Structural Surfaceomics: A Strategy for Immunotherapy Target Discovery

项目总结/摘要 工程细胞疗法，如CAR-T细胞，作为“活药物”有很大的希望。然而，在这方面， 这种方法在B细胞来源的恶性肿瘤之外的应用受到缺乏 肿瘤特异性细胞表面抗原靶标。目前识别新靶点的方法主要依赖于 来自RNA-seq数据的表达模式。然而，这种基于转录组的策略的局限性 迅速变得明显。我们被最近偶然发现的肿瘤所震惊- 血液癌症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。的 这一建议的中心假设是，许多额外的构象特异性肿瘤抗原可能 存在于各种癌症中，但目前还没有技术可以检测它们。在这里我们瞄准 将我们在细胞表面蛋白质组学方面的专业知识与交联技术相结合， 我们称之为“结构表面组学”虽然这种方法可以应用于 任何癌症，在这里我们首先探讨急性髓细胞白血病（AML），一种血液恶性肿瘤， 临床结果和缺乏高度特异性的细胞表面靶点。在初步数据中，利用 结构表面组学技术的初始版本，我们已经确定了一种新的 在AML中的构象特异性抗原，可能是一个有前途的治疗靶点。在这里，我们建议 两个具体目标：1）进一步发展结构表面组学方法。我们的初步 该方案仅限于具有足够赖氨酸交联的高表达表面抗原， 结构变化。为了扩大适用性，我们首先旨在实现最近描述的XL-MS 可以实现更高的蛋白质组覆盖率的策略。我们将进一步评估 我们的方法使用特定表面抗原的生化控制，以及额外的AML谱 用于新目标发现的线路。2)靶向构象特异性配体的新型CAR-T的开发 AML抗原。我们将使用标准scFv方法产生CAR-T相容性结合物， 基于现有的鼠抗体，以及通过酵母展示的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，在癌症发现的修订版中）。我们将在体外和体内进行初步研究 这些细胞疗法的有效性。总的来说，我们期望开发一种方法来识别 全新的免疫治疗靶点此外，我们的目标是证明我们的方法 可以提名一个有前途的细胞治疗候选人的AML特异性。在今后的工作中， 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， 以及我们的AML构象靶向CAR-T的更完整的临床前验证。

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.