A recombinant human interleukin-10 fusion protein and its application

Abstract

The present invention relates to the field of genetically engineered drugs, and particularly relates to a human interleukin 10-Fc fusion protein, its encoding gene and applications. The present invention provides an IL10-Fc fusion protein, wherein the C-terminus of IL-10 is directly or via a linker peptide linked to the N-terminus of the Fc protein of human IgG2 or human IgG4. The present invention also discloses a method for using the IL10-Fc fusion protein drug for treating diseases, the method comprising administering a therapeutically effective amount of the drug to an individual suffering from a disease, and the diseases include inflammatory conditions, immune-related disorders, fibrotic disorders and cancers, etc.

Description

Technical Field

[0001] The present invention relates to the field of genetically engineered drugs, and in particular to a recombinant human interleukin 10-Fc fusion protein, its encoding gene, and application. Background Art

[0002] Interleukin-10 (IL-10) is a cytokine discovered in 1991 that can regulate the body's inflammation and immune response. Initially reported, this cytokine can inhibit cytokine secretion, antigen presentation, and CD4+ cell activation. IL-10 can inhibit the immune response by inhibiting the expression of IL-1α, IL-1β, IL-6, IL-8, TNF-α, GM-CSF, and G-CSF in activated monocytes and activated macrophages, and it also inhibits IFN-γ production by NK cells. Although IL-10 is mainly expressed in macrophages, its expression has also been detected in activated T cells, B cells, mast cells, and monocytes. In addition to suppressing the immune response, IL-10 also exhibits immunostimulatory properties, including stimulating the proliferation of thymocytes treated with IL-2 and IL-4, enhancing the activity of B cells, and stimulating the expression of MHC II class.

[0003] However, recent clinical studies have shown that PEG-modified IL-10 can actually potently activate the human immune system, particularly CD8+ T cells, which are known to kill cancer cells. IL-10 can also co-stimulate B cell activation, prolong B cell survival, and facilitate class switching in B cells. In addition, it can co-stimulate natural killer (NK) cell proliferation and cytokine production and act as a growth factor to stimulate the proliferation of certain CD8+ T cell subsets (Mosser, DM & Yhang, X., Immunological Reviews 226, 205-218 (2008), high doses of IL-10 (20 and 25 μg / kg, respectively) can cause increased INFγ production in humans (Lauw, FN et al., 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, isoleucine, at position 87 in cellular IL-10 (Ding, Y. et al., J. Exp. Med. 191 (2), 213-223 (2000)).

[0004] Human IL-10 is a homodimeric protein, with each monomer comprising 178 amino acids, the first 18 of which comprise a signal peptide. Certain embodiments of the present disclosure include 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 monomer molecular weight of 18.7 kDa, and contains four cysteines forming disulfide bonds (12-108, 62-114). Its native active form is a 38 kDa homooligodimer linked by non-covalent bonds. This homodimer becomes biologically inactive upon disruption of the non-covalent interactions between the two monomeric subunits. Data from the published crystal structure of IL-10 indicate that the functional dimer exhibits some similarity to IFN-γ (Zdanov et al., 1995, Structure (Lond), 3:591-601). As a result of its pleiotropic activities, IL-10 has been linked to a wide variety of diseases, disorders, and conditions, including inflammatory conditions, immune-related disorders, fibrotic disorders, and cancer.

[0005] The half-life of recombinant human IL-10 in vivo is only 2-3 hours, and the protein is quickly cleared, 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 renal uptake, existing literature has disclosed that PEGylation can be used to extend its half-life in vivo (Mattos, A. et al., J. Control Release 162, 84-91(2012); Mumm, J. Be et al., Cancer Cell 20(6), 781-796(2011); Alvarez, H. Met al., Drug Metab. Dispos., 40(2), 360-373(2012); CN 201480024021.5, etc.). However, since there are multiple PEG modification sites, the products after PEGylation modification are heterogeneous, which brings trouble to the subsequent drug quality control.

[0006] Another approach involves fusing the IL-10 peptide to the Fc portion of an immunoglobulin. Immunoglobulins generally have a long circulatory half-life in vivo. For example, IgG molecules have a half-life of up to 23 days in humans. The Fc portion of the immunoglobulin is primarily responsible for this in vivo stability. While retaining the biological activity of the IL-10 molecule, the IL10-Fc fusion protein offers the advantage of retaining the biological activity of the IL-10 molecule while maintaining the stability provided by the Fc portion of the immunoglobulin.

[0007] Although this approach is feasible for IL-10 therapy, the potential risk of Fc fusion proteins becoming immunogenic when administered repeatedly over a long period of time is a potential concern for this type of drug. Furthermore, if the Fc portion retains unwanted biological effector functions, it could lead to additional therapeutic side effects, another potential concern for Fc fusion protein therapies. SUMMARY OF THE INVENTION

[0009] In one aspect, the present invention provides a fusion protein of human interleukin-10 (IL-10) and the Fc portion of human IgG (IL10-Fc fusion protein).

[0010] In one embodiment, the present invention provides an IL10-Fc fusion protein, wherein the C-terminus of IL-10 is directly or via a linker peptide linked to the N-terminus of a human IgG Fc protein; wherein the IL-10 sequence is consistent with that shown in Seq ID No: 1; and the general formula of the linker peptide sequence is (G4S) n 、(SG4) n or G4 (SG4) n , n is a number between 1 and 10; the human IgG Fc protein is selected from the native sequence Fc region or variant Fc region of human IgG2, IgG4.

[0011] In a preferred embodiment of the present invention, the human IgG2 Fc sequence is shown in SEQ ID NO: 10.

[0012] In another preferred embodiment of the present invention, the human IgG4 Fc sequence is shown in SEQ ID NO:11.

[0013] In one embodiment, the IL10-Fc fusion protein provided by the present invention has a preferred connecting peptide sequence of the formula [GlyGlyGlyGlySer] n , n is an integer from 1 to 4; more preferably, n is 3, and the sequence is [GlyGlyGlyGlySer]3. The in vivo function and stability of the fusion protein of the present invention are optimized by adding a small linker peptide to prevent potential unwanted domain interactions. Furthermore, the glycine-rich linker peptide provides structural flexibility, allowing the IL-10 moiety to effectively interact with the IL-10 receptor on target cells.

