CN111989340A

CN111989340A - Recombinant human interleukin 10 fusion protein and application thereof

Info

Publication number: CN111989340A
Application number: CN201880092332.3A
Authority: CN
Inventors: 汤甜甜; 许宝青
Original assignee: Hangzhou Bohu Biotechnology Co ltd
Current assignee: Hangzhou Bohu Biotechnology Co ltd
Priority date: 2018-11-18
Filing date: 2018-11-18
Publication date: 2020-11-24
Also published as: WO2020097946A1

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. The present invention provides IL10-Fc fusion proteins, wherein the C-terminus of IL-10 is linked, directly or via a linking peptide, to the N-terminus of an Fc protein of human IgG2 or human IgG 4. 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

[ 21.12.2018 corrected according to rule 26 ] recombinant human interleukin 10 fusion protein and application thereof

Technical Field

The invention relates to the field of genetic engineering medicines, in particular to a recombinant 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. Furthermore, it co-stimulates Natural Killer (NK) cell proliferation and cytokine production and acts as a growth factor to stimulate proliferation of certain subsets of CD8+ T cells (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 elevated INF γ production in humans (Lauw, F.N.et., J.Immunol.165,2783-2789 (2000); Tilg, H.et al., Gut 50,191-195 (2002)). the immunostimulatory activity of IL-10 is reported to be determined by a single amino acid at position 87 in IL-10 (MedDing, Y.et al, J.exp.191 (2),213-223 (2000)).

Human IL-10 is a homodimeric protein, each monomer comprising 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 a disulfide bond of 4 cysteines (12-108, 62-114), and its native active form is a non-covalently linked, 38kD molecular weight, homodimer that becomes biologically inactive after the non-covalent interaction between the two monomeric subunits is 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 sites for PEG modification, the product modified by PEGylation is heterogeneous, which is troublesome for the subsequent control of drug substance.

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 the primary 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.

Summary of The Invention

In one aspect, the invention provides a fusion protein of human interleukin-10 (IL-10) and a human IgG Fc portion (IL10-Fc fusion protein).

In one embodiment, the invention provides IL10-Fc fusion proteins, wherein the C-terminus of IL-10Is connected with the N end of the human IgG Fc protein directly or through a connecting peptide; wherein the IL-10 sequence is similar to Seq ID No:1 is consistent; the general formula of the connecting peptide sequence is (G)₄S) _n、(SG ₄) _nOr G₄(SG ₄) _nN is a number between 1 and 10; the human IgG Fc protein is selected from the native sequence Fc region or a variant Fc region of human IgG2, IgG 4.

In a preferred embodiment of the invention, the human IgG2 Fc sequence is as set forth in SEQ ID NO: shown at 10.

In another preferred embodiment of the invention, the human IgG4 Fc sequence is as set forth in SEQ ID NO: shown at 11.

In one embodiment, the present invention provides an IL10-Fc fusion protein, wherein the preferred linker peptide sequence has the general formula [ GlyGlyGlyGlySer ]] _nN is an integer of 1 to 4; more preferably, n is 3, having the sequence [ GlyGlyGlyGlySer ]] ₃. 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.

In a preferred embodiment, the present invention provides an IL10-Fc fusion protein in which the C-terminus of IL-10 is linked to the N-terminus of a human IgG Fc protein by a linker peptide; wherein the IL-10 sequence is similar to Seq ID No:1, the connecting peptide sequence is [ GlyGlyGlyGlySer ]3, and the human IgG Fc protein is selected from natural sequence Fc region or variant Fc region of human IgG2 and IgG 4.

In another preferred embodiment, the IL10-Fc fusion protein provided by the invention is IL 10-human IgG2 Fc fusion protein, the sequence of which is shown in SEQ ID NO: shown at 12.

In another preferred embodiment, the IL10-Fc fusion protein provided by the invention is IL 10-human IgG4 Fc fusion protein, and the sequence of the fusion protein is shown as SEQ ID NO. 13.

