CN114555636A - 针对犬cd20的单克隆抗体或抗体片段 - Google Patents
针对犬cd20的单克隆抗体或抗体片段 Download PDFInfo
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- CN114555636A CN114555636A CN202080069648.8A CN202080069648A CN114555636A CN 114555636 A CN114555636 A CN 114555636A CN 202080069648 A CN202080069648 A CN 202080069648A CN 114555636 A CN114555636 A CN 114555636A
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Abstract
本发明解决的问题在于提供在效果方面比现有抗体更优异的针对犬CD20的单克隆抗体。提供一种针对犬CD20的单克隆抗体或抗体片段,所述单克隆抗体包含:重链可变区,其包含由SEQ ID NO:1表示的氨基酸序列、或在由SEQ ID NO:1表示的氨基酸序列中缺失、置换或添加一个至多个氨基酸的氨基酸序列;轻链可变区,其包含由SEQ ID NO:2表示的氨基酸序列、或在由SEQ ID NO:2表示的氨基酸序列中缺失、置换或添加一个至多个氨基酸的氨基酸序列;和轻链恒定区,其包含由SEQ ID NO:3表示的氨基酸序列、或在由SEQ ID NO:3表示的氨基酸序列中缺失、置换或添加一个至多个氨基酸的氨基酸序列。
Description
技术领域
本发明涉及针对犬CD20的单克隆抗体或抗体片段。本发明更具体地涉及具有比现有抗体更优异的效果的针对犬CD20的单克隆抗体或抗体片段。
背景技术
CHOP疗法是多药抗癌药疗法并且已用于犬的B细胞淋巴瘤;然而,2年存活率低至20%,并且在缓解后的大部分病例中发生复发,使得B细胞淋巴瘤不可通过化学疗法来充分治疗。因此,近年来,已开发靶向CD20的抗体药物作为新的治疗方法。例如,专利文献1和2公开了针对犬CD20的胞外区的单克隆抗体或抗体片段,其具有由特定氨基酸序列组成的重链可变区和由特定氨基酸序列组成的轻链可变区。
专利文献3和4公开了结合至CD20并含有由特定氨基酸序列表示的L链可变区和H链可变区的人源化单克隆抗体或其抗原结合片段,和结合至CD20并含有具有CDR1、CDR2和QQCDR3的L链可变区和H链可变区的嵌合或人源化单克隆抗体或其抗原结合片段。专利文献5至7公开了在轻链恒定区和重链恒定区中也含有特定氨基酸序列的单克隆抗体。
尽管以这种方法开发了针对CD20的各种抗体,效果可取决于抗体而变化,并且不可以说已获得具有优异效果的抗体。因此,本发明人开发了针对犬CD20的大鼠-犬嵌合抗体(参见专利文献8),并且在本发明中试图产生具有更优异效果的针对犬CD20的单克隆抗体。
现有技术文献
专利文献
[专利文献1]日本特表2014-532649号公报
[专利文献2]日本特开2016-13104号公报
[专利文献3]日本特表2006-500904号公报
[专利文献4]日本特开2009-291197号公报
[专利文献5]日本特开2016-41750号公报
[专利文献6]日本特开2019-507128号公报
[专利文献7]日本特开2015-501146号公报
[专利文献8]日本特开2019-26625号公报
发明内容
发明要解决的问题
本发明所要解决的问题是提供具有比现有抗体更优异的效果的针对犬CD20的单克隆抗体。
用于解决问题的方案
作为为了解决本发明的问题而深入研究的结果,本发明人发现以下的针对犬CD20的单克隆抗体或抗体片段显示比现有抗体更优异的效果,从而完成本发明。该针对犬CD20的单克隆抗体或抗体片段是具有以下的针对犬CD20的单克隆抗体或抗体片段:重链可变区,其由SEQ ID NO:1的氨基酸序列、或其中在SEQ ID NO:1的氨基酸序列中缺失、置换、或添加一个或多个氨基酸的氨基酸序列组成,和轻链可变区,其由SEQ ID NO:2的氨基酸序列、或其中在SEQ ID NO:2的氨基酸序列中缺失、置换、或添加一个或多个氨基酸的氨基酸序列组成,并且具有轻链恒定区,其由SEQ ID NO:3的氨基酸序列、或其中在SEQ ID NO:3的氨基酸序列中缺失、置换、或添加一个或多个氨基酸的氨基酸序列组成。
因此,用于解决上述问题的本发明涉及下文中(1)至(11)中所描述的针对犬CD20的单克隆抗体、抗体片段等。
(1)一种针对犬CD20的单克隆抗体或抗体片段,其包含:重链可变区,其由SEQ IDNO:1的氨基酸序列、或其中在SEQ ID NO:1的氨基酸序列中缺失、置换或添加一个或多个氨基酸的氨基酸序列组成;和轻链可变区,其由SEQ ID NO:2的氨基酸序列、或其中在SEQ IDNO:2的氨基酸序列中缺失、置换或添加一个或多个氨基酸的氨基酸序列组成,和包含轻链恒定区,其由SEQ ID NO:3的氨基酸序列、或其中在SEQ ID NO:3的氨基酸序列中缺失、置换或添加一个或多个氨基酸的氨基酸序列组成。
(2)根据(1)所述的抗体或抗体片段,其包含重链可变区,所述重链可变区由SEQID NO:4或5的氨基酸序列、或其中在SEQ ID NO:4或5的氨基酸序列中缺失、置换或添加一个或多个氨基酸的氨基酸序列组成。
(3)根据(1)或(2)所述的抗体或抗体片段,其中所述抗体或抗体片段是去岩藻糖基化的(afucosylated)。
(4)一种用于减少犬B细胞的组合物,其包含根据(1)至(3)中任一项所述的抗体或抗体片段作为活性成分。
(5)一种用于治疗由于犬B细胞的增加而导致的疾病的治疗组合物,其包含根据(1)至(3)中任一项所述的抗体或抗体片段作为活性成分。
(6)根据(5)所述的治疗组合物,其中所述疾病是B细胞淋巴瘤、白血病、或自身免疫性疾病。
(7)一种犬B细胞检测试剂盒,其包含:根据(1)至(3)中任一项所述的抗体或抗体片段。
(8)一种用于诊断由于犬B细胞的增加而导致的疾病的试剂盒,其包含:根据(1)至(3)中任一项所述的抗体或抗体片段。
