Tumor-targeted superantigens for experimental immunotherapy of human leukemia/lymphoma
Sammanfattning: Bacterial superantigens (SAgs) have the property of stimulating high proportions of T cells expressing certain TCR Vβ sequences. The SAg first binds with high affinity to HLA class II molecules on the target cell and then stimulates the T cell to produce cytokines and to become cytotoxic. HLA class II+ target cells may be lysed by cytotoxic T cell cytokines such as TNFα and IFNγ but also by direct cell contact. Staphylococcus enterotoxin A (SEA) is a bacterial SAg produced by certain Staphylococcus areus strains. The HLA class II binding affinity of SEA was reduced by a point mutation (D227A, generating SEAm) not affecting T cell activating properties. This SEAm was first fused to protein A (PA) and later to the Fab-part of an anti-CD19 mAb. Using either fusion protein, cytotoxic T cells (CTLs) were successfully redirected from HLA class II molecules to a defined target structure.Cells from patients with chronic B-lymphocytic leukemia (B-CLL) were highly sensitive for SAg-dependent cell-mediated cytotoxicity (SDCC) using allogeneic SEA-reactive T cells in vitro. T cells targeted by the PA-SEAm fusion protein (FP) and B-cell reactive mAbs also killed the malignant cells. Activation of target cells with a phorbol ester, increased surface ICAM-1, LFA-1, LFA-3 and HLA-DR expression and enhanced their sensitivity for SDCC. The PA-SEAm FP was also used together with myeloid cell reactive mAbs and CTLs to kill 10 different acute or chronic myeloid leukemic (AML or CML) cell line cells in vitro.High concentrations of TNFα and IFNγ in vitro were not directly toxic to target cells exposed for 4h, arguing for other killing mechanisms. One likely mechanism was lytic killing by perforin and granzymes, since inhibition of granulae abolished the cytotoxic effect. MAbs against the Fas antigen did not interfere with killing.The anti-CD19-Fab-SEAm FP used together with CTLs efficiently killed CD19+ normal and malignant B cells. Malignant cells included both tumor B cell lines and tumor cells from patients with B non-Hodgkin's lymphoma (B-NHL). The sensitivity of target cells varied and was correlated to surface ICAM-1 expression. In vivo therapeutic effects were monitored in a humanized severe combined immunodeficiency disease (SCID) model. A significant reduction of tumor weight was registered in treated animals.In conclusion, antibody-superantigen FPs are attractive agents for treating human hematopoietic tumors resistant to conventional therapy. There are abundant numbers of potential effector T cells in lymphoma tissue and the bioaccessibility is good. The SAg activates about 1/5 of all T cells, leaving the majority unaffected. In this thesis, different immunotherapeutic concepts are discussed and compared with targeted SAg-therapy.
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