WO2013097748A1 - 白介素-22在治疗和预防神经损伤疾病或神经退行性疾病中的用途 - Google Patents

白介素-22在治疗和预防神经损伤疾病或神经退行性疾病中的用途 Download PDF

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WO2013097748A1
WO2013097748A1 PCT/CN2012/087694 CN2012087694W WO2013097748A1 WO 2013097748 A1 WO2013097748 A1 WO 2013097748A1 CN 2012087694 W CN2012087694 W CN 2012087694W WO 2013097748 A1 WO2013097748 A1 WO 2013097748A1
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monomer
interleukin
dimer
cells
formula
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PCT/CN2012/087694
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French (fr)
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武栋栋
黄智华
刘恒
黄予良
严孝强
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健能隆医药技术（上海）有限公司
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Priority to CN201280064852.6A priority Critical patent/CN104066440B/zh
Priority to US14/369,187 priority patent/US9352024B2/en
Publication of WO2013097748A1 publication Critical patent/WO2013097748A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/55—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6813—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin the drug being a peptidic cytokine, e.g. an interleukin or interferon
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia

Definitions

  • interleukin-22 in the treatment and prevention of neurological or neurodegenerative diseases
  • the invention relates to the field of biological and medical technology.
  • the invention relates to the use of interleukin-22 for the treatment and prevention of neurological or neurodegenerative diseases. Background technique
  • Interleukin-22 is a glycoprotein secreted by T cells, also known as IL- 10- related T cell-derived inducible factor (IL-TIF).
  • IL-TIF T cell-derived inducible factor
  • the expression of interleukin 22 mRNA was originally demonstrated in mouse IL-9-stimulated T cell lines and IL_9-stimulated mast cells, and spleen cells activated by concanaval inA.
  • Human interleukin 22 mRNA expression is mainly in peripherally isolated T cells and stimulated by anti-CD-3 antibodies or ConA.
  • Interleukin 22 mRNA is also expressed in activated NK cells.
  • the activated T cells are mainly CD4+, especially Thl cells from the CD28 pathway.
  • the interleukin-22 precursor consists of 179 amino acid residues (the mature peptide is 146 amino acid residues), and Dumoutier et al. first reported the cloned mouse and human interleukin-22 genes (Dumoutier et al., JI, 164:1814-1819). , 2000). In addition, Dumoutier has patented IL-22 (US6, 359, 117 and 6, 274, 710). Gurney also patented IL-22 for the treatment of human pancreatic lesions (US 6, 551, 799).
  • Interleukin-22 is mainly expressed in thymus, brain, activated T cells and mast cells, spleen cells stimulated by lectin (Duroutier 2002), and interleukin-2/interleukin-12 stimulated NK cells (Wolk, K / 2002). As well as expression in multiple organs and tissues after LPS stimulation (Dumoutier PNAS paper), elevated expression of IL-22 was detected in the intestine, liver, stomach, kidney, lung, heart, thymus, and spleen.
  • IL-22 exerts biological functions by binding to the IL-22RI receptor and the IL-10R2 receptor.
  • IL-22R1 is an IL-22-specific receptor that is expressed in the skin, kidney, digestive system (pancreas, small intestine, liver, large intestine, colon) as well as the respiratory system (lung, bronchi).
  • interleukin 22 is an immunomodulator.
  • the therapeutic use of IL-22 for lowering serum triglycerides and obesity has been reported (see WO2006/073508, CN200510023103.0).
  • it has not been found that interleukin-22 can play a positive role in the treatment of neurological or neurodegenerative diseases.
  • Neurodegenerative disease is a state of progressive degeneration and death of neuronal cells in the brain and spinal cord. It is a chronic, progressive neurological disease, mainly including Alzhemier's disease, AD. ), Parkinson's disease (PD), Huntington disease, amyotrophic lateral sclerosis, spinal muscular atrophy, spinal cord cerebellar Disorders (spinal cerebellar ataxias), etc., which are characterized by degenerative changes and apoptosis of neurons.
  • a neurological disease that causes abnormal behavior and dysfunction of the patient, causing premature death.
  • the pathogenesis of neurodegenerative diseases is still unclear, and there are no effective methods and drugs to prevent and treat.
  • Existing treatment methods include treating human PD by supplementing human brain neurotransmitters, such as levodopa, but left-handed.
  • Dopa does not effectively control the natural pathological process of PD, and can not affect the degeneration rate of dopaminergic neurons.
  • Long-term use also has a variety of adverse reactions, such as switching and dyskinesia, and its therapeutic effect can only last for about two years. Long-term use can also damage neurons and accelerate neuronal apoptosis.
  • the concentration of cholinesterase inhibitors is increased. This method is only symptomatic and cannot control the development of the disease.
  • the object of the present invention is an effective medicine for treating and preventing a nerve damage disease or a neurodegenerative disease and the use thereof, that is, Interleukin-22 (IL-22) for treating and preventing a neurological damage disease in a mammal or Use in neurodegenerative diseases.
  • IL-22 Interleukin-22
  • an interleukin-22 or a dimer or multimer thereof for the preparation of a medicament for the treatment and prevention of a neurological or neurodegenerative disease.
  • the nerve damage disease is selected from the group consisting of: stroke, spinal injury, and a nervous system disease associated with blood-brain barrier damage;
  • the neurodegenerative disease is selected from the group consisting of: Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, spinal muscular atrophy, primary lateral sclerosis, or spinal cord cerebellar Disorder.
  • the interleukin-22 comprises: human interleukin-22 or interleukin-22 of a mammal (e.g., mouse, rabbit, cow, or sheep).
  • a mammal e.g., mouse, rabbit, cow, or sheep.
  • the interleukin-22 comprises: recombinant interleukin-22, or native interleukin-22.
  • the dimer of interleukin-22 is a dimer of interleukin-22 of formula I:
  • Ml is the first monomer of interleukin-22;
  • M2 is the second monomer of interleukin-22
  • L is a joint member between the first monomer and the second monomer that connects the first monomer and the second monomer together
  • interleukin-22 dimer retains the biological activity of interleukin 22 and has a serum half-life of more than twice the serum half-life of the first monomer or the second monomer.
  • the serum half-life of the IL-22 dimer is more than 3 times, more than 5 times or more than 10 times the serum half life of the first monomer and/or the second monomer.
  • the joint element L is selected from the group consisting of: (i) a short peptide of 3-50 amino acids;
  • Y is a carrier protein
  • Z is a short peptide of no, or 1-30 amino acids
  • the first monomer and the second monomer are the same.
  • the first monomer and the second monomer are different.
  • the biological activity is one or more activities selected from the group consisting of:
  • the carrier protein is an Fc fragment of albumin or human IgG.
  • the carrier protein is formed by a disulfide bond between two Fc fragments of IgG. In another preferred embodiment, the number of the disulfide bonds is 2-4.
  • the "-" is a peptide bond.
  • the dimer is a dimer composed of a monomer having an amino acid sequence such as SEQ ID NOS: 2-5.
  • Ml is the first monomer of interleukin-22;
  • M2 is the second monomer of interleukin-22
  • L is a joint member between the first monomer and the second monomer that connects the first monomer and the second monomer together
  • interleukin-22 dimer retains the biological activity of interleukin 22 and has a serum half-life of more than twice the serum half-life of the first monomer or the second monomer.
  • a pharmaceutical composition for the treatment and prevention of a neurological or neurodegenerative disease comprising a pharmaceutically acceptable carrier and dimerization of interleukin-22 of formula I Body,
  • Ml is the first monomer of interleukin-22;
  • M2 is the second monomer of interleukin-22
  • L is a joint member between the first monomer and the second monomer that connects the first monomer and the second monomer together
  • interleukin-22 dimer retains the biological activity of interleukin 22 and has a serum half-life of more than twice the serum half-life of the first monomer or the second monomer.
  • the dimer is a dimer composed of a monomer having an amino acid sequence such as SEQ ID NO: 3 or 5.
  • the dimer of IL-22 is prepared by the following steps:
  • the invention uses the IL-22 dimer molecule, on the one hand, after the ischemic nerve damage in the animal, can protect the neurons, thereby recovering the damaged nerve function, and can effectively treat the nerve damage disease; on the other hand, it can be significant It inhibits the loss of dopaminergic neurons in the substantia nigra of PD model animals and enhances the function of dopaminergic neurons.
  • IL-22 dimer molecules can significantly improve the learning and memory ability of AD model rats, protect neurons, reduce neuronal cell apoptosis in hippocampus, and alleviate the symptoms of dementia.
  • the dimer IL-22 of the present invention has a prolonged serum half-life and effectively prevents the loss of neurons, thereby enabling more effective treatment of neurodegenerative diseases.
  • Figure 1 shows a schematic representation of an IL-22 dimer of the invention. Among them, "-" indicates a linker peptide, and IL-22 oval indicates an IL-22 monomer.
  • a sequence of the IL-22 dimer is shown in SEQ ID NO: 1, wherein the 1-146th position is IL-22, the 147th-162th position is a linker peptide, and the 163th-308th position is another IL- twenty two.
  • FIGS. 2A and 2B show schematic views of the IL-22 dimer of the present invention.
  • "-" indicates an amino acid linker peptide
  • IL-22 ellipse indicates an IL-22 monomer
  • an ellipse labeled "C” indicates a carrier protein
  • IL-22 is located at the N-terminus of the carrier protein.
  • a sequence of an IL-22 monomer having an Fc fragment for constituting the IL-22 dimer is shown in SEQ ID NO: 2, wherein the 1-146th position is IL-22, 147-162 The position is a linker peptide, and positions 163-385 are Fc fragments of human IgG2. Two IL-22 monomers bearing an Fc fragment form a dimer by pairing of Fc.
  • a sequence of an IL-22 monomer having an Fc fragment for constituting the IL-22 dimer is shown in SEQ ID NO: 3, wherein the 1-146th position is IL-22, 147-152 The position is a linker peptide, and positions 153-375 are Fc fragments of human IgG2. Two IL-22 monomers bearing an Fc fragment form a dimer by pairing of Fc.
  • FIGS 3A and 3B show schematic diagrams of the IL-22 dimer of the present invention.
  • "-" indicates an amino acid linker peptide
  • IL-22 ellipse indicates an IL-22 monomer
  • an ellipse labeled "C” indicates a carrier protein
  • IL-22 is located at the C-terminus of the carrier protein.
  • a sequence of an IL-22 monomer having an Fc fragment for constituting the IL-22 dimer is shown in SEQ ID NO: 4, wherein the 1-123 position is an Fc fragment of human IgG2, 224- Position 239 is the linker peptide and positions 240-385 are IL-22. Two IL-22 monomers with an Fc fragment form a dimer by pairing of Fc.
  • a sequence of an IL-22 monomer having an Fc fragment for constituting the IL-22 dimer is shown in SEQ ID NO: 5, wherein the 1-123 position is an Fc fragment of human IgG2, 224- Position 229 is the linker peptide and positions 230-375 are IL-22. Two IL-22 monomers with an Fc fragment form a dimer by pairing of Fc.
  • Figure 4 shows the effect of IL-22 and IL-22 dimer (IL-22-Fc) on activation of STAT3 in neuronal cells.
  • IL-22-Fc IL-22 dimer
  • Figure 5 shows the therapeutic effect of the IL-22 dimer of the present invention in a Focal cerebral schemia animal model.
  • Recombinant human IL-22 dimer can significantly reduce the volume of cerebral infarction.
  • Figure 6 shows the therapeutic effect of the IL-22 dimer of the present invention in a Focal cerebral schemia animal model.
  • Recombinant human IL-22 dimer can significantly improve the neural function of model animals.
  • Fig. 7 shows the results of in vitro activity analysis of the IL-22 dimer and IL-22 monomer of the present invention.
  • Figure 8A shows immunohistochemical staining of TH-positive neurons in the substantia nigra pars compacta of mice.
  • Figure 8B shows the results of TH positive cell count analysis in the substantia nigra pars compacta of mice. detailed description
  • interleukin-22 or interleukin-22 dimer exhibits a remarkable therapeutic effect in a neurological injury disease or a neurodegenerative disease.
  • the IL-22 dimer of the present invention is capable of prolonging the half-life in vivo, improving the kinetics of the drug, reducing the frequency of injection, and having significantly enhanced in vivo biological activity as compared to the IL-22 monomer.
  • Experiments on activation of STAT3 in neuronal cells indicate that IL-22 dimer can be made at a final concentration of IL-22 equivalent to 1/10 of the final concentration of IL-22 monomer relative to IL-22 monomer.
  • amino acid sequence is substantially identical refers to a difference in sequence or caused by one or more amino acid changes (deletion, addition, substitution), but such alteration does not substantially reduce its biological activity, ie, by binding to IL-22 Target cell receptors undergo biological functions.
  • any interleukin-22 that meets the "substantially identical" requirement is included in the present invention, whether it is glycosylated (ie, derived from natural or derived from eukaryotic expression systems) or non-glycosylated (ie, source) In prokaryotic expression systems or chemically synthesized).
  • treatment refers to the administration of the interleukin-22 of the present invention to a subject in need of treatment for the purpose of curing, alleviating, ameliorating, alleviating, affecting the disease, symptom, and disease of the subject.
  • terapéutica subject refers to rats, humans, and other mammals.
  • terapéuticaally effective amount refers to the amount of interleukin-22 which is capable of achieving therapeutic purposes in a subject. It will be understood by one of ordinary skill in the art that the “therapeutically effective amount” may vary depending on the route of administration of interleukin-22, the pharmaceutical excipients used, and the combination with other drugs. Interleukin 22 and its preparation
  • interleukin-22 refers to a protein which has substantially the same amino acid as human/mouse interleukin-22 as described by Dumoutier et al. in US359,117. The sequence and (b) have the same biological activity as native interleukin-22.
  • the interleukin-22 of the present invention includes, but is not limited to, human interleukin-22, recombinant human interleukin, murine interleukin-22, and/or recombinant murine interleukin-22.
  • the term "IL-22" may include IL-22 in the form of a monomer, a dimer or a multimer.
  • Interleukin-22 also includes PEGylated IL-22 and covalently modified IL-22 protein.
  • PEG polyethylene glycol
  • various activated polyethylene glycol (PEG) having a molecular weight of 5,000 to 100,000 can be modified to polymerize IL-22 and prolong its half-life.
  • PEG polyethylene glycol
  • the interleukin-22 of the present invention can be cloned and expressed by genetic recombination techniques.
  • Host cells for expression include prokaryotic cells, yeast cells, or higher eukaryotic cells.
  • Suitable prokaryotic host cells include, but are not limited to, G + or G-bacteria, such as E. coli A coli.
  • the £ co i. strains that can be obtained by public means include: K12 MM294 (ATCC 31, 446), X1776 (ATCC) 31, 537), W3110 (ATCC 27, 325) and K5 772 (ATCC 53, 635), etc.
  • E. coli. W3110 is recommended as a preferred host because it is often used as a fermentation host for recombinant DNA products.
