CN114853909A - 新型IL-2与INFα和Fc融合蛋白的设计、制备及用途 - Google Patents

新型IL-2与INFα和Fc融合蛋白的设计、制备及用途 Download PDF

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CN114853909A
CN114853909A CN202210521837.5A CN202210521837A CN114853909A CN 114853909 A CN114853909 A CN 114853909A CN 202210521837 A CN202210521837 A CN 202210521837A CN 114853909 A CN114853909 A CN 114853909A
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刘立明
韩镇
康平
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Nanjing Jisheng Aoma Biomedical Co ltd
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Abstract

本发明包含了针对人类白细胞介素‑2(IL‑2)和干扰素α(IFNα)的改构；改构后IL‑2、INFα或IL‑2/INFα双因子和Fc形成的融合蛋白；以及这些融合蛋白的设计、制备和用途；将突变型IL‑2和突变型INFα(双因子)分别融合在Fc的N端或C端；将突变型IL‑2融合在Fc的N端或C端；将突变型INFα分别融合在Fc的N端或C端；Fc段同时引入L234A/L235A突变，以降低与受体FcgRIII的结合能力；引入M252Y/S254T/T256E突变，以延长蛋白的半衰期；本发明通过基因突变，改变IL‑2与其受体、INFα与其受体的结合能力，得到比野生型更优的突变体，提高了与NK细胞和CD8+T细胞受体的结合能力，解决现有药物特异性弱和副作用大的缺点。

Description

新型IL-2与INFα和Fc融合蛋白的设计、制备及用途
技术领域
本发明属于生物医药技术领域，具体涉及新型IL-2与INFα和Fc融合蛋白的设计、制备 及用途。
背景技术
IL-2，也称为T细胞生长因子，基因位于4号染色体，包括7kb的序列，由133个氨基酸组成，分子量约15kD。IL-2通过IL-2R发生作用，IL-2R包括三个亚基，IL-2Rα(即CD25)、IL-2Rβ(即CD122)和IL-2Rγ(即CD132)。三个亚基可以形成三种受体形式：高结合力受体包含所有三个亚基IL-2Rα/β/γ，中结合力受体包含IL-2Rβ/γ两个亚基和低结合力受体IL-2Rα。 其中IL-2Rβ和IL-2Rγ是IL-2激活下游信号通路所必需的，当IL-2同时结合IL-2Rβ和IL-2Rγ 时，两个受体亚基形成异源二聚体，磷酸化细胞内的STAT5，进入细胞核导致相应的基因转 录和表达；IL-2Rα并非信号所必需，但可以促进IL-2与IL-2Rβ和IL-2Rγ的结合。IL-2Rγ在 所有免疫细胞中具有表达；IL-2Rβ在CD8+T细胞、NK细胞、调节性T细胞中均有表达；而 且在T细胞被激活后会提升表达水平；IL-2Rα在调节性T细胞持续高表达，在被激活的CD8+T 细胞中会有短暂表达，随即下调表达水平。IL-2是第二个批准的免疫疗法，用于治疗转移黑 色素瘤(1988)及肾细胞癌(1992)；INFα是第一个批准的免疫疗法(1986)，用于治疗毛细 胞白血病,后被批准治疗转移黑色素瘤、肾细胞癌、非霍奇金淋巴瘤、卡波济氏肉瘤。
IL-2药物的半衰期较短，所以已批准治疗肿瘤的IL-2药物需要较高剂量才有效果，而高 剂量的IL-2会引起明显的毒副作用而不能广泛得到应用。对表达IL-2Rβ和IL-2Rγ的NK细胞 和CD8+T细胞选择性较低，因此不能充分发挥NK细胞和CD8+T细胞杀伤肿瘤的能力。有 研究表明，IL-2的第38位氨基酸附近的区域对IL-2引起血管通透性增加起了关键作用，因 此对第38位氨基酸的改造可显著降低血管通透性。根据上述现有IL-2药物的缺点进行改构 可以提高治疗效果，降低毒副作用，提高治疗安全窗。
INFα是机体免疫细胞产生的一种细胞因子，是机体受到病毒感染时，免疫细胞通过抗病 毒应答反应而产生的一组结构类似、功能接近的低分子糖蛋白。干扰素在机体的免疫系统中 起着非常重要的作用。重组人干扰素α已批准用于治疗病毒性感染和肿瘤的治疗。由于干扰 素α受体分布较广，在很多正常细胞中均有表达，所以容易引起明显的毒副作用。有研究表 明，降低干扰素α与其受体的亲和力可显著降低干扰素α引起的毒副作用，同时保留其抗肿 瘤的功能。R144或R149对干扰素α与其受体结合起关键作用，突变后可引起亲和力显著降 低，但不影响其抗肿瘤功能。
发明内容
为解决上述问题，本发明公开了新型IL-2与INFα和Fc融合蛋白的设计、制备和用途， 通过基因突变的方式，改变IL-2与其受体、INFα与其受体的结合能力，解决现有药物特异 性弱、半衰期短和副作用大的缺点。
为达到上述目的，本发明的技术方案如下：
新型IL-2与INFα和Fc融合蛋白，包括：IL-2与INFα双因子Fc融合蛋白、IL-2与Fc融合蛋白、INFα与Fc融合蛋白。
将突变型IL-2和突变型INFα(双因子)分别融合在Fc的N端或C端，得到多肽链IL-2- Fc-INFα或INFα-Fc-IL-2；将突变型IL-2融合在Fc的N端或C端，得到多肽链Fc-IL-2或IL-2- Fc；将突变型INFα分别融合在Fc的N端或C端，得到多肽链Fc-INFα或INFα-Fc。
