Galectin-1 antibodies and methods of use thereof

Abstract

The present disclosure provides an anti-galectin-1 monoclonal antibody and methods of its use to detect intracellular or cell surface galectin-1. The disclosure also provides methods for using the anti-galectin-1 antibody to detect β-galactoside on a cell surface.

Description

GALECTIN-1 ANTIBODIES AND METHODS OF USE THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 583,053 filed on September 15, 2023, the content of which is incorporated by reference in its entirety.REFERENCE TO A SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (112624.01468.xml; Size: 15,428 bytes; and Date of Creation: September 11, 2024) is herein incorporated by reference in its entirety.BACKGROUND

[0003] Galectin-1 is a P-galactoside-binding lectin that has been implicated as a suppressive molecule in cancer and autoimmune diseases. Gal-1 has known immunomodulatory activity and was found to be expressed on regulatory T cells, leading to the potential for targeted immunotherapies. Therefore, mechanisms for detecting and targeting Gal-1 are of interest.SUMMARY

[0004] In an aspect, provided herein is an anti -galectin-1 (Gal-1) antibody, comprising: a heavy chain variable region (VH) complementary determining region (CDR) CDR1 comprising SEQ ID NO: 15; a VH CDR2 comprising SEQ ID NO: 16; a VH CDR3 comprising SEQ ID NO: 17; a light chain variable domain (VL) CDR1 comprising SEQ ID NO: 12; a VL CDR2 comprising SEQ ID NO: 13; and a VL CDR3 comprising SEQ ID NO: 14. The VH may comprise SEQ ID NO: 3 or a sequence having at least 90% identity thereto; and the VL may comprise SEQ ID NO: 1 or a sequence having at least 90% identity thereto. The Gal-1 antibody may be linked to a detectable label.

[0005] In another aspect, provided herein is a method for detecting at least one of intracellular Gal-1 and cell surface Gal-1 in a sample, the method comprising: contacting the sample with a Gal-1 antibody; and detecting binding of the antibody to Gal-1 in the sample; wherein the Gal-1 antibody comprises: a VH CDR1 comprising SEQ ID NO: 15; a VH CDR2 comprising SEQ IDNO: 16; a VH CDR3 comprising SEQ ID NO: 1 171 ; a light chain variable domain (VL) CDR1 comprising SEQ ID NO: 12; a VL CDR2 comprising SEQ ID NO: 13; and a VL CDR3 comprising SEQ ID NO: 14. The VH may comprise SEQ ID NO: 3 or a sequence having at least 90% identity thereto; and the VL may comprise SEQ ID NO: 1 or a sequence having at least 90% identity thereto. Detecting the binding of the antibody in the sample may be carried out using ELISA, Western blot, a lateral flow assay, immunostaining, immunoprecipitation, flow cytometry, sensor chips, or magnetic beads. The Gal-1 antibody may be linked to a detectable label. The Gal-1 antibody may be biotinylated.

[0006] The sample may comprise one or more of a blood sample, a serum sample, and a tissue sample. The sample may comprise immune cells. The sample may comprise cancer cells.

[0007] The method may comprise detecting only cell surface Gal-1. The method may comprise detecting only intracellular Gal-1. The method comprises detecting both cell surface Gal-1 and

[0008] The Gal-1 antibody may be linked to a solid support.

[0009] In another aspect, provided herein is a method for detecting -galactoside on the surface of a cell, the method comprising: contacting the cell with a Gal-1 protein; washing the cell to remove unbound Gal-1 protein; contacting the cell with a Gal-1 antibody; and detecting binding of the antibody to the Gal-1 protein; wherein the Gal-1 antibody comprises: a VH CDR1 comprising SEQ ID NO: 15; a VH CDR2 comprising SEQ ID NO: 16; a VH CDR3 comprising SEQ ID NO: 17; a light chain variable domain (VL) CDR1 comprising SEQ ID NO: 12; a VL CDR2 comprising SEQ ID NO: 13; and a VL CDR3 comprising SEQ ID NO: 14; wherein binding of the antibody indicates detection of [3-galactoside. The VH may comprise SEQ ID NO: 3 or a sequence having at least 90% identity thereto; and the VL may comprise SEQ ID NO: 1 or a sequence having at least 90% identity thereto. The Gal-1 protein may be a recombinant Gal-1 (rGal-1). The rGal-1 may comprise a sequence having at least 95% identity to SEQ ID NO: 5. The rGal-1 may comprise a detectable label.

[0010] In another aspect, provided is a kit comprising: the Gal-1 antibody described herein; and a detection reagent. The kit may further comprise a solid support. The antibody may be linked to the solid support. The detection reagent may comprise a secondary antibody linked to a detectable label.BRIEF DESCRIPTION OF THE DRAWINGS

[0011] FIGs. 1A-1B. (A) Coomassie blue stain of rGal-1. (B) Peptide mapping of rGal-1 by mass spectrometry. Tryptic digest of the expected bands (-14.5 and -30 kDa) were analyzed by LC-MS and results show 58.52% coverage of Gal-1. High confidence peptides are highlighted in dark gray. Amino acids with carbamidomethylation and oxidation sites are designated as C and O, respectively.

[0012] FIG. 2 shows Anti-Gal-1 mAh 6F3 binds to target recombinant Gal-1 on Western blot. rGal-1 was separated by SDS-PAGE (15%) and transferred to PVDF membrane. Membrane was probed with mAb 6F3, followed by detection with HRP -conjugated anti -mouse IgG secondary antibody. TrueBlue Peroxidase Substrate was used to visualize bands

[0013] FIG. 3 MAb 6F3 binds to SA-bound rGal-1 by ELISA. Serial dilutions of mAb 6F3 were incubated with rGal-1 immobilized by SA on ELISA plates. HRP-conjugated anti -mouse IgGFcy fragment specific secondary antibody was used to detect the interaction. Mean and standard deviation are plotted from three independent experiments.

[0014] FIG. 4 MAb 6F 3 binding is specific for Gal- 1. Competition ELISA was performed by preincubation of Ag:Ab a molar-fold excess. CTRL antigen was HL from the Coccidioides fungus. Ag:Ab was incubated before adding to SA-bound rGal-1 -coated plate. Mean and standard deviation are plotted from three independent experiments.

[0015] FIGs. 5A-5B. (A) Anti-Gal- 1 mAb 6F3 binds to cell surface and intracellular Gal-1 by flow cytometry. (B) MAb 6F3 detects rGal-1 bound to cell surface. MAb 6F3 staining (dark gray histogram) and secondary alone (light gray histogram) are shown. Cell surface and intracellular staining was performed for each cell type. For each histogram, DMFI values are indicated on the graph.

[0016] FIG. 6 Mab 6F3 does not cross-react with other galectins. Cross-reactivity to Galectin-3 and -9 was tested by ELISA. Serial dilutions of mAb 6F3 and commercial mAb were incubated with rGal-1, rGal-3, and rGal-9 coated ELISA plates. Mean and standard deviation are plotted from three independent experiments.DETAILED DESCRIPTION

[0017] The present disclosure provides antibodies for detecting galectin-1 (Gal-1), and methods of their use.

