CN113292635B - 靶向cd47的多肽及其应用 - Google Patents
靶向cd47的多肽及其应用 Download PDFInfo
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Abstract
本发明提供一种靶向CD47的多肽及其应用。本发明的多肽能特异性识别并结合CD47蛋白。本发明还提供该多肽及其形成的二价体、多价体和多肽偶联物及其在制备靶向杀伤肿瘤细胞的诊疗剂和显影剂中的应用。本发明的多肽采用化学方法合成,制备简单、特异性强、选择性高、安全可靠,弥补了抗体生物制剂生产周期长等缺点,可用于制备癌症的靶向探针。本发明可以通过免疫治疗法对CD47蛋白进行免疫阻断,从而激活体内免疫系统对肿瘤细胞的识别和吞噬作用,有效抑制肿瘤的生长和转移,实现良好的抗肿瘤效果。本发明的多肽可为免疫治疗提供有效临床依据和应用价值。
Description
技术领域
本发明涉及药物化学领域,具体地说,涉及一种靶向CD47的多肽及其应用。
背景技术
癌症是威胁人类生命的主要疾病之一,尽管在手术、化学治疗和放射治疗等常规策略方面取得了进展,但其治疗仍面临着严峻的挑战。传统的治疗方法除了直接杀伤肿瘤细胞以外,也会对人体的正常组织造成不可控制的损伤,使得肿瘤患者在治疗过程中要承受巨大的心理和生理痛苦,引发呕吐、脱发等不良反应,重要的是治疗手段并不能有效阻止肿瘤的复发,且治疗效果不佳。因此,医学和科研工作者希望寻求一种新的治疗方法来弥补传统治疗上的不足。随着近代生物化学、医学免疫学、分子生物学等有关学科理论和应用的迅速发展,肿瘤免疫治疗得到了深入研究。肿瘤免疫治疗手段具有较高的疗效性和安全性,其已经成为国内外肿瘤治疗领域当中颇具研究前景的方向之一,这也为肿瘤患者的治疗带来新的希望。与直接攻击肿瘤细胞相反,免疫疗法通过引发宿主的自然免疫反应,从而杀死癌细胞。当前的肿瘤免疫疗法主要包括免疫检查点治疗、过继性免疫细胞疗法以及肿瘤疫苗等多种方法。过继性免疫细胞疗法存在花费昂贵、条件要求较高等局限性;肿瘤疫苗的免疫原性较低,常常需要配合合适的免疫佐剂联合使用,因此两种方法仍有很大进步空间。相比之下,免疫检查点阻断法通过采用抑制分子或配体的拮抗剂来阻断信号通路,解除肿瘤患者体内对肿瘤的免疫抑制,进而刺激体内免疫细胞活化,激活其杀伤肿瘤细胞的能力。该疗法由于在疗效方面的优势和定点靶向的较低副作用而引起医学工作者的广泛关注。
CD47也称为整合素相关蛋白,属于免疫球蛋白超家族成员,相对分子质量约5.2×104Da,是一种广泛表达的高度糖基化跨膜蛋白。在其N端有1个IgV样结构域,并具有5个跨膜区段和可以选择性剪接高疏水性细胞质C端。CD47分子的亚型2表达最广泛,主要分布在造血细胞、血管内皮细胞和上皮细胞。CD47分子的配体主要是血小板反应蛋白1(thrombospondin 1,TSP1)和信号调节蛋白α(signal regulation protein,SIRPα),其中CD47与巨噬细胞表面上SIRPα结合激活胞内Src2酪氨酸磷酸酶结构域并抑制吞噬细胞突触中肌球蛋白的积累,最终向巨噬细胞发出“别吃我”的信号,从而躲避了巨噬细胞的吞噬作用,造成肿瘤免疫逃逸。CD47作为天然免疫重要的“免疫检查点”分子,在体内发挥着重要的生理和病理功能。一方面,能够维持体内正常红细胞的数量,及时清除衰老细胞;同时,在肿瘤发生发展过程中,CD47表现出至关重要的作用,其在肿瘤细胞表面广泛高表达,从而逃避天然免疫系统的识别和杀伤。研究证实,通过阻断CD47-SIRPα信号通路能有效增强体外巨噬细胞吞噬能力和体内抗肿瘤免疫应答。CD47不仅在天然免疫中发挥作用,还会影响T细胞功能,阻断CD47-SIRPα信号通路能促使T细胞介导的免疫原性肿瘤的消除。因此,基于在天然免疫中CD47分子的重要功能,研究者们致力于研究CD47-SIRPα信号通路相关作用机制并明确指出其在多种疾病特别是肿瘤相关疾病中的关键作用,因此CD47已成为重要的“新明星”肿瘤靶点之一。
