CN101054417B - 神经节苷脂相关的重组抗体及其在肿瘤的诊断和治疗中的应用 - Google Patents
神经节苷脂相关的重组抗体及其在肿瘤的诊断和治疗中的应用 Download PDFInfo
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Abstract
本发明涉及用DNA重组技术方法获得的修饰抗体,它是用在布达佩斯条约下保藏号ECACC94113026保藏的杂交瘤细胞系生产的鼠单克隆抗体P3(MAb P3)和保藏号ECACC97112901保藏的杂交瘤细胞系生产的其抗独特型鼠单克隆抗体1E10(MAbai1E10)来生产的,获得单克隆抗体的目的是,保持原始抗体的与抗原特异性结合的生物功能,然而又具有减少的免疫原性。本发明的嵌合抗体含有鼠免疫球蛋白的可变区和人免疫球蛋白的恒定区;除含有人免疫球蛋白的恒定区之外,并且将其人源化,其在鼠类构架区(FRs),尤其在那些处于T细胞抗原位点中的区域被修饰,因此,若干FRs的位置也是人的。该抗体可以在不同类型的肿瘤的诊断和治疗中应用。本发明还涉及用于治疗和诊断目的的抗体的应用。
Description
技术领域
本发明涉及生物技术领域,尤其是用基因工程获得的新的重组抗体,具体地说是从鼠单克隆抗体P3(MAb P3)和其抗独特型(anti-idiotype)鼠单克隆抗体1E10(MAbai 1E10)获得的嵌合和人源化抗体。
更具体地说,本发明涉及与含有N-乙醇酰化(N-glycolylated)唾液酸的神经节苷脂结合的抗体,但是,其既不与乙酰化形式的神经节苷脂也不与中性糖脂结合。含有N-乙醇酰化唾液酸的神经节苷脂是在乳腺癌和黑素瘤中广泛表达的抗原。另一方面,MAbai 1E10的抗肿瘤效果亦已经在实验模型中被证明。
本发明还涉及药物组合物,其含有前面所述的在癌症,尤其在乳腺癌和黑素瘤的诊断和治疗中有用的重组抗体。
现有技术
神经节苷脂是含有唾液酸的糖鞘脂并且存在于脊椎动物细胞的胞质膜中(Stults等人(1989):糖鞘脂:结构、生物学来源和特性(Glycosphingolipids:structure,biological source andproperties),Methods Enzymology,179:167-214)。在文献中已见某些该种分子作为肿瘤相关或肿瘤标记抗原的报道(Hakomori等人(1991):肿瘤相关碳水化合物类抗原的可能的功能(Possiblefunctions of tumor associated carbohydrate antigens),Curr.Opin.Immunol.,3:646-653),由于这个原因,抗神经节苷脂抗体的应用已经被认为在癌的诊断和治疗中是有用的(Hougton等人(1985):检测GD3神经节苷脂的鼠单克隆抗体IgG3抗体:在恶性黑素瘤患者中的第I阶段试验(Mouse monoclonal antibody IgG3antibody detecting GD3 ganglioside:to phase I trial inpatients with malignant melanoma),PNAS USA,82:1242-1246;Zhang等人(1997):应用免疫组织化学筛选作为免疫攻击目标的碳水化合物类肿瘤抗原,I.聚焦在神经节苷脂上(Selection ofcarbohydrate tumor antigens as targets for immune attackusing immunohistochemistry.I.Focus on gangliosides),Int.J.Cancer,73:42-49)。
在动物中更经常表达的唾液酸是N-乙酰化(NeuAc)和N-乙醇酰化(NeuGc)(Corfield等人(1982):唾液酸的发生(Occurrence ofsialic acids),Cell.Biol.Monogr.,10:5-50)。一般来说,NeuGc在正常人和鸡的组织中不表达,但在其它脊椎动物中分布广泛(Leeden和Yu,(1976):唾液酸化学和分析(Chemistry andanalysis of siali cacid).Biological Role of Sialic Acid.Rosemberg A和Shengtrund CL(Eds).Plenum Press,New York,1-48;Kawai等人(1991):用气相色谱质谱联用法对人癌组织和禽淋巴瘤细胞系中表达的作为肿瘤相关的唾液酸的N-羟乙酰神经氨酸进行定量测定(Quantitative determination of N-glycolylneuraminic acid expression in human canceroustissues and avian lymphoma cell lines as a tumor associatedsial icacid by gas chromatography-mass spectrometry),Cancer Research,51:1242-1246)。但是,有报道指出,抗-NeuGc抗体,可识别某些人的肿瘤和肿瘤细胞系(Higashi等人(1988):在人视网膜母细胞瘤细胞中作为N-羟乙酰神经氨酸特异的肿瘤相关Hanganutziu-Deicher抗原的神经节苷脂的检测(Detection of gangliosides as N-glycolylneuraminic acidspecific tumor—associated Hanganutziu-Deicher antigen inhumanret inoblastoma cells),Jpn.J.Cancer Res.,79:952-956;Fukui等人(1989):在人的胃癌细胞系NUGC4中作为肿瘤相关Hanganutziu-Deicher抗原的糖蛋白的检测(Detection ofglycoproteins as tumor-associated Hanganutziu-Deicherantigen in human gastric cancer cell line),Biochem.Biophys.Res.Commun.,160:1149-1154)。在人的乳腺癌中已发现GM3(NeuGc)神经节苷脂水平的提高(Marquina等人(1996):在人的乳腺癌中表达的神经节苷脂(Gangliosides expressed inhuman breast cancer),Cancer Research,1996;56:5165-5171),这一结果使该分子作为癌症治疗的靶位的应用受到注意。
