CN117362411A - 白细胞介素15蛋白突变体及其应用 - Google Patents
白细胞介素15蛋白突变体及其应用 Download PDFInfo
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Abstract
本发明公开了一种白细胞介素15蛋白突变体及其应用。本发明通过在野生型IL‑15分子中引入一对二硫键，提高IL‑15构象稳定性，在不依赖sushi结构域的情况下，显著增强了其对β和γ受体的亲和力和生物学活性以及高生产效能。本发明还提供了这种IL‑15突变体的其他分子形式，如融合Fc，偶联脂肪酸链，以提高IL‑15的半衰期，使其具有长效性。
Description
技术领域
本发明属于医药技术领域，具体而言，涉及白细胞介素15蛋白(IL-15)突变体及其应用。
背景技术
白细胞介素15(白介素15，IL-15)是一种分子量约为12-14kD的细胞因子，天然人白介素15成熟肽含有114个氨基酸，包括4个半胱氨酸残基，其中Cys35与Cys85、Cys42与Cys88连接，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。
白介素15受体(IL-15R)由α(又称CD215)、β(又称CD122)和γ(又称CD132)三个亚基组成。IL-2和IL-15的受体共用β和γ链，但IL-2和IL-15均有其各自特异性α受体链。IL-15Rα受体大量表达于抗原递呈细胞APC或巨噬细胞等表面，由于IL-15与IL-15Rα受体亲和力很高(KD～10-11M)，所以IL-15会大量的被抗原递呈细胞(APC)捕获，通过一种反式(trans)相互作用的形式，进一步再结合T细胞表面的β和γ受体而激活T细胞。IL-15单独结合α受体时并不能激活下游通路。IL-15与IL-15Rβγ异源二聚体的亲和力中等(KD～10-9M)，是传导下游信号通路的主要方式。2005年，法国的Erwan Mortier等人发现，IL-15在结合α受体中的sushi结构域后，能够发生一定的构象变化而增强对IL-15Rβγ异源二聚体的亲和力(大约提高10倍)，从而增强IL-15的生物学功能，因此，无论从生物学功能上，还是IL-15对βγ受体的结合能力上，IL-15结合其α受体均具有重要的意义。由于IL-2和IL-15的受体共用β和γ链，因此，IL-2和IL-15之间有许多相似的生物学功能，如都能促进NK细胞、B淋巴细胞及T淋巴细胞增殖并可维持这些免疫细胞功能，对于治疗一些恶性肿瘤存在一定疗效，具有抗肿瘤的作用。但是由于拥有不同α链受体，IL-15几乎不诱导Treg，并会抑制AICD。研究者认为IL-15在肿瘤治疗中具有更好的应用前景。
在大多数情况下IL-15和IL-15Rα受体在相同细胞中表达后，在胞内IL-15与IL-15Rα受体的sushi结构域以高亲和力结合后转运至膜表面，然后与其反应性细胞(如T细胞或NK细胞)膜表面的βγ异源二聚体复合物或αβγ异源三聚体复合物结合，β和γ受体可分别活化下游通路，最终达到激活T细胞和NK细胞的作用。因此，博际生物的BJ-001,Sushi-IL15-Apo、Immunity-Bio的N-803、Altor Bio-Science ALT-803、恒瑞的SHR-1501等利用α亚基的特点，用融合或非融合的方法将IL-15与α受体(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。虽然融合IL-15的α受体或其sushi结构域，能够显著的提高IL-15同βγ受体的亲和力，从而增强其抗肿瘤活性，但与天然的IL-15相比，融合α受体或其sushi结构域的IL-15衍生物不能够再次结合体内的α受体，因此该类分子不能够结合到APC上以反式相互作用的方式去激活T细胞，进而影响其抗肿瘤效果。同时，游离的高活性IL-15分子也可能产生药物毒性，比如(1)触发细胞因子级联反应，包括TNFα，IL1，IL6，GM-CSF以及促炎性细胞因子的产生；(2)对有些肿瘤进展是起促进作用，帮助肿瘤的增殖，存活和扩散；(3)可能会活化自身反应性T细胞并参与自身免疫性疾病过程。因此，通过蛋白质工程学改造的手段，使IL-15在不结合sushi结构域情况下，具备对β和γ受体的高亲和力，成为IL-15改造的一个新的方向。
此外，尽管IL-15较IL-2有着更高的安全性及活性，但作为蛋白类药物，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、半衰期短和容易形成异构体(isomer)等，从而难以进行产业化。
发明内容
本发明的目的在于针对现有技术的现状及存在的不足，提供一种IL-15蛋白突变体。本发明通过在天然野生型IL-15(SEQ ID NO:1)分子中引入一对二硫键，提高IL-15构象稳定性，在不依赖sushi结构域的情况下，显著增强了其对β和γ受体的亲和力和生物学活性以及高生产效能。本发明还提供了这种IL-15突变体的其他分子形式，如融合Fc，偶联脂肪酸链，以提高IL-15的半衰期，使其具有长效性。
本发明中所有氨基酸顺序标号均以天然野生型IL-15(SEQ ID NO:1)序列为准。由于本发明中的IL-15使用大肠杆菌表达，所以最终所有蛋白的氮端(N端)均带有一个起始氨基酸甲硫氨酸(Met，即第1位)，同时由于第77-78位存在Asn-Gly序列，导致第77位Asn易发生脱氨基反应，所以本发明中所有IL-15序列均含有G78A突变，以去除第77位Asn的脱氨基反应(wt-IL-15，SEQ ID NO:2)，以上突变均不影响IL-15的活性(参照Nellis DF,MichielDF,Jiang M-S,et al..Characterization of recombinant human IL-15deamidationand its practical elimination through substitution of asparagine 77.Pharm Res2012；29:722–38.)。因此，本发明中所述的野生型IL-15的氨基酸序列如SEQ ID NO:2所示。
为实现上述目的，本发明采用以下技术方案施行：
第一方面，本发明提供一种人白细胞介素15蛋白(IL-15)突变体，所述的突变体为在天然野生型IL-15的氨基酸序列中，第21位至29位之间任意选取一个氨基酸突变为Cys，在第91位至96位之间任意选取一个氨基酸突变为Cys，并在两个突变的Cys之间形成二硫键，所述天然野生型IL-15的氨基酸序列如SEQ ID NO.1所示，同时，在SEQ ID NO.1的N端添加一个甲硫氨酸，将第78位的Gly突变为Ala。
在本发明一个优选的实施方案中，所述的突变体为：将野生型IL-15的氨基酸序列的第24位Thr、第93位Glu，都突变为Cys，并在两个突变的Cys之间形成二硫键，所述IL-15突变体的氨基酸序列如SEQ ID NO.3所示。
第二方面，本发明提供一种人白介素15突变体-Fc的融合蛋白，所述融合蛋白的人白介素15突变体为第一方面所述的IL-15突变体，Fc片段包括部分铰链区、CH2区和CH3区，所述IL-15突变体与Fc序列之间为直接融合或通过连接序列融合，所述的Fc片段选自人或动物的IgG、IgM、IgD、IgA或者它们的亚型。
在本发明一个优选的实施方案中，所述融合蛋白的Fc片段选自人IgG4序列。
根据本发明，所述融合蛋白选自氨基酸序列如SEQ ID NO.8所示的IL-15(G78A,T27C,E92C)-Fc，氨基酸序列如SEQ ID NO.9所示的IL-15(G78A,T24C,E93C)-Fc，或氨基酸序列如SEQ ID NO.10的IL-15(G78A,L15C,S58C)-Fc。
在本发明一个优选的实施方案中，所述融合蛋白的氨基酸序列如SEQ ID NO.9所示，其中的第24位、第93位都是突变为Cys，并在两个突变的Cys之间形成二硫键。
第三方面，本发明提供能够表达第一方面所述的IL-15突变体或第二方面所述的融合蛋白的载体。
优选地，所述载体为原核载体，进一步优选地，所述原核载体为通用载体pET41a。
第四方面，本发明提供包含有第三方面所述载体的宿主细胞。
