EVOLVING HIGH AFFINITY SIRPa MUTANTS TO STIMULATE PHAGOCYTOSIS OF TUMOR CELLS

描述(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。通过巨噬细胞表面发现的抑制性受体SIRP发出信号，CD47能够抑制吞噬功能。许多癌细胞上调其表面CD47的表达，从而避免被巨噬细胞吞噬。然而，在一些临床前模型中，阻断CD47和SIRP之间相互作用的抗体可以增加吞噬功能并消除肿瘤。一种类似的封闭剂可以通过产生一种可溶性的SIRP来开发。使用这种分子可能会更有效，因为它会结合CD47的整个界面来阻断信号。然而，天然SIRP和CD47之间的弱相互作用限制了这种疗法的疗效。为此，酵母展示系统被用来生物工程Sirp突变体，使其与人CD47具有更高的结合亲和力。这项建议的目的是表征这些SIRP突变蛋白的生化特性，并评估它们作为免疫刺激癌症治疗药物的有效性。首先，将通过表面等离子体共振(目标1)来测量Sirp突变体的生化特性。接下来，将使用纯化的巨噬细胞和人类癌细胞在体外测试SIRP突变体的功能效力(AIM 2)。最后，将使用人类肿瘤的临床前异种移植小鼠模型(AIM 3)来评估SIRP突变蛋白的体内疗效。由于CD47是肿瘤细胞逃避免疫系统的常见机制，这项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，可以摄取碎片、细菌和其他病原体。这项提议的目标是开发新的疗法，刺激巨噬细胞攻击肿瘤细胞，作为消除多种人类癌症的一种方式。

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.