Rapid Generation and Molecular Docking Analysis of Single-Chain Fragment Variable (scFv) Antibody Selected by Ribosome Display Targeting Cholecystokinin B Receptor (CCK-BR) for Reduction of Chronic Neuropathic Pain.
Rapid Generation and Molecular Docking Analysis of Single-Chain Fragment Variable (scFv) Antibody Selected by Ribosome Display Targeting Cholecystokinin B Receptor (CCK-BR) for Reduction of Chronic Neuropathic Pain.
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DOI：
10.3390/ijms241311035
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发表时间：
2023-07-03
影响因子：
5.6
通讯作者：
中科院分区：
文献类型：
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利用一种强大的无细胞平台技术，核糖体展示结合克隆、表达和纯化来开发单链可变片段（scFv）抗体变体，作为针对小鼠胆囊收缩素B（CCK-B）受体的疼痛治疗。通过核糖体展示技术获得了3个有效的CCK-B肽特异性单链抗体。可溶性表达和ELISA分析表明，其中一种抗体scFv 77 -2具有最高的结合力，并且可以从细菌细胞中大量纯化。Octet测量进一步显示CCK-B scFv 77 -2抗体具有KD = 1.794 × 10-8 M的结合动力学。分子模拟和对接分析表明，scFv 77 -2抗体形成了一个适当的空腔，以嵌入整个CCK-B肽分子，并依赖于分子间的力，包括氢键，静电力，和疏水相互作用形成稳态复合物。因此，scFv抗体可应用于CCK-BR的分子间相互作用机制和体内功能研究。高亲和力scFv 77 -2抗体在慢性疼痛模型中测试的与CCK-BR的结合显示出良好的功效。体内研究验证了CCK-B受体（CCK-BR）scFv 77 -2抗体作为慢性三叉神经损伤诱导的疼痛的潜在疗法的功效。在诱导慢性三叉神经性疼痛模型3周后，在从急性疼痛向慢性疼痛的转变期间，给予小鼠单剂量的CCK-B受体（CCK-BR）scFv抗体。降低机械性超敏反应的长期有效性是明显的，持续数月。通常伴随持续性超敏反应的焦虑和抑郁相关行为随后在给予CCK-BR单链抗体的小鼠中从未出现。该抗体的有效性是进一步开发先导CCK-BR scFv作为慢性疼痛的有前途的非阿片类药物治疗和长期减少慢性疼痛和焦虑相关行为的基础。
A robust cell-free platform technology, ribosome display in combination with cloning, expression, and purification was utilized to develop single chain Fragment variable (scFv) antibody variants as pain therapy directed at the mouse cholecystokinin B (CCK-B) receptor. Three effective CCK-B peptide-specific scFvs were generated through ribosomal display technology. Soluble expression and ELISA analysis showed that one antibody, scFv77-2 had the highest binding and could be purified from bacterial cells in large quantities. Octet measurements further revealed that the CCK-B scFv77-2 antibody had binding kinetics of KD = 1.794 × 10–8 M. Molecular modeling and docking analyses suggested that the scFv77-2 antibody shaped a proper cavity to embed the whole CCK-B peptide molecule and that a steady-state complex was formed relying on intermolecular forces, including hydrogen bonding, electrostatic force, and hydrophobic interactions. Thus, the scFv antibody can be applied for mechanistic intermolecular interactions and functional in vivo studies of CCK-BR. The high affinity scFv77-2 antibody showed good efficacy with binding to CCK-BR tested in a chronic pain model. In vivo studies validated the efficacy of the CCK-B receptor (CCK-BR) scFv77-2 antibody as a potential therapy for chronic trigeminal nerve injury-induced pain. Mice were given a single dose of the CCK-B receptor (CCK-BR) scFv antibody 3 weeks after induction of a chronic trigeminal neuropathic pain model, during the transition from acute to chronic pain. The long-term effectiveness for the reduction of mechanical hypersensitivity was evident, persisting for months. The anxiety- and depression-related behaviors typically accompanying persisting hypersensitivity subsequently never developed in the mice given CCK-BR scFv. The effectiveness of the antibody is the basis for further development of the lead CCK-BR scFv as a promising non-opioid therapeutic for chronic pain and the long-term reduction of chronic pain- and anxiety-related behaviors.
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影响因子：
6.7
作者：
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通讯作者：
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