CN117186222A - Pd-l1结合分子及其用途 - Google Patents
Pd-l1结合分子及其用途 Download PDFInfo
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- CN117186222A CN117186222A CN202210611228.9A CN202210611228A CN117186222A CN 117186222 A CN117186222 A CN 117186222A CN 202210611228 A CN202210611228 A CN 202210611228A CN 117186222 A CN117186222 A CN 117186222A
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Abstract
本申请涉及与人PD‑L1特异结合的单域抗体、以及含有该单域抗体的结合分子。还提供编码该单域抗体和/或结合分子的核酸分子,以及用于表达该单域抗体和/或结合分子的表达载体、宿主细胞和方法。本申请还提供包含该单域抗体和/或结合分子的免疫交联物、双特异性分子、嵌合抗原受体、溶瘤病毒和药物组合物,以及使用本申请单域抗体和/或结合分子的治疗方法。
Description
技术领域
本申请涉及一种与人PD-L1特异结合的结合分子,如单域抗体、重链抗体或其抗原结合片段,及其制备和用途,尤其是其在治疗与PD-L1相关疾病中的用途,例如癌症和感染性疾病。
背景技术
免疫系统对癌细胞表现出一定的耐受性,这是由多种机制所介导的,包括调节性免疫细胞(如调节性T细胞(Treg))、免疫抑制性细胞因子和趋化因子、以及免疫检查点通路。PD-1/PD-L1和CTLA-4是研究最多的抑制性免疫检查点,已有若干种靶向这些分子的抗体上市,用于癌症治疗,如伊匹单抗、/>帕博丽珠单抗、和/>纳武单抗等。
CTLA-4主要在免疫活化的早期调节XD4+ T细胞的活化、以及CD4+ T细胞对CD8+T细胞致敏的协助,并加强Treg的免疫抑制活性,而PD-1主要作用于CD8+效应T细胞。在免疫激活过程中,初始T细胞在TCR识别抗原提呈细胞(APC)上的MHC肽后,经其表面CD28分子与抗原提呈细胞(APC)表面B7分子的结合,实现增殖、炎性细胞因子释放、迁移至抗原出现处等。随着T细胞的活化,CTLA-4的表达量上升,与CD28竞争结合B7分子,传递抑制性信号,且其结合亲和强度是CD28的数千倍。随后,在T细胞活化的数小时至数日内,T细胞表面逐渐表达PD-1,包括肿瘤浸润淋巴细胞(TIL)。在肿瘤微环境(TME)中,CD4+ Th1辅助细胞和CD8+ T细胞生成干扰素-γ(IFN-γ),这一方面激活巨噬细胞对肿瘤的杀伤,另一方面则诱导巨噬细胞和肿瘤细胞的PD-L1表达。例如,在实体瘤和血液瘤中,肿瘤细胞PD-L1表达上调。肿瘤特异CD8+ T细胞上PD-1与TME中各细胞表达的PD-L1结合,引起CD8+ T细胞的失能。TME中Treg所表达的CTLA-4会进一步抑制CD8+ T细胞的细胞因子释放和对肿瘤细胞的直接杀伤力(Topalian,S.et al.,(2016)Nat Rev Cancer 16(5):275-287)。近几年,PD-1/PD-L1通路因其显著的临床效果、持久反应性和低毒力得到众多科学家的热捧(Gong J et al.,(2018)J Immunother Cancer 6(1):8)。癌症治疗中,靶向PD-1/PD-L1通路目的不是增强免疫系统对肿瘤的反应性,而是让免疫系统保持正常活性,解除PD-1/PD-L1通路对其抑制。该方法也并非万能,只对部分病人有效。
此外,在病毒、细菌和寄生虫的急性或慢性感染方面,PD-1/PD-L1阻断也在临床和临床前研究中表现出良好的效果(Jubel JM et al.,(2020)Front Immunol.11:487)。例如,在乙肝病毒、丙肝病毒、人免疫缺陷病毒(HIV)和猴免疫缺陷病毒(SIV)的慢性感染后,PD-L1抗体处理增加IFN-γ、IL-2和TNFα的分泌,T细胞耗竭情况得到缓解,病毒血症得到控制。在感染性疾病的治疗方面,也需要更多的PD-L1抗体。
1993年,比利时科学家在单峰驼外周血中发现了一种特殊的单域抗体(sdAb,Hamers-Casterman C et al.,(1993)Nature.363(6428):446-448)。这种抗体天然缺失轻链,只有重链,且该重链只包含一个重链可变区(VHH),分子量只有15kDa,是目前所有抗体中分子量最小的结构,也被称作纳米抗体(nanobody,Nb)。与IgG相比,纳米抗体更易于克隆,表达水平更高;可溶性高且稳定;与抗原结合的亲和力和特异性高;CDR3较长,可以识别特异性的抗原表位;与人源VH同源性高(Muyldermans S.,(2001)Reviews in Molecu-larBiotechnology,74:277-302)。单域抗体的一系列特性,使得其在其他类型的抗体片段中脱颖而出,具有较好的应用前景。
发明内容
本申请的发明人发现了几种PD-L1结合分子,其与阿替利珠单抗相比,具有相当的PD-L1结合亲和力/活性、相当或更优的PD-1-PD-L1阻断活性、相当或更优的T细胞激活能力、和/或相当或更优的体内抗肿瘤效力。
因此,在一个方面,本申请提供一种分离的单域抗体,其与PD-L1(例如人和猴PD-L1)结合,可以含有重链可变区,该重链可变区可以含有CDR1区、CDR2区和CDR3区,其中CDR1区、CDR2区和CDR3区可以分别包含如(1)SEQ ID NOs:1、2(X1=T、X2=N、X3=A)和3(X=K);或(2)SEQ ID NOs:1、2(X1=L、X2=D、X3=V)和3(X=A)所示的氨基酸序列,或与上述序列具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%同一性的氨基酸序列。
本申请的分离的单域抗体的重链可变区可以包含与SEQ ID NOs:4、5、6、7、8、或9具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%同一性的氨基酸序列。
本申请的单域抗体可以包含重链可变区,该重链可变区可以含有CDR1区、CDR2区和CDR3区,其中,CDR1区、CDR2区和CDR3区可以分别与SEQ ID NOs:4、5、6、7、8、或9所示的重链可变区的CDR1区、CDR2区和CDR3具有同一性。
该单域抗体可以是骆驼源、嵌合、或人源化的。
在另一个方面,本申请提供一种结合分子,其包含本申请的单域抗体。
结合分子可以是本申请的单域抗体。
结合分子可以是本申请单域抗体与免疫球蛋白重链恒定区的融合蛋白。重链恒定区可以为IgG、IgD、IgA、IgM或IgE重链可变区,如IgG1、IgG2、IgG3或IgG4重链恒定区,或其功能片段(如Fc区)。在一些实施方式中,重链恒定区可以包含铰链区、CH2结构域和CH3结构域。在一些实施方式中,重链恒定区可以包含CH2结构域和CH3结构域。在一些实施方式中,重链恒定区是人IgG1重链恒定区或其功能片段,例如具有SEQ ID NOs:10所示的氨基酸序列。单域抗体的C端可以与重链恒定区或其功能片段的N端连接。
结合分子可以是包含两个上述单域抗体-免疫球蛋白重链恒定区融合蛋白(如单域抗体-Fc融合蛋白)的二聚体,两个融合蛋白通过例如一个或多个二硫键连接。
结合分子可以是重链抗体(HCAb)或其抗原结合片段,其包含本申请的单域抗体。在一些实施方式中,重链抗体或其抗原结合片段可以包含与上述免疫球蛋白重链恒定区连接的本申请单域抗体。重链抗体可以包含两条重链,或由两条重链构成,其中至少其中一条重链可以包含与上述免疫球蛋白重链恒定区连接的本申请单域抗体。重链抗体或其抗原结合片段可以是骆驼源、嵌合、或人源化的。在一些实施方式中,重链抗体或其抗原结合片段可以是上述的包含本申请单域抗体(例如,与上述免疫球蛋白重链恒定区连接的本申请单域抗体,如单域抗体-Fc融合蛋白)的二聚体。
本申请的单域抗体和结合分子可以a)与PD-L1结合,b)抑制PD-1-PD-L1结合,c)激活T细胞,和/或d)具有体内抗肿瘤活性。
本申请还提供含有本申请单域抗体或结合分子的免疫偶联物,该单域抗体或结合分子与治疗剂例如细胞毒素或抗癌剂相连接。本申请还提供含有本申请单域抗体或结合分子的双特异性分子,该单域抗体或结合分子连接有第二功能基团,例如,第二抗体,该第二功能基团具有不同于本申请单域抗体或结合分子的结合特异性。在另一方面,本申请的单域抗体或结合分子可以是嵌合抗原受体(CAR)或基因改造T细胞受体(TCR)的一部分。本申请还提供具有上述CAR和/或TCR的免疫细胞,包括T细胞、NK细胞等。本申请的单域抗体或结合分子还可以由溶瘤病毒编码或由溶瘤病毒承载。
本申请还包括编码本申请单域抗体、结合分子(包括重链抗体或其抗原结合片段)、免疫偶联物、双特异性分子、CAR、或TCR的核酸分子,以及包含该核酸的表达载体以及包含该表达载体的宿主细胞。
本申请还提供使用含有上述表达载体的宿主细胞来制备本申请单域抗体、结合分子、免疫偶联物、双特异性分子、CAR、或TCR的方法,包括:(i)在宿主细胞中表达单域抗体、结合分子、免疫偶联物、双特异性分子、CAR、或TCR,以及(ii)从宿主细胞或其培养物中分离单域抗体、结合分子、免疫偶联物、双特异性分子、CAR、或TCR。
本申请还提供药物组合物,其包含本申请的单域抗体或结合分子(包括重链抗体或其抗原结合片段)、免疫偶联物、双特异性分子、免疫细胞、溶瘤病毒、核酸分子、表达载体或宿主细胞,以及药学上可接受的载体。
在另一个方面,本申请提供在受试者中治疗或减缓PD-L1相关疾病的方法,包括向受试者施用治疗有效量的本申请药物组合物。
PD-L1相关疾病可以为肿瘤或感染性疾病。肿瘤可以为实体瘤和液体瘤,包括但不限于,黑色素瘤、肺癌(如非小细胞肺癌)、尿路上皮癌、肾细胞癌、头颈癌、霍奇金淋巴瘤、微卫星不稳定或错配修复缺陷癌、胃癌、结直肠癌、肝癌(如肝细胞癌)、和梅克尔(Merkel)细胞癌。在一些实施方式中,本申请的药物组合物可以与至少一种抗癌剂一起施用,例如PD-1抗体等。在另一个实施方式中,本申请的药物组合物与细胞因子(例如IL-2和/或IL-21)或共刺激抗体(例如CD137抗体和/或GITR抗体)一起施用。在另一个实施方式中,本申请的药物组合物可以与化疗剂一起施用,该化疗剂可以是细胞毒性剂。受试者可以为哺乳动物,特别是人。
PD-L1相关疾病可以为感染性疾病,特别是慢性感染疾病。在一些实施方式中,相关疾病可以为病毒造成的慢性感染疾病,如由乙肝病毒、丙肝病毒、人免疫缺陷病毒(HIV)或猴免疫缺陷病毒(SIV)引起的慢性感染疾病。在一些实施方式中,本申请的药物组合物可以与至少一种抗病毒剂一起施用。受试者可以为哺乳动物,特别是人。
