Key Points Engineered T-cell receptors can have redundant recognition of alternative protein motifs, resulting in severe cardiac toxicity. The use of induced pleuripotent stem cells (iPSCs) is a promising approach to identify potential off-target effects of engineered T cells.

Summary T-cell destiny during thymic selection depends on the affinity of the T-cell receptor (TCR) for autologous peptide ligands presented in the context of MHC molecules. This is a delicately balanced process; robust binding leads to negative selection, yet some affinity for the antigen complex is required for positive selection. All TCRs of the resulting repertoire thus have some intrinsic affinity for an MHC type presenting an assortment of peptides. Generally, TCR affinities of peripheral T cells will be low towards self-derived peptides, as these would have been presented during thymic selection, whereas, by serendipity, binding to pathogen-derived peptides which are encountered de novo could be stronger. A crucial question in assessing immunotherapeutic strategies for cancer is whether natural TCR repertoires have the capacity for efficiently recognizing tumor associated peptide antigens (TAPAs). Here, we report a comprehensive comparison of TCR affinities to a range of HLA-A2 presented antigens. TCRs which bind viral antigens (VAs) fall within a strikingly higher affinity range than those which bind cancer-related antigens. This difference may be one of the key explanations for tumor immune escape and for the deficiencies of T-cell vaccines against cancer.

Antigen-specific T cell receptor (TCR) gene transfer via patient-derived T cells is an attractive approach to cancer therapy, with the potential to circumvent immune regulatory networks. However, high-affinity tumour-specific TCR clonotypes are typically deleted from the available repertoire during thymic selection because the vast majority of targeted epitopes are derived from autologous proteins. This process places intrinsic constraints on the efficacy of T cell-based cancer vaccines and therapeutic strategies that employ naturally generated tumour-specific TCRs. In this study, we used altered peptide ligands and lentivirus-mediated transduction of affinity-enhanced TCRs selected by phage display to study the functional properties of CD8+ T cells specific for three different tumour-associated peptide antigens across a range of binding parameters. The key findings were: (i) TCR affinity controls T cell antigen sensitivity and polyfunctionality; (ii) supraphysiological affinity thresholds exist, above which T cell function cannot be improved; and (iii) T cells transduced with very high-affinity TCRs exhibit cross-reactivity with self-derived peptides presented by the restricting human leucocyte antigen. Optimal system-defined affinity windows above the range established for natural tumour-specific TCRs therefore allow the enhancement of T cell effector function without off-target effects. These findings have major implications for the rational design of novel TCR-based biologics underpinned by rigorous preclinical evaluation.

Superantigens are known to activate a large number of T cells. The SAg is presented by MHC class II on the APC and its classical feature is that it recognizes the variable region of the β-chain of the TCR. In this article, we report, by direct binding studies, that staphylococcal enterotoxin (SE) H (SEH), a bacterial SAg secreted by Staphylococcus aureus, instead recognizes the variable α-chain (TRAV27) of TCR. Furthermore, we show that different SAgs (e.g., SEH and SEA) can simultaneously bind to one TCR by binding the α-chain and the β-chain, respectively. Theoretical three-dimensional models of the penta complexes are presented. Hence, these findings open up a new dimension of the biology of the staphylococcal enterotoxins.

SummaryCD4+ T helper cells are a valuable component of the immune response towards cancer. Unfortunately, natural tumour‐specific CD4+ T cells occur in low frequency, express relatively low‐affinity T cell receptors (TCRs) and show poor reactivity towards cognate antigen. In addition, the lack of human leucocyte antigen (HLA) class II expression on most cancers dictates that these cells are often unable to respond to tumour cells directly. These deficiencies can be overcome by transducing primary CD4+ T cells with tumour‐specific HLA class I‐restricted TCRs prior to adoptive transfer. The lack of help from the co‐receptor CD8 glycoprotein in CD4+ cells might result in these cells requiring a different optimal TCR binding affinity. Here we compared primary CD4+ and CD8+ T cells expressing wild‐type and a range of affinity‐enhanced TCRs specific for the HLA A*0201‐restricted NY‐ESO‐1‐ and gp100 tumour antigens. Our major findings are: (i) redirected primary CD4+ T cells expressing TCRs of sufficiently high affinity exhibit a wide range of effector functions, including cytotoxicity, in response to cognate peptide; and (ii) optimal TCR binding affinity is higher in CD4+ T cells than CD8+ T cells. These results indicate that the CD4+ T cell component of current adoptive therapies using TCRs optimized for CD8+ T cells is below par and that there is room for substantial improvement.