Journal Articles

The Dendritic Cell-based Vaccine PROTEXI leverages Antiviral CD4 T cell Memory to boost anti-tumor immune responses in mice

The efficacy of dendritic cell (DC) cancer vaccines is linked to poor immunogenicity of tumor-associated antigens and failure to elicit robust MHC class II-restricted CD4_ T-cell responses. Here, we introduce PROTEXI, a DC vaccine platform that optimizes tumor immunity by co-presenting tumor-specific CD8_ T-cell epitopes alongside CD4_ T helper epitopes from the SARS-CoV-2 Spike protein, leveraging widespread anti-viral immunity. In preclinical mouse models of melanoma and breast cancer, PROTEXI significantly reduces tumor growth and improves survival by promoting robust T cell infiltration into immune-cold tumors, increasing cytotoxic T cell responses via epitope spreading, and activating genes linked to optimal DC, NK cell, and T cell function. Furthermore, PROTEXI elicits superior responses when combined with other immunotherapy agents in models of therapy-resistant tumors. Finally, in a humanized mouse model of melanoma, PROTEXI vaccine co-presenting CD4_ T-specific Spike epitopes with CD8_ T cell-restricted PRAME and MAGE-A3 antigens, significantly reduces tumor burden. Thus, these data underscore the potential of harnessing pre-existing viral-specific immunity to enhance the efficacy of DC vaccines in immune-cold tumors.

Author Info: (1) The Angie Fowler Adolescent & Young Adult Cancer Institute, University Hospitals Rainbow Babies & Children's Hospital, Cleveland, OH, USA. Department of Pediatric Hematology &

Author Info: (1) The Angie Fowler Adolescent & Young Adult Cancer Institute, University Hospitals Rainbow Babies & Children's Hospital, Cleveland, OH, USA. Department of Pediatric Hematology & Oncology, University Hospitals Cleveland Medical Center, Cleveland, OH, USA. (2) Department of Pediatrics, Case Western Reserve University, Cleveland, OH, USA. (3) Celloram Inc., 11000 Cedar Avenue STE 100F #23, Cleveland, OH, USA. (4) Department of Pediatrics, Case Western Reserve University, Cleveland, OH, USA. (5) Celloram Inc., 11000 Cedar Avenue STE 100F #23, Cleveland, OH, USA. (6) Celloram Inc., 11000 Cedar Avenue STE 100F #23, Cleveland, OH, USA. (7) MedPacto Inc., 92, Myeongdal-ro, Seocho-gu, Seoul, Republic of Korea. (8) The Angie Fowler Adolescent & Young Adult Cancer Institute, University Hospitals Rainbow Babies & Children's Hospital, Cleveland, OH, USA. John.Letterio@UHHospitals.org. Department of Pediatric Hematology & Oncology, University Hospitals Cleveland Medical Center, Cleveland, OH, USA. John.Letterio@UHHospitals.org. Department of Pediatrics, Case Western Reserve University, Cleveland, OH, USA. John.Letterio@UHHospitals.org. The Case Comprehensive Cancer Center, Case Western Reserve University School of Medicine, Cleveland, OH, USA. John.Letterio@UHHospitals.org. (9) The Angie Fowler Adolescent & Young Adult Cancer Institute, University Hospitals Rainbow Babies & Children's Hospital, Cleveland, OH, USA. SeunghwanLim@Celloram.com. Celloram Inc., 11000 Cedar Avenue STE 100F #23, Cleveland, OH, USA. SeunghwanLim@Celloram.com.

Tumor-specific antibodies elicited by engineered bacteria promote bladder cancer immunotherapy in preclinical mouse models Spotlight 

Rouanne et al. engineered probiotic E.coli (EcN) using a synchronized lysis integrated circuit to release human CXCL13 at critical population densities, without impeding normal bacterial growth. EcN CXCL13 release promoted B cell and splenocyte migration in vitro and high tumor expression after delivery into mouse bladders. In orthotopic bladder cancer models, EcN-colonized tumors selectively elicited GC responses in tdLNs. EcN boosted anti-PD-1-induced antitumor activity, tumor-specific antibody responses, and long-term survival in “cold” advanced bladder cancer models, and generated immune memory. Efficacy depended on CD8⁺ T and CD4⁺ TFH cells.

Contributed by Paula Hochman

Rouanne et al. engineered probiotic E.coli (EcN) using a synchronized lysis integrated circuit to release human CXCL13 at critical population densities, without impeding normal bacterial growth. EcN CXCL13 release promoted B cell and splenocyte migration in vitro and high tumor expression after delivery into mouse bladders. In orthotopic bladder cancer models, EcN-colonized tumors selectively elicited GC responses in tdLNs. EcN boosted anti-PD-1-induced antitumor activity, tumor-specific antibody responses, and long-term survival in “cold” advanced bladder cancer models, and generated immune memory. Efficacy depended on CD8⁺ T and CD4⁺ TFH cells.

Contributed by Paula Hochman

ABSTRACT: The intratumoral microbiome has recently emerged as a potential hallmark of cancer, with implications for response or resistance to therapy. Bacteria can either promote or inhibit cancer growth. However, intratumoral bacteria can also be engineered using synthetic biology to remodel the tumor microenvironment. Here, we engineered the probiotic bacterium Escherichia coli Nissle 1917 (EcN) to express the human chemokine CXCL13 (C-X-C motif chemokine ligand 13), a critical component of germinal center (GC) formation. Antibody affinity maturation and class switching are fundamental aspects of adaptive immune response. Both occur primarily in the GCs of secondary lymphoid organs for defense against pathogens. Immune checkpoint blockade (ICB) efficacy is primarily driven by T cells; however, recent studies in mice and humans have shown that humoral immune responses act as critical partners for ICB-mediated antitumor activity. Using orthotopic models of bladder cancer, intravesically delivered engineered CXCL13-expressing EcN colonized bladder tumors and elicited GC responses in bladder tumor-draining lymph nodes after intravesical delivery. When combined with programmed cell death protein 1 (PD-1) blockade, engineered EcN improved antitumor activity in two aggressive, fast-growing, and immunologically cold orthotopic mouse models of bladder cancer. Mechanistically, this antitumor effect was dependent on the presence of CD8(+) T cells and CD4(+) T follicular helper cells; combination therapy increased tumor-specific antibody responses and promoted long-term survival and protective immunity upon tumor rechallenge. Thus, we demonstrate that synthetically engineered CXCL13-expressing EcN can enhance the efficacy of PD-1 checkpoint blockade immunotherapy by amplifying tumor-specific humoral immunity.