用保藏号ECACC 94113026的细胞系生产单克隆抗体(Mab)P3(欧洲专利EP 0 657 471 B1),它是有着IgM同种型的鼠单克隆抗体,它是在融合用含有GM3(NeuGc)和破伤风类毒素的脂质体接种过的BALB/c小鼠的脾细胞和鼠骨髓瘤细胞系P3-X63-Ag8.653时获得。该Mab P3与含有N-乙醇酰化唾液酸的神经节苷脂起强烈反应但既不与乙酰化形式的神经节苷脂也不与中性糖脂起反应。用从良性的和恶性的肿瘤中得到的细胞系和组织进行免疫细胞化学和免疫组织化学研究证明,Mab P3识别乳腺癌(Vázquez等人(1995):,对N-羟乙酰神经氨酸特导的鼠单克隆抗体的产生,其亦识别硫酸糖脂(Generation of a murine monoclonal antibody specific for N-glycolylneuraminic acid-containing gangliosides that alsorecognizes sulfated glycolipids),Hybridoma,14:551-556)和黑素瘤。
即使不用佐剂和载体蛋白,Mab P3诱导了BALB/c小鼠(同基因模型)的抗独特型免疫反应(Vázquez等人(1998):抗含有抗NeuGc的神经节苷脂的单克隆抗体的同基因抗独特型单克隆抗体(Syngeneic antiidiotypic monoclonal antibodies to an anti-NeuGc-containing ganglioside monoclonal antibody);Hybridoma,17:527-534)。免疫化学分析提示了负电基团:唾液酸(对于神经节苷脂)和S03-(对于硫苷脂),在该抗体的识别特性中的作用(Moreno等人(1998):用抗含有N-羟乙酰神经氨酸的神经节苷脂的特异性抗体识别的表位的描述(Delineation of epitoperecognized by an antibody specific for N-glycolylneuraminican acid-containing gangliosides),Glycobiology,8:695-705)。
从用与KLH偶联的Mab P3免疫接种过的BALB/c小鼠获得IgG1亚型的抗独特型Mab 1E10(Mabai 1E10)(美国专利6,063,379,以保藏号ECACC 97112901保藏的细胞系)。Mabai 1E10可特异性地识别Mab P3而且其不与其它抗神经节苷脂IgM抗体结合。并且,Mabai 1E10阻止Mab P3与GM3(NeuGc)及与从乳腺导管癌(对于MabP3的结合为阳性)得到的细胞系MDA-MB-435的特异性结合。在给同基因或同种异基因(alogenic)模型中的小鼠免疫接种时,Mabai 1E10诱导Ab3抗体的强烈的免疫反应,即使在其携带的独特表位与Ab1抗体携带的相类似时,该Ab3抗体没有象Mab P3那样显示出同样的特异性(Vázquez等人(1998):抗含有抗NeuGc的神经节苷脂单克隆抗体的同基因抗独特型单克隆抗体(Syngeneic anti-idiotypicmonoclonal antibodies to an anti-NeuGc-containingganglioside monoclonal antibody),Hybridoma,17:527-534)。Mabai 1E10诱导了同基因的以及同种异基因的小鼠中的很强的抗肿瘤作用。当BALB/c小鼠被接种的情况下,用重复剂量的Freund佐剂中的与KLH偶联的Mabai 1E10可大大减少乳腺癌细胞系F3II的生长。接种疫苗之后,也减少了自发的肺转移的数量。在用黑素瘤细胞B16静脉接种之后10~14天,静脉给药Mabai1E10给接种了的C57BL/6小鼠,与用无关的IgG治疗的小鼠相比较,导致了肺转移的数量惊人地减少。该结果提示了一种以上抗肿瘤作用机制的启动。(Vázquez等人(2000):与含有N-乙醇酰的神经节苷脂有关的抗独特型单克隆抗体的抗肿瘤特性(Antitumor properties of ananti-idiotypic monoclonal antibody in relation to N-glycolyl-containing gangliosides),Oncol.Rep.,7:751-756,2000)。
在15年中杂交瘤技术已经得到发展(Koehler和Milstein(1975):分泌预定的特异性抗体的融合细胞的连续培养(Continuouscultures of fused cell ssecreting antibody of predefinedspecificity),Nature,256:495-497)并且当单克隆抗体在诊断和研究中仍然非常有用时,仍不能证明其对人治疗的有效性。这主要因为其在血液中短暂的半衰期以及鼠效应子功能对人免疫系统和人抗鼠的抗体免疫反应的失效(HAMA反应)。
另外,自从操纵免疫球蛋白基因,使获得降低抗原性的修饰抗体和用于特定病理的治疗或诊断而改进其效应子的功能成为可能以来,基因工程技术使MAb的潜在用途有了发展。降低免疫球蛋白免疫原性的方法有着一个根本的任务,就是缩小鼠抗体和人免疫球蛋白之间的差别,而不改变抗原识别的特异性(Morrison和Oi(1989):基因工程的抗体分子(Genetically engineered antibody molecules),Adv Immunol.,44:65-92)。
近来有几种方法已经被开发出以人源化小鼠或大鼠抗体,该方法减少异体蛋白注射到人体时对抗异体蛋白的异基因免疫反应。最早的减少抗原性的方法之一是嵌合抗体,在该方法中是将鼠蛋白质的可变区插入人分子的恒定区,它表现出相同的特异性但是与其的鼠类的相对应部分相比降低了免疫原性,人的效应子功能用嵌合抗体来保持,(Morrison等人(1984):嵌合人类抗体分子:鼠抗原结合区与人的恒定区(Chimeric human antibody molecules:Mouse antigen-binding domains with human constant region domains),PNASUSA,81:6851-6855)。甚至在嵌合抗体具有与鼠类的相对应部分相同特异性的时候,对啮齿类动物可变区的免疫反应常常可被观察到。
在进一步降低嵌合抗体免疫原性的尝试中,仅将来自啮齿类动物单克隆抗体的CDRs移植到人的构架区,该杂交可变区与人的恒定区一起表达(Jones等人(1986):用鼠类的互补决定区替换人的抗体中的互补决定区(Replacing the complementary-determiningregions in a human antibody with those from a mouse),Nature 321:522-524;Verhoeyen等人(1988):重构人的抗体:移植抗溶菌酶的活性(Reshaping human antibodies:graftingan antilysozyme activity),Science 239,1534-1536)。但是,该方法存在若干不足:所得抗体常常有着降低了的亲和力并且大量的构架残基必须被回复突变为对应的鼠类的残基以恢复结合能力(Rietchmann等人(1988):用于治疗的重构人类抗体(Reshapinghuman antibodies for therapy),Nature,332:323-327;Queen等人(1989):结合白细胞介素2受体的人源抗体(A humanizedantibody that binds to the interleukin 2 receptor),PNASUSA,86:10029-10033;Tempest等人(1991):重构人类单克隆抗体以阻止人的体内呼吸道合胞病毒感染(Reshaping a humanmonoclonal antibody to inhibit human respiratory syncytialvirus infection in vivo),Biotechnology,9:266-272)。此外,耐久的免疫原性在移植CDR的抗体中常常被观察到。
