Journal Articles

Radiotherapy synergizes with an inducible AAV-based immunotherapy platform to program local and systemic antitumor immunity

ABSTRACT: Radiotherapy (RT) can prime the immune system against cancer but often fails to generate effective antitumor responses due to concomitant induction of immunosuppressive factors. To overcome this limitation, we built upon the observation that RT enhances adeno-associated vectors (AAVs) tumor transduction through the epigenetic modification of vector episomes. We designed an AAV-based platform to deliver immunostimulatory cytokines through an interferon (IFN)-inducible promoter that allows for spatial control of transgene expression into irradiated tumors. As opposed to a constitutive system, local delivery of a vector encoding for inducible IL-12 (AAV-iIL12) achieves an efficient production of the cytokine without significant toxicity. Combination of RT and AAV-iIL12 generates a highly immunostimulatory tumor microenvironment (TME) leading to robust local and systemic antitumor responses in an IFNγ- and FAS-dependent manner, able to overcome common immune-evasion mechanisms. Our work shows that radiation coupled with AAV-based immune-gene delivery is an efficient and safe approach to treat cancer.

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Amino acid supplementation enhances in vivo efficacy of lipid nanoparticle-mediated mRNA delivery in preclinical models

Lipid nanoparticles (LNPs) play a critical role in the delivery of therapeutic messenger RNA (mRNA). Despite extensive efforts to optimize lipid formulations for in vivo delivery, efficacy of mRNA by LNPs remains suboptimal in many organs. Here, we demonstrate that LNP delivery efficacy is influenced by cellular metabolism, with the physiologic metabolome imposing constraints on mRNA expression from LNPs. Using an in vitro system, we found that simulated physiologic metabolic conditions led to the down-regulation of certain amino acid metabolic programs. Supplementation with an optimized formulation of methionine, arginine, and serine as an amino acid supplement (AAS) enhanced the uptake of LNPs and the expression of delivered mRNA cargo in epithelial cells in vitro. Coadministration of AAS with LNPs led to a 5- to 20-fold improvement in mRNA expression across various cell types and lipid formulations in vitro by promoting clathrin-independent carrier-mediated endocytosis. Delivery of mRNA by LNPs coadministered with AAS by multiple routes enhanced in vivo mRNA expression in preclinical models. Delivery of mRNA encoding growth hormone by LNPs with coadministration of AAS improved the liver growth hormone expression and the therapeutic outcomes in a model of inflammatory liver damage. Delivery of gene editing materials by LNP and AAS through an intratracheal route increased lung-targeted in vivo gene editing efficiency compared with LNP alone. The addition of an optimized AAS as a codelivered agent with LNPs may provide a simple strategy to broadly improve the efficacy of mRNA-based cell and gene therapies.

Author Info: (1) Biohub, Chicago, IL 60642, USA. Department of Biomedical Engineering, McCormick School of Engineering, Northwestern University, Evanston, IL 60208, USA. (2) Biohub, Chicago, IL

Author Info: (1) Biohub, Chicago, IL 60642, USA. Department of Biomedical Engineering, McCormick School of Engineering, Northwestern University, Evanston, IL 60208, USA. (2) Biohub, Chicago, IL 60642, USA. Department of Biomedical Engineering, McCormick School of Engineering, Northwestern University, Evanston, IL 60208, USA. (3) Biohub, Chicago, IL 60642, USA. (4) Biohub, Chicago, IL 60642, USA. (5) cTRL Therapeutics, Chicago, IL 60642, USA. (6) Biohub, Chicago, IL 60642, USA. Department of Biomedical Engineering, McCormick School of Engineering, Northwestern University, Evanston, IL 60208, USA. cTRL Therapeutics, Chicago, IL 60642, USA. Department of Chemistry, Weinberg College of Arts & Sciences, Northwestern University, Evanston, IL 60208, USA. Department of Biochemistry, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA. Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Chicago, IL 60611, USA. International Institute for Nanotechnology, Northwestern University, Evanston, IL 60208, USA. Simpson Querrey Institute, Northwestern University, Chicago, IL 60611, USA. (7) Biohub, Chicago, IL 60642, USA. Department of Biomedical Engineering, McCormick School of Engineering, Northwestern University, Evanston, IL 60208, USA.

Cancer cell-derived sialylated IgG interacting with Siglec-7/9/10 is a potential immunotherapeutic target in pancreatic cancer

The limited effectiveness of T cell-based immune checkpoint blockade (ICB) therapy in most patients with pancreatic ductal adenocarcinoma (PDAC) is largely due to poor CD8(+) T cell infiltration and a highly immunosuppressive microenvironment driven by excessive myeloid cell accumulation. This highlights the urgent need for new immunotherapy targets and strategies. In this study, an identified pro-cancer factor, cancer cell-derived sialylated IgG (SIA-IgG), is found to be significantly overexpressed in pancreatic cancer cells. SIA-IgG inhibits macrophage phagocytosis and induces an M2-like immunosuppressive phenotype through interactions with Siglec-7/9/10. SIA-IgG and TGF-_1, a key immunosuppressive factor, reinforce each other in a positive feedback loop, promoting immune evasion in PDAC. Blocking SIA-IgG with specific monoclonal antibodies shows significant therapeutic potential through reversal of PDAC's immunosuppressive microenvironment. Our findings identify the SIA-IgG/Siglec axis as an immunotherapeutic target for PDAC, offering a feasible approach for the development of immunotherapeutic strategies.