在某些实施方案中，所述Fc片段包括重链恒定区铰链区、重链恒定区第二个结构域CH2 和重链恒定区第三个结构域CH3。
在某些实施方案中，所述的Fc片段还引入L234A和L235A突变，同时引入M252Y、S254T 和T256E突变。
在某些实施方案中，IL-2突变体为通过分子生物学手段在野生型IL-2基础上引入突变得 到，所引入突变包括但不限于R38A、L80F、R81D、L85V、I86V和I92F。
在某些实施方案中，INFα衍生物为通过分子生物学手段在野生型INFα基础上引入突变 得到，所进入突变包括但不限于R144A和R149A。
在某些实施方案中，所述IL-2的氨基酸序列为：
APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEE ELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNR WITFCQSIISTLT(SEQ IDNO:1)
或为：
APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTFKFYMPKKATELKHLQCLEE ELKPLEEVLNLAQSKNFHFDPRDVVSNINVFVLELKGSETTFMCEYADETATIVEFLNR WITFCQSIISTLT(SEQ IDNO:2)。
在某些实施方案中，所述INFα的氨基酸序列为：
CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQ IFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMNEDSILAVR KYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKE(SEQ ID NO:5)
或为：
CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQ IFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMNEDSILAVRKYFQ RITLYLKEKKYSPCAWEVVAAEIMRSFSLSTNLQESLRSKE(SEQ ID NO:6)。
在某些实施方案中，所述柔性连接子的氨基酸序列为：
GGGGSGGGGSGGGGS(SEQ ID NO:11)。
在某些实施方案中，所述Fc段的氨基酸序列为：
EPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPK(SEQ ID NO:12)。
在某些实施方案中，所述IL-2融合蛋白的氨基酸序列为：
APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTFKFYMPKKATELKHLQCLEE ELKPLEEVLNLAQSKNFHFDPRDVVSNINVFVLELKGSETTFMCEYADETATIVEFLNR WITFCQSIISTLTGGGGSGGGGSGGGGSEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDT LYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPK(SEQ ID NO:3)
或为：
EPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPAGGGGSGGGGSGG GGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTFKFYMPKKATELKHLQC LEEELKPLEEVLNLAQSKNFHFDPRDVVSNINVFVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT(SEQ ID NO:4)。
在某些实施方案中，所述INFα融合蛋白的氨基酸序列为：
CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQ IFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMNEDSILAVRKYFQ RITLYLKEKKYSPCAWEVVAAEIMRSFSLSTNLQESLRSKEGGGGSGGGGSGGGGSEPKSC DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPK(SEQ ID NO:7)
或为：
EPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPAGGGGSGGGGSGG GGSCDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHE MIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMNEDSILAVRKYFQRITLYLKEKKYSPCAWEVVAAEIMRSFSLSTNLQESLRSKE(SEQ ID NO:8)
在某些实施方案中，所述IL-2与INFα融合蛋白的氨基酸序列为：
APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTFKFYMPKKATELKHLQCLEE ELKPLEEVLNLAQSKNFHFDPRDVVSNINVFVLELKGSETTFMCEYADETATIVEFLNR WITFCQSIISTLTGGGGSGGGGSGGGGSEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDT LYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPAGGGGSGGGGSGGGGSCDLPQTHSLGSRRTLMLLAQMRRISLFSCLKD RHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLND LEACVIQGVGVTETPLMNEDSILAVRKYFQRITLYLKEKKYSPCAWEVVAAEIMRSFSLSTN LQESLRSKE(SEQ ID NO:9)
或为：
CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQ IFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMNEDSILAVRKYFQ RITLYLKEKKYSPCAWEVVAAEIMRSFSLSTNLQESLRSKEGGGGSGGGGSGGGGSEPKSC DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPAGGGGSGGGGSGGGGS APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTFKFYMPKKATELKHLQCLEE ELKPLEEVLNLAQSKNFHFDPRDVVSNINVFVLELKGSETTFMCEYADETATIVEFLNR WITFCQSIISTLT(SEQ ID NO:10)。
本发明的另一个目的是提供一种制备上述的IL-2与INFα和Fc融合蛋白的方法，包括以 下步骤：
(1)将突变型IL-2和突变型INFα(双因子)分别融合在Fc的N端或C端，得到多肽 链IL-2-Fc-INFα或INFα-Fc-IL-2；将突变型IL-2融合在Fc的N端或C端，得到多肽链Fc-IL-2或IL-2-Fc；将突变型INFα分别融合在Fc的N端或C端，得到多肽链Fc-INFα或INFα-Fc；Fc段包括重链恒定区铰链区和重链恒定区第二个结构域CH2和重链恒定区第三个结构域 CH3，同时引入L234A/L235A突变，以降低与受体FcgRIII的结合能力，同时在Fc段引入 M252Y/S254T/T256E突变，以提高蛋白的半衰期；
(2)将步骤(1)得到的DNA片段克隆至pcDNA系列载体或其它用于哺乳动物细胞表达系统的载体，得到重组载体；表达载体中包括连接有合适的转录和翻译调节序列的融合DNA 序列；
(3)将步骤(2)中获得的重组载体转染至哺乳动物细胞或其他表达系统(如E.coli和酵 母)以进行融合蛋白的表达，纯化后得到IL-2与INFα和Fc融合蛋白；转染方式可以是化学 试剂转染或者电穿孔转染。
在某些实施方案中，步骤(3)中哺乳动物细胞为HEK293细胞、CHO(中国仓鼠卵巢，Chinese Hamster Ovary)细胞或上述细胞的衍生细胞或其他表达系统细胞(如E.coli和酵母)。
通过分子生物学手段在野生型IL-2基础上引入突变得到其衍生物，引入突变包括但不限 于R38A/L80F/R81D/L85V/I86V/I92F，得到的衍生物与野生型相比，与亚基IL-2Rβ的结合能 力提高。
通过分子生物学手段在野生型INFα基础上引入突变得到其衍生物，引入突变包括但不限 于R144A/R149A，得到的衍生物与野生型相比，与受体INFαR2的结合能力降低。
本发明还有一个目的是提供上述的IL-2与INFα和Fc融合蛋白在制备广谱抗肿瘤药物中 的用途。
本发明还有一个目的上提供上述的方法制备的IL-2与INFα和Fc融合蛋白在制备Fc融 合蛋白药物中的用途
本发明的特点为：
1.通过与Fc融合，延长药物的半衰期，减少细胞因子的用量，降低毒副作用。
2.通过对IL-2的改构，提高细胞因子对NK细胞和CD8+T细胞表面IL-2Rβ和IL-2Rγ的 亲和力，增强这些细胞对肿瘤的杀伤作用。
3.对IL-2第38位氨基酸的改造可显著降低IL-2引起的血管通透性增加的毒副作用。
4.对R144和R149的改造可的为降低其与受体INFαR2的结合能力，降低毒副作用，同 时保留其抗肿瘤活性。
5.综合以上特点，改构后的IL-2与INFα和Fc融合蛋白可以克服目前IL-2与INFα药物 的缺点，改善治疗效果，降低毒副作用，提高治疗安全窗。
附图说明
图1为本发明Fc融合蛋白结构示意图；
图2为本发明与受体IL-2Rα蛋白的结合能力检测图；
图3为本发明与受体IL-2Rβ蛋白的结合能力检测图；
图4为本发明与受体IFNαR2蛋白的结合能力检测图；
图5为本发明对Daudi B细胞的直接杀伤能力检测图。
具体实施方式
下面结合附图和具体实施方式，进一步阐明本发明，应理解下述具体实施方式仅用于说 明本发明而不用于限制本发明的范围。
实施例1
ELISA检测与受体IL-2Rα蛋白的结合能力
包被不同浓度(10000ng/ml、2500ng/ml、625ng/ml、156.25ng/ml、39.0625ng/ml、9.765625ng/ml、2.44140625ng/ml、0)的IL-2Rα蛋白(10165-H08H，义翘神州)，100ul/孔4℃过夜；用3％脱脂奶粉37℃封闭1h；每孔加入1ug/ml融合蛋白及其对照样品各100ul，37℃孵育1h；然后加入HRP标记的羊抗人IL-2Pab，37℃孵育1h，显色10min后，酶标仪上读 取OD450。结果见图2。
实施例2
ELISA检测与受体IL-2Rβ蛋白的结合能力