[0018] Antibodies

[0019] In a first aspect, provided herein is an anti-Gal-1 monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) complementary determining region (CDR) CDR1 comprising or consisting of SEQ ID NO: 15; a VH CDR2 comprising or consisting of SEQ ID NO: 16, a VH CDR3 comprising or consisting of SEQ ID NO: 17, a light chain variable domain (VL) CDR1 comprising or consisting of SEQ ID NO: 12, a VL CDR2 comprising or consisting of SEQ ID NO: 13, and a VL CDR3 comprising or consisting of SEQ ID NO: 14.

[0001] The VH may comprise or consist of SEQ ID NO: 3 or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto and the VL may comprise or consist of SEQ ID NO: 1 or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0002] Galectin-1 is a galactoside-binding lectin that belongs to a large family of galectins and has been implicated as a suppressive molecule in cancer and autoimmune diseases. The anti-Gal-1 antibody provided herein is highly specific to galectin-1 and exhibits low cross-reactivity with other galectins.

[0003] The term “antibody” includes “antibody fragments” or antibody-derived fragments” and “antigen binding fragments” which comprise an antigen binding domain (e.g., variable portions of the light chain and heavy chain). “Antibodies” also include chimeric antibodies, humanized antibodies, synthetic antibodies, recombinantly produced antibodies, as known to those skilled in the art, and produced using methods known in the art. An antibody of a particular IgG class may be referred to by its subclass (e.g., IgGl, IgG2, IgG3, and IgG4). Amino acid sequences are known to those skilled in the art for the Fc portion of antibodies of the respective IgG subclass. Examples of antibody fragments include, but are not limited to, Fab, Fab’, F(ab’)2, scFv, Fv, dimeric scFv, Fd, and Fd. Fragments may be synthesized or generated by enzymatic cleavage using methods known in the art. Antibodies can also be produced in either prokaryotic or eukaryotic in vitro translation systems using methods known in the art.

[0004] As used herein, the term “heavy chain” (VH) or “light chain” (VL) respectively refer to an antibody variable heavy (VH) and variable light (VL) domain consisting of a “framework” region interrupted by the three CDRs. The complementarity-determining regions (CDRs) of an antibody are subregions of the variable chains in antibodies involved in binding specific antigens. Anantibody may be referred to by the antibody’s CDRs, which is typically six CDRs total. Thus, an antibody may be referred to herein by the antibody’s CDRs of the heavy chain (VH CDR 1, VH CDR 2, and VH CDR 3) and the light chain (VL CDR 1, VL CDR 2, and VL CDR 3).

[0005] Antibodies may include “chimeric” antibodies (immunoglobulins), as well as fragments of such antibodies, as long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Set. USA 81 : 6851-6855 (1984); Oi et al., Biotechnologies 4(3): 214-221 (1986); and Liu et al., Proc. Natl. Acad. Sci. USA 84: 3439-43 (1987)).

[0006] “Humanized” or “CDR grafted” forms of non-human (e.g., murine) antibodies are human immunoglobulins (recipient antibody) in which hypervariable region residues of the recipient are replaced by hypervariable region residues from a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. The term “hypervariable region” when used herein refers to the amino acid residues of an antibody which are associated with its binding to antigen. The hypervariable regions encompass the amino acid residues of the “complementarity determining regions” or “CDRs”. In some instances, framework region (FW) residues of the human immunoglobulin are also replaced by corresponding non-human residues (so called “back mutations”). Furthermore, humanized antibodies may be modified to comprise residues which are not found in the recipient antibody or in the donor antibody, in order to further improve antibody properties, such as affinity. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable regions correspond to those of a non- human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321 : 522-525 (1986); and Reichmann et al., Nature 332: 323-329 (1988).

[0007] “Single-chain Fv” or “sFv” antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the sFv to form the desired structure for antigen binding. For a review of sFv see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994).

[0008] Variable regions and CDR sequences in an antibody can be identified according to general rules that have been developed in the art, such as, as set out above, for example using the Kabat nomenclature system, or by aligning the sequences against a database of known variable regions. CDRs can be defined using different systems such as, e.g., Kabat, Chothia, Kabat / Chothia, McCallum / Contact, IMGT, Gelfand, Honneger, Martin, North, and AbM.

[0009] Antibodies can be made by well-known methods, such as described in Harlow and Lane, Antibodies; A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1988). Monoclonal antibodies can be produced by immunizing inbred mice with a peptide antigen. As discussed in the Examples, Gal-1 antibody producing hybridoma cells disclosed herein were produced by immunizing mice with recombinant Gal-L

[0010] The sequences provided are only for the variable regions of the antibody. Those skilled in the art will appreciate that these regions determine the specificity of the antibody but that the effector function of the antibody is generally dependent on the constant regions (and the specific isotype) of the antibody. Those skilled in the art can engineer antibodies for specific purposes based on the variable regions provided herein. In some embodiments, the antibody comprises an Fc portion of a human or humanized antibody. In some embodiments, the antibody comprises an Fc region which has been engineered for a suitable effector function(s).

[0011] The term “identity”, as recognized by those skilled in the art, represents a comparison between two or more amino acid sequences performed using published methods and software known in the art. For example, the compared amino acid sequences are optimally aligned, and the number of amino acid differences are counted and converted to a percentage. For example, if a first amino acid sequence of 50 amino acids is optimally aligned with a second amino acid sequence of 50 amino acids, and 5 out of 50 amino acids differ from the second amino acid sequence, then the first amino acid sequence is said to have 10% identity with the second amino acid sequence.

[0012] Any of the antibodies provided herein may comprise a detectable label. Non-limiting examples of detectable labels include fluorophores (e.g. GFPs and fluoresceins), horseradish peroxidases, alkaline phosphatases, luminescent agents, luciferase, radioisotopes, biochemical tags (e.g. biotin or avidin), colorimetric reagents, and combinations thereof. In exemplary embodiments, the Gal-1 antibody is linked to biotin.

[0013] Methods for Detecting Gal-1

[0014] In a second aspect, provided herein is a method of detecting at least one of intracellular Gal-1 and cell surface Gal-1 in a cell sample, the method comprising contacting the sample with the anti-Gal-1 antibody disclosed herein and detecting binding of the antibody to Gal-1 in the sample.

[0015] The sample may comprise any sample isolated from a subject in which detecting the presence or absence of Gal-1 is desired. The sample may be a blood sample, a serum sample, a tissue sample, or cells extracted therefrom. The sample may be derived from a tumor. In preferred embodiments, the sample comprises at least one of immune cells and cancer cells. The sample may be taken from a subject having or suspected of having a disease or disorder related to expression of Gal-1. The disease or condition may be a cancer. The terms “subject” and “individual” are used interchangeably herein to refer to a mammal, preferably a human.

[0016] “Contacting” a cell or sample refers to adding the Gal-1 antibody or Gal-1 protein to the sample or a medium or buffer comprising the sample.

[0017] In embodiments, the method comprises detecting only cell surface Gal-1. In embodiments, the method comprises detecting only intracellular Gal-1. In embodiments in which only cell surface or intracellular Gal-1 are detected, the method may further comprise separating intracellular materials from the remaining cellular materials or separating cell membrane material from the remaining cellular materials by methods known in the art.

