Methods and Compositions for Screening And Detecting Biomarkers

Inventors

Nancy Tommye Ann Jordan

Assignees

1. EDP Biotech Corporation, Knoxville, TN (US)

Abstract

The present invention is concerned with a novel antigens associated with human tumors, including carcinomas of the colon or lung, as well as novel monoclonal antibodies which specifically bind to said antigen. The antibodies bind to normal human cells to a much lesser degree than to tumor cells. The antigens include 100 kDa glycoprotein that has a colorectal cancer membrane bound and a soluble form, has a UV absorbance peak at about 228 nm, an isoelectric point of about 3.5 to 4, a sialic acid content of about 20% and does not substantially bind to an antibody specific for ACT. The antibodies find use both in methods such as the detection of malignant cells associated with tumors and in monitoring therapeutic treatment of humans with tumors.

CovalX Technology Used

K200

HM3

Complex Tracker

Outcomes

Antibodies 5E5 and 5A1-1 have both been shown to identify a biomarker for colorectal cancer in serum obtained from patients. Antigen 168B is a partially glycosylated form of CEACAM5. Thus samples were obtained of Antibody 1 5E5-1: 0.2 mg/ml in 2 ml of glycine buffer, Antibody 2 5A1-1: 1.2 mg/ml in 2ml of glycine buffer and Antigen 168B: 0.8 mg/ml in 100 μL of PBS (pH 7.4). Three samples of mAb/Ag interactions were created.

• A Antigen:168B Antibody: 5E5-1 Mix 168B/5E5-1
  • 5 μL antigen (4 mM) mixed with 5 μL antibody (2 mM)
• B Antigen:168B Antibody: 5A1-1 Mix 168B/5A1-1
  • 5 μL antigen (4 mM) mixed with 5 μL of antibody (2 mM)
• C Antigen: 168B Antibody: 5A1-1/5E5-1 Mix 168B/5A1-1/5E5-1
  • 5 μL of antigen (4 mM) mixed with 2 μL of each antibody (2 mM)

1 μL of each mixture was mixed with 1 μL of sinapic acid matrix (recrystallized (10 mg/mL) in acetonitrile/water (1:1, v/v) with 0.1% TFA) from the CovalX K200 Kit. 1 μL of each final mixture was spotted on a MALDI plate and allowed to crystallize at room temperature before being analyzed by mass spectrometer. The remaining 9 μL of each mixture were cross-linked using the CovalX K200 kit. The 9 μL samples were mixed with 1 μL of K200 reagent (2 mg/mL) and allowed to crystallize at room temperature for 180 minutes before analyzed by MALDI mass spectrometry. The mass spectrometer used was modified with a CovalX HM3 detection system and the data obtained was analyzed using the CovalX Complex Tracker software. From the data, researchers were able to determine at what peaks the antigen and antibody were detected at.

• Mix A
  • Antigen 168B: 92.879 kDa
  • Antibody 5E5-1: 158.498 kDa
  • Crosslinked Antibody 5E5-1•B168: 258.945 kDa
  • Crosslinked Antibody 5E5-1•2B168: 354.164 kDa
  • Confirmed detection of two noncovalent complexes by Complex Tracker data overlay
    • [5E5-1•B168]
    • [5E5-1•2B168]
• Mix B
  • Antigen 168B: 92.743 kDa
  • Antibody 5A1-1: 159.125 kDa
  • Crosslinked Antibody 5A1-1•B168: 259.155 kDa
  • Crosslinked Antibody 5A1-1•2B168: 355.025 kDa
  • Confirmed detection of two noncovalent complexes by Complex Tracker overlay
    • [5A1-1•B168] (antibody binding with one antigen)
    • [5A1-1•2B168] (antibody binding with two antigens
• Mix C
  • Antigen 168B: 92.910 kDa
  • Antibodies 5E5, 5A1-1: 158.988 kDa
  • Crosslinked Peaks: 260.155 kDa and 356.012 kDa
  • Confirmed detection of two noncovalent complexes by Complex Tracker overlay
    • [5E5-1•B168]
    • [5E5-1•2B168]

Patent Number

US2015/0309028 A1