1033P - Anti-tumor efficacy and safety of conditionally activated armored CAR-T cells against gastrointestinal tumors

Background

Immunotherapies against tumor-associated antigens (TAAs) often lead to on-target off-tumor effects. To address this problem, we developed conditionally activated chimeric antigen receptor (CAR)-T cells in hypoxic tumors targeting epithelial glycoprotein-1, which is overexpressed in gastrointestinal tumors compared to normal tissues. The CAR-T cells were further armored with the dominant-negative TGFβRII (dnTGFβRII), which exhibited improved safety and effective killing against a broad spectrum of gastrointestinal tumors in vivo.

Methods

The prototype conditionally activated CAR, BTRP003H, a 2^nd-generation CAR bearing an oxygen-dependent degradation domain of HIF1a was modified with various hypoxia-responsive elements (HREs) to establish BTRP003I, BTRP003K, and BTRP003L. T cells isolated from human peripheral blood mononuclear cells were stimulated with anti-CD3/CD28 beads, lentivirally transduced, and expanded ex vivo. Expression of CAR and exhaustion markers were assessed by flow cytometry. IFN-γ ELISA and IncuCyte were used to analyze in-vitro activation and cytotoxicity. Anti-tumor efficacy was monitored in xenograft models. Safety evaluation was performed by infusing CAR-T cells targeting the murine orthologous TAA in immunocompromised mice.

Results

The HRE-enhanced constructs showed more controllable expression in response to different oxygen levels, with enhanced expression and cytokine release in a hypoxic environment but lower activation levels under normoxic conditions. BTRP003L with the murine PGK HRE showed the best anti-tumor activity among the various HRE-enhanced constructs. The co-expression of dnTGFβRII further enhanced its in-vivo proliferation and anti-tumor efficacy. Moreover, while the constitutively expressing 2^nd-generation CAR recognizing the murine orthologous TAA demonstrated a lethal weight loss phenotype in mice, the conditionally activated CAR-T-treated group showed no observable adverse events.

Conclusions

The hypoxia-regulated and dnTGFβRII-armored CAR-T cells demonstrated robust anti-tumor potential and improved safety profile, supporting further investigations in the clinical setting.

Clinical trial identification

Editorial acknowledgement

Legal entity responsible for the study

Biosyngen Pte Ltd.

Funding

Biosyngen Pte Ltd.

Disclosure

Z. Huang, X. Zhang, D. Han, J.P. Thiery: Financial Interests, Personal, Full or part-time Employment: Biosyngen Pte Ltd.