BCL-2-LIKE PROTEIN 11 SRM/MRM ASSAY

• US 20130302334A1
• Filed: 07/15/2013
• Published: 11/14/2013
• Est. Priority Date: 01/13/2011
• Status: Active Grant

• Current Assignee: Expression Pathology, Inc. (NantWorks, LLC)
• Original Assignee: Expression Pathology, Inc. (NantWorks, LLC)
• Inventors: Krizman, David, Hembrough, Todd, Thyparambil, Sheeno, Liao, Wei-Li
• Granted Patent: US 9,551,719 B2
• Time in Patent Office: Days
• US Class Current: 424/135.1
• CPC Class Codes: C07K 14/4747 Apoptosis related proteins
• CPC Class Codes: C07K 16/2863 against receptors for growth factors, growth regulators
• CPC Class Codes: C07K 16/32 against translation products of oncogenes
• CPC Class Codes: C07K 2317/76 Antagonist effect on antigen, e.g. neutralization or inhibition of binding
• CPC Class Codes: C12Q 1/485 involving kinase
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2600/158 Expression markers
• CPC Class Codes: G01N 2333/4703 Regulators; Modulating activity
• CPC Class Codes: G01N 2800/44 Multiple drug resistance
• CPC Class Codes: G01N 2800/52 Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
• CPC Class Codes: G01N 2800/56 Staging of a disease; Further complications associated with the disease
• CPC Class Codes: G01N 33/5088 of vertebrates
• CPC Class Codes: G01N 33/574 for cancer
• CPC Class Codes: G01N 33/6848 Methods of protein analysis involving mass spectrometry
• CPC Class Codes: G01N 33/6893 related to diseases not provided for elsewhere

Abstract

Specific peptides, and derived ionization characteristics of those peptides, from the Bcl-2-like protein 11 (BIM) are provided that are particularly advantageous for quantifying the BIM protein directly in biological samples that have been fixed in formalin by the method of Selected Reaction Monitoring (SRM) mass spectrometry, or what can also be termed as Multiple Reaction Monitoring (MRM). Such biological samples are chemically preserved and fixed where the biological sample is selected from tissues and cells treated with formaldehyde containing agents/fixatives including formalin-fixed tissue/cells, formalin-fixed/paraffin embedded (FFPE) tissue/cells, FFPE tissue blocks and cells from those blocks, and tissue culture cells that have been formalin fixed and or paraffin embedded. A protein sample is prepared from the biological sample using the Liquid Tissue™ reagents and protocol, and the BIM protein is quantitated in the Liquid Tissue™ sample by the method of SRM/MRM mass spectrometry by quantitating in the protein sample at least one or more of the peptides described. These peptides can be quantitated if they reside in a modified or an unmodified form. An example of a modified form of a BIM peptide is phosphorylation of a tyrosine, threonine, serine, and/or other amino acid residues within the peptide sequence.

Claims

1. A method for measuring the level of the Bcl-2-like protein 11 (BIM) in a biological sample, comprising detecting and/or quantifying the level of one or more modified or unmodified BIM fragment peptides in a protein digest prepared from the biological sample using mass spectrometry;

and calculating the level of modified or unmodified BIM protein in the sample; and

wherein the level is a relative level or an absolute level.

2. The method of claim 1, further comprising the step of fractionating the protein digest prior to detecting and/or quantifying the level of one or more modified or unmodified BIM fragment peptides.

3. The method of claim 2, wherein the fractionating step is selected from the group consisting of gel electrophoresis, liquid chromatography, capillary electrophoresis, nano-reversed phase liquid chromatography, high performance liquid chromatography, and reverse phase high performance liquid chromatography.

4. The method of claim 1, wherein the protein digest of the biological sample is prepared by the Liquid Tissue™

protocol.

5. The method of claim 1, wherein the protein digest comprises a protease digest.

6. The method of claim 5, wherein the protein digest comprises a trypsin digest.

7. The method of any of claim 1, wherein the mass spectrometry comprises tandem mass spectrometry, ion trap mass spectrometry, triple quadrupole mass spectrometry, MALDI-TOF mass spectrometry, MALDI mass spectrometry, or time of flight mass spectrometry, or any combination thereof.

8. The method of claim 7, wherein the mode of mass spectrometry used is Selected Reaction Monitoring (SRM), Multiple Reaction Monitoring (MRM), or multiple Selected Reaction Monitoring (mSRM), or any combination thereof.

9. The method of claim 1, wherein the BIM fragment peptide comprises an amino acid sequence as set forth as SEQ ID NO:

1, SEQ ID NO;

2, or SEQ ID NO;

3.

10. The method of claim 1, wherein the biological sample is a blood sample, a urine sample, a serum sample, an ascites sample, a sputum sample, lymphatic fluid, a saliva sample, a cell, or a solid tissue.

11. The method of claim 10, wherein the tissue is formalin fixed tissue.

12. The method of claim 10, wherein the tissue is paraffin embedded tissue.

13. The method of claim 10, wherein the tissue is obtained from a tumor.

14. The method of claim 13, wherein the tumor is a primary tumor.

15. The method of claim 13, wherein the tumor is a secondary tumor.

16. The method of claim 1, comprising quantifying a modified or unmodified BIM fragment peptide.

17. The method of claim 16, wherein quantifying a modified or unmodified fragment peptide comprises comparing the level of one or more BIM fragment peptides comprising an amino acid sequence of about 8 to about 45 amino acid residues of BIM as shown in SEQ ID NO:

1, SEQ ID NO;

2, and SEQ ID NO;

3 in one biological sample to the level of the same BIM fragment peptide in a different and separate biological sample.

