Abstract Autologous T cells engineered to express a chimeric antigen receptor (CAR) have transformed the standard of care for patients with B cell malignancies, but >50% of patients progress following therapy. Here, we sought to understand key T cell intrinsic factors impacting efficacy: CAR T cell expansion, persistence, and homing to the tumor. Using an endogenous T cell receptor (TCR) sequence as a ‘barcode’, we followed individual T cell clonotypes at the single-cell level from pre-manufacture apheresis and infusion products to tumor-involved lymph nodes and blood at peak and late expansion in 22 adult patients with relapsed or refractory large B cell lymphoma (LBCL) or acute lymphoblastic leukemia (ALL) treated with axicabtagene ciloleucel, an FDA-approved CD19-CAR T cell therapy, or bispecific CD19/CD22-CAR T cells on an investigator-initiated trial (NCT03233854). The resulting CAR T cell atlas comprises matched transcriptome (scRNA-seq) and surface protein expression (CITE-seq) for 846,344 cells from 97 samples, with 251,175 unique TCR clonotypes identified, including 17,479 clonotypes that could be traced across ≥2 timepoints in CAR+ cells, enabling lineage tracing at scale for the first time. We found that T cells present in apheresis that preferentially expand during manufacturing primarily comprise T central memory (Tcm) cells and are enriched for predicted specificity to common viruses, including influenza, but also include a subset of T regulatory (Treg) cells. In infusion products, CAR T conventional (non-Treg) cells with superior abilities to home to the tumor, expand, and persist post-infusion share features of activated T effector memory (Tem) phenotype enriched for expression of BATF3 and high TCF4 transcription factor activity. Baseline apheresis and infusion products were enriched for cells classified as Tregs in patients with poor response. This observation aligned with our recent work linking post-infusion CAR Tregs to progression following CD19-CAR T cell therapy. We therefore performed lineage tracing and methylation analysis of the Treg-specific demethylated region (TSDR) in the FOXP3 gene and found that post-infusion CAR Tregs are primarily derived from pre-existing Tregs. Of interest, flow cytometry analysis of baseline blood from a cohort of 53 patients treated with CD19-CAR for LBCL showed that Tregs are elevated in baseline blood relative to healthy donors. Removing Tregs from healthy donor T cells prior to manufacturing enhanced CAR T cell expansion and anti-tumor activity against JeKo-1 human lymphoma cell line, whereas adding 5% Tregs to mimic patient T cells rendered the resulting CAR T cells less active. These analyses pinpoint the identities of apheresis T cells and infusion CAR T cells with properties impacting efficacy, and also identify pre-existing Tregs as limiting efficacy of CAR T cell immunotherapies. Citation Format: Zinaida Good, Mark P. Hamilton, Jay Y. Spiegel, Moksha H. Desai, Zachary J. Ehlinger, Patrick J. Quinn, Yiyun Chen, Snehit Prabhu, Shin-Heng Chiou, Sreevidya Kurra, Eric Yang, Michael G. Ozawa, Matthew J. Frank, Lori Muffly, Gursharan K. Claire, Sushma Bharadwaj, Saurabh Dahiya, Katherine A. Kong, Mark M. Davis, Sylvia K. Plevritis, Elena Sotillo, Bita Sahaf, David B. Miklos, Crystal L. Mackall. Lineage tracing defines responding CAR T cells in patients with B cell malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6325.
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Apr 5, 2024
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