SBIR Phase I: Development of a Platform for Rapid "Plug and Play" Chemo-enzymatic Conjugation of Bio-therapeutics

这个小企业创新研究（SBIR）第一阶段项目建议开发一种平台技术，利用定向进化来设计转肽酶（tpase），该转肽酶将识别天然抗体中的序列，并通过肽键结合任何小分子，肽或感兴趣的蛋白质片段。目前生产抗体药物偶联物（adc）的方法要么生产难以制造和表征的混合物，要么使用位点特异性偶联技术，需要在插入可能具有免疫原性的肽标签或非天然氨基酸后重新制作感兴趣的抗体。在临床前研究中，这种方法将允许将几种不同的抗体与感兴趣的细胞毒素“即插即用”结合，从而快速确定最佳组合。此外，在生产环境中，这些变异的tpase将允许在特定的化学计量学下快速生产具有感兴趣的细胞毒素标记位点的adc，从cGMP制造的抗体开始。这将大大减少ADC制造和表征的时间和成本，并为患者提供更均匀、更好表征和更安全的产品。如果成功，这个项目的更广泛的影响/商业潜力将是药物输送和生物治疗的新应用。由于adc最近在临床中的显著成功，快速和位点特异性标记抗体的技术具有巨大的市场，并且被发现adc的公司以及制造adc的cmo所寻求。此外，随着adc单疗程治疗费用上升至10万美元，迫切需要找到既能满足这一日益增长的需求，又不会增加支付方成本的技术。除了adc之外，使用我们设计的TPases进行位点特异性标记将允许为抗体治疗产生伴随诊断，从而实现患者群体的有效分层以及手术环境中肿瘤边界的成像。此外，无需修饰或只需最小修饰即可结合蛋白质的能力也将允许产生免疫毒素/免疫酶，其中抗体与靶细胞内催化作用的强效蛋白质毒素或酶结合。最后，制造有效的设计蛋白连接酶的能力将允许开发试剂工具箱，就像DNA操作的限制性内切酶一样，这将为蛋白质工程领域提供有价值的构建工具包。

This Small Business Innovation Research (SBIR) Phase I project proposes to develop a platform technology utilizing directed evolution to engineer transpeptidases (TPases) that will recognize sequences within native antibodies, and conjugate any small molecule, peptide or protein moiety of interest through a peptide bond. Current approaches for production of Antibody Drug Conjugates (ADCs) either produce mixtures that are hard to manufacture and characterize, or use technology for site-specific conjugation that necessitates remaking the antibody of interest after inserting potentially immunogenic peptide tags or non-natural amino-acids. In pre-clinical research, this approach will allow "plug and play" conjugation of several different antibodies with cytotoxin(s) of interest, allowing rapid identification of the best combination(s). Furthermore, in a manufacturing setting, these variant TPases will allow rapid production of ADCs labeled site-specifically with the cytotoxin of interest at a defined stoichiometry, starting with cGMP manufactured antibodies. This will dramatically reduce the time and cost of ADC manufacturing and characterization, and provide more homogenous, better-characterized, and safer products to patients.The broader impact/commercial potential of this project, if successful, will be novel applications for drug delivery as well as biotherapeutics. Due to the remarkable recent success of ADCs in the clinic, technologies for rapid and site-specific labeling of antibodies have a large market and are sought by companies discovering ADCs as well as CMOs manufacturing ADCs. Moreover, with the cost of a single course of treatment with ADCs escalating to $100,000, there is an urgent need to find technologies that are able to meet this growing demand without increasing costs for payors. Beyond ADCs, site-specific labeling with our designer TPases will allow generation of companion diagnostics for antibody therapeutics to enable effective stratification of patient populations as well as imaging of tumor boundaries in surgical settings. In addition, the ability to conjugate proteins without modification or with minimal modification, will also allow the creation of immunotoxins/immunoenzymes where antibodies are tethered to potent protein toxins or enzymes that act catalytically within target cells. Lastly, the ability to make what are effectively designer protein ligases will allow the development of a toolbox of reagents, not unlike restriction enzymes for DNA manipulations, which will yield a valuable construction kit for the field of protein engineering.