(9)一种用于减少犬B细胞的方法,其使用以上述(1)至(3)中任一项所述的抗体或抗体片段作为活性成分的组合物。
(10)一种用于治疗由于犬B细胞的增加而导致的疾病的方法,其使用以上述(1)至(3)中任一项所述的抗体或抗体片段作为活性成分的组合物。
(11)根据上述(10)所述的治疗方法,其中,所述疾病是B细胞淋巴瘤、白血病或自身免疫性疾病。
发明的效果
本发明能够提供具有比现有抗体更优异的效果的针对犬CD20的单克隆抗体或抗体片段。使用该单克隆抗体或抗体片段作为活性成分,本发明还能够提供用于减少犬B细胞的组合物和用于减少犬B细胞的方法,或用于治疗由于犬B细胞的增加而导致的疾病如B细胞淋巴瘤、白血病、或自身免疫性疾病的治疗组合物、和用于治疗由于犬B细胞的增加而导致的疾病的方法。
附图说明
[图1]图1是显示4E1-7-B抗体和4E1-7-C抗体以浓度依赖性方式结合至CLBL-1细胞的图(实施例)。
[图2]图2是显示4E1-7-B抗体和4E1-7-C抗体结合至EL-4/cCD20细胞的图(实施例)。
[图3]图3是显示4E1-7-B_f抗体以与4E1-7-B抗体相同的方式结合至CLBL-1细胞的图(实施例)。
[图4]图4是显示4E1-7-B抗体和4E1-7-C抗体对CLBL-1细胞的增殖的效果的图(实施例)。
[图5]图5是显示4E1-7-B_f抗体对CLBL-1细胞的增殖的效果的图(实施例)。
[图6]图6是显示4E1-7-B抗体和4E1-7-C抗体对CLBL-1/luc细胞的细胞溶解的效果的图(实施例)。
[图7]图7是显示4E1-7-B抗体和4E1-7-B_f抗体对CLBL-1/luc细胞的细胞病变效果的图(实施例)。
[图8]图8是显示4E1-7-B抗体和4E1-7-C抗体对CLBL-1/luc细胞的细胞死亡诱导效果的图(实施例)。
[图9]图9是显示4E1-7-B抗体和4E1-7-B_f抗体对CLBL-1/luc细胞的细胞死亡诱导效果的图(实施例)。
[图10]图10的A是显示PBS的施用对小鼠中肿瘤生长的影响的图(实施例)。图10的B是显示PBMC的施用对小鼠中肿瘤生长的影响的图(实施例)。
[图11]图11的A是显示4E1-7-B抗体和PBMC的施用对小鼠中肿瘤生长的影响的图(实施例)。图11的B是显示4E1-7-B_f抗体和PBMC的施用对小鼠中肿瘤生长的影响的图(实施例)。
[图12]图12是显示4E1-7-B抗体和4E1-7-B_f抗体的施用对犬外周血中CD21+B细胞表达的效果的图(实施例)。
具体实施方式
本发明的“抗犬CD20单克隆抗体或抗体片段”是识别犬CD20分子的抗体或抗体片段,并且是指具有以下的针对犬CD20的单克隆抗体或抗体片段:重链可变区,其由SEQ IDNO:1的氨基酸序列、或其中在SEQ ID NO:1的氨基酸序列中缺失、置换或添加一个或多个氨基酸的氨基酸序列组成,和轻链可变区,其由SEQ ID NO:2的氨基酸序列、或其中在SEQ IDNO:2的氨基酸序列中缺失、置换或添加一个或多个氨基酸的氨基酸序列组成,和具有轻链恒定区,其由SEQ ID NO:3的氨基酸序列或其中在SEQ ID NO:3的氨基酸序列中缺失、置换或添加一个或多个氨基酸的氨基酸序列组成。
在本说明书中,多个意味着15个、14个、13个、12个、11个、10个、9个、8个、7个、6个、5个、4个、3个、或2个。多个意味着5个、4个、3个或2个等。
本发明的“抗犬CD20单克隆抗体或抗体片段”可良好结合至犬CD20的胞外区,并且可用于使用犬的活细胞和组织的FACS分析和通过荧光显微镜观察等的CD20阳性细胞的动力学等的分析。
本发明的“抗犬CD20单克隆抗体或抗体片段”可以是针对去岩藻糖基化的犬CD20的单克隆抗体或抗体片段。
本发明的“用于减少犬B细胞的组合物”是指含有本发明的“抗犬CD20单克隆抗体或抗体片段”作为活性成分的、用于减少犬B细胞的组合物。组合物可以含有除了这些活性成分以外的其它组分,只要该组分有利地用于减低犬B细胞即可,并且可进一步含有例如抗癌剂或放射性核素等药物。组合物可含有药学上可接受的添加剂,这样的添加剂的实例包括例如水等溶剂、表面活性剂、氯化钠、柠檬酸钠、无水柠檬酸、pH调节剂等。
本发明的“用于治疗由于犬B细胞的增加而导致的疾病的治疗组合物”是指含有本发明的“抗犬CD20单克隆抗体或抗体片段”作为活性成分的、用于治疗由于犬B细胞的增加而导致的疾病的治疗组合物。
这样的疾病的实例包括由于犬B细胞的增加而导致的犬B细胞淋巴瘤、与增加的B细胞的致瘤性转化相关的白血病、和由正常B细胞的过度增加而导致的自身免疫性疾病。
例如,当要治疗的疾病为B细胞淋巴瘤时,向患病犬施用本发明的“用于治疗由于犬B细胞的增加而导致的疾病的治疗组合物”允许活性成分“抗犬CD20单克隆抗体或抗体片段”结合至B细胞淋巴瘤细胞的表面上过表达的CD20,从而通过例如抗体依赖性细胞毒性(ADCC)活性、补体依赖性细胞毒性(CDC)活性、和细胞凋亡诱导等机制,使得犬B细胞淋巴瘤细胞的杀伤等成为可能。
本发明的“用于治疗由于犬B细胞的增加而导致的疾病的治疗组合物”可以是由活性成分“抗犬CD20单克隆抗体或抗体片段”构成的组合物,或者可以是除了这些活性成分以外进一步含有例如抗癌剂和放射性核素等其它组分和药物的组合物。
组合物可具有任何的药剂的形式,只要所述形式对于治疗由于犬B细胞的增加而导致的疾病为有效的即可,并且可作为用于静脉注射或输注的液体或粉末、片剂、胶囊剂等提供。
使用用于本发明的治疗的组合物的治疗方法可包括,例如,通过静脉注射、输注等、以约一周一次的间隔施用组合物,使得达到1至10mg/kg的本发明的“抗犬CD20单克隆抗体或抗体片段”。
本发明的“犬B细胞检测试剂盒”是指用于通过使用本发明的“抗犬CD20单克隆抗体或抗体片段”来检测样品中是否含有犬B细胞的试剂盒。本发明的“用于诊断由于犬B细胞的增加而导致的疾病的试剂盒”是指用于诊断感兴趣的犬是否患有由于犬B细胞的增加而导致的疾病如犬B细胞淋巴瘤、白血病、和自身免疫性疾病的试剂盒。
这些试剂盒至少包括本发明的“抗犬CD20单克隆抗体或抗体片段”作为构成成分之一。
包括在本发明的试剂盒中的“抗犬CD20单克隆抗体或抗体片段”可为非标记的或可为由常规已知标记物质如酶、荧光物质、或放射性物质标记的。
