CN114478800A - 基于血清白蛋白的融合蛋白、纳米组装体及其制备方法和应用 - Google Patents
基于血清白蛋白的融合蛋白、纳米组装体及其制备方法和应用 Download PDFInfo
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Classifications
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Abstract
本发明涉及一种融合蛋白,包括具有疏水区域的蛋白、肽接头、蛋白融合受体,所述肽接头将具有疏水区域的蛋白和蛋白融合受体连接而成;所述蛋白融合受体为特异性识别抗体Fc段的Fc受体片段,所述具有疏水区域的蛋白为血清白蛋白。本发明还涉及到由上述融合蛋白与疏水性可降解聚酯及其衍生物构成的纳米组装体。本发明还公开了上述具有优异的稳定性的纳米组装体在递送抗体平台中的应用。本发明首次将这种构建得到的纳米组装体平台创造性地应用于为肿瘤或者是自身免疫疾病、或者炎症制备免疫治疗药物或治疗药物中。
Description
技术领域
本发明涉及医药技术领域,具体是涉及基于血清白蛋白的融合蛋白、纳米组装体及其制备方法与应用。
背景技术
CTLA-4、PD-1、PD-L1等免疫检查点阻断抗体相继被批准用于多种类型肿瘤的治疗,取得了阶段性的成果,但随着研究的广泛深入,大量临床实验结果证明免疫检查点阻断等免疫疗法在不同类型肿瘤以及不同患者的同类肿瘤中的治疗效果差异很大,临床应答率总体偏低。许多单抗药物在临床应用中屡遭失败,亟需发展提高抗体药物抗肿瘤效果的新策略。
抗体联合给药或通过基因工程制备双/多特异性抗体被用于克服单克隆抗体药物效价不足的问题。目前研究人员已经开发出超过100种双特异性抗体建造模式,并且已有超过85个双特异性抗体处于临床开发阶段。虽然双特异性/多特异性抗体能够通过双重或者多重识别大幅提高抗体的效价和疾病治疗效果,但是其结构设计复杂度高,设计、制备、纯化等过程的复杂性相较于单克隆抗体大幅增加,而且其大多通过化学偶联和DNA重组技术制备获得,需要对产生效应的单抗进行化学修饰,不可避免的会影响抗体本身的抗原结合能力,还同时存在半衰期短、给药方式复杂、稳定性差、溶解性差、成本高等缺点,目前尚无双特异性/多特异性抗体被批准用于实体瘤的治疗。因此,如果能够利用双特异性/多特异性抗体的设计理念,开发新的简便策略,实现单克隆抗体的“多价化”、“多特异性化”和“多功能化”,有望大幅提高单克隆抗体的临床疗效,将更多的开发中或者已经临床的单克隆抗体应用到实体瘤的治疗中。
将多种单克隆抗体固定在纳米载体表面可以模拟双特异性/多特异性抗体的功能,实现单克隆抗体的“多价化”、“多特异性化”和“多功能化”。例如,美国约翰斯·霍普金斯大学Jonathan P.Schneck教授课题组将阻断型PD-L1单抗和激活型4-1BB单抗同时键合在右旋糖酐铁颗粒表面构建了具有“双靶向”功能的纳米粒,该纳米粒在阻断PD-L1/PD-1抑制性信号通路的同时,还能够激活4-1BBL/4-1BB通路,经瘤内给药后显著增强细胞毒性T细胞杀伤肿瘤细胞的能力。将多种单克隆抗体接在纳米载体表面是一种极具潜力的提高抗体疗效的策略。然而,已报道的固定抗体的方式主要是利用抗体药物分子上的氨基、羧基、巯基等基团将其键合到颗粒表面,这些方法存在诸多问题。首先,抗体和纳米颗粒的高分子量往往导致二者之间的反应效率较低,质量控制较难;其次,利用还原产生的巯基基团或者抗体表面丰富的氨基基团与颗粒进行反应,不仅反应和纯化过程复杂,还会破坏抗体的高级结构或封闭治疗型抗体的抗原识别区,显著降低抗体识别抗原的能力;再次,目前报道的抗体递送的载体多为聚苯乙烯纳米颗粒、四氧化三铁纳米颗粒等,生物相容性较差,这些都极大地阻碍了基于载体系统的“纳米抗体”的临床转化。
构筑具有临床转化前景的抗体递送载体,发展便捷、高效、可控的抗体药物结合方式,解决现有纳米载体固定抗体方式反应效率低、过程复杂的问题,实现抗体药物的“多价化”、“多特异性化”和“多功能化”,有望显著提高现有单克隆药物的抗肿瘤效果。
巨噬细胞等单核细胞表面存在多种Fc受体,其中FcγRI能够特异性和高亲和力地识别和结合抗体的Fc片段,利用FcγRI与单克隆抗体药物结合不涉及复杂的化学反应,对抗体药物的结构和功能几乎没有影响。
人血清白蛋白是一具有585个氨基酸的蛋白质,它是血清中维持渗透压的重要组成部分,起着运输内源和外源物质的载体功能。我们关注到白蛋白具有7个长链脂肪酸结合位点,且结合位点相对开放。其疏水性空腔通过精氨酸或赖氨酸残基,与酪氨酸或丝氨酸一起,以氢键和静电作用结合脂质的羧酸部分。于是,我们在前期的抗体递送平台的研发基础上,再次创新性地提出将FcγRI与白蛋白融合成重组蛋白,然后利用白蛋白与疏水性聚乳酸高分子材料构件成纳米颗粒,存在于颗粒表面的FcγRI识别和结合治疗性单克隆抗体药物,构建出新型双/多特异性抗体用于肿瘤、免疫相关疾病等治疗。
发明内容
基于此,本发明的目的之一在于提供一种融合蛋白,该融合蛋白可以用于至少一种抗体的递送。
包括以下技术方案:
一种用于递送至少一种抗体的融合蛋白,包括血清白蛋白和蛋白质受体,所述血清白蛋白与蛋白质受体直接或通过肽接头连接;所述蛋白质受体为Fc受体。
本发明的第二目的是提供一种用于递送至少一种抗体的纳米组装体。
一种用于递送至少一种抗体的纳米组装体,所述纳米组装体由上述融合蛋白与疏水性可降解聚酯或其衍生物通过疏水相互作用结合构成。
本发明的第三目的是提供一种上述的纳米组装体的制备方法,包括以下步骤:
(1)将所述融合蛋白与水或水溶液混合,得水相;将所述疏水性可降解聚酯及其衍生物与有机溶剂混合,得油相;
(2)将步骤(1)所述水相和油相制备成水包油的乳剂;
(3)将所述乳剂分离纯化,得纳米组装体。
本发明的第四目的是提供一种上述的纳米组装体在制备抗体递送的平台或系统中的应用。
本发明的第五目的是提供一种抗体递送平台或系统,包括上述的纳米组装体,以及至少一种所需递送的抗体。
本发明的第六目的是提供一种上述的纳米组装体作为免疫治疗药物的应用。
本发明的第七目的是提供上述融合蛋白上述的纳米组装体中的应用。
与现有技术相比,本发明具有以下有益效果:
本发明在基于前期大量的研发下,通过选择疏水性可降解聚酯或其衍生物和特定的具有疏水结构域的蛋白的融合蛋白制备用于递送至少一种单克隆抗体的纳米颗粒(组装体),疏水性可降解聚酯或其衍生物通过疏水相互作用与融合蛋白的疏水结构域进行缠绕组装,具有优异的稳定性。纳米组装体的蛋白-Fc受体融合蛋白所递送的特异性抗体仅仅通过简单的物理混合就可快捷、高效、可控地结合一种或多种类型治疗性单克隆抗体,可以在体内长循环过程中保持完整结构,从而简便地实现了抗体的“多价化”和“多特异性化”,使长期研发的这种多抗体递送系统具有临床应用的可能。本发明仅仅通过这种基于白蛋白的纳米颗粒与多种抗体物理混合的多抗体递送系统制备方法简单,且能在这递送系统或者平台下,多抗体的活性不受影响,并有效增强对肿瘤细胞的杀伤效果。
本发明首次将这种构建得到的纳米组装体平台创造性地应用于为肿瘤或者是自身免疫疾病、或者炎症制备免疫治疗药物或治疗药物中,将具有广阔的应用前景。
附图说明
图1为pPICZαA-mFcγRI-MSA质粒的构建过程。
图2为PCR鉴定目的基因-酵母菌载体
图3为酵母重组子的PCR鉴定。
图4为pcDNA3.1(+)-hFcγRI-HSA的质粒图谱。
图5为纯化的mFcγRI-MSA的SDS-PAGE和Western Blot分析分析。
图6为hFcγRI-HSA的Western Blot分析。
图7为纳米适配子制备示意图。
图8为5mg/mL浓度的纳米适配子NPmFcγRI-MSA的粒径。
图9为纳米适配子NPmFcγRI-MSA的扫描电子显微镜图片。
图10为纳米适配子NPmFcγRI-MSA的血清稳定性图片。
图11为ELISA测定NPmFcγRI-MSA结合效率图。
图12为随时间变化纳米适配子结合治疗性单克隆抗体的效率。
图13为体外刺激B16-F10黑色素瘤细胞和CD8+T细胞PD-L1、PD-1表达情况。
图14为NPmFcγRI-MSA@αPD-1+αPD-L1与B16-F10黑色素瘤细胞结合情况(A)胞外荧光强度随时间变化曲线;B)B16-F10细胞与imNAαPD-1&αPD-L1结合的CLSM图像,比例尺为5μm;C)台盼蓝淬灭前后荧光强度随时间变化的流式直方图:台盼蓝能够淬灭胞外荧光,故经淬灭后流式细胞术能检测到的荧光被认为是胞内荧光。FITC荧光标记在NP上)。
图15为NPmFcγRI-MSA@αPD-1+αPD-L1与CD8+T细胞结合情况。
图16为激光共聚焦观察双特异性纳米适配子介导下肿瘤细胞与CD8+T细胞的相互作用。
图17荧光素酶法实验测定B16-F10-luc黑色素瘤细胞活力。
图18为双特异性纳米抗体抑制原位乳腺癌生长的曲线图。
图19为双特异性纳米抗体治疗后小鼠的体重变化图。
图20为三特异性抗体纳米适配子抑制原位乳腺癌生长的曲线图。
图21为三特异性抗体纳米适配子抑制原位乳腺癌生长的生存曲线。
具体实施方式
本发明下列实施例中未注明具体条件的实验方法,通常按照常规条件,或按照制造厂商所建议的条件。实施例中所用到的各种常用化学试剂,均为市售产品。
除非另有定义,本发明所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本发明的说明书中所使用的术语只是为了描述具体的实施例的目的,不用于限制本发明。
本发明的术语“包括”和“具有”以及它们任何变形,意图在于覆盖不排他的包含。例如包含了一系列步骤的过程、方法、装置、产品或设备没有限定于已列出的步骤或模块,而是可选地还包括没有列出的步骤,或可选地还包括对于这些过程、方法、产品或设备固有的其它步骤。
在本发明中提及的“多个”是指两个或两个以上。“和/或”,描述关联对象的关联关系,表示可以存在三种关系,例如,A和/或B,可以表示:单独存在A,同时存在A和B,单独存在B这三种情况。字符“/”一般表示前后关联对象是一种“或”的关系。
同源:在生物学种系发生理论中,若两个或多个结构具有相同的祖先,则称它们同源(Homology)。
抗体亲和力(affinity of antibodies)指抗体的抗原结合簇同抗原的抗原决定簇的结合强度,或者是指抗体与抗原表位或抗原决定簇之间的结合力,本质是一种非共价作用力,包含了对氨基酸之间的吸引力,氢键、疏水性作用力等。
本发明一实施方式中涉及一种融合蛋白,包括具有疏水区域的蛋白、肽接头、蛋白质受体;蛋白融合受体包括Fc受体。
Fc受体是与抗体(IgG)的Fc片段结合的受体,包括FcγRI、FcγRII和FcγRIII,本发明所述Fc受体为特异性结合所递送的抗体的Fc段的受体,优选为FcγRI。进一步Fc受体与所递送的抗体具有相同或相似种属来源,优选mFcγRI(鼠FcγRI)或hFcγRI(人FcγRI)。
