WO2020200186A1 - 慢性炎症和病毒感染的诊断和治疗 - Google Patents

慢性炎症和病毒感染的诊断和治疗 Download PDF

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WO2020200186A1
WO2020200186A1 PCT/CN2020/082296 CN2020082296W WO2020200186A1 WO 2020200186 A1 WO2020200186 A1 WO 2020200186A1 CN 2020082296 W CN2020082296 W CN 2020082296W WO 2020200186 A1 WO2020200186 A1 WO 2020200186A1
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nmi
ifp35
antibody
seq
virus
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PCT/CN2020/082296
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English (en)
French (fr)
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梁欢欢
刘迎芳
于杨
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中山大学
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Priority claimed from CN202010122554.4A external-priority patent/CN113311164A/zh
Application filed by 中山大学 filed Critical 中山大学
Priority to EP20783545.5A priority Critical patent/EP3949978A4/en
Priority to JP2021560209A priority patent/JP7475366B2/ja
Priority to CN202080026317.6A priority patent/CN114025785A/zh
Priority to US17/600,842 priority patent/US20240043511A1/en
Publication of WO2020200186A1 publication Critical patent/WO2020200186A1/zh

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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • 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
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5035—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on sub-cellular localization
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2299/00—Coordinates from 3D structures of peptides, e.g. proteins or enzymes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/70—Mechanisms involved in disease identification
    • G01N2800/7095—Inflammation

Definitions

  • This application relates to the field of biomedicine, in particular to methods and products for the diagnosis and treatment of chronic inflammation and viral infections.
  • DAMPs damage-related molecular patterns
  • NMI N-myc and STAT interacting protein
  • IFP35 interferon-inducing protein 35
  • IFP35 and NMI can be secreted out of cells by monocytes and macrophages within one hour after being activated by LPS or interferon, and are used in lipopolysaccharide-induced sepsis models or acetaminophen-induced liver In the injury model, activated macrophages are released. Extracellular NMI and IFP35 can activate NF- ⁇ B through Toll-like receptor 4 (TLR4) to activate macrophages and release pro-inflammatory cytokines. In addition, in the serum of patients who died of severe inflammation, the level of NMI was significantly increased. NMI loss can reduce inflammation and reduce the mortality of mice in sepsis and liver injury models. Antibodies against IFP35 can reduce the expression level of inflammatory factors, thereby effectively improving the survival rate of septicemia mice.
  • TLR4 Toll-like receptor 4
  • MS multiple sclerosis
  • MS can cause sensory, mental, motor, physical, and cognitive dysfunctions, and is one of the main causes of disability in the young and middle-aged population.
  • the IFP35 protein family contains two homologous proteins: N-myc and STAT interacting protein (NMI) and interferon-inducing protein 35 (IFP35), which are both interferon-inducing genes (ISGs), and the human body and immune cells are being interfered After induction, the expression in the cell will increase. In addition, they are also damage-related molecular patterns (DAMP).
  • NMI and IFP35 can be released by activated macrophages, activate NF- ⁇ B through the TLR4 receptor signaling pathway, and promote macrophages to release pro-inflammatory cells. Knockout of factors, NMI and IFP35 can reduce the mortality of mice with sepsis and liver injury caused by bacterial or viral infection.
  • NMI and IFP35 can be quickly released from the cells into body fluids such as serum in. Injection of IFP35 protective antibody can protect mice from damage caused by LPS, Salmonella, and APAP.
  • the inflammatory response involves the participation of various immune cells and a large number of inflammation-related cytokines, and is a precisely regulated process. Inflammation is a double-edged sword. On the one hand, it is an important way for the body's immune defense to eliminate infection. On the other hand, inflammatory disorders are an important cause of many diseases, such as sepsis, chronic inflammatory diseases and autoimmune diseases. In the case of pathogen infection, the host's immune system produces an endogenous damage-related molecular pattern (Damage Associated Molecular Pattern, DAMP). At present, several DAMP inflammatory factors have been found in the human body, including HMGB1, IL1b, IL33, S100A and so on.
  • DAMP damage-related molecular Pattern
  • DAMP as an inflammatory factor, is further recognized by cell pattern recognition receptors (such as TLR4, etc.), activates downstream signaling pathways of natural immune cells (including JAK-STAT pathway, etc.), and activates inflammation-related transcription factors in cells, such as NF- ⁇ B and IRF3 /7, etc., induce the transcription and expression of a large number of inflammation-related genes, and activate the immune response. Therefore, DAMP inflammatory factors are a very important class of inflammatory response molecules in the immune system. These DAMP-type inflammatory factors are widely used in clinical testing and as targets for drug development such as antibodies and small molecule drugs.
  • One of the technical problems to be solved in this application is to provide a method and product for preventing and/or treating chronic inflammatory diseases and related antibodies, as well as a method and product for diagnosing chronic inflammatory diseases and related antibodies.
  • This application provides technical solutions for the application of reagents in the preparation of products for the prevention and/or treatment of chronic inflammatory diseases.
  • reagent in the preparation of drugs for preventing and/or treating chronic inflammatory diseases; the reagent is at least one of the following:
  • This application provides a method for preventing and/or treating chronic inflammatory diseases.
  • the method for preventing and/or treating chronic inflammatory diseases includes the following steps: applying an effective amount of the following reagents to the organism to achieve the purpose of preventing and/or treating chronic inflammatory diseases;
  • the reagent is at least one of the following:
  • the inhibition in (1)-(5) above can be direct inhibition, such as directly inhibiting the abnormal increase in the content and/or activity of IFP35 and/or NMI, or directly inhibiting the abnormal content of inflammatory factors that are secreted outside the cell
  • the activity of IFP35 and/or NMI such as directly inhibiting the secretion of IFP35 and/or NMI outside the cell as an inflammatory factor, such as inhibiting the content and/or activity of IFP35 and/or NMI that is secreted outside the cell as an inflammatory factor; It inhibits IFP35 and/or NMI indirectly by inhibiting the expression and/or activity of interferon.
  • the reagent is at least one of the following:
  • An antibody, polypeptide, or antigen-binding fragment that specifically binds to IFP35 and/or NMI, and has at least one of the functions (1) to (8) in claim 1;
  • the nucleic acid reagent has at least one of the functions (1) to (8) in claim 1.
  • the nucleic acid molecule can be siRNA, shRNA, or miRNA, which can target genes encoding IFP35 and/or NMI.
  • the small molecule compound may be a small molecule compound that inhibits the secretion, expression and/or activity of IFP35 and/or NMI.
  • the antibody, polypeptide, or antigen-binding fragment may be an antibody that specifically binds to the following epitopes: (1) The sequence of the epitope is located within amino acids 81-170, 177-268, or 136-216 of SEQ ID NO: 2 ; (2) The sequence of the epitope is located within the amino acids 81-168, 175-266, or 134-214 of SEQ ID NO: 4; (3) The sequence of the epitope is located within the 104-193, 202- of SEQ ID NO: 6 293, or within amino acids 151-250; (4) The sequence of the epitope is within the 103-192, 201-229, or 151-240 amino acids of SEQ ID NO: 8.
  • the antibody may be the following A or B or C or D antibody,
  • the antibody, polypeptide or antigen-binding fragment specifically binds to the epitope of IFP35/NMI, and the epitope is as follows (1) or (2): the epitope includes the following amino acid sites: IFP35 (SEQ ID NO: 2) Arg163, Asn164, Arg191, Gln194, Ile195, Gln197, Phe198, Thr199, Pro201, Gln206, Pro208, Arg210; (2) The epitope includes the following amino acid sites: NMI (SEQ ID NO: 8 ) Arg185, Asn186, Lys215, Lys218, Lys219, Glu221, Tyr222, Pro223, Tyr225, Cys230, Arg232, Thr234.
  • the antibody is a 35NIDmAb antibody, which has a light chain variable region and a heavy chain variable region.
  • the heavy chain variable region contains CDR1, CDR2 and CDR3, and the sequence of CDR1 is SEQ ID NO: 9 25-32 Amino acid (GYTFTNYG (SEQ ID NO: 13)), the sequence of CDR2 is amino acid 50-57 of SEQ ID NO: 9 (INTYTGEP (SEQ ID NO: 14)), and the sequence of CDR3 is SEQ ID NO: 9 Amino acids 98-106 (YGYSWAMDY (SEQ ID NO: 15)); the light chain variable region contains CDR1, CDR2 and CDR3, and the sequence of CDR1 is amino acid 26-31 of SEQ ID NO: 10 (SSSVSY (SEQ ID NO: 15)) ID NO: 16)), the sequence of CDR2 is amino acid 49-51 of SEQ ID NO: 10 (DTS (SEQ ID NO: 17)), and the sequence of CDR3 is amino acid 90-96
  • the antibody is an antibody obtained by mutating the remaining amino acids of 35NIDmAb except for the CDR; or, the antibody is an antibody obtained by humanizing 35NIDmAb.
  • the humanized antibody is at least one of the following: AE001-H1+L1, AE001-H2+L2 or AE001-H3+L3. These three antibodies all have a light chain variable region and a heavy chain variable region.
  • the heavy chain variable region contains CDR1, CDR2 and CDR3.
  • the sequence of CDR1 is SEQ ID NO: 9 amino acids 25-32 (GYTFTNYG)
  • the sequence of CDR2 is amino acid 50-57 of SEQ ID NO: 9 (INTYTGEP)
  • the sequence of CDR3 is amino acid 98-106 of SEQ ID NO: 9 (YGYSWAMDY);
  • the light chain variable region contains CDR1, CDR2 and CDR3
  • CDR1 sequence is SEQ ID NO: 10 amino acids 26-31 (SSSVSY)
  • CDR2 sequence is SEQ ID NO: 10 amino acids 49-51 (DTS)
  • CDR3 sequence is SEQ ID NO: 10 amino acids 90-96 (WSSNPPI).
  • the heavy chain constant region sequence of AE001-H1+L1 is AE001H1 (SEQ ID NO: 1), and the light chain constant region sequence is AE001L1 (SEQ ID NO: 3).
  • the heavy chain constant region sequence of AE001-H2+L2 is AE001H2 (SEQ ID NO: 5), and the light chain constant region sequence is AE001L2 (SEQ ID NO: 7).
  • the heavy chain constant region sequence of AE001-H3+L3 is AE001H3 (SEQ ID NO: 11), and the light chain constant region sequence is AE001L3 (SEQ ID NO: 12).
  • the antibody is an antibody obtained by mutating at least one CDR amino acid residue in 35NIDmAb or a humanized antibody thereof.
  • the mutation of at least one CDR in 35NIDmAb or its humanized antibody includes at least one of the following mutations:
  • the 25th, 26th, 27th, 28th, 29th and/or 32th amino acid residues of the heavy chain variable region CDR1 of 35NIDmAb were mutated; that is, the amino acid positions other than NY in (25GYTFT NY G 32) were mutated.
  • 35NIDmAb heavy chain variable region CDR3 98th, 99th, 101st, 103, 104, 105 and/or 106th amino acid residues are mutated; that is, (98 YG Y S W AMDY 106) except for the underlined amino acids in the remaining positions Amino acids are mutated.
  • amino acid residues 26, 27, 28 and/or 29 of the light chain variable region CDR1 of the 35NIDmAb were mutated; that is, the amino acids at the remaining positions except for the underlined amino acids in (26 SSSV SY 31) were mutated.
  • amino acid residues 90, 91, 94, 95 and/or 96 of the CDR3 of the light chain variable region of 35NIDmAb were mutated; that is, the amino acids at the remaining positions except for the underlined amino acids in (90 WS SN PPI 96) were mutated.
  • the two amino acids that bind to the antigen are mainly Asn30 and Tyr31; in the antibody heavy chain variable region CDR2 sequence (50 I N T YT GEP 57 ), the three amino acids that bind to the antigen are mainly Asn51, Tyr53 and Thr54; in the CDR3 (98 YG Y S W AMDY 106) sequence of the variable region of the antibody heavy chain, the two amino acids that bind to the antigen are mainly Tyr100 and Trp102. .
  • the above-mentioned residues can be referred to as the interaction residues between the antibody heavy chain and the antigen.
  • the main amino acids that bind to the antigen are Ser30 and Tyr31; in the antibody light chain variable region CDR2 sequence (49 D TS 51), the main amino acids that bind to the antigen are The amino acid is Asp49; in the CDR3 sequence of the variable region of the antibody light chain (90 WS SN PPI 96), the main amino acids that bind to the antigen are Ser92 and Asn93.
  • the above-mentioned residues can be referred to as the interaction residues between the antibody heavy chain and the antigen.
  • Other CDR residues do not or rarely bind to the antigen IFP35, so it is easier to make changes to obtain antibodies that do not significantly affect antibody activity.
  • the mutation of at least one CDR in 35NIDmAb or its humanized antibody includes at least one of the following mutations:
  • 35NIDmAb heavy chain variable region CDR1 amino acid residues 30 and/or 31 are mutated;
  • 35NIDmAb's heavy chain variable region CDR3 amino acid residue 100 and/or 102 is mutated
  • amino acid residues 92 and/or 93 of CDR3 in the light chain variable region of 35NIDmAb were mutated.
  • the antibody or antigen-binding fragment is an antibody or antigen-binding fragment capable of recognizing the following two or more amino acid residues: Asn30, Tyr31, Asn51, Tyr53, Thr54, Tyr100, and Trp102 of the heavy chain in 35NIDmAb or its humanized antibody Amino acid residues at position, which are involved in the recognition of antigens IFP35 and NMI.
  • the antibody or antigen-binding fragment is an antibody or antigen-binding fragment capable of recognizing the following two or more amino acid residues: the amino acid residues Ser30, Tyr31, Asp49, Ser92 and Asn93 of the light chain in 35NIDmAb or its humanized antibody , Which participates in the recognition of antigens IFP35 and NMI.
  • the two amino acids that bind to the antigen are mainly Asn30 and Tyr31; in the antibody heavy chain variable region CDR2 sequence (50 I N T YT GEP 57) The three amino acids that bind to the antigen are mainly Asn51, Tyr53 and Thr54; in the CDR3 (98 YG Y S W AMDY 106) sequence of the variable region of the antibody heavy chain, the two amino acids that bind to the antigen are mainly Tyr100 and Trp102. Therefore, changing some of the amino acids may be beneficial to the antigen-antibody binding ability.
  • the above-mentioned residues can be referred to as the interaction residues between the antibody heavy chain and the antigen. Changing any one or two or three of these antigen-interacting residues still maintains the amino acid binding to the corresponding epitope in the antigen-antibody complex structure of IFP35 or NMI, and should be within the protection scope of this application.
  • the main amino acids that bind to the antigen are Ser30 and Tyr31; in the antibody light chain variable region CDR2 sequence (49 D TS 51), the main amino acids that bind to the antigen are The amino acid is Asp49; in the CDR3 sequence of the variable region of the antibody light chain (90 WS SN PPI 96), the main amino acids that bind to the antigen are Ser92 and Asn93. Therefore, some of these amino acid changes may be beneficial to the antigen-antibody binding ability.
  • the above-mentioned residues can be referred to as the interaction residues between the antibody light chain and the antigen. Changing any one or two or three of these antigen-interacting residues still maintains the amino acid binding to the corresponding epitope in the antigen-antibody complex structure of IFP35 or NMI, and should be within the protection scope of this application.
  • the antibody obtained by mutating at least one CDR in 35NIDmAb or its humanized antibody is at least one of the following: AE001-5, AE001-6, AE001-7, AE001 -8, AE001-9.
  • the CDRs of the heavy chain variable region of AE001-5, AE001-6, AE001-7, AE001-8, and AE001-9 and the CDR of the light chain variable region are shown in the following table.
  • sequences of the heavy chain variable region and light chain variable region of AE001-5, AE001-6, AE001-7, AE001-8, and AE001-9 other than the CDR can be modified by those skilled in the art according to their needs.
  • a specific example, AE001-5, AE001-6, AE001-7, AE001-8, AE001-9 heavy chain variable regions and light chain variable regions other than CDR sequences can also be compared with the corresponding sequences of the 35NIDmAb antibody Same same.
  • the constant region sequences of AE001-5, AE001-6, AE001-7, AE001-8, and AE001-9 can be modified by those skilled in the art according to their needs.
  • the constant region sequence of AE001-5, AE001-6, AE001-7, AE001-8, and AE001-9 can be constant with the above-mentioned AE001-H1+L1, AE001-H2+L2 or AE001-H3+L3
  • the region sequence is the same.
  • the chronic inflammatory diseases include multiple sclerosis, arthritis, rheumatoid arthritis, psoriasis (psoriasis), various enteritis (such as IBD), asthma, chronic obstructive pulmonary disease, and systemic Lupus erythematosus, chronic hepatitis, chronic nephritis, chronic pancreatitis, encephalitis, malignant tumors, leukemia, Alzheimer's disease, Parkinson's syndrome, etc.
  • the chronic inflammatory diseases are all affected by the secreted abnormal content of IFP35/NMI inflammatory factors, including rheumatoid arthritis (Rheumatoid arthritis (RA)), osteoarthritis (OSTEOARTHRITIS (OA)) , Multiple sclerosis (MS), atherosclerosis (Myocardial Infarction), chronic obstructive pulmonary (COPD), chronic nephritis, chronic hepatitis, chronic pancreatitis, type 2 diabetes (type 2 diabetes) ), Systemic lupus erythematosus (SLE), Alzheimer's disease, Parkinson's disease (PD), malignant tumors, asthma (asthma), allergic diseases (Allergic diseases), heart disease Cardiovascular diseases, Musculoskeletal diseases, inflammatory bowel disease (IBD), obesity and diabetes (Obesity and diabetes), retinal inflammatory disease (AMD), periodontitis (Periodontitis), uveitis (Uveitis) )
  • RA rheum
  • IFP35 and/or NMI can also be secreted by cells into the extracellular matrix (blood, body fluids, etc.) to exert Activate the role of inflammation.
  • the multiple sclerosis exemplified in this application is a typical representative of chronic inflammatory diseases. The occurrence of multiple sclerosis is closely related to microglia. Microglia can play part of the immune cells (macrophages) of the nervous system, release inflammatory factors, and promote the occurrence of inflammatory reactions.
  • This phenomenon is equivalent to the inflammatory response triggered by immune cells such as macrophages when other tissues and organs of the body are inflammatory.
  • the inventors of the present application found that microglia secrete IFP35/NMI into the cell culture medium under the induction of LPS, and the content of IFP35/NMI in the serum of animals with multiple sclerosis also increased. It shows that IFP35/NMI is associated with multiple sclerosis.
  • immune cells such as macrophages and microglia release inflammatory factors to trigger an inflammatory response, which may lead to various chronic inflammatory diseases.
  • IFP35/NMI is the inflammatory factor among them.
  • This application also provides methods and applications for diagnosing chronic inflammatory diseases, which are the following technical solutions A, B or C or D:
  • the substance for detecting IFP35 and/or NMI is at least one of the following:
  • IFP35/NMI is secreted into the blood or body fluid of the organism (such as cerebrospinal fluid);
  • Technical Solution B Clinical testing products for disease diagnosis applications are used to detect IFP35 and/or NMI secreted into serum and body fluids;
  • the clinical application products for detecting IFP35 and/or NMI secreted into serum and body fluids include at least one of the following: fluorescence luminescence clinical detection reagent (kit), chemiluminescence clinical detection reagent (kit), Elisa detection reagent (kit), PCR clinical testing reagents (kits), etc.
  • the clinical application products for detecting IFP35 and/or NMI secreted into serum and body fluids include at least one of the following: fluorescence luminescence clinical detection reagent (kit), chemiluminescence clinical detection reagent (kit), Elisa detection reagent (kit), PCR clinical testing reagents (kits), etc.
  • a method for diagnosing whether a test organism has a chronic inflammatory disease including at least one of the following steps:
  • IFP35/NMI is secreted into the blood or body fluid of the organism (such as cerebrospinal fluid);
  • whether the organism to be tested has chronic inflammatory diseases can be diagnosed according to at least one of the following criteria:
  • test organism tissue such as spinal cord tissue
  • IFP35/NMI is secreted into the blood or body fluid of the organism (such as cerebrospinal fluid), it is determined that the organism to be tested has chronic inflammatory diseases;
  • IFP35/NMI is contained in the blood or body fluid (such as cerebrospinal fluid) of the organism, it is determined that the organism to be tested has chronic inflammatory disease.
  • IFP35/NMI The increased expression of IFP35/NMI is consistent with the expression and secretion of other inflammatory factors in the organism (interferon, TNF, IL1, IL6, etc.) in the blood or body fluids, or if it has clinical characteristics, the organism to be tested is judged Suffer from chronic inflammatory disease.
  • the clinical application products for detecting IFP35 and/or NMI secreted into serum and body fluids include at least one of the following: fluorescence luminescence clinical detection reagents (kits), chemiluminescence clinical detection reagents (kits), Elisa detection reagents (kits), PCR clinical detection reagents (kits), etc.
  • the detection of the content and/or activity of IFP35 and/or NMI can be performed at the level of DNA, RNA or protein.
  • nucleic acid probes can be used to detect DNA and/or RNA levels, PCR primers or chips can also be used.
  • antibodies are used for protein level detection.
  • PCT procalcitonin
  • CRP C-reactive protein
  • TNF TNF
  • IL6 IL6
  • PCT procalcitonin
  • CRP C-reactive protein
  • IL6 IL6
  • IFP35/NMI is abnormally secreted into the blood or body fluid of the mouse (such as in the cerebrospinal fluid), or the expression of IFP35/NMI is increased and other inflammatory factors in the mouse (The expression and secretion of interferon, TNF, IL1, IL6, etc.) are consistent with their secretion into the blood, causing obvious external symptoms such as changes in gait and paralysis of the limbs, which can basically diagnose the mice with MS.
  • the detection substance is an antibody, polypeptide or antigen-binding fragment of A or B or C or D:
  • the antibody, polypeptide or antigen-binding fragment specifically binds to the epitope of IFP35/NMI, and the epitope is as follows (1) or (2): the epitope includes the following amino acid sites: Arg163 of IFP35, Asn164, Arg191, Gln194, Ile195, Gln197, Phe198, Thr199, Pro201, Gln206, Pro208, Arg210; (2) The epitope includes the following amino acid sites: Arg185, Asn186, Lys215, Lys218, Lys219, Glu221, Tyr222, Pro223, Tyr225, Cys230, Arg232, Thr234.
  • the antibody has a light chain variable region and a heavy chain variable region.
  • the heavy chain variable region includes CDR1, CDR2, and CDR3.
  • the sequence of CDR1 is SEQ ID NO: 9 amino acids 25-32 (GYTFTNYG), CDR2 The sequence is amino acid 50-57 of SEQ ID NO: 9 (INTYTGEP), and the sequence of CDR3 is amino acid 98-106 of SEQ ID NO: 9 (YGYSWAMDY); the light chain variable region includes CDR1, CDR2 and CDR3,
  • the sequence of CDR1 is the amino acid 26-31 of SEQ ID NO: 10 (SSSVSY)
  • the sequence of CDR2 is the amino acid 49-51 (DTS) of SEQ ID NO: 10
  • the sequence of CDR3 is that of SEQ ID NO: 10.
  • Amino acids 90-96 (WSSNPPI).
  • the antibodies, polypeptides or antigen-binding fragments used in the above diagnosis are antibodies obtained by mutating amino acids at positions other than the CDR of 35NIDmAb; or, the antibodies are antibodies humanized with 35NIDmAb.
  • the humanized antibody is at least one of the following: AE001-H1+L1, AE001-H2+L2 or AE001-H3+L3. These three antibodies all have a light chain variable region and a heavy chain variable region.
  • the heavy chain variable region contains CDR1, CDR2 and CDR3.
  • the sequence of CDR1 is SEQ ID NO: 9 amino acids 25-32 (GYTFTNYG ), the sequence of CDR2 is amino acid 50-57 of SEQ ID NO: 9 (INTYTGEP), and the sequence of CDR3 is amino acid 98-106 of SEQ ID NO: 9 (YGYSWAMDY); the light chain variable region contains CDR1 , CDR2 and CDR3, CDR1 sequence is SEQ ID NO: 10 amino acids 26-31 (SSSVSY), CDR2 sequence is SEQ ID NO: 10 amino acids 49-51 (DTS), CDR3 sequence is SEQ ID NO: Amino acids 90-96 of 10 (WSSNPPI).
  • the heavy chain constant region sequence of AE001-H1+L1 is AE001H1 (SEQ ID NO: 1), and the light chain constant region sequence is AE001L1 (SEQ ID NO: 3).
  • the heavy chain constant region sequence of AE001-H2+L2 is AE001H2 (SEQ ID NO: 5), and the light chain constant region sequence is AE001L2 (SEQ ID NO: 7).
  • the heavy chain constant region sequence of AE001-H3+L3 is AE001H3 (SEQ ID NO: 11), and the light chain constant region sequence is AE001L3 (SEQ ID NO: 12).
  • the antibody, polypeptide or antigen-binding fragment in the above diagnosis is an antibody obtained by mutating an amino acid residue in at least one CDR of a 35NIDmAb or a humanized antibody thereof.
  • the mutation of at least one CDR in the 35NIDmAb or its humanized antibody is to perform at least one of the following mutations:
  • the 25th, 26th, 27th, 28th, 29th and/or 32th amino acid residues of the heavy chain variable region CDR1 of 35NIDmAb were mutated; that is, the amino acid positions other than NY in (25 GYTFT NY G 32) were mutated
  • 35NIDmAb heavy chain variable region CDR3 98th, 99th, 101st, 103, 104, 105 and/or 106th amino acid residues are mutated; that is, (98 YG Y S W AMDY 106) except for the underlined amino acids in the remaining positions Amino acid mutation
  • the 26th, 27th, 28th and/or 29th amino acid residues of the light chain variable region CDR1 of 35NIDmAb are mutated; that is, the amino acids at the remaining positions except for the underlined amino acids in (26 SSSV SY 31) are mutated
  • amino acid residues 90, 91, 94, 95 and/or 96 of the CDR3 of the light chain variable region of 35NIDmAb were mutated; that is, the amino acids at the remaining positions except for the underlined amino acids in (90 WS SN PPI 96) were mutated.
  • the two amino acids that bind to the antigen are mainly Asn30 and Tyr31; in the antibody heavy chain variable region CDR2 sequence (50 I N T YT GEP 57 ), the three amino acids that bind to the antigen are mainly Asn51, Tyr53 and Thr54; in the CDR3 (98 YG Y S W AMDY 106) sequence of the variable region of the antibody heavy chain, the two amino acids that bind to the antigen are mainly Tyr100 and Trp102. .
  • the above-mentioned residues can be referred to as the interaction residues between the antibody heavy chain and the antigen.
  • the main amino acids that bind to the antigen are Ser30 and Tyr31; in the antibody light chain variable region CDR2 sequence (49 D TS 51), the main amino acids that bind to the antigen are The amino acid is Asp49; in the CDR3 sequence of the variable region of the antibody light chain (90 WS SN PPI 96), the main amino acids that bind to the antigen are Ser92 and Asn93.
  • the above-mentioned residues can be referred to as the interaction residues between the antibody heavy chain and the antigen.
  • Other CDR residues do not or rarely bind to the antigen IFP35, so it is easier to make changes to obtain antibodies that do not significantly affect antibody activity.
  • the mutation of at least one CDR in the 35NIDmAb or its humanized antibody is to perform at least one of the following mutations:
  • 35NIDmAb heavy chain variable region CDR1 amino acid residues 30 and/or 31 are mutated;
  • 35NIDmAb's heavy chain variable region CDR3 amino acid residue 100 and/or 102 is mutated
  • amino acid residues 92 and/or 93 of CDR3 in the light chain variable region of 35NIDmAb were mutated.
  • the antibody, polypeptide or antigen-binding fragment in the above diagnosis is an antibody or antigen-binding fragment capable of recognizing two or more amino acid residues as follows: 35NIDmAb or Asn30 of the heavy chain in the humanized antibody , Tyr31, Asn51, Tyr53, Thr54, Tyr100 and Trp102 amino acid residues, which participate in the recognition of antigens IFP35 and NMI.
  • the two amino acids that bind to the antigen are mainly Asn30 and Tyr31; in the antibody heavy chain variable region CDR2 sequence (50 I N T YT GEP 57) The three amino acids that bind to the antigen are mainly Asn51, Tyr53 and Thr54; in the CDR3 (98 YG Y S W AMDY 106) sequence of the variable region of the antibody heavy chain, the two amino acids that bind to the antigen are mainly Tyr100 and Trp102. Therefore, changing some of the amino acids may be beneficial to the antigen-antibody binding ability.
  • the above-mentioned residues can be referred to as the interaction residues between the antibody heavy chain and the antigen. Changing any one or two or three of these antigen-interacting residues still maintains the amino acid binding to the corresponding epitope in the antigen-antibody complex structure of IFP35 or NMI, and should be within the protection scope of this application.
  • the main amino acids that bind to the antigen are Ser30 and Tyr31; in the antibody light chain variable region CDR2 sequence (49 D TS 51), the main amino acids that bind to the antigen are The amino acid is Asp49; in the CDR3 sequence of the variable region of the antibody light chain (90 WS SN PPI 96), the main amino acids that bind to the antigen are Ser92 and Asn93. Therefore, some of these amino acid changes may be beneficial to the antigen-antibody binding ability.
  • the above-mentioned residues can be referred to as the interaction residues between the antibody light chain and the antigen. Changing any one or two or three of these antigen-interacting residues still maintains the amino acid binding to the corresponding epitope in the antigen-antibody complex structure of IFP35 or NMI, and should be within the protection scope of this application.
