A bispecific single-chain antibody directed against EpCAM/CD3 in combination with the cytokines interferon alpha and interleukin-2 efficiently retargets T and CD3+CD56+ natural-killer-like T lymphocytes to EpCAM-expressing tumor cells.
Cytokine-induced killer cells (CIK), generated in vitro from peripheral blood mononuclear cells (PBMC) by addition of interferon gamma (IFNgamma), interleukin-2 (IL-2), IL-1 and a monoclonal antibody (mAb) against CD3, are highly efficient cytotoxic effector cells with the CD3+CD56+ phenotype. In this study, we evaluated whether the cytotoxicity of these natural-killer-like T lymphocytes against the colorectal tumor cell line HT29 can be enhanced by the addition of a bispecific single-chain antibody (bsAb) directed against EpCAM/CD3. For determination of bsAb-redirected cellular cytotoxicity we used a new flow-cytometric assay, which directly counts viable tumor cells and can assess long-term cytotoxicity. We found that this bsAb induced distinct cytotoxicity at a concentration above 100 ng/ml with both PBMC and CIK at an effector-to-target cell ratio as low as 1:1. CIK cells revealed higher bsAb-redirected cytotoxicity than PBMC. Cellular cytotoxicity appeared after 24 h whereas PBMC showed the highest bsAb-redirected cytotoxicity after 72 h. The addition of the cytokines IL-2 and IFNalpha but not granulocyte/macrophage-colony-stimulating factor enhanced bsAb-redirected cytotoxicity of both PBMC and CIK. When the bsAb was combined with the murine mAb BR55-2, which recognizes the Lewis(Y) antigen, bsAb-redirected cytotoxicity was partly augmented, whereas murine mAb 17-1A, which binds to EpCAM as well, slightly suppressed bsAb-redirected cytotoxicity induced by the bsAb. We conclude that CIK generated in vitro or in vivo combined with this new EpCAM/CD3 bsAb and the cytokine IL-2 should be evaluated for the treatment of EpCAM-expressing tumors.
Flieger
D
D
Medizinische Klinik und Poliklinik I, Allgemeine Innere Medizin, Universität Bonn, Germany. D.Flieger@uni-bonn.de
Kufer
P
P
Beier
I
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Sauerbruch
T
T
Schmidt-Wolf
I G
IG
D000818:Animals; D018033:Antibodies, Bispecific / Q000627:therapeutic use*; D000911:Antibodies, Monoclonal / Q000276:immunology; D000951:Antigens, Neoplasm / Q000276:immunology*; D017252:CD3 Complex / Q000276:immunology*; D019002:CD56 Antigen / Q000276:immunology*; D015815:Cell Adhesion Molecules / Q000276:immunology*; D002455:Cell Division / Q000187:drug effects / Q000276:immunology; D002469:Cell Separation; D004305:Dose-Response Relationship, Drug; D000071858:Epithelial Cell Adhesion Molecule; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000276:immunology / Q000378:metabolism; D006801:Humans; D016898:Interferon-alpha / Q000276:immunology* / Q000378:metabolism; D007376:Interleukin-2 / Q000276:immunology* / Q000378:metabolism; D007694:Killer Cells, Natural / Q000276:immunology*; D007700:Kinetics; D007963:Leukocytes, Mononuclear / Q000276:immunology / Q000378:metabolism; D051379:Mice; D013601:T-Lymphocytes / Q000276:immunology*; D013997:Time Factors; D014357:Trypsin / Q000494:pharmacology
D018033:Antibodies, Bispecific; D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D017252:CD3 Complex; D019002:CD56 Antigen; D015815:Cell Adhesion Molecules; D000071858:Epithelial Cell Adhesion Molecule; D016898:Interferon-alpha; D007376:Interleukin-2; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D014357:Trypsin