CN113831413A - 一种长效双功能蛋白及其制备方法和用途 - Google Patents

一种长效双功能蛋白及其制备方法和用途 Download PDF

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CN113831413A
CN113831413A CN202010508626.9A CN202010508626A CN113831413A CN 113831413 A CN113831413 A CN 113831413A CN 202010508626 A CN202010508626 A CN 202010508626A CN 113831413 A CN113831413 A CN 113831413A
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鞠佃文
范佳君
韩磊
胡晓植
梁嫣煦
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Abstract

本发明属生物药物技术领域，涉及一种同时靶向ANGPTL3和IL‑β治疗动脉粥样硬化的长效双功能蛋白，本发明通过抗人ANGPTL3单抗的筛选、序列优化；抗ANGPTL3单抗与人IL‑1RA双功能蛋白的制备及人源化，制备治疗动脉粥样硬化、高血脂等代谢性疾病的药物。所述长效双功能蛋白在实现降脂和抗炎两种功能的同时，实现其长效给药，从而持久全面地治疗动脉粥样硬化。具有较高的临床应用前景和应用价值。

Description

一种长效双功能蛋白及其制备方法和用途
技术领域
本发明涉及生物药物技术领域，涉及一种长效双功能蛋白其制备方法和用途，具体涉及一种同时靶向ANGPTL3和IL-β治疗动脉粥样硬化的长效双功能蛋白，所述的长效双功能蛋白可用于制备治疗动脉粥样硬化、高血脂等代谢性疾病的药物。
背景技术
现有技术公开了人血管生成素样蛋白3(Angiopoietin-like protein3,ANGPTL3)是血管生成素样蛋白的家族成员之一，一般情况下，它们的主要功能是通过C末端的纤维样结构域与血管内皮细胞上的酪氨酸激酶Tie1和Tie2受体相结合，从而加速新生血管的生成(Biochim Biophys Acta.2014；1841:919-33)，然而，与其他成员蛋白不同的是，ANGPTL3由于其C末端的纤维样结构域中缺少半胱氨酸和钙结合模块，导致它对血管生成的诱导作用较弱，而对脂质代谢的调节作用更显著。据报道，ANGPTL3能通过抑制脂蛋白脂肪酶(Lipoprotein lipase,LPL)的活性并上调载脂蛋白B(Apolipoprotein B,ApoB)与血液中的脂蛋白和胆固醇相互作用，导致血脂升高。有研究报道，血液中糖、脂以及胆固醇的水平过高能导致肝脏X受体(Liver-X receptor,LXR)的过表达，激活ANGPTL3基因的转录，从而导致ApoB的活化，使血糖、血脂和胆固醇进一步升高(JAmColl Cardiol.2017；69:2054-63；Biochim BiophysActa.2012；1821:782-9)，虽然目前有报道表明ANGPTL3与动脉粥样硬化、高血脂等代谢性的病程关系密切，然而临床实践中，迄今尚缺少靶向ANGPTL3治疗所述疾病的相关药物。
研究公开了白细胞介素-1受体拮抗剂(interleukin-1receptor antagonist,IL-1Ra)，与IL-1α、IL-1β三者共同属于IL-1家族成员，也是由单核细胞、巨噬细胞等产生(Immunity.2013；39:1003-18.)。IL-1Ra是IL-1α、IL-1β共同的拮抗剂，虽然结合后并不激活受体，但能阻断白细胞介素-1与该受体的结合及信号传导，达到特异性地封闭白细胞介素-1活性的目的；有报道表明阻断IL-1β信号通路有助于多种代谢性疾病的抗炎治疗。
基于现有技术的现状，本申请的发明人拟将调控代谢的ANGPTL3单抗与调控炎症的IL-1RA融合表达形成双功能蛋白药物，从调控脂代谢和炎症两个层面上控制动脉粥样硬化等代谢性疾病，获得更好的疗效，具体的，本发明拟提供一种长效双功能蛋白其制备方法和用途，所述的长效双功能蛋白可用于制备治疗动脉粥样硬化、高血脂等代谢性疾病的药物。
发明内容
本发明的目的在于基于现有技术的现状，本发明拟提供一种长效双功能蛋白其制备方法和用途，所述的长效双功能蛋白可用于制备治疗动脉粥样硬化、高血脂等代谢性疾病的药物。
本发明将调控代谢的ANGPTL3单抗与调控炎症的IL-1RA融合表达形成双功能蛋白药物。
本发明中，双功能蛋白药物是通过蛋白融合表达技术实现“一个蛋白，多个功能”的药物；作为一种全新的药物分子，双功能蛋白能根据融合表达的策略不同改变该药物在体内的代谢特征。
本发明中，通过免疫羊驼，获得抗ANGPTL3的纳米VHH单抗，并在此基础上将其与人IL-1RA和IgG Fc融合表达，形成抗ANGPTL3单抗与IL-1RA融合蛋白；一方面，在抗ANGTPL3单抗通过调控脂代谢的基础上，进一步通过IL-1RA赋予融合蛋白抗炎的功能，全方位地治疗动脉粥样硬化等代谢性疾病，另一方面，通过与IgG Fc蛋白融合表达，大幅度延长双功能蛋白的半衰期，实现高效全面持久地治疗动脉粥样硬化等代谢性疾病。
具体的，本发明解决的关键技术问题在于获得全新的抗ANGPLT3单抗序列，并将其与IL-1RA和IgG Fc端序列以多种方式融合表达，优化并筛选活性、半衰期最优的双功能蛋白药物；本发明通过实验筛选获得抗ANGPTL3的高亲和力单抗分子Fdu-ShCI-01A和Fdu-ShCI-01B，通过实验明确了通过在IL-1RA的C端通过蛋白连接肽[GGGS]5与抗ANGPTL3单抗IMT-AS-01A和IMT-AS-01B的N端相连，并通过抗ANGPTL3单抗的C端与IgG4的Fc端受体中的N端的双功能蛋白IL-1RA-Anti-ANGPTL3-AB-IgG4FC为最性能最优的融合蛋白。
更具体的，本发明提供了一种长效双功能蛋白，其特征在于，该双功能蛋白是抗人ANGPTL3单抗与IL-1RA双功能蛋白，其由IL-1RA、抗人ANGPTL3单抗(Anti-ANGPTL3-AB)、信号肽以及人IgG Fc融合表达制成长效融合蛋白，其中，IL-1RA的氨基酸序列如SEQ ID No.1中所示，人ANGPTL3的氨基酸序列如SEQ ID No.2中所示，人免疫球蛋白IgG的Fc端氨基酸序列如SEQ ID No.3中所示，信号肽的氨基酸序列如SEQ ID No.4所示。
