CN109689692B - 用于肿瘤治疗的双特异性抗体和抗体偶联物及其应用 - Google Patents
用于肿瘤治疗的双特异性抗体和抗体偶联物及其应用 Download PDFInfo
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- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
本发明提供一种双特异性抗体,其包含抗MUC1 VHH的抗体片段和抗CD16 VHH的抗体片段。本发明使用的抗体片段为来自骆驼重链抗体的可变区序列,与抗原具有高结合亲和性。本发明将识别MUC1和识别CD16的抗体片段构建在同一抗体分子中,可以特异性结合MUC1和CD16分子,促进NK细胞对MUC1阳性表达细胞的杀伤作用,对MUC1阳性肿瘤的生长起到抑制的作用。本发明还提供所述双特异性抗体的偶联物、相关的药物组合物和应用。
Description
技术领域
本发明涉及一种双特异性抗体及其制备,更具体地,涉及抗MUC1和抗CD16的双特异性抗体,用于肿瘤治疗领域。
背景技术
肿瘤免疫治疗中,双特异抗体(bispecific antibody,bsAb)在其中起到了非常重要的作用。双特异抗体是含有两种特异性抗原结合位点的人工抗体,通过结合位点,可同时结合效应细胞与靶细胞表面的特异性抗原,激活静止状态的效应细胞并募集到靶细胞周围,介导靶细胞的凋亡或裂解。已开发出多种靶向不同免疫效应细胞和肿瘤细胞的bsAb,其中在肿瘤免疫治疗中的主要免疫效应细胞为T细胞、NK细胞和巨噬细胞等。NK细胞和T细胞是目前研究较多的效应细胞。
在T细胞表面具有引发作用的分子,包括CD2、CD3、CD28等,因此在构建用于肿瘤治疗的双特异抗体时,通常都包括识别这些分子的特异性,其中基于CD3和CD28的双特异抗体研究较多。BiTE(双特异T细胞募集者)是一种双特异抗体的形式,能通过抗CD3的单链抗体片段最大程度的介导T靠近肿瘤细胞,进而发挥裂解肿瘤的作用。当前,有几种靶向BiTE正在研发之中,比如CD19(针对B细胞癌)、上皮细胞粘附分子(EpCAM,CD326),前列腺特异膜抗原(PSMA),以及MUC1。MEDI-565,又名MT111或AMG211,是一种BiTE抗体,能介导T细胞杀伤表达MUC1的肿瘤细胞,且与肿瘤细胞系的突变状态无关。现在该药物正处于临床I期实验(ClinicalTrails.gov,NCT02291614)中,用于治疗晚期结肠腺癌。虽然取得了这些进步,但是双特异抗体主要的挑战仍然存在,例如提高制造效率,保留免疫原性,降低毒性,和增加半衰期。
自然杀伤细胞(natural killer cell,NK)是体内免疫中的重要组成部分,在天然免疫中识别外界抗原,进而直接清除。其杀伤作用具有MHC非限制性,多种肿瘤细胞对NK细胞介导的杀伤作用敏感。其表面特异性分子CD16,是一种分布于NK细胞,中性粒细胞和单核细胞、巨噬细胞等表面的低亲和力Fc受体。CD16通过结合IgG的Fc区域,可以激活NK细胞产生ADCC。bsAb分子上的抗CD16抗体结合位点,可以在两个方面增加NK细胞基础上的免疫治疗作用:(1)作为肿瘤部位的锚定分子,募集NK细胞,使得局部NK细胞数量增加,延长肿瘤细胞与NK的接触时间,增强NK细胞的杀伤作用;(2)在肿瘤部位激活NK细胞,进而杀伤肿瘤细。目前已有多种结合CD16与肿瘤相关抗原的双特异抗体在研究之中,比如靶向Her2的乳腺癌,CD33,CD20等。其中靶向CD30和CD16的双特异抗体AFM13,用于治疗顽固性和/或再发性霍奇金淋巴瘤已经进入临床试验(clinicaltrials.gov identifier:NCT01221571)。
MUC1是Mucins黏蛋白家族成员,存在正常腺管上皮细胞及其来源的肿瘤细胞表面,由多肽核芯(核芯肽)和侧枝糖链构成。其核芯肽胞外段含有许多20个氨基酸(SAPDTRPAPGSTAPPAHGVT)的串联重复序列(vriable numbers tandem repeats,VNTRs)。正常组织MUC1与肿瘤组织不同,前者分布于腺管上皮细胞分泌极,与免疫细胞相对隔离,糖基化丰富;而后者广泛分布并异常丰富地表达于癌细胞表面,糖基化不完全,因此暴露出正常情况下隐蔽的表位,成为免疫细胞攻击的靶点。MUC1核芯肽的PDTRP表位既能被多种MUC1抗体识别,也可被CTL细胞识别和杀伤。
另外,双特异性抗体可能具有较差的药代动力学和物理性质(诸如免疫原性)以及和制造上的困难。因此,需要对这样的现有技术的改善或替代技术。
发明内容
本发明提供一种双特异性抗体,其包含抗MUC1 VHH的抗体片段和抗CD16 VHH的抗体片段,其中所述抗MUC1 VHH的抗体片段,其氨基酸序列如SEQ ID No.1所示,所述抗CD16VHH的抗体片段,其氨基酸序列如SEQ ID No.2或SEQ ID No.3所示。
在一些实施方式中,所述双特异性抗体中的抗MUC1 VHH的抗体片段和抗CD16 VHH的抗体片段之间是通过连接肽序列连接而成的。
在一些实施方式中,所述连接肽序列的氨基酸序列如SEQ ID No.4所示。
在一些实施方式中,所述双特异性抗体中的抗MUC1 VHH的抗体片段和抗CD16 VHH的抗体片段之间的连接方式为:抗MUC1 VHH-连接肽-抗CD16 VHH。
在另一些实施方式中,所述双特异性抗体中的抗MUC1 VHH的抗体片段和抗CD16VHH的抗体片段之间的连接方式为:抗CD16 VHH-连接肽-抗MUCl VHH。
本发明另一方面提供一种药物组合物,其包含本发明所述的双特异性抗体以及药学上可接受的附加剂,所述附加剂包括载体、稳定剂和/或赋形剂。
本发明另一方面提供一种编码本发明所述双特异性抗体的核苷酸序列、包含所述核苷酸序列的表达载体、或用所述表达载体转化或转染的宿主。
本发明另一方面还提供一种治疗患有癌症的对象方法,所述方法包括向有此需要的对象施用治疗有效量的本发明所述的双特异性抗体或其药物组合物。
本发明另一方面还提供本发明所述的双特异性抗体在制备治疗癌症的药物中的应用。
本发明的再一方面提供一种抗体偶联物,其包含偶联于纳米材料(例如为纳米微粒)上的如上所述的双特异性抗体。所述纳米微粒可以为生物可降解纳米材料,优选为聚乳酸-羟基乙酸、聚乳酸、聚己内酯、聚丁二醇丁二酸酯、聚苯胺、聚碳酸酯、乙丙交酯共聚物或乙交酯己内酯共聚物中的任意一种或至少两种的混合物,更优选为聚乳酸-羟基乙酸、聚乳酸和/或聚己内酯。
本发明的再一方面提供一种药物组合物,其包含本发明所述的抗体偶联物。所述药物组合物还可以包含细胞毒性效应细胞。所述细胞毒性效应细胞优选为白细胞,选白T细胞、NK细胞、NKT细胞、巨噬细胞、嗜中性粒细胞、嗜酸性粒细胞,优选为包含选自T细胞和NK细胞的细胞毒性效应细胞。所述药物组合物优选用于免疫治疗。
本发明的再一方面还提供一种治疗患有癌症的对象方法,所述方法包括向有此需要的对象施用治疗有效量的本发明所述的抗体偶联物或其药物组合物。
本发明的再一方面还提供本发明所述的抗体偶联物在制备治疗癌症的药物中的应用。
本发明提供的一种抗MUC1-CD16的双特异抗体,由抗CD16和抗MUC1的纳米抗体片段构建在同一抗体分子中,能够特异性地与这两者结合,引导NK细胞靠近MUC1表达阳性细胞,进而通过ADCC效应,杀伤或抑制肿瘤的生长。
本发明使用的抗体片段为来自骆驼重链抗体的可变区序列,与抗原具有高结合亲和性。本发明将识别MUC1和识别CD16的抗体片段构建在同一抗体分子中,可以特异性结合MUC1和CD16分子,促进NK细胞对MUC1阳性表达细胞的杀伤作用,对MUC1阳性肿瘤的生长起到抑制的作用。该分子能在原核表达系统中可溶性表达,有利于后续的分离纯化。该分子具有较好的热稳定性和高度可溶性。本发明使用的纳米抗体序列与人免疫球蛋白的众链可变区序列具有较高的同源性,抗原性弱。此外,本发明提供的双特异性抗体都已被人源化。
附图说明
图1:抗CD16-MUC1双特异纳米抗体的结构示意图。
图2:抗MUC1-抗CD16抗体的纯化电泳图。
图3:流式细胞术检测双特异抗体可结合肿瘤细胞表面的MUC1。
