ ID NO: 50 H CDR2 sequence and V comprising SEQ ID NO:51 H CDR3 sequence, and / or V L V comprising SEQ ID NO: 44 LCDR1 sequence and V comprising SEQ ID NO:45 L CDR2 sequence and V comprising SEQ ID NO:46 L and CDR3 sequence.

[0097] In certain embodiments, the anti-LGALS3 antibody or antigen-binding fragment thereof described herein comprises a VGALS3 antibody selected from the group consisting of SEQ ID NO:6, SEQ ID NO:18, SEQ ID NO:28, SEQ ID NO:38, and SEQ ID NO:48. H Includes the area.

[0098] In certain embodiments, the anti-LGALS3 antibody or antigen-binding fragment thereof described herein comprises a VGALS3 antibody selected from the group consisting of SEQ ID NO:1, SEQ ID NO:13, SEQ ID NO:23, SEQ ID NO:33, and SEQ ID NO:43. L Includes the area.

[0099] In some embodiments, V H comprises an amino acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:18, SEQ ID NO:28, SEQ ID NO:38, and SEQ ID NO:48, and / or V L comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 13, SEQ ID NO: 23, SEQ ID NO: 33, and SEQ ID NO: 43. Additionally or alternatively, in some embodiments, the antigen-binding fragment is a Fab, F(ab')2, Fab', Fv, or scFv.

[0100] Additionally or alternatively, in certain embodiments, the antibody or antigen-binding fragment is selected from the group consisting of SEQ ID NO:6 and SEQ ID NO:1, SEQ ID NO:18 and SEQ ID NO:13, SEQ ID NO:28 and SEQ ID NO:23, SEQ ID NO:38 and SEQ ID NO:33, and SEQ ID NO:48 and SEQ ID NO:43, respectively. H Amino acid sequence and V L Contains the amino acid sequence.

[0101] In certain embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein are selected from the group consisting of the V and VIII antibodies of the anti-LGALS3 antibodies shown in Table 6. HIn certain embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein comprise the V region of the anti-LGALS3 antibodies shown in Table 6. L Includes the area.

[0102] In certain embodiments, the anti-LGALS3 antibody or antigen-binding fragment thereof described herein has a V H Domain only or its V L By domain alone or its three Vs H By CDR alone or its three V L The CDRs may be described by themselves. For example, see Rader C et al. (1998) PNAS95:8910-8915, which describes the humanization of a murine anti-avP3 antibody, where identifying complementary light or heavy chains, respectively, from a human light or heavy chain library results in humanized antibody variants with affinities as high as or higher than those of the original antibody, and is incorporated herein by reference in its entirety. Also, certain V H Domain (or V L See Clackson T et al., (1991) Nature 352:624-628, which is incorporated by reference in its entirety, which describes a method for generating antibodies that bind to a specific antigen by screening libraries for complementary variable domains using complementary V domains. H domains to form a complementary V L Libraries for domains (e.g., human V L library), and then selecting the V L domains to generate additional complementary (e.g., human) V H See Kim and Hong (2007) J Microbiol 45:572-577, incorporated herein by reference in its entirety, which describes methods for generating antibodies that bind to specific antigens that can guide domain selection.

[0103] In certain embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein can be humanized antibodies, e.g., humanized forms of rodent antibodies. Humanized antibodies can be humanized using techniques such as, but not limited to, CDR-grafting (EP 239,400, WO 91 / 09967, and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089), chain shuffling (U.S. Pat. No. 5,565,332), veneering or resurfacing (EP 592,106 and 519,596, Padlan (1991) Molecular Immunology 28(4 / 5):489-498, Studnicka et al. (1994) Protein Engineering 7(6):805-814, and Roguska et al. (1994) PNAS91:969-973), as well as, for example, U.S. Pat. al.(1994) J.Mol.Biol.235(3):959-73, Couto et al.(1995) Cancer Res.55(8):1717-22, Roguska et al.(1996) Protein Eng.9(10):895 904, Baca et al. al. (1997) J. Biol. Chem. 272(16):10678-84, Couto et al. (1995) Cancer Res. 55(23Supp):5973s-5977s, Caldas et al. (2000) Protein Eng. 13(5):353-60, Morea et al. (2000) Methods 20(3):267-79, and Tan et al. (2002) J. Immunol. 169:1119-25. See also U.S. Patent Publication No. 2005 / 0042664 A1 (February 24, 2005), each of which is incorporated herein by reference in its entirety.

[0104] In certain embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein are composite human antibodies. Composite human antibodies can be generated, for example, by engineering variable region sequences from fragments of multiple human antibody variable region sequences in a manner that avoids T-cell epitopes, thereby minimizing the immunogenicity of the resulting antibody (see, for example, Baker et al. (2010) Self Nonself l(4):314-322, Bryson et al. (2010) BioDrugs 24(l):l-8, and Jones et al. (2009) Methods Mol Biol. 525:405-23). ​​Such antibodies can include human constant region sequences, for example, human light and / or heavy chain constant regions.

[0105] In certain embodiments, the anti-LGALS3 antibody or antigen-binding fragment thereof described herein can be a deimmunized antibody. A deimmunized antibody is an antibody with T cell epitopes removed. Methods for making deimmunized antibodies have been described. See, for example, Jones et al. Methods Mol Biol. 2009;525:405-23,xiv, and De Groot et al. (2006) Cell. Immunol. 244:148-153).

[0106] In certain embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein comprise any of the three V H CDR and 3 V L CDR (i.e., V H CDR1, V H CDR2, V H CDR3, V L CDR1, V L CDR2, and V LThe humanized immunoglobulin comprises a humanized CDR3, a human-derived framework region, and a human-derived constant region. Non-limiting examples of human framework regions are described in the art, see, for example, Kabat et al. (1991) Sequences of Proteins of Immunological Interest Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242). In certain embodiments, the anti-LGALS3 antibody or antigen-binding fragment thereof described herein comprises a framework region (e.g., V, VL ... L Domain and / or V H In certain embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein comprise framework regions (e.g., framework regions of the V domain) that are primate (e.g., non-human primate) framework regions or are derived from primate (e.g., non-human primate) framework regions. L Domain and / or V H For example, CDRs from an antigen-specific non-human antibody, typically from a rodent (e.g., mouse or rat), are grafted onto a homologous human or non-human primate (e.g., Old World apes, e.g., Pan troglodytes, Pan paniscus, or Gorilla, Pan troglodytes, Old World monkeys, e.g., monkeys from the genus Macaca, or cynomolgus Macaca cynomolgus). Non-human primate framework sequences are described in U.S. Patent Application Publication No. 2005 / 0208625.

[0107] In specific embodiments, the V H V in the region H CDR1, V H CDR2 and / or V H CDR3 position and / or V L V in the regionL CDR1, V L CDR2 and / or V L The CDR3 position may be varied by 1, 2, 3, 4, 5, 6 or more amino acid positions, so long as specific binding to LGALS3 or a portion thereof containing the carbohydrate binding domain is maintained (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, up to at least 95%).

[0108] In another embodiment, the V H V in the region H CDR1, V H CDR2 and / or V H CDR3 length and / or V L V in the region L CDR1, V L CDR2 and / or V L The length of the CDR3 can vary (e.g., be longer or shorter) by 1, 2, 3, 4, 5, 6 or more amino acids, so long as specific binding to LGALS3 or a portion thereof containing the carbohydrate binding domain is maintained (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, up to at least 95%).

[0109] In another embodiment, the V H CDR1, V H CDR2, V H CDR3, V L CDR1, V L CDR2 and / or V LThe amino and / or carboxy termini of CDR3 may be extended or shortened by 1, 2, 3, 4, 5, 6 or more amino acids compared to one or more of the CDRs described herein, so long as specific binding to LGALS3 or a portion thereof containing the carbohydrate binding domain is maintained (e.g., by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%).

[0110] Any method known in the art can be used to determine whether specific binding to LGALS3 is maintained (eg, ELISA binding assay, SPR analysis, or FACS analysis).

[0111] In specific aspects, provided herein are anti-LGALS3 antibodies or antigen-binding fragments thereof that include the heavy and / or light chains of the antibodies, e.g., only the heavy chain, only the light chain, or both the heavy and light chains.

[0112] In some embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein comprise a heavy chain, and the amino acid sequence of the variable region of the heavy chain is selected from the group consisting of any one of the V and VIII antibodies listed in Table 6. H CDR1, V H CDR2, and V H In certain embodiments, the anti-LGALS3 antibody or antigen-binding fragment thereof described herein comprises a heavy chain, and the heavy chain variable region has the amino acid sequence of SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, respectively; SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:21, respectively; SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:31, respectively; SEQ ID NO:39, SEQ ID NO:40, and SEQ ID NO:41, respectively; or SEQ ID NO:49, SEQ ID NO:50, and SEQ ID NO:51, respectively. H CDR1, V H CDR2, and V H The heavy chain constant region, including CDR3, is a human heavy chain constant region.

[0113] In specific embodiments, the anti-LGALS3 antibody or antigen-binding fragment thereof described herein comprises a heavy chain, wherein the amino acid sequence of the variable region of the heavy chain comprises the amino acid sequence of SEQ ID NO:6, SEQ ID NO:18, SEQ ID NO:28, SEQ ID NO:38, or SEQ ID NO:48, and the constant region of the heavy chain is a human heavy chain constant region. Non-limiting examples of human constant region sequences are described in the art, see, e.g., Kabat EA et al. (1991), supra.

[0114] In some embodiments, the heavy chain of the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein can be an alpha (α), delta (δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain. In another specific embodiment, the heavy chain of the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein can comprise a human alpha (α), delta (δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain. In some embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein comprise a heavy chain, and the amino acid sequence of the variable region of the heavy chain is selected from the group consisting of the V and VD of any one of the antibodies listed in Table 6. H CDR1, V H CDR2, and V H The heavy chain constant region, including the CDR3, is an alpha (α), delta (δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain.

[0115] In certain embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein comprise a heavy chain, wherein the amino acid sequences of the variable region of the heavy chain are V having the amino acid sequences of SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, respectively; SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:21, respectively; SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:31, respectively; SEQ ID NO:39, SEQ ID NO:40, and SEQ ID NO:41, respectively; or SEQ ID NO:49, SEQ ID NO:50, and SEQ ID NO:51, respectively. H CDR1, V H CDR2, and V Hand the heavy chain constant region comprises a human alpha (α), delta (δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain. In a specific embodiment, an anti-LGALS3 antibody or antigen-binding fragment thereof described herein comprises a heavy chain, the amino acid sequence of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:6, SEQ ID NO:18, SEQ ID NO:28, SEQ ID NO:38, or SEQ ID NO:48, and the heavy chain constant region is a human alpha (α), delta (δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain.

[0116] In certain embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein comprise a light chain, and the amino acid sequence of the variable region of the light chain is selected from the group consisting of the V and VL domains of any one of the antibodies listed in Table 6. L CDR1, V L CDR2, and V L In certain embodiments, the anti-LGALS3 antibody or antigen-binding fragment thereof described herein comprises a light chain, and the variable region of the light chain has the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, respectively; SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16, respectively; SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26, respectively; SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36, respectively; or SEQ ID NO:44, SEQ ID NO:45, and SEQ ID NO:46, respectively. L CDR1, V L CDR2, and V L CDR3, and the constant region of the light chain is a human light chain constant region. In a specific embodiment, the anti-LGALS3 antibody or antigen-binding fragment thereof described herein comprises a light chain, the amino acid sequence of the variable region of the light chain comprises the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 13, SEQ ID NO: 23, SEQ ID NO: 33, or SEQ ID NO: 43, and the constant region of the light chain is a human light chain constant region.

[0117] In some embodiments, the light chain of the anti-LGALS3 antibody or antigen-binding fragment thereof described herein is a kappa light chain. In another specific embodiment, the light chain of the anti-LGALS3 antibody or antigen-binding fragment thereof described herein is a lambda light chain. In yet another specific embodiment, the light chain of the anti-LGALS3 antibody or antigen-binding fragment thereof described herein is a human kappa light chain or a human lambda light chain. In certain embodiments, the anti-LGALS3 antibody or antigen-binding fragment thereof described herein comprises a light chain, and the amino acid sequence of the variable region of the light chain is selected from the group consisting of the V and VL chains of any one of the antibodies listed in Table 6. L CDR1, V L CDR2, and V L In certain embodiments, the anti-LGALS3 antibody or antigen-binding fragment thereof described herein comprises a light chain, and the variable region of the light chain has the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, respectively; SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16, respectively; SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26, respectively; SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36, respectively; or SEQ ID NO:44, SEQ ID NO:45, and SEQ ID NO:46, respectively. L CDR1, V L CDR2, and V L CDR3, and the constant region of the light chain is a kappa or lambda light chain constant region. In a specific embodiment, the anti-LGALS3 antibody or antigen-binding fragment thereof described herein comprises a light chain, the amino acid sequence of the variable region of the light chain comprises the amino acid sequence of SEQ ID NO:1, SEQ ID NO:13, SEQ ID NO:23, SEQ ID NO:33, or SEQ ID NO:43, and the constant region of the light chain is a kappa or lambda light chain constant region.

[0118] In specific embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein comprise a heavy chain variable region (V H ) and the light chain variable region (V L), wherein the constant region is of a type found in an IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, or a human IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule. In another specific embodiment, the anti-LGALS3 antibody or antigen-binding fragment thereof described herein comprises a V H and V L and the constant region is of the type found in an IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, of any class of immunoglobulin molecule (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or any subclass (e.g., IgG2a and IgG2b). In certain embodiments, the constant region is of the type found in a human IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, of any class of human immunoglobulin molecule (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or any subclass (e.g., IgG2a and IgG2).

[0119] In certain embodiments, the epitope of the antibody can be determined, for example, by NMR spectroscopy, X-ray diffraction crystallography studies, ELISA assays, hydrogen / deuterium exchange in combination with mass spectrometry (e.g., MALDI mass spectrometry), array-based oligopeptide scanning assays, and / or mutagenesis mapping (e.g., site-directed mutagenesis mapping). For X-ray crystallography, crystallization can be achieved using any of the methods known in the art (e.g., Giege R et al. (1994) Acta Crystallogr D Biol Crystallogr 50 (Pt 4): 339-350, McPherson A (1990) Eur J Biochem 189: 1-23, Chayen NE (1997) Structure 5: 1269-1274, McPherson A (1976) J Biol Chem 251: 6300-6303). Antibody:antigen crystals can be studied using well-known X-ray diffraction techniques and refined using computer software such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc., see e.g., Meth Enzymol (1985) volumes 114 & 115, eds. Wyckoff HW et al., U.S. Patent Application No. 2004 / 0014194) and BUSTER (Bricogne G (1993) Acta Crystallogr D Biol Crystallogr 49 (Pt 1): 37-60; Bricogne G (1997) Meth Enzymol 276A: 361-423, eds. Carter CW, Roversi P et al. (2000) Acta Crystallogr D Biol Crystallogr 56 (Pt 10): 1316-1323). Mutagenesis mapping studies can be accomplished using any method known to those skilled in the art. For a description of mutagenesis techniques, including alanine scanning mutagenesis techniques, see, for example, Champe M et al. (1995) and Cunningham BC & Wells JA (1989).In addition, antibodies that recognize and bind to the same or overlapping epitopes can be identified using conventional techniques such as immunoassays, for example, by showing the ability of one antibody to block the binding of another antibody to a target antigen (e.g., competitive binding assays). Competitive binding assays can also be used to determine whether two antibodies have similar binding specificity to an epitope. Competitive binding can be determined in an assay in which the immunoglobulin under test inhibits the specific binding of a reference antibody to a common antigen. Many types of competitive binding assays are available, such as solid phase direct or indirect radioimmunoassays (RIA), solid phase direct or indirect enzyme immunoassays (EIA), sandwich competition assays (see Stahli C et al. (1983) Methods Enzymol 9:242-253), solid phase direct biotin-avidin EIA (see Kirkland TN et al. (1986) J Immunol 137:3614-9), solid phase direct labeled assays, solid phase direct labeled sandwich assays (see Harlow E & Lane D, (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Press), solid phase direct labeled RIA using 1-125 label (Morel GA et al. (1988) Mol Immunol 25(1):7-15), solid phase direct biotin-avidin EIA (Cheung RC et al. (1990) Virology 176:546-52), and direct labeling RIA are known. (Moldenhauer G et al. (1990) Scand J Immunol 32:77-82). Typically such assays involve the use of purified antigen bound to a solid surface or cells containing either of these, an unlabeled test immunoglobulin and a labeled reference immunoglobulin. Competitive inhibition can be measured by determining the amount of label bound to the solid surface or cells in the presence of the test immunoglobulin. Usually the test immunoglobulin is present in excess.Typically, when present in excess, a competing antibody will inhibit specific binding of a reference antibody to a common antigen by at least 50-55%, 55-60%, 60-65%, 65-70%) 70-75%, or more. Competitive binding assays can be configured in a number of different formats using either labeled antigens or labeled antibodies. In a common aspect of this assay, the antigen is immobilized on a 96-well plate. The ability of unlabeled antibodies to block binding of the labeled antibody to the antigen is then measured using radioactive or enzymatic labels. For further details, see, e.g., Wagener C et al. (1983) J Immunol 130:2308-2315, Wagener C et al. (1984) J Immunol Methods 68:269-274, Kuroki M et al. (1990) Cancer Res 50:4872-4879, Kuroki M et al. (1992) Immunol Invest 21:523-538, Kuroki M et al. (1992) Hybridoma 11:391-407 and Antibodies: A Laboratory Manual, Ed Harlow E & Lane D editors supra, 386-389.

