Adoptive T cell therapy using T cell receptor (TCR)-engineered T (TCR-T) cells shows promise for treatment of cancer. Our lab employs mRNA electroporation to redirect T cell specificity towards HBV antigens expressed on HBV-related HCC, while exploiting mRNA's transient nature to limit toxicity. Here we first demonstrated that PBMCs from solid cancer patients are inferior manufacturing sources compared to healthy donors, showing reduced manufacturability and lower naïve T cell frequencies. Since these limitations necessitate shifting to allogeneic "off-the-shelf" TCR-T products from healthy donors, we developed a non-gene-editing platform to produce such TCR-T cells. We addressed host-versus-graft rejection by combining transient tacrolimus-induced immunosuppression with immunosuppressive drug-resistant armored (IDRA) TCR-T cells. To reduce the risk of graft-versus-host disease, we optimized manufacturing using a IL-2/4/7 cytokine cocktail. The resulting TCR-T cells exhibited reduced alloreactivity, a less NK-like phenotype and elevated IL-4/IL-10 secretion, while maintaining robust antigen-specific cytotoxicity and migratory capacity. This study provides an in vitro proof-of-concept for a feasible, multi-layered strategy to produce allogeneic TCR-T cell therapies, warranting further preclinical validation.
G47Δ, a triple-mutated oncolytic herpes simplex virus type 1 (HSV-1) marketing-approved for malignant glioma, has demonstrated high efficacy including survival benefits via robust oncolytic activities with efficient antitumor immunity induction. G47Δ-based, oncolytic HSV-1 expressing fusion-type interleukin 12 (IL-12), murine (T-mfIL12) and human (T-hIL12), were preclinically evaluated for the treatment of malignant melanoma. In DBA/2 mice harboring syngeneic clone M-3 melanoma, intratumoral T-mfIL12 not only suppressed the tumor growth but also upregulated the immune mediator gene expressions in both the injected and non-injected remote tumors. The efficacy was further enhanced by combined treatment with systemic PD-1 blockade. For clinical translation, safety evaluations were conducted in HSV-1-sensitive A/J mice. The toxicity was primarily tested by injecting T-mfIL12 or T-hIL12 into the brain. Also, after administering the viruses intradermally at high doses, clinical signs, body weight, histopathology, cytokine responses, and viral biodistribution were examined. No significant adverse effects were observed, and viral DNA remained confined to the injection site. Intratumoral T-mfIL12 injection caused a transient increase in the serum IL-12 level, with limited effects on the systemic cytokine/chemokine profiles. Based on these data, an investigator-initiated phase I/II clinical trial of T-hIL12 in patients with advanced malignant melanoma is currently underway (jRCT2033190086).
Non-small cell lung cancers (NSCLCs) with inactivation of the tumor suppressor LKB1 are common and respond poorly to current therapies. We previously identified aberrant activation of the CRTC-CREB transcriptional pathway as a key driver of malignancy in LKB1-deficient lung cancer. In this study, we developed stabilized α-helical peptides, termed Stabilized Alpha-Helices of the CREB Binding Domain (SAH-CBDs), which were designed to disrupt the CRTC-CREB protein-protein interaction. SAH-CBD peptides adopt a stable, bioactive α-helical conformation, bind directly to CREB, and effectively block CRTC-CREB complex formation in vitro. Importantly, SAH-CBDs penetrate cells without causing membrane disruption, inhibit CREB-dependent transcription, and selectively suppress the growth of LKB1-null lung cancer cells, with minimal effects on LKB1-wild-type cells. These findings establish SAH-CBDs as a mechanistic tool and a prototype therapeutic for targeting a key transcriptional vulnerability in LKB1-inactivated NSCLC.
Oncolytic virotherapy employing Coxsackievirus B3 has emerged as a promising and innovative strategy for cancer treatment. However, wild-type Coxsackievirus B3 is associated with significant organ toxicities, such as viral hepatitis, myocarditis, and pancreatitis, which have presented major obstacles to its clinical application. In this study, we engineered a second-generation recombinant Coxsackievirus B3 (CVB3-BHP) by incorporating target sequences for the pancreas-specific miR-217 and the miR-34a, which is predominantly expressed in normal tissues, to further enhance its safety profile. Preclinical evaluation demonstrated that CVB3-BHP exhibited potent and dose-dependent antitumor efficacy in murine tumor models, with complete tumor regression or marked shrinkage observed in the high-dose group. Biodistribution analysis showed that CVB3-BHP transiently accumulated in the spleen and lacrimal gland but was rapidly cleared from these tissues over time, with limited excretion in urine and feces. Toxicity assessment indicated that even repeated high-dose administration did not induce overt systemic toxicity or organ damage. Although mild inflammation and acinar atrophy were observed in the lacrimal gland, no clinically significant adverse events were observed. Collectively, these findings indicate that CVB3-BHP combines robust antitumor efficacy with an improved safety profile, supporting its potential as a strong candidate for clinical translation in oncolytic virotherapy.
