CN113321736A - 一种长效化白介素15融合蛋白及其制备方法和应用 - Google Patents
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Abstract
本发明公开了一种新型长效化白介素15(IL-15)融合蛋白及其制备方法和应用。天然IL-15分子量小,体内应用后半衰期短,不稳定,因而限制了其在体内生物学活性的发挥。本发明公开了一种新型长效化IL-15融合蛋白,其包含了利用合成生物学所开发的单体片段Fc(sFc)、IL-15功能性片段、IL-15受体α“sushi”结构域(IL-15RαSu),其中所述sFc与IL-15RαSu通过不同连接肽段连接。此新型长效化IL-15融合蛋白具有潜在的更好成药性,产量远高于Fc融合蛋白,并能够明显降低肿瘤组织中Treg细胞数量,有效激活免疫系统杀灭肿瘤细胞,提高IL-15体内肿瘤抑制效果,具有更好的体内抗肿瘤活性。
Description
技术领域
本发明属于生物技术领域,具体涉及一种长效化白介素15融合蛋白及其制备方法,以及构建该长效化蛋白所需的核酸、质粒、宿主细胞,相应的药用组合物及其应用。
背景技术
IL-15是Grabstein等人于1994年发现的一种约为12-14kD的细胞因子,可在机体正常的免疫应答中发挥作用,如促进T细胞、B细胞和NK细胞的增殖。IL-15和IL-2均属于IL-2家族,IL-2家族成员受体都含γc链:包括IL-2、IL-4、IL-7、IL-9、IL-15和IL-21。其中,IL-15和IL-2具有共同的IL-2/IL-15Rβ(CD122)和γc受体(CD132),IL-15的特异性受体为IL-15Rα(CD125)。
IL-15的信号传递可经由IL-15Rα、IL-15Rβ及γc的异三聚体复合物产生。近年发现,IL-15作用机制为转递呈作用机制,其中,IL-15及IL-15Rα由APC(单核及树突状细胞)协同表达,结合至IL-15Rα的IL-15转呈递至邻近的仅表达IL-15Rβγc受体的NK细胞或CD8T细胞。IL-15转递呈作用是目前体内IL-15发挥作用的主要机制,尤其在肿瘤免疫的监控上扮演了主要的角色。其中IL-15Rα内含一个Sushi结构域,能与IL-15结合,并且是使结合后的IL-15发挥生物学功能所必须的。而IL-15和IL-15Rα主要在树突状细胞和单核细胞膜表面表达,游离状态较少,因此IL-15信号通路的活化更多的是在细胞和细胞接触之后才能激活下游信号通路发挥作用。由于IL-15和IL-2具有相似的激活T细胞的功能,但是并不会激活Treg细胞。因此未来IL-15在临床使用中的副作用会更小,可能会成为IL-2的替代品。鉴于IL-15在肿瘤免疫治疗领域的良好预期,NIH最先进行了IL-15治疗肿瘤方面的研究,并尝试将其推入临床进行研究。但是由于天然IL-15分子量小、体内半衰期短,重复给药剂量很难控制及依从性差,且容易造成全身性免疫副反应等问题。因此本领域迫切需要一种提高IL-15体内半衰期,促进或者增强其体内生物学活性发挥的途径。
目前蛋白质药物的长效化技术主要有三种:PEG(聚乙二醇)修饰技术、HSA(人血清白蛋白)融合技术、Fc(人抗体Fc区)融合技术。这三种技术都有各自的弱点,总的来说,共有的关键缺点是会大大增加被融合或修饰的蛋白药物的分子量,且往往会显著降低被融合蛋白药物的产率和临床功效。本发明人在长效化蛋白领域创新性的利用合成生物学开发了基于抗体IgG Fc的新型单体Fc(CN109705211B;202011161007.3),为蛋白质药物的长效化提供了更优的可选方案。
发明内容
本申请的主要目的在于构建了一种长效化白介素15融合蛋白,该融合蛋白引入了单体Fc(sFc)、IL-15和IL-15受体αsushi结构域,利用了IL-15转递呈作用机制,具有潜在的更好成药性及更长的体内半衰期,以及更好的体内抗肿瘤活性。
为了实现上述目的,本发明提供如下技术方案:
本发明的第一方面,提供一种长效化白介素15融合蛋白,所述融合蛋白包含Fc单体,IL-15受体αsushi结构域和IL-15功能性片段,其中IL-15受体αsushi结构域一端与Fc单体连接,另一端与IL-15功能性片段连接。
上述一种长效化白介素15融合蛋白,作为一种优选的实施方案,IL-15受体αsushi结构域的C端与Fc单体连接,IL-15受体αsushi结构域与IL-15功能性片段以共价键形式连接;优选地,Fc单体与IL-15受体αsushi结构域通过连接肽连接,更优选地,所述连接肽为GGGGS或(GGGGS)3。
本发明所述共价键是指化学键的一种,两个或多个原子共同使用它们的外层电子,在理想情况下达到电子饱和的状态,由此组成比较稳定的化学结构,像这样由几个相邻原子通过共用电子并与共用电子之间形成的一种强烈作用叫做共价键。
