ANTIBODIES RELATING TO PIVKAII AND USES THEREOF

• US 20100233175A1
• Filed: 03/10/2009
• Published: 09/16/2010
• Est. Priority Date: 03/10/2009
• Status: Active Grant

• Current Assignee: Abbott Japan LLC (Abbott Laboratories)
• Original Assignee: Abbott Laboratories
• Inventors: Yoshimura, Toru, Ruan, Qiaoqiao, Beligere, Gangamani S., Dowell, Barry L.
• Granted Patent: US 8,283,162 B2
• Time in Patent Office: Days
• US Class Current: 424/139.100
• CPC Class Codes: A61P 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C07K 16/40 against enzymes
• CPC Class Codes: C07K 2317/92 Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
• CPC Class Codes: G01N 33/57438 of liver, pancreas or kidney

Abstract

The present invention relates to antibodies that may be used, for example, in the diagnosis, treatment and prevention of haepatocellular carcinoma (HCC), liver cancer and related conditions.

Claims

1. A hybridoma cell line designated by American Type Culture Collection (ATCC) deposit designation PTA-9638.

2. A monoclonal antibody produced by said hybridoma cell line of claim 1.

7. An isolated nucleic acid molecule encoding a binding protein which binds to PIVKA-II, wherein the amino acid sequence of the variable heavy chain of said binding protein has at least 70% identity the amino acid sequence of said monoclonal antibody of claim 2.

8. The isolated nucleic acid molecule of claim 7, wherein said binding protein binds to amino acids 13-27 of PIVKA-II.

9. A purified amino acid sequence having at least 70% identity to the amino acid sequence of said monoclonal antibody of claim 2.

10. A vector comprising said isolated nucleic acid molecule of claim 7.

11. An isolated host cell comprising said vector of claim 10.

12. A method of producing a binding protein capable of binding to PIVKA-II comprising the steps of:

a) constructing a vector comprising said nucleic acid molecule of claim 7 operably linked to a regulatory element;

b) transforming said resulting vector into a host cell; and

c) culturing said host cell for a time and under conditions sufficient to produce said binding protein.

13. An isolated binding protein produced according to the method of claim 12.

31. A kit comprising a container containing said monoclonal antibody of claim 2 or said binding protein of claim 3.

3. An isolated binding protein comprising an antigen binding domain that binds to amino acids 13-27 of Prothrombin Induced Vitamin K Antagonist II (PIVKA-II).

4. The isolated binding protein of claim 3, wherein said binding protein has a binding dissociation constant (K_D) of about 1.0×

10<sup>−

9</sup> M or lower.

5. The isolated binding protein of claim 4, wherein said binding protein has a K_D of about 1.0×

10<sup>−

9</sup> M to about 1.0×

10<sup>−

11</sup> M.

6. The isolated binding protein of claim 5, wherein said binding protein has a K_D of about 1.5×

10<sup>−

10</sup> M.

28. A pharmaceutical composition comprising the binding protein of claim 3 and a pharmaceutically acceptable carrier.

14. A method of detecting PIVKA-II antigen in a test sample comprising the steps of:

a) contacting said test sample with an antibody having an antigen binding domain which binds to amino acids 13-27 of PIVKA-II for a time and under conditions sufficient for the formation of antibody/antigen complexes; and

b) detecting presence of said complexes, presence of said complexes indicating presence of PIVKA-II antigen in said test sample.

15. The method of claim 14, wherein said antibody comprises an antigen-binding domain that binds to amino acids 13-27 of PIVKA-II.

16. The method of claim 15, wherein said antibody is a monoclonal antibody produced by a hybridoma cell line having ATCC deposit designation PTA-9638.

17. A method of detecting PIVKA-II antigen in a test sample comprising the steps of:

a) contacting said test sample with a first antibody having an antigen binding domain which binds to amino acids 13-27 of PIVKA-II for a time and under conditions sufficient for the formation of first antibody/antigen complexes;

b) adding a conjugate to said first antibody/antigen complexes, wherein said conjugate comprises a second antibody attached to a signal generating compound capable of generating a detectable signal, for a time and under conditions sufficient to form first antibody/antigen/second antibody complexes; and

c) detecting presence of a signal generating by said signal generating compound, presence of said signal indicating presence of PIVKA-II antigen in said test sample.

18. The method of claim 17, wherein said first antibody comprises an antigen binding domain that binds to amino acids 13-27 of PIVKA-II.

