Molecular Therapy Oncology

Oncolytic viruses are most commonly administered via intratumoral injection; however, their clinical efficacy in achieving tumor eradication remains limited by several challenges, including insufficient penetration into all tumor cells and the inability to elicit robust systemic antitumor immune responses capable of eliminating metastatic microtumors. Here, we report an oncolytic adenovirus, OAd-2B6, with an engineered adenoviral E1 region for tumor selectivity and carrying the prodrug- activating enzyme cytochrome P450 2B6 (CYP2B6) to activate the anticancer prodrug cyclophosphamide (Cytoxan, CTX). OAd-2B6 alone induced dose-dependent tumor cell killing across multiple human tumor cell lines and exhibited strong synergistic antitumor effects when combined with CTX. Importantly, OAd-2B6-mediated local activation of CTX resulted in a potent bystander killing effect that eliminated tumor cells not directly infected by the virus. In a H1299 lung cancer xenograft nude mouse model, intratumoral injection of OAd-2B6 combined with CTX significantly inhibited tumor growth and even achieved complete tumor regression, with markedly superior efficacy compared with monotherapy. In immunocompetent mice bearing 4T1 breast cancer xenografts, OAd-2B6 alone inhibited tumor growth and was accompanied by upregulation of IFN-{gamma} and GzmB expression in the tumor-infiltrated T cells. CTX combination therapy further enhances this anti-tumor immune response, promoting the activation of T cells to suppress non-injected tumors at a distal site. Collectively, this study demonstrates that OAd-2B6 exerts potent antitumor effects through multiple mechanisms, including direct oncolysis, intratumoral prodrug activation leading to bystander killing, and enhancement of systemic antitumor immunity. These findings provide a promising strategy for improving the therapeutic efficacy of oncolytic therapy.

Oncolytic virotherapy exploits viruses to selectively infect and destroy cancer cells while sparing normal tissues and represents a promising strategy in oncology. Human adenovirus type 5 (HAd5), although widely used, shows limited clinical efficacy due to high levels of preexisting immunity and suboptimal tumor selectivity. In this study, we evaluated novel gorilla-derived adenoviruses (GRAd) as alternative oncolytic vectors. Two distinct GRAd groups, GRAdBs and GRAdCs, were characterized for replication and cytopathic activity. GRAd25 (GRAdB group) exhibited robust replication in both tumor and normal cells, whereas GRAd32 (GRAdC group) demonstrated selective replication in tumor cells. To broaden tumor tropism while preserving selectivity, we generated a chimeric GRAd32 vector, GRAd32Fk25, by replacing its native fiber knob with that of GRAd25, potentially shifting receptor usage from CAR to CD46, which is more abundantly expressed in tumor cells. The vector was further armed with a therapeutic antibody by inserting the coding sequence for the single-chain Fc form (scFv-Fc) of EV20, a humanized anti-HER3 antibody, under endogenous viral regulatory control. In vitro analyses showed that GRAd32Fk25 maintained tumor-restricted replication and produced functional EV20 capable of binding HER3 and inhibiting downstream PI3K/Akt signaling. These results indicate that engineered GRAd vectors, exemplified by GRAd32Fk25 armed with EV20, provide a selective and versatile platform for oncolytic virotherapy with potential advantages over HAd5-based approaches.

Viroimmunotherapy leverages oncolytic viruses to induce antitumor immunity and is increasingly explored for solid tumors. Their activity can be enhanced by arming them with immunostimulatory payloads, but most approaches rely on protein-based transgenes that are constrained by viral genome packaging limits. Here, we establish a replication-competent Delta-24-RGD-based platform for localized production of immunotherapeutic RNA aptamers at the tumor site. RNA aptamers provide compact, highly specific ligands that can, in principle, target diverse immune receptors. As a model, we engineered a Delta-24-RGD derivative encoding circular 4-1BB targeting aptamers and show that infected tumor cells sustain aptamer transcription and release, which is associated with a pro-inflammatory remodeling of the tumor microenvironment and measurable antitumor activity in different mouse models with a comparable effect to that achieved with a 4-1BBL-expressing adenovirus used as a benchmark. Overall, this work delivers a proof of concept that replication-competent adenoviruses can serve as in situ factories for extracellularly active RNA aptamers, supporting their development as flexible platforms for localized non-coding cancer immunotherapy.

