CN113956346B - 一种重组白介素-15变体 - Google Patents
一种重组白介素-15变体 Download PDFInfo
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- CN113956346B CN113956346B CN202111246297.6A CN202111246297A CN113956346B CN 113956346 B CN113956346 B CN 113956346B CN 202111246297 A CN202111246297 A CN 202111246297A CN 113956346 B CN113956346 B CN 113956346B
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Abstract
本公开涉及一种重组白介素‑15变体，其在野生型白介素‑15氨基酸序列的末端添加、删除、取代一个或多个氨基酸残基，从而使得白介素‑15变体的羧基端较野生型白介素‑15有更加稳定的结构，使白介素‑15变体可以在大肠杆菌中高表达，更容易分离和纯化。本公开的白介素‑15变体的表达量为野生型白介素‑15的10至20倍。
Description
技术领域
本公开属于分子生物学领域，具体涉及一种重组白介素-15变体。
背景技术
白细胞介素-15(IL-15)是一种约为12-14kD的细胞因子。与IL-2、IL-4、IL-7、IL-9、粒细胞集落刺激因子、粒细胞-巨噬细胞集落刺激因子同属于4a螺旋细胞因子家族。
天然成熟人IL-15成熟肽含有114个氨基酸，包括4个半胱氨酸残基(Cys35与Cys85、Cys42与Cys88)，半胱氨酸残基形成的两对分子内二硫键对于保持IL-15的空间构象和生物学活性起重要作用。
IL-15受体由3个亚基组成(α、β和γ亚基)。IL-15受体的α亚基与IL-15有很高的亲和力，生理状态下IL-15与α亚基形成复合物(IL-15-Rα)，并增强IL-15对受体的β链和γ链亚基的亲和力，激活T细胞和NK细胞。因此，一些制药公司将IL-15与IL-15受体α亚基(或其部分)形成复合物，并在动物试验中显示了良好的生物效价及稳定性。
IL-15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、自然杀伤(NK)细胞的发育等。炎症与肿瘤的发生密切相连，由于IL-15可促进NK细胞、B淋巴细胞及T淋巴细胞的增殖并可维持这些免疫细胞功能，因此IL-15对于治疗一些恶性肿瘤存在一定疗效。在多种实验动物肿瘤模型(肺腺癌、黑素瘤、结肠癌、肝癌、淋巴瘤)中，利用IL-15治疗均可以促进肿瘤的消退，减少肿瘤的转移，提高存活率。
在感染性疾病模型的临床研究中发现IL-15是一种有效的疫苗佐剂。Steel等人开发了一种靶向性治疗neu+乳腺癌的DC疫苗(可组成型表达IL-15)。研究人员将IL-15作为一种刺激体内免疫细胞活性的免疫佐剂进行评估，现阶段已有的临床结果表明培养基中添加IL-15培养出的NK、CD8+T细胞、CD8+记忆T细胞、树突状细胞转移至动物体内后，生物活性显著提高。
随着对IL-15作用机制及临床应用研究的不断深入，IL-15作为药物及免疫佐剂其需求量也大大增加。然而，天然野生型的IL-15存在明显的药物开发瓶颈，包括原核生物和真核生物表达的表达量低、纯化困难、半衰期短等。市场上主要利用原核表达系统对其进行表达，大肠杆菌的表达产物通常以包涵体形式存在，需通过变性-复性的复杂操作才能得到活性形式的IL-15，此方法极大的限制了活性蛋白的产率。要得到高纯度的蛋白往往需要经过多步纯化操作，导致蛋白的产率下降，生物活性也会随之降低。所以，本领域有必要提供一种更加稳定的IL-15。
发明内容
IL-15变体
本文以下所用的氨基酸单字母代码时本领域公知的，如JBC，243，p3558，1968中所述。
本公开的一方面提供了一种IL-15变体，和野生型人IL-15相比，在野生型人IL-15的羧基端添加一段多肽，所述多肽由1至30个氨基酸残基组成。
在一些实施方案中，不限于具体的理论，在野生型人IL-15的羧基端添加不超过30个氨基酸长度的多肽时，有助于形成alpha螺旋；通过提高IL-15羧基端结构的稳定性来增加其在宿主(尤其是原核宿主)中的表达量。在一个示例中，表达量约为野生型IL-15的10至20倍，甚至更高。
在一些实施方案中，提供了一种IL-15变体，和野生型人IL-15相比，在野生型人IL-15的羧基端添加至少一个碱性亲水性氨基酸残基。
在一些实施方案中，所述碱性亲水性氨基酸选自以下任一个：H、R和K。
根据一些实施方案，提供了一种IL-15变体，其包含式I或式II所示的肽结构，从左至右按氨基端至羧基端方向：
X1-J-X2-X3(式I)
X1-X2-J-X3(式II)
其中：
X1表示野生型人IL-15；
-表示共价键，优选酰胺键；
X2表示随机序列或空缺，所述随机序列的长度是4至8个氨基酸长；所述随机序列不含亲水性肽残基；
X3表示碱性亲水性肽残基，其选自以下任一个或其组合：KK、HH、RH、KKK、WKK、HHK；
J表示接头或空缺。
接头和随机序列的顺序可互换，而不影响IL-15变体的改进的性能。
术语“接头(Linker)”在本公开中用于连接野生型IL-15和添加肽，以保证蛋白的正确折叠和稳定性。如果接头的序列太短，可能影响肽链的折叠，从而相互干扰；如果接头的序列太长，又涉及免疫原性的问题等。
