Journal Articles

Allogeneic CD19 CAR T cells armed with an anti-rejection CD70 CAR overcome antigen escape and evade alloimmune responses Spotlight 

Aiming to avoid allogeneic CAR-T rejection, Zhang and Li et al. found that a CD70 CAR depleted donor-mismatched, activated (CD70+) T and NK cells in coculture. Dual CD19/CD70 CAR T cells responded to CD19+ tumor cells comparably to single CD19 CAR-T, but also recognized CD70+ target cells and protected against allo-mediated killing. Dual CD19-CD70 CAR T cells transiently eliminated B cells in CD34-humanized mice, and depleted B cells and autoantibodies in lupus PBMC-humanized mice, with superior persistence of CD19 CAR-T cells, without lymphodepletion. CD70 CAR variants were optimized for expression and functionality.

Contributed by Alex Najibi

Aiming to avoid allogeneic CAR-T rejection, Zhang and Li et al. found that a CD70 CAR depleted donor-mismatched, activated (CD70+) T and NK cells in coculture. Dual CD19/CD70 CAR T cells responded to CD19+ tumor cells comparably to single CD19 CAR-T, but also recognized CD70+ target cells and protected against allo-mediated killing. Dual CD19-CD70 CAR T cells transiently eliminated B cells in CD34-humanized mice, and depleted B cells and autoantibodies in lupus PBMC-humanized mice, with superior persistence of CD19 CAR-T cells, without lymphodepletion. CD70 CAR variants were optimized for expression and functionality.

Contributed by Alex Najibi

ABSTRACT: Chimeric antigen receptor (CAR) T cells can achieve sustained clinical benefit in B cell malignancies and autoimmune diseases. Despite the many potential advantages over autologous products, allogeneic CAR T cells carry a higher risk of rejection, which may limit persistence and therapeutic efficacy. We report the design and evaluation of an optimized CD70 CAR that prevents rejection of allogeneic CAR T cells by targeting activated alloreactive lymphocytes. Co-expression of this CD70 CAR with a CD19 CAR resulted in sustained CAR T cell persistence in the presence of alloreactive lymphocytes and prolonged antitumor activity in a CD19 antigen escape model. In vivo, CD19/CD70 dual CAR T cells eliminated B cells and CD70(+) T cells derived from patients with systemic lupus erythematosus in humanized mouse models, resulting in reduced immunoglobulin production. An allogeneic CD19/CD70 dual CAR T cell therapy may therefore broaden clinical applicability while enabling the use of less intensive lymphodepleting conditioning regimens prior to CAR T cell infusion.

Author Info: (1) Allogene Therapeutics Inc., South San Francisco, CA, USA. (2) Allogene Therapeutics Inc., South San Francisco, CA, USA. (3) Allogene Therapeutics Inc., South San Francisco, CA,

Author Info: (1) Allogene Therapeutics Inc., South San Francisco, CA, USA. (2) Allogene Therapeutics Inc., South San Francisco, CA, USA. (3) Allogene Therapeutics Inc., South San Francisco, CA, USA. (4) Allogene Therapeutics Inc., South San Francisco, CA, USA. (5) Allogene Therapeutics Inc., South San Francisco, CA, USA. (6) Allogene Therapeutics Inc., South San Francisco, CA, USA. (7) Allogene Therapeutics Inc., South San Francisco, CA, USA. (8) Allogene Therapeutics Inc., South San Francisco, CA, USA. (9) Allogene Therapeutics Inc., South San Francisco, CA, USA. (10) Allogene Therapeutics Inc., South San Francisco, CA, USA. (11) Allogene Therapeutics Inc., South San Francisco, CA, USA. cesar.sommer@allogene.com. (12) Allogene Therapeutics Inc., South San Francisco, CA, USA. elvin.lauron@allogene.com.

In vivo CAR T cell generation using retargeted and functionalized lentiviral vectors with reduced immunogenicity Spotlight 

Measles virus (MeV) recognizes and fuses with target cells via hemagglutinin (H) and fusion (F) proteins, respectively. To achieve T cell-specific transduction, Ibrahim et al. produced a lentivirus (LV) expressing MEV-F and a re-targeted MEV-H linked to a targeting molecule (VHHs resulted in higher functional titers than scFvs). To avoid serum neutralization by anti-MeV antibodies, MeV-H/F proteins were redesigned as chimeras with dolphin morbillivirus-H/F. LVs expressing the chimeric proteins, CD7-targeting VHH, and anti-CD3 and CD80 (activation cues) generated CD19 CAR T cells in vivo and slowed Nalm6 tumor growth. CAR expression was largely restricted to T cells.