18. The method of claim 17, wherein quantifying one or more modified or unmodified BIM fragment peptides comprises determining the level of the each of the BIM fragment peptides in a biological sample by comparison to an added internal standard peptide of a known level, wherein each of the BIM fragment peptides in the biological sample is compared to an internal standard peptide having the same amino acid sequence.

19. The method of claim 18, wherein the internal standard peptide is an isotopically labeled peptide.

20. The method of claim 19, wherein the isotopically labeled internal standard peptide comprises one or more heavy stable isotopes selected from the group consisting of ¹⁸O, ¹⁷O, ³⁴S, ¹⁵N, ¹³C, and ²H, or any combinations thereof.

21. The method of claim 1, wherein detecting and/or quantifying the level of one or more modified or unmodified BIM fragment peptides in the protein digest indicates the presence of modified or unmodified BIM protein and an association with cancer in the subject.

22. The method of claim 21, further comprising correlating the results of detecting and/or quantifying levels of one or more modified or unmodified BIM fragment peptides, or the level of the BIM protein to the diagnostic stage/grade/status of the cancer.

23. The method of claim 22, wherein correlating the results of detecting and/or quantifying the level of one or more modified or unmodified BIM fragment peptides, or the level of the BIM protein to the diagnostic stage/grade/status of the cancer is combined with detected and/or quantified levels of other proteins, or peptides from other proteins, in a multiplex format to provide additional information about the diagnostic stage/grade/status of the cancer.

24. The method of claim 1, further comprising selecting for the subject, from which the biological sample is obtained, a treatment based on the presence, absence, or level of one or more BIM fragment peptides or the level of BIM protein.

25. The method of claim 1, further comprising administering to the patient or subject, from which the biological sample is obtained, a therapeutically effective amount of a therapeutic agent, wherein the therapeutic agent and/or amount of the therapeutic agent administered is based upon the level of one or more modified or unmodified BIM fragment peptides or the level of BIM protein.

26. The method of claim 24, wherein the treatment or the therapeutic agent is directed to cancer cells expressing BIM protein.

27. The method of claim 1, wherein the biological sample is formalin fixed tumor tissue that has been processed for quantifying the level of one or more modified or unmodified BIM fragment peptides employing the Liquid Tissue™

protocol and reagents.28. The method of any of claims 1-3, wherein said one or more modified or unmodified BIM fragment peptides is one or more of the peptides in Table 1.

29. The method of claim 1, comprising quantifying the amount of the peptides in Table 1.

30. A composition comprising one or more, two or more, or all three of the peptides in Table 1 and/or antibodies thereto.

31. The composition of claim 30 comprising nucleic acids coding for one, two or three of the peptides of Table 1, and/or the complement of nucleic acids coding for one, two or three of the peptides of Table 1.

32. A method of determining the resistance of a cancer to one or more kinase inhibitor(s), comprising determining the presence or level of BIM protein(s) in a cancer tissue or cancerous cells thereof employing an SRM/MRM assay;

wherein the presence of biologically active BIM protein(s) capable of mediating apoptosis of cells in said cancer tissue or cancerous cells thereof is indicative of the susceptibility of said cancer to one or more of said kinase inhibitor(s).

34. The method of claim 32, wherein one or more of said inhibitor(s) is an:

antibody, human antibody;

humanized antibody;

chimeric antibody;

monoclonal antibody;

monospecific antibody;

recombinant antibody;

antigen-binding antibody fragment;

single chain antibody;

diabody;

triabody;

tetrabody;

Fab fragment;

F(ab′

)2 fragment;

domain antibody;

IgD antibody;

IgE antibody;

IgM antibody;

IgG1 antibody;

IgG2 antibody;

IgG3 antibody;

or IgG4 antibody.

35. The method of claim 34, wherein said antibody is selected trastuzumab, cetuximab, and panitumumab.

37. The method of claim 32, wherein said SRM/MRM assay comprises detecting and/or quantifying the level of one or more modified or unmodified BIM fragment peptides in a protein digest.

38. The method of claim 37, wherein one or more of said fragment peptides is selected from a peptide of SEQ ID NO.:

1, SEQ ID NO.;

2, or SEQ ID NO.;

3.

33. A method of determining the resistance of a cancer to one or more EGFR, HER2, and/or PI3K inhibitor(s), comprising determining the presence or level of BIM proteins in a cancer tissue or cancerous cells thereof using an SRM/MRM assay;

wherein the presence of biologically active BIM protein(s) capable of mediating apoptosis of cells in said cancer tissue or cancerous cells thereof is indicative of the susceptibility of said cancer to one or more of said inhibitor(s).

36. A method of determining the resistance of a cancer to one or more therapeutics selected from Lapatinib, Erlotinib, Gefitinib, Vandetanib, Pelitinib, Canertinib, Foretinib, Critzotinib, Afatinib, Cabozantinib, Axitinib, Vatalanib, BMS-536924, OSI-906 Saracatinib, Ponatinib, or any combination of two, three or four of those therapeutics;

said method comprising determining the presence or level of BIM protein(s) in a cancer tissue or cancerous cells thereof using an SRM/MRM assay;

wherein the presence of biologically active BIM protein(s) capable of mediating apoptosis of cells in said cancer tissue or cancerous cells thereof is indicative of the susceptibility of said cancer to said therapeutics.