S909 AXL‐RTK INHIBITION DIRECTS THE FUNCTIONAL PHENOTYPE OF CHIMERIC ANTIGEN RECEPTOR T CELLS
Authors: Sakemura R. , Yang N. , Cox M. , Sinha S. , Hefazi M. , Hansen M. , Schick K. , Boysen J. , Mouritsen L. , Foulks J. , Warner S. , Parikh S. , Ding W. , Kay N. , Kenderian S. 
Published: June 2019

Background:

The expression of the receptor tyrosine kinase (RTK) Axl on cancer cells has been associated with poor prognosis and implicated in disease progression. Inhibition of Axl‐RTK with TP‐0903, a high affinity Axl‐RTK inhibitor induces robust apoptosis of several tumors, including chronic lymphocytic leukemia (CLL). Axl‐RTK inhibition has also been found to induce nonspecific immunomodulatory effects in syngeneic tumor models.

Aims:

We aimed to investigate the impact and downstream effects of Axl‐RTK inhibition on naïve T cells and chimeric antigen receptor T cells (CART), using CD19 directed CART cells (CART19) as a model.

Methods:

We utilized either PMA/Ionomycin or the CD19+ mantle cell lymphoma cell line, JeKo as a stimulant for naïve T cells and CART19, respectively. Cells were analyzed for cytokine profile, T cell phenotype, and kinetics after the stimulation in the presence or the absence of the Axl‐RTK inhibitor TP‐0903. Regarding in vivo experiment, a relapse JeKo xenograft model was used. Mice were treated with vehicle alone, TP‐0903 alone, CART19 alone, or TP‐0903 + CART19. Three weeks after the treatment, mice were re‐challenged with JeKo as a CART19 relapse model.

Results:

Stimulation of naïve T cells in the presence of the Axl‐RTK inhibitor TP‐0903 resulted in a significant reduction of Th2 cytokines and downregulation of inhibitory receptors. The differential expansion of either effector T cells or Tregs in the presence of TP‐0903 resulted in a preferential reduction of Tregs (Fig 1A). Axl‐RTK inhibition of CART19 led to Th1 polarization upon stimulation with the JeKo, as evident by the selective reduction of Th2 cytokine. Additionally, this resulted in a reduction of myeloid attracting cytokines, such as CXCL1, IL‐8, IL‐6, and IL‐10, cytokines implicated in the development cytokine release syndrome and neurotoxicity after CART cell therapy and in adaptive resistance to immunotherapy (Fig 1B). TP‐0903 also significantly downregulated inhibitory receptors on activated CARTs (Fig 1C). In our relapsed in vivo model, mice treated with CART19 + TP‐0903 rejected the JeKo challenge while mice received CART19 alone redeveloped disease, suggesting that Axl‐RTK inhibition enhanced CART cell persistence. Peripheral blood analysis of mice after the rechallenge showed a higher number of circulating T cells and lower level of MCP‐1. Western blotting was performed to investigate the downstream effect of Axl‐RTK inhibition on CART19 and it revealed a relatively selective inhibition of LCK on stimulated CART19. Transcriptome analysis of activated CARTs treated with TP‐0903 revealed >100 genes that were differentially expressed compared to untreated cells. Among these, Th cell and Treg polarization genes were significantly altered in activated CARTs treated with TP‐0903.

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Finally, we validated our findings in phase I clinical trial of TP‐0903 (NCT02729298). T cells isolated from 3 cancer patients showed a significant reduction of Tregs and Th1 polarization. This will be further investigated in a planned phase I clinical trial of TP‐0903 in relapsed/refractory CLL (NCT03572634).

Summary/Conclusion:

We showed for the first time that Axl‐RTK inhibition polarizes T cells into a Th1 functional phenotype, downregulates inhibitory receptors, and reduces myeloid associated cytokine production by CART19. The combination therapy yielded synergistic cytotoxicity against JeKo in vitro and in a CART19 relapse model. These findings encourage further study of Axl‐RTK inhibition with TP‐0903 as an enhancer of T cell immunotherapies.