在其中一些实施例中,蛋白质受体包括抗体的Fc受体,所述抗体的Fc受体包括但不限于:Fcγ受体(FcγR),例如小鼠Fc受体mFcγRI,人Fc受体hFcγRI。
在其中一些实施例中,本发明的FcγRI为天然蛋白质的胞外段。
FcγRI与所递送的单克隆抗体的Fc结构域非共价结合;所递送的特异性抗体与所述融合蛋白具有高度同源性。所递送的抗体与所述融合蛋白具有亲和力。
在其中一些实施例中,所述蛋白至少具有Fc受体和血清白蛋白片段,其能与疏水性可降解及其衍生物通过疏水相互作用结合,在本发明中,其为白蛋白,即血清白蛋白,可以是来自人血清白蛋白、牛血清白蛋白、小鼠血清白蛋白、小鼠血清白蛋白、大鼠血清白蛋白、兔血清白蛋白、鸡卵清白蛋白的至少一种。
所述血清白蛋白与所述Fc受体同源。
在其中一些优选的实施例中,所述融合蛋白包含白蛋白和Fc受体蛋白的全长或部分片段,或上述经取代、缺失、突变和/或添加一个或多个天然存在的、非天然存在的或修饰的氨基酸的蛋白,但相应功能或在递送抗体系统中所起的作用不丧失。在其中的一些实施例中,所述融合蛋白中,是由小鼠血清白蛋白MSA与小鼠Fc受体组成,或由人血清白蛋白HSA与人Fc受体组成;所述小鼠血清白蛋白MSA的序列为GENEBANK BC049971.1序列,去掉信号肽序列与终止密码子,如SEQ ID No.1所示,小鼠Fc受体mFcγRI的序列为GENEBANK NM_010186.5,去掉信号肽、跨膜区域与胞内段序列,如SEQ ID No.2所示,人血清白蛋白HSA的序列为GENEBANK HQ537426.1序列,去掉信号肽序列与终止密码子,如SEQ ID No.3所示,人Fc受体hFcγRI的序列为GENEBANK BC152383.1,去掉信号肽、跨膜区域与胞内段序列,如SEQ ID No.4所示。
肽接头可以是常规用于连接多肽的接头序列,其能够连接两个多肽并将其自然折叠成所期望结构,通常其是具有一段有疏水性和一定伸展性的短肽,在本发明中的目的是可将融合的两种蛋白分开,以缓解二者相互干扰作用。所述肽接头可以是柔性的。在某些实施方案中,柔性的肽接头可能是有利的,其能够连接两种蛋白/多肽成分,并且保持其各自的活性和功能。此类肽接头包括但不限于,(GGGGS)n。在其中一些实施例中,该肽接头使用[GlyGlyGlyGlySer]n,n为0-4的整数,更优选为1,2,3,4。当n为零时,即意味着所述融合蛋白可以由所述血清白蛋白与蛋白质受体直接连接而成。
在其中一些实施例中,所述融合蛋白从N端到C端依次为血清白蛋白、肽接头和蛋白质受体。
本发明的一些实施例中,涉及到所述融合蛋白的制备方法,包括以下步骤实现:(a)构建重组毕赤酵母细胞系;(b)融合蛋白在其生长培养基中经4天诱导表达,表达量达30mg/L;(c)纯化步骤(b)表达的蛋白质。
编码各种具有疏水结构域的蛋白,例如血清白蛋白的多聚核苷酸和编码FcγRI的多聚核苷酸可以用本领域周知的方法,如PCR、RT-PCR方法、人工合成的方法和构建筛选cDNA文库的方法等获得,用作PCR模板和用于构建cDNA文库的mRNA或cDNA可以来源于任何含有相应mRNA或cDNA的组织、细胞、及文库等,如从人肝胎cDNA文库获得。也可以用人工合成的方法获得,人工合成时可选用宿主偏爱的密码子,这样往往可以提高产物的表达。编码IL1ra的多聚核苷酸可以用RT-PCR的方法从人肝胎cDNA文库中获得。编码血清白蛋白的多聚核苷酸和编码FcγRI的多聚核苷酸的融合,在保持各自阅读框架不变的前提下,可以用本领域周知的各种方法,如通过PCR的方法,在编码序列的两侧引入限制性内切酶识别位点,通过酶切产生粘性末端,再将粘性末端用DNA连接酶连接,从而获得编码融合蛋白的基因;也可以通过overlap PCR的方法获得融合基因片段。如果需要可在本发明的编码融合蛋白基因的两侧引入多聚核苷酸,引入的多聚核苷酸可有限制性内切酶识别位点。可用本领域公知的方法将含编码融合蛋白序列的核酸克隆到各种表达载体中去。表达融合蛋白的宿主可以是酵母菌、哺乳动物细胞、细菌、动物、植物等。融合蛋白或多肽可以存在于宿主细胞内,也可以是从宿主中分泌出来,优选的是从宿主中分泌出来。分泌所用的信号肽,优选的是酵母α-factor信号肽或天然的血清白蛋白的信号肽,或这两种信号肽的类似物。更优选的用酵母α-factor信号肽,用该信号肽时融合蛋白表达水平较高。融合蛋白或多肽也可以不用信号肽,而在酵母中以胞内可溶形式表达。编码融合蛋白的核酸,可以插入至宿主染色体,或以游离质粒形式存在。
转化所需核酸至宿主细胞中去可用通常的方法,如:电穿孔、制备感受态的原生质球等。成功转化的细胞,即含有本发明DNA构建体的细胞,可通过人们熟知的技术加以鉴定,如细胞经收集并裂解,提取基因组,然后PCR方法鉴定,或者,细胞培养上清中或细胞破碎液中的蛋白可用抗血清白蛋白或抗的抗体检测。
可以通过培养含有本发明DNA构建体的宿主,如重组酵母、重组哺乳动物细胞、重组细菌、转基因动植物等,生产本发明的融合蛋白。具体的培养方法可以用摇瓶或生物反应器等,生产时优选为生物反应器。培养基应能提供菌体(或细胞)生长和产物表达所需的物质,应包含氮源、碳源、pH缓冲成分等,培养基配方一般应根据不同培养对象,通过试验获得。培养可分两个阶段,第一阶段主要用于菌体(或细胞)的生长,第二阶段主要用于表达产物。
通过离心收集细胞培养基,切向流装置浓缩培养基体积后,可以用各种蛋白分离的方法自含有本发明DNA构建体的细胞培养物中分离、纯化融合蛋白。如超滤、液相层析等技术及这些技术的组合。其中液相层析可以用凝胶排阻、亲和、离子交换、疏水、反相等层析技术。
本发明的一些实施例中,涉及到一种用于抗体递送的纳米组装体,所述纳米组装体由上述融合蛋白与疏水性可降解聚酯及其衍生物通过疏水相互作用结合构成。
所述疏水性可降解聚酯及其衍生物可是目前已知的可降解的生物材料,也包括将来进一步研发产生的新的可降解的生物材料,其能与上述融合蛋白中的蛋白部分的疏水区域结合。所述聚酯为脂肪族聚酯或其衍生物,或聚乙二醇修饰的脂肪族聚酯或其衍生物。
在其中一些实施例中,所述脂肪族聚酯为聚丙交酯、聚乙交酯、聚(乙交酯-co-丙交酯)和聚己内酯中的至少一种;或所述聚乙二醇修饰的脂肪族聚酯为聚乙二醇修饰的聚丙交酯、聚乙二醇修饰的聚乙交酯、聚乙二醇修饰的聚(乙交酯-co-丙交酯)和聚乙二醇修饰的聚己内酯中的至少一种。
在其中一些实施例中,所述脂肪族聚酯为聚丙交酯;所述聚丙交酯为左旋聚丙交酯、右旋聚丙交酯或外消旋聚丙交酯;所述聚丙交酯的端基为酯基、羧基和羟基中的至少一种。优选聚丙交酯的端基为酯基,其具有更强的疏水性。
在其中一些实施例中,所述聚丙交酯为左旋聚丙交酯,所述左旋聚丙交酯的端基为酯基。
在其中一些实施例中,所述左旋聚丙交酯的分子量范围为7200~1100000道尔顿,更进一步优选为137000~240000道尔顿。
在其中一些实施例中,纳米组装体为纳米颗粒,其粒径范围为80~200nm,优选范围为80~150nm。
本发明一些实施例中,涉及到一种上述的纳米组装体的制备方法,包括以下步骤:
(1)将所述融合蛋白与水或水溶液混合,得水相;将所述疏水性可降解聚酯及其衍生物与有机溶剂混合,得油相;
(2)将步骤(1)所述水相和油相制备成水包油的乳剂;
(3)将所述乳剂分离纯化,得纳米组装体。
本实施方式提供一种用于调控免疫反应的纳米适配子,由聚酯和具有疏水结构域的融合蛋白,所述融合蛋白的疏水结构域与所述聚酯通过疏水相互作用结合;所述融合蛋白为白蛋白-Fc受体中的至少一种。
其中,所述FcγRI能够与所递送的特异性抗体的Fc结构域非共价结合;所递送的特异性抗体与所述抗Fc段抗体或抗Fc段抗体片段具有相同的种属来源。
本发明所递送的特异性抗体与所述FcγRI具有相同的种属来源,如所递送的特异性抗体选择人源化抗PD-1抗体时,FcγRI选择人源FcγRI。
在其中一些实施例中,上述纳米颗粒的制备过程中不含额外的稳定剂。
在其中一些实施例中,纳米颗粒可以通过离心、切向流透析(通过切向流装置在切向剪切力的作用下透析)和排除色谱(根据纳米颗粒和游离蛋白的分子量大小)中的至少一种方法分离游离的蛋白和纳米颗粒。
在其中一些实施例中,将所述水相和油相制备成水包油的乳剂的方法包括超声乳化或高压均质乳化或微流控。
在其中一些实施例中,所述聚酯或其溶液与融合蛋白的重量比为1:0.1~1:30,优选为1:5~25,优选为1:5~15,更优选为1:7~11。
所述融合蛋白在水相中的浓度为0.5~20mg/mL,优选为5~10mg/ml;所述聚酯在油相中的浓度为0.5~10mg/mL,优选范围为1~5mg/mL。
优选地,所述水相与油相的体积比为1:1~10:1,优选为5-10:1,更优选为8:1~10:1。
在其中一些实施例中,所述有机溶剂是氯仿或二氯甲烷或者同类化合物。
本发明一实施方式中,涉及一种上述的纳米组装体在制备抗体递送的平台或系统中的应用。
本发明一实施方式中,抗体递送平台或系统,包括上述的纳米组装体,以及抗体。
在其中一些实施例中,所递送的抗体是至少一种,优选两种,或者三种,所述至少一种抗体包括有至少一种单克隆抗体,或特异性抗体或其抗原结合部分,优选包括两种或以上的单克隆抗体、多价抗体、人源化抗体、嵌合抗体、基因工程改造抗体。
至少一种抗体的的递送量,可以相同,也可以不同,例如可以为1-10:1-10,优选为1-5:1-5。
所述至少一种单克隆抗体是PD-1和PDL1。优选为,所述PD-1和PD-L1的量1-10:1-10,优选为1-5:1-5。
本发明一实施方式中,一种上述的纳米组装体作为免疫治疗药物的应用。
在其中一些实施例中,所述免疫治疗药物为肿瘤免疫治疗药物或自身免疫疾病治疗药物。
所述免疫治疗药物为肿瘤免疫治疗药物或自身免疫疾病治疗药物。
在其中一些实施例中,本发明所述纳米组装体可由经FDA批准的高分子聚酯和白蛋白融合蛋白组装而成,具有优异的生物相容性。
本发明所述融合蛋白的蛋白-Fc受体融合蛋白是通过受体-配体特异性识别的方式结合抗体,发明人发现,这种结构不会破坏抗体的结构,抗体之间也不会相互影响,克服了传统化学键合固定方式会破坏抗体药物的结构、封闭其抗体识别区、显著影响抗体药物功能、复杂度高、难度高等缺陷,为联合抗体治疗的发展提供了一种全新思路的简便结构设计。
此外,本发明的纳米组装体还能使得抗体的Fab段朝外暴露,从而可以最大程度地保留抗体的功能。
本发明一些实施例中,经大量体内外药理试验证明,纳米组装体与特异性抗体结合所得单克隆抗体递送系统NPmFcγR1@αPD-1+αPD-L1相比于游离单克隆抗体联合治疗具有显著的优越性,能够明显促进效-靶细胞的相互作用,增强T细胞所介导的抗肿瘤能力。
本发明一些实施例中,通过NPmFcγRI-MSA高效结合单克隆抗体,形成的双层抗体的纳米颗粒具备多价态、多特异性、多功能性的特点,并且可以快速进行不同治疗抗体的联合,以适应目前临床上精准治疗下个性化治疗方案的策略,具有巨大的临床应用潜能。