优选地，所述细胞选自真核细胞或原核细胞；进一步优选地，所述细胞为大肠杆菌。
第五方面，本发明提供一种人白细胞介素15蛋白突变体的衍生物，所述衍生物为第一方面所述的IL-15突变体的C端偶联脂肪酸链，所述偶联的脂肪酸链为(1)通式为GGG-linker-(CH2)X-COOH或(2)其它含有脂肪酸链的化学结构，其中(1)的通式中的linker部分包括但不仅限于PEG-linker(聚乙二醇)、多肽链、OEG等，(CH2)X中X的数目介于10-20之间；优选的x介于16-20之间；
进一步优选的，所述偶联的脂肪酸链包括：
GGG-PEG2-Lys-(CH2)16-COOH、
NHS-PEG2-PEG2-γ-Glu-(CH2)17-COOH、
GGG-PEG4-PEG4-PEG4-Lys-(CH2)17-COOH、
GGG-PEG4-γ-Glu-γ-Glu-Lys-(CH2)17-COOH、
HOOC-(CH2)16-γ-Glu-γ-Glu-Lys-GGG、
HOOC-(CH2)16-CONH-γ-Glu-γ-Glu-PEG2-Lys-Br、
GGG-γ-Glu-C2DA-2OEG-γ-Glu-(CH2)17-COOH、
GGG-γ-Glu-C2DA-2OEG-γ-Glu-(CH2)19-COOH、
GGG-OEG-C2DA-2OEG-γ-Glu-(CH2)19-COOH、
GGG-OEG-C2DA-2OEG-γ-Glu-Trx-(CH2)19-COOH、
CHO-PEG2-PEG2-γ-Glu-(CH2)17-COOH或
Mal-C2DA-2OEG-γ-Glu-Tn-(CH2)19-COOH任意一种。
在本发明一个优选的实施方案中，人白细胞介素15蛋白突变体的衍生物通过在IL-15突变体的C末端先添加含有LPXTG的多肽，所述X为任意氨基酸，然后偶联脂肪酸链；优选的，所述先添加的多肽序列为-GS-LPETG-GSGGS-HHHHHH，选用的脂肪酸链结构为GGG-PEG2-Lys-(CH2)16-COOH获得修饰后的序列为氨基酸序列如SEQ ID NO.7所示的IL-15-1303-FA。
第六方面，本发明还提供了一种C端偶联脂肪酸链的IL-15突变体的制备方法，所述方法通过在IL-15或其突变体的C末端添加含有LPXTG(X为任意氨基酸)实现，优选的，添加的序列为-GS-LPETG-GSGGS-HHHHHH，含有此序列的IL-15为氨基酸序列如SEQ ID NO.4所示的IL-15-781；和含有此序列的IL-15突变体为氨基酸序列如SEQ ID NO.5所示的IL-15-1303，所述IL-15-781与IL-15-1303均可以通过Sortase-A酶的催化，被第五方面所述脂肪酸链修饰偶连，修饰后的IL-15或IL-15突变体具备的结构为：IL-15-LPXT-GGG-脂肪酸链或IL-15突变体-LPXT-GGG-脂肪酸链。
在本发明一个优选的实施方案中，选用的脂肪酸链结构为GGG-PEG2-Lys-(CH2)16-COOH，修饰后的序列为IL-15-781-FA(SEQ ID NO.6)与IL-15-1303-FA(SEQ ID NO.7)。
第七方面，本发明提供一种药物组合物，其包含第一方面所述的人白细胞介素15蛋白突变体、或第二方面所述的人白介素15突变体-Fc融合蛋白、或第五方面所述的人白细胞介素15蛋白突变体的衍生物、和药学上可接受的赋形剂、稀释剂或载体。
第八方面，本发明提供一种用于刺激或抑制哺乳动物免疫应答的方法，其包括：向所述哺乳动物给予有效量的第一方面所述的人白细胞介素15蛋白突变体、或第二方面所述的人白介素15突变体-Fc融合蛋白、或第五方面所述的人白细胞介素15蛋白突变体的衍生物、或第七方面所述的药物组合物。
第九方面，本发明提供一种第一方面所述的人白细胞介素15蛋白突变体、或第二方面所述的人白介素15突变体-Fc融合蛋白、或第五方面所述的人白细胞介素15蛋白突变体的衍生物、或第七方面所述的药物组合物在制备用于治疗IL-15介导的疾病或病症的药物中的用途；其中所述的疾病为传染病、癌症、血液病和自身免疫性疾病。
所述的癌症优选黑色素瘤、结直肠癌、皮肤癌、淋巴瘤、肾细胞癌、实体瘤、肝癌、肺癌、胃癌、乳腺癌；所述的传染病优选天花病毒感染、HIV感染、细菌感染、真菌感染、HBV感染；所述的血液病优选贫血、急性髓系白血病、骨髓增生异常综合征、T-细胞大颗粒淋巴细胞性白血病；所述的自身免疫性疾病优选多发性硬化症、银屑病、风湿性关节炎、炎性疾病、胃炎、黏膜炎。
所述的人白细胞介素15蛋白突变体、或人白介素15突变体-Fc融合蛋白、或人白细胞介素15蛋白突变体的衍生物、或药物组合物可以单独使用，也可以与其它药物联合使用。
所述其它药物为小分子抑制剂或抗体类药物；所述的小分子抑制剂优选靶向化疗药物或放射治疗药物，更优选烷化剂；所述的抗体类药物优选单克隆抗体药物，更优选抗CD20、PD1、PDL1、Her2、EGFR、c-MET抗体。
第十方面，本发明提供一种治疗或预防疾病的方法，所述方法包括但不限于药物化疗、放射性治疗、手术治疗等方式；在所述疾病中，细胞表达疾病相关抗原，所述方法包括：向患者施予第一方面所述的人白细胞介素15蛋白突变体、或第二方面所述的人白介素15突变体-Fc融合蛋白、或第五方面所述的人白细胞介素15蛋白突变体的衍生物、或第七方面所述的药物组合物；在表达疾病相关抗原的细胞与表达IL-15Rα的免疫细胞之间形成足以活化所述免疫细胞的特异性结合复合物；以及通过所述免疫细胞杀死所述表达疾病相关抗原的细胞。
根据本发明，其中所述表达疾病相关抗原的细胞优选为肿瘤细胞或病毒感染细胞。其中所述的免疫细胞优选为T-细胞、LAK细胞或NK细胞。其中所述的疾病可以为传染病、癌症、血液病和自身免疫性疾病。所述的癌症优选黑色素瘤、结直肠癌、皮肤癌、淋巴瘤、肾细胞癌、实体瘤、肝癌、肺癌、胃癌、乳腺癌；所述的传染病优选天花病毒感染、HIV感染、细菌感染、真菌感染、HBV感染；所述的血液病优选贫血、急性髓系白血病、骨髓增生异常综合征、T-细胞大颗粒淋巴细胞性白血病；所述的自身免疫性疾病优选多发性硬化症、银屑病、风湿性关节炎、炎性疾病、胃炎、黏膜炎。
第十一方面，本发明提供一种联合用药治疗或预防疾病的方法，包括向患者施予第一方面所述的人白细胞介素15蛋白突变体、或第二方面所述的人白介素15突变体-Fc融合蛋白、或第五方面所述的人白细胞介素15蛋白突变体的衍生物、或第七方面所述的药物组合物，并联合施用其它药物，如小分子抑制剂或抗体类药物。所述的小分子抑制剂优选靶向化疗药物或放射治疗药物，更优选烷化剂；所述的抗体类药物优选单克隆抗体药物，更优选抗CD20、PD1、PDL1、Her2、EGFR、c-MET抗体。
为了更容易理解本发明，以下具体定义了某些技术和科学术语。除非在本文中另有明确定义，本文使用的所有其它技术和科学术语都具有本发明所属领域的普通技术人员通常理解的含义。
一、术语
本发明所用氨基酸三字母代码和单字母代码如J.biol.chem，243，p3558(1968)中所述。
本发明中“人白细胞介素15蛋白”或“白细胞介素15”或“白介素15”或“IL-15”均具备相同含义，指代人白细胞介素15蛋白或其特定突变体，该蛋白的天然野生型序列如SEQID NO.1所示。
本发明中的术语“突变”是指在多核苷酸的一个或多个(例如，若干个)位置处包含突变的核苷酸，并且保持多核苷酸的启动子活性。其中，本发明中的突变(包含，取代、插入和/或缺失)特指其中的取代和缺失，取代是指用不同的核苷酸置换占用一个位置的核苷酸。缺失是指去除占据某一位置的核苷酸。
在一些具体的实施方案中，本发明的“突变”包含在SEQ ID NO.3的第24位Thr、第93位Glu，都突变为Cys，并在两个突变的Cys之间形成二硫键，与未改造的SEQ ID NO：1相比，其构象更稳定，增加成药性，并增强其对β和γ受体的亲和力和生物学活性。
本发明所述的“融合蛋白”是指，通过用基因重组方法、化学方法或其它适当方法将两个或多个基因的编码区连接，在同一调控序列控制下表达基因重组所得的蛋白质产物。在本发明的一些实施方案中，人白细胞介素15蛋白突变体与生物活性多肽如Fc片段融合或非融合表达得到的单体蛋白质。本发明的融合蛋白中，两个或多个基因的编码区之间可由编码肽接头的序列于一个或数个位置融合。肽接头也可用于构建本发明的融合蛋白。