Author Info: (1) Department of Immunology, School of Basic Medical Sciences, Peking University, Beijing 100191, China; NHC Key Laboratory of Medical Immunology, Peking University, Beijing 10019

Author Info: (1) Department of Immunology, School of Basic Medical Sciences, Peking University, Beijing 100191, China; NHC Key Laboratory of Medical Immunology, Peking University, Beijing 100191, China. (2) Department of General Surgery, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730, China; Key Laboratory of Research in Pancreatic Tumor, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730, China; National Infrastructures for Translational Medicine, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730, China; State Key Laboratory of Complex, Severe, and Rare Diseases, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730, China. Electronic address: cuiming@pumch.cn. (3) Department of Immunology, School of Basic Medical Sciences, Peking University, Beijing 100191, China; NHC Key Laboratory of Medical Immunology, Peking University, Beijing 100191, China; Department of Respiratory and Critical Care Medicine, Nanjing Drum Tower Hospital, Affiliated Hospital of Nanjing University Medical School, Nanjing 210031, China. (4) Department of Immunology, School of Basic Medical Sciences, Peking University, Beijing 100191, China; NHC Key Laboratory of Medical Immunology, Peking University, Beijing 100191, China. (5) Department of General Surgery, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730, China; Key Laboratory of Research in Pancreatic Tumor, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730, China; National Infrastructures for Translational Medicine, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730, China; State Key Laboratory of Complex, Severe, and Rare Diseases, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730, China. (6) Department of Immunology, School of Basic Medical Sciences, Peking University, Beijing 100191, China; NHC Key Laboratory of Medical Immunology, Peking University, Beijing 100191, China. (7) Department of General Surgery, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730, China; Key Laboratory of Research in Pancreatic Tumor, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730, China; National Infrastructures for Translational Medicine, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730, China; State Key Laboratory of Complex, Severe, and Rare Diseases, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730, China. (8) Department of General Surgery, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730, China; Key Laboratory of Research in Pancreatic Tumor, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730, China; National Infrastructures for Translational Medicine, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730, China; State Key Laboratory of Complex, Severe, and Rare Diseases, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730, China. (9) State Key Laboratory of Complex, Severe, and Rare Diseases, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730, China; Research Center for Molecular Pathology, Department of Pathology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730, China. (10) Department of Immunology, School of Basic Medical Sciences, Peking University, Beijing 100191, China; NHC Key Laboratory of Medical Immunology, Peking University, Beijing 100191, China. (11) Department of Immunology, School of Basic Medical Sciences, Peking University, Beijing 100191, China; NHC Key Laboratory of Medical Immunology, Peking University, Beijing 100191, China. (12) Department of General Surgery, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730, China; Key Laboratory of Research in Pancreatic Tumor, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730, China; National Infrastructures for Translational Medicine, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730, China; State Key Laboratory of Complex, Severe, and Rare Diseases, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730, China. (13) Department of General Surgery, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730, China; Key Laboratory of Research in Pancreatic Tumor, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730, China; National Infrastructures for Translational Medicine, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730, China; State Key Laboratory of Complex, Severe, and Rare Diseases, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730, China. Electronic address: zhao8028@263.net. (14) Department of Immunology, School of Basic Medical Sciences, Peking University, Beijing 100191, China; NHC Key Laboratory of Medical Immunology, Peking University, Beijing 100191, China. Electronic address: qiuxy@bjmu.edu.cn.

A bispecific nanobody-drug conjugate targeting TROP2 and c-Met for low-concentration, single-dose treatment of pancreatic cancer

Pancreatic cancer remains highly lethal with limited treatment options. Although antibody-drug conjugates (ADCs) have emerged as promising therapeutic agents, their efficacy is often limited by heterogeneous antigen expression and poor tumor penetration. To address these limitations, we develop B6ADC, a nanobody-based bispecific ADC that simultaneously targets TROP2 and c-Met. In preclinical studies, B6ADC exhibits potent cytotoxicity in vitro across various TROP2/c-Met-expressing cancer cell lines and superior tumor inhibition in vivo compared with single-target ADC combination, including the clinically approved TROP2 ADC sacituzumab govitecan and c-Met ADC Teliso-V, as well as their combination. Notably, B6ADC eradicates giant tumors with a single dose at a low concentration of 2.2 mg/kg. We present a nanobody-based BsADC that simultaneously targets TROP2 and c-Met, with broad-spectrum antitumor activity, and improves selectivity for tumors with dual-positive or weakly positive antigen expression, offering a promising strategy for treating pancreatic cancer and other TROP2/c-Met-expressing malignancies.

Author Info: (1) State Key Laboratory of Vaccines for Infectious Diseases, Xiang An Biomedicine Laboratory, School of Public Health, Xiamen University, Xiamen 361102, China; National Institute

