CN116925206B - Gitr相关的多肽及其应用 - Google Patents
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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Abstract
本发明属于生物医学领域,具体涉及一种GITR相关的多肽及其应用,更具体的涉及抑制糖皮质激素诱导的TNF受体超家族中的肿瘤坏死因子受体相关蛋白(GITR)与其特异性结合的GITR配体(GITRL)结合的多肽及其应用。所述多肽的氨基酸序列如SEQ ID NO.1‑2所示,所述SEQ ID NO.1和SEQ ID NO.2的第1个氨基酸和第80个氨基酸通过肽键成环;所述的环肽能进一步通过解压缩技术得到一系列衍生的线性肽。本发明提供的多肽可用于治疗和/或预防多种自身免疫疾病、炎症性疾病和癌症。
Description
技术领域
本发明属于生物医学领域,具体涉及一种GITR相关的多肽及其应用。
背景技术
糖皮质激素诱导的肿瘤坏死因子受体相关蛋白(GITR/TNFRSF18)属于肿瘤坏死因子(TNF)受体超家族,由Nocentini等[Proc Natl Acad Sci USA,1997,94(12):6216-6221]于1997年从地塞米松处理的T淋巴细胞杂交瘤中发现,随后于1999年和2003年分别从人脐带内皮细胞[Curr Biol,1999,9(4):215-218]和小鼠脾细胞[Genes Immun,2003,4(8):564-569]中克隆出了能与GITR特异性结合的GITR配体(GITRL/TNFSF18)。作为重要的T淋巴细胞协同刺激分子,GITRL/GITR主要通过活化效应T淋巴细胞与调控调节性T淋巴细胞(Treg)参与炎症介导的疾病发生与进展过程,干预GITRL/GITR通路具有清除病毒与寄生虫感染、增强抗肿瘤治疗效果的作用,因而成为了备受关注的免疫分子。
GITR(TNFRSF18/CD357/AITR)是属于TNFRSF超家族的一种1型跨膜蛋白,其它成员还包括OX40、CD27、CD40和4-1BB。人GITR在CD4+CD25+FoxP3+Tregs上呈高水平表达,在初始和记忆性T细胞上呈低水平表达。在CD8+和CD4+效应T细胞的激活中,GITR在Tregs和效应T细胞上的表达迅速增加,在激活的Tregs上达到最高水平。GITR在自然杀伤细胞(NK)上也有表达,在B细胞、巨噬细胞和树突状细胞上也有低水平表达,并且可以通过激活上调,尤其是在NK细胞上。
GITRL是一种2型跨膜蛋白,也是TNFRSF的一个成员。它通常被认为是三聚体,尽管它也可以作为单体存在或组装成其他多聚体形式。GITRL主要由活化的抗原提呈细胞表达,包括巨噬细胞、B细胞、树突状细胞和内皮细胞。值得注意的是,GITR和GITRL的表达并不局限于造血细胞,GITR在表皮角质形成细胞和破骨细胞前体细胞上均有表达,而GITRL在内皮细胞上也有表达,尤其是在I型干扰素(IFN)作用后。
作为重要的T淋巴细胞协同刺激分子,GITRL可以直接与GITR特异性结合,也可以被酶切后形成可溶性胞外段与GITR特异性结合,不仅具有促进效应T淋巴细胞增殖与细胞因子分泌的作用,还具有调控Treg增殖和抑制效应T淋巴细胞的功能,从而发挥调控效应T淋巴细胞功能的作用。
鉴于GITR/GITRL潜在的抗炎、自身免疫性疾病及抗肿瘤作用,多家制药企业将目光投入GITR单抗的研发,大多数药物仍处于早期研究阶段,尚无产品获批上市。因而本领域仍然非常需要治疗和/或预防GITR相关和/或GITRL相关疾病和病症的多肽类药物。
发明内容
为解决现有疾病中存在的尚未得到满足的临床需求,本发明提供了一种抑制多肽及其应用,具体的涉及抑制GITR与GITRL结合的多肽及其应用。
一方面,本发明提供一种多肽或者其药学上可接受的盐,所述多肽氨基酸序列如SEQ ID NO.1或SEQ ID NO.2所示,其中,所述SEQ ID NO.1的第1个氨基酸和第80个氨基酸通过肽键成环;所述SEQ ID NO.2的第1个氨基酸和第80个氨基酸通过肽键成环。具体氨基酸序列如表1所示
表1本发明氨基酸序列
