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Background  Tumors and viruses have developed many mechanisms to evade the immune system, including down-regulation of target antigens and MHC molecules. These immune escape mechanisms may be able to be circumvented by adoptively transferring T cells engineered to express two different T cell receptors, each specific for a different antigen or MHC restriction molecule.    Methods  PBMC from the blood of normal healthy donors were stimulated for three days with an antigenic peptide from cytomegalovirus (CMV) pp65. These CMV reactive cultures were transduced with a encoding the TIL 5 T cell receptor (TCR) that mediates recognition of the dominant epitope of the melanoma antigen MART-1. Following selection for transduced cells, the cultures were evaluated for recognition of CMV pp65 and MART-1 expressing targets.    Results  We were able to rapidly create bifunctional T cells capable of recognizing both CMV pp65 and MART-1 using a combination of HLA-A2 tetramer staining and intracellular staining for interferon-γ. These bifunctional T cells were sensitive to very low levels of antigen, recognize MART-1 +  tumor cells, and maintained their bifunctionality for over 40 days in culture.    Conclusion  Bifunctional T cells can be engineered by transducing short term peptide stimulated T cell cultures. These bifunctional T cells may be more effective in treating patients with cancer or chronic virus infections because they would reduce the possibility of disease progression due to antigen and/or MHC loss variants.

journal of translational medicine

Retroviral transduction of peptide stimulated t cells can generate dual t cell receptor-expressing (bifunctional) t cells reactive with two defined antigens.