Author Info: (1) State Key Laboratory of Vaccines for Infectious Diseases, Xiang An Biomedicine Laboratory, School of Public Health, Xiamen University, Xiamen 361102, China; National Institute of Diagnostics and Vaccine Development in Infectious Diseases, National Innovation Platform for Industry-Education Integration in Vaccine Research, the Research Unit of Frontier Technology of Structural Vaccinology of Chinese Academy of Medical Sciences, Xiamen University, Xiamen 361102, China. (2) State Key Laboratory of Vaccines for Infectious Diseases, Xiang An Biomedicine Laboratory, School of Public Health, Xiamen University, Xiamen 361102, China; National Institute of Diagnostics and Vaccine Development in Infectious Diseases, National Innovation Platform for Industry-Education Integration in Vaccine Research, the Research Unit of Frontier Technology of Structural Vaccinology of Chinese Academy of Medical Sciences, Xiamen University, Xiamen 361102, China. (3) State Key Laboratory of Vaccines for Infectious Diseases, Xiang An Biomedicine Laboratory, School of Public Health, Xiamen University, Xiamen 361102, China; National Institute of Diagnostics and Vaccine Development in Infectious Diseases, National Innovation Platform for Industry-Education Integration in Vaccine Research, the Research Unit of Frontier Technology of Structural Vaccinology of Chinese Academy of Medical Sciences, Xiamen University, Xiamen 361102, China. (4) State Key Laboratory of Vaccines for Infectious Diseases, Xiang An Biomedicine Laboratory, School of Public Health, Xiamen University, Xiamen 361102, China; National Institute of Diagnostics and Vaccine Development in Infectious Diseases, National Innovation Platform for Industry-Education Integration in Vaccine Research, the Research Unit of Frontier Technology of Structural Vaccinology of Chinese Academy of Medical Sciences, Xiamen University, Xiamen 361102, China. (5) State Key Laboratory of Vaccines for Infectious Diseases, Xiang An Biomedicine Laboratory, School of Public Health, Xiamen University, Xiamen 361102, China; National Institute of Diagnostics and Vaccine Development in Infectious Diseases, National Innovation Platform for Industry-Education Integration in Vaccine Research, the Research Unit of Frontier Technology of Structural Vaccinology of Chinese Academy of Medical Sciences, Xiamen University, Xiamen 361102, China. (6) State Key Laboratory of Vaccines for Infectious Diseases, Xiang An Biomedicine Laboratory, School of Public Health, Xiamen University, Xiamen 361102, China; National Institute of Diagnostics and Vaccine Development in Infectious Diseases, National Innovation Platform for Industry-Education Integration in Vaccine Research, the Research Unit of Frontier Technology of Structural Vaccinology of Chinese Academy of Medical Sciences, Xiamen University, Xiamen 361102, China. (7) State Key Laboratory of Vaccines for Infectious Diseases, Xiang An Biomedicine Laboratory, School of Public Health, Xiamen University, Xiamen 361102, China; National Institute of Diagnostics and Vaccine Development in Infectious Diseases, National Innovation Platform for Industry-Education Integration in Vaccine Research, the Research Unit of Frontier Technology of Structural Vaccinology of Chinese Academy of Medical Sciences, Xiamen University, Xiamen 361102, China. (8) State Key Laboratory of Vaccines for Infectious Diseases, Xiang An Biomedicine Laboratory, School of Public Health, Xiamen University, Xiamen 361102, China; National Institute of Diagnostics and Vaccine Development in Infectious Diseases, National Innovation Platform for Industry-Education Integration in Vaccine Research, the Research Unit of Frontier Technology of Structural Vaccinology of Chinese Academy of Medical Sciences, Xiamen University, Xiamen 361102, China. (9) State Key Laboratory of Vaccines for Infectious Diseases, Xiang An Biomedicine Laboratory, School of Public Health, Xiamen University, Xiamen 361102, China; National Institute of Diagnostics and Vaccine Development in Infectious Diseases, National Innovation Platform for Industry-Education Integration in Vaccine Research, the Research Unit of Frontier Technology of Structural Vaccinology of Chinese Academy of Medical Sciences, Xiamen University, Xiamen 361102, China. (10) State Key Laboratory of Vaccines for Infectious Diseases, Xiang An Biomedicine Laboratory, School of Public Health, Xiamen University, Xiamen 361102, China; National Institute of Diagnostics and Vaccine Development in Infectious Diseases, National Innovation Platform for Industry-Education Integration in Vaccine Research, the Research Unit of Frontier Technology of Structural Vaccinology of Chinese Academy of Medical Sciences, Xiamen University, Xiamen 361102, China. (11) State Key Laboratory of Vaccines for Infectious Diseases, Xiang An Biomedicine Laboratory, School of Public Health, Xiamen University, Xiamen 361102, China; National Institute of Diagnostics and Vaccine Development in Infectious Diseases, National Innovation Platform for Industry-Education Integration in Vaccine Research, the Research Unit of Frontier Technology of Structural Vaccinology of Chinese Academy of Medical Sciences, Xiamen University, Xiamen 361102, China. (12) State Key Laboratory of Vaccines for Infectious Diseases, Xiang An Biomedicine Laboratory, School of Public Health, Xiamen University, Xiamen 361102, China; National Institute of Diagnostics and Vaccine Development in Infectious Diseases, National Innovation Platform for Industry-Education Integration in Vaccine Research, the Research Unit of Frontier Technology of Structural Vaccinology of Chinese Academy of Medical Sciences, Xiamen University, Xiamen 361102, China. (13) State Key Laboratory of Vaccines for Infectious Diseases, Xiang An Biomedicine Laboratory, School of Public Health, Xiamen University, Xiamen 361102, China; National Institute of Diagnostics and Vaccine Development in Infectious Diseases, National Innovation Platform for Industry-Education Integration in Vaccine Research, the Research Unit of Frontier Technology of Structural Vaccinology of Chinese Academy of Medical Sciences, Xiamen University, Xiamen 361102, China. (14) State Key Laboratory of Stress Biology, Fujian Provincial Key Laboratory of Innovative Drug Target Research, School of Pharmaceutical Sciences, Xiamen University, Xiamen 361102, China. (15) State Key Laboratory of Vaccines for Infectious Diseases, Xiang An Biomedicine Laboratory, School of Public Health, Xiamen University, Xiamen 361102, China; National Institute of Diagnostics and Vaccine Development in Infectious Diseases, National Innovation Platform for Industry-Education Integration in Vaccine Research, the Research Unit of Frontier Technology of Structural Vaccinology of Chinese Academy of Medical Sciences, Xiamen University, Xiamen 361102, China. Electronic address: yuanzhichen@xmu.edu.cn. (16) State Key Laboratory of Vaccines for Infectious Diseases, Xiang An Biomedicine Laboratory, School of Public Health, Xiamen University, Xiamen 361102, China; National Institute of Diagnostics and Vaccine Development in Infectious Diseases, National Innovation Platform for Industry-Education Integration in Vaccine Research, the Research Unit of Frontier Technology of Structural Vaccinology of Chinese Academy of Medical Sciences, Xiamen University, Xiamen 361102, China. Electronic address: nsxia@xmu.edu.cn. (17) State Key Laboratory of Vaccines for Infectious Diseases, Xiang An Biomedicine Laboratory, School of Public Health, Xiamen University, Xiamen 361102, China; National Institute of Diagnostics and Vaccine Development in Infectious Diseases, National Innovation Platform for Industry-Education Integration in Vaccine Research, the Research Unit of Frontier Technology of Structural Vaccinology of Chinese Academy of Medical Sciences, Xiamen University, Xiamen 361102, China. Electronic address: liuxue1108@xmu.edu.cn. (18) State Key Laboratory of Vaccines for Infectious Diseases, Xiang An Biomedicine Laboratory, School of Public Health, Xiamen University, Xiamen 361102, China; National Institute of Diagnostics and Vaccine Development in Infectious Diseases, National Innovation Platform for Industry-Education Integration in Vaccine Research, the Research Unit of Frontier Technology of Structural Vaccinology of Chinese Academy of Medical Sciences, Xiamen University, Xiamen 361102, China. Electronic address: wxluo@xmu.edu.cn.

