CN116751302B - 靶向Claudin18.2的抗体、衍生产品及其在肿瘤治疗中的应用 - Google Patents
靶向Claudin18.2的抗体、衍生产品及其在肿瘤治疗中的应用 Download PDFInfo
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Abstract
本发明公开了靶向Claudin18.2的抗体、衍生产品及其在肿瘤治疗中的应用,本发明提供了特异性靶向Claudin18.2的抗体、包含所述抗体的抗体药物偶联物,与IMAB362单抗相比,本发明提供的抗体具有更强的结合活性、内化率、ADCC活性和ADCP活性,且基于该抗体制备得到的抗体药物偶联物具有显著更优的体内外肿瘤杀伤活性,具有广阔的临床应用前景。
Description
技术领域
本发明属于生物医药技术领域,具体地,本发明涉及靶向Claudin18.2的抗体、衍生产品及其在肿瘤治疗中的应用。
背景技术
Claudin家族蛋白是位于上皮、内皮细胞层中细胞之间紧密连接(Tightjunctions,TJs)处的细胞间粘附分子,是构成TJs的跨膜蛋白中种类最丰富的家族。Claudin1是Claudin家族的一员,也是TJs的组成部分,参与调节细胞旁屏障功能及信号转导。其基因有两个可替代的第一个外显子,受不同的组织特异性启动子调控,通过可变剪接会产生两种蛋白质亚型:Claudin18.1及Claudin18.2,这两种蛋白亚型均是高度选择性的细胞谱系标记物,具有不同的细胞系特异性。在正常情况下,Claudin18.2蛋白在正常组织中表达量极低,而在胃癌、卵巢癌、食管癌、胰腺癌、肺癌等肿瘤组织中高度过表达,与这些癌症患者的不良预后和较差的临床治疗结果相关,是一种较为理想的肿瘤治疗靶点。
目前,针对Claudin18.2高表达的实体瘤的靶向治疗策略主要包括抗体药物偶联物(Antibody-drug conjugate,ADC)、嵌合抗原受体T细胞免疫疗法(Chimeric AntigenReceptor T-Cell,CAR-T)、单靶点抗体、双特异性抗体等,其中,ADC将单克隆抗体或者抗体片段通过稳定的化学接头化合物(连接子)与具有生物活性的细胞毒素相连,充分利用了抗体对正常细胞和肿瘤细胞表面抗原结合的特异性和细胞毒性物质的高效性,同时又避免了前者疗效偏低和后者毒副作用过大等缺陷。与以往传统的化疗药物相比,ADC能更精准地结合肿瘤细胞并降低对正常细胞的影响。虽然目前已有靶向Claudin18.2的抗体及ADC药物的相关报道,但是仍需要开发更有效且更具安全性的抗Claudin18.2的抗体及ADC药物,以更好地应用于Claudin18.2相关肿瘤的治疗中。
发明内容
有鉴于此,本发明的目的在于提供靶向Claudin18.2的抗体、衍生产品及其在肿瘤治疗中的应用。
为了实现上述目的,本发明采用了如下技术方案:
本发明的第一方面提供了一种靶向Claudin18.2的抗体或其抗原结合片段。
进一步,所述抗体或其抗原结合片段包含重链可变区和轻链可变区;
所述重链可变区包含HCDR1、HCDR2、HCDR3,所述HCDR1、HCDR2、HCDR3的氨基酸序列为如SEQ ID NO:4所示的重链可变区中的CDR1、CDR2、CDR3;
所述轻链可变区包含LCDR1、LCDR2、LCDR3,所述LCDR1、LCDR2、LCDR3的氨基酸序列为如SEQ ID NO:8所示的轻链可变区中的CDR1、CDR2、CDR3。
进一步,所述HCDR1、HCDR2、HCDR3的氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3所示或分别为与SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3具有至少90%、91%、92%、93%、94%、95%、96%、97%、98%、99%同源性的氨基酸序列;
所述LCDR1、LCDR2、LCDR3的氨基酸序列分别如SEQ ID NO:5、SEQ ID NO:6、SEQ IDNO:7所示或分别为与SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7具有至少90%、91%、92%、93%、94%、95%、96%、97%、98%、99%同源性的氨基酸序列。
在一些实施方案中,本发明所述HCDR1、HCDR2、HCDR3对应的氨基酸序列并不局限于如SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3所示的氨基酸序列,本发明所述LCDR1、LCDR2、LCDR3对应的氨基酸序列也并不局限于如SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7所示的氨基酸序列,采用任何CDR编号方案(现有的CDR编号方案或将来产生的新的CDR编号方案)分别对如SEQ ID NO:4所示的重链可变区中的CDR1、CDR2、CDR3进行定义、对如SEQ IDNO:8所示的轻链可变区中的CDR1、CDR2、CDR3进行定义得到的HCDR1、HCDR2、HCDR3、LCDR1、LCDR2、LCDR3对应的氨基酸序列或核苷酸序列均在本发明的保护范围内。
在一些实施方案中,所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2、LCDR3是根据IMGT编号方案、Chothia编号方案、Kabat编号方案、Martin(增强型Chothia)编号方案、AbM编号方案、Aho编号方案中的任意一种定义或任意多种(两种或两种以上)组合定义得到的,经上述定义方式定义得到的HCDR1、HCDR2、HCDR3、LCDR1、LCDR2、LCDR3对应的序列同样包含在本发明的保护范围内。
在一些实施方案中,本发明第一方面提供的抗体的功能性变体同样包含在本发明的保护范围内,所述“功能性变体”是指与亲本抗体相比具有明显或显著序列同一性或相似性的蛋白,所述功能性变体保留了亲本抗体的生物活性。功能性变体涵盖例如本文中所述抗体(亲本抗体)的以下变体,其与亲本抗体相比在类似程度上、在相同程度上或在更高程度上保留识别靶细胞的能力。参考亲本抗体,功能性变体可例如与亲本抗体在氨基酸序列方面具有至少约30%、50%、70%、75%、80%、85%、90%、95%或更高的同源性。