PD-1(程序性死亡受体1),也称为CD279(分化簇279),是一类重要的免疫抑制分子。通过向下调节免疫系统对人体细胞的反应,以及通过抑制T细胞炎症活动来调节免疫系统并促进自身耐受。这可以预防自身免疫性疾病,但它也可以防止免疫系统杀死癌细胞。
本发明中,包括各种已公开的PD-1抗体、PD-L1抗体以及任何在PD-1抗体、PD-L1抗体上做出改进的各种PD-1抗体或PD-L1抗体。
聚乳酸,又称聚丙交酯,polylactide,;polylactic acid,(C3H4O2)n是以乳酸为主要原料聚合得到的聚酯类聚合物,是一种新型的生物降解材料。
以下结合具体实施例对本发明作进一步详细的说明。
以下实施例中所用到的相关序列。
SEQ ID No.1
MSA
aggggtgtgtttcgccgagaagcacacaagagtgagatcgcccatcggtataatgatttgggagaacaacatttcaaaggcctagtcctgattgccttttcccagtatctccagaaatgctcatacgatgagcatgccaaattagtgcaggaagtaacagactttgcaaagacgtgtgttgccgatgagtctgccgccaactgtgacaaatcccttcacactctttttggagataagttgtgtgccattccaaacctccgtgaaaactatggtgaactggctgactgctgtacaaaacaagagcccgaaagaaacgaatgtttcctgcaacacaaagatgacaaccccagcctgccaccatttgaaaggccagaggctgaggccatgtgcacctcctttaaggaaaacccaaccacctttatgggacactatttgcatgaagttgccagaagacatccttatttctatgccccagaacttctttactatgctgagcagtacaatgagattctgacccagtgttgtgcagaggctgacaaggaaagctgcctgaccccgaagcttgatggtgtgaaggagaaagcattggtctcatctgtccgtcagagaatgaagtgctccagtatgcagaagtttggagagagagcttttaaagcatgggcagtagctcgtctgagccagacattccccaatgctgactttgcagaaatcaccaaattggcaacagacctgaccaaagtcaacaaggagtgctgccatggtgacctgctggaatgcgcagatgacagggcggaacttgccaagtacatgtgtgaaaaccaggcgactatctccagcaaactgcagacttgctgcgataaaccactgttgaagaaagcccactgtcttagtgaggtggagcatgacaccatgcctgctgatctgcctgccattgctgctgattttgttgaggaccaggaagtgtgcaagaactatgctgaggccaaggatgtcttcctgggcacgttcttgtatgaatattcaagaagacaccctgattactctgtatccctgttgctgagacttgctaagaaatatgaagccactctggaaaagtgctgcgctgaagccaatcctcccgcatgctacggcacagtgcttgctgaatttcagcctcttgtagaagagcctaagaacttggtcaaaaccaactgtgatctttacgagaagcttggagaatatggattccaaaatgccattctagttcgctacacccagaaagcacctcaggtgtcaaccccaactctcgtggaggctgcaagaaacctaggaagagtgggcaccaagtgttgtacacttcctgaagatcagagactgccttgtgtggaagactatctgtctgcaatcctgaaccgtgtgtgtctgctgcatgagaagaccccagtgagtgagcatgttaccaagtgctgtagtggatccctggtggaaaggcggccatgcttctctgctctgacagttgatgaaacatatgtccccaaagagtttaaagctgagaccttcaccttccactctgatatctgcacacttccagagaaggagaagcagattaagaaacaaacggctcttgctgagctggtgaagcacaagcccaaggctacagcggagcaactgaagactgtcatggatgactttgcacagttcctggatacatgttgcaaggctgctgacaaggacacctgcttctcgactgagggtccaaaccttgtcactagatgcaaagacgccttagccSEQ ID No.2
mFcγRI
gaagtggttaatgccaccaaggctgtgatcaccttgcagcctccatgggtcagtattttccagaaggaaaatgtcactttatggtgtgaggggcctcacctgcctggagacagttccacacaatggtttatcaacggaacagccgttcagatctccacgcctagttatagcatcccagaggccagttttcaggacagtggcgaatacaggtgtcagataggttcctcaatgccaagtgaccctgtgcagttgcaaatccacaatgattggctgctactccaggcctcccgcagagtcctcacagaaggagaacccctggccttgaggtgtcacggatggaagaataaactggtgtacaatgtggttttctatagaaatggaaaatcctttcagttttcttcagattcggaggtcgccattctgaaaaccaacctgagtcacagcggcatctaccactgctcaggcacgggaagacaccgctacacatctgcaggagtgtccatcacggtgaaagagctgtttaccacgccagtgctgagagcatccgtgtcatctcccttcccggaggggagtctggtcaccctgaactgtgagacgaatttgctcctgcagagacccggcttacagcttcacttctccttctacgtgggcagcaagatcctggagtacaggaacacatcctcagagtaccatatagcaagggcggaaagagaagatgctggattctactggtgtgaggtagccacggaggacagcagtgtccttaagcgcagccctgagttggagctccaagtgcttggtccccagtcatcagctcct。
SEQ ID No.3
HSA
gatgcacacaagagtgaggttgctcatcggtttaaagatttgggagaagaaaatttcaaagccttggtgttgattgcctttgctcagtatcttcagcagtgtccatttgaagatcatgtaaaattagtgaatgaagtaactgaatttgcaaaaacatgtgtagctgatgagtcagctgaaaattgtgacaaatcacttcataccctttttggagacaaattatgcacagttgcaactcttcgtgaaacctatggtgaaatggctgactgctgtgcaaaacaagaacctgagagaaatgaatgcttcttgcaacacaaagatgacaacccaaacctcccccgattggtcagaccagaggttgatgtgatgtgcactgcttttcatgacaatgaagagacatttttgaaaaaatacttatatgaaattgccagaagacatccttacttttatgccccggaactccttttctttgctaaaaggtataaagctgcttttacagaatgttgccaagctgctgataaagctgcctgcctgttgccaaagctcgatgaacttcgggatgaagggaaggcttcgtctgccaaacagagactcaaatgtgccagtctccaaaaatttggagaaagagctttcaaagcatgggcagtggctcgcctgagccagagatttcccaaagctgagtttgcagaagtttccaagttagtgacagatcttaccaaagtccacacggaatgctgccatggagatctgcttgaatgtgctgatgacagggcggaccttgccaagtatatctgtgaaaatcaggattcgatctccagtaaactgaaggaatgctgtgaaaaacctctgttggaaaaatcccactgcattgccgaagtggaaaatgatgagatgcctgctgacttgccttcattagctgctgattttgttgaaagtaaggatgtttgcaaaaactatgctgaggcaaaggatgtcttcctgggcatgtttttgtatgaatatgcaagaaggcatcctgattactctgtcgtgctgctgctgagacttgccaagacatatgaaaccactctagagaagtgctgtgccgctgcagatcctcatgaatgctatgccaaagtgttcgatgaatttaaacctcttgtggaagagcctcagaatttaatcaaacaaaactgtgagctttttgagcagcttggagagtacaaattccagaatgcgctattagttcgttacaccaagaaagtaccccaagtgtcaactccaactcttgtagaggtctcaagaaacctaggaaaagtgggcagcaaatgttgtaaacatcctgaagcaaaaagaatgccctgtgcagaagactatctatccgtggtcctgaaccagttatgtgtgttgcatgagaaaacgccagtaagtgacagagtcacaaaatgctgcacagagtccttggtgaacaggcgaccatgcttttcagctctggaagtcgatgaaacatacgttcccaaagagtttaatgctgaaacattcaccttccatgcagatatatgcacactttctgagaaggagagacaaatcaagaaacaaactgcacttgttgagcttgtgaaacacaagcccaaggcaacaaaagagcaactgaaagctgttatggatgatttcgcagcttttgtagagaagtgctgcaaggctgacgataaggagacctgctttgccgaggagggtaaaaaacttgttgctgcaagtcaagctgccttaggctta
