WO2020108426A1 - 一种人白细胞介素10变体及其衍生物 - Google Patents

一种人白细胞介素10变体及其衍生物 Download PDF

Info

Publication number
WO2020108426A1
WO2020108426A1 PCT/CN2019/120562 CN2019120562W WO2020108426A1 WO 2020108426 A1 WO2020108426 A1 WO 2020108426A1 CN 2019120562 W CN2019120562 W CN 2019120562W WO 2020108426 A1 WO2020108426 A1 WO 2020108426A1
Authority
WO
WIPO (PCT)
Prior art keywords
variant
peg
derivative
seq
cancer
Prior art date
Application number
PCT/CN2019/120562
Other languages
English (en)
French (fr)
Inventor
陈磊
葛虎
陆吉学
胡齐悦
唐任宏
张连山
Original Assignee
江苏恒瑞医药股份有限公司
上海恒瑞医药有限公司
上海盛迪医药有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 江苏恒瑞医药股份有限公司, 上海恒瑞医药有限公司, 上海盛迪医药有限公司 filed Critical 江苏恒瑞医药股份有限公司
Priority to CN201980072302.0A priority Critical patent/CN112955546B/zh
Publication of WO2020108426A1 publication Critical patent/WO2020108426A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor

Definitions

  • the present disclosure relates to variants or derivatives of human interleukin-10 with one or more amino acid mutations that allow human interleukin-10 to have enhanced stability and/or activity.
  • Interleukin-10 Human interleukin-10 (interleukin-10, IL-10), its gene is located on chromosome 1 (q31-32), a total of 5.1kb long.
  • the gene sequence of IL-10 contains 5 exons.
  • IL-10 consists of 178 amino acid residues, forming a "V" shaped homodimer with a molecular weight of about 35 kD.
  • 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 and so on.
  • IL-10 is a cytokine secreted by Th2 subtype cells of murine helper T cells, which can inhibit cytokines such as tumor necrosis factor ⁇ (TNF- ⁇ ) secreted by Th1 subtype cells, so It was originally named cytokine synthesis inhibitory factor (CSIF).
  • TNF- ⁇ tumor necrosis factor ⁇
  • IL-10 enhances the release of anti-inflammatory factors, such as IL-1 receptor antagonists and soluble TNF- ⁇ receptors; IL-10 inhibits several cytokines produced by type 1 helper T cells (eg, interferon-gamma, IL-2 and TNF- ⁇ ) synthesis, inhibit cell-mediated immune response regulators, and suppress antigen-presenting cell-dependent T cell responses, and 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- ⁇ ; IL-10 inhibits the synthesis of IL-12, thereby Impedes Th1 immune response.
  • type 1 helper T cells eg, interferon-gamma, IL-2 and TNF- ⁇
  • IL-10 inhibits the synthesis of IL-12, thereby Impedes 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 is involved in a variety of cells ( Such as immune cells, inflammatory cells, tumor cells) biological regulation.
  • IL-10 Due to its multi-effect activity, IL-10 has been studied in various clinical applications, including autoimmune diseases, severe infectious diseases, tumors, and transplantation immunity.
  • IL-10 includes mono- or di-pegylated IL-10, which may 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, papilloma virus-induced carcinoma, adenocarcinoma, carcinoma, melanoma, Sarcoma or teratocarcinoma, or for the treatment of lung tumors or metastatic lung cancer, or lymphoma (WO2008/054585; EP2468293B; EP2821078B); and can be administered to patients by an effective amount of PEGylated IL-10, folic acid analogs, 5-FU and platinum drugs to 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 be combined with immune checkpoint inhibitors (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 used in combination with IL-7 to treat disorders (WO2016/196211), as well as to regulate T cell-mediated immune responses with CAR-T cells (WO2016/191587).
  • IL-10 immunostimulatory activity of IL-10 is determined by the single amino acid isoleucine at position 87 (J. Exp. Med. 191(2), 213-223, 2000).
  • WO2012146628A1 discloses a fusion protein of antibody and IL-10, in which amino acid at position 87 is replaced with I87A, it is found that the modified fusion protein exhibits reduced affinity for IL-10 receptor, but improves the targeting efficiency, Reduces side effects caused by IL-10 immunostimulatory activity.
  • the prior art still lacks IL-10 variants with higher stability and higher activity. Providing such variants of IL-10 is an urgent problem in the art.
  • the present disclosure relates to variants or derivatives of human interleukin 10 (IL-10) sequence having one or more amino acid mutations.
  • IL-10 human interleukin 10
  • the present disclosure provides an IL-10 variant which is asparagine (N) at position 10 or/and methionine at position 39 in the wild-type human IL-10 sequence (SEQ ID NO: 2) (M) or/and the asparagine (N) position at position 92, the wild-type amino acid residue is mutated to other amino acids, wherein the sites are numbered according to the mature protein of human wild-type IL-10.
  • the variant has improved stability and/or activity compared to wild-type human IL-10.
  • 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) .
  • the IL-10 variant comprises Asn mutation at position 10 to Gln (N10Q) and/or Met mutation at position 39 to Thr (M39T) and/or Asn mutation to Gln at position 92 (N92Q) ; Or a combination selected from the group consisting of: N10Q and M39T, M39T and N92Q, N10Q and M39T and N92Q.
  • the IL-10 variant or derivative thereof may comprise a polypeptide selected from the following sequence: 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.
  • derivatives of IL-10 variants include full-length, partial proteins related to the disclosed IL-10 variants, or mutant proteins obtained by further mutation based on the variants, functional derivatives, and functionalities Fragments, biologically active peptides, fusion proteins, isotypes or salts thereof.
  • the IL-10 variant derivative includes a fusion protein of the IL-10 variant, a truncated body of the IL-10 variant, and a monomer containing the fusion protein of the truncated body, the IL-10 variant Or dimer or trimer or polymer, modified IL-10 variants (such as pegylation (PEGylation), glycosylation, albumin conjugation or fusion, hydroxyethylation, and antibody Or fusion of antigen-binding fragments thereof), and homologs of IL-10 variants in various species.
  • modified IL-10 variants such as pegylation (PEGylation), glycosylation, albumin conjugation or fusion, hydroxyethylation, and antibody Or fusion of antigen-binding fragments thereof
  • the antibody or antigen-binding fragment fused to the IL-10 variant is a murine antibody, human antibody, humanized antibody, chimeric antibody, or camel antibody or antigen-binding fragment thereof.
  • the antigen-binding fragment is a Fab fragment, Fab' fragment, F(ab') 2 fragment, single-chain Fv (sFv), VHH, or VH/VL domain.
  • 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 variants of mature IL-10 (as in US6217857) lacking a signal peptide, or variants of mature PEGylated IL-10.
  • the variant may exhibit activity comparable to or superior to wild-type human IL-10.
  • the variant may exhibit stability comparable to or higher than that of wild-type human IL-10.
  • the IL-10 variant or its derivative according to the present disclosure may not contain a signal peptide without (statistically) changing the desired activity, as a non-limiting example, in the sequence selected from Remove the signal peptide on the basis of: SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14.
  • IL-10 variants or derivatives thereof may be modified to adjust one or more properties (eg, pharmacokinetic parameters, efficacy, etc.), including whether they may be monomers or dimers Or in the form of a trimer, and/or PEGylated, and/or glycosylated, and/or conjugated and fused with albumin (eg, human serum albumin (HSA)), and/or hydroxyethyl Hesylation.
  • albumin eg, human serum albumin (HSA)
  • HSA human serum albumin
  • modified IL-10 may be less immunogenic than unmodified IL-10.
  • the IL-10 variant or derivative is PEGylated (denoted as PEG-IL-10), for example, a single or double PEGylated IL-10 variant or derivative.
  • PEG-IL-10 variants or derivatives include SC-PEG linkers.
  • the PEG-IL-10 variant or derivative includes a methoxy-PEG-aldehyde (mPEG-ALD) linker.
  • the average molecular weight of the PEG moiety is 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.
  • the mPEG-ALD linker includes PEG molecules with an average molecular weight selected from the group consisting of 5KDa, 12KDa, or 20KDa.
  • 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 proliferation activity, and provide a good basis for protein preparation.
  • the variant comprises N10Q/M39T or M39T/N92Q or a combination of N10Q/M39T/N92Q.
  • an isolated nucleic acid molecule which encodes an IL-10 variant or derivative thereof of the present disclosure.
  • the nucleic acid molecule may comprise the polynucleotides shown in 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 containing the above nucleic acid molecule is provided.
  • a host cell expressing the above vector is provided.
  • the host cell may be a prokaryotic or eukaryotic cell.
  • the host cell is a bacteria or yeast or mammalian cell (eg, CHO, COS, BHK21, NIH3T3, HeLa, C2C12, HEK), for example, Pichia pastoris or Saccharomyces cerevisiae.
  • the host cell does not contain cells capable of developing into plants, nor does it involve human fertilized eggs, human germ cells, nor cells capable of developing into animals.
  • composition comprising an IL-10 variant or derivative thereof of the present disclosure, which contains:
  • the unit dose of the pharmaceutical composition contains 1 mg to 1000 mg of an IL-10 variant or derivative thereof according to the present disclosure.
  • the concentration of the IL-10 variant or derivative thereof in the pharmaceutical composition is 1 mg/L to 1000 mg/L.
  • the pharmaceutical composition contains a buffering agent, and its content may be 1 mM to 1000 mM.
  • the pharmaceutical composition may be a lyophilized formulation or an injectable solution.
  • the proliferative disease may be a tumor or cancer, such as epithelial cell carcinoma, endothelial cell carcinoma, squamous cell carcinoma, papilloma virus-induced carcinoma, adenocarcinoma, carcinoma, melanoma, sarcoma, or teratoma Cancer, lung tumor or metastatic lung cancer, lymphoma, colon cancer, ovarian cancer, breast cancer, malignant glioma, and leukemia.
  • a tumor or cancer such as epithelial cell carcinoma, endothelial cell carcinoma, squamous cell carcinoma, papilloma virus-induced carcinoma, adenocarcinoma, carcinoma, melanoma, sarcoma, or teratoma Cancer
  • lung tumor or metastatic lung cancer lymphoma, colon cancer, ovarian cancer, breast cancer, malignant glioma, and leukemia.
  • the immune disease is diabetes, such as insulin-dependent diabetes.
  • the cardiovascular disorder is atherosclerosis, hypertension, and cardiomyopathy.
  • the thrombotic disorder is a disorder that can cause stroke or myocardial infarction.
  • the inflammatory disorder may be vasculitis.
  • the liver disease may be non-alcoholic fatty liver or hepatitis C.
  • 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 Rectal 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 (eg glioma, ganglion, central nervous system (CNS) and peripheral nervous system (PNS)) and immune system (eg spleen or thymus).
  • gastrointestinal cancer such as esophageal cancer, oropharyngeal cancer, gastric cancer, small or large intestine cancer, colon cancer or Rectal cancer
  • the tumor or cancer may also be an immunogenic tumor, a non-immunogenic tumor, a dormant tumor, a virus-induced cancer such as epithelial cell carcinoma, endothelial cell carcinoma, squamous cell carcinoma, papilloma virus, adenocarcinoma, lymphoma, carcinoma Tumor, melanoma, leukemia, myeloma, sarcoma, teratocarcinoma, chemically-induced cancer, metastatic lesions, and methods of angiogenesis.
  • a virus-induced cancer such as epithelial cell carcinoma, endothelial cell carcinoma, squamous cell carcinoma, papilloma virus, adenocarcinoma, lymphoma, carcinoma Tumor, melanoma, leukemia, myeloma, sarcoma, teratocarcinoma, chemically-induced cancer, metastatic lesions, and methods of angiogenesis.
  • a method of inhibiting or reducing the growth of a tumor or cancer and/or treating a patient suffering from cancer or tumor comprising subjecting the subject to an effective amount of an IL-10 variant of the present disclosure or Derivative contact.
  • the IL-10 variant or derivative can inhibit the growth of a tumor or cancer, or the IL-10 variant or derivative can reduce the size of a tumor or cancer.
  • T cells Treg
  • CD8+ T cells it is expected that the IL-10 variants or derivatives of the present disclosure may also reduce tolerance to tumor cell or cancer cell antigens.
  • PEGylated IL-10 variants or derivatives when compared to non-PEGylated IL-10 variants or derivatives, can increase the entry of CD8+ T cells into targets (eg, neoplasms, lesions) Permeability.
