肿瘤细胞治疗研究
英文原题：Engineering NKG2D ligand affinity transforms EGFR-targeted NK cell engagers into high-potency effectors against pancreatic cancer.
这些结果支持了在此评估的EGFR靶向、Fc功能性格式中NKG2D参与型ICEs的亲和力-活性关系。值得注意的是，提高ULBP6亲和力增强了NK细胞效应功能，并在NK重建的PDAC模型中提高了抗肿瘤疗效。总体而言，我们的发现为工程化高 potency、肿瘤锚定的NKG2D参与剂用于免疫冷肿瘤如PDAC的免疫治疗提供了设计框架。
胰腺导管腺癌（PDAC）常通过主要组织相容性复合体I类（MHC-I）抗原提呈受损来逃逸T细胞介导的免疫，导致其对基于T细胞的免疫疗法反应有限。由于自然杀伤（NK）细胞能够清除MHC-I低表达的肿瘤细胞，重定向NK细胞毒性对于PDAC等免疫冷肿瘤而言是一种有前景的策略。在激活性NK受体中，NKG2D已通过整合NKG2D配体（MICA/B和ULBP家族成员）被广泛用于NK细胞衔接平台。然而，NKG2D配体（NKG2DL）的身份和结合亲和力对NK细胞衔接器效力的影响尚未被定量明确。
我们设计了一组靶向表皮生长因子受体（EGFR）的异二聚体IgG1 Fc基αEGFR×NKG2DL免疫细胞衔接器（ICE），每个ICE将帕尼单抗来源的EGFR臂与六种人NKG2DL胞外域之一（MICA、MICB、ULBP1、ULBP2，以及ULBP6等位变体ULBP0601和ULBP0602）配对。所有ICE均特异性结合EGFR和NKG2D，且NKG2D结合亲和力与NK细胞对表达不同EGFR密度的PDAC细胞系的细胞毒效力直接相关。在天然配体中，ULBP0602表现出最强的NKG2D结合和最高的杀伤活性。基于酵母展示对ULBP0602进行亲和力改进，获得了变体ULBP6#2，其亲和力提高约13倍，主要归因于解离动力学减慢。亲和力改进的αEGFR×ULBP6#2使EC₅₀最多降低11倍，并比亲本构建体具有更大的最大裂解率，同时增强NK细胞分泌IFN-γ和TNF-α。在携带PDAC异种移植瘤的NK人源化NSG小鼠中，αEGFR×ULBP6#2在PANC-1（89%对57%）和BxPC-3（60%对25%）模型中均比αEGFR×ULBP0602实现了显著更强的肿瘤生长抑制，且未观察到毒性或体重减轻。
BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) often escapes T cell–mediated immunity through impaired major histocompatibility complex class I (MHC-I) antigen presentation, contributing to its limited responsiveness to T cell–based immunotherapies. Because natural killer (NK) cells are capable of eliminating MHC-I–low tumor cells, redirecting NK cytotoxicity represents a promising strategy for these immune-cold tumors such as PDAC. Among activating NK receptors, NKG2D has been widely exploited in NK cell–engaging platforms through the incorporation of NKG2D ligands (MICA/B and ULBP family members). However, the impact of NKG2D ligand (NKG2DL) identity and binding affinity on NK cell engager potency has not been quantitatively defined. RESULTS: We engineered a panel of epidermal growth factor receptor (EGFR)–targeted, heterodimeric IgG1 Fc-based αEGFR×NKG2DL immune cell engagers (ICEs), each pairing a panitumumab-derived EGFR arm with one of six human NKG2DL extracellular domains (MICA, MICB, ULBP1, ULBP2, and the ULBP6 allelic variants ULBP0601 and ULBP0602). All ICEs bound specifically to EGFR and NKG2D, and NKG2D binding affinity correlated directly with NK cell cytotoxic potency across PDAC cell lines expressing different EGFR densities. Among native ligands, ULBP0602 exhibited the strongest NKG2D binding and highest killing activity. Yeast display–based affinity improvement of ULBP0602 yielded the variant ULBP6#2, which showed approximately a 13-fold improvement in affinity due primarily to slower dissociation kinetics. The affinity-improved αEGFR×ULBP6#2 induced up to an 11-fold reduction in EC₅₀ and greater maximal lysis than the parental construct, along with enhanced IFN-γ and TNF-α secretion by NK cells. In NK-humanized NSG mice bearing PDAC xenografts, αEGFR×ULBP6#2 achieved markedly greater tumor growth inhibition compared with αEGFR×ULBP0602 in both PANC-1 (89% versus 57%) and BxPC-3 (60% versus 25%) models, without observable toxicity or weight loss. CONCLUSIONS: These results support an affinity–activity relationship for NKG2D-engaging ICEs within the EGFR-targeted, Fc-competent format evaluated here. Notably, improving ULBP6 affinity enhanced NK cell effector function and increased antitumor efficacy in NK-reconstituted PDAC models. Collectively, our findings provide a design framework for engineering high-potency, tumor-tethered NKG2D engagers for the immunotherapy of immune-cold tumors such as PDAC.
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