CN109069619A - 佐剂用于预防和/或治疗自身免疫病的用途 - Google Patents
佐剂用于预防和/或治疗自身免疫病的用途 Download PDFInfo
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Abstract
本发明提供了佐剂，其用于预防和/或治疗自身免疫病。
Description
技术领域
本发明涉及佐剂，具体而言涉及佐剂在预防和/或治疗自身免疫病中的用途。
发明背景
在许多疫苗中，抗原呈纯化或亚基蛋白的形式以改善其安全性，但这是以免疫原性降低为代价的。因此，有效的适应性免疫应答需要添加佐剂以引发早期炎症并活化抗原呈递细胞。
铝佐剂(明矾)广泛用于通过诱导Th2应答来促进抗体应答。基于角鲨烯的佐剂MF59和佐剂系统(AS) 03通过其诱导趋化因子和细胞因子释放的能力来促进Th1和Th2应答，导致免疫细胞的大量募集和活化。至于基于Toll样受体配体的佐剂AS04和AS01，它们通过直接活化先天免疫细胞来诱导Th1和Th2应答。
除了对效应子应答的影响之外，一些研究揭示某些佐剂在自身免疫病中的免疫调节性影响的有趣和矛盾的观察结果。仅用于兽医疫苗中的完全弗氏佐剂(CFA)的施用在非肥胖糖尿病小鼠中诱导保护和减轻1型糖尿病(Sadelain等人(1990) Diabetes 39: 583)。另外，脂多糖(LPS)(用于兽医疫苗的研究试验中的天然TLR4配体)的施用诱导保护免于实验性自身免疫性脑脊髓炎(EAE)，多发性硬化症的小鼠模型(Buenafe等人 (2007) J.Neuroimmunol. 182: 32)。尽管这些是有趣的观察结果，但由于毒性，CFA和LPS均不适合于人使用。
自身免疫病由针对身体自身组织成分的免疫应答引起。已知超过80种自身免疫病。自身免疫病的实例包括例如类风湿性关节炎(RA)、系统性红斑狼疮(狼疮)、炎性肠病(IBD)、多发性硬化症(MS)、1型糖尿病、格林-巴利综合征、克罗恩氏病和牛皮癣。许多这些疾病是慢性的并且可以引起显著的发病率和残疾。自身免疫病的治疗通常基于免疫抑制。尽管在自身免疫病的治疗中已经取得了重大进展，但仍需要改进的产品和方法，其增加效力，减少副作用，易于施用，安全且可用于长期或甚至持久治疗这些疾病。
发明概述
现在已令人惊讶地发现AS01和AS03，适用于且批准用于人用途的两种不含明矾的佐剂，可以在动物模型中诱导自身免疫病的几乎完全预防，支持它们在预防和/或治疗人中的自身免疫病中的用途。
因此，在第一个方面，本发明涉及佐剂，其用于预防和/或治疗自身免疫病，其中所述佐剂不包含铝并且适合用于人主体。
在一个进一步方面，提供了用于预防和/或治疗自身免疫病的方法，其包括将佐剂施用于主体，其中所述佐剂不包含铝并且适合用于人主体。
在一个甚至进一步方面，本发明涉及佐剂在制备用于预防和/或治疗自身免疫病的药物中的用途，其中所述佐剂不包含铝并且适合用于人主体。
附图简述
图1：a，皮下注射(足垫)疫苗佐剂后4天的腘引流淋巴结(dLN)的代表性图片。b，Treg细胞的典型流式细胞术分析。c，在皮下注射疫苗佐剂后4天和7天，在pLN中，与用PBS注射的小鼠中相比，CD4+细胞中Foxp3+细胞的百分比的倍数增加。来自3次独立实验的累积数据。d，在佐剂注射前过继性转移CD4+Foxp3-CD90.1+细胞，并在第4天评估供体细胞上Foxp3的诱导作为pTreg细胞的标志物。代表的是AS01治疗后Treg诱导的分析的一个实例。e，f，从4天前用AS01治疗的小鼠的dLN纯化的Treg细胞的离体抑制活性。1:2 Treg:Tconv细胞比率的代表性数据(e)和来自4次独立实验的不同Treg:Tconv细胞比率的平均值±SEM (f)。“Treg”细胞指调节性T细胞，且“Tconv”细胞指常规T细胞。
图2：a，皮下注射疫苗佐剂后4天dLN中CD45+细胞的绝对数量。来自3次独立实验的累积数据。b，皮下注射AS01后4天，来自dLN的Treg细胞中的CD44、ICOS和KI-67的代表性表达。c，皮下注射疫苗佐剂后3天从dLN纯化的Treg细胞的体外抑制活性。d，皮下注射AS01后3天，来自dLN的Treg细胞中的代表性Foxp3表达。e，皮下注射AS01后7天从dLN纯化的Treg细胞的体外抑制活性。“Treg”细胞指调节性T细胞。
图3：a，在第0天免疫以诱导EAE(实验性自身免疫性脑脊髓炎)并在第-3天和第0天用佐剂治疗的小鼠中EAE的临床评分。来自3次独立实验的累积数据。b，在免疫以诱导EAE(第0天)并用佐剂治疗(第-3天)的小鼠中转移后3天的2D2 MOG35–55特异性T细胞的代表性增殖(来自2次独立实验)。c，来自10天前免疫以诱导EAE(第0天)并用AS01或AS03治疗(第-3天)的小鼠的dLN的MOG反应性T细胞的INF-γ和IL-17产生。d，在第0天免疫以诱导EAE并在前一天用从PBS、AS01或AS03治疗的小鼠中纯化的Treg细胞转移的小鼠中EAE的临床评分。e，f 在第0天免疫以诱导EAE并在第-3天用AS03治疗的小鼠中在第10天在dLN中的Treg细胞(e)和Treg和Tconv细胞中的整联蛋白αL+和αM+(f)的百分比。“Treg”细胞指调节性T细胞，且“Tconv”细胞指常规T细胞。
