A groundbreaking study published in Nature Communications reveals a novel strategy to significantly enhance CAR-T cell therapy effectiveness against bladder cancer by modulating the PPAR-gamma pathway to upregulate NECTIN4 expression. The research, led by Chang, K., Delavan, H.M., Yip, E., and colleagues, addresses a critical challenge in solid tumor immunotherapy: the insufficient and heterogeneous expression of target antigens.
Engineering NECTIN4-Specific CAR-T Cells
The research team engineered multiple second-generation CAR constructs targeting NECTIN4, a cell adhesion molecule commonly overexpressed in bladder tumors. Using a previously validated EQ-28ζ CAR backbone containing an IgG4Fc(EQ) spacer, CD28 transmembrane domain, and CD28 costimulatory domain with CD3ζ signaling domain, they developed six different NECTIN4-binding scFv sequences.
The lead candidate, NECTIN4-CAR-1, demonstrated potent dose-dependent cytotoxicity against RT112 human bladder cancer cells across various effector-to-target ratios. Importantly, the CAR-T cells showed complete specificity, as they failed to kill NECTIN4 knockout cells generated using CRISPR/Cas9 technology. The researchers validated this specificity across multiple cell lines, including UMUC-9 and UMUC-3, confirming that NECTIN4-CAR-T cell activity directly correlates with target antigen expression levels.
PPAR-Gamma Pathway Controls NECTIN4 Expression
The investigators identified the peroxisome proliferator-activated receptor gamma (PPAR-γ) pathway as a key regulator of NECTIN4 expression. Treatment with thiazolidinedione drugs rosiglitazone and pioglitazone, both potent PPAR-γ agonists used for diabetes treatment, robustly increased NECTIN4 mRNA and surface protein expression across multiple bladder cancer cell lines.
Mechanistic studies revealed that PPAR-γ activation triggers transcriptional programs that enhance NECTIN4 gene transcription through direct promoter binding. Using RNA sequencing and chromatin immunoprecipitation assays, the team demonstrated that PPAR-γ binds to a predicted binding site upstream of the NECTIN4 transcriptional start site, leading to epigenetic changes that favor gene accessibility.
Conversely, treatment with T0070907, a PPAR-γ inverse agonist, decreased both NECTIN4 mRNA and protein levels. Genetic inhibition of PPARG using CRISPRi technology similarly reduced NECTIN4 expression, confirming the pathway's regulatory role.
Enhanced CAR-T Efficacy Through Tumor Priming
The researchers leveraged their discovery by pre-treating bladder cancer cells with rosiglitazone for 72 hours before CAR-T cell co-culture. This priming strategy significantly enhanced NECTIN4-CAR-T cell killing efficacy across multiple cell lines, including RT112, UMUC-1, and HT1197, particularly benefiting tumors with low to moderate NECTIN4 expression.
The enhanced cytotoxic activity was accompanied by increased interferon-gamma release, indicating stronger T cell activation. Importantly, rosiglitazone pre-treatment did not increase potential on-target, off-tumor toxicity, as NECTIN4-CAR-T cells remained inactive against normal skin keratinocytes and bladder epithelial cells despite their lower baseline NECTIN4 expression.
In Vivo Validation and Therapeutic Efficacy
In xenograft models using NOD scid gamma mice, systemic rosiglitazone administration for five days successfully increased NECTIN4 expression in RT112, HT1197, and UMUC-1 tumor xenografts. The combination of rosiglitazone pre-treatment followed by NECTIN4-CAR-T cell therapy demonstrated superior tumor growth inhibition compared to CAR-T cells alone.
Notably, the combination treatment improved overall survival without affecting body weight, suggesting minimal additional toxicity. The therapeutic benefit was observed across multiple tumor models and T cell donors, demonstrating the robustness and generalizability of the approach.
Overcoming Enfortumab Vedotin Resistance
A particularly significant finding addresses the clinical challenge of enfortumab vedotin (EV) resistance. The researchers generated EV-resistant RT112 cells through repeated exposure to escalating concentrations over nine months, resulting in a 10-fold higher IC50 compared to parental cells.
Crucially, EV-resistant cells maintained similar NECTIN4 expression levels and remained equally susceptible to NECTIN4-CAR-T cell killing. This finding was validated using both the original CAR construct and an enfortumab-derived NECTIN4-CAR, suggesting that antigen escape was not the primary resistance mechanism.
Clinical validation came from analyzing paired biopsies from bladder cancer patients treated with EV. Among 17 matched pre- and post-EV treatment samples, NECTIN4 expression was higher at the time of resistance in the majority of cases (9 of 17), supporting the potential for alternative NECTIN4-targeting strategies in the EV-resistant setting.
Clinical Translation Potential
The study's translational potential is enhanced by the use of clinically approved PPAR-γ agonists, which could accelerate the path to clinical trials by bypassing lengthy drug development timelines. The research team's strategy of transient rosiglitazone pulse treatment limits potential toxicities associated with long-term PPAR-γ agonist use, such as edema and cardiotoxicity.
The approach addresses fundamental challenges in solid tumor CAR-T therapy, including target antigen heterogeneity and immune evasion. By rendering NECTIN4 more uniformly expressed across tumor populations, PPAR-γ pathway modulation mitigates canonical resistance mechanisms that hamper immunotherapy success.
Broader Implications for Solid Tumor Immunotherapy
This work represents a paradigm shift from increasingly complex CAR designs toward strategic tumor cell modulation to enhance immune recognition. The convergence of metabolic signaling and immune surveillance opens new avenues for precision medicine approaches that combine molecular biology, immunology, and pharmacology.
The study also establishes NECTIN4 expression levels as potential dynamic biomarkers for monitoring therapeutic response and stratifying patients for personalized CAR-T therapy regimens. This adaptive biomarker model could facilitate real-time treatment adjustments to optimize clinical outcomes.
The research provides strong rationale for clinical translation of NECTIN4-CAR-T therapy in both frontline and EV-resistant settings, while guiding future combination strategies with other NECTIN4-targeting agents and immunomodulatory therapies.
