Expanding the design space of protein-small molecule conjugates

项目摘要/摘要 靶向治疗和靶向给药已经彻底改变了复杂疾病的临床护理 这些选择是可用的--然而，只有少数患者能够获得这些精准医疗 他们的护理是基于疾病的分子基础的方法。这种分子靶向导联 与传统的治疗方法相比，在一定范围内，更好的患者结局和更少的毒副作用 疾病的威胁。作为将精准医疗扩展到所有患有复杂疾病的患者的一部分，有一个关键的 需要满足目前不存在此类选择的临床护理中的分子靶向缺口。蛋白质- 药物结合物在靶向治疗和药物输送应用中具有巨大的前景，有许多 最近在抗体-药物结合物(ADCs)形式的癌症治疗方面取得了成功。然而，众多的设计 蛋白质-药物结合物的挑战仍然存在，包括治疗窗口狭窄和翻译困难 在肿瘤学方面取得成功，以适应其他临床适应症。即使在肿瘤学中，实体瘤仍然明显多于 比血液病癌症更难治疗。我们的假设是，当前的模块化组件集 设计空间是有限的，并导致了当前的许多挑战。因此，有一种未得到满足的需求需要创造 并验证目前正在使用的以外的蛋白质-药物结合成分和组合。这个 这项拟议研究的总体目标是扩大蛋白质-药物结合物的设计空间，使这一点成为可能 这类分子将在生物技术和医学的更广泛的应用中取得进一步的成功。我们 建议验证支架蛋白质和侧链反应性聚合物作为新的 蛋白质-药物共轭结构。作为这项工作的模型蛋白质支架，我们将使用Fn3多肽 Fold，这已经被证明是非常通用的工程分子识别使用Rational 和定向进化方法。我们建议用正则和非正则来修饰模型Fn3蛋白 使特定部位的生物结合反应成为可能的氨基酸(目标1)。作为一种新型的蛋白质-药物连接物系统 偶联物，我们建议使用侧链反应性聚(PFPA)作为合成蛋白质的模型聚合物。 聚合物-药物结合物。侧链反应性聚合物比目前使用的连接物在化学上更具通用性 适用于ADC，可提供多种功能，以克服当前ADC的限制。我们将使聚合物功能化 Poly(PFPA)能够连接到特定的蛋白质位置，并将可释放的小分子加载到聚合物上， 使用一系列药物负载率，结合小分子以实现联合治疗(AIM 2)。我们将测量和模拟偶联物的细胞和组织运输(目标3)，这是 治疗上的成功。这个应用的总体目标是扩展支架蛋白的能力和 侧链反应性聚合物填补了分子靶向挑战的空白。我们的工作将会有更广泛的 通过建立使用多种多肽折叠合成生物偶联物的方法和 聚合物连接物，朝着为每一个需要它们的患者创造有针对性的治疗的最终目标迈进。

PROJECT SUMMARY/ABSTRACT Targeted therapy and targeted drug delivery have revolutionized clinical care of complex disease whenever these options are available – and, yet, only a minority of patients have access to these precision medicine approaches where their care is based on the molecular basis of their disease. Such molecular targeting leads to better patient outcomes and fewer toxic side effects compared to traditional treatment approaches, for a range of diseases. As part of expanding precision medicine to all patients with complex diseases, there is a critical need to meet the molecular targeting gaps in clinical care where such options currently do not exist. Protein- drug conjugates have immense promise in targeted therapy and drug delivery applications, with a number of recent successes in the form of antibody-drug conjugates (ADCs) for cancer therapy. However, numerous design challenges for protein-drug conjugates remain, including narrow therapeutic windows and difficulty in translating success in oncology to other clinical indications. Even within oncology, solid tumors remain significantly more difficult to treat than hematological cancers. Our hypothesis is that the current set of modular components in the design space is limiting and leads to many of the current challenges. Therefore, there is an unmet need to create and validate protein-drug conjugate components and combinations beyond those currently being used. The overall goal of this proposed research is to expand the design space for protein-drug conjugates, enabling this class of molecules to have further success in a wider range of applications in biotechnology and medicine. We propose to validate scaffold proteins and side-chain reactive polymers as modular components for novel protein-drug conjugate structures. As a model protein scaffold for this work, we will use the Fn3 polypeptide fold, which has been demonstrated to be extremely versatile for engineering molecular recognition using rational and directed evolution approaches. We propose to modify model Fn3 proteins with canonical and non-canonical amino acids to enable site-specific bioconjugation reactions (Aim 1). As a novel linker system for protein-drug conjugates, we propose to use side-chain reactive poly(PFPA), as a model polymer for synthesizing protein- polymer-drug conjugates. Side-chain reactive polymers are more chemically versatile than current linkers in use for ADCs, and can serve multiple functions to overcome current ADC limitations. We will functionalize polymer poly(PFPA) to enable conjugation to specific protein sites, and load releasable small molecules onto the polymer, using a range of drug loading ratios, with combinations of small molecules to enable combination therapy (Aim 2). We will measure and model cellular and tissue trafficking of the conjugates (Aim 3), a key consideration for therapeutic success. The overall objective of this application is to extend the capacity of scaffold proteins and side-chain reactive polymers to fill the gap in molecular targeting challenges. Our work will have a broader positive impact by establishing methods for synthesizing bioconjugates using a myriad of polypeptide folds and polymer linkers, towards the ultimate goal of creating targeted treatments for every patient who needs them.