Contributed by Alex Najibi

Measles virus (MeV) recognizes and fuses with target cells via hemagglutinin (H) and fusion (F) proteins, respectively. To achieve T cell-specific transduction, Ibrahim et al. produced a lentivirus (LV) expressing MEV-F and a re-targeted MEV-H linked to a targeting molecule (VHHs resulted in higher functional titers than scFvs). To avoid serum neutralization by anti-MeV antibodies, MeV-H/F proteins were redesigned as chimeras with dolphin morbillivirus-H/F. LVs expressing the chimeric proteins, CD7-targeting VHH, and anti-CD3 and CD80 (activation cues) generated CD19 CAR T cells in vivo and slowed Nalm6 tumor growth. CAR expression was largely restricted to T cells.

Contributed by Alex Najibi

ABSTRACT: Despite striking efficacy against hematologic malignancies, the cost and complexity of CAR T manufacturing present significant barriers to broader patient access. Beyond manufacturing challenges, ex vivo expansion of T cells may be detrimental to their function and persistence. Thus, delivery of CARs to reprogram host cells in vivo would represent a significant advance towards a readily available therapy, but has been limited by low efficiency, low specificity, and immunogenicity of viral vectors. Here, we describe the design of pseudotyped lentiviral vectors (LV) with superior functionality and high target specificity. We show that LV pseudotyped with chimeric envelope glycoproteins from dolphin morbillivirus (DMV) can be engineered to selectively infect human T cells and evade neutralizing antibody responses in measles-vaccinated human serum. We further demonstrate that camelid-derived nanobodies are a superior retargeting domain, overcoming limitations inherent to the use of single-chain variable fragment antibodies. Using a chimeric DMV-pseudotyped virus targeting the CD7 receptor, we demonstrate efficient and highly specific infection of T cells both in vitro and in vivo, generating functional CAR T cells and inducing therapeutic efficacy in a preclinical B cell lymphoma model.

Author Info: (1) Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA. Krantz Family Center for Cancer Research and Department of Medicine, Massachusetts Gen

Author Info: (1) Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA. Krantz Family Center for Cancer Research and Department of Medicine, Massachusetts General Hospital, Boston, MA, USA. (2) Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA. Krantz Family Center for Cancer Research and Department of Medicine, Massachusetts General Hospital, Boston, MA, USA. (3) Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA. Krantz Family Center for Cancer Research and Department of Medicine, Massachusetts General Hospital, Boston, MA, USA. (4) Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA. Krantz Family Center for Cancer Research and Department of Medicine, Massachusetts General Hospital, Boston, MA, USA. Dana-Farber Cancer Institute, Gastrointestinal Cancer Center, Boston, MA, USA. (5) Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA. Krantz Family Center for Cancer Research and Department of Medicine, Massachusetts General Hospital, Boston, MA, USA. (6) Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA. Krantz Family Center for Cancer Research and Department of Medicine, Massachusetts General Hospital, Boston, MA, USA. (7) Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA. Krantz Family Center for Cancer Research and Department of Medicine, Massachusetts General Hospital, Boston, MA, USA. (8) Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA. Krantz Family Center for Cancer Research and Department of Medicine, Massachusetts General Hospital, Boston, MA, USA. (9) Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA. Krantz Family Center for Cancer Research and Department of Medicine, Massachusetts General Hospital, Boston, MA, USA. (10) Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA. Krantz Family Center for Cancer Research and Department of Medicine, Massachusetts General Hospital, Boston, MA, USA. (11) Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA. yates@broadinstitute.org. Krantz Family Center for Cancer Research and Department of Medicine, Massachusetts General Hospital, Boston, MA, USA. yates@broadinstitute.org. (12) Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA. rmanguso@broadinstitute.org. Krantz Family Center for Cancer Research and Department of Medicine, Massachusetts General Hospital, Boston, MA, USA. rmanguso@broadinstitute.org.

Lymphodepleting preconditioning impairs host antitumor immunity induced by adoptive T cell therapy in mouse models

Spotlight 

Figueroa et al. demonstrated that lasting efficacy of adoptive T cell therapy (ACT) against solid tumors relied not only on the antitumor activity of transferred T cells, but also on their ability to expand host CD8+ T cells in a TNF- and cDC1-dependent manner. Host CD8+ T cells protected against rechallenge with ACT-resistant antigen-negative tumor cells. Lymphodepleting preconditioning promoted transferred T cell expansion, but impaired host immunity against antigen-loss variants. In patients with melanoma, enrichment of cDC1, TNF signaling, Tpex and Tex gene signatures correlated with clinical responses to ACT and better overall survival.

