Anti-human lag-3 antibodies and their use in immunohistochemistry (ihc)

Abstract

The present invention provides chimeric or recombinant antibodies (Abs) or antigen binding fragments thereof, or monomeric or dimeric antigen binding proteins that can specifically bind to human LAG-3 polypeptides, including human LAG-3 polypeptides expressed on the surface of lymphocytes, such as activated T cells that have infiltrated a tumor or tumor infiltrating lymphocytes (TILs); and methods of making and using the same. In alternative embodiments, 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 diagnostics by virtue of their ability to specifically bind to activated T cells that have infiltrated a tumor or tumor infiltrating lymphocytes, for example in immunohistochemistry (IHC), for example for the diagnosis and / or treatment of cancer, such as bladder cancer, urothelial cancer, breast cancer, lung cancer, non-small cell lung cancer, kidney cancer, renal clear cell carcinoma, and / or melanoma or malignant melanoma.

Description

[0001] Related applications

[0002] This Patent Cooperation Treaty (PCT) international application claims priority to U.S. Provisional Patent Application Serial No. (USSN) 63 / 030,873, filed May 27, 2020, pursuant to 35 U.SC §119(e). The entire contents of the above application are expressly incorporated herein by reference for all purposes. Technical Field

[0003] This invention generally relates to immunohistochemistry (IHC) and cancer diagnosis and treatment. In alternative embodiments, chimeric or recombinant antibodies (Abs) or antigen-binding fragments thereof, or monomeric or dimeric antigen-binding proteins, are provided that specifically bind to human LAG-3 peptides, including human LAG-3 peptides expressed on the surface of lymphocytes (e.g., activated T cells infiltrating tumors) or on tumor-infiltrating lymphocytes (TILs). In alternative embodiments, manufacturing products and kits are provided comprising chimeric or recombinant Abs or antigen-binding fragments thereof, or monomeric or dimeric antigen-binding proteins, or nucleic acids encoding them, or cells expressing them, as provided herein, and methods for manufacturing and using them. 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 for in vitro diagnostics, such as in vitro diagnostics via immunohistochemistry (IHC). In alternative embodiments, chimeric or recombinant antibodies (Abs) or their antigen-binding fragments, or monomeric or dimeric antigen-binding proteins, as provided herein, are used in IHC protocols to diagnose and / or treat cancers, such as bladder cancer, urothelial carcinoma, breast cancer, lung cancer, renal cell carcinoma, renal clear cell carcinoma (RCC), and / or melanoma or malignant melanoma, by virtue of their ability to specifically bind to activated T cells or tumor-infiltrating lymphocytes (TILs) that have infiltrated tumors. Background Technology

[0004] The 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 expressed on various lymphocyte types. It is a marker of T cell activation and is expressed on CD4 T cells and CD8 T cells 3 to 4 days after activation [1]. In addition, LAG-3 is expressed on activated natural killer (NK) cells and plasmacytoid dendritic cells [2].

[0005] LAG-3-expressing lymphocytes, such as tumor-infiltrating lymphocytes, have been found in a variety of human tumors, such as melanoma, NSCLC, colorectal cancer, breast cancer, hepatocellular carcinoma, follicular lymphoma, head and neck squamous cell carcinoma, and renal cell carcinoma. These lymphocytes are significantly associated with aggressive tumor progression and clinicopathological features [3-17]. Summary of the Invention

[0006] In alternative embodiments, chimeric or recombinant antibodies (Abs) or antigen-binding fragments (Ag) thereof, or monomeric or dimeric antigen-binding proteins, are provided, which are capable of specifically binding to human lymphocyte activation gene 3 (LAG-3) peptides, including human LAG-3 peptides expressed on the surface of lymphocytes such as activated T cells that have infiltrated tumors, or human LAG-3 peptides expressed on tumor-infiltrating lymphocytes (TILs).

[0007] The chimeric or recombinant antibody (Ab) or its antigen-binding fragment, or monomeric or dimer antigen-binding protein specifically binds to a peptide or polypeptide or epitope comprising the following amino acid sequence:

[0008] GPPAAAPGHPLAPGPHPAAPSSWGPRPRR (SEQ ID NO: 1).

[0009] In an alternative embodiment, the chimeric or recombinant antibody (Ab) or its antigen-binding fragment, or monomeric or dimer antigen-binding protein, is manufactured in the following or the following forms:

[0010] Antigen-binding fragments (Fab, or Ab fragments having only one constant domain and one variable domain for each of the Ab heavy and light chains),

[0011] F(ab')2 (or Ab fragments produced by pepsin digestion, resulting in two fragments: F(ab')2 and pFc'),

[0012] Fab' (a single strand of the F(ab')2 fragment),

[0013] Single-chain variable fragment (scFv) (or a fusion protein in which the variable regions of the Ab heavy and light chains are linked together with a linker peptide, optionally of about 10 to about 25 amino acids in length),

[0014] (scFv)2, or di-scFv or bi-scFv, or a single peptide chain having two variable heavy regions and two variable light regions to produce tandem scFv,

[0015] Microantibodies (or fusion proteins in which the variable regions of the heavy and light chains of an antibody are linked together with alkyl groups, optionally including methyl or ethyl alkyl groups),

[0016] Biantibodies (or scFvs, the scFv having a linker peptide (optionally about 5 amino acids) that is too short for the two variable regions to fold together, thereby forcing the scFv to dimerize), triple-chain antibodies, or quadruple-chain antibodies (or scFvs, the scFv having a linker peptide (optionally about 1 or 2 amino acids) that is too short for the two variable regions to fold together, thereby forcing the scFv to trimerize or tetramerize).

[0017] Single-domain antibody (dAB) (or a single variable region of the Ab heavy chain or Ab light chain),

[0018] Multiple complementarity determining region (CDR) segments, or

[0019] Multispecific antibodies are formed from two or more antibody fragments.

[0020] In alternative embodiments such as chimeric or recombinant antibodies (Abs) or their antigen-binding fragments, or monomeric or dimer antigen-binding proteins, as provided herein:

[0021] -The sequence of the heavy chain variable region is or contains:

[0022] QSVKESEGGLFKPTDTLTLTCTVSGIDLSSGILVWVRQAPGSGLEWIGGIDANGRAYYASWAKSRSTIRNTNENTVTLKMTSLTAADTATYFCAGGAWNIWGPGTLVTVSS(SEQ ID NO:2);

[0023] -The sequence of the light chain variable region is or contains:

[0024] AQVLTQTPSPVSAAVGGTVTIKCQSSQSVYDSNTLAWFQQKPGQPPKLLMYSASTLAFGVPSRFSGSGSGTQFTLTISDLECADAATYYCLGSYDCSSVDCTAFGGGTEVVVK (SEQ ID NO: 3);

[0025] -The sequence of the heavy chain variable region is or contains:

[0026] QSVKESEGGLFKPTDTLTLTCTVSGIDLSSGILVWVRQAPGSGLEWIGGIDANGRAYYASWAKSRSTITRNTNENTVTLKMTSLTAADTATYFCAGGAWNIWGPGTLVTVSS (SEQ ID NO: 2), and

[0027] The sequence of the light chain variable region is or contains:

[0028] AQVLTQTPSPVSAAVGGTVTIKCQSSQSVYDSNTLAWFQQKPGQPPKLLMYSASTLAFGVPSRFSGSGSGTQFTLTISDLECADAATYYCLGSYDCSSVDCTAFGGGTEVVVK (SEQ ID NO: 3);

[0029] -The sequence of the heavy chain variable region comprises SEQ ID NO:2 having at least one, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve conserved amino acid substitutions, wherein the heavy chain variable region is capable of specifically binding the human LAG-3 polypeptide, the amino acid (SEQ ID NO:1), or the epitope when unpaired (alone) or paired with the light chain variable region.

[0030] - The sequence of the light chain variable region comprises SEQ ID NO:3 having at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve conserved amino acid substitutions, wherein the light chain variable region is capable of specifically binding the human LAG-3 polypeptide, the amino acid (SEQ ID NO:1), or the epitope when unpaired (alone) or paired with the heavy chain variable region.

[0031] - 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 with SEQ ID NO:2;

[0032] - The sequence of the variable region of the light chain has at least about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90% or 95% sequence identity with SEQ ID NO:3;

[0033] - When alignment is performed using distance matrix alignment, the Z-score of the heavy chain variable region sequence and the amino acid sequence SEQ ID NO:2 is about 2 to about 8, or the Z-score is at least 8;

[0034] - When alignment is performed using distance matrix alignment, the Z-score of the light chain variable region sequence and the amino acid sequence SEQ ID NO:3 is about 2 to about 8, or the Z-score is at least 8;

[0035] -The heavy chain variable region includes: the three CDR1, CDR2 and CDR3 complementarity-determining regions (CDRs) of SEQ ID NO:2, or the CDR1 amino acid (aa) residues 25-32, CDR2 aa residues 50-56 and CDR3 aa residues 95-101 of SEQ ID NO:2;

[0036] - The light chain variable region includes: the three CDR1, CDR2 and CDR3 complementarity-determining regions (CDRs) of SEQ ID NO:3, or the CDR1 amino acid (aa) residues 27-34, CDR2 aa residues 52-54 and CDR3 aa residues 91-102 of SEQ ID NO:3;

[0037] - A chimeric or recombinant Ab or its antigen-binding fragment, or a monomeric or dimer antigen-binding protein, comprising: (a) a heavy chain variable region comprising: the three CDR1, CDR2 and CDR3 complementarity-determining regions (CDRs) of SEQ ID NO:2, or the CDR1 amino acid (aa) residues 25-32, the CDR2 aa residues 50-56 and the 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 the CDR1 amino acid (aa) residues 27-34, the CDR2 aa residues 52-54 and the CDR3 aa residues 91-102 of SEQ ID NO:3;

[0038] - 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;

[0039] - The sequence of the light chain constant region is or contains: GDPGAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC (SEQ ID NO:4), or

[0040] GDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC(SEQ ID NO:5);

[0041] - The sequence of the light chain constant region includes SEQ ID NO:4 or SEQ ID NO:5 having at least one, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve or more conserved amino acid substitutions, wherein the light chain constant region having said conserved amino acid substitutions is capable of specifically binding to or associating with the heavy chain constant region.

[0042] - 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 with 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;

[0043] -The sequence of the heavy chain constant region is or contains:

[0044] GQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTA RPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK(SEQ ID NO:6);

[0045] -The sequence of the heavy chain constant region comprises SEQ ID NO:6 having at least one, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve or more conserved amino acid substitutions, wherein the heavy chain constant region having said conserved amino acid substitutions is capable of specifically binding to or associating with said light chain constant region.

[0046] 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 with SEQ ID NO:6, wherein the heavy chain constant region is capable of specifically binding to or associating with the light chain constant region;

[0047] - The antibody light chain sequence includes (the underlined part is the variable region):

[0048] AQVLTQTPSPVSAAVGGTVTIKCQSSQSVYDSNTLAWFQQKPGQPPKLLMYSASTLAFGVPSRFSGSG SGTQFTLTISDLECADAATYYCLGSYDCSSVDCTAFGGGTEVVVK GDPGAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC(SEQID NO:7), or

[0049] AQVLTQTPSPVSAAVGGTVTIKCQSSQSVYDSNTLAWFQQKPGQPPKLLMYSASTLAFGVPSRFSGSG SGTQFTLTISDLECADAATYYCLGSYDCSSVDCTAFGGGTEVVVK GDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC(SEQID NO:8);

[0050] - The sequence of the antibody heavy chain includes (the underlined part is the variable region): QSVKESEGGLFKPTDTLTLTCTVSGIDL SSGILVWVRQAPGSGLEWIGGIDANGRAYYASWAKSSRSTITRNTNENTVTLKMTSLTAADTATYFCAGGAWNIWGP GTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTA RPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK(SEQ ID NO:9),-

[0051] Chimeric or recombinant antibodies (Abs) or their antigen-binding fragments, or monomeric or dimer antigen-binding proteins, further comprise a detectable reagent or binding moiety, or are bound, paired, associated with, or covalently conjugated to said detectable reagent or binding moiety;

[0052] - The detectable reagents include: enzymes, biotin, fluorescent or chemiluminescent labels, fluorophores, cyanines such as sulfonylindocyanine, Nile red, rhodamine, perylene, fluorene, coumarin, 7-methoxycoumarin (Mca), dabcyl, [2-(4-nitro-2,1,3-benzoxadiazol-7-yl)aminoethyl]trimethylammonium (NBD), Nile blue, Tamra or tetramethylrhodamine (TMR), boron-dipyrrole methylene (BODIPY), HRP MAGENTA TM Chromogens (Dako Omnis, Agilent) or their derivatives, dyes, radioactive isotopes, quantum dots or photoluminescent aqueous nanocrystals, haptens or antibody-binding epitopes or domains;

[0053] - The enzyme is a peroxidase, alkaline phosphatase, or β-galactosidase, and the peroxidase may be horseradish peroxidase (HRP).

