No abstract

12T cell receptor (TCR) gene therapy is a promising cell therapy approach for the treatment of cancer. 13 However, most naturally occurring TCRs display low affinities to their peptide-MHC targets, and 14 engineering of TCRs for enhanced affinity is complicated by the risk of introducing cross-reactivity 15 and the poor correlation between affinity and function. Here we report the establishment of the TCR-16 accepting T cell (TnT) platform through five sequential CRISPR-Cas9 genome editing steps of a 17 human T cell line, and demonstrate its application for functional engineering of TCRs and prediction 18 of cross-reactivity. Using the TnT platform, we profile the mutational landscapes of tumor-specific 19 TCRs at high-throughput to reveal a substantial discordance between antigen binding and antigen-20 induced signaling. Furthermore, we combine CRISPR-targeting, functional selection and deep 21 sequencing to screen TCR mutagenesis libraries and identify variants with enhanced recognition of 22 the cancer-testis antigen MAGE-A3. Finally, functional cross-reactivity profiling using TnT cells was 23 able to accurately predict off-targets and identify engineered TCRs with exquisite specificity to 24 MAGE-A3. Thus, the TnT platform represents a valuable technology for the engineering of TCRs with 25 enhanced functional and safety profiles. Several technologies allowing the assessment of TCR specificity have been reported recently, highlighting 67 a strong interest in the development and safety screening of TCRs for gene therapy. Affinity-based methods 68 include the use of barcoded peptide-MHC multimer libraries for profiling TCRs displayed on primary T 69 cells 16,37,38 and the assessment of soluble TCR interaction with peptide-MHC libraries displayed on the 70 surface of yeast 18,39-41 . A potential limitation of such methods, however, is that they are unable to detect 71 very low-affinity interactions between TCR and peptide-MHC that are nevertheless functional 42 . Epitope 72 screening platforms based on cellular function include those relying on the display of peptide libraries by 73 cells expressing chimeric MHC receptors 43,44 , cells undergoing trogocytosis 45 , or cells harboring reporters 74 of exogenous granzyme activity 46,47 . Another method for the functional assessment of TCR cross-reactivity 75 involves measuring T cell reactivity to single amino acid mutants of the target peptide, which has been 76 recently utilized to profile the cross-reactivity of phage display-engineered TCRs reformatted for expression 77 in primary T cells 48,49 or a murine cell line 31 . While all of the above methods focus on specificity screening 78 of TCRs, they have not been reported to directly enable TCR engineering. 79 Here we report the development and application of the TCR-accepting T cell (TnT) platform for the functional 80 display, engineering and cross-reactivity profiling of TCRs. Reconstitution of Jurkat-derived TnT cells with 81 transgenic TCRs was targeted by CRISPR-Cas9 to the endogenous TCRβ genomic region, ...