CN113956352A - 新型冠状病毒中和性抗体及其制备方法与用途 - Google Patents
新型冠状病毒中和性抗体及其制备方法与用途 Download PDFInfo
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Abstract
本发明提供了一种特异性靶向SARS‑CoV‑2的刺突蛋白(spike蛋白)受体结合域(S‑RBD)的高亲和力中和抗体,中和性抗体对SARS‑CoV‑2假病毒的抑制率IC50为1.597μg/ml,亲和力KD值为10‑10M级别。中和性抗体可以阻断S‑RBD与人ACE2受体的相互反应,使得病毒无法进入到人体上皮细胞,抑制SARS‑CoV‑2病毒感染宿主的能力,可用于治疗SARS‑CoV‑2冠状病毒或者其它类似冠状病毒引起的感染性疾病。本发明还提供了编码本发明抗体的核酸分子、用于表达本发明抗体的表达载体、宿主细胞和方法,以及包含本发明抗体的药用组合物。
Description
技术领域
本发明涉及生物技术领域,具体涉及新型冠状病毒(SARS-CoV-2)中和性抗体及其制备方法与用途。
背景技术
冠状病毒是一个大型病毒家族,已知可引起感冒以及中东呼吸综合征 (MERS)和严重急性呼吸综合征(SARS)等较严重疾病。新型冠状病毒 (SARS-CoV-2)属于冠状病毒科的β-冠状病毒,它被认为是从蝙蝠身上衍生出来并感染人类,其常见体征有发热、乏力、干咳、逐渐出现呼吸困难,而部分患者病症状轻微,甚至存在无临床症状的无症状患者。具备人传人的特点,一般潜伏期1-14天,且潜伏期具有传染性,而无症状感染者也可能成为传染源,其主要通过呼吸道飞沫和密切接触传染传播,人群普遍易感。与SARS冠状病毒相比,SARS-CoV-2的感染率较高,但杀伤力相对较低。
感染SARS-CoV-2病毒的人会出现程度不同的症状,有的只是发烧或轻微咳嗽,有的会发展为肺炎,有的则更为严重甚至死亡。目前还没有预防和治疗新冠病毒的特效药物,已先后进入临床研究的候选药物包括洛匹那韦/利托那韦(lopinavir/ritonavir)、瑞德西韦(Remdesivir)、氯喹(Chloroquine)、血浆治疗、干细胞治疗、疫苗和中药研究等,但是目前为止披露的数据都乏善可陈。
新型冠状病毒通过动物宿主物种屏障,侵入呼吸道上皮细胞,特别是肺部,危害人类生命。研究发现,在康复期新冠病毒感染病人血中的中和抗体能够有效地阻断新冠病毒的入侵。当病毒入侵细胞时由人体B淋巴细胞产生中和作用的抗体,这种抗体能抢先与病毒表面的抗原结合,使得病毒无法进入人体细胞,从而被免疫系统清除。以往临床试验表明,单克隆中和抗体能降低埃博拉病人的病毒水平,起到有效中和病毒毒力、实质性改善临床症状、降低感染者死亡率等作用。
目前,还没有针对SARS-CoV-2的靶向药物和治疗性抗体。针对病毒表面尖峰蛋白的特异性抗体,特别是受体结合域(RBD)被认为是一种很有前途的中和性抗体,它可以抵抗进入人体的病毒,将有效治疗严重肺部感染患者的各种病毒。
发明概述
本发明提供了一种特异性靶向SARS-CoV-2的刺突蛋白(spike蛋白)受体结合域(S-RBD)中和抗体,可以阻断S-RBD与人ACE2受体的相互反应,使得病毒无法进入到人体上皮细胞。本发明提供了编码本发明抗体的核酸分子、用于表达本发明抗体的表达载体、宿主细胞和方法,以及包含本发明抗体的药用组合物。
本发明还提供了中和抗体短期预防和治疗COVID-19的医疗用途。该抗体也可用于新冠病毒的抗原检测和血清学检测。中和性抗体可以去除新冠病毒感染宿主的能力,保护暴露于病毒的未受感染宿主和解救重症病人的生命。因此,这种中和性抗体提供新冠病毒感染病人的治疗或者预防易感人群被新冠病毒侵袭,并且可能还提供SARS冠状病毒或者其它类似冠状病毒引起的感染性疾病。
在一方面,本发明提供了靶向新冠病毒SARS-CoV-2刺突蛋白受体结合域的单克隆抗体或其抗原结合部分,其包含:
(a)重链可变区CDR1,其包含与选自SEQ ID NO:1、SEQ ID NO:7、或SEQ ID NO:13所示一致的氨基酸序列;
(b)重链可变区CDR2,其包含与选自SEQ ID NO:2或SEQ ID NO:8所示一致的氨基酸序列;
(c)重链可变区CDR3,其包含与选自SEQ ID NO:3、SEQ ID NO:9、或SEQ ID NO:14所示一致的氨基酸序列;
(d)轻链可变区CDR1,其包含与选自SEQ ID NO:4或SEQ ID NO:10所示一致的氨基酸序列;
(e)轻链可变区CDR2,其包含与选自SEQ ID NO:5、SEQ ID NO:11、或SEQ ID NO:15所示一致的氨基酸序列;和
(f)轻链可变区CDR3,其包含与选自SEQ ID NO:6、SEQ ID NO:12或 SEQ ID NO:16所示一致的氨基酸序列。
在另一实施方案中,本发明提供了靶向新冠病毒SARS-CoV-2刺突蛋白受体结合域的单克隆抗体或其抗原结合部分,其重链可变区和轻链可变区的CDR序列同源性至少为90%,91%,92%,93%,94%,95%,96%,97%,98%, 99%或100%。
在又一个实施方案中,本发明提供靶向新冠病毒SARS-CoV-2刺突蛋白受体结合域的单克隆抗体或其抗原结合部分,其包含重链和轻链可变区序列:
(a)重链可变区,其包含与选自SEQ ID NO:17、SEQ ID NO:21或SEQ ID NO:25所示一致的氨基酸序列;
(b)轻链可变区,其包含与选自SEQ ID NO:19、SEQ ID NO:23或SEQ ID NO:27所示一致的氨基酸序列。
在另一实施方案中,本发明提供了靶向新冠病毒SARS-CoV-2刺突蛋白受体结合域的单克隆抗体或其抗原结合部分,其重链可变区与选自SEQ ID NO:17、SEQ ID NO:21或SEQID NO:25的氨基酸序列具有至少为90%, 91%,92%,93%,94%,95%,96%,97%,98%,99%或100%的序列同源性;其轻链可变区与选自SEQ ID NO:19、SEQ ID NO:23或SEQ IDNO:27的氨基酸序列具有至少为90%,91%,92%,93%,94%,95%,96%,97%,98%,99%或100%的序列同源性。
在另一实施方案中,所述抗体为人全长抗体。又一方面,所述人恒定区选自由IgGl、IgG2、IgG3、IgG4组成的组。在又一个具体方面中,所述人恒定区是IgG1。为提高抗体中和效应,本发明还对人恒定区是IgG1的 Fc段进行特定位点的突变,使得改造过的IgG1-Fc段由于突变使得FcR的结合能力丧失(主要指结合巨噬细胞和NK细胞的能力),目的是去除可能的抗体依赖的感染增强现象(ADE),但是保留补体激活通路(消灭被中和抗体结合的新冠病毒),上述改变还使得抗体的半衰期延长(FcRn再循环能力增强)。
在另一实施方案中,本发明抗体或抗体片段为人抗体或人抗体片段。
在另一实施方案中,本发明抗体片段为Fab、Fab’、Fab’-SH、Fv、 scFv或F(ab’)2抗体片段。
在另一实施方案中,本发明抗体片段为双抗体。
在另一实施方案中,本发明提供了靶向新冠病毒SARS-CoV-2刺突蛋白受体结合域的单克隆抗体,其包含:
(a)重链,其序列与SEQ ID NO:29、SEQ ID NO:33或SEQ ID NO: 37所示的氨基酸序列一致;以及
(b)轻链,其序列与SEQ ID NO:31、SEQ ID NO:35或SEQ ID NO: 39所示的氨基酸序列一致。
在本发明一个特别优选的实施方案中,本发明提供了IgG1-Fc经过突变的靶向新冠病毒SARS-CoV-2刺突蛋白受体结合域的单克隆抗体,其包含:
(a)重链,其序列与SEQ ID NO:41所示的氨基酸序列一致;以及
(b)轻链,其序列与SEQ ID NO:43所示的氨基酸序列一致。
本发明还提供了编码所述抗体或抗体片段的分离的多核苷酸。本发明提供了包含所述分离的多核苷酸的表达载体,以及包含所述表达载体的宿主细胞。
本发明提供了药物组合物,包含所述抗体或抗体片段以及可药用载体。
本发明提供了治疗疾病的方法,所述方法包括将治疗有效量的本发明的抗体或抗体片段给予需要治疗的COVID-19受试者或预防感染新冠病毒。
附图说明
附图1:中和性抗体R44的假病毒中和试验,横轴表示抗体的浓度,纵轴表示中和率。
附图2:Octet K2测定R44亲和力。横轴表示时间,纵轴表示结合高度。
附图3:Biacore 8K测定R44抗体亲和力。横轴表示时间,纵轴表示结合高度。
附图4:中和性抗体R44阻断ACE2和SARS-CoV-2S1蛋白结合试验,横轴表示时间,纵轴表示结合高度。
发明详述
为使本发明更易于理解,首先定义某些术语。别的定义将在整个详述中阐明。
除非另有说明,本发明的实施将采用分子生物学(包括重组技术)、微生物学、细胞生物学、生物化学和免疫学的常规技术,这些都在本领域的技术范围内,这些技术在本领域的技术文献和通用教科书中有充分解释,诸如Molecular Cloning:A Laboratory Manual(分子克隆:实验室手册)等。
术语“新冠病毒”和“新型冠状病毒”根据国际病毒分类委员会(ICTV) 命名规则正式名称为SARS-CoV-2,由新型冠状病毒感染引起的疾病的正式名称为“COVID-19”,其中“CO”代表“corona(冠状物)”,“VI”代表“virus(病毒)”,“D”代表“disease(疾病)”。
“S蛋白”即刺突糖蛋白,是一类很大的三聚体跨膜糖蛋白,其在病毒表面形成特殊的花冠结构,冠状病毒因此而得名。病原微生物入侵细胞时需要依赖病原体自身表达的特定分子与细胞上的受体结合,才能感染细胞,并进一步扩增。冠状病毒主要通过刺突糖蛋白(Spike glycoprotein,S glycoprotein)与宿主细胞受体结合介导病毒的入侵并决定病毒组织或宿主嗜性。S蛋白可识别宿主细胞受体并介导膜融合,对于病毒颗粒进入细胞至关重要,是病毒感染宿主细胞的关键因子。
“ACE2受体”是指人类宿主或其他动物的ACE2蛋白。SARS-CoV-2通过识别人类宿主的ACE2蛋白从而进入细胞的。
“RBD”指的是S蛋白结构域的细胞受体结合区(Receptor binding domain,RBD)直接参与了宿主受体的识别,该区域的氨基酸变异会导致病毒的种属嗜性和感染特性的变化。
“抗体”是指表现出所需生物学活性(例如抑制配体与其受体的结合或通过抑制配体诱导的受体信号转导)的抗体的任何形式。因此,“抗体”以其最广泛的意义来使用,并明确包括但不限于单克隆抗体(包括全长单克隆抗体)、多克隆抗体和多特异性抗体(例如双特异性抗体)。
“中和性抗体”是当病原微生物或其他抗原物质侵入机体时会产生相应的抗体。中和性抗体是B淋巴细胞产生的某些抗体,能够与病原微生物或其他抗原表面的抗原表位肽结合,从而阻止该病原微生物黏附靶细胞受体,防止侵入细胞。
“抗体片段”和“抗体结合片段”意即抗体的抗原结合片段及抗体类似物,包括但不限于:Fab、Fab′、F(ab′)2和Fv片段;双抗体;线性抗体(linear antibody);单链抗体分子,例如scFv、单抗体;纳米抗体;结构域抗体(Nanobody);和由抗体片段形成的多特异性抗体等。工程改造的抗体变体综述于Holliger和Hudson(2005)Nat.Biotechnol.23: 1126-1136中。
“Fab片段”由一条轻链和一条重链的CH1及可变区组成。Fab分子的重链不能与另一个重链分子形成二硫键。
“Fc”区含有包含抗体的CH1和CH2结构域的两个重链片段。两个重链片段由两个或多个二硫键并通过CH3结构域的疏水作用保持在一起。
“Fab′片段”含有一条轻链和包含VH结构域和CH1结构域以及CH1 和CH2结构域之间区域的一条重链的部分,由此可在两个Fab′片段的两条重链之间形成链间二硫键以形成F(ab′)2分子。
“F(ab′)2片段”含有两条轻链和两条包含CH1和CH2结构域之间的恒定区的部分的重链,由此在两条重链间形成链间二硫键。因此,F(ab′)2 片段由通过两条重链间的二硫键保持在一起的两个Fab′片段组成。
“Fv区”包含来自重链和轻链二者的可变区,但缺少恒定区。
“单链Fv抗体”(或“scFv抗体”)是指包含抗体的VH和VL结构域的抗体片段,其中这些结构域存在于单个多肽链中。一般而言,Fv多肽另外在VH和VL结构域之间包含多肽接头,该接头使得scFv能形成用于抗原结合的所需结构。
“双抗体”为具有两个抗原结合位点的小抗体片段。所述片段包含在相同的多肽链中与轻链可变结构域(VL)连接的重链可变结构域(VH)(VH-VL 或VL-VH)。通过使用短至不能在同一链的两个结构域之间配对的接头,迫使所述结构域与另一条链的互补结构域配对并形成两个抗原结合位点。
“分离的”抗体为业已被鉴定并与其天然环境组分相分离的抗体,其天然环境的污染组分是会干扰所述抗体的诊断性或治疗性应用的物质,可包括酶、激素和其它蛋白质溶质或非蛋白质溶质。在一些实施方案中,将所述抗体纯化到超过95%纯度,更优选超过99%纯度,其由Lowry法测定。分离的抗体通常由至少一个纯化步骤制备。
“分离的”核酸分子为被鉴定并与至少一种污染性核酸分子(通常在抗体核酸的天然来源中与其缔合)分离的核酸分子。分离的核酸分子不同于其天然存在的形式或环境。
术语“单克隆抗体””或“单克隆抗体组合物”在用于本文时指单一分子组合物的抗体分子的制备物。单克隆抗体组合物展现出对特定表位的单一结合特异性和亲和力。
本文所用序列“变体”是指在一个或多个氨基酸残基处不同于所示的序列但保留所得到的分子的生物学活性的序列。
本文所用术语“约”是指数值在由本领域一般技术人员所测定的具体值的可接受误差范围内,所述数值部分取决于怎样测量或测定(即测量体系的限度)。例如,“约”或“基本上包含”可意味着至多20%的范围。此外,特别对于生物学系统或过程而言,该术语可意味着至多一个数量级或数值的至多5倍。除非另外说明,否则当具体值在本申请和权利要求中出现时,“约”或“基本上包含”的含义应该假定为在该具体值的可接受误差范围内。