[0054] - The hapten includes biotin, theophylline, digoxin, carborane, fluorescein, or bromodeoxyuridine;

[0055] -The dyes include cyanine dyes; or Cy3 or Cy5;

[0056] - The fluorophore includes dansyl, fluorescein, or carboxyfluorescein (FAM) or 6-FAM; and / or

[0057] - The binding moiety includes: a glutathione S-transferase (GST) or ligand protein tag, a polyhistidine (poly-his) tag, a chitin-binding protein (CBP), and a STREP-TAG. TM Or the Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO:11) peptide tag, FLAG tag, or DYKDDDDK (SEQ ID NO:12) peptide tag, or maltose-binding protein.

[0058] In an alternative embodiment, a recombinant nucleic acid is provided that encodes a chimeric or recombinant antibody (Ab) or its antigen-binding fragment, or a monomeric or dimer antigen-binding protein, as provided herein.

[0059] In alternative implementations of recombinant nucleic acids as provided herein:

[0060] - The recombinant nucleic acid further includes and is operatively linked to a transcriptional regulatory element, and the transcriptional regulatory element may include a promoter, or the promoter may be an inducible promoter or a constitutive promoter;

[0061] - The recombinant nucleic acid further comprises a sequence encoding an additional protein or peptide portion or domain, and the additional protein or peptide portion or domain may include a purification portion or domain for assisting in the purification or isolation of a chimeric or recombinant antibody (Ab) or its antigen-binding fragment, or monomeric or dimer antigen-binding protein encoded by the recombinant nucleic acid.

[0062] - The additional protein or peptide portion or domain includes: glutathione S-transferase (GST) or ligand protein tag, polyhistidine tag, chitin-binding protein (CBP), STREP-TAG TM Or Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO:11) peptide tag, FLAG tag, or DYKDDDDK (SEQ ID NO:12) peptide tag, or maltose-binding protein; and / or

[0063] - The recombinant nucleic acid further includes a sequence encoding a protease cleavage site located between the purified portion or domain and a sequence encoding a chimeric or recombinant antibody (Ab) or its antigen-binding fragment, or a monomeric or dimer antigen-binding protein.

[0064] In alternative embodiments, chimeric or recombinant antibodies (Abs) as provided herein comprise:

[0065] (a) A light chain as shown in SEQ ID NO:7, said light chain being operatively bound, paired, associated, or conjugated with a heavy chain as shown in SEQ ID NO:9, wherein said chimeric or recombinant Ab is capable of selectively binding to human LAG-3 polypeptide; or

[0066] (b) A light chain as shown in SEQ ID NO:8, said light chain being operatively bound, paired, associated, or conjugated with a heavy chain as shown in SEQ ID NO:9, wherein said chimeric or recombinant Ab is capable of selectively binding human LAG-3 polypeptide.

[0067] In alternative embodiments, expression cassettes, vectors, recombinant viruses, artificial chromosomes, granules, or plasmids comprising recombinant nucleic acids as provided herein are provided.

[0068] In an alternative embodiment, a cell is provided comprising a chimeric or recombinant antibody (Ab) or an 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, viscera, or plasmid, as provided herein, and the cell may be a bacterial, fungal, mammalian, yeast, insect, avian, or plant cell.

[0069] In an alternative embodiment, a method is provided for generating a polyclonal antibody or a polyclonal immune serum that specifically binds to or binds to a human lymphocyte activation gene 3 (LAG-3) polypeptide, optionally specifically binding to a LAG-3 polypeptide expressed on the surface of tumor-infiltrating lymphocytes such as tumor-infiltrating activated T cells. The method comprises administering a peptide or polypeptide or epitope comprising the following amino acid sequence to a mammalian or avian species, or immunizing the mammalian or avian species with the peptide or polypeptide or epitope:

[0070] GPPAAAPGHPLAPGPHPAAPSSWGPRPRR (SEQ ID NO: 1).

[0071] In an alternative embodiment, a method is provided for detecting the presence of human LAG-3 protein in or on cells (optionally lymphocytes, or tumor-infiltrating lymphocytes, such as tumor-infiltrating activated T cells), tissues, organs, or any portion thereof, the method comprising: (a) contacting the cells, tissues, or organs, or any portion thereof, with a chimeric or recombinant antibody (Ab) encoded as provided herein or by a recombinant nucleic acid as provided herein, or an antigen-binding fragment thereof, or a monomeric or dimer antigen-binding protein; and (b) detecting the specific binding of the chimeric or recombinant antibody (Ab) or its antigen-binding fragment thereof, or the monomeric or dimer antigen-binding protein, to a human LAG-3 polypeptide or a polypeptide comprising GPPAAAPGHPLAPGPHPAAPSSWGPRPRR (SEQ ID NO:1) in the cells, tissues, or organs, or any portion thereof, thereby detecting the presence of human LAG-3 protein in the cells, tissues, organs, or any portion thereof, including contacting the cells, tissues, or organs, or any portion thereof.

[0072] In alternative embodiments of the methods provided herein for detecting the presence of human LAG-3 protein in cells (optionally lymphocytes, or tumor-infiltrating lymphocytes, such as tumor-infiltrating activated T cells), tissues, organs, or portions thereof:

[0073] - The contact included assays using immunohistochemistry (IHC);

[0074] - The method further includes contacting a chimeric or recombinant antibody (Ab) or its antigen-binding fragment, or monomeric or dimer antigen-binding protein, with a detectable reagent to indicate or signal the specific binding of the chimeric or recombinant antibody (Ab) or its antigen-binding fragment, or monomeric or dimer antigen-binding protein to human LAG-3 protein;

[0075] - The detectable reagent specifically binds to a chimeric or recombinant antibody (Ab) or its antigen-binding fragment, or monomeric or dimer antigen-binding protein; or, the detectable reagent is or contains an antibody or its antigen-binding fragment or secondary antibody, wherein the antibody or its antigen-binding fragment or secondary antibody specifically binds to a chimeric or recombinant antibody (Ab) or its antigen-binding fragment, or monomeric or dimer antigen-binding protein, and the chimeric or recombinant antibody or its antigen-binding fragment, or monomeric or dimer antigen-binding protein binds, pairs, or associates with human LAG-3 protein; or, the detectable reagent is or contains an antibody or its antigen-binding fragment or secondary antibody, wherein the antibody or its antigen-binding fragment or secondary antibody specifically binds to a hapten or tag, and the hapten or tag is linked or conjugated to the chimeric or recombinant antibody (Ab) or its antigen-binding fragment, or monomeric or dimer antigen-binding protein; and / or

[0076] - The antibody or antigen-binding fragment or secondary antibody further comprises or is linked to or conjugated to a second detectable reagent or enzyme, and the enzyme may be alkaline phosphatase, β-galactosidase, or peroxidase; or, the antibody or antigen-binding fragment or secondary antibody further comprises or is linked to or conjugated to biotin, fluorescent or chemiluminescent labeling, fluorophore, cyanine such as sulfonylindo-cyanine, Nile red, rhodamine, perylene, fluorene, coumarin, 7-methoxycoumarin (Mca), dabcyl, [2-(4-nitro-2,1,3-benzoxadiazol-7-yl)aminoethyl]trimethylammonium (NBD), Nile blue, HRP MAGENTA TM The chromogen (Dako Omnis, Agilent), Tamra or tetramethylrhodamine (TMR), boron-dipyrrole methylene (BODIPY) or its derivatives, dyes, radioactive isotopes, quantum dots or photoluminescent aqueous nanocrystals, haptens; and the dyes may include cyanine dyes, or Cy3 or Cy5; or the haptens may include biotin, theophylline, digoxin, carborane, fluorescein or bromodeoxyuridine.

[0077] In an alternative embodiment, a method is provided for detecting or diagnosing cancer expressing LAG-3 protein, or cancer tissue containing lymphocytes expressing LAG-3, or tumor-infiltrating lymphocytes expressing LAG-3 such as tumor-infiltrating activated T cells, the method comprising: 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 encoded as provided herein or 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, and the detection of specific binding indicating the expression or presence of human LAG-3 protein in the cell, tissue, or organ sample or a portion thereof.

[0078] In alternative embodiments of the methods provided herein for detecting or diagnosing cancers expressing the LAG-3 protein, or cancerous tissue containing lymphocytes expressing LAG-3, or tumor-infiltrating lymphocytes expressing LAG-3 such as tumor-infiltrating activated T cells:

[0079] - The cells are activated T cells, activated T cells that have infiltrated the tumor, or tumor-infiltrating lymphocytes (TILs);

[0080] - The specific binding detection indicates the expression or presence of human LAG-3 protein in the cell, tissue, or organ sample or a portion thereof, thereby diagnosing or detecting the cancer;

[0081] The cancers are selected from the group consisting of: renal cell carcinoma, clear cell renal cell carcinoma (RCC), adenocarcinoma, bladder cancer, urothelial carcinoma, breast cancer or malignant breast tumor or ductal carcinoma in situ (DCIS), carcinoid tumor, Hodgkin's lymphoma, chronic lymphocytic leukemia, colorectal cancer, ovarian cancer, renal cell carcinoma or kidney cancer, liver cancer or hepatocellular carcinoma, stomach cancer / 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 may be lung adenocarcinoma or colon adenocarcinoma;

[0082] - The detection includes the use or performance of immunohistochemistry (IHC) assays or flow cytometry;

[0083] - The use of flow cytometry includes the use of a fluorescence-activated cell sorter (FACS) or impedance flow cytometry; and / or

[0084] - The cell, tissue, or organ sample or a portion thereof is or is derived from a patient's biopsy.

[0085] In alternative embodiments, methods for treating, improving, or preventing cancer are provided, the methods comprising first detecting or diagnosing cancer in an individual in need using methods as provided herein, and then treating the individual in need.

[0086] In alternative embodiments of methods for treating, improving, or preventing cancer, the cancer is selected from the group consisting of: bladder cancer, urothelial carcinoma, breast cancer or malignant breast tumor or ductal carcinoma in situ (DCIS), lung cancer, non-small cell lung cancer (NSCLC), renal cell carcinoma, clear cell renal carcinoma (RCC), adenocarcinoma, malignant breast tumor or ductal carcinoma in situ (DCIS), carcinoid tumor, Hodgkin's lymphoma, chronic lymphocytic leukemia, colorectal cancer, ovarian cancer, kidney cancer, liver cancer or hepatocellular carcinoma, 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.

[0087] In alternative embodiments, chimeric or recombinant antibodies (Abs) encoded by recombinant nucleic acids as provided herein or encoded herein, or their antigen-binding fragments, or monomeric or dimeric antigen-binding proteins, are provided for the detection or diagnosis of cancer, or cancerous tissue containing lymphocytes expressing LAG-3, or tumor-infiltrating lymphocytes expressing LAG-3 such as tumor-infiltrating activated T cells, or for the treatment, improvement, or prevention of said cancer.