Author Info: (1) Department of Microbiology & Immunology, Columbia University, New York, NY 10032, USA. Herbert Irving Comprehensive Cancer Center, Columbia University, New York, NY 10032, USA.

Author Info: (1) Department of Microbiology & Immunology, Columbia University, New York, NY 10032, USA. Herbert Irving Comprehensive Cancer Center, Columbia University, New York, NY 10032, USA. (2) Department of Microbiology & Immunology, Columbia University, New York, NY 10032, USA. Herbert Irving Comprehensive Cancer Center, Columbia University, New York, NY 10032, USA. (3) Department of Microbiology & Immunology, Columbia University, New York, NY 10032, USA. Herbert Irving Comprehensive Cancer Center, Columbia University, New York, NY 10032, USA. (4) Department of Microbiology & Immunology, Columbia University, New York, NY 10032, USA. (5) Department of Microbiology & Immunology, Columbia University, New York, NY 10032, USA. Herbert Irving Comprehensive Cancer Center, Columbia University, New York, NY 10032, USA. (6) Department of Microbiology & Immunology, Columbia University, New York, NY 10032, USA. (7) Department of Microbiology & Immunology, Columbia University, New York, NY 10032, USA. (8) Department of Biomedical Engineering, Columbia University, New York, NY 10027, USA. (9) Department of Urology, Columbia University, New York, NY 10032, USA. (10) Herbert Irving Comprehensive Cancer Center, Columbia University, New York, NY 10032, USA. Department of Biomedical Engineering, Columbia University, New York, NY 10027, USA. Data Science Institute, Columbia University, New York, NY 10027, USA. (11) Department of Microbiology & Immunology, Columbia University, New York, NY 10032, USA. Herbert Irving Comprehensive Cancer Center, Columbia University, New York, NY 10032, USA.

Modulating the VIP-VIPR pathway reprograms CAR T cells for superior antitumor efficacy in preclinical cancer models Spotlight 

Inspired by clinical data that the neuropeptide vasoactive intestinal peptide (VIP), acting through its receptor (VIPR), was correlated with poor response in patients undergoing CAR therapy, Lin et al. used alpha-fold to design a similarly sized antagonistic peptide (VIPRa) for this immunosuppressive axis, and expressed it with a CAR. During manufacturing, VIPRa-expressing CAR T cells were less activated, but highly activatable, less exhausted, and had a memory phenotype, translating to improved tumor infiltration and in vivo efficacy. CAR/VIPRa T cells were metabolically superior, and more effectively engaged endogenous immunity.

Contributed by Ute Burkhardt

Inspired by clinical data that the neuropeptide vasoactive intestinal peptide (VIP), acting through its receptor (VIPR), was correlated with poor response in patients undergoing CAR therapy, Lin et al. used alpha-fold to design a similarly sized antagonistic peptide (VIPRa) for this immunosuppressive axis, and expressed it with a CAR. During manufacturing, VIPRa-expressing CAR T cells were less activated, but highly activatable, less exhausted, and had a memory phenotype, translating to improved tumor infiltration and in vivo efficacy. CAR/VIPRa T cells were metabolically superior, and more effectively engaged endogenous immunity.

Contributed by Ute Burkhardt

ABSTRACT: Clinical efficacy with chimeric antigen receptor (CAR) T cells is currently limited by numerous factors including poor initial product phenotypes and lack of engagement of endogenous immunity. Vasoactive intestinal peptide (VIP) is an immunosuppressive neuropeptide, and the antagonism of its receptor (VIPR) on T cells potentiates T cell activation. We demonstrated that VIP suppresses CAR T cell function and engineered CAR T cells to secrete a short peptide drug that antagonizes VIPR (CAR/VIPRa). Armored CAR/VIPRa T cells maintained a memory phenotype and were metabolically quiescent after manufacturing yet mounted a strong bioenergetic response after antigen stimulation. Moreover, CAR/VIPRa T cells potentiated endogenous antitumor immunity through the recruitment of host T cells. In syngeneic and xenogeneic mouse models of hematological and solid tumors, CAR/VIPRa T cells exhibited greater tumor infiltration and maintained a less exhausted memory phenotype, resulting in superior antitumor efficacy. Together, these data show that VIPRa peptides produced by armored CAR T cells can enhance T cell function and boost endogenous immunity, thereby improving tumor control.