术语“Fc片段”指的是人免疫球蛋白链恒定区，特别是免疫球蛋白重链恒定区的羧基端或其中的一部分，无抗原结合活性，是抗体分子与效应分子和细胞相互作用的部位。例如，免疫球蛋白Fc区可包括重链CH1、CH2、CH3、CH4的两个或更多结构域与免疫球蛋白铰链区的组合。根据重链恒定区的氨基酸序列，免疫球蛋白可以分为不同的种类，主要有5类免疫球蛋白：IgA、IgD、IgE、IgG和IgM。其中一些还可进一步分成亚类(同种型)，如IgG-1、IgG-2、IgG-3、IgG-4；IgA-1和IgA-2以及不同的基因型。
“Fc片段”优选包括至少一个免疫球蛋白绞链区或部分铰链区，以及IgG的CH2和CH3区。更优选包括IgG4的一个CH2结构域，一个CH3结构域和一个免疫球蛋白绞链区，铰链区起始氨基酸位置可以变动。
术语“连接肽(Linker)”在本发明中用于连接人白细胞介素15蛋白突变体与Fc片段，以保证蛋白的正确折叠和稳定性的肽。本发明的“连接肽”优选为(GAPQ)n，其中n可以为0、1、2、3、4、5或者更多，优选n为5。如果连接肽序列太短，可能影响两蛋白高级结构的折叠，从而相互干扰；如果连接肽序列太长，又涉及免疫原性的问题，因为连接肽序列本身就是新的抗原。
术语“脂肪酸链”指的是一端含有一个羧基的长的脂肪族碳氢链，是有机物，通式是C(n)H(2n+1)COOH。按碳链长度不同分类，它可被分成短链(含4～6个碳原子)脂肪酸；中链(含8～14个碳原子)脂肪酸；长链(含16～20个碳原子)脂肪酸和超长链(含20个或更多碳原子)脂肪酸四类。本发明的“脂肪酸链”优选为GGG-PEG2-Lys-(CH2)16-COOH。目前已上市的多种多肽药物均是通过增加脂肪酸侧链，实现与血浆白蛋白非共价结合的方式延长半衰期，如诺和诺德公司的利拉鲁肽和索马鲁肽等。本发明利用纯化后的带-GS-LPETG-GSGGS-HHHHHH的IL-15突变体(IL-15突变体-GS-LPETG-GSGGS-HHHHHH，SEQ ID NO:4和SEQ ID NO:5)，通过转肽酶Sortase-A酶催化的连接反应将N端带有GGG的脂肪酸链与IL-15突变体-GS-LPETG-GSGGS-HHHHHH相连接，形成了人白细胞介素15蛋白突变体的衍生物，即本发明人白细胞介素15蛋白突变体偶联脂肪酸链模式。本发明使用Biacore SPR-8K测试比较野生IL-15及带有24C-93C新二硫键的IL-15突变体IL-15-(T24C,E93C)，对IL-15Rβγ-Fc受体的结合强度，结果表明IL-15在新引入的一对二硫键下，不仅没有影响IL-15对受体的结合强度，反而使其和受体的结合里加强。而且其构象更加稳定，毒性低，活性比野生型增强。
“治疗”意指给予患者内用或外用治疗剂，诸如包含本发明的人白细胞介素15蛋白突变体、或人白介素15突变体-Fc融合蛋白、或人白细胞介素15蛋白突变体偶联脂肪酸链模式、或包含有它们的药物组合物。所述患者具有一种或多种选自“免疫”或“癌性”的疾病或病症。已知所述治疗剂对这些疾病或病症(疾病症状)具有治疗作用。通常，在受治疗患者或群体中以有效缓解一种或多种疾病或病症的量给予治疗剂，无论是通过诱导这类疾病或病症退化还是抑制这类疾病或病症发展到任何临床可测量的程度。有效缓解任何具体疾病或病症的治疗剂的量(也称作“治疗有效量”)可根据多种因素变化，例如患者的疾病状态、年龄和体重，以及药物在患者产生需要疗效的能力。
“免疫病症”或“免疫障碍”包括例如病理性炎症、炎性病症和自身免疫性疾病症或疾病。“免疫病症”还指感染、持续感染和增生性病症，例如癌症、肿瘤和血管发生。“癌性病症”包括例如癌症、癌细胞、肿瘤、血管发生和癌变前病症，例如发育异常。
本文使用的“聚合酶链式反应”或“PCR”是指例如美国专利号4，683，195中所述的扩增程序或技术。一般来说，需要获得来自目标区域末端或之外的序列信息，使得可以设计寡核苷酸引物。这些引物的序列与待扩增模板的对应链相同或相似。
“任选”或“任选地”意味着随后所描述地事件或环境可以但不必发生，该说明包括该事件或环境发生或不发生地场合。例如，“任选包含1-3个抗体重链可变区”意味着抗体重链可变区可以但不必须存在；存在时，可以是1个、2个或3个。
“药物组合物”表示含有一种或多种本文所述化合物或其生理学上/可药用的盐或前体药物与其它化学组分的混合物，以及其它组分例如生理学/可药用的载体和赋形剂。药物组合物的目的是促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。
本发明中所述的用重组DNA转化宿主细胞的步骤可用本领域技术人员熟知的常规技术进行。获得的转化子可以用常规方法培养，以及表达本发明的基因所编码的多肽。根据所用的宿主细胞，培养中所用的培养基可选自各种常规培养基。宿主细胞在适于宿主细胞生长的条件下进行培养。
与现有技术相比，本发明的有益效果：
1、本发明中所有的融合Fc蛋白模式，在添加一对二硫键的IL-15突变体IL-15(T27C,E92C)-Fc(SEQ ID NO:8)，IL-15(T24C,E93C)-Fc(SEQ ID NO:9)，IL-15(L15C,S58C)-Fc(SEQ ID NO:10)及wtIL-15-Fc(SEQ ID NO:11)的质粒转化至大肠杆菌宿主工程菌中进行表达，表达产生不可溶性包涵体。在变复性过程中，添加二硫键的IL-15(T24C,E93C)-Fc分子表现较为优良的变复性优势，通过对复性后条带在胶图上染色密度对比，可以看出添加二硫键的IL-15(T24C,E93C)-Fc比野生型wtIL-15-Fc，复性效率大大增加。但其它形式的IL-15衍生物IL-15(T27C,E92C)-Fc和IL-15(L15C,S58C)-Fc在复性过程中产生多条带或弥散条带，且纯化后的蛋白没有主要条带出现。
2.本发明中将携带新增一对二硫键(T24C,E93C)的IL-15突变体融合-GS-LPETG-GSGGS-HHHHHH得到的IL-15-1303，或wtIL-15融合-GS-LPETG-GSGGS-HHHHHH得到的IL-15-781。含有额外二硫键(T24C,E93C)的IL-15-1303同IL-15-781相比，其构象更稳定，复性产率为IL-15-781的1.3倍。另外，IL-15-1303分子的异构体产生比例也大大减少，而这部分异构体的减少，在后期的纯化过程中，为避免使用反相色谱纯化提供可能，从而减少对环境的污染，也减少后期的生产成本。同时，本发明还发现，IL-15-1303相比IL-15-781对IL-15受体的亲和力大大增强。通过体外MO7E和NK92细胞的增殖实验可以看到，在相同条件下，增加一对二硫键(T24C,E93C)的IL-15-1303分子，二硫键的引入不仅没有影响IL-15与受体的亲和力，甚至IL-15-1303的细胞增殖活性要比IL-15-781的活性高。
3、本发明中将携带新增一对二硫键(T24C,E93C)的IL-15突变体融合-GS-LPETG-GSGGS-HHHHHH得到的IL-15-1303，或wtIL-15融合-GS-LPETG-GSGGS-HHHHHH得到的IL-15-781。在该发明中，使用Sortase-A酶介导的偶连策略，在IL-15蛋白C末端偶连脂肪酸链，得到半衰期更长的分子IL-15-1303-FA和IL-15-781-FA。
4、本发明添加一对二硫键的IL-15突变体的偶联脂肪酸链模式IL-15-1303-FA和野生型IL-15的偶联脂肪酸链模式IL-15-781-FA对MO7E和NK92细胞的增殖效果表明，IL-15在偶联脂肪酸链的情况下，IL-15引入一对二硫键(如T24C,E93C)，生物活性比野生型增强。
5、本发明添加一对二硫键的IL-15突变体的偶联脂肪酸链模式IL-15-1303-FA和野生型IL-15的偶联脂肪酸链模式IL-15-781-FA均具有较低的毒性，在小鼠中给药剂量达到10mg/kg，一周两次剂量，持续14天的注射情况下，小鼠体重都没有下降，也未见其他明显异常，但是在动物体内抗肿瘤实验中，IL-15-1303-FA比IL-15-781-FA具备更优的抗肿瘤效果。
附图说明
图1.含有新加入二硫键T24C，E93C的IL-15突变体模拟结构示意图；
