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抗体适应人类使用以降低其免疫原性的常见步骤。虽然单链可变抗体（scFv）缺乏恒定结构域，但是所提出的人源化项目将用充分表征的人框架序列替换鼠胆囊收缩素B受体（CCKBR）scFv中的框架氨基酸。靶结合互补决定区（CDR）的亲和力成熟将确保人源化CCKBR scFv获得对啮齿动物和人CCKBR肽靶的高亲和力，其表现出85%的同一性。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功效和通过表面等离子体共振对结合动力学的彻底检查。人源化/亲和力成熟的CCKBR scFv候选物与亲本鼠scFv的比较将使得能够选择具有结合动力学（Kon和Koff）和靶亲和力（KD）的前导scFv候选物，预期其在神经性疼痛的动物模型中提供功效并且适合于商业开发和临床评价。总体目标是在达到以下里程碑后，使这项技术通过1期和2期人体试验。里程碑1 Year 1鉴定3-5个人源化scFv克隆，其表现出对人肽的KD <130 nM亲和力，其限定了亲本scFv的结合位点及其功效。将以足够的量（>90%纯度，低内毒素）提供表达和纯化以进行验证测定。里程碑2第1年使用hiPSC衍生的感觉神经元作为筛选平台，基于证明的体外疗效选择1-2个排名命中。第2年基于源自实验性疼痛模型的鼠三叉神经（TG）和背根神经节（DRG）初级感觉神经元的体外表征，选择1-2级命中。里程碑3第1-3年基于目的2中的体外功效，确定1-2 hCCKBR scFv治疗候选物在神经性疼痛和背痛的鼠模型中的剂量反应并验证其功效。比较将包括未经治疗的小鼠、假手术小鼠和神经性疼痛模型小鼠，两者均未经治疗和用鼻内施用一次的人源化scFv（n =10只小鼠/组）治疗。机械阈值增加>15倍以及从焦虑和抑郁样行为中完全恢复表示成功。与标准镇痛药吗啡和度洛沙汀的比较将作为概念验证。里程碑4第3-5年与NIH承包商一起表征和验证hCCKBR scFv先导物。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 Years 4-5巩固与NIH承包商和其他制药承包商在最终配方、升级、生产、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.