[0120] In certain aspects, competitive binding assays can be used to determine whether an antibody is competitively blocked (e.g., in a dose-dependent manner) by another antibody when two antibodies recognize the same or sterically overlapping epitopes in a competitive binding assay, such as a competitive ELISA assay, e.g., whether an antibody essentially binds to the same epitope as a reference antibody or to an overlapping epitope, which can be configured in any number of different formats using either labeled antigen or labeled antibody. In certain embodiments, antibodies can be tested in competitive binding assays with anti-LGALS3 antibodies or antigen-binding fragments thereof described herein.

[0121] In another aspect, provided herein is an antibody that does not compete (e.g., in a dose-dependent manner) with an anti-LGALS3 antibody or antigen-binding fragment thereof described herein from binding to LGALS3 as determined using an assay known to one of skill in the art or as determined using an assay described herein (e.g., ELISA). In another aspect, provided herein is an antibody that does not competitively inhibit (e.g., in a dose-dependent manner) an anti-LGALS3 antibody or antigen-binding fragment thereof described herein from binding to LGALS3 as determined using an assay known to one of skill in the art or as determined using an assay described herein (e.g., ELISA).

[0122] In certain embodiments, provided herein are antibodies that compete with the antibodies described herein for binding to the same extent that the anti-LGALS3 antibodies, or antigen-binding fragments thereof, described herein, self-compete for binding to LGALS3. In certain embodiments, provided herein are first antibodies that compete with the anti-LGALS3 antibodies, or antigen-binding fragments thereof, described herein, for binding to LGALS3, where the competition is demonstrated as a reduction in binding of the first antibody to the epitope by greater than 80% (e.g., 85%, 90%, 95%, or 98%, or 80%-85%, 80%-90%, 85%-90%, or 85%-95%). In specific aspects, provided herein are VGALS3 antibodies comprising the amino acid sequences set forth in Table 6. H CDR1, V H CDR2 and / or V H V containing CDR3 H and / or V comprising an amino acid sequence as set forth in Table 6 L CDR1, V L CDR2 and / or V L V containing CDR3 L In a specific embodiment, provided herein is an anti-LGALS3 antibody or antigen-binding fragment thereof that competes (e.g., in a dose-dependent manner) for specific binding to LGALS3 with an anti-LGALS3 antibody or antigen-binding fragment thereof comprising: H Domains and / or Vs listed in Table 6L The present invention provides an anti-LGALS3 antibody or antigen-binding fragment thereof that competes (eg, in a dose-dependent manner) for specific binding to LGALS3 with an anti-LGALS3 antibody or antigen-binding fragment thereof comprising:

[0123] In a specific embodiment, the present invention provides a VVLK antibody comprising an amino acid sequence as set forth in Table 6. H CDR1, V H CDR2 and / or V H V containing CDR3 H and / or V comprising an amino acid sequence as set forth in Table 6 L CDR1, V L CDR2 and / or V L V containing CDR3 L In a specific embodiment, an anti-LGALS3 antibody or antigen-binding fragment thereof is provided that binds to the same epitope as or an overlapping epitope of an antibody comprising: H Domains and / or Vs listed in Table 6 L The present invention provides an anti-LGALS3 antibody or antigen-binding fragment thereof that binds to the same epitope or an overlapping epitope of an antibody comprising:

[0124] Assays known to those of skill in the art or described herein (e.g., X-ray crystallography, ELISA assays, surface plasmon resonance (SPR) assays) can be used to determine whether two antibodies bind to the same epitope. Affinity can be measured by, but is not limited to, the equilibrium dissociation constant (K D ), and equilibrium association constant (KA). D can be determined by techniques known to those skilled in the art, such as biolayer interferometry.

[0125] In certain embodiments, the epitope bound by an anti-LGALS3 antibody or antigen-binding fragment thereof described herein is used as an immunogen to generate antibodies, which in some embodiments include all or a portion of the carbohydrate-binding domain of LGALS3.

[0126] Functional characterization of anti-LGALS3 antibodies In certain embodiments, the anti-LGALS3 antibody or antigen-binding fragment thereof described herein is administered in an amount of about 0.5×10 -3 / sec, 1×10 -3 / sec, 1.5×10 -3 / sec, 2×10 -3 / sec, 2.5×10 -3 / sec, 3×10 -3 / sec, 4×10 -3 / sec, 5×10 -3 / sec, 6×10 -3 / sec, 7×10 -3 / sec, 8×10 -3 / sec, 9×10 -3 / sec, l×10 -4 / sec, 2×10 -4 / sec, 3×10 -4 / sec, 4×10 -4 / sec, 5×10 -4 / sec, 6×10 -4 / sec, 7×10 -4 / sec, or 8 x 10 -4 k / sec d In some embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein bind to LGALS3 at about 0.5×10 -3 / sec~8×10 -4 Binds to LGALS3 with a kd of 100 / sec.

[0127] In certain embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein have a concentration of at least or about 2.5×10 4 / sec, 3×10 4 / sec, 3.5×10 4 / sec, 4×10 4 / sec, 4.5×10 4 / sec, 5×10 4 / sec, 5.5×10 4 / sec, 6×10 4 / sec, 6.5×10 4 / sec, 7×10 4 / sec, 7.5×10 4 / sec, 8×10 4 / sec, 9x10 4 / sec, or 9 x 105 In certain embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein bind to LGALS3 with a binding affinity of at least or about 2.5×10 4 / sec~9×10 5 Binds to LGALS3 at ka / sec.

[0128] In certain embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein have a K of about 1000 nM, 500 nM, 100 nM, 50 nM, 25 nM, 20 nM, 15 nM, 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.5 nM, 0.1 nM, or 0.05 nM or less. D In some embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein bind to LGALS3 with a K of about 1 nM to about 1000 nM. D In some embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein bind to LGALS3 with a K of about 1 nM to about 100 nM. D In some embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein bind to LGALS3 with a K of about 5 nM to about 50 nM. D In some embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein bind to LGALS3 with a K of about 5 nM to about 20 nM. D and binds to LGALS3.

[0129] In certain embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof provided herein bind to LGALS3 at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 250, 500, or 1000 times more than an isotype control antibody binds to LGALS3. An isotype control antibody is an art-recognized term that refers to an antibody that lacks specificity for a target but matches the class and type of a primary antibody (e.g., an anti-LGALS3 antibody or antigen-binding fragment thereof provided herein). An isotype control is used as a negative control to help distinguish non-specific background signals from specific antibody signals.

[0130] In certain embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein bind to LGALS3 with an affinity that is increased by 100-1,000 fold compared to the native ligand binding.

[0131] Assays for determining anti-LGALS3 antibody or antigen-binding fragment-mediated inhibition of Gal-3-PE binding to cancer cells are known to those skilled in the art. For example, OVCAR3 cells can be purchased from ATCC, Manassas, VA. PE-labeled Gal-3 (Gal-3-PE) can be purchased from Abcam, Cambridge, MA. Assays for inhibition of Gal-3-PE binding to OVCAR3 cells can be performed as follows. Briefly, cultured SKOV3 cells were trypsinized and washed with culture medium. 500, 1000, or 2000 cells were plated in a 24-well plate and grown at 37° C. in a 5% CO2 humidified incubator. After incubation, the cells were incubated with various concentrations of Gal-3-PE and test antibodies. Binding of Gal-3-PE to OVCAR3 cells was detected. The degree of inhibition was calculated using a standard curve such as that in FIG. 3A.

[0132] In specific embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein inhibit Gal-3-PE binding to cancer cells by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% as compared to mock-treated mice, as assessed by methods described herein or known to one of skill in the art. In specific embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein inhibit Gal-3-PE binding to cancer cells by at least about 25% or 35%, optionally by about 75%, as compared to mock-treated mice, as assessed by methods described herein or known to one of skill in the art. In specific embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein inhibit Gal-3-PE binding to cancer cells by at least about 1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold compared to mock-treated cancer cells as assessed by methods described herein or known to one of skill in the art. Mock-treated cancer cells can be treated with, for example, phosphate buffered saline or a control (e.g., an anti-IgG antibody).

[0133] Antibody conjugates In some embodiments, the anti-LGALS3 antibodies, or antigen-binding fragments thereof, provided herein are conjugated to another molecule, eg, an organic moiety, a detectable label, and / or an isotope.

[0134] In certain embodiments, provided herein is a conjugate of an anti-LGALS3 antibody or antigen-binding fragment thereof, in which the anti-LGALS3 antibody or antigen-binding fragment thereof is conjugated to one or more agents, such as imaging agents or cytotoxic agents. Also provided herein is a bispecific antibody conjugate, in which the bispecific antibody is conjugated to one or more agents, such as imaging agents or cytotoxic agents. Also provided herein is an antibody heavy chain conjugate, in which the antibody heavy chain is conjugated to one or more agents, such as imaging agents or cytotoxic agents. Also provided herein is an antibody light chain conjugate, in which the antibody light chain is conjugated to one or more agents, such as imaging agents or cytotoxic agents. Also provided herein is a fusion protein conjugate, in which the fusion protein is conjugated to an agent, such as imaging agents or cytotoxic agents. In certain embodiments, the agent is covalently or non-covalently conjugated. In certain embodiments, the imaging agent is a detectable label, such as a chromogenic, enzymatic, radioisotope, isotopic, fluorescent, toxic, chemiluminescent, nuclear magnetic resonance imaging agent, or other label.

[0135] Non-limiting examples of suitable chromogenic labels include diaminobenzidine and 4-hydroxyazo-benzene-2-carboxylic acid.

[0136] Non-limiting examples of suitable enzyme labels include malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast-alcohol dehydrogenase, alpha-glycerol phosphate dehydrogenase, triose phosphate isomerase, peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase, and acetylcholinesterase.

[0137] Non-limiting examples of suitable radioisotope labels include: 3 H, 111 In, 125 I, 131 I, 32 P, 35 S, 14 C. 51 Cr, 57 To, 58 Co, 59 Fe, 75 Se, 152 EU, 90 Y, 67 Cu, 217 Ci, 211 At, 212 Pb, 47 Sc, 223 Ra, 224 Ra, 89 Zr, 177 Lu, and 109 In certain embodiments, 111 In in the liver 125 I or 131 To avoid the problem of dehalogenation of the I-labeled anti-LGALS3 antibody or its antigen-binding fragment, it is used for in vivo imaging. 111 In has a more favorable gamma emission energy for imaging (Perkins et al. (1985) Eur. J. Nucl. Med. 70:296-301; Carasquillo et al. (1987) J. Nucl. Med. 25:281-287). For example, In linked to monoclonal antibodies with 1-(P-isothiocyanatobenzyl)-DPTA 111 In is poorly taken up by non-tumor tissues, especially the liver, thus enhancing the specificity of tumor localization (Esteban et al., (1987) J. Nucl. Med. 28:861-870).

[0138] Non-limiting examples of suitable non-radioactive isotope labels include: 157 Gd, 55 Mn, 162 Dy, 52 Tr, and 56 Fe is one example.

[0139] Non-limiting examples of suitable fluorescent labels include: 152 These include Eu labels, fluorescein labels, isothiocyanate labels, rhodamine labels, phycoerythrin labels, phycocyanin labels, allophycocyanin labels, green fluorescent protein (GFP) labels, o-phthaldehyde labels, and fluorescamine labels.

[0140] Non-limiting examples of chemiluminescent labels include a luminol label, an isoluminol label, an aromatic acridinium ester label, an imidazole label, an acridinium salt label, an oxalate ester label, a luciferin label, a luciferase label, and an aequorin label.

[0141] Non-limiting examples of nuclear magnetic resonance contrast agents include heavy metal nuclei such as Gd, Mn, and iron.

[0142] Techniques known to those skilled in the art for conjugating the above-mentioned labels to the above-mentioned anti-LGALS3 antibodies or antigen-binding fragments thereof, bispecific antibodies, antibody heavy chains, antibody light chains, and fusion proteins are described, for example, in Kennedy et al. (1976) Clin.Chm.Acta 70:1-31, and Schurs et al. (1977) Clin.Chm.Acta 81:1-40. The coupling techniques mentioned in the latter are the glutaraldehyde method, the periodate method, the dimaleimide method, the m-maleimide benzyl-N-hydroxy-succinimide ester method, all of which are incorporated herein by reference.

[0143] Non-limiting examples of cytotoxic agents include cytostatic or cytocidal agents, radioactive metal ions, e.g., alpha emitters, and toxins, e.g., Pseudomonas exotoxin, abrin, cholera toxin, ricin A, and diphtheria toxin.

[0144] In certain embodiments, the agent is a diagnostic agent. A diagnostic agent is an agent useful for diagnosing or detecting disease by locating cells containing an antigen. Useful diagnostic agents include, but are not limited to, radioisotopes, dyes (such as those containing biotin-streptavidin complexes), contrast agents, fluorescent compounds or molecules, and enhancement agents for magnetic resonance imaging (MRI) (e.g., paramagnetic ions). U.S. Patent No. 6,331,175 describes MRI techniques and the preparation of antibodies conjugated to MRI enhancement agents, and is incorporated by reference in its entirety. In some embodiments, the diagnostic agent is selected from the group consisting of radioisotopes, enhancement agents for use in magnetic resonance imaging, and fluorescent compounds. To incorporate radioactive metals or paramagnetic ions into an anti-LGALS3 antibody or antigen-binding fragment thereof, it may be necessary to react with a reagent that has a long tail to which multiple chelating groups are attached for binding to the ions. Such tails may be polymers such as polylysine, polysaccharides, or other derivatized or derivatizable chains having pendant groups that can be attached to chelating groups, e.g., ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), porphyrins, polyamines, crown ethers, bis-thiosemicarbazones, polyoximes, and other groups known to be useful for this purpose. The chelating agent is linked to the antibody using standard chemistries. The chelating agent is usually linked to the antibody by a group that allows for the formation of a bond to the molecule with minimal loss of immunoreactivity, minimal aggregation and / or other internal cross-linking; less conventional methods and reagents for conjugating chelating agents to antibodies are disclosed in U.S. Patent No. 4,824,659 to Hawthorne, entitled "Antibody Conjugates," issued April 25, 1989, the disclosure of which is incorporated herein by reference in its entirety. Particularly useful metal-chelate combinations include 2-benzyl-DTPA and its monomethyl and cyclohexyl analogs, which are used in conjunction with diagnostic isotopes for radioimaging.The same chelators are useful for MRI when used with the anti-LGALS3 antibodies or antigen-binding fragments provided herein when complexed with non-radioactive metals such as manganese, iron and gadolinium. Macrocyclic chelators such as NOTA, DOTA and TETA are useful when used with a variety of metals and radiometals, most particularly gallium, yttrium and copper radionuclides, respectively. Such metal-chelator complexes can be made very stable by adjusting the ring size to the metal of interest. Other ring-type chelators, such as macrocyclic polyethers, are included herein for stably binding nuclides such as 223Ra for RAIT.

[0145] In certain embodiments, the drug is an organic drug. Such organic drugs can produce conjugates with improved pharmacokinetic properties (e.g., increased in vivo serum half-life). The organic moiety can be a hydrophilic polymer group, a fatty acid group, or a fatty acid ester group. As used herein, the term "fatty acid" encompasses mono- and di-carboxylic acids. As used herein, "hydrophilic polymer group" refers to an organic polymer that is more soluble in water than in octane, such as polylysine. Suitable hydrophilic polymers for modifying the anti-LGALS3 antibodies or antigen-binding fragments thereof provided herein may be linear or branched, and include, for example, polyalkane glycols (e.g., polyethylene glycol, (PEG), monomethoxy-polyethylene glycol, and polypropylene glycol), carbohydrates (e.g., dextran, cellulose, oligosaccharides, and polysaccharides), polymers of hydrophilic amino acids (e.g., polylysine, polyarginine, and polyaspartate), polyalkane oxides (e.g., polyethylene oxide and polypropylene oxide), and polyvinylpyrrolidone. In certain embodiments, the hydrophilic polymer modifying an anti-LGALS3 antibody or antigen-binding fragment thereof, bispecific antibody, antibody heavy chain, antibody light chain, or fusion protein provided herein has a molecular weight of about 800 to about 150,000 daltons as a separate molecular entity. For example, PEG 5000 and PEG20,000 (where the subscript is the average molecular weight of the polymer in Daltons) can be used. The hydrophilic polymer group can be substituted with 1 to about 6 alkyl, fatty acid, or fatty acid ester groups. Hydrophilic polymers substituted with fatty acid or fatty acid ester groups can be prepared by using appropriate methods. For example, a polymer containing an amine group can be linked to a carboxylate of a fatty acid or fatty acid ester, and an activated carboxylate on the fatty acid or fatty acid ester (e.g., activated with N,N-carbonyldiimidazole) can be linked to a hydroxyl group on the polymer.

[0146] Fatty acids and fatty acid esters suitable for modifying the anti-LGALS3 antibody or antigen-binding fragment thereof, bispecific antibody, antibody heavy chain, antibody light chain, or fusion protein provided herein may be saturated or contain one or more unsaturated units. Fatty acids suitable for modifying the anti-LGALS3 antibody or antigen-binding fragment thereof, bispecific antibody, antibody heavy chain, antibody light chain, or fusion protein provided herein include, for example, n-dodecanoate, n-tetradecanoate, n-octadecanoate, n-eicosanoate, n-docosanoate, n-triacontanoate, n-tetracontanoate, cis-delta-9-octadecanoate, all cis-delta-5,8,11,14-eicosatetranoate, octanedioic acid, tetradecanedioic acid, octadecanedioic acid, docosanedioic acid, and the like. Suitable fatty acid esters include monoesters of dicarboxylic acids containing linear or branched lower alkyl groups. The lower alkyl group can contain 1 to about 12, preferably 1 to about 6, carbon atoms.

