Validating Technology To Optimize Antibody Affinity For Targeting Therapeutics

描述(申请人提供)：基于抗体的疗法现已在临床上成功用于各种癌症的治疗和诊断。靶标抗体的亲和力和特异性决定了治疗用药的特异性和非靶标副作用。发现和优化针对重要癌症靶点的高亲和力抗体对于开发新的临床方法至关重要，但仍然是一项非常具有挑战性的任务。一种强大的、可与噬菌体和酵母展示相媲美的真核技术将克服微生物的蛋白质翻译限制，从而提高展示抗体的多样性，这些抗体可以被筛选和优化，并更无缝地过渡到大规模哺乳动物表达系统用于临床生产。我们的长期目标是通过增加给药的特异性和 在靶组织中的分布，从而在减少/消除靶外副作用的同时提高疗效。本应用的总体目标是验证基于真核细胞逆转录病毒的新型抗体展示平台的实用性和有效性，以亲和力优化真核细胞中的抗体，促进抗体初始发现和文库筛选技术的发展。我们的中心假设是：ALV复制和ALV多肽展示的特点提供了一个强大的、真核版本的噬菌体展示，从而使经过适当处理和功能的ScFv变体的多样性能够得到改善，这些变体可以被筛选和亲和力优化，以改善有前景的抗体候选，与使用微生物的抗体展示和亲和力成熟相比。我们计划检验我们的中心假设，从而通过追求以下特定目标来实现应用的目标：目标1：验证ALV病毒展示技术对抗体亲和力优化的实用性。目的2：推动ALV抗体展示平台的发展。目的：评价亲和成熟抗体片段在相关肿瘤模型中的特异性、细胞毒性和生物分布。我们将亲和成熟一系列前导单链抗体和识别两个新出现的癌症靶点的单链抗体，Glypcan-3(肝癌)和Mesothelin(间皮瘤，卵巢癌)，并使用优化的抗体片段展示更好的靶向杀灭癌细胞。提议的实验的基本原理是解决对用于发现抗体和亲和力成熟的强大的真核多肽展示平台的需求，为使用微生物的现有展示技术提供一个强有力的补充。我们在这里做出贡献的意义就是提供了这样一种技术。本申请中提出的研究具有创新性，因为它代表了对使用基于微生物的平台的抗体展示现状的新的和实质性的偏离，通过在真核表达系统中提供病毒和细胞展示平台，提供了哺乳动物细胞展示的显著改进和扩展。

DESCRIPTION (provided by applicant): Antibody-based therapeutics have now had success in the clinic for the treatment and diagnosis of a variety of cancers. The affinity and specificityof the antibody for the target ligand determines the specificity of therapeutic delivery and off-targe side effects. The discovery and optimization of high affinity antibodies to important cancer targets is critical for the development of new clinical approaches but is still a very challenging task. A robust, eukaryotic technology that is comparable to phage and yeast display would overcome the protein translation limitations of microorganisms and thereby improve the diversity of the displayed antibodies that can be screened and optimized as well as more seamlessly transition into a large-scale mammalian expression sys- tem for clinical production. Our long-term goal is to improve the targeting of cancer therapeutics by increasing delivery specificity and distribution in the target tissue thereby improving efficacy while reducing/eliminating off-target side effects. The overall objective of this application is to validate the utility and efficiency o a novel antibody display platform based on the eukaryotic retrovirus, Avian Leukosis Virus (ALV), to affinity optimize anti- bodies in eukaryotic cells, and to advance the development of the technology for initial antibody discovery and improved library screening. Our central hypothesis is: the characteristics of ALV replication and ALV polypeptide display offer a robust, eukaryotic version of bacteriophage display, thereby enabling an improvement in the diversity of properly processed and functional scFv variants that can be screened and affinity optimized to improve promising antibody candidates compared to antibody display and affinity maturation using microorganisms. We plan to test our central hypothesis and, thereby, attain the objective of the application by pursuing the following specific aims: Aim 1: Validate the utility of ALV virus display technology for antibody affinity optimization. Aim 2: Advance the development of the ALV antibody display platform. Aim 3: Evaluate the specificity, cytotoxicity, and biodistribution o affinity-matured antibody fragments in relevant cancer models. We will affinity mature a series of lead scFvs and single-domains that recognize two newly emerging cancer targets, glypican-3 (liver cancers) and mesothelin (mesothelioma, ovarian cancer) and demonstrate superior cancer cell killing with targeting using the optimized antibody fragments. The rationale for the proposed experiments is to solve the need for a robust eukaryotic polypeptide display platform for the discovery and affinity maturation of antibodies, offering a powerful addition to the existig display technologies that use microorganisms. The significance of our contribution here is supplying just such a technology. The research proposed in this application is innovative because it represents a new and substantive departure from the status quo of antibody display using platforms based on microorganisms, providing a significant improvement and extension of mammalian cell display by offering both virus and cell display platforms in a eukaryotic expression system.