Structure-Function Studies of a Cell Penetrating Antibody that Inhibits DNA Repair

抑制 DNA 修复的细胞穿透抗体的结构功能研究

基本信息

批准号：
10633740
负责人：
Franziska Bleichert
金额：
$ 19.58万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-04-01 至 2025-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10633740
关键词：
Affinity Amino Acid Substitution Animals Anti-DNA Antibodies Anti-Idiotype Vaccine Antibodies Antineoplastic Agents BRCA2 gene Binding Biochemical Biochemistry Biological Assay Biotechnology Cell Nucleus Cells Clinic Clinical Clinical Treatment Crystallization DNA DNA Binding DNA Repair DNA Repair Inhibition DNA-Binding Proteins Generations Goals Health Human In Vitro Institution Licensing Lupus Malignant Neoplasms Measures Mediating Modeling Molecular Mus Mutagenesis Mutation Oligonucleotides PTEN gene Patients Penetration Phase I Clinical Trials Principal Investigator Property Rad51 recombinase Radiation Radiosensitization Reporter Resolution Solid Neoplasm Specificity Structure Systemic Lupus Erythematosus Testing Therapeutic Time Tissues Toxic effect Tumor Biology Variant Work X-Ray Crystallography anti-cancer cancer cell cancer therapy cell type clinical development clinical efficacy design extracellular improved in vivo inhibiting antibody inhibitor insight mouse model novel overexpression phase I trial preclinical development structural biology translational potential tumor tumor microenvironment

项目摘要

In this multi-PI R21 application, we propose to advance the anti-cancer efficacy of a tumor targeting and cell-penetrating antibody (3E10) that directly inhibits the DNA repair factor, RAD51, and thereby suppresses homology-directed DNA repair (HDR), sensitizes cancer cells to radiation, and is synthetically lethal to BRCA2- and PTEN-deficient cells. We will do so by a combination of structural, biochemical, cell-based and animal tumor studies. This proposal will leverage complementary expertise of the two PI’s in structural biology of DNA binding proteins and macromolecular machines (Bleichert) and in DNA repair, tumor biology and pre-clinical development of cancer therapeutics (Glazer). 3E10 was derived from a mouse model of systemic lupus erythematosus and was initially characterized as an anti-DNA antibody that could penetrate cells and localize in the nucleus. Our subsequent work further defined 3E10 as an inhibitor of the RAD51 recombinase via direct binding, providing a mechanistic explanation for its effect on DNA repair. Importantly, we have also established that 3E10 targets solid tumors in vivo in mice with extraordinarily high specificity compared to healthy tissues. This targeting specificity is due to the mechanism of cell penetration, which depends on engagement with the ENT2 transporter, which is highly over-expressed in human cancers compared to healthy tissues, and the presence of extracellular DNA in the tumor microenvironment, which promotes 3E10 interaction with ENT2. The ability of 3E10 to target tumors and directly penetrate cells and nuclei distinguishes it from all other antibodies currently approved for cancer therapy; as such, 3E10 potentially represents a totally new class of cancer therapy agents. In recent work studying variants of 3E10 with specific amino acid substitutions, we have discovered that the DNA binding, cell penetration, and tumor targeting properties of 3E10 are separable from RAD51 binding. This has led us to hypothesize that it should be possible to generate variants of 3E10 that have increased RAD51 binding affinity while retaining the cell penetration and tumor targeting properties. We further hypothesize that such a 3E10 variant would be an even more potent RAD51 inhibitor and a superior anti-cancer agent for DNA repair inhibition, radiation sensitization and synthetic lethal approaches. With increased synthetic lethality against BRCA2- and PTEN-deficient cancers, such a second-generation antibody would be particularly useful against the wide range of human cancers in which these factors are deficient. In this application, we propose to conduct structural studies of the 3E10 antibody to gain insight into the molecular basis of its DNA binding and RAD51 inhibition. This work will guide the design of novel 3E10 variants that will be tested for DNA binding, cell penetration, RAD51 inhibition, synthetic lethality, and tumor targeting, with the goal of identifying an optimized second-generation antibody to advance for clinical development. Consequently, the proposed work has a very high potential for translation into the clinic and could have a direct and substantial impact on human health.

在这个多PI R21应用中，我们建议提高肿瘤靶向和治疗的抗癌功效细胞穿透抗体 (3E10)，直接抑制 DNA 修复因子 RAD51，从而抑制同源定向 DNA 修复 (HDR)，使癌细胞对辐射敏感，并且对 BRCA2- 具有合成致死性我们将通过结构、生化、细胞和动物的结合来实现这一目标。该提案将利用两位 PI 在 DNA 结构生物学方面的互补专业知识。结合蛋白和大分子机器 (Bleichert) 以及 DNA 修复、肿瘤生物学和临床前癌症疗法的开发（Glazer）源自系统性狼疮小鼠模型。红斑狼疮，最初被定性为一种抗 DNA 抗体，可以穿透细胞并定位我们随后的工作进一步将 3E10 定义为 RAD51 重组酶的直接抑制剂。结合，为其对 DNA 修复的影响提供了机制解释。与健康组织相比，3E10 针对小鼠体内的实体瘤具有极高的特异性。这种靶向特异性是由于细胞渗透机制所致，该机制取决于与与健康组织相比，ENT2 转运蛋白在人类癌症中高度过度表达，肿瘤微环境中细胞外 DNA 的存在促进了 3E10 与 ENT2 的相互作用。 3E10 靶向细胞核肿瘤并直接穿透细胞的能力使其与所有其他药物区分开来目前已批准用于癌症治疗的抗体；因此，3E10 可能代表一类全新的抗体在最近对具有特定氨基酸取代的3E10的研究中，我们发现 3E10 的 DNA 结合、细胞渗透和肿瘤靶向特性是可分离的这使我们认识到应该有可能生成 3E10 的变体增加了 RAD51 结合亲和力，同时保留了细胞渗透和肿瘤靶向特性。此外，这样的 3E10 变体将是一种更有效的 RAD51 抑制剂，并且是一种更优异的 RAD51 抑制剂。用于 DNA 修复抑制、辐射增敏和合成致死方法的抗癌剂。增加针对 BRCA2 和 PTEN 缺陷癌症的合成致死率，例如第二代抗体对于对抗缺乏这些因子的多种人类癌症特别有用。在此应用中，我们建议对 3E10 抗体进行结构研究，以深入了解这项工作将指导新型 3E10 的设计。将测试 DNA 结合、细胞渗透、RAD51 抑制、合成致死性和肿瘤的变体靶向，旨在确定优化的第二代抗体以推进临床经过审查，拟议的工作具有很高的转化为临床和应用的潜力。可能对人类健康产生直接而重大的影响。