所述连接肽指含有柔性氨基酸残基的多肽链,所述柔性氨基酸残基为Gly,Ser,Ala,或Thr。所述多肽链应具有合适的距离,该距离适于连接两个分子使其互相而言具有正确构型以保持所需活性。为该目的的适宜长度包括至少一个以及不超过30个氨基酸残基。优选的,所述接头长度为约1-30个氨基酸,优选的接头长度为1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19和20个氨基酸。此外,被选用于包含在所述连接肽中的氨基酸残基所显示的性质不应明显影响两个被连接分子的活性。因此,所述连接肽总体上不显示与所述两个被连接分子不一致的电荷,或不影响内部折叠,或与一或多个单体中的氨基酸残基形成键合或其它相互作用,所述单体将严重阻碍受体单体结构域的结合。所述含有柔性氨基酸残基的连接肽包括甘氨酸-丝氨酸聚合物(如(GS)n、(GSGGS)n、(GGGGS)n、(GGGS)n,其中n是至少为1的整数),甘氨酸-丙氨酸聚合物,丙氨酸-丝氨酸聚合物,以及本领域已知的其它柔性连接肽诸如shaker钾通道的连接序列等。
本发明所述Fc单体指分子量仅为野生型Fc区域的一半,并保留了抗体Fc区的FcRn结合性能、Protein A/G结合性能,并可实现原核细胞中高效表达的Fc多肽。
上述一种长效化白介素15融合蛋白,作为一种优选的实施方案,所述Fc单体的序列包含SEQ ID NO:1所示的氨基酸序列;
X0选自L,S中的任一种氨基酸;X1选自C,G,S,L,N,D,F,I,V,Y,Q,K,E,M,T中的任一种氨基酸;X2选自L,Q,N,D,Y,R,C,G,S,F,T,I,V,A,K,M中的任一种氨基酸;X3选自P,N,T,I,S,M,Q,R,L,G,V,A,E,D,Y,F,H中的任一种氨基酸;X4选自K,N,S,I,M,E,Q,L,V,A,H,D,Y,F中的任一种氨基酸;X5选自M,Y中的任一种氨基酸;
优选地,所述Fc单体的序列还包含SEQ ID NO:2所示的氨基酸序列。
本发明所述IL-15受体αsushi结构域指IL-15受体α胞外区起始于第一外显子2编码的半胱氨酸残基(C1),并终止于第四外显子2编码的半胱氨酸残基(C4),残基C1和C4均包含在所述的sushi结构域中。经过一个或多个氨基酸残基的替换、缺失或添加而形成的并具有相应活性的IL-15受体αsushi结构域的氨基酸序列也包括在本发明中。
上述一种长效化白介素15融合蛋白,作为一种优选的实施方案,所述IL-15受体αsushi结构域的序列包含SEQ ID NO:3所示的氨基酸序列;优选地,所述IL-15功能性片段的序列包含SEQ ID NO:4所示的氨基酸序列。
SEQ ID NO:1(单体Fc氨基酸序列)
APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTX0PPSRDELTKNQVSLX1CX2VKGFYPSDIAVEWESNGQPENNYKTTX3PVLDSDGSFFLYSX4LTVDKSRWQQGNVFSCSVX5HEALHNHYTQKSLSLSPGK。
SEQ ID NO:2(单体Fc氨基酸序列)
APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTSPPSRDELTKNQVSLRCHVKGFYPSDIAVEWESNGQPENNYKTTKPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVYHEALHNHYTQKSLSLSPGK。
SEQ ID NO:3(IL-15RαSu的氨基酸序列)
ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR。
SEQ ID NO:4(IL-15mutants的氨基酸序列)
NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANDSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS。
本发明所述IL-15功能性片段指Grabstein等人于1994年发现的一种约为12-14kD的细胞因子(JG Girietal.EM BO J.1994 Jun 15;13(12):2822–2830.),可在机体正常的免疫应答中发挥作用,如促进T细胞、B细胞和NK细胞的增殖。经过一个或多个氨基酸残基的替换、缺失或添加而形成的并具有相应活性的IL-15功能性片段的氨基酸序列也包括在本发明中。
本发明的第二方面,提供一种构建上述长效化白介素15融合蛋白的方法,包含:
(a)Fc单体与IL-15受体αsushi结构域通过连接肽段连接;
(b)获得IL-15功能性片段;
(c)将(a)(b)共转染表达或分别表达后蛋白体外组装。
共转染表达是指将插有Fc单体与IL-15受体αsushi结构域连接肽段连接的载体与IL-15功能性片段载体按照合适比例来混合转染细胞(如Expi293细胞),经培养表达,纯化(如protein G一步纯化)获取IL-15融合蛋白。
蛋白体外组装方法是指将Fc单体与IL-15受体αsushi结构域连接肽段连接的载体与IL-15功能性片段载体以合适比例分别转染细胞(如Expi293细胞),经过培养诱导后,将诱导后细胞上清进行适当比例混合,经过纯化(如protein G一步纯化)得到高纯度目的蛋白。
本发明的第三方面,提供一种核酸分子,编码上述长效化白介素15融合蛋白。
本发明的第四方面,提供一种质粒,含有上述核酸分子。可操作地连接调控序列,如启动子、增强子等。
