CN106892982B

CN106892982B - Novel fusion polypeptide targeting IL-17 and TNF alpha and application thereof

Info

Publication number: CN106892982B
Application number: CN201710217125.3A
Authority: CN
Inventors: 李德山; 任桂萍; 刘芝航; 阚方明; 韩晓辉
Original assignee: Northeast Agricultural University
Current assignee: Northeast Agricultural University
Priority date: 2017-04-05
Filing date: 2017-04-05
Publication date: 2021-01-05
Anticipated expiration: 2037-04-05
Also published as: CN106892982A

Abstract

The invention discloses a novel fusion polypeptide targeting IL-17 and TNF alpha and application thereof. The novel fusion polypeptide provided by the invention is obtained by connecting a single-chain antibody of an anti-human IL-17 with a soluble tumor necrosis factor receptor 1 through a hinge region of the antibody and connecting an Fc fragment of immunoglobulin at the C end of the soluble tumor necrosis factor receptor 1. The fusion polypeptide Anti-IL-17scFv/sTNFR1-Fc provided by the invention can effectively inhibit the expression of collagen and various pro-inflammatory factors, and effectively relieve the clinical symptoms of rheumatoid arthritis. The invention has great application value for treating rheumatoid arthritis.

Description

Novel fusion polypeptide targeting IL-17 and TNF alpha and application thereof

Technical Field

The invention belongs to the field of biological medicine, relates to a novel fusion polypeptide of targeted IL-17 and TNF alpha and application thereof, and particularly relates to a novel fusion polypeptide of fusion of gene engineering bispecific multi-target fusion polypeptide with activity of resisting interleukin-17 and tumor necrosis factor alpha simultaneously and an immunoglobulin Fc fragment.

Background

IL-17 is a powerful proinflammatory cytokine and also a Fine-tuning cytokine for inflammatory responses. IL-17 stimulates the release of cytokines such as IL-6, IL-8, prostaglandin E2(PGE2), metalloproteinase-1 (MMP-1), granulocyte colony stimulating factor (G-CSF), and Monocyte Chemoattractant Protein (MCP) -1 from keratinocytes, fibroblasts, epithelial cells, and endothelial cells. It is currently believed that the mechanism by which IL-17 promotes bone destruction in Rheumatoid Arthritis (RA) may include: inducing the production of TNF alpha and IL-1, so that the destructive effect of joints is enhanced; increase the number of nuclear stimulating factor receptors and nuclear stimulating factor receptors, and destroy the balance of the bone protective protein and the nuclear stimulating factor receptors to aggravate bone destruction; IL-17 also acts directly on osteoclasts, stimulating differentiation and activation of osteoclasts.

IL-17 has a powerful role in recruiting neutrophils. When IL-17 is co-cultured with fibroblasts, the fibroblasts can continuously promote CD34⁺Proliferation and differentiation of hematopoietic stem cells into neutrophils; the antibodies of IL-6, IL-17 and MIP-2 all have the function of inhibiting the accumulation of neutrophils in the air channel. It is suggested that IL-17 is an early promoter of T cell dependent inflammatory responses and also plays an important role in linking the cytokine network of the hematopoietic system with the immune system.

TNF alpha is a cytokine mainly produced by activated macrophages, is an important bioactive factor, has strong tumor killing activity and wide biological activity, can activate macrophages and polymorphonuclear cells, induces the production of various cytokines and has synergistic effect, participates in immune regulation, promotes the expression of a histocompatibility complex, enhances the anti-infection capacity of an organism, participates in the absorption and remodeling of bone tissues and the like. However, excessive TNF α production and release can disrupt the immune balance of the body, cause severe inflammatory reactions, and can lead to fever, shock, multiple organ failure, and even death.

TNF α exerts its biological activity primarily through binding to the specific receptor TNFR on cell membranes, both TNFR1 and TNFR2, which bind both TNF α and TNF β, but mediate different biological functions. In vivo, a part of the extracellular domain of the TNF receptor is dissociated from the cell membrane and released in the blood to become soluble TNF receptor sTNFR. stnfrs do not mediate signaling, but bind to TNF α, neutralize TNF α activity, inhibit TNF α -induced cytotoxicity and autoimmune responses in vivo and in vitro, and are natural antagonists of TNF α.

The Fc fragment as part of an antibody may function to enhance antibody half-life, present antigen, and mediate ADCC and CDC. In addition, Fc fusion fragments can be added to optimize the purification process of the fusion protein. The antibodies and fusion proteins which are on the market at present mostly select different types of Fc fragments of immunoglobulins according to different requirements of own drugs.

The CHO is always the main carrier for expressing antibody drugs, which accounts for half of all antibody drugs on the market, and experiments also find that the fusion protein is superior to a prokaryotic expression carrier in activity and stability compared with the prokaryotic expression carrier.

As two important targets of autoimmune diseases, the antagonists and inhibitors of TNF alpha and IL-17 in the current foreign drug market are good. Three of the first 5 best marketed drugs in 2016 were inhibitors of TNF α, and at the same time, the development of IL-17 antibodies in 2016 reached a new height, and many pharmaceutical major companies focused on the study of IL-17 antagonists for the treatment of autoimmune diseases.

Disclosure of Invention

The invention aims to provide a novel fusion polypeptide targeting IL-17 and TNF alpha and application thereof.

The fusion polypeptide provided by the invention is obtained by connecting a single-chain antibody of an anti-human IL-17 with a soluble tumor necrosis factor receptor 1 through a hinge region of the antibody and connecting an Fc fragment (hinge region + CH2+ CH3) of immunoglobulin at the C end of the soluble tumor necrosis factor receptor 1.