In another embodiment, the invention provides that the IL10-Fc fusion protein is C-terminal to IL-10 directly or via a linker peptide to a human IgG4 Fc variant proteinThe white N ends are connected; wherein the IL-10 sequence is similar to Seq ID No:1 is consistent; the general formula of the connecting peptide sequence is (G)₄S) _n、(SG ₄) _nOr G₄(SG ₄) _nN is a number between 1 and 10; the human IgG4 Fc variant protein comprises 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.

In another preferred embodiment, the general formula of the preferred connecting peptide sequence in the IL10-IgG4 Fc fusion protein provided by the invention is [ GlyGlyGlyGlySer ]] _nAnd n is an integer of 1 to 4.

In another preferred embodiment, the general formula of the preferred connecting peptide sequence in the IL10-IgG4 Fc fusion protein provided by the invention is [ GlyGlyGlyGlySer ]] ₃. 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.

In another preferred embodiment, the present invention further modifies the wild-type IgG4 Fc sequence. 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).

In another preferred embodiment, preferred IL10-IgG4 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 invention further provides a polynucleotide encoding the IL10-Fc fusion protein of the invention. Further provided is a vector, particularly an expression vector, comprising a polynucleotide of the invention. In another aspect, the invention provides a host cell comprising a polynucleotide or vector of the invention. The present invention also provides a method for producing the IL10-Fc fusion protein of the invention, comprising the steps of: (i) culturing the host cell of the invention under conditions suitable for expression of the IL10-Fc fusion protein, and (ii) recovering the fusion protein.

In another aspect, the invention provides a pharmaceutical composition comprising an effective amount of an IL10-Fc fusion protein of the invention and a pharmaceutically acceptable carrier. Also provided are IL10-Fc fusion proteins or pharmaceutical compositions of the invention for use as medicaments and for the treatment or prevention of diseases in a subject in need thereof, including viral diseases, inflammatory diseases, immune-related disorders, fibrotic disorders and proliferative conditions, among others.

In a preferred embodiment, the IL10-Fc fusion proteins provided herein are used to treat or prevent 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, methods of using the IL10-Fc fusion protein medicaments for treating diseases are disclosed. 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.

Brief Description of Drawings

FIG. 1 is a schematic diagram of the structure of human interleukin 10-Fc fusion protein. The C-terminus of IL-10 is linked to the N-terminus of the human IgG Fc protein, either directly or via a linker peptide, and forms a dimer via disulfide bonding of the human IgG Fc region.

FIG. 2 is a graph showing the cell growth curves of different clones cultured in 7L tanks for 0-13 days. Ordinate unit is 10⁶/ml。

FIG. 3 shows the expression levels of different clones cultured in 7L tanks on days 10,12 and 13, in mg/ml on the ordinate.

FIG. 4 is an electrophoretogram of IL10-Fc fusion protein after affinity chromatography purification. 1 is cell line 1 culture supernatant; 2 is cell line 1 flow through; 3, purifying the cell line 1; 4 is cell line 2 culture supernatant; 5 is cell line 2 flow through; 6 is cell line 2 purification, 7 is cell line 3 culture supernatant; 8 is cell line 3 flow through; 9 is cell line 3 purification; 10 is a commercial standard Marker; the molecular weight of the IL10-Fc fusion protein is about 90 kD.

FIG. 5: IL10-Fc stimulates the production of cytotoxic factors by CD8+ cells. 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. 6: the tumor proliferation volume change of different groups is given for 6 times. Administering different antibodies according to groups, i.e. intraperitoneal injection, 250 ug/dose for the first time, and 200 ug/dose (10mg/kg) and q3dx6 times; IL10-Fc was administered subcutaneously around tumor bodies at 5ug (100ul solution) per tumor body, q3dx6 times; the tumor proliferation change and the mouse state after the administration treatment are observed, and the tumor proliferation is continuously observed and measured for 1-2 weeks after the administration is finished.

