Drug binding microprotein domains for targeted delivery of cytotoxic drugs

描述（由申请人提供）：将细胞毒性药物靶向递送到肿瘤组织是一种有效的策略，可以最大限度地减少正常组织的药物暴露，从而提高这些药物的毒性和疗效。肿瘤靶向系统由肿瘤识别片段与细胞毒性载荷相连接组成。抗体-药物结合物代表了这种方法的最先进形式。这些系统依赖于药物分子的化学偶联，通过各种连接化学靶向部分。化学修饰和耦合步骤的需要大大增加了制造过程的成本和复杂性。此外，人们仍然担心连接物可能具有不适当的稳定性特征，药物可能不会以活性状态或达到疗效所需的数量释放，并且偶联过程会干扰单抗的结合特性。因此，我们寻求一种通用的解决方案，这种解决方案将绕过复杂的化学偶联过程的需要，并将直接适用于各种各样的靶向方式。我们设想了一种基于蛋白质的结构域，它可以非共价地结合小分子药物。结合和稳定性特征可以根据药物释放的目标环境直接定制。重要的是，这些药物结合结构域可以在基因上融合到靶向结构域。微蛋白是一种非常小的蛋白质，具有很高的二硫键密度，具有独特的特性，使它们特别适合于这一目的。它们独特的结构允许容纳很大程度的序列和结构多样性。它们的小尺寸、稳定性和非免疫原性也是有吸引力的治疗特性。作为概念的初步证明，可以立即扩展到治疗产品概念，我们建议开发可以特异性结合常用抗癌药物阿霉素的微蛋白结构域，并在酸性或还原环境中释放它-在细胞内摄取而不是在体循环中普遍存在的条件。我们基于不同的微蛋白支架家族设计并构建了10个噬菌体展示文库。这些文库的总多样性超过1011个独特序列。我们计划通过以下系统的具体目标集来测试我们方法的可行性。1)泛噬菌体展示文库，用于展示与固定化阿霉素结合的微蛋白噬菌体。2)确认富集的微蛋白特异性结合固定化阿霉素的能力。我们的目标是找出至少5种不同的铅变体。3)表征所选微蛋白的结合特性和血清稳定性。这些I期里程碑的完成将使我们能够获得重要的概念证明，并验证我们开发微蛋白作为靶向递送癌症治疗药物的药物结合域的策略。我们的最终目标是推进优化的微蛋白药物结合结构域，将其融合到临床重要的靶向部分，对肿瘤抗原（如CD22、CD30或CD74）具有特异性，并用于临床研究。

DESCRIPTION (provided by applicant): Targeted delivery of cytotoxic drugs to tumor tissues is an effective strategy to minimize drug exposure of normal tissues and thus improve the toxicity and efficacy profiles of these agents. A tumor targeting system consists of a tumor recognition moiety linked to a cytotoxic payload. Antibody-drug conjugates represent the most advanced form of this approach. These systems are dependent on the chemical conjugation of drug molecules to the targeting moieties through various linker chemistries. The need for chemical modification and coupling steps adds significant cost and complexity to the manufacturing process. Additionally, there remain concerns that the linkers may have inappropriate stability profiles, the drugs may not be released in their active states or in quantities needed to achieve efficacy, and the conjugation process will perturb mAb binding characteristics. We thus seek a universal solution that would circumvent the need for complex chemical conjugation processes and that would be directly applicable to a wide variety of targeting modalities. We envision a protein-based domain that would bind small molecule drugs non-covalently. The binding and stability profile can be directly customized to the environment where the drug is targeted for release. Importantly, these drug-binding domains can be genetically fused to targeting domains. Microproteins, which are very small proteins with high disulfide bond densities, possess distinctive properties which make them particularly suited for this purpose. Their unique structure allows the accommodation of large degrees of both sequence and structural diversity. Their small sizes, stability and non-immunogenicity are also attractive therapeutic attributes. As an initial proof of concept, which can be immediately extended to a therapeutic product concept, we propose to develop microprotein domains that can specifically bind the commonly used cancer drug, doxorubicin, and release it in acidic or reducing environments - conditions which prevail after intracellular uptake and not within the systemic circulation. We have designed and constructed 10 phage display libraries based on different microprotein scaffold families. The total diversity in these libraries exceeds 1011 unique sequences. We plan to test the feasibility of our approach through the systematic set of specific aims below. 1) Pan phage display libraries for microprotein-displaying phages which bind to immobilized doxorubicin. 2) Confirm ability of enriched microproteins to specifically bind to immobilized doxorubicin. Our goal is to identify at least 5 different lead variants. 3) Characterize binding properties and serum stability of the selected microproteins. Completion of these Phase I milestones will enable us to obtain important proof of concept and validation of our strategy for developing microproteins as drug-binding domains for targeted delivery of cancer therapeutics. Our ultimate goal would be to advance optimized microprotein drug-binding domains, which are fused to clinically important targeting moieties with specificity for tumor antigens such as CD22, CD30, or CD74, into clinical studies.