本发明中，所述的抗人ANGPTL3单抗，包括但不限于VHH抗体与scFv抗体，优选VHH抗体。
本发明中，所述的人IgG Fc蛋白包括人IgG1、IgG2、IgG3和IgG4蛋白分子，其中优选IgG4分子。
本发明中，所述的抗人ANGPTL3单抗中，其CDR区关键特征序列如SEQ ID NO.5所示。
本发明中，所述的长效融合蛋白其融合表达的形式包括且不限于：IL-1RA-Anti-ANGPTL3-AB-IgGFc；IL-1RA-IgGFc-Anti-ANGPTL3-AB、IgGFc-Anti-ANGPTL3-AB-IL-1RA以及Anti-ANGPTL3-AB-IgGFc-IL-1RA，所述的融合蛋白的氨基酸序列如SEQ ID NO.6所示。
本发明中，所述的融合表达的形式为IL-1RA-Anti-ANGPTL3-AB-IgGFc。
本发明中，所述的融合蛋白中，其IL-1RA、Anti-ANGPTL3-AB和IgGFc两两之间可通过蛋白连接肽相连，其连接肽的氨基酸序列包括但不限于[GlyGlyGlyGlySer]n、[GlySer]n、Gly[GlySer]n(n为1-20的整数)，其中优选[GlyGlyGlyGlySer]n。
本发明中，所述的抗人ANGPTL3单抗，其基因序列如SEQ ID NO.7所示。
本发明中，所述的IL-1RA，其基因序列如SEQ ID NO.8所示。
本发明中，所述的抗人ANGPTL3单抗与IL-1RA双功能蛋白，其基因序列如SEQ IDNO.7所示。
本发明中，所述的抗人ANGPTL3单抗与IL-1RA双功能蛋白和抗人ANGPTL3单抗序列构建的任意药物分子；所述的任意药物分子组成药物组合物。
本发明中，所述的任意CDR区特征序列构建的任意药物分子；所述任意药物分子组成药物组合物。
本发明还提供了含有其连接肽的氨基酸序列包括但不限于[GlyGlyGlyGlySer]n、[GlySer]n、Gly[GlySer]n(n为1-20的整数)，以及其基因序列如SEQ ID NO.7所示抗人ANGPTL3单抗的任何载体。
本发明还提供了所述双功能蛋白在制备治疗脂代谢类疾病和炎症药物中的用途；所述脂代谢类疾病和炎症包括但不限于：动脉粥样硬化、高血脂、酒精性脂肪肝病、酒精性脂肪肝炎、非酒精性脂肪肝病、非酒精性脂肪肝炎、糖尿病肾病。所述的药物制成冻干粉针剂或rhPH20皮下制剂。
本发明还提供了所述的双功能蛋白在制备检测及预后脂代谢类疾病和炎症制剂中的应用；所述的脂代谢类疾病和炎症包括但不限于：动脉粥样硬化、高血脂、酒精性脂肪肝病、酒精性脂肪肝炎、非酒精性脂肪肝病、非酒精性脂肪肝炎、糖尿病肾病。
本发明中，所述的IL-1RA，其序列为SEQ ID No.1所示：
SEQ ID No.1
RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE
NS
抗ANGPTL3的VHH抗体序列为SEQ ID No.2所示，
SEQ ID No.2
Figure BDA0002527507680000041
人免疫球蛋白IgG的Fc端序列为SEQ ID No.3所示，
SEQ ID No.3
Figure BDA0002527507680000051
信号肽的氨基酸序列为SEQ ID No.4所示，
SEQ ID No.4
MGWSCIILFLVATATGVHS
抗人ANGPTL3单抗中，其特征序列如SEQ ID NO.5所示，
SEQ ID No.5
Figure BDA0002527507680000052
抗人ANGPTL3单抗与IL-1RA双功能蛋白，其氨基酸序列为SEQ ID NO.6所示，
SEQ ID No.6
Figure BDA0002527507680000061
Figure BDA0002527507680000071
Figure BDA0002527507680000081
Figure BDA0002527507680000091
Figure BDA0002527507680000101
抗人ANGPTL3单抗，其基因序列为SEQ ID NO.7所示，
SEQ ID NO.7
Figure BDA0002527507680000111
IL-1RA表达基因序列如SEQ ID NO.8所示，
SEQ ID NO.8
CGACCCTCTGGGAGAAAATCCAGCAAGATGCAAGCCTTCAGAATCTGGGATGTTAACCAGAAGACCTTCTATCTGAGGAACAACCAACTAGTTGCCGGATACTTGCAAGGACCAAATGTCAATTTAGAAGAAAAGATAGATGTGGTACCCATTGAGCCTCATGCTCTGTTCTTGGGAATCCATGGAGGGAAGATGTGCCTGTCCTGTGTCAAGTCTGGTGATGAGACCAGACTCCAGCTGGAGGCAGTTAACATCACTGACCTGAGCGAGAACAGAAAGCAGGACAAGCGCTTCGCCTTCATCCGCTCAGACAGTGGCCCCACCACCAGTTTTGAGTCTGCCGCCTGCCCCGGTTGGTTCCTCTGCACAGCGATGGAAGCTGACCAGCCCGTCAGCCTCACCAATATGCCTGACGAAGGCGTCATGGTCACCAAATTCTACTTCCAGGAGGACGAG
本发明提供了一种长效双功能蛋白，尤其是将调控代谢的ANGPTL3单抗与调控炎症的IL-1RA融合表达形成双功能蛋白药物。所述的长效双功能蛋白药物在抗ANGTPL3单抗通过调控脂代谢的基础上，进一步通过IL-1RA赋予融合蛋白抗炎的功能，全方位地治疗动脉粥样硬化等代谢性疾病，通过与IgG Fc蛋白融合表达，大幅度延长双功能蛋白的半衰期，实现高效全面持久地治疗动脉粥样硬化等代谢性疾病。
附图说明
图1显示了抗ANGPTL3 VHH抗体的BIACORE筛选，其中，(A)抗体的单循环动力学筛选，(B)抗体的梯度亲和力验证。
图2.是Fdu-ShCI-01A和Fdu-ShCI-02A分子的体外生物学活性检测。
图3.抗ANGPTL3单抗与IL-1RA的融合表达及其亲和力检测，其中显示了IL-1RA-Anti-ANGPTL3-AB-IgGFc；IL-1RA-IgGFc-Anti-ANGPTL3-AB、IgGFc-Anti-ANGPTL3-AB-IL-1RA以及Anti-ANGPTL3-AB-IgGFc-IL-1RA分别对于ANGPTL3(A)和IL-1β的亲和力检测。