一个方面,本申请提供在受试者中增强免疫应答的方法,包括向有需要的受试者施用有效量的本申请的药物组合物。增强免疫应答包括激活免疫细胞,包括T细胞等。受试者可以为哺乳动物,特别是人。
在本申请中引用或提及的所有文件(包括但不限于本文引用的所有文献、专利、公开的专利申请)(“本申请引用文件”),在本申请引用文件中引用或提及的所有文件,以及本申请或任意本申请引用文件中提及的任何产品的制造商手册、说明书、产品规格和产品页,均通过引用的方式并入本申请,且可能在实施本发明时采用。更具体而言,所有参考文件均通过引用的方式并入本申请,如同各文件通过引用的方式并入。在本文中提及的任何Genbank序列通过引用的方式并入本申请。
应当注意的是,在本申请中,特别是在权利要求中,术语例如“包含”、“包括”等可以具有中国专利法所赋予的意义;而术语例如“基本由......组成”具有中国专利法所赋予的意义,例如允许没有明确表述的元素的存在,但将现有技术中存在的元素、或影响本发明的基本或新的特性的元素排除在外。
基于以下具体描述和实施例,当前公开内容的其他特征和优势之处将会更加明晰,具体描述和实施例不应解读为限制性的。在本申请中引用的所有文献、Genbank记录、专利和已公开专利申请的内容通过引用的方式明确地包含在本文中。
附图说明
以下以示例方式给出但不意在将本发明限制于所述具体实施方式的具体描述,可以结合附图更好地进行理解。
图1示出9个骆驼源PD-L1纳米抗体-Fc融合蛋白(包括两个代表性纳米抗体10CA81和10CA192的Fc融合蛋白)以剂量依赖方式增加经抗原递呈细胞(APC)诱导的T细胞的IFN-γ分泌。
图2示出骆驼源PD-L1纳米抗体-Fc融合蛋白对HEK293A/人PD-L1(A)和HEK293A/猴PD-L1(B)的结合活性。
图3示出骆驼源PD-L1纳米抗体-Fc融合蛋白对PD-L1与PD-1结合的阻断活性。
图4示出骆驼源PD-L1纳米抗体-Fc融合蛋白以剂量依赖方式增加经APC细胞诱导的T细胞的IFN-γ分泌。
图5示出人源化10CA81纳米抗体-Fc融合蛋白对HEK293A/人PD-L1(A)和HEK293A/猴PD-L1(B)的结合活性。
图6示出人源化10CA192纳米抗体-Fc融合蛋白对HEK293A/人PD-L1(A)和HEK293A/猴PD-L1(B)的结合活性。
图7示出人源化PD-L1纳米抗体-Fc融合蛋白以剂量依赖方式增加经APC细胞诱导的T细胞的IFN-γ分泌。
图8示出人源化PD-L1小鼠经10CA81-VHH5-Fc融合蛋白、10CA81-VHH4-Fc融合蛋白、10CA192-VHH2-Fc融合蛋白、10CA192-VHH4-Fc融合蛋白和阿替利珠单抗处理后的肿瘤实物图(A)和重量(B)。
具体实施方式
为更好理解本申请,首先定义一些术语。其他定义则贯穿具体实施方式部分而列出。
术语“PD-L1”是指程序性死亡配体1,其为程序性死亡受体1(PD-1)的配体。该术语包括变体、同源物、直向同源物和平行同源物。例如,对人PD-L1特异的抗体可以在某些情况下与另一物种例如猴的PD-L1蛋白交叉反应。在其他实施方式中,对人PD-L1蛋白特异的抗体可以完全地对人PD-L1蛋白特异而不与其他物种或其他类型的蛋白交叉反应,或者可以与一些其他物种而非所有其他物种的PD-L1蛋白交叉反应。
术语“人PD-L1”是指具有人氨基酸序列的PD-L1蛋白,例如具有SEQ ID NO:11所示的氨基酸序列的PD-L1蛋白。术语“猴PD-L1”是指具有猴氨基酸序列的PD-L1蛋白,例如具有SEQ ID NO:12所示的氨基酸序列的PD-L1。术语“小鼠PD-L1”是指具有小鼠氨基酸PD-L1蛋白,例如具有SEQ ID NO:13所示的氨基酸序列的PD-L1蛋白。
本文中的术语“抗体”是指经抗原结合位点特异识别并结合靶点的免疫球蛋白分子,其中抗原结合位点通常在免疫球蛋白分子的可变区内。
在一些情况下,本文中的术语“抗体”具体指重链抗体或其抗原结合部分。术语“重链抗体”或“HCAb”是指包含重链但缺乏轻链的功能抗体。天然的重链抗体存在于骆驼科(骆驼、美洲驼、或羊驼)动物体内。各个骆驼源重链抗体包含重链可变区和重链恒定区,重链可变区称为VHH结构域、VHH片段或单域抗体(sdAb)。VHH与抗原相互作用。VHH包含3个互补决定区(CDR)和4个框架区(FR),从氨基端到羧基端以FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4的顺序排布。重链恒定区可以包含铰链区、CH2结构域和CH3结构域。缺失的CH1结构域可以被延长的铰链区所取代。在嵌合或人源化的重链抗体中,重链恒定区可以包含人IgG,例如IgG1、IgG2或IgG4恒定区。恒定区可以介导重链抗体与宿主组织或因子,包括免疫系统的多种细胞(如效应细胞)和补体系统第一成分(C1q),的结合。重链恒定区的“功能片段”是指恒定区中保留有使抗体与宿主组织或因子结合从而引导例如ADCC、CDC、ADCP等的活性的部分。
重链抗体的“抗原结合片段”或“抗原结合部分”是指重链抗体的一个或多个保留有与抗原(如PD-L1)特异结合的片段。已知重链抗体的抗原结合功能通过重链抗体的片段来实现。重链抗体的抗原结合片段或抗原结合部分的实例包括,但不限于,(i)分离的互补决定区(CDR);(ii)单价VHH片段;(iii)包含两个单价VHH片段的二价片段;(iv)包含VHH片段与重链恒定区或其功能片段相连的单价片段,例如连接重链恒定区CH2结构域、或CH2和CH3结构域的VHH片段;(v)包含两个分别与重链恒定区或其片段连接的VHH片段的二价片段;(vi)经接头,或直接连接的多个单价VHH结构域。
在一些情况下,本文中的“抗体”是指单域抗体、或纳米抗体。术语“单域抗体”或“sdAb”是指包含单个单体可变区的单个抗原结合多肽,其能够与抗原结合而无需与其他对应的含CDR的多肽配对,其中可变区包含三个互补决定区(CDR)。在一些情况下,由骆驼HCAb经改造得到单域抗体,也称为VHH结构域或HCAb片段。单域抗体是重链抗体的抗原结合部分。骆驼sdAb是已知的最小的抗原结合片段(Hamers-Casterman et al.,(1993)Nature363:446-8;Greenberg et al.,(1995)Nature 374:168-73;Hassanzadeh-Ghassabeh etal.,(2013)Nanomedicine(Lond),8:1013-26)。
抗体(包括重链抗体)的“Fc区”是抗体的与Fc受体以及一些补体系统蛋白相互作用以激活免疫系统的尾部区域。IgG、IgA和IgD的Fc区域由两个相同的得自抗体重链第二和第三恒定结构域(CH2和CH3)的片段构成,而IgM和IgE的Fc区包含3个重链恒定结构域(CH结构域2-4)。Fc区可以与补体成分C1q结合,激活经典补体级联反应,可以与吞噬细胞(即,巨噬细胞、粒细胞、和树突状细胞)上Fc受体结合,以引起对抗体结合细胞的吞噬,可以与免疫效应细胞(主要是自然杀伤细胞)的Fc受体结合,以使免疫效应细胞释放细胞毒性颗粒,导致抗体包裹细胞的死亡,以及可以与抗原提呈细胞例如树突状细胞的Fc受体结合,以诱导体液免疫和细胞免疫。
本文中的“结合分子”是指包含本申请单域抗体的分子,具有与PD-L1结合的亲和力/活性,包括单域抗体本身、单域抗体与重链可变区如Fc区的单价融合蛋白、该融合蛋白的二聚体(如重链抗体)等。
本文所用的术语“分离的”单域抗体或重链抗体是指基本不含具有不同抗原特异性的其他抗体的抗体。例如,与PD-L1蛋白特异结合的分离抗体基本不含特异结合PD-L1蛋白以外抗原的抗体。但是,特异结合人PD-L1蛋白的分离抗体可能对其他抗原例如其他物种的PD-L1蛋白具有交叉结合性。此外,分离的抗体基本不含其他细胞材料和/或化学物质。
术语“骆驼源抗体”是指可变区骨架和CDR区得自骆驼种系免疫球蛋白序列的抗体。本申请的骆驼源抗体可以包含不由人种系免疫球蛋白序列编码的氨基酸残基,例如通过体外随机突变或点突变或通过体内体细胞突变而导入的突变。然而,术语“骆驼源抗体”不包括在骆驼骨架序列中插入得自其他哺乳动物物种的CDR序列的抗体。
术语“嵌合抗体”是指通过组合非人源(如骆驼源)遗传物质与人源遗传物质而得来的抗体。或者更笼统地说,嵌合抗体是指组合有一个物种的遗传物质与另一物种遗传物质的抗体。
术语“人源化抗体”是指由非人源(例如,骆驼源)物种而来的抗体,其蛋白序列已改造成增加与人体内自然生成的抗体的相似度。
术语“识别抗原的抗体”以及“对抗原特异的抗体”在本文中与术语“特异结合抗原的抗体”交替使用。
在本文中,“特异结合人PD-L1”的抗体是指与人PD-L1(以及其他非人物种的PD-L1)结合但是基本不与非PD-L1蛋白结合的抗体。优选地,抗体以“高亲和力”结合人PD-L1蛋白,即KD值为5.0×10-9M以下。
术语“基本不结合”蛋白或细胞是指,不与蛋白或细胞结合,或者不以高亲和力与其结合,即结合蛋白或细胞的KD为1.0×10-6M以上,更优选1.0×10-5M以上,更优选1.0×10-4M以上、1.0×10-3M以上,更优选1.0×10-2M以上。
术语“EC50”,又叫半最大效应浓度,是指引起50%最大效应的抗体浓度。
术语“IC50”,是指半抑制浓度,即对指定的生物过程抑制一半时所需的药物或抑制剂的浓度。
术语“受试者”包括任何人或非人动物。术语“非人动物”包括所有脊椎动物,例如哺乳类和非哺乳类,例如非人灵长类、羊、狗、猫、牛、马、鸡、两栖类、和爬行类,尽管优选哺乳动物,例如非人灵长类、羊、狗、猫、牛和马。
术语“治疗有效量”是指足以防止或减缓与疾病或病症(例如癌症)相关的症状的本申请抗体量。治疗有效量与被治疗的疾病相关,其中本领域技术人员可以方便地判别出实际的有效量。
本申请的多个方面在以下更加具体地加以描述。
本申请的单域抗体和包含该单域抗体的结合分子,可以与人、猴等的PD-L1特异性结合,且结合活性/结合亲和性与现有技术抗体例如阿替利珠单抗相当或更佳。此外,与现有技术抗体例如阿替利珠单抗相比,本申请的单域抗体和结合分子具有相当或更佳的PD-1-PD-L1阻断活性、相当或更优的T细胞激活能力、和/或相当或更优的体内抗肿瘤效力。
优选的本申请单域抗体和/或重链抗体是单克隆抗体。此外,重链抗体可以是例如骆驼源的、嵌合的、或人源化的。
本申请单域抗体、和结合分子(包括重链抗体或其抗原结合部分)的可变区CDR通过Kabat编号系统确定,且CDR区氨基酸序列的SEQ ID NO列于表1中。领域内熟知,重链可变区CDR可以通过例如Chothia、IMGT、AbM或Contact编号系统/方法确定。
本申请中示例性单域抗体或结合分子(包括重链抗体或其抗原结合部分)的重链可变区序列的SEQ ID NO也列于表1中。
表1.重链可变区CDR和重链可变区的氨基酸序列SEQ ID NOs.
