Tumor-toxic CD47 mAb therapy for leukemia: a proof of concept study

描述(申请人提供)：CD47已成为血液病癌症的新治疗靶点。CD47表达水平最高的白血病预后最差。CD47表达的增加被认为是通过SIRPA向巨噬细胞发送“不要吃我”的信号来保护癌细胞免受吞噬清除。SIRPA是一种抑制受体，可以防止携带CD47的细胞吞噬。抗CD47单抗(CD47单抗)可以阻断CD47/SIRPA的相互作用，增强癌细胞的吞噬功能，有助于控制人-鼠异种移植白血病模型的肿瘤负荷()。然而，CD47单抗可以攻击尚未在抗癌战争中利用的转化细胞的机制是存在的。一种特殊的抗人CD47mAb(克隆1F7)对人类白血病具有直接的细胞毒作用。单抗1F7在没有补体或其他免疫细胞作用的情况下杀死携带CD47的肿瘤细胞。相反，mAb1F7通过一种非凋亡机制发挥作用，该机制涉及依赖CD47对线粒体的直接攻击，释放线粒体的膜电位(DYM)，破坏细胞的ATP生成能力，导致细胞快速死亡。值得注意的是，mAb1F7不会杀死也表达CD47的正常白细胞，而只会杀死那些通过转化而“激活”的细胞。因此，所有表达CD47的正常循环细胞都幸免于此，而癌细胞则被具有肿瘤毒性的CD47mAb选择性地杀死。重要的是，mAb1F7还阻断了SIRPA与CD47的结合，因此它可以通过两种机制发挥作用：(1)直接肿瘤细胞毒性；(2)促进死亡和濒临死亡的肿瘤细胞的吞噬。我们假设，一株能完成这两种功能的单抗将优于只阻断CD47/SIRPA结合的单抗。在这个第一阶段的项目中，我们将鉴定对小鼠白血病细胞具有肿瘤毒性活性的CD47mAbs，然后测试这些mAbs对小鼠急性早幼粒细胞白血病(APL)的疗效。在该计划的第二阶段，我们将人源化我们的候选CD47mAb，并评估其有效性和安全性。第一阶段的具体目标是：1.从一组与包括小鼠和人在内的多种物种的CD47发生反应的CD47单抗(系列400单抗)中筛选出一种具有肿瘤毒性的CD47mAb。2.AIM 1中鉴定的具有肿瘤毒性的CD47mAbs将在同基因小鼠APL模型中进行体内疗效测试。APL细胞将被荧光素酶和绿色荧光蛋白(GFP)标记以进行灵敏检测。我们将比较具有肿瘤毒性的CD47mAb和单纯阻断CD47/SIRPA结合的CD47mAb的抗癌效果。这项研究还将为我们提供体内安全性/耐受性的初步迹象，这些CD47mAb可以结合白血病和宿主正常细胞上的CD47，这一情况反映了人类癌症治疗的情况。

DESCRIPTION (provided by applicant): CD47 has emerged as a novel therapeutic target in hematologic cancers. Leukemias expressing the highest levels of CD47 have the worst prognosis. Increased CD47 expression is thought to protect cancer cells from phagocytic clearance by sending a "don't eat me" signal to macrophages via SIRPa, an inhibitory receptor that prevents phagocytosis of CD47-bearing cells. Anti-CD47 mAbs (CD47mAbs) that block the CD47/SIRPa interaction enhance phagocytosis of cancer cells and contribute to control of tumor burden in human to mouse xenograft leukemia models (). However, there are mechanisms by which CD47 mAbs can attack transformed cells that have not yet been exploited in the war on cancer. A particular anti-human CD47mAb (clone 1F7) has a direct, cytotoxic effect on human leukemias. MAb 1F7 kills CD47-bearing tumor cells without the action of complement or other immune cells. Instead, mAb 1F7 acts via a non-apoptotic mechanism that involves a direct CD47-dependent attack on mitochondria, discharging their membrane potential (DYm) and destroying the ATP-generating capacity of the cell leading to rapid cell death. It is noteworthy that mAb 1F7 does not kill normal leukocytes, which also express CD47, but only those cells that are "activated" by transformation. Thus normal circulating cells, all of which express CD47, are spared while cancer cells are selectively killed by the tumor-toxic CD47mAb. Importantly, mAb 1F7 also blocks binding of SIRPa to CD47 and thus it can act via two mechanisms: (1) direct tumor cytotoxicity and (2) promoting phagocytosis of the dead and dying tumor cells. We hypothesize that a single mAb that can accomplish both functions will be superior to one that only blocks CD47/SIRPa binding. In this phase I project, we will characterize CD47mAbs with tumor-toxic activity toward mouse leukemia cells and then test these mAbs for efficacy in treating mouse acute promyelocytic leukemias (APL). In phase II of this program, we will humanize our candidate CD47mAb and evaluate its efficacy and safety. The PHASE I SPECIFIC AIMS are: 1. Identification/characterization of a tumor-toxic CD47mAb selected from a panel of CD47 mAbs (series 400 mAbs) that react with CD47 from many species including mouse and human. 2. Tumor-toxic CD47mAbs identified in Aim 1 will be tested for efficacy in syngeneic mouse APL models in vivo. APL cells will be tagged with luciferase and green fluorescent protein (GFP) for sensitive detection. We will compare anti-cancer efficacy of the tumor-toxic CD47mAbs to a CD47mAb that simply blocks CD47/SIRPa binding. This study will also give us a preliminary indication of in vivo safety/tolerability of these CD47mAbs that can bind CD47 on both the leukemia and the host's normal cells, a situation reflecting human cancer therapy.