Drug binding microprotein domains for targeted delivery of cytotoxic drugs

描述(由申请人提供)：将细胞毒药物定向输送到肿瘤组织是一种有效的策略，可以最大限度地减少药物对正常组织的暴露，从而改善这些药物的毒性和疗效。肿瘤靶向系统由连接到细胞毒有效载荷的肿瘤识别部分组成。抗体-药物结合物代表了这一方法的最高级形式。这些体系依赖于药物分子通过各种连接物化学作用与靶向部分的化学偶联。对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，人们仍然担心接头可能具有不适当的稳定性，药物可能不会在其活性状态下释放或释放达到疗效所需的数量，以及结合过程将扰乱mAb结合特性。因此，我们寻求一种普遍的解决办法，避免复杂的化学结合过程的需要，并直接适用于各种靶向模式。我们设想了一个基于蛋白质的结构域，它将以非共价方式结合小分子药物。结合和稳定性曲线可以根据药物释放的目标环境直接定制。重要的是，这些药物结合域可以在基因上与靶向域融合。微蛋白是具有高二硫键密度的非常小的蛋白质，具有独特的性质，使它们特别适合于这一目的。它们独特的结构允许适应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属性。作为概念的初步证明，可以立即扩展到治疗产品概念，我们建议开发能够与常用抗癌药物阿霉素特异结合的微蛋白结构域，并在酸性或还原环境中释放它--这些条件在细胞内摄取后盛行，而不是在体循环中。我们根据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。我们计划通过以下一套系统的具体目标来测试我们方法的可行性。1)结合固定化阿霉素的微蛋白展示噬菌体展示文库。2)证实纯化的微蛋白与固定化阿霉素特异性结合的能力。我们的目标是确定至少5种不同的铅变种。3)鉴定所选微蛋白的结合特性和血清稳定性。这些第一阶段里程碑的完成将使我们能够获得重要的概念证明，并验证我们开发微型蛋白质作为靶向输送癌症治疗药物的药物结合域的战略。我们的最终目标将是推进优化的微蛋白药物结合域，这些结合域融合到具有肿瘤抗原特异性的临床重要靶向部分，如CD22、CD30或CD74，进入临床研究。

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.