Engineering lipid nanoparticle-stabilized emulsions for spatiotemporal mRNA delivery and enhanced T cell immunity

Achieving strong T cell responses remains a key challenge in mRNA vaccines and therapeutics. Here, we develop a colloid-engineered, lipid nanoparticle-stabilized emulsion (LSE) to study how spatiotemporal mRNA delivery influences immune dynamics. Multi-omic analyses (single-cell RNA sequencing [scRNA-seq], flow cytometry, and enzyme-linked immunosorbent assay [ELISA]) illustrate that LSE facilitates localizing mRNA to immunocytes, increasing antigen presentation while reducing off-target antigen secretion, and non-immune cell cross-presentation, which are key factors linked to T cell exhaustion in conventional LNP-based systems. This targeted delivery induces durable interferon (IFN)-_(+) and interleukin (IL)-2(+) T cell responses lasting up to 300 days and expands the T cell repertoire in mice compared to the AS01-adjuvanted Shingrix vaccine. Furthermore, LSE elicits potent protective and therapeutic effects against B16-OVA and LLC-NY-ESO1 inoculation in mice. These results indicate the potential of spatially controlled mRNA delivery for enhanced mRNA vaccinations.

Author Info: (1) National Key Laboratory of Biopharmaceutical Preparation and Delivery, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 10090, China; School of Chemical E

Author Info: (1) National Key Laboratory of Biopharmaceutical Preparation and Delivery, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 10090, China; School of Chemical Engineering, University of Chinese Academy of Sciences, Beijing 100049, China. (2) National Key Laboratory of Biopharmaceutical Preparation and Delivery, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 10090, China; School of Chemical Engineering, University of Chinese Academy of Sciences, Beijing 100049, China. (3) National Key Laboratory of Biopharmaceutical Preparation and Delivery, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 10090, China; School of Chemical Engineering, University of Chinese Academy of Sciences, Beijing 100049, China. (4) National Key Laboratory of Biopharmaceutical Preparation and Delivery, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 10090, China; School of Chemical Engineering, University of Chinese Academy of Sciences, Beijing 100049, China. (5) National Key Laboratory of Biopharmaceutical Preparation and Delivery, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 10090, China. (6) National Key Laboratory of Biopharmaceutical Preparation and Delivery, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 10090, China; School of Chemical Engineering, University of Chinese Academy of Sciences, Beijing 100049, China. (7) Thoracic Oncology Institute, Peking University People's Hospital, Beijing 100044, China; Department of Thoracic Surgery, Peking University People's Hospital, No. 11 Xizhimen South Street, Xicheng District, Beijing 100044, China; Institute of Advanced Clinical Medicine, Peking University, Beijing 100191, China. (8) Thoracic Oncology Institute, Peking University People's Hospital, Beijing 100044, China; Department of Thoracic Surgery, Peking University People's Hospital, No. 11 Xizhimen South Street, Xicheng District, Beijing 100044, China; Institute of Advanced Clinical Medicine, Peking University, Beijing 100191, China. (9) National Key Laboratory of Biopharmaceutical Preparation and Delivery, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 10090, China; School of Chemical Engineering, University of Chinese Academy of Sciences, Beijing 100049, China. (10) National Key Laboratory of Biopharmaceutical Preparation and Delivery, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 10090, China; School of Chemical Engineering, University of Chinese Academy of Sciences, Beijing 100049, China. Electronic address: yfxia@ipe.ac.cn.

Biodegradable targeted polymeric mRNA nanoparticles enable in vivo CD19 CAR T cell generation and lead to B cell depletion

While chimeric antigen receptor (CAR) T cell therapies have demonstrated therapeutic efficacy against B cell malignancies, widespread implementation of these therapies is hindered by a cumbersome, ex vivo manufacturing process. Delivery of CAR-encoding messenger RNA (mRNA) to endogenous T cells can generate these therapeutic cells in vivo and streamline this manufacturing workflow. To accomplish this, T cell-activating ligands were conjugated to a biodegradable polymeric mRNA nanoparticle to form T cell-targeted particles. By conjugating multiple activating ligands, T cell transfection and stimulation in vitro was increased, and greater T cell transfection and selectivity in vivo was achieved compared to an untargeted particle. These nanoparticles can flexibly encapsulate mRNA cargos and were used to deliver anti-CD19 CAR mRNA in vivo, enabling depletion of 95% of B cells in the peripheral blood and 50% depletion of splenic B cells in healthy mice. These findings regarding nanoparticle tropism and their potential therapeutic efficacy highlight the importance of this nonviral, polymeric platform to address key limitations associated with current CAR T practices.