  • the antibody obtained by mutating at least one CDR in 35NIDmAb or its humanized antibody is at least one of the following:
  • the CDRs of the heavy chain variable region of AE001-5, AE001-6, AE001-7, AE001-8, and AE001-9 and the CDR of the light chain variable region are shown in the following table.
  • sequences of the heavy chain variable region and light chain variable region of AE001-5, AE001-6, AE001-7, AE001-8, and AE001-9 other than the CDR can be modified by those skilled in the art according to their needs.
  • a specific example, AE001-5, AE001-6, AE001-7, AE001-8, AE001-9 heavy chain variable regions and light chain variable regions other than CDR sequences can also be compared with the corresponding sequences of the 35NIDmAb antibody Same same.
  • the constant region sequences of AE001-5, AE001-6, AE001-7, AE001-8, and AE001-9 can be modified by those skilled in the art according to their needs.
  • the constant region sequence of AE001-5, AE001-6, AE001-7, AE001-8, and AE001-9 can be constant with the above-mentioned AE001-H1+L1, AE001-H2+L2 or AE001-H3+L3
  • the region sequence is the same.
  • the chronic inflammatory disease is related to the increase in inflammation caused by abnormal secretion of IFP35/NMI into blood or body fluids, including arthritis, rheumatoid arthritis, psoriasis (psoriasis), various Enteritis (such as IBD), multiple sclerosis, asthma, chronic obstructive pulmonary disease, systemic lupus erythematosus, chronic hepatitis, chronic nephritis, chronic pancreatitis, encephalitis, malignant tumor, leukemia, Alzheimer's disease, Parkinson's syndrome , Allergic diseases, cardiovascular diseases, musculoskeletal diseases, inflammatory bowel disease, obesity and diabetes, retinal inflammatory diseases, periodontitis, uveitis, etc.
  • the chronic inflammatory disease is related to the increase in inflammation caused by abnormal secretion of IFP35/NMI into blood or body fluids, including arthritis, rheumatoid arthritis, psoriasis (psoriasis), various Enteritis (such as IBD), multiple sclerosis, asthma, chronic obstructive pulmonary disease, systemic lupus erythematosus, chronic hepatitis, chronic nephritis, chronic pancreatitis, encephalitis, malignant tumor, leukemia, Alzheimer's disease, Parkinson's syndrome , Allergic diseases, inflammatory enteritis, etc.
  • the above chronic inflammation has been repeatedly proved to be related to a variety of inflammatory factors.
  • the diagnosis is early diagnosis, disease diagnosis and prognosis judgment.
  • the epitope of IFP35 is at least one of the following amino acid residues: Arg163, Asn164, Arg191, Gln194, Ile195, Gln197, Phe198, Thr199, Pro201, Gln206, Pro208, Arg210 of IFP35.
  • the epitope of NMI is at least one of the following amino acid residues: Arg185, Asn186, Lys215, Lys218, Lys219, Glu221, Tyr222, Pro223, Tyr225, Cys230, Arg232, Thr234 of NMI.
  • This application also provides methods for preparing antibodies based on the above-mentioned epitopes.
  • the method for preparing antibodies against IFP35 or NMI provided in this application is to prepare antibodies against IFP35 or NMI by using any of the epitope information mentioned in the claims.
  • the above method for preparing antibodies against IFP35 or NMI is to modify the antibody sequence based on the following 35NIDmAb antibody-IFP35/NMI antigen complex structural information and sequence information, and then prepare the modified IFP35 or NMI antibody:
  • the structure information of the 35NIDmAb antibody-IFP35/NMI antigen complex is as follows:
  • the two amino acids that bind to the antigen are mainly Asn30 and Tyr31; in the antibody heavy chain variable region CDR2 sequence (50 I N T YT GEP 57) Among them, the three amino acids that bind to the antigen are mainly Asn51, Tyr53 and Thr54; in the CDR3 (98 YG Y S W AMDY 106) sequence of the variable region of the antibody heavy chain, the two amino acids that bind to the antigen are mainly Tyr100 and Trp102.
  • the main amino acids that bind to the antigen are Ser30 and Tyr31; in the antibody light chain variable region CDR2 sequence (49 DTS 51), the main amino acids that bind to the antigen are The amino acid is Asp49; in the CDR3 sequence of the variable region of the antibody light chain (90 WS SN PPI 96), the main amino acids that bind to the antigen are Ser92 and Asn93.
  • the original antibody is generally obtained through various screening techniques. Knowing the epitope (antigenic determinant) can also directly use this fragment to screen antibodies.
  • the structural information and sequence alignment information of the antibody-antigen complex mentioned above (1)
  • the amino acid residues located on the surface of the antigen can be seen intuitively. Since most of the antigenic determinants are distributed on the surface of the antigen, these surface residues can help to find Antigenic determinants; (2)
  • the interaction between antigen and antibody residues can be seen, so that the interaction characteristics between antigen and antibody amino acid residues can be analyzed, and the antibody can be modified, designed, and optimized for characteristic amino acid residues. (3) It can guide the modification of antibodies to obtain new antibodies against other homologous protein antigens.
  • Antibody genetic modification methods involve general gene-directed mutation technology and modified antibody genes or antibody gene fragments (such as antibody variable regions) can be expressed and purified by eukaryotic cells (such as mammalian cells) or prokaryotic cells to obtain modified Of purified monoclonal antibodies.
  • eukaryotic cells such as mammalian cells
  • prokaryotic cells to obtain modified Of purified monoclonal antibodies.
  • Antibodies prepared by any of the above methods also belong to the scope of protection of this application.
  • the 35NIDmAb antibody described herein has a light chain variable region and a heavy chain variable region.
  • the heavy chain variable region contains CDR1, CDR2 and CDR3.
  • the sequence of CDR1 is SEQ ID NO: 9 amino acids 25-32 ( GYTFTNYG)
  • CDR2 sequence is SEQ ID NO: 9 amino acids 50-57 (INTYTGEP)
  • CDR3 sequence is SEQ ID NO: 9 amino acids 98-106 (YGYSWAMDY);
  • the light chain variable region contains The sequence of CDR1, CDR2 and CDR3, CDR1 is the amino acid 26-31 of SEQ ID NO: 10 (SSSVSY), the sequence of CDR2 is the amino acid 49-51 (DTS) of SEQ ID NO: 10, and the sequence of CDR3 is SEQ ID NO: 10 amino acids 90-96 (WSSNPPI).
  • sequence of the variable region of the light chain is SEQ ID NO: 10
  • sequence of the variable region of the heavy chain is SEQ ID NO: 9.
  • the amino acid sequence of human IFP35 is SEQ ID NO: 2.
  • the amino acid sequence of mouse IFP35 is SEQ ID NO: 4.
  • amino acid sequence of the murine NMI is SEQ ID NO: 6.
  • amino acid sequence of human NMI is SEQ ID NO: 8.
  • the inventor of the present application has constructed some mouse models of inflammatory diseases, and detected the levels of IFP35 and NMI in the serum of these inflammatory disease animals, in order to find inflammatory diseases related to IFP35/NMI.
  • the IFP35 and NMI discovered by the inventor of the present application are closely related to some chronic inflammatory diseases, such as multiple sclerosis, and their high serum content is closely related to the onset of multiple sclerosis.
  • IFP35/NMI gene knockout or use of IFP35/NMI Harmonic antibodies can reduce the symptoms of multiple sclerosis. Therefore, the inventor of the present application believes that inhibiting the activity of IFP35 or NMI can be used to treat chronic diseases such as multiple sclerosis. Detection of IFP35 and NMI levels in serum or body fluids (such as cerebrospinal fluid) can be used for the diagnosis of chronic inflammatory diseases.
  • the inventor of the present application has developed a neutralizing antibody that can inhibit IFP35.
  • the inventor of the present application further analyzed the relationship between the neutralizing antibody and a NID domain of the antigen IFP35 Three-dimensional crystal structure of the complex.
  • the inventor of the present application can reveal the structure of the antibody, antigen structure and antibody recognition epitope, antigen-antibody binding mode and key interaction residues, etc., and can guide the optimization of the antibody through this structure , So that it can improve the binding capacity and the specificity of recognition, and improve the neutralizing activity of the antibody.
  • antibodies that improve the ability to recognize NMI proteins can also be designed and modified accordingly, so that specific antibodies against NMI and bispecific antibodies that can be directed against both IFP35 and NMI have been developed. .
  • Chronic inflammation The inventors of the present application have tested whether there is an increased expression of IFP35 or NMI in some inflammatory diseases.
  • the inventor of the present application found that in a mouse model of multiple sclerosis (MS) disease, the content of NMI in the serum was significantly increased. After gene knockout IFP35 and NMI, the symptoms of multiple sclerosis disease mice were significantly reduced.
  • the use of neutralizing antibodies against IFP35 can alleviate the symptoms of MS and achieve the purpose of treating or improving the disease.
  • the inventor of this application discovered a new type of DAMP-like inflammatory factor IFP35 family protein (including IFP35 and NMI) in the human body while studying influenza virus and host immune mechanism.
  • IFP35 and NMI DAMP-like inflammatory factor IFP35 family protein
  • these factors are very low or below the detectable level (level below the detection line) in normal human and mouse serum; in the case of pathogen infection, they can be quickly released from immune cells (macrophages) into the blood; they can quickly promote Immune cells secrete inflammatory factors such as TNF and IL6 to activate the body's inflammatory response; the content in the serum is positively correlated with the degree of inflammatory response; when testing samples from patients with sepsis, it is found that when the blood IFP35 and NMI levels reach high levels When the level (for example, IFP reaches hundreds of pg/ml), the mortality of patients is higher.
  • the inventor of the present application used influenza virus as a model and studied the relationship between such inflammatory factors and viral infection and found that compared with wild-type mice, mice with knockout of IFP35 and/or NMI family proteins lost weight The condition is greatly alleviated, the clinical symptoms are significantly reduced, the lung damage caused by influenza virus infection is significantly reduced, and the survival rate is significantly improved.
  • Neutralizing antibody treatment using IFP35 and/or NMI can achieve an effect equivalent to gene knockout, greatly reducing the symptoms of diseased mice, and significantly reducing the mortality caused by influenza virus infection.
  • IFP35 and NMI are significantly increased, indicating that IFP35 and NMI can be used as diagnostic or auxiliary diagnostic indicators for severe/critically ill patients, thereby helping medical care Staff provide warnings.
  • inhibitor drugs such as antibody drugs
  • IFP35 and NMI are expected to be used in the treatment of excessive inflammatory diseases caused by COVID-19. Whether it is the influenza virus or the new coronavirus COVID-19, it will cause IFP35 and NMI to be secreted into the blood and body fluids after infecting the human body.
  • IFP35 and NMI are related to the virus.
  • the severity of infection is related, which can be used as an indicator of the body's inflammatory response caused by these viruses, and can be a therapeutic target for inhibitors such as antibody drugs, peptide drugs, or chemical drugs.
  • Viruses in this article include viruses that can trigger an inflammatory response in the infected body, especially an excessive/severe inflammatory response (inflammatory factor storm), including but not limited to the coronavirus family (such as coronavirus, including COVID-19, SARS, MERS) Viruses), Orthomyxoviridae (for example, influenza viruses, such as type A (type A) influenza viruses), other viruses of the negative-strand RNA virus order (such as Ebola virus, Lassa virus, Marburg virus, Crimea -Congo hemorrhagic fever virus), retroviral family (such as human immunodeficiency virus, such as HIV-1 (also known as HTLV-III, LAV or HTLV-III/LAV or HIV-III); and other isolates, such as HIV -LP, Picornaviridae (e.g.
  • coronavirus family such as coronavirus, including COVID-19, SARS, MERS) Viruses
  • Orthomyxoviridae for example, influenza viruses
  • poliovirus hepatitis A virus, enterovirus, human coxsackie virus, rhinovirus, echovirus
  • Calciviridae e.g. strains that cause gastroenteritis
  • togavirus Family e.g. equine encephalitis virus, rubella virus
  • flaviviridae virus such as Zika virus, West Nile virus, dengue virus, Hanta virus, etc.
  • rhabdovirus family e.g. vesicular stomatitis virus, rabies virus
  • filoviridae e.g. Ebola virus
  • paramyxoviridae e.g.
  • RNA virus family e.g. bunga virus, phlebitis virus
  • Nairo virus nephritis virus family
  • nephritis virus family hemorrhagic fever virus
  • reovirus family rotavirus double antiviral family hepatitis virus family (hepatitis B virus), parvoviral family (parvovirus)
  • papillomavirus family Papillomavirus, Polyomavirus
  • Adenoviridae most adenoviruses
  • Herpesviridae herpes simplex virus (HSV) 1 and 2, varicella-zoster virus, cytomegalovirus (CMV), herpes virus)
  • Herpesviridae herpes simplex virus (HSV) 1 and 2, varicella-zoster virus, cytomegalovirus (CMV), herpes virus)
  • Pox virus smallpox virus, vaccinia virus, pox virus
  • iris virus for
  • biological sample herein refers to a sample containing cell secretions obtained from a virus-infected individual, including but not limited to blood, plasma, serum, cerebrospinal fluid, alveolar lavage fluid, urine, sweat, feces, etc.
  • the "individual” herein is not limited to a specific species or sample type.
  • the term “individual” can refer to a patient, and usually a human patient. However, the term is not limited to humans and therefore includes a variety of non-human animal or mammalian species.
  • “Mammal” refers to any species of mammalian species. Generally, the term “mammal” as used herein refers to a human, a human subject, or a human patient. “Mammal” also refers to any species of non-human mammal species, such as experimental, companion, or economic non-human mammals. Exemplary non-human mammals include mice, rats, rabbits, cats, dogs, pigs, cows, sheep, goats, horses, monkeys, gorillas, and chimpanzees.
  • therapeutically effective amount refers to the amount of the therapeutic agent that is effective when administered to cells, tissues, or subjects alone or in combination with other therapeutic agents to prevent or ameliorate abnormally high levels in the subject. IFP35 and/or NMI-related diseases or discomforts.
  • a therapeutically effective dose also refers to the amount of the therapeutic agent that is sufficient to cause improvement in symptoms, such as treating, curing, preventing or alleviating related medical discomfort, or increasing the speed of treating, curing, preventing, or alleviating this condition. When applied to a single active ingredient administered alone, the therapeutically effective dose refers to that ingredient alone.
  • a therapeutically effective dose refers to the combined amount of active ingredients that result in a therapeutic effect, whether administered in combination, consecutively or simultaneously.
  • an "effective amount of a compound for treating a specific disease” is an amount sufficient to alleviate or in some way reduce the symptoms associated with the disease. Such an amount can be administered as a single dose or can be administered according to a schedule so that it is effective. The amount can cure the disease, but is usually administered to improve the symptoms of the disease. Repeated administration may be required to achieve the desired symptom relief.
  • pharmaceutically acceptable carrier as used herein is meant to include any and all solvents, dispersion media, coatings, isotonic agents and absorption delaying agents that are compatible with drug administration.
  • the use of these media and agents for pharmaceutical active substances is well known in the art. See, for example, Remington, The Science and Practice of Pharmacy, 20th edition, (Lippincott, Williams & Wilkins 2003). Unless any conventional medium or agent is incompatible with the active compound, such use in the composition is considered.
  • inhibitor refers to any molecule that can negatively affect the biological effects of IFP35 and/or NMI. For example, it can interfere with the interaction of IFP35 and/or NMI with another molecule (for example, its substrate). Interact, or reduce the protein level of IFP35 and/or NMI, for example, by reducing the expression of genes encoding IFP35 and/or NMI.
  • Inhibitors can be "direct inhibitors" that interact with IFP35 and/or NMI or nucleic acids encoding IFP35 and/or NMI, or "indirect inhibitors” that do not interact with IFP35 and/or NMI or encoding IFP35 and / Or NMI nucleic acid interacts, but interacts with IFP35 and/or NMI upstream or downstream in the regulatory pathway.
  • the inhibitor can be a specific inhibitor. Those skilled in the art can understand that although the use of specific inhibitors is ideal, the use of multifunctional or universal protein inhibitors is also optional according to specific circumstances.
  • the inhibitor can be an expression inhibitor, a function inhibitor, or an inhibitor that can inhibit both expression and function.
  • the inhibitor is an inhibitor that can act on the transcription and/or translation level of IFP35 and/or NMI to reduce the amount of functional IFP35 and/or NMI produced.
  • inhibitors include, but are not limited to, dsRNA, microRNA, siRNA, shRNA, antisense RNA, or ribozymes.
  • RNA interference/silencing technology antisense nucleic acid technology or ribozyme technology to reduce the expression level of the target protein
  • a suitable design is designed according to the sequence structure of the target protein (for example, IFP35 and/or NMI) dsRNA, microRNA, siRNA, shRNA, antisense RNA or ribozyme molecules and the corresponding preparation and testing are also easily achieved by those skilled in the art.
  • the inhibitor is an inhibitor capable of inhibiting the function/activity of IFP35 and/or NMI.
  • inhibitors include, but are not limited to, antibodies against IFP35 and/or NMI, or antigen-binding fragments thereof, and small molecule compounds.
  • the antibodies that can be used in this application may include polyclonal antibodies or monoclonal antibodies.
  • the antibody may be a whole immunoglobulin derived from a natural source or from a recombinant source.
  • Antibodies can exist in various forms, including, for example, as whole antibodies, or as antibody fragments, or their other immunologically active fragments such as complementarity determining regions.
  • antibodies can exist as antibody fragments with functional antigen binding domains, namely heavy and light chain variable domains.
  • antibody fragments can be selected from but not limited to the following forms: Fv, Fab, F(ab)2, scFv (single-chain Fv), dAb (single domain antibody), bispecific antibody, diabody, and three Chain antibody.
  • the present application provides a method for diagnosing or evaluating the degree of inflammatory response in a virus-infected individual, including determining the interferon-inducible protein 35kD (IFP35) and/or N-Myc interacting protein in a biological sample obtained from the individual (NMI) amount.
  • IFP35 interferon-inducible protein 35kD
  • NPI N-Myc interacting protein
  • the method is used to diagnose or assist in the diagnosis of severely or critically ill individuals.
  • the virus is a virus of the Coronaviridae family, such as a novel coronavirus (COVID-19), SARS virus, and MERS virus; or a virus of the Orthomyxoviridae family, such as influenza virus (influenza A virus).
  • a virus of the Coronaviridae family such as a novel coronavirus (COVID-19), SARS virus, and MERS virus
  • COVID-19 novel coronavirus
  • SARS virus SARS virus
  • MERS virus a virus of the Orthomyxoviridae family
  • influenza virus influenza A virus
  • determining the amount of IFP35 and/or NMI in a biological sample is determining the protein content of IFP35 and/or NMI in the biological sample, or determining the nucleic acid level expression amount of IFP35 and/or NMI in the biological sample, such as mRNA content .
  • the individual is a mammal, such as a human.
  • the biological sample is blood, plasma, serum, cerebrospinal fluid, or alveolar lavage fluid.
  • the present application provides a kit for diagnosing or evaluating the degree of inflammatory response in a virus-infected individual, including a kit for determining interferon-inducible protein 35kD (IFP35) and/or N in a biological sample obtained from the individual -Reagent for the amount of Myc interacting protein (NMI).
  • IFP35 interferon-inducible protein 35kD
  • NPI Myc interacting protein
  • the kit is used to diagnose or assist in diagnosing severely or critically ill individuals.
  • the virus is a virus of the Coronaviridae family, such as a novel coronavirus (COVID-19), SARS virus, and MERS virus; or a virus of the Orthomyxoviridae family, such as influenza virus (influenza A virus).
  • a virus of the Coronaviridae family such as a novel coronavirus (COVID-19), SARS virus, and MERS virus
  • COVID-19 novel coronavirus
  • SARS virus SARS virus
  • MERS virus a virus of the Orthomyxoviridae family
  • influenza virus influenza A virus
  • determining the amount of IFP35 and/or NMI in a biological sample is determining the protein content of IFP35 and/or NMI in the biological sample, for example, the reagent includes an antibody that specifically binds to IFP35 and/or NMI.
  • determining the amount of IFP35 and/or NMI in a biological sample is determining the nucleic acid level expression amount of IFP35 and/or NMI in the biological sample, such as mRNA content, for example, the reagent includes cDNA against IFP35 and/or NMI Sequence designed specific amplification primers.
  • the individual is a mammal, such as a human.
  • the reagent includes a reagent for processing the biological sample to extract protein or nucleic acid material, for example, the biological sample is blood, plasma, serum, cerebrospinal fluid, or alveolar lavage fluid.
  • the present application provides a method for treating or alleviating the inflammatory response of a virus-infected individual, including administering to the individual a therapeutically effective amount of interferon-inducible protein 35kD (IFP35) and/or N-Myc interacting protein (NMI) Inhibitors.
  • IFP35 interferon-inducible protein 35kD
  • NPI N-Myc interacting protein
  • the individual is a severely or critically ill individual.
  • the virus is a virus of the Coronaviridae family, such as a novel coronavirus (COVID-19), SARS virus, and MERS virus; or a virus of the Orthomyxoviridae family, such as influenza virus (influenza A virus).
  • a virus of the Coronaviridae family such as a novel coronavirus (COVID-19), SARS virus, and MERS virus
  • COVID-19 novel coronavirus
  • SARS virus SARS virus
  • MERS virus a virus of the Orthomyxoviridae family
  • influenza virus influenza A virus
  • the inhibitor of IFP35 and/or NMI is a function inhibitor or expression inhibitor of IFP35 and/or NMI.
  • the functional inhibitor of IFP35 and/or NMI is an antibody of IFP35 and/or NMI or an antigen-binding fragment thereof, or a small molecule compound.
  • the expression inhibitor of IFP35 and/or NMI is an inhibitor that can act on the transcription and/or translation level of IFP35 and/or NMI to reduce the amount of functional IFP35 and/or NMI produced,
  • dsRNA dsRNA, microRNA, siRNA, shRNA, antisense RNA or ribozyme.
  • the individual is a mammal, such as a human.
  • the present application provides a pharmaceutical composition for treating or alleviating the inflammatory response of a virus-infected individual, including a therapeutically effective amount of interferon-inducing protein 35kD (IFP35) and/or N-Myc interacting protein (NMI) Inhibitors, and pharmaceutically acceptable carriers.
  • IFP35 interferon-inducing protein 35kD
  • NPI N-Myc interacting protein
  • the individual is a severely or critically ill individual.
  • the virus is a virus of the Coronaviridae family, such as a novel coronavirus (COVID-19), SARS virus, and MERS virus; or a virus of the Orthomyxoviridae family, such as influenza virus (influenza A virus).
  • a virus of the Coronaviridae family such as a novel coronavirus (COVID-19), SARS virus, and MERS virus
  • COVID-19 novel coronavirus
  • SARS virus SARS virus
  • MERS virus a virus of the Orthomyxoviridae family
  • influenza virus influenza A virus
  • the inhibitor of IFP35 and/or NMI is a function inhibitor or expression inhibitor of IFP35 and/or NMI.
  • the functional inhibitor of IFP35 and/or NMI is an antibody of IFP35 and/or NMI or an antigen-binding fragment thereof, or a small molecule compound.
  • the expression inhibitor of IFP35 and/or NMI is an inhibitor that can act on the transcription and/or translation level of IFP35 and/or NMI to reduce the amount of functional IFP35 and/or NMI produced,
  • dsRNA dsRNA, microRNA, siRNA, shRNA, antisense RNA or ribozyme.
  • the individual is a mammal, such as a human.
  • MOG and H37Ra induce NMI release from mouse macrophages.
  • MOG35-55 H37Ra and detecting in vitro stimulation of macrophages BMDM, Raw264.7 Thp1 three kinds of macrophages and cell lines
  • BMDM Raw264.7 Thp1 three kinds of macrophages and cell lines
  • MOG50ng / ml and / or H37Ra 100ng / ml were incubated for 8h ,
  • This figure illustrates that the combination of MOG35-55 and H37Ra can induce the secretion of NMI from mouse macrophages, and H37Ra plays the main role.
  • LPS induces the NMI release of microglia BV2.
  • This figure shows that the central nervous system is inflamed, and when microglia is activated, NMI can also be secreted.
  • mNMI protein induces M1 polarization of mouse microglia.
  • NMI secretory expression in mice at different times after EAE modeling The expression of NMI in sham treated mice and EAE mice in the pre-onset period (7 days), initial onset period (12 days), peak onset period (20 days), and disease remission period (30 days) were detected. This figure shows that the expression of NMI at each stage of the onset of MS in mice is closely related to the degree of MS.
  • FIG. 5 The expression of NMI and other inflammatory factors in mouse spinal cord tissue after EAE modeling. Detect the expression of NMI and main inflammatory factors iNOS, COX2, HMGB1 in mouse spinal cord tissue at different times after EAE modeling. INOS is almost undetectable in the spinal cord of normal mice. As MS enters the peak period, the expression of iNOS reaches the highest; COX2 and HMGB1 increase with the onset of MS; the normal expression of NMI is low, and the expression is highest when MS begins. This figure shows that the expression of NMI and other inflammatory factors can increase with the occurrence of MS, and NMI is one of the earlier up-regulated factors.
  • NMI knockout reduces the clinical symptoms of MS in mice.
  • NMI knockout reduces the MS clinical score of mice;
  • NMI knockout reduces spinal cord inflammatory cell infiltration;
  • NMI knockout reduces spinal cord demyelination symptoms in mice;
  • NMI knockout reduces spinal cord white matter in mice (d ) And gray matter (e) activation status of astrocytes and microglia. This figure shows that NMI can promote neuroinflammation and aggravate the clinical symptoms of MS. After knocking out NMI, the symptoms of MS in mice are reduced.
  • IFP35 antibody can reduce the symptoms of MS in mice.
  • IFP35 antibody was injected intravenously with 100 ⁇ g (5 mg/kg) for 10 days (12-22 days) to relieve the symptoms of MS in mice.
  • NMI knockout can reduce the infiltration and inflammation of spinal cord leukocytes in MS mice.
  • NMI knockout reduces the infiltration of CD45-positive leukocytes in spinal cord inflammation;
  • NMI knockout reduces the expression of spinal cord inflammatory factors iNOS and COX2.
  • FIG. 9 Comparison of NMI and PCT in mice with LPS-induced inflammation. After LPS (i.p., 10mg/kg) was used to establish LPS-induced inflammation model, NMI(a) and PCT(b) changed with time; 16h after CLP was established mouse inflammation model, NMI(c) and PCT(d) content. This figure shows that compared with PCT, NMI can detect inflammation earlier (1 ⁇ 2h), and the background expression of NMI is less than that of PCT, indicating that NMI can be used as a better clinical risk index detection indicator than PCT
  • FIG. 10 Influenza virus A/Puerto Rico/8/1934 (H1N1) strains infected mice after detecting the presence of secreted NMI/IFP35 in mouse serum.
  • the protein levels of IFP35 (also known as IFI35) and NMI in the serum increased significantly on the 3rd day after virus infection, indicating that influenza virus infection can cause inflammation in wild-type mice.
  • the protein levels of IFP35 (also known as IFI35) and NMI in the serum of NMI or IFP35 knockout mice did not increase significantly.
  • Figure 11 shows the antigen binding ability test of the starting murine antibody AE001-VH+VL (35NIDmAb) and the humanized antibody AE001-H1+L1, AE001-H2+L2, AE001-H3+L3.
  • Figure 12 shows the expression and purification results of various modified antibodies.
  • Figure 13 shows the antigen binding ability tests of various modified antibodies.
  • Figure 14 The structure of the complex of antigen IFP35NID and neutralizing antibody 35NIDmAb Fab.
  • Figure 15 shows a dot plot of the determination of IFP35 and NMI levels in the serum of healthy people and confirmed patients infected with the new coronavirus (COVID-19), where each dot indicates a healthy person or a patient's serum sample.
  • Figure 16 shows the verification that influenza virus causes NMI production in vivo and in vitro.
  • A NMI content in the serum of patients infected with influenza virus;
  • B NMI content in the supernatant of THP1 cells stimulated by influenza virus;
  • C NMI content in the supernatant of influenza virus stimulated A549 cells
  • D Serum NMI content of C57BL/6 wild-type mice infected with influenza virus strain PR8 or Mock;
  • E NMI content in the supernatant of RAW264.7 cells stimulated by influenza virus;
  • F Influenza virus The protein expression level of NMI/IFP35 in A549 cells infected with strain PR8 or Mock.
  • * means P ⁇ 0.05, ** means P ⁇ 0.01, *** means P ⁇ 0.001
  • Figure 17 shows the verification that influenza virus causes IFP35 production in vivo and in vitro.
  • A IFP35 content in the supernatant of RAW264.7 cells stimulated by influenza virus;
  • B IFP35 content in the serum of patients infected with influenza virus;
  • C C57BL/6 wild-type mice infected with PR8 or Mock Serum IFP35 content. * Means P ⁇ 0.05, ** means P ⁇ 0.01, *** means P ⁇ 0.001.
  • Figure 18 shows the NMI and IFP35 levels in the serum of 16 influenza patients and 10 healthy people. Patients in the intensive care unit (ICU) who develop severe pneumonia are marked with solid squares.
  • ICU intensive care unit
  • Figure 19 shows the various indicators of NMI knockout mice and wild-type mice after being infected with influenza virus strain PR8.
  • A Change in body weight (%);
  • B Clinical score, in which mice are monitored for sleepiness, creeping, wrinkled fur, hunched back, rapid shallow breathing, rales can be heard, (healthy) 0 ⁇ score ⁇ 5 (dying) ;
  • C H&E staining of lung tissue, the scale is 100 ⁇ m;
  • D survival rate (%).