当用“给予”和“治疗”提及动物、人、实验对象、细胞、组织、器官或生物液时,是指将外源性药物、治疗剂、诊断剂或组合物与动物、人、受治疗者、细胞、组织、器官或生物液接触。“给予”和“治疗”可指例如治疗方法、药动学方法、诊断方法、研究方法和实验方法。治疗细胞包括让试剂与细胞接触以及让试剂与流液接触,其中所述流液与细胞接触。“给予”和“治疗”还意味着例如通过试剂、诊断剂、结合组合物或通过其他细胞对细胞进行体外和离体治疗。
“有效量”包括足以改善或防止医学疾病的症状或病症的量。有效量还意指足以使得可以诊断或促进诊断的量。对具体受治疗者的有效量可视多种因素而变化,例如待治疗的疾病、患者的整体健康状况、给药的方法途径和剂量及副作用的严重性。有效量可为避免显著副作用或毒性作用的最大剂量或给药方案。
本发明的各个方面将在下述分部中进一步详细描述。
中和性抗体
目前,还没有针对SARS-CoV-2的靶向药物和治疗性抗体批准上市。针对SARS-CoV-2病毒表面尖峰蛋白的特异性抗体,特别是针对受体结合域 (RBD)被认为是一种很有前途的中和性抗体,它可以抵抗进入人体的病毒,将有效治疗严重肺部感染患者的各种病毒。
在一方面,本发明提供了靶向新冠病毒SARS-CoV-2刺突蛋白受体结合域的单克隆抗体或其抗原结合部分,其包含:
(a)重链可变区CDR1,其包含与选自SEQ ID NO:1、SEQ ID NO:7、或SEQ ID NO:13所示一致的氨基酸序列;
(b)重链可变区CDR2,其包含与选自SEQ ID NO:2或SEQ ID NO:8所示一致的氨基酸序列;
(c)重链可变区CDR3,其包含与选自SEQ ID NO:3、SEQ ID NO:9、或SEQ ID NO:14所示一致的氨基酸序列;
(d)轻链可变区CDR1,其包含与选自SEQ ID NO:4或SEQ ID NO:10所示一致的氨基酸序列;
(e)轻链可变区CDR2,其包含与选自SEQ ID NO:5、SEQ ID NO:11、或SEQ ID NO:15所示一致的氨基酸序列;和
(f)轻链可变区CDR3,其包含与选自SEQ ID NO:6、SEQ ID NO:12或 SEQ ID NO:16所示一致的氨基酸序列。
在另一实施方案中,本发明提供了分离的单克隆抗体或其抗原结合部分,其重链可变区和轻链可变区的CDR序列同源性至少为91%,92%,93%,94%,95%,96%,97%,98%,99%或100%。在本发明工程改造抗体的方法的某些实施方式中,可以向整个或部分中和性抗体编码序列中随机或者有选择的引入突变,所产生的经修饰的抗体可如本文所述的进行结合活性和/或其它功能特性的筛选。本领域已经记载了突变的方法。例如,Short的PCT 公开文本WO02/09278中描述了通过饱和诱变、合成的连接装配或其组合来创造和筛选抗体突变的方法。
其中,特别优选的中和性抗体经测序,所表达的抗体CDR区序列如下表所示:
在一个优选的实施方案中,本发明提供了靶向新冠病毒SARS-CoV-2 刺突蛋白受体结合域的单克隆抗体或其抗原结合部分,其包含:
(a)重链可变区CDR1,CDR2和CDR3的氨基酸序列分别与SEQ ID NO:1、 SEQ ID NO:2和SEQ ID NO:3所示一致;
(b)轻链可变区CDR1,CDR2和CDR3的氨基酸序列分别与SEQ ID NO:4、 SEQ ID NO:5和SEQ ID NO:6所示一致。
在另一优选的实施方案中,本发明提供了靶向新冠病毒SARS-CoV-2 刺突蛋白受体结合域的单克隆抗体或其抗原结合部分,其包含:
(a)重链可变区CDR1,CDR2和CDR3的氨基酸序列分别与SEQ ID NO:7、 SEQ ID NO:8和SEQ ID NO:9所示一致;
(b)轻链可变区CDR1,CDR2和CDR3的氨基酸序列分别与SEQ ID NO: 10、SEQ IDNO:11和SEQ ID NO:12所示一致。
在另一优选的实施方案中,本发明提供了靶向新冠病毒SARS-CoV-2 刺突蛋白受体结合域的单克隆抗体或其抗原结合部分,其包含:
(a)重链可变区CDR1,CDR2和CDR3的氨基酸序列分别与SEQ ID NO: 13、SEQ IDNO:2和SEQ ID NO:14所示一致;
(b)轻链可变区CDR1,CDR2和CDR3的氨基酸序列分别与SEQ ID NO:4、SEQ ID NO:15和SEQ ID NO:16所示一致。
在又一个实施方案中,本发明提供靶向新冠病毒SARS-CoV-2刺突蛋白受体结合域的单克隆抗体或其抗原结合部分,其包含重链和轻链可变区序列:
(a)重链可变区,其包含与选自SEQ ID NO:17、SEQ ID NO:21或SEQ ID NO:25所示一致的氨基酸序列;
(b)轻链可变区,其包含与选自SEQ ID NO:19、SEQ ID NO:23或SEQ ID NO:27所示一致的氨基酸序列。
在一个优选的实施方案中,本发明提供靶向新冠病毒SARS-CoV-2刺突蛋白受体结合域的单克隆抗体或其抗原结合部分,其包含重链和轻链可变区序列,其中重链可变区的氨基酸序列与SEQ ID NO:17所示一致,轻链可变区的氨基酸序列与SEQ ID NO:19所示一致。
在另一个优选的实施方案中,本发明提供靶向新冠病毒SARS-CoV-2刺突蛋白受体结合域的单克隆抗体或其抗原结合部分,其包含重链和轻链可变区序列,其中重链可变区的氨基酸序列与SEQ ID NO:21所示一致,轻链可变区的氨基酸序列与SEQ ID NO:23所示一致。
在另一个优选的实施方案中,本发明提供靶向新冠病毒SARS-CoV-2刺突蛋白受体结合域的单克隆抗体或其抗原结合部分,其包含重链和轻链可变区序列,其中重链可变区的氨基酸序列与SEQ ID NO:25所示一致,轻链可变区的氨基酸序列与SEQ ID NO:27所示一致。
在另一实施方案中,本发明提供了分离的单克隆抗体或其抗原结合部分,其重链可变区与选自SEQ ID NO:17、SEQ ID NO:21或SEQ ID NO: 25的氨基酸序列具有至少为90%,91%,92%,93%,94%,95%,96%,97%, 98%,99%或100%的序列同源性;其轻链可变区与选自SEQ ID NO:19、SEQ ID NO:23或SEQ ID NO:27的氨基酸序列具有至少为90%,91%,92%,93%, 94%,95%,96%,97%,98%,99%或100%的序列同源性。重链可变区(VH) 和轻链可变区(VL)与上述序列的VH和VL区具有高(即90%或更高)同源性的抗体通过保守序列修饰获得,包括氨基酸的取代、添加和缺失等。术语“保守序列修饰”意图指氨基酸修饰不会显著影响或改变含有该氨基酸序列的抗体的结合特征。修饰可以通过本领域已知的标准技术,例如定点诱变和PCR介导的诱变编码可变区序列的核酸分子。保守氨基酸取代指氨基酸残基用具有类似侧链的氨基酸残基替换。本领域中对具有类似侧链的氨基酸残基家族已有详细说明。这些家族包括具有碱性侧链(例如赖氨酸、精氨酸、组氨酸)、酸性侧链(例如天冬氨酸、谷氨酸)、不带电荷的极性侧链(例如甘氨酸、天冬酰胺、谷酰胺、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-分支侧链(例如苏氨酸、缬氨酸、异亮氨酸)和芳香侧链(例如酪氨酸、苯丙氨酸、色氨酸、组氨酸)的氨基酸。因此,可以用来自同一侧链家族的其它氨基酸残基替换本发明抗体CDR区域外的一个或多个氨基酸残基,并使用本文所述的功能测定法对改变后的抗体测试保留的功能。优选的定点诱变或PCR介导的诱变位点位于重链可变区 CDR1-CDR3和轻链可变区CDR1-CDR3之外的位点。
在另一实施方案中,所述抗体为人全长抗体。又一方面,所述人恒定区选自由IgGl、IgG2、IgG3、IgG4组成的组。在又一个具体方面中,所述人恒定区是IgG1。
在另一实施方案中,本发明抗体或抗体片段为人抗体或人抗体片段。
在另一实施方案中,本发明抗体片段为Fab、Fab’、Fab’-SH、Fv、 scFv或F(ab’)2抗体片段。
在另一实施方案中,本发明抗体片段为双抗体。
在另一实施方案中,本发明提供了分离的靶向新冠病毒SARS-CoV-2刺突蛋白受体结合域的单克隆抗体,其包含:
(a)重链,其序列与SEQ ID NO:29、SEQ ID NO:33或SEQ ID NO: 37所示的氨基酸序列一致;以及
(b)轻链,其序列与SEQ ID NO:31、SEQ ID NO:35或SEQ ID NO: 39所示的氨基酸序列一致。
在另一优选实施方案中,本发明提供了分离的靶向新冠病毒 SARS-CoV-2刺突蛋白受体结合域的单克隆抗体,其重链的氨基酸序列与SEQ ID NO:29所示一致,轻链的氨基酸序列与SEQ ID NO:31所示一致。
在另一优选实施方案中,本发明提供了分离的靶向新冠病毒 SARS-CoV-2刺突蛋白受体结合域的单克隆抗体,其重链的氨基酸序列与SEQ ID NO:33所示一致,轻链的氨基酸序列与SEQ ID NO:35所示一致。
在另一优选实施方案中,本发明提供了分离的靶向新冠病毒SARS-CoV-2刺突蛋白受体结合域的单克隆抗体,其重链的氨基酸序列与SEQ ID NO:37所示一致,轻链的氨基酸序列与SEQ ID NO:39所示一致。
在优选的实施方案中,本发明提供了分离的靶向新冠病毒SARS-CoV-2 刺突蛋白受体结合域的单克隆抗体,其重链(HC)与选自SEQ ID NO:29、 SEQ ID NO:33或SEQ ID NO:37的氨基酸序列具有至少为90%,91%,92%, 93%,94%,95%,96%,97%,98%,99%或100%的序列同源性;其轻链(LC) 与选自SEQ ID NO:31、SEQ ID NO:35或SEQ ID NO:39的氨基酸序列具有至少为90%,91%,92%,93%,94%,95%,96%,97%,98%,99%或100%的序列同源性。与上述序列的重链和轻链具有高(即90%或更高)同源性的抗体可以通过诱变(例如定点诱变或PCR介导的诱变)编码重链和轻链氨基酸的核酸分子,然后使用本文所述的功能测定法对所编码的改变后的抗体测试所保留的功能获得。优选的定点诱变或PCR介导的诱变位点位于重链可变区CDR1-CDR3和轻链可变区CDR1-CDR3之外的位点。
为提高抗体中和效应,本发明还对人恒定区是IgG1的Fc段进行特定位点的突变,使得改造过的IgG1-Fc段由于突变使得FcR的结合能力丧失,目的是去除可能的抗体依赖的感染增强现象(ADE),但是保留补体激活通路(消灭被中和抗体结合的新冠病毒),上述改变还使得抗体的半衰期延长。在一个优选的实施例中,本发明对IgG1-Fc段引入3个突变(T299L, K320E,Q386R),同时缺失IgG1-Fc段C末端的赖氨酸(K),以避免抗体表达过程中因蛋白酶切作用导致的产物不均一现象。其中T299L突变去除了N-糖基化位点,和其它突变K320E,Q386R使得抗体与FcgammaR的结合能力丧失(主要指结合巨噬细胞和NK细胞的能力)目的是去除可能的抗体依赖的感染增强现象(ADE,Antibody Dependent Enhancement),但是保留补体激活通路(消灭被中和抗体结合的新冠病毒),上述改变还使得抗体的半衰期延长(FcRn介导的再循环能力增强)。
在本发明一个特别优选的实施方案中,本发明提供了IgG1-Fc经过突变的靶向新冠病毒SARS-CoV-2刺突蛋白受体结合域的单克隆抗体,其包含:
(a)重链,其序列与SEQ ID NO:41所示的氨基酸序列一致;以及
(b)轻链,其序列与SEQ ID NO:43所示的氨基酸序列一致。
优选的,其中所述抗体或其抗原结合部分优选与新冠病毒SARS-CoV-2刺突蛋白受体结合域特异性结合,中和抗体的亲和力常数(KD)约在1×10-10M 级别或更小的KD与新冠病毒SARS-CoV-2刺突蛋白受体结合域结合。
编码本发明抗体的核酸分子
本发明的另一方面涉及编码本发明抗体的核酸分子。本发明的核酸可以是例如DNA或RNA,而且可以含有或不含内含子序列。在一优选的实施方式中,核酸是cDNA分子。
可以使用标准分子生物学技术来获得本发明的核酸分子。对于从免疫球蛋白基因库(例如使用噬菌体展示技术)中获得的抗体,可以从文库中回收编码抗体的核酸。
本发明优选的核酸分子是那些编码本发明中所示中和性抗体CDR区、可变区或者全长抗体的氨基酸序列的核酸分子。在获得编码本发明所述的中和性抗体的VH和VL区段的DNA片段后,进一步通过标准重组DNA技术操作这些DNA片段,例如将可变区基因转变为全长抗体链基因、Fab片段基因或scFv基因。在这些操作中,将编码VL或VH的DNA片段可操作的连接至编码另一蛋白质,诸如抗体恒定区或柔性接头的另一DNA片段。术语“可操作的连接”在用于本文时意图表示连接两DNA片段从而使得这两个DNA 片段所编码的氨基酸序列保持在同一读码框中。在一些实施方案中,编码抗体重链可变区的核苷酸序列如SEQ ID NO:18、SEQ ID NO:22或SEQ ID NO:26 所示,编码抗体轻链可变区的核苷酸序列如SEQ ID NO:20、SEQ ID NO:24 或SEQ ID NO:28所示。
通过将编码VH的DNA可操作的连接至编码重链恒定区(CH1、CH2 和CH3)的另一DNA分子可以将编码VH区的分离的DNA转变为全长重链基因。人重链恒定区基因的序列是本领域已知的(参见例如Kabat,E.A.等人 (1991),Sequences of Proteins of ImmunologicalInterest,Fifth Edition, U.S.Department of Health and Human Services,NIHPublication No.91-3242),包含这些区的DNA片段可以通过标准PCR扩增获得。重链恒定区可以是 IgG1、IgG2、IgG3、IgG4、IgA、IgE、IgM或IgD恒定区,但是最优选为 IgG1恒定区。为获得Fab片段重链基因,可以将编码VH的DNA可操作的连接至仅编码重链CH1恒定区的另一DNA分子。在一些实施方案中,编码抗体重链的核苷酸序列如SEQ ID NO:30、SEQ ID NO:34或SEQ ID NO:38 所示。
