Monoclonal anti-tk1 antibodies

Abstract

The embodiments relate to monoclonal antibodies or fragments capable of binding to a serum form of human TK1 and to kits and methods involving the use of such monoclonal antibodies or fragments.

1 . A monoclonal antibody or a fragment of a monoclonal antibody capable of binding to a serum form of human thymidine kinase 1, TK1, wherein said monoclonal antibody or fragment has specificity for an epitope selected from a group consisting of:
 GEAVAARKLF (SEQ ID NO: 2) of said human TK1;   at least one of NCPVPGKPGE (SEQ ID NO: 4), PVPGKPGEAV (SEQ ID NO: 5) and NCPVPGKPGEAV (SEQ ID NO: 3) of said human TK1; and   a conformation dependent epitope of said human TK1.   
     
     
         2 . The monoclonal antibody or fragment according to  claim 1 , wherein said monoclonal antibody or fragment is also capable of binding to human recombinant TK1 and to a cellular form of human TK1. 
     
     
         3 . The monoclonal antibody or fragment according to  claim 1 , wherein said monoclonal antibody or fragment is capable of binding to a peptide having amino acid sequence GQPAGPDNKENCPVPGKPGEAVAARKLFAPQ (SEQ ID NO: 1). 
     
     
         4 . The monoclonal antibody or fragment according to  claim 1 , obtainable by a process comprising:
 immunizing a non-human animal with a first peptide conjugate comprising a peptide having amino acid sequence GQPAGPDNKENCPVPGKPGEAVAARKLFAPQ (SEQ ID NO: 1) coupled to a first carrier protein or a second peptide conjugate comprising said peptide having amino acid sequence GQPAGPDNKENCPVPGKPGEAVAARKLFAPQ (SEQ ID NO: 1) coupled to a second carrier protein that is different from said first carrier protein;   isolating splenocytes from said non-human animal with blood titers for said first peptide conjugate or said second peptide conjugate;   forming hybridomas by fusing said splenocytes with myelomas;   screening supernatant from hydridomas, resulting from immunization with said first peptide conjugate, for titers for said second peptide conjugate and screening supernatant from hydridomas, resulting from said immunization with said second peptide conjugate, for titers for said first peptide conjugate;   selecting a hybridoma producing monoclonal antibodies capable of binding to said first peptide conjugate, to said second peptide conjugate and to human recombinant TK1; and   isolating said monoclonal antibody from supernatant of said selected hybridoma.   
     
     
         5 . The monoclonal antibody or fragment according to  claim 1 , wherein said monoclonal antibody or fragment has specificity for an epitope GEAVAARKLF (SEQ ID NO: 2) of said human TK1, and has:
 a variable heavy, VH, domain complementarity determining region 1, CDR1 having amino acid sequence (SEQ ID NO: 6);   a VH domain CDR2 having amino acid sequence (SEQ ID NO: 7);   a VH domain CDR3 having amino acid sequence (SEQ ID NO: 8);   a variable light, VL, domain CDR1 having amino acid sequence (SEQ ID NO: 9);   a VL domain CDR2 having amino acid sequence (SEQ ID NO: 10); and   a VL domain CDR3 having amino acid sequence (SEQ ID NO: 11).   
     
     
         6 . The monoclonal antibody or fragment according to  claim 1 , wherein said monoclonal antibody or fragment has specificity for said conformation dependent epitope of said human TK1, and has:
 a VH domain CDR1 having amino acid sequence (SEQ ID NO: 6);   a VH domain CDR2 having amino acid sequence (SEQ ID NO: 12);   a VH domain CDR3 having amino acid sequence (SEQ ID NO: 13);   a VL domain CDR1 having amino acid sequence (SEQ ID NO: 14);   a VL domain CDR2 having amino acid sequence (SEQ ID NO: 10); and   a VL domain CDR3 having amino acid sequence (SEQ ID NO: 11).   
     