本申请单域抗体可以与重链恒定区连接,例如可以是IgG1恒定区,或其功能片段,例如包含如SEQ ID NOs:10所示氨基酸序列的人IgG1恒定区(Fc区)。恒定区可以经改造为具有增强的FcR结合力。
领域内公知的是,CDR3结构域,独立于CDR1和/或CDR2,可单独确定抗体对同种抗原的结合特异性,且可以预测到基于该CDR3序列可生成具有相同结合特异性的多种抗体。参见,例如Klimka et al.,British J.of Cancer 83(2):252-260(2000);Beiboer etal.,J.Mol.Biol.296:833-849(2000);Rader et al.,Proc.Natl.Acad.Sci.U.S.A.95:8910-8915(1998);Barbas et al.,J.Am.Chem.Soc.116:2161-2162(1994);Barbas etal.,Proc.Natl.Acad.Sci.U.S.A.92:2529-2533(1995);Ditzel et al.,J.Immunol.157:739-749(1996);Berezov et al.,BIAjournal 8:Scientific Review 8(2001);Igarashiet al.,J.Biochem(Tokyo)117:452-7(1995);Bourgeois et al.,J.Virol 72:807-10(1998);Levi et al.,Proc.Natl.Acad.Sci.U.S.A.90:4374-8(1993);Polymenis andStoller,J.Immunol.152:5218-5329(1994)and Xu and Davis,Immunity 13:37-45(2000);U.S.Pat.Nos.6,951,646;6,914,128;6,090,382;6,818,216;6,156,313;6,827,925;5,833,943;5,762,905和5,760,185。这些参考文献均通过引用的方式全部并入本文。
本申请的单域抗体或结合分子(如重链抗体及其抗原结合部分)可以包含本申请单域抗体的重链可变区CDR2以及其他结合人PD-L1的单域抗体的CDR,例如重链可变区CDR1和/或CDR3。优选这些抗体(a)竞争结合PD-L1;(b)保留功能特性;(c)结合相同表位;和/或(d)具有与本申请PD-L1单域抗体和/或结合分子相似的结合亲和力。
在另一实施方式中,本申请的单域抗体或结合分子(如重链抗体及其抗原结合部分)可以包含与本申请PD-L1单域抗体或结合分子(如重链抗体及其抗原结合部分)存在一个或多个保守修饰的重链可变区序列或CDR1、CDR2和CDR3序列。本领域知道,一些保守序列修改不会使抗原结合性消失。参见,例如,Brummell et al.,(1993)Biochem 32:1180-8;deWildt et al.,(1997)Prot.Eng.10:835-41;Komissarov et al.,(1997)J.Biol.Chem.272:26864-26870;Hall et al.,(1992)J.Immunol.149:1605-12;Kelleyand O′Connell(1993)Biochem.32:6862-35;Adib-Conquy et al.,(1998)Int.Immunol.10:341-6 and Beers et al.,(2000)Clin.Can.Res.6:2835-43。
因此,在一个实施方式中,本申请的单域抗体或结合分子(如重链抗体及其抗原结合部分)包含重链可变区,重链可变区包含CDR1、CDR2和CDR3,其中:
(a)重链可变区CDR1包含表1列出的序列,和/或其保守修改;和/或
(b)重链可变区CDR2包含表1列出的序列,和/或其保守修改;和/或
(c)重链可变区CDR3包含表1列出的序列,和/或其保守修改;且
(d)该单域抗体特异结合人PD-L1。
本申请的单域抗体或结合分子(如重链抗体及其抗原结合部分)具有一个或多个以下功能特征,例如对人PD-L1的高亲和力和高特异结合性、PD-1-PD-L1阻断能力、T细胞激活能力、以及体内抗肿瘤活性。
在多个实施方式中,单域抗体、重链抗体或其抗原结合部分可以是例如骆驼源、嵌合或人源化的。
本文所用的术语“保守序列修饰”是指不会显著影响或改变抗体或结合分子的结合特性的氨基酸修饰。这样的保守修饰包括氨基酸替换、添加和删除。可以通过领域内已知的标准技术,例如点突变和PCR介导的突变,将修饰引入本申请抗体或结合分子中。保守氨基酸替换是氨基酸残基用具有相似侧链的氨基酸残基进行替换。具有相似侧链的氨基酸残基组在领域内已知。这些氨基酸残基组包括具有碱性侧链(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、色氨酸)、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、甲硫氨酸)、β-支链侧链(例如,苏氨酸、缬氨酸、异亮氨酸)和芳香族侧链(例如,酪氨酸、苯丙氨酸、色氨酸、组氨酸)的氨基酸。因此,本申请抗体的CDR区中的一个或多个氨基酸残基可以用同侧链组的其他氨基酸残基替换,且得到的抗体或结合分子可以使用本文所述的功能检测对其进行保留功能(即,上述的功能)的测试。
本申请的单域抗体或结合分子(如重链抗体或其抗原结合部分)可以以本申请PD-L1单域抗体或结合分子作为起始材料,制备成基因修饰的单域抗体或结合分子。单域抗体或结合分子可以通过修饰VHH内(例如,在一个或多个CDR区和/或一个或多个骨架区)的一个或多个残基来进行基因修饰,以改善结合亲和力和/或增加与某些物种中天然产生的抗体的相似性。或者,抗体可以通过修饰恒定区中的残基进行基因修饰,例如改变抗体的效应功能。
在某些实施方式中,CDR区植入可以用来基因修饰抗体的可变区。单域抗体主要通过位于3个重链互补决定区(CDR)中的氨基酸残基与靶标抗原进行相互作用。出于这个原因,CDR内的氨基酸残基比起CDR外的序列在个体抗体之间更加地多样。因为CDR序列负责主要的抗体-抗原相互作用,可以通过构建含有特定天然抗体的CDR序列植入到不同特性的不同抗体的骨架序列的表达载体,来表达模拟特定天然抗体的特性的重组抗体(Riechmannet al.,(1998)Nature 332:323-327;Jones et al.,(1986)Nature 321:522-525;Queenet al.,(1989)Proc.Natl.Acad;U.S.A.86:10029-10033;U.S.Pat.Nos.5,225,539;5,530,101;5,585,089;5,693,762和6,180,370)。
因此,本申请的另一实施方式涉及分离的单克隆单域抗体或包含该单域抗体的结合分子,如重链抗体或其抗原结合部分,其包含重链可变区VHH,VHH包含具有本申请上述序列的CDR1、CDR2和CDR3。尽管这些单域抗体或结合分子包含本申请的VHHCDR序列,它们可以含有不同的骨架序列。
这样的骨架序列可以从包括种系抗体基因序列的公开DNA数据库或公开参考文献中获得。例如,用于人重链可变区基因的种系DNA序列可以在Vbase人种系序列数据库(www.mrc-cpe.cam.ac.uk/vbase)以及Kabat et al.,(1991),同上;Tomlinson et al.,(1992)J.Mol.Biol.227:776-798;和Cox et al.,(1994)Eur.J.Immunol.24:827-836中获得。作为另一实施方式,用于人重链可变区基因的种系DNA序列可以在Genbank数据库中得到。例如,下列HCo7 HuMAb小鼠中的重链种系序列的Genbank登录号为1-69(NG--0010109,NT--024637&BC070333)、3-33(NG--0010109&NT--024637)和3-7(NG--0010109&NT--024637)。作为另一例子,以下来自Hco12 HuMAb小鼠的重链种系序列的Genbank登录号为1-69(NG--0010109,NT--024637&BC070333)、5-51(NG--0010109&NT--024637)、4-34(NG--0010109&NT--024637)、3-30.3(CAJ556644)和3-23(AJ406678)。
通过使用本领域公知的称为空格(gap)BLAST的序列相似性搜索方法之一(Altschul et al.,(1997)),将单域抗体蛋白序列与蛋白序列数据库进行比较。
用于本申请单域抗体、重链抗体或其抗原结合部分的优选骨架序列是结构上与本申请单域抗体、重链抗体或其抗原结合部分所用的骨架序列相似的那些。VHH CDR1、CDR2、和CDR3序列可以植入到与得到该骨架序列的种系免疫球蛋白基因具有相同序列的骨架区中,或者CDR序列可以植入到包含有与种系序列相比具有一个或多个突变的骨架区中。例如,在一些情况下,将骨架区中的残基进行突变是有益的,以保持或增强抗体的抗原结合性(参见例如U.S.Pat.Nos.5,530,101;5,585,089;5,693,762和6,180,370)。
另一类的可变区修饰是将VHH CDR1、CDR2和/或CDR3区内的氨基酸残基进行突变,从而改进目标抗体的一种或多种结合特性(例如,亲和力)。可以进行点突变或PCR介导的突变来引入突变,且其对于抗体结合或其他功能特性的影响可以在本领域所知的体外或体内检测中进行评价。优选地,引入本领域所知的保守修饰。突变可以是氨基酸替换、添加或缺失,但优选为替换。此外,通常改变CDR区内的不多于一个、两个、三个、四个或五个的残基。
此外,在另一实施方式中,本申请提供分离的PD-L1单域抗体、重链抗体或其抗原结合部分,包含重链可变区,其包含:(a)VHH CDR1区,包含本申请的序列,或一个、两个、三个、四个或五个氨基酸替换、缺失或添加的氨基酸序列;(b)VHH CDR2区,包含本申请的序列,或一个、两个、三个、四个或五个氨基酸替换、缺失或添加的氨基酸序列;和(c)VHH CDR3区,包含本申请的序列,或一个、两个、三个、四个或五个氨基酸替换、缺失或添加的氨基酸序列。
本申请的基因改造抗体包括在VHH的骨架残基中做出基因修饰以例如改变抗体特性的那些。骨架修饰包括对骨架区的、或者甚至一个或多个CDR区的一个或多个残基进行突变,以去除T细胞表位,从而减少抗体的可能导致的免疫原性。该方法也称为“去免疫化”,在美国专利公开20030153043中有更加详细的描述。
此外,作为骨架或CDR区内修饰之外的另一种选择,本申请的重链抗体可以基因改造成在Fc区包括基因修饰,通常来改变抗体的一个或多个功能特性,例如血清半衰期、补体结合、Fc受体结合、和/或抗体依赖的细胞毒性。此外,本申请的抗体可以进行化学修饰(例如,可以向抗体附加一个或多个化学功能基团),或者修饰成改变其糖基化,来改变抗体的一个或多个功能特性。
在一个实施方式中,CH1的铰链区进行修饰,改变,例如增加或减少铰链区的半胱氨酸残基的数量。该方法在美国专利5,677,425中进一步描述。改变CH1铰链区的半胱氨酸残基,来例如促进重链轻链的组装或增加/降低抗体的稳定性。
在另一个实施方式中,对重链抗体的Fc铰链区进行突变,以增加或降低抗体的生物半衰期。更加具体地,将一个或多个氨基酸突变引入Fc铰链片段的CH2H2-CH3H3连接区,从而抗体相对于天然Fc-铰链结构域SpA结合而言,具有减弱的SpA结合力。该方法在美国专利6,165,745中有更详细的描述。
