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WO2023035817A1 - 一种fgf21突变蛋白及其应用 - Google Patents

一种fgf21突变蛋白及其应用 Download PDF

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WO2023035817A1
WO2023035817A1 PCT/CN2022/110072 CN2022110072W WO2023035817A1 WO 2023035817 A1 WO2023035817 A1 WO 2023035817A1 CN 2022110072 W CN2022110072 W CN 2022110072W WO 2023035817 A1 WO2023035817 A1 WO 2023035817A1
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fgf21
amino acid
residue
residues
cysteine
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French (fr)
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高翔
赵耀
张建军
高展
张玉
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Leto Laboratories Co Ltd
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Leto Laboratories Co Ltd
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Priority to KR1020247007029A priority Critical patent/KR20240053591A/ko
Priority to AU2022341345A priority patent/AU2022341345A1/en
Priority to GB2403250.0A priority patent/GB2625224A/en
Priority to EP22866291.2A priority patent/EP4389763A4/en
Priority to JP2024513063A priority patent/JP2024531980A/ja
Priority to US18/689,432 priority patent/US20240376168A1/en
Publication of WO2023035817A1 publication Critical patent/WO2023035817A1/zh
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Definitions

  • the invention relates to an FGF21 mutant protein and its application, belonging to the field of biotechnology.
  • Human fibroblast growth factor 21 (Fibroblast growth factor 21, FGF21) is a polypeptide composed of 209 amino acids, in which the N-terminal of the FGF21 protein contains a signal peptide composed of 28 amino acids, so it is formed after cleavage of the signal peptide Mature FGF21 consists of 181 amino acids (SEQ ID NO: 1).
  • FGF21 is expressed in various human organs and tissues, such as liver, pancreas and adipose tissue.
  • FGF21 can be secreted into the blood circulation, and induce various signaling pathways and functional activities in the liver, pancreas, and adipose tissue, thereby realizing the physiological functions of regulating glucose and lipid metabolism and protecting pancreatic ⁇ cells.
  • Studies have shown that in obese mice induced by diet or genetic manipulation, injection of FGF21 protein can significantly reduce the body weight and blood sugar level of the mice, and the triglyceride content in the serum of the mice is also significantly reduced. Further studies showed that FGF21 can improve insulin sensitivity in the liver, thereby alleviating glucose intolerance.
  • FGF21 can reduce the body weight and body fat of experimental animals in a dose-dependent manner. Significantly lower.
  • FGF21 also activates signaling pathways in exocrine pancreatic cells and hepatocytes and inhibits hepatic glycogen export.
  • FGF21 is a member of the FGF (fibroblast growth factor) family. However, unlike most FGFs that have broad-spectrum mitogenic abilities, FGF21 has no obvious ability to promote cell proliferation. Studies have shown that in the whole life cycle of FGF21 transgenic mice, no abnormalities such as tumors and tissue hyperplasia were found in the body. At the same time, pharmacokinetic and drug safety experiments show that FGF21 exceeding the pharmacological dose will not cause hypoglycemia, which indicates that FGF21 is a potential ideal drug for treating diseases such as diabetes and obesity.
  • FGF fibroblast growth factor
  • FGF21 activates downstream signaling pathways through both FGF receptors (Fibroblast growth factor receptors, FGFRs) and co-receptor ⁇ -klotho, and neither ⁇ -klotho nor FGFR alone can activate FGF21 signaling .
  • FGF receptors Fibroblast growth factor receptors, FGFRs
  • FGFRs Fibroblast growth factor receptors
  • ⁇ -klotho nor FGFR alone can activate FGF21 signaling .
  • Structural biology studies have shown that the structure of FGF21 can be divided into two parts, of which the N-terminal domain is mainly related to the binding of FGFR, while the C-terminal domain is a relatively flexible sequence, which is related to the binding of ⁇ -klotho. Only after FGF21 forms a ternary complex with ⁇ -klotho and FGFR can it activate downstream related signaling pathways and exert its biological effects.
  • FGF21 The physiological functions of FGF21 in controlling blood sugar and reducing body weight have brought hope for the treatment of related diseases, but wild-type FGF21 has the disadvantages of being easily hydrolyzed by proteases and having a small molecular weight, and its half-life is only 0.5-2 hours, so it is not suitable for direct treatment. as medicine.
  • the C-terminus of FGF21 protein contains a flexible region responsible for binding to the ⁇ -klotho receptor, which is easily degraded by fibroblast activation protein (FAP), and its main degradation site is around Pro171.
  • FAP fibroblast activation protein
  • the FGF21 molecule When P171 is cleaved by FAP protease, the FGF21 molecule will no longer be able to bind to the ⁇ -klotho co-receptor, resulting in the inactivation of the FGF21 molecule. Faced with this difficulty, the medical community has increased the half-life of FGF21 by performing site-directed mutagenesis of amino acids at enzyme cleavage sites to prepare long-acting fusion proteins, or linking polyethylene glycol, fatty acid chains, etc. to the polypeptide backbone.
  • the natural wild-type human FGF21 protein has an amino acid sequence as shown in SEQ ID NO: 1, and the 171st proline residue (Pro) of the protein is the enzyme cleavage site of FAP protease.
  • the enzymatic cleavage site of FGF21 and/or its nearby structure can reduce the hydrolysis rate of FGF21 and prolong its half-life and drug efficacy in vivo.
