TW202033766A - The variant of human interleukin-10 and the derivatives thereof - Google Patents

Abstract

The present disclosure relates to a variant of human interleukin-10 and derivatives thereof. In particular, the present disclosure relates to human interleukin 10 having one or more amino acid mutations and the PEGylated derivative thereof, which increases the stability of the protein and/or increases the cell proliferative activity of human interleukin-10. The above derivatives provide good foundation for drug-forming properties of protein drug.

Description

A variant of human interleukin 10 and its derivatives

This application claims the priority of the Chinese patent application "A variant of human interleukin 10 or its derivative" (application number CN201811418270.9) filed on November 26, 2018, the content of which is incorporated herein by reference.

The present disclosure relates to variants of human interleukin-10 with one or more amino acid mutations or derivatives thereof, which mutations allow human interleukin-10 to have enhanced stability and/or activity.

Human interleukin-10 (IL-10), whose gene is located on chromosome 1 (q31-32), is 5.1kb long. The gene sequence of IL-10 contains 5 exons. IL-10 is composed of 178 amino acid residues, forming a "V"-shaped homodimer with a molecular weight of about 35kD.

IL-10 belongs to the IL-10 family, and family members include IL-10, IL-19, IL-20, IL-22/IL-TIF, IL-24/MDA-7, IL-26, etc.

Fiorentino first discovered in 1989 that IL-10 is a cytokine secreted by mouse helper T cells Th2 subtype cells, which can inhibit tumors secreted by Th1 subtype cells. Cytokines such as necrosis factor γ (TNF-γ) were originally named cytokine synthesis inhibitory factor (CSIF). IL-10 enhances the release of anti-inflammatory factors, such as IL-1 receptor antagonists and soluble TNF-α receptors; IL-10 inhibits several cytokines produced by type 1 helper T cells (such as interferon-γ, IL-2 and TNF-α) synthesis, inhibit cell-mediated immune response modifiers, and inhibit antigen-presenting cell-dependent T cell responses, inhibit other cytokines produced by monocytes/macrophages, such as IL-1 , IL-6, IL-8, granulocyte-macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF) and TNF-α; IL-10 inhibits the synthesis of IL-12, thereby Block Th1 immune response.

IL-10 is usually expressed in T cells, macrophages, monocytes, dendritic cells, mast cells, B cells, eosinophils, keratinocytes, epithelial cells and various tumors, and it participates in a variety of cells (such as immune Cells, inflammatory cells, tumor cells).

Due to its pleiotropic activity, IL-10 has been studied in a variety of clinical applications, including autoimmune diseases, severe infectious diseases, tumors and transplantation immunity.

The existing IL-10 includes mono- or di-pegylated IL-10, which can be PEGylated (WO2002/026265; WO2010/077853). PEGylated IL-10 can be used as a drug to treat tumors or cancers, the tumors are epithelial cell carcinoma, endothelial cell carcinoma, squamous cell carcinoma, cancer caused by papilloma virus, adenocarcinoma, carcinoma, melanoma, sarcoma or Teratocarcinoma, or for the treatment of lung tumors or metastatic lung cancer, or lymphoma (WO2008/054585; EP2468293B; EP2821078B); and by administering effective amounts of PEGylated IL-10, folic acid analogs, and 5 -FU and platinum drugs treat tumors (US9833514B). IL-10 can also be used to treat or prevent cardiovascular disorders, thrombotic disorders or inflammatory disorders, or hyperlipidemia (WO2015/031316; US20160193300A). IL-10 can also inhibit immune checkpoints Agents (such as PD-1) or IL-15 to treat or prevent cancer or tumors (WO2015/070060; WO2017/035232). IL-10 can also treat liver diseases, such as non-alcoholic fatty liver and hepatitis C (WO2015/187295; WO2015/108785). IL-10 can be combined with IL-7 to treat diseases (WO2016/196211), and with CAR-T cells to regulate T cell-mediated immune responses (WO2016/191587).

Existing IL-10 mutation studies have shown that by introducing cysteine into S51, E81, K88, A120 and other positions, IL-10 with immunosuppressive activity but no immunostimulatory activity can be obtained (WO2004/044006). The mutation at position I87A reduces the binding affinity of IL-10 to its receptor (WO2015/117930). The mutation at the F129S position can retain the anti-inflammatory properties of IL-10, but does not retain platelet cell regulation and cell proliferation activity (WO2006/130580). Mutations in Q62R, K58E, E160Q, and D162H reduce the binding and functional activity of IL-10 to the receptor, while maintaining immunogenicity (WO2012/135177). Ding Y et al. indicated that the immunostimulatory activity of IL-10 was determined by the 87th single amino acid isoleucine (J. Exp. Med. 191(2), 213-223, 2000). WO2012146628A1 discloses a fusion protein of an antibody and IL-10, in which the amino acid at position 87 is replaced by I87A, and it is found that the modified fusion protein exhibits reduced affinity for IL-10 receptor, but improves targeting efficiency , Reduce the side effects caused by IL-10 immunostimulatory activity. However, there is still a lack of IL-10 variants with higher stability and higher activity in the prior art. Providing such IL-10 variants is an urgent problem in this field.

The present disclosure relates to variants of human interleukin 10 (IL-10) sequence with one or more amino acid mutations or derivatives thereof.

In the first aspect, the present disclosure provides IL-10 variants, which are asparagine (N) or/and methionamine at position 10 of the wild-type human IL-10 sequence (SEQ ID NO: 2) The wild-type amino acid residues are mutated into other amino acids at the 92nd position of asparagine (M) or/and asparagine (N), where this position is based on the mature protein of human wild-type IL-10 Numbering. This variant has improved stability and/or activity compared to wild-type human IL-10.

In some embodiments, the IL-10 variant or derivative thereof comprises a mutation at position 10 to Gln(Q) and/or a mutation at position 39 to Thr(T) and/or a mutation at position 92 to Gln(Q).

In some specific embodiments, the IL-10 variant comprises a mutation of Asn at position 10 to Gln (N10Q) and/or a mutation of Met at position 39 to Thr (M39T) and/or a mutation of Asn at position 92 to Gln (N92Q) ; Or a combination selected from: N10Q and M39T, M39T and N92Q, N10Q and M39T and N92Q.

In some specific embodiments, the IL-10 variant or derivative thereof may comprise a polypeptide selected from the following sequences: SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO.12, SEQ ID NO.14. The amino acid sequence is shown in the table below.

Table 1. Human interleukin-10 wild-type and variant sequences

In the present disclosure, derivatives of IL-10 variants include full-length, partial proteins related to the IL-10 variants of the present disclosure, or muteins, functional derivatives, functional fragments, or mutant proteins obtained by further mutations on the basis of the variants. Biologically active peptide, fusion protein, isoform or salt thereof.

In some specific embodiments, IL-10 variant derivatives include IL-10 variant fusion proteins, IL-10 variant truncations, and fusion proteins containing the truncation, IL-10 variants Body or dimer or trimer or polymer, modified IL-10 variants (such as pegylation (PEGylation), glycosylation, albumin conjugation or fusion, hydroxyethylation, and Fusion of antibodies or antigen-binding fragments thereof), and homologs of IL-10 variants in various species.

In some embodiments, the antibody or antigen-binding fragment thereof fused to the IL-10 variant is a murine antibody, human antibody, humanized antibody, chimeric antibody, or camel antibody or antigen-binding fragment thereof. In some specific embodiments, the antigen binding fragment is a Fab fragment, Fab' fragment, F(ab')2 fragment, single chain Fv (sFv), VHH or VH/VL domain.

