CN117964709A - 非唾液酸化的抗炎多肽 - Google Patents
非唾液酸化的抗炎多肽 Download PDFInfo
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Abstract
本发明涉及非唾液酸化的抗炎多肽，具体而言，本发明涉及抗炎剂、组合物和用于治疗炎性疾病的方法。
Description
本申请是申请号为201280062837.8，申请日为2012年12月10日，发明名称为“非唾液酸化的抗炎多肽”的中国专利申请的分案申请。
相关申请的交叉引用
本申请要求2011年12月19日提交的美国临时申请号61/577,361的优先权。该申请的内容全文作为参考引入本发明。
政府的利益
本发明至少部分地是由美国国立卫生研究院资助号NIH AI035875提供的政府支持下获得的。因此，美国政府对本发明拥有一定的权利。
技术领域
本发明涉及抗炎剂、组合物和用于治疗炎性疾病的方法。
背景技术
炎性疾病，包括自身免疫性疾病，是涉及白细胞的异常活化和随后迁移到身体受影响部位的疾病。这些疾病包括范围广泛、影响数百万世界各地人民生活的疾病。虽然目前存在各种治疗方法，但许多方法有显著的副作用或不能非常有效地缓解症状。因此，存在用于治疗炎性疾病的抗炎剂的需要和用于识别和评估这些试剂的方法的需求。
长期以来认为免疫球蛋白(IgG)通过其Fc片段介导的相互作用介导既促炎又抗炎的活性。Fc-FcyR相互作用负责免疫复合物和细胞毒抗体的促炎特性，而静脉内γ球蛋白(IVIG)及其Fc片段是抗炎的并且被广泛用于抑制炎性疾病。已经提出IgG的糖基化对IgG的细胞毒性和炎性潜能的调节是至关重要的。例如，已经表明IVIG的抗炎活性是Fc片段及其连接的聚糖(glycan)的性质，需要末端α.2,6唾液酸连接，表明免疫抑制对特定的多肽骨架和多糖结构的组合要求。(Anthony,et al.,2008,Science 320:373-376和WO 2007/117505)。
但是，IVIG中只有一小群IgG具有末端为α2,6唾液酸的聚糖(sFc)及抗炎活性。其结果是，为了在多种临床条件下抑制自身抗体触发的炎症，患者不得不以高剂量(1-2g/kg)服用IVIG，以富集唾液酸化的IgG，或以其他方式提高IgG的唾液酸化水平(美国申请号20080206246和20090004179，以及Nimmerjahn et al.Annu Rev Immunol 26,513-533(2008))。
本发明通过鉴定无唾液酸化的抗炎多肽解决并满足上述需求。
发明内容
本发明涉及一种制剂，例如多肽和抗体，和治疗炎性疾病(如自身免疫性疾病)的方法。
因此，本发明的一个方面的特征在于一种分离的多肽，其包括与IgG Fc区具有至少75％同一性(例如，介于75％和100％之间的任意数字，包括，例如70％、80％、85％、90％、95％、99％和100％)的修饰的序列。修饰的序列是没有唾液酸化的并且多肽具有高于亲本多肽的抗炎活性。亲本多肽可以包括IgG Fc区，例如下列SEQ ID NO：1的序列。在一些实施方案中，多肽具有结合DC-SIGN并结合hFcγRIIA或RIIB的活性。在一个实施方案中，分离的多肽具有以2×10-5M或更低的KD(即5.0×104M-1或更高的KA)结合hFcγRIIA或RIIB的活性。优选地，修饰的序列具有FA241突变。修饰的序列可以与SEQ ID NO：2具有至少75％同一性(例如，介于75％和100％之间的任意数字，包括，例如70％、80％、85％、90％、95％、99％和100％)。在一些实施例中，修饰的序列包括或基本上由SEQ ID NO：2组成。
在另一个方面，本发明提供制备具有抗炎活性的多肽的方法。除其它之外，方法包括提供具有IgG Fc区的序列的亲本多肽或编码亲本多肽的第一核酸序列；以及修饰亲本多肽以获得修饰的多肽，使得修饰的多肽没有唾液酸化并模拟IgG Fc区的唾液酸化形式的结构。修饰步骤可以通过修饰第一核酸序列以获得编码修饰的多肽的第二核酸而进行。本发明还提供通过上述方法制得的多肽。
在第三个方面，本发明特征在于包括编码上述多肽的序列的分离的核酸；包括核酸的表达载体；和包括核酸的宿主细胞。本发明特征还在于生产多肽的方法。方法包括在允许由核酸编码的多肽表达的条件下、在培养基中培养宿主细胞，并从所培养的细胞或细胞的培养基中纯化多肽。
在第四个方面，本发明特征在于一种药物制剂，其包括(i)上述多肽或核酸，以及(ii)药学上可接受的载体。
在第五个方面，本发明提供一种治疗炎性疾病的方法。方法包括给有需要的受试者施用治疗有效量的上述多肽或编码多肽的核酸。还提供多肽或核酸在制备治疗炎性疾病的药物中的用途。本发明特征还在于一种分离的多肽、核酸、表达载体、宿主细胞、组合物或用于治疗实质上如本发明所示和记载的炎性疾病的方法。
本发明的一个或多个实施方案的详细内容如下所示。本发明的其它特征、目的和优点在说明书和权利要求中将是显而易见的。
附图说明
图1a-c是显示α2,6-连接的唾液酸所赋予的DC-SIGN对重组人IgG1 Fc的结合活性的图和照片。
图2A-B是显示破坏Fc-聚糖相互作用所赋予的DC-SIGN对重组人IgG1Fc的结合活性的图和照片。
图3是显示hIgG1 Fc的FA241突变重现了α2,6sFc的抗炎活性的一系列图。
图4的a-d是显示FA241抗炎活性特征性要求的图。
图5是显示FA241突变增加Fcγ受体结合的一系列图。
图6的a-b是显示由FA241诱导骨髓来源的巨噬细胞的IL-33mRNA的照片。
具体实施方式
本发明至少部分地基于一个意外的发现，即非唾液酸化的IgG Fc变体赋予抗炎活性并作为抗炎介质(mediator)模拟2,6唾液酸化Fc的作用。
IgG和Fc唾液酸化
