Replicor宣布发表研究显示NAP不直接刺激免疫反应

StephenW

Media Contact:

Natacha Dorget
[email protected]
514-733-1998

Replicor announces publication of study showing NAPs do not directly stimulate the immune response

NEW YORK, March 23, 2017 – Replicor Inc., a privately held biopharmaceutical company targeting a cure for patients with chronic hepatitis B virus (HBV) and chronic HBV and hepatitis delta virus (HDV) co-infection, announces the publication of its study on the immunostimulatory properties of its nucleic acid polymer (NAP technology) in the journal Scientific Reports (http://www.nature.com/articles/srep43838?WT.feed_name=subjects_immunology).

This study, conducted in collaboration with Dr. Ruth Broering’s group at the University Hospital at the University of Duisburg-Essen, reports on the immunostimulatory properties of clinically active NAPs in various liver and blood cells from non-infected human donors and shows that, even at high concentrations exceeding those present during therapy in clinical trials, that NAPs do not have any detectable immunostimulatory activity. “This study is the first published in a series of studies designed to improve our understanding of NAP pharmacology” said Dr. Andrew Vaillant, CSO “and allow us to focus our research efforts on identifying the molecular target(s) underlying the inhibition of HBsAg release by NAPs”.

Dr. Michel Bazinet, CEO, further commented “These results further demonstrate that the improved immune function and response to immunotherapy observed in patients receiving NAP-based combination therapy are driven by the clearance of HBsAg and not from direct exposure to NAPs”.

StephenW

媒体联系人：

Natacha Dorget
[email protected]
514-733-1998

Replicor宣布发表研究显示NAP不直接刺激免疫反应


纽约，2017年3月23日 - Replicor Inc.是一家私人持有的生物制药公司，旨在治愈慢性乙型肝炎病毒（HBV）和慢性乙型肝炎病毒（HBV）和丙型肝炎病毒（HDV）共感染患者，公布其研究报告关于其核酸聚合物（NAP技术）在“科学报告”（http://www.nature.com/articles/s ... subjects_immunology）杂志上的免疫刺激性质。

这项研究与杜伊斯堡 - 埃森大学大学医院的Ruth Broering博士合作进行了研究，报告了来自非感染人类捐赠者的各种肝细胞和临床活动性NAP的免疫刺激性质，在临床试验治疗期间高浓度超过那些，NAPs没有任何可检测的免疫刺激活性。 “这项研究是第一次发表在旨在提高我们对NAP药理学知识的一系列研究中的第一次发表。”CSO博士Andrew Vaillant博士说，“让我们将研究重点放在确定HBsAg抑制作用的分子靶点由国家行动方案发布“。

首席执行官Michel Bazinet博士进一步表示：“这些结果进一步表明，接受NAP联合治疗的患者观察到的免疫功能改善和对免疫治疗的反应是由HBsAg清除而不是直接暴露于NAP。

StephenW

Nucleic acid-based polymers effective against hepatitis B Virus infection in patients don’t harbor immunostimulatory properties in primary isolated liver cells

    Catherine Isabell Real, Melanie Werner, Andreas Paul, Guido Gerken, Joerg Friedrich Schlaak, Andrew Vaillant & Ruth Broering

    Scientific Reports 7, Article number: 43838 (2017)
    doi:10.1038/srep43838
    Download Citation
        Innate immunityViral hepatitis

Received:
    06 October 2016
Accepted:
    30 January 2017
Published online:
    08 March 2017

