CN117003851A - 一种白细胞介素-2突变蛋白 - Google Patents

一种白细胞介素-2突变蛋白 Download PDF

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CN117003851A
CN117003851A CN202210456852.6A CN202210456852A CN117003851A CN 117003851 A CN117003851 A CN 117003851A CN 202210456852 A CN202210456852 A CN 202210456852A CN 117003851 A CN117003851 A CN 117003851A
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宁金鹰
彭浩
郝锋
贺锋
吴国进
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Abstract

本发明提供了一种白细胞介素‑2(IL‑2)突变蛋白。相比于野生型IL‑2，本发明提供的IL‑2突变蛋白具有降低的对IL‑2受体α链和β链的亲和力，由此降低了对调节性T淋巴细胞(Treg细胞)增殖的诱导能力和对先天性和适应性效应细胞(Teff细胞)的激活作用。

Description

一种白细胞介素-2突变蛋白
技术领域
本发明属于生物医药领域，具体而言，本发明涉及白细胞介素-2突变 蛋白及其应用。
背景技术
白细胞介素-2(Interleukin-2，IL-2)又名T细胞生长因子，是主要由 活化的CD4+和CD8+T细胞产生的具有广泛生物活性的细胞因子。
IL-2具有抗肿瘤活性，抗肿瘤机理主要有：刺激T细胞的增殖和分化， 增强T细胞对肿瘤细胞的杀伤活性；刺激单核巨噬细胞的杀伤活性；刺激 NK细胞的增殖，增强NK细胞的杀瘤活性；刺激抗体的生成而发挥抗肿 瘤作用。IL-2扩增和激活先天性和适应性效应细胞的能力是其抗肿瘤活性的 基础，因此很多研究者在开发与挖掘其作为抗肿瘤药物的潜力；同时IL-2 作为可以防止过度免疫反应和自身免疫的调节因子，其也可以促进CD25+ 与CD4+阳性的调节性T淋巴细胞(Treg细胞)的增殖与扩增。
IL-2通过与细胞表面表达的白细胞介素-2受体(Interleukin-2receptor， IL-2R)特异性结合，触发信号级联反应，发挥免疫调节作用。T细胞、NK 细胞、B细胞及单核-巨噬细胞等的表面均可表达IL-2受体。通常认为，IL-2R 至少有三种不同的亚基，即α链、β链、γ链，参与构成IL-2R复合物。IL-2 与受体α链结合的亲和力是其与受体β链、γ链结合亲和力的50倍左右，IL-2 结合受体β链和γ链后会引起其信号传导，而结合受体α链后并不会引起信 号传导。受体β链和γ链表达在NK细胞、单核细胞、巨噬细胞以及CD4+ 和CD8+阳性的T细胞上。
通常认为，由于调节性T淋巴细胞(Treg细胞)IL-2Rα链的表达量高 于效应细胞(Teff细胞)，而IL-2与IL-2Rα链的亲和力又远高于其与β链 和γ链的亲和力，因此IL-2与调节性T淋巴细胞(Treg细胞)结合的亲和 力高于与效应细胞(Teff细胞)结合的亲和力，从而诱导其增殖，因而在抗 肿瘤作用中产生负面影响。同时，由于IL-2作为生命活动中非常重要的细胞 因子，其通过结合受体β链和γ链而诱导信号传导及激活先天性和适应性效应细胞的功能非常强，临床中的一些试验已经体现出其表现出的额外的毒副 作用。
发明内容
本发明的目的是提供一种新的白细胞介素-2(IL-2)突变蛋白。相比于野 生型IL-2，该突变蛋白与IL-2Rα链的亲和力降低，从而与调节性T淋巴细 胞(Treg细胞)结合的亲和力降低，使其诱导调节性T淋巴细胞(Treg细 胞)增殖的能力减弱，从而避免在抗肿瘤作用中产生负面影响；同时，该突 变蛋白与IL-2Rβ链的亲和力降低，从而其激活先天性和适应性效应细胞的 功能减弱，由此降低其作为药物的毒副作用。
本发明的技术方案如下。
一方面，本发明提供一种白细胞介素-2(IL-2)突变蛋白，所述IL-2突 变蛋白在对应于SEQ ID NO.32所示野生型IL-2的氨基酸序列的以下氨基 酸位点中的一个或多个处具有氨基酸突变：16、20、23、26、42、43、45、 61、62、72、82、83、84、95、126。
优选地，与SEQ ID NO.32所示野生型IL-2的氨基酸序列相比较，所 述IL-2突变蛋白的氨基酸序列具有以下氨基酸突变中的一个或多个： H16A，D20S，D20A，M23R，N26Q，F42S，F42A，K43E，K43A，K43R， Y45A，E61R，E61A，E61D，E62K，E62A，E62R，L72G，P82E，R83E，D84R，D84A，D84K，D84C，E95D，Q126A。
更优选地，所述IL-2突变蛋白的氨基酸序列具有所述氨基酸突变中的 1个、2个、3个、4个、5个或6个。
根据本发明的具体实施方式，与SEQ ID NO.32所示野生型IL-2的氨 基酸序列相比较，所述IL-2突变蛋白的氨基酸序列具有如下氨基酸突变： (1)H16A，F42S，K43E，E61R，E62K，D84R；(2)D20S，F42S，K43E， E61R，E62K；(3)H16A；(4)D20A；(5)F42A；(6)K43A；(7)E61A；(8)E62A； (9)D84A；(10)Q126A；(11)K43E；(12)D84R；(13)K43R；(14)E61D；(15)E95D；(16)E62R；(17)E61R；(18)K43E，E62R；(19)K43E；(20)K43A， E62A；(21)F42A，Y45A，L72G，D84R；(22)R83E；(23)P82E；(24)M23R； (25)E62A，D84R；(26)N26Q；D84R；(27)E62A，D84K；(28)N26Q，D84K； 或(29)D84C。
根据本发明的具体实施方式，本发明提供的IL-2突变蛋白包含SEQ ID NO.1至SEQID NO.29中任一个所示的氨基酸序列。
另一方面，本发明提供一种核酸分子，所述核酸分子包含编码所述IL-2 突变蛋白的核苷酸序列。
再一方面，本发明提供包含所述核酸分子的载体。所述载体可以为真核 表达载体、原核表达载体、人工染色体及噬菌体载体等。
又一方面，本发明提供包含所述核酸分子或载体的宿主细胞，或者被所 述核酸分子或载体转化或转染的宿主细胞。所述宿主细胞可以是任何原核或 真核细胞，例如细菌或昆虫、真菌、植物或动物细胞。
还一方面，本发明提供一种融合蛋白，所述融合蛋白包含本发明提供 的IL-2突变蛋白以及与其融合的第二部分，所述第二部分为抗体或其抗原 结合片段。优选地，所述抗体为单克隆抗体或单链抗体。优选地，所述抗 原结合片段为scFv、dsFv、(dsFv)2、Fab、Fab'、F(ab')2或Fv片段。
根据本发明的具体实施方式，所述融合蛋白为抗体构建体，所述抗体构 建体包含抗体的重链和轻链，所述抗体的轻链的C端与本发明提供的IL-2 突变蛋白的N端直接或经由连接肽间接融合。所述连接肽可以是柔性连接 肽或刚性连接肽。所述柔性连接肽例如为包含(GGGGS)n的重复序列，例 如n＝3～4；所述刚性连接肽例如为包含(EAAAK)m的重复序列，例如 m＝2～5。
在本发明提供的抗体构建体中，所述抗体可以为单克隆抗体。或者， 抗体构建体包含抗体的两条重链和两条轻链，其中两条轻链各自的C端与 本发明提供的IL-2突变蛋白的N端直接或经由连接肽间接融合(结构例 如示于图4)。根据本发明的具体实施方式，所述抗体为抗PD-L1抗体， 序列如下：
重链全长：SEQ ID NO.30；
重链可变区：SEQ ID NO.38；
重链CDR1至CDR3：SEQ ID NO.39；SEQ ID NO.40；SEQ ID NO.41。
轻链全长：SEQ ID NO.31；
轻链可变区：SEQ ID NO.42；
轻链CDR1至CDR3：SEQ ID NO.43；SEQ ID NO.44；SEQ ID NO.45。
另一方面，本发明还提供一种组合物，所述组合物包含本发明所述的 IL-2突变蛋白、核酸分子、载体、宿主细胞或融合蛋白。优选地，所述组合 物为药物组合物，其还任选地包含药学上可接受的载体、辅料或赋形剂。
又一方面，本发明还提供所述IL-2突变蛋白、核酸分子、载体、宿主 细胞、融合蛋白或组合物在制备疾病治疗药物中的用途，所述疾病为癌症 或自身免疫性疾病。
相应地，本发明还提供一种治疗疾病的方法，所述方法包括给有此需要 的受试者施用本发明提供的IL-2突变蛋白、核酸分子、载体、宿主细胞、 融合蛋白或组合物，所述疾病为癌症或自身免疫性疾病。优选地，所述受 试者为哺乳类动物；更优选地，所述受试者为人。
与现有技术相比，本发明提供了一种新型的IL-2突变蛋白，所述IL-2 突变蛋白相比于野生型IL-2与其受体IL-2Rα链和IL-2Rβ链的亲和力显著 降低，因此对调节性T淋巴细胞(Treg细胞)增殖的诱导能力显著下降，同 时激活先天性和适应性效应细胞(Teff细胞)的功能也被减弱，由此降低了 其作为药物的毒副作用。
附图说明
以下，结合附图来详细说明本发明的实施方案，其中：
图1示出了野生型IL-2与其受体的结合以及进行氨基酸突变的目标位 点。
图2示出了野生型IL-2在第84位Asp突变前后与IL-2Rβ的结合。
图3示出了野生型IL-2在第62位Glu突变前后与IL-2Rα的结合。
图4示出了抗体构建体PDL1×IL2的结构示意图。
图5示出了不同IL-2突变蛋白对Teff细胞的促增殖作用。
图6示出了不同IL-2突变蛋白对Teff细胞的促增殖作用。
图7示出了不同IL-2突变蛋白对IL-2信号通路的激活作用。
图8示出了不同IL-2突变蛋白对IL-2信号通路的激活作用。
图9示出了不同IL-2突变蛋白的促T细胞增殖活性，其中9A示出了不 同IL-2突变蛋白存在的条件下，Treg在CD4细胞中的比例；9B示出了不同 IL-2突变蛋白存在的条件下，发生增殖的Treg的比例。
具体实施方式
以下参照具体的实施例来说明本发明。本领域技术人员能够理解，这些 实施例仅用于说明本发明，其不以任何方式限制本发明的范围。
下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施 例中所用的原料、试剂材料等，如无特殊说明，均为市售购买产品。
采用了CN112759647A(中国专利申请CN2019109997884)中公开的抗 PD-L1抗体“14A7”，重链和轻链的序列如下：
>14A7-H(重链全长：SEQ ID NO.30；重链可变区：SEQ ID NO.38；重链 CDR1：SEQID NO.39；重链CDR2：SEQ ID NO.40；重链CDR3：SEQ ID NO.41)
QVQLVQSGAEVKKPGASVKVSCKASEYTFTNNWIAWVRQAPGQGLEWVGDIHPGGGFTNYNEKFKVRA TLTADTSTSTAYMELSSLRSEDTAVYYCAGSKTRDYDAWFAYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS LGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPPAAGPSVFLF
PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKG QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK
>14A7-L(轻链全长：SEQ ID NO.31；轻链可变区：SEQ ID NO.42；轻链 CDR1：SEQID NO.43；轻链CDR2：SEQ ID NO.44；轻链CDR3：SEQ ID NO.45)
DIQMTQSPSTLSASVGDRVTITCKSSQSLLYTGNQKNYLAWYQQKPGKAPKLLIYWASTRESGVPSRF SGSGSGTEFTLTISSLQPDDFATYYCQQYYTYRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC
采用了Kymab的IL-2突变蛋白和Synthekine的IL-2突变蛋白作为对照， 在本发明中分别命名为“mut0”和“STK-012”，序列如下：
>mut0(SEQ ID NO.36)
APTSSSTIGCTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKA TELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETT FMCEYADETATIVEFLNRWITFCQSIISTLT
>STK-012(SEQ ID NO.37)
PTSSSTKKTQLQLSQLLVLLKAILNGINNYKNPKLTRMLTFKFYMPKKATE LKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTF MCEYADETATIVEFLNRWITFCQSIISTLT
实施例1 IL-2突变蛋白的设计
对野生型IL-2(SEQ ID NO.32)与其受体alpha(IL-2Rα)、beta(IL-2Rβ)、 gamma(IL-2Rγ)的复合物进行结构分析(Protein Data Bank：2B5I)。鉴于 盐键在蛋白与蛋白或抗原与抗体相互作用的过程中起着非常重要的作用，因 此将IL-2与其受体形成的复合物中包含的盐键作为氨基酸突变的目标位点， 同时将IL-2与其受体结合的关键位置的氨基酸也作为氨基酸突变的目标位 点，通过对这些位点进行氨基酸突变，达到削弱IL2与其受体的结合能力的 目的。具体地，进行氨基酸突变的目的在于，降低IL-2与受体β链的亲和力， 从而减弱其激活先天性和适应性效应细胞的功能，降低其作为药物的毒副作 用；同时降低IL-2与受体α链的亲和力，从而降低其与调节性T淋巴细胞 (Treg细胞)的结合能力，使其不能诱导调节性T淋巴细胞(Treg细胞) 增殖，避免对抗肿瘤作用的负面影响。
>野生型IL-2(SEQ ID NO.32)：
APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKA TELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETT FMCEYADETATIVEFLNRWITFCQSIISTLT
>IL-2Rα(SEQ ID NO.33)
ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNS SHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQAS LPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCK MTHGKTRWTQPQLICTGEMETSQFPGEEKPQASPEGRPESETSCLVTTTDF QIQTEMAATMETSIFTTEYQVAVAGCVFLLISVLLLSGLTWQRRQRKSRRTI
>IL-2Rβ(SEQ ID NO.34)
AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTC ELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDF KPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPG HTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQ PLAFRTKPAALGKDTIPWLGHLLVGLSGAFGFIILVYLLINCRNTGPWLKK VLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVL ERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQ VYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLF SPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPD LVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV
>IL-2Rγ(SEQ ID NO.35)
LNTTILTPNGNEDTTADFFLTTMPTDSLSVSTLPLPEVQCFVFNVEYMNCT WNSSSEPQPTNLTLHYWYKNSDNDKVQKCSHYLFSEEITSGCQLQKKEIH LYQTFVVQLQDPREPRRQATQMLKLQNLVIPWAPENLTLHKLSESQLELN WNNRFLNHCLEHLVQYRTDWDHSWTEQSVDYRHKFSLPSVDGQKRYTF RVRSRFNPLCGSAQHWSEWSHPIHWGSNTSKENPFLFALEAVVISVGSMG LIISLLCVYFWLERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQ PDYSERLCLVSEIPPKGGALGEGPGASPCNQHSPYWAPPCYTLKPET
野生型IL-2蛋白中进行突变的位点示于图1。
例如，对于IL-2与其受体IL-2Rβ形成的复合物，针对IL-2的第84位 Asp与IL-2Rβ的第71位Lys形成的盐键，将IL-2的第84位Asp突变为Arg， 使其与IL-2Rβ的第71位Lys形成正电荷与正电荷的相互排斥作用力。见图 2(2A：野生型IL-2的Asp84与IL-2Rβ的Lys71形成了盐键；2B：突变后 IL-2与IL-2Rβ的Lys71的盐键被破坏)。
或者，针对IL-2的第62位Glu与IL-2Rα的第36位Arg形成的盐键， 将IL-2的第62位Glu突变为Ala，使其与IL-2Rα的第36位Arg形成正电 荷与正电荷的相互排斥作用力。见图3(3A：野生型IL-2的Glu62与IL-2Rα 的Arg36形成了盐键；3B：突变后IL-2与IL-2Rα的Arg36的盐键被破坏)。
得到多种IL-2突变蛋白，序列如下，其中以斜体和下划线示出了氨基酸 突变：
>mut1(SEQ ID NO.1)
>mut2(SEQ ID NO.2)
>mut3(SEQ ID NO.3)
>mut4(SEQ ID NO.4)
>mut5(SEQ ID NO.5)
>mut6(SEQ ID NO.6)
>mut7(SEQ ID NO.7)
>mut8(SEQ ID NO.8)
>mut9(SEQ ID NO.9)
>mut10(SEQ ID NO.10)
>mut11(SEQ ID NO.11)
>mut12(SEQ ID NO.12)
>mut13(SEQ ID NO.13)
>mut14(SEQ ID NO.14)
>mut15(SEQ ID NO.15)
>mut16(SEQ ID NO.16)
>mut17(SEQ ID NO.17)
>mut18(SEQ ID NO.18)
>mut19(SEQ ID NO.19)
>mut20(SEQ ID NO.20)
>mut21(SEQ ID NO.21)
>mut22(SEQ ID NO.22)
>mut23(SEQ ID NO.23)
>mut24(SEQ ID NO.24)
>mut25(SEQ ID NO.25)
>mut26(SEQ ID NO.26)
>mut27(SEQ ID NO.27)
>mut28(SEQ ID NO.28)
>mut29(SEQ ID NO.29)
实施例2抗体构建体(PDL1×IL2)的表达与纯化
将野生型IL-2和突变型IL-2(如上Mut0至Mut28)分别融合于抗PD-L1 抗体14A7轻链的C端，构建融合蛋白；不采用连接序列，而是将各个IL-2 蛋白的N端直接融合于抗体轻链的C端。
通过基因合成技术分别获得抗体重链和融合了IL-2突变蛋白的轻链的 编码基因，通过限制性酶切，将该编码基因插入到表达载体 pCDNA3.4pCDNA3.4中；转染宿主细胞E.coli DH5a，挑选不同单克隆菌落， 提取质粒，测序验证。挑选验证正确的克隆建库保存。
利用HEK293-F系统瞬时转染抗体重链和轻链质粒，来生产PDL1xIL2 蛋白。简言之，对于1L摇瓶，HEK293F细胞以0.5×106个细胞/mL密度接 种在250mL培养基中，110rpm，5％CO2温育；次日将预先准备的两种表达 质粒与转染试剂按照一定比例混合后，以1×106个细胞/mL细胞密度，将转 染复合物添加至细胞中，24h后补加营养物质及DNA合成抑制剂；细胞培 养5-7天后，收获分泌抗体的上清液；使用MabSelectSure亲和层析技术(GEHealthcare)，从细胞培养物上清中纯化PDL1×IL2蛋白。该融合蛋白即本发 明提供的抗体构建体(PDL1×IL2，结构示意图见图4)。根据IL-2突变蛋白 编号命名得到的不同抗体构建体。例如，抗PD-L1抗体14A7轻链的C端融 合了野生型IL-2得到的抗体构建体命名为“PDL1×IL2 WT”；融合了已知 突变型IL-2Mut0或STK-012得到的抗体构建体分别命名为“PDL1×IL2 Mut0”或“PDL1×IL2 STK-012”，以此类推。
实施例3抗体构建体对Teff细胞的促增殖作用
PBMC于前一天复苏，培养过夜。使用磁力分选方法分离Pan T细胞， 然后使用10mL全培养基(RPMI1640，含10％FBS)重悬细胞，接种于T25 培养瓶内。向瓶内加入终浓度为3μg/mL OKT3抗体，然后将培养瓶置于 37℃、5％CO2培养12-24h。
将Pan T细胞自培养瓶内取出，使用该全培养基洗涤细胞三次后计数。 加入90μL细胞悬液至96孔透明平底黑壁板对应孔内，每孔细胞数不小于 5×104个。将96孔板内的细胞置于37℃、5％CO2条件下继续培养。
将待测的抗体构建体以10μg/mL的起始浓度进行5倍稀释，得到10个 不同浓度的稀释液。将人IL-2(Acrobiosystems,P60568-1)使用该全培养基 配制成100ng/mL的浓度。将96孔板含Pan T细胞的不同孔分成试验孔(将 加入抗体构建体)，阳性对照孔(将加入人IL-2)，和阴性对照孔(将加入培 养基)，然后相应地分别加入10μL配制好的抗体构建体稀释液，加入10μL 配制好的人IL-2溶液，和等体积培养基。将该96孔板置于37℃、5％CO2继续培养72h。