  • one aspect of the present invention provides a FGF21 mutein, in a specific embodiment, it comprises an amino acid sequence with the following changes of a or b on the basis of the wild-type human FGF21 sequence:
  • a pair of disulfide bonds are formed at or around the proline residue at position 171 through substitution, deletion or addition of one or more amino acid residues.
  • a scheme of the present invention is to directly delete the proline residue at position 171 (such as the sequence shown in SEQ ID NO: 4, 10, 16 and 22), so that the FGF21 protein loses the enzyme cleavage site of FAP, thus also will not be hydrolyzed.
  • the FGF21 mutein comprises the amino acid sequence shown in SEQ ID NO:4.
  • Another solution of the present invention is to introduce two cysteine residues by substitution and/or addition at positions adjacent to the proline residue at position 171.
  • the disulfide bond can also be formed directly at position 171, that is, the proline residue at position 171 is replaced, and one or more amino acid residues at its adjacent positions are replaced or deleted Or after addition, a disulfide bond is formed between position 171 and its adjacent positions.
  • the adjacent sites refer to within three sites before and after; more preferably within two sites before and after.
  • the FGF21 mutein introduces two cysteine residues within three positions before and after the proline residue at position 171 by substitution and/or addition.
  • one or two amino acid residues are substituted and/or added within three positions before and after the proline residue at position 171, each introducing a cysteine residue.
  • the two cysteine residues forming the disulfide bond are separated by 1, 2 or 3 amino acids.
  • 1, 2 or 3 amino acids are separated between the two sites where the cysteine residue is substituted or added, or where the cysteine residue is replaced Or the two positions where the introduced cysteine residues are added are separated by 1, 2 or 3 amino acids by adding amino acid residues.
  • replace one amino acid residue with a cysteine residue within the first three positions of position 171, or within two positions before and after position 171, and within the three positions after position 171, or within two positions before and after position replace one amino acid residue with a cysteine residue within the dot; add one or two amino acid residues between positions 171 and One or two amino acid residues were added between position 171 and the position followed by a cysteine residue.
  • the one or two amino acid residues added are selected from glycine, alanine and serine.
  • the added amino acid residue is glycine.
  • 170G is replaced with 170C
  • 172S is replaced with 172C
  • one or two amino acid residues are inserted between 171P and 172C.
  • the FGF21 mutein comprises a protein selected from the group consisting of SEQ ID NO: 5, 6, 7, 8, 9, 11, 12, 13, 14, 15, 17, 18, 19, 20, 21, 23, 24, 25, Amino acid sequences shown in 26 and 27.
  • SEQ ID NO: 4, 16, and 22 are formed by adding one alanine, serine, and glycine residue before the first histidine in SEQ ID NO: 10 on the basis of SEQ ID NO: 10 Sequence, SEQ ID NO:5,17,23 and SEQ ID NO:11, SEQ ID NO:6,18,24 and SEQ ID NO:12, SEQ ID NO:7,19,25 and SEQ ID NO:13,
  • SEQ ID NO:8, 20, 26 and SEQ ID NO: 14, SEQ ID NO: 9, 21, 27 and SEQ ID NO: 15 is similar and will not be repeated.
  • replacing 121N with 121Q and 168M with 168L can weaken the deamination reaction at position 121 and the oxidation reaction at position 168.
  • adding an alanine, glycine or serine residue before the first histidine residue of the wild-type human FGF21 sequence can be used to remove the initiation of prokaryotic expression (such as E. coli expression) codon methionine.
  • the FGF21 mutein of the present invention also has chemical modifications, including but not limited to PEG and fatty acid chains.
  • the second aspect of the present invention provides a nucleic acid molecule encoding any one of the above FGF21 muteins.
  • the third aspect of the present invention provides a vector containing the above-mentioned nucleic acid molecule.
  • the fourth aspect of the present invention provides a genetically engineered cell containing the above-mentioned vector.
  • a fifth aspect of the present invention provides a method for preparing a protein, comprising the steps of:
  • the sixth aspect of the present invention provides a pharmaceutical composition, which contains the above-mentioned FGF21 mutein and pharmaceutically acceptable excipients.
  • the seventh aspect of the present invention provides the application of the FGF21 mutein in controlling blood sugar, blood lipid and reducing body weight.
  • the FGF21 mutein provided by the present invention can resist enzymatic cleavage and degradation of its C-terminus by FAP enzyme and maintain the activity of the C-terminus, that is, binding to the ⁇ -klotho (KLB) receptor, thereby prolonging its half-life and drug efficacy in vivo.
  • the FGF21 mutant in the specific embodiment of the present invention has a stronger ability to slow down the hydrolysis of FAP protease.
  • All amino acid sequence numbers involved in the present invention are based on wild-type FGF21 (SEQ ID NO: 1).
  • SEQ ID NO: 1 the first amino acid position is numbered 1, and the second is 2, so And so on down to the last amino acid.
  • Figure 1 is a schematic diagram of a molecular simulation structure of a disulfide bond formed on both sides of a proline residue at position 171 (taking 170C/173C as an example, SEQ ID NO: 5);
  • Fig. 2 is the SDS-PAGE electrophoresis image of the protein in Example 2 after expression and purification, wherein M is Marker;
  • FGF21 (121Q, 168L) mutant 1414 its amino acid sequence is shown in SEQ ID NO: 2, the mutation sites 121Q and 168L are to weaken the deamination reaction at position 121 and the oxidation reaction at position 168, which does not affect its activity or other functions (as disclosed in WO2011154349A2, CN113728013A), therefore, in the embodiment, mutant 1414 is used as a control, and the experimental results represent the results of wild-type FGF21.
