CN106008717B

CN106008717B - Long-acting recombinant GLP-1 fusion protein and preparation method and application thereof

Info

Publication number: CN106008717B
Application number: CN201610046012.7A
Authority: CN
Inventors: 宋海峰; 高新; 杨懿; 万德有; 冯红茹; 崔新玲; 刘蕴慧; 杨利; 陈方
Original assignee: BEIJING C&N INTERNATIONAL SCI-TECH Co Ltd
Current assignee: BEIJING C&N INTERNATIONAL SCI-TECH Co Ltd
Priority date: 2016-01-22
Filing date: 2016-01-22
Publication date: 2019-12-17
Anticipated expiration: 2036-01-22
Also published as: CN106008717A

Abstract

The invention belongs to the field of biological pharmacy, and provides a long-acting recombinant GLP-1 fusion protein, and a preparation method and application thereof. The long-acting recombinant GLP-1 fusion protein is obtained by screening through a cell surface display technology. The recombinant GLP-1 fusion protein has higher hypoglycemic activity, lower immunogenicity and longer in-vivo half-life, so the recombinant GLP-1 fusion protein can be used for treating diabetes mellitus and has good clinical application prospect.

Description

long-acting recombinant GLP-1 fusion protein and preparation method and application thereof

Technical Field

The invention belongs to the field of biological pharmacy. The invention provides a long-acting recombinant GLP-1 fusion protein, a preparation method and application thereof.

Background

Type II Diabetes (Type II Diabetes) is a chronic disease characterized by insulin resistance and insufficient insulin secretion, and is taken as a main Type of Diabetes, Type II Diabetes patients account for more than 90% of the total population of Diabetes, and are predicted to reach 3 hundred million of the population of the global Type II Diabetes patients in 2025 years, and become a disease which seriously harms human health after cancer, the Type II Diabetes in China has wide morbidity, and if the patients cannot be treated in time, various Diabetes complications are easily caused, and a great burden is caused to the patients and the country, so the demand for developing safe and efficient Type II Diabetes treatment medicines is extremely high.

GLP-1 receptor agonists are a new class of drugs developed in recent years for the treatment of type II diabetes.

Native GLP-1 isAn incretin hormone secreted by human body and transcribed by glucagon gene. It is secreted by intestinal L cells, synthesized and secreted by feeding stimuli. By linking to B family G protein-coupled receptors, GLP-1 can activate intracellular adenylate cyclase (adenylate cyclase), Protein Kinase A (PKA), guanylate converting factor (cAMP-regulated nucleotide exchange factor II, Epac2), thereby increasing intracellular Ca²⁺Concentration, promoting beta cell exocytosis of insulin. Pharmacological research shows that GLP-1 has the function of insulin secretion dependent on blood sugar, can inhibit the secretion of glucagon under the condition of hyperglycemia, and indirectly achieves the purpose of reducing weight by delaying gastric emptying and regulating appetite. Compared with insulin and other insulin secretagogues, GLP-1 has the most remarkable advantages of completing the function of reducing blood sugar strictly according to the blood sugar threshold and relieving the risk factors of diabetes by reducing body weight.

however, native GLP-1 is easily degraded and has a very short serum half-life. Researchers have employed a variety of strategies to extend the serum half-life of GLP-1, including amino acid mutations to resist degradation by Dipeptidyl-peptidase (DPP-iv) and Neutral Endopeptidase (NEP), fusion with albumin or IgG Fc fragments, nano-modification encapsulation, and the like. Among them, the strategy of using IgG Fc and oligopeptide or polypeptide to form fusion protein to prolong serum half-life has been applied more, and in the prior art, fusion protein of GLP-1 or its mutant and IgG4Fc or its mutant exists.

However, to obtain a long-acting GLP-1 fusion protein that can be clinically applied, the fusion Fc fragment is selected such that the bioactivity of GLP-1 is maintained and such that the half-life is as long as possible. Therefore, by screening the GLP-1 and Fc fragments used for constructing the fusion protein in combination by an appropriate research method, it is possible to obtain a more ideal GLP-1-Fc fusion protein.

The invention screens a GLP-1-Fc fusion protein coding sequence with high activity and long half-life from a GLP-1-Fc fusion protein coding gene mutation library by means of a mammal cell surface display technology, and further obtains an amino acid sequence of the GLP-1-Fc fusion protein coding sequence, and the obtained fusion protein is expected to be used for the treatment research of type II diabetes.

