Anti-human LAG-3 antibodies and their use in immunohistochemistry (IHC)

Abstract

Provided are chimeric or recombinant antibodies (Abs), or antigen-binding fragments thereof, or monomeric or dimeric antigen-binding proteins capable of specifically binding to human LAG-3 polypeptides, including human LAG-3 polypeptides expressed on the surface of lymphocytes, such as activated T cells or tumor-infiltrating lymphocytes (TILs), that have infiltrated tumors, and methods for producing and using the same. In an alternative embodiment, the chimeric or recombinant antibodies (Abs), or antigen-binding fragments thereof, or monomeric or dimeric antigen-binding proteins as provided herein are used for in vitro diagnosis, e.g., in immunohistochemistry (IHC), to diagnose and / or treat cancer, e.g., bladder cancer, urothelial carcinoma, breast cancer, lung cancer, non-small cell lung cancer, kidney cancer, renal clear cell carcinoma, and / or melanoma or malignant melanoma, due to their ability to specifically bind to activated T cells or tumor-infiltrating lymphocytes that have infiltrated tumors.

Description

[Technical Field]

[0001] Related Applications This Patent Convention Treaty (PCT) international application claims the benefit of priority under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application (USSN) 63 / 030,873, filed May 27, 2020. The above application is incorporated herein by reference in its entirety and for all purposes.

[0002] The present invention generally relates to immunohistochemistry (IHC) and cancer diagnosis and treatment. In an alternative embodiment, a chimeric or recombinant antibody (Ab), or an antigen-binding fragment thereof, or a monomeric or dimeric antigen-binding protein capable of specifically binding to a human LAG-3 polypeptide, including a human LAG-3 polypeptide expressed on the surface of lymphocytes, such as activated T cells, infiltrating tumors, or a human LAG-3 polypeptide expressed on tumor-infiltrating lymphocytes (TILs), is provided. In an alternative embodiment, articles of manufacture and kits comprising the chimeric or recombinant Ab, or an antigen-binding fragment thereof, or a monomeric or dimeric antigen-binding protein, as provided herein, or a nucleic acid encoding the same, or a cell expressing the same, and methods for producing and using the same are provided. In an alternative embodiment, the chimeric or recombinant antibody (Ab), or an antigen-binding fragment thereof, or a monomeric or dimeric antigen-binding protein, as provided herein, is used for in vitro diagnosis, e.g., by immunohistochemistry (IHC). In an alternative embodiment, a chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein as provided herein, is used in an IHC protocol for diagnosing and / or treating cancer, e.g., bladder cancer, urothelial carcinoma, breast cancer, lung cancer, renal cell carcinoma, renal clear cell carcinoma (RCC), and / or melanoma or malignant melanoma, due to its ability to specifically bind to tumor-infiltrating activated T cells or tumor-infiltrating lymphocytes (TILs). [Background technology]

[0003] Lymphocyte activation gene 3, or LAG-3 (or LAG3) protein, is encoded by the LAG3 gene and is also known as CD223. LAG-3 is a T cell activation marker expressed on various lymphoid cell types, and is expressed on both CD4 and CD8 T cells 3–4 days after activation [1]. In addition, LAG-3 is expressed on activated natural killer (NK) cells and plasmacytoid dendritic cells [2].

[0004] LAG-3-expressing lymphoid cells, such as tumor-infiltrating lymphoid cells, have been found in a variety of human tumors, including melanoma, NSCLC, colorectal cancer, breast cancer, hepatocellular carcinoma, follicular lymphoma, head and neck squamous cell carcinoma, and renal cancer, and are significantly associated with invasive tumor progression and clinicopathological characteristics [3–17]. Summary of the Invention

[0005] In an alternative embodiment, a chimeric or recombinant antibody (Ab), or antigen (Ag)-binding fragment thereof, or a monomeric or dimeric antigen-binding protein is provided that is capable of specifically binding to a human lymphocyte activation gene 3 (LAG-3) polypeptide, including a human LAG-3 polypeptide expressed on the surface of a lymphocyte, such as an activated T cell, that has infiltrated a tumor, or a human LAG-3 polypeptide expressed on a tumor-infiltrating lymphocyte (TIL), wherein the chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein has the amino acid sequence It specifically binds to a peptide or polypeptide, or epitope, comprising or consisting of the sequence GPPAAAPGHPLAPGPHPAAPSSWGPRPRR (SEQ ID NO: 1).

[0006] In an alternative embodiment, the chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein is antigen-binding fragments (Fab, or Ab fragments having only one constant domain and one variable domain of each of the Ab heavy and light chains); F(ab')2 (or Ab digested with pepsin to generate two fragments, F(ab')2 fragment and pFc' fragment), Fab' (single chain F(ab')2 fragment), single-chain variable fragments (scFv) (or fusion proteins of the variable regions of the Ab heavy and light chains, optionally linked together with a linker peptide of about 10 to about 25 amino acids in length); (scFv)2, or a single peptide chain with two variable heavy chain regions and two variable light chain regions resulting in a di-scFv or bi-scFv, or a tandem scFv; minibodies (or fusion proteins of Ab heavy and light chain variable regions optionally linked together with alkyl groups containing methyl or ethyl groups); diabodies (or scFvs that contain a linker peptide (optionally about 5 amino acids) that is too short for the two variable regions to fold together, causing the scFv to dimerize), triabodies or tetrabodies (or scFvs that contain a linker peptide (optionally about 1 or 2 amino acids) that is too short for the two variable regions to fold together, causing the scFv to trimerize or tetramerize); single domain antibodies (dABs) (or single variable regions of Ab heavy or Ab light chains); multiple complementarity determining region (CDR) fragments, or Multispecific antibodies formed from two or more antibody fragments It is made as or in the form of:

[0007] In alternative embodiments of the chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein as provided herein, - the sequence of the heavy chain variable region is is or comprises QSVKESEGGLFKPTDTLTLTCTVSGIDLSSGILVWVRQAPGSGLEWIGGIDANGRAYYASWAKSRSTITRNTNENTVTLKMTSLTAADTATYFCAGGAWNIWGPGTLVTVSS (SEQ ID NO: 2); - the sequence of the light chain variable region is is or comprises AQVLTQTPSPVSAAVGGTVTIKCQSSQSVYDSNTLAWFQQKPGQPPKLLMYSASTLAFGVPSRFSGSGSGTQFTLTISDLECADAATYYCLGSYDCSSVDCTAFGGGTEVVVK (SEQ ID NO: 3); - the sequence of the heavy chain variable region is QSVKESEGGLFKPTDTLTLTCTVSGIDLSSGILVWVRQAPGSGLEWIGGIDANGRAYYASWAKSRSTITRNTNENTVTLKMTSLTAADTATYFCAGGAWNIWGPGTLVTVSS (SEQ ID NO: 2); and The sequence of the light chain variable region is is or comprises AQVLTQTPSPVSAAVGGTVTIKCQSSQSVYDSNTLAWFQQKPGQPPKLLMYSASTLAFGVPSRFSGSGSGTQFTLTISDLECADAATYYCLGSYDCSSVDCTAFGGGTEVVVK (SEQ ID NO: 3); - the sequence of the heavy chain variable region comprises SEQ ID NO:2 with at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 conservative amino acid substitutions, wherein the heavy chain variable region is capable of specifically binding to a human LAG-3 polypeptide, amino acid (SEQ ID NO:1), or epitope when either unpaired (alone) or paired with a light chain variable region; - the sequence of the light chain variable region comprises SEQ ID NO:3 with at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 conservative amino acid substitutions, wherein the light chain variable region is capable of specifically binding to a human LAG-3 polypeptide, amino acid (SEQ ID NO:1), or epitope when either unpaired (alone) or paired with a heavy chain variable region; - the sequence of the heavy chain variable region has at least about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 2; - the sequence of the light chain variable region has at least about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 3; - the sequence of the heavy chain variable region and the amino acid sequence of SEQ ID NO: 2 have a Z-score of at least 8, and a Z-score of about 2 to about 8, when aligned using distance matrix alignment; - the sequence of the light chain variable region and the amino acid sequence of SEQ ID NO: 3 have a Z-score of at least 8, and a Z-score of about 2 to about 8, when aligned using distance matrix alignment; - the heavy chain variable region comprises the three CDR1, CDR2 and CDR3 complementarity determining regions (CDRs) of SEQ ID NO: 2, or CDR1 amino acid (aa) residues 25-32, CDR2 aa residues 50-56, and CDR3 aa residues 95-101 of SEQ ID NO: 2; - the light chain variable region comprises the three CDR1, CDR2 and CDR3 complementarity determining regions (CDRs) of SEQ ID NO: 3, or CDR1 amino acid (aa) residues 27-34, CDR2 aa residues 52-54, and CDR3 aa residues 91-102 of SEQ ID NO: 3; - a chimeric or recombinant Ab or antigen-binding fragment thereof, or a monomeric or dimeric antigen-binding protein, comprising: (a) a heavy chain variable region comprising three CDR1, CDR2, and CDR3 complementarity determining regions (CDRs) of SEQ ID NO: 2, or CDR1 amino acid (aa) residues 25-32, CDR2 aa residues 50-56, and CDR3 aa residues 95-101 of SEQ ID NO: 2; and (b) a light chain variable region comprising three CDR1, CDR2, and CDR3 complementarity determining regions (CDRs) of SEQ ID NO: 3, or CDR1 amino acid (aa) residues 27-34, CDR2 aa residues 52-54, and CDR3 aa residues 91-102 of SEQ ID NO: 3; - the antibody heavy chain is an IgM, IgG, IgA or IgE isotype heavy chain and / or the light chain is a kappa or lambda light chain; - the sequence of the light chain constant region is GDPGAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC (SEQ ID NO: 4), or is or comprises GDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC (SEQ ID NO: 5); - the sequence of the light chain constant region comprises SEQ ID NO:4 or SEQ ID NO:5 with at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 or more conservative amino acid substitutions, wherein the light chain constant region with the conservative amino acid substitutions is capable of specifically binding to or associating with a heavy chain constant region; - the sequence of the light chain constant region has at least about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO:4 or SEQ ID NO:5, wherein the light chain constant region is capable of specifically binding to or associating with the heavy chain constant region; - the sequence of the heavy chain constant region is is or comprises GQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK (SEQ ID NO: 6); - the sequence of the heavy chain constant region comprises SEQ ID NO: 6 with at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 or more conservative amino acid substitutions, wherein the heavy chain constant region with the conservative amino acid substitutions is capable of specifically binding to or associating with a light chain constant region; the sequence of the heavy chain constant region has at least about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO:6, wherein the heavy chain constant region is capable of specifically binding to or associating with the light chain constant region; - the sequence of the antibody light chain is AQVLTQTPSPVSAAVGGTVTIKCQSSQSVYDSNTLAWFQQKPGQPPKLLMYSASTLAFGVPSRFSGSGSGTQFTLTISDLECADAATYYCLGSYDCSSVDCTAFGGGGTEVVVK GDPGAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC (SEQ ID NO: 7), or AQVLTQTPSPVSAAVGGTVTIKCQSSQSVYDSNTLAWFQQKPGQPPKLLMYSASTLAFGVPSRFSGSGSGTQFTLTISDLECADAATYYCLGSYDCSSVDCTAFGGGGTEVVVKcomprising GDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC (SEQ ID NO: 8) (variable regions are underlined); - the sequence of the antibody heavy chain is QSVKESEGGLFKPTDTLTLTCTVSGIDLSSGILVWVRQAPGSGLEWIGGIDANGRAYYASWAKSRSTITRNTNENTVTLKMTSLTAADTATYFCAGGAWNIWGPGTLVTVSS GQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK (SEQ ID NO: 9), (variable regions are underlined); - the chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or the monomeric or dimeric antigen-binding protein, further comprises, or is bound to, paired with, associated with, or covalently conjugated to, a detectable agent or binding moiety; - detectable agents include enzymes, biotin, fluorescent or chemiluminescent labels, fluorophores, cyanines such as sulfoindo-cyanines, Nile red, rhodamine, perylene, fluorenyl, coumarin, 7-methoxycoumarin (Mca), dabcyl, [2-(4-nitro-2,1,3-benzoxadiazol-7-yl)aminoethyl]trimethylammonium (NBD), Nile blue, Tamra or tetramethylrhodamine (TMR), boron dipyrromethane (BODIPY), HRP MAGENTA™ chromogen (Dako Omnis, Agilent) or derivatives thereof, dyes, radioisotopes, quantum dots or photoluminescent aqueous nanocrystals, haptens or antibody binding epitopes or domains; - the enzyme is peroxidase, alkaline phosphatase, or β-galactosidase, and the peroxidase can be horseradish peroxidase (HRP); - the hapten comprises biotin, theophylline, digoxigenin, carborane, fluorescein or bromodeoxyuridine; - the dye is a cyanine dye; or comprises Cy3 or Cy5; - the fluorophore comprises dansyl, fluorescein or carboxyfluorescein (FAM) or 6-FAM; and / or - Binding moieties include glutathione-S-transferase (GST) or ligandin tags, polyhistidine (poly-his) tags, chitin-binding protein (CBP), STREP-TAG™ or Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 11) peptide tags, FLAG tags or DYKDDDDK (SEQ ID NO: 12) peptide tags, or maltose binding protein.

[0008] In an alternative embodiment, a recombinant nucleic acid encoding a chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or a monomeric or dimeric antigen-binding protein, as provided herein, is provided.

