PO.IM01.15 · 免疫学
利用VLPs和纳米盘为具有挑战性的膜蛋白解锁抗体发现
Harnessing VLPs and nanodiscs to unlock antibody discovery for challenging membrane proteins
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
多次跨膜蛋白（MP-TMPs），包括G蛋白偶联受体（GPCRs）、离子通道和转运体，是细胞通讯和信号传导的重要调控因子。它们占当前药物靶点的60%以上，然而其结构复杂性和对脂质环境的依赖性使得重组生产和抗体发现极为困难。传统的可溶性形式无法保持其构象完整性，限制了免疫原性和筛选相关性。为解决这些局限，KACTUS开发了一个多功能膜蛋白展示平台，整合了病毒样颗粒（VLPs）和纳米盘，以稳定处于天然构象的全长MP-TMPs。VLPs由缺乏遗传物质的自组装结构蛋白组成，提供天然的脂质包膜，支持正确折叠和高密度表面展示。使用该系统，KACTUS成功展示了全长STEAP1蛋白（一种前列腺癌相关抗原），并证明其与治疗性抗体Vandortuzumab特异性结合，EC₅₀为89.8 ng/mL。作为补充，在哺乳动物细胞中生产的基于SMA的纳米盘可在天然脂质双层内实现膜蛋白的无去污剂溶解。A2A腺苷受体（A2AR）纳米盘保持了结构完整性，并通过ELISA（EC₅₀ = 0.15 μg/mL）和SPR（K_D = 0.32 nM）表现出与抗A2AR单克隆抗体的强结合。此外，KACTUS开发了CXCR4 VLP免疫原，产生了功能性抗血清和单克隆抗体，其配体阻断能力可与商用CXCR4抗体Ulocuplumab相媲美。进一步拓展该方法，生物素化的VLPs和纳米盘实现了高效的噬菌体展示抗体淘选，如GPRC5D构建体所验证的（VLPs的EC₅₀ = 7.4 ng/mL；纳米盘的EC₅₀ = 0.28 μg/mL）。KACTUS还建立了首个TCR-CD3复合物纳米盘，保留了所有天然亚基（TCRα/β、CD3ε/γ/δ/ζ），用于准确的CD3抗体筛选和结合分析（SP34 K_D = 0.1 nM）。总之，这些结果表明，KACTUS的VLP和纳米盘平台提供了适用于抗体生成、配体结合和结构探究的类天然、生物活性的MP-TMPs。这一整合技术为克服膜蛋白免疫和筛选中长期存在的挑战提供了强有力的解决方案，为下一代治疗性抗体发现铺平了道路。
查看英文原文 English abstract
Multi-pass transmembrane proteins (MP-TMPs), including G protein-coupled receptors (GPCRs), ion channels, and transporters, are vital regulators of cellular communication and signaling. They represent over 60% of current drug targets, yet their structural complexity and dependence on lipid environments make recombinant production and antibody discovery extremely difficult. Traditional soluble formats fail to preserve their conformational integrity, limiting immunogenicity and screening relevance. To address these limitations, KACTUS developed a versatile membrane-protein display platform integrating virus-like particles (VLPs) and nanodiscs to stabilize full-length MP-TMPs in their native conformations. VLPs, composed of self-assembling structural proteins lacking genetic material, provide a natural lipid envelope that supports proper folding and high-density surface display. Using this system, KACTUS successfully presented the full-length STEAP1 protein (a prostate-cancer-associated antigen) and demonstrated specific binding to the therapeutic antibody Vandortuzumab with an EC₅₀ of 89.8 ng/mL. Complementing this, SMA-based nanodiscs produced in mammalian cells offer detergent-free solubilization of membrane proteins within native lipid bilayers. The A2A adenosine receptor (A2AR) nanodiscs retained structural integrity and exhibited strong binding to anti-A2AR monoclonal antibodies by ELISA (EC₅₀ = 0.15 µg/mL) and SPR (K_D = 0.32 nM). In addition, KACTUS developed CXCR4 VLP immunogens that generated functional antisera and monoclonal antibodies capable of ligand-blocking comparable to the commercial CXCR4 antibody Ulocuplumab. Further extending this approach, biotinylated VLPs and nanodiscs enabled efficient phage-display antibody panning , as validated with GPRC5D constructs (EC₅₀ = 7.4 ng/mL for VLPs; 0.28 µg/mL for nanodiscs). KACTUS also established the first TCR-CD3 complex nanodisc , preserving all native subunits (TCRalpha/beta, CD3ε/gamma/delta/ζ) for accurate CD3 antibody screening and binding analysis (SP34 K_D = 0.1 nM). Together, these results demonstrate that the KACTUS VLP and nanodisc platforms deliver native-like, bioactive MP-TMPs suitable for antibody generation, ligand binding, and structural interrogation. This integrated technology provides a powerful solution to overcome long-standing challenges in membrane-protein immunization and screening , paving the way for next-generation therapeutic antibody discovery.
利益披露 Disclosure
H. Flaherty Duncan, None..
S. Chen, None..
J. Cui, None.