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

Abstract

Provided are chimeric or recombinant antibodies (Ab), 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 tumors or tumor infiltrating lymphocytes (TILs), and methods for making and using them. In alternative embodiments, chimeric or a recombinant antibodies (Ab), or antigen binding fragments thereof, or monomeric or dimeric antigen binding proteins as provided herein are used for in vitro diagnostics, for example, in immuno-histochemistry (IHC), for example, to diagnose and/or treat a cancer, for example, bladder cancer, urothelial carcinoma, breast cancer, lung cancer, Non-Small Cell Lung Cancer, renal carcinoma, Renal Clear cell Carcinoma and/or melanoma or malignant melanoma, by their ability to specifically bind to activated T cells that have infiltrated tumors, or tumor infiltrating lymphocytes.

1 . (canceled) 
     
     
         2 . The recombinant nucleic acid of  claim 18 , wherein the chimeric or recombinant antibody (Ab), or antigen binding fragment thereof, or monomeric or dimeric antigen binding protein, is fabricated as or in the form of:
 an antigen-binding fragment (Fab, or an Ab fragment having just one constant and one variable domain of each of an Ab heavy and light chain),   a F(ab′) 2  (or an Ab digested by pepsin yielding two fragments: a F(ab′) 2  fragment and a pFc′ fragment),   a Fab′ (a single chain of a F(ab′) 2  fragment),   a single-chain variable fragment (scFv) (or a fusion protein of a variable region of an Ab heavy and light chain connected together with a linker peptide,   a (scFv) 2 , or a di-scFv or a bi-scFv, or a single peptide chain having two variable heavy and two variable light regions yielding tandem scFv,   a minibody (or a fusion protein of a variable region of an Ab heavy and light chain connected together with an alkyl group,   a diabody or an scFv with a linker peptide too short for the two variable regions to fold together forcing the scFvs to dimerize), a triabody or a tetrabody or an scFv with a linker peptide too short for the two variable regions to fold together forcing the scFvs to trimerize or tetramize),   a single-domain antibody (dAB) (or a single variable region of an Ab heavy or Ab light chain), or   a plurality of complementarity determining region (CDR) fragments, or a multi-specific antibody formed from two or more antibody fragments.   
     
     
         3 . The recombinant nucleic acid of  claim 18 , wherein
 the sequence of the heavy chain variable region is:   
       
         
           
                 
               
                   (SEQ ID NO: 2) 
                 
                   QSVKESEGGLFKPTDTLTLTCTVSGIDLSSGILVWVRQAPGSGLEWIGGI 
                 
                     
                 
                   DANGRAYYASWAKSRSTITRNTNENTVTLKMTSLTAADTATYFCAGGAWN 
                 
                     
                 
                   IWGPGTLVTVSS, 
                 
             
                
                
                
                
                
                
               
            
           
         
       
       and
 the sequence of the light chain variable region is: 
 
       
         
           
                 
               
                   (SEQ ID NO: 3) 
                 
                   AQVLTQTPSPVSAAVGGTVTIKCQSSQSVYDSNTLAWFQQKPGQPPKLLM 
                 
                     
                 
                   YSASTLAFGVPSRFSGSGSGTQFTLTISDLECADAATYYCLGSYDCSSVD 
                 
                     
                 
                   CTAFGGGTEVVVK. 
                 
             
                
                
                
                
                
                
               
            
           
         
       
     
     
         4 . The recombinant nucleic acid of  claim 18 , wherein:
 (a) 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 conservative amino acid substitutions, wherein the heavy chain variable region capable of specifically binding to the human LAG-3 polypeptide, the amino acid (SEQ ID NO: 1), or the epitope when either unpaired (alone) or paired with a light chain variable region;   (b) the sequence of the light chain variable region comprises SEQ ID NO: 3 having at least one, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve conservative amino acid substitutions, wherein the light chain variable region is capable of specifically binding to the human LAG-3 polypeptide, the amino acid (SEQ ID NO:1), or the epitope when either unpaired (alone) or paired with a heavy chain variable region;   (c) the sequence of the heavy chain variable region has at least about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 2;   (d) the sequence of the light chain variable region has at least about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO:3:   (e) the sequence of the heavy chain variable region and the amino acid sequence SEQ ID NO: 2 have a Z score of from about 2 to about 8, of a Z score of at least 8, when aligned using distance matrix alignment; or   (f) the sequence of the light chain variable region and the amino acid sequence SEQ ID NO: 3 have a Z score of from about 2 to about 8, of a Z score of at least 8, when aligned using distance matrix alignment.   
     