[0014] In a preferred embodiment, the present invention provides an IL10-Fc fusion protein in which the C-terminus of IL-10 is connected to the N-terminus of a human IgG Fc protein via a linker peptide; wherein the IL-10 sequence is consistent with that shown in Seq ID No: 1, the linker peptide sequence is [GlyGlyGlyGlySer]3, and the human IgG Fc protein is selected from the native sequence Fc region or variant Fc region of human IgG2 or IgG4.

[0015] In another preferred embodiment, the IL10-Fc fusion protein provided by the present invention is an IL10-human IgG2 Fc fusion protein, the sequence of which is shown in SEQ ID NO: 12.

[0016] In another preferred embodiment, the IL10-Fc fusion protein provided by the present invention is an IL10-human IgG4 Fc fusion protein, the sequence of which is shown in SEQ ID NO:13.

[0017] In another embodiment, the present invention provides an IL10-Fc fusion protein in which the C-terminus of IL-10 is directly or via a connecting peptide linked to the N-terminus of a human IgG4 Fc variant protein; wherein the IL-10 sequence is consistent with that shown in Seq ID No: 1; and the connecting peptide sequence is of the general formula (G4S) n 、(SG4) n or G4 (SG4) n , n is a number between 1 and 10; the human IgG4 Fc variant protein comprises the sequence of SEQ ID NO: 2, wherein:

[0018] 16-bit X1 is Pro or Glu;

[0019] X2 at position 17 is Phe, Val, or Ala;

[0020] X3 at position 18 is Leu, Glu, or Ala;

[0021] X4 at position 80 is Asn or Ala; and

[0022] X5 at position 230 is Lys or does not exist.

[0023] In another preferred embodiment, the preferred connecting peptide sequence in the IL10-IgG4 Fc fusion protein provided by the present invention is [GlyGlyGlyGlySer] n , n is an integer from 1 to 4.

[0024] In another preferred embodiment, the preferred linker peptide sequence in the IL10-IgG4 Fc fusion protein provided herein has the general formula [GlyGlyGlyGlySer] . The inclusion of a small linker peptide prevents potential unwanted domain interactions, thereby optimizing the in vivo function and stability of the fusion protein of the present invention. Furthermore, the glycine-rich linker peptide provides structural flexibility, enabling the IL-10 portion to effectively interact with the IL-10 receptor on target cells.

[0025] In another preferred embodiment, the wild-type IgG4 Fc sequence is further modified. The IgG4 Fc portion of the fusion protein of the present invention may contain one or more of the following substitutions: substitution of glutamine (Gln) with proline (Pro) or glutamic acid (Glu) at position 16 in SEQ ID NO: 2, substitution of phenylalanine (Phe) with alanine (Ala) or valine (Val) at position 17 in SEQ ID NO: 2, and substitution of leucine (Leu) with alanine (Ala) or glutamic acid (Glu) at position 18 in SEQ ID NO: 2.

[0026] In another preferred embodiment, the preferred IL10-IgG4 Fc fusion protein of the present invention comprises the following proteins:

[0027] 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 absent, and its sequence is shown in SEQ ID NO: 3.

[0028] 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 Ala, and X5 at position 230 does not exist, and its sequence is shown in SEQ ID NO: 4.

[0029] 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 Lys, and its sequence is shown in SEQ ID NO: 5.

[0030] IL10-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 Ala, and X5 at position 230 does not exist, and its sequence is shown in SEQ ID NO: 6.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] IL10-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 Ala, and X5 at position 230 is Lys.

[0036] 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.

[0037] The present invention further provides a polynucleotide encoding an IL10-Fc fusion protein of the present invention. A vector, particularly an expression vector, comprising the polynucleotide of the present invention is further provided. In another aspect, the present invention provides a host cell comprising the polynucleotide or vector of the present invention. The present invention also provides a method for producing the IL10-Fc fusion protein of the present invention, comprising the steps of: (i) culturing the host cell of the present invention under conditions suitable for expression of the IL10-Fc fusion protein, and (ii) recovering the fusion protein.

[0038] In another aspect, the present invention provides a pharmaceutical composition comprising an effective amount of an IL10-Fc fusion protein of the present invention and a pharmaceutically acceptable carrier. Also provided are the IL10-Fc fusion protein or pharmaceutical composition of the present invention for use as a medicament and for treating or preventing a disease in a subject in need thereof, including viral diseases, inflammatory diseases, immune-related disorders, fibrotic disorders, and proliferative conditions.

[0039] In a preferred embodiment, the IL10-Fc fusion protein provided by the present invention is used to treat or prevent proliferative conditions or disorders, including cancers, such as cancers of the uterus, cervix, breast, prostate, testicles, gastrointestinal tract, kidney, bladder, bone, bone marrow, skin, head or neck, skin, liver, gallbladder, heart, lung, pancreas, salivary glands, adrenal glands, thyroid gland, brain, ganglia, central nervous system (CNS) and peripheral nervous system (PNS), as well as cancers of the hematopoietic system and immune system. In specific embodiments, the tumor or cancer is colon cancer, ovarian cancer, breast cancer, melanoma, lung cancer, pancreatic cancer, glioblastoma or leukemia, etc.

[0040] In one embodiment, the IL10-Fc fusion protein pharmaceutical is used in a method for treating a disease. Depending on the type and severity of the disease, a serum trough concentration of IL10-Fc fusion protein greater than about 0.1 ng / mL (e.g., 0.1-2 ng / mL, 0.1-1 ng / mL, 0.5-1.5 ng / mL, or 1.1-2.1 ng / mL) can be a starting candidate dose for administration to a patient, whether by one or more separate administrations or by continuous infusion. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Attachment Figure 1 Schematic diagram of the human interleukin-10-Fc fusion protein structure. The C-terminus of IL-10 is linked directly or via a linker peptide to the N-terminus of the human IgG Fc protein, forming a dimer through disulfide bonds in the human IgG Fc region.