  • FGF21 (P171G) mutant 1407 its amino acid sequence is shown in SEQ ID NO: 3
  • FGF21 (Del- P171) mutant 14008 its amino acid sequence is shown in SEQ ID NO: 4
  • FGF21 (170C/Ins-172G/173C) mutant 1409 its amino acid sequence is shown in SEQ ID NO: 5
  • FGF21 (170C/172C) Mutant 1410 its amino acid sequence is shown in SEQ ID NO: 6
  • FGF21 (170C/173C) mutant 1411 its amino acid sequence is shown in SEQ ID NO: 7
  • FGF21 (170C/Ins-172A/173C) mutant 1412 its amino acid sequence is shown in SEQ ID NO: 8
  • FGF21 (170C/Ins-172S/173C) mutant 1413 its amino acid sequence is shown in SEQ ID NO:
  • the seeds cultivated overnight were transferred to a Erlenmeyer flask containing Kana-resistant 500mL TB medium at a volume ratio of 1:50.
  • the initial OD600 was about 0.1, 37°C, 220rpm, and cultivated until the OD600 was 2.0, adding IPTG (final concentration 0.5mmol/L), 37°C, 220rpm overnight cultivation and harvested bacteria.
  • the protein is expressed in the form of inclusion body (IB).
  • Example 2 The inclusion bodies obtained through cell disruption and washing in Example 1 were dissolved using 8M urea and 10mM DTT. Inclusion body dissolution was performed at room temperature for 4 hours.
  • the refolding solution which contains 2M urea, 10mM cysteine and 20mM Tris-Cl buffer solution with a pH of 8.0, and incubate overnight at room temperature. During renaturation, continuous stirring is required at a speed of 200 rpm.
  • the combined protein was diluted 10 times and then subjected to Source 30Q ion exchange chromatography, using NaCl solution gradient elution with a gradient of 0M to 1.0M to further remove nucleic acids, some protein polymers and foreign proteins.
  • the combined protein was concentrated, and then purified by molecular sieve Superdex 200pg, PBS was used as the buffer, and the protein purified in this step was used as the final sample.
  • FGF21 mutant 1414 and mutants 1407, 1408 and 1409 all exist in the form of monomers, of which FGF21 mutant
  • the additionally introduced disulfide bonds in 1409 have completely formed intramolecular disulfide bonds, and there is no mistaken formation of intermolecular disulfide bonds that lead to the formation of FGF21 dimers.
  • FGF21 mutant 1410 formed a large number of dimers, indicating that the additional disulfide bonds introduced by the molecule could not form correct intramolecular disulfide bonds, but formed a large number of intermolecular disulfide bonds by mistake.
  • 1409 inserts a glycine (Ins-G172) behind the P171 position, and the insertion of G172 allows more space between C170-C173 to form an intramolecular disulfide bond.
  • G172 glycine
  • FGF21 mutant 1412 FGF21 mutant 1412
  • S172 FGF21 mutant 1413
  • FGF21 mutant 1411 moves the mutation after P171 to position 173 (SEQ ID NO: 7), and this mutant only produces a small amount of wrong dimer.
  • FAP is a protease existing in the body, which can specifically cut the amino acid between 171-172 of FGF21, resulting in incomplete C-terminus of FGF21 injected into the body or existing in the body, and then unable to bind with the ⁇ -klotho receptor in vivo Binds, loses activity and tissue specificity.
  • an in vitro FAP purchased from Baipusaisi, Cat. No. FAP-H5244
  • enzyme digestion experiment was carried out to select FGF21 mutants that were not digested. The experiment was carried out according to the literature (http://dx.doi.org/10.1016/j.molmet.2016.07.003), and the results are shown in Table 2.
  • Human ⁇ -Klotho receptor extracellular protein (Poly-His tag fused at the C-terminus) was expressed and purified by our company, and the sequence was obtained from the Uniprot database. Affinity determination was carried out using Biacore-8K. Specifically, human ⁇ -Klotho protein was immobilized on an NTA chip with an amount of 1000 RU, and the flow buffer used was PBS (containing 0.05% Tween-20). During the experiment, FGF21 mutants of different concentrations were passed through to detect their binding to ⁇ -Klotho. The final data were fitted using kinetics and the dissociation constant KD was calculated. The specific affinity results are shown in Table 3.
  • HEK293 cells overexpressing ⁇ -Klotho (the cells were constructed by Pharmaron) were used to test the activation of FGF21 and mutants on the ERK signaling pathway.
  • the specific experimental steps are as follows: DMEM + 10% FBS + 1*PS cell culture medium to culture HEK293 cells overexpressing ⁇ -Klotho, and when the cells were cultured to the exponential phase, take 20 ⁇ L of cell suspension and add it to the white 384 assay plate, add appropriate medium and appropriate Cell density (5000/well), cultured at 37°C and 5% CO 2 .
  • FGF21 and its mutants start from 3uM/6uM/15uM in the culture medium, 3-fold serial dilution, and add 10ul compound/well to the cell experiment plate.