Oncolytic adenovirus (ADV) therapy faces heterogeneous responses, implying tumor-intrinsic resistance. We identify interleukin-17 (IL-17) signaling as a novel potential barrier associated with multi-modal cellular reprogramming. Transcriptomic analysis of ADV-treated 4T1 murine mammary carcinoma cells revealed specific upregulation of Il17rb, Il17rd, and Il17f, indicating viral induction of this inflammatory axis. The IL-17 signature correlates strongly with a cancer stemness phenotype. Metabolically, it associates with increased lipid metabolism and suppressed glycolysis, suggesting a state resistant to viral replication. Furthermore, it broadly negatively correlates with programmed cell death pathways (apoptosis, necrosis) while positively associating with pro-survival autophagy. IL-17 component expression effectively stratifies samples into distinct metastatic risk categories, underscoring its prognostic potential. Our findings reveal a previously unrecognized, tumor-intrinsic role for IL-17 signaling in ADV resistance, associated with enhanced stemness, altered metabolism, and impaired cell death. This nominates the IL-17 pathway as both a predictive biomarker and a therapeutic target for combination strategies. HighlightsO_LIOncolytic adenovirus infection selectively upregulates IL-17 receptor subunits (IL17RB, IL17RD) and IL17F ligand in 4T1 tumor cells C_LIO_LIIL-17 receptor expression strongly correlates with cancer stemness gene signatures, particularly through IL17RB and IL17RD C_LIO_LIThe IL-17 axis associates with broad suppression of lytic cell death pathways (apoptosis, necrosis, necroptosis) while positively correlating with autophagy C_LIO_LIIL-17 pathway activity correlates with metabolic reprogramming favoring lipid turnover over glycolysis C_LIO_LIIL-17 expression levels stratify samples into distinct metastatic risk categories, suggesting biomarker potential C_LI

Despite the clinical success of cancer immunotherapies, their efficacy is often compromised by antigen-related problems, including heterogeneity, downregulation, loss, and off-tumor toxicity. To overcome these limitations that challenge the current immunotherapies dependent on native antigens, we here describe a new cancer immunotherapy strategy, which artificially and specifically expresses a clinical validated antigen on variant tumors and thus repurposes clinical antibody drugs to treat cancers not belonging to their indications. To authenticate the strategy, we delivered a CD20 gene under a control of NF-{kappa}B-specific promoter to tumors by adeno-associated virus and then treated them with a CD20 antibody, rituximab. We found that CD20 was selectively expressed in tumors and the followed rituximab treatment activated natural killer (NK) cell to kill cancer cells by antibody-dependent cellular cytotoxicity. We demonstrated that this strategy is effective not only in variant cultivated cancer cells, HCT116 spheroids, and patient-derived organoids of human colorectal cancer, but also in humanized mouse with HCT116 xenograft and immunocompetent mouse with CT26 transplant. The strategy showed high cancer cell specificity in both in vitro and in vivo treatments, leading to high security in animal treatments. This strategy thus creates a new modality of cancer immune-redirection therapy by repurposing the clinical validated both antigen and antibody.

Hepatotoxicity poses a critical safety challenge for AAV-mediated gene therapy. To mitigate this, we evaluated strategies to minimize off-target hepatic transduction using an antibody expression model. We compared (i) muscle-restricted wild-type AAV9 expression and (ii) a novel myotropic capsid variant, AAV.eM. In humanized B-NDG mice bearing Raji-Luc lymphomas, intravenous administration of AAV9-MHCK7 encoding an CD19CD3 bispecific T-cell engager failed to reduce tumor burden. Conversely AAV.eM-MHCK7-CD19CD3 substantially alleviated tumor burden and achieved lymphoma clearance. By leveraging tissue-specific microRNAs, precise restriction of AAV.eM-mediated transgene expression to skeletal or cardiac muscle was achieved. Incorporating a heart-specific miR-208a binding site into the transgenes 3UTR did not compromise therapeutic efficacy when delivered via AAV.eM-MHCK7. Intramuscular delivery of AAV9-MHCK7-CD19CD3 or AAV.eM-MHCK7-CD19CD3 both cleared Raji-Luc tumors at a dose of 5 x 1012 vg/kg, underscoring the advantage of localized and targeted rAAV delivery over systemic administration. Notably, only AAV.eM-MHCK7-CD19CD3 achieved tumor eradication at a tenfold lower intramuscular dose (5 x 1011 vg/kg), reducing manufacturing costs and risks of dose-dependent immunogenicity and toxicity. Our findings demonstrate that combining tissue-specific targeting--via engineered capsids or tissue-selective promoters--with local delivery robustly reduces off-target hepatic expression, providing a strategic framework for enhancing the safety of AAV-based gene therapies.