  • eukaryotic cells such as filamentous fungi ( ⁇ or yeast (j3 ⁇ 4 ⁇ ) and the like are equally suitable for expression or cloning of the interleukin-22 of the present invention.
  • Saccharomyces cerevisiae 63 ⁇ 4c ⁇ a r 0 ⁇ C ⁇
  • other hosts such as Schizosaccharomyces cerevisiae; Kluyveromyces; Pichia pastoris.
  • Methylotrophic yeast e Ar 0 r 0 i C yeas ⁇
  • can also be used to express the interleukin-22 of the present invention including but not limited to various yeasts capable of growing in methanol such as Hansenula iMansenul, Rosary Candida, K. cerevisiae Udoecker, Pichia, Saccharoniyces, etc.
  • the host cell of the interleukin-22 of the present invention for expressing glycosylation may be derived from a multicellular organism.
  • invertebrate cells include insect cells such as Drosophila S2 and Spodoptera Sf9, plant cells.
  • suitable mammalian host cells include Chinese hamster ovary cells (CH0), COS cells. Specifically, monkey kidney CV1 cell line transformed with SV40 (C0S-7, ATCC CRL 1651); human embryonic kidney cell line 293 and the like.
  • CH0 Chinese hamster ovary cells
  • COS cells Specifically, monkey kidney CV1 cell line transformed with SV40 (C0S-7, ATCC CRL 1651); human embryonic kidney cell line 293 and the like.
  • the above host cells are transfected or transformed with an interleukin-22 expression vector or a cloning vector, and can be cultured in a conventional nutrient medium, which is modified to be suitable for inducing a promoter and a selective transformant. (selecting transf ormant) or amplifying the interleukin-22 encoding gene sequence. Selection of culture conditions such as medium, temperature, pH, etc. will be known to those of ordinary skill in the art. General principles, protocols, and techniques for maximizing cell culture fertility can be found in Mammal i an Cell Biotechnology: a Practical Approach, M. Butler, ed. (IRL Press, 1991) and Sambrook et al., supra.
  • CaCl 2 method calcium phosphate precipitation method, liposome medium method or electroporation method.
  • a corresponding standard transformation technique such as the CaCl 2 method (Sambrook et al, supra.) or electroporation, which is typically used for prokaryotic cells; for mammalian cells without cell walls.
  • calcium phosphate precipitation method can be used.
  • the nucleotide sequence (gPcDNA or genomic DNA) encoding the interleukin-22 of the present invention can be inserted into a replicable vector for gene cloning (DNA amplification) or expression.
  • Various vectors such as plasmids, cosmids, engineered plasmids, viral particles or phage can be obtained by public means.
  • the nucleotide sequence encoding the interleukin-22 of the present invention can be inserted into a suitable restriction endonuclease site on a replicable vector in a conventional manner using techniques well known in the art.
  • the interleukin-22 of the present invention can be directly expressed not only by genetic recombination, but also by forming a fusion polypeptide with a heterologous polypeptide, which may be a signal sequence located at the N-terminus of the mature protein or polypeptide, or may be located in a mature protein or polypeptide. N-terminal other polypeptide fragments with specific cleavage sites.
  • a heterologous polypeptide which may be a signal sequence located at the N-terminus of the mature protein or polypeptide, or may be located in a mature protein or polypeptide. N-terminal other polypeptide fragments with specific cleavage sites.
  • the signal sequence is part of the above replicable vector, or it may be part of the interleukin-22 encoding nucleotide sequence of the invention inserted into a replicable vector.
  • Both the expression vector and the cloning vector contain a nucleotide sequence which enables the vector to replicate in one or more corresponding host cells. Nucleotide sequence corresponding to various bacterial, yeast or viral host cells It is well known to those of ordinary skill in the art. For example, the origin of replication of plasmid pBR322 is applicable to most G-bacteria, 2. mu. The origin of plasmid replication is applicable to yeast cells, and various viral origins of replication (SV40, polyoma virus, adenovirus, VSV or BPV) apply. A cloning vector for use in mammalian cells.
  • Selection genes and cloning vectors usually contain a selection gene, also known as a "selection marker".
  • Selection genes encode proteins (a) that are resistant to certain antibiotics or toxins such as ampicillin, neomycin, methotrexate, tetracycline, etc.; (b) can compensate for auxotrophic deficiencies ( c) Supplementing the gene encoding the D-alanine racemase required for key nutrients such as Bacillus host cells that are not provided by the complex medium.
  • Selection genes suitable for use in mammalian host cells should be capable of distinguishing host cells capable of absorbing the interleukin-22 encoding gene of the present invention, such as DHFR or thymidine kinase.
  • Suitable host cells with wild-type DHFR as a selection gene are CHH cell lines without DHFR activity. For the preparation and propagation of this cell line, see Urlaub et al, Proc. Natl. Acad. Sci. USA, 77:4216 (1980) .
  • a selection gene suitable for use in yeast cells is the trpl gene expressed in the yeast plasmid Yrp7 (Stinchcomb et al, Nature, 282: 39 (1979); Kingsman et al, Gene, 7: 141 (1979); Tschemper et al, Gene, 10: 157 (1980)).
  • the trpl gene can be used to screen for mutant strains of yeast that cannot grow in tryptophan, such as ATCC ⁇ . 44047 or PEP4-1 (Jones, Genetics, 85:12 (1977)).
  • Expression vectors and cloning vectors typically contain a promoter operably linked to the interleukin-22 encoding nucleotide sequence of the invention to direct mRNA synthesis.
  • Promoters corresponding to various host cells are well known to those of ordinary skill in the art. Promoters suitable for use in prokaryotic host cells include beta-lactamase and lactose promoter systems and the like.
  • the bacterial host cell promoter also contains a Shine-Dalgarno (S. D.) sequence operably linked to the interleukin-22 encoding gene sequence of the present invention.
  • An enhancer is a cis-acting element of a DNA molecule, usually 10 to 300 bp, which enhances the transcription of DNA molecules by acting on a promoter.
  • Expression vectors in eukaryotic host cells also contain the nucleus required to stop transcription and stabilize mRNA. Glycosidic acid sequence. Such sequences are typically taken from the 5' end of the eukaryotic or viral DNA or cDNA untranslated region, and sometimes from the 3' end.
  • the nucleotide fragment contained in the "untranslated region” can be transcribed to produce a polyadenylation fragment located in the untranslated region of the interleukin-22 mRNA of the present invention.
  • Carrier proteins include, but are not limited to, Fc fragments of human IgG (1, 2, 3, 4), or human albumin (albumin).
  • IL-22 is located at the C-terminus of the available carrier protein, and may also be located at the N-terminus of the carrier protein.
  • linker peptide refers to a short peptide that acts as a linker between an IL-22 monomer and an IL-22 monomer.
  • the length of the linker peptide is not particularly limited. The length of the linker peptide is usually 5 to 50 amino acids. Generally, the linker peptide does not affect or significantly affect the formation of the correct folding and spatial conformation of the IL-22 monomer and the IL-22 monomer.
  • Some examples of linker peptides include (but are not limited to):
  • the linker peptide has an amino acid sequence selected from the group consisting of:
  • the amino acid sequence encoded by the multiple cloning site is usually 5-20, preferably 10-20 amino acids;
  • an amino acid sequence derived from a protein other than the IL-22 monomer such as an amino acid sequence derived from IgG or albumin;
  • Preferred linker peptides include: GSGGGSGGGGSGGGGS (positions 147-162 in SEQ ID NO: 1) and ASTKGP (positions 147-152 in SEQ ID NO: 3).
  • amino acid sequences which do not affect the activity of the IL-22 monomer may be added to the N-terminus or C-terminus of the fusion protein.
  • these added amino acid sequences facilitate expression (eg, signal peptides) and facilitate purification (eg, 6 X His sequence, Saccharomyces cerevisiae alpha-factor signal peptide cleavage site (Glu-Lys-Arg), or may facilitate fusion Protein activity.
  • the DNA sequence encoding the IL-22 dimer or fusion protein of the present invention can be all synthetically synthesized.
  • the DNA sequence encoding the IL-22 first monomer and/or the IL-22 second monomer can also be obtained by PCR amplification or synthetic methods, which are then spliced together to form a DNA sequence encoding the fusion protein of the present invention.
  • the IL-22 dimer coding sequence can be engineered, for example, using host cell-preferred codons to eliminate sequences that are detrimental to gene transcription and translation.
  • the yeast cell or mammalian cell-preferred codon can be used, and the IL-22 dimer gene can be detected by computer DNA software, and the sequence which is not conducive to gene transcription and translation in the gene is excluded, including the inclusion. Sub-shearing site, transcription termination sequence, and the like.
  • the DNA sequence encoding the novel fusion protein of the present invention After obtaining the DNA sequence encoding the novel fusion protein of the present invention, it is ligated into a suitable expression vector and transferred to a suitable host cell. Finally, the transformed host cells are cultured, and the novel fusion protein of the present invention is obtained by isolation and purification.
  • vector includes plasmids, cosmids, expression vectors, cloning vectors, viral vectors. Body and so on.
  • various carriers known in the art such as commercially available carriers can be used.
  • a commercially available vector is selected, and then a nucleotide sequence encoding a novel fusion protein of the present invention is operably linked to an expression control sequence to form a protein expression vector.
  • operably linked refers to a condition in which portions of a linear DNA sequence are capable of affecting the activity of other portions of the same linear DNA sequence. For example, if the signal peptide DNA is expressed as a precursor and is involved in the secretion of the polypeptide, then the signal peptide (secretion leader sequence) DNA is operably linked to the polypeptide DNA; if the promoter controls the transcription of the sequence, then it is operably linked to A coding sequence; if the ribosome binding site is placed at a position that enables translation, then it is operably linked to the coding sequence.
  • “operably linked to” means adjacent, and for secretory leader sequences means adjacent in the reading frame.
  • the term "host cell” includes prokaryotic cells and eukaryotic cells.
  • prokaryotic host cells include Escherichia coli, Bacillus subtilis and the like.
  • eukaryotic host cells include yeast cells, insect cells, and mammalian cells.
  • the host cell is a eukaryotic cell, more preferably a mammalian cell.
  • the cell After obtaining the transformed host cell, the cell can be cultured under conditions suitable for expression of the fusion protein of the present invention to express the fusion protein.
  • the expressed fusion protein is then isolated.
  • the IL-22 dimer of the present invention can produce a stronger receptor activation signal and has an excellent serum half-life
  • the composition can be used to treat and prevent a neurological or neurodegenerative disease.
  • the nerve damage diseases include: stroke, spinal injury and nervous system diseases accompanied by blood-brain barrier damage; the neurodegenerative diseases are selected from the group consisting of: Parkinson's disease, Alzheimer's disease, Huntington's disease , amyotrophic lateral sclerosis, spinal muscular atrophy, primary lateral sclerosis, or spinocerebellar ataxia.
  • the pharmaceutical compositions of the present invention comprise a safe or effective amount of the IL-22 or a dimer thereof of the present invention and a pharmaceutically acceptable excipient or carrier.
  • safe, effective amount means: The amount of the compound is sufficient to significantly improve the condition without causing serious side effects.
  • the pharmaceutical composition contains from 0.001 to 1000 mg of IL-22 or a dimer thereof, preferably from 0.05 to 300 mg of IL-22 or its dimer/agent, and more preferably, contains 0. 5-200 mg of IL-22 or its dimer/agent.
  • the compound of the present invention and a pharmacologically acceptable salt thereof can be formulated into various preparations, which comprise the IL-22 of the present invention or a dimer thereof or a pharmacologically acceptable salt thereof in a safe and effective amount, and pharmacologically acceptable Accepted excipient or carrier.
  • safe, effective amount means: The amount of the compound is sufficient to significantly improve the condition, rather than causing serious side effects.
  • the safe and effective amount of the compound is determined by the specific conditions such as the age, condition, and course of treatment of the subject.
  • “Pharmaceutically acceptable excipient or carrier” means: one or more compatible solid or liquid fills Materials or gel materials which are suitable for human use and which must be of sufficient purity and of sufficiently low toxicity.
  • “compatibility” it is meant herein that the components of the composition are compatible with the compounds of the invention and between them without significantly reducing the efficacy of the compounds.
  • Examples of pharmaceutically acceptable excipients or carriers are cellulose and its derivatives (such as sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as Tween) ®), wetting agents (such as sodium decyl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.
  • cellulose and its derivatives such as sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.
  • gelatin such as sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc
  • IL-22 or a dimer thereof of the present invention When IL-22 or a dimer thereof of the present invention is administered, it can be administered orally, rectally, parenterally (intravenously, intramuscularly or subcutaneously) or topically.
  • Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.
  • the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or mixed with: (a) a filler or compatibilizer, for example, Starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) moisturizing An agent, for example, glycerin; (d) a disintegrant, for example, agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) a slow solvent such as paraffin; f) absorption accelerators, for example, quaternary amine compounds; (g) wetting agents, such as cetyl alcohol
  • Solid dosage forms such as tablets, troches, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other materials known in the art. They may contain opacifying agents and the release of the active compound or compound in such compositions may be released in a portion of the digestive tract in a delayed manner. Examples of embedding components that can be employed are polymeric materials and waxy materials. If necessary, the active compound may also be in the form of microcapsules with one or more of the above excipients.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or elixirs.
  • the liquid dosage form may contain inert diluents conventionally employed in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1 , 3-butanediol, dimethylformamide and oils, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or a mixture of these substances.
  • inert diluents conventionally employed in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1 , 3-butanediol, dimethyl
  • compositions may contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents, and flavoring agents.
  • adjuvants such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents, and flavoring agents.
  • the suspension may contain a suspending agent, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan ester, microcrystalline cellulose, aluminum methoxide and agar or a mixture of these and the like.
  • a suspending agent for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan ester, microcrystalline cellulose, aluminum methoxide and agar or a mixture of these and the like.
  • compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or non-aqueous solutions, dispersed A liquid, suspension or emulsion, and a sterile powder for reconstitution into a sterile injectable solution or dispersion.
  • Suitable aqueous and nonaqueous vehicles, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.
  • Dosage forms of IL-22 or a dimer thereof of the present invention for topical administration include ointments, powders, patches, sprays, and inhalants.
  • the active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants which may be required if necessary.
  • the IL-22 or its dimer of the present invention may be administered alone or in combination with other pharmaceutically acceptable compounds.
  • microcapsules containing the interleukin-22 of the present invention or a dimer thereof can be used for the sustained release administration of the interleukin-22 of the present invention.
  • the microcapsule sustained release drug delivery technology of recombinant protein has been successfully applied to recombinant human growth hormone (rhGH), recombinant human interferon (rhIFN), interleukin-2 and MNrgpl20 (Johnson et al, Nat. Med., 2:795-799 ( 1996); Yasuda, Biomed. Ther 27: 1221-1223 (1993); W097/03692, W096/40072, WO 96/07399; US5654010.
  • the sustained release preparation of the interleukin-22 of the present invention or a dimer thereof can be produced by a lactic acid glycolic acid high polymer (PLGA) having good biocompatibility and broad biodegradability.