图4:Western blot检测双特异抗体可特异结合LS174T、HT29以及SKOV3等细胞表达的MUC1,而与CHO,HepG2中总蛋白无结合。
图5:Mucl-VHH-CD16-VHH-1(其中Mucl-VHH的序列如SEQ ID NO.1所示,CD16-VHH的序列如SEQ ID NO.2所示)介导NK/PBMCs细胞对肿瘤细胞的杀伤作用
图6:Muc1-VHH-CD16-VHH-2(其中Mucl-VHH的序列如SEQ ID NO.1所示,CD16-VHH的序列如SEQ ID NO.3所示)介导NK/PBMCs细胞对肿瘤细胞的杀伤作用。
图7:显示双特异性抗体BiTE(CD16-MUC1)及抗体偶联物BiTE(CD16-MUC1)-PLGA联合NK细胞对肺癌细胞A549(人非小细胞肺癌细胞)的杀伤率。
图8:显示双特异性抗体CD16-MUC1 BiTE或双特异性抗体偶联物CD16-MUC1 BiTE-PLGA NPs联合NK对肺癌细胞A549(人非小细胞肺癌细胞)的杀伤率,其中效靶比为4∶1。
具体实施方式
在描述本发明的抗体、抗体偶联物、方法和组合物之前,应当理解,本发明并不局限于所述的特定抗体、抗体偶联物、方法或组合物,因此当然可能会有所不同。还应当理解的是,本文所使用的术语仅用于描述特定的实施方案,并非限制性的。描述实施例是为本领域的普通技术人员提供如何制造和使用本发明的完整的公开内容和描述,并非旨在限制发明人视为其发明的范围,也并非旨在表示下面的实验是进行的全部或仅有的实验。已经努力确保所用数字(例如量、温度等)的准确度,但一些实验误差和偏差应该予以考虑。
除非另有定义,否则本文使用的所有技术和科学术语具有如由本发明所属领域的普通技术人员通常理解的相同的含义。现在描述一些潜在的和优选的方法和材料,尽管类似或等同于本文描述的任何方法和材料可以用于实施或测试本发明。本文提及的所有出版物在此通过引用并入,以公开和描述与所引用的出版物有关的方法和/或材料。可以理解的是,存在矛盾的情况下,以本公开内容取代所引用的出版物中的任何公开内容。
如在阅读本公开内容时对本领域技术人员显而易见的,每个本文所述和说明的单独的实施方案具有分立的组件和特征,其可容易地与其他若干实施方案中的特征分离或结合,而不脱离本发明的范围或实质。可以按所列举事件的顺序或逻辑上可能的任何其他顺序实施任何列举的方法。
定义
应当注意的是术语“一种(个)”实体是指一种(个)或多种(个)该实体,例如,“一种二价抗体”被理解为代表一种或多种二价抗体。同样地,这些术语“一种(个)”、“一种(个)或多种(个)”和“至少一种(个)”在此可互换使用。
“同源性”或“同一性”或者“相似性”是指两个多肽序列间的或两个核酸分子间的序列相似性。通过比较在每个序列的位置而测定同源性,每一个序列可能为了比较目的而被比对。当这种比较序列的位置被相同碱基或氨基酸占据时,那么这些分子在那个位置是同源的。序列间的同源性程度随着这些序列共有的匹配或同源位置的数量而变。“不相关的”或“非同源的”序列是指与本发明的序列之一具有低于40%的同一性,优选低于25%的同一性。
多核苷酸或多核苷酸区(或多肽、多肽区)与另一序列具有某种百分比(例如,60%、60%、65%、70%、75%、80%、85%、90%、95%、98%或99%)的“序列同一性”是指当比对时,在比较这两个序列时该百分比的碱基(或氨基酸)是相同的。使用本领域已知的软件程序可确定这种比对和同源性或序列同一性的百分比。
术语“等同的多核苷酸”是指与一种参照核酸序列或其互补序列具有一种一定程度同源性或序列同一性的核酸序列。在一个方面,核酸的同源体能够杂交至此核酸或其互补序列。同样地,“等同的多肽”是指与对照多肽的氨基酸序列具有一定程度同源性或序列同一性的多肽。在某些方面,序列的同一性至少约为70%、75%、80%、85%、90%、95%、98%或99%。在一些方面,这种等同序列保持对照序列的活性(例如表位结合)或结构(例如盐桥)。
在此公开的每一个多肽或多核苷酸,它的等同物也是预期的。在一个方面,一种多肽的等同物包括氨基酸残基的改变(即,缺失、增加或取代)。在一方面,一种多肽等同物包括不多于两种氨基酸残基的改变。在一方面,一种多肽的等同物包括不多于3、4或5种氨基酸残基的改变。在一些方面,这种氨基酸改变位于对参照的多肽活性不是关键的残基上。对多肽活性关键的残基能够通过位点特异性突变分析、甚至是序列比对(因为这种序列是高度保守的)而被轻易地测试。
本文所用的“抗体”或“抗原结合多肽”是指特异性地识别和结合到一种或多种抗原的多肽或多肽复合物。抗体是全抗体和其任何抗原结合片段或单链。因此,术语“抗体”包括至少含有免疫球蛋白一部分的分子的任何蛋白或肽,此免疫球蛋白分子的一部分具有结合至抗原的生物活性。这样的例子包括,但不限于,重/轻链或其配体结合部分的互补性决定区(CDR)、重链或轻链可变区、重链或轻链恒定区、构架(FR)区或其任何部分、或结合蛋白的至少一部分。术语抗体也包含一旦激活即具备抗原结合能力的多肽或多肽复合物。
本文所用的术语“抗体片段”或“抗原结合片段”是抗体的一部分,例如F(ab′)2、F(ab)2、Fab′、Fab、Fv、scFv等。无论结构如何,抗体片段都与完整抗体所识别的相同抗原结合。此术语“抗体片段”包括适配体、镜像体(spiegelmers)、双体。术语“抗体片段”也包括任何合成的或基因工程化的蛋白,这些蛋白通过结合至特异抗原以形成一种复合体而像抗体一样起作用。
本发明的抗体、抗原结合多肽、其变体或衍生物包括但不限于多克隆的、单克隆的、多特异性的、人类的、人源的、灵长化的(primatized)或嵌合的抗体、单链抗体、表位结合片段(例如,Fab、Fab′和F(ab′)2、Fd、Fvs、单链Fvs(scFv))、单链抗体、二硫键连接的Fvs(sdFv)、包含VK或VH域的片段、由Fab表达文库生成的片段、以及抗独特型(anti-Id)抗体(包括例如在此公开的anti-Id抗体至LIGHT抗体)。本发明的免疫球蛋白或抗体分子可以是任何类型(例如,IgG、IgE、IgM、IgD、IgA和IgY)、种类(例如,IgG1、IgG2、IgG3、IgG4、IgA1和IgA2)或亚类的免疫球蛋白分子。
轻链分为kappa或lambda(κ,λ)。每一个重链种类可与kappa或lambda轻链结合。总体来说,轻链和重链彼此共价结合,当通过杂交瘤、B细胞或基因工程的宿主细胞产生免疫球蛋白时,两个重链的“尾”部通过共价的二硫键或非共价键而彼此结合。在重链中,氨基酸序列从在Y构象叉状端的N端运行到在每一个链的底部的C端。
轻链和重链都被分为结构和功能的同源性的区。术语“恒定的”和“可变的”被功能性地使用。在这一方面,应当理解的是,轻链可变域(VK)和重链可变域(VH)都决定抗原识别和特异性。相反地,轻链(CK)和重链(CH1、CH2或CH3)的恒定域赋予重要的生物特性,例如分泌、经胎盘流动性、Fc受体结合、补体结合等。按照惯例,恒定区结构域的编号随着它们变得远离抗原结合位点或抗体氨基端时而增加。N端部分是可变域,而C端部分是恒定域;CH3和CK结构域实际上分别包含了轻链和重链的羧基端。
如上所述,可变区允许抗体选择性地识别及特异性地结合抗原上的表位。也就是说,抗体的VK结构域和VH结构域,或互补决定区(CDR)的亚类,结合以形成确定出三维的抗原结合位点的可变域。这种四元抗体结构形成了呈现在Y的每一个臂末端的抗原结合位点。更具体地,抗原结合位点由在每条VH和VK链上的三个CDR确定(即,CDR-H1、CDR-H2、CDR-H3、CDR-L1、CDR-L2和CDR-L3)。在一些例子中,例如,源自骆驼科或基于骆驼科免疫球蛋白的工程化的某些免疫球蛋白分子,完整免疫球蛋白分子可能仅由重链组成,而不具有轻链,参见例如Hamers-Casterman et al.,Nature 363:446-448(1993)。
