CN111978392B - 抗乙肝病毒抗体及其用途 - Google Patents
抗乙肝病毒抗体及其用途 Download PDFInfo
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- CN111978392B CN111978392B CN202010443062.5A CN202010443062A CN111978392B CN 111978392 B CN111978392 B CN 111978392B CN 202010443062 A CN202010443062 A CN 202010443062A CN 111978392 B CN111978392 B CN 111978392B
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Abstract
本发明涉及抗乙肝表面抗原(HBsAg)的抗体(特别是人源化抗体),编码它们的核酸分子,制备它们的方法,以及包含它们的药物组合物。本发明的抗HBsAg抗体在中性pH下结合HBsAg的亲和力比在酸性pH下更高,从而显著加强病毒清除效率、延长病毒抑制时间。本发明的抗体和药物组合物可用于预防和/或治疗HBV感染或与HBV感染相关的疾病(例如乙肝),用于在受试者(例如人)体内中和HBV的毒力,用于在受试者体内降低HBVDNA和/或HBsAg的血清水平,或用于激活受试者(例如慢性HBV感染者或慢性乙肝患者)针对HBV的体液免疫应答。
Description
技术领域
本发明涉及分子病毒学和免疫学领域,特别是治疗乙型肝炎病毒(Hepatitis Bvirus,HBV)感染的领域。具体而言,本发明涉及抗乙肝病毒表面抗原(HBsAg)的抗体和编码抗体的核酸,及其用途。本发明的抗HBsAg抗体在中性pH下结合HBsAg的亲和力比在酸性pH下更高。所述新型抗体可用于预防和/或治疗HBV感染或与HBV感染相关的疾病(例如乙肝),用于在受试者(例如人)体内中和HBV的毒力,或用于在受试者体内降低HBV DNA和/或HBsAg的血清水平。因此,本发明进一步涉及,所述抗体及其变体在制备药物组合物中的用途,所述药物组合物用于预防和/或治疗HBV感染或与HBV感染相关的疾病(例如乙肝),用于在受试者(例如人)体内中和HBV的毒力,用于在受试者体内降低HBV DNA和/或HBsAg的血清水平,或用于激活受试者(例如慢性HBV感染者或慢性乙肝患者)针对HBV的体液免疫应答。
背景技术
乙型肝炎病毒感染,尤其是慢性HBV感染是全球最为重要的公共卫生问题之一(Dienstag JL.Hepatitis B virus infection.N Engl J Med 2008Oct2;359(14):1486-1500)。慢性HBV感染可导致慢性乙型病毒性肝炎(Chronic hepatitis B,CHB)、肝硬化(Liver cirrhosis,LC)和原发性肝细胞癌(Hepatocellular carcinoma,HCC)等一系列肝脏疾病(Liaw YF,Chu CM.Hepatitis B virus infection.Lancet 2009Feb 14;373(9663):582-592)。据报道,目前全球约有20亿人曾经感染过HBV,现约有3.5亿的慢性乙型肝炎病毒感染者,这些感染者最终死于HBV感染相关肝脏疾病的风险可达15%-25%,全球每年超过100万人死于此类疾病(Dienstag JL.,同上;和Liaw YF等,同上)。
当前针对慢性HBV感染的治疗药物主要可分为干扰素类(Interferon,IFNs)和核苷/核苷酸类似物(nucleoside or nucleotide,NAs)(Dienstag JL.,同上;Kwon H,LokAS.Hepatitis B therapy.Nat Rev Gastroenterol Hepatol2011May;8(5):275-284;和Liaw YF等,同上)。对于HBV感染者(例如CHB患者)来说,上述药物的单独或联合治疗,已能较为有效地抑制体内病毒复制,大幅降低HBV DNA水平;特别地,52周或延长的此类治疗后,患者体内HBV DNA水平低于检测下限(病毒学应答)的应答率可达40-80%(Kwon H等,同上)。然而,上述药物的单独或联合治疗均不能完全清除感染者体内的HBV病毒,其导致的HBsAg阴转或HBsAg血清学转换(感染者体内HBV病毒彻底清除的标志)的应答率通常小于5%(Kwon H等,同上)。
基于免疫学手段发展新的治疗慢性HBV感染的药物是此领域的重要研究方向之一。慢性HBV感染的免疫治疗通常通过主动免疫治疗(其对应药物形式如疫苗等)和被动免疫治疗(其对应药物形式如抗体等)等两种方式来进行。主动免疫治疗则是指,通过施用治疗性疫苗(包括蛋白疫苗、多肽疫苗和核酸疫苗等),刺激慢性HBV感染者机体主动产生针对HBV的细胞免疫应答(CTL效应等)或/和体液免疫应答(抗体等),从而达到抑制或清除HBV的目的。目前尚无明确显著有效的、可用于治疗慢性HBV感染的主动免疫治疗药物/疫苗。被动免疫治疗(以抗体为例)是指,向HBV感染者被动施用具治疗特性的抗体,并利用抗体介导的病毒中和作用阻断HBV感染新生肝细胞,或利用抗体介导的免疫清除作用清除体内病毒和被感染的肝细胞,从而起到治疗的效果。目前,从预防性乙型肝炎疫苗免疫应答者或HBV感染恢复者血清/血浆中纯化获得的Anti-HBs多克隆抗体,即高效价的乙肝免疫球蛋白(HBIG)已广泛应用于阻断HBV的母婴垂直传播、预防慢性HBV感染者肝移植后的HBV再感染、以及预防意外暴露于HBV的人群的感染。然而,将HBIG直接用于HBV感染者(例如CHB患者)的治疗无明显疗效,且其存在例如高效价血浆来源较少、价格昂贵、性质不稳定、潜在的安全性问题等诸多局限性。
因此,针对HBV感染者发展创新的、能更为有效地清除HBV病毒、尤其是清除HBsAg的治疗方法和药物是迫切而必要的。
发明内容
本发明人前期已开发了具有优良性质的抗HBsAg人源化抗体,其能够在体内中和HBV的毒力、降低HBV DNA和/或HBsAg的血清水平。在前期研究的基础上,发明人付出了大量的创造性劳动,对该人源化抗体进行了深入的研究和改造,从而开发了具备pH依赖性抗原结合能力的抗HBsAg抗体:本发明的抗HBsAg抗体在中性pH下结合HBsAg的亲和力比在酸性pH下更高,从而实现抗体的循环使用,显著延长抗体半衰期,增强对HBV的清除效率。进一步,发明人通过对上述抗体的Fc区段引入突变,加强其在中性条件下与hFcRn或mFcγRII的亲和力,从而获得了清道夫抗体,进一步延长了抗体半衰期。
本发明的抗体是极为有利的,其不仅保留了降低HBV DNA和/或HBsAg的血清水平的活性,而且具有更长的抗原抑制时间,极大减轻治疗的注射剂量和给药频率,具有重大的临床价值。
本发明的抗体
因此,在一个方面,本发明提供了一种能够特异性结合HBsAg的抗体或其抗原结合片段,所述抗体或其抗原结合片段以在中性pH下比在酸性pH下更高的亲和力结合HBsAg。
在某些实施方案中,所述中性pH是pH6.7-pH7.5,例如pH7.4。
在某些实施方案中,所述酸性pH是pH4.0-pH6.5,例如pH6.0。
在某些实施方案中,所述抗体或其抗原结合片段在酸性pH(例如pH6.0)下结合HBsAg的KD与其在中性pH(例如pH7.4)下结合HBsAg的KD之比(即,KD(酸性pH)/KD(中性pH)的值)大于1,例如不小于1.5,不小于2,不小于3,不小于4,不小于5,不小于6,不小于7,不小于8,不小于9,不小于10,不小于15,不小于20,不小于30,不小于40,不小于50,不小于60,不小于70,不小于80,不小于90,不小于100,不小于300,不小于500,不小于800,不小于1000,不小于2000,不小于5000,或不小于10000。在某些实施方案中,所述KD(酸性pH)/KD(中性pH)的值大于1,且不大于10000,例如不大于5000,不大于2000,不大于1000,不大于900,不大于800,不大于700,不大于600,不大于500,不大于400,不大于300,不大于200,不大于100,不大于90,不大于80,不大于70,不大于60,不大于50,不大于40,不大于30,不大于20,或不大于10。所述KD可以通过领域里公知的技术测得,例如通过SPR技术(如Biacore)测得。
在某些实施方案中,所述抗体或其抗原结合片段在pH6.0下结合HBsAg的KD与其在pH7.4下结合HBsAg的KD之比大于1,例如不小于1.5,或不小于2。在某些实施方案中,本发明的抗体在中性pH的KD值可以为10-7M,10-8M,10-9M,10-10M,10-11M,10-12M或以下。在某些实施方案中,本发明的抗体在酸性pH的KD值可以为10-9M,10-8M,10-7M,10-6M或以上。
在某些实施方案中,所述抗体或其抗原结合片段在酸性pH(例如pH6.0)下结合HBsAg的EC50与其在中性pH(例如pH7.4)下结合HBsAg的EC50之比(即,EC50(酸性pH)/EC50(中性pH)的值)大于1,例如不小于1.5,不小于2,不小于3,不小于4,不小于5,不小于6,不小于7,不小于8,不小于9,不小于10,不小于15,不小于20,不小于30,不小于40,不小于50,不小于60,不小于70,不小于80,不小于90,不小于100,不小于300,不小于500,不小于800,不小于1000,不小于2000,不小于5000,或不小于10000。在某些实施方案中,所述EC50(酸性pH)/EC50(中性pH)的值大于1,且不大于10000,例如不大于5000,不大于2000,不大于1000,不大于900,不大于800,不大于700,不大于600,不大于500,不大于400,不大于300,不大于200,不大于100,不大于90,不大于80,不大于70,不大于60,不大于50,不大于40,不大于30,不大于20,或不大于10。在某些实施方案中,所述EC50通过ELISA法测得,例如通过ELISA法所产生的剂量反应曲线的回归分析来计算。
在某些实施方案中,所述抗体或其抗原结合片段在pH6.0下结合HBsAg的EC50与其在pH7.4下结合HBsAg的EC50之比大于1,例如不小于1.5,或不小于2。
在某些实施方案中,本发明的抗体或其抗原结合片段衍生自抗HBV的人源化抗体162(详细描述于中国专利申请201610879693.5)。
在某些实施方案中,所述抗体或其抗原结合片段以在中性pH下比在酸性pH下更高的亲和力结合HBsAg的aa121-124。
在某些实施方案中,所述抗体或其抗原结合片段包含含有HCDR1、HCDR2和HCDR3的重链可变区(VH),其具备以下特征中的一项或多项:
(i)HCDR1与SEQ ID NO:11所示的序列相比具有至少一个氨基酸(例如,1个,2个,3个,4个或5个氨基酸)被替换为组氨酸;
(ii)HCDR2与SEQ ID NO:12所示的序列相比具有至少一个氨基酸(例如,1个,2个,3个,4个或5个氨基酸)被替换为组氨酸;和/或,
(iii)HCDR3与SEQ ID NO:13所示的序列相比具有至少一个氨基酸(例如,1个,2个,3个,4个或5个氨基酸)被替换为组氨酸。
在某些实施方案中,所述抗体或其抗原结合片段包含含有LCDR1、LCDR2和LCDR3的重链可变区(VL),其具备以下特征中的一项或多项:
(i)LCDR1与SEQ ID NO:14所示的序列相比具有至少一个氨基酸(例如,1个,2个,3个,4个或5个氨基酸)被替换为组氨酸;
(ii)LCDR2与SEQ ID NO:15所示的序列相比具有至少一个氨基酸(例如,1个,2个,3个,4个或5个氨基酸)被替换为组氨酸;和/或,
(iii)LCDR3与SEQ ID NO:16所示的序列相比具有至少一个氨基酸(例如,1个,2个,3个,4个或5个氨基酸)被替换为组氨酸。
在某些实施方案中,所述抗体或其抗原结合片段包含:
(a)包含如下3个CDR的重链可变区(VH):
(i)序列为X1X2YHX3N(SEQ ID NO:26)的HCDR1,其中,X1选自Y或H,X2选自G或R,X3选自W或Y;
(ii)序列为YIX4X5DGSVX6YNPSLEN(SEQ ID NO:27)的HCDR2,其中,X4选自S、N或H,X5选自Y或H,X6选自L、H或Q;和
(iii)序列为GFDH(SEQ ID NO:13)的HCDR3;
和/或,
(b)包含如下3个CDR的轻链可变区(VL):
(iv)序列为RSSQSLVHSYGDX7YLH(SEQ ID NO:28)的LCDR1,其中,X7选自T或N;
(v)序列为KVSNRFS(SEQ ID NO:15)的LCDR2;和
(vi)序列为SQNTHX8PYT(SEQ ID NO:29)的LCDR3,其中,X8选自V、L或H。
在某些实施方案中,所述抗体或其抗原结合片段包含:
(a)包含如下3个CDR的重链可变区(VH):
(i)HCDR1,其由选自下列的序列组成:SEQ ID NOs:17、21、24;
(ii)HCDR2,其由选自下列的序列组成:SEQ ID NOs:18、20、22、12;和
(iii)HCDR3,其由SEQ ID NO:13所示的序列组成;
和/或,
(b)包含如下3个CDR的轻链可变区(VL):
(iv)LCDR1,其由选自下列的序列组成:SEQ ID NOs:14、25;
(v)LCDR2,其由SEQ ID NO:15所示的序列组成;和
(vi)LCDR3,其由选自下列的序列组成:SEQ ID NOs:19、16、23。
在某些实施方案中,X1选自H,X2选自G,X3选自W或Y,X4选自S或H,X5选自Y,X6选自L或H,X7选自T或N,X8选自V、L或H。
在某些实施方案中,所述抗体或其抗原结合片段包含:
(a)包含如下3个CDR的重链可变区(VH):
(i)HCDR1,其由选自下列的序列组成:SEQ ID NOs:17、24;
(ii)HCDR2,其由选自下列的序列组成:SEQ ID NOs:18、12;和
(iii)HCDR3,其由SEQ ID NO:13所示的序列组成;
和/或,
(b)包含如下3个CDR的轻链可变区(VL):
(iv)LCDR1,其由选自下列的序列组成:SEQ ID NOs:14、25;
(v)LCDR2,其由SEQ ID NO:15所示的序列组成;和
(vi)LCDR3,其由选自下列的序列组成:SEQ ID NOs:19、16、23。
在某些实施方案中,所述抗体或其抗原结合片段包含:
(1)包含如下3个CDR的VH:如SEQ ID NO:21所示的HCDR1,SEQ ID NO:22所示的HCDR2,SEQ ID NO:13所示的HCDR3;以及,包含如下3个CDR的VL:SEQ ID NO:14所示的LCDR1,SEQ ID NO:15所示的LCDR2,SEQ ID NO:23所示的LCDR3;
(2)包含如下3个CDR的VH:如SEQ ID NO:17所示的HCDR1,SEQ ID NO:18所示的HCDR2,SEQ ID NO:13所示的HCDR3;以及,包含如下3个CDR的VL:SEQ ID NO:14所示的LCDR1,SEQ ID NO:15所示的LCDR2,SEQ ID NO:19所示的LCDR3;