  • PLGA lactic acid glycolic acid high polymer
  • the degradation products of PLGA, lactic acid and hydroxyacetic acid can be quickly eliminated by the human body.
  • the degradation ability of the polymer can be extended from several months to several years depending on its molecular weight and composition (Lewis, "Controlled release of bioactive agents form lactide/ glycol ide polymer," in: M. Chasin and R Langer (Eds.), Biodegradable Polymers as Drug Delivery Systems (Marcel Dekker: New York, 1990), pp. 1-41)).
  • the dosage and concentration ranges of the pharmaceutical compositions of the invention may vary depending on the actual use.
  • One of ordinary skill in the art will know how to select the appropriate dosage and route of administration based on actual needs.
  • For different species such as the principle of adjustment of the drug dosage range between human and mouse, see Mordenti, J. and Chappell, W. "The use of interspecies scaling in toxicokinetics” In Toxicokinetics and New Drug Development, Yacobi et al; Pergamon Press , New York 1989, pp. 42-96.
  • a safe and effective amount of IL-22 or a dimer thereof of the present invention is applied to a mammal (e.g., a human) in need of treatment, wherein the dose at the time of administration is a pharmaceutically effective effective dose for 60 kg.
  • the dose is usually 0.01 to 300 mg, preferably 0.5 to 100 mg per administration.
  • specific doses should also consider factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled physician.
  • IL-22 or its dimer can activate neuronal STAT3 in vitro.
  • IL-22 dimers have been shown to be effective in the treatment of neurologically impaired diseases in animal models. 3. IL-22 dimer can prolong the half-life in vivo, improve the kinetics of the drug, reduce the frequency of injection, and significantly enhance the biological activity in vivo.
  • IL-22 or IL-22 dimer significantly inhibits the loss of dopaminergic neurons in the PD model animal and stimulates the function of dopaminergic neurons.
  • IL-22 or IL-22 dimer significantly inhibits the apoptosis of neuronal cells in the hippocampus and improves the learning and memory ability of AD model animals.
  • IL-22 or IL-22 dimer has significant neuroprotective effects in neurodegenerative diseases and is effective in the treatment of neurodegenerative diseases.
  • the invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are merely illustrative of the invention and are not intended to limit the scope of the invention.
  • the experimental methods in the following examples which do not specify the specific conditions are usually carried out according to the conditions described in conventional conditions such as Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer. The suggested conditions.
  • Example 1 Example 1
  • an IL-22 dimer having the structure shown in Figs. 1 to 3 (the sequence is SEQ ID NO: 1, or the monomer sequence is shown as SEQ ID NO: 2-5).
  • an IL-22 dimer is prepared from an IL-22-Fc complex, and the specific preparation method is as follows:
  • the cDNA sequence of the whole gene-synthesized IL-22-Fc complex (shown as SEQ ID NO: 6 or SEQ ID NO: 7, wherein SEQ ID NO.: 6 encodes the monomer represented by SEQ ID NO.: 2, SEQ ID NO.: 7 encodes the monomer shown in SEQ ID NO.: 3), the gene of human IL-22 monomer is ligated to the Fc gene fragment of IgG2, the 5' end is introduced into the EcoRI site, and the mammalian cell is expressed.
  • the desired elements such as the Kozak sequence and the signal peptide sequence, were introduced into the Xbal site at the 3' end, cloned into the commercially available pUC19 plasmid, designated pIL-22-Fc, and transformed into conventional E. col i TG1.
  • the pUC19 plasmid was digested with EcoRI and Xbal, and an IL-22-Fc fragment of about 1300 bp was recovered and ligated with the pcDNA3 (Invitrogen) expression plasmid digested with EcoRI and Xbal to construct the expression plasmid pEX-IL-22-Fc.
  • the expression plasmid pEX-IL-22-Fc was transfected into CH0 cells by linearization, and IL-22 dimer was expressed. The expression level was detected by ELISA, and the cell line with higher protein yield was selected, and the cell bank was prepared.
  • Recombinant CH0 cells are cultured by a conventional method to express a recombinant protein.
  • the cell supernatant (containing IL-22 complex, IL-22 dimer, IL-22 multimer and metabolite) was collected, and the supernatant was collected, filtered, and purified by multistage gel chromatography.
  • rProtein AS printed harose FF GE Healthcare, Cat. No. 17-1279-04
  • k364-01 with 20-50 mM NaAc/HAC, pH 3. 0 -5. 0
  • the buffer is eluted, the eluate inactivates the virus at a low pH, and the Nano20 membrane is filtered by a virus to obtain an IL-22 dimer.
  • the purity of the isolated IL-22 dimer was >95% (using reverse HPLC analysis). Electrophoresis showed that the IL-22 dimer (consisting of two monomers represented by SEQ ID NO.: 2) after purification had a molecular weight of 52 ⁇ 10 KD (analyzed by reproducible SDS-PAGE), which was consistent with the predicted value. The ultraviolet absorption spectrum was 280 nm. The IL-22 dimer stimulates Colo205 cells to produce IL-10 (ED50 10-1000 ng/mL) in vitro.
  • Rats were injected subcutaneously with IL-22 dimer (consisting of two sequences of IL-22-Fc monomer as shown in SEQ ID NO: 2) 100 g/kg.
  • the half-life of monomer IL-22 (recombinant human interleukin 22) in rats was about 1.3 hours.
  • the whole brain of the fetal rat of the SD rat at 17 days of pregnancy was placed in pre-cooled D-Hanks solution, and the cerebral cortex was removed under a dissecting microscope and the meninges were removed.
  • the brain tissue was cut into a size of 1 ⁇ 3 , 10 mL of O.125% trypsin was added, and it was digested in a 37 °C incubator for 15 min, and then the aspirate tissue was transferred to a centrifuge tube containing pre-cooled DMEM + 10% FBS. The digestion was terminated and pipetted several times with a pipette. After standing, the supernatant was taken and sucked into another centrifuge tube. Repeat the above steps 2 to 3 times.
  • the serum-free neuronal basal medium neurobasal (invitrogen, Cat. No. 21103049) + serum-free additive B27 (invitrogen, Cat. No. 17504044) was cultured for 8 days, and the solution was changed every 2 days.
  • IL-22 dimer Consisting of two sequences of IL-22-Fc monomer as shown in SEQ ID NO: 2
  • IL-22 dimer IL-22 dimer
  • Final concentration 0.3 ⁇ g/mL
  • final concentration of IL-22 1.2 ⁇ g/mL
  • the drug-containing medium supernatant was completely aspirated, and the cells were washed twice with PBS, and the cells were lysed according to the cell lysate instructions.
  • Cell lysate Cell Signaling Technology, Cat. No.
  • the supernatant was aspirated and the protein concentration was determined.
  • Another supernatant of ⁇ was used to detect changes in STAT phosphorylation levels using the STAT3 [pY705] ELISA kit (invitrogen, Cat. No. KH00481).
  • IL-22 and IL-22 dimers activate neuronal cell signaling and transcriptional activator 3 (STAT3) biological activity compared to the blank control group.
  • the IL-22 monomer final concentration of 1. 2 4 ⁇ /1 ⁇
  • the IL-22 dimer has a final concentration of only 0. 3 wg / mL, which is equivalent to 1/10 of the final concentration of IL-22 monomer, and can increase the signal level of neuronal p-STAT3 by 7.8 times, visible, IL-22 dimer to neuronal cells
  • the activation of STAT3 is significantly better than that of IL-22 monomer.
  • Example 4 Therapeutic effect of IL-22 dimer in an animal model of focal cerebral ischemia (Focal cerebral i schemia)
  • IL-22 dimer group MCA0+IL-22-D100 ⁇ g/kg
  • IL-22-D consisting of two sequences as SEQ ID NO: 2 shows the composition of IL-22-Fc monomer
  • the rat's right common carotid artery (CCA), right internal carotid artery (ICA), and right external carotid artery (ECA) were exposed through neck surgery. 5 ⁇
  • the head of the silicone-wrapped wire plug was inserted through the external carotid artery of the rat into the common carotid artery and then back to the internal carotid artery for occlusion, the insertion length of about 18. 5 ⁇ 0. 5mm. It feels slightly blocked after insertion, indicating that it has reached the middle artery and is able to block the blood flow supply to the middle artery. After 60 minutes of occlusion, the plug was pulled out about 10 mm for reperfusion.
  • the IL-22 dimer group was subcutaneously injected with IL-22 dimer 100 ⁇ g/kg at 0.5 h and 48 h, respectively, and the sham operation group and the model group were subcutaneously injected with the same volume of solvent.
  • the body temperature of the animal was maintained at 36.5.
  • the sham operation group only the neck skin was cut and the neck artery was separated, and no wire plug was inserted. After 21 days, the animals were euthanized to remove the brain.
  • Neurological functional scores were evaluated at 0 days, 1 day, 2 days, 3 days or 0, 1, 2, 3, 7, 14, 21 days of surgery. The scores were graded as follows: 0 - no neurological impairment 1- The left forelimb cannot stretch; 2-Discontinuously turn to the left; 3-Continuously turn to the left; 4-Dump to the left; 5-Nerve consciousness is lost, cannot be released.
  • IL-22 dimer 30 100 wg / kg administration group, wherein each group of administration group 4 Only / group, male and female.
  • the respective doses of IL-22 dimer (consisting of two sequences of IL-22-Fc monomer represented by SEQ ID NO.: 2) were administered by subcutaneous injection, respectively, and the administration volume was 0.2 ml/kg body weight, given once.
  • This example utilizes certain cells to produce IL-10 when stimulated by IL-22, and the activity of IL-22 is determined by detecting the corresponding 0D value.
  • Colo205 cells (ATCC No. CCL-222, human colon cancer cells) were cultured in RPMI164010% FBS medium. When the cells were grown to log phase, the supernatant was discarded, PBS was added to wash away the residual medium, and 2 to 5 mL of 0.25% pancreas was added. Digestion with protease-EDTA, add the medium and blow evenly, centrifuge at 1500 rpm for 5 min, collect the cells, then prepare a cell suspension of 5.0 ⁇ 10 5 Cell/ml with the basal medium, and add ⁇ holes to each well of the 96-well plate at 37°. C, 5% C0 2 incubator overnight.
  • the 96-well plate in the C0 2 incubator was taken out, centrifuged at 800 rpm for 5 minutes at 4 ° C, and 90 ⁇ L of the cell supernatant was withdrawn from each well, and 90 WL of 0.1% BSA/RPMI1640 was added, and IL was added.
  • -22 dimer consisting of two monomers represented by SEQ ID NO.: 2) to a final concentration of 1.4, 4.1, 12.3, 37.0, 111.1, 333.3, 1000, 3000 ng/mL, and adding IL-22 to a final concentration of 0.01 , 0.04, 0.12, 0.37, 1.1, 3.3, 10, 30 ng/mL, cultured at 37 ° C, 5% CO 2 incubator for 20 hr, collected cell supernatant, using IL-22 ELISA kit (R&D, item number S1000B) The 0D value is detected.
  • the results are shown in Figure 7.
  • the half effective concentration (ED50) value of the IL-22 dimer is The ED50 of IL-22 was 0. 54 ng/mL (32. 4 pM).
  • Examples 3 and 6 suggest that there is a large difference in the effect of IL-22 monomer or dimeric forms on different cells.
  • the activation of IL-22 dimer on nerve cells is significantly better than that of IL-22 monomer.
  • human colon cancer cells such as Colo205 cells
  • IL-22 monomer activity Slightly better than the activity of the IL-22 dimer.
  • Example 3 the in vitro assay of Example 3 is more suitable for determining the activity of the dimeric IL-22 (especially for the protection of nerve cells), and is also more suitable for reflecting the activity of the dimeric IL-22 in vivo.
  • Example 7 Protective effect of IL-22 or IL-22 dimer on MPP+ induced neurotoxicity in PC12 cells
  • PC12 cells are a rat pheochromocytoma cell line that can synthesize, metabolize, and deliver dopamine in vitro and can be used as an in vitro model to screen for active compounds.
  • PC12 cells were seeded in a 96-well plate at 40,000 cells/well.
  • the medium components were: DMEM, 10% horse serum + 5% FCS, and 1% penicillin-streptomycin.
  • MPP + Sigma
  • IL-22 was added to a final concentration of 0.04 ng/mL, 0.4 ng/mL, 4 ng/mL, 40 ng/mL IL- 22 dimer.
  • Consisting of two IL-22-Fc monomers selected from the sequence of SEQ ID NO: 2-5) to a final concentration of 0.1 ng/mL, Ing/m 10 ng/mL, 100 ng/mL.
  • a fluorescence cell survival assay Fluorimetric cell via assay
  • MPTP 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine
  • the experiment selected C57/BL6J mice, male, ⁇ 28g, 12-14 weeks, the animals were kept in the environment at room temperature 24 ⁇ 2 ° C, maintain light-dark 12-hour cycle alternate, give plenty of feed, free access to water.
  • mice were randomly divided into 5 groups, 10 in each group, which were solvent control group and MPTP model group.
  • MPTP+IL-22 40 ⁇ g/kg group, MPTP+IL - 22 - D 40 ⁇ g/kg group, MPTP+IL - 22 - D 100 ⁇ g/kg group, IL-22-D consists of two groups The IL-22 dimer consisting of the IL-22-Fc monomer represented by SEQ ID NOs: 2-5.
  • MPTP was intraperitoneally injected at a dose of 30 mg/kg for 5 consecutive days. After one day of recovery (from day 7), the MPTP+IL-22 40 ⁇ g/kg group was administered subcutaneously with IL-22 40 ⁇ g/kg. Administering IL-22 daily, per Once a day, 7 days in a row, from 7th to 13th day; MPTP+IL-22-D 40 ⁇ g/kg group was administered subcutaneously at 40 ⁇ g/kg, and administered on days 7, 9, and 11 respectively.
  • IL-22-D-time MPTP+IL-22-D 100 wg/kg group was administered by subcutaneous injection at 100 ⁇ g/kg, and IL-22-D was administered on days 7, 9, and 11 respectively;
  • the solvent control group received an equal volume of physiological saline by subcutaneous injection.
  • Behavioral testing was performed on the 10th day after the last dose of MPTP in this experiment.
  • METHODS The pole test was used to detect typical behavioral symptoms in PD - a slow-moving exercise (Matsuura et al., 1997; Araki et al., 2001; Kato et al., 2004).
  • T-turn t ime to turn
  • T-LA locomotion activity time
  • mice were sacrificed by decapitation.
  • the striatum was weighed out and placed in a 1.5 ml centrifuge tube and immediately placed in crushed ice.
  • the sample was added to a sample solution of 300 ⁇ l ice water bath (0. 2 M perchloric acid, 0.2 mM sodium metabisulfite, 0.01% EDTA-2Na, and 0.3 ⁇ m ⁇ ⁇ as an internal standard. ).
  • the above mixture was ultrasonically pulverized using an ultrasonic system, and then centrifuged at 10,000 g at 4 ° C for 20 mi to take the supernatant and filter with a 0.22 ⁇ ⁇ aqueous phase filter to detect the pattern by high performance liquid chromatography.
