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

这个小型企业创新研究(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)，这种转肽酶将识别天然抗体中的序列，并通过肽键连接任何感兴趣的小分子、多肽或蛋白质部分。目前生产抗体药物结合物(ADC)的方法要么是生产难以制造和表征的混合物，要么是使用定点连接技术，即在插入潜在的免疫原性多肽标签或非天然氨基酸后重新制造目标抗体。在临床前研究中，这种方法将允许几种不同的抗体与感兴趣的细胞毒素(S)“即插即用”结合，从而快速确定最佳组合(S)。此外，在制造环境中，这些变异的TPase将允许快速生产以特定化学计量比标记的ADC，从cGMP制造的抗体开始。这将大大减少ADC的制造和表征的时间和成本，并为患者提供更同质、更好和更安全的产品。如果该项目成功，其更广泛的影响/商业潜力将是药物输送和生物治疗的新应用。由于ADC最近在临床上取得了显著的成功，抗体的快速和定点标记技术拥有巨大的市场，并受到发现ADC的公司以及制造ADC的CMO的追捧。此外，随着ADC的单疗程治疗费用攀升至100,000美元，迫切需要找到能够满足这一日益增长的需求而不增加支付者成本的技术。除了ADC，我们设计的TPase的特定部位标记将允许生成抗体治疗的配套诊断，以实现患者群体的有效分层以及外科环境中肿瘤边界的成像。此外，不经修饰或只需最小限度的修饰就能结合蛋白质的能力，也将允许产生免疫毒素/免疫酶，其中抗体被拴在有效的蛋白质毒素或在靶细胞内催化作用的酶上。最后，制造有效设计的蛋白质连接酶的能力将允许开发试剂工具箱，就像用于DNA操作的限制酶一样，这将为蛋白质工程领域产生一个宝贵的构建工具。

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.