使用CTG法检测细胞活力(使用基于荧光素酶系统的细胞活力检测试 剂，购自南京诺唯赞，cat#DD1101-02)。
结果见图5和表1。
表1.不同IL2突变蛋白对Teff细胞的促增殖作用
其中，部分结果示于图6和表2。
表2.不同IL-2突变蛋白对Teff细胞的促增殖作用
PDL1×IL2 EC50(μg/mL)
PDL1×IL2 WT 0.00287
PDL1×IL2 Mut0 0.02179
PDL1×IL2 Mut12 0.04822
实施例4 PDL1×IL2对IL-2信号通路的激活作用
先将NK92细胞在无血清培养基中饥饿过夜；第二天用全培养基 (RPMI1640，含10％FBS)分别配制不同抗体构建体PDL1×IL2、抗PD-L1 抗体14A7以及人IL-2(His Tag)的稀释液：分别以100μg/mL的起始浓度进 行10倍稀释，得到6个不同浓度的稀释液。
调整经饥饿处理的细胞浓度为2-4×106个细胞/ml，在96孔板中每孔加 入24μl细胞，然后各孔加入6μl上述不同浓度的蛋白。混匀后，37℃、5％CO2孵育20分钟刺激p-STAT5的表达。然后用p-STAT5检测试剂盒 (PHOSPHO-STAT5(TYR694)KITS，Csbio，64AT5PEG)检测p-STAT5的 蛋白水平，计算HTRF比率：HTRF Ratio＝(Signal 665nm/Signal 620nm)×104，HTRF比率代表磷酸化STAT5的浓度。
结果见图7和表3。
表3.不同IL-2突变蛋白对IL-2信号通路的激活作用
其中，部分结果示于图8和表4。
表4.不同IL-2突变蛋白对IL-2信号通路的激活作用
蛋白 EC50(nM)
抗PD-L1抗体14A7 N/A
PDL1×IL2 WT 0.005
PDL1×IL2 Mut0 0.087
PDL1×IL2 Mut12 0.137
PDL1×IL2 Mut25 2.102
结果显示，相比于IL-2野生型的对照蛋白(包含于PDL1×IL2 WT中)， IL-2突变蛋白Mut25(包含于PDL1×IL2 Mut25中)的活性降低了～400倍 (EC50＝2.102vs 0.005)。
实施例5 T细胞增殖实验检测IL-2突变蛋白的活性
将抗人CD3抗体(OKT3，Biolegend，317302)在PBS中以10μg/ml 的浓度混匀，然后以每孔45μl加入平底96孔板中，4℃过夜包被。第二天 用全培养基(RPMI1640，含10％FBS)配制不同抗体构建体PDL1×IL2的 稀释液。
复苏冻存的PBMC，使用CD3分离试剂盒(Pan T Cell Isolation Kit,human,Miltenyibiotec,130-096-535)分离CD3细胞，然后用CD4分离试剂盒 (DynabeadsUntouched Human CD4 T Cell,Invitrogen,11346D)分离CD4细 胞(阴性部分)和CD8细胞(阳性部分)，得到的是CD3+CD4-CD8+细胞。 将细胞悬浮于PBS，调整细胞浓度为约1-2×107个细胞/ml，加入CFSE，使 其浓度为0.3μM，37℃放置10-15分钟。加入5ml全培养基离心洗涤；去除 培养基，加入1ml全培养基，将细胞浓度调整为约4×105个细胞/ml。
向包被有CD3抗体的96孔板中加入细胞悬液，每孔50μl。每孔加入上 述配制的抗体构建体和全培养基使抗体构建体浓度为20、2、0.2、0.02、0.002、 0.0002、0.00002μg/ml。混匀，培养3天后，使用eBioscienceTM Foxp3/转录 因子染色缓冲液套件(Thermofisher，00552300)进行细胞核内Foxp3染色。 流式检测，收集数据，分析低CFSE信号的为发生分裂的细胞。结果见图9。 CD4细胞中，Foxp3阳性部分低CFSE信号细胞的比例为发生分裂的Treg(9B)；同时计数Foxp3阳性细胞在CD4细胞的比例(9A)。
实验结果表明，相比于IL-2野生型的对照蛋白(包含于PDL1×IL2 WT 中)，IL-2突变蛋白Mut25(包含于PDL1×IL2 Mut25中)和IL-2突变蛋白 Mut12(包含于PDL1×IL2Mut12中)对Treg的促增殖活性更弱。
以上对本发明具体实施方式的描述并不限制本发明，本领域技术人员可 以根据本发明作出各种改变或变形，只要不脱离本发明的精神，均应属于本 发明所附权利要求的范围。
序列表
<110> 康源博创生物科技（北京）有限公司
<120> 一种白细胞介素-2突变蛋白
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Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys
35 40 45
Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys
50 55 60
Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu
65 70 75 80
Arg Pro Arg Ala Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu
85 90 95
Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala
100 105 110
Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile
115 120 125
Ile Ser Thr Leu Thr
130
<210> 10
<211> 133
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> IL-2突变蛋白
<400> 10
Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His
1 5 10 15
Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys
20 25 30
Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys
35 40 45
Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys
50 55 60
Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu
65 70 75 80
Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu
85 90 95
Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala
100 105 110
Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Ala Ser Ile
115 120 125
Ile Ser Thr Leu Thr
130
<210> 11
<211> 133
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> IL-2突变蛋白
<400> 11
Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His
1 5 10 15
Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys
20 25 30
Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Glu Phe Tyr Met Pro Lys
35 40 45
Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys
50 55 60
Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu
65 70 75 80
Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu
85 90 95
Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala
100 105 110
Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile
115 120 125
Ile Ser Thr Leu Thr
130
<210> 12
<211> 133
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> IL-2突变蛋白
<400> 12
Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His
1 5 10 15
Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys
20 25 30
Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys
35 40 45
Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys
50 55 60
Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu
65 70 75 80
Arg Pro Arg Arg Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu
85 90 95
Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala
100 105 110
Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile
115 120 125
Ile Ser Thr Leu Thr
130
<210> 13
<211> 133
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> IL-2突变蛋白
<400> 13
Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His
1 5 10 15
Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys
20 25 30
Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Arg Phe Tyr Met Pro Lys
35 40 45
Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys
50 55 60
Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu
65 70 75 80
Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu
85 90 95
Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala
100 105 110
Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile
115 120 125
Ile Ser Thr Leu Thr
130
<210> 14
<211> 133
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> IL-2突变蛋白
<400> 14
Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His
1 5 10 15
Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys
20 25 30
Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys
35 40 45
Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Asp Glu Leu Lys
50 55 60
Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu
65 70 75 80
Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu
85 90 95
Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala
100 105 110
Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile
115 120 125
Ile Ser Thr Leu Thr
130
<210> 15
<211> 133
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> IL-2突变蛋白
<400> 15
Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His
1 5 10 15
Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys
20 25 30
Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys
35 40 45
Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys
50 55 60
Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu
65 70 75 80
Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Asp Leu
85 90 95
Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala
100 105 110
Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile
115 120 125
Ile Ser Thr Leu Thr
130
<210> 16
<211> 133
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> IL-2突变蛋白
<400> 16
Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His
1 5 10 15
Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys
20 25 30
Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys
35 40 45
Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Arg Leu Lys
50 55 60
Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu
65 70 75 80
Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu
85 90 95
Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala
100 105 110
Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile
115 120 125
Ile Ser Thr Leu Thr
130
<210> 17
<211> 133
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> IL-2突变蛋白
<400> 17
Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His
1 5 10 15
Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys
20 25 30
Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys
35 40 45
Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Arg Glu Leu Lys
50 55 60
Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu
65 70 75 80
Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu
85 90 95
Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala
100 105 110
Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile
115 120 125
Ile Ser Thr Leu Thr
130
<210> 18
<211> 133
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> IL-2突变蛋白
<400> 18
Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His
1 5 10 15
Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys
20 25 30
Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Glu Phe Tyr Met Pro Lys
35 40 45
Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Arg Leu Lys
50 55 60
Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu
65 70 75 80
Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu
85 90 95
Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala
100 105 110
Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile
115 120 125
Ile Ser Thr Leu Thr
130
<210> 19
<211> 133
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> IL-2突变蛋白
<400> 19
Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His
1 5 10 15
Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys
20 25 30
Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Glu Phe Tyr Met Pro Lys
35 40 45
Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys
50 55 60
Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu
65 70 75 80
Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu
85 90 95
Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala
100 105 110
Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile
115 120 125
Ile Ser Thr Leu Thr
130
<210> 20
<211> 132
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> IL-2突变蛋白
<400> 20
Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His
1 5 10 15
Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys
20 25 30
Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Ala Phe Tyr Met Pro Lys
35 40 45
Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Ala Leu Lys
50 55 60
Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu
65 70 75 80
Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu
85 90 95
Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala
100 105 110
Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile
115 120 125
Ile Ser Thr Leu
130
<210> 21
<211> 133
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> IL-2突变蛋白
<400> 21
Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His
1 5 10 15
Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys
20 25 30
Asn Pro Lys Leu Thr Arg Met Leu Thr Ala Lys Phe Ala Met Pro Lys
35 40 45
Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys
50 55 60
Pro Leu Glu Glu Val Leu Asn Gly Ala Gln Ser Lys Asn Phe His Leu
65 70 75 80
Arg Pro Arg Arg Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu
85 90 95
Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala
100 105 110
Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile
115 120 125
Ile Ser Thr Leu Thr
130
<210> 22
<211> 133
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> IL-2突变蛋白
<400> 22
Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His
1 5 10 15
Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys
20 25 30
Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys
35 40 45
Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys
50 55 60
Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu
65 70 75 80
Arg Pro Glu Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu
85 90 95
Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala
100 105 110
Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile
115 120 125
Ile Ser Thr Leu Thr
130
<210> 23
<211> 133
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> IL-2突变蛋白
<400> 23
Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His
1 5 10 15
Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys
20 25 30
Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys
35 40 45
Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys
50 55 60
Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu
65 70 75 80
Arg Glu Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu
85 90 95
Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala
100 105 110
Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile
115 120 125
Ile Ser Thr Leu Thr
130
<210> 24
<211> 133
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> IL-2突变蛋白
<400> 24
Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His
1 5 10 15
Leu Leu Leu Asp Leu Gln Arg Ile Leu Asn Gly Ile Asn Asn Tyr Lys
20 25 30
Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys
35 40 45
Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys
50 55 60
Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu
65 70 75 80
Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu
85 90 95
Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala
100 105 110
Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile
115 120 125
Ile Ser Thr Leu Thr
130
<210> 25
<211> 133
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> IL-2突变蛋白
<400> 25
Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His
1 5 10 15
Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys
20 25 30
Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys
35 40 45
Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Ala Leu Lys
50 55 60
Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu
65 70 75 80
Arg Pro Arg Arg Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu
85 90 95
Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala
100 105 110
Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile
115 120 125
Ile Ser Thr Leu Thr
130
<210> 26
<211> 133
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> IL-2突变蛋白
<400> 26
Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His
1 5 10 15
Leu Leu Leu Asp Leu Gln Met Ile Leu Gln Gly Ile Asn Asn Tyr Lys
20 25 30
Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys
35 40 45
Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys
50 55 60
Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu
65 70 75 80
Arg Pro Arg Arg Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu
85 90 95
Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala
100 105 110
Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile
115 120 125
Ile Ser Thr Leu Thr
130
<210> 27
<211> 133
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> IL-2突变蛋白
<400> 27
Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His
1 5 10 15
Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys
20 25 30
Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys
35 40 45
Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Ala Leu Lys
50 55 60
Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu
65 70 75 80
Arg Pro Arg Lys Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu
85 90 95
Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala
100 105 110
Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile
115 120 125
Ile Ser Thr Leu Thr
130
<210> 28
<211> 133
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> IL-2突变蛋白
<400> 28
Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His
1 5 10 15