在天然抗体中,存在于每一个抗原结合域中的六个“互补决定域”或“CDR”是短的、非连续的氨基酸序列,它们被特异地定位以当抗体在水性环境中呈现三维构型时形成抗原结合域。抗原结合域中的剩余部分的氨基酸(称“构架区”)展现出较低的分子间可变性。构架区主要采用β-折叠构象,CDR形成环,这些环连接β-折叠结构,在某些情况下构成β-折叠结构的一部分)。因此,构架区用于形成支架,其通过链间的非共价的相互作用而将CDR以正确的方向定位。由排列的CDR形成的抗原结合域定义出在免疫反应性抗原上的表位互补的表面。这种互补表面促使抗体非共价地结合至它的同源表位。对于任何给定的重链或轻链的可变域,本领域普通技术人员能够轻易地分别鉴别包含CDR和构架区的这些氨基酸,因为它们已经被准确定义(参见“Sequences of Proteins of Immunological Interest,”Kabat,E.,et al.,U.S.Department of Health and Human Services,(1983);以及Chothia and Lesk,J.MoI.Biol.,196:901-917(1987),通过引用将它们整体并入本文中)。
如果在本领域中对于一个术语使用和/或接受两个或更多个定义,那么在本文中,除非另有说明,否则该术语的定义意在包括所有这样的涵义。一个具体的例子是,术语“互补决定区”(“CDR”)用来描述在重链多肽和轻链多肽的可变区内都找得到的非连续抗原结合位点。这个特定的区域已在以下文献中有描述:Kabat et al,U.S.Dept.of Health andHuman Services,“Sequences of Proteins of Immunological Interest”(1983)和Chothia et al.,J.MoI.Biol.196:901-917(1987),通过引用将它们合并至本文中。根据Kabat和Chothia对于CDR的定义,其包括彼此对比时的氨基酸残基的重叠或子群体。但是,使用任何一种关于抗体或其可变区的CDR的定义都被认为是在本发明定义和使用的术语的范围内。包含如以上任一引用文献所定义的CDR的合适氨基酸残基被作为对比而列于以下表格中。包含特定CDR的准确残基数量根据CDR的序列和大小而变化。对于抗体的可变区氨基酸序列来说,本领域技术人员可通过常规方法确定出包含特定CDR的残基。
Kabat等也定义了可适用于任何抗体的可变域序列的编号系统。本领域技术人员可明确地将该“Kabat编号”系统应用至任何可变域序列,而不需要依赖于超出序列本身的任何实验数据。如本文所使用的,“Kabat编号”是指在以下文献中阐述的编号系统:Kabatet al.,U.S.Dept.of Health and Human Services.“Sequence of Proteins ofImmunological Interest”(1983)。
“特异性结合”或“对……有特异性”通常是指抗体通过其抗原结合域结合至表位,并且该结合需要抗原结合域和表位之间的一定的互补性。根据这个定义,当抗体经由其抗原结合域相比于结合至随机的不相关的表位而言能够更快速地结合至特定表位,则称该抗体“特异性结合”至该表位。术语“特异性”被用来定量特定抗体结合特定表位的相对亲和性。例如,对于给定的表位,抗体“A”被视为比抗体“B”具有更高的特异性,或者抗体“A”结合至表位“C”的特异性高于其结合至相关的表位“D”的特异性。
本文使用的术语“治疗”是指治疗性或预防性手段,其中对象被预防或减慢(缓解)不期望的病理学变化或紊乱,例如癌症的进展。有益的或期望的临床结果包括,但不限于,症状的减轻、疾病程度的减小、疾病状态的稳定(即不恶化)、疾病进展的延迟或减慢、疾病状态的改善或缓和以及缓解(无论是局部的或整体的),无论这些结果是否是可检测的或不可检测的。“治疗”还指与不接受治疗预期的存活期相比存活期延长。需要治疗的对象包括已患有疾病或病症的对象以及倾向于患有该疾病或病症的对象或需要预防该疾病或病症的对象。
“对象”或“个体”或“动物”或“患者”或“哺乳动物”是指任何期望诊断、预后或治疗的对象,特别是哺乳动物对象。哺乳动物对象包括人、驯养动物、农畜以及动物园动物、竞技动物或宠物,例如狗、猫、豚鼠、兔、大鼠、小鼠、马、牛、奶牛等。
术语“有治疗需要的患者”或“有治疗需要的对象”包括可从施用本发明的抗体或组合物中受益的对象,例如哺乳动物对象,以实现例如检测、诊断程序和/或治疗的目的。
本发明的双特异性抗体还包括位于抗体片段之间的接头序列,通常为2-20个氨基酸构成的短肽。这些接头序列使得各组分之间被合理地定位而实现各组分的功能活性。
优选地,接头序列包括2至20个氨基酸序列,更优选为5至20个氨基酸。接头序列优选为柔性接头序列,因而其不会限制效应分子或多肽在单个不期望的构象中。接头序列优选主要由具有小侧链的氨基酸构成,例如甘氨酸、丙氨酸和丝氨酸,从而提供所述柔性。优选地,接头序列的约80以上或更多比例的氨基酸是甘氨酸、丙氨酸或丝氨酸残基,特别是甘氨酸和丝氨酸残基。合适的接头序列的例子有GGGGS(G4S),即Gly Gly Gly Gly Ser;或(G4S)3。也可以使用其他不同的接头序列,包括已被成功地用于连接不同抗体可变区的多种柔性接头设计。接头序列的大小和序列组成可通过常规的电脑建模及技术来确定。
以上描述的任何的抗体或多肽可进一步地包括额外的多肽,例如,指导所编码的多肽的分泌的信号肽、如本文所述的抗体恒定区或其他如本文所述的异源多肽。
本领域普通技术人员将理解,本文所公开的抗体可被修饰,使得它们在氨基酸序列上不同于它们所其衍生的天然结合多肽。例如,源自一种特定蛋白的多肽或氨基酸序列可以是相似的,例如,对起始序列具有一定百分数的一致性,例如它可能和起始序列具有60%、70%、75%、80%、85%、90%、95%、98%或99%的一致性。
此外,可在“非必需”氨基酸区进行核苷酸或氨基酸的保守性取代、缺失或插入。例如,源自特定蛋白多肽或氨基酸可以同起始序列一样,除了一个或多个单个的氨基酸取代、插入、或缺失(例如,一个、两个、三个、四个、五个、六个、七个、八个、九个、十个、十五个、二十个或更多个单独的氨基酸取代、插入或缺失)。在某些实施方式中,相对于起始序列,源自特定蛋白的多肽或氨基酸序列具有一至五、一至十、或一至二十个单独的氨基酸取代、插入或缺失。
在某些实施方式中,抗原结合多肽包含通常不与抗体相连的氨基酸序列或一个或多个部分。下文详细描述了示例性的修饰。例如,本发明的片段可包含柔性连接序列,或可被修饰以添加了功能部分(例如PEG、药物、毒素或标记)。
本发明的抗体、其变体或衍生物包括被修饰的衍生物,即任何类型分子共价连接至抗体,只要该共价连接不阻止抗体结合至表位。例如,但并非为了限制,抗体可以被修饰,例如糖基化、乙酰化、聚乙二醇化、磷酸化、酰胺化、通过已知的保护基或阻断基衍生化,蛋白酶切、连接至细胞配体或其他蛋白等。通过已知的技术可实施大量化学修饰中的任何修饰,包括但不限于特异的化学裂解、乙酰化、甲酰化、衣霉素的代谢合成等等。此外,抗体可以包含一个或多个非典型氨基酸。
在其他实施方式中,本发明的抗原结合多肽可以包含保守的氨基酸取代。
在“保守的氨基酸取代”中,一个氨基酸残基被具有相似侧链的氨基酸残基取代。具有相似侧链的氨基酸残基家族已经在本领域被定义,包括碱性侧链(例如赖氨酸、精氨酸、组氨酸)、酸性侧链(例如天冬氨酸、谷氨酸)、不带电荷的极性侧链(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸)、非极性侧链(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、蛋氨酸、色氨酸)、b分支侧链(例如苏氨酸、缬氨酸、异亮氨酸)及芳香族侧链(例如酪氨酸、苯丙氨酸、色氨酸、组氨酸)。因此,在免疫球蛋白多肽的非必须氨基酸残基更适宜由来自同样侧链家族的其他氨基酸残基取代。在另一个实施方式中,氨基酸链可以被结构相似但侧链家族成员的顺序或组成上不同的链取代。
下表提供了保守氨基酸取代的非限制的例子。在表中相似性分数0或更高来表明在两种氨基酸的保守取代。
在一些实施方式中,抗体可以被结合至治疗性的制剂、前药、肽、蛋白、酶、病毒、脂类、生物效应调节物、药物制剂或PEG。