其中,氨基酸序列如SEQ ID NO.1和2所示的多肽为从多肽库中,通过高通量筛选技术,检测多肽对GITR与GITRL结合的抑制率得到的80环肽,该环肽的发现包括多肽库溶解及稀释,利用TR-FRET筛选方法根据多肽对GITR与GITRL结合的抑制率结果从多肽库中筛选。
另一方面,本发明提供一种多肽或者其药学上可接受的盐,其特征在于,所述多肽分子是对SEQ ID NO.1或SEQ ID NO.2进行分析和拆解得到的环状肽或线性肽。对80环肽SEQ ID NO.1或SEQ ID NO.2进行氨基酸的序列进行分析和拆解工作,能够设计出10-80不同氨基酸序列的线性肽或者环肽。
优选地,所述环状肽的第1个氨基酸和最后一个氨基酸通过肽键成环。
优选地,所述环状肽具有30~35个氨基酸。
优选地,所述线性肽具有30-45个氨基酸;进一步优选地,所述线性肽具有40个氨基酸。
优选地,所述线性肽的氨基酸序列可选自SEQ ID NO.3至SEQ ID NO.12,具体氨基酸序列如表1所示。
另一方面,本发明提供一种多核苷酸分子,所述多核苷酸分子包含能够编码上述的多肽的一个或两个的多核苷酸。
优选地,本发明提供一种多核苷酸分子,所述多核苷酸分子包含能够编码上述SEQID NO.1至SEQ ID NO.12多肽。
另一方面,本发明提供一种药物组合物,它含有上述所述的多肽或其药学上可接受的盐或所述的多核苷酸分子。
通常,可将药物组合物制成可注射剂,例如液体溶液或悬液;还可制成在注射前适合配入溶液或悬液中、液体载体的固体形式。
一旦配成本发明的组合物,可将其通过常规途径进行给药,其中包括(但并不限于):瘤内、肌内、静脉内、肝动脉、口服、皮下、皮内、或局部给药。
当本发明的药物组合物被用于实际治疗时,可根据使用情况而采用各种不同剂型的药物组合物。较佳地为静脉用或肝动脉药制剂或瘤内用药注射剂。
这些药物组合物可根据常规方法通过混合、稀释或溶解而进行配制,并且偶尔添加合适的药物添加剂,如赋形剂、崩解剂、粘合剂、润滑剂、稀释剂、缓冲剂、等渗剂(isotonicities)、防腐剂、润湿剂、乳化剂、分散剂、稳定剂和助溶剂,而且该配制过程可根据剂型用惯常方式进行。
本发明的药物组合物还可以缓释剂形式给药。
另一方面,本发明提供一种上述的多肽或其药学上可接受的盐、多核苷酸分子或药物组合物在制备用于治疗与GITR/GITRL相关的疾病的药物上的应用。
优选地,与GITR/GITRL相关的疾病可以选自自身免疫性疾病、炎症、移植排斥、癌症;进一步的,所述自身免疫性疾病或炎症选自类风湿性关节炎、脑脊髓炎、骨关节炎、多发性硬化、自身免疫性胃炎、系统性红斑狼疮、牛皮鲜、哮喘、过敏或炎性肠疾病,包括克罗恩病和溃疡性结肠炎。
上述制备的用于治疗与GITR/GITRL相关的疾病的药物可以与一种或多种免疫学药物同时、分开或顺序地组合施用。
术语
除非本文中另外定义,否则本专利申请中所用的科学及技术术语应具有一般本领域技术人员通常所理解的含义。
本文所用“多肽库”是湖南中晟全肽生化有限公司利用PICT(PeptideInformation Compression Technology)专利技术,该技术利用生物学手段对多肽信息进行压缩,可将多个多肽的信息集成进一个多肽,从而实现以相对较小的库容包含较大的多肽信息量;通过PICT技术构建含有近73000条80个氨基酸的环肽库。其具体构建方法可以参见专利CN201580081102.3和专利CN201780089941.9。
本文所用“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。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。
本发明多肽可以是经过修饰的,修饰(通常不改变一级结构)形式包括:体内或体外的多肽的化学衍生形式包括但不限于乙酰化、羧基化、烷基化、酰基化、氨基甲酰化。修饰还包括糖基化,如那些在多肽的合成和加工中或进一步加工步骤中进行糖基化修饰而产生的多肽。这种修饰可以通过将多肽暴露于进行糖基化的酶(如哺乳动物的糖基化酶或去糖基化酶)而完成。修饰形式还包括具有磷酸化氨基酸残基(如磷酸酪氨酸,磷酸丝氨酸,磷酸苏氨酸)的序列。还包括被修饰从而提高了其抗蛋白水解性能或优化了溶解性能的多肽。