本发明的第五方面,提供一种宿主细胞,含有上述质粒。本发明所述宿主细胞可以是任何原核细胞或真核细胞,包括但不限于细菌细胞(例如大肠杆菌、枯草杆菌)、昆虫细胞(例如利用杆状病毒表达系统)、酵母或哺乳动物细胞(例如CHO或BHK细胞系)。其它合适的宿主细胞为本领域技术人员所知。
本发明的第六方面,提供一种药用组合物,含有有效预防或治疗剂量的上述长效化白介素15融合蛋白或上述核酸分子或上述质粒,和一种药学可接受载体。所述组合物包括但不限于冻干剂型、水溶液剂型、脂质体或胶囊剂型等。本发明的融合蛋白或其核酸分子、质粒的浓度可从约0.1%变化为100%(重量)。
本发明的第七方面,提供一种检测试剂盒,含有上述长效化白介素15融合蛋白,或上述核酸分子,或上述质粒;优选地,所述检测试剂盒用于检测病原体、肿瘤细胞。所述病原体包括病毒、细菌、真菌、寄生虫感染等,所述肿瘤细胞包括各种良性肿瘤细胞、恶性肿瘤细胞(即癌细胞)、实体瘤细胞、血源性癌细胞。
本发明的有益效果:本发明提供了一种诊断、预防或治疗疾病的方法,包括向受试者施用本发明所述的IL-15融合蛋白、核酸分子、质粒、以及药用组合物。
本发明所述疾病为自身免疫性疾病、炎症性疾病、神经退行性疾病、癌症或病原体感染。
优选的,本发明的IL-15融合蛋白可用于治疗癌症。本发明所述癌症包括但不限于淋巴瘤、胚细胞瘤、肉瘤(包括脂肉瘤)、神经内分泌肿瘤、间皮瘤、神经鞘瘤、脑膜瘤、腺瘤、黑素瘤以及非白血性白血病或淋巴恶性肿瘤。上述癌症更具体的实例包括鳞状细胞癌(如,鳞状上皮细胞癌)、肺癌、小细胞肺癌、非小细胞肺癌、肺腺癌以及肺鳞状细胞癌、腹膜癌、肝细胞癌、胃癌、胃肠癌、胰腺癌、恶性胶质瘤、宫颈癌、卵巢癌、肝癌、膀胱癌、肝细胞瘤、乳腺癌、结肠癌、直肠癌、结肠直肠癌、子宫内膜或子宫癌、唾液腺癌、肾癌、前列腺癌、外阴癌、甲状腺癌、肝癌、肛门癌、阴茎癌、睾丸癌、食道癌、胆管肿瘤,头癌、颈癌、骨髓基质瘤、破骨细胞瘤、多发性骨髓瘤、溶骨性癌(osteolytic bone cancers)、中枢神经系统肿瘤、脑肿瘤(神经胶质瘤、成神经细胞瘤、星细胞瘤、成神经管细胞瘤、室管膜细胞瘤和视网膜成神经细胞瘤)、鼻咽癌、基底细胞癌、胆管癌、卡波氏肉瘤、原发性肝癌或子宫内膜癌、以及血管系统肿瘤(血管肉瘤和hemagiopericytoma)。
优选的,本发明的IL-15融合蛋白可用于治疗病原体感染。本发明所述病原体包括但不限于细菌、真菌、病毒、寄生虫。
本发明的IL-15融合蛋白可用于治疗的病症具体包括但不限于:充血性心力衰竭(CHF)、血管炎、红斑痤疮、痤疮、湿疹、心肌炎和其它心肌病症、系统性红斑狼疮、糖尿病、脊椎病、滑液成纤维细胞增生、骨质丢失、变形性骨炎(paget’s disease)、失用型骨质减少、营养不良、牙周病、家族性脾性贫血、朗罕氏细胞组织细胞增多病、脊髓损伤、急性脓毒性关节炎、骨软化症、皮质醇增多症、单骨纤维性骨发育不良、多发性骨纤维性发育不良、牙周再建以及骨折、肉样瘤病、骨转移/骨痛治疗和体液恶性高钙血症、强直性脊椎炎和其它脊椎关节病、移植排斥、病毒感染、血液瘤、何杰金氏淋巴瘤、非何杰金淋巴瘤(Burkitt’s淋巴瘤、小淋巴细胞淋巴瘤/慢性淋巴细胞性白血病、蕈样肉芽肿病、外套细胞淋巴瘤、滤泡性淋巴瘤、弥漫性巨大B细胞淋巴瘤、边缘区淋巴瘤、毛细胞性白血病以及淋巴浆细胞性白血病)、淋巴细胞前体细胞肿瘤、B细胞急性成淋巴细胞性非白血性白血病/淋巴瘤、T细胞急性成淋巴细胞性非白血性白血病/淋巴瘤、胸腺瘤、成熟T和NK细胞肿瘤、外周T细胞非白血性白血病、成熟T细胞非白血性白血病/T细胞淋巴瘤、大颗粒状淋巴细胞性白血病、朗罕氏细胞组织细胞增多症、急性骨髓性粒细胞性白血病的骨髓瘤,成熟的急性骨髓性白血病(AML)、分化的急性骨髓性白血病、急性前髓细胞性白血病、急性骨髓单核细胞性白血病、急性单核细胞性白血病、脊髓发育不良综合征、慢性骨髓增生病,慢性髓细胞性白血病、骨质疏松症、肝炎、HIV、AIDS、脊椎关节炎、类风湿性关节炎、炎性肠疾病(IBD)、败血症和败血症性休克、节段性回肠炎、牛皮癣、硬皮病、移植物抗宿主病(GVHD)、异源胰岛移植排斥(allogenic isletgraftrejection)、血液恶性肿瘤诸如多发性骨髓瘤(MM)、骨髓增生异常综合征(MDS)和急性骨髓性白血病(AML)、肿瘤相关的炎症、周围神经损伤或脱髓鞘性病。
本发明的IL-15融合蛋白、核酸分子、质粒、以及药用组合物可以通过各种不同的给药途径给予人或动物受试者,所述给药途径通常取决于待治疗的疾病本身的特征。一般而言,可利用医学上可接受的任何给药模式来实施本发明的方法,所述给药模式包括经口、直肠、局部、眼内、脑池内、脑室内、气管内、鼻内滴入、透皮、皮下、鞘内、肌内、腹腔、腹膜内、颅内输注或者静脉输注。
附图说明
图1.显示新型长效化白介素15融合蛋白的结构模式图;
图2.显示新型长效化白介素15融合蛋白的还原SDS-PAGE分析并用考马斯亮蓝染色图;
图3.显示新型长效化白介素15融合蛋白的高效液相色谱分析(HPLC);分析物为FL115-2a(C)和FL115-2b(D),对照蛋白为sFc(A)和Fc(B);
图4.显示通过在CTLL-2上的增殖测定法来评价新型长效化白介素15融合蛋白的生物学活性。其中阳性对照为IL15与IL-15受体α的融合蛋白(positive control);
图5.显示新型长效化白介素15融合蛋白FL115-2a的小鼠体内抗肿瘤作用;