[0088] In alternative embodiments, chimeric or recombinant antibodies (Abs) encoded as provided herein or by recombinant nucleic acids as provided herein, or antigen-binding fragments thereof, or monomeric or dimer antigen-binding proteins, are provided for use in detecting or diagnosing cancer, or cancerous tissue containing lymphocytes expressing LAG-3, or tumor-infiltrating lymphocytes expressing LAG-3 such as tumor-infiltrating activated T cells, or for treating, improving, or preventing said cancer.

[0089] In alternative embodiments, kits are provided comprising chimeric or recombinant antibodies (Abs) encoded by recombinant nucleic acids as provided herein or encoded herein, or antigen-binding fragments thereof, or monomeric or dimer antigen-binding proteins. In alternative embodiments, kits as provided herein contain components required for immunohistochemical (IHC) assays; and / or instructions for use in practicing the methods as provided herein. In alternative embodiments of kits as provided herein, the chimeric or recombinant antibodies (Abs), their antigen-binding fragments, or monomeric or dimer antigen-binding proteins are substantially purified or isolated.

[0090] In alternative embodiments, a manufacturing product is provided comprising a chimeric or recombinant antibody (Ab) or its antigen-binding fragment, or monomeric or dimer antigen-binding protein, encoded by a recombinant nucleic acid as provided herein or as provided herein. In alternative embodiments, the manufacturing product comprises or is fabricated into or manufactured into a slide, well, chip, biochip, array, tray, dish, or microtiter plate or dish. In alternative embodiments of the manufacturing product, the chimeric or recombinant antibody (Ab), its antigen-binding fragment, or monomeric or dimer antigen-binding protein is substantially purified or isolated, or in the form of unpurified or partially purified culture supernatant.

[0091] In alternative embodiments, phages or phage particles are provided that contain or express on their surface a chimeric or recombinant antibody (Ab) or an antigen-binding fragment thereof, or a monomeric or dimer antigen-binding protein, as provided herein or encoded by recombinant nucleic acids as provided herein.

[0092] Details of one or more exemplary embodiments of the invention are set forth in the accompanying drawings and the following description. Other features, objects, and advantages of the invention will become apparent from the specification, drawings, and claims.

[0093] All publications, patents, and patent applications cited herein are expressly incorporated herein by reference in their entirety for all purposes. Attached Figure Description

[0094] The patent or application documents contain at least one color drawing. A copy of the color drawing disclosed in this patent or patent application will be provided by the patent office upon request and payment of the necessary fees.

[0095] The accompanying drawings are illustrations of exemplary embodiments provided herein and are not intended to limit the scope of the invention as covered by the claims.

[0096] The accompanying drawings are described in detail here.

[0097] Figure 1 Images of tonsils stained with ultrasensitive IHC using cell culture supernatant of exemplary clone 12H8 are shown.

[0098] Figure 2 Images of IHC staining of renal cell carcinoma (RCC) using the standard visualization system EnVision FLEX+ with DAB chromogen (brown) and exemplary clone 12H8 are shown. The images show LAG-3 staining of activated T cells in the lymphocyte-infiltrating region (tumor microenvironment (TME)) of tumor cells; the images show that LAG-3 clone 12H8 does not stain tumor cells, and the staining of activated T cells exhibits three morphological staining patterns: cytoplasmic staining (1), membrane staining (2), and Golgi apparatus staining (3).

[0099] Figures 3A to 3B , Figures 4A to 4B , Figures 5A to 5B , Figures 6A to 6B , Figures 7A to 7B and Figures 8A to 8B This demonstrates the use of the EnVision FLEX+ standard visualization system with DAB chromophore (brown) for malignant melanoma ( Figures 3A to 3B and Figures 4A to 4B ), lung NSCLC ( Figure 5A , Figure 5B , Figure 6A and Figure 6B ), lung adenocarcinoma ( Figure 7A and Figure 7B ) and renal cell carcinoma (RCC) Figure 8A and Figure 8B IHC staining images of tumor tissue samples, and: Figure 3A , Figure 4A , Figure 5A , Figure 6A , Figure 7A and Figure 8A It shows the same as Figure 3B , Figure 4B , Figure 5B , Figure 6B , Figure 7B and Figure 8B The exemplary LAG-3 antibody clone 12H8 is shown in comparison to the reference LAG-3 antibody clone 17B4 (Novus Bio); all images show LAG-3 staining of activated T cells in lymphocyte-infiltrating regions within tumor cells within the TME; the images show that when comparing Figure A and Figure B for different tumor types, neither the reference LAG-3 antibody clone 17B4 nor the exemplary clone 12H3 stained the tumor cells, and the exemplary clone 12H8 matched or even exceeded the reference clone 17B4 in terms of specific staining intensity, absence of unwanted background staining, and number of stained cells.

[0100] Figures 9 to 11 Double IHC staining of a tissue sample from a normal tonsil is shown:

[0101] Figure 9 Tumor tissue samples of squamous cell carcinoma (SQC) of the lung are shown. Figure 10 Tumor tissue samples of renal cell carcinoma (RCC) Figure 11 The double IHC staining method follows the scheme of Petersen et al., 2018

[18] : using EnVision FLEX+ in a sequential manner. TM The system, wherein the exemplary LAG-3 antibody and clone 12H8 (HRP DAB chromogen) constitute the first layer, followed by a sulfate blocking step, and an additional EnVision FLEX+ is added on top with PD1 antibody and clone NAT105 (magenta chromogen). TM staining layer; and Figure 9 This shows that LAG-3 and PD1 T cell markers are co-localized in the activated T cell subset within the germinal center of the tonsils. Other cells belonging to the activated T cell population are stained only by PD1. B lymphocytes in the germinal center are negative for both LAG-3 and PD1 antibodies, demonstrating the specificity of these two antibodies: (Red arrow: strong LAG-3 positivity, DAB obscures magenta PD1. Green arrow: weakly positive LAG-3, where the DAB membrane and DAB Golgi apparatus, as well as PD1 magenta staining, are visible). PD1-positive, LAG-3-negative T cells: (orange arrow).

[0102] Figure 10 and Figure 11The above method was used to perform double staining of LAG-3 and PD1 on the following lung non-small cell lung cancer (NSCLC). Figure 10 ) and clear cell renal cell carcinoma (RCC) Figure 11 LAG-3 and PD1 (a T-cell marker) are co-localized in activated T cells within the tumor microenvironment (TME) of both tumors; some of these T cells are positive for PD1 only, while all squamous cell tumors in clear cells of lung and kidney tumors are negative; these findings confirm the specificity of the exemplary LAG-3 antibody clone 12H8; for Figure 10 and Figure 11 (Red arrow: LAG-3 and PD1 co-localize in activated T cells within the tumor microenvironment (TME); Orange arrow: LAG-3 negative / PD1 positive T cells;) Figure 10 In the middle: Blue arrows: squamous tumor cells that are negative for LAG-3 and PD1; Figure 11 In the middle, blue arrows indicate RCC tumor cells that are negative for LAG-3 and PD1.

[0103] Figure 12 , Figure 13 and Figure 14 A normal tonsil is shown. Figure 12 Tumor tissue samples of squamous cell carcinoma of the lung (SQC) Figure 13 ) and renal cell carcinoma (RCC) ( Figure 14 Triple IHC staining; the triple IHC staining utilizes a high-sensitivity system with the above-described sulfuric acid blocking step between three layers of the following antibodies: exemplary LAG-3 antibody clone 12H8 (HRP DAB chromogen), polyclonal (Dako GA503) CD3 antibody (HRP MAGENTA) TM (Dako Omnis, Agilent) chromogen), CK-pan antibody clone AE1 / AE1 (yellow substrate); and Figure 12The images show the colocalization of LAG-3 and CD3 T cell markers in activated T cell subsets and T cells migrating through the tonsillar region beneath the tonsillar crypt epithelium; many T cells express only the CD3 marker, consistent with LAG-3 expression only in activated T cells; many other B lymphocytes in this region are neither stained by LAG-3 nor by CD3; CK-pan antibody stains the epithelial cells but not the lymphocytes; the images show the localization of LAG-3 positive T cells and their colocalization with the CD3 T cell marker, confirming the specificity of the LAG-3 antibody: (red arrow: strong LAG-3 positivity, DAB obscures magenta CD3; green arrow: weakly positive LAG-3, where the DAB membrane and DAB Golgi apparatus and CD3 magenta staining are visible); CD3 positive, LAG-3 negative T cells: (orange arrow); epithelial cells positive for CK-pan and negative for LAG-3 (blue arrow).

[0104] Figure 13 and Figure 14 This study illustrates the co-localization of LAG-3 and CD3 T cell markers in T cell subsets of tumor-infiltrating lymphocytes in the tumor-emerging lymphocytes (TME) of renal RCC tumors and lung SQC tumors. A minority of T cells expressed only CD3, and all renal and lung tumor cells were stained only with CK pan antibody, visually confirming that the exemplary LAG-3 antibody is confined to T cell subsets and not expressed on tumor cells; red arrows indicate activated T cells in the TME, double-positive for both LAG-3 and CD3; blue arrows indicate RCC tumors (…). Figure 13 ) and squamous cell carcinoma ( Figure 14 Cells are positive only for CK pan; green arrow: activated LAG-3 and CD3 double-positive T cells around tumor cells; orange arrow: T cells that are negative for LAG-3 and positive for CD3.

[0105] The same reference symbols in each figure represent the same element. Detailed Implementation

[0106] In alternative embodiments, chimeric or recombinant antibodies (Abs) or antigen-binding fragments thereof, or monomeric or dimeric antigen-binding proteins, are provided that specifically bind to human LAG-3 peptides, including human LAG-3 peptides expressed on the surface of lymphocytes such as activated T cells infiltrating tumors, or expressed on tumor-infiltrating lymphocytes (TILs). In alternative embodiments, manufacturing products and kits comprising chimeric or recombinant Abs or antigen-binding fragments thereof, or monomeric or dimeric antigen-binding proteins, or nucleic acids encoding them, as provided herein, are provided, along with methods for manufacturing and using them. In alternative embodiments, chimeric or recombinant antibodies (Abs) or their antigen-binding fragments, or monomeric or dimeric antigen-binding proteins, as provided herein, are used for in vitro diagnostics by their ability to specifically bind to activated T cells (e.g., including tumor-infiltrating lymphocytes (TILs)) that have infiltrated tumors, such as by immunohistochemistry (IHC), for example in IHC protocols for the diagnosis, detection, and / or treatment of cancers such as bladder cancer, urothelial carcinoma, breast cancer or malignant breast tumors or ductal carcinoma in situ (DCIS), carcinoid tumors, Hodgkin's lymphoma, chronic lymphocytic leukemia, colorectal cancer, ovarian cancer, non-small cell lung cancer (NSCLC), renal cancer or renal cell carcinoma or malignant renal tumors, clear cell renal carcinoma (RCC), mesothelioma or malignant pleural mesothelioma, squamous cell carcinoma, anal squamous cell carcinoma, pancreatic cancer, and melanoma or malignant melanoma. Therefore, chimeric or recombinant antibodies (Abs) or their antigen-binding fragments, or monomeric or dimeric antigen-binding proteins, as provided herein, can be used as companion diagnostics for the diagnosis and treatment of cancer by specifically staining for TILs.

[0107] Expression of recombinant chimeric antibodies

[0108] In alternative embodiments, chimeric and / or recombinant antibodies (Abs), their antigen-binding fragments, or monomeric or dimeric antigen-binding proteins, such as those provided herein, including exemplary chimeric or recombinant anti-human LAG-3Abs containing a heavy chain variable region SEQ ID NO:2 and a light chain variable region SEQ ID NO:3, with or without a signal peptide, can be expressed as recombinant Abs using, for example, plasmids or any expression vector encoding the corresponding heavy and light chains, or the heavy and light chains can be encoded in separate expression vectors.