本发明多肽可以是重组多肽或合成多肽。本发明的多肽可以是化学合成的,或重组的。相应地,本发明多肽可用常规方法人工合成,也可用重组方法生产。一种优选的方法是使用液相合成技术或固相合成技术。另一种方法是用重组技术产生本发明多肽。通过常规的重组DNA技术,可利用本发明的多核苷酸用来表达或生产重组的本发明多肽。由于本发明多肽较短,因此可以考虑将多个多肽串联在一起,重组表达后获得表达产物,然后通过酶切等方法形成所需的小肽。
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。
本文所用的“氨基酸”包括标准的20种遗传编码的氨基酸和它们的相应的“D”形式(与天然的“L”形式相比)的立体异构体、ω-氨基酸、其它天然存在的氨基酸、非常规氨基酸(例如α,α-双取代的氨基酸、N-烃基氨基酸,等等)和化学上衍生的氨基酸。
当明确列举氨基酸诸如“丙氨酸”或“Ala”或“A”时,该术语表示L-丙氨酸和D-丙氨酸,除非另外明确地阐明。其它非常规氨基酸也可以是本发明的多肽的合适组分,只要期望的功能特性被所述多肽保留。对于显示的肽,每种编码的氨基酸残基在适当的情况下用单字母名称表示,所述单字母名称对应于常规氨基酸的俗名。
本文所用的“包括”用于意谓“包括但不限于”。
本文所用的“药学上可接受的载体”指用于治疗剂给药的载体。该术语指这样一些药剂载体:它们本身不诱导产生对接受该组合物的个体有害的抗体,且给药后没有过分的毒性。这些载体是本领域普通技术人员所熟知的。在Remington's PharmaceuticalSciences(Mack Pub.Co.,N.J.1991)中可找到关于药学上可接受的赋形剂的充分讨论。这类载体包括(但并不限于):盐水、缓冲液、葡萄糖、水、甘油、乙醇、佐剂、及其组合。
本文所用的“编码”应用于多聚核苷酸时,是指被称为“编码”多肽的多聚核苷酸,在其天然状态或当通过本领域技术人员公知的方法操作时,经转录和/或翻译可以产生该多肽和/或其片段。
本文所用的“IC50”表示50%抑制浓度,即对指定的生物过程抑制一半时所需的药物或者抑制剂的浓度。
本文所用的“预防”表示预防疾病和/或它的附随症状的发作或者保护对象免于获得疾病的方法。
本文所用的“治疗”包括延缓和终止疾病的进展,或消除疾病,并不需要100%抑制、消灭和逆转。
与现有技术相比,本发明具有如下优点:
(1)本发明所用多肽库比传统化学合成多肽库信息量大得多的多肽化合物氨基酸序列,库内各化合物均为独立生产,均经过质谱鉴定和精确称量,保证了筛检的准确和稳定,避免了传统的噬菌体库等混合化合物库的失真(实际库容远低于理论值)问题。
(2)本发明提供了一系列抑制GITR与GITRL结合的多肽。本发明提供的多肽可以阻断人GITR与GITRL的相互作用,可用于治疗和/或预防类风湿性关节炎、脑脊髓炎、骨关节炎、多发性硬化、自身免疫性胃炎、系统性红斑狼疮、牛皮鲜、哮喘、过敏或炎性肠疾病,包括克罗恩病和溃疡性结肠炎等。
附图说明
图1为实施例1SEQ ID NO.1和2不同浓度抑制结果;
图2为实施例2SEQ ID NO.3至SEQ ID NO.12不同浓度抑制结果。
具体实施例
根据下述实施例,可以更好地理解本发明。然而,本领域的技术人员容易理解,实施例中描述的内容仅用于说明本发明,而不应当也不会限制权利要求书中所详细描述的本发明。
本发明所用主要实验试剂如表2所示
表2本发明实验试剂
名称 | 厂家 | 货号 |
Add&Read Human GITR/GITRL κit | 诺唯赞 | DD2228 |
实施例1
多肽库的溶解:将多肽库96孔深孔板放于离心机4000rpm离心2~3分钟。用自动分液仪向96孔深孔板中加入200μL/孔超纯水中。用硅胶盖密封,放置95℃水浴5分钟。注:此时多肽浓度约为:50μM。溶解后的96深孔板多肽放于离心机4000rpm离心2~3分钟。
多肽库稀释:将溶解后的多肽用工作站转移至384孔板中,用loading buffer(Tris-Hcl缓冲液,pH 7.4)稀释至10μM。
其中,多肽库是湖南中晟全肽生化有限公司自制。
应用TR-FRET筛选方法对大型实体多肽库进行验证,步骤如下:
步骤1:Add&ReadHuman GITR/GITRLκit试剂盒中试剂配制(1).Anti-Tag1-Eu和Anti-Tag2-A2工作液配制(储存液为50×)
