Methods of Producing Antibodies Specific for p95

• US 20150376293A1
• Filed: 06/12/2015
• Published: 12/31/2015
• Est. Priority Date: 12/01/2008
• Status: Active Grant

• Current Assignee: Laboratory Corporation Of America Holdings
• Original Assignee: Laboratory Corporation Of America Holdings
• Inventors: Sperinde, Jeff, Winslow, John William, Jin, Xueguang, Wallweber, Gerald J.
• Granted Patent: US 10,273,308 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C07K 16/32 against translation products of oncogenes
• CPC Class Codes: C07K 2317/30 characterized by aspects of specificity or valency
• CPC Class Codes: C07K 2317/34 Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
• CPC Class Codes: C07K 2317/73 Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
• 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 2333/71 for growth factors; for growth regulators
• 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 33/5005 involving human or animal cells immunoassay G01N33/56966; immunoassays of protozoa G01N33/56905; protozoa in screening assays C12Q1/025
• CPC Class Codes: G01N 33/57415 of breast
• CPC Class Codes: G01N 33/57484 involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
• CPC Class Codes: G01N 33/6893 related to diseases not provided for elsewhere
• CPC Class Codes: G01N 33/74 involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors

Abstract

The invention provides methods of measuring and/or quantifying the presence and/or amount of p95 and/or p95 complex in a sample. The invention also provides antibodies specific for p95, and methods of making such antibodies.

Claims

1-30. -30. (canceled)

31. A method of producing antibodies specific a p95Her-2 protein fragment having a first amino acid corresponding to methionine 611 of Her-2 protein (p95Her-2), comprising:

a) immunizing a mammal with an immunogen comprising a peptide comprising an amino acid sequence set forth in one of SEQ ID NOs 1-7;

b) generating one or more hybridoma cell lines expressing monoclonal antibodies from the immunized mammal;

c) screening the one or more hybridoma cell lines to identify a hybridoma cell line that express a monoclonal antibody that binds specifically to p95Her-2 but does not bind specifically to full length native Her-2 protein;

d) establishing monoclonal cultures for one or more specific hybridoma cell lines that express an antibody that binds specifically to p95Her-2; and

e) isolating one or more monoclonal antibodies that bind specifically to p95Her-2 but do not bind specifically to full length native Her-2 protein from the monoclonal cultures.

32. The method of claim 31, wherein the peptide of the immunogen is conjugated to keyhole limpet hemocyanin (KLH).

33. The method of claim 31, wherein the immunizing is performed over a period of about 5 weeks.

34. The method of claim 31, wherein the immunizing comprises twice-weekly administration of the immunogen to the mammal.

35. The method of claim 31, wherein the screening comprises analyzing culture supernatant from the one or more hybridoma cell lines using at least one of an enzyme-linked immunosorbent assay, Western blotting, or flow cytometry.

36. The method of claim 35, wherein the screening comprises using a peptide having an amino acid sequence as set forth in at least one of SEQ ID Nos:

1-7 to screen culture supernatant from the one or more hybridoma cell lines.

37. The method of claim 36, wherein the peptide is conjugated to bovine serum albumin (BSA).

38. The method of claim 31, wherein the screening comprises analyzing culture supernatant from the one or more hybridoma cell lines using an enzyme-linked immunosorbent assay for the ability to bind to at least one of a peptide having an amino acid sequence as set forth in at least one of SEQ ID Nos:

1-7 or full length native Her-2 protein.

39. The method of claim 35, wherein the screening comprises Western blot staining of cell lysates containing p95Her-2 or Her-2 using culture supernatant from the one or more hybridoma cell lines of step c).

40. The method of claim 35, wherein the screening comprises contacting cells expressing p95Her-2 or Her-2 with culture supernatant from the one or more hybridoma cell lines of step c) and analyzing the contacted cells by flow cytometry using a secondary antibody.

41. The method of claim 40, wherein the cells expressing p95Her-2 or Her-2 are cells that have been transiently transfected to express p95Her-2 or Her-2.

42. The method of claim 40, wherein the cells expressing p95Her-2 or Her-2 are fixed prior to analyzing by flow cytometry.

43. The method of claim 31, wherein the amino acid sequence of the peptide comprises SEQ ID NO:

5.

44. The method of claim 36, wherein the amino acid sequence of the peptide comprises SEQ ID NO:

5.

45. The method of claim 31, wherein the mammal is a mouse or a rabbit.