CN108948207A

CN108948207A - A kind of human interleukin 10-Fc fusion protein and its encoding gene and application

Info

Publication number: CN108948207A
Application number: CN201710362078.1A
Authority: CN
Inventors: 周亮
Original assignee: Hangzhou Bohu Biotechnology Co Ltd
Current assignee: Hangzhou Bohu Biotechnology Co Ltd
Priority date: 2017-05-22
Filing date: 2017-05-22
Publication date: 2018-12-07
Anticipated expiration: 2037-05-22
Also published as: CN112142859A; CN108948207B

Abstract

The present invention relates to genetically engineered drug fields, and in particular to a kind of human interleukin 10-Fc fusion protein and its encoding gene and application.Pass through the replacement to position amino acid multiple in the part IgG4Fc, so that improved IL10-Fc fusion protein has performance more superior than existing Fc fusion protein, such as increases internal stability, eliminates unnecessary effector function and reduce the immunogenicity of the fusion protein in vivo.The method for being used to treat disease the invention also discloses the IL10-Fc fusion protein drug, this method include the drug to the individual application therapeutically effective amount suffered from the disease, and the disease includes inflammatory condition, immune-related disorders, fibrotic conditions and cancer etc..

Description

A kind of human interleukin 10-Fc fusion protein and its encoding gene and application

Technical field

The present invention relates to genetically engineered drug fields, and in particular to a kind of human interleukin 10-Fc fusion protein and its Encoding gene and application.

Background technique

Interleukin 10 (Interleukin-10, IL-10) is the cell factor being found in 1991, can be adjusted Save body inflammatory and immune response.Originally report that the cell factor can inhibit cytokine secretion, antigen to offer and CD4+ cell Activation, IL-10 can by inhibit the monocyte of activation and the IL-1 α of macrophage of activation, IL-1 β, IL-6, IL-8, The expression of TNF-α, GM-CSF and G-CSF inhibits immune response, and it also inhibits the IFN-γ of NK cell to generate.Although IL-10 is mainly expressed in macrophage, but is also detected in the T cell of activation, B cell, mast cell and monocyte To expression.Other than inhibiting immune response, IL-10 also shows immunostimulatory properties, including stimulation IL-2 and IL-4 processing The proliferation of thymocyte enhances the vigor of B cell, and the expression of stimulation mhc class ii.

However, the polyethyleneglycol modified IL-10 of newest clinical studies show in fact can forcefully human activin exempt from Epidemic disease system function can especially be activated with the CD8+T cell for killing cancer cell effect.The activation of IL-10 energy costimulation B cell, Extend B cell survival and helps the class switch in B cell.In addition, it can costimulation natural killer (NK) cell Proliferation and thin Intracellular cytokine generate and play a part of growth factor stimulate certain CD8+T cell subsets be proliferated (Mosser, D.M.&Yhang, X., Immunological Reviews 226,205-218 (2008), the IL-10 (respectively 20 and 25 μ g/kg) of high dose is in people In can cause INF γ generate increase (Lauw, F.N.et al., J.Immunol.165,2783-2789 (2000)；Tilg,H.et al.,Gut50,191-195(2002))。

HIL-10 is homodimer and each monomer includes 178 amino acid, and preceding 18 amino acid includes letter Number peptide.The specific embodiment of the disclosure include lack signal peptide mature hIL-10 polypeptide (referring to U.S. Patent No. 6,217, No. 857).Mature IL-10 has 160 amino acid residues (Seq ID No.1), and monomer molecule amount is 18.7KD, contains 4 half The disulfide bond (12-108,62-114) that cystine is formed, natural activity form are to be by the molecular weight of non-covalent bond connection The homologous few dimer of 38KD, the noncovalent interaction between two monomer subunits become inanimate object after being destroyed Activity.Open crystal structure obtained from IL-10 statistics indicate that function dimer shows certain similitude with IFN-γ (Zdanov etc., 1995, Structure (Lond), 3:591-601).It is active as its pleiotropism as a result, IL-10 with crowd A variety of diseases, illness and symptom, including inflammatory condition, immune-related disorders, fibrotic conditions are associated with cancer.

The half-life period of Recombinant Human IL-10 in vivo, only 2-3h, albumen were removed quickly, and which has limited the biologies of IL-10 Availability (Braat, H.et al., Expert Opin.Biol.Ther.3 (5), 725-731 (2003)).In order to improve circulation Time, exposure, effect and kidney intake is reduced, existing document, which discloses, can be used PEGylated method of modifying and extend its Half-life in vivo (Mattos,A.et al.,J.Control Release 162,84-91(2012)；Mumm,J.B.et al.,Cancer Cell 20(6),781-796(2011)；Alvarez, H.M.et al., Drug Metab.Dispos., 40 (2), 360-373 (2012)；CN 201480024021.5 etc.).But due to PEG decorating site have it is multiple, using after PEGylated modification Product be it is inhomogenous, this makes troubles to subsequent Control of drug quality.

Another approach is related to merging IL-10 peptide with Fc portion of immunoglobulin.Immunoglobulin generally has in vivo There is long circulating half life.For example, IgG molecule has up to 23 days half lifes in human body.Fc portion of immunoglobulin is this The reason in part for internal stability.While retaining IL-10 molecular biology activity, IL10-Fc fused protein have by Stability that Fc portion of immunoglobulin provides and the advantages of retain the bioactivity of IL-10 molecule.