[0009] In an alternative embodiment of the recombinant nucleic acid as provided herein, - the recombinant nucleic acid further comprises and is operably linked to a transcriptional regulatory element, and the transcriptional regulatory element can comprise a promoter, or the promoter is an inducible promoter or a constitutive promoter; - the recombinant nucleic acid further comprises a sequence encoding an additional protein or peptide moiety or domain, and the additional protein or peptide moiety or domain can include a purification moiety or domain to aid in the purification or isolation of the chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein encoded by the recombinant nucleic acid; - the additional protein or peptide moiety or domain comprises a glutathione-S-transferase (GST) or ligandin tag, a polyhistidine (poly-his) tag, a chitin-binding protein (CBP), a STREP-TAG™ or Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 11) peptide tag, a FLAG tag or a DYKDDDDK (SEQ ID NO: 12) peptide tag, or a maltose-binding protein; and / or - the recombinant nucleic acid further comprises a sequence encoding a protease cleavage site located between the purification moiety or domain and the sequence encoding the chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein.

[0010] In an alternative embodiment, a chimeric or recombinant antibody (Ab) as provided herein comprises: (a) a light chain as set forth in SEQ ID NO:7 operably linked to, paired with, associated with, or configured together with a heavy chain as set forth in SEQ ID NO:9, wherein the chimeric or recombinant Ab is capable of selectively binding to a human LAG-3 polypeptide; or (b) a light chain as set forth in SEQ ID NO:8 operably linked to, paired with, associated with, or configured together with a heavy chain as set forth in SEQ ID NO:9, wherein the chimeric or recombinant Ab is capable of selectively binding to a human LAG-3 polypeptide. Includes.

[0011] In alternative embodiments, there is provided an expression cassette, vector, recombinant virus, artificial chromosome, cosmid, or plasmid comprising a recombinant nucleic acid as provided herein.

[0012] In alternative embodiments, a cell is provided that comprises a chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or a monomeric or dimeric antigen-binding protein as provided herein, a recombinant nucleic acid as provided herein, or an expression cassette, vector, recombinant virus, artificial chromosome, cosmid, or plasmid as provided herein, and the cell can be a bacterial, fungal, mammalian, yeast, insect, avian, or plant cell.

[0013] In an alternative embodiment, a method is provided for generating polyclonal antibodies specific for or specifically binding to human lymphocyte activation gene 3 (LAG-3) polypeptide, or for generating polyclonal immune serum, optionally specifically binding to LAG-3 polypeptide expressed on the surface of tumor-infiltrating lymphocytes, such as tumor-infiltrating activated T cells, the method comprising: The method comprises administering to a mammal or avian species a peptide or polypeptide, or epitope, comprising GPPAAAPGHPLAPGPHPAAPSSWGPRPRR (SEQ ID NO: 1), or immunizing the mammal or avian species with the peptide or polypeptide, or epitope.

[0014] In an alternative embodiment, a method is provided for detecting the presence of human LAG-3 protein in or on the surface of a cell (optionally a lymphocyte, or a tumor-infiltrating lymphocyte such as a tumor-infiltrating activated T cell), tissue, organ, or a portion of any of the above, the method comprising: (a) contacting the cell, tissue, or organ, or a portion of any of the above, with a chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or a monomeric or dimeric antigen-binding protein, as provided herein or encoded by a recombinant nucleic acid as provided herein; and (b) detecting in the cell, tissue, or organ, or a portion of any of the above, a human LAG-3 polypeptide or The method includes detecting specific binding of a chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or a monomeric or dimeric antigen-binding protein, to a GPPAAAPGHPLAPGPHPAAPSSWGPRPRR (SEQ ID NO: 1)-containing polypeptide, thereby detecting the presence of human LAG-3 protein in a cell, tissue, or organ, or a portion of any of the above, which comprises contacting the cell, tissue, or organ, or a portion of any of the above.

[0015] In an alternative embodiment of the method for detecting the presence of human LAG-3 protein in a cell (optionally a lymphocyte, or a tumor-infiltrating lymphocyte, such as a tumor-infiltrating activated T cell), tissue, organ or portion thereof as provided herein, the method comprises: - the contacting step involves the use of an immunohistochemistry (IHC) assay; - the method further comprises contacting the chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein, with a detectable agent to indicate or signal specific binding of the chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein to human LAG-3 protein; - the detectable agent specifically binds to a chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein; or the detectable agent is or comprises an antibody or antigen-binding fragment or secondary antibody that specifically binds to a chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein that is bound to, paired with, or associated with human LAG-3 protein; or the detectable agent is or comprises an antibody or antigen-binding fragment or secondary antibody that specifically binds to a hapten or tag that is linked or conjugated to a chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein; and / or - the antibody or antigen-binding fragment thereof or secondary antibody further comprises or is linked or conjugated to a second detectable agent or enzyme, and the enzyme can be alkaline phosphatase, β-galactosidase or peroxidase; or the antibody or antigen-binding fragment thereof or secondary antibody further comprises or is linked or conjugated to a second detectable agent or enzyme, and the enzyme can be alkaline phosphatase, β-galactosidase or peroxidase; or the antibody or antigen-binding fragment thereof or secondary antibody further comprises or is linked or conjugated to a second detectable agent or enzyme, and the enzyme can be alkaline phosphatase, β-galactosidase or peroxidase; or the antibody or antigen-binding fragment thereof or secondary antibody further comprises or is linked or conjugated to a second detectable agent or enzyme, and the second detectable agent or ... Omnis, Agilent), Tamra or tetramethylrhodamine (TMR), boron dipyrromethane (BODIPY), or derivatives thereof), dyes, radioisotopes, quantum dots, or photoluminescent aqueous nanocrystals, haptens, further comprising or linked or conjugated thereto; and the dyes can include cyanine dyes, or Cy3 or Cy5; or the haptens include biotin, theophylline, digoxigenin, carborane, fluorescein, or bromodeoxyuridine.

[0016] In an alternative embodiment, a method is provided for detecting or diagnosing LAG-3 protein-expressing cancer or cancer tissue containing LAG-3-expressing lymphocytes or LAG-3-expressing tumor-infiltrating lymphocytes, such as tumor-infiltrating activated T cells, comprising the steps of detecting the expression or presence of human LAG-3 protein in a cell, tissue or organ sample, or a portion thereof, by contacting the cell, tissue or organ sample with a chimeric or recombinant antibody as provided herein or encoded by a recombinant nucleic acid as provided herein, and detecting whether the chimeric or recombinant antibody specifically binds to human LAG-3 protein in the cell, tissue or organ sample, or a portion thereof, wherein detecting specific binding indicates the expression or presence of human LAG-3 protein in the cell, tissue or organ sample, or a portion thereof.

[0017] In an alternative embodiment of the method for detecting or diagnosing a LAG-3 protein-expressing cancer or cancerous tissue comprising LAG-3-expressing lymphocytes, or LAG-3-expressing tumor-infiltrating lymphocytes, such as tumor-infiltrating activated T cells, as provided herein, the method comprises: - the cell is an activated T cell, a tumor-infiltrating activated T cell, or a tumor-infiltrating lymphocyte (TIL); - detecting specific binding indicates expression or presence of human LAG-3 protein in the cell, tissue or organ sample, or a portion thereof, thereby diagnosing or detecting cancer; - the cancer is selected from the group consisting of renal cell carcinoma, renal clear cell carcinoma (RCC), adenocarcinoma, bladder cancer, urothelial carcinoma, breast cancer or mammary carcinoma or ductal carcinoma in situ (DCIS), carcinoid, Hodgkin's lymphoma, chronic lymphocytic leukemia, colorectal cancer, ovarian cancer, kidney cancer or renal cell carcinoma, liver cancer or hepatocellular carcinoma, stomach or gastric cancer, lymphoma or follicular lymphoma, prostate cancer, head and neck squamous cell carcinoma, lung cancer, non-small cell lung cancer (NSCLC), mesothelioma or malignant pleural mesothelioma, anal squamous cell carcinoma, pancreatic cancer, and melanoma or malignant melanoma; and the adenocarcinoma can be lung adenocarcinoma or colon adenocarcinoma; - detecting includes using or performing an immunohistochemistry (IHC) assay or flow cytometry; - performing or using flow cytometry includes the use of a fluorescence-activated cell sorter (FACS) or an impedance flow cytometer; and / or the cell, tissue or organ sample or part thereof is or is derived from a biopsy from a patient.

[0018] In an alternative embodiment, a method is provided for treating, ameliorating, or preventing cancer, comprising first detecting or diagnosing cancer in an individual in need thereof using a method as provided herein, and subsequently treating the individual in need thereof.

[0019] In alternative embodiments of the methods for treating, ameliorating, or preventing cancer, the cancer is selected from the group consisting of bladder cancer, urothelial carcinoma, breast cancer or mammary carcinoma or ductal carcinoma in situ (DCIS), lung cancer, non-small cell lung cancer (NSCLC), renal cell carcinoma, clear cell renal carcinoma (RCC), adenocarcinoma, breast cancer or mammary carcinoma or ductal carcinoma in situ (DCIS), carcinoid, Hodgkin's lymphoma, chronic lymphocytic leukemia, colorectal cancer, ovarian cancer, kidney cancer, liver cancer or hepatocellular carcinoma, stomach or gastric cancer, lymphoma or follicular lymphoma, prostate cancer, head and neck squamous cell carcinoma, mesothelioma or malignant pleural mesothelioma, anal squamous cell carcinoma, pancreatic cancer, and melanoma or malignant melanoma.

[0020] In an alternative embodiment, there is provided the use of a chimeric or recombinant antibody (Ab), or an antigen-binding fragment thereof, or a monomeric or dimeric antigen-binding protein, as provided herein, or encoded by a recombinant nucleic acid as provided herein, to detect or diagnose cancer or cancerous tissue containing LAG-3-expressing lymphocytes, or LAG-3-expressing tumor-infiltrating lymphocytes, such as tumor-infiltrating activated T cells, or to treat, ameliorate or prevent cancer.

[0021] In alternative embodiments, provided are chimeric or recombinant antibodies (Abs), or antigen-binding fragments thereof, or monomeric or dimeric antigen-binding proteins, as provided herein or encoded by recombinant nucleic acids as provided herein, for use in the detection or diagnosis of cancer or cancerous tissues containing LAG-3-expressing lymphocytes, or LAG-3-expressing tumor-infiltrating lymphocytes, such as tumor-infiltrating activated T cells, or in the treatment, amelioration, or prevention of cancer.

[0022] In alternative embodiments, kits are provided that include a chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or a monomeric or dimeric antigen-binding protein as provided herein or encoded by a recombinant nucleic acid as provided herein. In alternative embodiments, the kits as provided herein include components required for an immunohistochemistry (IHC) assay; and / or instructions for performing a method as provided herein. In alternative embodiments of the kits as provided herein, the chimeric or recombinant antibody (Ab), antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein is substantially purified or isolated.

[0023] In alternative embodiments, articles of manufacture are provided that include a chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or a monomeric or dimeric antigen-binding protein, as provided herein or encoded by a recombinant nucleic acid as provided herein. In alternative embodiments, the article of manufacture comprises, or is made or manufactured as, a slide, well, chip, biochip, array, tray, dish, or microtiter plate or dish. In alternative article of manufacture embodiments, the chimeric or recombinant antibody (Ab), antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein is substantially purified or isolated, or is in the form of an unpurified or partially purified culture supernatant.

[0024] In an alternative embodiment, a phage or phagemid is provided that comprises or expresses on its surface a chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or a monomeric or dimeric antigen-binding protein as provided herein or encoded by a recombinant nucleic acid as provided herein.

[0025] The details of one or more exemplary embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

[0026] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

[0027] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0028] The drawings presented herein are illustrations of exemplary embodiments provided herein and are not intended to limit the scope of the invention as encompassed by the claims.

[0029] The drawings are described in detail herein. [Brief explanation of the drawings]

[0030] [Figure 1] FIG. 1 illustrates images of tonsil staining using ultrasensitive IHC utilizing cell culture supernatant of exemplary clone 12H8. [Figure 2]

