ANTIBODIES TO TROPONIN I AND METHODS OF USE THEREOF

• US 20100216720A1
• Filed: 02/24/2009
• Published: 08/26/2010
• Est. Priority Date: 02/24/2009
• Status: Active Grant

• Current Assignee: Abbott Laboratories Incorporated
• Original Assignee: Abbott Laboratories Incorporated
• Inventors: Tyner, Lowell J., Brophy, Susan E., Tyner, Joan D., Tu, Bailin, Huang, Dagang, Ziemann, Robert N.
• Granted Patent: US 8,030,026 B2
• Time in Patent Office: Days
• US Class Current: 514/1.100
• CPC Class Codes: A61P 9/10 for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• CPC Class Codes: C07K 14/4716 Muscle proteins, e.g. myosin, actin
• CPC Class Codes: C07K 16/18 against material from animals or humans
• CPC Class Codes: C07K 2317/24 containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• CPC Class Codes: C07K 2317/565 Complementarity determining region [CDR]
• CPC Class Codes: C07K 2317/622 Single chain antibody (scFv)
• CPC Class Codes: C07K 2317/92 Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Abstract

The subject invention relates to antibodies to troponin I as well as methods of use thereof. In particular, such antibodies may be used to detect Troponin I in a patient and may also be used in the diagnosis of, for example, a myocardial infarction or acute coronary syndrome.

Claims

1. A Chinese Hamster Ovary (CHO) cell line, referred to as TnI 19C7 AM1 hG1 CHO 204, designated by American Type Culture Collection (ATCC) deposit number PTA-9816.

2. A recombinant antibody produced by said CHO cell line of claim 1.

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

3. An isolated binding protein comprising an antigen-binding domain which binds to Troponin I, said antigen-binding domain comprising at least one complementarity determining region (CDR) comprising an amino acid sequence selected from the group consisting of:

4. The binding protein of claim 3, wherein said binding protein comprises at least 3 CDRs.

5. The binding protein according to claim 4, wherein said binding protein comprises at least 6 CDRs.

6. The binding protein according to claim 3 further comprising a human acceptor framework.

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

35. The binding protein of claim 3, wherein said binding protein is selected from the group consisting of:

an immunoglobulin molecule, a monoclonal antibody, a chimeric antibody, a CDR-grafted antibody, a humanized antibody, a Fab, a Fab′

, a F(ab′

)2, a Fv, a disulfide linked Fv, a scFv, a single domain antibody, a diabody, a multispecific antibody, a dual specific antibody, an anti-idiotypic antibody, a bispecific antibody, and a functionally active epitope-binding fragment thereof.

7. An isolated nucleic acid molecule encoding a binding protein, wherein the amino acid sequence of the variable heavy chain of said binding protein has at least 70% identity to SEQ ID NO.:

25.

8. The isolated nucleic acid molecule of claim 7, wherein the amino acid sequence of the variable light chain of said binding protein has at least 70% identity to SEQ ID NO.:

28.

9. An isolated nucleic acid molecule encoding a binding protein, wherein the amino acid sequence of the variable heavy chain of said binding protein is SEQ ID NO.:

25.

10. An isolated nucleic acid molecule encoding a binding protein, wherein the amino acid sequence of the variable light chain of said binding protein is SEQ ID NO.:

28.

11. The isolated nucleic acid molecule of claim 10, wherein said binding protein further comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO.:

25.

14. A vector comprising said isolated nucleic acid molecule of claim 9 or claim 10.

15. An isolated host cell comprising said vector of claim 14.

16. A method of producing a binding protein capable of binding to Troponin I, comprising culturing said host cell of claim 15 for a time and under conditions sufficient to produce said binding protein.

17. An isolated protein produced according to the method of claim 16.

12. An isolated binding protein comprising the amino acid sequence of SEQ ID NO.:

25.

13. An isolated binding protein comprising the amino acid sequence of SEQ ID NO.:

28.

19. A method of detecting Troponin I antigen in a test sample comprising the steps of:

a) contacting said test sample with an antibody which binds to Troponin I and comprises the amino acid sequence of SEQ ID NO;

25 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 Troponin I antigen in said test sample.

20. The method of claim 19 wherein said antibody further comprises the amino acid sequence of SEQ ID NO:

28.

21. The method of claim 20 wherein said antibody is produced by CHO cell line TnI 19C7 AM1 hG1 CHO 204 having ATCC deposit designation PTA-9816

22. A method of detecting Troponin I antigen in a test sample comprising the steps of:

a) contacting said test sample with a first antibody which binds to Troponin I and comprises SEQ ID NO;

25 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 Troponin I antigen in said test sample.

23. The method of claim 22, wherein said first antibody further comprises SEQ ID NO:

28.

24. The method of claim 23, wherein said first antibody is produced by CHO cell line TnI 19C7 AM1 hG1 CHO 204 having ATCC deposit designation PTA-9816.

27. The method of claim 24 wherein said antibody is produced by CHO cell line TnI 19C7 AM1 hG1 CHO 204 cell line having ATCC deposit designation PTA-9816.

