CN116120405A - 一种抑制TSLP与TSLP/IL-7Rα受体结合的多肽及其用途 - Google Patents
一种抑制TSLP与TSLP/IL-7Rα受体结合的多肽及其用途 Download PDFInfo
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Abstract
本发明属于生物医药多肽技术领域,具体涉及一种抑制TSLP与TSLPR/IL‑7Rα受体结合的多肽或其药学上可接受的盐,进一步涉及所述多肽或其药学上可接受的盐在与TSLP相关的病症的药物中的应用。本发明所涉及的多肽具有较好的抑制TSLP与TSLPR/IL‑7Rα受体结合的IC50以及与TSLPR/IL‑7Rα的较好亲和力。
Description
技术领域
本发明属于生物医药多肽技术领域,具体涉及一种抑制TSLP与TSLP/IL-7Rα受体结合的多肽及其用途。
背景技术
胸腺基质淋巴细胞生成素(TSLP)是一类主要由上皮细胞分泌的、与IL-7类似的Ⅰ型细胞因子,其结构由4个螺旋束组成的短链,含有7个半胱氨酸残基和3个潜在的N-糖基化位点。TSLP的作用是支持B淋巴细胞的生长、分化及T细胞增殖,并可以在缺乏IL-7的条件下促进淋巴细胞的发育,并对树突状细胞的活化、成熟和迁移起着重要的调控作用。TSLP主要由上皮细胞表达,特别表达在经抗原激活的支气管上皮细胞、皮肤角化细胞、基质细胞、肥大细胞、平滑肌细胞和肺成纤维细胞。人类TSLP基因定位在染色体的5q22-14位置,与Th2细胞因子的基因簇置5q23-32非常接近。TSLPR也称为细胞因子受体样分子2(CRLM-2)或Ⅰ型细胞因子受体σ1,由TSLPR链和IL-7R-α链组成的二聚体。目前已鉴定了人、小鼠和大鼠TSLPR为Ⅰ型跨膜蛋白质,属于造血细胞因子受体家族成员,具有该族受体分子的典型结构特征。TSLP主要由活化的肺和肠上皮细胞、角质形成细胞和成纤维细胞表达,树突状细胞(DC)、肥大细胞等免疫细胞也可生成。人TSLP是由三对链内二硫键连接形成的四螺旋束细胞因子,编码基因位于5q22.1染色体。人TSLP主要存在短型(sfTSLP)和长型(lfTSLP)两种同源异构体。sfTSLP与lfTSLP的末端区域有部分重叠。sfTSLP主要由60个氨基酸组成,常于健康状态表达并发挥稳态作用。lfTSLP编码159个氨基酸,分子量约14.9kD,多于炎症时表达上调并发挥促炎作用。健康者肠道和皮肤组织sfTSLP表达较lfTSLP显著增高。一旦经Toll样受体3(TLR3)、TLR2和TLR6配体及多种细胞因子[如肿瘤坏死因子α(TNF-α)、IL-4和IL-13]刺激,lfTSLP水平显著上调,而sfTSLP水平相仿。其实,未经刺激的上皮细胞lfTSLP组成性表达显著低于sfTSLP,而过敏原(变应原)刺激后lfTSLP水平显著升高。sfTSLP尚可抑制多种细胞因子(包括TNF-α、IL-1β、IL-6)的产生。sfTSLP可抑制TSLP信号传导并降低气道炎症和气道高反应性,lfTSLP则通过增加IFN-γ释放并激活TSLPR而介导炎症反应。
TSLPR链类似于IL-2Rγ,分布于细胞内外,细胞外TSLPR链包括细胞因子识别同源结构域,细胞内包含一个BOX结构和一个单一的酪氨酸残基,信号转导后,细胞内外的TSLPR链均可与TSLP结合。对TSLPR的分子结构及功能研究表明,TSLPR本身与TSLP亲和力非常低,只有TSLPR与IL-7R-α共同作用时才可形成高亲和力的受体复合物。当人TSLP分子与TSLPR和IL-7R-α组成的异二聚体结合后,可激活信号转导子以及转录活化子5和3(STAT5,3),并启动下游基因的表达。TSLP与其特异性受体TSLPR及IL-7Rα结合,形成三元复合物,启动信号传导。比如,通过Janus激酶1(JAK1)、JAK2磷酸化,激活信号转导和转录因子(STAT)1、STAT3、STAT5,启动促炎信号,促进DC成熟和活化,诱导功能性Ⅱ型辅助性T细胞(Th2)、调节性T细胞(Treg)和滤泡辅助T细胞(Tfh)表达,调节皮肤、肺和肠道黏膜屏障的炎症过程。
在一些与TSLP相关的疾病中对TSLP亚型的表达模式也进行了研究。例如,在哮喘、溃疡性结肠炎、特应性皮炎和牛皮癣中观察到lfTSLP的过表达,而在麦胶性肠病中发现lfTSLP的表达降低。在克罗恩病、麦胶性肠病和特应性皮炎中sfTSLP的表达下调(KaterinaTsilingiri et al.,(2017)Cellular andMolecular Gastroenterology and Hepatology3(2):174-182;Zeuthen LH et al.,;Fornasa G et al.,(2015)J Allergy Clin Immunol136:413–422)。TSLP由于其与上述疾病具有相关性而成为临床靶标。有人建议恢复短型TSLP的表达以治疗难治性麦胶性肠病(Biancheri P et al.,(2015)Gut 65:1670–1680)。已上市的人单克隆抗体Tezepelumab被设计用于治疗哮喘和特应性皮炎,并已在临床试验中证明,其与安慰剂相比,可降低年哮喘急性发作率(JonathanCorren et al.,(2017)TheNew EnglandJournal of Medicine 377(10):936-946)。
