ENGINEERING HIGH-AFFINITY HUMANIZED ANTI-P185(HER2) ANTI-CD3 BISPECIFIC F(AB')(2) FOR EFFICIENT LYSIS OF P185(HER2) OVEREXPRESSING TUMOR-CELLS/

We previously constructed a humanized anti-p185(HER2)/anti-CD3 bispeci fic antibody variant, BsF(ab')(2) v1 which retargets the cytotoxic act ivity of human T cells in vitro against human breast tumor cells which overexpress the p185(HER2) product of the HER2/neu (c-erbB-2) protoon cogene. Subsequently we identified an improved anti-CD3 variant, v9, w hich binds to T cells with approx. 100-fold higher affinity than the o riginal variant, v1. Here we demonstrate that BsF(ab')(2) v9 is more p otent than BsF(ab')(2) v1 in stimulating the proliferation of both res ting peripheral blood lymphocytes (PBL) and IL-2-activated, long-term cultured T lymphocytes (ATL). In addition, at low concentrations (0.01 -1 ng/ml) BsF(ab')(2) v9 is much more efficient than BsF(ab')(2) v1 in directing lysis of p185(HER2)-overexpressing tumor cells by IL-2 acti vated PBL. In contrast, at higher concentration BsF(ab')(2) v9 and BsF (ab')(2) v1 have similar potency in retargeted cytotoxicity. At BsF(ab ')(2) v9 concentrations of greater than or equal to 1 ng/ml the suscep tibility of p185(HER2)-expressing tumor cells to lysis is apparently i ndependent of the level of p185(HER2) expression. At lower concentrati ons of BsF(ab')(2) v9 and/or lower ratios of effector to target cells the extent of lysis is reduced, in some cases improving the selectivit y of lysis of high p185(HER2) expressors over low expressors. Thus sel ection of a high affinity anti-CD3 arm is likely important in the desi gn of BsF(ab')(2) for retargeting the cytotoxicity of T cells to tumor s. The dose of BsF(ab')(2) v9 in any future clinical evaluation will r equire optimization to maximize anti-tumor efficacy whilst minimizing potential toxicity against normal tissue expressing p185(HER2). (C) 19 95 Wiley-Liss, Inc.