CN114573707A - 一种用于抗肿瘤的新型il-15融合蛋白 - Google Patents
一种用于抗肿瘤的新型il-15融合蛋白 Download PDFInfo
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- CN114573707A CN114573707A CN202011363875.XA CN202011363875A CN114573707A CN 114573707 A CN114573707 A CN 114573707A CN 202011363875 A CN202011363875 A CN 202011363875A CN 114573707 A CN114573707 A CN 114573707A
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Abstract
本发明公开了一种新型IL‑15融合蛋白，至少包括(1)IL‑15Rα多肽，(2)IL‑15多肽变体，(3)Fc结构域、Fc结构域衍生物或其功能性片段，(4)连接子或其衍生物，IL‑15多肽变体至少包括第一连接子或其衍生物，所述第一连接子或其衍生物连接IL‑15多肽的天然氨基端和羧基端。本发明的肿瘤治疗新型IL‑15融合蛋白，能够提高IL‑15的抗肿瘤功效。此外这种肿瘤治疗重组蛋白可以被高效地表达并纯化，为IL‑15治疗性蛋白产业化奠定基础，具有成为有效的免疫抗肿瘤疗法药物的巨大潜力。
Description
技术领域
本发明涉及分子生物学领域，尤其涉及一种用于抗肿瘤的新型IL-15融合蛋白。
背景技术
人白细胞介素-15(IL-15)基因位于染色体4q31，跨度约34kb，有7个内含子和9个外显子，其mRNA长1.5～1.8kb。IL-15是一种相对分子量约为12-14kD的糖基化细胞因子。IL-15的前体分子由162个氨基酸组成，先导序列较长，为48个氨基酸残基。其成熟蛋白含有114个氨基酸，包括4个半胱氨酸残基，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。
包括IL-15在内的细胞因子都具有广泛的免疫调节活性，IL-15作为一个有前途的抗肿瘤靶点已经被广泛研究。IL-15与IL-2虽然在一级结构上同源性很低，但二者在三级结构即空间构象上很相似，他们拥有共有的β和γ受体，但结合到不同的α受体。IL-15的作用主要是通过反式递呈方式，即IL-15与表达于抗原递呈细胞表面的IL-15Rα结合，然后与临近效应细胞表面的IL-15Rβγ复合物结合，最终激活效应细胞。IL-15能够参与调节多种免疫细胞的存活、增殖与功能，其中包括NK细胞、记忆性CD8+T细胞、DC细胞及巨噬细胞等。同时，与IL-2不同的是，IL-15不会导致激活诱导的细胞凋亡(AICD)，也不激活对免疫具有广泛抑制作用的调节性T细胞(Tregs)等优点，因此IL-15具有巨大的治疗潜力。
IL-15与其α受体有很高的亲和力，生理状态下IL-15大多与其α受体形成复合物，并增强IL-15对β和γ受体的亲和力，激活T细胞和NK细胞。研究表明，IL-15与可溶性IL-15Rα受体形成的复合物在促进记忆性CD8+T淋巴细胞和NT/NKT细胞增殖方面明显优于单独IL-15。因此，Cytune Pharma，Altor BioScience，博际生物医药和恒瑞医药等公司，用融合或非融合的方法将IL-15与其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。
尽管IL-15是肿瘤免疫治疗领域最有希望的候选药物之一，但是IL-15仍然有一定的不足。首先，天然的IL-15由于表达量低、纯化困难的制约，难以进行产业化，存在明显的药物开发瓶颈。因此，有必要对其分子结构进行重新设计以获得高表达和高稳定性的突变体。IL-15的作用是全身性，而不是肿瘤特异性的，易引起全身免疫副作用。
因此，本领域的技术人员致力于开发一种高活性、高稳定性的新型IL-15融合蛋白。
