Ma Xuqian, He Huixia, Zhu Yuankui, Zuo Dianbao, Wang FangLin, Feng Mingqian, Ji Kangkang, Chen Xin
College of Life Science and Technology, Huazhong Agricultural University, Wuhan, Hubei, China.
College of Biomedicine and Health, Huazhong Agricultural University, Wuhan, Hubei, China.
Front Immunol. 2025 Aug 12;16:1625813. doi: 10.3389/fimmu.2025.1625813. eCollection 2025.
B7-H6, a tumor-specific immune checkpoint molecule within the B7 family, represents a promising therapeutic target due to its selective overexpression in malignancies and negligible expression in normal tissues.
Here, we developed bispecific antibodies (BsAbs) targeting B7-H6 to redirect T and NK cells against solid tumors. Through phage display, 15 high-affinity B7-H6 monoclonal antibodies were generated.
Two optimized BsAbs, B7-H6M4-OKT3 (T cell-engaging) and B7-H6M4-LC21 (NK cell-engaging), were constructed in and scFv-hFc-scFv format. Both demonstrated nanomolar affinity (EC50: 0.04-1.22 nM) and selective cytotoxicity against B7-H6+ cells (H446, Huh-7, HepG2), while showing minimal cytotoxicity against B7-H6-negative cells (A431). B7-H6M4LC21 exhibited enhanced tumor-killing efficacy (IC50: 5 ng/mL) compared to B7H6M4-OKT3(IC50: 1 ng/mL) when combined with an IL-15/IL-15Ra sushi fusion protein, which augmented NK cell proliferation and cytotoxicity. In H446 xenograft models, both BsAbs suppressed tumor growth in a dose-dependent manner (0.1-20 mg/kg) without significant toxicity. Combination therapy with B7-H6M4-LC21 (10 mg/kg) and B7-H6M18/IL-15/IL-15Ra sushi (0.03 mg/kg) achieved synergistic tumor inhibition (p<0.05), surpassing the efficacy of T cell-based combinations.
These findings establish B7-H6-targeted BsAbs combined with cytokine engineering as a viable strategy for treating refractory solid tumors.
B7-H6是B7家族中的一种肿瘤特异性免疫检查点分子，由于其在恶性肿瘤中选择性过表达而在正常组织中表达可忽略不计，因此是一个有前景的治疗靶点。
在此，我们开发了靶向B7-H6的双特异性抗体（BsAbs），以重定向T细胞和NK细胞对抗实体瘤。通过噬菌体展示，产生了15种高亲和力的B7-H6单克隆抗体。
构建了两种优化的BsAbs，即B7-H6M4-OKT3（T细胞接合型）和B7-H6M4-LC21（NK细胞接合型），采用scFv-hFc-scFv形式。两者均表现出纳摩尔亲和力（EC50：0.04 - 1.22 nM），对B7-H6+细胞（H446、Huh-7、HepG2）具有选择性细胞毒性，而对B7-H6阴性细胞（A431）的细胞毒性最小。与B7H6M4-OKT3（IC50：1 ng/mL）相比，B7-H6M4LC21与IL-15/IL-15Ra寿司融合蛋白联合使用时表现出增强的肿瘤杀伤效力（IC50：5 ng/mL），该融合蛋白可增强NK细胞增殖和细胞毒性。在H446异种移植模型中，两种BsAbs均以剂量依赖性方式（0.1 - 20 mg/kg）抑制肿瘤生长，且无明显毒性。B7-H6M4-LC21（10 mg/kg）与B7-H6M18/IL-15/IL-15Ra寿司（0.03 mg/kg）联合治疗实现了协同肿瘤抑制（p<0.05），超过了基于T细胞的联合治疗效果。
这些发现确立了靶向B7-H6的BsAbs与细胞因子工程相结合作为治疗难治性实体瘤的可行策略。