图4：a，b 在第0天免疫以诱导EAE(实验性自身免疫性脑脊髓炎)并在第-3天(a)或第0天(b)用AS01或AS03治疗的小鼠中EAE的临床评分。来自3次独立实验的累积数据。c，d在第0天免疫以诱导EAE并在第0天和第-3天用AS03治疗的小鼠中在第10天在dLN中CCR6+细胞和CXCR3+细胞(c)以及Treg细胞和Tconv细胞中的整联蛋白α4+细胞和整联蛋白αL+细胞(d)的比例。“Treg”细胞指调节性T细胞，且“Tconv”细胞指常规T细胞。
详述
如上所述，在第一个方面，本发明涉及佐剂，其用于预防和/或治疗自身免疫病，其中所述佐剂不包含铝并且适合用于人主体。
通常，本发明的用途或本发明的方法的目的是预防自身免疫病，包括延迟疾病的发作，和/或治疗这种疾病，即降低这种疾病的严重程度，例如通过减少自身免疫病的病因和/或减少其症状。在一个实施方案中，实现了如根据本文实施例测定的超过50％、例如超过75％的症状减少。
用于本发明中的佐剂
在一些实施方案中，所述佐剂包含免疫活性皂苷，例如，QS21。本领域已描述了包含皂苷的佐剂。皂苷例如描述于：Lacaille-Dubois和Wagner (1996) (A review of thebiological and pharmacological activities of saponins. Phytomedicine Vol. 2:363)。皂苷已知作为疫苗中的佐剂。例如，Quil A(衍生自南美洲树木皂皮树(QuillajaSaponaria Molina)的树皮)由Dalsgaard等人在1974年在"Saponin adjuvants" (Archiv.fur die gesamte Virusforschung, Vol. 44, Springer Verlag, Berlin, 243)中描述为具有佐剂活性。已通过HPLC分离了Quil A的纯化级分，其保留了佐剂活性而无与Quil A相关的毒性(Kensil等人(1991) J. Immunol. 146: 431)。Quil A级分也描述于US 5,057,540和"Saponins as vaccine adjuvants" (Kensil, C. R., Crit. Rev. Ther. DrugCarrier Syst., 1996, 12 (1-2): 1-55)。
适合用于本发明的两种此类级分是QS7和QS21(也称为QA-7和QA-21)。QS21是用于本发明的优选的免疫活性皂苷级分。QS21已综述于 “Vaccine Adjuvants: preparationmethods and research protocols” (Humana Press, Totowa, New Jersey, Edited byDerek T. O’Hagan, Chapter 15: “QS21 Adjuvant”)。包含Quil A级分例如QS21和QS7的微粒佐剂系统例如描述于WO 96/33739、WO 96/11711和WO 07/068907中。
除了皂苷组分之外，佐剂优选包含固醇。固醇的存在可以进一步降低包含皂苷的组合物的反应原性，参见例如EP0822831。合适的固醇包括β-谷固醇、豆固醇、麦角固醇、麦角钙化固醇和胆固醇。胆固醇是特别合适的。合适地，免疫活性皂苷级分为QS21，并且QS21:固醇的比率为1:100至1:1w/w，例如1:10至1:1w/w，例如1:5至1:1 w/w。
在一些实施方案中，本发明中使用的佐剂包含TLR(Toll样受体)激动剂，诸如TLR4激动剂，例如3D-MPL。TLR激动剂在佐剂中的使用是本领域众所周知的，并且已例如被Lahiri等人(2008 Vaccine 26:6777)综述。可以被刺激以实现佐剂效应的TLR包括TLR2、TLR4、TLR5、TLR7、TLR8和TLR9。TLR2、TLR4、TLR7和TLR8激动剂，特别是TLR4激动剂是优选的。
合适的TLR4激动剂包括脂多糖，例如单磷酰脂质A(MPL)和3-O-脱酰基单磷酰脂质A(3D-MPL)。US 4,436,727公开了MPL及其制备。US 4,912,094和复审证书B1 4,912,094公开了3D-MPL及其制备方法。另一种TLR4激动剂是吡喃葡萄糖基脂质佐剂(GLA)，一种合成的脂质A样分子(参见例如Fox等人(2012) Clin. Vaccine Immunol 19:1633)。在进一步的实施方案中，TLR4激动剂可以是合成的TLR4激动剂，例如在结构上与MPL和3D-MPL相似的合成二糖分子，或者可以是合成单糖分子，例如WO 98/50399、WO 01/34617、WO 02/12258、WO03/065806、WO 04/062599、WO 06/016997、WO 06/12425、WO 03/066065和WO01/90129中公开的例如氨基烷基氨基葡糖苷磷酸酯(AGP)化合物。这些分子在科学和专利文献中也已被描述为脂质A模拟物。脂质A模拟物适当地与脂质A共享一些功能和/或结构活性，并且在一个方面由TLR4受体识别。如本文所述的AGP有时在本领域中被称为脂质A模拟物。在一个优选实施方案中，TLR4激动剂是3D-MPL。TLR4激动剂，例如3-O-脱酰基单磷酰脂质A(3D-MPL)，并且它们在疫苗中作为佐剂的用途已例如描述于WO 96/33739和WO 07/068907中，并且综述于Alving等人 (2012, Curr. Opin. in Immunol. 24: 310)中。