Author Info: (1) Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA. Institute for NanoBioTechnology, and Translational Tissue Engineeri

Author Info: (1) Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA. Institute for NanoBioTechnology, and Translational Tissue Engineering Center, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA. Johns Hopkins Translational ImmunoEngineering Center, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA. (2) Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA. Institute for NanoBioTechnology, and Translational Tissue Engineering Center, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA. Johns Hopkins Translational ImmunoEngineering Center, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA. (3) Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA. Institute for NanoBioTechnology, and Translational Tissue Engineering Center, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA. Johns Hopkins Translational ImmunoEngineering Center, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA. (4) Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA. Institute for NanoBioTechnology, and Translational Tissue Engineering Center, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA. Johns Hopkins Translational ImmunoEngineering Center, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA. (5) Institute for NanoBioTechnology, and Translational Tissue Engineering Center, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA. Johns Hopkins Translational ImmunoEngineering Center, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA. Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD 21218, USA. (6) Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA. Institute for NanoBioTechnology, and Translational Tissue Engineering Center, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA. Johns Hopkins Translational ImmunoEngineering Center, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA. (7) Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA. Institute for NanoBioTechnology, and Translational Tissue Engineering Center, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA. Johns Hopkins Translational ImmunoEngineering Center, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA. (8) Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA. Institute for NanoBioTechnology, and Translational Tissue Engineering Center, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA. Johns Hopkins Translational ImmunoEngineering Center, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA. (9) Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA. Institute for NanoBioTechnology, and Translational Tissue Engineering Center, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA. Johns Hopkins Translational ImmunoEngineering Center, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA. (10) Division of Rheumatology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21224, USA. Center for Autoimmunity and Immuno-Oncology, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA. (11) Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA. Institute for NanoBioTechnology, and Translational Tissue Engineering Center, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA. Johns Hopkins Translational ImmunoEngineering Center, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA. (12) Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA. Institute for NanoBioTechnology, and Translational Tissue Engineering Center, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA. Johns Hopkins Translational ImmunoEngineering Center, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA. Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA. Departments of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA. Department of Oncology, the Sidney Kimmel Comprehensive Cancer Center, and the Bloomberg~Kimmel Institute for Cancer Immunotherapy, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA. (13) Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA. Institute for NanoBioTechnology, and Translational Tissue Engineering Center, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA. Johns Hopkins Translational ImmunoEngineering Center, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA. Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD 21218, USA. Department of Oncology, the Sidney Kimmel Comprehensive Cancer Center, and the Bloomberg~Kimmel Institute for Cancer Immunotherapy, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA. Department of Materials Science & Engineering, Johns Hopkins University, Baltimore, MD 21218, USA. Departments of Ophthalmology and Neurosurgery, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA.

IL-7/IL-15/IL-21 cytokine-fusion scaffold generates highly functional CAR T cells enriched in long-lived T memory stem cells

Functional persistence of chimeric antigen receptor T cells (CAR T cells) is limited by conventional CAR T cell manufacturing using anti-CD3/CD28 (_CD3/28) stimulation, which generates terminally differentiated and shorter-lived CAR T cells. We demonstrated that HCW9206, a unique protein scaffold linking interleukin-7 (IL-7), an IL-15/IL-15 receptor _ (IL-15R_) complex, and IL-21, generates CAR T cells without requiring _CD3/28 activation, which are highly enriched in long-lived T memory stem cells (T(SCM) cells) (>50%) and display potent activity across distinct disease models, HIV-1 or B cell leukemia. In a humanized mouse HIV infection model, HCW9206-generated anti-HIV duoCAR T cells suppressed viremia more effectively than _CD3/28-generated anti-HIV duoCAR T cells. In a xenograft leukemia mouse model, a recall proliferative response and complete clearance of leukemia rechallenge were displayed by HCW9206-generated but not by _CD3/28-generated anti-CD19 CAR T cells. HCW9206, a first-in-class cytokine scaffold-based platform, enables production of more potent CAR T cell-based immunotherapies by generating a CAR T cell population, which is highly functional and also markedly enriched for long-lived T(SCM) cells. This strategy is broadly applicable to increase persistence and functionality of CAR T cells, enhancing their efficacy for treating infectious disease and cancer.

Author Info: (1) Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461, USA. (2) Department of Microbiology and Immunology, Albert Einstein College of

Author Info: (1) Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461, USA. (2) Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461, USA. (3) Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461, USA. (4) Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461, USA. (5) Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461, USA. (6) RUH Bioinformatics, Center for Clinical and Translational Science, Rockefeller University Hospital, New York, NY 10065, USA. (7) RUH Bioinformatics, Center for Clinical and Translational Science, Rockefeller University Hospital, New York, NY 10065, USA. (8) HCW Biologics Inc., Miramar, FL 33025, USA. (9) Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461, USA. (10) Caring Cross, Gaithersburg, MD 20878, USA. (11) Caring Cross, Gaithersburg, MD 20878, USA. (12) Department of Physiology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA. (13) HCW Biologics Inc., Miramar, FL 33025, USA. (14) HCW Biologics Inc., Miramar, FL 33025, USA. (15) HCW Biologics Inc., Miramar, FL 33025, USA. (16) Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461, USA. Department of Pediatrics, Albert Einstein College of Medicine, 1300 Morris Park Ave, Bronx, NY 10461, USA.

Modulating AP-1 enables CAR T cells to establish an intratumoral stemlike reservoir and overcomes resistance to PD-1 blockade Spotlight 

ROR1 CAR T cells infiltrated ROR1+ NSCLC mouse tumors, but lost TCF1 expression and failed to maintain a progenitor exhausted (Tpex) population. The addition of anti-PD-L1 did not improve CAR T cell counts or efficacy, and further drove exhaustion. Snyder et al. found that co-delivery of c-Jun by the ROR1 CAR transiently increased CAR-T tumor accumulation and Tpex phenotype, and combining with anti-PD-L1 further improved tumor T cell counts, c-Jun expression, phenotype, and efficacy. Spatial transcriptomics found that c-Jun CAR-T were distributed throughout lung tumors, proximal to PD-L1+ myeloid cells, and Tpex-enriched relative to standard CAR-T.