在一些实施方案中，所述接头包含选自G和S的至少一个氨基酸残基。
在一些实施方案中，所述接头选自以下任一个或其组合：(G)n、(GGGGS)n、(GS)n、(GGS)n，其中n为1至10的整数(1、2、3、4、5、6、7、8、9、10)。
在一些具体的实施方案中，提供了一种IL-15变体，其包含选自以下的任一个结构(或如以下结构所示)：
X1-HPLT(SEQ ID NO：13)-WKK、
X1-LRLISG(SEQ ID NO：14)-RH、
X1-LIER(SEQ ID NO：15)-HHK、
X1-HVESG(SEQ ID NO：16)-KKK、
X1-HSQLETG(SEQ ID NO：17)-KK；
其中，X1表示野生型人IL-15。
在本申请中，变体是指在母体多肽的基础上引入氨基酸残基的取代、添加、缺失或修饰后所产生的多肽。相较于母体多肽的物理、化学或生物活性，变体具有改进的活性。
在一些实施方案中，野生型人IL-15选自以下的任一项：天然存在的人IL-15、天然存在的人IL-15剪接变体。
在一些具体的实施方案中，所述野生型人IL-15是野生型人IL-15的成熟形式。在一些具体的实施方案中，所述野生型人IL-15是SEQ ID NO：1所示(首位甲硫氨酸是任选的，SEQ ID NO：2)。
在本申请中，术语“白介素-15”、“白细胞介素-15”、“IL-15”指白介素-15蛋白或多肽，尤其是人白介素-15蛋白或多肽。未成熟形式的IL-15包含162个氨基酸，其中第1-29位氨基酸构成信号肽，第30-48位氨基酸构成前肽；IL-15的成熟形式对应于氨基酸49至162。未成熟形式的IL-15可在UniProtKB登录号P40933获得。人成熟IL-15的氨基酸序列对应于SEQ ID NO：1或去除首位M的SEQ ID NO：2。术语“IL-15”还包括由细胞天然表达的IL-15的任何变体或同种型。值得注意的是，已经报道了IL-15的若干种剪接的转录变体。
在本申请中，“多肽”理解为由至少两个氨基酸通过肽键连接而形成的线性或环状聚合物。在一些情况下，多肽包含天然的20种氨基酸；在另一些情况下，多肽可以包含除遗传密码定义的20种氨基酸以外的氨基酸；在又一些情况下，多肽可包含修饰的氨基酸(如翻译后修饰、或通过化学过程)。这些修饰可以出现在多肽的任何位置：在肽骨架中、在侧链中或在羧基或氨基末端；例如但不限于酰化、核糖基化、二硫键形成、脱甲基化、甲酰化、甲基化、肉豆蔻酰化、氧化、羟基化、磷酸化、糖基化、聚乙二醇化、GPI锚形成、硫酸化、泛素化、共价或非共价交联、环化、固定至脂质或脂质衍生物。
在一些实施方案中，本公开的IL-15变体包含选自以下任一项所示的多肽：SEQ IDNO：3、SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：6、SEQ ID NO：7、SEQ ID NO：8、SEQ ID NO：9、SEQ ID NO：10、SEQ ID NO：11、SEQ ID NO：12。
在一些实施方案中，本公开的IL-15变体与SEQ ID NO：3至12中任一个具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的同一性。
多核苷酸
根据在一些实施方案中，本公开提供了一种多核苷酸，其编码本公开的IL-15变体。
术语“多核苷酸”或“核酸分子”是指包含核苷酸的聚合物。多核苷酸的示例是，例如但不限于DNA、RNA、LNA、cDNA。
技术人员理解，编码相同氨基酸序列的核苷酸序列可以不同，由于密码子的兼并性和不同宿主的密码子偏好性，不同的核苷酸序列能够编码相同的氨基酸序列，这些序列均属于本公开的范畴内。
表达载体
根据在一些实施方案中，本公开提供了一种重组表达载体，其包含本公开的多核苷酸。
表达载体可以是原核或真核表达载体，也可以是穿梭载体。
表达载体是本领域熟知的，通常可以市售获得。在用于表达本申请的活性分子时，技术人员将编码序列插入表达载体的适当位点。
表达载体的示例，例如但不限于：PET表达载体，例如pET Dsb 39b、pETExpression System 33b、pET GST Fusion Systems 41、pET NusA Fusion Systems 43.1；PGEX表达载体，例如pGEX-2T、pGEX-2TK、pGEX-3X、pGEX-4T-1、pGEX-4T-2、pGEX-4T-3、pGEX-5X-1、pGEX-5X-2、pGEX-5X-3、pGEX-6P-1、pGEX-6P-2、pGEX-6P-3；PTYB表达载体，例如PTYB1、PTYB2、PTYB11、PTYB12；真核表达载体，例如pCDNA3.1(-)、pCDNA3.1(+)、pPICZalpha A、pGAPZαA、PYES2.0、pBI121、pEGFP-N1、pEGFP-C1、pPIC9K、pPIC3.5K。
本申请对表达载体没有特别的限制，技术人员根据公知常识有能力根据后续应用、宿主类型、生产规模等因素，而选择适当的表达载体。
宿主细胞
本公开提供一种宿主细胞，其包含根据本公开的表达载体、或表达根据本公开的IL-15变体。
一些实施方案中，宿主细胞包含(如已被转化或转染有)表达载体，所述表达载体包含根据本公开的多核苷酸。
一些实施方案中，所述宿主细胞是原核细胞。
一些实施方案中，所述宿主细胞是真核细胞。
一些具体实施方案中，宿主细胞为细菌、酵母菌或哺乳动物细胞，具体可以为巴斯德毕赤酵母或酿酒酵母。