SEQ ID No.4
hFcγRI
caagtggacaccacaaaggcagtgatcactttgcagcctccatgggtcagcgtgttccaagaggaaaccgtaaccttgcattgtgaggtgctccatctgcctgggagcagctctacacagtggtttctcaatggcacagccactcagacctcgacccccagctacagaatcacctctgccagtgtcaatgacagtggtgaatacaggtgccagagaggtctctcagggcgaagtgaccccatacagctggaaatccacagaggctggctactactgcaggtctccagcagagtcttcacggaaggagaacctctggccttgaggtgtcatgcgtggaaggataagctggtgtacaatgtgctttactatcgaaatggcaaagcctttaagtttttccactggaattctaacctcaccattctgaaaaccaacataagtcacaatggcacctaccattgctcaggcatgggaaagcatcgctacacatcagcaggaatatctgtcactgtgaaagagctatttccagctccagtgctgaatgcatctgtgacatccccactcctggaggggaatctggtcaccctgagctgtgaaacaaagttgctcttgcagaggcctggtttgcagctttacttctccttctacatgggcagcaagaccctgcgaggcaggaacacatcctctgaataccaaatactaactgctagaagagaagactctgggttatactggtgcgaggctgccacagaggatggaaatgtccttaagcgcagccctgagttggagcttcaagtgcttggcctccagttaccaactcctgtctggtttcat
primer:
MSA-F ggtggtggtggttctgaagcacacaagagt SEQ ID NO.5
MSA-R gactctagaggctaaggcgtctttgcatct SEQ ID NO.6
mFcγRI-F gcctcgagaaaagagaagtggttaatgccaccaaggc SEQ ID NO.7
mFcγRI-R acagaaccaccaccaccaggagctgatga SEQ ID NO.8.
实施例中所用原料及来源:
mFcγRⅠ-MSA融合蛋白:由重组酵母菌表达,经AKTA蛋白纯化仪纯化所得。
mFcγRⅠ-GS4-MSA融合蛋白:由重组酵母菌表达,经AKTA蛋白纯化仪纯化所得。
hFcγRⅠ-(GS4)2-HSA融合蛋白:由重组HEK293T细胞表达,经AKTA蛋白纯化仪纯化所得。
聚乳酸PLA137K,分子量为137000Da、封端为酯基的左旋聚乳酸:购自济南岱罡生物科技有限公司。
二氯甲烷:购自广州化学试剂厂。
无水乙醇:购自国药集团化学试剂有限公司。
小鼠来源的IgG1抗体:购自美国Bio X Cell公司。
羊抗小鼠IgG的金标抗体:购自美国Sigma-Aldrich公司。
透射电镜铜网:购自海德创业(北京)生物科技有限公司。
无蛋白封闭液:购自上海生工生物工程股份有限公司。
His-tag antibody(HRP,mouse antibody):购自北京义翘神州生物技术有限公司。
CD64 antibody(mouse antibody):购自美国Thermo Fisher公司。
Albumin antibody(mouse antibody):购自美国Abcam公司。
ELISA显色液:购自北京义翘神州生物技术有限公司。
PD-L1抗原:购自北京义翘神州生物技术有限公司。
大鼠来源的抗PD-L1抗体:购自购自美国Bio X Cell公司。
羊抗大鼠IgG的HRP抗体:购自北京义翘神州生物技术有限公司。
ELISA所用聚苯乙烯板:购自美国Corning公司。
实施例中所用实验仪器及型号公司:
超声波细胞破碎仪:VCX130,美国Sonics公司。
旋转蒸发仪:RV 10digital V数显型,德国IKA公司。
微通道反应器:1300SERIES A2,美国Corning公司。
纳米粒度及Zeta电位仪:Nano ZSE,英国Malvern公司。
台式微量冷冻离心机:Microfuge 20R,美国Beckman Coulter公司。
透射电子显微镜:Talos L120C,美国赛默飞世尔科技公司。
酶标仪:美国BioTek公司。
实施例1 MSA cDNA的克隆
用PCR方法从小鼠肝胎cDNA文库中获得不带有信号肽编码序列的MSA(小鼠血清白蛋白,Mouse Serum Albumin)cDNA,所用的引物MSA F(SEQ ID NO.5)和MSA R(SEQ IDNO.6)用寡聚核苷酸合成仪合成,下游引物引入XbaI酶切位点和保护碱基,划线处为内切酶识别序列。
50μL PCR反应体系:2x Mix 25μL,DNA模板<200ng,Primer MSA F(10pmol/μL)1μL,Primer MSA R(10pmol/μL)1μL,剩余用ddH2O补足,反应体系可按需求等倍缩小或放大。轻柔混匀后进行PCR,PCR反应条件为94℃热变性1min;94℃变性30s;58℃退火30s;72℃延伸1.5min;共30个循环;再72℃延伸5min。通过1%琼脂糖凝胶检测分析得到预期为1.6kb的条带,胶回收,定量。
实施例2mFcγRI cDNA的克隆
用基因合成的方法获得不带有信号肽编码序列的mFcγRI cDNA,所用的引物mFcγRI-F(SEQ ID NO.7)和mFcγRI-R(SEQ ID NO.8)用寡聚核苷酸合成仪合成,下游引物引入XhoI酶切位点和保护碱基。
50μL PCR反应体系:2x Mix 25μL,DNA模板<200ng,Primer mFcγRI F(10pmol/μL)1μL,Primer mFcγRI R(10pmol/μL)1μL,剩余用ddH2O补足,反应体系可按需求等倍缩小或放大。轻柔混匀后进行PCR,PCR反应条件为94℃热变性1min;94℃变性30s;57℃退火30s;72℃延伸1.5min;共30个循环;再72℃延伸5min。通过1%琼脂糖凝胶检测分析得到预期为1.7kb的条带,胶回收,定量。
实施例3Overlap PCR融合目的基因
50μL PCR反应体系:2×Mix 25μL,Primer mFcγRI F(10pmol/μL)1μL,PrimerMSA R(10pmol/μL)1μL,剩余用ddH2O补足,轻柔混匀后进行PCR,PCR反应条件为94℃热变性1min;94℃变性30s;66℃(-0.5℃/cycle)延伸1.5min;共17个循环;再94℃变性30s;58℃(-0.5℃/cycle)退火30s,72℃延伸1.5min;共5个循环;再72℃延伸5min。
实施例4构建融合基因-酵母菌载体
Xhol和XbaI双酶切mFcγRI-MSA融合片段、酵母菌质粒,50μL酶切反应体系:mFcγRI-MSA片段和酵母菌质粒1μg,Xhol和XbaI内切酶各1μL,CutSmart buffer 5μL,剩余用ddH2O补足,37℃酶切2h以上(无星号活性最好过夜),65℃20min热失活。琼脂糖凝胶电泳,切割目的条带后胶回收。T4DNA ligase连接胶回收后的插入片段与质粒,20μL连接反应体系:T4 Reaction Buffer 2μL,Vector DNA,XμL,Insert DNA YμL,ddH2O ZμL,T4 DNALigase 1μL,25℃反应20min或16℃过夜,质粒载体的构建流程如图1。
实施例5酵母菌载体转化大肠杆菌
使用无菌水或TE缓冲液稀释1μL质粒(1μg/μL)到50ng/μL。E.coli DH5αCompetentCells(100μL)使用前在冰上融化,加入质粒1μL(<50ng),冰中放置30min,42℃放置45s,立刻放入冰中放置1-2min,避免摇动离心管,添加无抗生素LB培养基(预先在37℃保温)至1mL,混匀后37℃振荡培养1h(200rpm),取适量(100mm平板的<100μL)涂布于选择培养基(含25μg/mL Zeocin的低盐LB培养基),正面放置半小时待菌液吸收后,37℃过夜倒置培养12-16h,挑斑,含25μg/mL Zeocin的低盐LB液体培养基进行扩增,提取质粒。
实施例6菌落PCR鉴定大肠杆菌
用无菌枪头挑取单个菌落(colon,并进行编号),置于20uL 0.1%Triton X-100中搅和一下,将装有20uL 0.1%Triton X-100的EP管在100℃下煮沸3min,稍微离心1min;取1uL上清为模板,20uL反应PCR体系为:2x Mix 10μL,DNA模板1μL,Primer 5‘AOX(10pmol/μL)0.5μL,Primer 3‘AOX(10pmol/μL)
0.5μL,ddH2O 8μL。轻柔混匀后进行PCR,PCR反应条件为94℃热变性1min;94℃变性30s;54℃退火30s;72℃延伸1.5min;共30个循环;再72℃延伸5min。通过1%琼脂糖凝胶检测分析得到预期为3.2kb的条带,胶回收,定量。参见图2。LB(含抗生素)液体培养基进行培养扩增,培养18小时后,取1mL菌液送样测序。
实施例7化学转化酵母菌
线性化质粒DNA并脱磷酸化处理,50μL酶切反应体系为质粒DNA 5μg,CutSmartBuffer(10X)5μL,PmeI 1μL,快速CIP 1μL,补充ddH2O至50μL,PCR仪37℃酶切2h以上,65℃热灭活20min;琼脂糖凝胶鉴定酶切完全。
在室温下解冻一管感受态细胞,加3μg线性化的DNA载体到感受态细胞中。向DNA/细胞混合物中加入1mL溶液II,通过涡旋或轻弹离心管进行混合。将转化混合物在30℃的水浴或培养箱中培养1小时。每隔15分钟通过涡旋或轻弹离心管混合转化反应。在42℃的热块或水浴中10分钟来热休克细胞。将细胞分成2管(大约525μL每管)并各加1mL YPD培养基。将细胞在30℃下孵育1小时,以表达Zeocin抗性基因。在室温下以3000×g离心5分钟使细胞成球。弃上清。每管细胞用500μL溶液III重悬,并将两管细胞整合到一管。在室温下以3000×g离心5分钟使细胞成球。弃上清。细胞用100-150μL溶液III重悬。用无菌涂布器将整个转化子置于适当的平板上筛选。在30℃下培养3至10天,每次转化应产生约50个菌落。选择6-10个Zeocin抗性的毕赤酵母转化子,使用PCR分析插入物的存在情况。参见图3。
