Serologic mapping and biochemical characterization of the carcinoembryonic antigen epitopes using fourteen distinct monoclonal antibodies

Abstract

A series of 14 monoclonal anti-carcinoembryonic antigen (CEA, Mr 180,000) antibodies (MAbs) that show a strong degree of selective reactivity for human colon carcinomas versus normal adult tissues were used to construct a serological map of the CEA molecule. The MAbs were generated using extracts of colon carcinomas as immunogen and are thus given a COL designation. None of the 14 COL-MAbs tested were reactive with purified non-specific cross-reacting antigen (NCA, Mr 55,000) from normal lung, although some showed reactivity to human granulocytes. All the COL-MAbs tested were reactive with normal fecal antigen-2 (NFA-2, Mr 170,000); however, many of the COL-MAbs demonstrated a higher affinity constant to CEA than to NFA-2. Cross-competition radioimmunoassays classified the 14 COL-MAbs into 5 groups. The chemical nature of the COL-binding domains was tested using chemically or enzymatically treated CEA; all reacted with periodate-treated CEA and deglycosylated CEA, indicating that the COL-reactive epitopes appear to be of a proteinaceous nature. Heat treatment, reduction, alkylation, pepsin digestion or pronase treatment of CEA, however, gave differential results with respect to COL binding. Antibody titration experiments were carried out to define differential reactivities to colorectal carcinoma versus NCA-containing granulocyte extracts; these results were compared with results obtained using several anti-CEA MAbs that have been used in clinical trials. Granulocyte binding and biochemical studies showed that the COL MAbs may distinguish as many as 7 to 10 CEA epitopes.

Monoclonal antibody COL-1 reacts with restricted epitopes on carcinoembryonic antigen: an immunohistochemical study.

“…The epitope recognized by MAb COG1 is resistant to periodate and neuraminidase, sensitive to pepsin, and partially resistant to heat, reduction alkylation, and pronase digestion (15). These data, and the reactivity of COL1 with deglycosylated CEA (15), suggest that COL1 reacts with a peptide determinant on CEA.…”

“…Previous reports have shown that MAb COLl reacts with NCA-2 and NFA-2, although both of these antigens had a much lower affinity for COLl than CEA (15). Since NCA-2 is present in meconium and NFA-2 is found in normal adult feces, this crossreactivity should not present a problem in applying COL1 in the differential diagnosis of colon neoplasms (9,ll).…”

“…Smooth muscle (15) Cardiac muscle (2) Urinary system Kidney ( 5 ) Bladder (3) Prostate (3) Testes (2) Breast (2) Uterus (1) Nervous system Cortex (2) Cerebellum (3) Peripheral nerve (2) Endocrine system Thyroid gland (3) Parathyroid ( with graded alcohols. Endogenous peroxidase activity was quenched by bathing slides in 3% H202 in methanol for 10 min at room temperature (RT).…”

Carcinoembryonic antigen regulation in human colorectal tumor cells by a site‐selective cyclic amp analogue: A comparison with interferon‐gamma

“…COL-1 (IgG,), COL-4 (IgG,), and COL-12 (IgG,,) were purified from ascites fluid by ammonium sulphate precipitation and ionexchange chromatography (Colcher et al, 1984). In a previous study (Kuroki et al, 1989), it was shown that COL-1, COL-4, and COL-12 all recognize separate and distinct epitopes on the CEA molecule. The description of MAb D612 has been published (Muraro et al, 1989).…”

Grafting of “Abbreviated” Complementarity-Determining Regions Containing Specificity-Determining Residues Essential for Ligand Contact to Engineer a Less Immunogenic Humanized Monoclonal Antibody

“…The mAb COL-1, an IgG2a, has a high affinity for CEA and has no detectable reactivity to granulocytes or to the CEA-related Ags NCA-1 and normal fecal Ag-1 (28). Murine COL-1 (mCOL-1) reacts with biopsies of colon and gastric carcinomas (80 and 88%, respectively) and with biopsies of human mammary and nonsmall-cell lung carcinomas (17,29).…”