25. A method of detecting Troponin I antigen in a test sample comprising the steps of:

a) contacting Troponin I antigen with an antibody to Troponin I for a time and under conditions sufficient to form Troponin I antigen/antibody complexes, wherein said antibody comprises the amino acid sequence of SEQ ID NO;

25 and is labeled with a signal-generating compound capable of generating a detectable signal;

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

c) detecting presence of a signal generating by said signal generating compound, presence of said signal indicating presence of Troponin I antigen in said test sample.

26. The method of claim 25 wherein said antibody further comprises the amino acid sequence of SEQ ID NO:

28.

28. A method of detecting Troponin I antigen in a test sample comprising the steps of:

a) contacting said test sample with

     1) a Troponin I reference antigen, wherein said antigen is attached to a signal generating compound capable of generating a detectable signal and

     2) an antibody to Troponin I antigen wherein said antibody comprises the amino acid sequence of SEQ ID NO;

25, for a time and under conditions sufficient to form Troponin I reference antigen/antibody complexes; and

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

29. The method of claim 28 wherein said antibody further comprises the amino acid sequence of SEQ ID NO:

28.

30. The method of claim 29 wherein said antibody is produced by CHO cell line TnI 19C7 AM1 hG1 CHO 204 having ATCC deposit designation PTA-9816.

31. A method of diagnosing acute coronary syndrome or myocardial infarction 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 which binds to Troponin I and comprises the amino acid sequence of SEQ ID NO;

25, for a time and under conditions sufficient for formation of Troponin I antigen/antibody complexes; and

c) detecting presence of said Troponin I antigen/antibody complexes;

d) dissociating said Troponin I antigen present in said complexes from said antibody present in said complexes; and

e) measuring the amount of dissociated Troponin I antigen, wherein an amount of Troponin I antigen greater than approximately 1-5 times the Troponin I value of the 99^th percentile of a normal population indicates a diagnosis of acute coronary syndrome or myocardial infarction in said patient.

32. A method of diagnosing acute coronary syndrome or myocardial infarction 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 which binds to Troponin I and comprises the amino acid sequence of SEQ ID NO;

25 for a time and under conditions sufficient for the formation of Troponin I antigen/antibody complexes;

c) adding a conjugate to the resulting Troponin I antigen/antibody complexes for a time and under conditions sufficient to allow said conjugate to bind to the bound Troponin I 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 Troponin I 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 Troponin I antigen present in said test sample by measuring the intensity of said signal, an amount of Troponin I antigen greater than approximately 1-5 times the value of the 99^th percentile of a normal population indicating a diagnosis of acute coronary syndrome or myocardial infarction in said patient.

34. An isolated binding protein comprising an antigen-binding domain, wherein said antigen-binding domain comprises at least one CDR comprising an amino acid sequence selected from the group consisting of:

CDR-VH1. X₁—

X₂—

X₃—

X₄—

X₅—

X₆—

X₇—

X₈—

X₉—

X₁₀ (SEQ ID NO;

63), wherein;

X₁ is G;

X₂ is Y;

X₃ is T or S;

X₄ is F;

X₅ is T;

X₆ is D;

X₇ is Y;

X₈ is N;

X₉ is I or L; and

X₁₀ is H.

CDR-VH2. X₁—

X₂—

X₃—

X₄—

X₅—

X₆—

X₇—

X₈—

X₉—

X₁₀—

X₁₁—

X₁₂—

X₁₃—

X₁₄—

X₁₅—

X₁₆—

X₁₇ (SEQ ID NO;

64), wherein;

X₁ is Y;

X₂ is I;

X₃ is Y;

X₄ is P;

X₅ is Y;

X₆ is N;

X₇ is G;

X₈ is I;

X₉ is T;

X₁₀ is G;

X₁₁ is Y;

X₁₂ is N;

X₁₃ is Q;

X₁₄ is K;

X₁₅ is F;

X₁₆ is K; and

X₁₇ is S.

CDR-VH3. X₁—

X₂—

X₃—

X₄—

X₅—

X₆—

X₇—

X₈—

X₉—

X₁₀ (SEQ ID NO;

65), wherein;

X₁ is D;

X₂ is A or F;

X₃ is Y;

X₄ is D;

X₅ is Y or S;

X₆ is D;

X₇ is W, Y or A;

X₈ is L;

X₉ is A or T; and

X₁₀ is Y or D.

CDR-VL1. X₁—

X₂—

X₃—

X₄—

X₅—

X₆—

X₇—

X₈—

X₉—

X₁₀—

X₁₁ (SEQ ID NO;

66), wherein;

X₁ is R;

X₂ is A or T;

X₃ is S;

X₄ is Q or K;

X₅ is S or N;

X₆ is I or V;

X₇ is G;

X₈ is T;

X₉ is N;

X₁₀ is I; and

X₁₁ is Y or H.

CDR-VL2. X₁—

X₂—

X₃—

X₄—

X₅—

X₆—

X₇ (SEQ ID NO;

67), wherein;

X₁ is Y;

X₂ is A or G;

X₃ is S or T;

X₄ is E;

X₅ is S or R;

X₆ is I, L or V; and

X₇ is S, P or F.

and

CDR-VL3. X₁—

X₂—

X₃—

X₄—

X₅—

X₆—

X₇—

X₈—

X₉ (SEQ ID NO;

68), wherein;

X₁ is Q;

X₂ is Q;

X₃ is S;

X₄ is N;

X₅ is N;

X₆ is W;

X₇ is P;

X₈ is Y; and

X₉ is T.