CN112142859A

CN112142859A - Human interleukin 10-Fc fusion protein and coding gene and application thereof

Info

Publication number: CN112142859A
Application number: CN202011086483.3A
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: 2020-12-29
Also published as: CN108948207B; CN108948207A

Abstract

The invention relates to the field of genetic engineering medicines, in particular to human interleukin 10-Fc fusion protein, and a coding gene and application thereof. Through the substitution of amino acids at multiple positions in an IgG4 Fc part, the modified IL10-Fc fusion protein has more excellent performance than the existing Fc fusion protein, such as increased in vivo stability, elimination of unnecessary effector functions and reduction of the immunogenicity of the fusion protein in organisms. The invention also discloses a method for using the IL10-Fc fusion protein medicine for treating diseases, which comprises the step of administering a therapeutically effective amount of the medicine to an individual suffering from the diseases, wherein the diseases comprise inflammatory symptoms, immune-related diseases, fibrotic diseases, cancer and the like.

Description

Human interleukin 10-Fc fusion protein and coding gene and application thereof

The application is a divisional application of Chinese patent application with the application number of 201710362078.1, the application date of 2017, 5 and 22 months, and the invention name of 'human interleukin 10-Fc fusion protein and coding gene and application thereof'.

Technical Field

The invention relates to the field of genetic engineering medicines, in particular to human interleukin 10-Fc fusion protein, and a coding gene and application thereof.

Background

Interleukin-10 (IL-10) is a cytokine discovered in 1991 and can regulate inflammation and immune response of the body. It was originally reported that this cytokine could inhibit cytokine secretion, antigen presentation and CD4+ cell activation, IL-10 could inhibit immune responses by inhibiting the expression of IL-1 α, IL-1 β, IL-6, IL-8, TNF- α, GM-CSF and G-CSF of activated monocytes and activated macrophages, and it also inhibited IFN- γ production by NK cells. Although IL-10 is expressed primarily in macrophages, expression has also been detected in activated T cells, B cells, mast cells and monocytes. In addition to suppressing immune responses, IL-10 also exhibits immunostimulatory properties, including stimulation of proliferation of IL-2 and IL-4 treated thymocytes, enhancement of B cell viability, and stimulation of MHC class II expression.

However, recent clinical studies have shown that PEG-modified IL-10 can in fact strongly activate the immune system of humans, and in particular CD8+ T cells with a cancer cell killing effect. IL-10 is able to co-stimulate B cell activation, prolong B cell survival, and aid in class switching in B cells. In addition, it co-stimulates Natural Killer (NK) cell proliferation and cytokine production and acts as a growth factor to stimulate proliferation of certain CD8+ T cell subsets (Mosser, D.M. & Yohang, X., Immunological Reviews 226,205-218(2008), high doses of IL-10 (20 and 25. mu.g/kg, respectively) can cause increased INF γ production in humans (Lauw, F.N.et al, J.Immunol.165,2783-2789 (2000); Tilg, H.et al, Gut 50,191-195 (2002)).

Human IL-10 is a homodimer and each monomer comprises 178 amino acids, the first 18 of which comprise a signal peptide. Particular embodiments of the present disclosure comprise mature human IL-10 polypeptides lacking a signal peptide (see U.S. patent No. 6,217,857). Mature IL-10 has 160 amino acid residues (Seq ID No.1), a monomeric molecular weight of 18.7kD, contains 4 cysteine-forming disulfide bonds (12-108, 62-114), and its native active form is a non-covalently linked, 38kD molecular weight, homodimer which becomes biologically inactive after the non-covalent interactions between the two monomeric subunits are disrupted. Data from published crystal structures of IL-10 indicate that functional dimers exhibit some similarity to IFN- γ (ZDanov et al, 1995, Structure (Lond), 3: 591-. As a result of its pleiotropic activity, IL-10 has been associated with a wide variety of diseases, disorders and conditions, including inflammatory conditions, immune-related disorders, fibrotic disorders and cancer.

Recombinant human IL-10 has a half-life of only 2-3h in vivo and the protein is cleared very rapidly, which limits the bioavailability of IL-10 (Braat, H.et al, Expert Opin. biol. Ther.3(5),725-731 (2003)). In order to improve circulation time, exposure, efficacy and reduce kidney uptake, it has been disclosed in the literature that PEG-modification can be used to extend the in vivo half-life (Mattos, a.et al, j.control Release 162,84-91 (2012); mummm, j.b.et al, Cancer Cell 20(6), 781-. However, since there are a plurality of PEG modification sites, the product modified by pegylation is not uniform, which is troublesome for the subsequent drug substance control.