[0018] The step of detecting the binding of the antibody may be done using any suitable detection methods known in the art, including, but not limited to, ELISA, Western blot, immunostaining, immunoprecipitation, flow cytometry, and use of sensor chips, magnetic beads, nanoparticles and the like. The Gal-1 antibody may be linked to a detectable label. The Gal-1 antibody may be biotinylated. The Gal-1 antibody may be linked to a solid support.

[0019] A “solid support” is any suitable material for the immobilization of peptides (including antibodies) of the present disclosure. For example, the solid support may be a substrate, a bead, a particle, a tube, a tissue culture plate, a probe, a dipstick, a slide, a fiber, a membrane, a paper, a natural or modified cellulose, polyacrylamide, agarose, glass, polypropylene, polyethylene, polystyrene, dextran, nylon, amylases, plastics, magnetite or any other suitable material readily known to one of skill in the art.

[0020] The method may further comprise measuring the levels and / or concentrations of bound antibody-Gal-1 complexes.

[0021] Method for Detecting p-galactoside

[0022] In a third aspect, provided herein is a method for detecting P-galactoside on a cell surface, the method comprising contacting the cell with a Gal-1 protein, contacting the cell with the Gal-1 antibody disclosed herein, and detecting binding of the antibody to the Gal-1 protein. The binding of the antibody indicates detection of P-galactoside on the cell surface. The step of detecting the binding of the antibody may be done using any suitable detection methods known in the art, including, but not limited to, ELISA, Western blot, a lateral flow assay, immunostaining, immunoprecipitation, flow cytometry, and use of sensor chips, magnetic beads, nanoparticles and the like. For lateral flow assays, the Gal-1 antibody may be provided at about 2 pg / 100 pL. For Western blots, the Gal-1 antibody may be provided at about 5 pg / mL. The Gal-1 antibody may be linked to a detectable label. The Gal-1 antibody may be biotinylated.

[0023] The Gal-1 may be a recombinant Gal-1 (rGal-1). The rGal-1 may be derived from Human Galectin-1 expression plasmid (Galectin-1 cDNA ORF Clone, Human, C-His tag in pCMV3) (Sino Biological (Catalog #HG10290-CH)). The rGal-1 may comprise a sequence having at least 95% identity to SEQ ID NO: 5. The Gal-1 antibody or rGal-1 may be linked to a solid support.

[0024] Kits

[0001] In a fourth aspect, provided herein is a kit comprising components for carrying out the methods described herein. The kit may comprise the Gal-1 antibody disclosed herein and a detection reagent. The kit may comprise a solid support to which the Gal-1 antibody may be linked. The solid support may be a lateral flow device. The detection reagent may comprise a secondary antibody linked to a detectable label. The secondary antibody may be capable of binding the Fc portion of the Gal-1 antibody. A suitable kit may be an ELISA kit. The kit may include a dropper for dispensing controlled volumes of liquid, such as a controlled volume disposable Pasteur pipette. The kit may further include a written insert component comprising instructions for detecting Gal-1 in a sample according to methods of this disclosure.

[0025] Miscellaneous

[0026] The present disclosure is not limited to the specific details of construction, arrangement of components, or method steps set forth herein. The compositions and methods disclosed herein are capable of being made, practiced, used, carried out and / or formed in various ways that will be apparent to one of skill in the art in light of the disclosure that follows. The phraseology and terminology used herein is for the purpose of description only and should not be regarded aslimiting to the scope of the claims. Ordinal indicators, such as first, second, and third, as used in the description and the claims to refer to various structures or method steps, are not meant to be construed to indicate any specific structures or steps, or any particular order or configuration to such structures or steps. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to facilitate the disclosure and does not imply any limitation on the scope of the disclosure unless otherwise claimed. No language in the specification, and no structures shown in the drawings, should be construed as indicating that any non-claimed element is essential to the practice of the disclosed subject matter.

[0027] The use herein of the terms “including,” “comprising,” or “having,” and variations thereof, is meant to encompass the elements listed thereafter and equivalents thereof, as well as additional elements. Embodiments recited as “including,” “comprising,” or “having” certain elements are also contemplated as “consisting essentially of’ and “consisting of’ those certain elements.

[0028] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure. Use of the word “about” to describe a particular recited amount or range of amounts is meant to indicate that values very near to the recited amount are included in that amount, such as values that could or naturally would be accounted for due to manufacturing tolerances, instrument and human error in forming measurements, and the like. All percentages referring to amounts are by weight unless indicated otherwise.

[0029] In those instances where a convention analogous to “at least one of A, B and C, etc.” is used, in general such a construction is intended in the sense of one having ordinary skill in the art would understand the convention (e.g., “a system having at least one of A, B and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and Ctogether, B and C together, and / or A, B, and C together ). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description or figures, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or ‘B or “A and B.”

[0030] No admission is made that any reference, including any non-patent or patent document cited in this specification, constitutes prior art. In particular, it will be understood that, unless otherwise stated, reference to any document herein does not constitute an admission that any of these documents forms part of the common general knowledge in the art in the United States or in any other country. Any discussion of the references states what their authors assert, and the applicant reserves the right to challenge the accuracy and pertinence of any of the documents cited herein. All references cited herein are fully incorporated by reference, unless explicitly indicated otherwise. The present disclosure shall control in the event there are any disparities between any definitions and / or description found in the cited references.

[0031] Preferred aspects of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred aspects may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect a person having ordinary skill in the art to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0032] The following examples are meant only to be illustrative and are not meant as limitations on the scope of the invention or of the appended claims.

[0033] EXAMPLES

[0034] The following examples are meant only to be illustrative and are not meant as limitations on the scope of the invention or of the appended claims.

[0035] Galectin-1 (Gal-1) is a 14.5kDa protein encoded by LGALS1 gene belonging to a family of conserved lectins that bind to b-galactoside-containing glycoproteins. These lectins share conserved carbohydrate recognition domains (CRD) that are responsible for binding to b-galactoside1. Gal-1 functions as a homodimer, contains one CRD and can be found both intra- and extracellularly. Although it does not contain a canonical signal sequence, it is known to be secreted and bind to cell surfaces, but the exact mechanism is unknown2. Intra- and extracellular Gal-1 have distinct functions. Extracellular Gal-1 predominately exists as a homodimer and functions by binding carbohydrate moi eties containing b-galactoside, while the function of intracellular Gal-1 does not require carbohydrate binding1. Intracellular Gal-1 can be found in the cytosol and nucleus and plays a role in signal transduction and transcription to enhance cell proliferation3’4. Extracellular Gal-1 homodimers mediate cell-cell interactions by binding to cell surface receptors via b-galactosides which induces cross-linking due to bivalent binding activity5,6. These interactions with cell surface glycoproteins induce signaling pathways that modulate cell proliferation3. Extracellular Gal-1 has been shown to exert strong immunosuppressive activity, specifically targeting and inducing apoptosis of activated T cells7,8, making it a potential biological target for immunotherapy.