人们越来越期望并追求更多具有更好疗效更高安全性的药物,包括对lfTSLP具有特异性并与sfTSLP相互作用最小化的药物,从而可能使临床结果得到更多改善。而多肽类药物很好的综合了小分子化药和蛋白质药物的优点,具有稳定性好、特异性强、杂质低、疗效好、毒副作用小等优势,能够广泛作用于内分泌系统、免疫系统、消化系统、心血管系统、血液系统、肌肉骨骼系统等。因而有必要开发有效的针对TSLP靶点相关的多肽药物。
发明内容
以下仅概况说明本发明的一些方面,并不局限于此。这些方面和其他部分在后面有更完整的说明。本说明书中的所有参考文献通过整体引用与此。当本说明书的公开内容与引用文献有差异时,以本说明书的公开内容为准。
本发明的第一方面,提供一种多肽或其药学上可接受的盐,其特征在于,所述多肽的氨基酸序列为任选SEQ ID NO:1~SEQ ID NO:10氨基酸序列之一或与SEQ ID NO:1~SEQID NO:10具有至少90%序列同一性的氨基酸序列或经修饰的SEQ ID NO:1~SEQ ID NO:10所示的氨基酸序列的衍生物。
在一些实施方式中,所述经修饰的氨基酸序列的衍生物包括选自以下的一种或更多种修饰:N端和/或C端修饰;以一个或更多个天然和/或非天然氨基酸残基取代一个、二个或更多个氨基酸残基。
在一些实施方式中,所述多肽或其药学上可接受的盐,包含与所述多肽SEQ IDNO:1~SEQ ID NO:10任一的氨基酸序列具有至少60%、65%、70%、75%、80%、85%、90%、93%、94%或95%同一性的氨基酸序列。
在一些实施方式中,所述多肽或其药学上可接受的盐可通过插入蛋白标签进行分离纯化,进一步的,所述蛋白标签选自His、Flag、GST、Myc、eGFP/eCFP/eYFP/mCherryeGFP、HA、SUMO;优选的,蛋白标签为His。
在一些实施方式中,所述插入多肽序列的蛋白标签位于SEQ ID NO:1~SEQ IDNO:10序列的C端、N端或序列中苯丙氨酸的N端。
第二方面,本发明还涉及一种药物组合物,其包含本发明任一所述的多肽或其药学上可接受的盐。
在一些实施方案中,本发明所述的药物组合物进一步包含药学上可接受的载体、赋形剂、稀释剂、辅剂和媒介物的至少一种。
另一方面,本发明涉及所述的多肽或其药学上可接受的盐或所述的药物组合物在制备用于预防、处理、治疗或减轻TSLP相关病症的药物中的用途;所述TSLP相关病症选自TSLP相关炎性病症或自身免疫性疾病。
在一些实施方式中,所述TSLP相关炎性病症选自过敏性炎症、哮喘、特发性肺纤维化、慢性阻塞性肺病、特应性皮炎或嗜酸性粒细胞食道炎;进一步的所述过敏性炎症选自过敏性鼻炎、过敏性鼻窦炎或过敏性结膜炎;所述哮喘为嗜酸细胞性哮喘或非嗜酸细胞性哮喘,进一步的所述哮喘为低嗜酸细胞性哮喘。
最后一方面,本发明还涉及一种测试上述多肽活性的方法,包括如下步骤:
a)应用TR-FRET技术建立筛选体系,以Streptavidin-Eu作为荧光供体,GoatAnti-Human IgG Fc-Alexa Fluor647作为荧光受体;选取生物素标记TSLP和偶联人IgG Fctag的IL-7RA&TSLPR二聚体;
b)当TSLP与IL-7RA&TSLP R结合后,加入荧光供体和受体,荧光供体Streptavidin-Eu与TSLP上的生物素结合,荧光受体Goat Anti-Human IgG Fc-AlexaFluor647与IL-7RA&TSLP R上的Fc tag结合,使得荧光供体Eu与荧光受体Alexa Fluor647靠近,以发生FRET;
c)加入TSLP Monoclonal Antibody抗体后,阻断TSLP与IL-7RA&TSLP R结合,使FRET信号减弱;
d)将多肽库96孔深孔板放于离心机离心,用自动分液仪向96孔深孔板中加入超纯水中,密封,放置≥90℃水浴中溶解,溶解后的96深孔板多肽放于离心机再次离心;
e)将再次离心后的上清液用工作站转移至384孔板中,用loadingbuffer稀释;
f)应用TR-FRET技术建立筛选方法对大型实体多肽库进行高通量筛选选取抑制率大于50%的多肽。
附图说明
图1:人源TSLP胞外区蛋白SDS-PAGE结果