[0147] The conjugates provided herein can be prepared using suitable methods, for example, by reaction with one or more modifying agents. As used herein, an "activating group" is a chemical moiety or functional group that can react with a second chemical group under appropriate conditions, thereby forming a covalent bond between the modifying agent and the second chemical group. For example, amine-reactive activating groups include electrophilic groups such as tosylate, mesylate, halo (chloro, bromo, fluoro, iodo), N-hydroxysuccinimidyl ester (NHS), and the like. Activating groups that can react with thiols include, for example, maleimide, iodoacetyl, acryloyl, pyridyl disulfide, 5-thiol-2-nitrobenzoic acid thiol (TNB-thiol), and the like. Aldehyde functional groups can be linked to amine or hydrazine-containing molecules, and azide groups can be reacted with trivalent phosphorus groups to form phosphoramidate or phosphorimide bonds. Suitable methods for introducing activating groups into molecules are known in the art (see, for example, Hernanson, GT, Bioconjugate Techniques, Academic Press: San Diego, Calif. (1996)). Activating groups can be attached directly to an organic group (e.g., hydrophilic polymer, fatty acid, fatty acid ester) or to a linker moiety, e.g., a divalent C-C 12The linker moiety may be linked via a group, and one or more carbon atoms may be replaced with a heteroatom such as oxygen, nitrogen, or sulfur. Suitable linker moieties include, for example, tetraethylene glycol, (CH2)3, and NH. Modifiers containing linker moieties can be generated, for example, by reacting mono-Boc-alkyldiamines (e.g., mono-Boc-ethylenediamine or mono-Boc-diaminohexane) with fatty acids in the presence of l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) to form amide bonds between the free amine and the fatty acid carboxylate. The Boc protecting group can be removed from the product by treatment with trifluoroacetic acid (TFA) to expose a primary amine, which can be coupled with another carboxylate as shown, or reacted with maleic anhydride, and the resulting product can be cyclized to generate an activated maleimide derivative of a fatty acid. (See, for example, Thompson, et al., WO92 / 16221, the teachings of which are incorporated herein by reference in their entirety.)

[0148] A "modifying agent" can refer to a suitable organic group (e.g., hydrophilic polymers, fatty acids, and fatty acid esters) that contains an activating group. For example, an organic moiety can be attached to an anti-LGALS3 antibody or antigen-binding fragment thereof in a non-site-specific manner by using an amine-reactive modifying agent, such as an N-hydroxysuccinimide ester of PEG. A modified anti-LGALS3 antibody or antigen-binding fragment thereof can also be prepared by reducing disulfide bonds (e.g., intrachain disulfide bonds) of an anti-LGALS3 antibody or antigen-binding fragment thereof, a bispecific antibody, an antibody heavy chain, an antibody light chain, or a fusion protein. The reduced anti-LGALS3 antibody or antigen-binding fragment thereof, a bispecific antibody, an antibody heavy chain, an antibody light chain, or a fusion protein can then be reacted with a thiol-reactive modifying agent to generate a conjugate provided herein. Conjugates containing organic moieties that are attached to specific sites on the anti-LGALS3 antibodies or antigen-binding fragments thereof provided herein can be prepared using suitable methods, such as reverse proteolysis (Fisch et al. (1992) Bioconjugate Chem., 3:147-153; Werlen et al. (1994) Bioconjugate Chem., 5:41 1-417; Kumaran et al. (1997) Protein Sci. 6(10):2233-2241; Itoh et al. (1996) Bioorg. Chem., 24(1):59-68; Capellas et al. (1997) Biotechnol. Bioeng. 56(4):456-463) and the methods described in Hermanson, GT, Bioconjugate Techniques, Academic Press: San Diego, Calif. (1996).

[0149] antibody generation Antibody generation and screening In another aspect, provided herein is a method for producing an anti-LGALS3 antibody or antigen-binding fragment. The antibody or antigen-binding fragment thereof described herein can be produced by any method known in the art for the synthesis of antibodies, for example, by chemical synthesis or by recombinant expression technology. The methods described herein employ conventional techniques in molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and related fields within the art, unless otherwise indicated. These techniques are described, for example, in the references cited herein and are fully explained in the literature.See, for example, Maniatis T et al. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; Sambrook J et al. (1989) Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press; Sambrook J et al. (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel FM et al. Current Protocols in Molecular Biology, John Wiley & Sons (1987 and annually updated); Current Protocols in Immunology, John Wiley & Sons (1987 and annually updated); Gait (ed.) (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein (ed.) (1991) Oligonucleotides and Analogues: A Practical Approach, IRL Press; Birren B et al. (ed.) (1999) Genome See Analysis: A Laboratory Manual, Cold Spring Harbor Laboratory Press.

[0150] In specific embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein are antibodies (e.g., recombinant antibodies) that are prepared, expressed, generated, or isolated by any means involving, for example, synthesis, generation via genetic engineering of DNA sequences. In certain embodiments, such antibodies comprise sequences encoded by DNA sequences that do not naturally occur in the antibody germline repertoire of an animal or mammal (e.g., human) in vivo. In specific embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein are made by a method that includes using the carbohydrate-binding domain of LGALS3 or a portion thereof. For example, see Examples 1-2 for a detailed description of exemplary methods of how to generate the antibodies described herein.

[0151] In certain aspects, the anti-LGALS3 antibodies provided herein are generated using immunogenic peptides including: ADNFSLHDALSGSGNPNPQGWPGAWGNQPAGAGGYPGASYPGAYPGQAPPGAYPGQAPPGAYPGAPGAYPGAPAPGVYPGPPSGPGAYPSSGQPSATGAYPATGPYGAPAGPLIVPYNLPLPGGVVPRMLITILGTVKPNANRIALDFQRGNDVAFHFNPRFNENNRRVIVCNTKLDNNWGREERQSVFPFESGKPFKIQVLVEPDHFKVAVNDAHLLQYNHRVKKLNEISKLGISGDIDLTSASYTMI (sequence number 53).

[0152] In certain aspects, the anti-LGALS3 antibodies provided herein are generated using an immunogenic peptide comprising an Fc fusion protein comprising the Fc region (CH2 and CH3 domains) of a human IgG1 heavy chain and amino acids 117-224 of LGALS3 linked to a hinge region. Amino acids 117-224 of LGALS3 are represented by the amino acid sequence: PYNLPLPGGVVPRMLITILGTVKPNANRIALDFQRGNDVAFHFNPRFNENNRRVIVCNTKLDNNWGREERQSVFPFESGKPFKIQVLVEPDHFKVAVNDAHLLQYNHRVKKLNEISKLGISGDIDLTS (sequence number 54).

[0153] In some embodiments, amino acids 117-224 of LGALS3 can be encoded by the following exemplary nucleotide sequence: CCTTATAACCTGCCTTTGCCTGGGGGAGTGGTGCCTCGCATGCTGATAACAATTCTGGGCACGGTGAAGCCCAATGCAAACAGAATTGCTTTAGATTTCCAAAGAGGGAATGATGTTGCCTTCCACTTTAACCCACGCTTCAATGAGAACAACAGGAGAGTCATTGTTTGCAATACAAAGCTGGATAATAACTGGG GAAGGGAAGAAAGACAGTCGGTTTTCCCATTTGAAAGTGGGAAACCATTCAAAATACAAGTACTGGTTGAACCTGACCACTTCAAGGTTGCAGTGAATGATGCTCACTTGTTGCAGTACAATCATCGGGTTAAAAAAACTCAATGAAATCAGCAAACTGGGAATTTCTGGTGACATAGACCTCACCAGT (SEQ ID NO: 55).

[0154] In certain embodiments, immunogenic peptides are conjugated to immunogenic carrier proteins. In most cases, small antigens (e.g., short peptides or small haptens) are not complex enough to induce the production of antibodies. Due to their large size and complex structure, immunogenic carrier proteins may confer immunogenicity to the conjugated small antigens, resulting in antibodies being generated against epitopes on the small antigens and immunogenic carrier proteins. Therefore, small antigens are always chemically conjugated to immunogenic carrier proteins to enhance the immune response for successful generation of antibodies. Commonly used immunogenic carrier proteins include, but are not limited to, keyhole limpet hemocyanin (KLH), Concholepas concholepas hemocyanin (CCH), bovine serum albumin (BSA), and ovalbumin (OVA). In a specific embodiment, immunogenic peptides are conjugated to KLH. KLH is a copper-containing polypeptide that belongs to a group of non-heme proteins called hemocyanins found in arthropods and mollusks. KLH is isolated from keyhole limpet (Megathura crenulata). Due to its evolutionary distance from mammals, high molecular weight, complex structure, and large surface containing hundreds of lysine groups that provide primary amines as targets for conjugation, KLH is a highly immunogenic and effective carrier protein in mammals.

[0155] In certain embodiments, the immunogenic peptide is 10-60 amino acid residues in length. In some embodiments, the immunogenic peptide is 10-30 amino acid residues in length. In some embodiments, the immunogenic peptide is 15-25 amino acid residues in length. In some embodiments, the immunogenic peptide is 15-20 amino acid residues in length. In specific embodiments, the immunogenic peptide is 15-18 amino acid residues in length.

[0156] In certain embodiments, the immunogenic peptide comprises at least a 10 amino acid portion of the LGALS3CBD domain. In certain embodiments, the immunogenic peptide comprises at least a 10 amino acid portion of the amino acid sequence of SEQ ID NO: 53. In certain other embodiments, the immunogenic peptide comprises at least a 15, 20, 25, or 30 amino acid portion of the amino acid sequence of SEQ ID NO: 53. In specific embodiments, the immunogenic peptide consists of 15-30 contiguous amino acid residues of SEQ ID NO: 53.

[0157] In another aspect, provided herein is a method for generating an antibody or antigen-binding fragment thereof that specifically binds to LGALS3, comprising immunizing a subject with the immunogenic peptide described above. The subject immunized according to the methods described herein may be, but is not limited to, a goat, sheep, donkey, chicken, guinea pig, rat, rabbit, or mouse. In some embodiments, the subject immunized according to the methods described herein is a rat, rabbit, or mouse. In some embodiments, the subject immunized according to the methods described herein is the AlivaMab® Mouse platform (Ablexis). In a specific embodiment, the subject immunized according to the methods described herein is a mouse. In some embodiments, the subject immunized according to the methods described herein is a transgenic mouse that expresses chimeric forms of various immunoglobulin chains. In some embodiments, the transgenic mouse expresses a human variable region linked to a mouse constant region. (See, e.g., U.S. Patent Nos. 8502018, 9580491, U.S. Patent Publication Nos. 2017 / 0218090, 2016 / 0222093, and 2013 / 0167256, each of which is incorporated by reference in its entirety. See also the AlivaMab® Mouse platform (Ablexis).)

[0158] Immunization of a subject can be carried out by any method known in the art, for example, by administering to the subject an immunogenic peptide and an adjuvant.

[0159] In another aspect, the present specification also provides a method for preparing the immunogenic peptide described herein. In certain embodiments, the method for preparing the immunogenic peptide comprises synthesizing a peptide portion. The peptide portion of the immunogenic peptide can be synthesized by any method known in the art, for example, by Fmoc solid phase peptide synthesis.

[0160] Methods for producing the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein are known to those skilled in the art, for example, by chemical synthesis, by purification from biological sources, or by recombinant expression techniques, including, for example, from mammalian cells or transgenic preparations. The methods described herein employ, unless otherwise indicated, conventional techniques in molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and related fields within the art. These techniques are described, for example, in the references cited herein and are fully explained in the literature.See, e.g., Maniatis et al. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press; Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons (1987 and annually updated); Current Protocols in Immunology, John Wiley & Sons (1987 and annually updated); Gait (ed.) (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein (ed.) (1991) Oligonucleotides and Analogues: A Practical Approach, IRL Press; Birren et al. (ed.) (1999) Genome Analysis: A Laboratory Manual See Manual, Cold Spring Harbor Laboratory Press.

[0161] Various methods exist in the art for producing the anti-LGALS3 antibody or antigen-binding fragment thereof described herein. For example, the anti-LGALS3 antibody or antigen-binding fragment thereof can be produced by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567. One or more DNAs encoding the anti-LGALS3 antibody or antigen-binding fragment thereof provided herein can be easily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of mouse antibodies, or such chains from human, humanized, or other sources). Once isolated, the DNA can be placed into an expression vector and then transformed into a host cell, such as an NSO cell, monkey COS cell, Chinese hamster ovary (CHO) cell, yeast cell, algae cell, egg, or myeloma cell, that does not otherwise produce immunoglobulin protein, to obtain the synthesis of the anti-LGALS3 antibody or antigen-binding fragment thereof in the recombinant host cell. The DNA can also be modified, for example, by substituting coding sequences for human heavy and light chain constant domains of the desired species in place of the homologous human sequences (U.S. Pat. No. 4,816,567, Morrison et al., supra), or by covalently linking all or part of the coding sequence for a non-immunoglobulin polypeptide to the immunoglobulin coding sequence. Such a non-immunoglobulin polypeptide can be substituted for the constant domain of an anti-LGALS3 antibody or antigen-binding fragment thereof provided herein. In certain embodiments, DNA encoding an anti-LGALS3 antibody or antigen-binding fragment thereof provided herein can also be prepared using a polynucleotide encoding at least one anti-LGALS3 antibody or antigen-binding fragment thereof to provide a transgenic animal or mammal, such as a goat, cow, horse, sheep, etc., that produces such an antibody in its milk. Such an animal can be provided using known methods.See, for example, but not by way of limitation, U.S. Pat. Nos. 5,827,690, 5,849,992, 4,873,316, 5,849,992, 5,994,616, 5,565,362, and 5,304,489, each of which is incorporated herein by reference in its entirety.

[0162] In certain embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof provided herein can be additionally prepared using a polynucleotide encoding at least one anti-LGALS3 antibody or antigen-binding fragment thereof provided herein to provide transgenic plants and cultured plant cells (e.g., but not limited to, tobacco and corn), plant parts, or specific parts or variants in cells cultured therefrom that produce such antibodies. As a non-limiting example, transgenic tobacco leaves expressing recombinant proteins have been successfully used to provide large amounts of recombinant proteins, for example, using inducible promoters. See, e.g., Cramer et al. (1999) Curr. Top. Microbol. Immunol. 240:95-118 (1999) and references cited therein. Transgenic corn has also been used to express mammalian proteins at commercial production levels with biological activity equivalent to those produced in other recombinant systems or purified from natural sources. See, e.g., Hood et al. (1999) Adv. Exp. Med. Biol. 464:127-147, and references cited therein. Antibodies have also been produced in large quantities from transgenic plant seeds containing antibody fragments, e.g., scFvs, including tobacco seeds and potato tubers. See, e.g., Conrad et al. (1998) Plant Mol. Biol. 38:101-109, and references cited therein. Thus, transgenic plants can also be used to produce anti-LGALS3 antibodies or antigen-binding fragments thereof, according to known methods. See also, e.g., Fischer et al. (1999) Biotechnol. Appl. Biochem. 30:99-108, Ma et al. (1995) Trends Biotechnol. 13:522-7, Ma et al, (1995) Plant Physiol. 109:341-6, Whitelam et al. (1994) Biochem Soc. Trans. 22:940-944, and references cited therein.Each of the above references is incorporated herein by reference in its entirety.

[0163] In certain embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof provided herein can be prepared using a polynucleotide encoding at least one anti-LGALS3 antibody or antigen-binding fragment thereof provided herein, and bacteria producing such anti-LGALS3 antibodies or antigen-binding fragments can be provided. As a non-limiting example, E. coli expressing recombinant proteins has been successfully used to provide large amounts of recombinant proteins. See, e.g., Verma et al. (1998) 216(1-2):165-181 and references cited therein.

[0164] Methods for making multispecific (e.g., bispecific) antibodies have been described, see, e.g., U.S. Pat. Nos. 7,951,917, 7,183,076, 8,227,577, 5,837,242, 5,989,830, 5,869,620, 6,132,992, and 8,586,713.

[0165] In certain embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof provided herein are utilized to generate bispecific antibodies. Bispecific antibodies can be generated by fusing two hybridomas to create a hybrid immunoglobulin molecule with two binding sites. Bispecific antibodies not only restrict tumor movement to T cells, but also crosslink CD3 on T cells, initiating an activation cascade. In this way, T cell receptor-based cytotoxicity is redirected to the desired tumor target circumventing MHC restriction. Arming polyclonal activated T cells (ATC) with anti-CD3 anti-LGALS3 bispecific binding molecules combines the targeting specificity of anti-LGALS3 antibodies with the non-MHC-restricted perforin / granzyme-mediated cytotoxicity of T cells. Bispecific binding molecules BsAb or BiTE can arm activated T cells expanded ex vivo before infusion into patients. This strategy converts any ATC into a specific CTL (Thakur and Lum (2010) Curr Opin Mol Ther 12, 340-349; Grabert et al. (2006) Clin Cancer Res 12, 569-576).

[0166] The bispecific binding molecule may be composed of an anti-LGALS3 antibody, where the anti-LGALS3 antibody is an immunoglobulin and each light chain of the immunoglobulin is a fusion protein, where the fusion protein is an immunoglobulin light chain linked via a peptide linker to a scFv that targets CD3. The N297A mutation in the CH2 domain results in glycosylation that does not result in FcR or Clq binding.

[0167] The anti-LGALS3 antibodies or antigen-binding fragments thereof provided herein may be utilized to generate CARs. CARs are most commonly composed of a single-chain variable fragment length antibody (scFv), e.g., derived from a monoclonal antibody targeting a given tumor-associated antigen and / or variants thereof, a transmembrane domain (e.g., a transmembrane domain derived from a T cell surface molecule, e.g., a costimulatory molecule, e.g., CD8, CD28, OX-40, and 4-1BB), a signaling portion of the TCR complex, e.g., the intracellular domain of the TCR zeta (ζ) chain, and / or additional portions, e.g., the cytoplasmic signaling domain thereof.

