Characterization and comparative analysis of multifunctional natural killer cell engagers (NKCEs) during antitumor responses

T cell engagers (TCEs) are transformational oncology therapies but limited in use due to the induction of cytokine release syndrome (CRS). In comparison to T cells, natural killer (NK) cells produce less cytokines upon activation, leading to the exploration of NK cell engagers (NKCEs). However, why NK cells secrete less cytokines such as tumor necrosis factor (TNF) and how NKCEs perform directly against TCEs remain unclear. Here we report that relative to T cells, NK cells have reduced trafficking and processing of TNF. Systematic development and benchmarking studies show that NKCEs can be optimized to engage multiple activating receptors and incorporate the interleukin (IL)-2, thereby increasing their potency and durability. Furthermore, comparative studies of NKCE, IL-2, and TCE therapy in animal tumor models reveal both common and different therapeutic benefits. Our results provide a blueprint for development of multifunctional NKCEs that may serve as an alternative to current TCE therapies. Overall design: CD4+ T, CD8+ T, and natural killer (NK) cells were treated either with PBS (control) or various T-cell engager (TCE) or NK-cell engager (NKCE) molecules, respectively. One molecule was tested on T-cells while several others were tested on NK cells. RNA pellets were collected both at 16H and 64H post-treatment.

T细胞衔接蛋白（T cell engagers, TCEs）是具有变革性的肿瘤治疗手段，但因可诱导细胞因子释放综合征（cytokine release syndrome, CRS）而应用受限。相较于T细胞，自然杀伤细胞（natural killer, NK）激活后产生的细胞因子更少，这推动了NK细胞衔接蛋白（NK cell engagers, NKCEs）的研发。然而，NK细胞为何分泌的肿瘤坏死因子（tumor necrosis factor, TNF）等细胞因子更少，以及NKCEs相较于TCEs的直接作用机制仍不明确。本研究发现，相较于T细胞，NK细胞的肿瘤坏死因子（TNF）转运与加工过程均存在减弱现象。通过系统性开发与基准测试研究表明，NKCEs可被优化以结合多种激活受体，并整合白细胞介素（interleukin, IL）-2，从而提升其效力与持久性。此外，在动物肿瘤模型中开展的NKCE、IL-2与TCE疗法对比研究显示，三种疗法兼具共同与差异化的治疗获益。本研究结果为多功能NKCEs的开发提供了蓝图，有望成为当前TCE疗法的替代方案。
研究整体设计：分别以磷酸盐缓冲液（PBS，对照组）、多种T细胞衔接蛋白（TCE）或NK细胞衔接蛋白（NKCE）分子处理CD4+ T细胞、CD8+ T细胞与自然杀伤（NK）细胞。其中一种分子在T细胞中进行测试，其余多种分子则在NK细胞中开展实验。分别于处理后16小时与64小时收集RNA沉淀样本。