Project Summary/Abstract Renal Cell Carcinoma (RCC) accounts for approximately 90% of kidney cancers in adults. Despite the advancements made in cytokine, immune checkpoint, and targeted therapies, the rate of complete remission is low and nearly all patients with metastatic disease experience disease progression. CAR-T therapy showed clinically curative outcomes in hematological malignancies. However, there are multiple roadblocks for CAR-T efficacy in solid tumors. Our company and founder have developed novel unbiased large/whole-genome T cell CRISPR screening approaches and identified several universal modifications that enhanced CAR-T efficacy against several tumor models in mice. We have developed a novel unbiased loss-of-function (LOF) screen, enabled by site-specific, high-throughput massive knock-in engineering platform. Through this unbiased evolution platform, we have identified a key gene PRDM1 mutation (exon 3 deletion) that modifies the epigenetic state of T cells and significantly enhances immune characteristics of CAR-T cells, including increased T cell proliferation, reduced exhaustion with improved memory and persistence, and high intra-tumor infiltration. In addition, we have exploited the endocytic feature of the cytotoxic T-lymphocyte associated antigen-4 (CTLA-4) cytoplasmic tail (CT) to reprogram CAR-T function and substantially enhance CAR-T anti-tumor activity in vivo. CAR-T cells with CTLA-4 CT fused to the CAR C-terminus exhibit a progressive increase in cytotoxicity under repeated stimulation, accompanied by reduced production of proinflammatory cytokines. ENPP3 is highly expressed in >90% RCC cases, and expression is highly restricted in normal tissues and typically located apically in normal tissues and thus relatively inaccessible to CAR-T. We have generated and screened multiple scFvs against ENPP3, developed ENPP3 gene-enhanced CAR-Ts, and showed promising in vitro and in vivo results in our kidney cancer animal model with no toxicity. The goal of this proposal is to evaluate whether the combination of CAR-CTLA4 tail fusion and PRDM1-exon 3 knockout (KO) results in superior CAR-T–activity against RCC tumor relative to either modification alone, and to nominate the best performing ENPP3 CAR-T plus genetic modification(s) for IND and Phase 1 clinical trials. Specifically, we will evaluate ENPP3 CAR-Ts with PRDM1 △exon3 alone (CIB-101), CTLA4 tail fusion alone (CIB-102), both PRDM1 △exon3 + CTLA4 tail fusion (CIB-103), and CAR-T with no modifications (CIB-100). We will select our final clinical candidate by performing side-by-side in vitro as well as pre-clinical in vivo evaluation of CIB-100 to 103 (aim 1, phase1), followed by process development, GMP manufacturing, pre-clinical development, and IND filing. These studies include efficacy, PK/PD, GLP toxicology, and pathology analysis on our final autologous ENPP3 CAR-T clinical candidate (aim 2, phase 2). By the end of the study period, we anticipate having selected ...