一些具体实施方案中，宿主细胞是原核微生物(如大肠杆菌)。
一些具体实施方案中，宿主细胞真核生物细胞。
一些实施方案中，使用表达糖基化多肽的宿主细胞，例如植物和昆虫细胞。脊椎动物细胞也可以用作宿主细胞，例如，悬浮生长的哺乳动物细胞系、猴肾CV1系(COS-7)、人胚胎肾系(293或293T细胞)、幼仑鼠肾细胞(BHK)、小鼠塞托利(sertoli)细胞(TM4细胞)、猴肾细胞(CV1)、VERO-76、人宫颈癌细胞(HELA)、犬肾细胞(MDCK)、buffalo大鼠肝细胞(BRL3A)、人肺细胞(W138)、人肝细胞(Hep G2)、小鼠乳房肿瘤细胞(MMT060562)、MRC5细胞、FS4细胞、CHO细胞、骨髓瘤细胞系(如YO、NS0、P3X63和Sp2/0)。
本申请中，宿主细胞不能发育成动物个体或植物个体。
生产方法
本公开提供一种生产IL-15变体的方法。
一些实施方案中，将编码IL-15变体的多核苷酸与表达载体相连，转入表达宿主中，通过诱导而表达IL-15变体。
一些实施方案中，IL-15变体以包涵体的方式表达，对包涵体进行复性。
一些实施方案中，包涵体溶解于尿素溶液(例如，8M)中，通过离子交换和/或反相色谱法纯化IL-15变体。
一些具体的实施方案中，表达载体为原核pET41a，表达宿主为大肠杆菌BL21(DE3)或C41(DE3)。
药物组合物
本公开提供一种药物组合物，其含有根据本公开的IL-15变体；任选地还包含药学上可接受的稀释剂、载体或赋形剂。
术语“药学上可接受的”是指不具有不期望的体内或体外作用。
术语“药学上可接受的稀释剂、载体或赋形剂”是指存在于药物制剂(或药物组合物)中而不是活性成分的任何组分。因此，可以提及稀释剂、粘合剂、润滑剂、崩解剂、填充剂、着色剂、润湿剂、乳化剂、pH缓冲剂、防腐剂等。药学上可接受的稀释剂、载体或赋形剂在教科书Remington's The Science and Practice of Pharmacy中有详细描述。
在示例性组合物中，可包含一种或多种药学上可接受的成分，例如稳定剂、抗微生物剂、缓冲剂、着色剂、调味剂、佐剂等。
本公开的活性分子(IL-15变体)与常规使用的载体(或稀释剂、或赋形剂)一起制成药物组合物及其单位剂量的形式。
组合物可以是固体形式，例如片剂或填充胶囊、冷冻干燥形式。
本公开的组合物是固体制剂时，在使用前用合适的媒介物重构。
组合物可以是液体形式(例如注射剂、溶液、悬浮液、乳液)。示例性组合物用于口服、或胃肠外(包括皮下)施用的无菌注射溶液。本公开的组合物是液体制剂时，包括但不限于水性或油性悬浮液、溶液、乳液、糖浆。适于口服给药的液体形式可包括含有缓冲剂、悬浮剂和分散剂、着色剂、调味剂等水性或非水性媒介物。悬浮剂包括但不限于山梨糖醇浆、甲基纤维素、葡萄糖、明胶、羟乙基纤维素、羧甲基纤维素、硬脂酸铝凝胶。乳化剂包括但不限于卵磷脂、脱水山梨糖醇单油酸酯、阿拉伯胶。非水性媒介物包括但不限于食用油、丙二醇、乙醇。防腐剂包括但不限于对羟基苯甲酸甲酯、对羟基苯甲酸丙酯、山梨酸。
在一些具体实施方案中，所述药物组合物可以为冻干制剂或可注射溶液。
在一些具体实施方案中，可注射的组合物通常基于可注射的无菌盐水或磷酸盐缓冲盐水。
在一些具体实施方案中，药物组合物单位计量中可含有按质量计1％至10％的IL-15变体；具体而言1％±10％、2％±10％、3％±10％、4％±10％、5％±10％、6％±10％、7％±10％、8％±10％、9％±10％、10％±10％。
在一些具体实施方案中，药物组合物单位剂量中含有的IL-15变体的量为0.25μg/kg至100μg/kg；例如但不限于1μg/kg、5μg/kg、10μg/kg、20μg/kg、25μg/kg、30μg/kg、35μg/kg、40μg/kg、45μg/kg、50μg/kg、55μg/kg、60μg/kg、65μg/kg、70μg/kg、75μg/kg、80μg/kg、85μg/kg、90μg/kg、95μg/kg、100μg/kg±10％。
在一些实施方案中，本公开的IL-15变体或药物组合物分多次施用。在一些实施方案中，第一周期(28天/周期)的剂量为0.25μg/kg，然后连续周期的剂量水平分别为0.5μg/kg、1μg/kg、2μg/kg、4μg/kg和8μg/kg。
在一些实施方案中，第一周期的剂量为1μg/kg，第二周期中的剂量为2μg/kg，第三周期中的剂量为4μg/kg，第四周期中的剂量为8μg/kg。例如，受试者可以持续两周中每周三次接受相同的剂量，然后在每个治疗周期之间，间隔两周作为休息。在另一些实施方案中，可以皮下s.c.施用本公开的IL-15变体或药物组合物。
用途、治疗方法
本公开提供一种IL-15变体，其用于预防或治疗感染性疾病。在一些实施方案中，所述感染性疾病选自：天花病毒感染、HIV感染、细菌感染、真菌感染、HBV感染。
本公开提供一种IL-15变体，其用于预防或治疗癌症。在一些实施方案中，所述癌症选自：黑色素瘤、结直肠癌、皮肤癌、淋巴瘤、肾细胞癌、肝癌、肺癌、胃癌、乳腺癌。
本公开提供一种IL-15变体，其用于预防或治疗血液病。在一些实施方案中，所述血液病选自：贫血、白血病、骨髓增生异常综合征。
本公开提供一种IL-15变体，其用于预防或治疗炎性疾病。在一些实施方案中，所述炎性疾病选自：自身免疫性疾病、乳糜泻、结节病、溃疡性结肠炎、克罗恩病、胆管炎、葡萄膜炎、皮炎；在一些实施方案中，所述自身免疫性疾病选自：多发性硬化症、银屑病、风湿性关节炎、胃炎、黏膜炎。