[0168] In a specific embodiment, the heavy and light chain variable regions of the monoclonal anti-LGALS3 antibodies described herein are isolated from a hybridoma cell line that produces the monoclonal anti-LGALS3 antibody. For example, RNA is extracted from the hybridoma cell line and cDNA is produced from the RNA by reverse transcription PCR. H and V L The V chain variable regions are cloned by standard PCR using primers specific for such variable regions. H and V L The fragments are subcloned into a shuttle vector, such as the TopoTA PCR 2.1 cloning vector (Invitrogen), and sequenced. H and V LThe fragment is then ligated to a (Gly4Ser)3 spacer domain (sequence number 56) to generate the anti-LGALS3 antibody scFv, and fused to the human CD8 leader peptide (CD8L) by overlap PCR (CD8L-anti-LGALS3 antibody scFv) (see, e.g., Maher et al. (2002) Nat Biotechnol 20(1):70-5 and Gong et al. (1999) Neoplasia 1(2):123-7). The coding region for the CD8L-anti-LGALS3 antibody scFv is fused to the human CD8 hinge and transmembrane domains, or alternatively, to the CD28 transmembrane and cytoplasmic signaling domains and to the T cell receptor 0)3-zeta signaling domain (see, e.g., Maher et al. (2002) Nat Biotechnol 20(1):70-5; Brentjens et al. (2003) Nat Med 9(3):279-86; and Brentjens et al. (2007) Clin Cancer Res 13(18Pt 1):5426-35).

[0169] Also provided herein are T cells expressing the CARs described herein. Methods for the generation of T cells expressing CARs are known in the art. For example, the CAR construct can be subcloned into a modified MMLV retroviral vector SFG (see, e.g., Riviere et al. (1995) Proc Natl Acad Sci USA 92(15):6733-7), or other suitable retroviral vector. In some embodiments, the retroviral vector is a lentiviral vector, e.g., an HIV-based vector. VSV-G pseudotyped retroviral supernatant from transduced gpg29 fibroblasts can be used to construct a cell line producing stable PG13 gibbon ape leukemia virus (GaLV) envelope pseudotyped retrovirus (see, e.g., Gong et al. (1999) Neoplasia 1(2):123-7). Isolated healthy donor peripheral blood mononuclear cells (PBMCs) can be activated with 2 μg / ml phytohemagglutinin (PHA) (Sigma. St. Louis, MO) and retrovirally transduced on retronectin-coated non-tissue culture plates (Quintas-Cardama A, et al. (2007) Hum Gene Ther 18(12):1253-60) to generate T cells recombinantly expressing the CAR. Gene transfer of the CAR into the T cells can be assessed by FACS.

[0170] Single domain antibodies, e.g., antibodies lacking light chains, can be generated by methods well known in the art, see, e.g., Riechmann and Muyldermans (1999) J Immunol 231:25-38, Nuttall SD et al. (2000) Curr Pharm Biotechnol 1(3):253-263, Muyldermans S, (2001) J Biotechnol 74(4):277-302, U.S. Patent No. 6,005,079, and WO 94 / 04678, WO 94 / 25591, and WO 01 / 44301, each of which is incorporated by reference in its entirety.

[0171] In certain embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein bind to the same epitope or an overlapping epitope as the anti-LGALS3 antibodies described herein and are human anti-LGALS3 antibodies or antigen-binding fragments thereof. In certain embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein competitively block (e.g., in a dose-dependent manner) any of the antibodies described herein from binding to LGALS3 and are human anti-LGALS3 antibodies or antigen-binding fragments thereof.

[0172] Human antibodies can be produced using any method known in the art. For example, transgenic mice that are incapable of expressing functional endogenous immunoglobulins, but can express human immunoglobulin genes, can be used. In particular, human heavy and light chain immunoglobulin gene complexes can be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, human variable, constant, and diversity regions can be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes. The mouse heavy and light chain immunoglobulin genes can be made non-functional separately or simultaneously with the introduction of the human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the JH region prevents endogenous antibody production. The modified embryonic stem cells are expanded and microinjected into blastocysts to generate chimeric mice. The chimeric mice are then bred to generate homozygous progeny that express human antibodies. The transgenic mice are immunized in the usual manner with a selected antigen, e.g., all or a portion of the antigen. Monoclonal antibodies directed against an antigen can be obtained from immunized transgenic mice using conventional hybridoma technology. The human immunoglobulin transgenes carried by the transgenic mice rearrange during B cell differentiation and subsequently undergo class switching and somatic mutation. Thus, using such technology, it is possible to generate therapeutically useful IgG, IgA, IgM and IgE antibodies. For an overview of this technology for generating human antibodies, see, e.g., Lonberg and Huszar (1995) Int Rev Immunol 13:65-93. For a detailed description of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., WO 98 / 24893, WO 96 / 34096, and WO 96 / 33735, as well as U.S. Pat. Nos. 5,413,923, 5,625,126, 5,633,425, 5,569,825, 5,661,016, 5,545,806, 5,814,318, and 5,939,598.Examples of mice capable of producing human antibodies include the Xenomouse™ (Abgenix, Inc., U.S. Pat. Nos. 6,075,181 and 6,150,184), HuAb-Mouse™ (Mederex, Inc. / Gen Pharm, U.S. Pat. Nos. 5,545,806 and 5,569,825), Trans Chromo Mouse™ (Kirin), and KM Mouse™ (Medarex / Kirin).

[0173] Human antibodies that specifically bind to LGALS3 can be produced by various methods known in the art, including the above-mentioned phage display method, using antibody libraries derived from human immunoglobulin sequences. See also U.S. Patent Nos. 4,444,887, 4,716,111, and 5,885,793, and WO 98 / 46645, 98 / 50433, 98 / 24893, 98 / 16654, 96 / 34096, 96 / 33735, and 91 / 10741.

[0174] In some embodiments, human antibodies can be produced using mouse-human hybridomas. For example, Epstein-Barr Virus (EBV) transformed human peripheral blood lymphocytes can be fused with mouse myeloma cells to produce mouse-human hybridomas that secrete human monoclonal antibodies, and these mouse-human hybridomas can be screened to determine which secrete human monoclonal antibodies that specifically bind to target antigens. Such methods are known and described in the art, see, for example, Shinmoto H et al. (2004) Cytotechnology 46:19-23, Naganawa Y et al. (2005) Human Antibodies 14:27-31.

[0175] Exemplary methods for generating antibodies or antigen-binding fragments thereof that specifically bind to LGALS3, as well as methods for screening and selecting antibodies or antigen-binding fragments thereof that specifically bind to LGALS3, are described in Example 2. In exemplary embodiments, monoclonal antibodies secreted by hybridomas are screened for binding to the LGALS3 carbohydrate-binding domain peptide (CBD), the full-length LGALS3 protein. In some embodiments, the LGALS3 N-terminus, which contains the polymerization domain, is used as a control. In some embodiments, monoclonal antibodies are further screened against human Gal3 (or other members of the galectin family, e.g., LGAL1, LGAL7, LGAL8, and LGAL9). In some embodiments, monoclonal antibodies are further screened by using a functional assay for inhibition of binding of human Gal-3 to suitable cancer cells.

[0176] Once the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein are produced, they can be purified by any method known in the art for the purification of immunoglobulin molecules, such as, for example, by chromatography (e.g., by ion exchange, affinity, particularly affinity for a specific antigen following Protein A and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. Additionally, to facilitate purification, the antibodies described herein can be fused to heterologous polypeptide sequences described herein or known in the art.

[0177] In specific embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein are isolated or purified. Generally, an isolated antibody is one that is substantially free of other antibodies having antigenic specificity different from that of the isolated antibody. For example, in certain embodiments, preparations of the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein are substantially free of cellular material and / or chemical precursors. The language "substantially free of cellular material" includes preparations of the anti-LGALS3 antibodies or antigen-binding fragments thereof in which the antibodies are separated from cellular components of the cells from which they are isolated or recombinantly produced. Thus, an anti-LGALS3 antibody or antigen-binding fragment thereof that is substantially free of cellular material includes preparations of antibodies that have less than about 30%, 20%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% (by dry weight) of heterologous proteins (also referred to herein as "contaminating proteins") and / or variants of the anti-LGALS3 antibody or antigen-binding fragment thereof, such as different post-translationally modified forms of the anti-LGALS3 antibody or antigen-binding fragment thereof, or other different aspects of the anti-LGALS3 antibody or antigen-binding fragment thereof (e.g., antibody fragments). When the antibody is recombinantly produced, it is also generally substantially free of culture medium, i.e., culture medium represents less than about 20%, 10%, 2%, 1%, 0.5%, or 0.1% of the volume of the protein preparation. When the antibody is produced by chemical synthesis, it is generally substantially free of chemical precursors or other chemicals, i.e., separated from chemical precursors or other chemicals involved in the synthesis of the protein. Accordingly, such preparations of antibodies have less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors or compounds other than the antibody of interest. In specific embodiments, the antibodies described herein are isolated or purified.

[0178] Polynucleotides In certain embodiments, provided herein is a polynucleotide comprising a nucleotide sequence encoding an anti-LGALS3 antibody or an antigen-binding fragment thereof. Also provided herein is a vector comprising such a polynucleotide. Also provided herein is a polynucleotide encoding an antigen of the anti-LGALS3 antibody or antigen-binding fragment thereof described herein. Also provided herein is a polynucleotide that hybridizes under stringent or less stringent hybridization conditions to a polynucleotide encoding an anti-LGALS3 antibody or antigen-binding fragment thereof described herein.

[0179] The language "purified" includes preparations of polynucleotides or nucleic acid molecules that have less than about 15%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% (particularly less than 10%) of other materials, e.g., cellular material, culture medium, other nucleic acid molecules, chemical precursors, and / or other chemicals. In specific embodiments, nucleic acid molecules encoding anti-LGALS3 antibodies or antigen-binding fragments thereof described herein are isolated or purified.

[0180] The nucleic acid molecules provided herein may be in the form of RNA, such as mRNA, hnRNA, tRNA, or any other form, or in the form of DNA, including but not limited to cDNA, and genomic DNA obtained by cloning or produced synthetically, or any combination thereof. The DNA may be triple-stranded, double-stranded, or single-stranded, or any combination thereof. Any portion of at least one strand of the DNA or RNA may be the coding strand, also known as the sense strand, or the non-coding strand, also referred to as the antisense strand.

[0181] In certain embodiments, provided herein is a polynucleotide comprising a nucleotide sequence encoding an anti-LGALS3 antibody or antigen-binding fragment thereof described herein. In certain aspects, provided herein is a polynucleotide comprising a nucleotide sequence encoding an anti-LGALS3 antibody or antigen-binding fragment thereof, which specifically binds to LGALS3 and comprises an amino acid sequence described herein, as well as an antibody that competes with such an anti-LGALS3 antibody or antigen-binding fragment thereof for binding to LGALS3 or binds to the same epitope as such an antibody.

[0182] The polynucleotides provided herein can be obtained by any method known in the art. For example, if the nucleotide sequence encoding the anti-LGALS3 antibody or antigen-binding fragment thereof described herein is known, the polynucleotide encoding the anti-LGALS3 antibody or antigen-binding fragment thereof can be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al. (1994) BioTechniques 17:242), which briefly involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligating these oligonucleotides, and then amplifying the ligated oligonucleotides by PCR.

[0183] Alternatively, polynucleotides encoding anti-LGALS3 antibodies or antigen-binding fragments thereof can be generated from nucleic acids from a suitable source. If a clone containing a nucleic acid encoding a particular anti-LGALS3 antibody or antigen-binding fragment thereof is not available, but the sequence of the anti-LGALS3 antibody or antigen-binding fragment thereof is known, the nucleic acid encoding the anti-LGALS3 antibody or antigen-binding fragment thereof may be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated therefrom, or a nucleic acid, preferably polyA+RNA isolated therefrom, any tissue or cell expressing an antibody, e.g., a hybridoma cell selected to express an anti-LGALS3 antibody or antigen-binding fragment thereof provided herein) by PCR amplification using synthetic primers hybridizing to the 3' and 5' ends of the sequence, or by cloning using an oligonucleotide probe specific for a particular gene sequence, for example, to identify a cDNA clone from a cDNA library encoding the antibody. The amplified nucleic acid generated by PCR may then be cloned into a replicable cloning vector using any method well known in the art. In such embodiments, polynucleotides encoding such anti-LGALS3 antibodies or antigen-binding fragments thereof can be manipulated using methods well known in the art for the manipulation of nucleotide sequences, such as recombinant DNA technology, site-directed mutagenesis, PCR, etc. (e.g., the techniques described in Sambrook et al. (19900 Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, and Ausubel et al., eds. (1998) Current Protocols in Molecular Biology, John Wiley & Sons, NY, both of which are incorporated by reference in their entireties) to generate anti-LGALS3 antibodies or antigen-binding fragments thereof having different amino acid sequences, e.g., making amino acid substitutions, deletions, and / or insertions.For example, such manipulations can be carried out to deglycosylate the encoded amino acids, or to destroy the ability of the antibody to bind Clq, Fc receptors, or to activate the complement system.

[0184] The isolated nucleic acid molecules provided herein may include nucleic acid molecules that optionally include an open reading frame (ORF) with at least one specified portion of at least one complementarity determining region (CDR), including, but not limited to, one or more introns, such as CDR1, CDR2 and / or CDR3 of at least one heavy or light chain; nucleic acid molecules that include a coding sequence or variable region for an anti-LGALS3 antibody; and nucleic acid molecules that are substantially different from those described above, but that, due to the degeneracy of the genetic code, still encode at least one anti-LGALS3 antibody or antigen-binding fragment thereof described herein.

[0185] Also provided herein are isolated nucleic acids that hybridize under selective hybridization conditions to the polynucleotides described herein. Thus, the polynucleotides of this embodiment can be used to isolate, detect, and / or quantify nucleic acids that contain such polynucleotides. For example, the polynucleotides provided herein can be used to identify, isolate, or amplify partial or full-length clones in a deposited library. In some embodiments, the polynucleotides are genomic or cDNA sequences isolated from a human or mammalian nucleic acid library or otherwise complementary to cDNAs from a human or mammalian nucleic acid library.

[0186] The nucleic acid may advantageously contain sequences in addition to the polynucleotides provided herein. For example, a multicloning site containing one or more endonuclease restriction sites may be inserted into the nucleic acid to aid in the isolation of the polynucleotide. In addition, a translatable sequence may be inserted to aid in the isolation of the translated polynucleotides provided herein. For example, a hexa-histidine marker sequence (SEQ ID NO:57) provides a convenient means for purifying the polypeptides provided herein. The nucleic acids provided herein (excluding coding sequences) are optionally vectors, adapters, or linkers for the cloning and / or expression of the polynucleotides provided herein.

[0187] Additional sequences can also be added to such cloning and / or expression sequences to optimize their function in cloning and / or expression, aid in isolation of the polynucleotide, or improve introduction of the polynucleotide into a cell. The use of cloning vectors, expression vectors, adapters, and linkers is well known in the art. (See, e.g., Ausubel, supra, or Sambrook, supra).

[0188] In a specific embodiment, one or more of the CDRs of the anti-LGALS3 antibody or antigen-binding fragment thereof described herein can be inserted into a known framework region using routine recombinant DNA techniques. The framework region can be naturally occurring or a consensus framework region, preferably a human framework region (see, for example, Chothia et al., (1998) J. Mol. Biol. 278:457-479 for a list of human framework regions). In some embodiments, the polynucleotide generated by the combination of the framework regions and the CDRs encodes an anti-LGALS3 antibody or antigen-binding fragment thereof that specifically binds to LGALS3. One or more amino acid substitutions may be made within the framework region, and preferably, the amino acid substitutions improve the binding of the antibody to its antigen. Additionally, such methods can be used to make amino acid substitutions or deletions of one or more variable region cysteine ​​residues involved in intrachain disulfide bonds to generate an antibody molecule lacking one or more intrachain disulfide bonds. Other modifications to the polynucleotide are provided herein and within the skill of the art.

[0189] In certain embodiments, the isolated or purified nucleic acid molecule, or a fragment thereof, when linked to another nucleic acid molecule, may encode a fusion protein. The generation of fusion proteins is within the skill of the art and may involve the use of restriction enzymes or recombinant cloning techniques (see, e.g., Gateway™ (Invitrogen)). See also U.S. Patent No. 5,314,995.

[0190] In certain embodiments, the polynucleotides provided herein are in the form of a vector (e.g., an expression vector). In certain aspects, provided herein are polynucleotides comprising an anti-LGALS3 antibody or antigen-binding fragment thereof described herein, or an antigen-binding fragment thereof that specifically binds to LGALS3 (e.g., a variable light chain region and / or a variable heavy chain region), as well as vectors, e.g., vectors comprising such polynucleotides for their efficient expression in host cells (e.g., E. coli and mammalian cells). In some embodiments, the polynucleotides are isolated or purified.

[0191] In certain aspects, provided herein are polynucleotides comprising a nucleotide sequence encoding an anti-LGALS3 antibody or antigen-binding fragment thereof, comprising an amino acid sequence described herein, as well as an antibody that competes with such an antibody (e.g., in a dose-dependent manner) for binding to LGALS3 or binds to the same or overlapping epitope as such an antibody.

[0192] In certain aspects, provided herein is a polynucleotide comprising a nucleic acid sequence encoding a light or heavy chain of an anti-LGALS3 antibody or antigen-binding fragment thereof described herein. The polynucleotide may be any of the V and VL2 sequences described herein. H The polynucleotide may comprise a nucleotide sequence encoding a heavy chain comprising the CDRs (see, e.g., Table 6). L The nucleic acid sequence may include a nucleotide sequence encoding a light chain comprising the CDRs (see, for example, Table 6).