在本发明的一个优选实施方案中，所述佐剂包含免疫活性皂苷和TLR4激动剂，例如QS21和3D-MPL。
在一个进一步优选的实施方案中，所述佐剂包含脂质体制剂中的免疫活性皂苷和TLR4激动剂，例如QS21和3D-MPL。
当在本文中使用时，术语“脂质体”指包封水性内部的单层或多层脂质结构。脂质体和脂质体制剂是本领域中众所周知的。脂质体呈递例如描述于WO 96/33739和WO 07/068907中。能够形成脂质体的脂质包括具有脂肪或脂肪样性质的所有物质。
取决于磷脂组成及用于其制备的方法，脂质体大小可以从30 nm至几μm变化。在本发明的具体实施方案中，脂质体大小将在50nm至500nm的范围内，并且在进一步的实施方案中在50nm至200nm的范围内。动态激光散射是本领域技术人员众所周知的用于测量脂质体大小的方法。
在特别合适的实施方案中，用于本发明的脂质体包含DOPC和固醇，特别是胆固醇。因此，在一个具体实施方案中，本发明的佐剂包含以脂质体形式的以本文所述任何量的QS21，其中所述脂质体包含DOPC和固醇，特别是胆固醇。
在一个实施方案中，所述佐剂包含5至100，诸如10至75，例如25或50μg的QS21/每剂量，和5至100，诸如10至75，例如25或50μg的3D-MPL/每剂量。
众所周知，对于肠胃外施用，溶液应当是生理学上等渗的(即具有药学上可接受的重量摩尔渗透压浓度)以避免细胞变形或裂解。“等渗剂”是生理学耐受的化合物，并且对制剂(例如本发明的免疫原性组合物)赋予合适的张力，以防止水跨越与制剂接触的细胞膜的净流动。已知含有100 mM氯化钠或更多的水性佐剂组合物，例如WO 05/112991和WO 08/142133中的佐剂系统A(AS)或WO 07/068907中公开的脂质体佐剂。
在一些实施方案中，用于组合物的等渗剂是盐。然而，在其他实施方案中，组合物包含非离子等渗剂，且组合物中的氯化钠浓度或离子强度小于100 mM，例如小于80 mM，例如小于30 mM，例如小于10 mM或小于5mM。在一个优选实施方案中，非离子等渗剂是多元醇，例如山梨醇。山梨醇的浓度可以是例如约3%至约15%(w/v)，例如约4%至约10%(w/v)。包含免疫活性皂苷级分和TLR4激动剂的佐剂(其中等渗剂是盐或多元醇)已描述于WO 10/142685，参见例如WO 10/142685中的实施例1和2。
在一个实施方案中，本发明中使用的佐剂包含水包油乳液。合适地所述乳液含有以总体积的0.5%-20% 的量的可代谢油，术语可代谢油的含义是本领域众所周知的。可代谢的可以定义为‘能够通过新陈代谢来转化’(Dorland's Illustrated MedicalDictionary, W.B.Sanders Company, 第25版 (1974))。油可以是任何植物油、鱼油、动物油或合成油，其对于受体是无毒的并且能够通过新陈代谢来转化。坚果、种子和谷物是植物油的常见来源。合成油也是本发明的部分，并且可以包括可商购的油诸如NEOBEE®及其他。特别合适的可代谢油是角鲨烯。角鲨烯(2,6,10,15,19,23-六甲基-2,6,10,14,18,22-二十四碳六烯)是不饱和油，其大量发现于鲨鱼肝油中，以较低量于橄榄油、麦胚芽油、米糠油和酵母中，并且是用于本发明的尤其合适的油。角鲨烯是借助于以下的事实的可代谢油，即其是胆固醇的生物合成的中间体(Merck 索引, 第10版, 登录号8619)。
水包油乳液本身在本领域中是众所周知的，并且已被暗示可用为佐剂组合物(EP399843; WO 95/17210)。合适地，可代谢油以免疫原性组合物总体积的0.5%-20%(终浓度)的量存在，合适地以总体积的1.0%-10%的量，合适地以总体积的2.0%-6.0%的量。
在一个具体实施方案中，可代谢油以免疫原性组合物总体积的约0.5%、1%、3.5%或 5%的最终量存在。在另一个具体实施方案中，可代谢油以免疫原性组合物总体积的0.5%、1%、3.57%或5%的最终量存在。角鲨烯合适的量为每剂量约10.7 mg，合适地为每剂量10.4至11.0 mg。
合适地，本发明中使用的水包油乳液系统具有亚微米范围的小油滴大小。合适地，微滴大小将在120-750 nm范围中，合适地，大小为直径120-600 nm。通常，水包油乳液包含其中以强度(intensity)计至少70％为直径小于500 nm，具体而言以强度计至少80％为直径小于300 nm，合适地以强度计至少90％为直径范围在120-200 nm的油滴。
根据本发明的水包油乳液可以含有固醇或生育酚，例如α生育酚。固醇在本领域内众所周知，例如胆固醇是众所周知的，并公开于例如Merck Index, 第11版, 第341页中，其作为发现于动物脂肪中的天然存在的固醇。其他合适的固醇包括β-谷固醇、豆固醇、麦角固醇和麦角钙化固醇。合适地，存在α-生育酚或其衍生物例如α-生育酚琥珀酸酯。合适地，α-生育酚以免疫原性组合物总体积的0.2%至5.0%(v/v)的量存在，合适地以在0.5ml剂量体积中2.5% (v/v)的量，或在0.5 ml剂量体积中0.5% (v/v) 或在0.7 ml剂量体积中1.7-1.9%(v/v)，合适地为1.8% 。在此说明，以v/v给出的浓度可以通过应用以下转换因子转换为以w/v计的浓度：5% (v/v)的α-生育酚浓度等于4.8% (w/v)α-生育酚浓度。α-生育酚的合适量为每剂量约11.9 mg，合适地每剂量11.6 至 12.2 mg。