Abstract

Nucleic acid polymers (NAPs) block the release of subviral particles from hepatocytes, a mechanism consistent with their antiviral activity against hepatitis B virus (HBV) in patients. Analysis of immunostimulatory properties of NAPs were conducted with several NAP species: REP 2006, the prototypic degenerate NAP [dN]40, containing TLR9-stimulatory CpG; REP 2055 a clinically active NAP with a sequence [dAdC]20 devoid of CpG content; REP 2139 (also clinically active) and REP 2165 (REP 2055 analogues further rendered immunologically inactive by replacing cytidine with 5-methylcytidine and incorporating 2′-O methylation of riboses). These analyses revealed pro-inflammatory responses in human peripheral blood mononuclear cells with REP 2006 and with REP 2139 and REP 2165 only at high dose but displayed no significant antiviral activity. In primary isolated human hepatocytes and liver sinusoidal endothelial cells no significant inflammatory or antiviral responses were detected for any NAPs. In human Kupffer cells pro-inflammatory activity was observed with REP 2006 and REP 2055, whereas a weak but significant induction of interferon genes was only observed with REP 2006 at the highest concentration. We therefore hypothesize that the antiviral activity of NAPs optimized to treat HBV infection in patients cannot be explained by direct induction of innate antiviral responses.
Introduction

Nucleic acid polymers (NAPs) act through size dependent and sequence independent amphipathic interactions of single stranded phosphorothioated oligonucleotides. NAPs have been shown to have antiviral activity in vitro and in vivo in a broad spectrum of viruses where they act as entry inhibitors similar to sulfated glycans by interfering with amphipathic alpha helices conserved in viruses with type 1 fusion glycoproteins (human immunodeficiency virus, herpes simplex virus, cytomegalovirus, lymphocytic choriomeningitis virus) or putatively by interfering with apolipoprotein interactions required for viral fusion with the host cell (hepatitis C virus)1. In hepadnaviruses, NAPs have both entry and post-entry antiviral effects2 but in vivo the entry-inhibitory properties of NAPs do not appear to contribute to their antiviral effects in vivo3. NAPs have the unique ability to clear the hepatitis B surface antigen (HBsAg) from the circulation, an effect which is derived from the blockage of secretion of HBsAg from infected hepatocytes, potentially by interfering with the release of subviral particles4. Both in vivo and more recently in human patients with chronic HBV infection, clearance of serum HBsAg leads to unmasking of anti-HBsAg antibodies, clearance of HBV DNA and more importantly the apparent enhancement of the efficacy of immunotherapy to achieve functional control of chronic HBV infection5.

Oligonucleotides have the ability to stimulate the innate immune response through a variety of pattern recognition receptors (PRR) including TLR3 (dsRNA), TLR7/8 (ssRNA), TLR9 (CpG DNA), RIG-I (ss and dsRNA), MDA5 (dsRNA) and DAI (dsDNA)6,7 which function as sensors for viral and bacterial infection. The role of innate immunity in chronic viral hepatitis, primarily focusing on parenchymal and non-parenchymal murine liver cells have been described previously, indicating a diversification of TLR signaling pathways in Kupffer cells (KCs) and liver sinusoidal endothelial cells (LSECs) compared to ‘classical’ antigen-presenting cells, such as myeloid dendritic cells8. Assuming, that TLR agonist-induced expression of pro-inflammatory (TNF, IL6, IL1b), antiviral (IFNB1) and anti-inflammatory cytokines (IL10) in murine KCs, LSECs, and hepatocytes is cell-type specific8,9. It has been demonstrated that activation of the local innate immune system of the liver through TLR ligands has the potential to control HBV replication in a co-culture model in vitro10,11. Although inflammatory responses were observed early in vivo experiments with degenerate NAPs (i.e. REP 2006), consistent with activation of the innate response2,12 the antiviral activities of NAPs containing sequences and naturally occurring nucleotide modifications designed to block recognition by pattern receptors13,14,15,16,17,18,19, persist and are not accompanied by pro-inflammatory effects in vivo or in human patients3,5,12,20. However since many of the antiviral effects of NAP therapy in HBV infection are similar to those observed with immunotherapy, a more rigorous examination of immunostimulatory effects of NAPs optimized for therapeutic use was conducted in primary cultures of human parenchymal and non-parenchymal liver cells and peripheral blood mononuclear cells. Experimental setup and description of NAPs are depicted in (Fig. 1).

