Abstract 6399: Two-stage CD8+ CAR T-cell differentiation in patients with large B-cell lymphoma

Abstract Background: Chimeric antigen receptor (CAR) T-cell therapy has expanded therapeutic options for patients with relapsed/refractory diffuse large B-cell lymphoma (r/r DLBCL). However, progress in improving clinical outcomes has been limited by an incomplete understanding of CAR T-cell differentiation in patients. Methods: To comprehensively investigate CAR T-cell differentiation in vivo, we performed single-cell, multi-modal, and longitudinal analyses of CD28-costimulated CAR T cells from infusion product and peripheral blood (day 8-28) of seven patients with r/r DLBCL who exhibited a complete response with axicabtagene ciloleucel. Results: Our clonotypic and phenotypic analyses indicate that CD8+ CAR T cells undergo two distinct waves of clonal expansion in vivo. The first wave is dominated by CAR T cells with an exhausted-like effector memory transcriptomic profile and CD39+CD57−CX3CR1lo flow phenotype during the peak expansion period (day 8-14). The second wave is dominated by CAR T cells with a terminal effector transcriptomic profile and CD39−CD57+CX3CR1hi flow phenotype during the post-peak persistence period (day 21-28). The two waves exhibit non-overlapping clonotypes and phenotypes. Our observation of two waves biologically uncouples CAR T-cell expansion from persistence, by designating these characteristics to different in vivo cell populations. Moreover, lineage tracing analysis via each CAR T cell’s endogenous TCR clonotype demonstrates that the two waves originate from different effector precursors in the infusion product. Precursors of the first wave exhibit more effector-like signatures, whereas precursors of the second wave exhibit more stem-like signatures. These findings suggest that pre-infusion heterogeneity mediates the two waves of in vivo clonal expansion. Conclusion: Our findings support a two-stage model for CD8+ CAR T-cell differentiation. Although two-stage differentiation is a novel phenomenon that has not yet been reported in the literature, the individual elements of our model are consistent with current understanding in the field. Importantly, our finding implies that manipulating the phenotypic composition of the infusion product may allow more precise control over in vivo CAR T-cell differentiation for modulating therapeutic efficacy. Lastly, our findings provide evidence against the intuitive idea that the post-peak contraction in CAR abundance is solely apoptosis or extravasation of short-lived CAR T cells from peak expansion. Rather, even as total CAR abundance contracts after peak expansion, a distinct subset of CAR T-cell clones simultaneously expands to eventually dominate the peripheral blood CAR T-cell repertoire. In conclusion, our findings demonstrate that CAR T-cell expansion and persistence are mediated by clonally, phenotypically, and ontogenically distinct CAR T-cell populations that serve complementary clinical purposes. Citation Format: Guoshuai Cao, Yifei Hu, Tony Pan, Erting Tang, Nicholas Asby, Thomas Althaus, Jun Wan, Peter Riedell, Michael Bishop, Justin Kline, Jun Huang. Two-stage CD8+ CAR T-cell differentiation in patients with large B-cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6399.