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

• Published in: Cancer research (Chicago, Ill.) Ill.), 2017-07, Vol.77 (13_Supplement), p.2990-2990
• Main 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
• Format: Article
• Language: English
• ISSN: 0008-5472; 1538-7445
• DOI: 10.1158/1538-7445.AM2017-2990

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