Rhabdomyosarcoma (RMS), the most common pediatric soft tissue sarcoma, shows dismal survival in relapsed or metastatic alveolar disease. Chimeric antigen receptor (CAR) T cells are promising but limited by scarce tumor-selective antigens and suboptimal efficacy at low antigen density. We investigated L1 cell adhesion molecule (L1CAM) as a therapeutic target by profiling its expression by flow cytometry, immunoblotting, and immunohistochemistry in cell lines, patient-derived xenografts, and healthy tissues. Using the scFv derived from the CE7 antibody, we engineered L1CAM-CARs with distinct hinge and costimulatory domains and tested them in vitro and in orthotopic RMS mouse models against clinically tested CE7- and B7-H3-CARs. L1CAM was consistently expressed at moderate levels in RMS, especially alveolar subtypes, but very weakly expressed in healthy tissues. Flow cytometry revealed a moderate density typically limiting CAR activity. Among constructs, L1CAM.III (CE7-CAR with long hinge and CD28 domain) showed the strongest cytotoxicity and IFN-{gamma} release. In vivo, L1CAM.III-CAR T cells regressed tumors, prolonged survival, and persisted in orthotopic RMS models, showing greater efficacy in alveolar RMS and no off-tumor activity. These findings establish L1CAM as a rational RMS therapeutic target. Optimized L1CAM.III-CAR T cells overcome moderate antigen density, achieving potent and persistent antitumor activity comparable to B7-H3-CARs but with improved safety. This work supports CAR optimization for clinical translation to broaden pediatric sarcoma immunotherapy.

For mRNA-based cancer gene therapy, we engineered a membrane-bound fusion protein combining interferon-{gamma} (IFN{gamma}) with the Fas intracellular domain (FasICD) to couple local IFN{gamma} signaling with Fas-driven apoptotic tumor cell death. IFN{gamma}-FasICD was robustly expressed on the plasma membrane after mRNA transfection. In murine cancer cell lines, IFN{gamma}-FasICD mRNA reduced viability within 24 h, resulting in [~]50% cell death in MC38 cells and [~]75% in B16OVA cells, exceeding the cytotoxicity of the FasICD-deleted control (IFN{gamma}-Fas{Delta}). Mechanistically, IFN{gamma}-FasICD induced predominantly apoptotic rather than necrotic cell death. IFN{gamma}-FasICD also activated IFN{gamma} receptor signaling in both cancer and the immune cells, inducing IFN{gamma}-responsive genes in IFN{gamma}R-high B16OVA cells and triggering STAT1 phosphorylation in co-cultured splenocytes. For in vivo delivery, IFN{gamma}-FasICD mRNA was formulated in lipid nanoparticles (LNPs), enabling strong intratumoral expression that peaked at [~]3 h and persisted for more than 48 h. Repeated intratumoral injections of LNP-formulated IFN{gamma}-FasICD mRNA suppressed the growth of established B16OVA and MC38 tumors and improved survival, with [~]40% and [~]20% of mice surviving beyond 30 days, respectively. IFN{gamma}-FasICD treatment remodeled the tumor microenvironment by increasing tumor-infiltrating CD45+ cells and CD8+ T cells, while further reducing FOXP3+ regulatory T cells. Moreover, NK/NKT cells and cDC1/cDC2 populations were increased, and their activation was enhanced. In tumor-draining lymph nodes, IFN{gamma}-FasICD mRNA promoted dendritic cell migration and increased priming and differentiation of CD8+ T cells toward effector and memory phenotypes, accompanied by enhanced functional activation of IFN{gamma}-producing CD8+ T cells and highly cytotoxic NK cells in peripheral blood. Overall, our findings provide a mechanistic foundation for cytokine-death receptor fusion proteins as an in vivo antitumor strategy that can reprogram tumor cells into localized sources of both apoptotic signals and immune-activating cues.