为提高抗体中和效应,本发明还对人恒定区是IgG1的Fc段进行特定位点的突变,使得改造过的IgG1-Fc段由于突变使得FcR的结合能力丧失 (主要指结合巨噬细胞和NK细胞的能力),目的是去除可能的抗体依赖的感染增强现象(ADE),但是保留补体激活通路(消灭被中和抗体结合的新冠病毒),上述改变还使得抗体的半衰期延长(FcRn再循环能力增强)。在本发明已有特别优选的实施例中,本发明提供了IgG1-Fc经过突变的抗体,其重链序列与SEQ ID NO:41一致,编码该抗体重链的核苷酸序列如 SEQ ID NO:42所示。
通过将编码VL的DNA可操作的连接至编码轻链恒定区CL另一DNA 分子可以将编码VL区的分离的DNA转变为全长轻链基因(以及Fab轻链基因)。人轻链恒定区基因的序列是本领域已知的(参见例如Kabat,E.A.等人 (1991),Sequences of Proteins ofImmunological Interest,Fifth Edition, U.S.Department of Health and HumanServices,NIH Publication No.91-3242),包含这些区的DNA片段可以通过标准PCR扩增获得。轻链恒定区可以是κ或λ恒定区。在一些实施方案中,编码抗体轻链的核苷酸序列如SEQID NO:32、SEQ ID NO:36或SEQ ID NO:40所示。
为创造scFv基因,将编码VH和VL的DNA片段可操作的连接至编码柔性接头,例如编码氨基酸序列(Gly4-Ser)3的另一片段,从而使得VH和VL 序列可以表达成相邻的单链蛋白质,其中VL和VH区通过柔性接头连接(参见例如Bird等人(1988)Science 242:423-426;Huston等人 (1988)Proc.Natl.Acad.Sci.USA 85:5879-5883;McCafferty等人 (1990)Nature 348:552-554)。
本发明提供了编码所述抗体或抗体片段的分离的多核苷酸。在一些实施方案中,编码抗体重链的核苷酸序列如SEQ ID NO:30、SEQ ID NO:34或 SEQ ID NO:38或SEQ IDNO:42所示,编码抗体轻链的核苷酸序列如SEQ ID NO:32、SEQ ID NO:36或SEQ ID NO:40所示。
表达载体及宿主细胞
本发明提供了包含所述分离的多核苷酸的表达载体,以及包含所述表达载体的宿主细胞。
适宜载体的选择将主要取决于将要插入载体的核酸的大小和将要用载体转化的具体宿主细胞。为表达抗体或其抗体片段,可以通过标准分子生物学技术获得编码部分或全长轻链和重链的DNA,并且可以将DNA插入到表达载体中,从而使得基因与转录和翻译调控序列可操作的连接。
选择适合所用表达宿主细胞的表达载体和表达调控序列。可将抗体轻链基因和抗体重链基因插入到不同的载体中,或者更通常的,将两个基因插入到同一表达载体中。通过标准方法将抗体基因插入到表达载体中。可以使用本文所述抗体的轻链和重链可变区来创造任何抗体同种型的全长抗体基因,其通过将它们插入已编码期望同种型的重链恒定区和轻链恒定区的表达载体中,从而使得VH区段与载体中的CH区段可操作的连接,VK区段与载体中的CL区段可操作的连接。或者,重组表达载体可以编码信号肽,其利于宿主细胞中抗体链的分泌。可将抗体链基因克隆到载体中以使信号肽与抗体链基因的氨基末端连接于同一读码框中。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)。
除了抗体链基因,本发明的重组表达载体还携带调控序列,诸如源自巨细胞病毒(CMV)、猿病毒40(SV40)、腺病毒(例如腺病毒主要晚期启动子 (AdMLP))和多瘤病毒的启动子和/或增强子,其调控抗体链基因在宿主细胞中的表达。术语“调控序列”意图包括启动子、增强子和其它表达调控元件(例如聚腺苷酸化信号),其调控抗体链基因的转录或翻译。此类调控序列记载于例如Goeddel,Gene Expression Technology.Methodsin Enzymology185,Academic Press,San Diego,CA(1990)中。
为表达轻链和重链,通过标准技术将编码重链和轻链的表达载体转染到宿主细胞中。各种形式的术语“转染”意图涵盖多种通常用于将外源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-右旋糖苷转染等。虽然理论上在原核或真核宿主细胞中都有可能表达本发明的抗体,但是最优选在真核细胞中(最优选在哺乳动物宿主细胞中)表达抗体。用于表达本发明重组抗体的优选哺乳动物宿主细胞包括中国仓鼠卵巢细胞(CHO细胞)、NSO骨髓瘤细胞、COS细胞和SP2细胞等,优选CHO细胞。
当将编码抗体基因的重组表达载体导入哺乳动物宿主细胞时,通过培养宿主细胞一段足以使宿主细胞中抗体表达的时间生产抗体,或者更优选的是,将抗体分泌到培养宿主细胞的培养基中。可以使用标准蛋白质纯化方法从培养物的培养液中回收抗体。
药物组合物
本发明提供了药物组合物,包含所述的中和性抗体和药学可接受的载体。
一方面,本发明提供了一种药用组合物,其包含一种或一组本发明的靶向新冠病毒SARS-CoV-2刺突蛋白受体结合域单克隆抗体或其抗原结合部分,与药学可接受载体配制在一起。药剂学可接受载体包括任何和所有溶剂、分散介质、包衣、抗细菌和抗真菌剂、等渗和吸收延迟剂等生理学相容的载体。优选的是,载体适于静脉内、肌肉内、皮下、胃肠外、脊髓或表皮施用(例如通过注射或输注)。
药物组合物在生产和贮存条件下通常必须是无菌的和稳定的。可以将组合物配制成溶液、微乳液、脂质体或冻干粉针等剂型。本发明药物组合物的优选给药途径包括静脉内、肌肉内、皮内、腹膜内、皮下、脊髓/脊柱或其它胃肠外施用路径,例如通过注射或输注。
本发明的抗体的给药剂量的范围为大约0.0001-100mg/kg,更加通常的为0.01-5mg/kg宿主体重。例如,剂量可以是0.3mg/kg体重、1mg/kg体重、 3mg/kg体重、5mg/kg体重或10mg/kg体重或在1-10mg/kg的范围内。例示性的治疗方案要求每周施用一次、每两周一次、每三周一次、每四周一次、每月一次、每3个月一次或每3-6个月一次。对于本发明的中和性抗体而言,优选的剂量方案包括经静脉内施用1mg/kg体重-3mg/kg体重。
本发明药用组合物中的活性成分的实际剂量水平可以变化,从而获得对于特定患者、组合物和施用模式有效实现期望治疗性应答而对患者是无毒的活性成分量。“治疗有效量”的本发明中和性抗体优选导致疾病症状严重性降低、无疾病症状期的频率和持续时间提高、或对患病所造成的损害或残疾的预防。可以在动物模型系统中评估抗体中和新冠病毒感染靶细胞的能力,所述动物模型系统可以预测在人感染患者中的功效。本领域普通技术人员将会能够基于诸如受试者体型大小、受试者症状的严重性、及所选择的特定组合物或施用路径的因素来确定此类量。
本发明的用途和方法
本发明还提供了中和抗体短期预防和治疗COVID-19的医疗用途。该抗体也可用于新冠病毒的抗原检测和血清学检测。中和性抗体可以去除新冠病毒感染宿主的能力,保护暴露于病毒的未受感染宿主和解救重症病人的生命。因此,这种中和性抗体提供新冠病毒感染病人的治疗或者预防易感人群被新冠病毒侵袭,并且可能还提供SARS冠状病毒或者其它类似冠状病毒引起的感染性疾病。
在一些实施方案中,本发明提供了治疗COVID-19受试者的中和性抗体。中和抗体是一种治疗性抗体,可以通过中和或抑制病原体的生物学活性来保护细胞免受侵害。凭借特异性和高亲和力特点,中和抗体能够抢先与病毒刺突蛋白(S蛋白)结合,从而阻断病毒与宿主细胞结合,病毒无法感染正常细胞,就很容易被免疫系统清除。
ACE2在Ⅱ型肺泡细胞中高表达,新型冠状病毒通过S-RBD与人ACE2受体的相互作用进入人肺泡上皮细胞,导致COVID-19。此外,ACE2在肠道,特别是十二指肠上皮细胞中也高度表达,但很少有SARS-CoV-2通过消化道感染的报道。抗冠状病毒的中和抗体主要针对与ACE2相互作用并使冠状病毒进入宿主细胞的三聚体spike糖蛋白S蛋白。S蛋白有两个功能亚单位,介导细胞粘附(S1亚单位,存在于S1A到S1D的四个核心区域)和病毒与细胞膜融合(S2亚单位)。有效的中和抗体常常阻断S-RBD与人ACE2受体的结合,使病毒无法进入人上皮细胞。新型冠状病毒S蛋白含有1273个氨基酸残基(GenBank:QHD43416.1),与SARS冠状病毒S蛋白的同源性为 77.5%。通常,人ACE2蛋白作为宿主受体通过其S1b结构域结合。已知受体间的相互作用可以触发冠状病毒棘突蛋白不可逆的构象变化,从而实现膜融合。
为了鉴定和筛选新冠病毒的中和抗体,对来自恢复期新冠肺炎病人血中B细胞进行高通量的测序和筛选。凡是与新冠病毒外壳刺突蛋白的亚单位S1会起结合的抗体基因(可变区重链和轻链)经过噬菌体展示初步筛选出来。使用单细胞测序技术从恢复期患者外周血单个核细胞中(PBMC)分离出157个SARS-CoV-2中和性单克隆抗体(mAb),经酶联免疫吸附(ELISA) 进行亲和力分析,对18个单克隆抗体进行筛选,5个系列(R11、R14、R29、 R44、R157)抗新冠病毒S1蛋白的抗体呈现出阻断S蛋白与人ACE2之间 (S1残基318-510)相互反应的抗体挑选出来,其中(5个抗体)呈新冠病毒假病毒中和活性。重组抗体并重组表达为全人类IgG1等型抗体以进一步鉴定,经过多轮筛选最终获得本发明所述的中和能力比较强的抗体R14, R44和R157作为候选抗体。
在SARS-CoV-2假病毒中和实验中,SARS-CoV-2假病毒可感染 HEK293T细胞,而中和性抗体可特异性结合到S-RBD区域并因此阻断 SARS-CoV-2刺突蛋白与细胞表面ACE2受体的结合,阻断了SARS-CoV-2 假病毒对细胞的感染。假病毒含有荧光素酶LUC报告基因,可以使用化学发光酶标仪检测荧光信号以表征假病毒感染被阻断的程度。R44在细胞实验中表现出良好的中和活性。R44中和性抗体对假病毒的的抑制率IC50为 1.597μg/ml。同时,生物膜干涉技术(OCTET法)测定其KD值为 2.715×10-10M。而基于SPR技术测定mAb结合亲和力常数,R44和 SARS-CoV-2S1蛋白的亲和力KD值为1.34×10-9M,R44和SARS-CoV-2 S-RBD蛋白的亲和力KD值为2.05×10-10M。而根据文献报道(Shi,R.,Shan, C.,Duan,X.et al.A humanneutralizing antibody targets the receptor-binding site of SARS-CoV-2.Nature(2020)),其所筛到的两株中和性抗体CA1 and CB6的亲和力(KD)分别为4.68±1.64nM和2.49±1.65nM,本发明的中和性抗体亲和力要比文献报道的抗体高10倍左右。
进一步的试验证明,R44抗体能直接阻断ACE2和SARS-CoV-2S1蛋白之间的结合,且此阻断作用是特异性的。中和性抗体可以阻断S-RBD与人ACE2受体的相互反应,亲和力越高意味着抗体与S-RBD的结合越紧密,阻止病毒感染肺泡上皮细胞的能力就越强,使得病毒无法进入到人体上皮细胞,从而抑制新冠病毒感染宿主的能力。
对抗新冠病毒的第一战场在呼吸道和肺部,根据Medimmune同样作用于呼吸道的RSV抗体的数据表明,单抗的血液浓度和对应的肺部器官的分布有将近1000倍的浓度差。如何在有限的注射频率和剂量下延长抗体的保护期是一个关键因素。为解决抗体分布的浓度差问题,本发明通过抗体工程延长单抗的半衰期以保持单抗的有效浓度,对IgG1-Fc段引入3个突变 (T299L,K320E,Q386R),同时缺失IgG1-Fc段C末端的赖氨酸(K),使得抗体与FcgammaR的结合能力丧失(主要指结合巨噬细胞和NK细胞的能力),目的是去除可能的抗体依赖的感染增强现象(ADE,Antibody Dependent Enhancement),但是保留补体激活通路(消灭被中和抗体结合的新冠病毒),上述改变还使得抗体的半衰期延长(FcRn介导的再循环能力增强)。
在另一实施方案中,本发明制备的中和性抗体可与其他新冠病毒中和抗体组合作为鸡尾酒疗法,同时且非竞争性地与病毒刺突蛋白的关键受体结合域(RBD)结合削弱病毒因突变造成的逃逸,并可起到治疗和暴露后预防的作用,可为老年人、免疫功能低下等对疫苗不响应人群提供保护。
本发明提供了靶向新冠病毒SARS-CoV-2刺突蛋白受体结合域的单克隆抗体或其抗原结合部分,包括如下的技术方案:
1、靶向新冠病毒SARS-CoV-2刺突蛋白受体结合域的单克隆抗体或其抗原结合部分,其包含:
(a)重链可变区CDR1,其包含与选自SEQ ID NO:1、SEQ ID NO:7、或SEQ ID NO:13所示一致的氨基酸序列;
(b)重链可变区CDR2,其包含与选自SEQ ID NO:2或SEQ ID NO:8所示一致的氨基酸序列;
(c)重链可变区CDR3,其包含与选自SEQ ID NO:3、SEQ ID NO:9、或SEQ ID NO:14所示一致的氨基酸序列;
(d)轻链可变区CDR1,其包含与选自SEQ ID NO:4或SEQ ID NO:10所示一致的氨基酸序列;
(e)轻链可变区CDR2,其包含与选自SEQ ID NO:5、SEQ ID NO:11、或SEQ ID NO:15所示一致的氨基酸序列;和
(f)轻链可变区CDR3,其包含与选自SEQ ID NO:6、SEQ ID NO:12或 SEQ ID NO:16所示一致的氨基酸序列。
2、根据技术方案1所述的单克隆抗体或其抗原结合部分,其特征在于,其包含:
(a)重链可变区CDR1,CDR2和CDR3的氨基酸序列分别与SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示一致;
(b)轻链可变区CDR1,CDR2和CDR3的氨基酸序列分别与SEQ ID NO:4、 SEQ ID NO:5和SEQ ID NO:6所示一致。
3、根据技术方案1所述的单克隆抗体或其抗原结合部分,其特征在于,其包含:
(a)重链可变区CDR1,CDR2和CDR3的氨基酸序列分别与SEQ ID NO:7、 SEQ ID NO:8和SEQ ID NO:9所示一致;
(b)轻链可变区CDR1,CDR2和CDR3的氨基酸序列分别与SEQ ID NO: 10、SEQ IDNO:11和SEQ ID NO:12所示一致。