这些抗体可以被结合或融合至治疗性的制剂,这种治疗性制剂可包括可检测标记(例如放射性标记)、免疫调节剂、激素、酶、寡核苷酸、光敏治疗剂或诊断剂、细胞毒性制剂(药物或毒素)、超声增强剂、非放射性标记、它们的组合和其他本领域已知的制剂。
C | G | P | S | A | T | D | E | N | Q | H | K | R | V | M | I | L | F | Y | W | |
W | -8 | -7 | -6 | -2 | -6 | -5 | -7 | -7 | -4 | -5 | -3 | -3 | 2 | -6 | -4 | -5 | -2 | 0 | 0 | 17 |
Y | 0 | -5 | -5 | -3 | -3 | -3 | -4 | -4 | -2 | -4 | 0 | -4 | -5 | -2 | -2 | -1 | -1 | 7 | 10 | |
F | -4 | -5 | -5 | -3 | -4 | -3 | -6 | -5 | -4 | -5 | -2 | -5 | -4 | -1 | 0 | 1 | 2 | 9 | ||
L | -6 | -4 | -3 | -3 | -2 | -2 | -4 | -3 | -3 | -2 | -2 | -3 | -3 | 2 | 4 | 2 | 6 | |||
I | -2 | -3 | -2 | -1 | -1 | 0 | -2 | -2 | -2 | -2 | -2 | -2 | -2 | 4 | 2 | 5 | ||||
M | -5 | -3 | -2 | -2 | -1 | -1 | -3 | -2 | 0 | -1 | -2 | 0 | 0 | 2 | 6 | |||||
V | -2 | -1 | -1 | -1 | 0 | 0 | -2 | -2 | -2 | -2 | -2 | -2 | -2 | 4 | ||||||
R | -4 | -3 | 0 | 0 | -2 | -1 | -1 | -1 | 0 | 1 | 2 | 3 | 6 | |||||||
K | -5 | -2 | -1 | 0 | -1 | 0 | 0 | 0 | 1 | 1 | 0 | 5 | ||||||||
H | -3 | -2 | 0 | -1 | -1 | -1 | 1 | 1 | 2 | 3 | 6 | |||||||||
Q | -5 | -1 | 0 | -1 | 0 | -1 | 2 | 2 | 1 | 4 | ||||||||||
N | -4 | 0 | -1 | 1 | 0 | 0 | 2 | 1 | 2 | |||||||||||
E | -5 | 0 | -1 | 0 | 0 | 0 | 3 | 4 | ||||||||||||
D | -5 | 1 | -1 | 0 | 0 | 0 | 4 | |||||||||||||
T | -2 | 0 | 0 | 1 | 1 | 3 | ||||||||||||||
A | -2 | 1 | 1 | 1 | 2 | |||||||||||||||
S | 0 | 1 | 1 | 1 | ||||||||||||||||
P | -3 | -1 | 6 | |||||||||||||||||
G | -3 | 5 | ||||||||||||||||||
C | 12 |
通过耦合至化学发光的化合物来标记抗体,则该抗体可以被检测。然后,通过检测荧光(出现在化学反应过程中)的存在,来确定化学发光标记的抗原结合多肽的存在。极其有用的化学发光标记化合物的例子是鲁米诺、异鲁米诺、theromatic吖啶酯、咪唑、吖啶盐和草酸酯。
编码抗体的多核苷酸及制备抗体的方法
本发明也提供了分离的编码双特异抗体、其变体或衍生物的多核苷酸或核酸分子。
本发明的多核苷酸可在相同的多核苷酸分子上或在独立的多核苷酸分子上编码全部的VHH、其变体或衍生物。此外,本发明的多核苷酸可在相同的多核苷酸分子上或在独立的多核苷酸分子上编码抗体的或VHH、其变体或衍生物的一部分。
在某些实施方式中,制备的抗体将不会在受试动物(例如人体)上引起有害的免疫反应。在一个实施方式中,使用领域认可的技术修饰本发明的抗原结合多肽、其变体或衍生物以降低它们的免疫原性。例如,抗体可以是人源化的、灵长化的、去免疫性的,或者可制备嵌合抗体。
本发明的双特异抗体的结合特异性能通过体外分析(例如,免疫沉淀法、放射免疫测定(RIA)或免疫酶联吸附法(ELISA))来确定。
生产系统和方法
本发明还提供了用于生产本发明的双特异性抗体的方法和系统。本领域公知的适于生产抗体的细胞包括人类细胞(例如CHO细胞)、哺乳动物细胞和细菌细胞。用细菌细胞来生产双特异抗体存在重大挑战。然而,正如在实施例中所展示的,当在细菌细胞中表达抗体时,即使是两个肽链都表达于相同细胞中时,产生的抗体很大程度上也是可溶的。
因此,在一个实施方式中,本发明提供了包含一个或多个编码本发明的双特异抗体的两个链的多核苷酸的宿主细胞。在一个方面,单核苷酸构建体(例如质粒)包括两个编码序列。在另一个方面,本发明提供两种不同多核苷酸构建体,每一个编码其中一个多核苷酸链。在一个实施方式中,本发明还提供了包含本发明的双特异抗体的两个多肽链的宿主细胞。
在一些方面,所述宿主细胞是人类细胞。在一些方面,所述宿主细胞是哺乳动物细胞。在一些方面,所述宿主细胞是酵母细胞。在一些方面,所述宿主细胞是细菌细胞,包括G+和G-细菌细胞。代表性的细菌细胞包括,但不限于,大肠杆菌和鼠伤寒沙门氏菌。
在某些方面,本发明还提供制备本发明的双特异性抗体的方法。在一个方面,该方法需要在宿主细胞中表达该抗体的两个肽链并且从细胞裂解液中提取抗体。此外,本发明还提供由这些方法得到的双特异性抗体。
本发明所用的纳米材料可以是可药用的纳米材料,优选为生物可降解纳米材料,例如为纳米微粒。所述纳米材料更优选为聚乳酸-羟基乙酸、聚乳酸、聚己内酯、聚丁二醇丁二酸酯、聚苯胺、聚碳酸酯、乙丙交酯共聚物或乙交酯己内酯共聚物中的任意一种或至少两种的混合物,最优选为聚乳酸-羟基乙酸(PLGA)、聚乳酸(PLA)和/或聚己内酯(PCL)。所述纳米微粒的平均粒径可以例如为大约10-990nm,例如为约900nm、约850nm、约800nm、约750nm、约700nm、约650nm、约600nm、约550nm、约500nm、约450nm、约400nm、约350nm、约300nm、约250nm、约200nm、约150nm、约100nm或小于100nm,例如大小为约90nm、约80nm、约70nm、约60nm、约50nm、约40nm、约30nm、约20nm或约10nm。优选纳米颗粒的平均粒径在10-500nm范围内,更优选在10-300nm、50-250nm、80-250nm、100-250nm或100-200nm范围内。
本发明偶联物的制备
以下描述本发明的双特异性抗体偶联物的制备方法。所述制备方法包括以下步骤:
(1)纳米材料的制备、收集和活化;
(2)将步骤(1)得到的纳米材料与双特异性抗体进行连接。
在步骤(1)中,所述纳米材料的制备包括:利用溶剂将纳米材料完全溶解,搅拌,加水,形成均匀的乳浊液。其中所述搅拌可以在500-20000rpm/min的转速下进行,例如转速可以是500rpm/min、700rpm/min、800rpm/min、1000rpm/min、1100rpm/min、1200rpm/min、1300rpm/min、1400rpm/min、1480rpm/min、1500rpm/min、2000rpm/min、2200rpm/min、2500rpm/min、3000rpm/min、3500rpm/min、4000rpm/min、4200rpm/min、4500rpm/min、5000rpm/min、5500rpm/min、6000rpm/min、6500rpm/min、7000rpm/min、7500rpm/min、8000rpm/min、8500rpm/min、9000rpm/min、9500rpm/min、10000rpm/min、11000rpm/min、12000rpm/min、13000rpm/min、14000rpm/min、15000rpm/min、16000rpm/min、17000rpm/min、18000rpm/min、19000rpm/min或20000rpm/min。必要时,可以采用更高的转速。