Specifically, the fusion polypeptide consists of an A fragment, a B fragment and a C fragment;

the fragment A sequentially consists of a heavy chain variable region (VH) of an anti-human IL-17 antibody, a connecting peptide (GS linker), a light chain variable region (VL) of the anti-human IL-17 antibody and an antibody hinge region (hinge region) positioned between CH1 and CH2 from upstream to downstream, and the amino acid sequence of the fragment A is (a) or (b):

(a) a sequence 1 in a sequence table;

(b) a sequence which is derived from the sequence 1 and has the same function after the amino acid sequence of the sequence 1 is substituted and/or deleted and/or added by one or more amino acid residues;

the fragment B is soluble tumor necrosis factor receptor I, and the amino acid sequence thereof is (c) or (d):

(c) a sequence 3 in a sequence table;

(d) and (b) a sequence which is derived from the sequence 3 and has the same function after the amino acid sequence of the sequence 3 is subjected to substitution and/or deletion and/or addition of one or more amino acid residues.

The C fragment is an Fc fragment (hinge region, CH2 and CH3) of immunoglobulin IgG4, and the amino acid sequence of the C fragment is (e) or (f)

(e) Sequence 5 in the sequence table;

(f) and (b) the sequence derived from the sequence 5, which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence of the sequence 5 and has the same function.

In the present invention, the a fragment, the B fragment and the C fragment in the fusion polypeptide are sequentially seamlessly connected.

Nucleic acid molecules encoding such fusion polypeptides are also within the scope of the invention.

The nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule can also be an RNA, such as an mRNA, hnRNA, or tRNA, and the like.

In the present invention, the nucleic acid molecule is specifically a DNA composition. The DNA composition consists of a DNA molecule A for coding the A segment, a DNA molecule B for coding the B segment and a DNA molecule C for coding the C segment.

The DNA molecule A is specifically the DNA molecule of the following (1), 2 or 3):

(1) DNA molecule shown in sequence 2 in the sequence table;

(2) a DNA molecule which is hybridized with the DNA sequence defined in (1) under strict conditions and encodes the A segment;

(3) a DNA molecule which has more than 90% of homology with the DNA sequence defined in (1) or (2) and encodes the A segment;

the DNA molecule B is specifically the following DNA molecule (4), or (5) or (6):

(4) DNA molecule shown in sequence 4 in the sequence table;

(5) a DNA molecule which is hybridized with the DNA sequence defined in (4) under strict conditions and encodes the B segment;

(6) a DNA molecule which has more than 90% of homology with the DNA sequence defined in (4) or (5) and encodes the B segment;

the DNA molecule C is specifically the following DNA molecule (7), or (8) or (9):

(7) DNA molecule shown in sequence 6 in the sequence table;

(8) a DNA molecule which is hybridized with the DNA sequence defined in (7) under strict conditions and encodes the C segment;

(9) and (3) a DNA molecule which has more than 90% homology with the DNA sequence defined in (7) or (8) and encodes the C segment.

The stringent conditions may be hybridization in a solution of 6 XSSC, 0.5% SDS at 65 ℃ and washing the membrane once with each of 2 XSSC, 0.1% SDS, and 1 XSSC, 0.1% SDS.

Recombinant vectors, expression cassettes, transgenic cell lines or recombinant bacteria containing the nucleic acid molecules also belong to the scope of protection of the invention.

The application of the fusion polypeptide or the nucleic acid molecule or the recombinant vector, the expression cassette, the transgenic cell line or the recombinant bacterium in any one of the following also belongs to the protection scope of the invention:

(1) for the preparation of a medicament for the treatment and/or prevention of autoimmune diseases;

(2) preparing a preparation for blocking inflammatory pathways caused by IL-17 and/or TNF alpha.

Products having the functions shown in the following (1) and (2) also belong to the scope of the present invention. The active component of the product is the fusion polypeptide or the nucleic acid molecule or the recombinant vector, the expression cassette, the transgenic cell line or the recombinant bacterium;

(1) treatment and/or prevention of autoimmune diseases;

(2) blocking inflammatory pathways induced by IL-17 and/or TNF alpha.

In the present invention, the autoimmune disease is in particular rheumatoid arthritis.

In the present invention, the blocking of inflammatory pathways caused by IL-17 and/or TNF α is embodied in: reducing the expression of inflammatory factors caused by IL-17 and/or TNF alpha; the inflammatory factors caused by IL-17 and/or TNF alpha include IL-6, IL-8, IL-1 beta, MMP3, etc.

The product may in particular be a medicament. The curative effect of the medicine can be embodied as blocking the inflammatory reaction of rheumatoid arthritis and relieving bone injury.

The invention also claims a method for preparing the fusion polypeptide.

A method of making the fusion polypeptide may comprise the steps of: and introducing the nucleic acid molecule into a CHO-K1SV cell line for eukaryotic expression, and purifying by using a Protein A chromatographic column by using the Fc part of the nucleic acid molecule to obtain the fusion polypeptide.

In the present invention, the method for preparing the fusion polypeptide specifically comprises the steps of: (1) connecting double-stranded DNA molecules shown in a sequence 2 and a sequence 4 in a sequence table by overlapping PCR, then connecting the double-stranded DNA molecules by using a connecting sequence 6 by using an overlapping PCR method, and inserting the connected fragments into a Pee12.4 vector; (2) transfecting the eukaryotic expression vector obtained in the step (1) into a CHO-K1SV cell line for culture; (3) screening out cell strains capable of stably expressing the fusion polypeptide by using an MSX pressure screening system and a flow screening system; (4) selecting the cell strain with high expression in the step (3) for culturing, and collecting cell supernatant; (5) purifying cell supernatant by using an AKTA purification system and a Protein A gel column to obtain the fusion polypeptide (named Anti-IL-17scFv/sTNFR 1-Fc); (6) the fusion polypeptide is then subjected to a buffer displacement treatment to render it present in phosphate.