[0109] In some implementations, the heavy and light chains can be derived from any plasmid, granule, recombinant virus, or equivalent vector, for example, from pTT5. TM Expression via vector (one or more) (National Research Council Canada, NRC-CNRC, Canada) or equivalent (cis or trans).

[0110] In alternative embodiments, expression vectors (e.g., plasmids) containing one or more exemplary nucleic acids encoding Abs as provided herein are expressed in an in vitro expression system or in cultured tissues, cells, or organoids, which may be bacterial, fungal, mammalian, yeast, insect, or plant cell expression systems, or hybrid or synthetic expression systems. For example, exemplary nucleic acids encoding Abs may be expressed in human embryonic kidney (HEK) cells such as HEK293-6E cells. In alternative embodiments, one or more vectors expressing exemplary nucleic acids encoding Abs, such as exemplary heavy chains and / or light chains, are either free or chromosomally integrated into stable cell lines, for example, capable of synthesizing, optionally induced to synthesize, said heavy chains and / or light chains.

[0111] In alternative embodiments, nucleic acids encoding chimeric or recombinant antibodies as provided herein are provided. Nucleic acids as provided herein can be prepared, isolated, and / or manipulated, for example, by cloning and expression of cDNA libraries, PCR amplification of messenger or genomic DNA, etc. Nucleic acids used to practice the embodiments provided herein, whether RNA, cDNA, genomic DNA, vectors, viruses, or hybrids thereof, can be isolated from a variety of sources, genetically engineered, amplified, and / or recombinantly expressed / produced. Recombinant polypeptides derived from these nucleic acids can be isolated or cloned individually 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.

[0112] Alternatively, these nucleic acids can be synthesized in vitro using well-known chemical synthesis techniques, as described in, for example, 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; and U.S. Patent No. 4,458,066.

[0113] Techniques for manipulating nucleic acids, such as subcloning, labeled probes (e.g., random primer labeling using Klenow polymerase, nick translation, amplification), sequencing, hybridization, etc., are described in detail in scientific and patent literature, see, for example, 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).

[0114] Another useful method for obtaining and manipulating nucleic acids for practicing the embodiments provided herein includes screening and recloning insert sequences isolated or amplified from, for example, genomic clones or cDNA clones. Nucleic acid sources include, for example, recombinant nucleic acid sequences contained and / or expressed in mammalian artificial chromosomes (MACs), genomic or cDNA libraries, see, for example, U.S. Patent Nos. 5,721,118; 6,025,155; human artificial chromosomes, see, for example, Rosenfeld (1997) Nat. Genet. 15:333-335; yeast artificial chromosomes (YACs); bacterial artificial chromosomes (BACs); P1 artificial chromosomes, see, for example, Woon (1998) Genomics 50:306-316; P1-derived vectors (PACs), see, for example, Kern (1997) Biotechniques 23:120-124; granules, recombinant viruses, bacteriophages, or plasmids.

[0115] In alternative embodiments, nucleic acids, as provided herein, are operatively linked to transcriptional regulatory elements, which include promoters and may be constitutive or inducible transcriptional regulatory elements.

[0116] Alternatively, "expression cassettes" comprising nucleotide sequences as provided herein are provided, for example, encoding chimeric or recombinant antibodies as provided herein. The expression cassette may include at least one transcriptional regulatory element, such as a promoter, operatively linked to the antibody-coding sequence, and optionally may also include a transcription termination signal. Other factors, such as enhancers, that are necessary or helpful in influencing expression may also be used.

[0117] Alternatively, expression cassettes used in practicing the embodiments provided herein include plasmids, expression vectors, recombinant viruses, recombinant “naked DNA” vectors of any form, etc. Alternatively, a “vector” used in practicing the embodiments provided herein may contain nucleic acids capable of infecting, transfecting, transiently or permanently transducing cells. Alternatively, a vector used in practicing the embodiments provided herein may be naked nucleic acid, or nucleic acid complexed with a protein or lipid. Alternatively, a vector used in practicing the embodiments provided herein may contain viral or bacterial nucleic acids and / or proteins, and / or membranes (e.g., cell membranes, viral lipid envelopes, etc.). Alternatively, a vector used in practicing the embodiments provided herein may include, but is not limited to, replicons (e.g., RNA replicons, bacteriophages), to which DNA fragments may attach and be replicated. Therefore, vectors include, but are not limited to, RNA, autonomously replicating circular or linear DNA or RNA (e.g., plasmids, viruses, etc., see, for example, U.S. Patent No. 5,217,879), and may include both expression plasmids and non-expression plasmids. In alternatives, vectors used to practice the embodiments provided herein can be stably replicated by the cell as autonomous structures during mitosis, or can be integrated into the host genome.

[0118] In alternatives, the “promoter” used in practicing the embodiments provided herein includes all sequences capable of driving transcription of coding sequences in cells, such as bacteria, yeast, fungi, plants, insects (e.g., baculoviruses), or mammalian cells. Therefore, promoters used in constructs include cis-acting transcriptional control elements and regulatory sequences that participate in regulating or modulating the timing and / or rate of gene transcription. For example, promoters used in practicing the embodiments provided herein may be cis-acting transcriptional control elements, including enhancers, promoters, transcription terminators, origins of replication, chromosomal integration sequences, 5' and 3' untranslated regions, or intron sequences that participate in transcriptional regulation. These cis-acting sequences can interact with proteins or other biomolecules to perform transcription (on / off, regulation, etc.).

[0119] A “constitutive” promoter used in practicing the embodiments provided herein can be a promoter that continuously drives expression under most environmental conditions and developmental or cell differentiation states. An “inducible” or “tunable” promoter used in practicing the embodiments provided herein can direct the expression of nucleic acids as described herein under the influence of environmental or developmental conditions. Examples of environmental conditions that can affect transcription by an inducible promoter used in practicing the embodiments provided herein include the presence of inducible factors applied to the cell.

[0120] In alternative embodiments, antibodies used to practice the embodiments provided herein may comprise any “mimic” and / or “peptide mimic” form. In alternative embodiments, peptides and polypeptides used to practice the embodiments provided herein may comprise synthetic chemical compounds having substantially the same structural and / or functional characteristics as natural polypeptides, such as chimeric or recombinant antibodies as provided herein. Mimics used to practice the embodiments provided herein may consist entirely of synthetic non-natural amino acid analogs, or chimeric molecules comprising a portion of natural peptide amino acids and a portion of non-natural amino acid analogs. The mimics may also incorporate any amount of conserved substitutions of natural amino acids, provided that such substitutions do not substantially alter the structure and / or activity of the mimic. Conventional experiments will determine whether the mimic is effective for practicing the invention, for example, whether the mimic composition effectively and specifically binds 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 mimics for practicing the compositions and / or methods provided herein.

[0121] Peptide mimicry compositions used to practice the embodiments provided herein can comprise any combination of non-natural structural components. Alternatively, mimicry compositions used to practice the embodiments provided herein can comprise one or all of three structural groups: a) residue-linking groups other than natural amide bonds (“peptide bonds”); b) non-natural residues that substitute for naturally occurring amino acid residues; or c) residues that induce or stabilize secondary structures, such as β-turns, γ-turns, β-sheets, α-helical conformations, etc. For example, a peptide can be characterized as a mimicry when all or some of its residues are linked by chemical means other than natural peptide bonds.

[0122] Purification and isolation of recombinant proteins

[0123] In an alternative embodiment, the chimeric or recombinant antibody, its antigen-binding fragment, or monomeric or dimer antigen-binding protein is substantially purified or isolated, and optionally, the substantially purified or isolated form is used in immunohistochemical methods and / or as a reagent, kit, and / or manufacturing product as provided herein.

[0124] In alternative embodiments, the chimeric or recombinant antibody, its antigen-binding fragment, or monomeric or dimer antigen-binding protein is substantially purified or separated using: physicochemical fractionation, such as differential precipitation, size exclusion, or immobilization based on the size, charge, or other common chemical characteristics of antibodies in a typical sample; class-specific affinity, such as immobilization of a specific antibody class (e.g., IgG or IgM) by immobilization of biological ligands with specific affinity for immunoglobulins (e.g., proteins, lectins, etc.), which can purify all antibodies of the target class regardless of antigen specificity; or antigen-specific affinity, such as affinity purification of only those antibodies in the sample that bind to a specific antigen molecule through their specific antigen-binding domain, wherein this purifies all antibodies bound to the antigen regardless of antibody class or isotype.

[0125] In an alternative embodiment, the chimeric or recombinant antibody, its antigen-binding fragment, or monomeric or dimer antigen-binding protein is substantially purified or separated using standard separation methods, such as chromatography, for example ion exchange (IEX) chromatography, hydrophobic interaction chromatography (HIC), countercurrent chromatography, immunoaffinity and / or size exclusion chromatography.

[0126] In alternative embodiments, chimeric or recombinant antibodies, their antigen-binding fragments, or monomeric or dimer antigen-binding proteins are produced in a bioreactor, such as a perfusion bioreactor, using continuous expression and purification methods, for example, as described by Vogg et al., Methods Mol Biol. 2018; Vol. 1850: 147-178; or produced by purification after using a stirred tank or oscillating bioreactor system.

[0127] Manufacturing products and reagent kits

[0128] Manufacturing products and kits are provided that contain chimeric or recombinant anti-human LAG-3Ab as provided herein and are for practicing the methods provided herein using chimeric or recombinant anti-human LAG-3Ab as provided herein; and optionally, the manufacturing products and kits may further contain some or all of the reagents required for performing IHC, and may optionally contain instructions for use for practicing the methods provided herein.

[0129] In alternative embodiments, the manufactured product has been attached or immobilized (optionally covalently bound) to a chimeric or recombinant antibody (Ab) or its antigen-binding fragment, or a monomeric or dimer antigen-binding protein as provided herein, and optionally, the manufactured product as provided herein is or comprises an array, biochip, slide, tray, dish (e.g., microtiter dish), phage, or phage particle.

[0130] Immunohistochemistry

[0131] In alternative embodiments, immunohistochemical methods and / or reagents used to practice the compositions, manufacturing products, kits, or methods provided herein may include or incorporate any IHC protocol, IHC medical device, apparatus, and / or image or data analysis system for practicing IHC or IHC reagents known in the art, for example, as described in U.S. Patent Nos. (USPN) 10,565,479 (describing a method for identifying blurred regions in digital images of stained tissue); 10,564,076 (describing a system for analyzing (or IHC) sample preparation); 10,551,395 (describing an automated histological staining system); 10,551,378 (describing a tissue staining method); 10,504,224 (describing a digital tissue image analysis system for IHC); 10,501,777 (describing a method for analyzing protein expression in IHC). (Simultaneous, multiplex detection and quantification); 10,488,340 (Describes a method for extracting images of target fluorophores in biological materials); 10,453,195 (Describes a method for detecting target tissue regions using digital pathological imaging); 10,438,381 (Describes apparatus, systems, and methods for generating digital images of tissue sections); 10,416,176 (Describes a method for processing specimens in an automated tissue staining system); 10,393,633 (Describes a method for processing and inhibiting the degradation of IHC samples); 10,217,011 (Describes the disposal of IHC slides); 10,209,165 (Describes an automated or semi-automated method for assessing the staining quality of specimens containing cells); 10,126,216 (Describes a method for fixing tissue samples for IHC); as described in 9,423,322.

[0132] In alternative embodiments, the chimeric or recombinant antibodies, their antigen-binding fragments, or monomeric or dimer antigen-binding proteins in the IHC protocols or kits provided herein are substantially purified or isolated, or are in the form of unpurified or partially purified culture supernatants.

[0133] In alternative embodiments, the methods provided herein may use or include reagents for detecting or visualizing antibody-antigen interactions using, for example, any product or method known in the art, such as IHC protocols or reagents.