[0036] The immunomodulatory activity of Gal-1 has been implicated in numerous cancers5,9,10and autoimmune diseases8. Gal-1 expression and subsequent secretion has been found to be upregulated in tumors compared to normal cells, influencing tumor progression via immunosuppression, and by promoting cell adhesion, apoptosis and angiogenesis5,11. Increased expression of Gal-1 is found in highly invasive and poorly differentiated tumors and correlated with poor patient prognosis1,5,11. The potential role of Gal-1 in immune suppression and cancer progression makes it a significant protein of interest.

[0037] Evidence of the role of Gal-1 in tumorigenesis has led to an increased interest in investigating novel approaches to inhibit its function. Targeting strategies include using oligosaccharides, synthetic peptides, small molecules and monoclonal antibodies that specifically block the CRD and prevent Gal-l-glycan interactions3. Several Gal-1 inhibitors are currently under development. OTX008 compound was derived from a peptide that was shown to inhibit Gal-1 and reduces tumor growth and angiogenesis in vivo3. An allosteric inhibitor is being evaluated that targets both Gal-1 and Gal-312. Neutralizing mAbs targeting Gal-1 are also being investigated. An anti-Gal-1 neutralizing antibody was developed and shown to block Gal-l-induced apoptosis9,13. Another anti-Gal-1 neutralizing mAb is currently being tested in tumor models for immunomodulatory activity.14

[0038] We became interested in Gal-1 as it has been identified on human CD4+CD25highFoxP3+ Tregs and may play a role in Treg cell expansion and suppressive activity12 15. Previously, we found that human CD4+CD25highFoxP3+ regulatory T cells (Tregs) suppress CD4+ PBMCs by an uncharacterized cell contact-dependent mechanism16. However, Tregs are difficult to purify and expand due to high heterogeneity and lack of a Treg-specific cell surface molecule. To study Tregs, we identified a human Treg-like cell line that is phenotypically similar and suppresses activated CD4+ PBMCs16. However, the mechanisms of Treg-mediated immune suppression are not fully understood. In vitro, Tregs suppress via cell-cell contact and suppression is not reversed by blocking cytokines or immune checkpoint pathways that are known to play a role in Treg suppression in vzvo15 16. Proteomic analysis by our group found Gal-1 to be expressed on Tregs.

[0039] To aid in the study of Gal-1 mediated suppression by Tregs and tumor cells, we developed a non-neutralizing, non-blocking monoclonal antibody against Gal-1. This antibody may be useful in research and / or diagnostic settings for the study of Gal-l-T cell interactions and immunosuppression. Herein, we describe the generation and characterization of a Gal-1 specific monoclonal antibody compatible with flow cytometry and intracellular staining.

[0040] Methods

[0041] Cell lines

[0042] Tregs were isolated using EasySep Human CD4+CD127lowCD25+ Regulatory T Cell Isolation Kit from whole PBMCs. MoT cells were a gift from the late David Golde (ATCC CRL- 8066; Manassas, V A, USA) and are a CD4+ human T-lymphoblast cell line derived from apatient with a T-cell variant of hairy-cell leukemia17. Jurkat cells (ATCC TIB- 152) are a human T- lymphocyte cell line from a patient with acute T cell leukemia. MT-2 cells (RRID:CVCL 2631) area T cell line established by co-incubation with HTLV-I-infected leukemic cells from a patient with adult T cell leukemia. MT-4 cells (RRID:CVCL_2632) are a T cell line derived from coincubation with cells from a patient with adult T cell leukemia. H9 cells (HTB-176) are a human cutaneous T-lymphocyte line isolated from a patient with lymphoma. MDA-MB-231 (ATCC HTB-26) is a human triple-negative breast adenocarcinoma (TNBC) cell line and MCF7 (ATCC HTB-22) is a human non-invasive breast cancer cell line18. All cell lines were maintained in DMEM (Gibco; Grand Island, NY, USA) supplemented with 5% heat-inactivated fetal bovine serum (Atlanta Biologicals; Norcross, GA, USA). Cell lines underwent short tandem repeat validation to confirm their identities (University of Arizona Genetics Core).

[0043] Production and purification of recombinant Galectin-1

[0044] Human Galectin-1 expression plasmid (Galectin-1 cDNA ORF Clone, Human, C-His tag in pCMV3), was purchased from Sino Biological (Catalog #HG10290-CH) and transfected into FreeStyle 293F cells (Catalog R79007; Thermo Fisher). Transfected 293F cells were grown shaking at 37°C, 8% CO2 for seven days. Cell supernatants were harvested by centrifugation and recombinant Gal-1 (rGal-1) was purified by nickel column via C-terminal 10X histidine tag. Purified protein was concentrated and dialyzed in IX phosphate-buffered saline (PBS) with an Amicon filter unit (Sigma), quantified using the Pierce BCA Protein Assay Kit (Thermo Fisher) and stored at -80°C. lOpg of protein was excised from a polyacrylamide gel after electrophoresis (SDS-PAGE) and subjected to in-gel tryptic digestion protocol for LC-MS / MS. Tryptic digest was submitted to the ASU proteomics core facility for analysis. Mass spectra were searched against a human proteome FASTA database from UniProt (Proteome ID: UP000005640) using Proteome Discover.

[0045] Immunization and hybridoma production

[0046] BALB / c mice were immunized by subcutaneous (SQ) injection with rGal-1 (15pg) in Magic Mouse Adjuvant (Creative Diagnostics) under an I ACUC -approved protocol at Arizona State University. Serum was collected to monitor anti-Gal-1 antibody titers by indirect enzyme linked immunosorbent assay (ELISA). Once sufficient antibody titers were reached, a final SQ boost was given, mice were sacrificed and splenocytes were fused with myeloma cells (P3X63Ag8.653; ATCC CRL-1580) by a previously described hybridoma generation technique19. Briefly, splenocytes were fused with myeloma cells at a ratio of 1 :2 using 50% polyethylene glycol solution (Sigma-Aldrich). The fused cells were resuspended in hypoxanthine-aminopterin- thymidine (HAT) media (Sigma-Aldrich) to select for successfully fused clones and plated at 5 x 104splenocytes per well in 96-well flat bottom plates. Plates were incubated for ten days at 37°C, 5% CO2.

[0047] Detection and purification of anti-Gal-1 monoclonal antibodies

[0048] Mouse antisera and hybridoma supernatants were screened by indirect ELISA for anti- Gal- 1 IgG antibodies. Briefly, streptavidin at 10 pg / ml was coated onto a 96-well high-binding plate at 37°C for 1 hour, washed three times with IX PBS + 0.05% Tween-20 (PBST) andblocked in 1% bovine serum albumin (BSA) in PBS for 1 h. Next, biotinylated rGal-1 was added to the plate at 1 pg / ml in 1% BSA / PBS and allowed to bind overnight at 4°C. To monitor antibody titers, mouseantisera was two-fold serially diluted starting at 1 :2,000, added to the plate, and incubated for 1 h at room temperature. To test hybridoma supernatants, undiluted cell supernatant was added to the plate and incubated for 1 h at room temperature. To detect primary antibody bound to rGal-1, plates were washed with PBST three times and incubated with HRP-conjugated Goat Anti-Mouse IgG, Fey fragment specific antibody (Jackson ImmunoResearch) at 1 : 10,000 dilution for 1 h at room temperature. Plates were then washed with PBST four times and TMB substrate was added to develop for 15 minutes. The reaction was stopped with 0.16 M sulfuric acid, and absorbance was measured at 450 nm. Positive wells were subcloned by limiting dilution and re-screened after ten days using the same procedure. Positive subclones were cultured in DMEM (Gibco; Grand Island, NY, USA) supplemented with 10% heat-inactivated fetal bovine serum (Atlanta Biologicals; Norcross, GA, USA). Monoclonal antibodies were purified from hybridoma supernatants by Protein A chromatography (MabSelect; Cytiva). Multiple mAbs against rGal-1 were identified, but mAb 6F3 performed well in several assays.

