Parker Institute for Cancer Immunotherapy

Cytokines dimerize two receptor chains to activate Janus kinases and STAT transcription factors that regulate immune cells but have therapeutic liabilities. We engineered “Trikines” to compel cis formation of three-chain cytokine receptor complexes at the cell surface that induce bespoke STAT transcriptional signaling programs optimized for therapeutic efficacy. Designed Trikines co-activated pSTAT5 and pSTAT3 signatures distinct from any natural cytokines, by assembling trimeric combinations of Interleukin-2 (IL-2), Interleukin-10 (IL-10), and Interleukin-21 (IL-21) receptors. An IL-2-based-Trikine restrained terminal differentiation of T cells, promoted stemness, and enhanced durability of tumor control without toxicity. Unexpectedly, an IL-10-based Trikine induced immune infiltration into poorly immunogenic tumors, showing striking efficacy in small cell lung cancer and pancreatic cancer models. Trikines obviate the need for cell engineering to customize STAT signatures for immunotherapy.

Fibroblasts play critical roles in regulating cellular relationships during tissue homeostasis, immunity, and tumor biology at multiple sites. However, tools to perturb fibroblasts at just one site in vivo are limited, restricting our understanding of how these cellular relationships act locally. We optimized local gene editing of fibroblasts in mouse tumor models to investigate how fibroblast perturbations affect the tumor microenvironment (TME). By knocking out receptors Osmr, Tgfbr2, or Il1r1 on cancer-associated fibroblasts (CAFs), we uncover that TGFBR2 signaling loss induces the emergence of a new Col18a1hi CAF cell state that is associated with worse survival in pancreatic cancer patients. Combinatorial gene KOs in CAFs reveals a circuit where these Col18a1hi CAFs reshape the TME by recruiting Siglec-Fhi neutrophils via Cxcl5 expression, and where this Col18a1hi CAF cell state is dependent on TNFR1 and canonical Wnt signaling. Together, a fast, affordable, and modular engineering method is demonstrated, allowing discovery of modified fibroblast identities and local intercellular relationships in the TME.

Conventional type 1 dendritic cells (cDC1s) are specialized for cross-presenting tumor antigens and determining the efficacy of immunotherapies, including immune checkpoint blockade and adoptive cell therapy. However, their rarity and tumor-induced dysfunction severely limit CD8 T cell priming and represent a central bottleneck to therapeutic efficacy. While strategies such as anti-DEC-205-mediated antigen delivery and Flt3L-driven DC expansion can enhance host DC function, their reliance on functional cDC1s remains a significant constraint. We developed Tim-3-targeted vaccines by conjugating tumor antigens or neoantigens to anti-Tim-3 antibodies. These vaccines delivered antigens to both cDC1s and cDC2s, and elicited robust, durable CD8 T cell responses. Remarkably, Tim-3-targeted vaccines endowed cDC2s with efficient cross-presentation capacity that matched that of cDC1s. In tumor-bearing mice or in CD11c-β-cateninactive mice, which model β-catenin-driven DC dysfunction, Tim-3-targeted vaccination restored cross-priming and counteracted tumor- and DC-mediated immunosuppression. In Batf3-/- mice lacking cDC1s, anti-Tim-3-based vaccines still elicited significant CD8 T cell cross-priming and tumor control-albeit both were reduced compared to wild-type mice- demonstrating that cDC1s contribute to but are not essential for Tim-3-targeted vaccine-induced CD8 T cell priming and anti-tumor efficacy. Strikingly, a single dose of anti-Tim-3-neoantigen vaccination eradicated large established MC38 tumors in a CD8 T cell-dependent manner. Together, these data identify Tim-3-targeted vaccines as a next-generation cancer vaccine platform that broadens DC engagement, reduces reliance on cDC1s, and overcomes tumor- and DC-mediated immunosuppression, addressing key limitations of current DC-based cancer vaccines.

Chimeric antigen receptor (CAR) T cell therapy has shown limited efficacy against solid tumors, which often reside in highly immunosuppressive tumor microenvironments (TMEs). TMEs can be highly abundant in vascular endothelial growth factor A (VEGF), which contributes to immunosuppression and abnormal tumor vasculature. Here, we found that CAR T cells engineered to secrete an anti-VEGF single-chain variable fragment (CAR-αVEGF T cells) achieved superior antitumor efficacy against multiple in vivo models of ovarian cancer and glioma, outperforming conventional CAR T cells with and without combination anti-VEGF antibody therapy. Microscopy, flow cytometry, and transcriptomic analyses revealed that armoring the CAR T cells with anti-VEGF single-chain variable fragments enhanced their activation and mitochondrial fitness and enriched immune-stimulatory signatures among endogenous immune cells in the tumor-bearing brain. Moreover, CAR-αVEGF T cells circumvented multiple detrimental effects associated with on-target CAR T cell therapy, including infiltration of suppressive myeloid cells, exaggerated vasculature abnormalities, and hypoxia. Together, our results provide rationale for the clinical translation of CAR-αVEGF T cells as a safe and potent therapy for solid tumors characterized by elevated VEGF.