Leu Leu Leu Asp Leu Gln Met Ile Leu Gln Gly Ile Asn Asn Tyr Lys
20 25 30
Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys
35 40 45
Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys
50 55 60
Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu
65 70 75 80
Arg Pro Arg Lys Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu
85 90 95
Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala
100 105 110
Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile
115 120 125
Ile Ser Thr Leu Thr
130
<210> 29
<211> 133
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> IL-2突变蛋白
<400> 29
Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His
1 5 10 15
Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys
20 25 30
Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys
35 40 45
Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys
50 55 60
Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu
65 70 75 80
Arg Pro Arg Cys Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu
85 90 95
Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala
100 105 110
Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile
115 120 125
Ile Ser Thr Leu Thr
130
<210> 30
<211> 450
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> 抗PD-L1抗体，重链
<400> 30
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Glu Tyr Thr Phe Thr Asn Asn
20 25 30
Trp Ile Ala Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Val
35 40 45
Gly Asp Ile His Pro Gly Gly Gly Phe Thr Asn Tyr Asn Glu Lys Phe
50 55 60
Lys Val Arg Ala Thr Leu Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Gly Ser Lys Thr Arg Asp Tyr Asp Ala Trp Phe Ala Tyr Trp Gly
100 105 110
Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
115 120 125
Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
130 135 140
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
145 150 155 160
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
165 170 175
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
180 185 190
Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
195 200 205
Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys
210 215 220
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Pro Ala Ala Gly
225 230 235 240
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
260 265 270
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
275 280 285
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
290 295 300
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
305 310 315 320
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Ala Ala Pro Ile Glu
325 330 335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
340 345 350
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
355 360 365
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
385 390 395 400
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
405 410 415
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445
Gly Lys
450
<210> 31
<211> 219
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> 抗PD-L1抗体，轻链
<400> 31
Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Leu Tyr Thr
20 25 30
Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys
35 40 45
Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
85 90 95
Tyr Tyr Thr Tyr Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105 110
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
115 120 125
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
130 135 140
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
145 150 155 160
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
165 170 175
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
180 185 190
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
195 200 205
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215
<210> 32
<211> 133
<212> PRT
<213> human
<400> 32
Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His
1 5 10 15
Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys
20 25 30
Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys
35 40 45
Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys
50 55 60
Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu
65 70 75 80
Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu
85 90 95
Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala
100 105 110
Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile
115 120 125
Ile Ser Thr Leu Thr
130
<210> 33
<211> 251
<212> PRT
<213> human
<400> 33
Glu Leu Cys Asp Asp Asp Pro Pro Glu Ile Pro His Ala Thr Phe Lys
1 5 10 15
Ala Met Ala Tyr Lys Glu Gly Thr Met Leu Asn Cys Glu Cys Lys Arg
20 25 30
Gly Phe Arg Arg Ile Lys Ser Gly Ser Leu Tyr Met Leu Cys Thr Gly
35 40 45
Asn Ser Ser His Ser Ser Trp Asp Asn Gln Cys Gln Cys Thr Ser Ser
50 55 60
Ala Thr Arg Asn Thr Thr Lys Gln Val Thr Pro Gln Pro Glu Glu Gln
65 70 75 80
Lys Glu Arg Lys Thr Thr Glu Met Gln Ser Pro Met Gln Pro Val Asp
85 90 95
Gln Ala Ser Leu Pro Gly His Cys Arg Glu Pro Pro Pro Trp Glu Asn