由于CD47分子特殊的作用机制,开发靶向CD47抑制剂能够抑制肿瘤的免疫逃逸,起到有效抗肿瘤作用。抗体具有分子量大、穿透性低、生产周期长和人源化困难等局限性。与抗体相比,多肽是一类内源性小分子,具有显著的优势:易于合成,靶向性好、选择性高、穿透能力强且稳定性好。由于多肽的天然成分使其具有良好的生物相容性,通过合理设计产生具有可控形态的纳米结构,并开发用于疾病诊断和治疗的功能性生物材料。利用分子间的非共价相互作用,自组装成具有纳米粒子、纳米纤维、纳米带、纳米管等不同形态的纳米结构。基于多肽组装体的形态转变,多肽纳米载体也可以增加其在体内的循环时间,有效增强与靶向受体的亲和力。此外,多肽纳米结构的主动靶向能力促进了治疗剂在肿瘤部位的积累。多肽及其组装体已用于不同种类的癌症免疫治疗,迄今为止,已经开发了相当数量的多肽作为检查点阻断剂和癌症疫苗佐剂。许多临床研究发现基于多肽免疫阻断疗法具有显著调节免疫反应和抑制肿瘤生长的能力,证明了肽基生物材料作为疫苗佐剂增强抗原免疫应答或作为免疫治疗药物传递平台的潜力巨大。
发明内容
本发明的目的是提供一种新型的靶向CD47的多肽及其应用。
为了实现本发明目的,第一方面,本发明提供一种靶向CD47的多肽,所述多肽包含如下的氨基酸序列或由其组成:
i)X1X2X3X4X5X6X7X8X9X10;或
ii)在i)的N端和/或C端连接标签得到的氨基酸序列;
其中,X1为赖氨酸、组氨酸或精氨酸;X2为脯氨酸、色氨酸、酪氨酸或苯丙氨酸;X3为谷氨酸、天冬氨酸、丝氨酸、谷氨酰胺、天冬酰胺或苏氨酸;X4为脯氨酸、色氨酸、酪氨酸或苯丙氨酸;X5为脯氨酸、色氨酸、酪氨酸或苯丙氨酸;X6为谷氨酸、天冬氨酸、丝氨酸、谷氨酰胺、天冬酰胺、苏氨酸或亮氨酸;X7为谷氨酸、天冬氨酸、丝氨酸、谷氨酰胺、天冬酰胺、苏氨酸或亮氨酸;X8为赖氨酸、组氨酸、精氨酸或甘氨酸;X9为赖氨酸、组氨酸、精氨酸或甘氨酸;X10为赖氨酸、组氨酸、精氨酸或甘氨酸。
优选地,本发明靶向CD47的多肽的氨基酸序列为HFEYWEERHK(SEQ ID NO:1)。
第二方面,本发明提供编码所述多肽的核酸分子。
第三方面,本发明提供含有所述核酸分子的生物材料,所述生物材料包括但不限于重组DNA、表达盒、转座子、质粒载体、病毒载体、工程菌或转基因细胞系。
第四方面,本发明提供一种靶向CD47的由所述多肽形成的二价体或多价体。
所述二价体或多价体是多肽单体之间通过连接分子共价连接形成的。其中,所述连接分子可选自四苯基乙烯(TPE)、1-乙基-3-(3-二甲基氨丙基)-碳化二亚胺(EDC)或N-羟基琥珀酰亚胺(NHS)等中的至少一种。
所述二价体或多价体也可以是将多肽单体与多聚体混合,通过非共价连接形成的。其中,所述多聚体可选自聚乙二醇(PEG)、聚乙烯醇(PVA)、环糊精、聚酰胺-胺型树枝状高分子(PAMAM)、聚乳酸(PLA)、聚乳酸-乙醇胺(PLGA)等中的至少一种。
第五方面,本发明提供一种药物组合物,包括能杀伤癌细胞的制剂以及所述多肽或由所述多肽形成的二价体或多价体。
所述能杀伤癌细胞的制剂选自化学药物、生物药物、纳米药物、放射性药物、光热治疗或光动力治疗药物等,以及包裹这些药物的载体中的至少一种。