优选地,所述纳米材料为聚乳酸-羟基乙酸(PLGA)、聚乳酸(PLA)、聚己内酯(PCL)、聚丁二醇丁二酸酯、聚苯胺、聚碳酸酯、乙丙交酯共聚物或乙交酯己内酯共聚物中的任意一种或至少两种的混合物。
优选地,所述溶剂为丙酮、丁酮、甲醇、乙醇或异丙醇中的任意一种或至少两种的混合物。
优选地,所述纳米材料的收集包括:通过离心收集制备的纳米材料,然后用去离子水重悬,重复操作2次洗涤纳米材料。所述离心可以在8000-15000rpm/min的转速下进行,例如转速可以是8000rpm/min、9000rpm/min、10000rpm/min、11000rpm/min、12000rpm/min、13000rpm/min、14000rpm/min、14500rpm/min、14800rpm/min、15000rpm/min。必要时,可以采用更高的转速。可以采用其他方法收集或进一步纯化纳米材料(纳米微粒)。纳米微粒可以具有如上所述的平均粒径
优选地,所述纳米材料的活化包括:用1-10mg/mL的1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐(EDS)和N-羟基琥珀酰亚胺(NHS)混合溶剂于室温活化纳米材料0.5-5h。
本发明的步骤(2)中,所述连接包括:活化后的纳米材料通过离心收集,然后用连接反应液洗涤纳米材料1次。将需要连接的双特异性抗体加入到连接反应液,然后用含有所述双特异性抗体的连接反应液重悬纳米材料,于室温进行接反应0.5-5h。反应结束后离心收集纳米材料,用杜氏磷酸盐缓冲溶液洗涤纳米材料2次,然后再重悬到杜氏磷酸盐缓冲溶液(D-PBS)中放4℃保存备用。可以采用其他方法进行纳米材料的活化。
本发明双特异性抗体偶联物的制备方法例如具体包括以下步骤:
(1)纳米材料的制备:利用丙酮将纳米材料完全溶解至浓度为5-30mg/mL,按照丙酮和去离子水1∶4的体积比,在500-1500rpm/min磁力搅拌的状态下将纳米材料与丙酮的溶液加入去离子水中,形成均匀的乳浊液,然后继续搅拌至丙酮挥发;
(2)纳米材料的收集:8000-15000rpm/min离心收集制备的纳米材料,然后用去离子水重悬,重复操作2次洗涤纳米材料;也可以进一步纯化纳米材料,以获得适当大小的纳米材料;
(3)纳米材料的活化:利用1-10mg/mL的1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐和N-羟基琥珀酰亚胺混合溶剂室温活化纳米材料0.5-5h;
(4)纳米材料连接抗体:将活化后的纳米材料离心收集,然后用0.1M、pH=8.0的杜氏磷酸盐缓冲溶液洗涤纳米材料1次,将需要连接的双特异性抗体加入到连接反应液,然后用含有所述双特异性抗体的连接反应液重悬纳米材料,室温连接反应0.5-5h,反应结束后离心收集纳米材料,用杜氏磷酸盐缓冲溶液洗涤纳米材料2次,然后再重悬到杜氏磷酸盐缓冲溶液中放4℃保存备用。
治疗和诊断方法
如上所述,本发明的双特异性抗体、其变体或衍生物可用于与癌症或感染性疾病相关的某些治疗和诊断方法中。
本发明进一步涉及基于抗体的疗法,其涉及将本发明的双特异性抗体施用至患者,例如动物、哺乳动物和人体,以治疗本文所述的一种或多种疾病或症状。本发明的治疗性组合物包括,但不限于,本发明的抗体(包括本文所述的其变体和衍生物)和编码本发明的抗体(包括本文所述的其变体和衍生物)的核酸或多核苷酸。
本发明的抗体可被用来治疗、抑制或预防以下疾病、失调或病症,包括与例如与增加细胞存活或抑制细胞凋亡相关的疾病或失调(如癌症)相关的恶性疾病、失调或病症,所述癌症包括但不限于滤泡性淋巴瘤、带有p53突变的癌症以及激素依赖的肿瘤(包括但不限于结肠癌、心脏肿瘤、胰腺癌、黑色素瘤、成视网膜瘤、恶性胶质瘤、肺癌、大肠癌、睾丸癌、胃癌、成神经母细胞瘤、粘液瘤、肌瘤、淋巴瘤、内皮瘤、成骨细胞瘤、破骨细胞瘤、骨肉瘤、软骨肉瘤、腺癌、乳腺癌、前列腺癌、Kaposi肉瘤);自体免疫失调(例如多发性硬化症、Sjogren综合征、Grave病、Hashimoto甲状腺炎、自体免疫糖尿病、胆汁性肝硬变、Behcet病、Crohn病、多肌炎、系统性红斑狼疮、免疫相关的肾小球肾炎、自体免疫的胃炎、自体免疫的血小板减少性紫癜及类风湿性关节炎);和病毒性感染(例如疱疹病毒、痘病毒及腺病毒)、炎症、移植物抗宿主病(急性和/或慢性)、急性移植物排斥和慢性移植物排斥。本发明的抗原结合多肽、其变体或衍生物被用于抑制癌症的发展、演进和/或转移,特别是在上文或随后的段落中列出的癌症。
可用本发明的抗体或其变体或衍生物治疗、预防、诊断和/或预后与细胞存活增加相关的其他的疾病或病症,包括但不限于,以下疾病的进展和/或转移:恶性肿瘤及相关疾病,例如白血病(急性白血病(如急性淋巴细胞白血病、急性髓细胞白血病(包括成髓细胞、早幼粒细胞白血病、髓单核细胞、单核细胞和白血病细胞))和慢性白血病(如慢性髓细胞(粒细胞)白血病和慢性淋巴细胞白血病))、真性红细胞增多、淋巴瘤(如Hodgkin病和非Hodgkin病)、多发性骨髓瘤、Waldenstrom巨球蛋白血症、重链病和实体肿瘤(包括但不限于:肉瘤和癌(如纤维肉瘤、粘液肉瘤、脂肪肉瘤、软骨肉瘤、骨原性肉瘤、脊锁瘤、血管肉瘤、内皮肉瘤、淋巴肉瘤、淋巴内皮肉瘤、滑膜瘤、间皮瘤、Ewing瘤、平滑肌瘤、横纹肌肉瘤、结肠癌、胰腺癌、胸腺癌、卵巢癌、前列腺癌、鳞状细胞癌、基底细胞癌、腺癌、汗腺瘤、乳头状癌、乳头状腺癌、囊腺癌、髓样癌、支气管癌、肾细胞癌、肝癌、胆管癌、绒毛膜癌、精原细胞癌、胚胎性癌、Wilm肿瘤、宫颈癌、睾丸瘤、肺癌、小细胞肺癌、膀胱癌、上皮癌、胶质瘤、形状细胞瘤、成神经管细胞瘤、颅咽管瘤、室管膜瘤、松果体瘤、成血管细胞瘤、听神经瘤、间胶质瘤、脑膜瘤、黑色素瘤、成神经细胞瘤和成视网细胞膜瘤))。
本发明的抗体也可通过靶定微生物和免疫细胞来影响微生物的消除而用于治疗由微生物引起的感染性疾病或杀灭微生物。在一个方面,该微生物是病毒(包括RNA和DNA病毒)、革兰氏阳性菌、革兰氏阴性菌、原生动物或真菌。
对于任何特定病人特异的剂量和治疗方案将取决于多种因素(包括所使用的特异抗原结合多肽、其变体或衍生物、抗体偶联物,患者的年龄、体重、总体健康、性别、饮食、施用时间、排泄率、药物的联合以及被治疗的特定疾病的严重程度)。医护人员对这样的因素的判断属于本领域普通技术人员判断范围内。该剂量还会基于被治疗的个体病人、施用途径、配方的类型、所使用的组合物的特点、疾病的严重程度及所期待的效果。使用的剂量可通过本领域所公知的药理学和药代动力学的原则决定。
双特异抗体、变体、抗体偶联物的施用方法包括但不限于:皮内的、肌内的、腹腔的、静脉的、皮下的、鼻内的、硬膜外的和口腔途径。抗原结合多肽或组合物可以通过任何方便途径施用,例如,通过输注或弹丸式注射、通过上皮或黏膜保护层的吸收(例如口腔黏膜、直肠和肠黏膜等等);它可同其他生物活性制剂一起使用。因此,包含本发明的抗原结合多肽的药用组合物可以经口腔、直肠、肠外、阴道内、腹腔、局部(如通过粉剂、软膏、滴剂或皮肤药贴)、含服施用或是通过口腔或鼻的喷雾施用。
本文所用的术语“肠胃外的”是指施用的方式,这种方式包括静脉内的、肌内的、腹腔的、胸骨内的、皮下的和关节内的注射和输注。
施用可以是系统的或局部的。此外,可预期通过任何合适途径将本发明的抗体引入至中枢神经系统,这些途径包括:脑室内和鞘内的注射;通过脑室内导管(例如连接至储存器(例如Ommaya储存器))可以促进脑室内的注射。也可采用肺部给药,例如通过使用吸入器或喷雾器及带有气雾剂的配方给药。
将本发明的双特异抗体、抗体偶联物或组合物局部施用至需要治疗的区域可能是合乎需要的;这可通过例如(但不限于)以下方式实现:手术中局部输注、局部施用(例如在术后联合使用伤口敷料)、注射、通过导管、通过栓剂或通过植入物(植入物为多孔的、非多孔的、非渗透的或凝胶状的材料,包括薄膜(例如硅胶膜)或纤维))。优选地,当给予包括本发明的蛋白(包括抗体)时,需注意使用此蛋白不吸附的材料。
