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

 描述（由申请方提供）：有强有力的证据支持"紧张性信号传导"是从前B细胞到成熟、完全感受态B细胞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，并且对于前B细胞保护免于凋亡是必需的。该提议基于中心假设，即B细胞前体急性成纤维细胞白血病（BCP-ALL）母细胞也需要这种前BCR存活机制，这可能有助于抵抗标准化疗挑战并调节增殖率。此外，提出来自前BCR的紧张性信号传导通过1）通过替代轻链的15组分的自缔合发生的瞬时二聚化和2）半乳糖凝集素介导的前BCR交联，形成更稳定的前BCR二聚体和潜在的更高级寡聚体。为了测试这些概念，这项研究将采用最先进的成像方法，包括单粒子跟踪，FRAP和高光谱成像。这种前BCR二聚化引发了前BCR信号传导组分（IG α和IG β）胞质尾部上免疫受体酪氨酸基活化基序（ITAM）的lyn介导的磷酸化。ITAM磷酸化导致脾酪氨酸激酶（Syk）的募集和参与细胞命运决定的下游信号级联的激活。这些关键事件的读数将基于免疫共沉淀、抗PY蛋白质印迹和邻近连接。该提案的另一个主要目标是开发肽模拟物抑制剂以阻断前BCR二聚化和紧张性信号传导。该项目的这一阶段将使用分子动力学方法，使用严格的评分标准设计抑制肽，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传导测定法检测前BCR自缔合和/或半乳糖凝集素结合界面的破坏。随后，产生的肽将用于筛选噬菌体和酵母文库，以开发能够以高亲和力结合前BCR亚组分的scFv。总体目标是开发以高亲和力结合前BCR并阻断促生存信号传导的单价生物制剂（肽模拟物，scFv-Fc）。将评价scFv-Fc募集NK细胞和巨噬细胞用于抗体介导的细胞毒性（ADCC）或吞噬作用（ADCP）的潜力，这是开发抗前BCR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.