肿瘤细胞治疗研究
英文原题：Linker-dependent modulation of anti-CD22 scFv antibody stability and avidity: Combined structural and experimental insights.
这些发现支持将连接子工程作为优化 scFv 性能的策略，并为合理设计具有更高治疗潜力的 CAR-T 受体提供依据。
靶向CD22的单克隆抗体在急性淋巴细胞白血病（ALL）等血液系统恶性肿瘤中显示出靶向免疫治疗的潜力，CD22是一种在这些恶性肿瘤中过表达的B细胞表面抗原。单链可变片段（scFv）为CAR-T平台提供了增强的模块化特性，而VH与VL结构域之间的连接肽长度已知会影响其构象和结合能力。在此，我们系统比较了两种抗CD22 scFv，分别带有短连接肽（GGGGS）或长连接肽（(GGGGS)）。通过分子动力学模拟（在水相和膜环境中）、生物膜层干涉技术（BLI）和流式细胞术，我们发现膜邻近性会影响结合，短连接肽scFv表现出更高的亲和力（Kd = 5.1 nM vs. 42.1 nM）、更慢的解离速率以及对CD22更强的亲合力。结合自由能分解和RMSD分析揭示，短连接肽构建体具有更稳定的界面，尤其是在膜附近。SEC分析显示短连接肽scFv的二聚体比例略高，尽管流式细胞术表明单体和二聚体形式的CD22结合能力相当。这些发现支持连接肽工程作为优化scFv性能的策略，并为合理设计具有改善治疗潜力的CAR-T受体提供依据。
Monoclonal antibodies targeting CD22, a B-cell surface antigen overexpressed in hematologic malignancies such as acute lymphoblastic leukemia (ALL), have shown promise for targeted immunotherapy. Single-chain variable fragments (scFvs) offer enhanced modularity for CAR-T platforms, and linker length between VH and VL domains is known to influence their conformation and binding. Here, we systematically compared two anti-CD22 scFvs with short (GGGGS) or long ((GGGGS) ) linkers. Using molecular dynamics simulations (in aqueous and membrane environments), bio-layer interferometry (BLI), and flow cytometry, we found that membrane proximity influences binding, and the short-linker scFv exhibited higher affinity (Kd = 5.1 nM vs. 42.1 nM), slower dissociation, and greater avidity for CD22. Binding free energy decomposition and RMSD analyses revealed a more stable interface for the short-linker construct, especially near the membrane. SEC analysis showed a modestly higher dimeric fraction for the short-linker scFv, although flow cytometry indicated comparable CD22 binding across monomeric and dimeric forms. These findings support linker engineering as a strategy to optimize scFv performance and inform the rational design of CAR-T receptors with improved therapeutic potential.
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