Although this approach is feasible, human body when Fc fused protein long-term applies repeatedly for IL-10 therapy It is the potential hidden danger of such drug that immunogenicity, which can be generated,.In addition, if the part Fc retains unwanted biological effect function, meeting Lead to additional treatment side reaction, this is also the potential misgivings of Fc fusion protein therapy.

Summary of the invention

The present invention obtains a kind of human interleukin 10-Fc fusion protein (IL10-Fc by the transformation to Fc protein sequence Fusion protein), by the replacement to position amino acid multiple in the part Fc, so that improved IL10-Fc fusion protein has Performance more superior than existing Fc fusion protein, such as increase internal stability, eliminate unnecessary effector function and drop The immunogenicity of the low fusion protein in vivo.

IL10-Fc fusion protein of the present invention, wherein the C-terminal of IL-10 is directly or by link peptide and Fc albumen N-terminal be connected；Wherein, consistent shown in IL-10 sequence and Seq ID No.1；Connecting peptide sequence general formula is [GlyGlyGlyGlySer] n, n are the integer of 1-5；Fc protein part includes the sequence of SEQ ID NO.2, wherein:

16 X1 are Pro or Glu；

17 X2 are Phe, Val or Ala；

18 X3 are Leu, Glu or Ala；

80 X4 are Asn or Ala；And

230 X5 are Lys or are not present.

IL10-Fc fusion protein of the present invention, wherein preferably connection peptide sequence general formula is [GlyGlyGlyGlySer] n, n are the integer of 1-3；It is furthermore preferred that n is 3, sequence Gly-Gly-Gly-Gly-Ser- Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser.It is potential unnecessary to prevent by the way that small link peptide is added Structural domain interaction optimize the in vivo functionality and stability of fusion protein of the invention.In addition, being rich in the company of glycine It connects peptide and provides certain structural flexibility, make the part IL-10 that can effectively interact with IL-10 receptor on target cell.

Fc protein part of the invention comes from human IgG 4, but compared with wild-type human sequence includes one or more The part Fc of amino acid substitution.The part Fc is by two light chain constants by noncovalent interaction and the antibody of disulfide-bonded District's groups at.The part Fc may include hinge area, and extend to antibody C-terminal through CH2 and CH3 structural domain.The part Fc can also wrap Containing one or more glycosylation sites.

There are five types of the human immunoglobulins with different effect subfunction and pharmacokinetic properties of type for human body. IgG be it is most stable of in five seed types, there is about 23 days serum half-lifes in human body.There are four subclass by human body IgG: IgG1, IgG2, IgG3 and IgG4, each subclass have the function of the different biological of referred to as effector function.These effector functions usually by Interaction with Fc receptor (Fc γ R) or by combining Clq and complement-fixing to mediate.And the combination of Fc γ R can cause to resist The cell cracking that body dependent cells mediate, and and the combination of complement factor can lead to the cell cracking of complement-mediated.

It is important by effector function minimum in the design for the Fc fusion protein for extending half-life period merely with the part Fc 's.For the antibody of some pure antagonisms, such as soluble cell hormone such as TNF α, IL17A etc. or immunologic test point are such as For the antibody of PD-1, the effect of Fc γ Rs bring effect is not needed, and the brings cytotoxicity such as can prevent ADCC, because This Fc effect weak IgG2 and IgG4 is selected to as skeleton.On existing 4 IgG2 and 6 IgG4 monoclonal antibodies go through now City, such as anti-PD1 monoclonal antibody nivolumab and pembrolizumab, anti-IL17A monoclonal antibody ixekizumab and anti- PCSK9 monoclonal antibody evolocumab etc. uses IgG2 or IgG4 hypotype.Therefore, the portion Fc in Fc fusion protein structure of the invention Divide and be preferred from the region human IgG 4Fc, because it combines the ability of Fc γ R and complement factor to reduce compared with other IgG hypotypes.

To further decrease its effector function, the region IgG4Fc of wild type is further transformed in the present invention. The part IgG4Fc of fusion protein of the invention can contain one or more following replacements: corresponding in SEQ ID NO:2 With proline (Pro) or glutamic acid (Glu) replacement glutamine (Gln) at 16th, corresponding to the 17th in SEQ ID NO:2 With alanine (Ala) or valine (Val) replacement phenylalanine (Phe) at position, at the 18th in SEQ ID NO:2 With alanine (Ala) or glutamic acid (Glu) replacement leucine (Leu).

The position N297 of the part human IgG molecule Fc can glycosylate, which has a significant impact to the activity of IgG. If site glycosylation is removed, the conformation of CH2 top half will affect, so that the binding ability to Fc γ Rs is lost, Influence the relevant bioactivity of antibody.But for the fusion protein that the present invention constructs, due to not needing Fc γ Rs band The effect effect come, and the brings cytotoxicity such as ADCC for needing to prevent fusion protein, it is therefore desirable to which nothing is carried out to the part Fc It is glycosylation engineered.Based on this consideration, inventor's hair now corresponds at SEQ ID NO:2 the 80th and replaces Asn with Ala, can The glycosylation site of N- connection in the region IgG4Fc is removed, which can reduce the biologies such as the ADCC of fusion protein Learn effect.

