Abstract 2317: Dose control of CAR-like T cell activity through post-translational covalent loading of ligands to a universal immune receptor
Authors: Minutolo Nicholas G. , Sharma Prannda , Tsourkas Andrew , Powell Daniel J. 
Published: August 2019

Abstract
The use of chimeric antigen receptor (CAR) T cells for the treatment of CD19+ hematologic malignancies has shown great success in the clinic, leading to FDA approval for various forms of leukemia. However, their broad use is limited since a CAR targets a single tumor associated antigen (TAA), which is not effective against tumors with heterogeneous TAA expression or emerging antigen loss variants. Further, stably engineered CAR T cells can continually proliferate and activate in response to antigen, potentially causing fatal cytokine release syndrome (CRS) or neurotoxicity without a means of quantifiable control. These toxicities and limitations have prompted the development of an alternative CAR format called universal immune receptors (UIRs). UIRs allow multi-antigen targeting with a single receptor to prevent and address antigen escape due to heterogeneity and loss, targeting ligand (TL) dose dependent control of CAR activity, control of CRS, and normal B cell reconstitution following CD19 targeting. However, all current formats rely on non-covalent engagement of the TL which can lead to issues with sub-optimal affinity and specificity.
We have developed a next generation UIR system that allows for post-translational covalent attachment of TLs to the receptor. This is accomplished through the use of the SpyCatcher-SpyTag protein system. When combined at physiologic pH and temperature, these two proteins form a covalent bond. The SpyCatcher immune receptor (Spy-IR) contains the SpyCatcher protein as its extracellular domain attached to standard second-generation CAR intracellular signaling domains. Addition of TAA-specific TLs labeled with SpyTag allows for on-demand CAR generation through covalent bond formation between the TL and the receptor at the T cell surface. Here, primary human T cells expressing the Spy-IR are quantitatively loaded with targeting ligands, allowing for dose-titratable control of redirected T cell effector function and tumor cell lysis. Even as a single cell product, Spy-IR T cells can recognize an array of tumor antigens, (e.g. Her2, EGFR, EpCAM, and CD20) either simultaneously or sequentially to address the evolving tumor antigen landscape. Further, dose-dependent arming of Spy-IR T cells with TL allows for controlled immune recognition of cells expressing either high or low level antigen to distinguish between tumor and normal cells, thus providing an additional means of safety. Finally, preclinical efficacy of the Spy-IR system was achieved in NSG mouse models of aggressive solid tumor and intraperitoneal disease. In conclusion, the SpyCatcher immune receptor is the first UIR designed for post-translational covalent attachment of targeting ligands that permits timed “on demand” redirection of T cells against a diverse array of antigens, to address limitations of safety and antigen diversity by conventional CAR T cell therapy.
Citation Format: Nicholas G. Minutolo, Prannda Sharma, Andrew Tsourkas, Daniel J. Powell. Dose control of CAR-like T cell activity through post-translational covalent loading of ligands to a universal immune receptor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2317.