实施例8Mut+重组酵母的诱导表达(摇瓶培养)
挑选单菌落,置于装有25mL BMGY培养基的250ml摇瓶中,于28-30℃250-300rpm培养至OD600=2-6(16-18h),取1mL冻存;室温下1500-3000g离心5min,收集菌体,用BMMY重悬菌体,使OD600=1.0左右(约100-200mL),开始诱导表达;将所得的菌液置于1L的摇瓶中,用双层纱布或粗棉布封口,放置于20-30℃,转速为250-300rpm的摇床上继续生长;每24h向培养基中添加100%甲醇至终浓度为0.5~1.0%;按时间点分别取菌液样品,取样量为1mL,置于1.5mL EP管中,最大转速离心2~3min,分别收集上清和菌体,分析目的蛋白的表达量和菌液最佳收获时间。时间点一般取:0、6、12、24、36、48、60、72、84和96h。
实施例9Mut+重组酵母的诱导表达(发酵罐培养)
将Mut+重组酵母接种于100mL YPD培养基(酵母抽提物10g/L,胰蛋白胨20g/L,甘油10g/L),摇床30℃,280转/分钟培养24h。接种至装有2L基础盐培养基的5L发酵罐,其中基础盐培养基的配制方法为:浓磷酸3.5mL/L,CaSO4·2H2O 0.15g/L,K2SO4 2.4g/L,MgSO4.7H2O 1.95g/L,KOH 0.65g/L,121℃高压灭菌30分钟,再加入40mL/L甘油(单独121℃高压灭菌30分钟),1mL/L PTM1(配方为CuSO4·5H2O 6.0g/L,CoCl2·6H2O,MnSO4·H2O3.0g/L,H3BO3 0.02g/L,FeSO4·7H2O 65.0g/L,NaMoO4·2H2O 0.2g/L,ZnSO4·7H2O 20.0g/L,Kl 0.1g/L,浓硫酸5ml/L,0.02%生物素0.5ml/L,过滤除菌)。接种前用氨水将培养基pH调至5.0。发酵过程控制温度为25℃,溶氧始终大于30%饱和度,培养至甘油耗尽后,开始流加甘油(50%甘油,含12mL/L PTM1),继续培养至密度OD600值约为150时,开始补加甲醇(分析纯甲醇,含12mL/L PTM1)诱导培养72小时。
实施例10构建pcDNA3.1(+)-hFcγRI-HSA载体
通过双酶切hFcγRI-HSA融合片段、酵母菌质粒,50μL酶切反应体系:hFcγRI-HSA片段和pcDNA3.1(+)质粒1μg,NheI和XbaI内切酶各1μL,CutSmart buffer 5μL,剩余用ddH2O补足,37℃酶切2h以上(无星号活性最好过夜),65℃20min热失活。琼脂糖凝胶电泳,切割目的条带后胶回收。T4DNA ligase连接胶回收后的插入片段与质粒,20μL连接反应体系:T4 Reaction Buffer 2μL,Vector DNA,XμL,Insert DNA YμL,ddH2O ZμL,T4 DNALigase1μL,25℃反应20min或16℃过夜,pcDNA3.1-hFcγRI-HSA质粒载体图谱如图4。
实施例11hFcγRI-HSA-pcDNA3.1载体转染HEK293T细胞
将20μg质粒与不含血清的RPMI 1640培养基混匀至500μL,60μg PEI与不含血清的RPMI 1640培养基混匀至500μL。将F12-K/PEI混合液逐滴加入至质粒混合液中,混合均匀,室温孵育20min,期间轻轻弹EP管。孵育完成后,将混合液与约1×107细胞混合,37℃培养6-8h,更换为无血清的RPMI 1640培养基,培养72h,收取上清。
实施例12切向流浓缩培养基上清
将培养96h的1L菌液5250×g离心5-10min,收集上清;切向流将菌液浓缩至100mL,再次加入500mL PBS,再次浓缩至50mL。
实施例13融合蛋白的纯化
将镍柱用ddH2O平衡5个柱体积,再用Native Binding Buffer平衡10个柱体积,将浓缩后的培养基上样,用Native Wash Buffer洗涤10个柱体积,再用Native ElutionBuffer洗脱蛋白,收集馏分,获得纯化后的融合蛋白的mFcγRⅠ-MSA。mFcγRI-MSA的SDS-PAGE、Western-Blot表征结果,结果参见图5,hFcγRI-HSA的表征结果,结果参见图6。
实施例14、白蛋白融合蛋白-聚乳酸纳米颗粒的制备
(1)超声乳化法
将纯化所得mFcγRⅠ-MSA融合蛋白(由Nanodrop One超微量紫外分光光度计定量,以确定浓度)用超纯水配制为5mg/mL溶液,并配制5mg/mL聚乳酸(PLA137k)的氯仿溶液。取1mL的5mg/mL mFcγRⅠ-MSA融合蛋白水溶液于15mL离心管中,加入100μL的5mg/mL聚乳酸(PLA137k)氯仿溶液(即mFcγRⅠ-MSA融合蛋白与PLA137k的质量比为10:1),在冰水浴中通过超声波细胞破碎仪进行超声乳化。其中,超声功率为130W,振幅为50%,超声时间为1.5min,超声5s停2s(中断时间不计入超声时间)。超声结束后将乳液转移至100mL圆底烧瓶中,并用超纯水洗出离心管中残余乳液,将洗涤液一并转入100mL圆底烧瓶中,旋转蒸发仪按照真空度200/100/50/30/20mbar依次旋蒸,每个真空度下保持10min。其中,在真空度为30/20mbar时将圆底烧瓶浸入32℃水浴锅中,以充分去除氯仿,并蒸发一定体积的水,以浓缩纳米颗粒溶液的体积。旋蒸结束后收集mFcγRⅠ-MSA融合蛋白-聚乳酸纳米颗粒备用,纳米颗粒示意图如图7所示。其他不同分子量、不同种类聚酯与mFcγRⅠ-MSA融合蛋白以及聚酯与mFcγRⅠ-MSA融合蛋白不同比例的纳米颗粒制备方法参考上述制备方法。
(2)微流控技术
将纯化所得mFcγRⅠ-MSA融合蛋白(由Nanodrop One超微量紫外分光光度计定量,以确定浓度)用超纯水配制为5mg/mL溶液,并用氯仿配制2.5mg/mL的聚乳酸溶液。选择微通道反应器的第二、三进样泵进行纳米颗粒的制备,其中,PLA137k氯仿溶液从第二进样泵进样;mFcγRⅠ-MSA融合蛋白水溶液从第三进样泵进样。进样前首先使用无水乙醇以最大流速清洗管路,而后使用所进样的样品溶剂(氯仿和水)以最大流速分别洗涤各自的进样管路。洗涤结束后,设定PLA137k氯仿溶液的进样速度为1.6mL/min,mFcγRⅠ-MSA融合蛋白水溶液的进样速度为6.4mL/min(即水相与有机相的体积比为4:1,mFcγRⅠ-MSA融合蛋白与PLA137k氯仿的质量比为8:1)。待出样口产生的乳液均一且稳定时进行收样,收集到100/250mL圆底烧瓶中,使用旋转蒸发仪按照真空度200/100/50/30/20mbar依次旋蒸,每个真空度下保持10min。其中,在真空度为30/20mbar时将圆底烧瓶浸入32℃水浴锅中,以充分去除氯仿,并蒸发一定体积的水,以浓缩纳米颗粒溶液的体积。旋蒸结束后收集mFcγRⅠ-MSA融合蛋白-聚乳酸纳米颗粒备用。
实施例15、mFcγRⅠ-MSA融合蛋白-聚乳酸纳米颗粒的纯化方法(离心法)
通过台式微量冷冻离心机对实施例12所制得的纳米颗粒进行低速离心(3000rpm,5min,4℃)以去除未组装的聚乳酸;将上清液转移至新的EP管中进行高速离心(15000rpm,2h,4℃)以沉淀出纳米颗粒,去除上清中的游离蛋白,将下层颗粒沉淀用1×PBS重悬备用。
实施例16、mFcγRⅠ-MSA融合蛋白-聚乳酸纳米颗粒的粒径表征
取100μL实施例13纯化重悬后的颗粒溶液于粒径池中,通过纳米粒度及Zeta电位仪测定纳米颗粒的水化直径和分散度变化,测得的纳米颗粒粒径在130~140nm左右,且分布均一,所对应的纳米颗粒粒径分布图如图8所示,不同分子量、不同种类聚酯与mFcγRⅠ-MSA融合蛋白以及聚酯与mFcγRⅠ-MSA融合蛋白不同比例的纳米颗粒的粒径总结如下:
序号 | 脂肪族聚酯种类 | 融合蛋白:聚酯(w:w) | 粒径 |
1 | PLA<sub>7.2k</sub> | 5:1 | 162.5±5.47 |
2 | PLA<sub>36k</sub> | 5:1 | 154.8±2.92 |
3 | PLA<sub>240k</sub> | 5:1 | 190.7±6.89 |
4 | PLA<sub>240k</sub> | 10:1 | 147.2±3.50 |
5 | PLGA(LA/GA=50/50)<sub>30k</sub> | 10:1 | 120.5±12.3 |
实施例17、通过透射电子显微镜对mFcγRⅠ-MSA融合蛋白-聚乳酸纳米颗粒进行形貌表征
取实施例13纯化重悬后的颗粒溶液,加入小鼠来源的IgG1抗体于4℃过夜孵育(8~10h),孵育结束后进行离心(15000rpm,2h,4℃)以去除游离未结合的抗体,并将下层沉淀出的结合了抗体的黑色颗粒沉淀用1×PBS重悬。而后向重悬的颗粒溶液中加入羊抗小鼠IgG的金标抗体,于4℃孵育8h,孵育结束后离心(15000rpm,20min,4℃)去除未结合的金标抗体,将下层结合了金标抗体的红色颗粒沉淀用超纯水重悬。将重悬后的颗粒溶液进行适当稀释(通过纳米粒度及Zeta电位仪,将颗粒溶液稀释至attenuator为8,count rate约为200kcps),取2μL滴到透射电子显微镜(TEM)铜网上使其自然风干8h,而后于TEM下观察。如图9所示,mFcγRⅠ-MSA融合蛋白-聚乳酸纳米颗粒呈圆球状形貌。
实施例18、mFcγRⅠ-MSA融合蛋白-聚乳酸纳米颗粒的蛋白组装率及蛋白释放行为的测定
取实施例13纯化重悬后的颗粒溶液等分为7份,并于37℃摇床中放置,每个时间点(0、4、8、12、24、48、72h)取出一份离心(15000rpm,2h,4℃),离心后取上清于-20℃中储存,待收取完所有时间点的上清后进行ELISA实验,以测定各时间点上清中的融合蛋白含量。
ELISA法:以mFcγRⅠ-MSA融合蛋白作为标准品,对各时间点取得的上清进行适当的稀释作为样品,将标准品和样品进行铺板(每孔100μL),并于4℃过夜孵育,孵育结束后使用PBST洗涤以去除未结合到板上的蛋白;而后将无蛋白封闭液用超纯水1:1混合,每孔加入200μL,于37℃孵育1h后使用PBST洗涤以去除残余的封闭液;之后于37℃孵育His-tagantibody(HRP)45min,PBST洗涤去除未结合的His-tag antibody(HRP)后进行显色。显色时A、B液1:1混合,每孔100μL,避光显色8~10min后,加入2mol/L H2SO4终止显色,立即使用酶标仪检测OD450nm、OD630nm的值。
对标准曲线的线性区域进行线性拟合,并依此计算各上清样品中的蛋白含量,从而确定mFcγRⅠ-MSA融合蛋白-聚乳酸纳米颗粒的蛋白组装率为47%左右。如图10所示,在72h的时间内,纳米颗粒具有良好的稳定性,无明显的蛋白释放。