The invention provides a novel bispecific multi-target polypeptide drug (Anti-IL-17scFv/sTNFR1-Fc fusion polypeptide) which takes IL-17 and TNF alpha as targets and is fused with an immunoglobulin Fc fragment, has the capability of simultaneously blocking inflammatory reaction caused by two cytokines, because the fusion protein adopts a eukaryotic expression mode of CHO-K1SV cell expression, compared with the Anti-IL-17scFv/sTNFR1 which needs to be denatured and renatured to obtain prokaryotic expression of the protein, the Anti-IL-17scFv/sTNFR1-Fc has better capability of combining antigen, the Fc fragment not only increases the half life of the protein, but also proves through experiments that the Fc fragment of the immunoglobulin can also stabilize the structure of the novel fusion protein so as to enhance the biological activity of the novel fusion protein, in addition, the purification strategy of the novel fusion protein is optimized, so that the produced protein is purer and has better biological activity.

The fusion polypeptide Anti-IL-17scFv/sTNFR1-Fc provided by the invention can effectively inhibit the expression of collagen and various proinflammatory factors, effectively relieve the clinical symptoms of rheumatoid arthritis, can make up the defects of the currently clinically used single-target antibody medicament or fusion protein through direct application or optimized modification, or can replace the existing antibody medicament or fusion protein to become a new generation of multi-target fusion protein medicament to improve the treatment effect on the rheumatoid arthritis, and has great application value for the treatment of the rheumatoid arthritis.

Drawings

FIG. 1 is a schematic diagram showing the structural composition of the Anti-IL-17scFv/sTNFR1-Fc novel fusion polypeptide.

FIG. 2 is an SDS-PAGE pattern of Anti-IL-17scFv/sTNFR1-Fc novel fusion polypeptides.

FIG. 3 shows the results of the Anti-IL-17scFv/sTNFR1-Fc novel fusion polypeptide tested for antigen binding by ELISA, compared to the ability of a prokaryotic expression of the Anti-IL-17scFv/sTNFR1 fusion polypeptide to bind antigen.

FIG. 4 is the Real-time PCR results of Anti-IL-17scFv/sTNFR1-Fc novel fusion polypeptide blocking the inflammatory pathway in THP-1 cells.

FIG. 5 is the Real-time PCR results of Anti-IL-17scFv/sTNFR1-Fc novel fusion polypeptide blocking the inflammatory pathway in RA-FLS cells.

FIG. 6 shows the effect of Anti-IL-17scFv/sTNFR1-Fc novel fusion polypeptide on the pathogenesis of CIA mouse arthritis in mouse RA model.

FIG. 7 shows the effect of Anti-IL-17scFv/sTNFR1-Fc novel fusion polypeptide on the expression level of inflammatory factor protein in the serum of CIA model mice (ELISA).

FIG. 8 shows the effect of Anti-IL-17scFv/sTNFR1-Fc novel fusion polypeptide on the expression level of inflammatory factor mRNA in spleen of CIA model mouse (Real-time-PCR).

FIG. 9 shows the effect of Anti-IL-17scFv/sTNFR1-Fc novel fusion polypeptide on the pathogenesis of CIA mouse arthritis in mouse RA model.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

In the quantitative tests in the following examples, three replicates were set up and the results averaged.

Etanercept (sTNFRI-Fc): shanghai Xinguo Jianyao stockings (Shanghai, 12.5 mg/count).

pKappa vector: biovector plasmid vector Strain cell Gene Collection. The method is recorded in Zhanya Kun, Wangweifei, He Kun and the like, the new method for rapidly screening the high-water stable expression to the fibroblast growth factor-21 eukaryotic cell strain is established, the article of Chinese biochemistry and molecular biology, 2012,28(8):768-774, the public can obtain the method from the applicant, and the method can only be used for the repeated invention experiments.

pee12.4 vector: LONZA products

pIRES2-EGFP plasmid: products of Clontech, USA.

CHO-K1SV cells: american Type Culture Collection (ATCC) product.

RA-FLS cells (synovial cells of rheumatoid patients): purchased at a second hospital affiliated with Guangzhou medical university. The subject of PRP on migration and invasion of RA-FLS cells is described in "environmental university, 2015, Master paper," publicly available from the Applicant, and only available for repeated experiments.

THP-1 cells: american Type Culture Collection (ATCC) product.

Example 1 preparation of the fusion polypeptide Anti-IL-17scFv/sTNFR1-Fc

Structural description of Anti-IL-17scFv/sTNFR1-Fc fusion polypeptide

The fusion polypeptide aiming at hIL-17 and hTNF alpha provided by the invention is named as IL-17scFv/sTNFRI-Fc and consists of an A fragment, a B fragment and a C fragment. The fragment A sequentially consists of a heavy chain variable region (VH) of an anti-human IL-17 antibody, a connecting peptide (GS linker), a light chain variable region (VL) of the anti-human IL-17 antibody and a hinge region (hinge) of the antibody between CH1 and CH2 from upstream to downstream. The amino acid sequence of the fragment A is shown as the sequence 1 in the sequence table, and the coding gene is shown as the sequence 2 in the sequence table. The B fragment is soluble tumor necrosis factor receptor I. The amino acid sequence of the fragment B is shown as a sequence 3 in a sequence table, and the coding gene is shown as a sequence 4 in the sequence table. The C fragment is Fc fragment (hinge region, CH2, CH3) of immunoglobulin IgG 4. The amino acid sequence of the C fragment is shown as the sequence 5 in the sequence table, and the coding gene is shown as the sequence 6 in the sequence table.