  • the concentration of dopamine in the body was obtained by decapitation.
  • the primary antibody was monoclonal mouse anti-caries (1: 1000, CHEMIC0N), and was incubated with brain slices of the striatum and midbrain at 4 ° C overnight, washed with PBS three times and then incubated with biotinylated secondary antibody at room temperature. Lh. The SABC complex was incubated for 1 h at room temperature. DAB color development, ethanol gradient dehydration, xylene transparent, neutral gum seal. Black matter TH positive cell count, striatum TH positive staining optical density scan.
  • serum-free neuronal basal medium purchased from invitrogen, Cat. No. 21103049
  • serum-free additive B27 invitrogen, Cat. No. 17504044
  • IL-22 dimer Consisting of two IL-22-Fc monomers selected from the sequences as shown in SEQ ID NO: 2-5) and IL-22 (IL-, respectively) were used.
  • Final concentration of 22 dimer 1, 10, 100 ng/mL; final concentration of IL-22 monomer: 0.4, 4, 40 ng/mL
  • the medium was completely aspirated, the cells were washed twice with PBS, and the cells were lysed according to the cell lysate instructions.
  • Cell lysate (Cel l Signaling Technology, Cat. No.
  • IL-22 dimer (IL-22-D) and IL-22 monomer can activate the biological activity of STAT3 in hippocampal neurons.
  • Example 10 Protective effect of IL-22 or IL-22 dimer on ⁇ -induced apoptosis of PC12 cells
  • PC12 cells When PC12 cells are induced by nerve growth factor (NGF), they can grow protuberances with neuronal characteristics.
  • ⁇ ⁇ amyloid
  • ⁇ induces apoptosis in PC12 cells and can be used as an in vitro model for modeling Eltzheimer (AD).
  • PC12 cells were cultured in basal medium (DMEM, 10% FCS, 1% penicillin-streptomycin), trypsinized, resuspended in medium containing NGF 50 ng/mL, and the cell concentration was adjusted to 2 ⁇ 10 4 cells/well.
  • the cells were added to a 96-well plate and cultured at 37 ° C in a 5% CO 2 incubator for 24 hr.
  • the IL-22-Fc monomer composition was treated.
  • the final concentration of IL-22 monomer was 0.4, 4, 40 ng/mL, and the final concentration of IL-22-D was 1, 10, 100 ng/mL, respectively.
  • the model well was added with an equal volume of PBS, and the negative control well was not added with ⁇ , and the culture was continued for 24 hr. Cell morphology was observed by Hochest staining, or proliferation of PC12 cells was detected by MTT assay.
  • ⁇ 1-methyl-4-phenyl-1, 2,3,6-tetrahydropyridine ( ⁇ ), which can specifically damage dopaminergic energy Neurons, which cause massive loss of nigral dopaminergic neurons, cause symptoms similar to Parkinson's disease (PD).
  • Tyrosine hydroxylase (TH) is a specific marker for dopaminergic neurons that can be used to quantify the number of dopaminergic neurons in the substantia nigra.
  • the molecular weight ratio of IL-22 monomer to IL-22-D is about 1:5, and one IL-22-D contains two IL-22 monomers, in order to compare the test results, equimolar is used in this test.
  • MPTP+IL-22 40 ⁇ g/kg group MPTP was injected intraperitoneally at a dose of 30 mg/kg. After 5 days of administration, after one day of animal recovery (ie from day 7), IL-22 (recombinant human interleukin) was administered subcutaneously at 40 ⁇ g/kg.
  • MPTP+IL-22-D 100 ⁇ g/kg group MPTP was intraperitoneally injected at a dose of 30 mg/kg for 5 consecutive days, after one day of recovery (ie from day 7), subcutaneous injection at 100 ⁇ g/kg IL-22-D was administered once on days 7, 9, and 11 respectively;
  • MPTP model group MPTP was intraperitoneally injected at a dose of 30 mg/kg for 5 consecutive days. After one day of recovery, an equal volume of solvent (0.5 mM mouse serum/PBS) was given from day 7;
  • Normal control group An equal volume of physiological saline was administered for 5 days, and after the animal was recovered for one day, an equal volume of solvent (0.5% mouse serum/PBS) was administered from the 7th day.
  • the IL-22 dimer (IL-22-D) consists of two sequences such as IL-22-Fc as shown in SEQ ID NO: 2.
  • IL-22-D may also be composed of two IL-22-Fc monomers selected from the group consisting of SEQ ID NO: 3-5.
  • Figure 8A shows immunohistochemical staining of TH-positive neurons in the substantia nigra pars compacta of mice. After the mice were injected with MPTP for 5 consecutive days, the TH-positive neurons in the substantia nigra pars compacta were largely lost, and the number of TH-positive neurons was significantly restored after administration of IL-22 or IL-22 dimer.
  • Figure 8B shows the results of TH positive cell count analysis in the substantia nigra parsing of mice. Compared with the normal control group, the number of TH-positive neurons in the substantia nigra pars compacta was significantly reduced in the MPTP model group, which was approximately the normal control group. 51%, indicating that MPTP causes a large loss of dopaminergic neurons in the substantia nigra.
  • TH-positive dopamine neurons in the substantia nigra were approximately 80% of the normal control group, and in the MPTP+IL-22-D 100 g/kg group, TH-positive dopamine in the substantia nigra The neurons were approximately 86% of the normal control group.
  • IL-22 and IL-22 dimers have protective effects on MPTP-induced massive loss of dopaminergic neurons (the protective effect of IL-22 dimer is more significant), and thus the MPTP-induced Parkinson's disease model.
  • Degenerative lesions of mouse brain dopamine neurons have therapeutic effects.

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Abstract

本发明公开了白介素-22在治疗和预防神经损伤疾病或神经退行性疾病中的用途。具体地，本发明公开了白介素-22或白介素-22二聚体的如下用途：（1）在动物缺血性神经损伤后，保护神经元，从而使损伤的神经功能得到恢复；（2）能够显著抑制黑质多巴胺能神经元的丢失，提高多巴胺能神经元的功能，减少海玛区神经元细胞的凋亡，从而治疗神经退行性疾病。

Description

白介素 -22在治疗和预防神经损伤疾病或神经退行性疾病中的用途 技术领域
本发明涉及生物和医药技术领域。 具体地， 本发明涉及白介素 -22在治疗和预 防神经损伤疾病或神经退行性疾病中的用途。 背景技术
白介素 -22(Interleukin-22， IL-22)是一种 T 细胞分泌的糖蛋白， 又名白介 素 10 诱导的 T 细胞因子（IL- 10- related T cell-derived inducible factor, IL-TIF)。白介素 22 mRNA的表达最初证明在小鼠的 IL-9剌激后的 T细胞株和 IL_9 剌激的肥大细胞株（mast cells), 以及 concanaval inA 激活后的脾细胞。 人白介 素 22 mRNA的表达主要在外周自分离的 T细胞并通过抗 CD-3的抗体或 ConA剌激。 白介素 22 mRNA在激活后的 NK细胞也有表达。 活化的 T细胞主要是 CD4+, 特别是 由 CD28通路的 Thl细胞。
白介素 -22 前体由 179 个氨基酸残基组成（成熟肽为 146 个氨基酸残基）， Dumoutier 等最先报道了克隆小鼠和人的白介素- 22 基因（Dumoutier 等， JI， 164:1814-1819， 2000)。 此外， Dumoutier 还取得了 IL-22 的专利（US6， 359， 117 和 6， 274， 710)。 Gurney 也取得了 IL-22 在治疗人胰腺病变方面的应用专利（US 6， 551， 799)。
白介素 -22主要在胸腺、 大脑、 活化的 T细胞和肥大细胞、 lectin剌激后的 脾细胞（Duroutier 2002)、 白介素 _2/白介素 -12剌激后的 NK细胞（Wolk， K / 2002), 以及 LPS剌激后的多个器官和组织中表达（Dumoutier PNAS paper), 包括 肠道、 肝、 胃、 肾、 肺、 心脏、 胸腺、 脾都可测到 IL-22的表达升高。
IL-22通过结合 IL-22RI受体和 IL-10R2受体， 发挥生物学功能。 IL-22R1是 IL-22特异的受体， 它表达在皮肤， 肾， 消化系统（胰腺、 小肠、 肝、 大肠、 结肠） 以及呼吸系统（肺、 支气管）。 已公开的研究表明白介素 22是一个免疫调节剂。 在 专利申请白介素 -22的医药用途中， 已报道 IL-22对降低血清甘油三酯， 肥胖的医 疗用途（见 W02006/073508, CN200510023103.0)。 然而， 目前并未发现白介素 -22 可以在治疗神经损伤疾病或神经退行性疾病中可发挥积极的作用。