图6.显示本发明新型长效化白介素15融合蛋白对背部皮下接种有B16F10黑色素瘤肿瘤小鼠体重的影响;
图7.显示本发明新型长效化白介素15融合蛋白对背部皮下接种有B16F10黑色素瘤肿瘤小鼠生存率的影响;
图8.显示新型长效化白介素15融合蛋白的小鼠体内抗肿瘤作用及对背部皮下接种有B16F10黑色素瘤肿瘤小鼠体重的影响;阳性对照为IL-15的Fc融合蛋白(简写为PC)、IL15KIH(IL-15&IL15Rα的Fc融合蛋白)及WT-IL15,阴性对照为PBS;
图9.显示新型长效化白介素15融合蛋白的针对每组5只小鼠体内抗肿瘤作用;
图10.显示新型长效化白介素15融合蛋白治疗每组每只小鼠的抗肿瘤效果。称重每只老鼠的肿瘤重量,通过one-way ANOVA分析统计分析组间是否存在统计学差异;
图11.显示流式细胞术分析接种有B16F10黑色素瘤肿瘤小鼠接受新型长效化白介素15融合蛋白后体内Treg细胞数量减少,具体表现为Foxp3+CD25+减少;
图12.显示流式细胞术分析接种有B16F10黑色素瘤肿瘤小鼠接受新型长效化白介素15融合蛋白后体内NK细胞的数量增加及其所分泌的IFN-γ和穿孔素增加;
图13.显示流式细胞术分析接种有B16F10黑色素瘤肿瘤小鼠接受新型长效化白介素15融合蛋白后体内IL6增加。
具体实施方式
为了使本技术领域的人员更好地理解本申请方案,下面将结合案例对本申请实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本申请一部分的实施例,而不是全部的实施例。基于本申请中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都应当属于本申请保护的范围。
为了能更彻底地理解发明,以下列出一些定义。上述定义意在包含语法等同成分。
融合蛋白(fusion protein)有两种不同的含义,一种是通过DNA重组技术得到的两个基因重组后的表达产物,另一种是介导两个细胞质膜融合的一组蛋白,如在仙台病毒脂双层外侧小叶中含有的两种糖蛋白之一,介导病毒被膜与宿主细胞质膜的融合作用。另一种糖蛋白是血细胞凝集素神经酰胺酶。两个不同的蛋白质既可以通过化学方法也可以通过基因的融合来连成一个大分子。
IL-15是一种可溶性细胞因子,作为多种免疫细胞的趋化因子参与并调节机体炎症反应及免疫反应。生物学作用与IL-2相似,由多种细胞产生。
本文使用的“单体”意指一种可以进行聚合从而为高分子的基本结构提供结构单元的分子。大量单体结合形成聚合物的过程称为聚合。
本文中使用的“Fc”、“Fc单体”包括包含除免疫球蛋白结构域第一恒定区外的抗体恒定区的多肽。因此,Fc指IgA、IgD和IgG免疫球蛋白结构域的最后两个恒定区,IgE和IgM免疫球蛋白结构域的最后三个恒定区,和这些结构域的N-末端连接的柔性铰链。对于IgA和IgM而言,Fc可包括J链。对于IgG来说,Fc包含免疫球蛋白结构域Cγ2和Cγ3以及Cγ1和Cγ2之间的铰链。尽管Fc区的边界可改变,人IgG重链Fc区通常规定为在其羧基末端包含残基C226或P230,其中编号方式依照Kabat的EU标引方式。Fc可指分离的该区域,或在上下文的抗体、抗体片段或Fc融合体中的该区域。Fc可以是抗体、Fc融合体,或包含Fc的蛋白质或蛋白质结构域。具体优选的是Fc变体,其是非天然存在的利用合成生物学获取的Fc变体。
功能性片段是分子结构介于氨基酸和蛋白质之间的一类化合物,是蛋白质的结构与结构片段,本身具有很强的生物活性。
本文中使用的“N端”也称为氨基末端,NH2-末端,N-末端或胺末端,是蛋白质或多肽的起始,指的是位于多肽末端的游离胺基团(-NH2)。“C端”也称为羧基末端,羧基末端,C-末端尾部,C-末端或COOH-末,是蛋白质或多肽的末端,由游离羧基(-COOH)末端终止。
本文中使用的“氨基酸”意指20种天然存在的氨基酸之一或任一非天然类似物,它们可位于具体规定的位置。本文中“蛋白质”意指至少两个共价连接的氨基酸,其包括蛋白质、多肽、寡肽和肽。蛋白质可由天然存在的氨基酸和肽键构成,或由合成的肽模拟物结构构成,该肽模拟物即“类似物”。因此本文中使用的“氨基酸”或“肽残基”意指天然存在和合成的氨基酸。举例来说,对于本发明目的而言,高苯基丙氨酸、瓜氨酸和降亮氨酸被认为是用于本发明目的的氨基酸。“氨基酸”也包括诸如脯氨酸和羟脯氨酸的亚氨基酸残基。侧链可以是(R)或(S)构型。在优选的实施方案中,氨基酸以(S)或L-构型存在。如果使用非天然存在的侧链,可使用非氨基酸取代,例如以阻止或延迟体内降解。
本文所使用的“核酸”意指由核苷酸单元(核糖核苷酸,脱氧核糖核苷酸,相关的天然存在的结构变体及其合成的非天然存在的类似物)通过磷酸二酯键组成的聚合物。因此,该术语包括核苷酸聚合物,其中核苷酸和它们之间的键包括非天然存在的合成类似物,例如但不限于硫代磷酸酯,氨基磷酸酯,甲基磷酸酯,手性甲基磷酸酯,2'-O-甲基核糖核苷酸,肽核酸(PNA)等。例如,可以使用自动DNA合成仪合成这些多核苷酸。术语“寡核苷酸”通常是指短多核苷酸,通常不大于约50个核苷酸。应当理解,当核苷酸序列由DNA序列(即A,T,G,C)表示时,这也包括其中“U”取代“T”的RNA序列(即A,U,G,C)。