Chimeric antigen receptor (CAR) T cell therapy has transformed the treatment of hematologic malignancies, but its efficacy in solid tumors remains constrained by poor infiltration, metabolic stress, and limited persistence. Short-chain fatty acids (SCFAs), particularly butyrate and pentanoate, offer a way to influence CAR T metabolism and chromatin state during manufacturing. Butyrate combines class I histone deacetylase inhibition with acetyl-CoA metabolism and AMP-activated protein kinase (AMPK)-associated restraint of mTORC1, whereas pentanoate can reinforce effector programs through mTOR signaling and a distinct TCA-ATP-citrate lyase carbon-routing pathway. Direct CAR T studies and clinical associations now support the biological relevance of both metabolites, although their effects depend on dose, exposure schedule, cell composition, and experimental context. Building on their complementary actions, we propose sequential butyrate-pentanoate conditioning, with early butyrate exposure used to support oxidative and progenitor-associated features and later pentanoate exposure used to reinforce effector function. This review develops the mechanistic basis for that strategy, defines the experiments needed to distinguish cooperation from antagonism, and considers its manufacturing and translational implications.
Chimeric antigen receptor (CAR) therapies have shown great success in hematological malignancies but remain largely ineffective against solid tumors such as pancreatic ductal adenocarcinoma (PDAC). A key obstacle among various aspects, is the dense stromal barrier formed by cancer-associated fibroblasts (CAFs), providing a rationale for simultaneously targeting stroma and tumor cells. Using immunohistochemistry of primary PDAC tumors and liver metastases, we confirmed high mesothelin (MSLN) expression on tumor cells, and CD70 expression on tumor cells and predominantly CAFs. Based on these results and the favorable safety profile of CAR natural killer (NK) cells over CAR T cells, we generated MSLN- and CD70-targeting IL-15-armored CAR NK cells. Both constructs mediated cytotoxicity against different pancreatic cancer and CAF cell lines with varying antigen expression in vitro, demonstrating that both, the CAR-molecule and IL-15 were required to increase functionality against more treatment-resistant cell lines. Interestingly, pooled MSLN- and CD70-CAR NK cells did not significantly improve cytolysis compared to monotherapies in an advanced 3D in vitro model or in vivo. Together these findings highlight the limitations of dual-targeting approaches and underscore the need for advanced engineering strategies to improve CAR NK cells beyond antigen targeting and cytokine support in the PDAC microenvironment.
StagX1 {Ethyl 2-[[2-[2-[(2,3-Dihydro-1,4-benzodioxin-6-yl)amino]-2-oxoethyl]-1,2-dihydro-1-oxo-5-isoquinolinyl]oxy]propanoate} is a derivative of isoquinolinone, possessing an ethyl propionate. StagX1 exhibits growth-inhibitory activity in multiple Ewing sarcoma cell lines. To advance StagX1 as a potential lead, we conducted experiments to examine its metabolism and stability in tissue culture media, plasma, liver microsomes, cells and mice. Our studies demonstrate that StagX1 is metabolically unstable and undergoes rapid hydrolysis to its corresponding acid metabolite (StagX1-acid) through cleavage of the ethyl ester group. We identified carboxylesterase 1 (CES1) as the primary enzyme responsible for this conversion. Notably, cells expressing CES1 are sensitive to StagX1, whereas CES1-deficient cells show minimal response, indicating that metabolic activation is required for its activity. In contrast, StagX1-acid is metabolically stable. These findings suggest that StagX1 functions as a prodrug that is enzymatically converted to its active metabolite, StagX1-acid, within cells. This metabolic conversion likely underlies its mechanism of action and contributes to its selective anticancer activity in Ewing sarcoma. Our findings provide insight into the metabolism of StagX1 and the role of CES1 in mediating its effects and demonstrate that StagX1 is a promising compound with growth inhibitory effects in CES1 positive Ewing sarcoma cells.
Antigen (signal 1) is the ignition and fuel for T cell responses. Chimeric antigen receptors (CARs) co-opt T cell receptor (TCR) signaling domains (e.g., ITAM elements) to redirect T cell responses to specific antigens. Most efforts to enhance potency have added elements intended to preserve antigen dependence while boosting sensitivity, persistence, etc. However, solid tumors pose unique challenges compared to blood cancers. For example, access to tumor tissues that express target antigen is highly restricted by the blood vessel walls and, with limiting antigen, it is unclear how antigen-dependent boosters can be brought into action. Here, we describe a simple circuit that addresses this problem by mimicking an antigen stimulus with a small molecule to boost signaling downstream of the CAR. These signal 1 boosters are variants of the previously identified MyD88-CD40 fusion protein but mitigate the excessive antigen sensitization of this molecule. We identified a booster that, when expressed with CAR or Tmod, produces small-molecule-inducible T cell expansion and activation while maintaining a favorable safety profile in a surrogate normal-tissue mouse model.