[0043] Figure 2 The cell growth curves of different clones cultured in 7L tanks from day 0 to day 13. The vertical axis is 10 6 / ml.

[0044] Figure 3 The expression levels of different clones on days 10, 12, and 13 of culture in 7 L jars. The unit of the vertical axis is mg / ml.

[0045] Figure 4 This is an electrophoresis diagram of IL10-Fc fusion protein after affinity chromatography purification. 1: cell line 1 culture supernatant; 2: cell line 1 flowthrough; 3: cell line 1 purification; 4: cell line 2 culture supernatant; 5: cell line 2 flowthrough; 6: cell line 2 purification; 7: cell line 3 culture supernatant; 8: cell line 3 flowthrough; 9: cell line 3 purification; 10: commercial standard marker. The molecular weight of IL10-Fc fusion protein is approximately 90 kD.

[0046] Attachment Figure 5 IL10-Fc stimulates CD8+ cells to produce cytotoxic factors. CD8+ cells isolated from mouse spleens, cultured and activated in vitro, and then treated with varying concentrations of IL10-Fc (using IL-10 as a control) stimulate the cells to produce cytotoxic effects (increased granzyme / perforin expression) and IFNγ expression.

[0047] Attachment Figure 6 Tumor volume changes in different groups after six dosings. Different antibodies were administered intraperitoneally according to the group, with an initial dose of 250 μg / mouse followed by 200 μg / mouse / time (10 mg / kg) every three days for six doses. IL10-Fc was administered subcutaneously around the tumor, with 5 μg (100 μl solution) per tumor administered every three days for six doses. Tumor proliferation changes and the status of the mice were observed after treatment, and tumor proliferation was observed and measured for 1-2 weeks after the end of dosing. Detailed Description of the Invention

[0049] To make the present invention easier to understand, some terms are first defined. Other definitions will be explained throughout the detailed description.

[0050] Unless otherwise indicated, the practice of the present invention will employ conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are fully explained in the literature, such as

[0051] Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, 1984); Animal Cell Culture (RI Freshney, 1987); Methods in Enzymology (Academic Press, Inc.); Current Protocols in Molecular Biology (FM Ausubel et al., 1987); PCR: The Polymerase Chain Reaction (Mullis et al., 1994); A Practical Guide to Molecular Cloning

[0052] (A Practical Guide to Molecular Cloning) (Perbal Bernard V., 1988); Phage Display: A Laboratory Manual (Barbas et al., 2001), etc.

[0053] 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 linker peptide. For clarity, the individual peptide chains of the human IgG Fc portion of the fusion protein can be non-covalently linked, for example, by disulfide bonds.

[0054] "Fused" means that the components are linked by peptide bonds, either directly or via one or more linker peptides.

[0055] Native IL-10 is a homodimer composed of two α-helical monomer domains. The sequence of the native human IL-10 monomer domain is shown in SEQ ID NO: 1.

[0056] The term "Fc domain" or "Fc region" is used herein to define the C-terminal region of an antibody heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. The IgG Fc region comprises the IgG CH2 and IgG CH3 domains. The "CH2 domain" of a human IgG Fc region typically extends from the amino acid residue at about position 231 to the amino acid residue at about position 340. In one embodiment, a carbohydrate chain is attached to the CH2 domain. The CH2 domain herein may be a native sequence CH2 domain or a variant CH2 domain. The "CH3 domain" comprises a stretch of residues at the C-terminus of the CH2 domain in the Fc region (i.e., from the amino acid residue at about position 341 to the amino acid residue at about position 447 in 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 called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0057] The term "variant" encompasses naturally occurring variants and non-naturally occurring variants and refers broadly 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 completely synthetic genes.

[0058] The terms "DNA," "nucleic acid," "nucleic acid molecule," "polynucleotide," and the like are used interchangeably herein to refer to a polymeric form of nucleotides of any length, either 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.

[0059] The term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that integrate into the genome of a host cell into which they are introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0060] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells.

[0061] The term "pharmaceutical composition" refers to a preparation that is in such form as to permit the biological activity of the active ingredient contained therein to be effective, and that contains no additional ingredients that would be unacceptably toxic to a subject to which the formulation would be administered.

[0062] The term "pharmaceutically acceptable carrier" refers to a component other than the active ingredient in a pharmaceutical composition that is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, preservatives, and the like.

[0063] A "therapeutically effective amount" of an agent, such as a pharmaceutical composition, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

[0064] The terms "patient" or "subject" are used interchangeably to refer to a human or a non-human animal (eg, a mammal).

[0065] The terms "treat," "treat," "treat," and the like refer to clinical intervention (e.g., administration of an IL10-Fc fusion protein or a pharmaceutical composition comprising an IL10-Fc fusion protein) performed after the disease, disorder, or condition or its symptoms have been diagnosed, observed, etc., in order to temporarily or permanently eliminate, alleviate, suppress, relieve, or improve the disease, disorder, or condition afflicting a subject.

[0066] IL10-Fc fusion protein of the present invention

[0067] In one embodiment, the present invention provides an IL10-Fc fusion protein, wherein the C-terminus of IL-10 is directly or via a linker peptide linked to the N-terminus of a human IgG Fc protein; wherein the IL-10 sequence is consistent with that shown in Seq ID No: 1; and the general formula of the linker peptide sequence is (G4S) n 、(SG4) n or G4 (SG4) n , n is a number between 1 and 10; the human IgG Fc protein is selected from the native sequence Fc region or variant Fc region of human IgG2, IgG4.

[0068] 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 approximately 23 days in humans. Human IgG has four subclasses: IgG1, IgG2, IgG3, and IgG4, each with distinct biological functions known as effector functions. These effector functions are typically mediated by interactions with Fc receptors (FcγRs) or by binding to Clq and complement fixation. Binding to FcγRs can lead to antibody-dependent cell-mediated cytolysis, while binding to complement factors can lead to complement-mediated cytolysis.