Contributed by Ute Burkhardt

Figueroa et al. demonstrated that lasting efficacy of adoptive T cell therapy (ACT) against solid tumors relied not only on the antitumor activity of transferred T cells, but also on their ability to expand host CD8+ T cells in a TNF- and cDC1-dependent manner. Host CD8+ T cells protected against rechallenge with ACT-resistant antigen-negative tumor cells. Lymphodepleting preconditioning promoted transferred T cell expansion, but impaired host immunity against antigen-loss variants. In patients with melanoma, enrichment of cDC1, TNF signaling, Tpex and Tex gene signatures correlated with clinical responses to ACT and better overall survival.

Contributed by Ute Burkhardt

ABSTRACT: Adoptive T cell therapy (ACT) is effective against hematologic cancers, but the mechanisms underlying durable responses in solid tumors remain unclear. We show that adoptively transferred CD8(+) T cells that eradicate established murine tumors promote expansion of host CD8(+) T cells exhibiting tumor-reactive and tissue-resident phenotypes that contribute to tumor elimination. Mechanistically, tumor necrosis factor (TNF) from transferred cells induces dendritic cell (DC)-dependent expansion of host CD8(+) T cells, conferring protection against ACT-resistant tumor cells lacking the targeted antigen. Lymphodepleting preconditioning promotes expansion of transferred cells and primary tumor eradication but impairs host antitumor immunity and abrogates protection against ACT-resistant tumors. In human tumors, increased TNF/DC/CD8(+) T cell profiles correlate with favorable ACT responses and improved survival. These findings reveal a TNF-dependent interplay between transferred and host CD8(+) T cells underlying durable antitumor immunity that is impaired by lymphodepleting preconditioning in mouse models, suggesting an underappreciated mechanism of ACT resistance.

Author Info: (1) Centro Basal Ciencia & Vida, Fundaci—n Ciencia & Vida, Santiago, Chile. (2) Centro Basal Ciencia & Vida, Fundaci—n Ciencia & Vida, Santiago, Chile. (3) Centro Basal Ciencia & V

Author Info: (1) Centro Basal Ciencia & Vida, Fundaci—n Ciencia & Vida, Santiago, Chile. (2) Centro Basal Ciencia & Vida, Fundaci—n Ciencia & Vida, Santiago, Chile. (3) Centro Basal Ciencia & Vida, Fundaci—n Ciencia & Vida, Santiago, Chile. Centro de Investigaci—n e Innovaci—n en C‡ncer, Fundaci—n Arturo L—pez PŽrez OECI Cancer Center, Santiago, Chile. (4) Centro Basal Ciencia & Vida, Fundaci—n Ciencia & Vida, Santiago, Chile. (5) Centro Basal Ciencia & Vida, Fundaci—n Ciencia & Vida, Santiago, Chile. Centro de Investigaci—n e Innovaci—n en C‡ncer, Fundaci—n Arturo L—pez PŽrez OECI Cancer Center, Santiago, Chile. (6) Centro Basal Ciencia & Vida, Fundaci—n Ciencia & Vida, Santiago, Chile. (7) Centro Basal Ciencia & Vida, Fundaci—n Ciencia & Vida, Santiago, Chile. (8) Centro Basal Ciencia & Vida, Fundaci—n Ciencia & Vida, Santiago, Chile. (9) Centro Basal Ciencia & Vida, Fundaci—n Ciencia & Vida, Santiago, Chile. (10) Laboratory of Immunology and Cellular Stress, Facultad de Medicina, Universidad de Chile, Santiago, Chile. (11) Laboratory of Immune Regulation, NDM Centre for Immuno-Oncology, University of Oxford, Oxford, UK. (12) Centro Basal Ciencia & Vida, Fundaci—n Ciencia & Vida, Santiago, Chile. Department of Anatomy, University of California San Francisco, San Francisco, CA, USA. (13) Centro Basal Ciencia & Vida, Fundaci—n Ciencia & Vida, Santiago, Chile. Centro de Biolog’a Celular y Biomedicina (CEBICEM), Facultad de Ciencias, Universidad San Sebasti‡n, Santiago, Chile. (14) Centro Basal Ciencia & Vida, Fundaci—n Ciencia & Vida, Santiago, Chile. Laboratory of Immunology, Facultad de Ciencias, Universidad de Chile, Santiago, Chile. (15) Centro Basal Ciencia & Vida, Fundaci—n Ciencia & Vida, Santiago, Chile. Laboratory of Immunology and Cellular Stress, Facultad de Medicina, Universidad de Chile, Santiago, Chile. (16) Centro Basal Ciencia & Vida, Fundaci—n Ciencia & Vida, Santiago, Chile. vincenzo.borgna@uss.cl. Servicio de Urolog’a, Hospital Barros Luco Trudeau, Santiago, Chile. vincenzo.borgna@uss.cl. Facultad de Medicina, Universidad San Sebasti‡n, Santiago, Chile. vincenzo.borgna@uss.cl. (17) Centro Basal Ciencia & Vida, Fundaci—n Ciencia & Vida, Santiago, Chile. alladser@cienciavida.org. Facultad de Medicina, Universidad San Sebasti‡n, Santiago, Chile. alladser@cienciavida.org.