图2.IL-15突变体-Fc融合蛋白变复性后经亲和层析的蛋白检测SDS-PAGE胶图；黑色箭头处为正确分子量；
图3.含有新加入二硫键的IL-15-1303和野生型IL-15-781经过亲和层析后样品的UPLC-反相分析图；
图4.含有新加入二硫键的IL-15-1303-FA和野生型IL-15-781-FA的质谱分析图；
图5.PBS、IL-15-1303-FA、IL-15-781-FA给药后小鼠体重监测实验；
图6.PBS、IL-15-1303-FA、IL-15-781-FA给药后小鼠的MC38肿瘤体积检测结果。
具体实施方式
下文将结合具体实施例对本发明的技术方案做更进一步的详细说明。应当理解，下列实施例仅为示例性地说明和解释本发明，而不应被解释为对本发明保护范围的限制。凡基于本发明上述内容所实现的技术均涵盖在本发明旨在保护的范围内。
除非另有说明，以下实施例中使用的原料和试剂均为市售商品，或者可以通过已知方法制备。下列实施例中未注明具体条件的实验方法，通常按照常规条件如Sambrook等人，分子克隆：实验室手册(New York: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。
实施例1：IL-15新加入二硫键的设计方法
本发明首先对野生型IL-15中可能加入二硫键的位置进行了预测和计算，在设计这些新加入二硫键的位置时，充分考虑了突变氨基酸beta-碳原子之间的距离(一般小于0.6nm，见表1)，且突变的氨基酸侧链处于方向临近的构象上。本发明在设计新加入的二硫键时，还充分考虑了新加入二硫键对野生型IL-15已有的两对二硫键的影响，使新加入的二硫键尽量远离原有的二硫键。最终，综合上述考虑，该发明设计的三对新加入二硫键如表1所示。
表1新加入二硫键突变氨基酸beta-碳原子的距离
| 二硫键突变位点 | Beta-碳原子距离(单位：nm) |
| T24C，E93C | 0.41 |
| T27C,E92C | 0.56 |
| L15C,S58C | 0.57 |
实施例2.IL-15及其突变体质粒构建及蛋白表达
2.1、表达质粒构建
委托北京擎科生物科技有限公司合成IL-15及其突变体基因，包括wt-IL-15(SEQI D NO:2)，IL-15(T24C,E93C)(SEQ ID NO:3)，IL-15-781(SEQ ID NO:4)，IL-15-1303(SEQID NO:5)，IL-15(T27C,E92C)-Fc(SEQ ID NO:8)，IL-15(T24C,E93C)-Fc(SEQ ID NO:9)，IL-15(L15C,S58C)-Fc(SEQ ID NO:10)，wtIL-15-Fc(SE Q ID NO:11)。按照《分子克隆》中的操作方法，进行重叠PCR得到目的片段。然后进行片段和通用载体pET41a的重组连接、转化、测序和保菌，从而得到能够表达相应蛋白的质粒。
2.2、质粒提取
按照《Qiagen Mini-prep Kit》中的操作方法，制备含wt-IL-15(SEQ ID NO:2)，IL-15(T24C,E93C)(SEQ ID NO:3)，IL-15-781(SEQ ID NO:4)，IL-15-1303(SEQ ID NO:5)，IL-15(T27C,E92C)-Fc(SEQ ID NO:8)，IL-15(T24C,E93C)-Fc(SEQ ID NO:9)，IL-15(L15C,S58C)-Fc(SEQ ID NO:10)，wtIL-15-Fc(SEQ ID NO:11)的质粒。
2.3、大肠杆菌的表达
将2.2构建成功的质粒转化入大肠杆菌表达菌株BL21(DE3)中，挑取单克隆进行过夜培养，作为种子，将种子按1:50的体积比转接到含Kana抗性的500mL TB培养基的三角瓶中，初始OD600大约为0.1，37℃，220rpm，培养至OD600为2.0，加入IPTG(终浓度为0.5mmol/L)，37℃、220rpm、4h培养后收菌。使用SDS-PAGE分析蛋白表达情况，所有蛋白均以包涵体(inclusion body，IB)的形式成功表达，且表达量较高。
实施例3.IL-15及其突变体蛋白的制备过程
本发明将实施2中构建好的蛋白的质粒转化至大肠杆菌宿主工程菌中进行表达，表达产生不可溶性包涵体。经菌体高压破碎，洗涤后，回收包涵体。
3.1蛋白制备
取上述IL-15及其突变体蛋白的包涵体，溶解在8M尿素溶液中，加入一定量的DTT，变性一小时后，用水稀释，室温过夜，抽取样品进行检测，同时通过亲和层析法或离子交换层析发进行纯化，获得目的蛋白。蛋白纯化的具体信息如下表所示(表2)。
表2该发明中IL-15及其突变蛋白制备信息表
| 蛋白名称 | 蛋白序列 | 纯化后蛋白浓度 | 蛋白纯度 |
| wt-IL-15 | SEQ ID NO:2 | 1mg/ml | 95％ |
| IL-15(T24C,E93C) | SEQ ID NO:3 | 1.2mg/ml | 95％ |
| IL-15-781 | SEQ ID NO:4 | 3mg/ml | 95％ |
| IL-15-1303 | SEQ ID NO:5 | 1.6mg/ml | 95％ |
| IL-15(T27C,E92C)-Fc | SEQ ID NO:8 | 无法检测 | <10％ |
| IL-15(T24C,E93C)-Fc | SEQ ID NO:9 | 0.85mg/ml | 85％ |
| IL-15(L15C,S58C)-Fc | SEQ ID NO:10 | 无法检测 | <10％ |
| Wt-IL-15-Fc | SEQ ID NO:11 | 0.5mg/ml | 85％ |
3.2样品结果分析
该发明在制备IL-15及其突变体的Fc融合蛋白(SEQ ID NO:8，SEQ ID NO:9，SEQID NO:10，SEQ ID NO:11)时发现，在变复性过程中，添加一对二硫键的IL-15(T24C,E93C)-Fc(SEQ ID NO:9)分子表现较为优良的变复性优势，通过对复性后条带在胶图上染色密度对比，可以看出添加一对二硫键的IL-15(T24C,E93C)-Fc相比野生型wtIL-15-Fc，复性效率大大增加，如图2所示。但其它形式的IL-15突变体-Fc融合蛋白IL-15(T27C,E92C)-Fc和IL-15(L15C,S58C)-Fc在复性过程中产生多条带或弥散条带，且纯化后的蛋白没有主要条带出现，由此推断这两个分子由于引入的一对新二硫键位置并不合适，在复性中产生大量二硫键错配或分子错误折叠，因此，不能作为候选分子做进一步的开发。同时，还观察到wtIL-15-Fc在正确分子量处有两条分子量非常接近的条带出现，这表明wtIL-15-Fc在refolding中出现了异构体isomer，而IL-15(T24C,E93C)-Fc在正确分子量处没有发现异构体的出现。
同样的，本发明在制备wt-IL-15与IL-15(T24C,E93C)，IL-15-781与IL-15-1303均发现，二硫键T24C,E93C引入可以消除野生型IL-15在refolding过程中产生的异构体isomer(图3，以IL-15-781与IL-15-1303为例)，且refolding的效率亦有提升。异构体isomer属于IL-15生产过程中难以去除的杂质蛋白，需要使用有机溶剂(如乙腈等)加反相层析去除，因此在制药工业中会因环保等原因受到限制而无法实现大规模生产。二硫键T24C,E93C的引入，消除了IL-15生产过程中的异构体isomer的形成，使IL-15纯化不再使用反相层析，大大提高了IL-15的制备工艺。
基于上述发现，本发明对含有T24C,E93C位置二硫键的分子进行了进一步的研究。
实施例4.IL-15及突变体体外细胞活性实验
NK92,MO7E细胞的增殖实验是普遍应用的细胞水平测定白介素刺激免疫细胞活性的实验。因此，此处通过wtIL-15-Fc和IL-15(T24C,E93C)-Fc，以及IL-15-781和IL-15-1303对这两种细胞的增殖效果来验证人白介素15突变体-Fc融合蛋白的生物活性。具体实验步骤如下：
1)所有细胞株于37℃，5％CO2条件下培养于完全培养基。使用PBS洗涤细胞两次，洗掉细胞因子，然后用不含细胞因子的培养基重悬细胞，随后接种于96孔细胞培养板，每孔接种90μL，共5000个细胞继续培养，待用。