Contributed by Alex Najibi

ROR1 CAR T cells infiltrated ROR1+ NSCLC mouse tumors, but lost TCF1 expression and failed to maintain a progenitor exhausted (Tpex) population. The addition of anti-PD-L1 did not improve CAR T cell counts or efficacy, and further drove exhaustion. Snyder et al. found that co-delivery of c-Jun by the ROR1 CAR transiently increased CAR-T tumor accumulation and Tpex phenotype, and combining with anti-PD-L1 further improved tumor T cell counts, c-Jun expression, phenotype, and efficacy. Spatial transcriptomics found that c-Jun CAR-T were distributed throughout lung tumors, proximal to PD-L1+ myeloid cells, and Tpex-enriched relative to standard CAR-T.

Contributed by Alex Najibi

ABSTRACT: Chimeric antigen receptor T (CAR T) cell therapy has shown limited synergy with immune checkpoint inhibitors, but the mechanisms underlying resistance remain unclear. Stemlike T cells coexpressing programmed cell death protein 1 (PD-1) and T cell factor 1 (TCF1) mediate responses to PD-1-PD-L1 (programmed death ligand 1) blockade and are maintained by major histocompatibility complex (MHC)-dependent interactions with dendritic cells in lymphoid tissues. Because CAR T cells recognize intact antigen rather than peptide-MHC, their activation is restricted to tumors, potentially limiting maintenance of this critical subset. In murine models of lung cancer, CAR T cells down-regulated TCF1, became exhausted, and were not enhanced by PD-L1 blockade. Overexpression of the transcription factor c-Jun increased intratumoral PD-1(+)TCF1(+) CAR T cells but did not prevent exhaustion, given that PD-1 induced posttranscriptional c-Jun down-regulation. PD-L1 blockade restored c-Jun levels, markedly increased CAR T cells, and enabled near-complete tumor clearance, revealing a mechanism by which MHC-independent CAR T cells can be engineered to overcome resistance to PD-1-PD-L1 blockade.

Author Info: (1) Human Biology Division, Fred Hutchinson Cancer Center, Seattle, WA, USA. Molecular and Cellular Biology Graduate Program, University of Washington, Seattle, WA, USA. Medical Sc

Author Info: (1) Human Biology Division, Fred Hutchinson Cancer Center, Seattle, WA, USA. Molecular and Cellular Biology Graduate Program, University of Washington, Seattle, WA, USA. Medical Scientist Training Program, University of Washington, Seattle, WA, USA. (2) Human Biology Division, Fred Hutchinson Cancer Center, Seattle, WA, USA. (3) Human Biology Division, Fred Hutchinson Cancer Center, Seattle, WA, USA. Molecular and Cellular Biology Graduate Program, University of Washington, Seattle, WA, USA. Medical Scientist Training Program, University of Washington, Seattle, WA, USA. (4) Human Biology Division, Fred Hutchinson Cancer Center, Seattle, WA, USA. Molecular and Cellular Biology Graduate Program, University of Washington, Seattle, WA, USA. (5) Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Center, Seattle, WA, USA. (6) Translational Science and Therapeutics, Fred Hutchinson Cancer Center, Seattle, WA, USA. (7) Human Biology Division, Fred Hutchinson Cancer Center, Seattle, WA, USA. Department of Immunology, University of Washington, Seattle, WA, USA. (8) Public Health Sciences Division, Fred Hutchinson Cancer Center, Seattle, WA,USA. (9) Genomics and Bioinformatics Shared Resources, Fred Hutchinson Cancer Center, Seattle, WA, USA. (10) Translational Science and Therapeutics, Fred Hutchinson Cancer Center, Seattle, WA, USA. (11) Human Biology Division, Fred Hutchinson Cancer Center, Seattle, WA, USA. Molecular and Cellular Biology Graduate Program, University of Washington, Seattle, WA, USA. Medical Scientist Training Program, University of Washington, Seattle, WA, USA. (12) Human Biology Division, Fred Hutchinson Cancer Center, Seattle, WA, USA. (13) Human Biology Division, Fred Hutchinson Cancer Center, Seattle, WA, USA. Translational Science and Therapeutics, Fred Hutchinson Cancer Center, Seattle, WA, USA. (14) Human Biology Division, Fred Hutchinson Cancer Center, Seattle, WA, USA. (15) Human Biology Division, Fred Hutchinson Cancer Center, Seattle, WA, USA. Invent Program, Seattle Children's Research Institute, Seattle, WA, USA. (16) Human Biology Division, Fred Hutchinson Cancer Center, Seattle, WA, USA. (17) Comparative Medicine, Translational Research Model Services, Fred Hutchinson Cancer Center, Seattle, WA, USA. (18) Translational Science and Therapeutics, Fred Hutchinson Cancer Center, Seattle, WA, USA. (19) Translational Science and Therapeutics, Fred Hutchinson Cancer Center, Seattle, WA, USA. (20) Human Biology Division, Fred Hutchinson Cancer Center, Seattle, WA, USA. (21) Lyell Immunopharma, South San Francisco, CA, USA. (22) Lyell Immunopharma, South San Francisco, CA, USA. (23) Fred Hutch Innovation Lab, Immunotherapy Integrated Research Center, Fred Hutchinson Cancer Center, Seattle, WA, USA. (24) Fred Hutch Innovation Lab, Immunotherapy Integrated Research Center, Fred Hutchinson Cancer Center, Seattle, WA, USA. (25) Human Biology Division, Fred Hutchinson Cancer Center, Seattle, WA, USA. Public Health Sciences Division, Fred Hutchinson Cancer Center, Seattle, WA,USA. Center for Metastasis Research eXcellence (MET-X), Fred Hutchinson Cancer Center, Seattle, WA, USA. (26) Translational Science and Therapeutics, Fred Hutchinson Cancer Center, Seattle, WA, USA. (27) Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Center, Seattle, WA, USA. (28) Human Biology Division, Fred Hutchinson Cancer Center, Seattle, WA, USA. Department of Immunology, University of Washington, Seattle, WA, USA.

