Structural Surfaceomics: A Strategy for Immunotherapy Target Discovery

项目摘要/摘要 工程细胞疗法，如CAR-T细胞，被认为是“活药”，前景广阔。然而， 这种方法的应用超出了B细胞起源的恶性肿瘤的范围，因为缺乏 肿瘤特异性细胞表面抗原靶标。目前识别新目标的方法很大程度上依赖于 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 已经迅速变得明显起来。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符合CAR-T靶向的特异性表面抗原 与正常的造血细胞相比，不是通过它的表达，而是通过它的结构构象。这个 这一提议的中心假设是，许多额外的构象特异性肿瘤抗原可能 存在于癌症之间，但目前还没有可用的技术来检测它们。在这里我们的目标是 为了开发这样一种技术，将我们在细胞表面蛋白质组学方面的专业知识与交联剂结合起来 质谱学，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系白血病(AML)，这是一种恶性血液病 临床结果和缺乏高度特异的细胞表面靶点。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 AML中的构象特异性抗原可能是一个有希望的治疗靶点。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。我们的预赛 协议仅限于具有足够赖氨酸交联度的高表达表面抗原以供报告 结构性变化。为了扩大适用性，我们首先致力于实现最近描述的XL-MS 可以实现更高的蛋白质组覆盖率的策略。我们将进一步评估其表现。 我们的方法使用特定表面抗原的生化控制，以及对额外的AML进行剖析 新奇目标发现的台词。2)针对特定构象的新型CAR-T的开发 急性髓系白血病抗原。我们将使用标准的scFv方法生成与CAR-T兼容的绑定器， 基于现有的小鼠抗体，以及通过酵母展示在体外完全选择的纳米体。 我们的实验室最近展示了后一种策略，作为一种有希望的细胞治疗方法 急性白血病(Nix等人，《癌症发现》修订版)。我们将在体外和体内进行初步的实验 对这些细胞疗法的验证。总体而言，我们预计将开发一种方法来识别 一类全新的免疫治疗靶点。此外，我们的目标是证明我们的方法 可以提名一种有前途的针对AML的细胞治疗候选药物。在今后的工作中，除此之外， 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概况，如 以及更完整的针对CAR-T的AML构象的临床前验证。

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.