PO.ET02.06 · 实验与分子治疗
靶向多个LGR5表位的多样化VHH抗体用于癌症治疗
Diverse VHH antibodies targeting multiple LGR5 epitopes for cancer therapy
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摘要 Abstract
中文摘要
背景：LGR5是一个经过验证的治疗靶点，在癌症干细胞和实体瘤中高表达。Petosemtamab是一种EGFR/LGR5双特异性抗体，证实了其成药性。单域抗体（VHH）凭借小尺寸、高组织渗透性和可扩展的生产，相较于传统抗体具有优势。我们旨在创建一组针对LGR5的VHH结合物，具有广泛的表位多样性和有利于治疗应用的生物物理特性。
方法：使用基于AI的结构和序列引导建模设计VHH，靶向重组人LGR5胞外结构域。应用思维链优化以同时改进多个参数，包括亲和力、热稳定性（Tm）、表达产量和疏水相互作用色谱（HIC）行为。对顶级候选物进行了评估，包括通过表面等离子体共振测定动力学、使用N-糖基敲除变体通过冷冻电镜进行表位定位、通过差示扫描荧光法测定热稳定性，以及在哺乳动物系统中的表达。针对相关受体LGR4和LGR6测试了特异性。
结果：多个VHH克隆表现出对LGR5的高亲和力结合，KD值处于纳摩尔范围。冷冻电镜定位揭示了跨LGR5胞外结构域的不同、非重叠表位，提供了对潜在结合位点的完整结构覆盖。该组合既包括LGR5选择性结合物，也包括泛LGR结合物，为治疗设计提供了灵活性。R-spondin竞争试验鉴定出靶向不同表位的配体阻断型和非竞争型VHH。所有先导分子均表现出强稳定性（Tm >65℃）和稳健表达，支持高可制造性。这些特性使得既能实现通路抑制，又能实现靶向有效载荷递送的方法，以清除LGR5+癌症干细胞。
结论：AI驱动的蛋白质工程产生了一组多样化、高质量的LGR5靶向VHH，具有多表位覆盖、广泛的亲和力范围和强大的可制造性。思维链优化并行改进了多个参数，加速了VHH开发。该文库能够合理设计双特异性和多特异性治疗药物，以增强肿瘤靶向，并代表了下一代LGR5导向的ADC、双特异性抗体和CAR-T疗法的基础。
查看英文原文 English abstract
Background: LGR5 is a validated therapeutic target highly expressed in cancer stem cells and solid tumors. Petosemtamab, an EGFR/LGR5 bispecific antibody, confirms its druggability. Single-domain antibodies (VHHs) provide advantages over conventional antibodies through small size, high tissue penetration, and scalable manufacturing. We aimed to create a panel of VHH binders against LGR5 with broad epitope diversity and favorable biophysical traits for therapeutic use.
Methods: VHHs were designed using AI-based structure and sequence-guided modeling targeting recombinant human LGR5 extracellular domain. Chain-of-thought optimization was applied to improve multiple parameters simultaneously, including affinity, thermal stability (Tm), expression yield, and hydrophobic interaction chromatography (HIC) behavior. Top candidates were evaluated for kinetics via surface plasmon resonance, epitope mapping by cryo-EM using N-glycan knockout variants, thermal stability by differential scanning fluorimetry, and expression in mammalian systems. Specificity was tested against related receptors LGR4 and LGR6.
Results: Multiple VHH clones showed high-affinity binding to LGR5, with KD values in the nanomolar range. Cryo-EM mapping revealed distinct, non-overlapping epitopes across the LGR5 extracellular domain, providing full structural coverage of potential binding sites. The panel included both LGR5-selective and pan-LGR binders, offering flexibility in therapeutic design. R-spondin competition assays identified ligand-blocking and non-competing VHHs targeting alternate epitopes. All lead molecules displayed strong stability (Tm >65°C) and robust expression, supporting high manufacturability. These characteristics enable both pathway inhibition and targeted payload delivery approaches to eliminate LGR5+ cancer stem cells.
Conclusions: AI-driven protein engineering produced a diverse, high-quality panel of LGR5-targeted VHHs with multi-epitope coverage, broad affinity range, and strong manufacturability. Chain-of-thought optimization improved multiple parameters in parallel, accelerating VHH development. This library enables rational design of bispecific and multispecific therapeutics for enhanced tumor targeting and represents a foundation for next-generation LGR5-directed ADCs, bispecifics, and CAR-T therapies.
利益披露 Disclosure
C. Xu,
BioMap Employment.
Y. Li,
BioMap Employment.
T. Nguyen,
BioMap Employment.
R. Shek,
BioMap Employment.
L. Chong,
BioMap Employment.
T. H. Lee,
BioMap Employment.
Y. Lang,
BioMap Employment.
L. Yi,
BioMap Employment.
P. Greisen,
BioMap Inc Employment.