Another approach involves fusing the IL-10 peptide to an Fc portion of an immunoglobulin. Immunoglobulins generally have a long circulating half-life in vivo. For example, IgG molecules have a half-life in humans of up to 23 days. The immunoglobulin Fc portion is part of the reason for this in vivo stability. While retaining the biological activity of IL-10 molecules, IL10-Fc fusion proteins have the advantage of retaining the biological activity of IL-10 molecules with the stability provided by the Fc portion of immunoglobulins.

Although this route is feasible for IL-10 therapy, the potential for this class of drugs is that the human body develops immunogenicity upon repeated administration of Fc fusion proteins over extended periods of time. Furthermore, if the Fc portion retains unwanted biological effector functions, additional therapeutic side effects may result, which is also a potential concern for Fc fusion protein therapy.

Disclosure of Invention

The invention obtains a human interleukin 10-Fc fusion protein (IL10-Fc fusion protein) by modifying an Fc protein sequence, and the modified IL10-Fc fusion protein has more excellent performance than the prior Fc fusion protein by replacing amino acids at a plurality of positions in an Fc part, such as increasing the in vivo stability, eliminating unnecessary effector functions and reducing the immunogenicity of the fusion protein in organisms.

The IL10-Fc fusion protein provided by the invention, wherein the C terminal of IL-10 is connected with the N terminal of the Fc protein directly or through a connecting peptide; wherein the IL-10 sequence is consistent with that shown in Seq ID No. 1; the general formula of the connecting peptide sequence is [ GlyGlyGlySer ] n, and n is an integer from 1 to 5; the Fc protein portion comprises the sequence of SEQ ID No.2, wherein:

x1 at position 16 is Pro or Glu;

x2 at position 17 is Phe, Val, or Ala;

x3 at position 18 is Leu, Glu, or Ala;

x4 at position 80 is Asn or Ala; and

x5 at position 230 is Lys or absent.

The general formula of the preferable connecting peptide sequence of the IL10-Fc fusion protein is [ GlyGlyGlyGlySer ] n, wherein n is an integer from 1 to 3; more preferably, n is 3 with the sequence Gly-Gly-Gly-Gly-Ser. The in vivo function and stability of the fusion proteins of the invention is optimized by adding small linker peptides to prevent potential unwanted domain interactions. In addition, glycine-rich linker peptides provide some structural flexibility to allow the IL-10 moiety to interact efficiently with the IL-10 receptor on target cells.

The Fc protein portion of the invention is derived from human IgG4, but includes one or more amino acid substitutions in comparison to the wild-type human sequence. The Fc portion consists of the two heavy chain constant regions of the antibody bound by non-covalent interactions and disulfide bonds. The Fc portion may comprise a hinge region and extend to the C-terminus of the antibody via the CH2 and CH3 domains. The Fc portion may also comprise one or more glycosylation sites.

The human body has five types of human immunoglobulins with different effector functions and pharmacokinetic properties. IgG is the most stable of the five types, with a serum half-life of about 23 days in humans. Human IgG has four subclasses: IgG1, IgG2, IgG3, and IgG4, each subclass having a different biological function called effector function. These effector functions are typically mediated by interaction with Fc receptors (Fc γ R) or by binding Clq and fixing complement. Binding to Fc γ R can result in antibody-dependent cell lysis, while binding to complement factors can result in complement-mediated cell lysis.

In the design of Fc fusion proteins that extend half-life using only the Fc portion, it is important to minimize effector function. For some antibodies with pure antagonism, such as soluble cytokines such as TNF α, IL17A, etc., or antibodies with immune checkpoints such as PD-1, the effector effects of Fc γ Rs are not required and cytotoxicity by ADCC, etc. can be prevented, so IgG2 and IgG4, which have weak Fc effects, are selected as the backbone. At present, 4 IgG2 and 6 IgG4 monoclonal antibodies are approved to be marketed, for example, anti-PD1 monoclonal antibodies nivolumab and pembrolizumab, anti-IL17A monoclonal antibodies ixekizumab and anti-PCSK9 monoclonal antibodies evolocumab and the like adopt IgG2 or IgG4 subtypes. Thus, the Fc portion of the Fc fusion protein structures of the invention is preferably derived from the human IgG4 Fc region, due to its reduced ability to bind Fc γ R and complement factors compared to other IgG subtypes.

To further reduce its effector function, the present invention further modifies the wild-type IgG4 Fc region. The IgG4 Fc portion of the fusion proteins of the invention may contain one or more of the following substitutions: corresponding to SEQ ID NO: 2 with proline (Pro) or glutamic acid (Glu) at position 16 in a position corresponding to SEQ ID NO: 2 by alanine (Ala) or valine (Val) at position 17 in SEQ ID NO: 2 by replacement of leucine (Leu) with alanine (Ala) or glutamic acid (Glu).

