框架人源化通过降低强直信号增强靶向 GM3(Neu5Gc) 的 CAR-T 细胞功能

GM3(Neu5Gc), a tumor-associated ganglioside absent in normal human tissues due to a CMP-N-acetylneuraminic acid hydroxylase (CMAH) mutation, is an attractive target for solid tumor immunotherapy. To advance the clinical potential of GM3(Neu5Gc)-targeted CAR-T cells, we systematically evaluated antibody humanization by comparing CARs based on the murine 14F7 antibody and its humanized variant 14F7hT. Within each scFv framework, we further assessed three hinge domains (CD8 , CD28, IgG4) to optimize CAR design.

While hinge selection influenced in vitro cytotoxicity-favoring CD28-the humanized 14F7hT-based CARs consistently outperformed their murine counterparts in vivo . The optimized 14F7hT-CD28 CAR-T cells demonstrated superior expansion, persistence, tumor infiltration, and antitumor efficacy in xenograft models.

To further characterize the basis of this in vivo advantage, we performed a repeated tumor stimulation assay mimicking chronic antigen exposure in solid tumors. In this setting, hGM3/CD28 CAR-T cells exhibited enhanced cytotoxicity, degranulation, and proliferation, supporting improved functional durability.

Mechanistically, this was linked to reduced tonic signaling: CAR-Toner predicted a near-optimal signal strength for hGM3/CD28 (score 59) versus excessive signaling in mGM3/CD28 (score 65). This was confirmed experimentally by lower basal cytokine secretion and activation marker expression in cytokine- and antigen-free conditions. Despite preserving complementarity-determining regions, scFv humanization induced subtle structural changes that attenuated tonic signaling and enhanced CAR-T functionality.

These findings underscore a critical, previously underappreciated role for antibody framework regions in modulating CAR signaling and therapeutic efficacy.

Our study establishes 14F7hT-CD28 as a promising candidate for GM3(Neu5Gc)-positive tumors and highlights framework humanization as a key strategy to improve CAR-T cell performance.