     
         5 . The recombinant nucleic acid of  claim 18 , wherein the chimeric or recombinant antibody (Ab), or antigen binding fragment thereof, or monomeric or dimeric antigen binding protein, comprises:
 (a) a heavy chain variable region comprising the three CDR1, CDR2 and CDR3 complementarity determining regions (CDRs) of SEQ ID NO: 2, or CDR1 amino acid (aa) residues 25-32, CDR2 aa residues 50-56, and CDR3 aa residues 95-101, of SEQ ID NO: 2; or   (b) a light chain variable region comprising the three CDR1, CDR2 and CDR3 complementarity determining regions (CDRs) of SEQ ID NO: 3, or CDR1 amino acid (aa) residues 27-34, CDR2 aa residues 52-54, and CDR3 aa residues 91-102, of SEQ ID NO: 3.   
     
     
         6 . The recombinant nucleic acid of  claim 18 , wherein the chimeric or recombinant antibody (Ab), or antigen binding fragment thereof, or monomeric or dimeric antigen binding protein, comprises:
 (a) a heavy chain variable region comprising: the three CDR1, CDR2 and CDR3 complementarity determining regions (CDRs) of SEQ ID NO:2, or CDR1 amino acid (aa) residues 25-32, CDR2 aa residues 50-56, and CDR3 aa residues 95-101, of SEQ ID NO:2; and   (b) a light chain variable region comprising: the three CDR1, CDR2 and CDR3 complementarity determining regions (CDRs) of SEQ ID NO:3, or CDR1 amino acid (aa) residues 27-34, CDR2 aa residues 52-54, and CDR3 aa residues 91-102, of SEQ ID NO: 3.   
     
     
         7 . The recombinant nucleic acid of  claim 18 , wherein the antibody heavy chain is an IgM, IgG, IgA or IgE isotype heavy chain, and/or the light chain is a kappa or a lambda light chain. 
     
     
         8 . The recombinant nucleic acid of  claim 18 , wherein the sequence of the light chain constant region is or comprises: 
       
         
           
                 
               
                   (SEQ ID NO: 4) 
                 
                   GDPGAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTG 
                 
                   IENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFN 
                 
                   RGDC, 
                 
                   or 
                 
                     
                 
                   (SEQ ID NO: 5) 
                 
                   GDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTG 
                 
                   IENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFN 
                 
                   RGDC. 
                 
             
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         9 . The recombinant nucleic acid of  claim 18 , 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 conservative amino acid substitutions, wherein the light chain constant region with the conservative amino acid substitutions is capable of specifically binding to or associating with a heavy chain constant region: or   (b) the sequence of the light chain constant region has at least about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 4 or SEQ ID NO: 5, wherein the light chain constant region is capable of specifically binding to or associating with a heavy chain constant region.   
     
     
         10 . The recombinant nucleic acid of  claim 18 , wherein the sequence of the heavy chain constant region is or comprises: 
       
         
           
                 
               
                   (SEQ ID NO: 6) 
                 
                   GQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGV 
                 
                     
                 
                   RTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTC 
                 
                     
                 
                   SKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQ 
                 
                     
                 
                   FTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVH 
                 
                     
                 
                   NKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYP 
                 
                     
                 
                   SDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFT 
                 
                     
                 
                   CSVMHEALHNHYTQKSISRSPGK. 
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         11 . The recombinant nucleic acid of  claim 18 , 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 conservative amino acid substitutions, wherein the heavy chain constant region with the conservative amino acid substitutions is capable of specifically binding to or associating with a light chain constant region; or   (b) the sequence of the heavy chain constant region has at least about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO:6, wherein the heavy chain constant region is capable of specifically binding to or associating with a light chain constant region.   
     