It is present in naturally in addition, can be lacked in the part Fc derived from the IgG4 of IL10-Fc fusion protein discussed herein C-terminal lysine residue in molecule (Seq ID NO.2 the 230th；The lysine of missing is known as des-K).Certain cells Such as the C-terminal of NS0 cell expression is inhomogenous, the C-terminal amino of certain fusion proteins for the Fc fusion protein of lysine Acid is lysine, and the C-terminal of a part of fusion protein will lack lysine, which is due to certain form of mammal The effect of protease during cell expression.Therefore, in order to avoid this heterogeneity, when Fc fusion protein construction preferred C-terminal Lack lysine.

For convenience of understanding, provides common amino acid single-letter and three-letter codes correspond to table, as follows.

Currently preferred IL10-Fc fusion protein includes following protein:

IL10-[GlyGlyGlyGlySer]₃- IgG4Fc, wherein Fc the 16th X1 is Glu, and 17 X2 are Ala, 18 The X3 of position is Ala, and the X5 that 80 X4 are Asn and 230 is not present, and sequence is as shown in SEQ ID NO:3.

IL10-[GlyGlyGlyGlySer]₃- IgG4Fc, wherein Fc the 16th X1 is Glu, and 17 X2 are Ala, 18 The X3 of position is Ala, and the X5 that 80 X4 are Ala and 230 is not present, shown in sequence SEQ ID NO:4.

IL10-GlyGlyGlyGlySer-IgG4Fc, wherein Fc the 16th X1 is Glu, and 17 X2 are Ala, 18 X3 be Ala, the X5 that 80 X4 are Asn and 230 is Lys, shown in sequence SEQ ID NO:5.

IL10-IgG4Fc, wherein Fc the 16th X1 is Glu, and 17 X2 are Ala, and 18 X3 are Ala, 80 X4 is that Ala and 230 X5 is not present, shown in sequence SEQ ID NO:6.

IL10-[GlyGlyGlyGlySer]₃- IgG4Fc, wherein Fc the 16th X1 is Glu, and 17 X2 are Ala, 18 The X3 of position is Ala, and the X5 that 80 X4 are Asn and 230 is Lys.

IL10-GlyGlyGlyGlySer-IgG4Fc, wherein Fc the 16th X1 is Glu, and 17 X2 are Ala, 18 X3 be Ala, 80 X4 are that Asn and 230 X5 is not present.

IL10-[GlyGlyGlyGlySer]₃- IgG4Fc, wherein Fc the 16th X1 is Pro, and 17 X2 are Phe, 18 The X3 of position is Ala, and the X5 that 80 X4 are Ala and 230 is not present.

IL10-[GlyGlyGlyGlySer]₃- IgG4Fc, wherein Fc the 16th X1 is Pro, and 17 X2 are Val, 18 The X3 of position is Ala, and the X5 that 80 X4 are Ala and 230 is not present.

IL10-IgG4Fc, wherein Fc the 16th X1 is Glu, and 17 X2 are Ala, and 18 X3 are Ala, 80 The X5 that X4 is Ala and 230 is Lys.

IL10-[GlyGlyGlyGlySer]₂- IgG4Fc, wherein Fc the 16th X1 is Pro, and 17 X2 are Ala, 18 The X3 of position is Ala, and the X5 that 80 X4 are Asn and 230 is Lys.

The structure of IL10-Fc fusion protein is as shown in Figure 1.

Wild type human's IgG4 protein can be obtained from a variety of sources.For example, mesh can be expressed from detectable level The cell of mRNA prepare cDNA library to obtain these protein.Use the disclosed DNA or albumen of specific purpose protein The probe of matter sequence design can screen library.For example, in Adams et al., (1980) Biochemistry 19: 2711-2719；Goughet et al., (1980) Biochemistry 19:2702-2710；Dolby et al., (1980) Proc.Natl.Acad.Sci.USA 77:6027-6031；Rice et al., (1982) Proc.Natl.Acad.Sci.USA 79: 7862-7862；Falkner et al., (1982) Nature 298:286-288；And Morrison et al., (1984) Immunoglobulin light or heavy chain constant region are described in Ann.Rev.Immunol.2:239-256.

The DNA for encoding IL-10 and IgG4Fc of the invention can be generated by a variety of different methods, the method includes The molecular cloning method of standardization program and chemically synthesized DNA.May then pass through will encode the DNA of IL-10 in frame and this Locate the DNA connection of the coding IgG4Fc protein and constructs the gene of encoding fusion protein.It can in the pre-connection or compile The DNA of encoding wild type IgG4Fc segment is mutated in the cDNA of the entire fused protein of code.A variety of induced-mutation techniques are wide in this field It is well known.The gene for encoding IL-10 gene and coding IgG4Fc analog protein can also be by encoding the link peptide rich in G DNA connected in frame.

The present invention provides the gene that can encode IL10-Fc fusion protein, such as IL10- shown in coding SEQ ID NO:3 [GlyGlyGlyGlySer]₃The gene order of-IgG4Fc fusion protein is as shown in SEQ ID NO:7.When coding IL10-Fc In the nucleic acid molecules insertion suitable carrier of fusion protein, it can be expressed when the carrier is introduced suitable host cell IL10-Fc fusion protein.The protokaryon for the various commercially viable purchases that suitable carrier is well known to those skilled in the art is true Nuclear expression carrier, prokaryotic expression carrier such as pET serial carrier, pQE serial carrier；Yeast expression carrier pPICZ- α-A, pHIL- D2, pPIC9, pHIL-S1 (Invitrogen Corp.San Diego.California.USA)；Animal cell expression vectors PIRES plasmid, pSVK3, pMSG (Amersham Pharmacia Biotech Inc.USA) etc..

