CN117603361A - 包含angptl3单抗的融合蛋白 - Google Patents
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Abstract
本申请公开了包含ANGPTL3单抗的融合蛋白ANGPTL3单抗‑B,其所述融合蛋白ANGPTL3单抗‑B由ANGPTL3单抗与序列B融合构成;本申请公开的融合蛋白ANGPTL3单抗‑B用于治疗其介导的疾病或病症,如糖尿病及其并发症如:糖尿病肾病、糖尿病性视网膜病变、糖尿病足、糖尿病心血管并发症、糖尿病性脑血管病(脑动脉硬化、无症状脑卒中)、糖尿病神经病变、肾脏相关疾病如膜性肾病、IgA肾病、紫癜肾、狼疮肾等,肿瘤相关疾病如肝细胞癌、肾细胞癌、卵巢癌、***、口腔癌、食管癌等。
Description
技术领域
本发明涉及医学免疫学和分子生物学领域。具体涉及包含血管生成素样蛋白3(ANGPTL3)的单抗的融合蛋白及其医学用途。
背景技术
血管生成素样蛋白3(ANGPTL3)是一种分泌性糖蛋白,其结构包括氨基端卷曲螺旋结构域(CCD)、分泌信号肽、短的连接肽和羧基端球状纤维蛋白原样结构域(FLD)。ANGPTL3氨基端的卷曲螺旋区域通过抑制脂蛋白酯酶LPL的活性使血浆甘油三酯(triglyceride,TG)水平升高,羧基端FLD(207-460aa)与整合素αvβ3受体结合促进血管生成(Camenisch G,Pisabarro MT,Sherman D,et al.ANGPTL3 stimulates endothelial cell adhesion andmigration via integrinαvβ3and induces blood vessel formation in vivo.J BiolChem,2002,277(19):17281-17290),氨基端螺旋样结构域参与调节脂质代谢(Ono M,Shimizugawa T,Shimamura M,et al.Protein region important for regulation oflipid metabolism inangiopoietin-like 3(ANGPTL3):ANGPTL3 is cleaved andactivated in vivo[J].J Biol Chem,2003,278(43):41804-41809),同时在足细胞损伤(Liu J,Gao X,Zhai Y,et al.A novel role of angiopoietin-like-3associated withpodocyte injury[J].Pediatr Res,2015,77(6):732-739.)、胰岛素抵抗(Robciuc MR,Maranghi M,Lahikainen A,et al.Angptl3 deficiency is associated with increasedinsulin sensitivity,lipoprotein lipase activity,and decreased serum freefatty acids[J].Arterioscler Thromb Vasc Biol,2013,33(7):1706-1713.)、动脉粥样硬化(Dewey FE,Gusarova V,Dunbar RL,et al.Genetic and pharmacologicinactivation of ANGPTL3 and cardiovascular disease[J].N Engl JMed,2017,377(3):211-221.)及肿瘤的发生及转移(Koyama T,Ogawara K,Kasamatsu A,et al.ANGPTL3is a novel biomarker as it activates ERK/MAPK pathway in oral cancer[J].Cancer Med,2015,4(5):759-769)、肥胖、糖尿病、家族性低β脂蛋白血症等方面也发挥着重要作用。此外,研究表明ANGPTL3与其他代谢疾病如肾脏疾病发生发展有一定的相关性(Liu J,Gao X,Zhai Y,et al.A novel role of angiopoietin-like-3associated withpodocyte injury.Pediatr Res,2015,77(6):732-739),ANGPTL3在功能正常的肾脏中仅微量表达,而在多种以蛋白尿为主要表现的肾脏疾病中表达量显著增多(张强,尹卫东,唐朝克;ANGPTL3与代谢紊乱相关疾病的研究进展[J]生命的化学,2018,38(1):30-34)。