The N297 position of the Fc part of human IgG molecules can be glycosylated, which has a large effect on IgG activity. If this site is removed, glycosylation will affect the conformation of the upper half of CH2, thereby losing the ability to bind Fc γ Rs, affecting the biological activity associated with the antibody. However, the fusion protein constructed according to the present invention does not require effector action by Fc γ Rs, and it is necessary to prevent cytotoxicity by ADCC and the like of the fusion protein, and therefore, it is necessary to modify the Fc portion without glycosylation. Based on this consideration, the inventors found that the sequences corresponding to SEQ ID NOs: 2, substitution of Ala for Asn at position 80, removes the N-linked glycosylation site in the Fc region of IgG4, and this aglycosylation-free modification reduces the biological effects of ADCC and the like of the fusion protein.

Furthermore, the C-terminal lysine residue present in the native molecule may be deleted in the IgG 4-derived Fc portion of the IL10-Fc fusion proteins discussed herein (position 230 of Seq ID No. 2; the deleted lysine is referred to as des-K). Certain cells, such as NS0 cells, express Fc fusion proteins with lysine at the C-terminus that are heterogeneous, with some fusion proteins having lysine at the C-terminal amino acid, while some fusion proteins will lack lysine at the C-terminus due to the action of proteases during expression in certain types of mammalian cells. Therefore, to avoid this heterogeneity, the Fc fusion protein is preferably constructed with a C-terminal lysine deletion.

For ease of understanding, a table of common single letter and three letter code correspondences for amino acids is provided, as shown below.

Preferred IL10-Fc fusion proteins of the invention include the following proteins:

IL10-[GlyGlyGlyGlySer]₃-IgG4 Fc, wherein X1 at position 16 of the Fc is Glu, X2 at position 17 is Ala, X3 at position 18 is Ala, X4 at position 80 is Asn, and X5 at position 230 is absent, having the sequence set forth in SEQ ID NO: 3, respectively.

IL10-[GlyGlyGlyGlySer]₃-IgG4 Fc, wherein X1 at position 16 of the Fc is Glu, X2 at position 17 is Ala, X3 at position 18 is Ala, X4 at position 80 is Ala, and X5 at position 230 is absent, having the sequence of SEQ ID NO: 4, respectively.

IL 10-glyglyglygiser-IgG 4 Fc, wherein X1 at position 16 of the Fc is Glu, X2 at position 17 is Ala, X3 at position 18 is Ala, X4 at position 80 is Asn, and X5 at position 230 is Lys, having the sequence of SEQ ID NO: 5, respectively.

IL10-IgG4 Fc, wherein X1 at position 16 of the Fc is Glu, X2 at position 17 is Ala, X3 at position 18 is Ala, X4 at position 80 is Ala, and X5 at position 230 is absent, having the sequence of SEQ ID NO: and 6.

IL10-[GlyGlyGlyGlySer]₃-IgG4 Fc, wherein X1 at position 16 of Fc is Glu, X2 at position 17 is Ala, X3 at position 18 is Ala, X4 at position 80 is Asn, and X5 at position 230 is Lys.

IL10-GlyGlyGlyGlySer-IgG4 Fc, wherein X1 at position 16 of Fc is Glu, X2 at position 17 is Ala, X3 at position 18 is Ala, X4 at position 80 is Asn, and X5 at position 230 is absent.

IL10-[GlyGlyGlyGlySer]₃-IgG4 Fc, wherein X1 at position 16 of Fc is Pro, X2 at position 17 is Phe, X3 at position 18 is Ala, X4 at position 80 is Ala, and X5 at position 230 is absent.

IL10-[GlyGlyGlyGlySer]₃-IgG4 Fc, wherein X1 at position 16 of Fc is Pro, X2 at position 17 is Val, X3 at position 18 is Ala, X4 at position 80 is Ala, and X5 at position 230 is absent.

IL10-IgG4 Fc, wherein X1 at position 16 of the Fc is Glu, X2 at position 17 is Ala, X3 at position 18 is Ala, X4 at position 80 is Ala, and X5 at position 230 is Lys.

IL10-[GlyGlyGlyGlySer]₂-IgG4 Fc, wherein X1 at position 16 of Fc is Pro, X2 at position 17 is Ala, X3 at position 18 is Ala, X4 at position 80 is Asn, and X5 at position 230 is Lys.

The structure of the IL10-Fc fusion protein is shown in FIG. 1.

Wild-type human IgG4 protein can be obtained from a variety of sources. For example, a cDNA library can be prepared from cells expressing the mRNA of interest at detectable levels to obtain these proteins. Libraries can be screened using probes designed using published DNA or protein sequences for a particular protein of interest. 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-; and Morrison et al, (1984) ann. rev. immunol.2: 239-, 256, describe immunoglobulin light or heavy chain constant regions.