Suitable host cell includes but is not limited to bacterium, yeast, insect and mammalian cell.The IL10-Fc containing coding The recombinant cell of the exogenous nucleic acid of fusion protein can be prepared by any suitable technology, for example, with naked DNA plasmid vector, Viral vectors, invasive bacterial cell carrier or other full cell carriers etc. are transfected/transformed, including turning by calcium phosphate precipitation Dye, receptor-mediated positioning and transfection, biolistics hit delivering, electroporation, the transfection that glucan mediates, liposome-mediated turn IL10-Fc fusion protein coded sequence is delivered in cell and prepares by the methods of change, protoplast fusion, direct microinjection.Turn Change/transfection cell method be it is known in the art, referring to Sambrook et al.Molecular Cloning:A Laboratory Manual, Cold Spring Harbor Laboratory Press (2d Edition, 1989 or 3rd Edition, 2001).

The expression of nucleic acid molecule of the present invention can be regulated and controled by another nucleotide sequence, thus the molecule is with recombinant DNA It is expressed in the host of molecule conversion.For example, expression can pass through any promoter/enhancer element control known in the art System.Can be used for controlling chimeric polyeptides developed by molecule promoter include but is not limited to long terminal repeats (Squinto etc., 1991, Cell, 65:1-20)；SV40 early promoter area, CMV, M-MuLV, thymidine kinase promoter, metallothionein (metallothionine) regulating and controlling sequence of gene；Prokaryotic expression carrier such as b- iactamase promoter or tac promoter (see Scientific American (1980), 242:74-94)；Promoter element from yeast or other fungies such as Gal 4 is opened Mover, ADH, PGK, alkaline phosphatase and derived from the tissue-specific transcription control zone of gene such as elastase I gene.

As host for recombinant protein cell line be it is well known in the art, including it is a variety of can be from American Type culture The immortalized cell line that collection (ATCC) obtains.In these cell lines include Chinese hamster ovary (CHO) cell, NSO, SP2 cell, HeLa cell, baby hamster kidney (BHK) cell, MK cells (COS), human liver cancer cell (such as Hep G2), A549 Cell and a variety of other cell lines.In a preferred technical solution, (dhfr-CHO is thin in Chinese hamster ovary celI for fusion protein antibody Born of the same parents, using DHFR as selection markers) expression.Another embodiment of expression system is GS (glutamate synthetase) gene expression system System, specifically refers to WO87/04462, WO89/01036 and EP338841 etc..When the core of coding such as IL10-Fc fusion protein It, can be thin in host by being enough host cell culture when sour (or carrier containing nucleic acid) imported into mammalian host cell IL10-Fc fusion protein is expressed in born of the same parents or fusion protein is secreted into the culture medium of host cell growth.

IL10-Fc fusion protein can be recycled from culture medium with any standard protein purification method known in the art, such as Affine in immunity column purification, sulfate precipitation, ion-exchange chromatography, hydrophobic interaction chromatograph, reverse chromatograms or gel filtration etc. Purification technique or its arbitrary combination.Physical condition for purifying specific protein will partly depend on factor, as net charge, Hydrophobicity, hydrophily etc..For the affinitive layer purification of fusion protein of the invention, can be used with albumin A or Protein G Matrix.

On the other hand, the present invention provides the pharmaceutical composition comprising any IL10-Fc fusion protein provided herein, this The pharmaceutical composition of invention includes the IL10-Fc fusion protein and pharmaceutically acceptable carrier of therapeutically effective amount.It is pharmaceutically acceptable Carrier refer to used dosage and concentration generally to recipient nontoxicity, i.e., in due course to animal (such as people) apply When do not generate the molecular entity and composition of unfavorable, allergia or other improper reactions.Pharmaceutically acceptable carrier includes any With all solvents, buffer, decentralized medium, coating material, surfactant, antioxidant, preservative (such as antibacterium Agent, antifungal agent), isotonic agent, absorb delayer, salt, preservative, antioxidant, protein, drug, drug stabilizing agent, polymerization Object, gel, adhesive, excipient, disintegrating agent, lubricant, sweetener, aromatic, dyestuff, such similar material and its group It closes.

Pharmaceutical composition of the present invention, can intravenous, intradermal, intra-arterial, in peritonaeum, encephalic, the side such as intra-articular Formula administration.Fusion protein of the invention is particularly suitable for parenteral, especially by injection apply, such as subcutaneously, it is intradermal, Intravenously, intra-arterial, the application of intramuscular, intrathecal or intraperitoneal injection.It, can be by fusion protein of the invention aqueous for injection Solution is preferably prepared in the buffer of physiological compatible.Alternatively, fusion protein can be powder type, for using Preceding suitable medium such as sterile water dissolves.