  • the mice were infected with 2x 10 6 pfu PR8 virus. Log-rank test was used to analyze whether there was a significant difference in the survival rate of mice with different treatments. ** means P ⁇ 0.01.
  • Figure 20 shows the indicators of IFP35 knockout mice and wild-type mice after being infected with influenza virus strain PR8.
  • A Changes in body weight (%);
  • B H&E staining of lung tissue, with a scale of 100 ⁇ m;
  • C survival rate (%).
  • the mice were infected with 2x10 6 pfu PR8 virus.
  • Log-rank test was used to analyze whether there was a significant difference in the survival rate of mice with different treatments. * Indicates P ⁇ 0.05.
  • Figure 21 shows the therapeutic effect of IFP35 neutralizing antibodies.
  • A Experimental protocol;
  • B Weight change (%);
  • C Clinical score;
  • D Survival rate (%).
  • the mice were infected with 2x10 6 pfu PR8 virus. Log-rank test was used to analyze whether there was a significant difference in the survival rate of mice with different treatments. ** means P ⁇ 0.01.
  • Example 1 Using antibodies to treat MS in mice
  • a mouse MS model was established, and then treated with IFP35 antibody. The result is shown in Figure 7. After treatment with IFP35 antibody, the symptoms of MS in mice were significantly reduced.
  • Example 3 Using IFP35/NMI as serum or body fluid (cerebrospinal fluid, etc.) markers to detect MS mice
  • MS animal model experiments show that (see experimental methods), normal mice compared with MS mouse model, the expression of IFP35/NMI in MS mouse model is significantly increased, and is secreted into the serum, and IFP35/NMI as An inflammatory factor is consistent with the high expression and release of other MS characteristic inflammatory factors TNF, iNOS, and IL1 ⁇ in the MS mouse model. It shows that IFP35/NMI may be used as a characteristic biomarker and detection index of MS in serum or body fluid. As shown in Figure 4 and Figure 5. This result is consistent with the results of LPS-induced animal models and influenza virus-induced viral animal models. These acute and chronic inflammatory diseases all lead to the secretion of IFP35 and NMI. As shown in Figure 9, Figure 10. But the PCT behaves differently on this point. PCT generally does not increase serum levels in the case of viral infection.
  • IFP35/NMI in the spinal cord tissue of mice is significantly increased, or whether IFP35/NMI is secreted into the blood or body fluids of the mice (such as cerebrospinal fluid) and secreted into the blood or body fluids. Increase the correlation between them to help diagnose whether the mice may have MS and the inflammatory factors that cause the disease.
  • IFP35/NMI is abnormally secreted into the blood or body fluid of the mouse (such as in the cerebrospinal fluid), or the expression of IFP35/NMI is increased and other inflammatory factors in the mouse (The expression and secretion of interferon, TNF, IL1, IL6, etc.) are consistent with their secretion into the blood, causing obvious external symptoms such as changes in gait and paralysis of the limbs, which can basically diagnose the mice with MS.
  • mice were killed by pulling the neck. Two hind legs of the mice were soaked in 70% alcohol for 1 minute, and the muscles on the receding bones were removed as much as possible.
  • the PBS containing double antibody (penicillin) was sucked into the syringe to flush the bone marrow cavity. Centrifuge at 400g for 10 minutes, remove the supernatant, 1ml red blood cell lysate to lyse red blood cells for 30 seconds, then add 10ml PBS to neutralize the red blood cell lysate.
  • Thp1 cell culture
  • MOG and H37Ra stimulate cells:
  • MOG and H37Ra were added to the above cells at a final concentration of 100ng/ml to stimulate for 8h.
  • mNMI stimulates BV2 cells
  • mice recombinant protein mNMI Express the mouse recombinant protein mNMI, stimulate mouse primary microglia in vitro, adjust the cell concentration to 2 ⁇ 10 6 , mNMI 5 ⁇ g/ml and incubate for 6 hours.
  • QPCR detects the polarization markers of macrophages.
  • PrimeScript TM II 1st Strand cDNA Synthesis Kit Cat.No.6210A and Premix Ex Taq TM (Tli RNaseH Plus), ROX plus Q-PCR kit Cat.NO.RR42LR are all from TAKARA company
  • mice selected for the experiment were female mice with a C57BL/6 background and 8-12 weeks old, which were purchased in combination from Vital River Laboratory Animal Technology Co., Ltd. Emulsify with oligodendrocyte protein MOG35-55 and Freund’s complete adjuvant CFA.
  • Each mouse is injected subcutaneously with MOG35-55 and 0.2mg of CFA each, combined with pertussis toxin PTX, 500ng is injected into the intraperitoneal cavity of each mouse, and PTX is repeated at 48h once.
  • the sham treatment group was given PBS instead of MOG35-55, and the rest was treated with the same amount of CFA and PTX. The disease and life status of the mice were observed and recorded daily.
  • mice were scored after the onset of the disease.
  • the scoring standards were as follows: 0, no clinical symptoms; 1, paralysis of the tail of the mouse; 2, paralysis of one hind limb or weakness of both hind limbs of the mouse; 3, paralysis of both hind limbs of the mouse; 4 , The mouse's hind limbs are paralyzed and the forelimb is affected; 5. the mouse is dying. Score and evaluate the diseased mice according to the scoring standard.
  • C57BL/6WT, IFI35 -/- and NMI -/- were infected with 300 pfu of A/PR8 strain as the challenge experimental group, and C57BL/6 wild-type mice were inoculated with the same amount of PBS as the negative control group. Detect whether the protein levels of IFP35 and NMI in the serum of mice change on the 3rd day after virus infection, and detect the levels of TNF and IL6 in the serum at the same time.
  • mice were injected intraperitoneally with IFP35 monoclonal hybridoma cells, 7-10 days later, mouse ascites were collected, and the ascites antibody was purified by Protein G Agrose beads (GE):
  • the ultrafiltration tube concentrates the antibody and determines the antibody concentration.
  • mice Establish a mouse EAE model, divide the IFP35 antibody treatment group and IgG control group, start to administer the antibody on the 12th day, and inject 100 ⁇ g (5mg/kg) of antibody intravenously per mouse every day for 10 consecutive days. Compare the two groups of mice Clinical symptoms.
  • Termination Add 50 ⁇ l of 2Mol/L H 2 SO4 to each well.
  • Preparation method of knockout mice C57BL/6 mice were used. 8-12 weeks old. All mice used were killed by cervical dislocation. C57BL/6 wild-type mice (000664) were purchased from Vital River Laboratory Animal Technology Co., Ltd. Use CRISPR-Cas9 to generate NMI and Ifp35 knockout mice technology. Fertilized eggs were collected from the fallopian tubes of superovulation, and female C57BL/6 mice were mated with male C57BL/6 mice. Cas9mRNA (150ng ml-1) is mixed with sgRNA (100ng ml-1) produced by transcription and microinjected into the cells. The cytoplasm of the recipient egg has a recognized pronucleus (Sigma, M7167-100ML) in M2 medium.
  • the sgRNA sequence of NMI is 5'-AAAACAAAGAACTAGACGAGG-3'
  • the sgRNA sequence of IFP35 is 5'-CAGCTCAAAAGGGAGCGCACAGG-3'.
  • the frameshift mutations of NMI and Ifp35 genes produced by CRISPR technology result in the failure of NMI and IFP35.
  • Each corresponding sgRNA was injected with about 100-250 fertilized eggs, and then transferred into the uterus of surrogate ICR female mice to obtain F1 generation mice.
  • FIG Method 1 adjusting BMDM, RAW267.4 cell concentration and Thp1 106 per ml, induced mouse macrophages with MOG 35-55 (50ng / ml) and H37Ra (100ng / ml), were incubated for 8h, then detection The amount of NMI secreted in the cell supernatant.
  • the error bars in the figure represent three repeated experiments ⁇ sem; significant differences are detected by unpaired t-test; *P ⁇ 0.05, **P ⁇ 0.01.
  • MOG 35-55 and H37Ra are the main reagents used to induce MS in mice
  • MOG 35-55 is a glycoprotein on the surface of oligodendrocytes , used to cause immune cells to attack self-antigens
  • H37Ra is inactivated
  • the Mycobacterium tuberculosis is used to activate an expanded immune response.
  • MOG 35-55 and H37Ra to stimulate mouse peripheral macrophages in combination, it can be detected that NMI is secreted in large quantities, and the main role is H37Ra.
  • the inducing effect of H37Ra is much greater than that of MOG 35-55 , indicating that H37Ra activates macrophages and is the main promoter of inflammation.
  • MOG 35-55 is not as strong as H37Ra in inducing activation of macrophages, and the combination of the two has a high stimulating effect. In one of them alone. It shows that the agent that induces the onset of MS in mice can induce the release of NMI from macrophages. This experiment can prove that in the induced macrophage activation model, NMI expression increases and is released outside the cell.
  • Figure 2 Method: a, use 100ng/ml LPS to induce BV2 in microglia, and detect the amount of NMI secreted by cells into the supernatant at 0, 2, 4, 6, and 8 hours; b, use different concentrations of LPS (0, 10, 20, 50, 100 ng/ml) stimulated BV2 cells for 8 hours, and the NMI content in the supernatant was detected.
  • the error bars in the figure represent three repeated experiments ⁇ s.e.m; significant differences are detected by unpaired t-test; *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001.
  • Microglia are the main immune cells of the central nervous system and are involved in central nervous inflammatory diseases, including the occurrence and development of MS.
  • LPS lipopolysaccharide or endotoxin
  • Experimental results show that after LPS-induced activation of microglia, a large amount of NMI is secreted, and the amount of secretion is positively correlated with the time and intensity of LPS stimulation, indicating that NMI may be a DAMP in the pathogenesis of MS, and central macrophages NMI is released upon activation.
  • mNMI protein induces M1 polarization of mouse microglia
  • Figure 3 Use purified murine NMI (mNMI) protein (5 ⁇ g/ml) to induce mouse microglia for 6 hours, and detect microglia M1 type marker molecules TNF, iNOS, IL1 ⁇ (a) and M2 type marker molecules Arg1, IL10, TGF- ⁇ , CD206(b) mRNA changes under LPS or NMI stimulation; the error bars in the figure represent three repeated experiments ⁇ sem; significant differences are detected by unpaired t-test; *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001.
  • mNMI murine NMI
  • FIG. 3 Results: As a kind of macrophages, microglia are polarized into M1 and M2 types. Among them, M1 type microglia are mainly involved in the pro-inflammatory response, mediating phagocytosis, secretion of inflammatory factors and tissue damage, and M1 type microglia play a role in promoting disease development in the pathogenesis of MS. M2 type microglia mediate anti-inflammatory response and participate in the tissue repair process. The experimental results in Figure 3 show that mNMI can induce microglia to polarize toward the pro-inflammatory M1 type, and promote the MS inflammatory response.
  • Figure 4 shows that the NMI secretion expression detection of mice at different times after EAE modeling is in the pre-onset period (7 days), the initial onset period (12 days), and the peak period of onset (20 days) in the control experimental group of mice and EAE mice. , NMI expression in the remission period (30 days). The results showed that the expression of NMI at various stages of the onset of MS in mice is closely related to the onset of MS.
  • Figure 5 shows that the detection of the expression of NMI and the main inflammatory factors iNOS, COX2, HMGB1 in the mouse spinal cord tissue at different times after EAE modeling shows that iNOS is almost undetectable in the spinal cord of normal mice.
  • iNOS the main inflammatory factors
  • COX2 and HMGB1 increased with the onset of MS
  • the normal expression level of NMI was low, and the expression level was highest when MS began to develop.
  • the expression of NMI and other inflammatory factors can increase with the occurrence of MS, and NMI is one of the factors that are up-regulated at an early stage. Since the cerebrospinal fluid of mice is more difficult to obtain, the spinal cord tissue is tested here.
  • the expression of iNOS reaches the highest level; COX2 and HMGB1 increase with the onset of MS; the expression level of NMI in normal spinal cord tissue is lower, and the onset of MS begins The expression level is highest when the disease is in remission, and the expression of NMI and other inflammatory factors can be increased with the onset of MS.
  • NMI is one of the inflammatory factors that are up-regulated at an early stage.
  • Figure 6 Method: a, construct NMI knockout mice (NMI -/- ) and wild-type mice (WT) MS animal models, and compare their clinical symptoms. The scoring criteria are as follows: 0, no clinical symptoms; 1, mouse tail paralysis; 2, mouse paralysis of one hind limb or weakness of both hind limbs; 3. mouse paralysis of both hind limbs; 4. mouse paralysis of both hind limbs and forelimb involvement; 5 , The mouse is dying. Score and evaluate the diseased mice according to the scoring standard.
  • b HE pathological sections of spinal cord tissue of NMI -/- and WT mice.
  • Figure 6 Results Use NMI knockout mice to construct an MS animal model, and compare its clinical symptoms with the MS model prepared by wild-type mice.
  • Figure a shows that although NMI -/- mice and WT mice have basically the same time to enter MS, the MS symptoms of NMI -/- mice are subsequently lighter than wild-type mice.
  • figure b HE staining of spinal cord tissue sections was used to observe the infiltration of inflammatory cells in the spinal cord of NMI -/- mice and WT mice. It can be seen that NMI knockout reduced the infiltration of inflammatory cells in the spinal cord of MS mice.
  • Figure c shows that the spinal cord demyelination symptoms of NMI -/- mice are alleviated by LFB staining analysis of spinal cord tissue sections.
  • IFP35 antibody can reduce the symptoms of MS in mice
  • Figure 7 shows that since gene knockout can significantly improve the symptoms of MS mice, the inventors of the present application used neutralizing antibodies of IFP35 to observe whether the symptoms of MS mice can be reduced.
  • Figure a represents the use of purified IFP35 neutralizing antibody described in this application, and SDS-PAGE detection shows that the position of 55kD is the antibody heavy chain and the position of 25kD is the light chain of the antibody. Shows that the antibody is well purified.
  • Figure b shows that the purified IFP35 antibody was injected intravenously with 100 ⁇ g (5 mg/kg) antibody for 10 days (12-22 days) to observe and score the symptoms of MS mice. It can be found that the antibody can significantly alleviate the symptoms of MS in mice.
  • Mouse MS modeling adopts recognized evaluation standards.
  • the scoring standards are as follows: 0, no clinical symptoms; 1, mouse tail paralysis; 2, mouse paralysis of one hind limb or weakness of both hind limbs; 3, mouse paralysis of both hind limbs; 4 , The mouse's hind limbs are paralyzed and the forelimb is affected; 5. the mouse is dying. Score and evaluate the diseased mice according to the scoring standard.
  • the corresponding antibody in this experiment is 35NIDmAb, the light chain variable region is SEQ ID NO: 10, and the heavy chain variable region is SEQ ID NO: 9.
  • the heavy chain variable region includes CDR1, CDR2 and CDR3, and the sequence of CDR1 is the 25-32th amino acid (GYTFTNYG) of SEQ ID NO: 9, and the CDR2 sequence is the 50-57th amino acid of SEQ ID NO: 9 ( INTYTGEP), the sequence of CDR3 is amino acid 98-106 of SEQ ID NO: 9 (YGYSWAMDY).
  • the light chain variable region includes CDR1, CDR2 and CDR3, and the sequence of CDR1 is the amino acid 26-31 of SEQ ID NO: 10 (SSSVSY), and the sequence of CDR2 is the amino acid 49-51 of SEQ ID NO: 10 ( DTS), the sequence of CDR3 is SEQ ID NO: 10 amino acids 90-96 (WSSNPPI), and the naming system is Kabat.
  • NMI knockout can reduce the infiltration and inflammation of spinal cord leukocytes in MS mice
  • NMI knockout reduces the infiltration of CD45 positive leukocytes in spinal cord inflammation; b. NMI knockout reduces the expression of spinal cord inflammatory factors iNOS and COX2.
  • NMI(a) and PCT(b) changed over time; similarly, 16h after intestinal ligation CLP established a mouse inflammation model, NMI(c) and PCT( d) Content.
  • NMI can detect inflammation earlier (1 ⁇ 2h), and the background expression of NMI is less than that of PCT, indicating that NMI can be used as a better clinical risk index detection indicator than PCT.
  • influenza virus A/Puerto Rico/8/1934 (PR8) strains infect mice detect the presence of secreted NMI/IFP35 in the mouse serum.
  • the inventors of the present application also compared whether IFP35 and NMI were secreted in the case of virus infection.
  • Research method C57BL/6WT, IFI35 -/- and NMI -/- were infected with PR8 strain at a dose of 300 pfu as the challenge experimental group, and C57BL/6 wild-type mice were inoculated with the same amount of PBS as the negative control group.
  • Figure 10 Figures A and B, the protein levels of IFP35 (also known as IFI35) and NMI in the serum increased significantly on day 3 after virus infection, indicating that influenza virus infection can cause inflammation in wild-type mice.
  • influenza virus infection can increase the secretion of IL-6 and TNF- ⁇ in serum ( Figure C and Figure D), while IL-6 and TNF- ⁇ in serum of NMI or IFP35 knockout mice
  • TNF- ⁇ protein level was significantly lower than that of wild-type mice.
  • inflammation and infection can lead to the secretion of NMI and IFP35, and inhibition (gene knockout) of IFP35 or NMI can reduce inflammation.
  • This result is different from PCT, which generally does not increase in serum content in the case of virus infection.
  • the above results suggest that whether it is viruses, bacteria, or chronic inflammatory diseases, IFP35 and NMI can be secreted into body fluids (including blood). Therefore, it can be used as a detection index for chronic inflammatory diseases.
  • Experimental method Use PCR site-directed mutagenesis to introduce mutations in the DNA sequence at specific amino acid residue positions of the mouse antibody, so as to change some amino acids on the mouse antibody to transform it into a human antibody sequence, thereby realizing antibody humanization . Then clone the modified antibody gene into the corresponding antibody expression vector, such as pCDNA3.1, pCDNA3.4 and so on. Transform into human kidney cell line eExpi293F or HEK293T cells for expression, and then purify the expressed antibody secreted outside the cell. The expression and purification of the modified antibody and antigen IFP35 were used to detect the binding ability. The equipment used is BiACo or ITC, etc. The Elisa method can also be used to detect the binding ability of the humanized antibody to the antigen. The antigen is human IFP35 protein.
  • the sequence of the mouse antibody (35NIDmAb) before modification and the sequence of the antibody after modification are as follows:
  • the light chain constant region sequence of AE001VL (pre-modified mouse antibody (35NIDmAb) is SEQ ID NO: 24; the humanized modified mouse light chain constant region sequence AE001L1 is SEQ ID NO: 3, and AE001L2 is SEQ ID NO: 7. AE001L3 is SEQ ID NO: 12.
  • the heavy chain constant region sequence of AE001VH (pre-modified mouse antibody (35NIDmAb) is SEQ ID NO: 25; the humanized modified mouse heavy chain constant region sequence AE001H1 is SEQ ID NO: 1, AE001H2 is SEQ ID NO: 5. AE001H3 is SEQ ID NO: 11.
  • AE001-VH+VL 35NIDmAb
  • AE001-H1+L1 AE001-H2+L2
  • AE001-H3+L3 are three groups of humanized antibodies. It can be seen that all humanized antibodies can bind antigen. Since AE001-H3+L3 is closer to human antibodies, this group of antibodies was selected for the following antibody affinity maturity modification.
  • the results showed that the amino acid sequence of the murine antibody increased the degree of humanization, making the antibody a sequence closer to humanization. This experiment also shows that, based on the differences between murine and human antibodies, further humanization of antibodies can be achieved.
  • the backbone sequence recognized by the antibody such as the variable region sequence of the antibody, is the main segment that recognizes the antigen.
  • the more critical region is the CDR region in the antibody variable region, which is more critical for antigen recognition.
  • the core region of these sequences determines the specificity of antigen recognition.
  • Experimental method The experiment adopts general mutant plasmid construction method and protein antibody expression method.
  • random mutations are introduced into the DNA sequence of the CDR region of a humanized antibody combination AE001-H3+L3 by PCR random mutation method, thereby changing the humanized antibody CDR sequence.
  • the modified antibody sequence is cloned into the corresponding antibody expression vector, such as pCDNA3.1 or pCDNA3.4.
  • the library plasmids were transferred into human kidney cell line 293T or Expi293F cells for expression, and then the expressed antibodies secreted outside the cells were purified.
  • the expression and purification of the modified antibody and the in vitro purified antigen IFP35 or NMI or IFP35/NMI complex were used to detect the binding ability.
  • the equipment used is BiACo or ITC, etc.
  • the Elisa method can also be used to detect the binding ability of the antibody to the antigen after the CDR region is modified.
  • the antigen is IFP35, and the result is shown in Figure 13.
  • an antibody with optimized antigen-antibody binding ability can be obtained by changing some amino acid residues of the CDR sequence, and then a neutralizing antibody with better activity can be obtained. It can be seen from the above list that on the basis of the original antibodies, the inventors have screened and obtained some antibodies that can bind to the antigen IFP35 by changing part of the CDR sequence. The conclusion is that changing the amino acid sequence of the above CDR part helps to obtain antibodies with better activity. Changing part of the CDR sequence is an important method commonly used internationally to obtain more optimized antibodies.
  • an antibody that has a homology of 50% or more with the CDR sequence of the antibody disclosed in the present application (the same amino acid at the same CDR position and the same sequence number position) and which binds to IFP35 should fall within the protection scope of the present application. .
  • Example 5 The fine three-dimensional crystal structure of the complex of the neutralizing antibody and the antigen IFP35 reveals the key residues for the antibody to recognize the antigen and the amino acid residue characteristics of the IFP35 epitope
  • mice were immunized, and a monoclonal antibody against IFP35 was obtained from the mouse spleen, which was designated as 35NIDmAb.
  • the monoclonal antibody 35NIDmAb is a monoclonal antibody targeting IFP35 NID (fragment 124 to 220) with neutralizing activity.
  • In vitro purification experiments showed that 35NIDmAb and IFP35 NID were mixed and purified by gel filtration chromatography, and the two were still combined. These results indicate that 35NIDmAb targeting IFP35 NID can bind to IFP35 NID strongly, and experiments show that the antibody also has neutralizing activity. The aforementioned experiments have shown that the monoclonal antibody can protect mice from multiple sclerosis (MS) symptoms.
  • MS multiple sclerosis
  • the inventor of the present application analyzed the crystal structure of 35NIDmAbFab and IFP35NID complex with a resolution of 2.9 angstroms and IFP35 NID structure.
  • 35NIDmAb Fab-IFP35 NID IFP35 exists as a monomer, while in the structure of IFP35 NID alone, IFP35 NID exists as a twosome.
  • the structure of a single IFP35 NID in these two structures is similar.
  • 35NIDmAb Fab-IFP35 NID structure there are four 35NIDmAb Fab-IFP35 NID complexes in an asymmetric unit.
  • Each 35NIDmAb Fab consists of a heavy chain (Ab_VH) and a light chain (Ab_VL) respectively, and interacts with an IFP35NID.
  • 35NIDmAb Fab mainly recognizes and binds to the c-terminal of IFP35 NID.
  • the main amino acid residues on IFP35 NID involved are Arg163, Asn164, Arg191, Gln194, Ile195, Gln197, Phe198, Thr199, Pro201, Gln206, Pro208, Arg210.
  • amino acids that interact with the antigen through the structure.
  • amino acids include:
  • the two amino acids that bind to the antigen are mainly Asn30 and Tyr31; in the antibody heavy chain variable region CDR2 sequence (50 INTYTGEP 57), the main binding to the antigen is It is the three amino acids of Asn51, Tyr53 and Thr54; in the CDR3 (98 YGYSWAMDY 106) sequence of the antibody heavy chain variable region, the two amino acids that bind to the antigen are mainly Tyr100 and Trp102.
  • the main amino acids that bind to the antigen are Ser30 and Tyr31; in the antibody light chain variable region CDR2 sequence (49 DTS 51), the main amino acids that bind to the antigen are Asp49: In the CDR3 sequence of the variable region of the antibody light chain (90 WSSNPPI 96), the main amino acids that bind to the antigen are Ser92 and Asn93.
  • Figure 14 shows the structure of the complex of antigen IFP35 NID and neutralizing antibody 35NIDmAb Fab.
  • Picture A is the complete picture of antibody and antigen structure.
  • the antibody heavy chain (Ab-VH) is light blue, and the antibody light chain (Ab-VL) is green.
  • the antigen (IFP35 NID) is purple.
  • Figure B is a structural diagram of the antibody itself viewed from two angles, in which the residues that interact with the antigen are represented by sticks. The key residues are marked on the diagram.
  • the antibody heavy chain (Ab-VH) is light blue, and the antibody light chain (Ab-VL) is green.
  • Panel C is a schematic diagram of the interaction between the antibody heavy chain and the antigen. The key residues are represented by short sticks.
  • Panel D is a schematic diagram of the interaction between the antibody light chain and the antigen. The key residues are represented by short sticks.
  • NMI NID In addition to the structure of IFP35 NID and the structure of 35NIDmAb Fab and IFP35 NID complex, the inventor of the present application also analyzed the structure of NMI NID.
  • the overall structure of NMI NID is very similar to that of IFP35 NID.
  • the amino acids at the corresponding positions on NMINID are different from IFP35NID (see the sequence alignment diagram below), and the corresponding residues are Arg185 and Asn186 respectively. Lys215, Lys218, Lys219, Glu221, Tyr222, Pro223, Tyr225, Cys230, Arg232, Thr234.
  • These amino acids may affect the interaction of NMI NID and 35NIDmAb.
  • these NMI amino acid residues represent an antibody binding epitope on NMI, which helps to screen and obtain neutralizing antibodies.
  • IFP35 NID The gene fragment encoding IFP35 NID (residues 124 to 220) was cloned into pGEX-6p-1 and expressed in E. coli.
  • the plasmid of IFP35 NID was transformed into BL21(DE3). Add 100 mg/L ampicillin to LB medium, and cultivate bacteria at 37°C. When the OD600 reached 0.8-1.0, the final concentration of 0.5mM isopropyl- ⁇ -D-thioglactosidase was induced at 16°C for 20h. After that, the bacteria were collected by centrifugation at 4000 rpm for 15 minutes.
  • lysis buffer (20Mm Tris at pH 8.0, 400mM NaCl, 5% Glycerol), break the bacteria with an ultrasonic disruptor, and centrifuge at 16000rpm for 30 minutes at high speed. The supernatant after centrifugation was combined with the GST affinity column for 1h, and then the GST affinity column was washed.
  • the recombinant protein was eluted with elution buffer (20mM Tris at Ph 8.0, 150mM NaCl, 30mM Glutathione), the eluted protein was digested with PPase, and further used with ion exchange column (high salt buffer: 20mM Tris at Ph 8.0 ,1M NaCl, 5% Glycerol,; low salt buffer: 20mM Tris at Ph 8.0, 100mM NaCl, 5% Glycerol) and gel filtration chromatography column Superdex 200 (buffer: 20mM Tris at Ph 8.0, 150mM NaCl) purification .
  • elution buffer 20mM Tris at Ph 8.0, 150mM NaCl, 30mM Glutathione
  • NMI NID from 155 to 240 gene fragments are the same as IFP35 NID.
  • 35NIDmAb hybrid tumor cells After obtaining 35NIDmAb, add 1mM EDTA and 1mM cysteine to PBS, and use this buffer to dissolve papain.
  • the purified 35nidmab was cleaved with papain at a mass ratio of 100:1 (35nidmab: papain) and reacted at 37°C for 6-8 hours to obtain 35NIDmAb Fab.
  • 35NIDmAb Fab and IFP35NID were mixed at a ratio of 1:1, and then separated and purified by gel filtration chromatography using superdex 200.
  • the hanging drop method was used for crystal screening, and the crystals were spotted at 16°C with 0.2ul protein (10 mg/ml) + 0.2ul pool solution.
  • the crystals of 35NIDmAb Fab and IFP35 NID were grown in a pool containing 0.1M Tris (pH 8.5), 0.2M MgCl2, and 18% PEG 3350. Add 15% DMSO to the crystallization solution to protect the crystal at low temperature, and diffract the crystal on the beamline BL17U and BL18U of the Shanghai Synchrotron Radiation Facility. Use HKL3000 to process the diffraction data.
  • the single-wavelength anomalous dispersion method is used to solve the structure of IFP35 NID and NMI NID.
  • Use coot and phenix to correct the structure Taking the structure of HIV neutralizing monoclonal antibody YZ23 (PDB code 3CLF) and IFP35 NID as a model, the structure of 35NIDmAbFab-IFP35NID complex was solved by molecular replacement method.
  • the initial model of the structure was constructed with Phenix.Autobuild. Use coot and phenix to correct the structure.
  • Sample collection 4mL of venous blood is drawn on an empty stomach and placed in a disposable vacuum tube without anticoagulant, centrifuged at 3000r/min for 15min, after separating the serum, put the serum sample tube in a 56°C water bath for at least half an hour to inactivate the virus . After that, the sample tube is stored in a refrigerator at -20°C or -80°C for testing, avoiding repeated freezing and thawing.
  • Sample detection Use enzyme-linked immunosorbent assay (ELISA) to detect the concentration of IFP35 and/or NMI in the serum. The detection steps are as follows:
  • HEK293T (ATCC, CRL-11268), A549 (ATCC, CRM-CRL-185) and MDCK (NBL-2) (ATCC CCL-34TM) cell lines were cultured in DMEM medium and added with 10% fetal bovine serum (Gibco ).
  • THP1 (ATCC, TIB-202TM) and RAW264.7 cells (ATCCTIB-71TM) were fed in RPMI1640 medium (Gibco, C1875500BT).
