Engineering of macrophage phagocytosis for cancer and stem cell immunotherapy

描述（由申请人提供）：巨噬细胞是吞噬细胞，其识别并“吃掉”外来细胞微生物，以及垂死细胞和异常细胞如癌症。它们有各种各样的感受器，通过这些感受器，它们可以识别“吃我”和“别吃我”的信号。 靶细胞当造血干细胞（HSC）通过血窦时，巨噬细胞检查迁移的HSC。巨噬细胞还检查癌细胞，癌细胞表达促吞噬细胞“吃我”信号，以确保异常细胞在称为程序性细胞清除的过程中被摄入。然而，成功的癌细胞通过表达CD 47来逃避程序性细胞清除，CD 47是一种显性的“不要吃我”信号，是SIRP的配体，SIRP是一种在巨噬细胞上表达的抑制性受体。通过CD 47连接SIRP可阻断巨噬细胞吞噬肿瘤细胞。CD 47/SIRP轴代表一个拨动开关，可以被阻断或刺激以实现不同的治疗目标。阻断CD 47与SIRP的结合可促进癌细胞的吞噬作用。相反，CD 47/SIRP相互作用是造血细胞移植（HCT）中植入成功的关键决定因素：HSC上的CD 47表达水平与其相对植入能力相关。不幸的是，骨髓HSC表达低水平的CD 47，这是它们在移植中相对低效的原因。因此，根据临床情况，SIRP/CD 47系统为抗肿瘤和移植治疗提供了一个令人兴奋的新轴心。然而，有效利用SIRP或CD 47的可溶性胞外域作为激动剂或拮抗剂受到野生型CD 47/SIRP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SIRP和CD 47胞外域作为癌症和HCT的治疗剂。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联合收割机结合起来，以靶向CD 47/SIRP轴。对于目标#1，我们已经设计了高亲和力SIRP？单体，其防止内源性CD 47和SIRP？之间的相互作用，并且在体内刺激肿瘤细胞的吞噬中与临床建立的治疗性单克隆抗体显著协同。这种对巨噬细胞的靶向致敏的“1-2”冲击，然后通过抗肿瘤mAb进行细胞溶解是一种全新的策略。对于目标#2，我们正在工程化高亲和力CD 47变体，其激活SIRP抑制性信号传导并减少巨噬细胞活化，从而增强HSC植入。最后，在目标#3中，我们希望重建和分子表征由CD 47和SIRP组成的细胞表面“不要吃我”复合物，其具有推定的替代配体，包括血小板反应蛋白和整合素，以充分理解该系统用于调节巨噬细胞吞噬作用的治疗潜力。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 SIRP¿, an inhibitory receptor expressed on macrophages. Ligation of SIRP¿ by CD47 blocks macrophages from phagocytosing the tumor cells. The CD47/SIRP¿ axis represents a toggle switch that can be blocked or stimulated for different therapeutic goals. Blocking binding of CD47 to SIRP¿ promotes phagocytosis of cancer cells. Conversely, the CD47/SIRP¿ 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 SIRP¿/CD47 system offers an exciting new axis for both anti-tumor and transplant therapy. However, effective utilization of the soluble ectodomains of either SIRP¿ or CD47 as agonists, or antagonists is limited by the low affinity of the wild-type CD47/SIRP¿ interaction. We wish to execute a structure-based engineering approach to creating high-affinity SIRP¿ 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/SIRP¿ axis. For Aim #1, we have engineered high-affinity SIRP¿ monomers, that prevent the interaction between endogenous CD47 and SIRP¿, 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 SIRP¿ 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 SIRP¿ 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.