Recent clinical trials show that CAR-T cell therapies can initially blunt tumor growth in glioblastoma (GBM) patients. However, the tumor microenvironment activates mechanisms that inhibit tumor-killing potential of the CAR-T cells and limit their therapeutic efficacy. To counteract this, we have utilized oncolytic adenovirus (OV) Ad5-{Delta}24-RGD as a platform to overexpress a bispecific T cell engager (BiTE) targeting both T cell marker CD3 and GBM specific tumor associated antigen IL-13R2. We first demonstrated that OV-BiTE could enhance recruitment of T cells to GBM in vitro and in vivo. We then showed that intratumoral injection of OV-BiTE followed by infusion of combined EGFR- and EGFRvIII-CAR-T cells was more effective than OV-BiTE supplemented with either CAR-T therapy alone, and led to significant tumor eradication in a GBM xenograft mouse model. In conclusion, our multimodal OV-BiTE & CAR-T cell immunotherapy is capable of overcoming immunosuppressive tumor microenvironment and GBM resistance to treatment. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=160 SRC="FIGDIR/small/667708v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@c55b15org.highwire.dtl.DTLVardef@deffb2org.highwire.dtl.DTLVardef@64ff90org.highwire.dtl.DTLVardef@c65fc2_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSO_LIOncolytic adenovirus encoding bispecific T cell engager (OV-BiTE) combines two immunotherapeutic agents into one. C_LIO_LIOV-BiTE strategy modifies tumor microenvironment and enhances recruitment of T cells to glioblastoma (GBM) in vitro and in vivo. C_LIO_LIMultimodal OV-BiTE & CAR-T cell immunotherapy effectively reduced tumor mass in a GBM xenograft mouse model and is superior to either immunotherapy alone. C_LI

Oncolytic viruses (OVs) are promising cancer therapeutics that selectively infect and lyse tumor cells while sparing normal tissues and stimulating antitumor immunity. However, their efficacy remains limited by suboptimal cytolytic activity and insufficient immune stimulation, highlighting the need for improved designs. Here, we introduce a synthetic virology platform leveraging transformation-associated recombination (TAR) in yeast to generate infectious chimeric poxviruses with enhanced therapeutic potential. Using TAR, we first cloned the Vaccinia virus (VACV) genome into a yeast plasmid and rescued it in human cancer cells. This plasmid was then co-transformed with Cowpox virus (CPXV) and Rabbitpox virus (RPXV) genomic DNA to promote recombination and create chimeric constructs. Subsequent rescue with Modified Vaccinia virus Ankara (MVA) yielded five infectious chimeric viruses. Phenotypic characterization revealed diverse plaque morphologies, comet-like spreading, and variable oncolytic activity across multiple cancer cell lines, indicating functional diversity arising from genome shuffling. Whole-genome sequencing confirmed recombination between VACV, CPXV, RPXV, and MVA. This study represents the first demonstration of TAR cloning for chimeric virus generation, establishing a versatile platform for designing next-generation oncolytic viruses.

Glioblastoma (GBM) remains one of the most challenging and lethal brain cancers, with limited treatment options. While CAR-T cells have shown promise in some patients, sustaining T-cell activity and overcoming the immunosuppressive tumour microenvironment (TME) remain significant hurdles. Here, we present an armoured CAR-T cell design to address these challenges and enhance persistence in GBM tumours. We developed a highly specific humanised single-domain antibody (VHH) targeting IL13R2 and included it alongside four additional modular elements in a single retroviral vector for CAR-T generation. Our results demonstrate that this single-cassette CAR-T cell design possesses high resilience against TGF-{beta}-mediated immunosuppression, enhanced tumour-killing capacity through IL-12 secretion while maintaining a favourable safety profile, extended persistence in the host, and an additional layer of safety control through the incorporation of a suicide switch. Importantly, despite its complexity, the construct can still be manufactured efficiently. These advancements represent a significant step forward in addressing key challenges associated with CAR-T cell therapy in solid tumours.

Adeno-associated virus (AAV) has found immense success as a delivery system for gene therapy, yet the small 4.7 kb packaging capacity of the AAV sharply limits the scope of its application. In addition, high doses of AAV are frequently required to facilitate therapeutic effects, leading to acute toxicity issues. While dual and triple AAV approaches have been developed to mitigate the packaging capacity problem, these necessitate even higher doses to ensure that co-infection occurs at sufficient frequency. To address these challenges, we herein describe a novel delivery system consisting of adenovirus (Ad) covalently linked to multiple adeno-associated virus capsids as a new way of more efficiently co-infecting cells with lower overall amounts of AAVs. We utilize the DogTag-DogCatcher (DgT-DgC) molecular glue system to construct our AdAAVs and we demonstrate that these hybrid virus complexes achieve enhanced co-transduction of cultured cells, including physiologically relevant primary cells. On this basis, AdAAV technology may eventually facilitate therapeutic co-delivery of multiple transgenes at low virus doses for treating complex ailments.

