CN103788212A - 双特异性抗原结合蛋白复合物以及制备双特异性抗体的方法 - Google Patents
双特异性抗原结合蛋白复合物以及制备双特异性抗体的方法 Download PDFInfo
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Abstract
本发明提供了双特异性抗原结合蛋白复合物,双特异性抗体,制备所述双特异性抗体的方法和所述双特异性抗体的药物用途。应用根据本发明一个方面的蛋白复合物,有可能有效制得识别两种抗原、或相同抗原上两种表位的双特异性抗体。所述双特异性抗体可以用于诊断、预防和/或治疗诸如细胞增生性疾病或免疫性疾病之类的疾病。
Description
相关申请的交叉参考
本申请要求2012年10月31日向韩国专利局提交的韩国专利申请10-2012-0122559的权益,该申请全文引入本文作为参考。
技术领域
本申请涉及双特异性抗原结合蛋白复合物,制备双特异性抗体的方法,以及所述双特异性抗体的药用目的。
背景技术
单克隆抗体已成为上市新药的领导者,因此已被研发作为针对多种标靶的药物。但在很多情况中,新药研发受限;例如效力不令人满意,用于产生抗体的花费高昂,等等。作为解决这些问题的方案之一,对双特异性抗体的研究自上世纪八十年代中期起就逐步发展,虽然付出了很大努力,不过仍未有占优势的技术。
在制备双特异性抗体的常规方法中,对于大量制备同质的双特异性抗体仍有困难,或者因效率低和副作用而有实际操作的困难。近年来,基于对抗体改造技术的大力研发,出现了一些有竞争性的新抗体平台,但它们仍处于验证阶段。
因此,即使是利用常规技术,仍需要研发新平台来制备对至少两种异质性抗原具有特异性的抗体,并需要制备所述蛋白的方法。
发明概述
根据本发明的一个方面,在此提供了双特异性抗原结合蛋白复合物,其包括两个抗体结合位点。
根据本发明的另一方面,在此提供了编码所述双特异性抗原结合蛋白复合物的多核苷酸。
本发明还提供了用经重组表达载体转化的宿主细胞制备双特异性抗体的方法。
本发明还提供了包括所述双特异性抗体的药物组合物。
本发明还提供了包括所述双特异性抗体的诊断组合物。
其它方面在下文的说明书中描述了一部分,另外的部分基于说明书是很明显的,或者可以通过实施所述各方面而理解到。
附图说明
这些和/或其它方面在参照下文对各个方面的描述,并结合所附的附图后将是非常明显和更易于理解到的:
图1和图2示意根据本发明一个方面的双特异性抗原结合蛋白复合物和双特异性抗体;
图3示意根据本发明一个方面的双特异性抗原结合蛋白复合物的氨基酸序列结构;
图4示意根据本发明一个方面表达并纯化双特异性抗体的离子交换层析结果;
图5示意根据本发明一个方面,未经β-巯基乙醇处理(-)和经过其处理(+)的双特异性抗体的SDS-PAGE;和
图6是传感图(sensogram),示意了双特异性抗体的双重抗原结合反应。
发明详述
现在会详细提到各实施方案,附图中举例说明了它们的实例,其中同样的参考号指代全文上下的相同要素。在这点上,当前的实施方案可以具有不同的形式且不应解释为限于本文中所列的描述。因而,下文仅仅为了解释本说明书的各方面而参照附图来描述各实施方案。如本文中所使用的,术语“和/或”包括一个或多个所列项的任何和所有组合。象“至少一个”这样的表述置于一系列元件之前时,修饰的是所列出的所有元件,而不是修饰列表中的单个元件。
应理解,本文描述的例示性的各个方面应仅视为描述性质而不是限制。对每个方面中多个特征或项的描述一般应视为也能适用于其它方面中的类似特征或项。
根据本发明一个方面,提供了双特异性抗原结合蛋白复合物,其包括第一多肽、第二多肽和接头,所述第一多肽在N端包括第一抗原结合位点,所述第二多肽在N端包括第二抗原结合位点,所述接头连接所述第一多肽和所述第二多肽,其中所述接头在两末端包括第一标签和第二标签,且所述第一标签连接至所述第一多肽C端,所述第二标签连接至所述第二多肽N端,所述第一标签和第二标签各自包括可切割的氨基酸序列。
根据本发明另一方面,提供了双特异性抗原结合蛋白复合物,其包括第一多肽、第二多肽和接头,所述第一多肽在N端包括第一抗原结合位点,所述第二多肽在N端包括第二抗原结合位点,所述接头连接所述第一多肽和所述第二多肽,其中所述接头包括位于一个末端的标签,且所述标签连接至所述第一多肽C端或所述第二多肽N端、并包括可切割的氨基酸序列。
术语“双特异性”在本文中是指两种不同抗原,或甚至当这两者是相同抗原时,它们每一个都具有针对不同表位的结合特异性。所述表位可以源自不同抗原或相同抗原。术语“双特异性抗原结合蛋白复合物”和“双特异性抗体”在本文中是指所有制得的具有全长抗体或带抗原结合位点的片段的产物。所述抗体可以是人抗体,非人抗体,人源化抗体,或嵌合抗体。
术语“抗原结合位点”在本文中是指免疫球蛋白分子中结合抗原或表位的位点,所述抗原结合位点可包括互补决定区(CDR)。CDR是指免疫球蛋白重链或轻链高变区中发现的氨基酸。重链和轻链各自可包含三个CDR(例如,CDRH1,CDRH2,CDRH3,以及CDRL1,CDRL2,CDRL3)。CDR可为所述抗体与抗原或表位结合而提供主要的接触残基。术语“重链”在本文中理解为包括全长重链,该重链包含具有决定抗原特异性的氨基酸序列的可变区(VH)和具有三个恒定结构域(CH1,CH2,和CH3)的恒定区,以及它们的片段。同样,术语“轻链”在本文中包括全长轻链,该轻链包含具有决定抗原特异性的氨基酸序列的可变区(VL),还包含恒定区(CL),以及它们的片段。