In some embodiments, the IL-10 variant or derivative is a homodimer of IL-10, and each monomer contains 178 amino acids, of which the first 18 amino acids are signal peptides. Some specific embodiments include a variant of mature IL-10 (such as in US6217857) lacking a signal peptide, or a variant of mature PEGylated IL-10. This variant can display activity comparable to or superior to wild-type human IL-10. This variant can exhibit stability comparable to or higher than that of wild-type human IL-10. Therefore, the skilled person can understand that the IL-10 variant or derivative thereof according to the present disclosure may not contain a signal peptide, and (statistically) does not change the desired activity, as a non-limiting As an illustrative example, the signal peptide is removed based on a sequence selected from the following: SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.14 .

In some embodiments, the IL-10 variant or derivative thereof may be modified to adjust one or more properties (eg, pharmacokinetic parameters, efficacy, etc.), including that it may be a monomer or a dimer Or in the form of trimers, and/or PEGylated, and/or glycosylated, and/or albumin (for example, human serum albumin (HSA)) conjugated and fused, and/or hydroxyethyl Hesylation. The modification of IL-10 does not cause treatment-related adverse effects on immunogenicity, and in some embodiments, modified IL-10 may be less immunogenic than unmodified IL-10.

In some embodiments, the IL-10 variant or derivative is PEGylated (denoted as PEG-IL-10), such as a mono- or di-PEGylated IL-10 variant or derivative. PEG-IL-10 variants or derivatives include SC-PEG linkers. In other embodiments, the PEG-IL-10 variant or derivative includes a methoxy-PEG-aldehyde (mPEG-ALD) linker. In certain embodiments, the average molecular weight of the PEG moiety ranges from about 5KD to about 50KD, specifically 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 , 24, 25, 30, 35, 40, 45, 50KD; or about 5KD to about 40KD, or about 10KD to about 30KD, or about 10KD to about 20KD, or about 10KD to about 15KD. In certain specific embodiments, the mPEG-ALD linker includes a PEG molecule having an average molecular weight selected from the group consisting of 5KDa, 12KDa, or 20KDa. In some embodiments, the aldehyde group of mPEG-ALD may be acetaldehyde, propionaldehyde, butyraldehyde, or the like.

IL-10 variants with mutations at position 10 and/or 39 and/or 92 have improved stability and/or activity relative to wild-type IL-10. These mutations reduce or prevent the deamination and oxidation of IL-10, increase the stability of the protein, while the cell proliferation activity does not decrease or increase the cell size. Cell proliferation activity provides a good basis for protein medicine. In some specific solutions, the variant includes a combination of N10Q/M39T or M39T/N92Q or N10Q/M39T/N92Q.

In the second aspect, an isolated nucleic acid molecule is provided, which encodes an IL-10 variant or derivative thereof of the present disclosure. The nucleic acid molecule may include the polynucleotides shown in SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, and SEQ ID NO.

In some embodiments, an expression vector containing the aforementioned nucleic acid molecule is provided.

In some embodiments, a host cell expressing the aforementioned vector is provided. The host cell can be a prokaryotic or eukaryotic cell. In some specific embodiments, the host cell is a bacterium or yeast or mammalian cell (e.g., CHO, COS, BHK21, NIH3T3, HeLa, C2C12, HEK), for example, Pichia pastoris or Saccharomyces cerevisiae. Host cells do not include cells that can develop into plants, human fertilized eggs, human germ cells, or cells that can develop into animals.

In a third aspect, a pharmaceutical composition is provided, which comprises an IL-10 variant or derivative thereof of the present disclosure, which contains:

-IL-10 variants or derivatives thereof according to the present disclosure, and

-If necessary, pharmaceutically acceptable excipients, diluents or carriers.

In some embodiments, the unit dose of the pharmaceutical composition contains 1 mg to 1000 mg of the IL-10 variant or derivative thereof according to the present disclosure. In some embodiments, the concentration of the IL-10 variant or derivative thereof in the pharmaceutical composition is 1 mg/L to 1000 mg/L. In some embodiments, the pharmaceutical composition contains a buffer, the content of which may be 1 mM to 1000 mM. In some embodiments, the pharmaceutical composition may be a lyophilized formulation or an injectable solution.

In a fourth aspect, IL-10 variants or derivatives or pharmaceutical compositions thereof of the present disclosure are provided for preparing and treating proliferative diseases, immune diseases, cardiovascular diseases, thrombotic diseases, hyperlipidemia, inflammatory diseases, Liver disease, use in drugs for regulating T cell-mediated immune response.

In some embodiments, the proliferative disease may be tumor or cancer, such as epithelial cell carcinoma, endothelial cell carcinoma, squamous cell carcinoma, cancer caused by papilloma virus, adenocarcinoma, carcinoma, melanoma, sarcoma or teratoma, Lung tumors or metastatic lung cancer, lymphoma, colon cancer, ovarian cancer, breast cancer, malignant glioma and leukemia.

In some embodiments, the immune disease is diabetes, such as insulin-dependent diabetes.

In some embodiments, the cardiovascular disorder is atherosclerosis, hypertension, cardiomyopathy.

In some embodiments, the thrombotic disorder is a disorder that can cause stroke or myocardial infarction.

In some embodiments, the inflammatory disorder may be vasculitis.

In some embodiments, the liver disease may be non-alcoholic fatty liver or hepatitis C.

In some embodiments, the tumor or cancer is uterine cancer, cervical cancer, breast cancer, prostate cancer, testicular cancer, penile cancer, gastrointestinal cancer, such as esophageal cancer, oropharyngeal cancer, gastric cancer, small or large intestine cancer, colon cancer, or rectum Cancer, kidney cancer, renal cell carcinoma, bladder cancer, bone cancer, bone marrow cancer, skin cancer, brain or neck cancer, skin cancer, liver cancer, gallbladder cancer, heart cancer, lung cancer, pancreatic cancer, salivary gland cancer, adrenal cancer, thyroid cancer , Brain cancer (such as glioma, ganglion, central nervous system (CNS) and peripheral nervous system (PNS)) and immune system (such as spleen or thymus). The tumor or cancer can also be an immunogenic tumor, a non-immunogenic tumor, a dormant tumor, Virus-induced cancers such as epithelial cell carcinoma, endothelial cell carcinoma, squamous cell carcinoma, papilloma virus, adenocarcinoma, lymphoma, carcinoma, melanoma, leukemia, myeloma, sarcoma, teratoma, chemically-induced cancer, Methods of metastatic disease and angiogenesis.

In a fifth aspect, there is provided a method for inhibiting or reducing the growth of tumors or cancers and/or treating patients suffering from cancer or tumors, the method comprising administering an effective amount of the IL-10 variant or derivative of the present disclosure to the subject contact. The IL-10 variant or derivative can inhibit the growth of tumor or cancer, or the IL-10 variant or derivative can reduce the size of tumor or cancer. For example, by regulating the activity of regulatory T cells (Treg) and/or CD8+ T cells, it is expected that the IL-10 variants or derivatives of the present disclosure can also reduce the tolerance to tumor cells or cancer cell antigens.

In some embodiments, when compared with non-PEGylated IL-10 variants or derivatives, PEGylated IL-10 variants or derivatives can increase CD8+ T cell entry into targets (e.g. neoplasms, lesions) Permeability. In other embodiments, the PEGylated IL-10 variant or derivative can increase the expression of at least one inflammatory cytokine, which can be selected from IFN γ, IL-4, IL-6, IL-10 and RANK- Ligand (RANK-L). In certain embodiments, the PEGylated IL-10 variant or derivative is co-administered with at least one chemotherapeutic agent. The chemotherapeutic agent can be administered before, concurrently with, or after the administration of the PEGylated IL-10 variant or derivative.

In some embodiments, the patient or subject being treated is a human.