进一步,功能性变体可例如包含具有至少一个保守性氨基酸替换的亲本抗体的氨基酸序列。作为替选或补充,功能性变体可包含具有至少一个非保守性氨基酸替换的亲本抗体的氨基酸序列。在这种情况下,非保守性氨基酸替换优选不干扰或抑制功能性变体的生物活性。非保守性氨基酸替换可增强功能性变体的生物活性,使得与亲本抗体相比,功能性变体的生物活性提高。
进一步,保守性氨基酸替换是本领域已知的,并且包括其中将一个具有特定物理和/或化学特性的氨基酸交换为另一个具有相同或相似化学或物理特性的氨基酸替换。例如,保守性氨基酸替换可以是酸性/带负电荷的极性氨基酸替换另一个酸性/带负电荷的极性氨基酸(例如,Asp或Glu)、具有非极性侧链的氨基酸替换另一个具有非极性侧链的氨基酸(例如,Ala、Gly、Val、He、Leu、Met、Phe、Pro、Trp、Cys、Val等)、碱性/带正电荷的极性氨基酸替换另一个碱性/带正电荷的极性氨基酸(例如,Lys、His、Arg等)、具有极性侧链的不带电荷氨基酸替换另一个具有极性侧链的不带电荷的氨基酸(例如,Asn、Gin、Ser、Thr、Tyr等)、具有β支化侧链的氨基酸替换另一个具有β支化侧链的氨基酸(例如,He、Thr和Val)、具有芳香族侧链的氨基酸替换另一个具有芳香族侧链的氨基酸(例如,His、Phe、Trp和Tyr)。
本领域普通技术人员可以很容易地采用已知的方法,例如采用定向进化和点突变的方法,对本发明所述抗体对应的核苷酸序列进行突变。那些经过人工修饰的,具有与本发明所述抗体对应的核苷酸序列75%或75%以上同源性的核苷酸,只要编码本发明第一方面所述的抗体或其抗原结合片段,均是衍生于本发明的核苷酸序列并且等同于本发明的序列,同样包含在本发明的保护范围内。
本发明的第二方面提供了一种多特异性抗体。
进一步,所述多特异性抗体包含本发明第一方面所述的抗体或其抗原结合片段,以及一个或多个与其他抗原特异性结合的第二抗体或其抗原结合片段。
在一些实施方案中,所述其他抗原包括但不限于:Trop-2、Her2、Her3、Her4、EGF、EGFR、CD2、CD3、CD5、CD7、CD13、CD19、CD20、CD21、CD23、CD30、CD33、CD34、CD38、CD46、CD55、CD59、CD69、CD70、CD71、CD97、CD117、CD123、CD127、CD134、CD137、CD138、CD146、CD147、CD152、CD154、CD174、CD195、CD200、CD205、CD212、CD223、CD227、CD253、CD272、CD274、CD276、CD278、CD279、CD309、CD319、CD326、CD340、MUC1、uPA、MAGE3、MUC16、KLK3、PSMA、BCMA、GPNMB、EphA2、EphB2、TMEFF2、B7H3、B7H4、CDH6、HAVCR1、STEAP-1、STEAP-2、UPK2。
在一些实施方案中,所述多特异性抗体具有针对多于一种抗原的结合特异性(例如,具有两种、三种或更多种抗原结合特异性的抗体,其中一种抗原为本发明所述的Claudin18.2)。在一些实施方案中,所述多特异性抗体是双特异性抗体。在一些实施方案中,所述双特异性抗体包含对两种不同抗原的结合特异性(其中一种抗原为本发明所述的Claudin18.2)。在一些实施方案中,所述双特异性抗体包含针对同一抗原(例如Claudin18.2)的两种不同的结合特异性(例如,针对同一抗原具有不同的结合亲和力和/或特异性表位)。
本发明的第三方面提供了一种核酸分子或表达载体。
进一步,所述核酸分子编码本发明第一方面所述的抗体或其抗原结合片段或本发明第二方面所述的多特异性抗体,所述表达载体包含所述核酸分子。
在一些实施方案中,所述核酸分子可包含天然的、非天然的或被改变的核苷酸;并且其可包含天然的、非天然的或被改变的核苷酸间连接,例如氨基磷酸酯连接或硫代磷酸酯连接,代替在未经修饰寡核苷酸的核苷酸之间存在的磷酸二酯。在一些实施方案中,核酸不包含任何插入、缺失、倒位和/或替换。然而,在一些情况下对核酸而言,包含一个或更多个插入、缺失、倒位和/或替换可以是合适的,因此,这些插入、缺失、倒位和/或替换形成的核酸同样在本发明的保护范围内。
在一些实施方案中,可用于本发明的载体的实例包括但不限于:质粒、噬菌粒、粘粒、人工染色体、病毒来源的载体。可选用本领域已知的各种载体,例如选用市售的载体,然后将编码本发明第一方面所述的抗体或其抗原结合片段或本发明第二方面所述的多特异性抗体的核苷酸序列可操作性地连于表达调控序列上形成表达载体。在一些实施方案中,所述病毒来源的载体包括但不限于:慢病毒载体、逆转录病毒载体、腺病毒载体、腺相关病毒载体、痘病毒载体、疱疹病毒载体、杆状病毒载体、乳头状瘤病毒载体、乳头多瘤空泡病毒载体。
在一些实施方案中,所述表达载体可包含表达调节序列,例如转录和翻译起始和终止密码子,所述表达调节序列对需向其中引入载体的宿主细胞的类型(例如,细菌、真菌、植物或动物)具有特异性,这视情况而定并且考虑载体是基于DNA还是基于RNA。重组表达载体可包含限制性位点以利于克隆。
在一些实施方案中,所述表达载体还可包含一个或更多个允许选择转化或转染的宿主细胞的标记基因。标记基因包括杀生物剂抗性(例如针对抗生素、重金属等的抗性);营养缺陷型宿主中提供原养型的互补等等。用于本发明表达载体的合适的标记基因包括例如新霉素/G418抗性基因、潮霉素抗性基因、组氨醇抗性基因、四环素抗性基因、氨苄青霉素抗性基因、卡那霉素抗性基因、嘌呤霉素抗性基因等。
本发明的第四方面提供了一种经基因改造的宿主细胞。
进一步,所述经基因改造的宿主细胞包含本发明第三方面所述的核酸分子或表达载体。
在一些实施方案中,所述宿主细胞包括真核细胞、原核细胞。在一些实施方案中,所述真核细胞包括但不限于:哺乳动物细胞、昆虫细胞、植物细胞、酵母细胞。所述原核细胞包括但不限于:细菌、放线菌、蓝细菌、支原体、衣原体、立克次氏体。在一些实施方案中,用于表达本发明所述抗体的宿主细胞包括大肠杆菌、酵母细胞、昆虫细胞、COS细胞、CHO细胞等。较佳地,该宿主细胞是真核细胞,更佳地是哺乳动物细胞。
