TARGETING OF T-LYMPHOCYTES AGAINST EGF-RECEPTOR(-CELLS BY BISPECIFIC MONOCLONAL-ANTIBODIES - REQUIREMENT OF CD3 MOLECULE CROSS-LINKING FOR T-CELL ACTIVATION() TUMOR)

Targeting of T lymphocytes against epidermal growth-factor-receptor (E GF-R)+ tumor cells was achieved by constructing a hybrid hybridoma whi ch secretes an anti-EGF-R/anti-CD3 bispecific monoclonal antibody (biM Ab) of hybrid isotype (IgG1/IgG2a). Purification of biMAb molecules fr om parental anti-EGF-R and anti-CD3 MAbs was performed by protein-A ch romatography. The purified biMAb was able to trigger the lysis of EGF- R+ tumor cell lines (A431, IGROV-1, MDA-468 and U-87) and of NIH-3T3 t ransfectants expressing human EGF-R by cytolytic T lymphocytes, but it was ineffective in the case of EGF-R-negative tumor targets. Normal E GF-R+ cells (keratinocytes and endometrial cells) were also susceptibl e to biMAb-targeted cytolysis. However, the amount of biMAb required t o induce half-maximal cytolysis of tumor cells over-expressing the EGF -R molecule (A43 1) was considerably lower than that required to induc e lysis of EGF-R+ tumor or normal cells which express EGF-R at conside rably lower density. The ability of such biMAbs to deliver activation signals to T cells was evaluated by Ca++ mobilization and lymphokine p roduction experiments. The soluble anti-EGF-R/anti-CD3 biMAb failed to induce intracellular Ca++ increases, which occurred only after cross- linking induced by an anti-mouse IgG antibody. Secretion of lymphokine s (IFN-gamma, TNF-alpha and GM-CSF) was induced by contact of the biMA b-coated effector cells with the relevant tumor target, whereas the so luble biMAb was virtually ineffective. In addition, biMAb-coated effec tor cells retained the ability to recognize and to lyse EGF-R+ tumor c ells for a prolonged period of time. Our data indicate that activation of effector cells targeted by biMAbs can only occur at the tumor site , where cross-linking of surface CD3 molecules is induced by contact w ith the tumor cells. (C) 1993 Wiley-Liss, Inc.