100 105 110
Glu Ala Thr Glu Arg Ile Tyr His Phe Val Val Gly Gln Met Val Tyr
115 120 125
Tyr Gln Cys Val Gln Gly Tyr Arg Ala Leu His Arg Gly Pro Ala Glu
130 135 140
Ser Val Cys Lys Met Thr His Gly Lys Thr Arg Trp Thr Gln Pro Gln
145 150 155 160
Leu Ile Cys Thr Gly Glu Met Glu Thr Ser Gln Phe Pro Gly Glu Glu
165 170 175
Lys Pro Gln Ala Ser Pro Glu Gly Arg Pro Glu Ser Glu Thr Ser Cys
180 185 190
Leu Val Thr Thr Thr Asp Phe Gln Ile Gln Thr Glu Met Ala Ala Thr
195 200 205
Met Glu Thr Ser Ile Phe Thr Thr Glu Tyr Gln Val Ala Val Ala Gly
210 215 220
Cys Val Phe Leu Leu Ile Ser Val Leu Leu Leu Ser Gly Leu Thr Trp
225 230 235 240
Gln Arg Arg Gln Arg Lys Ser Arg Arg Thr Ile
245 250
<210> 34
<211> 525
<212> PRT
<213> human
<400> 34
Ala Val Asn Gly Thr Ser Gln Phe Thr Cys Phe Tyr Asn Ser Arg Ala
1 5 10 15
Asn Ile Ser Cys Val Trp Ser Gln Asp Gly Ala Leu Gln Asp Thr Ser
20 25 30
Cys Gln Val His Ala Trp Pro Asp Arg Arg Arg Trp Asn Gln Thr Cys
35 40 45
Glu Leu Leu Pro Val Ser Gln Ala Ser Trp Ala Cys Asn Leu Ile Leu
50 55 60
Gly Ala Pro Asp Ser Gln Lys Leu Thr Thr Val Asp Ile Val Thr Leu
65 70 75 80
Arg Val Leu Cys Arg Glu Gly Val Arg Trp Arg Val Met Ala Ile Gln
85 90 95
Asp Phe Lys Pro Phe Glu Asn Leu Arg Leu Met Ala Pro Ile Ser Leu
100 105 110
Gln Val Val His Val Glu Thr His Arg Cys Asn Ile Ser Trp Glu Ile
115 120 125
Ser Gln Ala Ser His Tyr Phe Glu Arg His Leu Glu Phe Glu Ala Arg
130 135 140
Thr Leu Ser Pro Gly His Thr Trp Glu Glu Ala Pro Leu Leu Thr Leu
145 150 155 160
Lys Gln Lys Gln Glu Trp Ile Cys Leu Glu Thr Leu Thr Pro Asp Thr
165 170 175
Gln Tyr Glu Phe Gln Val Arg Val Lys Pro Leu Gln Gly Glu Phe Thr
180 185 190
Thr Trp Ser Pro Trp Ser Gln Pro Leu Ala Phe Arg Thr Lys Pro Ala
195 200 205
Ala Leu Gly Lys Asp Thr Ile Pro Trp Leu Gly His Leu Leu Val Gly
210 215 220
Leu Ser Gly Ala Phe Gly Phe Ile Ile Leu Val Tyr Leu Leu Ile Asn
225 230 235 240
Cys Arg Asn Thr Gly Pro Trp Leu Lys Lys Val Leu Lys Cys Asn Thr
245 250 255
Pro Asp Pro Ser Lys Phe Phe Ser Gln Leu Ser Ser Glu His Gly Gly
260 265 270
Asp Val Gln Lys Trp Leu Ser Ser Pro Phe Pro Ser Ser Ser Phe Ser
275 280 285
Pro Gly Gly Leu Ala Pro Glu Ile Ser Pro Leu Glu Val Leu Glu Arg
290 295 300
Asp Lys Val Thr Gln Leu Leu Leu Gln Gln Asp Lys Val Pro Glu Pro
305 310 315 320
Ala Ser Leu Ser Ser Asn His Ser Leu Thr Ser Cys Phe Thr Asn Gln
325 330 335
Gly Tyr Phe Phe Phe His Leu Pro Asp Ala Leu Glu Ile Glu Ala Cys
340 345 350
Gln Val Tyr Phe Thr Tyr Asp Pro Tyr Ser Glu Glu Asp Pro Asp Glu
355 360 365
Gly Val Ala Gly Ala Pro Thr Gly Ser Ser Pro Gln Pro Leu Gln Pro
370 375 380
Leu Ser Gly Glu Asp Asp Ala Tyr Cys Thr Phe Pro Ser Arg Asp Asp
385 390 395 400
Leu Leu Leu Phe Ser Pro Ser Leu Leu Gly Gly Pro Ser Pro Pro Ser
405 410 415
Thr Ala Pro Gly Gly Ser Gly Ala Gly Glu Glu Arg Met Pro Pro Ser
420 425 430
Leu Gln Glu Arg Val Pro Arg Asp Trp Asp Pro Gln Pro Leu Gly Pro
435 440 445
Pro Thr Pro Gly Val Pro Asp Leu Val Asp Phe Gln Pro Pro Pro Glu
450 455 460
Leu Val Leu Arg Glu Ala Gly Glu Glu Val Pro Asp Ala Gly Pro Arg
465 470 475 480
Glu Gly Val Ser Phe Pro Trp Ser Arg Pro Pro Gly Gln Gly Glu Phe
485 490 495
Arg Ala Leu Asn Ala Arg Leu Pro Leu Asn Thr Asp Ala Tyr Leu Ser
500 505 510
Leu Gln Glu Leu Gln Gly Gln Asp Pro Thr His Leu Val
515 520 525
<210> 35
<211> 347
<212> PRT
<213> human
<400> 35
Leu Asn Thr Thr Ile Leu Thr Pro Asn Gly Asn Glu Asp Thr Thr Ala
1 5 10 15
Asp Phe Phe Leu Thr Thr Met Pro Thr Asp Ser Leu Ser Val Ser Thr
20 25 30
Leu Pro Leu Pro Glu Val Gln Cys Phe Val Phe Asn Val Glu Tyr Met
35 40 45
Asn Cys Thr Trp Asn Ser Ser Ser Glu Pro Gln Pro Thr Asn Leu Thr
50 55 60
Leu His Tyr Trp Tyr Lys Asn Ser Asp Asn Asp Lys Val Gln Lys Cys
65 70 75 80
Ser His Tyr Leu Phe Ser Glu Glu Ile Thr Ser Gly Cys Gln Leu Gln
85 90 95
Lys Lys Glu Ile His Leu Tyr Gln Thr Phe Val Val Gln Leu Gln Asp
100 105 110
Pro Arg Glu Pro Arg Arg Gln Ala Thr Gln Met Leu Lys Leu Gln Asn
115 120 125
Leu Val Ile Pro Trp Ala Pro Glu Asn Leu Thr Leu His Lys Leu Ser
130 135 140
Glu Ser Gln Leu Glu Leu Asn Trp Asn Asn Arg Phe Leu Asn His Cys
145 150 155 160
Leu Glu His Leu Val Gln Tyr Arg Thr Asp Trp Asp His Ser Trp Thr
165 170 175
Glu Gln Ser Val Asp Tyr Arg His Lys Phe Ser Leu Pro Ser Val Asp
180 185 190
Gly Gln Lys Arg Tyr Thr Phe Arg Val Arg Ser Arg Phe Asn Pro Leu
195 200 205
Cys Gly Ser Ala Gln His Trp Ser Glu Trp Ser His Pro Ile His Trp
210 215 220
Gly Ser Asn Thr Ser Lys Glu Asn Pro Phe Leu Phe Ala Leu Glu Ala
225 230 235 240
Val Val Ile Ser Val Gly Ser Met Gly Leu Ile Ile Ser Leu Leu Cys
245 250 255
Val Tyr Phe Trp Leu Glu Arg Thr Met Pro Arg Ile Pro Thr Leu Lys
260 265 270
Asn Leu Glu Asp Leu Val Thr Glu Tyr His Gly Asn Phe Ser Ala Trp
275 280 285
Ser Gly Val Ser Lys Gly Leu Ala Glu Ser Leu Gln Pro Asp Tyr Ser
290 295 300
Glu Arg Leu Cys Leu Val Ser Glu Ile Pro Pro Lys Gly Gly Ala Leu
305 310 315 320
Gly Glu Gly Pro Gly Ala Ser Pro Cys Asn Gln His Ser Pro Tyr Trp
325 330 335