4、根据技术方案1所述的单克隆抗体或其抗原结合部分,其特征在于,其包含:
(a)重链可变区CDR1,CDR2和CDR3的氨基酸序列分别与SEQ ID NO: 13、SEQ IDNO:2和SEQ ID NO:14所示一致;
(b)轻链可变区CDR1,CDR2和CDR3的氨基酸序列分别与SEQ ID NO:4、SEQ ID NO:15和SEQ ID NO:16所示一致。
5、根据技术方案1-4任一项所述的单克隆抗体或其抗原结合部分,其特征在于,其重链可变区和轻链可变区的CDR序列同源性至少为91%,92%,93%, 94%,95%,96%,97%,98%,99%或100%。
6、靶向新冠病毒SARS-CoV-2刺突蛋白受体结合域的单克隆抗体或其抗原结合部分,其包含重链和轻链可变区序列:
(a)重链可变区,其包含与选自SEQ ID NO:17、SEQ ID NO:21或SEQ ID NO:25所示一致的氨基酸序列;
(b)轻链可变区,其包含与选自SEQ ID NO:19、SEQ ID NO:23或SEQ ID NO:27所示一致的氨基酸序列。
7、根据技术方案6所述的单克隆抗体或其抗原结合部分,其特征在于,其中重链可变区的氨基酸序列与SEQ ID NO:17所示一致,轻链可变区的氨基酸序列与SEQ ID NO:19所示一致。
8、根据技术方案6所述的单克隆抗体或其抗原结合部分,其特征在于,其中重链可变区的氨基酸序列与SEQ ID NO:21所示一致,轻链可变区的氨基酸序列与SEQ ID NO:23所示一致。
9、根据技术方案6所述的单克隆抗体或其抗原结合部分,其特征在于,其中重链可变区的氨基酸序列与SEQ ID NO:25所示一致,轻链可变区的氨基酸序列与SEQ ID NO:27所示一致。
10、根据技术方案6-9任一项的单克隆抗体或其抗原结合部分,其特征在于,其重链可变区与选自SEQ ID NO:17、SEQ ID NO:21或SEQ ID NO:25的氨基酸序列具有至少为90%,91%,92%,93%,94%,95%,96%,97%, 98%,99%或100%的序列同源性;其轻链可变区与选自SEQ ID NO:19、SEQ ID NO:23或SEQ ID NO:27的氨基酸序列具有至少为90%,91%,92%,93%, 94%,95%,96%,97%,98%,99%或100%的序列同源性。
11、根据技术方案1-10任一项所述的单克隆抗体或其抗原结合部分,其特征在于,所述抗体为人全长抗体。
12、根据技术方案11所述的单克隆抗体或其抗原结合部分,其特征在于, 所述人抗体恒定区选自由IgGl、IgG2、IgG3、IgG4组成的组。
13、根据技术方案12所述的单克隆抗体或其抗原结合部分,其特征在于, 所述人抗体恒定区是IgG1。
14、根据技术方案11所述的单克隆抗体或其抗原结合部分,其特征在于, 所述抗体或抗体片段为人抗体或人抗体片段。
15、根据技术方案14所述的单克隆抗体或其抗原结合部分,其特征在于, 所述抗体片段为Fab、Fab’、Fab’-SH、Fv、scFv或F(ab’)2抗体片段。
16、根据技术方案15所述的单克隆抗体或其抗原结合部分,其特征在于, 所述抗体片段为双抗体。
17、分离的靶向新冠病毒SARS-CoV-2刺突蛋白受体结合域的单克隆抗体,其包含:
(a)重链,其序列与SEQ ID NO:29、SEQ ID NO:33或SEQ ID NO: 37所示的氨基酸序列一致;以及
(b)轻链,其序列与SEQ ID NO:31、SEQ ID NO:35或SEQ ID NO: 39所示的氨基酸序列一致。
18、根据技术方案17所述的单克隆抗体,其特征在于,其重链的氨基酸序列与SEQID NO:29所示一致,轻链的氨基酸序列与SEQ ID NO:31所示一致。
19、根据技术方案17所述的单克隆抗体,其特征在于,其重链的氨基酸序列与SEQID NO:33所示一致,轻链的氨基酸序列与SEQ ID NO:35所示一致。
20、根据技术方案17所述的单克隆抗体,其特征在于,其重链的氨基酸序列与SEQID NO:37所示一致,轻链的氨基酸序列与SEQ ID NO:39所示一致。
21、根据技术方案17-20任一项所述的单克隆抗体,其特征在于,其重链 (HC)与选自SEQ ID NO:29、SEQ ID NO:33或SEQ ID NO:37的氨基酸序列具有至少为90%,91%,92%,93%,94%,95%,96%,97%,98%,99%或 100%的序列同源性;其轻链(LC)与选自SEQ ID NO:31、SEQ ID NO:35 或SEQ ID NO:39的氨基酸序列具有至少为90%,91%,92%,93%,94%, 95%,96%,97%,98%,99%或100%的序列同源性。
22、根据技术方案17-20任一项所述的单克隆抗体,其特征在于,对所述抗体人恒定区是IgG1的Fc段引入3个突变(T299L,K320E,Q386R),同时缺失IgG1-Fc段C末端的赖氨酸(K)。
23、根据技术方案22所述的单克隆抗体,其特征在于,其包含:
(a)重链,其序列与SEQ ID NO:41所示的氨基酸序列一致;以及
(b)轻链,其序列与SEQ ID NO:43所示的氨基酸序列一致。
24、编码技术方案1-23任一项所述的单克隆抗体或其抗原结合部分的核酸分子。
25、根据技术方案24所述的核酸分子,其特征在于,核酸是cDNA分子。
25、根据技术方案24所述的核酸分子,其特征在于,编码抗体重链可变区的核苷酸序列如SEQ ID NO:18、SEQ ID NO:22或SEQ ID NO:26所示,编码抗体轻链可变区的核苷酸序列如SEQ ID NO:20、SEQ ID NO:24或SEQ ID NO:28所示。
26、根据技术方案24所述的核酸分子,其特征在于,编码抗体重链的核苷酸序列如SEQ ID NO:30、SEQ ID NO:34或SEQ ID NO:38或SEQ ID NO:42 所示,编码抗体轻链的核苷酸序列如SEQ ID NO:32、SEQ ID NO:36或SEQ ID NO:40所示。
27、一种表达载体,其包含技术方案24-26任一项所述的核酸分子。
28、根据技术方案27所述的表达载体,其特征在于,所述表达载体还携带调控序列,诸如源自巨细胞病毒(CMV)、猿病毒40(SV40)、腺病毒(例如腺病毒主要晚期启动子(AdMLP))和多瘤病毒的启动子和/或增强子,其调控抗体链基因在宿主细胞中的表达。
29、包含技术方案27所述表达载体的宿主细胞。
30、一种药用组合物,其包含有效治疗量的技术方案1-23任一项所述的靶向新冠病毒SARS-CoV-2刺突蛋白受体结合域单克隆抗体或其抗原结合部分以及药学可接受的载体。
31、根据技术方案30所述的药用组合物,其给药途径包括静脉内、肌肉内、皮内、腹膜内、皮下、脊髓/脊柱或其它胃肠外施用路径
32、根据技术方案30所述的药用组合物,其特征至于:所述抗体的给药剂量的范围为0.0001-100mg/kg。
33、根据技术方案32所述的药用组合物,其特征至于:所述抗体的给药剂量的范围为0.01-5mg/kg宿主体重。
34、根据技术方案30所述的药用组合物,其特征至于:所述抗体为每周施用一次、每两周一次、每三周一次、每四周一次、每月一次、每3个月一次或每3-6个月一次。
35、技术方案1-23任一项所述的单克隆抗体或抗原结合部分在制备用于治疗或预防COVID-19的药物中的用途。
通过下列实施例进一步说明本发明,所述实施例不应解释为进一步限制。在此将整篇申请中引用的所有附图和所有参考文献、专利和已公开专利申请的内容明确收入本文作为参考。
具体实施方式
实施例1:中和性抗体的获得
根据文献Cao,Y.,et al;Potent neutralizing antibodies against SARS-CoV-2 identified by high-throughput single-cell sequencing of convalescentpatients’B cells,Cell(2020)所描述的方法并做适当修改:
1.从人外周血(PBMC)中富集B细胞
根据试剂盒厂家(EasySepTM人B细胞富集试剂盒)提供的方法,通过免疫磁阴性选择,从先前冷冻的PBMC(来自福州市疾病控制中心获得3 个新冠肺炎恢复期患者的血样)中分离出B细胞。先用磁珠标记非B细胞,并使用EasySepTM磁体分离;再用PBS中洗涤纯化的B细胞,并保存在在含有2%(v/v)胎牛血清(FBS)和1mM EDTA中。根据厂家(Thermofisher) 提供的方法,使用0.4%(w/v)台盼蓝染色剂和Countess自动细胞计数器对纯化的B细胞进行计数。
2.CD27+记忆性B细胞的富集
根据厂家(EasySepTM人类记忆B细胞分离试剂盒,STEMCELL)提供的方法,通过免疫磁阳性选择从纯化的B细胞中,分离出CD27+记忆性B 细胞。将用与CD27抗体结合的磁珠标记CD27+B细胞,并使用EasySep TM磁体分离。用含有2%(v/v)胎牛血清(FBS)和1mM EDTA的PBS 洗涤纯化CD27+B细胞。根据厂家提供的方法,使用0.4%(w/v)台盼蓝染色剂和Countess自动细胞计数器对CD27+B细胞进行计数。
3.单细胞测序
分选所得B细胞按照10X Genomics标准方案操作。进行RNA和BCR 库的制备。使用Qubit 4.0(ThermoFisher),Fragment Analyzer和qPCR定量并质检所有文库。符合要求的文库在Illumina Novaseq平台上进行测序。
4.单克隆抗体的体外表达
对选定的成对的重链和轻链cDNA进行密码子优化,并将其克隆到含有人IgG1恒定区的表达载体中。通过用等量的重链和轻链质粒转染 HEK293细胞来表达18个IgG mAb。
表1中和性抗体序列
抗体编号 | 抗体名称 | 抗体编号 | 抗体名称 |
1 | R-12 | 10 | R-44 |
2 | R-14 | 11 | R-11 |
3 | R-1 | 12 | S-16 |
4 | R-28 | 13 | R-63 |
5 | R-29 | 14 | R-72 |
6 | R-23 | 15 | R-157 |
7 | R-216 | 16 | R202 |
8 | R-86 | 17 | R-239 |
9 | R-214 | 18 | R-277 |
实施例2:ELISA法检测中和性抗体
在4℃下用0.01μg/mL的SARS-CoV-2RBD蛋白在PBS中包被ELISA 平板过夜。标准洗涤和封闭后,将100μL的1μg/mL抗体添加到每个孔中。在室温下孵育2小时后,将板洗涤并与0.08μg/mL山羊抗人IgG(H +L)/HRP(JACKSON)一起在室温下孵育1小时。使用发色团溶液作为底物,并通过酶标仪测量450nm处的吸光度。当使用1μg/mL RBD使 OD450饱和时,mAb被定义为ELISA阳性。选择R14,R44和R157为下一轮候选抗体。
表2:ELISA法检测中和性抗体
编号 | 名称 | ELISA值 | 编号 | 名称 | ELISA值 |
1 | R-12 | 0.400 | 10 | R-44 | 1.174 |
2 | R-14 | 1.006 | 11 | R-11 | 1.108 |
3 | R-1 | 0.451 | 12 | S-16 | 0.166 |
4 | R-28 | 0.672 | 13 | R-63 | 0.101 |
5 | R-29 | 1.064 | 14 | R-72 | 0.093 |
6 | R-23 | 0.339 | 15 | R-157 | 1.139 |
7 | R-216 | 0.089 | 16 | R202 | 0.326 |
8 | R-86 | 0.303 | 17 | R-239 | 0.129 |
9 | R-214 | 0.642 | 18 | R-277 | 0.103 |
表3测定的R14,R44和R157抗体CDR区序列
表4 R14抗体可变区序列
表5 R44抗体可变区序列
表6 R157抗体可变区序列
表7 R14抗体全长序列
表8 R44抗体全长序列
表9 R157抗体全长序列
实施例3:中和性抗体的假病毒中和试验(R44抗体)
根据试剂厂家(义翘神州)提供的方法,通过使用新冠Spike假病毒与待检抗体孵育后侵染293T-ACE2细胞,采用化学发光法检测Luciferase发光值RLU,根据RLU读值计算待检血清的假病毒抑制率,评价待检抗体的中和效果。将293T-ACE2细胞,以30000cells/孔铺在96孔板上,培养基为: DMEM+10%FBS,DMEM培养基母液;铺板后细胞放入37℃,5%CO2培养箱中继续培养。样品组:稀释后的R44抗体和假病毒取等体积混合,37 度孵育1小时,加入到提前铺板的293T-ACE2细胞上,每个梯度2个复孔。阳性对照:假病毒和DMEM培养基母液取等体积混合后,37度孵育1小时,加入到提前铺板的293T-ACE2细胞上,2个复孔。阴性对照:DMEM培养基母液37度孵育1小时,加入到提前铺板的293T-ACE2细胞上,2个复孔。将样品和阴阳性对照添加到细胞上后,37℃,5%CO2培养箱中继续培养 48-72小时。用化学发光仪对96孔板中的样品进行Luciferase发光值检测。
假病毒的抑制率试验见表10,中和抗体的抑制效果图见图1。R44的抑制率IC50为1.597μg/ml。
表10:中和性抗体对假病毒的抑制率(R44)
实施例4:mAb结合亲和力常数的测量(R44)
用生物膜干涉技术BLI测定中和性抗体的亲和常数KD。具体方法为使用Fortebio公司Octet K2系统,SA传感器,第一步结合经生物素标记的 SARS-CoV-2S1蛋白(Acrobiosystems,cat.#S1N-C52H3),固化高度约0.5nm,基线平衡后与1:1梯度稀释的纯化抗体样品结合,浓度100nM至3.125nM,共6个浓度。结合时间300秒,解离时间1800秒,解离缓冲液PBST(PBS pH7.4+0.02%Tween-20)。数据用Octet数据分析软件V11.0分析(图2), 1:1模型拟合,R44的亲和力KD值为2.715×10-10M。