In some embodiments, at least twice a day, at least once a day, at least once every 48 hours, at least once every 72 hours, at least once a week, at least once every 2 weeks, at least once every month, every 2 A method of administering an IL-10 variant or derivative to a subject at least once a month or at least once every 3 months. The IL-10 variant or derivative can be administered by any effective route, for example, by parenteral injection (including subcutaneous injection). Specific embodiments relate to a variant or derivative of IL-10 comprising a pharmaceutically acceptable amount (eg, a therapeutically effective amount) together with One or more pharmaceutically acceptable diluents, carriers or excipients (e.g., isotonic injection solution) pharmaceutical composition. The medical composition is usually a medical composition suitable for human administration. Furthermore, in some embodiments, the pharmaceutical composition includes at least one additional prophylactic or therapeutic agent. Some embodiments contain: one of the aforementioned pharmaceutical compositions, optionally one or more additional components, and a sterile container.

In a sixth aspect, there is provided a method for preparing IL-10 variants or derivatives, including:

-Introduce a mutation selected from the group consisting of N10Q and M39T, M39T and N92Q, N10Q and M39T and N92Q into wild-type human IL-10; or

-To express the aforementioned nucleic acid molecule; or

-Express the aforementioned expression vector; or

-Make the aforementioned host cells express.

In the seventh aspect, a PEGylated IL-10 variant or its derivative structure and preparation method are provided.

In some embodiments, a mono-PEGylated IL-10 variant or derivative thereof is provided, which comprises:

A PEG molecule covalently attached to the IL-10 variant or its derivative via a linker. PEG can be attached to the lysine residue or the N-terminal amino acid residue of the IL-10 variant or derivative thereof. In some specific embodiments, the IL-10 variant or derivative thereof is a homodimer, and a 20KD or 12KD single PEG is attached to one of the monomers of the homodimer.

In other embodiments, there is provided a di-PEGylated IL-10 variant or derivative thereof, which comprises:

With a linker, two PEG molecules covalently attached to the IL-10 variant or its derivative. PEG can be attached to the lysine residue or the N-terminal amino acid residue of the IL-10 variant or derivative thereof. Some tools In other embodiments, the IL-10 variant or derivative thereof is a homodimer, and a 20KD or 12KD single PEG is connected to two monomers of the homodimer, respectively.

In some embodiments, the structure of the PEG-IL-10 variant or derivative is as follows:

[XO-(CH ₂ CH ₂ O) _n ] _b -L-NH-IL-10 variant or derivative,

Where X is H or C _1-4 alkyl, n is 20 to 2300, b is 1 to 9, L is an α-π alkyl linker (also used as a linker), and L is covalently attached to the IL-10 variant Or the amine terminal of its derivative, when b is greater than 1, the sum of n does not exceed 2,300. In some specific schemes, L is "-CH ₂ CH ₂ CH ₂ -".

In some specific embodiments, the PEG structure is CH ₃ O-(CH ₂ CH ₂ O)n-CH ₂ CH ₂ CH ₂ -CHO.

In other specific embodiments, the aldehyde group of PEG-butyraldehyde forms a bond with the amine group of the N-terminal methionine of IL-10 variants or derivatives thereof, and the structure is as follows:

CH ₃ O-(CH ₂ CH ₂ O)n-CH ₂ CH ₂ CH ₂ -CH ₂ -NH-IL-10 variant or derivative.

In some embodiments, a method for preparing single and double PEG-IL-10 is provided, which includes the steps:

The IL-10 variant or derivative is contacted with an activated PEG-aldehyde linker in the presence of a reducing agent to form a single or double PEG-IL-10, wherein the single PEG-aldehyde linker is one of the IL-10 variant or derivative The amino acid residues are covalently linked.

Figure 1 shows the proliferation experiment results of IL-10 variants and 20KD-, 12KD-mono- and double-PEGylated IL-10 variants on human primary CD8+ T cells. Among them, 20KD mono-PEGylation refers to the IL-10 dimer conjugated with a single PEG, the average molecular weight of the PEG is 20KD, which is conjugated to one of the monomers in the dimer; 20KD double-PEGylation refers to conjugation IL-10 with two PEGs Dimer, the average molecular weight of the PEG is 20KD, and two PEGs are respectively conjugated to two monomers in the dimer; 12KD mono-PEGylation refers to the IL-10 dimer conjugated with a single PEG. The average molecular weight of the PEG is 12KD, which is conjugated to one of the monomers in the dimer; 12KD diPEGylation refers to the IL-10 dimer conjugated with two PEGs. The average molecular weight of the PEG is 12KD. Respectively conjugate and to the two monomers in the dimer.

Figure 2 shows the activation experiment results of IL-10 variants and 20KD-, 12KD-mono- and double-PEGylated IL-10 variants on human primary CD8+ T cells.

Figure 3 shows the results of plasma concentrations of IL-10 variants after subcutaneous administration in mice.

Figure 4 shows the results of IL-10 variants and their 20KD and 12KD mono- and di-PEGylated derivatives inhibiting the growth of CT26 mouse colon cancer xenografts.

Figure 5 shows the results of the effects of IL-10 variants and their 20KD and 12KD mono- and di-PEGylated derivatives on the body weight of CT26 mice.

In order to make the present disclosure easier to understand, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have meanings commonly understood by those of ordinary skill in the art to which this disclosure belongs.

The three-letter codes and one-letter codes of amino acids used in the present disclosure are as described in J. Biol. Chem, 243, p3558 (1968).

the term

"Derivatives" are intended to be interpreted broadly and include any IL-10 related products. Including but not limited to human and non-human IL-10 homologs, fragments or truncations, fusion proteins (such as fusion with signal peptide or other active or inactive ingredients, active ingredients such as antibodies or their Antigen-binding fragments), modified forms (such as PEGylation, glycosylation, albumin conjugation/fusion, hydroxyethylation, etc.), and conservatively modified proteins.

"Antibody" refers to immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds. The amino acid composition and sequence of the constant region of the immunoglobulin heavy chain are different, so their antigenicity is also different. Accordingly, immunoglobulins can be divided into five categories, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE. The corresponding heavy chains are μ chain, δ chain, γ chain, Alpha chain and epsilon chain. The same type of Ig can be divided into different subclasses according to the amino acid composition of the hinge area and the number and position of heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. The light chain is classified into a kappa chain or a lambda chain by the difference in the constant region. Each of the five types of Ig can have a kappa chain or a lambda chain. Antibodies include murine antibodies, human antibodies, humanized antibodies, chimeric antibodies, and camel antibodies.

"Antigen-binding fragments" refer to Fab fragments, Fab' fragments, F(ab')2 fragments, single-chain Fv (i.e. sFv), nano-antibodies (i.e. VHH), and VH/VL domains with antigen-binding activity. The Fv fragment contains the variable region of the antibody heavy chain and the variable region of the light chain, but does not have the constant region, and has the smallest antibody fragment with all antigen binding sites. Generally, Fv antibodies also include a polypeptide linker between the VH and VL domains, and can form the structure required for antigen binding. Different linkers can also be used to connect the variable regions of two antibodies to form a polypeptide chain, which is called single chain antibody or single chain Fv (sFv).

"Conservative modifications" apply to amino acid and nucleotide sequences. For a specific nucleotide sequence, conservative modification refers to those nucleic acids that encode the same or substantially the same amino acid sequence, or in the case where the nucleotide does not encode the amino acid sequence, refers to the substantially same nucleoside Acid sequence. For the amino acid sequence, "conservative modification" refers to the substitution of other amino acids with similar characteristics (such as charge, side chain size, hydrophobicity/hydrophilicity, main chain conformation and rigidity, etc.) in the protein, so that Changes can be made frequently without changing the biological activity of the protein. Those skilled in the art know that, generally speaking, the substitution of a single amino acid in a non-essential region of a polypeptide does not substantially change the biological activity (see, for example, Watson et al. (1987) Molec μ lar Biology of the Gene, The Benjamin/Cummings Pub .Co., page 224, (4th edition)).