[0049] Western blotting

[0050] rGal-1 was subjected to SDS-PAGE under non-reducing conditions using 15% polyacrylamide gels at 130 V for 1 h (Bio-Rad) and subsequently stained with Coomassie Blue dye. For Western blot analysis, proteins were transferred to PVDF membranes at 90V for 1 h. Membranes were then blocked in 5% BSA / PBS overnight at 4°C. Next, anti-Gal-1 6F3 at 5 pg / ml was added and incubated with the membrane for 1 h at room temperature, then washed three times in Tris-buffered saline + 0.1% Tween-20 (TBST). After washing, secondary antibody (peroxidase conjugated goat anti-mouse IgG, Fey fragment specific; Jackson ImmunoResearch) was added at 1 : 10,000 and incubated for 1 h at room temperature. Membranes were then washed four times with TBST followed by addition of KPL TrueBlue Peroxidase Substrate (Sera Care) and developed for 10 minutes.

[0051] Indirect ELISA

[0052] To evaluate mAb 6F3 binding by ELISA, biotinylated rGal-1 was bound to streptavidin (SA-bound) as described above. Then, the plates were washed three times with PBST and blocked in 1% BSA / PBS for 1 h. To test binding, mAb 6F3 was added at 16 pg / ml and diluted two-fold in 1% BSA / PBS for 1 h. The plates were washed three times with PBST and then incubated with HRP-conjugated Goat Anti -Mouse IgG, Fey fragment specific antibody (Jackson ImmunoResearch) at 1 : 10,000 dilution for 1 h at room temperature. Plates were then washed withPBST four times and developed for 15 minutes with TMB substrate. The reaction was stopped with 0.16 M sulfuric acid, and absorbance was measured at 450 nm.

[0053] Flow cytometry

[0054] 2 x 105cells per 12x75 mm flow tube were washed twice in PBS and blocked inflow cytometry staining buffer (5% FBS / PBS) for 1 h at room temperature. Next, primary antibody anti- Gal-1 mAb 6F3 or anti-Gal-1 mAb control (Galectin-1 Monoclonal Antibody, clone 6C8.4-1; Invitrogen) was added at 2 pg / 100 pl for 1 h at room temperature. Cells were washed two times in 5% FBS / PBS and secondary antibody was added (PE-conjugated Goat Anti-Mouse Ig; BD Pharmingen) at 1 :50 for 1 h at room temperature. Next, cells were washed three times in 5% FBS / PBS and analyzed on CytoFlex LX flow cytometer (Beckman Coulter). For intracellular staining, cells were fixed and permeabilized with True-Nuclear Transcription Factor Buffer Set (BioLegend) then stained as described above to detect intracellular Gal-1. For Treg staining, biotinylated mAb 6F3 was used and detected with AlexaFluor 555-conjugated streptavidin (Invitrogen). Fluorescence was quantified and expressed as difference in geometric mean fluorescence intensity (DMFI) between the primary antibody staining and secondary alone control.

[0055] Results

[0056] Production and purification of recombinant Galectin-1

[0057] Recombinant human Gal-1 was produced using pCMV3 mammalian expression plasmid. After purification, rGal-1 was separated by SDS-PAGE and stained with Coomassie blue stain (FIG. 1A). Bands were observed at the expected molecular weight of monomeric (~14.5kDa) and dimeric (~30kDa) Gal-1. Further analysis using mass spectrometry confirmed the identity of these bands as Gal-1, accession no. P09382 (FIG. IB).

[0058] Anti-Gal-1 mAb binds specifically to recombinant Galectin-1

[0059] Mice were immunized with rGal-1, followed by generation of hybridomas. Anti-Gal-1 IgG antibodies were detected in spent hybridoma supernatants by ELISA (described above). Clone 6F3 was identified by screening supernatants for binding to rGal-1 and purified by Protein A affinity chromatography. Western blot analysis was performed to evaluate binding of purified mAb 6F3 to rGal-1 (FIG. 2).

[0060] Since immobilization of protein on plastic can sterically hinder the ability of antibody to bind antigen20, we tested the binding ability of mAb 6F3 to SA-bound biotinylated rGal-1 by ELISA (FIG. 3). Control antigen (CTRL) was used as a negative control. HL antigen, derived fromthe Coccidioides fungus, was used as a control recombinant protein and was produced in 293F cells under the same conditions as rGal-1. This protein was chosen due to its similar molecular weight ~17kDa and because it contains a C-terminal his-tag.

[0061] To further support that mAb 6F3 specifically binds to rGal-1, a competition ELISA was performed (FIG. 4). MAb 6F3 was pre-incubated with rGal-1 at molar fold-excess of antigen starting at 200: 1 and two-fold serially diluted. When rGal-1 was allowed to pre-incubate with mAb 6F3, we observed reduced binding to SA-bound rGal-1 in a dose-dependent manner. Preincubation of mAb 6F3 with control antigen had no effect on binding.

[0062] Since Galectins-3 and -9 are also implicated in immune modulation, are known to be secreted, and interact with T cell surface proteins12,21,22, we tested mAb 6F3 for cross-reactivity. We found neither mAb 6F3 or the commercial mAb show cross-reactivity by ELISA (FIG. 6).

[0063] Anti-Gal-1 mAb binding to cell surface and intracellular Gal-1 detected by flow cytometry

[0064] Gal-1 has been found to be expressed on Tregs and may play a role in Treg-mediated suppression of inflammatory T cells13. We previously characterized a Treg-like cell line, MoT cells, which expresses Treg-associated markers and suppresses CD4+ T cell proliferation, similarly to human Tregs isolated from peripheral blood16. Therefore, we wanted to evaluate mAb 6F3 binding to Gal-1 on PBMC-derived Tregs and MoT cells. We found that mAb 6F3 binds to Tregs and MoT cell surface but not to Jurkat cells, a non-suppressive T cell line (FIG. 5A). In contrast, a commercially available anti-Gal-1 mAb bound poorly to MoT cells and also demonstrated minor non-specific binding on Jurkat cells. We also evaluated other Treg-like cell lines: MT-223, MT-4 and H9. MAb 6F3 detected intracellular Gal-1 in Tregs, MoT, MT-2, andMT-4 but not H9 cells. Since Gal-1 has been implicated in metastatic breast cancer4, we also evaluated mAb 6F3 binding to MDA-MB-231 (highly aggressive) and MCF7 (non-metastatic) breast cancer cell lines. MAb 6F3 detected higher levels of intracellular Gal-1 in MDA-MB-231 compared to MCF7. In contrast, we observed shifts of all cell lines relative to the secondary control when using the commercial mAb. We then wanted to evaluate the ability of mAb 6F3 to detect endogenous versus recombinant Gal-1 on the cell surface. Briefly, Jurkat cells (Gal- 1 -negative) were pre-incubated with rGal-1 at increasing concentrations to allow rGal-1 to bind to the cell surface. After incubation, the cells were washed to remove unbound rGal-1 and subsequently surface stained with mAb 6F3 as described. Commercially available Gal-1 mAb was used as a control. MAb 6F3 was able to detectrGal-1 bound to Jurkat cells suggesting that Jurkat cells do not produce Gal-1 but possess the b- galactoside carbohydrate (FIG. 5B).