炎症因子是由免疫细胞或组织细胞分泌的在细胞间发挥相互调控作用的一类高效性的小分子可溶性蛋白质,常见的炎症因子类型有白细胞介素(interleukin,IL)、干扰素(interferon,IFN)和肿瘤坏死因子(tumornecrosisfactor,TNF)等,它们可调节影响免疫细胞的发育分化、参与固有免疫和适应性免疫应答、免疫调节,在炎症反应中扮演着重要角色(EKTanYX.Chao,A.WestLL.ChanWPoeweJ.JankovicParkinson diseaseand theimmune system-associations,mechanisms and therapeuticsJ].NatRevNeurol202016(6):303-318)。
糖尿病是一个***性疾病,可引起多种并发症,其中糖尿病肾病为糖尿病并发症之一,糖尿病肾病是由糖尿病继发的肾脏病变。我国近十几年来,糖尿病肾病引起的终末期肾病发生率占首位(赵延香.早期糖尿病肾病患者血清TNF-a、IL-6的变化及其与尿白蛋白/肌酐的相关性分析[U].中国医学创新,2020,17(4):121-124.)邵长娟,沈萌,孙丽丽.血清IL-6、TNF-a、超敏C反应蛋白在糖尿病肾病患者中的水平变化及意义[J].中国卫生工程学,2020,19(3):444-446.)。近年来多项研究指出,血清炎症因子在糖尿病肾病的发病机制中起着重要作用,由于前炎症细胞因子作用于机体,诱导促炎细胞因子的产生,促进炎症因子反应,进而促进糖尿病肾病的发展(曹娟终末期糖尿病肾病患者实施血液透析联合血液灌流对血清炎症因子水平的影响[J].临床研究,2020,28(3):76-77.)。
足细胞损伤是肾病综合征(NS)常见病理变化。足细胞作为肾小球滤过屏障的重要组成成分,其损伤会引起蛋白尿,而蛋白尿是N S发展的主要推动因素,当蛋白尿达到特定阈值后,即出现低蛋白血症、水肿等表现(MacéC,Chugh SS.Nephrotic syndrome:components,connections,and angiopoietin-like-4-related therapeutics[J].J AmSoc Nephrol,2014,25(11):2393-2398),蛋白尿是肾脏疾病常见的临床表现;慢性肾疾病发展到一定阶段可引起肾纤维化。肾纤维化可由多种因素诱导产生,包括肾受到严重创伤和感染,肾血液循环阻塞或者免疫反应等。各种因素引起肾组织受损后,大量胶原纤维在间质沉积,瘢痕形成,导致肾结构和功能发生改变,从而引起肾纤维化,迄今关于肾纤维化的发生机制尚不完全明确。
目前NS病理诊断主要依靠肾活检,但其有创性限制了其广泛使用。而激素及免疫抑制剂作为治疗NS的基本药物,亦有诸多毒副作用。虽然在一种抗ANGPTL3单克隆抗体及其在制备治疗肾病综合征药物中的用途(专利号:201911177503.5)中制备了ANGPTL3-FLD的鼠源单克隆抗体,其在体内、外有效减轻足细胞损伤和降低蛋白尿的治疗效果,但没有起到组织糖尿病肾病治疗过程中肾纤维化的一大关键问题。
目前需要开发一种能够达到保护足细胞、降低尿蛋白、减轻肾脏炎症、抑制肾脏纤维化的多维度、多时空药理效应的治疗方法及药剂,本发明可满足该需求。
发明内容
本申请公开了包含ANGPTL3单抗的融合蛋白ANGPTL3单抗-B,其所述融合蛋白ANGPTL3单抗-B由ANGPTL3单抗与序列B融合构成;所述B选自细胞因子。
进一步的,细胞因子可选自白细胞介素(interleukin,IL)、干扰素(interferon,IFN)和肿瘤坏死因子(tumornecrosisfactor,TNF)等。在一些实施案例中,细胞因子进一步选自:IL-22,IL-1RA,IL-4,IL-10,IL-13,TGF-β和/或EPO等。进一步优选人源IL-22(SEQ IDNo:5)和或鼠源IL-22(SEQ ID No:6),在一些实施方案中,ANGPTL3单抗与序列B可以通过连接子连接,所述连接子可选自(GGGGS)2,(GGGGS)3,(GGGGS)4,(EAAAK)2,和/或(EAAAK)3等。
本申请还公开了药物组合物,所述药物组合物包含所公开的融合蛋白ANGPTL3单抗-B和药学上可接受的载体。