"PEGylation" refers to the attachment of at least one PEG molecule to another molecule (eg, a therapeutic protein). For example, Adagen (a PEGylated formulation of adenosine deaminase) is approved for the treatment of severe combined immunodeficiency. It has been shown that polyethylene glycol linkage can prevent proteolysis (see, for example, Sada et al. (1991) J. Fermentation Bioengineering 71:137-139).

In the most common form, PEG is a linear or branched polyether attached to a hydroxyl group at one end, and has the following general structure: HO-(CH ₂ CH ₂ O) _n -CH ₂ CH ₂ -OH. In order to couple PEG to molecules (polypeptides, polysaccharides, polynucleotides and small organic molecules), PEG can be activated by preparing derivatives of PEG with functional groups at one or both ends. The common approach for PEG conjugation of proteins is to activate PEG with a functional group that is suitable for the reaction with lysine and the N-terminal amino acid group. In particular, the common reactive group involved in conjugation is the alpha or epsilon amine group of lysine. The reaction of the PEGylated linker with the protein can lead to the attachment of the PEG moiety mainly at the following positions: the alpha amine group on the N-terminus of the protein, the epsilon amine group on the side chain of the lysine residue, or histidine The imidazole group on the side chain of the residue. Since most recombinant proteins have a single α and many ε amine groups and imidazole groups, many positional isomers can be produced according to the chemical properties of the linking group.

The first generation of activated monomethoxy PEGs (mPEGs) are succinimidyl carbonate PEG (SC-PEG; see, for example, Zalipsky et al., (1992) Biotehnol. Appl. Biochem 15: 100-114; and Miron and Wilcheck, (1993) Bioconjug.Chem. 4:568-569) and benzotriazole carbonate PEG (BTC-PEG; see, for example, US 5650234), which preferentially react with lysine residues to form carbamates connection Group, but it is also known to react with histidine and tyrosine residues. The linker of histidine residues on IFN α has been shown to be a hydrolytically unstable imidazolyl carbamate linker (see, for example, US5985263).

The second generation of PEGylation technology avoids unstable linkers and lack of selectivity in residue reactions. Using PEG-aldehyde linkers can target a single site on the N-terminus of the polypeptide by reductive amination. . Different types of linkers and pH values can be used to PEGylate IL-10 to obtain various forms of PEGylated molecules (see, for example, US5252714, US5643575, US5919455, US5932462, US5985263, US7052686).

"Administration", "administration" and "treatment" when applied to animals, humans, experimental subjects, cells, tissues, organs or biological fluids refer to exogenous drugs, therapeutic agents, diagnostic agents or compositions and animals , Human, subject, cell, tissue, organ or biological fluid contact. "Administration", "administration" and "treatment" can refer to, for example, treatment, pharmacokinetics, diagnosis, research, and experimental methods. The treatment of cells includes contact of reagents with cells, and contact of reagents with fluids, where the fluids contact the cells. "Administration", "administration" and "treatment" also mean the treatment of, for example, cells by reagents, diagnostics, binding compositions, or by another cell in vitro and ex vivo. "Administration" and "treatment" when applied to human, veterinary or research subjects refer to therapeutic treatment, preventive or preventive measures, research and diagnostic applications.

"Treatment" means administering an internal or external therapeutic agent to a subject, such as comprising any IL-10 variant and derivative thereof or a composition comprising the variant or derivative of the present disclosure, and the subject is diagnosed with Has, is suspected of suffering from, or is susceptible to one or more disease symptoms, and the therapeutic agent is known to have a therapeutic effect on these symptoms. Generally, the therapeutic agent is administered in the subject or population to be treated in an amount effective to alleviate one or more symptoms of the disease, whether by inducing the regression of such symptoms or inhibiting the development of such symptoms to any clinically unmeasurable degree.

The amount of therapeutic agent (also referred to as "therapeutically effective amount") that is effective to alleviate the symptoms of any particular disease can vary depending on various factors, such as the subject's disease state, age and weight, and the ability of the drug to produce the desired therapeutic effect in the subject . By any clinical testing methods commonly used by doctors or other professional health care professionals to evaluate the severity or progression of the symptoms, it can be evaluated whether the symptoms of the disease have been alleviated. As far as the embodiments of the present disclosure (such as treatment methods or products) may be ineffective in alleviating the symptoms of the target disease that each patient has, according to any statistical test methods known in the art such as Student t test, chi-square test, and basis Mann and Whitney's U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test determined that it should reduce the symptoms of the target disease in a statistically significant number of subjects.

"Effective amount" includes an amount sufficient to improve or prevent the symptoms or conditions of the medical condition. An effective amount also means an amount sufficient to allow or facilitate diagnosis. The effective amount for a particular subject or veterinary subject may vary depending on factors such as the condition to be treated, the general health of the subject, the method of administration and dosage, and the severity of side effects. The effective amount can be the maximum dose or dosing schedule that avoids significant side effects or toxic effects.

"Chemotherapeutic agents" are compounds used to treat neoplastic diseases. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN); alkyl sulfonates such as busulfan, Improsulfan and bailfen; aziridine, For example, benzodopa, carboquinone, meturedopa and uredopa; aziridine and methyl melamine, including hexamethylmelamine, tritamide, and triethylene Phosphasamide, triethylene thiophosphamide and trimethyl melamine mustard such as chlorambucil, chlorambucil, cholophosphamide, estramustine, ifosphate Amine, dichloromethanediethylamine, dichloromethanediethylamine oxide hydrochloride, chlorambucil, chlorambucil, chlorambucil phenylacetate, cholesteryl esters, pinewood, chlorpheniramine, Uracil Nitrogen Mustard; nitrosoureas, such as carmustine, pyrogluconamide, formustine, cyclohexylnitrosourea, pyrimidine nitrosourea, ramustine (Ranimustine); antibiotics, such as aclamycin , Nathromycin, authramycin, azoserine, bleomycin, actinomycin C, calicheamicin, nordaunorubicin, carmine, carcinophilin, Chromomycin, actinomycin, daunorubicin, ditorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin , Idarubicin, methiromycin, mitomycin, mycophenolic acid, nogamycin, olivemycin, pelomycin sulfate, porphyromycin (potfiromycin), puromycin, triiron adriamycin , Rhodoubicin, streptozotocin, streptozotocin, tuberculin, ubiquitin, neocarcinogen, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil ( 5-FU); Folic acid analogues, such as dimethicone, methotrexate, pterorxine, trimethoate; Purine analogues, such as fludarabine, 6-mercaptopurine, thiomipurine, thioguanine; Pyrimidine analogues, such as ancitabine, azacitidine, 6-azuridine, carmofur, cytarabine, dideoxyuridine, deoxyfluridine, enoxabine, azuridine, 5 -FU; Androgens, such as cap testosterone, drosterone propionate, thiosterol, meandrostane, testosterone; anti-adrenergics, such as amiluminide, mitotane, tripostane; Folic acid supplements, such as leucovorin; acetoglucurolide; aldophosphamide glycoside; alanine propionic acid; amsacrine; bestrabucil; bisantrene; idatril Sand; colchicamine; diacridinone; elfornithine; elfornithine; ammonium elitonate; Etoglu; gallium nitrate; hydroxyurea; mushroom polysaccharide; clonidamine; propionylhydrazone; mitoxantrone; Mo Piridol; nitraerine; pentostatin; methionine; pirarubicin; podophyllic acid; 2-ethylisonicotinyl hydrazine; procarbazine; PSK.; C Imine; Cizonan; Germanspiramine; Alternaria tenuinic acid; Triethyleneimine benzoquinone; 2,2′,2″-Trichlorotriethylamine; Urethane; Deacetylvinblastamide ; Dacarbazine; Mannitol mustard; Dibromomannitol; Dibromodulcitol; Dibromopropylpiperazine; Gacytosine; Arabinoside ("Ara-C"); Cyclophosphamide; Thiotepa; taxanes such as paclitaxel (TAXOL Bristol-Myers Squibb Oncology, Princeton, NJ) and doxetaxel (Taxotere, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate ; Platinum analogues, such as cisplatin and carboplatin; Vinca rosea; Platinum; Etoposide (VP-16); Ifosfamide; Mitomycin C; Mitoxantrone; Vinca rosea; Catharanthus roseus Rebin; Winnopine; Noanto; Epipodophyllotoxin thiophene glycoside; Daunorubicin; Aminopterin; Xeloda (xeloda); Ibandronate; CPT11; Topical isomerase inhibitor RFS2000 ; Difluoromethylornithine (DMFO); retinoic acid; esperamicins; capecitabine; and a pharmaceutically acceptable salt, acid or derivative of any of the above. Also included in this definition are anti-hormonal agents that regulate or inhibit hormone effects on tumors, such as anti-estrogens, including, for example, tamoxifen, ranoxifen, and aromatase that inhibits 4(5)-imidazole, 4- Hydroxytamoxifen, trivoxifene, keoxifene, LY117018, onapristone, and toremifene citrate (fareston); and antiandrogens, such as flutamide , Nilutamide, bicalutamide, leuprolide, and goserelin; and a pharmaceutically acceptable salt, acid or derivative of any of the above.