[0065] Discussion

[0066] In this study, mAbs against Gal-1 were generated by fusion of B cells from mice immunized with rGal-1 to mouse myeloma line P3X63. Anti-Gal-1 IgG antibodies were detected in the resulting hybridoma supernatants. MAb 6F3 was isolated and found to bind to rGal-1 by Western blot (FIG. 2). In FIGs. 3 and 4, we show mAh 6F3 is specific for rGal-1 by indirect and competition ELISA techniques. MAb 6F3 does not demonstrate cross-reactivity with other galectins (FIG. 4).

[0067] MAb 6F3 was used to detect cell surface and intracellular Gal-1 in Tregs, T cell lines and breast cancer lines by flow cytometry analysis. Interestingly, mAb 6F3 detects Gal-1 on the cell surface of PBMC-derived Tregs and MoT cells but not on other Treg-like or other T cell lines (FIG. 5 A). MAb 6F3 also detected higher levels of intracellular Gal-1 in MDA-MB-231 compared to MCF7. This finding supports Gal-1 expression in highly invasive, metastatic breast cancer as has been reported5. In comparison, the commercial mAb showed positive intracellular staining of all cell lines, even in Gal-1 negative / low cells. Mab 6F3 detected low and high concentrations of rGal-1 in a dose-dependent manner on the cell surface of a Gal- 1 -negative cell line (FIG. 5B).

[0068] The generation of mAb 6F3 provides a new tool in studying Gal-1 function and interactions with other proteins. Gal-1 is known to interact with several binding partners including extracellular matrix proteins, cell surface glycoproteins, cytoplasmic and nuclear proteins. Flow cytometry showed that mAb 6F3 has the ability to bind to tumor cells and human Tregs. This is significant as Gal-1 is known to bind to the cell surface and interact with ECM proteins, integrins, and T cell glycoproteins. MAb 6F3 can also detect intracellular Gal-1 (FIG. 5 A), which can be used as a tool when studying interactions between Gal-1 and other intracellular proteins. Gal-1 has been shown to interact with and influence the function of nuclear proteins FoxP34and Genim41 3and cytoplasmic protein Ras1,3. These proteins that have significant biological functions in cell growth signaling pathways, RNA splicing, and gene activation.

[0069] Gal-1 promotes an immunosuppressive environment by increasing the number of Tregs12and inducing T cell apoptosis, although the mechanism of Gal- 1 -mediated apoptosis is not well understood11,25. Development of an anti-Gal-1 mAb can be used to evaluate Gal-1 function, including Gal- 1 -mediated apoptosis of T cells. We tested mAb 6F3 in a suppression assay todetermine if it reversed Gal-1 -mediated Treg suppression as previously suggested15andfound that it did not block suppressive activity (data not shown). This led to the conclusion thatmAb 6F3 either does not block Gal-1 activity or Gal-1 is not mediating Treg cell contact-dependent suppression. Herein, we demonstrate that mAb 6F3 may be used to evaluate Gal-1 expression in different tumor cell types and study its function in the context of cancer and otherimmune diseases.

[0070] References1. Camby I, Le Mercier M, Lefranc F, Kiss R. Galectin-1 : A small protein with major functions. Glycobiology. 2006; 16(11). doi: 10.1093 / glycob / cwl0252. Hughes RC. Secretion of the galectin family of mammalian carbohydrate-binding proteins. Biochim Biophys Acta - Gen Subj. 1999; 1473(1): 172-185. doi:10.1016 / S0304- 4165(99)00177-43. Raymond E. OTX008, a selective small-molecule inhibitor of galectin-1, downregulates cancer cell proliferation, invasion and tumour angiogenesis. Eur J Cancer. 2014;50(14):2463- 2477. doi: 10.1016 / j.ejca.2014.06.0154. Gao Y, Li X, Shu Z, et al. Nuclear galectin- 1-FOXP3 interaction dampens the tumorsuppressive properties of FOXP3 in breast cancer. Cell Death Dis. 2018;9(4). doi : 10.1038 / s41419-018-0448-65. Astorgues-Xerri L, Riveiro ME, Tijeras-Raballand A, et al. Unraveling galectin-1 as a novel therapeutic target for cancer. Cancer Treat Rev. 2014;40(2):307-319. doi: 10.1016 / j.ctrv.2013.07.0076. Cedeno-Laurent F, Dimitroff CJ. Galectin-1 research in T cell immunity: Past, present and future. Clin lmmunol. 2012; 142(2): 107-116. doi: 10.1016 / j.clim.2011.09.0117. Perillo Nl, Pace Ke, Seilhamer Jj, Baum Lg. Apoptosis of T cells mediated by galectin-1. Nature. 1995;378(6558):736-739.8. Deak M, Hornung A, Novak J, et al. Novel role for galectin-1 in T-cells under physiological and pathological conditions. Immunobiology. 2015;220(4):483-489. doi: 10.1016 / j.imbio.2014.10.023

[0071] Embodiments

[0072] 1. An anti -galectin-1 (Gal-1) antibody, comprising: a heavy chain variable region (VH) complementary determining region (CDR) CDR1 comprising SEQ ID NO: 15; a VH CDR2 comprising SEQ ID NO: 16; a VH CDR3 comprising SEQ ID NO: 17; a light chain variabledomain (VL) CDR1 comprising SEQ ID NO: 12; a VL CDR2 comprising SEQ ID NO: 13; and a VL CDR3 comprising SEQ ID NO: 14.

[0073] 2. The Gal-1 antibody of embodiment 1, wherein the VH comprises SEQ ID NO: 3 or a sequence having at least 90% identity thereto; and wherein the VL comprises SEQ ID NO: 1 or a sequence having at least 90% identity thereto.

[0074] 3. The Gal-1 antibody of embodiment 1 or 2, wherein the Gal-1 antibody is linked to a detectable label.

[0075] 4. A method for detecting at least one of intracellular Gal-1 and cell surface Gal-1 in a sample, the method comprising: contacting the sample with a Gal-1 antibody; and detecting binding of the antibody to Gal-1 in the sample; wherein the Gal-1 antibody comprises: a VH CDR1 comprising SEQ ID NO: 15; a VH CDR2 comprising SEQ ID NO: 16; a VH CDR3 comprising SEQ ID NO: 17; a light chain variable domain (VL) CDR1 comprising SEQ ID NO: 12; a VL CDR2 comprising SEQ ID NO: 13; and a VL CDR3 comprising SEQ ID NO: 14.