包被不同浓度(10000ng/ml、2500ng/ml、625ng/ml、156.25ng/ml、39.0625ng/ml、9.765625ng/ml、2.44140625ng/ml、0)的IL-2Rβ蛋白(10696-H05H，义翘神州)，100ul/孔4℃过夜；用3％脱脂奶粉37℃封闭1h；每孔加入1ug/ml融合蛋白及其对照样品各100ul，37℃孵育1h；然后加入HRP标记的羊抗人IL-2Pab，37℃孵育1h，显色10min后，酶标仪上读 取OD450。结果见图3。
实施例3
ELISA检测与受体IFNαR2蛋白的结合能力
包被不同浓度(10000ng/ml、2500ng/ml、625ng/ml、156.25ng/ml、39.0625ng/ml、9.765625ng/ml、2.44140625ng/ml、0)的IFNαR2蛋白(10359-H02H，义翘神州)，100ul/孔4℃过夜；用3％脱脂奶粉37℃封闭1h；每孔加入1ug/ml融合蛋白及其对照样品各100ul，37℃孵育1h；然后加入兔抗人干扰素2，37℃孵育1h，然后加入HRP标记的羊抗兔H+L，37℃ 孵育1h，显色10min后，酶标仪上读取OD450。结果见图4。
实施例4
对Daudi B细胞的直接杀伤能力检测
培养扩增Daudi B cells至所需细胞量，按7500Cell/Well将Daudi B cell铺板至96孔板 中，按照试验设计加入不同浓度(62.5nM、12.5nM、2500pM、500pM、100pM、20pM、4pM、1.25pM、0.25pM、0.05pM、0)蛋白，继续培养72h；采用化学发光法测定细胞ATP水平， 进而评价细胞活力。具体操作按照说明书操作,培养结束后加入50μL CTG溶液，混匀后，裂 解混合物转移到酶标板中，5-10分钟后，在酶标仪中采集化学发光数据。使用Excel软件分 析处理数据，使用GraphPad Prism 7软件，根据化学发光数据拟合药效剂量曲线，计算IC50。 结果见图5。
实施例5
对Daudi B细胞的间接杀伤能力检测
培养扩增Daudi B cells至所需细胞量，从Donor处采集分离Fresh PBMC，按10000Cell/Well 将Daudi B cell铺板至96孔板中,设置两个复孔，PBMC(效靶比设置为10：1和5：1)及 不同浓度IAMA005(16nM、160nM)按实验排版设计加入对应孔板中,共3块板，按不同检 测时间分别取用，Co-Culture培养24h、48h、72h后分布留取培养上清至LDH检测保存液 中，并存放于-80度冰箱。每孔细胞分别重悬后按流式检测要求染色并进行检测，采集并分析 流式数据，进行数据汇总并分析。
需要说明的是，以上内容仅仅说明了本发明的技术思想，不能以此限定本发明的保护范 围，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干 改进和润饰，这些改进和润饰均落入本发明权利要求书的保护范围之内。
序列表
<110> 南京吉盛澳玛生物医药有限公司
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<210> 9
<211> 559
<212> PRT
<213> 人工序列(Artificial Sequence)
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Gly Gly Gly Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro
145 150 155 160
Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe
165 170 175
Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile Thr Arg Glu Pro Glu Val
180 185 190
Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
195 200 205
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
210 215 220
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
225 230 235 240
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
245 250 255
Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
260 265 270
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
275 280 285
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
290 295 300
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
305 310 315 320
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
325 330 335
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
340 345 350
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
355 360 365