[0069] When designing Fc-fusion proteins that utilize only the Fc portion to extend half-life, minimizing effector functions is crucial. For some purely antagonistic antibodies, such as those targeting soluble cytokines like TNFα and IL17A, or immune checkpoints like PD-1, the effector functions of FcγRs are unnecessary and can prevent cytotoxicity caused by ADCC. Therefore, IgG2 and IgG4, with their weak Fc effects, are chosen as backbones. Currently, four IgG2 and six IgG4 mAbs have been approved for marketing, including the anti-PD1 mAbs nivolumab and pembrolizumab, the anti-IL17A mAb ixekizumab, and the anti-PCSK9 mAb evolocumab, all of which utilize the IgG2 or IgG4 subtype.

[0070] One objective of the present invention is to extend the half-life of recombinant human IL-10 in vivo, improve circulation time, exposure, efficacy, and reduce renal uptake, while reducing unnecessary immune effects such as ADCC and CDC. Therefore, the Fc portion of the Fc fusion protein structure of the present invention is preferably derived from a human IgG2 Fc sequence or an IgG4 Fc sequence, as its ability to bind to FcγR and complement factors is reduced compared to other IgG subtypes.

[0071] In a preferred embodiment, the present invention provides an IL10-Fc fusion protein comprising an IL-10 C-terminal linker peptide linked to the N-terminus of an Fc protein; wherein the IL-10 sequence is consistent with that shown in Seq ID No: 1, the linker peptide sequence is [GlyGlyGlyGlySer]3, and the human IgG Fc protein is selected from a native sequence Fc region or a variant Fc region of human IgG2 or IgG4. In another preferred embodiment, the IL10-Fc fusion protein provided herein is an IL10-human IgG2 Fc fusion protein, the sequence of which is shown in SEQ ID NO: 12. In another preferred embodiment, the IL10-Fc fusion protein provided herein is an IL10-human IgG4 Fc fusion protein, the sequence of which is shown in SEQ ID NO: 13.

[0072] In another embodiment, the present invention provides an IL10-Fc fusion protein in which the C-terminus of IL-10 is directly or via a connecting peptide linked to the N-terminus of a human IgG4 Fc variant protein; wherein the IL-10 sequence is consistent with that shown in Seq ID No: 1; and the connecting peptide sequence is of the general formula (G4S) n 、(SG4) n or G4 (SG4) n , n is a number between 1 and 10; the human IgG4 Fc region or variant protein comprises the sequence of SEQ ID NO: 2, wherein:

[0073] 16-bit X1 is Pro or Glu;

[0074] X2 at position 17 is Phe, Val, or Ala;

[0075] X3 at position 18 is Leu, Glu, or Ala;

[0076] X4 at position 80 is Asn or Ala; and

[0077] X5 at position 230 is Lys or does not exist.

[0078] In another preferred embodiment, the preferred connecting peptide sequence in the IL10-Fc fusion protein provided by the present invention has the general formula [GlyGlyGlyGlySer]n, where n is an integer of 1-5.

[0079] In another preferred embodiment, the preferred linker peptide sequence in the IL10-Fc fusion protein provided herein has the general formula [GlyGlyGlyGlySer] . The in vivo function and stability of the fusion protein of the present invention are optimized by incorporating a small linker peptide to prevent potential unwanted domain interactions. Furthermore, the glycine-rich linker peptide provides structural flexibility, enabling the IL-10 portion to effectively interact with the IL-10 receptor on target cells.

[0080] In another preferred embodiment, the present invention further modifies the wild-type IgG4 Fc sequence. By modifying the IgG4 Fc protein sequence, the present invention produces a novel human interleukin 10-IgG4 Fc fusion protein (IL10-IgG4 Fc fusion protein). By replacing amino acids at multiple positions in the Fc portion, the modified IL10-Fc fusion protein exhibits superior properties compared to existing Fc fusion proteins, such as increased in vivo stability, elimination of unnecessary effector functions, and reduced in vivo immunogenicity.

[0081] The IL10-IgG4 Fc fusion protein of the present invention comprises an IL-10 C-terminus linked to the N-terminus of the Fc protein directly or via a linker peptide; wherein the IL-10 sequence is consistent with that shown in Seq ID No: 1; the linker peptide sequence has the general formula [GlyGlyGlyGlySer]n, where n is an integer from 1 to 5; and the Fc protein portion comprises the sequence of SEQ ID NO: 2, wherein:

[0082] 16-bit X1 is Pro or Glu;

[0083] X2 at position 17 is Phe, Val, or Ala;

[0084] X3 at position 18 is Leu, Glu, or Ala;

[0085] X4 at position 80 is Asn or Ala; and

[0086] X5 at position 230 is Lys or does not exist.

[0087] The IL10-IgG4 Fc fusion protein of the present invention preferably has a connecting peptide sequence of the general formula [GlyGlyGlyGlySer]n, where n is an integer from 1 to 3; more preferably, n is 3, and the sequence is Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser. The in vivo function and stability of the fusion protein of the present invention are optimized by incorporating a small connecting peptide to prevent potential unwanted domain interactions. Furthermore, the glycine-rich connecting peptide provides structural flexibility, enabling the IL-10 portion to effectively interact with the IL-10 receptor on target cells.

[0088] The Fc protein portion of the present invention is derived from human IgG4, but includes an Fc portion with one or more amino acid substitutions compared to the wild-type human sequence. The Fc portion is composed of two heavy chain constant regions of an antibody bound by non-covalent interactions and disulfide bonds. The Fc portion may include a hinge region and extend to the C-terminus of the antibody via the CH2 and CH3 domains. The Fc portion may also include one or more glycosylation sites.