(3)包含如下3个CDR的VH:如SEQ ID NO:17所示的HCDR1,SEQ ID NO:20所示的HCDR2,SEQ ID NO:13所示的HCDR3;以及,包含如下3个CDR的VL:SEQ ID NO:14所示的LCDR1,SEQ ID NO:15所示的LCDR2,SEQ ID NO:16所示的LCDR3;
(4)包含如下3个CDR的VH:如SEQ ID NO:24所示的HCDR1,SEQ ID NO:12所示的HCDR2,SEQ ID NO:13所示的HCDR3;以及,包含如下3个CDR的VL:SEQ ID NO:25所示的LCDR1,SEQ ID NO:15所示的LCDR2,SEQ ID NO:16所示的LCDR3;
(5)包含如下3个CDR的VH:如SEQ ID NO:17所示的HCDR1,SEQ ID NO:12所示的HCDR2,SEQ ID NO:13所示的HCDR3;以及,包含如下3个CDR的VL:SEQ ID NO:25所示的LCDR1,SEQ ID NO:15所示的LCDR2,SEQ ID NO:23所示的LCDR3;或
(6)包含如下3个CDR的VH:如SEQ ID NO:17所示的HCDR1,SEQ ID NO:12所示的HCDR2,SEQ ID NO:13所示的HCDR3;以及,包含如下3个CDR的VL:SEQ ID NO:25所示的LCDR1,SEQ ID NO:15所示的LCDR2,SEQ ID NO:16所示的LCDR3。
在某些实施方案中,所述抗体或其抗原结合片段进一步包含人免疫球蛋白的框架区(例如,人胚系抗体基因所编码的氨基酸序列中所包含的框架区),所述框架区任选地包含一个或多个(例如,1个、2个、3个、4个、5个、6个、7个、8个、9个或10个)从人源残基至鼠源残基的回复突变。
在某些实施方案中,所述抗体或其抗原结合片段包含:人重链胚系基因所编码的氨基酸序列中所包含的重链框架区,和/或人轻链胚系基因所编码的氨基酸序列中所包含的轻链框架区。
在某些实施方案中,所述抗体或其抗原结合片段包含:人重链胚系基因4-28-02(SEQ ID NO:38)所编码的氨基酸序列中所包含的重链框架区,和人轻链胚系基因2D-28-01(SEQ ID NO:39)所编码的氨基酸序列中所包含的轻链框架区,所述重链框架区和/或轻链框架区任选地包含一个或多个(例如,1个、2个、3个、4个、5个、6个、7个、8个、9个或10个)从人源残基至鼠源残基的回复突变。
在某些实施方案中,所述抗体或其抗原结合片段的VH包含:如SEQ ID NO:30所示的VH FR1、如SEQ ID NO:31所示的VH FR2、如SEQ ID NO:32所示的VH FR3、和如SEQ ID NO:33所示的VH FR4。
在某些实施方案中,所述抗体或其抗原结合片段的VL包含:如SEQ ID NO:34所示的VL FR1、如SEQ ID NO:35所示的VL FR2、如SEQ ID NO:36所示的VL FR3、和如SEQ ID NO:37所示的VL FR4。
在某些实施方案中,所述抗体或其抗原结合片段包含:
(a)重链可变区(VH),其包含选自下列的氨基酸序列:
(i)SEQ ID NOs:3、5、6、8任一项所示的序列;
(ii)与SEQ ID NOs:3、5、6、8任一项所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个,2个,3个,4个或5个氨基酸的置换、缺失或添加)的序列;或
(iii)与SEQ ID NOs:3、5、6、8任一项所示的序列具有至少80%、至少85%、至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%、至少99%、或100%的序列同一性的序列;
和
(b)轻链可变区(VL),其包含选自下列的氨基酸序列:
(iv)SEQ ID NOs:4、2、7、9、10任一项所示的序列;
(v)与SEQ ID NOs:4、2、7、9、10任一项所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个,2个,3个,4个或5个氨基酸的置换、缺失或添加)的序列;或
(vi)与SEQ ID NOs:4、2、7、9、10任一项所示的序列具有至少80%、至少85%、至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%、至少99%、或100%的序列同一性的序列。
优选地,(ii)或(v)中所述的置换是保守置换。
在某些实施方案中,所述抗体或其抗原结合片段包含:
(1)具有如SEQ ID NO:3所示的序列的VH和具有如SEQ ID NO:4所示的序列的VL;
(2)具有如SEQ ID NO:5所示的序列的VH和具有如SEQ ID NO:2所示的序列的VL;
(3)具有如SEQ ID NO:6所示的序列的VH和具有如SEQ ID NO:7所示的序列的VL;
(4)具有如SEQ ID NO:8所示的序列的VH和具有如SEQ ID NO:9所示的序列的VL;
(5)具有如SEQ ID NO:3所示的序列的VH和具有如SEQ ID NO:10所示的序列的VL;或
(6)具有如SEQ ID NO:3所示的序列的VH和具有如SEQ ID NO:9所示的序列的VL。
在某些实施方案中,所述抗体或其抗原结合片段进一步包含来源于人免疫球蛋白的恒定区。
在某些实施方案中,所述抗体或其抗原结合片段的重链包含来源于人免疫球蛋白(例如IgG1、IgG2、IgG3或IgG4)的重链恒定区,所述抗体或其抗原结合片段的轻链包含来源于人免疫球蛋白(例如κ或λ)的轻链恒定区。
在某些实施方案中,所述抗体或其抗原结合片段包含:
(a)人免疫球蛋白的重链恒定区(CH)或其变体,所述变体与其所源自的野生型序列相比具有一个或多个氨基酸的置换、缺失或添加或其任意组合(例如,至多20个、至多15个、至多10个、或至多5个氨基酸的置换、缺失或添加或其任意组合;例如1个,2个,3个,4个或5个氨基酸的置换、缺失或添加或其任意组合);和/或
(b)人免疫球蛋白的轻链恒定区(CL)或其变体,所述变体与其所源自的野生型序列相比具有一个或多个氨基酸的置换、缺失或添加或其任意组合(例如,至多20个、至多15个、至多10个、或至多5个氨基酸的置换、缺失或添加或其任意组合;例如1个,2个,3个,4个或5个氨基酸的置换、缺失或添加或其任意组合)。
在某些实施方案中,所述抗体或其抗原结合片段包含人IgG1或IgG4重链恒定区。在某些实施方案中,所述抗体或其抗原结合片段包含如SEQ ID NO:40所示的重链恒定区(CH)。
在某些实施方案中,本发明的抗体或其抗原结合片段包含人免疫球蛋白的重链恒定区(CH)的变体,所述变体与其所源自的野生型序列相比具有增强的在中性pH(例如pH7.4)下与hFcRn或mFcγRII的亲和力。在此类实施方案中,所述变体与其所源自的野生型序列相比通常具有至少一个氨基酸的置换。
在某些实施方案中,所述抗体或其抗原结合片段包含人IgG1重链恒定区的变体,所述变体与其所源自的野生型序列相比具有以下置换:(i)M252Y、N286E、N434Y;或,(ii)K326D、L328Y;其中,以上提及的氨基酸位置是根据Kabat编号系统的位置。在某些实施方案中,所述抗体或其抗原结合片段包含如SEQ ID NO:42或43所示的重链恒定区(CH)。
在某些实施方案中,所述轻链恒定区是κ轻链恒定区。在某些实施方案中,所述抗体或其抗原结合片段包含如SEQ ID NO:41所示的轻链恒定区(CL)。
在某些实施方案中,所述抗体或其抗原结合片段包含:
(1)包括SEQ ID NO:3所示的VH和SEQ ID NO:40所示的CH的重链,和,包括SEQ IDNO:4所示的VL和SEQ ID NO:41所示的CL的轻链;
(2)包括SEQ ID NO:3所示的VH和SEQ ID NO:47所示的CH的重链,和,包括SEQ IDNO:4所示的VL和SEQ ID NO:41所示的CL的轻链;
(3)包括SEQ ID NO:3所示的VH和SEQ ID NO:48所示的CH的重链,和,包括SEQ IDNO:4所示的VL和SEQ ID NO:41所示的CL的轻链;
(4)包括SEQ ID NO:5所示的VH和SEQ ID NO:40所示的CH的重链,和,包括SEQ IDNO:2所示的VL和SEQ ID NO:41所示的CL的轻链;
(5)包括SEQ ID NO:5所示的VH和SEQ ID NO:47所示的CH的重链,和,包括SEQ IDNO:2所示的VL和SEQ ID NO:41所示的CL的轻链;
(6)包括SEQ ID NO:5所示的VH和SEQ ID NO:48所示的CH的重链,和,包括SEQ IDNO:2所示的VL和SEQ ID NO:41所示的CL的轻链;
(7)包括SEQ ID NO:6所示的VH和SEQ ID NO:40所示的CH的重链,和,包括SEQ IDNO:7所示的VL和SEQ ID NO:41所示的CL的轻链;
(8)包括SEQ ID NO:6所示的VH和SEQ ID NO:47所示的CH的重链,和,包括SEQ IDNO:7所示的VL和SEQ ID NO:41所示的CL的轻链;
(9)包括SEQ ID NO:6所示的VH和SEQ ID NO:48所示的CH的重链,和,包括SEQ IDNO:7所示的VL和SEQ ID NO:41所示的CL的轻链;
(10)包括SEQ ID NO:8所示的VH和SEQ ID NO:40所示的CH的重链,和,包括SEQ IDNO:9所示的VL和SEQ ID NO:41所示的CL的轻链;
(11)包括SEQ ID NO:8所示的VH和SEQ ID NO:47所示的CH的重链,和,包括SEQ IDNO:9所示的VL和SEQ ID NO:41所示的CL的轻链;
(12)包括SEQ ID NO:8所示的VH和SEQ ID NO:48所示的CH的重链,和,包括SEQ IDNO:9所示的VL和SEQ ID NO:41所示的CL的轻链;
(13)包括SEQ ID NO:3所示的VH和SEQ ID NO:40所示的CH的重链,和,包括SEQ IDNO:10所示的VL和SEQ ID NO:41所示的CL的轻链;
(14)包括SEQ ID NO:3所示的VH和SEQ ID NO:47所示的CH的重链,和,包括SEQ IDNO:10所示的VL和SEQ ID NO:41所示的CL的轻链;
(15)包括SEQ ID NO:3所示的VH和SEQ ID NO:48所示的CH的重链,和,包括SEQ IDNO:10所示的VL和SEQ ID NO:41所示的CL的轻链;
(16)包括SEQ ID NO:3所示的VH和SEQ ID NO:40所示的CH的重链,和,包括SEQ IDNO:9所示的VL和SEQ ID NO:41所示的CL的轻链;
(17)包括SEQ ID NO:3所示的VH和SEQ ID NO:47所示的CH的重链,和,包括SEQ IDNO:9所示的VL和SEQ ID NO:41所示的CL的轻链;或
(18)包括SEQ ID NO:3所示的VH和SEQ ID NO:48所示的CH的重链,和,包括SEQ IDNO:9所示的VL和SEQ ID NO:41所示的CL的轻链。
抗体的制备
本发明的抗体可以本领域已知的各种方法来制备,例如通过基因工程重组技术来获得。例如,通过化学合成或PCR扩增获得编码本发明抗体的重链和轻链基因的DNA分子。将所得DNA分子插入表达载体内,然后转染宿主细胞。然后,在特定条件下培养转染后的宿主细胞,并表达本发明的抗体。
本发明的抗原结合片段可以通过水解完整的抗体分子获得(参见Morimoto etal.,J.Biochem.Biophys.Methods 24:107-117(1992)and Brennan et al.,Science 229:81(1985))。另外,这些抗原结合片段也可以直接由重组宿主细胞产生(reviewed inHudson,Curr.Opin.Immunol.11:548-557(1999);Little et al.,Immunol.Today,21:364-370(2000))。比如,Fab’片段可以直接从宿主细胞中获得;可以将Fab’片段化学偶联形成F(ab’)2片段(Carter et al.,Bio/Technology,10:163-167(1992))。另外,Fv、Fab或F(ab’)2片段也可以直接从重组宿主细胞培养液中直接分离得到。本领域的普通技术人员完全知晓制备这些抗原结合片段的其它技术。
因此,在另一个方面,本发明提供了一种分离的核酸分子,其包含编码本发明的抗体或其抗原结合片段,或其重链可变区和/或轻链可变区的核苷酸序列。在某些优选的实施方案中,所述分离的核酸分子编码本发明的抗体或其抗原结合片段,或其重链可变区和/或轻链可变区。
在另一个方面,本发明提供了一种载体(例如克隆载体或表达载体),其包含本发明的分离的核酸分子。在某些优选的实施方案中,本发明的载体是例如质粒,粘粒,噬菌体等。
在另一个方面,本发明提供了一种宿主细胞,其包含本发明的分离的核酸分子或本发明的载体。此类宿主细胞包括但不限于,原核细胞例如大肠杆菌细胞,以及真核细胞例如酵母细胞,昆虫细胞,植物细胞和动物细胞(如哺乳动物细胞,例如小鼠细胞、人细胞等)。在某些优选的实施方案中,本发明的宿主细胞是哺乳动物细胞,例如CHO(例如CHO-K1、CHO-S、CHO DG44)。
在另一个方面,提供了制备本发明的抗体或其抗原结合片段的方法,其包括,在允许所述抗体或其抗原结合片段表达的条件下,培养本发明的宿主细胞,和从培养的宿主细胞培养物中回收所述抗体或其抗原结合片段。
衍生的抗体
本发明的抗体或其抗原结合片段可进行衍生化,例如被连接至另一个分子(例如另一个多肽或蛋白)。通常,抗体或其抗原结合片段的衍生化(例如,标记)不会不利影响其对HBsAg的结合。因此,本发明的抗体或其抗原结合片段还意欲包括此类衍生化的形式。例如,可以将本发明的抗体或其抗原结合片段功能性连接(通过化学偶合、基因融合、非共价连接或其它方式)于一个或多个其它分子基团,例如另一个抗体(例如,形成双特异性抗体),检测试剂,药用试剂,和/或能够介导抗体或抗原结合片段与另一个分子结合的蛋白或多肽(例如,抗生物素蛋白或多组氨酸标签)。
因此,在某些实施方案中,本发明的抗体或其抗原结合片段带有标记。在某些实施方案中,本发明的抗体或其抗原结合片段带有可检测的标记,例如酶、放射性核素、荧光染料、发光物质(如化学发光物质)或生物素。本发明所述的可检测的标记可以是可通过荧光、光谱、光化学、生物化学、免疫学、电学、光学或化学手段检测的任何物质。这类标记是本领域熟知的,其实例包括但不限于,酶(例如,辣根过氧化物酶、碱性磷酸酶、β-半乳糖苷酶、脲酶、葡萄糖氧化酶,等)、放射性核素(例如,3H、125I、35S、14C或32P)、荧光染料(例如,异硫氰酸荧光素(FITC)、荧光素、异硫氰酸四甲基罗丹明(TRITC)、藻红蛋白(PE)、德克萨斯红、罗丹明、量子点或花菁染料衍生物(例如Cy7、Alexa 750))、发光物质(例如化学发光物质,如吖啶酯类化合物)、磁珠(例如,)、测热标记物例如胶体金或有色玻璃或塑料(例如,聚苯乙烯、聚丙烯、乳胶,等)珠、以及用于结合上述标记物修饰的亲和素(例如,链霉亲和素)的生物素。在某些实施方案中,此类标记能够适用于免疫学检测(例如,酶联免疫测定法、放射免疫测定法、荧光免疫测定法、化学发光免疫测定法等)。在某些实施方案中,可通过不同长度的接头(linker)将如上所述的可检测的标记连接至本发明的抗体或其抗原结合片段,以降低潜在的位阻。