Regardless of whether it is conjugated with polyethylene glycol or in a non-conjugated form, the "interleukin-10" or "IL-10" used in the present disclosure is composed of two identical non-covalently linked subunits forming a dimer protein. Unless otherwise stated, "Interleukin-10" and "IL-10" as used herein may refer to human or mouse IL-10 (Genbank Accession Nos. NP 000563; M37897; or US6217857), which is also called It is "hIL-10" or "mIL-10".

"PEGylated IL-10" or "PEG-IL-10" is an IL-10 molecule with one or more polyethylene glycol molecules (whether in the form of monomer or dimer). The linker is covalently linked to one or more amino acid residues of IL-10 protein, so the link is stable.

The terms "mono-PEGylated IL-10" and "mono-PEG-IL-10" refer to a polyethylene glycol molecule with a single amino acid residue on the subunit of the IL-10 dimer via a linker Covalently attached (regardless of whether IL-10 is in monomer or dimeric form). The average molecular weight of the PEG moiety is between about 5KD to about 50KD, or about 5KD to about 40KD, or about 10KD to about 30KD, or about 10KD to about 20KD, or about 10KD to about 15KD. There is no restriction on the method or site of PEG and IL-10 connection, but the skilled person understands that the connection of PEG and IL-10 should not change (or only minimally change) the activity of IL-10 variants or derivatives .

This disclosure introduces the IL-10 PEGylation method and the PEGylation structure, activity and function detection method described in US7052686 in full in this disclosure, which can be used to PEGylate the IL-10 variant of the present disclosure and obtain the corresponding PEGylated IL-10 variant.

"Half-life" or "serum half-life" refers to the elimination half-life, that is, the time required for the serum concentration of the analyte to reach half of its initial or maximum value. For synthetic drugs, the term "increased half-life" used in this disclosure means that synthetic drugs are eliminated at a slower rate than non-synthetic, endogenous drugs or their recombinantly produced forms.

Example

The present disclosure is further described below in conjunction with embodiments, but these embodiments do not limit the scope of the present disclosure.

Example 1: Construction and expression of wild-type IL-10 and variants

1. Gene synthesis and recombinant expression vector construction

The wild-type IL-10 nucleic acid sequence is shown in SEQ ID NO. 1, with an Nde I restriction site added at the 5'end and a BamH I restriction site at the 3'end .

(SEQ ID NO.1)；

(SEQ ID NO.1);

(The parts in italics are Nde I and BamH I restriction sites respectively)

The corresponding protein sequence is as follows:

(SEQ ID NO.2)；

(SEQ ID NO. 2);

The expression vector used was the E. coli expression vector pET-9a (Novagen, Cat.69431-3) purchased from Novagen. After the IL-10 nucleic acid sequence is synthesized, a recombinant expression vector is constructed to obtain a wild-type IL-10 expression vector.

2. Introduce the amino acid mutations defined in this disclosure into the wild-type IL-10 amino acid sequence

In a variant, asparagine (N) at position 10 is replaced with glutamine (Q), and the codon AAC at positions 28-30 of the corresponding nucleotide sequence is changed to CAG. The nucleic acid sequence after mutation is as follows:

(SEQ NO ID.3)；

(SEQ NO ID. 3);

The corresponding amino acid sequence is shown below, that is, the IL-10-01 variant sequence:

(SEQ NO ID.4)；

(SEQ NO ID. 4);

In a variant, methionine (M) at position 39 is replaced with threonine (T), and the codon ATG at positions 115-117 of the corresponding nucleotide sequence is changed to ACC. The nucleic acid sequence after mutation is as follows:

(SEQ NO ID.5)；

(SEQ NO ID.5);

The corresponding amino acid sequence is shown below, that is, the IL-10-02 variant sequence:

(SEQ NO ID.6)；

(SEQ NO ID. 6);

In a variant, the asparagine (N) at position 92 is replaced with glutamine (Q); the codon AAC at positions 274-276 of its corresponding nucleotide sequence is changed to CAA. The nucleic acid sequence after mutation is as follows:

(SEQ NO ID.7)；

(SEQ NO ID. 7);

The corresponding amino acid sequence is shown below, that is, the IL-10-03 variant sequence:

(SEQ NO ID.8)；

(SEQ NO ID. 8);

In a variant, asparagine (N) at position 10 is replaced with glutamine (Q), and the codon AAC at positions 28-30 of the corresponding nucleotide sequence is changed to CAG; Methionine (M) was replaced with threonine (T), and the codon ATG at positions 115-117 of the corresponding nucleotide sequence was changed to ACC. The nucleic acid sequence after mutation is as follows:

(SEQ NO ID.9)；

(SEQ NO ID. 9);

The corresponding amino acid sequence is shown below, that is, the IL-10-04 variant sequence:

(SEQ NO ID.10)；

(SEQ NO ID. 10);

In a variant, methionine (M) at position 39 is replaced with threonine (T), and the codon ATG at position 115-117 of the corresponding nucleotide sequence is changed to ACC; Butyramide (N) was replaced with glutamine (Q); the corresponding nucleotide sequence 274-276 codon AAC was changed to CAA. The nucleic acid sequence after mutation is as follows:

(SEQ NO ID.11)；

(SEQ NO ID.11);

The corresponding amino acid sequence is shown below, that is, the IL-10-05 variant sequence:

(SEQ NO ID.12)；

(SEQ NO ID. 12);

In a variant, asparagine (N) at position 10 is replaced with glutamine (Q), and the codon AAC at positions 28-30 of the corresponding nucleotide sequence is changed to CAG; Methionine (M) was replaced with threonine (T), and the codon ATG at position 115-117 of the corresponding nucleotide sequence was replaced with ACC; the asparagine (N) at position 92 was replaced with valley Amidoamine (Q); the codon AAC at positions 274-276 of its corresponding nucleotide sequence was changed to CAA. The nucleic acid sequence after mutation is as follows:

(SEQ NO ID.13)；

(SEQ NO ID. 13);

The corresponding amino acid sequence is shown below, that is, the IL-10-06 variant sequence:

(SEQ NO ID.14)；

(SEQ NO ID. 14);

After the gene sequence of the above variant is synthesized, it is connected with the expression vector pET-9a to form a recombinant expression vector.