发明内容
有鉴于现有技术的上述缺陷，本发明所要解决的技术问题是如何提高IL-15在体内的抗肿瘤活性。
为实现上述目的，本发明提供了一种新型IL-15融合蛋白，其特征在于，至少包括(1)IL-15Rα多肽、IL-15Rα多肽衍生物或其功能性片段，(2)IL-15多肽变体，(3)Fc结构域、Fc结构域衍生物或其功能性片段，(4)连接子或其衍生物，
其中，IL-15多肽变体至少包括第一连接子或其衍生物，第一连接子或其衍生物连接IL-15多肽的天然氨基端和羧基端形成环状体，并且选取N77，G78，N79，V80，T81，E82，S83，和G84组成组中的一个氨基酸残基后面的肽键作为断裂点，形成新氨基端和羧基端。
进一步的，各组分的排列顺序为IL-15Rα多肽-第二连接子-IL-15多肽变体-Fc结构域。
进一步的，各组分的排列顺序为第三连接子-Fc结构域-第二连接子-IL-15Rα多肽-IL-15多肽变体。
进一步的，第二连接子与IL-15Rα多肽或IL-15多肽变体共价连接。
进一步的，第一连接子为SEQ ID NO.1所示序列，或该序列经过取代、添加或缺失一个或多个氨基酸的衍生序列。
进一步的，第一连接子为SEQ ID NO.2所示序列，或该序列经过取代、添加或缺失一个或多个氨基酸的衍生序列。
进一步的，第三连接子为SEQ ID NO.3所示序列，或该序列经过取代、添加或缺失一个或多个氨基酸的衍生序列。
进一步的，第二连接子为SEQ ID NO.4所示序列，或该序列经过取代、添加或缺失一个或多个氨基酸的衍生序列。
进一步的，IL-15多肽变体的氨基酸序列如SEQ ID NO.5所示，或SEQ ID NO.5所示序列经过取代、添加或缺失一个或多个氨基酸且具有IL-15多肽相同相似功能的衍生序列。
进一步的，IL-15多肽变体的氨基酸序列如SEQ ID NO.6所示，或SEQ ID NO.6所示序列经过取代、添加或缺失一个或多个氨基酸且具有IL-15多肽相同相似功能的衍生序列。
进一步的，Fc结构域的序列如SEQ ID NO.7所示，或SEQ ID NO.7所示序列经过取代、添加或缺失一个或多个氨基酸且具有Fc结构域相同相似功能的衍生序列。
进一步的，Fc结构域或其衍生序列选自人类IgG1 Fc结构域、人类IgG2 Fc结构域、人类IgG3 Fc结构域、人类IgG4 Fc结构域所成群组的Fc结构域。
进一步的，IL-15Rα多肽的序列SEQ ID NO.8所示，或SEQ ID NO.8所示序列经过取代、添加或缺失一个或多个氨基酸且具有IL-15Rα相同相似功能的衍生序列
本发明另一方面还提供一种核酸序列，编码上述IL-15融合蛋白。
进一步的，包括操作性连结至编码IL-15融合蛋白的序列的启动子、转录起始信号及前导序列。
本发明还提供一种DNA载体，包括含有上述的核酸序列。
本发明提供一种宿主，包括含有如权利要求16的核酸序列。
本发明另一方面提供一种制备如上述的IL-15融合蛋白的方法，包括如下步骤：
(1)提供上述的核酸序列，
(2)收获包含步骤(1)中的核酸序列的质粒，
(3)将步骤(2)中的质粒转入细胞中，
(4)IL-15融合蛋白在细胞中表达。
进一步的，步骤(3)的详细步骤为：
(3.1)试剂制备
A)制备G418溶液：称取250mg GeneticinTM加入4.5mL超纯水溶解，超纯水定容至5mL，0.22μm滤膜过滤，-20℃保存，
B)制备PEI溶液：称取50mg PEI加入45mL超纯水溶解，1M NaOH调节pH至7.0，超纯水定容至50mL，0.22μm滤膜过滤，-20℃保存，
C)制备培养基：在1L FreeStyleTM 293Expression Medium中加入10mLPluronicdTM F-68和500μL G418，
D)质粒提前准备在2mL去内毒素离心管中，
E)根据转染需要体积准备好新鲜传代至(1-1.2)×10^6个/mL的细胞悬液，
(3.2)配制转染试剂-质粒复合物
将1μg/mL质粒与33.3μL/mL Opti-MEMTM混合得到A液，
