Abstract 2790: The monocyte lineage is required for production of clinically relevant interleukin-6 (IL-6) during chimeric antigen receptor (CAR) T cell cytotoxicity
Authors: Barrett David M. , Singh Nathan , Levine Bruce , Grupp Stephan A. 
Published: May 2015

BACKGROUND CARs combine a single chain variable fragment (scFv) of an antibody with intracellular signaling domains into a single chimeric protein. We previously reported on CAR T cells (CTL019) recognizing CD19 combined with the CD137 and CD3zeta intracellular activation domain, including 1 sustained CR in a patient with ALL (Grupp, et al. NEJM 2013). This CR was associated with significant cytokine release syndrome (CRS) characterized by high IL-6, and toxicity was reversed promptly with targeted IL-6 blockade via tocilizumab. We investigated the mechanism of this unexpected CRS using clinical samples.

METHODS T cells were transduced with a CAR composed of anti-CD19 scFv/4-1BB/CD3ζ, activated/expanded ex-vivo with anti-CD3/anti-CD28 beads. NSG mice were given 106 of a primary pediatric leukemia IV on Day 0 followed by 107 CTL019 cells from the clinical product of the patient reported in the NEJM (CHP100) or a normal donor (ND). We gave tocilizumab at 100 mcg IP before or after CTL019 infusion and examined human cytokine production in the mouse serum by Luminex.

For the in vitro experiment, CTL019 cells were mixed with Nalm-6 target cells and either autologous monocytes, immature dendritic cells (iDC), mature dendritic cells (mDC) or macrophages and cytokines measured by Luminex at 18 and 48 hours.

RESULTS The xenograft model showed efficacy with CTL019 cells extending overall survival in whether from CHP100 or a ND. Cytokines significantly elevated in mouse serum (p&lt;0.05) included interferon-gamma (IFNg), tumor necrosis factor alpha (TNFa), granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-2 (IL-2). No IL-6 was detected in any mouse and pre or post-CTL019 tocilizumab had no effect on survival. Given the lack of IL-6 in a model that includes only ALL targets and CTL019, we investigated in vitro the role of the monocyte lineage in IL-6 secretion. When CTL019 and Nalm-6 targets are co-cultured, IFNg, TNFa, IL-2 and GM-CSF are again elevated but not IL-6. When any monocyte lineage is present, the IL-2 was elevated 100-fold.IL-6 rose from &lt;10 pg/mL to 250 to 300 pg/mL, and was highest with mDC.

Controls pairing monocyte lineages with T cells or Nalm-6 only produced no cytokines.

CONCLUSIONS:

CTL019 cells can undergo robust in-vivo expansion and can persist for 18 mo or longer in pts with relapsed ALL, and is associated with a significant CRS that responds rapidly to IL-6-targeted anticytokine treatment.

These data using a clinical product shows the importance of host factors in the CRS, and indicates the monocyte lineage is required for IL-6 secretion and maintenance of a feedback loop that is key for toxicity. Further study into models that include the monocyte lineage will be needed to fully address the importance of IL-6 blockade in the therapeutic efficacy of CTL019 and the timing of blockade to alleviate or prevent toxicity.

Citation Format: David M. Barrett, Nathan Singh, Bruce Levine, Stephan A. Grupp. The monocyte lineage is required for production of clinically relevant interleukin-6 (IL-6) during chimeric antigen receptor (CAR) T cell cytotoxicity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2790. doi:10.1158/1538-7445.AM2014-2790