Mateo和其同事(美国专利号US 5 712 120)叙述了一种降低鼠抗体免疫原性的方法。根据该方法,修饰限于可变区,特别是嵌合抗体的鼠FRs。此外,替换只在FRs具有两性序列的那些区域进行,因此它们是被T细胞识别的潜在表位。该方法包括几个氨基酸残基被最同源的人类相应序列审慎地替换,被替换的氨基酸残基位于潜在免疫原性表位中,主要对规范的结构起作用的氨基酸以及与CDRs紧密相邻的或Vernier区中的残基必须保留。
所得抗体保留其抗原结合特异性并且与它的鼠的或嵌合的前体相比免疫原性更小(Mateo等人(2000):从基因工程抗体去除T细胞表位:生产被修饰了的降低了免疫原性的免疫球蛋白(Removal of Tcell epitopes from genetically engineered antibodies:Production of modified immunoglobulins with reducedimmunogenicity),Hybridoma 19:463-71),该特性提高了其治疗效用。采用该新方法,仅有几种突变需要完成,当然,就只要进行较少的基因操作。
发明详述
本发明涉及由基因工程技术获得的重组抗体。具体地说,本发明涉及从鼠单克隆抗体P3得到的嵌合抗体,它是用保藏号ECACC94113026的杂交瘤细胞系生产。MAB P3识别在乳腺癌细胞和黑素瘤中表达的抗原。MAB P3的特征在于重链和轻链高度可变区(CDRs)的下述序列:
重链
CDR1:RYSVH
CDR2:MIWGGGSTDYNSALKS
CDR3:SGVREGRAQAWFAY
CADENA LIGERA
CDR1:KASQDVSTAVA
CDR2:SASYRYT
CDR3:QQHYSTPWT
优选的重链和轻链的FRs序列如下:
重链
FR1:QVQLKESGPGLVAPSQSLSITCTVSGFSLS
FR2:WVRQPPGKGLEWLG
FR3:RLSISKDNSKSQVFLKMNSLQTDDTAMYYCAR
FR4:WGQGTLV
轻链
FR1:DIVMTQSHKFMSTSVGDRVSITC
FR2:WYQQKPGQSPKLLIY
FR3:GVPDRFTGSGSGTDFTFTISSVQAEDLAVYYC
FR4:FGGGTKL
在优选的实施方案中,本发明的嵌合抗体,含有人的IgG1重链的恒定区和人Ck轻链的恒定区。在另一方面,本发明涉及由用保藏号ECACC 94113026的杂交瘤细胞系生产的Mab P3得到的人源化抗体,其特征在于含有人IgG1重链的恒定区和人轻链Ck的恒定区并且其轻链的FRs区含有任一下面的点突变:
轻链
位置8:用Pro替换His
位置9:用Ser替换Lys
位置10:用Ser替换Phe
位置11:用Leu替换Met
位置13:用Ala替换Thr
在另一方面,本发明涉及由用保藏号ECACC 97112901的杂交瘤细胞系生产的鼠单克隆抗体1E10得到的嵌合抗体,并且它是能识别Mab P3的抗独特型抗体。MAbai 1E10的特征在于重链和轻链高度可变区(CDRs)的下述序列:
重链
CDR1:SYDIN
CDR2:WIFPGDGSTKYNEKFKG
CDR3:EDYYDNSYYFDY
轻链
CDR1:RASQDISNYLN
CDR2:YTSRLHSG
CDR3:QQGNTLPWT
优选的重链和轻链的FRs序列如下:
重链
FR1:QVQLQQSGAELVKPGASVKLSCKASGYTFT
FR2:WVRQRPEQGLEWIG
FR3:KATLTTDKSSSTAYMQLSRLTSEDSAVYFCAR
FR4:WGQGTTLTV
轻链
FR1:DIQMTQTTSSLSASLGDRVTISC
FR2:WYQQKPDGTVKLLIY
FR3:VPSRFSGSGSGTDYSLTISNLEQEDIATYFC
FR4:FGGGTKLESK
在优选的实施方案中,本发明的嵌合抗体,含有人IgG1重链的恒定区和人Ck轻链的恒定区。在另一方面,本发明涉及由用保藏号ECACC 97112901的杂交瘤细胞系生产的Mab 1E10得到的人源化抗体,其特征在于它含有人IgG1重链的恒定区和人Ck轻链的恒定区并且重链和轻链FRs区含有任一下面的点突变:
轻链
位置7:用Ser替换Thr
位置8:用Pro替换Thr
位置15:用Val替换Leu
重链
位置5:用Val替换Gln
位置40:用Ala替换Arg
位置42:用Gly替换Glu
位置87(根据Kabat编号为83):用Arg替换Thr
在另一方面,本发明涉及表达所述嵌合和人源化抗体的细胞系;另外本发明涉及含有所述抗体的药物组合物。
优选的是,涉及含有所述的抗体和适当的赋形剂的用于治疗乳腺、肺、消化系统、泌尿生殖系统、黑素瘤、肉瘤和神经外胚层的肿瘤、以及它们的转移和复发的药物组合物。
在本发明的另一表述中,含有所述的抗体的药物组合物,可以用于对乳腺、肺、消化系统、泌尿生殖系统、黑素瘤、肉瘤和神经外胚层的肿瘤、以及它们的转移和复发进行的体内定位和诊断。
用PCR(聚合酶链式反应)进行Mab P3和Mabai 1E10可变区的
cDNA的合成和基因扩增。
从大约106的P3(鼠IgM MAb,可识别GM3N-乙醇酰化神经节苷脂)或1E10(抗P3的抗独特型抗体)杂交瘤细胞中提取胞质RNA。使用TRIZOL试剂(GIBCO BRL,Grand Island,NY),根据生产者的说明书来提取RNA。
混合5μg RNA、25皮摩尔Vh(与VHP3的鼠IgM的恒定区互补,并且具有VH 1E10的鼠IgG1恒定区)或Vk(与两种抗体的鼠κ恒定区互补)、2.5mM各种dNTP、pH值7.5的Tris-Hcl 50mM、75mMKCl、10mM DTT、8mM MgCl2,并且在每50μl反应混合物中加入15单位RNA酶抑制剂以进行cDNA的合成反应。在70℃加热10分钟,缓慢冷却至37℃。然后,加入100单位MLV逆转录酶并且在42℃下连续培育一小时。
用PCR扩增可变区VK和VH的cDNAs。简言之,将5μl VH或VK的cDNA与25皮摩尔的特异性引物、2.5mM各种dNTP、5μl10倍Taq DNA聚合酶的缓冲液和1单位该酶的组成物混合。试样须经在94℃下30秒、50℃下30秒、72℃下1分钟的25次热循环,最后在72℃下培育5分钟。