The structural composition of the Anti-IL-17scFv/sTNFR1-Fc fusion polypeptide is shown in FIG. 1. The fusion protein in FIG. 1 is in a dimeric form (as detected by non-reducing SDS-PAGE), and the two domains do not affect their respective functions, and the binding mode is shown schematically.

II, preparing and purifying Anti-IL-17scFv/sTNFR1-Fc fusion polypeptide

1. The sequence 2 of the sequence table is passed through primer K1 (5-GGATCCACTGGTCAGGTGCAGCTGGTGCAGTCTGGG-3') and a primer K2(5'-ACACTATCTCTCTTCTCCCTGGGTTTTGGGGGGAACAGGAAGA-3'), the sequence 4 is amplified by a primer K3(5'-CTGTTCCCCCCAAAACCCAGGGAGAAGAGAGATAGTGTGTGTC-3') and a primer K4(5'-GCATGGGGGACCATATTTGGATAGGCACAACTTCGTGCACTCCAGG-3'), and then the sequence 2 and the sequence 4 are connected by the primers K1 and K4; then using the primer K5(5'-GGAGTGCACGAAGTTGTGCCTATCCAAATATGGTCCCCCATGCCCA-3') and the primer K6 (5-GCTAGCTTATTTACCCAGAGACAGGGAGAGGCT-3'), and then the ligation product of the sequences 2 and 4 is ligated to the sequence 6 using primers K1 and K6, and the ligation is performed atThe upstream of the ligation product has a BamHI cleavage site, and the downstream has a NheI cleavage site. Then carrying out enzyme digestion on the ligated product and a pKApp vector by BamHI and NheI at the same time, and constructing the ligated product on the pKApp vector through the cohesive end of the same enzyme digestion site to obtain a recombinant plasmid. Then carrying out double enzyme digestion by using Hind III and NheI through a genetic engineering technology, recovering an Anti-IL-17scFv/sTNFR1-Fc fragment containing Kappa leader signal peptide in the recombinant plasmid, carrying out double enzyme digestion on pIRES2-EGFP plasmid by using NheI and EcoRI to obtain an IRES-EGFP fragment with sticky ends of NheI and EcoRI, then carrying out double enzyme digestion on the Pee12.4 vector by using Hind III and EcoRI, and finally connecting the Kappa leader-Anti-IL-17scFv/sTNFR1-Fc fragment and the IRES-EGFP fragment with the Pee12.4 vector through corresponding restriction enzymes to obtain the recombinant plasmid. The recombinant plasmid which is verified to be correct by sequencing is named Pee-anti-IL-17/TNF-EGFP. The recombinant plasmid can simultaneously and respectively express a novel fusion protein (Anti-IL-17scFv/sTNFR1-Fc) and EGFP.

2. Then, the recombinant plasmid Pee-Anti-IL-17/TNF-EGFP constructed in the step 1 is transfected into CHO-K1SV cells by utilizing liposome 2000(Invitrogen, USA), after MSX is cultured under pressure for a period of time, the surviving cells are screened by a flow cytometer in 3 rounds, cell strains which stably express a novel fusion protein (Anti-IL-17scFv/sTNFR1-Fc) and EGFP can be screened out, then the cell strains are collected and cultured continuously.

3. The cell strain in 2 is subjected to conventional culture, when the cell density of the culture after screening reaches 80%, the MSX culture medium is changed into CD OptiCHO^TM AGT^TM(GIBCO, USA) culture medium, in this medium cultured for 3 days, collected serum-free medium supernatant.

4. The cell supernatant of 3 was purified by Protein-A column chromatography using AKTA Protein purifier. The Protein-A column was equilibrated with an equilibration solution (20mM Na)₃PO₄150mM NaCl, pH 7.0), and the cell supernatant was subjected to elution with an eluent (20mM Na-Citrate, pH3.0), and the protein obtained from the elution peak was collected and replaced in a phosphate buffer at pH 7.0.

5. The concentration of Anti-IL-17scFv/sTNFR1-Fc fusion polypeptide (concentration of fusion polypeptide is based on protein concentration) was adjusted with physiological saline.

FIG. 2 is an SDS-PAGE image in which the order of spotting is cell supernatant (lane 1), purified flow through (lane 2), purified fusion polypeptide (lane 3), and target protein in the arrow.

Example 2 biological functional identification and detection of blocking Activity of Anti-IL-17scFv/sTNFR1-Fc fusion Polypeptides

First, Anti-IL-17scFv/sTNFR1-Fc fusion polypeptide ability to bind antigen simultaneously

Coating of 96-well plates with 1nmol (excess) of humanized IL-17 and 1nmol (excess) of humanized TNF α (with 0.1M NaCO)₃-NaHCO₃Diluted at pH 9.6), overnight at 4 ℃; followed by PBST (PBS + 0.05% Tween-20) wash, skim milk Powder (PBS) blocking; incubation with 100. mu.L of different concentrations of the novel fusion polypeptide Anti-IL-17scFv/sTNFR1-Fc (100pmol/mL, 10pmol/mL, 1pmol/mL, 0.1pmol/mL) and BSA (negative control) for 1 hour at 37 ℃; PBST was washed, followed by 100. mu.L goat anti-human secondary antibody (R)&Product of company D, catalog number G-102-C) for 1 hour (1:7500 dilution); PBST was washed, and a color developing solution was added to the mixture to react for 15 minutes, followed by addition of a stop reaction solution (H)₂SO₄) Readings were taken with a microplate reader at a wavelength of 450 nm.