水包油乳液可以包含乳化剂。乳化剂可以以免疫原性组合物的0.01重量％至 5.0重量% (w/w)的量存在，合适地以0.1重量％至 2.0重量% (w/w)的量存在。合适的浓度为总组合物的0.5重量％至 1.5重量%(w/w)。乳化剂可以合适地为聚氧乙烯山梨醇酐单油酸酯(聚山梨醇酯80或Tween 80)。在一个具体实施方案中，0.5 ml剂量体积含有1% (w/w)Tween 80，并且0.7 ml剂量体积含有0.7% (w/w) Tween 80。在另一个具体实施方案中，Tween 80浓度为 0.2% (w/w)。聚山梨醇酯80的合适量为每剂量约4.9 mg，合适地每剂量4.6至5.2 mg。
Span 85(聚氧乙烯山梨糖醇酐三油酸酯)也可以存在，例如以1%的水平。水包油乳液佐剂的实例包含用于本发明的Span 85，其在EP0399843B中给出并详述，也称为MF59。
在一个优选实施方案中，所述佐剂是包含角鲨烯、α-生育酚和表面活性剂的水包油乳液，即所述乳液包含水相中的角鲨烯、α-生育酚和表面活性剂，例如聚山梨醇酯80。此类佐剂的制备例如描述于WO 95/17210和WO 06/100109中。在一个实施方案中，所述乳液包含2-10% (v/v)角鲨烯、2-10% (v/v)α-生育酚和0.3-3% (v/v) Tween 80。优选地，所述乳液包含2.5％角鲨烯(v/v)、2.5％ α-生育酚(v/v)、0.9％聚氧乙烯山梨醇酐单油酸酯(v/v)(Tween 80)。
待预防和/或治疗的自身免疫病
如上所解释，本发明涉及佐剂用于预防和/或治疗自身免疫病的用途。以下疾病被归类为自身免疫病：
急性播散性脑脊髓炎、艾迪生氏病、血中丙球蛋白贫乏、斑秃、淀粉样变性、强直性脊柱炎、抗GBM/抗TBM肾炎、抗磷脂综合征(APS)、自身免疫性肝炎、自身免疫性内耳疾病(AIED)、轴突和神经元神经病变(AMAN)、贝切特氏病、大疱性类天疱疮、Castleman病(CD)、乳糜泻、查加斯病、慢性炎性脱髓鞘性多发性神经病(CIDP)、慢性复发性多灶性骨髓炎(CRMO)、Churg-Strauss、瘢痕性类天疱疮/良性粘膜类天疱疮、Cogan氏综合征、冷凝集素病、先天性心脏传导阻滞、柯萨奇心肌炎、CREST综合征、克罗恩氏病、疱疹样皮炎、皮肌炎、德维克氏病(视神经脊髓炎)、盘状狼疮、德雷斯勒综合征、子宫内膜异位症、嗜酸细胞性食管炎(EoE)、嗜酸细胞性筋膜炎、结节性红斑、必需混合性冷球蛋白血症、埃文斯综合征、纤维肌痛、纤维化肺泡炎、巨细胞动脉炎(颞动脉炎)、巨细胞心肌炎、肾小球肾炎、Goodpasture氏综合征、肉芽肿病伴多血管炎、格雷夫斯氏病、格林-巴利综合征、桥本氏甲状腺炎、溶血性贫血、亨-舍二氏紫癜(HSP)、妊娠期疱疹或妊娠性类天疱疮(PG)、低丙种球蛋白血症、IgA肾病、IgG4相关硬化病、包涵体肌炎(IBM)、间质性膀胱炎(IC)、青少年关节炎、青少年肌炎(JM)、川崎病、Lambert-Eaton综合征、白细胞破碎性血管炎、扁平苔藓、硬化性苔藓、木质性结膜炎、线性IgA病(LAD)、狼疮、慢性莱姆病、梅尼埃氏病、显微镜下多血管炎(MPA)、混合性结缔组织病(MCTD)、Mooren氏溃疡、Mucha-Habermann病、多发性硬化症(MS)、重症肌无力、肌炎、发作性睡病、视神经脊髓炎、中性粒细胞减少症、眼瘢痕性类天疱疮、视神经炎、复发性风湿病(PR)、PANDAS(与链球菌相关的儿科自身免疫性神经精神病症)、副肿瘤性小脑变性(PCD)、阵发性睡眠性血红蛋白尿症(PNH)、Parry Romberg综合征、睫状体扁平部炎(外周葡萄膜炎)、Parsonnage-Turner综合征、天疱疮、外周神经病变、静脉周围脑脊髓炎、恶性贫血(PA)、POEMS综合征(多发性神经病、器官巨大症、内分泌病、单克隆丙种球蛋白病、皮肤变化)、结节性多动脉炎、风湿性多肌痛、多肌炎、心肌梗塞后综合征、心包切开术后综合征、原发性胆汁性肝硬化、原发性硬化性胆管炎、孕酮性皮炎、牛皮癣、牛皮癣关节炎、纯红细胞再生障碍(PRCA)、坏疽性脓皮病、雷诺氏现象、反应性关节炎、反射性交感神经营养不良、瑞特氏综合征、复发性多软骨炎、多动腿综合征(RLS)、腹膜后纤维化、风湿热、类风湿性关节炎(RA)、结节病、施密特综合征、巩膜炎、硬皮病、Sjogren氏综合征、精子和睾丸自身免疫、僵硬人综合征(SPS)、亚急性细菌性心内膜炎(SBE)、Susac氏综合征、交感性眼炎(SO)、高安氏动脉炎、颞动脉炎/巨细胞动脉炎、血小板减少性紫癜(TTP)、Tolosa-Hunt综合征(THS)、横贯性脊髓炎、1型糖尿病、溃疡性结肠炎(UC)、未分化结缔组织病(UCTD)、葡萄膜炎、血管炎、白癜风和韦格纳肉芽肿病(现称为肉芽肿病伴多血管炎(GPA))。
待根据本发明预防和/或治疗的优选的疾病包括：
• 类风湿性关节炎