本公开提供前述IL-15变体在制备药物中的用途，所述药物用于预防或治疗选自以下的疾病：感染性疾病、癌症、血液病、炎性疾病。在一些实施方案中，所述感染性疾病选自：天花病毒感染、HIV感染、细菌感染、真菌感染、HBV感染。在一些实施方案中，所述癌症选自：黑色素瘤、结直肠癌、皮肤癌、淋巴瘤、肾细胞癌、肝癌、肺癌、胃癌、乳腺癌。在一些实施方案中，所述血液病选自：贫血、白血病、骨髓增生异常综合征。在一些实施方案中，所述炎性疾病选自：自身免疫性疾病、乳糜泻、结节病、溃疡性结肠炎、克罗恩病、胆管炎、葡萄膜炎、皮炎；在一些实施方案中，所述自身免疫性疾病选自：多发性硬化症、银屑病、风湿性关节炎、胃炎、黏膜炎。
本公开提供一种预防或治疗以下疾病的方法：感染性疾病、癌症、血液病、炎性疾病。所述方法包括步骤：向受试者施用预防或治疗有效量的本公开的IL-15变体。在一些实施方案中，所述感染性疾病选自：天花病毒感染、HIV感染、细菌感染、真菌感染、HBV感染。在一些实施方案中，所述癌症选自：黑色素瘤、结直肠癌、皮肤癌、淋巴瘤、肾细胞癌、肝癌、肺癌、胃癌、乳腺癌。在一些实施方案中，所述血液病选自：贫血、白血病、骨髓增生异常综合征。在一些实施方案中，所述炎性疾病选自：自身免疫性疾病、乳糜泻、结节病、溃疡性结肠炎、克罗恩病、胆管炎、葡萄膜炎、皮炎；在一些实施方案中，所述自身免疫性疾病选自：多发性硬化症、银屑病、风湿性关节炎、胃炎、黏膜炎。
一些实施方案中，需要治疗的受试者(如患者或个体)通常为哺乳动物，例如人。
一些实施方案中，每日至少2次、每日至少1次、每48小时至少1次、每72小时至少一次、每周至少一次、每2周至少一次、每个月至少一次、每2个月至少一次、或每3个月至少一次向受试者施用本公开的IL-15变体、药物组合物。
一些实施方案中，通过任何途径施用本公开的IL-15变体、药物组合物。例如，通过肠胃外注射(例如，皮下、静脉注射)施用。
附图说明
图1：野生型IL-15在不同大肠杆菌单克隆中诱导表达的SDS-PAGE电泳图。
图2A至图2E：本公开的IL-15变体在大肠杆菌单克隆中表达的SDS-PAGE电泳图。图2A至图2E分别表示变体1至变体5。
图3：HPLC定量结果。
图4：本公开的IL-15变体的立体结构图。在水溶液的极性环境中，IL-15变体能稳定保持链内氢键，降低水分子造成的解螺旋效应。
具体实施方式
以下将结合实施例对本公开作进一步地说明，应理解这些实施例仅作为例证的目的，不用于限制本公开的保护范围。
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，例如Sambrook等分子克隆：实验室手册，Cold Spring Harbor Laboratory Press，1989中所述的条件，或按照制造厂商所建议的条件。所采用的试剂，若无特殊说明，均为市售或公开渠道可以获得的试剂。
实施例
实施例1.野生型IL-15及IL-15变体在大肠杆菌中的表达
1.表达载体的构建
野生型IL-15及IL-15变体的核苷酸序列由天津卡梅德生物技术有限公司合成。通过在反向引物中引入需要添加的序列来修饰野生型IL-15的羧基端。当技术人员知道所需添加的氨基酸序列时，可以根据公知的引物设计原则，使用任何可用的引物设计软件来设计并合成引物，这属于本领域技术人员能力范围内。
野生型IL-15、构建的IL-15变体的氨基酸序列如表1所示。
表1.野生型IL-15、IL-15变体的氨基酸序列
2.按常规方法进行PCR扩增。
3.对表达载体进行酶切：反应体系为30μL，和限制性酶混匀后37℃过夜。
4.回收PCR产物和酶切载体。
5.载体与片段的摩尔比为2:1，37℃反应30min，立即置于冰上冷却，转化入DH5α中。
6.在1.5mL的管中加入0.5mL含Amp抗性的LB培养基，向其中接种单菌落，共5管；在37℃摇床中进行振荡培养。培养3h以后，取1μL做模板，进行PCR。PCR后，用琼脂糖凝胶电泳进行检测，选择阳性克隆进行测序。
7.阳性菌液保存及质粒的提取：将测序正确的阳性克隆接种于5mL Amp抗性的LB液体培养基中，接种量为10μL。取500μL的菌液加入1.5mL的管中，加入500μL 40％甘油放入-80℃保存。离心菌液，收集菌体，用于质粒提取。
8.野生型IL-15和IL-15变体的表达
a)取10μl质粒加入100μl BL21(DE3)感受态细胞中，立即混匀，冰上放置30min；
b)42℃热激90s，迅速冰浴5min；涂布LB(含50μg/ml卡那霉素)平板，37℃培养14h后挑取单克隆进行诱导培养。
9.小规模表达
a)从LB平板中挑取单克隆，加入2ml LB液体培养基(含50μg/ml卡那霉素)活化；
b)将过夜培养的菌液按1:50比例转接到20ml含Amp的LB培养基中，37℃，220rpm，培养至至菌体OD600为0.6-0.8；转接到5ml LB液体培养基(含50μg/ml卡那霉素)培养至菌体OD600为0.6-0.8；加入浓度为1mM的IPTG进行37℃诱导表达4h。
10.SDS-PAGE鉴定表达量
a)测定培养液的OD600，取10OD菌液，10000rpm，2min离心，去掉上清；
b)用1mL裂解液(10mM Tris-HCl，pH 8.0)重悬菌体，置于冰上进行超声裂解细胞，超声条件：130W、4min、on 3s、off 3s；
c)超声结束后，取80μl样品加入20μl 5×上样缓冲液，95℃加热5min，每个样品取12.5μl(0.1OD)进行SDS-PAGE电泳。