在另一实施方式中,修饰抗体的糖基化。例如,可以制备去糖基化的抗体(即,抗体缺少糖基化)。可以改变糖基化,来例如增加抗体对抗原的亲和性。这样的糖化修饰可以通过例如改变抗体序列中的一个或多个糖基化位点来达成。例如,可以做出一个或多个氨基酸替换,以消除一个或多个可变区骨架糖基化位点,从而消除该位置的糖基化。这样的去糖基化可以增加抗体对抗原的亲和性。参见,例如美国专利5,714,350和6,350,861。
此外,可以制备具有改变的糖基化类型的抗体,例如岩藻糖残基量减少的低岩藻糖基抗体,或者具有增加的平分型GlcNac结构的抗体。改变的糖基化形式被证明能增加抗体的ADCC活性。这样的糖化修饰可以通过例如在糖基化系统改变的宿主细胞中表达抗体而进行。具有改变的糖基化系统的细胞在领域中已知,包括但不限于,FUT8剔除细胞系、变异CHO细胞系Lec13、大鼠融合瘤细胞系YB2/0、包含特异性地针对FUT8基因的小干扰RNA的细胞系、共表达β-1,4-N-乙酰基葡糖胺基转移酶III和高尔基体α-甘露糖苷酶II的细胞系。它们可以用作表达本申请重组抗体的宿主细胞,以制备具有改变的糖基化的抗体。例如,
本文抗体的另一修饰是聚乙二醇化(PEG化)。抗体可以PEG化,例如来增加抗体的生物(例如,血清)半衰期。为使抗体PEG化,抗体或其片段通常与聚乙二醇(PEG),例如PEG的反应性酯或醛类衍生物,在使一个或多个PEG基团附于抗体或抗体片段的条件下反应。优选地,PEG化通过与反应性PEG分子(或类似的有反应性的水溶性聚合物)的酰化反应或烷化反应进行。本文中所用的术语“聚乙二醇”包括任何形式的用于衍生其他蛋白的PEG,例如单(C1-C10)烷氧基-或芳氧基聚乙二醇或聚乙二醇马来酰亚胺。在某些实施方式中,需要PEG化的抗体是去糖基化的抗体。PEG化蛋白的方法在领域内已知,且可以应用到本申请的抗体。参见,例如EPO 154 316和EP 0 401 384。
本申请的抗体可以用它们的多种物理特性进行表征,以检测和/或区别其分类。
例如,抗体可以在重链可变区包含一个或多个糖基化位点。这些糖基化位点可能引起增加的抗体免疫原性,或由于改变的抗原结合而引起改变的抗体pK值(Marshall etal(1972)Annu Rev Biochem 41:673-702;Gala and Morrison(2004)J Immunol 172:5489-94;Wallick et al(1988)J Exp Med 168:1099-109;Spiro(2002)Glycobiology 12:43R-56R;Parekh et al(1985)Nature 316:452-7;Mimura et al.,(2000)Mol Immunol37:697-706)。糖基化已知发生在含有N-X-S/T序列的基序中。在一些情况下,优选PD-L1抗体不包含可变区糖基化。这可以通过选择不在可变区包含糖基化基序的抗体或通过突变糖基化区域的残基来实现。
在优选实施方式中,抗体不包含天冬酰胺异构位点。天冬酰胺的脱酰胺可能出现在N-G或D-G序列,创建出异天冬氨酸残基,其向多肽链中引入扭结并降低其稳定性(异天冬氨酸效果)。
各抗体将具有独特的等电点(pI),基本落在6-9.5的pH范围内。IgG抗体的pI通常落在7-9.5的pH范围内,而IgG4抗体的pI基本落在6-8的pH范围内。推测pI在正常范围外的抗体可能在体内条件下具有一些展开结构且不稳定。因此,优选PD-L1单域抗体或结合分子的pI值落在正常范围内。这可以通过选择pI在正常范围内的抗体或通过突变不带电的表面残基来实现。
在另一方面,本申请提供编码本申请单域抗体或结合分子(包括重链抗体或其抗原结合部分)的重链可变区、CDR、和/或其他片段的核酸分子。核酸分子可以存在整细胞中,在细胞裂解液中,或处于部分纯化或基本纯的形式。当通过标准技术从其他细胞组分或其他污染物例如其他细胞核酸或蛋白中纯化出来后,核酸是“分离的”或“基本纯的”。本申请的核酸可以为例如DNA或RNA,且可能包含或可能不包含内含子序列。在优选实施方式中,核酸是cDNA分子。
本申请的核酸分子可以使用标准的分子生物学技术获得。对于由杂交瘤(例如,由携带人免疫球蛋白基因的转基因小鼠制备的杂交瘤)表达的抗体,编码杂交瘤制备的抗体的重链的cDNA可以通过标准PCR扩增或cDNA克隆技术获得。对于(例如使用噬菌体展示技术)从免疫球蛋白基因库获得的抗体,编码这类抗体的核酸可以从基因库中收集。
优选的本申请核酸分子包括编码PD-L1单域抗体的VHH序列、和/或CDR的那些。一旦获得了编码VHH的DNA片段,这些DNA片段可以进一步通过标准的重组DNA技术进行操作,例如将可变区基因转变为全长抗体链(重链抗体)基因。术语“可操作地连接”是指两个DNA片段连接在一起,从而两个DNA片段编码的氨基酸序列都在阅读框内。
编码VHH区的分离DNA可以通过可操作地连接VHH编码DNA与编码重链恒定区(CH1、CH2和CH3)的另一DNA分子而转变成全长重链基因。人重链恒定区基因的序列在领域内已知,且包括这些区域的DNA片段可以通过标准PCR扩增而获得。重链恒定区可以是IgG1、IgG2、IgG3、IgG4、IgA、IgE、IgM或IgD恒定区,但是优选为IgG1或IgG4恒定区。
本申请的单克隆单域/重链抗体可以使用Kohler and Milstein(1975)Nature256:495的体细胞杂交(杂交瘤)技术进行制备。制备单克隆单域/重链抗体的其他实施方式包括B淋巴细胞的病毒或致癌性转化以及噬菌体展示技术。嵌合或人源化抗体也在领域内熟知。参见,例如美国专利4,816,567;5,225,539;5,530,101;5,585,089;5,693,762和6,180,370。
本申请的单域抗体、重链抗体或其抗原结合部分还可以使用例如重组DNA技术结合基因转染方法,在宿主细胞转染瘤中生成(例如Morrison,S.(1985)Science 229:1202)。在一个实施方式中,将由标准分子生物技术得到的编码部分或全长重链的DNA插入一个或多个表达载体中,从而基因与转录和翻译调控序列可操作地连接。在该情况下,术语“可操作地连接”是指抗体基因连接到载体中,从而载体内的转录和翻译控制序列行使它们既定的调控抗体基因转录和翻译的功能。
术语“调控序列”包括控制抗体基因转录或翻译的启动子、增强子和其他表达控制元件(例如,多腺苷酸化信号)。这样的调控序列在例如Goeddel(Gene ExpressionTechnology.Methods in Enzymology 185,Academic Press,San Diego,Calif.(1990))中有过描述。优选的用于哺乳动物宿主细胞表达的调控序列包括引导在哺乳动物细胞中的高水平蛋白表达的病毒元件,例如得自巨细胞病毒(CMV)、猿猴病毒40(SV40)、腺病毒的启动子和/或增强子,如腺病毒主要晚期启动子(AdMLP)和多瘤病毒。或者,可以使用非病毒调控序列,例如泛素启动子或β-珠蛋白启动子。另外,调控元件由不同来源的序列构成,例如SRα启动子系统,其包含来自SV40早期启动子的序列和人T细胞白血病I型病毒的长末端重复(Takebe et al.,(1988)Mol.Cell.Biol.8:466-472)。表达载体和表达控制序列选为与所使用的表达宿主细胞相容。
重链可变区通过插入到已经编码所需亚型的重链恒定区的表达载体中而构建重链抗体基因,从而VHH与载体中的CH可操作地连接。或者,重组表达载体可以编码促进抗体链从宿主细胞分泌的信号肽。抗体链基因可以克隆到载体中,从而信号肽在阅读框内连接到抗体链基因的氨基端。信号肽可以是免疫球蛋白信号肽或异源信号肽(即,来自非免疫球蛋白的信号肽)。
除抗体链基因和调控序列外,本申请的重组表达载体可以携带其他序列,例如调控载体在宿主细胞中复制的序列(例如,复制起始点)和可选择标记物基因。可选择标记物基因可用于选择已导入载体的宿主细胞(参见,例如,美国专利4,399,216;4,634,665和5,179,017)。例如,通常可选择标记物基因赋予已导入载体的宿主细胞以药物抗性,例如G418、潮霉素、或氨甲喋呤抗性。优选的可选择标记物基因包括二氢叶酸还原酶(DHFR)基因(用于dhfr宿主细胞的氨甲喋呤选择/扩增)和neo基因(用于G418选择)。
对于重链的表达,编码重链的表达载体通过标准技术转染到宿主细胞中。多个形式的术语“转染”包括多种常用于将外源DNA导入原核或真核宿主细胞的技术,例如,电穿孔、磷酸钙沉淀、DEAE-右旋糖转染等。尽管在原核或真核宿主细胞中表达本申请抗体在理论上是可行的,优选抗体在真核细胞中表达,最优选在哺乳动物宿主细胞中表达,因为真核细胞,特别是哺乳动物细胞,比原核细胞更可能组装并分泌适当折叠且有免疫活性的抗体。
优选的用于表达本申请重组抗体的哺乳动物宿主细胞包括FUT8剔除细胞系、变异CHO细胞系Lec13、大鼠融合瘤细胞系YB2/0、包含特异性地针对FUT8基因的小干扰RNA的细胞系、共表达β-1,4-N-乙酰基葡糖胺基转移酶III和高尔基体α-甘露糖苷酶II、中国仓鼠卵巢(CHO细胞)(包括与DHFR可选择标记物一起施用的dhff-CHO细胞,在Urlaub and Chasin,(1980)Proc.Natl.Acad.Sci.USA 77:4216-4220中有过描述,DHFR可选择标记物在例如R.J.Kaufman and P.A.Sharp(1982)J.Mol.Biol.159:601-621中描述)、NSO骨髓瘤细胞、COS细胞和SP2细胞。当编码抗体基因的重组表达载体导入哺乳动物宿主细胞时,通过将宿主细胞培养足以使得宿主细胞中抗体表达、或优选地足以使得使抗体分泌到宿主细胞生长的培养基中的一段时间,从而制备抗体。抗体可以使用蛋白纯化方法从培养基中回收。
本申请的单域抗体或结合分子(包括重链抗体或其抗原结合部分)可以与治疗剂偶联,形成免疫偶联物,例如抗体-药物偶联物(ADC)。合适的治疗剂包括细胞毒素、烷化剂、DNA小沟结合分子、DNA嵌入剂、DNA交联剂、组蛋白去乙酰化酶抑制剂、核输出抑制剂、蛋白酶体抑制剂、拓扑异构酶I或II的抑制剂、热激蛋白抑制剂、酪氨酸激酶抑制剂、抗生素和抗有丝分裂剂。在ADC中,抗体或结合分子和治疗剂优选地通过接头交联,该接头可切割,例如肽类接头、二硫类接头或腙类接头。更优选地,接头是肽类接头,例如Val-Cit、Ala-Val、Val-Ala-Val、Lys-Lys、Ala-Asn-Val、Val-Leu-Lys、Ala-Ala-Asn、Cit-Cit、Val-Lys、Lys、Cit、Ser或Glu。ADC可以如美国专利7,087,600;6,989,452;和7,129,261;PCT公开WO 02/096910;WO 07/038,658;WO 07/051,081;WO 07/059,404;WO 08/083,312和WO 08/103,693;美国专利公开20060024317;20060004081和20060247295中描述般进行制备。
另一方面,本申请涉及包含与至少一个其他功能分子如另一种肽或蛋白(例如,另一抗体或受体配体)相连接的一种或多种本申请或结合分子(包括重链抗体或其抗原结合部分)的双特异性分子,以生成与至少两个不同结合位点或靶向分子结合的双特异性分子。术语“双特异性分子”包括具有三种或更多种特异性的分子。
