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

项目概要/摘要 儿童癌症的成功免疫疗法通常针对谱系，而不是癌症特异性 标记物，B 细胞特异性 CD19 就是最好的例子。这些成功在最近的 FDA 批准双特异性 T 细胞接合器和嵌合抗原受体 (CAR) 武装 T 细胞用于治疗 B 细胞急性 淋巴细胞白血病（B-ALL）。然而，接受 CD19- 治疗的患者经常出现复发。 定向免疫疗法，通常是由于表位丢失。虽然基于双抗原靶向的策略是 开始出现，它们仍然基于针对经典 B 细胞标记，但不可避免的一面 正常 B 细胞完全消融的影响。儿科癌症中明显缺乏肿瘤特异性靶点 （包括白血病）可能会限制未来的免疫疗法。我们假设选择性剪接 可能既是 B-ALL 中表位丢失的机制，也是新抗原的丰富来源。确实，使用 通过计算和生化方法，我们已经识别出数百种局部剪接变异 (LSV)，映射到具有突出胞外结构域的跨膜蛋白（例如 CD19 和 CD22） （胞外域）。事实上，在我们之前的工作中，我们描述了一种 CART-19 获得性耐药机制 基于 CD19 胞外域的选择性丢失，主要是通过外显子 2 跳跃。两大目标 该U01如下。目标 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 细胞构建专用的“剪接代码”。这样的 代码将预测顺式作用遗传变异以及参与选择性剪接的反式作用因子 并允许我们识别所有选择性剪接的胞外域。然后在目标 2 中，我们将研究效果 B-ALL 免疫疗法中的选择性剪接的研究。仅以 CD22 为例，我们将确定如何 截短的蛋白质亚型赋予对 CD22 靶向免疫治疗的耐药性，包括抗体- 药物偶联物，例如伊诺珠单抗奥佐米星 (inotuzumab ozogamicin)，最近被 FDA 批准用于治疗复发或 难治性B-ALL。我们还将产生针对跨越新外显子连接的肽的抗体，生成 抗体-药物偶联物，并测试其对 B-ALL 细胞系和患者来源的异种移植物的功效。在 总之，这个基于白血病的 U01 将创建新的计算和概念框架， 将与儿科免疫疗法发现和开发网络（PI-DDN）高度协同 总体目标，包括鉴定儿童时期独特表达的抗原表位 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.