[0023] Figure 1 illustrates images of IHC staining for renal cell carcinoma (RCC) using the standard visualization system EnVision FLEX+ with DAB chromogen (brown) and exemplary clone 12H8. The images illustrate LAG-3 staining of activated T cells in areas of lymphocytic infiltration among tumor cells (tumor microenvironment (TME)); the images show that LAG-3 clone 12H8 does not stain tumor cells, and that the staining of activated T cells morphologically has three different staining patterns: cytoplasmic (1), membrane (2), and Golgi staining (3). [Figure 3A]3A and 3B illustrate IHC staining images of melanoma tumor tissue samples using the standard visualization system EnVision FLEX+ with DAB chromogen (brown), where FIG. 3A shows reference LAG-3 antibody clone 17B4 (Novus Bio) compared to exemplary LAG-3 antibody clone 12H8 as shown in FIG. 3B; all images illustrate LAG-3 staining of activated T cells in areas of lymphocytic infiltration among tumor cells within the TME; the images show that neither reference LAG-3 antibody clone 17B4 nor exemplary clone 12H3 stain tumor cells, and that exemplary clone 12H8 matches or even exceeds reference clone 17B4 in terms of specific staining intensity, lack of unwanted background staining, and number of stained cells when comparing FIG. A to FIG. B in various tumor types. [Figure 3B] 3A and 3B illustrate IHC staining images of melanoma tumor tissue samples using the standard visualization system EnVision FLEX+ with DAB chromogen (brown), where FIG. 3A shows reference LAG-3 antibody clone 17B4 (Novus Bio) compared to exemplary LAG-3 antibody clone 12H8 as shown in FIG. 3B; all images illustrate LAG-3 staining of activated T cells in areas of lymphocytic infiltration among tumor cells within the TME; the images show that neither reference LAG-3 antibody clone 17B4 nor exemplary clone 12H3 stain tumor cells, and that exemplary clone 12H8 matches or even exceeds reference clone 17B4 in terms of specific staining intensity, lack of unwanted background staining, and number of stained cells when comparing FIG. A to FIG. B in various tumor types. [Figure 4A]4A and 4B illustrate IHC staining images of melanoma tumor tissue samples using the standard visualization system EnVision FLEX+ with DAB chromogen (brown), where FIG. 4A shows reference LAG-3 antibody clone 17B4 (Novus Bio) compared to exemplary LAG-3 antibody clone 12H8 as shown in FIG. 4B; all images illustrate LAG-3 staining of activated T cells in areas of lymphocytic infiltration among tumor cells within the TME; the images show that neither reference LAG-3 antibody clone 17B4 nor exemplary clone 12H3 stain tumor cells, and that exemplary clone 12H8 matches or even exceeds reference clone 17B4 in terms of specific staining intensity, lack of unwanted background staining, and number of stained cells when comparing FIG. A to FIG. B in various tumor types. [Figure 4B] 4A and 4B illustrate IHC staining images of melanoma tumor tissue samples using the standard visualization system EnVision FLEX+ with DAB chromogen (brown), where FIG. 4A shows reference LAG-3 antibody clone 17B4 (Novus Bio) compared to exemplary LAG-3 antibody clone 12H8 as shown in FIG. 4B; all images illustrate LAG-3 staining of activated T cells in areas of lymphocytic infiltration among tumor cells within the TME; the images show that neither reference LAG-3 antibody clone 17B4 nor exemplary clone 12H3 stain tumor cells, and that exemplary clone 12H8 matches or even exceeds reference clone 17B4 in terms of specific staining intensity, lack of unwanted background staining, and number of stained cells when comparing FIG. A to FIG. B in various tumor types. [Figure 5A]5A and 5B illustrate IHC staining images of lung NSCLC tumor tissue samples using the standard visualization system EnVision FLEX+ with DAB chromogen (brown), where FIG. 5A shows reference LAG-3 antibody clone 17B4 (Novus Bio) compared to exemplary LAG-3 antibody clone 12H8 as shown in FIG. 5B; all images illustrate LAG-3 staining of activated T cells in areas of lymphocytic infiltration among tumor cells within the TME; the images show that neither reference LAG-3 antibody clone 17B4 nor exemplary clone 12H3 stain tumor cells, and that exemplary clone 12H8 matches or even exceeds reference clone 17B4 in terms of specific staining intensity, lack of undesirable background staining, and number of stained cells when comparing FIG. A to FIG. B in various tumor types. [Figure 5B] 5A and 5B illustrate IHC staining images of lung NSCLC tumor tissue samples using the standard visualization system EnVision FLEX+ with DAB chromogen (brown), where FIG. 5A shows reference LAG-3 antibody clone 17B4 (Novus Bio) compared to exemplary LAG-3 antibody clone 12H8 as shown in FIG. 5B; all images illustrate LAG-3 staining of activated T cells in areas of lymphocytic infiltration among tumor cells within the TME; the images show that neither reference LAG-3 antibody clone 17B4 nor exemplary clone 12H3 stain tumor cells, and that exemplary clone 12H8 matches or even exceeds reference clone 17B4 in terms of specific staining intensity, lack of undesirable background staining, and number of stained cells when comparing FIG. A to FIG. B in various tumor types. [Figure 6A]6A and 6B illustrate IHC staining images of lung NSCLC tumor tissue samples using the standard visualization system EnVision FLEX+ with DAB chromogen (brown), where FIG. 6A shows reference LAG-3 antibody clone 17B4 (Novus Bio) compared to exemplary LAG-3 antibody clone 12H8 as shown in FIG. 6B; all images illustrate LAG-3 staining of activated T cells in areas of lymphocytic infiltration among tumor cells within the TME; the images show that neither reference LAG-3 antibody clone 17B4 nor exemplary clone 12H3 stain tumor cells, and that exemplary clone 12H8 matches or even exceeds reference clone 17B4 in terms of specific staining intensity, lack of undesirable background staining, and number of stained cells when comparing FIG. A to FIG. B in various tumor types. [Figure 6B] 6A and 6B illustrate IHC staining images of lung NSCLC tumor tissue samples using the standard visualization system EnVision FLEX+ with DAB chromogen (brown), where FIG. 6A shows reference LAG-3 antibody clone 17B4 (Novus Bio) compared to exemplary LAG-3 antibody clone 12H8 as shown in FIG. 6B; all images illustrate LAG-3 staining of activated T cells in areas of lymphocytic infiltration among tumor cells within the TME; the images show that neither reference LAG-3 antibody clone 17B4 nor exemplary clone 12H3 stain tumor cells, and that exemplary clone 12H8 matches or even exceeds reference clone 17B4 in terms of specific staining intensity, lack of undesirable background staining, and number of stained cells when comparing FIG. A to FIG. B in various tumor types. [Figure 7A]7A and 7B illustrate IHC staining images of tumor tissue samples from lung adenocarcinoma using the standard visualization system EnVision FLEX+ with DAB chromogen (brown), where FIG. 7A shows reference LAG-3 antibody clone 17B4 (Novus Bio) compared to exemplary LAG-3 antibody clone 12H8 as shown in FIG. 7B; all images illustrate LAG-3 staining of activated T cells in areas of lymphocytic infiltration among tumor cells within the TME; the images show that neither reference LAG-3 antibody clone 17B4 nor exemplary clone 12H3 stain tumor cells, and that exemplary clone 12H8 rivals or even exceeds reference clone 17B4 in terms of specific staining intensity, lack of undesirable background staining, and number of stained cells when comparing FIG. A to FIG. B in various tumor types. [Figure 7B] 7A and 7B illustrate IHC staining images of tumor tissue samples from lung adenocarcinoma using the standard visualization system EnVision FLEX+ with DAB chromogen (brown), where FIG. 7A shows reference LAG-3 antibody clone 17B4 (Novus Bio) compared to exemplary LAG-3 antibody clone 12H8 as shown in FIG. 7B; all images illustrate LAG-3 staining of activated T cells in areas of lymphocytic infiltration among tumor cells within the TME; the images show that neither reference LAG-3 antibody clone 17B4 nor exemplary clone 12H3 stain tumor cells, and that exemplary clone 12H8 rivals or even exceeds reference clone 17B4 in terms of specific staining intensity, lack of undesirable background staining, and number of stained cells when comparing FIG. A to FIG. B in various tumor types. [Figure 8A]8A and 8B illustrate IHC staining images of renal cell carcinoma (RCC) tumor tissue samples using the standard visualization system EnVision FLEX+ with DAB chromogen (brown), where FIG. 8A shows reference LAG-3 antibody clone 17B4 (Novus Bio) compared to exemplary LAG-3 antibody clone 12H8 as shown in FIG. 8B; all images illustrate LAG-3 staining of activated T cells in areas of lymphocytic infiltration among tumor cells within the TME; the images show that neither reference LAG-3 antibody clone 17B4 nor exemplary clone 12H3 stain tumor cells, and that exemplary clone 12H8 rivals or even exceeds reference clone 17B4 in terms of specific staining intensity, lack of undesirable background staining, and number of stained cells when comparing FIG. A to FIG. B in various tumor types. [Figure 8B] 8A and 8B illustrate IHC staining images of renal cell carcinoma (RCC) tumor tissue samples using the standard visualization system EnVision FLEX+ with DAB chromogen (brown), where FIG. 8A shows reference LAG-3 antibody clone 17B4 (Novus Bio) compared to exemplary LAG-3 antibody clone 12H8 as shown in FIG. 8B; all images illustrate LAG-3 staining of activated T cells in areas of lymphocytic infiltration among tumor cells within the TME; the images show that neither reference LAG-3 antibody clone 17B4 nor exemplary clone 12H3 stain tumor cells, and that exemplary clone 12H8 rivals or even exceeds reference clone 17B4 in terms of specific staining intensity, lack of undesirable background staining, and number of stained cells when comparing FIG. A to FIG. B in various tumor types. [Figure 9]Figure 9 illustrates double IHC staining of normal tonsil tissue samples. Figure 9 illustrates lung squamous cell carcinoma (SQC) (Figure 10) and kidney renal cell carcinoma (RCC) (Figure 11) tumor tissue samples; the double IHC staining method followed the protocol of Petersen et al., 2018

[18] using the EnVision FLEX+™ system in a sequential manner, where an exemplary LAG-3 antibody, clone 12H8 (HRP DAB chromogen), constitutes the first layer, followed by a sulfuric acid blocking step and the addition of an additional EnVision FLEX+™ staining layer on top using a PD1 antibody, clone NAT105 (magenta chromogen); Figure 9 illustrates that LAG-3 colocalizes with the PD1 T cell marker in a subpopulation of activated T cells in the germinal centers of tonsils. Other cells belonging to the activated T cell population are stained only by PD1. Germinal center B lymphocytes were negative for both LAG-3 and PD1 antibodies, illustrating the specificity of both of these antibodies (red arrows: strongly LAG-3 positive, DAB obscures magenta PD1; green arrows: weakly LAG-3 positive, with both DAB membrane and DAB Golgi staining and visible PD1 magenta staining). PD1-positive, LAG-3-negative T cells (orange arrows). [Figure 10] Figure 10 illustrates double IHC staining of normal tonsil tissue samples. Figure 10 illustrates double staining of LAG-3 and PD1 in lung non-small cell lung cancer (NSCLC) using the above methodology. LAG-3 co-localizes with PD1 (a T cell marker) in activated T cells within the tumor microenvironment (TME) of both tumors; some of these T cells are positive only for PD1, while all squamous tumor cells in the lung and renal tumor clear cell are negative; these findings confirm the specificity of the exemplary LAG-3 antibody, clone 12H8. Regarding Figures 10 and 11 (red arrows: LAG-3 co-localizes with PD1 in activated T cells within the tumor microenvironment (TME); orange arrows: LAG-3-negative / PD1-positive T cells; in Figure 10, blue arrows: squamous tumor cells negative for LAG-3 and PD1). [Figure 11]FIG. 11 illustrates double IHC staining of normal tonsil tissue samples. FIG. 11 illustrates LAG-3 and PD1 double staining of renal clear cell carcinoma (RCC) using the above methodology. LAG-3 co-localizes with PD1 (a T cell marker) on activated T cells within the tumor microenvironment (TME) of both tumors; some of these T cells are positive only for PD1, while all squamous tumor cells in the lung and renal tumor clear cell are negative. These findings confirm the specificity of the exemplary LAG-3 antibody, clone 12H8. Regarding FIG. 10 and FIG. 11 (red arrows: LAG-3 co-localizes with PD1 on activated T cells within the tumor microenvironment (TME); orange arrows: LAG-3-negative / PD1-positive T cells; in FIG. 11, blue arrows: RCC tumor cells negative for LAG-3 and PD1). [Figure 12] 11 illustrates triple IHC staining of normal tonsils; the triple IHC staining utilized the ultrasensitive system with the sulfuric acid blocking step described above between three layers of antibodies: an exemplary LAG-3 antibody, clone 12H8 (HRP DAB chromogen), a polyclonal (Dako GA503) CD3 antibody (HRP MAGENTA™ (Dako Omnis, Agilent) chromogen), a CK pan-antibody, and clone AE1 / AE1 (yellow substrate); FIG. 12 demonstrates that LAG-3 binds to CD3 in a subpopulation of activated T cells in the region of the tonsil beneath the tonsillar crypt epithelium and in T cells migrating through the epithelium. colocalization with T cell markers is illustrated; many T cells express only the CD3 marker, consistent with the exclusive expression of LAG-3 on activated T cells; many other B lymphocytes in the area do not stain for either LAG-3 or CD3; the CK pan antibody stains epithelial cells but not lymphocytes; the image illustrates the localization of LAG-3-positive T cells, and colocalization with the CD3 T cell marker confirms the specificity of the LAG-3 antibody (red arrow: strongly LAG-3 positive, DAB obscures magenta CD3; green arrow: weakly LAG-3 positive, with both DAB membrane and DAB Golgi and visible CD3 magenta staining); CD3-positive, LAG-3-negative T cells (orange arrow); epithelial cells positive for CK pan and negative for LAG-3 (blue arrow). [Figure 13] 13 and 14 illustrate the colocalization of LAG-3 and CD3 T cell markers in a subpopulation of T cells among tumor-infiltrating lymphocytes in the TME in renal RCC and lung SQC tumors. Only a small number of T cells expressed CD3, and all renal and lung tumor cells stained only with the CK pan-antibody, visually confirming that the exemplary LAG-3 antibody is restricted to only a subpopulation of T cells and not expressed on tumor cells; red arrows indicate activated T cells in the TME that are double-positive for LAG-3 and CD3; blue arrows: RCC tumor cells that are only positive for CK pan-antibody; green arrows: activated LAG-3 and CD3 double-positive T cells surrounding tumor cells; orange arrows: T cells that are negative for LAG-3 and positive for CD3. [Figure 14]13 and 14 illustrate the colocalization of LAG-3 and CD3 T cell markers in a subpopulation of T cells among tumor-infiltrating lymphocytes in the TME in kidney RCC and lung SQC tumors. Only a small number of T cells expressed CD3, and all renal and lung tumor cells stained only with the CK pan antibody, visually confirming that the exemplary LAG-3 antibody is restricted to only a subpopulation of T cells and not expressed on tumor cells; red arrows indicate activated T cells in the TME that are double-positive for LAG-3 and CD3; blue arrows: squamous tumor cells that are only positive for CK pan; green arrows: activated LAG-3 and CD3 double-positive T cells surrounding tumor cells; orange arrows: T cells that are negative for LAG-3 and positive for CD3. DETAILED DESCRIPTION OF THE INVENTION

[0031] Like reference symbols in the various drawings indicate like elements.

