SINGLE-CHAIN FV FOLATE CONJUGATES MEDIATE EFFICIENT LYSIS OF FOLATE-RECEPTOR-POSITIVE TUMOR-CELLS/

Bispecific antibodies that bind to a tumor antigen and the T cell rece ptor (TCR) redirect cytotoxic T lymphocytes (CTL) to lyse tumor cells which have escaped normal immune recognition mechanisms. One well-char acterized tumor antigen, the folate receptor (FR), is expressed on mos t ovarian carcinomas and some types of brain cancer. Recently, it was shown that conjugates of folate and anti-TCR antibodies are extremely potent bispecific agents that target tumor cells expressing the high-a ffinity folate receptor, but not normal cells expressing only the redu ced folate carrier protein. In this paper, it is shown that the size o f these conjugates can be reduced to the smallest bispecific agent yet described (30 kDa) by attaching folate to a single-chain antibody, sc Fv, of the anti-TCR antibody KJ16. The scFv/folate conjugates are as e ffective as IgG/folate conjugates in mediating lysis of FR; tumor cell s by CTL. The optimal folate density was in the range of 5-15 folate m olecules per scFv or IgG molecule, which yielded half-maximal lysis va lues (EC50) of approximately 40 pM (1.2 ng/mL for scFv). Finally, the scFv/folate conjugates could efficiently target tumor cells even in th e presence of free folic acid at concentrations that are normally foun d in serum. Compared to conventional bispecific antibodies, the small size of scFv/folate conjugates may prove advantageous in the ability t o penetrate tumors and in reduced immunogenicity.