Modification of B-CLL Cells Via Infection with a Replication-Defective MVA Virus Encoding Three Costimulatory Molecules: A Potential Approach to Tumor Cell Immunotherapy of B-CLL.

Chronic lymphocytic leukemia of B-cell origin (B-CLL) is a lymphoproliferative disorder characterized by the accumulation of slowly dividing, apoptosis-defective, clonal CD5⁺ B cells that typically involve the bone marrow, lymph nodes, spleen and blood of affected individuals. New therapeutic approaches are necessary to be investigated for the treatment of B-CLL. Several characteristics of the neoplastic B cells make B-CLL a good candidate for immunotherapy; evidence exists that B-CLL cells can be recognized by autologous anti-tumor T cells in an MHC-restricted manner. B-CLL cells, however, are themselves ineffective antigen-presenting cells mainly because of their inadequate costimulatory capacity. We have investigated the ability of in vitro manipulated B-CLL cells that express a triad of costimulatory molecules, designated TRICOM, to stimulate effective anti-tumor T-cell responses and their potential as cancer vaccines. Delivery of transgenes to the B-CLL cells could be accomplished through the use of a modified vaccinia virus strain Ankara designated MVA. This is a highly attenuated, non-replicative virus that has been demonstrated to be safe and well tolerated in both animals and humans. Here we report the use of a recombinant MVA vector to deliver the simultaneous expression of three human costimulatory molecules (B7-1, ICAM-1, LFA-3) on the surface of B-CLL cells. Infection was conducted using various multiplicity-of-infection (MOI) of MVA-TRICOM and the efficiency of the vector to enhance the expression of the encoded transgenes was evaluated by FACS analysis. The infection of B-CLL cells with MVA-TRICOM markedly increased the expression of B7-1, ICAM-1 and LFA-3 on the cell surface. The ability of the MVA-TRICOM-infected B-CLL cells to activate specific T cells was subsequently analyzed in proliferation assays, cytotoxic, and cytokine production assays. Proliferation of both autologous and allogeneic T cells was observed when MVA-TRICOM-infected B-CLL cells were used as stimulators in mixed lymphocyte reactions. Autologous CTLs were then generated in vitro using MVA-TRICOM-infected B-CLL cells as APC; these CTLs were capable of lysing uninfected autologous as well as allogeneic B-CLL cells. From our results we conclude that MVA-TRICOM-infected B-CLL cells can be used to induce B-CLL-specific CTLs that, in turn, can mediate the lysis of unmodified B-CLL cells. Our findings have implications for a potential use of MVA-TRICOM-infected B-CLL cells as tumor vaccines in the immunotherapy of B-CLL.