将2μg/mL PEI与33.3μL/mL Opti-MEMTM混合得到B液，
将B液倒入A液混匀孵育10min后，加入细胞悬液，
(3.3)换液转染
在115rpm，36.8℃，5％CO2培养4h后，800g 5min离心，换成未添加F68和G418的FreeStyleTM293 Expression Medium。
进一步的，步骤(4)具体为：在115rpm，36.8℃，5％CO2培养5天后，8500rpm离心15min，收集细胞上清，纯化IL-15融合蛋白。
进一步的，根据Fc结构域标签，先后通过亲和纯化和凝胶过滤层析去除聚合体得到IL-15融合蛋白。
进一步的，细胞为哺乳动物细胞，选自HEK293或CHO细胞。
本发明还提供一种使用上述的IL-15融合蛋白制造在患者中用以抗肿瘤的药物的用途。
进一步的，肿瘤为进行性肿瘤、晚期肿瘤或转移性肿瘤。
本发明还提供一种药物组合物，包含上述的IL-15融合蛋白以及药学上可接受的辅料。
进一步的，上述药物组合物在制备治疗肿瘤药物中的应用。
本发明另一方面提供一种药物组合物，包含上述的IL-15融合蛋白以及另一种抗癌剂。
进一步的，上述药物组合物在制备治疗肿瘤药物中的应用。
本发明还提供一种试剂，包含上述的IL-15融合蛋白、上述的核酸序列、上述的表达载体或者上述的宿主。
进一步的，还包含溶解上述的IL-15融合蛋白、上述的核酸序列、上述的表达载体或者上述的宿主的缓冲液。
技术效果
本发明基于结构，将IL-15蛋白进行重新切割和重组，并在合适的位置融合IL-15Rα受体及Fc结构域，形成的复合物分子可以提高蛋白表达量，分子稳定性，同时也简化了生产过程。两个肿瘤治疗重组蛋白IL-15 M1和IL-15 M2具有全新的，改进或增强与天然受体的类似的蛋白相关的生物功能或活性。这些改进包括但不限于增加结合亲和力、增加活性、增加活性部位的柔韧性、增加稳定性，改进的药代动力学参数，改进的物理性质，以及它们的组合。
一方面能够提高IL-15的抗肿瘤功效，主要是通过促进T细胞的增殖，另一方面能够有效延长IL-15半衰期。此外这种肿瘤治疗重组蛋白可以被高效地表达并纯化，为IL-15治疗性蛋白产业化奠定基础，具有成为有效的免疫抗肿瘤疗法药物的巨大潜力。
以下将结合附图对本发明的构思、具体结构及产生的技术效果作进一步说明，以充分地了解本发明的目的、特征和效果。
附图说明
图1是本发明实施例的IL-15Rα-IL-15变体的三维结构示意图；
图2a，2b是本发明实施例的重组蛋白IL-15 M1和IL-15 M2的示意图；
图3是本发明实施例的IL-15 M1和IL-15 M2在HEK293中表达上清液的SDS-PAGE电泳；其中，还原为上样缓冲液中加入还原剂β-巯基乙醇，非还原为不加还原剂；
图4是本发明实施例中纯化后的IL-15 M1和IL-15 M2的SDS-PAGE电泳；其中，还原为上样缓冲液中加入还原剂β-巯基乙醇，非还原为不加还原剂；
图5是本发明实施例中用qPCR的方法比较IL-15 M1，IL-15M2与RDB1408的热稳定性。
具体实施方式
以下参考说明书附图介绍本发明的多个优选实施例，使其技术内容更加清楚和便于理解。本发明可以通过许多不同形式的实施例来得以体现，本发明的保护范围并非仅限于文中提到的实施例。
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(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。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。
本文中，“IL-15Rα”是指白介素-15受体α，也称为“α受体亚基”；“IL-15Rβ”是指白介素-15受体β，也称为“β受体亚基”；“IL-15Rβγ”白介素-15受体β和受体γ形成的复合物，也称为“β和γ受体亚基复合物”。
在本发明中，“IL-15 M1”为M1型IL-15融合蛋白，各组分的排列顺序为IL-15Rα多肽-第二连接子-IL-15多肽变体-Fc结构域。“IL-15 M2”为M2型IL-15融合蛋白，各组分的排列顺序为第三连接子-Fc结构域-第二连接子-IL-15Rα多肽-IL-15多肽变体。