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Ala Gly Gly Gly Gly Ser
370 375 380
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Cys Asp Leu Pro Gln Thr
385 390 395 400
His Ser Leu Gly Ser Arg Arg Thr Leu Met Leu Leu Ala Gln Met Arg
405 410 415
Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp Arg His Asp Phe Gly Phe
420 425 430
Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln Lys Ala Glu Thr Ile Pro
435 440 445
Val Leu His Glu Met Ile Gln Gln Ile Phe Asn Leu Phe Ser Thr Lys
450 455 460
Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu Leu Asp Lys Phe Tyr Thr
465 470 475 480
Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu Ala Cys Val Ile Gln Gly
485 490 495
Val Gly Val Thr Glu Thr Pro Leu Met Asn Glu Asp Ser Ile Leu Ala
500 505 510
Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu Tyr Leu Lys Glu Lys Lys
515 520 525
Tyr Ser Pro Cys Ala Trp Glu Val Val Ala Ala Glu Ile Met Arg Ser
530 535 540
Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser Leu Arg Ser Lys Glu
545 550 555
<210> 10
<211> 559
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 10
Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met
1 5 10 15
Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp
20 25 30
Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln
35 40 45
Lys Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe
50 55 60
Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu
65 70 75 80
Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu
85 90 95
Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu Met Asn
100 105 110
Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu
115 120 125
Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Ala
130 135 140
Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser
145 150 155 160
Leu Arg Ser Lys Glu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
165 170 175
Gly Gly Gly Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro
180 185 190
Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe
195 200 205
Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile Thr Arg Glu Pro Glu Val
210 215 220
Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
225 230 235 240
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
245 250 255
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
260 265 270
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
275 280 285
Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
290 295 300
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