第六方面,本发明提供一种多肽偶联物,所述多肽偶联物由所述多肽与载体偶联得到;所述载体可选自细胞因子、放射性元素、载体蛋白、抗体、酶、荧光基团、量子点或高吸光系数发色团等中的至少一种。
优选地,所述载体为荧光基团,如异硫氰酸荧光素。
第七方面,本发明提供所述多肽、由所述多肽形成的二价体或多价体、或者所述多肽偶联物的以下任一应用:
1)制备用于癌症免疫治疗的药物或显像剂;
2)制备癌症诊断试剂或试剂盒;
3)用于癌症治疗;
4)用于癌症诊断及预后监测。
前述的应用,所述癌症为CD47蛋白高表达相关的癌症,如急性淋巴细胞白血病、骨髓瘤、膀胱癌、结直肠癌、乳腺癌、肝癌或胰腺癌等。
借由上述技术方案,本发明至少具有下列优点及有益效果:
(一)本发明多肽对CD47阳性肿瘤细胞具有靶向作用,选择性强,且本发明多肽可以采用化学合成的方法制备得到,纯度高,特异性强,无免疫原性,安全可靠。弥补了现有技术中抗体药物制备繁琐,稳定性差等缺点。
(二)本发明的多肽具有靶向CD47阳性肿瘤细胞的特性,因而在实际应用中,可以将本发明的多肽作为靶向多肽,与能杀伤癌细胞的制剂相缀合或混合,用于肿瘤的靶向治疗和成像。本发明的多肽偶联物用于实体瘤中可实现对CD47阳性肿瘤细胞进行高灵敏度监测和靶向治疗。
(三)CD47多肽介导的靶向纳米载体相比抗体用于免疫诊疗具有独特优势。多肽作为内源性小分子,可通过特定的序列设计或者通过共价偶联特定序列形成规整有序的纳米结构。多肽功能化的纳米载体在肿瘤分子诊断和靶向治疗中具有重要应用价值,为结直肠癌等多种肿瘤早期诊断、靶向治疗等提供有力工具,具有广阔的应用前景。
附图说明
图1为本发明CD47靶向多肽微芯片筛选示意图。
图2为本发明较佳实施例中表面等离子共振(SPRi)检测阳性多肽与CD47蛋白的亲合力。其中,1为20μg/mL,2为10μg/mL,3为5μg/mL,4为2.5μg/mL,5为1.25μg/mL,6为0.625μg/mL。
图3为本发明较佳实施例中多肽TR与CD47高表达HT29细胞,CD47低表达HEK-293T细胞,CD47 siRNA转染HT29细胞相互作用图。
图4为本发明较佳实施例中多肽TNTR与CD47高表达CT26细胞,CD47低表达HEK-293T细胞相互作用图。
图5为本发明较佳实施例中多肽TNTR负载量子点与CD47高表达CT26细胞相互作用图。
具体实施方式
本发明提供一种靶向CD47的多肽,特别是一种能分别与乳腺癌、结直肠癌等肿瘤标志物CD47蛋白结合的多肽和由该肽衍生的且能与CD47蛋白结合的产品在制备抗癌药物或显像制剂中的用途。该多肽和荧光基团连接,靶向CD47肿瘤标志物,实现了体内无创成像;此外,该多肽与KLVFF短肽缀合能够自组装成纳米管,包载化疗药物,制备成CD47多肽纳米化疗药物载药纳米系统。
本发明采用如下技术方案:
第一方面,本发明提供一种靶向CD47的多肽,该多肽能与CD47蛋白相结合,所述多肽包括如下氨基酸序列:X1X2X3X4X5X6X7X8X9X10。
其中,X1为赖氨酸、组氨酸或精氨酸;X2为脯氨酸、色氨酸、酪氨酸或苯丙氨酸;X3为谷氨酸、天冬氨酸、丝氨酸、谷氨酰胺、天冬酰胺或苏氨酸;X4为脯氨酸、色氨酸、酪氨酸或苯丙氨酸;X5为脯氨酸、色氨酸、酪氨酸或苯丙氨酸;X6为谷氨酸、天冬氨酸、丝氨酸、谷氨酰胺、天冬酰胺、苏氨酸或亮氨酸;X7为谷氨酸、天冬氨酸、丝氨酸、谷氨酰胺、天冬酰胺、苏氨酸或亮氨酸;X8为赖氨酸、组氨酸、精氨酸或甘氨酸;X9为赖氨酸、组氨酸、精氨酸或甘氨酸;X10为赖氨酸、组氨酸、精氨酸或甘氨酸。
本发明所述的氨基酸残基可以是L-型,也可以是D-型,或者是L-、D-型的混合。