药物组合物及治疗用途
本发明的抗体或其抗原结合片段可用于预防或治疗受试者(例如人)的HBV感染或与HBV感染相关的疾病(例如乙肝),用于体外或在受试者(例如人)体内中和HBV的毒力,用于在受试者(例如人)体内降低HBV DNA和/或HBsAg的血清水平,以及用于激活受试者(例如慢性HBV感染者或慢性乙肝患者)针对HBV的体液免疫应答。
因此,在另一个方面,本发明提供了一种药物组合物,其含有本发明的抗体或其抗原结合片段,以及药学上可接受的载体和/或赋形剂。本发明的药物组合物还可包含另外的药学活性剂。在某些实施方案中,所述另外的药学活性剂是用于预防或治疗HBV感染或与HBV感染相关的疾病(例如乙肝)的药物,例如干扰素类药物,如干扰素或聚乙二醇干扰素。
在另一个方面,提供了本发明的抗体或其抗原结合片段或本发明的药物组合物在制备药物中的用途,所述药物用于预防和/或治疗受试者(例如人)的HBV感染或与HBV感染相关的疾病(例如乙肝),用于体外或在受试者(例如人)体内中和HBV的毒力,用于在受试者(例如人)体内降低HBV DNA和/或HBsAg的血清水平,和/或用于激活受试者(例如慢性HBV感染者或慢性乙肝患者)针对HBV的体液免疫应答。
在另一个方面,本发明提供了一种方法,其用于预防或治疗受试者(例如人)的HBV感染或与HBV感染相关的疾病(例如乙肝),用于在受试者(例如人)体内中和HBV的毒力,用于在受试者(例如人)体内降低HBV DNA和/或HBsAg的血清水平,和/或用于激活受试者(例如慢性HBV感染者或慢性乙肝患者)针对HBV的体液免疫应答,所述方法包括,给有此需要的受试者施用有效量的根据本发明的抗体或其抗原结合片段或根据本发明的药物组合物。
本发明所提供的药物和药物组合物可以单独使用或联合使用,也可以与另外的药学活性剂(例如其它抗病毒试剂,例如干扰素类药物,如干扰素或聚乙二醇干扰素)联合使用。
本发明的抗体或其抗原结合片段或者本发明的药物组合物的施用方式可以是传统的施用途径,包括但不限于,口服、口腔、舌下、眼球、局部、肠胃外、直肠、叶鞘内、内胞浆网槽内、腹股沟、膀胱内、局部(如,粉剂、药膏或滴剂),或鼻腔途径。本发明的抗体或其抗原结合片段可通过本领域已知的多种方法给药。但是,对于许多治疗用途而言,优选的给药途径/方式是胃肠外给药(例如静脉注射,皮下注射,腹膜内注射,肌内注射)。技术人员应理解,给药途径和/或方式将根据预期目的而发生变化。在一个优选的实施方案中,本发明的抗体或其抗原结合片段通过静脉输注或注射给予。
本发明的抗体或其抗原结合片段或者本发明的药物组合物可以配制成多种剂型,例如液体、半固体和固体剂型,例如溶液剂(例如注射剂)、分散剂或悬浮剂、片剂、粉剂、颗粒剂、乳剂、丸剂、糖浆剂、粉剂、脂质体、胶囊剂和栓剂。优选剂型取决于预期的给药方式和治疗用途。
例如,一种优选的剂型是注射剂。此类注射剂可以是无菌注射溶液。例如,可通过下述方法来制备无菌注射溶液:在适当的溶剂中掺入必需剂量的本发明的抗体或其抗原结合片段,以及任选地,同时掺入其他期望的成分(包括但不限于,pH调节剂,表面活性剂,佐剂,离子强度增强剂,等渗剂、防腐剂、稀释剂,或其任何组合),随后过滤除菌。此外,可以将无菌注射溶液制备为无菌冻干粉剂(例如,通过真空干燥或冷冻干燥)以便于储存和使用。此类无菌冻干粉剂可在使用前分散于合适的载体中,例如无菌无热原水。
另一种优选的剂型是分散剂。可通过下述方法来制备分散剂:将本发明的抗体或其抗原结合片段掺入无菌载体中,其含有一种基础分散介质以及任选的其他期望的成分(包括但不限于,pH调节剂,表面活性剂,佐剂,离子强度增强剂,等渗剂、防腐剂、稀释剂,或其任何组合)。此外,还可以在分散剂中掺入可延迟吸收的试剂,例如单硬脂酸盐和明胶,以获得期望的药代动力学特征。
另一种优选的剂型是口服固相剂型,包括胶囊、片剂、粉剂、颗粒剂等。此类固相剂型通常含有下述中的至少一种:(a)惰性药物赋形剂(或载体),如柠檬酸钠、磷酸钙;(b)填充剂,如淀粉、乳糖、蔗糖、甘露糖和硅酸;(c)粘合剂,如羧甲基纤维素、藻酸盐、明胶、聚乙烯吡咯烷酮、蔗糖和阿拉伯树胶;(d)湿润剂,如甘油;(e)碎裂剂,如琼脂,碳酸钙,马铃薯粉或木薯粉;(f)缓凝剂,如石蜡;(g)促吸收剂,如四氨基混合物;(h)保湿剂,如十六烷基醇和单硬脂酸甘油酯;(i)吸附剂,如高岭土和斑脱土;(j)润滑剂,如滑石,硬脂酸钙,硬脂酸镁,固体聚乙二醇,硫酸十二烷醇钠,或其任何组合。在片剂和胶囊剂型的情况下,还可以含有缓冲剂。
此外,还可以在口服固相剂型中添加释放速率改造剂(即,能改变药物释放速率的试剂),以获得改良释放或脉冲释放剂型。此类释放速率改造剂包括但不限于,羧丙基甲基纤维素,甲基纤维素,碳甲基纤维素钠,纤维素乙烷,醋酸纤维素,聚乙烯氧化物,黄原胶糖,异丙烯酸氨共聚物,氢化调味油,巴西棕榈蜡,石蜡,邻苯二酸醋酸纤维素,邻苯二甲酸羧丙基甲基纤维素,甲基丙烯酸共聚物,或其任何组合。改良释放和脉冲释放剂型可含有一种或一组释放速率改造剂。
另一种优选的剂型是口服的液体剂型,包括例如乳剂、溶液剂、悬浮剂、糖浆剂等。除了活性成分之外,此类口服的液体剂型还可含有本领域常用的一些惰性溶液,例如水或其它溶剂,如乙烷基醇,异丙基醇,丙烯乙二醇,1,3-丁烯乙二醇,油(例如棉籽油,落花生油,玉米油,橄榄油,调味油和芝麻油),甘油,聚乙二醇和脂肪酸山梨醇酯,以及其任何组合。除了这些惰性溶液之外,此类口服的液体剂型还可包括保湿剂、乳化剂、悬浮剂、糖化剂、调味剂和香味剂等。
此外,本发明的抗体或其抗原结合片段可以以单位剂量形式存在于药物组合物中,以便于施用。本发明的药物组合物应当是无菌的并在生产和储存条件下稳定。
本发明所提供的药物和药物组合物可以单独使用或联合使用,也可以与另外的药学活性剂(例如其它抗病毒试剂,例如干扰素类药物,如干扰素或聚乙二醇干扰素)联合使用。在某些优选的实施方案中,将本发明的抗体或其抗原结合片段与其它抗病毒试剂联合使用,以预防和或治疗乙型肝炎病毒感染相关疾病。本发明的抗体或其抗原结合片段可以和此类抗病毒试剂同时、分开或连续给药。此类抗病毒试剂包括但不限于,干扰素类药物、利巴韦林、金刚烷、羧基脲、IL-2、L-12和五羧链胞酸等。
本发明的药物组合物可包括“治疗有效量”或“预防有效量”的本发明抗体或抗原结合片段。“预防有效量”是指,足以预防,阻止,或延迟疾病(例如HBV感染或与HBV感染相关的疾病)的发生的量。“治疗有效量”是指,足以治愈或至少部分阻止已患有疾病的患者的疾病和其并发症的量。本发明抗体或抗原结合片段的治疗有效量可根据如下因素发生变化:待治疗的疾病的严重度、患者自己的免疫系统的总体状态、患者的一般情况例如年龄,体重和性别,药物的施用方式,以及同时施用的其他治疗等等。
可调整给药方案以获得最佳目的反应(例如治疗或预防反应)。例如,可以单次给药,可以在一段时间内多次给药,或者可以随治疗情况的紧急程度按比例减少或增加剂量。
本发明抗体或抗原结合片段的治疗或预防有效量的典型非极限范围是0.025到50mg/kg,更优选0.1到50mg/kg,更优选0.1-25mg/kg,0.1-10mg/kg。应注意的是,剂量可随需要治疗的症状的类型和严重性不同而发生变化。此外,本领域技术人员理解,对于任一特定患者,特定的给药方案应根据患者需要和医生的专业评价而随时间调整;此处给出的剂量范围只用于举例说明目的,而不限定本发明药物组合物的使用或范围。
试剂盒及检测用途
本发明的抗体或其抗原结合片段能够特异性结合HBsAg,从而可用于检测HBsAg在样品中的存在或其水平。
因此,在另一个方面,本发明提供了一种试剂盒,其包括本发明的抗体或其抗原结合片段。在一些实施方案中,本发明的抗体或其抗原结合片段带有可检测的标记。在另一些实施方案中,所述试剂盒还包括第二抗体,其特异性识别本发明的抗体或其抗原结合片段。优选地,所述第二抗体还包括可检测的标记。此类可检测的标记是本领域技术人员熟知的,包括但不限于,放射性同位素,荧光物质,发光物质,有色物质和酶(例如辣根过氧化物酶)等。
在另一个方面,本发明提供了检测HBsAg蛋白在样品中的存在或其水平的方法,其包括:使用本发明的抗体或其抗原结合片段。在一些实施方案中,本发明的抗体或其抗原结合片段还包括可检测的标记。在另一些实施方案中,所述方法还包括,使用携带可检测的标记的第二抗体来检测本发明的抗体或其抗原结合片段。所述方法可以用于诊断目的,或者非诊断目的(例如,所述样品是细胞样品,而非来自患者的样品)。
在某些实施方案中,所述方法包括:(1)将所述样品与本发明的抗体或其抗原结合片段接触;(2)检测所述抗体或其抗原结合片段与HBsAg蛋白之间复合物的形成或检测所述复合物的量。所述复合物的形成表明存在HBsAg蛋白和/或HBV。
在另一个方面,本发明提供了诊断受试者是否感染了HBV的方法,其包括:使用本发明的抗体或其抗原结合片段检测HBsAg蛋白在来自所述受试者的样品中的存在。在一些实施方案中,本发明的抗体或其抗原结合片段还包括可检测的标记。在另一些实施方案中,所述方法还包括,使用携带可检测的标记的第二抗体来检测本发明的抗体或其抗原结合片段。
在另一个方面,提供了本发明的抗体或其抗原结合片段在制备试剂盒中的用途,所述试剂盒用于检测HBsAg蛋白在样品中的存在或其水平,或用于诊断受试者是否感染了HBV。
术语定义
在本发明中,除非另有说明,否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且,本文中所用的细胞培养、生物化学、核酸化学、免疫学实验室等操作步骤均为相应领域内广泛使用的常规步骤。同时,为了更好地理解本发明,下面提供相关术语的定义和解释。
如本文中所使用的,术语“抗体”是指,通常由两对多肽链组成的免疫球蛋白分子,每对具有一条轻链(LC)和一条重链(HC)。抗体轻链可分类为κ(kappa)和λ(lambda)轻链。重链可分类为μ、δ、γ、α或ε,并且分别将抗体的同种型定义为IgM、IgD、IgG、IgA和IgE。在轻链和重链内,可变区和恒定区通过大约12或更多个氨基酸的“J”区连接,重链还包含大约3个或更多个氨基酸的“D”区。各重链由重链可变区(VH)和重链恒定区(CH)组成。重链恒定区由3个结构域(CH1、CH2和CH3)组成。各轻链由轻链可变区(VL)和轻链恒定区(CL)组成。轻链恒定区由一个结构域CL组成。恒定结构域不直接参与抗体与抗原的结合,但展现出多种效应子功能,如可介导免疫球蛋白与宿主组织或因子,包括免疫系统的各种细胞(例如,效应细胞)和经典补体系统的第一组分(C1q)的结合。VH和VL区还可被细分为具有高变性的区域(称为互补决定区(CDR)),其间散布有较保守的称为构架区(FR)的区域。各VH和VL由按下列顺序:FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4从氨基末端至羧基末端排列的3个CDR和4个FR组成。各重链/轻链对的可变区(VH和VL)分别形成抗原结合部位。氨基酸在各区域或结构域的分配可遵循Kabat,Sequences of Proteins of Immunological Interest(NationalInstitutes of Health,Bethesda,Md.(1987and 1991)),或Chothia&Lesk(1987)J.Mol.Biol.196:901-917;Chothia等人(1989)Nature 342:878-883的定义。
如本文中所使用的,术语“互补决定区”或“CDR”是指抗体可变区中负责抗原结合的氨基酸残基。在重链和轻链的可变区中各含有三个CDR,命名为CDR1、CDR2和CDR3。这些CDR的精确边界可根据本领域已知的各种编号系统进行定义,例如可按照Kabat编号系统(Kabat et al.,Sequences of Proteins of Immunological Interest,5th Ed.PublicHealth Service,National Institutes of Health,Bethesda,Md.,1991)、Chothia编号系统(Chothia&Lesk(1987)J.Mol.Biol.196:901-917;Chothia等人(1989)Nature 342:878-883)或IMGT编号系统(Lefranc et al.,Dev.Comparat.Immunol.27:55-77,2003)中的定义。对于给定的抗体,本领域技术人员将容易地鉴别各编号系统所定义的CDR。并且,不同编号系统之间的对应关系是本领域技术人员熟知的(例如,可参见Lefranc et al.,Dev.Comparat.Immunol.27:55-77,2003)。
在本发明中,本发明的抗体或其抗原结合片段含有的CDR可根据本领域已知的各种编号系统确定。在某些实施方案中,本发明的抗体或其抗原结合片段含有的CDR优选地通过Kabat、Chothia或IMGT编号系统确定。在某些实施方案中,本发明的抗体或其抗原结合片段含有的CDR优选地通过Kabat编号系统确定。
如本文中所使用的,术语“构架区”或“FR”残基是指,抗体可变区中除了如上定义的CDR残基以外的那些氨基酸残基。
术语“抗体”不受任何特定的产生抗体的方法限制。例如,其包括,重组抗体、单克隆抗体和多克隆抗体。抗体可以是不同同种型的抗体,例如,IgG(例如,IgG1,IgG2,IgG3或IgG4亚型),IgA1,IgA2,IgD,IgE或IgM抗体。
如本文中所使用的,术语抗体的“抗原结合片段”是指包含全长抗体的片段的多肽,其保持特异性结合全长抗体所结合的相同抗原的能力,和/或与全长抗体竞争对抗原的特异性结合,其也被称为“抗原结合部分”。通常参见,Fundamental Immunology,Ch.7(Paul,W.,ed.,第2版,Raven Press,N.Y.(1989),其以其全文通过引用合并入本文,用于所有目的。可通过重组DNA技术或通过完整抗体的酶促或化学断裂产生抗体的抗原结合片段。抗原结合片段的非限制性实例包括Fab、Fab’、F(ab’)2、Fd、Fv、互补决定区(CDR)片段、scFv、双抗体(diabody)、单域抗体(single domain antibody)、嵌合抗体、线性抗体(linear antibody)、纳米抗体(技术来自Domantis)、、probody和这样的多肽,其包含足以赋予多肽特异性抗原结合能力的抗体的至少一部分。工程改造的抗体变体综述于Holliger等,2005;Nat Biotechnol,23:1126-1136中。