  • 9-day-old SPF chicken embryos were purchased from Guangdong Dahuanong Biotechnology Co., Ltd.
  • IFP35 monoclonal antibody (H00003430-M01) was purchased from Abnova.
  • NMI antibody (ab183724) was purchased from abcam.
  • Influenza A virus NS1 antibody (sc-130568) was purchased from Santa Cruz.
  • Anti-influenza A virus nucleoprotein antibody was purchased from abcam (ab128193).
  • E6446 (dihydrochloride) (HY-12756A) (TLR7/9 inhibitor) and CU-CPT-9b (HY-112051) (TLR8 inhibitor) were purchased from Med ChemExpress (MCE, USA).
  • TLR3 dsRNA inhibitor (614310) was purchased from Millipore. Resatorvid (TAK-242) was purchased from Selleck (USA).
  • C57BL/6(B6) wild-type mice were purchased from Guangdong Medical Experimental Animal Center (GDMLAC). According to published literature reports, the C57BL/6 mouse model was used to construct NMI -/- or IFP35 -/- homozygous knockout mice. All mice used in the experiment were 8 to 12 weeks old and gender matched. The mice are kept in an environment free of specific pathogens, and the breeding conditions meet the standards of the Animal Welfare and Use Committee of Sun Yat-sen University.
  • TRIzol LS reagent USA to extract total RNA from cells or lung tissues. Then use the specific primer U12A: AGCAAAAGAGG retroviral genomic RNA (1 ⁇ g), and use the PrimeScript II first-strand cDNA synthesis kit (Takara) to configure the reverse transcription system. Viral mRNA was first purified with an mRNA isolation kit (China Yesen Biotechnology Co., Ltd.), and then oligo(d T)18 was used as a primer for reverse transcription reaction. Using the cDNA product as a template, SYBR Green Mix (Applied Biosystems) was used for qPCR amplification. Use Quant Studio 5 (Applied Biosystems, Thermo Fisher Scientific) for quantitative PCR reaction. Use the standard curve method to analyze the data.
  • Mouse mIFP35 (E10460m) ELISA kit was purchased from EIAab company.
  • Human IFP35 ELISA kit (OKEH02088) was purchased from AVIVA SYSTEM BIOLOGY.
  • Human hNMI (CSB-EL015893HU) and mouse mNMI (CSB-EL015893MO) ELISA kits were purchased from CUSABIO. Perform ELISA experiments according to the operating manual. A brief description is as follows: add 100 ⁇ l of the standard, test sample, negative and blank control diluted in multiples to the corresponding test ELISA plate, and set 2 replicates for each sample. After covering the membrane, incubate at 37°C for 2 hours.
  • mice All mouse lung tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 4 ⁇ m slices. Then all sections were stained with hematoxylin and eosin (H&E), and tissue damage, necrosis, and inflammatory cell infiltration were checked under a microscope, and different areas were randomly selected to take pictures.
  • H&E hematoxylin and eosin
  • Quantitative data are shown as mean ⁇ SD, and unpaired Student's t test is used to determine whether the differences between groups are statistically significant. Log-rank test was used to analyze whether there was a significant difference in the survival rate of mice with different treatments. When the P value is less than 0.05, it is considered to be statistically significant, when the P value is less than 0.001, it is considered to have a significant difference#,P>0.05; *,P ⁇ 0.05; **,P ⁇ 0.01;***,P ⁇ 0.001).
  • Example 6 Identification of IFP35 and/or NMI as serological indicators of novel coronavirus pneumonia (COVID-19)
  • the inventor of the present application tested the plasma samples of 5 severe/critically ill patients with novel coronavirus infection collected from designated hospitals for novel coronavirus pneumonia, and found that the levels of IFP35 and NMI of all patients were significantly increased. Similarly, further analysis using more (tens of) patient serum samples yielded the same results. What is more noteworthy is that combined with the clinical manifestations and prognosis of patients, IFP35 and NMI levels are highly correlated, that is, the higher the IFP35 and NMI levels, the more severe the patient’s clinical manifestations and the worse the prognosis (IFP35 and NMI in this study). The patient with the highest NMI level eventually died).
  • Figure 15 shows the serum IFP35 and NMI results of 5 patients.
  • the IFP35 content in some samples has reached the level of 500-700pg/mL, which has reached a level close to the sepsis patients previously discovered by the inventor of the present application.
  • the lethal level (approximately 700pg/mL), which indicates that IFP35 and/or NMI are related to the inflammatory response of the new coronavirus infection, especially in severe/critically ill patients, so they can be treated as COVID-19 infected patients Blood test markers to help medical staff judge the severity and prognosis of inflammatory diseases in patients.
  • Example 7 Identification of IFP35 and/or NMI as serological indicators of influenza virus infection
  • influenza virus PR8 stimulates human monocytes (THP1), epithelial cells (A549), and RAW264.7 cells. Caused cells to release Nmi into the culture medium ( Figure 16 B, C, E); in PR8 infected C57BL/6 mice, serum NMI was significantly higher than the control group ( Figure 16 D); cell NMI and IFP35 PR8 gradually increased after infection, and showed different abundances in A549 cells ( Figure 16 in F panel).
  • IFP35 The results observed for IFP35 are similar to NMI.
  • the serum NMI of patients infected with influenza A virus is higher than that of healthy donors (figure B in Figure 17); RAW264.7 cells stimulated by influenza virus PR8 can cause the cells to release Nmi into the culture medium (figure 17 A panel); In PR8 infected C57BL/6 mice, serum IFP35 was significantly higher than the control group (Figure 17 C panel);
  • Figure 18 shows the levels of NMI and IFP35 in the serum of 16 influenza patients and 10 healthy people.
  • the levels of NMI and IFP35 in the serum of influenza patients are much higher than those of healthy people, and the intensive care unit (ICU) has developed severe illness
  • the content of pneumonia patients (marked by solid squares) is particularly high.
  • Example 8 Verification of the protective effect of NMI and IFP35 deletion on influenza virus infection
  • NMI -/- knockout mice were established in the C57BL/6 mouse model. A group of C57BL/6 wild-type or NMI -/- knockout mice were tested with 2 ⁇ 10 6 pfu PR8 virus, and 90% of the infected mice were killed. All mice are monitored daily to determine whether they are alive, lose weight, and have clinical symptoms of the disease (for example, lethargy, hair loss, wrinkled fur, hunched posture, rapid shallow breathing, audible Rales). The daily clinical score of each mouse ranges from 0 (asymptomatic) to 5 (dying). As shown in panels A and B in Fig. 19, NNMI -/- mice showed mild clinical symptoms and lost weight (%).
  • IFP35 -/- knockout mice had no obvious lung injury (B panel); compared with wild-type mice, the survival rate (%) slightly increased (C panel); interestingly, body weight The change (%) was not significantly different from the control group (Panel A).
  • IFP35 neutralizing antibody was used as a drug to treat 12 C57BL/6 wild-type mice 1 day before virus infection, and at the same time, the same amount of mouse IgG was taken as a negative control Another group of C57BL/6 wild-type mice were treated. Each mouse was injected intravenously with 200 ⁇ g antibody for 5 consecutive days, and then all mice were inoculated with LD 90 PR8 virus on day 0. Each mouse was weighed and monitored to monitor for wrinkled fur, lethargy, crawling, hunchback, shortness of breath, and audible rales. The clinical symptoms were scored daily for 2 weeks. For a schematic diagram of the experimental scheme, refer to Figure 21 in Panel A.
  • IFP35 neutralizing antibody-treated mice can significantly protect them from the lethal PR8 infection (LD 90 dose), while most of the mice given mIgG died (Figure 21, D panel); clinical scores are consistent with the survival rate results (Panel C in Figure 21); however, there was no significant difference in body weight changes (Panel A in Figure 21), which is consistent with the results in Figure A in Figure 20, and further research is needed to determine its mechanism.
  • IFP35 neutralizing antibodies alleviated clinical symptoms and protected mice from lethal influenza virus infection.
  • Influenza viruses such as the pandemic H1N1, highly pathogenic H5N1 and new recombinant H7N9, can directly infect humans and cause severe acute lung injury, acute respiratory distress syndrome, and ultimately respiratory failure. This usually leads to secondary bacterial infections, leading to a variety of infectious pneumonia, encephalitis, myocarditis, and eventually multiple organ failure.
  • influenza virus infection can lead to an increase in the levels of IFP35 and/or NMI in the blood of infected individuals, and this result can be used as a characteristic index for detecting influenza virus infection for clinical testing.
  • exogenous administration methods such as antibody drugs and chemical drugs to inhibit IFP35 and/or NMI can be used for the treatment of influenza virus.

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Abstract

本申请提供一种慢性炎症的诊断和治疗方法。本申请提供的一种技术方案是试剂在制备预防和/或治疗慢性炎症疾病的产品中的应用：所述试剂为抑制被分泌到细胞外作为炎症因子的异常含量的IFP35和/或NMI的活性的物质。实验证明用抗体等抑制被分泌到细胞外作为炎症因子的异常含量的IFP35和/或NMI的活性，可以有效治疗慢性炎症疾病。本申请还以IFP35和/或NMI作为靶点，为病毒感染、特别是新型冠状病毒(COVID-19)感染，提供了诊断/辅助诊断和治疗的方法和工具。

Description

慢性炎症和病毒感染的诊断和治疗
相关申请
本申请要求2019年4月1日提交的中国专利申请201910256220.3号以及2020年2月27日提交的中国专利申请202010122554.4的优先权，通过引用的方式将这两篇专利申请的内容整体并入本文，用于所有目的。
技术领域
本申请涉及生物医药领域，具体涉及用于慢性炎症和病毒感染的诊断和治疗的方法和产品。
背景技术
机体在感染微生物后或收到组织损伤等情况下引起体液中多种细胞因子如TNF、IL-6、IL-8、IL-12、IFN-γ和MCP-1等迅速并大量产生，甚至可能引起细胞因子风暴以及急性呼吸窘迫综合症、多器官衰竭等多种严重的临床疾病。炎症因子与各类急性及慢性炎症疾病密切相关。而目前为止，针对这些疾病在临床上并没有有效的治疗方法。
有生存压力的细胞会释放内源性细胞因子，启动免疫应答反应，发挥有效清除受损伤或被感染的细胞的作用。在组织感染或损伤时，一类被称为损伤相关分子模式(DAMPs)的细胞因子释放并激活邻近的吞噬细胞，最终清除丧失正常功能的细胞。DAMP可以启动天然免疫应答并加剧炎症反应，进而对抗感染和细胞损伤。因此，DAMP的鉴定和其功能的揭示至关重要。在过去，本申请发明人发现两个可以作为DAMPs发挥作用的蛋白，即N-myc和STAT相互作用蛋白(NMI)和干扰素诱导蛋白35(IFP35)。研究发现，IFP35和NMI可以在被LPS或者干扰素激活后一小时内，被单核细胞和巨噬细胞分泌到细胞外，并在脂多糖诱导的脓毒症模型或对乙酰氨基酚诱发的肝损伤模型中，被活化的巨噬细胞释放。胞外NMI和IFP35可以通过Toll样受体4(TLR4)激活NF-κB进而激活巨噬细胞并释放促炎性细胞因子。此外，在死于严重炎症反应的患者血清中，NMI的水平显著升高。NMI缺失可减轻炎症反应，并降低脓毒症模型和肝损伤模型中小鼠的死亡率。针对IFP35的抗体可以降低炎症因子的表达水平，从而有效提高败血病小鼠的存活率。
以多发性硬化症(multiple sclerosis，MS)为例。多发性硬化是慢性炎症疾病中的一种。MS可引起患者感觉、精神、运动、肢体活动和认知功能障碍，是引起青壮年人口致残的主要原因之一。
IFP35蛋白家族包含两种同源蛋白：N-myc和STAT相互作用蛋白(NMI)和干扰素诱导蛋白35(IFP35)，它们都是干扰素诱导基因(ISGs),人体机体及免疫细胞在受到干扰素诱导后，细胞中的表达量会升高。此外，它们还是损伤相关分子模式(DAMP)，在感染或器官损伤中，NMI和IFP35可以被激活的巨噬细胞释放，通过TLR4受体信号通路激活NF-κB，促进巨噬细胞释放促炎细胞因子，NMI和IFP35敲除可以降低小鼠由于细菌或病毒感染导致的脓毒症和肝损伤模型的死亡率。LPS、沙门氏菌等病菌、病毒等诱导的小鼠败血症小鼠模型以及由于毒性物质(如对乙酰氨基酚APAP)侵入体内的小鼠动物模型中，NMI和IFP35能够由细胞内快速释放到血清等体液中。注射IFP35保护性抗体，能够保护LPS和沙门氏菌、以及APAP对小鼠造成的机体损伤。
长久以来，病毒感染性疾病对中国及整个人类社会构成极大威胁。在难以开发出有效的特异性抗病毒药物的情况下，控制病毒感染患者体内的过度炎症反应对减轻机体炎性损伤及 降低死亡率尤为重要。找到导致炎症因子风暴的关键细胞因子，开发新型诊疗药物对于应对突发病毒性疾病十分必要。
[根据细则9.2改正08.06.2020]　
与SARS病毒、流感病毒和其它呼吸道病毒类似，COVID-19病毒感染的靶器官为肺脏，可造成感染患者肺衰竭并导致急性呼吸窘迫综合征。病毒感染宿主后，往往导致病患体内发生强烈的或失调的炎症反应。炎症失调和炎症过度被认为是SARS等病毒致死的重要原因，推测这也是COVID-19病毒感染致死的重要原因。因此，病毒感染疾病需要从两方面进行治疗：一方面是直接的抗病毒药物治疗；另一方面则是需要平衡机体的免疫反应，抑制免疫过度。在没有针对性的抗病毒药物的情况下，调控机体的炎症反应对减轻疾病损伤程度、降低死亡率尤其重要，临床上迫切需要研发精准调控炎症反应的药物。
炎症反应涉及各类免疫细胞和大量炎症相关细胞因子的参与，是一个被精确调控的过程。炎症反应是一把双刃剑。一方面它是机体免疫防御的重要途径，用以清除感染。另一方面，炎症失调是很多疾病发生的重要原因，如脓毒症、慢性炎症疾病及自身免疫性疾病等。病原侵染情况下，宿主的免疫系统产生内源性的损伤相关分子模式(Damage Associated Molecular Pattern,DAMP)。目前人体中发现了几种DAMP类炎症因子，包括HMGB1、IL1b、IL33、S100A等。DAMP作为炎症因子进一步被细胞的模式识别受体(如TLR4等)识别，激活天然免疫细胞下游信号通路(包括JAK-STAT通路等)，激活细胞中炎症相关的转录因子，如NF-κB和IRF3/7等，诱导大量炎症相关基因的转录与表达，激活免疫反应。所以，DAMP炎症因子是免疫系统中一类非常重要的炎症反应分子。这些DAMP类炎症因子普遍被应用于临床检测及作为抗体、小分子化药等药物研发的靶标。
目前，针对爆发的COVID-19病毒感染，尚缺乏临床确定有效的疫苗和抗病毒药物。根据报道，一些抗HIV及SARS/MERS的药物，如洛匹那韦、瑞德西韦(Remdesivir)等可能在COVID-19病毒感染病例中产生治疗效果。临床上，以往治疗SARS以及目前的COVID-19病毒感染的危重症患者时中采取的措施会包括大剂量使用激素类药物控制过度炎症反应。但是，大量的激素使用往往带来严重的副作用，过度的免疫抑制也不利于机体清除病毒。因此，寻找关键的调控炎症反应细胞因子，筛选能够有效抑制导致宿主过度炎症反应的细胞因子药物是解决新发、突发传染病的有效治疗手段，可用于临床及预后的精准干预治疗。这不仅对目前的COVID-19病毒的防治具有重要意义，且在未来应对各类缺乏针对性药物的突发传染性疾病也具有普遍的意义。
发明内容
本申请索要解决的技术问题之一是，提供一种预防和/或治疗慢性炎症疾病的方法和产品及相关抗体，以及提供一种诊断慢性炎症疾病的方法和产品及相关抗体。
本申请提供了试剂在制备预防和/或治疗慢性炎症疾病的产品中的应用的技术方案。
该技术方案如下：
试剂在制备预防和/或治疗慢性炎症疾病的药物中的应用；所述试剂为如下中的至少一种：
(1)抑制被分泌到细胞外作为炎症因子的异常含量的IFP35和/或NMI的活性的物质；
(2)抑制被分泌到细胞外作为炎症因子的IFP35和/或NMI的含量和/或活性的物质；
(3)抑制IFP35和/或NMI被分泌到细胞外作为炎症因子的物质；
(4)抑制IFP35和/或NMI的含量和/或活性的异常增高的物质；
(5)抑制IFP35和/或NMI被分泌到细胞外的物质；
(6)抑制IFP35和/或NMI作为炎症因子引起慢性炎症疾病的物质或抑制IFP35和/或NMI作为DAMPs引起慢性炎症疾病的物质；
(7)抑制IFP35和/或NMI上调一些炎症因子的表达和分泌的物质；所述一些炎症因子为干扰素、TNF、IL1和/或IL6；
(8)抑制IFP35和/或NMI与TLR4作用通过TLR4/MD2激活NF-κB的物质。
本申请提供了一种预防和/或治疗慢性炎症疾病的方法。
本申请所提供的预防和/或治疗慢性炎症疾病的方法，包括如下步骤，向生物体施加有效量的如下试剂，以达到预防和/或治疗慢性炎症疾病的目的；
所述试剂为如下中的至少一种：
(1)抑制被分泌到细胞外作为炎症因子的异常含量的IFP35和/或NMI的活性的物质；
(2)抑制被分泌到细胞外作为炎症因子的IFP35和/或NMI的含量和/或活性的物质；
(3)抑制IFP35和/或NMI被分泌到细胞外作为炎症因子的物质；
(4)抑制IFP35和/或NMI的含量和/或活性的异常增高的物质；
(5)抑制IFP35和/或NMI被分泌到细胞外的物质；
(6)抑制IFP35和/或NMI作为炎症因子引起慢性炎症疾病的物质或抑制IFP35和/或NMI作为DAMPs引起慢性炎症疾病的物质；
(7)抑制IFP35和/或NMI上调一些炎症因子的表达和分泌的物质；所述一些炎症因子为干扰素、TNF、IL1和/或IL6；
(8)抑制IFP35和/或NMI与TLR4作用通过TLR4/MD2激活NF-κB的物质。
现有技术中任何能够实现上述(1)-(8)任一所述功能的试剂在制备预防和/或治疗慢性炎症疾病的产品中的应用都属于本申请的保护范围。
上述(1)-(5)中的抑制，可以是直接抑制，比如直接抑制IFP35和/或NMI的含量和/或活性的异常增高，或直接抑制被分泌到细胞外作为炎症因子的异常含量的IFP35和/或NMI的活性，比如直接抑制IFP35和/或NMI被分泌到细胞外作为炎症因子，比如抑制被分泌到细胞外作为炎症因子的IFP35和/或NMI的含量和/或活性；也可以是间接地通过抑制干扰素的表达和/或活性进行抑制IFP35和/或NMI。
上述治疗方法和治疗应用中，所述试剂为如下中至少一种：
抗体或多肽或抗原结合片段，其特异结合IFP35和/或NMI，其具有权利要求1中(1)-(8)至少一种功能；
小分子化合物，其具有权利要求1中(1)-(8)至少一种功能；
核酸试剂，具有权利要求1中(1)-(8)至少一种功能。
核酸分子可为siRNA,shRNA,或miRNA，可以靶向IFP35和/或NMI的编码基因的。
小分子化合物，可以是抑制IFP35和/或NMI的分泌、表达和/或活性的小分子化合物。
所述抗体或多肽或抗原结合片段可以是特异结合如下抗原表位的抗体：(1)抗原表位的序列位于SEQ ID NO:2的第81-170,177-268,或136-216位氨基酸之内；(2)抗原表位的序列位于SEQ ID NO:4第81-168,175-266,或134-214位氨基酸之内；(3)抗原表位的序列位于SEQ ID NO:6第104-193,202-293,或151-250位氨基酸之内；(4)抗原表位的序列位于SEQ ID NO:8第103-192,201-292,或151-240位氨基酸之内。