本发明中,采用氨基修饰的TentaGel树脂作为固相载体,利用Fmoc合成策略进行混合均分合成库容量为106的“一珠一物”肽库。利用微流控芯片的方法进行高通量一珠一物肽库筛选,阳性肽珠经MALDI-TOF-MS鉴定,获得了一系列能特异性结合CD47的活性多肽。
作为优选方案,本发明多肽的氨基酸序列为HFEYWEERHK。该多肽对CD47蛋白高特异性亲和。
第二方面,本发明还提供一种二价体或多价体,由本发明第一方面所述多肽组装而成。
本发明中的二价体或多价体具有靶向CD47阳性肿瘤细胞的特性。
作为优选方案,本发明的二价体或多价体是通过连接分子共价连接形成或通过与多聚体混合、非共价连接形成的。
优选地,所述连接分子为四苯基乙烯(TPE)、1-乙基-3-(3-二甲基氨丙基)-碳化二亚胺(EDC)或N-羟基琥珀酰亚胺(NHS)。该连接分子是一种新型无毒、生物相容性良好的交联剂。
可以根据具体需要来选择多聚体,例如可以是聚乙二醇(PEG)、聚乙烯醇(PVA)、环糊精、聚酰胺-胺型树枝状高分子(PAMAM)、聚乳酸(PLA)、聚乳酸-乙醇胺(PLGA)中的任意一种或至少两种的混合。
第三方面,本发明还提供一种药物组合物,包括本发明第一方面所述多肽或本发明第二方面所述二价体或多价体作为靶向多肽,以及能杀伤癌细胞的制剂。
进一步地,所述药物组合物还包括与所述的多肽或所述二价体或多价体相缀合或混合可制备靶向药物的载体。
优选地,所述制剂为能杀伤癌细胞的化学药物、生物药物、纳米药物、放射性药物、光热治疗或光动力治疗药物;或包裹这些药物的载体中的任意一种。
更优选地,所述制剂为烷化剂、抗代谢药物、抗肿瘤天然药物、抗肿瘤抗生素、激素、金属络合物或肿瘤放射靶向标记物中的任意一种。
更优选地,所述载体为纳米材料、脂质体、聚合物中的任意一种。
本发明采用将第一方面所述多肽、第二方面所述二价体或多价体与纳米材料、脂质体等高分子材料缀合,本发明的多肽、二价体或多价体可以使缀合后生成的化合物在机体内更稳定地被运输到靶细胞。
第四方面,本发明还提供一种多肽偶联物,所述多肽偶联物由前述多肽与载体偶联获得;所述载体为药物、细胞因子、放射性元素、载体蛋白、抗体、酶、荧光基团、量子点或高吸光系数发色团。
所述载体可以根据具体的目的进行选择,例如,载体可以为药物或细胞因子等,可将药物或细胞因子靶向癌细胞,也可用放射性元素、载体蛋白、抗体、酶、凝集素、荧光基团、量子点或高吸光系数发色团作为载体。
优选地,所述载体为荧光基团;更优选地,所述荧光基团为异硫氰酸荧光素。
第五方面,本发明还提供另一种药物组合物,所述药物组合物包括本发明第一方面所述多肽或本发明第二方面所述二价体或多价体,以及显像制剂。
优选地,所述多肽、二价体或多价体与显像制剂相缀合或混合。
优选地,所述显像制剂为放射性核素、放射性核素标记物或分子影像制剂中的任意一种。
第六方面,本发明还提供本发明第一方面所述多肽或本发明第二方面所述二价体或多价体在制备用于治疗、预防或诊断癌症的药物或显像制剂中的用途。
作为优选方案,本发明所述癌症为CD47高表达相关的癌症,包括急性淋巴细胞白血病、骨髓瘤、膀胱癌、结直肠癌、乳腺癌、肝癌和胰腺癌。
本发明所述的多肽、二价体或多价体具有靶向CD47蛋白的作用,可以作为载有药物的载体如纳米材料、脂质体等在CD47阳性细胞中的含量,再添加药学上可接受的辅料或佐剂制成新型的更有效的靶向抗癌药物。
本发明多肽和化疗药物联用,制备成多肽纳米药物载药纳米系统,利用CD47多肽增强了药物对肿瘤组织的靶向渗透能力。
以下实施例用于说明本发明,但不用来限制本发明的范围。若未特别指明,实施例中所用的技术手段为本领域技术人员所熟知的常规手段,所用原料均为市售商品。