IL-15 M1和IL-15 M2的各组分可以被替代为它们的衍生物。术语“衍生物”是指与母体物质如IL-15Rα多肽、Fc结构域具有至少92.5％，优选的至少95％，更优选的至少98.5％或至少99％同一性百分数的氨基酸序列。
“第一连接子”连接天然IL-15多肽的氨基端和羧基端。“第二连接子”使得IL-15与IL-15Rα能够相互作用形成复合物，在一些实施例中，IL-15或者IL-15Rα，Fc结构域通过第二连接子合理的定位从而发挥每个结构域的功能活性。第二连接子通常为4-20个氨基酸构成的短肽。“第三连接子”可以引导融合的蛋白大分子药物在肿瘤组织部位选择性富集，增加局部浓度来增强肿瘤杀伤作用并限制全身性毒性。
现有技术中，为了能够使IL-15稳定或增强和βγ受体的结合，都是将天然的IL-15与α受体以共价或非共价方式进行融合或共表达。
本发明的原理是通过将IL-15的天然氨基端和羧基端用第一连接子连接，并在IL-15的C螺旋和D螺旋连接loop处进行切割和重组，从而产生新的氨基和羧基末端。新的融合蛋白的一级序列被重新排布，而二三级结构和蛋白活性被保留或增强。在非C螺旋和D螺旋连接loop处进行切割和重组，不能形成稳定的蛋白结构。由于新的氨基端相对于天然的氨基端可以为IL-15Rα蛋白的融合提供更好的折叠空间，所以用第二连接子将IL-15Rα融合在新的氨基端，进而形成结构稳定的复合物，即IL-15Rα-IL-15变体融合蛋白，如图1所示。
实施例1
在IL-15 M1实施方式中，是通过将IL-15的天然氨基端和羧基端用第一连接子如SEQ ID NO.1所示序列进行连接，并在IL-15的E82残基后面的肽键处进行切割，从而产生新的氨基和羧基末端。
将IL-15Rα多肽通过第二连接子融合连接到IL-15多肽变体的新的氨基端，之后将该新的融合蛋白再直接融合到hIgG1 Fc结构域的氨基端，如图2a所示。本领域技术人员能够根据本发明给出的教导以及现有技术设计出其它满足本发明的IL-15 M1变体，如Fc结构域-IL-15Rα多肽-第二连接子-IL-15多肽变体。
实施例2
在IL-15 M2实施方式中，是通过将IL-15的天然氨基端和羧基端用第一连接子如SEQ ID NO.2所示序列进行连接，并在IL-15的E82残基后面的肽键处进行切割，从而产生新的氨基和羧基末端。
将IL-15Rα多肽直接融合连接到IL-15多肽变体的新的氨基端，之后将该新的融合蛋白通过第二连接子如SEQ ID NO.4所示序列融合到hIgG1 Fc结构域的羧基端并在Fc结构域的氨基端融合第三连接子SEQ ID NO.3所示序列，如图2b所示。
实施例3
将IL-15 M1和IL-15 M2分别进行基因合成及表达，并依靠分子Fc结构域标签进行纯化和制备。断裂点为E82残基后面的肽键。表达宿主为哺乳动物细胞(HEK293或CHO)。
i.表达质粒合成
委托苏州金唯智生物科技有限公司合成编码IL-15 M1和IL-15 M2的基因，然后按照《分子克隆实验指南》提到的操作方法，进行转化DH10B、测序和保菌，从而得到相应的质粒。
ii.质粒提取及HEK293细胞准备
ii-1质粒提取
按照《分子克隆实验指南》提到的操作方法，转化DH10B、测序、保菌和菌液培养。按照《Qiagen Mini-prep Kit》和《Qiagen Endofree Maxi-prep Kit》提到的操作方法，进行IL-15 M1和IL-15 M2的质粒的制备。
ii-2HEK293细胞准备
新鲜传代的密度为1-1.2×10^6/mL的HEK293细胞(National Research Council，Canada)用于瞬时表达。
iii.HEK293瞬时表达
iii-1试剂制备
A)G418溶液：称取250mg GeneticinTM加入4.5mL超纯水溶解，超纯水定容至5mL，0.22μm滤膜过滤，-20℃保存；