2)使用不含细胞因子的培养基配制10倍药物浓度，使得最后工作浓度为：a)MO7E细胞最高浓度为20μg/mL，3.16倍稀释比例，稀释9个浓度，每个药物浓度设置三个复孔；b)NK92细胞最高浓度为10μg/mL，5倍稀释比例，稀释9个浓度，每个药物浓度设置三个复孔，然后转移连续稀释样品各10μL至96孔细胞板的相应实验孔中。
3)将已加药的96孔板中的细胞置于37℃、5％CO2条件下继续培养72小时，之后进行CTG分析。
4)每孔加入与孔中液体等体积的室温CTG溶液(Promega，G7573)，震摇培养板5分钟使细胞裂解。
5)使用酶标仪读取冷光值，收集数据。
使用GraphPad Prism 7.0软件分析数据，利用非线性S曲线回归来拟合数据得出剂量-效应曲线，并由此计算EC50值。细胞存活率(％)＝(Lum待测药-Lum培养基对照)/(Lum溶剂对照-Lum培养基对照)×100％。具体数据见表3：
表3 wtIL-15-Fc和IL-15(T24C,E93C)-Fc，以及IL-15-781和IL-15-1303在NK92,MO7E细胞的增殖实验中的活性比较
通过体外细胞的增殖实验，如表3所示，在相同条件下，增加一对二硫键(T24C,E93C)的IL-15突变体-Fc融合蛋白IL-15(T24C,E93C)-Fc和融合-GS-LPETG-GSGGS-HHHHHH的IL-15-1303分子均比相应的野生型wtIL-15-Fc或者IL-15-781活性提高(以NK92细胞实验计算分别提高18倍和3倍)，表明二硫键的引入不仅没有影响IL-15的活力，甚至大大增强了其细胞活性。
实施例5.IL-15突变体的偶联脂肪酸链模式(IL-15-1303-FA)和野生型IL-15的偶联脂肪酸链模式(IL-15-781-FA)的制备过程
为了进一步验证IL-15(T24C,E93C)-Fc分子中二硫键对IL-15分子活性的提高，本发明在此基础上，使用Sortase-A酶促反应，在C端偶联脂肪酸链，同样也获得另一种长效的且活力增强的IL-15衍生物，其比野生型IL-15的偶联脂肪酸链模式更有生产优势。具体操作如下。
本发明将上述新增一对二硫键(T24C,E93C)的IL-15突变体IL-15(T24C,E93C)及野生型wt-IL-15,分别融合-GS-LPETG-GSGGS-HHHHHH，得到IL-15-1303(SEQ ID NO:5)和IL-15-781(SEQ ID NO:4)，这两个蛋白制备过程在实施例2中已有表述。
利用纯化后的IL-15-1303和IL-15-781，通过转肽酶Sortase-A酶催化的连接反应将N端带有GGG的脂肪酸链GGG-PEG2-Lys-(CH2)16-COOH分别与IL-15-1303或IL-15-781相连接。反应按照Sortase A:IL-15-1303或IL-15-781:脂肪酸链比例为1:6:30进行，反应缓冲液为50mM Tris-HCl,1mM CaCl2，150mM NaCl pH8.0，在室温下反应3小时后，进行纯化。纯化采用反相色谱C8或者离子交换法，将未偶联的IL-15-1303或IL-15-781、未反应的脂肪酸链以及偶联后的产物分开，最终产物经过UPLC检测纯度和LC-MS(图4)鉴定表明，IL-15突变体IL-15-1303和野生型IL-15-781已偶联上脂肪酸链，形成IL-15-1303-FA(SEQ ID NO:7)和IL-15-781-FA(SEQ ID NO:6)。
实施例6.IL-15突变体IL-15-1303-FA和IL-15-781-FA对MO7E和NK92细胞增殖的实验
为了进一步验证经过脂肪酸链修饰后IL-15-1303-FA和IL-15-781-FA的细胞学活性，本发明将这两个分子对MO7E和NK92细胞增殖的活性进行了测试，实验方法同实施例4中所述方法保持一致，具体结果见表4所示。
表4 IL-15-1303-FA和IL-15-781-FA对MO7E和NK92细胞增殖的实验
通过对脂肪酸链修饰后IL-15-1303-FA和IL-15-781-FA的MO7E和NK92增殖细胞学活性测试可得知，与实施例4中观察到的现象类似，二硫键T24C,E93C的引入同样可以增强脂肪酸链修饰后的IL-15的活性(提高大于20倍)。
实施例7.IL-15突变体IL-15-781，IL-15-1303，IL-15-781-FA和IL-15-1303-FA对IL-15受体亲和力的测试
在本发明的实施例2-6中，通过对IL-15不同突变体制备和细胞活性的测试表明，二硫键T24C,E93C的引入可以增强IL-15细胞学活性。为了研究二硫键T24C,E93C增强IL-15细胞学活性的机制，我们使用Biacore-SPR 8K对IL-15-781、IL-15-1303、IL-15-781-FA和IL-15-1303-FA对IL-15的受体IL-15-Rβγ-Fc的亲和力进行了测定，具体方法如下：
1)使用Protein-A芯片(GE healthcare)将带有IL-15-Rβγ-Fc固定在芯片表面，固定量为1000RU，使用的流动相为含有0.05％Tween20的PBS缓冲液。
2)流过不同浓度的上述IL-15突变体，并检测其响应值，使用的浓度范围包括0～50nM。
3)实验结束后，使用Biacore SPR 8K自带的数据处理软件分析数据，结果采用动力学拟合方法进行，具体数据见表5。结果表明IL-15在新引入的一对二硫键下(IL-15-1303和IL-15-1303-FA)，使其和受体IL-15-Rβγ的结合力加强，这很好的解释了前述二硫键T24C,E93C的引入可以增强IL-15的细胞活性的现象。
表5.IL-15突变体IL-15-1303及野生型IL-15-781体外与受体结合力的检测
实施例8.蛋白偶联脂肪酸链模式下的IL-15突变体IL-15-1303-FA及野生型IL-15-781-FA在小鼠体内的毒性实验
本实施例中使用的IL-15突变体IL-15-1303-FA及野生型IL-15-781-FA为实施例5中制备。6只6～8周龄的C57BL/6小鼠，随机分为3组，分别为试剂对照组(PBS)、野生型长效IL-15-781-FA组和长效IL-15突变体IL-15-1303-FA组，每组2只小鼠。通过小鼠尾静脉注射，一周两次，每次10mg/kg。给药后观察药物对实验动物造成的影响，以及给药后小鼠的体重变化，在实验终点，进行解剖，检查脏器有无肉眼可见病变。其中体重检测实验结果如图5所示。
如图5所示，和对照组相比，长效IL-15突变体IL-15-1303-FA具有明显的低毒性，在10mg/kg，一周两次剂量，持续14天的注射情况下，小鼠体重没有下降，未见其他明显异常。而Altor BioScience公司的ALT-803，报道在4mg/kg的情况下，小鼠体重已经出现明显下降，建议小鼠的治疗剂量为1mg/kg，一周一次，持续4周(DOI:10.1158/2326-6066.CIR-15-0093-T)。博际生物的BJ-001(DOI:10.1200/JCO.2021.39.15_s uppl.e14545 Journalof Clinical Oncology)，报道BJ-001目前在人体内最佳忍受剂量为6μg/kg。对比这些数据，本发明的长效IL-15突变体IL-15-1303-FA已具有非常低的毒性，这为将来的药物开发提供良好的基础。
实施例9.蛋白偶联脂肪酸链模式下的IL-15突变体IL-15-1303-FA及野生型IL-15-781-FA在小鼠皮下肿瘤模型中的抗肿瘤实验