作为这样的标记物质的酶的实例包括过氧化物酶、β-半乳糖苷酶、碱性磷酸酶、葡萄糖氧化酶、乙酰胆碱酯酶、乳酸脱氢酶、淀粉酶等。荧光物质的实例包括异硫氰酸荧光素(FITC)、四甲基罗丹明异硫氰酸酯(TRITC)、Cy3、Cy5、R-藻红蛋白、B-藻红蛋白、AlexaFluor 488或Alexa Fluor 647等,放射性物质的实例包括氚、碘-125、碘-131等。可使用生物素、链霉亲和素等。
在使用本发明的“犬B细胞检测试剂盒”和“用于诊断由于犬B细胞的增加而导致的疾病的试剂盒”时,可使用有利地用于检测犬B细胞和诊断由于犬B细胞的增加而导致的疾病的任何常规已知试剂、设备等。
从例如要诊断的犬的血液等活组织分离淋巴细胞,并使包括在这些试剂盒中的“抗犬CD20单克隆抗体或抗体片段”作用于淋巴细胞。通过由FACS分析、荧光显微镜观察等来观察CD20阳性细胞的动力学和形态,可进行犬B细胞的检测和由于犬B细胞的增加而导致的疾病的诊断。
通过使用这些试剂盒,活细胞可用作要检测或要诊断的对象,并且不需要细胞固定操作等,使得可准确地且容易地进行检测或诊断。
下文中描述本发明的实施例;然而,本发明不限于此。
实施例
抗犬CD20单克隆抗体的制备
<样品制备>
1)细胞的制备和培养
(1)使用的犬B细胞淋巴瘤细胞系为CLBL-1(Rutgen博士(维也纳兽医大学(University of Veterinary Medicine Vienna))分发),使用的犬T细胞淋巴瘤细胞系为UL-1细胞(山口大学(Yamaguchi University))、Nody-1细胞(山口大学)、Ema细胞(山口大学)、CLK细胞(山口大学)、CLC细胞(山口大学)、CLGL-90(Wellman博士(俄亥俄州立大学(Ohaio State University))分发)、17-71(Kurzman博士(威斯康星大学(WisconsinUniversity))分发)、人类淋巴母细胞细胞系(Jurkat细胞)(ATCC:美国典型培养物保藏中心(American Type Culture Collection))、小鼠类淋巴母细胞细胞系(EL-4细胞)(ATCC)、和小鼠骨髓瘤细胞系(P3U1细胞)(ATCC)。通过使用加湿培养箱(5%CO2、37℃)、在R10培养基(补充有10%FBS、100U/ml青霉素、100μg/ml链霉素、和55μMβ-巯基乙醇的RPMI 1640培养基)中培养所有这些细胞。
大鼠肾脏细胞系(NRK细胞)(Cosmo Bio)、人肾脏细胞系(HEK293T细胞)(CosmoBio)和包装细胞系(PLAT-E细胞系,PLAT-gp细胞系,和PG13细胞系)(Cosmo Bio)全部通过使用加湿培养箱(5%CO2、37℃)、在D10培养基(补充有10%FBS、100U/ml青霉素、100μg/ml链霉素、和55μMβ-巯基乙醇的DMEM培养基)中培养。
在山口大学动物医疗中心(Yamaguchi University Animal Medical Center),通过使用LymphoprepTM(Axis-Shield Ltd.)、根据常规方法从健康比格犬分离犬外周血单核细胞(PBMC)。对一些分离的PBMC直接进行流式细胞术染色或接种至小鼠作为效应细胞。在加湿培养箱(5%CO2、37℃)中、在补充有1,000IU/ml人重组IL-2(ChironTherapeutics,Emeryville)的R10培养基中培养细胞7天用于ADCC检验作为淋巴因子激活杀伤(LAK)效应细胞。
(2)肿瘤细胞
从为了诊断和治疗而访问山口大学动物医疗中心的犬的增大的淋巴结分离原发性淋巴瘤细胞。在通过细胞学诊断来确证所有样品含有90%以上的成淋巴细胞后,离心样品并在取得所有者的同意的情况下用于流式细胞术染色。
2)分子克隆
通过使用健康比格犬的子宫颈淋巴结和脾脏来进行犬CD20(下文中,有时简称为CD20)、IgG重链恒定区、和轻链恒定区的分子克隆。
通过PCR、使用犬CD20的核苷酸序列(NCBI登记号:AB210085)作为模板和表1中所描述的引物来扩增CD20、IgG重链区、和IgGκ轻链区。用于本实施例的所有引物通过寄售方式获得。
使用KOD DNA聚合酶-Plus(TOYOBO)、通过95℃下2分钟的变性步骤、接着95℃下30秒、56℃下30秒、和72℃下1至1.5分钟的30个循环来进行PCR。其后,在存在Taq DNA聚合酶的情况下,在72℃下进行培养10分钟。
通过使用TOPO TA克隆试剂盒(Thermo Fisher Scientific)将扩增产物连接至载体,连接至CD20的质粒命名为pCR-cCD20;连接至IgG重链区的质粒中的、连接至IgGB的质粒和连接至IgGC的质粒分别命名为pCR-H-B和pCR-H-C;和连接至IgGκ轻链区的质粒命名为pBS-κ。通过使用ABI PRISM 3100-Avant测序仪(山口大学遗传研究中心DNA核心设施(DNACore Facility,Genetic Research Center,Yamaguchi University))来分析这些质粒中的扩增产物的碱基序列。
表1
3)表达质粒的制备
(1)FLAG标记的犬CD20表达质粒的制备
通过PCR、使用表2中所描述的YTM1233引物和YTM1234引物来扩增2)中制备的pCR-cCD20,然后通过使用YTM1233引物和YTM838引物来进行再次的PCR。
用BamHI切割扩增产物并连接至pMXs-IP(Kurzman博士(威斯康星大学)分发)的BamHI和SnaBI位点以获得pMx-IP-cCD20-flag#4。将该pMx-IP-cCD20-flag#4的BamHI-HincII片段插入至CSII-CMV-MCS-IRES2-Bsd(由DNA Bank,Ibaraki,RIKEN BioResourceCenter分发)的BamHI-HpaI位点以制备CSII-CMV-cCD20-Flag-IP#4作为慢病毒表达载体。
(2)用于荧光素酶的逆转录病毒表达质粒的制备