根据本发明一个方面,所述蛋白复合物和双特异性抗体可包括结合不同抗原或不同表位的第一抗原结合位点和第二抗原结合位点。可结合抗原结合位点的抗原在正常情况下可以不表达或低水平表达;但该抗原在特定的疾病条件下,例如,在瘤形成疾病或在免疫性疾病中,可以显示增加的表达。这种抗原可选自:VEGF,EGFR,EpCAM,CCR5,CD19,HER-2neu,HER-3,HER-4,PSMA,CEA,MUC-1(粘蛋白),MUC2,MUC3,MUC4,MUC5AC,MUC5B,MUC7,βhCG,Lewis-Y,CD20,CD33,CD30,神经节苷脂GD3,9-O-乙酰基-GD3,GM2,Globo H,岩藻糖基GM1,poly SA,GD2,碳酸酐水化酶IX(MN/CA IX),CD44v6,音猬因子(Sonic Hedgehog,Shh),Wue-1,浆细胞抗原,(膜结合)IgE,黑素瘤硫酸软骨素蛋白聚糖(Melanoma ChondroitinSulfate Proteoglycan,MCSP),CCR8,TNF-α前体,STEAP,间皮素(mesothelin),A33抗原,前列腺干细胞抗原(PSCA),Ly-6,桥粒芯蛋白4,E-钙粘着蛋白新表位(neoepitope),胎儿乙酰胆碱受体,CD25,CA19-9标记物,CA-125标记物和缪氏抑制物质(Mullerian Inhibitory Substance,MIS)Ⅱ,sTn(唾液酸化Tn抗原;TAG-72),FAP(成纤维细胞激活蛋白),内皮唾液酸蛋白(endosialin),EGFRvIII,LG,SAS和CD63。
为了实现均衡的(uniform)生理效应,所述结合不同抗原的蛋白复合物和双特异性抗体可以使用抗原组合,所述组合诱导该两种抗原-抗体反应的协同效应、或允许一系列连锁作用。针对抗原组合的双特异性抗体可包括,例如,靶向肿瘤细胞抗原和细胞毒触发分子抗原的双特异性抗体(BsAb),例如,抗-FcγRI/抗-CD15,抗-p185HER2/FcγRIII(CD16),抗-CD3/抗-恶性-B细胞(10),抗-CD3/抗-p185HER2,抗-CD3/抗-p97,抗-CD3/抗-肾细胞癌,抗-CD3/抗-OVCAR-3,抗-CD3/L-D1(抗-结肠直肠癌),抗-CD3/抗-黑色素刺激性激素类似物,抗-EGFR/抗-CD3,抗-CD3/抗-CAMA1,抗-CD3/抗-CD19,抗-CD3/MoV18,抗-神经细胞粘附分子(NCAM)/抗-CD3,抗-叶酸结合蛋白(FBP)/抗-CD3,抗-泛癌相关抗原(AMOC-31)/抗-CD3;靶向肿瘤细胞抗原和抗毒素抗原的BsAb,例如,抗-皂素/抗-Id-1,抗-CD22/抗-皂素,抗-CD7/抗-皂素,抗-CD38/抗-皂素,抗-CEA/抗-赖氨酸A链,抗-干扰素-α(IFN-α)/抗-杂交瘤独特型,抗-CEA/抗-长春花生物碱(Vinca alkaloid);改变被酶激活的前药的BsAb,例如,抗-CD30/抗–碱性磷酸酶(催化丝裂霉素磷酸酶前药变为丝裂霉素醇);用作纤维蛋白分解物(fibrin decomposer)的BsAb,例如,抗-纤维蛋白/抗-组织纤溶酶原激活剂(tPA),抗-纤维蛋白/抗-尿激酶型纤维蛋白溶酶原激活剂(uPA);靶向细胞表面受体中免疫复合物的BsAb,例如,抗-低密度脂蛋白(LDL)/抗-Fc受体(例如:FcγRI,FcγR11或cγRIII);用于治疗传染病的BsAb,例如,抗-CD3/抗-单纯疱疹病毒(HSV),抗-T-细胞受体:CD3复合物/抗-流感,抗-FcγR/抗-HIV;用于体外或体内检测肿瘤的BsAb,例如,抗-CEA/抗-EOTUBE,抗-CEA/抗-DPTA,抗-p185HER2/抗-半抗原);作为疫苗佐剂的BsAb;和作为诊断工具的BsAb,例如,抗-兔IgG/抗-铁蛋白,抗-马辣根过氧化物酶(HRP)/抗-激素,抗-生长抑素(somatostatin)/抗-物质P,抗-HRP/抗-FITC,和抗-CEA/抗-β-半乳糖苷酶。
根据本发明一个方面,所述包括抗原结合位点的多肽可以是完整抗体或者是完整抗体的片段(抗原结合片段)。
完整抗体具有以下结构:两条全长轻链和两条全长重链,每对轻链和重链由二硫键(S-S键)相连。抗体的恒定区分为重链恒定区和轻链恒定区,重链恒定区有gamma(γ)、mu(μ)、alpha(α)、delta(δ)、和epsilon(ε)型,还有gamma1(γ1)、gamma2(γ2)、gamma3(γ3)、gamma4(γ4)、alpha1(α1)、和alpha2(α2)亚型。轻链恒定区有kappa(κ)和lambda(λ)型。
术语"抗原结合片段"在本文中是指完整抗体中因有抗原结合位点而具有抗原结合能力的一个部分。该定义中的抗原结合片段可包括(i)轻链可变区(VL),带有轻链恒定区(CL)的Fab片段,重链可变区(VH)和重链第一恒定区(CH1);(ii)Fab’片段,是在CH1结构域的C端有至少一个半胱氨酸的Fab片段;(iii)带有VH和CH1结构域的Fd片段;(iv)在VH、和CH1结构域、和CH1结构域C端有至少一个半胱氨酸的Fd’片段;(v)Fv片段,是在抗体单臂上有VL和VH结构域的最小抗体片段(两链Fv通过该抗体重链可变区和轻链可变区之间的非共价键相连),单链Fv(scFv)一般是重链可变区和轻链可变区之间由肽接头经共价键而连成,或者,由于单链Fv直接连至C端,因而可以形成类似于双链Fv的二聚体;(vi)dAb片段,由VH结构域构成(Ward et al.,Nature341,544-546(1989));(vii)分离的CDR区;(viii)F(ab’)2片段,是双价片段,包括由位于绞链区的二硫桥连接的两个Fab’片段;(ix)单链抗体分子(例如,单链Fv;scFv(Bird et al.,Science242:423-426(1988);Huston et al.,PNAS(USA)85:5879-5883(1988));(x)双体抗体(diabody),在同一多肽链上有两个抗原结合位点,所述位点包括轻链可变区和重链可变区(Hollinger et al.,Proc.Natl.Acad.Sci.USA,90:6444-6448(1993));(xi)线性抗体,包括一对串联Fd节段(VH-CH1-VH-CH1),与互补的轻链多肽一起形成一对抗原结合区(Zapata etal.Protein Eng.8(10):1057-1062(1995));和(xii)单域抗体,仅包括由VH、CH2、和CH3重链组成的重链。抗原结合片段可以用蛋白酶获得(例如,Fab可以由木瓜蛋白酶对完整抗体进行限制性片段化而获得,F(ab')2可以由胃蛋白酶进行片段化获得),所述片段也可以用重组DNA技术制得。
