Project Summary This proposal describes a unique SUPRA CAR system in human T cells to address in vivo off-tumor toxici- ty, using an established liver transgene expression mouse model. We will use our recently developed split, universal and programmable (SUPRA) CAR system that simultaneously encompasses multiple critical up- grades, such as the ability to finely tune T cell activation strength, sense and logically respond to multiple antigens, and independently regulate two T cell subtypes simultaneously. Specifically, we will evaluate two types of circuits: (1) AND and (2) NIMPLY (A AND NOT B) logic CAR circuits, which have been determined bioinformatically as the best type for circuits for discriminating tumors from healthy cells. The proposed exper- iments will test the hypotheses that (1) our modular and tunable logic CAR T systems are uniquely suited for preventing on-target off-tumor toxicity in vivo, even when healthy tissues expressing a tar- get antigen are near the tumor, and (2) the tumor specificity and activity of our SUPRA CAR systems is enhanced by controlled delivery. Importantly, preliminary data support these hypotheses, and well- characterized materials and rigorous experimental designs are established in this proposal with essential cross-disciplinary collaborations and expertise that encompass synthetic biology, immunology, animal tumor biology, and biomaterials. The specific aims of this five-year proposal are as follows: Aim 1 evaluates AND log- ic SUPRA CAR specificity in vivo. Aim 2 investigate the tumor specificity and toxicity of NIMPLY logic SUPRA CAR in vivo. Aim 3 determine the effect of adaptor protein delivery mechanism on tumor-targeting specificity. Outcomes from this work will establish the clinical potential of logic CAR systems and expand their use to addi- tional cancers, including solid tumors.

Project Narrative While CAR T cells therapy are very promising, the precise targeting to cancer cells with minimal off-tumor tox- icity remains a significant challenge. Thus, we will use our novel SUPRA CAR system in human T cells and a liver transgene expression mouse model to interrogate human therapeutics while addressing in vivo off-tumor toxicity in animals. Successful completion of the project aims will afford material and designs for improved CAR T cell treatments for a wide array of cancers, including solid tumors.