The invention content is as follows:

In view of the above technical problems, the present invention aims to provide a screening method for obtaining a sequence of a GLP-1-Fc fusion protein by means of activity screening, and further provides a pharmaceutical composition comprising the GLP-1-Fc fusion protein and a use thereof.

To achieve the above and other related objects, in a first aspect, the present invention provides a method for constructing and screening a cDNA encoding a GLP-1-Fc fusion protein, which comprises preparing 3 parts of the coding sequence of the GLP-1-Fc fusion protein: a cDNA sequence encoding GLP-1; a cDNA sequence encoding a flexible linker peptide, and a mutant cDNA sequence encoding Fc. The flexible connecting peptide consists of glycine and serine, namely, Gly containing 2-5 groups₄Ser; the cDNA sequence of the coding flexible connecting peptide is shown as SEQ ID NO 2-5; the Fc-encoding mutant cDNA sequence was subjected to error-prone PCR amplification using 3 Fc sequences of IgG1, IgG2, and IgG4 as mixed templates using universal primers to obtain a prepared cDNA library. And (3) connecting the cDNA sequence of the GLP-1, the cDNA sequence of the flexible connecting peptide and the cDNA sequence of different Fc mutations by using a type IIs restriction endonuclease to form a cDNA library containing the different Fc mutation sequences. The library is ligated with a mammalian surface Display vector p-Display to construct a surface Display library for screening of candidate sequences. And (3) transfecting the plasmid library into 293T cells, culturing for 60 hours, and then carrying out magnetic bead sorting and flow sorting. Firstly, magnetic bead sorting: co-incubating the transfected 293T cells and magnetic beads fixed with Fc gamma R, and collecting unbound cells for flow sorting; goat anti-human IgG with FITC label was then used to co-incubate with the magnetic bead sorted cells. Plasmids were extracted from the sorted cells and transformed into e.coli DH5 α for amplification as a sub-library for the next round of screening. In the next round of screening, the concentration of FITC label was reduced to 1/10 in the first round. After three rounds of elutriation and screening, the obtained positive plasmid is sequenced and analyzed to determine the DNA sequence.

In one embodiment of the present invention, the DNA sequence encoding GLP-1-Fc contained in plasmid No. 11-04 to be screened is shown in SEQ ID NO 13.

In another aspect, the invention provides a method for preparing the GLP-1-Fc fusion protein, wherein cDNA encoding the GLP-1-Fc fusion protein is connected into a stable expression vector of a mammalian cell, and a CHO-K1 cell is transfected to establish a stable expression cell strain. The recombinant GLP-1-Fc fusion protein is secreted and expressed in cell supernatant, and the recombinant GLP-1-Fc fusion protein is prepared by protein A affinity chromatography purification.

In one embodiment of the invention, the amino acid sequence of the GLP-1-Fc fusion protein has the amino acid sequence shown in SEQ ID NO. 14.

In one aspect, the invention provides a recombinant GLP-1-Fc fusion protein comprising GLP-1, a linker peptide, and a human immunoglobulin Fc variant. The connecting peptide consists of Gly of 2-5 groups₄Ser, and 3 groups are preferred.

In a preferred embodiment of the invention, the recombinant GLP-1-Fc fusion protein has the amino acid sequence shown in SEQ ID NO. 14.

In another aspect, the invention provides a GLP-1-Fc fusion protein-encoding gene comprising a cDNA encoding GLP-1, a cDNA encoding a flexible linker, and a mutated cDNA sequence encoding Fc.

Wherein the cDNA for coding GLP-1 has a nucleotide sequence shown as SEQ ID NO. 1.

the cDNA for coding the flexible connecting peptide has a nucleotide sequence shown in SEQ ID NO. 2-5, and preferably SEQ ID NO. 3.

in a preferred embodiment of the present invention, the GLP-1-Fc fusion protein encoding gene has a nucleotide sequence as shown in SEQ ID NO 13.

The test for measuring the hypoglycemic effect and the half-life period proves that: compared with the present Duraglutide, the recombinant GLP-1-Fc fusion protein prepared by the invention has better hypoglycemic effect, lower immunogenicity and longer half-life by 10%.

Therefore, the invention provides a pharmaceutical composition for reducing blood sugar and treating type II diabetes, which comprises the recombinant GLP-1-Fc fusion protein and a pharmaceutically acceptable carrier.

In another aspect, the invention provides the use of the recombinant GLP-1-Fc fusion protein in the preparation of a medicament for preventing and treating type II diabetes.

