384 Systematic, antigen-agnostic discovery of tumor-reactive TCRs powered by repertoire-scale TCR gene synthesis and the use of banked allogeneic cancer cell lines

Background Both neoantigen-reactive and wildtype tumor-associated antigen- (TAA) reactive TCRs contribute to anti-tumor immunity. While neoantigens can be identified by deep sequencing, the spectrum of TAA is harder to define. Although some TAAs are well studied, the majority of TAAs are likely undiscovered. Therefore it is difficult to survey the landscape of TAA-reactive TCRs for a given patient. Antigen-agnostic approaches have been reported,1,2 where patient-derived TCR genes are synthesized and used to make TCR-transgenic T cells, which are co-cultured with autologous tumor cells to observe cytokine secretion. However, these approaches are limited by the number of TCRs tested and the availability of autologous tumor cells. Methods We have developed a new approach to solve both problems. First, we developed an ultrahigh-throughput TCR gene synthesis technology called PathFinder DNA AssemblyTM 3 (figure 1, figure 2a), which bring the cost of TCR gene synthesis to <$1 per TCR (100-folder lower than existing technologies). Second, we use banked cancer cell lines, which express a large swath of known and undiscovered TAAs. This method is justified in part by the similar gene expression profiles between many tumor tissues and many cell lines.4,5 We developed a simple method to inactivate the endogenous HLA genes of the cell lines and replace them with the HLA genes from the patient, a process we call ‘HLA-personalization’ (figure 2b, figure 2c). Results For example, we have synthesized 1,575 TCRs from a liver cancer patient and screened them against HLA-personalized liver cancer cell lines HepG2, Huh6 and Huh7. We have discovered several tumor-reactive TCRs including one that reacts strongly to HepG2, restricted by HLA-C*03:04 (figure 3a). We further confirmed that this TCR can sense and kill HLA-C*03:04-expressing HepG2 cells and autologous tumor cells, but not PBMC of a C*03:04+ donor (figure 3b). We have similarly synthesized 1,053 TCRs from a cervical cancer patient and identified 4 TCRs reactive to HLA-personalized cervical cancer cell lines SiHA or C33A (figure 4). Two of these TCRs were confirmed to be reactive to HPV E7, restricted by HLA-B*38:15. Using this approach, we have synthesized ~50,000 TCRs from 9 patients and identified >70 tumor-reactive TCRs, restricted by a wide array of HLAs, from every patient tested. Conclusions To our knowledge, this work represents the largest scale of TCR functional screening. The TCRs discovered may be used both as fully personalized TCR-T therapy, and as a repository of TAA-reactive TCRs for conventional TCR-T development after their deorphanization.6, 7 References Scheper W, Kelderman S, Fanchi LF, Linnemann C, Bendle G, de Rooij MAJ, Hirt C, Mezzadra R, Slagter M, Dijkstra K, Kluin RJC, Snaebjornsson P, Milne K, Nelson BH, Zijlmans H, Kenter G, Voest EE, Haanen JBAG, Schumacher TN. Low and variable tumor reactivity of the intratumoral TCR repertoire in human cancers. Nat Med. 2019; 25:89-94. Oliveira G, Stromhaug K, Klaeger S, Kula T, Frederick DT, Le PM, Forman J, Huang T, Li S, Zhang W, Xu Q, Cieri N, Clauser KR, Shukla SA, Neuberg D, Justesen S, MacBeath G, Carr SA, Fritsch EF, Hacohen N, Sade-Feldman M, Livak KJ, Boland GM, Ott PA, Keskin DB, Wu CJ. Phenotype, specificity and avidity of antitumour CD8+ T cells in melanoma. Nature. 2021; 596:119-125. Chen X, Porter E, Compositions and methods for t-cell receptor gene assembly, WO2020206238A2 Yu K, Chen B, Aran D, Charalel J, Yau C, Wolf DM, van’t Veer LJ, Butte AJ, Goldstein T, Sirota M. Comprehensive transcriptomic analysis of cell lines as models of primary tumors across 22 tumor types. Nat Commun. 2019; 10:3574 Warren A, Chen Y, Jones A, Shibue T, Hahn WC, Boehm JS, Vazquez F, Tsherniak A, McFarland JM. Global computational alignment of tumor and cell line transcriptional profiles. Nat Commun. 2021; 12:22 Joglekar AV, Leonard MT, Jeppson JD, Swift M, Li G, Wong S, Peng S, Zaretsky JM, Heath JR, Ribas A, Bethune MT, Baltimore D. T cell antigen discovery via signaling and antigen-presenting bifunctional receptors. Nat Methods. 2019;16:191-198. Kula T, Dezfulian MH, Wang CI, Abdelfattah NS, Hartman ZC, Wucherpfennig KW, Lyerly HK, Elledge SJ. T-Scan: A Genome-wide method for the systematic discovery of T cell epitopes. Cell. 2019;178:1016-1028. Ethics Approval The study obtained ethics approval by ethics committees at Shenzhen People’s Hospital and Zhongshan Hospital. Consent Written informed consent was obtained from the patient for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.