A multitude of tools now exist that allow us to precisely manipulate the human genome in a myriad of different ways. However, successful delivery of these tools to the cells of human patients remains a major barrier to their clinical implementation. Here we introduce a new cellular approach for in vivo genetic engineering, Secreted Particle Information Transfer (SPIT) that utilizes human cells as delivery vectors for in vivo genetic engineering. We demonstrate the application of SPIT for cell-cell delivery of Cre recombinase and CRISPR-Cas9 enzymes, we show that genetic logic can be incorporated into SPIT and present the first demonstration of human cells as a delivery platform for in vivo genetic engineering in immunocompetent mice. We successfully applied SPIT to genetically modify multiple organs and tissue stem cells in vivo including the liver, spleen, intestines, peripheral blood, and bone marrow. We anticipate that by harnessing the large packaging capacity of a human cells nucleus, the ability of human cells to engraft into patients long term and the capacity of human cells for complex genetic programming, that SPIT will become a paradigm shifting approach for in vivo genetic engineering.

BackgroundCD19-targeted chimeric antigen receptor therapies (CAR19) have driven a paradigm shift in the treatment of relapsed/refractory B-cell malignancies. However, >50% of CAR19-treated patients experienced progressive disease mainly due to antigen escape and low persistence. Clinical prognosis is heavily influenced by CAR-T cell function and systemic cytokine toxicities. Furthermore, it remains a challenge to efficiently, cost-effectively, and consistently manufacture clinically relevant number of virally engineered CAR-T cells. MethodsUsing a highly efficient piggyBac transposon-based vector, Quantum pBac, we developed a virus-free cell engineering system, Quantum CART (qCART), for development and production of multiplex CAR-T therapies. ResultsHere, we demonstrated in vitro and in vivo that consistent, robust, and functional CD20/CD19 dual-targeted CAR-T stem cell memory (TSCM) cells can be efficiently manufactured using the qCART system for clinical application. qCART-manufactured CAR-T cells from cancer patients expanded efficiently, rapidly eradicated tumors, and can be safely controlled via an iCasp9 suicide gene-inducing drug. ConclusionsThe qCART system is an elegant system for the manufacturing of CAR-T products having all the desired CAR-T therapy attributes. We believe that the simplicity of manufacturing multiplex CAR-T cells using the qCART system will not only significantly enhance the accessibility of CAR-T therapy but also unlock the full potential of armored CAR-T therapy for the treatment of solid tumors in the future. What is already known on this topicDespite the considerable success which has been achieved with CD19-targeted chimeric antigen receptor therapies (CAR19), >50% of CAR19-treated patients still experienced progressive disease. Therefore, there is a need to further improve CAR19 therapies. Current CAR19 therapies commonly utilize virus-based cell engineering methods. CAR-T production using these methods face multiple hurdles, including difficulties to efficiently, cost-effectively, and consistently manufacture clinically relevant number of CAR-T cells. We have previously used a highly efficient piggyBac transposon-based vector, Quantum pBac, to establish Quantum CART (qCART) which is a virus-free cell engineering system for development and production of multiplex CAR-T therapies. What this study addsIn this report, we further demonstrate in vitro and in vivo that consistent, robust, and functional iCasp9-regulatable, CD20/CD19 dual-targeted CAR-T stem cell memory (TSCM) cells can be efficiently manufactured using the qCART system for clinical application. These cells possess all the desired attributes for ensuring therapeutic efficacy in CAR-T therapy, including high CAR-TSCM, balanced CD8/CD4 ratio, low exhaustion and senescence marker expressions, and high ex vivo and in vivo expansion capacity. Importantly, we show that qCART-manufactured CAR-T cells from hematological cancer patients expanded efficiently, effectively eradicated tumors, and can be safely controlled via an iCasp9 suicide gene-inducing drug. We believe that the simplicity of manufacturing multiplex CAR-T cells using the qCART system will not only significantly enhance the accessibility of CAR-T therapy but also unlock the full potential of armored CAR-T therapy for the treatment of solid tumors in the future. How this study might affect research, practice or policyOur findings demonstrate that qCART is a virus-free CAR-T engineering system for manufacturing CAR-TSCM cells from either healthy donors or hematological cancer patients, that possess all the desired attributes for a successful CAR-T therapy. These cells expanded efficiently, rapidly eradicated tumors, and can be safely controlled via activation of iCasp9. We expect that this simple yet robust system for manufacturing multiplex CAR-T cells will advance the CAR-T field.