通过使用表2中所描述的引物YTM912和YTM913来扩增荧光素酶基因,用AgeI切割,并连接至具有pGL4.50luc2的HindIII-AgeI片段的pBluescript SK(-)质粒(Promega KK)的HindIII-EcoRV位点以获得PBS-luc2。切离该pBS-luc2的XhoI-NotI片段并插入至pMX-IP(Kurzman博士(威斯康星大学)分配)的XhoI-NotI位点以制备pMX-luc-IP#9作为用于荧光素酶的逆转录表达质粒。
表2
4)细胞系的建立
将上述3)的(1)中制备的具有pCAGGS-VSVG的pMx-IP-cCD20-flag#4、或上述3)的(1)中制备的具有pCVSVG和p8.9QV的CSII-CMV-cCD20-Flag-IP#4导入包装细胞系PLAT-gp或HEK293T细胞。
通过使用制备的逆转录病毒来感染NRK细胞、Jurkat细胞、和EL-4细胞,并在存在嘌呤霉素(10μg/ml和4μg/ml)的情况下培养,以获得NRK/cCD20细胞、Jurkat/cCD20细胞、和EL-4/cCD20细胞作为犬CD20过表达细胞。
为了获得表达荧光素酶基因的CLBL-1细胞系,将上述3)的(2)中制备的pMX-luc-IP#9导入PLAT-E细胞。将产生的逆转录病毒添加至PG13包装细胞系(PG13/luc),将通过PG13产生的逆转录病毒进一步添加至CLBL-1细胞,然后在存在0.5μg/ml嘌呤霉素的情况下选择并获得CLBL-1/luc细胞。
5)单克隆抗体的制备
如本发明人的先前专利申请(日本特开2019-26625号公报),制备4E1-7抗体作为针对犬CD20的单克隆抗体。
具体地,用4)中建立的NRK/cCD20细胞免疫SD大鼠。具体地,将NRK/cCD20细胞与作为佐剂的TiterGold(CytRx)混合,并皮下注射至6周龄Sprague-Dawley(SD)大鼠(Kyudo)。基于山口大学动物福利指南(Yamaguchi University Animal WelfareGuidelines),使大鼠生活在无特定病原体的场所中。免疫后10天,将1×107个细胞进一步注射至足垫(footpad),2天后,切除腘窝淋巴结(popliteal lymph node)。从淋巴结收集淋巴细胞并与小鼠骨髓瘤细胞系的P3U1细胞融合。
收集获得的杂交瘤培养上清液,并通过流式细胞术(FACS)来筛选产生对NRK/cCD20细胞和Jurkat/cCD20细胞有反应性的单克隆抗体的杂交瘤。通过有限稀释法来最终分离单个杂交瘤克隆(4E1-7),并将培养基更换为无血清培养基,杂交瘤SFM(ThermoFisher Scientific K.K.)。合并上清液,并通过HiTrap蛋白A HP柱(GE Healthcare UKLtd)纯化单克隆抗体。最后,通过流式细胞术确定抗体的亚类。
结果,确证了所获得的抗犬CD20单克隆抗体(4E1-7)的亚类为大鼠IgG2a。4E1-7与NRK/cCD20细胞反应,但不与NRK细胞反应,以浓度依赖性方式结合至犬B细胞淋巴瘤细胞系CLBL-1,但不结合至其它犬淋巴瘤细胞系(GL-1、CL-1、Ema、UL-1、CLC、CLK、CLGL90和17-71细胞系)。此外,4E1-7与Jurkat/cCD20细胞反应,但不与Jurkat细胞反应。
通过抗Flag抗体,在Jurkat/cCD20细胞的细胞裂解物中检测到犬CD20的适当的分子量(37kDa)的条带。还在来自Jurkat/cCD20细胞的4E1-7免疫沉淀细胞裂解物中检测到类似的条带。然而,4E1-7不在来自Jurkat/cCD20细胞的细胞裂解物中检测到条带,因此表明4E1-7识别非线性表位。
从这些结果来看,其确证了4E1-7是对于犬CD20分子为特异性的单克隆抗体。
6)嵌合抗CD20抗体表达载体的制备
为了获得5)中所获得的单克隆抗体的重链和轻链的可变区,通过使用基因特异性引物来进行杂交瘤克隆(4E1-7)的5'RACE。
具体地,提取例如4E-7等的总RNA,并通过使用Superscript II(Thermo FisherScientific KK)和表3中所描述的基因特异性引物YTM171(用于κ轻链)来进行逆转录。使用的引物的序列描述于下表3。
YTM172(用于重链)引物用于5'RACE。向所获得的cDNA的5’末端,通过使用末端转移酶(TOYOBO)来添加dCTP,并且通过使用在3’末端处具有poly G的引物YTM166和YTM171或YTM172来进行PCR反应。通过使用引物YTM166和YTM173或YTM174来进一步扩增该扩增产物。
将PCR产物连接至pBluescript SK(-)质粒(Promega KK)的SmaI位点,并通过基因分析器(ABI PRISM 3100-Avant测序仪(山口大学遗传研究中心DNA核心设施))来确定碱基序列。
通过重叠PCR来组装4E1-7(κ链)可变区(SEQ ID NO:2)和犬IgG轻链(κ链)恒定区(pBS-κ)(SEQ ID NO:3),并连接至pCAGGS-MCS载体以获得pCAGGS-4E1-7VκCκ#25。
通过重叠PCR来组装4E1-7(重链)可变区(SEQ ID NO:1)和犬IgG重链的IgG-B(pBS-HB)恒定区(SEQ ID NO:4)或IgG-C(pBS-HC)恒定区(SEQ ID NO:5),并连接至pCAGGS-MCS载体以获得pCAGGS-4E1-7VH-CHB#31或pCAGGS-4E1-7VH-CHC#6。
为了获得已知抗体(1E4-B)作为抗体功能的比较,基于下文中的参考文献1和专利文献1的描述,合成1E4重链和1E4轻链的可变区(GenScript)。
通过1E4 scFv质粒、犬IgG轻链(κ链)恒定区(pBS-κ)(SEQ ID NO:3)、或犬IgG重链的IgG-B(pBS-HB)恒定区(SEQ ID NO:4)的重叠PCR来构建编码嵌合抗体1E4-B的质粒。
参考文献1:
Rue SM,Eckelman BP,Efe JA,Bloink K,Deveraux QL,Lowery D,etal.Identification of a candidate therapeutic antibody for treatment of canineB-cell lymphoma.Vet Immunol Immunopathol 2015;164:148-159.