双特异性分子可以以多种形式和尺寸出现。在尺寸谱的一端,双特异性分子保持传统抗体形式,除其具有两个结合臂且各臂具有不同特异性外,替代具有两个特异性相同的结合臂的情况。在另一极端的是双特异性分子,由两个经肽链连接的单域抗体构成。中间尺寸的双特异性分子包括由肽类接头连接的一个F(ab)片段和一个单域抗体。这些和其他形式的双特异性分子可以通过基因改造、体细胞杂交或化学法进行制备。参见,例如Caoand Suresh,Bioconjugate Chemistry,9(6),635-644(1998);和van Spriel et al.,Immunology Today,21(8),391-397(2000)。
本申请还提供包含PD-L1单域抗体或结合分子(包括重链抗体或其抗原结合部分)的嵌合抗原受体,该单域抗体或结合分子包含本申请中所述的重链CDR、或重链可变区。
PD-L1嵌合抗原受体可以包含(a)含有PD-L1单域抗体或结合分子的胞外抗原结合域;(b)跨膜结构域;和(c)胞内信号转导结构域。
溶瘤病毒偏好地侵染并杀灭癌细胞。本申请的单域抗体或结合分子(包括重链抗体或其抗原结合部分)可以与溶瘤病毒一起使用。此外,编码本申请单域抗体或结合分子(包括重链抗体或其抗原结合部分)的溶瘤病毒可以引入人体中。
在另一方面,本申请提供一种药物组合物,其包含本申请的单域抗体或结合分子(包括重链抗体或其抗原结合部分)、免疫偶联物、免疫细胞、双特异抗体、溶瘤病毒、核酸分子、表达载体、和/或表达宿主细胞,与药学上可接受的载体配制在一起。组合物可以任选地包含一种或多种其他药学上的有效成分,例如另一抗肿瘤抗体、或免疫增强抗体,或者非抗体类抗肿瘤剂、或免疫增强剂。本申请的药学组合物可以与例如另一抗癌剂、或另一免疫增强剂联合使用。
药学组合物可以包含任何数量的赋形剂。可以使用的赋形剂包括载体、表面活性剂、增稠或乳化剂、固体粘合剂、分散或混悬剂、增溶剂、染色剂、矫味剂、涂层、崩解剂、润滑剂、甜味剂、防腐剂、等渗剂及其组合。合适赋形剂的选择和使用在Gennaro,ed.,Remington:The Science and Practice of Pharmacy,20th Ed.(Lippincott Williams&Wilkins 2003)中有教导。
优选地,药物组合物适合于静脉内、肌内、皮下、肠道外、脊柱或表皮施用(例如通过注射或推注)。基于施用途径的不同,有效成分可以包在材料中,以保护其不受酸和可能使其失活的其他自然条件的影响。“肠道外施用”是指不同于肠道和局部外用的方式,通常通过注射进行,包括但不限于静脉内、肌内、动脉内、膜内、囊内、眶内、心脏内、皮内、腹膜内、经气管、皮下、表皮下、关节内、囊下、蛛网膜下、脊柱内、硬脑膜上和胸骨内注射和推注。或者,本申请的抗体可以通过非肠道外路径施用,例如外用、表皮施用或粘膜施用,例如鼻内、经口、阴道、直肠、舌下、或局部外用。
药物组合物可以为无菌水溶液或分散液的形式。它们也可以配制在微乳剂、脂质体或其他适于高浓度药物的有序结构中。
与载体材料一起制备成单剂型的有效成分的量将随着治疗主体和特定施用模式而变,且基本上而言是产生疗效的组合物的量。以百分比计,该量为约0.01-约99%的与药学上可接受载体结合的有效成分。
给药方案经调整提供最佳的所需反应(例如,治疗反应)。例如,可以施用快速灌注剂,可以随时间推移施用多个分剂量,或者剂量可以随治疗情况的危急程度成比例降低或提高。特别有利的是,以方便施用和剂量均匀的剂量单位型配置肠道外组合物。剂量单位型是指物理上分开的单位,适于治疗主体的单次给药;各单位包含计算出来与药学载体一起产生所需疗效的预定量的有效成分。或者,抗体可以以缓释剂施用,这种情况下所需的施用频率降低。
对于单域抗体或结合分子(包括重链抗体或其抗原结合部分)的施用,剂量可以为约0.001-100mg/kg宿主体重。示例性的治疗方案涉及每周施用一次。本申请药物组合物的优选给药方案包括静脉内施用。
“治疗有效量”的本申请药物组合物引起疾病症状严重程度的降低、无症状期频率和持续时间的增加。例如,对于带瘤受试者的治疗,“治疗有效量”优选地,与未治疗受试者相比,将肿瘤生长抑制至少约20%、更优选抑制至少约40%,甚至更优选地抑制至少约60%,且更优选地抑制至少约80%。治疗有效量的治疗抗体可以减小肿瘤尺寸,或者减轻受试者的症状,受试者可以是人或另一哺乳动物。
药物组合物可以是缓释试剂,包括植入体、和微胶囊递送系统。可以使用生物可降解、生物相容的聚合物,例如乙烯-醋酸乙烯、聚酸酐、聚乙醇酸、胶原蛋白、聚原酸酯、和聚乳酸。参见,例如,Sustained and Controlled Release Drug Delivery Systems,J.R.Robinson,ed.,Marcel Dekker,Inc.,New York,1978。
药学组合物可以经医学设备来给药,例如(1)无针皮下注射设备(例如,美国专利5,399,163;5,383,851;5,312,335;5,064,413;4,941,880;4,790,824;和4,596,556);(2)微量输液泵(美国专利4,487,603);(3)经皮给药设备(美国专利4,486,194);(4)推注设备(美国专利4,447,233和4,447,224);和(5)渗透设备(美国专利4,439,196和4,475,196)。
在某些实施方式中,本申请的单域抗体或结合分子可以经配制,以确保合适的体内分布。例如,为确保本申请的治疗抗体或结合分子穿越血脑屏障,抗体可以配制在脂质体中,其还可以额外地包含靶向功能基团,以增强对特定细胞或器官的选择性输送。参见,例如美国专利4,522,811;5,374,548;5,416,016;和5,399,331;V.V.Ranade(1989)J.Clin.Pharmacol.29:685;Umezawa et al.,(1988)Biochem.Biophys.Res.Commun.153:1038;Bloeman et al.,(1995)FEBS Lett.357:140;M.Owais et al.,(1995)Antimicrob.Agents Chemother.39:180;Briscoe et al.,(1995)Am.J.Physiol.1233:134;Schreier et al.,(1994)J.Biol.Chem.269:9090;Keinanen and Laukkanen(1994)FEBS Lett.346:123;和Killion and Fidler(1994)Immunomethods 4:273。
本申请的药物组合物具有多种体外和外内应用,涉及例如癌症的治疗,或者更笼统地讲,用于癌症等疾病患者的免疫增强。药物组合物可以施用至人受试者,以例如体内抑制肿瘤生长。
考虑到本申请药物组合物的抑制肿瘤细胞增殖和存活的能力,本申请提供抑制受试者中肿瘤细胞生长的方法,包括向该受试者施用本申请的药物组合物,从而在该受试者中肿瘤生长被抑制。可以由本申请抗体治疗的肿瘤的非限制性例子包括但不限于色素瘤、肺癌(如非小细胞肺癌)、尿路上皮癌、肾细胞癌、头颈癌、霍奇金淋巴瘤、微卫星不稳定或错配修复缺陷癌、胃癌、结直肠癌、肝癌(如肝细胞癌)、和梅克尔(Merkel)细胞癌,不管是原发还是转移的。此外,难治的或复发的恶性肿瘤也可能可以用本申请的药物组合物进行治疗。
本申请提供本申请药物组合物与一种或多种其他抗体或非抗体类治疗剂一起施用的联合疗法,其能有效抑制受试者中的肿瘤生长。在一个实施方式中,本申请提供在受试者中抑制肿瘤生长的方法,包括向受试者施用本申请药物组合物以及一种或多种其他抗体,例如PD-1抗体和/或PD-L1抗体。在某些实施方式中,受试者是人。在另一个方面,本申请提供癌症治疗方法,其中本申请的药物组合物与化疗剂一起施用,化疗剂可以是细胞毒性剂。其他可以与本申请药物组合物联合的疗法包括但不限于免疫原性剂施用、白介素2(IL-2)施用、放疗、手术或激素去除。
本申请的药物组合物可以用于在受试者中增强免疫应答,例如激活免疫细胞如NK细胞、T细胞等。
本申请的药物组合物也可以用于在受试者中治疗感染性疾病,特别是慢性感染。在一些实施方式中,相关疾病可以为病毒造成的慢性感染疾病,如由乙肝病毒、丙肝病毒、人免疫缺陷病毒(HIV)或猴免疫缺陷病毒(SIV)引起的慢性感染疾病。在一些实施方式中,本申请的药物组合物可以与至少一种抗病毒剂一起施用。
本文讨论的治疗剂的组合可以作为在药学可接受载体中的单一组合物同时施用,或者作为分开的组合物同时施用,其中各药剂处于药学可接受载体中。在另一个实施方式中,治疗剂的组合可以按序施用。
此外,如果进行多次联合疗法施用,且药剂按序施用,则在各时间点的按序施用的次序可以反转或保持相同,按序施用可以与同时施用或其任何组合相结合。
本申请还通过以下实施例进行进一步描述,实施例不应当解读为限制性的。所有附图和在本申请中通篇引用的所有参考文献、Genebank序列、专利和公开专利申请都通过引用的方式全部并入本文。
实施例
实施例1.稳定表达人、猴或小鼠PD-L1的HEK293A细胞株的构建
使用HEK293A细胞来构建稳定过表达人、猴或小鼠PD-L1的细胞系。简单而言,合成人、猴或小鼠PD-L1(氨基酸序列分别如SEQ ID NOs:11、12和13所示)的cDNA序列,并经酶切克隆到pLV-EGFP(2A)-Puro载体(北京英茂盛业生物科技有限公司,中国)中。将得到的pLV-EGFP(2A)-Puro-PD-L1与psPAX和pMD2.G质粒通过脂质体转染的方式转染到HEK293T细胞(南京科佰公司,中国)中,产生慢病毒,具体转染方法与Lipofectamine 3000试剂盒(Thermo Fisher Scientific,美国)说明书步骤一致。转染三天后,从HEK293T细胞的细胞培养基(DMEM培养基(Cat#:SH30022.01,Gibco),补充有10%FBS(Cat#:FND500,Excell))中收获慢病毒。然后用慢病毒转染HEK293A细胞(南京科佰公司,中国),得到稳定表达人、猴、或小鼠PD-L1的HEK293A细胞(分别为HEK293A/人PD-L1、HEK293A/猴PD-L1、HEK293A/小鼠PD-L1)。转染的HEK293A细胞培养在含有0.2μg/ml嘌呤毒素(Cat#:A11138-03,Gibco)的DMEM+10%FBS培养基中7天。人和猴PD-L1的表达使用市售的PD-L1抗体(PE-人PD-L1抗体,Biolegend,美国,Cat#:393607)通过流式分析仪经FACS分析。相似地,小鼠PD-L1的表达通过市售的小鼠PD-L1抗体(PE-小鼠PD-L1抗体,Biolegend,美国,Cat#:124307)经FACS确证。
实施例2.制备产生骆驼抗人PD-L1纳米抗体的噬菌体文库
骆驼抗人PD-L1纳米抗体通过构建噬菌体展示文库技术获得。
免疫
表2.免疫方案
用重组人PD-L1(ECD)-hFc(义翘神州,中国,Cat#:10084-H02H)和重组猴PD-L1(ECD)-hFc(义翘神州,中国,Cat#:90251-C02H),交叉注射免疫新疆双峰驼,具体免疫流程如表2所示。人PD-L1(ECD)-hFc蛋白和猴PD-L1(ECD)-hFc蛋白用等体积的完全Freund佐剂(Cat#:F5881-10*10ML,Sigma,美国)或不完全Freund佐剂(Cat#:F5506-6*10ML,Sigma,美国)超声乳化。第六次免疫后1周,从新疆双峰驼颈部取5ml血清,使用重组人PD-L1(ECD)-his(Cat#:10084-H08H,义翘神州,中国)和猴PD-L1(ECD)-his(Cat#:10377-H08H,义翘神州,中国),经ELISA检测滴度。
噬菌体文库的制备
通过噬菌体文库展示技术制备噬菌体文库。