On the other hand, the invention provides the application of the GLP-1-Fc fusion protein coding gene in preparing a medicament for preventing and treating type II diabetes.

In another aspect, the present invention provides a method for constructing a display-type GLP-1-Fc mutation library, comprising: (a) synthesizing a cDNA sequence for coding GLP-1; (b) synthesizing a cDNA sequence encoding a flexible linker peptide; (c) amplifying Fc-encoding mutant cdnas, (i) synthesizing IgG1, IgG2, and IgG4 cdnas; (ii) synthesizing a primer; (iii) performing PCR amplification using the IgG1, IgG2, and IgG4cDNA mixture as a template (e.g., 1: 1: 1 mixture) using the primers synthesized in (ii) to obtain Fc mutated cDNAs; (d) and (3) carrying out restriction enzyme cutting on the cDNA sequence coding the GLP-1, the cDNA sequence coding the flexible connecting peptide and the different Fc mutant cDNA sequences, and then connecting to obtain a GLP-1-Fc cDNA library containing different Fc mutant sequences, wherein the used restriction enzyme is type IIs restriction enzyme.

In the present invention, the main objective of fusion Fc is to obtain a fusion protein sequence with longer half-life while maintaining GLP-1 activity. The introduction of Fc sequences, which may mediate antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), requires the mutation and screening of Fc sequences to obtain new molecules with long half-lives and low immunotoxicity. As shown in FIG. 1, the mutation is realized by using 3 subtypes of IgG-Fc as a mixed template and combining error-prone PCR with DNA shuffling, and the obtained mutation library is connected with flexible connecting peptide sequences with different lengths by IIs type restriction endonuclease and then spliced with GLP-1 to generate a cDNA library containing different Fc mutants.

On the other hand, the invention provides a method for preparing GLP-1-Fc fusion protein, which comprises the steps of utilizing a mammal surface display carrier p-DisplayS to express and display GLP-1-Fc fusion protein coded by a mutant cDNA library, and finally obtaining a fusion protein coding sequence with a target function through magnetic bead and flow cytometer sorting. The coding sequence is further constructed into mammalian cell expression vectors (such as SGLs), and mammalian host cells (such as CHO-K1 cells are transfected to prepare the recombinant GLP-1-Fc fusion protein.

Drawings

FIG. 1 is a schematic diagram illustrating the construction of a screening library of the present invention, wherein G is GLP-1, L is flexible linker peptides of different lengths, and F is an Fc fragment. The GLP-1 molecule adopts a general GLP-1 mutant sequence and has the function of resisting the degradation of DDP-IV; the number of amino acids of the flexible connecting peptide may influence the activity exertion of GLP-1, so that connecting peptides with different lengths are designed; in the present invention, the main objective of fusion Fc is to obtain a fusion protein sequence with longer half-life while maintaining GLP-1 activity. The introduction of Fc sequences, which may mediate antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), requires the mutation and screening of Fc sequences to obtain new molecules with long half-lives and low immunotoxicity. As shown in figure 1, mutation is realized by taking 3 subtypes of IgG-Fc as a mixed template and combining error-prone PCR with DNA shuffling, and the obtained mutation library is connected with flexible connecting peptide sequences with different lengths through IIs type restriction endonuclease and then spliced with GLP-1 to generate a cDNA library containing different Fc mutants. GLP-1-Fc fusion protein encoded by the mutant cDNA library is expressed and displayed by using a mammalian surface display vector p-DisplayS.

FIG. 2 shows the results of the homology alignment between Fc-encoding DNA sequences of IgG1, IgG2, and IgG 4. Wherein a and b are regions for designing the upstream primer of PCR, and a part of the homologous part (the part marked by a frame line in FIG. 2) of b corresponds to the 3' end of the upstream primer; part a corresponds to the 5' end of the primer.

FIG. 3 is a graph showing the results of the pharmacological effects of the recombinant GLP-1-Fc fusion protein of the present invention in mice.