IL-15野生型的表达如图1所示：无论是在裂解液、上清或沉淀中，都检测不到野生型IL-15的表达。图1的1、2、3、4代表四种不同的表达载体。相比于野生型白介素-15，IL-15的变体1至变体17的表达水平在大肠杆菌大中有统计学上显著的改善(p<0.05)，约提高10至20倍(图2A至图2E所示)。
11.HPLC鉴定表达量
a)表达后收取10OD细胞，用1ml 10mM Tris-HCl，pH8.0的缓冲液重悬；
b)超声破碎；
c)破碎后的样品，经12000rpm离心10min，弃上清；
d)沉淀用1ml新鲜配制的8M尿素/10mM Tris-HCl，pH8.0，10mM DTT室温振荡溶解约1h；
e)用0.2μm过滤器过滤，上样进行高效液相色谱分析。
利用C4分析柱，流动相A为含0.1％TFA的去离子水，流动相B为含0.1％TFA的乙腈；用15分钟从20％B到60％B的梯度来进行分析。HPLC定量结果如图3所示，IL-15变体1至5的表达量要高于野生型IL-15的表达量20倍左右。
实施例2.IL-15变体在水溶液中的分子动力学
对于IL-15变体1至变体5，利用SIWSS-model进行同源建模。使用同源建模得到的结构作为初始结构，使用GROMACS软件进行200ns的分子动力学模拟。立场选用OPLS-AA，水分子模型选用TIP4P模型。
经过长达200ns的长程动力学模拟，得到最终的IL-15变体的平均结构。使用pymol软件分析蛋白质结构，可以看到在蛋白质羧基端添加的HHH特征序列在长时间水溶液环境下的动力学模拟中保持了alpha螺旋的结构。这说明本公开中设计的添加肽在水溶液的极性环境中能稳定保持链内氢键结构，使得水分子造成的解螺旋效应降低(图4所示变体4，其他变体结果一致)。
序列表
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Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu
50 55 60
Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val
65 70 75 80
Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile
85 90 95
Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn
100 105 110
Thr Ser
<210> 3
<211> 122
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<221> VARIANT
<222>
<223> IL-15变体
<400> 3
Met Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu
1 5 10 15
Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val
20 25 30
His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu
35 40 45
Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val
50 55 60
Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Ala Asn
65 70 75 80
Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn
85 90 95
Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile
100 105 110
Asn Thr Ser His Pro Leu Thr Trp Lys Lys
115 120
<210> 4
<211> 121
<212> PRT
<213> 人工序列(artificial sequence)
<400> 4
Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile
1 5 10 15
Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His
20 25 30
Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln
35 40 45
Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu
50 55 60
Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Ala Asn Val
65 70 75 80
Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile
85 90 95
Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn
100 105 110
Thr Ser His Pro Leu Thr Trp Lys Lys
115 120
<210> 5
<211> 123