[0193] In certain embodiments, provided herein is a polynucleotide comprising three V H CDRs, e.g., V H CDR1, V H CDR2, and V HPolynucleotides are provided that include a nucleotide sequence encoding an anti-LGALS3 antibody containing CDR3, where the antibody specifically binds to LGALS3. In a specific embodiment, the polynucleotides described herein include the V-terminal sequence of the 46H02 antibody (i.e., SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, respectively), the 12H07 antibody (i.e., SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:21, respectively), the 20F08 antibody (i.e., SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:31, respectively), the 38E05 antibody (i.e., SEQ ID NO:39, SEQ ID NO:40, and SEQ ID NO:41, respectively), or the 39F02 antibody (i.e., SEQ ID NO:49, SEQ ID NO:50, and SEQ ID NO:51, respectively). H CDR1, V H CDR2, and V H In certain embodiments, the polynucleotides described herein comprise a nucleotide sequence encoding an anti-LGALS3 antibody described herein, comprising a heavy chain variable region comprising an amino acid sequence described herein (e.g., SEQ ID NO:6, SEQ ID NO:18, SEQ ID NO:28, SEQ ID NO:38, or SEQ ID NO:48), and the antibody specifically binds to LGALS3.

[0194] In certain embodiments, provided herein is a polynucleotide comprising three V L CDRs, e.g., V L CDR1, V L CDR2, and V LPolynucleotides are provided that include a nucleotide sequence encoding an anti-LGALS3 antibody containing CDR3, where the antibody specifically binds to LGALS3. In a specific embodiment, the polynucleotides described herein include the V-terminal sequence of the 46H02 antibody (i.e., SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, respectively), the 12H07 antibody (i.e., SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16, respectively), the 20F08 antibody (i.e., SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26, respectively), the 38E05 antibody (i.e., SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36, respectively), or the 39F02 antibody (i.e., SEQ ID NO:44, SEQ ID NO:45, and SEQ ID NO:46, respectively). L CDR1, V L CDR2, and V L In certain embodiments, the polynucleotides described herein comprise a nucleotide sequence encoding an anti-LGALS3 antibody described herein comprising a light chain variable region comprising an amino acid sequence described herein (e.g., SEQ ID NO:1, SEQ ID NO:13, SEQ ID NO:23, SEQ ID NO:33, or SEQ ID NO:43), and the antibody specifically binds to LGALS3.

[0195] In certain embodiments, the polynucleotides described herein comprise a nucleotide sequence encoding an anti-LGALS3 antibody described herein comprising a heavy chain variable region comprising an amino acid sequence described herein (e.g., SEQ ID NO:6, SEQ ID NO:18, SEQ ID NO:28, SEQ ID NO:38, or SEQ ID NO:48) and a light chain variable region comprising an amino acid sequence described herein (e.g., SEQ ID NO:1, SEQ ID NO:13, SEQ ID NO:23, SEQ ID NO:33, or SEQ ID NO:43), wherein the antibody specifically binds to LGALS3.

[0196] In specific aspects, provided herein are polynucleotides comprising nucleotide sequences encoding an anti-LGALS3 antibody or antigen-binding fragment thereof comprising a light chain and a heavy chain, e.g., a separate light chain and heavy chain. With respect to the heavy chain, in specific embodiments, the polynucleotides provided herein comprise a nucleotide sequence encoding an alpha (a), delta (δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain. With respect to the light chain, in specific embodiments, the polynucleotides provided herein comprise a nucleotide sequence encoding a kappa (κ) or lambda (λ) light chain.

[0197] Cells and Vectors In certain embodiments, provided herein are cells (e.g., isolated cells or ex vivo cells) that (e.g., recombinantly) express one or more anti-LGALS3 antibodies or antigen-binding fragments thereof. Also provided herein are vectors (e.g., expression vectors) that include nucleotide sequences encoding the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein for recombinant expression in a host cell, preferably a mammalian cell. Also provided herein are cells (e.g., isolated cells or ex vivo cells) that include such vectors or nucleotide sequences for recombinantly expressing the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein. Also provided herein are methods for producing the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein, comprising expressing such anti-LGALS3 antibodies or antigen-binding fragments thereof from a cell (e.g., isolated cells or ex vivo cells).

[0198] A vector (e.g., an expression vector) is a DNA molecule that contains a gene to be expressed in a cell (e.g., an ex vivo cell). Typically, gene expression is placed under the control of certain regulatory elements, including constitutive or inducible promoters, tissue-specific regulatory elements, and enhancers. Such a gene is said to be "operably linked" to a regulatory element, e.g., a promoter. A recombinant host can be any prokaryotic or eukaryotic cell that contains either a cloning vector or an expression vector. The term also includes prokaryotic or eukaryotic cells that have been genetically engineered to contain a cloned gene in the chromosome or genome of the host cell, or in the cells of the host cell (e.g., an ex vivo cell), and transgenic animals. In one embodiment, the promoter is a CMV promoter.

[0199] In certain embodiments, provided herein are vectors comprising one or more polynucleotides described herein. In certain embodiments, the polynucleotides described herein can be cloned into a suitable vector, which can be used to transform or transfect a suitable host. Vectors and methods for constructing such vectors are known to those skilled in the art and described in general technical references (see generally, "Recombinant DNA Part D," Methods in Enzymology, Vol. 153, Wu and Grossman, eds., Academic Press (1987)). In certain embodiments, the vector comprises regulatory sequences, e.g., initiation and termination codons for transcription and translation, that are specific for the type of host into which the vector is to be introduced (e.g., bacteria, fungi, plants, insects, or mammals), taking into account whether the vector is DNA or RNA, as appropriate. In certain embodiments, the vector comprises regulatory sequences that are specific for the genus of the host. In certain embodiments, the vector comprises regulatory sequences that are specific for the species of the host. In certain embodiments, the vector comprises one or more marker genes that allow for the selection of transformed or transfected hosts. Non-limiting examples of marker genes include biocide resistance, resistance to e.g., antibiotics, heavy metals, etc., complementation in auxotrophic hosts to provide prototrophy, etc. In certain embodiments, the vector contains ampicillin and hygromyci selectable markers.

[0200] In certain embodiments, the expression vector may comprise a native promoter or a canonical promoter operably linked to the polynucleotide described herein. For example, the selection of strong, weak, inducible, tissue-specific, and development-specific promoters is within the skill of the art. Similarly, as described above, it is also within the skill of the art to combine a nucleic acid molecule or a fragment thereof with a promoter.

[0201] Non-limiting examples of suitable vectors include those designed for propagation or propagation, or for expression, or both. For example, cloning vectors can be selected from the group consisting of pUC series, pBluescript series (Stratagene, LaJolla, Calif), pET series (Novagen, Madison, Wis.), pGEX series (Pharmacia Biotech, Uppsala, Sweden), and pEX series (Clontech, Palo Alto, Calif). Bacteriophage vectors, such as lambda-GTIO, lambda-GTl 1, lambda-ZapII (Stratagene), lambda-EMBL4, and lambda-NMl 149, can also be used. Non-limiting examples of plant expression vectors include pBIl 10, pBI101.2, pBI101.3, pBI121, and pBIN19 (Clontech). Non-limiting examples of animal expression vectors include pEUK-Cl, pMAM, and pMAMneo (Clontech). The TOPO cloning system (Invitrogen, Carlsbad, Calif.) can also be used following the manufacturer's recommendations.

[0202] In certain embodiments, the vector is a mammalian vector. In certain embodiments, the mammalian vector contains at least one promoter element that mediates the initiation of transcription of the sequence encoding the mRNA, anti-LGALS3 antibody or antigen-binding fragment thereof, and signals necessary for the termination of transcription and polyadenylation of the transcript. In certain embodiments, the mammalian vector contains additional elements, such as enhancers flanking donor and acceptor sites for RNA splicing, Kozak sequences, and intervening sequences. In certain embodiments, highly efficient transcription can be achieved using, for example, early and late promoters from SV40, long terminal repeats (LTRS) from retroviruses, such as RSV, HTLVI, HIVI, and early promoters from cytomegalovirus (CMV). However, cellular elements can also be used (e.g., human actin promoter). Non-limiting examples of mammalian expression vectors include vectors such as pIRESlneo, pRetro-Off, pRetro-On, PLXSN, or pLNCX (Clonetech Labs, Palo Alto, Calif), pcDNA3.1(+ / -), pcDNA / Zeo(+ / -), or pcDNA3.1 / Hygro(+ / -) (Invitrogen), PSVL and PMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC37152), pSV2dhfr (ATCC37146), and pBC12MI (ATCC67109). Non-limiting examples of mammalian host cells that can be used in combination with such mammalian vectors include human Hela293, H9 and Jurkat cells, mouse NIH3T3 and C127 cells, Cos1, Cos7, and CV1, quail QCl-3 cells, mouse L cells, and Chinese hamster ovary (CHO) cells.

[0203] In certain embodiments, the vector is a viral vector, such as a retroviral vector, a parvovirus-based vector, such as an adeno-associated virus (AAV)-based vector, an AAV-adenovirus chimeric vector, and an adenovirus-based vector, and a lentiviral vector, such as a herpes simplex (HSV)-based vector. In certain embodiments, the viral vector is engineered to be defective in viral replication. In certain embodiments, the viral vector is engineered to eliminate toxicity to the host. These viral vectors can be prepared using standard recombinant DNA techniques, for example, as described in Sambrook et al., Molecular Cloning, a Laboratory Manual, 2d edition, Cold Spring Harbor Press, Cold Spring Harbor, NY (1989), and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley&Sons, New York, NY (1994).

[0204] In certain embodiments, the vectors or polynucleotides described herein can be transferred into cells (e.g., ex vivo cells) by conventional techniques, and the resulting cells can be cultured by conventional techniques to produce the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein. Thus, provided herein is a cell comprising a polynucleotide encoding an anti-LGALS3 antibody or antigen-binding fragment thereof, a heavy or light chain thereof, or a light chain fusion polypeptide thereof, operably linked to a promoter for expression of such sequences in a host cell. In certain embodiments, a vector encoding a heavy chain operably linked to a promoter and a vector encoding a light chain operably linked to a promoter can be co-expressed in a cell for expression of the entire anti-LGALS3 antibody. In certain embodiments, a vector encoding a heavy chain operably linked to a promoter and a vector encoding a light chain fusion polypeptide operably linked to a promoter can be co-expressed in a cell for expression of the entire bispecific binding molecule. In certain embodiments, a cell comprises a vector comprising a polynucleotide encoding both the heavy and light chain polypeptides of the anti-LGALS3 antibodies described herein operably linked to a promoter. In certain embodiments, the cell comprises a vector comprising a polynucleotide encoding both the heavy and light chain fusion polypeptides of a bispecific binding molecule described herein operably linked to a promoter. In certain embodiments, the cell comprises two different vectors, a first vector comprising a polynucleotide encoding a heavy chain operably linked to a promoter, and a second vector comprising a polynucleotide encoding a light chain polypeptide operably linked to a promoter. In certain embodiments, the cell comprises two different vectors, a first vector comprising a polynucleotide encoding a heavy chain operably linked to a promoter, and a second vector comprising a polynucleotide encoding a light chain fusion polypeptide operably linked to a promoter.In certain embodiments, the first cell comprises a first vector comprising a polynucleotide encoding a heavy chain of an anti-LGALS3 antibody described herein, and the second cell comprises a second vector comprising a polynucleotide encoding a light chain polypeptide of an anti-LGALS3 antibody described herein. In certain embodiments, provided herein is a mixture of cells comprising such a first cell and such a second cell. In certain embodiments, the first cell comprises a first vector comprising a polynucleotide encoding a heavy chain of a bispecific binding molecule described herein, and the second cell comprises a second vector comprising a polynucleotide encoding a light chain fusion polypeptide of a bispecific binding molecule described herein. In certain embodiments, provided herein is a mixture of cells comprising such a first cell and such a second cell. In certain embodiments, the cell expresses the vector or vectors such that the polynucleotides are efficiently transcribed and translated by the cell.

[0205] In some embodiments, the cell expresses the vector, such that the polynucleotide, or a fragment thereof, is both transcribed and efficiently translated by the cell.

[0206] In certain embodiments, the cell is present in a host, which may be an animal, e.g., a mammal. Exemplary cells include, but are not limited to, human cells, human cell lines, E. coli (e.g., E. coli TB-1, TG-2, DH5a, XL-Blue MRF (Stratagene), SA2821, and Y1090), B. subtilis, P. aeruginosa, S. cerevisiae, N. crassa, insect cells (e.g., Sf9, Ea4), and others described below. In certain embodiments, the cell is a CHO cell. In another particular embodiment, the cell is a CHO-S cell.

[0207] In certain embodiments, the polynucleotides described herein can be expressed in a stable cell line comprising the polynucleotide integrated into a chromosome by introducing the polynucleotide into the cell. In certain embodiments, the polynucleotide is introduced into the cell, for example, by electroporation. In certain embodiments, the polynucleotide is introduced into the cell, for example, by transfecting a vector comprising the polynucleotide into the cell. In certain embodiments, the vector is co-transfected with a selectable marker, such as DHFR, GPT, neomycin, or hygromycin, to allow identification and isolation of transfected cells. In certain embodiments, the transfected polynucleotide can also be amplified to express large amounts of the encoded anti-LGALS3 antibody or antigen-binding fragment thereof. For example, a DHFR (dihydrofolate reductase) marker can be utilized to develop cell lines carrying hundreds or even thousands of copies of the polynucleotide of interest. Another example of a selectable marker is the enzyme glutamine synthase (GS) (Murphy, et al., Biochem. J. 227:277-279 (1991); Bebbington, et al., Bio / Technology 10:169-175). Using these markers, cells are grown in selective medium and the cells with the highest resistance are selected. These cell lines contain an amplified gene integrated into the chromosome. Chinese Hamster Ovary (CHO) and NSO cells are often used for the production of antibodies.

[0208] In some embodiments, the vector comprises (i) a first polynucleotide sequence encoding an immunoglobulin light chain that binds to LGALS3 operably linked to a first promoter, and (ii) a second polynucleotide sequence encoding an immunoglobulin heavy chain that binds to LGALS3 operably linked to a second promoter. In certain embodiments, the vector is a viral vector.

[0209] In some embodiments, the vector comprises (i) a first polynucleotide sequence encoding a light chain fusion polypeptide comprising an immunoglobulin light chain fused to an scFv via a peptide linker, the light chain binding to LGALS3 and the scFv binding to CD3, operably linked to a first promoter, and (ii) a second polynucleotide encoding an immunoglobulin heavy chain binding to LGALS3, operably linked to a second promoter. In certain embodiments, the vector is a viral vector.

[0210] Pharmaceutical Compositions In certain embodiments, provided herein are compositions (e.g., pharmaceutical compositions) and kits comprising a pharma- ceutical effective amount of one or more anti-LGALS3 antibodies or antigen-binding fragments thereof. In certain embodiments, the pharmaceutical compositions comprise immune cells, e.g., T cells, recombinantly expressing the antibodies, antigen-binding fragments thereof, and / or CARs described herein. The compositions can be used to prepare individual single unit dosage forms. The compositions provided herein can be formulated for parenteral, subcutaneous, intramuscular, intravenous, intraarticular, intrabronchial, intraperitoneal, intracapsular, intrachondral, intracavitary, intracelial, intracerebral, intraventricular, intraocular, intravitreal, intracolonic, intracervical, intragastric, intrahepatic, intramyocardial, intraosseous, intrapelvic, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal, intrathecal, intraventricular, intraparenchymal, or transdermal administration.

[0211] In certain embodiments, provided herein is a composition comprising one or more polynucleotides comprising a nucleotide sequence encoding an anti-LGALS3 antibody or antigen-binding fragment thereof described herein. In certain embodiments, provided herein is a composition comprising a cell, wherein the cell comprises one or more polynucleotides comprising a nucleotide sequence encoding an anti-LGALS3 antibody or antigen-binding fragment thereof described herein. In certain embodiments, provided herein is a composition comprising a vector, wherein the vector comprises one or more polynucleotides comprising a nucleotide sequence encoding an anti-LGALS3 antibody or antigen-binding fragment thereof described herein. In certain embodiments, provided herein is a composition comprising a cell, wherein the cell comprises a vector, wherein the vector comprises one or more polynucleotides comprising a nucleotide sequence encoding an anti-LGALS3 antibody or antigen-binding fragment thereof described herein. In certain embodiments, provided herein is a composition comprising a cell, wherein the cell comprises a vector, wherein the vector comprises one or more polynucleotides comprising a nucleotide sequence encoding an anti-LGALS3 antibody or antigen-binding fragment thereof described herein.