[0089] To further reduce its effector function, the present invention further modifies the wild-type IgG4 Fc region. The IgG4 Fc portion of the fusion protein of the present invention may contain one or more of the following substitutions: substitution of glutamine (Gln) with proline (Pro) or glutamic acid (Glu) at position 16 in SEQ ID NO: 2, substitution of phenylalanine (Phe) with alanine (Ala) or valine (Val) at position 17 in SEQ ID NO: 2, and substitution of leucine (Leu) with alanine (Ala) or glutamic acid (Glu) at position 18 in SEQ ID NO: 2.

[0090] The N297 position (EU numbering system) of the Fc portion of the human IgG molecule can be glycosylated, and this glycosylation has a significant impact on the activity of IgG. If the glycosylation at this site is removed, it will affect the conformation of the upper half of CH2, thereby losing the ability to bind to FcγRs and affecting the biological activity associated with the antibody. However, for the fusion protein constructed in the present invention, since the effector effects brought by FcγRs are not required and the cytotoxicity caused by ADCC of the fusion protein needs to be prevented, the Fc portion needs to be modified without glycosylation. Based on this consideration, the inventors found that replacing Asn with Ala at position 80 corresponding to SEQ ID NO: 2 can remove the N-linked glycosylation site in the IgG4 Fc region. This modification without glycosylation can reduce the biological effects of the fusion protein, such as ADCC.

[0091] Furthermore, the IgG4-derived Fc portion of the IL10-Fc fusion protein discussed herein can be deleted from the C-terminal lysine residue present in the native molecule (Seq ID NO: 2, position 230; the deleted lysine is referred to as des-K). Expression of C-terminal lysine-containing Fc fusion proteins in certain cells, such as NS0 cells, is heterogeneous: some fusion proteins have a lysine at the C-terminus, while others lack the lysine at the C-terminus. This deletion is due to the action of proteases during expression in certain mammalian cell types. Therefore, to avoid this heterogeneity, it is preferred to construct Fc fusion proteins with a C-terminal lysine deletion.

[0092] For ease of understanding, a table of common amino acid single-letter and three-letter codes is provided below.

[0093]

[0094] Preferred IL10-Fc fusion proteins of the present invention include the following proteins:

[0095] 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 absent, and its sequence is shown in SEQ ID NO: 3.

[0096] 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 Ala, and X5 at position 230 does not exist, and its sequence is shown in SEQ ID NO: 4.

[0097] 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 Lys, and its sequence is shown in SEQ ID NO: 5.

[0098] IL10-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 Ala, and X5 at position 230 does not exist, and its sequence is shown in SEQ ID NO: 6.

[0099] 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.

[0100] 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.

[0101] 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.

[0102] 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.

[0103] IL10-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 Ala, and X5 at position 230 is Lys.

[0104] 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.

[0105] The structure of IL10-Fc fusion protein is as follows Figure 1 shown.

[0106] Wild-type human IgG2 or IgG4 proteins can be obtained from a variety of sources. For example, cDNA libraries can be prepared from cells expressing the target mRNA at detectable levels to obtain these proteins. The libraries can be screened using probes designed using the published DNA or protein sequences of the specific target protein. Immunoglobulin light or heavy chain constant regions are described, for example, in Adams et al., (1980) Biochemistry 19:2711-2719; Goughet et al., (1980) Biochemistry 19:2702-2710; Dolby et al., (1980) Proc. Natl. Acad. Sci. USA 77:6027-6031; Rice et al., (1982) Proc. Natl. Acad. Sci. USA 79:7862-7862; Falkner et al., (1982) Nature 298:286-288; and Morrison et al., (1984) Ann. Rev. Immunol. 2:239-256.

[0107] polynucleotides

[0108] The present invention also provides polynucleotides encoding an IL10-Fc fusion protein as described herein.

[0109] Polynucleotides of the present invention also include those having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% homology to the polynucleotide sequence encoding an IL10-Fc fusion protein as described herein.

[0110] DNA encoding the IL-10 and IgG Fc (IgG2Fc or IgG4Fc) of the present invention can be generated by a variety of different methods, including standard molecular cloning methods and chemically synthesized DNA. The gene encoding the fusion protein can then be constructed by ligating the DNA encoding the IL-10 in frame with the DNA encoding the IgG Fc protein 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 the IL-10 gene and the gene encoding the IgG Fc analog protein can also be linked in frame via DNA encoding a G-rich connecting peptide.

[0111] The present invention provides a gene encoding an IL10-Fc fusion protein, for example, the gene sequence encoding the IL10-[GlyGlyGlyGlySer]3-IgG4 Fc fusion protein shown in SEQ ID NO: 3 is shown in SEQ ID NO: 7.

[0112] Recombination methods

[0113] Fusion proteins 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 present invention is provided. Methods known to those skilled in the art can be used to construct an expression vector containing the coding sequence for the 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.

[0114] The present invention also provides a method for producing the IL10-Fc fusion protein of the present invention, comprising the following steps:

[0115] (i) culturing the host cell of the present invention under conditions suitable for expressing the IL10-Fc fusion protein;

[0116] (ii) recovering the fusion protein.

[0117] 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 include various commercially available prokaryotic or eukaryotic expression vectors known to those skilled in the art, such as the pET series vectors and the pQE series vectors; yeast expression vectors pPICZ-α-A, pHIL-D2, pPIC9, and pHIL-S1 (Invitrogen Corp., San Diego, California, USA); and animal cell expression vectors such as the pIRES plasmid, pSVK3, and pMSG (Amersham Pharmacia Biotech Inc., USA).

[0118] Suitable host cells include, but are not limited to, bacteria, yeast, insect, and mammalian cells. Recombinant cells containing exogenous nucleic acid encoding the IL10-Fc fusion protein can be prepared by any suitable technique, for example, by transfection / transformation using naked DNA plasmid vectors, viral vectors, invasive bacterial cell vectors, or other whole-cell vectors, including transfection by calcium phosphate precipitation, receptor-mediated targeting 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 and are described in Sambrook et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (2nd Edition, 1989 or 3rd Edition, 2001).