本申请还公开了编码本文所公开的融合蛋白ANGPTL3单抗-B分离的核苷酸,本发明还涵盖所包含公开的核苷酸的载体,及包含该载体或编码本发明融合蛋白ANGPTL3单抗-B的多核苷酸的宿主细胞。
本申请还公开了融合蛋白ANGPTL3单抗-B在制备治疗其介导的疾病或病症的药物中的用途,该用途包括向有需要的患者施用包含本文所公开的融合蛋白ANGPTL3单抗-B的药物组合物。
附图说明
图1.SDS-PAGE检测融合蛋白的分子量
图2.SEC-HPLC检测融合蛋白的纯度
图3.SPR检测融合蛋白的亲和力
图4.热稳定性分析检测融合蛋白的熔点
图5.体外荧光实验验证融合蛋白ANGPTL3单抗端体外活性
图6.Western Blot验证融合蛋白IL-22端体外活性
图7.糖尿病肾病小鼠模型中融合蛋白干预减少尿蛋白
图8.糖尿病肾病小鼠模型中融合蛋白干预保护肾功能
图9.糖尿病肾病小鼠模型中融合蛋白干预降低血糖血脂
图10.糖尿病肾病小鼠模型中融合蛋白干预减轻足细胞损伤
图11.糖尿病肾病小鼠模型中融合蛋白干预抑制肾脏纤维化
图12.糖尿病肾病小鼠模型中融合蛋白干预减轻炎症反应
图13.糖尿病肾病小鼠模型中融合蛋白通过抑制NF/κB/NLRP3信号通路保护肾脏
图14.阿霉素肾病小鼠模型中融合蛋白干预减少尿蛋白
图15.阿霉素肾病小鼠模型中融合蛋白干预保护肾功能
图16.阿霉素肾病小鼠模型中融合蛋白干预改善低白蛋白血症
图17.阿霉素肾病小鼠模型中融合蛋白干预改善高脂血症
图18.阿霉素肾病小鼠模型中融合蛋白干预减轻足细胞损伤
图19.阿霉素肾病小鼠模型中融合蛋白干预抑制肾脏纤维化
图20.阿霉素肾病小鼠模型中融合蛋白通过改善线粒体功能保护肾脏
图21.阿霉素肾病小鼠模型中融合蛋白通过改善溶酶体自噬保护肾脏
具体实施方式
本申请公开了融合蛋白ANGPTL3单抗-B,所述融合蛋白ANGPTL3单抗-B通过ANGPTL3单抗和细胞因子连接,可保护足细胞、降低尿蛋白、减轻肾脏炎症、抑制肾脏纤维化。
定义和通用技术
除非本文另作定义,否则结合本发明使用的科技术语应当具有本领域普通技术人员通常所了解的意义。一般来说,与本文所描述的细胞和组织培养、分子生物学、免疫学、微生物学、遗传学以及蛋白质和核酸化学及杂交学结合使用的命名法及其技术是本领域中众所周知并日常用的那些。
如本文所述术语ANGPTL3是血管生成素样蛋白(ANGPTL)家族的重要组成成员,是一种与血管生成素结构相似的分泌性糖蛋白,在调节血管生成中起重要作用。ANGPTL3的结构包括氨基端卷曲螺旋结构域、分泌信号肽、短的连接肽或羧基端球状纤维蛋白原样结构域(fibrinogen-like domain,FLD),术语“融合蛋白ANGPTL3单抗-B”是指ANGPTL3蛋白或其蛋白片段(氨基端卷曲螺旋结构域、分泌信号肽、短的连接肽或羧基端球状纤维蛋白原样结构域)做抗原制备的单抗与其它序列的融合。所述ANGPTL3单抗能够靶向作用于ANGPTL3蛋白或其蛋白片段如氨基端卷曲螺旋结构域、分泌信号肽、短的连接肽或羧基端球状纤维蛋白原样结构域FLD,优选ANGPTL3单抗靶向作用于FLD结构域。目前已有的ANGPTL3单抗如SEQID No:1、SEQ ID No:2和SEQ ID No:3SEQ ID No:4也都适用于本发明。
在本文可互换使用的术语“多肽”、“肽”和“蛋白质”是指具有任何长度的氨基酸的聚合形式,其可包括遗传编码和非遗传编码的氨基酸、化学或生物化学修饰的或衍生的氨基酸以及具有修饰的肽主链的多肽。所述术语包括融合蛋白,包括但不限于具有异源氨基酸序列的融合蛋白、具有异源和同源前导序列的融合体等。
融合蛋白ANGPTL3单抗-B可以通过ANGPTL3单抗融合异源序列而得到。如本文所描述,异源序列可以是含氨基酸序列或不含氨基酸序列的聚合物。异源序列可以与ANGPTL3单抗直接融合、以化学方式或通过由单一多核苷酸重组表达来与ANGPTL3单抗融合,或其可以经由连接子或接头分子接合。肽基连接子或接头分子可以是一个或多个氨基酸残基(或mer),例如1、2、3、4、5、6、7、8或9个残基(或mer)。连接子或接头分子还可以被设计成具有DNA限制性核酸内切酶或蛋白酶的裂解位点以允许分离融合的部分。