DNA encoding the IL-10 and IgG4 Fc of the invention can be produced by a variety of different methods, including molecular cloning methods and chemically synthesized DNA by standard procedures. Genes encoding fusion proteins can then be constructed by ligating DNA encoding IL-10 in frame with the DNA encoding IgG4 Fc protein described herein. The DNA encoding the wild-type IgG4 Fc fragment can be mutated prior to ligation or in the cDNA encoding the entire fusion protein. Various mutagenesis techniques are well known in the art. The gene encoding IL-10 and the gene encoding the IgG4 Fc analog protein may also be linked in frame by DNA encoding a G-rich linker peptide.

The present invention provides genes encoding IL10-Fc fusion proteins, such as genes encoding the amino acid sequence of SEQ ID NO: 3 of IL10- [ GlyGlyGlyGlySer ]]₃The gene sequence of the IgG4 Fc fusion protein is shown in SEQ ID NO: shown at 7. When a nucleic acid molecule encoding an IL10-Fc fusion protein is inserted into a suitable vector, the IL10-Fc fusion protein can be expressed when the vector is introduced into a suitable host cell. Suitable vectors are various commercially available prokaryotic or eukaryotic expression vectors, such as pET series vectors, pQE series vectors; yeast expression vectors, pPICZ-alpha-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 cells include, but are not limited to, bacterial, yeast, insect and mammalian cells. Recombinant cells containing exogenous nucleic acid encoding an IL10-Fc fusion protein can be prepared by any suitable technique, e.g., transfection/transformation with naked DNA plasmid vectors, viral vectors, invasive bacterial cell vectors, or other whole cell vectors, including by calcium phosphate precipitation transfection, receptor-mediated localization and transfection, biolistic delivery, electroporation, dextran-mediated transfection, liposome-mediated transformation, protoplast fusion, direct microinjection, and the like, to deliver the IL10-Fc fusion protein coding sequence into the cell. Methods for transforming/transfecting cells are known in the art, see Sambrook et al molecular Cloning: a Laboratory Manual, Cold Spring Harbor Laboratory Press (2d Edition, 1989 or 3rd Edition, 2001).

Expression of the nucleotide molecule of the invention may be regulated by another nucleotide sequence, such that the molecule is expressed in a host transformed with the recombinant DNA molecule. For example, expression may be controlled by any promoter/enhancer element known in the art. Promoters useful for controlling the expression of the chimeric polypeptide molecule include, but are not limited to, the long terminal repeat (Squinto et al, 1991, Cell, 65: 1-20); SV40 early promoter region, CMV, M-MuLV, thymidine kinase promoter, metallothionein (metallothionein) gene regulatory sequences; prokaryotic expression vectors such as the b-lactamase promoter or the tac promoter (see Scientific American (1980), 242: 74-94); promoter elements from yeast or other fungi such as the Gal 4 promoter, ADH, PGK, alkaline phosphatase and tissue-specific transcriptional control regions derived from genes such as the elastase I gene.

Cell lines useful as hosts for recombinant proteins are well known in the art and include a variety of immortalized cell lines available from the American Type Culture Collection (ATCC). These cell lines include Chinese Hamster Ovary (CHO) cells, NSO, SP2 cells, HeLa cells, Baby Hamster Kidney (BHK) cells, monkey kidney Cells (COS), human hepatoma cells (e.g., Hep G2), a549 cells, and various other cell lines. In a preferred embodiment, the fusion protein antibody is expressed in CHO cells (DHFR-CHO cells, using DHFR as a selection marker). Another example of an expression system is the GS (glutamate synthetase) gene expression system, to which reference is made in particular to WO87/04462, WO89/01036 and EP338841, among others. When a nucleic acid (or nucleic acid-containing vector) encoding, for example, an IL10-Fc fusion protein is introduced into a mammalian host cell, the fusion protein can be produced by culturing the host cell in a medium sufficient to express the IL10-Fc fusion protein in the host cell or secrete the fusion protein into the medium in which the host cell is grown.

IL10-Fc fusion protein can be recovered from the culture medium by any standard protein purification method known in the art, such as immunoaffinity column purification, sulfate precipitation, ion exchange chromatography, hydrophobic interaction chromatography, reverse phase chromatography, or gel filtration, or any combination thereof. The actual conditions used to purify a particular protein will depend in part on factors such as net charge, hydrophobicity, hydrophilicity, and the like. For affinity chromatography purification of the fusion protein of the present invention, a matrix having protein a or protein G may be used.