StephenW

对乙型肝炎有效的基于核酸的聚合物患者的病毒感染在原代分离的肝细胞中不具有免疫刺激性质

    凯瑟琳·伊莎贝尔·真实，梅兰妮·维纳，安德烈亚斯·保罗，吉多·格肯，乔尔·弗里德里希·施拉克，安德鲁·威兰特和露丝·雷根

    科学报告7，文章编号：43838（2017）
    doi：10.1038 / srep43838
    下载引文
        先天性免疫性病毒性肝炎

收到：
    2016年10月06日
公认：
    2017年1月30日
在线发布：
    2017年3月8日

抽象

核酸聚合物（NAP）阻断肝细胞中亚病毒颗粒的释放，这与其对患者乙型肝炎病毒（HBV）的抗病毒活性一致。使用几种NAP物种进行NAP的免疫刺激性质分析：REP 2006，原型简并NAP [dN] 40，含有TLR9-刺激性CpG; REP 2055具有缺乏CpG含量的序列[dAdC] 20的临床活性NAP; REP 2139（也是临床活动）和REP 2165（REP 2055类似物进一步通过用5-甲基胞苷替代胞苷并引入核糖的2'-O甲基化进一步使其无免疫活性）。这些分析仅在高剂量时显示了REP 2006和REP 2139和REP 2165在人外周血单核细胞中的促炎反应，但没有显示出显着的抗病毒活性。在初级分离的人肝细胞和肝窦内皮细胞中，对于任何NAP都没有检测到显着的炎症或抗病毒反应。在人类Kupffer细胞中，REP 2006和REP 2055观察到促炎活性，而干细胞基因的弱但显着的诱导仅在最高浓度的REP 2006中观察到。因此，我们假设，优化用于治疗患者HBV感染的NAP的抗病毒活性不能通过直接诱导天然抗病毒反应来解释。
介绍

核酸聚合物（NAP）通过单链硫代磷酸化寡核苷酸的大小依赖性和序列无关的两亲性相互作用起作用。已经显示NAP在体外和体内在广谱病毒中具有抗病毒活性，其中它们通过干扰1型融合糖蛋白（人免疫缺陷病毒，单纯疱疹病毒）中保护的两亲性α螺旋而作为类似于硫酸化聚糖的入口抑制剂病毒，巨细胞病毒，淋巴细胞性脉络丛脑膜炎病毒）或推测通过干扰与宿主细胞（丙型肝炎病毒）病毒融合所需的载脂蛋白相互作用1。在肝炎病毒中，NAP具有进入和进入后的抗病毒作用2，但在体内，NAP的进入抑制性质似乎不会对其在体内的抗病毒作用有贡献3。 NAP具有从循环中清除乙型肝炎表面抗原（HBsAg）的独特能力，这是由于阻碍HBsAg从感染的肝细胞分泌而产生的影响，潜在地可能通过干扰亚病毒颗粒的释放4。在体内和最近在人类慢性HBV感染患者中，血清HBsAg的清除导致抗HBsAg抗体的揭开，HBV DNA的清除，更重要的是免疫治疗实现慢性HBV感染功能控制的功效的明显增强5 。