19. The method of claim 18, wherein said first antibody is produced by a hybridoma cell line having ATCC deposit designation PTA-9638.

20. A method of detecting PIVKA-II antigen in a test sample comprising the steps of:

a) contacting PIVKA-II antigen with an antibody to PIVKA-II antigen for a time and under conditions sufficient to form PIVKA-II antigen/antibody complexes, wherein said antibody is labeled with a signal-generating compound capable of generating a detectable signal;

b) adding said test sample to said PIVKA-II antigen/antibody complexes for a time and under conditions sufficient to form PIVKA-II antigen/antibody/PIVKA-II test sample antigen complexes; and

c) detecting presence of a signal generating by said signal generating compound, presence of said signal indicating presence of PIVKA-II antigens in said test sample.

21. The method of claim 20 wherein said antibody to PIVKA-II antigen comprises an antigen binding domain that binds to amino acids 13-27 of PIVKA-II.

22. The method of claim 21 wherein said antibody is produced by a hybridoma cell line having ATCC deposit designation PTA-9638.

23. A method of detecting PIVKA-II antigen in a test sample comprising the steps of:

a) contacting said test sample with

     1) a PIVKA-II reference antigen, wherein said antigen is attached to a signal generating compound capable of generating a detectable signal and

     2) an antibody to PIKVA-II antigen, for a time and under conditions sufficient to form PIVKA-II reference antigen/antibody complexes;

b) detecting a signal generated by said signal generating compound, wherein the amount of PIVKA-II antigen detected in said test sample is inversely proportional to the amount of PIVKA-II reference antigen bound to said antibody.

24. The method of claim 23 wherein said antibody comprises an antigen-binding domain that binds to amino acids 13-27 of PIVKA-II.

25. The method of claim 24 wherein said antibody is produced by a hybridoma cell line having ATCC deposit designation PTA-9638.

26. A method of producing a hybridoma cell line which expresses a binding protein comprising an antigen-binding domain which binds to amino acids 13-27 of PIVKA-II comprising the steps of:

a) immunizing a GANP mouse with an antigen comprising amino acids 13-27 of PIVKA-II for a time and under conditions sufficient for said mouse to produce antibodies against said antigen;

b) harvesting and purifying B cells from the spleen of said mouse;

c) fusing said spleen cells with myeloma cells in order to produce hybridomas; and

d) selecting a hybridoma cell line which expresses said binding protein comprising an antigen-binding domain which binds to amino acids 13-27 of PIVKA-II.

27. The method of claim 26, wherein said hybridoma cell line has ATCC deposit designation PTA-9638.

29. A method of diagnosing hepatocellular carcinoma (HCC) or liver cancer in a patient suspected of having one of these conditions comprising the steps of:

a) isolating a biological sample from said patient;

b) contacting said biological sample with an antibody comprising an antigen binding domain that binds to amino acids 13-27 of PIVKA-II antigen for a time and under conditions sufficient for formation of PIVKA-II antigen/antibody complexes; and

c) detecting presence of said PIVKA-II antigen/antibody complexes;

d) dissociating said PIVKA-II antigen present in said complexes from said antibody present in said complexes; and

d) measuring the amount of dissociated PIVKA-II antigen, wherein an amount of PIVKA-II antigen greater than approximately 40 mAU/mL indicates a diagnosis of HCC or liver cancer in said patient.

30. A method of diagnosing HCC or liver cancer in a patient suspected of having one of these conditions comprising the steps of:

a) isolating a biological sample from said patient;

b) contacting said biological sample with a first antibody comprising an antigen bind domain that binds to amino acids 13-27 of PIVKA-II antigen for a time and under conditions sufficient for the formation of PIVKA-II antigen/antibody complexes;

c) adding a conjugate to the resulting PIVKA-II antigen/antibody complexes for a time and under conditions sufficient to allow said conjugate to bind to the bound PIVKA-II antigen, wherein said conjugate comprises a second antibody attached to a signal generating compound capable of generating a detectable signal;

d) detecting the presence of PIVKA-II antigen which may be present in said biological sample by detecting a signal generated by said signal generating compound; and

e) measuring the amount of PIVKA-II antigen present in said test sample by measuring the intensity of said signal, an amount of PIVKA-II antigen greater than approximately 40 mAU/mL indicating a diagnosis of HCC or liver cancer in said patient.