Dynamic reprogramming of the tumor immune network via multicycle checkpoint degradation for cancer immunotherapy Spotlight 

Chi et al. engineered a recyclable PD-L1 degrader (RECYC) by fusing a moderate-affinity peptide binder of PD-L1 to an aptamer against the ubiquitous endolysosomal trafficking receptor CI-M6PR. RECYC robustly degraded PD-L1 in tumor and myeloid cells, and exhibited pH-sensitive dissociation from PD-L1, enabling repeated knockdown following membrane recycling. I.v. RECYC outperformed anti-PD-L1 in multiple tumor models. and drove greater PD-L1 knockdown in tumor and myeloid cells, which correlated with efficacy. RECYC enhanced T cell infiltration and early activation, and uniquely promoted M1/N1-like skewing in tumor-associated macrophages and neutrophils.

Contributed by Morgan Janes

Chi et al. engineered a recyclable PD-L1 degrader (RECYC) by fusing a moderate-affinity peptide binder of PD-L1 to an aptamer against the ubiquitous endolysosomal trafficking receptor CI-M6PR. RECYC robustly degraded PD-L1 in tumor and myeloid cells, and exhibited pH-sensitive dissociation from PD-L1, enabling repeated knockdown following membrane recycling. I.v. RECYC outperformed anti-PD-L1 in multiple tumor models. and drove greater PD-L1 knockdown in tumor and myeloid cells, which correlated with efficacy. RECYC enhanced T cell infiltration and early activation, and uniquely promoted M1/N1-like skewing in tumor-associated macrophages and neutrophils.

Contributed by Morgan Janes

ABSTRACT: The efficacy of immune checkpoint blockade is often limited by intrinsic immunosuppressive networks within the tumor immune microenvironment (TIME). Despite progress in cancer treatment, current extracellular targeted protein degradation approaches often overlook the multicellular distribution and crosstalk of immune checkpoints. Here we reported a Receptor-mediated Endolysosomal recYcling Chimera (RECYC) platform. RECYC employs a CI-M6PR-targeting aptamer that remains stable across late endosomal pH and a protein-binding peptide with moderate affinity and pH responsiveness, which together drive recycling and sustained checkpoint clearance. In ex vivo co-culture and in vivo murine models, RECYC efficiently eliminated programmed death-ligand 1 (PD-L1) expression from both tumor cells and tumor-associated myeloid cells (macrophages, neutrophils and dendritic cells). By converting an immunosuppressive TIME to an immunostimulatory state, RECYC remodeled the tumor-immune network in an anti-tumor direction, thereby enhancing CD8(+) T cell response and repolarizing immunosuppressive myeloid cells. Moreover, in both immune-cold and immune-hot murine cancer models, RECYC demonstrated superior anti-tumor effect compared to PD-L1 blockade treatment. Collectively, we propose an effective strategy to induce recycling and broad checkpoint clearance in the TIME, which in turn reprograms the multicellular tumor-immune network to achieve durable immunotherapy responses.

Author Info: (1) Department of Oncology, Nanjing Drum Tower Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing 210008, China. (2) Institute of Chemical Biology and Nan

Author Info: (1) Department of Oncology, Nanjing Drum Tower Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing 210008, China. (2) Institute of Chemical Biology and Nanomedicine, State Key Laboratory of Chemo and Biosensing, Hunan Provincial Key Laboratory of Biomacromolecular Chemical Biology, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, China. (3) Department of Oncology, Nanjing Drum Tower Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing 210008, China. (4) Department of Oncology, Nanjing Drum Tower Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing 210008, China. (5) Department of Oncology, Nanjing Drum Tower Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing 210008, China. (6) Department of Oncology, Nanjing Drum Tower Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing 210008, China. (7) Institute of Chemical Biology and Nanomedicine, State Key Laboratory of Chemo and Biosensing, Hunan Provincial Key Laboratory of Biomacromolecular Chemical Biology, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, China. (8) Department of Oncology, Nanjing Drum Tower Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing 210008, China. (9) Department of Oncology, Nanjing Drum Tower Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing 210008, China. (10) Institute of Chemical Biology and Nanomedicine, State Key Laboratory of Chemo and Biosensing, Hunan Provincial Key Laboratory of Biomacromolecular Chemical Biology, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, China. (11) Institute of Chemical Biology and Nanomedicine, State Key Laboratory of Chemo and Biosensing, Hunan Provincial Key Laboratory of Biomacromolecular Chemical Biology, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, China. (12) Institute of Chemical Biology and Nanomedicine, State Key Laboratory of Chemo and Biosensing, Hunan Provincial Key Laboratory of Biomacromolecular Chemical Biology, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, China. (13) Institute of Chemical Biology and Nanomedicine, State Key Laboratory of Chemo and Biosensing, Hunan Provincial Key Laboratory of Biomacromolecular Chemical Biology, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, China. (14) Department of Oncology, Nanjing Drum Tower Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing 210008, China. ChemBioMed Interdisciplinary Research Center, Nanjing University, Nanjing 210061, China. Collaborative Innovation Center for Personalized Cancer Medicine, Nanjing Medical University, Nanjing 211166, China.

VISTA drives pancreatic tumor progression through modulation of the tumor-associated macrophage polarity Spotlight 

Shin and Kim et al. demonstrated that VISTA deletion enhanced macrophage and CD8+ T cell infiltration and reduced tumor growth in orthotopic Pan02 and KPC pancreatic mouse tumor models. VISTA deficiency reprogrammed TAMs from a suppressive SPP1+ to a stimulatory CXCL9+ phenotype. CXCL9+ TAMs exhibited enhanced antigen processing and cross-presentation, and increased recruitment of CXCR3+ CD8+ T cells with sustained cytotoxicity and reduced exhaustion. Anti-VISTA plus gemcitabine produced a synergistic antitumor response. In human PDAC datasets, expression of VSIR (encoding VISTA) correlated with immunosuppressive macrophage states.