表3
7)抗体的制造和纯化
通过使用xpi293表达系统(Thermo Fisher Scientific KK),将6)中获得的各表达质粒导入HEK293细胞或PDIS-5(核心岩藻糖KO CHO-S)细胞(Sigma-Aldrich Japan)。
在导入各质粒后,收集培养的细胞的上清液,通过使用HiTrap蛋白A HP柱(GEHealthcare)纯化,并使用PD10柱(GE Healthcare)进一步脱盐。
由4E1-7表示纯化抗体,由4E1-7-B表示具有犬IgG重链的IgG-B恒定区的抗体,由4E1-7-C表示具有IgG-C恒定区的抗体,和由1E4-B表示用于比较的抗体。由4E1-7-B_f表示去岩藻糖基化抗体。
8)流式细胞术
在收集7)中培养的细胞并用缓冲液(含有2%FBS和0.1%NaN3的PBS)后,用一抗(PE标记的抗CD21抗体(Biorad,Hercules,CA))染色2×105个细胞。大鼠IgG2a(BioLegendJapan)用作同种型对照。然后用二抗(PE标记的抗大鼠IgG(SouthernBiotech)、Dylight649标记的抗大鼠IgG(BioLegend)、或Alexa 647标记的抗犬IgG(JacksonImmunoresearch))染色细胞。
为了确定4E1-7的亚类,首先用抗犬CD20抗体(4E1-7)染色NRK/cCD20细胞,然后用生物素标记的抗大鼠IgG-κ或抗大鼠IgG-λ染色。其后,添加抗大鼠IgG1抗体、抗大鼠IgG2a抗体、或抗大鼠IgG2b抗体,并用链霉亲和素-PE(ebioscience)培养。反应后,通过BDAccuri C5(BD Biosciences)和FlowJo v.10软件(Tree Star Inc.)来分析结果。
结果,如图1所示,确证了4E1-7-B和4E-1-7-C抗体二者以浓度依赖性方式结合至CLBL-1细胞系。此外,如图2中所示,确证了这些抗体比1E4-B更强地结合至EL-4/cCD20细胞。
此外,如图3中所示,4E1-7-B_f也以与4E1-7-B相同的方式结合至CLBL-1细胞系。
9)免疫印迹和免疫沉淀
在4℃下在1%NP40缓冲液中裂解7)中培养的细胞15分钟。在4℃下以15,000×g离心细胞裂解物15分钟。通过Pierce BCA蛋白检验(Thermo Fisher Scientific K.K.)来检测蛋白浓度并用于免疫印迹(Western blotting)和免疫沉淀。
基于参考文献2中的描述,用常规方法进行SDS-PAGE和免疫印迹。抗Flag M2抗体(Sigma-Aldrich Japan)和抗肌动蛋白小鼠单克隆抗体(Sigma-Aldrich Japan)用作一抗,HRP结合抗大鼠Ab(Zymed)或抗小鼠Ab(Biorad)用作二抗。反应后,将膜浸渍在WesternLightning化学发光试剂(Perkin Elmer)中,并通过使用Luminescent Image AnalyzerLAS 3000mini(FIJIFILM)确证结果。
对于免疫沉淀,在旋转的同时,在4℃下、用10μl的蛋白A/G琼脂糖(Santa CruzBiotechnology,Inc.)预澄清上述制备的细胞裂解物1小时。将10μl的该上清液、1μg的7)中获得的各抗体的混合物和蛋白A/G琼脂糖在4℃下混合过夜。然后用PBS洗涤3次免疫沉淀物并用于如上所述的免疫印迹。
参考文献2:
Okawa T,Kurio Y,Morimoto M,Hayashi T,Nakagawa T,Sasaki N,etal.Calreticulin expression in neoplastic versus normal dog mammary glands:AcDNA subtraction-based study.Res Vet Sci 2012;92:80-91.
10)细胞增殖检验
将CLBL-1细胞(2×104个细胞/100μl)接种在含有7)中获得的各抗体的96孔微滴定板上。然后在冰上培养CLBL-1细胞和各抗体15分钟,然后添加10μg/ml抗犬IgG Fc特异性抗体(Jackson Immunoresearch)。在培养其72小时后,将10μl的CCK-8溶液(DOJINDO)添加至各孔,并进一步培养2小时。其后,用ARVO X4酶标仪(microplate reader)(PerkinElmer)测定450nm处的吸光度。
结果,如图4中所示,当用4E1-7-B和4E1-7-C处理CLBL-1细胞时,与4E1-7相比,表现非常弱的细胞增殖的抑制。通过4E1-7-B和4E1-7-C与抗犬抗体的交联来增强该效果。另一方面,即使在存在抗犬IgG交联的情况下,1E4-B也不表现直接细胞杀伤。
如图5中所示,4E1-7-B_f表现CLBL-1细胞的细胞增殖的抑制,这通过添加抗犬IgG来增强。细胞增殖抑制率略高于4E1-7-B_f处理的CLBL-1细胞中的。
11)ADCC检验
将CLBL-1/luc细胞(5×103个细胞/孔)接种在含有大鼠IgG2a(ebioscience)或7)中获得的各抗体的96孔微滴定板上。在冰上培养其15分钟后,以20:1的效应物/靶比添加作为效应细胞的用IL-2刺激的PBL。进一步培养培养物4小时,然后通过使用ONE-Glo荧光素酶检验系统(Promega KK)来进行细胞溶解。通过ARVO X4系统(PerkinElmer)检测荧光素酶活性。
根据以下等式计算细胞介导的细胞毒性。通过取3个孔的平均值来确定溶解的百分比。
[公式(math)1]
特异性溶解(%)=(自然死亡RLu–试验RLU)/(自然死亡RLu–最大杀伤RLU)
结果,如图6中所示,4E1-7-B和4E1-7-C二者以浓度依赖性方式诱导CLBL-1/luc细胞的细胞溶解。该活性在4E1-7-B中比在4E1-7-C中更强。相反,1E4-B抗体不表现ADCC活性。