本发明的抗体、抗体偶联物在炎症、免疫或恶性疾病、失调或病症的治疗、抑制和预防中的有效量,可以通过标准的临床技术来确定。此外,可以选择性地采用体外测定来帮助识别最佳剂量范围。在配方中采用的准确的剂量也将取决于给药的途径以及疾病、失调、或病症的严重性,并且应当根据执业医生的判断和每一个患者的情况来决定。有效剂量可以从源自体外的或动物模型测试系统的剂量-反应曲线推论出。
作为一般性的建议,给予患者的本发明的抗原结合多肽的剂量通常为0.1mg/kg至100mg/kg患者体重、0.1mg/kg至20mg/kg患者体重,或1mg/kg至10mg/kg患者体重。总体米说,由于对外源的多肽的免疫反应,在人体内,人的抗体比米自其他种属的抗体具有更长的半衰期。因此,人抗体的较低给药剂量和更低给药频率通常是可能的。而且,本发明的抗体的给药频率和剂量可通过修饰(例如脂化)而增强这些抗体的摄入和组织穿透力(例如进入大脑)来降低。
所述的偶联物用于人类的施用剂量将取决于诸如患者年龄、体重、身高、性别、一般医学病状和既往病史的因素而变化。可能需要给接受者提供在约01μg/kg-25mg/kg范围内的作为单次静脉输注的偶联物剂量(以多特异性抗体偶联物中的第一结合部分和第二结合部分的总量计),但也可以施用更低或更高的剂量,视情况决定。举例来说,对于70kg患者,0.1μg/kg-20mg/kg的剂量是0.7μg-1400mg。需要时剂量可以重复,例如每周一次持续4-10周、每周一次持续8周或每周一次持续4周。在维持疗法中,需要时其还可以较低频率给予,诸如每隔一周一次持续若干个月,或者每月或每季度一次持续许多个月。也可以每个疗程连续施用2次、3次、4次、5次或6次,在连续施用后间隔例如约10天、15天、20天、25天、30天、35天、40天、45天、50、55天或60天或更长时间再进行下一个疗程。
所述的偶联物可以与效应细胞如白细胞(NK细胞)一起施用,例如一起静脉回输。当偶联物与效应细胞一起施用时,例如每周一次持续4-10周、每周一次持续8周或每周一次持续4周。在维持疗法中,需要时偶联物与效应细胞还可以较低频率给予,诸如每隔一周或数周一次持续若干个月,或者每月或每季度一次持续许多个月。也可以每个疗程连续施用2次、3次、4次、5次或6次,在连续施用后间隔例如约10天、15天、20天、25天、30天、35天、40天、45天、50、55天或60天或更长时间再进行下一个疗程。
用于治疗感染或恶性疾病、病症或失调的方法(包含给予本发明的抗体、其变体或衍生物、抗体偶联物),在用于人体前,通常在体外被测试,然后在可接受动物模型中进行体内测试,以获得期待的治疗或预防活性。合适的动物模型(包括转基因动物)对本领域普通技术人员是熟知的。例如,表明本文所述的抗原结合多肽的治疗效用的体外实验包括抗原结合多肽在细胞系或病人组织样品上的效果。利用本领域技术人员所知的技术(例如在本文其他地方公开的试验)可确定抗原结合多肽在细胞系和/或组织样品上的效果。根据本发明,可被用来确定是否需要使用特异性的抗原结合多肽的体外试验包括体外细胞培养试验(其中病人组织样品在培养基中生长并被暴露于或以其他方式给予抗体)以及观察这种抗体在该组织样品上的效果。
在进一步的实施方式中,本发明的组合物与抗肿瘤剂、抗病毒剂、抗菌剂或抗生素制剂或抗真菌制剂联合施用。任何这些在本领域已知的制剂都可以在本发明的组合物中被给予。
在另一个实施方式中,本发明的组合物与化疗制剂联合给予。可与本发明的组合物一起给予的化疗制剂包括但不限于:抗生素衍生物(例如阿霉素、博来霉素、道诺霉素、防线菌素)、抗雌激素(例如他莫昔芬)、抗代谢物(例如氟尿嘧啶、5-FU、氨甲嘌呤、氟尿苷、干扰素a-2b、谷氨酸、普卡霉素、巯嘌呤和6-巯基鸟嘌呤)、细胞毒剂(例如卡莫司汀、BCNU、洛莫司汀、CCNU、阿糖胞苷、环磷酰胺、雌莫司汀、羟基脲、甲苄肼、丝裂霉素、白消安、顺铂及长春新碱硫酸盐)、激素(例如甲羟孕酮、雌莫司汀磷酸钠、乙炔雌二醇、雌二醇、醋酸甲地孕酮、甲羟睾酮、二磷酸己烯雌酚、氯烯雌醚及睾内酯)、氮芥衍生物(苯丙氨酸氮芥、苯丁酸氮芥、二氯甲基二乙胺(氮芥)、噻替哌)、类固醇类及制品(倍他米松磷酸钠)及其他(例如达卡巴嗪、天冬酰胺酶、米托坦、长春新碱硫化物、长春花碱硫化物和依托泊苷)。
在另外的实施方式中,本发明的组合物与细胞因子联合给予。可与本发明的组合物一起被给予的细胞因子包括但不限于:IL-2、IL-3、IL-4、IL-5、IL-6、IL-7、IL-10、IL-12、IL-13、IL-15、抗-CD40、CD40L和TNF-a。
在另外的实施方式中,本发明的组合物与其他治疗或预防疗法(例如放射治疗)联合给予。
组合物
本发明还提供了药物组合物。这样的组合物包含有效量的抗体和/或抗体偶联物及可接受的载体。在一个具体实施方式中,术语“药学上可接受的”意为由联邦的或州政府的监管机构许可的、或在美国药典或其他通常公认的药典所列的用于动物,更具体来说,用于人的。进一步地,“药学上可接受载体”通常将是无毒的固态的、半固态的或液态的填充剂、稀释剂、包封材料或任何类型的辅料。
术语“载体”是指药物使用所借助的稀释剂、佐剂、赋形剂或载体。这样的药物载体可以是无菌的液体,例如水和油,包括石油、动物、植物或合成来源的油,例如花生油、大豆油、矿物油、芝麻油等。当药物组合物被静脉注射地给药时,水是首选载体。盐溶液及水性的葡萄糖与甘油溶液也可以作为液相的载体被采用,特别是对可注射的溶液来说。合适的药物赋形剂包括:淀粉、葡萄糖、乳糖、蔗糖、明胶、麦芽、米、面粉、白垩、硅胶、硬脂酸钠、单硬脂酸甘油酯、滑石、氯化钠、脱脂奶粉、丙三醇、丙烯、乙二醇、水、乙醇等。如果需要,组合物也可以包含少量的润湿或乳化剂或pH缓冲剂,例如醋酸盐、柠檬酸盐或磷酸盐。也可预期加入抗菌制剂(例如苄醇或苯甲酸甲酯);抗氧化剂(例如抗坏血酸或亚硫酸氢钠);螯合剂(乙二胺四乙酸)和用于等张性调节的制剂(例如氯化钠或右旋糖)。这些组合物可以采取溶液、悬浮液、药片、药丸、胶囊、粉末、缓释配方等形式。该组合物可使用惯常的粘合剂和载体(例如甘油三酯)而作为栓剂配制。口服配方可包括标准的载体,例如,药物级的甘露醇、乳糖、淀粉、硬脂酸镁、糖精钠、纤维素、碳酸镁等等。在Remington′s Pharmaceutical Sciences中由E.W.Martin描述合适的药物载体的例子(通过引用将其合并至本中)。这样的组合物将包含治疗上有效量的抗原结合多肽(较好为纯化的形式)与合适数量的载体,以便为患者提供合适的给药方式。这种配方应适合给药的方式。这种亲代(parental)制剂可装于安瓿瓶、一次性注射器或是玻璃或塑料制成的多剂量瓶中。
在一种实施方式中,根据例行程序将该组合物配制成适于静脉给药于人类的药物组合物。通常,用于静脉给药的组合物为在无菌等渗的水性缓冲液中的溶液。在必要时,组合物也可包括增溶剂和局部麻醉药(例如利多卡因,以减轻注射部位疼痛)。组分通常是单独或是混合在一起以单位剂量形式提供,例如,在密闭容器(例如标明活性制剂的数量的安瓿或sachette)中的冻干粉剂或无水浓缩物。当组合物通过输注给予时,它可分散于含有无菌的药物级水或盐水的输液瓶中。当组合物通过注射给予时,可提供一个安瓿的用于注射的无菌水或盐水,以便在给药前此成分可以被混合。
本发明的组合物可以被配制成中性的或盐的形式。药学上可接受的盐包括由阴离子形成的盐,例如那些源于盐酸、磷酸、乙酸、草酸、酒石酸等的盐,及由阳离子形成的盐,例如那些源于钠、钾、铵、钙、氢氧化铁、异丙胺、三乙胺、乙基羟乙胺、组氨酸、普鲁卡因等的盐。
实施例1-抗CD16-MUC1双特异纳米抗体核苷酸序列的设计和合成
根据该双特异抗体的序列和连接形式,重新设计和优化核苷酸序列,并在该序列的5’端加入NcoI酶切位点,在其3’端加入HindIII酶切位点。直接合成DsbA-anti-MUClVHH-GS-anti-CD16 VHH-6His,并通过双酶切连接到载体pETDuet中,形成表达质粒。抗CD16-MUC1双特异纳米抗体的结构示意图如图1所示。
实施例2-载体转化BL21(DE3)