如本文所述术语“连接子”当形成本发明的融合蛋白时,可以(但未必)采用连接子。连接子可以由通过肽键(即肽基连接子)连接在一起的氨基酸构成。在本发明的一些实施方案中,连接子是由通过肽键连接的1至20个氨基酸构成,其中这些氨基酸选自20种天然存在的氨基酸。在一些实施方案中,适合连接子包括(GGGGS)2,(GGGGS)3,(GGGGS)4,(EAAAK)2,(EAAAK)3等。
如本文所述术语“药物组合物”通过混合具有所需纯度的本发明的融合蛋白ANGPTL3单抗-B和药学上可接受的载体来制备,所述载体为冻干制剂或水溶液的形式。药学上可接受的载体在使用的剂量和浓度下对接受者通常是无毒的,配制用于以液体形式、固体形式、气溶胶形式或其它口服摄入形式。在某些方面,本公开中提供的药物组合物可以用于治疗受试者的疾病的方法,其中所述方法包括向受试者施用:融合蛋白和载体,其包含编码融合蛋白的多核苷酸;修饰的表达融合蛋白的宿主细胞;或其药物组合物,其中所述疾病与融合蛋白结合的抗原的存在有关。
如本文所述术语“细胞因子”是指参与炎症反应的各种细胞因子,本文中所述细胞因子主要有白细胞介素(interleukin,IL)、干扰素(interferon,IFN)和肿瘤坏死因子(tumornecrosisfactor,TNF)中的一种或多种。在一种实施例中,细胞因子进一步选自:IL-22,IL-1RA,IL-4,IL-10,IL-13,TGF-β,EPO等。
如本文所述术语“载体”也可以是能够转运另一种核酸的核酸分子。载体可以是例如质粒、粘粒、病毒、噬菌体、RNA载体或线性或环状DNA或RNA分子,其可以包括染色体、非染色体、半合成或合成核酸分子。在一些实施方案中,载体是“质粒”,即,可以连接其它DNA区段的环状双链DNA环。此外,某些载体能够引导其可操作地连接的基因的表达。此类载体在本文中称为“重组表达载体”(或简称为“表达载体”)。
如本文所述术语“重组宿主细胞”(或简称“宿主细胞”)意思指引入外源核酸和/或重组载体的细胞。应了解,“重组宿主细胞”和“宿主细胞”不仅是指特定受试细胞,而且还指此类细胞的子代。由于某些修饰可能因突变或环境影响而出现在后代中,故事实上,此子代可能不同于亲代细胞,但仍包括在如本文中所使用的术语“宿主细胞”的范围内。转化的宿主细胞可以包括但不限于原核细胞、真核细胞以及哺乳动物、植物、昆虫、真菌或细菌来源的细胞。另外,对于哺乳动物细胞,可以使用CHO细胞、F2N细胞、CSO细胞、BHK细胞、鲍氏黑色素瘤(Bowes melanoma)细胞、HeLa细胞、911细胞、AT1080细胞、A549细胞、HEK 293细胞、HEK293T细胞等。然而,哺乳动物细胞不限于此,并且可以使用本领域技术人员已知可用作哺乳动物宿主细胞的任何细胞。
如本文所用术语“治疗”等是指获得所需药理学和/或生理学作用。所述作用就完全或部分预防疾病或其症状而言可为防治性的和/或部分或完全治愈疾病和/或可归因于所述疾病的不利作用而言可为治疗性的。如本文所用的“治疗(Treatment)”涵盖对哺乳动物,特别是人的疾病的任何治疗,并且包括:(a)预防所述疾病在可易患所述疾病但尚未诊断为患有所述疾病的受试者中发生;(b)抑制所述疾病,即遏止其发展;以及(c)减轻所述疾病,即导致所述疾病消退。
可在本文中互换使用的术语“患者”是包括人和非人动物,例如哺乳动物,例如小鼠,大鼠,豚鼠,狗,猫,兔,牛,马,绵羊,山羊和猪。所述术语还包括鸟类,鱼类,爬行动物,水陆两栖动物等。应当理解,更具体的患者是人。同样,更具体的患者和受试者是非人哺乳动物,例如小鼠,大鼠和狗。
可在本文中互换使用的术语“施用”是指本发明公开内容的融合蛋白足以改善被治疗疾病的一种或多种症状的融合蛋白的量及给药方式,具有统计意义的方式。在一种实施例中,注射给药,ANGPTL3单抗/IL-22融合蛋白5-100mg/kg、20-100mg/kg、30-100mg/kg、40-100mg/kg、50-100mg/kg、60—100mg/kg、70-100mg/kg、80-100mg/kg、90-100mg/kg。
如本文所述术语“单克隆抗体”通常是指一群基本同源的抗体,包含于该群的各个抗体除了可能的以微量存在的天然发生的突变之外可以是相同的。单克隆抗体是高度特异性的,直接针对单个抗原性位点。
“分离的核苷酸”是指不含基因的核酸(例如DNA),该核苷酸在本发明中是指融合蛋白ANGPTL3单抗-B的核酸分子。因此,该术语包括,例如,掺入载体的重组DNA;进入自主复制的质粒或病毒;或进入原核生物或真核生物的基因组DNA;或者作为独立于其它序列的单独分子(例如,cDNA或通过PCR或限制性核酸内切酶消化产生的基因组或cDNA片段)存在。此外,该术语包括从DNA分子转录的RNA分子,以及编码附加多肽序列的杂交基因的一部分的重组DNA。
