CN116836241B - 抑制CD47与SIRPα结合的多肽及其应用 - Google Patents
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Abstract
本发明属于生物医药领域,公开了一种抑制CD47与SIRPα结合的多肽及其应用。所述多肽的氨基酸序列如SEQ ID NO.1~SEQ ID NO.10中的任意一个所示。本发明应用高通量药物筛选技术筛选出可能具有抑制作用的80环肽,然后通过TR‑FRET筛选方法进行验证得到能够抑制CD47与SIRPα结合的SEQ ID NO.1。通过分析和拆解SEQ ID NO.1得到10~80个多肽,利用TR‑FRET筛选方法对拆解的多肽进行验证,得到SEQ ID NO.2~SEQ ID NO.10,这一组多肽同样能够抑制CD47与SIRPα结合。多肽通过抑制CD47与SIRPα结合可以治疗CD47‑SIRPα信号通路相关的癌症,例如急性髓系白血病、非霍奇金淋巴瘤、膀胱癌、乳腺癌、胃癌和肺癌等。
Description
技术领域
本发明属于生物医药领域,具体涉及一种抑制CD47与SIRPα结合的多肽及其应用。
背景技术
CD47(cluster ofdifferentiation 47)又名整合素相关蛋白(integrinassociatedprotein,IAP)和卵巢癌抗原(ovarian cancer antigen,OA3),是一种跨膜蛋白,分子量大约为50kDa,属于免疫球蛋白超家族,其分子结构包括1个V型Ig样的细胞外可变区域,5个疏水的跨膜螺旋结构,以及一个非常短的C末端的细胞内信号序列。CD47参与的生物学作用包括:细胞的凋亡、细胞的增殖、细胞的粘附、细胞的迁移、调节炎症反应和巨噬细胞吞噬作用的抑制。
CD47几乎广泛表达于所有的正常细胞表面,在肿瘤细胞高表达,包括骨髓瘤、平滑肌肉瘤、急性淋巴细胞白血病、非霍奇金淋巴瘤,乳腺癌,骨肉瘤,头颈部鳞状细胞癌。目前已知的CD47的天然配体有三种:整合素(integrin)、血小板反应蛋白-1(thrombospondin-1,TSP-1)和信号调节蛋白α(signal-regulatory protein alpha,SIRPα)。
SIRPα又称为src homology 2domain-containing protein tyrosinephosphatase substrate-1(SHPS-1),也是一种跨膜蛋白,在髓系造血细胞中高表达,例如巨噬细胞和树突状细胞。SIRPα其胞外域由三个Ig样结构域组成,分别是一个V样和两个C1样结构域,胞内域的C端含有两个酪氨酸磷酸化位点。
SIRPα的IgV样结构域能与CD47的IgV样结构域反式结合,并促进SIRPα胞内域中的酪氨酸磷酸化,SIRPα的酪氨酸磷酸化位点能与蛋白质酪氨酸磷酸酶SHP-1和SHP-2结合,从而激活这些磷酸酶。SIRPα主要表达在巨噬细胞表面,与其他细胞上的CD47结合后,可向巨噬细胞传递抑制性信号,抑制巨噬细胞对靶细胞的吞噬作用。因此,这条通路可用来识别己细胞(self)与非己细胞(non-self)。一些肿瘤细胞会利用这个机制,以高表达CD47来向巨噬细胞发出“别吃我(Don’t eat me)”的信号,来进行免疫逃逸。
CD47-SIRPα信号通路具有巨大的治疗潜力,在癌症免疫治疗中,CD47已成为继PD-1/PD-L1之后另一个高度竞争的靶点。目前,靶向该通路的在研药物主要分三类,包括抗CD47单克隆抗体(靶向CD47)、SIRPα融合蛋白(靶向CD47)以及抗SIRPα抗体(靶向SIRPα)。作用机制主要包括:阻断CD47与SIRPα的结合,切断“别吃我”信号,促进巨噬细胞吞噬肿瘤细胞;抗CD47单克隆抗体可通过促进树突状细胞对肿瘤细胞的吞噬作用,随后将抗原呈递给T细胞,刺激抗肿瘤适应性应答;抗CD47单克隆抗体通过自然杀伤细胞介导的抗体依赖性细胞介导的细胞毒作用(ADCC)和补体依赖的细胞毒性作用(CDC)作用杀伤肿瘤细胞;抗CD47单克隆抗体可激活肿瘤细胞凋亡途径,直接诱导肿瘤细胞凋亡。
目前,一些CD47靶向抗体或药物已进入临床试验,包括Hu5F9-G4(Forty-Seven)、CC-90002(Celgene)、TTI-621(Trillium)、ALX148(Alexo Therapeutics)、SRF231(SurfaceOncology)、SHR-1603(恒瑞)和IBI188(Innovent Biologics)等。其中Hu5F9-G4、CC-90002和IBI188是抗CD47单克隆抗体,TTI-621和ALX148是SIRPα-Fc融合蛋白。
