Background Immunotherapies against wide-spectrum tumor-associated antigens (TAA) can target multiple cancers, but their clinical use is confined by the potential on-target off-tumor effects. To address this problem, we developed a hypoxia-activated chimeric antigen receptor (CAR) T cell that targeted a TAA, epithelial glycoprotein-1 (EGP-1), which is often overexpressed in gastrointestinal tumors compared to normal tissues.

Methods BTRP003 was a 2^nd-gen CAR against EGP-1. BTRP003H was constructed by adding an oxygen-dependent degradation domain of HIF1α, and BVHTRP010 and BVHTRP010H were constructed by replacing the scFv of BTRP003 and BTRP003H with a VHH targeting the murine ortholog (figure 1A). T cells isolated from peripheral blood mononuclear cells were stimulated with CD3/CD28 beads, transduced with lentiviral vectors, and expanded ex vivo. CAR expression and exhaustion markers were assessed by flow cytometry before and after co-culture with tumor cells at hypoxic and normoxic conditions. IFN-γ ELISA and IncuCyte were used to analyze the in-vitro activation and cytotoxicity. Anti-tumor efficacy and safety evaluation were evaluated in NOG mice. An investigator-initiated trial was conducted by using 2×10⁸ BTRP003H cells which were manufactured in strict compliance with GMP requirements.

Results The hypoxia-activated BTRP003H cells had a significantly higher CAR expression at 1% O₂ than at 5% O₂ (figure 1B). When co-cultured with 293T overexpressing EGP-1, BTRP003H released a higher level of IFN-γ release at the hypoxic condition (figure 1C), and the PD-1 expression of BTRP003H was significantly lower than the constitutively expressing BTRP003 cells (figure 1D). The robust killing of BTRP003H against various gastrointestinal cancer cells was observed both in vitro and in vivo (figures 1E and 1F). NOG mice infused with constitutively expressing murine CAR-T cells, BVHTRP010, had lethal weight loss, while mice infused with the hypoxia-activated CAR-T cells, BVHTRP010H, showed no observable adverse events (figure 1G). As a proof-of-concept trial (NCT06104215), 2×10⁸ BTRP003H cells were infused in a rectal cancer patient with liver metastasis failing standard chemotherapy, anti-VEGF, and anti-PD1 treatments. The level of CAR-T cells in peripheral blood significantly increased in the first two weeks (figure 2A), and the liver lesion shrunk by 18.9% within 28 days post-infusion (figure 2B), with controllable adverse effects such as skin rash and fever.

Conclusions The hypoxia-activated CAR-T cells demonstrated robust anti-tumor potential and improved clinically tolerable safety profile. These data support further investigations of these CAR-T cells in the clinical setting.

Trial Registration NCT06104215.

Ethics Approval All animal studies were conducted by Biosyngen staff at Guangzhou Regenerative Medicine and Health, Guangdong Laboratory (GRMH-GDL) as approved by the Ruiye model animal (Guangzhou) Biotechnology Co., Ltd facility as approved by its Experimental Animal Care and Use Committee (approval No. RYEth20220516-1). The investigator initiated clinical study was approved by the Affiliated Hospital of Xuzhou Medical University’s Ethics Board (approval number XYF2023-KL287-02).

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Abstract 340 Figure 1

The design and pre-clinical testing of BTRP003H. (A) Constructs. (B) Flow cytometric analysis. (C) IFN-γ ELISA. 293T-OE, 293T cells overexpressing EGP-1. (D) Flow cytometric analysis. (E) In vitro killing. (F) In-vivo test in xenograft NOG mice. (G) In-vivo test in non-tumor-bearing NOG mice

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Abstract 340 Figure 2

Clinical case of BTRP003H in an advanced rectal cancer patient. (A) CAR-T cell number in the peripheral blood (qPCR). EGP-1 transcript was detected by qRT-PCR. (B) Representative tumor response images for the patient before and after BTRP003H infusion