The multifaceted dysfunction of tumour-infiltrating T cells, including exhaustion and mitochondrial dysfunction, remains a major obstacle in cancer immunotherapy1-6. Transcriptomic and epigenomic regulation of T cell dysfunction have been extensively studied7-9, but the role of proteostasis in regulating these obstacles remains less defined. Here we combined computational analyses of atlases of T cell exhaustion and mitochondrial fitness with performed targeted in vivo CRISPR screens, which identified the E3 ubiquitin ligase KLHL6 as a dual-negative regulator of both T cell exhaustion and mitochondrial dysfunction. Mechanistically, KLHL6 expression promoted TOX poly-ubiquitination and subsequent proteasomal degradation, thereby attenuating the transition of progenitor exhausted T cells towards terminal exhaustion. Simultaneously, KLHL6 maintained mitochondrial fitness by constraining the excessive mitochondrial fission that occurs during chronic T cell receptor stimulation by means of post-translational regulation of the PGAM5-Drp1 axis. However, KLHL6 is naturally downregulated by T cell receptor ligation, mitigating its potentially beneficial ubiquitin ligase activities during exposure to chronic stimulation. Enforcing KLHL6 expression in T cells markedly improved efficacy and long-term persistence against tumours and during viral infections in vivo. These findings uncover KLHL6 as a multifunctional, clinically actionable target for cancer immunotherapy, and highlight the potential of modulating proteostasis and ubiquitin modification to improve immunotherapy.

BackgroundEngineering chimeric antigen receptor (CAR) T cells with logic-gated synthetic Notch (synNotch) receptor circuits can enhance specificity and mitigate on-target/off-tumor toxicity. However, the conventional synNotch system uses two lentiviral vectors encoding the synNotch receptor and inducible CAR, requiring dual transduction and cell sorting, which limits clinical translation. Integrating the synNotch-CAR circuit into a single lentiviral vector could overcome this limitation, yet manufacturing CAR T cells with large transgenes remains challenging, as increasing transgene size drastically reduces lentiviral titers and T cell transduction efficiency. Current production workflows compensate for low transduction efficiency by sorting transduced cells, further impeding clinical translation. Consequently, these constraints have limited the broader development of synNotch-CAR T cell therapies.MethodsWe engineered a single-vector synNotch (svsNotch) system that integrates all components of the conventional dual-vector circuit into one lentiviral vector to facilitate clinical translation. To overcome the low lentiviral titers and T cell transduction efficiency caused by the large svsNotch transgene, we established an optimized CAR T cell production workflow for effector T cells with large lentiviral transgenes.ResultsOur optimized workflow increased T cell transduction rates by up to 14.8-fold and enabled the production of effector T cells with lentiviral transgenes exceeding the effective packaging capacity limit of 9.2 kb. As a proof of concept, we engineered human epidermal growth factor receptor 2 (HER2)-mesothelin (MSLN) svsNotch (9.2 kb), in which a synNotch receptor targeting HER2 regulates the expression of a second-generation 4-1BBζ CAR against MSLN to enable selective targeting of double-positive HER2+MSLN+ ovarian tumors. In vitro, HER2-MSLN svsNotch T cells demonstrated superior specificity to conventional dual-vector synNotch-CAR T cells, with selective cytotoxicity against HER2+MSLN+ but not HER2koMSLN+ tumor cells. To enable in vivo monitoring, we engineered HER2-MSLN-click beetle green (CBG) svsNotch (10.1 kb) incorporating CBG luciferase. In mouse models using constitutive CAR T cells as controls, HER2-MSLN-CBG svsNotch T cells exhibited minimal cytotoxicity in the absence of HER2 and superior efficacy against HER2lowMSLNhigh and HER2highMSLNhigh tumors.ConclusionThese data establish a framework for engineering logic-gated single-vector immunotherapies and provide an optimized workflow for generating CAR T cells with transgenes that exceed current size limitations.