CD20 is a molecular target for scFvFc:zeta receptor redirected T cells: implications for cellular immunotherapy of CD20+ malignancy.
The CD20 molecule was evaluated as a B-cell lymphoma target epitope for T cells expressing a CD20-specific single-chain FvFc-zeta (scFvFc:zeta) chimeric receptor. A cDNA construct consisting of a murine kappa leader sequence, CD20-specific scFv, human immunoglobulin (Ig) G1 hinge-C(H)2-C(H)3, the human CD4 transmembrane, and the intracellular signaling domain of the human CD3 complex's zeta chain was synthesized by polymerase chain reaction splice-overlap extension. The human CD4+ Jurkat cell line was electroporated with the CD20-specific scFvFc:zeta construct cloned into the mammalian expression vector pcDNAneo. Western blot analysis of transfectant whole cell lysate with an anti-zeta antibody demonstrated the expression of both endogenous zeta and the chimeric receptor protein, with a mobility consistent with the expected molecular weight of 66 kD under reducing conditions; nonreduced lysate revealed a chimeric receptor complex of approximately 132 kD. The scFvFc:zeta receptor was present on the cell surface as detected by flow cytometry of T-cell transfectants stained with an anti-mouse Fab-specific antibody and anti-human Fc gamma-specific monoclonal antibody. Coculture of Jurkat transfectants with CD20+ lymphoma cells resulted in the accumulation of interleukin (IL)-2 in culture supernatants as detected by ELISA. IL-2 production was triggered by the specific interaction between the CD20 molecule and the scFvFc:zeta as IL-2 was not detected in cultures with mock transfected Jurkat cells or CD20- stimulator cells. Furthermore, IL-2 production was inhibited by the addition of a soluble anti-CD20 monoclonal antibody to cocultured Jurkat transfectants and CD20+ stimulator cells. The capacity of CD20 to trigger the lytic machinery of scFvFc:zeta-expressing cytotoxic T lymphocytes (CTLs) was assessed using the murine allo-specific CD8+ CTL clone 2c. CD20-specific redirected cytolytic activity against human lymphoma targets was observed with 2c transfectants in a 4-hour chromium release assay. These results demonstrate that CD20 can serve as a target epitope for scFvFc:zeta receptor-expressing T cells.
Beckman Research Institute, City of Hope National Medical Center, Duarte, CA 91010-3000, USA.
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Jensen
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Tan
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Forman
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Wu
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Raubitschek
D000911:Antibodies, Monoclonal / Q000627:therapeutic use; D000918:Antibody Specificity; D018951:Antigens, CD20 / Q000097:blood*; D006801:Humans; D007167:Immunotherapy; D019169:Jurkat Cells; D016393:Lymphoma, B-Cell / Q000276:immunology / Q000628:therapy*; D011993:Recombinant Fusion Proteins / Q000276:immunology*; D013601:T-Lymphocytes / Q000276:immunology*; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D018951:Antigens, CD20; D011993:Recombinant Fusion Proteins