IMAT-ITCR Collaboration - Identification and development of T cell receptor mimic antibodies for high value neoantigen targets in triple negative breast cancer

IMAT-ITCR 合作 - 针对三阴性乳腺癌高价值新抗原靶标的 T 细胞受体模拟抗体的鉴定和开发

• 批准号: 10460807
• 负责人: Georgios Alexandrakis
• 金额: $ 7.7万
• 依托单位: UNIVERSITY OF TEXAS ARLINGTON
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10460807
• 关键词: Administrative Supplement; Affinity; Antibodies; Antibody Therapy; Antigens; Antitumor Response; Autoimmunity; Avidity; Binding; Biological Assay; Biopsy Specimen; Bypass; Cancer Vaccines; Cell Count; Cells; Cellular immunotherapy; Charge; Clinical; Code; Collaborations; Complement; Complex; Data; Detection; Development; Devices; Engineering; Enzyme-Linked Immunosorbent Assay; Evaluation; Flow Cytometry; Funding; Glycoproteins; Goals; HLA Antigens; Health; Heterogeneity; Human; Immunohistochemistry; Individual; Informatics; Intervention; Laboratories; Ligands; Liquid Chromatography; Major Histocompatibility Complex; Malignant Neoplasms; Mass Spectrum Analysis; Measurement; Mediating; Methods; Microfluidics; Molecular Analysis; Optics; Parents; Peptides; Performance; Proteins; Proteomics; Recombinants; Scanning; Signal Transduction; Somatic Mutation; Surface; T cell therapy; T-Cell Development; T-Cell Receptor; T-Lymphocyte; Technology; Testing; Texas; Tissues; Tumor Antigens; Tumor Tissue; Universities; Vaccines; Validation; Washington; Work; anticancer research; base; cancer cell; cell killing; fighting; immune checkpoint blockade; innovation; insight; nanosensors; neoantigens; patient biomarkers; personalized immunotherapy; receptor; sensor; sensor technology; success; tandem mass spectrometry; therapeutic target; triple-negative invasive breast carcinoma; tumor; tumor xenograft

Abstract, as submitted in the parent (R21 CA240220) application Major histocompatibility complexes (MHC), also termed Human Leukocyte Antigens (HLA) in humans are glycoproteins expressed on the surface of nucleated cells that act as proteomic scanning chips by providing insight into the status of cellular health. The recognition of antigen-presenting receptors by recombinant T-cell receptor (TCR)-like antibodies that mediate specific cancer cell killing forms the basis for newly emerging and very promising approaches to fight cancer that include antibody therapies, vaccines and cell-based immunotherapy. The success of these interventions depends on their personalization to a patient's biomarkers such as peptides presented by MHC-I molecules (pMHCs). The higher affinity binding of TCR-like antibodies to multiple pMHCs (higher avidity) can augment antitumor response significantly, up to a limit set by autoimmunity. The cell copy number of pMHCs targeted by specific TCR-like antibodies, is an important determinant of avidity and therefore of antitumor response. There is no easy way to quantify with current analytical technologies the number of pMHCs per cancer cell targeted by a specific TCR-like antibody. Common current methods for identifying antibody-ligand targeting include liquid chromatography with tandem mass spectrometry, ELISA, flow cytometry, immunohistochemistry and complement assays. These assays are challenged by low pMHC copy numbers often found in heterogeneous tumors. Our goal here is to develop a high-sensitivity nanosensor to quantify the copy number of pMHCs targeted by candidate TCR-like antibodies, enriched from only a few thousand (~104) cancer cells per assay. Working with low cell numbers will be essential for testing tumor pMHC heterogeneity from limited biopsy samples. Microfluidic isotachophoresis (ITP) will be integrated to the nanosensor to enable bound complex enrichment before detection. The nanosensor enables simultaneous quantification of size (optical signal) and effective charge (electrical signal). These bimodal data will provide independent measurements to verify whether an antibody forms a complex with the target ligand. In this proof of concept work, our overall hypothesis is that microfluidic ITP enrichment integrated with our sensor can detect individual TCR-like antibody-pMHC complexes isolated from ~104 cells derived from heterogeneous human tumor xenograft (PDX) tissues and distinguish specific binding from unbound protein, non-specific binding and aggregates to estimate targeted pMHC copy number per cancer cell. Accordingly, our Specific Aims are to (1a) determine sensor sensitivity limits to detect targeted complexes in pure protein solution, (1b) Implement ITP- based concentration enhancement and separation of antibody-pMHC complexes, and (2) Quantify copy number of targeted pMHCs enriched from cancer cells and PDX tumor tissues. Nanosensor sensitivity will be compared to ELISA, and mass spectroscopy will be used for pMHC target validation. Successful implementation of our Specific Aims will demonstrate how any feasibility gaps will be bridged to develop a clinical laboratory device in subsequent work for quantifying tumor pMHC expression heterogeneity to guide personalized immunotherapy.

