Abstract 2990: Polyfunctional anti-CD19 CAR T cells determined by single-cell multiplex proteomics associated with clinical activity in patients with advanced non-Hodgkin’s lymphoma

Abstract

Introduction: Autologous anti-CD19 CAR T cells have shown promising clinical efficacy in B cell malignancies, with T cell expansion and blood levels for IL-15, IL-10 and Granzyme B as correlates of objective response and toxicity (Kochenderfer et al. J Clin Oncol 2016; 34:LBA3010). It is unclear, however, which key immune programs in CAR T cells impact their in vivo expansion and clinical outcome. We evaluated in detail the functionality of anti-CD19 CAR T cells by using single-cell proteomics analysis (Lu et al. PNAS 2015;113:607-615). We explored how the polyfunctionality of pre-infusion CAR T cell products, post-stimulation with the CD19 antigen in vitro, associated with CAR T cell expansion in vivo and objective response. Methods: Product T cells were separated into CD4+ or CD8+ T cell subsets using microbeads. CD4+ or CD8+ fractions were then co-cultured with CD19-K562 targets or NGFR-K562 control cells, at a 1:2 ratio for 20 hrs. Single cells were then analyzed using a 32-plex panel of secreted cytokines, chemokines, and cytotoxic molecules. Specifically, T cells were loaded onto a single-cell barcode chip capable of assaying 32 secreted proteins/cell. The polyfunctional profile and strength (pSI) of each sample was determined (Ma et al. Cancer Discov 2013;3:418-429) and analyzed relative to in vivo expansion of the CAR T cells and patient response to the CAR T cell therapy. CAR T cell expansion in blood was measured by quantitative PCR. Results: Single-cell pSI of patient CAR T cells showed a statistically significant association (p = 0.011) with objective response (complete or partial response) to the therapy. While product pSI showed variability across patients, the median pSI was 2+ times higher for responders versus non-responders. The polyfunctional profiles for both CD4+ and CD8+ cells were dominated by effector molecules, stimulatory cytokines and chemokines. Polyfunctional CD4+ and CD8+ subsets with IFN-γ, IL-8 and/or MIP-1α correlated best with patient outcome, with CD8+ T cells showing co-expression of Granzyme B, and CD4+ T cells also comprising IL-17A+IL8+ and IL5+IL8+ subsets. While CAR expansion in vivo also correlated with objective response (p = 0.032), the association between product pSI and CAR cell expansion in vivo did not reach statistical significance (p = 0.079), suggesting that they bring independent contributions to predicting objective response. In support of that, a composite index integrating pSI and CAR T cell expansion in vivo associated best with clinical response (p = 0.005). Conclusion: Polyfunctionality of CAR T cells, in conjunction with their expansion in vivo, correlates with clinical outcome in an anti-CD19 CAR T cell clinical trial. Single-cell multiplexed proteomics measurements may provide powerful insight into the clinical performance of CAR T cell products. [J.R. and P.P. contributed equally to this study.] Citation Format: John Rossi, Patrick Paczkowski, Yueh-wei Shen, Kevin Morse, Brianna Flynn, Alaina Kaiser, Colin Ng, Kyle Gallatin, Tom Cain, Rong Fan, Sean Mackay, James Heath, Steven A. Rosenberg, James N. Kochenderfer, Jing Zhou, Adrian Bot. Polyfunctional anti-CD19 CAR T cells determined by single-cell multiplex proteomics associated with clinical activity in patients with advanced non-Hodgkin’s lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2990. doi:10.1158/1538-7445.AM2017-2990

Single-Cell Analysis of CAR-T Cell Activation Reveals A Mixed TH1/TH2 Response Independent of Differentiation

“…The cytokine response is minimally associated with differentiation states such as naïve, central memory, effector memory, and effector cells. All the results suggest that the activation of CAR-engineered T cells is a canonical process associated with a highly mixed multi-functional response, supporting the notion that polyfunctional CAR-T cells correlate with objective response of non-Hodgkin’s lymphoma patients reported in a CD19 CAR-T clinical trial [19].…”

Concise Review: Emerging Principles from the Clinical Application of Chimeric Antigen Receptor T Cell Therapies for B Cell Malignancies

“…Once manufactured, the T cell composition of the CAR T product is also important and influenced by the starting material. For example, the presence of a CCR7+ naïve/central memory subset correlates with peak CAR T expansion and optimal function, while patient outcomes are best when utilizing CAR T cell subsets that are capable of secreting multiple classes of cytokines . As a specific example of a treatment that affects T cells, in CLL the kinase inhibitor ibrutinib inhibits malignant B cell proliferation through bruton's tyrosine kinase (BTK), but also affects non‐malignant T cells and shifts T helper cells toward the Th1 immune subset .…”

“…There is evidence that the functionality of manufactured CAR T cells is influenced by the composition of the T cell subsets that are circulating in the patient's blood at the time of collection. For example, Sommermeyer et al enriched various T cell subsets from patient blood and tested their anti-tumor function as CAR T 56,86,87]. As a specific example of a treatment that affects T cells, in CLL the kinase inhibitor ibrutinib inhibits malignant B cell proliferation through bruton's tyrosine kinase (BTK), but also affects non-malignant T cells and shifts T helper cells toward the Th1 immune subset [88].…”