本文所用术语“可变轻链”(VL)是指抗体轻链可变结合区。可变结合区由离散的,定义明确的子区域组成,这些子区域称为“互补决定区”(CDR,也称为HVR(高变区))和“框架区”(FR)。CDR指的是抗体可变区内的赋予抗原特异性和/或结合亲和力的氨基酸,被FR隔开。每个抗体轻链可变区(LCDR1、LCDR2、LCDR3)中有三个CDR,每个抗体重链可变区(HCDR1、HCR2、HCDR3)中有三个CDR。
如本文所述术语“CL”是指“免疫球蛋白轻链恒定区”或“轻链恒定区”,即来自抗体轻链的恒定区。
如本文所用术语“表达”是指基于核酸分子例如基因的编码序列产生多肽的过程。该过程可以包括转录、转录后控制、转录后修饰、翻译、翻译后控制、翻译后修饰或其任何组合。
如本文所用术语“病症或疾病”包括糖尿病及其并发症如:糖尿病肾病、糖尿病性视网膜病变、糖尿病足、糖尿病心血管并发症、糖尿病性脑血管病(脑动脉硬化、无症状脑卒中)、糖尿病神经病变、肾脏相关疾病如膜性肾病、IgA肾病、紫癜肾、狼疮肾等,肿瘤相关疾病如肝细胞癌、肾细胞癌、卵巢癌、***、口腔癌、食管癌等。
如本文所述术语“瞬时转染”是指将构建好的质粒通过某种方式导入到哺乳动物细胞内,该质粒上的外源基因不整合到细胞自身的基因组上,这一整个快速转染得到蛋白的过程就称为瞬时转染表达。
实施例1融合蛋白表达和纯化
1.1以pTT5为载体分别构建2种质粒
1.1.1在HindIII和BamHI酶切位点中***单抗重链可变区和恒定区序列(VH+CH1-CH3)、接头序列linker(GGGGS)3、鼠源IL-22序列(mIL-22);
1.1.2在EcoRI和BamHI酶切位点中***单抗轻链可变区和恒定区序列(VL+CL),如图1所示。构建好的质粒经Sanger测序无误后进行下一步操作。
1.2转化感受态细胞DH5α扩增质粒
将质粒和感受态细胞DH5α混匀共同冰浴,经42℃水浴热激后,迅速转至冰上静置,随后加入LB液体培养基在37℃的摇床上复苏,取少量复苏后液体涂布到LB固体培养基平板上,37℃倒置培养过夜,挑选单克隆入摇瓶中扩大培养后,采用天根大提试剂盒抽提质粒备用。
1.3将扩增后的2种质粒共同瞬时转染ExpiCHO-S细胞以表达蛋白
将ExpiCHO-S细胞在5%CO2的恒温培养箱中培养至密度为3-6×106cells/mL,将细胞浓度调至3.5×106cells/mL,之后在相同的培养条件下培养18-20h至细胞密度在7-9×106cells/mL、活率大于95%时,用预热的培养基将细胞稀释到6×106cells/mL,使用赛默飞ExpiFectamineTM CHO转染试剂盒,将质粒与转染试剂混合静置1min后,缓慢加入细胞中,边加边晃动摇瓶(避免局部浓度过高),充分混匀后继续在之前的条件下培养,并于第一天和第五天添加补料培养基,期间检测细胞密度,培养12天后,离心收集上清用于后续的蛋白纯化。
1.4蛋白纯化和超滤浓缩
使用ProteinA亲和层析柱纯化蛋白,配制Buffer A(1×TBS溶液)和BufferB(0.1M甘氨酸溶液),打开纯化仪,1mL的Protein A亲和层析柱在用Buffer A平衡10个柱体积之后,将上述细胞培养物上清液进样,上样结束后用Buffer A冲洗至少10个柱体积,之后用Buffer B洗脱5个柱体积,收集洗脱液,用Tris-HCl缓冲液将洗脱液调至中性。使用50KD超滤管对洗脱液进行超滤,加PBS溶液补齐至15ml后3500rpm、10min离心3次,收集终末600-1000ul超滤管内液体至干净1.5mlEP管中,-80℃保存。
实施例2融合蛋白特性鉴定方法
2.1SDS-PAGE检测融合蛋白的分子量
配置10%的Tris-甘氨酸凝胶,在还原和非还原条件下上样20ul进行电泳,设置电压80V用以浓缩及120V用以分离,电泳结束后取出凝胶使用考马斯亮蓝染色10min,再用30%甲醇和10%乙酸组成的脱色液漂洗过夜,通过Biorad凝胶成像仪拍摄成像,根据条带位置确定分子量。
2.2SEC-HPLC检测融合蛋白的纯度
采用Agilent 1260Infinity II SFC体系,用PBS缓冲液稀释100μg纯化蛋白后,以