此外,如图7中所示,4E1-7-B和4E1-7-B_f抗体二者在CLBL-1/luc细胞中诱导细胞病变效果;然而,4E1-7-B_f表现与4E1-7-B相比几乎10倍更高的细胞毒性效果。
12)CDC检验
将CLBL-1细胞(1×105个细胞/100μl)接种在含有大鼠IgG2a抗体或7)中获得的4E1-7的96孔微滴定板上。在冰上培养其20分钟后,向各孔添加-H兔补体(CEDALANE;最终浓度1:40)并进一步培养90分钟。其后,通过台盼蓝染料排斥检验(trypanblue dye exclusion assay)来计数活细胞和死细胞。
使用CLBL-1/luc1细胞(1×105个细胞/100μl)并接种在含有4E1-7B或4E1-7的96孔微滴定板上,并以如上所述相同的方式进行处理,通过11)的ADCC检验中所描述的方法来检测活细胞和死细胞。
结果,如图8中所示,4E1-7-C与1E4-B同样地不诱导CLBL-1/luc细胞的细胞死亡;然而,确证了4E1-7-B以浓度依赖性方式诱导细胞死亡。
如图9中所示,确证了4E1-7-B和4E1-7-B_f二者以浓度依赖性方式诱导细胞死亡。
13)细胞凋亡检验
将CLBL-1细胞(1×105个细胞/100μl)接种在含有大鼠IgG2a或4E1-7抗体的24孔板上。在培养72小时后,通过FITC Annexin V细胞凋亡检测试剂盒I(BD Bioscience)染色细胞并通过流式细胞术分析。
14)体内试验(小鼠)
使六至八周龄的NOD.CB17-Prkdcscid/J(NOD/SCID)小鼠(Charles riverlaboratories Japan,Inc.)生活在无特定病原体的场所中。将CLBL-1细胞(1×106个细胞/30μl PBS)皮下移植至NOD/SCID小鼠的侧腹部。接种后,用卡尺每日测定肿瘤大小(2个垂直尺寸)。根据以下等式计算肿瘤体积。当肿瘤达到150至250mm3时,每4天腹腔内注射7)中获得的各抗体(150μg/500μl PBS)。与抗体注射一同,将从健康比格犬新鲜分离的犬PBMC(5×106个细胞)悬浮于60μl的PBS并在肿瘤的周围注射以补充NK细胞活性。当肿瘤达到2,000mm3或自开始处理13天后,处死小鼠。
[公式2]
肿瘤体积(mm3)=1/2×长度(mm)×宽度2(mm2)
结果,如图10A和10B中所示,与作为对照的仅向其施用PBS的小鼠相比(图10A),仅向其施用PBMC的小鼠(图10B)中表现肿瘤生长的略微延迟。还确证了肿瘤生长在向其施用4E1-7-B和PBMC的(图11A)四只小鼠的两只中延迟,并且肿瘤生长在向其施用4E1-7-Bf和PBMC的(图11B)全部四只小鼠中被抑制。
15)体内试验(犬)
将健康比格犬(Nippon Zenyaku Kogyo Co.,Ltd.)分为4组(每组中4只犬),通过静脉注射、以0.5mg/kg、5.0mg/kg、或25mg/kg施用7)中获得的4E1-7-B或4E1-7-B_f。通过流式细胞术分析(Animal Allergy Clinical LABORATORIES)监测外周血中CD21+B细胞的百分比。
在抗体施用后第14天和第28天,从各组中的一只犬获得淋巴结,切成3μm的厚度,用抗CD79a抗体(克隆HM57,稀释1:50,Dako)染色,和进行免疫组织病理学分析。
结果,如图12中所示,无论施用哪种抗体,施用后第1天的CD21+B细胞的百分比降低至几乎为0,并且犬在施用后第3天不具有CD21+B细胞。
在向其施用0.5mg/kg的4E1-7-B的组中的犬中,该情况持续直至施用后第7天,并且即使在施用后第14天,一只犬仍表现无CD21+B细胞。在向其施用5.0mg/kg的4E1-7-B的组中的犬中,在施用后第14天不表现CD21+B细胞,并且尽管在施用后第21天中、在两只狗中观察到略微的增加,一只狗即使在施用后第28天也不具有CD21+B细胞。在所有组中,CD21+B细胞的百分比不恢复至原始百分比直至施用后第119天,并且B细胞的值(淋巴细胞的%)在所有组中都保持减少。因此,该结果表明抗体的效果长时间持续。在向其施用4E1-7-B_f的组中表现类似的反应。
作为免疫组织化学染色的结果,确证了当施用5.0mg/kg的4E1-7-B时,样品中的阳性区域在施用后第14天和第28天减少。
16)统计分析
通过使用JMP14.0软件(JMP Japan)进行所有数据分析。通过使用单因素方差分析、接着图基-克雷默(Tukey-Kramer)多重比较检验来分析数据。结果,确认如果P<0.05则存在显著性差异。
经过上述1)至16)的步骤提供了具有以下的针对犬CD20的单克隆抗体或抗体片段:重链可变区,其由SEQ ID NO:1的氨基酸序列、或其中在SEQ ID NO:1的氨基酸序列中缺失、置换或添加一个或多个氨基酸的氨基酸序列组成,和轻链可变区,其由SEQ ID NO:2的氨基酸序列、或其中在SEQ ID NO:2的氨基酸序列中缺失、置换或添加一个或多个氨基酸的氨基酸序列组成,和具有轻链恒定区,其由SEQ ID NO:3的氨基酸序列、或其中在SEQ IDNO:3的氨基酸序列中缺失、置换或添加一个或多个氨基酸的氨基酸序列组成。
产业上的可利用性
本发明可提供具有比现有抗体更优异的效果的针对犬CD20的单克隆抗体或抗体片段。使用该单克隆抗体或抗体片段作为活性成分,本发明还可提供用于减少犬B细胞的组合物和用于减少犬B细胞的方法、或用于治疗由于犬B细胞的增加而导致的疾病如B细胞淋巴瘤、白血病、或自身免疫性疾病的治疗组合物、和用于治疗由于犬B细胞的增加而导致的疾病的方法。
序列表
<110> 日本全药工业株式会社(Nippon Zenyaku Kogyo Co., Ltd.)
