OrthoCARs: Engineered human IL-2/IL-2Rb orthogonal pairs selectively enhance CAR T cell antitumorefficacy
Authors: Aspuria Paul-Joseph , Bauer Michael A , vivona Sandro , de Waal Malefyt Rene , Kastelein Rob , Oft Martin , Emmerich Jan , Rokkam Deepti , Kauder Steven E , McCauley Scott , Riener Romina , Verma Rakesh 
Published: November 2020

CAR T cell therapy has demonstrated remarkable clinical efficacy against relapsed and refractory hematological malignancies, such as B cell non-Hodgkin lymphoma (NHL) and acute lymphoblastic leukemia (ALL) (Neelapu et al. NEJM, 2017; Schuster et al. NEJM, 2018; Turtle et al. Sci Trans Med, 2016). Despite these advances, prominent barriers including poor T cell effector function, lack of proliferation, and limited CAR T cell persistence prevent CAR T cell therapies from reaching their full curative potential (Srivastava and Riddell, Journal of Immunology, 2019). Interleukin-2 (IL-2) is a potent stimulator of CD4 and CD8 T cell proliferation, survival, and cytotoxic function, thereby making it an attractive molecule to support CAR T cell therapy. However, therapeutic use of IL-2 is limited by systemic toxicity due its promiscuous activation of undesired immune cell populations, including non-tumor reactive T cells and NK cells (Rosenberg et al. Journal of Immunology, 2014).
To facilitate selective ex vivo and in vivo expansion of engineered T cells we have developed a human orthogonal (ortho) ligand/receptor system consisting of a pegylated, IL-2 mutein (STK- 009) that does not significantly activate the wild type receptor and a mutated IL-2 Receptor Beta (orthoIL-2Rβ) that does not significantly respond to its native ligand, wild type IL-2. This system enables in vivo IL-2 signaling in engineered cells that express the orthoIL-2Rβ while avoiding signaling in bystander T cells and NK cells. Here, we demonstrate the ability of the STK-009/orthoIL-2Rb ligand/receptor pair to selectively potentiate human orthoIL-2Rb (hoRb) expressing CD19 CAR T cells (CD19 orthoCAR T cells) in vitro and in vivo. We also demonstrate that STK-009 is selective for the orthogonal IL-2Rb and in a non-human primate model does not potentiate wild type T or NK cells and shows no evidence of toxicity.
The STK-009/CD19 orthoCAR T platform was evaluated in a disseminated Raji mouse model of aggressive lymphoma. Subcutaneous administration of STK-009 dramatically expanded the CD19 orthoCAR T cells possessing a clinically favorable TSCM and TEMRA immunophenotype and significant antitumor efficacy was observed even at doses of CAR T cells typically regarded as sub-efficacious. When STK-009 dosing was stopped after complete tumor responses, CD19 orthoCAR T cells contracted as expected. Subsequent redosing of STK-009 in these tumor free mice re-expanded CD19 orthoCAR T cell levels demonstrating the on-demand control of the STK-009/orthoCAR T cell platform.
Given the deep and durable responses we observed in the disseminated Raji model, we subsequently invested investigated the efficacy of the STK-009/orthoCAR T cell platform in a subcutaneous Raji model of lymphoma characteristically resistant to CAR T cell therapy. No significant anti-tumor effect was observed in mice treated with either CAR T cells alone or the combination of high dose wild-type IL-2 and CAR T cells. The subcutaneous administration of STK-009 in combination with a sub-efficacious dose of CD19 orthoCAR T cells demonstrated significant expansion of the CD19 orthoCAR T cells with the clinically favorable TSCM and TEMRA immunophenotype and potent anti-tumor efficacy in this subcutaneous lymphoma model, demonstrating the selective potentiation of the CD19 orthoCAR T cells in response to STK-009.
The toxicity of STK-009 was evaluated in a non-human primate dose-escalation study. Subcutaneous administration STK-009 at anticipated therapeutic doses showed no evidence of toxicity or biological effect on immune cells expressing the wild-type IL-2 receptor. Pharmacokinetic analysis of STK-009 in this study showed 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 CAR T cell therapies.

Disclosures
Aspuria: Synthekine: Current Employment. Bauer:Synthekine: Current Employment. vivona:Synthekine: Current Employment. de Waal Malefyt:Synthekine: Current Employment. Kastelein:Synthekine: Current Employment. Oft:Synthekine: Current Employment. Emmerich:Synthekine: Current Employment. Rokkam:Synthekine: Current Employment. Kauder:Synthekine: Current Employment. McCauley:Synthekine: Current Employment. Riener:Synthekine: Current Employment. Verma:Synthekine: Current Employment.