Generation and characterization of a single gene-encoded single-chain-tetravalent antitumor antibody

Monoclonal antibody (mAb) CC49, a murine IgG1, reacts with the tumor-associ ated glycoprotein-72 expressed on a variety of carcinomas. In clinical tria ls, radiolabeled CC49 has shown excellent tumor localization to a variety o f carcinomas. To minimize the immunogenicity of CC49 mAb in patients, a hum anized CC49 (HuCC49) was generated by complementarity-determining region (C DR) grafting. The relative affinity of HuCC49 was 2-3-fold less than that o f the murine mAb. With the aim of improving tumor targeting, attempts have been made to enhance the avidity of the HuCC49 mAb. Previous research has y ielded a single gene-encoded immunoglobulin, SCIgcCC49 Delta CH1, which is a dimer of a single chain consisting of CC49 single-chain Fv linked to the NH2 terminus of the human gamma 1 Fc through the hinge region, This molecul e is comparable to the mouse-human chimeric CC49 in terms of in vitro antig en binding properties, cytolytic activity, and rate of plasma clearance in athymic mice bearing human tumor xenografts. Recently, a single gene encodi ng a single-chain immunoglobulin consisting of a HuCC49 diabody attached to human gamma 1 Fe via the hinge region was constructed. The diabody, a biva lent antigen-binding structure, is made up of variable heavy (V-H)/ variabl e light (V-L) domains and V-L/V-H domains. In each of the variable domain p airs, the V-H and V-L domains are linked through a short linker peptide. Me anwhile, the two pairs are linked via a 30-residue Gly-Ser linker peptide t o yield two antigen-binding sites by lateral and noncovalent association of the V-L of one pair with the V-H of the other. Transfectomas expressing th e single-gene immunoglobulin secrete a homodimer of about M-r 160,000 that reacts to tumor-associated glycoprotein-72, This tetravalent humanized anti tumor immunoglobulin molecule may potentially be an efficacious therapeutic and diagnostic reagent against a wide range of human carcinomas.