上述治疗方法和治疗应用中，所述抗体可为如下A或B或C或D的抗体，
A、该抗体、多肽或抗原结合片段特异结合IFP35/NMI的抗原表位，所述抗原表位为如下(1)或(2)：所述抗原表位包含如下氨基酸位点：IFP35(SEQ ID NO:2)的Arg163，Asn164,Arg191,Gln194,Ile195,Gln197,Phe198,Thr199,Pro201,Gln206,Pro208,Arg210；(2)所述抗原表位包含如下氨基酸位点：NMI(SEQ ID NO:8)的Arg185,Asn186Lys215,Lys218，Lys219，Glu221,Tyr222,Pro223,Tyr225,Cys230，Arg232,Thr234。
B、任何能够结合上述(1)中抗原表位中的至少一个氨基酸残基并抑制IFP35的活性抗体或小分子或多肽物质；
C、任何能够结合上述(2)中抗原表位中的至少一个氨基酸残基并抑制NMI的活性抗体或小分子或多肽物质。
D、所述抗体为35NIDmAb抗体，其具有轻链可变区和重链可变区，重链可变区中含有CDR1,CDR2和CDR3,CDR1的序列是SEQ ID NO：9的第25-32位氨基酸(GYTFTNYG(SEQ  ID NO：13)),CDR2的序列是SEQ ID NO：9的第50-57位氨基酸(INTYTGEP(SEQ ID NO：14)),CDR3的序列是SEQ ID NO：9的第98-106位氨基酸(YGYSWAMDY(SEQ ID NO：15))；轻链可变区中含有CDR1,CDR2和CDR3,CDR1的序列是SEQ ID NO：10的第26-31位氨基酸(SSSVSY(SEQ ID NO：16)),CDR2的序列是SEQ ID NO：10的第49-51位氨基酸(DTS(SEQ ID NO：17)),CDR3的序列是SEQ ID NO：10的第90-96位氨基酸(WSSNPPI(SEQ ID NO：18))。
上述的抗体、多肽或抗原结合片段中，所述抗体为将35NIDmAb除CDR外其余位置氨基酸进行突变得到的抗体；或，所述抗体为将35NIDmAb进行人源化的抗体。
本领域技术人员可以根据本领域公知常识和惯用手段，将上述任一所述抗体，除了CDR序列之外的其它氨基酸进行改造突变，得到的抗体也属于本申请的保护范围。
所述人源化抗体为如下中至少一种：AE001-H1+L1、AE001-H2+L2或AE001-H3+L3。这三种抗体均具有轻链可变区和重链可变区，重链可变区中含有CDR1,CDR2和CDR3,CDR1的序列是SEQ ID NO：9的第25-32位氨基酸(GYTFTNYG),CDR2的序列是SEQ ID NO：9的第50-57位氨基酸(INTYTGEP),CDR3的序列是SEQ ID NO：9的第98-106位氨基酸(YGYSWAMDY)；轻链可变区中含有CDR1,CDR2和CDR3,CDR1的序列是SEQ ID NO：10的第26-31位氨基酸(SSSVSY),CDR2的序列是SEQ ID NO：10的第49-51位氨基酸(DTS),CDR3的序列是SEQ ID NO：10的第90-96位氨基酸(WSSNPPI)。
AE001-H1+L1的重链恒定区序列为AE001H1(SEQ ID NO：1)，轻链恒定区序列为AE001L1(SEQ ID NO：3)。
AE001-H2+L2的重链恒定区序列为AE001H2(SEQ ID NO：5)，轻链恒定区序列为AE001L2(SEQ ID NO：7)。
AE001-H3+L3的重链恒定区序列为AE001H3(SEQ ID NO：11)，轻链恒定区序列为AE001L3(SEQ ID NO：12)。
上述的抗体、多肽或抗原结合片段中，所述抗体为将35NIDmAb或其人源化抗体中的至少一个CDR中氨基酸残基进行突变得到的抗体。
上述的抗体、多肽或抗原结合片段中，所述将35NIDmAb或其人源化抗体中的至少一个CDR进行突变为进行如下至少一种突变：
35NIDmAb的重链可变区CDR1的第25、26、27、28、29和/或32位氨基酸残基进行突变；即(25GYTFT NYG 32)中除了NY其余氨基酸位置进行突变。
35NIDmAb的重链可变区CDR2的第50、52、55、56和/或57位氨基酸残基进行突变；即(50 I NT YTGEP 57)中除了下划线氨基酸外其余位置的氨基酸进行突变。
35NIDmAb的重链可变区CDR3的第98、99、101、103、104、105和/或106位氨基酸残基进行突变；即(98 YG YS WAMDY 106)中除了下划线氨基酸外其余位置的氨基酸进行突变。
35NIDmAb的轻链可变区CDR1的第26、27、28和/或29位氨基酸残基进行突变；即(26 SSSV SY 31)中除了下划线氨基酸外其余位置的氨基酸进行突变。
35NIDmAb的轻链可变区CDR3的第90、91、94、95和/或96位氨基酸残基进行突变；即(90 WS SNPPI 96)中除了下划线氨基酸外其余位置的氨基酸进行突变。
在35NIDmAb的抗体重链可变区CDR1序列(25 GYTFT NYG 32)中，与抗原结合的主要是Asn30和Tyr31两个氨基酸；在抗体重链可变区CDR2序列(50 I NT YTGEP 57)中，与抗原结合的主要是Asn51，Tyr53以及Thr54三个氨基酸；在抗体重链可变区CDR3(98 YG YS WAMDY 106)序列中，与抗原结合的主要是Tyr100和Trp102两个氨基酸。以上所述残基可以称为抗体重链与抗原相互作用残基。其它CDR残基不与或者很少与抗原IFP35结合，因此更易进行更改而获得不显著影响抗体活性的抗体。因此更改任何以上所述CDR中不与抗原相互作用的残基，仍然可能保持与IFP35或NMI的抗原-抗体复合体结构中对应的抗原表位氨基酸结合，应在本申请保护范围内。
在抗体轻链可变区CDR1序列(26 SSSV SY 31)中，与抗原结合的主要氨基酸是Ser30和Tyr31；在抗体轻链可变区CDR2序列(49  DTS 51)中，与抗原结合的主要氨基酸是Asp49；在抗体轻链可变区CDR3序列(90 WS SNPPI 96)中，与抗原结合的主要氨基酸是Ser92和Asn93。以上所述残基可以称为抗体重链与抗原相互作用残基。其它CDR残基不与或者很少与抗原IFP35结合，因此更易进行更改而获得不显著影响抗体活性的抗体。因此更改任何以上所述CDR中不与抗原相互作用的残基，仍然可能保持与IFP35或NMI的抗原-抗体复合体结构中对应的抗原表位氨基酸结合，应在本申请保护范围内。
上述的抗体、多肽或抗原结合片段中，所述将35NIDmAb或其人源化抗体中的至少一个CDR进行突变为进行如下至少一种突变：
35NIDmAb的重链可变区CDR1的第30和/或31位氨基酸残基进行突变；
35NIDmAb的重链可变区CDR2的第51、53和/或54位氨基酸残基进行突变；
35NIDmAb的重链可变区CDR3的第100和/或102位氨基酸残基进行突变；
35NIDmAb的轻链可变区CDR1的第30和/或31位氨基酸残基进行突变；
35NIDmAb的轻链可变区CDR3的第92和/或93位氨基酸残基进行突变。
所述抗体或抗原结合片段是能够识别如下两个或以上氨基酸残基的抗体或抗原结合片段：35NIDmAb或其人源化抗体中的重链的Asn30、Tyr31、Asn51、Tyr53、Thr54、Tyr100和Trp102位氨基酸残基，其参与与抗原IFP35及NMI的识别。
所述抗体或抗原结合片段是能够识别如下两个或以上氨基酸残基的抗体或抗原结合片段：35NIDmAb或其人源化抗体中的轻链的Ser30、Tyr31、Asp49、Ser92和Asn93位氨基酸残基，其参与与抗原IFP35及NMI的识别。
在抗体重链可变区CDR1序列(25 GYTFT NYG 32)中，与抗原结合的主要是Asn30和Tyr31两个氨基酸；在抗体重链可变区CDR2序列(50 I NT YTGEP 57)中，与抗原结合的主要是Asn51，Tyr53以及Thr54三个氨基酸；在抗体重链可变区CDR3(98 YG YS WAMDY 106)序列中，与抗原结合的主要是Tyr100和Trp102两个氨基酸。因此，改变其中部分氨基酸都可能有利于抗原抗体的结合能力。以上所述残基可以称为抗体重链与抗原相互作用残基。更改这些与抗原相互作用残基的任何1个或2个或3个，仍然保持与IFP35或NMI的抗原-抗体复合体结构中对应的抗原表位氨基酸结合，应在本申请保护范围内。
在抗体轻链可变区CDR1序列(26 SSSV SY 31)中，与抗原结合的主要氨基酸是Ser30和Tyr31；在抗体轻链可变区CDR2序列(49  DTS 51)中，与抗原结合的主要氨基酸是Asp49；在抗体轻链可变区CDR3序列(90 WS SNPPI 96)中，与抗原结合的主要氨基酸是Ser92和Asn93。因此，部分这些氨基酸的改变都可能有利于抗原抗体的结合能力。以上所述残基可以称为抗体轻链与抗原相互作用残基。更改这些与抗原相互作用残基的任何1个或2个或3个，仍然保持与IFP35或NMI的抗原-抗体复合体结构中对应的抗原表位氨基酸结合，应在本申请保护范围内。
上述的抗体、多肽或抗原结合片段中，所述将35NIDmAb或其人源化抗体中的至少一个CDR进行突变得到的抗体为如下至少一种：AE001-5、AE001-6、AE001-7、AE001-8、AE001-9。
AE001-5、AE001-6、AE001-7、AE001-8、AE001-9的重链可变区的CDR和轻链可变区的CDR如下表所示。
重链可变区CDR：
Figure PCTCN2020082296-appb-000001
Figure PCTCN2020082296-appb-000002
轻链可变区CDR：
Figure PCTCN2020082296-appb-000003
AE001-5、AE001-6、AE001-7、AE001-8、AE001-9的重链可变区和轻链可变区除CDR以外的序列，本领域技术人员可以根据自己的需要进行改造。具体的一个例子，AE001-5、AE001-6、AE001-7、AE001-8、AE001-9的重链可变区和轻链可变区除CDR以外的序列，还可以与35NIDmAb抗体的相应序列相同相同。
AE001-5、AE001-6、AE001-7、AE001-8、AE001-9的恒定区序列，本领域技术人员可以根据自己的需要进行改造。具体的一个例子，AE001-5、AE001-6、AE001-7、AE001-8、AE001-9的恒定区序列可与上述AE001-H1+L1、AE001-H2+L2或AE001-H3+L3的恒定区序列相同。
上述治疗方法和治疗应用中，所述慢性炎症疾病包括多发性硬化症、关节炎、类风湿关节炎、银屑病(牛皮癣)、各种肠炎(如IBD)、哮喘、慢阻肺和系统性红斑狼疮、慢性肝炎、慢性肾炎、慢性胰腺炎、脑炎、恶性肿瘤、白血病、阿尔兹海默症、帕金森综合征等。
上述治疗方法和治疗应用中，所述慢性炎症疾病都由分泌的异常含量IFP35/NMI炎症因子发挥作用，包括类风湿关节炎(Rheumatoid arthritis(RA))、骨性关节炎(OSTEOARTHRITIS(OA))、多发性硬化(multiple sclerosis，MS)、动脉粥样硬化(atherosclerosis)、心肌梗死(Myocardial Infarction)、慢阻肺(COPD)、慢性肾炎、慢性肝炎、慢性胰腺炎、II型糖尿病(type 2 diabetes)、系统性红斑狼疮(Systemic lupus erythematosus(SLE))、阿兹海默(Alzheimer's disease)、帕金森(Parkinson's disease(PD))恶性肿瘤、哮喘(asthma)、过敏性疾病(Allergic diseases)、心血管疾病(Cardiovascular diseases)、肌肉骨骼疾病(Musculoskeletal diseases)、炎性肠炎(IBD)、肥胖及糖尿病(Obesity和diabetes)、视网膜炎症疾病(AMD)、牙周炎(Periodontitis)、葡萄膜炎(Uveitis)等。
慢性炎症疾病的病理过程有多种炎症因子参与。作为可以被免疫细胞等分泌到细胞外基质(血液、体液等)、发挥促进炎症反应作用的炎症因子，IFP35和/或NMI也可以被细胞分泌到细胞外基质(血液、体液等)中，发挥激活炎症反应的作用。本申请中示例性研究的多发性硬化症是慢性炎症疾病的一个典型代表。多发性硬化症的发生与小胶质细胞密切相关。小胶质细胞可以发挥部分神经系统的免疫细胞(巨噬细胞)的作用，释放炎症因子，促进炎症反应的发生。这一现象相当于机体其它组织和器官发生炎症反应时由巨噬细胞等免疫细胞引发的炎症反应。本申请的发明人发现，LPS等诱导情况下小胶质细胞分泌IFP35/NMI到细 胞培养基质中，且患有多发性硬化症的动物血清中IFP35/NMI含量也有所上升。表明IFP35/NMI与多发性硬化症存在着关联。总体来说，巨噬细胞、小胶质细胞等免疫细胞释放炎症因子引发炎症反应，可能导致各类慢性炎症疾病，IFP35/NMI作为其中的炎症因子。本申请还提供了诊断慢性炎症疾病的方法和应用，为如下技术方案A、B或C或D：
技术方案A、检测IFP35和/或NMI的物质在制备诊断慢性炎症疾病的产品中的应用；
所述检测IFP35和/或NMI的物质为如下至少一种：
(1)检测生物体脊髓组织中IFP35/NMI的表达量是否显著升高；
(2)检测IFP35/NMI是否被分泌到生物体的血液中或体液中(如脑脊液中)；
(3)检测生物体的血液中或体液中(如脑脊液中)是否含有IFP35/NMI，和/或检测生物体的血液中或体液中(如脑脊液中)的IFP35/NMI的含量；
(4)检测被分泌到生物体的血液中或体液中(如脑脊液中)的IFP35/NMI的含量用于协助临床炎症疾病的医学诊断；
(5)检测分泌的IFP35/NMI与生物体中其它炎症因子(如TNF、IL1、IL6等)以及生物标志物降钙素原(PCT)、C反应蛋白(CRP)的表达提高和分泌到血液中含量增多之间的差异及相关性；
技术方案B、为疾病诊断应用的临床检测产品用于检测分泌到血清及体液中的IFP35和/或NMI；
所述检测分泌到血清及体液中的IFP35和/或NMI的临床应用产品包括如下至少一种：荧光发光临床检测试剂(盒)、化学发光临床检测试剂(盒)、Elisa检测试剂(盒)、PCR临床检测试剂(盒)等。
检测分泌的IFP35/NMI与生物体中其它炎症因子(如TNF、IL1、IL6等)以及生物标志物降钙素原(PCT)、C反应蛋白(CRP)的表达提高和分泌到血液中含量增多之间的差异及相关性；
技术方案C、为慢性炎症疾病诊断应用的临床检测产品用于检测分泌到血清及体液中的IFP35和/或NMI；
所述检测分泌到血清及体液中的IFP35和/或NMI的临床应用产品包括如下至少一种：荧光发光临床检测试剂(盒)、化学发光临床检测试剂(盒)、Elisa检测试剂(盒)、PCR临床检测试剂(盒)等。
检测分泌的IFP35/NMI与生物体中其它炎症因子(如TNF、IL1、IL6等)以及生物标志物降钙素原(PCT)、C反应蛋白(CRP)的表达提高和分泌到血液中含量增多之间的差异及相关性。
技术方案D、一种诊断待测生物体是否患有慢性炎症疾病的方法，包括如下中的至少一种的步骤：
(1)检测生物体组织中(如脊髓组织)中IFP35/NMI的表达量是否显著升高；
(2)检测IFP35/NMI是否被分泌到生物体的血液中或体液中(如脑脊液中)；
(3)检测生物体的血液中或体液中(如脑脊液中)是否含有IFP35/NMI，和/或检测生物体的血液中或体液中(如脑脊液中)的IFP35/NMI的含量；
(4)检测被分泌到生物体的血液中或体液中(如脑脊液中)的IFP35/NMI的含量用于协助临床炎症疾病的医学诊断；
(5)检测分泌的IFP35/NMI与生物体中其它炎症因子(如TNF、IL1、IL6等)以及生物标志物降钙素原(PCT)、C反应蛋白(CRP)的表达提高和分泌到血液中含量增多之间的差异及相关性；
上述诊断方法和应用中，可以根据如下至少一种的判断标准来诊断待测生物体是否患有慢性炎症疾病：
(1)若待测生物体组织中(如脊髓组织)中IFP35/NMI的表达量显著升高，则判断待测生物体患有慢性炎症疾病；
(2)若检测到IFP35/NMI被分泌到生物体的血液中或体液中(如脑脊液中)，则判断待测生物体患有慢性炎症疾病；
(3)若检测到生物体的血液中或体液中(如脑脊液中)中含有IFP35/NMI，则判断待测生物体患有慢性炎症疾病。
(4)IFP35/NMI表达提高与生物体中其它炎症因子(干扰素、TNF、IL1、IL6等)的表达和分泌到血液或体液中相一致，或具有临床表征性状，则判断待测生物体患有慢性炎症疾病。
上述诊断方法和应用中，所述检测分泌到血清及体液中的IFP35和/或NMI的临床应用产品包括如下至少一种：荧光发光临床检测试剂(盒)、化学发光临床检测试剂(盒)、Elisa检测试剂(盒)、PCR临床检测试剂(盒)等。
上述诊断方法和应用中，检测IFP35和/或NMI的含量和/或活性可以从DNA,RNA或蛋白水平进行检测。
上述诊断方法和应用中，DNA和/或RNA水平检测可以使用特异性核酸探针，也可以使用PCR引物或芯片等。
上述诊断方法和应用中，蛋白水平检测用抗体。
上述诊断方法和应用中，目前，临床上常用的炎症疾病检测指标包括降钙素原(PCT)、C反应蛋白(CRP)、TNF和IL6等。但是这些检测指标存在着局限性。比如PCT不能用来指示由于病毒造成的感染。另外，对于不同疾病，不同的炎症因子在不同病人上表现不同。因此，一方面需要开发其它合适的检测指标；另一方面应该对已知的炎症因子进行普遍检测。对于慢性炎症疾病，临床上可以检测IFP35和NMI并与其它指标比较。
上述诊断方法和应用中，可以通过检测小鼠脊髓组织中IFP35/NMI的表达量是否显著升高、或者检测IFP35/NMI是否被分泌到小鼠血液中或体液中(如脑脊液中)、或者检测IFP35/NMI与小鼠中其它炎症因子(如TNF、IL1、IL6等)的表达提高和分泌到血液中含量增多之间的相关性，来帮助诊断小鼠是否患有MS。若小鼠组织中IFP35/NMI的表达量异常升高、IFP35/NMI并被异常分泌到小鼠血液中或体液中(如脑脊液中)、或者IFP35/NMI表达提高与小鼠中其它炎症因子(干扰素、TNF、IL1、IL6等)的表达和分泌到血液中相一致，造成小鼠从步态改变、四肢是否有瘫痪等外在症状十分明显，基本可以诊断小鼠患有MS。
上述诊断方法和应用中，所述检测物质为A或B或C或D的抗体、多肽或抗原结合片段：
A、该抗体、多肽或抗原结合片段特异结合IFP35/NMI的抗原表位，所述抗原表位为如下(1)或(2)：所述抗原表位包含如下氨基酸位点：IFP35的Arg163，Asn164,Arg191,Gln194,Ile195,Gln197,Phe198,Thr199,Pro201,Gln206,Pro208,Arg210；(2)所述抗原表位包含如下氨基酸位点：NMI的Arg185,Asn186Lys215,Lys218，Lys219，Glu221,Tyr222,Pro223,Tyr225,Cys230，Arg232,Thr234。
B、任何能够结合上述(1)中抗原表位中的至少一个氨基酸残基并抑制IFP35的活性抗体或小分子或多肽物质；
C、任何能够结合上述(2)中抗原表位中的至少一个氨基酸残基并抑制NMI的活性抗体或小分子或多肽物质。
D、为如下35NIDmAb抗体：
所述抗体具有轻链可变区和重链可变区，重链可变区包括CDR1,CDR2和CDR3,CDR1的序列是SEQ ID NO：9的第25-32位氨基酸(GYTFTNYG),CDR2的序列是SEQ ID NO：9的第50-57位氨基酸(INTYTGEP),CDR3的序列是SEQ ID NO：9的第98-106位氨基酸(YGYSWAMDY)；轻链可变区包括CDR1,CDR2和CDR3,CDR1的序列是SEQ ID NO：10的第26-31位氨基酸(SSSVSY),CDR2的序列是SEQ ID NO：10的第49-51位氨基酸(DTS),CDR3的序列是SEQ ID NO：10的第90-96位氨基酸(WSSNPPI)。
上述诊断中的抗体、多肽或抗原结合片段，为将35NIDmAb除CDR外其余位置氨基酸 进行突变得到的抗体；或，所述抗体为将35NIDmAb进行人源化的抗体。
本领域技术人员可以根据本领域公知常识和惯用手段，将上述任一所述抗体，除了CDR序列之外的其它氨基酸进行改造突变，得到的抗体也属于本申请的保护范围。
所述人源化抗体为如下中至少一种：AE001-H1+L1、AE001-H2+L2或AE001-H3+L3。这三种抗体的均具有轻链可变区和重链可变区，重链可变区中含有CDR1,CDR2和CDR3,CDR1的序列是SEQ ID NO：9的第25-32位氨基酸(GYTFTNYG),CDR2的序列是SEQ ID NO：9的第50-57位氨基酸(INTYTGEP),CDR3的序列是SEQ ID NO：9的第98-106位氨基酸(YGYSWAMDY)；轻链可变区中含有CDR1,CDR2和CDR3,CDR1的序列是SEQ ID NO：10的第26-31位氨基酸(SSSVSY),CDR2的序列是SEQ ID NO：10的第49-51位氨基酸(DTS),CDR3的序列是SEQ ID NO：10的第90-96位氨基酸(WSSNPPI)。
AE001-H1+L1的重链恒定区序列为AE001H1(SEQ ID NO：1)，轻链恒定区序列为AE001L1(SEQ ID NO：3)。
AE001-H2+L2的重链恒定区序列为AE001H2(SEQ ID NO：5)，轻链恒定区序列为AE001L2(SEQ ID NO：7)。
AE001-H3+L3的重链恒定区序列为AE001H3(SEQ ID NO：11)，轻链恒定区序列为AE001L3(SEQ ID NO：12)。
上述诊断中的抗体、多肽或抗原结合片段，所述抗体为将35NIDmAb或其人源化抗体中的至少一个CDR中氨基酸残基进行突变得到的抗体。
上述诊断中的抗体、多肽或抗原结合片段，所述将35NIDmAb或其人源化抗体中的至少一个CDR进行突变为进行如下至少一种突变：
35NIDmAb的重链可变区CDR1的第25、26、27、28、29和/或32位氨基酸残基进行突变；即(25 GYTFT NYG 32)中除了NY其余氨基酸位置进行突变
35NIDmAb的重链可变区CDR2的第50、52、55、56和/或57位氨基酸残基进行突变；即(50 I NT YTGEP 57)中除了下划线氨基酸外其余位置的氨基酸进行突变
35NIDmAb的重链可变区CDR3的第98、99、101、103、104、105和/或106位氨基酸残基进行突变；即(98 YG YS WAMDY 106)中除了下划线氨基酸外其余位置的氨基酸进行突变
35NIDmAb的轻链可变区CDR1的第26、27、28和/或29位氨基酸残基进行突变；即(26 SSSV SY 31)中除了下划线氨基酸外其余位置的氨基酸进行突变
35NIDmAb的轻链可变区CDR3的第90、91、94、95和/或96位氨基酸残基进行突变；即(90 WS SNPPI 96)中除了下划线氨基酸外其余位置的氨基酸进行突变。
在35NIDmAb的抗体重链可变区CDR1序列(25 GYTFT NYG 32)中，与抗原结合的主要是Asn30和Tyr31两个氨基酸；在抗体重链可变区CDR2序列(50 I NT YTGEP 57)中，与抗原结合的主要是Asn51，Tyr53以及Thr54三个氨基酸；在抗体重链可变区CDR3(98 YG YS WAMDY 106)序列中，与抗原结合的主要是Tyr100和Trp102两个氨基酸。以上所述残基可以称为抗体重链与抗原相互作用残基。其它CDR残基不与或者很少与抗原IFP35结合，因此更易进行更改而获得不显著影响抗体活性的抗体。因此更改任何以上所述CDR中不与抗原相互作用的残基，仍然可能保持与IFP35或NMI的抗原-抗体复合体结构中对应的抗原表位氨基酸结合，应在本申请保护范围内。
在抗体轻链可变区CDR1序列(26 SSSV SY 31)中，与抗原结合的主要氨基酸是Ser30和Tyr31；在抗体轻链可变区CDR2序列(49  DTS 51)中，与抗原结合的主要氨基酸是Asp49；在抗体轻链可变区CDR3序列(90 WS SNPPI 96)中，与抗原结合的主要氨基酸是Ser92和Asn93。以上所述残基可以称为抗体重链与抗原相互作用残基。其它CDR残基不与或者很少与抗原IFP35结合，因此更易进行更改而获得不显著影响抗体活性的抗体。因此更改任何以上所述CDR中不与抗原相互作用的残基，仍然可能保持与IFP35或NMI的抗原-抗体复合体结构中对应的抗原表位氨基酸结合，应在本申请保护范围内。
上述诊断中的抗体、多肽或抗原结合片段，所述将35NIDmAb或其人源化抗体中的至少一个CDR进行突变为进行如下至少一种突变：
35NIDmAb的重链可变区CDR1的第30和/或31位氨基酸残基进行突变；
35NIDmAb的重链可变区CDR2的第51、53和/或54位氨基酸残基进行突变；
35NIDmAb的重链可变区CDR3的第100和/或102位氨基酸残基进行突变；
35NIDmAb的轻链可变区CDR1的第30和/或31位氨基酸残基进行突变；
35NIDmAb的轻链可变区CDR3的第92和/或93位氨基酸残基进行突变。
上述诊断中的抗体、多肽或抗原结合片段，所述抗体或抗原结合片段是能够识别如下两个或以上氨基酸残基的抗体或抗原结合片段：35NIDmAb或其人源化抗体中的重链的Asn30、Tyr31、Asn51、Tyr53、Thr54、Tyr100和Trp102位氨基酸残基，其参与与抗原IFP35及NMI的识别。
上述诊断中的抗体、多肽或抗原结合片段，述抗体或抗原结合片段是能够识别如下两个或以上氨基酸残基的抗体或抗原结合片段：35NIDmAb或其人源化抗体中的轻链的Ser30、Tyr31、Asp49、Ser92和Asn93位氨基酸残基，其参与与抗原IFP35及NMI的识别。
在抗体重链可变区CDR1序列(25 GYTFT NYG 32)中，与抗原结合的主要是Asn30和Tyr31两个氨基酸；在抗体重链可变区CDR2序列(50 I NT YTGEP 57)中，与抗原结合的主要是Asn51，Tyr53以及Thr54三个氨基酸；在抗体重链可变区CDR3(98 YG YS WAMDY 106)序列中，与抗原结合的主要是Tyr100和Trp102两个氨基酸。因此，改变其中部分氨基酸都可能有利于抗原抗体的结合能力。以上所述残基可以称为抗体重链与抗原相互作用残基。更改这些与抗原相互作用残基的任何1个或2个或3个，仍然保持与IFP35或NMI的抗原-抗体复合体结构中对应的抗原表位氨基酸结合，应在本申请保护范围内。
在抗体轻链可变区CDR1序列(26 SSSV SY 31)中，与抗原结合的主要氨基酸是Ser30和Tyr31；在抗体轻链可变区CDR2序列(49  DTS 51)中，与抗原结合的主要氨基酸是Asp49；在抗体轻链可变区CDR3序列(90 WS SNPPI 96)中，与抗原结合的主要氨基酸是Ser92和Asn93。因此，部分这些氨基酸的改变都可能有利于抗原抗体的结合能力。以上所述残基可以称为抗体轻链与抗原相互作用残基。更改这些与抗原相互作用残基的任何1个或2个或3个，仍然保持与IFP35或NMI的抗原-抗体复合体结构中对应的抗原表位氨基酸结合，应在本申请保护范围内。
上述诊断中的抗体、多肽或抗原结合片段，所述将35NIDmAb或其人源化抗体中的至少一个CDR进行突变得到的抗体为如下至少一种：
AE001-5、AE001-6、AE001-7、AE001-8、AE001-9的重链可变区的CDR和轻链可变区的CDR如下表所示。
重链可变区CDR：
Figure PCTCN2020082296-appb-000004
轻链可变区CDR：
Figure PCTCN2020082296-appb-000005
Figure PCTCN2020082296-appb-000006
AE001-5、AE001-6、AE001-7、AE001-8、AE001-9的重链可变区和轻链可变区除CDR以外的序列，本领域技术人员可以根据自己的需要进行改造。具体的一个例子，AE001-5、AE001-6、AE001-7、AE001-8、AE001-9的重链可变区和轻链可变区除CDR以外的序列，还可以与35NIDmAb抗体的相应序列相同相同。
AE001-5、AE001-6、AE001-7、AE001-8、AE001-9的恒定区序列，本领域技术人员可以根据自己的需要进行改造。具体的一个例子，AE001-5、AE001-6、AE001-7、AE001-8、AE001-9的恒定区序列可与上述AE001-H1+L1、AE001-H2+L2或AE001-H3+L3的恒定区序列相同。
上述诊断方法和应用中，所述慢性炎症疾病为与IFP35/NMI异常分泌到血液或体液中导致的炎症反应升高有关，包括关节炎、类风湿关节炎、银屑病(牛皮癣)、各种肠炎(如IBD)、多发性硬化症、哮喘、慢阻肺、系统性红斑狼疮、慢性肝炎、慢性肾炎、慢性胰腺炎、脑炎、恶性肿瘤、白血病、阿尔兹海默症、帕金森综合征、过敏性疾病、心血管疾病、肌肉骨骼疾病、炎性肠炎、肥胖及糖尿病、视网膜炎症疾病、牙周炎、葡萄膜炎等。
上述诊断方法和应用中，所述慢性炎症疾病为与IFP35/NMI异常分泌到血液或体液中导致的炎症反应升高有关，包括关节炎、类风湿关节炎、银屑病(牛皮癣)、各种肠炎(如IBD)、多发性硬化症、哮喘、慢阻肺、系统性红斑狼疮、慢性肝炎、慢性肾炎、慢性胰腺炎、脑炎、恶性肿瘤、白血病、阿尔兹海默症、帕金森综合征、过敏性疾病、炎性肠炎等。以上慢性炎症已被反复证明与多种炎症因子有关。
上述诊断方法和应用中，所述诊断为早期诊断、病情诊断和预后判断。
本申请还提供了IFP35的抗原表位。IFP35的抗原表位，为如下氨基酸残基中的至少一个：IFP35的Arg163，Asn164,Arg191,Gln194,Ile195,Gln197,Phe198,Thr199,Pro201,Gln206,Pro208,Arg210。
本申请还提供了NMI的抗原表位。NMI的抗原表位，为如下氨基酸残基中的至少一个：NMI的Arg185,Asn186Lys215,Lys218，Lys219，Glu221,Tyr222,Pro223,Tyr225,Cys230，Arg232,Thr234。
本申请还提供了基于上述抗原表位制备抗体的方法。
本申请还提供的制备IFP35或NMI的抗体的方法，为利用权利要求上述任一述抗原表位信息制备得到IFP35或NMI的抗体。
上述制备IFP35或NMI的抗体的方法，为，基于以下35NIDmAb抗体-IFP35/NMI抗原复合体结构信息及序列信息进行抗体序列改造，然后制备得到改造的IFP35或NMI的抗体：
所述35NIDmAb抗体-IFP35/NMI抗原复合体结构信息如下：
在抗体35NIDmAb重链可变区CDR1序列(25 GYTFT NYG 32)中，与抗原结合的主要是Asn30和Tyr31两个氨基酸；在抗体重链可变区CDR2序列(50 I NT YTGEP 57)中，与抗原结合的主要是Asn51，Tyr53以及Thr54三个氨基酸；在抗体重链可变区CDR3(98 YG YS WAMDY 106)序列中，与抗原结合的主要是Tyr100和Trp102两个氨基酸。
在抗体35NIDmAb轻链可变区CDR1序列(26 SSSV SY 31)中，与抗原结合的主要氨基酸是Ser30和Tyr31；在抗体轻链可变区CDR2序列(49 DTS 51)中，与抗原结合的主要氨 基酸是Asp49；在抗体轻链可变区CDR3序列(90 WS SNPPI 96)中，与抗原结合的主要氨基酸是Ser92和Asn93。
本领域技术人员公知，根据特定的抗原表位结构可以帮助设计、改造已有的原始抗体。原始抗体一般还是要通过各种筛选技术获得。知道了抗原表位(抗原决定簇)还可以直接用此片段筛选抗体。