Detailed Description

To make the invention easier to understand, certain terms are first defined. Additional definitions will be set forth throughout the detailed description.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. These techniques are explained fully in the literature, such as Molecular Cloning: a Laboratory Manual (molecular cloning: A Laboratory Manual), second edition (Sambrook et al, 1989); oligonucleotide Synthesis (m.j. gait, 1984); animal Cell Culture (r.i. freshney, 1987); methods in Enzymology (Methods in Enzymology) (Academic Press, Inc.); current Protocols in Molecular Biology (contemporary Molecular Biology Protocols) (F.M. Ausubel et al, 1987); and (3) PCR: the Polymerase Chain Reaction (PCR), (Mullis et al, 1994); a Practical Guide to Molecular Cloning (Practical Guide for Molecular Cloning) (Perbal Bernard V., 1988); phage Display: a Laboratory Manual (phage display: A Laboratory Manual) (Barbas et al, 2001) and the like.

As used herein, the term "fusion protein" refers to a fusion polypeptide molecule comprising an IL-10 molecule and a human IgG Fc portion, wherein the components of the fusion protein are linked to each other by peptide bonds, either directly or via a linking peptide. For clarity, the individual peptide chains of the human IgG Fc portion of the fusion protein may be non-covalently linked, e.g., by disulfide bonds.

By "fusion" is meant that the components are linked by peptide bonds, either directly or via one or more linking peptides.

Native IL-10 is a homodimer consisting of two alpha-helical monomer domains. The sequence of the native human IL-10 monomer domain is shown in SEQ ID NO: 1.

the term "Fc domain" or "Fc region" is used herein to define a C-terminal region of an antibody heavy chain that contains at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. The IgG Fc region comprises IgG CH2 and IgG CH3 domains. The "CH 2 domain" of the human IgG Fc region typically extends from approximately the amino acid residue at position 231 to approximately the amino acid residue at position 340. In one embodiment, the carbohydrate chain is attached to a CH2 domain. The CH2 domain herein may be a native sequence CH2 domain or a variant CH2 domain. The "CH 3 domain" comprises a stretch of residues C-terminal to the CH2 domain in the Fc region (i.e., from about the amino acid residue at position 341 to about the amino acid residue at position 447 in an IgG). The CH3 region herein may be a native sequence CH3 domain or a variant CH3 domain, see U.S. patent No.5,821,333, expressly incorporated herein by reference). Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as EU index, as described in Kabat et al, Sequences of Proteins of Immunological Interest,5th ed.

The term "variant" encompasses naturally occurring variants and non-naturally occurring variants, broadly refers to mutated recombinant proteins, which typically carry single or multiple amino acid substitutions and are often derived from cloned genes that have been subjected to site-directed or random mutagenesis or from entirely synthetic genes.

The terms "DNA," "nucleic acid molecule," "polynucleotide," and the like are used interchangeably herein to refer to a polymeric form of nucleotides of any length (deoxyribonucleotides or ribonucleotides or analogs thereof). Non-limiting examples of polynucleotides include linear and circular nucleic acids, messenger rna (mrna), complementary dna (cdna), recombinant polynucleotides, vectors, probes, primers, and the like.

The term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors which are self-replicating nucleic acid structures and vectors which integrate into the genome of a host cell into which they are introduced. Certain vectors are capable of directing the expression of a nucleic acid to which they are operably linked. Such vectors are referred to herein as "expression vectors".

The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to a cell into which an exogenous nucleic acid has been introduced, including the progeny of such a cell.

The term "pharmaceutical composition" refers to a preparation in a form that allows the biological activity of the active ingredient contained therein to be effective, and that is free of other ingredients that have unacceptable toxicity to the subject to whom the formulation will be administered.

The term "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical composition other than the active ingredient that is not toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, preservatives, and the like.

A "therapeutically effective amount" of an agent, e.g., a pharmaceutical composition, refers to an amount (in a necessary dose and for a necessary time) effective to achieve the desired therapeutic or prophylactic result.

The terms "patient" or "subject" are used interchangeably to refer to a human or non-human animal (e.g., a mammal).

The terms "treat," "treatment," or the like, refer to clinical intervention (e.g., administration of IL10-Fc fusion protein or a pharmaceutical composition comprising IL10-Fc fusion protein) that is performed in order to transiently or permanently eliminate, alleviate, suppress, palliate, or ameliorate a disease, disorder, or condition afflicting a subject, after the disease, disorder, or condition, or symptoms thereof, has been diagnosed, observed, or the like.

