Background: Adoptive T-cell therapy with engineered T-cell receptors (eTCR) has demonstrated promising, yet modest clinical benefit to date. Key obstacles for this technology are 1) competition with the endogenous TCR for CD3 components that are required for surface expression of a functional TCR complex and 2) mispairing of endogenous and exogenous TCRα and TCRβ chains. Substituting murine TCR constant domains has been shown to enhance eTCR expression, but increases the risk of immunologic rejection. Knockout or knockdown of the endogenous TCR has also been shown to improve eTCR expression. Current eTCR delivery approaches use semi-randomly integrating lentivirus or retrovirus to generate an eTCR expressing T-cell product. An alternative approach is to combine TCR knockout with targeted integration of eTCRs into the T-cell receptor alpha constant (TRAC) locus. In this study, we evaluate newly discovered eTCRs specific for human papillomavirus (HPV) type 16 oncogenic protein E7, expressed via these various gene engineering approaches, to optimize engineered T-cell functionality. Methods: HPV E7-specific eTCRs were introduced into primary human T-cells by lentiviral transduction or a dual CRISPR-Cas9/AAV eTCR delivery platform for targeted insertion into the TRAC locus using homology directed repair (HDR). Comparisons were made among TCR sufficient and TCR knockouT-cells with gene delivery by lentivirus or HDR. Engineered T-cell function was assessed both in vitro and in vivo against HPV-16+ head and neck squamous cell carcinoma cell lines. Results: CRISPR/Cas9-mediated TRAC editing eliminated endogenous TCR expression in >85% of T-cells. The impact of TRAC editing on eTCR expression and engineered T-cell function was variable across multiple eTCRαβ sequences. With many eTCRs, TRAC editing in lentivirus-derived populations resulted in increased expression and improved cytokine and killing responses. Targeted insertion of these eTCRs into the TRAC locus by HDR increased engineered cell product homogeneity and enhanced function compared to lentiviral integration. However, there were also examples of eTCRs that were minimally impacted by TRAC locus engineering. Conclusions: Our results demonstrate that elimination of the endogenous TCR, alone or combined with targeted insertion at the TRAC locus, improves eTCR expression and engineered T-cell function for the majority of eTCRs tested. While infrequent, some eTCRs were less impacted by TRAC locus engineering, suggesting a sequence-specific property that enables these eTCRs to out-compete the endogenous TCR for CD3 componentry. The cause for this variable response to TRAC locus engineering is still being explored.

Citation Format: Alexandra Croft, Cameron Brandt, Stephen Burleigh, Eric Chadwick, Melissa Chin, Dean Toy, Bailey Donahue, Clay Patton, Stephen Goldfless, Brian Belmont, Ruth Salmon, Grant Welstead, Blythe D. Sather, David J. Huss. Targeted insertion of an HPV-16 E7-specific engineered T-cell receptor into the TRAC locus [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A027.