实施例19、mFcγRⅠ-MSA融合蛋白-聚乳酸纳米颗粒的体外稳定性
(1)PBS中的稳定性
取实施例13纯化重悬后的颗粒溶液等分为7份,并于37℃摇床中放置,每个时间点(0、4、8、12、24、48、72h)取出一份通过纳米粒度及Zeta电位仪检测颗粒粒径。如图11-1所示,在72h的时间内,mFcγRⅠ-MSA融合蛋白-聚乳酸纳米颗粒的粒径未发生显著变化,表明本发明的融合蛋白纳米颗粒在PBS中具有良好的稳定性。
(2)血清稳定性
通过台式微量冷冻离心机对实施例12所制得的纳米颗粒进行低速离心(3000rpm,5min,4℃)以去除未组装的聚乳酸;将上清液转移至新的EP管中进行高速离心(15000rpm,2h,4℃)以沉淀出纳米颗粒,去除上清中的游离蛋白,将下层沉淀用DMEM培养基(加入10%FBS)重悬,然后等分为8份,并于37℃摇床中放置,在不同时间点(0、6、18、24、32、48、72、96h)取出一份通过纳米粒度及Zeta电位仪检测颗粒粒径。如图11-2所示,在96h的时间内,mFcγRⅠ-MSA融合蛋白-聚乳酸纳米颗粒的粒径未发生显著变化,表明本发明的融合蛋白纳米颗粒在细胞培养基中也具有良好的稳定性。
实施例20、mFcγRⅠ-MSA融合蛋白-聚乳酸纳米颗粒的抗体结合效率
固定αPD-L1抗体的量一致(10μg),按照颗粒与抗体不同的质量比(250:1、100:1、50:1、25:1、10:1、5:1、2:1、1:1)投入不同量的实施例13纯化重悬后的颗粒溶液,然后用PBS将各组样品的体积补至500μL,并设置相同体积的游离抗体(无颗粒)组,于4℃过夜孵育。孵育结束后离心(15000rpm,2h),取上清,通过ELISA测定上清中的抗体浓度。
ELISA法:以αPD-L1抗体作为标准品,对各时间点取得的上清稀释2000倍作为样品。使用PD-L1抗原铺板(每孔100μL),并于4℃过夜孵育,孵育结束后使用PBST洗涤以去除未结合到板上的抗原;而后将无蛋白封闭液用超纯水1:1混合,每孔加入200μL,于37℃孵育1h后使用PBST洗涤以去除残余的封闭液;之后将αPD-L1抗体标准品及稀释后的上清样品作为一抗于37℃孵育(每孔100μL)1h,PBST洗涤去除未结合的一抗后加入羊抗大鼠IgG的HRP抗体于37℃孵育45min,PBST洗涤去除未结合的羊抗大鼠IgG的HRP抗体后进行显色。显色时A、B液1:1混合,每孔100μL,避光显色8~10min后,加入2mol/L H2SO4终止显色,立即使用酶标仪检测OD450nm、OD630nm的值。
对标准曲线的线性区域进行线性拟合,并依此计算各上清样品中的抗体含量。将游离抗体(无颗粒)组测定的抗体浓度作为原始投入量,从而计算不同颗粒与抗体质量比条件下抗体的结合效率。如图12所示,本发明的融合蛋白纳米颗粒具有优异的抗体结合能力。
实施例21、血清白蛋白融合蛋白双特异性纳米抗体与肿瘤细胞、CD8+T细胞的结合
小鼠B16-F10黑色素瘤细胞系和小鼠4T1原位乳腺癌细胞系均来源于美国标准生物品收藏中心(ATCC)。SPF级C57BL/6小鼠和雌性BALB/C小鼠,5-6周龄,购自湖南斯莱克景达实验动物有限公司。小鼠饲养在华南理工大学实验动物中心,动物实验流程遵循华南理工大学实验动物管理规范条例的相关规定。
大鼠抗小鼠PD-1(CD279)抗体(αPD-1),大鼠抗小鼠PD-L1(B7-H1)抗体(αPDL1):均购自美国Bio X Cell公司。
1、我们用10ng/mL IFN-γ刺激诱导B16-F10细胞的PD-L1的高表达(1.0×105细胞/孔),以及使用5μg/mLαCD3ε诱导自脾脏分选的CD8+T细胞活化(5.0×105细胞/孔)在体外模拟肿瘤微环境。通过流式细胞术检测观察到B16-F10细胞的PD-L1表达显著上调和CD8+T细胞的PD-1表达上调(图13)。这两种刺激活化后的细胞可以作为体外实验中模拟肿瘤微环境的效-靶细胞。
2、为了评估该纳米抗体的优越性,我们首先对血清白蛋白融合蛋白纳米颗粒双特异性抗体递送平台与细胞相互作用的能力进行探究。利用实施例11所述的融合蛋白mFcγRⅠ-MSA(5mg/mL)和聚乳酸高分子材料PLLA137k(5mg/mL)为基本组分,通过超声乳化的方法制备出融合蛋白-聚乳酸复合物NPmFcγRI-MSA;将NPmFcγRI-MSA与抗小鼠PD-1、PD-L1抗体(二者比例1:1)按照质量比25:1进行混合制备(参照实施例15)出双特异性纳米抗体NPmFcγRI-MSA@αPD-1&αPD-L1。利用BSA(5mg/mL)和聚乳酸高分子材料PLLA137k(5mg/mL)为基本组分,通过超声乳化的方法制备出融合蛋白-聚乳酸复合物NPBSA;将NPBSA与抗小鼠PD-1、PD-L1抗体(二者比例1:1)按照质量比25:1进行混合制备出双特异性纳米抗体NPBSA@αPD-1&αPD-L1。将PD-L1 high B16-F10细胞(5.0×104细胞/孔和1.0×104细胞/皿)和PD-1high CD8+T细胞(5.0×104细胞/孔和1.0×104细胞/皿)分别与FITC标记的NPBSA@αPD-1&αPD-L1和NPmFcγRI-MSA@αPD-1&αPD-L1共孵育(αPD-1&αPD-L1浓度为20μg/mL),并通过流式细胞仪和激光扫描共聚焦显微镜(CLSM)评价NPmFcγRI-MSA@αPD-1&αPD-L1的靶向结合能力。如图14A所示,B16-F10细胞与NPmFcγRI-MSA@αPD-1&αPD-L1荧光强度随着孵育时间的延长而增加;并且我们以台盼蓝淬灭细胞外荧光的方法,确认了颗粒在细胞膜表面而非进入胞内。同时我们还设置了不同浓度抗体的NPBSA@αPD-1&αPD-L1和NPmFcγRI-MSA@αPD-1&αPD-L1处理组,通过流式细胞术检测发现,在抗体浓度大于6.25μg/mL时,B16-F10细胞和CD8+T细胞的NPmFcγRI-MSA@αPD-1&αPD-L1平均荧光强度(meanfluorescence intensity,MFI)随着浓度的增加而增加(图14B)。CLSM图像还显示大量的NPmFcγRI-MSA@αPD-1&αPD-L1结合在B16-F10细胞(表达mCherry荧光蛋白,NP上的蛋白质用FITC标记)的表面(图14C)。对于CD8+T细胞,NPmFcγRI-MSA@αPD-1&αPD-L1也呈时间剂量依赖性的结合,并且几乎没有颗粒进入到CD8+T细胞中(图15)。与之相反的,对照组NPBSA@αPD-1&αPD-L1与两种细胞均呈现较弱的相互作用(图14和图15),说明NPmFcγRI-MSA@αPD-1&αPD-L1与细胞的结合取决于携带的单克隆抗体的与抗原特异性的识别与结合。以上结果证明,NPmFcγRI-MSA可以特异性结合共抑制分子αPD-1&αPD-L1,而NPBSA不可以特异性结合共抑制分子αPD-1&αPD-L。
3、为了探究结合有治疗抗体的血清白蛋白融合蛋白纳米颗粒与细胞的相互作用,我们选取了小鼠黑色素瘤细胞系B16-F10,对脾脏分离的CD8+T细胞进行标记CFSE后,与B16-F10细胞(表达mCherry荧光蛋白)共同培养,设置PBS对照组,游离αPD-1与αPD-L1混合组、NPBSA同步携载αPD-1和αPD-L1组(NPBSA@αPD-1&αPD-L1)、血清白蛋白融合蛋白双特异性纳米抗体组即NPmFcγRI-MSA同步携载αPD-1和αPD-L1组(NPmFcγRI-MSA@αPD-1&αPD-L1)([αPD-1]、[αPD-L1]各10μg/mL)四个实验组。分别对各组细胞进行相应处理,培养4h后,洗去未结合的纳米颗粒、抗体及未与肿瘤细胞作用的CD8+T细胞,如图16所示,NPmFcγRI-MSA@αPD-1&αPD-L1相比其他组,更多的CD8+T细胞(绿色)与肿瘤细胞(红色)存在共定位现象,说明该纳米抗体能够促进两种细胞的相互作用。
实施例22、血清白蛋白融合蛋白双特异性纳米抗体的体外细胞杀伤实验
1、为了解NPmFcγRI-MSA@αPD-1&αPD-L1是否能够在体外进一步激活CD8+T细胞,并促进其介导的细胞毒效应,分选得到的T细胞经过αCD3ε抗体活化,与B16-F10细胞(表达luciferase荧光)共培养。设置阳性对照组(加入1%Triton),阴性对照组(加入等体积的培养基),PBS对照组,游离αPD-1与αPD-L1混合组,NPBSA同步携载αPD-1和αPD-L1组(NPBSA@αPD-1&αPD-L1),血清白蛋白融合蛋白双特异性纳米抗体组即NPmFcγRI-MSA同步携载αPD-1和αPD-L1组(NPmFcγRI-MSA@αPD-1&αPD-L1)四个实验组,NPmFcγRI-MSA@αPD-1&αPD-L1还设置不同浓度抗体处理组。分别对各组细胞进行相应处理,于37℃5%CO2环境下培养24h。加入150μg/mL荧光素,立即使用多功能酶标仪检测化学发光,并根据公式:T细胞活力(%)=[(实验组OD值-阳性组OD值)/(阴性组OD值-阳性组OD值)],计算细胞活力。如图17所示,经NPmFcγRI-MSA@αPD-1&αPD-L1参与的实验组检测到更多的肿瘤细胞内荧光,显示出更有效的杀伤效果;且随着颗粒浓度的增加,多价态抗体的浓度相应增加,有效增强T细胞对肿瘤细胞的杀伤效果。
实施例23、动物水平抗肿瘤治疗实验
将15只植有4T1原位乳腺癌的BALB/C小鼠,随机分为3组,每组5只小鼠,分别进行尾静脉注射200μL的PBS,αPD-1&αPD-L1(100μg/只;FreeαPD-1&αPD-L1组),NPmFcγRI-MSA@αPD-1&αPD-L1(mFcγRI-MSA 2mg/只,αPD-1&αPD-L1 100μg/只;NPmFcγRI-MSA@αPD-1&αPD-L1组),每隔三天给一次药,共三次。在整个治疗过程中,每两天称量一次小鼠体重及使用游标卡尺测量肿瘤大小,肿瘤体积按以下公式计算:体积(mm3)=0.5×长×宽2。如图18所示,PBS对照组和游离抗体组肿瘤迅速生长,NPmFcγRI-MSA@αPD-1&αPD-L1组肿瘤生长明显被抑制,这是由于该双特异性纳米抗体能够携带抗体药物递送到体内同时增强肿瘤细胞与T细胞的相互作用。如图19所示,在整个治疗过程中,各组小鼠体重均无明显变化,表明各组组分对小鼠生存无严重毒性。