将质粒转化大肠杆菌DH5α感受态细胞株,选取阳性克隆,在含有100μg/ml氨苄青霉素的LB培养基(3mL)中37℃过夜培养,菌液5000rpm常温离心弃上清,所得的大肠杆菌用Qiagen公司的质粒提取试剂盒裂解并提取质粒,获得表达载体。
实施例3-抗MUC1-抗CD16的表达与纯化
纯化后的pETDuet-bsAb表达载体,转化大肠杆菌BL21(DE3)菌株,选取阳性克隆,在含有100μg/mL氨苄青霉素的LB培养基(3mL)中37℃过夜培养,转移到含有100μg/mL氨苄青霉素的300mL LB中,37℃培养至OD600在0.6-08,加入终浓度为005mM的IPTG,16℃诱导16小时。4000rpm,离心弃上清,沉淀按照1∶4的重量体积比加入20mM Tris-HCl,pH8.0,25%蔗糖,1mM EDTA溶液,重悬后冰浴30min,8500g,4℃离心20分钟,保留上清。沉淀按照1∶5的重量体积比加入5mM MgCl2,1mg/mL的溶菌酶溶液重悬,冰浴20min,8500g,4℃离心20分钟,取上清。
结合:合并两次上清,过2mL Ni-NTA(Qiagen公司)重力沉降柱。
除杂:依次以20mL 20mM Tris-HCl pH8.0,15mM咪唑,1M NaCl和20mL 20mM Tris-HCl pH8.0,25mM咪唑,1M NaCl进行除杂。
洗脱:依次以10mL 20mM Tris-HCl pH8.0,50mM咪唑,0.15M NaCl、10mL 20mMTris-HCl pH8.0,100mM咪唑,0.15M NaCl、10mL 20mM Tris-HCl pH8.0,200mM咪唑,0.15MNaCl、10mL20mM Tris-HCl pH8.0,500mM咪唑,0.15MNaCl进行洗脱。
合并50mM咪唑、100mM咪唑以及200mM咪唑洗脱组分,以20mM PB,pH7.6,10%甘油4℃透析过夜。
1mL Q-HP纯化:透析过夜后含目标组分溶液,20000g,4℃离心20min后上样,上样流速1mL/min,收集流穿组分进行超滤浓缩。
实验结果见图2。
实施例4-双特异抗体体外结合MUC1测试
方法:
1)流式细胞术检测双特异抗体对MUC1的结合
1.体外培养MUC1阳性细胞LS174T,HT29,SKOV3和阴性细胞CHO,HepG2细胞;0.25%胰蛋白酶消化成单细胞,1000rpm离心10min后,收集细胞沉淀,以冰冷PBS+0.2%BSA重悬。
2.离心,4℃,1000rpm,5分钟,弃上清
3.以冰冷PBS+0.2%BSA重悬,配置成浓度为2*106/mL的细胞悬液。
4.按下表分别加入
一抗:mouse anti-MUCl/CD66e mAb(1∶200);bsAb(10ug/mL)后4℃孵育1小时。
管号 | 一抗 | 二抗 |
A | None | Goat-Anti-mouse IgG-FITC(1∶500) |
B | Anti-MUC1(1∶200) | Goat-Anti-mouse IgG-FITC(1∶500) |
C | None | Anti-His-FITC(1∶500) |
D | bsAb(10ug/mL) | Anti-His-FITC(1∶500) |
5.加入5mL冰冷PBS+02%BSA;
6.1000rpm,离心5分钟,4℃;
7.细胞沉淀以冰冷1mL PBS+02%BSA重悬;
8.1000rpm,离心5分钟,4℃;
9.细胞沉淀以05mL冰冷PBS+02%BSA重悬;
10.依上述表格,对应加入二抗,4℃孵育1小时;
11.1000rpm,离心5分钟,4℃;
12.细胞沉淀以冰冷1mL PBS+02%BSA重悬;
13.1000rpm,离心5分钟,4℃;
14.细胞沉淀以1mL冰冷PBS+0.2%BSA重悬;
15.1000rpm,离心5分钟,4℃;
16.细胞沉淀以1mL冰冷PBS+0.2%BSA重悬,上机测试。
实验结果:bsAb可结合肿瘤细胞表面的MUC1,结果见图3。
2).Western blot检测双特异抗体对MUC1的结合
体外培养MUC1阳性细胞LS174T,HT29,SKOV3和阴性细胞CHO,HepG2细胞,RIPA裂解后用BCA法测定蛋白质含量。
SDS-PAGE电泳:分离胶浓度8%。每种细胞系裂解物上样量为30μg,120V,恒压45分钟后转膜。
转膜:取SDS-PAGE电泳后的分离胶,放在负极,PVDF膜放在正极,进行湿转,100V,90分钟。
封闭:取转膜后的PVDF膜,置于含5%脱脂奶粉TBST中,封闭1小时;
一抗孵育:双特异抗体(1mg/mL)以1∶1000比例稀释于含5%脱脂奶粉TBST中,室温孵育1小时;阳性对照用商品化抗MUC1兔单克隆抗体(Abcam公司,货号:),稀释比例1∶1000,孵育1小时。内参用兔抗GAPDH单克隆抗体(Abcam公司,货号:)
洗膜:TBST洗涤三次,每次10分钟;
二抗孵育:抗His IgG-HRP以1∶3000比例稀释于TBST中,室温孵育1小时;阳性对照二抗用抗兔IgG HRP,稀释比例为1∶5000,孵育1小时。
洗膜:TBST洗涤三次,每次10分钟;
显色:将膜置于伯乐的化学发光成像系统中,均匀滴加millipore显色剂,避光进行显影拍照。
实验结果:双特异抗体可特异结合LS174T、HT29以及SKOV3等细胞表达的MUC1,而与CHO,HepG2中总蛋白无结合。结果见图4。
实施例5-体外细胞毒性实验
1.体外细胞毒性试验
1)将SKOV3、HT29以及LS174T肿瘤细胞,加0.25%胰蛋白酶消化成单个细胞后,铺96孔板,每孔5000个细胞。37℃,5%二氧化碳培养6个小时。
2)每孔加入5*104个NK细胞,分别加入0,0.28μg/mL,2.8μg/mL的bsAb。37℃,5%二氧化碳培养48个小时。
3)吸取培养基,以PBS轻轻清洗两次。
4)加入CCK8试剂,孵育2小时
5)用酶标仪以620nm为参比,测定OD450的读数。
6)裂解率用如下公式进行计算:
裂解率=1-(As-Ab)/(A0-Ab),其中Ab为空白对照吸收值,As为实验组吸收值,A0为未加药孔吸收值。
实验结果见图5和图6。
2.EC50测试
1)将LS174T肿瘤细胞,加025%胰蛋白酶消化成单个细胞后,铺96孔板,每孔5000个细胞。37℃,5%二氧化碳培养6个小时。
2)每孔加入5*104个NK细胞,分别加入0,0.28μg/mL,28μg/mL的bsAb。37℃,5%二氧化碳培养48个小时。
3)吸取培养基,以PBS轻轻清洗两次。
4)加入CCK8试剂,孵育2小时
5)用酶标仪以620nm为参比,测定OD450的读数。
6)裂解率用如下公式进行计算:
裂解率=1-(As-Ab)/(A0-Ab),其中Ab为空白对照吸收值,As为实验组吸收值,A0为未加药孔吸收值。
双特异纳米抗体 | EC<sub>50</sub> |
Muc1-VHH-CD16-VHH-1 | 016uM |
Mucl-VHH-CD16-VHH-2 | 0.08uM |
实施例6.双特异性抗体偶联物的制备
制备了分别具有偶联于PLGA纳米颗粒的双特异性抗体CD16-MUC1 BiTE的双特异性抗体偶联物(BiTE(CD16-MUC1)-PLGA)。其中“CD16-MUC1 BiTE”为Muc1 VHH(其序列示于SEQ ID NO:1)和CD16 VHH-2(其序列示于SEQ ID NO:3)通过连接肽(其序列示于SEQ IDNO:3)连接的双特异性抗体。
双特异性抗体偶联物的具体制备方法如下:
(1)PLGA纳米颗粒的制备:用丙酮将PLGA完全溶解至浓度为5mg/mL,按照丙酮和去离子水1∶4的体积比,在1000rpm/min磁力搅拌的状态下将PLGA与丙酮的溶液加入去离子水中,形成均匀的乳浊液,然后继续搅拌至丙酮挥发;
(2)PLGA纳米颗粒的收集:以8000rpm/min离心10min收集制备的较大颗粒的纳米颗粒;再以15000rpm/min离心10min收集得到较小颗粒的纳米颗粒。弃去较大颗粒的纳米颗粒,较小颗粒的纳米颗粒用去离子水重悬,重复操作2次洗涤纳米颗粒。可以进一步纯化纳米材料,以获得更小的纳米颗粒。使用较小颗粒的纳米颗粒分别进行如下操作;