     
         12 . The recombinant nucleic acid of  claim 18 , wherein the sequence of the antibody light chain comprises: 
       
         
           
                 
               
                   (SEQ ID NO: 7) 
                 
                   AQVLTQTPSPVSAAVGGTVTIKCQSSQSVYDSNTLAWFQQKPGQPPKLLM 
                 
                   YSASTLAFGVPSRFSGSGSGTQFTLTISDLECADAATYYCLGSYDCSSVD 
                 
                   CTAFGGGTEVVVKGDPGAPTVLIFPPAADQVATGTVTIVCVANKYFPDVT 
                 
                   VTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCK 
                 
                   VTQGTTSVVQSFN RGDC, 
                 
                   or 
                 
                     
                 
                   (SEQ ID NO: 8) 
                 
                   AQVLTQTPSPVSAAVGGTVTIKCQSSQSVYDSNTLAWFQQKPGQPPKLLM 
                 
                   YSASTLAFGVPSRFSGSGSGTQFTLTISDLECADAATYYCLGSYDCSSVD 
                 
                   CTAFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVT 
                 
                   VTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCK 
                 
                   VTQGTTSVVQSFN RGDC. 
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         13 . The recombinant nucleic acid of  claim 18 , wherein the sequence of the antibody heavy chain comprises: 
       
         
           
                 
               
                   (SEQ ID NO: 9) 
                 
                   QSVKESEGGLFKPTDTLTLTCTVSGIDLSSGILVWVRQAPGSGLEWIGGI 
                 
                     
                 
                   DANGRAYYASWAKSRSTITRNTNENTVTLKMTSLTAADTATYFCAGGAWN 
                 
                     
                 
                   IWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVT 
                 
                     
                 
                   VTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATN 
                 
                     
                 
                   TKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCV 
                 
                     
                 
                   VVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQD 
                 
                     
                 
                   WLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRS 
                 
                     
                 
                   VSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSV 
                 
                     
                 
                   PTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK. 
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         14 . The recombinant nucleic acid of  claim 18 , wherein the chimeric or recombinant Ab comprises:
 (a) a light chain as set forth in SEQ ID NO: 7 operatively bound to, paired with, associated with, or configured with a heavy chain as set forth in SEQ ID NO: 9, wherein the chimeric or recombinant Ab is capable of selectively binding to a human LAG-3 polypeptide; or   (b) a light chain as set forth in SEQ ID NO: 8 operatively bound to, paired with, associated with, or configured with a heavy chain as set forth in SEQ ID NO: 9, wherein the chimeric or recombinant Ab is capable of selectively binding to a human LAG-3 polypeptide.   
     
     
         15 . The recombinant nucleic acid of  claim 14 , wherein the chimeric or recombinant Ab comprises:
 a light chain as set forth in SEQ ID NO: 7 operatively bound to, paired with, associated with, or configured with a heavy chain as set forth in SEQ ID NO: 9, wherein the chimeric or recombinant Ab is capable of selectively binding to a human LAG-3 polypeptide.   
     
     
         16 . The recombinant nucleic acid of  claim 14 , wherein the chimeric or recombinant Ab comprises:
 a light chain as set forth in SEQ ID NO: 8 operatively bound to, paired with, associated with, or configured with a heavy chain as set forth in SEQ ID NO: 9, wherein the chimeric or recombinant Ab is capable of selectively binding to a human LAG-3 polypeptide.   
     