305 310 315 320
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
325 330 335
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
340 345 350
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
355 360 365
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
370 375 380
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
385 390 395 400
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Ala Gly Gly Gly Gly Ser
405 410 415
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser
420 425 430
Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln
435 440 445
Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Ala
450 455 460
Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys
465 470 475 480
His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu
485 490 495
Asn Leu Ala Gln Ser Lys Asn Phe His Phe Asp Pro Arg Asp Val Val
500 505 510
Ser Asn Ile Asn Val Phe Val Leu Glu Leu Lys Gly Ser Glu Thr Thr
515 520 525
Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu
530 535 540
Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile Ser Thr Leu Thr
545 550 555
<210> 11
<211> 15
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 11
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
1 5 10 15
<210> 12
<211> 231
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 12
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
1 5 10 15
Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
20 25 30
Lys Asp Thr Leu Tyr Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val
35 40 45
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
50 55 60
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
65 70 75 80
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
85 90 95
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
100 105 110
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
115 120 125
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr
130 135 140
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
145 150 155 160
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
165 170 175
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
180 185 190
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
195 200 205
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
210 215 220
Ser Leu Ser Leu Ser Pro Lys
225 230

Claims (18)

1.新型IL-2与INFα和Fc融合蛋白，其特征在于，包括：IL-2与INFα双因子Fc融合蛋白、IL-2与Fc融合蛋白、INFα与Fc融合蛋白；
IL-2与INFα双因子Fc融合蛋白：将突变型IL-2和突变型INFα分别融合在Fc的N端或C端，得到多肽链IL-2-Fc-INFα或INFα-Fc-IL-2；
