Session: 703. Adoptive Immunotherapy: In Vitro, Correlative, and Early Phase Studies to Improve Safety and Efficacy of CAR-T Cells
Hematology Disease Topics & Pathways:
Biological, Therapies, CAR-Ts
To understand the fate of the infused CAR-T cells we determined the phenotype, function, and clonal nature of the persisting CTL019 cells. Flow cytometric CART19 cell analyses demonstrated that early during the anti-leukemia response, activated, HLA-DR-expressing CD8+ CAR-T cells rapidly expanded, followed by similarly activated CD4+ CAR-T cells. With tumor clearance the CAR-T cell population contracted, but an activated CD4+ CAR-T cell population was maintained and was still detectable at the last follow-up of 7 years. The CD8+ CAR-T cell pool remained present at low frequencies. Both populations had acquired and maintained an effector memory phenotype, a phenotype most consistent with active disease control. Furthermore, the analysis of the classical immune checkpoint inhibitory markers PD1, TIM3, LAG3, and CTLA4 showed that only PD1 was expressed from the earliest to the latest time point on >80% of all CAR-T cells, whereas LAG3 and TIM3 were expressed only early on but lost after tumor clearance. These data suggest that the initial tumor clearance was mediated by CD8+ CAR-T cells, but sustained by a CD4+ CAR-T cell population that still actively engages with target cells. To understand the clonal nature of these long-term persisting CAR-T cells we used two complementary methods: a) CAR T cells were sorted from post-infusion aliquots during the first two years for T cell receptor-beta deep-sequencing (TCR-seq); b) the CAR integration sites in the genome were sequenced in the infusion product and in circulating CAR-T cells. TCR-seq analysis of early post-infusion time points demonstrated that the circulating CAR-T cell populations consisted of hundreds to thousands of distinct clones which in patient 1 and 2 displayed clonal focusing by 21 and 1 month post-infusion, respectively, with some clones making up as much as 12% (patient 1) and 48% (patient 2) of the CAR-T cell repertoire. The analysis of clonotype sharing at the various time points via Morisita’s overlap index analysis similarly showed repertoire stabilization late (21 months; patient 1) and early (1 month; patient 2) after infusion. Lastly, fate mapping of the infused CART19 cells via CAR integration site analysis in the infusion product until the latest time point indicated that the infusion products for both patients had a very diverse, non-clonal make-up, containing over 8,000 and 3,700 integration sites in patients 1 and 2, respectively. The higher degree of clonality in patient 2 but not 1 CAR-T cells as seen by TCR-seq was confirmed by integration site analysis, as was the sharing of CAR-T cell clones over time. Importantly, whereas the CAR integration site repertoire in patient 1 was diverse in the first two years, it stabilized and trended towards oligoclonality 21 months after infusion. Lastly, CAR integration site analysis revealed a high degree of clonal persistence, suggesting that tumor control and B cell aplasia were maintained by few, highly functional CD4+ CAR-T cell clones.
In summary, we demonstrate that in both patients with the longest persistence of CAR-T cells reported thus far, early and late phases of the anti-CLL response are dominated by highly activated CD8+ and CD4+ CAR-T cells, respectively, largely comprised of a small number of persisting CD4+ CAR-T cell clones.
Disclosures: Melenhorst: Parker Institute for Cancer Immunotherapy: Research Funding; Incyte: Research Funding; Casi Pharmaceuticals: Consultancy; novartis: Patents & Royalties, Research Funding; Shanghai UNICAR Therapy, Inc: Consultancy. Porter: Genentech: Other: Spouse employment; Novartis: Other: Advisory board, Patents & Royalties, Research Funding; Kite Pharma: Other: Advisory board. Lacey: Novartis Pharmaceuticals Corporation: Research Funding; Tmunity: Research Funding; Novartis Pharmaceuticals Corporation: Patents & Royalties; Parker Foundation: Research Funding. Fraietta: Novartis: Patents & Royalties: WO/2015/157252, WO/2016/164580, WO/2017/049166. Frey: Novartis: Consultancy; Servier Consultancy: Consultancy. Young: Novartis: Patents & Royalties, Research Funding. Siegel: Novartis: Research Funding. June: Novartis Pharmaceutical Corporation: Patents & Royalties, Research Funding; Immune Design: Membership on an entity's Board of Directors or advisory committees; Tmunity Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Immune Design: Membership on an entity's Board of Directors or advisory committees; Celldex: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceutical Corporation: Patents & Royalties, Research Funding; Tmunity Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.
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