2022年12月26日,宜明昂科宣布其自主研发的国内首个靶向人CD47的SIRPαFc融合蛋白药物IMM01,在联合阿扎胞苷(AZA)针对初治高危骨髓增生异常综合症(MDS)以及初治的AML适应症的两个II期临床研究,已完成患者入组。IMM01具有双重机制,能够同时阻断来自肿瘤的“别吃我”信号,并通过IgG1激活患者免疫系统的“吃我”信号。差异化的设计思路使得IMM01在体外实验中完全不与红细胞结合,不会引起严重贫血事件;同时,由于糖基化修饰,大大降低了药物的免疫原性,显著提高了药物的生物利用度。从而完美解决了CD47靶点药物研发中的核心痛点,并具有“Best-In-Class”的潜力。
在小鼠和猕猴上使用抗CD47单克隆抗体进行的临床前研究表明,这些疗法具有良好的耐受性。然而,2017年,Arch Oncology终止了抗CD47单克隆抗体Ti-061的I/II期临床试验,2018年,Celgene终止了抗CD47单克隆抗体CC-90002治疗急性髓系白血病(AML)的临床试验。鉴于CD47在造血系统的非恶性细胞表达,包括正常红细胞、衰老红细胞和血小板,使用抗CD47单克隆抗体作为抗癌治疗可能存在一定的问题,例如Buatois等人表明Hu47F9-G4单独或与其他抗体结合可能导致正常红细胞意外死亡,可能导致贫血。
CD47的普遍表达意味着一种药物可能需要大的起始剂量和/或频繁的给药来实现对CD47的有效阻断。与CD47相比,SIRPα的组织学分布更为有限,这可能使其在靶向治疗时毒性更小并有更大的阻断作用。但是,SIRPα在神经细胞中也有表达,对于中枢神经系统的副作用应该考虑到。
目前研究出的阻止CD47与SIRPα结合的药物数量较少,大部分在临床阶段,或者存在药物的副作用较大的问题,CD47-SIRPα信号通路方面仍需要研发大量药物。且近年来,多肽合成技术不断发展和成熟,多肽药物的稳定性较好,使得多肽药物在抗肿瘤治疗领域中越来越重要,已成为新药研发的重要方向之一。因此,研究出抑制CD47与SIRPα结合的多肽对治疗肿瘤领域具有重要意义。
发明内容
为解决现有技术的不足,本发明提供了一种抑制CD47与SIRPα结合的多肽及其应用。
一方面,本发明提供一种抑制CD47与SIRPα结合的多肽或其药学上可接受的盐或其溶剂化物,其特征在于为首尾相接的环肽,长度为80个氨基酸。
如SEQ ID NO.1所示的80环肽是通过根据多肽信息压缩技术(PICT)搭建的高通量新药筛选平台筛选,然后应用TR-FRET筛选方法对其进行抑制CD47与SIRPα结合的抑制率验证得到的。
进一步的,所述氨基酸序列如SEQ ID NO.1所示,具体氨基酸序列可见表1,其中SEQ ID NO.1的第1个氨基酸与第80个氨基酸通过脱水缩合形成肽键得到首尾相接的环肽。
另一方面,本发明提供一种抑制CD47与SIRPα结合的多肽或其药学上可接受的盐或其溶剂化物,其特征在于所述多肽分子是通过对SEQ ID NO.1进行分析和拆解得到的多肽。
对SEQ ID NO.1进行氨基酸序列的分析和拆解可以得到10~80条多肽,使用TR-FRET筛选方法检验多肽抑制CD47与SIRPα结合的抑制率。
进一步的,所述多肽分子为环状肽,第一个氨基酸与最后一个氨基酸通过脱水缩合形成肽键得到首尾相接的环肽。
进一步的,所述环肽分子的长度为30~40个氨基酸。
进一步的,所述环肽分子的长度为32个氨基酸。
进一步的,所述环肽分子的氨基酸序列可选自SEQ ID NO.2~SEQ ID NO.10,具体氨基酸序列可见表1。
表1本发明氨基酸序列
另一方面,本发明提供一种多核苷酸分子,其特征在于所述多核苷酸分子能够编码出上述多肽分子。
另一方面,本发明提供一种药物组合物,其特征在于包含(a)安全有效量的的本发明多肽或其药学上可接受的盐或其溶剂化物;(b)药学上可接受的载体或赋形剂。
用于本发明的方法中的药物组合物可含有任何药学上可接受的赋形剂。赋形剂的实例包括但不限于淀粉、糖、微晶纤维素、稀释剂、粒化剂、润滑剂、粘合剂、崩解剂、湿润剂、乳化剂、着色剂、释放剂、包覆剂、抗氧化剂、塑化剂、胶凝剂、增稠剂、硬化剂、凝固剂、混悬剂、表面活性剂、保湿剂、载体、稳定剂、以及它们的组合。
用于本发明的方法中的药物组合物可含有任何药学上可接受的载体。举例来说,载体可为液体或固体填充剂、稀释剂、赋形剂、溶剂、或囊封物质、或它们的组合。
各种实施方案中,本发明的药物组合物可被配制以通过任何施用途径递送。这可包括例如气雾剂、经鼻、口服、经粘膜、经皮、胃肠外或经肠。