在一些实施方案中,所述宿主细胞通过如下方法制备得到:将本发明第三方面所述的核酸分子或表达载体引入到宿主细胞中,所述引入的方法包括但不限于:物理方法、化学方法、生物方法。所述物理方法包括但不限于:磷酸钙沉淀、脂质转染法、粒子轰击、微注射、电穿孔;所述化学方法包括但不限于:胶体分散系统、基于脂质的系统;所述胶体分散系统包括但不限于:大分子复合物、纳米胶囊、微球、珠;所述基于脂质的系统包括但不限于:水包油乳剂、胶束、混合胶束、脂质体;所述生物方法包括但不限于:DNA载体、慢病毒载体、痘病毒载体、单纯疱疹病毒载体、腺病毒载体、腺相关病毒载体。
在一些实施方案中,可以通过各种合适的方式将本发明第三方面所述的核酸分子或表达载体引入到宿主细胞中,并不局限于本发明中所列举出的方法,例如磷酸钙转染、DEAE-葡聚糖介导的转染、显微注射、电穿孔、TALEN方法、ZFN方法、非病毒载体介导的转染(例如脂质体)或病毒载体介导的转染(例如慢病毒感染,逆转录病毒感染,腺病毒感染),以及其他用于转移入细胞的物理、化学或生物学手段,如转座子技术,CRISPR-Cas9等技术。
本发明的第五方面提供了一种嵌合抗原受体或工程化免疫细胞。
进一步,所述嵌合抗原受体包含本发明第一方面所述的抗体或其抗原结合片段或本发明第二方面所述的多特异性抗体,所述工程化免疫细胞包含所述嵌合抗原受体。
在一些实施方案中,所述嵌合抗原受体还包含铰链区和跨膜区、共刺激信号结构域、胞内信号传导结构域。其中,所述铰链区和跨膜区包括但不限于下列分子的铰链区和跨膜区:CD8、4-1BB、IgG1、IgG4、PD-1、CD28、CD34、OX40、CD3ε;所述共刺激信号结构域包括但不限于下列分子的共刺激信号结构域:HVEM、CD27、CD19、CD28、ICOS、CD4、CD8α、CD8β、CD40、4-1BB、OX40、CD226、CD278;所述胞内信号传导结构域包括但不限于下列分子的胞内信号传导结构域:CD3ζ、CD3γ、CD3δ、CD3ε、CD278、CD21、CD22、FcεRI、FcRγ、FcRβ。在一些实施方案中,所述嵌合抗原受体中还可包含启动子、信号肽等。
在一些实施方案中,所述免疫细胞包括但不限于:T细胞、B细胞、NK细胞、iNKT细胞、CTL细胞、树突状细胞、髓样细胞、单核细胞、巨噬细胞或其任意组合。
本发明的第六方面提供了一种抗体药物偶联物。
进一步,所述抗体药物偶联物包含本发明第一方面所述的抗体或其抗原结合片段或本发明第二方面所述的多特异性抗体,以及与所述抗体偶联的细胞毒性剂。
进一步,所述细胞毒性剂选自以下组:MMAE、MMAF、美登素DM1、美登素DM4、奥瑞他汀E、奥瑞他汀F、美登素醇、山德霉素、吡咯并苯并二氮杂卓、吡咯并苯并二氮杂卓二聚体、蒽环类、卡奇霉素、多拉司他汀10、多卡霉素、多柔比星、泰兰斯他汀A、钩霉素、鹅膏菌素、蓖麻毒素、白喉毒素、艾立布林、131I、白细胞介素、肿瘤坏死因子、趋化因子、伊立替康、依喜替康、纳米颗粒。
在一些实施方案中,本发明所述的细胞毒性剂并不局限于MMAE,任何能够用于和本发明所述的抗体偶联并发挥细胞毒性作用的小分子化合物均在本发明的保护范围内。在一些实施方案中,本发明所述的连接子并无特别限制,任何能够将本发明第一方面所述的抗体或其抗原结合片段或本发明第二方面所述的多特异性抗体与所述细胞毒性剂共价偶联形成能够发挥有效肿瘤治疗作用的抗体药物偶联物的连接子均在本发明的保护范围内。
本发明的第七方面提供了一种用于治疗和/或预防Claudin18.2阳性相关疾病的药物组合物。
进一步,所述药物组合物包含治疗和/或预防有效量的本发明第一方面所述的抗体或其抗原结合片段、本发明第二方面所述的多特异性抗体、本发明第五方面中所述的工程化免疫细胞、本发明第六方面所述的抗体药物偶联物、抗PD1抗体中的任意一种或多种。
在一些实施方案中,所述药物组合物包含本发明第一方面所述的抗体或其抗原结合片段,以及抗PD1抗体,本发明通过实验验证发现所述抗体和抗PD1抗体联合具有显著更优的抑瘤效果。在一些实施方案中,所述药物组合物还可包含其他能够用于治疗肿瘤的药物中的任意一种或多种。
在一些实施方案中,合适的药物组合物的施用形式包括适用于肠胃外施用的形式,例如通过注射或输注,例如通过快速注射或连续输液、静脉内、可吸入或皮下形式。在产品用于注射或输注的情况下,其可采用在油性或水性媒介物中的悬浮液、溶液或乳液的形式并且其可含有配制试剂,诸如悬浮剂、防腐剂、稳定剂和/或分散剂。
在一些实施方案中,所述药物组合物还可包含药学上可接受的载体和/或辅料适合的药学上可接受的载体和/或辅料在Remington's Pharmaceutical Sciences(19thed.,1995)中有详细的记载,这些物质根据需要用于帮助配方的稳定性或有助于提高活性或活性物质的生物有效性。在一些实施方案中,使用药物组合物时,是将安全有效量的本发明所述的药物组合物施用于人。
在一些实施方案中,所述药学上可接受的载体和/或辅料可以包含无菌可注射液体(如水性或非水性悬浮液或溶液)。在某些示例性实施方案中,此类无菌可注射液体选自注射用水(WFI)、抑菌性注射用水(BWFI)、氯化钠溶液(例如0.9% (w/v)NaCl)、葡萄糖溶液(例如5%葡萄糖)、含有表面活性剂的溶液(例如0.01%聚山梨醇20)、pH缓冲溶液(例如磷酸盐缓冲溶液)、Ringer氏溶液及其任意组合。
在一些实施方案中,所述药物组合物可根据需要制成各种剂型,并可由医师根据患者种类、年龄、体重和大致疾病状况、给药方式等因素确定对病人有益的剂量进行施用,熟练的医生通常能够容易地决定处方及处方对所希望的治疗和/或预防有效的给药剂量。给药方式例如可以采用注射或其它治疗方式。
本发明的第八方面提供了一种用于检测Claudin18.2的检测试剂或用于诊断Claudin18.2阳性相关疾病的试剂盒。
进一步,所述检测试剂包含本发明第一方面所述的抗体或其抗原结合片段或本发明第三方面所述的多特异性抗体,所述试剂盒包含所述检测试剂。
