Human tumour-associated cell adhesion protein MN/CA IX: identification of M75 epitope and of the region mediating cell adhesion

Abstract

MN/CA IX is a cell surface protein, strongly associated with several types of human carcinomas. It exerts activity of carbonic anhydrase and capacity of binding to cell surface receptors. In the present work, we used affinity purified MN/CA IX protein to demonstrate that the cells adhere to immobilized MN/CA IX and that the monoclonal antibody M75 abrogates cell attachment to MN/CA IX. Using synthetic oligopeptides, we identified M75 epitope and located it in the proteoglycan domain, which contains a sixfold tandem repeat of six amino acids GEEDLP. From phage display library of random heptapeptides we identified and chemically synthesized those which compete for the epitope with M75 and inhibit adhesion of cells to MN/CA IX. These heptapeptides might serve as lead compounds for drug design. © 2000 Cancer Research Campaign

Soluble form of carbonic anhydrase IX (CA IX) in the serum and urine of renal carcinoma patients

“…One of them is enzymatic activity; CA IX belongs to carbonic anhydrases with the highest activity (Wingo et al, 2001). In addition, CA IX is a cell adhesion molecule, most likely involved in cell-to-cell communication; its binding site overlaps with the M75 epitope (Závada et al, 2000).…”

“…The isotype of mAb M75 is IgG 2b and that of V-10 is IgG 2a. The IgG was purified from hybridoma TC fluid by affinity chromatography as previously (Závada et al, 2000). For the concentration of immune complexes formed by the mAbs with CA IX, antigen from TC media or cell extracts Protein A-Sepharose (Sigma) was used: the mAb-CA IX complexes from human sera or urine were adsorbed to anti-mouse IgG-Agarose (Sigma).…”

“…Western blot analysis Sodium dodecyl sulphate -polyacrylamide gel electrophoresis and Western blotting were performed as described before (Závada et al, 2000). Carbonic anhydrase IX antigen on the blots was visualised by M75-Px conjugate, followed by enhanced chemiluminescence (ECL) and exposure to X-ray film Medix XG (FOMA, Czech Republic), with maximum sensitivity to yellow-green light.…”

“…the V-10 antibody used in this report). The epitope of our original M75 antibody is located in the PG domain of CA IX and its amino-acid sequence is PGEEDLP (Závada et al, 2000).…”

Therapeutic Mechanism and Efficacy of the Antibody–Drug Conjugate BAY 79-4620 Targeting Human Carbonic Anhydrase 9

“…By contrast, the expression of CAIX on normal tissues is largely restricted to the apical surface of cells of the stomach, bile duct mucosa (4,11), and small intestine (4,12). The extracellular domain of this type I transmembrane protein comprises both a proteoglycan domain implicated in cell adhesion through homotypic interaction (13) and the carbonic anhydrase domain that catalyzes the reversible hydration of carbon dioxide to bicarbonate and protons (14) and is involved in the regulation of the pH within the tumor environment (15).…”