  • the cells were placed in a 5% CO2 incubator at 37°C for 0.5 hours.
  • After removing the cell supernatant add 16 ⁇ L of cell lysis buffer and shake at room temperature for 30-60 minutes.
  • Add 4 ⁇ L of the premixed antibody solution prepared in the detection buffer seal the experimental plate with a sealing mold, and read the fluorescence emission at 665nm and 620nm after incubation at room temperature overnight, and measure the activity of the mutant by comparing its EC50 value.
  • Table 4 the 1407-1413 mutant (1410 not tested) has very similar biological activity to the FGF21 control mutant 1414.

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Abstract

本发明提供一种FGF21突变蛋白及其应用。该突变蛋白在野生型人类FGF21序列的基础上具有一些改变：171位的脯氨酸残基缺失，或通过一个或多个氨基酸残基的取代、缺失或添加而在171位的脯氨酸残基前后形成一对二硫键。本发明的FGF21突变蛋白可以抵抗成纤维细胞活化蛋白酶(FAP)对其C端的酶切降解，保持C端与β-klotho受体结合的活性，从而可以延长其体内半衰期和药效。

Description

一种FGF21突变蛋白及其应用 技术领域
本发明涉及一种FGF21突变蛋白及其应用，属于生物技术领域。
背景技术
人类成纤维细胞生长因子21(Fibroblast growth factor 21，FGF21)是一种由209个氨基酸组成的多肽，其中FGF21蛋白的N末端含有28个氨基酸构成的信号肽，因此经过信号肽剪切后形成的成熟FGF21由181个氨基酸组成(SEQ ID NO:1)。
FGF21在人类多种器官和组织中有表达，如肝脏、胰腺和脂肪组织中等。在人体中，FGF21可以分泌进入血液循环，并诱导肝脏、胰腺和脂肪组织中的多种信号通路和功能活动，进而实现对糖脂代谢的调节和对胰岛β细胞保护的生理功能。研究表明，在通过饮食诱导或者基因操作产生的肥胖小鼠中，注射FGF21蛋白能够显著的降低小鼠体重和血糖水平，同时小鼠血清中的甘油三酯(triglyceride)含量也显著降低。进一步的研究表明，FGF21可以改善肝脏的胰岛素敏感性，从而缓解葡萄糖不耐受。在更接近人类的哺乳类动物上的研究表明，FGF21可剂量依赖性地减少实验动物体重和体脂，如向患糖尿病的恒河猴施用FGF21，发现其空腹血浆葡萄糖和甘油三酯水平均有显著降低。此外，FGF21还能激活外分泌胰腺细胞和肝细胞信号通路，抑制肝糖原输出。
FGF21属于FGF(成纤维细胞生长因子)家族成员，然而，不同于大多数FGF所具有广谱的促有丝分裂的能力，FGF21并没有明显的促进细胞增殖的能力。有研究表明，FGF21转基因鼠在整个生命周期内，体内未发现肿瘤以及组织增生等异常状况。同时，药代动力学和药物安全性实验表明，超过药理学剂量的FGF21并不会出现低血糖症，这表明FGF21是一种潜在的理想治疗糖尿病以及肥胖等疾病的药物。
不同于大多数的FGF家族成员，FGF21激活下游信号通路需要同时通过FGF受体(Fibroblast growth factor receptors，FGFRs)和辅助受体β-klotho来实现，单独的β-klotho或者FGFR都不能激活FGF21信号。结构生物学研究表明，FGF21的结构可以分为两个部分，其中N端结构域主要FGFR的结合有关，而C端结构域则为一个相对柔性的序列，与β-klotho的结合有关。FGF21只有与β-klotho、FGFR形成一个三元复合体后，才能激活下游相关的信号通路，进而发挥其生物效应。
FGF21在控制血糖和降低体重等方面的生理功能为治疗相关疾病带来了希望，但是野生型FGF21存在着易被蛋白酶水解，分子量过小等缺点，其半衰期仅为0.5-2小 时，不适合直接作为药物。同时，FGF21蛋白的C末端含有的负责和β-klotho受体结合的柔性区域，很容易被成纤维细胞活化蛋白酶(Fibroblast Activation Protein，FAP)降解，其主要降解位点为Pro171周围。当P171被FAP蛋白酶切割以后，FGF21分子将不能再与β-klotho辅助受体结合，进而导致FGF21分子失活。面对这种困难，医药界通过进行酶切位点氨基酸定点突变制备长效融合蛋白，或将聚乙二醇、脂肪酸链等连接到多肽骨架等方法来提高FGF21的半衰期。在US 8,034,770 B2中描述了一些基于人野生型FGF21多肽序列的突变体，其中P171G或者P171A可有效地减慢FAP蛋白酶对FGF21蛋白C末端的水解，但无论是根据该专利的报道还是在我们的体外实验中，P171G仍然存在着一定程度的被FAP水解而失活的形式，寻找新的更有效的突变体减弱FAP蛋白酶的水解对于进一步提高FGF21蛋白的成药性具有很大的意义。
发明内容