“胃肠外”是指通常与注射相关的施用途径,包括眶内、输注、动脉内、囊内、心内、真皮内、肌肉内、腹膜内、肺内、脊柱内、胸骨内、鞘内、子宫内、静脉内、蛛网膜下、囊下、皮下、经粘膜或经气管。通过胃肠外途径,组合物可呈用于输注或用于注射的溶液或混悬液形式,或呈冻干粉剂形式。通过胃肠外途径,组合物可呈用于输注或用于注射的溶液或混悬液形式。通过经肠途径,药物组合物可呈片剂、凝胶胶囊、糖包衣片剂、糖浆、混悬液、溶液、粉剂、颗粒剂、乳液、允许控制释放的微球体或纳米球体或脂质囊泡或聚合物囊泡形式。通常,组合物通过注射施用。用于这些施用的方法为本领域技术人员所知。
另一方面,本发明提供的多肽分子或其药学上可接受的盐或其溶剂化物、多核苷酸分子、药物组合物可用于治疗与CD47-SIRPα信号通路相关的疾病。
进一步的,与CD47-SIRPα信号通路相关的疾病可选自癌症,包括急性髓系白血病、非霍奇金淋巴瘤、膀胱癌、乳腺癌、胃癌和肺癌等。
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。
本文提到的“PICT(Peptide Information Compression Technology)”为湖南中晟全肽独家拥有的专利技术,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。其具体构建方法可以参见专利CN201580081102.3和专利CN201780089941.9。
本文提到的“高通量新药筛选平台”是利用湖南中晟全肽生化有限公司的PICT(Peptide Information Compression Technology)专利技术搭建而成的,该平台可利用公司自主构建的超大型多肽库针对已知靶点或新兴靶点进行筛选,将显著加快多肽新药的发现进程,降低多肽新药研发成本。
与现有技术相比,本发明具有以下优点:
(1)本发明利用PICT技术对多肽药物进行筛选,加快了找到抑制CD47与SIRPα结合的多肽的进程,大大节约的经济成本和时间成本。
(2)本发明提供了一种抑制CD47与SIRPα结合的多肽。本发明提供的多肽可以切断肿瘤细胞对吞噬细胞发出的“别吃我”信号,从而阻止肿瘤细胞的增值,达到治疗与肿瘤相关疾病的效果,如急性髓系白血病、非霍奇金淋巴瘤、膀胱癌、乳腺癌、胃癌和肺癌等。
附图说明
图1为实施例1中不同浓度的SEQ ID NO.1对抑制CD47与SIRPα结合的结果。
图2为实施例2中不同浓度的SEQ ID NO.2至SEQ ID NO.6对抑制CD47与SIRPα结合的结果。
图3为实施例2中不同浓度的SEQ ID NO.7至SEQ ID NO.10对抑制CD47与SIRPα结合的结果。
具体实施方式
为更好理解本发明,下面结合附图和实施例对本发明作进一步的详细说明,但不以任何方式对本发明加以限制,基于本发明教导所作的任何变换或改进,均落入本发明的保护范围。
本发明需要用到的试剂如表2所示。
表2本发明使用试剂
名称 | 厂家 | 货号 |
RecombinantHumanCD47ProteinBiotinylated | SinoBiological | 12283-HCCH-B |
SIRPalphaProtein,Human,Recombinant(ECD,hFcTag) | SinoBiological | 11612-H02H1 |
CD47Antibody,RabbitPab,AntigenAffinityPurified | SinoBiological | 12283-T26 |
Streptavidin-Eu(SA-Eu) | ATTBio | 16925 |
Anti-humanFcantibody-AlexaFluor647 | Jackson | 109-605-170 |
实施例1
阻止CD47与SIRPα结合的80环肽的筛选。
确定好靶标后应用TR-FRET筛选方法利用湖南中晟全肽生化有限公司的高通量筛选平台筛选出一定数量可能抑制CD47与SIRPα结合的80环肽。
(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。
多肽库的稀释: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,pH7.4)稀释至10μM。
(2)应用TR-FRET筛选方法对大型实体多肽库进行验证。在384孔板中依次加入不同浓度的80环肽、20nM SIRPα和5nM CD47,以及荧光供体Streptavidin-Eu和荧光受体GoatAnti-Human IgG Fc-AlexaFluor647,室温孵育2小时后,检测TR-FRET信号。