根据本发明一个方面,包含抗原结合位点的多肽可以是单域抗体。术语“单域抗体”在本文中是指具有单个可变区(VH)单体的肽链,由不含重链和轻链CH1区的约110个氨基酸组成。所述单域抗体可以是,重链抗体,天然单域抗体(一种天然不具有轻链的抗体),由常规的4链抗体衍生的单域抗体,或人工抗体;或单域支架,是并非来源于抗体的单域支架。单域抗体分子是非常小(只有IgG分子的约1/10)和非常稳定(在极端的pH或温度条件下都维持稳定性)的单链多肽。而且,与常规抗体不同,单域抗体分子具有对蛋白酶活性的耐受,能够在体外以较高的产量大量生成。单域抗体可包括抗原结合区或可结晶片段(fragment crystallizable,Fc)区。抗原结合位点可以是,例如,当结合的抗原是VEGF时具有序列表中编号为2或3的氨基酸序列,当结合的抗原是EFGR时具有序列表中编号为5或6的氨基酸序列。Fc区可包括绞链区或两个恒定区(CH2和CH3),例如,可具有序列表中编号为4的氨基酸序列。
根据本发明一个方面,包含抗原结合位点的多肽可以选自序列号8-44的氨基酸序列。
术语"标签"在本文中是指结合在融合蛋白末端的蛋白或多肽,所述标签是连接不同融合蛋白的媒介。所述标签可以连接至多肽的N端或C端。根据本发明一个方面,所述标签可以在体外或体内切割。所述体外或体内切割可通过蛋白酶来进行。
根据本发明一个方面,所述标签可以选自泛素、泛素-样蛋白、和TEV切割肽。泛素(Ub)是自然界发现的最保守的蛋白,其序列中有76个氨基酸,可溶于水,在诸如昆虫、虹鳟和人类等多个进化上各异的物种之间有非常大的同源性。而且,泛素已知是在pH改变时显示稳定的蛋白,在高温时不容易变性,有蛋白酶时也显示稳定。
泛素或泛素-样蛋白可选自野生型泛素、野生型泛素-样蛋白、突变的泛素、和突变的泛素-样蛋白。根据本发明一个方面,泛素可以由序列号7的氨基酸序列组成。泛素-样泛素蛋白是具有与泛素相似的性质的蛋白,可选自,例如,Nedd8,SUMO-1,SUMO-2,NUB1,PIC1,UBL3,UBL5,和ISG15。突变的泛素是指野生型泛素的氨基酸序列变成另一种氨基酸序列,例如,突变的泛素包括,野生型泛素的Lys被Arg取代,以及野生型泛素的C端RGG被RGA取代。根据本发明一个方面,考虑到Lys被Arg取代的突变泛素,所述取代可见于序列号6,11,27,29,33,48,和63所示的野生型泛素中的Lys,且所述取代可以独立地发生或组合地发生。
根据本发明一个方面,泛素或泛素-样蛋白可包括能在体外或体内在C端被蛋白酶切割的氨基酸序列。能被蛋白酶切割的氨基酸序列可在本领域已知的搜索数据库中找到。例如,蛋白酶和可切割的氨基酸序列可见于http://www.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html。当包括可切割的氨基酸序列时,蛋白复合物可以在体外或体内切割其标签,从而该蛋白复合物可形成双特异性抗体的三级结构,由此行使其功能。
根据本发明一个方面,蛋白复合物可包括连接所述第一多肽和所述第二多肽的接头。所述接头可以是肽接头。可以采用本领域已知的多种接头,例如,所述接头可以由多个氨基酸组成。根据本发明一个方面,所述接头可以是,例如,由约1-100个或约2-50个随机氨基酸组成的多肽。
所述肽接头可以通过充分分隔所述第一多肽和所述第二多肽而折叠成功能性的二级或三级结构。例如,肽接头可包括Gly、Asn、和Ser残基,并可包括诸如Thr和Ala等中性氨基酸。适于肽接头的氨基酸序列是本领域已知的。另一方面,接头的长度可以多种多样,只要该长度不影响融合蛋白的功能。
根据本发明一个方面,所述接头可以在至少一个末端包括标签。而且,所述标签连接至接头的该末端,并可以包括可切割的氨基酸序列。
根据本发明另一方面,蛋白复合物还可以包括分泌信号序列。
分泌信号序列是指,通过连接至编码序列位于细胞膜外侧或细胞外侧的N端而诱导所表达的蛋白或肽的分泌的序列,所述信号序列可以是由约18-30个氨基酸组成的肽序列。所有能转运到细胞膜外侧的蛋白有不同的信号序列,所述信号序列被细胞膜上的信号肽酶切割。通常,对于并非宿主细胞天然表达的外来蛋白而言,可以采用能将该蛋白分泌到细胞周质或培养基中的分泌信号序列,或采用修饰的序列。
根据本发明一个方面,蛋白复合物的氨基酸序列可以适当改变,只要目标功能或特性如抗原特异性未真正改变。氨基酸中的变化基于氨基酸残基取代产物的相似性而发生,例如,基于疏水特性、亲水特性、电荷、和/或大小,为此,可以考虑氨基酸疏水指数。所述变化可以是,例如,部分地取代、缺失、和/或添加氨基酸,特别地,所述取代可以是保守取代。术语“保守取代”在本文中是指不改变所得分子的生物活性的取代,使得取代的氨基酸不影响蛋白的三级结构或局部荷电状态。不完全影响分子活性的氨基酸取代是本领域已知的,例如可包括以下氨基酸取代:Ala/Ser,Val/Ile,Asp/Glu,Thr/Ser,Ala/Gly,Ala/Thr,Ser/Asn,Ala/Val,Ser/Gly,Thy/Phe,Ala/Pro,Lys/Arg,Asp/Asn,Leu/Ile,Leu/Val,Ala/Glu,和/或Asp/Gly。