The invention also discloses IL10-Fc fusion proteins to manufacture or prepare the purposes in drug, and the drug is for controlling The disease in individuals in need is treated, the disease includes viral disease, inflammatory disease, immune-related disorders, fiber Change illness and cancer etc..IL-10 is the cell factor in immunological regulation and inflammation with pleiotropic effects.It is by mast cell It generates, clears up these cells in the inflammatory effect at anaphylactoid position.Although it is able to suppress pro-inflammatory cytokine such as The synthesis of IFN-γ, IL-2, IL-3, TNF α and GM-CSF, but IL-10 also has irritation to certain T cells and mast cell And mature B cell, proliferation and antibody is stimulated to generate.IL-10 can block NF- kB activity and participate in JAK-STAT signal transduction The regulation of approach.It also induces the cellular cytoxicity activity of CD8+T cell and the antibody of B cell to generate, and it inhibits macrophage thin Cytoactive and rush tumour inflammation.The adjusting of CD8+T cell be it is dose-dependent, wherein higher dosage induces stronger cell toxicant Property reaction.

IL-10 plays multiple effects in the activation of CD8+T cell.For example, IL-10 is induced in memory CD8+T cell Effector molecule (IFN γ, perforin and granzyme B).Such memory CD8+T cell is responsible for providing the long-term disease-resistant of subject The cell of malicious protective effect.Although the generation and amplification of memory CD (3) 8+T cell can occur in the absence of the IL-10 (Vicari, A. and Trinchieri, G., (2004) Immuno.Rev.202:223-236), but the fact that the such cell of IL-10 direct activation Unique and alternative treatment method is provided.

IL10-Fc fusion protein has the similar biological activity of IL-10.In view of above-mentioned, the embodiment base of the disclosure Contacting between CD8+T cell and cancer and virus infection.Therefore, treat and/or prevent cancer-related diseases, illness and disease Certain methods of shape, such as maintenance is greater than 0.5ng/mL, the average IL10-Fc greater than 1ng/mL or greater than 0.1ng/mL melts Hop protein serum-concentration should be also applied for the treatment of such disease.The disclosure covers IL10-Fc fusion protein as described herein Purposes in the extensive disease for the treatment of or prevention, illness or symptom and/or its symptom.According to the disclosure, IL10-Fc merges egg It is white to be used to treat or prevent proliferative symptom or illness, including cancer, such as uterus, uterine neck, mammary gland, prostate, testis, stomach and intestine Road, kidney, bladder, bone, marrow, skin, head or neck, skin, liver, gall-bladder, heart, lung, pancreas, salivary gland, adrenal gland, first shape Gland, brain, neuromere, central nervous system (CNS) and peripheral nervous system (PNS) cancer and hemopoietic system and siberian crabapple The cancer of system).In specific embodiments, tumour or cancer are colon cancer, oophoroma, breast cancer, melanoma, lung cancer, pancreas Gland cancer, glioblastoma or leukaemia etc..

In one embodiment, method of the IL10-Fc fusion protein drug for treating disease, the party are disclosed Method include to suffer from the disease individual application therapeutically effective amount drug, the disease include inflammatory condition, immune-related disorders, Fibrotic conditions and cancer etc..The individual is mammal, preferably people.According to the type and severity of disease, greatly It is dense in the serum paddy of about 0.1ng/mL (for example, 0.1-2ng/mL, 0.1-1ng/mL, 0.5-1.5ng/mL or 1.1-2.1ng/mL) The IL10-Fc fusion protein of degree can be the initial candidate dosage for applying to patient, either for example by primary or more Secondary separated application, or carried out by continuous infusion.

It preferably, can be to be greater than 2.0 μ g/kg/ day, be greater than 2.5 μ g/kg/ days, greater than 3.0 μ when subject is a human G/kg/ days, be greater than 5 μ g/kg/ day, be greater than 8 μ g/kg/ day, greater than 10 μ g/kg/ days, greater than 12 μ g/kg/ days, 15 μ g/kg/ days, Greater than 18 μ g/kg/ days, be greater than 20 μ g/kg/ day, be greater than 21 μ g/kg/ day, be greater than 22 μ g/kg/ days, greater than 23 μ g/kg/ days, greatly IL10-Fc fusion protein is applied in 24 μ g/kg/ days or greater than 25 g/kg/ days dosage of μ.Using techniques well known in the art, Predose can be estimated from vitro data such as animal model.Those skilled in the art can be easily based on animal The data-optimized application to people.

Detailed description of the invention

Attached drawing 1: human interleukin 10-Fc fusion protein structural schematic diagram

Attached drawing 2:IL10-Fc stimulates CD8+ cell to generate cytotoxic factor

Specific embodiment

The building of embodiment 1:IL10-Fc antigen-4 fusion protein gene

SEQ ID NO:3 is translated into DNA sequence dna, and is optimized according to the codon preference of Chinese hamster ovary celI, IL10- is obtained The expressed sequence SEQ ID NO:7 of Fc fusion protein.In the good end of sequence 5 ' the addition NheI restriction enzyme site of the optimization and kozac sequence It arranges (SEQ ID No.8), in 3 ' end addition terminator codons and XhoI restriction enzyme site (SEQ ID No.9), obtains fusion protein Expressed intact frame.Artificial synthesized expressed intact frame sequence, is inserted between NheI the and XhoI restriction enzyme site of pIRES plasmid, Obtain pIRES-IL10-Fc expression plasmid.After the plasmid is linearized, electrotransfection to CHO-s cell is added MSX and filters out sun Property clone.