在一些实施方案中,所述检测试剂还可包含与所述抗体偶联的诊断剂,所述诊断剂包括但不限于放射性核素、化学发光剂、生物发光剂、顺磁性离子、酶、和/或光敏诊断剂。在一些实施方案中,所述放射性核素包括18F、52Fe、62Cu、64Cu、67Cu、86Y、90Y、89Zr、120I、123I、13N、15O、186Re;所述化学发光剂包括鲁米诺、异鲁米诺、芳族吖啶酯、咪唑;所述生物发光剂包括荧光素、荧光素酶、水母发光蛋白;所述顺磁性离子包括铬(III)、锰(II)、铁(III)、铁(II);所述酶包括辣根过氧化物酶、碱性磷酸酯酶、葡萄糖氧化酶;所述光敏诊断剂包括二羟基硅酞菁、亚甲基蓝、原卟啉、血卟啉、光卟啉。
在一些实施方案中,所述试剂盒还包括容器、使用说明书、缓冲剂等,在另一些实施方案中,所述试剂盒还包含用于溶解待测样品的裂解介质、检测所需的通用试剂和缓冲液,如各种缓冲液、检测标记、检测底物等。该检测试剂盒可以是体外诊断装置。
本发明的第九方面提供了如下任一种方法:
(1) 一种生产本发明第一方面所述的抗体或其抗原结合片段的方法,所述方法包括如下步骤:培养本发明第四方面所述的经基因改造的宿主细胞,从宿主细胞培养产物中分离纯化得到本发明第一方面所述的抗体或其抗原结合片段;
(2) 一种非诊断目的地检测Claudin18.2蛋白的方法,所述方法包括如下步骤:将待测样品与本发明第一方面所述的抗体或其抗原结合片段或本发明第二方面所述的多特异性抗体接触,检测Claudin18.2蛋白与抗体免疫复合物的形成;
(3) 一种本发明第四方面所述的经基因改造的宿主细胞的制备方法,所述方法包括如下步骤:将本发明第三方面中所述的表达载体引入到宿主细胞中;
(4) 一种体外抑制Claudin18.2蛋白活性的方法,所述方法包括如下步骤:将本发明第一方面所述的抗体或其抗原结合片段、本发明第二方面所述的多特异性抗体、本发明第三方面所述的核酸分子或表达载体引入到生物体细胞中,通过表达本发明第一方面所述的抗体或其抗原结合片段或本发明第二方面所述的多特异性抗体抑制Claudin18.2蛋白的活性。
在一些实施方案中,所述引入的方式包括但不限于:脂质转染法、微注射、电穿孔、DNA载体、逆转录病毒载体、慢病毒载体、痘病毒载体、单纯疱疹病毒载体、腺病毒载体、腺相关病毒载体。
本发明还提供了一种诊断和/或辅助诊断Claudin18.2阳性相关疾病的方法。
进一步,所述方法包括如下步骤:采用本发明第一方面所述的抗体或其抗原结合片段、本发明第二方面所述的多特异性抗体和/或本发明第七方面所述的检测试剂或试剂盒对受试者来源的待测样品进行检测,通过抗原抗体反应来检测待测样品中Claudin18.2蛋白的存在情况,以诊断和/或辅助诊断受试者是否患有Claudin18.2阳性相关疾病和/或患Claudin18.2阳性相关疾病的风险。
本发明还提供了一种治疗和/或预防Claudin18.2阳性相关疾病的方法。
进一步,所述方法包括如下步骤:给有需要的受试者施用治疗和/或预防有效量的本发明第一方面所述的抗体或其抗原结合片段、本发明第二方面所述的多特异性抗体、本发明第五方面中所述的工程化免疫细胞、本发明第六方面所述的抗体药物偶联物和/或本发明第七方面所述的药物组合物。
本发明的第十方面如下任一方面应用,所述应用包括:
(1) 本发明第一方面所述的抗体或其抗原结合片段、和/或本发明第二方面所述的多特异性抗体在制备用于检测Claudin18.2蛋白的检测试剂中的应用;
(2) 本发明第一方面所述的抗体或其抗原结合片段、本发明第二方面所述的多特异性抗体、和/或本发明第八方面中所述的检测试剂在制备用于检测Claudin18.2蛋白的试剂盒中的应用;
(3) 本发明第一方面所述的抗体或其抗原结合片段、本发明第二方面所述的多特异性抗体、本发明第八方面所述的检测试剂或试剂盒在制备用于诊断和/或辅助诊断Claudin18.2阳性相关疾病的产品中的应用;
(4) 本发明第一方面所述的抗体或其抗原结合片段、本发明第二方面所述的多特异性抗体、本发明第八方面所述的检测试剂或试剂盒在非诊断目的地检测Claudin18.2蛋白中的应用;
(5) 本发明第一方面所述的抗体或其抗原结合片段、本发明第二方面所述的多特异性抗体、本发明第三方面所述的核酸分子或表达载体在制备用于治疗和/或预防Claudin18.2阳性相关疾病的经基因改造的宿主细胞中的应用;
(6) 本发明第一方面所述的抗体或其抗原结合片段、本发明第二方面所述的多特异性抗体、本发明第三方面所述的核酸分子或表达载体、和/或本发明第五方面中所述的嵌合抗原受体在制备用于治疗和/或预防Claudin18.2阳性相关疾病的工程化免疫细胞中的应用;
(7) 本发明第一方面所述的抗体或其抗原结合片段、本发明第二方面所述的多特异性抗体、和/或本发明第三方面中所述的核酸分子在制备用于治疗和/或预防Claudin18.2阳性相关疾病的抗体药物中的应用;
(8) 本发明第一方面所述的抗体或其抗原结合片段、本发明第二方面所述的多特异性抗体、本发明第三方面所述的核酸分子或表达载体、本发明第四方面所述的经基因改造的宿主细胞、本发明第五方面所述的嵌合抗原受体或工程化免疫细胞、和/或本发明第六方面所述的抗体药物偶联物在制备用于治疗和/或预防Claudin18.2阳性相关疾病的药物或生物制剂中的应用;
(9) 本发明第一方面所述的抗体或其抗原结合片段或本发明第二方面所述的多特异性抗体和抗PD1抗体联合在制备用于治疗和/或预防Claudin18.2阳性相关疾病的药物中的应用。
进一步,所述Claudin18.2阳性相关疾病包括胃癌、卵巢癌、食管癌、胰腺癌、肺癌、头颈鳞状细胞癌、神经胶质瘤、神经母细胞瘤、咽喉癌、鼻咽癌、甲状腺癌、恶性胸膜间皮瘤、肝癌、结直肠癌、肾癌、子宫内膜癌、子宫颈癌、膀胱癌、前列腺癌、睾丸癌、皮肤癌、黑色素瘤、白血病、淋巴瘤。
在本发明的具体实施方案中,所述Claudin18.2阳性相关疾病并不局限于本发明所列举出的疾病,任何和Claudin18.2过表达相关的疾病均在本发明的保护范围内。
相对于现有技术,本发明具有的优点和有益效果如下:
本发明提供了一种全新的特异性靶向Claudin18.2的抗体,与临床上第一个针对Claudin18.2靶点的单抗药物IMAB362(即Zolbetuximab)相比,本发明提供的抗体具有更强的结合活性、内化率、ADCC活性和ADCP活性,本发明提供的抗体可应用于Claudin18.2阳性相关疾病的药物制备中,具有广阔的临床应用前景。
附图说明