IL10-Fc fusion protein of the invention

In one embodiment, the present invention provides an IL10-Fc fusion protein wherein the C-terminus of IL-10 is linked to the N-terminus of a human IgG Fc protein, either directly or via a linker peptide; wherein the IL-10 sequence is similar to Seq ID No:1 is consistent; the general formula of the connecting peptide sequence is (G)₄S) _n、(SG ₄) _nOr G₄(SG ₄) _nN is a number between 1 and 10; the human IgG Fc protein is selected from the native sequence Fc region or a variant Fc region of human IgG2, IgG 4.

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.

It is an object of the present invention to extend the half-life of recombinant human IL-10 in vivo, improve circulation time, exposure, efficacy and reduce renal uptake, while reducing unwanted immune effects such as ADCC, CDC and the like, and therefore the Fc portion of the Fc fusion protein structure of the present invention is preferably derived from the human IgG2 Fc sequence or the IgG4 Fc sequence, due to its reduced ability to bind Fc γ R and complement factors compared to other IgG subtypes.

In a preferred embodiment, the present invention provides an IL10-Fc fusion protein in which the C-terminal linker peptide of IL-10 is linked to the N-terminus of the Fc protein; wherein the IL-10 sequence is similar to Seq ID No:1, the connecting peptide sequence is [ GlyGlyGlyGlySer ]3, and the human IgG Fc protein is selected from a natural sequence Fc region or a variant Fc region of human IgG2 or IgG 4. In another preferred embodiment, the IL10-Fc fusion protein provided by the invention is IL 10-human IgG2 Fc fusion protein, and the sequence of the fusion protein is shown as SEQ ID NO. 12. In another preferred embodiment, the IL10-Fc fusion protein provided by the invention is IL 10-human IgG4 Fc fusion protein, and the sequence of the fusion protein is shown as SEQ ID NO. 13.

In another embodiment, the invention provides that the IL10-Fc fusion protein is such that the C-terminus of IL-10 is linked, either directly or via a linker peptide, to the N-terminus of a human IgG4 Fc variant protein; wherein the IL-10 sequence is similar to Seq ID No:1 is consistent; the general formula of the connecting peptide sequence is (G)₄S) _n、(SG ₄) _nOr G₄(SG ₄) _nN is a number between 1 and 10; the human IgG4 Fc region or variant protein comprises 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.

In another preferred embodiment, the preferred linker peptide sequence in the IL10-Fc fusion protein provided by the present invention has the general formula [ GlyGlyGlyGlySer ] n, wherein n is an integer of 1-5.

In another preferred embodiment, the preferred linker peptide sequence in the IL10-Fc fusion protein provided by the present invention has the general formula [ GlyGlyGlyGlySer ] 3. 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.

In another preferred embodiment, the present invention further modifies the wild-type IgG4 Fc sequence. The invention obtains a novel human interleukin 10-IgG4 Fc fusion protein (IL10-IgG4 Fc fusion protein) by modifying an IgG4 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-IgG4 Fc fusion protein provided by the invention is characterized in that 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 similar to Seq ID No:1 is consistent; 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 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-IgG4 Fc fusion protein is [ GlyGlyGlyGlySer ] n, wherein n is an integer of 1-3; 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.

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).

Glycosylation at position N297 (EU numbering system) of the Fc part of human IgG molecules can occur, 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 (Seq ID NO: 2, position 230; 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:

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

Wild-type human IgG2 or IgG4 proteins 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.usa77: 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.

Polynucleotide

The invention also provides polynucleotides encoding IL10-Fc fusion proteins as described herein.

Polynucleotides of the invention also include those that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% homologous to a polynucleotide sequence encoding an IL10-Fc fusion protein as described herein.