本文使用常规符号来描述核苷酸序列:单链核苷酸序列的左手末端5'末端;双链核苷酸序列的左手方向称5'方向。向新生RNA转录物添加5'至3'核苷酸的方向称为转录方向。具有与mRNA相同序列的DNA链被称为编码链。
本文中所使用的“编码”意指多核苷酸中特定核苷酸序列的固有特性,例如基因,cDNA或mRNA,用作在具有确定的核苷酸序列的生物过程中合成其他聚合物和大分子的模板,或确定的氨基酸序列和由此产生的生物学特性。因此,如果由该基因产生的mRNA的转录和翻译在细胞或其他生物系统中产生蛋白质,则基因编码蛋白质。编码链(其核苷酸序列与mRNA序列相同并且通常在序列表中提供)和非编码链(用作转录模板,基因或cDNA)可以被称为编码蛋白质。或该基因或cDNA的其他产物。除非另有说明,否则“编码氨基酸序列的核苷酸序列”包括彼此简并形式且编码相同氨基酸序列的所有核苷酸序列。编码蛋白质和RNA的核苷酸序列可包括内含子。
本文中使用的“质粒”意指在天然质粒的基础上为适应实验室操作而进行人工构建的质粒。可将核酸分子导入宿主细胞,从而产生转化的宿主细胞。载体可包括允许其在宿主细胞中复制的核酸序列,例如复制起点,还可以包括一种或多种选择标记基因和本领域已知的其他遗传元件。
本文所使用的“宿主细胞”也称为受体细胞,是指在转化和转导(感染)中接受外源基因的宿主细胞。
本文中使用的“药学可接受载体”意指常规的药学上可接受的载体。Remington'sPharmaceuticalSciences,EW Martin,Mack Publishing Co.,Easton,Pa.,第15版(1975),描述了适用于药物递送一种或多种治疗化合物或分子(例如一种或多种抗体)的组合物和制剂,以及另外的药剂。
本文中所使用的“诊断”疾病是指在给病人做检查之后判定病人的病症及其发展情况。“预防”疾病是指抑制疾病的完全发展。“治疗”是指在其开始发展后改善疾病或病理状况的体征或症状的治疗性干预。
本文中“施用”意指选择合适的途径将所述物质引入受试者。例如,如果所选择的途径是静脉内的,则通过将所述物质引入受试者的静脉来施用组合物。
本文中“有效预防/治疗剂量”意指足以在用该药剂治疗的受试者中达到所需效果的一定量的特定药剂。精确的剂量将依赖于治疗的目的,并可为本领域技术人员通过使用公知技术所确定。剂量范围可为0.01-100mg/kg体重或更大,例如0.1、1、10或50mg/kg体重,优选1-10mg/kg。如本领域所公知,对于抗体或Fc融合体降解、全身性或局部性递药和新蛋白酶合成速率,以及年龄、体重、大致健康状况、性别、饮食、给药时间、药物相互作用以及病症的严重程度而言,调整可以是必需的,并可由本领域那些技术人员通过常规的实验方法来确定。此类试剂包括本文所述的单体Fc结构域分子。在一个非限制性实例中,这可以是用于预防,治疗或改善HIV感染的HIV特异性单体Fc结构域(或HIV特异性CH3结构域分子)的量。理想地,治疗有效量的抗体是足以预防,治疗或改善感染或疾病的量,例如由受试者中的HIV感染引起而不会在受试者中引起显着的细胞毒性作用。用于预防,改善和/或治疗受试者的治疗有效量的药剂将取决于所治疗的受试者,痛苦的类型和严重程度,以及治疗组合物的施用方式。
本文中“自身免疫性疾病”指一种疾病,其中免疫系统针对正常宿主的一部分抗原(即自身抗原)产生免疫应答(例如,B细胞或T细胞应答),随后对组织造成损伤。自身抗原可以源自宿主细胞,或者可以衍生自共生生物,例如通常定殖于粘膜表面的微生物(称为共生生物)。影响哺乳动物的自身免疫疾病包括但不限于类风湿性关节炎,幼年型少关节炎,胶原诱导的关节炎,佐剂诱导的关节炎,斯耶格伦综合征,多发性硬化症,实验性自身免疫性脑脊髓炎,炎性肠病(例如,克罗恩病,溃疡性结肠炎),自身免疫性胃萎缩,寻常型天疱疮,牛皮癣,白癜风,1型糖尿病,非肥胖性糖尿病,重症肌无力,格雷夫斯病,桥本氏甲状腺炎,硬化性胆管炎,硬化性唾液腺炎,系统性红斑狼疮,自身免疫性血小板减少性紫癜,Goodpasture综合征,艾迪生病,系统性硬化症,多发性肌炎,皮肌炎,自身免疫性溶血性贫血,恶性贫血等。
本文中“病毒”来自但不限于以下家族的病毒:逆转录病毒科(例如,人免疫缺陷病毒(HIV)、人T细胞白血病病毒(HTLV));小核糖核酸病毒科(例如,脊髓灰质炎病毒、甲型肝炎病毒、丙型肝炎病毒、肠道病毒、人类柯萨奇病毒、鼻病毒、埃可病毒、口蹄疫病毒);钙粘病毒科(如引起胃肠炎的病毒株);披膜病毒科(例如马脑炎病毒、风疹病毒);黄病毒科(例如,登革病毒、黄热病病毒、西尼罗河病毒、圣路易斯脑炎病毒、日本脑炎病毒和其他脑炎病毒);冠状病毒科(例如冠状病毒、严重急性呼吸道综合征(SARS)病毒);弹状病毒科(例如,水疱性口炎病毒、狂犬病病毒);副粘病毒科(例如,副流感病毒、腮腺炎病毒、麻疹病毒、呼吸道合胞病毒(RSV));正粘病毒科(例如,流感病毒);布尼亚病毒科(例如汉坦病毒、SinNombre病毒、裂谷热病毒、bunya病毒、phleboviruses和Nairo病毒);沙粒病毒科(例如出血热病毒、Machupo病毒、Junin病毒);呼肠孤病毒科(例如呼肠孤病毒、orbiviurse和轮状病毒);双核糖核酸病毒科;嗜肝病毒科(例如乙型肝炎病毒);细小病毒科(例如细小病毒);乳多空病毒科(例如乳头瘤病毒、多瘤病毒、BK病毒);腺病毒科(例如,大多数腺病毒,如腺相关病毒);疱疹病毒科(例如,单纯疱疹病毒(HSV-1和HSV-2)、巨细胞病毒(CMV)、爱泼斯坦-巴尔病毒(EBV)、水痘带状疱疹病毒(VZV)、和其他疱疹病毒、包括HSV-6);痘病毒科(例如天花病毒、痘苗病毒、痘病毒);和虹膜病毒科(如非洲猪瘟病毒);病毒科(例如,埃博拉病毒、马尔堡病毒);杯状病毒科(例如,诺沃克病毒)和未分类的病毒(例如,海绵状脑病的病原体、三角洲肝炎的病原体(被认为是乙型肝炎病毒的有缺陷的卫星)和星状病毒)。
