EVOLVING HIGH AFFINITY SIRPa MUTANTS TO STIMULATE PHAGOCYTOSIS OF TUMOR CELLS

描述（由申请人提供）：CD47是一种细胞表面分子，对宿主免疫系统起到“不要吃我”信号的作用。通过SIRP？（巨噬细胞表面发现的抑制性受体）的信号传导，CD47能够抑制吞噬作用。许多癌细胞上调其表面的CD 47表达，使其能够避免被巨噬细胞摄入。然而，阻断CD47和SIRP之间相互作用的抗体在许多临床前模型中增加吞噬作用并消除肿瘤。类似的阻断剂可以通过产生可溶形式的SIRP来开发。使用这种分子可能更有效，因为它会结合CD47的整个界面以阻断信号传导。然而，天然SIRP和CD47之间的弱相互作用限制了这种疗法的功效。出于这个原因，酵母展示系统被用于生物工程改造SIRP突变体，其对人CD47的结合亲和力增加。该提案的目标是表征这些SIRP突变蛋白的生化特性，并评估其作为免疫刺激癌症治疗剂的功效。首先，将通过表面等离子体共振测量SIRP突变体的生物化学性质（目的1）。接下来，将使用纯化的巨噬细胞和人癌细胞在体外测试SIRP突变体的功能功效（目的2）。最后，将使用人肿瘤的临床前异种移植小鼠模型评估SIRP突变蛋白的体内功效（目的3）。由于CD47是肿瘤细胞逃避免疫系统的一种常见机制，因此本研究中评估的分子可能是许多类型癌症的有效免疫疗法。 公共卫生相关性：巨噬细胞是人体组织中发现的免疫细胞，可摄取碎片，细菌和其他病原体。该提案的目标是开发新的疗法，刺激巨噬细胞攻击肿瘤细胞，作为消除多种类型人类癌症的一种方法。

DESCRIPTION (provided by applicant): CD47 is a cell-surface molecule that acts as a "don't eat me" signal to the host immune system. By signaling through SIRP¿, an inhibitory receptor found on the surface of macrophages, CD47 is able to inhibit phagocytosis. Many cancer cells upregulate CD47 expression on their surface, allowing them to avoid ingestion by macrophages. However, antibodies that block the interaction between CD47 and SIRP¿ increase phagocytosis and eliminate tumors in a number of pre-clinical models. A similar blocking reagent could be developed by producing a soluble form of SIRP¿. The use of such a molecule could be more efficacious as it would bind the entire interface of CD47 to block signaling. However, the weak interaction between native SIRP¿ and CD47 limits the efficacy of such a therapy. For this reason, a yeast-display system was used to bioengineer SIRP¿ mutants with increased binding affinity for human CD47. The goal of this proposal is to characterize the biochemical properties of these SIRP¿ mutant proteins and evaluate their efficacy as immunostimulatory cancer therapeutics. First, the biochemical properties of the SIRP¿ mutants will be measured by surface plasmon resonance (Aim 1). Next, the functional efficacy of the SIRP¿ mutants will be tested in vitro using purified macrophages and human cancer cells (Aim 2). Last, in vivo efficacy of the SIRP¿ mutant proteins will be evaluated using pre-clinical xenotransplantation mouse models of human tumors (Aim 3). Since CD47 is a common mechanism that tumor cells use to evade the immune system, the molecules evaluated in this study could be effective immune-based therapies for many types of cancer. PUBLIC HEALTH RELEVANCE: Macrophages are immune cells found in human tissues that ingest debris, bacteria, and other pathogens. The goal of this proposal is to develop novel therapies that stimulate macrophages to attack tumor cells as a way of eliminating multiple types of human cancer.