  • PEGylated IL-10 variants or derivatives can increase the expression of at least one inflammatory cytokine, which can be selected from IFN ⁇ , IL-4, IL-6, IL-10, and RANK- Ligand (RANK-L).
  • the PEGylated IL-10 variant or derivative is co-administered with at least one chemotherapeutic agent.
  • the chemotherapeutic agent can be administered before, simultaneously with, or after administration of the PEGylated IL-10 variant or derivative.
  • the patient or subject being treated is a human.
  • IL-10 variants or derivatives can be administered by any effective route, for example, by parenteral injection (including subcutaneous injection).
  • a pharmaceutically acceptable amount of an IL-10 variant or derivative eg, a therapeutically effective amount
  • one or more pharmaceutically acceptable diluents, carriers, or excipients eg, etc. Osmotic injection solution.
  • the pharmaceutical composition is generally a pharmaceutical composition suitable for human administration.
  • the pharmaceutical composition comprises 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.
  • a method for preparing a variant or derivative of IL-10 including:
  • a single PEGylated IL-10 variant or derivative thereof which comprises:
  • a PEG molecule covalently linked to the IL-10 variant or its derivative via a linker can be linked to lysine residues or N-terminal amino acid residues of IL-10 variants or derivatives thereof.
  • the IL-10 variant or derivative thereof is a homodimer, and 20KD or 12KD monoPEG is linked to one of the monomers of the homodimer.
  • a double-PEGylated IL-10 variant or derivative thereof comprising:
  • IL-10 variant or its derivative covalently linked to the IL-10 variant or its derivative.
  • PEG can be linked to lysine residues or N-terminal amino acid residues of IL-10 variants or derivatives thereof.
  • the IL-10 variant or derivative thereof is a homodimer, and a single PEG of 20KD or 12KD is respectively connected to two monomers of the homodimer.
  • the structure of the PEG-IL-10 variant or derivative is as follows:
  • n 20 to 2300
  • b 1 to 9
  • L is an ⁇ - ⁇ alkyl linker (also used as a linker), and L is covalently linked to the IL-10 variant The amino terminus of its derivatives, when b is greater than 1, the sum of n does not exceed 2300.
  • L is "-CH 2 CH 2 CH 2 -".
  • the PEG structure is CH 3 O-(CH 2 CH 2 O)n-CH 2 CH 2 CH 2 -CHO.
  • the aldehyde group of PEG-butyraldehyde is bonded to the amino group of the N-terminal methionine of the IL-10 variant or derivative thereof, and the structure is as follows:
  • a method for preparing single and double PEG-IL-10 including the steps of:
  • 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, where a single PEG-aldehyde linker is in contact with one of the IL-10 variant or derivative
  • the amino acid residues are covalently linked.
  • Figure 1 Proliferation experiment results of IL-10 variants and 20KD-, 12KD- single and double PEGylated IL-10 variants on human primary CD8+ T cells.
  • 20KD single PEGylation refers to IL-10 dimer conjugated with single PEG, the average molecular weight of the PEG is 20KD, conjugated to one of the monomers in the dimer
  • 20KD double PEGylation refers to conjugation An IL-10 dimer combined with two PEGs, the average molecular weight of the PEG is 20KD, and two PEGs are conjugated to two monomers in the dimer
  • 12KD single PEGylation means conjugated with a single PEG IL-10 dimer, the average molecular weight of the PEG is 12KD, one of the monomers conjugated to the dimer
  • 12KD double PEGylation refers to the IL-10 dimer conjugated with two PEGs, The average molecular weight of the PEG is 12KD, and the two PEG
  • Figure 2 IL-10 variants and 20KD-, 12KD- single and double PEGylated IL-10 variants activate human primary CD8+ T cells.
  • Figure 3 Plasma concentration results of IL-10 variants after subcutaneous administration in mice.
  • Figure 4 IL-10 variant and its 20KD, 12KD single and double PEGylated derivatives inhibit the growth of colon cancer xenografts in CT26 mice.
  • Figure 5 Graph showing the effect of IL-10 variants and its 20KD and 12KD mono- and double-PEGylated derivatives on the body weight of CT26 mice.
  • “Derivatives” are intended to be interpreted broadly, including any IL-10 related products. Including but not limited to human and non-human IL-10 homologs, fragments or truncates, fusion proteins (such as fusion with signal peptides or other active, inactive ingredients, active ingredients such as antibodies or antigen-binding fragments thereof), modifications Forms (such as PEGylation, glycosylation, albumin conjugation/fusion, hydroxyethylation, etc.), and conservatively modified proteins, etc.
  • Antibody refers to immunoglobulin, which is a tetrapeptide chain structure formed by connecting two identical heavy chains and two identical light chains through interchain disulfide bonds.
  • the immunoglobulin heavy chain constant region has different amino acid composition and arrangement order, so its antigenicity is also different. According to this, immunoglobulins can be divided into five categories, or isotypes called immunoglobulins, namely IgM, IgD, IgG, IgA and IgE, and their corresponding heavy chains are ⁇ chain, ⁇ chain and ⁇ chain, respectively. , ⁇ chain and ⁇ chain.
  • IgG can be divided into IgG1, IgG2, IgG3 and IgG4.
  • the light chain is divided into a kappa chain or a lambda chain by different constant regions.
  • Each of the five types of Ig can have a ⁇ chain or a ⁇ chain.
  • Antibodies include murine antibodies, human antibodies, humanized antibodies, chimeric antibodies, camel antibodies.
  • Antigen-binding fragment refers to a Fab fragment having an antigen-binding activity, a Fab' fragment, an F(ab') 2 fragment, a single-chain Fv (i.e., sFv), a Nanobody (i.e., VHH), and a VH/VL domain.
  • the Fv fragment contains the antibody heavy chain variable region and light chain variable region, but has no constant region, and has the smallest antibody fragment with all antigen binding sites.
  • Fv antibodies also contain a polypeptide linker between the VH and VL domains, and are capable of forming the structure required for antigen binding. Different linkers can also be used to connect the variable regions of two antibodies into one polypeptide chain, called single chain antibody (single chain antibody) or single chain Fv (sFv).
  • Constant modifications apply to amino acid and nucleotide sequences.
  • 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 an amino acid sequence, to substantially the same nucleotide sequence.
  • conservative modification refers to the replacement of amino acids in a protein by other amino acids with similar characteristics (such as charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation, and rigidity, etc.) so that frequent changes can be made. Does not change the biological activity of the protein.
  • PEGylated means that at least one PEG molecule is linked to another molecule (eg, a therapeutic protein).
  • Adagen PEGylated adenosine deaminase
  • Adagen is approved for the treatment of severe combined immunodeficiency diseases. It has been shown that the attachment of polyethylene glycol can prevent proteolysis (see, for example, Sada et al. (1991) J. Fermentation Bioengineering 71:137-139).
  • PEG is a linear or branched polyether connected 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.
  • PEG can be activated by preparing derivatives of PEG with functional groups at one or both ends.
  • a common way of PEG conjugation of proteins is to activate PEG with a functional group that is suitable for reaction with lysine and N-terminal amino acid groups.
  • the common reactive group involved in conjugation is the alpha or epsilon amino 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 sites: the ⁇ -amino group at the N-terminus of the protein, the ⁇ -amino group on the side chain of the lysine residue, or the histidine residue Imidazolyl on the side chain. Since most recombinant proteins have a single alpha and many epsilon amino and imidazolyl groups, many positional isomers can be generated according to the chemical nature of the linking group.
  • the first generation of activated monomethoxy PEGs is 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 eg US5650234), which preferentially reacts with lysine residues to form carbamate linkages Group, but it is also known to react with histidine and tyrosine residues.
  • the linker for the histidine residue on IFN ⁇ has been shown to be a hydrolytically unstable imidazole carbamate linker (see, for example, US5985263).
  • the second-generation pegylation technique is to avoid unstable linkers and lack of selectivity in the residue reaction.
  • the use of PEG-aldehyde linkers can target a single site on the N-terminus of the polypeptide by reductive amination.
  • Different types of linkers and pH, PEGylated IL-10 can be used to derive various forms of PEGylated molecules (see, for example, US5252714, US5643575, US5919455, US5932462, US5985263, US7052686).
  • administering 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 , Humans, subjects, cells, tissues, organs or biological fluids.
  • administering may refer to, for example, treatment, pharmacokinetics, diagnosis, research, and experimental methods.
  • the treatment of cells includes the contact of reagents with cells and the contact of reagents with fluids, wherein the fluids are in contact with cells.
  • administering “administering,” and “treating” also mean in vitro and ex vivo treatment of, for example, cells by an agent, diagnosis, binding composition, or by another cell.
  • administering when applied to human, veterinary or research subjects refer to therapeutic treatment, prophylactic or preventative measures, research and diagnostic applications.
  • Treatment means administration to a subject of an internal or external therapeutic agent, such as a composition comprising any one of the IL-10 variants of the present disclosure and derivatives thereof or a composition comprising the variant or derivative, the subject
  • an internal or external therapeutic agent such as a composition comprising any one of the IL-10 variants of the present disclosure and derivatives thereof or a composition comprising the variant or derivative
  • the patient is diagnosed with, suspected of having, or susceptible to one or more disease symptoms, and the therapeutic agent is known to have a therapeutic effect on these symptoms.
  • the therapeutic agent is administered in an amount or amount effective to relieve one or more disease symptoms in the subject or population being treated, whether by inducing the regression of such symptoms or inhibiting the development of such symptoms to any clinically unmeasurable extent.
  • the amount of therapeutic agent effective to relieve the symptoms of any specific disease may vary according to various factors, such as the disease state, age and weight of the subject, and the effect of the drug in the subject ability. It is possible to assess whether the symptoms of the disease have been alleviated by any clinical testing methods commonly used by doctors or other health care professionals to assess the severity or progression of the symptoms.
  • the embodiments of the present disclosure may be ineffective in relieving the symptoms of the target disease that each patient has, but according to any statistical test methods known in the art such as Student test, chi-square test, and evidence Mann and Whitney’s U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test determined that they should alleviate the target disease symptoms in a statistically significant number of subjects.
  • Effective amount includes an amount sufficient to improve or prevent the symptoms or conditions of a 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 can vary depending on factors such as the condition to be treated, the general health of the subject, the route of administration and dosage, and the severity of side effects.
  • the effective amount may be the maximum dose or dosing regimen 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 (Improsulfan) and baifefen; aziridine, such as Benzodopa, carboquinone, meturedopa and uredopa; aziridine and methylmelamine, including hexamethylmelamine, tromethamine, triethylenephosphine Amide, triethylene thiophosphoramide and trimethyl melamine nitrogen mustards such as chiorambucil, naphthalene mustard, cholophosphamide, estramustine, ifosfamide, dichloromethane Amine, dichloromethylenediamine oxide hydrochloride, phenylalanine nitrogen mustard, neonitrogen mustard, pheny