扩增的cDNA的克隆和序列测定
将VH和VK(各为P3和1E10的)的PCR产物克隆到TA载体中(TA克隆试剂盒.Promega,USA)。对所得克隆产物用T7DNA聚合酶以双脱氧的方法测序(T7测序试剂盒.Pharmacia,Sweden)。
嵌合基因的构建
用酶消化的方法从TA载体上将VH VK基因切下,并且将其克隆到各自的表达载体中(Coloma等人(1992):用于表达使用以聚合酶链式反应生产的可变区的抗体分子的新型载体(Novel vectors forthe expression of antibody molecules using variable regionsgenerated by polymerase chain reaction),J.Immunol.Meth.,152:89-104)。
用EcoRV和Nhel以酶消化的方法从TA载体中将VH基因切下,并且克隆到包含人的IgG1可变区和组氨醇抗性基因的表达载体(PAH4604)中。结果产生的构建体是P3VH-PAH4604和1E10VH-PAH4604。用EcoRV和SaII以酶消化的方法从TA载体中将VK基因切下,并且克隆到表达载体(PAG4622)中。该载体包括霉酚酸抗性基因和人的κ恒定区。结果产生的构建体是P3VK-PAG4622和1E10VK-PAG4622。
从Mab P3和Mabid 1E10获得的嵌合抗体的表达
将NS-O细胞用10μg P3VK-PAG4622或1E10VK-PAG4622电穿孔,将表达人的κ轻链的克隆用10μg P3VH-PAH4604或1E10VH-PAH4604转染。
用Pvul酶通过消化把DNA直线化,用乙醇沉淀并溶解在50μlPBS中。用离心法得到大约107的细胞,将其与电穿孔杯中消化过的DNA一起在0.5ml PBS中重新悬浮。置于冰上10分钟后,给细胞一个200 Volts 960 μF的脉冲电流,并且在冰上再放置10分钟。将细胞与加入10%胎牛血清的D'MEM F12一起分置到96孔板中。二或四天后,加入选择培养基(分别含有霉酚酸0,45μg/ml或组氨醇10mM的D′MEM F12)。14天后,转染的克隆可以在肉眼下看到。
用ELISA测定含有转染克隆的孔中培养基中存在的人类抗体。用羊抗人κ轻链(对于生产人的κ链的克隆)或抗人IgG(γ链特异的)(对于生产完全抗体的克隆)抗体覆盖微量滴定板孔。用PBST(含0.05% Tween20的磷酸盐缓冲溶液)洗涤后,在37℃把稀释的含有转染产物的培养基加入到每一微量滴定板孔中一小时。孔用PBS-T洗涤,并加入与辣根过氧化物酶共轭(peroxidase of spicyradish-conjugated)的羊抗人κ轻链或与碱性磷酸酶共轭的羊抗人IgG(γ链特异的),之后在37℃培育一小时。孔用PBS-T洗涤过之后,分别加入含有邻苯二胺或对硝基苯磷酸盐(p-nitrophenylphosphate)的底物缓冲液。半小时后,分别测定在492或405nm下的吸收。
通过人源化T细胞表位构建人源化抗体P3hu和1E10 hu。T细胞表位
的预测。
用AMPHI算法分析P3和1E10可变区的序列(Margalit等人(1987):从一级序列预测免疫决定辅助T细胞抗原位点(Predictionof immunodominant helper T cell antigenic sites from theprimary sequence),J.Immunol.,138:2213-2229)。检出有着7或11个氨基酸残基的与T免疫原性有关的两亲性(amphipatic)螺旋片段。程序SOHHA还预测了疏水的螺旋片段。(Elliot等人(1987).An hypothesis on the binding of an amphipatic,alpha helical sequence in li to the desotope of class IIantigen,J.Immunol.,138:2949-2952)。两种算法都预测抗体P3和1E10可变区中序列的何种片段能够在MHC II类分子环境中呈递给辅助T细胞。
与人免疫球蛋白的同源性分析
将鼠可变区的氨基酸序列与GeneBank和EMBL数据库(从因特网中可获得)中包含的免疫球蛋白序列比较。对于每个抗体,确定了最同源的人类可变区序列。序列同源性检测使用的软件是PC-TWOHIBIO PROSIS 06-00(Hitachi)。
免疫原性减小的分析
本方法的目的是用最小的变化来减少免疫原性,破坏或人源化潜在的有免疫原性的T表位。方法包括位于两亲性螺旋片段的几个氨基酸残基的审慎的替换。对规范的结构起主要作用的氨基酸以及与CDRs紧密相邻的或Vernier区中的残基必须保留。
根据本方法,将鼠可变区序列与最同源的人类序列比较,并且将在每一个位置上的鼠MAb和最同源的人类序列之间不同的氨基酸残基确定下来,仅考虑FRs中的残基(Kabat(1991),Sequences ofproteins of immunological interest,第五版,NationalInstitute of Health),把前面确定的残基用在最同源的人类序列中存在的那些残基替换。替换过程由定向诱变技术来完成。
涉及结合部位的三维结构的残基不被突变;它可能影响抗原识别。关于替换在三级结构中的影响的附加信息能够从抗原结合部位的分子模拟来获得。
两亲性螺旋片段中脯氨酸残基的存在和特定的鼠的残基并不出现在人类最同源序列的相同位置但在其它的人免疫球蛋白中频繁出现的事实,是必须牢记的。出于这个原因,替换到构架中的鼠的氨基酸集合不是唯一的。用不同数量替换以获得不同形式的修饰抗体是可能的。该突变是通过重叠PCR来进行。
克隆和表达人源化抗体P3hu和1E10hu。
按照所叙述的关于嵌合抗体的方法,与P3hu和1E10hu对应的遗传构建体被克隆到表达载体中。结果产生的构建体是P3VKhu-PAG4622或1E10Vkhu-PAG4622和P3VHhu-PAH4604和1E10VHhu-PAH4604。按照关于嵌合抗体的前述方案将它们转染到NS-O细胞中。
重组抗体的纯化。
使用蛋白质A(Pharmacia,Upssala,Sweden)用亲和层析提纯重组抗体。
生物活性。
用ELISA测定的与抗原的特异性结合情况检测了重组抗体的生物活性。
对于重组MAb P3,用甲醇中的GM3(NeuGc)神经节苷脂覆盖微量滴定板。干燥一小时后,用Tris-HCl缓冲液中的1%牛血清白蛋白(BSA)阻断非特异性结合,并在37℃培育一小时。用PBS洗涤孔并且与纯化的重组Mab P3一起在37℃培育一小时。用tris-HCl洗涤孔并且加入与碱性磷酸酶共轭的羊抗人类抗体,之后在37℃培育一小时。最后,洗涤孔,加入含有对硝基苯磷酸的底物缓冲液。半小时后分别测定在405或492nm下的吸收。