神经退行性疾病（neurodegenerative disease)是大脑和脊髓的神经元细胞渐 进性变性、 死亡的状态， 是一种慢性、 进行性神经系统疾病， 主要包括阿尔茨海 默氏病（Alzhemier' s disease, AD)、 巾白金森氏病（Parkinson' s disease, PD)、 亨廷顿舞蹈病 (Huntington disease)、 肌萎缩侧索硬化症 (amyotrophic lateral sclerosis) , 脊髓肌萎缩病（spinal muscular atrophy)、 脊髓小脑共济失调 (spinal cerebellar ataxias)等， 其共同特征是发生神经元的退行性病变和凋亡， 导致病人行为异常和功能障碍， 引起过早死亡的神经性疾病。 神经退行性疾病的 发病机制尚不清楚， 至今还没有有效的方法和药物来防治， 现有治疗方法包括， 通过服用或静脉注射补充人大脑神经递质治疗 PD， 如左旋多巴， 但是， 左旋多巴 并不能有效的控制 PD的自然病变过程， 不能影响多巴胺能神经元的变性速度， 长 期使用还具有多种不良反应， 如开关现象和运动障碍， 其治疗作用也只能维持两 年左右， 长期使用还可损害神经元， 加速神经元的凋亡； 针对 AD脑内乙酰胆碱不 足， 用胆碱酯酶抑制剂提高其浓度， 该方法只是对症治疗， 不能控制疾病的发展。
综上， 本领域迫切需要开发有效的治疗和预防神经损伤疾病或神经退行性疾 病的药物和方法。 发明内容
本发明的目的就是一种有效的治疗和预防神经损伤疾病或神经退行性疾病的 药物及其用途， 即白介素 -22 (Interleukin-22， 即 IL-22)在治疗和预防哺乳动物 神经损伤疾病或神经退行性疾病中的用途。
在本发明的第一方面， 提供了一种白介素 -22或其二聚体或多聚体的用途， 它用 于制备治疗和预防神经损伤疾病或神经退行性疾病的药物。
在另一优选例中， 所述的神经损伤疾病选自： 中风、 脊柱损伤和伴有血脑屏障损 伤的神经系统疾病；
所述神经退行性疾病选自： 帕金森氏病、 阿尔茨海默氏病、 亨廷顿舞蹈病、 肌萎 缩侧索硬化病、 脊髓肌萎缩病、 原发性侧索硬化病、 或脊髓小脑共济失调。
在另一优选例中， 所述白介素 -22包括： 人白介素 -22或哺乳动物 (如鼠、 兔、 牛、 或羊）的白介素 -22。
在另一优选例中， 所述白介素 -22包括： 重组的白介素 -22、 或天然的白介素 -22。 在另一优选例中， 所述的白介素 -22的二聚体是式 I所示的白介素 -22的二聚体：
M1-L-M2 式 I
式中，
Ml是白介素 -22的第一单体；
M2是白介素 -22的第二单体；
L是位于所述的第一单体和第二单体之间的，将所述第一单体和第二单体连接在一 起的接头元件，
其中，所述的白介素 -22二聚体保持了白介素 22的生物活性， 并且其血清半衰期是 所述第一单体或第二单体的血清半衰期的 2倍以上。
在另一优选例中， 所述的 IL-22二聚体的血清半衰期是所述第一单体和 /或第二单 体的血清半衰期的 3倍以上、 5倍以上或 10倍以上。
在另一优选例中， 所述的接头元件 L选自下组： (i) 3-50个氨基酸的短肽；
(ϋ) 式 II所示的多肽元件：
-Z-Y-Z- 式 II
式中，
Y为载体蛋白；
Z为无、 或 1-30个氨基酸的短肽；
"-"为化学键或共价键。
在另一优选例中， 所述的第一单体和第二单体是相同的。
在另一优选例中， 所述的第一单体和第二单体是不同的。
在另一优选例中， 所述的生物活性是选自下组的一种或多种活性：
(a) 在体外激活神经元细胞 STAT3;
(b) 在体内脑缺血性损伤后， 能保护神经元， 减少脑梗死体积；
(c) 在体外激活多巴胺能神经元细胞 STAT3或海马神经元细胞 STAT3;
(d) 在体内能显著抑制 PD模型动物黑质多巴胺能神经元的丢失；
(e) 在体内能显著减少 AD模型动物海马区神经元细胞的凋亡。
在另一优选例中， 所述的载体蛋白是白蛋白或人 IgG的 Fc片段。
在另一优选例中， 所述的载体蛋白是二个 IgG的 Fc片段之间通过二硫键连接而形 成。 在另一优选例中， 所述二硫键的数量为 2-4个。
在另一优选例中， 所述的 "-"是肽键。
在另一优选例中， 所述二聚体是由氨基酸序列如 SEQ ID NO: 2-5所示的单体所构 成的二聚体。
在本发明的第二方面， 提供了式 I所示的白介素 -22的二聚体：
M1-L-M2 式 I
式中，
Ml是白介素 -22的第一单体；
M2是白介素 -22的第二单体；
L是位于所述的第一单体和第二单体之间的，将所述第一单体和第二单体连接在一 起的接头元件，
其中，所述的白介素 -22二聚体保持了白介素 22的生物活性， 并且其血清半衰期是 所述第一单体或第二单体的血清半衰期的 2倍以上。
在本发明的第三方面，提供了一种可用于治疗和预防神经损伤疾病或神经退行 性疾病的药物组合物， 它含有药学上可接受的载体以及式 I所示的白介素 -22的二聚 体，
M1-L-M2 式 I 式中，
Ml是白介素 -22的第一单体；
M2是白介素 -22的第二单体；
L是位于所述的第一单体和第二单体之间的，将所述第一单体和第二单体连接在一 起的接头元件，
其中，所述的白介素 -22二聚体保持了白介素 22的生物活性， 并且其血清半衰期是 所述第一单体或第二单体的血清半衰期的 2倍以上。
在另一优选例中， 所述二聚体是由氨基酸序列如 SEQ ID NO : 3或 5所示的单体所构 成的二聚体。
在另一优选例中， 所述 IL-22的二聚体采用以下步骤制备：
(a)采用包含编码 IL-22-Fc复合物的 DNA序列的表达载体转化哺乳动物细胞。
(b)培养所述哺乳动物细胞， 和
(c)分离纯化所述 IL-22二聚体。
本发明使用 IL-22二聚体分子， 一方面在动物体内缺血性神经损伤后， 可以保 护神经元， 从而使损伤的神经功能得到恢复， 能够有效地治疗神经损伤疾病； 另 一方面能够显著抑制 PD模型动物黑质多巴胺能神经元的丢失， 提高多巴胺能神经 元的功能。 此外， IL-22二聚体分子能够显著改善 AD模型大鼠学习记忆能力， 保护 神经元， 减少海马区神经元细胞的凋亡， 减轻痴呆的症状。 本发明的二聚体 IL-22 的血清半衰期延长， 有效阻止神经元的丢失， 从而能够更有效地治疗神经退行性 疾病。
应理解， 在本发明范围内中， 本发明的上述各技术特征和在下文（如实施例） 中具体描述的各技术特征可以互相组合， 从而构成新的或优选的技术方案。 限于 篇幅， 在此不再一一累述。 附图说明
图 1显示了本发明一种 IL-22二聚体的示意图。其中， " -" 表示连接肽， IL-22 椭圆形表示 IL-22单体。
该 IL-22二聚体的一种序列如 SEQ ID N0 : 1所示， 其中第 1-146位为 IL-22， 第 147-162位为连接肽， 第 163- 308位为另一 IL- 22。
图 2A和 2B显示了本发明 IL-22 二聚体的示意图。 其中， " -" 表示氨基酸连接 肽， IL-22椭圆形表示 IL-22单体， 标记为 " C " 的椭圆形表示载体蛋白， 并且 IL-22 位于载体蛋白的 N-末端。
一种用于构成该 IL-22二聚体的、 带有 Fc片段的 IL-22单体的序列如 SEQ ID NO : 2所示， 其中第 1-146位为 IL-22 , 第 147-162位为连接肽， 第 163-385位为人 IgG2 的 Fc片段。 二个带有 Fc片段的 IL-22单体， 通过 Fc的配对作用构成二聚体。 一种用于构成该 IL-22二聚体的、 带有 Fc片段的 IL-22单体的序列如 SEQ ID NO : 3所示， 其中第 1-146位为 IL-22 , 第 147-152位为连接肽， 第 153-375位为人 IgG2 的 Fc片段。 二个带有 Fc片段的 IL-22单体， 通过 Fc的配对作用构成二聚体。
图 3A和 3B显示了本发明 IL-22二聚体的示意图。 其中， " - " 表示氨基酸连接 肽， IL-22椭圆形表示 IL-22单体， 标记为 " C " 的椭圆形表示载体蛋白， 并且 IL-22 位于载体蛋白的 C-末端。
一种用于构成该 IL-22二聚体的、 带有 Fc片段的 IL-22单体的序列如 SEQ ID N0 : 4所示， 其中第 1-223位为人 IgG2的 Fc片段， 第 224-239位为连接肽， 第 240-385 位为 IL-22。 二个带有 Fc片段的 IL-22单体， 通过 Fc的配对作用构成二聚体。
一种用于构成该 IL-22二聚体的、 带有 Fc片段的 IL-22单体的序列如 SEQ ID N0 : 5所示， 其中第 1-223位为人 IgG2的 Fc片段， 第 224-229位为连接肽， 第 230-375 位为 IL-22。 二个带有 Fc片段的 IL-22单体， 通过 Fc的配对作用构成二聚体。
图 4显示了 IL-22和 IL-22二聚体（IL-22-Fc)对激活神经元细胞 STAT3的影响。 结果显示， IL-22和 IL-22 二聚体均具有激活神经元细胞 STAT3的生物活性， 而且， IL-22 二聚体比 IL-22具有更显著的生物活性。
图 5显示了本发明的 IL-22二聚体在局灶性脑缺血动物模型（Focal cerebral i schemia)中的治疗作用。 重组人 IL-22二聚体可以显著地减少脑梗死体积。
图 6显示了本发明的 IL-22二聚体在局灶性脑缺血动物模型（Focal cerebral i schemia)中的治疗作用。 重组人 IL-22二聚体可以显著地改善造模动物的神经功 能。
图 7显示了本发明的 IL-22二聚体、 IL-22单体的体外活性分析结果。
图 8A显示了小鼠黑质致密部 TH阳性神经元免疫组化染色图片。
图 8B显示了小鼠黑质致密部 TH阳性细胞计数分析结果。 具体实施方式
本发明人经过广泛而深入的研究， 首次意外地发现： 白介素 -22或白介素 -22 二聚体在神经损伤疾病或神经退行性疾病中表现出明显的治疗效果。 此外， 与 IL-22单体相比， 本发明的 IL-22二聚体能够延长体内半衰期， 改善药物的动力学， 减少注射频率， 并且具有显著增强的体内生物活性。 对神经元细胞 STAT3的激活作 用实验表明， 相对于 IL-22单体， IL-22二聚体在 IL-22摩尔终浓度相当于 IL-22单 体的终浓度的 1/10时， 可以使神经元 P-STAT3的信号水平上升更明显， 因此二聚体 有更明显的神经保护作用， 能够更有效地治疗神经损伤疾病； 还可有效地阻止神 经元的丢失， 从而能够更有效地治疗神经退行性疾病。 在此基础上完成了本发明。 术语 术语 "氨基酸序列基本相同" 是指序列相同或由一个或多个氨基酸改变（缺 失、 增加、 取代）引起的不同， 但这种改变基本上不降低其生物学活性， 即可以通 过结合 IL-22靶细胞受体而发生生物学功能。 任何符合 "基本相同" 要求的白介素 -22均包括在本发明内， 无论它是糖基化的（即来源于天然的或来源于真核生物表 达系统的）或是非糖基化的（即来源于原核生物表达系统或化学合成的）。
术语 "治疗" 是指基于治愈、 缓解、 改善、 减轻、 影响治疗对象疾病、 症状、 疾病体质（predisposition)的目的而给予需要治疗的对象本发明的白介素 -22。
术语 "治疗对象" 是指鼠、 人及其他哺乳动物。
术语 "治疗有效量" 是指能够在治疗对象体内达到治疗目的的白介素 -22的 量。 本领域的普通技术人员应理解， 所述 "治疗有效量" 可随白介素 -22的给药途 径、 所用药物辅料以及与其他药物联合用药情况的不同而有所不同。 白介素 22及其制备
如本发明所用， "白介素 -22 " 或 " IL-22 " 是指一种蛋白质， 该蛋白质（a)具 有与 Dumout ier等人在 US359, 117中描述的人 /鼠白介素 -22基本相同的氨基酸序列 和（b)具有与天然白介素 -22相同的生物学活性。 本发明的白介素 -22包括， 但不限 于： 人白介素 -22、 重组人白介素、 鼠白介素 -22和 /或重组鼠白介素 -22。 在本发 明中， 术语 " IL-22 " 可包括单体、 二聚体或多聚体形式的 IL-22。
"白介素 -22 " 还包括 PEG化的 IL-22以及共价修饰的 IL-22蛋白质。 例如， 可 用各种活化的分子量为 5， 000〜 100， 000的聚乙二醇（PEG)修饰以使 IL-22高分子 化，延长其半衰期。具体操作可参见 Greenwald等， Bioorg. Med. Chem. Lett. 1994， 4， 2465； Cal icet i等， IL Farmaco , 1993， 48， 919； Zal ipsky and Lee , 《聚 乙二醇化学： 生物技术与生物医学应用》 ， J. M. Harri s编， Plenum Press , N. Y. ， 1992。优选使用多臂树杈型活性 PEG (CNZL02101672. 0、 W09932139、 PCT/US95/0755, PCT/US94/13013、 US4， 640， 835、 US 4， 496， 689、 US 4， 301， 144、 US 4， 670， 417、 US 4， 791， 192、 US 4， 179， 337)。
本发明的白介素 -22可用基因重组技术克隆表达的。 表达的宿主细胞包括原核 细胞、 酵母细胞或者高等真核生物细胞。 适用的原核宿主细胞包括， 但不限于： G+或 G—菌， 如大肠杆菌 A coli. , 能够通过公共途径得到的 £ co i.菌株包括： K12 MM294 (ATCC 31, 446)、 X1776 (ATCC 31, 537)、 W3110 (ATCC 27， 325)和 K5 772 (ATCC 53， 635)等。 其他可用的原核细胞包括但不限于： 欧式杆菌（^^i w'a)、 克雷伯杆 mUebsiella)、 变形杆菌（ ^O e^)等。 E. coli. W3110因其常被用作重组 DNA 产品的发酵宿主而被推荐为优选。
除了原核细胞， 真核细胞如丝状真菌（ Ϊ 或酵母菌（j¾^ ) 等同样适用于表达或者克隆本发明的白介素 -22。 酿酒酵母菌（6¾c^ar0 ^C^)就 是一种常用的低等真核宿主微生物， 其他宿主如非洲粟酒裂殖酵母菌； 克鲁维酵 母菌； 巴氏毕赤酵母菌等。 甲基营养酵母菌（ e Ar 0 r0 iC yeas^)同样可用于 表达本发明的白介素 -22， 包括但不限于各种能够在甲醇中生长的酵母菌如汉逊酵 母菌 iMansenul )， 念珠菌（ Candida)， 克勒克酵母菌 Udoecker )， 毕赤酵母菌 {Pichia)， 酿酒酵母菌 ^Saccharoniyces)等。
用于表达糖基化的本发明的白介素 -22的宿主细胞可来源于多细胞有机体。 无 脊椎动物细胞的例子包括昆虫细胞如 DrosophilaS2和 SpodopteraSf9， 植物细胞。 适用的哺乳动物宿主细胞的例子包括中华仓鼠卵巢细胞（CH0)， COS细胞。 特别是， 经 SV40转化的猴肾 CV1细胞株（C0S-7, ATCC CRL 1651)； 人胚胎肾细胞株 293等。 本领域的普通技术人员应知如何选择合适的宿主细胞。