本发明另一方面中,提供了编码所述蛋白复合物的多核苷酸。
术语“多核苷酸”在本文中是指以单链或双链形式存在的脱氧核糖核酸或核糖核酸聚合物。所述多核苷酸包括RNA基因组序列,DNA(gDNA和cDNA)或从DNA转录的RNA序列,而且,除非特别指明,所述多肽还包括天然多核苷酸、糖、或碱基改变的类似物。根据本发明一个方面,所述多核苷酸是轻链多核苷酸。
所述多核苷酸包括编码蛋白复合物氨基酸序列的核苷酸序列,也包括与其互补的核苷酸序列。所述互补序列包括完全互补的序列和基本上互补的序列,这是指能在本领域已知的严谨条件下与编码蛋白复合物氨基酸序列的核苷酸序列杂交的序列。
而且,编码蛋白复合物氨基酸序列的核苷酸序列可以被改变或突变。所述改变包括添加、缺失、或非保守取代或保守取代。编码蛋白复合物氨基酸序列的多核苷酸可以被解释为,包括相对于该多核苷酸有实质性同一性的核苷酸序列。所述实质性同一性将该核苷酸序列与另外的随机序列以使得它们最大对应的方式进行比对,当用本领域常见的算法分析所比对的序列时,所述序列可显示大于80%的同源性,大于90%的同源性,或大于95%的同源性。
根据本发明一个方面,所述多核苷酸可具有选自序列号45-81的碱基序列。
制备所述蛋白复合物可以使用所述多核苷酸,并可以涉及本领域已知的遗传工程技术和/或化学合成。所述遗传工程技术可涉及制备克隆载体或表达载体以便用该载体转化宿主细胞,并培养所述宿主细胞以便表达目标蛋白。
因此,本发明一个方面提供制备双特异性抗体的方法,所述方法包括,制备重组表达载体,其中插入了编码上述蛋白复合物的多核苷酸,用所述重组表达载体转化宿主细胞,培养所转化的宿主细胞,以及收集在宿主细胞中表达的双特异性抗体。
术语“载体”在本文中是指表达目标基因的工具,将它导入宿主细胞后,它产生多个拷贝的外来DNA,所述DNA是独立地克隆并插入所述载体和引入所述细胞中的。术语“重组表达载体”在本文中是指插入了外来DNA片段用以扩增目标蛋白的载体,所述外来DNA片段可以是编码蛋白复合物的多核苷酸。产生用于表达或克隆的载体系统的方法是本领域已知的。
所述载体可包括与所述多核苷酸序列可操作相连的调控序列。
术语“调控序列”在本文中是指用于表达编码序列的核酸序列,调控序列的特性可以因宿主生物而有不同。在原核生物中,调控序列一般包括启动子、核糖体结合位点、以及转录/翻译终止子。在真核生物中,调控序列一般包括启动子、终止子,在某些情况下,还包括增强子,反式活化因子,或转录因子。术语“可操作相连”在本文中是指因功能性结合所致的连接,使得各组分能按照预想的进行操作。可操作相连至编码序列的调控序列是在编码序列的表达能与调控序列并存的情况下进行连接的。
以原核细胞作为宿主时,重组载体可包括:能处理转录的强启动子(例如,tac启动子,lac启动子,lacUV5启动子,lpp启动子,pLλ启动子,pRλ启动子,rac5启动子,amp启动子,recA启动子,SP6启动子,trp启动子,和/或T7启动子);用于启动翻译的核糖体结合位点;和转录/翻译终止子。以大肠杆菌(例如,HB101,BL21,或DH5α)作为宿主细胞时,可以采用大肠杆菌启动子和色氨酸生物合成途径的多个操纵子(Yanofsky,C.(1984),J.Bacteriol.,158:1018-1024),和/或噬菌体λ的左向启动子(pLλ启动子,Herskowitz,I.和Hagen,D.(1980),Ann.Rev.Genet.,14:399-445)作为调控区。以真核细胞为宿主时,可以采用源自哺乳动物细胞前体的启动子(例如:金属硫蛋白启动子)或源自哺乳动物病毒的启动子(例如:腺病毒晚期启动子,豆苗病毒启动子7.5K,SV40启动子或巨细胞病毒启动子,以及HSV的atk启动子),并可以有聚腺苷酸化序列作为转录终止序列。
除了调控序列,所述重组表达载体还可包括限制性位点,诸如耐药基因等标记基因、分泌信号序列、或先导序列。限制性位点是指被限制性酶特异性识别的特定碱基序列。限制性位点可以是被诸如下列的限制性酶特异性识别的序列:例如,EcoRI、BamHI、HindⅢ、kpn Ⅰ、Not Ⅰ、Pst Ⅰ、Sma Ⅰ、和/或Xho I。标记基因起可选择标记的作用,可以是针对诸如氨苄青霉素、庆大霉素、羧苄青霉素、氯霉素、链霉素、卡那霉素、遗传霉素、新霉素、和/或四环素等药物的耐药基因。分泌信号序列或先导序列都是诱导所合成的蛋白移动至细胞隔室(例如,周质空间)的序列,或诱导所合成的蛋白分泌至细胞外的培养基中的序列,所述序列可以包括在多核苷酸序列的编码序列中。所述序列可以由本领域普通技术人员适当地选择,以便对应于所引入的DNA、宿主细胞类型、和/或培养基的各方面情况。
包含所述多种因子的合适载体已知有,例如,Okayama-Berg cDNA表达载体pcDV1(Pharmacia),pCDM8,pRc/CMV,pcDNA1,pcDNA3(Invitrogen)),pEF-DHFR,pEF-ADA或pEF-neo,或pSPORT1(GIBCO BRL)。
根据本发明一个方面,宿主细胞可以通过用所述重组表达载体或双特异性抗原结合蛋白复合物转化或转染宿主来制得。