In another aspect, the invention provides a pharmaceutical composition comprising any of the IL10-Fc fusion proteins provided herein, the pharmaceutical composition of the invention comprising a therapeutically effective amount of IL10-Fc fusion protein and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers refer to molecular entities and compositions that are generally non-toxic to recipients, i.e., do not produce an adverse, allergic, or other untoward reaction when administered to an animal (e.g., human) where appropriate, at the dosages and concentrations employed. Pharmaceutically acceptable carriers include any and all solvents, buffers, dispersion media, coating materials, surfactants, antioxidants, preservatives (e.g., antibacterial, antifungal), isotonic agents, absorption retardants, salts, preservatives, antioxidants, proteins, drugs, drug stabilizers, polymers, gels, binders, excipients, disintegrants, lubricants, sweeteners, fragrances, dyes, such similar materials, and combinations thereof.

The pharmaceutical composition of the present invention can be administered intravenously, intradermally, intraarterially, intraperitoneally, intracranially, intraarticularly, etc. The fusion proteins of the invention are particularly suitable for parenteral administration, in particular by injection, for example subcutaneous, intradermal, intravenous, intraarterial, intramuscular, intrathecal or intraperitoneal injection. For injection, the fusion proteins of the invention may be formulated in aqueous solution, preferably in a physiologically compatible buffer. Alternatively, the fusion protein may be in powder form for dissolution in a suitable vehicle, e.g., sterile water, prior to use.

The invention also discloses the use of an IL10-Fc fusion protein in the manufacture or manufacture of a medicament for the treatment of a disease in an individual in need thereof, including viral diseases, inflammatory diseases, immune-related disorders, fibrotic disorders, cancer and the like. IL-10 is a cytokine with pleiotropic effects in immune regulation and inflammation. It is produced by mast cells, counteracting the inflammatory effects of these cells at the site of the allergic reaction. Although it is capable of inhibiting the synthesis of proinflammatory cytokines such as IFN- γ, IL-2, IL-3, TNF α, and GM-CSF, IL-10 is also stimulatory for certain T cells and mast cells and stimulates B cell maturation, proliferation, and antibody production. IL-10 blocks NF-. kappa.B activity and is involved in the regulation of the JAK-STAT signal transduction pathway. It also induces the cytotoxic activity of CD8+ T cells and antibody production by B cells, and it inhibits macrophage activity and pro-tumor inflammation. Modulation of CD8+ T cells is dose-dependent, with higher doses inducing stronger cytotoxic responses.

IL-10 plays multiple roles in the activation of CD8+ T cells. For example, IL-10 induces effector molecules (IFN γ, perforin and granzyme B) in memory CD8+ T cells. Such memory CD8+ T cells are the cells responsible for providing long-term antiviral protection to a subject. Although the production and expansion of memory CD, 8+ T cells can occur when IL-10 is absent (Vicari, A. and Trinchieri, G., (2004) immunity. Rev.202:223-236), the fact that IL-10 directly activates such cells provides a unique and alternative therapeutic approach.

IL10-Fc fusion protein has similar biological activity to IL-10. In view of the above, embodiments of the present disclosure are based on the association between CD8+ T cells and cancer and viral infection. Thus, certain methods of treating and/or preventing cancer-related diseases, disorders and conditions, such as maintaining a mean IL10-Fc fusion protein serum concentration, e.g., greater than 0.5ng/mL, greater than 1ng/mL, or greater than 0.1ng/mL, should also be applicable to the treatment of such diseases. The present disclosure encompasses the use of IL10-Fc fusion proteins described herein in the treatment or prevention of a wide range of diseases, disorders or conditions and/or symptoms thereof. According to the present disclosure, IL10-Fc fusion proteins are useful for treating or preventing proliferative conditions or disorders, including cancers, such as cancers of the uterus, cervix, breast, prostate, testis, gastrointestinal tract, kidney, bladder, bone marrow, skin, head or neck, skin, liver, gall bladder, heart, lung, pancreas, salivary glands, adrenal gland, thyroid, brain, ganglia, Central Nervous System (CNS) and Peripheral Nervous System (PNS), and cancers of the hematopoietic and immune systems. In particular embodiments, the tumor or cancer is colon cancer, ovarian cancer, breast cancer, melanoma, lung cancer, pancreatic cancer, glioblastoma, leukemia, or the like.

In one embodiment, a method of using the IL10-Fc fusion protein drug for treating a disease is disclosed, the method comprising administering a therapeutically effective amount of the drug to an individual suffering from a disease, including inflammatory conditions, immune-related disorders, fibrotic disorders, cancer, and the like. The subject is a mammal, preferably a human. Depending on the type and severity of the disease, a serum trough concentration of IL10-Fc fusion protein of greater than about 0.1ng/mL (e.g., 0.1-2ng/mL, 0.1-1ng/mL, 0.5-1.5ng/mL, or 1.1-2.1ng/mL) can be a starting candidate dose for administration to a patient, whether, for example, by one or more divided administrations, or by continuous infusion.

