CONSTRUCTION, EXPRESSION, AND CHARACTERIZATION OF BD1-G28-5 SFV, A SINGLE-CHAIN ANTI-CD40 IMMUNOTOXIN CONTAINING THE RIBOSOME-INACTIVATING PROTEIN BRYODIN-1

The major limitation to the use of immunotoxins in the clinic is the t oxicity associated with the toxin moiety, BD1-G28-5 single-chain Fv (s Fv) is a single-chain immunotoxin targeted to human CD40 and consists of bryodin 1 (BD1), a plant ribosome-inactivating protein that is 20-3 0-fold less toxic in animals than commonly used toxins, fused to the s Fv region of the anti-CD40 monoclonal antibody G28-5, This immunotoxin was expressed in Escherichia coli and purified from refolded inclusio n bodies. BD1-G28-5 sFv retained the full protein synthesis inhibition activity of recombinant BD1 and specifically bound to CD40 with a bin ding affinity, k(d), of 1.5 nM, within 10-fold of the bivalent parenta l monoclonal antibody, BD1-G28-5 sFv was potently cytotoxic against CD 40-expressing B lineage non-Hodgkin's lymphoma and multiple myeloma ce ll lines, with EC50 values in the ng/ml range, but not against a CD40- negative T cell line, Interestingly, BD1-G28-5 sFv was not cytotoxic a gainst CD40-expressing carcinoma cell lines that were sensitive to a B D1-based immunotoxin conjugate targeted to the Le(y) carbohydrate anti gen, These data represent the first report indicating that BD1 can be used in the construction of potent single-chain immunotoxins, Addition ally, although BD1-G28-5 sFv effectively killed CD40-expressing hemato logic malignancies, its lack of activity against CD40-expressing carci nomas suggests that CD40-mediated trafficking of BD1 differs in the tw o cancer types.