[0076] 5. The method of embodiment 4, wherein the VH comprises SEQ ID NO: 3 or a sequence having at least 90% identity thereto; and wherein the VL comprises SEQ ID NO: 1 or a sequence having at least 90% identity thereto.

[0077] 6. The method of embodiment 4 or 5, wherein detecting the binding of the antibody in the sample is carried out using ELISA, Western blot, a lateral flow assay, immunostaining, immunoprecipitation, flow cytometry, sensor chips, or magnetic beads.

[0078] 7. The method of any one of embodiments 4-6, wherein the Gal-1 antibody is linked to a detectable label.

[0079] 8. The method of any one of embodiments 4-6, wherein the Gal-1 antibody is biotinylated.

[0080] 9. The method of any one of embodiments 4-8, wherein the sample comprises one or more of a blood sample, a serum sample, and a tissue sample.

[0081] 10. The method of any one of embodiments 4-9, wherein the sample comprises immune cells.

[0082] 11. The method of any one of embodiments 4-9, wherein the sample comprises cancer cells.

[0083] 12. The method of any one of embodiments 4-11, wherein the method comprises detecting only cell surface Gal-1.

[0084] 13. The method of any one of embodiments 4-1 1, wherein the method comprises detecting only intracellular Gal-1.

[0085] 14. The method of any one of embodiments 4-11, wherein the method comprises detecting both cell surface Gal-1 and intracellular Gal-1.

[0086] 15. The method of any one of embodiments 4-14, wherein the Gal-1 antibody is linked to a solid support.

[0087] 16. A method for detecting [3-galactoside on the surface of a cell, the method comprising: contacting the cell with a Gal-1 protein; washing the cell to remove unbound Gal-1 protein; contacting the cell with a Gal-1 antibody; and detecting binding of the antibody to the Gal-1 protein; wherein the Gal-1 antibody comprises: a VH CDR1 comprising SEQ ID NO: 15; a VH CDR2 comprising SEQ ID NO: 16; a VH CDR3 comprising SEQ ID NO: 17; a light chain variable domain (VL) CDR1 comprising SEQ ID NO: 12; a VL CDR2 comprising SEQ ID NO: 13; and a VL CDR3 comprising SEQ ID NO: 14; wherein binding of the antibody indicates detection of -galactoside.

[0088] 17. The method of embodiment 16, wherein the VH comprises SEQ ID NO: 3 or a sequence having at least 90% identity thereto; and wherein the VL comprises SEQ ID NO: 1 or a sequence having at least 90% identity thereto.

[0089] 18. The method of embodiment 16 or 17, wherein the Gal-1 protein is a recombinant Gal-1 (rGal-1).

[0090] 19. The method of embodiment 18, wherein the rGal-1 comprises a sequence having at least 95% identity to SEQ ID NO: 5.

[0091] 20. The method of embodiment 18 or 19, wherein the rGal-1 comprises a detectable label.

[0092] 21. A kit comprising: the antibody of any one of embodiments 1-3; and a detection reagent.

[0093] 22. The kit of embodiment 21, further comprising: i.a solid support.

[0094] 23. The kit of embodiment 22, wherein the antibody is linked to the solid support.

[0095] 24. The kit of any one of embodiments 21-23, wherein the detection reagent comprises a secondary antibody linked to a detectable label.

[0096] 25. An anti -gal ectin- 1 (Gal-1) antibody, comprising: a heavy chain variable region (VH) comprising SEQ ID NO: 3 or a sequence having at least 90% identity thereto; and a light chain variable domain (VL) comprising SEQ ID NO: 1 or a sequence having at least 90% identity thereto.

[0097] 26. The Gal-l antibody of embodiment 25, wherein the Gal-l antibody is linked to a detectable label.

[0098] 27. A method for detecting at least one of intracellular Gal-1 and cell surface Gal-1 in a sample, the method comprising: contacting the sample with a Gal-1 antibody; and detecting binding of the antibody to Gal-1 in the sample; wherein the Gal-1 antibody comprises: a heavy chain variable region (VH) comprising SEQ ID NO: 3 or a sequence having at least 90% identity thereto; and a light chain variable region (VL) comprising SEQ ID NO: 1 or a sequence having at least 90% identity thereto.

[0099] 28. The method of embodiment 27, wherein detecting the binding of the antibody in the sample is carried out using ELISA, Western blot, a lateral flow assay, immunostaining, immunoprecipitation, flow cytometry, sensor chips, or magnetic beads.

[0100] 29. The method of embodiment 27 or 28, wherein the Gal-1 antibody is linked to a detectable label.

[0101] 30. The method of embodiment 27 or 28, wherein the GaLl antibody is biotinylated.

[0102] 31. The method of any one of embodiments 27-30, wherein the sample comprises one or more of a blood sample, a serum sample, and a tissue sample.

[0103] 32. The method of any one of embodiments 27-31, wherein the sample comprises immune cells.

[0104] 33. The method of any one of embodiments 27-31, wherein the sample comprises cancer cells.

[0105] 34. The method of any one of embodiments 27-33, wherein the method comprises detecting only cell surface Gal-l.

[0106] 35. The method of any one of embodiments 27-33, wherein the method comprises detecting only intracellular GaLl.

[0107] 36. The method of any one of embodiments 27-33, wherein the method comprises detecting both cell surface Gal-1 and intracellular Gal-l.

[0108] 37. The method of any one of embodiments 27-36, wherein the Gal-1 antibody is linked to a solid support.

[0109] 38. A method for detecting [3-galactoside on the surface of a cell, the method comprising: contacting the cell with a Gal-1 protein; washing the cell to remove unbound Gal-1 protein; contacting the cell with a Gal-1 antibody; and detecting binding of the antibody to the Gal-1 protein; wherein the Gal-1 antibody comprises: a heavy chain variable region (VH) comprising SEQ ID NO: 3 or a sequence having at least 90% identity thereto; and a light chain variable domain (VL) comprising SEQ ID NO: 1 or a sequence having at least 90% identity thereto; wherein binding of the antibody indicates detection of P-galactoside.

[0110] 39. The method of embodiment 38, wherein the Gal-1 protein is a recombinant Gal-1 (rGal-1).

[0111] 40. The method of embodiment 39, wherein the rGal-1 comprises a sequence having at least 95% identity to SEQ ID NO: 5.

[0112] 41. The method of embodiment 39 or 40, wherein the rGal-1 comprises a detectable label.

[0113] 42. A kit comprising: the antibody of embodiment 25 or 26; and a detection reagent.

[0114] 43. The kit of embodiment 42, further comprising: a solid support.

[0115] 44. The kit of embodiment 43, wherein the antibody is linked to the solid support.

[0116] 45. The kit of any one of embodiments 42-44, wherein the detection reagent comprises a secondary antibody linked to a detectable label.

