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，如父母（R21 CA240220）中提交的摘要 主要的组织相容性复合物（MHC），也称为人类白细胞抗原（HLA） 糖蛋白在核细胞表面表达，该细胞表面充当蛋白质组学扫描芯片 深入了解细胞健康状况。通过重组T细胞识别抗原呈递受体 介导特定癌细胞杀伤的受体（TCR）类似于新出现和的抗体是 抗击癌症的非常有前途的方法，包括抗体疗法，疫苗和基于细胞的癌症 免疫疗法。这些干预措施的成功取决于它们对患者的生物标志物的个性化 例如MHC-I分子（PMHC）提出的肽。 TCR样抗体与 多个PMHC（较高的自发）可以显着增加抗肿瘤反应，直至自身免疫设定的极限。 由特定TCR样抗体靶向的PMHC的细胞拷贝数是亲戚的重要决定因素 因此具有抗肿瘤反应。没有简单的方法可以用当前的分析技术量化 由特定TCR样抗体靶向的每个癌细胞的PMHC数量。常见的当前方法 识别抗体 - 配体靶向靶向包括液相色谱法，串联质谱法，ELISA，流动 细胞仪，免疫组织化学和补体测定法。这些测定受到低PMHC副本的挑战 在异质肿瘤中经常发现的数字。我们的目标是开发高敏性纳米传感器 量化由候选TCR样抗体靶向的PMHC的拷贝数，仅从少数几个 每种测定千（〜104）个癌细胞。使用低细胞数对于测试肿瘤PMHC至关重要 有限活检样品的异质性。微流体等渗（ITP）将集成到 纳米传感器可在检测前实现结合的复合物富集。纳米传感器同时启用 量化尺寸（光信号）和有效电荷（电信号）。这些双峰数据将提供 独立测量以验证抗体是否与靶配体形成复合物。在这个证明 概念工作，我们的总体假设是与传感器集成的微流体ITP富集可以检测到 从源自异质人的〜104个细胞中分离出的单个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 骨料估算每个癌细胞的靶向PMHC拷贝数。因此，我们的具体目的是（1a） 确定传感器敏感性极限以检测纯蛋白质溶液中的靶向复合物，（1b）实现ITP- 抗体-PMHC复合物的基于浓度增强和分离，（2）量化拷贝数 从癌细胞和PDX肿瘤组织中富含的靶向PMHC。将比较纳米传感器灵敏度 到ELISA，质谱将用于PMHC目标验证。成功实施我们 具体目的将证明将如何桥接任何可行性差距以在 随后量化肿瘤PMHC表达异质性的工作以指导个性化的免疫疗法。

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