B)PEI溶液：称取50mgPEI加入45mL超纯水溶解，1M NaOH调节pH至7.0，超纯水定容至50mL，0.22μm滤膜过滤，-20℃保存；
C)培养基：在1L FreeStyleTM 293Expression Medium中加入10mL PluronicdTMF-68和500μL G418；
D)质粒提前准备在2mL去内毒素离心管中；
E)根据转染需要体积准备好新鲜传代至1-1.2×10^6个/mL的细胞悬液。
iii-2配制转染试剂-质粒复合物
A液：质粒1μg/mL+Opti-MEMTM 33.3μL/mL
B液：PEI 2μg/mL+Opti-MEMTM 33.3μL/mL
将B液倒入A液混匀孵育10min后，加入细胞悬液。
iii-3转染换液
在115rpm，36.8℃，5％CO2培养4h后，800g 5min离心，换成未添加F68和G418的FreeStyleTM293 Expression Medium
iv.表达培养和收获
在115rpm，36.8℃，5％CO2培养5天后，离心8500rpm 15min，收集细胞上清。按照《分子克隆实验指南》描述的方法进行SDS-PAGE分析。
v.结果
如图3所示，表明质粒转染细胞后，表达生成了IL-15 M1和IL-15 M2重组蛋白，而且表达量较高。
实施例4
由于IL-15 M1和IL-15 M2重组蛋白都带有Fc结构域标签，因此利用Mabselectsure(Protein A,GE healthcare)进行亲和纯化，纯化方法可见纯化填料说明书；然后再经过凝胶过滤层析(superdex200，GE Healthcare)进行进一步纯化得到纯度较高的蛋白，并去除聚合体。按照《分子克隆实验指南》描述的方法进行SDS-PAGE分析，利用高效液相色谱分子筛柱子进行纯度分析。
结果如图4所示，表明两个融合蛋白纯化成功，且纯化后的融合蛋白纯度较好，聚合物和片段整体要小于5％。
实施例5
通过生物膜干涉技术(biolayer interferomeory，BLI)测定IL-15 M1和IL-15 M2融合蛋白与IL-15Rα，IL-15Rβ和IL-15Rβγ的亲和力
(1)实验材料
实验所用蛋白均为北京志道生物科技有限公司生产，其中IL-15Rβ-His，IL-15 Rβ-Fc，IL-15Rβγ-Fc，RLI-15(IL-15激活剂)是通过HEK293瞬时表达以及亲和纯化后获得，IL-15 WT是通过E.coli包涵体表达及变复性纯化后获得。缓冲液配方：10mM HEPES，150mM氯化钠，3mM EDTA，0.1％BSA和0.05％吐温20；ProA传感器(Pall ForteBio公司，货号#18-5010)；AR2G传感器(Pall ForteBio公司，货号#18-5092)；BLI设备为Pall ForteBio公司生产的Octet RED96；数据获取和分析工作分别采用Data acquisition 11.0和Dataanalysis 11.0软件进行。
(2)实验方法
1)IL-15Rβ-His，IL-15Rβ-Fc和IL-15Rβγ-Fc准备
用缓冲液稀释IL-15Rβ-His，IL-15Rβ-Fc和IL-15Rβγ-Fc至浓度10μg/mL，加入96孔测定板的1列中，控制程序中设为Loading，600s。
2)样品准备
用缓冲液将IL-15 M1，IL-15 M2，IL-15 WT，RLI-15和RDB1408,RDB1408的序列如SEQ ID NO.9所示(都稀释到200nM，然后1：1向下系列稀释7个梯度(共8个梯度)至浓度为3.125nM以及0浓度，分别加入96孔测定板，控制程序中设定为Association，200s。在96孔测定板加入4列缓冲液，1列加入pH1.7的甘氨酸，上述样品和溶液的加样量均为200μL。
4)检测与IL-15Rβ-His的亲和力
取8个AR2G传感器分别置于传感器支架第1列的A-H，在Data acquisition 11.0软件中将检测条件设置如下：
1预湿，Baseline，60s；2循环检测，loading，600s；Baseline1，60s；Association，200s；Dissociation，600s；中和；再生。5)检测与IL-15Rβ-Fc或IL-15Rβγ-Fc的亲和力