[0212] In certain embodiments, the compositions described herein are stable or preserved formulations. In certain embodiments, the stable formulations include phosphate buffer with saline or selected salts. In certain embodiments, the compositions described herein are versatile preserved formulations suitable for pharmaceutical or veterinary use. In certain embodiments, the compositions described herein include a preservative. Preservatives are known to those skilled in the art. Non-limiting examples of preservatives include phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, phenylmercuric nitrate, phenoxyethanol, formaldehyde, chlorobutanol, magnesium chloride (e.g., hexahydrate), alkyl parabens (methyl, ethyl, propyl, butyl, etc.), benzalkonium chloride, benzethonium chloride, and sodium dehydroacetate and thimerosal, or mixtures thereof in an aqueous diluent. Any suitable concentration or mixture, such as, for example, 0.001-5%, or any range or value therein, such as, but not limited to, 0.001, 0.003, 0.005, 0.009, 0.01, 0.02, 0.03, 0.05, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2 ...6, 2.7, 2.8, 2.9, 2.0, 2.1, 2.2, 2.3, 2.4, 6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.3, 4.5, 4.6, 4.7, 4.8, 4.9%, or any range or value therein, can be used as known in the art.Non-limiting examples include no preservatives, 0.1-2% m-cresol (e.g., 0.2, 0.3, 0.4, 0.5, 0.9, 1.0%), 0.1-3% benzyl alcohol (e.g., 0.5, 0.9, 1.1, 1.5, 1.9, 2.0, 2.5%), 0.001-0.5% thimerosal (e.g., 0.005, 0.01), 0.001-2.0% phenol (e.g., Examples of suitable alkylparabens include 0.05, 0.25, 0.28, 0.5, 0.9, 1.0%), 0.0005 to 1.0% alkylparaben (for example, 0.00075, 0.0009, 0.001, 0.002, 0.005, 0.0075, 0.009, 0.01, 0.02, 0.05, 0.075, 0.09, 0.1, 0.2, 0.3, 0.5, 0.75, 0.9, 1.0%), and the like.

[0213] It may be desirable to deliver the compositions provided herein to a subject over an extended period of time, for example, from one week to a year or more from a single administration. A variety of sustained release, depot, or implant dosage forms can be utilized. For example, the dosage form may contain a pharma- ceutically acceptable non-toxic salt of a compound having a low degree of solubility in bodily fluids, such as (a) an acid addition salt with a polybasic acid, such as phosphoric acid, sulfuric acid, citric acid, tartaric acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalene mono- or disulfonic acid, polygalacturonic acid, (b) a salt with a polyvalent metal ion, such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, or an organic cation, such as formed from N,N'-dibenzyl-ethylenediamine or ethylenediamine, or (c) a combination of (a) and (b), such as zinc tannate. In addition, the compositions provided herein, preferably the relatively insoluble salts as described immediately above, can be formulated in gels, such as aluminum monostearate gels, including, for example, sesame oil, suitable for injection. Particular salts are zinc salts, zinc tannate salts, pamoate salts, and the like. Another type of sustained release depot formulation for injection can contain the compound or salt dispersed for encapsulation in a slowly degrading, non-toxic, non-antigenic polymer, such as polylactic acid / polyglycolic acid polymer, as described, for example, in U.S. Pat. No. 3,773,919. The compound, or preferably the relatively insoluble salts as described above, can also be formulated in cholesterol matrix silastic pellets, particularly for use in animals. Additional sustained release, depot, or implant compositions, such as gas or liquid liposomes, are known in the literature (U.S. Pat. No. 5,770,222, and "Sustained and Controlled Release Drug Delivery Systems", JR Robinson ed., Marcel Dekker, Inc., NY, 1978).

[0214] The range of compositions of at least one anti-LGALS3 antibody or antigen-binding fragment thereof provided herein includes amounts that, when in a wet / dry system, provide a concentration of about 1.0 micrograms / ml to about 1000 mg / ml upon reconstitution, although lower and higher concentrations are operable and will depend on the intended delivery vehicle, e.g., solution formulations will differ from transdermal patch, pulmonary, transmucosal, or osmotic or micropump methods.

[0215] In certain embodiments, the compositions provided herein include at least one of any suitable auxiliary substances, such as, but not limited to, diluents, binders, stabilizers, buffers, salts, lipophilic solvents, preservatives, adjuvants, etc. In certain embodiments, the pharma- ceutically acceptable auxiliary substances are specific. Non-limiting examples of such sterile solutions and methods for preparation are well known in the art, such as, but not limited to, Gennaro, Ed., Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Co. (Easton, Pa.) 1990.

[0216] A pharma- ceutically acceptable carrier suitable for the mode of administration, solubility, and / or stability of the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein can be routinely selected.

[0217] In certain embodiments, the compositions provided herein contain one or more pharmaceutical excipients and / or additives. Non-limiting examples of pharmaceutical excipients and additives are proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars including monosaccharides, disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides, derivatized sugars such as alditols, aldonic acids, esterified sugars, and polysaccharides or sugar polymers), which may be present alone or in combination, and comprise 1-99.99% by weight or volume, alone or in combination. Non-limiting examples of protein excipients include serum albumins, such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like. Non-limiting examples of amino acids / antibody components that may also function in a buffering capacity include alanine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and the like. In certain embodiments, the amino acid is glycine. Non-limiting examples of carbohydrate excipients include monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, disaccharides such as lactose, sucrose, trehalose, cellobiose, polysaccharides such as raffinose, melezitose, maltodextrin, dextran, starch, and alditols such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), myo-inositol, and the like. In certain embodiments, the carbohydrate excipient is mannitol, trehalose, or raffinose.

[0218] In certain embodiments, the compositions provided herein include one or more buffering agents or pH adjusting agents, typically the buffering agent is a salt prepared from an organic acid or base. Non-limiting examples of buffering agents include organic acid salts such as salts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid, Tris, tromethamine hydrochloride, or phosphate buffers. In certain embodiments, the buffering agent is an organic acid salt such as citrate. Other excipients, such as isotonicity agents, buffering agents, antioxidants, preservative enhancers, may be added, optionally, but preferably, to the diluent. Isotonicity agents, such as glycerin, are generally used at known concentrations. It is preferred to add a physiologically tolerable buffering agent to provide improved pH control. The compositions can cover a wide range of pH, such as, for example, from about pH 4 to about pH 10, with a specific range of about pH 5 to about pH 9, and a most specific range of about 6.0 to about 8.0. In some embodiments, the compositions provided herein have a pH of about 6.8 to about 7.8. Preferred buffers include phosphate buffers, most preferably sodium phosphate, particularly phosphate buffered saline (PBS).

[0219] In certain embodiments, the compositions provided herein comprise one or more polymeric excipients / additives, such as polyvinylpyrrolidone, Ficoll (a polymeric sugar), dextrates (e.g., cyclodextrins, e.g., 2-hydroxypropyl-beta-cyclodextrin), polyethylene glycol, flavoring agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents, surfactants (e.g., polysorbates such as "TWEEN 20" and "TWEEN 80"), lipids (e.g., phospholipids, fatty acids), steroids (e.g., cholesterol), and / or chelating agents (e.g., EDTA).

[0220] Other additives, such as Tween 20 (polyoxyethylene (20) sorbitan monolaurate), Tween 40 (polyoxyethylene (20) sorbitan monopalmitate), Tween 80 (polyoxyethylene (20) sorbitan monooleate), Pluronic F68 (polyoxyethylene polyoxypropylene block copolymer), and PEG (polyethylene glycol), or pharma- ceutically acceptable solubilizers such as polysorbate 20 or 80 or poloxamer 184 or 188, non-ionic surfactants such as Pluronic® polyols, other block copolymers, and chelating agents such as EDTA and EGTA, can be optionally added to the composition to reduce aggregation. These additives are particularly useful when using a pump or plastic container to administer the composition. The presence of a pharma-ceutically acceptable surfactant mitigates the tendency of the protein to aggregate.

[0221] Additional pharmaceutical excipients and / or additives suitable for use in the compositions provided herein are known to those skilled in the art and are referred to, for example, in "Remington: The Science & Practice of Pharmacy", 19.sup.th ed., Williams & Williams, (1995), and "Physician's Desk Reference", 52nd ed., Medical Economics, Montvale, NJ (1998), which are incorporated herein by reference in their entireties. In certain particular embodiments, the carrier or excipient material is a carbohydrate (e.g., sugars and alditols) and a buffering agent (e.g., citrate) or a polymeric agent.

[0222] In some embodiments, the aqueous diluent optionally further comprises a pharma- ceutically acceptable preservative. Exemplary preservatives include those selected from the group consisting of phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkyl parabens (methyl, ethyl, propyl, butyl, etc.), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate, and thimerosal, or mixtures thereof. The concentration of the preservative used in the composition is sufficient to provide an antibacterial effect. Such concentration depends on the preservative selected and can be easily determined by those skilled in the art.

[0223] The compositions provided herein can be prepared by a process that includes mixing at least one anti-LGALS3 antibody or antigen-binding fragment thereof described herein and a preservative selected from the group consisting of phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkyl parabens (methyl, ethyl, propyl, butyl, etc.), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate, and thimerosal, or mixtures thereof, in an aqueous diluent. Mixing at least one anti-LGALS3 antibody or antigen-binding fragment thereof described herein and a preservative in an aqueous diluent is performed using conventional dissolution and mixing procedures. To prepare a suitable composition, for example, a measured amount of at least one anti-LGALS3 antibody or antigen-binding fragment thereof described herein in a buffer solution is combined with the desired preservative in a buffer solution in an amount sufficient to provide the anti-LGALS3 antibody or antigen-binding fragment thereof described herein and the preservative at the desired concentration. The compositions provided herein can be prepared by a process that includes mixing at least one anti-LGALS3 antibody or antigen-binding fragment thereof described herein and a selected buffer, preferably a phosphate buffer containing a salt or a selected salt. Mixing at least one anti-LGALS3 antibody or antigen-binding fragment thereof described herein and a buffer in an aqueous diluent is performed using conventional dissolution and mixing procedures. To prepare a suitable composition, for example, a measured amount of at least one anti-LGALS3 antibody or antigen-binding fragment thereof described herein in water or buffer is combined with a desired buffer in water in an amount sufficient to provide the protein and buffer at the desired concentration. Variations of these processes will be recognized by those skilled in the art. For example, the order in which the components are added, whether additional additives are used, the temperature and pH at which the composition is prepared are all factors that can be optimized for the concentration and administration means used.

[0224] Parenteral Formulations In certain embodiments, the compositions provided herein are formulated for parenteral injectable administration. As used herein, the term "parenteral" includes intravenous, intravascular, intramuscular, intradermal, subcutaneous, and intraocular. For parenteral administration, the compositions can be formulated as a solution, suspension, emulsion, or lyophilized powder in association with a pharma- ceutically acceptable parenteral vehicle, or can be provided separately. Non-limiting examples of such vehicles are water, saline, Ringer's solution, dextrose solution, glycerol, ethanol, and 1-10% human serum albumin. Non-aqueous vehicles such as liposomes and fixed oils can also be used. The vehicle or lyophilized powder can contain additives that maintain isotonicity (e.g., sodium chloride, mannitol) and chemical stability (e.g., buffers and preservatives). The formulation is sterilized by known or suitable techniques.

[0225] Suitable pharmaceutical carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, A. Osol, a standard reference text in this field.

[0226] Preparations for parenteral administration may contain, as common excipients, sterile water or saline, polyalkylene glycols such as polyethylene glycol, vegetable oils, hydrogenated naphthalenes, etc. Aqueous or oily suspensions for injection can be prepared by using appropriate emulsifiers or moisturizers and suspending agents according to known methods. The medicament for injection may be a non-toxic parenterally administrable diluent, for example, an aqueous solution, or a sterile injection solution or suspension in a solvent. Water, Ringer's solution, isotonic saline, etc. are acceptable as usable vehicles or solvents. Sterile non-volatile oils can be used as normal solvents or suspension solvents. For these purposes, any kind of non-volatile oils and fatty acids can be used, including natural, synthetic, or semi-synthetic aliphatic oils or fatty acids, natural, synthetic, or semi-synthetic monoglycerides, diglycerides, or triglycerides. Parental administration is known in the art and includes, but is not limited to, conventional injection means, gas pressurized needleless injection devices such as those described in U.S. Pat. No. 5,851,198, and laser perforation devices such as those described in U.S. Pat. No. 5,839,446, which is incorporated by reference in its entirety herein.

[0227] Pulmonary preparations In certain embodiments, compositions comprising the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein are formulated for pulmonary administration. For pulmonary administration, the compositions are delivered in a particle size effective to reach the lower airways or paranasal sinuses of the lungs.

[0228] Compositions for pulmonary administration can be delivered by any of a variety of inhalation or nasal devices known in the art for administration of therapeutic agents by inhalation. These devices, which can deposit aerosolized formulations in the sinuses or alveoli of a patient, include metered dose inhalers, nebulizers, dry powder generators, sprayers, and the like. Other devices suitable for effecting pulmonary or nasal administration of the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein are also known in the art. All such devices use formulations suitable for administration of the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein for dispensing in an aerosol. Such aerosols may be composed of either solutions (both aqueous and non-aqueous) or solid particles. Metered dose inhalers, such as the Ventolin® metered dose inhaler, typically use a propellant gas and require actuation during inhalation (see, e.g., WO94 / 16970, WO98 / 35888). Dry powder inhalers such as Turbuhaler™ (Astra), Rotahaler® (Glaxo), Diskus® (Glaxo), and devices marketed by Inhale Therapeutics, to name a few, use breath actuation of a mixed powder (U.S. Pat. No. 4,668,218 Astra, EP 237507 Astra, WO 97 / 25086 Glaxo, WO 94 / 08552 Dura, U.S. Pat. No. 5,458,135 Inhale, WO 94 / 06498 Fisons, which are incorporated herein by reference in their entireties).

[0229] Nebulizers such as the Ultravent® nebulizer (Mallinckrodt) and the Acorn II® nebulizer (Marquest Medical Products) (U.S. Pat. No. 5,404,871 Aradigm, WO 97 / 22376), the above references being fully incorporated herein by reference, generate aerosols from solutions, while metered dose inhalers, dry powder inhalers, and the like generate aerosols of small particles. These examples of commercially available inhalation devices are non-limiting examples and are not intended to be limiting in scope.

[0230] In certain embodiments, a spray comprising an anti-LGALS3 antibody or antigen-binding fragment thereof described herein can be generated by forcing a suspension or solution of at least one anti-LGALS3 antibody or antigen-binding fragment thereof described herein through a nozzle under pressure. The size and configuration of the nozzle, the applied pressure, and the liquid feed rate can be selected to achieve the desired output and particle size. An electrospray can be created, for example, by an electric field in combination with a capillary or nozzle feed. Advantageously, the particles of the composition comprising at least one anti-LGALS3 antibody or antigen-binding fragment thereof described herein delivered by the sprayer have a particle size of less than about 10 μm, for example, in the range of about 1 μm to about 5 μm, for example, about 2 μm to about 3 μm.

[0231] Formulations of compositions comprising at least one anti-LGALS3 antibody or antigen-binding fragment thereof described herein suitable for use with a nebulizer typically comprise at least one anti-LGALS3 antibody or antigen-binding fragment thereof described herein in aqueous solution at a concentration of about 0.1 mg to about 100 mg per ml of solution, or mg / gm, or any range or value therein, such as, but not limited to, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90, or 100 mg / ml or mg / gm. The formulation may include agents such as excipients, buffers, isotonicity agents, preservatives, surfactants, preferably zinc. The formulation may also include excipients or agents for stabilization of the anti-LGALS3 antibody or antigen-binding fragment thereof composition, such as buffers, reducing agents, bulk proteins, or carbohydrates. Bulk proteins useful in formulating such compositions include albumin, protamine, and the like. Exemplary carbohydrates useful in formulating antibody composition proteins include sucrose, mannitol, lactose, trehalose, glucose, and the like. The composition may also include a surfactant, which may reduce or prevent surface-induced aggregation of the composition caused by atomization of the solution in forming the aerosol. A variety of conventional surfactants may be used, such as polyoxyethylene fatty acid esters and alcohols, as well as polyoxyethylene sorbitol fatty acid esters. The amount generally ranges from 0.001% to 14% by weight of the formulation. Preferred surfactants are polyoxyethylene sorbitan monooleate, polysorbate 80, polysorbate 20, and the like.

[0232] In certain embodiments, the composition is administered via a nebulizer, such as a jet nebulizer or ultrasonic nebulizer. Typically, a jet nebulizer uses a compressed air source to generate a high-velocity air jet through an orifice. As the gas expands beyond the nozzle, a low-pressure region is created that pulls the antibody composition protein solution through a capillary tube connected to a liquid container. The liquid stream from the capillary tube is sheared into unstable filaments and droplets as it exits the tube, generating an aerosol. Various configurations, flow rates, and baffle types can be used to achieve the desired performance characteristics from a given jet nebulizer. In an ultrasonic nebulizer, high-frequency electrical energy is used to generate vibrational mechanical energy, typically using a piezoelectric transducer. This energy is transferred to the antibody composition protein formulation, either directly or through a connecting fluid, to generate an aerosol containing the antibody composition protein. Advantageously, the antibody composition protein particles delivered by the nebulizer have a particle size of less than about 10 um, preferably in the range of about 1 um to about 5 um, and most preferably about 2 um to about 3 um.

[0233] In certain embodiments, the compositions are administered via a metered dose inhaler (MDI), in which the propellant, at least one anti-LGALS3 antibody or antigen-binding fragment thereof described herein, and any excipients or other additives are contained in a canister as a mixture with a liquefied compressed gas.

[0234] Actuation of the metering valve releases the dye mixture as an aerosol, which preferably contains particles in the size range of less than about 10 um, preferably about 1 um to about 5 um, and most preferably about 2 um to about 3 um. The desired aerosol particle size can be obtained by using formulations of the antibody composition protein produced by a variety of methods known to those skilled in the art, including jet mill mixing, spray drying, critical point condensation, and the like. Preferred metered dose inhalers include those manufactured by 3M or Glaxo, which use hydrofluorocarbon propellants.

[0235] Formulations of the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein for use with metered dose inhalation devices generally comprise a finely divided powder containing at least one anti-IL-6 antibody as a suspension in a non-aqueous medium, for example, suspended in a propellant with the aid of a surfactant. The propellant can be any conventional material used for this purpose, for example, a chlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, HFA-134a (hydrofluoroalkane-134a), HFA-227 (hydrofluoroalkane-227), and the like. In some embodiments, the propellant is a hydrofluorocarbon. The surfactant can be selected to stabilize at least one anti-LGALS3 antibody or antigen-binding fragment thereof described herein as a suspension in the propellant, such as to protect the active agent from chemical degradation. Suitable surfactants include sorbitan trioleate, soybean lecithin, oleic acid, and the like. In some cases, the solution aerosol uses a solvent, such as ethanol, among others.

