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.

项目总结和摘要 治疗性抗体的人源化是使鼠源抗体适应人类使用的常见步骤 以降低其免疫原性。虽然单链片段可变抗体（scFv）缺乏恒定结构域， 所提出的人源化项目将取代鼠胆囊收缩素B受体中的框架氨基酸 具有良好表征的人框架序列的（CCKBR）scFv。总的目标是把这个 在达到以下里程碑后，通过第1和第2期人体试验使用该技术。在这个多样性 作为我们资助的UG3赠款的补充，我们提出了以下机制研究，以支持Aleyah Goins女士 我们领先的人源化CCKBR scFv： 里程碑A。人源化CCKBR scFv对神经元过度兴奋的功能研究。膜片钳 来自神经病小鼠的鼠感觉神经元的电生理学和钙成像研究， 将进行用和不用人源化CCKBR scFv处理的致敏的hiPSC衍生的感觉神经元。 里程碑B。细胞因子和外泌体分析。外泌体将从以下细胞的培养上清液中分离： 用人源化CCKBR scFv或媒介物处理的致敏的hiPSC衍生的感觉神经元。外泌体会 裂解，分离microRNA，然后使用RNA测序进行分析。 里程碑C。神经病小鼠组织和血液分析：免疫细胞群的表征 将进一步了解我们的人源化CCKBR scFv如何介导慢性疼痛缓解。全血， 脑、脊髓将从用人源化CCKBR scFv或 在安乐死和灌注后给予溶剂。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。已知染色 将进行损伤标记物、兴奋性和抑制性神经元标记物。 戈因斯女士研究项目的这3个核心目标将阐明我们的人性化的作用机制。 CCKBR scFv对人类神经元的作用及其对小鼠模型中慢性疼痛和焦虑样行为的逆转

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