1.0ml/min的流速注入TOSOH TSKgel G3000WXL色谱柱(7.8mm×30cm,5μm),在37℃、280nm紫外照射下检测30分钟,通过了解融合蛋白的聚集和降解程度判断其纯度。
2.3SPR检测融合蛋白的亲和力
采用Biacore T200***(GE Healthcare,USA)SPR分析,将人IgG捕获抗体预固定在CM5芯片(GE Healthcare,USA)上,通过调节捕获时间,在大约60RU的时间将融合蛋白捕获到芯片上,随后在芯片上流经抗原hANGPTL3(S17-E460)或抗原mANGPTL3(S17-T455),结合120秒,解离600秒,通过***生成的结合和解离参数来计算亲和力。
2.4热稳定性分析检测融合蛋白的熔点
采用Nano Temper PR.48仪器(Nano Temper Scientific,Germany),在20-95℃温度范围内,将融合蛋白用PBS缓冲液稀释至1.03mg/ml、0.5mg/ml和2.2mg/ml,通过监测色氨酸残基微变化引起的荧光强度变化,将原始数据拟合为S形曲线,其拐点即为熔点,借此判断融合蛋白的热稳定性。
2.5体外荧光实验验证融合蛋白ANGPTL3单抗端体外活性
高糖条件下,足细胞高表达ANGPTL3,能与整合素ανβ3结合,导致PSI表位暴露,可被特异性抗体AP5结合和显示。为测定融合蛋白ANGPTL3单抗端活性,将肾小球足细胞(MPC5)接种于6孔板,密度为1×106/ml、温度为37℃、浓度为5%CO2过夜,细胞贴壁后加入126ng/ml融合蛋白孵育1-2小时,再加入30mM高糖溶液进行损伤干预4-6小时,最后应用PSI结构域特异性抗体AP5检测融合蛋白是否能与ANGPTL3竞争结合以阻断PSI表位的暴露。
2.6WesternBlot验证融合蛋白IL-22端体外活性
IL-22能够诱导小鼠近端肾小管上皮细胞(mPTC)中STAT3的磷酸化。首先将mPTC接种于6孔细胞培养板中,密度为1×106/ml、温度为37℃、浓度为5%CO2过夜,随后使用20mm融合蛋白处理细胞2h、4h和6h,收集细胞后用50ul弱RIPA裂解30min,4℃、12000rpm离心15min后收集裂解液上清,最后用兔抗小鼠磷酸化STAT3抗体(Biolegend)和HPR标记的山羊抗兔抗体(Merck)进行Western blot检测。
实施例3动物实验中的功能验证
3.1构建糖尿病肾病小鼠模型及给药方案
选用常州Cavins动物实验有限公司(中国江苏)生产的db/m和db/db小鼠(C57BKS/Lepr,雄性,6-7周龄),饲养在特定无病原体(SPF)条件下,对照组为db/m小鼠,饲喂普通饲料,模型组为db/db小鼠,饲喂高脂(脂肪含量40%)饲料4-5周。4周左右成功建立DN小鼠模型,开始给药,每周2次腹腔注射,连续注射8周,正常对照组及模型组注射相同剂量的生理盐水。实验分组:对照组(7只),模型组(7只),ANGPTL3单抗治疗组(7只,ANGPTL3单抗20mg/kg),IL-22Fc治疗组(7只,IL-22Fc 12mg/kg),ANGPTL3单抗/IL-22融合蛋白治疗组(7只,融合蛋白25.3mg/kg),ANGPTL3单抗/IL-22Fc联合治疗组(7只,ANGPTL3单抗20mg/kg+IL-22Fc12mg/kg),阳性药物氯沙坦治疗组(7只,氯沙坦20mg/kg/d,每日灌胃)。
3.2构建阿霉素肾病小鼠模型及给药方案
选取6周龄雄性Balb/c小鼠共48只,饲养在特定无病原体(SPF)条件下,试验期间自由进水、进食。喂养1周后(7周龄),用生理盐水稀释至2mg/ml的阿霉素一次性尾静脉注射10.5mg/kg造模。造模后第2天开始给药,每周2次腹腔注射,连续注射12周,正常对照组及模型组注射相同剂量的生理盐水。实验分组:对照组(8只),模型组(8只),ANGPTL3单抗治疗组(8只,ANGPTL3单抗20mg/kg),IL-22Fc治疗组(8只,IL-22Fc 12mg/kg),ANGPTL3单抗/IL-22融合蛋白治疗组(8只,融合蛋白25.3mg/kg),ANGPTL3单抗/IL-22Fc联合治疗组(8只,ANGPTL3单抗20mg/kg+IL-22Fc 12mg/kg)。
3.3检测血液、尿液样本中各生物大分子或代谢产物水平
分别于给药后的不同时间点收集样本,血液通过内眼角毛细血管采集,尿液通过24小时小鼠代谢笼采集,保存于-80℃冰箱中。后续使用相关检测试剂盒(中国南京建成)检测血清肌酐、血清尿素氮、血清总胆固醇、血清甘油三酯、血糖、尿肌酐和24小时尿蛋白水平等;使用ELISA试剂盒(中国南京建成)检测尿微量白蛋白、血清TNF-α、IL-6和IL-1β等。