3. Recombinant expression and preparation of wild-type IL-10 and its variants

The above-mentioned recombinant expression vector was transformed into BL21(DE3) strain (Novagen, Cat.69450-3) purchased from Novagen to obtain wild-type IL-10 and variant recombinant bacteria. The recombinant bacteria were spread on Kan-resistant Screen on LB plate.

Pick a single colony selected by Kan in 10mL LB medium, culture at 37℃, 220rpm to OD ₆₀₀ of 1.2±0.2, add 50% glycerol to a final concentration of 10%, and distribute it to cryopreservation tubes (1mL/tube). Store at -80°C.

Take a frozen glycerol tube, resuscitate it in a 37°C water bath, inoculate it in 1L LB medium, cultivate at 37°C, 220rpm to an OD ₆₀₀ of 0.6 to 1.0, add 1M IPTG to make the final concentration 1mM, 37°C, 220rpm Induction culture for 4h. After the induction, the cells were collected.

After the bacterial cell is broken, the inclusion body is denatured, renatured, and purified to obtain the target protein.

Example 2: Stability study of wild-type IL-10 and its variants

The wild-type IL-10 sample (buffer system is 10mM Tris-HCl, pH7.4) was placed at 37°C for stability study, and peptide maps were drawn on the samples by LC/MS to analyze the degradation.

Detection method: Take 0.2mL sample, add 0.2mL denaturing solution (8mol/L guanidine hydrochloride, 0.2mol/L Tris-HCl, pH7.5), add 5 μl 1mol/L DTT (dithiothreitol) and mix well, Water bath at 25℃ for 1h. Then add 30 μl of 0.5mol/L IAM (iodoacetamide) in a water bath at 25°C for 1 hour. The sample was replaced with enzymolysis buffer (50mmol/L ammonium bicarbonate, 1mol/L guanidine hydrochloride, pH8.0) through PD-10 column. 0.4mL samples taken after replacement buffer is added to 40 μ g V8 protease, a water bath at 25 deg.] C for 18 h, hydrochloric acid was added to terminate the reaction by LC-MS peptide map drawing.

The test results found that the fragments containing N10 and M39 were degraded.

In the process of variant research, the stability of the two variants containing N10Q and M39T mutation sites respectively was investigated. Sampling to detect degradation, the method is the same as wild-type IL-10. The results are shown in Table 2. The experimental results showed that the mutations at the N10 and M39 sites respectively reduced the deamination and oxidation of the fragments at the two sites, indicating that the mutant was more stable than the wild-type, which met the design expectations.

Table 2. Stability results of IL-10 variants

Among them, D represents the sky.

Example 3: Activity determination of wild-type IL-10 and variants

Under different IL-10 concentrations, the cell-dependent MC-9 (Master Cell-9) cell survival rate is different, so as to detect the biological activity of IL-10.

Basic medium: DMEM (high glucose) + 10% FBS + 1% P/S (penicillin) + 0.05mM beta ME;

Complete medium: DMEM+10%FBS+ 1%P/S (penicillin)+5ng/mL mIL-4+2.5ng/mL hIL-10+0.05mM beta ME (beta-mercaptoethanol);

MTT solution: Weigh 0.1g of MTT, add sterile PBS (phosphate buffered saline, pH7.4) to dissolve and dilute to 20mL, filter and sterilize through a 0.22 μm filter membrane;

Lysis solution: 15% sodium lauryl sulfate solution.

Preparation of reference solution: take the recombinant human interleukin-10 reference substance and dilute it with basic culture medium (containing 30ng/mL mIL-4) to 30ng per 1mL. In a 96-well cell culture plate, make a 3-fold serial dilution, a total of 8 gradients, and make 2 wells for each dilution. Were left per well was 50 μ L, more wells was discarded, the above operations are carried out under sterile conditions.

Preparation of test sample solution: Take the recombinant human IL-10 test sample and dilute it with basic medium (containing 30ng/mL mIL-4) to contain 30ng per 1mL. In a 96-well cell culture plate, make a 3-fold serial dilution, a total of 8 dilutions, each with 2 wells. Were left in each well 50 μ L test sample solution, excess solution was discarded and the wells, the above operation was conducted under sterile conditions.

IL-10 activity determination method: MC-9 cells are cultured in complete culture medium at 37°C and 5% carbon dioxide to a sufficient amount; MC-9 cells are collected by centrifugation, washed 3 times with DMEM medium; resuspended in basic medium It was prepared into a cell suspension containing 5.0×10 ⁵ cells per 1 mL; kept at 37°C and 5% carbon dioxide for use. In a 96-well cell culture plate with standard solution and test sample solution, add 50 μl of cell suspension to each well and incubate at 37°C and 5% carbon dioxide for 42 to 48 hours; then add MTT to each well 20 μl of solution, after incubating at 37°C and 5% carbon dioxide for 4-6 hours, add 150 μl of lysate to each well, and incubate at 37°C and 5% carbon dioxide for 18-24 hours. The above operations are all sterile Under the conditions. Mix the liquid in the cell plate, put it into the microplate reader, measure the absorbance at 570nm with 630nm as the reference wavelength, and record the measurement results.

The computer program or data using a four-parameter regression calculation processing, and calculates the results for the following formula: relative biological activity of the sample to be tested (%) = Reference Sample EC ₅₀ / sample to be tested EC ₅₀ (EC _50: half-maximal effect concentration).

Table 3 shows the activity data of the mutants in Example 1.

Table 3. Activity of IL-10 wild type and its variants

Example 4: Preparation of PEGylated IL-10 variants

1. The experimental instruments used are all conventional, such as:

Cell viability analyzer (Cellometer, model NC200); flow cytometer (Invitrogen, model Attune NxT).

2. The materials used are all conventional, such as:

DMEM, FBS, RPMI1640 (all purchased from Gibco); Trypsin-EDTA (0.25%), CellTrace ^TM Violet (all purchased from Invitrogen); DMSO (purchased from Sigma); Rat T-STIM (purchased from Becton Dickenson); IL- 4 (purchased from Peprotech); CellTiter-Glo (purchased from Promega); Pen-Strep (purchased from Gibco); FITC mouse anti-human CD8, Alexa Fluor® 647 mouse anti-human granzyme (Granzyme) B, human IL-10 ELISA KIT2 (both purchased from BD); Fc receptor blocker (Human TruStain FcX), anti-mouse CD20 antibody (both purchased from biolegend); ImmunoCult ^TM human CD3/CD28 T cell activator, EasySep human CD8+ T cell isolation kit ( All purchased from Stemcell); Brefeldin A solution, permeabilization buffer (10×Permeabilization Buffer), IC fixation buffer (all purchased from eBioscience); PMBC (purchased from Schbio).

3. PEG modification of IL-10 variants

-Take IL-10 wild type or its variants, the protein concentration is 3-4mg/mL, the buffer system is 50mM PB/0.1M NaCl, pH 6.0-6.3;

-Add mPEG-butyraldehyde aqueous solution with a concentration of 2mM to the IL-10 solution. The amount of mPEG butyraldehyde is 1 time the amount of IL-10, and stir well;

-Add 500mM sodium cyanoborohydride aqueous solution to the reaction system to make the final concentration 15mM;

-After stirring the reaction at 25°C for about 16 hours, add a 1M aqueous solution of glycine to terminate the reaction. The final concentration of glycine is 30mM.