In vivo generation of CAR myeloid cells through erythrocyte-mediated mRNA delivery for cancer immunotherapy Spotlight 

Nie, Liu, Song, and Yao et al. developed a spleen delivery platform of mRNA-loaded lipid nanoparticles (LNPs) covalently bound to erythrocytes (mRNA-LNP-Ery), which naturally target splenic CD11b+ myeloid cells. Unlike conventional LNPs, mRNA-LNP-Ery entered cells via phagocytosis, avoiding lysosomal degradation and efficiently delivering mRNA. CAR myeloid cells (HER2 or CD19) adopted a proinflammatory antigen-presenting phenotype, migrated to tumors, and stimulated T and NK cell influx, potent antitumor activity, and systemic immunity, which was spleen-dependent. Repeated doses of mRNA-LNP-Ery resulted in superior efficacy at 1/10 the dose of LNPs.

Contributed by Katherine Turner

Nie, Liu, Song, and Yao et al. developed a spleen delivery platform of mRNA-loaded lipid nanoparticles (LNPs) covalently bound to erythrocytes (mRNA-LNP-Ery), which naturally target splenic CD11b+ myeloid cells. Unlike conventional LNPs, mRNA-LNP-Ery entered cells via phagocytosis, avoiding lysosomal degradation and efficiently delivering mRNA. CAR myeloid cells (HER2 or CD19) adopted a proinflammatory antigen-presenting phenotype, migrated to tumors, and stimulated T and NK cell influx, potent antitumor activity, and systemic immunity, which was spleen-dependent. Repeated doses of mRNA-LNP-Ery resulted in superior efficacy at 1/10 the dose of LNPs.

Contributed by Katherine Turner

ABSTRACT: Engineering myeloid cells with chimeric antigen receptors (CARs) holds great therapeutic promise, but their generation in vivo remains challenging. Here, we developed an erythrocyte-mediated messenger RNA (mRNA) delivery platform, termed mRNA-LNP-Ery, in which mRNA-loaded lipid nanoparticles (LNPs) are covalently anchored onto erythrocytes. Exploiting erythrocytes' intrinsic splenic homing capacity and unique biocompatibility, mRNA-LNP-Ery enables highly selective and efficient mRNA delivery to CD11b(+) myeloid cells in the spleen, with minimal uptake by hepatocytes. We also demonstrated that mRNA-LNP-Ery is internalized through phagocytosis and avoids lysosomal degradation, resulting in enhanced cytosolic mRNA translation. Delivery of mRNAs encoding CARs targeting human epidermal growth factor receptor 2 (HER2) or CD19 generated functional CAR myeloid cells in vivo that adopted a proinflammatory, antigen-presenting phenotype. These cells migrated to tumors, eliminated cancer cells, and remodeled the tumor microenvironment, leading to increased infiltration of effector T and natural killer (NK) cells. The antitumor effect was abolished in splenectomized mice and partially diminished in nude mice, indicating that therapeutic activity depends on both CAR myeloid cell formation within the spleen and their cross-talk with adaptive immunity. Furthermore, repeated administration of mRNA-LNP-Ery achieved superior antitumor efficacy to conventional mRNA-LNPs at one-tenth the mRNA dose, with minimal systemic toxicity, underscoring the high efficiency and safety of spleen-targeted delivery. Together, our findings established a clinically translatable erythrocyte-based mRNA platform that enables direct in vivo immune cell programming and advances CAR myeloid therapies for solid tumors.

Author Info: (1) Key Laboratory of Growth Regulation and Translational Research of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou, Zhejiang 310030, China. Westlake La

Author Info: (1) Key Laboratory of Growth Regulation and Translational Research of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou, Zhejiang 310030, China. Westlake Laboratory of Life Sciences and Biomedicine, Hangzhou, Zhejiang 310024, China. (2) Key Laboratory of Growth Regulation and Translational Research of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou, Zhejiang 310030, China. Westlake Laboratory of Life Sciences and Biomedicine, Hangzhou, Zhejiang 310024, China. Research Center for Industries of the Future and School of Life Sciences, Westlake University, Hangzhou, Zhejiang 310030, China. (3) Westlake Therapeutics, Hangzhou, Zhejiang 310024, China. (4) Key Laboratory of Growth Regulation and Translational Research of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou, Zhejiang 310030, China. Westlake Laboratory of Life Sciences and Biomedicine, Hangzhou, Zhejiang 310024, China. (5) Westlake Therapeutics, Hangzhou, Zhejiang 310024, China. (6) Peking-Tsinghua Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing 100871, China. Ministry of Education Key Laboratory of Cell Proliferation and Differentiation, School of Life Sciences, Peking University, Beijing 100871, China. (7) Key Laboratory of Growth Regulation and Translational Research of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou, Zhejiang 310030, China. Westlake Laboratory of Life Sciences and Biomedicine, Hangzhou, Zhejiang 310024, China. Research Center for Industries of the Future and School of Life Sciences, Westlake University, Hangzhou, Zhejiang 310030, China.