3.4借助光镜或电镜观察肾脏组织形态学改变
小鼠处死后摘取肾脏纵向切割,在4%多聚甲醛溶液中固定过夜,石蜡包埋,切成4μm厚的切片,分别使用H&E染色、PAS染色、Masson染色,在200×、400×显微镜下观察肾组织病理损伤及纤维化程度,利用Image J软件对整个图像中正区域的百分比进行量化和分析。为观察肾小球超微结构的变化,取适当大小的肾组织在4℃下固定过夜,随后在60kv下进行电镜观察,每组取3-5只小鼠肾组织,观察足细胞超微结构、肾小球基底膜厚度、线粒体及溶酶体的变化。
实施例4:实验结果
1.SDS-PAGE检测融合蛋白的分子量
SDS-PAGE显示了还原条件下ANGPTL3单抗-IL22融合蛋白的分子量为重链72.39KD、轻链25.94KD,与预期相符,并显示了其高纯度(图1)
2.SEC-HPLC检测融合蛋白的纯度
SEC-HPLC显示蛋白主峰位于5.583min处,仅在8min前后存在一微弱小峰,表明ANGPTL3单抗-IL22融合蛋白未发生明显的聚集和解离,进一步证实了其纯度良好。(见图2)
3.SPR检测融合蛋白的亲和力
SPR分析显示ANGPTL3单抗-IL22融合蛋白与鼠源ANGPTL3的KD值为1.040E-8,与人源ANGPTL3的KD值为5.572E-9,呈现出和ANGPTL3单抗本身相近的亲和力。(见图3)
4.热稳定性分析检测融合蛋白的熔点
热稳定性分析显示ANGPTL3单抗-IL22融合蛋白、ANGPTL3单抗、mIL22Fc三种蛋白的Tm1值分别为66.1℃、66.6℃和66.5℃,表明融合蛋白的构建并不影响其原先的熔点,呈现出良好的热稳定性。(见图4)
5.体外荧光实验验证融合蛋白ANGPTL3单抗端体外活性
体外荧光实验显示ANGPTL3单抗-IL22融合蛋白与ANGPTL3单抗相似,可有效阻断ANGPTL3与足细胞表面整合素ανβ3的结合抑制PSI表位的暴露,证实了其ANGPTL3单抗端的体外活性。(见图5)
6.WesternBlot验证融合蛋白IL-22端体外活性
WesternBlot显示ANGPTL3单抗-IL22融合蛋白在对小鼠近段肾小管上皮细胞作用2小时后即可明显升高STAT3的磷酸化水平,证实了其IL-22端的体外活性。(见图6)
7.糖尿病肾病小鼠模型中融合蛋白干预减少尿蛋白
糖尿病肾病小鼠模型使用ANGPTL3单抗-IL22融合蛋白治疗8周后相比模型组和mIL22Fc治疗组24小时尿蛋白显著降低(P<0.05)。(见图7)
8.糖尿病肾病小鼠模型中融合蛋白干预保护肾功能
糖尿病肾病小鼠模型使用ANGPTL3单抗-IL22融合蛋白治疗8周后相比模型组和ANGPTL3单抗治疗组血清尿素氮水平显著降低(P<0.05)。(见图8)
9.糖尿病肾病小鼠模型中融合蛋白干预降低血糖血脂
糖尿病肾病小鼠模型使用ANGPTL3单抗-IL22融合蛋白治疗8周后相比模型组血糖、血清甘油三酯、血清总胆固醇水平均有显著降低(P<0.05),同时可以看到,ANGPTL3单抗-IL22融合蛋白相比ANGPTL3和IL22联用在降低血清甘油三酯上显示出一定的优势。(见图9)
10.糖尿病肾病小鼠模型中融合蛋白干预减轻足细胞损伤
糖尿病肾病小鼠模型使用ANGPTL3单抗-IL22融合蛋白治疗8周后相比模型组、ANGPTL3单抗治疗组、mIL22Fc治疗组足突融合和基底膜增厚得到了明显改善。(见图10)
11.糖尿病肾病小鼠模型中融合蛋白干预抑制肾脏纤维化
糖尿病肾病小鼠模型使用ANGPTL3单抗-IL22融合蛋白治疗8周后相比模型组、ANGPTL3单抗治疗组、mIL22Fc治疗组肾小球及肾间质区域的胶原沉积显著减少,肾脏纤维化得到了显著改善。(见图11)
12.糖尿病肾病小鼠模型中融合蛋白干预减轻炎症反应
糖尿病肾病小鼠模型使用ANGPTL3单抗-IL22融合蛋白治疗8周后相比模型组和ANGPTL3单抗治疗组血清炎症因子TNF-α、IL-6和IL-1β水平均有显著降低(P<0.05),同时可以看到,ANGPTL3单抗-IL22融合蛋白相比ANGPTL3和IL22联用在降低TNF-α和IL-1β上显示出显著的优势。(见图12)
13.糖尿病肾病小鼠模型中融合蛋白通过抑制NF-κB/NLRP3信号通路保护肾脏