取8个ProA传感器分别置于传感器支架第1列的A-H，在Data acquisition 11.0软件中将检测条件设置如下：
1预湿，Baseline，60s；2循环检测，loading，600s；Baseline1，60s；Association，200s；Dissociation，600s；中和；再生。
6)数据分析
使用Data analysis11.0软件对数据进行分析。以0浓度为对照扣减背景，通过Fitting curve计算KD值。
(3)实验结果
如表1所示，IL-15 M1，IL-15 M2与IL-15Rβ或IL-15Rβγ的结合力远远强于IL-15WT或RLI-15与IL-15Rβ或IL-15Rβγ的结合力，由此表明和验证了IL-15 M1和IL-15M2两种重组蛋白中的IL-15变体能与重组蛋白上的IL-15Rα形成稳定复合体结构，并且增强了和IL-15Rβ的结合。也从侧面说明，IL-15的作用主要是通过反式递呈方式，即IL-15与表达于抗原递呈细胞表面的IL-15Rα结合，然后与临近效应细胞表面的IL-15Rβγ复合物结合，最终激活效应细胞。
表1本发明实施例中使用ForteBio检测IL-15 M1，IL-15 M2，RDB1408，IL-15 WT和RLI-15与IL-15 Rβ受体或IL-15 Rβγ受体的结合结果
| KD(M) | IL-15 WT | RLI-15 | IL-15 M1 | IL-15 M2 | RDB1408 |
| 与IL-15Rβ受体 | ～1.92x10<sup>-7</sup> | ～7.34x10<sup>-8</sup> | ～9.44x10<sup>-11</sup> | ～6.84x10<sup>-11</sup> | ～1.4x10<sup>-10</sup> |
| 与IL-15Rβγ受体 | ～1.0x10<sup>-12</sup> | 8.0x10<sup>-10</sup> | ～1.51x10-11 | ～3.4x10<sup>-11</sup> | ～2.71x10<sup>-10</sup> |
两种重组蛋白IL-15 M1和IL-15 M2对IL-15受体β的结合亲和力比起天然IL-15蛋白对IL-15受体β提高至少10倍、至少20倍或更多。实施例6
本实施例所用到的蛋白与实施例1的区别在于，断裂点为N77。经过实施例5中的亲和力实验，发现该蛋白不与外源性的IL-15Rα受体结合。对IL-15受体β的结合亲和力比起IL-15 M1对IL-15受体β明显降低，但是优于天然IL-15蛋白对IL-15受体β的亲和力。与IL15βγ复合物具有相似的高亲和力。
本实施例还用到另一种蛋白，与实施例1的区别在于，断裂点为G84。经过实施例5中的亲和力实验，发现该蛋白不与外源性的IL-15Rα受体结合。对IL-15受体β的结合亲和力比起IL-15 M1对IL-15受体β明显降低，但是优于天然IL-15蛋白对IL-15受体β的亲和力。与IL15βγ复合物具有相似的高亲和力。
实施例7
本实施例采用蛋白RDB1408作为对比例，利用实施例5中的实验方法验证其与IL-15Rβ和IL-15Rβγ的亲和力。结果如表1所示，IL-15M1和IL-15M2比RDB1408对IL-15Rβ更有亲和力，高近10倍。而在IL-15Rβγ的亲和力相似。
本实施例还采用qPCR仪器测定RDB1408，IL-15M1和IL-15M2的热熔解温度Tm，结果如图5所示，IL-15 M1和IL-15 M2的Tm值相当，但明显高于RDB1408的Tm值，说明比RDB1408热稳定性更好。
实施例8
本实施例的两个蛋白能够促进T细胞的增殖，从而起到抗肿瘤的作用。
以上详细描述了本发明的较佳具体实施例。应当理解，本领域的普通技术无需创造性劳动就可以根据本发明的构思作出诸多修改和变化。因此，凡本技术领域中技术人员依本发明的构思在现有技术的基础上通过逻辑分析、推理或者有限的实验可以得到的技术方案，皆应在由权利要求书所确定的保护范围内。