Overcoming T cell tolerance to tumor self-antigens through catch-bond engineering Spotlight 

To improve the potency of a prostate TAA-specific TCR, Chen and Mao et al. screened for CDR hotspot mutations that could increase catch-bond formation and thus TCR sensitivity, without modifying TCR affinity (and the potential for off-target toxicity). Several variants increased TCR–pHLA bond lifetime, which correlated with TCR response to cognate peptide. These variants increased T cell proliferation, cytotoxicity, in vivo tumor efficacy, and effector/proliferative gene expression among TILs. Crystal structures and in silico modeling revealed alterations to water inclusion and hydrogen-bonding, supporting HLA, TCR, or peptide interactions.

Contributed by Alex Najibi

To improve the potency of a prostate TAA-specific TCR, Chen and Mao et al. screened for CDR hotspot mutations that could increase catch-bond formation and thus TCR sensitivity, without modifying TCR affinity (and the potential for off-target toxicity). Several variants increased TCR–pHLA bond lifetime, which correlated with TCR response to cognate peptide. These variants increased T cell proliferation, cytotoxicity, in vivo tumor efficacy, and effector/proliferative gene expression among TILs. Crystal structures and in silico modeling revealed alterations to water inclusion and hydrogen-bonding, supporting HLA, TCR, or peptide interactions.

Contributed by Alex Najibi

ABSTRACT: T cells are often weakly responsive to tumor self-antigens because of central tolerance, constraining their ability to eliminate tumors. We exploited mechanical force to engineer a weakly reactive T cell receptor (TCR) specific for a nonmutated tumor-associated antigen (TAA), prostatic acid phosphatase (PAP). We identified a catch-bonding "hotspot" whose mutation enhanced T cell activity by increasing TCR-pMHC (peptide-major histocompatibility complex) bond lifetime while preserving physiological affinities and antigen fine specificities. T cells expressing these engineered TCRs showed vastly superior expansion in the tumor, effector phenotypes, and tumor elimination. Crystal structures and molecular dynamics simulations revealed a single amino acid mutation at the catch-bond hotspot primes the TCR for peptide interaction through water reorganization at the TCR-pMHC interface. Catch-bond engineering is a viable biophysically based strategy for transforming tolerized antitumor T cells into potent TCR-T cell therapy killers.

Author Info: (1) Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA. (2) Department of Microbiology, Immunology, and Molecular Genetics,

Author Info: (1) Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA. (2) Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles (UCLA), Los Angeles, CA, USA. Department of Molecular and Medical Pharmacology, UCLA, Los Angeles, CA, USA. (3) Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT, USA. (4) Department of Biochemistry and Molecular Biophysics, Columbia University Irving Medical Center, New York, NY, USA. (5) Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA. (6) Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA. (7) Department of Medicine, Division of Immunology and Rheumatology, Stanford University School of Medicine, Stanford, CA, USA. Department of Medicine, Center for Biomedical Informatics Research, Stanford University School of Medicine, Stanford, CA, USA. (8) Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles (UCLA), Los Angeles, CA, USA. (9) Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles (UCLA), Los Angeles, CA, USA. (10) Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA. (11) Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA. (12) Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles (UCLA), Los Angeles, CA, USA. (13) Department of Molecular and Medical Pharmacology, UCLA, Los Angeles, CA, USA. (14) Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA. (15) Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA. (16) Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA. Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA, USA. (17) Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles (UCLA), Los Angeles, CA, USA. (18) Department of Molecular and Medical Pharmacology, UCLA, Los Angeles, CA, USA. Department of Urology, UCLA, Los Angeles, CA, USA. (19) Department of Molecular and Medical Pharmacology, UCLA, Los Angeles, CA, USA. Department of Medicine, Division of Hematology/Oncology, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA. (20) Department of Medicine, Division of Immunology and Rheumatology, Stanford University School of Medicine, Stanford, CA, USA. Department of Medicine, Center for Biomedical Informatics Research, Stanford University School of Medicine, Stanford, CA, USA. Parker Institute for Cancer Immunotherapy, Stanford University, Stanford, CA, USA. (21) Department of Biochemistry and Molecular Biophysics, Columbia University Irving Medical Center, New York, NY, USA. (22) Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT, USA. (23) Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles (UCLA), Los Angeles, CA, USA. Department of Molecular and Medical Pharmacology, UCLA, Los Angeles, CA, USA. Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, UCLA, Los Angeles, CA, USA. Parker Institute for Cancer Immunotherapy, UCLA, Los Angeles, CA, USA. Jonsson Comprehensive Cancer Center, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA. Molecular Biology Institute, UCLA, Los Angeles, CA, USA. (24) Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA. Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA, USA. Parker Institute for Cancer Immunotherapy, Stanford University, Stanford, CA, USA. Department of Structural Biology, Stanford University School of Medicine, Stanford, CA, USA.

