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The immune system is a sophisticated network of different cell types performing complex biocomputation at single-cell and consortium levels. The ability to reprogram such an interconnected multicellular system holds enormous promise in treating various diseases, as exemplified by the use of chimeric antigen receptor (CAR) T cells as cancer therapy. However, most CAR designs lack computation features and cannot reprogram multiple immune cell types in a coordinated manner. Here, leveraging our split, universal, and programmable (SUPRA) CAR system, we develop an inhibitory feature, achieving a three-input logic, and demonstrate that this programmable system is functional in diverse adaptive and innate immune cells. We also create an inducible multi-cellular NIMPLY circuit, kill switch, and a synthetic intercellular communication channel. Our work highlights that a simple split CAR design can generate diverse and complex phenotypes and provide a foundation for engineering an immune cell consortium with user-defined functionalities.

BackgroundThe application of CRISPR/Cas9-gene-editing to enhance the anti-tumor activity of T cell Adoptive Cell Therapies (ACT) is a promising approach in the treatment of patients with solid tumors. We developed an in vivo CRISPR^2 screening approach and interrogated the top dual-edit combinations enhancing T cell anti-tumor function. We discovered that across all possible dual-edit combinations of T cell targets, inactivation of Regnase-1 and SOCS1 led to the greatest enhancement in anti-tumor T cell potency in vivo. We applied these findings to discover KSQ-004, a Regnase-1/SOCS1 dual-edited human CRISPR/Cas9-engineered TIL (eTIL) therapy currently under development for therapeutic use.MethodsWe generated randomly paired CRISPR guide libraries (CRISPR2) targeting top hits from previous single-gene Immune CRISPRomics® screens. CRISPR^2 libraries with over 1200 gene pairs were screened in primary mouse OT1 and PMEL-TCR-Tg-T cells in the relevant syngeneic tumor models. Top dual-edit combinations were then evaluated in the immunotherapy-refractory B16F10 metastatic lung tumor model. The efficacy of the top combo was further evaluated in a mouse TIL model wherein TIL from B16-Ova tumors were expanded and engineered ex vivo and adoptively transferred into tumor bearing hosts for efficacy assessment.ResultsOf the 1200+ combinations tested in the CRISPR^2 screens, the Regnase-1/SOCS1 combination ranked amongst the top dual-edits, with this combination enhancing T cell infiltration into tumors >3500-fold in comparison to controls. Studies conducted in the checkpoint therapy refractory B16F10 lung metastasis model revealed that Regnase-1/SOCS1 dual-edited PMEL-TCR-Tg-T cells conferred remarkable survival benefit relative to controls, significantly extending median survival of animals from 21 days to 53 days. Furthermore, Regnase-1+SOCS1-edited mouse TIL isolated and expanded from B16-Ova tumors exerted complete control of tumors upon re-infusion into hosts, suggesting rejuvenation of tumor-experienced TILs by this edit combination. To apply these insights for therapeutic use, we discovered KSQ-004, a human Regnase-1/SOCS1 dual-edited CRISPR/Cas9-engineered TIL (eTIL). Methods were developed to manufacture KSQ-004 from melanoma and NSCLC tumor samples, with eTIL demonstrating robust expansion and viability comparable to unedited control TIL with over 90% knockout of both targets. Importantly, KSQ-004 produced elevated IFNγ upon autologous tumor stimulation and exerted greater control of tumor spheroids in vitro.ConclusionsWe used a novel CRISPR^2 screen approach to identify Regnase-1/SOCS1 as the top dual edit combination enhancing T cell function in the tumor microenvironment. We translated these findings to therapeutic use with the discovery of KSQ-004, a dual-edited eTIL therapy engineered that possesses enhanced anti-tumor potency and persistence against solid tumors.

BackgroundAdoptive cell therapy with ex vivo expanded tumor infiltrating lymphocytes (TIL) offers a potentially curative treatment for cancer. However, the immunosuppressive tumor microenvironment limits the effectiveness of TIL therapy. To address this medical need, we used our Immune-CRISPRomics® Platform to perform a series of genome-wide CRISPR/Cas9 screens to identify targets enhancing the ability of T cells to infiltrate and kill solid tumors in an in vivo setting. These screens identified SOCS1 as a top target that restrains T cell anti-tumor immunity. Based on these findings, we developed KSQ-001, an engineered TIL (eTIL) therapy created via CRISPR/Cas9-mediated editing of SOCS1 for the treatment of solid tumors.MethodsGenome-wide CRISPR/Cas9 screens were conducted in in vitro primary human T cells and TIL cultures and in in vivo primary mouse OT1 and PMEL-TCR-Tg T cells in syngeneic tumor models. The efficacy of surrogate murine KSQ-001 (mKSQ-001), in which the SOCS1 gene is inactivated by CRISPR/Cas9 in OT1 or PMEL-TCR-Tg T cells, was evaluated in both the B16-Ova and CRC-gp100 syngeneic tumor models, with memory formation and efficacy evaluated both in the presence and absence of cyclophosphamide-mediated lymphodepletion. KSQ-001 was manufactured from human TIL using SOCS1-targeting sgRNAs selected for therapeutic use based on potency and selectivity, with KSQ-001 characterized for in vitro function and in vivo anti-tumor efficacy.ResultsUpon adoptive transfer of a single dose into solid tumor-bearing hosts, mKSQ-001 was found to robustly enhance anti-tumor efficacy and eradicate tumors in 7/10 mice in the PD1-sensitive OT1/B16-Ova model and to drive responses in the PD-1 refractory PMEL/CRC-gp100 syngeneic tumor model. mKSQ-001 also showed a ten-fold increase in anti-tumor potency in vivo compared to unengineered T-cell product and established durable anti-tumor memory by persisting in the form of T central memory cells detectable at high frequency in the peripheral blood of complete responder mice. In the setting of lymphodepletion, mKSQ-001 also displayed heightened anti-tumor potency, accumulation, and memory formation in comparison to inactivation of PD-1. Importantly, human KSQ-001 displayed a transcriptional signature indicative of increased anti-tumor function, produced increased amounts of pro-inflammatory cytokines, exhibited a hypersensitivity to IL-12 signaling, and demonstrated increased anti-tumor function both in vitro and in vivo solid tumor models.ConclusionsBased on insights from our Immune-CRISPRomics® platform and demonstrated efficacy across multiple preclinical tumor models, we have developed KSQ-001, a novel eTIL therapy. These preclinical data support clinical testing of KSQ-001 in a variety of solid tumor indications.

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