  • anti-hormonal agents that act as regulators or inhibitors of tumors, such as anti-estrogens, including, for example, tamoxifen, ranoxifol, aromatase that inhibits 4(5)-imidazole, 4 -Hydroxy tamoxifen, trovaxifen, keoxifene, LY117018, onapristone, and toremifene citrate (fareston); and anti-androgens, such as flutamide Amines, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing.
  • interleukin-10 or "IL-10” used in this disclosure is composed of two identical non-covalently linked subunits that form a dimer protein.
  • interleukin-10 and IL-10 as used herein may refer to human or mouse IL-10 (Genbank Accession Nos. NP 000563; M37897; or US6217857), also known as It is “hIL-10” or "mIL-10”.
  • PEGylated IL-10 or “PEG-IL-10” is an IL-10 molecule (whether in monomer or dimer form) with one or more polyethylene glycol molecules, linked by polyethylene glycol The group is covalently linked to one or more amino acid residues of the IL-10 protein, so the connection is stable.
  • the terms “mono-PEGylated IL-10” and “mono-PEG-IL-10” refer to a polyethylene glycol molecule covalently linked to a single amino acid residue on the subunit of the IL-10 dimer via a linker (Whether IL-10 is in monomeric or dimeric form).
  • the average molecular weight of the PEG moiety is from about 5KD to about 50KD, or from about 5KD to about 40KD, or from about 10KD to about 30KD, or from about 10KD to about 20KD, or from about 10KD to about 15KD.
  • the entire disclosure of this disclosure introduces the PEGylation method of IL-10 described in US7052686 and the PEGylation structure and activity, function detection method, which can be used to PEGylate the IL-10 variant of the disclosure and obtain the corresponding PEGylated IL-10 variant.
  • Hyf-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.
  • the term "increased half-life” used in this disclosure means that synthetic agents are cleared at a slower rate than non-synthetic, endogenous agents or their recombinantly produced forms.
  • Example 1 Construction and expression of wild-type IL-10 and variants
  • the wild-type IL-10 nucleic acid sequence is shown in SEQ ID NO.1, an Nde I cleavage site was added at the 5'end, and a BamH I cleavage site was added at the 3'end.
  • 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 an expression vector for wild-type IL-10.
  • asparagine (N) at position 10 is replaced with glutamine (Q), and the codon AAC at positions 28-30 of its corresponding nucleotide sequence is changed to CAG.
  • the nucleic acid sequence after mutation is as follows:
  • IL-10-01 variant sequence The corresponding amino acid sequence is shown below, namely the IL-10-01 variant sequence:
  • methionine (M) at position 39 is replaced with threonine (T), and the codon ATG at positions 115-117 of its corresponding nucleotide sequence is changed to ACC.
  • the nucleic acid sequence after mutation is as follows:
  • 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:
  • asparagine (N) at position 10 is replaced with glutamine (Q), and the codon AAC at positions 28-30 of its corresponding nucleotide sequence is changed to CAG;
  • the 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:
  • methionine (M) at position 39 is replaced with threonine (T), and the codon ATG at positions 115-117 of its corresponding nucleotide sequence is changed to ACC; position 92 Asparagine (N) was replaced with glutamine (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:
  • asparagine (N) at position 10 is replaced with glutamine (Q), and the codon AAC at positions 28-30 of its corresponding nucleotide sequence is changed to CAG;
  • 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:
  • IL-10-06 variant sequence The corresponding amino acid sequence is shown below, that is, the IL-10-06 variant sequence:
  • the gene sequences of the above variants are synthesized and connected to the expression vector pET-9a to form a recombinant expression vector.
  • the above recombinant expression vector was transformed into BL21 (DE3) strain (Novagen, Cat. 69450-3) purchased from Novagen, to obtain recombinant bacteria of wild type IL-10 and variants, and the recombinant bacteria were coated on Kan-containing LB plates to be screened.
  • the inclusion body is denatured, renatured and purified to obtain the target protein.
  • Example 2 Stability study of wild-type IL-10 and variants
  • Wild-type IL-10 samples (buffer system is 10 mM Tris-HCl, pH 7.4), placed at 37 °C, stability study was carried out, peptide sampling was drawn by liquid-mass spectrometry to analyze the degradation.
  • Detection method take 0.2mL of sample and add 0.2mL of denaturing solution (8mol/L guanidine hydrochloride, 0.2mol/L Tris-HCl, pH7.5), add 5 ⁇ l 1mol/L DTT (dithiothreitol) and mix well, 25°C Water bath 1h. Then add 30 ⁇ l 0.5mol/L IAM (iodoacetamide) water bath at 25°C for 1h. The sample was replaced in the digestion buffer (50 mmol/L ammonium bicarbonate, 1 mol/L guanidine hydrochloride, pH 8.0) through the PD-10 column. Take 0.4mL of the sample after replacement buffer, add 40 ⁇ g of V8 protease, add water at 25°C for 18h, add hydrochloric acid to stop the reaction, and draw a peptide map by LC/MS.
  • denaturing solution 8mol/L guanidine hydrochloride, 0.2mol/L Tris-HCl, pH7.5
  • D represents the day.
  • Example 3 Activity determination of wild-type IL-10 and variants
  • the cell-dependent strain MC-9 (Master Cell-9) cell survival rate is different, in order to detect the biological activity of IL-10.
  • Basic culture medium DMEM (high glucose) + 10% FBS + 1% P/S (penicillin) + 0.05 mM beta ME;
  • MTT solution Weigh 0.1g of MTT, add sterile PBS (phosphate buffer, PH7.4) to dissolve and dilute to 20mL, filter and sterilize through 0.22 ⁇ m filter membrane;
  • sterile PBS phosphate buffer, PH7.4
  • Lysis solution 15% sodium dodecyl sulfate solution.
  • Preparation of reference substance solution take recombinant human interleukin-10 reference substance and dilute with basic culture solution (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. Leave 50 ⁇ L of solution in each well and discard more than the solution in the well. The above operations are performed under sterile conditions.
  • sample solution to be tested Take recombinant human IL-10 sample to be tested and dilute it with basal medium (containing 30 ng/mL of mIL-4) to contain 30 ng per 1 mL.
  • basal medium containing 30 ng/mL of mIL-4.
  • IL-10 activity measurement method MC-9 cells are cultured to a sufficient amount under complete culture medium at 37°C and 5% carbon dioxide; MC-9 cells are collected by centrifugation and washed 3 times with DMEM culture medium; resuspended in basic culture medium It is prepared into a cell suspension containing 5.0 ⁇ 10 5 cells per 1 mL; used at 37°C and 5% carbon dioxide.
  • the experimental instruments used are conventional, such as:
  • DMEM fetal calf serum
  • FBS 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 purchasedd from Promega
  • Pen-Strep purchased from Gibco
  • FITC mouse anti-human CD8, Alexa 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/ CD28T cell activator both purchased from Stemcell
  • Brefeldin A solution permeabilization buffer (10 ⁇ Permeabilization Buffer)
  • IC fixation buffer both purchased from Stemcell
  • protein concentration is 3-4mg/mL
  • the buffer system is 50mM PB/0.1M NaCl, pH 6.0-6.3;
  • mPEG-butyraldehyde aqueous solution with a concentration of 2mM to the IL-10 solution, the amount of mPEG butyraldehyde is twice the amount of IL-10, and stir evenly;
  • the reaction was terminated by adding a 1 M glycine aqueous solution, and the final concentration of glycine was 30 mM.
  • the aldehyde group of PEG-butyraldehyde forms a bond with the amino group of the N-terminal methionine of IL-10.
  • the packing for purification is Q Sepharose TM High Performance (GE lifescience);
  • Buffer used for purification Buffer A is 10mM Tris-HCl pH8.7, Buffer B is 10mM Tris-HCl/0.5M NaCl pH8.7.
  • Buffer B is pre-equilibrated with high salt, and Buffer A is re-equilibrated. After the UV, conductivity, and pH are stable, the UV will be zeroed, 150cm/h;
  • Loading loading the sample after filtration, 150cm/h, load 1-2mg/mL;
  • IL-10 can not only promote the proliferation of activated CD8+ T cells, but also promote the function of CD8+ T cells, such as the release of graninomycin.
  • the unmodified IL-10 mutant IL-10-06 can stimulate CD8+ T cells to secrete granulin
  • the EC 50 is 1.63ng/mL
  • PEGylated IL-10-06 can promote human dose-dependently. CD8+ T cells secrete granulin.
  • the EC 50 of 20KD-single PEG-IL-10-06 is 16.1ng/mL; the EC 50 of 20KD-dual PEG-IL-10-06 is 105ng/mL; the EC 50 of 12KD-single PEG-IL-10-06 EC 50 of 13.7ng / mL; 20KD- bis PEG-IL-10-06 of the EC 50 is 33.4ng / mL.
  • mice 6-8 weeks C57BL/6 mice, male (Shanghai Lingchang Biotechnology Co., Ltd.), the breeding environment of mice is SPF level.
  • the ambient temperature is controlled at 20-26°C, the humidity is 40-70%, 12 hours of darkness and 12 hours of light, and the mice can freely eat (standard feed) and drink water. Only animals that pass the health check will be used for this experiment.
  • IL-10 samples 60 ⁇ g IL-10 samples were administered subcutaneously. Blood was taken before administration and at 4, 8, 24, 48, 72, 96 and 120 hours after administration. Heparin sodium was added for anticoagulation and immediately stored in the -80 degree refrigerator. After all plasma collection was completed, the content of IL-10 in mouse plasma was detected by ELISA. The results are shown in Figure 3.
  • the unmodified IL-10-06 has a very short half-life in vivo. After 24 hours of administration, IL-10 in plasma has been lower than the detection limit, and PEGylation can significantly prolong the half-life of IL-10-06 in vivo.
  • the half-life of 12KD-mono-PEG-IL-10-06 in vivo is 7.9 hours
  • the half-life of 12KD-bis-PEG-IL-10-06 in vivo is 10.5 hours
  • the half-life of 20KD-single PEG-IL-10-06 single modification in vivo is 9.37 hours.
  • Example 8 In vivo efficacy of mouse colon cancer CT-26 cell subcutaneous allograft tumor model in BALB/c mice
  • mice Mouse colon cancer CT-26 cells (CT-26-C2, ATCC), the culture conditions are RPMI1640 medium plus 10% fetal bovine serum, 37°C 5% CO 2 incubator culture. When the cell saturation is 80%-90%, and the quantity reaches the requirement, the cells are collected and counted.
  • 0.1 mL (0.3 ⁇ 10 6 ) CT-26 cells were subcutaneously inoculated subcutaneously on the right back of each mouse (BALB/c mice, female, 6-8 weeks old, purchased from Shanghai Lingchang Biotechnology Co., Ltd.) .
  • the feeding environment of mice is SPF level.
  • mice can freely eat (standard feed) and drink water. Only animals that pass the health check will be used for this experiment.
  • group administration was started.
  • 200 ⁇ g of mouse CD20 antibody was administered to each mouse 24 hours before the formal administration.
  • test samples were administered subcutaneously twice a day at different concentrations. Observed for 19 days, the changes in tumor volume and body weight of mice were measured twice a week. The results are shown in Figures 4 and 5.
  • the results showed that the administration of PEGylated IL-10-06 after 19 days could inhibit the growth of colon cancer xenografts in CT26 mice.
  • the tumor volume of the 12KD double PEG-IL-10-06 0.1mpk administration group was 61.4% of the control group tumor volume (T/C), the 12KD double PEG-IL-10-06 0.3mpk, T after 19 days of administration
  • the /C value was 83.6%, and the tumor of one of the six mice completely resolved.
  • T/C value is 49.93% after 19 days of administration
  • T/C value is 18.72% after 19 days of administration, of which 6 Tumors of 3 of the mice completely resolved.
  • IL-10-06 without PEG modification tended to inhibit tumors about 10 days after administration, but after 19 days of administration, the T/C was 102.9%, and there was no antitumor activity.
  • mice in the control group and the IL-10-06 administration group continued to gain weight during the administration period (Figure 5), which may be due to the continued growth of the tumor.
  • the weight gain was weaker, which may be caused by the inhibition of tumor growth.
  • the administration group there was no significant weight loss 19 days after administration compared with before administration, suggesting that the administration group did not produce intolerable toxic and side effects.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biophysics (AREA)
  • Wood Science & Technology (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Physics & Mathematics (AREA)
  • Toxicology (AREA)
  • Plant Pathology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)
  • Medicinal Preparation (AREA)