宿主细胞可以是本领域已知有可能稳定重组载体并有可能持续地克隆和表达的原核细胞或真核细胞。原核生物是指可以用DNA分子或RNA分子转化以便表达蛋白的细菌。例如,大肠杆菌(Escherichia coli)、芽孢杆菌属的菌株如枯草芽孢杆菌(Bacillus subtilis)和苏云金芽孢杆菌(Bacillusthuringiensis)、链霉菌(Streptomyces)、假单胞菌(Pseudomonas)(例如,铜绿假单胞菌(Pseudomonas putida))、奇异变形杆菌(Proteus mirabilis)、葡萄球菌(Staphylococcus)(例如,肉葡萄球菌(Staphylococcus carnosus))、大鼠伤寒(rattyphus)(鼠伤寒沙门氏菌(S.typhimurium))、或粘质沙雷氏菌(Serratiamarcescens)。真核细胞包括酵母、高等植物、昆虫或哺乳动物细胞。根据本发明一个方面,宿主细胞可以是哺乳动物细胞。有用的哺乳动物细胞的例子有猴肾细胞,用SV40转化的CV1细胞系(COS-7,ATCC CRL1651);人胚肾细胞系(HEK-293或亚克隆的hEK-293细胞用于在悬浮培养中的生长,Graham etal.,J.Gen Virol.36:59(1977));仓鼠幼鼠肾细胞(BHK,ATCC CCL10);中国仓鼠卵巢细胞/-DHFR(CHO,Urlaub et al.,Proc.Natl.Acad.Sci.USA77:4216(1980));小鼠sertoli细胞(TM4,Mather,Biol.Reprod.23:243-251(1980));猴肾细胞(CV1ATCC CCL70);非洲绿猴肾细胞(VERO-76,ATCC CRL-1587);人宫颈癌细胞(HELA,ATCC CCL2);犬肾细胞(MDCK,ATCC CCL34);buffalo大鼠肝细胞(BRL3A,ATCC CRL1442);人肺细胞(W138,ATCC CCL75);人肝细胞(Hep G2,HB8065);小鼠乳腺癌细胞(MMT060562,ATCCCCL51);TR1细胞(Mather et al.,Annals N.Y.Acad.Sci.383:44-68(1982));MRC5细胞;FS4细胞;和/或人肝肿瘤细胞系(Hep G2)。
将重组表达载体转化至宿主细胞中可以通过,例如,DEAE-葡聚糖介导的转染、电穿孔、转到、磷酸钙转染、阳离子脂质介导的转染、刮拭加载(scrapeloading)、和/或感染来进行。
培养宿主细胞可通过用本领域已知的合适培养基和培养条件来进行。可以采用商品化培养基,例如,Ham's F10(Sigma),MEM(Minimal EssentialMedium,Sigma),RPMI-1640(Sigma),和/或DMEM(Dulbecco's ModifiedEagle's Medium,Sigma)。需要时,可以根据已知的合适浓度添加激素或其它生长因子,盐,缓冲液,核苷酸,抗生素,痕量元素和/或葡萄糖。培养条件例如温度和/或pH可以取决于本领域普通技术人员所选定的宿主细胞。
宿主细胞所表达的双特异性抗原结合蛋白复合物可以通过分泌信号肽分泌到细胞外,在这种情况下,可以通过从培养液或培养基中回收而获得该复合物。例如,通过用蛋白滤器浓缩培养上清,可以分离出抗体蛋白。但是,无分泌信号序列时表达的蛋白复合物可以从细胞裂解物直接获得,因为该蛋白复合物存在于细胞的周质中。将分泌至周质中的抗体分离出的方法是本领域已知的,所述抗体通常可以通过离心颗粒片段(宿主细胞的片段或解体的宿主细胞)或通过超离心来获得。
选择性地,从培养中获得的双特异性抗体可以进一步用本领域已知方法纯化。例如,根据所回收的抗体,对双特异性抗体的纯化可以用通常已知的蛋白纯化方法诸如(例如,离子交换,亲水,疏水,和/或大小排阻),层析聚焦,SDS-PAGE,和/或溶液分级分离(例如,硫酸铵沉淀)。根据本发明一个方面,双特异性抗体可以用亲和层析来纯化。作为亲和配体,蛋白A的合适性对应于抗体中的Fc区类型和免疫球蛋白同种型。附着有亲和配体的基质可以是琼脂糖,但不限于此,并且,机械方面稳定的基质(例如,调孔玻璃(regulated pore glass)或聚(苯乙烯二乙烯基)苯)可以比琼脂糖更改进流速和处理时间。
根据本发明另一方面,提供了包括上述双特异性抗体的药物组合物,和可药用载体,赋形剂,或稳定剂。
所述药物组合物可以通过双特异性抗原-抗体结合反应所致的生理效应作为治疗机制来预防和治疗疾病,或者所述组合物可以靶向因抗原-抗体反应导致的损伤。根据本发明一个方面,所述疾病可以是例如,增生性疾病,瘤形成疾病,炎性疾病,自身免疫病,传染病,病毒性疾病,过敏,移植物抗宿主病,和/或宿主抗移植物病。
例如,就特异性结合VEGF和EGFR的抗体而言,所述包含双特异性抗体的药物组合物可用于预防和/或治疗因抑制血管生成和/或抑制表皮生长而改善的疾病,例如,瘤形成病(neoplastic disease)。所述瘤形成病可以是,肺鳞状细胞癌,肺癌(包括小细胞肺癌,非小细胞肺癌,肺腺癌,或肺鳞状细胞癌),腹膜癌(peritoneal cancer),肝细胞瘤(hepatoma),胃腺癌(包括胃肠癌),胰腺癌,胶质瘤(glioma),胶质母细胞瘤,宫颈癌,卵巢癌,肝癌(liver cancer),膀胱癌,肝肿瘤(hepatic tumor),乳腺癌,结肠癌,结直肠癌,子宫内膜癌或子宫癌,唾液腺肿瘤,肾细胞癌,前列腺癌,外阴癌(vulva cancer),甲状腺癌,肝癌(hepatic carcinoma),以及各种形式的头颈癌;B-细胞淋巴瘤(低级/滤泡性非何杰金氏淋巴瘤(NHL);小淋巴细胞淋巴瘤(SL);非何杰金氏淋巴瘤;中级/滤泡性非何杰金氏淋巴瘤;中级分化型非何杰金氏淋巴瘤;高级免疫母细胞性非何杰金氏淋巴瘤;高级淋巴母细胞性非何杰金氏淋巴瘤;高级非裂解小细胞型非何杰金氏淋巴瘤;大肿块(bulky disease)非何杰金氏淋巴瘤;套细胞淋巴瘤;AIDS-相关淋巴瘤;和Waldenstrom氏巨球蛋白血症;慢性淋巴细胞白血病(CLL);急性淋巴母细胞白血病(ALL);毛发细胞白血病;慢性髓细胞白血病;移植后淋巴增生性疾病(PTLD);和/或与斑痣性错构瘤(phacomatosis)有关的血管内皮细胞异常增生,水肿(与脑瘤有关的水肿),和/或麦杰综合征(Meige syndrome)。
根据本发明一个方面,药物组合物中的双特异性抗体可以是与第二种激活剂(功能分子)结合的形式。所述第二种激活剂可以是能预防或治疗目标疾病的随机功能分子,可包括化合物,肽,多肽,核酸,碳水化合物,脂质,或无机粒子。在所述药物组合物中,双特异性抗体可以本身具有治疗活性;但它可以发挥将所述第二种激活剂靶向特异性疾病区的功能。所述疾病区可以是与抗原特异性结合的双特异性抗体所聚集和分布的那些器官,组织,或细胞。靶向所述疾病区的药物以高浓度存在,使得药物效应相比注射的量增加。因此,药物组合物可以用于治疗耐药性肿瘤,并可以减少因非特异性药物分布所致的副作用和不利的药物反应。
药物组合物可以通过将具有所需纯度的双特异性抗体与可药用载体、赋形剂、或稳定剂混合来制备。所用的可药用载体、赋形剂、或稳定剂都是在剂量和浓度方面对受体无毒的,可包括磷酸,柠檬酸,和其它有机酸;抗氧化剂(例如,抗坏血酸和甲硫氨酸);抗菌剂(例如,十八烷基二甲基苯氯化铵,氯化六烃季铵,苯扎氯铵,酚,丁醇或苯甲醇,烷基尼泊金,邻苯二酚,间苯二酚,环己醇,3-戊醇,或间甲酚);低分子量(不到约10kDa)多肽;蛋白,例如,血清白蛋白,明胶,或免疫球蛋白;亲水性聚合物,例如,聚乙烯吡咯烷酮;氨基酸(例如,甘氨酸,谷氨酰胺,天冬酰胺,组氨酸,精氨酸,或赖氨酸);单糖,二糖和其它碳水化合物(包括例如,葡萄糖,甘露糖,或葡聚糖);螯合剂(例如,EDTA);糖(sugar)(例如,蔗糖,甘露醇,海藻糖,或山梨醇);成盐反离子;金属复合物;和/或非离子型表面活性剂(例如,包括TWEENTM,PLURONICSTM,或聚乙二醇(PEG))。此外,根据配制方法,可以由本领域普通技术人员适当选择常用的填充剂,稀释剂,结合剂,增湿剂,崩解剂,和/或表面活性剂。
药物组合物中包含双特异性抗体的激活剂可以容纳在微胶囊中,或容纳在胶体性质的药物运送系统(如脂质体,白蛋白小球体,微乳剂,纳米颗粒及纳米胶囊)中,或者容纳在大乳剂(macroemulsions)中,所述微胶囊可以通过诸如凝聚(coacervation)技术或界面聚合作用来制备,例子分别有羟甲基纤维素或明胶微胶囊和聚-(异丁烯酸甲酯)微胶囊。
而且,双特异性抗体可以配制为延迟释放的(extended-release)片剂。所述延迟释放的片剂可以是,例如,包含抗体的固体疏水聚合物的半透性基质。所述基质可以是膜或微胶囊形式,并且可以是聚酯,水凝胶(例如,聚(2-羟乙基-甲基丙烯酸酯),或聚(乙烯醇)),聚乳酸(美国专利3,773,919),L-谷氨酸和γ乙基-L-谷氨酸的共聚物,非可降解的乙烯乙酸乙酯(ethylene-vinylacetate),乳酸-羟基乙酸的可降解型共聚物,例如,LUPRON DEPOTTM(一种可注射型微球,包括乳酸-羟基乙酸的共聚物,以及乙酸亮丙瑞林),和/或聚-D-(-)-3-羟基丁酸。当被包裹的抗体蛋白长时间留在体内时,因暴露于37℃的潮湿环境,该抗体可能会变性或聚集,从而失去生物活性并引起免疫原性改变,因此可以考虑稳定所述抗体的适当方法。例如,当凝聚剂是经由巯基-二硫键交换而在细胞间形成的S-S键时,抗体可以通过重新形成含巯基的片段、从酸性溶液冻干、控制湿度、用合适的添加剂和/或通过开发特异性聚合物基质来稳定。
根据本发明另一方面,提供了预防和/或治疗和施用治疗有效量的药物组合物以预防和/或治疗疾病的方法,所述疾病可选自增生性疾病,瘤形成疾病,炎性疾病,自身免疫病,传染病,病毒性疾病,过敏,移植物抗宿主病,和宿主抗移植物病。
药物组合物可通过多种途径注射至实体中,所述实体包括大鼠、小鼠、家养动物、和/或人类。所有注射方法都可以预期,例如,口服,直肠,静脉,鼻,腹部,皮下,或局部注射都是有可能的。组合物可以用本领域已知的其它方法(例如,最新版的Remington's Pharmaceutical Science中介绍的方法)来注射。
“治疗有效量”在本文中是指,根据合理的益损比来看,能治疗疾病的足够量。治疗有效量可以因患者引起的多种原因而有不同,所述原因例如,疾病类型、严重程度、发作、实体的年龄、体重、排泄速度、反应易感性、健康状态、和/或并发症;和/或药物活性、注射途径、注射周期和注射次数、和/或药物组合;也可以由本领域普通技术人员根据治疗目的进行适当选择。例如,注射量可以随机分为多次,使得该量为约0.001-100mg/kg成人体重。
本发明另一方面中,提供了用于疾病的包含双特异性抗体的诊断组合物,所述疾病可选自增生性疾病,瘤形成疾病,炎性疾病,自身免疫病,传染病,病毒性疾病,过敏,移植物抗宿主病,和宿主抗移植物病
根据本发明一个方面,诊断组合物应用在生物样品上,可以用于检测疾病特有的抗原。术语“生物样品”可以包括细胞,组织,全血,血浆,组织尸检样品(脑,皮肤,淋巴结,和脊柱),细胞培养上清,和/或受损的真核细胞。就收集的生物样品而言,可以体外应用所述组合物,或者可以将组合物注射到被研究的实体中而进行体内应用。
术语“检测”在本文中是指,通过使诊断组合物中的双特异性抗体与生物样品反应来证实抗原-抗体组合物的形成,其可以利用可检测的标记和检测方法来进行。检测方法可以是比色法,电化学法,荧光测量法,发光法,颗粒计数法,目测评估或闪烁计数法。可检测的标记可以是酶,荧光物质,发光物质,配体,纳米颗粒,或放射性同位素。用作检测标记的酶可包括乙酰胆碱酯酶,碱性磷酸酶,β-D-半乳糖苷酶,辣根过氧化物酶,和/或β-内酰胺酶。荧光物质可包括荧光素,Eu3+,Eu3+螯合物或穴合物(cryptate)。发光物质可包括吖啶酯和/或异鲁米诺衍生物,配体可包括生物素衍生物,纳米颗粒可包括胶体或金色乳胶,放射性同位素可包括57Co,3H,125I,125I-Bonton,和/或Hunter样品。根据本发明一个方面,对抗原-抗体复合物的检测可以用酶联免疫吸附实验(ELISA)进行。而且,当通过将诊断组合物注射至实体中来检测抗原-抗体反应时,可检测的标记可以通过结合或偶联至双特异性抗体而被注射。