图2:人源TSLP蛋白Western Blot结果
图3:人源TSLPR/IL-7Rα胞外区SDS-PAGE结果
图4:人源TSLPR/IL-7Rα胞外区蛋白Western Blot结果
图5:多肽阻断TSLP与IL-7RA&TSLP R结合活性的结果(TR-FRET测试)
图6:多肽阻断TSLP与IL-7RA&TSLP R结合活性的结果(ELISA测试)
图7:序列1所示多肽与IL-7RA&TSLPR的亲和力测试
图8:序列2所示多肽与IL-7RA&TSLPR的亲和力测试
图9:序列3所示多肽与IL-7RA&TSLPR的亲和力测试
图10:序列5所示多肽与IL-7RA&TSLPR的亲和力测试
具体实施方式
术语“胸腺基质淋巴细胞生成素(Thymic Stromal Lymphopoietin,TSLP)”是四α-螺旋束Ⅰ型细胞因子,也是响应促炎症刺激而产生的上皮细胞衍生的细胞因子,与白细胞介素-7(IL-7)密切相关,其通过刺激树突细胞(DC)起始变态反应,是调节人体免疫反应的重要因子。术语“TSLP”包括TSLP的变体、同种型、同系物、直系同源物和旁系同源物。
术语“肽”或“多肽”的含义是被本专业领域的技术人员所熟知的。通常情况下,肽或多肽是两个或多个氨基酸由酰胺键链接,酰胺键则由一个氨基酸的氨基与相邻氨基酸的羧基构成。本文所述的多肽可包含天然存在的氨基酸或者非天然存在的氨基酸。可被修饰成其类似物,衍生物,功能模拟物,伪肽等诸如此类包含至少两个氨基酸的化合物。除非指明N-端或C-末端具有特定的修饰,否则一个包含特定氨基酸序列的多肽,则包括不加修饰的和修饰的氨基和/或羧基末端,这是被本领域的专业技术人员所熟知的。一个特定的氨基酸序列的多肽可以包括修饰的氨基酸和/或额外的氨基酸,除非N-和/或C-末端包含妨碍进一步添加氨基酸的修饰。这样的修改包括,例如,N-末端的乙酰化和/或C-末端的酰胺化。
本发明的多肽可以通过改造修饰,形成多肽衍生物。正如本领域技术人员所熟知的,可以对多肽进行各种改造修饰。典型的改造修饰包含但不限于,N-末端乙酰化、C-末端酰胺化、d型氨基酸替换、非天然氨基酸替换、脂肪酸修饰或以上各种修饰改造的组合。本发明包括任何被众所周知的多肽的修饰改造。例如,多肽衍生物可以包括对于多肽的化学修饰,如烷基化、酰基化、氨基甲酰化、碘化或其他任何产生多肽衍生物的改造修饰。多肽的改造修饰可以包含改造过的氨基酸,例如,羟基脯氨酸或羧基谷氨酸,并且可以包括以非肽键相连的氨基酸。
对于本发明的多肽的其它修饰改造可采用非天然氨基酸对多肽中的天然氨基酸进行取代,非天然氨基酸包含但不限于,2-氨基脂肪酸(Aad)、3-氨基脂肪酸(βAad)、β-丙氨酸,β-氨基丙酸(βAla)、2-氨基丁酸(Abu)、4-氨基丁酸、哌啶羧酸(4Abu)、6-氨基己酸(Acp)、2-氨基庚酸(Ahe)、2-氨基异丁酸(Aib)、3-氨基异丁酸(βAib)、2-氨基庚二酸(Apm)、2,4-二氨基丁酸(Dbu)、锁链素(Des),2,2'-二氨基庚二酸(Dpm),2,3-二氨基丙酸(Dpr),N乙基甘氨酸(EtGly)、N-乙基天冬酰胺(EtAsn),羟赖氨酸(Hyl)、异羟赖氨酸(aHyl)、3-羟脯氨酸(3Hyp)、4-羟基脯氨酸(4Hyp)、异锁链素(Ide)、异-异亮氨酸(aIle)、N-甲基甘氨酸(MeGly)、N-甲基异亮氨酸(MeIle)、6-N-甲基赖氨酸(MeLys)、N-甲基缬氨酸(MeVal)、正缬氨酸(Nva)、正亮氨酸(Nle)和鸟氨酸(Orn)。当然,所有被修饰改造的α-氨基酸可以被相应的β-,γ-或ω-氨基羧酸所取代。
术语“氨基酸”是指含有氨基和羧基的分子。合适的氨基酸包括但不限于天然存在的氨基酸的D-和L-异构体,以及通过有机合成或其它代谢途径制备的非天然存在的氨基酸。如本文所用,术语氨基酸包括但不限于α-氨基酸、天然氨基酸、非天然氨基酸和氨基酸类似物。
术语“天然存在的氨基酸”是指在自然界中合成的肽中常见的20种L-氨基酸中的任何一种,即丙氨酸(Ala或A)、精氨酸(Arg或R)、天冬酰胺(Asn或N)、天冬氨酸(Asp或D)、半胱氨酸(Cys或C)、谷氨酸(Glu或E)、谷氨酰胺(Glu或Q)、甘氨酸(Gly或G)、组氨酸(His或H)、异亮氨酸(Ile或I)、亮氨酸(Leu或L)、赖氨酸(Lys或K)、甲硫氨酸(Met或M)、苯丙氨酸(Phe或F)、脯氨酸(Pro或P)、丝氨酸(Ser或S)、苏氨酸(Thr或T)、色氨酸(Trp或W)、酪氨酸(Tyr或Y)和缬氨酸(Val或V)的L-异构体。