Similarly, the fusion protein Anti-IL-17scFv/sTNFR1 expressed in pronucleus was tested to compare the antigen binding ability. Wherein the fusion protein Anti-IL-17scFv/sTNFR1 which is prokaryotic expressed is expressed by using Escherichia coli, and the amino acid sequence of the fusion protein Anti-IL-17scFv/sTNFR1-Fc fusion polypeptide lacks the Fc fragment of IgG4 shown in sequence 5.

The results are shown in fig. 3 (the differences between the group Anti-IL-17scFv/sTNFR1 and the group Anti-IL-17scFv/sTNFR1-Fc fusion polypeptides are significantly expressed as p <0.05 and p < 0.01). The result shows that the immunoglobulin Fc fragment added by the novel fusion polypeptide Anti-IL-17scFv/sTNFR1-Fc can effectively increase the binding capacity.

II, Anti-IL-17scFv/sTNFR1-Fc fusion polypeptide can block inflammatory pathway caused by IL-17 and TNF alpha in RA-FLS and THP-1 cells

The RA-FLS cells (rheumatoid patients have slippery joints)Membrane cells), THP-1 cells were seeded in 6-well plates; when the density of RA-FLS reaches 2X 10⁴THP-1 cells reached 1X 10 per well⁶Each well was incubated with "IL-17 (20. mu.g/mL) + TNF α (20. mu.g/mL)" or "IL-17 (20. mu.g/mL) + TNF α (20. mu.g/mL) + Anti-IL-17scFv/sTNFR1-Fc (200. mu.g/mL) fusion polypeptide" for 24 hours. After incubation, cellular total RNA was extracted, reverse transcribed into cDNA and tested for changes in mRNA expression levels of factors of the relevant inflammatory pathway (IL-6, IL-8 and IL-1. beta.) by realtime-PCR.

Wherein, the primers used for detecting IL-6 are 5'-ACTCACCTCTTCAGAACGAATTG-3' and 5'-CCATCTTTGGAAGGTTCAGGTTG-3'; primers for detecting IL-8 were 5'-TTTTGCCAAGGAGTGCTAAAGA-3' and 5'-AACCCTCTGCACCCAGTTTTC-3'; the primers used for detection of IL-1 β were 5'-ATGATGGCTTATTACAGTGGCAA-3' and 5'-GTCGGAGATTCGTAGCTGGA-3'. The internal reference is beta-actin, and the detection primers are 5'-CATGTACGTTGCTATCCAGGC-3' and 5'-CTCCTTAATGTCACGCACGAT-3'.

Untreated RA-FLS cells and THP-1 cells were also used as controls (control).

The experiment was also set up with a positive control of an equal amount of etanercept instead of the Anti-IL-17scFv/sTNFR1-Fc fusion polypeptide.

The experiment was also set up with a control in which the Anti-IL-17scFv/sTNFR1-Fc fusion polypeptide was replaced by an equivalent amount of the prokaryotic expressed fusion protein Anti-IL-17scFv/sTNFR 1. Wherein the fusion protein Anti-IL-17scFv/sTNFR1 which is prokaryotic expressed is expressed by using Escherichia coli, and the amino acid sequence of the fusion protein Anti-IL-17scFv/sTNFR1-Fc fusion polypeptide lacks the Fc fragment of IgG4 shown in sequence 5.

FIG. 4 is the Real-time PCR results of Anti-IL-17scFv/sTNFR1-Fc novel fusion polypeptide blocking the inflammatory pathway in THP-1 cells. FIG. 5 is the Real-time PCR results of Anti-IL-17scFv/sTNFR1-Fc novel fusion polypeptide blocking the inflammatory pathway in RA-FLS cells. In FIGS. 4 and 5, the fusion protein is Anti-IL-17scFv/sTNFR1, the novel fusion protein is Anti-IL-17scFv/sTNFR1-Fc, and Etanercept is Etanercept. In the figure, p <0.05 compared to the "IL-17 + TNF α" group; represents p <0.01 compared to the "IL-17 + TNF α" group; # represents p <0.05 compared to the "IL-17 + TNF α + Etanercept" group; # indicates p <0.01 compared to the "IL-17 + TNF α + Etanercept" group.

As can be seen, the Anti-IL-17scFv/sTNFR1-Fc fusion polypeptide, the Anti-IL-17scFv/sTNFR1 fusion polypeptide and etanercept as a positive control can block inflammatory pathways in RA-FLS cells and THP-1 cells, wherein the Anti-IL-17scFv/sTNFR1-Fc fusion polypeptide has the best effect and the effect is obviously better than that of the Anti-IL-17scFv/sTNFR1 fusion polypeptide and the etanercept.

Example 3 use of Anti-IL-17scFv/sTNFR1-Fc fusion Polypeptides

CII, chicken type 2 collagen: sigma, Inc., cat # 9007-43-5.

The mice used in this example were 6-8 week old SPF-grade male C57/BL6 mice: from Vinca department of hundred million (animal Certification number: SCXK (Jing) 2012-0001).

Dissolving CII in 0.1mol/L acetic acid aqueous solution (stirring at 4 ℃ to fully dissolve the CII) to obtain CII solution with CII concentration of 2g/L, standing at 4 ℃ for 12 hours, and mixing with Freund's complete adjuvant in equal volume, and emulsifying to obtain CII emulsion (CII concentration in the CII emulsion is 1 g/L).

Establishing mouse Rheumatoid Arthritis (RA) model and performing grouped drug delivery

Since modeling and group administration were continuous operations, the number of experimental days was counted from the start of modeling.