The expression and purifying of embodiment 2:IL10-Fc fusion protein

The positive colony obtained in embodiment 1 filters out expression quantity and preferably clones, through expanding by two-wheeled limiting dilution Big culture is inoculated into 7L fermentor and carries out feed-batch culture, express express target protein.4500rpm is centrifuged 6min after fermentation, collects Supernatant adjusts 4 DEG C of preservations after pH to 4.0.

Supernatant first uses the ultrafiltration membrane packet of 10KDa, is concentrated by ultrafiltration；Then preliminary affine layer is carried out with Mabselect Sure Fusion protein is collected in analysis.Affinity chromatography mobile phase are as follows: A1:25mM PB+50mM Nacl, pH7.0, B1:20mM Gly, PH3.0, B2:20mM citrate buffer solution, pH3.0.Chromatographic column first uses mobile phase A 1 to balance, and after loading, is first washed with Mobile phase B 1 Removing impurities matter, then fusion protein is eluted with Mobile phase B 2, the elution being collected into also is adjusted in pH with 1M Tris-His pH8.0 Property.This walks the thick pure sample being collected into, consummate by Capto adhere chromatographic column.Sample is used after the combination of pH7.0 loading PH4.0 affords purer sample (95% or more).

Embodiment 3: Half-life in vivo measurement

By tail vein injection, merged by rhIL-10 (Rochy Hill company) and by the IL10-Fc that embodiment 2 obtains Albumen is injected separately into the SD rat of average weight about 200g by 200ng/Kg body weight dose).After injection, in different time points (0, 1,2,4,6,8,12,24,36,48,60,72,96 hours) by cutting tail blood taking method collection blood sample, heparin sodium is anticoagulant, by acquisition Blood sample 12000g is centrifuged 5min, collects serum.

It detects blood sample employment IL-10ELISA kit (being purchased from Bender Medsystem company) and referring to specification The content of fusion protein in serum, results are averaged.The results show that the present invention prepares disappearing in vivo for IL10-Fc fusion protein Except half-life period be 22.6 hours, and employment rhIL-10 is eliminated after tail vein injection half-life period be 4 hours, show system of the present invention 5-6 times is extended for the half-life period of IL10-Fc fusion protein in vivo is obtained relative to rhIL-10 reference substance.

Embodiment 4: anti-tumor activity experiment

Research has shown that IL-10 not only can play anti-tumor effect by activated NK, can also pass through activating T cell Inhibit the development of tumour.Research shows that the tumor-bearing mice of IL-10 treatment can induce the expression of IFN-γ and granzyme.The effect May be by having the IL-10 signal path of specificity to mediate in CD8+T cell in tumour: IL-10 activates phosphorus in CD8+T cell The STAT1 and STAT3 of acidification, to induce the proliferation and IFN-γ of CD8+T cell, cytotoxic protein perforin and particle egg The expression of white enzyme；IFN-γ can induce MHC- class Ⅰ antigens in tumour cell and mononuclear macrophage to offer the expression of molecule, association CD8+T cell is helped to kill most of antigentic specificity tumour cell；TCR in activation CD8+T cell can effectively induce anti- Antiapoptotic signals and cell proliferation signals.In short, IL-10 can not only be enhanced by improving the cellular cytoxicity activity of NK cell Tumor cytotoxicity, can also by the infiltration and activation of mediate tumor internal specific cytotoxicity CD8+T cell, IFN-γ and The tumor-killing ability and IFN-γ of CD8+T cell in tumor are offered etc. and improved to the expression of granule protein enzyme and enhancing tumour antigen The HLA-II antigen of induction, to enhance the function of antineoplastic immune escape.

In order to detect the cytotoxicity of IL10-Fc stimulation CD8+T, CD8+ cell is isolated from mouse spleen, in vitro After cultivating and activating, after IL10-Fc (with IL-10 for control) effect of various concentration is added, cell can be stimulated to generate cell toxicant Effect (granzyme/perforin expression is promoted) simultaneously stimulates IFN γ to express (such as Fig. 2).Although in terms of external activity, IL10-Fc is only There is the 30%-40% or so of IL10, it is contemplated that IL10-Fc Half-life in vivo significantly extends, Biological acdtivity in vivo is simultaneously It is not remarkably decreased.

Sequence table

<110>Hangzhou Bo Hu Biotechnology Co., Ltd

<120>a kind of human interleukin 10-Fc fusion protein and its encoding gene and application

<160> 9

<170> PatentIn version 3.3

<210> 1

<211> 160

<212> PRT

<213> Homo sapiens (Human)

<400> 1

SPGQGTQSEN SCTHFPGNLP NMLRDLRDAF SRVKTFFQMK DQLDNLLLKE SLLEDFKGYL 60

GCQALSEMIQ FYLEEVMPQA ENQDPDIKAH VNSLGENLKT LRLRLRRCHR FLPCENKSKA 120

VEQVKNAFNK LQEKGIYKAM SEFDIFINYI EAYMTMKIRN 160

<210> 2

<211> 230

<212> PRT

<213>artificial sequence

<400> 2

AESKYGPPCP PCPAPX1X2X3GG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS QEDPEVQFNW 60

YVDGVEVHNA KTKPREEQFX4 STYRVVSVLT VLHQDWLNGK EYKCKVSNKG LPSSIEKTIS 120

KAKGQPREPQ VYTLPPSQEE MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV 180