SEQUENCE LISTING
<110> 北京志道生物科技有限公司
<120> 一种用于抗肿瘤的新型IL-15融合蛋白
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Claims (27)
1.一种新型的IL-15融合蛋白，其特征在于，至少包括(1)IL-15Rα多肽、IL-15Rα多肽衍生物或其功能性片段，(2)IL-15多肽变体，(3)Fc结构域、Fc结构域衍生物或其功能性片段，(4)连接子或其衍生物，
其中，所述IL-15多肽变体至少包括第一连接子或其衍生物，所述第一连接子或其衍生物连接IL-15多肽的天然氨基端和羧基端形成环状体，并且选取N77，E82，和G84组成组中的一个氨基酸残基后面的肽键作为断裂点从而形成新氨基端和新羧基端，各组分的第一排列顺序为IL-15Rα多肽-第二连接子-IL-15多肽变体-Fc结构域，或者第二排列顺序为第三连接子-Fc结构域-第二连接子-IL-15Rα多肽-IL-15多肽变体。
2.如权利要求1所述的IL-15融合蛋白，其特征在于，所选取的断裂点为E82氨基酸后面的肽键。
3.如权利要求2所述的IL-15融合蛋白，其特征在于，所述第二连接子与所述IL-15Rα多肽或IL-15多肽变体共价连接。
4.如权利要求3所述的IL-15融合蛋白，其特征在于，在各组分的第一排列顺序中，所述第一连接子为SEQ ID NO.1所示序列，或该序列经过取代、添加或缺失一个或多个氨基酸的衍生序列，所述IL-15多肽变体的氨基酸序列如SEQ ID NO.5所示，或SEQ ID NO.5所示序列经过取代、添加或缺失一个或多个氨基酸且具有所述IL-15多肽相同相似功能的衍生序列。
5.如权利要求3所述的IL-15融合蛋白，其特征在于，在各组分的第二排列顺序中，所述第一连接子为SEQ ID NO.2所示序列，或该序列经过取代、添加或缺失一个或多个氨基酸的衍生序列，所述IL-15多肽变体的氨基酸序列如SEQ ID NO.6所示，或SEQ ID NO.6所示序列经过取代、添加或缺失一个或多个氨基酸且具有所述IL-15多肽相同相似功能的衍生序列。
6.如权利要求5所述的IL-15融合蛋白，其特征在于，所述第三连接子为SEQ ID NO.3所示序列，或该序列经过取代、添加或缺失一个或多个氨基酸的衍生序列。
7.如权利要求1-6任一项所述的IL-15融合蛋白，其特征在于，所述第二连接子为SEQID NO.4所示序列，或该序列经过取代、添加或缺失一个或多个氨基酸的衍生序列。
8.如权利要求7所述的IL-15融合蛋白，其特征在于，所述Fc结构域的序列如SEQ IDNO.7所示，或SEQ ID NO.7所示序列经过取代、添加或缺失一个或多个氨基酸且具有所述Fc结构域相同相似功能的衍生序列。
9.如权利要求8所述的IL-15融合蛋白，其特征在于，所述Fc结构域或其衍生序列选自人类IgG1 Fc结构域、人类IgG2 Fc结构域、人类IgG3 Fc结构域、人类IgG4 Fc结构域所成群组的Fc结构域。
10.如权利要求7所述的IL-15融合蛋白，其特征在于，所述IL-15Rα多肽的序列SEQ IDNO.8所示，或SEQ ID NO.8所示序列经过取代、添加或缺失一个或多个氨基酸且具有所述IL-15Rα相同相似功能的衍生序列。
11.一种核酸序列，编码权利要求1-6任一项的IL-15融合蛋白。
12.如权利要求11所述的核酸序列，其特征在于，包括操作性连结至编码所述IL-15融合蛋白的所述序列的启动子、转录起始信号及前导序列。
13.一种DNA载体，包括含有如权利要求11所述的核酸序列。
14.一种宿主，包括含有如权利要求11所述的核酸序列。
15.一种制备如权利要求1所述的IL-15融合蛋白的方法，其特征在于，包括如下步骤：
(1)提供如权利要求11所述的核酸序列，
(2)收获包含步骤(1)中的核酸序列的质粒，
(3)将步骤(2)中的质粒转入细胞中，
(4)IL-15融合蛋白在细胞中表达。
16.如权利要求15所述的方法，其特征在于，步骤(3)的详细步骤为：