IgG是主要的血清免疫球蛋白。它是由两条相同的重链和两条轻链组成的糖蛋白，重链和轻链依次由可变区和恒定区组成。IgG在其两条重链的每一个的CH2结构域的Asn297位置上含有单个的、N-连接的聚糖。该共价连接的、复杂的碳水化合物(carbohydrate)是由一个核心的、双分支的含有N-乙酰葡糖胺(GlcNAc)和甘露糖(man)的戊多糖(penta-polysaccharide)组成。在血清抗体中观察到核心碳水化合物结构进一步修饰存在有可变存在(variably found)的岩藻糖、支链GlcNAc、半乳糖(gal)和末端唾液酸(sa)部分(moieties)。因此，已经检测到超过40种不同的糖型被共价连接到该单个糖基化位点(Fujii et al.,J.Biol.Chem.265,6009,1990)。已经显示IgG的糖基化对于结合至所有FcyRs以维持两条重链的开放构象是至关重要的。Jefferis和Lund,Immune.1Lett.82,57(2002),Sondermann et al.,J.Mol.Biol.309,737(2001)。据信，FcyR结合的该IgG糖基化解释了脱糖基化的IgG抗体不能介导体内触发的炎症反应，例如ADCC、吞噬作用和炎症介质的释放。Nimmerjahn和Ravetch,Immunity 24,19(2006)。进一步观察到IgG的个体糖型可能有助于调节炎症反应，这通过所报道的个体FcyRs对含有或缺乏岩藻糖的IgG抗体的改变的亲和性和它们随之对细胞毒作用的影响而得以证实。Shields et al.,J.Biol.Chem.277,26733(2002),Nimmerjahn和Ravetch,Science310,1510(2005)。在患有类风湿关节炎和一些自身免疫性血管炎的患者中已经观察到自身免疫状态和IgG抗体的特定糖基化模式之间的联系，其中已报道了IgG抗体降低的糖基化和唾液酸化。Parekh et al.,Nature 316,452(1985),Rademacher et al.,Proc.Natl.Acad.Sci.USA 91,6123(1994),Matsumoto etal.,128,621(2000),Holland et al.,Biochim.Biophys.Acta 12月27日。也已经报道IgG糖型的变化与衰老和免疫相关，尽管还没有确定这些变化在体内的意义。Shikata et al.,Glycoconj.J.15,683(1998),Lastra,et al.,Autoimmunity 28,25(1998)。
如本发明所公开的，某些非唾液酸化的IgG Fc变体也令人惊讶地赋予抗炎活性。这些变体，包括FA241变体，由于凭借模拟唾液酸化的Fc的结构和生物学特性但不需要唾液酸化而代表了较大类分子内的品类，，能够被开发作抗炎治疗剂。
多肽和核酸
多肽
如本发明所公开的，本发明提供分离的多肽，其具有人IgG Fc变体的序列，其缺乏具有位于前述Asn237位点、通过α2,6连接连接到半乳糖部分的末端唾液酸的多糖链。该非唾液酸化的IgG Fc变体可源自天然存在的抗体或表达于细胞系。
在一个实施方案中，Fc区包括一个或多个hIgG1氨基酸序列的取代。尽管不局限于此，示例性的IgG1 Fc区提供如下：
hIgG1的Fc(从Kabat系统210位氨基酸开始)：
(SEQ ID NO：1；下划线为F241和F243)
hIgG1 FA241的Fc：
(SEQ ID NO：2)
hIgG1 FA243的Fc：
(SEQ ID NO：3)
本发明交互使用术语“肽”、“多肽”和“蛋白质”，描述聚合物中氨基酸残基的结构(arrangement)。除了罕见的氨基酸和合成的氨基酸类似物，肽、多肽或蛋白质可以由标准的20种天然存在的氨基酸组成。它们可以是任何氨基酸链，而不管长度或翻译后修饰(例如糖基化或磷酸化)。“本发明的”肽、多肽或蛋白质包括重组或合成生产的具有特定的结合DC-SIGN、FcγRIIA和FcγRIIB的结构域或部分。该术语还包括具有附加的氨基末端甲硫氨酸的多肽(在原核细胞中表达时有用)。
“分离”的多肽或蛋白质指已经被从与其一起天然相关的其他蛋白质、脂质和核酸中分离的多肽或蛋白质。多肽/蛋白质可以占纯化制剂干重的至少10％(即介于10％和100％之间的任意百分比，例如20％、30％、40％、50％、60％、70％、80％、85％、90％、95％和99％)。纯度可以通过任何合适的标准方法来测定，例如，通过柱层析、聚丙烯酰胺凝胶电泳或HPLC分析。本发明所述的分离的多肽/蛋白质可以从天然来源纯化、通过重组DNA技术生产或通过化学方法生产得到。IgG Fc的功能等价物指IgG Fc的多肽衍生物，例如，具有一个或多个点突变、插入、删除、截短的蛋白、融合蛋白，或其组合。它基本上保留IgG Fc的活性，即，结合至相应受体并触发相应细胞反应的能力。分离的多肽可以含有SEQ ID NO：2。通常，功能等价物与SEQ ID NO：2具有至少75％(例如，介于75％和100％之间的任意数字，包括，例如70％、80％、85％、90％、95％和99％)的同一性。
两个氨基酸序列或两个核酸的“同一性百分比”采用Karlin和AltschulProc.Natl.Acad.Sci.USA 87:2264-68,1990所述并如Karlin和AltschulProc.Natl.Acad.Sci.USA 90:5873-77,1993修改的算法确定。该算法整合入Altschul,etal.J.Mol.Biol.215:403-10,1990的NBLAST和XBLAST程序(2.0版本)。BLAST核苷酸检索可以通过NBLAST程序、分数＝100、字长-12来进行以获得与本发明的核酸分子同源的核苷酸序列。BLAST蛋白质检索可以通过XBLAST程序、分数＝50、字长＝3来进行以获得与本发明的蛋白质分子同源的氨基酸序列。当两个序列之间存在缺口(gap)时，可以采用Altschul etal.,Nucleic Acids Res.25(17):3389-3402,1997所述的缺口BLAST。当采用BLAST和缺口BLAST程序时，可以使用相应程序(例如XBLAST和NBLAST)的缺省参数。
