Targeting of T lymphocytes against EGF-receptor+ tumor cells by bispecific monoclonal antibodies: requirement of CD3 molecule cross-linking for T-cell activation.
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/IgG2a). 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+ cells (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 (A431) 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-gamma, TNF-alpha 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.
Ferrini
S
S
Istituto Nazionale per la Ricerca sul Cancro, Genoa, Italy.
Cambiaggi
A
A
Sforzini
S
S
Marciano
S
S
Canevari
S
S
Mezzanzanica
D
D
Colnaghi
M I
MI
Grossi
C E
CE
Moretta
L
L
D018033:Antibodies, Bispecific / Q000276:immunology*; D017252:CD3 Complex / Q000276:immunology*; D004306:Dose-Response Relationship, Immunologic; D066246:ErbB Receptors / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D007074:Immunoglobulin G / Q000276:immunology*; D008213:Lymphocyte Activation / Q000276:immunology*; D008222:Lymphokines / Q000378:metabolism; D013601:T-Lymphocytes / Q000276:immunology* / Q000378:metabolism
D018033:Antibodies, Bispecific; D017252:CD3 Complex; D007074:Immunoglobulin G; D008222:Lymphokines; D066246:ErbB Receptors