Abstract

本公开涉及一种人白细胞介素10变体及其衍生物。具体而言，本公开涉及具有一个或多个氨基酸突变的人白细胞介素10及其PEG化衍生物，这些突变增加了蛋白的稳定性和/或提高了人白介素10的细胞增殖活性，为蛋白的成药性提供了良好的基础。

Description

一种人白细胞介素10变体及其衍生物
本申请要求2018年11月26日提交的中国专利申请《一种人白细胞介素10变体或其衍生物》(申请号CN201811418270.9)的优先权，其内容通过引用并入此处。
技术领域
本公开涉及具有一个或多个氨基酸突变的人白细胞介素-10变体或其衍生物，所述突变使得人白细胞介素-10具有增强的稳定性和/或活性。
背景技术
人白细胞介素-10(interleukin-10，IL-10)，其基因位于1号染色体(q31-32)，共5.1kb长。IL-10的基因序列包含5个外显子。IL-10由178个氨基酸残基组成，构成“V”字型的同型二聚体，分子量约35kD。
IL-10属于IL-10家族，家族成员包括IL-10、IL-19、IL-20、IL-22/IL-TIF、IL-24/MDA-7、IL-26等。
Fiorentino最初在1989年发现，IL-10是由鼠辅助性T细胞Th2亚型细胞分泌的一种细胞因子，能抑制Th1亚型细胞分泌的肿瘤坏死因子γ(TNF-γ)等细胞因子，所以最初被命名为细胞因子合成抑制因子(cytokine synthesis inhibitory factor，CSIF)。IL-10增强抗炎性因子的释放，如IL-1受体拮抗剂和溶解性TNF-α受体；IL-10抑制1型辅助T细胞产生的几种细胞因子(例如γ-干扰素、IL-2和TNF-α)的合成，抑制细胞介导的免疫应答调节剂，并抑制抗原呈递细胞依赖性T细胞应答，抑制单核细胞/巨噬细胞产生的其它细胞因子，例如IL-1、IL-6、IL-8、粒细胞-巨噬细胞集落刺激因子(GM-CSF)、粒细胞集落刺激因子(G-CSF)和TNF-α；IL-10抑制IL-12的合成，从而阻碍Th1免疫反应。
IL-10通常在T细胞、巨噬细胞、单核细胞、树突细胞、肥大细胞、B细胞、嗜酸性粒细胞、角质形成细胞、上皮细胞以及各种肿瘤中表达，其参与多种细胞(如免疫细胞、炎症细胞、肿瘤细胞)的生物调节。
由于IL-10的多效活性，已对其进行多种临床应用的研究，包括自身免疫性疾病、严重感染性疾病、肿瘤及移植免疫等。
目前已有的IL-10包括单-或双-聚乙二醇化的IL-10，所述IL-10可以是PEG化的(WO2002/026265；WO2010/077853)。PEG化的IL-10可用作药物治疗肿瘤或癌症，所述肿瘤为上皮细胞癌、内皮细胞癌、鳞状细胞癌、乳头状瘤病毒引起的癌、腺癌、癌肿、黑素瘤、肉瘤或畸胎癌，或用于治疗肺部肿瘤或转移性肺癌，或淋巴瘤(WO2008/054585；EP2468293B；EP2821078B)；以及可通过给予患者有效量的PEG化的IL-10、叶酸类似物、5-FU和铂类药物治疗肿瘤(US9833514B)。 IL-10还可以用于治疗或预防心血管病症、血栓性病症或炎性病症，或高血脂(WO2015/031316；US20160193300A)。IL-10还可以与免疫检查点抑制剂(如PD-1)或IL-15联合治疗或预防癌症或肿瘤(WO2015/070060；WO2017/035232)。IL-10还可治疗肝病，如非酒精性脂肪肝、丙肝(WO2015/187295；WO2015/108785)。IL-10可以与IL-7联合治疗病症(WO2016/196211)，以及与CAR-T细胞一起调节T细胞介导的免疫应答(WO2016/191587)。
已有的IL-10突变研究显示，S51、E81、K88、A120等位置引入半胱氨酸，可获得具有免疫抑制活性而没有免疫刺激活性的IL-10(WO2004/044006)。I87A位置的突变使得IL-10与其受体的结合亲和力降低(WO2015/117930)。F129S位置的突变能保留IL-10的抗炎特性，但不保留血小板细胞调节和细胞增殖活性(WO2006/130580)。Q62R、K58E、E160Q、D162H的突变使得IL-10与受体结合和功能活性降低，同时保持免疫原性(WO2012/135177)等。Ding Y等人指明IL-10的免疫刺激活性是由第87位的单一氨基酸异亮氨酸决定的(J.Exp.Med.191(2),213-223，2000)。WO2012146628A1公开了一种抗体和IL-10的融合蛋白，其中将第87位的氨基酸替代为I87A，发现经改造的融合蛋白展现降低的对IL-10受体的亲和力，但是改善了靶向效率、降低了由IL-10免疫刺激活性所引起的副作用。然而，现有技术中仍然缺少具有更高稳定性、更高活性的IL-10变体。提供这样的IL-10变体，是本领域中亟需解决的问题。
发明内容
本公开涉及人白介素10(IL-10)序列具有一个或多个氨基酸突变的变体或其衍生物。
第一方面，本公开提供IL-10变体，其是在野生型人IL-10序列(SEQ ID NO:2)的第10位天冬酰胺(N)或/和第39位甲硫氨酸(M)或/和第92位天冬酰胺(N)位点上，将野生型氨基酸残基突变为其它氨基酸，其中所述位点按人野生型IL-10的成熟蛋白进行编号。所述变体相较于野生型人IL-10具有提高的稳定性和/或活性。
一些实施方案中，所述IL-10变体或其衍生物包含第10位突变为Gln(Q)和/或第39位突变为Thr(T)和/或第92位突变为Gln(Q)。
一些具体实施方案中，IL-10变体包含第10位Asn突变为Gln(N10Q)和/或第39位是Met突变为Thr(M39T)和/或第92位是Asn突变为Gln(N92Q)；或包含选自以下的组合：N10Q和M39T、M39T和N92Q、N10Q和M39T和N92Q。
一些具体实施方案中，IL-10变体或其衍生物可以包含选自以下序列所示的多肽：SEQ ID NO.4、SEQ ID NO.6、SEQ ID NO.8、SEQ ID NO.10、SEQ ID NO.12、SEQ ID NO.14。所述的氨基酸序列如下表所示。
表1.人白介素10野生型及变体序列
Figure PCTCN2019120562-appb-000001
Figure PCTCN2019120562-appb-000002
本公开中，IL-10变体的衍生物包括涉及本公开IL-10变体的全长、部分蛋白或在所述变体的基础上进一步突变获得的突变蛋白、功能性衍生物、功能性片段、生物活性肽、融合蛋白、同种型或其盐。
一些具体实施方案中，IL-10变体衍生物包括IL-10变体的融合蛋白、IL-10变体的截断体、及包含所述截断体的融合蛋白、IL-10变体的单体或二聚物或三聚物或多聚物、经修饰的IL-10变体(如聚乙二醇化(PEG化)、糖基化、白蛋白缀合或融合、羟乙基化、与抗体或其抗原结合片段融合)、以及IL-10变体在各物种中的同源物。
一些实施方案中，与IL-10变体融合的抗体或其抗原结合片段是鼠源抗体、人抗体、人源化抗体、嵌合抗体或骆驼抗体或其抗原结合片段。一些具体的实施方案中，抗原结合片段是Fab片段、Fab’片段、F(ab’)2片段、单链Fv(sFv)、VHH或VH/VL结构域。
一些实施方案中，IL-10变体或衍生物是IL-10的同二聚体，每一个单体包含178个氨基酸，其中的前18个氨基酸为信号肽。一些具体实施方案中，包括缺少信号肽的成熟IL-10(如US6217857中的)的变体，或成熟的PEG化IL-10的变体。所述变体可展示与野生型人IL-10相当或比其优越的活性。所述变体可展示比与野生型人IL-10相当或比其高的稳定性。因而，技术人员能够理解，根据本公开的IL-10变体或其衍生物可以不含信号肽，而(统计学意义上)不改变期望的活性，作为非限制性示例，在选自以下序列的基础上去除信号肽：SEQ ID NO.4、SEQ ID NO.6、SEQ ID NO.8、SEQ ID NO.10、SEQ ID NO.12、SEQ ID NO.14。
一些实施方案中，IL-10变体或其衍生物可以是经修饰的，以调节一个或多个性质(例如，药代动力学参数、功效等)，包括其可以是单体或二聚物或三聚物的形式，和/或是PEG化的，和/或糖基化的，和/或白蛋白(例如，人血清白蛋白(HSA))缀合和融合，和/或羟乙基化(hesylation)的。所述IL-10的修饰不会导致对免疫原性的治疗相关的不利影响，并且在部分实施方案中，经修饰的IL-10可以比未修饰的IL-10的免疫原性低。
在一些实施方案中，IL-10变体或衍生物是PEG化的(表示为PEG-IL-10)，例如是单或双PEG化的IL-10变体或衍生物。PEG-IL-10变体或衍生物包括SC-PEG连接基。在另一些实施方案中，PEG-IL-10变体或衍生物包括甲氧基-PEG-醛(mPEG-ALD)连接基。在某些实施方案中，PEG部分的平均分子量在约5KD至约50KD，具体地5、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、30、35、40、45、50KD；或约5KD至约40KD，或约10KD至约 30KD，或约10KD至约20KD之间，或约10KD至约15KD之间。某些具体实施方案中，mPEG-ALD连接基包括具有选自下列平均分子量的PEG分子：5KDa、12KDa或20KDa。在某些实施方案中，mPEG-ALD的醛基可以是乙醛、丙醛或丁醛等。
具有10位和/或39位和/或92位突变的IL-10变体相对于野生型IL-10具有提高的稳定性和/或活性。这些突变减少或防止了IL-10的脱氨和氧化，增加了蛋白的稳定性同时细胞增殖活性不降低或提高了细胞增殖活性，为蛋白成药提供了良好的基础。一些具体方案中，所述变体包含N10Q/M39T或M39T/N92Q或N10Q/M39T/N92Q的组合。
第二方面，提供分离的核酸分子，其编码本公开IL-10变体或其衍生物。所述核酸分子可以包含SEQ ID NO.3、SEQ ID NO.5、SEQ ID NO.7、SEQ ID NO.9、SEQ ID NO.11、SEQ ID NO.13所示的多核苷酸。
一些实施方案中，提供含有上述核酸分子的表达载体。
一些实施方案中，提供表达上述载体的宿主细胞。所述宿主细胞可以是原核或真核细胞。一些具体实施方案中，宿主细胞为细菌或酵母菌或哺乳动物细胞(例如CHO、COS、BHK21、NIH3T3、HeLa、C2C12、HEK)，例如，为巴斯德毕赤酵母或酿酒酵母。宿主细胞不包含能够发育成植株的细胞，不涉及人受精卵、人生殖细胞，也不涉及能够发育成动物的细胞。
第三方面，提供一种药物组合物，其包含本公开IL-10变体或其衍生物，其含有：
-根据本公开的IL-10变体或其衍生物，和
-任选，可药用的赋形剂、稀释剂或载体。
在一些实施方案中，所述药物组合物的单位剂量含有1mg至1000mg根据本公开的IL-10变体或其衍生物。在一些实施方案中，所述药物组合物中的IL-10变体或其衍生物的浓度为1mg/L至1000mg/L。在一些实施方案中，所述药物组合物含有缓冲剂，其含量可以为1mM至1000mM。在一些实施方案中，所述药物组合物可以为冻干制剂或可注射溶液。
第四方面，提供本公开的IL-10变体或其衍生物或其药物组合物用于制备治疗增生性疾病、免疫性疾病、心血管病症、血栓性病症、高血脂症、炎性病症、肝病、调节T细胞介导的免疫应答的药物中的用途。
一些实施方案中，增生性疾病可以是肿瘤或癌，例如上皮细胞癌、内皮细胞癌、鳞状细胞癌、乳头状瘤病毒引起的癌、腺癌、癌肿、黑素瘤、肉瘤或畸胎癌、肺部肿瘤或转移性肺癌、淋巴瘤、结肠癌、卵巢癌、乳腺癌、恶性胶质瘤和白血病。
一些实施方案中，免疫性疾病是糖尿病，例如是胰岛素依赖型糖尿病。
一些实施方案中，心血管病症为动脉粥样硬化、高血压、心肌病。
一些实施方案中，血栓性病症是可以引起中风或心肌梗塞的病症。
一些实施方案中，炎性病症可以为脉管炎。
一些实施方案中，肝病可以为非酒精性脂肪肝或丙肝。