[0117] Table 1. Sequences

Claims

CLAIMSWhat is claimed is:

1. An anti-galectin-1 (Gal-1) antibody, comprising: a heavy chain variable region (VH) complementary determining region (CDR) CDR1 comprising SEQ ID NO: 15; a VH CDR2 comprising SEQ ID NO: 16; a VH CDR3 comprising SEQ ID NO: 17; a light chain variable domain (VL) CDR1 comprising SEQ ID NO: 12; a VL CDR2 comprising SEQ ID NO: 13; and a VL CDR3 comprising SEQ ID NO: 14.

2. The Gal-1 antibody of claim 1, wherein the VH comprises SEQ ID NO: 3 or a sequence having at least 90% identity thereto; and wherein the VL comprises SEQ ID NO: 1 or a sequence having at least 90% identity thereto.

3. The Gal-1 antibody of claim 1 or 2, wherein the Gal-1 antibody is linked to a detectable label.

4. A method for detecting at least one of intracellular Gal-1 and cell surface Gal-1 in a sample, the method comprising: contacting the sample with a Gal-1 antibody; and detecting binding of the antibody to Gal-1 in the sample; wherein the Gal-1 antibody comprises: a VH CDR1 comprising SEQ ID NO: 15; a VH CDR2 comprising SEQ ID NO: 16; a VH CDR3 comprising SEQ ID NO: 17; a light chain variable domain (VL) CDR1 comprising SEQ ID NO: 12; a VL CDR2 comprising SEQ ID NO: 13; and a VL CDR3 comprising SEQ ID NO: 14.

5. The method of claim 4, wherein the VH comprises SEQ ID NO: 3 or a sequence having at least 90% identity thereto; and wherein the VL comprises SEQ ID NO: 1 or a sequence having at least 90% identity thereto.

6. The method of claim 4 or 5, wherein detecting the binding of the antibody in the sample is carried out using ELISA, Western blot, a lateral flow assay, immunostaining, immunoprecipitation, flow cytometry, sensor chips, or magnetic beads.

7. The method of claim 6, wherein the Gal-1 antibody is linked to a detectable label.

8. The method of claim 6, wherein the Gal-1 antibody is biotinylated.

9. The method of claim 6, wherein the sample comprises one or more of a blood sample, a serum sample, and a tissue sample.

10. The method of claim 6, wherein the sample comprises immune cells.

11. The method of claim 6, wherein the sample comprises cancer cells.

12. The method of claim 6, wherein the method comprises detecting only cell surface Gal-1.

13. The method of claim 6, wherein the method comprises detecting only intracellular Gal-1.

14. The method of claim 6, wherein the method comprises detecting both cell surface Gal-1 and intracellular Gal-1.

15. The method of claim 6, wherein the Gal-1 antibody is linked to a solid support.

16. A method for detecting 0-galactoside on the surface of a cell, the method comprising: contacting the cell with a Gal-1 protein; washing the cell to remove unbound Gal-1 protein; contacting the cell with a Gal-1 antibody; and detecting binding of the antibody to the Gal-1 protein; wherein the Gal-1 antibody comprises: a VH CDR1 comprising SEQ ID NO: 15; a VH CDR2 comprising SEQ ID NO: 16; a VH CDR3 comprising SEQ ID NO: 17; a light chain variable domain (VL) CDR1 comprising SEQ ID NO: 12; a VL CDR2 comprising SEQ ID NO: 13; and a VL CDR3 comprising SEQ ID NO: 14; wherein binding of the antibody indicates detection of P -galactoside.

17. The method of claim 16, wherein the VH comprises SEQ ID NO: 3 or a sequence having at least 90% identity thereto; and wherein the VL comprises SEQ ID NO: 1 or a sequence having at least 90% identity thereto.

18. The method of claim 16 or 17, wherein the Gal-1 protein is a recombinant Gal-1 (rGal-1).

19. The method of claim 18, wherein the rGal-1 comprises a sequence having at least 95% identity to SEQ ID NO: 5.

20. The method of claim 18, wherein the rGal-1 comprises a detectable label.

21. A kit compri si n : the antibody of claim 1; and a detection reagent.

22. The kit of claim 21, further comprising: a solid support.

23. The kit of claim 22, wherein the antibody is linked to the solid support.

24. The kit of claim 21, wherein the detection reagent comprises a secondary antibody linked to a detectable label.

25. An anti-galectin-1 (Gal-1) antibody, comprising: a heavy chain variable region (VH) comprising SEQ ID NO: 3 or a sequence having at least 90% identity thereto; and a light chain variable domain (VL) comprising SEQ ID NO: 1 or a sequence having at least 90% identity thereto.

26. The Gal-1 antibody of claim 25, wherein the Gal-1 antibody is linked to a detectable label.

27. A method for detecting at least one of intracellular Gal-1 and cell surface Gal-1 in a sample, the method comprising: contacting the sample with a Gal-1 antibody; and detecting binding of the antibody to Gal-1 in the sample; wherein the Gal-1 antibody comprises: a heavy chain variable region (VH) comprising SEQ ID NO: 3 or a sequence having at least 90% identity thereto; and a light chain variable region (VL) comprising SEQ ID NO: 1 or a sequence having at least 90% identity thereto.

28. The method of claim 27, wherein detecting the binding of the antibody in the sample is carried out using ELISA, Western blot, a lateral flow assay, immunostaining, immunoprecipitation, flow cytometry, sensor chips, or magnetic beads.

29. The method of claim 27 or 28, wherein the Gal-1 antibody is linked to a detectable label.

30. The method of claim 27 or 28, wherein the Gal-1 antibody is biotinylated.

31. The method of claim 29, wherein the sample comprises one or more of a blood sample, a serum sample, and a tissue sample.

32. The method of claim 29, wherein the sample comprises immune cells.

33. The method of claim 29, wherein the sample comprises cancer cells.

34. The method of claim 29, wherein the method comprises detecting only cell surface Gal-1.

35. The method of claim 29, wherein the method comprises detecting only intracellular Gal-1.

36. The method of claim 29, wherein the method comprises detecting both cell surface Gal-1 and intracellular Gal-1.

37. The method of claim 29, wherein the Gal-1 antibody is linked to a solid support.

38. A method for detecting P-galactoside on the surface of a cell, the method comprising: contacting the cell with a Gal-1 protein; washing the cell to remove unbound Gal-1 protein; contacting the cell with a Gal-1 antibody; and detecting binding of the antibody to the Gal-1 protein; wherein the Gal-1 antibody comprises: a heavy chain variable region (VH) comprising SEQ ID NO: 3 or a sequence having at least 90% identity thereto; and a light chain variable domain (VL) comprising SEQ ID NO: 1 or a sequence having at least 90% identity thereto; wherein binding of the antibody indicates detection of P-galactoside.

39. The method of claim 38, wherein the Gal-1 protein is a recombinant Gal-1 (rGal-1).

40. The method of claim 39, wherein the rGal-1 comprises a sequence having at least 95% identity to SEQ ID NO: 5.

41. The method of claim 39 or 40, wherein the rGal-1 comprises a detectable label.

42. A kit comprising: the antibody of claim 25 or 26; and a detection reagent.

43. The kit of claim 42, further comprising: a solid support.

44. The kit of claim 43, wherein the antibody is linked to the solid support.

45. The kit of claim 42, wherein the detection reagent comprises a secondary antibody linked to a detectable label.

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