本发明另一方面中,提供了包含上述双特异性抗体的试剂盒。所述含上述组分的试剂盒可以是用于诊断、预防、和/或治疗疾病的医用试剂盒,所述疾病选自增生性疾病,瘤形成疾病,炎性疾病,自身免疫病,传染病,病毒性疾病,过敏,移植物抗宿主病,和宿主抗移植物病。
利用根据本发明一个方面的蛋白复合物,能有效制备识别两种抗原或相同抗原的两种表位的双特异性抗体。所述双特异性抗体可以用于诊断、预防和/或治疗疾病的目的,所述疾病诸如细胞增生性疾病或免疫性疾病。
图1和2示意了根据本发明一个方面的双特异性抗原结合蛋白复合物(包含抗原结合位点)和双特异性抗体。
如图1所示,第一标签102和第二标签202分别连接第一多肽100(包括第一抗原结合位点101)和第二多肽200(包括第二抗原结合位点201),且第一标签102和第二标签202都连接至由多肽组成的接头300的末端。第一标签102和第二标签202都能在体外或体内切割,因为它们由诸如泛素或泛素-样蛋白之类的蛋白组成。不管是体外还是体内,所述第一多肽100(包括第一抗原结合位点101)和所述第二多肽201(包括第二抗原结合位点201)可以形成通过完全自发地结合而各具有不同抗原结合位点的双特异性抗体。
图2示意了一例蛋白复合物,其包括两个或多个根据图1中一实施方案所示的多肽(含有抗原结合位点),但不具有第二标签202。如上述,所述蛋白复合物通过体外或体内切割形成包括不同抗原结合位点的双特异性抗体,但由于图2的蛋白复合物不具有第二标签202,其变成所述接头300与所述第二多肽200(包括第二抗原结合位点201)结合的形式;但由于所述接头300包含约2-50个氨基酸的短小氨基酸序列,使得它不影响所述第二多肽200(包括第二抗原结合位点201)的功能。
实施例1:制备抗-VEGF-EGFR双特异性抗体表达载体
为了制备双特异性抗体,使其包括针对血管内皮细胞生长因子(VEGF)和内皮细胞生长因子受体(EGFR)的特异性结合位点,请求GeneArt制得了该双特异性抗体蛋白复合物的表达载体,用pCDNA3.1myc/his A(Invitrogen)作为蛋白过表达的载体。
具体地,如图3(A)和(B)所示,合成了:信号序列(ss,序列号1)、VEGF结合位点V1或V2(序列号2或3)、由包括绞链的Fc区(序列号4)组成的单域抗体、EGFR结合位点E1或E2(序列号5或6)、由包括绞链的Fc区(序列号4)组成的单域抗体、至少一个泛素标签(序列号7)、以及与蛋白复合物中由接头(Gly-Gly或(Gly-Gly-Gly-Gly-Ser)n肽)组成的氨基酸序列对应的单链DNA(总共37种蛋白复合物,对应于ss的长度,V1/V2和E1/E2的长度、接头的长度、多个泛素的长度,以及两个含绞链的Fc区的长度的总和)。将DNA片段的核苷酸序列插入质粒中,以表达由SEQ ID No:45-81表示的蛋白复合物。所插入的DNA片段包括,在5’端可被EcoRI切割的核苷酸序列,以及在3’端可被Xhol切割的核苷酸序列,因此该DNA片段可插入pcDNA3.1myc/his A载体的EcoRI-Xho1限制性位点。
实施例2.表达和纯化双特异性VEGF-EGFR
按实施例1所述,得到携带DNA片段SEQ ID No:78的重组载体,利用脂质体将该载体转染至HEK-293细胞系(人胚肾-293细胞,Korean Cell LineBank),由此表达并纯化抗-VEGF-EGFR双特异性抗体。
在500mL Erlenmeyer烧瓶中,对HEK-293细胞接种100mL FreestyleTM293培养基,浓度是1x106细胞/mL,用FreestyleTMMAX制备DNA-脂质体混合物。为制备DNA-脂质体复合物,使该混合物在室温反应10分钟,然后将复合物混合物添加至HEK-293细胞。将细胞在37℃、8%CO2摇床培养箱中培养7天,来诱导蛋白表达。
表达所述双特异性抗体的细胞的培养基用0.2μm滤膜过滤。用蛋白A亲和柱(GE healthcare)对细胞培养基进行层析。将该细胞培养基中包括的双特异性抗体与蛋白A柱结合,用磷酸缓冲盐水(PBS,pH7.4)洗柱,用洗脱剂(100mM甘氨酸-HCl,pH2.7)从蛋白A柱上将抗体洗脱。向洗脱剂中加入1/10体积的Tris缓冲液(1M Tris-HCl,pH9.0),以中和该洗脱剂。用脱盐柱将洗脱剂更换为缓冲液(30mM Tris-HCl,pH9.0),然后加载至MonoS柱(GEhealthcare),进行离子交换层析。结果见图4,双特异性抗体被洗脱。
洗脱物中双特异性抗体的存在通过SDS-PAGE来证实。所述双特异性抗体用β-巯基乙醇处理,以确认形成双特异性抗体的单体的分子量。结果见图5,它证实,包括VEGF结合位点的单臂抗体和包括EGFR结合位点的单臂抗体都以单体形式被检测到。
实施例3:验证抗-VEGF-EGFR双特异性抗体的抗原结合能力
为确认实施例2制得的双特异性抗体的双特异性抗原-抗体反应,用BiacoreT100仪(GE Healthcare Bio-Sciences AB)验证对VEGF和EGFR的抗体结合能力。将人VEGF(R&D Systems)以约2000RU(反应单位)的浓度通过氨基偶联化学反应结合到CM5芯片上。将按照实施例2制得的双特异性抗体以10μl/分钟的流速流动1分钟。在证实偶联后,将人EGFR胞外域(Prospec)以10μl/分钟的流速流动1分钟。在证实偶联后,将甘氨酸-HCl(GE Healthcare)溶液(pH2.0)以10μl/分钟的流速流动1分钟。
以上分析的结果是,证实所述双特异性抗体具有同时结合人VEGF和人EGFR的能力(图6)。
Claims (14)
1.双特异性抗原结合蛋白复合物,包含:
第一多肽,其在N端包含第一抗原结合位点;
第二多肽,其在N端包含第二抗原结合位点;和