实施例1靶向CD47的多肽筛选系统的构建和筛选
1、实验仪器与材料
N-甲基吗啉(NMM),哌啶,三氟乙酸(TFA),二氯甲烷(DCM),茚三酮,维生素C,苯酚,四甲基脲六氟磷酸盐(HBTU),六氢吡啶,三异丙基硅烷(TIS),乙二硫醇(EDT),N,N二甲基甲酰胺(DMF),N,N-二异丙基乙胺(DIPEA),5-异硫氰酸荧光素(FITC),无水乙醚,树脂,甲醇,各种Fmoc保护氨基酸,(4,7-双(5-溴-2-噻吩基)-2,1,3-苯并噻二唑)(DBT),MB-Streptavidin(链霉亲和素磁珠),多肽合成管,摇床,真空水泵,旋转蒸发仪,激光共聚焦显微镜(Olympus FV1000-IX81),上述试剂和材料均从商业途径获得。
2、“一珠一物”多肽文库的合成
采用Fmoc固相肽合成方法合成多肽文库,具体方法如下:称取200mg的Tentagel-NH2树脂,按照上述固相多肽合成程序循环,依次加入Lys、His、Arg、Gly依次进行反应,脱保护后进行混合均分合成。
(1)待反应完成后,将树脂均分4份,向每管分别加入Lys、His、Arg、Gly与等量的HBTU进行偶联,待偶联完毕后,将4管树脂脱保护后混合,再将树脂相应均分为若干份,依次循环合成;
(2)分别加入Lys、His、Arg、Gly偶联方法同(1);
(3)分别加入Glu、Asp、Ser、Gln、Asn、Thr、Leu偶联方法同(1);
(4)分别加入Glu、Asp、Ser、Gln、Asn、Thr、Leu偶联方法同(1);
(5)分别加入Pro、Trp、Tyr、Phe偶联方法同(1);
(6)分别加入Pro、Trp、Tyr、Phe偶联方法同(1);
(8)分别加入Glu、Asp、Ser、Gln、Asn、Thr偶联方法同(1);
(9)分别加入Pro、Trp、Tyr、Phe偶联方法同(1);
(11)分别加入Lys、His、Arg、Gly偶联方法同(1);
(12)分别分五步在四个合成管中加入Phe、Phe、Val、Leu、Lys偶联方法同(1),待偶联完毕后,树脂TFA脱保护后混合。经过甲醇置换和收缩步骤,真空抽干,得到加载有肽库的干燥树脂备用。
3、CD47阳性多肽的筛选
(1)取干燥肽库用1×PBS洗3次,加入5%的脱脂牛奶在混旋仪上37℃对肽珠表面封闭2h,再用1×PBS洗3次;
(2)根据生物素标记试剂盒标记CD47蛋白,取生物素(biotin)标记的CD47蛋白与多肽库混合,37℃孵育2h后用1×PBS洗3次;
(3)然后取100μL MB-Streptavidin加入肽库在混旋仪上37℃避光混合孵育2h。将孵育后含多肽库EP管置于磁力架上。阳性多肽受磁力影响吸附于EP管侧壁,而阴性多肽由于重力沉降在EP管底。
图1为筛选CD47靶向多肽原理示意图,当阳性多肽珠与生物素标记的受体蛋白孵育后,阳性肽珠特异性识别蛋白,标记链霉亲和素的磁球通过识别生物素而识别阳性肽珠。阳性肽珠表面将包覆一层磁珠具有磁性从而被磁场捕获。首先,挑选出其中包覆磁珠最多,作用力较强的几条多肽,并按照新的序列重新合成,通过溴化氰裂解,进行二级质谱鉴定,继而运用Mascot数据库解出相应序列信息。按序列重新合成阳性多肽部分标记荧光,MALDI-TOF鉴定和HPLC纯化用于后续试验。经化学合成制得本发明多肽命名为TR,其氨基酸序列为HFEYWEERHK。
实施例2利用表面等离子共振(SPRi)方法检测CD47阳性多肽与CD47蛋白的亲和作用