本文中的“细菌”来自但不限于:幽门螺杆菌、Borelia burgdorferi、嗜肺军团菌、分枝杆菌(如M.tuberculosis,M.avium,M.intracellulare,M.kansaii,M.gordonae)、金黄色葡萄球菌、淋病奈瑟菌,脑膜炎奈瑟菌、单核细胞增生李斯特菌、化脓性链球菌(A组链球菌)、无乳链球菌(B组链球菌)、链球菌(草绿蝇群)、粪链球菌、牛链球菌、链球菌(厌氧菌)、肺炎链球菌、致病性弯曲杆菌、肠球菌、流感嗜血杆菌、炭疽芽孢杆菌、白喉棒状杆菌、棒状杆菌、猪瘟病毒、产气荚膜梭菌、破伤风梭菌、产气肠杆菌、肺炎克雷伯菌、多杀巴斯德氏菌、拟杆菌属、梭状芽孢杆菌、麦芽链霉菌、梅毒螺旋体、密螺旋体、钩端螺旋体或放线菌(Actinomyces israelli)。
本文中的“真菌”来自但不限于:新型隐球菌、夹膜组织胞浆菌(Histoplasmacapsulatum)、粗球孢子菌(Coccidioides immitis)、皮炎芽生菌(Blastomycesdermatitidis)、沙眼衣原体(Chlamydia trachomatis)或白念珠菌(Candida albicans)。
本文中的“寄生虫”来自但不限于:恶性疟原虫(Plasmodium falciparum)或弓形虫(Toxoplasma gondii)。
本文中的“癌症”为实体瘤或血源性癌。本发明所述的实体瘤是肉瘤或癌,例如纤维肉瘤,粘液肉瘤,脂肪肉瘤,软骨肉瘤,成骨肉瘤,或另一种肉瘤,滑膜瘤,间皮瘤,尤文氏瘤,平滑肌肉瘤,横纹肌肉瘤,结肠癌,淋巴恶性肿瘤,胰腺癌,乳腺癌,肺癌,卵巢癌,前列腺癌,肝细胞癌,鳞状细胞癌,基底细胞癌,腺癌,汗腺癌,皮脂腺癌,乳头状癌,乳头状腺癌,髓样癌,支气管癌,肾细胞癌,肝细胞癌,胆管癌,绒毛膜癌,肾母细胞瘤,宫颈癌,睾丸肿瘤,膀胱癌或中枢神经系统肿瘤(如胶质瘤,星形细胞瘤,成神经管细胞瘤,颅咽管瘤,室管膜瘤,松果体,血管母细胞瘤,听神经瘤,少突神经胶质瘤,血管瘤,黑素瘤,神经母细胞瘤或视网膜母细胞瘤)。本发明所述的血源性癌症是白血病,如急性白血病(如急性淋巴细胞白血病,急性髓细胞白血病,急性髓性白血病和成髓细胞,早幼粒细胞,髓单核细胞,单核细胞和红白血病);慢性白血病(如慢性粒细胞性(粒细胞)白血病,慢性粒细胞白血病和慢性淋巴细胞白血病),真性红细胞增多症,淋巴瘤,霍奇金病,非霍奇金淋巴瘤(惰性和高级形式),多发性骨髓瘤,瓦尔登斯特伦巨球蛋白血症,重链性疾病,骨髓增生异常综合征,多毛细胞白血病或骨髓增生异常。
除非另外说明,否则本文使用的所有技术和科学术语具有与本公开所属领域的普通技术人员通常理解的含义相同的含义。除非上下文另有明确说明,否则单数术语“一”,“一个”和“该”包括复数指示物。还应理解,对于核酸或多肽给出的所有碱基大小或氨基酸大小,以及所有分子量或分子量值是近似的,并且提供用于描述。尽管与本文描述的那些类似或等同的方法和材料可用于本公开的实践或测试,但下文描述了合适的方法和材料。术语“包含”表示“包括”。本文提及的所有出版物、专利申请、专利和其他参考文献均通过引用整体并入。如果发生冲突,将以本说明书(包括术语解释)为准。另外,材料,方法和实施例仅是说明性的而不是限制性的。
实施例中使用的标准的重组DNA技术和分子克隆技术是本领域所熟知的(Ausubel,F.M等人,Current Protocols in Molecular Biology,Greene PublishingAssoc.和Wiley-Interscience出版),适用于微生物生长的材料和方法是本领域熟知的。主要化学、生物试剂购自KAPA Biosystems,New England Biolabs,TransGen Biotech,Thermo Fisher Scientific,OMEGA bio-tek等。
下面结合具体实施例对本发明进行详细说明。
实施例1新型长效化白介素15融合蛋白设计及制备
设计的新型长效化白介素15融合蛋白的模式图见图1。将编码IL15Rα-GGGGS-sFc(SEQ ID NO:5),IL15Rα-(GGGGS)3-sFc(SEQ ID NO:6),IL15Rα-Fc(SEQ ID NO:7),IL15(N72D)(序列为SEQ ID NO:4)基因由南京金斯瑞(南京,Genescript)公司合成。并将基因克隆到真核表达载体PTT(购自Thermo Fisher公司)中。将构建成功的质粒大提后瞬时共转Expi293细胞(购自Thermo Fisher公司)进行表达(其中IL15Rα-(GGGGS)3-sFc与IL15(N72D)共转获得FL115-2a,IL15Rα-GGGGS-sFc与IL15(N72D)共转获得FL115-2b,IL15Rα-Fc与IL15(N72D)共转获得IL-15的Fc融合蛋白),通过protein G树脂(购自GE Healthcare公司)进行纯化(具体转化、表达、纯化方法见:应天雷等,JOURNAL OF BIOLOGICALCHEMISTRY,2012,287(23):19399-19408)。纯化的蛋白通过SDS-PAGE胶进行纯度验证,结果显示蛋白纯度良好,目的条带正确,无杂带(见图2)。
