Fournier Léxane, Arras Paul, Pekar Lukas, Kolmar Harald, Zielonka Stefan, Toleikis Lars, Becker Stefan
Early Protein Supply and Characterization, Merck Healthcare KGaA, 64293 Darmstadt, Germany.
Institute for Organic Chemistry and Biochemistry, Technical University of Darmstadt, 64287 Darmstadt, Germany.
Mol Ther Oncol. 2024 Dec 6;33(1):200917. doi: 10.1016/j.omton.2024.200917. eCollection 2025 Mar 20.
In this work, we report the discovery and engineering of allosteric variable domains of the heavy chain (VHHs) derived from camelid immunization targeting NKp30, an activating receptor on natural killer (NK) cells. The aim was to enhance NK cell-mediated killing capacities by identifying VHHs that do not compete with the natural ligand of NKp30:B7-H6, thereby maximizing the recognition of B7-H6 tumor cells. By relying on the DuoBody technology, bispecific therapeutic antibodies were engineered, creating a panel of bispecific antibodies against NKp30xEGFR (cetuximab moiety) or NKp30xHER2 (trastuzumab moiety), called natural killer cell engagers (NKCEs). These NKCEs were assessed for their killing capacities on B7-H6-expressing tumor cells. The results demonstrated an enhancement in NK killing capacities for both EGFR-expressing (HeLa) and HER2-expressing (SK-BR-3) cells, indicating the significance of the natural NKp30/B7-H6 axis in tumor recognition by the immune system. Notably, engineering NKCEs to allow natural recognition of B7-H6 was found to be more effective in promoting NKCE-mediated killing of B7-H6 tumor cells via enhancement of cytokine release. This study highlights the potential of an enhanced-targeting approach, wherein tumor cell surface antigens are targeted while still enabling the natural recognition of the activating ligand (B7-H6) by the immune cells.
在本研究中，我们报告了源自骆驼科动物免疫的重链可变区（VHHs）的发现与工程改造，这些VHHs靶向自然杀伤（NK）细胞上的激活受体NKp30。目的是通过鉴定不与NKp30的天然配体B7-H6竞争的VHHs来增强NK细胞介导的杀伤能力，从而最大限度地识别B7-H6肿瘤细胞。借助双体技术，构建了双特异性治疗性抗体，创建了一组针对NKp30xEGFR（西妥昔单抗部分）或NKp30xHER2（曲妥珠单抗部分）的双特异性抗体，称为自然杀伤细胞衔接器（NKCEs）。评估了这些NKCEs对表达B7-H6的肿瘤细胞的杀伤能力。结果表明，对于表达EGFR的（HeLa）细胞和表达HER2的（SK-BR-3）细胞，NK杀伤能力均有所增强，这表明天然NKp30/B7-H6轴在免疫系统识别肿瘤中的重要性。值得注意的是，通过增强细胞因子释放，发现对NKCEs进行工程改造以实现对B7-H6的天然识别在促进NKCE介导的对B7-H6肿瘤细胞的杀伤方面更有效。这项研究突出了增强靶向方法的潜力，即靶向肿瘤细胞表面抗原，同时仍能使免疫细胞天然识别激活配体（B7-H6）。
