Neuroimmune mechanisms of a humanized CCK-B receptor scFv as therapy for chronic pain patients

人源化CCK-B受体scFv治疗慢性疼痛的神经免疫机制

• 批准号: 10571425
• 负责人: Sascha R Alles
• 金额: $ 7.78万
• 依托单位: UNIVERSITY OF NEW MEXICO HEALTH SCIS CTR
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10571425
• 关键词: Afferent Neurons; Affinity; Amino Acids; Antibodies; Anxiety; Blood; Brain; CCKBR gene; Calcium; Cells; Cholecystokinin; Cholecystokinin B Receptor; Chronic; Electrophysiology (science); Euthanasia; Flow Cytometry; Funding; Goals; Grant; Human; Immune; Injury; Lead; Mediating; Mental Depression; MicroRNAs; Mus; Nerve; Neuroimmune; Neuroimmunomodulation; Neurons; Neuropathy; Pain; Penetrance; Perfusion; Phase; Population; Research Project Grants; Safety; Specificity; Spinal Cord; Stains; Technology; Therapeutic antibodies; Tissues; Trigeminal nerve structure; Whole Blood; anxiety-like behavior; chronic pain; chronic pain management; chronic pain patient; cytokine; effective therapy; excitatory neuron; exosome; imaging study; immunogenicity; inhibitory neuron; meetings; mouse model; murine antibody; nerve injury; painful neuropathy; patch clamp; transcriptome sequencing

PROJECT SUMMARY AND ABSTRACT Humanization of therapeutic antibodies is a common step in adapting murine-derived antibodies for human use to reduce their immunogenicity. While single chain Fragment variable antibodies (scFvs) lack constant domains, the proposed humanization project will replace framework amino acids in the murine cholecystokinin B rector (CCKBR) scFvs with well-characterized human framework sequences. The overall goal is to bring this technology through Phase 1 and 2 human trials after meeting the following milestones. In this Diversity Supplement to our funded UG3 grant to support Ms. Aleyah Goins, we propose the following mechanistic studies of our lead humanized CCKBR scFv: Milestone A. Functional studies of humanized CCKBR scFv on neuronal hyperexcitability. Patch-clamp electrophysiological and calcium imaging studies of murine sensory neurons from neuropathic mice and sensitized hiPSC-derived sensory neurons treated with and without humanized CCKBR scFv will be performed. Milestone B. Cytokine and exosomal analysis. Exosomes will be isolated from culture supernatant of sensitized hiPSC-derived sensory neurons treated with humanized CCKBR scFv or vehicle. The exosomes will be lysed, microRNAs isolated and then analyzed using RNA sequencing. Milestone C. Neuropathic mice tissue and blood analysis: Characterization of the immune cell populations will advance understanding of how our humanized CCKBR scFv mediates chronic pain alleviation. Whole blood, brain, spinal cord will be obtained from mice with neuropathic pain treated with humanized CCKBR scFv or vehicle after euthanasia and perfusion. Flow cytometry analysis of blood and tissues, as well as immunohistochemical staining of trigeminal nerve, brain, and spinal cord will be performed. Staining of known injury markers, excitatory and inhibitory neuron markers will be performed. These 3 core goals of Ms. Goins research project will elucidate the mechanism of action of our humanized CCKBR scFv on human neurons and its reversal of chronic pain- and anxiety-like behaviors in mouse models.

项目摘要和摘要 治疗性抗体的人源化是使鼠源性抗体适用于人类的常见步骤 以降低它们的免疫原性。虽然单链片段可变抗体(ScFv)缺乏恒定结构域， 拟议的人源化项目将取代小鼠胆囊收缩素B受体中的框架氨基酸 (CCKBR)具有特征良好的人骨架序列的单链抗体。总体目标是实现这一点 在达到以下里程碑之后，技术通过了第一阶段和第二阶段的人体试验。在这种多样性中 为了支持Aleyah Goins女士，我们建议进行以下机械研究 我们领先的人性化CCKBR scFv： 里程碑A.人源化CCKBR单链抗体对神经元超兴奋性的功能研究。膜片钳 神经病小鼠和小鼠感觉神经元的电生理和钙成像研究 用人源化CCKBR scFv和不用人源化CCKBR scFv处理敏化的HiPSC来源的感觉神经元将进行实验。 里程碑B.细胞因子和外切体分析。Exosome将从培养上清液中分离出来 人源化CCKBR单链抗体或赋形剂处理致敏的HiPSC来源的感觉神经元。外体将会 裂解，分离microRNAs，然后进行RNA测序分析。 里程碑C.神经病变小鼠的组织和血液分析：免疫细胞群体的特征 将增进对我们人性化的CCKBR scFv如何介导慢性疼痛缓解的理解。全血， 人源化CCKBR单链抗体或人源化CCKBR单链抗体治疗神经病理性疼痛小鼠的脑、脊髓 安乐死和灌流后的交通工具。血液和组织的流式细胞术分析以及 将进行三叉神经、脑和脊髓的免疫组织化学染色。已知的染色 将进行损伤标记物、兴奋性和抑制性神经元标记物。 Goins女士研究项目的这三个核心目标将阐明我们人性化的 CCKBR单链抗体对人类神经元的影响及其对小鼠慢性疼痛和焦虑样行为的逆转作用。

项目成果

Peroxisome proliferator-activated receptor gamma agonist ELB00824 suppresses oxaliplatin-induced pain, neuronal hypersensitivity, and oxidative stress.

• DOI: 10.1016/j.neuropharm.2022.109233
• 发表时间: 2022-11-01
• 期刊: NEUROPHARMACOLOGY
• 影响因子: 4.7
• 作者: Zhang, Morgan;Hu, Min;Alles, Sascha R. A.;Montera, Marena A.;Adams, Ian;Santi, Maria D.;Inoue, Kenji;Tu, Nguyen Huu;Westlund, Karin N.;Ye, Yi
• 通讯作者: Ye, Yi

Chronic pregabalin treatment protects against spreading depolarization and alters hippocampal synaptic characteristics in a model of familial hemiplegic migraine-type 1.

• DOI: 10.1186/s13041-023-01062-6
• 发表时间: 2023-11-03
• 期刊: Molecular brain
• 影响因子: 3.6

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.

• DOI: 10.3390/ijms241311035
• 发表时间: 2023-07-03
• 期刊: International journal of molecular sciences
• 影响因子: 5.6

Neuronal allodynic mechanisms of Slc7a5 (LAT1) in the spared nerve injury rodent model of neuropathic pain.

• DOI: 10.1007/s00424-021-02653-9
• 发表时间: 2022-04
• 期刊: Pflugers Archiv : European journal of physiology
• 作者: Goins AE;Gomez K;Ran D;Afaghpour-Becklund M;Khanna R;Alles SRA
• 通讯作者: Alles SRA