     
         17 . The recombinant nucleic acid of  claim 18 , wherein the chimeric or recombinant antibody (Ab), or antigen binding fragment thereof, or monomeric or dimeric antigen binding protein, further comprises or is bound to, paired with, associated with, or covalently conjugated to, a detectable agent or a binding moiety,
 wherein optionally the detectable agent comprises: an enzyme, a biotin, a fluorescent or chemiluminescent label, a fluorophore, a cyanine or sulfoindo-cyanine, nile red, rhodamine, perylene, fluorenyl, coumarin, 7-methoxycoumarin (Mca), dabcyl, [2-(4-nitro-2,1,3-benzoxadiazol-7-yl)aminoethyl]trimethylammonium (NBD), Nile blue, Tamra or tetramethylrhodamine (TMR), HRP MAGENTA™ chromogen (Dako Omnis, Agilent), boron-dipyrromethene (BODIPY), or derivatives thereof), a dye, a radioisotope, a quantum dot or photoluminescent aqueous nanocrystal, a hapten or an antibody binding epitope or domain,   and optionally the enzyme is a peroxidase, an alkaline phosphatase, or a beta-galactosidase, and optionally the peroxidase is a horse radish peroxidase (HRP),   and optionally the hapten comprises a biotin, theophylline, digoxigenin, carborane, fluorescein or bromodeoxyuridine,   
       and optionally the dye comprises a cyanine dye; or Cy3 or Cy5,
 and optionally the fluorophore comprises dansyl, fluorescein or carboxyfluorescein (FAM) or 6-FAM, 
 and optionally the binding moiety comprises: a glutathione S-transferase (GST) or ligandin tag, a polyhistidine (poly-his) tag, a chitin binding protein (CBP), a STREP-TAG™ or a Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO:10) peptide tag, a FLAG tag or DYKDDDDK (SEQ ID NO:11) peptide tag, or a maltose binding protein. 
 
     
     
         18 . A recombinant nucleic acid encoding a chimeric or a recombinant antibody (Ab), or an antigen binding fragment thereof, or a monomeric or dimeric antigen binding protein, capable of specifically binding to a human Lymphocyte-Activation Gene 3 (LAG-3) polypeptide, comprising:
 a heavy chain variable region comprising:   
       
         
           
                 
               
                   (SEQ ID NO: 2) 
                 
                   
                     QSVKESEGGLFKPTDTLTLTCTVSGIDLSSGILVWVRQAPGSGLEWIGGI 
                   
                 
                     
                 
                   
                     DANGRAYYASWAKSRSTITRNTNENTVTLKMTSLTAADTATYFCAGGAWN 
                   
                 
                     
                 
                     IWGPGTLVTVSS ; 
                 
             
                
                
                
                
                
                
               
            
           
         
       
       or
 a light chain variable region comprising: 
 
       
         
           
                 
               
                   (SEQ ID NO: 3) 
                 
                   
                     AQVLTQTPSPVSAAVGGTVTIKCQSSQSVYDSNTLAWFQQKPGQPPKLLM 
                   
                 
                     
                 
                   
                     YSASTLAFGVPSRFSGSGSGTQFTLTISDLECADAATYYCLGSYDCSSVD 
                   
                 
                     
                 
                     CTAFGGGTEVVVK . 
                 
             
                
                
                
                
                
                
               
            
           
         
       
     
     
         19 . A method for detecting the presence of a human LAG-3 protein in a cell, a tissue, an organ or a portion of any of the foregoing, comprising:
 contacting the cell, tissue or organ or portion of any of the foregoing with a recombinant nucleic acid of  claim 18 .   
     
     
         20 . A method for detecting or diagnosing a LAG-3 protein-expressing cancer, or a cancer tissue having contained therein a LAG-3 expressing lymphocyte or a LAG-3 expressing tumor infiltrating lymphocyte (TIL), and optionally the TIL comprises a tumor infiltrating activated T cell, comprising: detecting the expression or presence of a human LAG-3 protein in or on a cell, tissue or organ sample or portion thereof by contacting the cell, tissue or organ sample with a recombinant nucleic acid of  claim 18 , and detecting whether or not the chimeric or recombinant antibody specifically binds to a human LAG-3 protein in the cell, tissue or organ sample or portion thereof, and the detecting of specific binding indicates the expression or presence of the human LAG-3 protein in the cell, tissue or organ sample, or portion thereof.