[0032] In alternative embodiments, chimeric or recombinant antibodies (Abs), or antigen-binding fragments thereof, or monomeric or dimeric antigen-binding proteins capable of specifically binding to human LAG-3 polypeptides, including human LAG-3 polypeptides expressed on the surface of lymphocytes, such as activated T cells infiltrating tumors, or human LAG-3 polypeptides expressed on tumor-infiltrating lymphocytes (TILs), are provided. In alternative embodiments, articles of manufacture and kits comprising the chimeric or recombinant Abs, or antigen-binding fragments thereof, or monomeric or dimeric antigen-binding proteins, or nucleic acids encoding same, as provided herein, and methods for their production and use are provided. In alternative embodiments, chimeric or recombinant antibodies (Abs), or antigen-binding fragments thereof, or monomeric or dimeric antigen-binding proteins as provided herein, are used in, e.g., by immunohistochemistry (IHC), e.g., in IHC protocols for diagnosing, detecting, and / or treating cancer, e.g., bladder cancer, urothelial carcinoma, breast cancer or mammary carcinoma or ductal carcinoma in situ (DCIS), carcinoid, Hodgkin's lymphoma, chronic lymphocytic leukemia, colorectal cancer, ovarian cancer, lung cancer, non-small cell lung cancer (NSCLC), kidney cancer or renal cell carcinoma or renal carcinoma, renal clear cell carcinoma (RCC), mesothelioma or malignant pleural mesothelioma, squamous cell carcinoma, anal squamous cell carcinoma, pancreatic cancer, and melanoma or malignant melanoma, e.g., by their ability to specifically bind to activated T cells infiltrating tumors, including tumor-infiltrating lymphocytes (TILs). Thus, chimeric or recombinant antibodies (Abs), or antigen-binding fragments thereof, or monomeric or dimeric antigen-binding proteins as provided herein, can be used as accompanying diagnostic agents for the diagnosis and treatment of cancer by specifically staining TILs.

[0033] Expression of recombinant chimeric antibodies In alternative embodiments, chimeric or recombinant antibodies (Abs), or antigen-binding fragments thereof, or monomeric or dimeric antigen-binding proteins as provided herein, including the exemplary chimeric or recombinant anti-human LAG-3 Ab comprising the heavy chain variable region SEQ ID NO:2 and the light chain variable region SEQ ID NO:3, with or without a signal peptide, can be expressed as recombinant Abs, e.g., using plasmids or any expression vehicle encoding the respective heavy and light chains, or the heavy and light chains can be encoded in separate expression vehicles.

[0034] In some embodiments, the heavy and light chains can be expressed (in cis or trans) from any plasmid, cosmid, recombinant virus or equivalent vector, for example, from a pTT5™ vector (National Research Council Canada, NRC-CNRC, Canada) or equivalent.

[0035] In alternative embodiments, the expression vehicle (such as plasmid) comprising exemplary Ab-encoding nucleic acid as provided herein is expressed in in vitro expression system, or expressed in cultured tissue, cell or organoid, which can be bacterial, fungal, mammalian, yeast, insect or plant cell expression system, or hybrid or synthetic expression system.For example, exemplary Ab-encoding nucleic acid can be expressed in human embryonic kidney (HEK) cell (such as HEK293-6E cell).In alternative embodiments, exemplary Ab-encoding nucleic acid, for example, one or more vectors that express exemplary heavy chain and / or light chain, are episomal or chromosomally integrated, for example, in a stable cell line that can synthesize, optionally inducibly synthesize, heavy chain and / or light chain.

[0036] In alternative embodiments, nucleic acids encoding chimeric or recombinant Abs as provided herein are provided. Nucleic acids as provided herein can be produced, isolated, and / or manipulated, for example, by cloning and expression of cDNA libraries, amplification of message or genomic DNA by PCR, etc. Nucleic acids used to practice the embodiments as provided herein can be isolated, genetically engineered, amplified, and / or expressed / recombinantly produced from a variety of sources, whether RNA, cDNA, genomic DNA, vectors, viruses, or hybrids thereof. Recombinant polypeptides produced from these nucleic acids can be individually isolated or cloned and tested for the desired activity. Any recombinant expression system can be used, including bacterial, fungal, mammalian, yeast, insect, or plant cell expression systems, or hybrid or synthetic expression systems.

[0037] Alternatively, these nucleic acids can be prepared using methods described, for example, in Martin et al., ACS Synth. Biol. (2017) 6, 7, 1370-1379; Adams (1983) J. Am. Chem. Soc. 105:661; Belousov (1997) Nucleic Acids Res. 25:3440-3444; Frenkel (1995) Free Radic. Biol. Med. 19:373-380; Blommers (1994) Biochemistry 33:7886-7896; Narang (1979) Meth. Enzymol. 68:90; Brown (1979) Meth. Enzymol. 68:109; Beaucage (1981) Tetra. Lett. 22:1859; or in vitro synthesis by well-known chemical synthesis techniques, as described in U.S. Pat. No. 4,458,066.

[0038] Techniques for the manipulation of nucleic acids, such as, for example, subcloning, labeling probes (e.g., random primer labeling using Klenow polymerase, nick translation, amplification), sequencing, hybridization, etc., are well described in the scientific and patent literature, see, e.g., Sambrook (ed.), MOLECULAR CLONING: A LABORATORY MANUAL (2nd ed.), Vols. 1-3, Cold Spring Harbor Laboratory, (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel (ed.), John Wiley & Sons, Inc., New York (1997); LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULAR BIOLOGY: HYBRIDIZATION WITH NUCLEIC ACID PROBES, Part I. Theory and Nucleic Acid Preparation, Tijssen (ed.), Elsevier, NY (1993).

[0039] Another useful means for obtaining and manipulating the nucleic acid used to carry out the embodiments provided herein includes, for example, screening and recloning the insert that is isolated or amplified from genomic clone or cDNA clone.Nucleic acid sources include, for example, mammalian artificial chromosome (MAC) (see, for example, U.S. Patent No. 5,721,118; U.S. Patent No. 6,025,155); human artificial chromosome (see, for example, Rosenfeld (1997) Nat. Genet. 15:333-335); yeast artificial chromosome (YAC); bacterial artificial chromosome (BAC); P1 artificial chromosome (see, for example, Woon (1998) Genomics 50:306-316); P1-derived vector (PAC) (see, for example, Kern (1997) Biotechniques 23:120-124); recombinant nucleic acid sequence, genome or cDNA library, that is contained in and / or expressed in cosmid, recombinant virus, phage or plasmid.

[0040] In alternative embodiments, the nucleic acids as provided herein are operably linked to a transcriptional regulatory element, including a promoter, which may be a constitutive or an inducible transcriptional regulatory element.

[0041] In alternative aspects, "expression cassettes" are provided that include a nucleotide sequence as provided herein, e.g., encoding a chimeric or recombinant antibody as provided herein. Expression cassettes can include at least a transcriptional regulatory element, e.g., a promoter, operably linked to the antibody coding sequence, and can optionally include a transcription termination signal. Additional factors necessary or helpful in achieving expression can also be used, e.g., enhancers.

[0042] In alternative aspects, expression cassettes used to practice embodiments as provided herein include plasmids, expression vectors, recombinant viruses, any form of recombinant "naked DNA" vector, and the like. In alternative aspects, "vectors" used to practice embodiments as provided herein can comprise nucleic acids capable of infecting, transfecting, transiently, or permanently transducing cells. In alternative aspects, vectors used to practice embodiments as provided herein can be naked nucleic acids or nucleic acids complexed with proteins or lipids. In alternative aspects, vectors used to practice embodiments as provided herein can comprise viral or bacterial nucleic acids and / or proteins, and / or membranes (e.g., cell membranes, viral lipid envelopes, etc.). In alternative aspects, vectors used to practice embodiments as provided herein include, but are not limited to, replicons (e.g., RNA replicons, bacteriophages) to which fragments of DNA can be ligated and replicated. Thus, vectors include, but are not limited to, RNA, autonomous self-replicating circular or linear DNA or RNA (e.g., plasmids, viruses, etc., see, e.g., U.S. Pat. No. 5,217,879), and include both expression and non-expression plasmids. In alternative aspects, vectors used to practice the embodiments as provided herein may be stably replicated by cells during mitosis as autonomous structures, or may be integrated into the host's genome.

[0043] In alternative aspects, a "promoter" used to practice the embodiments as provided herein includes any sequence capable of driving transcription of a coding sequence in a cell, such as a bacterial, yeast, fungal, plant, insect (e.g., baculovirus), or mammalian cell. Thus, a promoter used in a construct includes cis-acting transcriptional control elements and regulatory sequences involved in regulating or altering the timing and / or rate of transcription of a gene. For example, a promoter used to practice the embodiments as provided herein can be a cis-acting transcriptional control element including an enhancer, promoter, transcription terminator, origin of replication, chromosomal integration sequence, 5' and 3' untranslated region, or intron sequence involved in transcriptional regulation. These cis-acting sequences can interact with proteins or other biomolecules to effectuate (turn on / off, regulate, alter, etc.) transcription.

[0044] A "constitutive" promoter used to practice the embodiments as provided herein can be one that continuously drives expression under most environmental conditions and developmental or cell differentiation situations. An "inducible" or "regulatable" promoter used to practice the embodiments as provided herein can direct expression of a nucleic acid as provided herein under the influence of an environmental or developmental condition. An example of an environmental condition that can affect transcription by an inducible promoter used to practice the embodiments as provided herein includes the presence of an inducer administered to a cell.

[0045] In alternative embodiments, antibodies used to practice the embodiments as provided herein can include any "mimetic" and / or "peptidomimetic" form. In alternative embodiments, peptides and polypeptides used to practice the embodiments as provided herein can include synthetic compounds that have substantially the same structural and / or functional properties as naturally occurring polypeptides, e.g., chimeric or recombinant antibodies as provided herein. Mimetics used to practice the embodiments as provided herein can either be entirely composed of synthetic, non-natural analogs of amino acids, or be chimeric molecules that are partially natural peptide amino acids and partially non-natural analogs of amino acids. Mimetics can also incorporate any amount of naturally occurring amino acid conservative substitutions, so long as such substitutions do not also substantially alter the structure and / or activity of the mimetic. Routine experimentation will determine whether a mimetic is effective for practicing the invention, e.g., whether a mimetic composition is effective in specifically binding to human LAG-3 protein. The methodologies detailed herein and other methodologies known to those skilled in the art can be used to select or guide the selection of effective mimetics for practicing the compositions and / or methods as provided herein.

[0046] Polypeptide mimetic compositions for practicing embodiments as provided herein can include any combination of non-natural structural components. In alternative aspects, mimetic compositions for practicing embodiments as provided herein can include one or all of three structural groups: (a) residue linking groups other than natural amide bond ("peptide bond") linkages; (b) non-natural residues in place of naturally occurring amino acid residues; or (c) residues that induce secondary structure mimicry, i.e., to induce or stabilize secondary structure, e.g., β-turn, γ-turn, β-sheet, α-helical conformation, etc. For example, a polypeptide can be characterized as a mimetic if all or some of its residues are linked by chemical means other than natural peptide bonds.

[0047] Recombinant protein purification and isolation In alternative embodiments, the chimeric or recombinant antibody, antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein is substantially purified or isolated, optionally in substantially purified or isolated form, in a form that is used in immunohistochemistry methodologies and / or as reagents, kits and / or articles of manufacture as provided herein.

[0048] In alternative embodiments, chimeric or recombinant antibodies, antigen-binding fragments thereof, or monomeric or dimeric antigen-binding proteins are substantially purified or isolated using, for example, physicochemical fractionation using differential precipitation, size exclusion, or solid phase binding of immunoglobulins based on the size, charge, or other shared chemical properties of the antibodies in a representative sample; class-specific affinity, for example, solid phase binding of a particular antibody class (e.g., IgG or IgM) with a solid-phased biological ligand (e.g., protein, lectin, etc.) that has specific affinity for immunoglobulins (which can purify all antibodies of a target class regardless of antigen specificity); or antigen-specific affinity, for example, affinity purification of only antibodies in a sample that bind to a particular antigen molecule through their specific antigen-binding domain (which purifies all antibodies that bind to an antigen regardless of antibody class or isotype).

[0049] In alternative embodiments, chimeric or recombinant antibodies, antigen-binding fragments thereof, or monomeric or dimeric antigen-binding proteins are substantially purified or isolated using standard isolation methodologies such as chromatography, e.g., ion exchange (IEX) chromatography, hydrophobic interaction chromatography (HIC), counter-current chromatography, immunoaffinity and / or size exclusion chromatography.

[0050] In alternative embodiments, chimeric or recombinant antibodies, antigen-binding fragments thereof, or monomeric or dimeric antigen-binding proteins are produced in a bioreactor, e.g., a perfusion bioreactor, using a continuous expression and purification process, e.g., as described by Vogg et al., Methods Mol Biol. 2018; 1850:147-178, or using a stirred tank or rocking bioreactor system and subsequent purification.

[0051] Manufacturing Products and Kits Articles of manufacture and kits are provided that include chimeric or recombinant anti-human LAG-3 Abs as provided, and for practicing the methods as provided herein using chimeric or recombinant anti-human LAG-3 Abs as provided herein; optionally, the articles of manufacture and kits can further include some or all of the reagents necessary to perform IHC, and optionally include instructions for practicing the methods as provided herein.

[0052] In alternative embodiments, the article of manufacture is linked to, immobilized (optionally covalently attached) on or to a chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein as provided herein, and optionally, the article of manufacture as provided herein is or comprises an array, biochip, slide, tray, dish (e.g., microtiter dish), phage, or phagemid.