六次免疫结束一周后,基于ELISA结果,从新疆双峰驼颈部取100ml血清,提取总RNA,合成cDNA,并利用套式PCR扩增VHH。利用限制性内切酶PstI及NotI酶切20μg pMECS噬菌体展示载体及10μg VHH,并连接两种片段。将连接产物转化至电转感受态细胞TG1中,构建纳米抗体噬菌体展示文库并测定库容,库容的大小约为1.2×109。
噬菌体文库的富集
取200μL重组TG1细胞至2×TY培养基中培养,期间加入40μL辅助噬菌体VCSM13侵染TG1细胞,并培养过夜以扩增噬菌体。次日,利用PEG/NaCl沉淀噬菌体,离心收集扩增噬菌体。将溶于100mM pH 8.2NaHCO3中的PD-L1蛋白200μg偶联在酶标板上,4℃放置过夜,同时设立阴性对照。第二天加入100μl的3%BSA,室温封闭2h。2h后,加入100μl扩增噬菌体(2×1011tfu免疫骆驼纳米抗体噬菌体展示基因库),室温作用1h。板用PBS+0.05%Tween-20洗5遍,以洗掉未结合的噬菌体。用终浓度为25mg/ml的胰蛋白酶将与PD-L1蛋白特异性结合的噬菌体解离下来,并感染处于对数生长期的大肠杆菌TG1细胞,37℃培养1h,产生并收集噬菌体,用于下一轮的筛选。相同的筛选过程重复3轮,使噬菌体逐步得到富集。
通过ELISA筛选噬菌体阳性克隆
从上述3轮筛选后的细胞培养板中,挑选200个单菌落分别接种于含100μg/mL氨苄青霉素的TB培养基的96深孔板中,并设置空白对照。37℃培养至对数期后,加入终浓度为1mM的IPTG,28℃培养过夜。利用渗透胀破法获得粗提抗体,并将抗体转移至经抗原包被的ELISA板上,室温放置1h。用PBST洗去未结合的抗体,加入100μl经1∶2000稀释后的小鼠抗-HA标签抗体(购自科文斯),在室温放置1h。用PBST洗去未结合的抗体,加入100μl经1∶2000稀释后的抗小鼠碱性磷酸酶结合物(购自于西格玛),在室温放置1h。用PBST洗去未结合的碱性磷酸酶结合物,加入碱性磷酸酶显色液,反应5-10min后于酶标仪上450nm波长处,读取吸收值。当样品孔OD值大于对照孔5倍以上时,判定为阳性克隆孔。将阳性克隆孔的菌转摇在含有100μg/μl氨苄青霉素的LB培养基中以便提取质粒并进行测序。
根据序列比对软件VectorNTI分析各个克隆株的基因序列,把FR1、FR2、FR3、FR4、CDR1、CDR2、CDR3序列相同的株视为同一克隆株,而序列不同的株视为不同克隆株,最终获得9株产生PD-L1抗体的噬菌体阳性克隆。
实施例3.骆驼PD-L1纳米抗体-Fc融合蛋白的表达和纯化
对实施例2得到的9个克隆,进行进一步的研究。首先将9个所选克隆的纳米抗体与Fc融合,进行融合蛋白的表达和纯化,其中纳米抗体重链可变区的C端与Fc的N端连接。首先通过PCR方法对这个抗体的噬菌体克隆进行重链可变区序列的克隆,并测序。序列总结在表1和表7中。通过将编码可变区和人IgG1 Fc区(SEQ ID NO:10)的序列插入到pCDNA3.1(Invitrogen,美国)的限制性酶切位点XhoI/BamHI之间来构建表达载体。
将上述获得的表达载体PEI转染HEK-293F细胞(Cobioer,中国)。具体而言,HEK-293F细胞在Free StyleTM 293表达培养基(Cat#:12338-018,Gibco)中培养,并用聚乙烯亚胺(PEI)转染的方式将各表达载体转染至细胞,DNA与PEI的比例是1∶3,每毫升细胞培养液中加入DNA的量是1.5μg。转染后的HEK-293F细胞在37℃、5%CO2的培养箱中以120RPM转速培养。10-12天后,收集细胞培养上清,3500rpm离心5分钟,并通过0.22μm滤膜过滤除去细胞碎片。融合蛋白通过预平衡的蛋白-A亲和柱(Cat#:17040501,GE,美国)来富集纯化。后用洗脱缓冲液(20mM柠檬酸,pH3.0-pH3.5)进行洗脱。之后,抗体保存在PBS(pH 7.0)中,并通过NanoDrop检测抗体浓度。
实施例4.骆驼PD-L1抗体-Fc融合蛋白促进T细胞激活
PD-L1纳米抗体-Fc融合蛋白对APC介导的T细胞激活的作用通过混合淋巴细胞反应(MLR)检测进行研究。
简单而言,通过梯度密度离心收集健康人供体血样中的PBMC,重悬于RPMI1640培养基中。PBMC在37℃孵育箱中培养2小时,收集贴壁细胞,即得到分离的单核细胞。单核细胞在补充有100ng/ml重组人GM-CSF(Cat#:7954-GM,R&D,美国)、100ng/ml重组人IL-4(Cat#:6507-IL,R&D,美国)和10%FBS的RPMI1640培养基中培养。三天后,将一半的培养基替换为新鲜培养基。在培养的第6天,将培养基替换为含有100ng/ml重组人GM-CSF、100ng/ml重组人IL-4、10ng/ml rhTNF-α(Cat#:210-TA-100,R&D,US)、1000U/ml rhIL-6(Cat#:7270-IL-025,R&D,US)、1μg/ml PGE2(Cat#363-24-6,TOCRIS,US)和10ng/ml IL-1β(Cat#:210-LB-025,R&D,US)的培养基。细胞再孵育2天。之后,通过梯度密度离心收集另一健康人供体血样中的PBMC,重悬于RPMI1640培养基中。使用Invitrogen Dynabeads不接触人CD4+ T细胞分离试剂盒(Cat#:11346D,Thermal Fisher Scientific,美国)从PBMC中分离出CD4+ T细胞。在96孔U形底检测板中,将来自第一供体的树突细胞和来自第二供体的CD4+ T细胞以2.5×104细胞/孔和5×104细胞/孔的密度进行铺板,培养基总体积为150μl。向孔中加入50μl PD-L1纳米抗体-Fc融合蛋白(0.1-10μg/ml)、或对照抗体Hel(Cat#:LT12031,LifeTein,美国),板再孵育72小时。使用制造商的方法步骤,经ELISA(Cat#:SIF50,R&D,美国)确定IFN-γ浓度。实验设三个重复。
如图1所示,在用10CA81和10CA192的PD-L1纳米抗体-Fc融合蛋白处理的孔中,检测到最高量的IFN-γ。
实施例5.PD-L1纳米抗体-Fc融合蛋白与HEK293A细胞表达的人和猴PD-L1结合
为确定PD-L1纳米抗体-Fc融合蛋白是否与HEK293A细胞表达的人、猴或小鼠PD-L1结合,分别使用实施例1构建的稳定过表达人、猴或小鼠PD-L1的HEK293A细胞进行FACS细胞结合检测。简单而言,将100μl培养基中的105个HEK293A细胞在96孔板上铺板,并加入50μl梯度稀释的PD-L1纳米抗体-Fc融合蛋白。4℃孵育1个小时后,96孔板用PBST清洗3遍。之后,加入500倍稀释的PE-山羊抗人IgG(Cat#:PAI-86078,Thermofisher,美国)。4℃孵育1小时后,96孔板用PBS清洗3遍,然后使用FACS检测仪(BD)检测细胞荧光。
表3.PD-L1纳米抗体-Fc融合蛋白与人、猴以及小鼠PD-L1的结合力
数据表明,所有PD-L1纳米抗体-Fc融合蛋白均对人以及猴PD-L1表现出高结合力,但不与小鼠PD-L1结合。两种代表性抗体10CA81、10CA192的结合力的EC50总结在表3中。图2示出10CA81和10CA192对HEK293A/人PD-L1(A)和HEK293A/猴PD-L1(B)有较强的结合能力,且呈梯度依赖关系。
实施例6.PD-L1纳米抗体-Fc融合蛋白抑制人PD-L1-PD-1相互作用
使用实施例1制备的稳定过表达人PD-L1的HEK293A细胞,经FACS进行阻断检测。简单而言,将100μl培养基中的105个HEK293A/人PD-L1细胞在96孔板上铺板,并加入50μl梯度稀释的PD-L1纳米抗体-Fc融合蛋白。4℃孵育1个小时后,板用PBST清洗3遍。之后,加入100μl 200μg/ml PD1-mFc融合蛋白(义翘神州,中国,Cat#:10377-H05H),4℃孵育1个小时后,板用PBST清洗3遍,加入500倍稀释的PE-山羊抗小鼠IgG(Cat#:405307,BioLegend,美国)。4℃孵育1个小时后,板用PBST清洗3遍,使用FACS仪(BD)检测细胞荧光度。
表4.PD-L1抗体对PD-1-PD-L1相互作用的阻断力
抗体 | PD-1阻断检测EC50(M/L) |
阿替利珠单抗 | 1.8E-10 |
10CA81 | 1.1E-10 |
10CA192 | 1.8E-10 |
数据示出,9种抗体中仅有两种抗体无法阻断PD-L1-PD-1之间的相互作用,其他7种抗体都能显著地阻断PD-1-PD-L1的相互作用,其中,两种代表性抗体10CA81、10CA192的EC50值总结在表4中。图3示出10CA81和10CA192阻断PD-L1与PD1的相互作用,且呈梯度依赖关系,与阿替利珠单抗相比有更好或相当的阻断效果。
实施例7.PD-L1纳米抗体-Fc融合蛋白促进T细胞激活
PD-L1抗体对APC介导的T细胞激活的作用通过混合淋巴细胞反应(MLR)检测进行研究。具体地,使用实施例4的检测方法,对两个代表性融合蛋白10CA81和10CA192的PD-L1纳米抗体-Fc融合蛋白与阳性对照组阿替利珠单抗进行对比。如图4所示,相对于对照,这些抗体增加T细胞分泌IFN-γ,且为剂量依赖的方式,且在某些浓度下,10CA81和10CA192的活性要优于阿替利珠单抗。
实施例8.PD-L1纳米抗体的人源化改造
基于上述相关的功能试验,对10CA81和10CA192这2种抗体进行人源化改造和进一步研究。抗体的人源化改造通过互补决定区(CDR)移植法(美国专利5,225,539)进行,具体方法详见下文。
为选出这2种抗体的人源化受体框架,将其VHH序列与NCBI网站的人免疫球蛋白基因数据库(http://www.ncbi.nlm.nih.gov/igblast/)进行比对。选择与其同源度最高的人种系IGVH和IGVK作为人源化改造的框架。所选择的重链种系受体序列是人IGHV1-46*01。对2种抗体的可变结构域进行三维结构模拟,以确定可能对于维持CDR环状结构起重要作用的关键框架氨基酸残基,从而设计人源化抗体的回复突变。
表5.人源化突变氨基酸总结
基于上述的结构,10CA81 VHH鉴定出10个潜在的回复突变(M48I、M70L、R72V、R87T、R38K、A40R、T28S、Y95F、R67K、V68A)。10CA192 VHH鉴定出7个潜在的回复突变(E1Q、T28A、F29V、A97S、K98Q、W108K、I70T)。
如表5所示,对于10CA81,共设计出5个人源化重链可变区,共得到5个人源化抗体。对于10CA192,共设计出5个人源化重链可变区,共得到5个人源化抗体。所有序列信息总结在表1、表5和表7中。
合成编码人源化重链可变区加人IgG1恒定区(SEQ ID NO:10)的序列,并分别利用EcoR I/Xho I和Cla I/Hind III限制性酶切位点克隆到GS表达载体(Invitrogen,美国)中。所有表达构建均经测序证实。用表达载体转染EXPiCHO表达系统(Invitrogen,美国),并瞬时表达10个人源化PD-L1抗体,方法步骤如实施例3所述。人源化抗体按照实施例3所述进行纯化。
实施例9.人源化PD-L1纳米抗体-Fc融合蛋白的PD-L1结合力/亲和力鉴定
根据实施例5的方法步骤,进一步通过FACS检测人源化纳米抗体-Fc融合蛋白与实施例1制备的HEK293A/人PD-L1细胞、HEK293A/猴PD-L1细胞以及HEK293A/小鼠PD-L1细胞的结合力。结果分别显示在图5和图6。