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

To enhance nanoparticle (NP) uptake by, target gene delivery to, and activation of T cells, Jain et al. generated stable (to lyophilization and freeze/thaw), biodegradable, beta-­amino ester polymer-based NPs with PEG-lipid-­anchored ligands/Abs (tPNPs). Anti-CD3/anti-CD28-expressing tPNPs with CAR-encoding mRNA cargo achieved high CAR expression by and stimulation of primary murine T cells in vitro, and enhanced lymphoid selectivity and T cell activation, proliferation, and effector and memory T cell generation upon i.v. delivery to mice. Anti-CD19 CAR-encoding tPNP safely and robustly depleted B cells in the peripheral blood and spleens of healthy mice.

Contributed by Paula Hochman

To enhance nanoparticle (NP) uptake by, target gene delivery to, and activation of T cells, Jain et al. generated stable (to lyophilization and freeze/thaw), biodegradable, beta-­amino ester polymer-based NPs with PEG-lipid-­anchored ligands/Abs (tPNPs). Anti-CD3/anti-CD28-expressing tPNPs with CAR-encoding mRNA cargo achieved high CAR expression by and stimulation of primary murine T cells in vitro, and enhanced lymphoid selectivity and T cell activation, proliferation, and effector and memory T cell generation upon i.v. delivery to mice. Anti-CD19 CAR-encoding tPNP safely and robustly depleted B cells in the peripheral blood and spleens of healthy mice.

Contributed by Paula Hochman

ABSTRACT: 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 Spotlight 

Cole et al. used HCW9206 – a soluble tissue factor fusion protein that links IL-7, IL-15/IL-15Rα superagonist, and IL-21 – for the generation of T stem cell memory (TSCM)-enriched polyfunctional CAR T cells, without requiring anti-CD3/CD28 activation. In a humanized mouse model of HIV infection, HCW9206-stimulated duoCAR T cells (simultaneously targeting two HIV epitopes) showed superior viremia suppression compared to duoCAR TαCD3/CD28 cells. CD19 CAR THCW9206 cells exhibited increased functional persistence and elimination of an initial and subsequent rechallenge with NALM-6 leukemia cells in vivo compared to CD19 CAR TαCD3/CD28 cells.

Contributed by Ute Burkhardt

Cole et al. used HCW9206 – a soluble tissue factor fusion protein that links IL-7, IL-15/IL-15Rα superagonist, and IL-21 – for the generation of T stem cell memory (TSCM)-enriched polyfunctional CAR T cells, without requiring anti-CD3/CD28 activation. In a humanized mouse model of HIV infection, HCW9206-stimulated duoCAR T cells (simultaneously targeting two HIV epitopes) showed superior viremia suppression compared to duoCAR TαCD3/CD28 cells. CD19 CAR THCW9206 cells exhibited increased functional persistence and elimination of an initial and subsequent rechallenge with NALM-6 leukemia cells in vivo compared to CD19 CAR TαCD3/CD28 cells.

Contributed by Ute Burkhardt

ABSTRACT: Functional persistence of chimeric antigen receptor T cells (CAR T cells) is limited by conventional CAR T cell manufacturing using anti-CD3/CD28 (αCD3/28) 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 α (IL-15Rα) complex, and IL-21, generates CAR T cells without requiring αCD3/28 activation, which are highly enriched in long-lived T memory stem cells (TSCM 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 αCD3/28-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 αCD3/28-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 TSCM 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.

Engineering CAR T cells to secrete VEGF-neutralizing scFvs enhances antitumor activity against solid tumors Spotlight 

To improve anti-angiogenic therapies in VEGF-overexpressing solid tumors, Gao et al. engineered CAR T cells to secrete anti-VEGF scFvs (CAR-αVEGF T cells) and compared their efficacy with standard CAR T cell therapy alone or combined with anti-VEGF Ab. αVEGF-scFv secretion resulted in superior CAR T cell efficacy in ovarian cancer and orthotopic glioma models. Mechanistically, CAR-αVEGF T cells prevented treatment-induced angiogenesis and hypoxia, promoted CD8+ T cell activation and mitochondrial fitness, and boosted immune-stimulatory myeloid phenotypes, while decreasing infiltration of suppressive, VEGF-expressing myeloid cells.