分别将1mg/mL的多肽TR及1×PBS点到芯片上,在4℃湿润条件下孵育过夜,然后用10×PBS清洗10min,再用1×PBS清洗10min,最后用去离子水清洗2次,每次10min,浸入含5%牛奶的1×PBS中,4℃条件下孵育过夜,然后用10×PBS清洗10min,1×PBS清洗10min,最后用去离子水清洗2次,每次10min,用氮气吹干,装芯片上机(Plexera HT表面等离子共振成像系统)。
流动相依次通过1×PBS、2×PBS、0.625μg/mL、1.25μg/mL、2.5μg/mL、5μg/mL、10μg/mL和20μg/mL的CD47纯化蛋白,记录分析SPRi信号。
由图2可以看出,TR的SPRi信号随着蛋白浓度的增加逐渐增强,说明本发明的CD47阳性多肽能够强结合CD47,亲和解离常数达3.18×10-7M。此外,我们根据筛选出来结果合成了另一条阳性肽(HFEYWTNEYR),测定其亲和解离常数为2.07×10-5M,通过综合比较,将TR作为靶向CD47的多肽用于肿瘤相关的靶向治疗应用潜力巨大。
实施例3TR分别与高表达CD47的HT29细胞和低表达CD47的HEK-293T细胞相互作用
CD47阳性人结直肠癌细胞系HT29(CD47高表达)和CD47阴性人肾上皮细胞系293T(CD47低表达)分别用含10%胎牛血清及1%青霉素和链霉素的RPMI 1640/High glucose培养基和DMEM/High glucose培养基在37℃恒温培养箱(5%CO2)中培养。
以1×105/mL的细胞浓度植入圆形玻底培养皿(35mm),37℃,5%CO2细胞培养箱中培养24h后,弃去培养液,两种细胞中分别加入含1μmol/L Hoechst 33342,4℃避光孵育8min后,用预冷1×PBS洗涤2次,分别加入50μM的FITC标记多肽TR,4℃避光孵育10min后,用预冷1×PBS洗涤2次。用激光扫描共聚焦显微镜(Olympus FV1000-IX81)检测细胞中的荧光分布。
结果如图3中a和b所示,加入TR的HT29的细胞膜上观察到明显的绿色荧光,而293T细胞几乎没有荧光。结果表明TR多肽能够结合在CD47阳性细胞的细胞膜上,说明TR多肽与阳性细胞系的识别具有专一性,而且特异性与靶标蛋白的表达量呈正相关,可以作为靶向分子用于相关的诊断和检测。由此进一步说明本发明实施例1制得的TR多肽能特异性靶向CD47。
实施例4CD47基因敲除实验
在含10%胎牛血清及1%青霉素和链霉素的RPMI 1640/High glucose培养基中培养HT29细胞,过夜培养24h。然后将培养基换成含10%胎牛血清的RPMI 1640/High glucose培养基(不含抗生素)。用50μL的Opti-MEM培养液稀释2.5μMCD47 siRNA,然后在室温下静置5分钟。用50μL Opti-MEM稀释2.0μL LipofectamineTM2000,并将混合物在室温下静置5分钟。混合转染试剂和siRNA稀释液,并在室温下静置20分钟。随后,立即将转染复合物添加到含有HT29细胞的培养皿中,在37℃含5%CO2的细胞培养箱中转染6h。然后,丢弃含有转染试剂的培养基,并添加胰蛋白酶消化细胞。然后用含10%胎牛血清及1%青霉素和链霉素的1640/High glucose培养基培养经siRNA转染的HT29细胞。
实施例5TR多肽与经过CD47 siRNA转染的低表达CD47的HT29细胞相互作用
CD47 siRNA转染的HT29细胞用含10%胎牛血清及1%青霉素和链霉素的1640/High glucose培养基在37℃恒温培养箱(5%CO2)中培养。