图4是抗ANGPTL3单抗Fdu-ShCI-01A、IL-1RA以及双功能蛋白Fdu-ShCI-01A-BS1抑制ApoE-/-小鼠血脂(A)和炎性细胞因子(B)的作用，其中，ApoE-/-小鼠先通过高脂饲料喂养4周后通过抗ANGPTL3单抗Fdu-ShCI-01A、IL-1RA以及双功能蛋白Fdu-ShCI-01A-BS1治疗4周，给药剂量为i.p.注射(10mg/kg)，每周两次(其中IL-1RA每天一次，10mg/kg)。
图5是抗ANGPTL3单抗与双功能蛋白的药代动力学研究结果，其中，
(A)Fdu-ShCI-01A与Fdu-ShCI-01A-BS1的血浆半衰期测定；
(B)Fdu-ShCI-01B与Fdu-ShCI-01B-BS1的血浆半衰期测定，
其中，各组药物注射剂量均为10mg/kg。
具体实施方式
实施例1噬菌体展示技术筛选抗ANGPTL3的VHH单抗
(1)抗ANGPTL3抗体表位噬菌体文库的淘选
根据ANGPTL3分子浓度用0.5％的NaHCO3缓冲液(pH＝9.6)稀释至终浓度为5μg/mL，按100μL/孔加入酶标孔中，每个靶分子包被2孔，4℃包被过夜。随后，用PBS洗涤3次，每孔加入300μL 3％BSA-PBS封闭液，37℃封闭1h。封闭完成后加入PBS洗涤3次，吸尽残余液体，并加入100μL噬菌体文库，在37℃孵育1h。接着，我们从每个孔中吸出未结合的噬菌体，用PBST洗涤6次，PBS洗涤2次，同时加入100μLGly-HCl洗脱液，在37℃下孵育8min，洗脱特异性结合的噬菌体，并将该洗脱液转移至1.5mL无菌离心管中，迅速用15μLTris-HCl中和缓冲液中和。最后取10μL洗脱液进行梯度稀释，测定滴度，并用于接下来的扩增实验。
(2)噬菌体文库的扩增和鉴定
将淘选洗脱物与处于对数生长前期的E.coli TG1培养物20mL混匀，在37℃下静置30min并加入1mL20％葡萄糖和4μLAmp+，在37℃下180r/min振荡培养30min。随后按cell：phage＝1：20的比例加入M13K07噬菌体，于37℃的黄静下静置30min后，补加20mL2×YT，在37℃180r/min的条件下继续振荡培养30min。之后，将培养物分装于离心管中，于25℃，5000r/min的条件下离心10min，收集细胞沉淀以50mL2×YT-AK液体培养基重悬，并于30℃，230r/min的条件下振荡培养过夜。
将过夜培养物4℃，10000r/min离心20min，将上清转移至新离心管，加入1/5体积的PEG/NaCl，混匀后置于4℃并静置2h。之后在4℃环境下，以10000r/min离心20min去除上清，并将沉淀重悬于1mL PBS中，加入1/5体积的PEG/NaCl，混匀后在4℃放置1h。
将淘选3次并扩增后的噬菌体滴于LB平板上，待菌落生长后随机挑出10-20个克隆至1mL培养基中放大培养8h，随后按cell：phage＝1：20的比例加入M13K07噬菌体，于37℃静置15min，后在220r/min条件下振荡培养45min。随后取出培养物补加800μL体积的2×YT-AK，30℃培养过夜。第二天，在常温下，以12000g的离心力离心10min，取上清，用于单克隆ELISA鉴定，从而筛选阳性的抗ANGPTL3单抗的表位序列。
最后通过测序获得抗ANGPTL3单抗的序列并通过pEE6.4质粒在CHO细胞株进行表达。所获得的单抗分子通过BIACORE进行亲和力检测，其结果如附图1所示。
实施例2抗ANGPTL3单抗体外生物学活性的检测
抗ANGPTL3单抗的体外生物学功能由脂蛋白酯酶(Lipoprotein Lipase，LDL)活性实验进行检测，这是由于ANGPTL3能介导脂蛋白酯酶的功能抑制。在实验开始前，实验组的ANGPTL3与抗ANGPTL3单抗以1：1的比例预孵育1h。随后加入20nM的骨髓LPL、0.23μM的ApoCII以及2mg/mL的BSA，并用PBS将检测样品的体积稀释至25μL。将这25μL的待检测液加入已用荧光素包被好的酯酶检测板中，并振荡混匀，于37℃避光反应4h。最后通过荧光酶标仪在340/400波长处检测荧光读数。各分子所得结果如图2所示。选取了其中亲和力较高的分子Fdu-ShCI-01A和Fdu-ShCI-01B进行检测，发现这两个分子都能显著抑制脂蛋酶的活性。
实施例3抗ANGPTL3单抗与IL-1RA的融合表达
将抗ANGPTL3单抗的基因序列与IL-1RA的序列融合并转入pEE6.4质粒在CHO细胞株进行表达。随后，通过ELISA检测各分子分别对IL-1β以及ANGPTL3的亲和力，所得结果如图3所示。结果显示IL-1RA-Anti-ANGPTL3-AB-IgG4Fc分子(即Fdu-ShCI-01A-BS1分子和Fdu-ShCI-02A-BS1分子)对于IL-1β和ANGPTL3的亲和力最高，几乎与融合前一致，其他融合条件均有损失。
实施例4抗ANGPTL3单抗与IL-1RA融合蛋白对于脂代谢和炎症的调控作用
通过6-8周龄的雄性ApoE-/-以及LDLR-/-小鼠用高脂饮食(含0.25％胆固醇和15％可可油的饲料粒)饲养4周后。抽取小鼠眼眶血液进行TG、TC和LDL-C以及炎性细胞因子IL-1β、TNF-α和IL-6的检测，并通过Fdu-ShCI-01A-BS1和Fdu-ShCI-02A-BS1两种双功能蛋白治疗4周后在此检测血液中脂代谢指标和炎性细胞因子指标，研究其双功能蛋白同时调控脂代谢和炎症的作用，所得结果如图4所示。结果显示Fdu-ShCI-01A-BS1和Fdu-ShCI-02A-BS1两种双功能蛋白不仅能下调血脂，还能抑制炎性细胞因子的分子，由于大多数代谢性疾病如动脉粥样硬化、糖尿病肾病在代谢紊乱的同时经常伴随炎症的发生，故而推测其对于动脉粥样硬化等代谢型疾病具有显著的治疗作用。
实施例5抗ANGPTL3单抗与IL-1RA融合蛋白的初步药代动力学研究
本申请对Fdu-ShCI-01A-BS1和Fdu-ShCI-02A-BS1两种双功能蛋白通过静脉注射给药、皮下给药和腹腔给药的药物血浆半衰期进行了检测，所得结果如图5所示，结果显示所述的双功能蛋白能通过皮下注射进入血液，其血浆半衰期长于腹腔给药和静脉给药的血浆半衰期，由于单纯的IL-1RA血浆半衰期仅为6h，故而所述的双功能蛋白的疗效将强于同等剂量的Fdu-ShCI-01A或Fdu-ShCI-02A与IL-1RA的联用。
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370 375 380
Gly Arg Lys Ser Ser Lys Met Gln Ala Phe Arg Ile Trp Asp Val Asn
385 390 395 400