对于重组Mabai 1E10,除用Mab P3覆盖孔和用PBS-0.05%Tween20进行洗涤之外,其它ELISA实验条件相似。
实施例
在下面实施例中使用的全部酶,以及试剂和材料均从商业来源获得,除非特别指明。
实施例1.嵌合MAb P3的获得。
如前所述,cDNA的合成是通过使用逆转录酶的反应来获得的,它是用从生产Mab P3的杂交瘤获得的RNA开始的。在该反应中所用的特异性引物的序列如下所示:
对于VH:
5'AGGTCTAGAA(CT)CTCCACACACAGG(AG)(AG)CCAGTGGATAGAC3'
对于VK:
5'GCGTCTAGAACTGGATGGTGGGAAGATGG3'
用Taq聚合酶和特异性的引物通过PCR将cDNA VHP3和cDNAVKP3扩增。引物中包含的限制位点,对于VH是ECORV/NHEI,对于VK是ECORV/SALI。所用引物的序列如下:
对于VH:
引物1(信号肽):
5'GGGGATATCCACCATGG(AG)ATG(CG)AGCTG(TG)GT(CA)AT(CG)CTCTT3'
引物2(CH1):
5'GGGGCTAGCTGCAGAGACAGTGACCAGAGT3'
对于VK:
引物1(信号肽):
5'GGGGATATCCACCATGGAG(TA)CACA(GT)(TA)CTCAGGTCTTT(GA)T3'
引物2(Ck):
5'AGCGTCGACTTACGTTT(TG)ATTTCCA(GA)CTT(GT)GTCCC3'
将PCR产物克隆到TA载体中(TA cloning kit,Invitrogen)。对十二个独立克隆使用T7 DNA Pol(Pharmacia)以双脱氧的方法测序。用同源检索分析,确定了VHP3和VKP3的最同源序列组。根据Kabat分类,VHP3和VKP3序列(图1和图2)分别与IB和V组有很高的同源性。
对于VHP3用限制酶ECORV和NHEI而对于VKP3用限制酶ECORV和SALI消化后,它们被克隆到事先用相同的酶消化过的表达载体中,对于VH和VK分别为PAH4604和PAG4622。该表达载体由SherieMorrison(UCLA,California,USA)提供,它适合于在哺乳动物细胞中的免疫球蛋白的表达。载体PAH 4604已经包含了人IgG1的恒定区和人的PAG 4622(Coloma等人(1992):用于表达使用以聚合酶链式反应生产的可变区的抗体分子的新型载体(Novel vectors forthe expression of antibody molecules using variable regionsgenerated by polymerase chain reaction),J.Immunol.Meth.,152:89-104)。结果产生的构建体是P3VH-PAH4604和P3VK-PAG4622。
将NS-O细胞用10μg P3VK-PAG4622转染,将表达轻链的克隆用10μg P3VH-PAH4604转染,在这两种情况下,在转染前,都用PvuI把DNA直线化,用乙醇沉淀并溶解在50μl PBS中。
用离心法得到大约107的细胞,将其与电穿孔杯中消化过的DNA一起在0.5ml PBS中重新悬浮。置于冰上10分钟后,给细胞一个200 Volts 960 μF的脉冲电流,并且在冰上再放置10分钟。将细胞与加入10%胎牛血清的D'MEM F12一起分置到96孔板中。二或四天后,加入选择培养基(分别含有霉酚酸0,45μg/ml或组氨醇10mM的D'MEM F12)。14天后,转染的克隆可以在肉眼下看到。
用ELISA测定含有转染克隆的孔中培养基中存在的人类抗体。用羊抗人κ轻链(对于生产人κ链的克隆)或抗人IgG(γ链特异的)(对于生产完全抗体的克隆)抗体覆盖微量滴定板孔。用PBST(含0.05% Tween20的磷酸盐缓冲溶液)洗涤后,在37℃把稀释的含有转染产物的培养基加入到每一微量滴定板孔中一小时。孔用PBS-T洗涤,并加入辣根过氧化物酶共轭的羊抗人κ轻链或与碱性磷酸酶共轭的羊抗人IgG(γ链特异的),之后在室温培育一小时。孔用PBS-T洗涤过之后,分别加入含有邻苯二胺或对硝基苯磷酸盐的底物缓冲液。半小时后,分别测定在492或405nm下的吸收。
实施例2.不同形式的人源化抗体P3的获得。
将鼠VHP3和VKP3序列(图1和图2)与人的序列比较。图3和图4显示了最同源的人序列。在鼠P3可变区序列中检测两亲性螺旋片段或潜在的T细胞表位,根据所用方法制定氨基酸替换的审慎的策略,以破坏或人源化鼠序列中的潜在T细胞表位。
对于VHP3的分析得到了(图3)2个两亲性片段,第一个片段包括CDR1、FR2和CDR2的某些残基,第二个片段包括FR3的末端和CDR3。在CDR或涉及结合部位的三维结构的残基中发现了与最同源人序列和鼠的序列的主要差别。由于这个原因,决定不替换任何鼠的VHP3的氨基酸。
关于VKP3的分析也得到了2个两亲性片段(图4),第一个片段包括FR1,第二个片段包括CDR2和FR3的某些残基。决定用最同源人序列中相同位置的残基替换在位置8、9、10、11和13的残基。分别用Pro、Ser、Ser、Leu和Ala替换氨基酸His、Lys、Phe、Met和Thr。替换使用引物1和2和3和4通过重叠PCR进行(Kammann等人(1989)用DNA聚合酶链式反应(PCR)快速插入诱变(Rapidinsertional mutagenesis of DNA polymerase chain reaction(PCR)),Nucleic Acids Res.,17:5404),所用引物的序列如下:
引物1:
5'ATGACCCAGTCTCCTTCTTCTCTTTCCGCGTCAGTAGGAGAC3'
引物2:
5'AGCGTCGACTTACGTTT(TG)ATTTCCA(GA)CTT(GT)GTCCC3'
引物3:
5'GTCTCCTACTGACGCGGAAAGAGAAGAAGGAGACTGGGTCAT3'
引物4:
5'GGGGATATCCACCATGGAG(TA)CACA(GT)(TA)CTCAGGTCTTT(GA)T3'
以序列测定检验点突变。结果产生的构建体是P3Vkhu,并将其克隆到PAG4622表达载体上。结果产生的构建体是P3VKhu-PAG4622。为表达人源化抗体P3,将NS-O细胞用P3VH-PAH4604和P3VKhu-PAG4622转染。
按照前述用于嵌合抗体的电穿孔和检测方法,转染P3hu抗体。
实施例3:嵌合MAb P3的生物活性。
用ELISA测定与抗原的特异性结合情况来检测嵌合MAb P3的生物活性。
对于重组MAb P3,用甲醇中的GM3(NeuGc)神经节苷脂覆盖微量滴定板。在37℃干燥一小时后,用Tris-HCl缓冲液中的1%牛血清白蛋白(BSA)阻断非特异性结合,并在37℃培育一小时。用PBS洗涤孔并且与纯化的重组Mab P3一起在37℃培育一小时。用tris-HCl洗涤孔并且加入与碱性磷酸酶共轭的羊抗人类抗体,之后在37℃培育一小时。最后,用tris-HCl洗涤孔,加入含有对硝基苯磷酸的底物缓冲液。半小时后测定在405nm下的吸收。