LDSDGSFFLY SRLTVDKSRW QEGNVFSCSV MHEALHNHYT QKSLSLSLGX5 230

<210> 3

<211> 404

<212> PRT

<213>artificial sequence

<400> 3

SPGQGTQSEN SCTHFPGNLP NMLRDLRDAF SRVKTFFQMK DQLDNLLLKE SLLEDFKGYL 60

GCQALSEMIQ FYLEEVMPQA ENQDPDIKAH VNSLGENLKT LRLRLRRCHR FLPCENKSKA 120

VEQVKNAFNK LQEKGIYKAM SEFDIFINYI EAYMTMKIRN GGGGSGGGGS GGGGSAESKY 180

GPPCPPCPAP EAAGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSQEDPE VQFNWYVDGV 240

EVHNAKTKPR EEQFNSTYRV VSVLTVLHQD WLNGKEYKCK VSNKGLPSSI EKTISKAKGQ 300

PREPQVYTLP PSQEEMTKNQ VSLTCLVKGF YPSDIAVEWE SNGQPENNYK TTPPVLDSDG 360

SFFLYSRLTV DKSRWQEGNV FSCSVMHEAL HNHYTQKSLS LSLG 404

<210> 4

<211> 404

<212> PRT

<213>artificial sequence

<400> 4

SPGQGTQSEN SCTHFPGNLP NMLRDLRDAF SRVKTFFQMK DQLDNLLLKE SLLEDFKGYL 60

GCQALSEMIQ FYLEEVMPQA ENQDPDIKAH VNSLGENLKT LRLRLRRCHR FLPCENKSKA 120

VEQVKNAFNK LQEKGIYKAM SEFDIFINYI EAYMTMKIRN GGGGSGGGGS GGGGSAESKY 180

GPPCPPCPAP EAAGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSQEDPE VQFNWYVDGV 240

EVHNAKTKPR EEQFASTYRV VSVLTVLHQD WLNGKEYKCK VSNKGLPSSI EKTISKAKGQ 300

PREPQVYTLP PSQEEMTKNQ VSLTCLVKGF YPSDIAVEWE SNGQPENNYK TTPPVLDSDG 360

SFFLYSRLTV DKSRWQEGNV FSCSVMHEAL HNHYTQKSLS LSLG 404

<210> 5

<211> 404

<212> PRT

<213>artificial sequence

<400> 5

SPGQGTQSEN SCTHFPGNLP NMLRDLRDAF SRVKTFFQMK DQLDNLLLKE SLLEDFKGYL 60

GCQALSEMIQ FYLEEVMPQA ENQDPDIKAH VNSLGENLKT LRLRLRRCHR FLPCENKSKA 120

VEQVKNAFNK LQEKGIYKAM SEFDIFINYI EAYMTMKIRN GGGGSAESKY GPPCPPCPAP 180

EAAGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSQEDPE VQFNWYVDGV EVHNAKTKPR 240

EEQFNSTYRV VSVLTVLHQD WLNGKEYKCK VSNKGLPSSI EKTISKAKGQ PREPQVYTLP 300

PSQEEMTKNQ VSLTCLVKGF YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLYSRLTV 360