IL-2与Fc融合蛋白：将突变型IL-2融合在Fc的N端或C端，得到多肽链Fc-IL-2或IL-2-Fc；
INFα与Fc融合蛋白：将突变型INFα分别融合在Fc的N端或C端，得到多肽链Fc-INFα或INFα-Fc。
2.根据权利要求1所述的新型IL-2与INFα和Fc融合蛋白，其特征在于，所述IL-2与INFα双因子Fc融合蛋白中，Fc片段包括重链恒定区铰链区、重链恒定区第二个结构域CH2和重链恒定区第三个结构域CH3。
3.根据权利要求1所述的新型IL-2与INFα和Fc融合蛋白，其特征在于，所述IL-2与Fc
融合蛋白中，Fc片段包括重链恒定区铰链区、重链恒定区第二个结构域CH2和重链恒定区第三个结构域CH3。
4.根据权利要求1所述的新型IL-2与INFα和Fc融合蛋白，其特征在于，所述INFα与Fc融合蛋白，Fc片段包括重链恒定区铰链区、重链恒定区第二个结构域CH2和重链恒定区第三个结构域CH3。
5.根据权利要求1-2任一项所述的新型IL-2与INFα和Fc融合蛋白，其特征在于，所述IL-2与INFα双因子Fc融合蛋白，Fc片段还引入L234A和L235A突变，同时引入M252Y、S254T和T256E突变。
6.根据权利要求1或3任一项所述的新型IL-2与INFα和Fc融合蛋白，其特征在于，所述的IL-2与Fc融合蛋白，Fc片段还引入L234A和L235A突变，同时引入M252Y、S254T和T256E突变。
7.根据权利要求1或4任一项所述的新型IL-2与INFα和Fc融合蛋白，其特征在于，所述的INFα与Fc融合蛋白，Fc片段还引入L234A和L235A突变，同时引入M252Y、S254T和T256E突变。
8.根据权利要求1所述的新型IL-2与INFα和Fc融合蛋白，其特征在于，所述IL-2与INFα双因子Fc融合蛋白，IL-2突变体为通过分子生物学手段在野生型IL-2基础上引入突变得到，所引入突变包括R38A、L80F、R81D、L85V、I86V和I92F。
9.根据权利要求3所述的新型IL-2与INFα和Fc融合蛋白，其特征在于，所述IL-2与Fc融合蛋白，IL-2突变体为通过分子生物学手段在野生型IL-2基础上引入突变得到，所引入突变包括但不限于R38A、L80F、R81D、L85V、I86V和I92F。
10.根据权利要求1所述的新型IL-2与INFα和Fc融合蛋白，其特征在于，所述IL-2与INFα双因子Fc融合蛋白，INFα衍生物为通过分子生物学手段在野生型INFα基础上引入突变得到，所进入突变包括R144A或R149A。
11.根据权利要求4所述的新型IL-2与INFα和Fc融合蛋白，其特征在于，所述INFα与Fc融合蛋白，INFα衍生物为通过分子生物学手段在野生型INFα基础上引入突变得到，所进入突变包括但不限于R144A或R149A。
12.制备如权利要求1-2中任一项所述的新型IL-2与INFα和Fc融合蛋白的方法，其特征在于，IL-2与INFα双因子Fc融合蛋白的制备方法，包括以下步骤：
(1)将IL-2突变体通过柔性连接子连接在Fc片段N端或C端，将INFα衍生物通过柔性连接子连接在Fc片段C端或N端，得到多肽链IL-2-Fc-INFα或INFα-Fc-IL-2；在Fc片段引入突变L234A、L235A、M252Y、S254T和T256E；
(2)将步骤(1)得到的DNA片段克隆至pcDNA系列载体或其它用于哺乳动物细胞表达系统的载体，得到重组载体；
(3)将步骤(2)中获得的重组载体转染至哺乳动物细胞以进行融合蛋白的表达，纯化后得到IL-2与INFα双因子Fc融合蛋白。
13.制备如权利要求1或3中任一项所述的新型IL-2与INFα和Fc融合蛋白的方法，其特征在于，IL-2与Fc融合蛋白的制备方法，包括以下步骤：
(1)将IL-2突变体通过柔性连接子连接在Fc片段N端或C端，得到多肽链IL-2-Fc或Fc-IL-2；在Fc片段引入突变L234A、L235A、M252Y、S254T和T256E；
(2)将步骤(1)得到的DNA片段克隆至pcDNA系列载体或其它用于哺乳动物细胞表达系统的载体，得到重组载体；
(3)将步骤(2)中获得的重组载体转染至哺乳动物细胞以进行融合蛋白的表达，纯化后得到IL-2与Fc融合蛋白。
14.制备如权利要求1或4中任一项所述的新型IL-2与INFα和Fc融合蛋白的方法，其特征在于，INFα与Fc融合蛋白的制备方法，包括以下步骤：
(1)将INFα衍生物通过柔性连接子连接在Fc片段C端或N端，得到多肽链Fc-INFα或INFα-Fc；在Fc片段引入突变L234A、L235A、M252Y、S254T和T256E；
(2)将步骤(1)得到的DNA片段克隆至pcDNA系列载体或其它用于哺乳动物细胞表达系统的载体，得到重组载体；
(3)将步骤(2)中获得的重组载体转染至哺乳动物细胞以进行融合蛋白的表达，纯化后得到INFα与Fc融合蛋白。
15.根据权利要求12-14任一项所述的制备新型IL-2与INFα和Fc融合蛋白的方法，其特征在于，步骤(3)中哺乳动物细胞包括HEK293细胞、CHO细胞或上述细胞的衍生细胞。
16.根据权利要求12-14任一项所述的制备新型IL-2与INFα和Fc融合蛋白的方法，其特征在于，步骤(1)中Fc片段引入突变具体为：在Fc片段引入L234A和L235A突变，同时在Fc片段引入M252Y、S254T和T256E突变。
17.所述权利要求1-11中任一项所述的新型IL-2与INFα和Fc融合蛋白在制备广谱抗肿瘤药物中的用途。
18.权利要求12-16中任一项所述的方法制备的新型IL-2与INFα和Fc融合蛋白在制备Fc融合蛋白药物中的用途。
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