Validation of the Pre-BCR Signaling Complex in pre-B ALL Cell Model by Two-Color Single Particle Tracking and Peptidomimetic Inhibition

 描述(由申请人提供)：有强有力的证据支持‘紧张性信号’是导致从前B细胞到成熟的、完全有能力的B细胞的发育过程中的中心特征。这些信号被认为是通过前B细胞受体(Pre-BCR)介导的，对于保护前B细胞免受凋亡是必不可少的。这一建议是基于一个中心假设，即B细胞前体急性淋巴细胞性白血病(BCP-ALL)原始细胞也需要这种BCR前存活机制，这可能有助于抵抗标准的化疗挑战并调节增殖率。此外，来自Pre-BCR的强直信号被认为是通过1)通过代理轻链的L5组分的自结合而发生的瞬时二聚和2)Galectin介导的Pre-BCR的交联而发生的，从而形成更稳定的Pre-BCR二聚体和潜在的高阶低聚物。为了测试这些概念，这项研究将结合最先进的成像方法，包括单粒子跟踪、FRAP和高光谱成像。这种bcr前二聚体启动Lyn介导的免疫受体酪氨酸激活基序(ITAM)在bcr前信号成分(Igα和Igβ)的细胞质尾部的磷酸化。ITAM的磷酸化导致脾酪氨酸激酶(Syk)的募集和下游信号级联的激活，参与细胞命运的决定。这些关键事件的读数将基于免疫共沉淀、抗PY蛋白印迹和邻近结扎。这项提案的另一个主要目标是开发模拟多肽的抑制剂来阻断BCR前二聚化和紧张性信号。该项目的这一阶段将使用分子动力学方法设计抑制肽，使用严格的评分标准，然后进行实验以优化结合，并使用先进的成像技术和细胞信号分析测试BCR前自结合和/或Galectin结合界面的破坏。随后，所产生的多肽将用于筛选噬菌体和酵母库，以开发能够与BCR前亚组分高亲和力结合的单链抗体。总体目标是开发与前BCR高亲和力结合并阻断生存信号的单价生物制剂(多肽模拟物，ScFv-Fc)。ScFv-Fc将被评估招募NK细胞和巨噬细胞进行抗体介导的细胞毒(ADCC)或吞噬(ADCP)的潜力，这是开发抗BCR前治疗性抗体的关键第一步。

 DESCRIPTION (provided by applicant): There is strong evidence to support 'tonic signaling' as central feature in the developmental process leading from pre-B cells to mature, fully competent B cells. These signals are thought to be mediated through the pre-B Cell Receptor (pre-BCR) and essential for pre-B cell protection from apoptosis. This proposal is based upon the central hypothesis that B Cell Precursor Acute Lympoblastic Leukemia (BCP-ALL) blasts also require this pre-BCR survival mechanism, which may contribute to resistance to standard chemotherapeutic challenges and modulate proliferative rates. Moreover, tonic signaling from the pre-BCR is proposed to occur through 1) transient dimerization that occurs through self-association of the l5 component of the surrogate light chain and 2) galectin-mediated crosslinking of pre-BCR, forming more stable pre-BCR dimers and potentially higher- order oligomers. To test these concepts, this study will incorporate state-of-the-art imaging methods, including single particle tracking, FRAP and hyperspectral imaging. This pre-BCR dimerization initiates lyn-mediated phosphorylation of Immunoreceptor Tyrosine-based Activation Motifs (ITAMs) on the cytoplasmic tails of pre- BCR signaling components (Igα and Igβ). ITAM phosphorylation leads to recruitment of spleen tyrosine kinase (Syk) and activation of downstream signaling cascades involved in cell fate decisions. Readouts of these key events will be based on co-immunoprecipitation, anti-PY western blotting and proximity ligation. Another major goal of this proposal is to develop peptidomimetic inhibitors to block pre-BCR dimerization and tonic signaling. This phase of the project will use molecular dynamic approaches to design inhibitory peptides using a stringent scoring criteria, followed by experiments to optimize binding and to test for disruption of the pre-BCR self- association and/or galectin-binding interfaces using the advanced imaging technologies and cell signaling assays. Later, the generated peptides will be used to screen phage and yeast libraries to develop scFv that can bind to the pre-BCR subcomponents with high affinity. The overall goal is to develop monovalent, biologic agents (peptidomimetics, scFv-Fc) that bind with high-affinity to the pre-BCR and block prosurvival signaling. The scFv-Fc will be evaluated for the potential to recruit NK cells and macrophages for antibody-mediated cytotoxicity (ADCC) or phagocytosis (ADCP), a critical first step for development of anti-pre-BCR therapeutic antibodies.