如本文中所使用的,术语“全长抗体”意指,由两条“全长重链”和两条“全长轻链”组成的抗体。其中,“全长重链”是指这样的多肽链,其在N端到C端的方向上由重链可变区(VH)、重链恒定区CH1结构域、铰链区(HR)、重链恒定区CH2结构域、重链恒定区CH3结构域组成;并且,当所述全长抗体为IgE同种型时,任选地还包括重链恒定区CH4结构域。优选地,“全长重链”是在N端到C端方向上由VH、CH1、HR、CH2和CH3组成的多肽链。“全长轻链”是在N端到C端方向上由轻链可变区(VL)和轻链恒定区(CL)组成的多肽链。两对全长抗体链通过在CL和CH1之间的二硫键和两条全长重链的HR之间的二硫键连接在一起。本发明的全长抗体可以来自单一物种,例如人;也可以是嵌合抗体或人源化抗体。本发明的全长抗体包含分别由VH和VL对形成的两个抗原结合部位,这两个抗原结合部位特异性识别/结合相同的抗原。
如本文中所使用的,术语“Fd”意指由VH和CH1结构域组成的抗体片段;术语“dAb片段”意指由VH结构域组成的抗体片段(Ward等人,Nature 341:544 546(1989));术语“Fab片段”意指由VL、VH、CL和CH1结构域组成的抗体片段;术语“F(ab’)2片段”意指包含通过铰链区上的二硫桥连接的两个Fab片段的抗体片段;术语“Fab’片段”意指还原连接F(ab’)2片段中两个重链片段的二硫键后所获片段,由一条完整的轻链和重链的Fd片段(由VH和CH1结构域组成)组成。
如本文中所使用的,术语“Fv”意指由抗体的单臂的VL和VH结构域组成的抗体片段。Fv片段通常被认为是,能形成完整的抗原结合位点的最小抗体片段。一般认为,六个CDR赋予抗体的抗原结合特异性。然而,即便是一个可变区(例如Fd片段,其仅仅含有三个对抗原特异的CDR)也能够识别并结合抗原,尽管其亲和力可能低于完整的结合位点。
如本文中所使用的,术语“Fc”意指,由抗体的第一重链的第二、第三恒定区与第二重链的第二、第三恒定区经二硫键结合而形成的抗体片段。抗体的Fc片段具有多种不同的功能,但不参与抗原的结合。
如本文中所使用的,术语“scFv”是指,包含VL和VH结构域的单个多肽链,其中所述VL和VH通过接头(linker)相连(参见,例如,Bird等人,Science 242:423-426(1988);Huston等人,Proc.Natl.Acad.Sci.USA85:5879-5883(1988);和Pluckthun,ThePharmacology of Monoclonal Antibodies,第113卷,Roseburg和Moore编,Springer-Verlag,纽约,第269-315页(1994))。此类scFv分子可具有一般结构:NH2-VL-接头-VH-COOH或NH2-VH-接头-VL-COOH。合适的现有技术接头由重复的GGGGS氨基酸序列或其变体组成。例如,可使用具有氨基酸序列(GGGGS)4的接头,但也可使用其变体(Holliger等人(1993),Proc.Natl.Acad.Sci.USA90:6444-6448)。可用于本发明的其他接头由Alfthan等人(1995),Protein Eng.8:725-731,Choi等人(2001),Eur.J.Immunol.31:94-106,Hu等人(1996),Cancer Res.56:3055-3061,Kipriyanov等人(1999),J.Mol.Biol.293:41-56和Roovers等人(2001),Cancer Immunol.描述。在一些情况下,scFv的VH与VL之间还可以存在二硫键。
如本文中所使用的,术语“双抗体”意指,其VH和VL结构域在单个多肽链上表达,但使用太短的连接体以致不允许在相同链的两个结构域之间配对,从而迫使结构域与另一条链的互补结构域配对并且产生两个抗原结合部位(参见,例如,Holliger P.等人,Proc.Natl.Acad.Sci.USA90:6444-6448(1993),和Poljak R.J.等人,Structure 2:1121-1123(1994))。
上述各个抗体片段均保持了特异性结合全长抗体所结合的相同抗原的能力,和/或与全长抗体竞争对抗原的特异性结合。
可使用本领域技术人员已知的常规技术(例如,重组DNA技术或酶促或化学断裂法)从给定的抗体(例如本发明提供的抗体)获得抗体的抗原结合片段(例如,上述抗体片段),并且以与用于完整抗体的方式相同的方式就特异性筛选抗体的抗原结合片段。
在本文中,除非上下文明确指出,否则当提及术语“抗体”时,其不仅包括完整抗体,而且包括抗体的抗原结合片段。
如本文中所使用的,术语“单克隆抗体”、“单抗”、“mAb”具有相同的含义且可互换使用可互换,其是指,来自一群高度同源的抗体分子中的一个抗体或抗体的一个片段,也即,除可能自发出现的自然突变外,一群完全相同的抗体分子。单抗对抗原上的单一表位具有高特异性。多克隆抗体是相对于单克隆抗体而言的,其通常包含至少2种或更多种的不同抗体,这些不同的抗体通常识别抗原上的不同表位。此外,修饰语“单克隆”仅表明该抗体的特征为从高度同源的抗体群中获得,不能理解为需要通过任何特定方法来制备所述抗体。
如本文中所使用的,术语“嵌合抗体(Chimeric antibody)”是指,这样的抗体,其轻链或/和重链的一部分源自一个抗体(其可以源自某一特定物种或属于某一特定抗体类或亚类),且轻链或/和重链的另一部分源自另一个抗体(其可以源自相同或不同的物种或属于相同或不同的抗体类或亚类),但无论如何,其仍保留对目标抗原的结合活性(U.S.P4,816,567to Cabilly et al.;Morrison et al.,Proc.Natl.Acad.Sci.USA,81:68516855(1984))。例如,术语“嵌合抗体”可包括这样的抗体(例如人鼠嵌合抗体),其中抗体的重链和轻链可变区来自第一抗体(例如鼠源抗体),而抗体的重链和轻链恒定区来自第二抗体(例如人抗体)。为制备嵌合抗体,可使用本领域已知的方法将免疫动物的免疫球蛋白可变区连接至人免疫球蛋白恒定区(参见例如Cabilly等人的美国专利No.4,816,567)。例如,将编码VH的DNA可操作的连接至编码重链恒定区的另一DNA分子以获得全长重链基因。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 ofProteins of Immunological Interest,Fifth Edition,U.S.Department of Health andHuman Services,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。重链恒定区可以是IgG1、IgG2、IgG3、IgG4、IgA、IgE、IgM或IgD恒定区,但是通常优选为IgG1或IgG4恒定区。例如,将编码VL的DNA可操作的连接至编码轻链恒定区CL的另一DNA分子以获得全长轻链基因(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 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。轻链恒定区可以是κ或λ恒定区,但通常优选为κ恒定区。
如本文中所使用的,术语“人源化抗体”是指,经基因工程改造的非人源抗体,其氨基酸序列经修饰以提高与人源抗体的序列的同源性。通常而言,人源化抗体的全部或部分CDR区来自于非人源抗体(供体抗体),全部或部分的非CDR区(例如,可变区FR和/或恒定区)来自于人源免疫球蛋白(受体抗体)。在某些实施方案中,人源化抗体的CDR区来自于非人源抗体(供体抗体),全部或部分的非CDR区(例如,可变区FR和/或恒定区)来自于人源免疫球蛋白(受体抗体)。人源化抗体通常保留了供体抗体的预期性质,包括但不限于,抗原特异性、亲和性、反应性等。供体抗体可以是有预期性质(例如,抗原特异性、亲和性、反应性等)的小鼠、大鼠、兔或非人灵长类动物(例如,食蟹猴)抗体。为制备人源化抗体,可以使用本领域已知的方法将免疫动物的CDR区插入人源框架序列(参见Winter的美国专利No.5,225,539;Queen等人的美国专利Nos.5,530,101;5,585,089;5,693,762和6,180,370;以及Lo,Benny,K.C.,editor,in Antibody Engineering:Methods and Protocols,volume 248,Humana Press,New Jersey,2004)。
如本文中所使用的,术语“胚系抗体基因(germline antibody gene)”或“胚系抗体基因片段(germline antibody gene segment)”是指,存在于生物体的基因组中的编码免疫球蛋白的序列,其没有经历过能够导致表达特异性免疫球蛋白的遗传学重排及突变的成熟过程。在本发明中,表述“重链胚系基因”是指,编码免疫球蛋白重链的胚系抗体基因或基因片段,其包括V基因(variable)、D基因(diversity)、J基因(joining)和C基因(constant);类似地,表述“轻链胚系基因”是指,编码免疫球蛋白轻链的胚系抗体基因或基因片段,其包括V基因(variable)、J基因(joining)和C基因(constant)。在本发明中,由所述胚系抗体基因或胚系抗体基因片段所编码的氨基酸序列也称为“胚系序列(germlinesequence)”。胚系抗体基因或胚系抗体基因片段及其相应的胚系序列是本领域技术人员熟知的,并且可从专业数据库(例如,IMGT、UNSWIg、NCBI或VBASE2)获得或查询。
如本文中所使用的,术语“特异性结合”是指,两分子间的非随机的结合反应,如抗体和其所针对的抗原之间的反应。特异性结合相互作用的强度或亲和力可以该相互作用的平衡解离常数(KD)表示。在本发明中,术语“KD”是指特定抗体-抗原相互作用的解离平衡常数,其用于描述抗体与抗原之间的结合亲和力。平衡解离常数越小,抗体-抗原结合越紧密,抗体与抗原之间的亲和力越高。两分子间的特异性结合性质可使用本领域公知的方法进行测定,例如使用表面等离子体共振术(SPR)在BIACORE仪中测定。
如本文中所使用的,表述“在中性pH下比在酸性pH下更高的亲和力结合”或等价表述“pH依赖性的结合”是指本发明的抗体在酸性pH下结合HBsAg的KD值或EC50值高于其在中性pH下结合HBsAg的KD值或EC50值。所述KD可以通过领域里公知的技术测得,例如通过SPR技术(如Biacore)测得。在本发明中,术语“EC50”是指抗体-抗原半数最大效应浓度,即特定抗体-抗原之间达到最大结合效应的50%所需要的抗体浓度,其用于描述抗体与抗原之间的结合能力。EC50越小,抗体与抗原之间的结合能力越高。抗体-抗原半数最大效应浓度(EC50)可使用本领域公知的方法进行测定,例如使用以抗原结合固相载体,抗体与抗原特异性结合的酶联免疫吸附测定法(ELISA)进行测定。
如本文中所使用的,“中和抗体”是指,能够显著降低或完全抑制目标病毒的毒力(例如,感染细胞的能力)的抗体或其抗原结合片段。通常而言,中和抗体能够识别并结合目标病毒,并且阻止目标病毒进入/感染受试者的细胞。本发明的抗体即是中和抗体。
然而,应当理解的是,在本申请中,抗体的中和病毒的能力并不直接等同于抗体的清除病毒的能力。如本文中所使用的,“中和病毒”是指,通过抑制目标病毒进入/感染受试者的细胞的过程,从而中和目标病毒的毒力(即,显著降低或完全抑制目标病毒的毒力)。如本文中所使用的,“清除病毒”是指,机体内的目标病毒(无论其是否感染了细胞)被从机体中消除,从而机体朝向未被病毒感染之前的状态转变(例如,病毒的血清学检测结果转变为阴性)。因此,在一般情况下,中和抗体并不一定具备清除病毒的能力。然而,在本申请中,发明人出人意料地发现,本发明的抗体不仅具有中和HBV的能力,而且具有清除病毒的能力(即,能够清除体内的HBV DNA和/或HBsAg,清除体内的HBV和被HBV感染的细胞),从而具有重大的临床价值。
如本文中所使用的,术语“分离的”是指,经人工手段从天然状态下获得。如果自然界中出现某一种“被分离”的物质或成分,那么可能是其所处的天然环境发生了改变或从天然环境下离分出该物质,或二者情况均有发生。比如,对于某一活体动物体内天然存在的某种未被分离的多聚核苷酸或多肽而言,从这种天然状态下分离出来的高纯度的相同的多聚核苷酸或多肽即称之为“分离的”。术语“分离的”不排除人工或合成的物质的存在,也不排除不影响物质活性的其它不纯物质的存在。
如本文中所使用的,术语“载体(vector)”是指,可将多聚核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸编码的蛋白获得表达时,载体称为表达载体。载体可以通过转化,转导或者转染导入宿主细胞,使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的,包括但不限于:质粒;噬菌粒;柯斯质粒;人工染色体,例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC);噬菌体如λ噬菌体或M13噬菌体及动物病毒等。可用作载体的动物病毒包括但不限于,逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。一种载体可以含有多种控制表达的元件,包括但不限于,启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外,载体还可含有复制起始位点。
如本文中所使用的,术语“宿主细胞”是指,可用于导入载体的细胞,其包括但不限于,如大肠杆菌或枯草菌等的原核细胞,如酵母细胞或曲霉菌等的真菌细胞,如S2果蝇细胞或Sf9等的昆虫细胞,或者如纤维原细胞,CHO细胞,COS细胞,NSO细胞,HeLa细胞,BHK细胞,HEK 293细胞或人细胞等的动物细胞。
如本文中所使用的,术语“同一性”用于指两个多肽之间或两个核酸之间序列的匹配情况。当两个进行比较的序列中的某个位置都被相同的碱基或氨基酸单体亚单元占据时(例如,两个DNA分子的每一个中的某个位置都被腺嘌呤占据,或两个多肽的每一个中的某个位置都被赖氨酸占据),那么各分子在该位置上是同一的。两个序列之间的“百分数同一性”是由这两个序列共有的匹配位置数目除以进行比较的位置数目×100的函数。例如,如果两个序列的10个位置中有6个匹配,那么这两个序列具有60%的同一性。例如,DNA序列CTGACT和CAGGTT共有50%的同一性(总共6个位置中有3个位置匹配)。通常,在将两个序列比对以产生最大同一性时进行比较。这样的比对可通过使用,例如,可通过计算机程序例如Align程序(DNAstar,Inc.)方便地进行的Needleman等人(1970)J.Mol.Biol.48:443-453的方法来实现。还可使用已整合入ALIGN程序(版本2.0)的E.Meyers和W.Miller(Comput.ApplBiosci.,4:11-17(1988))的算法,使用PAM120权重残基表(weight residue table)、12的缺口长度罚分和4的缺口罚分来测定两个氨基酸序列之间的百分数同一性。此外,可使用已整合入GCG软件包(可在www.gcg.com上获得)的GAP程序中的Needleman和Wunsch(J MoIBiol.48:444-453(1970))算法,使用Blossum 62矩阵或PAM250矩阵以及16、14、12、10、8、6或4的缺口权重(gap weight)和1、2、3、4、5或6的长度权重来测定两个氨基酸序列之间的百分数同一性。