天然的野生型人类FGF21蛋白具有如SEQ ID NO:1所示的氨基酸序列，该蛋白的171位脯氨酸残基(Pro)是FAP蛋白酶的酶切位点，本发明的目的是通过改变野生型FGF21的酶切位点和/或其附近的结构来降低FGF21的水解率，延长其体内半衰期和药效。
为了实现上述目的，本发明一个方面提供了一种FGF21突变蛋白，在一个具体实施方式中，其包含在野生型人类FGF21序列的基础上具有以下a或b的改变的氨基酸序列：
a.删除171位的脯氨酸残基；
b.通过一个或多个氨基酸残基的替换、删除或添加，在171位的脯氨酸残基处或其前后形成一对二硫键。
本发明的一种方案是直接删除171位点的脯氨酸残基(例如SEQ ID NO:4、10、16和22所示的序列)，使FGF21蛋白失去FAP的酶切位点，因而也就不会被水解。可选地，FGF21突变蛋白包含如SEQ ID NO:4所示的氨基酸序列。
本发明的另一种方案是在171位的脯氨酸残基邻近位点通过替换和/或添加引入两个半胱氨酸残基。
可选地，通过Pro171两边邻近的氨基酸残基的替换、删除或添加，在Pro171前后形成一对跨越该位点的二硫键，阻挡住Pro171的酶切位点，从而使其不易被水解。
可选地，所述二硫键也可以直接形成在171位点上，即对171位的脯氨酸残基进行替换，以及对其邻近位点的一个或多个氨基酸残基进行替换、删除或添加后，在171位以及其邻近位点之间形成二硫键。
可选地，所述邻近位点是指前后三个位点以内；进一步优选为前后两个位点以内。
在本发明的另一实施方式中，所述FGF21突变蛋白在171位的脯氨酸残基前后三个位点以内通过替换和/或添加各引入两个半胱氨酸残基。
可选地，在171位的脯氨酸残基前后三个位点以内通过一个或两个氨基酸残基替换和/或添加各引入一个半胱氨酸残基。
可选地，形成二硫键的两个半胱氨酸残基之间相隔1个、2个或3个氨基酸。
可选地，替换为半胱氨酸残基或添加引入的半胱氨酸残基的两个位点之间相隔1个、2个或3个氨基酸，或者在替换为半胱氨酸残基或添加引入的半胱氨酸残基的两个位点之间通过添加氨基酸残基的方式使之相隔1个、2个或3个氨基酸。
可选地，在171位前三个位点以内、或前后两个位点以内将一个氨基酸残基替换为半胱氨酸残基，在171位后三个位点以内、或前后两个位点以内将一个氨基酸残基替换为半胱氨酸残基；在171位与171位前替换为半胱氨酸残基的位点之间添加一或两个氨基酸残基，和/或在171位与171位后替换为半胱氨酸残基的位点之间添加一或两个氨基酸残基。
可选地，添加的所述一或两个氨基酸残基选自甘氨酸、丙氨酸和丝氨酸。优选地，添加的氨基酸残基为甘氨酸。
通过使形成二硫键的两个半胱氨酸残基之间间隔2个或3个氨基酸残基，形成前述间隔引入的氨基酸为甘氨酸、丙氨酸或丝氨酸(优选为甘氨酸)时，能够减少FGF21突变体形成错配的二聚体。
可选地，将170G替换为170C，将172S替换为172C，在171P和172C之间插入一个或两个氨基酸残基。
可选地，FGF21突变蛋白包含选自如SEQ ID NO:5、6、7、8、9、11、12、13、14、15、17、18、19、20、21、23、24、25、26和27所示的氨基酸序列。其中，SEQ ID NO:4、16、22是在SEQ ID NO:10的基础上在SEQ ID NO:10的第1位组氨酸之前分别增加1个丙氨酸、丝氨酸、甘氨酸残基形成的序列，SEQ ID NO:5、17、23与SEQ ID NO:11，SEQ ID NO:6、18、24与SEQ ID NO:12，SEQ ID NO:7、19、25与SEQ ID NO:13，SEQ ID NO:8、20、26与SEQ ID NO:14、SEQ ID NO:9、21、27与SEQ ID NO:15的关系类似，不再赘述。
在另一个实施方案中，对171位的脯氨酸残基进行替换，以及对其邻近位点的一个或多个氨基酸残基进行替换、删除或添加后，在171位以及其邻近位点之间形成二硫键。
可选地，将121N替换为121Q，将168M替换为168L，可以减弱121位的脱氨基反应和168位的氧化反应。
可选地，在野生型人类FGF21序列的第一位的组氨酸残基前添加1个丙氨酸、甘氨酸或丝氨酸残基，可以用于去除原核表达(如大肠杆菌表达)时的起始密码子甲硫氨酸。
进一步，本发明所述FGF21突变蛋白还具有化学修饰，所述修饰包括但不限于PEG和脂肪酸链。
本发明的第二方面提供了编码任意一个上述FGF21突变蛋白的核酸分子。
本发明的第三方面提供了一种载体，其含有上述的核酸分子。
本发明的第四方面提供了一种基因工程细胞，其含有上述的载体。
本发明的第五方面提供了一种蛋白的制备方法，包括步骤：
1)构建如上所述的载体；
2)将构建的载体转染入宿主细胞；
3)在适合表达的条件下，培养所述宿主细胞；
4)从培养物中分离出本发明所述的FGF21突变蛋白。
本发明的第六方面提供了一种药物组合物，该组合物含有如上所述的FGF21突变蛋白以及药学上可接受的赋形剂。
本发明的第七方面提供了所述的FGF21突变蛋白在控制血糖、血脂和降低体重方面的应用。
本发明所提供的FGF21突变蛋白可以抵抗FAP酶对其C端的酶切降解并保持C端的活性，即与β-klotho(KLB)受体的结合，从而可以延长其体内半衰期和药效。且与突变体P171G相比，本发明具体实施方式中的FGF21突变体具备更强的减慢FAP蛋白酶水解的能力。
本发明中所涉及的所有氨基酸序列编号均以野生型FGF21(SEQ ID NO:1)为准，在SEQ ID NO:1中第一个氨基酸位点编号为1，第二个为2，以此类推至最后一个氨基酸。