1. Establishment of mouse RA model

On day 1 of the experiment, mice were immunized with CII emulsion by intradermal multiple injection into the tail roots (0.1 mL CII emulsion per mouse, for the purpose of causing inflammation); on day 21 of the experiment, mice were immunized with CII emulsion by tail root intradermal multiple injection (0.1 mL CII emulsion per mouse); on days 0-56 after the second immunization, the mice were observed and scored for joint swelling, with the following scoring criteria for each foot of the mice: 0 minute: no red swelling; 1 minute: red swelling of the little toe joint; and 2, dividing: swelling of the toe joints and toes; and 3, dividing: swollen paw below the ankle joint; and 4, dividing: all paws, including the ankle, were swollen. And adding the scores of the limbs of the mouse to obtain the total score of the mouse.

2. From the 21 st day of the experiment, the model mice were divided into 6 groups (8 mice each) and treated as follows:

Anti-IL-17scFv/sTNFR1-Fc fusion polypeptide group: the fusion polypeptide was administered intraperitoneally every 2 days (3 mg Anti-IL-17scFv/sTNFR1-Fc fusion polypeptide per kg mouse, 100. mu.l per mouse per administration);

Anti-IL-17scFv/sTNFR1 fusion polypeptide group: the fusion polypeptide was administered intraperitoneally every 2 days (3 mg Anti-IL-17scFv/sTNFR1 fusion polypeptide per kg mouse, 100 microliters per mouse per administration volume); wherein the Anti-IL-17scFv/sTNFR1 fusion polypeptide is expressed in E.coli and has an amino acid sequence which lacks the Fc fragment of IgG4 shown in sequence 5 compared to the Anti-IL-17scFv/sTNFR1-Fc fusion polypeptide.

Etanercept group (Etanercept): etanercept was administered intraperitoneally every 2 days (3 mg etanercept per kg mouse at a dose of 100 microliters per mouse);

CIA model group (PBS): PBS was administered every 2 days (100 μ l per mouse dose volume);

meanwhile, a normal group (blank) consisting of 10 SPF male Kunming mice of 6-8 weeks old was set without any treatment.

II, Effect of Anti-IL-17scFv/sTNFR1-Fc fusion polypeptide on mouse RA pathogenesis

Mice were scored for joint swelling every 4 days, starting on day 21 of the experiment. The results are shown in FIG. 6 (. alpha. indicates p < 0.05;. alpha. indicates p < 0.01. alpha. compared to the CIA model group,. alpha. alpha. indicates p < 0.05. alpha. compared to the novel fusion protein group,. alpha. alpha. indicates p < 0.01. compared to the novel fusion protein group). The result shows that the Anti-IL-17scFv/sTNFR1-Fc fusion polypeptide has obvious inhibition effect on the multiple arthritis characterization of mice, and the effect is better than that of etanercept with the same dose.

Influence of Anti-IL-17scFv/sTNFR1-Fc fusion polypeptide on expression level of inflammatory factor in serum of RA mouse

On the 77 th day of the experiment, blood was collected from the eyeball, and serum was collected, and the expression change amounts of IL-17, TNF α, IL-6, and CCP in the serum of mice of different groups were measured according to the procedure of ELISA kits (R & D, USA) for IL-17, TNF α, IL-6, and CCP. The specific operation is carried out according to the kit instructions.

FIG. 7 shows the effect of Anti-IL-17scFv/sTNFR1-Fc novel fusion polypeptide on the expression level of inflammatory factor protein in the serum of CIA model mice (ELISA). In the figure, the fusion protein represents Anti-IL-17scFv/sTNFR1, the novel fusion protein represents Anti-IL-17scFv/sTNFR1-Fc, and Etanercept is Etanercept. In the figure, p <0.05 compared to the CIA model group; represents p <0.01 compared to the group of CIA models; # represents p <0.05 compared to the Etanercept group; # indicates p <0.01 compared to Etanercept group.

As can be seen, the Anti-IL-17scFv/sTNFR1-Fc fusion polypeptide, the Anti-IL-17scFv/sTNFR1 fusion polypeptide and the etanercept serving as a positive control can obviously reduce the level of inflammatory factors in the blood serum of the CIA mice and block inflammatory reaction to a certain extent. The Anti-IL-17scFv/sTNFR1-Fc fusion polypeptide has the best effect, and particularly has better effects on IL-17, TNF alpha and IL-6 than etanercept.

IV, Effect of Anti-IL-17scFv/sTNFR1-Fc fusion polypeptide on cytokine expression level in RA mice

56 days after the secondary immunization, the spleen of the mouse is taken, total RNA is extracted and is reversely transcribed into cDNA, the cDNA is used as a template, and the relative expression quantity of each cytokine is detected by Real time PCR (adopting beta-actin gene as an internal reference).

The primers used to identify the relative expression of IL-1 β were as follows:

an upstream primer: 5'-CCATGGCACATTCTGTTCAAA-3', respectively;

a downstream primer: 5'-GCCCATCAGAGGCAAGGA-3' are provided.

The primers used to identify the relative expression of IL-6 were as follows:

an upstream primer: 5'-ACAACCACGGCCTTCCCTACTT-3', respectively;

a downstream primer: 5'-CACGATTTCCCAGAGAACATGTG-3' are provided.

The primers used to identify the relative expression of IL-17 were as follows:

an upstream primer: 5'-GGACTCTCCACCGCAATGA-3', respectively;

a downstream primer: 5'-GACCAGGATCTCTTGCTGGA-3' are provided.

The primers used to identify the relative expression of TNF- α were as follows:

an upstream primer: 5'-GGAAACCCAGAGGCATTGAC-3', respectively;

a downstream primer: 5'-TCAGGATCTGGCCCTTGAAC-3' are provided.