Contributed by Katherine Turner

To improve anti-angiogenic therapies in VEGF-overexpressing solid tumors, Gao et al. engineered CAR T cells to secrete anti-VEGF scFvs (CAR-αVEGF T cells) and compared their efficacy with standard CAR T cell therapy alone or combined with anti-VEGF Ab. αVEGF-scFv secretion resulted in superior CAR T cell efficacy in ovarian cancer and orthotopic glioma models. Mechanistically, CAR-αVEGF T cells prevented treatment-induced angiogenesis and hypoxia, promoted CD8+ T cell activation and mitochondrial fitness, and boosted immune-stimulatory myeloid phenotypes, while decreasing infiltration of suppressive, VEGF-expressing myeloid cells.

Contributed by Katherine Turner

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

Author Info: (1) Department of Chemical and Biomolecular Engineering, University of California, Los Angeles, Los Angeles, CA 90095, USA. Department of Microbiology, Immunology, and Molecular Ge

Author Info: (1) Department of Chemical and Biomolecular Engineering, University of California, Los Angeles, Los Angeles, CA 90095, USA. Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA 90095, USA. (2) Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA 90095, USA. Molecular Biology Institute, University of California, Los Angeles, Los Angeles, CA 90095, USA. (3) Department of Chemical and Biomolecular Engineering, University of California, Los Angeles, Los Angeles, CA 90095, USA. Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA 90095, USA. (4) Institute of Cellular and Organismic Biology, Academia Sinica, Taipei 115201, Taiwan. (5) Department of Obstetrics and Gynecology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA. Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, CA 90095, USA. (6) Department of Obstetrics and Gynecology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA. Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, CA 90095, USA. (7) Department of Obstetrics and Gynecology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA. Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, CA 90095, USA. (8) Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA 90095, USA. (9) Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA 90095, USA. (10) Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA 90095, USA. (11) Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA 90095, USA. (12) Department of Molecular and Medical Pharmacology, University of California, Los Angeles, Los Angeles, CA 90095, USA. (13) Department of Molecular and Medical Pharmacology, University of California, Los Angeles, Los Angeles, CA 90095, USA. (14) Molecular Biology Institute, University of California, Los Angeles, Los Angeles, CA 90095, USA. Department of Obstetrics and Gynecology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA. Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, CA 90095, USA. Department of Molecular and Medical Pharmacology, University of California, Los Angeles, Los Angeles, CA 90095, USA. VA Greater Los Angeles Healthcare System, Los Angeles, CA 90095, USA. Jonsson Comprehensive Cancer Center, University of California, Los Angeles, Los Angeles, CA 90095, USA. (15) Institute of Cellular and Organismic Biology, Academia Sinica, Taipei 115201, Taiwan. (16) Department of Chemical and Biomolecular Engineering, University of California, Los Angeles, Los Angeles, CA 90095, USA. Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA 90095, USA. Molecular Biology Institute, University of California, Los Angeles, Los Angeles, CA 90095, USA. Jonsson Comprehensive Cancer Center, University of California, Los Angeles, Los Angeles, CA 90095, USA. Parker Institute for Cancer Immunotherapy Center at UCLA, Los Angeles, CA 90095, USA.

NR2F6 deletion revives CAR-T cell function and induces antigen-agnostic immune memory in solid tumors Spotlight 

Dominik and Victoria et al. identified NR2F6 as a T cell-intrinsic metabolic checkpoint for CAR T cells in solid tumors. CRISPR/Cas9-mediated deletion of NR2F6 sustained a TCF1+ progenitor exhausted state and maintained metabolic fitness during chronic antigen stimulation. NR2F6 deletion in CAR T cells increased cytotoxicity, cytokine production, resistance to functional exhaustion, and tumor control in immunocompetent Panc02-EpCAM tumor models. DC-mediated cross-priming with epitope spreading and activation of endogenous immunity generated durable protection against CAR antigen-positive and -negative tumor rechallenge.

Contributed by Shishir Pant

Dominik and Victoria et al. identified NR2F6 as a T cell-intrinsic metabolic checkpoint for CAR T cells in solid tumors. CRISPR/Cas9-mediated deletion of NR2F6 sustained a TCF1+ progenitor exhausted state and maintained metabolic fitness during chronic antigen stimulation. NR2F6 deletion in CAR T cells increased cytotoxicity, cytokine production, resistance to functional exhaustion, and tumor control in immunocompetent Panc02-EpCAM tumor models. DC-mediated cross-priming with epitope spreading and activation of endogenous immunity generated durable protection against CAR antigen-positive and -negative tumor rechallenge.