一些实施方案中，肿瘤或癌是子宫癌、宫颈癌、乳房癌、前列腺癌、睾丸癌、阴茎癌、胃肠道癌，例如食道癌、口咽癌、胃癌、小或大肠癌、结肠癌或直肠癌、肾癌、肾细胞癌、膀胱癌、骨骼癌、骨髓癌、皮肤癌、脑或颈癌、皮肤癌、肝癌、胆囊癌、心脏癌、肺癌、胰腺癌、唾液腺癌、肾上腺癌、甲状腺癌、脑癌(例如神经胶质瘤、神经节、中枢神经系统(CNS)和周围神经系统(PNS))和免疫系统(例如脾或胸腺)。肿瘤或癌还可以是免疫原性肿瘤、非免疫原性肿瘤、休眠肿瘤、病毒引起的癌例如上皮细胞癌、内皮细胞癌、鳞状细胞癌、乳头状瘤病毒、腺癌、淋巴瘤、癌肿、黑素瘤、白血病、骨髓瘤、肉瘤、畸胎癌、化学引起的癌、转移病变和血管生成的方法。
第五方面，提供一种抑制或降低肿瘤或癌的生长和/或治疗患有癌或肿瘤的患者的方法，所述方法包括使受试者与有效量的本公开的IL-10变体或衍生物接触。所述IL-10变体或衍生物可以抑制肿瘤或癌的生长，或所述IL-10变体或衍生物可以降低肿瘤或癌的大小。例如，通过调节调控T细胞(Treg)和/或CD8+T细胞的活性，预计本公开的IL-10变体或衍生物还可以降低对肿瘤细胞或癌细胞抗原的耐受性。
一些实施方案中，当与非PEG化的IL-10变体或衍生物相比时，PEG化的IL-10变体或衍生物可以提高CD8+T细胞进入靶标(例如赘生物、病灶)的渗透性。在另一些实施方案中，PEG化的IL-10变体或衍生物可以提高至少一种炎性细胞因子的表达，其可以选自IFNγ、IL-4、IL-6、IL-10和RANK-配体(RANK-L)。在某些实施方案中，PEG化的IL-10变体或衍生物是与至少一种化学治疗剂共同给予的。化学治疗剂可以在给予PEG化的IL-10变体或衍生物之前、与其同时、或在其之后给予。
一些实施方案中，被治疗的患者或受试者是人。
一些实施方案中，每日至少2次、每日至少1次、每48小时至少1次、每72小时至少一次、每周至少一次、每2周至少一次、每个月至少一次、每2个月至少一次或每3个月至少一次向受试者施用IL-10变体或衍生物的方法。可通过任何有效途径施用IL-10变体或衍生物，例如，通过肠胃外注射(包括皮下注射)施用IL-10。具体实施方案涉及包含药学上可接受的量的IL-10变体或衍生物(例如，治疗有效量)连同一种或多种药学上可接受的稀释剂、载体或赋形剂(例如，等渗注射溶液)的药物组合物。所述药物组合物通常为适于人施用的药物组合物。此外，在一些实施方案中，所述药物组合物包含至少一种另外的预防剂或治疗剂。一些实施方式含有：上述药物组合物之一、任选地一种或多种另外的组分、以及无菌容器。
第六方面，提供了一种制备IL-10变体或衍生物的方法，包括：
-向野生型人IL-10中引入选自以下的突变：N10Q和M39T、M39T和N92Q、N10Q和M39T和N92Q；或
-使前述核酸分子进行表达；或
-使前述表达载体进行表达；或
-使前述宿主细胞进行表达。
第七方面，提供PEG化IL-10变体或其衍生物结构及其制备方法。
一些实施方案中，提供单PEG化IL-10变体或其衍生物，其包含：
通过接头，共价连接至IL-10变体或其衍生物的一个PEG分子。PEG可与IL-10变体或其衍生物的赖氨酸残基或N端氨基酸残基连接。一些具体实施方案中，所述IL-10变体或其衍生物为同二聚体，20KD或12KD的单PEG连接至同二聚体的单体之一。
另一些实施方案中，提供双PEG化IL-10变体或其衍生物，其包含：
通过接头，共价连接至IL-10变体或其衍生物的两个PEG分子。PEG可与IL-10变体或其衍生物的赖氨酸残基或N端氨基酸残基连接。一些具体实施方案中，所述IL-10变体或其衍生物为同二聚体，20KD或12KD的单PEG分别连接至同二聚体的两个单体。
一些实施方案中，PEG-IL-10变体或衍生物的结构如下：
[XO-(CH 2CH 2O) n] b-L-NH-IL-10变体或衍生物，
其中，X是H或C 1-4烷基，n是20至2300，b是1至9，L是α-π烷基连接体(也作接头)，L共价连接在IL-10变体或其衍生物的氨基末端，当b大于1时，n的总和不超过2300。一些具体方案中，L是“-CH 2CH 2CH 2-”。
一些具体的实施方案中，PEG结构是CH 3O-(CH 2CH 2O)n-CH 2CH 2CH 2-CHO。
在另一些具体的实施方案中，PEG-丁醛的醛基与IL-10变体或其衍生物的N端甲硫氨酸的氨基成键，结构如下：
CH 3O-(CH 2CH 2O)n-CH 2CH 2CH 2-CH 2-NH-IL-10变体或衍生物。
一些实施方案中，提供制备单、双PEG-IL-10的方法，包括步骤：
在还原剂存在下使IL-10变体或衍生物与活化的PEG-醛接头接触，形成单或双PEG-IL-10，其中单个PEG-醛接头与IL-10变体或衍生物的一个氨基酸残基共价连接。
附图说明
图1：IL-10变体及20KD-、12KD-的单、双PEG化IL-10变体对人原代CD8+T细胞的增殖实验结果。其中，20KD单PEG化是指缀合有单PEG的IL-10二聚体，所述PEG的平均分子量为20KD，缀合至二聚体中的单体之一；20KD双PEG化是指缀合有两个PEG的IL-10二聚体，所述PEG的平均分子量为20KD，两个PEG 分别缀和至二聚体中的两个单体；12KD单PEG化是指缀合有单PEG的IL-10二聚体，所述PEG的平均分子量为12KD，缀合至二聚体中的单体之一；12KD双PEG化是指缀合有两个PEG的IL-10二聚体，所述PEG的平均分子量为12KD，两个PEG分别缀和至二聚体中的两个单体。
图2：IL-10变体及20KD-、12KD-的单、双PEG化IL-10变体对人原代CD8+T细胞的激活实验结果。
图3：小鼠皮下给药后，IL-10变体的血浆浓度结果图。
图4：IL-10变体及其20KD、12KD的单、双PEG化衍生物抑制CT26小鼠结肠癌移植瘤生长的结果图。
图5：IL-10变体及其20KD、12KD的单、双PEG化衍生物对CT26小鼠体重影响的结果图。
具体实施方式
为了更容易理解本公开，以下具体定义了某些技术和科学术语。除显而易见在本文件中的它处另有明确定义，否则本文使用的所有其它技术和科学术语都具有本公开所属领域的一般技术人员通常理解的含义。
本公开所用氨基酸三字母代码和单字母代码如J.Biol.Chem，243，p3558(1968)中所述。
术语
“衍生物”旨在被广义地解释，包括任意IL-10相关的产品。包括但不限于人和非人的IL-10同系物、片段或截短体、融合蛋白(如与信号肽融合或其他活性、非活性成份融合，活性成份例如抗体或其抗原结合片段)、修饰形式(如PEG化、糖基化、白蛋白缀合/融合、羟乙基化等)、和保守修饰的蛋白等。
“抗体”指免疫球蛋白，是由两条相同的重链和两条相同的轻链通过链间二硫键连接而成的四肽链结构。免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同，故其抗原性也不同。据此，可将免疫球蛋白分为五类，或称为免疫球蛋白的同种型，即IgM、IgD、IgG、IgA和IgE，其相应的重链分别为μ链、δ链、γ链、α链和ε链。同一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类，如IgG可分为IgG1、IgG2、IgG3、IgG4。轻链通过恒定区的不同分为κ链或λ链。五类Ig中第每类Ig都可以有κ链或λ链。抗体包括鼠源抗体、人抗体、人源化抗体、嵌合抗体、骆驼抗体。
“抗原结合片段”，指具有抗原结合活性的Fab片段，Fab’片段，F(ab’)2片段，单链Fv(即sFv)，纳米抗体(即VHH)，VH/VL结构域。Fv片段含有抗体重链可变区和轻链可变区，但没有恒定区，并具有全部抗原结合位点的最小抗体片段。一般地，Fv抗体还包含在VH和VL结构域之间的多肽接头，且能够形成抗原结合所需的结构。也可以用不同的连接物将两个抗体可变区连接成一条多肽 链，称为单链抗体(single chain antibody)或单链Fv(sFv)。
“保守修饰”适用于氨基酸和核苷酸序列。对于特定的核苷酸序列，保守改性是指编码相同或基本相同的氨基酸序列的那些核酸，或在核苷酸不编码氨基酸序列的情况下，是指基本上相同的核苷酸序列。对于氨基酸序列，“保守修饰”是指具有类似特征(例如电荷、侧链大小、疏水性/亲水性、主链构象和刚性等)的其它氨基酸置换蛋白中的氨基酸，使得可频繁进行改变而不改变蛋白的生物学活性。本领域技术人员知晓，一般而言，多肽的非必需区域中的单个氨基酸置换基本上不改变生物学活性(参见例如Watson等(1987)Molecμlar Biology of the Gene，The Benjamin/Cummings Pub.Co.，第224页，(第4版))。
“PEG化”是指至少一个PEG分子与另一个分子(例如治疗性蛋白)连接。例如，批准Adagen(腺苷脱氨酶的PEG化制剂)用于治疗严重联合免疫缺陷病。已经显示，聚乙二醇的连接可以防止蛋白水解作用(参见，例如，Sada等人，(1991)J.Fermentation Bioengineering 71：137-139)。
在最常见形式中，PEG是在一端与羟基连接的直链或支链聚醚，并且具有下列常规结构：HO-(CH 2CH 2O) n-CH 2CH 2-OH。为了使PEG与分子(多肽、多糖、多核苷酸和小的有机分子)偶联，可以通过制备在一个或两个末端具有官能团的PEG的衍生物来活化PEG。蛋白的PEG缀合的常见途径是用官能团活化PEG，该官能团适合与赖氨酸和N-末端氨基酸基团的反应。尤其是，参与缀合的常见反应基团是赖氨酸的α或ε氨基。聚乙二醇化连接基与蛋白的反应可导致PEG部分主要在下列位点处的连接：蛋白的N-末端的α氨基、赖氨酸残基侧链上的ε氨基、或组氨酸残基侧链上的咪唑基。由于大部分重组蛋白质具有单个α和许多ε氨基和咪唑基，可以根据连接基团的化学性质，产生许多位置异构体。
第一代活化单甲氧基PEGs(mPEGs)是琥珀酰亚胺基碳酸酯PEG(SC-PEG；参见例如，Zalipsky等人，(1992)Biotehnol.Appl.Biochem 15：100-114；和Miron和Wilcheck，(1993)Bioconjug.Chem.4：568-569)和苯并三唑碳酸酯PEG(BTC-PEG；参见例如US 5650234)，其优先与赖氨酸残基反应，形成氨基甲酸酯连接基，但还已知其与组氨酸和酪氨酸残基反应。IFNα上的组氨酸残基的连接基已经被证明是一种水解不稳定的咪唑氨基甲酸酯连接基(参见例如US5985263)。
第二代聚乙二醇化技术是避免不稳定的连接基以及在残基反应中缺乏选择性，使用PEG-醛连接基可以通过还原胺化来靶向多肽的N-末端上的单个位点。可以使用不同类型的连接基和pH值，PEG化IL-10，以得出各种形式的PEG化的分子(参见例如US5252714，US5643575，US 5919455，US5932462，US5985263，US7052686)。
“施用”、“给予”和“处理”当应用于动物、人、实验受试者、细胞、组织、器官或生物流体时，是指外源性药物、治疗剂、诊断剂或组合物与动物、人、受试者、细胞、组织、器官或生物流体的接触。“施用”、“给予”和“处理”可以指例如治 疗、药物代谢动力学、诊断、研究和实验方法。细胞的处理包括试剂与细胞的接触，以及试剂与流体的接触，其中所述流体与细胞接触。“施用”、“给予”和“处理”还意指通过试剂、诊断、结合组合物或通过另一种细胞体外和离体处理例如细胞。“施用”、“处理”当应用于人、兽医学或研究受试者时，是指治疗处理、预防或预防性措施，研究和诊断应用。
“治疗”意指给予受试者内用或外用治疗剂，诸如包含本公开的任一种IL-10变体及其衍生物或包含所述变体或衍生物的组合物，所述受试者确诊患有、疑似患有、易感于一种或多种疾病症状，而已知所述治疗剂对这些症状具有治疗作用。通常，在受治疗受试者或群体中以有效缓解一种或多种疾病症状的量给予治疗剂，无论是通过诱导这类症状退化还是抑制这类症状发展到任何临床不可测量的程度。