Preferably, when the subject is a human, IL10-Fc fusion protein can be administered at a dose of greater than 2.0 μ g/kg/day, greater than 2.5 μ g/kg/day, greater than 3.0 μ g/kg/day, greater than 5 μ g/kg/day, greater than 8 μ g/kg/day, greater than 10 μ g/kg/day, greater than 12 μ g/kg/day, 15 μ g/kg/day, greater than 18 μ g/kg/day, greater than 20 μ g/kg/day, greater than 21 μ g/kg/day, greater than 22 μ g/kg/day, greater than 23 μ g/kg/day, greater than 24 μ g/kg/day, or greater than 25 μ g/kg/day. Initial doses can be estimated from in vitro data, such as animal models, using techniques well known in the art. One of ordinary skill in the art can readily optimize administration to humans based on animal data.

Drawings

FIG. 1 is a schematic diagram of the structure of human interleukin 10-Fc fusion protein

FIG. 2 is a drawing: IL10-Fc stimulation of CD8+ cells to produce cytotoxic factors

Detailed Description

Example 1: construction of IL10-Fc fusion protein Gene

Converting SEQ ID NO: 3, translating into a DNA sequence, and optimizing according to the codon preference of CHO cells to obtain an expression sequence SEQ ID NO: 7. adding NheI enzyme cutting site and kozac sequence (SEQ ID No.8) at the 5 'end of the optimized sequence, and adding stop codon and XhoI enzyme cutting site (SEQ ID No.9) at the 3' end to obtain a complete expression frame of the fusion protein. And (3) artificially synthesizing a complete expression frame sequence, and inserting the complete expression frame sequence between NheI and XhoI enzyme cutting sites of the pIRES plasmid to obtain the pIRES-IL10-Fc expression plasmid. After linearization, the plasmid is transfected to CHO-s cell electrically, and MSX is added to select positive clone.

Example 2: expression and purification of IL10-Fc fusion protein

The positive clones obtained in example 1 were subjected to two rounds of limiting dilution to select clones with superior expression level, and then inoculated into a 7L fermentor for fed-batch culture to express the target protein through amplification culture. Centrifuging at 4500rpm for 6min after fermentation is finished, collecting supernatant, adjusting pH to 4.0, and storing at 4 deg.C.

The supernatant is firstly processed by ultrafiltration and concentration by using a 10KDa ultrafiltration membrane; then, primary affinity chromatography was performed using Mabselect Sure to collect the fusion protein. The affinity chromatography mobile phase is: a1 25mM PB +50mM NaCl, pH7.0, B1 20mM Gly, pH3.0, B2 20mM citrate buffer, pH 3.0. The column was equilibrated with mobile phase a1, after loading, impurities were eluted with mobile phase B1 first, then fusion protein was eluted with mobile phase B2, and the collected eluate was also adjusted to pH neutral with 1M Tris-His pH 8.0. The crude pure sample collected in this step was purified by passing through a Capto adhere column. After binding of the sample by loading at pH7.0, a purer sample (more than 95%) was obtained by elution at pH 4.0.

Example 3: in vivo half-life assay

rhIL-10(Rochy Hill Co., Ltd.) and IL10-Fc fusion protein obtained in example 2 were each injected into SD rats having an average body weight of about 200g at a dose of 200ng/Kg body weight by tail vein injection). After injection, blood samples were collected by tail-clipping at various time points (0, 1, 2, 4, 6, 8, 12, 24, 36, 48, 60, 72, 96 hours), anticoagulated with heparin sodium, and serum was collected by centrifugation at 12000g for 5 min.

The blood samples were tested for the amount of fusion protein in serum using human IL-10ELISA kit (available from Bender Medsystem) and according to the instructions, and the results were averaged. The results show that the in vivo elimination half-life of the IL10-Fc fusion protein prepared by the invention is 22.6 hours, while the elimination half-life of human rhIL-10 after tail vein injection is 4 hours, which indicates that the in vivo half-life of the IL10-Fc fusion protein prepared by the invention is prolonged by 5-6 times compared with that of the rhIL-10 control.

