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T cell receptor (TCR) gene modified T cells are a promising form of adoptive cellular therapy against human malignancies and viral infections. Since the first human clinical trial was carried out in 2006, several strategies have been developed to improve the efficacy and safety of TCR engineered T cells by enhancing the surface expression of the introduced therapeutic TCRs whilst reducing the mis-pairing with endogenous TCR chains. In this study, we explored how modifications of framework residues in the TCR variable domains affect TCR expression and function. We used bioinformatic and protein structural analyses to identify candidate amino acid residues in the framework of the variable β domain predicted to drive high TCR surface expression. Changes of these residues in poorly expressed TCRs resulted in improved surface expression and boosted target cell specific killing by engineered T cells expressing the modified TCRs. Overall, these results indicate that small changes in the framework of the TCR variable domains can result in improved expression and functionality, while at the same time reducing the risk of toxicity associated with TCR mis-pairing.

NKG2D is a major regulator of the activity of cytotoxic cells and interacts with eight different ligands (NKG2DL) from two families of MIC and ULBP proteins. The selective forces that drove evolution of NKG2DL are uncertain, but are likely to have been dominated by infectious disease and cancer. Of interest, NKG2DL are some of the most polymorphic genes outside the MHC locus and the study of these is uncovering a range of novel observations regarding the structure and function of NKG2DL. Polymorphism is present within all NKG2DL members and varies markedly within different populations. Allelic variation influences functional responses through three major mechanisms. First, it may drive differential levels of protein expression, modulate subcellular trafficking, or regulate release of soluble isoforms. In addition, it may alter the affinity of interaction with NKG2D or modulate cytotoxic activity from the target cell. In particular, ligands with high affinity for NKG2D are associated with down regulation of this protein on the effector cell, effectively limiting cytotoxic activity in a negative-feedback circuit. Given these observations, it is not surprising that NKG2DL alleles are associated with relative risk for development of several clinical disorders and the critical role of the NKG2D:NKG2DL interaction is demonstrated in many murine models. Increased understanding of the biophysical and functional consequences of this polymorphism is likely to provide insights into novel immunotherapeutic approaches.