Author Info: (1) Department of Hematology and Medical Oncology, Emory University School of Medicine, Atlanta, GA, USA. (2) Department of Hematology and Medical Oncology, Emory University School

Author Info: (1) Department of Hematology and Medical Oncology, Emory University School of Medicine, Atlanta, GA, USA. (2) Department of Hematology and Medical Oncology, Emory University School of Medicine, Atlanta, GA, USA. (3) Department of Hematology and Medical Oncology, Emory University School of Medicine, Atlanta, GA, USA. (4) Department of Hematology and Medical Oncology, Emory University School of Medicine, Atlanta, GA, USA. (5) George W. Woodruff School of Mechanical Engineering, Georgia Institute of Technology, Atlanta, GA, USA. (6) Department of Hematology and Medical Oncology, Emory University School of Medicine, Atlanta, GA, USA. (7) Department of Surgery, Emory University School of Medicine, Atlanta, GA, USA. (8) Department of Hematology and Medical Oncology, Emory University School of Medicine, Atlanta, GA, USA. (9) Department of Pharmacology and Chemical Biology, Emory University School of Medicine, Atlanta, GA, USA. (10) Department of Pharmacology and Chemical Biology, Emory University School of Medicine, Atlanta, GA, USA. (11) Department of Hematology and Medical Oncology, Emory University School of Medicine, Atlanta, GA, USA. (12) George W. Woodruff School of Mechanical Engineering, Georgia Institute of Technology, Atlanta, GA, USA. (13) George W. Woodruff School of Mechanical Engineering, Georgia Institute of Technology, Atlanta, GA, USA. (14) Department of Hematology and Medical Oncology, Emory University School of Medicine, Atlanta, GA, USA. (15) Department of Hematology and Medical Oncology, Emory University School of Medicine, Atlanta, GA, USA. (16) Biostatistics Shared Resource, Emory University School of Medicine, Atlanta, GA, USA. (17) Department of Hematology and Medical Oncology, Emory University School of Medicine, Atlanta, GA, USA. (18) Department of Hematology and Medical Oncology, Emory University School of Medicine, Atlanta, GA, USA. (19) Cambium Oncology, Atlanta, GA, USA. (20) Achieve Clinics, Los Angeles, CA, USA. (21) Achieve Clinics, Los Angeles, CA, USA. Eldred Advisors, Los Angeles, CA, USA. (22) Department of Pediatrics, Emory University School of Medicine, Atlanta, GA, USA. (23) Department of Pediatrics, Emory University School of Medicine, Atlanta, GA, USA. Winship Cancer Institute of Emory University, Atlanta, GA, USA. Aflac Cancer & Blood Disorders Center, Children's Healthcare of Atlanta, Atlanta, GA, USA. (24) Department of Hematology and Medical Oncology, Emory University School of Medicine, Atlanta, GA, USA. Winship Cancer Institute of Emory University, Atlanta, GA, USA. (25) Department of Surgery, Emory University School of Medicine, Atlanta, GA, USA. Winship Cancer Institute of Emory University, Atlanta, GA, USA. Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA, USA. (26) Department of Surgery, Emory University School of Medicine, Atlanta, GA, USA. Winship Cancer Institute of Emory University, Atlanta, GA, USA. (27) Department of Pharmacology and Chemical Biology, Emory University School of Medicine, Atlanta, GA, USA. Winship Cancer Institute of Emory University, Atlanta, GA, USA. (28) George W. Woodruff School of Mechanical Engineering, Georgia Institute of Technology, Atlanta, GA, USA. Winship Cancer Institute of Emory University, Atlanta, GA, USA. Parker H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GA, USA. Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA, USA. (29) Department of Hematology and Medical Oncology, Emory University School of Medicine, Atlanta, GA, USA. Winship Cancer Institute of Emory University, Atlanta, GA, USA. (30) Department of Hematology and Medical Oncology, Emory University School of Medicine, Atlanta, GA, USA. Winship Cancer Institute of Emory University, Atlanta, GA, USA.

Cytotoxic CD39+ tumor-associated NK cells respond to NKG2A blockade in lung cancer

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Serger et al. profiled NK cells in NSCLC, and identified two tumor-associated NK cell (taNK; CD103+CD49a+) populations. These cells were cytotoxic, but showed hallmarks of dysfunction. Trajectory analysis showed a transition from the CD56bright phenotype through an interferon response and GZMB induction, leading to the expression of genes related to tissue residency, dysfunction, and cytotoxicity. The CD39-expressing subset of taNK cells had the highest tumor killing capacity and responded to anti-NKG2A therapy.

Serger et al. profiled NK cells in NSCLC, and identified two tumor-associated NK cell (taNK; CD103+CD49a+) populations. These cells were cytotoxic, but showed hallmarks of dysfunction. Trajectory analysis showed a transition from the CD56bright phenotype through an interferon response and GZMB induction, leading to the expression of genes related to tissue residency, dysfunction, and cytotoxicity. The CD39-expressing subset of taNK cells had the highest tumor killing capacity and responded to anti-NKG2A therapy.

ABSTRACT: Natural killer (NK) cell-targeting immunotherapies are emerging, yet the differentiation and functional states of tumor-infiltrating NK cells remain poorly understood. Using matched single-nucleus RNA and ATAC sequencing of samples from patients with non-small cell lung cancer (NSCLC), we resolved the transcriptional and epigenetic landscape of intratumoral NK cells. We identified two tumor-associated NK (taNK) cell subsets marked by expression of ITGAE (CD103) and ITGA1 (CD49a) that display features of tissue residency and dysfunction while preserving cytotoxic function. Trajectory and regulon analyses revealed an inflammation-driven transition from early granzyme K (GZMK)(+) NK cells toward an ENTPD1(+) (CD39(+)) effector state characterized by interferon-stimulated gene (ISG) programs. Functional profiling established CD39(+) taNK cells as the dominant cytotoxic NK cell population with superior killing capacity that was further potentiated by NKG2A blockade. This study offers mechanistic insights into NK cell differentiation in NSCLC and establishes CD39(+) taNK cells as a targetable effector population for immunotherapy.