Clinical efficacy of chimeric Antigen Receptor (CAR) T cell therapy is limited by tumor antigen escape and heterogeneity, leading to cancer relapse. To address this limitation, we developed a focused ultrasound (FUS) controllable and cis-activated CAR (FUS-CisCAR) platform, in which CAR T cells are engineered to express their own target antigen upon FUS stimulation. This self-supplied antigen triggers a ‘cis-activation’ loop, initiating a localized bystander effect to eliminate nearby tumor cells, including those that lack the target antigen. We demonstrate that primary human FUS-CisCAR T cells effectively kill local antigen-negative tumor cells in both in vitro and in vivo models. The FUS-CisCAR platform offers a controllable strategy that has the potential to overcome relapse caused by tumor heterogeneity and antigen escape, thus enhancing the durability of CAR T cell therapy.
Prostate cancer kills ≈350,000 yearly. While localized cases have 99% five-year survival, metastatic prostate cancer is hard to treat, with few options for tumors resistant to androgen deprivation. To leverage the immune system to fight prostate cancer, we developed a non-integrative lentiviral vector, namely "Lenti-PROST-02", which encodes clusters of T cell immunodominant regions of human prostatic acid phosphatase and prostate-specific antigen. Immunotherapy with Lenti-PROST-02 in a preclinical virulent prostate tumor model resulted in complete tumor eradication in vast majority of the treated animals. This antitumor effect was concomitant with induction of poly-functional CD8+ T splenocyte effectors against numerous T cell epitopes of the antigens encoded by Lenti-PROST-02, increased proportions of tumor-infiltrating CD8+ T cells with activated/differentiated/effector phenotype and a "cold-to-hot" inflammatory switch of the tumor microenvironment. Immunity induced by Lenti-PROST-02 was long-lasting and prevented tumor relapse. It was characterized by the persistence of CD44+ CD62L- CD127+ KLRG1- CD8+ memory T cells in secondary lymphoid organs, as well as an antigen-diversified memory response. Therefore, Lenti-PROST-02 therapeutic vaccine is a promising approach for prostate immuno-oncotherapy.
Glioblastoma multiforme (GBM) is a lethal malignancy with limited therapeutic options. Its aggressive progression and resistance to standard therapy necessitate the development of novel therapeutic strategies. Plant-based compounds have emerged as promising candidates for therapeutic development modulating key targets in cancer. The Ruta graveolens displays anti-inflammatory, analgesic, and antimicrobial properties. Herein, we report the anticancer potential of R. graveolens water extract (RGWE) on long-term cultures of human glioblastoma and elucidate the regulatory mechanisms driving its effect. Secondary stabilized cell cultures (U-87 MG, T98MG, and U-138 MG) and primary cell culture (FCN, MZC, and GL18-15) were either treated with RGWE and/or cis-diamminedichloroplatinum (cisplatin, CDDP). Cytostatic responses were assessed by cell viability (TB exclusion test), cell cycle (propidium iodide [PI] staining), and apoptosis (Annex-V and caspase-3) assays. We demonstrate that RGWE slows U-87 MG growth without affecting apoptosis and arrests the cell cycle in the G2/M phase. Mechanistically, RGWE interferes with PKC/MEK/ERK signaling pathway, as assessed by western blot analysis, through PKC inhibition in its active form. Furthermore, the combination of RGWE and cisplatin enables the use of a sublethal dose of the latter (0.2 μg/mL), thereby reducing cytotoxicity and the resistance to the drug. These findings reveal a novel mechanism by which RGWE controls glioblastoma growth, highlighting the therapeutic potential of targeting the PKC/MEK/ERK axis in glioma treatment.
Autologous T cells engineered to express a HER2 chimeric antigen receptor (CAR) have shown limited clinical efficacy. To broaden antitumor activity, we co-expressed a chimeric costimulatory receptor (CCR) targeting ICAM1 (ICCR) in HER2 CAR T cells. ICAM1 is upregulated by inflammatory cytokines, reinforces immune-synapse formation, and is elevated in aggressive or dedifferentiated tumors, including those with heterogeneous or low HER2 expression. HER2 CAR/ICCR T cells preserved potent cytotoxicity against HER2-high targets and showed significantly enhanced killing of HER2-low cell lines compared with HER2 CAR T cells alone. ICCR augmented NF-κB activation particularly under low HER2 conditions. Upon repeated stimulation with HER2-negative tumor cells, HER2 CAR/ICCR T cells underwent nearly 10-fold expansion with superior cytotoxicity, whereas HER2 CAR T cells failed to expand. In vivo, both constructs rapidly eliminated HER2-high tumors, but only HER2 CAR/ICCR T cells achieved complete clearance of HER2-low tumors, with delayed kinetics consistent with clonally expanded, tumor-specific T cells. TCR sequencing confirmed substantially greater clonal expansion in HER2 CAR/ICCR T cells across in vivo and in vitro studies. This dual-receptor platform (HMJ01) has supported IND authorization for a first-in-human trial in patients with HER2-positive and HER2-low gastric cancer.
