Anti-CD3 x anti-tumor F(ab')₂ bifunctional antibody activates and retargets tumor-infiltrating lymphocytes

Bifunctional Abs (BFA) with specificity for the TCR/CD3 complex of T cells and tumor Ag can bridge T lymphocytes and tumor cells and, thereby, trigger activation events. The ability of intact and F(ab')₂ anti-CD3 (500A2) x anti-p97 (96.5) BFA to induce activation of T lymphocytes in the presence of murine melanoma tumor cells (CL-62) expressing human melanoma-associated Ag (p97) was investigated in vitro and in vivo. Intact and F(ab')₂ BFA induced significant proliferation of T lymphocytes in the presence of p97⁺ tumor cells. Incubation of splenocytes with intact or F(ab')₂ BFA and p97⁺ tumor cells increased BFA-mediated cytotoxicity against relevant tumor cells. Intact BFA, in contrast to F(ab')₂ BFA, induced some activation of T cells in vitro even in the absence of p97⁺ target cells. In nontumor-bearing mice, administration of F(ab')₂ BFA, in contrast to intact BFA, did not increase cytotoxic activity of lymph node (LN) cells and splenocytes. However, when F(ab')₂ BFA was administrated into CL-62-bearing mice, an increase of BFA- mediated cytotoxicity of tumor-infiltrating lymphocytes, but not splenocytes nor LN cells, was observed. Moreover, in D-galactosamine-sensitized mice, injection of intact BFA (1 μg/mice) induced 100% lethality, whereas the same dose of F(ab')₂ BFA was not toxic. These results demonstrate that F(ab')₂ BFA can induce activation of cytotoxic lymphocytes only in the presence of relevant tumor cells, both in vitro and in vivo. That these activated lymphocytes can be redirected to lyse relevant tumor cells by the same BFA has important implications for the clinical application of BFA anti-tumor therapies.