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
Authors: Rossi John , Paczkowski Patrick , Shen Yueh-wei , Morse Kevin , Flynn Brianna , Kaiser Alaina , Ng Colin , Gallatin Kyle , Cain Tom , Fan Rong , Mackay Sean , Heath James , Rosenberg Steven A. , Kochenderfer James N. , Zhou Jing , Bot Adrian 
Published: July 2017

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