[0053] immunohistochemistry In alternative embodiments, immunohistochemistry methodologies and / or reagents used to practice the compositions, articles of manufacture, kits, or methods as provided herein include those described, for example, in U.S. Patent (USPN) Nos. 10,565,479 (describing methods for identifying blurred areas in digital images of stained tissue); 10,564,076 (describing systems for analytical (or IHC) sample preparation); 10,551,395 (describing automated histological staining systems); 10,551,378 (describing tissue staining methods); 10,504,224 (describing digital tissue image analysis systems for IHC); 10,501,777 (describing simultaneous multiplexed detection and quantification of protein expression in IHC); 10,488,340 (describing methods for extracting images of target fluorophores in biological material); 10,453,195 (describing methods for detecting tissue regions of interest using digital pathology imaging). No. 10,438,381 (describing devices, systems and methods for generating digital images of tissue sections); No. 10,416,176 (describing methods for processing specimens in automated histological staining systems); No. 10,393,633 (describing methods for processing IHC samples and inhibiting their degradation); No. 10,217,011 (describing handling of IHC slides); No. 10,209,165 (describing automated or semi-automated methods for assessing the quality of staining of cell-containing specimens); No. 10,126,216 (describing methods for fixing tissue samples for IHC); No. 9,423,322 (describing methods for fixing tissue samples for IHC) or any IHC protocol, IHC equipment, device and / or image or data analysis system for performing IHC or IHC reagents known in the art, as described in the above.

[0054] In alternative embodiments, the chimeric or recombinant antibodies, antigen-binding fragments thereof, or monomeric or dimeric antigen-binding proteins in the IHC protocols or kits as provided herein are substantially purified or isolated, or in the form of crude or partially purified culture supernatants.

[0055] In alternative embodiments, the methods as provided herein can use or include reagents for detecting or visualizing antibody-antigen interactions, e.g., using any products or methods known in the art, and IHC protocols or reagents.

[0056] In an alternative embodiment, the methods as provided herein comprise the use of chromogenic immunohistochemistry (CIH), wherein a primary antibody (e.g., a chimeric or recombinant antibody (Ab) as provided herein, or an antigen-binding fragment thereof, or a monomeric or dimeric antigen-binding protein) or a secondary antibody (e.g., where the secondary antibody binds to a (primary antibody) chimeric or recombinant antibody (Ab), or an antigen-binding fragment thereof, or a monomeric or dimeric antigen-binding protein as provided herein after it specifically binds to, pairs with, or associates with a LAG-3 epitope or polypeptide) is conjugated to an enzyme, such as a peroxidase (or immunoperoxidase), e.g., horseradish peroxidase (HRP), that can catalyze a color-developing reaction.

[0057] In alternative embodiments, the methods as provided herein include the use of immunofluorescence, wherein a primary or secondary antibody is tagged with a fluorophore, such as fluorescein or fluorescein isothiocyanate (FITC), triarylmethane dyes (rhodamine or rhodamine derivatives (e.g., tetramethylrhodamine (TRITC), rhodamine 6G, rhodamine 123, rhodamine B, carboxytetramethylrhodamine (TAMRA), tetramethylrhodamine (TMR), sulforhodamine 101), etc.), aminomethylcoumarin acetate (AMCA), ALEXA™, or DYLIGHT™ fluor. 3,3'-diaminobenzidine (DAB) can also be used.

[0058] In an alternative embodiment, the methods as provided herein involve the use of a direct or one-step staining method in which a primary antibody (e.g., a chimeric or recombinant antibody (Ab) as provided herein, or an antigen-binding fragment thereof, or a monomeric or dimeric antigen-binding protein) is labeled and reacts directly with the antigen, e.g., in a tissue section. While this technique utilizes only one type of antibody and is therefore simple and rapid, sensitivity may be relatively low due to little signal amplification.

[0059] In an alternative embodiment, the method as provided herein involves the use of an indirect method, in which an unlabeled primary antibody (first layer) binds to a target antigen (LAG-3), for example, in a tissue or organ, and a labeled secondary antibody (second layer) subsequently reacts with the primary antibody. The secondary antibody can be directed against the isotype of the animal species from which the primary antibody is derived, for example, IgG. This method can be more sensitive than direct detection strategies when the secondary antibodies are conjugated to a detection agent such as a fluorescent or enzyme reporter, due to signal amplification resulting from the binding of several secondary antibodies to each primary antibody.

[0060] In an alternative embodiment, further amplification is achieved when the secondary antibody is conjugated to a biotin molecule that can recruit a complex of several detection molecules, such as avidin, streptavidin, or NEUTRAVIDIN™ protein-linked enzymes.

[0061] In alternative embodiments, IHC is performed on tissue sections or tissue biopsies, e.g., paraformaldehyde (PFA)-fixed tissues or organs, or formalin-fixed, paraffin-embedded tissues. In alternative embodiments, tissues are sliced ​​or used whole. Prior to sectioning, tissue samples can be embedded in media, e.g., paraffin wax or freezing media. Tissue sections can be sliced ​​with a variety of instruments, most commonly using a microtome, cryostat, or vibratome. Specimens can be sliced ​​in the range of approximately 3 μm to 5 μm. Slices can be mounted on slides, dehydrated using alcohol washes of increasing concentrations (e.g., 50%, 75%, 90%, 95%, 100%), and cleared using a detergent such as xylene, before being imaged under a microscope.

[0062] Depending on the method of fixation and tissue preservation, samples may require additional steps to make LAG-3 epitopes available for antibody binding, including deparaffinization and antigen retrieval. For formalin-fixed, paraffin-embedded tissue, antigen retrieval is often necessary and can include pre-treating the sections with heat or proteases.

[0063] In an alternative embodiment, IHC is performed using the ENVISION DUOFLEX DOUBLESTAIN SYSTEM™ (Agilent, San Jose, CA), which allows staining of two or more markers on a single slide. In an alternative embodiment, IHC is performed using the EnVision FLEX HRP Magenta, High pH (Dako Omnis) system, and binding can be visualized with the EnVision FLEX HRP Magenta chromogen. In an alternative embodiment, IHC is performed using the EnVision FLEX Mini Kit, High pH, ​​a highly sensitive visualization system intended for use in IHC with the Dako AUTOSTAINER™ instrument; this dual-link system detects primary mouse and rabbit antibodies, and the reaction is visualized with 3,3′-diaminobenzidine (DAB) chromogen (DAB forms a water-insoluble brown precipitate when oxidized, for example, by peroxidase).

[0064] Any of the above aspects and embodiments may be combined with any other aspect or embodiment as disclosed herein in the Summary, Drawings and / or Detailed Description sections.

[0065] As used in this specification and the claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0066] Unless specifically stated otherwise or clear from context, as used herein, the term "or" is understood to include and encompass both "or" and "and."

[0067] Unless specifically stated otherwise or clear from the context, the term "about" as used herein is understood to mean within the range of normal acceptance in the art, for example, within two standard deviations of the mean. About can be understood to mean within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the specified value. Unless otherwise clear from the context, all numerical values ​​provided herein are modified by the term "about."

[0068] Unless specifically stated or clear from context, as used herein, the terms "substantially all," "substantially a majority of," "substantially all of," or "the majority of" include at least about 90%, 95%, 97%, 98%, 99%, or 99.5%, or more, of the referenced amount of a composition.

[0069] Each patent, patent application, publication, and document referenced herein is incorporated herein by reference in its entirety. Citation of the above patents, patent applications, publications, and documents is not an admission that any of the above is pertinent prior art, and does not constitute any admission as to the contents or date of these publications or documents. The incorporation by reference of these documents should not be construed as an assertion or admission that, independently, any portion of the contents of any document is deemed essential material to satisfy any national or local statutory disclosure requirements for patent applications. The right is nevertheless reserved to rely on any such documents, where appropriate, to provide material deemed essential to claimed subject matter by an examining office or court.

[0070] Modifications to the above can be made without departing from the basic aspects of the invention. Although the present invention has been described in considerable detail with reference to one or more specific embodiments, those skilled in the art will recognize that changes can be made to the embodiments specifically disclosed in this application, and that these modifications and improvements will still fall within the scope and spirit of the invention. The invention illustratively described herein as appropriate can be practiced in the absence of any element not specifically disclosed herein. Thus, for example, in each example herein, either of the terms "comprising," "essentially consisting of," and "consisting of" can be replaced with either of the other two terms. Therefore, the terms and expressions used are used as terms of description, not as terms of limitation, and do not exclude equivalents of the features shown and described, or portions thereof, and it is recognized that various modifications are possible within the scope of the invention. Embodiments of the invention are set forth in the following claims.

[0071] The present invention will be further illustrated with reference to the examples described herein; however, it should be understood that the invention is not limited to such embodiments. [Example]

[0072] Unless otherwise specified in the examples, all recombinant DNA techniques are performed according to standard protocols, for example, as described in Sambrook et al. (2012) Molecular Cloning: A Laboratory Manual, 4th Edition, Cold Spring Harbor Laboratory Press, NY and Ausubel et al. (1994) Current Protocols in Molecular Biology, Current Protocols, USA, Volumes 1 and 2. Other references for standard molecular biology techniques include Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press, NY, and Brown (1998) Molecular Biology LabFax, 2nd Edition, Academic Press (UK), Volumes I and II. Standard materials and methods for the polymerase chain reaction can be found in Dieffenbach and Dveksler (1995) PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press, and McPherson et al. (2000) PCR-Basics: From Background to Bench, 1st ed., Springer Verlag, Germany.

[0073] Example 1: Development of an exemplary anti-LAG antibody This example describes the development of an exemplary anti-LAG antibody as provided herein.

[0074] The antigen used to immunize rabbits was a synthetic peptide sequence representing amino acids 70-98 of human LAG-3. GPPAAAPGHPLAPGPHPAAPSSWGPRPRR (SEQ ID NO: 1). The peptide was conjugated to KLH and used to immunize five rabbits.

[0075] The antibody titers of the rabbits were tested against the peptides and the bleeds were tested in IHC for LAG-3 specific staining in IHC. All five rabbits showed LAG-3 specific staining in IHC.

[0076] B cell selection was performed in one rabbit to obtain several promising B cell clones producing antibodies specific for LAG-3. The antibody coding sequences were cloned into expression plasmids.

[0077] Sequence of rabbit anti-human LAG-3 antibody, clone 12H8; The heavy chain variable region is QSVKESEGGLFKPTDTLTLTCTVSGIDLSSGILVWVRQAPGSGLEWIGGIDANGRAYYASWAKSRSTITRNTNENTVTLKMTSLTAADTATYFCAGGAWNIWGPGTLVTVSS (SEQ ID NO: 2).

[0078] Sequence of rabbit anti-human LAG-3 antibody, clone 12H8; The light chain variable region is AQVLTQTPSPVSAAVGGTVTIKCQSSQSVYDSNTLAWFQQKPGQPPKLLMYSASTLAFGVPSRFSGSGSGTQFTLTISDLECADAATYYCLGSYDCSSVDCTAFGGGTEVVVK (SEQ ID NO: 3).

[0079] A recombinant antibody was generated and tested by standard IHC showing LAG-3 specific staining. This antibody was further tested to confirm its specificity for LAG-3.

[0080] Recombinant antibody expression was achieved using the HEK293-6E cell line and pTT5-based vectors. This transient expression of antibodies requires approximately 10 days after transfection, making it a rapid and high-yield method compared to hybridoma technology.

[0081] Other methods can be used for transient transfection and generation of stable cell lines for expression of antibodies. For example, in one embodiment, the dihydrofolate reductase (DHFR)-deficient cell line CHO DG44 cells are used (e.g., using the FREEDOM™ DG44 kit (Gibco)).

[0082] The creation of stable cell lines requires cell selection and cloning to produce good expression and stable cell lines. It is important that the stable cell line created is monoclonal to ensure the production of homogeneous monoclonal antibodies. Other cell lines can be used for both transient and / or stable transfection.

[0083] LAG-3 antibody clone development For the development of anti-human LAG-3 antibodies, different antigens were designed and generated. The antigen used for the final clone 12H8 was a synthetic peptide covering amino acids 70-98; The sequence was GPPAAAPGHPLAPGPHPAAPSSWGPRPRR (SEQ ID NO: 1).

[0084] This sequence is part of the extracellular domain of the human LAG-3 protein and creates an additional loop compared to CD4, which has high structural homology to LAG-3 and was previously used to generate LAG-3 antibodies

[19] .

[0085] Rabbits were immunized and titers were tested by ELISA. Subsequently, specificity testing was performed using serum samples against a tissue array including normal tonsils, reactive lymph nodes, malignant melanoma (clinical tissue), normal liver, carcinoid tumor, breast cancer, colon cancer, cerebellum, normal prostate, normal kidney, and normal pancreas. All rabbit sera demonstrated some degree of specificity for the LAG-3 protein in IHC. The rabbits producing the best-performing serum samples were selected for B cell selection. Blood samples were obtained from the rabbits and subjected to B cell selection to isolate B cells producing antibodies that bound to the LAG-3 antigen. The B cells were cultured monoclonally under stimulatory conditions, and the resulting cell culture supernatants were tested by ELISA to identify wells containing B cells producing antigen-binding antibodies. ELISA-positive cell culture supernatants were further tested by ultrasensitive IHC to identify cell culture supernatants with antibodies specific to LAG-3 in IHC. Ten clones were identified as exhibiting LAG-3-specific staining in IHC.

[0086] The two B cell clones with the best IHC performance were selected for cloning. Selection and prioritization of B cell clones were performed using ultrasensitive IHC on both normal and tumor tissues as listed above. Tissues were selected by IHC screening using a reference LAG-3 antibody (clone 17B4 Novus bio) to select both normal and tumor tissues with high LAG-3 expression. All B cell clones were then compared with the LAG-3 reference antibody in ultrasensitive IHC. Clones with the correct specificity, correct morphological expression (membrane, cytoplasm, and Golgi), and highest sensitivity (signal-to-noise ratio) were selected and prioritized.