在具有类风湿性关节炎的人中，免疫系统主要靶向覆盖各种关节的覆膜(lining)(滑膜)。滑膜的炎症通常是对称的(在身体的两侧同样发生)并引起关节的疼痛、肿胀和僵硬。这些特征将类风湿性关节炎与骨关节炎区分开，所述骨关节炎是一种更常见和退行性的“磨损性(wear-and-tear)”关节炎。目前可用的疗法集中于用抗炎或免疫抑制药物减少关节的炎症。有时，免疫系统也可以靶向肺、血管或眼睛；偶尔，患者也可以发展其他自身免疫病的症状，诸如Sjogren的炎症、瘙痒和脱屑。
• 多发性硬化症
多发性硬化症是免疫系统靶向中枢神经系统的神经组织的疾病。最通常，间歇性地发生对中枢神经系统的损害，允许人过还算正常的生活。在另一个极端，症状可能变得恒定，导致疾病进展，伴随可能的失明、瘫痪和过早死亡。一些药物诸如β干扰素对具有多发性硬化症的间歇性形式的患者有帮助。在年轻人中，多发性硬化症是神经系统的最常见的致残疾病。
• 免疫介导的或1型糖尿病
1型糖尿病由胰腺的产生胰岛素的细胞的自身免疫性破坏引起。身体需要胰岛素来控制血糖(葡萄糖)水平。高水平的葡萄糖是该疾病的症状和并发症的原因。然而，在患者发展糖尿病的症状之前，大多数产生胰岛素的细胞被破坏。症状包括疲劳、尿频、口渴增加和可能的突然混乱。1型糖尿病通常在30岁之前被诊断出，并且可以早至在出生后的第一个月被诊断出。与2型糖尿病(不被认为是自身免疫病)一起，糖尿病是肾损伤、视力丧失和腿截肢的主要原因。糖水平的密切控制降低这些事件发生的速度。
• 炎性肠病
该医学术语用于克罗恩氏病和溃疡性结肠炎，两种免疫系统攻击肠道(肠)的疾病。患者可以具有腹泻、恶心、呕吐、腹部痉挛和可以难以控制的疼痛。患病个体的疾病可以由肠道炎症和用于该疾病的药物的副作用引起。例如，控制克罗恩氏病的严重症状所需的高剂量皮质类固醇(泼尼松)疗法的每日使用可使患者倾向于感染、骨质变薄(骨质疏松症)和骨折。
• 系统性红斑狼疮
具有系统性红斑狼疮的患者最通常经历严重的疲劳、皮疹和关节疼痛。在严重的情况下，免疫系统可以会攻击并损坏几种器官诸如肾、脑或肺。对于许多个体，可以用可用的抗炎药物控制疾病的症状和损害。
• 牛皮癣
• 牛皮癣是一种免疫系统病症，其影响皮肤，且偶尔影响眼睛、指甲和关节。牛皮癣可以影响非常小的皮肤区域，或覆盖整个身体，伴随称为斑块的红色鳞屑的形成。斑块具有不同的大小、形状和严重程度，并且可以是痛苦以及难看的。细菌感染和对皮肤的压力或创伤可以加重牛皮癣。大多数治疗聚焦于局部皮肤护理，以缓解炎症、瘙痒和脱屑。
• 硬皮病
这种自身免疫病导致皮肤和血管的增厚。几乎每个具有硬皮病的患者都具有雷诺氏症，这是手指和脚趾的血管的痉挛。雷诺氏症的症状包括手指和脚趾对寒冷的敏感性增加，皮肤颜色的变化，疼痛，以及偶尔指尖或脚趾的溃疡。在具有硬皮病的人中，皮肤和血管的增厚可以导致运动的丧失和呼吸的短促，或者更少见地导致肾、心脏或肺衰竭。
• 自身免疫性甲状腺疾病
桥本氏甲状腺炎和格雷夫氏病由甲状腺组织的免疫系统破坏或刺激导致。低(低)或过度活跃(超)甲状腺功能的症状是非特异性的，并且可以缓慢或突然发展；这些包括疲劳、紧张、冷或热不耐受、虚弱、头发质地或数量的变化以及重量增加或减少。用适当的实验室检查容易地进行甲状腺疾病的诊断。甲状腺功能减退症的症状用替代甲状腺激素丸剂控制；然而，可以发生来自激素替代过度或不足的并发症。甲状腺功能亢进症的治疗需要长期抗甲状腺药物疗法或用放射性碘或外科手术破坏甲状腺。这两种治疗方法都具有某些风险和长期副作用。
在一个优选实施方案中，待预防和/或治疗的自身免疫病是影响神经系统(诸如中枢神经系统)的疾病。在一个甚至更优选实施方案中，所述自身免疫病是多发性硬化症。在一个进一步更优选的实施方案中，所述自身免疫病是1型糖尿病。在一个进一步优选的实施方案中，所述自身免疫病是急性播散性脑脊髓炎。
具体而言，包含水包油乳液的佐剂，例如包含水相中的角鲨烯、α-生育酚和聚山梨醇酯80的佐剂，优选用于预防和/或治疗影响神经系统(诸如中枢神经系统)的疾病，例如多发性硬化症。
治疗选项
在优选实施方案中，本发明的方法或本发明的用途包括多次施用佐剂，例如，至少2次、至少3次、至少4次、至少5次或至少10次施用佐剂。在一个实施方案中，每次施用之间的时间间隔是1天至6个月，例如每次施用之间的间隔可以是1周至1个月。
在一个优选实施方案中，所述主体是人主体。待使用本发明的方法治疗的人主体可以是任何年龄的。然而，在一个实施方案中，当开始治疗时，人主体超过18岁。在进一步实施方案中，所述主体超过40岁，诸如超过50岁，例如超过60岁。所述主体可以是男性或女性。
在一个实施方案中，所述佐剂用于预防倾向于发展自身免疫病的人主体、例如倾向于发展多发性硬化症的主体或倾向于发展1型糖尿病的主体中的自身免疫病。倾向于发展自身免疫病的人主体可以例如是显示自身免疫病的早期临床症状的主体，具有发展自身免疫病的遗传风险的主体或具有特定自身抗体的主体。
倾向于发展多发性硬化症的人是例如这样的人，其具有CIS (临床孤立综合症) -在中枢神经系统中持续至少24小时的第一次和单次炎症或脱髓鞘的神经发作，特别是当在MRI上看到与多发性硬化症一致的病变时。
倾向于发展1型糖尿病的人是例如具有自身抗体(诸如自身抗体GADA、IA-2A和/或mIAA)的人(Sosenko等人(2013) Diabetes Care 36: 2615)。