本文涉及的二十个常规氨基酸的编写遵循常规用法。参见例如,Immunology-ASynthesis(2nd Edition,E.S.Golub and D.R.Gren,Eds.,Sinauer Associates,Sunderland,Mass.(1991)),其以引用的方式并入本文中。在本发明中,术语“多肽”和“蛋白质”具有相同的含义且可互换使用。并且在本发明中,氨基酸通常用本领域公知的单字母和三字母缩写来表示。例如,丙氨酸可用A或Ala表示。此外,如本文中所使用的,术语“单克隆抗体”和“单抗”具有相同的含义且可互换使用;术语“多克隆抗体”和“多抗”具有相同的含义且可互换使用。
如本文中所使用的,术语“药学上可接受的载体和/或赋形剂”是指在药理学和/或生理学上与受试者和活性成分相容的载体和/或赋形剂,其是本领域公知的(参见例如Remington's Pharmaceutical Sciences.Edited by Gennaro AR,19thed.Pennsylvania:Mack Publishing Company,1995),并且包括但不限于:pH调节剂,表面活性剂,佐剂,离子强度增强剂,稀释剂,维持渗透压的试剂,延迟吸收的试剂,防腐剂。例如,pH调节剂包括但不限于磷酸盐缓冲液。表面活性剂包括但不限于阳离子,阴离子或者非离子型表面活性剂,例如Tween-80。离子强度增强剂包括但不限于氯化钠。防腐剂包括但不限于各种抗细菌试剂和抗真菌试剂,例如对羟苯甲酸酯,三氯叔丁醇,苯酚,山梨酸等。维持渗透压的试剂包括但不限于糖、NaCl及其类似物。延迟吸收的试剂包括但不限于单硬脂酸盐和明胶。
如本文中所使用的,术语“预防”是指,为了阻止或延迟疾病或病症或症状(例如,HBV感染或与HBV感染相关的疾病)在受试者体内的发生而实施的方法。如本文中所使用的,术语“治疗”是指,为了获得有益或所需临床结果而实施的方法。为了本发明的目的,有益或所需的临床结果包括(但不限于)减轻症状、缩小疾病的范围、稳定(即,不再恶化)疾病的状态,延迟或减缓疾病的发展、改善或减轻疾病的状态、和缓解症状(无论部分或全部),无论是可检测或是不可检测的。此外,“治疗”还可以指,与期望的存活期相比(如果未接受治疗),延长存活期。在本申请中,本发明的抗体具有中和HBV的能力,从而可用于预防/阻止未患病受试者或其细胞感染HBV。此外,本发明的抗体具有清除HBV的能力(即,能够清除体内的HBV DNA和/或HBsAg,清除体内的HBV和被HBV感染的细胞),从而可用于治疗患病受试者的HBV感染或与HBV感染相关的疾病。
如本文中使用的,术语“受试者”是指哺乳动物,例如灵长类哺乳动物,例如人。
如本文中所使用的,术语“有效量”是指足以获得或至少部分获得期望的效果的量。例如,预防疾病(例如HBV感染或与HBV感染相关的疾病)有效量是指,足以预防,阻止,或延迟疾病(例如HBV感染或与HBV感染相关的疾病)的发生的量;治疗疾病有效量是指,足以治愈或至少部分阻止已患有疾病的患者的疾病和其并发症的量。测定这样的有效量完全在本领域技术人员的能力范围之内。例如,对于治疗用途有效的量将取决于待治疗的疾病的严重度、患者自己的免疫系统的总体状态、患者的一般情况例如年龄,体重和性别,药物的施用方式,以及同时施用的其他治疗等等。
发明的有益效果
本发明的抗体不仅能够特异性识别/结合HBsAg,能够中和HBV的毒力,能够在受试者体内降低HBV DNA和/或HBsAg的血清水平,能够有效清除体内的HBV和被HBV感染的细胞,而且具有显著加强的抗原清除效果和抗原抑制时间。尤其令人意外的是,本领域已知慢性乙肝患者由于体内高水平的HBsAg往往产生针对HBV的免疫耗竭(耐受)进而使感染迁延不愈,而本发明的抗体能够激活受试者(例如慢性HBV感染者,或慢性乙肝患者)重新产生针对HBV的体液免疫应答,进而提高临床治愈率。因此,本发明的抗体特别适用于预防和治疗HBV感染以及与HBV感染相关的疾病(例如乙肝)。此外,本发明的抗体具有pH依赖抗原结合特性,能够一分子抗体结合多分子抗原,因此本发明的抗体还能够降低给药频率和剂量,具有重大的临床价值。
下面将结合附图和实施例对本发明的实施方案进行详细描述,但是本领域技术人员将理解,下列附图和实施例仅用于说明本发明,而不是对本发明的范围的限定。根据附图和优选实施方案的下列详细描述,本发明的各种目的和有利方面对于本领域技术人员来说将变得显然。
附图说明
图1显示了具备pH依赖性抗原结合活性的抗体的工作原理示意图:人血浆为中性,pH 7.4左右,而胞内环境为酸性,pH 6.0左右,具备pH依赖性抗原结合活性的抗体能在血浆中结合抗原,抗原抗体复合物被内化进细胞后,在内涵体酸性的环境下,该pH依赖性抗体会与抗原解离,解离抗原的抗体被FcRn捕获循环到胞外,在胞外中性的环境下,FcRn将抗体释放,回到血浆中的抗体可以再结合别的抗原,实现抗体的循环使用。
图2显示了162解析的Fab晶体结构与抗原模拟短肽对接的结果,其中蓝色结构为抗原短肽,红色结构为部分162抗体结合区域。
图3显示了编码scFv抗体的重组载体(pCGMT-scFv)的示意图,其中scFv抗体的结构为:NH2-VH-linker-VL-COOH。
图4A-4D显示了展示衍生自162的pH依赖性scFv抗体的噬菌体文库与抗原HBsAg的ELISA检测结果。其中,图4A:衍生自162的噬菌体文库经第三轮筛选后,与HBsAg在pH7.4和pH6.0下的结合检测结果,横坐标表示噬菌体抗体编号,纵坐标表示OD值,结果显示,这些单克隆都具有较强的抗原结合活性且在pH 6.0下结合活性的显著的下降。图4B:第三轮中pH7.4下OD(450/630)值高,同时与pH 6.0下OD(450/630)值差异最大的13个单克隆进行8梯度,3倍稀释后与HBsAg的pH依赖结合的检测结果,其中横坐标代表稀释倍数,纵坐标表示OD值,结果显示,经过梯度稀释后,pH依赖抗原结合的效果有更好的呈现,其中C32、C27、C26和C19表现最好,其中C27分子效果最好(其余9个未展示)。图4C:衍生自162的噬菌体文库经第四轮筛选后,与HBsAg在pH7.4和pH6.0下的结合检测结果,横坐标表示噬菌体抗体编号,纵坐标表示OD值。图4D:第四轮中pH 7.4下OD(450/630)值高,同时与pH 6.0下OD(450/630)值差异最大的8个单克隆进行8梯度,3倍稀释后与HBsAg的pH依赖结合的检测结果,其中横坐标代表稀释倍数,纵坐标表示OD值,结果显示,经过梯度稀释后,pH依赖抗原结合的效果有更好的呈现,其中D3、D4和D5表现最好,其中D5分子效果最好(其余5个未展示)。
图5显示了C26,C27,C32,D3,D4和D5的突变位点汇总。
图6A显示了实施例3中C32、C27、C26真核小量转染后,细胞上清经定量后与HBsAg在pH7.4和pH6.0下的结合检测结果,图6B显示了实施例3中D3、D4、D5真核小量转染后,细胞上清经定量后与HBsAg在pH7.4和pH6.0下的结合检测结果。其中,横坐标表示抗体浓度(Log10 ng/ml),纵坐标表示OD值。结果显示C32、C27、C26、D3、D4和D5均能维持亲本抗体162中性pH下相当的抗原结合活性,且均在pH 6.0的条件下,与抗原的结合活性有明显的下降。
图7显示了清道夫抗体的工作原理图。pH依赖抗原结合活性发挥作用在胞内,若入胞这一第一限制因素未被打破,后续pH依赖抗原结合特性就无“施展”机会,改造效果将大打折扣。通过Fc区段的氨基酸的进一步突变,获得清道夫抗体,能够加强中性pH下与hFcRn受体的结合,或者加强与FcγRs受体的结合,使其铆定在细胞外,扮演一个往复运送抗原入胞的“帮运工”的角色,从而使抗体半衰期得到突破性的延长,同时可以再次结合抗原,提高抗原入胞效率,使清除效率得到极大提高。
图8A-8B显示了pH依赖性抗体及DY改造抗体的蛋白胶结果。图8A:pH依赖性抗体的蛋白胶图,162为阳性对照。结果显示表达的C26、D3、D4、D5抗体成分单一。图8B:DY改造抗体的蛋白胶图,162为阳性对照。结果显示表达的C26 DY、D3 DY、D4 DY和D5 DY抗体成分单一。
图9A-9D显示了实施例4中pH依赖性抗体及DY改造抗体与HBsAg在pH7.4和pH6.0下的结合检测结果,其中,横坐标表示抗体浓度(Log10ng/ml),纵坐标表示OD值。结果显示,D26、D3、D4、D5能维持亲本(162)中性pH下相当的抗原结合活性,且在pH 6.0的条件下,与抗原的结合活性有明显的下降,相应的DY改造抗体也能维持亲本中性pH下相当的抗原结合活性以及pH依赖抗原结合活性。
图10A显示了实施例4中pH依赖性抗体及DY改造抗体的鼠原代巨噬细胞的免疫荧光实验,其中绿色荧光代表hFcRn,蓝色荧光代表细胞核,红色荧光代表HBsAg。结果显示DY改造增强了鼠巨噬细胞对抗原抗体复合物的吞噬。图10B显示了实施例4中pH依赖性抗体及DY改造抗体的基于人THP-1吞噬细胞的吞噬实验。结果显示,DY改造增强了人THP-1吞噬细胞对抗原抗体复合物的吞噬。
图11A-11B显示了实施例4中C26 DY清道夫抗体及162,以单针尾静脉5mg/kg的剂量注射后,HBV转基因鼠的治疗效果,图11C显示了实施例4中D3 DY、D4 DY和D5 DY,以单针尾静脉5mg/kg的剂量注射后,HBV转基因鼠的治疗效果。其中,图11A:横坐标表示注射抗体后的天数(d),纵坐标表示小鼠血清中HBsAg的清除后水平(log10 IU/ml)。图11B:小鼠血清中抗体的浓度的变化情况,其中横坐标表示注射抗体后的天数(d),纵坐标表示抗体浓度(ng/ml)。图11C:横坐标表示注射抗体后的天数(d),纵坐标表示小鼠血清中HBsAg的清除后水平(log10 IU/ml)。结果显示,DY改造的清道夫抗体C26 DY、D3 DY、D4 DY和D5 DY在抗原清除能力上比162强一个数量级以上。说明在5mg/kg的低的注射剂量下,DY改造的清道夫抗体C26 DY、D3 DY、D4 DY和D5 DY发挥了循环往复结合抗原的功能,加强了抗原清除的效果。
序列信息
本发明涉及的部分序列的信息提供于下面的表1中。
表1:序列的描述
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具体实施方式
现参照下列意在举例说明本发明(而非限定本发明)的实施例来描述本发明。
除非特别指明,本发明中所使用的分子生物学实验方法和免疫检测法,基本上参照J.Sambrook等人,分子克隆:实验室手册,第2版,冷泉港实验室出版社,1989,以及F.M.Ausubel等人,精编分子生物学实验指南,第3版,John Wiley&Sons,Inc.,1995中所述的方法进行;限制性内切酶的使用依照产品制造商推荐的条件。本领域技术人员知晓,实施例以举例方式描述本发明,且不意欲限制本发明所要求保护的范围。
实施例1:pH依赖性抗HBsAg抗体的噬菌体筛选
1.1pH依赖抗体改造突变位点的确定
使用实验室前期研发的抗HBV的人源化抗体162(详细描述于中国专利申请201610879693.5)为亲本抗体,对其可变区进行pH依赖抗原结合改造,如图1所示,改造后的162能保持中性条件下的抗原结合活性而酸性条件下的抗原结合活性大幅降低,解离的改造后的162,通过和胞内的FcRn结合,从而可以返回到血浆中,再次与抗原结合,达到一分子pH依赖抗原结合改造后的162可以反复结合,中和多分子抗原。组氨酸在酸性条件下会发生质子化,是带来pH依赖抗原结合特性的关键氨基酸。162Fab已有解析的晶体结构,将解析的晶体结构与抗原短肽进行模拟对接,部分结果示于图2,蓝色结构为抗原短肽,红色结构为部分162抗体结合区域。根据对接结果,找到抗原抗体相互结合的关键氨基酸共14个,考虑到模拟对接结果提供的参考性更大,选择交界面上氨基酸及两侧氨基酸进行突变,确定位点为26个。
1.2衍生自162的pH依赖性scFv抗体的噬菌体文库构建
将162抗体轻重链可变区为模板,对抗体可变区CDR中最终确定的pH依赖改造突变位点,按表2中的引物,对目的片段进行扩增,获得编码衍生自162的pH依赖抗原结合改造的scFv抗体的基因片段。PCR条件为:95℃,5min;95℃,30s;57℃,30s;72℃,30s;72℃,10min;扩增循环数为25个;SOE-PCR反应条件为:95℃,5min;95℃,30s;57℃,30s;72℃,30s;72℃,10min;扩增循环数为5个;通过琼脂糖凝胶电泳分析扩增产物,并用DNA纯化回收试剂盒(TianGen,DP214-03)回收/纯化扩增产物,从而获得编码衍生自162的人源化scFv抗体的基因片段H-K。scFv抗体的结构为:NH2-VH-linker-VL-COOH,其中linker的序列可以为(G4S)3。将各个基因片段H-K用SfiI酶切,然后按照10∶1(基因片段:载体)的摩尔比分别与载体pCGMT(来自Scripps,Making chemistry selectable by linking it to infectivity)进行连接。通过电穿孔法(电转条件:25μF、2.5KV、200Ω),将连接产物转化入感受态大肠杆菌ER2738。经转化的大肠杆菌在SOC培养基中恢复45min,然后取出200μL菌液用于涂布LB平板(含100g/L的氨苄青霉素+四环素+2g/mL葡萄糖),并在37℃静置培养过夜。板上的所有菌落为可变区确定的突变位点,随机突变成组氨酸的文库,用于后续的筛选。从平板上挑取单克隆菌落,并进行测序,以确保编码scFv抗体的重组载体的序列正确性。编码scFv抗体的重组载体(pCGMT-scFv)的示意图如图3所示。
表2:衍生自162的pH依赖性scFv抗体的突变引物
引物名称 | 引物序列 |
VH-F | SEQ ID NO:44 |
HCDR1-R | SEQ ID NO:45 |
HCDR2-F | SEQ ID NO:46 |
HCDR3-R | SEQ ID NO:47 |
VH-R | SEQ ID NO:48 |
VK-F | SEQ ID NO:49 |
KCDR1-R | SEQ ID NO:50 |
KCDR2-F | SEQ ID NO:51 |
KCDR3-R | SEQ ID NO:52 |
VK-R | SEQ ID NO:53 |
1.3人源化的scFv抗体的检测
将前一步骤获得的文库进行多轮筛选,将筛选中获得的阳性单克隆菌落用含氨苄青霉素(100g/L)和葡萄糖(2g/mL)的2×YT培养基培养至OD=0.6,然后添加M13KO7进行辅助超感染。2h后,添加100g/L的卡那霉素,并在37℃进行超感染。2h后,以4000rpm离心培养物10min,弃去上清,并收集细胞沉淀。将细胞沉淀用含氨苄青霉素和卡那霉素(100g/L)的双抗性培养基重悬,并在30℃振荡培养过夜。随后,以12000rpm离心培养物10min,收集菌体和上清,4℃保存用于检测。