本实施例中使用的IL-15突变体IL-15-1303-FA及野生型IL-15-781-FA为实施例5中制备，具体实验方法为：15只6～8周龄的C57BL/6雌性小鼠，随机分为3组，每组共5只小鼠，分别为试剂对照组(PBS)、野生型长效IL-15-781-FA组和长效IL-15突变体IL-15-1303-FA组。本实施例中选取的分子主要用于验证活性增强的IL-15-1303-FA分子对抗肿瘤效果的影响。该实验每组5只小鼠，在小鼠右侧肩部皮下按照5*10E5的细胞量接种MC38肿瘤细胞。接种肿瘤细胞后，第二天开始通过小鼠尾静脉注射PBS、IL-15-781-FA和IL-15-1303-FA，一周两次，给药剂量均为10mg/kg。给药后观察药物对实验动物造成的影响，以及给药后小鼠的肿瘤体积变化，在实验终点，进行解剖，检查脏器有无肉眼可见病变。每周使用游标卡尺对肿瘤体积进行2次测量并用电子天平称量小鼠体重，测量肿瘤的长径和短径，肿瘤体积计算公式为：肿瘤体积TV(mm3)＝0.5×长径(mm)×短径(mm)2。瘤重抑制率TGITW％＝(1－T/C)×100％，T/C＝治疗组瘤重均值/对照组瘤重均值。其中小鼠的肿瘤体积检测结果如图6所示。在整个药物给药期间，未观察到和药物相关的毒性，小鼠体重未见明显下降，解剖后脏器无肉眼可见病变，表明该实验中测试的IL-15-1303-FA和IL-15-781-FA均具有良好的安全性，但相对IL-15-781-FA而言，IL-15-1303-FA具备更强的抗肿瘤活性(IL-15-781-FA肿瘤抑制率TGITW为20％，而IL-15-1303-FA肿瘤抑制率TGITW为32％)。
以上，对本发明的实施方式进行了说明。但是，本发明不限定于上述实施方式。凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
序列表
<110> 北京志道生物科技有限公司
<120> 白细胞介素15蛋白突变体及其应用
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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 Cys 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
115
<210> 4
<211> 133
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
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 Gly Ser Leu Pro Glu Thr Gly Gly Ser Gly Gly Ser His
115 120 125
His His His His His
130
<210> 5
<211> 133
<212> PRT
<213> 人工序列(Artificial Sequence)
<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 Cys 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 Cys 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 Gly Ser Leu Pro Glu Thr Gly Gly Ser Gly Gly Ser His
115 120 125
His His His His His
130
<210> 6
<211> 124
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221> LIPID
<222> (124)..(124)
<223> PEG2-Lys-(CH2)16-COOH
<400> 6
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 Gly Ser Leu Pro Glu Thr Gly Gly Gly
115 120
<210> 7
<211> 124
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221> LIPID
<222> (124)..(124)
<223> PEG2-Lys-(CH2)16-COOH
<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 Cys 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 Cys 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 Gly Ser Leu Pro Glu Thr Gly Gly Gly
115 120
<210> 8
<211> 356
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 8
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 Cys 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 Cys 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 Gly Ala Pro Gln Gly Ala Pro Gln Gly Ala Pro Gln Gly
115 120 125
Ala Pro Gln Gly Ala Pro Gln Ala Gly Pro Cys Pro Ala Pro Glu Ala
130 135 140
Lys Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
145 150 155 160
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
165 170 175
Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
180 185 190
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Glu Ser
195 200 205
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
210 215 220
Gln Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser
225 230 235 240
Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
245 250 255
Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln
260 265 270
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
275 280 285
Val Glu Trp Glu Ser Gln Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
290 295 300
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu
305 310 315 320
Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser
325 330 335
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
340 345 350
Leu Ser Leu Gly
355
<210> 9
<211> 356
<212> PRT
<213> 人工序列(Artificial Sequence)
<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 Cys 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 Cys 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 Gly Ala Pro Gln Gly Ala Pro Gln Gly Ala Pro Gln Gly
115 120 125
Ala Pro Gln Gly Ala Pro Gln Ala Gly Pro Cys Pro Ala Pro Glu Ala
130 135 140