DNA encoding IL-10 and IgG Fc (IgG2 Fc or IgG4 Fc) of the invention can be produced by a variety of different methods, including standard procedures for molecular cloning and chemically synthesized DNA. Genes encoding fusion proteins can then be constructed by ligating DNA encoding IL-10 in frame with DNA encoding IgG Fc protein as described herein. The DNA encoding the wild-type IgG 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 IgG 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, and the gene sequence of the IL10- [ GlyGlyGlyGlySer ]3-IgG4 Fc fusion protein is shown as SEQ ID NO: shown at 7.

Recombination method

The fusion protein of the present invention can be obtained, for example, by recombinant production. For recombinant production, one or more polynucleotides encoding the IL10-Fc fusion protein (e.g., as described above) are isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such polynucleotides can be readily isolated and sequenced using conventional procedures. In one embodiment, a vector (preferably an expression vector) comprising one or more polynucleotides of the invention is provided. Methods well known to those skilled in the art can be used to construct expression vectors containing the coding sequence for IL10-Fc fusion protein and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo recombination/genetic recombination.

The present invention also provides a method for producing the IL10-Fc fusion protein of the invention, comprising the steps of:

(i) culturing a host cell of the invention under conditions suitable for expression of an IL10-Fc fusion protein;

(ii) recovering the fusion protein.

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 delivery of the IL10-Fc fusion protein coding sequence into cells by transfection of calcium phosphate precipitates, receptor-mediated localization and transfection, biolistic delivery, electroporation, dextran-mediated transfection, liposome-mediated transformation, protoplast fusion, direct microinjection, and the like. Methods for transforming/transfecting cells are known in the art, see Sambrook et al molecular Cloning: the 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.

Pharmaceutical composition

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.

Method of treatment

Any of the IL10-Fc fusion proteins provided herein can be used in a method of treatment.

For use in a method of treatment, the fusion proteins of the invention will be formulated, administered and administered in a manner consistent with clinical medical practice. Factors considered in this context include the particular condition being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the condition, the site of delivery of the agent, the method of administration, the timing of administration, and other factors known to medical practitioners.

In certain embodiments, IL10-Fc fusion proteins or pharmaceutical compositions of the present invention are provided for use as a medicament and for treating or preventing diseases including viral diseases, inflammatory diseases, immune-related disorders, fibrotic disorders and proliferative conditions, among others, in a subject in need thereof.

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 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 memory CD8+ T cell production and expansion 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), as well as 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.

Examples

The following are examples of the methods and compositions of the present invention. It is to be understood that various other embodiments may be practiced in view of the general description provided above.

Example 1: construction of IL10-IgG4 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-IgG4 Fc fusion protein

The positive clones obtained in example 1 were subjected to two rounds of limiting dilution, and 3 clones with the best expression level were selected, and inoculated into a 7L fermentor for fed-batch culture (FIG. 2) to express the target protein (FIG. 3). 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 ultrafiltered and concentrated by an ultrafiltration membrane package of 10 KDa; 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, the sample was loaded and then the impurities were eluted with mobile phase B1 followed by mobile phase B2, and the collected eluate was also adjusted to neutral pH 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. The electrophoretogram after affinity chromatography purification is shown in FIG. 4, and the molecular weight of the IL10-IgG4 Fc fusion protein is about 90 kD.

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 the collected blood samples were centrifuged at 12000g for 5min to collect serum.

The blood samples were tested for the amount of fusion protein in serum using human IL-10 ELISA kit (available from Bender Medsystem) and according to the instructions, and the results were averaged. The result shows that the in vivo elimination half-life of the IL10-Fc fusion protein prepared by the invention is more than 22.6 hours, while the elimination half-life of the human rhIL-10 injected by tail vein is 2-4 hours, which indicates that the in vivo half-life of the IL10-Fc fusion protein prepared by the invention is prolonged by more than 5-6 times compared with the rhIL-10 reference substance.

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. 5). 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.

