131 Genetic disruption of negative immune regulators to enhance CAR-T efficacy against solid tumors (2021)

<h3>Background</h3> CAR-T cell therapy has demonstrated remarkable success in hematological malignancies but displays limited efficacy in solid tumors, which comprise most cancer cases. Recent studies suggest that CAR-T cell failure via T cell exhaustion is characterized by decreased surface CAR expression, cytotoxicity, and Th1 cytokine production, leading to reduced antitumor functionality.<sup>1 2 3</sup> To address these issues, studies have turned to genetically knocking out or overexpressing targets associated with an exhaustion or effector phenotype, such as PD-1 knockout (KO) and c-Jun overexpression, among other candidates that are typically receptors or transcription factors.<sup>4 5 6</sup> However, there are other underexplored factors that mediate various aspects of immune regulation. While genome-wide CRISPR screens may discover such factors, they are unlikely to reveal phenotypes for genes whose function is partially redundant, therefore promising candidates may be missed. Such candidates include post-transcriptional regulators (PTRs) that coordinate immune responses, which are less well-studied in the context of CAR-T cell function. We hypothesized that KO of these PTRs may increase CAR-T cell cytokine activity, phenotype, and persistence, potentially under long-term or exhaustion-inducing conditions, leading to increased tumor control. Ultimately, disruption of negative immune regulators could produce CAR-T cells with enhanced activity and persistence, narrowing the gap between efficacy in hematological and solid tumors. <h3>Methods</h3> To explore whether the disruption of two target PTRs improves solid tumor efficacy, we used CRISPR-Cas9 to genetically delete one or both PTRs in mesothelin-targeting human CAR-T cells and assayed their function in vitro and in vivo in NSG mice. <h3>Results</h3> We show successful genetic deletion of these genes in post-thymic human T cells and that their disruption does not affect primary expansion (figure 1) or transduction efficiency (figure 2). These KO CAR-T cells display increased expression of co-stimulatory receptors and various cytokines (figure 3). While KO CAR-T cells are functionally similar to WT CAR-T cells in in vitro assays (figure 4), KO CAR-T cells demonstrate superior activity in vivo and can clear large, established tumors compared to WT CAR-T cells at low dose (figure 5). <h3>Conclusions</h3> These results indicate that KO of our target PTRs may improve the potency of CAR-T cells in solid tumors and may have important implications on the development of effective solid-tumor cell therapies. <h3>References</h3> JE Wherry and M Kurachi, Molecular and cellular insights into T cell exhaustion, <i>Nature Reviews Immunology</i> 2015;<b>15</b>:486–499. EW Weber, et al. Transient rest restores functionality in exhausted CAR-T cells through epigenetic remodeling. <i>Science</i> 2021;<b>372</b>:6537. S Kuramitsu et al. Induction of T cell dysfunction and NK-like T cell differentiation in vitro and in patients after CAR T cell treatment. <i>Cell</i>, in revision. BD Choi et al, CRISPR-Cas9 disruption of PD-1 enhances activity of university EGFRvIII CAR T cells in a preclinical model of human glioblastoma. <i>Journal for ImmunoTherapy of Cancer</i> 2019;<b>7</b>:304. RC Lynn et al. c-Jun overexpression in CAR T cells induces exhaustion resistance. <i>Nature</i> 2019;<b>576</b>:293–300. LJ Rupp et al. CRISPR/Cas9-mediated PD-1 disruption enhances anti-tumor efficacy of human chimeric antigen receptor T cells. <i>Scientific Reports</i> 2017;<b>7</b>:737.