Engineering high affinity tumor-targeting peptides against carbonic anhydrase IX

针对碳酸酐酶 IX 设计高亲和力肿瘤靶向肽

• 批准号: 7772227
• 负责人: JENNIFER R COCHRAN
• 金额: $ 17.14万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-03-01 至 2012-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7772227
• 关键词: Address; Affinity; Animal Model; Antibodies; Area; Binding; Binding Proteins; Biodistribution; Biological Assay; Blood; Boxing; Breast; Cancer Biology; Cell Culture Techniques; Cell Survival; Cell membrane; Cell surface; Cells; Cervix Uteri; Chemicals; Clinical; Clinical Research; Colon; Cyclic Peptides; Cystine; Development; Diagnostic; Diffuse; Disease; Disease Progression; Drug Design; Drug Kinetics; Engineering; Environment; Epitopes; Esophagus; Extracellular Domain; Figs - dietary; Flow Cytometry; Flowcharts; Foundations; Future; Goals; Human; Hypoxia; Image; Immunohistochemistry; Integrins; Kidney; Label; Laboratories; Libraries; Life; Ligands; Malignant Neoplasms; Measures; Membrane; Methods; Modeling; Molecular Target; Monitor; Monoclonal Antibodies; Mus; Mutation; Neoplasm Metastasis; Oligonucleotides; Organ; Outcome; Patients; Penetration; Peptide Synthesis; Peptides; Phage Display; Phase; Play; Positron-Emission Tomography; Property; Protein Engineering; Protein Isoforms; Proteins; Radiation therapy; Radioisotopes; Radiolabeled; Recombinants; Relative (related person); Research; Role; SHFM1 gene; Serum; Solid; Structure; Surface; Techniques; Technology; Testing; Therapeutic; Therapeutic Agents; Tissues; Toxic effect; Translations; Treatment Protocols; Work; Xenograft Model; Yeasts; base; cancer cell; cancer diagnosis; cancer imaging; cancer therapy; carbonate dehydratase; chemical conjugate; combinatorial; imaging modality; imaging probe; improved; in vivo; inhibitor/antagonist; lung Carcinoma; magnetic beads; molecular imaging; mutant; neoplastic cell; novel; novel strategies; overexpression; preclinical study; prevent; public health relevance; radiotracer; receptor; scaffold; small molecule; tumor; tumorigenesis; uptake

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.

描述（由申请人提供）：碳酸酐酶IX（CA IX）是一种在缺氧环境中癌细胞表面过表达的膜结合蛋白。CA IX参与肿瘤细胞存活和转移，并且增加的表达与不良临床结果相关，然而没有针对CA IX的批准的疗法。单克隆抗体已被用于靶向CA IX，但它们的大尺寸限制了贯穿血管化不良的肿瘤的渗透，并且由于高背景和毒性，它们的缓慢血液清除限制了它们作为肿瘤成像剂或放射治疗剂的用途。抑制CA IX的小有机分子是高度非特异性的，并且可以扩散穿过细胞膜以结合到健康组织中丰富的细胞内碳酸酐酶同种型。在这里，我们提出了几种策略，工程高度稳定的约束肽（knottins）和小分子共轭物，选择性地结合到CA IX的细胞外结构域与低纳摩尔亲和力。这项工作将确定新的CA-IX靶向肽作为诊断和治疗剂用于临床翻译，并将产生可广泛应用于靶向癌症和其他疾病的膜受体的技术。 目的1：通过设计与CA IX高亲和力结合的knottin肽，开发肿瘤靶向药物。我们将使用酵母表面展示工程knottin肽结合CA IX在低纳摩尔范围内的高亲和力。我们将测量工程knottin肽与肿瘤细胞表面上表达的CA IX的相对结合亲和力。 目的2：通过化学偶联小分子CA IX抑制剂和knottin肽来开发肿瘤靶向药物。我们将化学偶联小分子CA IX抑制剂与结蛋白肽，以将已知小分子的CA IX靶向性质与结蛋白肽提供的有利组织生物分布联合收割机组合。除了产生新的CA IX靶向分子外，该目标还将导致开发通用技术平台以改善小分子肿瘤靶向剂的生物分布，并将导致产生双特异性肿瘤靶向剂的新方法。 目的3：测量工程化CA IX靶向剂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。将在人荷瘤小鼠异种移植模型中使用64 Cu标记的结蛋白肽和小分子缀合物进行MicroPET成像、生物分布研究和代谢物稳定性研究。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。 公共卫生相关性：靶向碳酸酐酶IX（CA IX）（一种在癌细胞表面过表达的膜结合蛋白）的工程肽的可用性将在肿瘤发生、癌症生物学、分子成像和基于结构的药物设计中开辟新的研究领域。我们提出的临床前研究将验证CA IX作为癌症成像的分子靶点，以确定从靶向治疗中获益最多的患者，并监测他们的疾病进展。此外，这项工作将奠定了基础，为未来的发展工程CA IX结合肽作为靶向剂的肿瘤特异性输送化疗药物和放射性核素。