Brian K. Link, Sheri A. Kostelny, Michael S. Cole, William P. Fusselman, J. Yun Tso, George J. Weiner

Anti-CD3 x anti-B-cell antigen bispecific monoclonal antibodies (bsAbs) can redirect T-cell-mediated lysis toward malignant B cells. Clinical trials with CD3-based bsAbs have shown toxicity in patients which is likely related to nonspecific T-cell activation and targeting. Our current studies were designed to explore the mechanisms responsible for the observed in vivo toxicity by evaluating the immunologic effects of 2 different bsAb preparations in vitro. 1D10 was used as the tumor specific arm of the bsAbs. This antibody reacts with a variant of HLA-DR found on a majority of pre-B- and B-cell malignancies, and normal B cells in some individuals. Anti-CD3 served as the T-cell specific arm. A 1D10 x anti-CD3 bispecific IgG (bsIgG) produced using the hybrid-hybridoma method was compared to a 1D10 x anti-CD3 bispecific F(ab')2 [bsF(ab')2] produced using the leucine zipper technique. In cytotoxicity assays, both bsIgG and bsF(ab')2 induced lysis by pre-activated T cells of 1D10 (+) malignant B cells. bsIgG at high concentrations also induced lysis of 1D10 (-) tumor cells, while bsF(ab')2 did not. Proliferation of T cells induced by bsIgG and bsF(ab')2 was also evaluated. Both forms of bsAbs induced T-cell proliferation in the presence of antigen (+) Raji cells, while only bsIgG did so in the presence of antigen (-) malignant B cells. bsF(ab')2 induced T-cell activation in the absence of any tumor cells when testing was performed on samples where the 1D10 target antigen was present on normal peripheral blood B cells. We conclude that non-specific T-cell activation from bsAbs can occur in an antigen-independent manner due to the Fc/Fc receptor (FcR) interaction, or in an antigen-dependent manner when antigen is expressed on normal or tumor cells. Both mechanisms may have been responsible for the toxicity observed in prior clinical studies.