“保守氨基酸取代”是其中氨基酸残基被具有相似侧链的氨基酸残基所取代的氨基酸取代。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,K、R、H)、酸性侧链(例如,D、E)、不带电荷的极性侧链(例如,G、N、Q、S、T、Y、C)、非极性侧链(例如,A、V、L、I、P、F、M、W)、β-分支侧链(例如,T、V、I)和芳族侧链(例如,Y、F、W、H)的氨基酸。因此,例如,多肽中预测非必需氨基酸残基优选被来自相同侧链家族的另一个氨基酸残基取代。可接受的取代的其它实例是基于电子等排考虑的取代(例如,正亮氨酸取代甲硫氨酸)或其它性质(例如,2-噻吩基丙氨酸取代苯丙氨酸)。
本发明的多肽可以使用本领域技术人员所熟知的方法制备,包括众所周知的化学合成的方法。因此,当多肽或其衍生物包含一个或多个非标准氨基酸,则极有可能是通过化学合成法制备而来。除了使用化学合成的方法制备多肽或其衍生物,还可以通过编码核酸表达来制备。这对于制备只含有天然氨基酸的多肽或其衍生物特别适用,在这种情况下可以使用众所周知的核酸编码多肽序列的制备方法(参见Sambrook etal.,Molecula rCloning:ALa bora tory Manua l,Third Ed.,Cold Spring Ha rbor Laboratory,NewYork(2001);Ausubel et al.,Current Protocols in Molecular Biology,JohnWiley and Sons,Baltimore,MD(1999))。多肽可以在生物体中表达,并通过公知的纯化技术进行纯化。
术语“类似物”是指与参考物质共有一个或多个特定结构特征、要素、组分或部分的物质。通常,“类似物”显示出与参考物质的显著的结构相似性,例如共有核心或共有结构,而且在某些离散方式上也有所不同。在一些实施方案中,类似物是可以例如通过参考物质的化学操纵而从参考物质产生的物质。在一些实施方案中,类似物是可以通过与产生参考物质的合成过程基本上类似(例如,与它们共有多个步骤)的合成过程的进行来产生的物质。在一些实施方案中,类似物通过或可以通过与用于产生参考物质的合成过程不同的合成过程的进行来产生。
关于序列同一性。根据本领域已知的方法,序列同一性是通过序列比对计算的。为了确定两个氨基酸序列的同一性百分比,比对序列以进行最佳比较。例如,可以在第一氨基酸序列的序列中引入空位以便与第二氨基酸序列最佳比对。然后比较相应氨基酸位置的氨基酸残基。当第一序列中的位置被第二序列中相应位置的相同氨基酸残基占据时,分子在该位置是相同的。两个序列之间的百分比同一性是序列共有的相同位置数的函数。因此%同一性=相同位置数/重叠位置的总数乘以100。在该比较中,序列可以是相同的长度或可以是不同的长度。用于确定比较窗口的最佳序列比对可以通过Smith和Waterman的局部同源性算法(J.Theor.Biol.,1981),通过Needleman和Wunsch的同源性比对算法(J.Mol.Biol,1972),通过Pearson和Lipman的方法寻找相似性(Proc.Natl.Acad.Sci.U.S.A.,1988)来进行,通过这些算法的计算机化实施(威斯康星遗传学软件包7.0版中的GAP、BESTFIT、FASTA和TFASTA,Genetic Computer Group,575,Science Drive,Madison,Wisconsin)或者例如使用公共可用的计算机软件例如BLAST。当使用这种软件时,优选使用默认参数,例如空位罚分或延伸罚分。选择由各种方法产生的最佳比对(即整个比较窗口范围内产生最高的同一性百分比)。
术语“同源性”是指两个多核苷酸序列之间或两个多肽之间的序列相似性。当两个比较序列中的位置均被相同碱基或氨基酸单体亚基占据时,例如如果两个DNA分子的每一个位置都被腺嘌呤占据时,那么所述分子在该位置是同源的。两个序列之间的同源性百分率是两个序列共有的匹配或同源位置数除以比较的位置数×100的函数。例如,在序列最佳比对时,如果两个序列中的10个位置有6个匹配或同源,那么两个序列为60%同源;如果两个序列中的100个位置有95个匹配或同源,那么两个序列为95%同源。通常,当比对两个序列时进行比较以给出最大百分比同源性。例如,可以通过BLAST算法执行比较,其中选择算法的参数以在各个参考序列的整个长度上给出各个序列之间的最大匹配。以下参考文献涉及经常用于序列分析的BLAST算法:BLAST算法(BLAST ALGORITHMS):Altschul,S.F.等人,(1990)J.Mol.Biol.215:403-410;Gish,W.等人,(1993)NatureGenet.3:266-272:Madden,TL.等人,(1996)Meth.Fnzymol.266:131-141;Altschul,S.F.等人,(1997)Nucleic AcidsRes.25:3389-3402;Zhang,J.等人,(1997)Genome Res.7:649-656。其他如NCBI BLAST提供的常规BLAST算法也为本领域技术人员所熟知。
