Discussion
cancer, tumour
SOLID TUMOUR, CANCER, SYNDROMES, TUMOUR, EVENTS, METASTASES
Here we developed and utilized several technologies to achieve the following: efficiently define the landscape of T cell responses to mutational neoantigens presented by over 60 HLA class I alleles; clone neoTCR genes from individual T cells circulating in peripheral blood; and genetically engineer them back into autologous T cells using non-viral gene-editing techniques. In doing so, we demonstrated that it is feasible to generate personalized T cell therapies with neoantigen-specific TCRs for ACT. When infused into patients, these neoTCR transgenic T cells circulate through the blood and were detected in tumour metastases at frequencies higher than baseline native T cells with the same neoantigen-specific TCRs.The technology described herein also demonstrated the feasibility and safety of non-viral precision genome-engineering for the manufacture of clinical-grade gene-engineered ACT products. A similar approach has been recently used to insert a CD19 chimeric antigen receptor into the The clinical trial approach described herein has several limitations. A major challenge of an approach that uses targeted personalized neoantigens is the limited ability and time to characterize each of them for protein expression and neoepitope presentation. Here we assessed gene expression and mutation truncality parameters to select neoantigens for targeting. Favouring mutations induced by oncogenic events in the first cancer clone (for example, from carcinogens such as tobacco or ultraviolet light) should result in truncal mutations, as opposed to targeting mutations induced by DNA repair syndromes or APOBEC, which have a higher likelihood for divergent heterogeneous evolutionAnother complexity of the personalized approach results from the different affinities of the neoTCRs selected for each patient. In the current work, we initially cast a wide net based on literature that suggested that low-affinity TCRs on T cells could be beneficial in chronic viral infectionsLast, the personalized neoTCR isolation, cloning, validation and selection steps resulted in a lengthy process that was heavily dependent on the quality of the tumour and PBMC samples available for analyses. The time taken to obtain additional samples and repeated analyses, particularly for screening of TCRs, delayed the ability to infuse the neoTCR cell products into patients. This could be mitigated in the future through streamlined sample acquisition and process automation. A potential solution for issues regarding TCR and neoantigen variability and the lengthy product selection process would be to use the same technology for TCR discovery and validation to generate a pre-established library of TCRs specific for common mutations and viral antigens that would cover multiple HLA specificities.Limited in vivo expansion of the infused neoTCR gene-edited T cells was observed in participants who received the original conditioning chemotherapy and cell products manufactured using processes v.2.0 and v.2.1 and the two low-cell doses. This in turn probably resulted in a low probability of clinical benefit. The dose-escalation study started with cell doses that may be lower than would be needed for the potential of a clinical response, especially if we consider that the total transgenic cell dose was divided by the three TCRs in many patients. In the solid tumour setting, TCR transgenic T cell clinical trials conducted by other groups have shown clinical activity in the 5–10 × 10In conclusion, we demonstrated the ability to use CRISPR-based, non-viral knockout and knock-in editing to genetically redirect T cells to mutational neoantigens in humans. This work builds on research on the use of genome editing to redirect T cell specificity with transgenic TCR engineering