上述宿主细胞经白介素 -22表达载体或克隆载体转染或者转化后可在传统的 营养基（nutrient media) 中培养， 所述营养基经修饰后适于诱导启动子 (promoter)、 选择性转化体（selecting transf ormant)或者扩增白介素 -22编码基 因序列。 培养条件如培养基、 温度、 pH等的选择对本领域的普通技术人员来说则 是应知的。 如何使细胞培养繁殖力最大化的一般原则、 方案以及操作技术可参见 Mammal i an Cell Biotechnology： a Practical Approach, M. Butler, ed. (IRL Press, 1991) 禾口 Sambrook 等， supra。
本领域的普通技术人员应熟知真核细胞转染和原核细胞转化的方法， 例如
CaCl2法、 磷酸钙沉淀法、 脂质体媒介法或电穿孔法。 根据使用的宿主细胞的不同， 本领域的普通技术人员可选择相应的标准转化技术， 例如 CaCl2法（Sambrook等， supra. )或电穿孔法通常用于原核细胞； 对于没有细胞壁的哺乳动物细胞， 则可以 使用磷酸钙沉淀法。
编码本发明的白介素 -22的核苷酸序列（gPcDNA或基因组 DNA)可被插入一可复 制载体（replicable vector)进行基因克隆（DNA扩增）或者表达。 各种载体如质粒、 粘粒（cosmic!, 装配型质粒）、 病毒颗粒或噬菌体等均可通过公共途径获得。 运用 本领域的公知技术， 可将本发明的白介素 -22的编码核苷酸序列按常规步骤插入可 复制载体上适宜的限制性核酸内切酶位点。
本发明的白介素 -22不但可以通过基因重组直接表达， 也可以通过与异种多肽 形成融和多肽的方式生产， 后者可以是一段位于成熟蛋白质或多肽 N端的信号序 列， 也可以是位于成熟蛋白质或多肽 N端的具有特异性切割位点的其他多肽片段。 通常情况下， 这段信号序列是上述可复制载体的一部分， 或者它也可以是插入可 复制载体的本发明的白介素 -22编码核苷酸序列的一部分。
表达载体和克隆载体均含有一段核苷酸序列， 该序列使载体能够在一个或多 个相应的宿主细胞内复制。 与各种细菌、 酵母或病毒宿主细胞对应的核苷酸序列 为本领域普通技术人员所公知。例如，质粒 pBR322的复制起点适用于大多数 G—细菌， 2. mu.质粒复制起点适用于酵母细胞， 而各种病毒复制起点（SV40， 多形瘤病毒， 腺病毒， VSV或 BPV)则适用于哺乳动物细胞内的克隆载体。
表达载体和克隆载体通常含有一段选择基因， 又名 "选择标记" 。 典型的选 择基因编码产生的蛋白质（a)对某些抗生素或毒素如氨苄青霉素， 新霉素， 氨甲蝶 呤，四环素等具有抗性； （b)能弥补营养缺陷型缺乏（auxotrophic deficiencies) (c) 补充复合型培养基不能提供的关键营养物质如杆菌宿主细胞需要的 D-丙氨酸消旋 酶的编码基因。
适用于哺乳动物宿主细胞的选择基因应该可以将有能力吸纳本发明的白介素 -22编码基因的宿主细胞区分出来， 如 DHFR或胸（腺嘧啶核）苷激酶。 适用的以野生 型 DHFR为选择基因的宿主细胞是无 DHFR活性的 CH0细胞株， 该细胞株的制备及繁殖 方法可参见 Urlaub等， Proc. Natl. Acad. Sci. USA, 77:4216(1980)。 适用于酵 母细胞的选择基因是在酵母质粒 Yrp7中表达的 trpl基因（Stinchcomb等， Nature, 282:39(1979) ； Kingsman等， Gene , 7:141(1979) ； Tschemper等， Gene , 10:157(1980))。 trpl基因可用于筛选不能在色氨酸中生长的酵母菌突变株如 ATCC Νο· 44047或 PEP4-1 (Jones, Genetics, 85:12 (1977))。
表达载体和克隆载体通常含有一个可人工操纵地连接到本发明的白介素 -22 编码核苷酸序列上的启动子， 以指导 mRNA的合成。 与各种宿主细胞对应的启动子 为本领域普通技术人员所公知。 适用于原核宿主细胞的启动子包括 β -内酰胺酶和 乳糖启动子系统等。 细菌宿主细胞启动子同样包含一段可人工操纵地连接到本发 明的白介素 -22编码基因序列的 Shine-Dalgarno (S. D. )序列。
通过在可复制载体中插入增强子可增强本发明的白介素 -22的编码核苷酸序 列在高等真核生物表达体系中的转录。 增强子是一种 DNA分子的顺式作用元件， 通 常为 10到 300个 bp， 通过作用于启动子而增强 DNA分子的转录。
真核宿主细胞（酵母细胞、 真菌细胞、 昆虫细胞、 植物细胞、 动物细胞、 人类 细胞， 或者来源于其他多细胞有机体的有核细胞）中的表达载体同样含有中止转录 和稳定 mRNA所需的核苷酸序列。 此类序列通常取自真核或者病毒 DNA或 cDNA非翻译 区的 5' 端， 有时也可取自 3' 端。 所述 "非翻译区" 包含的核苷酸片段可转录产 生位于本发明的白介素 -22 mRNA非翻译区的聚腺苷酰化片段。
其他可在重组脊椎动物培养体系中用于合成本发明的白介素 -22的方法、 载体 和宿主细胞可参见 Gething等， Nature, 293:620-625 (1981)； Mantei等， Nature, 281:40-46 (1979)； EP 117, 060； 以及 EP 117,058。
IL-22二聚体
本发明的 IL-22二聚体的结构如式 I所示。代表性的结构如图 1-3中所示。其中， 载体蛋白包括（但并不限于）是指人 IgG (l，2，3，4)的 Fc片段、 或人白蛋白 (albumin)。
IL-22位于可用载体蛋白的 C-末端， 也可用位于载体蛋白的 N-末端。
如本文所用， 术语 "连接肽" （l inker)指位于 IL-22单体和 IL-22单体之间的、 起连接作用的短肽。 连接肽的长度没有特别限制。 连接肽的长度通常为 5-50个氨 基酸。 通常， 连接肽不影响或不显著影响 IL-22单体和 IL-22单体形成正确的折叠 和空间构象。 一些连接肽的例子包括（但并不限于）：
较佳地， 所述的连接肽具有选自下组的氨基酸序列：
(a) 疏水性氨基酸 Gly和 Pro构成 的 3-16个氨基酸序列 ， 例 如 Gly - Pro - Gly - Pro - Gly - Pro；
(b)多克隆位点所编码的氨基酸序列。 该序列通常为 5-20个， 较佳地 10-20个 氨基酸；
(c)来自于 IL-22单体之外的蛋白的氨基酸序列， 例如来自于 IgG或白蛋白的氨 基酸序列；
(d)由（a)、 （b)和（c)组合形成的氨基酸序列。
优选的连接肽包括： GSGGGSGGGGSGGGGS (SEQ ID NO : 1中第 147- 162位）和 ASTKGP (SEQ ID NO : 3中第 147- 152位）。
此外， 在融合蛋白的 N端或 C末端还可添加其他不影响 IL-22单体活性的氨基酸 序列。 较佳地， 这些添加的氨基酸序列有利于表达（如信号肽）， 有利于纯化（如 6 X His序列、 酿酒酵母 α -因子信号肽切割位点（Glu-Lys-Arg)， 或可促进融合蛋 白的活性。 二聚体的制备方法
编码本发明 IL-22二聚体或融合蛋白的 DNA序列， 可以全部人工合成。 也可用 PCR扩增或合成的方法获得 IL-22第一单体和 /或 IL-22第二单体的编码 DNA序列， 然 后将其拼接在一起， 形成编码本发明融合蛋白的 DNA序列。
为了提高宿主细胞的表达量， 可以对 IL-22二聚体编码序列进行改造， 例如采 用宿主细胞偏好的密码子， 消除不利于基因转录及翻译的序列。 在本发明中， 可 以采用酵母细胞或哺乳动物细胞偏好的密码子， 并采用计算机 DNA软件对 IL-22二 聚体基因进行检测， 排除在基因中不利于基因转录及翻译的序列， 包括内含子剪 切位点， 转录终止序列等。
在获得了编码本发明新融合蛋白的 DNA序列之后， 将其连入合适的表达载体， 再转入合适的宿主细胞。 最后， 培养转化后的宿主细胞， 通过分离纯化得到本发 明的新的融合蛋白。
如本文所用， 术语 "载体" 包括质粒、 粘粒、 表达载体、 克隆载体、 病毒载 体等。
在本发明中， 可选用本领域已知的各种载体如市售的载体。 比如， 选用市售 的载体， 然后将编码本发明新融合蛋白的核苷酸序列可操作地连于表达调控序列， 可以形成蛋白表达载体。
如本文所用， "可操作地连于" 指这样一种状况， 即线性 DNA序列的某些部分 能够影响同一线性 DNA序列其他部分的活性。 例如， 如果信号肽 DNA作为前体表达 并参与多肽的分泌， 那么信号肽（分泌前导序列） DNA就是可操作地连于多肽 DNA ;如 果启动子控制序列的转录， 那么它是可操作地连于编码序列；如果核糖体结合位点 被置于能使其翻译的位置时， 那么它是可操作地连于编码序列。 一般， "可操作 地连于" 意味着相邻近， 而对于分泌前导序列则意味着在阅读框中相邻。
在本发明中， 术语 "宿主细胞" 包括原核细胞和真核细胞。 常用的原核宿主 细胞的例子包括大肠杆菌、 枯草杆菌等。 常用的真核宿主细胞包括酵母细胞， 昆 虫细胞、 和哺乳动物细胞等。 较佳地， 该宿主细胞是真核细胞， 更佳地是哺乳动 物细胞。
在获得转化的宿主细胞后， 可在适合表达本发明融合蛋白的条件下培养该细 胞， 从而表达出融合蛋白。 然后再分离出表达的融合蛋白。 药物组合物和施用方法
由于本发明 IL-22二聚体可产生更强的受体激活信号并具有优异的血清半衰 期， 因此本发明 IL-22二聚体以及含有本发明 IL-22二聚体为主要活性成分的药物组 合物可用于治疗和预防神经损伤疾病或神经退行性疾病。 其中， 所述的神经损伤疾 病包括： 中风、 脊柱损伤和伴有血脑屏障损伤的神经系统疾病； 所述神经退行性疾病 选自： 帕金森氏病、 阿尔茨海默氏病、 亨廷顿舞蹈病、 肌萎缩侧索硬化病、 脊髓肌萎 缩病、 原发性侧索硬化病、 或脊髓小脑共济失调。
本发明的药物组合物包含安全、 有效量范围内的本发明 IL-22或其二聚体及药 理上可以接受的赋形剂或载体。 其中 "安全、 有效量" 指的是： 化合物的量足以明 显改善病情， 而不至于产生严重的副作用。 通常， 药物组合物含有 0. 001-lOOOmg 的 IL-22或其二聚体 /剂， 较佳地 0. 05-300 mg的 IL-22或其二聚体 /剂， 更佳地， 含 有 0. 5-200mg的 IL-22或其二聚体 /剂。
本发明的化合物及其药理上可接受的盐可制成各种制剂， 其中包含安全、 有效 量范围内的本发明 IL-22或其二聚体或其药理上可接受的盐及药理上可以接受的赋 形剂或载体。 其中 "安全、 有效量" 指的是： 化合物的量足以明显改善病情， 而不 至于产生严重的副作用。 化合物的安全、 有效量根据治疗对象的年龄、 病情、 疗程 等具体情况来确定。
"药理上可以接受的赋形剂或载体"指的是： 一种或多种相容性固体或液体填 料或凝胶物质， 它们适合于人使用， 而且必须有足够的纯度和足够低的毒性。 "相 容性"在此指的是组合物中各组份能与本发明的化合物以及它们之间相互惨和， 而 不明显降低化合物的药效。 药理上可以接受的赋形剂或载体部分例子有纤维素及其 衍生物（如羧甲基纤维素钠、 乙基纤维素钠、 纤维素乙酸酯等）、 明胶、 滑石、 固体 润滑剂（如硬脂酸、 硬脂酸镁）、 硫酸钙、 植物油（如豆油、 芝麻油、 花生油、 橄榄 油等）、 多元醇（如丙二醇、 甘油、 甘露醇、 山梨醇等）、 乳化剂（如吐温 ®)、 润湿剂 (如十二垸基硫酸钠）、 着色剂、 调味剂、 稳定剂、 抗氧化剂、 防腐剂、 无热原水等。
施用本发明 IL-22或其二聚体时， 可以口服、 直肠、 肠胃外（静脉内、 肌肉内或 皮下）、 局部给药。
用于口服给药的固体剂型包括胶囊剂、 片剂、 丸剂、 散剂和颗粒剂。 在这些固 体剂型中， 活性化合物与至少一种常规惰性赋形剂（或载体)混合， 如柠檬酸钠或磷 酸二钙， 或与下述成分混合： （a) 填料或增容剂， 例如， 淀粉、 乳糖、 蔗糖、 葡萄 糖、 甘露醇和硅酸； （b) 粘合剂， 例如， 羟甲基纤维素、 藻酸盐、 明胶、 聚乙烯基 吡咯垸酮、 蔗糖和阿拉伯胶； （c)保湿剂， 例如， 甘油； （d)崩解剂， 例如， 琼脂、 碳酸钙、 马铃薯淀粉或木薯淀粉、 藻酸、 某些复合硅酸盐、 和碳酸钠； （e)缓溶剂， 例如石蜡； （f) 吸收加速剂， 例如， 季胺化合物； （g) 润湿剂， 例如鲸蜡醇和单硬 脂酸甘油酯； （h) 吸附剂， 例如， 高岭土； 和（i)润滑剂， 例如， 滑石、 硬脂酸钙、 硬脂酸镁、 固体聚乙二醇、 十二垸基硫酸钠， 或其混合物。 胶囊剂、 片剂和丸剂中， 剂型也可包含缓冲剂。
固体剂型如片剂、 糖丸、 胶囊剂、 丸剂和颗粒剂可采用包衣和壳材制备， 如肠 衣和其它本领域公知的材料。 它们可包含不透明剂， 并且， 这种组合物中活性化合 物或化合物的释放可以延迟的方式在消化道内的某一部分中释放。 可采用的包埋组 分的实例是聚合物质和蜡类物质。 必要时， 活性化合物也可与上述赋形剂中的一种 或多种形成微胶囊形式。
用于口服给药的液体剂型包括药学上可接受的乳液、 溶液、 悬浮液、 糖浆或酊 剂。 除了活性化合物外， 液体剂型可包含本领域中常规采用的惰性稀释剂， 如水或 其它溶剂， 增溶剂和乳化剂， 例知， 乙醇、 异丙醇、 碳酸乙酯、 乙酸乙酯、 丙二醇、 1，3-丁二醇、 二甲基甲酰胺以及油， 特别是棉籽油、 花生油、 玉米胚油、 橄榄油、 蓖麻油和芝麻油或这些物质的混合物等。
除了这些惰性稀释剂外， 组合物也可包含助剂， 如润湿剂、 乳化剂和悬浮剂、 甜味剂、 娇味剂和香料。
除了活性化合物外， 悬浮液可包含悬浮剂， 例如， 乙氧基化异十八垸醇、 聚氧 乙烯山梨醇和脱水山梨醇酯、 微晶纤维素、 甲醇铝和琼脂或这些物质的混合物等。
用于肠胃外注射的组合物可包含生理上可接受的无菌含水或无水溶液、 分散 液、 悬浮液或乳液， 和用于重新溶解成无菌的可注射溶液或分散液的无菌粉末。 适 宜的含水和非水载体、 稀释剂、 溶剂或赋形剂包括水、 乙醇、 多元醇及其适宜的混 合物。
用于局部给药的本发明 IL-22或其二聚体的剂型包括软膏剂、 散剂、 贴剂、 喷 射剂和吸入剂。 活性成分在无菌条件下与生理上可接受的载体及任何防腐剂、 缓冲 剂， 或必要时可能需要的推进剂一起混合。
本发明 IL-22或其二聚体可以单独给药， 或者与其他药学上可接受的化合物联 合给药。
含有本发明的白介素 -22或其二聚体的微胶囊可用于本发明的白介素 -22的缓 释给药。 重组蛋白的微囊缓释给药技术已成功应用于重组人生长激素（rhGH)、 重 组人干扰素（rhIFN)、 白介素 - 2和 MNrgpl20(Johnson等， Nat. Med. , 2:795-799 (1996)； Yasuda, Biomed. Ther 27:1221-1223 (1993)； W097/03692, W096/40072, WO 96/07399； US5654010。
本发明的白介素 -22或其二聚体的缓释制剂可用具有良好生物兼容性和宽泛 生物可降解性的乳酸羟基乙酸高聚物（PLGA)制备。 PLGA的降解产物， 乳酸和羟基 乙酸可被人体很快清除。 而且， 该高聚物的降解能力可随其分子量和组成的不同， 从几个月延长到几年 (Lewis, "Controlled release of bioactive agents form lactide/ glycol ide polymer, " in: M. Chasin and R. Langer (Eds. ) ， Biodegradable Polymers as Drug Delivery Systems (Marcel Dekker： New York, 1990)， pp. 1-41))。