[0119] The expression of the nucleic acid molecules of the present invention can be regulated by another nucleotide sequence, thereby allowing the molecule to be expressed in a host transformed with the recombinant DNA molecule. For example, expression can be controlled by any promoter / enhancer element known in the art. Promoters that can be used to control the expression of chimeric polypeptide molecules include, but are not limited to, long terminal repeat sequences (Squinto et al., 1991, Cell, 65: 1-20); the SV40 early promoter region, CMV, M-MuLV, thymidine kinase promoter, regulatory sequences of metallothionine genes; prokaryotic expression vectors such as the β-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 transcription control regions from genes such as the elastase I gene.

[0120] Cell lines used as hosts for recombinant proteins are well known in the art and include various 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 (COS) cells, 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, for details, see WO87 / 04462, WO89 / 01036, and EP338841. When a nucleic acid encoding a fusion protein such as IL10-Fc (or a vector containing the nucleic acid) is introduced into a mammalian host cell, the IL10-Fc fusion protein can be expressed in the host cell by culturing the host cell or secreted into the culture medium in which the host cell is grown.

[0121] The IL10-Fc fusion protein can be recovered from the culture medium using 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, etc. For affinity chromatography purification of the fusion proteins of the present invention, a matrix containing Protein A or Protein G can be used.

[0122] Pharmaceutical composition

[0123] In another aspect, the present invention provides pharmaceutical compositions comprising any of the IL10-Fc fusion proteins provided herein, the pharmaceutical compositions comprising a therapeutically effective amount of the 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 at the dosages and concentrations employed, i.e., do not produce adverse, allergic, or other untoward reactions when administered to animals (e.g., humans), as appropriate. Pharmaceutically acceptable carriers include any and all solvents, buffers, dispersion media, coating materials, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, antioxidants, proteins, drugs, drug stabilizers, polymers, gels, binders, excipients, disintegrants, lubricants, sweeteners, fragrances, dyes, and similar materials and combinations thereof.

[0124] Pharmaceutical composition of the present invention can be administered in a manner such as intravenous, intradermal, intraarterial, intraperitoneal, intracranial, intraarticular. Fusion protein of the present invention is particularly suitable for parenteral administration, particularly by injection, for example, subcutaneous, intradermal, intravenous, intraarterial, intramuscular, intrathecal or intraperitoneal injection. For injection, fusion protein of the present invention can be prepared in aqueous solution, preferably in a physiologically compatible buffer. Alternatively, fusion protein can be in powder form, for example, dissolved in sterile water with a suitable vehicle before use.

[0125] Treatment

[0126] Any of the IL10-Fc fusion proteins provided herein can be used in therapeutic methods.

[0127] For use in therapeutic methods, the fusion protein of the present invention will be formulated, administered, and used in a manner consistent with clinical medical practice. Factors considered in this context include the specific condition being treated, the specific mammal being treated, the clinical condition of the individual patient, the cause of the condition, the delivery site of the medicament, the method of administration, the time of administration, and other factors known to medical practitioners.

[0128] In certain embodiments, the IL10-Fc fusion protein or pharmaceutical composition of the present invention is provided for use as a medicament and for treating or preventing a disease in a subject in need thereof, including viral diseases, inflammatory diseases, immune-related disorders, fibrotic disorders, and proliferative conditions.

[0129] IL-10 is a cytokine with pleiotropic effects in immune regulation and inflammation. It is produced by mast cells and eliminates the inflammatory effects of these cells at the site of allergic reactions. Although it can inhibit the synthesis of proinflammatory cytokines such as IFN-γ, IL-2, IL-3, TNFα and GM-CSF, IL-10 is also stimulating to certain T cells and mast cells and stimulates B cell maturation, proliferation and antibody production. IL-10 can block NF-κB activity and participate in the regulation of the JAK-STAT signal transduction pathway. It also induces the cytotoxic activity of CD8+ T cells and antibody production of B cells, and it inhibits macrophage activity and tumor-promoting inflammation. The regulation of CD8+ T cells is dose-dependent, with higher doses inducing stronger cytotoxic reactions.

[0130] IL-10 plays multiple roles in the activation of CD8+T cells. For example, IL-10 induces effector molecules (IFNγ, perforin and granzyme B) in memory CD8+T cells. Such memory CD8+T cells are cells responsible for providing long-term antiviral protection of subjects. Although the generation and expansion of memory CD8+T cells can occur when IL-10 is not present (Vicari, A. and Trinchieri, G., (2004) Immuno. Rev. 202: 223-236), the fact that IL-10 directly activates such cells provides unique and alternative treatment methods.

[0131] IL10-Fc fusion protein has biological activity similar to IL-10. In view of the above, embodiments of the present disclosure are based on the connection between CD8+ T cells and cancer and viral infection. Therefore, certain methods for treating and / or preventing cancer-related diseases, disorders and conditions, such as maintaining, for example, an average IL10-Fc fusion protein serum concentration greater than 0.5 ng / mL, greater than 1 ng / mL or greater than 0.1 ng / mL should also be applicable to the treatment of such diseases. The present disclosure encompasses the use of the IL10-Fc fusion protein described herein in treating or preventing a wide range of diseases, disorders or conditions and / or their symptoms. According to the present disclosure, the IL10-Fc fusion protein is 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, 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), as well as cancers of the hematopoietic system and immune system. In certain embodiments, the tumor or cancer is colon cancer, ovarian cancer, breast cancer, melanoma, lung cancer, pancreatic cancer, glioblastoma, or leukemia, among others.

[0132] In one embodiment, a method for treating a disease using an IL10-Fc fusion protein pharmaceutical is disclosed, comprising administering a therapeutically effective amount of the pharmaceutical to a subject suffering from the disease, wherein the disease includes inflammatory conditions, immune-related disorders, fibrotic disorders, and cancer, among others. The subject is a mammal, preferably a human. Depending on the type and severity of the disease, a serum trough concentration of an IL10-Fc fusion protein of greater than about 0.1 ng / mL (e.g., 0.1-2 ng / mL, 0.1-1 ng / mL, 0.5-1.5 ng / mL, or 1.1-2.1 ng / mL) can be a starting candidate dose for administration to a patient, whether by one or more separate administrations or by continuous infusion.

[0133] Preferably, when the subject is a human, the IL10-Fc fusion protein can be administered at a dose of greater than 2.0 μg / kg / day, greater than 2.5 μg / kg / day, greater than 3.0 μg / kg / day, greater than 5 μg / kg / day, greater than 8 μg / kg / day, greater than 10 μg / kg / day, greater than 12 μg / kg / day, 15 μg / kg / day, greater than 18 μg / kg / day, greater than 20 μg / kg / day, greater than 21 μg / kg / day, greater than 22 μg / kg / day, greater than 23 μg / kg / day, greater than 24 μg / kg / day, or greater than 25 μg / kg / day. The initial dose can be estimated from in vitro data, such as animal models, using techniques known in the art. One of ordinary skill in the art can readily optimize administration to humans based on animal data. Example

[0134] The following are examples of methods and compositions of the present invention. It is understood that various other embodiments may be practiced, given the general description provided above.

[0135] Example 1: Construction of IL10-IgG4 Fc fusion protein gene

[0136] SEQ ID NO:3 was translated into a DNA sequence and optimized based on the codon preference of CHO cells to obtain the expression sequence for the IL10-Fc fusion protein, SEQ ID NO:7. An NheI restriction site and a Kozac sequence (SEQ ID No. 8) were added to the 5' end of the optimized sequence, and a stop codon and an XhoI restriction site (SEQ ID No. 9) were added to the 3' end to obtain the complete expression cassette for the fusion protein. The complete expression cassette sequence was artificially synthesized and inserted between the NheI and XhoI restriction sites of the pIRES plasmid to generate the pIRES-IL10-Fc expression plasmid. This plasmid was linearized and electroporated into CHO-S cells, and positive clones were screened using MSX.

[0137] Example 2: Expression and purification of IL10-IgG4 Fc fusion protein

[0138] The positive clones obtained in Example 1 were subjected to two rounds of limiting dilution to screen out three clones with better expression levels. After expansion culture, they were inoculated into a 7 L fermenter for fed-batch culture ( Figure 2 ), express the target protein ( Figure 3 After the fermentation, the mixture was centrifuged at 4500 rpm for 6 min, the supernatant was collected, the pH was adjusted to 4.0, and the supernatant was stored at 4°C.

[0139] The supernatant was first concentrated by ultrafiltration using a 10KDa ultrafiltration membrane; then Mabselect Sure was used for preliminary affinity chromatography to collect the fusion protein. The affinity chromatography mobile phase was: A1: 25mM PB+50mM Nacl, pH7.0, B1: 20mM Gly, pH3.0, B2: 20mM citric acid buffer, pH3.0. The chromatography column was first balanced with mobile phase A1. After loading the sample, impurities were eluted with mobile phase B1, and then the fusion protein was eluted with mobile phase B2. The collected elution was also adjusted to a neutral pH with 1M Tris-His pH8.0. The crude pure sample collected in this step was purified by Capto adhere chromatography column. After the sample was loaded and bound at pH7.0, it was eluted at pH4.0 to obtain a purer sample (more than 95%). The electrophoresis diagram after affinity chromatography purification is as follows. Figure 4 As shown, the molecular weight of the IL10-IgG4 Fc fusion protein was determined to be approximately 90 kD.

[0140] Example 3: In vivo half-life determination

[0141] rhIL-10 (Rochy Hill) and the IL10-Fc fusion protein obtained in Example 2 were injected into the tail vein of SD rats weighing approximately 200 g at a dose of 200 ng / kg body weight. After injection, blood samples were collected by tail-snipping at different time points (0, 1, 2, 4, 6, 8, 12, 24, 36, 48, 60, 72, and 96 hours). The blood samples were anticoagulated with sodium heparin and centrifuged at 12,000 g for 5 minutes to collect serum.

[0142] Blood samples were assayed for serum fusion protein levels using a human IL-10 ELISA kit (purchased from Bender Medsystem) according to the manufacturer's instructions, and the results were averaged. The results showed that the in vivo elimination half-life of the IL10-Fc fusion protein prepared in the present invention was greater than 22.6 hours, compared to the 2-4 hours elimination half-life of human rhIL-10 following tail vein injection. This indicates that the in vivo half-life of the IL10-Fc fusion protein prepared in the present invention is extended by more than 5-6 times compared to the rhIL-10 control.

[0143] Example 4: Antitumor activity experiment

[0144] Studies have demonstrated that IL-10 not only exerts anti-tumor effects by activating NK cells but also inhibits tumor progression by activating T cells. Studies have shown that IL-10 treatment of tumor-bearing mice induces the expression of IFN-γ and granzymes. This effect may be mediated by an IL-10 signaling pathway specific to CD8+ T cells within the tumor: IL-10 activates phosphorylated STAT1 and STAT3 in CD8+ T cells, thereby inducing CD8+ T cell proliferation and the expression of IFN-γ, the cytotoxic protein perforin, and granzymes. IFN-γ can induce the expression of MHC class I antigen-presenting molecules on tumor cells and monocytes and macrophages, assisting CD8+ T cells in killing a large number of antigen-specific tumor cells. Activation of the TCR in CD8+ T cells effectively induces anti-apoptotic and cell proliferation signals. In summary, IL-10 can not only enhance the tumor killing effect by increasing the cytotoxic activity of NK cells, but also enhance the tumor killing ability of intratumoral CD8+ T cells and IFN-γ-induced antigen presentation ability by mediating the infiltration and activation of specific cytotoxic CD8+ T cells in the tumor, the expression of IFN-γ and granule proteases, and enhancing tumor antigen presentation, thereby enhancing the function of anti-tumor immune escape.

[0145] In order to detect the cytotoxic effect of IL10-Fc on CD8+ T cells, CD8+ cells were isolated from mouse spleens, cultured and activated in vitro, and then added with different concentrations of IL10-Fc (with IL-10 as a control). The cells were stimulated to produce cytotoxic effects (enhanced expression of granzymes / perforin) and stimulated to express IFNγ (e.g. Figure 5 Although IL10-Fc has only about 30%-40% of the in vitro activity of IL10, its in vivo biological activity has not been significantly reduced, considering that the half-life of IL10-Fc in vivo is significantly prolonged.