(3.1)试剂制备
A)制备G418溶液：称取250mg GeneticinTM加入4.5mL超纯水溶解，超纯水定容至5mL，0.22μm滤膜过滤，-20℃保存，
B)制备PEI溶液：称取50mg PEI加入45mL超纯水溶解，1M NaOH调节pH至7.0，超纯水定容至50mL，0.22μm滤膜过滤，-20℃保存，
C)制备培养基：在1L FreeStyleTM 293Expression Medium中加入10mL PluronicdTM F-68和500μL G418，
D)质粒提前准备在2mL去内毒素离心管中，
E)根据转染需要体积准备好新鲜传代至(1-1.2)×10^6个/mL的细胞悬液，
(3.2)配制转染试剂-质粒复合物
将1μg/mL质粒与33.3μL/mL Opti-MEMTM混合得到A液，
将2μg/mL PEI与33.3μL/mL Opti-MEMTM混合得到B液，
将B液倒入A液混匀孵育10min后，加入细胞悬液，
(3.3)换液转染
在115rpm，36.8℃，5％CO2培养4h后，800g 5min离心，换成未添加F68和G418的FreeStyleTM293 Expression Medium。
17.如权利要求16所述的方法，其特征在于，步骤(4)具体为：在115rpm，36.8℃，5％CO2培养5天后，8500rpm离心15min，收集细胞上清，纯化IL-15融合蛋白。
18.如权利要求17所述的方法，其特征在于，根据Fc结构域标签，先后通过亲和纯化和凝胶过滤层析去除聚合体得到IL-15融合蛋白。
19.如权利要求15-18任一项所述的方法，其特征在于，细胞为哺乳动物细胞，选自HEK293或CHO细胞。
20.一种使用如权利要求1-6任一项所述的IL-15融合蛋白制造在患者中用以抗肿瘤的药物的用途。
21.如权利要求20所述的用途，其特征在于，所述肿瘤为进行性肿瘤、晚期肿瘤或转移性肿瘤。
22.一种药物组合物，其特征在于，包含如权利要求1-6任一项所述的IL-15融合蛋白以及药学上可接受的辅料。
23.如权利要求22所述的药物组合物在制备治疗肿瘤药物中的应用。
24.一种药物组合物，其特征在于，包含如权利要求1-6任一项所述的IL-15融合蛋白以及另一种抗癌剂。
25.如权利要求24所述的药物组合物在制备治疗肿瘤药物中的应用。
26.一种试剂盒，其特征在于，包含如权利要求1-6任一项所述的IL-15融合蛋白、如权利要求11所述的核酸序列、如权利要求13所述的表达载体或者如权利要求14所述的宿主。
27.如权利要求26所述的试剂盒，其特征在于，还包含溶解如权利要求1-6任一项所述的IL-15融合蛋白、如权利要求11所述的核酸序列、如权利要求13所述的表达载体或者如权利要求14所述的宿主和缓冲液。
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|---|---|---|---|---|
| CN113956346A (zh) * | 2021-10-26 | 2022-01-21 | 西安龙腾景云生物科技有限公司 | 一种重组白介素-15变体 |
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2020
- 2020-11-28 CN CN202011363875.XA patent/CN114573707A/zh active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113956346A (zh) * | 2021-10-26 | 2022-01-21 | 西安龙腾景云生物科技有限公司 | 一种重组白介素-15变体 |
| CN113956346B (zh) * | 2021-10-26 | 2024-03-01 | 西安龙腾景云生物科技有限公司 | 一种重组白介素-15变体 |
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