Development and characterization of a bispecific single-chain antibody directed against T cells and ovarian carcinoma

Bispecific antibodies with specificity for tumor antigen and CD3 have been shown to redirect the cytotoxicity of T cells against relevant tumor, Our o bjective was to generate single-chain bispecific antibodies (bsSCA) that co uld retarget mouse cytotoxic T lymphocytes (CTL) to destroy human ovarian c arcinoma in a xenogeneic setting. A bsSCA, 2C11 x B43.13, was constructed b y genetic engineering and expressed in mammalian cells. Molecular character istics, binding properties, and ability to retarget CTL were studied. Weste rn blot analysis showed that the product is a 65-kDa protein. Purification of antibodies could be done by single-step affinity chromatography using pr otein L-agarose with an unoptimized yield of 200 mu g/L. BsSCA 2C11 x B43.1 3 was capable of binding to mouse CD3 and human CA125 as detected by FAGS a nalysis of EL4 and OVCAR Nu3H2 cells, respectively. It could also bridge ac tivated splenic T cells and human ovarian carcinoma as demonstrated by a br idge FAGS assay. Redirected mouse CTL could mediate human target cell lysis in a 20-h Cr-51 release assay despite that they are xenogeneic. Prolonged incubation of redirected CTL and tumor targets resulted in a dramatic reduc tion in tumor cell number. CD28 co-stimulation enhanced redirected CTL func tion in both types of assays. BsSCA 2C11 x B43.13 thus can be used as a pre clinical immunotherapeutic model for human ovarian cancer in a xenogeneic s etting.