Author Info: (1) Department of Biomedicine, University Hospital and University of Basel, Basel, Switzerland. (2) Aix Marseille UniversitŽ, CNRS, INSERM, Centre d'Immunologie de Marseille-Luminy

Author Info: (1) Department of Biomedicine, University Hospital and University of Basel, Basel, Switzerland. (2) Aix Marseille UniversitŽ, CNRS, INSERM, Centre d'Immunologie de Marseille-Luminy, Marseille, France. (3) Department of Biomedicine, University Hospital and University of Basel, Basel, Switzerland. (4) Institute of Transplant Immunology, Hannover Medical School, Hannover, Germany. (5) Department of Biomedicine, University Hospital and University of Basel, Basel, Switzerland. (6) Department of Biomedicine, University Hospital and University of Basel, Basel, Switzerland. (7) Institute of Transplant Immunology, Hannover Medical School, Hannover, Germany. (8) Institute of Transplant Immunology, Hannover Medical School, Hannover, Germany. (9) Institute of Transplant Immunology, Hannover Medical School, Hannover, Germany. (10) Department of Biomedicine, University Hospital and University of Basel, Basel, Switzerland. (11) Department of Biomedicine, University Hospital and University of Basel, Basel, Switzerland. (12) Institute of Pathology, University Hospital and University of Basel, Basel, Switzerland. (13) Department of Biomedicine, University Hospital and University of Basel, Basel, Switzerland. (14) Department of Biomedicine, University Hospital and University of Basel, Basel, Switzerland. (15) Department of Biomedicine, University Hospital and University of Basel, Basel, Switzerland. Roche Pharma Research and Early Development pRED, Roche Innovation Center, Basel, Switzerland. (16) Department of Biomedicine, University Hospital and University of Basel, Basel, Switzerland. (17) Department of Thoracic Surgery, University Hospital Basel, Basel, Switzerland. (18) Department of Thoracic Surgery, University Hospital Basel, Basel, Switzerland. (19) German Centre for Lung Diseases (DZL), BREATH site, Hannover, Germany. Institute of Pathology, Hannover Medical School, Hannover, Germany. (20) Department of Biomedicine, University Hospital and University of Basel, Basel, Switzerland. Medical Oncology, University Hospital Basel, Basel, Switzerland. (21) Institute of Pathology, University Hospital and University of Basel, Basel, Switzerland. (22) Department of Biomedicine, University Hospital and University of Basel, Basel, Switzerland. (23) Department of Biomedicine, University Hospital and University of Basel, Basel, Switzerland. (24) Department of Biomedicine, University Hospital and University of Basel, Basel, Switzerland. (25) Institute of Pathology, University Hospital and University of Basel, Basel, Switzerland. (26) Department of Biomedicine, University Hospital and University of Basel, Basel, Switzerland. Medical Oncology, University Hospital Basel, Basel, Switzerland. (27) Institute of Transplant Immunology, Hannover Medical School, Hannover, Germany. German Centre for Lung Diseases (DZL), BREATH site, Hannover, Germany. German Centre for Infection Research (DZIF), TTU-IICH, Hannover/Braunschweig site, Hannover, Germany. (28) Department of Biomedicine, University Hospital and University of Basel, Basel, Switzerland. (29) Aix Marseille UniversitŽ, CNRS, INSERM, Centre d'Immunologie de Marseille-Luminy, Marseille, France. APHM, H™pital de la Timone, Marseille-Immunop™le Profiling Platform, Marseille, France. Paris-Saclay Cancer Cluster, Villejuif, France. Ecole Polytechnique, Palaiseau, France. (30) Roche Pharma Research and Early Development pRED, Roche Innovation Center, Basel, Switzerland. (31) Department of Biomedicine, University Hospital and University of Basel, Basel, Switzerland. Medical Oncology, University Hospital Basel, Basel, Switzerland.

Drilling dendritic cell activation- Engineering interfacial mechano-biochemical cues for enhanced immunotherapy

Spotlight 

Ming et al. generated oil-water emulsions stabilized by alum particles (ASPEs) to mechanically activate DCs. Increasing alum crystallinity increased ASPE interfacial stiffness, in turn increasing DC contact area, membrane tension, and internalization, leading to PIEZO1-induced calcium flux and MAPK activation, altogether improving DC activation. Admixed with Ag, ASPEs induced a stronger Th1 responses in C57 mice compared to standard alum, increasing with ASPE stiffness. A high-stiffness ASPE incorporating MPLA adjuvant also induced strong, Th1-biased immune responses and improved efficacy over MPLA alone when used to prepare a DC vaccine.

Contributed by Alex Najibi

Ming et al. generated oil-water emulsions stabilized by alum particles (ASPEs) to mechanically activate DCs. Increasing alum crystallinity increased ASPE interfacial stiffness, in turn increasing DC contact area, membrane tension, and internalization, leading to PIEZO1-induced calcium flux and MAPK activation, altogether improving DC activation. Admixed with Ag, ASPEs induced a stronger Th1 responses in C57 mice compared to standard alum, increasing with ASPE stiffness. A high-stiffness ASPE incorporating MPLA adjuvant also induced strong, Th1-biased immune responses and improved efficacy over MPLA alone when used to prepare a DC vaccine.

Contributed by Alex Najibi

ABSTRACT: A key challenge in immunotherapy is enhancing immune responses without introducing new molecular entities that trigger regulatory hurdles. While the size, shape, and composition of approved adjuvants have been optimized, their mechanical properties remain underexplored. Here, we repurpose approved aluminum-based adjuvants (alum) by engineering alum-stabilized Pickering emulsions (ASPEs) to synergize mechanical (PIEZO1) and biochemical (TLR4) cues. ASPEs, featuring interfacial alum with optimal rigidity, were heralded to promote an enlarged contact area with dendritic cells (DCs) during endocytosis, transmitting localized stress that activates PIEZO1-mediated calcium/mitogen-activated protein kinase (MAPK) signaling. This enhances antigen cross-presentation and Th1 immunity. Co-delivering a TLR4 agonist (monophosphoryl lipid A [MPLA]) further boosted immunogenicity in a varicella-zoster virus vaccine among aged mice, outperforming alum+MPLA (AS04). In antigen-pulsed DC therapy combined with PD-1 blockade, ASPE-M-treated DCs achieved a 2.11-fold greater tumor suppression compared with tumor lysate-M-based clinical approaches. These findings demonstrate how tuning the interfacial mechanics of approved materials can unlock mechano-immunotherapy with translational potential.