[0134] In alternative embodiments, the methods provided herein include the use of chromogenic immunohistochemistry (CIH), wherein a primary antibody (e.g., a chimeric or recombinant antibody (Ab) or its antigen-binding fragment, or monomeric or dimer antigen-binding protein, as provided herein) or a secondary antibody (e.g., wherein the secondary antibody binds to the primary antibody (primary antibody) after the chimeric or recombinant antibody (Ab) or its antigen-binding fragment, or monomeric or dimer antigen-binding protein, as provided herein, has been specifically bound, paired, or associated with a LAG-3 epitope or polypeptide) is conjugated to an enzyme capable of catalyzing a color-producing reaction, such as a peroxidase (or immunoperoxidase), for example, horseradish peroxidase (HRP).

[0135] In alternative embodiments, the methods provided herein include the use of immunofluorescence, wherein a primary or secondary antibody is labeled with a fluorophore such as fluorescein or fluorescein isothiocyanate (FITC), a triarylmethane dye such as rhodamine or rhodamine derivatives (e.g., tetramethylrhodamine (TRITC), rhodamine 6G, rhodamine 123, rhodamine B, carboxytetramethylrhodamine (TAMRA), tetramethylrhodamine (TMR), sulfonylrhodamine 101), aminomethylcoumarin acetate (AMCA), Alexa... TM Or Dylight TM Fluorescent dyes. 3,3'-Diaminobenzidine (DAB) can also be used.

[0136] In alternative embodiments, methods as provided herein include the use of direct or one-step staining methods, wherein, for example, in tissue sections, a primary antibody (e.g., a chimeric or recombinant antibody (Ab) or its antigen-binding fragment, or a monomeric or dimer antigen-binding protein, as provided herein) is labeled and reacts directly with the antigen. While this technique utilizes only one antibody and is therefore simple and rapid, it suffers from low sensitivity due to minimal signal amplification.

[0137] In alternative implementations, methods such as those provided herein include the use of an indirect approach, wherein an unlabeled primary antibody (first layer) binds to a target antigen (LAG-3) in, for example, a tissue or organ, and then a labeled secondary antibody (second layer) reacts with the primary antibody. For example, the secondary antibody may target an isotype of IgG from the animal species from which the primary antibody originates. This approach can be more sensitive than direct detection strategies because the signal amplification results from the binding of the secondary antibody to each primary antibody if several secondary antibodies are conjugated to a detection reagent such as a fluorescent or enzyme reporter molecule.

[0138] In an alternative embodiment, if the secondary antibody is conjugated to several detection molecules, such as biotin molecules, the biotin molecules can recruit avidin-conjugating enzymes, streptavidin-conjugating enzymes, or NeutrAvidin. TM The protein-binding enzyme complex then enabled further amplification.

[0139] In alternative embodiments, IHC is performed on tissue sections or biopsy sections, such as paraformaldehyde (PFA)-fixed tissues or organs or formalin-fixed paraffin-embedded tissues. In alternative embodiments, the tissue is used either as a section or as a whole. Before sectioning, the tissue sample may be embedded in a medium such as paraffin or a freezing medium. Tissue sections can be sectioned on various instruments, most commonly a microtome, a cryostat, or a vibratory microtome. Samples can be sectioned in the range of approximately 3 μm to 5 μm. Sections can be mounted on glass slides, dehydrated using alcohol washes of varying concentrations (e.g., 50%, 75%, 90%, 95%, 100%), cleaned with a detergent such as xylene, and then imaged under a microscope.

[0140] Depending on the fixation and tissue preservation methods, samples may require additional steps to make the LAG-3 epitopes available for antibody binding, including dewaxing and antigen retrieval. For formalin-fixed paraffin-embedded tissues, antigen retrieval is typically required and may include pretreatment of sections with heat or proteases.

[0141] In an alternative implementation, the EnVision DuoFLEX Doublestain System is used. TMIHC is performed using the EnVisionDuoFLEX dual staining 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 (DakoOmnis) system, and the combination can be visualized using the EnVision FLEX HRP Magenta chromogen. In another alternative embodiment, IHC is performed using the EnVision FLEX Microkit, High pH, ​​a highly sensitive visualization system designed to work with the Dako Autostainer. TM The instruments are used together in IHC; this dual-linkage system detects mouse and rabbit primary antibodies and visualizes the reaction via 3,3'-diaminobenzidine (DAB) chromogen (DAB forms a water-insoluble brown precipitate when oxidized, for example, by peroxidase).

[0142] Any of the foregoing aspects and implementations may be combined with any other aspects or implementations disclosed herein in the Summary of the Invention, the Drawings and / or the Detailed Description sections.

[0143] As used in this specification and the appended claims, unless the context clearly indicates otherwise, the singular forms “an,” “a,” and “the” include the plural designation.

[0144] Unless otherwise specified or obvious from the context, as used herein, the term “or” is understood to be inclusive and encompasses both “or” and “and”.

[0145] Unless otherwise specified or obvious from the context, as used herein, the term "about" is understood to mean within the normal tolerance range in the field, such as within 2 standard deviations of the mean. "About" can be understood as 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 clearly stated from the context, all numerical values ​​provided herein are modified by the term "about".

[0146] Unless specifically stated or obvious from the context, as used herein, the terms “substantially all,” “substantially most,” “substantially all,” or “most” cover at least about 90%, 95%, 97%, 98%, 99%, or 99.5% or more of the reference amount of the composition.

[0147] The entire contents of each patent, patent application, publication, and document cited herein are incorporated herein by reference. Reference to any of the aforementioned patents, patent applications, publications, and documents does not constitute an admission that any of them is applicable prior art, nor does it constitute any admission of the content or dates of such publications or documents. A separate reference to these documents should not be construed as an assertion or admission that any portion of any document is considered necessary material to satisfy the statutory disclosure requirements of any national or regional patent application. Nevertheless, the right to rely on any such documents where appropriate to provide material that the examining authority or court deems essential to the claimed subject matter is reserved.

[0148] Modifications to the foregoing can be made without departing from the basic aspects of the invention. Although the 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 herein, and such modifications and improvements remain within the scope and spirit of the invention. The invention described herein can be suitably practiced without any element(s) not specifically disclosed herein. Therefore, for example, in each instance herein, any of the terms “comprising,” “substantially consisting of,” and “consisting of” can be replaced by any of the other two terms. Thus, the terms and expressions used are used as descriptive rather than limiting terms, and equivalents of the features shown and described or portions thereof are not excluded, and it should be recognized that various modifications are possible within the scope of the invention. Embodiments of the invention are set forth in the following claims.

[0149] The invention will be further described with reference to the embodiments described herein; however, it should be understood that the invention is not limited to such embodiments.

[0150] Example

[0151] Unless otherwise stated in the examples, all recombinant DNA techniques were performed according to standard protocols, such as those described in Sambrook et al., (2012) Molecular Cloning: A Laboratory Manual, 4th Edition, Cold Spring Harbor Laboratory Press, NY, and in Ausubel et al., (1994) Current Protocols in Molecular Biology, Current Protocols, USA, Volumes 1 and 2. Other references to 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 1 and 2. Standard materials and methods for 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, First Edition, Springer Verlag, Germany.

[0152] Example 1: Development of an exemplary anti-LAG antibody

[0153] This embodiment describes the development of an exemplary anti-LAG antibody as provided herein.

[0154] The antigen used to immunize rabbits is a synthetic peptide sequence GPPAAAPGHPLAPGPHPAAPSSWGPRPRR (SEQ ID NO: 1), representing amino acids 70-98 of human LAG-3. This peptide is conjugated to KLH and used to immunize five rabbits.

[0155] Antibody titers of the peptide in rabbits were tested, and LAG-3-specific staining for hemorrhage was assessed in IHC. All five rabbits showed LAG-3-specific staining in IHC.

[0156] B cell selection was performed using a rabbit, resulting in several promising B cell clones that produced antibodies specific to LAG-3. The antibody coding sequence was then cloned into an expression plasmid.

[0157] Rabbit anti-human LAG-3 antibody, clone 12H8, the sequence of the heavy chain variable region is:

[0158] QSVKESEGGLFKPTDTLTLTCTVSGIDLSSGILVWVRQAPGSG LEWIGGIDANGRAYYASWAKSSRSTITRNTNENTVTLKMTSLTAA DTATYFCAGGAWNIWGPGTLVTVSS(SEQ ID NO:2)

[0159] Rabbit anti-human LAG-3 antibody, clone 12H8, the sequence of the light chain variable region is:

[0160] AQVLTQTPSPVSAAVGGTVTIKCQSSQSVYDSNTLAWFQQK PGQPPKLLMYSASTLAFGVPSRFSGSGSGTQFTLTISDLECADAAT YYCLGSYDCSSVDCTAFGGGTEVVVK(SEQ ID NO:3)

[0161] A recombinant antibody was generated and tested in a standard IHC assay, showing LAG-3-specific staining. Further testing of this antibody confirmed its specificity for LAG-3.

[0162] Recombinant antibodies were expressed using the HEK293-6E cell line and a pTT5-based vector. Transient expression of this antibody takes approximately 10 days post-transfection and is a rapid and high-yield method compared to hybridoma techniques.

[0163] Other methods can be used for transient transfection and the preparation of stable cell lines for antibody expression. For example, in one embodiment, the dihydrofolate reductase (DHFR) deficient cell line CHO DG44 cells (e.g., using Freedom) is used. TM DG44 kit (Gibco)).

[0164] Establishing stable cell lines requires the selection and cloning of cells to produce well-expressing and stable cell lines. Importantly, the resulting stable cell lines must be monoclonal to ensure the production of homologous monoclonal antibodies. Other cell lines can be used for both transient and / or stable transfection.

[0165] LAG-3 antibody cloning development

[0166] To develop anti-human LAG-3 antibodies, different antigens were designed and produced. The antigen used for the final clone 12H8 was a synthetic peptide containing amino acids 70 to 98; GPPAAAPGHPLAPGPHPAAPSSWGPRPRR (SEQ ID NO:1).

[0167] This sequence is part of the extracellular domain of the human LAG-3 protein and forms an additional loop compared to CD4, which has a high structural homology with LAG-3 previously used to generate LAG-3 antibodies

[19] .

[0168] Rabbits were immunized, and titers were tested using ELISA. Subsequently, serum samples were used to perform IHC specificity tests on tissue arrays containing the following different tissues: normal tonsils, reactive lymph nodes, malignant melanoma (clinical tissue), normal liver, carcinoid tumors, malignant breast tumors, colon cancer, cerebellum, normal prostate, normal kidney, and normal pancreas. All rabbit sera showed some degree of specificity for LAG-3 protein in the IHC. Rabbits producing the best serum samples were selected for B cell selection. Blood samples were taken from rabbits, and B cell selection was performed to isolate B cells producing antibodies binding to the LAG-3 antigen. B cells were cultured as monoclonal clones under stimulation conditions, and the resulting cell culture supernatants were tested in ELISA to identify wells containing B cells producing antigen-binding antibodies. ELISA-positive cell culture supernatants were further tested in ultrasensitive IHC to identify cell culture supernatants with LAG-3-specific antibodies in the IHC. Ten clones showing LAG-3-specific staining were identified in the IHC.

[0169] Two B-cell clones exhibiting the best IHC performance were selected for cloning. As described above, ultrasensitive IHC was used to select and prioritize B-cell clones from both normal and tumor tissues. Tissues were selected as follows: IHC screening was performed using a reference LAG-3 antibody (clone 17B4Novus bio) to select both normal and tumor tissues with high LAG-3 expression. All B-cell clones were then compared to the LAG-3 reference antibody in ultrasensitive IHC. Clones with correct specificity, correct morphological expression (membrane, cytoplasm, and Golgi apparatus), and optimal sensitivity (signal-to-noise ratio) were selected and prioritized.