实施例2新型长效化白介素15融合蛋白理化性质
新型长效化白介素15融合蛋白(FL115-2a和FL115-2b)按照实施例1方法进行表达纯化。同时将IL-15的Fc融合蛋白进行同样设计进行构建及统一表达体系进行Fc融合蛋白的表达纯化,获取相关蛋白。通过protein G树脂进行纯化(具体转化、表达、纯化方法见:应天雷等,JOURNAL OF BIOLOGICAL CHEM ISTRY,2012,287(23):19399-19408),新型长效化白介素15融合蛋白产量高达10mg/L,相较于IL-15的Fc融合蛋白1mg/L的产量具有明显的优势,理论上在工业生产成本上占据显著优势。同时我们取等体积100ul的上述蛋白进行高效液相色谱(HPLC)进行蛋白均一性的验证,如图3所示,新型长效化白介素15融合蛋白性质均一,单体形式存在。其中FL115-2a和FL115-2b代表sFc与IL15Rα之间分别利用GGGGS,(GGGGS)3肽段连接。上述结果说明新型长效化白介素15融合蛋白具有潜在的更好成药性。
实施例3在CTLL-2细胞系中评价新型长效化白介素15融合蛋白的生物学活性
为了评价新型长效化白介素15融合蛋白的生物学活性,使用在CTLL-2细胞系上的增殖测定法(Santos-Savio A.等人,BiotecnAplic.2000,17:221-4)。按照下面所描述的程序,采用用溴化3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四唑鎓(MTT)(购自Sigma公司)进行的线粒体染色(具体方法见:MossmanT.J.Immunol.Methods.1983,65(1-2):55-63),通过这些细胞的增殖的刺激来测量生物学活性。在96-孔培养板(购自Costar,USA)中,在50μL的体积的补充有10%胎牛血清(FBS)(购自Gibco)和50μg/mL庆大霉素(购自Sigma公司)的RPMI培养基(购自Gibco)中,从4μg/mL的浓度开始,进行FL115-2a、FL115-2b、阳性对照(IL15&IL15Rα)和sFc的系列稀释。事先将CTLL-2细胞(购自ATCC)用RPMI培养基洗涤5次,并且在50μL的体积中按照5×103个细胞/孔添加至平板。在37℃、5%CO2和98%的相对湿度下温育72小时。通过用MTT进行染色来测定细胞生存力。sFc不是在生物学上有活性的,因为它不诱导CTLL-2细胞的增殖,这与FL115-2a、FL115-2b、阳性对照(IL15&IL15Rα)(它们以剂量依赖性方式刺激增殖)不同(见图4)。
实施例4新型长效化白介素15融合蛋白体内药效学验证
为了评估新型长效化白介素15融合蛋白的药效学,我们在6-8周龄雌性C57BL/6小鼠(购自江苏集萃药康生物科技有限公司)于右侧背部被注射5×105B16F10小鼠黑色素瘤细胞。接种之后第1,8天开始给予不同剂量的FL115-2a-1、阳性对照(IL15&IL15Rα)、野生型IL15(WT-IL15)或阴性对照(PBS),相应的FL115-2a-2则是肿瘤接种后每隔一天给药。并在指定日期测量小鼠肿瘤大小。实验结果为平均值加减标准差,每组有5只小鼠。通过one-way ANOVA分析实验数据统计学显著性,**代表p<0.01。其中阳性对照为IL15与IL-15受体α的融合蛋白(positive control),WT-IL15及PBS组为对照,其中FL115-2a-1代表第1,8天给药,FL115-2a-2代表每个1天给药一次。
实验结果显示FL115-2a-1,FL115-2a-2给药方式不影响新型长效化白介素15融合蛋白的体内抗肿瘤功效,且两组具有非常明显的抗肿瘤功效,与阴性对照组具有明显的统计学差异,我们猜测与IL15体内半衰期延长相关,如图5所示。同时我们检测了每组老鼠的体重,证实了融合蛋白的安全性良好,如图6所示,且FL115-2a-1,FL115-2a-2生存率良好,如图7所示。
实施例5新型长效化白介素15融合蛋白具有高效的体内抗肿瘤活性
为了评估新型长效化白介素15融合蛋白的体内抗肿瘤功效,我们根据上述药效学验证时实验设计,在6-8周龄雌性C57BL/6小鼠(购自江苏集萃药康生物科技有限公司)于背部皮下移植B16F10小鼠黑色素瘤细胞,建立小鼠移植瘤模型。模型建立后于第1,8天尾静脉注射FL115-2a,PC(IL-15的Fc融合蛋白),IL15KIH(IL-15&IL15Rα的Fc融合蛋白),WT-IL15和阴性对照PBS,每隔1天测量小鼠肿瘤大小。FL115-2a表现出与PC相当的体内肿瘤生长抑制效果,与阴性对照组具有明显的统计学差异,如图8。且各组小鼠体重均未受到影响,说明了新型长效化白介素15融合蛋白小鼠体内的安全性,如图8所示。同时我们也分析了每组小鼠中5只小鼠分别的肿瘤抑制效果分析,发现FL115-2a组每只小鼠的肿瘤抑制效果具有一致性,抗肿瘤抑制效果明显,抑制率达到62%,如图9所示。我们将老鼠进行了解剖,取出了每只小鼠的肿瘤进行称重,FL115-2a因为具有高效的体内抗肿瘤活性,小鼠肿瘤抑制效果明显,不管大小还是体重均与阴性对照组呈现显著性差异,如图10所示。