以1×105/mL的细胞浓度植入圆形玻底培养皿(35mm),37℃,5%CO2细胞培养箱中培养24h后,弃掉培养液,两种细胞中分别加入含1μmol/L Hoechst 33342,4℃避光孵育8min后,用预冷1×PBS洗涤2次,分别加入50μM的FITC标记多肽TR,4℃避光孵育10min后,用预冷1×PBS洗涤2次。用激光扫描共聚焦显微镜(Olympus FV1000-IX81)检测细胞中的荧光分布。
结果如图3中c所示,CD47 siRNA转染HT29细胞的细胞膜上荧光明显减弱。该结果表明TR是特异性地靶向CD47。
实施例6TR多价体的制备
多肽自组装的纳米材料通过分子组装形成的规则结构能进一步赋予多肽作为药物载体和成像剂的功能,因此选择性地在其氮端与KLVFF短肽连接,并连接TPE荧光基团来指示组装行为(TPE具有聚集诱导发光(AIE)性质,并且当分子发生聚集时就会发光)。称量200mg的Tentagel-NH2树脂,参照实施例1中固相多肽合成的程序步骤,按照设计依次加入Lys、His、Arg、Glu、Glu、Trp、Tyr、Glu、Phe、His、Phe、Phe、Val、Leu、Lys合成,之后将TPE和HBTU加入到合成管中进行偶联。偶联反应结束后,脱保护,清洗,透析纯化,然后冻干。通过MALDI-TOF-MS分析产物(TNTR)。然后将纯化产品溶解在水溶液中并静置48h,以备后续反应使用。
实施例7TR多肽荧光偶联物的制备
将TR多肽(0.05umol)和0.1umol的5-异硫氰酸荧光素(FITC)放入棕色反应瓶中,加入200μL N,N二甲基甲酰胺(DMF)溶解上述固体,并用1.0N的N,N-二异丙基乙胺(DIPEA)溶液将体系pH调至10。在室温下用磁力搅拌器搅拌避光反应16小时后,高效液相分离反应液,纯化得到荧光分子偶联的多肽荧光偶联物用于后续实验。
实施例8TNTR分别与高表达CD47的CT26细胞和低表达CD47的HEK-293T细胞相互作用
CD47阳性鼠结直肠癌细胞系CT26(CD47高表达)和CD47阴性人肾上皮细胞系HEK-293T(CD47低表达)分别用含10%胎牛血清及1%青霉素和链霉素的RPMI1640/Highglucose培养基和DMEM/High glucose培养基在37℃恒温培养箱(5%CO2)中培养。
以1×105/mL的细胞浓度植入圆形玻底培养皿(35mm),37℃,5%CO2细胞培养箱中培养24h后,弃去培养液,两种细胞中分别加入含5μmol/L DRAQ 5,4℃避光孵育8min后,用预冷1×PBS洗涤2次,分别加入50μM的实施例6制备的多肽多价体TNTR,4℃避光孵育10min后,用激光扫描共聚焦显微镜(Olympus FV1000-IX81)检测细胞中的荧光分布。
结果如图4所示,加入TNTR的CT26的细胞膜上观察到明显的蓝色荧光,而293T细胞膜上几乎没有荧光。结果表明TNTR多肽依然能够有效结合在CD47阳性细胞的细胞膜上,说明经过组装的多肽与阳性细胞系的识别仍具有专一性,可以作为靶向分子用于CD47相关的诊断和检测,进一步表明本发明的TR多肽能特异性靶向CD47。
实施例9TNTR多肽负载量子点实验
CD47阳性鼠源结直肠癌细胞系CT26(CD47高表达)用含10%胎牛血清及1%青霉素和链霉素的RPMI 1640/High glucose培养基在37℃恒温培养箱(5%CO2)中培养。
以1×105个/mL的细胞浓度植入圆形玻底培养皿(35mm),37℃,5%CO2细胞培养箱中培养24h后,弃去培养液,CT26细胞中分别加入含1μmol/L Hoechst 33342,4℃避光孵育8min后,用预冷1×PBS洗涤2次,加入50μM的实施例6制备的多肽多价体TNTR包裹的Ag2S量子点溶液和纯Ag2S量子点溶液,4℃避光孵育10min后,用预冷1×PBS洗涤2次。用激光扫描共聚焦显微镜(Olympus FV1000-IX81)检测细胞中的荧光分布。