嵌合Mab T1被作为阴性对照来应用。
图5显示了嵌合MAb P3与抗原的特异性结合的情况。
实施例4.嵌合MAb 1E10的获得。
如前所述,cDNA的合成是通过使用逆转录酶的反应来获得的,它是用从生产Mab 1E10的杂交瘤获得的RNA开始的。在该反应中所用的特异性引物的序列如下所示:
对于VH:
5'GGGGCTAGCTGAGGAGACTGTGAGAGTGGT3'
对于VK:
5'GCGTCTAGAACTGGATGGTGGGAAGATGGA3'
用Taq聚合酶和特异性的引物通过PCR将cDNA VH1E10和cDNAVK1E10扩增。
对于VH:
引物1(信号肽):
5'GGGGATATCCACCATGG(AG)ATG(CG)AGCTG(TG)GT(CA)AT(CG)CTCTT3'
引物2(CH1):
5'GGGGCTAGCTGAGGAGACTGTGAGAGTGGT3'
对于VK:
引物1(信号肽):
5'GGGGTTAACCACCATGAGG(GT)CCCC(AT)GCTCAG(CT)T(CT)CT(TG)GG(GA)3'
引物2(Ck):
5'AGCGTCGACTTACGTTT(TG)ATTTCCA(GA)CTT(GT)GTCCC3'
将PCR产物克隆到TA载体中(TA cloning kit,Invitrogen)。对十二个独立克隆使用T7 DNA Pol(Pharmacia)以双脱氧的方法测序(图7和图8)。通过同源检索分析,确定了VH1E10和VK1E10的最同源序列组。根据Kabat分类,VH1E10和VK1E10序列分别与混杂的组和V组有很高的同源性。
对于VH1E10用限制酶ECORV和NHEI而对于VK1E10用限制酶HincII和SALI消化后,它们被克隆到事先用相同的酶消化过的表达载体中,对于VH和VK分别为PAH4604和PAG4622。该表达载体由Sherie Morrison(UCLA,California,USA)提供,它适合于在哺乳动物细胞中的免疫球蛋白的表达。载体PAH 4604已经包括了人IgG1的恒定区和人的PAG 4622(Coloma等人(1992):用于表达使用以聚合酶链式反应生产的可变区的抗体分子的新型载体(Novelvectors for the expression of antibody molecules usingvariable regions generated by polymerase chain reaction),J.Immunol.Meth.,152:89-104)。结果产生的构建体是1E10VH-PAH4604和1E10VK-PAG4622。
将NS-O细胞用10μg 1E10VK-PAG4622转染,将表达轻链的克隆用10μg 1E10VH-PAH4604转染,在这两种情况中,在转染前,都用PvuI把DNA直线化,用乙醇沉淀并溶解在50μl PBS中。
用离心法得到大约107的细胞,将其与电穿孔杯中消化过的DNA一起在0.5ml PBS中重新悬浮。置于冰上10分钟后,给细胞一个200 Volts 960 μF的脉冲电流,并且在冰上再放置10分钟。将细胞与加入10%胎牛血清的D'MEM F12一起分置到96孔板中。二或四天后,加入选择培养基(分别含有霉酚酸0,45μg/ml或组氨醇10mM的D'MEM F12)。14天后,转染的克隆可以在肉眼下看到。
用ELISA测定含有转染克隆的孔中培养基中存在的人类抗体。用羊抗人κ轻链(对于生产人κ链的克隆)或抗人IgG(γ链特异的)(对于生产完全抗体的克隆)抗体覆盖微量滴定板孔。用PBST(含0.05%Tween20的磷酸盐缓冲溶液)洗涤后,在37℃把稀释的含有转染产物的培养基加入到每一微量滴定板孔中一小时。孔用PBS-T洗涤,并加入与辣根过氧化物酶共轭的羊抗人κ轻链或与碱性磷酸酶共轭的羊抗人IgG(γ链特异的),之后在室温培育一小时。孔用PBS-T洗涤过之后,分别加入含有邻苯二胺或对硝基苯磷酸盐的底物缓冲液。半小时后,分别测定在492或405nm下的吸收。
实施例5.不同形式的人源化抗体1E10的获得。
将鼠VH 1E10和VK 1E10序列(图6和图7)与人的序列比较。图8和图9显示了最同源人序列。在鼠1E10可变区序列中检测两亲性螺旋片段或潜在的T细胞表位,根据所用方法制定氨基酸替换的审慎的策略,以破坏或人源化鼠序列中的潜在T细胞表位。
对于VH1E10的分析得到了(图8)3个两亲性片段,第一个片段包括FR1,第二个片段包括FR2,第三个片段包括FR3。决定用最同源人序列中相同位置的残基替换在位置5、40、42和87(根据Kabat的编号为83)的残基。氨基酸Gln、Arg、Glu分别被Val、Ala、Gly和Arg替换。
替换使用一系列不同的引物通过重叠PCR进行(Kammann等人(1989)用DNA聚合酶链式反应(PCR)快速插入诱变(Rapidinsertional mutagenesis of DNA polymerase chain reaction(PCR)),Nucleic Acids Res.,17:5404)。
用于在重链位置5突变的引物是1和2和3和4,其序列如下
引物1:
5'CAGGTTCAGCTGGTGCAGTCTGGAGCT3'
引物2:
5'GGGGCTAGCTGAGGAGACTGTGAGAGTGGT3'
引物3:
5'AGCTCCAGACTGCACCAGCTGAACCTG3'
引物4:
5'GGGGATATCCACCATGG(AG)ATG(CG)AGCTG(TG)GT(CA)AT(CG)CTCTT3'
用测序检查位置5的点突变后,位置40和42的突变被引入。
用于重链位置40和42突变的引物:
引物1:
5'TGGGTGAGGCAGGCGCCTGGGCAGGGACTTGAG3'
引物2:
5'GGGGCTAGCTGAGGAGACTGTGAGAGTGGT3'
引物3:
5'CTCAAGTCCCTGCCCAGGCGCCTGCCTCACCCA3'
引物4:
5'GGGGATATCCACCATGG(AG)ATG(CG)AGCTG(TG)GT(CA)AT(CG)CTCTT3'
用测序检查位置40和42的点突变后,位置87(根据Kabat的编号为83)的突变被引入。
用于重链位置87(根据Kabat的编号为83)突变的引物:
引物1:
5'CTCAGCAGGCTGCGGTCTGAGGACTCT3'
引物2:
5'GGGGCTAGCTGAGGAGACTGTGAGAGTGGT3'
引物3:
5'AGAGTCCTCAGACCGCAGCCTGCTGAG3'
引物4:
5'GGGGATATCCACCATGG(AG)ATG(CG)AGCTG(TG)GT(CA)AT(CG)CTCTT3'
因为残基涉及结合部位的三维结构,因此不进行其他替换。
用测序核实点突变。结果产生的构建体是1E10VHhu,并将它克隆到PAH4604表达载体中。结果产生的构建体是1E10VH-PAH4604。