糖尿病肾病小鼠模型使用ANGPTL3单抗-IL22融合蛋白治疗8周后相比模型组、ANGPTL3单抗治疗组,肾脏组织中NLRP3和Pro-caspase-1的水平均有显著降低(P<0.05),证实了其通过抑制NF-κB/NLRP3信号通路发挥肾脏保护作用。(见图13)
14.阿霉素肾病小鼠模型中融合蛋白干预减少尿蛋白
阿霉素肾病小鼠模型使用ANGPTL3单抗-IL22融合蛋白治疗8周后相比模型组和mIL22Fc治疗组尿白蛋白/肌酐比值显著降低(P<0.05),同时可以看到,ANGPTL3单抗-IL22融合蛋白相比ANGPTL3和IL22联用在降低尿白蛋白/肌酐比值上显示出显著的优势。(见图14)
15.阿霉素肾病小鼠模型中融合蛋白干预保护肾功能
阿霉素肾病小鼠模型使用ANGPTL3单抗-IL22融合蛋白治疗8周后相比模型组血清肌酐水平显著降低(P<0.05),同时可以看到,ANGPTL3单抗-IL22融合蛋白相比ANGPTL3和IL22联用在降低血清肌酐上显示出显著的优势。(见图15)
16.阿霉素肾病小鼠模型中融合蛋白干预改善低白蛋白血症
阿霉素肾病小鼠模型使用ANGPTL3单抗-IL22融合蛋白治疗8周后相比模型组血清白蛋白水平显著升高(P<0.05)。(见图16)
17.阿霉素肾病小鼠模型中融合蛋白干预改善高脂血症
阿霉素肾病小鼠模型使用ANGPTL3单抗-IL22融合蛋白治疗8周后相比模型组血清总胆固醇水平显著降低(P<0.05)。(见图17)
18.阿霉素肾病小鼠模型中融合蛋白干预减轻足细胞损伤
阿霉素肾病小鼠模型使用ANGPTL3单抗-IL22融合蛋白治疗8周后相比模型组足突融合、基底膜增厚、微绒毛变性等病理学改变均有明显改善。(见图18)
19.阿霉素肾病小鼠模型中融合蛋白干预抑制肾脏纤维化
阿霉素肾病小鼠模型使用ANGPTL3单抗-IL22融合蛋白治疗8周后相比模型组肾小球及肾间质区域的胶原沉积显著减少,肾脏纤维化显著改善。(见图19)
20.阿霉素肾病小鼠模型中融合蛋白通过改善线粒体功能保护肾脏
阿霉素肾病小鼠模型使用ANGPTL3单抗-IL22融合蛋白治疗8周后相比模型组线粒体长度缩短、排列紊乱、内嵴断裂等病理损伤表现均有明显改善。(见图20)
21.阿霉素肾病小鼠模型中融合蛋白通过改善溶酶体自噬保护肾脏
阿霉素肾病小鼠模型使用ANGPTL3单抗-IL22融合蛋白治疗8周后相比模型组溶酶体数量增多、体积变大的病理改变得到明显缓解,揭示了溶酶体自噬行为的改善(见图21)。
Claims (10)
1.一种包含ANGPTL3单抗的融合蛋白ANGPTL3单抗-B,其所述融合蛋白ANGPTL3单抗-B由ANGPTL3单抗与序列B融合构成。
2.如权利要求1所述的融合蛋白ANGPTL3单抗-B,所述B选自细胞因子,所述细胞因子可选自白细胞介素(interleukin,IL)、干扰素(interferon,IFN)和肿瘤坏死因子(tumornecrosisfactor,TNF)。
3.如权利要求2所述的融合蛋白ANGPTL3单抗-B,所述细胞因子选自:IL-22,IL-1RA,IL-4,IL-10,IL-13,TGF-β和/或EPO。
4.如权利要求1-3任一项所述的融合蛋白ANGPTL3单抗-B,其特征在于ANGPTL3单抗与序列B可以通过连接子连接,所述连接子可选自(GGGGS)2,(GGGGS)3,(GGGGS)4,(EAAAK)2和或(EAAAK)3。
5.包含权利要求1-4任一项所述融合蛋白ANGPTL3单抗-B的药物组合物,所述药物组合物进一步包含药学上可接受的载体。
6.编码权利要求1-4任一项所述融合蛋白ANGPTL3单抗-B的多核苷酸。
7.表达权利要求1-4任一项所述融合蛋白ANGPTL3单抗-B的多核苷酸的宿主细胞。
8.权利要求1-4任一项所述融合蛋白ANGPTL3单抗-B在制备治疗其介导的疾病或病症的药物中的用途。
9.如权利要求8所述的融合蛋白ANGPTL3单抗-B在制备治疗其介导的疾病或病症的药物中的用途,其中所述疾病或病症包括糖尿病肾病、糖尿病性视网膜病变、糖尿病足、糖尿病心血管并发症、糖尿病性脑血管病、脑动脉硬化、无症状脑卒中、糖尿病神经病变、膜性肾病、IgA肾病、紫癜肾、狼疮肾。
10.如权利要求8所述的融合蛋白ANGPTL3单抗-B在制备治疗其介导的疾病或病症的药物中的用途,其中所述疾病或病症包括肝细胞癌、肾细胞癌、卵巢癌、***、口腔癌、食管癌。
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