[0170] Cultured cells from the corresponding wells of the two clones were lysed, and RNA was extracted and used to generate cDNA. The variable heavy and light chains were amplified separately by PCR using custom primers, and the PCR products were cloned into custom expression vectors (using the pTT5 backbone) containing the corresponding rabbit constant heavy and light IgG chains, thereby generating functional antibody-encoding sequences. The heavy and light chain plasmids were transfected into the HEK293-6E cell line, and recombinant antibodies were generated and tested using the standard IHC (Envision FLEX) protocol.

[0171] Antibody clone 12H8 exhibited good fragility and specificity in IHC, with correct morphological expression (membrane, cytoplasm, and Golgi apparatus). It showed excellent sensitivity in both high-expression tissues (tonsils) and low-expression tissues (melanoma). Figure 1 Images of tonsils stained with ultrasensitive IHC using cell culture supernatant of exemplary clone 12H8 are shown.

[0172] Furthermore, this clone showed no adverse staining or nonspecific background staining in any of the other included tissues (liver, colon adenocarcinoma, breast malignancy, carcinoid tumor, normal colon, cerebellum, prostate, kidney, and pancreas).

[0173] The recombinant monoclonal rabbit anti-human LAG-3 antibody was then further tested. A 6-point titration was performed on the aforementioned tissue array using the EnVisionFLEX (Agilent) IHC system, supplemented with three additional clinical tissues (non-small cell lung cancer (NSCLC), clear cell renal cell carcinoma (RCC), and malignant melanoma). Preliminary optimal concentrations were obtained to match the histological localization, morphological expression, staining intensity, and optimal signal-to-noise ratio of both normal and tumor tissues with the reference antibody. The optimal version of this exemplary protocol was determined to be:

[0174] Exemplary IHC protocol for LAG-3 cloned using LAG-3:

[0175] The optimal antibody concentration in the S3022 antibody dilution buffer is 1.75 μg / mL. Target acquisition was performed in high pH TR buffer. Visualization system: EnVision FLEX+ with rabbit adapter.

[0176] As confirmation of 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 melanomas, and 2 kidney RCCs) and two negative clinical tissues (1 lung cancer and 1 melanoma).

[0177] LAG-3 multiplexing

[0178] LAG-3 blockade is underway in multiple clinical trials, many of which combine LAG-3 with other targets, including PD-1. Figures 9 to 11 As shown and as described above, the exemplary LAG-3 antibody provided herein was subjected to dual testing to demonstrate the extent of co-localization with PD-1 and CD3, respectively.

[0179] LAG-3 expression on tumor-infiltrating lymphocytes (TILs)

[0180] like Figures 12 to 14 As shown above, LAG-3 is expressed on tumor-infiltrating lymphocyte (TIL) subsets in clinical tissues but not on tumor cells (

[32] ).

[0181] Sequencing data

[0182] Sequencing data for an exemplary anti-human LAG-3 antibody clone 12H8:

[0183] Heavy chain variable region

[0184] QSVKESEGGLFKPTDTLTLTCTVS GIDLSSGI LVWVRQAPGSGLEWIGG IDANGRA YYASWAKSSRSTITRNTNENTVTLKMTSLTAADTATYFC AGGAWNI WGPGTLVTVSS(SEQ ID NO:2)

[0185] The underlined regions indicate the CDR regions. SEQ ID NO:2 contains CDR1 amino acid (aa) residues 25-32, CDR2 aa residues 50-56, and CDR3 aa residues 95-101. CDR regions are numbered according to the IMGT numbering system (http: / / www.imgt.org / ).

[0186] Light chain variable region:

[0187] AQVLTQTPSPVSAAVGGTVTIKCQSS QSVYDSNT LAWFQQK PGQPPKLLMY SAS TLAFGVPSRFSGSGSGTQFTLTISDLECADAAT YYC LGSYDCSSVDCT AFGGGTEVVVK(SEQ ID NO:3)

[0188] The underlined regions indicate the CDR regions. SEQ ID NO:3 includes CDR1 aa residues 27-34, CDR2 aa residues 52-54, and CDR3 aa residues 91-102. These are CDR regions numbered according to the IMGT numbering system (http: / / www.imgt.org / ).

[0189] The published content of each of the following references is incorporated herein by reference in its entirety:

[0190] References

[0191] 1.Andrews, LP, et al., LAG3(CD223) as a cancer immunotherapy target. Immunol Rev, 2017.276(1):p.80-96.

[0192] 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.

[0193] 3.Workman,CJ,et al.,LAG-3regulates plasmacytoid dendritic cellhomeostasis.J Immunol,2009.182(4):p.1885-91.

[0194] 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.

[0195] 5.Gandhi,M.K.,et al.,Expression of LAG-3by tumor-infiltratinglymphocytes 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.

[0196] 6.Chen,J.and Z.Chen,The effect of immune microenvironment on theprogression and prognosis of colorectal cancer.Med Oncol,2014.31(8):p.82.

[0197] 7.Matsuzaki,J.,et al.,Tumor-infiltrating NY-ESO-1-specific CD8+Tcells are negatively regulated by LAG-3and PD-1in human ovarian cancer.ProcNatl Acad Sci U S A,2010.107(17):p.7875-80.

[0198] 8.Li,F.J.,et al.,Expression of LAG-3is coincident with the impairedeffector function of HBV-specific CD8(+)T cell in HCC patients.Immunol Lett,2013.150(1-2):p.116-22.

[0199] 9.Giraldo,N.A.,et al.,Orchestration and Prognostic Significance ofImmune Checkpoints in the Microenvironment of Primary and Metastatic RenalCell Cancer.Clin Cancer Res,2015.21(13):p.3031-40.

[0200] 10.Takaya,S.,H.Saito,and M.Ikeguchi,Upregulation of Immune CheckpointMolecules,PD-1 and LAG-3,on CD4+and CD8+T Cells after Gastric CancerSurgery.Yonago Acta Med,2015.58(1):p.39-44.

[0201] 11.Yang,Z.Z.,et al.,Expression of LAG-3 defines exhaustion ofintratumoral PD-1(+)T cells and correlates with poor outcome in follicularlymphoma.Oncotarget,2017.8(37):p.61425-61439.

[0202] 12.Norstrom,M.M.,et al.,Progression of benign prostatic hyperplasiais associated with pro-inflammatory mediators and chronic activation ofprostate-infiltrating lymphocytes.Oncotarget,2016.7(17):p.23581-93.

[0203] 13.Deng,G.,et al.,BRAF mutation is frequently present in sporadiccolorectal cancer with methylated hMLH1,but not in hereditary nonpolyposiscolorectal cancer.Clin Cancer Res,2004.10(1 Pt 1):p.191-5.

[0204] 14.He,Y.,et al.,LAG-3 Protein Expression in Non-Small Cell LungCancer and Its Relationship with PD-1 / PD-L1 and Tumor-InfiltratingLymphocytes.J Thorac Oncol,2017.12(5):p.814-823.

[0205] 15.Marcq,E.,et al.,Abundant expression of TIM-3,LAG-3,PD-1and PD-L1as immunotherapy checkpoint targets in effusions of mesotheliomapatients.Oncotarget,2017.8(52):p.89722-89735.

[0206] 16.Burugu,S.,et al.,LAG-3+tumor infiltrating lymphocytes in breastcancer:clinical correlates and association with PD-1 / PD-L1+tumors.Ann Oncol,2017.28(12):p.2977-2984.

[0207] 17.Yanik,E.L.,et al.,Association of HIV Status With Local ImmuneResponse to Anal Squamous Cell Carcinoma:Implications for Immunotherapy.JAMAOncol,2017.3(7):p.974-978.

[0208] 18.Meng,Q.,et al.,Expansion of Tumor-reactive T Cells From PatientsWith Pancreatic Cancer.J Immunother,2016.39(2):p.81-9.

[0209] 19.Woo, SR, et al., Immune inhibitory molecules LAG-3and PD-1 synergistically regulate T-cell function to promote tumoral immune escape. Cancer Res, 2012.72(4):p.917-27.

[0210] 20. Huang, RY, et al., LAG3 and PD1 co-inhibitory molecules collaborate to limit CD8+T cell signaling and dampen antitumor immunity in a murineovarian cancer model. Oncotarget, 2015.6(29):p.27359-77.

[0211] 21. Petersen, KH, J. Lohse, and L. Ramsgaard, Automated sequentialchromogenic IHC double staining with two HRP substrates. PLoS One, 2018.13(11):p.e0207867.

[0212] 22.Baixeras,E.,et al.,Characterization of the lymphocyte activationgene 3-encoded protein.A new ligand for human leukocyte antigen class IIantigens.J Exp Med, 1992.176(2):p.327-37.

