Structural Surfaceomics: A Strategy for Immunotherapy Target Discovery

结构表面组学：免疫治疗靶点发现的策略

• 批准号: 10434121
• 负责人: Arun P. Wiita
• 金额: $ 22.11万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10434121
• 关键词: Acute Myelocytic Leukemia; Acute leukemia; American; Antibody-drug conjugates; Antigen Targeting; Antigens; B-Lymphocytes; Basic Science; Biochemical; Bispecific Antibodies; CAR T cell therapy; Cell Line; Cell Therapy; Cell surface; Cells; Clinical; Communicable Diseases; Data; Detection; Development; Diagnosis; Disease; Elements; Engineering; Funding; Future; Generations; Genetic Transcription; Goals; Grain; Hematologic Neoplasms; Hematopoietic; Hematopoietic Neoplasms; Hybridomas; ITGB2 gene; Immune system; Immunotherapy; In Vitro; Integrins; Investigation; Leukocytes; Libraries; Lysine; Malignant Neoplasms; Mass Spectrum Analysis; Membrane Proteins; Methods; Molecular Conformation; Multiple Myeloma; Mus; New Agents; Normal Cell; Outcome; Pathway interactions; Pattern; Performance; Pharmaceutical Preparations; Prognosis; Protein Conformation; Proteins; Proteomics; Protocols documentation; Reporting; Rest; Risk; Safety; Sampling; Shapes; Specificity; Surface Antigens; System; Technology; Toxic effect; Tumor Antigens; Validation; Work; Yeasts; acute myeloid leukemia cell; base; cancer cell; cancer type; cellular engineering; chimeric antigen receptor T cells; crosslink; exhaust; experimental study; in vitro Assay; in vivo; murine antibody; nanobodies; neoplastic cell; new technology; novel; novel strategies; novel therapeutic intervention; pre-clinical; preclinical evaluation; therapeutic candidate; therapeutic target; transcriptome; transcriptome sequencing; tumor; tumor immunology

PROJECT SUMMARY/ABSTRACT Engineered cellular therapies, such as CAR-T cells, hold great promise as “living drugs”. However, the application of this approach beyond B-cell origin malignancies has been hampered by a lack of tumor-specific cell surface antigen targets. Current methods to identify new targets rely largely on expression patterns from RNA-seq data. However, the limitations of this transcriptome-based strategy have rapidly become apparent. We were struck by the recent serendipitous discovery of a tumor- specific surface antigen in the blood cancer multiple myeloma, amenable to CAR-T targeting, defined not by its expression but by its structural conformation compared to normal hematopoietic cells. The central hypothesis of this proposal is that many additional conformation-specific tumor antigens likely exist, across cancers, but currently there is no technology yet available to detect them. Here we aim to develop such a technology, combining our expertise in cell surface proteomics with crosslinking mass spectrometry, which we call “structural surfaceomics”. While this approach could be applied to any cancer, here we first explore acute myeloid leukemia (AML), a hematologic malignancy with poor clinical outcomes and a lack of highly-specific cell surface targets. In preliminary data, utilizing an initial version of the structural surfaceomics technology, we have already identified a novel conformation-specific antigen in AML that may be a promising therapeutic target. Here, we propose two Specific Aims: 1) Further development of the structural surfaceomics approach. Our preliminary protocol is restricted to highly-expressed surface antigens with sufficient lysine crosslinks to report on structural changes. To broaden applicability, we first aim to implement recently-described XL-MS strategies that can achieve much higher proteomic coverage. We will further assess the performance of our method using biochemical control of specific surface antigens, as well as profile additional AML lines for novel target discovery. 2) Development of novel CAR-T's targeting a conformation-specific AML antigen. We will generate CAR-T compatible binders both using a standard scFv approach, based on an existing murine antibody, as well as fully in vitro-selected nanobodies via yeast display. Our lab has recently demonstrated the latter strategy as a promising approach for cellular therapy in acute leukemia (Nix et al., in revision for Cancer Discovery). We will perform initial in vitro and in vivo validation of these cellular therapies. Overall, we anticipate developing an approach to identify an entirely new class of immunotherapy targets. Furthermore, we aim to demonstrate that our approach can nominate a promising cellular therapeutic candidate specific for AML. In future work, beyond this pilot funding, we anticipate applying structural surfaceomics to broader profiling of malignancies, as well as more complete preclinical validation of our AML conformation-targeting CAR-T's.

项目概要/摘要 工程化细胞疗法，例如 CAR-T 细胞，作为“活体药物”前景广阔。 这种方法在 B 细胞起源的恶性肿瘤之外的应用因缺乏 目前识别新靶点的方法很大程度上依赖于肿瘤特异性细胞表面抗原靶点。 然而，这种基于转录组的策略的局限性。 我们对最近偶然发现的一种肿瘤感到震惊—— 血癌多发性骨髓瘤中的特异性表面抗原，适合 CAR-T 靶向，定义 不是通过其表达，而是通过其与正常造血细胞相比的结构构象。 该提案的中心假设是许多其他构象特异性肿瘤抗原可能 跨癌症都存在，但目前还没有技术可以检测它们。 开发这样的技术，将我们在细胞表面蛋白质组学方面的专业知识与交联相结合 质谱分析，我们称之为“结构表面组学”，而这种方法可以应用于。 对于任何癌症，我们首先探讨急性髓系白血病 (AML)，这是一种恶性程度低的血液系统恶性肿瘤。 在初步数据中，利用了临床结果和缺乏高度特异性的细胞表面靶点。 结构表面组学技术的初始版本，我们已经确定了一种新颖的 在此，我们提出 AML 中的构象特异性抗原可能是一个有前途的治疗靶点。 两个具体目标：1）进一步发展结构表面组学方法。 方案仅限于具有足够赖氨酸交联的高表达表面抗原以进行报告 为了扩大适用性，我们首先旨在实施最近描述的 XL-MS。 我们将进一步评估可以实现更高蛋白质组覆盖率的策略。 我们的方法使用特定表面抗原的生化控制，以及分析其他 AML 2) 开发针对构象特异性的新型 CAR-T 我们将使用标准 scFv 方法生成 CAR-T 兼容结合物， 基于现有的鼠类抗体，以及通过酵母展示完全体外选择的纳米抗体。 我们的实验室最近证明了后一种策略是细胞治疗的一种有前途的方法 急性白血病（Nix 等人，癌症发现修订版）我们将进行初步的体外和体内实验。 总体而言，我们期望开发一种方法来识别细胞疗法。 此外，我们的目标是证明我们的方法。 可以在未来的工作中提名一种有前途的针对 AML 的细胞治疗候选药物。 通过试点资金，我们预计将结构表面组学应用于更广泛的恶性肿瘤分析，如 以及对我们的 AML 构象靶向 CAR-T 进行更完整的临床前验证。