关于VK1E10的分析也得到了3个两亲片段(图9),第一个片段包括FR1,第二个片段包括CDR1,第三个片段包括FR3。决定用最同源人序列中相同位置的残基替换在位置7、8和15的残基。分别用Ser、Pro和Val替换氨基酸Thr、Thr和Leu。替换使用引物1和2和3和4通过重叠PCR进行(Kammann等人(1989)用聚合酶链反应(PCR)快速插入诱变DNA(Rapid insertional mutagenesis ofDNA by polymerase chain reaction(PCR)),Nucleic AcidsRes.,17:5404),所用引物的序列如下:
用于轻链位置7、8和15突变的引物:
引物1:
5'CAGATGACACAGTCTCCTTCCTCCCTGTCTGCCTCTGTGGGAGACAGAGTC3'
引物2:
5'AGCGTCGACTTACGTTT(TG)ATTTCCA(GA)CTT(GT)GTCCC3'
引物3:
5'GACTCTGTCTCCCACAGAGGCAGACAGGGAGGAAGGAGACTGTGTCATCTG3'
引物4:
5'GGGGTTAACCACCATGAGG(GT)CCCC(AT)GCTCAG(CT)T(CT)CT(TG)GG(GA)3'
用测序核实点突变。结果产生的构建体是1E10Vkhu,并将其克隆到PAG 4622表达载体中。结果产生的构建体是1E10VKhu-PAG4622。
为表达人源化抗体1E10,将NS-O细胞用1E10VHhu-PAH4604和1E10VKhu-PAG4622转染。
按照前述用于嵌合抗体的电穿孔和检测方法,转染1E10hu抗体。
实施例6:嵌合MAb1E10的生物活性。
用ELISA测定与抗原的特异性结合情况来检测嵌合MAb 1E10的生物活性。
对于重组MAb 1E10,用Mab P3覆盖微量滴定板。用PBST(含0.05%吐温20的磷酸缓冲盐溶液)洗涤后,用PBST中的1%牛血清白蛋白(BSA)阻断非特异性结合,在37℃培育一小时。洗涤孔并且与纯化的重组Mab 1E10一起在37℃培育一小时。用PBST洗涤孔并且加入与碱性磷酸酶共轭的羊抗人抗体,之后在37℃培育一小时。最后,用PBST洗涤孔,加入含有对硝基苯磷酸的底物缓冲液。半小时后测定在405nm下的吸收。
嵌合Mab C5被作为阴性对照来应用。
图10显示了嵌合MAb1E10与Mab P3的特异性结合的情况。
附图简述:
图1:VHP3DNA和推出的氨基酸序列。序列根据Kabat的编号排列(Kabat等人(1991),Sequences of proteins ofimmunological interest,第五版,National Institute ofHealth),出现的CDR用虚线标明。
图2:VKP3 DNA和推出的氨基酸序列。序列根据Kabat的编号排列(Kabat和其同事(1991),Sequences of proteins ofimmunological interest,第五版,National Institute ofHealth),出现的CDR用虚线标明。
图3:VHP3与最同源的人的序列比对。两亲性片段加下划线并且CDR为黑体字。
图4:VKP3与最同源的人的序列比对。两亲性片段加下划线并且CDR为黑体字。
图5:嵌合Mab P3与GM3(NeuGc)的特异性结合。用ELISA测定了Mab P3和Mab T1(阴性对照)的不同浓度。将微量滴定板用甲醇中的GM3(NeuGc)和GM3(NeuAc)(阴性对照)神经节苷脂覆盖,并测定特异性结合情况。
图6:VH1E10 DNA和推出的氨基酸序列。序列根据Kabat的编号排列(Kabat和其同事(1991),Sequences of proteins ofimmunological interest,第五版,National Institute ofHealth),出现的CDR用虚线标明。
图7:VK1E10 DNA和推出的氨基酸序列。序列根据Kabat的编号排列(Kabat等人(1991),Sequences of proteins ofimmunological interest,第五版,National Institute ofHealth),出现的CDR用虚线标明。
图8:VH1E10与最同源的人的序列比对。两亲性片段加下划线并且CDR为黑体字。
图9:VK1E10与最同源的人的序列比对。两亲性片段加下划线并且CDR为黑体字。
图10:嵌合Mab 1E10与鼠类Mab P3的特异性结合。用ELISA测定了Mab 1E10和MAb C5(阴性对照)的不同浓度。将微量滴定板用Mab P3和Mab A3(阴性对照)覆盖,并测定其特异性结合情况。
Claims (6)
1.一种人源化的单克隆抗体,它从可识别鼠类单克隆抗体P3的鼠类抗独特型单克隆抗体1E10得到,所述单克隆抗体1E10由保藏号为ECACC 97112901的杂交瘤细胞系生产,其中其重链和轻链的高度可变区的序列如下:
重链
CDR1:SYDIN
CDR2:WIFPGDGSTKYNEKFKG
CDR3:EDYYDNSYYFDY
轻链
CDR1:RASQDISNYLN
CDR2:YTSRLHSG
CDR3:QQGNTLPWT
其重链和轻链的构架区(FRs)的序列如下:
重链
FR1:QVQLQQSGAELVKPGASVKLSCKASGYTFT
FR2:WVRQRPEQGLEWIG
FR3:KATLTTDKSSSTAYMQLSRLTSEDSAVYFCAR
FR4:WGQGTTLTV
轻链
FR1:DIQMTQTTSSLSASLGDRVTISC
FR2:WYQQKPDGTVKLLIY
FR3:VPSRFSGSGSGTDYSLTISNLEQEDIATYFC
FR4:FGGGTKLESK
并且其包括下列点突变:
轻链
位置7:用Ser替换Thr
位置8:用Pro替换Thr
位置15:用Val替换Leu
重链
位置5:用Val替换Gln
位置40:用Ala替换Arg
位置42:用Gly替换Glu
位置87(根据Kabat的编号为83):用Arg替换Thr。
2.根据权利要求1所述的单克隆抗体,其中重链恒定区包括γ-1链的氨基酸序列,轻链恒定区包括κ链的氨基酸序列,γ-1链和κ链都源自人的免疫球蛋白。
3.一种细胞系,它可用于生产权利要求1-2的任意一种单克隆抗体。
4.一种药物组合物,它用于恶性乳腺肿瘤或黑素瘤或它们之一的转移或复发的治疗,该药物组合物包括权利要求1-2的任意一种单克隆抗体。
5.一种药物组合物,它用于恶性乳腺肿瘤或黑素瘤或它们之一的转移或复发的“体内”定位和鉴定,该药物组合物包括权利要求1-2的任意一种单克隆抗体。
6.权利要求1-2的任意一种单克隆抗体在制备用于恶性乳腺肿瘤或黑素瘤或它们之一的转移或复发的治疗的药物中的应用。
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FI20055398A0 (fi) | 2005-07-08 | 2005-07-08 | Suomen Punainen Risti Veripalv | Menetelmä solupopulaatioiden evaluoimiseksi |