Author Info: (1) State Key Laboratory of Biopharmaceutical Preparation and Delivery, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, P.R. China (2) University of

Author Info: (1) State Key Laboratory of Biopharmaceutical Preparation and Delivery, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, P.R. China (2) University of Chinese Academy of Sciences, Beijing 100049, P.R. China (3) Department of Thoracic Surgery, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450003, P.R. China (4) Lead contact

Development of a high-affinity anti-ROR1 variable region for broad anti-cancer immunotherapy

Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is an emerging target in cancer immunotherapy, recognized for its consistent and elevated expression across several epithelial tumors, including triple-negative breast cancer (TNBC). TNBC is an aggressive and difficult-to-treat cancer, with limited effective therapeutic options currently available. Therapeutic approaches centered on targeting ROR1 have therefore become increasingly popular, with ROR1 chimeric antigen receptor (CAR) T cells currently in clinical trials to treat TNBC patients. While ROR1-targeting therapies have shown promising preclinical results, single arm treatment has often shown low efficacy as well as off-target toxicity. Natural killer (NK) cell-based immunotherapies, such as antibody-dependent cell cytotoxicity-inducing monoclonal antibodies and CAR NK cells, have also been shown to induce cancer cell cytotoxicity; however, with less toxicity compared with CAR T cells. Here, we developed and characterized a phage-derived single-chain fragment variable (scFv) against a highly specific ROR1 region and generated scFv-derived chimeric monoclonal antibodies and anti-ROR1-CAR NK cells, which show anti-cancer efficacy against TNBC cells. Additionally, we found TGF-_ inhibition using either small-molecule inhibitors or CRISPR-Cas9-edited NK cells could further enhance ROR1-targeting therapy persistence and efficacy in controlling TNBC tumor growth.

Author Info: (1) Frazer Institute, The University of Queensland, Woolloongabba, QLD 4102, Australia. (2) Frazer Institute, The University of Queensland, Woolloongabba, QLD 4102, Australia. (3)

Author Info: (1) Frazer Institute, The University of Queensland, Woolloongabba, QLD 4102, Australia. (2) Frazer Institute, The University of Queensland, Woolloongabba, QLD 4102, Australia. (3) Frazer Institute, The University of Queensland, Woolloongabba, QLD 4102, Australia. (4) Frazer Institute, The University of Queensland, Woolloongabba, QLD 4102, Australia. (5) Frazer Institute, The University of Queensland, Woolloongabba, QLD 4102, Australia. (6) Frazer Institute, The University of Queensland, Woolloongabba, QLD 4102, Australia. (7) Queensland Cyber Infrastructure Foundation Ltd (QCIF) Bioinformatics, Brisbane, QLD 4072, Australia. (8) Australian Institute for Bioengineering and Nanotechnology, University of Queensland, St Lucia, QLD 4067, Australia; BASE Facility, University of Queensland, St Lucia, QLD 4067, Australia. (9) Australian Institute for Bioengineering and Nanotechnology, University of Queensland, St Lucia, QLD 4067, Australia. (10) Laborat—rio de Patologia Experimental, Curitiba, Queensland 80215-901, Brazil. (11) Australian Institute for Bioengineering and Nanotechnology, University of Queensland, St Lucia, QLD 4067, Australia; BASE Facility, University of Queensland, St Lucia, QLD 4067, Australia. (12) Mater Research Institute, The University of Queensland, Brisbane, QLD 4102, Australia. (13) Cancer Immunology Program, Peter MacCallum Cancer Centre, Melbourne, VIC 3000, Australia; Sir Peter MacCallum Department of Oncology, The University of Melbourne, Parkville, VIC 3010, Australia. (14) Olivia Newton-John Cancer Research Institute, Heidelberg, VIC 3084, Australia. (15) Olivia Newton-John Cancer Research Institute, Heidelberg, VIC 3084, Australia. (16) Frazer Institute, The University of Queensland, Woolloongabba, QLD 4102, Australia. (17) UQ Centre for Clinical Research, Faculty of Health, Medicine and Behavioural Sciences, The University of Queensland, Herston, QLD 4029, Australia. (18) UQ Centre for Clinical Research, Faculty of Health, Medicine and Behavioural Sciences, The University of Queensland, Herston, QLD 4029, Australia. (19) UQ Centre for Clinical Research, Faculty of Health, Medicine and Behavioural Sciences, The University of Queensland, Herston, QLD 4029, Australia; School of Biomedical Sciences, Faculty of Health, Medicine and Behavioural Sciences, The University of Queensland, Saint Lucia, QLD 4067, Australia. (20) Frazer Institute, The University of Queensland, Woolloongabba, QLD 4102, Australia. (21) Frazer Institute, The University of Queensland, Woolloongabba, QLD 4102, Australia; School of Science & Technology, University of New England, Armidale NSW 2351, Australia. (22) Australian Institute for Bioengineering and Nanotechnology, University of Queensland, St Lucia, QLD 4067, Australia. (23) Frazer Institute, The University of Queensland, Woolloongabba, QLD 4102, Australia. (24) Frazer Institute, The University of Queensland, Woolloongabba, QLD 4102, Australia. Electronic address: f.guimaraes@uq.edu.au.