The primers used for identifying the relative expression quantity of the beta-actin reference gene are as follows:

an upstream primer: 5'-GAGACCTTCAACCCC-3', respectively;

a downstream primer: 5'-GTGGTGGTGAAGCTGTAGCC-3' are provided.

The results are shown in FIG. 8, and it can be seen that compared with the CIA model PBS treatment group, the mRNA expression levels of IL-1 β, IL-6, IL-17 and TNF α in the spleen of mice in the Anti-IL-17scFv/sTNFR1-Fc novel fusion polypeptide group, the Anti-IL-17scFv/sTNFR1 fusion polypeptide group and the etanercept group are all significantly reduced, and the reduction of the Anti-IL-17scFv/sTNFR1-Fc novel fusion polypeptide group is greatest, i.e., the therapeutic effect of Anti-IL-17scFv/sTNFR1-Fc novel fusion polypeptide is better than that of the fusion polypeptides Anti-IL-17scFv/sTNFR1 and etanercept, and the difference is significantly achieved. Results are the average of each group of mice; represents p <0.01 compared to the group of CIA models; # represents p <0.05 compared to etanercept group.

Fifth, observation of mouse tissue section

The normal group of mice had no redness and swelling of the joints of the four limbs during the test period, and had flexible movements. After the mice in the model group are immunized for the second time, the plantar skin congestion appears, and the foot pad is raised; then the swelling of the ankle and toe joints is obvious, and the toes are in a drumstick shape. After the mice are sacrificed, the toe joints of the mice are taken for tissue section and HE staining, and the fusion polypeptide Anti-IL-17scFv/sTNFR1, the novel fusion polypeptide Anti-IL-17scFv/sTNFR1-Fc and Etanercept (Etanercept) treatment groups are found to reduce the typical joint inflammation; moreover, the Anti-IL-17scFv/sTNFR1-Fc novel fusion polypeptide is found to have a more obvious therapeutic effect than the therapeutic effect of the fusion polypeptide (Anti-IL-17scFv/sTNFR1) and etanercept with the same dosage. The result is shown in figure 9, and the fusion polypeptide Anti-IL-17scFv/sTNFR1-Fc has obvious therapeutic effect on the mouse rheumatoid arthritis.

<110> northeast university of agriculture

<120> novel fusion polypeptide targeting IL-17 and TNF alpha and application thereof

<130> GNCLN170549

<160> 6

<170> PatentIn version 3.5

<210> 1

<211> 280

<212> PRT

<213> Artificial sequence

<220>

<223>

<400> 1

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr

20 25 30

His Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Val Ile Asn Pro Met Tyr Gly Thr Thr Asp Tyr Asn Gln Arg Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Glu 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 Arg Tyr Asp Tyr Phe Thr Gly Thr Gly Val Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

