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，如母申请（R21 CA 240220）中提交的 主要组织相容性复合物（MHC），在人类中也称为人类白细胞抗原（HLA）， 在有核细胞表面上表达的糖蛋白，其通过提供 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 介导特异性癌细胞杀伤的TCR样抗体形成了新出现的和 非常有前途的方法来对抗癌症，包括抗体疗法，疫苗和细胞为基础的 免疫疗法。这些干预措施的成功取决于它们对患者生物标志物的个性化 例如由MHC-I分子（pMHC）呈递的肽。TCR样抗体与人T淋巴细胞的更高亲和力结合， 多个pMHC（更高的亲合力）可显著增强抗肿瘤应答，直至自身免疫设定的极限。 特异性TCR样抗体靶向的pMHC的细胞拷贝数是亲和力的重要决定因素 因此也是抗肿瘤反应。用目前的分析技术没有简单的方法来量化 每个癌细胞被特异性TCR样抗体靶向的pMHC数量。目前常用的方法 鉴定抗体-配体靶向的方法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。这些检测受到低pMHC拷贝的挑战 在异质性肿瘤中常见。我们的目标是开发一种高灵敏度的纳米传感器， 定量由候选TCR样抗体靶向的pMHC的拷贝数， 每项检测可检测1000（~104）个癌细胞。使用低细胞数的工作对于检测肿瘤pMHC至关重要 来自有限活检样本的异质性。微流控等速电泳（ITP）将整合到 纳米传感器，以使结合的复合物富集检测前。纳米传感器可以同时 尺寸（光信号）和有效电荷（电信号）的定量。这些双峰数据将提供 通过独立测量来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，微流体ITP富集与我们的传感器集成可以检测 从来源于异质性人的~104个细胞分离的单个TCR样抗体-pMHC复合物 肿瘤异种移植物（PDX）组织，并区分特异性结合与未结合的蛋白质、非特异性结合和 聚集以估计每个癌细胞的靶向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靶向复合物，（1b）实施ITP- 基于抗体-pMHC复合物的浓度增强和分离，和（2）定量拷贝数 从癌细胞和PDX肿瘤组织中富集的靶向pMHC。将比较纳米传感器灵敏度 ELISA和质谱法将用于pMHC靶标验证。成功实施我们的 具体目标将证明如何弥合任何可行性差距，以开发临床实验室器械， 随后的工作用于定量肿瘤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