WO2018072751A1 - 高稳定性和高亲和力的dmic及其制法 - Google Patents

高稳定性和高亲和力的dmic及其制法 Download PDF

Info

Publication number
WO2018072751A1
WO2018072751A1 PCT/CN2017/107145 CN2017107145W WO2018072751A1 WO 2018072751 A1 WO2018072751 A1 WO 2018072751A1 CN 2017107145 W CN2017107145 W CN 2017107145W WO 2018072751 A1 WO2018072751 A1 WO 2018072751A1
Authority
WO
WIPO (PCT)
Prior art keywords
mica
seq
amino acid
dmic
acid residue
Prior art date
Application number
PCT/CN2017/107145
Other languages
English (en)
French (fr)
Inventor
李懿
蔡文旋
Original Assignee
中国科学院广州生物医药与健康研究院
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 中国科学院广州生物医药与健康研究院 filed Critical 中国科学院广州生物医药与健康研究院
Priority to CN201780065101.9A priority Critical patent/CN109890837B/zh
Publication of WO2018072751A1 publication Critical patent/WO2018072751A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70539—MHC-molecules, e.g. HLA-molecules
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/1774—Immunoglobulin superfamily (e.g. CD2, CD4, CD8, ICAM molecules, B7 molecules, Fc-receptors, MHC-molecules)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/70539—MHC-molecules, e.g. HLA-molecules
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to the field of biotechnology, and more particularly to a hydrophobic, highly stable, high affinity soluble DMIC of a hydrophobic core region.
  • the invention also relates to a method of preparation and use of the DMIC.
  • NKG2D Natural Killer Group 2D
  • MICA MHC class I-related chain A
  • MICA MHC class I-related chain A
  • melanoma glioma
  • colon cancer kidney cancer, lung cancer, ovarian cancer, breast cancer, prostate.
  • cancer cells such as cancer, but not expressed or underexpressed in normal cells and mature tissues. Studies have shown that the interaction of NKG2D with MICA plays an important role in tumor immune surveillance.
  • NKG2D with MICA can directly activate NK cells to kill tumor cells, and at the same time, act as co-stimulatory molecules of TCR, activate CD8+ ⁇ T cells and ⁇ + T cells.
  • Glioblastoma cells overexpressing MICA can enhance the activity of NK cells and T cells in vitro, and the growth of gliomas overexpressing MICA in nude mice and VMDK mice is also significantly delayed.
  • NKG2D-mediated cytotoxicity of NK cells is closely related to the density of MICA expressed on the surface of the tumor.
  • MICA on the surface of the tumor plays a role in tumor immune surveillance and anti-tumor, while free, soluble MICA (sMICA) damages the body's immune response and promotes tumor immune escape.
  • sMICA free, soluble MICA
  • Erp5 endoplasmic reticulum protease 5
  • MPs metalloproteinases
  • MICA is involved in both the immune surveillance of tumors and the immune escape of tumors, the dual identity makes MICA a potential target for immunotherapy.
  • In-depth study of MICA can better exert its anti-tumor effect.
  • