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

治疗性抗体的人源化是使鼠源性抗体用于人类以降低其免疫原性的常见步骤。虽然单链片段可变区(ScFv)缺乏恒定结构域，但拟议的人源化计划将用具有良好特征的人骨架序列取代小鼠胆囊收缩素B受体(CCKBR)scFv中的骨架氨基酸。靶结合互补决定区(CDRs)的亲和力成熟将确保人源化CCKBR scFv与啮齿动物和人CCKBR多肽靶标获得高亲和力，这两个靶点表现出85%的同源性。CHO细胞的表达和前10个候选克隆的纯化将提供支持有效性的蛋白质，并通过表面等离子体共振彻底检查结合动力学。将人源化/亲和力成熟的CCKBR scFv候选单链抗体与亲本鼠源ScFv进行比较，将能够选择具有结合动力学(Kon和Koff)和靶向亲和力(Kd)的领先单链抗体候选者(S)，预计将在神经病理性疼痛的动物模型中提供疗效，并适合于商业开发和临床评估。总体目标是在达到以下里程碑后，使这项技术通过第一阶段和第二阶段的人体试验。里程碑1年1鉴定了3-5个人源化单链抗体克隆，这些克隆对人源多肽的Kd为130 nM，人源多肽定义了亲本单链抗体的结合部位及其效力。将提供足够数量的表达和纯化(&gt；90%纯度，低内毒素)以进行验证分析。里程碑2年1选择1-2基于HiPSC来源的感觉神经元作为筛选平台的体外疗效排名的HITS。第2年根据实验疼痛模型衍生的小鼠三叉神经(TG)和背根神经节(DRG)初级感觉神经元的体外特征，选择1-2个排序的HITS。里程碑3年1-3年根据Aim 2的体外疗效，确定1-2 hCCKBR scFv候选治疗方案在神经病理性和背部疼痛小鼠模型中的剂量反应并验证有效性。比较将包括未治疗和人源化scFv(n=10只/组)鼻腔注射一次的天真、假和神经病理性疼痛模型小鼠。机械阈值增加15倍，并完全从焦虑和抑郁样行为中恢复表示成功。与标准止痛药吗啡和度洛西汀的比较将作为概念的证明。里程碑4年3-5与NIH承包商一起表征和验证hCCKBR scFv Lead。我们将专注于IND使能研究，包括支持cGMP生产和GLP临床前Tox/PK/PD所需的CMC活动。里程碑5年4-5巩固了与NIH承包商和其他制药承包商在最终配方、扩大规模、制造、临床试验批准和商业化方面的互动。

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.