以上所述抗体-抗原复合体结构信息及序列比对信息，(1)可以直观的看到位于抗原表面的氨基酸残基，由于抗原决定簇大多分布在抗原表面，所以这些表面残基可以帮助寻找抗原决定簇；(2)可以看到抗原与抗体相互作用的残基，从而可以分析抗原、抗体氨基酸残基之间相互作用特点，帮助针对特点的氨基酸残基进行改造、设计、及优化抗体。(3)可以指导改造抗体，从而获得针对其它同源蛋白抗原的新的抗体。
抗体基因改造方法涉及通用的基因定点突变技术及改造后的抗体基因或抗体基因片段(如抗体可变区)可以通过真核细胞(如哺乳动物细胞)或原核细胞进行表达、纯化，获得改造后的纯化的单克隆抗体。
由上述任一所述方法制备得到的抗体也属于本申请的保护范围。
本文所述的35NIDmAb抗体，具有轻链可变区和重链可变区，重链可变区中含有CDR1,CDR2和CDR3,CDR1的序列是SEQ ID NO：9的第25-32位氨基酸(GYTFTNYG),CDR2的序列是SEQ ID NO：9的第50-57位氨基酸(INTYTGEP),CDR3的序列是SEQ ID NO：9的第98-106位氨基酸(YGYSWAMDY)；轻链可变区中含有CDR1,CDR2和CDR3,CDR1的序列是SEQ ID NO：10的第26-31位氨基酸(SSSVSY),CDR2的序列是SEQ ID NO：10的第49-51位氨基酸(DTS),CDR3的序列是SEQ ID NO：10的第90-96位氨基酸(WSSNPPI)。
本文所述的35NIDmAb抗体，轻链可变区的序列是SEQ ID NO：10，重链可变区的序列是SEQ ID NO：9。
人源IFP35的氨基酸序列为SEQ ID NO：2。
鼠源IFP35的氨基酸序列为SEQ ID NO：4。
鼠源的NMI的氨基酸序列为SEQ ID NO：6。
人源的NMI的氨基酸序列为SEQ ID NO：8。
在此申请中，本申请发明人构建了一些炎症疾病的小鼠模型，并检测了这些炎症疾病动物血清中IFP35及NMI含量，以此来寻找与IFP35/NMI相关的炎症疾病。本申请发明人发现的IFP35和NMI与一些慢性炎症疾病，如多发性硬化症密切相关，其血清含量高与多发性硬化症发病密切相关，IFP35/NMI基因敲除或使用针对IFP35/NMI的中和性抗体可以减轻多发性硬化症的症状。因此，本申请发明人认为抑制IFP35或NMI活性可以用于治疗多发性硬化症等慢性疾病。检测血清或体液中(如脑脊液中)的IFP35和NMI含量可以用于慢性炎症疾病的诊断。
据此，本申请发明人开发了可以抑制IFP35的中和性抗体，为了更清楚的揭示抗体的抑制作用机制，本申请发明人进而解析了该中和性抗体与抗原IFP35的一个NID结构域的复合体三维晶体结构。通过这一复合体结构，本申请发明人可以揭示抗体的结构、抗原结构及抗体识别表位、抗原-抗体结合方式及关键相互作用残基等，并可以通过这一结构指导进行抗体的优化改造，以使其提高结合能力及增强识别的特异性，提高抗体的中和性活性。另外，由于IFP35与NMI序列上的高度同源性，还可以据此设计改造提高识别NMI蛋白能力的抗体，从而也开发出针对NMI的特异性抗体以及可以同时针对IFP35及NMI的双特异性抗体。
慢性炎症部分：本申请发明人检测了一些炎症疾病中是否存在IFP35或NMI表达提高的情况。本申请发明人发现，在多发性硬化(Multiple sclerosis,MS)疾病小鼠模型中，NMI在血清中含量显著提高。基因敲除IFP35及NMI后，多发性硬化(Multiple Sclerosis)疾病小鼠的症状显著减轻。使用针对IFP35的中和性抗体可以缓解MS症状，达到治疗或改善疾病的目的。
在本申请的另外一部分内容中，本申请的发明人在研究流感病毒以及宿主免疫机制中，发现了人体中一种新型的DAMP类炎症因子IFP35家族蛋白(包含IFP35和NMI)。这类因子在正常人和鼠血清中很低或低于可检测水平(检测线以下水平)；在病原感染情况下，能迅速从免疫细胞(巨噬细胞)中释放到血液中；能迅速促进免疫细胞分泌TNF、IL6等炎症因子，激活机体的炎症反应；在血清中的含量与炎症反应程度正相关；在脓毒症病患样品进行检测时发现，当血液中IFP35和NMI含量达到较高水平(例如IFP达到数百pg/ml)时，病患死亡率较高。
本申请的发明人以流感病毒为模型，研究了此种炎症因子与病毒感染之间的关系后发现，与野生型小鼠相比，IFP35和/或NMI家族蛋白基因敲除的小鼠体重减轻状况大大缓解，临床症状显著减轻，流感病毒感染导致的肺损伤显著减轻，存活率显著提高。使用IFP35和/或NMI的中和性抗体治疗可以达到与基因敲除相当的效果，大大减轻患病小鼠的症状，显著降低流感病毒感染导致的死亡率。
基于上述研究成果，在中国以及世界均在寻找对抗新型冠状病毒肺炎(COVID-19)的大形势下，本申请的发明人探索此种炎症因子能够作为COVID-19感染的血液检测标志物，用以帮助医护人员判断患者疾病严重程度及预后，并可以作为抑制过度炎症反应的靶标应用于病毒导致的过度炎症反应(或称脓毒症)的治疗，并由此建立了本申请的一项或多项发明。
研究结果表明：感染新型冠状病毒COVID-19的重症/危重症患者的血清中IFP35和NMI的水平显著提高，表明IFP35和NMI可以作为重症/危重症患者的诊断或辅助诊断指标，从而为帮助医护人员提供警示。此外，结合以IFP35和NMI作为靶标的流感病毒研究结果，针对IFP35和NMI的抑制剂药物(例如抗体药物)有望用于COVID-19导致的过度炎症反应疾病的治疗。无论是流感病毒或是新型冠状病毒COVID-19，在感染人体后都会引起IFP35及NMI分泌到血液及体液中。当敲除这两种炎症因子IFP35和NMI中的任一基因或使用中和抗体对其中一个基因进行抑制后，感染的机体炎症反应降低，死亡率下降，这些结果说明分泌的IFP35和NMI与病毒(以流感病毒和新型冠状病毒COVID-19为例)的感染严重程度相关，可以作为这些病毒导致的机体炎症反应的指标，并且可以成为抗体药物、多肽药物或化学药物等抑制剂的治疗靶标。
定义
除非另有定义，否则本文使用的所有技术和科学术语具有与本发明所属领域的普通技术人员通常理解的相同的含义。本文引用的所有专利、专利申请(公开的或未公开的)和其它出版物的通过引用以其整体并入本文。如果本部分中阐述的定义与通过引用并入本文的专利、申请、公开的申请和其它出版物中阐述的定义相反或不一致，则以本部分中阐述的定义为准。
本文中的病毒包括能够引发被感染的机体中炎症反应、特别是过度/重度炎症反应(炎症因子风暴)的病毒，包括但不限于冠状病毒科(例如冠状病毒，包括COVID-19、SARS、MERS病毒)、正粘病毒科(例如，流感病毒，例如甲型(A型)流感病毒)、负链RNA病毒目的其它病毒(如埃博拉病毒、拉沙病毒、马尔堡病毒、克里米亚-刚果出血热病毒)、逆转录病毒科(例如人免疫缺陷病毒，例如HIV-1(也称为HTLV-III、LAV或HTLV-III/LAV或HIV-III)；以及其他分离株，例如HIV-LP、小核糖核酸病毒科(例如脊髓灰质炎病毒、甲型肝炎病毒、肠道病毒、人柯萨奇病毒、鼻病毒、回声病毒)、Calciviridae(例如引起肠胃炎的菌株)、披膜病毒科(例如马脑炎病毒、风疹病毒)、黄病毒科病毒，如寨卡病毒、西尼罗病毒、登革热病毒、汉坦病毒等)、弹状病毒科(例如水泡性口炎病毒、狂犬病毒)、丝状病毒科(例如埃博拉病毒)、副粘病毒科(例如副流感病毒、腮腺炎病毒、麻疹病毒、呼吸道合胞病毒)、双链RNA病毒科(例如bunga病毒、静脉炎病毒和Nairo病毒)、肾炎病毒科(出血热病毒)、呼肠孤病毒科轮状病毒)、双抗病毒科肝炎病毒科(乙型肝炎病毒)、细小病毒科(细小病毒)、乳头状病毒科(乳头瘤病毒、多瘤病毒)、腺病毒科(大多数腺病毒)、疱疹病毒科(单纯疱疹病毒(HSV)1和2、水痘带状疱疹病毒、巨细胞病毒(CMV)、疱疹病毒)、痘病毒(天花病毒、牛痘病毒、痘病毒)和虹彩病毒(例如，非洲猪瘟病毒)、丙型肝炎病毒和未分类的病毒(例如，δ型肝炎(被认为是乙 型肝炎病毒的有缺陷的卫星)、诺瓦克和相关病毒，和星状病毒)。
本文中的“生物样品”是指感染病毒的个体中获取的含有细胞分泌物的样品，包括但不限于血液、血浆、血清、脑脊液、肺泡灌洗液、尿液、汗液、粪便等。
本文中的“个体”不限于特定物种或样品类型。例如，术语“个体”可以指患者，并且通常是人类患者。然而，该术语不限于人，因此包括多种非人动物或哺乳动物物种。“哺乳动物”是指任何哺乳动物种类的物种。通常，本文所用的术“哺乳动物”是指人、人类受试者或人类患者。“哺乳动物”还指任何非人哺乳动物种类的物种，例如实验、伴侣或经济性的非人哺乳动物。示例性非人哺乳动物包括小鼠、大鼠、兔、猫、狗、猪、牛、绵羊、山羊、马、猴、大猩猩和黑猩猩。
本文中的“治疗有效量”或“有效量”是指当单独或与其它治疗剂组合施用于细胞、组织或受试者时有效的治疗剂的量预防或改善受试者中与异常高水平的IFP35和/或NMI相关的疾病或不适。治疗有效剂量还指治疗剂的量足以导致症状的改善，例如治疗、治愈、预防或缓解相关医学不适，或提高治疗、治愈、预防或缓解这种病况的速度。当应用于单独施用的单个活性成分时，治疗有效剂量是指单个的该成分。当应用于组合时，治疗有效剂量是指导致治疗效果的活性成分的组合量，无论是组合、连续或同时施用。在一些实施方案中，“治疗特定疾病的化合物的有效量”是足以缓解或以某种方式减少与疾病相关的症状的量。这样的量可以作为单一剂量施用或可以根据方案施用，从而其是有效的。所述量可治愈疾病，但通常是为了改善疾病的症状而施用。可能需要重复给药以实现期望的症状缓解。
本文中的术语“药学上可接受的载体”指在包括与药物施用相容的任何和所有溶剂、分散介质、包衣、等渗剂和吸收延迟剂等。这些介质和试剂用于药物活性物质的用途是本领域熟知的。见，例如Remington，The Science and Practice of Pharmacy，第20版，(Lippincott，Williams &Wilkins 2003)。除非任何常规介质或试剂与活性化合物不相容，否则考虑在组合物中的这种用途。
本文所用的术语“抑制剂”指能对IFP35和/或NMI的生物学作用产生负性影响的任何分子，例如，可通过干扰IFP35和/或NMI与另一分子(例如，其底物)的相互作用，或降低IFP35和/或NMI的蛋白水平，例如通过降低编码IFP35和/或NMI的基因的表达。抑制剂可为“直接的抑制剂”，其与IFP35和/或NMI或编码IFP35和/或NMI的核酸相互作用，或者“间接的抑制剂”，其不与IFP35和/或NMI或编码IFP35和/或NMI的核酸相互作用，但与调控通路中IFP35和/或NMI的上游或下游相互作用。抑制剂可以为特异性抑制剂。本领域技术人员能够理解，虽然利用特异性抑制剂是比较理想的，但是根据具体情况，使用多功能或通用型蛋白抑制剂也是可选的。如上文所讨论的，抑制剂可以为表达抑制剂、功能抑制剂、或同时能抑制表达和功能的抑制剂。在一些实施方案中，抑制剂为能作用于IFP35和/或NMI的转录和/或翻译水平从而降低生成的功能性IFP35和/或NMI的量的抑制剂。在一些相关实施方案中，抑制剂包括，但不限于，dsRNA、microRNA、siRNA、shRNA、反义RNA或核酶。利用RNA干扰/沉默技术、反义核酸技术或核酶技术降低目标蛋白的表达水平是本领域技术人员所公知的，并且根据目标蛋白(例如，IFP35和/或NMI)的序列结构设计出合适的dsRNA、microRNA、siRNA、shRNA、反义RNA或核酶分子以及相应的制备和测试检验也是本领域技术人员容易实现的。在一些实施方案中，抑制剂为能抑制IFP35和/或NMI功能/活性的抑制剂。在一些相关实施方案中，抑制剂包括但不限于，针对IFP35和/或NMI的抗体或其抗原结合片段、小分子化合物。可用于本申请的抗体可包括多克隆抗体或单克隆抗体。此外，抗体可为来源于天然来源或来自重组来源的整个免疫球蛋白。抗体可以各种形式存在，包括例如作为整个抗体，或作为抗体片段，或它们的其他免疫活性片段如互补决定区。类似地，抗体可作为具有功能性抗原结合结构域，即重链和轻链可变域的抗体片段存在。同样，抗体片段可以选自但不限于如下的形式存在：Fv、Fab、F(ab)2、scFv(单链Fv)、dAb(单结构域抗体)、双特异性抗体、双链抗体和三链抗体。
另一方面，本申请提供了诊断或评估感染病毒的个体的炎症反应程度的方法，包括 测定获自所述个体的生物样品中干扰素诱导蛋白35kD(IFP35)和/或N-Myc相互作用蛋白(NMI)的量。
在一些实施方案中，所述方法用于诊断或辅助诊断重症或危重症患病个体。
在一些实施方案中，所述病毒为冠状病毒科的病毒，例如新型冠状病毒(COVID-19)、SARS病毒、MERS病毒；或正粘病毒科的病毒，例如流感病毒(甲型流感病毒)。
在一些实施方案中，测定生物样品中IFP35和/或NMI的量为测定生物样品中IFP35和/或NMI的蛋白含量，或测定生物样品中IFP35和/或NMI的核酸水平表达量，例如mRNA含量。
在一些实施方案中，个体为哺乳动物，例如人类。
在一些实施方案中，生物样品为血液、血浆、血清、脑脊液或肺泡灌洗液。
另一方面，本申请提供了用于诊断或评估感染病毒的个体的炎症反应程度的试剂盒，包括用于测定获自所述个体的生物样品中干扰素诱导蛋白35kD(IFP35)和/或N-Myc相互作用蛋白(NMI)的量的试剂。
在一些实施方案中，试剂盒用于诊断或辅助诊断重症或危重症患病个体。
在一些实施方案中，所述病毒为冠状病毒科的病毒，例如新型冠状病毒(COVID-19)、SARS病毒、MERS病毒；或正粘病毒科的病毒，例如流感病毒(甲型流感病毒)。
在一些实施方案中，测定生物样品中IFP35和/或NMI的量为测定生物样品中IFP35和/或NMI的蛋白含量，例如所述试剂包括特异性结合IFP35和/或NMI的抗体。
在一些实施方案中，测定生物样品中IFP35和/或NMI的量为测定生物样品中IFP35和/或NMI的核酸水平表达量，例如mRNA含量，例如所述试剂包括针对IFP35和/或NMI的cDNA序列设计的特异性扩增引物。
在一些实施方案中，个体为哺乳动物，例如人类。
在一些实施方案中，所述试剂包括用于处理所述生物样品以提取蛋白或核酸物质的试剂，例如，所述生物样品为血液、血浆、血清、脑脊液或肺泡灌洗液。
另一方面，本申请提供了治疗或缓解感染病毒的个体的炎症反应的方法，包括给予所述个体治疗有效量的干扰素诱导蛋白35kD(IFP35)和/或N-Myc相互作用蛋白(NMI)的抑制剂。
在一些实施方案中，所述个体为重症或危重症患病个体。
在一些实施方案中，所述病毒为冠状病毒科的病毒，例如新型冠状病毒(COVID-19)、SARS病毒、MERS病毒；或正粘病毒科的病毒，例如流感病毒(甲型流感病毒)。
在一些实施方案中，所述IFP35和/或NMI的抑制剂为IFP35和/或NMI的功能抑制剂或表达抑制剂。
在一些实施方案中，所述IFP35和/或NMI的功能抑制剂为IFP35和/或NMI的抗体或其抗原结合片段，或小分子化合物。
在一些实施方案中，所述IFP35和/或NMI的表达抑制剂为能作用于IFP35和/或NMI的转录和/或翻译水平从而降低生成的功能性IFP35和/或NMI的量的抑制剂，例如dsRNA、microRNA、siRNA、shRNA、反义RNA或核酶。
在一些实施方案中，所述个体为哺乳动物，例如人类。
另一方面，本申请提供了用于治疗或缓解感染病毒的个体的炎症反应的药物组合物，包括治疗有效量的干扰素诱导蛋白35kD(IFP35)和/或N-Myc相互作用蛋白(NMI)的抑制剂，以及药学上可接受的载体。
在一些实施方案中，所述个体为重症或危重症患病个体。
在一些实施方案中，所述病毒为冠状病毒科的病毒，例如新型冠状病毒(COVID-19)、SARS病毒、MERS病毒；或正粘病毒科的病毒，例如流感病毒(甲型流感病毒)。
在一些实施方案中，所述IFP35和/或NMI的抑制剂为IFP35和/或NMI的功能抑制剂或表达抑制剂。
在一些实施方案中，所述IFP35和/或NMI的功能抑制剂为IFP35和/或NMI的抗体或其抗原结合片段，或小分子化合物。
在一些实施方案中，所述IFP35和/或NMI的表达抑制剂为能作用于IFP35和/或NMI的转录和/或翻译水平从而降低生成的功能性IFP35和/或NMI的量的抑制剂，例如dsRNA、microRNA、siRNA、shRNA、反义RNA或核酶。
在一些实施方案中，所述个体为哺乳动物，例如人类。
应当理解，以上详细描述仅为了使本领域技术人员更清楚地了解本申请的内容，而并非意图在任何方面加以限制。本领域技术人员能够对所述实施方案进行各种改动和变化。
附图说明
图1.MOG和H37Ra诱导小鼠巨噬细胞NMI释放。检测MOG35-55和H37Ra体外刺激巨噬细胞(BMDM、Raw264.7和Thp1三种巨噬细胞细胞系)，调节细胞浓度每毫升10 6个,MOG50ng/ml和/或H37Ra 100ng/ml共孵育8h,检测细胞上清NMI分泌。此图说明MOG35-55和H37Ra联合可以诱导小鼠巨噬细胞分泌NMI，其中起主要作用的是H37Ra。
图2.LPS诱导小胶质细胞BV2的NMI释放。(a)100ng/ml LPS与BV2细胞共培养，检测0、2、4、6、8hNMI的分泌表达；(b)检测0、10、20、50、100ng/ml LPS刺激BV2细胞8h，检测上清NMI表达。此图说明中枢神经系统发生炎症，小胶质细胞活化时，也能分泌NMI。
图3.mNMI蛋白诱导小鼠小胶质细胞M1型极化。(a)小胶质细胞M1型标志分子TNF、iNOS、IL1β的mRNA在LPS和NMI刺激下表达增高；(b)小胶质细胞M2型标志分子Arg1、IL10、TGF-β、CD206的mRNA在LPS和NMI刺激变化不大；此图说明mNMI可诱导小胶质细胞向促炎M1型方向极化。
图4.EAE建模后小鼠不同时间NMI分泌表达。检测假处理小鼠、EAE小鼠在发病前期(7天)、初始发病期(12天)、发病高峰期(20天)、疾病缓解期(30天)NMI表达。此图说明NMI在小鼠MS的发病各个时期表达量与MS发病程度密切相关。
图5.EAE建模后小鼠脊髓组织NMI和其它炎性因子表达。检测EAE建模后小鼠不同时间内小鼠脊髓组织内NMI和主要炎性因子iNOS、COX2、HMGB1的表达。正常小鼠脊髓几乎检测不到iNOS，随着MS进入高峰期，iNOS表达量达到最高；COX2和HMGB1随MS发病升高；NMI正常表达量较低，在MS开始发病时表达量最高。此图说明NMI和其他炎性因子表达均能随着MS发生而升高，NMI是较为早期上调的因子之一。
图6.NMI敲除减轻小鼠MS临床症状。(a)NMI敲除减轻小鼠MS临床评分；(b)NMI敲除减轻脊髓炎症细胞浸润；(c)NMI敲除减轻小鼠脊髓脱髓鞘症状；NMI敲除减轻小鼠脊髓白质(d)和灰质(e)内星形胶质细胞和小胶质细胞活化状况。此图说明NMI可以促进神经炎症，加重MS临床症状，敲除NMI后小鼠MS症状减轻。
图7.IFP35抗体能减轻小鼠MS症状。(a)纯化的IFP35抗体，其中55kD位置为抗体重链，25kD位置为抗体轻链。(b)IFP35抗体静脉注射100μg(5mg/kg)10天(12-22天)缓解小鼠MS症状。
图8NMI敲除能减轻MS小鼠脊髓白细胞浸润和炎症。(a)NMI敲除减轻脊髓炎症CD45阳性白细胞浸润；(b)NMI敲除减轻脊髓炎症因子iNOS和COX2表达。
图9.LPS诱导的炎症小鼠NMI与PCT比较。以LPS(i.p.,10mg/kg)建立LPS诱导的炎症模型后NMI(a)和PCT(b)随时间变化；CLP建立小鼠炎症模型后16h，NMI(c)和PCT(d)含量。此图显示，相较PCT，NMI可以更早(1～2h)的检测到炎症发生，且NMI的本底表达较PCT更少，说明NMI可作为比PCT更好的临床危险指数检测指标
图10.流感病毒A/Puerto Rico/8/1934(H1N1)毒株感染小鼠后检测小鼠血清中存在分泌的NMI/IFP35。如图A及B所示，在病毒感染后的第3天血清中IFP35(又称IFI35)和NMI的蛋白水平显著升高，表明流感病毒感染可引起野生型小鼠的炎症反应。而在NMI或IFP35 基因敲除小鼠血清中IFP35(又称IFI35)和NMI的蛋白水平没有显著提升。同时发现在IFI35 -/-基因敲除小鼠血清中NMI的蛋白水平也显著低于WT小鼠；在NMI -/-基因敲除小鼠血清中IFI35的蛋白水平也显著低于WT小鼠(图A和图B)。表明IFI35与NMI二者之间的分泌存在着相互关联、相互作用。同时，本申请发明人发现流感病毒感染可使血清中IL-6和TNF-α的分泌水平增加(图C和图D)，而在NMI或IFP35基因敲除小鼠血清中的IL-6和TNF-α蛋白水平显著低于野生型小鼠。
图11显示了起始鼠源抗体AE001-VH+VL(35NIDmAb)和人源化改造的抗体AE001-H1+L1、AE001-H2+L2、AE001-H3+L3的抗原结合能力测试。
图12显示了各种改造后抗体的表达纯化结果。
图13显示了各种改造后的抗体的抗原结合能力测试。
图14抗原IFP35NID与中和性抗体35NIDmAb Fab复合体结构图。
图15显示了健康人及新型冠状病毒(COVID-19)感染的确诊患者血清中IFP35及NMI含量测定的点状图，其中每一个点指示一个健康人或者一个患者的血清样品。
图16显示了流感病毒在体内和体外导致NMI产生的验证。(A)感染流感病毒的病患血清中的NMI含量；(B)流感病毒刺激的THP1细胞的上清液中的NMI含量；(C)流感病毒刺激的A549细胞的上清液中的NMI含量；(D)流感病毒毒株PR8或Mock感染的C57BL/6野生型小鼠的血清NMI含量；(E)流感病毒刺激的RAW264.7细胞的上清液中的NMI含量；(F)流感病毒毒株PR8或Mock感染的A549细胞中NMI/IFP35的蛋白表达水平。(*表示P<0.05，**表示P<0.01，***表示P<0.001)
图17显示了流感病毒在体内和体外导致IFP35产生的验证。(A)流感病毒刺激的RAW264.7细胞的上清液中的IFP35含量；(B)感染流感病毒的病患血清中的IFP35含量；(C)PR8或Mock感染的C57BL/6野生型小鼠的血清IFP35含量。*表示P<0.05，**表示P<0.01，***表示P<0.001。
图18显示了16例流感患者和10例健康人的血清中的NMI和IFP35含量。重症监护室(ICU)中发展为重症肺炎的患者用实心方块标记。
图19显示了NMI基因敲除小鼠与野生型小鼠被流感病毒毒株PR8感染后的各项指标。(A)体重变化(％)；(B)临床评分，其中监测小鼠嗜睡、匍匐、皱褶皮毛、驼背、快速浅呼吸，可听到啰音，(健康)0≤分数≤5(垂死)；(C)肺组织的H&E染色，标尺为100μm；(D)存活率(％)。用2x 10 6pfu PR8病毒感染小鼠。用log-rank test分析不同处理小鼠存活率是否具有显著性差异。**表示P<0.01。
图20显示了IFP35基因敲除小鼠与野生型小鼠被流感病毒毒株PR8感染后的各项指标。(A)体重变化(％)；(B)肺组织的H&E染色，标尺为100μm；(C)存活率(％)。用2x10 6pfu PR8病毒感染小鼠。用log-rank test分析不同处理小鼠存活率是否具有显著性差异。*表示P<0.05。
图21显示了IFP35中和抗体的治疗效果。(A)实验方案；(B)体重变化(％)；(C)临床评分；(D)存活率(％)。用2x10 6pfu PR8病毒感染小鼠。用log-rank test分析不同处理小鼠存活率是否具有显著性差异。**表示P<0.01。
实施例
提供以下实施例仅仅是对本申请的一些实施方案进行举例说明，没有任何限制的目的或性质。
实施例1：利用抗体治疗小鼠MS
建立小鼠MS模型，然后用IFP35抗体进行治疗。结果如图7所示。经过IFP35抗体的治疗，小鼠的MS症状显著减轻。
实施例2：利用核酸药物治疗小鼠MS
敲除小鼠的NMI基因。再观察NMI敲除小鼠的MS症状。结果如图6和图8所示。敲除NMI后小鼠MS症状减轻。
实施例3：将IFP35/NMI作为血清或体液(脑脊液等)标志物检测MS小鼠
细胞实验表明，正常细胞与LPS、MOG和H37Ra诱导的巨噬细胞(类似于MS动物模型)相比，诱导后巨噬细胞中的IFP35/NMI表达量显著升高，并且被分泌到细胞外，从而可以在血清中检测到IFP35/NMI含量的提升。同时，可以发现其它一些炎症细胞因子的表达及在血清中含量也有了提高，比如小胶质细胞M1型标志分子TNF、iNOS、IL1βmRNA在LPS和NMI刺激下表达增高；iNOS、HMGB1、TNF、iNOS、IL1β等β。如图1、图2和图3所示。
MS的动物模型实验表明(参见实验方法)，正常小鼠与MS小鼠模型相比，MS小鼠模型中的IFP35/NMI表达量显著升高，并且被分泌到血清中，并且IFP35/NMI作为一种炎症因子与MS小鼠模型中的其它MS特征性炎症因子TNF、iNOS、IL1β的高表达和释放表现一致。表明IFP35/NMI可能作为MS在血清中或者体液中的特征性生物标志物及检测指标。如图4、图5所示。此结果与LPS诱导的动物模型及与流感病毒诱导的病毒动物模型结果一致，这些急性、慢性炎症疾病都导致了IFP35和NMI的分泌。如图9、图10所示。但这一点上PCT表现不同。PCT一般不会在病毒感染情况下血清含量增高。
因此，可以通过检测小鼠脊髓组织中IFP35/NMI的表达量是否显著升高、或者检测IFP35/NMI是否被分泌到小鼠血液中或体液中(如脑脊液中)以及分泌到血液或体液中含量增多之间的相关性，来帮助诊断小鼠是否可能患有MS及导致患病的炎症因子因素。若小鼠组织中IFP35/NMI的表达量异常升高、IFP35/NMI并被异常分泌到小鼠血液中或体液中(如脑脊液中)、或者IFP35/NMI表达提高与小鼠中其它炎症因子(干扰素、TNF、IL1、IL6等)的表达和分泌到血液中相一致，造成小鼠从步态改变、四肢是否有瘫痪等外在症状十分明显，基本可以诊断小鼠患有MS。
实验方法：
BMDM培养：
拉颈处死小鼠，取小鼠两只后腿，70％酒精浸泡1分钟，并尽量剔除后退骨上的肌肉，注射器吸入含双抗(青链霉素)的PBS，冲洗骨髓腔。离心400g,10分钟，去上清，红细胞裂解液1ml裂解红细胞，30秒，然后加入10mlPBS中和红细胞裂解液。离心400g,10分钟，添加10％FBS(Gibco)，1％青链霉素，1％L-谷氨酰胺的DMEM(Gibico)完全培养液再加入终浓度为20ng/ml MCSF(peprotech)，37℃,5％CO2培养箱培养
Raw264.7细胞培养：
10％FBS(Gibco)，1％青链霉素，1％L-谷氨酰胺的DMEM(Gibico)完全培养液，37℃,5％CO2培养箱培养
Thp1细胞培养：
10％FBS(Gibco)，1％青链霉素，1％L-谷氨酰胺的1640(Gibico)完全培养液，37℃,5％CO2培养箱培养
BV2细胞培养：
10％FBS(Gibco)，1％青链霉素，1％L-谷氨酰胺的DMEM(Gibico)完全培养液，37℃,5％CO2培养箱培养
MOG和H37Ra刺激细胞：
MOG和H37Ra加入到上述细胞中，终浓度为100ng/ml刺激8h。
mNMI刺激BV2细胞：
表达小鼠重组蛋白mNMI，体外刺激小鼠原代小胶质细胞，调节细胞浓度2×10 6,mNMI 5μg/ml 共孵育6h，QPCR检测巨噬细胞极化标志。
细胞Total RNA提取：
BMDM细胞Total RNA提取用康为世纪生物公司的RNA提取试剂盒Cat No.CW 0597 BV2细胞Total RNA提取用Trizol(invitrogen)
mRNA反转录成cDNA：
PrimeScript TM II 1st Strand cDNA Synthesis Kit Cat.No.6210A和
Figure PCTCN2020082296-appb-000007
Premix Ex Taq TM(Tli RNaseH Plus),ROX plus Q-PCR试剂盒Cat.NO.RR42LR均来自TAKARA公司
MS小鼠模型(EAE)建立：
实验所选小鼠为C57BL/6背景，8-12周的雌性小鼠，从Vital River Laboratory Animal Technology有限公司结合购买。使用少突胶质细胞蛋白MOG35-55和弗氏完全佐剂CFA乳化，每只小鼠皮下注射MOG35-55和CFA各0.2mg，联合百日咳毒素PTX，每只腹腔注射500ng，第48h重复注射PTX一次。假处理组给予PBS替代MOG35-55，其余等量CFA和PTX处理，每天观察记录小鼠发病情况和生活状态。小鼠发病后对小鼠发病进行评分，评分标准如下：0，无临床症状；1，小鼠尾巴瘫痪；2，小鼠一只后肢瘫痪或两后肢无力；3，小鼠两后肢瘫痪；4，小鼠两后肢瘫痪加前肢受累；5，小鼠濒死。根据评分标准对发病小鼠打分评价。
小鼠脊髓组织全蛋白提取：
取灌注去血的小鼠脊髓组织100mg,加入RIPA(Proteinase inhibitor cocktail,Roche)冰上裂解30分钟，12,000rpm,4℃离心10分钟，5X SDS-PAGE loading buffer，95℃,5分钟。
流感病毒感染小鼠后检测小鼠血清中存在分泌的NMI/IFP35
用300pfu剂量的A/PR8毒株感染C57BL/6WT、IFI35 -/-和NMI -/-作为攻毒实验组，同时以等量的PBS接种C57BL/6野生型小鼠作为阴性对照组。检测在病毒感染后的第3天小鼠血清中的IFP35和NMI的蛋白水平含量是否变化，并同时检测血清中TNF和IL6的含量。
IFP35中和性抗体制备：
小鼠腹腔注射IFP35单克隆杂交瘤细胞，7-10天后收集小鼠腹水，Protein G Agrose beads(GE)纯化腹水中抗体：
(1)1ml Protein G Agrose beads slurry,加入到蛋白纯化填充柱中
(2)10ml磷酸盐缓冲液洗涤纯化柱中的基质
(3)1ml加入到5ml磷酸盐缓冲液中，加入纯化柱中，4℃孵育2小时
(4)洗涤：20ml磷酸盐缓冲液洗柱
(5)洗脱：用适量的0.1M Glycine(PH＝2.5-3.0)Buffer洗脱抗体，事先在洗脱管中加入1M PH＝10.0的Tris-HCl Buffer中和洗脱液
超滤管浓缩抗体并测定抗体浓度。
IFP35抗体治疗MS小鼠：
建立小鼠EAE模型，分组IFP35抗体治疗组和IgG对照组，第12天开始给与抗体，每天每只小鼠静脉注射100μg(5mg/kg)抗体，连续给药10天，比较两组小鼠临床症状。
Elisa实验：
(1)取出酶标版，恢复室温，加入稀释好的标准品和样品，每孔100μl，室温2h。标准品和样品用1％BSA的PBST(0.05％Tween-20)稀释。
(2)洗涤：PBST(0.05％Tween-20)洗涤3次，去离子水洗2次
(3)加检测抗体：每孔100μl，室温2h。用1％BSA的PBST(0.05％Tween-20)稀释。
(4)加底物显色：每孔加入TMB 100μl，避光，脱色摇床上10-20分钟
(5)终止：每孔加入2Mol/L H 2SO4 50μl。
酶标板，450nm扫描OD值。
基因敲除小鼠的制备方法：使用C57BL/6小鼠。8-12周龄。使用的所有小鼠都被通过颈椎脱臼杀死。C57BL/6野生型小鼠(000664)购自Vital River实验动物技术有限公司。使用CRISPR-Cas9产生NMI和Ifp35敲除小鼠技术。从超排卵的输卵管收集受精卵，雌性C57BL/6小鼠与雄性C57BL/6小鼠交配。Cas9mRNA(150ng ml-1)与转录产生的sgRNA(100ng ml-1)混合并显微注射到细胞内。受体卵的细胞质在M2培养基中具有公认的原核(Sigma，M7167-100ML)。NMI的sgRNA序列是5'-AAAACAAAGAACTAGACGAGG-3'，而IFP35的sgRNA序列是5'-CAGCTCAAAAGGGAGCGCACAGG-3'。CRISPR技术产生的移码突变的NMI和Ifp35基因，从而导致NMI和IFP35不能产生。每种相应的sgRNA注射大约100-250个受精卵，随后转入代孕的ICR雌鼠的子宫，从而获得F1代小鼠。
结果：
1、MOG和H37Ra诱导小鼠巨噬细胞NMI释放.