取包被有HBsAg(200ng/mL)抗原的ELISA板,每孔加入100μL
待测上清,并在37℃温育1h(每个上清两孔)。随后,用PBST洗涤ELISA板1次,然后分别添加120μL pH 7.4和pH 6.0的PBS到每一上清的两个孔中37℃温育30min。分别用相应pH的PBST洗涤5遍,添加以1∶5000稀释的anti M13-HRP 100μL,并在37℃温育30min。随后,用PBST洗涤ELISA板5次,并添加底物TMB溶液。显色15min后,用H2SO4终止显色反应,并于OD450/630测定读值。第三轮ELISA检测结果如图4A-4D所示。结果显示,展示这些scFv抗体的噬菌体均具有ELISA检测反应活性且在pH 6.0下与抗原有弱的结合;初步获得6株效果良好的pH依赖性噬菌体抗体,分别命名为C-26、C-27、C-32、D3、D4、D5。
实施例2:pH依赖性抗HBsAg抗体的制备
2.1用于真核表达的重组载体的构建
本发明中,需要进行大量的抗体重组,因此需要构建一套可以高效重组抗体的轻重链载体。本发明对实验室现有的真核表达载体pTT5进行了特殊改造,构建一套供双质粒共转染用轻重链重组载体。轻重链信号肽采用MGWSCIILFLVATATGVHS(SEQ ID NO:54)。信号肽下游分别接入编码人抗体轻重链恒定区的序列,构建成一套方便抗体重组的pTT5-CH,pTT5-Cκ和pTT5-Cλ真核表达载体。
将1.3中得到的6株scFv抗体,用表3中的引物对轻重链可变区片段进行扩增。具体扩增反应条件为:95℃,5min;95℃,30s;57℃,30s;72℃,30s;72℃,10min;扩增循环数为25个,扩增后产物进行切胶回收。
利用实验室自制的Gibson装配液将上述构建好的真核表达载体与回收到的抗体可变区基因PCR产物连接(引物带有与载体同源序列),得到重组载体VH+pTT5-CH(包含SEQID NO:40所示的CH)和VH+pTT5-Cκ(包含SEQ ID NO:41所示的CL)。将重组载体转化入到大肠杆菌DH5α菌株,涂布LB平板,并在37℃培养箱培养过夜。从平板上挑取单克隆菌落,并进行测序,测序结果使用MEGA对序列进行比对,以确认其基因正确性,排除假基因。
表3:真核表达载体构建的引物
引物名称 | 引物序列 |
VH-F | SEQ ID NO:55 |
VH-R | SEQ ID NO:56 |
VK-F | SEQ ID NO:57 |
VK-R | SEQ ID NO:58 |
2.2抗体基因的小量及大量表达
将构建好的重组载体VH+pTT5-CH和VH+pTT5-Cκ共转染HEK293-细胞,小量表达双质粒共转染至每孔500μL的24孔板中,如小量表达的细胞上清具有抗原活性,将转染体系放大到100mL(根据抗体用量决定)的FreeStyleTM 293F悬浮细胞(细胞密度为约2×106cells/ml)。转染后的细胞在32℃,5%CO2培养箱摇瓶培养,表达7天后收上清。
2.3抗体纯化
收集细胞表达上清,根据制造商的说明书,用Protein A层析柱进行纯化。具体步骤如下:收获的细胞培养上清,8000rpm,离心10min,保留上清,利用干粉Na2HPO4调节其PH值为8.4,然后用0.22μm孔径滤膜过滤。取偶联上Protein A的Sepharose 4B介质10mL装柱,连接至AKTA Explorer100系统,将A泵接入0.2M磷酸氢二钠溶液,B泵接入0.2M柠檬酸溶液。选取检测波长UV 280nm,流速5mL/min,调节A/B泵进样比例,先取100%B(pH 2.3)冲洗柱子出去杂蛋白,取10%B(pH 8.0)平衡pH,待检测波长稳定后归零,上样,待穿透峰过后取10%B继续平衡至检测波长降至零点并稳定后,用70%B(pH 4.0)洗脱,收取洗脱峰。将洗脱峰样品透析至PBS缓冲液中,并测定浓度及SDS-PAGE或HPLC分析,以确定IgG抗体的纯度。
实施例3:pH依赖性抗HBsAg抗体的性质分析及功能评估
通过实施例1的方法,初步筛选获得了6株pH依赖性结合HBsAg的噬菌体抗体,分别命名为C26,C27,C32,D3,D4和D5。进一步,通过实施例2的方法对6株噬菌体抗体进行了小量真核表达及纯化。6株抗体的VH和VL氨基酸序列如下表4所示。另外,还确定了6株抗体的CDR序列,其重链可变区和轻链可变区的CDR的氨基酸序列如表5所示。将赋予C26,C27,C32,D3,D4和D5与HBsAg pH依赖抗原结合特性的突变位点进行汇总,汇总结果示于图5中。
表4:C26/C27/C32/D3/D4/D5轻重链可变区氨基酸序列
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表5:C26/C27/C32/D3/D4/D5轻重链CDR的序列
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发明人对6株单克隆抗体先进行小量转染,对转染上清定量后通过ELISA法分别检测了与HBsAg的pH依赖抗原结合能力,将抗体的浓度统一稀释到1111.11ng/mL。随后,用20%NBS进行抗体浓度梯度稀释3倍浓度梯度稀释,共稀释8个浓度梯度。随后,将稀释好的抗体加入商业化的HBsAg板中(购于北京万泰),并在37℃孵育1h(每个上清两孔)。随后,用PBST洗涤ELISA板1次,甩干。然后分别添加120μL pH 7.4和pH 6.0的PBS到每一上清的两个孔中37℃温育30min。分别用相应pH的PBST洗涤5遍,甩干。随后,加入GAH-HRP酶标二抗,孵育30min,用PBST洗板5次,甩干。并添加底物TMB溶液。显色15min后,用H2SO4终止显色反应,并于OD450/630测定读值。
结果示于图6A-6B,结果可以看出,候选分子均能维持亲本抗体162中性pH下相当的抗原结合活性,且均在pH 6.0的条件下,与抗原的结合活性有明显的下降。EC50汇总于表6。
表6:C26,C27,C32,D3,D4和D5 pH依赖活性检测的EC50
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实施例4:清道夫抗体的构建及功能评估
pH依赖性抗体需要进入胞内发挥其pH依赖抗原结合活性,因此若入胞这一第一限制因素未被打破,后续pH依赖抗原结合特性就无“施展”机会。因此,本实施例通过对Fc区段的氨基酸的进一步突变,获得清道夫抗体,能够加强中性pH下与hFcRn受体的结合,或者加强与FcγRs受体的结合,如图7显示,使其铆定在细胞外,扮演一个往复运送抗原入胞的“帮运工”的角色,从而使抗体半衰期得到突破性的延长,同时可以再次结合抗原,提高抗原入胞效率,使清除效率得到极大提高。
选用C26、D3、D4和D5为后续评估抗体,并对其进行Fc的DY(K326D、L328Y)突变,增强中性条件下与mFcγRII的亲和力(其中C26Fc改造工作委托通用生物公司,订单号G122413),获得结合mFcγRII的清道夫抗体C26 DY、D3 DY、D4 DY和D5 DY,将上述抗体进行大量真核表达纯化,具体操作步骤见实施例1.2和1.3。
图8A-8B显示了原始抗体与改造抗体的蛋白胶结果。图8A:原始抗体的蛋白胶图,162为阳性对照。结果显示表达的原始抗体成分单一。图8B:DY改造抗体的蛋白胶图,162为阳性对照。结果显示表达的DY改造抗体成分单一。
4.1结合mFcγRII的清道夫抗体的pH依赖抗原结合能力活性评价
发明人对表达纯化后的原始抗体和DY改造抗体,通过ELISA法分别检测了与HBsAg的pH依赖抗原结合能力。首先,用BCA蛋白定量试剂盒测定纯化后的抗体的浓度,并将抗体的浓度统一稀释到1111.11ng/mL。随后,用20%NBS进行抗体浓度梯度稀释3倍浓度梯度稀释,共稀释8个浓度梯度。随后,将稀释好的抗体加入商业化的HBsAg板中(购于北京万泰),并在37℃孵育1h(每个上清两孔)。随后,用PBST洗涤ELISA板1次,甩干。然后分别添加120μLpH 7.4和pH 6.0的PBS到每一上清的两个孔中37℃温育30min。分别用相应pH的PBST洗涤5遍,甩干。随后,加入GAH-HRP酶标二抗,孵育30min,用PBST洗板5次,甩干。并添加底物TMB溶液。显色15min后,用H2SO4终止显色反应,并于OD450/630测定读值。
结果如图9A-9D所示,C26、D3、D4、D5及其DY改造抗体均具有与162相当的抗原结合活性,但在pH 6.0下与抗原弱结合,具有良好的pH依赖抗原结合活性。
4.2结合mFcγRII的清道夫抗体细胞水平的功能验证
4.2.1HBsAg标记488荧光
以标记1mg HBsAg为例,全程避光操作。
(1)1mL,1mg/mL的HBsAg透析到硼酸盐缓冲液中(PH 8.5,500mL),4℃,4h;
(2)HBsAg与488标记的摩尔比为1:5,经计算需要488荧光0.1988mg;
(3)用DMF配置10mg/mL的488荧光液,混合均匀;
(4)19.88μL,488荧光加入到透析后的1mL,HBsAg中,混合均匀,室温孵育1h;
(5)将孵育后混合物透析至PBS中,4℃,过夜。
4.2.2基于鼠原代巨噬细胞的免疫荧光实验
(1)实验前4天,向每只鼠腹腔内注入1.5mL 3%的硫代乙醇酸钠溶液,勿注入肠内;
(2)将2只小鼠处死,浸泡75%酒精3min;
(3)将鼠仰卧式固定于泡沫板上,暴露出腹部;用组织剪剪开腹部皮肤,消毒腹膜后,切开并进入腹腔,左手持两把有齿钳拉开腹部切口皮肤并固定,右手持巴氏吸管吸取1640培养液,灌洗腹腔,4mL/次,共两次,用吸管轻柔又充分的搅拌腹腔,使灌洗更加充分彻底,充分搅拌约2min后,静置约5min,使巨噬细胞充分游离出来,然后吸取灌洗液于离心管中;
(4)4℃,1100g,5min;
(5)小心弃除上清,1640培养基洗涤细胞2次,4℃,1100g,5min弃上清,RPM1640重悬细胞;
(6)细胞计数后,调整成106个/mL密度的细胞,250μL/孔培养于24孔玻璃底细胞成像培养板,2h后换液,并用RPM1640洗涤一次,弃去未贴壁的细胞后,37℃、CO2培养箱培养过夜;
(7)将标记相应荧光的抗体和抗原用无血清培养基稀释成:抗原800ng/mL、抗体20μg/mL;
(8)抗原和抗体各125μL混合均匀后,37℃、CO2培养箱静置1h;
(9)弃除细胞成像培养板中的细胞上清,加入抗原抗体复合物,稍微晃匀后,37℃、CO2培养箱静置2h;
(10)弃除上清,用1mL提前37℃温育的无菌PBS,“刷洗”细胞面3~5次,用移液枪吸除干净;
(11)1:2000稀释Dio,加入没过细胞的量,室温静置20min;
(12)弃除上清,用1mL提前37℃温育的无菌PBS,“刷洗”细胞面3~5次,用移液枪吸除干净;
(13)加入活细胞核染料(1mL体积加2滴),室温静置20min,置于高内涵成像仪中成像。
实验结果示于图10A。从结果可以看出,DY改造增强了鼠巨噬细胞对抗原抗体复合物的吞噬,带来更多抗原的降解。
4.2.3基于人THP-1吞噬细胞的化学发光法验证
(1)贴壁THP-1细胞以2×105/孔铺板,加入含有10%血清的1640培养基中,放置于37℃二氧化碳培养箱中培养24h;
(2)以无血清1640培养基将HBsAg稀释是800ng/mL,且待测抗体以20ug/mL为初始浓度,2倍梯度稀释,共11个梯度。各取300uL稀释好的HBsAg和待测抗体,1:1混合,37℃放置1h;
(3)弃除THP-1细胞上清,取250uL的HBsAg-抗体混合液加入THP-1细胞中,放置于37℃二氧化碳培养箱反应1h;
(4)弃除THP-1细胞上清,用无菌PBS洗涤3遍;
(5)每孔THP-1细胞中加入120uLDDM细胞裂解液,放置于4℃反应1h;
(6)取裂解液上清,用乙肝表面抗原定量检测试剂盒(北京万泰)检测HBsAg浓度
实验结果示于图10B。从结果可以看出,DY改造增强了人THP-1吞噬细胞对抗原抗体复合物的吞噬。
4.3结合mFcγRII的清道夫抗体在动物模型中的治疗效果测定
选用HBV转基因鼠为动物模型。选用6~8周龄的HBV转基因鼠,将C26 DY、D3 DY、D4DY和D5 DY清道夫抗体及162,以单针尾静脉5mg/kg的剂量注射(每组4只)。通过检测血清中HBsAg、抗体浓度及HBV DNA的浓度,分析清道夫抗体在体内的抗原清除速率和抗体半衰期的情况。
HBsAg的定量检测
(1)反应板的制备:将小鼠单克隆抗体HBs-45E9用20mM PB缓冲液(Na2HPO4/NaH2PO4缓冲液,pH 7.4)稀释到2μg/mL,在化学发光板中每孔添加100μL的包被液,并在2-8℃下包被16-24h,随后再37℃包被2h,用PBST洗涤液洗涤平板一次,甩干。洗涤后,在各孔中加入200μL的封闭液,并在37℃封闭2h。随后,弃去封闭液,将平板放入干燥间干燥,于2-8℃保存备用。
(2)样品稀释:将采集的小鼠血清用含20%NBS(新生牛血清)的PBS溶液,稀释成1:30和1:150两个梯度,用于后续的定量检测。
(3)样品变性处理:取15μL经上述稀释的血清样品与7.5μL变性缓冲液(15%SDS,溶于20mM PB7.4)充分混匀,并在37℃反应1h。随后,加入90μL的中和缓冲液(4%CHAPS,溶于20mM PB7.4),充分混匀。
(4)样品反应:在反应板中加入100μL经上述变性处理的血清样品,37℃反应1h。随后用PBST洗涤反应板5次,甩干。
(5)酶标记物反应:向化学发光板中按100μL/孔,加入HBs-A6A7-HRP反应液,并在37℃反应1h。随后用PBST洗涤平板5次,甩干。
(6)发光反应和测量:向化学发光板中加入发光液(100μL/孔),并进行光强检测。
(7)小鼠血清样品中的HBsAg浓度的计算:使用标准品进行平行实验,根据标准品的测定结果绘制标准曲线。然后,将小鼠血清样品的光强测量值代入标准曲线,计算出待检血清样品中的HBsAg浓度。
血清中HBsAg的检测情况结果示于图11A与图11C。从图11A可以看出,DY改造的清道夫抗体C26 DY在抗原清除能力上比162强一个数量级以上,这与血清中抗体半衰期的检测结果是一致的(图11B)。对比血清中抗体的浓度,C26 DY的半衰期较162延长了近12天的时间,这说明清道夫抗体C26 DY发挥了循环往复结合抗原的功能,进而增加了抗原清除的时间。图11C的实验结果显示D3 DY、D4 DY和D5 DY的抗原清除能力与C26 DY相当,且持续时间久于C26 DY,说明在5mg/kg的低的注射剂量下,DY改造的清道夫抗体D3 DY、D4 DY和D5DY具有更优的循环往复结合抗原的功能,更好的进行了抗原清除。
实施例5:162和C26的亲和力测定
以5μg/mL的HBsAg溶解于醋酸钠(pH 4.5)在Biacore 3000设备上运行包被芯片程序,将HBsAg包被至CM5芯片上,HBsAg的包被量为2400RU。将分析物从100nM,2倍梯度稀释,制备7个浓度的样品。在Biacore 3000设备上运行亲和力测定程序,设置流速为50μl/min,结合时间为90s,解离时间为600s,样品室的温度设置为10℃,再生液为50mM NaOH,再生流速为50μL/min,再生时间为60s。结果汇总于表7。
