Engineering high affinity tumor-targeting peptides against carbonic anhydrase IX

描述（申请人提供）：碳酸酐酶IX（CA IX）是一种膜结合蛋白，在缺氧环境下在癌细胞表面过度表达。 CA IX 参与肿瘤细胞存活和转移，表达增加与不良临床结果相关，但目前还没有批准的针对 CA IX 的疗法。单克隆抗体已用于靶向 CA IX，但它们的大尺寸限制了对血管化不良肿瘤的渗透，并且由于高背景和毒性，它们缓慢的血液清除限制了它们作为肿瘤显像剂或放射治疗剂的用途。抑制 CA IX 的有机小分子具有高度非特异性，可以扩散穿过细胞膜，与健康组织中丰富的细胞内碳酸酐酶亚型结合。在这里，我们提出了几种策略来设计高度稳定的约束肽（结蛋白）和小分子缀合物，以低纳摩尔亲和力选择性地结合 CA IX 的胞外结构域。这项工作将确定新型 CA-IX 靶向肽，用于临床转化作为诊断和治疗剂，还将产生可广泛应用于癌症和其他疾病的靶向膜受体的技术。 目标 1：通过改造以高亲和力与 CA IX 结合的结蛋白肽来开发肿瘤靶向药物。我们将使用酵母表面展示来设计结蛋白肽，使其在低纳摩尔范围内以高亲和力与 CA IX 结合。我们将测量工程结蛋白肽与肿瘤细胞表面表达的 CA IX 的相对结合亲和力。 目标 2：通过将小分子 CA IX 抑制剂与打结肽化学结合来开发肿瘤靶向药物。我们将通过化学方法将小分子 CA IX 抑制剂与纽蛋白肽结合，将已知小分子的 CA IX 靶向特性与纽蛋白肽提供的有利组织生物分布相结合。除了产生新的 CA IX 靶向分子外，该目标还将开发一个通用技术平台，以改善小分子肿瘤靶向剂的生物分布，并将产生一种创建双特异性肿瘤靶向剂的新方法。 目标 3：测量工程 CA IX 靶向药物在活体受试者中的生物分布和肿瘤摄取。将测试工程化的 CA IX 结合肽在体内靶向缺氧肿瘤的能力。 MicroPET 成像、生物分布研究和代谢稳定性研究将在人类荷瘤小鼠异种移植模型中使用 64Cu 标记的纽结蛋白肽和小分子缀合物进行。这一目标将进一步确立 CA IX 作为癌症诊断和治疗的靶点，并验证工程化的 CAIX 结合结蛋白肽用于其他临床研究。 公共健康相关性：针对碳酸酐酶 IX (CA IX)（一种在癌细胞表面过度表达的膜结合蛋白）的工程肽的出现，将为肿瘤发生、癌症生物学、分子成像和基于结构的药物设计开辟新的研究领域。我们提议的临床前研究将验证 CA IX 作为癌症成像的分子靶标，以确定最能从靶向治疗中受益的患者并监测他们的疾病进展。此外，这项工作将为未来开发工程化的 CA IX 结合肽作为肿瘤特异性递送化疗药物和放射性核素的靶向剂奠定基础。

DESCRIPTION (provided by applicant): Carbonic anhydrase IX (CA IX) is a membrane bound protein overexpressed on the surface of cancer cells in a hypoxic environment. CA IX is involved in tumor cell survival and metastasis, and increased expression correlates with poor clinical outcome, however there are no approved therapies against CA IX. Monoclonal antibodies have been used to target CA IX, but their large size limits penetration throughout a poorly vascularized tumor, and their slow blood clearance limits their use as tumor imaging agents or radiotherapeutics due to high background and toxicity. Small organic molecules that inhibit CA IX are highly non-specific, and can diffuse across cell membranes to bind to intracellular carbonic anhydrase isoforms abundant in healthy tissue. Here, we propose several strategies to engineer highly stable constrained peptides (knottins) and small molecule conjugates that selectively bind to the extracellular domain of CA IX with low nanomolar affinity. This work will identify novel CA-IX targeting peptides for clinical translation as diagnostic and therapeutic agents, and will also generate technology that could broadly be applied to target membrane receptors in cancer and other diseases. Aim 1: Develop tumor-targeting agents by engineering knottin peptides that bind to CA IX with high affinity. We will use yeast surface display to engineer knottin peptides that bind to CA IX with high affinities in the low nanomolar range. We will measure the relative binding affinities of engineered knottin peptides to CA IX expressed on the surface of tumor cells. Aim 2: Develop tumor-targeting agents by chemically conjugating small molecule CA IX inhibitors to knottin peptides. We will chemically couple small molecule CA IX inhibitors to knottin peptides to combine the CA IX targeting properties of known small molecules with the favorable tissue biodistribution afforded by knottin peptides. In addition to generating new CA IX targeting molecules, this aim will result in the development of a general technology platform to improve the biodistribution of small molecule tumor-targeting agents and will result in a novel approach for creating bi-specific tumor targeting agents. Aim 3: Measure biodistribution and tumor uptake of engineered CA IX targeting agents in living subjects. Engineered CA IX binding peptides will be tested for their ability to target hypoxic tumors in vivo. MicroPET imaging, biodistribution studies, and metabolite stability will be performed with 64Cu-labeled knottin peptides and small molecule conjugates in human tumor-bearing mouse xenograft models. This aim will further establish CA IX as a target for cancer diagnosis and therapy, and validate engineered CAIX-binding knottin peptides for additional clinical studies. PUBLIC HEALTH RELEVANCE: The availability of engineered peptides that target carbonic anhydrase IX (CA IX), a membrane bound protein overexpressed on the surface of cancer cells, will open up new research areas in tumorigenesis, cancer biology, molecular imaging, and structure-based drug design. The preclinical studies we are proposing will validate CA IX as a molecular target for cancer imaging to identify patients who would benefit most from targeted therapies and to monitor their disease progression. Moreover, this work will lay a foundation for future development of engineered CA IX-binding peptides as targeting agents for tumor-specific delivery of chemotherapeutics and radionuclides.