Gln Lys Thr Phe Tyr Leu Arg Asn Asn Gln Leu Val Ala Gly Tyr Leu
405 410 415
Gln Gly Pro Asn Val Asn Leu Glu Glu Lys Ile Asp Val Val Pro Ile
420 425 430
Glu Pro His Ala Leu Phe Leu Gly Ile His Gly Gly Lys Met Cys Leu
435 440 445
Ser Cys Val Lys Ser Gly Asp Glu Thr Arg Leu Gln Leu Glu Ala Val
450 455 460
Asn Ile Thr Asp Leu Ser Glu Asn Arg Lys Gln Asp Lys Arg Phe Ala
465 470 475 480
Phe Ile Arg Ser Asp Ser Gly Pro Thr Thr Ser Phe Glu Ser Ala Ala
485 490 495
Cys Pro Gly Trp Phe Leu Cys Thr Ala Met Glu Ala Asp Gln Pro Val
500 505 510
Ser Leu Thr Asn Met Pro Asp Glu Gly Val Met Val Thr Lys Phe Tyr
515 520 525
Phe Gln Glu Asp Glu
530
<210> 13
<211> 533
<212> PRT
<213> Fdu-ShCI-01B-BS1
<400> 13
Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly
1 5 10 15
Val His Ser Arg Pro Ser Gly Arg Lys Ser Ser Lys Met Gln Ala Phe
20 25 30
Arg Ile Trp Asp Val Asn Gln Lys Thr Phe Tyr Leu Arg Asn Asn Gln
35 40 45
Leu Val Ala Gly Tyr Leu Gln Gly Pro Asn Val Asn Leu Glu Glu Lys
50 55 60
Ile Asp Val Val Pro Ile Glu Pro His Ala Leu Phe Leu Gly Ile His
65 70 75 80
Gly Gly Lys Met Cys Leu Ser Cys Val Lys Ser Gly Asp Glu Thr Arg
85 90 95
Leu Gln Leu Glu Ala Val Asn Ile Thr Asp Leu Ser Glu Asn Arg Lys
100 105 110
Gln Asp Lys Arg Phe Ala Phe Ile Arg Ser Asp Ser Gly Pro Thr Thr
115 120 125
Ser Phe Glu Ser Ala Ala Cys Pro Gly Trp Phe Leu Cys Thr Ala Met
130 135 140
Glu Ala Asp Gln Pro Val Ser Leu Thr Asn Met Pro Asp Glu Gly Val
145 150 155 160
Met Val Thr Lys Phe Tyr Phe Gln Glu Asp Glu Gly Gly Gly Ser Gly
165 170 175
Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gln
180 185 190
Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Pro Pro Gly Gly Ser
195 200 205
Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Asn Phe Arg Phe Ile Ser
210 215 220
Leu Gly Trp Tyr Arg Arg Ala Pro Gly Lys Gln Arg Glu Leu Val Ala
225 230 235 240
Gln Ile Thr Ser Arg Ala Asp Thr Asn Tyr Ala Asp Ser Val Lys Gly
245 250 255
Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg Glu Thr Val Tyr Leu Gln
260 265 270
Met Asn Gly Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn Ile
275 280 285
Leu His Thr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
290 295 300
Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe
305 310 315 320
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
325 330 335
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
340 345 350
Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
355 360 365
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser
370 375 380
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
385 390 395 400
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser
405 410 415
Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
420 425 430
Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln
435 440 445
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
450 455 460