Lys Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
145 150 155 160
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
165 170 175
Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
180 185 190
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Glu Ser
195 200 205
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
210 215 220
Gln Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser
225 230 235 240
Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
245 250 255
Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln
260 265 270
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
275 280 285
Val Glu Trp Glu Ser Gln Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
290 295 300
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu
305 310 315 320
Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser
325 330 335
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
340 345 350
Leu Ser Leu Gly
355
<210> 10
<211> 356
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 10
Met Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Cys
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 Cys 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 Gly Ala Pro Gln Gly Ala Pro Gln Gly Ala Pro Gln Gly
115 120 125
Ala Pro Gln Gly Ala Pro Gln Ala Gly Pro Cys Pro Ala Pro Glu Ala
130 135 140
Lys Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
145 150 155 160
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
165 170 175
Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
180 185 190
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Glu Ser
195 200 205
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
210 215 220
Gln Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser
225 230 235 240
Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
245 250 255
Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln
260 265 270
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
275 280 285
Val Glu Trp Glu Ser Gln Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
290 295 300
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu
305 310 315 320
Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser
325 330 335
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
340 345 350
Leu Ser Leu Gly
355
<210> 11
<211> 356
<212> PRT
<213> 人工序列(Artificial Sequence)
<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 Gly Ala Pro Gln Gly Ala Pro Gln Gly Ala Pro Gln Gly
115 120 125
Ala Pro Gln Gly Ala Pro Gln Ala Gly Pro Cys Pro Ala Pro Glu Ala
130 135 140
Lys Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
145 150 155 160
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
165 170 175
Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
180 185 190
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Glu Ser
195 200 205
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
210 215 220
Gln Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser
225 230 235 240
Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
245 250 255
Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln
260 265 270
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
275 280 285
Val Glu Trp Glu Ser Gln Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
290 295 300
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu
305 310 315 320
Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser
325 330 335
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
340 345 350
Leu Ser Leu Gly
355
Claims (11)
