Human VH-antibody-based CAR T cells targeting folate receptor-alpha show enhanced persistence and reduced T-cell exhaustion against ovarian cancer

Folate receptor-alpha (FOLR1) is highly expressed in ovarian cancer and correlates with poor clinical outcomes, making it an attractive target for adoptive cell therapy. Single-domain antibodies (V H domains) have gained increasing interest as antigen-binding domains for CAR T-cell engineering. We generated a second-generation FOLR1-specific CAR incorporating a fully human V H -only antibody and compared its in vitro functional activity with a MOv19-derived scFV CAR. CAR expression, memory phenotype, cytotoxicity, cytokine secretion, and exhaustion markers were evaluated following antigen stimulation. Antitumor efficacy was further assessed in 3D spheroids and repeated tumor-rechallenge assays. Both V H -based and scFV-based FOLR1 CAR T cells demonstrated potent and antigen-specific cytotoxicity against FOLR1-positive ovarian cancer cells. Intriguingly, FOLR1-V H CAR T cells showed lower activation and cytokine release upon initial stimulation, accompanied by reduced expression of exhaustion markers including PD-1 and LAG-3. FOLR1-V H CAR T cells preferentially preserved a central-memory phenotype and displayed superior persistence and tumor control during multiple rounds of antigen rechallenge. Both CAR formats achieved comparable cytotoxicity in 3D spheroid models. Human FOLR1-V H CAR T cells demonstrated potent antitumor activity with reduced exhaustion and enhanced persistence. These properties highlight the V H domain as a promising targeting module for next-generation CAR T-cell therapies in ovarian cancer. • Human VH-based FOLR1 CAR T cells were generated for targeting ovarian cancer. • FOLR1-VH CAR T cells preserved central memory with potent cytotoxicity and reduced exhaustion. • FOLR1-VH CAR T cells showed superior tumor control and enhanced T-cell persistence after rechallenge.