In the above process, the aldehyde group of PEG-butyraldehyde forms a bond with the amine group of the N-terminal methionine of IL-10.

4. Purification of PEGylated-IL-10 variants

After the PEGylation reaction is terminated, the reaction system is replaced with 10 mM Tris-HCl pH 8.7 buffer, and chromatographic purification is started.

The packing for purification is Q sepharose ^TM High Performance (GE lifescience);

Buffer used for purification: buffer A is 10mM Tris-HCl pH8.7, and buffer B is 10mM Tris-HCl/0.5M NaCl pH8.7.

Equilibrium: Buffer B is pre-equilibrated with high salt, and then Buffer A is rebalanced. After the UV, conductivity, and pH are stable, the UV is set to zero, 150cm/h;

Load sample: load the sample after filtration, 150cm/h, load 1-2mg/mL;

Gradient extraction parameters: 0-20% B, gradient extraction, 20CV, 75cm/h; collect the target protein peak after peaking. RP-HPLC and SE-HPLC detect the purity of the target protein. After testing, a PEGylated IL-10 variant was obtained.

Example 5: MC-9 cell proliferation experiment

The experimental method refers to Example 3, and the results are shown in Table 4.

Table 4. MC-9 cell proliferation experiment of IL-10

The results show that the unmodified IL-10 mutant IL-10-06 and its PEGylated samples can promote the proliferation of MC-9 cells to varying degrees.

Example 6: CD8+ T cell proliferation and granulomycin release experiment

1. Experimental procedure: Use EasySep Human CD8+ T cell isolation kit to negatively select the frozen PBMC to obtain CD8+ T cells, add CD3/CD28 T cell activator (25ul/1E6 cells/mL), culture for 48 hours, and collect by centrifugation CD8+ T cells. Adjust the T cell density to 2.5E6 cells/mL and plant them in a 96-well plate with 200 μl culture medium per well. The culture medium is 1640 containing 1% penicillin-streptomycin and 10% FBS, and then add different concentrations of IL- 10 The test sample is incubated for 72 hours. In the CD8+T cell proliferation experiment, directly add Cell Titer-Glo and measure the number of living cells. The results are shown in Figure 1.

2. In the granomycin release experiment, before using flow cytometry to check granomycin release, add protein transport inhibitor Brefeldin A solution and CD3/CD28 T cell activation to CD8+ T cells 72 hours after IL-10 action Agent for 16 hours. Collect CD8+ T cells; use FITC mouse anti-human CD8 and Alexa Fluor® 647 mouse anti-human granzyme B to label cells in sequence according to the two-step method of intracellular staining, and flow cytometry; the results are CD8 positive T cells granzyme B flow The formula MFI value is used as an indicator of cell activation. The EC ₅₀ value of IL-10 to promote the release of granulomycin from CD8+ T cells uses the four-parameter logit method of formula I above. The results are shown in Figure 2.

The results show that since IL-10 receptors are only expressed on activated CD8 cells, in this example, after activating human CD8+ T cells, IL-10 was added for action. It can be seen from Figure 1 that the unmodified IL-10 mutant IL-10-06 and its PEGylated derivatives can promote the proliferation of activated CD8+ T cells in a dose-dependent manner to varying degrees.

In addition, IL-10 can not only promote the proliferation of activated CD8+ T cells, but also promote the functions of CD8+ T cells, such as the release of granulomycin. In Figure 2, the unmodified IL-10 mutant IL-10-06 can stimulate CD8+ T cells to secrete granulomycin, with an EC ₅₀ of 1.63ng/mL, and PEGylated IL-10-06 can promote it in a dose-dependent manner. Human CD8+ T cells secrete granulomycin. Among them, the EC ₅₀ of 20KD-monoPEG-IL-10-06 is 16.1ng/mL; the EC ₅₀ of 20KD-diPEG-IL-10-06 is 105ng/mL; the EC ₅₀ of 12KD-monoPEG-IL-10-06 The EC ₅₀ is 13.7 ng/mL; the EC ₅₀ of 20KD-bisPEG-IL-10-06 is 33.4 ng/mL.

Example 7: Pharmacokinetics experiment in mice

Experimental procedure: 6-8 weeks of C57BL/6 mice, male (Shanghai Lingchang Biological Technology Co., Ltd.), the rearing environment of the mice is SPF level. The environment temperature is controlled at 20-26℃, humidity is 40-70%, 12 hours of darkness and 12 hours of light, and the mice are free to eat (standard feed) and drink water. Only animals that have passed the health check will be used in this experiment.

60 micrograms of IL-10 samples were administered subcutaneously, and blood was taken before administration and 4, 8, 24, 48, 72, 96, and 120 hours after administration, added heparin sodium for anticoagulation, and immediately stored in a -80 degree refrigerator. After all the plasma was collected, the IL-10 content in mouse plasma was detected by ELISA method. The result is shown in Figure 3.

In Figure 3, the half-life of unmodified IL-10-06 in vivo is very short. After 24 hours of administration, IL-10 in plasma is already below the detection limit, and PEGylation can significantly extend the half-life of IL-10-06 in vivo. Among them, the in vivo half-life of 12KD-monoPEG-IL-10-06 is 7.9 hours, the in vivo half-life of 12KD-diPEG-IL-10-06 is 10.5 hours, and the in vivo half-life of 20KD-monoPEG-IL-10-06 is 9.37 hours.

Example 8: In vivo efficacy of mouse colon cancer CT-26 cell subcutaneous allograft tumor model in BALB/c mice

Experimental procedure: mouse colon cancer CT-26 cells (CT-26-C2, ATCC), culture conditions are RPMI1640 medium plus 10% fetal bovine serum, 37ºC 5% CO ₂ incubator culture. When the cell saturation is 80%-90% and the number reaches the requirement, the cells are collected and counted. 0.1 mL (0.3×10 ⁶ cells) of CT-26 cells were subcutaneously inoculated into the right back of each mouse (BALB/c mice, female, 6-8 weeks old, purchased from Shanghai Lingchang Biotechnology Co., Ltd.). The breeding environment of mice is SPF level. The environment temperature is controlled at 20-26℃, humidity is 40-70%, 12 hours of darkness and 12 hours of light, and the mice are free to eat (standard feed) and drink water. Only animals that have passed the health check will be used in this experiment. When the average tumor volume of the mice reaches about 0-100mm ³ , group administration will be started. Mice in order to prevent anti-human IL-10 antibody to thereby antagonizing its antitumor activity, mice were administered per 200 μ g CD20 antibody mouse 24 hours before dosing formal. Then the test samples of different concentrations were administered subcutaneously twice a day. Observation for 19 days, the tumor volume and mouse body weight changes were measured twice a week. The results are shown in Figures 4 and 5.

The results showed that administration of PEGylated IL-10-06 could inhibit the growth of colon cancer transplanted tumors in CT26 mice after 19 days. The tumor volume (T/C) of the 12KD double PEG-IL-10-06 0.1mpk administration group was 61.4% compared with the tumor volume (T/C) of the mice in the control group, and the 12KD double PEG-IL-10-06 0.3mpk, 19 days after administration The T/C value was 83.6%, and the tumor in one of the six mice completely resolved. 20KD single PEG-IL-10-06 0.1mpk, 19 days after administration, T/C value is 49.93%, 20KD single PEG-IL-10-06 0.3mpk, 19 days after administration, T/C value is 18.72%, Among the 6 mice, 3 mice tumors completely resolved. However, IL-10-06 without PEG modification has a tendency to inhibit tumors after 10 days of administration, but after 19 days of administration, the T/C is 102.9% and has no anti-tumor activity.