接头,其连接所述第一多肽和所述第二多肽;
其中,所述接头在至少一个末端包括标签,所述标签连接至所述第一多肽C端和所述第二多肽N端这两者中的至少一个,且所述标签包括可切割的氨基酸序列。
2.权利要求1的蛋白复合物,
其中,所述接头在其一端包括第一标签并在其另一端包括第二标签,所述第一标签连接至所述第一多肽的C端,所述第二标签连接至所述第二多肽的N端,且所述第一标签和所述第二标签各包括可切割的氨基酸序列。
3.权利要求1或2的蛋白复合物,其中所述包含抗原结合位点的第一多肽和第二多肽中的至少一个是选自Fab片段、Fab'片段、Fv片段、和scFv片段的抗体重链或轻链或其片段,或单域抗体。
4.权利要求1或2的蛋白复合物,其中所述标签选自泛素,泛素-样蛋白,和TEV切割肽。
5.权利要求1或2的蛋白复合物,其中所述第一抗原结合位点和所述第二抗原结合位点各自独立地包含与选自下组的靶抗原特异性结合的位点:EpCAM,CCR5,CD19,HER-2neu,HER-3,HER-4,EGFR,PSMA,CEA,MUC-1(粘蛋白),MUC2,MUC3,MUC4,MUC5AC,MUC5B,MUC7,βhCG,Lewis-Y,CD20,CD33,CD30,神经节苷脂GD3,9-O-乙酰基-GD3,GM2,Globo H,岩藻糖基GM1,poly SA,GD2,碳酸酐水化酶IX(MN/CA IX),CD44v6,音猬因子(Shh),Wue-1,浆细胞抗原,(膜结合)IgE,黑素瘤硫酸软骨素蛋白聚糖(MCSP),CCR8,TNF-alpha前体,STEAP,间皮素,A33抗原,前列腺干细胞抗原(PSCA),Ly-6,桥粒芯蛋白4,E-钙粘着蛋白新表位,胎儿乙酰胆碱受体,CD25,CA19-9标记物,CA-125标记物,缪氏抑制物质(MIS)Ⅱ受体,sTn(唾液酸化Tn抗原;TAG-72),FAP(成纤维细胞激活抗原),内皮唾液酸蛋白,EGFRvIII,LG,SAS,和CD63。
6.权利要求1或2的蛋白复合物,其中所述接头由约1-100个随机氨基酸组成。
7.权利要求1或2的蛋白复合物,所述第一和第二多肽具有选自序列号8至序列号44的氨基酸序列。
8.多核苷酸,编码权利要求1或2的蛋白复合物。
9.权利要求8的多核苷酸,其中所述多核苷酸具有选自序列号45至序列号81的碱基序列。
10.制备双特异性抗体的方法,包括:
制备重组表达载体,其中插入了权利要求8的多核苷酸;
用所述重组表达载体转化宿主细胞;
培养所转化的宿主细胞;和
收集宿主细胞中表达的双特异性抗体。
11.用于预防或治疗疾病的药物组合物,包含:
治疗有效量的由权利要求10制得的双特异性抗体;和可药用载体,赋形剂,或稳定剂,其中所述疾病选自增生性疾病、瘤形成疾病、炎性疾病、自身免疫病、传染病、病毒性疾病、过敏性疾病、移植物抗宿主疾病、以及宿主抗移植物疾病。
12.权利要求11的药物组合物,其中所述双特异性抗体以与第二种激活剂结合的形式存在,并将所述第二种激活剂靶向疾病部位。
13.组合物,包含权利要求10所制得的双特异性抗体,是用于诊断疾病的组合物,所述疾病选自增生性疾病、瘤形成疾病、炎性疾病、自身免疫病、传染病、病毒性疾病、过敏性疾病、移植物抗宿主疾病、以及宿主抗移植物疾病。
14.权利要求13的组合物,其是用于体内注射的组合物,包含附着至所述双特异性抗体的可检测的标记。
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- 2013-10-30 EP EP13190818.8A patent/EP2727940A1/en not_active Ceased
- 2013-10-31 CN CN201310529748.6A patent/CN103788212A/zh active Pending
- 2013-10-31 US US14/069,091 patent/US9932412B2/en active Active
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CN110621814A (zh) * | 2017-05-09 | 2019-12-27 | 安升(上海)医药科技有限公司 | 多特异性蛋白药物及其文库、以及制备方法和应用 |
CN110621814B (zh) * | 2017-05-09 | 2023-09-15 | 安升(上海)医药科技有限公司 | 多特异性蛋白药物及其文库、以及制备方法和应用 |
CN110669770A (zh) * | 2018-07-02 | 2020-01-10 | 盛禾(中国)生物制药有限公司 | 人源化单克隆抗体、其制备方法及其用途 |
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WO2023241141A1 (zh) * | 2022-06-17 | 2023-12-21 | 南京北恒生物科技有限公司 | 靶向ccr8的嵌合抗原受体及其用途 |
Also Published As
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US9932412B2 (en) | 2018-04-03 |
KR101911438B1 (ko) | 2018-10-24 |
US20140127210A1 (en) | 2014-05-08 |
EP2727940A1 (en) | 2014-05-07 |
JP2014090721A (ja) | 2014-05-19 |
KR20140055536A (ko) | 2014-05-09 |
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