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
465 470 475 480
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu
485 490 495
Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser
500 505 510
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
515 520 525
Leu Ser Leu Gly Lys
530
<210> 14
<211> 533
<212> PRT
<213> Fdu-ShCI-01B-BS2
<400> 14
Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly
1 5 10 15
Val His Ser Arg Pro Ser Gly Arg Lys Ser Ser Lys Met Gln Ala Phe
20 25 30
Arg Ile Trp Asp Val Asn Gln Lys Thr Phe Tyr Leu Arg Asn Asn Gln
35 40 45
Leu Val Ala Gly Tyr Leu Gln Gly Pro Asn Val Asn Leu Glu Glu Lys
50 55 60
Ile Asp Val Val Pro Ile Glu Pro His Ala Leu Phe Leu Gly Ile His
65 70 75 80
Gly Gly Lys Met Cys Leu Ser Cys Val Lys Ser Gly Asp Glu Thr Arg
85 90 95
Leu Gln Leu Glu Ala Val Asn Ile Thr Asp Leu Ser Glu Asn Arg Lys
100 105 110
Gln Asp Lys Arg Phe Ala Phe Ile Arg Ser Asp Ser Gly Pro Thr Thr
115 120 125
Ser Phe Glu Ser Ala Ala Cys Pro Gly Trp Phe Leu Cys Thr Ala Met
130 135 140
Glu Ala Asp Gln Pro Val Ser Leu Thr Asn Met Pro Asp Glu Gly Val
145 150 155 160
Met Val Thr Lys Phe Tyr Phe Gln Glu Asp Glu Glu Ser Lys Tyr Gly
165 170 175
Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser
180 185 190
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
195 200 205
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro
210 215 220
Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
225 230 235 240
Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val
245 250 255
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
260 265 270
Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr
275 280 285
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
290 295 300
Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
305 310 315 320
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
325 330 335
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
340 345 350
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser
355 360 365
Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
370 375 380
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys
385 390 395 400
Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser
405 410 415
Gly Gly Gly Ser Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val
420 425 430
Pro Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Asn
435 440 445
Phe Arg Phe Ile Ser Leu Gly Trp Tyr Arg Arg Ala Pro Gly Lys Gln
450 455 460
Arg Glu Leu Val Ala Gln Ile Thr Ser Arg Ala Asp Thr Asn Tyr Ala
465 470 475 480
Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg Glu
485 490 495
Thr Val Tyr Leu Gln Met Asn Gly Leu Lys Pro Glu Asp Thr Ala Val
500 505 510
Tyr Tyr Cys Asn Ile Leu His Thr Asp Tyr Trp Gly Gln Gly Thr Gln
515 520 525
Val Thr Val Ser Ser
530
<210> 15
<211> 553
<212> PRT
<213> Fdu-ShCI-01B-BS3
<400> 15
Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly
1 5 10 15
Val His Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala
20 25 30
Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
35 40 45
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
50 55 60
Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val
65 70 75 80
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
85 90 95
Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
100 105 110
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly
115 120 125
Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
130 135 140
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr
145 150 155 160
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
165 170 175
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
180 185 190
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
195 200 205
Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe
210 215 220
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
225 230 235 240
Ser Leu Ser Leu Ser Leu Gly Lys Gly Gly Gly Ser Gly Gly Gly Ser
245 250 255
Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gln Leu Gln Leu
260 265 270
Val Glu Ser Gly Gly Gly Leu Val Pro Pro Gly Gly Ser Leu Arg Leu
275 280 285
Ser Cys Ala Ala Ser Gly Ser Asn Phe Arg Phe Ile Ser Leu Gly Trp
290 295 300
Tyr Arg Arg Ala Pro Gly Lys Gln Arg Glu Leu Val Ala Gln Ile Thr
305 310 315 320
Ser Arg Ala Asp Thr Asn Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr
325 330 335
Ile Ser Arg Asp Asn Ala Arg Glu Thr Val Tyr Leu Gln Met Asn Gly
340 345 350
Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn Ile Leu His Thr
355 360 365
Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly
370 375 380
Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly
385 390 395 400
Ser Arg Pro Ser Gly Arg Lys Ser Ser Lys Met Gln Ala Phe Arg Ile
405 410 415
Trp Asp Val Asn Gln Lys Thr Phe Tyr Leu Arg Asn Asn Gln Leu Val
420 425 430
Ala Gly Tyr Leu Gln Gly Pro Asn Val Asn Leu Glu Glu Lys Ile Asp
435 440 445
Val Val Pro Ile Glu Pro His Ala Leu Phe Leu Gly Ile His Gly Gly
450 455 460
Lys Met Cys Leu Ser Cys Val Lys Ser Gly Asp Glu Thr Arg Leu Gln
465 470 475 480
Leu Glu Ala Val Asn Ile Thr Asp Leu Ser Glu Asn Arg Lys Gln Asp
485 490 495
Lys Arg Phe Ala Phe Ile Arg Ser Asp Ser Gly Pro Thr Thr Ser Phe
500 505 510
Glu Ser Ala Ala Cys Pro Gly Trp Phe Leu Cys Thr Ala Met Glu Ala
515 520 525
Asp Gln Pro Val Ser Leu Thr Asn Met Pro Asp Glu Gly Val Met Val
530 535 540
Thr Lys Phe Tyr Phe Gln Glu Asp Glu
545 550
<210> 16
<211> 533
<212> PRT
<213> Fdu-ShCI-01B-BS4
<400> 16
Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly
1 5 10 15
Val His Ser Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Pro
20 25 30
Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Asn Phe
35 40 45
Arg Phe Ile Ser Leu Gly Trp Tyr Arg Arg Ala Pro Gly Lys Gln Arg