图1方法：调节BMDM、RAW267.4和Thp1细胞浓度每毫升10 6个，用MOG 35-55(50ng/ml)和H37Ra(100ng/ml)诱导小鼠巨噬细胞，共孵育8h，然后检测细胞上清中NMI分泌量。图中的误差线表示三个重复实验±s.e.m；显著性差异通过不配对的t检验检测；*P<0.05，**P<0.01。
图1结果：MOG 35-55和H37Ra是用来诱导小鼠发生MS的主要试剂，MOG 35-55是少突胶质细胞表面糖蛋白 ，用来引起免疫细胞对自身抗原攻击，H37Ra是灭活的结核杆菌，用来激活扩大免疫反应。用MOG 35-55和H37Ra联合刺激小鼠外周巨噬细胞，可以检测到NMI被大量分泌出来，其中起主要作用的是H37Ra成分。H37Ra的诱导效应远大于MOG 35-55，说明H37Ra激活了巨噬细胞，是炎症发生的主要推动者，MOG 35-55对巨噬细胞的诱导激活效应不如H37Ra强，二者联合的刺激作用高于其中单独一种。表明诱导小鼠MS发病的试剂可以诱导NMI被巨噬细胞释放到细胞外。该实验可以证明诱导的巨噬细胞活化模型中，NMI表达量升高，且被释放到细胞外。
2、LPS诱导小胶质细胞BV2的NMI释放
图2方法：a,使用100ng/ml的LPS诱导小胶质细胞BV2，检测0、2、4、6、8小时时NMI被细胞分泌到上清液中的量；b,检测使用不同浓度的LPS(0、10、20、50、100ng/ml)刺激BV2细胞8小时，检测上清中NMI的含量。图中的误差线表示三个重复实验±s.e.m；显著性差异通过不配对的t检验检测；*P<0.05，**P<0.01，***P<0.001。
图2结果：小胶质细胞是中枢神经系统的主要免疫细胞，参与了中枢神经炎症性疾病，包括MS发生发展过程。LPS(脂多糖或内毒素)可以诱导小胶质细胞活化。实验结果表明LPS诱导的小胶质细胞活化后，有大量的NMI分泌，并且分泌量与LPS刺激的时间和强度呈正相关，说明NMI可能是MS发病中的一种DAMP发挥作用，中枢巨噬细胞活化有NMI释放。
3、mNMI蛋白诱导小鼠小胶质细胞M1型极化
图3方法：使用纯化的鼠源NMI(mNMI)蛋白(5μg/ml)诱导小鼠小胶质细胞6h，检测小胶质细胞M1型标志分子TNF、iNOS、IL1β(a)和M2型标志分子Arg1、IL10、TGF-β、CD206(b)mRNA在LPS或NMI刺激下变化；图中的误差线表示三个重复实验±s.e.m；显著性差异通过不配对的t检验检测；*P<0.05，**P<0.01，***P<0.001。
图3结果：小胶质细胞作为巨噬细胞的一种，极化方式分为M1型和M2型。其中M1型小胶质细胞主要参与促炎反应，介导吞噬、炎症因子分泌和组织损伤，M1型小胶质细胞在MS发病中起到促进疾病发展作用。M2型小胶质细胞介导抗炎反应，参与组织修复过程。图3实验结果说明mNMI可诱导小胶质细胞向促炎M1型方向极化，促进MS炎症反应。
4、EAE建模后小鼠不同时间NMI分泌表达
图4表明，EAE建模后小鼠不同时间NMI分泌表达检测假处理对照实验组小鼠、EAE小鼠在发病前期(7天)、初始发病期(12天)、发病高峰期(20天)、疾病缓解期(30天)NMI表达。结果表明，NMI在小鼠MS的发病各个时期表达量与MS发病密切相关.
5、EAE建模后小鼠脊髓组织NMI和其它炎性因子表达
图5表明，检测EAE建模后小鼠不同时间内小鼠脊髓组织内NMI和主要炎性因子iNOS、COX2、HMGB1的表达，可见正常小鼠脊髓几乎检测不到iNOS，随着MS进入高峰期，iNOS表达量达到最高；COX2和HMGB1随MS发病升高；NMI正常表达量较低，在MS开始发病时表达量最高。说明NMI和其他炎性因子表达均能随着MS发生而升高，NMI是较为早期上调的因子之一。由于小鼠的脑脊液较难取到，因此在此检测的是脊髓组织。
图4及图5结果总结：血液中的炎症因子升高是免疫炎症性疾病的特点之一，为了检测NMI是否可以作为MS的biomarker，以及作为biomarker的潜在功能，本申请发明人检测了MS小鼠模型的血液(a)和脊髓组织内部(b)NMI表达情况。结果表明，NMI在MS小鼠发病各个时期表达量与MS发病程度密切相关，随着MS发病到达高峰期(第20天)，NMI表达量达到最高。在脊髓组织中，正常小鼠脊髓几乎检测不到iNOS，随着MS进入高峰期，iNOS表达量达到最高；COX2和HMGB1随MS发病升高；NMI正常脊髓组织表达量较低，在MS开始发病时表达量最高，疾病缓解期下调，说明NMI和其他炎性因子表达均能随着MS发病而升高，NMI是较为早期上调的炎症因子之一。
这个实验可以证明，MS疾病的动物模型中，NMI表达量升高、且被释放到血液，但不能说NMI促进了其他炎症因子的释放，因为NMI和其它炎性因子iNOS、COX2、HMGB1是在一起检测的，没有先后和因果关系。
6、NMI敲除减轻小鼠MS临床症状
图6方法：a,构建NMI敲除小鼠(NMI -/-)和野生型小鼠(WT)MS动物模型，比较其临床症状。评分标准如下：0，无临床症状；1，小鼠尾巴瘫痪；2，小鼠一只后肢瘫痪或两后肢无力；3，小鼠两后肢瘫痪；4，小鼠两后肢瘫痪加前肢受累；5，小鼠濒死。根据评分标准对发病小鼠打分评价。b,NMI -/-和WT小鼠脊髓组织HE病理切片。c,NMI -/-和WT小鼠脊髓组织LFB病理切片；IF实验，以GFAP标记星形胶质细胞，以IBA1标记小胶质细胞，比较NMI -/-和WT小鼠脊髓组织白质(d)和灰质(e)内星形胶质细胞和小胶质细胞活化状况。
图6结果：使用NMI敲除小鼠来构建MS动物模型，并与野生型小鼠制备的MS模型进行比较其临床症状。图a显示，NMI -/-小鼠和WT小鼠虽然进入MS时间基本一致，但NMI -/-小鼠MS病症随后轻于野生型小鼠。b图通过脊髓组织切片HE染色观察NMI -/-小鼠和WT小鼠的脊髓炎症细胞浸润情况，可见NMI敲除减轻了MS小鼠脊髓炎症细胞浸润。c图通过脊髓组织切片LFB染色分析，NMI -/-小鼠脊髓脱髓鞘症状减轻。IF实验，NMI -/-小鼠脊髓白质(d)和灰质(e)内星形胶质细胞和小胶质细胞活化减少。综合以上结果说明，NMI可以促进神经炎症，加重MS临床症状，敲除NMI后小鼠MS症状减轻。
7、IFP35抗体能减轻小鼠MS症状
图7表明，由于基因敲除可以显著改善MS小鼠症状，因此，本申请发明人使用IFP35的中和性抗体来观察是否可以减轻MS小鼠症状。图a代表使用纯化的本申请所述的IFP35中和性抗体，进行SDS-PAGE检测，可见其中55kD位置为抗体重链，25kD位置为抗体轻链。表明抗体纯化良好。图b表明使用该纯化的IFP35抗体静脉注射100μg(5mg/kg)抗体10天(12-22天)观察MS小鼠的症状并评分，可以发现该抗体可以显著缓解小鼠MS症状。这一结果表明，抑制IFP35家族蛋白的单克隆抗体可以用于治疗多发性硬化症(MS)。小鼠MS建模采用公认的评价标准，评分标准如下：0，无临床症状；1，小鼠尾巴瘫痪；2，小鼠一只后肢瘫痪或两后肢无力；3，小鼠两后肢瘫痪；4，小鼠两后肢瘫痪加前肢受累；5，小鼠濒死。根据评分标准对发病小鼠打分评价。
此实验中对应的抗体为35NIDmAb,轻链可变区为SEQ ID NO:10，重链可变区为SEQ ID NO:9。
重链可变区包括CDR1,CDR2和CDR3,和CDR1的序列是SEQ ID NO：9的第25-32位氨基酸(GYTFTNYG),CDR2的序列是SEQ ID NO：9的第50-57位氨基酸(INTYTGEP),CDR3的序列是SEQ ID NO：9的第98-106位氨基酸(YGYSWAMDY)。
轻链可变区包括CDR1,CDR2和CDR3,和CDR1的序列是SEQ ID NO：10的第26-31位氨基酸(SSSVSY),CDR2的序列是SEQ ID NO：10的第49-51位氨基酸(DTS),CDR3的序列是SEQ ID NO：10的第90-96位氨基酸(WSSNPPI),and命名系统是Kabat。
本实验中发现NMI/IFP35在MS动物模型中以及诱导MS的试剂MOG等诱导的巨噬细胞实验中，发现IFP35/NMI被释放到血清中，因此，本项目就是研究被释放的IFP35/NMI与疾病的关系。由于IFP35/NMI已被本申请发明人之前证明一旦释放到血清中就会发挥DAMPs作用，因此，本研究实际上就是说被释放到血清中的IFP35/NMI在疾病中的作用，也就是DAMP的功能。
8、NMI敲除能减轻MS小鼠脊髓白细胞浸润和炎症
结果如图8所示。a.NMI敲除减轻脊髓炎症CD45阳性白细胞浸润；b.NMI敲除减轻脊髓炎症因子iNOS和COX2表达。
9、LPS诱导的炎症小鼠血清中NMI与PCT的比较
结果如图9所示。以LPS(i.p.,10mg/kg)建立LPS诱导的炎症模型后NMI(a)和PCT(b)随时间变化；同样的，肠结扎CLP建立小鼠炎症模型后16h，NMI(c)和PCT(d)含量。此图显示，相较PCT，NMI可以更早(1～2h)的检测到炎症发生，且NMI的本底表达较PCT更少，说明NMI可作为比PCT更好的临床危险指数检测指标。
10、流感病毒A/Puerto Rico/8/1934(PR8)毒株感染小鼠后检测小鼠血清中存在分泌的NMI/IFP35。
作为对照，本申请发明人同时比较了病毒感染情况下IFP35和NMI是否分泌。研究方法：用300pfu剂量的PR8毒株感染C57BL/6WT、IFI35 -/-和NMI -/-作为攻毒实验组，同时以等量的PBS接种C57BL/6野生型小鼠作为阴性对照组。如图10，图A及B所示，在病毒感染后的第3天血清中IFP35(又称IFI35)和NMI的蛋白水平显著升高，表明流感病毒感染可引起野生型小鼠的炎症反应。而在NMI或IFP35基因敲除小鼠血清中IFP35(又称IFI35)和NMI的蛋白水平没有显著提升。同时发现在IFI35 -/-基因敲除小鼠血清中NMI的蛋白水平也显著低于WT小鼠；在NMI -/-基因敲除小鼠血清中IFI35的蛋白水平也显著低于WT小鼠(图A和图B)。表明IFI35与NMI二者之间的分泌存在着相互关联、相互作用。同时，本申请发明人发现流感病毒感染可使血清中IL-6和TNF-α的分泌水平增加(图C和图D)，而在NMI或IFP35基因敲除小鼠血清中的IL-6和TNF-α蛋白水平显著低于野生型小鼠。进一步说明炎症反应及感染可以导致NMI和IFP35的分泌，且抑制(基因敲除)IFP35或NMI可以减轻炎症反应。这一结果与PCT不同，后者在病毒感染情况下血清中含量一般不会增加。以上结果提示：无论是病毒、细菌、或慢性炎症疾病都可导致IFP35和NMI分泌到体液(包括血液)中去。因此，可以成为慢性炎症疾病等的检测指标。
实施例4：抗体的改进
一、抗体人源化：
1、实验方法：通过PCR定点突变方法在鼠源抗体特定氨基酸残基位置的DNA序列上引入突变，从而实现改变鼠源抗体上一些氨基酸，使得变换为人源抗体序列，从而实现抗体人源化改造。然后将序列改造后的抗体基因克隆到相应的抗体表达载体上，如pCDNA3.1、pCDNA3.4等。转入人肾细胞系eExpi293F或HEK293T细胞中进行表达，然后纯化分泌到细胞外的表达的抗体。使用表达纯化的改造后抗体与抗原IFP35进行结合力检测。使用设备为 BiACo或ITC等。也可通过使用Elisa法检测人源化改造后的抗体与抗原结合能力。抗原是人源的IFP35蛋白。
改造前小鼠抗体(35NIDmAb)序列及改造后抗体序列如下：
AE001VL(改造前小鼠抗体(35NIDmAb)的轻链恒定区序列为SEQ ID NO：24；人源化改造后的小鼠轻链恒定区序列AE001L1为SEQ ID NO：3，AE001L2为SEQ ID NO：7，AE001L3为SEQ ID NO：12。
AE001VH(改造前小鼠抗体(35NIDmAb)的重链恒定区序列为SEQ ID NO：25；人源化改造后的小鼠重链恒定区序列AE001H1为SEQ ID NO：1，AE001H2为SEQ ID NO：5，AE001H3为SEQ ID NO：11。
实验结果如图11所示。AE001-VH+VL(35NIDmAb)为起始鼠源抗体。AE001-H1+L1以及AE001-H2+L2以及AE001-H3+L3分别为三组人源化改造的抗体，由此可见，人源化改造的抗体都可以结合抗原。由于AE001-H3+L3更接近于人源抗体，因此选择此组抗体进行如下抗体亲和力成熟度改造。结果表明鼠源抗体氨基酸序列提高了人源化程度，使得抗体成为具有更接近于人源化的抗体序列。本实验也说明，基于鼠源抗体与人源抗体的差异，对抗体进一步人源化改造是可以实现的。但是其抗体识别的主干序列，如抗体的可变区序列是识别抗原的主要区段。更关键的区域是抗体可变区中的CDR区域，与抗原识别更为关键。这些序列的核心区域决定抗原识别特异性。
二、抗体亲和力成熟改造
1、实验方法：实验采用通用的突变质粒构建方法以及蛋白质抗体表达方法。首先通过PCR随机突变方法在人源化改造后的一个抗体组合AE001-H3+L3的CDR区的DNA序列上随机引入突变，从而改变人源化后的抗体CDR序列。然后改造后的抗体序列克隆到相应的抗体表达载体上，如pCDNA3.1或pCDNA3.4等。构建随机突变抗体文库。将文库质粒转入人肾细胞系293T或Expi293F细胞中进行表达，然后纯化分泌到细胞外的表达的抗体。使用表达纯化的改造后的抗体与体外纯化的抗原IFP35或NMI或IFP35/NMI复合体进行结合力检测。使用设备为BiACo或ITC等。也可通过使用Elisa法检测CDR区改造后抗体与抗原的结合能力。
2、结果
1)Elisa法随机突变抗体亲和力检测初筛结果：
Figure PCTCN2020082296-appb-000008
由上结果选择结合能力较好的五组抗体(以上下划线标注的抗体)，分别编号为AE001-5，AE001-6，AE001-7，AE001-8，AE001-9。情况列表如下：
2)初筛的突变抗体重链和轻链配对情况：
Figure PCTCN2020082296-appb-000009
3)抗体表达纯化情况如图12所示。
4)改造后的抗体与抗原结合力检测：抗原是IFP35，结果如图13所示。
5)获得5组抗体及与抗原亲和力测定数据：
Loading Sample ID KD(M) kon(1/Ms) kdis(1/s) Full R^2
AE001-H3+L3 1.39E-08 1.45E+05 2.01E-03 0.9953
AE001-6HC+LC 3.35E-09 1.52E+05 5.08E-04 0.9982
AE001-6HC+8LC 2.99E-09 1.47E+05 4.40E-04 0.9985
AE001-6HC+9LC 3.04E-09 1.49E+05 4.54E-04 0.9986
AE001-6HC+7LC 3.46E-09 1.51E+05 5.24E-04 0.9980
AE001-5HC+LC 5.47E-09 1.69E+05 9.22E-04 0.9964
结论：获得的5个(编号为5-9)优选抗体都有不同程度的亲和力提高，经亲和力动力学测定(fortebio)，各个抗体有2.5-4.6倍的亲和力提升；其中亲和力提升主要为Koff得到了很大改善，且AE001-6HC+8LC(AE001-8号抗体)克隆亲和力改善最好。
重链序列改变：
Figure PCTCN2020082296-appb-000010
轻链序列变化：
Figure PCTCN2020082296-appb-000011
图示说明：cyan标记的残基为抗原结合的残基。
结论：在原始筛选出来的抗体基础上，可以通过改变CDR序列部分氨基酸残基，可以获得抗原-抗体结合能力优化的抗体，进而获得活性更好的中和性抗体。由以上列表可见，在原有抗体的基础上，通过改变部分CDR序列，发明人筛选得到了一些能够与抗原IFP35结合的抗体。结论是改变以上CDR部分氨基酸序列有助于获得活性更好的抗体。改变CDR部分序列是国际上普遍采用的获得更优化抗体的重要方法。因此，与本申请披露的抗体所述CDR序列同源性(在相同CDR位置、相同序列编号位置相同的氨基酸)达到50％及以上、并与IFP35相结合的抗体应在本申请保护范围之内。
实施例5：中和性抗体与抗原IFP35的复合体精细三维晶体结构揭示了抗体识别抗原的关键残基以及IFP35抗原表位氨基酸残基特征
以IFP35的NID结构域(124-220位氨基酸片段)为免疫原，免疫小鼠，并从小鼠脾脏中筛选制备得到针对IFP35的单克隆抗体，记作35NIDmAb。
该单抗35NIDmAb是具有中和活性的靶向IFP35 NID(片段124至220)的单克隆抗体。靶向IFP35 NID的35NIDmAb与IFP35 NID结合很强(Kd＝28-10 -9M)。体外纯化实验显示35NIDmAb与IFP35 NID混合后再经凝胶过滤层析纯化，两者依然结合在一起。这些结果表明靶向IFP35 NID的35NIDmAb可较强地结合IFP35 NID，实验表明该抗体并具有中和活性。前述实验中已经证明该单克隆抗体可以保护小鼠减轻多发性硬化症(MS)症状。
为阐明35NIDmAb对IFP35 NID具有中和活性的结构基础，在35NIDmAb与IFP35 NID可高亲和力结合的基础上，本申请发明人解析了分辨率为2.9埃的35NIDmAb Fab与IFP35 NID复合体的晶体结构以及IFP35 NID的结构。在35NIDmAb Fab-IFP35 NID结构中，IFP35 以单体的形式存在，而在IFP35 NID单独的结构中，IFP35 NID以二体的形成存在。此外，单个IFP35 NID在这两个结构中的结构类似。在35NIDmAb Fab-IFP35 NID结构中，一个不对称单位中有四个35NIDmAb Fab-IFP35 NID复合体。每个35NIDmAb Fab分别由重链(Ab_VH)和轻链(Ab_VL)组成，并与一个IFP35 NID相互作用。35NIDmAb Fab主要识别和结合IFP35 NID的c-端。在该相互作用界面中，主要涉及的IFP35 NID上的氨基酸残基有Arg163，Asn164,Arg191,Gln194,Ile195,Gln197,Phe198,Thr199,Pro201,Gln206,Pro208,Arg210。
另外，本申请发明人可以通过结构揭示出与抗原相互作用的关键抗体氨基酸。这些氨基酸包括：
在抗体重链可变区CDR1序列(25 GYTFTNYG 32)中，与抗原结合的主要是Asn30和Tyr31两个氨基酸；在抗体重链可变区CDR2序列(50 INTYTGEP 57)中，与抗原结合的主要是Asn51，Tyr53以及Thr54三个氨基酸；在抗体重链可变区CDR3(98 YGYSWAMDY 106)序列中，与抗原结合的主要是Tyr100和Trp102两个氨基酸。
在抗体轻链可变区CDR1序列(26 SSSVSY 31)中，与抗原结合的主要氨基酸是Ser30 和Tyr31；在抗体轻链可变区CDR2序列(49 DTS 51)中，与抗原结合的主要氨基酸是Asp49；在抗体轻链可变区CDR3序列(90 WSSNPPI 96)中，与抗原结合的主要氨基酸是Ser92和Asn93。
如图14，抗原IFP35 NID与中和性抗体35NIDmAb Fab复合体结构图。A图是抗体与抗原结构全图。抗体重链(Ab-VH)为浅蓝色，抗体轻链(Ab-VL)为绿色。抗原(IFP35 NID)为紫色。B图是从两个角度观察抗体自身的结构图，其中与抗原相互作用的残基以短棒图示(stick)表示出来。关键的残基在图上有标识。抗体重链(Ab-VH)为浅蓝色，抗体轻链(Ab-VL)为绿色。图C为抗体重链与抗原相互作用图示。关键残基以短棒状(stick)表示。图D为抗体轻链与抗原相互作用图示。关键残基以短棒状(stick)表示。
NMI NID的结构
除了IFP35 NID的结构和35NIDmAb Fab与IFP35 NID复合物的结构，本申请发明人还解析了NMI NID的结构。NMI NID的整体结构与IFP35 NID的结构非常相似。与IFP35 NID中参与35NIDmAb Fab的相互作用中所涉及的氨基酸相比，NMI NID上对应位置上的氨基酸与IFP35 NID有所不同(参见如下序列比对图)，对应的残基分别是Arg185,Asn186 Lys215,Lys218，Lys219，Glu221,Tyr222,Pro223,Tyr225,Cys230，Arg232,Thr234。这些氨基酸可能影响NMI NID与35NIDmAb的相互作用。但是这些NMI的氨基酸残基代表着NMI上的一个抗体结合表位，有助于筛选获得中和性抗体。
在IFP35上的抗体结合位点序列与NMI同源区段的序列比对：
IFP35_人156EIFFGKTRNGGGDVDVREL--LPGSVMLGFARDGVAQRLCQIGQFTVPLGGQQVPLRV 211
NMI__人178ELSFSKSRNGGGEVDRVDYDRQSGSAVITFVEIGVADKILKKKEYPLYINQTCHRVTV 235
实验方法：
(1)IFP35 NID和NMI NID的表达和纯化
将编码IFP35 NID(从124到220的残基)的基因片段克隆到pGEX-6p-1并在大肠杆菌中表达。将IFP35 NID的质粒转化到BL21(DE3)中。在LB培养基添加100毫克/L氨苄青霉素，并在37℃培养细菌。当OD600达到0.8--1.0时,用终浓度为0.5mM的isopropyl-β-D-thioglactosidase在16℃诱导20h。之后以4000rpm,15分钟的方式离心收集细菌。用裂解缓冲液(20Mm Tris at pH 8.0,400mM NaCl,5％Glycerol)重悬菌体，并用超声破碎仪破碎细菌,之后以16000rpm 30分钟高速离心。将离心后的上清液与GST亲和柱结合1h,随后清洗GST亲和柱。用洗脱缓冲液(20mM Tris at Ph 8.0,150mM NaCl,30mM Glutathione)洗脱重组蛋白，洗脱后的蛋白用PPase酶切，并进一步用离子交换柱(高盐缓冲液:20mM Tris at Ph 8.0,1M NaCl,5％Glycerol,；低盐缓冲液:20mM Tris at Ph 8.0,100mM NaCl,5％Glycerol)和凝胶过滤层析柱Superdex 200(缓冲液:20mM Tris at Ph 8.0,150mM NaCl)纯化。使用SDS-PAGE蛋白胶和考马斯亮蓝染色检验重组蛋白的纯度和完整性。
NMI NID(从155到240)基因片段的表达和纯化方法与IFP35 NID相同。
(2)35NIDmAb Fab的生产和纯化
用文献已报道的方法制作35NIDmAb杂化瘤细胞和生产35NIDmAb抗体。获得35NIDmAb后，在PBS中添加1mM EDTA和1mM半胱氨酸，用此缓冲液溶解木瓜蛋白酶。纯化后的35nidmab用木瓜蛋白酶裂解以100:1(35nidmab:木瓜蛋白酶)的质量比在37℃反应6-8小时并获得35NIDmAb Fab。
(3)结晶和结构解析
在结晶过程中，35NIDmAb Fab与IFP35 NID以1:1的比例混合，再用superdex 200进行凝胶过滤层析分离纯化。晶体筛选采用悬滴法，在16℃以0.2ul蛋白(10毫克/毫升)+0.2ul池液混合的方法点晶体。35NIDmAb Fab与IFP35 NID的结晶体在含有0.1M Tris(pH 8.5)、 0.2M MgCl2、18％PEG 3350的池液中生长。在结晶液中加入15％的DMSO对晶体进行低温保护，并在上海同步辐射装置的beamline BL17U和BL18U上衍射晶体。用HKL3000对衍射数据进行处理。
采用单波长异常色散法求解IFP35 NID和NMI NID的结构。使用SHELX C/D,Phenix.Autosol和Phenix.Autobuild获得两者的初始结构模型。使用coot和phenix对结构进行修正。以HIV中和单克隆抗体YZ23(PDB code 3CLF)和IFP35 NID的结构为模型并采用分子置换法求解35NIDmAb Fab-IFP35 NID复合体的结构。该结构的初始模型是用Phenix.Autobuild构建的。使用coot和phenix对结构进行修正。
以下是对于COVID-19和流感病毒感染的研究。
材料和方法
人体(患者或健康正常人)血清中IFP35和/或NMI检测方法
(1)样品采集：空腹状态抽取静脉血4mL置于无抗凝剂一次性真空采血管，3000r/min离心15min，分离血清后将血清样品管放入56℃水浴中保温至少半小时灭活病毒。之后样品管置于-20℃或-80℃冰箱中保存待测，避免反复冻融。
(2)样品检测：采用酶联免疫吸附法(ELISA)检测血清中IFP35和/或NMI的浓度，检测步骤如下：
2.1试剂、样品、标准品准备：样品及试剂缓慢溶解至室温(18-25℃)。将标准品依次倍比稀释为5000pg/mL，2500pg/mL，1250pg/mL，625pg/mL，312pg/mL，156pg/mL，78pg/mL。标准品稀释液(0pg/mL)直接作为空白孔。为保证检测结果有效性，每次检测对照品应现配现用。
2.2检测：分别设标准孔、待测样品孔、空白孔。酶标板加上覆膜，37℃温育2小时。弃去液体，甩干，加入待测工作液A，酶标板加上覆膜，37℃温育1小时。弃去孔内液体，甩干，洗板5次。加入待测工作液B,37℃温育1小时,弃去孔内液体，甩干，洗板5次。每孔加入底物溶液90uL,酶标板加上覆膜，37℃避光显色(反应时间控制在15-25分钟，当标准孔的前3-4孔有明显的梯度蓝色。后3-4孔梯度不明显时，即可终止)每孔加终止溶液50uL，终止反应，终止液的加入顺序应尽量与底物溶液加入顺序相同，如出现颜色不均一，请轻轻晃动酶标板使溶液尽量混匀。在确保酶标板底无水滴及孔内无气泡后，使用酶标仪测量各孔的光密度。
2.3结果处理：通过标准曲线计算血清中IFP35和/或NMI的浓度，采用SPSS统计软件处理数据，采用平均值±标准差表示，对样品组和对照组数据进行t检验，P＜0.05为差异有统计学意义。
细胞系和病毒株
HEK293T(ATCC，CRL-11268)、A549(ATCC，CRM-CRL-185)和MDCK(NBL-2)(ATCC CCL-34TM)细胞系用DMEM培养基来培养，并添加10％胎牛血清(Gibco)。将THP1(ATCC，TIB-202TM)和RAW264.7细胞(ATCCTIB-71TM)饲养在RPMI1640培养基中(Gibco，C1875500BT)。9日龄无特定病原体(SPF)鸡胚购自于广东大华农生物技术有限公司。A型流感病毒A/Puerto Rico/8/1934(H1N1)(PR8)毒株在SPF鸡胚中扩繁，经蔗糖密度梯度离心纯化后，用MOCK细胞测定病毒滴度。
抗体和试剂
IFP35单克隆抗体(H00003430-M01)购自Abnova公司。NMI抗体(ab183724)购于abcam。A型流感病毒NS1抗体(sc-130568)购于Santa Cruz。抗A型流感病毒核蛋白抗体购于abcam(ab128193)。E6446(二氢氯化物)(HY-12756A)(TLR7/9抑制剂)和CU-CPT-9b(HY-112051)(TLR8抑制剂)从Med ChemExpress(MCE，美国)购买。TLR3 dsRNA抑制剂(614310) 购于Millipore。Resatorvid(TAK-242)购于Selleck(美国)。
小鼠
C57BL/6(B6)野生型小鼠购于广东省医学实验动物中心(GDMLAC)。根据已发表的文献报道，使用C57BL/6小鼠模型构建NMI -/-或IFP35 -/-纯合子基因敲除小鼠。实验中使用的所有小鼠均为8至12周龄，性别匹配。小鼠饲养在无特定病原体环境，饲养条件符合中山大学动物福利与使用委员会标准。
质粒构建与转染
所有载体均经测序验证，使用ExFect2000转染试剂(Vazyme，T202-1)转染细胞进行瞬时表达。简述如下，在病毒感染前24h，用每个质粒瞬时转染293T细胞，然后将5MOI PR8病毒接种到6孔板的细胞中。细胞孵育1h后，用PBS冲洗一次后，更换为无FBS的正常DMEM培养基。然后收集上清液测定病毒滴度，并收集不同时间点样品经Western印记电泳分析。
实时-荧光定量PCR