115 120 125

Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Thr Pro Leu Ser

130 135 140

Leu Ser Val Thr Pro Gly Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser

145 150 155 160

Arg Ser Leu Val His Ser Arg Gly Asn Thr Tyr Leu His Trp Tyr Leu

165 170 175

Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn

180 185 190

Arg Phe Ile Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr

195 200 205

Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val

210 215 220

Tyr Tyr Cys Ser Gln Ser Thr His Leu Pro Phe Thr Phe Gly Gln Gly

225 230 235 240

Thr Lys Leu Glu Ile Lys Arg Thr Glu Pro Lys Ser Ser Lys Tyr Gly

245 250 255

Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser

260 265 270

Val Phe Leu Phe Pro Pro Lys Pro

275 280

<210> 2

<211> 840

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 2

caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc agtgaaggtt 60

tcctgcaagg catctggtta ctcattcact gactaccata ttcattgggt gcgacaggcc 120

cctggacaag ggcttgagtg gatgggagta attaatccta tgtatggtac tactgactac 180

aatcagaggt tcaagggcag agtcaccatt accgcggacg aatccacgag cacagcctac 240

atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc gagatatgat 300

tactttactg ggacgggtgt gtactggggc caaggaaccc tggtcaccgt ctcctcaggt 360

ggcggtggct ccggcggtgg tgggtcgggt ggcggcggat ctgatattgt gatgactcag 420

actccactct ccctgtccgt cacccctgga cagccggcct ccatctcctg cagatctagt 480

aggagccttg tacacagtcg tggaaacacc tatttacatt ggtacctgca gaagccaggc 540

caatctccac agctcctaat ttataaagtt tccaaccggt ttattggggt cccagacaga 600

ttcagcggca gtgggtcagg cacagatttc acactgaaaa tcagcagggt ggaggccgaa 660

gatgttgggg tttattactg ctctcaaagt acacatcttc cattcacgtt tggccaaggg 720

accaagctgg agatcaaacg aactgagccc aaatcttcca aatatggtcc cccatgccca 780

ccctgcccag cacctgagtt cctgggggga ccatcagtct tcctgttccc cccaaaaccc 840

<210> 3

<211> 160

<212> PRT

<213> Artificial sequence

<220>

<223>

<400> 3

Arg Glu Lys Arg Asp Ser Val Cys Pro Gln Gly Lys Tyr Ile His Pro

1 5 10 15

Gln Asn Asn Ser Ile Cys Cys Thr Lys Cys His Lys Gly Thr Tyr Leu

20 25 30

Tyr Asn Asp Cys Pro Gly Pro Gly Gln Asp Thr Asp Cys Arg Glu Cys

35 40 45

Glu Ser Gly Ser Phe Thr Ala Ser Glu Asn His Leu Arg His Cys Leu

50 55 60

Ser Cys Ser Lys Cys Arg Lys Glu Met Gly Gln Val Glu Ile Ser Ser

65 70 75 80

Cys Thr Val Asp Arg Asp Thr Val Cys Gly Cys Arg Lys Asn Gln Tyr

85 90 95

Arg His Tyr Trp Ser Glu Asn Leu Phe Gln Cys Phe Asn Cys Ser Leu

100 105 110

Cys Leu Asn Gly Thr Val His Leu Ser Cys Gln Glu Lys Gln Asn Thr

115 120 125

Val Cys Thr Cys His Ala Gly Phe Phe Leu Arg Glu Asn Glu Cys Val

130 135 140

Ser Cys Ser Asn Cys Lys Lys Ser Leu Glu Cys Thr Lys Leu Cys Leu

145 150 155 160

<210> 4

<211> 480

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 4

agggagaaga gagatagtgt gtgtccccaa ggaaaatata tccaccctca aaataattcg 60

atttgctgta ccaagtgcca caaaggaacc tacttgtaca atgactgtcc aggcccgggg 120

caggatacgg actgcaggga gtgtgagagc ggctccttca ccgcttcaga aaaccacctc 180

agacactgcc tcagctgctc caaatgccga aaggaaatgg gtcaggtgga gatctcttct 240

tgcacagtgg accgggacac cgtgtgtggc tgcaggaaga accagtaccg gcattattgg 300

agtgaaaacc ttttccagtg cttcaattgc agcctctgcc tcaatgggac cgtgcacctc 360

tcctgccagg agaaacagaa caccgtgtgc acctgccatg caggtttctt tctaagagaa 420

aacgagtgtg tctcctgtag taactgtaag aaaagcctgg agtgcacgaa gttgtgccta 480

<210> 5

<211> 228

<212> PRT

<213> Artificial sequence

<220>

<223>

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Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe Leu

1 5 10 15

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

20 25 30

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

35 40 45

Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu

50 55 60

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr

65 70 75 80

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

85 90 95

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser

100 105 110

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

115 120 125

Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val

130 135 140

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

145 150 155 160

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

165 170 175

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr

180 185 190

Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val

195 200 205

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

210 215 220

Ser Leu Gly Lys

225

<210> 6

<211> 684

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 6

tccaaatatg gtcccccatg cccatcatgc ccagcacctg agttcctggg gggaccatca 60

gtcttcctgt tccccccaaa acccaaggac actctcatga tctcccggac ccctgaggtc 120

acgtgcgtgg tggtggacgt gagccaggaa gaccccgagg tccagttcaa ctggtacgtg 180

gatggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagtt caacagcacg 240

taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaacgg caaggagtac 300

aagtgcaagg tctccaacaa aggcctcccg tcctccatcg agaaaaccat ctccaaagcc 360

aaagggcagc cccgagagcc acaggtgtac accctgcccc catcccagga ggagatgacc 420

aagaaccagg tcagcctgac ctgcctggtc aaaggcttct accccagcga catcgccgtg 480

gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac 540

tccgacggct ccttcttcct ctacagcagg ctaaccgtgg acaagagcag gtggcaggag 600

gggaatgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacacagaag 660

agcctctccc tgtctctggg taaa 684

Claims

1. A fusion polypeptide is obtained by connecting a single-chain antibody of an anti-human IL-17 with a soluble tumor necrosis factor receptor 1 through a hinge region of the antibody and connecting an Fc fragment of immunoglobulin at the C end of the soluble tumor necrosis factor receptor 1; the Fc fragment of the immunoglobulin is the Fc fragment of immunoglobulin IgG 4;

the hinge region of the anti-human IL-17 single-chain antibody and the antibody is an A fragment;

the soluble tumor necrosis factor receptor I is a B fragment;

the Fc fragment of the immunoglobulin IgG4 is a C fragment;

the amino acid sequence of the fragment A is a sequence 1 in a sequence table;

the amino acid sequence of the fragment B is a sequence 3 in a sequence table;

the amino acid sequence of the C fragment is sequence 5 in the sequence table.

2. A nucleic acid molecule encoding the fusion polypeptide of claim 1.

3. The nucleic acid molecule of claim 2, wherein: the nucleic acid molecule is a DNA composition; the DNA composition comprises a DNA molecule A encoding the A fragment of claim 1, a DNA molecule B encoding the B fragment of claim 1, and a DNA molecule C encoding the C fragment of claim 1.

4. The nucleic acid molecule of claim 3, wherein: the DNA molecule A is a DNA molecule shown in a sequence 2 in a sequence table;

the DNA molecule B is a DNA molecule shown as a sequence 4 in the sequence table;

the DNA molecule C is a DNA molecule shown as a sequence 6 in a sequence table.

5. A recombinant vector, expression cassette, transgenic cell line or recombinant bacterium comprising the nucleic acid molecule of any one of claims 2 to 4.

6. Use of the fusion polypeptide of claim 1 or the nucleic acid molecule of any one of claims 2 to 4 or the recombinant vector, expression cassette, transgenic cell line or recombinant bacterium of claim 5 in any one of:

(2) preparing an agent for blocking inflammatory pathways induced by IL-17 and/or TNF alpha;

the autoimmune disease is rheumatoid arthritis.

7. A product having the functions of (1) and (2), wherein the active ingredient of the product is the fusion polypeptide of claim 1 or the nucleic acid molecule of any one of claims 2-4 or the recombinant vector, expression cassette, transgenic cell line or recombinant bacterium of claim 5;

(1) treatment and/or prevention of autoimmune diseases;

(2) block inflammatory pathways induced by IL-17 and/or TNF alpha;

the autoimmune disease is rheumatoid arthritis.

8. A method of making the fusion polypeptide of claim 1, comprising the steps of: introducing the nucleic acid molecule of any one of claims 2-4 into a CHO-K1SV cell for eukaryotic expression to obtain the fusion polypeptide.