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是在171位的脯氨酸残基两边(以170C/173C为例，SEQ ID NO:5)形成二硫键的分子模拟结构示意图；
图2是实施例2中蛋白经过表达纯化后SDS-PAGE电泳图，其中M为Marker；
具体实施方式
实施例1 重组表达载体的构建以及表达
1.1、表达质粒构建
委托苏州金唯智生物科技有限公司分别合成能够表达如下所示编号为1414、1407、1408、1409、1410、1411、1412和1413的蛋白的基因，并将基因克隆到pET21b载体 中，然后转化到BL21(DE3)菌株中。
FGF21(121Q，168L)突变体1414，其氨基酸序列如SEQ ID NO:2所示，突变位点121Q与168L是为了减弱121位的脱氨基反应和168位的氧化反应，本身并不影响其活力或其它功能(如WO2011154349A2、CN113728013A中所公开的)，因此，实施例中以突变体1414作为对照，其实验结果代表野生型FGF21的结果，另外，在FGF21野生型的第1位组氨酸残基前添加1个丙氨酸残基，用于去除原核表达时的启示密码子甲硫氨酸；FGF21(P171G)突变体1407，其氨基酸序列如SEQ ID NO:3所示；FGF21(Del-P171)突变体1408，其氨基酸序列如SEQ ID NO:4所示；FGF21(170C/Ins-172G/173C)突变体1409，其氨基酸序列如SEQ ID NO:5所示；FGF21(170C/172C)突变体1410，其氨基酸序列如SEQ ID NO:6所示；FGF21(170C/173C)突变体1411，其氨基酸序列如SEQ ID NO:7所示；FGF21(170C/Ins-172A/173C)突变体1412，其氨基酸序列如SEQ ID NO:8所示；FGF21(170C/Ins-172S/173C)突变体1413，其氨基酸序列如SEQ ID NO:9所示；
1.2、表达
过夜培养的种子按1:50的体积比转接到含Kana抗性的500mL TB培养基的三角瓶中，初始OD600大约为0.1，37℃，220rpm，培养至OD600为2.0，加入IPTG(终浓度为0.5mmol/L)，37℃、220rpm过夜培养后收菌。蛋白以包涵体(inclusion body，IB)的形式表达。
实施例2 FGF21及突变体蛋白的复性和纯化
2.1包涵体变性溶解
使用8M尿素和10mM DTT溶解实施例1中经过细胞破碎和清洗得到的包涵体。包涵体溶解在室温进行，时间为4小时。
2.2复性
将以上溶解的包涵体溶液加入复性液中，复性溶液中含有2M尿素，10mM cysteine以及pH为8.0的20mM Tris-Cl缓冲液，室温孵育过夜。复性期间需要连续搅拌，转速为200转/分钟。
2.3 FGF21及突变体蛋白进行疏水作用层析Phenyl纯化
加入终浓度为2M NaCl和20mM Tris-Cl缓冲液后流过疏水层析phenyl柱(购于GE)，然后进行NaCl溶液梯度洗脱，梯度为2M至0M。根据SDS-PAGE选择目的蛋白所在的收集管合并保存，此步可以捕获目的蛋白并去除一些杂蛋白。
2.4 FGF21及突变体蛋白进行离子交换层析Source 30Q纯化
疏水作用层析后合并的蛋白10倍稀释后进行Source 30Q离子交换层析，采用NaCl 溶液梯度洗脱，梯度为0M到1.0M，以进一步去除核酸和一些蛋白高聚体以及杂蛋白。
2.5 FGF21及突变体蛋白进行分子筛精细纯化
离子交换层析source 30Q纯化后将合并的蛋白浓缩，然后进行分子筛Superdex 200pg精细纯化，PBS作为缓冲液，此步纯化的蛋白作为最终样品。
纯化信息如表1所示，所有蛋白都达到了电泳纯的纯度。
表1 纯化得到的FGF21及突变体浓度表
蛋白编号 浓度(mg/mL)
1414 3.2
1407 2.9
1408 3.3
1409 1.6
1410 1.78
1411 1.25
1412 2.2
1413 2.5
从最终FGF21突变体1414及突变体纯化得到的蛋白的SDS-PAGE分析图(图2)可得知，FGF21突变体1414和突变体1407、1408及1409均以单体形式存在，其中FGF21突变体1409中额外引入的二硫键已经完全形成分子内二硫键，并没有错误的形成分子间二硫键而导致FGF21二聚体的形成。FGF21突变体1410则大量形成二聚体，表明该分子额外引入的二硫键并不能形成正确的分子内二硫键，而是错误的形成了大量的分子间二硫键。对比1409和1410的分子设计，1409在P171位后面插入了一个甘氨酸(Ins-G172)，G172的插入可使得C170-C173之间有更多的空间形成分子内二硫键。如将插入的氨基酸G172置换为A172(FGF21突变体1412)或S172(FGF21突变体1413)能起到类似的技术效果，但仍可形成少量的错误的二聚体。FGF21突变体1411是将P171后的突变挪至173位(SEQ ID NO:7)，该突变体仅产生微量的错误二聚体。
实施例3体外进行FAP酶切测试
FAP是体内存在的一种蛋白酶，它能特异性地切割FGF21第171-172之间的氨基酸，造成注射进体内或是体内存在的FGF21的C端不完整，进而不能与体内β-klotho受体结合，失去活性和组织特异性。本实施例利用此特性，进行体外FAP(购自百普 赛斯，货号FAP-H5244)酶切实验，挑选出不被酶切的FGF21突变体。实验按照文献(http://dx.doi.org/10.1016/j.molmet.2016.07.003)进行，结果如表2所示，当阴性对照FGF21突变体1414完全被FAP酶切完毕时，FGF21突变体P171G(1407)在该实验条件下，也有约一半被酶切，而该发明中设计的突变体1408-1413(1410未检测)仅有微量酶切或不被酶切。