EP2594591B1 (en) * | 2005-08-11 | 2018-06-06 | Arpi Matossian-Rogers | Tcr-v-beta related peptides for treatment and diagnosis of autoimmune disease |
JP5415071B2 (ja) * | 2005-08-19 | 2014-02-12 | ワイス・エルエルシー | Gdf−8に対するアンタゴニスト抗体ならびにalsおよびその他のgdf−8関連障害の処置における使用 |
ITFI20060163A1 (it) * | 2006-06-29 | 2006-09-28 | Menarini Internat Operations Luxembourg Sa | Composizione farmaceutica contenente un anticorpo monoclonale anti idiotipico anti-ca-125 ed alluminio |
TWI434855B (zh) | 2006-11-21 | 2014-04-21 | Hoffmann La Roche | 結合物及其在免疫分析中作為參考標準之用途 |
WO2009104649A1 (ja) * | 2008-02-22 | 2009-08-27 | 片山化学工業株式会社 | 合成糖脂質含有リポソーム |
EP2166085A1 (en) | 2008-07-16 | 2010-03-24 | Suomen Punainen Risti Veripalvelu | Divalent modified cells |
TWI516501B (zh) | 2008-09-12 | 2016-01-11 | 禮納特神經系統科學公司 | Pcsk9拮抗劑類 |
NZ594985A (en) * | 2009-03-10 | 2013-07-26 | Biogen Idec Inc | Anti-bcma (b-cell maturation antigen, cd269, tnfrsf17) antibodies |
SG174992A1 (en) | 2009-04-01 | 2011-11-28 | Genentech Inc | Anti-fcrh5 antibodies and immunoconjugates and methods of use |
CU23736A1 (es) * | 2009-05-04 | 2011-11-15 | Centro Inmunologia Molecular | Anticuerpos que reconocen sulfatidos y proteoglicanos sulfatados y su uso |
WO2011026242A1 (en) * | 2009-09-03 | 2011-03-10 | Vancouver Biotech Ltd. | Monoclonal antibodies against gonadotropin-releasing hormone receptor |
WO2012096994A2 (en) * | 2011-01-10 | 2012-07-19 | Emory University | Antibodies directed against influenza |
CU24070B1 (es) * | 2011-12-27 | 2015-01-29 | Ct De Inmunología Molecular | Composiciones farmacéuticas para el tratamiento de tumores que expresan regf y gangliósidos n-glicolilados gm3 (neugcgm3) |
EP2641916A1 (en) * | 2012-03-23 | 2013-09-25 | Centre National de la Recherche Scientifique (C.N.R.S) | Novel antibodies anti-sPLA2-IIA and uses thereof |
MY177331A (en) | 2012-06-15 | 2020-09-12 | Pfizer | Improved antagonist antibodies against gdf-8 and uses therefor |
AR096687A1 (es) | 2013-06-24 | 2016-01-27 | Genentech Inc | Anticuerpos anti-fcrh5 |
UA120753C2 (uk) | 2013-12-17 | 2020-02-10 | Дженентек, Інк. | Біспецифічне антитіло до сd3 та cd20 |
JP2017512759A (ja) * | 2014-04-10 | 2017-05-25 | オービーアイ ファーマ インコーポレイテッド | 抗体、前記抗体を産生するハイブリドーマ、前記抗体を含む薬学的組成物及びその用途 |
TWI715587B (zh) | 2015-05-28 | 2021-01-11 | 美商安可美德藥物股份有限公司 | Tigit結合劑和彼之用途 |
CA2986928A1 (en) | 2015-06-16 | 2016-12-22 | Genentech, Inc. | Humanized and affinity matured antibodies to fcrh5 and methods of use |
WO2018017864A2 (en) * | 2016-07-20 | 2018-01-25 | Oncomed Pharmaceuticals, Inc. | Pvrig-binding agents and uses thereof |
CA3044664C (en) | 2016-11-30 | 2022-11-22 | Oncomed Pharmaceuticals, Inc. | Methods for treatment of cancer comprising tigit-binding agents |
CU20170173A7 (es) * | 2017-12-27 | 2019-11-04 | Ct Inmunologia Molecular | Nano-partículas que contienen el gangliósido gm3 como inmunomoduladoras |
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