实施例5:基于SPR技术的mAb结合亲和力常数的测量(R44)
用表面等离子共振技术SPR测定中和性抗体的亲和常数KD。具体方法为使用Cytiva公司Biacore 8K系统,Protein A芯片,第一步结合待测抗体,再与1:1梯度稀释的SARS-CoV-2S1蛋白(Acrobiosystems,cat.# S1N-C52H3)或SARS-CoV-2S-RBD蛋白(Acrobiosystems,cat.#SPD-C52H3) 结合,浓度50nM至0.78125nM,共7个浓度,结合时间180秒,解离时间 900秒。数据用BIAevaluation数据分析软件分析(图3),1:1Langmuir模型拟合,R44和SARS-CoV-2S1蛋白的亲和力KD值为1.34×10-9M,R44 和SARS-CoV-2S-RBD蛋白的亲和力KD值为2.05×10-10M。
实施例6:中和性抗体阻断ACE2和SARS-CoV-2S1蛋白结合能力的测量 (R44)
用生物膜干涉技术BLI测定中和性抗体阻断ACE2和SARS-CoV-2S1 蛋白的结合能力。具体方法为使用Fortebio公司Octet K2系统,将SA传感器结合经AvitagTM技术在羧基末端标记了单一生物素分子的ACE2蛋白 (Acrobiosystems,cat.#AC2-H82E6),另取中和性抗体或无关抗体(与中和性抗体相同种型)与SARS-CoV-2S1蛋白(Acrobiosystems,cat.#S1N-C52H3)溶液混匀(混合液中抗体浓度250nM,SARS-CoV-2S1蛋白浓度50nM)后于室温孵育30min。再将结合了生物素化ACE2蛋白后的SA传感器与中和性抗体-SARS-CoV-2S1蛋白混合物进行结合测试,同时设置单独中和性抗体溶液,单独SARS-CoV-2S1蛋白溶液的对照。结合时间300秒。结果(图4) 显示R44抗体能直接阻断ACE2和SARS-CoV-2S1蛋白之间的结合,且此阻断作用是特异性的。
实施例7 R44抗体Fc序列的改造
对抗新冠病毒的第一战场在呼吸道和肺部,根据Medimmune同样作用于呼吸道的RSV抗体的数据表明,单抗的血液浓度和对应的肺部器官的分布有将近1000倍的浓度差。如何在有限的注射频率和剂量下延长抗体的保护期是一个关键因素。为解决抗体分布的浓度差问题,通过抗体工程对R44 进行进一步改造,在IgG1-Fc段引入3个突变(T299L,K320E,Q386R),同时缺失IgG1-Fc段C末端的赖氨酸(K),以避免抗体表达过程中因蛋白酶切作用导致的产物不均一现象(其全长序列如表11所示)。其中T299L突变去除了N-糖基化位点,和其它突变K320E,Q386R使得抗体与FcgammaR 的结合能力丧失(主要指结合巨噬细胞和NK细胞的能力)目的是去除可能的抗体依赖的感染增强现象(ADE,Antibody DependentEnhancement),但是保留补体激活通路(消灭被中和抗体结合的新冠病毒),上述改变还使得抗体的半衰期延长(FcRn介导的再循环能力增强)。
表11 R44抗体突变体全长序列
序列表
<110> 福建医科大学附属协和医院
<120> 新型冠状病毒中和性抗体及其制备方法与用途
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<210> 27
<211> 113
<212> PRT
<213> R157 light chain variable(Artificial Sequence)
<400> 27
Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln
1 5 10 15
Arg Val Thr Val Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly
20 25 30
Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu
35 40 45
Leu Ile Tyr Gly Asn Thr Asn Arg Pro Ser Gly Val Pro Asp Arg Phe
50 55 60
Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu
65 70 75 80
Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Ser
85 90 95
Leu Ser Asp Val Val Phe Gly Gly Gly Thr Gln Leu Thr Val Leu Pro
100 105 110
Arg
<210> 28
<211> 339
<212> DNA
<213> R157 light chain variable(Artificial Sequence)
<400> 28
cagtctgtct tgacgcagcc gccctcagtg tctggggccc cagggcagag ggtcaccgtc 60
tcctgcactg ggagcagctc caacatcggg gcaggttatg atgtacactg gtaccagcag 120
cttccaggaa cagcccccaa actcctcatc tatggtaaca ccaatcggcc ctcaggggtc 180
cctgaccgat tctctggctc caagtctggc acctcagcct ccctggccat cactgggctc 240
caggctgagg atgaggctga ttattactgc cagtcctatg acagcagcct gagtgatgtg 300
gtattcggcg gaggcaccca gctgaccgtc ctccctagg 339
<210> 29
<211> 454
<212> PRT
<213> R14 heavy chain full length-IgG1(Artificial Sequence)
<400> 29
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr
20 25 30
Thr Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Arg Ile Ile Pro Ile Leu Gly Ile Ala Asn Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Glu Ile Gly Tyr Ser Ser Ser Gly Ser Asn Tyr Tyr Met Asp
100 105 110
Val Trp Gly Lys Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys
115 120 125
Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly
130 135 140
Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
145 150 155 160
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
165 170 175
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
180 185 190
Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
195 200 205
Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro
210 215 220
Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
225 230 235 240
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
245 250 255
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
260 265 270
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
275 280 285
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
290 295 300
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
305 310 315 320
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
325 330 335
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
340 345 350
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
355 360 365
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
370 375 380
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
385 390 395 400
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
405 410 415
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
420 425 430
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
435 440 445
Ser Leu Ser Pro Gly Lys
450
<210> 30
<211> 1362
<212> DNA
<213> R14 heavy chain full length-IgG1(Artificial Sequence)
<400> 30
caggtccagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc 60
tcctgcaagg cttctggagg caccttcagc agttatacta tcagctgggt gcgacaggcc 120
cctggacaag ggcttgagtg gatgggaagg atcatcccca tccttggtat agcaaactac 180
gcacagaagt tccagggcag agtcacgatt accgcggaca aatccacgag cacagcctac 240
atggagctga gcagcctgag atctgaggac acggccgtgt attactgcgc gagagagatc 300
gggtatagca gcagcgggtc caattactac atggacgtct ggggcaaagg gaccacagtc 360
accgtctcct cagctagcac caagggccca tcggtcttcc ccctggcacc ctcctccaag 420
agcacctctg ggggcacagc ggccctgggc tgcctggtca aggactactt ccccgaaccg 480
gtgacggtgt cgtggaactc aggcgccctg accagcggcg tgcacacctt cccggctgtc 540
ctacagtcct caggactcta ctccctcagc agcgtggtga ccgtgccctc cagcagcttg 600
ggcacccaga cctacatctg caacgtgaat cacaagccca gcaacaccaa ggtggacaag 660
aaagttgagc ccaaatcttg tgacaaaact cacacatgcc caccgtgccc agcacctgaa 720
ctcctggggg gaccgtcagt cttcctcttc cccccaaaac ccaaggacac cctcatgatc 780
tcccggaccc ctgaggtcac atgcgtggtg gtggacgtga gccacgaaga ccctgaggtc 840
aagttcaact ggtacgtgga cggcgtggag gtgcataatg ccaagacaaa gccgcgggag 900
gagcagtaca acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg 960
ctgaatggca aggagtacaa gtgcaaggtc tccaacaaag ccctcccagc ccccatcgag 1020
aaaaccatct ccaaagccaa agggcagccc cgagaaccac aggtgtacac cctgcctcca 1080
tctcgggatg agctgaccaa gaaccaggtc agcctgacct gcctggtcaa aggcttctat 1140
cccagcgaca tcgccgtgga gtgggagagc aatgggcagc cggagaacaa ctacaagacc 1200
acgcctcccg tgctggactc cgacggctcc ttcttcctct atagcaagct caccgtggac 1260
aagagcaggt ggcagcaggg gaacgtcttc tcatgctccg tgatgcatga ggctctgcac 1320
aaccactaca cgcagaagag cctctccctg tctccgggta aa 1362
<210> 31
<211> 218
<212> PRT