有效缓解任何具体疾病症状的治疗剂的量(也称作“治疗有效量”)可根据多种因素变化，例如受试者的疾病状态、年龄和体重，以及药物在受试者产生需要疗效的能力。通过医生或其它专业卫生保健人士通常用于评价该症状的严重性或进展状况的任何临床检测方法，可评价疾病症状是否已被减轻。尽本公开的实施方案(例如治疗方法或制品)在缓解每个患都有的目标疾病症状方面可能无效，但是根据本领域已知的任何统计学检验方法如Student t检验、卡方检验、依据Mann和Whitney的U检验、Kruskal-Wallis检验(H检验)、Jonckheere-Terpstra检验和Wilcoxon检验确定，其在统计学显著数目的受试者中应当减轻目标疾病症状。
“有效量”包含足以改善或预防医学病症的症状或病症的量。有效量还意指足以允许或促进诊断的量。用于特定受试者或兽医学受试者的有效量可依据以下因素而变化：如待治疗的病症、受试者的总体健康情况、给药的方法途径和剂量以及副作用严重性。有效量可以是避免显著副作用或毒性作用的最大剂量或给药方案。
“化学治疗剂”是用于治疗赘生性疾病的化合物。化学治疗剂的例子包括烷基化剂，例如硫替派和环磷酰胺(CYTOXAN)；烷基磺酸盐，例如白消安，英丙舒凡(Improsulfan)和保释芬；氮丙啶，例如苯佐替哌(benzodopa)，卡波醌，美妥替派(meturedopa)和乌瑞替派(uredopa)；氮丙啶和甲基蜜胺，包括六甲蜜胺，曲他胺，三亚乙基磷酰胺，三亚乙基硫代磷酰胺和三甲基三聚氰胺氮芥例如瘤可宁(chiorambucil)，萘氮芥，胆磷酰胺(cholophosphamide)，雌莫司汀，异环磷酰胺，二氯甲二乙胺，二氯甲二乙胺氧化物盐酸盐，苯丙氨酸氮芥，新氮芥，苯乙酸氮芥胆甾醇酯，松龙苯芥，氯乙环磷酰胺，尿嘧啶氮芥；亚硝基脲，例如卡莫司汀，吡葡亚硝脲，福莫司汀，环己亚硝脲，嘧啶亚硝脲，雷莫司汀(Ranimustine)；抗生素，例如阿克拉霉素，纳霉素，氨茴霉素(authramycin)，偶氮丝氨酸，博来霉素，放线菌素C，刺孢霉素，去甲柔红霉素，洋红霉素，嗜癌霉素，色霉素，放线菌素，柔红霉素，地托比星，6-重氮基-5-氧代-L-正亮氨酸，多柔比星，表柔比星，依索比星，伊达比星，麻西罗霉素，丝裂霉素，霉酚酸，诺加霉素，橄榄霉素， 硫酸培洛霉素，紫菜霉素(potfiromycin)，嘌呤霉素，三铁阿霉素，罗多比星，链黑菌素，链脲霉素，杀结核菌素，乌苯美司，新制癌菌素，佐柔比星；抗代谢物例如氨甲喋呤和5-氟尿嘧啶(5-FU)；叶酸类似物，例如二甲叶酸，氨甲喋呤，蝶罗呤，三甲曲沙；嘌呤类似物，例如氟达拉滨，6-巯基嘌呤，硫咪嘌呤，硫鸟嘌呤；嘧啶类似物，例如安西他滨，阿扎胞苷，6-氮尿苷，卡莫氟，阿糖胞苷，二脱氧尿苷，去氧氟尿苷，依诺他滨，氮尿苷，5-FU；雄激素，例如卡普睾酮，屈他雄酮丙酸盐，环硫雄醇，美雄烷，睾内酯；抗肾上腺药，例如氨鲁米特，米托坦，曲洛司坦；叶酸补充剂，例如亚叶酸；醋葡醛内酯；醛磷酰胺糖苷；氨基乙酰丙酸；安吖啶；bestrabucil；比生群(bisantrene)；依达曲沙；秋水仙胺；地吖醌；依氟鸟氨酸(elfornithine)；依利醋铵；依托格鲁；硝酸镓；羟基脲；蘑菇多糖；氯尼达明；丙脒腙；米托蒽醌；莫哌达醇；二胺硝吖啶(nitraerine)；喷司他丁；蛋氨氮芥；吡柔比星；鬼臼酸；2-乙基异烟酰肼；普鲁苄肼；PSK.；丙亚胺；西佐喃；锗螺胺；细交链孢菌酮酸；三乙撑亚胺苯醌；2，2′，2″-三氯三乙胺；尿烷；去乙酰长春酰胺；达卡巴嗪；甘露醇氮芥；二溴甘露醇；二溴卫矛醇；双溴丙基哌嗪；gacytosine；阿拉伯糖苷(″Ara-C″)；环磷酰胺；硫替派；紫杉类药物，例如太平洋紫杉醇(TAXOLBristol-Myers Squibb Oncology，Princeton，N.J.)和多西他赛(doxetaxel)(Taxotere，Rhone-Poulenc Rorer，Antony，France)；苯丁酸氮芥；吉西他滨；6-硫代鸟嘌呤；巯基嘌呤；氨甲喋呤；铂类似物，例如顺铂和卡铂；长春花碱；铂；依托泊苷(VP-16)；异环磷酰胺；丝裂霉素C；米托蒽醌；长春花新碱；长春瑞宾；温诺平；诺安托；表鬼臼毒噻吩糖苷；柔毛霉素；氨基蝶呤；希罗达(xeloda)；依班膦酸盐；CPT11；局部异构酶抑制剂RFS2000；二氟甲基鸟氨酸(DMFO)；视黄酸；埃斯培拉霉素(esperamicins)；卡培他滨；和上述任一项的可药用盐、酸或衍生物。还包括在该定义中的是对肿瘤起到调节或抑制激素作用的抗激素药剂，例如抗雌激素，包括例如它莫西芬，雷诺昔酚，抑制4(5)-咪唑的芳香酶，4-羟基它莫西芬，曲沃昔芬，雷洛西芬(keoxifene)，LY117018，奥那司酮(onapristone)，和枸橼酸托瑞米芬(fareston)；和抗雄激素，例如氟他胺，尼鲁米特，比卡鲁胺，亮丙瑞林，和戈舍瑞林；和上述任一项的可药用盐、酸或衍生物。
不论是否与聚乙二醇缀合或非缀合形式，本公开中使用的“白细胞介素-10”或“IL-10”是包含两个相同的非共价连接形成二体的亚单元的蛋白。除非另有陈述，本文中使用的“白细胞介素-10”和“IL-10”可以指的是人或小鼠IL-10(Genbank Accession Nos.NP 000563；M37897；或US6217857)，还称其为“hIL-10”或“mIL-10”。
“PEG化的IL-10”或“PEG-IL-10”是具有一个或多个聚乙二醇分子的IL-10分子(无论是单体还是二聚体形式)，聚乙二醇通过连接基与一个或一个以上IL-10蛋白的氨基酸残基共价连接，因此该连接是稳定的。
术语“单PEG化的IL-10”和“单-PEG-IL-10”是指一个聚乙二醇分子通过连接基 与IL-10二聚体的亚单元上的单个氨基酸残基共价连接(而无论IL-10是单体还是二聚体形式)。PEG部分的平均分子量在约5KD至约50KD，或约5KD至约40KD，或约10KD至约30KD，或约10KD至约20KD之间，或约10KD至约15KD之间。对PEG与IL-10连接的方法或位点不做限制性要求，然而技术人员理解PEG与IL-10的连接不应改变(或仅仅最低限度地改变)IL-10变体或衍生物的活性。
本公开全文引入US7052686中所描述的IL-10的PEG化方法和PEG化结构和活性、功能检测方法，可用于PEG化本公开的IL-10变体并获得相应PEG化IL-10变体。
“半衰期”或“血清半衰期”是指消除半衰期，即待测物的血清浓度达到其初始或最大值的一半所需要的时间。对于合成药剂，本公开中使用的术语“半衰期提高”是指合成药剂比非合成的、内源性的药剂或其重组产生的形式，以更慢的速度被清除。
实施例
以下结合实施例进一步描述本公开，但这些实施例并非限制本公开的范围。
实施例1:野生型IL-10及变体的构建和表达
1.基因合成及重组表达载体构建
野生型IL-10核酸序列如SEQ ID NO.1所示，在5’端加了一个Nde I酶切位点，在3’端添加了一个BamH I酶切位点。
Figure PCTCN2019120562-appb-000003
(斜体部分分别是NdeI和BamHI酶切位点)
对应的蛋白序列如下所示：
Figure PCTCN2019120562-appb-000004
所使用的表达载体是购自Novagen公司的大肠杆菌表达载体pET-9a(Novagen，Cat.69431-3)。IL-10核酸序列合成后，构建重组表达载体，得到野 生型IL-10的表达载体。
2.在野生型IL-10氨基酸序列中引入本公开中所限定的氨基酸突变
在一种变体中，10位的天冬酰胺(N)置换成谷氨酰胺(Q)，将其对应的核苷酸序列28-30位处的密码子AAC变为CAG。突变后的核酸序列如下：
Figure PCTCN2019120562-appb-000005
其对应的氨基酸序列如下所示，即IL-10-01变体序列：
Figure PCTCN2019120562-appb-000006
在一种变体中，将39位的甲硫氨酸(M)置换成苏氨酸(T)，将其对应的核苷酸序列115-117位处的密码子ATG变为ACC。突变后的核酸序列如下所示：
Figure PCTCN2019120562-appb-000007
其对应的氨基酸序列如下所示，即IL-10-02变体序列：
Figure PCTCN2019120562-appb-000008
在一种变体中，将92位的天冬酰胺(N)置换成谷氨酰胺(Q)；将其对应的核苷酸序列274-276位处的密码子AAC变为CAA。突变后的核酸序列如下所示：
Figure PCTCN2019120562-appb-000009
Figure PCTCN2019120562-appb-000010
其对应的氨基酸序列如下所示，即IL-10-03变体序列：
Figure PCTCN2019120562-appb-000011
在一种变体中，10位的天冬酰胺(N)置换成谷氨酰胺(Q)，将其对应的核苷酸序列28-30位处的密码子AAC变为CAG；39位的甲硫氨酸(M)置换成苏氨酸(T)，将其对应的核苷酸序列115-117位处的密码子ATG变为ACC。突变后的核酸序列如下所示：
Figure PCTCN2019120562-appb-000012
其对应的氨基酸序列如下所示，即IL-10-04变体序列：
Figure PCTCN2019120562-appb-000013
在一种变体中，39位的甲硫氨酸(M)置换成苏氨酸(T)，将其对应的核苷酸序列115-117位处的密码子ATG变为ACC；92位的天冬酰胺(N)置换成谷氨酰胺(Q)；将其对应的核苷酸序列274-276位处的密码子AAC变为CAA。突变后的核酸序列如下所示：
Figure PCTCN2019120562-appb-000014
Figure PCTCN2019120562-appb-000015
其对应的氨基酸序列如下所示，即IL-10-05变体序列：
Figure PCTCN2019120562-appb-000016
在一种变体中，10位的天冬酰胺(N)置换成谷氨酰胺(Q)，将其对应的核苷酸序列28-30位处的密码子AAC变为CAG；39位的甲硫氨酸(M)置换成苏氨酸(T)，将其对应的核苷酸序列115-117位处的密码子ATG变为ACC；92位的天冬酰胺(N)置换成谷氨酰胺(Q)；将其对应的核苷酸序列274-276位处的密码子AAC变为CAA。突变后的核酸序列如下所示：
Figure PCTCN2019120562-appb-000017
其对应的氨基酸序列如下所示，即IL-10-06变体序列：
Figure PCTCN2019120562-appb-000018
上述变体的基因序列经合成后，与表达载体pET-9a相连，形成重组表达载体。
3.野生型IL-10及变体的重组表达与制备
将上述重组表达载体转化到从Novagen公司购买的BL21(DE3)菌株(Novagen，Cat.69450-3)，得到野生型IL-10及变体的重组菌，将重组菌涂布在含Kan抗性的LB平板进行筛选。
挑取Kan筛选后的单菌落于10mL LB培养基，37℃，220rpm培养至OD 600 为1.2±0.2，加入50％甘油至终浓度为10％，分装至冻存管中(1mL/管)，保存于-80℃。
取冻存的甘油管1支，于37℃水浴中复苏，接种至1L LB培养基中，37℃，220rpm培养至OD 600为0.6至1.0，加入1M IPTG使其终浓度为1mM，37℃，220rpm诱导培养4h。诱导结束后，收集菌体。
菌体经破碎，包涵体变性、复性、纯化获得目的蛋白。
实施例2:野生型IL-10及变体的稳定性研究
野生型IL-10样品(缓冲体系为10mM Tris-HCl，pH7.4)，置于37℃，进行稳定性研究，通过液质联用对取样绘制肽图，分析降解情况。
检测方法：取样品0.2mL加入0.2mL变性溶液(8mol/L盐酸胍，0.2mol/L Tris-HCl，pH7.5)，加入5μl 1mol/L DTT(二硫苏糖醇)混匀，25℃水浴1h。然后加入30μl 0.5mol/L IAM(碘乙酰胺)25℃水浴1h。经PD-10柱将样品置换在酶解缓冲液(50mmol/L碳酸氢铵，1mol/L盐酸胍，pH8.0)中。取置换缓冲液后的样品0.4mL加入40μg V8蛋白酶，25℃水浴18h，加盐酸终止反应，通过液质联用绘制肽图。
