Engineering of macrophage phagocytosis for cancer and stem cell immunotherapy

描述（由申请人提供）：巨噬细胞是一种吞噬细胞，可以识别并“吃掉”外来细胞微生物，以及垂死细胞和异常细胞（如癌症）。它们展示了多种受体，通过这些受体，它们可以识别目标细胞上的“吃我”和“不要吃我”信号。巨噬细胞检查迁移的造血干细胞（HSC），当它们通过鼻窦。巨噬细胞也检查癌细胞，癌细胞表达吞噬前“吃掉我”的信号，以确保在称为程序性细胞清除的过程中摄入异常细胞。然而，成功的癌细胞通过表达CD47来逃避程序性细胞移除，CD47是一种显性的“不要吃我”信号，是巨噬细胞上表达的抑制受体SIRPα的配体。CD47连接SIRPα可阻断巨噬细胞吞噬肿瘤细胞。CD47/SIRPα轴代表一个开关，可以阻断或刺激不同的治疗目标。阻断CD47与SIRPα的结合可促进癌细胞的吞噬。相反，CD47/SIRPα相互作用是造血细胞移植（HCT）中移植成功的关键决定因素：HSC上CD47的表达水平与它们的相对可移植性相关。不幸的是，骨髓HSC表达低水平的CD47，导致它们在移植中相对低效。因此，根据临床情况，SIRPα/CD47系统为抗肿瘤和移植治疗提供了一个令人兴奋的新轴。然而，SIRPv或CD47的可溶性外结构域作为激动剂或拮抗剂的有效利用受到野生型CD47/SIRPα相互作用的低亲和力的限制。我们希望执行基于结构的工程方法来创建高亲和力的SIRPα和CD47外结构域，作为癌症和HCT的治疗方法。我们建议将Garcia实验室在结构生物学、蛋白质工程和免疫干预方面的专业知识与Weissman和Shizuru实验室在体内癌症和造血细胞移植生物学方面的优势结合起来，以靶向CD47/SIRPα轴。针对Aim #1，我们设计了高亲和力的SIRPα单体，可以阻止内源性CD47和SIRPα之间的相互作用，并与临床建立的治疗性单克隆抗体在体内刺激肿瘤细胞的吞噬作用。这种对巨噬细胞进行“1-2”致敏，然后用抗肿瘤单抗进行细胞溶解的方法是一种全新的策略。对于Aim #2，我们正在设计高亲和力CD47变体，激活SIRPα抑制信号并减少巨噬细胞激活，从而增强HSC植入。最后，在Aim #3中，我们希望重建和分子表征细胞表面“不要吃我”复合物，该复合物由CD47和SIRPα与包括血小板反应蛋白和整合素在内的假定替代配体组成，以充分了解该系统调节巨噬细胞吞噬的治疗潜力。我们预计这些研究将产生新的生物治疗药物，应用于许多类型的人类癌症和移植治疗。

DESCRIPTION (provided by applicant): Macrophages are phagocytic cells that recognize and 'eat' foreign cellular microbes, as well as dying cells and aberrant cells such as cancers. They display a variety of receptors by which they recognize 'eat me' and 'don't eat me' signals on their target cells. Macrophages inspect migrating hematopoietic stem cells (HSC) as they pass through the sinusoids. Macrophages also inspect cancer cells, which express pro-phagocytic 'eat me' signals to ensure that aberrant cells are ingested in a process termed programmed cell removal. However, successful cancer cells evade programmed cell removal by expressing CD47, a dominant 'don't eat me' signal that is a ligand for SIRPα, an inhibitory receptor expressed on macrophages. Ligation of SIRPα by CD47 blocks macrophages from phagocytosing the tumor cells. The CD47/SIRPα axis represents a toggle switch that can be blocked or stimulated for different therapeutic goals. Blocking binding of CD47 to SIRPα promotes phagocytosis of cancer cells. Conversely, the CD47/SIRPα interaction is a critical determinant of engraftment success in hematopoietic cell transplantation (HCT): CD47 expression levels on HSC correlate with their relative engraftability. Unfortunately, bone marrow HSC express low levels of CD47, accounting for their relative inefficiency in transplantation. Thus, depending on the clinical scenario, the SIRPα/CD47 system offers an exciting new axis for both anti-tumor and transplant therapy. However, effective utilization of the soluble ectodomains of either SIRPv or CD47 as agonists, or antagonists is limited by the low affinity of the wild-type CD47/SIRPα interaction. We wish to execute a structure-based engineering approach to creating high-affinity SIRPα and CD47 ectodomains as therapeutics for cancer and HCT. We propose to combine the expertise of the Garcia lab in structural biology, protein engineering and immune intervention, with the strengths of the Weissman and Shizuru labs in in vivo cancer, and hematopoietic cell transplantation biology, to target the CD47/SIRPα axis. For Aim #1, we have engineered high-affinity SIRPα monomers, that prevent the interaction between endogenous CD47 and SIRPα, and dramatically synergize with clinically-established therapeutic monoclonal antibodies in stimulating phagocytosis of tumor cells in vivo. This "1-2" punch of target sensitization to macrophages followed by cytolysis by anti-tumor mAb is a completely novel strategy. For Aim #2, we are engineering high-affinity CD47 variants that activate SIRPα inhibitory signaling and decrease macrophage activation, thus enhancing HSC engraftment. Finally, in Aim #3 we wish to reconstitute and molecularly characterize the cell surface 'don't eat me' complex composed of CD47 and SIRPα with putative alternative ligands including thrombospondin and integrins in order to fully understand the therapeutic potential of this system for modulating macrophage phagocytosis. We anticipate these studies will yield novel classes of biotherapeutics with applications in many types of human cancers, and for transplant therapy.