使用TRIzol LS试剂(美国)从细胞或肺组织中提取总RNA。然后使用特异性引物U12A：AGCAAAAGAGG逆转录病毒基因组RNA(1μg)，使用PrimeScript II第一链cDNA合成试剂盒(Takara)配置逆转录体系。病毒mRNA首先用mRNA分离试剂盒(中国叶森生物技术有限公司)纯化，然后用oligo(d T)18作引物进行逆转录反应。以cDNA产物为模板，用SYBR Green Mix(Applied Biosystems)进行qPCR扩增。使用Quant Studio5(Applied Biosystems,Thermo Fisher Scientific)进行定量PCR反应。采用标准曲线的方法对数据进行分析。
酶联免疫吸附实验
鼠源mIFP35(E10460m)ELISA试剂盒购买于EIAab公司。人源IFP35 ELISA试剂盒(OKEH02088)购于AVIVA SYSTEM BIOLOGY。人源hNMI(CSB-EL015 893HU)、鼠源mNMI(CSB-EL015893MO)ELISA试剂盒均从CUSABIO购买。根据操作手册进行ELISA实验。简述如下：将100μl倍比稀释的标准品、检测样品、阴性及空白对照分别加入到相应的检测ELISA板中，每个样品设置2个重复。盖好覆膜后在37℃孵育2个小时。取下覆膜，弃掉孔中的液体，倒扣在纸巾上拍打以弃掉残余液体。向每孔中加入100μl生物素标记(1×)的检测抗体，盖好覆膜后在37℃孵育1个小时。取下覆膜，弃掉孔中的液体，倒扣在纸巾上拍打以弃掉残余液体。用1×洗涤缓冲液洗板3次。向每孔中加入100μl HRP标记的检测二抗后，盖好覆膜后在37℃孵育1个小时。取下覆膜，弃掉孔中的液体，倒扣在纸巾上拍打以弃掉残余液体。用1×洗涤缓冲液洗板5次。在每孔中加入90μl TMB底物，在37℃下避光孵育15-30分钟。在每孔中加入50μl的终止液，用ELISA读板机读取OD450吸光值。
肺脏组织病理学
所有小鼠肺组织固定在4％多聚甲醛中，石蜡包埋，并切成4μm薄片。然后用苏木精和伊红(H&E)对所有切片进行染色，并在显微镜下检查组织损伤、坏死和炎症细胞浸润情况，随机选取不同区域进行拍照。
患者血清样品
流感病毒研究中病人血液及正常志愿者对照样品来源于中山大学医学院、中山大学附属第六医院、中山大学附属第一医院、广州医科大学附属第一医院、广东省妇女儿童等。分离血清后，置于-80℃保存。血清样品的检测获得所有参与者的知情同意，并经相关医院人体研究伦理委员会批准(201701093)。
伦理描述
依照中华人民共和国国务院批准的《实验动物事务管理条例》开展动物实验。该动物实验已获得中山大学动物福利与使用委员会(IACUC)批准，许可号SYXK2016-0112。所有涉及流感病毒的操作均在生物安全三级实验室完成。
数据分析
定量数据以平均值±SD来显示，用未配对学生t检验判定组间差异是否具有统计学意义。用log-rank test分析不同处理小鼠存活率是否具有显著性差异。当P值小于0.05时认为其具有统计学意义，当P值小于0.001时认为其具有显著性差异#,P>0.05；*,P<0.05；**,P<0.01；***,P<0.001)。
实施例6：IFP35和/或NMI作为新型冠状病毒肺炎(COVID-19)的血清学指标的鉴定
本申请的发明人对新型冠状病毒肺炎定点医院收集的5例新型冠状病毒感染重症/危重症患者的血浆样本进行了检测，发现所有患者的IFP35和NMI水平均显著升高。相似的，进一步使用更多(数十例)病患血清样本的分析得出同样的结果。更值得注意的是，结合患者的临床表现以及预后来看，IFP35和NMI水平与之高度相关，即，IFP35和NMI水平越高，患者的临床表现越重，预后越差(本研究中IFP35和NMI水平最高的患者最终死亡)。图15显示了其中5位病患的血清IFP35和NMI结果，部分样本中的IFP35含量已经达到500-700pg/mL的水平，这已经达到级接近本申请的发明人之前发现的脓毒症病患致死的水平(约700pg/mL)，这表明IFP35和/或NMI与新型冠状病毒的感染的炎症反应相关，特别是在重症/危重症患者中大幅升高，因此可以作为COVID-19感染病患的血液检测标志物，用以帮助医护人员判断患者发生的炎症疾病的严重程度及预后。
本申请的发明人的其他研究已经证明(例如参见后文的流感病毒研究)，由于这种炎症因子在血清中的提高激活炎症反应，引发过度炎症反应等，抑制这些炎症因子可以降低炎症反应程度，降低病毒感染导致的患病程度及死亡。由此可以推断使用药物(抗体药物或化学药物)抑制血液中的IFP35和/或NMI可以抑制新型冠状病毒COVID-19感染导致的过度炎症反应的发生。
实施例7：IFP35和/或NMI作为流感病毒感染的血清学指标的鉴定
本申请的发明人的前期研究表明，LPS刺激能诱导巨噬细胞表达和释放NMI，因此在本研究中作为阳性对照，PBS作为阴性对照。
对于NMI而言，A型流感病毒感染患者的血清NMI高于健康供体(图16中A图)；流感病毒PR8刺激人单核细胞(THP1)、上皮细胞(A549)、RAW264.7细胞能导致细胞向培养液释放Nmi(图16中B、C、E图)；在PR8感染的C57BL/6小鼠中，血清NMI明显高于对照组(图16中D图)；细胞NMI和IFP35在PR8感染后逐渐增加，在A549细胞中表现出不同的丰度(图16中F图)。
对于IFP35观察到的结果与NMI相似，A型流感病毒感染患者的血清NMI高于健康供体(图17中B图)；流感病毒PR8刺激RAW264.7细胞能导致细胞向培养液释放Nmi(图17中A图)；在PR8感染的C57BL/6小鼠中，血清IFP35明显高于对照组(图17中C图)；
图18显示了16例流感患者和10例健康人的血清中的NMI和IFP35含量，其中流感患者的血清中的NMI和IFP35含量大大高于健康人，并且重症监护室(ICU)中发展为重症肺炎的患者(实心方块标记)的含量尤其高。
所有这些结果表明，流感病毒感染在体外和体内都能导致NMI/IFP35的产生。
实施例8：NMI和IFP35缺失对流感病毒感染的保护作用验证
在C57BL/6小鼠模型中建立了NMI -/-敲除小鼠。将一组C57BL/6野生型或NMI -/-敲除小 鼠分别用2×10 6pfu的PR8病毒进行试验，可杀死90％的感染小鼠。所有小鼠每天都要接受监测，以确定是否存活、体重减轻，以及是否有患病的临床症状(例如，嗜睡、毛发脱落、皱褶的皮毛、驼背的姿势、急促的浅呼吸、可听到的啰音)。每只小鼠每天的临床评分从0(无症状)到5(垂死)不等。如图19中的A和B图所示，NNMI -/-小鼠表现出轻微的临床症状，减轻了体重(％)。相比之下，野生型小鼠的临床评分要高得多，体重下降了近30％。H&E染色结果显示(图19中C图)：PR8感染野生型小鼠肺组织受损，炎性渗出液及充血充盈肺泡间隙及肺泡间隔。而NMI -/-小鼠的肺组织基本保持完整，无明显病变，与PBS感染对照小鼠相似。实验结束时，与野生型小鼠相比，NNMI -/-小鼠在很大程度上受到保护，不受致命的PR8感染(LD 90)(图19中D图)。
为了研究同源蛋白IFP35，采用CRISPR Cas9技术将C57BL/6小鼠中的IFP35基因敲除。分别用LD 90剂量的PR8病毒感染C57BL/6野生型或IFP35 -/-基因敲除小鼠进行试验。如图20所示，IFP35 -/-敲除小鼠没有明显的肺损伤(B图)；与野生型小鼠相比，存活率(％)略有增加(C图)；有趣的是，体重变化(％)与对照组无显著性差异(A图)。
以上证据表明，降低NMI和/或IFP35的体内水平对于流感病毒感染具有保护作用。
实施例9：外源抑制剂对流感病毒感染的保护作用验证
为了验证外源抑制IFP35能否实现相似的保护作用，使用IFP35中和抗体作为药物处理病毒感染前1天的12只C57BL/6野生型小鼠，同时，取等量的小鼠IgG作为阴性对照处理另一组C57BL/6野生型小鼠，每只小鼠连续5天静脉注射200μg抗体，然后所有小鼠在第0天接种LD 90的PR8病毒。对每只小鼠进行称重和监测，监测有皱褶的毛皮、嗜睡、匍匐、驼背、呼吸急促、可听到的啰音，每天对临床症状进行评分持续2周。实验方案的示意图参见图21中A图。
结果表明：IFP35中和抗体治疗的小鼠可显著保护其免受致命的PR8感染(LD 90剂量)，而给予mIgG小鼠的大多死亡(图21中D图)；临床评分与存活率结果一致(图21中C图)；然而，体重变化却没有显著性差异(图21中A图)，这与图20中A图中的结果一致，需要进一步的研究来确定其机制。总的来说，IFP35中和抗体减轻了临床症状，保护小鼠免受致死性流感病毒感染。
流感病毒，如造成大流行的H1N1、高致病性H5N1和新型重组H7N9可直接感染人，并引起严重急性肺损伤，导致急性呼吸窘迫综合征，并最终导致呼吸系统衰竭。这通常会引发继发性细菌感染，导致多种感染性肺炎、脑炎、心肌炎，并最终导致多器官衰竭。
本申请的流感病毒研究支持以下结论：流感病毒感染可以导致IFP35和/或NMI在感染个体的血液中含量升高，这一结果可以被作为检测流感病毒感染特征指标，用于临床检测。同时，使用抗体药物、化学药物等外源给药方法抑制IFP35和/或NMI可以用于流感病毒的治疗。
在不偏离本申请公开的实质和范围的情况下，可对本申请公开的各实施方案进行多种改变和用等同替换。除非上下文中另有说明，否则本公开的实施方案的任何特征、步骤或实施方案都可以与任何其他特征、步骤或实施方案组合使用。

Claims (49)

  1. 试剂在制备预防和/或治疗慢性炎症疾病的产品中的用途，所述试剂为如下中的至少一种：
    (1)抑制被分泌到细胞外作为炎症因子的异常含量的IFP35和/或NMI的活性的物质；
    (2)抑制被分泌到细胞外作为炎症因子的IFP35和/或NMI的含量和/或活性的物质；
    (3)抑制IFP35和/或NMI被分泌到细胞外作为炎症因子的物质；
    (4)抑制IFP35和/或NMI的含量和/或活性的异常增高的物质；
    (5)抑制IFP35和/或NMI被分泌到细胞外的物质；
    (6)抑制IFP35和/或NMI作为炎症因子引起慢性炎症疾病的物质或抑制IFP35和/或NMI作为DAMPs引起慢性炎症疾病的物质；
    (7)抑制IFP35和/或NMI上调一些炎症因子的表达和分泌的物质；所述一些炎症因子为干扰素、TNF、IL1和/或IL6；
    (8)抑制IFP35和/或NMI与TLR4作用通过TLR4/MD2激活NF-κB的物质。
  2. 一种预防和/或治疗慢性炎症疾病的方法，包括向生物体施加有效量的如下试剂的步骤，以达到预防和/或治疗慢性炎症疾病的目的；所述试剂为如下中的至少一种：
    (1)抑制被分泌到细胞外作为炎症因子的异常含量的IFP35和/或NMI的活性的物质；
    (2)抑制被分泌到细胞外作为炎症因子的IFP35和/或NMI的含量和/或活性的物质；
    (3)抑制IFP35和/或NMI被分泌到细胞外作为炎症因子的物质；
    (4)抑制IFP35和/或NMI的含量和/或活性的异常增高的物质；
    (5)抑制IFP35和/或NMI被分泌到细胞外的物质；
    (6)抑制IFP35和/或NMI作为炎症因子引起慢性炎症疾病的物质或抑制IFP35和/或NMI作为DAMPs引起慢性炎症疾病的物质；
    (7)抑制IFP35和/或NMI上调一些炎症因子的表达和分泌的物质；所述一些炎症因子为干扰素、TNF、IL1和/或IL6；
    (8)抑制IFP35和/或NMI与TLR4作用通过TLR4/MD2激活NF-κB的物质。
  3. 根据权利要求1所述的用途或权利要求2所述的方法，其特征在于，所述试剂为如下中至少一种：
    抗体或多肽或抗原结合片段，其特异结合IFP35和/或NMI，其具有权利要求1中(1)-(8)至少一种功能；
    小分子化合物，其具有权利要求1中(1)-(8)至少一种功能；
    核酸试剂，具有权利要求1中(1)-(8)至少一种功能。
  4. 根据权利要求1-3任一所述的用途或方法，其特征在于，所述抗体为权利要求12-20中任一项所述的抗体，
    或，所述抗体具有轻链可变区和重链可变区，重链可变区中含有的CDR1,CDR2和CDR3的序列分别为SEQ ID NO：13、14、15，轻链可变区中含有的CDR1,CDR2和CDR3的序列分别为SEQ ID NO：16、17、18。
  5. 根据权利要求1-4任一所述的用途或方法，其特征在于，所述慢性炎症疾病包括多发性硬化症、关节炎、类风湿关节炎、银屑病(牛皮癣)、各种肠炎(如IBD)、哮喘、慢阻肺和系统性红斑狼疮、慢性肝炎、慢性肾炎、慢性胰腺炎、脑炎、恶性肿瘤、白血病、阿尔兹海默症、帕金森综合征等。
  6. 根据权利要求5所述的用途或方法，其特征在于，所述慢性炎症疾病都由分泌的异常含量IFP35/NMI炎症因子发挥作用，包括类风湿关节炎(Rheumatoid arthritis(RA))、骨性关节炎(OSTEOARTHRITIS(OA))、多发性硬化(multiple sclerosis，MS)、动脉粥样硬化 (atherosclerosis)、心肌梗死(Myocardial Infarction)、慢阻肺(COPD)、慢性肾炎、慢性肝炎、慢性胰腺炎、II型糖尿病(type 2diabetes)、系统性红斑狼疮(Systemic lupus erythematosus(SLE))、阿兹海默(Alzheimer's disease)、帕金森(Parkinson's disease(PD))恶性肿瘤、哮喘(asthma)、过敏性疾病(Allergic diseases)、心血管疾病(Cardiovascular diseases)、肌肉骨骼疾病(Musculoskeletal diseases)、炎性肠炎(IBD)、肥胖及糖尿病(Obesity和diabetes)、视网膜炎症疾病(AMD)、牙周炎(Periodontitis)、葡萄膜炎(Uveitis)等。
  7. 检测IFP35和/或NMI的物质在制备诊断慢性炎症疾病的产品中的用途，例如，所述产品为荧光发光检测试剂或试剂盒、化学发光检测试剂或试剂盒、Elisa检测试剂或试剂盒、PCR检测试剂或试剂盒。
  8. 一种诊断生物体是否患有慢性炎症疾病的方法，包括如下中的至少一个步骤：
    (1)检测生物体组织中(如脊髓组织)中IFP35/NMI的表达量是否显著升高；
    (2)检测IFP35/NMI是否被分泌到生物体的血液中或体液中(如脑脊液中)；
    (3)检测生物体的血液中或体液中(如脑脊液中)是否含有IFP35和/或NMI，和/或检测生物体的血液中或体液中(如脑脊液中)的IFP35和/或NMI的含量；
    (4)检测被分泌到生物体的血液中或体液中(如脑脊液中)的IFP35和/或NMI的含量用于协助临床炎症疾病的医学诊断；
    (5)检测分泌的IFP35和/或NMI与生物体中其它炎症因子(如TNF、IL1、IL6等)以及生物标志物降钙素原(PCT)、C反应蛋白(CRP)的表达提高和分泌到血液中含量增多之间的差异及相关性；
  9. 根据权利要求7所述的用途或权利要求8所述的方法，其特征在于，检测IFP35和/或NMI的物质为权利要求12-20中任一项所述的抗体，
    或，所述抗体具有轻链可变区和重链可变区，重链可变区中含有的CDR1,CDR2和CDR3的序列分别为SEQ ID NO：13、14、15，轻链可变区中含有的CDR1,CDR2和CDR3的序列分别为SEQ ID NO：16、17、18。
  10. 根据权利要求7-9任一所述的用途或方法，其特征在于，所述慢性炎症疾病为与IFP35和/或NMI异常分泌到血液或体液中导致的炎症反应升高有关，包括关节炎、类风湿关节炎、银屑病(牛皮癣)、各种肠炎(如IBD)、多发性硬化症、哮喘、慢阻肺、系统性红斑狼疮、慢性肝炎、慢性肾炎、慢性胰腺炎、脑炎、恶性肿瘤、白血病、阿尔兹海默症、帕金森综合征、过敏性疾病、心血管疾病、肌肉骨骼疾病、炎性肠炎、肥胖及糖尿病、视网膜炎症疾病、牙周炎、葡萄膜炎等。
  11. 根据权利要求7-10任一所述的用途或方法，其特征在于，所述慢性炎症疾病为与IFP35和/或NMI异常分泌到血液或体液中导致的炎症反应升高有关，包括关节炎、类风湿关节炎、银屑病(牛皮癣)、各种肠炎(如IBD)、多发性硬化症、哮喘、慢阻肺、系统性红斑狼疮、慢性肝炎、慢性肾炎、慢性胰腺炎、脑炎、恶性肿瘤、白血病、阿尔兹海默症、帕金森综合征、过敏性疾病、炎性肠炎等。以上慢性炎症已被反复证明与多种炎症因子有关。
  12. 根据权利要求7-11任一所述的用途或方法，其特征在于，所述诊断为早期诊断、病情诊断和预后判断。
  13. 一种抗体、多肽或抗原结合片段，为如下A或B或C所定义：
    A、该抗体、多肽或抗原结合片段特异结合IFP35和/或NMI的抗原表位，所述抗原表位为如下(1)或(2)：所述抗原表位包含如下氨基酸位点：IFP35(SEQ ID NO：2)的Arg163，Asn164,Arg191,Gln194,Ile195,Gln197,Phe198,Thr199,Pro201,Gln206,Pro208,Arg210；(2)所述抗原表位包含如下氨基酸位点：NMI(SEQ ID NO：8)的Arg185,Asn186Lys215,Lys218，Lys219，Glu221,Tyr222,Pro223,Tyr225,Cys230，Arg232,Thr234；
    B、任何能够结合上述(1)中抗原表位中的至少一个氨基酸残基并抑制IFP35的活性抗体或小分子或多肽物质；
    C、任何能够结合上述(2)中抗原表位中的至少一个氨基酸残基并抑制NMI的活性抗体 或小分子或多肽物质。
  14. 根据权利要求13所述的抗体、多肽或抗原结合片段，其中所述抗体具有轻链可变区和重链可变区，重链可变区中含有的CDR1,CDR2和CDR3的序列分别为SEQ ID NO：13、14、15；轻链可变区中含有的CDR1,CDR2和CDR3的序列分别为SEQ ID NO：16、17、18。
  15. 根据权利要求14所述的抗体、多肽或抗原结合片段，其特征在于，所述抗体的重链可变区序列为SEQ ID NO：9，所述抗体的轻链可变区序列为SEQ ID NO：10。
  16. 根据权利要求14所述的抗体、多肽或抗原结合片段，其特征在于，
    所述抗体的重链恒定区序列为SEQ ID NO：25，所述抗体的轻链恒定区序列为SEQ ID NO：24；或
    所述抗体的重链恒定区序列为SEQ ID NO：1，所述抗体的轻链恒定区序列为SEQ ID NO：3；或
    所述抗体的重链恒定区序列为SEQ ID NO：5，所述抗体的轻链恒定区序列为SEQ ID NO：7；或
    所述抗体的重链恒定区序列为SEQ ID NO：11，所述抗体的轻链恒定区序列为SEQ ID NO：12。
  17. 根据权利要求13所述的抗体或抗原结合片段，其特征在于，所述抗体具有轻链可变区和重链可变区，重链可变区中含有的CDR1,CDR2和CDR3的序列分别为SEQ ID NO：13、14、15；轻链可变区中含有的CDR1,CDR2和CDR3的序列分别为SEQ ID NO：16、17、18，并且SEQ ID NO：13-18的至少一个CDR中氨基酸残基进行突变。
  18. 根据权利要求17所述的抗体或抗原结合片段，其特征在于，所述将SEQ ID NO：13-18的至少一个CDR进行突变为进行如下至少一种突变：
    SEQ ID NO：13，GYTFT NYG，中除了NY其余氨基酸位置进行突变；
    SEQ ID NO：14，I NT YTGEP，中除了下划线氨基酸外其余位置的氨基酸进行突变；
    SEQ ID NO：15，YG YS WAMDY，中除了下划线氨基酸外其余位置的氨基酸进行突变；
    SEQ ID NO：16，SSSV SY，中除了下划线氨基酸外其余位置的氨基酸进行突变；
    SEQ ID NO：17，WS SNPPI，中除了下划线氨基酸外其余位置的氨基酸进行突变。
  19. 根据权利要求13-18任一所述的抗体或抗原结合片段，其特征在于，所述抗体或抗原结合片段是能够识别如下两个或以上氨基酸残基的抗体或抗原结合片段：SEQ ID NO：9的Asn30、Tyr31、Asn51、Tyr53、Thr54、Tyr100和Trp102位氨基酸残基，其参与与抗原IFP35及NMI的识别。
  20. 根据权利要求13-19任一所述的抗体或抗原结合片段，其特征在于，述抗体或抗原结合片段是能够识别如下两个或以上氨基酸残基的抗体或抗原结合片段：SEQ ID NO：10的Ser30、Tyr31、Asp49、Ser92和Asn93位氨基酸残基，其参与与抗原IFP35及NMI的识别。
  21. 根据权利要求18所述的抗体或抗原结合片段，其特征在于，
    所述抗体的重链可变区中的CDR1,CDR2和CDR3的序列分别为SEQ ID NO：19、14、15，轻链可变区的CDR1,CDR2和CDR3的序列分别为SEQ ID NO：16、17、18；或
    重链可变区中含有的CDR1,CDR2和CDR3的序列分别为SEQ ID NO：13、14、15；轻链可变区中含有的CDR1,CDR2和CDR3的序列分别为SEQ ID NO：16、17、20；或
    重链可变区中含有的CDR1,CDR2和CDR3的序列分别为SEQ ID NO：13、14、15；轻链可变区中含有的CDR1,CDR2和CDR3的序列分别为SEQ ID NO：16、17、21；或
    重链可变区中含有的CDR1,CDR2和CDR3的序列分别为SEQ ID NO：13、14、15；轻链可变区中含有的CDR1,CDR2和CDR3的序列分别为SEQ ID NO：16、17、22；或
    重链可变区中含有的CDR1,CDR2和CDR3的序列分别为SEQ ID NO：13、14、15；轻链可变区中含有的CDR1,CDR2和CDR3的序列分别为SEQ ID NO：16、17、23。
  22. 编码权利要求14-21任一所述抗体的核酸分子。
  23. 诊断或评估感染病毒的个体的炎症反应程度的方法，包括测定获自所述个体的 生物样品中干扰素诱导蛋白35kD(IFP35)和/或N-Myc相互作用蛋白(NMI)的量。
  24. 如权利要求23所述的方法，其中所述方法用于诊断或辅助诊断重症或危重症患病个体。
  25. 如权利要求23或24所述的方法，其中所述病毒为冠状病毒科的病毒，例如新型冠状病毒(COVID-19)、SARS病毒、MERS病毒；或正粘病毒科的病毒，例如流感病毒(甲型流感病毒)。
  26. 如权利要求23-25中任一项所述的方法，其中测定生物样品中IFP35和/或NMI的量为测定生物样品中IFP35和/或NMI的蛋白含量，或测定生物样品中IFP35和/或NMI的核酸水平表达量，例如mRNA含量。
  27. 如权利要求23-26中任一项所述的方法，其中所述个体为哺乳动物，例如人类。
  28. 如权利要求23-27中任一项所述的方法，其中所述生物样品为血液、血浆、血清、脑脊液或肺泡灌洗液。
  29. 用于诊断或评估感染病毒的个体的炎症反应程度的试剂盒，包括用于测定获自所述个体的生物样品中干扰素诱导蛋白35kD(IFP35)和/或N-Myc相互作用蛋白(NMI)的量的试剂。
  30. 如权利要求29所述的试剂盒，其用于诊断或辅助诊断重症或危重症患病个体。
  31. 如权利要求29或30所述的试剂盒，其中所述病毒为冠状病毒科的病毒，例如新型冠状病毒(COVID-19)、SARS病毒、MERS病毒；或正粘病毒科的病毒，例如流感病毒(甲型流感病毒)。
  32. 如权利要求29-31中任一项所述的试剂盒，其中测定生物样品中IFP35和/或NMI的量为测定生物样品中IFP35和/或NMI的蛋白含量，例如所述试剂包括特异性结合IFP35和/或NMI的抗体。
  33. 如权利要求29-31中任一项所述的试剂盒，其中测定生物样品中IFP35和/或NMI的量为测定生物样品中IFP35和/或NMI的核酸水平表达量，例如mRNA含量，例如所述试剂包括针对IFP35和/或NMI的cDNA序列设计的特异性扩增引物。
  34. 如权利要求29-33中任一项所述的试剂盒，其中所述个体为哺乳动物，例如人类。
  35. 如权利要求29-34中任一项所述的试剂盒，其中所述试剂包括用于处理所述生物样品以提取蛋白或核酸物质的试剂，例如，所述生物样品为血液、血浆、血清、脑脊液或肺泡灌洗液。
  36. 治疗或缓解感染病毒的个体的炎症反应的方法，包括给予所述个体治疗有效量的干扰素诱导蛋白35kD(IFP35)和/或N-Myc相互作用蛋白(NMI)的抑制剂。
  37. 如权利要求36所述的方法，其中所述个体为重症或危重症患病个体。
  38. 如权利要求36或37所述的方法，其中所述病毒为冠状病毒科的病毒，例如新型冠状病毒(COVID-19)、SARS病毒、MERS病毒；或正粘病毒科的病毒，例如流感病毒(甲型流感病毒)。
  39. 如权利要求36-38中任一项所述的方法，其中所述IFP35和/或NMI的抑制剂为IFP35和/或NMI的功能抑制剂或表达抑制剂。
  40. 如权利要求39所述的方法，其中所述IFP35和/或NMI的功能抑制剂为IFP35和/或NMI的抗体或其抗原结合片段，或小分子化合物。
  41. 如权利要求39所述的方法，其中所述IFP35和/或NMI的表达抑制剂为能作用于IFP35和/或NMI的转录和/或翻译水平从而降低生成的功能性IFP35和/或NMI的量的抑制剂，例如dsRNA、microRNA、siRNA、shRNA、反义RNA或核酶。
  42. 如权利要求36-41中任一项所述的方法，其中所述个体为哺乳动物，例如人类。
  43. 用于治疗或缓解感染病毒的个体的炎症反应的药物组合物，包括治疗有效量的干扰素诱导蛋白35kD(IFP35)和/或N-Myc相互作用蛋白(NMI)的抑制剂，以及药学上可接受的载体。
  44. 如权利要求43所述的药物组合物，其中所述个体为重症或危重症患病个体。
  45. 如权利要求43或44所述的药物组合物，其中所述病毒为冠状病毒科的病毒，例如新型冠状病毒(COVID-19)、SARS病毒、MERS病毒；或正粘病毒科的病毒，例如流感病毒(甲型流感病毒)。
  46. 如权利要求43-45中任一项所述的药物组合物，其中所述IFP35和/或NMI的抑制剂为IFP35和/或NMI的功能抑制剂或表达抑制剂。
  47. 如权利要求46所述的药物组合物，其中所述IFP35和/或NMI的功能抑制剂为IFP35和/或NMI的抗体或其抗原结合片段，或小分子化合物。
  48. 如权利要求46所述的药物组合物，其中所述IFP35和/或NMI的表达抑制剂为能作用于IFP35和/或NMI的转录和/或翻译水平从而降低生成的功能性IFP35和/或NMI的量的抑制剂，例如dsRNA、microRNA、siRNA、shRNA、反义RNA或核酶。
  49. 如权利要43-48中任一项所述的药物组合物，其中所述个体为哺乳动物，例如人类。
PCT/CN2020/082296 2019-04-01 2020-03-31 慢性炎症和病毒感染的诊断和治疗 WO2020200186A1 (zh)

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US11466061B2 (en) 2014-08-22 2022-10-11 Yingfang Liu Methods and compositions for treating and/or preventing a disease or disorder associated with abnormal level and/or activity of the IFP35 family of proteins
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CN116064763A (zh) * 2021-07-09 2023-05-05 广州恩迈生物科技有限公司 以ifp35和nmi为靶点的疾病诊断和治疗
WO2023008503A1 (ja) * 2021-07-28 2023-02-02 慶應義塾 重症化予測装置、重症化予測方法、及びプログラム
CN114432285A (zh) * 2022-03-06 2022-05-06 江苏省人民医院（南京医科大学第一附属医院） Resatorvid上调EFTUD2表达和抑制HBV药物中的用途

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