Example 4: experiment on antitumor Activity

Studies prove that IL-10 not only can exert an anti-tumor effect by activating NK cells, but also can inhibit the development of tumors by activating T cells. Studies have shown that IL-10 treated tumor-bearing mice can induce the expression of IFN-gamma and granzyme. This effect may be mediated by an IL-10 signaling pathway specific for intratumoral CD8+ T cells: IL-10 activates phosphorylated STAT1 and STAT3 in CD8+ T cells, thereby inducing proliferation of CD8+ T cells and expression of IFN- γ, the cytotoxic protein perforin, and the granule protease; IFN-gamma can induce the expression of MHC-I antigen presenting molecules in tumor cells and mononuclear macrophages, and assists CD8+ T cells to kill most of antigen-specific tumor cells; activation of TCR in CD8+ T cells can effectively induce anti-apoptotic and cell proliferative signals. In a word, the IL-10 can not only enhance the tumor killing effect by improving the cytotoxic activity of NK cells, but also enhance the tumor killing capability of CD8+ T cells in tumors and the antigen presenting capability induced by IFN-gamma by mediating the infiltration and activation of specific cytotoxic CD8+ T cells in tumors, the expression of IFN-gamma and granular protease, the enhancement of tumor antigen presenting and the like, thereby enhancing the anti-tumor immune escape function.

To test the cytotoxic effect of IL10-Fc on CD8+ T, CD8+ cells were isolated from mouse spleen and cultured in vitro and activated, and after the addition of different concentrations of IL10-Fc (with IL-10 as a control), the cells were stimulated to produce cytotoxicity (increased granzyme/perforin expression) and IFN γ expression (FIG. 2). Although IL10-Fc only accounts for about 30% -40% of IL10 in terms of in vitro activity, IL10-Fc has no significant decrease in vivo biological activity in view of its significantly prolonged in vivo half-life.

Sequence listing

<110> Hangzhou Bohu Biotechnology Ltd

<120> human interleukin 10-Fc fusion protein, and coding gene and application thereof

<160> 9

<170> SIPOSequenceListing 1.0

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<211> 160

<212> PRT

<213> Homo sapiens (Human)

<400> 1

Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro

1 5 10 15

Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg

20 25 30

Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu

35 40 45

Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala

50 55 60

Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala

65 70 75 80

Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly Glu

85 90 95

Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu

100 105 110

Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala Phe

115 120 125

Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp

130 135 140

Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg Asn

145 150 155 160

<210> 2

<211> 230

<212> PRT

<213> Artificial Sequence

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Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Xaa