术语“药物组合物”,本发明涉及包括治疗有效量的本发明的多肽以及药学可接受载体或赋形剂的药物组合物。如本发明所用,“药学可接受载体”或“药学可接受赋形剂”包括任何和所有的溶剂、分散介质、涂层、抗菌和抗真菌剂、等渗和吸收延迟剂和生理学可相容的类似物。药学可接受载体或赋形剂的例子包括以下的一种或更多种:水、盐水、磷酸缓冲盐水、葡萄糖、甘油、乙醇和类似物以及其组合。在任何情况下,优选地在组合物中其包括等渗剂,例如,糖,多元醇,例如甘露醇、山梨糖醇,或氯化钠。也可以包括药学可接受的物质,例如润湿量或微量的辅助物质,例如提高抗体或抗体部分的存放时间和有效性的润湿或乳化剂、防腐剂或缓冲剂。任选地,可以包括崩解剂,例如交联聚乙烯基吡咯烷酮、琼脂、海藻酸或其盐,例如海藻酸钠。除赋形剂之外,药物组合物还可以包括以下的一种或更多种:载体蛋白质例如血清白蛋白、缓冲剂、结合剂、甜味剂和其他调味剂;着色剂和聚乙二醇。
组合物可以是许多种形式,例如,液体、半固体和固体剂型,例如液体溶液(如可注射溶液和可输注溶液)、分散液或悬浮液、片剂、丸药、粉末、脂质体和栓剂。优选的形式将取决于既定的给药途径和治疗应用。在一个实施方式中,组合物是可注射或可输注液体的形式,例如类似于用抗体对人进行被动免疫使用的那些形式。在一个实施方式中,给药方式是肠胃外(例如静脉内、皮下、腹膜内、肌肉内)、在一个实施方式中,通过静脉内注射或输注给予多肽。在另一实施方式中,通过肌肉内或皮下注射给予多肽。
其他适合用于该药物组合物的给药途径包括,但不限于,直肠、透皮、阴道、透粘膜或肠内给药。
术语“药学上可接受的载体”指适合用于制剂中用于递送抗体或抗原结合片段的任何无活性物质。载体可以是抗粘附剂、粘合剂、包衣、崩解剂、充填剂或稀释剂、防腐剂(如抗氧化剂、抗菌剂或抗真菌剂)、增甜剂、吸收延迟剂、润湿剂、乳化剂、缓冲剂等。合适的药学上可接受的载体的示例包括水、乙醇、多元醇(例如甘油、丙二醇、聚乙二醇等)右旋糖、植物油(例如橄榄油)、盐水、缓冲液、缓冲的盐水和等渗剂例如糖、多元醇、山梨糖醇和氯化钠。
用于合成多肽(如本文描述的)的方法在本领域中是已知的。在一些肽合成方法中,一个氨基酸(或氨基酸衍生物)的氨基连接于另一个氨基酸(或氨基酸衍生物)的羧基,该羧基通过它和试剂如二环己基碳二亚胺(DCC)的反应来活化。当自由氨基和活化羧基起化学反应时,会形成肽键并释放二环己基脲。在这样的方法中,可以封闭(“保护”)其它潜在活性基团(如N端氨基酸或氨基酸衍生物的α–氨基和C端氨基酸或氨基酸衍生物的羧基),以使其避免参与化学反应。因而,仅特定的活性基团进行反应,以致形成所期望的产物。可用于此目的保护基团包括但不限于用来保护胺基的叔丁氧炭基(t-Boc)和苯甲酰氧基)基;以及用来保护羧基的简单酯(如甲基和乙基)和酯。通常可以借助于留下完好的肽键的处理(例如,用稀酸进行的处理)来随后除去保护基团可以重复保护反应基团(其不应反应)、耦合以形成肽键、和对反应基团进行去保护的过程。可以通过将氨基酸依次加入一个生长的肽链来合成肽。按照本发明,液相和固相肽合成方法均是适用的。在固相肽合成方法中,通常通过将C端氨基酸连接于基质来将生长肽链连接于不溶性基质(如,例如,聚苯乙烯珠)。在合成结束时,可以利用并不破坏肽键的剪切试剂,如氢氟酸(HF)来从基质释放肽。此时,还通常除去保护基团。按照本发明,还可以使用自动化的、高通量、和/或平行肽合成方法。关于肽合成方法的更多信息,参见,例如,Merrifield(1969)“Solid-phase peptidesynthesis,”Adv Enzymol RelatAreas Mol Biol.,32:221-96;Fridkin et al.(1974)Annu Rev Biochem.,43(0):419-43;Merrifield(1997)“Concept and Early Developmentof Solid Phase PeptideSynthesis,”Methods in Enzymology,289:3-13;Sabatino etal.(2009)“Advances inautomatic,manual and microwave-assisted solid-phasepeptide synthesis,"Curr0pin Drug Discov Devel.,11(6):762-70,上述各自的全部内容以引用方式结合于本文。
另外,本发明公开的多肽、包括它们的盐,也可以以它们的水合物形式或包含其溶剂(例如乙醇,DMSO,等等)的形式存在,并可用于结晶。本发明公开化合物可以与药学上可接受的溶剂(包括水)固有地或通过设计形成溶剂化物;因此,本发明化合物包括溶剂化的和未溶剂化的形式。
