Cassette exons in neoplastic pro-B-cells: implications for immunotherapy

项目摘要/摘要 成功的儿童癌症免疫疗法通常针对的是谱系特异性，而不是癌症特异性 标记物，B细胞特异性CD19是最好的例子。这些成功在最近的FDA达到了顶峰 双特异性T细胞激活剂和嵌合抗原受体(CAR)臂T细胞在B细胞急性白血病中的应用 淋巴细胞白血病(B-ALL)。然而，使用CD19-1治疗的患者经常会复发。 定向免疫疗法，通常是由于表位丢失所致。而基于双重抗原靶向的策略是 开始出现时，它们仍然是以规范的B细胞标记物为目标的，有不可避免的一面 完全消融正常B细胞的效果。肿瘤特异性靶点在儿童癌症中的明显缺乏 (包括白血病)可能会限制未来的免疫疗法。我们假设另一种剪接 这可能既是B-ALL表位丢失的一种机制，也是新抗原的丰富来源。事实上，使用 计算和生化方法，我们已经确定了数百种局部剪接变异 (LSVs)，定位于具有显著胞外区的跨膜蛋白(例如，CD19和CD22) (外域)。事实上，在我们之前的工作中，我们描述了一种对CART-19的获得性抗性机制 基于CD19胞外区的选择性丢失，主要是通过跳过外显子2。的两大目标 这款U01如下所示。目的1：确定B-ALL细胞剪接改变的程序和决定因素 表面抗原。我们的首要目标是为白血病B细胞构建一种专门的“剪接密码”。是这样的 密码将预测顺式作用的遗传变异以及参与选择性剪接的反式作用因子。 并使我们能够识别所有选择性剪接的胞外结构域。然后在目标2中，我们将调查这些影响 B-ALL免疫疗法的选择性剪接。仅以CD22为例，我们将确定如何 截短的蛋白亚型使人对CD22靶向免疫疗法产生抵抗性，包括抗体- 药物结合物，如inotuzumab ozogamicin，最近被FDA批准用于治疗复发或 难治性B-ALL。我们还将提高针对跨越新的外显子连接的多肽的抗体，产生 抗体-药物结合物，并测试它们对B-ALL细胞系和患者来源的异种移植的有效性。在……里面 总结，基于白血病的U01将创建新的计算和概念框架，其中 将与儿科免疫疗法发现和发展网络(PI-DDN)高度协同 总体目标，包括确定在儿童时期独有表达的抗原表位 癌症和癌细胞--免疫逃避的内在机制。

PROJECT SUMMARY/ABSTRACT Successful immunotherapies for childhood cancers typically target lineage-, rather than cancer-specific markers, B-cell specific CD19 being the prime example. These successes culminated in the recent FDA approval of bi-specific T-cell engagers and chimeric antigen receptor (CAR)-armed T-cells for B-cell acute lymphoblastic leukemia (B-ALL). However, relapses frequently occur in patients treated with CD19- directed immunotherapies, often due to epitope loss. While strategies based on dual antigen targeting are beginning to emerge, they are still based on targeting canonical B-cell markers, with the unavoidable side effect of total ablation of normal B-cells. The apparent paucity of tumor-specific targets in pediatric cancers (including leukemias) is likely to limit future immunotherapies. We hypothesize that alternative splicing could be both a mechanism of epitope loss and a rich source of a neo-antigens in B-ALL. Indeed, using computational and biochemical approaches, we have identified hundreds of local splicing variations (LSVs), mapping to transmembrane proteins (e.g., CD19 and CD22) with prominent extracellular domains (ectodomains). In fact, in our prior work we described a mechanism of acquired resistance to CART-19 based on selective loss of the CD19 ectodomain, primarily through exon 2 skipping. The two large Aims of this U01 are as follows. Aim 1: To identify programs and determinants of altered splicing of B-ALL cell surface antigens. Our overarching goal is to construct a dedicated “splicing code” for leukemic B-cells. Such a code will predict cis-acting genetic variants as well as trans-acting factors involved in alternative splicing and allow us to identify all alternatively spliced ectodomains. Then in Aim 2, we will investigate the effects of alternative splicing on B-ALL immunotherapy. Using CD22 as just one example, we will determine how truncated protein isoforms confer resistance to CD22-targeting immunotherapeutics, including antibody- drug conjugates such as inotuzumab ozogamicin, which was recently approved by FDA to treat relapsed or refractory B-ALL. We will also raise antibodies against peptides spanning novel exon junctions, generate antibody-drug conjugates, and test their efficacy against B-ALL cell lines and patient-derived xenografts. In summary, this leukemia-based U01 will create new computational and conceptual frameworks, which would be highly synergistic with Pediatric Immunotherapy Discovery & Development Network (PI-DDN) overall goals, including identification of antigenic epitopes that are uniquely expressed on childhood cancers and of cancer cell-intrinsic mechanisms of immune evasion.