The weight of the mice in the control group and the IL-10-06 administration group continued to increase during the administration period (Figure 5), which may be due to the continued growth of tumors. The weight gain was weaker in the 12KD double PEG-IL10-06 and 20KD single PEG-IL10-06 administration group, which may be Tumor growth is inhibited. In addition, the administration group did not show any significant weight loss 19 days after administration compared with before administration, indicating that the administration group did not produce intolerable side effects.

<110> Jiangsu Hengrui Pharmaceutical Co., Ltd. Shanghai Hengrui Pharmaceutical Co., Ltd. Shanghai Shengdi Pharmaceutical Co., Ltd.

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<220>

<221> peptide

<222> (1)..(161)

<223> IL-10-05 amino acid sequence

<400> 12

<210> 13

<211> 498

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> Gene

<222> (1)..(498)

<223> IL-10-6 nucleotide sequence, 5'end with Nde I restriction site, 3'end with BamH I restriction site

<400> 13

<210> 14

<211> 161

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> peptide

<222> (1)..(161)

<223> IL-10-06 amino acid sequence

<400> 14

Claims (34)

An IL-10 (human interleukin 10) variant or a derivative thereof, which comprises amino acid mutations at one or more positions selected from the following: position 10, position 39, and position 92. The IL-10 variant or its derivative as described in the first item of the scope of patent application, wherein the amino acid residue at position 10 is mutated to glutamine residue Q. The IL-10 variant or derivative thereof according to the first item of the scope of patent application, wherein the amino acid residue at position 39 is mutated to threonine residue T. The IL-10 variant or its derivative as described in the first item of the scope of patent application, wherein the amino acid residue at position 92 is mutated to glutamine residue Q. The IL-10 variant or derivative thereof according to any one of items 1 to 4 in the scope of the patent application, which comprises an amino acid mutation selected from the following combination: 10Q and 39T; 39T and 92Q; 10Q and 39T and 92Q. The IL-10 variant or derivative thereof according to item 5 of the scope of patent application, wherein, compared with wild-type human IL-10, the IL-10 variant or derivative comprises an amine group selected from the following combination Acid mutation: N10Q and M39T; M39T and N92Q; N10Q and M39T and N92Q. The IL-10 variant or derivative thereof according to any one of items 1 to 6 in the scope of the patent application, which comprises or consists of a polypeptide selected from any one of the following: SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.14. The IL-10 variant or derivative thereof according to any one of items 1 to 7 in the scope of the patent application, wherein: The IL-10 variant or derivative thereof is a form selected from any one of the following: monomer, dimer, trimer; The derivative of the IL-10 variant is embodied in a form selected from any one of the following or a combination thereof: PEGylated, glycosylated, hydroxyethylated, conjugated with albumin, fused to albumin, fused to an antibody or antigen-binding fragment thereof, truncated. The IL-10 variant or its derivative as described in item 8 of the scope of patent application, wherein the IL-10 variant or its derivative is a PEGylated IL-10 variant or its derivative. The IL-10 variant or derivative thereof according to item 9 of the scope of patent application, wherein the PEGylation is located at the N-terminus of the IL-10 variant or derivative. The IL-10 variant or derivative thereof according to item 10 of the scope of patent application, wherein the PEGylated IL-10 variant or derivative is any one selected from the following: -IL-10 dimer conjugated with PEG, wherein the PEG has an average molecular weight of 20KD, and the PEG is a single PEG, which is conjugated to one of the monomers in the dimer; -IL-10 dimer conjugated with PEG, wherein the average molecular weight of the PEG is 20KD, the PEG is two PEGs, and the two PEGs are respectively conjugated to two monomers in the dimer; -IL-10 dimer conjugated with PEG, wherein the PEG has an average molecular weight of 12KD, and the PEG is a single PEG, which is conjugated to one of the monomers in the dimer; -IL-10 dimer conjugated with PEG, wherein the average molecular weight of the PEG is 12KD, the PEG is two PEGs, and the two PEGs are respectively conjugated to the two monomers in the dimer. The IL-10 variant or derivative thereof according to item 8 of the scope of patent application, wherein the dimer is a homodimer. The IL-10 variant or derivative thereof according to item 8 of the scope of patent application, wherein the truncation refers to the lack of a signal peptide. The IL-10 variant or derivative thereof as described in any one of items 8 to 13 of the scope of patent application, which is mono-PEGylated or di-PEGylated. The IL-10 variant or derivative thereof according to item 14 of the scope of patent application, wherein the PEGylation is located at the N-terminus of the IL-10 variant or derivative. The IL-10 variant or derivative thereof according to the 14th patent application, wherein the average molecular weight of the PEG is about 5KD to about 50KD. The IL-10 variant or derivative thereof according to the 16th item of the scope of patent application, wherein the average molecular weight of the PEG is about 10KD to about 30KD. A medicinal composition containing: -The IL-10 variant or derivative thereof described in any one of items 1 to 17 in the scope of patent application, -If necessary, a pharmaceutically acceptable diluent, carrier or adjuvant. An isolated nucleic acid molecule that encodes the IL-10 variant or derivative thereof described in any one of items 1 to 17 in the scope of the patent application. The isolated nucleic acid molecule described in item 19 of the scope of the patent application comprises or consists of a polynucleotide selected from any one of the following: SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7. SEQ ID NO.9, SEQ ID NO.11, SEQ ID NO.13. An expression vector comprising the isolated nucleic acid molecule described in item 19 or 20 of the scope of patent application. A host cell expressing the expression vector described in item 21 of the scope of patent application; The host cell is a prokaryotic or eukaryotic cell. The host cell according to claim 22, wherein the host cell is a bacterium, or yeast, or mammalian cell. The host cell according to claim 23, wherein the host cell is Escherichia coli. A use of the IL-10 variant or derivative thereof according to any one of the 1st to 17th items of the patent application, and the pharmaceutical composition according to the 18th item of the patent application in the preparation of medicines; The medicine is used to treat diseases selected from the group consisting of proliferative diseases, immune diseases, cardiovascular diseases, thrombotic diseases, hyperlipidemia, inflammatory diseases, liver diseases, and regulating T cell-mediated immune responses. The use according to item 25 of the scope of patent application, wherein the proliferative disease is tumor or cancer. The use according to item 26 of the scope of patent application, wherein the tumor or cancer is selected from the group consisting of epithelial cell carcinoma, endothelial cell carcinoma, squamous cell carcinoma, cancer caused by papilloma virus, adenocarcinoma, carcinoma, melanoma, Sarcoma, teratocarcinoma, lung tumor, metastatic lung cancer, lymphoma. The use described in item 25 of the scope of patent application, wherein the immune disease is diabetes. The use according to item 28 of the scope of patent application, wherein the immune disease is insulin-dependent diabetes. The use according to item 25 of the scope of patent application, wherein the cardiovascular disease is selected from the group consisting of atherosclerosis, hypertension, and cardiomyopathy. The use according to item 25 of the scope of patent application, wherein the thrombotic disorder is a disorder that causes stroke or myocardial infarction. The use according to item 25 of the scope of patent application, wherein the inflammatory disease is vasculitis. The use according to item 25 of the scope of patent application, wherein the liver disease is non-alcoholic fatty liver or hepatitis C. A method for preparing IL-10 variants or derivatives thereof, including: Introduce into wild-type human IL-10 the mutation contained in the IL-10 variant or its derivative described in any one of items 1 to 7 in the scope of patent application, or Use the nucleic acid molecule described in item 19 or 20 of the scope of the patent application for expression, or Use the expression vector described in item 21 of the scope of the patent application for expression, or Use the host cell described in item 22 of the scope of patent application for expression.

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