此外,本发明公开的TSLP相关的疾病没有限制,只要它是与TSLP相关的疾病即可,例如利用本公开的分子诱导的治疗反应可通过结合人类TSLP,然后阻遏TSLP与其受体结合,或杀伤过表达TSLP的细胞。
在以上说明书中提出了本公开一种或多种实施方式的细节。虽然可使用与本文所述类似或相同的任何方法和材料来实施或测试本发明,但是以下描述优选的方法和材料。通过说明书和权利要求书,本公开的其他特点、目的和优点将是显而易见的。在说明书和权利要求书中,除非上下文中有清楚的另外指明,单数形式包括复数指代物的情况。除非另有定义,本文使用的所有技术和科学术语都具有本发明所属领域普通技术人员所理解的一般含义。说明书中引用的所有专利和出版物都通过引用纳入。提出以下实施例是为了更全面地说明本发明的优选实施方式。这些实施例不应以任何方式理解为限制本发明的范围。
实施例
本公开提供的多肽化合物及其衍生物采用固相合成的方法合成。合成载体为Fmoc-Cys(Trt)-2-Chlotrityl Resin树脂。合成过程中,首先将Fmoc-Cys(Trt)-2-Chlotrityl Resin树脂在N,N-二甲基甲酰胺(DMF)中充分溶胀,然后该固相载体与活化后氨基酸衍生物重复缩合→洗涤→去保护Fmoc→洗涤→下一轮氨基酸缩合的操作以达到所要合成的多肽链长度,最后用三氟乙酸:水:三异丙基硅烷:苯甲硫醚(90:2.5:2.5:5:,v:v:v:v)的混合溶液与树脂反应将多肽从固相载体上裂解下来,再由冷冻甲基叔丁基醚沉降后得到直链前体的固体粗品。多肽粗品在0.1%三氟乙酸的乙腈/水的体系由C-18反相制备色谱柱纯化分离后得到多肽及其衍生物的纯品。所得氨基酸序列如表1所示。
表1实施例中所用的氨基酸序列
实施例1.生物表达及纯化人源TSLP蛋白和TSLPR/IL-7Rα胞外区蛋白
1)主要实验材料
序号 | 物料名称 | 来源 |
1 | Expi293F细胞及其细胞培养基 | 赛默飞世尔科技有限公司 |
2 | PEI | 翌圣生物科技有限公司 |
3 | 生物素 | 阿拉丁试剂有限公司 |
4 | Anti-DYKDDDDK G1 Affinity Resin | 金斯瑞生物科技有限公司 |
5 | 3X Flag多肽 | 碧云天生物技术有限公司 |
6 | BCA法蛋白浓度检测试剂盒 | 碧云天生物技术有限公司 |
7 | Streptavidin-HRP | 美国英杰生命技术公司 |
2)人源TSLP和TSLPR/IL-7Rα胞外区重组载体构建
选择表达TSLP和TSLPR/IL-7Rα的胞外区片段,且在胞外区末端加入Avi及FLAG融合表达标签,分别将目的基因片段插入pcDNA3.4质粒中,构建真核表达载体pcDNA3.4-hTSLP及pcDNA3.4-hTSLPR/IL-7Rα。
3)真核细胞转染及蛋白表达
(a)培养Expi293F细胞,在转染前一天取对数生长期的细胞进行传代;
(b)转染当天,将细胞稀释至1x106 cells/ml进行转染。按照DNA:转染试剂=1:3(ω/ω)的比例进行混合,静置15min后,将DNA/转染试剂混合物逐滴加入细胞培养液中;
(c)144h后,收集转染后的细胞培养液进行纯化。
4)蛋白生物素化及纯化
(a)细胞培养液高速离心,获得细胞培养上清。在上清中加入Tris缓冲液(50mMTris-HCl,150mM NaCl,pH 7.4),再加入等摩尔比的生物素,将蛋白质进行体外生物素化,10℃,120rpm,摇床孵育过夜。
(b)使用3倍柱体积的平衡缓冲液(50mM Tris-HCl,150mM NaCl,pH 7.4)平衡Anti-DYKDDDDK G1Affinity Resin,重复3次。将其加入生物素化后的细胞培养上清中,10℃,120rpm,摇床孵育3h。
(c)参考柱层析法,将孵育混合物进行上柱,使用10~20倍柱体积的平衡buffer进行洗涤。最后加入含3xFLAG多肽的竞争洗脱缓冲液(150μg/ml 3xFLAG多肽,50mM Tris-HCl,150mM NaCl,pH 7.4)进行洗脱并收集。
5)目标蛋白鉴定和分析
(a)SDS-PAGE和考马斯亮蓝染色检测蛋白纯度
取洗脱样品加入上样buffer,105℃加热5min后上样至SDS-PAGE凝胶中进行电泳。用考马斯亮兰染色10min,再用脱色液脱色至蛋白条带清晰即可拍照。
(b)Western Blot检测纯化的重组蛋白
取SDSPAGE电泳后的凝胶,电转至NC膜上,用25mg/ml的BSA低温封闭过夜,洗涤后,再用1:2500稀释的Streptavidin-HRP于37℃孵育1.5h后显色并拍照记录结果。
6)结果
(a)人源TSLP胞外区蛋白
SDS-PAGE结果如图1所示,纯化获得的人源TSLP胞外区蛋白纯度>80%,经BCA法检测得蛋白浓度为1.82mg/ml;
