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Background Off-the-shelf allogeneic CAR-NK cell therapies hold promise for replacing expensive autologous CAR-T cell therapies, whose undependable manufacturability caters to a fraction of clinically eligible patients. Nonetheless, CAR-NK cell therapies are themselves disadvantaged by scanty availability of NK cells in blood, stunted ex vivo expansion, reduced lentiviral tranducibility and importantly, subpar in vivo persistence.Methods To address each of these challenges, we iteratively optimized a scalable, closed, semi-automated CAR-NK manufacturing platform, using peripheral blood NK cells, that is Good Manufacturing Practices (GMP)-ready. To better reflect the persistence of CAR-NK cells, we evolved a long-term in vitro tumor rechallenge assay, with semiweekly tumor challenges, in tumor-conducive media. We systematically designed multiple IL-15 constructs varying in their domain and linker compositions, for self-sustenance and prolonged cytotoxicity of CAR-NK cells.Results We obtained an exceptional cell expansion (50,000-120,000-fold) in a duration of 17 days, while maintaining uncompromised potency, purity, viability and activated phenotype ( figure 1). We have standardized a closed-system lentiviral transduction process, to obtain >50% CAR positivity with ~1 integration per CAR-positive cell. One manufacturing batch would yield >1 trillion CAR-NK cells (>750 usable doses), greatly reducing cost.Unlike in several reported in vitro cytotoxicity assays lasting 1-2 weeks,1–3 we show that our anti-CD19-CAR-NK cells could repeatedly kill CD19+ Raji lymphoma cells for staggering 3 months and more, albeit with exogenous IL-15 supplementation (figure 1). In attempts to reduce this dependency, we found that CAR-NK cells armored with literature-derived IL-15 constructs4–6 could eliminate tumor cells for merely 1-3 weeks, in the absence of exogenous IL-15, only marginally better than control CAR-NK cells. Conversely, CAR-NK cells armored with our novel engineered IL-15 modules eliminated tumor cells for an astounding duration of >2 months, without exogenous IL-15 support (figure 2). Furthermore, when rechallenged after a 2-week hiatus to mimic relapse, CAR-NK cells still cleared tumor cells, suggesting that tumor-mediated activation was dispensable. Interestingly, CAR-NK cells expressing only the membrane-versions of IL-15 got substantially enriched, indicating cis signaling and avoidance of systemic toxicities. In vivo evaluations will be conducted to validate the same.Conclusions Our novel IL-15 modules confer CAR-NK cells unmatched persistence, confuting qualms about NK cells’ long-term persistence and cytotoxic potency. We also describe a robust CAR-NK manufacturing process to cater to hundreds of patients with malignancies and autoimmune disorders. Conceived with cost-sensitive countries in mind, our CAR-NK therapy is expected to be priced much lower than the CAR-T cell therapy cost in developed countries.References Marin D, Li Y, Basar R, Rafei H, Daher M, Dou J, Mohanty V, Dede M, Nieto Y, Uprety N, Acharya S, Liu E, Wilson J, Banerjee P, Macapinlac HA, Ganesh C, Thall PF, Bassett R, Ammari M, Rao S, Cao K, Shanley M, Kaplan M, Hosing C, Kebriaei P, Nastoupil LJ, Flowers CR, Moseley SM, Lin P, Ang S, Popat UR, Qazilbash MH, Champlin RE, Chen K, Shpall EJ, Rezvani K. Safety, efficacy and determinants of response of allogeneic CD19-specific CAR-NK cells in CD19+ B cell tumors: a phase 1/2 trial. Nat Med. 2024 Mar;30(3):772-784. doi: 10.1038/s41591-023-02785-8. Epub 2024 Jan 18. PMID: 38238616; PMCID: PMC10957466Egli L, Kaulfuss M, Mietz J, Picozzi A, Verhoeyen E, Münz C, Chijioke O. CAR T cells outperform CAR NK cells in CAR-mediated effector functions in head-to-head comparison. Exp Hematol Oncol. 2024 May 14;13(1):51. doi: 10.1186/s40164-024-00522-6. PMID: 38745250; PMCID: PMC11092129Williams MD, Chen AT, Stone MR, Guo L, Belmont BJ, Turk R, Bogard N, Kearns N, Young M, Daines B, Darnell M. TRAFfic signals: High-throughput CAR discovery in NK cells reveals novel TRAF-binding endodomains that drive enhanced persistence and cytotoxicity. bioRxiv [Preprint]. 2023 Aug 5:2023.08.02.551530. doi: 10.1101/2023.08.02.551530. PMID: 37577560; PMCID: PMC10418287Silvestre RN, Eitler J, de Azevedo JTC, Tirapelle MC, Fantacini DMC, de Souza LEB, Swiech K, Covas DT, Calado RT, Montero PO, Malmegrim KCR, Figueiredo ML, Tonn T, Picanço-Castro V. Engineering NK-CAR.19 cells with the IL-15/IL-15Rα complex improved proliferation and anti-tumor effect in vivo. Front Immunol. 2023 Sep 25;14:1226518. doi: 10.3389/fimmu.2023.1226518. PMID: 37818365; PMCID: PMC10561086Imamura M, Shook D, Kamiya T, Shimasaki N, Chai SM, Coustan-Smith E, Imai C, Campana D. Autonomous growth and increased cytotoxicity of natural killer cells expressing membrane-bound interleukin-15. Blood. 2014 Aug 14;124(7):1081-8. doi: 10.1182/blood-2014-02-556837. Epub 2014 Jul 8. PMID: 25006133Liu E, Tong Y, Dotti G, Shaim H, Savoldo B, Mukherjee M, Orange J, Wan X, Lu X, Reynolds A, Gagea M, Banerjee P, Cai R, Bdaiwi MH, Basar R, Muftuoglu M, Li L, Marin D, Wierda W, Keating M, Champlin R, Shpall E, Rezvani K. Cord blood NK cells engineered to express IL-15 and a CD19-targeted CAR show long-term persistence and potent antitumor activity. Leukemia. 2018 Feb;32(2):520-531. doi: 10.1038/leu.2017.226. Epub 2017 Jul 20. PMID: 28725044; PMCID: PMC6063081Abstract 288 Figure 1Properties of CAR-NK cells manufactured using our indigenous platform (a) Properties of CAR-NK cells at harvest. (b) Transduction evaluated across multiplicities of infection (MOI). (c) Long-term tumor rechallenge assay without or with secretory IL-15 armoring, over 96 days, with 27 semiweekly challenges with Raji cellsAbstract 288 Figure 2Long-term in vitro tumor rechallenge assays using IL-15-armored CAR-NK cells. (a) CAR-NK cells without (Control) or with different IL-15 armors, challenged semiweekly with Raji cells, ± exogenous IL-15. (b,c) Enrichment of CAR-NK cells co-cultured for >2 months (b) or 3 months (c), with regular or interrupted tumor rechallenges