Abstract 1512: OrthoCARs: Engineered human IL-2/IL-2Rb orthogonal pairs selectively enhance CAR T cell antitumor efficacy by driving T cell expansion and fitness
Authors: Aspuria Paul-Joseph P. , Vivona Sandro , Bauer Michele , Semana Marie , Ratti Navneet , McCauley Scott , Riener Romina , Malefyt Rene de Waal , Rokkam Deepti , Emmerich Jan , Lupardus Patrick J. , Kastelein Rob A. , Oft Martin 
Published: July 2021

Abstract
CAR T cell therapy (CAR) has demonstrated remarkable clinical efficacy in hematological malignancies. However, barriers such as poor T cell effector function, lack of proliferation, and limited persistence prevent CARs from reaching their full curative potential. IL-2 is a potent stimulator of T cell proliferation, survival, and cytotoxic function, making it an attractive cytokine to support CARs. However, therapeutic use of IL-2 is limited by systemic toxicity due its promiscuous activation of undesired immune cell populations.
To facilitate selective expansion and activation of CARs we have developed a human orthogonal ligand/receptor system consisting of a pegylated IL-2 mutein (STK-009) that does not significantly activate the wild type IL-2 receptor and a mutated IL-2 Receptor Beta (hoRb) that is fully activated by STK-009, but does not respond to the native IL-2 ligand. This system enables in vivo IL-2 signaling in CARs engineered to express hoRb while avoiding signaling bystander T cells and NK cells. Here, we demonstrate the ability of the STK-009/hoRb receptor pair to selectively enhance the anti-tumor efficacy of hoRb expressing CD19 CARs (SYNCAR-001) in preclinical lymphoma mouse models at will. We also demonstrate, in non-human primates (NHP), that STK-009 is selective for hoRb expressing cells.
SYNCAR-001 + STK-009 can lead to complete responses in subcutaneous Raji mouse models, even with SYNCAR-001 dosed at sub-efficacious levels (400,000 CAR-Ts/mouse). Subcutaneous dosing of STK-009 expands SYNCAR-001 systemically and drives infiltration of SYNCAR-001 into tumors. UMAP analysis of STK-009 treated SYNCAR-001 reveal a small PD1+LAG3+ subpopulation during tumor rejection which disappeared once tumors are controlled. The majority of T cells showed no exhaustion markers. Further, an IL-7R+ population arises and remains after tumor control, indicating long term memory development. Nanostring analysis confirms upregulation of IL-7R and other critical genes involved in cytotoxic activity and persistence in SYNCAR-001 when treated with STK-009. These data demonstrate that STK-009 treatment expands SYNCAR-001 and delivers a gene signature indicative of enhanced T cell fitness and activity.
Subcutaneous administration of STK-009 in NHP shows no evidence of toxicity or physiological IL-2 mediated activity on immune cells, including Teffs, Tregs, and NK cells. Pharmacokinetic analysis of STK-009 shows stable exposure with minimal clearance, demonstrating the selectivity of STK-009.
These findings validate an orthogonal platform that selectively drives potent T cell effector functions of engineered cells without the toxicities mediated by NK cells or non-tumor specific T cells associated with high dose IL-2 therapy. These results demonstrate the ability of this orthogonal platform to improve the efficacy and durability of CARs.
Citation Format: Paul-Joseph P. Aspuria, Sandro Vivona, Michele Bauer, Marie Semana, Navneet Ratti, Scott McCauley, Romina Riener, Rene de Waal Malefyt, Deepti Rokkam, Jan Emmerich, Patrick J. Lupardus, Rob A. Kastelein, Martin Oft. OrthoCARs: Engineered human IL-2/IL-2Rb orthogonal pairs selectively enhance CAR T cell antitumor efficacy by driving T cell expansion and fitness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1512.