Engineering of macrophage phagocytosis for cancer and stem cell immunotherapy

描述(申请人提供)：巨噬细胞是吞噬细胞细胞，识别和‘吃掉’外来细胞微生物，以及死亡细胞和变异细胞，如癌症。它们在目标细胞上显示出各种受体，通过这些受体识别“吃我”和“不要吃我”的信号。巨噬细胞在迁移的造血干细胞(HSC)通过血窦时对其进行检查。巨噬细胞还检查癌细胞，癌细胞表达吞噬细胞的信号，以确保异常细胞在被称为程序性细胞去除的过程中被吞噬。然而，成功的癌细胞通过表达cd47来逃避程序性细胞去除，cd47是一种主要的“不要吃我”信号，是表达在巨噬细胞上的抑制性受体Sirpα的配基。CD47连接Sirpα可阻断巨噬细胞吞噬肿瘤细胞。CD47/Sirpα轴代表一个触发开关，可以被阻止或刺激以达到不同的治疗目的。阻断CD47与Sirpα的结合可促进癌细胞的吞噬功能。相反，CD47/Sirpα的相互作用是造血细胞移植植入成功的关键决定因素：造血细胞上CD47的表达水平与它们的相对植入性相关。不幸的是，骨髓HSC表达低水平的CD47，这是它们在移植中相对低效的原因。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，由于野生型CD47/Sirpα相互作用的低亲和力，有效地利用SIRPv或CD47胞外域作为激动剂或拮抗剂是有限的。我们希望实施一种基于结构的工程方法来创建高亲和力的Sirp、α和CD47胞外域，作为治疗癌症和红细胞压积的药物。我们建议结合加西亚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，以及魏斯曼和静鲁实验室在体内癌症和造血细胞移植生物学方面的优势，以CD47Sirpα轴为靶点。对于第一个目标，我们设计了高亲和力的Sirpα单体，它可以防止内源性α和Sirp CD47之间的相互作用，并与临床建立的治疗性单抗在刺激体内肿瘤细胞吞噬方面显著协同。这种“1-2”式的靶向增敏巨噬细胞，然后用抗肿瘤单抗进行细胞溶解，是一种全新的策略。对于第二个目标，我们正在设计高亲和力的CD47变异体，激活Sirpα抑制信号并减少巨噬细胞的激活，从而增强造血干细胞的植入。最后，在目标#3中，我们希望重建由CD47和Sirpα组成的细胞表面‘不要吃我’复合体，并对其进行分子表征，以充分了解该系统在调节巨噬细胞吞噬功能方面的治疗潜力。我们预计，这些研究将产生新的生物疗法类别，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.