Human ovarian carcinoma lysis by cytotoxic T cells targeted by bispecific monoclonal antibodies: analysis of the antibody components.
In the perspective of in vivo therapeutic applications, the monoclonal antibody (MAb) MOv18 was selected for its restricted reactivity with human ovarian carcinoma. Using the pH 2.8 desorption assay, we found that the antigen recognized by MOv18 had a high stability on the cell membrane and poor internalization. Therefore, a therapeutic approach which does not require internalization, i.e., the re-targeting of cytotoxic T lymphocytes (CTL) by bispecific MAbs, was investigated. MOv18 and anti-CD3 MAbs were used to produce bispecific reagents, obtained either by chemical cross-linkage (hetero-conjugates) or by somatic hybridization techniques (hybrid MAbs). The maintenance of the binding reactivity and specificity of the bispecific MAbs was analyzed by solid-phase radioimmunoassay, immunofluorescence and cross-competition tests on the relevant target cells (ovarian carcinoma cell line OVCA 432 for MOv18 and PHA-stimulated peripheral blood mononuclear cells for anti-CD3 MAbs), and on 2 irrelevant tumor cell lines. Bv a 51Cr-release assay the bispecific MAbs were found to efficiently promote, at picomolar concentration, cell lysis by CTL clones, but the specificity pattern was wider than that predicted by the binding studies. The F(ab')2 fragment of one hybrid MAb mediated a lysis which was just as efficient as the entire MAb on the relevant target cells and allowed specific lysis to be distinguished from Fc-receptor-mediated lysis. Human immunoglobulins were unable to compete with the Fc receptor binding of the hybrid MAbs and therefore, in the perspective of in vivo applications, Fc fragment removal seems to be an essential step. Analysis of the bispecific reagents indicated that hybrid MAbs are superior to the heteroconjugate as far as storage stability is concerned.
Mezzanzanica
D
D
Division of Experimental Oncology E, Istituto Nazionale per lo Studio e la Cura dei Tumori, Milan, Italy.
Canevari
S
S
Ménard
S
S
Pupa
S M
SM
Tagliabue
E
E
Lanzavecchia
A
A
Colnaghi
M I
MI
D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis* / Q000276:immunology; D000945:Antigens, Differentiation, T-Lymphocyte / Q000276:immunology; D000951:Antigens, Neoplasm / Q000032:analysis; D003602:Cytotoxicity, Immunologic; D005260:Female; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000276:immunology; D007136:Immunoglobulins / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D010051:Ovarian Neoplasms / Q000276:immunology*; D011961:Receptors, Fc / Q000276:immunology; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000945:Antigens, Differentiation, T-Lymphocyte; D000951:Antigens, Neoplasm; D007140:Immunoglobulin Fab Fragments; D007136:Immunoglobulins; D011961:Receptors, Fc