本发明多肽的氨基酸组成可以在不破坏多肽结合至相应受体并触发相应细胞反应的能力的情况下发生改变。例如，它可以含有一个或多个保守氨基酸替换。“保守氨基酸替换”是一个氨基酸残基被替换为一个具有相似侧链的氨基酸残基。本领域已经定义了具有相似侧链的氨基酸残基家族。这些家族包括具有碱性侧链(例如赖氨酸、精氨酸、组氨酸)、酸性侧链(例如天冬氨酸、谷氨酸)、不带电荷的极性侧链(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸)、非极性侧链(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸，苯丙氨酸、甲硫氨酸、色氨酸)、β-支链侧链(例如苏氨酸、缬氨酸、异亮氨酸)和芳香族侧链(例如酪氨酸、苯丙氨酸、色氨酸、组氨酸)的氨基酸。因此，在例如SEQ ID NO：2中的一个预测的非必需氨基酸残基优选由来自相同侧链家族的另一个氨基酸残基来替换。可选的，突变可以沿着序列全长或部分随机引入，例如通过饱和突变，并且由此产生的突变体被筛选结合至相应受体并触发相应细胞反应的能力，以鉴定保留以下实施例所述的活性的突变体。
本发明的多肽可以作为重组多肽获得。为了制备重组多肽，编码它的核酸(例如FA241、SEQ ID NO：2)可以连接至编码融合伴侣的另一个核酸，所述融合伴侣例如谷胱甘肽-s-转移酶(GST)、6x-组氨酸表位标签或M13基因3蛋白。由此产生的融合核酸在合适的宿主细胞中表达可以通过本领域已知的方法分离的融合蛋白。所分离的融合蛋白可以被进一步处理，例如，通过酶消化，以去除融合伴侣并获得本发明的重组多肽。
核酸
本发明的另一个方面特征在于分离的核酸，其包括编码上述多肽或蛋白质的序列。核酸指DNA分子(例如cDNA或基因组DNA)、RNA分子(例如mRNA)或DNA或RNA类似物。DNA或RNA类似物可以由核苷酸类似物合成。核酸分子可以是单链或双链的，但优选双链的DNA。“分离的核酸”指一种核酸，其结构与任何天然存在的核酸或天然存在的基因组核酸的任何片段的结构都不同。因此，该术语包括，例如，(a)DNA，其具有天然存在的基因组DNA分子的部分的序列，但其两侧并不连接(is not flanked by)位于其天然存在的生物体的基因组的分子的部分两侧的两个编码序列；(b)核酸，其整合入载体或原核或真核基因组DNA，其整合方式使得所得的分子与任何天然存在的载体或基因组DNA都不同；(c)分离的分子，例如cDNA、基因组片段、由聚合酶链式反应(PCR)生产的片段或酶切片段；以及(d)重组核苷酸序列，其是杂合体基因的一部分，即编码融合蛋白的基因。上述核酸可以用于表达本发明的融合蛋白。为此目的，可以有效地将核酸与合适的调节序列连接以产生表达载体。
载体指能够运送与其相连的另一核酸的核酸分子。载体可以能够自住复制或整合入宿主DNA中。载体的例子包括质粒、粘粒或病毒载体。载体包括处于适于在宿主细胞内表达核酸的形式的核酸。优选地，载体包括一个或多个有效地连接至待表达的核酸序列的调节序列。
“调节序列”包括启动子、增强子和其它表达控制元件(例如多聚腺苷酸化信号)。调节序列包括那些指导核苷酸序列组成型表达的，以及组织特异性调节和/或诱导型的序列。表达载体的设计可取决于这样的因素：所转化的宿主细胞的选择、所期望的蛋白质或RNA的表达水平等。表达载体可被引入宿主细胞以生产本发明的多肽。启动子定义为指导RNA聚合酶结合DNA并起始RNA合成的DNA序列。强启动子是导致mRNA高频起始的启动子。
上述任何多核苷酸或本领域技术人员为了相同的目的可用的生物学等价的多核苷酸可被插入到合适的表达载体并与其它DNA分子相连，以形成表达该接受体(receptor)的“重组DNA分子”。这些载体可以包括DNA或RNA；对大多数克隆目的，优选DNA载体。典型的载体包括质粒、修饰的病毒、噬菌体和粘粒、酵母人工染色体和其它形式的附加型或整合的DNA。确定某一特定用途的合适载体是本领域技术人员公知的。
有各种哺乳动物表达载体可用于在哺乳动物细胞中表达上述IgG Fc。如上面所指出的，表达载体可以是在合适的宿主中转录克隆的DNA并翻译mRNA所需要的DNA序列。这些载体可用于在各种宿主(例如细菌、蓝绿藻、植物细胞、昆虫细胞和动物细胞)中表达真核DNA。特定设计的载体允许DNA在宿主之间(例如细菌-酵母或细菌-动物细胞)穿梭。合适构建的表达载体应当含有：在宿主细胞中自主复制的复制起点、选择性标记、限定数目的有用的限制性酶切位点、高拷贝数的潜力，和活跃的启动子。表达载体可以包括但不限于克隆载体、修饰的克隆载体、特定设计的质粒或病毒。合适的市售的哺乳动物表达载体包括但不限于pcDNA3.neo(Invitrogen),pcDNA3.1(Invitrogen),pCI-neo(Promega),pLITMUS28,pLITMUS29,pLITMUS38和pLITMUS39(New England Bioloabs),pcDNAI,pcDNAIamp(Invitrogen),pcDNA3(Invitrogen),pMC1neo(Stratagene),pXTl(Stratagene),pSG5(Stratagene),EBO-pSV2-neo(ATCC 37593)pBPV-1(8-2)(ATCC 37110),pdBPV-MMTneo(342-12)(ATCC 37224),pRSVgpt(ATCC 37199),pRSVneo(ATCC 37198),pSV2-dhfr(ATCC37146),pUCTag(ATCC 37460),和IZD35(ATCC 37565)。
还落在本发明范围内的是含有上述核酸的宿主细胞。例子包括大肠杆菌细胞、昆虫细胞(例如使用杆状病毒表达载体)、酵母细胞或哺乳动物细胞。参见，例如Goeddel,(1990)Gene Expression Technology:Methods in Enzymology 185,Academic Press,SanDiego,Calif。为了生产本发明的多肽，可以在允许表达由本发明核酸编码的多肽的条件下、在培养基中培养宿主细胞，并从所培养的细胞或细胞的培养基中纯化多肽。可选的，本发明的核酸可以在体外转录和翻译，例如使用T7启动子调节序列和T7聚合酶。