结果如图5中a和b所示,加入TNTR的CT26的细胞膜上观察到明显的红色荧光,而Ag2S量子点孵育的CT26细胞膜上几乎没有荧光。结果表明TNTR多肽能够有效包裹量子点,可以作为靶向成像分子用于相关的诊断。由此进一步说明本发明实施例6中制备的TR多肽多价体具有潜在成像治疗性能。
从以上实验结果可以得出,本发明的多肽TR具有靶向表达CD47阳性肿瘤细胞的特性,在临床上对相关肿瘤具有诊断和治疗的应用价值。在实际应用中,可以将本发明的多肽作为靶向肽,与能杀伤癌细胞的制剂相缀合或混合,用于肿瘤的成像、靶向治疗和免疫治疗反应标志物的检测。
由TR多肽构成的纳米组装体能实现药物的定点释放,提高药物治疗效果,同时还能降低肿瘤细胞的耐药性;利用微流控芯片系统筛选出来的靶向CD47的高亲和多肽,能有效阻断CD47和SIRPα的通路,激活免疫细胞的免疫功能,诱导免疫细胞吞噬肿瘤细胞,最终实现杀伤肿瘤细胞的目的。
多肽KRFYVVMWKK为TSP配体分子的末端结构域,其亲和解离常数为19μM,结果表明该条多肽与受体CD47的亲和力较弱。本发明多肽TR的亲和解离常数是通过等离子体共振技术测定的,测定结果显示多肽TR与CD47的结合力更高,且多肽TR可以根据实际需求进行不同的修饰,实现与受体的多价识别与结合。此外,TR多肽可以根据需求连接其他组装单元、荧光单元和靶向单元,构建形貌可控的纳米组装体,实现肿瘤高效靶向治疗和精准成像。
虽然,上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述,但在本发明基础上,可以对之做一些修改或改进,这对本领域技术人员而言是显而易见的。因此,在不偏离本发明精神的基础上所做的这些修改或改进,均属于本发明要求保护的范围。
序列表
<110> 北京理工大学
<120> 靶向CD47的多肽及其应用
<130> KHP211115809.8
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 10
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
His Phe Glu Tyr Trp Glu Glu Arg His Lys
1 5 10
Claims (7)
1.靶向CD47的多肽,其特征在于,其氨基酸序列为HFEYWEERHK。
2.含有编码权利要求1所述多肽的核酸分子的生物材料,所述生物材料为重组DNA、表达盒、转座子、质粒载体、病毒载体、工程菌或转基因细胞系。
3.多肽偶联物,其特征在于,所述多肽偶联物由权利要求1所述多肽与载体偶联得到;所述载体选自细胞因子、放射性元素、载体蛋白、抗体、酶、荧光基团、量子点或高吸光系数发色团中的至少一种。
4.根据权利要求3所述的多肽偶联物,其特征在于,所述载体为荧光基团。
5.根据权利要求4所述的多肽偶联物,其特征在于,所述荧光基团为异硫氰酸荧光素。
6.权利要求1所述多肽或者权利要求3-5任一项所述多肽偶联物的以下任一应用:
1)制备用于癌症免疫治疗的药物或显像剂;
2)制备癌症诊断试剂或试剂盒;
所述癌症为CD47蛋白高表达相关的癌症。
7.根据权利要求6所述的应用,其特征在于,所述癌症为急性淋巴细胞白血病、骨髓瘤、膀胱癌、结直肠癌、乳腺癌、肝癌或胰腺癌。
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