Final Report Summary - ROLE OF TCR AFFINITY (Study and modification of T-cell receptor structure to enhance anti-tumor activity)
It has now being established that the cellular immune response against tumor cells is central to the elimination of cancer. While several factors such as the tumor micro-environment or tumor-escape mechanisms are of importance, cytotoxic CD8+ T-lymphocytes are amongst the major players in the anti-tumor response. In that regard, adoptive cell transfer of tumor-reactive lymphocytes has been shown to mediate the regression of large solid tumors in cancer patients. To overcome the need to isolate and expand tumor-reactive lymphocytes that pre-exist in the patient, it is possible to engineer cells to express a tumor-specific T-cell receptor - TCR, thus reprogramming their specificity. This pioneering therapeutic approach has been demonstrated to cause cancer regression in terminal melanoma patients as well as synovial cell cancer that were refractory to previous treatments. However, little is known about TCR affinity requirements for mounting an optimal immune response in this context. While high affinity TCR may confer to the host lymphocyte excellent cytotoxic ability, several studies have shown that a too-high affinity may actually be detrimental to T-cell function. We therefore hypothesize that T-cell receptor affinity can influence the cytotoxic activity of lymphocytes in TCR-transfer treatment and that it exists an optimal range or threshold of TCR affinity for mounting an efficient anti-tumor response. Thus, the major objective of the proposed research was to study the influence of TCR affinity on the overall cytotoxic anti-tumor response in a TCR-gene transfer model. To test this, we propose to use the ovalbumin specific OT1 TCR model and to mutate specific CDR-residues in order to generate TCRs with the same specificity (OVA/H2Kb-restricted) but with different affinities. We have therefore generated mutations in all of the CDRs of both the alpha and beta chains and screened them. We have isolated several TCRs that display a differential recognition of the OVA/H2Kb complex. We have further characterized the activity of several mutants by means of cytokine section and upregulation of activation markers on T-cells and it correlated with differential functional avidity of the TCRs. A retroviral platform for the simultaneous expression of both TCR chains has been constructed and validated. We have performed an extensive biochemical and functional characterization of our mutants, tested potential combinations of mutated alpha and beta chains and perfected our transduction approach into murine splenocytes. Using the B16-OVA melanoma model, we designed an original in ovo cytotoxic assays based on the chick-embryo chorioallantoic CAM model in which we adoptively transferred lymphocyte populations expressing mutant TCRs with different affinities and compare their activity in order to study the influence of TCR affinity on anti-tumor cytotoxic response. We clearly showed that above a certain threshold, not only that affinity improvement does not yield any benefit in terms of immune response; on the contrary we observed a considerable diminution of the cytotoxic potential of T-cells expressing the high affinity receptor which did not correlated with its level of expression. We also have initiated in vivo experiments model which demonstrate the importance of TCR affinity in mediating tumor regression. Thus, this demonstrates that both fundamental and translational issues are related to the ability of the immune system to recognize and eliminate tumor cells in the context of TCR-transfer treatment. In turn, we believe that our findings will help to improve existing immunotherapies in order to target more efficiently cancer or viral infected cells.