所有天然存在的IgG Fc、遗传工程化的IgG Fc和化学合成的IgG Fc都可以用于实施本发明所公开的发明。通过重组DNA技术获得的IgG Fc可以具有与[FA241]SEQ ID NO：2)或与其功能等价物相同的氨基酸序列。术语“IgG Fc”还包括化学修饰的版本。化学修饰的IgG Fc的例子包括发生构象变化、添加或缺失糖链的IgG Fc和已经结合化合物(例如聚乙二醇)的IgG Fc。
可以使用如下所示的动物模型(例如转基因小鼠)来验证这样制得的多肽/蛋白质的功效。在体内嗜碱细胞IL-33表达或效应子巨噬细胞上的FcγRIIB受体表达的任何统计学上显著的增加都表明该多肽/蛋白质是用于治疗下文所述疾病的候选者。在一个实施方案中，上述分析可以基于结合至DC-SIGN蛋白或DC-SIGN(+)细胞的测量。本领域充斥着本领域技术人员可用的各种技术，其适于测量化合物至DC-SIGN或DC-SIGN(+)细胞的能力以及由DC-SING通路调控的基因的表达的相关改变，例如IL-33。本领域技术人员不需要进行过度的实验就能够调配并匹配这些各种研究工具。一旦通过标准方法或根据以下实施例所述的分析和方法进行了纯化和检测，非唾液酸化的IgG Fc变体可被包含于用于治疗炎性疾病的药物组合物中。
如本发明所使用的，“抗体”以其最广泛的含义使用并具体包括单克隆抗体(包括全长单克隆抗体)、多克隆抗体、多特异性抗体(例如双特异性抗体)和抗体片段，只要它们展示所期望的生物学活性。
如本发明所使用的，“抗体片段”可以包括完整抗体的一部分，通常包括完整抗体的抗原结合或可变区或抗体的保留FcR结合能力的Fc区。抗体片段的例子包括线性抗体；单链抗体分子和由抗体片段形成的多特异性抗体。抗体片段优选保留至少部分铰链区和可选的IgG重链的CH1区。更优选地，抗体片段保留IgG重链的完整恒定区并包括IgG轻链。
如本发明所使用的，术语“Fc片段”或“Fc区”用于定义免疫球蛋白重链的C-末端区。“Fc区”可以是天然序列Fc区或变体Fc区。虽然免疫球蛋白重链的Fc区的边界可能发生变化，人IgG重链Fc区通常定义为从Cys226位或从Pro230位氨基酸残基至其羧基末端的片段(stretch)。
“天然序列Fc区”包括与自然界中发现的Fc区的氨基酸序列相同的氨基酸序列。本领域普通技术人员知晓的“变体Fc区”包括与天然序列Fc区由于至少一个“氨基酸修饰”而不同的氨基酸序列。优选地，与天然序列Fc区或亲本多肽的Fc区相比，变体Fc区具有至少一个氨基酸替换，例如在天然序列Fc区或亲本多肽的Fc区中大约一至大约十个氨基酸替换，并优选从大约一个至大约五个氨基酸替换。本发明的变体Fc区优选与天然序列Fc区和/或与亲本多肽的Fc区具有至少大约75或80％同源性，并且更优选至少大约90％同源性，更优选至少大约95％同源性，甚至更优选至少大约99％同源性。
术语“Fc受体”或“FcR”用于描述结合抗体的Fc区的受体。在本发明的一个实施方案中，FcR是天然序列人FcR。在另一个实施方案中，FcR(包括人FcR)结合IgG抗体(γ受体)并包括FcγRI、FcγRII和FcγRIII亚类的受体，包括等位的变体和可选的这些受体的剪接形式。FcγRII受体包括FcγRIIA(“激活受体”)和FcγRIIB(“抑制受体”)，其具有相似的氨基酸序列而主要在其胞质结构域不同。激活受体FcγRIIA在其胞质结构域含有基于免疫受体酪氨酸的激活基序(immunoreceptor tyrosine-based activation motif，ITAM)。抑制受体FcγRIIB在其胞质结构域含有基于免疫受体酪氨酸的抑制基序(immunoreceptortyrosine-based inhibition motif，ITIM)(综述参见Daron,Annu Rev Immunol,15,203-234(1997)；FcRs综述于Ravetch和Kinet,Annu Rev Immunol,9,457-92(1991)；Capel etal.,Immunomethods,4,25-34(1994)；和de Haas et al.,J Lab Clin Med,126,330-41(1995),Nimmerjahn和Ravetch 2006,Ravetch Fc Receptors in FundementalImmunology,ed William Paul 5th Ed。其每一个的全文作为参考引入本发明)。
术语“天然”或“亲本”指包括Fc氨基酸序列的未修饰的多肽。亲本多肽可以包括天然序列Fc区或带有预存在的氨基酸序列修饰(例如添加、缺失和/或替换)的Fc区。
组合物
在本发明范围内的是含有合适的载体和一种或多种上述试剂的组合物，例如非唾液酸化的IgG Fc变体。组合物可以是含有药学上可接受的载体的药物组合物或含有美容学上可接受的载体的化妆品组合物。
术语“药物组合物”指活性剂与惰性或活性载体的组合，使得该组合物特别适合于体内的或离体的诊断或治疗用途。“药学上可接受的载体”在给予受试者后不导致不希望的生理效应。药物组合物中的载体必须是“可接受的”的意义还在于它是与活性成分相容的并且可以能够稳定它。一种或多种增溶剂可以用作药物载体以用于递送活性化合物。药学上可接受的载体的例子包括但不限于生物相容性载体、佐剂、添加剂和稀释剂，以获得可作为一种剂型的组合物。其它载体的例子包括胶态硅氧化物、硬脂酸镁、纤维素和月桂基硫酸钠。
处于上述任何形式的上述组合物可用于治疗特征为炎症的疾病。有效量指赋予一个治疗的受试者治疗效果所需要的活性化合物/试剂的量。如本领域技术人员所知晓的，有效剂量会发生变化，这取决于所治疗疾病的类型、给药途径、赋形剂的使用和与其他治疗性处理共同使用的可能性。
本发明的药物组合物可以经肠胃外、经口、经鼻、经直肠、局部或经颊给药。本发明所使用的术语“经肠胃外”指皮下、皮内、静脉内、肌肉内、关节内、动脉内、滑膜内、胸骨内、鞘内、病灶内或颅内注射，以及任何合适的输注技术。
无菌可注射组合物可以是在无毒的肠胃外可接受的稀释剂或溶剂中的溶液或悬浮液。这些溶液包括但不限于1,3-丁二醇、甘露糖醇、水、林格氏溶液和等渗氯化钠溶液。此外，不挥发性油通常用作溶剂或悬浮介质(例如合成的单或双甘油酯)。脂肪酸(例如但不限于油酸及其甘油酯衍生物)可用于注射剂的制备，天然的药学上可接受的油(例如但不限于橄榄油或蓖麻油、其聚氧乙烯化的版本)也可以使用。这些油溶液或悬浮液还可以含有长链醇稀释剂或分散剂，例如但不限于羟甲基纤维素或类似的分散剂。为了制剂目的，也可以使用其它常用的表面活性剂，例如但不限于吐温或SPANS或其它相似的乳化剂或生物利用度增强剂，其通常用于药学上可接受的固体、液体或其它剂型的制备。