图1为人源抗体噬菌体库建库及淘选示意图;
图2为3轮噬菌体淘洗的富集结果图;
图3为人源抗体库抗体还原条件下SDS-PAGE凝胶电泳结果图;
图4为4个候选抗体的流式结合活性图,其中,A图:与Claudin18.2过表达细胞系HEK293(Claudin 18.2)的结合活性图;B图:与Claudin18.1过表达细胞系HEK293(Claudin18.1)的结合活性图;
图5为4个候选抗体的细胞内化结果图;
图6为流式细胞术验证4A7、IMAB362抗体的结合活性图;
图7为抗体4A7与IMAB372的ADCC活性评价及肿瘤细胞杀活性结果图,其中,A图:利用荧光素酶报告基因细胞系Jurkat FcγRIII 评价抗体的ADCC活性;B图:评价4A7、IMAB362抗体靶向肿瘤细胞并利用人PBMC细胞介导的杀伤活性;
图8为利用荧光素酶报告基因细胞系Jurkat FcγRII评价抗体4A7与IMAB362的ADCP活性;
图9为抗体4A7、IMAB362单药治疗在NOD-SCID小鼠中的肿瘤抑制效果图;
图10为抗体4A7、IMAB362单药治疗或与鼠抗PD-1抗体联合治疗在C57BL6/J小鼠中的肿瘤抑制效果图及解剖后小鼠质量图,其中,A图:各治疗组肿瘤体积变化图,*P<0.05,**P<0.01,***P<0.001,****P<0.0001,n=5,mean±SEC;B图:各治疗组小鼠处死后解剖肿瘤重量。
具体实施方式
下面结合具体实施例,进一步阐述本发明,具体实施例仅用于解释本发明,而不能理解为对本发明的限制。本领域的普通技术人员可以理解为:在不脱离本发明的原理和宗旨的情况下可以对这些实施例进行多种变化、修改、替换和变型,本发明的范围由权利要求及其等同物限定。下列实施例中未注明具体条件的实验方法,通常按照常规条件或按照厂商所建议的条件实施检测。
实施例1 抗Claudin18.2抗体4A7的筛选
1、天然人抗体噬菌体库的筛选
首先,如图1所示,利用天然人源噬菌体抗体展示库质粒制备和扩增靶向胃癌过表达抗原Claudin18.2的人源化抗体库,共经3轮的负向和正向淘选以获得与表达Claudin18.1的细胞弱结合并同时与表达Claudin18.2的细胞强结合的阳性噬菌体,图2为3轮噬菌体淘洗的富集情况分析,第3轮生物淘洗的阳性率显著增加,说明阳性噬菌体得到了有效的富集,可用于进一步的候选分子的筛选。从第3轮淘洗的阳性克隆挑取4个克隆进行测序。
2、候选抗体的制备
将上述4套抗体库序列构建至真核表达载体pCDNA3.1上,转染至处于对数生长期的CHO悬浮细胞中,培养7天左右收获培养上清,并利用镍柱进行蛋白纯化。利用SDS-PAGE、HPLC-SEC来评价纯化后目的蛋白的相对纯度。
结果如图3所示,在还原状态下,抗体分子量均约为25 KDa和50 KDa,与理论预期相符合,且蛋白纯度均大于95%,表明抗体制备成功。各个候选抗体的表达量、蛋白纯度等见表1。
表1 候选抗体表达量、分子大小、浓度(mg/L)值及纯度总结
3、候选抗体的特异性分析
抗体的特异性是指抗体只能与其靶标抗原结合的专一性能。本发明筛选制备了4个抗体,为验证候选抗体的特异性,本实施例利用流式细胞术试验测定了4株候选抗体分别与过表达Claudin 18.1、Claudin 18.2抗原的HEK293细胞的结合活性。
从结果图4A可以看出,4株候选抗体4A7、4A8、2D2、2H9与Claudin18.2蛋白的结合活性在高浓度剂量(50 μg/mL)、低浓度剂量(5 μg/mL、0.5 μg/mL)下均明显优于临床对照抗体IMAB362;但由图4B可知,只有抗体4A7与Claudin18.1蛋白不结合,而其余3株候选抗体与Claudin18.1蛋白均有一定的结合。以上结果证明了4A7与Claudin18.2的识别具有特异性(所述抗体4A7的序列信息如下表2中所示),因此,抗体4A7具有更优的特异性靶向性,这一结论也将为后续抗体活性评价提供依据。
表2 抗体4A7的序列信息
4、候选抗体内化率评价
抗体内化实验:高内化效率的抗体筛选是ADC药物靶向性和安全性的重要保证,多数ADC药物的开发依赖于抗体的内化作用。具体实验步骤如下:
①细胞准备:将用胰酶消化后的NUGC4(人胃癌细胞系)细胞的密度调整为2×107个/mL,50 μL/孔加至1.5 mL Ep管中,FACS缓冲液清洗后并去掉上清。②样本准备:将抗体稀释至10 μg/mL。③将步骤②中的样本以每孔100 μL的体积与细胞孵育,4℃,1 h。④FACS缓冲液洗1次,离心,去掉上清。⑤37℃孵育使结合抗体内化:每孔加入200 μL的FACS缓冲液,37℃,孵育2h。⑥设置对照组:每个时间点,使用另一个在4℃孵育相同时间的样品管作为无内化的阴性对照,并设置不加抗体的二抗对照。⑦终止内化:将孵育至对应时间的37℃孵育管和4℃孵育管同时加入1 mL冰浴的FACS缓冲液,离心,去掉上清。⑧加检测抗体:APC-Anti Human IgG Fc,20 μL/孔,4℃,30 min,FACS缓冲液洗2次。⑨细胞固定:1%多聚甲醛,300 μL/孔。⑩数据获取和抗体内化率计算:流式细胞仪测定APC荧光数值,使用以下公式计算抗体内化率:tχ时间点的%MFI=37℃孵育样品的MFI×100/4℃孵育对照样品的MFI。
结果如图5所示,4株候选抗体,4A7、4A8、2D2和2H9的内化率分别为21.45%、19.07%、10.62%、11.56%,临床对照抗体IMAB362没有发现内化作用。这表明抗体4A7的内化率更高,为后续抗体偶联药物(ADC)的开发及评价提供有力依据。
5、候选抗体稳定性评价
利用Uncle高通量多参数蛋白稳定性分析仪,按仪器说明书完成蛋白上样及操作,鉴定候选抗体的理化性质,包括蛋白溶解温度(Tm)、聚集温度(Tagg)。理论上,Tm值反应某种蛋白构象稳定性,通常Tm越高,结构热稳定性越好;Tagg值反映蛋白胶体稳定性,通常Tagg越高,蛋白胶体热稳定性越好;平均粒径(Z-Ave.Dia.)代表颗粒粒径的平均值;众数粒径(Pk1 Mode Dia.)代表主峰的粒径值;样品的平均粒径均在10 nm左右;PDI为多分散性系数,反映溶液的均一性,PDI<0.1:单分散,窄分布,0.1<PDI<0.2:轻微聚集,PDI>0.2:多分散,明显聚集。