表2 FGF21及突变体被FAP酶切实验
FGF21及突变体 酶切产生片段的百分比
1407 46％
1408 <2％
1409 0
1410 未测试
1411 0
1412 0
1413 0
1414 100％
实施例4 FGF21突变体与β-Klotho受体的结合测试
人源的β-Klotho受体胞外端蛋白(C末端融合有Poly-His tag)由本公司表达和纯化，序列来自Uniprot数据库。亲和力测定使用Biacore-8K进行，具体是将人源β-Klotho蛋白固定于NTA芯片上，固定量为1000RU，使用的流动缓冲液为PBS(含有0.05％Tween-20)。实验时通过流过不同浓度的FGF21突变体，检测其对β-Klotho结合的情况。最终数据使用动力学进行拟合并计算解离常数KD。具体亲和力结果如表3所示。可以看到1407-1413突变体(1410未测试)均与FGF21对照突变体1414表现出了相似的对β-Klotho的亲和力。这表明各突变体的亲和力活性未受到抑制，并且每种突变体的活性彼此相似。
表3 FGF21及突变体同β-Klotho结合活性测试
FGF21及突变体 同β-Klotho的结合强度(M)
1407 4.25E-08
1408 6.75E-08
1409 5.94E-08
1410 未测试
1411 6.94E-08
1412 6.99E-08
1413 6.46E-08
1414 5.12E-08
实施例5 FGF21及突变体在β-Klotho-ERK细胞活性实验
为了进一步验证该发明中的FGF21及突变体的活性，使用过量表达β-Klotho的HEK293细胞(细胞由康龙化成构建)测试了FGF21及突变的对ERK信号通路的激活作用，具体实验步骤为使用DMEM+10％FBS+1*PS细胞培养基培养过表达β-Klotho的HEK293细胞，并在细胞培养至指数期时取20μL细胞悬液加入白色384测定板中，加入适当的培养基和适当的细胞密度(5000/孔)中，置于37℃5％CO 2下培养。FGF21及突变体在培养基中从3uM/6uM/15uM起始，3倍梯度稀释，在细胞实验板中加入10ul化合物/孔。加入待测蛋白后的细胞置于37℃5％CO 2培养箱中0.5小时。去除细胞上清液后加入16μL细胞裂解缓冲液，在室温下振荡30～60分钟。加入4μL检测缓冲液中制备的预混抗体溶液，用封板模封好实验板，在室温下培养过夜后读取665nm和620nm处的荧光发射，通过比较其EC50值来测量突变体的活性。结果如表4所示，1407-1413突变体(1410未测试)相对于FGF21对照突变体1414，具备非常类似的生物学活性。
表4 FGF21及突变体细胞活性测试
FGF21及突变体 在β-Klotho-ERK细胞活性(nM)
1407 1.152
1408 1.484
1409 2.140
1410 未检测
1411 0.999
1412 2.516
1413 2.093
1414 0.850
以上详细描述了本发明的较佳具体实施例。应当理解，本领域的普通技术人员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。

Claims (17)

  1. 一种FGF21突变蛋白，其特征在于，包含在野生型人类FGF21序列的基础上具有以下a或b的改变的氨基酸序列：
    a.删除171位的脯氨酸残基；
    b.通过一个或多个氨基酸残基的替换、删除或插入，在171位的脯氨酸残基处或其前后形成一对二硫键。
  2. 如权利要求1所述的FGF21突变蛋白，其特征在于，包含选自如SEQ ID NO:4、10、16和22所示的氨基酸序列。
  3. 如权利要求1所述的FGF21突变蛋白，其特征在于，在171位的脯氨酸残基前后三个位点以内通过替换和/或添加引入两个半胱氨酸残基。
  4. 如权利要求3所述的FGF21突变蛋白，其特征在于，形成二硫键的两个半胱氨酸残基之间相隔1个、2个或3个氨基酸。
  5. 如权利要求4所述的FGF21突变蛋白，其特征在于，替换为半胱氨酸残基或添加引入的半胱氨酸残基的两个位点之间相隔1个、2个或3个氨基酸，或者在替换为半胱氨酸残基或添加引入的半胱氨酸残基的两个位点之间通过添加氨基酸残基的方式使之相隔1个、2个或3个氨基酸。
  6. 如权利要求3所述的FGF21突变蛋白，其特征在于：
    a.在171位前三个位点以内将一个氨基酸残基替换为半胱氨酸残基；
    b.在171位后三个位点以内将一个氨基酸残基替换为半胱氨酸残基；
    c.在171位与171位前替换为半胱氨酸残基的位点之间添加一或两个氨基酸残基，和/或在171位与171位后替换为半胱氨酸残基的位点之间添加一或两个氨基酸残基。
  7. 如权利要求6所述的FGF21突变蛋白，其特征在于，添加的所述一或两个氨基酸残基选自甘氨酸、丙氨酸或丝氨酸。
  8. 如权利要求6所述的FGF21突变蛋白，其特征在于:
    a.将170G替换为170C；
    b.将172S替换为172C；
    c.在171P和172C之间插入一个或两个氨基酸残基。
  9. 如权利要求3所述的FGF21突变蛋白，其特征在于，包含选自如SEQ ID NO:5、6、7、8、9、11、12、13、14、15、17、18、19、20、21、23、24、25、26和27所示的氨基酸序列。