用于口服给药的组合物可以是任何口服可接受的剂型，包括胶囊剂、片剂、乳剂和水性悬浮剂、分散剂和溶液剂。
在片剂的情况下，常用的载体包括但不限于乳糖和玉米淀粉。还通常加入润滑剂，例如但不限于硬脂酸镁。对于胶囊形式的口服给药，有用的稀释剂包括但不限于乳糖和干燥的玉米淀粉。当水性悬浮剂或乳剂被口服给药时，活性成分可悬浮或溶解于与乳化剂或悬浮剂组合的油相中。如果需要，可以加入某些甜味剂、矫味剂或着色剂。
根据本发明的用于局部给药的药物组合物可以配制成溶液剂、软膏剂、霜剂、悬浮剂、洗剂、粉剂、糊剂、凝胶剂、喷雾剂、气雾剂或油剂。可选地，局部用制剂可以是浸渍有活性成分的贴剂或者敷料的形式，其可任选地包括一种或多种赋形剂或稀释剂。在一些优选的实施方案中，局部用制剂包括能够增强活性剂通过皮肤或其他感染区域的吸附或渗透的材料。局部用组合物对于治疗皮肤的炎性疾病，包括但不限于湿疹、痤疮、红斑痤疮、牛皮癣、接触性皮炎和对野藤的反应。
局部用组合物含有安全和有效量的适于施用到皮肤上的皮肤学上可接受的载体。“美容学上可接受的”或“皮肤学上可接受的”组合物或成分指适合用于接触人皮肤而没有不适当的毒性、不相容性、不稳定性、变态反应等的组合物或成分。载体使得活性剂和可选成分被以合适的浓度递送至皮肤。因此，载体可以作为稀释剂、分散剂、溶剂等发挥作用以确保活性材料被施用并以合适的浓度平均分布在所选目标上。载体可以为固体、半固体或液体。载体可以是乳液、乳膏或凝胶的形式，尤其是具有足够厚度或屈服点(yield point)以阻止活性材料沉淀的一种。载体可以上惰性的或具有皮肤学上的益处。它还可以与本发明的活性成分在生理和化学上相容，并且不应当不适当地损害与该组合物相关的稳定性、有效性或其它用途的益处。局部用组合物可以是本领域中已知的用于局部或经皮施用形式的化妆品或皮肤病学的产品，所述形式包括溶液剂、气雾剂、乳膏剂、凝胶剂、贴剂、软膏剂、洗剂或泡沫剂。
治疗方法
本发明提供治疗受试者炎性疾病的方法。术语“炎性疾病”指特征为异常的或不希望的炎症的疾病，例如自身免疫性疾病。自身免疫性疾病是特征为在非激活条件下免疫细胞被慢性激活的疾病。例子包括牛皮癣、炎性肠病(例如克罗恩病和溃疡性结肠炎)、类风湿关节炎、银屑病关节炎、多发性硬化症、狼疮、I型糖尿病、原发性胆汁性肝硬化和移植。
可以通过本发明的方法治疗的炎性疾病的其它例子包括哮喘、心肌梗塞、中风、炎性皮肤病(例如皮炎、湿疹、特应性皮炎、过敏性接触皮炎、荨麻疹、坏死性血管炎、皮肤血管炎、超敏性血管炎、嗜酸细胞性肌炎、多发性肌炎、皮肤肌炎和嗜酸细胞性筋膜炎)、急性呼吸窘迫综合症、爆发性肝炎、超敏性肺疾病(例如过敏性肺炎、嗜酸细胞性肺炎、迟发型过敏、间质性肺疾病(ILD)、特发性肺纤维化和类风湿关节炎相关的ILD)和过敏性鼻炎。另外的例子还包括重症肌无力、青少年发病型糖尿病、肾小球肾炎、自身免疫性甲状腺炎、强直性脊柱炎、系统性硬化症、急性和慢性炎性疾病(如全身性过敏和超敏反应、药物过敏、昆虫叮咬过敏、移植排斥和移植物抗宿主病)和干燥综合征。
“受试者”指人或非人类动物。非人类动物的例子包括所有脊椎动物，例如哺乳动物，例如非人类哺乳动物、非人类灵长类动物(尤其是高级灵长类动物)、狗、啮齿类动物(例如小鼠或大鼠)、豚鼠、猫和兔，以及非哺乳动物，例如鸟、两栖动物、爬行动物等。在一个技术方案中，受试者是人。在另一个实施方案中，受试者是实验性、非人类动物或合适作为疾病模型的动物。
要被治疗炎性疾病的受试者可以通过诊断该疾病的标准技术来鉴定。任选地，受试者可以通过本领域已知的方法检查从受试者获得的试验样品的一种或多种细胞因子或细胞的水平或百分比。如果水平或百分比位于或低于阈值(其可从正常受试者获得)，受试者是本发明治疗的候选者。为确认抑制或治疗，可以评估和/或验证治疗后受试者中一种或多种上述细胞因子或细胞的水平或百分比。
“治疗(treating)”或“治疗(treatment)”指向受试者施用化合物或试剂，所述受试者患有疾病，施用目的是为了治愈、减轻、缓解、补救、延缓发病、预防或改善疾病、疾病的症状、疾病继发的疾病状态或对疾病的易感性。
“有效量”或“治疗有效量”指能够在所治疗的受试者中产生医学上所想要的结果的化合物或试剂的量。治疗方法可以在体内或离体进行，单独地或者与其它药物或治疗联合。治疗有效量可以一次或多次给药、施用或剂量被施用，且不应被限于特定的制剂或给药途径。
试剂可以体内或离体施用，单独或与其它药物或治疗联合施用，即，鸡尾酒疗法。如本发明所使用的，术语“共同施用(co-administration)”或“共同施用(co-administered)”指给受试者施用至少两种试剂或治疗。在一些实施方案中，两种或更多种试剂/治疗的共同施用是同时进行的。在其它实施方案中，第一试剂/治疗在第二试剂/治疗之前施用。本领域技术人员理解，可以改变所使用的各种试剂/治疗的剂型和/或给药途径。
在体内方法中，给受试者施用化合物或试剂。通常，化合物或试剂悬浮于药学上可接受的载体(如，例如但不限于生理盐水)并口服或通过静脉内输注给药，或经皮下、肌内、鞘内、腹膜内、直肠内、阴道内、鼻内、胃内、气管内或肺内注射或植入。
所需剂量(dosage)取决于所选择的给药途径；制剂的性质；患者疾病的性质；受试者的大小、重量、表面积、年龄和性别；施用的其它药物；和主治医师的判断。合适的剂量是在0.01-100mg/kg的范围内。所需剂量的变化考虑到可用的化合物/试剂种类和各种给药途径的不同效率而是可以预期的。例如，可以预期口服给药比通过静脉注射给药需要更高的剂量。这些剂量水平的变化可以使用本领域公知的标准的经验性的优化途径进行调整。在合适的递送载体(例如聚合微粒或植入装置)中的化合物的封装可以提高递送效率，尤其对于口服递送而言。
实施例1：方法和材料
本实施例描述了实施例2-7所使用的通用方法和材料。
小鼠