<213> R14 light chain full length(Artificial Sequence)
<400> 31
Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln
1 5 10 15
Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly
20 25 30
Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu
35 40 45
Leu Ile Tyr Gly Asn Ser Asn Arg Pro Ser Gly Val Pro Asp Arg Phe
50 55 60
Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu
65 70 75 80
Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Ser
85 90 95
Leu Ser Gly Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Pro
100 105 110
Arg Thr Ala Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser
115 120 125
Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp
130 135 140
Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro
145 150 155 160
Val Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn
165 170 175
Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys
180 185 190
Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val
195 200 205
Glu Lys Thr Val Ala Pro Thr Glu Cys Ser
210 215
<210> 32
<211> 654
<212> DNA
<213> R14 light chain full length(Artificial Sequence)
<400> 32
cagtctgtct tgacgcagcc gccctcagtg tctggggccc cagggcagag ggtcaccatc 60
tcctgcactg ggagcagctc caacatcggg gcaggttatg atgtacactg gtaccagcag 120
cttccaggaa cagcccccaa actcctcatc tatggtaaca gcaatcggcc ctcaggggtc 180
cctgaccgat tctctggctc caagtctggc acctcagcct ccctggccat cactgggctc 240
caggctgagg atgaggctga ttattactgc cagtcctatg acagcagcct gagtggcgtg 300
gtattcggcg gagggaccaa gctgaccgtc ctacctagga cagccaaggc cgctccctct 360
gtgaccctgt ttcctccctc ttccgaggag ctgcaggcta acaaggccac actggtgtgc 420
ctgatctccg acttctaccc tggcgctgtg accgtggcct ggaaggctga ctcctctccc 480
gtgaaggctg gcgtggagac aaccactccc tctaagcagt ccaacaacaa gtacgctgcc 540
tcttcctacc tgtccctgac tcccgagcag tggaagtctc accggtccta ctcctgccag 600
gtgacccacg agggttccac cgtggagaag accgtggctc ccaccgagtg ctcc 654
<210> 33
<211> 445
<212> PRT
<213> R44 heavy chain full length-IgG1(Artificial Sequence)
<400> 33
Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Glu Ile Thr Val Ser Ser Asn
20 25 30
Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Val Leu Tyr Ser Gly Gly Ser Thr Asp Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Glu Val Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr
100 105 110
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
115 120 125
Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val
130 135 140
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
145 150 155 160
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
165 170 175
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
180 185 190
Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys
195 200 205
Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys
210 215 220
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
225 230 235 240
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
245 250 255
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
260 265 270
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
275 280 285
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
290 295 300
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
305 310 315 320
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
325 330 335
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
340 345 350
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
355 360 365
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
370 375 380
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
385 390 395 400
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
405 410 415
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
420 425 430
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 34
<211> 1335
<212> DNA
<213> R44 heavy chain full length-IgG1(Artificial Sequence)
<400> 34
caggtccagc tggtgcagtc tgggggaggc ttggtccagc ctggggggtc cctgagactc 60
tcctgtgcag cctctgaaat caccgtcagt agcaactaca tgagctgggt ccgccaggct 120
ccagggaagg ggctggagtg ggtctcagtt ctttatagcg gtggtagcac agactatgca 180
gactccgtga agggccgatt caccatctcc agagacaatt ccaagaacac gctgtatctt 240
caaatgaaca gcctgagagc tgaggacacg gctgtgtatt actgtgcgag agaggtgtac 300
ggtatggacg tctggggcca agggaccaca gtcaccgtct cctcagctag caccaagggc 360
ccatcggtct tccccctggc accctcctcc aagagcacct ctgggggcac agcggccctg 420
ggctgcctgg tcaaggacta cttccccgaa ccggtgacgg tgtcgtggaa ctcaggcgcc 480
ctgaccagcg gcgtgcacac cttcccggct gtcctacagt cctcaggact ctactccctc 540
agcagcgtgg tgaccgtgcc ctccagcagc ttgggcaccc agacctacat ctgcaacgtg 600
aatcacaagc ccagcaacac caaggtggac aagaaagttg agcccaaatc ttgtgacaaa 660
actcacacat gcccaccgtg cccagcacct gaactcctgg ggggaccgtc agtcttcctc 720
ttccccccaa aacccaagga caccctcatg atctcccgga cccctgaggt cacatgcgtg 780
gtggtggacg tgagccacga agaccctgag gtcaagttca actggtacgt ggacggcgtg 840
gaggtgcata atgccaagac aaagccgcgg gaggagcagt acaacagcac gtaccgtgtg 900
gtcagcgtcc tcaccgtcct gcaccaggac tggctgaatg gcaaggagta caagtgcaag 960
gtctccaaca aagccctccc agcccccatc gagaaaacca tctccaaagc caaagggcag 1020
ccccgagaac cacaggtgta caccctgcct ccatctcggg atgagctgac caagaaccag 1080
gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt ggagtgggag 1140
agcaatgggc agccggagaa caactacaag accacgcctc ccgtgctgga ctccgacggc 1200
tccttcttcc tctatagcaa gctcaccgtg gacaagagca ggtggcagca ggggaacgtc 1260
ttctcatgct ccgtgatgca tgaggctctg cacaaccact acacgcagaa gagcctctcc 1320
ctgtctccgg gtaaa 1335
<210> 35
<211> 214
<212> PRT
<213> R44 light chain full length(Artificial Sequence)
<400> 35
Asp Ile Val Leu Thr Gln Thr Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Glu Leu Ser Thr Tyr Pro Val
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 36
<211> 642
<212> DNA
<213> R44 light chain full length(Artificial Sequence)
<400> 36
gatattgtgc tgacccagac tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60
atcacttgcc gggccagtca gggcattagc agttatttag cctggtatca gcaaaaacca 120
gggaaagccc ctaagctcct gatctatgct gcatccactt tgcaaagtgg ggtcccatca 180
aggttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240
gaagattttg caacttatta ctgtcaagag cttagtactt acccggtcac ttttggccag 300
gggaccaagg tggaaatcaa acgtacggtg gctgcaccat ctgtcttcat cttcccgcca 360
tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 420
cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 480
gagagtgtca cagagcaaga cagcaaggac agcacctaca gcctcagcag caccctgacg 540
ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 600
ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gt 642
<210> 37
<211> 454
<212> PRT
<213> R157 heavy chain full length-IgG1(Artificial Sequence)
<400> 37
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr
20 25 30
Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Gly Ile Ile Pro Ile Leu Gly Ile Ala Asn Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Glu Gln Gly Tyr Ser Gly Ser Gly Ala Asn Tyr Tyr Phe Asp
100 105 110
Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys
115 120 125
Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly
130 135 140
Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
145 150 155 160
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
165 170 175
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
180 185 190
Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
195 200 205
Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro
210 215 220
Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
225 230 235 240
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
245 250 255
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
260 265 270
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
275 280 285
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
290 295 300
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
305 310 315 320
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
325 330 335
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
340 345 350
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
355 360 365
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
370 375 380
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
385 390 395 400
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
405 410 415
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
420 425 430
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
435 440 445
Ser Leu Ser Pro Gly Lys
450
<210> 38
<211> 1362
<212> DNA
<213> R157 heavy chain full length-IgG1(Artificial Sequence)
<400> 38
caggtccagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc agtgaaggtc 60
tcctgcaagg cttctggagg caccttcagc agctatgcta tcaactgggt gcgacaggcc 120
cctggacaag ggcttgagtg gatgggaggg atcatcccta tccttggtat agcaaactac 180
gcacagaagt tccagggcag agtcacgatt accgcggaca aatccacgag cacagcctac 240
atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc gagagagcag 300
ggttactctg gttcgggagc taattactac tttgactact ggggccaggg aaccctggtc 360
accgtctcct cagctagcac caagggccca tcggtcttcc ccctggcacc ctcctccaag 420
agcacctctg ggggcacagc ggccctgggc tgcctggtca aggactactt ccccgaaccg 480
gtgacggtgt cgtggaactc aggcgccctg accagcggcg tgcacacctt cccggctgtc 540
ctacagtcct caggactcta ctccctcagc agcgtggtga ccgtgccctc cagcagcttg 600
ggcacccaga cctacatctg caacgtgaat cacaagccca gcaacaccaa ggtggacaag 660
aaagttgagc ccaaatcttg tgacaaaact cacacatgcc caccgtgccc agcacctgaa 720
ctcctggggg gaccgtcagt cttcctcttc cccccaaaac ccaaggacac cctcatgatc 780
tcccggaccc ctgaggtcac atgcgtggtg gtggacgtga gccacgaaga ccctgaggtc 840
aagttcaact ggtacgtgga cggcgtggag gtgcataatg ccaagacaaa gccgcgggag 900
gagcagtaca acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg 960
ctgaatggca aggagtacaa gtgcaaggtc tccaacaaag ccctcccagc ccccatcgag 1020
aaaaccatct ccaaagccaa agggcagccc cgagaaccac aggtgtacac cctgcctcca 1080
tctcgggatg agctgaccaa gaaccaggtc agcctgacct gcctggtcaa aggcttctat 1140
cccagcgaca tcgccgtgga gtgggagagc aatgggcagc cggagaacaa ctacaagacc 1200
acgcctcccg tgctggactc cgacggctcc ttcttcctct atagcaagct caccgtggac 1260
aagagcaggt ggcagcaggg gaacgtcttc tcatgctccg tgatgcatga ggctctgcac 1320
aaccactaca cgcagaagag cctctccctg tctccgggta aa 1362
<210> 39
<211> 218
<212> PRT
<213> R157 light chain full length(Artificial Sequence)
<400> 39
Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln
1 5 10 15
Arg Val Thr Val Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly
20 25 30
Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu
35 40 45
Leu Ile Tyr Gly Asn Thr Asn Arg Pro Ser Gly Val Pro Asp Arg Phe
50 55 60
Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu
65 70 75 80
Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Ser
85 90 95
Leu Ser Asp Val Val Phe Gly Gly Gly Thr Gln Leu Thr Val Leu Pro
100 105 110
Arg Thr Ala Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser
115 120 125
Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp
130 135 140
Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro
145 150 155 160
Val Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn
165 170 175
Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys
180 185 190
Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val
195 200 205
Glu Lys Thr Val Ala Pro Thr Glu Cys Ser
210 215
<210> 40
<211> 654
<212> DNA
<213> R157 light chain full length(Artificial Sequence)
<400> 40
cagtctgtct tgacgcagcc gccctcagtg tctggggccc cagggcagag ggtcaccgtc 60
tcctgcactg ggagcagctc caacatcggg gcaggttatg atgtacactg gtaccagcag 120
cttccaggaa cagcccccaa actcctcatc tatggtaaca ccaatcggcc ctcaggggtc 180
cctgaccgat tctctggctc caagtctggc acctcagcct ccctggccat cactgggctc 240
caggctgagg atgaggctga ttattactgc cagtcctatg acagcagcct gagtgatgtg 300
gtattcggcg gaggcaccca gctgaccgtc ctccctagga cagccaaggc cgctccctct 360
gtgaccctgt ttcctccctc ttccgaggag ctgcaggcta acaaggccac actggtgtgc 420
ctgatctccg acttctaccc tggcgctgtg accgtggcct ggaaggctga ctcctctccc 480
gtgaaggctg gcgtggagac aaccactccc tctaagcagt ccaacaacaa gtacgctgcc 540
tcttcctacc tgtccctgac tcccgagcag tggaagtctc accggtccta ctcctgccag 600
gtgacccacg agggttccac cgtggagaag accgtggctc ccaccgagtg ctcc 654
<210> 41
<211> 444
<212> PRT
<213> R44 heavy chain full length-mutant(Artificial Sequence)
<400> 41
Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Glu Ile Thr Val Ser Ser Asn
20 25 30
Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Val Leu Tyr Ser Gly Gly Ser Thr Asp Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Glu Val Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr
