Abstract 3252: OrthoCARs: Engineered human IL-2/IL-2Rb orthogonal pairs selectively enhance CAR T cell anti-tumor efficacy and durability of response
Authors: Aspuria Paul-Joseph , Bauer Michele , Vivona Sandro , Kauder Steven E. , McCauley Scott , Riener Romina , Malefyte Rene De Waal , Ratti Navneet , Rokkam Deepti , Emmerich Jan , Lupardus Patrick J. , Kastelein Rob A. , Oft Martin 
Published: September 2020

Abstract
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). 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. Interleukin-2 (IL-2) is a potent stimulator of CD4 and CD8 effector 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. To facilitate selective in vivo expansion of engineered T cells we have developed an orthogonal (ortho) ligand/receptor system consisting of a pegylated, IL-2 mutein (STK-009) and a mutated IL-2 Receptor Beta (orthoIL-2Rβ) that selectively bind each another, but do not interact with their wild type receptor and cytokine counterparts. This system allows for in-vivo IL-2 signaling in engineered adoptive cell therapies that express the orthoIL-2Rβ while avoiding signaling in non-tumor reactive T cells and NK cells. Here, we demonstrate the ability of STK-009/orthoIL-2Rβ pair to selectively potentiate human orthoIL-2Rβ expressing CD19 CAR T cells in vitro and in vivo. We incorporated orthoIL-2Rβ into a CD19 directed CAR lentiviral construct utilizing a T2A peptide linker, allowing the use of a single lentiviral plasmid to generate orthoCAR T cells. Transduction of donor T cells with the CAR+orthoIL-2Rβ lentivirus and the use of STK-009 during in vitro manufacturing enabled selective proliferation and enrichment of orthoCAR transduced T cells. orthoCAR T cells grown in STK-009 maintained a similar immunophenotype and cytotoxic function compared to T cells manufactured with wild type IL-2. Subcutaneous administration of STK-009 dramatically enhanced the expansion (&gt;100-fold) and efficacy of orthoCAR T cells in an aggressive pre-clinical mouse model of B cell malignancy (Raji), demonstrating 100% durable complete responses, compared to 50% relapsed and refractory lymphoma in CD19 CAR alone. Immunoprofiling of in vivo orthoCAR T cells revealed that STK-009 treatment significantly increased both naïve and effector memory RA (TEMRA) populations. RNAseq and IHC analysis also showed that STK-009 treatment increases expression of cytotoxic molecules such as Granzyme B and perforin specifically in the CAR T cells, without inducing GranzymeB in other cells. These findings validate a potent platform that selectively harnesses the potent anti-tumoral T cell effector functions of IL-2 to improve the efficacy and durability CAR T cell therapy.
Citation Format: Paul-Joseph Aspuria, Michele Bauer, Sandro Vivona, Steven E. Kauder, Scott McCauley, Romina Riener, Rene De Waal Malefyte, Navneet Ratti, 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 anti-tumor efficacy and durability of response [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3252.