野生型C57BL/6小鼠购自Jackson Laboratories。SIGNR1-/-小鼠由A.McKenzie提供。CD11c-DC-SIGN+转基因小鼠由T.Sparwasser提供。SIGNR1-/-背景的hDC-SIGN BAC转基因小鼠由发明人的实验室按先前所述方法产生。KRN TCR C57BL/6小鼠(由D.Mathis和C.Benoist赠送)与NOD小鼠一起饲养以产生K/BxN小鼠。收集K/BxN小鼠(6-12周龄)血并且含有致关节炎抗体的血清合并到一起。200μL K/BxN血清通过静脉注射被动转移至首次用于实验的小鼠(8-12周龄)来诱导关节炎。对每个爪子进行炎症评分0-3并加到一起得到每个个体小鼠的总临床得分。
重组Fc制备
IDEC-114(全长人IgG1单克隆抗体的重组来源)用木瓜蛋白酶于37℃过夜消化以裂解Fab和Fc片段。消化后，加入2.5mg/mL的碘乙酰胺终止反应。为了从未消化的抗体中分离裂解的片段，将样品通过HiPrep 26/60S-200HR体积排阻柱(GE HEALTHCARE)。随后，Fc片段用蛋白G琼脂糖珠纯化。样品纯度通过SDS-聚丙烯酰胺凝胶的考马斯亮蓝染色验证。可选地，重组Fc可以通过人IgG1 Fc表达质粒瞬时转染至293T细胞随后通过上清部分的硫酸铵沉淀和蛋白G纯化生产得到。通过标准PCR实验步骤从4-4-20IgG1扩增编码人IgG1的Fc区的基因序列并连入pSecTag2(INVITROGEN)。通过标准的定点突变技术向Fc编码序列引入点突变并通过DNA测序进行验证。用于在241位Phe至Ala替换(FA241)的PCR引物是
5'-ggggaccgtcagtcgccctcttccccccaa-3'(SEQ ID NO:4)，和
5'-ttggggggaagagggcgactgacggtcccc-3'(SEQ ID NO:5)。
用抗人Fc抗体的免疫印迹和/或SDS-聚丙烯酰胺凝胶的考马斯亮蓝染色确认蛋白的表达和纯度。
两步体外唾液酸化反应
纯化后，10-50mg/mL Fc片段的缓冲液被交换成半乳糖化反应缓冲液(50mM MOPS,pH 7.2；20mM MnCl2)并用50mg UDP-半乳糖和0.75Uβ1,4-半乳糖转移酶37℃过夜孵育。使用ECL通过凝集素印迹识别末端半乳糖残基来证实半乳糖化。然后，半乳糖化的Fc的缓冲液被交换成唾液酸化反应缓冲液(100mM MOPS,0.2mg/mL BSA,0.5％ Triton X-100,pH 7.4)并用50mg CMP-唾液酸和0.75Uα2,6-唾液酸转移酶37℃过夜孵育。使用SNA通过凝集素印迹识别带有α2-6连接的末端唾液酸残基来证实唾液酸化。
骨髓衍生的巨噬细胞的过继转移
骨髓细胞从DC-SIGNtg或SIGNR1-/-小鼠的胫骨和股骨冲下，并接种于补充有10％FBS、1％青/链霉素、IL-3(5ng/mL,PEPROTECH)和M-CSF(5ng/mL,PEPROTECH)的RPMI 1640培养基的非组织培养物处理的10厘米平板。37℃过夜孵育后，回收未吸附的细胞并转至补充有IL-3/M-CSF的RPMI培养基的非组织培养物处理的10厘米平板，并于37℃培养5-7天。成熟的巨噬细胞用胰蛋白酶消化并以2xl06细胞/孔的密度接种在6孔板中，并使其附着过夜。第二天，巨噬细胞于37℃用所示重组Fc制剂脉冲处理30min。回收细胞，用冷PBS洗涤，1xl06细胞静脉施用给野生型C57BL/6小鼠。注射一小时后，受体小鼠接受K/BxN血清的攻毒(challenge)。
可溶性人DC-SIGN的表达与纯化
含有人DC-SIGN胞外结构域(ECD)的cDNA序列的质粒由K.Drickamer提供。通过标准PCR技术修饰DC-SIGN ECD的编码序列以引入N-末端链霉素标签并连接至pET28b(+)。pET28b-strepDCSIGN转化大肠杆菌菌株BL21/DE3并在3L TB培养基中37℃培养直至细菌培养物达到OD6000.7-0.8。加入100mg/L的IPTG诱导蛋白表达并且培养物37℃培养3.5h。4℃4000xg离心10min来沉淀细菌。细菌沉淀重悬于10mM Tris-HCl，pH7.8，并超声裂解。4℃10,000xg离心15min来沉淀包涵体并溶于100mL的6M盐酸胍；100mM Tris-HCl,pH 7.8；0.2％ TRITON X-100。4℃ 20,000xg离心30min来去除颗粒物质，并且上清部分用250mMNaCl；25mM Tris-HCl,pH 7.8；25mM CaCl2透析。透析后，4℃ 20,000xg离心30min来去除不溶性沉淀，上清组分施加到strep-tactin树脂(NOVAGEN)以拉下链霉素标记的DC-SIGNECD。用制造商(NOVAGEN)提供的洗脱缓冲液将结合的蛋白从树脂上洗脱下来。组分用SDS-PAGE分析并且合并阳性组分并装到甘露糖-琼脂糖柱以选择活性受体。用250mM NaCl；25mMTris-HCl,pH 7.8；5mM EDTA洗脱DC-SIGN ECD。组分用SDS-PAGE分析。
表面等离子体共振
为了确定各种重组Fc制剂对可溶性hDC-SIGN或hFcγR的相互作用，稳态亲和测量记录于Biacore T100传感器。于NaOAc pH 5.0中稀释至20-50μg/mL的受体通过标准胺偶联高密度地(2000RU)固定于CM5芯片。对hDC-SIGN相互作用，用调整至pH 9.0并用2mM CaCl2和500mM NaCl补充的市售HBS-P+缓冲液，以流速20μL/min进行注射。对hFcγR相互作用，用市售的HBS-EP+缓冲液以流速20μL/min进行注射。用50mM NaOH的短脉冲再生表面。采用Biacore评价软件计算去除结合至对照流动池(flow cell)的背景后的Kd值。
RT-PCR