Example 5: test experiment of in vivo efficacy of IL10-Fc

Firstly, an animal model with SPC immune repertoire tolerance is constructed, an SPC-PDL1 lung cancer cell line and PBMC extracted from human peripheral blood are mixed and inoculated to the right armpit of a mouse, and 3x10 is inoculated to each armpit⁶About 7-9 days later, the majority of tumor bodies are proliferated to 90-250mm³Time (average value at 120-³). Then excluding tumor body less than 90mm³Or more than 250mm³The remaining mice were assigned equally to human IgG4 antibody (HuIgG4) blank group, PD-1 antibody positive control group, and IL10-Fc group (10 each) according to a random assignment method, and then were subjected to 6 antibody treatments or IL10-Fc as required for different groups.

The dosing regimen for the mice was: administering different antibodies according to groups, i.e. intraperitoneal injection, 250 ug/dose for the first time, and 200 ug/dose (10mg/kg) and q3dx6 times; IL10-Fc was administered subcutaneously around tumor bodies at 5ug (100ul solution) per tumor body, q3dx6 times; the tumor proliferation change and the mouse state after the administration treatment are observed, and the tumor proliferation is continuously observed and measured for 1-2 weeks after the administration is finished. The administration was performed 6 times per group, during which the PD-1 antibody and IL10-Fc groups were comparable in tumor volume and proliferation rate overall and significantly slower than the control HuIgG4 group, with statistically significant differences (p < 0.05), as shown in fig. 6. Preliminary animal experiments prove that the effect of IL10-Fc on inhibiting the growth of tumor cells in mice is close to that of a PD-1 antibody, and the potential medical value of the drug in tumor treatment is shown.

Claims

An IL10-Fc fusion protein wherein the C-terminus of IL-10 is linked to the N-terminus of a human IgG Fc protein, either directly or via a linking peptide; wherein the IL-10 sequence is similar to Seq ID No:1 is consistent; the general formula of the connecting peptide sequence is (G)₄S) _n、(SG ₄) _nOr G₄(SG ₄) _nN is a number between 1 and 10; the human IgG Fc protein is selected from the native sequence Fc region or a variant Fc region of human IgG2, IgG 4.
The IL10-Fc fusion protein according to claim 1, characterized in that: the human IgG2 Fc sequence is shown in SEQ ID NO: shown at 10.
The IL10-Fc fusion protein according to claim 1, characterized in that: the human IgG4 Fc sequence is shown in SEQ ID NO: shown at 11.
The IL10-Fc fusion protein according to claim 1, characterized in that: the general formula of the connecting peptide sequence is [ GlyGlyGlyGlySer ]] _nAnd n is an integer of 1 to 4.
The IL10-Fc fusion protein according to claim 1, characterized in that: the IL10-Fc fusion protein is formed by connecting peptide [ GlyGlyGlyGlySer ] at the C terminal of IL-10] ₃Linked to the N-terminus of a human IgG Fc protein; wherein the IL-10 sequence is similar to Seq ID No:1, the human IgG Fc protein is selected from the native sequence Fc region or a variant Fc region of human IgG2, IgG 4.
IL10-Fc fusion protein according to claim 5, characterized in that: the IL10-Fc fusion protein is IL 10-human IgG2 Fc fusion protein, and the sequence of the fusion protein is shown in SEQ ID NO: shown at 12.
IL10-Fc fusion protein according to claim 5, characterized in that: the IL10-Fc fusion protein is IL 10-human IgG4 Fc fusion protein, and the sequence of the fusion protein is shown as SEQ ID NO. 13.
The IL10-Fc fusion protein according to claim 1, characterized in that: the IL10-Fc fusion protein is formed by connecting the C terminal of IL-10 with the N terminal of human IgG4 Fc variant protein directly or through a connecting peptide; wherein the IL-10 sequence is similar to Seq ID No:1 is consistent; the general formula of the connecting peptide sequence is (G)₄S) _n、(SG ₄) _nOr G₄(SG ₄) _nN is a number between 1 and 10; the human IgG4 Fc variant protein comprises 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 IL10-Fc fusion protein according to claim 1, characterized in that: the general formula of the connecting peptide sequence in the IL10-IgG4 Fc fusion protein is [ GlyGlyGlyGlySer ]] _nAnd n is an integer of 1 to 4.
The IL10-Fc fusion protein according to claim 9, characterized in that: the general formula of the connecting peptide sequence in the IL10-IgG4 Fc fusion protein is [ GlyGlyGlyGlySer ]] ₃。
The IL10-Fc fusion protein according to claim 1, characterized in that: the IL10-IgG4 Fc fusion protein comprises 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 is shown in the specification;