表7:162和C26的亲和力测定
尽管本发明的具体实施方式已经得到详细的描述,但本领域技术人员将理解:根据已经公布的所有教导,可以对细节进行各种修改和变动,并且这些改变均在本发明的保护范围之内。本发明的全部分为由所附权利要求及其任何等同物给出。
SEQUENCE LISTING
<110> 厦门大学
养生堂有限公司
<120> 抗乙肝病毒新型抗体及其用途
<130> IDC200207
<150> CN 201910432602.7
<151> 2019-05-23
<160> 58
<170> PatentIn version 3.5
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Ser Gln Asn Thr His Val Pro Tyr Thr
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His Gly Tyr His Trp Asn
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<211> 6
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Tyr Arg Tyr His Trp Asn
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Tyr Ile Asn Tyr Asp Gly Ser Val His Tyr Asn Pro Ser Leu Glu Asn
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<210> 23
<211> 9
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Ser Gln Asn Thr His Leu Pro Tyr Thr
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His Gly Tyr His Tyr Asn
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Arg Ser Ser Gln Ser Leu Val His Ser Tyr Gly Asp Asn Tyr Leu His
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<212> PRT
<213> 人工序列
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<221> MISC_FEATURE
<222> (1)..(1)
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<220>
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<221> MISC_FEATURE
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Xaa Xaa Tyr His Xaa Asn
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<212> PRT
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<223> HCDR2通式
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<221> MISC_FEATURE
<222> (3)..(3)
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<220>
<221> MISC_FEATURE
<222> (4)..(4)
<223> X = Y或H
<220>
<221> MISC_FEATURE
<222> (9)..(9)
<223> X = L、H或Q
<400> 27
Tyr Ile Xaa Xaa Asp Gly Ser Val Xaa Tyr Asn Pro Ser Leu Glu Asn
1 5 10 15
<210> 28
<211> 16
<212> PRT
<213> 人工序列
<220>
<223> LCDR1通式
<220>
<221> MISC_FEATURE
<222> (13)..(13)
<223> X = T或N
<400> 28
Arg Ser Ser Gln Ser Leu Val His Ser Tyr Gly Asp Xaa Tyr Leu His
1 5 10 15
<210> 29
<211> 9
<212> PRT
<213> 人工序列
<220>
<223> LCDR3通式
<220>
<221> MISC_FEATURE
<222> (6)..(6)
<223> X = V、L或H
<400> 29
Ser Gln Asn Thr His Xaa Pro Tyr Thr
1 5
<210> 30
<211> 30
<212> PRT
<213> 人工序列
<220>
<223> C26 C27 C32 D3 D4 D5 HFR1
<400> 30
Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Ser Ser Ile Thr
20 25 30
<210> 31
<211> 14
<212> PRT
<213> 人工序列
<220>
<223> C26 C27 C32 D3 D4 D5 HFR2
<400> 31
Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp Ile Gly
1 5 10
<210> 32
<211> 32
<212> PRT
<213> 人工序列
<220>
<223> C26 C27 C32 D3 D4 D5 HFR3
<400> 32
Arg Val Thr Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe Leu Lys
1 5 10 15
Leu Ser Ser Val Thr Ala Glu Asp Thr Ala Lys Tyr Tyr Cys Ala Ser
20 25 30
<210> 33
<211> 11
<212> PRT
<213> 人工序列
<220>
<223> C26 C27 C32 D3 D4 D5 HFR4
<400> 33
Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser
1 5 10
<210> 34
<211> 23
<212> PRT
<213> 人工序列
<220>
<223> C26 C27 C32 D3 D4 D5 LFR1
<400> 34
Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly
1 5 10 15
Glu Pro Ala Ser Ile Ser Cys
20
<210> 35
<211> 15
<212> PRT
<213> 人工序列
<220>
<223> C26 C27 C32 D3 D4 D5 LFR2
<400> 35
Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr
1 5 10 15
<210> 36
<211> 32
<212> PRT
<213> 人工序列
<220>
<223> C26 C27 C32 D3 D4 D5 LFR3
<400> 36
Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
1 5 10 15
Leu Lys Ile Ser Arg Val Glu Thr Glu Asp Leu Gly Val Tyr Tyr Cys
20 25 30
<210> 37
<211> 10
<212> PRT
<213> 人工序列
<220>
<223> C26 C27 C32 D3 D4 D5 LFR4
<400> 37
Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
1 5 10
<210> 38
<211> 98
<212> PRT
<213> 人工序列
<220>
<223> 4-28-02
<400> 38
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Ser Ser
20 25 30
Asn Trp Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp
35 40 45
Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Ile Tyr Tyr Asn Pro Ser Leu
50 55 60
Lys Ser Arg Val Thr Met Ser Val Asp Thr Ser Lys Asn Gln Phe Ser
65 70 75 80
Leu Lys Leu Ser Ser Val Thr Ala Val Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg
<210> 39
<211> 93
<212> PRT
<213> 人工序列
<220>
<223> 2D-28-01
<400> 39
Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly
1 5 10 15
Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser
20 25 30
Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys
85 90
<210> 40
<211> 330
<212> PRT
<213> 人工序列
<220>
<223> 人IgG1重链恒定区
<400> 40
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
225 230 235 240
Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
325 330
<210> 41
<211> 107
<212> PRT
<213> 人工序列
<220>
<223> 人κ轻链恒定区
<400> 41
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
1 5 10 15
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
20 25 30
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Ser Ala Leu Gln
35 40 45
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
50 55 60
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
65 70 75 80
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
85 90 95
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
100 105
<210> 42
<211> 330
<212> PRT
<213> 人工序列
<220>
<223> 具有V4突变的人IgG1重链恒定区
<400> 42
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125
Lys Pro Lys Asp Thr Leu Tyr Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160
Tyr Val Asp Gly Val Glu Val His Glu Ala Lys Thr Lys Pro Arg Glu
165 170 175
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
225 230 235 240
Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Tyr His Tyr Thr
305 310 315 320
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
325 330
<210> 43
<211> 330
<212> PRT
<213> 人工序列
<220>
<223> 具有DY突变人IgG1重链恒定区
<400> 43
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205
Asp Ala Tyr Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
225 230 235 240
Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
325 330
<210> 44
<211> 51
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 44
gttattactc gtggcccagc cggccatggc agaggtgcag ctgcaggagt c 51
<210> 45
<211> 69
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 45
ctccagcttg ttccctggga actgccggat ccagttsyrg tggtrgysgt rggtgatgga 60
gctaccaga 69
<210> 46
<211> 74
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 46
gttcccaggg aacaagctgg agtggattgg gyacmwcmrc yacsacggca gcswycwsya 60
caatccatct ctcg 74
<210> 47
<211> 51
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 47
gactgtgaga gttgtgcctt ggccccagtg gtsgwracca ctcgcacagt a 51