Ala Pro Pro Cys Tyr Thr Leu Lys Pro Glu Thr
340 345
<210> 36
<211> 134
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> IL-2突变蛋白
<400> 36
Ala Pro Thr Ser Ser Ser Thr Ile Gly Cys Thr Gln Leu Gln Leu Glu
1 5 10 15
His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr
20 25 30
Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro
35 40 45
Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu
50 55 60
Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His
65 70 75 80
Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu
85 90 95
Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr
100 105 110
Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser
115 120 125
Ile Ile Ser Thr Leu Thr
130
<210> 37
<211> 132
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> IL-2突变蛋白
<400> 37
Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Ser Gln Leu
1 5 10 15
Leu Val Leu Leu Lys Ala Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn
20 25 30
Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys
35 40 45
Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro
50 55 60
Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg
65 70 75 80
Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys
85 90 95
Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr
100 105 110
Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile
115 120 125
Ser Thr Leu Thr
130
<210> 38
<211> 121
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> 抗PD-L1抗体，重链可变区
<400> 38
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Glu Tyr Thr Phe Thr Asn Asn
20 25 30
Trp Ile Ala Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Val
35 40 45
Gly Asp Ile His Pro Gly Gly Gly Phe Thr Asn Tyr Asn Glu Lys Phe
50 55 60
Lys Val Arg Ala Thr Leu Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Gly Ser Lys Thr Arg Asp Tyr Asp Ala Trp Phe Ala Tyr Trp Gly
100 105 110
Gln Gly Thr Thr Val Thr Val Ser Ser
115 120
<210> 39
<211> 10
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> 抗PD-L1抗体，重链CDR1
<400> 39
Glu Tyr Thr Phe Thr Asn Asn Trp Ile Ala
1 5 10
<210> 40
<211> 17
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> 抗PD-L1抗体，重链CDR2
<400> 40
Asp Ile His Pro Gly Gly Gly Phe Thr Asn Tyr Asn Glu Lys Phe Lys
1 5 10 15
Val
<210> 41
<211> 12
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> 抗PD-L1抗体，重链CDR3
<400> 41
Ser Lys Thr Arg Asp Tyr Asp Ala Trp Phe Ala Tyr
1 5 10
<210> 42
<211> 112
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> 抗PD-L1抗体，轻链可变区
<400> 42
Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Leu Tyr Thr
20 25 30
Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys
35 40 45
Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
85 90 95
Tyr Tyr Thr Tyr Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105 110
<210> 43
<211> 17
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> 抗PD-L1抗体，轻链CDR1
<400> 43
Lys Ser Ser Gln Ser Leu Leu Tyr Thr Gly Asn Gln Lys Asn Tyr Leu
1 5 10 15
Ala
<210> 44
<211> 7
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> 抗PD-L1抗体，轻链CDR2
<400> 44
Trp Ala Ser Thr Arg Glu Ser
1 5
<210> 45
<211> 8
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> 抗PD-L1抗体，轻链CDR3
<400> 45
Gln Gln Tyr Tyr Thr Tyr Arg Thr
1 5

Claims (10)

1.一种白细胞介素-2(IL-2)突变蛋白，与SEQ ID NO.32所示野生型IL-2的氨基酸序列相比较，所述IL-2突变蛋白的氨基酸序列具有以下氨基酸突变中的一个或多个：H16A，D20S，D20A，M23R，N26Q，F42S，F42A，K43E，K43A，K43R，Y45A，E61R，E61A，E61D，E62K，E62A，E62R，L72G，P82E，R83E，D84R，D84A，D84K，D84C，E95D，Q126A。
2.根据权利要求1所述的IL-2突变蛋白，其特征在于，所述IL-2突变蛋白的氨基酸序列具有如下氨基酸突变：
(1)H16A，F42S，K43E，E61R，E62K，D84R；
(2)D20S，F42S，K43E，E61R，E62K；
(3)H16A；
(4)D20A；
(5)F42A；
(6)K43A；
(7)E61A；
(8)E62A；
(9)D84A；
(10)Q126A；
(11)K43E；
(12)D84R；
(13)K43R；
(14)E61D；
(15)E95D；
(16)E62R；
(17)E61R；
(18)K43E，E62R；
(19)K43E；
(20)K43A，E62A；
(21)F42A，Y45A，L72G，D84R；
(22)R83E；
(23)P82E；
(24)M23R；
(25)E62A，D84R；
(26)N26Q；D84R；
(27)E62A，D84K；
(28)N26Q，D84K；或
(29)D84C；
优选地，所述IL-2突变蛋白包含SEQ ID NO.1至SEQ ID NO.29中任一个所示的氨基酸序列。
3.一种核酸分子，所述核酸分子包含编码根据权利要求1或2所述的IL-2突变蛋白的核苷酸序列。
4.一种载体，所述载体包含权利要求3所述的核酸分子。
5.一种宿主细胞，所述宿主细胞包含权利要求3所述的核酸分子和/或权利要求4所述的载体，或者所述宿主细胞被权利要求3所述的核酸分子和/或权利要求4所述的载体转化或转染。
6.一种融合蛋白，所述融合蛋白包含根据权利要求1或2所述的IL-2突变蛋白以及与其融合的第二部分，所述第二部分为抗体或其抗原结合片段。
7.根据权利要求6所述的融合蛋白，其特征在于，所述融合蛋白为抗体构建体，其中，所述抗体构建体包含抗体的重链和轻链，所述抗体的轻链的C端与权利要求1或2所述的IL-2突变蛋白的N端直接或经由连接肽间接融合；
优选地，所述抗体构建体中，所述抗体为抗PD-1或PD-L1抗体。
8.根据权利要求6或7所述的融合蛋白，其特征在于，所述融合蛋白为抗体构建体，所述抗体构建体包含抗体的两条重链和两条轻链；
优选地，所述抗体为抗PD-L1抗体，其重链依次包含具有SEQ ID NO.39、SEQ ID NO.40、SEQ ID NO.41所示氨基酸序列的重链CDR1至CDR3；其轻链依次包含具有SEQ ID NO.43、SEQID NO.44、SEQ ID NO.45所示氨基酸序列的轻链CDR1至CDR3。
9.一种组合物，所述组合物包含权利要求1或2所述的IL-2突变蛋白、权利要求3所述的核酸分子、权利要求4所述的载体、权利要求5所述的宿主细胞或权利要求6至8中任一项所述的融合蛋白；
优选地，所述组合物为药物组合物，其还包含任选的药学上可接受的辅料。
10.权利要求1或2所述的IL-2突变蛋白、权利要求3所述的核酸分子、权利要求4所述的载体、权利要求5所述的宿主细胞、权利要求6至8中任一项所述的融合蛋白或权利要求9所述的组合物在制备疾病治疗药物中的用途，所述疾病为癌症或自身免疫性疾病。
CN202210456852.6A 2022-04-27 2022-04-27 一种白细胞介素-2突变蛋白 Pending CN117003851A (zh)

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