     
         7 . The monoclonal antibody or fragment according to  claim 1 , wherein said monoclonal antibody or fragment has specificity for at least one epitope selected from a group consisting of NCPVPGKPGE (SEQ ID NO: 4), PVPGKPGEAV (SEQ ID NO: 5) and NCPVPGKPGEAV (SEQ ID NO: 3) of said human TK1, and has:
 a VH domain CDR1 having amino acid sequence (SEQ ID NO: 15);   a VH domain CDR2 having amino acid sequence (SEQ ID NO: 16);   a VH domain CDR3 having amino acid sequence (SEQ ID NO: 17);   a VL domain CDR1 having amino acid sequence (SEQ ID NO: 18);   a VL domain CDR2 having amino acid sequence (SEQ ID NO: 19); and   a VL domain CDR3 having amino acid sequence (SEQ ID NO: 20).   
     
     
         8 . The monoclonal antibody or fragment according to  claim 1 , wherein said monoclonal antibody or fragment is capable of binding to said serum form of said human TK1 with a dissociation constant, K d , of less than 5 nM. 
     
     
         9 . The monoclonal antibody or fragment according to  claim 1 , wherein said monoclonal antibody or fragment ha a heavy chain of IgG1 isotype and a light chain of K isotype. 
     
     
         10 . A kit for determining a level of cellular and/or serum TK1 material in a body sample, comprising:
 a first monoclonal antibody according to  claim 1  immobilized to a solid support or intended to be immobilized to said solid support; and   a second monoclonal antibody according to  claim 1 .   
     
     
         11 . The kit according to  claim 10 , wherein
 said first monoclonal antibody has specificity for a first epitope selected from a group consisting of:
 GEAVAARKLF (SEQ ID NO: 2) of human TK1; 
 at least one of NCPVPGKPGE (SEQ ID NO: 4), PVPGKPGEAV (SEQ ID NO: 5) and NCPVPGKPGEAV (SEQ ID NO: 3) of said human TK1; and 
 a conformation dependent epitope of said human TK1, and 
   said second monoclonal antibody has specificity for a second, different epitope selected from said group.   
     
     
         12 . The kit according to  claim 11 , wherein
 said first epitope to which said first monoclonal antibody has specificity is one of (i) GEAVAARKLF (SEQ ID NO: 2) of said human TK1, and (ii) said conformation dependent epitope of said human TK1, and   said second epitope to which said second monoclonal antibody has specificity is the other of (i) GEAVAARKLF (SEQ ID NO: 2) of said human TK1 and (ii) said conformation dependent epitope of said human TK1.   
     
     
         13 . The kit according to  claim 10 , wherein said kit a sandwich assay kit. 
     
     
         14 . The kit according to  claim 10 , wherein said kit is an Enzyme-Linked Immunosorbent Assay, ELISA, kit. 
     
     
         15 . The kit according to  claim 14 , wherein said second monoclonal antibody has a covalently attached biotin or a covalently attached strepatvidin or avidin. 
     
     
         16 . The kit according to  claim 15 , further comprising:
 a horseradish peroxidase, HRP, labeled streptavidin or avidin or a HRP labeled biotin; and   a HRP substrate selected from a group consisting of a 3,3′,5,5′-tetramethylbenzidine, TMB, substrate, a 3,3′-diaminobenzidine, DAB, substrate and a 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid, ABTS, substrate.   
     
     
         17 . The kit according to  claim 10 , further comprising a microtiter plate as said solid support. 
     
     
         18 . The kit according to  claim 10 , further comprising agarose beads or magnetic beads as said solid support. 
     
     
         19 . A method for determining a level of cellular and/or serum TK1 material in a body sample, comprising:
 contacting said body sample with a monoclonal antibody or fragment according to  claim 1 ; and   detecting an amount of bound monoclonal antibody or fragment.   
     
     
         20 . The method according to  claim 19 , wherein said body sample is selected from a group consisting of a cell sample, a tissue sample, a blood sample, a serum sample, a cerebrospinal fluid sample, a pleural fluid sample, a synovial fluid sample and a peritoneal cavity fluid sample. 
     