摘要，如在父 (R21 CA240220) 申请中提交的那样 人类主要组织相容性复合体 (MHC)，也称为人类白细胞抗原 (HLA) 在有核细胞表面表达的糖蛋白，通过提供蛋白质组扫描芯片的作用 深入了解细胞健康状况。重组T细胞对抗原呈递受体的识别 介导特定癌细胞杀伤的受体（TCR）样抗体构成了新兴和 非常有前途的抗癌方法，包括抗体疗法、疫苗和基于细胞的疗法 免疫疗法。这些干预措施的成功取决于其针对患者生物标志物的个性化 例如 MHC-I 分子 (pMHC) 呈递的肽。 TCR 样抗体的更高亲和力结合 多种 pMHC（更高的亲和力）可以显着增强抗肿瘤反应，直至达到自身免疫设定的极限。 特定 TCR 样抗体靶向的 pMHC 的细胞拷贝数是亲合力的重要决定因素 从而影响抗肿瘤反应。使用当前的分析技术没有简单的方法来量化 每个癌细胞被特定 TCR 样抗体靶向的 pMHC 数量。目前常见的方法 识别抗体-配体靶向包括液相色谱串联质谱法、ELISA、流式细胞术 细胞计数、免疫组织化学和补体测定。这些检测方法受到 pMHC 拷贝数低的挑战 数量经常在异质性肿瘤中发现。我们的目标是开发一种高灵敏度纳米传感器 量化候选 TCR 样抗体靶向的 pMHC 的拷贝数，仅从少数抗体中富集 每次检测数千 (~104) 个癌细胞。使用低细胞数对于测试肿瘤 pMHC 至关重要 有限活检样本的异质性。微流控等速电泳 (ITP) 将集成到 纳米传感器可在检测前实现结合复合物富集。纳米传感器能够同时 尺寸（光信号）和有效电荷（电信号）的量化。这些双峰数据将提供 独立的测量来验证抗体是否与目标配体形成复合物。在这个证明中 概念工作，我们的总体假设是微流控 ITP 富集与我们的传感器集成可以检测 从来自异质人类的约 104 个细胞中分离出单个 TCR 样抗体-pMHC 复合物 肿瘤异种移植 (PDX) 组织并区分特异性结合与未结合蛋白、非特异性结合和 聚合来估计每个癌细胞的目标 pMHC 拷贝数。因此，我们的具体目标是 (1a) 确定传感器灵敏度极限以检测纯蛋白质溶液中的目标复合物，(1b) 实施 ITP- 基于抗体-pMHC复合物的浓度增强和分离，以及(2)量化拷贝数 从癌细胞和 PDX 肿瘤组织中富集的靶向 pMHC。将比较纳米传感器的灵敏度 ELISA 和质谱将用于 pMHC 靶标验证。我们的成功实施 具体目标将展示如何弥合任何可行性差距，以开发临床实验室设备 随后的工作是量化肿瘤 pMHC 表达异质性以指导个性化免疫治疗。

项目成果

Nanopore Data Analysis: Baseline Construction and Abrupt Change-Based Multilevel Fitting

• DOI: 10.1021/acs.analchem.1c01646
• 发表时间: 2021-08-17
• 期刊: ANALYTICAL CHEMISTRY
• 影响因子: 7.4
• 作者: Bandara, Y. M. Nuwan D. Y.;Saharia, Jugal;Kim, Min Jun
• 通讯作者: Kim, Min Jun

Self-Induced Back-Action Actuated Nanopore Electrophoresis (SANE) Sensor for Label-Free Detection of Cancer Immunotherapy-Relevant Antibody-Ligand Interactions.

• DOI: 10.1007/978-1-0716-1811-0_20
• 发表时间: 2022
• 期刊: Methods in molecular biology (Clifton, N.J.)

Use of a solid‐state nanopore for profiling the transferrin receptor protein and distinguishing between transferrin receptor and its ligand protein

使用固态纳米孔分析转铁蛋白受体蛋白并区分转铁蛋白受体及其配体蛋白

• DOI: 10.1002/elps.202200147
• 发表时间: 2022
• 期刊: ELECTROPHORESIS
• 影响因子: 2.9
• 作者: O'Donohue, Matthew;Saharia, Jugal;Bandara, Nuwan;Alexandrakis, Georgios;Kim, Min Jun
• 通讯作者: Kim, Min Jun