[0236] Additional agents known in the art for formulation of proteins can also be included in the formulation.

[0237] Oral Formulations In certain embodiments, the compositions provided herein are formulated for oral administration. In certain embodiments, the compositions and methods for administering at least one anti-LGALS3 antibody or antigen-binding fragment thereof described herein for oral administration rely on the co-administration of adjuvants, such as resorcinol and non-ionic surfactants, such as polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether, to artificially increase the permeability of the intestinal wall, and the co-administration of enzyme inhibitors, such as pancreatic trypsin inhibitor, diisopropyl fluorophosphate (DFF) and trasylol, to inhibit enzymatic degradation. The active component compound of the solid dosage form for oral administration can be mixed with at least one additive, including sucrose, lactose, cellulose, mannitol, trehalose, raffinose, maltitol, dextran, starch, agar, alginate, chitin, chitosan, pectin, tragacanth gum, gum arabic, gelatin, collagen, casein, albumin, synthetic or semi-synthetic polymer, and glyceride. These dosage forms may also contain other types of additives, such as, for example, inert diluents, lubricants such as magnesium stearate, preservatives such as parabens, sorbic acid, ascorbic acid, alpha-tocopherol, antioxidants such as cysteine, disintegrants, binders, thickeners, buffers, sweetening agents, flavoring agents, and perfuming agents.

[0238] In certain embodiments, tablets and pills for oral administration can be further processed into enteric coated preparations. In certain embodiments, liquid preparations for oral administration include, for example, emulsions, syrups, elixirs, suspensions, and solution preparations that are acceptable for medical use. These preparations may contain inert diluents that are commonly used in the art, such as water. Liposomal preparations can be used for oral administration preparations, for example, as described for insulin and heparin (U.S. Pat. No. 4,239,754). In addition, microparticles of artificial polymers of mixed amino acids (proteinoids) can be used for oral administration of pharmaceuticals, for example, as described in U.S. Pat. No. 4,925,673. Furthermore, carrier compounds, such as those described in U.S. Pat. Nos. 5,879,681 and 5,871,753, are used for oral administration of biologically active agents.

[0239] Mucosal preparations In certain embodiments, the compositions provided herein are formulated for absorption through a mucosal surface. In certain embodiments, compositions and methods for administering at least one anti-LGALS3 antibody or antigen-binding fragment thereof described herein for absorption through a mucosal surface include an emulsion comprising a plurality of submicron particles, a mucoadhesive polymer, a bioactive peptide, and an aqueous continuous phase, which achieves mucoadhesion of the emulsion particles, thereby promoting absorption through the mucosal surface (U.S. Pat. No. 5,514,670). Mucous surfaces suitable for application of the emulsions provided herein may include, for example, corneal, conjunctival, oral, sublingual, nasal, vaginal, pulmonary, gastric, intestinal, and rectal routes of administration. Formulations for vaginal or rectal administration, such as suppositories, may contain excipients, such as polyalkylene glycols, petrolatum, cocoa butter, and the like. Formulations for intranasal administration may be solid, may contain excipients, such as lactose, or may be aqueous or oily solutions of nasal drops. For buccal administration, excipients include, for example, sugars, calcium stearate, magnesium stearate, pregelatinized starch, and the like (US Pat. No. 5,849,695).

[0240] Transdermal formulations In certain embodiments, the compositions provided herein are formulated for transdermal administration.In certain embodiments, for transdermal administration, the compositions comprise at least one anti-LGALS3 antibody or its antigen-binding fragment as described herein, which is encapsulated in a delivery device, such as liposome or polymer nanoparticle, microparticle, microcapsule, or microparticle (collectively referred to as microparticle unless otherwise stated).For transdermal administration, some suitable devices are known, including microparticles made from polyhydroxy acids such as polylactic acid, polyglycolic acid, and their copolymers, synthetic polymers such as polyorthoesters, polyanhydrides, and polyphosphazenes, and natural polymers such as collagen, polyamino acids, albumin, and other proteins, alginates and other polysaccharides, and combinations thereof (US Pat. No. 5,814,599).

[0241] kit Also disclosed herein are kits for the detection and / or treatment of galectin-3-associated diseases or conditions (e.g., galectin-3-associated cancers, cardiovascular disease, fibrosis, rheumatic diseases, and inflammation), comprising at least one immunoglobulin-related composition of the present technology (e.g., any of the antibodies, antigen-binding fragments, antibody conjugates, bispecific antibodies or antibody conjugates, scFvs, scFv conjugates, CARs, T cells, recombinant nucleic acid sequences, vectors, or isolated cells described herein) and instructions for use. In certain embodiments, the immunoglobulin-related composition is linked to one or more detectable labels. In one embodiment, the one or more detectable labels comprise a radioactive label, a fluorescent label, or a chromogenic label. Additionally or alternatively, in some embodiments, the kit further comprises a secondary antibody that specifically binds to the immunoglobulin-related composition described herein. In some embodiments, the secondary antibody is linked to at least one detectable label selected from the group consisting of a radioactive label, a fluorescent label, or a chromogenic label.

[0242] In specific embodiments, provided herein is a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions described herein, e.g., one or more antibodies or antigen-binding fragments thereof described herein, In some embodiments, the kit contains a pharmaceutical composition described herein and a prophylactic or therapeutic agent.

[0243] Optionally, associated with such container may be a notice, dosage form, and / or instructions for its use in a format prescribed by a government agency regulating the manufacture, use, or sale of pharmaceutical or biological products. In certain embodiments, instructions included in the kit provide guidance regarding dosages and / or dosing regimens for administration of the pharmaceutical composition.

[0244] Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, packets, sachets, tubes, inhalers, pumps, bags, vials, containers, syringes, and any packaging material suitable for the selected pharmaceutical composition and intended mode of administration and treatment.

[0245] The kits provided herein may further comprise a device used to administer the active ingredient. Examples of such devices include, but are not limited to, syringes, needleless syringes, drip bags, patches, and inhalers.

[0246] The kits provided herein may further include a pharma- ceutically acceptable vehicle that can be used to administer the components. For example, if the components are provided in a solid form that must be reconstituted for parenteral administration, the kit may include a sealed container of a suitable vehicle in which the components can be dissolved to form a particulate-free sterile solution suitable for parenteral administration, or reconstituted as a suspension for oral administration. Examples of pharma- ceutically acceptable vehicles include, but are not limited to, aqueous vehicles (including, but not limited to, Water for Injection USP, Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, and the like).

[0247] Injection, and Lactated Ringer's Injection), water miscible vehicles (including, but not limited to, ethyl alcohol, polyethylene glycol, and polypropylene glycol), and non-aqueous vehicles (including, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate).

[0248] Use and Method Therapeutic Uses and Methods In certain embodiments, provided herein is a method for treating an LGALS3-associated disease or condition, comprising administering to a subject in need thereof a therapeutically effective amount of an anti-LGALS3 antibody or antigen-binding fragment thereof. Exemplary LGALS3-associated diseases or conditions include, but are not limited to, cancer, tumor metastasis, tumor angiogenesis, heart failure, pulmonary fibrosis, renal glomerular disease, inflammatory disease, and rheumatic disease.

[0249] In specific embodiments, the anti-LGALS3 antibody or antigen-binding fragment thereof is administered in combination with one or more additional therapeutic agents. In some embodiments, the one or more additional therapeutic agents inhibit or treat one or more symptoms of an LGALS3-related disease or condition.

[0250] In certain embodiments, provided herein is a method for treating cancer in a subject, particularly a LGALS3-positive cancer in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of an anti-LGALS3 antibody or an antigen-binding fragment thereof. In specific embodiments, the LGALS3-positive cancer is ovarian cancer, lung cancer, pancreatic cancer, breast cancer, fallopian tube cancer, uterine (e.g., endometrial) cancer, primary peritoneal cancer, or cancer of any other tissue that expresses LGALS3.

[0251] For use of anti-LGALS3 antibody or its fragment in a subject of a particular species, an anti-LGALS3 antibody or its fragment that binds to the LGALS3 of that particular species is used. For example, to treat humans, an anti-LGALS3 antibody or its antigen-binding fragment that binds to human LGALS3 is used. In a specific embodiment, the anti-LGALS3 antibody or its antigen-binding fragment is an immunoglobulin.

[0252] In addition, for use of an anti-LGALS3 antibody or fragment thereof in a subject of a particular species, the constant region of the anti-LGALS3 antibody, in particular embodiments, the anti-LGALS3 antibody or antigen-binding fragment thereof, is derived from that particular species. For example, to treat a human, the anti-LGALS3 antibody or fragment thereof may include an anti-LGALS3 antibody or antigen-binding fragment thereof that is an immunoglobulin, and the immunoglobulin includes a human constant region. In a specific embodiment, the subject is a human.

[0253] In specific embodiments, treatment can be achieving beneficial or desired clinical results, whether detectable or undetectable, including but not limited to alleviating symptoms, reducing the extent of disease, stabilizing (i.e., not worsening) the disease state, delaying or slowing disease progression, improving or mitigating the disease state, and remission (whether partial or complete). In specific embodiments, "treatment" can also prolong survival compared to expected survival in the absence of treatment. In specific embodiments, administration of an anti-LGALS3 antibody or antigen-binding fragment thereof described herein, or a pharmaceutical composition described herein, to a subject with cancer (e.g., ovarian cancer, lung cancer, pancreatic cancer, breast cancer, fallopian tube cancer, uterine (e.g., endometrial) cancer, or primary peritoneal cancer, or cancer of any other tissue expressing LGALS3) achieves at least one, two, three, four, or more of the following effects:(i) reducing or ameliorating the severity of one or more symptoms of cancer, (ii) reducing the duration of one or more symptoms associated with cancer, (iii) preventing the recurrence of a symptom associated with cancer, (iv) reducing hospitalization of a subject, (v) reducing the length of hospitalization, (vi) increasing survival rate of a subject, (vii) enhancing or improving the therapeutic efficacy of another therapy, (viii) inhibiting the onset or occurrence of one or more symptoms associated with cancer, (ix) reducing the number of symptoms associated with cancer, (x) improving quality of life as assessed by methods known in the art, (x) inhibiting recurrence of a tumor, (xi) reversing the progression of a tumor and / or one or more symptoms associated therewith, (xii) inhibiting the progression of a tumor and / or one or more symptoms associated therewith, (xiii) reducing tumor growth, (xiv) decreasing tumor size (e.g., (xv) reduction in the formation of newly formed tumors, (xvi) prevention, eradication, removal, or control of primary, regional, and / or metastatic tumors, (xvii) reduction in the number or size of metastases, (xviii) reduction in mortality, (xix) increase in recurrence-free survival, (xx) maintenance of tumor size, no increase in tumor size, or an increase that is less than that seen following standard therapy, as measured by conventional methods available to those skilled in the art, such as magnetic resonance imaging (MRI), dynamic contrast-enhanced MRI (DCE-MRI), X-ray, and computed tomography (CT) scan, or positron emission tomography (PET) scan, and / or (xxi) increase in the length of remission in a patient. Treatment can be the achievement of one or more of the foregoing.

[0254] Diagnostic Use In certain embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein can be used for diagnostic purposes to detect, diagnose, or monitor a condition described herein (e.g., a condition involving LGALS3-positive cancer cells). In certain embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof for use in diagnostic purposes are labeled.

[0255] In certain embodiments, provided herein is a method for detection of a condition described herein, comprising: (a) assaying expression of LGALS3 or a fragment thereof in a cell or tissue sample from a subject using one or more anti-LGALS3 antibodies or antigen-binding fragments thereof described herein; and (b) comparing the level of LGALS3 or a fragment thereof expression to a control level of LGALS3 or a fragment thereof expression, e.g., by comparing the level in a normal tissue sample (e.g., from a subject not having a condition described herein, or from the same patient prior to onset of the condition), wherein an increase or decrease in the assayed level of LGALS3 or a fragment thereof expression is indicative of a condition described herein.

[0256] The antibodies described herein can be used to assay levels of LGALS3 or fragments thereof in biological samples using classical immunohistological methods described herein or known to those of skill in the art (see, e.g., Jalkanen et al., 1985, J. Cell. Biol. 101:976-985, and Jalkanen et al. (1987) J. Cell. Biol. 105:3087-3096). Other antibody-based methods useful for detecting protein gene expression include immunoassays, such as enzyme-linked immunosorbent assays (ELISAs) and radioimmunoassays (RIAs). Suitable antibody assay labels are known in the art and include enzyme labels, such as glucose oxidase; radioisotopes, such as iodine ( 125 I, 121I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), Indium ( 121 In), and technetium ( 99 Tc); luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin.

[0257] In certain embodiments, monitoring of a condition described herein (eg, LGALS3-positive cancer) is performed by repeating the method for diagnosis over a period of time after initial diagnosis.

[0258] The presence of the labeled molecule can be detected in a subject using methods known in the art for in vivo scanning. Those skilled in the art will be able to determine the appropriate method for detecting a particular label. Methods and devices that can be used in the diagnostic methods of the present disclosure include, but are not limited to, whole body scans such as computed tomography (CT), position emission tomography (PET), magnetic resonance imaging (MRI), and sonography.

[0259] Dosage and Regimen The anti-LGALS3 antibody or antigen-binding fragment thereof, or composition, or cell expressing the antibody or antigen-binding fragment thereof, described herein, may be delivered to a subject by various routes. These routes include, but are not limited to, parenteral, intranasal, intratracheal, oral, intradermal, topical, intramuscular, intraperitoneal, transdermal, intravenous, intratumoral, conjunctival, and subcutaneous routes. Pulmonary administration can also be used, for example, by using an inhaler or nebulizer, and formulation with an aerosolizing agent for use as a spray. In one embodiment, the anti-LGALS3 antibody or antigen-binding fragment thereof, or composition described herein, is parenterally administered to a subject. In a specific embodiment, the parenteral administration is intravenous, intramuscular, or subcutaneous.

[0260] The amount of the anti-LGALS3 antibody or antigen-binding fragment thereof, or composition that will be effective in the treatment and / or prevention of a condition will depend on the nature of the disease, and can be determined by standard clinical techniques.

[0261] The exact dosage used in the composition also depends on the route of administration and the type of cancer, and should be determined according to the judgment of the physician and each subject's circumstances.For example, the effective dosage may also vary depending on the means of administration, the target site, the physiological condition of the patient (including age, weight, and health), whether the patient is human or animal, other medicines administered, or whether the treatment is preventive or therapeutic.The treatment dosage is optimally titrated to optimize safety and effectiveness.

[0262] In certain embodiments, in vitro assays are used to help identify optimal dosage ranges, and effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0263] For anti-LGALS3 antibodies or antigen-binding fragments thereof, dosages can range from about 0.0001 to 100 mg / kg, more usually 0.01 to 15 mg / kg, of patient body weight. For example, dosages can be 1 mg / kg body weight, 10 mg / kg body weight, or in the range of 1 to 10 mg / kg, or in other words, 70 mg or 700 mg, or in the range of 70 to 700 mg, respectively, for a 70 kg patient. In general, human antibodies have a longer half-life in the human body than antibodies from other species due to the immune response to foreign polypeptides. Thus, lower dosages of human antibodies and less frequent administration are often possible.

[0264] In certain embodiments, e.g., upon administration of an antibody or antigen-binding fragment thereof, or an engineered cell expressing a CAR, a subject is administered a range of about 1 million to about 100 billion cells, e.g., 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the above values), e.g., about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, , about 70 million cells, about 80 million cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or a range defined by any two of the above values), in some cases about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells), or any value between these ranges. In some embodiments, the dose of total cells and / or the dose of individual subpopulations of cells is administered in the range of 10 4 Or about 10 4 ~10 9 Or about 10 9 cells / kilogram (kg) body weight, e.g., 10 5 ~10 6 in the range of cells / kg body weight, e.g., 1 x 10 5 Or about 1 x 10 5 Cells / kg, 1.5 x 10 5 Or about 1.5 x 10 5 Cells / kg, 2 x 10 5 Or about 2 x 10 5 cells / kg, or 1 x 10 6 Or about 1 x 10 6 Cells / kg, 2 x 10 6 Or about 2 x 10 6 Cells / kg, 5 x 10 6 Or about 5 x 106 cells / kg, or 10 x 10 6 Or about 10 x 10 6 For example, in some embodiments, the cells are 10 4 Or about 10 4 ~10 9 Or about 10 9 T cells / kilogram (kg) body weight, e.g., 10 5 ~10 7 T cells / kg body weight, or within their specified margin of error.

[0265] The anti-LGALS3 antibody or antigen-binding fragment thereof can be administered multiple times. The interval between doses can be, for example, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 6 months, 1 year, or 2 years.

[0266] Combination therapy In specific embodiments, the methods provided herein for treating cancer in a subject (e.g., ovarian cancer, pancreatic cancer, lung cancer, breast cancer, fallopian tube cancer, uterine (e.g., endometrial) cancer, or primary peritoneal cancer) comprise administering to a subject in need thereof a pharmaceutical composition comprising an anti-LGALS3 antibody or antigen-binding fragment thereof described herein, and further comprise administering to the subject one or more additional therapeutic agents. In specific embodiments, the additional therapeutic agent is for treating cancer in the subject (e.g., ovarian cancer, pancreatic cancer, lung cancer, breast cancer, fallopian tube cancer, uterine (e.g., endometrial) cancer, and primary peritoneal cancer). In specific embodiments, the additional therapeutic agent is for treating any side effects of treatment with the anti-LGALS3 antibody or antigen-binding fragment thereof described herein.