a.Anti-Tag1-Eu工作液配制:将Anti-Tag1-Eu从-20冰箱取出,室温放置使其溶解;向1体积Anti-Tag1-Eu中加入49体积的Detection buffer,混合均匀。
b.Anti-Tag2-A2工作液配制:将Anti-Tag2-A2从-20冰箱取出,室温放置使其溶解;向1体积Anti-Tag2-A2中加入49体积的Detection buffer,混合均匀。(2)Tag1-GITR和Tag2-GITRL工作液配制(储存液为50×)
a.Tag1-GITR工作液配制:将Tag1-GITR从-80冰箱取出,室温放置使其溶解;向1体积Tag1-GITR中加入49体积的Diluent,混合均匀。
b.Tag2-GITRL工作液配制:将Tag2-GITRL从-80冰箱取出,室温放置使其溶解;向1体积Tag2-GITRL中加入49体积的Diluent,混合均匀。
步骤2:筛选过程:
(1)将多肽样品用pH 7.4的buffer稀释至实验浓度;
(2)向384孔板中加入4μL的多肽样品或者标准品;
(3)加入4μLTag2-GITRL工作液,离心混匀;
(4)加入4μLTag1-GITR工作液,离心混匀;
(5)将Anti-Tag1-Eu和Anti-Tag2-A2工作液以1:1混合均匀,向体系中加入8μL,离心混匀;
(6)室温孵育2小时,放于酶标仪中检测,激发光为320nm,检测两个波长665nm和620nm的发射光。计算抑制率,计算IC50值。
通过对初筛到的多肽进行重复实验确认,最终确定从自制的80环肽库中筛选到的SEQ ID NO.1和2的样品的抑制率较高,后进行浓度依赖性验证。计算抑制率,作图。实验结果如表3所示,浓度响应曲线如图1所示。
表3:80环肽SEQ ID NO.1和2的筛选结果
SEQ ID NO. | IC50(μM) |
1 | 0.42 |
2 | 1.25 |
从实验结果可以看出,本发明的80环肽对GITR与GITRL结合有抑制作用,且抑制作用随着80环肽的浓度增大而增强。
实施例2
应用内部环肽解压缩技术,对实施例1中筛选出的80环肽SEQ ID NO.1和2的氨基酸序列进行分析和拆解工作,设计出10-80条不同氨基酸序列的线性肽或者环肽。按照实施例1筛选过程对设计出的10-80条不同氨基酸序列的线性肽或者环肽进行筛选,并对设计出的10-80条不同氨基酸序列中具有活性的线性肽或者环肽进行IC50验证。
对设计出的10-80条不同氨基酸序列IC50验证之后,进一步对活性多肽进行浓度依赖验证,实验结果如表4所示,浓度响应曲线如图2所示。
表4:活性多肽的筛选结果
SEQ ID NO. | IC50(μM) | SEQ ID NO. | IC50(μM) |
3 | 0.84 | 8 | 0.37 |
4 | 0.44 | 9 | 0.20 |
5 | 0.27 | 10 | 0.17 |
6 | 0.31 | 11 | 0.50 |
7 | 0.65 | 12 | 0.39 |
从实验结果可以看出,本发明的80环肽拆解之后得到的线性肽对GITR与GITRL结合有抑制作用。
应当指出,以上所述仅是本发明的优选实施方式,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (4)
1.一种分离的多肽或者其药学上可接受的盐,其特征在于,所述多肽的氨基酸序列如SEQ ID NO.1或SEQ ID NO.2所示,其中,所述SEQ ID NO.1的第1个氨基酸和第80个氨基酸通过肽键成环;所述SEQ ID NO.2的第1个氨基酸和第80个氨基酸通过肽键成环。
2.一种分离的多肽或者其药学上可接受的盐,其特征在于,所述多肽是对SEQ ID NO.1或SEQ ID NO.2进行分析和拆解得到的线性肽,所述线性肽的氨基酸序列如SEQ ID NO.3~SEQ ID NO.12中任一项所示。
3.多核苷酸分子,其特征在于,所述多核苷酸分子包含编码权利要求2所述的多肽的一个或两个多核苷酸。
4.一种药物组合物,包含权利要求1或2所述的多肽或其药学上可接受的盐或权利要求3所述的多核苷酸分子。
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