实施例6流式细胞分析细胞表型
小鼠麻醉后从眼眶静脉采集外周静脉血,并立即进行抗凝血和红细胞裂解处理。小鼠人道处死后,取出脾脏并立即采集脾脏细胞,并通过70uM尼龙筛(购自FALCON)的过滤和红细胞裂解处理后得到脾脏单细胞悬液。同时取出肿瘤组织,用镊子轻轻破坏肿瘤组织结构后在37℃下用0.2mg/ml Collagenase IV(购自Sigma-Aldrich公司)和为0.1mg/ml的DNAse I(购自Sigma-Aldrich公司)消化15分钟。收集单细胞悬液并且将剩余的肿瘤组织继续用上述酶溶液消化25分钟。合并得到的单细胞悬液用70微米的尼龙网过滤。外周血单细胞,脾脏细胞和肿瘤细胞用流式抗体(购自BD)染色后用4%多聚甲醛溶液(购自Sigma-Aldrich公司)固定并避光保存或立即上流式细胞仪(购自BD)检测细胞表型。经过抗体染色后用流式细胞术分析可发现,经过FL115-2a治疗的小鼠肿瘤组织中的Treg细胞数量减少,具体表现为Foxp3+CD25+减少,如图11所示。同时NK细胞的数量增加及其所分泌的IFN-γ和穿孔素增加,该实验结果提示新型长效化白介素15融合蛋白不仅可以降低Treg细胞数量,减少其抑制作用,同时还能促进NK细胞能多的渗透进入肿瘤组织,IFN-γ和穿孔素的分泌显著增多,这进一步提示新型长效化白介素15融合蛋白能够有效激活免疫系统从而杀灭肿瘤细胞,提高IL-15体内肿瘤抑制效果,如图12所示。另外,流式细胞术还显示,新型长效化白介素15融合蛋白可以提高IL6的表达,如图13所示。
对所公开的实施例的上述说明,使本领域专业技术人员能够实现或使用本发明。以上所述仅是本发明的优选实施方式,应当指出,对于本领域的技术人员,在不脱离本发明原理的前提下,还可以做出若干改进和补充,这些改进和补充也应当视为本发明的保护范围。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的,本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下,在其它实施例中实现。因此,本发明将不会被限制于本文所示的这些实施例,而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。
Claims (10)
1.一种长效化白介素15融合蛋白,其特征在于,所述融合蛋白包含Fc单体,IL-15受体αsushi结构域和IL-15功能性片段,其中IL-15受体αsushi结构域一端与Fc单体连接,另一端与IL-15功能性片段连接。
2.根据权利要求1所述的长效化白介素15融合蛋白,其特征在于,IL-15受体αsushi结构域的C端与Fc单体连接,IL-15受体αsushi结构域与IL-15功能性片段以共价键形式连接;优选地,Fc单体与IL-15受体αsushi结构域通过连接肽连接,更优选地,所述连接肽为GGGGS或(GGGGS)3。
3.根据权利要求1-2所述的任一长效化白介素15融合蛋白,其特征在于,所述Fc单体的序列包含SEQ ID NO:1所示的氨基酸序列;
X0选自L,S中的任一种氨基酸;X1选自C,G,S,L,N,D,F,I,V,Y,Q,K,E,M,T中的任一种氨基酸;X2选自L,Q,N,D,Y,R,C,G,S,F,T,I,V,A,K,M中的任一种氨基酸;X3选自P,N,T,I,S,M,Q,R,L,G,V,A,E,D,Y,F,H中的任一种氨基酸;X4选自K,N,S,I,M,E,Q,L,V,A,H,D,Y,F中的任一种氨基酸;X5选自M,Y中的任一种氨基酸;
优选地,所述Fc单体的序列还包含SEQ ID NO:2所示的氨基酸序列。
4.根据权利要求1-3所述的任一长效化白介素15融合蛋白,其特征在于,所述IL-15受体αsushi结构域的序列包含SEQ ID NO:3所示的氨基酸序列;优选地,所述IL-15功能性片段的序列包含SEQ ID NO:4所示的氨基酸序列。
5.一种构建如权利要求1-4中所述的任一长效化白介素15融合蛋白的方法,其特征在于,包含:
(a)Fc单体与IL-15受体αsushi结构域通过连接肽段连接;
(b)获得IL-15功能性片段;
(c)将(a)(b)共转染表达或分别表达后蛋白体外组装。
6.一种核酸分子,其特征在于,编码权利要求1-5中所述的任一长效化白介素15融合蛋白。
7.一种质粒,其特征在于,含有权利要求6所述的核酸分子。
8.一种宿主细胞,其特征在于,含有权利要求7所述的质粒。
9.一种药用组合物,其特征在于,含有有效预防或治疗剂量的如权利要求1-5中所述的任一长效化白介素15融合蛋白或如权利要求6中所述的核酸分子或如权利要求7中所述的质粒,和一种药学可接受载体。
10.一种检测试剂盒,其特征在于,含有如权利要求1-5中所述的任一长效化白介素15融合蛋白,或权利要求6所述的核酸分子,或权利要求7所述的质粒;优选地,所述检测试剂盒用于检测病原体、肿瘤细胞。
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