Examples

Example 1: construction of display type GLP-1-Fc mutation library

1.1 Synthesis of library construction templates and primers:

Respectively synthesizing GLP-1 coding cDNA shown as SEQ ID NO. 1 (Sfi restriction enzyme cutting site is introduced at the upstream of the cDNA, BsmBI restriction enzyme cutting site is introduced at the downstream of the cDNA), flexible connecting peptide coding cDNA shown as SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 and SEQ ID NO. 5 (BsmBI restriction enzyme cutting site is contained at the upstream of each sequence, BsaI restriction enzyme cutting site is contained at the downstream of each sequence), and coding cDNA of Fc fragments of IgG1, IgG2 and IgG4 shown as SEQ ID NO. 6, SEQ ID NO. 7 and SEQ ID NO. 8;

primers for error-prone PCR mutagenesis of Fc fragment were designed as follows:

The upstream primers used in PCR were divided into two parts a and b, based on homologous alignment of the Fc-encoding DNA sequences of IgG1, IgG2 and IgG4, wherein part b is located at the 3 'end of the primers and contains a universal sequence that matches the 5' homologous sequence of the different Fc-encoding DNAs (see the boxed portion in FIG. 2); part a is positioned at the 5 'end of the primer and corresponds to nucleotide sequences of different Fc, a BsaI enzyme cutting site is introduced at the 5' end of the primer so as to be spliced with the flexible peptide, and 3 upstream primers are shown as SEQ ID NO 9, SEQ ID NO 10 and SEQ ID NO 11.

The downstream primer is a universal primer, is complementary with a 3' end homologous sequence of an Fc coding DNA sequence, and is introduced into a SalI enzyme cutting site so as to be connected with a P-displayS vector, and the sequence is shown as SEQ ID NO. 12.

All DNA sequences and primers were synthesized by Genescript.

1.2 PCR mutation amplification

Prepared and PCR amplified as in table 1 below:

Table 1: error-prone PCR reaction liquid preparation table

PCR amplification conditions were 94 ℃ for 3 min; 1min at 94 ℃, 1min at 53 ℃, 2min at 72 ℃,30 cycles, and finally 10min at 72 ℃. And purifying the error-prone PCR amplification product by using a DNA purification kit.

Respectively adopting restriction endonuclease SfiI/SalI double-enzyme digestion pDisplay display vector, SfiI/BmbI double-enzyme digestion GLP-1 sequence SEQ ID NO:1, BsaI/BmbI double-enzyme digestion flexible connecting peptide coding sequence SEQ ID NO:2-5 mixture and BsaI/SalI double-enzyme digestion error-prone PCR product, purifying all enzyme digestion products, connecting the enzyme digestion products together through DNA ligase, transforming E.coliTOP10 competent cells, coating transformed bacterial liquid on an LB plate (containing 100g/L Amp), transferring a transformant to an LB liquid culture medium for culturing after 12h, and obtaining mutant plasmid library pDisplay-GLP-1-Fcs.

Example 2: screening of mutant plasmids

2.1 transfection of the mutant plasmid library into 293T:

The mutant plasmid library pDisplay-GLP-1-Fcs obtained in example 1 was transiently transfected into 293T cells using PEI reagent (Invitrogen), and after 60 hours, the cells were digested into individual cells using trypsin-free digestion solution (Invitrogen), and washed 3 times with PBS for use.

2.2 magnetic bead screening to remove cells reactive with Fc γ I-III:

Biotinylated recombinant human CD64, CD32a, CD16a proteins were purchased from beijing yi qian shenzhou biotechnology limited. Streptavidin magnetic bead sorting column VarioMACS is a product of american and whirly companies, germany.

Cells transfected with the mutant plasmid library were counted at about 1X 10⁸Cells are centrifuged at 300 Xg for 10min to remove supernatant, then resuspended by using a labeling buffer solution, and labeled by adding biotinylated recombinant human CD64, CD32a and CD16a proteins at 1 mu g/ml for 10 min; the cells were washed 3 times with 3ml of labeling buffer by centrifugation, resuspended in 900. mu.L of labeling buffer, 100. mu.L of streptavidin magnetic beads were added, incubated for 15min at 4 ℃ on a rocking platform, washed 3 times with 3ml of labeling buffer by centrifugation, and the cells were resuspended in 500. mu.L of separation buffer.

The vario macs was placed on a magnetic sorting rack, rinsed with 60ml of separation buffer, then the cell suspension was loaded onto a column and unbound cells in the permeate were collected; the permeate was then eluted with 30ml of separation buffer and collected. The combined transudate solutions were centrifuged at 300 Xg for 10min to collect cells for a one-step flow sorting.

