Highlight
- Targeting oncofetal chondroitin sulfate (ofCS) with humanized C9-66 CAR-T cells offers tumor-specific immunotherapy in pancreatic ductal adenocarcinoma (PDAC).
- Charge optimization of CAR constructs reduces T cell exhaustion and enhances sustained antitumor activity in vivo.
- Metabolic reprogramming using inosine and Nr5a2 overexpression improves CAR-T cell functionality and mitochondrial fitness.
- Sequential tumor microenvironment (TME) modulation via GLP-1R, CSF-1R blockade, and PD-1 inhibition augments intratumoral CAR-T infiltration and prolongs survival.
Study Background
Pancreatic ductal adenocarcinoma (PDAC) stands among the deadliest malignancies owing to its aggressive nature and profound resistance to conventional therapies including chemotherapy, radiotherapy, and even most targeted therapies or checkpoint inhibitors. Despite incremental advances, the 5-year survival remains dismal, underscoring an unmet clinical need for more effective, tumor-selective immunotherapeutic strategies. One central challenge has been the lack of truly tumor-restricted antigens to direct immune effector cells against PDAC cells while sparing normal tissues.
Oncofetal chondroitin sulfate (ofCS) is a glycosaminoglycan epitope predominantly expressed in various solid tumors but minimally present in normal adult tissues, making it an appealing target for next-generation chimeric antigen receptor (CAR)-T cell therapies. Prior efforts at CAR-T cell therapy in solid tumors, including PDAC, suffered from limited tumor antigen specificity and the immunosuppressive tumor microenvironment that induces exhaustion and functional impairment in infused T cells.
Study Design
This study by Jiang et al. employed a multimodal engineering approach focused on optimizing C9-based CAR-T cells targeting ofCS in both murine and human PDAC models. The investigators first developed a humanized single-chain antibody fragment specifically binding ofCS and integrated it into CAR constructs. A charge-optimized variant, C9-66 CAR-T, was generated to improve T cell persistence and reduce exhaustion.
They examined key functional endpoints including cytotoxicity, cytokine production, memory differentiation, and metabolic fitness using preclinical murine PDAC and patient-derived xenograft PDAC models. Enhancement strategies integrated metabolic reprogramming with inosine supplementation, genetic overexpression of the nuclear receptor Nr5a2 to boost mitochondrial respiration, and sequential tumor microenvironment (TME) modulation combining GLP-1 receptor (GLP-1R) on/off agonism, colony stimulating factor-1 receptor (CSF-1R) blockade, and programmed cell death protein-1 (PD-1) checkpoint inhibition.
Key Findings
Potent Antitumor Efficacy and Survival Benefit: C9 CAR-T cells demonstrated marked cytotoxicity against PDAC cells, significantly delaying tumor growth and extending survival in treated mice compared to controls.
Improved T Cell Function with Charge Optimization: The charge-optimized C9-66 CAR-T cells exhibited reduced exhaustion markers and more durable antitumor activity in vivo. This modification appeared key to maintaining functional persistence within immunosuppressive TME conditions.
Metabolic Enhancements: Inosine supplementation enhanced cytokine production and favored differentiation into central-memory T cell subsets, mitigating functional exhaustion. Nr5a2 overexpression led to increased mitochondrial respiration and further enhanced cytotoxic function of CAR-T cells, suggesting improved metabolic fitness as a vital adjunct to CAR design.
Sequential TME Reprogramming Synergizes with CAR-T Activity: Modulating GLP-1R signaling in a phased manner coordinated with macrophage CSF-1R blockade and PD-1 checkpoint inhibition enhanced intratumoral CAR-T cell infiltration and functionality, translating into prolonged survival advantages in PDAC models.
Human Translational Relevance: Human C9-66 CAR-T cells maintained specific recognition of ofCS and effectively lysed patient-derived PDAC cells in vitro and in vivo, supporting clinical translatability.
Expert Commentary
This study represents a sophisticated example of integrating tumor antigen targeting with CAR-T cell engineering and tumor microenvironment modulation to tackle a notably challenging solid tumor. The utilization of oncofetal chondroitin sulfate as a tumor-restricted antigen leverages biological specificity critical for safety and efficacy. The enhancements in CAR-T cell metabolic programming—often an underappreciated factor—highlight the complex biology required to sustain T cell function within hostile tumor microenvironments.
The layered TME reprogramming combining GLP-1R modulation, macrophage targeting, and checkpoint inhibition reflects a holistic approach to overcoming multiple immunosuppressive mechanisms. This multimodal strategy aligns with emerging evidence that single immunotherapies rarely suffice in solid tumors without addressing stromal and suppressive niche factors. While promising, the translational leap to clinical application must carefully assess potential off-tumor toxicities, optimal dosing, and long-term persistence of engineered T cells.
Limitations include reliance on preclinical models, which although robust, may incompletely capture human disease complexity. Moreover, the metabolic and TME modulations require further refinement to balance efficacy with safety in humans.
Conclusion
The development of charge-optimized C9-66 CAR-T cells targeting oncofetal chondroitin sulfate with adjunctive metabolic and tumor microenvironment reprogramming provides a compelling and clinically translatable immunotherapeutic platform for pancreatic cancer. This multimodal approach overcomes major hurdles including poor antigen specificity, T cell exhaustion, and TME immunosuppression to demonstrate enhanced antitumor efficacy and survival benefits in preclinical models. Future clinical trials are warranted to validate safety and effectiveness in PDAC patients. Success could introduce a novel CAR-T paradigm for this deadly cancer and other solid tumors expressing ofCS.
Funding and Clinical Trials
The original study was supported by institutional and research grants acknowledged in the publication (Jiang et al., 2026). No specific clinical trials were yet registered as this remains a preclinical investigation.
References
- Jiang K, Lai CH, Dong S, et al. Multimodal C9-66 CAR-T cell immunotherapy improves outcome in preclinical models of pancreatic cancer. Gut. 2026 Jul 15. PMID: 42457620.
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