Specificity and properties of MAb RS7-3G11 and the antigen defined by this pancarcinoma monoclonal antibody

Abstract

The murine monoclonal antibody (MAb) RS7-3G11 is an IgG1 with pancarcinoma reactivity, which has been raised against human squamous-cell carcinoma of the lung. Immunoperoxidase staining of frozen tissue sections demonstrated that the antigen defined by RS7-3G11 is present in tumors of the lung, stomach, bladder, breast, ovary, uterus and prostate. The rate and extent of internalization of RS7-3G11 into Calu-3, an adenocarcinoma of the lung cell line, was investigated using unconjugated MAb, followed by fluorescence labelling, and by binding 125I-RS7-3G11 followed by acid removal of surface-bound antibody. Rapid internalization of MAb RS7-3G11 into target cells was observed. Antibody internalization was noted at 30 min, and by 2 hr virtually all MAb RS7-3G11 was internal. Although MAb RS7-3G11 was raised against non-small-cell carcinoma of the lung, ME-180, a cervical-carcinoma cell line, expresses higher quantities of the antigen than the lung-carcinoma cell lines. Due to the higher antigen density in ME-180 cells, this line was used for immunoprecipitation studies and antigen purification. Immunoprecipitation studies using the ME-180 cervical-carcinoma cell line metabolically labeled with [3H]leucine or [3H]glucosamine demonstrated that the antigen defined by RS7-3G11 is a glycoprotein of M(r) 46 kDa. Deglycosylation by treatment with endoglycosidase-F resulted in a protein with a M(r) of 35 kDa. RS7-3G11-antigen was purified from ME-180 tissue-culture cells using affinity-column chromatography. By SDS-PAGE it was seen that the antigen was highly purified. The major band appeared at M(r) of 45 to 48 kDa. This result is in agreement with the immunoprecipitation studies. The broad band observed in the SDS-PAGE is typical of many glycoproteins, and suggests heterogeneity of glycosylation. Chemical and enzymatic treatments of the antigen, followed by Western blot analyses, suggest that the RS7-3G11 antigenic determinant is composed of a conformation-dependent peptide.