[0267] In a specific embodiment, the additional agent is an agent used to treat ovarian cancer. In a specific embodiment, the additional agent is an agent used to treat pancreatic cancer. In a specific embodiment, the additional agent is an agent used to treat lung cancer. In a specific embodiment, the additional agent is an agent used to treat breast cancer. In a specific embodiment, the additional agent is an agent used to treat fallopian tube cancer. In a specific embodiment, the additional agent is an agent used to treat uterine (e.g., endometrial) cancer. In a specific embodiment, the additional agent is an agent used to treat primary peritoneal cancer.

[0268] The anti-LGALS3 antibodies or antigen-binding fragments thereof described herein may be administered simultaneously with or sequentially (before and / or after) the additional therapeutic agent. The antibodies or antigen-binding fragments thereof and the additional therapeutic agent may be administered in the same composition or different compositions and may be administered by the same or different routes of administration. A first therapy (which is an anti-LGALS3 antibody or antigen-binding fragment thereof described herein, or an additional therapeutic agent) may be administered within (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 28 hours, 30 minutes, 45 minutes, 1 hour, 2 hours, 30 minutes, 50 minutes, 60 minutes, 70 minutes, 80 minutes, 90 minutes, 100 minutes, 120 minutes, 140 minutes, 160 minutes, 180 minutes, 200 minutes, 240 minutes, 280 minutes, 30 minutes, 45 minutes, 50 minutes, 60 minutes, 120 minutes, 160 minutes, 18 ...40 minutes, 240 minutes, 240 minutes, 240 minutes, 240 minutes, 240 minutes, 240 minutes, 240 minutes, 240 minutes, 240 minutes, 240 minutes, 240 minutes, 240 minutes, 240 minutes, 240 The additional therapeutic agent may be administered to the subject in combination with the anti-LGALS3 antibody or antigen-binding fragment thereof described herein in the same composition (pharmaceutical composition). In other embodiments, the additional therapeutic agent may be administered to the subject in combination with the anti-LGALS3 antibody or antigen-binding fragment thereof described herein in a different composition (e.g., two or more pharmaceutical compositions are used) than the anti-LGALS3 antibody or antigen-binding fragment thereof described herein.

[0269] Patient population The subject treated according to the methods provided herein can be any mammal, such as a rodent, cat, dog, horse, cow, pig, monkey, primate, or human. In certain embodiments, the subject is a human. In another specific embodiment, the subject is a dog. As used herein, the terms "subject" and "patient" are used interchangeably.

[0270] In certain embodiments, the subject treated in accordance with the methods provided herein has been diagnosed with an LGALS3-positive cancer, including but not limited to, ovarian, lung, pancreatic, breast, uterine, fallopian tube, or primary peritoneal cancer, or cancer of any other tissue that expresses LGALS3.

[0271] The following examples are offered by way of illustration and not by way of limitation. EXAMPLES

[0272] Example 1. Generation of anti-galectin-3 hybridomas using AlivaMab® Mouse The amino acid sequence of the Gal-3 immunogen used to generate antibodies in Ablexis AlivaMab® Mouse is below. ADNFSLHDALSGSGNPNPQGWPGAWGNQPAGAGGYPGASYPGAYPGQAPPGAYPGQAPPGAYPGAPGAYPGAPAPGVYPGPPSGPGAYPSSGQPSATGAYPATGPYGAPAGPLIVPYNLPLPGGVVPRMLITILGTVKPNANRIALDFQRGNDVAFHFNPRFNENNRRVIVCNTKLDNNWGREERQSVFPFESGKPFKIQVLVEPDHFKVAVNDAHLLQYNHRVKKLNEISKLGISGDIDLTSASYTMI (SEQ ID NO: 53)

[0273] To elicit anti-Gal-3 antibodies in AlivaMab® Mouse, multiple tolerance-breaking strategies were used to enhance antigen presentation or stimulate T helper cells. Once the titers in the animals' serum reached high levels, splenocytes from several mice were used in fusions to generate hybridomas.

[0274] Example 2. Screening of hybridomas As a primary screen, ELISA using human Gal-3 was used, and 10 enriched clones specific for Gal-3 were obtained.

[0275] An assay for Gal-3-PE binding to cancer cells was developed as a secondary screen. huGal-3 (Abcam ab89487) was labeled with PE (Innova Biosciences) according to the manufacturer's instructions. An assay for Gal-3-PE binding to OVCAR3 cells was performed as follows: Cultured SKOV3 cells were trypsinized and washed with culture medium. 500, 1000, or 2000 cells were incubated with various concentrations of Gal-3-PE. The indicated number of OVCAR3 cells was pipetted into a 96-well plate with the indicated amount of Gal-3-PE and incubated on the bench in the dark for 2 hours before being read on a Mirrorball® device (TTP Labtech). Binding of Gal-3-PE to OVCAR3 cells was detected using a Mirrorball® flow cytometer. As shown in Figure 3A, OVCAR3 cells showed dose-dependent binding to Gal-3-PE. Similarly, MOLT-4 also demonstrated dose-dependent binding to Gal-3-PE, as shown in FIG. 3B.

[0276] The binding assay described above was converted to an assay for inhibition of Gal-3-PE binding to OVCAR3 cells as follows. Binding reactions to measure binding of Gal-3-PE to OVCAR3 cells were set up in the presence of PBS (negative control) or the indicated antibodies obtained by enrichment (crude purification) from clones selected in the primary binding screen. The extent of binding was measured by flow cytometry and plotted. As shown in Figure 4, five antibodies (12H7, 20F08, 38E05, 39F02, and 46H02) blocked Gal-3-PE binding to OVCAR3 cells. Clone 14D11 (also known as 14D11.2D3) did not block Gal-3-PE binding to OVCAR3 cells in this assay and therefore serves as a negative control.

[0277] Example 3. Characterization of antibodies To determine the species cross-reactivity of the obtained antibodies, binding to human Gal-3 or mouse Gal-3 was detected using ELISA. As shown in Figures 5A-5B, clones 39F02, 38E05, and 46H02 bound to mouse Gal-3, but clones 20F08 and 12H07 did not bind to mouse Gal-3.

[0278] The clones were then subjected to a dose response determination of blocking Gal-3-PE binding to OVCAR3 cells. Assays for inhibition of Gal-3-PE binding to OVCAR3 cells were performed in the presence of increasing concentrations of antibody. Binding reactions to measure binding of Gal-3-PE to OVCAR3 cells were set up in the presence of PBS (negative control) or increasing amounts of purified antibody. As shown in Figure 6A, clones 20F08 and 12H07 showed very similar dose responses of blocking Gal-3-PE binding to OVCAR3 cells. Interestingly, 20F08 and 12H07 were found to have identical sequences. As shown in Figure 6B, clone 46H02 showed a unique dose response of blocking Gal-3-PE binding to OVCAR3 cells. Clones 38E05 and 39F02, which also have identical sequences, showed very similar dose responses in blocking Gal-3-PE binding to OVCAR3 cells, as shown in Figure 6C. Clone 14D11 did not block Gal-3-PE binding to OVCAR3 cells in this assay (Figure 6D).

[0279] Binding parameters were then determined for clones 46H02 and 20F08 using surface plasmon resonance (SPR) in the presence of increasing concentrations of these antibodies. As shown in Figures 7A-7B, monoclonal antibodies (mAbs) 46H02 (Figure 7A) and 20F08 (Figure 7B) exhibited characteristic binding kinetics to human Gal-3. These data demonstrated that mAbs 46H02 and 20F08 exhibited K D The results show that the antibody bound to human Gal-3 at 100 ng / g.

[0280] Since the first generation AlivaMab® Mouse (Ablexis) used here produces a mixture of antibodies with human heavy and light chains, or hybrid antibodies with human heavy and mouse light chains, clones 12H07, 20F08, 38E05, 39F02, and 46H02 were subjected to light chain type determination by ELISA using anti-mouse kappa, anti-mouse lambda, anti-human kappa, and anti-human lambda antibodies. As shown in Figure 8, 46H02 has a fully human F(ab)2, while the other hits have a human HC and a mouse LC.

[0281] To analyze whether 46H02 and 14D11.2D3 bind to overlapping epitopes on Gal-3, an epitope binning assay was performed using a premix epitope binning assay strategy (Figure 9A). In this strategy, the test antibody (analyte) is immobilized on a capture surface, the ligand is flowed over the analyte, and binding is detected by SPR. The ligand is either antigen alone or a premixed solution of antigen complexed with a second antibody. As shown, antigen alone generates a characteristic SPR profile that indicates binding of the analyte to the antigen (left panel). When the ligand is a premixed solution of antigen complexed with a second antibody, if the second antibody binds to a distinct epitope on the antigen, an SPR profile is obtained that indicates binding of the analyte to the antigen (center panel). If the second antibody blocks binding of the analyte, the SPR profile shows no binding, indicating that the second antibody blocks binding of the analyte (right panel). The test antibody (analyte) itself was used as a control to mimic a second antibody blocking the binding of the analyte. As shown in Figure 9B, when mAb2D3 was used as the analyte, the Gal-3+14D11.2D3 complex completely inhibited the binding. In contrast, Gal-3 or Gal-3+46H02 complex did not inhibit the binding (Figure 9B). Similarly, as shown in Figure 9C, when mAb46H02 was used as the analyte, the Gal-3+46H02 complex completely inhibited the binding. In contrast, Gal-3 or Gal-3+14D11.2D3 complex did not inhibit the binding (Figure 9C). Thus, mAb46H02 and 14D11 do not compete with each other and therefore bind to distinct epitopes. [Table 1] TIFF2024501581000003.tif197142 TIFF2024501581000004.tif166139 TIFF2024501581000005.tif161139 TIFF2024501581000006.tif161139 TIFF2024501581000007.tif182140 TIFF2024501581000008.tif166140 TIFF2024501581000009.tif151139

[0282] All publications, patents, and patent applications cited in this specification are incorporated by reference herein as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. Although the foregoing technology of the present invention has been described in some detail by way of illustration and example for clarity of understanding, it will be readily apparent to those skilled in the art in light of the teachings of the technology that certain changes and modifications can be made without departing from the spirit or scope of the appended claims.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to galectin-3 (LGALS3), The antibody or antigen-binding fragment thereof comprises a heavy chain variable region (V H ) and the light chain variable region (V L ), (a) The V H V includes SEQ ID NO:7 H Complementarity determining region (CDR) 1 sequence and V comprising SEQ ID NO:8 H CDR2 sequence and V comprising SEQ ID NO:9 H and / or said V L V includes SEQ ID NO:2 L CDR1 sequence and V comprising SEQ ID NO:3 L CDR2 sequence and V comprising SEQ ID NO:4 L and a CDR3 sequence, (b) the V H V comprising SEQ ID NO: 19 H Complementarity determining region (CDR) 1 sequence and V comprising SEQ ID NO: 20 H CDR2 sequence and V comprising SEQ ID NO:21 H and / or said V L V comprising SEQ ID NO: 14 L CDR1 sequence and V comprising SEQ ID NO:15 L CDR2 sequence and V comprising SEQ ID NO: 16 L and a CDR3 sequence, (c) Said V H V comprising SEQ ID NO:29 H Complementarity determining region (CDR) 1 sequence and V comprising SEQ ID NO: 30 H CDR2 sequence and V comprising SEQ ID NO:31 H and / or said V L V comprising SEQ ID NO:24 L CDR1 sequence and V comprising SEQ ID NO:25 L CDR2 sequence and V comprising SEQ ID NO:26 L and a CDR3 sequence, (d) the V H V comprising SEQ ID NO:39 H Complementarity determining region (CDR) 1 sequence and V comprising SEQ ID NO: 40 H CDR2 sequence and V comprising SEQ ID NO:41 H and / or said V L V comprising SEQ ID NO:34 L CDR1 sequence and V comprising SEQ ID NO:35 L CDR2 sequence and V comprising SEQ ID NO:36 L and a CDR3 sequence, or (e) the V H V comprising SEQ ID NO:49 H Complementarity determining region (CDR) 1 sequence and V comprising SEQ ID NO:50 H CDR2 sequence and V comprising SEQ ID NO:51 H and / or said V L V comprising SEQ ID NO:44 L CDR1 sequence and V comprising SEQ ID NO:45 L CDR2 sequence and V comprising SEQ ID NO:46 L and CDR3 sequence, optionally produced by immunizing a transgenic mouse.

2. The V H comprises an amino acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:18, SEQ ID NO:28, SEQ ID NO:38, and SEQ ID NO:48; or 2. The antibody or antigen-binding fragment of claim 1, wherein the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:13, SEQ ID NO:23, SEQ ID NO:33, and SEQ ID NO:

43.

3. V selected from the group consisting of SEQ ID NO:6 and SEQ ID NO:1, SEQ ID NO:18 and SEQ ID NO:13, SEQ ID NO:28 and SEQ ID NO:23, SEQ ID NO:38 and SEQ ID NO:33, and SEQ ID NO:48 and SEQ ID NO:43, respectively. H Amino acid sequence and V L The antibody or antigen-binding fragment of any one of claims 1 to 2, comprising an amino acid sequence.

4. The antibody or antigen-binding fragment thereof comprises a human or humanized heavy chain variable domain (V H ) and / or a human or humanized light chain variable domain (V L or the antibody or antigen-binding fragment thereof is a humanized or fully human antibody, or antigen-binding fragment thereof, or the antibody comprises heavy and light chain constant regions of human origin, optionally the heavy chain constant region having an isotype selected from the group consisting of gamma 1, gamma 2, gamma 3, and gamma 4, or the light chain constant region having an isotype selected from the group consisting of kappa and tamda, or the antibody is an immunoglobulin comprising two identical heavy chains and two identical light chains, optionally the immunoglobulin is an IgG, or the antibody or antigen-binding fragment thereof inhibits Gal-3-phycoerythrin (PE) binding to ovarian tumor cells, optionally the ovarian tumor cells are OVCAR3 cells, or the antibody or antigen-binding fragment thereof inhibits binding of LGALS3 to a glycosylated cell surface receptor. or the antibody or antigen-binding fragment inhibits binding of LGALS3 to a glycosylated growth factor receptor, or the antibody or antigen-binding fragment inhibits binding of LGALS3 to one or more of glycosylated mucin-1 (MUC1), mucin-4 (MUC4), mucin-16 (MUC16), disialoganglioside, GD2, epidermal growth factor receptor (EGFR), platelet derived growth factor receptor (PDGFR), insulin-like growth factor receptor (IGFR), integrins, or CTLA4, optionally wherein the glycosylated MUC16 is N-glycosylated at Asn1800 or Asn1806, or the antibody or antigen-binding fragment inhibits growth of a tumor expressing a glycosylated form of MUC16, or the antibody is a monoclonal antibody, or the antigen-binding fragment is a Fab, F(ab') 2, Fab', Fv, or scFv; An antibody or antigen-binding fragment according to any one of claims 1 to 3.

5. 5. An antibody conjugate comprising the antibody or antigen-binding fragment of any one of claims 1 to 4 conjugated to an agent, or a scFv conjugate comprising a scFv conjugated to an agent, optionally wherein the agent may be an imaging agent or a cytotoxic agent.

6. 5. The antibody or antigen-binding fragment of any one of claims 1 to 4, wherein the antibody or antigen-binding fragment is a bispecific antibody, optionally wherein the bispecific antibody specifically binds to CD3, or wherein the bispecific antibody comprises an immunoglobulin that specifically binds to LGALS3, the light chain of the immunoglobulin being conjugated via a peptide linker to a single chain variable fragment (scFv) that specifically binds to CD3.

7. A bispecific antibody conjugate comprising the bispecific antibody of claim 6 conjugated to an agent, optionally wherein the agent is an imaging agent or a cytotoxic agent.

8. A chimeric antigen receptor (CAR) comprising the antibody or antigen-binding fragment of any one of claims 1 to 4, or the scFv of claim 4.

9. A T cell recombinantly expressing the CAR of claim 8.

10. 10. A polynucleotide comprising a nucleic acid sequence encoding the antibody or antigen-binding fragment of any one of claims 1 to 6, the scFv of claim 4, and / or the CAR of claim 8, which may optionally comprise the polynucleotide of any one of SEQ ID NO:5, SEQ ID NO:17, SEQ ID NO:27, SEQ ID NO:37, SEQ ID NO:47, SEQ ID NO:10, SEQ ID NO:22, SEQ ID NO:32, SEQ ID NO:42, or SEQ ID NO:52, or a portion thereof encoding at least one CDR sequence.

11. 11. A vector or host cell comprising the polynucleotide of claim 10, optionally wherein the polynucleotide is operably linked to a promoter.

12. A cell comprising the vector of claim 11.

13. 13. A pharmaceutical composition comprising a therapeutically effective amount of the antibody or antigen-binding fragment of any one of claims 1 to 4, the antibody conjugate of claim 5, the bispecific antibody of claim 6, the bispecific antibody conjugate of any one of claim 7, the scFv of claim 4, the scFv conjugate of claim 5, the CAR of claim 8, the T cell of claim 9, the polynucleotide of claim 10, the vector of claim 11, or the cell of claim 11 or 12.

14. The pharmaceutical composition of claim 13 for treating cancer in a patient.

15. 15. The pharmaceutical composition of claim 14, wherein the cancer is metastatic cancer or cancer of the ovary, lung, pancreas, breast, uterus, fallopian tube, or primary peritoneum.

16. 16. The pharmaceutical composition of claim 14 or 15, which inhibits metastasis in a patient.

17. The pharmaceutical composition according to any one of claims 14 to 16, wherein the patient is a human patient.

18. A pharmaceutical composition according to any one of claims 14 to 17, for administration in combination with a therapeutically effective amount of an additional therapeutic agent.