50 55 60
Glu Leu Val Ala Gln Ile Thr Ser Arg Ala Asp Thr Asn Tyr Ala Asp
65 70 75 80
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg Glu Thr
85 90 95
Val Tyr Leu Gln Met Asn Gly Leu Lys Pro Glu Asp Thr Ala Val Tyr
100 105 110
Tyr Cys Asn Ile Leu His Thr Asp Tyr Trp Gly Gln Gly Thr Gln Val
115 120 125
Thr Val Ser Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro
130 135 140
Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
145 150 155 160
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
165 170 175
Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr
180 185 190
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
195 200 205
Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
210 215 220
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
225 230 235 240
Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
245 250 255
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met
260 265 270
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
275 280 285
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
290 295 300
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
305 310 315 320
Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val
325 330 335
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
340 345 350
Lys Ser Leu Ser Leu Ser Leu Gly Lys Gly Gly Gly Ser Gly Gly Gly
355 360 365
Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Arg Pro Ser
370 375 380
Gly Arg Lys Ser Ser Lys Met Gln Ala Phe Arg Ile Trp Asp Val Asn
385 390 395 400
Gln Lys Thr Phe Tyr Leu Arg Asn Asn Gln Leu Val Ala Gly Tyr Leu
405 410 415
Gln Gly Pro Asn Val Asn Leu Glu Glu Lys Ile Asp Val Val Pro Ile
420 425 430
Glu Pro His Ala Leu Phe Leu Gly Ile His Gly Gly Lys Met Cys Leu
435 440 445
Ser Cys Val Lys Ser Gly Asp Glu Thr Arg Leu Gln Leu Glu Ala Val
450 455 460
Asn Ile Thr Asp Leu Ser Glu Asn Arg Lys Gln Asp Lys Arg Phe Ala
465 470 475 480
Phe Ile Arg Ser Asp Ser Gly Pro Thr Thr Ser Phe Glu Ser Ala Ala
485 490 495
Cys Pro Gly Trp Phe Leu Cys Thr Ala Met Glu Ala Asp Gln Pro Val
500 505 510
Ser Leu Thr Asn Met Pro Asp Glu Gly Val Met Val Thr Lys Phe Tyr
515 520 525
Phe Gln Glu Asp Glu
530
<210> 17
<211> 339
<212> DNA
<213> Fdu-ShCI-01A
<400> 17
cagctgcagc tggtggaatc aggaggagga ctggtgccac caggaggatc tctgagactg 60
tcttgcgccg ccagcggaag caacttcaga ttcatcagcc tgggttggta ccggagggct 120
ccaggaaaac agagggagct ggtggctcag atcagcagca gagccgacac caactacgcc 180
gatagcgtga agggccggtt caccatcagc agagacaacg cccaggagac cgtgtacctg 240
cagatgaacg gcctgaagcc cgaggatacc gccgtctact actgcaacat cctgcacacc 300
gactattggg gccagggaac acaggtcacc gtgtccagc 339
<210> 18
<211> 339
<212> DNA
<213> Fdu-ShCI-01B
<400> 18
caggtgcagc tggtggaatc aggaggagga ctggtgccac caggaggatc tctgagactg 60
tcttgcgccg ccagcggaag caacttcaga ttcatcagcc tgggttggta ccggagggct 120