<120> 针对犬CD20的单克隆抗体或抗体片段
<130> P1910
<150> JP 2019-183079
<151> 2019-10-03
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<170> PatentIn 版本 3.5
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Asp Thr Gly Ile Gln Glu Ser Val Thr Glu Gln Asp Lys Asp Ser Thr
50 55 60
Tyr Ser Leu Ser Ser Thr Leu Thr Met Ser Ser Thr Glu Tyr Leu Ser
65 70 75 80
His Glu Leu Tyr Ser Cys Glu Ile Thr His Lys Ser Leu Pro Ser Thr
85 90 95
Leu Ile Lys Ser Phe Gln Arg Ser Glu Cys Gln Arg Val Asp
100 105 110
<210> 4
<211> 334
<212> PRT
<213> 犬
<400> 4
Ser Thr Thr Ala Pro Ser Val Phe Pro Leu Ala Pro Ser Cys Gly Ser
1 5 10 15
Thr Ser Gly Ser Thr Val Ala Leu Ala Cys Leu Val Ser Gly Tyr Phe
20 25 30
Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ser Leu Thr Ser Gly
35 40 45
Val His Thr Phe Pro Ser Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu
50 55 60
Ser Ser Met Val Thr Val Pro Ser Ser Arg Trp Pro Ser Glu Thr Phe
65 70 75 80
Thr Cys Asn Val Ala His Pro Ala Ser Lys Thr Lys Val Asp Lys Pro
85 90 95
Val Pro Lys Arg Glu Asn Gly Arg Val Pro Arg Pro Pro Asp Cys Pro
100 105 110
Lys Cys Pro Ala Pro Glu Met Leu Gly Gly Pro Ser Val Phe Ile Phe
115 120 125
Pro Pro Lys Pro Lys Asp Thr Leu Leu Ile Ala Arg Thr Pro Glu Val
130 135 140
Thr Cys Val Val Val Asp Leu Asp Pro Glu Asp Pro Glu Val Gln Ile
145 150 155 160
Ser Trp Phe Val Asp Gly Lys Gln Met Gln Thr Ala Lys Thr Gln Pro
165 170 175
Arg Glu Glu Gln Phe Asn Gly Thr Tyr Arg Val Val Ser Val Leu Pro
180 185 190
Ile Gly His Gln Asp Trp Leu Lys Gly Lys Gln Phe Thr Cys Lys Val
195 200 205
Asn Asn Lys Ala Leu Pro Ser Pro Ile Glu Arg Thr Ile Ser Lys Ala
210 215 220
Arg Gly Gln Ala His Gln Pro Ser Val Tyr Val Leu Pro Pro Ser Arg
225 230 235 240
Glu Glu Leu Ser Lys Asn Thr Val Ser Leu Thr Cys Leu Ile Lys Asp
245 250 255
Phe Phe Pro Pro Asp Ile Asp Val Glu Trp Gln Ser Asn Gly Gln Gln
260 265 270
Glu Pro Glu Ser Lys Tyr Arg Thr Thr Pro Pro Gln Leu Asp Glu Asp
275 280 285
Gly Ser Tyr Phe Leu Tyr Ser Lys Leu Ser Val Asp Lys Ser Arg Trp
290 295 300
Gln Arg Gly Asp Thr Phe Ile Cys Ala Val Met His Glu Ala Leu His
305 310 315 320
Asn His Tyr Thr Gln Glu Ser Leu Ser His Ser Pro Gly Lys
325 330
<210> 5
<211> 332
<212> PRT
<213> 犬
<400> 5
Ser Thr Thr Ala Pro Ser Val Phe Pro Leu Ala Pro Ser Cys Gly Ser
1 5 10 15
Gln Ser Gly Ser Thr Val Ala Leu Ala Cys Leu Val Ser Gly Tyr Ile
20 25 30
Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ser Leu Thr Ser Gly
35 40 45
Val His Thr Phe Pro Ser Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu
50 55 60
Ser Ser Met Val Thr Val Pro Ser Ser Arg Trp Pro Ser Glu Thr Phe
65 70 75 80
Thr Cys Asn Val Ala His Pro Ala Thr Asn Thr Lys Val Asp Lys Pro
85 90 95
Val Val Lys Glu Cys Glu Cys Lys Cys Asn Cys Asn Asn Cys Pro Cys
100 105 110
Pro Gly Cys Gly Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro
115 120 125
Lys Pro Lys Asp Ile Leu Val Thr Ala Arg Thr Pro Thr Val Thr Cys
130 135 140
Val Val Val Asp Leu Asp Pro Glu Asn Pro Glu Val Gln Ile Ser Trp
145 150 155 160
Phe Val Asp Ser Lys Gln Val Gln Thr Ala Asn Thr Gln Pro Arg Glu
165 170 175
Glu Gln Ser Asn Gly Thr Tyr Arg Val Val Ser Val Leu Pro Ile Gly
180 185 190
His Gln Asp Trp Leu Ser Gly Lys Gln Phe Lys Cys Lys Val Asn Asn
195 200 205
Lys Ala Leu Pro Ser Pro Ile Glu Glu Ile Ile Ser Lys Thr Pro Gly
210 215 220
Gln Ala His Gln Pro Asn Val Tyr Val Leu Pro Pro Ser Arg Asp Glu
225 230 235 240
Met Ser Lys Asn Thr Val Thr Leu Thr Cys Leu Val Lys Asp Phe Phe
245 250 255
Pro Pro Glu Ile Asp Val Glu Trp Gln Ser Asn Gly Gln Gln Glu Pro
260 265 270
Glu Ser Lys Tyr Arg Met Thr Pro Pro Gln Leu Asp Glu Asp Gly Ser
275 280 285
Tyr Phe Leu Tyr Ser Lys Leu Ser Val Asp Lys Ser Arg Trp Gln Arg
290 295 300
Gly Asp Thr Phe Ile Cys Ala Val Met His Glu Ala Leu His Asn His
305 310 315 320
Tyr Thr Gln Lys Ser Leu Ser His Ser Pro Gly Lys
325 330
<210> 6
<211> 32
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 6
gcgcggccgc tctcaggagt tcagagggtg ag 32
<210> 7
<211> 28
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 7
cagaattctc aggaaacagg ggtggata 28
<210> 8
<211> 30
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 8
ccaggtgacc ccattcagtg ctcaggacac 30
<210> 9
<211> 28
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 9
gggtgggggg cttgctgggt gccgggcg 28
<210> 10
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 10
cactgtccgt gtctgtcagc 20
<210> 11
<211> 19
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 11
ccaaggcctg agctaggag 19
<210> 12
<211> 32
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 12
acggatccat gacaacaccc agaaattcaa tg 32
<210> 13
<211> 38
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 13