[0087] Cultured cells from each well of the two clones were lysed, and RNA was extracted and used to generate cDNA. The variable heavy and light chains were amplified by PCR using custom-made primers, and the PCR products were cloned into custom-made expression vectors (using a pTT5 backbone) containing rabbit constant IgG heavy and light chains, respectively, to obtain functional antibody coding sequences. The heavy and light chain plasmids were transfected into HEK293-6E cell lines, and recombinant antibodies were produced and tested using standard IHC (Envision FLEX) protocols.

[0088] Antibody clone 12H8 demonstrated good, clear, and specific performance in IHC, with correct morphological expression (both membrane, cytoplasm, and Golgi). Sensitivity was excellent in both high-expressing (tonsil) and low-expressing (melanoma) tissues. Figure 1 illustrates images of tonsil staining using ultrasensitive IHC utilizing cell culture supernatant of exemplary clone 12H8.

[0089] Furthermore, this clone had no counterstaining in any of the other included tissues (liver, colon adenocarcinoma, breast carcinoma, carcinoid, normal colon, cerebellum, prostate, kidney and pancreas) and was free of any nonspecific background staining.

[0090] The recombinant monoclonal rabbit anti-human LAG-3 antibody was then subjected to further testing. Six-point titration using the IHC system EnVision FLEX (Agilent) was performed on the above-mentioned tissue array supplemented with three additional clinical tissues (non-small cell lung cancer (NSCLC), renal clear cell carcinoma (RCC), and malignant melanoma). A preliminary optimal concentration was obtained, which corresponded to tissue localization, morphological expression, staining intensity, and the best signal-to-noise ratio for both normal and tumor tissues using the reference antibody. The optimal version of this exemplary protocol was: LAG-3 Exemplary IHC Protocol Using LAG-3 Clone: Optimal antibody concentration: 1.75 μg / mL in S3022 antibody dilution buffer. Target retrieval in high pH TR buffer. Visualization system: EnVision FLEX+ with rabbit linker. It was decided as follows.

[0091] To confirm the optimal protocol, the LAG-3 antibody was tested on a small tissue package consisting of seven positive clinical tissues (2 lung NSCLC, 1 lung adenocarcinoma, 2 malignant melanoma, 2 renal RCC) and two negative clinical tissues (1 lung carcinoma, 1 melanoma).

[0092] LAG-3 multiplexing LAG-3 blockade is in several clinical trials, many of which use LAG-3 in combination with other targets, notably PD-1. Exemplary LAG-3 antibodies as provided herein were tested in duplicate to demonstrate the degree of colocalization with PD-1 and CD3, respectively, as illustrated in Figures 9-11, as described above.

[0093] LAG-3 expression on tumor-infiltrating lymphocytes (TIL) In clinical tissues, LAG-3 is expressed on a subpopulation of tumor-infiltrating lymphocytes (TILs) and not on tumor cells, as shown in Figures 12-14 (

[32] ).

[0094] Sequencing data Sequencing data for an exemplary anti-human LAG-3 antibody, clone 12H8: Heavy chain variable region: JPEG0007776446000001.jpg12170 The CDR regions are underlined. CDR1 amino acid (aa) residues 25-32, CDR2 aa residues 50-56, and CDR3 aa residues 95-101 of SEQ ID NO: 2. CDR regions according to numbering according to IMGT numbering (http: / / www.imgt.org / ).

[0095] Light chain variable region: JPEG0007776446000002.jpg11170 CDR regions are underlined. CDR1 aa residues 27-34, CDR2 aa residues 52-54, CDR3 aa residues 91-102 of SEQ ID NO: 3. CDR regions according to IMGT numbering (http: / / www.imgt.org / ).

[0096] The disclosures of each of the following references are incorporated herein by reference in their entirety.

[0097] References 1. Andrews, LP, et al., LAG3 (CD223) as a cancer immunotherapy target. Immunol Rev, 2017. 276(1): p. 80-96. 2. Huard, B., et al., Cellular expression and tissue distribution of the human LAG-3-encoded protein, an MHC class II ligand. Immunogenetics, 1994. 39(3): p. 213-7. 3. Workman, CJ, et al., LAG-3 regulates plasmacytoid dendritic cell homeostasis. J Immunol, 2009. 182(4): p. 1885-91. 4. Hemon, P., et al., MHC class II engagement by its ligand LAG-3 (CD223) contributes to melanoma resistance to apoptosis. J Immunol, 2011. 186(9): p. 5173-83. 5. Gandhi, M.K., et al., Expression of LAG-3 by tumor-infiltrating lymphocytes is coincident with the suppression of latent membrane antigen-specific CD8+ T-cell function in Hodgkin lymphoma patients. Blood, 2006. 108(7): p. 2280-9. 6. Chen, J. and Z. Chen, The effect of immune microenvironment on the progression and prognosis of colorectal cancer. Med Oncol, 2014. 31(8): p. 82. 7. Matsuzaki, J., et al., Tumor-infiltrating NY-ESO-1-specific CD8+ T cells are negatively regulated by LAG-3 and PD-1 in human ovarian cancer. Proc Natl Acad Sci U S A, 2010. 107(17): p. 7875-80. 8. Li, F.J., et al., Expression of LAG-3 is coincident with the impaired effector function of HBV-specific CD8(+) T cell in HCC patients. Immunol Lett, 2013. 150(1-2): p. 116-22. 9. Giraldo, N.A., et al., Orchestration and Prognostic Significance of Immune Checkpoints in the Microenvironment of Primary and Metastatic Renal Cell Cancer. Clin Cancer Res, 2015. 21(13): p. 3031-40. 10. Takaya, S., H. Saito, and M. Ikeguchi, Upregulation of Immune Checkpoint Molecules, PD-1 and LAG-3, on CD4+ and CD8+ T Cells after Gastric Cancer Surgery. Yonago Acta Med, 2015. 58(1): p. 39-44. 11. Yang, Z.Z., et al., Expression of LAG-3 defines exhaustion of intratumoral PD-1(+) T cells and correlates with poor outcome in follicular lymphoma. Oncotarget, 2017. 8(37): p. 61425-61439. 12. Norstrom, M.M., et al., Progression of benign prostatic hyperplasia is associated with pro-inflammatory mediators and chronic activation of prostate-infiltrating lymphocytes. Oncotarget, 2016. 7(17): p. 23581-93. 13. Deng, G., et al., BRAF mutation is frequently present in sporadic colorectal cancer with methylated hMLH1, but not in hereditary nonpolyposis colorectal cancer. Clin Cancer Res, 2004. 10(1 Pt 1): p. 191-5. 14. He, Y., et al., LAG-3 Protein Expression in Non-Small Cell Lung Cancer and Its Relationship with PD-1 / PD-L1 and Tumor-Infiltrating Lymphocytes. J Thorac Oncol, 2017. 12(5): p. 814-823. 15. Marcq, E., et al., Abundant expression of TIM-3, LAG-3, PD-1 and PD-L1 as immunotherapy checkpoint targets in effusions of mesothelioma patients. Oncotarget, 2017. 8(52): p. 89722-89735. 16. Burugu, S., et al., LAG-3+ tumor infiltrating lymphocytes in breast cancer: clinical correlates and association with PD-1 / PD-L1+ tumors. Ann Oncol, 2017. 28(12): p. 2977-2984. 17. Yanik, E.L., et al., Association of HIV Status With Local Immune Response to Anal Squamous Cell Carcinoma: Implications for Immunotherapy. JAMA Oncol, 2017. 3(7): p. 974-978. 18. Meng, Q., et al., Expansion of Tumor-reactive T Cells From Patients With Pancreatic Cancer. J Immunother, 2016. 39(2): p. 81-9. 19. Woo, S.R., et al., Immune inhibitory molecules LAG-3 and PD-1 synergistically regulate T-cell function to promote tumoral immune escape. Cancer Res, 2012. 72(4): p. 917-27. 20. Huang, R.Y., et al., LAG3 and PD1 co-inhibitory molecules collaborate to limit CD8+ T cell signaling and dampen antitumor immunity in a murine ovarian cancer model. Oncotarget, 2015. 6(29): p. 27359-77. 21. Petersen, K.H., J. Lohse, and L. Ramsgaard, Automated sequential chromogenic IHC double staining with two HRP substrates. PLoS One, 2018. 13(11): p. e0207867. 22. Baixeras, E., et al., Characterization of the lymphocyte activation gene 3-encoded protein. A new ligand for human leukocyte antigen class II antigens. J Exp Med, 1992. 176(2): p. 327-37.