IL-12 and GM-CSF engineered dendritic cells enhance the enrichment and selection of tumor-reactive T cells for cancer immunotherapy

The use of tumor-reactive T cells in targeted tumor elimination holds significant potential for cancer immunotherapy, such as Tumor-Infiltrating Lymphocyte (TIL) therapy and TCR-T adoptive immunotherapy. Critical aspects of the effective clinical application of these immunotherapies include the enrichment and selection of tumor antigens and their corresponding reactive T cells. However, current in vitro methods for expanding and screening tumor antigen-reactive T cells remain inefficient. One reason for this inefficiency is the dysfunctional state of tumor-reactive T cells, which limits their expansion and activation. To address this challenge, we developed an optimized dendritic cell-based culture system, in which dendritic cells simultaneously express interleukin-12 and granulocyte-macrophage colony-stimulating factor (12GM-DCs), to enhance the expansion of tumor-reactive T cells. We found that 12GM-DCs can enrich reactive T cells targeting various tumor antigens, including virus-associated tumor antigens, tumor-associated antigens, mutant tumor neoantigens, and patient-specific tumor neoantigens. Moreover, 12GM-DCs increased the proportion of antigen-specific T cells, enhanced the activation of those T cells, and promoted the maintenance of a memory phenotype. The cytotoxicity of these antigen-reactive T cells was increased after co-culture with 12GM-DCs, likely due to the increased secretion of interferon-_ and granzyme B. Importantly, these functions and phenotypic advantages of tumor antigen-reactive T cells derived from the 12GM-DC culture system could be effectively maintained and the antitumor activity was also enhanced in tumor-burden mice. Our 12GM-DC coculture system effectively enriches antigen-specific T cells and has the potential to advance the clinical application of cancer immunotherapy by targeting tumor antigens and their reactive T cells.

Author Info: (1) Department of Cell Biology and Stem Cell Research Center, School of Basic Medical Sciences, Peking University Health Science Center, Peking University, Beijing, China. (2) Depa

Author Info: (1) Department of Cell Biology and Stem Cell Research Center, School of Basic Medical Sciences, Peking University Health Science Center, Peking University, Beijing, China. (2) Department of Cell Biology and Stem Cell Research Center, School of Basic Medical Sciences, Peking University Health Science Center, Peking University, Beijing, China. The Second Affiliated Hospital of Chongqing Medical University, Chongqing, China. (3) Department of Gastrointestinal Surgery, Peking University Shougang Hospital, Beijing, China. (4) School of Life Sciences, Center for Bioinformatics, Center for Statistical Science, Peking University, Beijing, China. (5) School of Life Sciences, Center for Bioinformatics, Center for Statistical Science, Peking University, Beijing, China. (6) Department of Cell Biology and Stem Cell Research Center, School of Basic Medical Sciences, Peking University Health Science Center, Peking University, Beijing, China. (7) Department of Cell Biology and Stem Cell Research Center, School of Basic Medical Sciences, Peking University Health Science Center, Peking University, Beijing, China. (8) Department of Gastrointestinal Surgery, Peking University Shougang Hospital, Beijing, China. (9) School of Life Sciences, Peking University, Beijing, China. (10) Department of Gastrointestinal Surgery, Peking University Shougang Hospital, Beijing, China. (11) Department of Cell Biology and Stem Cell Research Center, School of Basic Medical Sciences, Peking University Health Science Center, Peking University, Beijing, China. (12) School of Life Sciences, Center for Bioinformatics, Center for Statistical Science, Peking University, Beijing, China. (13) Department of Cell Biology and Stem Cell Research Center, School of Basic Medical Sciences, Peking University Health Science Center, Peking University, Beijing, China. (14) Department of Cell Biology and Stem Cell Research Center, School of Basic Medical Sciences, Peking University Health Science Center, Peking University, Beijing, China. School of Life Sciences, Peking University, Beijing, China. (15) Department of Cell Biology and Stem Cell Research Center, School of Basic Medical Sciences, Peking University Health Science Center, Peking University, Beijing, China. Department of Cell Biology and Stem Cell Research Center, School of Basic Medical Sciences, State Key Laboratory of Natural and Biomimetic Drugs, Peking University Health Science Center, Peking University, Beijing, China. Changping Laboratory, MOE Key Laboratory of Cell Proliferation and Differentiation, College of Life Sciences, Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China.

CD8+ T cell antitumor immunity via human iNKT-DC conjugates Spotlight 

Baiu et al. showed that CD4+ invariant natural killer T cells (iNKT) and autologous or allogeneic monocyte-derived DCs formed stable complexes, with enhanced expression of MHC I, 4-1BBL, OX40L, and IL-15Ra in DCs, and CD70 in iNKT cells. Complexes generated sustained DC signaling and created a platform for antigen-specific CD8+ T cell activation. In a xenograft model of B cell lymphoma, iNKT-DC induced T cell effector differentiation, reduced tumor burden, and remained effective at late disease stages that were resistant to ICB. Patient-derived DCs formed similar conjugates with allogeneic CD4+ iNKT cells and activated tumor antigen-specific CD8+ T cells.

Contributed by Shishir Pant

Baiu et al. showed that CD4+ invariant natural killer T cells (iNKT) and autologous or allogeneic monocyte-derived DCs formed stable complexes, with enhanced expression of MHC I, 4-1BBL, OX40L, and IL-15Ra in DCs, and CD70 in iNKT cells. Complexes generated sustained DC signaling and created a platform for antigen-specific CD8+ T cell activation. In a xenograft model of B cell lymphoma, iNKT-DC induced T cell effector differentiation, reduced tumor burden, and remained effective at late disease stages that were resistant to ICB. Patient-derived DCs formed similar conjugates with allogeneic CD4+ iNKT cells and activated tumor antigen-specific CD8+ T cells.