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 is shown in the specification;

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 is shown in the specification;

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: 6 is shown in the specification;

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; IL10- [ GlyGlyGlyGlySer ]2-IgG4 Fc, wherein X1 at position 16 of the 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.
A polynucleotide encoding an IL10-Fc fusion protein according to any one of claims 1-11.
A polynucleotide having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% homology to a polynucleotide sequence encoding an IL10-Fc fusion protein of any one of claims 1-11.
The polynucleotide of claim 12, having a gene sequence as set forth in SEQ ID NO: shown at 7.
A vector, in particular an expression vector, comprising a polynucleotide according to any one of claims 12 to 14.
The carrier of claim 15, wherein: the vector is selected from prokaryotic expression vectors such as pET series vectors and pQE series vectors; yeast expression vector pPICZ-alpha-A, pHIL-D2, pPIC9 or pHIL-S1; animal cell expression vector pIRES plasmid, pSVK3 or pMSG;
a host cell comprising the polynucleotide of claim 12 or the vector of claim 15.
The host cell of claim 17, wherein: the host cell is selected from bacterial, yeast, insect or mammalian cells.
The host cell of claim 18, wherein: the host cell is selected from Chinese Hamster Ovary (CHO) cells, NSO cells, SP2 cells, HeLa cells, Baby Hamster Kidney (BHK) cells, monkey kidney Cells (COS) cells, human liver cancer cells Hep G2 or A549 cells.
A method for producing an IL10-Fc fusion protein according to any one of claims 1-11, comprising the steps of:

(i) culturing the host cell of claim 17 under conditions suitable for expression of IL10-Fc fusion protein, and

(ii) recovering the IL10-Fc fusion protein.
The method of claim 20, wherein: step (ii) the method for recovering IL10-Fc fusion protein comprises immunoaffinity column purification, sulfate precipitation, ion exchange chromatography, hydrophobic interaction chromatography, reverse phase chromatography or gel filtration, or any combination thereof.
A pharmaceutical composition comprising a therapeutically effective amount of an IL10-Fc fusion protein according to any one of claims 1-11 and a pharmaceutically acceptable carrier.
An IL10-Fc fusion protein according to any one of claims 1 to 11 or a pharmaceutical composition according to claim 22 for use as a medicament.
An IL10-Fc fusion protein according to any one of claims 1-11 or a pharmaceutical composition according to claim 22 for use in the treatment or prevention of a disease in a subject in need thereof, said disease comprising a viral disease, an inflammatory disease, an immune-related disorder, a fibrotic disorder or a proliferative condition, and the like.
An IL10-Fc fusion protein according to any one of claims 1-11 or a pharmaceutical composition according to claim 22 for use in the treatment or prevention of a proliferative condition or disorder, including cancer.
The IL10-Fc fusion protein of any one of claims 1-11 or the pharmaceutical composition of claim 22 for use in treating or preventing cancer, including cancer 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 glands, thyroid, brain, ganglia, Central Nervous System (CNS) and Peripheral Nervous System (PNS), and cancers of the hematopoietic and immune systems.
The IL10-Fc fusion protein according to any one of claims 1-11 or the pharmaceutical composition according to claim 22 for use in the treatment or prevention of cancer, wherein the tumor or cancer is colon cancer, ovarian cancer, breast cancer, melanoma, lung cancer, pancreatic cancer, glioblastoma, leukemia, or the like.
A method of treating a disease comprising administering to an individual suffering from a disease comprising an inflammatory condition, an immune-related disorder, a fibrotic disorder, cancer, etc., a therapeutically effective amount of an IL10-Fc fusion protein of any one of claims 1-11.
The method of claim 28, wherein the subject is a mammal.
The method of claim 29, wherein the subject is a human.
The method of claim 30, wherein the individual is a human, the IL10-Fc fusion protein is 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.