将36只植有4T1原位乳腺癌的BALB/c小鼠,随机分为3组,每组12只小鼠,分别进行尾静脉注射200μL的PBS,αPD-1&αPD-L1&αNKG2A(100μg/抗体/只,多种抗体物理混合;FreeαPD-1&αPD-L1&αNKG2A组),NPmFcγRI-MSA@αPD-1&αPD-L1&αNKG2A(mFcγRI-GS4-MSA 3mg/只,αPD-1&αPD-L1&αNKG2A 100μg/抗体/只,纳米组装体(纳米颗粒)与抗体混合物物理混合;NPmFcγRI-GS4-MSA@αPD-1&αPD-L1&αNKG2A组),颗粒制备方式参照实施例14,每隔三天给一次药,共两次。在整个治疗过程中,每两天称量一次小鼠体重及使用游标卡尺测量肿瘤大小,肿瘤体积按以下公式计算:体积(mm3)=0.5×长×宽2。如图20所示,PBS对照组和游离抗体组肿瘤迅速生长,NPmFcγRI-GS4-MSA@αPD-1&αPD-L1&αNKG2A组肿瘤生长明显被抑制,这是由于该NPmFcγRI-GS4-MSA@αPD-1&αPD-L1&αNKG2A抗体递送体系能够携带抗体药物递送到体内同时增强肿瘤细胞与T细胞、NK细胞的相互作用。如图21所示,NPmFcγRI-GS4-MSA@αPD-1&αPD-L1&αNKG2A能够有效延长荷瘤小鼠的生存期。
以上所述实施例的各技术特征可以进行任意的组合,为使描述简洁,未对上述实施例中的各个技术特征所有可能的组合都进行描述,然而,只要这些技术特征的组合不存在矛盾,都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。因此,本发明专利的保护范围应以所附权利要求为准。
序列表
<110> 华南理工大学
<120> 基于血清白蛋白的融合蛋白、纳米组装体及其制备方法和应用
<150> 2021101643751
<151> 2021-02-05
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1770
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
aggggtgtgt ttcgccgaga agcacacaag agtgagatcg cccatcggta taatgatttg 60
ggagaacaac atttcaaagg cctagtcctg attgcctttt cccagtatct ccagaaatgc 120
tcatacgatg agcatgccaa attagtgcag gaagtaacag actttgcaaa gacgtgtgtt 180
gccgatgagt ctgccgccaa ctgtgacaaa tcccttcaca ctctttttgg agataagttg 240
tgtgccattc caaacctccg tgaaaactat ggtgaactgg ctgactgctg tacaaaacaa 300
gagcccgaaa gaaacgaatg tttcctgcaa cacaaagatg acaaccccag cctgccacca 360
tttgaaaggc cagaggctga ggccatgtgc acctccttta aggaaaaccc aaccaccttt 420
atgggacact atttgcatga agttgccaga agacatcctt atttctatgc cccagaactt 480
ctttactatg ctgagcagta caatgagatt ctgacccagt gttgtgcaga ggctgacaag 540
gaaagctgcc tgaccccgaa gcttgatggt gtgaaggaga aagcattggt ctcatctgtc 600
cgtcagagaa tgaagtgctc cagtatgcag aagtttggag agagagcttt taaagcatgg 660
gcagtagctc gtctgagcca gacattcccc aatgctgact ttgcagaaat caccaaattg 720
gcaacagacc tgaccaaagt caacaaggag tgctgccatg gtgacctgct ggaatgcgca 780
gatgacaggg cggaacttgc caagtacatg tgtgaaaacc aggcgactat ctccagcaaa 840
ctgcagactt gctgcgataa accactgttg aagaaagccc actgtcttag tgaggtggag 900
catgacacca tgcctgctga tctgcctgcc attgctgctg attttgttga ggaccaggaa 960
gtgtgcaaga actatgctga ggccaaggat gtcttcctgg gcacgttctt gtatgaatat 1020
tcaagaagac accctgatta ctctgtatcc ctgttgctga gacttgctaa gaaatatgaa 1080
gccactctgg aaaagtgctg cgctgaagcc aatcctcccg catgctacgg cacagtgctt 1140
gctgaatttc agcctcttgt agaagagcct aagaacttgg tcaaaaccaa ctgtgatctt 1200
tacgagaagc ttggagaata tggattccaa aatgccattc tagttcgcta cacccagaaa 1260
gcacctcagg tgtcaacccc aactctcgtg gaggctgcaa gaaacctagg aagagtgggc 1320
accaagtgtt gtacacttcc tgaagatcag agactgcctt gtgtggaaga ctatctgtct 1380
gcaatcctga accgtgtgtg tctgctgcat gagaagaccc cagtgagtga gcatgttacc 1440
aagtgctgta gtggatccct ggtggaaagg cggccatgct tctctgctct gacagttgat 1500
gaaacatatg tccccaaaga gtttaaagct gagaccttca ccttccactc tgatatctgc 1560
acacttccag agaaggagaa gcagattaag aaacaaacgg ctcttgctga gctggtgaag 1620
cacaagccca aggctacagc ggagcaactg aagactgtca tggatgactt tgcacagttc 1680
ctggatacat gttgcaaggc tgctgacaag gacacctgct tctcgactga gggtccaaac 1740
cttgtcacta gatgcaaaga cgccttagcc 1770
<210> 2
<211> 819
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
gaagtggtta atgccaccaa ggctgtgatc accttgcagc ctccatgggt cagtattttc 60
cagaaggaaa atgtcacttt atggtgtgag gggcctcacc tgcctggaga cagttccaca 120
caatggttta tcaacggaac agccgttcag atctccacgc ctagttatag catcccagag 180
gccagttttc aggacagtgg cgaatacagg tgtcagatag gttcctcaat gccaagtgac 240
cctgtgcagt tgcaaatcca caatgattgg ctgctactcc aggcctcccg cagagtcctc 300
acagaaggag aacccctggc cttgaggtgt cacggatgga agaataaact ggtgtacaat 360
gtggttttct atagaaatgg aaaatccttt cagttttctt cagattcgga ggtcgccatt 420
ctgaaaacca acctgagtca cagcggcatc taccactgct caggcacggg aagacaccgc 480
tacacatctg caggagtgtc catcacggtg aaagagctgt ttaccacgcc agtgctgaga 540
gcatccgtgt catctccctt cccggagggg agtctggtca ccctgaactg tgagacgaat 600
ttgctcctgc agagacccgg cttacagctt cacttctcct tctacgtggg cagcaagatc 660
ctggagtaca ggaacacatc ctcagagtac catatagcaa gggcggaaag agaagatgct 720
ggattctact ggtgtgaggt agccacggag gacagcagtg tccttaagcg cagccctgag 780
ttggagctcc aagtgcttgg tccccagtca tcagctcct 819
<210> 3
<211> 1755
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
gatgcacaca agagtgaggt tgctcatcgg tttaaagatt tgggagaaga aaatttcaaa 60
gccttggtgt tgattgcctt tgctcagtat cttcagcagt gtccatttga agatcatgta 120
aaattagtga atgaagtaac tgaatttgca aaaacatgtg tagctgatga gtcagctgaa 180
aattgtgaca aatcacttca tacccttttt ggagacaaat tatgcacagt tgcaactctt 240
cgtgaaacct atggtgaaat ggctgactgc tgtgcaaaac aagaacctga gagaaatgaa 300
tgcttcttgc aacacaaaga tgacaaccca aacctccccc gattggtcag accagaggtt 360
gatgtgatgt gcactgcttt tcatgacaat gaagagacat ttttgaaaaa atacttatat 420
gaaattgcca gaagacatcc ttacttttat gccccggaac tccttttctt tgctaaaagg 480
tataaagctg cttttacaga atgttgccaa gctgctgata aagctgcctg cctgttgcca 540
aagctcgatg aacttcggga tgaagggaag gcttcgtctg ccaaacagag actcaaatgt 600
gccagtctcc aaaaatttgg agaaagagct ttcaaagcat gggcagtggc tcgcctgagc 660