  10. 如权利要求1所述的FGF21突变蛋白，其特征在于，对171位的脯氨酸残基进行替换，以及对其邻近位点的一个或多个氨基酸残基进行替换、删除或添加后，在171位以及其邻近位点之间形成二硫键。
  11. 如权利要求1所述的FGF21突变蛋白，其特征在于，将121N替换为121Q，将168M替换为168L；和/或在野生型人类FGF21序列的第1位的组氨酸残基前添加1个丙氨酸、甘氨酸或丝氨酸残基。
  12. 如权利要求1所述的FGF21突变蛋白，其特征在于，还具有化学修饰。
  13. 一种核酸分子，其特征在于，其编码如权利要求1-12中任一项所述的FGF21突变蛋白。
  14. 一种载体，其特征在于，含有如权利要求13所述的核酸分子。
  15. 一种基因工程细胞，其特征在于，其含有如权利要求14所述的载体。
  16. 一种药物组合，其特征在于，含有如权利要求1-12中任一项所述的FGF21突变蛋白以及药学上可接受的赋形剂。
  17. 如权利要求1-12中任一项所述的FGF21突变蛋白或权利要求16所述的药物组合物在控制血糖、血脂和降低体重方面的应用。
PCT/CN2022/110072 2021-09-08 2022-08-03 一种fgf21突变蛋白及其应用 Ceased WO2023035817A1 (zh)

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Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8034770B2 (en) 2008-06-04 2011-10-11 Amgen Inc. FGF21 polypeptides comprising two or more mutations
WO2011154349A2 (en) 2010-06-08 2011-12-15 Novo Nordisk A/S Fgf21 analogues and derivatives
CN107056925A (zh) * 2017-04-28 2017-08-18 中国科学院合肥物质科学研究院 人fgf21突变体、其制备方法及用途
CN107188950A (zh) * 2009-05-05 2017-09-22 安姆根有限公司 Fgf21突变体及其用途
CN108602869A (zh) * 2015-12-02 2018-09-28 赛诺菲 Fgf21变体
CN110128525A (zh) * 2018-02-08 2019-08-16 广东东阳光药业有限公司 Fgf21变体、融合蛋白及其应用
CN113366024A (zh) * 2020-01-08 2021-09-07 上海翰森生物医药科技有限公司 Fgf21突变体蛋白及其融合蛋白
CN113728013A (zh) 2020-01-11 2021-11-30 北京质肽生物医药科技有限公司 Glp-1和fgf21的融合蛋白的缀合物

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2358749B1 (en) * 2008-10-10 2018-07-18 Amgen, Inc Fgf21 mutants and uses thereof
AU2010246038A1 (en) * 2009-05-05 2011-12-01 Amgen Inc. FGF21 mutants and uses thereof
CN107108710B (zh) * 2014-10-24 2022-02-15 百时美施贵宝公司 修饰的fgf-21多肽及其用途
KR102427527B1 (ko) * 2014-12-23 2022-08-01 노보 노르디스크 에이/에스 Fgf21 유도체 및 그것의 용도
US20180280474A1 (en) * 2015-10-01 2018-10-04 Amgen Inc. Treatment of bile acid disorders

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8034770B2 (en) 2008-06-04 2011-10-11 Amgen Inc. FGF21 polypeptides comprising two or more mutations
CN107188950A (zh) * 2009-05-05 2017-09-22 安姆根有限公司 Fgf21突变体及其用途
WO2011154349A2 (en) 2010-06-08 2011-12-15 Novo Nordisk A/S Fgf21 analogues and derivatives
CN108602869A (zh) * 2015-12-02 2018-09-28 赛诺菲 Fgf21变体
CN107056925A (zh) * 2017-04-28 2017-08-18 中国科学院合肥物质科学研究院 人fgf21突变体、其制备方法及用途
CN110128525A (zh) * 2018-02-08 2019-08-16 广东东阳光药业有限公司 Fgf21变体、融合蛋白及其应用
CN113366024A (zh) * 2020-01-08 2021-09-07 上海翰森生物医药科技有限公司 Fgf21突变体蛋白及其融合蛋白
CN113728013A (zh) 2020-01-11 2021-11-30 北京质肽生物医药科技有限公司 Glp-1和fgf21的融合蛋白的缀合物

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