Cancer treatment remains challenging due to heterogeneous responses to immunotherapy across patients and tumor types. Innovative strategies are required to overcome immune evasion. We have identified the splicing factor SLU7 as essential for the survival of cancer cells from diverse origins. SLU7 knockdown induces R-loop accumulation, transcription-dependent genomic instability, DNA damage, and replication catastrophe, together with aberrant splicing and inhibition of nonsense-mediated mRNA decay (NMD) and/or DNA methylation. These alterations lead to the expression of neoantigens, interferon B1, endogenous retroviruses, and cancer-testis antigens, which would enhance tumor immunogenicity. Therefore, we propose SLU7 targeting as a dual-action therapy, combining direct tumor suppression with immune activation. Using various murine cancer models, including orthotopic liver tumors, and multiple molecular strategies--such as inducible CRISPR/Cas9, systemic delivery of chimeric siSLU7-nucleolin aptamers (APTASLU), and intratumoral injection of siSLU7-loaded nanoparticles--we show that distinct siSLU7 sequences and delivery platforms effectively inhibit tumor growth. Furthermore, SLU7 silencing synergizes with immune checkpoint inhibitors, amplifying anti-tumor responses. Our in vivo data demonstrate that SLU7 is a promising, versatile target for diverse cancers. Its multimodal mechanism offers potential to overcome tumor heterogeneity, reverse immune tolerance, and enhance immunotherapy efficacy.

Fibroblast activation protein (FAP) is an attractive target for the development of cancer theranostics due to its selective expression on cancer-associated fibroblasts (CAFs). While a number of small-molecule FAP inhibitors (FAPIs) have been developed, few biologics have been investigated as FAP targeting vectors. Camelid-derived single-domain antibodies, or variable-heavy-heavy domains (VHHs), offer a compelling alternative, combining high affinity with versatile engineering options. In this study, we first identified a novel anti-FAP VHH, F7, from an affinity-matured camelid phage display library. To investigate how valency and molecular weight affected target engagement and in vivo properties, F7 was engineered into three formats: a monomer (F7), a tethered dimer (F7D), and an Fc-fusion protein (F7-Fc). All three were specific for FAP with the two bivalent constructs demonstrating picomolar affinity. Positron emission tomography imaging in FAP-positive xenograft models revealed distinct pharmacokinetic profiles across constructs with notable differences in tumor uptake and clearance. F7 had rapid uptake and clearance resulting in significantly higher tumor uptake than FAPI-46. Low molecular weight bivalent F7D demonstrated similar kinetics but was retained by the tumor resulting in a high tumor-to-blood ratio with secondary uptake limited to clearance organs. The largest construct, F7-Fc, resulted in the highest tumor uptake and allowed for longitudinal imaging. Absorbed dose calculations confirmed that tumors received significantly higher radiation doses compared to normal tissues. These findings demonstrate that tuning VHH scaffold size and valency can improve biodistribution and retention, establishing F7-based constructs as promising targeting vectors for FAP.

Cancer immunotherapy has made significant advancements over the past few decades, with immune checkpoint and cytokine-based drugs being successfully implemented in clinical settings. Nonetheless, the effective and safe clinical application of these therapies is hindered by critical issues, such as severe toxicity to healthy tissues due to on-target off-tumor effects. In this study, we have developed a novel immunogene therapy characterized by high tumor selectivity and safety in vivo, effectively mitigating the off-tumor effects associated with current antibody-based immune checkpoint therapies. We engineered a gene expression vector that is specifically activated by NF-{kappa}B activity to co-express artificial microRNAs targeting two key immune checkpoints (PD-L1 and CD47) and cytokine IL-15. This vector is capable of selectively and effectively down regulating the expression of PDL1 and CD47 while over expressing IL-15 just exclusively in cancer cells, both in vitro and in vivo. Through this mechanism, both adaptive and innate immune responses can be simultaneously activated and enhanced via the transfection of this vector. The in vivo administration of this vector via recombinant adeno-associated virus (AAV) demonstrated significant antitumor activity, high tumor selectivity, and safety in murine models. Consequently, this vector may offer a potential more effective and safer alternative to the current immune checkpoint inhibitors in future clinical applications.