所述佐剂可以经由各种合适的途径施用，包括肠胃外施用，诸如肌肉内施用、皮内施用或皮下施用。合适地，本发明中使用的佐剂组合物具有0.05 ml至1 ml，诸如0.1至0.5ml的人剂量体积，具体而言约0.5 ml或0.7 ml的剂量体积。
所述佐剂可以作为单一疗法或与其他物质(例如，已知对自身免疫病具有治疗或预防作用的其他物质)组合给予。
本申请中所有参考文献包括专利申请和授权的专利的教导，均通过引用完整地并入本文。本文中的术语‘包含(comprising)’、‘包含(comprise)’和‘包含(comprises)’可任选分别由术语‘由……组成(consisting of)’、‘由……组成(consist of)’和‘由……组成(consists of)’所替换。本发明将通过参考以下非限制性实施例进一步描述。
实施例
实施例1 - 方法
小鼠
WT小鼠购自Janvier Labs(法国)。髓鞘少突胶质细胞糖蛋白特异性的2D2 T细胞受体转基因小鼠购自Jackson Laboratory。Foxp3-IRES-GFP敲入(Foxp3GFP)小鼠由BernardMalissen教授友情提供。将2D2和Foxp3GFP小鼠与CD90.1同类系动物回交。所有小鼠均在C57Bl/6背景上。将小鼠圈养在无特定病原体的条件下，并在7-14周龄时进行研究。所有实验方案均由当地伦理委员会批准，并符合欧盟指南。
试剂
AS01由MPL (1 mg/ml)、QS-21 (1 mg/ml)和脂质体构成。AS03由α-生育酚(23.72 mg/ml)、角鲨烯油(21.38 mg/ml)和聚山梨醇酯80 (9.72 mg/ml)构成。AS04由MPL (0.1 mg/ml)和明矾(1mg/ml)构成。弗氏佐剂购自Sigma-Aldrich。不完全形式(IFA)含有85％的石蜡油和15％的失水甘露糖醇单油酸酯。完全形式(CFA)添加热杀死并干燥的以1 mg/ml的浓度的结核分枝杆菌(H37Ra)。
佐剂的施用
小鼠在左后足垫中通过皮下途径接受30μl疫苗佐剂或PBS。为了分析它们的作用，在不同的时间点收集左侧腘引流淋巴结(dLN)和非引流右侧肱淋巴结。在经免疫以诱导EAE(实验性自身免疫性脑脊髓炎)的小鼠中，它们在EAE(实验性自身免疫性脑脊髓炎)诱导前3天和EAE(实验性自身免疫性脑脊髓炎)诱导当天在尾基部和上背部通过皮下途径接受100μl疫苗佐剂或PBS。
流式细胞术分析
将来自淋巴结的细胞机械解离并重悬浮于PBS 3% SVF中。它们首先用2.4G2抗体处理以阻断Fc受体并用以下抗体染色：抗CD45 (30-F11)、抗CD3 (145-2C11)、抗CD4 (RM4-5)、抗CD8 (53-6.7)、抗CD25 (PC61)、抗Foxp3 (MF23)、抗CTLA-4 (UC10-4F10-11)、抗GITR(DTA-1)、抗ICOS (17G9)、抗Ki-67 (B56)。所有抗体均获得自BD Biosciences。使用来自eBioscience的细胞内固定和透化缓冲液集合进行细胞内染色。在BD LSRII细胞计数器上获取细胞并使用FlowJo软件分析。
Treg外周诱导评价
分离Foxp3GFP CD90.1小鼠的淋巴结(肱、腋、颈和腹股沟)和脾细胞，如上所述处理并用抗CD4抗体染色。使用BD FACSAria II纯化CD4+GFP-细胞(Tconv)，然后静脉内注射于WT小鼠中(106个细胞/小鼠)。第二天，小鼠在左后足垫中通过皮下途径接受30μl疫苗佐剂或PBS。三天后，收集dLN并用抗CD4、抗CD90.1 (OX-7)、抗CD90.2 (30-H12)和抗Foxp3抗体染色细胞。通过流式细胞术评估CD90.1转移的细胞中的Foxp3诱导。
体外Treg抑制测定
Foxp3GFP小鼠在尾基部和上背部通过皮下途径接受100μl疫苗佐剂或PBS。在3天或7天收集肱和腹股沟淋巴结。如上所述分离细胞并染色。然后使用BD FACSAria II分选CD4+GFP-细胞(Tconv)和CD4+GFP+细胞(Treg)。将Tconv细胞用CellTrace Violet增殖试剂盒(Lifetechnologies)标记，并以2.5 x 104个细胞/孔与以7.5 x 104个细胞/孔的来自CD3-/-小鼠的脾细胞铺板于96孔板中。用来自BioXCell的抗CD3 (KT3)以0.05μg/ml补充培养基。然后以1:1至1:16的不同比率添加Treg细胞。在第3天，通过流式细胞术评价CellTrace稀释度。
EAE诱导
通过皮下注射在100μl补充有50μg热灭活的结核分枝杆菌H37Ra (BD Biosciences)的CFA (Sigma-Aldrich)中乳化的100μg MOG35-55肽(PolyPeptide)来免疫小鼠。在免疫时和免疫后两天，另外向动物静脉内注射200 ng百日咳博德特氏菌(Bordetella pertussis)毒素(Enzo)。临床评估通过5分量表每天进行，所述5分量表范围为0，无临床体征；1，跛尾；2，跛尾，翻正反射受损，和一肢轻瘫；3，后肢麻痹；4，后肢和前肢麻痹；5，垂死。
细胞因子测量