ccaggaaaac agagggagct ggtggctcag atcacctcca gagccgacac caactacgcc 180
gatagcgtga agggccggtt caccatcagc agagacaacg ccagggagac cgtgtacctg 240
cagatgaacg gcctgaagcc cgaggatacc gccgtctact actgcaacat cctgcacacc 300
gactattggg gccagggaac acaggtcacc gtgtccagc 339
<210> 19
<211> 456
<212> DNA
<213> IL-1RA
<400> 19
cgaccctctg ggagaaaatc cagcaagatg caagccttca gaatctggga tgttaaccag 60
aagaccttct atctgaggaa caaccaacta gttgccggat acttgcaagg accaaatgtc 120
aatttagaag aaaagataga tgtggtaccc attgagcctc atgctctgtt cttgggaatc 180
catggaggga agatgtgcct gtcctgtgtc aagtctggtg atgagaccag actccagctg 240
gaggcagtta acatcactga cctgagcgag aacagaaagc aggacaagcg cttcgccttc 300
atccgctcag acagtggccc caccaccagt tttgagtctg ccgcctgccc cggttggttc 360
ctctgcacag cgatggaagc tgaccagccc gtcagcctca ccaatatgcc tgacgaaggc 420
gtcatggtca ccaaattcta cttccaggag gacgag 456

Claims (18)

1.一种长效双功能蛋白，其特征在于，该双功能蛋白是抗人ANGPTL3单抗与IL-1RA双功能蛋白，其由IL-1RA、抗人ANGPTL3单抗(Anti-ANGPTL3-AB)、信号肽以及人IgG Fc融合表达制成长效融合蛋白，其中，IL-1RA的氨基酸序列如SEQ ID No.1中所示，人ANGPTL3的氨基酸序列如SEQ ID No.2中所示，人免疫球蛋白IgG的Fc端氨基酸序列如SEQ ID No.3中所示，信号肽的氨基酸序列如SEQ ID No.4所示。
2.按权利要求1所述的长效双功能蛋白，其特征在于，所述的抗人ANGPTL3单抗，包括但不限于VHH抗体与scFv抗体，优选VHH抗体。
3.按权利要求1所述的长效双功能蛋白，其特征在于，所述的人IgG Fc蛋白包括人IgG1、IgG2、IgG3和IgG4蛋白分子，其中优选IgG4分子。
4.按权利要求1所述的长效双功能蛋白，其特征在于，所述的抗人ANGPTL3单抗中，其CDR区关键特征序列如SEQ ID NO.5所示。
5.按权利要求1所述的长效双功能蛋白，其特征在于，所述的长效融合蛋白其融合表达的形式包括且不限于：IL-1RA-Anti-ANGPTL3-AB-IgGFc；IL-1RA-IgGFc-Anti-ANGPTL3-AB、IgGFc-Anti-ANGPTL3-AB-IL-1RA以及Anti-ANGPTL3-AB-IgGFc-IL-1RA，所述的融合蛋白的氨基酸序列如SEQ ID NO.6所示。
6.按权利要求5所述的长效双功能蛋白，其特征在于，所述的融合表达的形式为IL-1RA-Anti-ANGPTL3-AB-IgGFc。
7.按权利要求5所述的长效双功能蛋白，其特征在于，所述的融合蛋白中，其IL-1RA、Anti-ANGPTL3-AB和IgGFc两两之间可通过蛋白连接肽相连，其连接肽的氨基酸序列包括但不限于[GlyGlyGlyGlySer]n、[GlySer]n、Gly[GlySer]n(n为1-20的整数)，其中优选[GlyGlyGlyGlySer]n。
8.按权利要求1所述的长效双功能蛋白，其特征在于，所述的抗人ANGPTL3单抗，其基因序列如SEQ ID NO.7所示。
9.按权利要求1所述的长效双功能蛋白，其特征在于，所述的IL-1RA，其基因序列如SEQID NO.8所示。
10.按权利要求1所述的长效双功能蛋白，其特征在于，所述的抗人ANGPTL3单抗与IL-1RA双功能蛋白，其基因序列如SEQ ID NO.7所示。
11.由权利要求1中所述的抗人ANGPTL3单抗与IL-1RA双功能蛋白和抗人ANGPTL3单抗序列构建的任意药物分子。
12.由权利要求11中所述的任意药物分子所组成的药物组合物。
13.由权利要求项4中所述的任意CDR区特征序列构建的任意药物分子。
14.由权利要求项13中所述任意药物分子组成的药物组合物。
15.含有权利要求7和权利要求8中所述基因序列的任何载体。
16.权利要求1中所述的抗人ANGPTL3单抗、IL-1RA以及抗人ANGPTL3单抗与IL-1RA双功能蛋白在制备治疗脂代谢类疾病和炎症药物中的用途；所述脂代谢类疾病和炎症包括但不限于：动脉粥样硬化、高血脂、酒精性脂肪肝病、酒精性脂肪肝炎、非酒精性脂肪肝病、非酒精性脂肪肝炎、糖尿病肾病。
17.权利要求1中所述的抗人ANGPTL3单抗、IL-1RA以及抗人ANGPTL3单抗与IL-1RA双功能蛋白在制备检测及预后脂代谢类疾病和炎症制剂中的应用；所述的脂代谢类疾病和炎症包括但不限于：动脉粥样硬化、高血脂、酒精性脂肪肝病、酒精性脂肪肝炎、非酒精性脂肪肝病、非酒精性脂肪肝炎、糖尿病肾病。
18.按权利要求16所述的用途，其特征在于，所述的药物制成冻干粉针剂或rhPH20皮下制剂。
CN202010508626.9A 2020-06-06 2020-06-06 一种长效双功能蛋白及其制备方法和用途 Pending CN113831413A (zh)

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WO2024041236A1 (zh) * 2022-08-22 2024-02-29 复旦大学附属儿科医院 包含angptl3单抗的融合蛋白

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WO2024041236A1 (zh) * 2022-08-22 2024-02-29 复旦大学附属儿科医院 包含angptl3单抗的融合蛋白

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