2.3 flow-type sorting:

Incubating goat anti-human IgG labeled with FITC at 10nM with the cells sorted by the magnetic beads, washing with PBS for 3 times at room temperature for 1hr, sorting with a flow cytometer, and collecting the 0.2% -0.4% cells with the strongest fluorescence. Extracting plasmids from the cells obtained by the first round of screening, transforming the plasmids into escherichia coli DH5 alpha, instantly transforming the plasmids into 293T cells again after extraction, and carrying out second round of screening, wherein the concentration of the goat anti-human IgG is reduced to 1nM so as to improve the screening specificity. After two rounds of sorting, the sequence analysis is carried out on the recovered plasmid to obtain the coding DNA sequence of the GLP-1-Fc fusion protein, wherein the sequence with the highest frequency of occurrence is shown as SEQ ID NO. 13.

Example 3: expression and identification of recombinant GLP-1-Fc fusion protein

3.1 construction and screening of stably expressing cell lines:

HindIII and EcoRI restriction sites are respectively introduced into the upstream and downstream of a GLP-1-Fc fusion protein coding DNA sequence by means of PCR, and are connected into SGLs vector which is also subjected to double restriction by HindIII and EcoRI, so that an expression plasmid is constructed. The correct expression plasmid was identified by digestion and sequencing, linearized with PvuI digestion, and transfected into CHOS cells using electroporation. After transfection, the cells are paved in a 96-well plate, 25 mu M MSX is added for pressurized screening, and monoclonal cells are obtained for expression and purification of the recombinant GLP-1-Fc fusion protein.

3.2 expression of recombinant GLP-1-Fc fusion proteins

The cell strain obtained by screening was inoculated in a shake flask, cultured continuously in a CO2 shaker for 12 days, and the cell culture supernatant was collected, and the fusion Protein was purified by Protein A affinity chromatography (purification system: 10mM PBS equilibration solution at pH7.4, 50mM citrate buffer solution at pH3.0, and Tris-HCl neutralization solution at pH 9.0) and the purified Protein concentrated by ultrafiltration was quantified using BCA quantification kit.

Example 4 assay of the hypoglycemic Effect of recombinant GLP-1-Fc fusion proteins

the hypoglycemic effect of the recombinant GLP-1-Fc fusion protein prepared in example 3 is tested by taking a spontaneous II type diabetes animal model KKAY mouse as a model animal and adopting a subcutaneous administration mode. A blank control group and a positive control group (Dulagutide drug group, 1mg/kg body weight) are respectively set, and a low dose group, a medium dose group and a high dose group (0.3/1/3mg/kg body weight) are respectively set by the recombinant GLP-1-Fc fusion protein, and the results are shown in figure 3. From the results, it can be seen that: the recombinant GLP-1-Fc fusion protein of the invention maintains the hypoglycemic activity of a group of 0.3mg/kg which is not less than that of a dolaglutide contrast medicament, and the group of 1mg/kg which has the same dosage with the dolaglutide contrast medicament shows the hypoglycemic effect which is obviously better than that of the contrast medicament.

example 5 half-life assay of recombinant GLP-1-Fc fusion proteins

See w.glaesener et al, Diabetes meta Res Rev 2010; 26: 287-296. The recombinant GLP-1-Fc fusion protein is injected subcutaneously into a macaque at a single time according to the weight of 0.1mg/kg, the same dose of similar marketed drug dilaglutide is injected in the same mode to serve as a positive control, 0.6mL of venous blood is collected at the 2 nd, 4 th, 8 th, 12 th hours, 2 rd, 3 th, 4 th, 8 th, 10 th and 12 th days after administration, the GLP-1-Fc content in a sample is determined by sandwich ELISA, the PK parameter of the GLP-1-Fc in the macaque serum is calculated by adopting WinNonlin software, and the results are shown in the following table 2. Pharmacokinetic parameters show that the recombinant GLP-1-Fc fusion protein has a higher area under a drug-hour curve, has a half-life of about 57hr, and is about 10% longer than that of the similar marketed drug dilaglutide (51.6 hr).

Table 2: recombinant GLP-1-Fc fusion protein drug-induced parameter determination

Claims

1. A GLP-1-Fc recombinant fusion protein has an amino acid sequence shown in SEQ ID NO. 14.

2. A GLP-1-Fc fusion protein coding gene has a nucleotide sequence shown in SEQ ID NO. 13.

3. A pharmaceutical composition for treating type II diabetes by lowering blood glucose, comprising the recombinant fusion protein of claim 1 and a pharmaceutically acceptable carrier.

4. Use of a GLP-1-Fc recombinant fusion protein according to claim 1 or a gene encoding a GLP-1-Fc fusion protein according to claim 2 for the manufacture of a medicament for the treatment of type II diabetes.