寡核苷酸具有通过各种模式识别受体（PRR）刺激先天免疫应答的能力，包括TLR3（dsRNA），TLR7 / 8（ssRNA），TLR9（CpG DNA），RIG-1（ss和dsRNA），MDA5（ dsRNA）和DAI（dsDNA）6,7，其作为病毒和细菌感染的传感器。先前已经描述了主要集中在实质和非实质鼠肝细胞的慢性病毒性肝炎中的先天免疫的作用，表明在Kupffer细胞（KCs）和肝窦内皮细胞（LSEC）中TLR信号通路的多样化与“经典的抗原呈递细胞，如骨髓树突细胞8。假设TLR激动剂诱导的小鼠KCs，LSECs和肝细胞中促炎症（TNF，IL6，IL1b），抗病毒（IFNB1）和抗炎细胞因子（IL10）的表达是细胞型特异性的。已经证明，通过TLR配体激活肝脏的局部先天免疫系统具有在体外共培养模型中控制HBV复制的潜力10,11。尽管在退化NAP早期体内实验中发现炎症反应（即REP 2006），与先天反应激活一致2,12含有序列的NAP的抗病毒活性和设计用于阻断模式受体识别的天然存在的核苷酸修饰13,14,15 ，16,17,18,19，持续存在，不伴有体内或人类患者的促炎症作用3,5,12,20。然而，由于NAP治疗在HBV感染中的许多抗病毒作用与用免疫治疗观察到的相似，所以在人实质和非实质肝细胞和外周血的原代培养物中进行了针对治疗用途优化的NAP的免疫刺激作用的更严格的检查 血液单核细胞。 NAP的实验设置和描述如图1所示。

齐欢畅2

arc520的失败也证明了这一点。

齐欢畅2

低谷中。

kite2002005

齐欢畅2 发表于 2017-3-23 22:33
低谷中。

意思是不是说，无法治愈
?

齐欢畅2

我的猜测一种可能是，人体因为对乙肝病毒的各种抗原形成免疫耐受，已经极度缺乏可以产生免疫反应的特异性免疫细胞，如果可以从正常人的身体中提取一些免疫细胞，然后在持续的表抗（hbsag）持续抑制中，把正常人身上提取的免疫细胞，通过克隆的办法在输入人体内，也许可以激活免疫系统。不知道这是否是个办法。（抛砖引玉）

齐欢畅2

齐欢畅2 发表于 2017-3-23 22:47
我的猜测一种可能是，人体因为对乙肝病毒的各种抗原形成免疫耐受，已经极度缺乏可以产生免疫反应的特异性免 ...

为何这么猜测呢？因为人体对某种抗原的免疫耐受是免疫系统的一种自我保护机制，比如人感染了乙肝病毒，一时间被病毒安营扎寨后，无法清除病毒，只好和病毒和平相处，这有点像清政府被八国联军侵华一样，你打不过八国联军，只好“量中华之物力，结于国之欢心”，只要我清政府还在，江山还在就可以了，那些反对八国联军的各种民间团练，统统干掉。以免再次挑起事端，当年义和团扶清灭洋，最后还是被清政府自己个剿杀的。就是这个道理。所以人体中那些对乙肝病毒有特异性免疫的细胞都在战役中阵亡了，或者说是被免疫系统自己给干掉的，怎么干掉的，我也不知道。这是个很大的课题。
所以，包括rep公司的还有arc520都是这个问题，以前大家都想，既然表面抗原抑制了免疫系统，那我把表面抗原干掉，结果还是没用。这好比义和团被干掉了，扶清灭洋的人也没了，除非还有几个幸存的义和团成员再次雄起，招兵买马才行，所以，乙肝自愈的很少，但确实有的人，他身体里的免疫细胞还是有幸存者的，比如有些休眠的免疫细胞，他逃过了免疫耐受期的免疫监视，到了一定年龄，当条件适当，就活了过来，获得了大量繁殖的机会。从而一举带动整个机体的免疫复苏。
所以我觉得，如果可以从正常人身体找到免疫细胞，大量克隆后，持续的输入人体，就有可能激活免疫。

StephenW

齐欢畅2 发表于 2017-3-23 22:30
arc520的失败也证明了这一点。

这项研究不关系arc520的失败.

“这些结果进一步表明，接受NAP联合治疗的患者观察到的免疫功能改善和对免疫治疗的反应是因为HBsAg清除而不是直接暴露于NAP。"

REP9AC是一种NAP (不要混同 NUCs).

ARC520 不是失败! - ARC520的RNAi 能够i减少HBsAg, 因此可以恢复对HBV的免疫作用, 正如 REP9AC. ARC520 的传递方式有问题, 因此新的 Arrowhead hbv RNAi 用新的传递方式.