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

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

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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.

在此多PI R21应用中，我们建议提高肿瘤靶向的抗癌效率和细胞穿透抗体（3E10）直接抑制DNA修复因子RAD51，从而抑制同源指导的DNA修复（HDR），敏感的癌细胞对辐射，并在brca2-- 和缺乏PTEN的细胞。我们将通过结构，生化，基于细胞和动物的结合来做到这一点肿瘤研究。该建议将利用DNA结构生物学的两个PI的完整专业知识结合蛋白和大分子机器（Bleichert）以及在DNA修复中，肿瘤生物学和临床前癌症治疗的发展（Glazer）。 3E10源自全身性狼疮的鼠标模型红斑座，最初被表征为一种可以穿透细胞并定位的抗DNA抗体我们随后的工作进一步将3E10定义为Rad51重组酶的抑制剂结合，为其对DNA修复的影响提供了一种机械解释。重要的是，我们也建立了与健康组织相比，该3E10靶向具有极高特异性的小鼠体内实体瘤。这种靶向特异性是由于细胞渗透机制引起的，这取决于与 ENT2转运蛋白，与健康组织相比，人类癌症高表达肿瘤微环境中存在细胞外DNA，从而促进了与ENT2的3E10相互作用。 3E10靶向肿瘤并直接穿透细胞和核的能力将其与所有其他区分开目前已批准用于癌症治疗的抗体；因此，3E10可能代表了一个全新的一类癌症治疗剂。在最近研究3E10和特定氨基酸取代的变异的工作中，我们已经发现3E10的DNA结合，细胞穿透和肿瘤靶向特性是独立的来自rad51结合。这使我们假设应该产生3e10的变体在保留细胞渗透和肿瘤靶向特性的同时，rad51结合亲和力增加。我们进一步假设，这种3e10变体将是更大的潜在rad51抑制剂，并且是上等的用于DNA修复抑制，辐射敏感性和合成致命方法的抗癌剂。和对BRCA2和PTEN缺陷癌的合成致死性增加，此类第二代抗体对于这些因素不足的广泛人类癌症，将特别有用。在此应用中，我们建议对3E10抗体进行结构研究，以深入了解其DNA结合和RAD51抑制的分子基础。这项工作将指导小说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，目的是确定优化的第二代抗体以促进临床发展。因此，拟议的工作具有转化为诊所的很高潜力可能对人类健康有直接而实质性的影响。