[0146] Example 5: In vivo efficacy test of IL10-Fc

[0147] First, an animal model of SPC immune repertoire tolerance was constructed. The SPC-PDL1 lung cancer cell line and PBMC extracted from human peripheral blood were mixed and inoculated into the right armpit of mice, with 3x10 6 After about 7-9 days, when most tumors grow to 90-250 mm 3 When (average value is 120-140mm 3 ). Then exclude the tumor smaller than 90mm 3 or greater than 250mm 3For mice with abnormalities or other abnormal conditions, the remaining mice were randomly allocated and equally divided into a human IgG4 antibody (HuIgG4) blank group, a PD-1 antibody positive control group, and an IL10-Fc group (10 mice in each group), and then received 6 antibody treatments or IL10-Fc according to the requirements of different groups.

[0148] The dosing regimen for mice was as follows: different antibodies were administered according to the groups, intraperitoneally, with the first dose being 250ug / mouse, followed by 200ug / mouse / time (10mg / kg), q3dx6 times; IL10-Fc was administered subcutaneously around the tumor, with each tumor receiving 5ug (100ul solution) each time, q3dx6 times; the changes in tumor proliferation and the status of the mice after treatment were observed, and tumor proliferation was continued to be observed and measured for 1-2 weeks after the end of the treatment. According to the experimental design, each group was administered 6 times. During the administration period, the overall tumor volume and proliferation rate of the PD-1 antibody and IL10-Fc groups were similar, and significantly slower than the control HuIgG4 group, with statistically significant differences (p﹤0.05). Figure 6 Preliminary animal studies have shown that IL10-Fc's ability to inhibit tumor cell growth in mice is similar to that of PD-1 antibodies, demonstrating the drug's potential therapeutic value in tumor treatment.

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Claims

1. Use of an IL10-Fc fusion protein in the preparation of a medicament for treating or preventing a disease in an individual in need thereof, said disease being lung cancer, wherein the C-terminus of IL-10 is directly or via a linker peptide linked to the N-terminus of a human IgG Fc protein; wherein, The IL-10 sequence is identical to that shown in Seq ID No: 1; the general formula of the linker peptide sequence is (G4S) n , (SG4) n or G4(SG4) n , where n is a number between 1 and 10; the human IgG Fc protein is selected from the natural sequence Fc region or variant Fc region of human IgG4 as shown in SEQ ID NO: 11, and the human IgG4 variant Fc region contains 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 use according to claim 1, wherein: The general formula of the connecting peptide sequence is [GlyGlyGlyGlySer] n , where n is an integer from 1 to 4.

3. The use according to claim 1, characterized in that: The IL10-Fc fusion protein is such that the C-terminus of IL-10 is linked to the N-terminus of human IgG Fc protein through the linker peptide [GlyGlyGlyGlySer]3; wherein, the IL-10 sequence is the same as that shown in Seq ID No: 1, and the human IgG Fc protein is selected from the native sequence Fc region or variant Fc region of human IgG4.

4. The use according to claim 3, characterized in that: The IL10-Fc fusion protein is an IL10-human IgG4Fc fusion protein, and its sequence is as shown in SEQ ID NO:

13.

5. The use according to claim 1, characterized in that: The IL10-IgG4 Fc fusion protein includes 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 absent, and its sequence is as shown in SEQ ID NO: 3; 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 Ala, and X5 at position 230 is absent, and its sequence is as shown in SEQ ID NO: 4; 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 Lys, and its sequence is as shown in SEQ ID NO: 5; IL10-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 Ala, and X5 at position 230 is absent, and its sequence is as shown in SEQ ID NO: 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 the 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.

6. The use according to claim 5, wherein when the individual is a human, the IL10-Fc fusion protein is administered at a dose greater than 2.0 μg / kg / day.

7. The use according to claim 6, wherein when the individual is a human, the IL10-Fc fusion protein is administered at a dose greater than 2.5 μg / kg / day.

8. The use according to claim 7, wherein when the individual is a human, the IL10-Fc fusion protein is administered at a dose greater than 3.0 μg / kg / day.

9. The use according to claim 8, wherein when the individual is a human, the IL10-Fc fusion protein is administered at a dose greater than 5 μg / kg / day.

10. The use according to claim 9, wherein when the individual is a human, the IL10-Fc fusion protein is administered at a dose greater than 8 μg / kg / day.

11. The use according to claim 10, wherein when the individual is a human, the IL10-Fc fusion protein is administered at a dose greater than 10 μg / kg / day.

12. The use according to claim 11, wherein when the individual is a human, the IL10-Fc fusion protein is administered at a dose greater than 12 μg / kg / day.

13. The use according to claim 12, wherein when the individual is a human, the IL10-Fc fusion protein is administered at a dose greater than 15 μg / kg / day.

14. The use according to claim 13, wherein when the individual is a human, the IL10-Fc fusion protein is administered at a dose greater than 18 μg / kg / day.

15. The use according to claim 14, wherein when the individual is a human, the IL10-Fc fusion protein is administered at a dose greater than 20 μg / kg / day.

16. The use according to claim 15, wherein when the individual is a human, the IL10-Fc fusion protein is administered at a dose greater than 21 μg / kg / day.

17. The use according to claim 16, wherein when the individual is a human, the IL10-Fc fusion protein is administered at a dose greater than 22 μg / kg / day.

18. The use according to claim 17, wherein when the individual is a human, the IL10-Fc fusion protein is administered at a dose greater than 23 μg / kg / day.

19. The use according to claim 18, wherein when the individual is a human, the IL10-Fc fusion protein is administered at a dose greater than 24 μg / kg / day.

20. The use according to claim 19, wherein when the individual is a human, the IL10-Fc fusion protein is administered at a dose greater than 25 μg / kg / day.

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