Abstract
BackgroundTumors 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.MethodsPBMC 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.ResultsWe 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.ConclusionBifunctional 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.
Highlights
Tumors and viruses have developed many mechanisms to evade the immune system, including down-regulation of target antigens and MHC molecules
Recognition of Peptides and Tumor Cells by TIL 5 TCRtransduced CMV peptide stimulated T cells Bifunctional T cells reactive with CMV and MART-1 were engineered by first stimulating donor Peripheral blood mononuclear cells (PBMC) with CMV pp65:495–503 peptide for three days transducing the T cell cultures with a retrovirus encoding a T cell receptor (TCR) specific for MART-1:27–35 presented by HLA-A2
Significant amounts of interferon-γ were released when the TIL 5 TCR-transduced CMV peptide stimulated T cells were cocultured with CMV pp65:495–503 or MART-1:27–35 peptide-loaded T2 cells, COS cells engineered to express HLAA2 with a CMV pp65:495–503 mini-gene, or HLA-A2+ MART-1+ tumor cells (Figure 1)
Summary
Tumors and viruses have developed many mechanisms to evade the immune system, including down-regulation of target antigens and MHC molecules. While every T cell that is transduced to express a second TCR expresses its own TCR capable of recognizing some antigen, it has only recently been shown that "bifunctional" T cells capable of recognizing two known antigens can be generated [5] Using this technology, it may be possible to treat patients with T cells bearing two functional T cell receptors (TCRs) with each TCR being specific for a different tumor-associated antigen (TAA) or viral antigen restricted by one or more HLA molecule. It may be possible to treat patients with T cells bearing two functional T cell receptors (TCRs) with each TCR being specific for a different tumor-associated antigen (TAA) or viral antigen restricted by one or more HLA molecule These bifunctional T cells would retain effectiveness against single antigen-loss variants or HLA loss variants and may have improved efficacy over monospecific T cells for the treatment of tumors or viruses
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