Novel Anti-CD19/Idiotype Bispecific Diabody Vaccine for B-Cell Lymphoma.

Abstract 2712

Poster Board II-688

The unique immunoglobulin idiotype (Id) on B-cell lymphoma is an excellent target for a “custom-made” vaccine therapy for individual patients. Previous studies have shown that vaccine-induced anti-Id humoral responses are effective in protecting mice from the aggressive 38C13 lymphoma. Therefore, we explore a novel strategy of direct activation of Id-specific B cells using a bispecific diabody containing the Id variable fragments (Fv) of 38C13, and those of an anti-CD19 monoclonal antibody. This molecule is designed to cross-link the Id-specific B cell receptor to the CD19/CD21/CD81 co-stimulatory complex, an interaction that has been shown to lower the activation threshold of B cells and result in a dramatic increase in antibody production. The αCD19/Id diabody is a 52 kDa heterodimer of two single chain Fv's (scFv), αCD19VH-38C13VL and 38C13VH -αCD19VL, held together by non-covalent interactions between the cognate VH and VL domains. It was produced in an E. coli extract-based Cell-Free Protein Synthesis (CFPS) System developed by our group, which has been used successfully to rapidly produce cytokine-Id fusion vaccines in previous studies. Within 2 hours, the two diabody scFv's were co-expressed and self-assembled in vitro. The heterodimer product was purified by metal affinity chromatography. Correct folding was confirmed by a flow cytometry assay in which the diabody bound simultaneously to the CD19 on the surface of B cells and also to an anti-38C13 Id antibody labeled with FITC. Most importantly, as a vaccine the αCD19/Id diabody generated a robust anti-Id IgG response comparable to that induced by 38C13 IgM conjugated to the carrier protein keyhole limpet hemocyanin (KLH). Diabody vaccinated mice also showed improved survival compared to control groups when both were challenged with a lethal dose of tumor. Further optimization of the therapeutic efficacy and the development of improved methods for the rapid and economical production of αCD19/Id diabody by the CFPS technology may make it possible to use individualized diabody-based therapy as a frontline treatment, before the use of toxic chemotherapy and radiation therapy.