Contributed by Shishir Pant

Shin and Kim et al. demonstrated that VISTA deletion enhanced macrophage and CD8+ T cell infiltration and reduced tumor growth in orthotopic Pan02 and KPC pancreatic mouse tumor models. VISTA deficiency reprogrammed TAMs from a suppressive SPP1+ to a stimulatory CXCL9+ phenotype. CXCL9+ TAMs exhibited enhanced antigen processing and cross-presentation, and increased recruitment of CXCR3+ CD8+ T cells with sustained cytotoxicity and reduced exhaustion. Anti-VISTA plus gemcitabine produced a synergistic antitumor response. In human PDAC datasets, expression of VSIR (encoding VISTA) correlated with immunosuppressive macrophage states.

Contributed by Shishir Pant

ABSTRACT: Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest malignancies due to its highly immunosuppressive tumor microenvironment (TME), which limits effective therapeutic interventions. Here, we demonstrate that V-domain immunoglobulin suppressor of T cell activation (VISTA) plays a crucial role in orchestrating macrophage polarity within the PDAC TME. Using murine PDAC models, we show that VISTA deficiency markedly impairs tumor growth, leading to prolonged survival. Functionally, VISTA deficiency is linked to a shift in tumor-associated macrophages (TAMs) from an immunosuppressive phenotype marked by secreted phosphoprotein 1 (SPP1), to one enriched for C-X-C motif chemokine ligand 9 (CXCL9), indicative of a pro-inflammatory state. This shift is accompanied by enhanced recruitment of CXCR3⁺ CD8⁺ T cells with sustained cytotoxic potential, among which terminal exhaustion-like CD8+ T cell states are less prevalent. Additionally, VISTA-deficient TAMs exhibit increased antigen cross-presentation, further amplifying CD8+ T cell response against tumors. These findings are corroborated by human PDAC data, which reflect similar immune reprogramming trends. By defining the role of VISTA in controlling Cxcl9:Spp1 ratio and modulating CD8⁺ T cell dynamics, this study positions VISTA inhibition as a promising strategy to reshape the TME and potentiate anti-tumor immunity in PDAC.

Author Info: (1) Department of Biomedical Sciences, Cancer Research Institute, Seoul National University College of Medicine, Seoul, Republic of Korea. Wide River Institute of Immunology, Seoul

Author Info: (1) Department of Biomedical Sciences, Cancer Research Institute, Seoul National University College of Medicine, Seoul, Republic of Korea. Wide River Institute of Immunology, Seoul National University, Gangwon, Republic of Korea. Convergence Research Center for Dementia, Seoul National University Medical Research Center, Seoul, Republic of Korea. BK21 FOUR Biomedical Science Project, Seoul National University College of Medicine, Seoul, Republic of Korea. (2) Department of Biomedical Sciences, Cancer Research Institute, Seoul National University College of Medicine, Seoul, Republic of Korea. Medical Research Center, Seoul National University College of Medicine, Seoul, Republic of Korea. (3) Department of Biomedical Sciences, Cancer Research Institute, Seoul National University College of Medicine, Seoul, Republic of Korea. Wide River Institute of Immunology, Seoul National University, Gangwon, Republic of Korea. Convergence Research Center for Dementia, Seoul National University Medical Research Center, Seoul, Republic of Korea. BK21 FOUR Biomedical Science Project, Seoul National University College of Medicine, Seoul, Republic of Korea. (4) Department of Biomedical Sciences, Cancer Research Institute, Seoul National University College of Medicine, Seoul, Republic of Korea. Department of Pharmacology, Ischemic/Hypoxic Disease Institute, Seoul National University College of Medicine, Seoul, Republic of Korea. (5) Department of Biomedical Sciences, Cancer Research Institute, Seoul National University College of Medicine, Seoul, Republic of Korea. Wide River Institute of Immunology, Seoul National University, Gangwon, Republic of Korea. BK21 FOUR Biomedical Science Project, Seoul National University College of Medicine, Seoul, Republic of Korea. Department of Pharmacology, Ischemic/Hypoxic Disease Institute, Seoul National University College of Medicine, Seoul, Republic of Korea. (6) Department of Pathology, Seoul National University College of Medicine, Seoul, Republic of Korea. (7) Department of Biomedical Sciences, Cancer Research Institute, Seoul National University College of Medicine, Seoul, Republic of Korea. BK21 FOUR Biomedical Science Project, Seoul National University College of Medicine, Seoul, Republic of Korea. (8) Department of Biomedical Sciences, Cancer Research Institute, Seoul National University College of Medicine, Seoul, Republic of Korea. hyunmu.shin@snu.ac.kr. Wide River Institute of Immunology, Seoul National University, Gangwon, Republic of Korea. hyunmu.shin@snu.ac.kr. BK21 FOUR Biomedical Science Project, Seoul National University College of Medicine, Seoul, Republic of Korea. hyunmu.shin@snu.ac.kr. Medical Research Center, Seoul National University College of Medicine, Seoul, Republic of Korea. hyunmu.shin@snu.ac.kr. (9) Samsung Precision Genome Medicine Institute, Research Institute for Future Medicine, Samsung Medical Center, Seoul, Republic of Korea. hangrae.kim@skku.edu. Department of Health Sciences and Technology, Samsung Advanced Institute for Health Sciences & Technology (SAIHST), Sungkyunkwan University, Seoul, Republic of Korea. hangrae.kim@skku.edu. (10) Department of Biomedical Sciences, Cancer Research Institute, Seoul National University College of Medicine, Seoul, Republic of Korea. dlee5522@snu.ac.kr. Convergence Research Center for Dementia, Seoul National University Medical Research Center, Seoul, Republic of Korea. dlee5522@snu.ac.kr. BK21 FOUR Biomedical Science Project, Seoul National University College of Medicine, Seoul, Republic of Korea. dlee5522@snu.ac.kr.

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