In Vitro and In Vivo Characterization of Axicabtagene Ciloleucel Identifies Features Associated with Treatment Resistance in Patients, Including a Dysfunctional CD8+ T Cell State Characterized By Overexpression of GATA3 Transcript

Abstract

Background

Autologous anti-CD19 CAR T cell therapy is a curative-intent treatment for B cell malignancies. Still, over 50% of patients either fail to respond or relapse. Tumor intrinsic factors like high disease burden, low antigen expression, or an immune-suppressive microenvironment are associated with disease progression. A less-differentiated, naïve-like T cell phenotype and CAR T cell expansion are linked to favorable outcome. These factors, however, could only partially explain treatment outcome, and understanding of product features associated with resistance remains limited.

Methods

We performed genomic and functional assessments of axicabtagene ciloleucel (axi-cel) products from 36 LBCL patients in ZUMA-1. Genomic evaluation included both single-cell RNA sequencing (scRNAseq) and epigenetic sequencing (scATACseq). Functional assays measured cytokine secretion after CAR T cell stimulation with CD19-coated red blood cells (RBC) or CD19+ cancer cells. Additionally, tumor bearing NSG-MHC I/II DKO mice were treated with axi-cel from 19 patients to evaluate in vivo efficacy, including CAR T-cell pharmacokinetics and cytokine levels. Correlative analyses between assay results and matched patients’ clinical outcomes were performed.

Results

CD19 antigen stimulation in vitro induced production of Th1 (IL2, IFNγ) and Th2 (IL4, IL5, IL13) cytokines, highlighting differences in Th1 polarization. A scaled Th1-Th2 index was strongly linked to clinical efficacy (P < 0.05), especially with CD19-cross-linked RBC stimulation. scRNA-seq of axi-cel products from ZUMA-1 patients revealed 12 transcriptionally distinct T cell clusters. We identified strong association (P=0.029) between disease progression and a CD8+ cluster overexpressing Th2 master regulator, GATA3. Additionally, a hypoproliferative exhausted T cell cluster was found to be associated with lack of response. Both clusters showed enrichment of exhaustion marker, TIGIT. Matched scATAC-seq confirmed these findings and presented an enrichment in chromatin accessibility in the GATA3 promoter of CD8+ T cells from non-responders. Lastly, tumor-bearing mice treated with CAR T products from patients with durable responses showed superior tumor clearance and survival, compared to those treated with products from relapsed or non-responder patients. Patient clinical response was associated with mouse survival (P = 0.08). Further, tumor control in vivo significantly correlated with CAR T cell expansion in both mice and patients (P = 0.01), as well as with the Th1-Th2 index in mice and patients.

Conclusions

Functional assays, as well as transcriptomic and epigenetic profiling with axi-cel, demonstrate the importance and relative ranking of product features in driving treatment outcomes. These findings can inform on mechanisms of resistance and development of next generation CAR T-cell therapies.