结果如表3所示,2株候选抗体4A7、2D2的PDI多分散系数均小于0.1,表明4A7、2D2抗体溶液有较高的均一性,无明聚集;进一步,抗体4A7的Tm、Tagg均值比抗体2D2高0.3~0.6℃,具有更高的热稳定性,且与临床对照抗体IMAB362相比,虽然4A7的Tm、Tagg值有所降低,但是仍然处于蛋白药物正常Tm、Tagg值范围。因此,4A7不仅具有更优的生物学特性,同时具有良好的稳定性,为后续药物开发及评价奠定良好基础。
表3 候选抗体稳定性评价
实施例2 抗体4A7的生物活性评价
1、基于前期筛选获得的抗体4A7,理论上相比较IMAB362单抗(临床上第一个针对Claudin18.2靶点的单抗药物)具有更高的亲和力和抗原靶向性。具体实验步骤如下:①细胞准备:将用胰酶消化后的HEK293(表达Claudin18.2)细胞的密度调整为5×106个/mL,100μL/孔加至1.5 mL Ep管中,FACS缓冲液清洗并去掉上清。②样品准备:将抗体4A7和对照抗体IMAB362稀释8个浓度,依次为10 μg/mL,5 μg/mL,2.5 μg/mL,1.25 μg/mL,0.625 μg/mL,0.3125 μg/mL,0.156 μg/mL,0.078 μg/mL。③将步骤②中的样品与细胞孵育,4℃,30 min。④FACS缓冲液洗1次,去掉上清。⑤加检测抗体:APC-Anti Human IgG Fc,100 μL/孔,4 ℃,30 min,FACS缓冲液洗2次。⑥细胞固定:1%多聚甲醛,300 μL/孔。⑦数据获取:流式细胞仪测定APC荧光数值,保存数据。
结果如图6所示,结果表明抗体4A7、抗体IMAB362与细胞上Claudin18.2抗原结合的半数最大效应浓度分别为0.78 μg/mL和1.53 μg/mL,抗体4A7具有更好的结合活性,并且结合具有一定的剂量依赖性。
2、抗体ADCC活性测定
1)荧光素酶报告细胞激活试验:①细胞准备与铺板:稳定表达FcγRIIIa受体(高亲和力158V)和NFAT反应元件驱动的萤火虫荧光素酶的人外周血白血病T细胞Jurkat效应细胞系和HEK293(表达Claudin18.2)细胞系分别作为效应细胞和靶细胞培养,两种细胞均被消化,并用MEM(1×)培养基以一定密度(4×105/mL和2.4×106/mL)重悬。将效应细胞和靶细胞分别以50 μL体积,2×104细胞/孔和1.2×105细胞/孔的比例接种到平底96孔板中,使其效靶比为1:6。②样品准备:将抗体4A7和对照抗体IMAB362稀释8个浓度,依次为80 μg/mL,10 μg/mL,1.25 μg/mL,0.156 μg/mL,0.0195 μg/mL,0.0024 μg/mL,0.0003 μg/mL,0.00004 μg/mL。100 μL/孔加至步骤①中的效应细胞与靶细胞的混合液中。③将样品板放置于细胞孵育箱中,37℃,培养6 h。④利用Bright-Lumi™II萤火虫萤光素酶报告基因检测试剂盒测定细胞的RLU值。
结果如图7A所示,抗体4A7和IMAB362均能以剂量依赖的方式有效地激活JurkatFcγRIII荧光素酶报告基因细胞系,但与IMAB362抗体对照相比(EC50约0.84 μg/mL),抗体4A7的EC50值低至0.03 μg/mL,其ADCC活性提高约26倍。
2)PBMC杀伤试验:为能更加直观地评价抗体依赖的细胞介导的细胞毒性作用,本发明使用人PBMC作为效应细胞,MC-38(Claudin18.2)(过表达人Claudin18.2抗原小鼠结直肠癌细胞系)作为靶细胞,通过乳酸脱氢酶(LDH)释放法定量测定药物激活PBMC对肿瘤细胞的杀伤活性。①靶细胞准备与铺板:MC-38(Claudin18.2)细胞消化并用ADCC缓冲液(MEM(1×),1% FBS)重悬,并将其密度调整至5×105/mL,以每孔50 μL体积,2.5×104个细胞/孔接种到96孔板中。②样品准备:抗体4A7、对照抗体IMAB362和已知具有ADCC活性但不与Cluadin18.2抗原结合的商业抗体Herceptin以30 μg/mL作为起始浓度,10倍稀释,共获得8个梯度。以每孔100 μL体积与①中的靶细胞孵育,37℃,培养30 min。③效应细胞准备:用缓冲液重新悬浮人PBMC细胞(提前1天用终浓度为100 ng/mL的抗CD3和抗CD28抗体预刺激),并将细胞密度调整至5×106/mL,以每孔50 μL体积,2.5×105个细胞/孔加入对应孔,使效应细胞与靶细胞的比例为10:1。④对照组设置:对照1(背景对照):ADCC缓冲液;对照2:MC-38(Claudin18.2)细胞+ADCC缓冲液;对照3(阳性对照):MC-38(Claudin18.2)细胞+裂解物3#(试剂盒供应)+ADCC缓冲液;对照4:MC-38(Claudin18.2)细胞+hPBMC细胞+ADCC缓冲液。⑤96孔板置于37°C、5% CO2的细胞培养箱中3 h。在细胞培养结束前15 min,裂解物3#(5 μL/孔)加入到对照3中。然后,吸取100 μL混合上清液与100 μL反应溶液混合(1#和2#混合物,试剂盒供应),并将孔板放置在37°C下孵育10 min。最后,以50 μL/孔的体积加入试剂终止溶液4#。⑥数据获取并分析:微板读取器测量OD492值。在减去相应的对照1数据背景后,所有OD492数据用于数据分析。特异性细胞毒性裂解(%)=(实验组-对照4)/(对照3-对照2)×100%。GraphPad Prism Version 5用于剂量效应曲线的数据分析。
结果如图7B所示,在靶向杀伤肿瘤细胞方面,抗体4A7、IMAB362的最大杀伤率在30%左右,相比之下,阴性对照Herceptin由于不具有肿瘤靶向性,因此并没有诱导肿瘤杀伤。4A7和IMAB362的EC50值约为0.017 μg/mL和0.14 μg/mL,4A7的肿瘤杀伤活性提高100倍左右,与前期结果一致。
3、抗体ADCP活性测定
荧光素酶报告细胞激活试验:基于前期实验结果,本发明利用荧光素酶报告细胞比较了抗体依赖的细胞介导的细胞吞噬活性。首先,①细胞准备:稳定表达FcγRII受体和NFAT反应元件驱动的萤火虫荧光素酶的人外周血白血病T细胞Jurkat效应细胞系和HEK293(Claudin18.2)细胞系分别作为效应细胞和靶细胞培养。②其余步骤参考实施例2中抗体ADCC活性测定中的“1)荧光素酶报告细胞激活试验”。