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Ser Leu Ser Leu Gly Xaa

225 230

<210> 3

<211> 404

<212> PRT

<213> Artificial Sequence

<400> 3

Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro

1 5 10 15

Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg

20 25 30

Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu

35 40 45

Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala

50 55 60

Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala

65 70 75 80

Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly Glu

85 90 95

Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu

100 105 110

Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala Phe

115 120 125

Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp

130 135 140

Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg Asn

145 150 155 160

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

Leu Ser Leu Gly

<210> 4

<211> 404

<212> PRT

<213> Artificial Sequence

<400> 4

Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro

1 5 10 15

Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg

20 25 30

Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu

35 40 45

Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala

50 55 60

Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala

65 70 75 80

Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly Glu

85 90 95

Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu

100 105 110

Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala Phe

115 120 125

Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp

130 135 140

Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg Asn

145 150 155 160

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

Leu Ser Leu Gly

<210> 5

<211> 395

<212> PRT

<213> Artificial Sequence

<400> 5

Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro

1 5 10 15

Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg

20 25 30

Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu

35 40 45

Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala

50 55 60

Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala

65 70 75 80

Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly Glu

85 90 95

Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu

100 105 110

Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala Phe

115 120 125

Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp

130 135 140

Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg Asn

145 150 155 160

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

165 170 175

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

180 185 190

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

195 200 205

Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn

210 215 220

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

225 230 235 240

Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

245 250 255

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

260 265 270

Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys

275 280 285

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu

290 295 300

Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

305 310 315 320

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

325 330 335

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

340 345 350

Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly

355 360 365

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

370 375 380

Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

385 390 395

<210> 6

<211> 389

<212> PRT

<213> Artificial Sequence

<400> 6

Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro

1 5 10 15

Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg

20 25 30

Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu

35 40 45

Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala

50 55 60

Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala

65 70 75 80

Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly Glu

85 90 95

Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu

100 105 110

Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala Phe

115 120 125

Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp

130 135 140

Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg Asn

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

Ser Leu Ser Leu Gly

385

<210> 7

<211> 972

<212> DNA

<213> Artificial Sequence

<400> 7

gagaaccagg accccgacat caaggcccac gtgaacagcc tgggcgagaa cctgaagacc 60

ctgaggctga ggctgaggag gtgccacagg ttcctgccct gcgagaacaa gagcaaggcc 120

gtggagcagg tgaagaacgc cttcaacaag ctgcaggaga agggcatcta caaggccatg 180

agcgagttcg acatcttcat caactacatc gaggcctaca tgaccatgaa gatcaggaac 240

ggcggcggcg gcagcggcgg cggcggcagc ggcggcggcg gcagcgccga gagcaagtac 300

ggccccccct gccccccctg ccccgccccc gaggccgccg gcggccccag cgtgttcctg 360

ttccccccca agcccaagga caccctgatg atcagcagga cccccgaggt gacctgcgtg 420

gtggtggacg tgagccagga ggaccccgag gtgcagttca actggtacgt ggacggcgtg 480

gaggtgcaca acgccaagac caagcccagg gaggagcagt tcaacagcac ctacagggtg 540

gtgagcgtgc tgaccgtgct gcaccaggac tggctgaacg gcaaggagta caagtgcaag 600

gtgagcaaca agggcctgcc cagcagcatc gagaagacca tcagcaaggc caagggccag 660

cccagggagc cccaggtgta caccctgccc cccagccagg aggagatgac caagaaccag 720

gtgagcctga cctgcctggt gaagggcttc taccccagcg acatcgccgt ggagtgggag 780

agcaacggcc agcccgagaa caactacaag accacccccc ccgtgctgga cagcgacggc 840

agcttcttcc tgtacagcag gctgaccgtg gacaagagca ggtggcagga gggcaacgtg 900

ttcagctgca gcgtgatgca cgaggccctg cacaaccact acacccagaa gagcctgagc 960

ctgagcctgg gc 972

<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. An IL10-Fc fusion protein wherein the C-terminus of IL-10 is linked to the N-terminus of the Fc protein either directly or via a linking peptide; wherein the IL-10 sequence is consistent with that shown in Seq ID No. 1; the general formula of the connecting peptide sequence is [ GlyGlyGlySer ] n, and n is an integer from 1 to 5; the Fc protein portion comprises the sequence of SEQ ID No.2, wherein:

x1 at position 16 is Pro or Glu;

x2 at position 17 is Phe, Val, or Ala;

x3 at position 18 is Leu, Glu, or Ala;

x4 at position 80 is Asn or Ala; and

x5 at position 230 is Lys or absent.

2. The IL10-Fc fusion protein according to claim 1, characterized in that: the general formula of the connecting peptide sequence in the fusion protein is [ GlyGlyGlyGlySer ]]_nAnd n is an integer of 1 to 3.

3. The IL10-Fc fusion protein according to claim 2, characterized in that: the sequence of the connecting peptide in the fusion protein is [ GlyGlyGlyGlySer ]]₃。

4. The IL10-Fc fusion protein according to claim 1, characterized in that: in the fusion protein, SEQ ID NO: 2, amino acid X4 at position 80 is Ala to remove the N-linked glycosylation site in the IgG4 Fc region.

5. The IL10-Fc fusion protein according to claim 1, characterized in that: in the fusion protein, SEQ ID NO: 2, amino acid X5 at position 230.

6. The IL10-Fc fusion protein according to claim 1, characterized in that: the amino acid sequence of the fusion protein is similar to that of SEQ ID NO: 4. SEQ ID NO: 5 or SEQ ID NO: 6 are identical.

7. The IL10-Fc fusion protein according to claim 1, characterized in that: the fusion protein is selected from the following proteins:

IL10-[GlyGlyGlyGlySer]₃-IgG4 Fc, wherein X1 at position 16 of Fc is Glu, X2 at position 17 is Ala, X3 at position 18 is Ala, X4 at position 80 is Asn, and X5 at position 230 is Lys;

IL 10-glyglyglygiser-IgG 4 Fc, wherein X1 at position 16 of Fc is Glu, X2 at position 17 is Ala, X3 at position 18 is Ala, X4 at position 80 is Asn, and X5 at position 230 is absent;

IL10-[GlyGlyGlyGlySer]₃-IgG4 Fc, wherein X1 at position 16 of Fc is Pro, X2 at position 17 is Phe, X3 at position 18 is Ala, X4 at position 80 is Ala, and X5 at position 230 is absent;

IL10-[GlyGlyGlyGlySer]₃-IgG4 Fc, wherein X1 at position 16 of Fc is Pro, X2 at position 17 is Val, X3 at position 18 is Ala, X4 at position 80 is Ala, and X5 at position 230 is absent;

IL10-IgG4 Fc, wherein X1 at position 16 of the Fc is Glu, X2 at position 17 is Ala, X3 at position 18 is Ala, X4 at position 80 is Ala, and X5 at position 230 is Lys;

8. A gene encoding an IL10-Fc fusion protein according to any one of claims 1 to 7.

9. A pharmaceutical composition comprising a therapeutically effective amount of an IL10-Fc fusion protein according to any one of claims 1-7 and a pharmaceutically acceptable carrier.

10. Use of an IL10-Fc fusion protein according to any one of claims 1 to 7 for the manufacture of a medicament for the treatment of viral diseases, inflammatory conditions, immune related disorders, fibrotic disorders and cancer.