Contributed by Shishir Pant

ABSTRACT: Invariant Natural Killer T (iNKT) cells are a conserved T lymphocyte population capable of acting on dendritic cells (DCs) to potently amplify downstream immune responses. However, the processes underlying such iNKT adjuvancy remain poorly understood. Here, we showed that allogeneic human CD4+ iNKT cells form stably adhered bi-cellular complexes with monocyte-derived DCs that migrated together as pairs and showed extended DC calcium signaling. Compared to DCs treated with the synthetic adjuvant monophosphoryl lipid A (MPLA), DCs complexed with iNKT cells had elevated expression of MHC class I and multiple costimulatory molecules including 4-1BBL, OX40L, and IL-15R_, while the iNKT cells expressed CD70. Consistent with this distinctive co-stimulatory profile, iNKT-DC complexes were efficient activators of CD8+ T cells. Administering iNKT-DC complexes as a cellular immunotherapy in a xenograft model of aggressive human B cell lymphoma resulted in rapid reduction in tumor mass, antigen-specific B cell clearance, and transcriptional activation indicative of enhanced T cell proliferation and effector responses. iNKT-DC immunotherapy was effective at late stages of tumor progression that were refractory to immune checkpoint blockade immunotherapy, suggesting that the consortium of activating signals provided by iNKT-DC complexes rejuvenates exhausted antitumor immunity. Finally, allogeneic CD4+ iNKT cells formed similar complexes with monocyte-derived DCs from Head and Neck Cancer patients and promoted tumor antigen-dependent CD8+ T cell activation. These results show that monocyte-derived DCs paired with allogeneic CD4+ iNKT cells act as a potent antitumor cellular immunotherapy that activates antigen-specific CD8+ T cell immunity.

Author Info: (1) University of Wisconsin-Madison, Madison, WI, United States. (2) University of Wisconsin-Madison, Madison, WI, United States. (3) University of Wisconsin-Madison, Madison, WI,

Author Info: (1) University of Wisconsin-Madison, Madison, WI, United States. (2) University of Wisconsin-Madison, Madison, WI, United States. (3) University of Wisconsin-Madison, Madison, WI, United States. (4) University of Wisconsin-Madison, Madison, WI, United States. (5) University of Wisconsin-Madison, Madison, WI, United States. (6) University of Wisconsin-Madison, United States. (7) University of Wisconsin-Madison, Madison, WI, United States. (8) University of Wisconsin-Madison, Madison, WI, United States. (9) (10) University of Wisconsin-Madison, Madison, WI, United States.

Dendritic cell progenitors engineered to express extracellular-vesicle-internalizing receptors enhance cancer immunotherapy in mouse models Spotlight 

Ghasemi et al. engineered DC progenitors expressing IL-12 and a non-signaling receptor (EVIR) targeting GD2, which promoted uptake of GD2+ cancer cells and their EVs. T cell activation by the DCs was dependent on MHC-I presentation by cancer cells, indicating cross-dressing as the primary mode for DC antigen display. IL-12+EVIR+ progenitor DCs (but not moDCs) synergized with anti-PD-1 in ICB-non-responsive models, enhancing CD8+ T cell infiltration and M1 polarization. In a B16 model with heterogeneous antigen expression, GD2-targeted EVIR+ DCs enhanced T cell activation against non-target cells to control tumor growth.

Contributed by Morgan Janes

Ghasemi et al. engineered DC progenitors expressing IL-12 and a non-signaling receptor (EVIR) targeting GD2, which promoted uptake of GD2+ cancer cells and their EVs. T cell activation by the DCs was dependent on MHC-I presentation by cancer cells, indicating cross-dressing as the primary mode for DC antigen display. IL-12+EVIR+ progenitor DCs (but not moDCs) synergized with anti-PD-1 in ICB-non-responsive models, enhancing CD8+ T cell infiltration and M1 polarization. In a B16 model with heterogeneous antigen expression, GD2-targeted EVIR+ DCs enhanced T cell activation against non-target cells to control tumor growth.

Contributed by Morgan Janes

ABSTRACT: Cancer immunotherapy using dendritic cells (DC) pulsed ex vivo with tumour antigens is considered safe, but its clinical efficacy is generally modest. Here we engineer DC progenitors (DCP), which can replenish conventional type 1 DCs (cDC1) in mice, to constitutively express IL-12 together with a non-signalling chimeric receptor, termed extracellular vesicle-internalizing receptor (EVIR). By binding to a bait molecule (GD2 disialoganglioside) expressed on cancer cells and their EVs, the EVIR enforces EV internalization by cDC1 to promote their cross-dressing with preformed, tumour-derived MHCI-peptide complexes. Upon systemic deployment to mice, the engineered DCPs cause only mild and transient elevation of liver enzymes, acquire tumour-derived material, engage tumour-specific T cells, and enhance the efficacy of PD-1 blockade in an immunotherapy-resistant melanoma model comprising both GD2-positive and -negative cancer cells, without the need for ex vivo antigen pulsing. These results indicate that EVIR-engineered DCPs may avert the positive selection of antigen-negative cancer cells, potentially addressing a critical limitation of immunotherapies targeting defined tumour antigens.

Author Info: (1) Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, Swiss Federal Institute of Technology in Lausanne (EPFL), Lausanne, Switzerland. Agora Cancer