     
         21 . A method for estimating the likelihood of recurrence of a tumor in a subject, comprising:
 determining a level of serum TK1 material in a body sample from said subject using a method according to  claim 19 , and   comparing said level of serum TK1 material in said body sample with a level of serum TK1 material representative of a population of healthy subjects or with a level of serum TK1 material previously determined in said subject.   
     
     
         22 . A method for determining cell proliferation in a subject, comprising:
 determining a level of serum TK1 material in a body sample from said subject using a method according to  claim 19 , and   determining said cell proliferation based on said level of serum TK1 material in said body sample.   
     
     
         23 . A method for determining a proliferation process response in a subject suffering from a malignant disease, comprising:
 determining a level of serum TK1 material in a body sample from said subject using a method according to  claim 19 , and   determining said proliferation process response based on said level of serum TK1 material in said body sample.   
     
     
         24 . A method for determining a level of inflammation, infection, or tumor cell proliferation in a subject, comprising:
 determining a level of serum TK1 material in a body sample from said subject using a method according to  claim 19 , and   determining said level of inflammation, infection or tumor cell proliferation based on said level of serum TK1 material in said body sample.   
     
     
         25 . A method of producing a monoclonal antibody capable of binding to a serum form of human thymidine kinase 1, TK1, said method comprising:
 immunizing a non-human animal with a first peptide conjugate comprising a peptide having amino acid sequence GQPAGPDNKENCPVPGKPGEAVAARKLFAPQ (SEQ ID NO: 1) coupled to a first carrier protein or a second peptide conjugate comprising said peptide having amino acid sequence GQPAGPDNKENCPVPGKPGEAVAARKLFAPQ (SEQ ID NO: 1) coupled to a second carrier protein that is different from said first carrier protein;   isolating splenocytes from said non-human animal with blood titers for said first peptide conjugate or said second peptide conjugate;   forming hybridomas by fusing said splenocytes with myelomas;   screening supernatant from hydridomas, resulting from immunization with said first peptide conjugate, for titers for said second peptide conjugate and screening supernatant from hydridomas, resulting from immunization with said second peptide conjugate, for titers for said first peptide conjugate;   selecting a hybridoma producing a monoclonal antibody capable of binding to said first peptide conjugate, to said second peptide conjugate and to human rTK1; and   isolating said monoclonal antibody from supernatant of said selected hybridoma.   
     
     
         26 . The method according to  claim 25 , wherein selecting said hybridoma comprises selecting a hybridoma producing a monoclonal antibody capable of binding to said first peptide conjugate, to said second peptide conjugate, to said human rTK1 and to TK1 present in a serum sample obtained from a human subject suffering from cancer. 
     
     
         27 . An isolated peptide consisting of the amino acid sequence GEAVAARKLF (SEQ ID NO: 2), NCPVPGKPGE (SEQ ID NO: 4), PVPGKPGEAV (SEQ ID NO: 5) or NCPVPGKPGEAV (SEQ ID NO: 3). 
     
     
         28 . A method for estimating the likelihood of recurrence of a tumor in a subject, comprising:
 determining a level of serum TK1 material in a body sample from said subject using a kit according to  claim 10 , and   comparing said level of serum TK1 material in said body sample with a level of serum TK1 material representative of a population of healthy subjects or with a level of serum TK1 material previously determined in said subject.   
     
     
         29 . A method for determining cell proliferation in a subject, comprising:
 determining a level of serum TK1 material in a body sample from said subject using a kit according to  claim 10 , and   determining said cell proliferation based on said level of serum TK1 material in said body sample.   
     
     
         30 . A method for determining a proliferation process response in a subject suffering from a malignant disease, comprising:
 determining a level of serum TK1 material in a body sample from said subject using a kit according to  claim 10 , and   determining said proliferation process response based on said level of serum TK1 material in said body sample.   
     
     
         31 . A method for determining a level of inflammation, infection, or tumor cell proliferation in a subject, comprising:
 determining a level of serum TK1 material in a body sample from said subject using a kit according to  claim 10 , and   determining said level of inflammation, infection or tumor cell proliferation based on said level of serum TK1 material in said body sample.