Western Blot如图2所示,人源TSLP胞外区蛋白纯化成功。
(b)人源TSLPR/IL-7Rα胞外区蛋白
SDS-PAGE结果如图3所示,纯化获得的人源TSLPR/IL-7Rα胞外区蛋白纯度>90%,经HPLC检测得浓度为0.51mg/ml;
Western Blot如图4所示,人源TSLPR/IL-7Rα胞外区蛋白纯化成功。
生物学评价
实施例2.多肽抑制TSLP及其受体IL-7RA&TSLPR结合的IC50值
1)主要实验材料
2)TR-FRET方法
在384孔板中依次加入不同浓度的待测多肽(最终检测浓度30μM,3倍梯度稀释8-12个浓度)、1nM TSLP和1nM IL-7RA&TSLP R,以及荧光供体Streptavidin-Eu和荧光受体GoatAnti-Human IgG Fc-Alexa Fluor647,室温孵育2小时后,检测TR-FRET信号。阳性对照:不含多肽,只含1nM TSLP和1nM IL-7RA&TSLPR,以及荧光供体Streptavidin-Eu和荧光受体GoatAnti-Human IgG Fc-AlexaFluor647;阴性对照为:不含多肽,只含1nM TSLP与1nMIL-7RA&TSLP R其中一个组分或者2者都不含,以及荧光供体Streptavidin-Eu和荧光受体GoatAnti-Human IgG Fc-Alexa Fluor647。计算抑制率,计算多肽抑制TSLP及其受体IL-7RA&TSLPR相互作用的IC50值,如图5所示。
3)ELISA方法
将IL-7RA&TSLP R用包被液(0.05mol/L碳酸盐缓冲液pH 9.6)稀释至0.25μg/ml,包被25μl/孔加入至384孔板(Thermo或者Greiner),放于4℃过夜包被。第二天,用洗涤液(0.05%Tween-20in TBS,pH7.4)洗涤3-5次。加入封闭液(2%(w/v)BSAin TBS,pH 7.4。)放于37℃进行封闭。洗涤3-5次后,加入梯度待测多肽(最终检测浓度30μM,3倍梯度稀释8-12个浓度)、以及0.03125μg/ml TSLP,放于37℃孵育1小时后,洗涤液洗去未结合部分,再加入SA-HRP在37℃孵育1小时,洗涤后续加入TMB显色,最后用HCL终止反应,检测OD450。计算抑制率,计算多肽抑制TSLP及其受体IL-7RA&TSLPR相互作用的IC50值,如图6所示。
4)结果
通过ELISA和TR-FRET方法进行浓度梯度测试多肽对TSLP及其受体IL-7RA&TSLPR结合的抑制作用,本发明发现多肽可以较好的抑制TSLP及其受体IL-7RA&TSLPR相互作用,IC50值小于10μM。表明如表2所示。
表2:多肽抑制TSLP及其受体IL-7RA&TSLPR结合的IC50值
多肽编号 | ELISA竞争抑制测试(μM) | TR-FRET竞争抑制测试(μM) |
1 | 2.32 | 2.6 |
2 | 2.89 | 0.19 |
3 | 0.98 | 2.41 |
4 | 4.05 | 9.56 |
5 | 6.64 | 17.63 |
6 | 1.48 | 1.2 |
7 | 8.02 | 0.6 |
8 | 6.4 | 5.03 |
9 | 2.92 | 0.78 |
10 | 1.19 | 0.86 |
实施例3.BLI测试多肽与IL-7RA&TSLPR结合的亲和力测试
1)主要实验材料
BLI是生物膜干涉技术(Bio-Layer Interferometry,简称BLI)是一种无标记的、实时监测的光学检测技术,主要用于生物分子间相互作用的全方位定量分析以及蛋白浓度测定。BLI可实时监控整个分子间的结合过程,并计算出分子之间的亲和力(KD)、结合速率(ka)、解离速率(kd)等重要数据。
2)不含蛋白标签(tag)的多肽
对与不含tag的多肽,用K buffer(PBS pH7.4+0.02%吐温)测IL-7RA&TSLPR稀释为5或者10μg/ml,用K buffer(PBS pH7.4+0.02%吐温)将多肽稀释至浓度为10000nM、3333nM、1111nM、370nM、123nM、41nM。将蛋白和多肽放96孔板中,于25℃条件下,震荡100-120s,将IL-7RA&TSLPR固定在生物传感器hFc探针或者proteinA探针,后将传感器浸入KBuffer震荡120s进行平衡,再将传感器浸入稀释后的不同浓度多肽溶液震荡120s进行IL-7RA&TSLPR蛋白和多肽的结合反应,后将传感器浸入K Buffer震荡120s进行IL-7RA&TSLPR蛋白和多肽的解离反应,通过计算出Kon和Koff,应用1:1拟合的方式计算出亲和力KD值。
3)含蛋白标签(tag)的多肽