[0213] Several embodiments of the invention have been described. However, it will be understood that various modifications can be made without departing from the spirit and scope of the invention. Therefore, other embodiments are within the scope of the following claims. sequence list <110> Agilent Technologies, Inc. <120> Anti-human LAG-3 antibody and its use in immunohistochemistry (IHC) <130> 6363.139818PCT / 20200046 <140> Pending allocation <141> 2021-05-26 <150> 63 / 030,873 <151> 2020-05-27 <160> 11 <170> PatentIn version 3.5 <210> 1 <211> 29 <212> PRT <213> Homo sapiens <400> 1 Gly Pro Pro Ala Ala Ala Pro Gly His Pro Leu Ala Pro Gly Pro His 1 5 10 15 Pro Ala Ala Pro Ser Ser Trp Gly Pro Arg Pro Arg Arg 20 25 <210> 2 <211> 112 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 2 Gln Ser Val Lys Glu Ser Glu Gly Gly Leu Phe Lys Pro Thr Asp Thr 1 5 10 15 Leu Thr Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Ser Ser Gly Ile 20 25 30 Leu Val Trp Val Arg Gln Ala Pro Gly Ser Gly Leu Glu Trp Ile Gly 35 40 45 Gly Ile Asp Ala Asn Gly Arg Ala Tyr Tyr Ala Ser Trp Ala Lys Ser 50 55 60 Arg Ser Thr Ile Thr Arg Asn Thr Asn Glu Asn Thr Val Thr Leu Lys 65 70 75 80 Met Thr Ser Leu Thr Ala Ala Asp Thr Ala Thr Tyr Phe Cys Ala Gly 85 90 95 Gly Ala Trp Asn Ile Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser 100 105 110 <210> 3 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 3 Ala Gln Val Leu Thr Gln Thr Pro Ser Pro Val Ser Ala Ala Val Gly 1 5 10 15 Gly Thr Val Thr Ile Lys Cys Gln Ser Ser Gln Ser Val Tyr Asp Ser 20 25 30 Asn Thr Leu Ala Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu 35 40 45 Leu Met Tyr Ser Ala Ser Thr Leu Ala Phe Gly Val Pro Ser Arg Phe 50 55 60 Ser Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Asp Leu 65 70 75 80 Glu Cys Ala Asp Ala Ala Thr Tyr Tyr Cys Leu Gly Ser Tyr Asp Cys 85 90 95 Ser Ser Val Asp Cys Thr Ala Phe Gly Gly Gly Thr Glu Val Val Val 100 105 110 Lys <210> 4 <211> 104 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 4 Gly Asp Pro Gly Ala Pro Thr Val Leu Ile Phe Pro Pro Ala Ala Asp 1 5 10 15 Gln Val Ala Thr Gly Thr Val Thr Ile Val Cys Val Ala Asn Lys Tyr 20 25 30 Phe Pro Asp Val Thr Val Thr Trp Glu Val Asp Gly Thr Thr Gln Thr 35 40 45 Thr Gly Ile Glu Asn Ser Lys Thr Pro Gln Asn Ser Ala Asp Cys Thr 50 55 60 Tyr Asn Leu Ser Ser Thr Leu Thr Leu Thr Ser Thr Gln Tyr Asn Ser 65 70 75 80 His Lys Glu Tyr Thr Cys Lys Val Thr Gln Gly Thr Thr Ser Val Val 85 90 95 Gln Ser Phe Asn Arg Gly Asp Cys 100 <210> 5 <211> 104 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 5 Gly Asp Pro Val Ala Pro Thr Val Leu Ile Phe Pro Pro Ala Ala Asp 1 5 10 15 Gln Val Ala Thr Gly Thr Val Thr Ile Val Cys Val Ala Asn Lys Tyr 20 25 30 Phe Pro Asp Val Thr Val Thr Trp Glu Val Asp Gly Thr Thr Gln Thr 35 40 45 Thr Gly Ile Glu Asn Ser Lys Thr Pro Gln Asn Ser Ala Asp Cys Thr 50 55 60 Tyr Asn Leu Ser Ser Thr Leu Thr Leu Thr Ser Thr Gln Tyr Asn Ser 65 70 75 80 His Lys Glu Tyr Thr Cys Lys Val Thr Gln Gly Thr Thr Ser Val Val 85 90 95 Gln Ser Phe Asn Arg Gly Asp Cys 100 <210> 6 <211> 323 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 6 Gly Gln Pro Lys Ala Pro Ser Val Phe Pro Leu Ala Pro Cys Cys Gly 1 5 10 15 Asp Thr Pro Ser Ser Thr Val Thr Leu Gly Cys Leu Val Lys Gly Tyr 20 25 30 Leu Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Thr Leu Thr Asn 35 40 45 Gly Val Arg Thr Phe Pro Ser Val Arg Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Ser Val Thr Ser Ser Ser Gln Pro Val Thr Cys 65 70 75 80 Asn Val Ala His Pro Ala Thr Asn Thr Lys Val Asp Lys Thr Val Ala 85 90 95 Pro Ser Thr Cys Ser Lys Pro Thr Cys Pro Pro Pro Glu Leu Leu Gly 100 105 110 Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 115 120 125 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln 130 135 140 Asp Asp Pro Glu Val Gln Phe Thr Trp Tyr Ile Asn Asn Glu Gln Val 145 150 155 160 Arg Thr Ala Arg Pro Pro Leu Arg Glu Gln Gln Phe Asn Ser Thr Ile 165 170 175 Arg Val Val Ser Thr Leu Pro Ile Ala His Gln Asp Trp Leu Arg Gly 180 185 190 Lys Glu Phe Lys Cys Lys Val His Asn Lys Ala Leu Pro Ala Pro Ile 195 200 205 Glu Lys Thr Ile Ser Lys Ala Arg Gly Gln Pro Leu Glu Pro Lys Val 210 215 220 Tyr Thr Met Gly Pro Pro Arg Glu Glu Leu Ser Ser Arg Ser Val Ser 225 230 235 240 Leu Thr Cys Met Ile Asn Gly Phe Tyr Pro Ser Asp Ile Ser Val Glu 245 250 255 Trp Glu Lys Asn Gly Lys Ala Glu Asp Asn Tyr Lys Thr Thr Pro Ala 260 265 270 Val Leu Asp Ser Asp Gly Ser Tyr Phe Leu Tyr Ser Lys Leu Ser Val 275 280 285 Pro Thr Ser Glu Trp Gln Arg Gly Asp Val Phe Thr Cys Ser Val Met 290 295 300 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Ile Ser Arg Ser 305 310 315 320 Pro Gly Lys <210> 7 <211> 217 <212> PRT <213> Synthetic sequence <220> <223> Synthetic polypeptide <400> 7 Ala Gln Val Leu Thr Gln Thr Pro Ser Pro Val Ser Ala Ala Val Gly 1 5 10 15 Gly Thr Val Thr Ile Lys Cys Gln Ser Ser Gln Ser Val Tyr Asp Ser 20 25 30 Asn Thr Leu Ala Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu 35 40 45 Leu Met Tyr Ser Ala Ser Thr Leu Ala Phe Gly Val Pro Ser Arg Phe 50 55 60 Ser Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Asp Leu 65 70 75 80 Glu Cys Ala Asp Ala Ala Thr Tyr Tyr Cys Leu Gly Ser Tyr Asp Cys 85 90 95 Ser Ser Val Asp Cys Thr Ala Phe Gly Gly Gly Thr Glu Val Val Val 100 105 110 Lys Gly Asp Pro Gly Ala Pro Thr Val Leu Ile Phe Pro Pro Ala Ala 115 120 125 Asp Gln Val Ala Thr Gly Thr Val Thr Ile Val Cys Val Ala Asn Lys 130 135 140 Tyr Phe Pro Asp Val Thr Val Thr Trp Glu Val Asp Gly Thr Thr Gln 145 150 155 160 Thr Thr Gly Ile Glu Asn Ser Lys Thr Pro Gln Asn Ser Ala Asp Cys 165 170 175 Thr Tyr Asn Leu Ser Ser Thr Leu Thr Leu Thr Ser Thr Gln Tyr Asn 180 185 190 Ser His Lys Glu Tyr Thr Cys Lys Val Thr Gln Gly Thr Thr Ser Val 195 200 205 Val Gln Ser Phe Asn Arg Gly Asp Cys 210 215 <210> 8 <211> 217 <212> PRT <213> Synthetic Sequence <220> <223> Synthetic Polypeptide <400> 8 Ala Gln Val Leu Thr Gln Thr Pro Ser Pro Val Ser Ala Ala Val Gly 1 5 10 15 Gly Thr Val Thr Ile Lys Cys Gln Ser Ser Gln Ser Val Tyr Asp Ser 20 25 30 Asn Thr Leu Ala Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu 35 40 45 Leu Met Tyr Ser Ala Ser Thr Leu Ala Phe Gly Val Pro Ser Arg Phe 50 55 60 Ser Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Asp Leu 65 70 75 80 Glu Cys Ala Asp Ala Ala Thr Tyr Tyr Cys Leu Gly Ser Tyr Asp Cys 85 90 95 Ser Ser Val Asp Cys Thr Ala Phe Gly Gly Gly Thr Glu Val Val Val 100 105 110 Lys Gly Asp Pro Val Ala Pro Thr Val Leu Ile Phe Pro Pro Ala Ala 115 120 125 Asp Gln Val Ala Thr Gly Thr Val Thr Ile Val Cys Val Ala Asn Lys 130 135 140 Tyr Phe Pro Asp Val Thr Val Thr Trp Glu Val Asp Gly Thr Thr Gln 145 150 155 160 Thr Thr Gly Ile Glu Asn Ser Lys Thr Pro Gln Asn Ser Ala Asp Cys 165 170 175 Thr Tyr Asn Leu Ser Ser Thr Leu Thr Leu Thr Ser Thr Gln Tyr Asn 180 185 190 Ser His Lys Glu Tyr Thr Cys Lys Val Thr Gln Gly Thr Thr Ser Val 195 200 205 Val Gln Ser Phe Asn Arg Gly Asp Cys 210 215 <210> 9 <211> 435 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 9 Gln Ser Val Lys Glu Ser Glu Gly Gly Leu Phe Lys Pro Thr Asp Thr 1 5 10 15 Leu Thr Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Ser Ser Gly Ile 20 25 30 Leu Val Trp Val Arg Gln Ala Pro Gly Ser Gly Leu Glu Trp Ile Gly 35 40 45 Gly Ile Asp Ala Asn Gly Arg Ala Tyr Tyr Ala Ser Trp Ala Lys Ser 50 55 60 Arg Ser Thr Ile Thr Arg Asn Thr Asn Glu Asn Thr Val Thr Leu Lys 65 70 75 80 Met Thr Ser Leu Thr Ala Ala Asp Thr Ala Thr Tyr Phe Cys Ala Gly 85 90 95 Gly Ala Trp Asn Ile Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser 100 105 110 Gly Gln Pro Lys Ala Pro Ser Val Phe Pro Leu Ala Pro Cys Cys Gly 115 120 125 Asp Thr Pro Ser Ser Thr Val Thr Leu Gly Cys Leu Val Lys Gly Tyr 130 135 140 Leu Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Thr Leu Thr Asn 145 150 155 160 Gly Val Arg Thr Phe Pro Ser Val Arg Gln Ser Ser Gly Leu Tyr Ser 165 170 175 Leu Ser Ser Val Val Ser Val Thr Ser Ser Ser Gln Pro Val Thr Cys 180 185 190 Asn Val Ala His Pro Ala Thr Asn Thr Lys Val Asp Lys Thr Val Ala 195 200 205 Pro Ser Thr Cys Ser Lys Pro Thr Cys Pro Pro Pro Glu Leu Leu Gly 210 215 220 Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 225 230 235 240 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln 245 250 255 Asp Asp Pro Glu Val Gln Phe Thr Trp Tyr Ile Asn Asn Glu Gln Val 260 265 270 Arg Thr Ala Arg Pro Pro Leu Arg Glu Gln Gln Phe Asn Ser Thr Ile 275 280 285 Arg Val Val Ser Thr Leu Pro Ile Ala His Gln Asp Trp Leu Arg Gly 290 295 300 Lys Glu Phe Lys Cys Lys Val His Asn Lys Ala Leu Pro Ala Pro Ile 305 310 315 320 Glu Lys Thr Ile Ser Lys Ala Arg Gly Gln Pro Leu Glu Pro Lys Val 325 330 335 Tyr Thr Met Gly Pro Pro Arg Glu Glu Leu Ser Ser Arg Ser Val Ser 340 345 350 Leu Thr Cys Met Ile Asn Gly Phe Tyr Pro Ser Asp Ile Ser Val Glu 355 360 365 Trp Glu Lys Asn Gly Lys Ala Glu Asp Asn Tyr Lys Thr Thr Pro Ala 370 375 380 Val Leu Asp Ser Asp Gly Ser Tyr Phe Leu Tyr Ser Lys Leu Ser Val 385 390 395 400 Pro Thr Ser Glu Trp Gln Arg Gly Asp Val Phe Thr Cys Ser Val Met 405 410 415 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Ile Ser Arg Ser 420 425 430 Pro Gly Lys 435 <210> 10 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 10 Trp Ser His Pro Gln Phe Glu Lys 1 5 <210> 11 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 11 Asp Tyr Lys Asp Asp Asp Asp Lys 1 5

Claims

1. A chimeric or recombinant antibody (Ab) or an antigen-binding fragment thereof, said chimeric or recombinant antibody or antigen-binding fragment thereof being capable of specifically binding to human lymphocyte activation gene 3 (LAG-3) polypeptide, said chimeric or recombinant antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, The chimeric or recombinant antibody or its antigen-binding fragment specifically binds to a peptide containing the following amino acid sequence: GPPAAAPGHPLAPGPHPAAPSSWGPRPRR (SEQ ID NO:1), 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).

2. The chimeric or recombinant antibody (Ab) or its antigen-binding fragment according to claim 1, wherein the chimeric or recombinant antibody (Ab) or its antigen-binding fragment is any one of the following: F(ab')2, Fab', and single-stranded variable fragments.

3. The chimeric or recombinant antibody (Ab) or its antigen-binding fragment according to claim 1 or 2, wherein the chimeric or recombinant antibody (Ab) or its antigen-binding fragment comprises: (a) The heavy chain variable region, which includes the three CDR1, CDR2, and CDR3 complementary determinant regions (CDRs) of SEQ ID NO:2; and (b) Light chain variable region, the light chain variable region comprising the three CDR1, CDR2 and CDR3 complementary determination regions (CDRs) of SEQ ID NO:

3.

4. The chimeric or recombinant antibody (Ab) or its antigen-binding fragment according to claim 1 or 2, wherein the chimeric or recombinant antibody (Ab) or its antigen-binding fragment comprises: (a) a heavy chain variable region comprising CDR1 represented by amino acid (aa) residues 25-32 of SEQ ID NO:2, CDR2 represented by aa residues 50-56 of SEQ ID NO:2, and CDR3 represented by aa residues 95-101 of SEQ ID NO:2; and (b) a light chain variable region comprising CDR1 represented by amino acid (aa) residues 27-34 of SEQ ID NO:3, CDR2 represented by aa residues 52-54 of SEQ ID NO:3, and CDR3 represented by aa residues 91-102 of SEQ ID NO:

3.