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<221> VARIANT
<222>
<223> IL-15变体
<400> 5
Met Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu
1 5 10 15
Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val
20 25 30
His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu
35 40 45
Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val
50 55 60
Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Ala Asn
65 70 75 80
Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn
85 90 95
Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile
100 105 110
Asn Thr Ser Leu Arg Leu Ile Ser Gly Arg His
115 120
<210> 6
<211> 122
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<221> VARIANT
<222>
<223> IL-15变体
<400> 6
Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile
1 5 10 15
Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His
20 25 30
Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln
35 40 45
Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu
50 55 60
Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Ala Asn Val
65 70 75 80
Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile
85 90 95
Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn
100 105 110
Thr Ser Leu Arg Leu Ile Ser Gly Arg His
115 120
<210> 7
<211> 124
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<221> VARIANT
<222>
<223> IL-15变体
<400> 7
Met Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu
1 5 10 15
Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val
20 25 30
His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu
35 40 45
Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val
50 55 60
Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Ala Asn
65 70 75 80
Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn
85 90 95
Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile
100 105 110
Asn Thr Ser His Ser Gln Leu Glu Thr Gly Lys Lys
115 120
<210> 8
<211> 123
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<221> VARIANT
<222>
<223> IL-15变体
<400> 8
Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile
1 5 10 15
Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His
20 25 30
Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln
35 40 45
Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu
50 55 60
Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Ala Asn Val
65 70 75 80
Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile
85 90 95
Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn
100 105 110
Thr Ser His Ser Gln Leu Glu Thr Gly Lys Lys
115 120
<210> 9
<211> 122
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<221> VARIANT
<222>
<223> IL-15变体
<400> 9
Met Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu
1 5 10 15
Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val
20 25 30
His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu
35 40 45
Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val
50 55 60
Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Ala Asn
65 70 75 80
Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn
85 90 95
Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile
100 105 110
Asn Thr Ser Leu Ile Glu Arg His His Lys
115 120
<210> 10
<211> 121
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<221> VARIANT
<222>
<223> IL-15变体
<400> 10
Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile
1 5 10 15
Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His
20 25 30
Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln
35 40 45
Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu
50 55 60
Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Ala Asn Val
65 70 75 80
Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile
85 90 95
Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn
100 105 110
Thr Ser Leu Ile Glu Arg His His Lys
115 120
<210> 11
<211> 123
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<221> VARIANT
<222>
<223> IL-15变体
<400> 11
Met Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu
1 5 10 15
Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val
20 25 30
His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu
35 40 45
Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val
50 55 60
Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Ala Asn
65 70 75 80
Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn
85 90 95
Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile
100 105 110
Asn Thr Ser His Val Glu Ser Gly Lys Lys Lys
115 120
<210> 12
<211> 122
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<221> VARIANT
<222>
<223> IL-15变体
<400> 12
Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile
1 5 10 15
Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His
20 25 30
Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln
35 40 45
Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu
50 55 60
Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Ala Asn Val
65 70 75 80
Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile
85 90 95
Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn
100 105 110
Thr Ser His Val Glu Ser Gly Lys Lys Lys
115 120
<210> 13
<211> 4
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<221> UNSURE
<222>
<223> 随机序列
<400> 13
His Pro Leu Thr
1
<210> 14
<211> 6
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<221> UNSURE
<222>
<223> 随机序列
<400> 14
Leu Arg Leu Ile Ser Gly
1 5
<210> 15
<211> 4
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<221> UNSURE
<222>
<223> 随机序列
<400> 15
Leu Ile Glu Arg
1
<210> 16
<211> 5
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<221> UNSURE
<222>
<223> 随机序列
<400> 16
His Val Glu Ser Gly
1 5
<210> 17
<211> 7
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<221> UNSURE
<222>
<223> 随机序列
<400> 17
His Ser Gln Leu Glu Thr Gly
1 5
Claims (9)
1. 一种白介素-15变体，其是选自以下的任一个结构所示，从左至右按氨基端至羧基端方向：
X1-HPLT-WKK、
X1-LIER-HHK、
X1-HVESG-KKK、
X1-LRLISG-RH、
X1-HSQLETG-KK；
X1表示野生型人白介素-15；
所述野生型人白介素-15是SEQ ID NO：1或2所示。
2.根据权利要求1所述的白介素-15变体，其是选自以下任一项所示的多肽：SEQ IDNO：3、SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：6、SEQ ID NO：7、SEQ ID NO：8、SEQ ID NO：9、SEQ ID NO：10、SEQ ID NO：11、SEQ ID NO：12。
3.一种多核苷酸，其编码权利要求1至2中任一项所述的白介素-15变体。
4.一种重组表达载体，其包含权利要求3所述的多核苷酸。
5.一种宿主细胞，其：
- 包含权利要求4所述的重组表达载体；或
- 表达权利要求1至2中任一项所述的白介素-15变体；
所述宿主细胞不能发育成动物个体或植物个体。
6.一种药物组合物，其包含：
权利要求1至2中任一项所述的白介素-15变体；
任选地，可药用载体或赋形剂或稀释剂。
7.一种蛋白复合物，其包含权利要求1至2中任一项所述的白介素-15变体。
8.权利要求1至2中任一项所述的白介素-15变体在制备药物中的用途；所述药物用于预防或治疗选自以下的疾病：感染性疾病、癌症、血液病、炎性疾病；
所述感染性疾病选自：天花病毒感染、HIV感染、细菌感染、真菌感染、HBV感染；
所述癌症选自：黑色素瘤、结直肠癌、皮肤癌、淋巴瘤、肾细胞癌、肝癌、肺癌、胃癌、乳腺癌；
所述血液病选自：贫血、白血病、骨髓增生异常综合征；
所述炎性疾病选自：自身免疫性疾病、乳糜泻、结节病、溃疡性结肠炎、克罗恩病、胆管炎、葡萄膜炎、皮炎；所述自身免疫性疾病选自：多发性硬化症、银屑病、风湿性关节炎、胃炎、黏膜炎。
9.一种提高白介素-15在宿主细胞中表达量的方法，包括步骤：
- 在培养介质中，培养权利要求5所述的宿主细胞；
- 收获权利要求1至2中任一项所述的白介素-15变体；
所述宿主细胞是大肠杆菌。
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| CN114573707A (zh) * | 2020-11-28 | 2022-06-03 | 北京志道生物科技有限公司 | 一种用于抗肿瘤的新型il-15融合蛋白 |
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| CN107636015A (zh) * | 2015-03-06 | 2018-01-26 | 德国癌症研究中心 | 包含结合蛋白和对IL‑15Rα具有降低亲和力的白介素‑15多肽的融合蛋白及其治疗用途 |
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