结果如图8所示,4A7激活FcγRII受体效应细胞的最大活性以荧光素酶荧光值(RLU)表示约为2×104,EC50值约为0.068 μg/mL,而IMAB362的最大ADCP活性以荧光值(RLU)表示约为1×104,降低1倍,EC50值约为1.242 μg/mL,这表明4A7也具有更强的ADCP活性。
实施例3 抗体4A7的体内活性评价
1、NOD-SCID免疫缺陷小鼠评价抗体4A7体内抑瘤效果
鉴于前期实验结果,本发明已经证明4A7在体外具有更强的结合活性、内化率、ADCC活性、ADCP活性,且4A7-MMAE抗体偶联物具有更优的肿瘤杀伤活性。为了进一步验证4A7的抗肿瘤疗效,首先,①将6周龄雌性NOD-SCID小鼠于SPF级动物房中饲养7-10天,记录体重并观察有无脱毛等异常,以适应饲养环境。②将NOD-SCID小鼠左侧皮下接种人胃癌细胞系N87(Claudin18.2)细胞(1×106个)。③分组方案:皮下接种65天后,当肿瘤体积达到约100 mm3时,分为PBS组、4A7(15 mg/kg)组、IMAB362(15 mg/kg)组,每组5只。③给药及测量方案:将待测样品4A7、IMAB362用PBS稀释成对应浓度,腹腔注射200 μL,一周给药2次,共6周,并监测小鼠肿瘤体积及体重。利用GraphPad Prism 9软件分析数据,采用的统计学方法为Two-way ANOVA。
结果如图9所示,第6周给药结束后测量各组小鼠肿瘤体积,PBS对照组小鼠肿瘤体积为600 mm3,相比较而言,4A7治疗组肿瘤体积均值约为350 mm3(P<0.001),IMAB362治疗组小鼠肿瘤体积约为500 mm3。进一步地,相比较IMAB362治疗组,4A7治疗组具有统计学意义上更优的抑瘤效果(P<0.05)。
2、C57BL/6免疫正常小鼠评价抗体4A7体内抑瘤效果
鉴于前期的结果,本发明进一步在C57BL/6免疫正常小鼠构建了过表达Claudin18.2抗原的结直肠癌模型,首先,①将6周龄雌性C57BL/6小鼠于SPF级动物房饲养7-10天,记录体重并观察有无脱毛等异常,以适应饲养环境。②C57BL/6小鼠左侧皮下接种过表达人Claudin18.2抗原的结直肠癌细胞系MC-38(Claudin18.2)(1×106个)。③分组方案:皮下接种7天后,当肿瘤体积达到约100 mm3时,分为Vehicle组、IMAB362(10 mg/kg)组、4A7(10mg/kg)组、anti-mPD1(购自于InvivoGen公司,货号:mpd1-mab15-10)(3 mg/kg)组、anti-mPD1+IMAB362(3 mg/kg+10 mg/kg)组、anti-mPD1+4A7(3 mg/kg+10 mg/kg)组,每组5只。③给药及测量方案:将待测样品4A7、IMAB362、anti-mPD1用PBS稀释成对应浓度,单独或联合腹腔注射200 μL,一周给药2次,共3周,并监测小鼠肿瘤体积及体重。利用 GraphPad Prism9软件分析数据,采用的统计学方法为Two-way ANOVA。
结果如图10A所示,根据动物福利3R原则,对给药后第17天的4A7组的部分肿瘤体积大于2000 mm3的小鼠进行了安乐死并解剖其肿瘤进行称重。给药后21天,相比较Vehicle组,IMAB362组、anti-mPD1组、anti-mPD1+IMAB362、anti-mPD1+4A7组抑瘤率分别为-1.42%(无显著性差异)、47.69%(P<0.01)、39.88%(P<0.05)、86.6%(P<0.0001),进一步,相比较anti-mPD1治疗组,anti-mPD1+4A7依然具有统计学意义上显著更优的抑瘤效果,与前期结论一致。值得注意的是,末次治疗结束后,anti-mPD1+4A7抗体治疗组有2只小鼠肿瘤完全消退。且各治疗组(Vehicle组、IMAB362组、4A7组、anti-mPD1组、anti-mPD1+IMAB362组、anti-mPD1+4A7组)末次治疗后解剖小鼠的肿瘤质量均值约为3.0035 g、2.70082 g、2.23014 g、2.425375 g、0.6135 g(图10B)。相比较Vehicle组而言,只有anti-mPD1+4A7抗体治疗组小鼠的肿瘤质量具有统计学意义上的减小(P<0.05)。以上实验结果表明,相比较单抗IMAB362、anti-mPD1单药治疗组或其联合组,4A7与anti-mPD1联合治疗,具有显著更优的抑瘤疗效,且小鼠具有更长的生存率。
Claims (31)
1.一种靶向Claudin18.2的抗体或其抗原结合片段,其特征在于,所述抗体或其抗原结合片段包含重链可变区和轻链可变区;
所述重链可变区包含HCDR1、HCDR2、HCDR3,所述轻链可变区包含LCDR1、LCDR2、LCDR3;
所述HCDR1、HCDR2、HCDR3的氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3所示;
所述LCDR1、LCDR2、LCDR3的氨基酸序列分别如SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7所示。
2.一种多特异性抗体,其特征在于,所述多特异性抗体包含权利要求1所述的抗体或其抗原结合片段,以及一个与其他抗原特异性结合的第二抗体或其抗原结合片段。
3.一种核酸分子,其特征在于,所述核酸分子编码权利要求1所述的抗体或其抗原结合片段或权利要求2所述的多特异性抗体。
4.一种表达载体,其特征在于,所述表达载体包含权利要求3所述的核酸分子。
5.一种经基因改造的宿主细胞,其特征在于,所述经基因改造的宿主细胞包含权利要求3所述的核酸分子或权利要求4所述的表达载体。
6.一种嵌合抗原受体,其特征在于,所述嵌合抗原受体包含权利要求1所述的抗体或其抗原结合片段或权利要求2所述的多特异性抗体。
7.一种工程化免疫细胞,其特征在于,所述工程化免疫细胞包含权利要求6所述的嵌合抗原受体。
8.一种抗体药物偶联物,其特征在于,所述抗体药物偶联物包含权利要求1所述的抗体或其抗原结合片段或权利要求2所述的多特异性抗体,以及与所述抗体偶联的细胞毒性剂。