检测结果发现，含有N10和M39的片段出现降解。
在变体研究的过程中，考察了分别含有N10Q和M39T突变位点的两个变体的稳定性。取样检测降解情况，方法同野生型IL-10。结果见表2。实验结果表明，N10和M39位点的突变分别降低了两个位点所在片段的脱氨和氧化情况，说明突变体比野生型更加稳定，符合设计预期。
表2.IL-10变体的稳定性结果
Figure PCTCN2019120562-appb-000019
其中，D代表天。
实施例3:野生型IL-10及变体的活性测定
在不同IL-10的浓度下，其细胞依赖株MC-9(Master Cell-9)细胞存活率不同，以此检测IL-10的生物学活性。
基础培养液：DMEM(高糖)+10％FBS+1％P/S(青链霉素)+0.05mM beta ME；
完全培养基：DMEM+10％FBS+1％P/S(青链霉素)+5ng/mL的mIL-4+2.5ng/mL hIL-10+0.05mM beta ME(beta-巯基乙醇)；
MTT溶液：称取MTT 0.1g，加无菌PBS(磷酸盐缓冲液，PH7.4)溶解并稀释至20mL，经0.22μm滤膜过滤除菌；
裂解液：15％十二烷基硫酸钠溶液。
对照品溶液的制备：取重组人白介素-10对照品，用基础培养液(含30ng/mL的mIL-4)稀释至每1mL含30ng。在96孔细胞培养板中，做3倍系列稀释，共8个梯度，每个稀释度做2孔。每孔分别留50μL溶液，弃去孔中多于溶液，以上操作均在无菌条件下进行。
待测样品溶液的制备：取重组人IL-10待测样品，用基础培养基(含30ng/mL的mIL-4)稀释成每1mL含30ng。在96孔板细胞培养板中，做3倍系列稀释，共8个稀释度，每个稀释度2孔。每孔分别留50μL待测样品溶液，弃去孔中多余溶液，以上操作在无菌条件下进行。
IL-10活性测定方法：MC-9细胞用完全培养液于37℃、5％二氧化碳条件下培养至足够量；离心收集MC-9细胞，用DMEM培养液洗涤3次；重悬于基础培养液中配制成每1mL含5.0×10 5个细胞的细胞悬液；于37℃、5％二氧化碳条件下备用。在加有标准品溶液和待测样品溶液的96孔板细胞培养板中，每孔加入细胞悬液50μl，于37℃、5％二氧化碳条件下培养42至48小时；然后每孔加入MTT溶液20μl，于37℃、5％二氧化碳条件下培养4-6小时后，每孔加入裂解液150μl，于37℃、5％二氧化碳条件下保温18-24小时，以上操作均在无菌条件下进行。混匀细胞板中的液体，放入酶标仪，以630nm为参比波长，在波长570nm处测定吸光度，记录测定结果。
数据采用计算机程序或四参数回归计算法进行处理，并按下式进行计算结果：待测样品的相对生物学活性(％)＝对照品EC 50/待测样品EC 50(EC 50：半最大效应浓度)。
实施例1中突变体的活性数据见表3。
表3.IL-10野生型及其变体的活性
名称 对应氨基酸序列号 突变位置 相对活性
IL-10 SEQ ID NO:2 野生型 100％
IL-10-01 SEQ ID NO:4 N10Q 110％
IL-10-02 SEQ ID NO:6 M39T 111％
IL-10-03 SEQ ID NO:8 N92Q 194％
IL-10-04 SEQ ID NO:10 N10Q/M39T 100％
IL-10-05 SEQ ID NO:12 M39T/N92Q 188％
IL-10-06 SEQ ID NO:14 N10Q/M39T/N92Q 258％
实施例4:PEG化的IL-10变体的制备
1.所使用的实验仪器均为常规，如：
细胞活力分析仪(Cellometer，型号NC200)；流式细胞仪(Invitrogen，型号Attune NxT)。
2.所使用的材料均为常规，如：
DMEM、FBS、RPMI1640(均购自Gibco)；Trypsin-EDTA(0.25％)、CellTrace TMViolet(均购自Invitrogen)；DMSO(购自Sigma)；Rat T-STIM(购自Becton Dickenson)；IL-4(购自Peprotech)；CellTiter-Glo(购自Promega)；Pen-Strep(购自Gibco)；FITC鼠抗人CD8、Alexa
Figure PCTCN2019120562-appb-000020
647鼠抗人颗粒酶(Granzyme)B、人IL-10ELISA KIT2(均购自BD)；Fc受体封闭剂(Human TruStain FcX)、抗鼠CD20抗体(均购自biolegend)；ImmunoCult TM人CD3/CD28T细胞激活剂、EasySep人CD8+T细胞分离试剂盒(均购自Stemcell)；Brefeldin A溶液，透化缓冲液(10×Permeabilization Buffer)，IC固定缓冲液(均购自eBioscience)；PMBC(购自Schbio)。
3.IL-10变体的PEG修饰
-取IL-10野生型或其变体，蛋白浓度为3-4mg/mL，所处的缓冲体系为50mM PB/0.1M NaCl，pH 6.0-6.3；
-向IL-10溶液中加入浓度为2mM的mPEG-丁醛水溶液，mPEG丁醛的物质的量为IL-10量的1倍，搅拌均匀；
-向反应体系中加入500mM的氰基硼氢化钠水溶液，使其终浓度为15mM；
-25℃搅拌反应约16小时后，加入浓度为1M的甘氨酸水溶液终止反应，甘氨酸的终浓度为30mM。
上述过程中，PEG-丁醛的醛基与IL-10的N端甲硫氨酸的氨基成键。
4.PEG化-IL-10变体的纯化
PEG化反应终止后，将反应体系对10mM Tris-HCl pH8.7缓冲液进行置换，开始层析纯化。
纯化用填料为Q Sepharose TMHigh Performance(GE lifescience)；
纯化所用缓冲液：缓冲液A为10mM Tris-HCl pH8.7，缓冲液B为10mM Tris-HCl/0.5M NaCl pH8.7。
平衡：缓冲液B先用高盐预平衡，缓冲液A再平衡，待紫外、电导、pH均稳定，将紫外归零，150cm/h；
上样：样品过滤后上样，150cm/h，载量1-2mg/mL；
梯度洗脱参数：0-20％B，梯度洗脱，20CV，75cm/h；出峰后收集目的蛋白峰。RP-HPLC和SE-HPLC检测目的蛋白纯度。经检测获得了PEG化的IL-10变体。
实施例5:MC-9细胞增殖实验
实验方法参考实施例3，结果如表4所示。
表4.IL-10的MC-9细胞增殖实验
Figure PCTCN2019120562-appb-000021
结果显示，未修饰的IL-10突变体IL-10-06及其PEG化后的样品均能不同程度促进MC-9细胞的增殖。
实施例6:CD8+T细胞增殖及颗粒霉素释放实验
1.实验步骤：将冻存的PBMC用EasySep Human CD8+T细胞分离试剂盒负选得到CD8+T细胞，加入CD3/CD28T细胞激活剂(25ul/1E6cells/mL)培养48小时后离心收集CD8+T细胞。调整T细胞密度为2.5E6细胞/mL，种入96孔板中，每孔200μl培养液，培养液为1640含有1％青霉素-链霉素和10％FBS，再加入不同浓度的IL-10待测样品培养72小时。在CD8+T细胞增殖实验中，直接加入Cell Titer-Glo后测定活细胞数目。结果见图1所示。
2.在颗粒霉素释放实验中，在用流式检颗粒霉素释放前，在IL-10作用72小时后的CD8+T细胞中加入蛋白转运抑制剂Brefeldin A溶液和CD3/CD28T细胞激活剂，作用16小时。收集CD8+T细胞；按细胞内染色方法两步法依次用FITC鼠抗人CD8和Alexa
Figure PCTCN2019120562-appb-000022
647鼠抗人颗粒酶B标记细胞，流式检测；结果以CD8阳性的T细胞颗粒酶B流式MFI值作为细胞激活指标。IL-10促进CD8+T细胞颗粒霉素释放的EC 50值用如上公式I的四参数logit方法。结果见图2所示。
结果显示，由于IL-10受体只在活化的CD8细胞上表达，因此本实施例将人的CD8+T细胞激活后，加入IL-10作用。由图1可知，未修饰的IL-10突变体IL-10-06及其PEG化衍生物均能不同程度地，剂量依赖性地促进活化CD8+T细胞的增殖。
此外，IL-10不仅能促进活化CD8+T细胞的增殖，并且能促进CD8+T细胞的功能，如颗粒霉素的释放。图2中，未修饰的IL-10突变体IL-10-06能刺激CD8+T细胞分泌颗粒霉素，EC 50为1.63ng/mL，PEG化IL-10-06均能剂量依赖地促进人CD8+T细胞分泌颗粒霉素。其中，20KD-单PEG-IL-10-06的EC 50为16.1ng/mL；20KD-双PEG-IL-10-06的EC 50为105ng/mL；12KD-单PEG-IL-10-06的EC 50为13.7ng/mL；20KD-双PEG-IL-10-06的EC 50为33.4ng/mL。
实施例7:小鼠体内药代动力学实验
实验步骤：6-8周C57BL/6小鼠，雄性(上海灵畅生物科技有限公司)，小鼠的饲养环境为SPF级别。环境温度控制在20-26℃，湿度40-70％，12小时黑暗和12小时光照，小鼠自由取食(标准饲料)和饮水。只有经过健康检查合格的动物将用于本实验。
皮下给予60微克IL-10样品，分别在给药前和给药后4、8、24、48、72、96和120小时取血加入肝素钠抗凝血，立即存入-80度冰箱。所有血浆收集完成后用ELISA的方法分别检测小鼠血浆内的IL-10含量。结果如图3所示。
图3中，未修饰的IL-10-06体内半衰期很短，给药24小时后，血浆中的IL-10已经低于检测限，而PEG化可以显著延长IL-10-06体内半衰期，其中12KD-单PEG-IL-10-06体内半衰期是7.9小时，12KD-双PEG-IL-10-06体内半衰期是10.5小时，20KD-单PEG-IL-10-06单修饰体内半衰期9.37小时。
实施例8：小鼠结肠癌CT-26细胞皮下同种移植肿瘤模型在BALB/c小鼠的体内药效
实验步骤：小鼠结肠癌CT-26细胞(CT-26-C2，ATCC)，培养条件为RPMI1640培养基中加10％胎牛血清，37℃ 5％CO 2孵箱培养。当细胞饱和度为80％-90％，数量到达要求时，收取细胞，计数。0.1mL(0.3×10 6个)CT-26细胞皮下接种于每只小鼠的右后背皮下(BALB/c小鼠，雌性，6-8周龄，购自上海灵畅生物科技有限公司)。小鼠的饲养环境为SPF级别。环境温度控制在20-26℃，湿度40-70％，12小时黑暗和12小时光照，小鼠自由取食(标准饲料)和饮水。只有经过健康检查合格的动物将用于本实验。当小鼠肿瘤平均体积达到约0-100mm 3时开始分组给药。为了防止小鼠产生抗人IL-10的抗体从而拮抗其抗肿瘤活性，在正式给药前24小时给予每只小鼠200μg小鼠CD20抗体。然后一天两次皮下给予不同浓度的待测样品。观测19天，每周两次测量肿瘤体积及小鼠体重的变化。结果如图4和图5所示。
结果显示，给予PEG化的IL-10-06 19天后能够抑制CT26小鼠结肠癌移植瘤的生长。其中12KD双PEG-IL-10-06 0.1mpk给药组瘤体积比对照组小鼠瘤体积(T/C)为61.4％，12KD双PEG-IL-10-06 0.3mpk，给药19天后T/C值为83.6％，六只小鼠中一只小鼠的肿瘤完全消退。20KD单PEG-IL-10-06 0.1mpk，给药19天后T/C值为49.93％，20KD单PEG-IL-10-06 0.3mpk，给药19天后T/C值为18.72％，其中6只小鼠中3只小鼠肿瘤完全消退。但是未PEG修饰的IL-10-06在给药10天左右有抑制肿瘤的趋势，但是在给药19天后，T/C为102.9％，并无抗肿瘤活性。
对照组小鼠和IL-10-06给药组在给药期间体重持续上升(图5)，可能由于肿瘤的持续生长造成的体重增长。在12KD双PEG-IL10-06和20KD单PEG-IL10-06给药组体重增长程度较弱，可能是肿瘤生长被抑制造成的。并且给药组在给药后19天与给药前相比，并未出现明显的体重下降，提示给药组并未产生难以耐受的毒副作用。