另外,通过BIAcoreTM8K(GE Life Sciences,美国)来定量测定嵌合或人源化PD-L1纳米抗体-Fc融合蛋白对人和猴PD-L1的结合亲和力。具体地,将100-200 RU(反应单位)的人PD-L1(ECD)-his蛋白(Cat#:10084-H08H,义翘神州,中国)或猴PD-L1(ECD)-his蛋白(Cat#:90251-C08H,义翘神州,中国)耦联到CM5生物芯片(Cat#:BR-1005-30,GE LifeSciences,美国)上,随后用1M氨基乙醇封闭芯片上的未反应基团。梯度稀释(浓度从0.3μM到10μM)的抗体注入到SPR反应液(HBS-EP缓冲液,pH7.4,Cat#:BR-1006-69,GE LifeSciences,美国)中,速度控制在30 μL/分钟。纳米抗体-Fc融合蛋白的结合力计算时,扣减空白对照孔的RU。结合速率(ka)和解离速率(kd)使用BIA评估软件中的1∶1配对模型的公式进行计算。平衡解离常数KD通过kd/ka计算得到。通过BIAcoreTM测量的人源化抗体-Fc融合蛋白的结合亲和力显示在表6中。
表6.PD-L1纳米抗体-Fc融合蛋白对人PD-L1的结合亲和力
如图5和图6所示,人源化PD-L1纳米抗体-Fc融合蛋白与人和猴PD-L1都有很强的亲和力,其结合力与各自母本相当。
如表6所示,人源化10CA81和10CA192纳米抗体-Fc融合蛋白与阿替利珠单抗相比,有相当的人PD-L1结合亲和力。
实施例10.人源化PD-L1纳米抗体-Fc融合蛋白抑制人PD-L1-PD-1相互作用
根据实施例6的方法步骤,使用实施例1制备的稳定过表达人PD-L1的HEK293A细胞,经FACS进行阻断检测。简单而言,将100μl培养基中的105个HEK293A/人PD-L1细胞在96孔板上铺板,并加入50μl梯度稀释的PD-L1抗体。4℃孵育1个小时后,板用PBST清洗3遍。之后,加入100μl 200μg/ml PD1-mFc融合蛋白(义翘神州,中国,Cat#:10377-H05H)4℃孵育1个小时后,板用PBST清洗3遍,加入500倍稀释的PE-山羊抗小鼠IgG(Cat#:405307,BioLegend,美国)。4℃孵育1个小时后,板用PBST清洗3遍,使用FACS仪(BD)检测细胞荧光度。
数据示出,人源化PD-L1纳米抗体-Fc融合蛋白,能显著地阻断PD-1-PD-L1的相互作用,其阻断力与各自母本相当。
实施例11.人源化PD-L1纳米抗体-Fc融合蛋白促进T细胞激活
进一步检测PD-L1人源化纳米抗体-Fc融合蛋白对T细胞的激活效果,采取实施例4中的实验方法。IFN-γ的分泌通过商品化检测试剂盒(R&D,US,Cat#:STA00C)按照说明书进行操作检测。
实验结果如图7所示,所有检测的人源化纳米抗体-Fc融合蛋白都能剂量依赖地促进T细胞的活性,增加IFN-γ的分泌。其中,抗体10CA81-VHH5、10CA81-VHH4、10CA192-VHH2和10CA192-VHH4的Fc融合蛋白具备最好的T细胞激活效应。
实施例12.人源化纳米抗体-Fc融合蛋白的体内抗肿瘤效果
对10CA81和10CA192人源化纳米抗体-Fc融合蛋白的体内抗肿瘤效果进行研究,所使用的动物模型通过对PD-L1靶点人源化的转基因小鼠(GemPharmatech Co.Ltd,中国)植入MC38小鼠肠腺癌而建立。小鼠在第0天在侧腹部皮下注射1×106MC38细胞,随机分成6组,每组10只。第1-6组小鼠分别在第0、4、7、11、14和18天腹腔注射PBS(20mg/kg,G1)、10CA81-VHH5-Fc融合蛋白(20mg/kg,G2)、10CA81-VHH4-Fc融合蛋白(20mg/kg,G3)、10CA192-VHH2-Fc融合蛋白(20mg/kg,G4)、10CA192-VHH4-Fc融合蛋白(20mg/kg,G5)、和阿替利珠单抗(20mg/kg,G6),其中阿替利珠单抗按照专利WO2013079174A1中的氨基酸序列合成表达。
随时间追踪肿瘤大小和小鼠体重。用游标卡尺测量肿瘤的长边(D)和短边(d),并通过公式TV=0.5x D x d2计算肿瘤体积。在抗体组肿瘤达到3.5cm3前停止实验。用单因素方差分析来确定肿瘤体积差异。
如图8所示,所有PD-L1抗体均能显著抑制人源化PD-L1小鼠中肿瘤的生长,且抗体10CA192-VHH4-Fc融合蛋白的体内抗肿瘤效果优于阿替利珠单抗。
本申请中提及的序列总结在表7中。
表7.序列
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尽管本发明已结合一个或多个实施方式进行了描述,应当理解的是,本发明不限于这些实施方式,且上述描述意在涵盖包括在所附权利要求的精神和范围内的所有其他可选择形式、修饰和等同物。本文引用的所有文献均通过引用的方式全部并入本文。
序列表
<110> 北京天广实生物技术股份有限公司
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Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
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Ser Thr Ile Asn Ser Gly Gly Gln Ser Ser Ser Tyr Leu Asp Ser Val
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85 90 95
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100 105 110
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65 70 75 80
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100 105 110
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<210> 9
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<213> 人工序列
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Gln Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
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20 25 30
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35 40 45
Ser Thr Ile Asn Ser Gly Gly Gln Ser Ser Ser Tyr Leu Asp Ser Val
50 55 60
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65 70 75 80
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<400> 10
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
1 5 10 15
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
20 25 30
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
35 40 45
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
50 55 60
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
65 70 75 80
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
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Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
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Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
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Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr
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Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
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Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
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Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
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Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
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Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
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Ser Leu Ser Leu Ser Pro Gly Lys
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<212> PRT
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Met Arg Ile Phe Ala Val Phe Ile Phe Met Thr Tyr Trp His Leu Leu
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Asn Ala Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr
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Gly Ser Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu
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Asp Leu Ala Ala Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile
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Ile Gln Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser
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Cys Thr Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu
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Val Ile Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Thr His
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Leu Val Ile Leu Gly Ala Ile Leu Leu Cys Leu Gly Val Ala Leu Thr
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Gly Ile Gln Asp Thr Asn Ser Lys Lys Gln Ser Asp Thr His Leu Glu
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Glu Thr
290
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<400> 12
Met Arg Ile Phe Ala Val Phe Ile Phe Thr Ile Tyr Trp His Leu Leu
1 5 10 15
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20 25 30
Gly Ser Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu
35 40 45
Asp Leu Thr Ser Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile
50 55 60
Ile Gln Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Asn
65 70 75 80
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85 90 95
Ala Ala Leu Arg Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr
100 105 110
Arg Cys Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val
115 120 125
Lys Val Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val
130 135 140
Asp Pro Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr
145 150 155 160
Pro Lys Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser
165 170 175
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180 185 190
Val Thr Ser Thr Leu Arg Ile Asn Thr Thr Ala Asn Glu Ile Phe Tyr
195 200 205
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210 215 220
Val Ile Pro Glu Leu Pro Leu Ala Leu Pro Pro Asn Glu Arg Thr His
225 230 235 240
Leu Val Ile Leu Gly Ala Ile Phe Leu Leu Leu Gly Val Ala Leu Thr
245 250 255
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260 265 270
Gly Ile Arg Val Thr Asn Ser Lys Lys Gln Arg Asp Thr Gln Leu Glu
275 280 285
Glu Thr
290
<210> 13
<211> 290
<212> PRT
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<223> 小鼠PD-L1
<400> 13
Met Arg Ile Phe Ala Gly Ile Ile Phe Thr Ala Cys Cys His Leu Leu
1 5 10 15
Arg Ala Phe Thr Ile Thr Ala Pro Lys Asp Leu Tyr Val Val Glu Tyr
20 25 30
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35 40 45
Asp Leu Leu Ala Leu Val Val Tyr Trp Glu Lys Glu Asp Glu Gln Val
50 55 60
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65 70 75 80
Phe Arg Gly Arg Ala Ser Leu Pro Lys Asp Gln Leu Leu Lys Gly Asn
85 90 95
Ala Ala Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr
100 105 110
Cys Cys Ile Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Leu
115 120 125
Lys Val Asn Ala Pro Tyr Arg Lys Ile Asn Gln Arg Ile Ser Val Asp
130 135 140
Pro Ala Thr Ser Glu His Glu Leu Ile Cys Gln Ala Glu Gly Tyr Pro
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165 170 175
Lys Arg Ser Val Thr Thr Ser Arg Thr Glu Gly Met Leu Leu Asn Val
180 185 190
Thr Ser Ser Leu Arg Val Asn Ala Thr Ala Asn Asp Val Phe Tyr Cys
195 200 205
Thr Phe Trp Arg Ser Gln Pro Gly Gln Asn His Thr Ala Glu Leu Ile
210 215 220
Ile Pro Glu Leu Pro Ala Thr His Pro Pro Gln Asn Arg Thr His Trp
225 230 235 240
Val Leu Leu Gly Ser Ile Leu Leu Phe Leu Ile Val Val Ser Thr Val
245 250 255
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260 265 270
Gly Val Glu Asp Thr Ser Ser Lys Asn Arg Asn Asp Thr Gln Phe Glu
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Glu Thr
290
Claims (16)
1.一种分离的结合分子,其与PD-L1结合,包含重链可变区,该重链可变区含有CDR1区、CDR2区和CDR3区,其中,CDR1区、CDR2区和CDR3区分别包含与(1)SEQ ID NOs:1、2(X1=T、X2=N、X3=A)和3(X=K);或(2)SEQ ID NOs:1、2(X1=L、X2=D、X3=V)和3(X=A)具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%同一性所示的氨基酸序列。
2.根据权利要求1所述的结合分子,其中重链可变区包含与SEQ ID NOs:4、5、6、7、8、或9具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%同一性所示的氨基酸序列。
3.一种分离的结合分子,其与PD-L1结合,包含重链可变区,该重链可变区含有SEQ IDNOs:4、5、6、7、8、或9中的CDR1区、CDR2区和CDR3区。
4.根据权利要求1或3所述的结合分子,还包含免疫球蛋白重链恒定区,与所述重链可变区形成融合蛋白。
5.根据权利要求4所述的结合分子,其中免疫球蛋白重链恒定区为IgG1恒定区或其Fc区。
6.根据权利要求5所述的结合分子,其中免疫球蛋白重链恒定区包含SEQ ID NO:10所示的氨基酸序列。
7.根据权利要求4所述的结合分子,其为该融合蛋白的二聚体。
8.根据权利要求1或7所述的分离的结合分子,其(a)与人PD-L1结合;(b)与猴PD-L1结合;(c)不与小鼠PD-L1结合;(d)阻断PD-1-PD-L1结合;(e)能够激活免疫细胞;和/或(f)具有体内抗肿瘤效果。
9.一种双特异性分子、免疫偶联物、嵌合抗原受体、基因改造T细胞受体、或溶瘤病毒,包含权利要求1-8中任一项所述的分离的结合分子。
10.一种核酸分子,编码权利要求1-8中任一项所述的结合分子。
11.一种药物组合物,包含权利要求1-8中任一项所述的结合分子、或权利要求10所述的核酸分子,以及药学上可接受的载体。
12.权利要求1-8中任一项所述的结合分子、权利要求10所述的核酸分子、或权利要求11所述的药物组合物在制备治疗PD-L1相关疾病的药物中的用途。
13.根据权利要求13所述的用途,其中PD-L1相关疾病为肿瘤。
14.根据权利要求14所述的用途,其中肿瘤为实体瘤或液体瘤。
15.根据权利要求14所述的用途,其中肿瘤选自黑色素瘤、肺癌、尿路上皮癌、肾细胞癌、头颈癌、霍奇金淋巴瘤、微卫星不稳定或错配修复缺陷癌、胃癌、结直肠癌、肝癌、和梅克尔细胞癌。
16.权利要求1-8中任一项所述的结合分子、或权利要求10所述的核酸分子、或权利要求11所述的药物组合物在制备用于增强免疫应答的药物中的用途。
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US18/323,645 US20230382995A1 (en) | 2022-05-31 | 2023-05-25 | Molecules binding pd-l1 and uses thereof |
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