<210> 48
<211> 58
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 48
ccagatccgc cacctccact cccgcctcca cctgaggaga ctgtgagagt tgtgcctt 58
<210> 49
<211> 52
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 49
gtggaggtgg cggatctgga gggggtggta gcgatgttgt gatgacccaa tc 52
<210> 50
<211> 63
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 50
ctttggagac tggcctggct tctgcaggta ccaatgswgg trgkkgtctc catagykgtg 60
rws 63
<210> 51
<211> 54
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 51
agccaggcca gtctccaaag ctcctgatct acaaagtttc caaccgattt tctg 54
<210> 52
<211> 64
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 52
tttccagctt ggtcccccct ccgaagkkgt rgkgrwsatg gkkgtkstga gagcagtaat 60
aaac 64
<210> 53
<211> 48
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 53
tagtcgacca ggcccccgag gccttttatt tccagcttgg tcccccct 48
<210> 54
<211> 19
<212> PRT
<213> 人工序列
<220>
<223> 信号肽
<400> 54
Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly
1 5 10 15
Val His Ser
<210> 55
<211> 48
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 55
agtagcaact gcaaccggtg tacattctca ggtgcagctg caggagtc 48
<210> 56
<211> 40
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 56
gatgggccct tggtcgacgc tgaagagacg gtgacggtgg 40
<210> 57
<211> 50
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 57
agtagcaact gcaaccggtg tacattctga catacagatg acgcagtctc 50
<210> 58
<211> 39
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 58
atggtgcagc caccgtacgt ttgatttcca ccttggtcc 39
Claims (24)
1.能够特异性结合HBsAg的抗体或其抗原结合片段,所述抗体或其抗原结合片段以在中性pH下比在酸性pH下更高的亲和力结合HBsAg,其中,所述抗体或其抗原结合片段包含:
(1)包含如下3个CDR的重链可变区(VH):如SEQ ID NO:21所示的HCDR1,SEQ ID NO:22所示的HCDR2,SEQ ID NO:13所示的HCDR3;以及,包含如下3个CDR的轻链可变区(VL):SEQID NO:14所示的LCDR1,SEQ ID NO:15所示的LCDR2,SEQ ID NO:23所示的LCDR3;
(2)包含如下3个CDR的重链可变区(VH):如SEQ ID NO:17所示的HCDR1,SEQ ID NO:18所示的HCDR2,SEQ ID NO:13所示的HCDR3;以及,包含如下3个CDR的轻链可变区(VL):SEQID NO:14所示的LCDR1,SEQ ID NO:15所示的LCDR2,SEQ ID NO:19所示的LCDR3;
(3)包含如下3个CDR的重链可变区(VH):如SEQ ID NO:17所示的HCDR1,SEQ ID NO:20所示的HCDR2,SEQ ID NO:13所示的HCDR3;以及,包含如下3个CDR的轻链可变区(VL):SEQID NO:14所示的LCDR1,SEQ ID NO:15所示的LCDR2,SEQ ID NO:16所示的LCDR3;
(4)包含如下3个CDR的重链可变区(VH):如SEQ ID NO:24所示的HCDR1,SEQ ID NO:12所示的HCDR2,SEQ ID NO:13所示的HCDR3;以及,包含如下3个CDR的轻链可变区(VL):SEQID NO:25所示的LCDR1,SEQ ID NO:15所示的LCDR2,SEQ ID NO:16所示的LCDR3;
(5)包含如下3个CDR的重链可变区(VH):如SEQ ID NO:17所示的HCDR1,SEQ ID NO:12所示的HCDR2,SEQ ID NO:13所示的HCDR3;以及,包含如下3个CDR的轻链可变区(VL):SEQID NO:25所示的LCDR1,SEQ ID NO:15所示的LCDR2,SEQ ID NO:23所示的LCDR3;或
(6)包含如下3个CDR的重链可变区(VH):如SEQ ID NO:17所示的HCDR1,SEQ ID NO:12所示的HCDR2,SEQ ID NO:13所示的HCDR3;以及,包含如下3个CDR的轻链可变区(VL):SEQID NO:25所示的LCDR1,SEQ ID NO:15所示的LCDR2,SEQ ID NO:16所示的LCDR3。
2.权利要求1所述的抗体或其抗原结合片段,其中,所述抗体或其抗原结合片段进一步包含人免疫球蛋白的框架区,所述框架区任选地包含从人源残基至鼠源残基的回复突变。
3.权利要求1所述的抗体或其抗原结合片段,其中,所述抗体或其抗原结合片段包含:人重链胚系基因所编码的氨基酸序列中所包含的重链框架区,和人轻链胚系基因所编码的氨基酸序列中所包含的轻链框架区。
4.权利要求1所述的抗体或其抗原结合片段,其中,所述抗体或其抗原结合片段包含:如SEQ ID NO:38所示的人重链胚系基因4-28-02所编码的氨基酸序列中所包含的重链框架区,和如SEQ ID NO:39所示的人轻链胚系基因2D-28-01所编码的氨基酸序列中所包含的轻链框架区,所述重链框架区和/或轻链框架区任选地包含从人源残基至鼠源残基的回复突变。
5.权利要求1所述的抗体或其抗原结合片段,其中,所述抗体或其抗原结合片段的VH包含:如SEQ ID NO:30所示的VH FR1、如SEQ ID NO:31所示的VH FR2、如SEQ ID NO:32所示的VH FR3、和如SEQ ID NO:33所示的VH FR4;
所述抗体或其抗原结合片段的VL包含:如SEQ ID NO:34所示的VL FR1、如SEQ ID NO:35所示的VL FR2、如SEQ ID NO:36所示的VL FR3、和如SEQ ID NO:37所示的VL FR4。
6.权利要求1所述的抗体或其抗原结合片段,其中,所述抗体或其抗原结合片段包含:
(1)具有如SEQ ID NO:3所示的序列的VH和具有如SEQ ID NO:4所示的序列的VL;
(2)具有如SEQ ID NO:5所示的序列的VH和具有如SEQ ID NO:2所示的序列的VL;
(3)具有如SEQ ID NO:6所示的序列的VH和具有如SEQ ID NO:7所示的序列的VL;
(4)具有如SEQ ID NO:8所示的序列的VH和具有如SEQ ID NO:9所示的序列的VL;
(5)具有如SEQ ID NO:3所示的序列的VH和具有如SEQ ID NO:10所示的序列的VL;或
(6)具有如SEQ ID NO:3所示的序列的VH和具有如SEQ ID NO:9所示的序列的VL。
7.权利要求1-6任一项所述的抗体或其抗原结合片段,其中,所述抗体或其抗原结合片段进一步包含来源于人免疫球蛋白的恒定区。
8.权利要求7所述的抗体或其抗原结合片段,其中,所述抗体或其抗原结合片段的重链包含来源于人IgG1、IgG2、IgG3或IgG4重链恒定区,所述抗体或其抗原结合片段的轻链包含来源于人κ或λ轻链恒定区。
9.权利要求7所述的抗体或其抗原结合片段,其中,所述抗体或其抗原结合片段包含如SEQ ID NO:41所示的轻链恒定区(CL)。
10.权利要求7所述的抗体或其抗原结合片段,其中,所述抗体或其抗原结合片段包含人IgG1重链恒定区的变体,所述变体与其所源自的野生型序列相比具有以下置换:(i)M252Y、N286E、N434Y;或,(ii)K326D、L328Y;其中,以上提及的氨基酸位置是根据Kabat编号系统的位置。
11.权利要求10所述的抗体或其抗原结合片段,其中,所述抗体或其抗原结合片段包含如SEQ ID NO:42或43所示的重链恒定区(CH)。
12.权利要求7所述的抗体或其抗原结合片段,其中,所述抗体或其抗原结合片段包含如SEQ ID NO:40所示的重链恒定区(CH)。
13.权利要求1所述的抗体或其抗原结合片段,其中,所述抗体或其抗原结合片段包含:
(1)包括SEQ ID NO:3所示的VH和SEQ ID NO:40所示的CH的重链,和,包括SEQ ID NO:4所示的VL和SEQ ID NO:41所示的CL的轻链;
(2)包括SEQ ID NO:3所示的VH和SEQ ID NO:47所示的CH的重链,和,包括SEQ ID NO:4所示的VL和SEQ ID NO:41所示的CL的轻链;
(3)包括SEQ ID NO:3所示的VH和SEQ ID NO:48所示的CH的重链,和,包括SEQ ID NO:4所示的VL和SEQ ID NO:41所示的CL的轻链;
(4)包括SEQ ID NO:5所示的VH和SEQ ID NO:40所示的CH的重链,和,包括SEQ ID NO:2所示的VL和SEQ ID NO:41所示的CL的轻链;
(5)包括SEQ ID NO:5所示的VH和SEQ ID NO:47所示的CH的重链,和,包括SEQ ID NO:2所示的VL和SEQ ID NO:41所示的CL的轻链;
(6)包括SEQ ID NO:5所示的VH和SEQ ID NO:48所示的CH的重链,和,包括SEQ ID NO:2所示的VL和SEQ ID NO:41所示的CL的轻链;
(7)包括SEQ ID NO:6所示的VH和SEQ ID NO:40所示的CH的重链,和,包括SEQ ID NO:7所示的VL和SEQ ID NO:41所示的CL的轻链;
(8)包括SEQ ID NO:6所示的VH和SEQ ID NO:47所示的CH的重链,和,包括SEQ ID NO:7所示的VL和SEQ ID NO:41所示的CL的轻链;
(9)包括SEQ ID NO:6所示的VH和SEQ ID NO:48所示的CH的重链,和,包括SEQ ID NO:7所示的VL和SEQ ID NO:41所示的CL的轻链;
(10)包括SEQ ID NO:8所示的VH和SEQ ID NO:40所示的CH的重链,和,包括SEQ ID NO:9所示的VL和SEQ ID NO:41所示的CL的轻链;
(11)包括SEQ ID NO:8所示的VH和SEQ ID NO:47所示的CH的重链,和,包括SEQ ID NO:9所示的VL和SEQ ID NO:41所示的CL的轻链;
(12)包括SEQ ID NO:8所示的VH和SEQ ID NO:48所示的CH的重链,和,包括SEQ ID NO:9所示的VL和SEQ ID NO:41所示的CL的轻链;
(13)包括SEQ ID NO:3所示的VH和SEQ ID NO:40所示的CH的重链,和,包括SEQ ID NO:10所示的VL和SEQ ID NO:41所示的CL的轻链;
(14)包括SEQ ID NO:3所示的VH和SEQ ID NO:47所示的CH的重链,和,包括SEQ ID NO:10所示的VL和SEQ ID NO:41所示的CL的轻链;
(15)包括SEQ ID NO:3所示的VH和SEQ ID NO:48所示的CH的重链,和,包括SEQ ID NO:10所示的VL和SEQ ID NO:41所示的CL的轻链;
(16)包括SEQ ID NO:3所示的VH和SEQ ID NO:40所示的CH的重链,和,包括SEQ ID NO:9所示的VL和SEQ ID NO:41所示的CL的轻链;
(17)包括SEQ ID NO:3所示的VH和SEQ ID NO:47所示的CH的重链,和,包括SEQ ID NO:9所示的VL和SEQ ID NO:41所示的CL的轻链;或
(18)包括SEQ ID NO:3所示的VH和SEQ ID NO:48所示的CH的重链,和,包括SEQ ID NO:9所示的VL和SEQ ID NO:41所示的CL的轻链。
14.权利要求1所述的抗体或其抗原结合片段,其中,所述抗体或其抗原结合片段选自scFv、Fab、Fab’、(Fab’)2、Fv片段、双抗体(diabody)、双特异性抗体、多特异性抗体、probody、嵌合抗体或人源化抗体。
15.权利要求1所述的抗体或其抗原结合片段,其中,所述抗体或其抗原结合片段能够特异性结合HBsAg,中和HBV的毒力,和/或降低HBV DNA和/或HBsAg在受试者体内的血清水平。
16.分离的核酸分子,其编码权利要求1-15任一项所述的抗体或其抗原结合片段,或其重链可变区和轻链可变区。
17.载体,其包含权利要求16所述的核酸分子。
18.宿主细胞,其包含权利要求16所述的核酸分子或权利要求17所述的载体。
19.制备权利要求1-15任一项所述的抗体或其抗原结合片段的方法,其包括,在允许所述抗体或其抗原结合片段表达的条件下,培养权利要求18所述的宿主细胞,和从培养的宿主细胞培养物中回收所述抗体或其抗原结合片段。
20.药物组合物,其含有权利要求1-15任一项所述的抗体或其抗原结合片段,以及药学上可接受的载体和/或赋形剂。
21.权利要求1-15任一项所述的抗体或其抗原结合片段或权利要求20所述的药物组合物在制备药物中的用途,所述药物用于预防和/或治疗受试者的HBV感染或与HBV感染相关的疾病,用于体外或在受试者体内中和HBV的毒力,用于在受试者体内降低HBV DNA和/或HBsAg的血清水平,和/或用于激活受试者针对HBV的体液免疫应答。
22.权利要求21所述的用途,其中,所述受试者为人。
23.权利要求21所述的用途,其中,所述受试者为慢性HBV感染者或慢性乙肝患者。
24.权利要求21所述的用途,其中,所述与HBV感染相关的疾病为乙肝。
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