使用RNeasy迷你试剂盒(QIAGEN)从骨髓来源的巨噬细胞提取总RNA。一毫克总RNA用于通过RT-PCR采用OneStep RT-PCR试剂盒(QIAGEN)分析IL-33mRNA表达。GAPDH表达作为上样对照。mIL-33的PCR引物是5'-gaagatcccaacagaagacc-3'(SEQ ID NO:6)和5'-ttccggaggcgagacgtcac-3'(SEQ ID NO:7)；以及mGAPDH的引物是5'-gccgcctggagaaacctgc-3'(SEQ ID NO:8)和5'-tgaggtccaccaccctgttg-3'(SEQ ID NO:9)。PCR条件是94℃ 30s；55℃ 30s；72℃ 60s x 35个循环(IL-33)或25个循环(GAPDH)。
实施例2：α2,6-连接的唾液酸赋予DC-SIGN与重组人IgG1Fc的结合活性
IVIG制剂中一小群抗体抑制自身抗体诱导的炎症。这些在Fc聚糖上含有末端α2,6-连接的唾液酸的抗体通过结合边缘区巨噬细胞上的SIGNR1或其髓样细胞上的人直系同源物DC-SIGN来介导抗炎反应。
为了研究sFc与DC-SIGN的相互作用，从细菌纯化了DC-SIGN的胞外结构域(DC-SIGN ECD)的可溶性形式并固定于CM5芯片上。sFc从全长IDEC-114抗体制备并体外唾液酸化(图1b)，并特异性结合至DC-SIGN缀合的表面(图1a)。进行稳态亲和测量并且计算该相互作用的KD值为～1.3x10-6M(图1c)。与此相反，IDEC-114Fc的非唾液酸化糖型没有显示与DC-SIGN的结合活性，这表明唾液酸化诱导Fc骨架上的构象改变以呈现DC-SIGN结合位点。
如图1a所示，如通过表面等离子体共振测量(SPR)所测量的，发现重组α2,6-sFc结合至可溶性DC-SIGN。木瓜蛋白酶裂解全长人单克隆IgG1抗体(IDEC-114)并随后进行体外半乳糖化和唾液酸化反应来制备Fc。如上述实施例1所述，抗体结合固定的DC-SIGN的SPR传感图显示了hIgG1 Fc的唾液酸化和半乳糖化糖型。发现流过DC-SIGN ECD的Fc浓度范围为3-0.8μΜ。如图1b所示，与SNA的凝集素印迹证实带有2,6-连接的唾液酸在Fc上的连接(上图)；考马斯染色的上样对照显示于下图。通过Biacore评价软件计算sFc结合至DC-SIGN的稳态KD测量值(如a.所示)。
实施例3破坏Fc-聚糖相互作用的突变赋予DC-SIGN与重组人IgG1
Fc的结合活性
连接至Asn297的核心寡糖链得到与Fc氨基酸骨架的广泛非共价相互作用。由连接至核心聚糖的不同糖残基诱导的Fc内的构象变化由这些蛋白-碳水化合物相互作用介导。消除Fc骨架与聚糖残基间的关键接触点的丙氨酸替换似乎损害(impart)DC-SIGN结合活性。
如图2A所示，FA241和FA243突变显示没有体外酶法处理的DC-SIGN结合活性。表观KD值范围从FA241的6x10-7 M至FA243的3x10-7 M。之前的报道显示这些突变提高抗体的唾液酸化，推测可能是通过表达于哺乳动物细胞时使得聚糖更接近糖基转移酶。为了验证这一点，进行凝集素印迹以确定是否FA241和FA243结合至DC-SIGN是因为瞬时表达的蛋白的提高的唾液酸化。如图2B所示，SNA印迹并未检测到纯化的FA241和FA243内的末端唾液酸残基，这表明DC-SIGN相互作用独立于唾液酸修饰。
更具体地，沿着IgG1 Fc氨基酸骨架的残基F241、F243、D265和R301替换为丙氨酸以破坏与寡糖残基的非共价相互作用。从293T细胞表达并纯化Fc并用上述表面等离子体共振分析DC-SIGN结合活性。发现具有突变FA241或FA243的Fc相对于sFc的亲和性测量显示出对DC-SIGN的增加的亲和性(图1a)。进行与ECL(图1b，中间图)和SNA(上图)的凝集素印迹以确定从293T细胞纯化的Fc上的末端糖部分。作为唾液酸化Fc的阳性对照，如图1a所述，FA241体外唾液酸化。考马斯染色的上样对照显示于图1b的下图。
实施例4hIgG1Fc的FA241突变重现了α2,6sFc的抗炎活性
如果FA241和FA243突变模拟sFc的DC-SIGN结合活性，则进行试验以检查这些突变是否能够复制sFc的体内抗炎活性。用致关节炎的K/BxN血清攻击(challenge)年龄和性别匹配的SIGNR1-/-和hDC-SIGN+/SIGNR1-/-小鼠并以有效剂量0.033g/kg的sFc、FA241或FA243处理。与先前的发现一致，sFc抑制DC-SIGN+小鼠而不是SIGNR1-/-小鼠的足垫肿胀。相似地，证实FA241在hDC-SIGN+/SIGNR1-/-小鼠中具有可与sFc相比的抗炎活性。施用FA243的小鼠没有显示出关节炎症的减轻。这些发现表明，具有F241A突变的重组Fc(FA241)在没有唾液酸修饰的情况下重现了sFc的DC-SIGN结合和抗炎活性。
如图3所示，hDC-SIGN+/SIGNR1-/-(白方块)和SIGNR1-/-(黑方块)小鼠通过静脉注射施用0.7mg/小鼠的sFc、FA241或FA243。1h后，随后用K/BxN血清挑战小鼠。监测并评分足垫肿胀好几天。如先前报道的，sFc的抗炎活性是DC-SIGN依赖性的(左图)。FA241也以DC-SIGN依赖性的方式抑制K/BxN血清挑战(challenge)的小鼠的关节炎。在第6天，FA243没有显著降低足垫肿胀。第6天绘制每组4-5只小鼠的临床评分的均值和SEM。
实施例5表征FA241抗炎活性的要求
为了鉴定FA241的抗炎活性的决定因素，来自CD11c.DC-SIGN+和SIGNR1-/-小鼠的骨髓衍生的巨噬细胞(BMMΦ)用FA241或其它Fc制剂刺激并转至用K/BxN血清挑战的WTC57BL/6受体小鼠。
简言之，于IL-3(5ng/mL)和M-CSF(5ng/mL)中培养来自CD11c.DC-SIGN+和SIGNR1-/-小鼠的骨髓衍生的巨噬细胞5-7天。如图4所示，DC-SIGN+BMMΦ用所示Fc制剂的0.5mg/mL非唾液酸化的(黑色条)或唾液酸化(白色条)糖型脉冲处理。Fc处理的BMMΦ转WTC57BL/6受体小鼠，然后K/BxN挑战。如图4的b所示，SIGNR1-/-(黑色条)和DC-SIGN+(白色条)BMMΦ用0.5mg/mL所示Fc制剂脉冲处理并转至WT C57BL/6受体小鼠，然后K/BxN挑战。相似地，DC-SIGN+BMMΦ用0.5mg/mL的FA241或脱糖基化的FA241(图4的c，白色条)或PBS(图4的c，黑色条)脉冲处理并转至WT C57BL/6受体小鼠，然后K/BxN挑战。FA241用PNGase F脱糖基化并且通过凝集素印迹证实聚糖的去除。DC-SIGN+BMMΦ用所示非唾液酸化Fc制剂或PBS(图4的d，黑色圆)脉冲处理并转至WT C57BL/6受体小鼠，然后K/BxN挑战。在所有情况下，监测并评分足垫肿胀好几天。绘制每组4-5只小鼠的临床评分的均值和SEM。*P<0.05，如通过方差(ANOVA)检验和随后的Tukey post hoc检验的分析所确定的。
