Targeting of T lymphocytes against egf‐receptor+ tumor cells by bispecific monoclonal antibodies: Requirement of CD3 molecule cross‐linking for t‐cell activation

Abstract

Targeting of T lymphocytes against epidermal growth-factor-receptor (EGF-R)⁺ tumor cells was achieved by constructing a hybrid hybridoma which secretes an anti-EGF-R/anti-CD3 bispecific monoclonal antibody (biMAb) of hybrid isotype (IgG₁/ IgG_2a). Purification of biMAb molecules from parental anti-EGF-R and anti-CD3 MAbs was performed by protein-A chromatography. 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 transfectants expressing human EGF-R by cytolytic T lymphocytes, but it was ineffective in the case of EGF-R-negative tumor targets. Normal EGF-R⁺ (keratinocytes and endometrial cells) were also susceptible to biMAb-targeted cytolysis. However, the amount of biMAb required to induce half-maximal cytolysis of tumor cells over-expressing the EGF-R molecule (A43I) was considerably lower than that required to induce lysis of EGF-R⁺ tumor or normal cells which express EGF-R at considerably lower density. The ability of such biMAbs to deliver activation signals to T cells was evaluated by Ca⁺⁺ mobilization and lymphokine production 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 lymphokines (IFN-γ, TNF-α and GM-CSF) was induced by contact of the biMAb-coated effector cells with the relevant tumor target, whereas the soluble biMAb was virtually ineffective. In addition, biMAb-coated effector cells retained the ability to recognize and to lyse EGF-R⁺ tumor cells 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 with the tumor cells.