Bispecific F(ab'gamma)3 antibody derivatives for redirecting unprimed cytotoxic T cells

Abstract

Bispecific F(ab'gamma)3 derivatives have been constructed by selective coupling of monoclonal antibody Fab'gamma fragments, using the cross-linker o-phenylenedimaleimide attached to hinge-region sulfhydryl groups. All the derivatives were designed to redirect the cytotoxic activity of circulating human T lymphocytes, via their CD2, CD3 or CD5 molecules, against 51Cr-labeled chicken red blood cells (CRBC) or the human lymphoblastoid cell line, Namalwa. The composition of F(ab'gamma)3 molecules was controlled by the selection of Fab'gamma for initial attachment of cross-linker, allowing production of derivatives with either two anti-effector and one anti-target Fab'gamma arms or vice versa. Bispecific F(ab'gamma)3 derivatives, recruiting peripheral blood lymphocytes (PBL) via CD3 as effectors against CRBC, gave titers in redirected cellular cytotoxicity assays which were consistently 25-100 times higher than those given by an equivalent F(ab'gamma)2 reagent, with full activity being retained down to antibody concentrations of 1 ng/ml. The allocation of valencies--two anti-target/one anti-effector or the converse--was not important in determining the increase in titer. No significant lysis was seen when recruiting PBL via CD2 or CD5. It was notable that these CD3-specific derivatives yielded good tumor-cell lysis when using fresh, unprimed PBL as effectors. The increased activity over a bispecific F(ab'gamma)2 was apparent both in titer and the maximum level of lysis achieved. Blocking studies with excess Fab'gamma suggested that the potency of F(ab'gamma)3 derivatives lie in the strength of bridging between effector and target cells. This high avidity contact could favor metabolic activation in either the target or effector cell, or could increase the efficiency of lytic machinery already triggered.