Author Info: (1) Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, Swiss Federal Institute of Technology in Lausanne (EPFL), Lausanne, Switzerland. Agora Cancer Research Center, Lausanne, Switzerland. Swiss Cancer Center LŽman (SCCL), Lausanne, Switzerland. (2) Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, Swiss Federal Institute of Technology in Lausanne (EPFL), Lausanne, Switzerland. Agora Cancer Research Center, Lausanne, Switzerland. Swiss Cancer Center LŽman (SCCL), Lausanne, Switzerland. (3) Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, Swiss Federal Institute of Technology in Lausanne (EPFL), Lausanne, Switzerland. Agora Cancer Research Center, Lausanne, Switzerland. Swiss Cancer Center LŽman (SCCL), Lausanne, Switzerland. (4) Laboratory of Metabolic Signaling, Institute of Bioengineering, EPFL, Lausanne, Switzerland. (5) Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, Swiss Federal Institute of Technology in Lausanne (EPFL), Lausanne, Switzerland. Agora Cancer Research Center, Lausanne, Switzerland. Swiss Cancer Center LŽman (SCCL), Lausanne, Switzerland. (6) Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, Swiss Federal Institute of Technology in Lausanne (EPFL), Lausanne, Switzerland. Agora Cancer Research Center, Lausanne, Switzerland. Swiss Cancer Center LŽman (SCCL), Lausanne, Switzerland. (7) Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, Swiss Federal Institute of Technology in Lausanne (EPFL), Lausanne, Switzerland. Agora Cancer Research Center, Lausanne, Switzerland. Swiss Cancer Center LŽman (SCCL), Lausanne, Switzerland. (8) Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, Swiss Federal Institute of Technology in Lausanne (EPFL), Lausanne, Switzerland. Agora Cancer Research Center, Lausanne, Switzerland. Swiss Cancer Center LŽman (SCCL), Lausanne, Switzerland. (9) Animal Modeling Facility, Netherlands Cancer Institute (NKI), Amsterdam, The Netherlands. (10) Animal Modeling Facility, Netherlands Cancer Institute (NKI), Amsterdam, The Netherlands. (11) Department of Oncology, University of Lausanne (UNIL), Lausanne, Switzerland. Department of Oncology, Lausanne University Hospital (CHUV), Lausanne, Switzerland. (12) Agora Cancer Research Center, Lausanne, Switzerland. Swiss Cancer Center LŽman (SCCL), Lausanne, Switzerland. Department of Oncology, Geneva University Hospital (HUG), Geneva, Switzerland. Center for Translational Research in Onco-Hematology, University of Geneva (UNIGE), Geneva, Switzerland. (13) Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, Swiss Federal Institute of Technology in Lausanne (EPFL), Lausanne, Switzerland. michele.depalma@epfl.ch. Agora Cancer Research Center, Lausanne, Switzerland. michele.depalma@epfl.ch. Swiss Cancer Center LŽman (SCCL), Lausanne, Switzerland. michele.depalma@epfl.ch.

Antibody-gamma/delta T cell receptors targeting GPC2 regress neuroblastoma with low antigen density Spotlight 

To treat neuroblastoma expressing the oncofetal antigen GPC2, Quan and Huo et al. generated "AbTCR-T cells" expressing (1) anti-GPC2 Fab linked to TCRγδ and (2) anti-GPC2 scFv linked to a CD30 costimulatory domain. The GPC2-binding domain was humanized from the murine CT3 antibody, and retained specific GPC2 binding. Compared to CAR-T, AbTCR-T had superior cytotoxicity, tumor T cell infiltration, and in vivo efficacy against tumors with high or, in particular, low antigen expression. AbTCR-T also maintained a less exhausted and more stem-like phenotype, improving serial cytotoxicity, and augmented endogenous TCR and NFAT signaling.

Contributed by Alex Najibi

To treat neuroblastoma expressing the oncofetal antigen GPC2, Quan and Huo et al. generated "AbTCR-T cells" expressing (1) anti-GPC2 Fab linked to TCRγδ and (2) anti-GPC2 scFv linked to a CD30 costimulatory domain. The GPC2-binding domain was humanized from the murine CT3 antibody, and retained specific GPC2 binding. Compared to CAR-T, AbTCR-T had superior cytotoxicity, tumor T cell infiltration, and in vivo efficacy against tumors with high or, in particular, low antigen expression. AbTCR-T also maintained a less exhausted and more stem-like phenotype, improving serial cytotoxicity, and augmented endogenous TCR and NFAT signaling.

Contributed by Alex Najibi

ABSTRACT: Chimeric antigen receptor (CAR) T cells have shown promise in hematological cancers but face challenges in solid tumors, partly due to heterogeneous antigen density. Glypican-2 (GPC2) is an oncofetal antigen highly expressed in neuroblastoma and under evaluation in phase 1 clinical trials. Here, we engineer T cells with antibody-T cell receptors (AbTCRs) targeting GPC2. We generate autologous AbTCR T cells using CT3 or humanized CT3 (hCT3) antigen-binding fragments (Fab) linked to _/_ T cell receptors (TCRs), along with a CD30 co-stimulatory domain. Both CT3 and hCT3 AbTCR T cells show superior antitumor efficacy compared to CT3 CAR T cells, with hCT3 AbTCR T cells inducing significant regression in neuroblastoma with low GPC2 antigen density. Enhanced efficacy is associated with stronger TCR signaling, expansion of stem cell-like memory T cells, and improved CD8(+) T cell infiltration. These results highlight the potential of hCT3 AbTCR T cells for neuroblastoma and indicate broad application of AbTCR T cells in solid tumors.

Author Info: (1) Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4264, USA. (2) Laboratory of Molecular

Author Info: (1) Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4264, USA. (2) Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4264, USA. (3) Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4264, USA. (4) Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4264, USA. (5) Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4264, USA. (6) Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4264, USA. (7) Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4264, USA. (8) Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4264, USA. (9) Molecular Histopathology Laboratory, Frederick National Laboratory for Cancer Research, Frederick, MD 21702, USA. (10) Spatomics LLC, 246 Goose Ln, Ste 202A, Guilford, CT 06437, USA. (11) Spatomics LLC, 246 Goose Ln, Ste 202A, Guilford, CT 06437, USA. (12) Spatomics LLC, 246 Goose Ln, Ste 202A, Guilford, CT 06437, USA. (13) Eureka Therapeutics Inc., 5858 Horton Street, Suite 370, Emeryville, CA 94608, USA. (14) Eureka Therapeutics Inc., 5858 Horton Street, Suite 370, Emeryville, CA 94608, USA. (15) Eureka Therapeutics Inc., 5858 Horton Street, Suite 370, Emeryville, CA 94608, USA. (16) Eureka Therapeutics Inc., 5858 Horton Street, Suite 370, Emeryville, CA 94608, USA. (17) Eureka Therapeutics Inc., 5858 Horton Street, Suite 370, Emeryville, CA 94608, USA. (18) Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4264, USA. (19) Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4264, USA. (20) Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4264, USA. Electronic address: homi@mail.nih.gov.

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