Background: Autologous anti-CD19 CAR T cell therapy is a curative-intent treatment for patients with B cell malignancies. Still, more than 50% of patients either do not respond or relapse after an initial response to the treatment. Tumor intrinsic factors including elevated disease burden, low antigen expression, or an immune suppressive microenvironment have been associated with disease progression [Scholler et al., Nat Med, 2022; Locke, Filosto et al., Nat Med, 2024]. A less differentiated, naïve-like, product T cell phenotype and CAR T cell expansion has been associated with favorable outcome [Filosto et al., Blood Canc Disc 2024, Locke et al., Blood Adv. 2020]. These features, however, could only partially explain the outcome following CAR T cell therapy and our knowledge of product features associated with and possibly predictive of treatment resistance remains limited.
Methods: We performed genomic and functional assessments of Axicabtagene Ciloleucel (axi-cel) products from 36 LBCL patients (11 ongoing responders, 19 relapsed, 6 non-responders by data cut off) enrolled in cohorts 1 and 2 of the Zuma-1 trial. CAR T cell products (N = 36) were profiled by both single-cell RNA sequencing (sc-RNAseq) and single-cell epigenetic sequencing (sc-ATACseq). Functional assays included measurement of secreted cytokines following CAR T cell stimulation with CD19+ cancer cells (N=36) as well as with synthetic target cells composed of red blood cells (RBC) coated with specific densities of recombinant CD19 (N=34). NSG-MHC I/II DKO mice (n=105) inoculated intravenously with Raji tumor cells were treated with axi-cel from 19 ZUMA1 patients for efficacy evaluation in vivo. The in vivo studies included analysis of peripheral blood CAR T-cell kinetics (by dd-PCR) and cytokine levels (by Luminex). Correlative analyses were performed between the in vitro or in vivo assay readouts and matched patients' clinical outcome.
Results: In vitro, multiple modes of CD19 antigen stimulation induced production of select Th1 (IL2 and Interferon gamma) and Th2 (IL4, IL5, and IL13) cytokines at various ratios, suggesting differences in Th1 polarization. A scaled Th1-Th2 index strongly associated (descriptive P < 0.05) with clinical efficacy, particularly when product cells were stimulated with RBC cross-linked with CD19. Leiden clustering of sc-RNAseq profiles revealed 12 major product T cell clusters. We identified a strong association (P = 0.029) between poor efficacy and a CD8 + T cell cluster, characterized by the overexpression of the Th2 master regulator, GATA3 (P = 0.0037). This cluster resembled the dysfunctional CD8+ infiltrating lymphocytes previously observed in solid tumors (Singer et al., Cell, 2016). These associations with clinical outcome were stronger, compared to the trend observed for naïve-like T-cell product phenotype in the same patients (P = 0.44). An association with lack of response was further found for a hypo-proliferative, exhausted T cell cluster (P = 0.049). The expression of the exhaustion and immune checkpoint marker, TIGIT, was common to both clusters associated with disease progression. Consistent associations were observed for progression-free survival analyses. Matched sc-ATACseq analysis presented an enrichment in chromatin accessibility in the GATA3 promoter of CD8+ T cells from non-responder patients, consistent with results from the sc-RNAseq analysis. In vivo, tumor-bearing mice treated with products derived from patients in durable ongoing response had superior tumor clearance and survival compared to those treated with products from relapsed and non-responder patients. Clinical response in patients showed association with mouse survival in vivo by Fisher's exact test (P = 0.08). Further, a significant correlation was observed between tumor control in vivo with CAR T expansion in mice (P < 0.005) and in patients (P = 0.01), as well as Th1-Th2 index measured in mice (P = 0.003) and in patients (P = 0.02).
Conclusions: In vitro and in vivo functional assays, as well as transcriptomic and epigenetic profiling with axi-cel from ZUMA1 patients, demonstrate the importance and relative ranking of certain product features in driving treatment outcomes. These findings can inform on mechanisms of resistance and development of next generation CAR T-cell therapies.
Poddar:Kite, a Gilead Company: Current Employment, Other: Travel Support; Gilead Sciences: Current equity holder in publicly-traded company; UCLA: Patents & Royalties. Balderrama-Gutierrez:Kite, A Gilead Company: Current Employment, Current equity holder in publicly-traded company. Williams:Kite, A Gilead Company: Current Employment, Current equity holder in publicly-traded company. Trinh:Kite, A Gilead Company: Current Employment, Current equity holder in publicly-traded company. Cao:Kite, A Gilead Company: Current Employment, Current equity holder in publicly-traded company. Kumar:Kite, a Gilead company: Current Employment, Current equity holder in publicly-traded company. Walker:Kite, a Gilead company: Current Employment, Current equity holder in publicly-traded company. Davis:Kite, A Gilead Company: Current Employment, Current equity holder in publicly-traded company. Calo:Kite, A Gilead Company: Current Employment, Current equity holder in publicly-traded company. Valny:Kite, A Gilead Company: Current Employment, Current equity holder in publicly-traded company. Yoder:Kite, a Gilead company: Current Employment, Current equity holder in publicly-traded company. Huang:Kite, A Gilead Company: Current Employment, Current equity holder in publicly-traded company. Budka:Kite, A Gilead Company: Current Employment. Kim:Kite Pharma: Current Employment; Gilead Sciences: Current equity holder in publicly-traded company. Shen:Kite, a Gilead Company: Current Employment, Patents & Royalties; Gilead Sciences: Current equity holder in publicly-traded company; Atara: Patents & Royalties. Filosto:Kite, A Gilead Company: Current Employment. Viaud:Kite, a Gilead company: Current Employment, Current equity holder in publicly-traded company; Gustave Roussy Institute: Patents & Royalties; Calibr: Patents & Royalties. Andrade:Kite, A Gilead Company: Current Employment, Current equity holder in publicly-traded company. Bedognetti:Kite, A Gilead Company: Current Employment, Current equity holder in publicly-traded company. Mattie:Kite, A Gilead Company: Current Employment.
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