Immune checkpoint blockade therapy (ICBT) has resulted in extended overall survival for some patients with certain types of cancer, most prominently including colorectal cancer (CRC) patients associated with microsatellite instability (MSI). However, most patients with CRC whose phenotypes have microsatellite stability (MSS) are unresponsive to ICBT. In efforts to understand the responsiveness of CRC tumors to ICBT, genotypic and phenotypic signatures of CRC tumors are now being investigated. The MSI and MSS classification has been clinically validated as helpful in predicting response vs. nonresponse to ICBT in CRC patients. Other potential predictive markers include mutational and neoantigen loads, T-cell receptor diversity, and the immune score system, all of which have mechanistic connections to ICBT response. These novel predictive signatures could provide unprecedented insights into patients with CRC associated with MSS. Clinical trials or prospective cohort studies using standardized methodologies for biomarker quantification should be illuminating. Further validation of these novel predictive signatures will be essential to tailoring treatment of patients whose CRC is most likely to respond to ICBT.

Introduction: Data on survival of patients with MSI mCRC treated with systemic non-immunotherapy are scarce, which complicates the interpretation of recent immunotherapy trials. Our study examined survival in MSI mCRC patients in a retrospective observational cohort. Methods: We performed a study involving 186 mCRC MSI patients (n=54 from the phase 3 CAIRO, CAIRO2 and CAIRO3 trials and n=132 from The Netherlands Cancer Registry 2015 and 2016). Overall survival from diagnosis of mCRC (OS), from start of first-line (OS1) and second-line therapy (OS2) to death of any cause were examined. Progression-free survival from start of first line (PFS1) and second-line therapy (PFS2) to progression or death of any cause were examined. Results: Of the 186 patients, 57% were females, median age 67 years, 19% had liver-only metastasis, 31% peritoneal metastasis and 23% underwent metastasectomy. In 113 patients with known BRAF status, 51% had BRAF mutated tumors. The proportion of patients receiving first, second and third-line systemic treatment was 68%, 27% and 7%, respectively. For all patients, median OS was 13.8 months (95% confidence interval [C.I.] 11.8 - 16.3). In patients receiving at least one line of treatment median OS was 15.6 months (13.8 - 19.3), OS1 was 13.9 months (11.6-16.6) and PFS1 was 6.4 months (5.5 - 9.2). In the 48 patients receiving second-line therapy, median OS was 16.5 months (13.9 - 21.5), OS2 was 6.1 months (5.4 - 9.7) and PFS2 was 2.2 months (1.9 - 4.2). Patients starting second-line treatment with a performance score of 0-1, thus eligible for CHECKMATE-142, had an OS2 of 6.7 months (5.0 - 12.1). Discussion: A direct comparison between our cohort and the CHECKMATE-142 cohort is not possible due to key differences in the patient populations: CHECKMATE-142 patients often received more than one therapy line prior to inclusion and displayed fewer BRAF mutant tumors. However, the survival from start of second-line in our cohort of approximately 7 months is inferior to the published CHECKMATE-142 results, where median OS was not reached after a median follow-up of 1 year in the nivolumab and the nivolumab and ipilimumab cohorts. Conclusion: We present data on systemic non-immunotherapy in the largest cohort of trial and population-based mCRC MSI patients to date. Our results suggest that immunotherapy may offer a survival benefit for mCRC MSI patients. Citation Format: G. E. Wensink, M. A. Elferink, A. M. May, L. Mol, P. A. Hamers, C. J. Punt, G. R. Vink, J. M. Roodhart, M. Koopman. Survival of patients with microsatellite instable (MSI) metastatic colorectal cancer (mCRC) upon systemic non-immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4467.

T cell therapies for cancer have shown striking clinical efficacy in different tumor settings. Integration of a transgene cassette expressing an antigen receptor into the genome enables T cells to recognize specific tumor antigens. Viral vectors and transposons can introduce transgenes into primary human T cells. However these approaches cannot control genomic location and have limited control over the copy number of cassette insertions. Here, we used epigenetic analysis, transcriptional profiling, high-throughput gene-editing and T cell functional experiments to identify the optimal genomic locus amenable to non-viral genome targeting. First we analyzed the epigenetic landscape of primary human T cells to identify regions (“gene deserts”) that are accessible but devoid of annotated regulatory elements or coding sequences. Candidate loci were validated using CRISPR/Cas9 genome editing to directly cut and a marker gene cassette was inserted into these sites. Knock-in efficiency and transgene expression stability in primary human T cells were evaluated for all loci. Evaluation of 40 loci demonstrated substantial variability (10 fold) in transgene expression and confirmed that not all sites were equivalent in their ability to sustain transgene expression. Next, we evaluated 8 most promising candidate loci for compatibility with complex T cell programs embodied by integrated circuits, containing a priming receptor (PrimeR) that triggers expression of a CAR in response to a priming antigen. We identified one integration site (termed “GS94”) that supported: 1) stable and high PrimeR expression; 2) high and inducible CAR expression; and 3) a superior T cell cytotoxic and cytokine secretion profile. Finally, we assessed off-target cutting mediated by the CRISPR sgRNA targeting GS94. The sgRNA is the most specific out of the candidates evaluated with iGUIDE and targeted PCR. We were unable to detect any measurable off-target activity by targeted PCR on predicted sites. The GS94 site is a component of the integrated circuit T cell (ICT) therapy, AB-1015, which is now under development for the treatment of high-grade serous carcinoma ovarian tumors. AB-1015 includes an inserted cassette that contains a logic gate and two shRNAs that enhance the potency and solid tumor microenvironment resistance of the AB-1015 ICT cells. These computational and experimental approaches can be generalized to identify new safe harbor sites for different cell types, expanding the genome engineering toolkit for diverse cell therapy applications. Citation Format: Grace X.Y. Zheng, Somya Khare, Brendan Galvin, Robby Moot, Aaron Cooper, Michelle Nguyen, Michelle Tan, Shan Sabri, Audre May, Jun Fung, Anzhi Yao, Andrea Liu, Matt Drever, Steve Santoro, W Nicholas Haining, Tarjei Mikkelsen. Identification of a safe harbor CRISPR-Cas9 integration site for improved cell therapy safety and potency [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2840.