Contributed by Shishir Pant

ABSTRACT: CAR-T cell therapy is effective in hematologic malignancies but remains challenging in solid tumors owing to antigen heterogeneity and tumor microenvironment-induced exhaustion. Here, gene editing of the nuclear receptor NR2F6 restores CAR-T cell functionality, sustaining a TCF1⁺ progenitor-exhausted phenotype, enhancing metabolic fitness, and preserving cytotoxic potency under chronic antigen exposure. In immunocompetent models, Nr2f6-deficient CAR-T cells suppress solid tumor growth and induce robust, polyclonal host antitumor responses that persist after CAR-T clearance, as demonstrated by tumor re-challenge protection. Although infused CAR-T cells disappear within 2 weeks, durable tumor control coincides with epitope spreading and secondary immune responses, likely via dendritic cell reactivation. Protection against antigen-negative tumors and transferable immunity reveal a dual mode of direct cytotoxicity followed by durable immune reprogramming. This broadened host immunity may offset immune escape driven by antigen heterogeneity or loss, establishing NR2F6 inhibition as a promising CAR-T engineering strategy for durable, antigen-agnostic solid-tumor immunotherapy.

Author Info: (1) Institute for Cell Genetics, Medical University of Innsbruck, Innsbruck, Austria. (2) Institute for Cell Genetics, Medical University of Innsbruck, Innsbruck, Austria. victoria

Author Info: (1) Institute for Cell Genetics, Medical University of Innsbruck, Innsbruck, Austria. (2) Institute for Cell Genetics, Medical University of Innsbruck, Innsbruck, Austria. victoria.klepsch@i-med.ac.at. (3) Biocenter, Institute of Bioinformatics, Medical University of Innsbruck, Innsbruck, Austria. (4) Institute for Cell Genetics, Medical University of Innsbruck, Innsbruck, Austria. (5) Institute for Cell Genetics, Medical University of Innsbruck, Innsbruck, Austria. (6) Institute for Cell Genetics, Medical University of Innsbruck, Innsbruck, Austria. (7) Institute for Cell Genetics, Medical University of Innsbruck, Innsbruck, Austria. (8) Institute for Cell Genetics, Medical University of Innsbruck, Innsbruck, Austria. Department of Internal Medicine V, Haematology & Oncology, Comprehensive Cancer Center Innsbruck and Tyrolean Cancer Research Institute, Medical University of Innsbruck, Innsbruck, Austria. (9) Institute for Cell Genetics, Medical University of Innsbruck, Innsbruck, Austria. (10) Institute for Cell Genetics, Medical University of Innsbruck, Innsbruck, Austria. (11) Institute for Cell Genetics, Medical University of Innsbruck, Innsbruck, Austria. Institute of Molecular Immunology, School of Medicine and Health, Technical University of Munich, Munich, Germany. (12) Tyrolean Cancer Research Institute, Innsbruck, Austria. Department of Therapeutic Radiology and Oncology, Medical University Innsbruck, Innsbruck, Austria. (13) Institute of Hygiene and Medical Microbiology Medical University of Innsbruck, Innsbruck, Austria. (14) Institute of Hygiene and Medical Microbiology Medical University of Innsbruck, Innsbruck, Austria. (15) Tyrolean Cancer Research Institute, Innsbruck, Austria. Department of Therapeutic Radiology and Oncology, Medical University Innsbruck, Innsbruck, Austria. (16) Biocenter, Institute of Bioinformatics, Medical University of Innsbruck, Innsbruck, Austria. (17) Department of Internal Medicine V, Haematology & Oncology, Comprehensive Cancer Center Innsbruck and Tyrolean Cancer Research Institute, Medical University of Innsbruck, Innsbruck, Austria. (18) Institute for Clinical Pharmacology, Klinikum der UniversitŠt MŸnchen, Munich, Germany. German Cancer Consortium, a partnership between LMU Hospital and the DKFZ, Munich, Germany. (19) Biocenter, Institute of Bioinformatics, Medical University of Innsbruck, Innsbruck, Austria. (20) Institute for Cell Genetics, Medical University of Innsbruck, Innsbruck, Austria. (21) Institute for Cell Genetics, Medical University of Innsbruck, Innsbruck, Austria. (22) Institute for Cell Genetics, Medical University of Innsbruck, Innsbruck, Austria. (23) Department of Internal Medicine V, Haematology & Oncology, Comprehensive Cancer Center Innsbruck and Tyrolean Cancer Research Institute, Medical University of Innsbruck, Innsbruck, Austria. (24) Institute for Cell Genetics, Medical University of Innsbruck, Innsbruck, Austria. gottfried.baier@i-med.ac.at.

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