5. The chimeric or recombinant antibody (Ab) or its antigen-binding fragment 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 κ or λ light chain.

6. The chimeric or recombinant antibody (Ab) or its antigen-binding fragment according to claim 1 or 2, wherein the sequence of the light chain constant region is or comprises the following sequence: GDPGAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC (SEQ ID NO:4), or GDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC (SEQ ID NO: 5).

7. The chimeric or recombinant antibody (Ab) or its antigen-binding fragment according to claim 1 or 2, wherein: (a) The sequence of the light chain constant region comprises SEQ ID NO:4 or SEQ ID NO:5 having at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve or more conserved amino acid substitutions, wherein the light chain constant region having said conserved amino acid substitutions is capable of specifically binding to or associating with the heavy chain constant region; or (b) The sequence of the light chain constant region has at least 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90% or 95% sequence identity with 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.

8. The chimeric or recombinant antibody (Ab) or its antigen-binding fragment according to claim 1 or 2, wherein the sequence of the heavy chain constant region is or comprises: GQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTA RPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK (SEQ ID NO:6).

9. The chimeric or recombinant antibody (Ab) or its antigen-binding fragment according to claim 1 or 2, wherein: (a) The sequence of the heavy chain constant region comprises SEQ ID NO:6 having at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve or more conserved amino acid substitutions, wherein the heavy chain constant region having said conserved amino acid substitutions is capable of specifically binding to or associating with said light chain constant region; or (b) The sequence of the heavy chain constant region has at least 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90% or 95% sequence identity with SEQ ID NO:6, wherein the heavy chain constant region is capable of specifically binding to or associating with the light chain constant region.

10. The chimeric or recombinant antibody (Ab) or its antigen-binding fragment according to claim 1 or 2, wherein the sequence of the antibody light chain comprises: AQVLTQTPSPVSAAVGGTVTIKCQSSQSVYDSNTLAWFQQKPGQPPKLLMYSASTLAFGVPSRFSGSGSGTQFTLTISDLECADAATYYCLGSYDCSSVDCTAFGGGTEV VVKGDPGAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC (SEQ IDNO:7), or AQVLTQTPSPVSAAVGGTVTIKCQSSQSVYDSNTLAWFQQKPGQPPKLLMYSASTLAFGVPSRFSGSGSGTQFTLTISDLECADAATYYCLGSYDCSSVDCTAFGGGTEV VVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC (SEQ IDNO:8).

11. The chimeric or recombinant antibody (Ab) or its antigen-binding fragment according to claim 1 or 2, wherein the sequence of the antibody heavy chain comprises: QSVKESEGGLFKPTDTLTLTCTVSGIDLSSGILVWVRQAPGSGLEWIGGIDANGRAYYASWAKSRSTITRNTNENTVTLKMTSLTAADTATYFCAGGAWNIWGPGTLVT VSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPP PELLGGPSVFIFPPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARG QPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK (SEQ ID NO:9).

12. The chimeric or recombinant antibody (Ab) or its antigen-binding fragment according to claim 1 or 2, wherein the chimeric or recombinant Ab comprises: (a) The light chain as shown in SEQ ID NO:7 and the heavy chain as shown in SEQ ID NO:9, wherein the chimeric or recombinant Ab is capable of selectively binding to human LAG-3 peptide; or (b) The light chain as shown in SEQ ID NO:8 and the heavy chain as shown in SEQ ID NO:9, wherein the chimeric or recombinant Ab is capable of selectively binding human LAG-3 polypeptide.

13. The chimeric or recombinant antibody (Ab) or its antigen-binding fragment according to claim 12, wherein the chimeric or recombinant Ab comprises: The light chain, as shown in SEQ ID NO:7, and the heavy chain, as shown in SEQ ID NO:9, wherein the chimeric or recombinant Ab is capable of selectively binding human LAG-3 polypeptide.

14. The chimeric or recombinant antibody (Ab) or its antigen-binding fragment according to claim 12, wherein the chimeric or recombinant Ab comprises: The light chain as shown in SEQ ID NO:8 and the heavy chain as shown in SEQ ID NO:9, wherein the chimeric or recombinant Ab is capable of selectively binding human LAG-3 polypeptide.

15. The chimeric or recombinant antibody (Ab) or its antigen-binding fragment according to claim 1 or 2, wherein the chimeric or recombinant antibody or its antigen-binding fragment further comprises a detectable reagent or binding moiety. wherein optionally the detectable agent comprises: Enzymes, biotin, fluorescent or chemiluminescent labels, radioactive isotopes, antibody-binding epitopes, Optionally, the fluorescent label is selected from dyes, quantum dots, and photoluminescent aqueous nanocrystals. And optionally, the enzyme is a peroxidase, alkaline phosphatase, or β-galactosidase. And optionally, the peroxidase is horseradish peroxidase (HRP). Optionally, the dyes include cyanine dyes; optionally, the cyanine dyes are Cy3 or Cy5. Optionally, the fluorescent or chemiluminescent label includes a fluorophore, which includes dansyl or fluorescein, and optionally the fluorescein is carboxyfluorescein (FAM). The binding components include: glutathione S-transferase (GST), poly-histag, chitin-binding protein (CBP), STREP-TAG™, FLAG tag, or maltose-binding protein; Optionally, the antigen-binding epitope is a hapten, which may include biotin, carborane, fluorescein, or bromodeoxyuridine.

16. The chimeric or recombinant antibody (Ab) or its antigen-binding fragment according to claim 15, wherein the detectable agent comprises: Cyanide, Nile Red, Rhodamine, Perylene, Fluorene, Coumarin, Dabcyl, [2-(4-nitro-2,1,3-benzoxadiazol-7-yl)aminoethyl]trimethylammonium (NBD), Nile Blue, Boron-dipyrrole methylene (BODIPY), HRP MAGENTA™ chromogen or its derivatives; Optionally, the cyanine is sulfonylindo-cyanine, the coumarin is 7-methoxycoumarin (Mca), and the rhodamine is tetramethylrhodamine (TMR).

17. A recombinant nucleic acid encoding a chimeric or recombinant antibody (Ab) or an antigen-binding fragment thereof as described in any one of claims 1 to 16.

18. Use of any one of claims 1 to 16 or of any one of claims 17, a chimeric or recombinant antibody (Ab) encoded by a recombinant nucleic acid, or an antigen-binding fragment thereof, in a kit for preparing a method for detecting the presence of human LAG-3 protein in a cell, tissue, organ, or a portion thereof, wherein the method comprises: (a) Contacting the cell, tissue, or organ, or a portion thereof, with the chimeric or recombinant antibody (Ab) or its antigen-binding fragment, and (b) Detecting the specific binding of the chimeric or recombinant antibody (Ab) or its antigen-binding fragment to the human LAG-3 polypeptide or a polypeptide containing GPPAAAPGHPLAPGPHPAAPSSWGPRPRR (SEQ ID NO:1) in the cell, tissue, or organ or a portion thereof. This allows for the detection of the presence of the human LAG-3 protein in the cells, tissues, organs, or a portion thereof, including contact with the cells, tissues, organs, or a portion thereof. Optionally, the contact includes assays using immunohistochemistry (IHC). Optionally, the method further includes contacting the chimeric or recombinant antibody (Ab) or its antigen-binding fragment with another detectable reagent to indicate specific binding of the chimeric or recombinant antibody (Ab) or its antigen-binding fragment to the human LAG-3 protein, and optionally signaling to indicate specific binding of the chimeric or recombinant antibody (Ab) or its antigen-binding fragment to the human LAG-3 protein. Optionally, the other detectable reagent specifically binds to the chimeric or recombinant antibody (Ab) or its antigen-binding fragment. Optionally, the other detectable reagent is a secondary antibody, or the other detectable reagent comprises a secondary antibody, which specifically binds to the chimeric or recombinant antibody (Ab) or its antigen-binding fragment, which binds to the human LAG-3 protein. Optionally, the other detectable reagent is a secondary antibody, or the other detectable reagent comprises a secondary antibody, the secondary antibody specifically binding to a hapten or tag, the hapten or tag being linked to or conjugated to the chimeric or recombinant antibody (Ab) or its antigen-binding fragment. Optionally, the secondary antibody further comprises a second detectable reagent or is linked to or conjugated with a second detectable reagent. Optionally, the secondary antibody further comprises or has an enzyme, biotin, fluorescent or chemiluminescent label, radioactive isotope, or hapten attached or conjugated thereto. Optionally, the enzyme is an alkaline phosphatase, β-galactosidase, or peroxidase. Optionally, the fluorescent label is selected from dyes, quantum dots, and photoluminescent aqueous nanocrystals. Optionally, the fluorescent or chemiluminescent label includes a fluorophore. Optionally, the dye includes a cyanine dye; optionally, the cyanine dye is Cy3 or Cy5. Optionally, the hapten includes biotin, carborane, fluorescein, or bromodeoxyuridine.

19. The use according to claim 18, The second detectable reagent includes: Cyanide, Nile Red, Rhodamine, Perylene, Fluorene, Coumarin, Dabcyl, [2-(4-nitro-2,1,3-benzoxadiazol-7-yl)aminoethyl]trimethylammonium (NBD), Nile Blue, Boron-dipyrrole methylene (BODIPY), HRP MAGENTA™ chromogen or its derivatives; Optionally, the cyanine is sulfonylindo-cyanine, the coumarin is 7-methoxycoumarin (Mca), and the rhodamine is tetramethylrhodamine (TMR).

20. Use of the chimeric or recombinant antibody according to any one of claims 1 to 16, or the chimeric or recombinant antibody encoded by the recombinant nucleic acid according to claim 17, in a kit used in a method for detecting or diagnosing cancers expressing LAG-3 protein, or in a method for detecting whether lymphocytes expressing LAG-3 are present in cancer tissue, wherein the method comprises: The expression or presence of human LAG-3 protein in or on a cell, tissue, or organ sample or a portion thereof is detected by: contacting the cell, tissue, or organ sample with the chimeric or recombinant antibody; 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 the detection of specific binding indicates the expression or presence of human LAG-3 protein in the cell, tissue, or organ sample or a portion thereof. Optionally, the LAG-3-expressing lymphocytes are activated T cells. Optionally, the specific binding of the detection indicates the expression or presence of the human LAG-3 protein in the cell, tissue, or organ sample or a portion thereof, thereby diagnosing the cancer. The cancers mentioned are selected from the group consisting of: adenocarcinoma, bladder cancer, urothelial carcinoma, breast cancer, chronic lymphocytic leukemia, colorectal cancer, ovarian cancer, kidney cancer, liver cancer, stomach cancer, lymphoma, lung cancer, mesothelioma, and melanoma. Optionally, the renal cell carcinoma includes renal cell carcinoma. Optionally, the liver cancer includes hepatocellular carcinoma. Optionally, the adenocarcinoma is lung adenocarcinoma or colon adenocarcinoma. Optionally, the detection includes the use or performance of immunohistochemistry (IHC) assays or flow cytometry. Optionally, performing or using the flow cytometry includes using a fluorescence activated cell sorter (FACS) or an impedance flow cytometer. Optionally, the cell, tissue, or organ sample or a portion thereof is or is derived from a patient's biopsy.

21. The use according to claim 20, wherein the cancer is selected from the group consisting of: clear cell renal cell carcinoma (RCC), malignant breast tumors or ductal carcinoma in situ (DCIS), Hodgkin lymphoma, follicular lymphoma, non-small cell lung cancer (NSCLC), malignant pleural mesothelioma, pancreatic cancer, and malignant melanoma.

22. In the use according to claim 20 or 21, the LAG-3-expressing lymphocytes are activated T cells that have infiltrated the tumor.

23. In the use according to claim 20 or 21, the LAG-3-expressing lymphocytes are tumor-infiltrating lymphocytes (TILs).

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