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.

项目摘要/摘要 工程的细胞疗法（例如CAR-T细胞）具有“活药”的巨大希望。然而， 超出B细胞起源的这种方法的应用受到了缺乏 肿瘤特异性细胞表面抗原靶标。当前识别新目标的方法很大程度上依赖 RNA-Seq数据的表达模式。但是，基于转录组的策略的局限性 迅速变得明显。最近发现肿瘤的偶然发现使我们感到震惊。 血液癌多发性骨髓瘤中的特定表面抗原，可以定义为CAR-T靶标 与正常的造血细胞相比，不是通过其表达而是其结构考虑。 该提议的中心假设是许多其他构象特异性肿瘤抗原可能 存在于整个癌症中，但目前尚无可用技术来检测它们。我们在这里 要开发这样的技术，将我们在细胞表面蛋白质组学方面的专业知识与交联的链接相结合 质谱法，我们称之为“结构表面学”。虽然这种方法可以应用于 任何癌症，我们首先探索急性髓细胞性白血病（AML），这是一种较差的血液恶性肿瘤 临床结果和缺乏高度特异性的细胞表面靶标。在初步数据中，使用 结构表面学技术的初始版本，我们已经确定了一种小说 AML中的构象特异性抗原可能是一个有前途的治疗靶标。在这里，我们建议 两个具体的目的：1）进一步发展结构表面学方法。我们的初步 方案仅限于具有足够赖氨酸交联的高度表达的表面抗原以报告 结构变化。为了扩大适用性，我们首先要实施最近描述的XL-MS 可以实现更高蛋白质组学覆盖的策略。我们将进一步评估性能 我们的方法使用特定表面抗原的生化控制以及剖面 新颖目标发现的线条。 2）开发新颖的Car-T针对特定构象的目标 AML抗原。我们将使用标准SCFV方法生成CAR-T兼容粘合剂， 基于现有的鼠抗体，以及通过酵母显示器完全体外选择的纳米体。 我们的实验室最近证明了后来的策略是一种有前途的细胞治疗方法 急性白血病（Nix等人，在癌症发现修订中）。我们将在体外和体内执行初始 这些细胞疗法的验证。总体而言，我们预计开发一种方法来确定 全新的免疫疗法靶标。此外，我们旨在证明我们的方法 可以提名AML特有的有希望的细胞治疗候选者。在以后的工作中 飞行员资助，我们预计将结构表面组学应用于更广泛的恶性肿瘤，作为 以及对我们的AML构型CAR-T的更完整的临床前验证。