对与带有tag的多肽,用K buffer(PBS pH7.4+0.02%吐温)将待测多肽稀释为10~50μg/ml,用K buffer(PBS pH7.4+0.02%吐温)将IL-7RA&TSLPR蛋白稀释至浓度为1000nM、333nM、111nM、37nM、12.4nM、4.12nM。将蛋白和多肽放96孔板中,于25℃条件下,震荡100-120s,将10~50μg/ml多肽固定在生物传感器His探针,后将传感器浸入K Buffer震荡120s进行平衡,再将传感器浸入稀释后的不同浓度IL-7RA&TSLPR溶液震荡120s进行IL-7RA&TSLPR蛋白和多肽的结合反应,后将传感器浸入KBuffer震荡120s进行IL-7RA&TSLPR蛋白和多肽的解离反应,通过计算出Kon和Koff,应用1:1拟合的方式计算出亲和力KD值。
4)结果
通过BLI分子互作技术进行验证,本发明发现的多肽与IL-7RA&TSLPR具有较好的亲和力,所述多肽能够特异性的与受体结合,进一步验证了多肽的活性。详见表3。
表3:多肽与IL-7RA&TSLPR结合的亲和力测试
以上已对本发明创造的较佳实施例进行了具体说明,但本发明创造并不限于所述的实施例,熟悉本领域的技术人员在不违背本发明创造精神的前提下还可做出种种的等同的变形或替换,这些等同的变形或替换均包含在本申请权利要求所限定的范围内。
Claims (10)
1. 一种多肽或其药学上可接受的盐,其特征在于,所述多肽的氨基酸序列为任选SEQID NO:1~SEQ ID NO:10氨基酸序列之一或与SEQ ID NO:1~SEQ ID NO:10具有至少90%序列同一性的氨基酸序列或经修饰的SEQ ID NO:1~SEQ ID NO:10所示的氨基酸序列的衍生物。
2.根据权利要求1所述的多肽或其药学上可接受的盐,其特征在于,所述经修饰的氨基酸序列的衍生物包括选自以下的一种或更多种修饰:N端和/或C端修饰;以一个或更多个天然和/或非天然氨基酸残基取代一个、二个或更多个氨基酸残基。
3.根据权利要求1所述的多肽或其药学上可接受的盐,其特征在于,所述多肽可通过插入蛋白标签进行分离纯化,进一步的,所述蛋白标签选自His、Flag、GST、Myc、eGFP/eCFP/eYFP/mCherryeGFP、HA、SUMO;优选的,蛋白标签为His。
4.根据权利要求3所述的多肽或其药学上可接受的盐,其特征在于,所述蛋白标签位于SEQ ID NO:1~SEQ ID NO:8序列的C端、N端或序列中苯丙氨酸的N端。
5.一种药物组合物包含权利要求1-4任一项所述的多肽或其药学上可接受的盐。
6.根据权利要求5所述的药物组合物,进一步包含药学上可接受的载体、赋形剂、稀释剂、辅剂或媒介物中的一种或多种。
7.权利要求1-4任一项所述的多肽或其药学上可接受的盐或权利要求5-6所述的药物组合物在制备用于预防、处理、治疗或减轻TSLP相关病症的药物中的用途。
8.根据权利要求7所述的用途,其特征在于,所述TSLP相关病症选自TSLP相关炎性病症或自身免疫性疾病。
9.根据权利要求8所述的用途,其特征在于,所述TSLP相关炎性病症选自过敏性炎症、哮喘、特发性肺纤维化、慢性阻塞性肺病、特应性皮炎或嗜酸性粒细胞食道炎;进一步的所述过敏性炎症选自过敏性鼻炎、过敏性鼻窦炎或过敏性结膜炎;所述哮喘为嗜酸细胞性哮喘或非嗜酸细胞性哮喘,进一步的所述哮喘为低嗜酸细胞性哮喘。
10.一种生物活性检测如上权利要求1-4任一项所述的多肽的方法,包括如下步骤:
a)应用TR-FRET技术建立筛选体系,以Streptavidin-Eu作为荧光供体,Goat Anti-Human IgG Fc-Alexa Fluor647作为荧光受体;选取生物素标记TSLP和偶联人IgG Fc tag的IL-7RA&TSLPR 二聚体;
b)当TSLP与IL-7 RA&TSLP R结合后,加入荧光供体和受体,荧光供体Streptavidin-Eu与TSLP上的生物素结合,荧光受体Goat Anti-Human IgG Fc-Alexa Fluor647与IL-7 RA&TSLP R上的Fc tag结合,使得荧光供体Eu与荧光受体Alexa Fluor647靠近,以发生FRET;
c)加入TSLP Monoclonal Antibody抗体后,阻断TSLP与IL-7 RA&TSLP R结合,使FRET信号减弱;
d)将多肽库96孔深孔板放于离心机离心,用自动分液仪向96孔深孔板中加入超纯水中,密封,放置≥90℃水浴中溶解,溶解后的96深孔板多肽放于离心机再次离心;
e)将再次离心后的上清液用工作站转移至384孔板中,用loading buffer稀释;
f)应用TR-FRET技术建立筛选方法对大型实体多肽库进行高通量筛选选取抑制率大于50%的多肽。
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