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适应于人类使用以降低其免疫原性的常见步骤。虽然单链片段可变抗体（scFvs）缺乏固定结构域，但拟建的人源化项目将用具有良好特征的人类框架序列取代小鼠胆囊收缩素B载体（CCKBR） scFvs中的框架氨基酸。靶结合互补决定区（cdr）的亲和成熟将确保人源化CCKBR scFv对啮齿动物和人CCKBR肽靶点都具有高亲和性，具有85%的同源性。前10个候选克隆的CHO细胞表达和纯化将提供蛋白质，以支持有效性和表面等离子体共振结合动力学的彻底检查。将人源化/亲和成熟的CCKBR scFv候选物与亲本小鼠scFv进行比较，将有助于选择具有结合动力学（Kon和Koff）和目标亲和性（KD）的主要scFv候选物，以期在神经性疼痛的动物模型中提供疗效，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确定3-5个人源化scFv克隆，这些克隆对确定亲本scFv结合位点及其效力的人肽具有<130 nM的亲和力。将提供足够数量的表达和纯化（>纯度90%，低内毒素）进行验证试验。使用hipsc来源的感觉神经元作为筛选平台，根据已证明的体外功效选择1-2个排名的命中。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表征，选择1-2个排序命中。里程碑3年1-3根据Aim 2的体外疗效，确定1-2种hCCKBR scFv治疗候选药物在神经性和背痛小鼠模型中的剂量反应和有效性。比较将包括naïve，假手术和神经性疼痛模型小鼠，未经治疗和人源化scFv治疗（n =10只小鼠/组）一次鼻内给药。机械阈值增加15倍，焦虑和抑郁样行为完全恢复是成功的标志。与标准镇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年表征和验证hCCKBR scFv铅与NIH承包商一起。我们将专注于IND启用研究，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年巩固与NIH承包商和其他制药承包商的最终配方，升级，生产，临床试验批准和商业化的互动。

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.