100 105 110
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
115 120 125
Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val
130 135 140
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
145 150 155 160
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
165 170 175
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
180 185 190
Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys
195 200 205
Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys
210 215 220
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
225 230 235 240
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
245 250 255
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
260 265 270
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
275 280 285
Pro Arg Glu Glu Gln Tyr Asn Ser Leu Tyr Arg Val Val Ser Val Leu
290 295 300
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Glu Cys Lys
305 310 315 320
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
325 330 335
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
340 345 350
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
355 360 365
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Arg
370 375 380
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
385 390 395 400
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
405 410 415
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
420 425 430
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440
<210> 42
<211> 1332
<212> DNA
<213> R44 heavy chain full length-mutant(Artificial Sequence)
<400> 42
caggtccagc tggtgcagtc tgggggaggc ttggtccagc ctggggggtc cctgagactc 60
tcctgtgcag cctctgaaat caccgtcagt agcaactaca tgagctgggt ccgccaggct 120
ccagggaagg ggctggagtg ggtctcagtt ctttatagcg gtggtagcac agactatgca 180
gactccgtga agggccgatt caccatctcc agagacaatt ccaagaacac gctgtatctt 240
caaatgaaca gcctgagagc tgaggacacg gctgtgtatt actgtgcgag agaggtgtac 300
ggtatggacg tctggggcca agggaccaca gtcaccgtct cctcagctag caccaagggc 360
ccatcggtct tccccctggc accctcctcc aagagcacct ctgggggcac agcggccctg 420
ggctgcctgg tcaaggacta cttccccgaa ccggtgacgg tgtcgtggaa ctcaggcgcc 480
ctgaccagcg gcgtgcacac cttcccggct gtcctacagt cctcaggact ctactccctc 540
agcagcgtgg tgaccgtgcc ctccagcagc ttgggcaccc agacctacat ctgcaacgtg 600
aatcacaagc ccagcaacac caaggtggac aagaaagttg agcccaaatc ttgtgacaaa 660
actcacacat gcccaccgtg cccagcacct gaactcctgg ggggaccgtc agtcttcctc 720
ttccccccaa aacccaagga caccctcatg atctcccgga cccctgaggt cacatgcgtg 780
gtggtggacg tgagccacga agaccctgag gtcaagttca actggtacgt ggacggcgtg 840
gaggtgcata atgccaagac aaagccgcgg gaggagcagt acaacagcct gtaccgtgtg 900
gtcagcgtcc tcaccgtcct gcaccaggac tggctgaatg gcaaggagta cgagtgcaag 960
gtctccaaca aagccctccc agcccccatc gagaaaacca tctccaaagc caaagggcag 1020
ccccgagaac cacaggtgta caccctgcct ccatctcggg atgagctgac caagaaccag 1080
gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt ggagtgggag 1140
agcaatgggc ggccggagaa caactacaag accacgcctc ccgtgctgga ctccgacggc 1200
tccttcttcc tctatagcaa gctcaccgtg gacaagagca ggtggcagca ggggaacgtc 1260
ttctcatgct ccgtgatgca tgaggctctg cacaaccact acacgcagaa gagcctctcc 1320
ctgtctccgg gt 1332
<210> 43
<211> 214
<212> PRT
<213> R44 light chain full length-mutant(Artificial Sequence)
<400> 43
Asp Ile Val Leu Thr Gln Thr Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Glu Leu Ser Thr Tyr Pro Val
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 44
<211> 642
<212> DNA
<213> R44 light chain full length-mutant(Artificial Sequence)
<400> 44
gatattgtgc tgacccagac tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60
atcacttgcc gggccagtca gggcattagc agttatttag cctggtatca gcaaaaacca 120
gggaaagccc ctaagctcct gatctatgct gcatccactt tgcaaagtgg ggtcccatca 180
aggttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240
gaagattttg caacttatta ctgtcaagag cttagtactt acccggtcac ttttggccag 300
gggaccaagg tggaaatcaa acgtacggtg gctgcaccat ctgtcttcat cttcccgcca 360
tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 420
cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 480
gagagtgtca cagagcaaga cagcaaggac agcacctaca gcctcagcag caccctgacg 540
ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 600
ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gt 642
Claims (10)
1.靶向新冠病毒SARS-CoV-2刺突蛋白受体结合域的单克隆抗体或其抗原结合部分,其包含:
(a)重链可变区CDR1,其包含与选自SEQ ID NO:1、SEQ ID NO:7、或SEQ ID NO:13所示一致的氨基酸序列;
(b)重链可变区CDR2,其包含与选自SEQ ID NO:2或SEQ ID NO:8所示一致的氨基酸序列;
(c)重链可变区CDR3,其包含与选自SEQ ID NO:3、SEQ ID NO:9、或SEQ ID NO:14所示一致的氨基酸序列;
(d)轻链可变区CDR1,其包含与选自SEQ ID NO:4或SEQ ID NO:10所示一致的氨基酸序列;
(e)轻链可变区CDR2,其包含与选自SEQ ID NO:5、SEQ ID NO:11、或SEQ ID NO:15所示一致的氨基酸序列;和
(f)轻链可变区CDR3,其包含与选自SEQ ID NO:6、SEQ ID NO:12或SEQ ID NO:16所示一致的氨基酸序列。
2.根据权利要求1所述的单克隆抗体或其抗原结合部分,其特征在于,其包含:
(a)重链可变区CDR1,CDR2和CDR3的氨基酸序列分别与SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示一致;
(b)轻链可变区CDR1,CDR2和CDR3的氨基酸序列分别与SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示一致。
3.根据权利要求1所述的单克隆抗体或其抗原结合部分,其特征在于,其包含:
(a)重链可变区CDR1,CDR2和CDR3的氨基酸序列分别与SEQ ID NO:7、SEQ ID NO:8和SEQ ID NO:9所示一致;
(b)轻链可变区CDR1,CDR2和CDR3的氨基酸序列分别与SEQ ID NO:10、SEQ ID NO:11和SEQ ID NO:12所示一致。
4.根据权利要求1所述的单克隆抗体或其抗原结合部分,其特征在于,其包含:
(a)重链可变区CDR1,CDR2和CDR3的氨基酸序列分别与SEQ ID NO:13、SEQ ID NO:2和SEQ ID NO:14所示一致;
(b)轻链可变区CDR1,CDR2和CDR3的氨基酸序列分别与SEQ ID NO:4、SEQ ID NO:15和SEQ ID NO:16所示一致。
5.靶向新冠病毒SARS-CoV-2刺突蛋白受体结合域的单克隆抗体或其抗原结合部分,其包含重链和轻链可变区序列:
(a)重链可变区,其包含与选自SEQ ID NO:17、SEQ ID NO:21或SEQ ID NO:25所示一致的氨基酸序列;
(b)轻链可变区,其包含与选自SEQ ID NO:19、SEQ ID NO:23或SEQ ID NO:27所示一致的氨基酸序列。
6.根据权利要求5所述的单克隆抗体或其抗原结合部分,其特征在于,所述人抗体恒定区选自由IgGl、IgG2、IgG3、IgG4组成的组。
7.分离的靶向新冠病毒SARS-CoV-2刺突蛋白受体结合域的单克隆抗体,其包含:
(a)重链,其序列与SEQ ID NO:29、SEQ ID NO:33或SEQ ID NO:37所示的氨基酸序列一致;以及
(b)轻链,其序列与SEQ ID NO:31、SEQ ID NO:35或SEQ ID NO:39所示的氨基酸序列一致。
8.根据权利要求7所述的单克隆抗体,其特征在于,其包含:
(a)重链,其序列与SEQ ID NO:41所示的氨基酸序列一致;以及
(b)轻链,其序列与SEQ ID NO:43所示的氨基酸序列一致。
9.编码权利要求1-8任一项所述的单克隆抗体或其抗原结合部分的核酸分子。
10.权利要求1-8任一项所述的单克隆抗体或抗原结合部分在制备用于治疗或预防COVID-19的药物中的用途。
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CN117304317A (zh) * | 2022-06-28 | 2023-12-29 | 四川大学 | Ace2受体特异性结合肽及其应用 |
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