在EAE诱导后10天，收集肱和腹股沟dLN。将细胞与1μg/ml MOG35-55肽一起在96孔板中以2 x 105个细胞/孔培养。在第3天，收获上清液以通过ELISA (eBioscience)测量INF-γ和IL-17分泌。
T细胞引发评估
来自2D2 CD90.1小鼠的淋巴结和脾细胞用以下生物素标记的抗体染色：抗CD19(6D5)、抗CD11b (M1/70)、抗CD11c (N418)、抗CD8 (53-6.7)和抗CD25 (7D4)，且然后用抗生物素微珠(Miltenyi Biotec)包被。在磁性分选之后，将富含CD4+的阴性级分的细胞用CellTrace Violet增殖试剂盒标记，并在未处理小鼠中静脉内注射(106个细胞/小鼠)。第二天，用MOG35-55肽免疫小鼠，用于EAE诱导。在第3天，通过流式细胞术评价来自肱和腹股沟淋巴结的CD4+CD90.1+Vβ11+ (T细胞受体转基因)细胞的CellTrace稀释液。在接受佐剂的小鼠中，将它们在尾基部和上背部通过皮下途径注射100μl疫苗佐剂或PBS。
Treg细胞过继性转移
Foxp3GFP CD90.1小鼠在尾基部和上背部通过皮下途径接受100μl疫苗佐剂或PBS。在第3天，收集肱和腹股沟淋巴结，并用抗CD4抗体染色细胞。然后使用BD FACSAria II纯化CD4+GFP+细胞(Treg)，并静脉内注射于WT小鼠中(1 x 106个细胞/小鼠)。第二天，如前面在EAE诱导中所述用MOG35-55肽免疫小鼠。
统计分析
使用GraphPad Prism软件进行统计学分析。使用双尾非参数Mann-Whitney U检验确定统计学显著性。*p<0.05，**p<0.01，***p<0.001。在整个附图中使用平均值±SEM。
实施例2 - 疫苗佐剂诱导Treg细胞的瞬时优先扩增
将CFA、不完全弗氏佐剂(IFA)、AS01、AS03和AS04皮下施用于小鼠。4天后，观察到引流淋巴结(dLN)的大量炎症和肿胀(图1a)。这些dLN的细胞性增加11至20倍(图2a)。相比之下，注射明矾对dLN的大小和细胞数没有影响。
然后通过流式细胞术分析Treg细胞(图1b)。除了明矾以外，我们观察到注射不同佐剂后CD4+细胞中Treg细胞比例的快速增加(第4天)，与注射PBS的小鼠相比最高达1.9倍(图1c)。该Treg细胞扩增与活化的表型相关，如由CD44、ICOS和Ki-67的表达增加所示(图2b)。这些Treg细胞扩增和活化是瞬时的，因为它在第7天不再存在，除了IFA(图1c)。由佐剂注射驱动的Treg细胞扩增是由于胸腺Tregs (tTregs)积累，而不是由于外周Treg的诱导增加。实际上，在过继性转移的CD4+Foxp3-细胞中，佐剂不诱导Foxp3表达(图1d)。这些数据揭示新一代佐剂对tTreg细胞的体内活化。
然后，我们通过经典的体外抑制测定评价疫苗佐剂施用对Treg细胞活性的影响。明矾、弗氏佐剂、AS03和AS04对Treg细胞功能没有影响(图2c)，而AS01部分降低其抑制活性和Foxp3表达(图1e和1f和2d)。在第4天观察到这种作用，但在第7天不再观察到(图2e)。因此，新一代佐剂的施用不会损害或仅轻微地损害Treg细胞的抑制功能。
实施例3 - 通过施用AS01和AS03佐剂预防EAE
为了评估疫苗佐剂在病理生理学背景下对耐受诱导的作用，我们在EAE自身免疫模型中测试了佐剂治疗。引人注目的是，在免疫前3天和免疫时2次注射AS01或AS03，诱导了疾病发展的几乎完全预防。平均临床评分低于0.14-0.17，相比之下，对照中为1.17。相反，AS04和明矾对EAE没有作用(图3a)。在免疫前3天或在免疫时进行的仅一次AS01或AS03注射后，EAE分别显著延迟或降低(图4a和4b)。因此，AS01和AS03对EAE具有强烈的预防作用。
我们接下来研究自身反应性Tconv细胞的引发和细胞因子极化。在EAE诱导前表达MOG35–55肽特异性的转基因I-Ab–限制性T细胞受体的2D2转基因小鼠(Bettelli等人(2003)J. Exp. Med. 197:1073)的过继性转移之后评价T细胞增殖。AS01施用在第3天在dLN中略微降低MOG35–55-特异性T细胞的增殖，而AS03没有作用(图3b)。通过在通过MOG35–55免疫肽特异性的自身反应性Tconv细胞的EAE诱导后在第10天在dLN中测量IFNγ和IL-17(EAE中的2种主要致病性细胞因子)的释放来评价T细胞极化。AS01施用与IFNγ产生的减少相关，而IFNγ和IL-17不受AS03施用的影响(图3c)。因此，尽管AS01和AS03施用预防EAE的发展，但只有AS01破坏MOG35–55反应性T细胞的引发和极化。
然后，我们进行过继性转移实验，以进一步分析Treg细胞在佐剂驱动的EAE预防中的作用。在EAE诱导前，将来自佐剂处理的小鼠的dLN的纯化Treg细胞注射至未处理小鼠中。令人惊讶的是，接受来自AS03处理的小鼠的Treg细胞的小鼠被完全保护免于疾病。相反，来自AS01处理的小鼠的Treg细胞没有作用，因为用这些细胞转移的受体小鼠与用PBS或来自未处理小鼠的Treg转移的对照小鼠发生相同的EAE(图3d)。这些数据揭示，AS03施用强烈增强Treg细胞控制EAE的能力。
