Here we report the successful engineering of T cells that are able to respond independently to two unrelated known antigens via both an endogenous and a retrovirally-transduced T cell receptor. These T cells were able to respond to low concentration of peptide, and were able to recognize antigen-positive tumor cells. By utilizing the initial antigen response as the activation for transduction, our 12-day protocol represents an efficient technique for generating bifunctional T cells from donor blood, and theoretically can be applied to any tumor or viral antigen in the context of one or more MHC restricting elements.
Many previous efforts at creating TCR transductants used non-specific activation of bulk or clonal populations [7, 8, 12] or, for the creation of bifunctional T cells, specific activation of semi-clonal populations with peptide-loaded autologous PBMC . Non-specific T cell activation fails to expand T cell populations with known reactivity hence making it virtually impossible engineer T cells reactive with two know antigens. Engineering clonal or semi-clonal populations of T cells will create T cells reactive with two known antigens . However, this process necessitates the establishment of antigen reactive T cell clones or long term T cell cultures prior to transduction. Although technically feasible, the creation of bifunctional T cells from T cell clones (this study) or long term T cell cultures  is time consuming and in our experience has a comparatively low yield of bifunctional cells. Furthermore, it is likely that the reactivity and therapeutic efficacy of T cells are diminished with extended culturing (13). Therefore, any method capable of rapidly producing bifunctional T cells will be better suited to clinical applications.
In contrast to using anti-CD3 mAb, in vitro stimulation with antigenic peptides will preferentially activate antigen reactive T cells to expand. These proliferating antigen reactive T cells can be transduced to express a second TCR. Based on our tetramer analysis, only 0.7% of the unstimulated donor PBL stained with the CMV tetramer (data not shown), compared to 44.6% of our peptide stimulated populations (data not shown). This profound expansion allowed for more efficient transduction, and 2.7% of the resulting culture was measurably bifunctional (figure 4). As retroviral transduction and in vitro selection for transduced T cells becomes more efficient, the frequency of bifunctional T cells in these cultures will increase to the point where it is feasible to treat patients.
The combination tumor/viral bifunctional cells we have generated here may have novel uses in immunotherapy, such as bypassing tumor- or viral-induced T cell unresponsiveness. Fossati and colleagues demonstrated that naïve bifunctional T cells "preactivated" via one TCR prior to adoptive transfer would then mediate cytotoxicity via the second TCR . Animal and in vitro studies have shown that peripherally-induced tolerance can be reversed, resulting in regained T cell responsiveness [15, 16]. It may be possible to reactivate tolerized T cells in vitro or in vivo by activating a second T cell receptor specific for a non-tolerized antigen [16, 17]. In addition, viral antigens such as those associated with influenza, trigger alternate T cell activation pathways  and have been shown to elicit a strong T cell immune response . Redirecting the vigorous anti-viral T cells which have not been exposed to the immunologic tolerance associated with most tumor-reactive T cells may be effective in eradicating tumor burden.
The substantial proliferation in response to strong immunogens such as viral antigens can also be used to improve the localization of T cells that also have anti-tumor activity. Using murine bifunctional T cells created by retroviral transfer of a chimeric immunoglobulin receptor specific for an ovarian cancer-associated tumor antigen to alloreactive T cells, Kershaw and colleagues were able to demonstrate in vivo expansion in response to alloimmunization and demonstrated anti-tumor activity . It is possible that tumor/viral bifunctional cells would also behave in this way, and we are currently working on murine models with human/mouse chimeric TCRs to test this hypothesis.
In addition, some current immunotherapy protocols for the treatment of metastatic melanoma involve immunodepletion prior to adoptive cell transfer . Such protocols are similar to solid organ and stem cell transplantation in that the patients are temporarily immunosuppressed and at risk for reactivation of latent viruses such as CMV and Epstein-Barr virus. Tumor/viral bifunctional T cells may be particularly useful in this setting, where the anti-viral activity may help treat reactivation, and the reactivation of the virus may further boost the anti-tumor activity of the T cells by inducing additional stimulation of the bifunctional cells.
Another consideration to bear in mind with the creation of bifunctional T cells is alternate pairing of the alpha and beta chains resulting in the combination of novel T cell receptors within a bifunctional cell. These T cells could have undesirable autoimmune properties. This could be circumvented by identifying T cells within a bifunctional population that have maximal expression of both the endogenous and introduced TCRs, indicating minimal cross-pairing of chains . Screening for these T cells and selectively expanding them would reduce the risk of untoward autospecificity.
In our experiences, it has been difficult to transduce PBL-derived T cells from normal donors that are stimulated with antigenic peptides derived from self-antigens (data not shown). This is likely due to the low precursor frequency and/or the state of immunologic tolerance of T cells reactive with antigens such as gp100 or tyrosinase [20, 22, 23]. These limitations do not preclude generating T cells capable of recognizing two different tumor antigens, for we have demonstrated here that a T cell clone reactive with gp100:209–217 can be engineered to also recognize MART-1. However, transducing T cell clones is more time consuming since it is first necessary to isolate the T cells clones prior to transduction. There are two potential strategies for overcoming the limitations of transducing T cells with low precursor frequencies or that are immunologically tolerant. First, is transducing actively expanding tumor infiltrating lymphocyte cultures which contain tumor antigen-reactive T cells . Second, patients vaccinated against tumor associated self antigens often have increased frequencies of antigen reactive T cells in their peripheral blood , and these T cells may lend themselves to activation and expansion in vitro to enable efficient retroviral transduction.