gtcgatgtca tgatctttat aatcagggat gctgtcgt 38
<210> 14
<211> 39
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 14
tcactacttg tcatcgtcat ccttgtagtc gatgtcatg 39
<210> 15
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 15
gctaaggtgg tggacttgga 20
<210> 16
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 16
ccgccccgac tctagaatta 20
<210> 17
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 17
tgccatcaat cttccacttg aca 23
<210> 18
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 18
aaytttcttg tccaccttgg 20
<210> 19
<211> 36
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 19
ggccacgcgt cgactagtac gggggggggg gggggg 36
<210> 20
<211> 28
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 20
gttgttcawg argcacacga ctgaggca 28
<210> 21
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 21
aatagccctt gaccaggcat 20
Claims (8)
1.一种针对犬CD20的单克隆抗体或抗体片段,其包含:重链可变区,其由SEQ ID NO:1的氨基酸序列、或其中在SEQ ID NO:1的氨基酸序列中缺失、置换或添加一个或多个氨基酸的氨基酸序列组成;和轻链可变区,其由SEQ ID NO:2的氨基酸序列、或其中在SEQ ID NO:2的氨基酸序列中缺失、置换或添加一个或多个氨基酸的氨基酸序列组成,和包含轻链恒定区,其由SEQ ID NO:3的氨基酸序列、或其中在SEQ ID NO:3的氨基酸序列中缺失、置换或添加一个或多个氨基酸的氨基酸序列组成。
2.根据权利要求1所述的抗体或抗体片段,其包含重链可变区,所述重链可变区由SEQID NO:4或5的氨基酸序列、或其中在SEQ ID NO:4或5的氨基酸序列中缺失、置换或添加一个或多个氨基酸的氨基酸序列组成。
3.根据权利要求1或2所述的抗体或抗体片段,其中所述抗体或抗体片段是去岩藻糖基化的。
4.一种用于减少犬B细胞的组合物,其包含根据权利要求1至3中任一项所述的抗体或抗体片段作为活性成分。
5.一种用于治疗由于犬B细胞的增加而导致的疾病的治疗组合物,其包含根据权利要求1至3中任一项所述的抗体或抗体片段作为活性成分。
6.根据权利要求5所述的治疗组合物,其中所述疾病是B细胞淋巴瘤、白血病、或自身免疫性疾病。
7.一种犬B细胞检测试剂盒,其包含:根据权利要求1至3中任一项所述的抗体或抗体片段。
8.一种用于诊断由于犬B细胞的增加而导致的疾病的试剂盒,其包含:根据权利要求1至3中任一项所述的抗体或抗体片段。
Applications Claiming Priority (3)
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JP2019183079A JP2021059499A (ja) | 2019-10-03 | 2019-10-03 | イヌcd20に対するモノクローナル抗体又は抗体フラグメント |
JP2019-183079 | 2019-10-03 | ||
PCT/JP2020/037514 WO2021066134A1 (ja) | 2019-10-03 | 2020-10-02 | イヌcd20に対するモノクローナル抗体又は抗体フラグメント |
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CN114555636A true CN114555636A (zh) | 2022-05-27 |
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US (1) | US20220348676A1 (zh) |
EP (1) | EP4039273A4 (zh) |
JP (1) | JP2021059499A (zh) |
CN (1) | CN114555636A (zh) |
WO (1) | WO2021066134A1 (zh) |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20100061988A1 (en) * | 2008-09-04 | 2010-03-11 | Hansen Genevieve | Monoclonal antibodies |
AU2014200771A1 (en) * | 2008-09-04 | 2014-03-06 | Vet Therapeutics, Inc. | Monoclonal antibodies |
AU2016216708A1 (en) * | 2008-09-04 | 2016-09-08 | Vet Therapeutics, Inc. | Monoclonal antibodies |
CN110770348A (zh) * | 2017-06-01 | 2020-02-07 | 上海斯丹赛生物技术有限公司 | 嵌合抗原受体细胞的制备及其用途 |
Family Cites Families (7)
Publication number | Priority date | Publication date | Assignee | Title |
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EP2295468B1 (en) | 2002-02-14 | 2015-07-08 | Immunomedics, Inc. | Anti-CD20 antibodies and fusion proteins thereof and methods of use |
US9580496B2 (en) | 2011-05-06 | 2017-02-28 | Nexvet Australia Pty Ltd | Therapeutic canine immunoglobulins and methods of using same |
WO2013054127A1 (en) | 2011-10-13 | 2013-04-18 | Nvip Pty Ltd | Canine/feline cd20 binding epitope and compositions for binding thereto |
WO2013063186A2 (en) | 2011-10-26 | 2013-05-02 | Novartis Animal Health Us, Inc. | Monoclonal antibodies and methods of use |
JP2016013104A (ja) | 2014-07-02 | 2016-01-28 | 学校法人日本大学 | 抗イヌcd20モノクローナル抗体又は抗体フラグメント、キット、診断方法、治療用組成物、治療方法、核酸、ベクター、キメラ抗原受容体及びt細胞 |
KR102131544B1 (ko) | 2016-02-18 | 2020-07-07 | 엘랑코 유에스 인코포레이티드 | 키메라 개 항-cd20 항체 |
JP2019026625A (ja) | 2017-08-03 | 2019-02-21 | 日本全薬工業株式会社 | 抗イヌcd20モノクローナル抗体 |
-
2019
- 2019-10-03 JP JP2019183079A patent/JP2021059499A/ja active Pending
-
2020
- 2020-10-02 US US17/762,407 patent/US20220348676A1/en active Pending
- 2020-10-02 WO PCT/JP2020/037514 patent/WO2021066134A1/ja unknown
- 2020-10-02 CN CN202080069648.8A patent/CN114555636A/zh active Pending
- 2020-10-02 EP EP20871889.0A patent/EP4039273A4/en active Pending
Patent Citations (4)
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US20100061988A1 (en) * | 2008-09-04 | 2010-03-11 | Hansen Genevieve | Monoclonal antibodies |
AU2014200771A1 (en) * | 2008-09-04 | 2014-03-06 | Vet Therapeutics, Inc. | Monoclonal antibodies |
AU2016216708A1 (en) * | 2008-09-04 | 2016-09-08 | Vet Therapeutics, Inc. | Monoclonal antibodies |
CN110770348A (zh) * | 2017-06-01 | 2020-02-07 | 上海斯丹赛生物技术有限公司 | 嵌合抗原受体细胞的制备及其用途 |
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EP4039273A1 (en) | 2022-08-10 |
EP4039273A4 (en) | 2023-12-20 |
US20220348676A1 (en) | 2022-11-03 |
JP2021059499A (ja) | 2021-04-15 |
WO2021066134A1 (ja) | 2021-04-08 |
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