为了获得AS03预防EAE的机制的更深入见解，我们进一步研究Treg细胞和参与T细胞迁移的分子。在第10天分析来自免疫以诱导EAE并用AS03处理的小鼠的dLN的细胞。与对照相比，在AS03处理的小鼠中Treg细胞比例显著增加(图3e)。Tconv细胞(其在EAE期间在致病性T细胞进入中枢神经系统(CNS)中发挥关键作用(Reboldi等人(2009) Nat. Immunol.10 514; Sporici和Issekutz (2010) Eur. J. Immunol. 40 2751))表达CCR6和CXCR3趋化因子受体不受AS03处理的影响(图4c)。有趣的是，参与CNS归巢的整联蛋白的表达水平(Yednock等人(1992) Nature 356:63; Rothhammer et al.(2011) J. Exp. Med. 208:2465)被佐剂显著修饰。Treg细胞表达更高水平的整联蛋白αL，而Tconv细胞显示降低的整联蛋白αM的水平(图3f)。此外，Tconv细胞的整联蛋白α4和αL的表达水平未受影响(图4d)。这些结果表明，AS03施用影响CNS中的Treg和Tconv细胞的迁移，这可以解释其抑制EAE的能力。
结论
该工作表明，一些新一代疫苗佐剂具有强烈的免疫调节特性且影响Treg细胞活化和功能，因此揭示它们在治疗自身免疫病中的潜力。AS01和AS03施用均诱导保护免于EAE。有趣的是，它们的抑制机制看起来不同。AS01改变脑炎致病性Tconv细胞的引发和细胞因子极化，而AS03强烈增加EAE中的Treg细胞的保护能力，并可改变Tconv和Treg细胞的迁移。
Claims (21)
1.佐剂，其用于预防和/或治疗自身免疫病，其中所述佐剂不包含铝并且适合用于人主体。
2.权利要求1的佐剂，其中所述佐剂包含免疫活性皂苷。
3.权利要求2的佐剂，其中所述佐剂包含QS21。
4.权利要求2或3的佐剂，其中所述佐剂包含固醇。
5.前述权利要求中任一项的佐剂，其中所述佐剂包含TLR激动剂，诸如TLR4激动剂。
6.前述权利要求中任一项的佐剂，其中所述佐剂包含3D-MPL。
7.前述权利要求中任一项的佐剂，其中所述佐剂包含脂质体制剂中的QS21和3D-MPL。
8.前述权利要求中任一项的佐剂，其中所述佐剂包含水包油乳液。
9.权利要求8的佐剂，其中所述乳液包含角鲨烯。
10.权利要求9的佐剂，其中所述乳液进一步包含水相中的α-生育酚和聚山梨醇酯80。
11.前述权利要求中任一项的佐剂，其中所述自身免疫病是选自以下的疾病：多发性硬化症、1型糖尿病、类风湿性关节炎、炎性肠病、系统性红斑狼疮、牛皮癣、硬皮病和自身免疫性甲状腺疾病。
12.前述权利要求中任一项的佐剂，其中所述自身免疫病是影响神经系统、诸如中枢神经系统的疾病。
13.前述权利要求中任一项的佐剂，其中所述自身免疫病是多发性硬化症。
14.前述权利要求中任一项的佐剂，其中所述自身免疫病是急性播散性脑脊髓炎。
15.前述权利要求1至11中任一项的佐剂，其中所述自身免疫病是1型糖尿病。
16.前述权利要求中任一项的佐剂，其中所述佐剂用于人患者。
17.前述权利要求中任一项的佐剂，其中所述佐剂用于预防倾向于发展自身免疫病的人患者中的自身免疫病。
18.用于预防和/或治疗自身免疫病的方法，其包括将佐剂施用于主体，其中所述佐剂不包含铝并且适合用于人主体。
19.权利要求18的方法，其包括如权利要求2至17中所记载的另外特征中的一种或多种。
20.佐剂在制备用于预防和/或治疗自身免疫病的药物中的用途，其中所述佐剂不包含铝并且适合用于人主体。
21.权利要求20的用途，其包括如权利要求2至17中所记载的另外特征中的一种或多种。
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| CN111617107A (zh) * | 2020-04-10 | 2020-09-04 | 王全栋 | 治疗结肠炎的药物组合物 |
Also Published As
| Publication number | Publication date |
|---|---|
| MX2018007408A (es) | 2018-11-09 |
| BE1023966A1 (fr) | 2017-09-25 |
| EP3389709A1 (en) | 2018-10-24 |
| WO2017102939A1 (en) | 2017-06-22 |
| JP7060507B2 (ja) | 2022-04-26 |
| BE1023966B1 (fr) | 2017-09-26 |
| JP2019504020A (ja) | 2019-02-14 |
| US11311618B2 (en) | 2022-04-26 |
| BR112018012126A2 (pt) | 2018-12-04 |
| GB201522329D0 (en) | 2016-02-03 |
| JP2022058449A (ja) | 2022-04-12 |
| US20200261569A1 (en) | 2020-08-20 |
| CA3008333A1 (en) | 2017-06-22 |
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