如图4的a所示，与sFc相比，非唾液酸化的或唾液酸化的FA241制剂对抑制关节炎同样有效。但是，WT Fc制剂要求α2,6-连接的唾液酸，因为用非唾液酸化的WT Fc脉冲处理的DC-SIGN+BMMΦ没有将保护传递给受体小鼠。与图3所示结果相对应的，sFc和FA241均要求在BMMΦ上的DC-SIGN表达以传递保护(图4的c)。虽然FA241不要求唾液酸来传递保护，用PNGase F脱糖基化终止了FA241的抗炎特性(图4的c)，这表明Fc聚糖仍然是必需的。此外，为了显示所观察到的抗炎活性是特异性针对F241A突变的，DC-SIGN+BMMΦ用具有没有赋予增强的DC-SIGN结合的替代突变的Fc脉冲处理。只有FA241刺激的BMMΦ保护K/BxN挑战后的受体小鼠。
实施例6FA241突变增强Fcγ受体结合
如果241位的丙氨酸替换诱导Fc中的构象变化，那么可能会改变对人Fcγ受体的亲和性。此前报道，唾液酸化降低IgG对FcγR的亲和力，从而减弱体内ADCC活性。
通过表面等离子体共振(SPR)测量重组IgG1 Fc与可溶性FcγR的结合。以上述方式制备Fc。图5显示了针对WT hIgG1 Fc的非唾液酸化和唾液酸化糖型和对非唾液酸化的FA241 Fc的抗体结合固定的hFcγRIIA131R和hFcγRIIB的SPR传感图。
如图5所示，观察到的WT Fc的非唾液酸化糖型结合hFcγRIIA和RIIB的KD值为～2-3x10-5 M。但是，sFc没有显示出结合hFcγRIIA或RIIB。令人惊讶的是，FA241显示出以更强的亲和性数量级结合hFcγRIIA和RIIB二者(KD＝～2x10-6 M)。
实施例7FA241诱导骨髓来源的巨噬细胞中的IL-33mRNA
sFc诱导TH2依赖的抗炎通路，其要求从FcεRI+白细胞群分泌IL-4，可能是嗜碱性粒细胞，以上调调节性巨噬细胞上的FcγRIIB。体内或体外施用IL-33刺激嗜碱性粒细胞释放存储的IL-4。在用sFc或IVIG处理的WT C57BL/6小鼠的脾脏中IL-33mRNA表达上调，但在SIGNR1-/-小鼠中没有。这表明sFc可能诱导SIGNR1+或DC-SIGN+细胞中的IL-33表达。在该实施例中，进行试验并显示用FA241刺激DC-SIGN+BMMΦ显示上调IL-33表达。
更具体地，以上述方式培养来自CD11c.DC-SIGN+和SIGNR1-/-小鼠的骨髓衍生的巨噬细胞。BMMΦ在无血清RPMI培养基中接种至12孔板并37℃过夜吸附。第二天，细胞用在无血清RPMI培养基中的0.5mg/mL的所示Fc于37℃脉冲处理1h(图6的a)或4h(图6的b)。于指定的时间点从细胞中收获mRNA并且1μg总RNA用于IL-33mRNA的RT-PCR扩增(上图)。GAPDH扩增作为上样对照(下图)。共表达DA265 Fc和人唾液酸转移酶(ST6Gall)的质粒转染293T细胞，获得高度唾液酸化的重组Fc(ST6-DA265)。
如图6所示，用FA241刺激DC-SIGN+BMMΦ显示上调IL-33表达。尽管与DC-SIGN+BMMΦ相比具有更高基础表达水平的IL-33，SIGNR1-/-BMMΦ对FA241处理的反应是下调IL-33mRNA表达。
上述实施例和优选实施方案的描述应被视为说明性的，而不应视为限制权利要求所限定的本发明。本发明引用的所有出版物的全文在此通过引用并入本发明。容易理解的是，可以在不脱离权利要求所限定的本发明的情况下利用上述特征的许多变化和组合。这样的变化不被视为脱离本发明的范围，并且所有这样的变化也包括在以下权利要求的范围之内。
Claims (12)
1.一种分离的多肽，其由与SEQ ID NO：2至少95％同一的氨基酸序列组成，其中多肽的对应于SEQ ID NO：2的氨基酸残基32的氨基酸残基241(根据Kabat的EU索引编号)是丙氨酸(A)。
2.如权利要求1所述的分离的多肽，其中氨基酸序列与SEQ ID NO：2至少99％同一。
3.如权利要求1所述的分离的多肽，其中多肽具有SEQ ID NO：2所示的氨基酸序列。
4.一种药物制剂，其包含(i)权利要求1-3任一项所述的分离的多肽，和(ii)药学上可接受的载体。
5.一种权利要求1-3任一项所述的分离的多肽在制备用于治疗炎性疾病的药物中的用途。
6.如权利要求5所述的用途，其中炎性疾病是自身免疫性疾病。
7.如权利要求5所述的用途，其中自身免疫性疾病是炎性肠病，炎性皮肤病，超敏性肺疾病，或急性或慢性炎性疾病。
8.如权利要求5所述的用途，其中炎性疾病是牛皮癣、类风湿关节炎、银屑病关节炎、多发性硬化症、狼疮、I型糖尿病、原发性胆汁性肝硬化、哮喘、急性呼吸窘迫综合症、爆发性肝炎、重症肌无力、青少年发病型糖尿病、肾小球肾炎、自身免疫性甲状腺炎、强直性脊柱炎、系统性硬化症或干燥综合征。
9.如权利要求7所述的用途，其中炎性肠病是克罗恩病或溃疡性结肠炎。
10.如权利要求7所述的用途，其中炎性皮肤病是皮炎、湿疹、特应性皮炎、过敏性接触皮炎、荨麻疹、坏死性血管炎、皮肤血管炎、超敏性血管炎、嗜酸细胞性肌炎、多发性肌炎、皮肤肌炎或嗜酸细胞性筋膜炎。
11.如权利要求7所述的用途，其中超敏性肺疾病是过敏性肺炎、嗜酸细胞性肺炎、迟发型过敏、间质性肺疾病(ILD)、特发性肺纤维化或类风湿关节炎相关的ILD。
12.如权利要求7所述的用途，其中急性和慢性炎性疾病是全身性过敏和超敏反应、移植排斥或移植物抗宿主病。
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