CONSTRUCTION AND EVALUATION OF A MEMBRANE-LYTIC IMMUNOCONJUGATE SELECTIVE FOR COLON CANCER CELLS

DÍAZ, I.(1), FIGUEREDO, R.(2), ROQUE, L.(2), PAZOS, F.(1), MARTÍNEZ, D.(1), IZNAGA-ESCOBAR, N.(2), PÉREZ, R.(2), ALVAREZ, C.(1), LANIO, M. E.(1), TEJUCA, M.(1)

(1)Centro de Estudios de Proteínas y Departamento de Bioquímica, Facultad de Biologia, Universidad de La Habana, Ciudad de La Habana, Cuba, (2)Centro de Inmunología Molecular, Ciudad de La Habana, Cuba.

Sticholysin I, a potent cytolysin isolated from the sea anemone Stichodactyla helianthus, was linked to the monoclonal antibody iorC5. Sticholysin I acts by forming oligomeric hydrophilic pores in the membrane of the attacked cells, impairing cellular functions and leading to osmotic lysis. The monoclonal antibody iorC5 is a murine IgG₁, which recognizes the tumor associated antigen ior C2, a glycoprotein expressed at the cell surface in more than 83 % of primary colorectal carcinomas. The cytolysin and the monoclonal antibody were coupled by using the heterobifunctional cross-linking reagent Sulfosuccinimidyl 4-(N-Maleimidomethyl)-cyclohexane-1-carboxylate (SMCC). Two hybrid molecules composed by one ior C5 and one or two Sticholysin I molecules were obtained, as demonstrated by electrophoresis. The conjugates called I and II, respectively, were purified by hydrophobic interaction chromatography in a Progel-TSK Phenyl-5PW column. The purified conjugates were evaluated by a binding affinity assay against an iorC2-positive colon cancer cell line (SW948), and compared with the unconjugate dior C5. The hybrid molecules were able to recognize their specific antigen in the same way that ior C5 does. Both conjugates lost most of their hemolytic activity but their residual activity was very similar. Nevertheless, when we studied their cytotoxicity on the colon cancer cell line SW948, only the conjugate II killed efficiently the cells, indicating a specific antigen-monoclonal antibody interaction of this hybrid molecule. This suggests that this could be a useful approach for immunotoxin design.

CORRESPONDENCE TO:

María Eliana Lanio, Centro de Estudio de Proteínas, Facultad de Biología, Universidad de la Habana, Calle 25, entre J e I, Vedado, Ciudad Habana, Cuba, Email: mlanio@infomed.sld.cu