[0098] Although numerous embodiments of the present invention have been described, it will be understood that various modifications can be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims. The scope of the patent claims at the time of filing is as follows: [Claim 1] A chimeric or recombinant antibody (Ab), or an antigen-binding fragment thereof, or a monomeric or dimeric antigen-binding protein, capable of specifically binding to a human lymphocyte activation gene 3 (LAG-3) polypeptide, The chimeric or recombinant antibody, or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein has the amino acid sequence GPPAAAPGHPLAPGPHPAAPSSWGPRPRR (SEQ ID NO: 1) or a chimeric or recombinant antibody (Ab), or an antigen-binding fragment thereof, or a monomeric or dimeric antigen-binding protein, which specifically binds to an epitope comprising: [Claim 2] antigen-binding fragments (Fab, or Ab fragments having only one constant domain and one variable domain of each of the Ab heavy and light chains); F(ab')2 (or Ab digested with pepsin to generate two fragments, F(ab')2 fragment and pFc' fragment), Fab' (single chain F(ab')2 fragment), single-chain variable fragments (scFv) or fusion proteins of the variable regions of the Ab heavy and light chains linked together with a linker peptide, optionally about 10 to about 25 amino acids in length; (scFv)2, or a single peptide chain with two variable heavy chain regions and two variable light chain regions resulting in a di-scFv or bi-scFv, or a tandem scFv; Minibodies or fusion proteins of Ab heavy and light chain variable regions linked together with alkyl groups, optionally containing methyl or ethyl groups; diabodies, or scFvs that contain a linker peptide that is too short for the two variable regions to fold together (optionally a linker peptide of about 5 amino acids) that causes the scFv to dimerize; triabodies or tetrabodies, or scFvs that contain a linker peptide that is too short for the two variable regions to fold together (optionally a linker peptide of about 1 or 2 amino acids) that causes the scFv to trimerize or tetramerize; single domain antibodies (dABs) (or single variable regions of Ab heavy or Ab light chains); multiple complementarity determining region (CDR) fragments, or Multispecific antibodies formed from two or more antibody fragments 2. The chimeric or recombinant antibody (Ab) of claim 1, or an antigen-binding fragment thereof, or a monomeric or dimeric antigen-binding protein, produced as or in the form of: [Claim 3] The sequence of the heavy chain variable region is QSVKESEGGLFKPTDTLTLTCTVSGIDLSSGILVWVRQAPGSGLEWIGGIDANGRAYYASWAKSRSTITRNTNENTVTLKMTSLTAADTATYFCAGGAWNIWGPGTLVTVSS (SEQ ID NO: 2) 3. The chimeric or recombinant antibody (Ab) of claim 1 or 2, or an antigen-binding fragment thereof, or a monomeric or dimeric antigen-binding protein, [Claim 4] The sequence of the light chain variable region is AQVLTQTPSPVSAAVGGTVTIKCQSSQSVYDSNTLAWFQQKPGQPPKLLMYSASTLAFGVPSRFSGSGSGTQFTLTISDLECADAATYYCLGSYDCSSVDCTAFGGGTEVVVK (SEQ ID NO: 3) 4. The chimeric or recombinant antibody (Ab) according to claim 1, or an antigen-binding fragment thereof, or a monomeric or dimeric antigen-binding protein, wherein: [Claim 5] The sequence of the heavy chain variable region is QSVKESEGGLFKPTDTLTLTCTVSGIDLSSGILVWVRQAPGSGLEWIGGIDANGRAYYASWAKSRSTITRNTNENTVTLKMTSLTAADTATYFCAGGAWNIWGPGTLVTVSS (SEQ ID NO: 2), and The sequence of the light chain variable region is AQVLTQTPSPVSAAVGGTVTIKCQSSQSVYDSNTLAWFQQKPGQPPKLLMYSASTLAFGVPSRFSGSGSGTQFTLTISDLECADAATYYCLGSYDCSSVDCTAFGGGTEVVVK (SEQ ID NO: 3) 5. The chimeric or recombinant antibody (Ab) according to claim 1, or an antigen-binding fragment thereof, or a monomeric or dimeric antigen-binding protein, wherein: [Claim 6] (a) the sequence of the heavy chain variable region comprises SEQ ID NO:2 with at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 conservative amino acid substitutions, and the heavy chain variable region is capable of specifically binding to a human LAG-3 polypeptide, amino acid (SEQ ID NO:1), or epitope when either unpaired (alone) or paired with a light chain variable region; (b) the sequence of the light chain variable region comprises SEQ ID NO:3 with at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 conservative amino acid substitutions, and the light chain variable region is capable of specifically binding to a human LAG-3 polypeptide, amino acid (SEQ ID NO:1), or epitope when either unpaired (alone) or paired with a heavy chain variable region; (c) the sequence of the heavy chain variable region has at least about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, or 95% sequence identity to SEQ ID NO:2; (d) the sequence of the light chain variable region has at least about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, or 95% sequence identity to SEQ ID NO:3; (e) the sequence of the heavy chain variable region and the amino acid sequence of SEQ ID NO: 2, when aligned using distance matrix alignment, have a Z-score of at least 8, of about 2 to about 8; or (f) when the sequence of the light chain variable region and the amino acid sequence of SEQ ID NO: 3 are aligned using distance matrix alignment, the sequence has a Z score of at least 8, and a Z score of about 2 to about 8; 6. The chimeric or recombinant antibody (Ab) according to any one of claims 1 to 5, or an antigen-binding fragment thereof, or a monomeric or dimeric antigen-binding protein. [Claim 7] (a) the heavy chain variable region comprises the three CDR1, CDR2 and CDR3 complementarity determining regions (CDRs) of SEQ ID NO:2, or CDR1 amino acid (aa) residues 25-32, CDR2 aa residues 50-56, and CDR3 aa residues 95-101 of SEQ ID NO:2; or (b) the light chain variable region comprises three CDR1, CDR2 and CDR3 complementarity determining regions (CDRs) of SEQ ID NO: 3, or CDR1 amino acid (aa) residues 27-34, CDR2 aa residues 52-54, and CDR3 aa residues 91-102 of SEQ ID NO: 3; 7. The chimeric or recombinant antibody (Ab) according to any one of claims 1 to 6, or an antigen-binding fragment thereof, or a monomeric or dimeric antigen-binding protein. [Claim 8] 8. The chimeric or recombinant antibody (Ab) or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein according to any one of claims 1 to 7, comprising: (a) a heavy chain variable region comprising three CDR1, CDR2, and CDR3 complementarity determining regions (CDRs) of SEQ ID NO: 2, or CDR1 amino acid (aa) residues 25 to 32, CDR2 aa residues 50 to 56, and CDR3 aa residues 95 to 101 of SEQ ID NO: 2; and (b) a light chain variable region comprising three CDR1, CDR2, and CDR3 complementarity determining regions (CDRs) of SEQ ID NO: 3, or CDR1 amino acid (aa) residues 27 to 34, CDR2 aa residues 52 to 54, and CDR3 aa residues 91 to 102 of SEQ ID NO: 3. [Claim 9] 9. The chimeric or recombinant antibody (Ab) or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein according to any one of claims 1 to 8, wherein the antibody heavy chain is an IgM, IgG, IgA or IgE isotype heavy chain and / or the light chain is a κ or λ light chain. [Claim 10] The sequence of the light chain constant region is GDPGAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC (SEQ ID NO: 4), or GDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC (SEQ ID NO: 5) 10. The chimeric or recombinant antibody (Ab) of claim 1, or an antigen-binding fragment thereof, or a monomeric or dimeric antigen-binding protein comprising: [Claim 11] (a) the sequence of the light chain constant region comprises SEQ ID NO:4 or SEQ ID NO:5 with at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve or more conservative amino acid substitutions, and the light chain constant region with the conservative amino acid substitutions is capable of specifically binding to or associating with a heavy chain constant region; or (b) the sequence of the light chain constant region has at least about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, or 95% sequence identity to SEQ ID NO:4 or SEQ ID NO:5, and the light chain constant region is capable of specifically binding to or associating with a heavy chain constant region; 11. The chimeric or recombinant antibody (Ab) according to any one of claims 1 to 10, or an antigen-binding fragment thereof, or a monomeric or dimeric antigen-binding protein. [Claim 12] The sequence of the heavy chain constant region is GQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK (SEQ ID NO: 6) 12. The chimeric or recombinant antibody (Ab) of claim 1, or an antigen-binding fragment thereof, or a monomeric or dimeric antigen-binding protein comprising: [Claim 13] (a) the sequence of the heavy chain constant region comprises SEQ ID NO: 6 with at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve or more conservative amino acid substitutions, and the heavy chain constant region with the conservative amino acid substitutions is capable of specifically binding to or associating with a light chain constant region; or (b) the sequence of the heavy chain constant region has at least about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 6, and the heavy chain constant region is capable of specifically binding to or associating with a light chain constant region; 13. The chimeric or recombinant antibody (Ab) according to any one of claims 1 to 12, or an antigen-binding fragment thereof, or a monomeric or dimeric antigen-binding protein. [Claim 14] The sequence of the antibody light chain is AQVLTQTPSPVSAAVGGTVTIKCQSSQSVYDSNTLAWFQQKPGQPPKLLMYSASTLAFGVPSRFSGSGSGTQFTLTISDLECADAATYYCLGSYDCSSVDCTAFGGGTEVVVKGDPGAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC (SEQ ID NO: 7), or AQVLTQTPSPVSAAVGGTVTIKCQSSQSVYDSNTLAWFQQKPGQPPKLLMYSASTLAFGVPSRFSGSGSGTQFTLTISDLECADAATYYCLGSYDCSSVDCTAFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC (SEQ ID NO: 8) 14. The chimeric or recombinant antibody (Ab) of claim 1, or an antigen-binding fragment thereof, or a monomeric or dimeric antigen-binding protein, comprising: [Claim 15] The sequence of the antibody heavy chain is QSVKESEGGLFKPTDTLTLTCTVSGIDLSSGILVWVRQAPGSGLEWIGGIDANGRAYYASWAKSRSTITRNTNENTVTLKMTSLTAADTATYFCAGGAWNIWGPGTLVTV SSGQPKAPSVFPLAPCCGDTPSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPE LLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK (SEQ ID NO: 9) 15. The chimeric or recombinant antibody (Ab) of claim 1, or an antigen-binding fragment thereof, or a monomeric or dimeric antigen-binding protein, comprising: [Claim 16] the chimeric or recombinant Ab is (a) a light chain as set forth in SEQ ID NO:7 operably linked to, paired with, associated with, or configured together with a heavy chain as set forth in SEQ ID NO:9, wherein the chimeric or recombinant Ab is capable of selectively binding to a human LAG-3 polypeptide; or (b) a light chain as set forth in SEQ ID NO:8 operably linked to, paired with, associated with, or configured together with a heavy chain as set forth in SEQ ID NO:9, wherein the chimeric or recombinant Ab is capable of selectively binding to a human LAG-3 polypeptide. 16. The chimeric or recombinant antibody (Ab) of claim 1, or an antigen-binding fragment thereof, or a monomeric or dimeric antigen-binding protein, comprising: [Claim 17] the chimeric or recombinant Ab is A light chain as set forth in SEQ ID NO:7 operably linked to, paired with, associated with, or configured together with a heavy chain as set forth in SEQ ID NO:9, wherein the chimeric or recombinant Ab is capable of selectively binding to a human LAG-3 polypeptide. 17. The chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein of claim 16, comprising: [Claim 18] the chimeric or recombinant Ab is A light chain as set forth in SEQ ID NO:8 operably linked to, paired with, associated with, or configured together with a heavy chain as set forth in SEQ ID NO:9, wherein the chimeric or recombinant Ab is capable of selectively binding to a human LAG-3 polypeptide. 17. The chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein of claim 16, comprising: [Claim 19] 19. The chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein of any one of claims 1 to 18, further comprising, or bound to, paired with, associated with, or covalently conjugated to, a detectable agent or binding moiety, Optionally, the detectable agent comprises an enzyme, biotin, a fluorescent or chemiluminescent label, a fluorophore, cyanine or sulfoindo-cyanine, Nile red, rhodamine, perylene, fluorenyl, coumarin, 7-methoxycoumarin (Mca), dabcyl, [2-(4-nitro-2,1,3-benzoxadiazol-7-yl)aminoethyl]trimethylammonium (NBD), Nile blue, Tamra or tetramethylrhodamine (TMR), boron dipyrromethane (BODIPY), HRP MAGENTA™ chromogen (Dako Omnis, Agilent), or a derivative thereof, a dye, a radioisotope, a quantum dot or photoluminescent aqueous nanocrystal, a hapten, or an antibody binding epitope or domain; and optionally, the enzyme is peroxidase, alkaline phosphatase, or β-galactosidase; and optionally, the peroxidase is horseradish peroxidase (HRP); and optionally, the hapten comprises biotin, theophylline, digoxigenin, carborane, fluorescein, or bromodeoxyuridine; and optionally, the dye comprises a cyanine dye; or Cy3 or Cy5; and optionally, the fluorophore comprises dansyl, fluorescein, or carboxyfluorescein (FAM) or 6-FAM; and optionally, the binding moiety comprises a glutathione-S-transferase (GST) or ligandin tag, a polyhistidine (poly-his) tag, a chitin-binding protein (CBP), a STREP-TAG™ or Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 11) peptide tag, a FLAG tag or a DYKDDDDK (SEQ ID NO: 12) peptide tag, or a maltose binding protein, A chimeric or recombinant antibody (Ab), or an antigen-binding fragment thereof, or a monomeric or dimeric antigen-binding protein. [Claim 20] 20. A recombinant nucleic acid encoding the chimeric or recombinant antibody (Ab) of any one of claims 1 to 19, or an antigen-binding fragment thereof, or a monomeric or dimeric antigen-binding protein. [Claim 21] (a) contacting a cell, tissue, or organ, or a portion of any of the above, with a chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or a monomeric or dimeric antigen-binding protein according to any one of claims 1 to 19, or encoded by a recombinant nucleic acid according to claim 20; and (b) detecting specific binding of the chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein, to a human LAG-3 polypeptide or a polypeptide containing GPPAAAPGHPLAPGPHPAAPSSWGPRPRR (SEQ ID NO: 1) in a cell, tissue, or organ, or a portion of any of the above; thereby detecting the presence of human LAG-3 protein in a cell, tissue, organ, or part of any of the above, comprising contacting the cell, tissue, or organ, or part of any of the above. 1. A method for detecting the presence of human LAG-3 protein in a cell, tissue, organ or part of any of the above, comprising: and optionally, the contacting step comprises the use of an immunohistochemistry (IHC) assay; and optionally, the method further comprises contacting the chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein, with a detectable agent to indicate or signal specific binding of the chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein to human LAG-3 protein; and optionally, the detectable agent specifically binds to a chimeric or recombinant antibody (Ab), or an antigen-binding fragment thereof, or a monomeric or dimeric antigen-binding protein; and optionally, the detectable agent is or comprises the chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or an antibody or antigen-binding fragment thereof that specifically binds to the monomeric or dimeric antigen-binding protein, or a secondary antibody that is bound to, paired with, associated with, or configured with human LAG-3 protein; and optionally, the detectable agent is or comprises an antibody or antigen-binding fragment thereof or a secondary antibody that specifically binds to a hapten or tag linked or conjugated to the chimeric or recombinant antibody (Ab) or antigen-binding fragment thereof, or the monomeric or dimeric antigen-binding protein; and optionally, the antibody or antigen-binding fragment or secondary antibody further comprises or is linked or conjugated to a second detectable agent or enzyme; and optionally, the enzyme is alkaline phosphatase, β-galactosidase, or peroxidase; and optionally, the antibody or antigen-binding fragment or secondary antibody further comprises or is linked or conjugated to biotin, a fluorescent or chemiluminescent label, a fluorophore, cyanine or sulfoindo-cyanine, Nile red, rhodamine, perylene, fluorenyl, coumarin, 7-methoxycoumarin (Mca), dabcyl, [2-(4-nitro-2,1,3-benzoxadiazol-7-yl)aminoethyl]trimethylammonium (NBD), Nile blue, Tamra or tetramethylrhodamine (TMR), HRP MAGENTA™ chromogen (Dako Omnis, Agilent), boron dipyrromethane (BODIPY), or derivatives thereof, a dye, a radioisotope, a quantum dot or photoluminescent aqueous nanocrystal, a hapten; and optionally, the dye comprises a cyanine dye, or Cy3 or Cy5; and optionally, the hapten comprises biotin, theophylline, digoxigenin, carborane, fluorescein, or bromodeoxyuridine; method. [Claim 22] 1. A method for detecting or diagnosing a LAG-3 protein-expressing cancer or cancer tissue containing therein LAG-3-expressing lymphocytes or LAG-3-expressing tumor-infiltrating lymphocytes (TILs), optionally wherein the TILs comprise tumor-infiltrating activated T cells, the method comprising the steps of detecting expression or presence of human LAG-3 protein in or on the surface of a cell, tissue or organ sample, or a portion thereof, by contacting the cell, tissue or organ sample with a chimeric or recombinant antibody according to any one of claims 1 to 17, or encoded by a recombinant nucleic acid according to claim 38 or any one of claims 1 to 21, and detecting whether the chimeric or recombinant antibody specifically binds to human LAG-3 protein in the cell, tissue or organ sample, or a portion thereof, wherein detection of specific binding indicates expression or presence of human LAG-3 protein in the cell, tissue or organ sample, or a portion thereof; and optionally, the cell is an activated T cell, a tumor-infiltrating activated T cell, or a tumor-infiltrating lymphocyte (TIL); and optionally, detecting said specific binding indicates expression or presence of human LAG-3 protein in the cell, tissue or organ sample, or a portion thereof, thereby diagnosing cancer; and optionally, the cancer is selected from the group consisting of renal cell carcinoma, renal clear cell carcinoma (RCC), adenocarcinoma, bladder cancer, urothelial carcinoma, breast cancer or mammary carcinoma or ductal carcinoma in situ (DCIS), carcinoid, Hodgkin's lymphoma, chronic lymphocytic leukemia, colorectal cancer, ovarian cancer, kidney cancer or renal cell carcinoma, liver cancer or hepatocellular carcinoma, stomach or gastric cancer, lymphoma or follicular lymphoma, prostate cancer, head and neck squamous cell carcinoma, lung cancer, non-small cell lung cancer (NSCLC), mesothelioma or malignant pleural mesothelioma, anal squamous cell carcinoma, pancreatic cancer, and melanoma or malignant melanoma; and optionally, the adenocarcinoma is lung adenocarcinoma or colon adenocarcinoma; and optionally, said detecting comprises using or performing an immunohistochemistry (IHC) assay or flow cytometry; and optionally, performing or using flow cytometry includes using a fluorescence activated cell sorter (FACS) or an impedance flow cytometer; and optionally, said cell, tissue or organ sample or portion thereof is or is derived from a biopsy from a patient. method.