9.一种用于治疗和/或预防Claudin18.2阳性疾病的药物组合物,其特征在于,所述药物组合物包含权利要求1所述的抗体或其抗原结合片段、权利要求2所述的多特异性抗体、权利要求7所述的工程化免疫细胞、权利要求8所述的抗体药物偶联物中的任意一种或多种。
10.根据权利要求9所述的药物组合物,其特征在于,所述药物组合物还包含抗PD1抗体。
11.一种用于检测Claudin18.2的检测试剂,其特征在于,所述检测试剂包含权利要求1所述的抗体或其抗原结合片段或权利要求2所述的多特异性抗体。
12.一种用于诊断Claudin18.2阳性疾病的试剂盒,其特征在于,所述试剂盒包含权利要求11所述的检测试剂。
13.一种生产权利要求1所述的抗体或其抗原结合片段的方法,其特征在于,所述方法包括如下步骤:培养权利要求5所述的经基因改造的宿主细胞,从宿主细胞培养产物中分离纯化得到权利要求1所述的抗体或其抗原结合片段。
14.一种非诊断目的地检测Claudin18.2蛋白的方法,其特征在于,所述方法包括如下步骤:将待测样品与权利要求1所述的抗体或其抗原结合片段或权利要求2所述的多特异性抗体接触,检测Claudin18.2蛋白与抗体免疫复合物的形成。
15.一种权利要求5所述的经基因改造的宿主细胞的制备方法,其特征在于,所述方法包括如下步骤:将权利要求4所述的表达载体引入到宿主细胞中。
16.一种体外抑制Claudin18.2蛋白活性的方法,其特征在于,所述方法包括如下步骤:将权利要求3所述的核酸分子或权利要求4所述的表达载体引入到生物体细胞中,通过表达权利要求1所述的抗体或其抗原结合片段或权利要求2所述的多特异性抗体抑制Claudin18.2蛋白的活性。
17.权利要求1所述的抗体或其抗原结合片段、和/或权利要求2所述的多特异性抗体在制备用于检测Claudin18.2蛋白的检测试剂中的应用。
18.权利要求1所述的抗体或其抗原结合片段、权利要求2所述的多特异性抗体、和/或权利要求11所述的检测试剂在制备用于检测Claudin18.2蛋白的试剂盒中的应用。
19.权利要求1所述的抗体或其抗原结合片段、权利要求2所述的多特异性抗体、权利要求11所述的检测试剂、和/或权利要求12所述的试剂盒在制备用于诊断和/或辅助诊断Claudin18.2阳性疾病的产品中的应用。
20.根据权利要求19所述的应用,其特征在于,所述Claudin18.2阳性疾病包括胃癌、卵巢癌、食管癌、胰腺癌、肺癌、头颈鳞状细胞癌、神经胶质瘤、神经母细胞瘤、咽喉癌、鼻咽癌、甲状腺癌、恶性胸膜间皮瘤、肝癌、结直肠癌、肾癌、子宫内膜癌、子宫颈癌、膀胱癌、前列腺癌、睾丸癌、皮肤癌、黑色素瘤、白血病或淋巴瘤。
21.权利要求1所述的抗体或其抗原结合片段、权利要求2所述的多特异性抗体、权利要求11所述的检测试剂、和/或权利要求12所述的试剂盒在非诊断目的地检测Claudin18.2蛋白中的应用。
22.权利要求1所述的抗体或其抗原结合片段、权利要求2所述的多特异性抗体、权利要求3所述的核酸分子、和/或权利要求4所述的表达载体在制备用于治疗和/或预防Claudin18.2阳性疾病的经基因改造的宿主细胞中的应用。
23.根据权利要求22所述的应用,其特征在于,所述Claudin18.2阳性疾病包括胃癌、卵巢癌、食管癌、胰腺癌、肺癌、头颈鳞状细胞癌、神经胶质瘤、神经母细胞瘤、咽喉癌、鼻咽癌、甲状腺癌、恶性胸膜间皮瘤、肝癌、结直肠癌、肾癌、子宫内膜癌、子宫颈癌、膀胱癌、前列腺癌、睾丸癌、皮肤癌、黑色素瘤、白血病或淋巴瘤。
24.权利要求1所述的抗体或其抗原结合片段、权利要求2所述的多特异性抗体、权利要求3所述的核酸分子、权利要求4所述的表达载体、和/或权利要求6所述的嵌合抗原受体在制备用于治疗和/或预防Claudin18.2阳性疾病的工程化免疫细胞中的应用。
25.根据权利要求24所述的应用,其特征在于,所述Claudin18.2阳性疾病包括胃癌、卵巢癌、食管癌、胰腺癌、肺癌、头颈鳞状细胞癌、神经胶质瘤、神经母细胞瘤、咽喉癌、鼻咽癌、甲状腺癌、恶性胸膜间皮瘤、肝癌、结直肠癌、肾癌、子宫内膜癌、子宫颈癌、膀胱癌、前列腺癌、睾丸癌、皮肤癌、黑色素瘤、白血病或淋巴瘤。
26.权利要求1所述的抗体或其抗原结合片段、权利要求2所述的多特异性抗体、和/或权利要求3所述的核酸分子在制备用于治疗和/或预防Claudin18.2阳性疾病的抗体药物偶联物中的应用。
27.根据权利要求26所述的应用,其特征在于,所述Claudin18.2阳性疾病包括胃癌、卵巢癌、食管癌、胰腺癌、肺癌、头颈鳞状细胞癌、神经胶质瘤、神经母细胞瘤、咽喉癌、鼻咽癌、甲状腺癌、恶性胸膜间皮瘤、肝癌、结直肠癌、肾癌、子宫内膜癌、子宫颈癌、膀胱癌、前列腺癌、睾丸癌、皮肤癌、黑色素瘤、白血病或淋巴瘤。
28.权利要求1所述的抗体或其抗原结合片段、权利要求2所述的多特异性抗体、权利要求3所述的核酸分子、权利要求4所述的表达载体、权利要求5所述的经基因改造的宿主细胞、权利要求6所述的嵌合抗原受体、权利要求7所述的工程化免疫细胞、和/或权利要求8所述的抗体药物偶联物在制备用于治疗和/或预防Claudin18.2阳性疾病的药物或生物制剂中的应用。
29.根据权利要求28所述的应用,其特征在于,所述Claudin18.2阳性疾病包括胃癌、卵巢癌、食管癌、胰腺癌、肺癌、头颈鳞状细胞癌、神经胶质瘤、神经母细胞瘤、咽喉癌、鼻咽癌、甲状腺癌、恶性胸膜间皮瘤、肝癌、结直肠癌、肾癌、子宫内膜癌、子宫颈癌、膀胱癌、前列腺癌、睾丸癌、皮肤癌、黑色素瘤、白血病或淋巴瘤。
30.权利要求1所述的抗体或其抗原结合片段或权利要求2所述的多特异性抗体和抗PD1抗体联合在制备用于治疗和/或预防Claudin18.2阳性疾病的药物中的应用。
31.根据权利要求30所述的应用,其特征在于,所述Claudin18.2阳性疾病包括胃癌、卵巢癌、食管癌、胰腺癌、肺癌、头颈鳞状细胞癌、神经胶质瘤、神经母细胞瘤、咽喉癌、鼻咽癌、甲状腺癌、恶性胸膜间皮瘤、肝癌、结直肠癌、肾癌、子宫内膜癌、子宫颈癌、膀胱癌、前列腺癌、睾丸癌、皮肤癌、黑色素瘤、白血病或淋巴瘤。
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