Chimeric antigen receptor (CAR)-T cell therapies have achieved remarkable success in treating hematologic malignancies, yet their clinical utility remains limited by safety concerns, poor persistence, and T-cell exhaustion driven by continuous receptor signaling. Although switchable CAR systems offer external control, most existing designs are irreversible, binary, or compromising CAR-T potency. Here, we introduce a chemically switchable CAR platform that enables graded, reversible regulation of CAR-T activity while retaining full therapeutic capacity. Using engineered CAR-T cells, we evaluate drug-controlled activation, cytotoxicity, and cytokine release against CD19 tumor cells and screened clinically approved NS3/4A inhibitors to identify optimal small-molecule controllers. Compared with conventional CAR-T cells, switchable CAR-T cells exhibited minimal background activity in the OFF state, preventing antigen-driven activation and cytokine release in the absence of drug. Upon drug addition, CAR expression was rapidly restored, with full-length CAR detectable within 1 hour and [~]80% of maximal expression achieved by 4 hours. Reversible suppression of CAR expression protected normal CD19 B cells once malignant cells were eliminated, addressing the clinical challenge of persistent CD19 CAR-T activity that can lead to B-cell aplasia, hypogammaglobulinemia, and recurrent infections. Furthermore, switchable CAR-T cells displayed reduced exhaustion, enhanced persistence, stable CAR expression, and preferential central memory differentiation following tumor clearance. Together, these findings establish the switchable CAR-T system as a next-generation, reversible, and clinically compatible CAR-T platform. Key PointsO_LIOptimized switchable CAR enables precise control of functional CAR expression, T-cell activation, cytokine release, and cytotoxicity. C_LIO_LIExternal regulation of CAR-T cells enhances safety and promotes sustained persistence in chronic stimulation models. C_LI

Chimeric antigen receptor T (CAR-T) cell therapy has the potential to transform cancer treatment. However, CAR-T therapy application is currently limited to certain types of relapsed/refractory B cell lymphomas. To unlock the full potential of CAR-T therapy, technologic breakthroughs will be needed in multiple areas, including optimization of autologous CAR-T development, shortening the innovation cycle, and further manufacturing advancement of next-generation CAR-T therapies. Here, we established a simple and robust virus-free multiplex Quantum CART system that seamlessly and synergistically integrates four platforms: 1. GTailor for rapid identification of lead CAR construct design, 2. Quantum Nufect for effective but gentle electroporation-based gene delivery, 3. Quantum pBac, featuring a virus-free transposon-based vector with large payload capacity and integration profile similar to retrovirus, and 4. iCellar for robust and high-quality CAR+ T memory stem cell expansion. This robust, virus-free multiplex Quantum CART system is expected to unleash the full potential of CAR-T therapy for treating diseases.

Chimeric antigen receptor (CAR)-modified V{delta}2 T cells are an attractive therapeutic cell platform for cancer immunotherapy. However, their clinical efficacy is limited by short in vivo persistence due to insufficient cytokine support and high susceptibility to activation-induced cell death (AICD). Through comparison of membrane-bound (mb) cytokines, we identified mbIL-18 to support superior anti-tumor activity of CAR-V{delta}2 T cells in vitro and in vivo. To reduce constitutive surface exposure of IL-18 and enable antigen-driven signal 3, we fused MyD88 - the key IL-18R signaling mediator - to an extracellular domain of Fas (Fas88). Antigen stimulation-induced FasL engagement of Fas88 triggered IL-18 signaling while simultaneously protecting V{delta}2 T cells from AICD. Fas88-armed human CAR-V{delta}2 T cells produced superior yet stimulation-dependent in vivo expansion and functional persistence in xenograft models of hematologic and solid malignancies. Together, these findings highlight the importance of IL-18 signaling and AICD resistance for CAR-V{delta}2 T cell activity, enabling a single-transgene modification to limit inflammatory risk and facilitate clinical translation.