(3)增加阳性对照:不含80环肽,只含20nM SIRPα和5nM CD47,以及荧光供体Streptavidin-Eu和荧光受体GoatAnti-Human IgG Fc-Alexa Fluor647,目的是检测多肽是否具有抑制CD47与SIRPα结合的功能。
(4)增加阴性对照:不含80环肽,只含20nM SIRPα和5nM CD47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Goat Anti-Human IgG Fc-AlexaFluor647,目的是排除实验中会影响实验结果的变量。
(5)通过对初筛到的80环肽进行重复实验确认,最终确定初筛的的80环肽库中筛选到的SEQ ID NO.1样品抑制率较高,后进行浓度依赖性验证。计算抑制率,用graphpad作图。实验结果如表3所示,不同浓度的环肽对抑制CD47与SIRPα结合的结果曲线如图1所示。
从实验结果可以看出,本发明的80环肽对CD47与SIRPα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,但当80环肽的浓度较低时,其对CD47与SIRPα结合没有抑制作用。
表3 80环肽SEQ ID NO.1的筛选结果
SEQ ID NO. | IC50(μM) |
1 | 2.17 |
实施例2
抑制CD47与SIRPα结合的环肽的筛选。
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环肽。
对80环肽SEQ ID NO.1进行解压缩,得到一组可能具有抑制CD47与SIRPα结合的环肽,按照实施例1的实验步骤对该组环肽进行复筛处理,确定SEQ ID NO.2至SEQ ID NO.10多肽对CD47与SIRPα结合有抑制作用,后进行浓度依赖性验证,计算抑制率后用graphpad作图。实验结果如表4所示,不同浓度的环肽对抑制CD47与SIRPα结合的结果曲线如图2和图3所示。
表4线性肽的筛选结果
SEQ ID NO. | IC50(μM) |
2 | 7.04 |
3 | 5.10 |
4 | 3.77 |
5 | 9.48 |
6 | 3.21 |
7 | 8.82 |
8 | 9.34 |
9 | 6.71 |
10 | 3.58 |
从实验结果可以看出,本发明的环肽对CD47与SIRPα结合有抑制作用。
Claims (4)
1.一种抑制CD47与SIRPα结合的多肽或其药学上可接受的盐,其特征在于,所述多肽为长度为80个氨基酸的环肽,所述环肽的氨基酸序列为SEQ ID NO.1,第1个氨基酸与第80个氨基酸通过肽键首尾相接。
2.一种抑制CD47与SIRPα结合的多肽或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进行分析和拆解得到的多肽,所述拆解得到的多肽的氨基酸序列选自SEQ ID NO.2~SEQ ID NO.10中的一种,多肽的第一个氨基酸与最后一个氨基酸通过肽键首尾相接。
3.一种多核苷酸分子,其特征在于所述多核苷酸分子能够编码出权利要求1或2所述的多肽。
4.一种药物组合物,其特征在于包含(a)安全有效量的权利要求1或2所述的多肽或其药学上可接受的盐;(b)药学上可接受的载体或赋形剂。
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Country or region after: China Address after: Building 5, Modern Service Industry Headquarters Park, No. 1769 Yunlong Avenue, Yunlong Demonstration Zone, Zhuzhou City, Hunan Province, 412000 Applicant after: Hunan Zhongsheng Whole Peptide Biotechnology Co.,Ltd. Address before: Building 5, Modern Service Industry Headquarters Park, No. 1769 Yunlong Avenue, Yunlong Demonstration Zone, Zhuzhou City, Hunan Province, 412000 Applicant before: HOHAI University Country or region before: China |
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