1.一种人白细胞介素15蛋白IL-15突变体，其特征在于，所述的突变体为在天然野生型IL-15的氨基酸序列中，第21位至29位之间任意选取一个氨基酸突变为Cys，在第91位至96位之间任意选取一个氨基酸突变为Cys，并在两个突变的Cys之间形成二硫键，所述天然野生型IL-15的氨基酸序列如SEQ ID NO.1所示；同时，在SEQ ID NO.1的N端添加一个甲硫氨酸，将第78位的Gly突变为Ala；
优选地，所述的突变体为：将天然野生型IL-15的氨基酸序列的第24位Thr、第93位Glu，都突变为Cys，并在两个突变的Cys之间形成二硫键，所述IL-15突变体的氨基酸序列如SEQID NO.3所示。
2.一种人白介素15突变体-Fc的融合蛋白，其特征在于，所述融合蛋白的人白介素15突变体为权利要求1所述的IL-15突变体，Fc片段包括部分铰链区、CH2区和CH3区，所述IL-15突变体与Fc序列之间为直接融合或通过连接序列融合，所述的Fc片段选自人或动物的IgG、IgM、IgD、IgA或者它们的亚型；
优选地，所述融合蛋白的Fc片段选自人IgG4序列；
进一步优选地，所述融合蛋白选自氨基酸序列如SEQ ID NO.8所示的IL-15(G78A,T27C,E92C)-Fc，氨基酸序列如SEQ ID NO.9所示的IL-15(G78A,T24C,E93C)-Fc，或氨基酸序列如SEQ ID NO.10的IL-15(G78A,L15C,S58C)-Fc；
更进一步优选地，所述融合蛋白的氨基酸序列如SEQ ID NO.9所示，其中的第24位、第93位都是突变为Cys，并在两个突变的Cys之间形成二硫键。
3.一种能够表达权利要求1所述的IL-15突变体或权利要求2所述的融合蛋白的载体；
优选地，所述载体为原核载体，进一步优选地，所述原核载体为通用载体pET41a。
4.一种包含权利要求3所述载体的宿主细胞；
优选地，所述细胞选自真核细胞或原核细胞；进一步优选地，所述细胞为大肠杆菌。
5.一种人白细胞介素15蛋白突变体的衍生物，其特征在于，所述衍生物为权利要求1所述的IL-15突变体的C端偶联脂肪酸链，所述脂肪酸链为：(1)通式为GGG-linker-(CH2)X-COOH，或，(2)其它含有脂肪酸链的化学结构，其中(1)通式中的linker部分包括但不仅限于PEG-linker(聚乙二醇)、多肽链、OEG等，(CH2)X中x的数目介于10-20之间；优选的，x介于16-20之间；
进一步优选的，所述偶联的脂肪酸链包括：
GGG-PEG2-Lys-(CH2)16-COOH、
NHS-PEG2-PEG2-γ-Glu-(CH2)17-COOH、
GGG-PEG4-PEG4-PEG4-Lys-(CH2)17-COOH、
GGG-PEG4-γ-Glu-γ-Glu-Lys-(CH2)17-COOH、
HOOC-(CH2)16-γ-Glu-γ-Glu-Lys-GGG、
HOOC-(CH2)16-CONH-γ-Glu-γ-Glu-PEG2-Lys-Br、
GGG-γ-Glu-C2DA-2OEG-γ-Glu-(CH2)17-COOH、
GGG-γ-Glu-C2DA-2OEG-γ-Glu-(CH2)19-COOH、
GGG-OEG-C2DA-2OEG-γ-Glu-(CH2)19-COOH、
GGG-OEG-C2DA-2OEG-γ-Glu-Trx-(CH2)19-COOH、
CHO-PEG2-PEG2-γ-Glu-(CH2)17-COOH或
Mal-C2DA-2OEG-γ-Glu-Tn-(CH2)19-COOH任意一种；
优选地，所述人白细胞介素15蛋白突变体的衍生物是通过在IL-15突变体的C末端先添加含有LPXTG的多肽，所述X为任意氨基酸，然后偶联脂肪酸链；
进一步优选的，所述先添加的多肽序列为-GS-LPETG-GSGGS-HHHHHH，选用的偶联脂肪酸链结构为GGG-PEG2-Lys-(CH2)16-COOH获得修饰后的序列为氨基酸序列如SEQ ID NO.7所示的IL-15-1303-FA。
6.一种C端偶联脂肪酸链的IL-15突变体的制备方法，其特征在于，所述方法通过在IL-15或其突变体的C末端添加含有LPXTG(X为任意氨基酸)实现，优选的，添加的序列为-GS-LPETG-GSGGS-HHHHHH，含有此序列的IL-15突变体为氨基酸序列如SEQ ID NO.5所示的IL-15-1303，IL-15-1303通过Sortase-A酶的催化，被权利要求5所述的脂肪酸链修饰偶连，修饰后的IL-15突变体的结构为：IL-15突变体-LPXT-GGG-脂肪酸链；
优选地，选用的脂肪酸链结构为GGG-PEG2-Lys-(CH2)16-COOH，修饰后的序列为氨基酸序列如SEQ ID NO.7所示的IL-15-1303-FA。
7.一种药物组合物，其特征在于，所述组合物包含权利要求1所述的人白细胞介素15蛋白IL-15突变体、或权利要求2所述的人白介素15突变体-Fc融合蛋白、或权利要求5所述的人白细胞介素15蛋白突变体的衍生物和药学上可接受的赋形剂、稀释剂或载体。
8.一种用于刺激或抑制哺乳动物免疫应答的方法，其特征在于，包括：向所述哺乳动物给予有效量的权利要求1所述的人白细胞介素15蛋白突变体、或权利要求2所述的人白介素15突变体-Fc融合蛋白、或权利要求5所述的人白细胞介素15蛋白突变体的衍生物、或权利要求7所述的药物组合物。
9.一种权利要求1所述的人白细胞介素15蛋白突变体、或权利要求2所述的人白介素15突变体-Fc融合蛋白、或权利要求5所述的人白细胞介素15蛋白突变体的衍生物、或权利要求7所述的药物组合物在制备用于治疗IL-15介导的疾病或病症的药物中的用途，其特征在于，其中所述的疾病可以为传染病、癌症、血液病和自身免疫性疾病。所述的癌症优选黑色素瘤、结直肠癌、皮肤癌、淋巴瘤、肾细胞癌、实体瘤、肝癌、肺癌、胃癌、乳腺癌；所述的传染病优选天花病毒感染、HIV感染、细菌感染、真菌感染、HBV感染；所述的血液病优选贫血、急性髓系白血病、骨髓增生异常综合征、T-细胞大颗粒淋巴细胞性白血病；所述的自身免疫性疾病优选多发性硬化症、银屑病、风湿性关节炎、炎性疾病、胃炎、黏膜炎；所述的人白细胞介素15蛋白突变体、或人白介素15突变体-Fc融合蛋白、或人白细胞介素15蛋白突变体的衍生物、或药物组合物可以单独使用，也可以与其它药物联合使用，所述其它药物为小分子抑制剂或抗体类药物，所述的小分子抑制剂优选靶向化疗药物或放射治疗药物，进一步优选烷化剂；所述的抗体类药物优选单克隆抗体药物，进一步优选抗CD20、PD1、PDL1、Her2、EGFR、c-MET抗体。
10.一种治疗或预防疾病的方法，其特征在于，所述方法包括但不仅限于药物化疗、放射性治疗、手术治疗等方式；在所述疾病中，细胞表达疾病相关抗原，所述方法包括：向患者施予权利要求1所述的人白细胞介素15蛋白突变体、或权利要求2所述的人白介素15突变体-Fc融合蛋白、或权利要求5所述的人白细胞介素15蛋白突变体的衍生物、或权利要求7所述的药物组合物；在表达疾病相关抗原的细胞与表达IL-15Rα的免疫细胞之间形成足以活化所述免疫细胞的特异性结合复合物；以及通过所述免疫细胞杀死所述表达疾病相关抗原的细胞；其中所述表达疾病相关抗原的细胞优选为肿瘤细胞或病毒感染细胞；其中所述的免疫细胞优选为T-细胞、LAK细胞或NK细胞；其中所述的疾病可以为传染病、癌症、血液病和自身免疫性疾病；所述的癌症优选黑色素瘤、结直肠癌、皮肤癌、淋巴瘤、肾细胞癌、实体瘤、肝癌、肺癌、胃癌、乳腺癌；所述的传染病优选天花病毒感染、HIV感染、细菌感染、真菌感染、HBV感染；所述的血液病优选贫血、急性髓系白血病、骨髓增生异常综合征、T-细胞大颗粒淋巴细胞性白血病；所述的自身免疫性疾病优选多发性硬化症、银屑病、风湿性关节炎、炎性疾病、胃炎、黏膜炎。
11.一种联合用药治疗或预防疾病的方法，其特征在于，包括向患者施予权利要求1所述的人白细胞介素15蛋白突变体、或权利要求2所述的人白介素15突变体-Fc融合蛋白、或权利要求5所述的人白细胞介素15蛋白突变体的衍生物、或权利要求7所述的药物组合物，并联合施用其它药物，如小分子抑制剂或抗体类药物；所述的小分子抑制剂优选靶向化疗药物或放射治疗药物，更优选烷化剂；所述的抗体类药物优选单克隆抗体药物，更优选抗CD20、PD1、PDL1、Her2、EGFR、c-MET抗体。
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