(3)PLGA纳米颗粒的活化:用5mg/mL的1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐和N-羟基琥珀酰亚胺混合溶剂于室温下活化PLGA纳米颗粒1h;
(4)PLGA纳米颗粒连接抗体:活化后的纳米材料通过离心收集,然后用0.1M、pH=8.0的杜氏磷酸盐缓冲溶液洗涤纳米材料1次。将需要连接的BiTE(CD16-MUCl)或BiTE(CD16-CEA)加入到连接反应液,然后用含有所述BiTE的连接反应液重悬纳米材料,于室温下连接反应进行0.5h。反应结束后离心收集纳米材料,用杜氏磷酸盐缓冲溶液洗涤纳米材料2次,然后再重悬到杜氏磷酸盐缓冲溶液中放4℃保存备用。
实施例7.双特异性抗体偶联物对癌细胞的杀伤效力
对实施例6中制备的双特异性抗体偶联物杀伤肿瘤细胞的能力进行了评估。
具体而言,于96孔板中,以5000个靶细胞/孔培养12h后,弃原培养基。用无细胞因子的X-vivo 15培养基(购于lonza公司)调整NK细胞(用健康献血者的外周血单核细胞诱导扩增获得)密度,使100μl体积NK细胞数量为靶细胞4倍(效靶比为4∶1)。加100μlNK细胞悬液到癌细胞培养板中,并加入10μl制备的双特异性抗体偶联物(双特异性抗体偶联物含量为0.2mg,含有BiTE 0.2μg)或者双特异性抗体(BiTE(CD16-MUC1)(0.2μg),于培养箱中孵育8h。然后加入CCK-8试剂,按照试剂说明进行孵育。用酶标仪检测450nm处吸光值。对数据进行统计分析,按照以下公式计算DC-CIK细胞对癌细胞的杀伤率。
杀伤率=[1-(实验组-效应对照组)/(靶对照组-空白对照组)]×100%
其中,空白对照表示加入培养基;靶对照组表示加入靶细胞+培养基;效应对照组表示加入效应细胞+培养基;实验组表示加入效应细胞+靶细胞+培养基+双特异性抗体偶联物。
结果示于表1和图7中。图7显示双特异性抗体BiTE(CD16-MUC1)及抗体偶联物BiTE(CD16-MUC1)-PLGA联合NK细胞对肺癌细胞A549(人非小细胞肺癌细胞)的杀伤率。其中对照表示加入效应细胞+靶细胞+培养基;BiTE表示加入效应细胞+靶细胞+培养基+BiTE;BiTE-PLGA表示加入效应细胞+靶细胞+培养基+BiTE-PLGA(即双特异性抗体偶联物),其中PLGA纳米颗粒的平均粒径约为50nm。
表1.双特异性抗体和双特异性抗体偶联物对肿瘤细胞的杀伤能力(效靶比为4∶1)
实施例8
按照实施例6的方法,使用同样的双特异性抗体,制备了PLGA纳米颗粒平均粒径约为100-150nm的双特异性抗体偶联物。另外,使用同样的PLGA纳米颗粒制备Mucl VHH(其序列示于SEQ ID NO:1)偶联物作为对照。
对所制备的双特异性抗体偶联物杀伤肿瘤细胞的能力进行了评估。
具体而言,于96孔板中,以5000个靶细胞/孔培养12h后,弃原培养基。用无细胞因子的X-vivo 15培养基(购于lonza公司)调整NK细胞(用健康献血者的外周血单核细胞诱导扩增获得)密度,使100μl体积NK细胞数量为靶细胞4倍(效靶比为4∶1)。加100μlNK细胞悬液到癌细胞培养板中,并加入10μl制备的双特异性抗体偶联物CD16-MUC1 BiTE+PLGA NPs(偶联物含量为02mg,含有CD16-MUC1 BiTE 0.2μg)或者双特异性抗体(BiTE)(0.2μg),或者加入作为对照的Muc1+PLGA NPs(偶联物含量为0.2mg,含有Muc1 VHH 0.1μg)或PLGA纳米颗粒即PLGA NPs(02mg)。于培养箱中孵育48h。然后加入CCK-8试剂,按照试剂说明进行孵育。用酶标仪检测450nm处吸光值。对数据进行统计分析,按照以下公式计算DC-CIK细胞对癌细胞的杀伤率。
杀伤率=[1-(实验组-效应对照组)/(靶对照组-空白对照组)]×100%
其中,空白对照表示加入培养基;靶对照组表示加入靶细胞+培养基;效应对照组表示加入效应细胞+培养基;实验组表示加入效应细胞+靶细胞+培养基+双特异性抗体偶联物。
结果示于表2和图8中。表2和图8显示双特异性抗体BiTE及抗体偶联物BiTE-PLGA联合NK细胞对肺癌细胞A549(人非小细胞肺癌细胞)的杀伤率。其中对照(control)表示加入效应细胞+靶细胞+培养基;MUC1+PLGA NPs表示加入效应细胞+靶细胞+培养基+PLGA纳米颗粒;BiTE表示加入效应细胞+靶细胞+培养基+双特异性抗体;BiTE-PLGA NPs表示加入效应细胞+靶细胞+培养基+双特异性抗体偶联物。
表2.双特异性抗体或双特异性抗体偶联物对肿瘤细胞A549的杀伤能力(效靶比为4∶1)
实验分组 | 杀伤率 |
对照(control) | 31.52% |
PLGA纳米颗粒(PLGA NPs) | 32.47% |
MUC1+PLGA NPs | 3250% |
CD16-MUC1 BiTE | 73.90% |
CD16-MUC1 BiTE+PLGA NPs | 8340% |
本领域技术人员会容易意识到,本发明可容易改造而获得本文所述的那些目的和优点以及隐含在本文中的那些目的和优点。在本文中以当前优选实施方式的代表的形式描述的方法、变体和组合物是示例性的,并不意在限制本发明的范围。对于本领域技术人员来说,可对它们做出改变或将其用于其他用途,但这都包括在如所附权利要求定义的本发明的范围内。
虽然本发明已通过优选实施方式和任选的特征具体公开,但本领域技术人员可对本文公开的想法做出修改和变化,而这些修改和变化仍属于所附权利要求定义出的本发明的范围之内。
Claims (19)
1.一种双特异性抗体,其包含抗MUC1 VHH的抗体片段和抗CD16 VHH的抗体片段,其中所述抗MUC1 VHH的抗体片段,其氨基酸序列如SEQ ID No.1所示,所述抗CD16 VHH的抗体片段,其氨基酸序列如SEQ ID No.2或SEQ ID No.3 所示。
2.根据权利要求1所述的双特异性抗体,其中抗MUC1 VHH的抗体片段和抗CD16 VHH的抗体片段之间是通过连接肽序列连接而成的。
3.根据权利要求2所述的双特异性抗体,其中所述连接肽序列的氨基酸序列如SEQ IDNo. 4所示。
4.根据权利要求2或3所述的双特异性抗体,其中抗MUC1 VHH的抗体片段和抗CD16 VHH的抗体片段之间的连接方式为:抗MUC1 VHH -连接肽-抗CD16 VHH。
5.根据权利要求2或3所述的双特异性抗体,其中抗MUC1 VHH的抗体片段和抗CD16 VHH的抗体片段之间的连接方式为:抗CD16 VHH-连接肽-抗MUC1 VHH。
6.一种药物组合物,包含权利要求1-5任一项所述的双特异性抗体以及药学上可接受的辅料。
7.一种编码权利要求1-5任一项所述的双特异性抗体的多核苷酸。
8.一种包含权利要求7所述的多核苷酸的表达载体。
9.一种用权利要求8所述的表达载体转化或转染的宿主细胞。
10.权利要求1-5任一项所述的双特异性抗体在制备治疗癌症的药物中的应用。
11.一种抗体偶联物,其包含偶联于纳米微粒上的权利要求1-5中任一项的双特异性抗体。
12.权利要求11的抗体偶联物,其中所述纳米微粒为生物可降解纳米材料。
13.权利要求11的抗体偶联物,其中所述纳米微粒为聚乳酸-羟基乙酸、聚乳酸、聚己内酯、聚丁二醇丁二酸酯、聚苯胺、聚碳酸酯、乙丙交酯共聚物或乙交酯己内酯共聚物中的任意一种或至少两种的混合物。
14.权利要求11的抗体偶联物,其中所述纳米微粒为聚乳酸-羟基乙酸、聚乳酸和/或聚己内酯。
15.一种药物组合物,其包含权利要求11-14中任一项所述的偶联物。
16.根据权利要求15所述的药物组合物,其还包含白细胞。
17.根据权利要求16所述的药物组合物,其中所述白细胞包含NK细胞。
18.权利要求11-14中任一项所述的抗体偶联物在制备治疗癌症的药物中的应用。
19.抗MUC1 VHH,其氨基酸序列如SEQ ID No.1所示。
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