Claims (13)

  1. 一种人白细胞介素10(IL-10)变体或其衍生物，其在选自以下的一个或多个位点上包含氨基酸突变：第10位、第39位、第92位；
    优选地，在第10位上的氨基酸残基突变为谷氨酰胺残基Q；
    优选地，在第39位上的氨基酸残基突变为苏氨酸残基T；
    优选地，在第92位上的氨基酸残基突变为谷氨酰胺残基Q。
  2. 根据权利要求1所述的IL-10变体或其衍生物，其包含选自以下组合的氨基酸突变：
    10Q和39T；
    39T和92Q；
    10Q和39T和92Q；
    优选地，相较于野生型人IL-10，所述IL-10变体或其衍生物包含选自以下组合的氨基酸突变：
    N10Q和M39T；
    M39T和N92Q；
    N10Q和M39T和N92Q。
  3. 根据权利要求1或2所述的IL-10变体或其衍生物，其包含选自以下任一项所示的多肽或由其组成：
    SEQ ID NO.4、SEQ ID NO.6、SEQ ID NO.8、SEQ ID NO.10、SEQ ID NO.12、SEQ ID NO.14。
  4. 根据权利要求1至3中任意一项所述的IL-10变体或其衍生物，其中：
    所述的IL-10变体或其衍生物是选自以下任一项的形式：单体、二聚物、三聚物；
    所述的IL-10变体的衍生物体现为选自以下任一项的形式或其组合：
    PEG化的、糖基化的、羟乙基化的、缀合有白蛋白、融合至白蛋白、融合至抗体或其抗原结合片段、截短的；
    优选地，所述IL-10变体或其衍生物是PEG化的IL-10变体或其衍生物；
    更优选地，所述PEG化位于所述IL-10变体或其衍生物的N末端；
    最优选地，所述PEG化的IL-10变体或其衍生物是选自以下的任一项：
    -缀合有PEG的IL-10二聚体，其中所述PEG的平均分子量为20KD，所述PEG为单PEG，其缀合至所述二聚体中的单体之一；
    -缀合有PEG的IL-10二聚体，其中所述PEG的平均分子量为20KD，所述PEG为两个PEG，两个PEG分别缀和至所述二聚体中的两个单体；
    -缀合有PEG的IL-10二聚体，其中所述PEG的平均分子量为12KD，所述PEG为单PEG，其缀合至所述二聚体中的单体之一；
    -缀合有PEG的IL-10二聚体，其中所述PEG的平均分子量为12KD，所述PEG为两个PEG，两个PEG分别缀和至所述二聚体中的两个单体；
    优选地，所述二聚体是同二聚体；
    更优选地，所述截短是指缺少信号肽。
  5. 根据权利要求4所述的IL-10变体或其衍生物，其是单PEG化的或双PEG化的；
    优选地，所述PEG化位于所述IL-10变体或其衍生物的N末端；
    优选地，所述PEG的平均分子量为约5KD至约50KD，
    更优选地，所述PEG的平均分子量为约10KD至约30KD。
  6. 一种药物组合物，其含有：
    -权利要求1至5中任意一项所述的IL-10变体或其衍生物，
    -任选地，药学上可接受的稀释剂、载体或助剂。
  7. 一种分离的核酸分子，其编码权利要求1至5中任意一项所述的IL-10变体或其衍生物。
  8. 根据权利要求7所述的分离的核酸分子，其包含选自以下任一项所示的多核苷酸或由其组成：SEQ ID NO.3、SEQ ID NO.5、SEQ ID NO.7、SEQ ID NO.9、SEQ ID NO.11、SEQ ID NO.13。
  9. 一种表达载体，其包含权利要求7或8所述分离的核酸分子。
  10. 一种宿主细胞，其表达权利要求9所述的表达载体；
    所述的宿主细胞为原核或真核细胞，
    优选地，其为细菌、或酵母菌、或哺乳动物细胞；
    更优选地，其为大肠杆菌。
  11. 选自以下的任一项在制备药物中的用途：
    权利要求1-5任意一项所述的IL-10变体或其衍生物、权利要求6所述的药物组合物；
    所述的药物用于治疗选自以下的疾病：增生性疾病、免疫性疾病、心血管病症、血栓性病症、高血脂症、炎性病症、肝病、调节T细胞介导的免疫应答；
    优选地，所述增生性疾病是肿瘤或癌；
    优选地，所述免疫性疾病是糖尿病，更优选胰岛素依赖型糖尿病；
    优选地，所述心血管病症选自：动脉粥样硬化、高血压、心肌病；
    优选地，所述血栓性病症是引起中风或心肌梗塞的病症；
    优选地，所述炎性病症为脉管炎；
    优选地，所述肝病为非酒精性脂肪肝或丙肝；
    更优选地，所述肿瘤或癌选自：上皮细胞癌、内皮细胞癌、鳞状细胞癌、乳头状瘤病毒引起的癌、腺癌、癌肿、黑素瘤、肉瘤、畸胎癌、肺部肿瘤、转移性肺癌、淋巴瘤。
  12. 一种制备IL-10变体或衍生物的方法，包括：
    向野生型人IL-10中引入权利要求1-3任意一项中所限定的突变，或
    使用权利要求7或8所述的核酸分子进行表达，或
    使用权利要求9所述的表达载体进行表达，或
    使用权利要求10所述的宿主细胞进行表达。
  13. 一种治疗疾病的方法，其包括步骤：
    使受试者接触治疗有效量的权利要求1-5任意一项所述的IL-10变体或其衍生物或接触治疗有效量的权利要求6所述的药物组合物；
    所述疾病选自：增生性疾病、免疫性疾病、心血管病症、血栓性病症、高血脂症、炎性病症、肝病、调节T细胞介导的免疫应答；
    优选地，所述增生性疾病是肿瘤或癌；
    优选地，所述免疫性疾病是糖尿病，更优选胰岛素依赖型糖尿病；
    优选地，所述心血管病症选自：动脉粥样硬化、高血压、心肌病；
    优选地，所述血栓性病症是引起中风或心肌梗塞的病症；
    优选地，所述炎性病症为脉管炎；
    优选地，所述肝病为非酒精性脂肪肝或丙肝；
    更优选地，所述肿瘤或癌选自：上皮细胞癌、内皮细胞癌、鳞状细胞癌、乳头状瘤病毒引起的癌、腺癌、癌肿、黑素瘤、肉瘤、畸胎癌、肺部肿瘤、转移性肺癌、淋巴瘤。
PCT/CN2019/120562 2018-11-26 2019-11-25 一种人白细胞介素10变体及其衍生物 WO2020108426A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201980072302.0A CN112955546B (zh) 2018-11-26 2019-11-25 一种人白细胞介素10变体及其衍生物

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201811418270.9 2018-11-26
CN201811418270 2018-11-26

Publications (1)

Publication Number Publication Date
WO2020108426A1 true WO2020108426A1 (zh) 2020-06-04

Family

ID=70853748

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2019/120562 WO2020108426A1 (zh) 2018-11-26 2019-11-25 一种人白细胞介素10变体及其衍生物

Country Status (3)

Country Link
CN (1) CN112955546B (zh)
TW (1) TW202033766A (zh)
WO (1) WO2020108426A1 (zh)

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001058950A1 (en) * 2000-02-11 2001-08-16 Maxygen Aps Improved interleukin 10
WO2006130580A2 (en) * 2005-05-31 2006-12-07 Avigen, Inc. Mutant il-10
CN102256625A (zh) * 2008-12-17 2011-11-23 先灵公司 单和双peg il10的生产和用途
WO2012135177A2 (en) * 2011-03-29 2012-10-04 Uab Research Foundation Methods and compositions for cytomegalovirus il-10 protein
WO2014023673A1 (en) * 2012-08-08 2014-02-13 Roche Glycart Ag Interleukin-10 fusion proteins and uses thereof
WO2014176373A2 (en) * 2013-04-24 2014-10-30 Armo Biosciences, Inc. Interleukin-10 compositions and uses thereof

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030186386A1 (en) * 2000-02-11 2003-10-02 Hansen Christian Karsten Interleukin 10

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001058950A1 (en) * 2000-02-11 2001-08-16 Maxygen Aps Improved interleukin 10
WO2006130580A2 (en) * 2005-05-31 2006-12-07 Avigen, Inc. Mutant il-10
CN102256625A (zh) * 2008-12-17 2011-11-23 先灵公司 单和双peg il10的生产和用途
WO2012135177A2 (en) * 2011-03-29 2012-10-04 Uab Research Foundation Methods and compositions for cytomegalovirus il-10 protein
WO2014023673A1 (en) * 2012-08-08 2014-02-13 Roche Glycart Ag Interleukin-10 fusion proteins and uses thereof
WO2014176373A2 (en) * 2013-04-24 2014-10-30 Armo Biosciences, Inc. Interleukin-10 compositions and uses thereof

Also Published As

Publication number Publication date
CN112955546A (zh) 2021-06-11
CN112955546B (zh) 2024-04-16
TW202033766A (zh) 2020-09-16

Similar Documents

Publication Publication Date Title
JP6905024B2 (ja) モノ−およびジ−ｐｅｇ ｉｌ１０の製造および用途
JP6440776B2 (ja) 癌を処置するためのペグ化ｉｌ−１０の使用
US11413332B2 (en) Methods of using interleukin-10 for treating diseases and disorders
CN108697776A (zh) 在产生抗原特异性cd8+t细胞中的白介素-10及其使用方法
BR112021010983A2 (pt) Variante de interleucina-2 humana ou seu derivado
JP2020519676A (ja) 長時間作用型インターロイキン−１５受容体作動薬並びに関連する免疫治療組成物及び方法
EP3706779B1 (en) Compositions and methods of use of interleukin-10 in combination with immune checkpoint pathway inhibitors
US10618970B2 (en) Method of treating cancer with IL-10 and antibodies that induce ADCC
KR20180038553A (ko) 질환 및 장애를 치료하기 위한 인터류킨-10을 사용하는 방법
KR20220008323A (ko) 면역 세포를 증식하는 il-2 돌연변이 단백질
WO2020108426A1 (zh) 一种人白细胞介素10变体及其衍生物
RU2799437C2 (ru) Вариант человеческого интерлейкина-2 или его производное
EA043667B1 (ru) Длительно действующие агонисты рецептора интерлейкина-15, связанные с ними композиции и способы их получения и применения

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19888984

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19888984

Country of ref document: EP

Kind code of ref document: A1