Abstract 1847: Engineered cell surface tag-targeted IL-2 and IL-21 selectively and safely enhance CAR-T anti-tumor activity via different mechanisms
Authors: Mathewson Nathan D. , Chen Wei , Bessette Paul , Sleiman Sara , Sukthankar Meghana , Moynihan Kelly D. , Kimberlin Chris , Park Terrence , Hollingsworth Audrey , Gill Saar , Yeung Andy , Djuretic Ivana 
Published: June 2023

Abstract
The treatment of some patients with hematological malignancies has been transformed by chimeric antigen receptor T (CAR-T) cells. However, a large clinical need remains for more effective CAR-T therapies and to expand their use to broader patient groups including those with solid tumors. The exogenous administration of IL-2 and IL-21 were shown to enhance the engraftment, persistence, and functionality of CAR-Ts in preclinical models. However, clinical use of such cytokines is limited due to their pleiotropic nature that can result in toxicity and undesired effects on endogenous immune cells. To overcome this, we applied our cis-targeting technology to develop CAR-T specific IL-2 and IL-21 molecules that selectively activate CAR-Ts and exhibit minimal activity on non-CAR cells. Cis-targeted IL-2 and IL-21 molecules are comprised of a targeting antibody fused to an affinity-attenuated cytokine mutein, the activity of which is rescued due to the avidity provided by the targeting arm. This enables the highly selective delivery of cytokine support to human CAR-T cells. We engineered two types of fusions targeting either 1) the CAR directly without blocking CAR antigen recognition (CAR-IL2) or 2) an exogenous cell surface tag (non-signaling truncated epidermal growth factor receptor [EGFRt]) co-expressed with the CAR (EGFRt-IL2 and EGFRt-IL21). We characterized these molecules in vitro and in vivo. Both CAR- and EGFRt-targeted molecules demonstrated &gt;100-fold preferential STAT activation of CAR-T cells over non-CAR cells. Unexpectedly, CAR-targeted fusions induced substantial antigen independent cytokine release, whereas fusions targeting the EGFRt tag did not. We found that antigen independent cytokine release was more suppressed by a LCK inhibitor rather than a JAK inhibitor, suggesting that downstream CAR signaling was the major contributor, compared to cytokine receptor signaling. In vivo studies examined the anti-tumor activity of the tag-targeted cytokine fusions given their desired safety profile. A single dose of either EGFRt-IL2 or EGFRt-IL21 on day 1 or day 7 following a sub-optimal infusion of CD19 CAR-T cells (0.1 x106) induced strong tumor regression and relapse free survival for &gt;70 days in an aggressive NALM-6 leukemia model. EGFRt-IL21 induced a sustained 10-fold expansion of CAR-T numbers over PBS in the blood by day 7. In contrast, EGFRt-IL2 induced a delayed but greater expansion (1000-fold over PBS) by day 14 suggestive of a mechanistic difference of the two cis-targeted molecules. Analysis of EGFRt-IL2 and EGFRt-IL21 in a solid tumor model using CAR-T cells targeting endogenous tumor antigen showed similar anti-tumor effects. Cis-targeted IL-2 and IL-21 fusion molecules directed by anti-tag EGFRt antibodies confer selective enhancement of CAR-T cells with a minimal safety risk, representing a promising approach as an adjuvant CAR-T therapy.
Citation Format: Nathan D. Mathewson, Wei Chen, Paul Bessette, Sara Sleiman, Meghana Sukthankar, Kelly D. Moynihan, Chris Kimberlin, Terrence Park, Audrey Hollingsworth, Saar Gill, Andy Yeung, Ivana Djuretic. Engineered cell surface tag-targeted IL-2 and IL-21 selectively and safely enhance CAR-T anti-tumor activity via different mechanisms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1847.