cagagatttc ccaaagctga gtttgcagaa gtttccaagt tagtgacaga tcttaccaaa 720
gtccacacgg aatgctgcca tggagatctg cttgaatgtg ctgatgacag ggcggacctt 780
gccaagtata tctgtgaaaa tcaggattcg atctccagta aactgaagga atgctgtgaa 840
aaacctctgt tggaaaaatc ccactgcatt gccgaagtgg aaaatgatga gatgcctgct 900
gacttgcctt cattagctgc tgattttgtt gaaagtaagg atgtttgcaa aaactatgct 960
gaggcaaagg atgtcttcct gggcatgttt ttgtatgaat atgcaagaag gcatcctgat 1020
tactctgtcg tgctgctgct gagacttgcc aagacatatg aaaccactct agagaagtgc 1080
tgtgccgctg cagatcctca tgaatgctat gccaaagtgt tcgatgaatt taaacctctt 1140
gtggaagagc ctcagaattt aatcaaacaa aactgtgagc tttttgagca gcttggagag 1200
tacaaattcc agaatgcgct attagttcgt tacaccaaga aagtacccca agtgtcaact 1260
ccaactcttg tagaggtctc aagaaaccta ggaaaagtgg gcagcaaatg ttgtaaacat 1320
cctgaagcaa aaagaatgcc ctgtgcagaa gactatctat ccgtggtcct gaaccagtta 1380
tgtgtgttgc atgagaaaac gccagtaagt gacagagtca caaaatgctg cacagagtcc 1440
ttggtgaaca ggcgaccatg cttttcagct ctggaagtcg atgaaacata cgttcccaaa 1500
gagtttaatg ctgaaacatt caccttccat gcagatatat gcacactttc tgagaaggag 1560
agacaaatca agaaacaaac tgcacttgtt gagcttgtga aacacaagcc caaggcaaca 1620
aaagagcaac tgaaagctgt tatggatgat ttcgcagctt ttgtagagaa gtgctgcaag 1680
gctgacgata aggagacctg ctttgccgag gagggtaaaa aacttgttgc tgcaagtcaa 1740
gctgccttag gctta 1755
<210> 4
<211> 831
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
caagtggaca ccacaaaggc agtgatcact ttgcagcctc catgggtcag cgtgttccaa 60
gaggaaaccg taaccttgca ttgtgaggtg ctccatctgc ctgggagcag ctctacacag 120
tggtttctca atggcacagc cactcagacc tcgaccccca gctacagaat cacctctgcc 180
agtgtcaatg acagtggtga atacaggtgc cagagaggtc tctcagggcg aagtgacccc 240
atacagctgg aaatccacag aggctggcta ctactgcagg tctccagcag agtcttcacg 300
gaaggagaac ctctggcctt gaggtgtcat gcgtggaagg ataagctggt gtacaatgtg 360
ctttactatc gaaatggcaa agcctttaag tttttccact ggaattctaa cctcaccatt 420
ctgaaaacca acataagtca caatggcacc taccattgct caggcatggg aaagcatcgc 480
tacacatcag caggaatatc tgtcactgtg aaagagctat ttccagctcc agtgctgaat 540
gcatctgtga catccccact cctggagggg aatctggtca ccctgagctg tgaaacaaag 600
ttgctcttgc agaggcctgg tttgcagctt tacttctcct tctacatggg cagcaagacc 660
ctgcgaggca ggaacacatc ctctgaatac caaatactaa ctgctagaag agaagactct 720
gggttatact ggtgcgaggc tgccacagag gatggaaatg tccttaagcg cagccctgag 780
ttggagcttc aagtgcttgg cctccagtta ccaactcctg tctggtttca t 831
<210> 5
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
ggtggtggtg gttctgaagc acacaagagt 30
<210> 6
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
gactctagag gctaaggcgt ctttgcatct 30
<210> 7
<211> 37
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
gcctcgagaa aagagaagtg gttaatgcca ccaaggc 37
<210> 8
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
acagaaccac caccaccagg agctgatga 29
Claims (21)
1.一种用于递送至少一种抗体的融合蛋白,其特征在于,包括血清白蛋白和蛋白质受体,所述血清白蛋白与蛋白质受体直接或通过肽接头连接;所述蛋白质受体为Fc受体。
2.根据权利要求1所述的融合蛋白,其特征在于,所述Fc受体为特异性结合所递送的抗体的Fc段的受体,Fc受体与所递送的抗体具有相同或相似种属来源,优选为FcγRI,更优选mFcγRI或hFcγRI。
3.根据权利要求1所述的融合蛋白,其特征在于,所述血清白蛋白是哺乳动物血清白蛋白,优选为人血清白蛋白、牛血清白蛋白、小鼠血清白蛋白、小鼠血清白蛋白、大鼠血清白蛋白、兔血清白蛋白、鸡卵清白蛋白中的至少一种,更优选为小鼠血清白蛋白或人血清白蛋白;
和/或所述血清白蛋白与所述Fc受体同源。
4.根据权利要求1所述的融合蛋白,其特征在于,Fc受体与所递送抗体的Fc结构域非共价结合;和/或所递送的抗体与所述融合蛋白具有亲和力。
5.根据权利要求1-4任一项所述的融合蛋白,其特征在于,所述融合蛋白包含血清白蛋白和Fc受体蛋白的全长或部分片段,或上述经取代、缺失、突变和/或添加一个或多个天然存在的、非天然存在的或修饰的氨基酸的蛋白。
6.根据权利要求1所述的融合蛋白,其特征在于,所述融合蛋白从N端到C端依次为血清白蛋白、肽接头和蛋白质受体;优选地,所述肽接头的残基可选[GlyGlyGlyGlySer]n,n为1-4的整数。
7.一种用于递送至少一种抗体的纳米组装体,其特征在于,所述纳米组装体由权利要求1-6任一项所述的融合蛋白与疏水性可降解聚酯或其衍生物通过疏水相互作用结合构成。
8.根据权利要求7所述的纳米组装体,其特征在于,所述疏水性可降解聚酯或其衍生物是聚酯,优选所述聚酯为脂肪族聚酯或其衍生物,或聚乙二醇修饰的脂肪族聚酯或其衍生物。
9.根据权利要求8所述的纳米组装体,其特征在于,所述脂肪族聚酯为聚丙交酯、聚乙交酯、聚(乙交酯-co-丙交酯)和聚己内酯中的至少一种。
10.根据权利要求9所述的纳米组装体,其特征在于,
所述脂肪族聚酯为聚丙交酯;优选地,所述聚丙交酯为左旋聚丙交酯、右旋聚丙交酯或外消旋聚丙交酯;所述聚丙交酯的端基为酯基、羧基和羟基中的至少一种。
11.根据权利要求10所述的纳米组装体,其特征在于,
所述聚丙交酯为左旋聚丙交酯,优选地,所述左旋聚丙交酯的端基为酯基。
12.根据权利要求11所述的纳米组装体,其特征在于,
所述左旋聚丙交酯的分子量范围为7200~1100000道尔顿,优选为137000~240000道尔顿。
13.根据权利要求7所述的纳米组装体,其特征在于,
所述纳米组装体为纳米颗粒,其粒径范围为80~200nm,优选范围为80~150nm。
14.一种权利要求7所述的纳米组装体的制备方法,其特征在于,包括以下步骤:
(1)将权利要求1-6任一项所述融合蛋白与水或水溶液混合,得水相,其浓度为0.5~20mg/mL,优选为5~10mg/mL;
将所述疏水性可降解聚酯及其衍生物与有机溶剂混合,其浓度为0.5~10mg/mL,优选范围为1~5mg/mL,得油相;
(2)将步骤(1)所述水相和油相制备成水包油的乳剂,优选地,所述水相与油相的体积比为1:1~10:1,优选为5:1~10:1;
(3)将所述乳剂分离纯化,得纳米组装体。
15.权利要求7-14所述的纳米组装体在制备至少一种抗体递送的平台或系统中的应用。
16.一种抗体递送平台或系统,其特征在于,包括权利要求7-13任一项所述的纳米组装体,以及至少一种需要递送的抗体。
17.根据权利要求16所述的抗体递送平台或系统,其特征在于,所述需要递送的抗体至少有一种抗体,优选两种,或者三种;和/或所述抗体包括有至少一种单克隆抗体,或特异性抗体或其抗原结合部分,优选包括两种或以上的单克隆抗体、多价抗体、人源化抗体、嵌合抗体、基因工程改造抗体。
18.一种权利要求16所述的抗体递送平台或系统的制备方法,其特征在于,将所述纳米组装体,以及至少一种需要递送的抗体进行物理混合,即得。
19.权利要求7-14任一项所述的纳米组装体在制备免疫治疗药物的应用。
20.根据权利要求19所述的应用,所述免疫治疗药物为肿瘤免疫治疗药物或自身免疫疾病治疗药物。
21.权利要求1-6任一项所述融合蛋白在制备权利要求7-14所述的纳米组装体中的应用。
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