Development of a CCKBR-targeting scFv as Therapy for Chronic Pain Patients

开发 CCKBR 靶向 scFv 作为慢性疼痛患者的治疗方法

• 批准号: 10304029
• 负责人: Sascha R Alles
• 金额: $ 99.91万
• 依托单位: UNIVERSITY OF NEW MEXICO HEALTH SCIS CTR
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-30 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10304029
• 关键词: Absence of pain sensation; Address; Afferent Neurons; Affinity; Amino Acids; Analgesics; Animal Model; Anti-Anxiety Agents; Antibodies; Anxiety; Back Pain; Behavior; Binding; Binding Sites; Biodistribution; Biological; Biological Assay; Brain; Chinese Hamster Ovary Cell; Cholecystokinin; Chronic; Clinical Trials; Cognitive; Complementarity Determining Regions; Contractor; Development; Dizziness; Dose; Endotoxins; Ensure; Exhibits; Formulation; Goals; Human; Hypersensitivity; In Vitro; Kinetics; Lead; Mechanics; Mental Depression; Monoclonal Antibodies; Morphine; Mus; Nerve; Nervous system structure; Neurons; Neuropeptides; Pain; Pain management; Panic Attack; Parents; Penetrance; Peptide Receptor; Peptides; Pharmaceutical Preparations; Pharmacologic Substance; Phase; Proteins; Quality of life; Recovery; Rodent; Safety; Specificity; Spinal Ganglia; Surface Plasmon Resonance; Technology; Therapeutic antibodies; Time; Trigeminal System; United States National Institutes of Health; Validation; Ventilatory Depression; base; cGMP production; chronic neuropathic pain; chronic pain; chronic pain patient; commercialization; disability; dosage; duloxetine; efficacy validation; experience; immunogenicity; induced pluripotent stem cell; meetings; mouse model; murine antibody; nerve injury; non-opioid analgesic; nonhuman primate; pain model; painful neuropathy; pharmacokinetics and pharmacodynamics; placebo controlled trial; pre-clinical; prevent; receptor; research clinical testing; response; screening; side effect; success; therapeutic candidate

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. Affinity maturation of the target-binding complementarity-determining regions (CDRs) will ensure that the humanized CCKBR scFv acquires high affinity to both the rodent and human CCKBR peptide targets, which exhibit 85% identity. CHO cell expression and purification of top 10 candidate clones will provide protein to support efficacy and a thorough examination of binding kinetics by surface plasmon resonance. Comparison of humanized/affinity matured CCKBR scFv candidates to the parent murine scFv will enable selection of a lead scFv candidate(s) with binding kinetics (Kon and Koff) and target affinity (KD) anticipated to provide efficacy in animal models of neuropathic pain and be suitable for commercial development and clinical evaluation. The overall goal is to bring this technology through Phase 1 and 2 human trials after meeting the following milestones. Milestone 1 Year 1 Identify 3-5 humanized scFv clones that exhibit a KD of <130 nM affinity for the human peptide that defines the binding site for the parent scFv and its efficacy. Expression and purification will be provided in sufficient quantity (>90% purity, low endotoxin) to conduct validation assays. Milestone 2 Year 1 Select 1-2 ranked hits based on demonstrated in vitro efficacy using hiPSC-derived sensory neurons as the screening platform. Year 2 Select 1-2 ranked hits based on in vitro characterization in murine trigeminal (TG) and dorsal root ganglia (DRG) primary sensory neurons derived from experimental pain models. Milestone 3 Year 1-3 Determine dose response and validate efficacy of 1-2 hCCKBR scFv therapeutic candidates in murine models of neuropathic and back pain based on in vitro efficacy in Aim 2. Comparisons will include naïve, sham, and neuropathic pain model mice, both untreated and treated with humanized scFv (n =10 mice/group) administered once intranasally. Mechanical threshold increases of >15-fold and full recovery from anxiety and depression-like behaviors denote success. Comparisons with standard analgesics morphine and duloxetine will serve as proof of concept. Milestone 4 Year 3-5 Characterize and validate hCCKBR scFv lead in conjunction with NIH contractors. We will focus on IND enabling studies including CMC activities needed to support cGMP production and GLP pre-clinical Tox/PK/PD. Milestone 5 Years 4-5 Solidify interactions with NIH contractors and other pharmaceutical contractors for final formulation, upscaling, manufacturing, approvals for clinical trials, and commercialization.

治疗性抗体的人源化是将鼠源抗体改造为人用以降低其免疫原性的常见步骤，虽然单链片段可变抗体 (scFv) 缺乏恒定结构域，但拟议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。 (CCKBR) scFv 具有充分表征的人类框架序列，靶标结合互补决定区 (CDR) 的亲和力成熟将确保人源化。 CCKBR scFv 与啮齿动物和人类 CCKBR 肽靶标具有高亲和力，其 CHO 细胞表达和前 10 个候选克隆的纯化将提供蛋白质以支持功效并通过表面等离振子共振对结合动力学进行彻底检查。将人源化/亲和力成熟的CCKBR scFv候选物与亲本鼠scFv进行比较将能够选择具有结合动力学（Kon和Koff）和目标亲和力（KD）预计将在神经性疼痛的动物模型中发挥功效，并适合商业开发和临床评估，总体目标是在达到以下里程碑后使该技术通过第一阶段和第二阶段的人体试验。第 1 年 鉴定 3-5 个人源化 scFv 克隆，这些克隆对定义亲本 scFv 结合位点的人肽表现出 KD <130 nM 的亲和力，并提供充分的表达和纯化。数量（> 90% 纯度，低内毒素）进行验证分析 里程碑 2 第 1 年，根据使用 hiPSC 衍生的感觉神经元作为筛选平台，选择 1-2 个排名的命中。基于来自实验性疼痛模型的小鼠三叉神经 (TG) 和背根神经节 (DRG) 初级感觉神经元的体外特征 里程碑 3 第 1-3 年 确定剂量反应并验证疗效。 1-2 hCCKBR scFv 治疗候选药物在神经性和背痛小鼠模型中基于目标 2 中的体外功效。比较将包括未经治疗和经人源化 scFv 治疗的幼稚小鼠、假小鼠和神经性疼痛模型小鼠（n = 10 只小鼠/与标准镇痛药相比，鼻内给药一次后，机械阈值增加>15倍，并且从焦虑和抑郁样行为中完全恢复。吗啡和度洛西汀将作为概念验证，第 4 年 3-5 年与 NIH 承包商一起表征和验证 hCCKBR scFv 药物。我们将重点关注 IND 启用研究，包括支持 cGMP 生产和 GLP 临床前毒理学/ GLP 所需的 CMC 活动。 PK/PD 里程碑 5 年 4-5 巩固与 NIH 承包商和其他制药承包商的互动，以实现最终配方、升级、生产、临床试验批准等。商业化。