本发明的药物组合物的剂量和浓度范围可因实际使用情况的不同而变化。 本 领域的普通技术人员应知如何根据实际需求去选择合适的剂量和给药途径。 对于 不同种系， 例如人和鼠之间药物剂量范围的调整原则， 可参见 Mordenti, J. and Chappell, W. "The use of interspecies scaling in toxicokinetics" In Toxicokinetics and New Drug Development, Yacobi等； Pergamon Press, New York 1989， pp. 42-96。
使用药物组合物时， 是将安全有效量的本发明 IL-22或其二聚体适用于需要治 疗的哺乳动物（如人）， 其中施用时剂量为药学上认为的有效给药剂量， 对于 60kg 体重的人而言， 每次给药剂量通常为 0.01〜300mg， 优选 0.5〜100mg。 当然， 具体 剂量还应考虑给药途径、 病人健康状况等因素， 这些都是熟练医师技能范围之内 的。
本发明的主要优点在于：
1. IL-22或其二聚体在体外能激活神经细胞 STAT3。
2. IL-22二聚体在动物模型已证实可有效治疗神经损伤疾病。 3. IL-22 二聚体能够延长体内半衰期， 改善药物的动力学， 减少注射频率； 能显著增强体内生物活性。
4. IL-22或 IL-22二聚体在显著抑制 PD模型动物黑质多巴胺能神经元的丢失， 激发多巴胺能神经元的功能。
5. IL-22或 IL-22二聚体显著抑制海马区神经元细胞的凋亡， 改善 AD模型动 物的学习记忆能力。
6. IL-22或 IL-22二聚体在神经退行性疾病中具有显著的神经保护作用， 有效 治疗神经退行性疾病。 下面结合具体实施例， 进一步阐述本发明。 应理解， 这些实施例仅用于说明 本发明而不用于限制本发明的范围。 下列实施例中未注明具体条件的实验方法， 通常按照常规条件如 Sambrook等人，分子克隆：实验室手册（New York： Cold Spring Harbor Laboratory Press , 1989)中所述的条件， 或按照制造厂商所建议的条件。 实施例 1
用常规方法制备和纯化， 获得结构如图 1-图 3所示的 IL-22二聚体（序列为 SEQ ID NO : 1， 或单体序列如 SEQ ID NO : 2-5所示）。 例如， 由 IL-22-Fc复合物制备 IL-22 二聚体， 具体制备方法如下：
a. IL-22 二聚体表达细胞株的构建
采用全基因合成 IL-22-Fc复合物的 cDNA序列（如 SEQ ID NO : 6或 SEQ ID NO : 7 所示， 其中 SEQ ID NO.： 6编码 SEQ ID NO.： 2所示的单体， SEQ ID NO.： 7编码 SEQ ID NO.： 3所示的单体）， 将人 IL-22单体的基因与 IgG2的 Fc基因片段连接， 5 ' 端 引入 EcoRI位点， 以及哺乳动物细胞表达所需要的元件如 Kozak序列和信号肽序列， 3 ' 端引入 Xbal位点， 克隆到市售的 pUC19质粒， 命名为 pIL-22-Fc， 转化常规的 E. col i TG1。 用 EcoRI和 Xbal酶切 pUC19质粒， 回收约 1300bp的 IL-22-Fc片段， 与经 EcoRI和 Xbal酶切后的 pcDNA3 (Invitrogen)表达质粒相连接， 构建表达质粒 pEX-IL-22-Fc。 表达质粒 pEX-IL-22-Fc通过线性化转染 CH0细胞， 表达 IL-22二聚 体， ELISA法检测表达量， 筛选出蛋白产量较高的细胞株， 并制备细胞库。
b . IL-22二聚体的分离纯化
将重组 CH0细胞用常规方法进行培养从而表达重组蛋白。 培养结束后， 收集细 胞上清（含有 IL-22复合物、 IL-22二聚体、 IL-22多聚体及代谢物）， 细胞上清收集 后经过滤、 多级凝胶层析纯化， 例如， rProtein A S印 harose FF (GE Healthcare, 货号： 17- 1279- 04)捕获， 用 20- 50 mM柠檬酸缓冲液， 0. 1- 2M NaCl , pH 3. 5-3. 8 的缓冲液洗脱， 得到纯度〉 90%的 IL-22二聚体， 接下来采用 PPA复合介质的凝胶层 析（PALL Life Sciences 货号： k364- 01)， 用 20- 50 mM NaAc/HAC, pH 3. 0-5. 0的 缓冲液洗脱， 洗脱液经低 pH灭活病毒， Nano20膜除病毒过滤等， 最终获得 IL-22二 聚体。
分离纯化得到的 IL-22二聚体的纯度 >95% (采用反向 HPLC分析）。 电泳显示， 纯 化后 IL-22二聚体（由二个 SEQ ID NO.： 2所示单体构成）分子量为 52 ± 10KD (采用还 原型 SDS-PAGE分析）， 与预测值相符。 紫外吸收光谱为 280nm。 IL-22二聚体在体外 能剌激 Colo205细胞产生 IL-10(ED50为 10-1000 ng/mL)。
实施例 2 IL-22二聚体体内半衰期
大鼠皮下单次注射 IL-22二聚体（由二个序列如 SEQ ID N0:2所示的 IL-22-Fc 单体构成） 100 g/kg, 药代动力学参数如下表 1所示（n=6)。 而单体 IL-22 (重组 人白介素 22) 在大鼠的半衰期约 1.3小时。
表 1
参数 单位 平均值 SD
AUC(O-t) ng/mL*h 4216.7 638.3
MRT(O-t) H 22.6 1.6
Figure imgf000015_0001
CLz/F L/h/kg 0.028 0.003
Cmax ng/mL 153.2 26.2 实施例 3 IL-22或 IL-22二聚体对神经元细胞 STAT3的激活作用
取孕 17天 SD大鼠的胎鼠的全脑放入预冷的 D-Hanks液中， 在解剖显微镜下取大 脑皮层并除去脑膜。 剪碎脑组织成 1匪3大小， 加入 10 mLO.125%胰酶， 并放入 37°C 孵箱内消化 15 min, 随后吸出组织转移到装有预冷的 DMEM+10%FBS的离心管内终 止消化， 用移液枪吹打数次， 静置后取上清并吸到另一支离心管内。 重复上述步 骤 2〜3次。
用无血清神经元基础培养基 neurobasal(invitrogen， 货号: 21103049)+无血 清添加剂 B27(invitrogen， 货号： 17504044)培养 8天， 每 2天换液一次。
培养至第八天， 分别用不同浓度 IL-22二聚体（由二个序列如 SEQ ID N0:2所示 的 IL-22-Fc单体构成）和 IL-22(IL-22二聚体的终浓度： 0.3 μ g/mL; IL-22的终浓 度： 1.2 μ g/mL)处理神经元 15分钟。 将含药物的培养基上清完全吸出， 细胞用 PBS 洗两遍， 按照细胞裂解液使用说明步骤裂解细胞。 用细胞裂解液（Cell Signaling Technology, 货号： 9803； 主要成分： 20 mM Tris- HC1 (pH 7.5)， 150 mM NaCl， 1 mM Na2EDTA， 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM beta- glycerophosphate, 1 mM Na3V04， 1 g/ml leupeptin, 1 mM PMSF)冰上裂 解细胞 20分钟， 用细胞刮子刮取细胞。 收集细胞裂解液， 12000rpm， 4°C离心 10分 钟。
吸取上清， 测定蛋白浓度。 另取 Ιθθμΐ的上清用 STAT3 [pY705] ELISA试剂盒 (invitrogen, 货号： KH00481)检测 STAT磷酸化水平变化。
结果如图 4所示： 与空白对照组相比， IL-22、 IL-22二聚体均能激活神经元细 胞信号转导及转录活化因子 3 (STAT3)的生物活性。 其中， IL-22单体（终浓度为 1. 2 4 §/1^)可使神经元 -51 13的信号水平上升5. 4倍， 而 IL-22二聚体， 其终浓度 仅为 0. 3 w g/mL， 相当于 IL-22单体的终浓度的 1/10， 而且可以使神经元 p-STAT3的 信号水平上升 7. 8倍， 可见， IL-22二聚体对神经元细胞 STAT3的激活作用显著优于 IL-22单体。 实施例 4 IL-22二聚体在局灶性脑缺血动物模型（Focal cerebral i schemia) 中的治疗作用
雄性 SD大鼠， 体重为 250〜300g。 动物分成 3组：模型组（MCA0+溶剂， n=12)， IL- 22二聚体组（MCA0+IL- 22- D100 μ g/kg, IL- 22- D由二个序列如 SEQ ID N0 : 2 所 示的 IL-22-Fc单体构成）， 假手术组（手术 +溶剂， n=12)。
麻醉后， 大鼠的右颈总动脉（CCA)、 右颈内动脉（ICA)、 右颈外动脉（ECA)通过 颈部手术暴露在外。 一种头部硅胶包裹的线栓通过大鼠颈外动脉插入颈总动脉再 回拨至颈内动脉进行阻塞， 插入长度大约 18. 5 ± 0. 5mm。 插入后感觉略微受阻， 表 明已到达中动脉处，能够阻塞中动脉的血流供应。阻塞 60min后，线栓被拔出约 10mm 左右进行再灌注。 IL-22二聚体组分别于再灌注 0. 5h和 48h时， 皮下注射 IL-22二聚 体 100 μ g/kg, 假手术组和模型组皮下注射相同体积的溶剂。 整个手术过程中， 动物体温被保持在 36. 5 。 假手术组只切开颈部皮肤分离颈部动脉， 不插入线栓。 21天后动物被安乐处死取出大脑。
用 2mm的冠状切片模具将大脑切成 6片， 放在 2%TTC溶液中， 37°C避光温孵 20-30min,最后固定在 10%福尔马林溶液中。固定后将这些脑切片用数码相机拍照， 白色未染上的部分为梗死区。 然后计算其体积百分比％= (对侧脑半球面积-同侧脑 半球未梗死部位面积）/对侧脑半球面积 *100%。 梗死面积的测量用 Photoshop 7. 0 软件完成。
神经学症状的临床评价
神经功能学分数在手术的 0天， 1天， 2天， 3天或者第 0、 1、 2、 3、 7、 14、 21 天被评价， 其分数分级标准为： 0-无神经功能性损伤； 1-左前肢不能伸展； 2-不 连续地向左边转圈； 3-连续的向左边转圈； 4-向左侧倾倒； 5-神经意识丧失， 不 能自发行走。
结果如图 5-6所示， 模型组梗死体积为 54. 7%。 与模型组比较， 注射 IL-22二聚 体后显示出了显著的治疗效果（P<0. 01)， 其梗死体积为 33. 8%， 相对于模型组， IL-22二聚体组的梗死体积减少了 20.9%。 在术后第三天， IL-22二聚体组与模型组 相比， 神经功能评分有显著性差异（P<0.05)， 提示 IL-22二聚体可以保护神经的坏 死， 促进神经功能的恢复。 实施例 5 IL-22二聚体在食蟹猴体内的药代动力学
成年健康猕猴， 8只， 雌雄各半， 体重 3-5公斤， 按动物体重随机分成 2组， 分 别为 IL-22二聚体 30、 100 w g/kg给药组， 其中给药组各组 4只 /组， 雌雄各半。 分 别皮下注射给予相应剂量的 IL-22二聚体（由二个序列如 SEQ ID NO.： 2所示的 IL-22-Fc单体构成）， 给药体积 0.2 ml/kg体重， 单次给药， 于给药前、 给药后 0.5、 1、 2、 4、 8、 16、 24、 48、 72、 96、 120、 144、 168小时下肢隐静脉取血 0.6ml， 室温静置 30min后， 分离血清， 采用 ELISA试剂盒（Biolegend, 货号: 434507)检测 血清中的 IL-22二聚体浓度， 检测结果采用非房室模型分析药代动力学参数， 结果 如下表 2所示。 IL-22在食蟹猴体内的半衰期（tl/2z)约为 2hr。
表 2 药代动力学参数（均值士 SD， n=4)
给药 AUC (0 - t) MRT (0 - t) Tmax CLz/F Cmax 剂量 mg/L*hr hr hr hr mL/h/kg ng/mL
3(^g/kg 11.92±0· 91 50.5±5 63.3±38· 9 17±9· 5 2±1 172.3±17.1
10(^g/kg 39.9±6· 2 51.1±4· 7 65.6±10.9 24±0 2±0 506.9±115.7 实施例 6 IL-22二聚体、 IL-22的体外活性分析
本实施例利用某些细胞在受到 IL-22剌激时， 会产生 IL-10, 通过检测相应的 0D值， 从而测定 IL-22的活性。 方法如下：
Colo205细胞（ATCC号 CCL-222, 人结肠癌细胞）培养于 RPMI164010%FBS培养 基中，细胞生长至对数期时，弃上清，加入 PBS洗去残留培养基，加入 2〜5mL 0.25% 胰蛋白酶 -EDTA消化， 加入培养基吹打均匀， 1500rpm离心 5min， 收集细胞， 然后 用基础培养基配制成 5.0X105Cell/ml 的细胞悬液， 于 96 孔板各孔分别添加 ΙΟΟμΐν孔， 于 37°C、 5%C02培养箱中过夜。 次日， 取出 C02培养箱中的 96孔板， 于 4°C条件下， 800rpm离心 5分钟， 从各孔抽出细胞上清液 90 μ L， 并补入 90wL 0.1%BSA/RPMI1640, 加入 IL-22二聚体（由二个 SEQ ID NO.： 2所示单体构成）至 终浓度 1.4、 4.1、 12.3、 37.0、 111.1、 333.3、 1000、 3000ng/mL, 加入 IL- 22至 终浓度 0.01、 0.04、 0.12、 0.37、 1.1、 3.3、 10、 30ng/mL, 于 37°C， 5%C02培养 箱中培养 20hr， 收集细胞上清液， 采用 IL-22ELISA试剂盒（R&D， 货号 S1000B)检 测 0D值。
结果如 图 7 所示 ， IL-22 二聚体的半数有效浓度 （ED50) 值为 229ng/mL (2675pM)， IL-22的 ED50为 0. 54ng/mL (32. 4pM)。
以上结果显示， 虽然体外实验中 IL-22活性略优于 IL-22二聚体的活性， 而 IL-22二聚体在体内的药代动力学参数和有效活性远远优于 IL-22 ,可见评价 IL-22 二聚体的生物活性宜采用体内实验模型。
实施例 3和 6的结果提示， IL-22单体或二聚体形式对不同细胞的作用存在很 大差异。 对神经细胞而言， IL-22二聚体对神经细胞的激活作用明显优于 IL-22单 体的作用， 对人结肠癌类细胞（如 Colo205细胞）而言， IL-22单体的活性略优于较 IL-22二聚体的活性。
因此， 实施例 3的体外测定方法更适合用于测定二聚体 IL-22的活性（尤其是 针对神经细胞的保护作用）， 也更适合反映二聚体 IL-22在体内的活性。 实施例 7 IL-22或 IL-22二聚体对 MPP+诱导的 PC12细胞神经毒性的保护作用
PC12细胞是一种大鼠嗜铬细胞瘤细胞系， 体外培养能合成、 代谢、 传递多巴 胺， 可作为体外模型筛选具有活性的化合物。
实验将 PC12细胞按 40000个 /孔种于 96孔板， 培养基成分： DMEM, 10%马血清 +5%FCS, 1%青霉素-链霉素。 MPP+ (Sigma)加入至 30〜3000 μ m终浓度，分别加入 IL-22 至终浓度为 0. 04 ng/mL、 0. 4ng/mL、 4ng/mL、 40ng/mL IL- 22二聚体（由两个选自 序列如 SEQ ID N0 : 2-5所示的 IL-22-Fc单体构成）至终浓度为 0. 1 ng/mL、 Ing/m 10ng/mL、 100ng/mL。 培养 24hr后， 采用荧光细胞存活分析（Fluorimetric cell viabi l ity assay)。