DKSRWQEGNV FSCSVMHEAL HNHYTQKSLS LSLGK 395

<210> 6

<211> 404

<212> PRT

<213>artificial sequence

<400> 6

SPGQGTQSEN SCTHFPGNLP NMLRDLRDAF SRVKTFFQMK DQLDNLLLKE SLLEDFKGYL 60

GCQALSEMIQ FYLEEVMPQA ENQDPDIKAH VNSLGENLKT LRLRLRRCHR FLPCENKSKA 120

VEQVKNAFNK LQEKGIYKAM SEFDIFINYI EAYMTMKIRN AESKYGPPCP PCPAPEAAGG 180

PSVFLFPPKP KDTLMISRTP EVTCVVVDVS QEDPEVQFNW YVDGVEVHNA KTKPREEQFA 240

STYRVVSVLT VLHQDWLNGK EYKCKVSNKG LPSSIEKTIS KAKGQPREPQ VYTLPPSQEE 300

MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SRLTVDKSRW 360

QEGNVFSCSV MHEALHNHYT QKSLSLSLG 389

<210> 7

<211> 1212

<212> DNA

<213>artificial sequence

<400> 7

GAGAACCAGG ACCCCGACAT CAAGGCCCAC GTGAACAGCC TGGGCGAGAA CCTGAAGACC 300

CTGAGGCTGA GGCTGAGGAG GTGCCACAGG TTCCTGCCCT GCGAGAACAA GAGCAAGGCC 360

GTGGAGCAGG TGAAGAACGC CTTCAACAAG CTGCAGGAGA AGGGCATCTA CAAGGCCATG 420

AGCGAGTTCG ACATCTTCAT CAACTACATC GAGGCCTACA TGACCATGAA GATCAGGAAC 480

GGCGGCGGCG GCAGCGGCGG CGGCGGCAGC GGCGGCGGCG GCAGCGCCGA GAGCAAGTAC 540

GGCCCCCCCT GCCCCCCCTG CCCCGCCCCC GAGGCCGCCG GCGGCCCCAG CGTGTTCCTG 600

TTCCCCCCCA AGCCCAAGGA CACCCTGATG ATCAGCAGGA CCCCCGAGGT GACCTGCGTG 660

GTGGTGGACG TGAGCCAGGA GGACCCCGAG GTGCAGTTCA ACTGGTACGT GGACGGCGTG 720

GAGGTGCACA ACGCCAAGAC CAAGCCCAGG GAGGAGCAGT TCAACAGCAC CTACAGGGTG 780

GTGAGCGTGC TGACCGTGCT GCACCAGGAC TGGCTGAACG GCAAGGAGTA CAAGTGCAAG 840

GTGAGCAACA AGGGCCTGCC CAGCAGCATC GAGAAGACCA TCAGCAAGGC CAAGGGCCAG 900

CCCAGGGAGC CCCAGGTGTA CACCCTGCCC CCCAGCCAGG AGGAGATGAC CAAGAACCAG 960

GTGAGCCTGA CCTGCCTGGT GAAGGGCTTC TACCCCAGCG ACATCGCCGT GGAGTGGGAG 1020

AGCAACGGCC AGCCCGAGAA CAACTACAAG ACCACCCCCC CCGTGCTGGA CAGCGACGGC 1080

AGCTTCTTCC TGTACAGCAG GCTGACCGTG GACAAGAGCA GGTGGCAGGA GGGCAACGTG 1140

TTCAGCTGCA GCGTGATGCA CGAGGCCCTG CACAACCACT ACACCCAGAA GAGCCTGAGC 1200

CTGAGCCTGG GC 1212

<210> 8

<211> 22

<212> DNA

<213>artificial sequence

<400> 8

GCTAGCCGCC ACCATGCATA TG 22

<210> 9

<211> 9

<212> DNA

<213>artificial sequence

<400> 9

TAACTCGAG 9

Claims

1. a kind of IL10-Fc fusion protein, wherein the C-terminal of IL-10 is connected directly or by link peptide with the N-terminal of Fc albumen； Wherein, consistent shown in IL-10 sequence and Seq ID No.1；Connection peptide sequence general formula is [GlyGlyGlyGlySer] n, n 1-5 Integer；Fc protein part includes the sequence of SEQ ID NO.2, wherein:

16 X1 are Pro or Glu；

17 X2 are Phe, Val or Ala；

18 X3 are Leu, Glu or Ala；

80 X4 are Asn or Ala；And

230 X5 are Lys or are not present.

2. IL10-Fc fusion protein according to claim 1, it is characterised in that: connect peptide sequence in the fusion protein General formula is [GlyGlyGlyGlySer]_n, n is the integer of 1-3.

3. IL10-Fc fusion protein according to claim 2, it is characterised in that: connect peptide sequence in the fusion protein For [GlyGlyGlyGlySer]₃。

4. IL10-Fc fusion protein according to claim 1, it is characterised in that: in the fusion protein, SEQ ID NO: Amino acid X4 at 2 the 80th is Ala, to remove the glycosylation site of N- connection in the region IgG4Fc.

5. IL10-Fc fusion protein according to claim 1, it is characterised in that: in the fusion protein, SEQ ID NO: Amino acid X5 missing at 2 the 230th.

6. IL10-Fc fusion protein according to claim 1, it is characterised in that: the amino acid sequence of the fusion protein It is consistent with sequence shown in SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6.

7. IL10-Fc fusion protein according to claim 1, it is characterised in that: the fusion protein egg chosen from the followings White matter:

IL10-[GlyGlyGlyGlySer]₃- IgG4Fc, wherein Fc the 16th X1 is Glu, and 17 X2 are Ala, 18 X3 is Ala, and the X5 that 80 X4 are Asn and 230 is Lys；

IL10-GlyGlyGlyGlySer-IgG4Fc, wherein Fc the 16th X1 is Glu, and 17 X2 are Ala, 18 X3 It is not present for Ala, the X5 that 80 X4 are Asn and 230；

IL10-[GlyGlyGlyGlySer]₃- IgG4Fc, wherein Fc the 16th X1 is Pro, and 17 X2 are Phe, 18 X3 is Ala, and the X5 that 80 X4 are Ala and 230 is not present；

IL10-[GlyGlyGlyGlySer]₃- IgG4Fc, wherein Fc the 16th X1 is Pro, and 17 X2 are Val, 18 X3 is Ala, and the X5 that 80 X4 are Ala and 230 is not present；

IL10-IgG4Fc, wherein Fc the 16th X1 is Glu, and 17 X2 are Ala, and 18 X3 are Ala, and 80 X4 are Ala and 230 X5 is Lys；

IL10-[GlyGlyGlyGlySer]₂- IgG4Fc, wherein Fc the 16th X1 is Pro, and 17 X2 are Ala, 18 X3 is Ala, and the X5 that 80 X4 are Asn and 230 is Lys.

8. encoding the gene of the described in any item IL10-Fc fusion proteins of claim 1-7.

9. a kind of pharmaceutical composition, the described in any item IL10-Fc fusion proteins of the claim 1-7 comprising therapeutically effective amount and Pharmaceutically acceptable carrier.

10. the described in any item IL10-Fc fusion proteins of claim 1-7 are preparing for viral disease, inflammatory condition, are exempting from Purposes in the disease therapeuticing medicines such as epidemic disease associated disease, fibrotic conditions and cancer.