Claims

1. A chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or a monomeric or dimeric antigen-binding protein, capable of specifically binding to a human lymphocyte activation gene 3 (LAG-3) polypeptide, comprising: The chimeric or recombinant antibody, or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein has the amino acid sequence GPPAAAPGHPLAPGPHPAAPSSWGPRPRR (SEQ ID NO: 1) or a peptide or polypeptide comprising the chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein, (a) a heavy chain variable region comprising the three CDR1, CDR2, and CDR3 complementarity determining regions (CDRs) of SEQ ID NO:2, or CDR1 amino acid (aa) residues 25-32, CDR2 aa residues 50-56, and CDR3 aa residues 95-101 of SEQ ID NO:2; and (b) a light chain variable region comprising the three CDR1, CDR2, and CDR3 complementarity determining regions (CDRs) of SEQ ID NO:3, or CDR1 amino acid (aa) residues 27-34, CDR2 aa residues 52-54, and CDR3 aa residues 91-102 of SEQ ID NO:

3. A chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or a monomeric or dimeric antigen-binding protein, comprising:

2. antigen-binding fragments (Fab, or Ab fragments having only one constant domain and one variable domain of each of the Ab heavy and light chains); F(ab') ２ (or digested with pepsin to give F(ab') ２ an Ab that produces two fragments, a pFc′ fragment and a pFc′ fragment; Fab' (F(ab') ２ single stranded fragments), single chain variable fragments (scFv) or fusion proteins of the variable regions of the Ab heavy and light chains linked together with a linker peptide, optionally from about 10 to about 25 amino acids in length; (scFv) ２ or a single peptide chain with two variable heavy and two variable light regions resulting in a di-scFv or bi-scFv, or a tandem scFv; Minibodies or fusion proteins of Ab heavy and light chain variable regions linked together with alkyl groups, optionally containing methyl or ethyl groups; diabodies, or scFvs that contain a linker peptide that is too short for the two variable regions to fold together (optionally a linker peptide of about 5 amino acids) that causes the scFv to dimerize, triabodies or tetrabodies, or scFvs that contain a linker peptide that is too short for the two variable regions to fold together (optionally a linker peptide of about 1 or 2 amino acids) that causes the scFv to trimerize or tetramerize; single domain antibodies (dABs) (or single variable regions of Ab heavy or Ab light chains); a plurality of complementarity determining region (CDR) fragments, or Multispecific antibodies formed from two or more antibody fragments 2. The chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein of claim 1, produced as or in the form of:

3. 3. A chimeric or recombinant antibody (Ab), or an antigen-binding fragment thereof, or a monomeric or dimeric antigen-binding protein, according to claim 1 or 2, wherein the antibody heavy chain is an IgM, IgG, IgA or IgE isotype heavy chain and / or the light chain is a K or λ light chain.

4. The sequence of the light chain constant region is GDPGAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC (SEQ ID NO: 4), or GDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC (SEQ ID NO: 5) 4. The chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein according to any one of claims 1 to 3, which is or comprises:

5. (a) the sequence of the light chain constant region comprises SEQ ID NO:4 or SEQ ID NO:5 with at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve or more conservative amino acid substitutions, and the light chain constant region with the conservative amino acid substitutions is capable of specifically binding to or associating with a heavy chain constant region; or (b) the sequence of the light chain constant region has at least about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, or 95% sequence identity to SEQ ID NO:4 or SEQ ID NO:5, and the light chain constant region is capable of specifically binding to or associating with a heavy chain constant region; A chimeric or recombinant antibody (Ab), or an antigen-binding fragment thereof, or a monomeric or dimeric antigen-binding protein according to any one of claims 1 to 4.

6. The sequence of the heavy chain constant region is GQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK (SEQ ID NO: 6) 6. The chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein according to any one of claims 1 to 5, which is or comprises:

7. (a) the sequence of the heavy chain constant region comprises SEQ ID NO: 6 with at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve or more conservative amino acid substitutions, and the heavy chain constant region with the conservative amino acid substitutions is capable of specifically binding to or associating with a light chain constant region; or (b) the sequence of the heavy chain constant region has at least about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 6, and the heavy chain constant region is capable of specifically binding to or associating with a light chain constant region; A chimeric or recombinant antibody (Ab), or an antigen-binding fragment thereof, or a monomeric or dimeric antigen-binding protein according to any one of claims 1 to 6.

8. The sequence of the antibody light chain is AQVLTQTPSPVSAAVGGTVTIKCQSSQSVYDSNTLAWFQQKPGQPPKLLMYSASTLAFGVPSRFSGSGSGTQFTLTISDLECADAATYYCLGSYDCSSVDCTAFGGGTEVVVKGDPGAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC (SEQ ID NO: 7), or AQVLTQTPSPVSAAVGGTVTIKCQSSQSVYDSNTLAWFQQKPGQPPKLLMYSASTLAFGVPSRFSGSGSGTQFTLTISDLECADAATYYCLGSYDCSSVDCTAFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC (SEQ ID NO: 8) 8. The chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein of any one of claims 1 to 7, comprising:

9. The sequence of the antibody heavy chain is QSVKESEGGLFKPTDTLTLTCTVSGIDLSSGILVWVRQAPGSGLEWIGGIDANGRAYYASWAKSRSTITRNTNENTVTLKMTSLTAADTATYFCAGGAWNIWGPGTLVTV SSGQPKAPSVFPLAPCCGDTPSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPE LLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK (SEQ ID NO: 9) 9. The chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein of any one of claims 1 to 8, comprising:

10. the chimeric or recombinant Ab is (a) a light chain as set forth in SEQ ID NO:7 operably linked to, paired with, associated with, or configured together with a heavy chain as set forth in SEQ ID NO:9, wherein the chimeric or recombinant Ab is capable of selectively binding to a human LAG-3 polypeptide; or (b) a light chain as set forth in SEQ ID NO:8 operably linked to, paired with, associated with, or configured together with a heavy chain as set forth in SEQ ID NO:9, wherein the chimeric or recombinant Ab is capable of selectively binding to a human LAG-3 polypeptide.

10. The chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein of any one of claims 1 to 9, comprising:

11. the chimeric or recombinant Ab is A light chain as set forth in SEQ ID NO:7 operably linked to, paired with, associated with, or configured together with a heavy chain as set forth in SEQ ID NO:9, wherein the chimeric or recombinant Ab is capable of selectively binding to a human LAG-3 polypeptide.

11. The chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein of claim 10, comprising:

12. the chimeric or recombinant Ab is A light chain as set forth in SEQ ID NO:8 operably linked to, paired with, associated with, or configured together with a heavy chain as set forth in SEQ ID NO:9, wherein the chimeric or recombinant Ab is capable of selectively binding to a human LAG-3 polypeptide.

11. The chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein of claim 10, comprising:

13. 13. The chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein of any one of claims 1 to 12, further comprising, or bound to, paired with, associated with, or covalently conjugated to, a detectable agent or binding moiety, Optionally, the detectable agent comprises an enzyme, biotin, a fluorescent or chemiluminescent label, a fluorophore, cyanine or sulfoindo-cyanine, Nile red, rhodamine, perylene, fluorenyl, coumarin, 7-methoxycoumarin (Mca), dabcyl, [2-(4-nitro-2,1,3-benzoxadiazol-7-yl)aminoethyl]trimethylammonium (NBD), Nile blue, Tamra or tetramethylrhodamine (TMR), boron dipyrromethane (BODIPY), HRP MAGENTA™ chromogen (Dako Omnis, Agilent, or a derivative thereof), a dye, a radioisotope, a quantum dot or photoluminescent aqueous nanocrystal, a hapten, or an antibody binding epitope or domain; and optionally, the enzyme is peroxidase, alkaline phosphatase, or β-galactosidase; and optionally, the peroxidase is horseradish peroxidase (HRP); and optionally, the hapten comprises biotin, theophylline, digoxigenin, carborane, fluorescein, or bromodeoxyuridine; and optionally, the dye comprises a cyanine dye; or Cy3 or Cy5; and optionally, the fluorophore comprises dansyl, fluorescein, or carboxyfluorescein (FAM) or 6-FAM; and optionally, the binding moiety comprises a glutathione-S-transferase (GST) or ligandin tag, a polyhistidine (poly-his) tag, a chitin-binding protein (CBP), a STREP-TAG™ or Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 11) peptide tag, a FLAG tag or a DYKDDDDK (SEQ ID NO: 12) peptide tag, or a maltose binding protein; A chimeric or recombinant antibody (Ab), or an antigen-binding fragment thereof, or a monomeric or dimeric antigen-binding protein.

14. A recombinant nucleic acid encoding the chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein of any one of claims 1 to 13.

15. (a) contacting a cell, tissue or organ, or a part of any of the above, with a chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or a monomeric or dimeric antigen-binding protein according to any one of claims 1 to 13, or encoded by a recombinant nucleic acid according to claim 14; and (b) detecting specific binding of the chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein, to a human LAG-3 polypeptide or a GPPAAAPGHPLAPGPHPAAPSSWGPRPRR (SEQ ID NO: 1)-containing polypeptide in a cell, tissue, or organ, or a portion of any of the above; thereby detecting the presence of human LAG-3 protein in a cell, tissue, organ, or part of any of the above, comprising contacting the cell, tissue, or organ, or part of any of the above.

1. An in vitro method for detecting the presence of human LAG-3 protein in a cell, tissue, organ, or part of any of the above, comprising: and optionally, the contacting step comprises the use of an immunohistochemistry (IHC) assay; and optionally, the method further comprises contacting the chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein, with a detectable agent to indicate or signal specific binding of the chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein to human LAG-3 protein; and optionally, the detectable agent specifically binds to a chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or a monomeric or dimeric antigen-binding protein; and optionally, the detectable agent is or comprises the chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or an antibody or antigen-binding fragment thereof that specifically binds to the monomeric or dimeric antigen-binding protein, or a secondary antibody that is bound to, paired with, associated with, or configured with human LAG-3 protein; and optionally, the detectable agent is or comprises an antibody or antigen-binding fragment thereof or a secondary antibody that specifically binds to a hapten or tag that is linked or conjugated to the chimeric or recombinant antibody (Ab) or antigen-binding fragment thereof, or the monomeric or dimeric antigen-binding protein; and optionally, the antibody or antigen-binding fragment or secondary antibody further comprises or is linked or conjugated to a second detectable agent or enzyme; and optionally, the enzyme is alkaline phosphatase, β-galactosidase, or peroxidase; and optionally, the antibody or antigen-binding fragment or secondary antibody further comprises or is linked or conjugated to biotin, a fluorescent or chemiluminescent label, a fluorophore, cyanine or sulfoindo-cyanine, Nile red, rhodamine, perylene, fluorenyl, coumarin, 7-methoxycoumarin (Mca), dabcyl, [2-(4-nitro-2,1,3-benzoxadiazol-7-yl)aminoethyl]trimethylammonium (NBD), Nile blue, Tamra or tetramethylrhodamine (TMR), HRP MAGENTA™ chromogen (Dako Omnis, Agilent), boron dipyrromethane (BODIPY, or derivatives thereof), a dye, a radioisotope, a quantum dot or photoluminescent aqueous nanocrystal, a hapten; and optionally, the dye comprises a cyanine dye, or Cy3 or Cy5; and optionally, the hapten comprises biotin, theophylline, digoxigenin, carborane, fluorescein, or bromodeoxyuridine; In vitro methods.

16. 14. An in vitro method for detecting or diagnosing a LAG-3 protein-expressing cancer or cancer tissue comprising therein LAG-3-expressing lymphocytes or LAG-3-expressing tumor-infiltrating lymphocytes (TILs), optionally wherein the TILs comprise tumor-infiltrating activated T cells, the method comprising the steps of detecting expression or presence of human LAG-3 protein in or on the surface of a cell, tissue, or organ sample, or a portion thereof, by contacting the cell, tissue, or organ sample with a chimeric or recombinant antibody according to any one of claims 1 to 11 or encoded by a recombinant nucleic acid according to claim 14, and detecting whether the chimeric or recombinant antibody specifically binds to human LAG-3 protein in the cell, tissue, or organ sample, or a portion thereof, wherein detection of specific binding indicates expression or presence of human LAG-3 protein in the cell, tissue, or organ sample, or a portion thereof; and optionally, the cell is an activated T cell, a tumor-infiltrating activated T cell, or a tumor-infiltrating lymphocyte (TIL); and optionally, said step of detecting specific binding indicates expression or presence of human LAG-3 protein in the cell, tissue or organ sample, or a portion thereof, thereby diagnosing cancer; and optionally, the cancer is selected from the group consisting of renal cell carcinoma, renal clear cell carcinoma (RCC), adenocarcinoma, bladder cancer, urothelial carcinoma, breast cancer or mammary carcinoma or ductal carcinoma in situ (DCIS), carcinoid, Hodgkin's lymphoma, chronic lymphocytic leukemia, colorectal cancer, ovarian cancer, kidney cancer or renal cell carcinoma, liver cancer or hepatocellular carcinoma, stomach or gastric cancer, lymphoma or follicular lymphoma, prostate cancer, head and neck squamous cell carcinoma, lung cancer, non-small cell lung cancer (NSCLC), mesothelioma or malignant pleural mesothelioma, anal squamous cell carcinoma, pancreatic cancer, and melanoma or malignant melanoma; and optionally, the adenocarcinoma is lung adenocarcinoma or colon adenocarcinoma; and optionally, said detecting comprises using or performing an immunohistochemistry (IHC) assay or flow cytometry; and optionally, performing or using flow cytometry includes using a fluorescence activated cell sorter (FACS) or an impedance flow cytometer; and optionally, said cell, tissue or organ sample or portion thereof is or is derived from a biopsy from a patient. In vitro methods.

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