结果显示， MPP+处理后， PC12细胞的存活率随着 MPP+浓度的增加而降低， IL-22 和 IL-22二聚体对 PC12细胞均显示出显著的保护作用。 实施例 8 IL-22或 IL-22二聚体对 MPTP诱导的动物帕金森氏病（PD)模型的治 疗作用
1-甲基 -4-苯基 -1， 2， 3， 6-四氢吡啶（MPTP) , 可以特异性的损伤多巴胺能神 经元， 使黑质多巴胺能神经元大量丢失， 出现类似帕金森氏病的症状。
实验选取 C57/BL6J小鼠， 雄性， 〜28g， 12-14周， 动物饲养于室温 24± 2 ° C 的环境中， 维持光-暗 12小时循环交替， 给以充足的饲料， 自由饮水。
50只小鼠随机分成 5组， 每组 10只， 分别为溶剂对照组、 MPTP模型组、
MPTP+IL-22 40 μ g/kg组、 MPTP+IL - 22 - D 40 μ g/kg组、 MPTP+IL - 22 - D 100 μ g/kg 组， 其中 IL-22-D为由二个序列如 SEQ ID NO : 2-5所示的 IL-22-Fc单体构成的 IL-22 二聚体。
MPTP以 30mg/kg的剂量腹腔注射， 连续给予 5天， 恢复一天后（从第 7天 起） MPTP+IL-22 40 μ g/kg组按 IL-22 40 μ g/kg皮下注射给药， 每天给药 IL-22 , 每 天一次， 连续 7天， 即从第 7〜13天； MPTP+IL-22-D 40 μ g/kg组按 40 μ g/kg皮下注 射给药， 于第 7、 9、 11天分别给药 IL-22-D—次； MPTP+IL-22-D 100 w g/kg组按 100 μ g/kg皮下注射给药， 于第 7、 9、 11天分别给药 IL-22-D—次； 溶剂对照组皮 下注射给予等体积的生理盐水。
第 14天对动物进行以下评价。
a. PD小鼠的行为学评价
在本实验于 MPTP末次给药后第 10天进行行为学检测。方法：爬杆法（pole test) 用于检测 PD中典型行为学症状一运动徐缓（Matsuura 等， 1997; Araki等， 2001 ; Kato等， 2004)。
将小鼠头向上轻柔的放在粗糙的杆顶（直径 8 mm, 高 55cm)。 小鼠从头向上调 整至头完全向下的时间记录为 T-turn (t ime to turn) , 小鼠从向下运动至四肢全 部到达杆底的时间记录为 T-LA (locomotion activity time) , 超过 30 s按照 30 s 记录。 每只小鼠重复检测 5次取平均值。
结果显示， IL-22和 IL-22二聚体均能显著改善 MPTP诱导的小鼠行为学异常。 b.检测紋状体内多巴胺的浓度
方法： 小鼠断头处死， 将纹状体取出称重后装在 1. 5 ml的离心管中， 并立即 置于碎冰中。 样本每 10 mg加入 300 μ 1冰水浴中的样品处理液（0. 2 M高氯酸、 0. 2 mM焦亚硫酸钠、 0. 01% EDTA-2Na， 同时含有 0. 3 μ Μ ϋΗΒΑ作为内标）。 以上混合液 使用超声仪超声粉碎， 随后将其在 4°C下 10， 000 g离心 20 mi n e 取其上清液并用 0. 22 μ Μ水相滤膜过滤， 采用高效液相色谱法检测纹状体多巴胺的浓度。
结果显示， IL-22和 IL-22二聚体能显著抑制 MPTP诱导的小鼠纹状体多巴胺浓 度的降低。
c.黑质内多巴胺能神经元的情况观察
方法： 10%水合氯醛麻醉， 4%多聚甲醛灌流后取脑。 4%多聚甲醛后固定 24h， 再将样本转入 10%， 20%, 30%的蔗糖溶液中梯度脱水至样本沉底， 在 -20 ° C冰冻切 片机中做中脑与纹状体部位冠状切片， 小鼠脑切片厚度为 15 μ πι。 ΤΗ为多巴胺能能 神经元的特异性标记。 一抗为单克隆小鼠抗 ΤΗ (1 : 1000， CHEMIC0N) , 与纹状体和 中脑部位的脑片 4° C共同孵育过夜， PBS洗三遍后用生物素化二抗， 室温孵育 lh。 SABC复合物室温孵育 lh。 DAB显色， 乙醇梯度脱水， 二甲苯透明， 中性树胶封片。 黑质 TH阳性细胞计数， 纹状体 TH阳性染色光密度扫描。
结果显示， IL-22和 IL-22二聚体可以明显抑制 MPTP诱导的多巴胺能神经元的 大量丢失。 实施例 9 IL-22或 IL-22二聚体对海马神经元细胞 STAT3的激活作用
取孕 17天 SD大鼠的胎鼠的全脑放入预冷的 D-Hanks液中， 在解剖显微镜下取海 马。 剪碎成 1 mm3大小， 加入 10 mL 0. 125%胰酶， 并放入 37°C孵箱内消化 15 min, 随后吸出组织转移到装有预冷的 DMEM+ 10%FBS的离心管内终止消化， 用移液枪吹 打数次， 静置后取上清并吸到另一支离心管内。 重复上述步骤 2〜3次。
用无血清神经元基础培养基（neurobasal ,购自 invitrogen,货号: 21103049) + 无血清添加剂 B27 (invitrogen， 货号： 17504044)培养 8天， 每 2天换液一次。
培养至第八天， 分别用不同浓度 IL-22二聚体（由两个选自序列如 SEQ ID N0 : 2-5所示的 IL-22-Fc单体构成）和 IL-22 (IL-22二聚体的终浓度： 1、 10、 100 ng/mL; IL-22单体的终浓度： 0. 4、 4、 40 ng/mL)处理神经元 15分钟。 将培养基完 全吸出， 细胞用 PBS洗两遍， 按照细胞裂解液使用说明步骤裂解细胞。 用细胞裂解 液（Cel l Signal ing Technology,货号： 9803； 主要成分： 20 mM Tri s-HCl (ρΗ 7· 5)， 150 mM NaCl， 1 mM Na2EDTA， 1 mM EGTA， 1% Triton, 2. 5 mM焦磷酸钠， 1 πιΜ β - 甘油磷酸盐， 1 mM Na3V04， 1 μδ/πι1 亮抑酶肽， 1 mM PMSF)冰上裂解细胞 20分钟， 用细胞刮子刮取细胞。 收集细胞裂解液， 12000rpm， 4°C离心 10分钟。 吸取上清， 测定蛋白浓度。 另取 Ιθθμΐ的上清用 STAT3 [pY705] ELISA 试剂盒（invitrogen, 货号： KH00481)检测 STAT磷酸化水平变化。
结果显示， IL-22二聚体（IL-22-D)与 IL-22单体均能激活海马神经元 STAT3的 生物学活性。 实施例 10 IL-22或 IL-22二聚体对 Α β诱导的 PC12细胞凋亡的保护作用
PC12细胞用神经生长因子（NGF)诱导后可长出突起， 具有神经细胞特性， Α β ( β淀粉样蛋白）诱导 PC12细胞凋亡可作为体外模拟埃尔茨海默 (AD)模型。
将 PC12细胞培养于基础培养基（DMEM， 10%FCS， 1%青霉素 -链霉素）， 胰酶消化， 重悬于含 NGF 50ng/mL的培养基中， 细胞浓度调整为 2X104cells/孔加入 96孔板， 于 37°C , 5%C02培养箱中培养 24hr。 加入 A β至终浓度 1-100 mol/L， 分别用不同浓度 的 IL-22单体和 IL-22-D (IL-22-D由二个选自序列如 SEQ ID NO : 2-5所示的 IL-22-Fc 单体构成）处理， IL-22单体的终浓度分别为 0. 4、 4、 40ng/mL, IL-22-D的终浓度 分别为 1、 10、 lOOng/mL, 模型孔加入等体积的 PBS， 阴性对照孔不加 Α β， 继续培 养 24hr。 Hochest染色观察细胞形态学， 或 MTT法检测 PC12细胞的增殖。
与阴性对照孔相比， 模型孔的 PC12细胞细胞核荧光染色明显不均匀， 可见到 细胞凋亡引起的致密浓染的高荧光细胞核， IL-22单体和 IL-22二聚体处理的 PC12 细胞细胞核荧光染色均匀，未见到明显的致密浓染的高荧光细胞核。 IL-22和 IL-22 二聚体均能抑制 NGF分化后 Α β 诱导所致的 PC12细胞的凋亡， 保护神经细胞。 实施例 11 IL-22或 IL-22二聚体对 ΜΡΤΡ诱导的 PD动物模型的治疗作用
1-甲基 -4-苯基 -1， 2， 3， 6-四氢吡啶 （ΜΡΤΡ) ， 可以特异性的损伤多巴胺能 神经元， 使黑质多巴胺能神经元大量丢失， 从而出现类似帕金森氏病 （PD) 的症 状。 酪氨酸羟化酶 （TH) 是多巴胺能神经元的特异性标记， 可以用于定量检测黑 质多巴胺能神经元的数量。
因 IL-22单体与 IL-22-D的分子量之比为约 1 : 5，且一个 IL-22-D含 2个 IL-22单 体， 故为了比较试验结果， 本试验中选用等摩尔用量（按 IL-22单体计）， 即 IL-22 用量为 40 g/kg, 而 IL- 22- D用量为 100 μ g/kg。
实验选取雄性 C57/BL6J 小鼠， 22〜30g， 12-14周， 随机分成 4组， 分别为： MPTP+IL-22 40 μ g/kg组: 以 30 mg/kg的剂量腹腔注射 MPTP, 连续给予 5天， 动物恢复一天后 （即从第 7天起） 按 40 μ g/kg皮下注射给予 IL-22 (重组人白介素
22)， 连续给药 7天；
MPTP+IL-22-D 100 μ g/kg组： 以 30 mg/kg的剂量腹腔注射 MPTP, 连续给予 5天， 动物恢复一天后 （即从第 7天起） 按 100 μ g/kg皮下注射给予 IL-22-D, 分别于第 7、 9、 11天分别给药一次；
MPTP模型组： 以 30 mg/kg的剂量腹腔注射 MPTP, 连续给予 5天， 动物恢复一天 后， 从第 7天起给予等体积的溶剂 （0. 5%鼠血清 /PBS ) ；
正常对照组： 给予等体积的生理盐水， 连续给予 5天， 动物恢复一天后， 从第 7天起给予等体积的溶剂 （0. 5%鼠血清 /PBS ) 。
所述 IL-22二聚体 （IL-22-D) 由二个序列如 SEQ ID N0 : 2所示的 IL-22-Fc构成。 IL-22-D也可以由二个选自序列如 SEQ ID NO : 3-5所示的 IL-22-Fc单体构成。
第 14天处死动物， 检测黑质内多巴胺能神经元的情况。
黑质内多巴胺能神经元的情况观察
方法： 10%水合氯醛麻醉， 4%多聚甲醛灌流后取脑。 4%多聚甲醛后固定 24h， 再将样本转入 10%， 20%, 30%的蔗糖溶液中梯度脱水至样本沉底， 在一 20°C 冰冻 切片机中做中脑部位冠状切片， 小鼠脑切片厚度为 20 μ πι， TH免疫组化分析。 一抗 为单克隆小鼠抗 TH抗体 （1 : 1000， Sigma) ， 与中脑部位的脑片 4°C共同孵育过夜， PBS 洗三遍后用生物素化二抗（羊抗鼠）， 室温孵育 lh。 SABC复合物室温孵育 lh。 DAB显色， 乙醇梯度脱水， 二甲苯透明， 中性树胶封片。 进行黑质致密部 TH阳性细 胞计数分析。
结果如图 8A、 图 8B所示：
图 8A显示了小鼠黑质致密部 TH阳性神经元免疫组化染色图片。 小鼠连续 5天注 射 MPTP后， 黑质致密部 TH阳性神经元大量丢失， 在施用 IL-22或者 IL-22二聚体后， 明显恢复了 TH阳性神经元数量。
图 8B显示了小鼠黑质致密部 TH阳性细胞计数分析结果。 与正常对照组相比， MPTP模型组中， 黑质致密部 TH阳性神经元的数量显著减少， 大约为正常对照组的 51%， 说明 MPTP导致黑质内多巴胺能神经元的大量丢失。 MPTP+IL-22 40 μ g/kg组 中， 黑质内 TH阳性多巴胺神经元大约为正常对照组的 80%， MPTP+IL-22-D 100 g/kg组中， 黑质内 TH阳性多巴胺神经元大约为正常对照组的 86%。
可见， IL-22、 IL-22二聚体对 MPTP诱导的多巴胺能神经元的大量丢失有保护 作用（IL-22二聚体的保护作用更为显著）， 从而对 MPTP诱导的帕金森疾病模型小鼠 脑多巴胺神经元的退行性病变具有治疗作用。 在本发明提及的所有文献都在本申请中引用作为参考， 就如同每一篇文献被 单独引用作为参考那样。 此外应理解， 在阅读了本发明的上述讲授内容之后， 本 领域技术人员可以对本发明作各种改动或修改， 这些等价形式同样落于本申请所 附权利要求书所限定的范围。

Claims

权 利 要 求
1. 一种白介素 -22或其二聚体或多聚体的用途， 其特征在于， 用于制备治疗和预 防神经损伤疾病或神经退行性疾病的药物。
2. 如权利要求 1所述的用途， 其特征在于， 所述的神经损伤疾病选自： 中风、 脊 柱损伤和伴有血脑屏障损伤的神经系统疾病；
所述神经退行性疾病选自： 帕金森氏病、 阿尔茨海默氏病、 亨廷顿舞蹈病、 肌萎 缩侧索硬化病、 脊髓肌萎缩病、 原发性侧索硬化病、 或脊髓小脑共济失调。
3. 如权利要求 1所述的用途， 其特征在于， 所述的白介素 -22的二聚体是式 I所示 的白介素 -22的二聚体，
M1-L-M2 式 I
式中，
Ml是白介素 -22的第一单体；
M2是白介素 -22的第二单体；
L是位于所述的第一单体和第二单体之间的，将所述第一单体和第二单体连接在一 起的接头元件，
其中，所述的白介素 -22二聚体保持了白介素 22的生物活性， 并且其血清半衰期是 所述第一单体或第二单体的血清半衰期的 2倍以上。
4. 如权利要求 3所述的用途， 其特征在于， 所述的接头元件 L选自下组：
(i) 3-50个氨基酸的短肽；
(ϋ) 式 II所示的多肽元件：
-Z-Y-Z- 式 II
式中，
Y为载体蛋白；
Z为无、 或 1-30个氨基酸的短肽；
"-"为化学键或共价键。
5. 如权利要求 3所述的用途， 其特征在于， 所述的第一单体和第二单体是相同的。
6. 如权利要求 3所述的用途， 其特征在于， 所述的生物活性是选自下组的一种或 多种活性：
(a) 在体外激活神经元细胞 STAT3;
(b) 在体内脑缺血性损伤后， 能保护神经元， 减少脑梗死体积；
(c) 在体外激活多巴胺能神经元细胞 STAT3或海马神经元细胞 STAT3;
(d) 在体内能显著抑制 PD模型动物黑质多巴胺能神经元的丢失；
(e) 在体内能显著减少 AD模型动物海马区神经元细胞的凋亡。
7. 如权利要求 4所述的用途， 其特征在于， 所述的载体蛋白是白蛋白或人 IgG的 Fc 片段。
8. 如权利要求 4所述的用途，其特征在于，所述二聚体是由氨基酸序列如 SEQ ID NO: 2-5所示的单体所构成的二聚体。
9. 一种白介素 -22的二聚体， 其特征在于， 所述二聚体的结构如式 I所示的白介 素 -22的二聚体，
M1-L-M2 式 I
式中，
Ml是白介素 -22的第一单体；
M2是白介素 -22的第二单体；
L是位于所述的第一单体和第二单体之间的，将所述第一单体和第二单体连接在一 起的接头元件，
其中，所述的白介素 -22二聚体保持了白介素 22的生物活性， 并且其血清半衰期是 所述第一单体或第二单体的血清半衰期的 2倍以上。
10. 一种可用于治疗和预防神经损伤疾病或神经退行性疾病的药物组合物， 其特 征在于， 所述药物含有药学上可接受的载体以及式 I所示的白介素 -22的二聚体，
M1-L-M2 式 I
式中，
Ml是白介素 -22的第一单体；
M2是白介素 -22的第二单体；
L是位于所述的第一单体和第二单体之间的，将所述第一单体和第二单体连接在一 起的接头元件，
其中，所述的白介素 -22二聚体保持了白介素 22的生物活性， 并且其血清半衰期是 所述第一单体或第二单体的血清半衰期的 2倍以上。
PCT/CN2012/087694 2011-12-27 2012-12-27 白介素-22在治疗和预防神经损伤疾病或神经退行性疾病中的用途 WO2013097748A1 (zh)

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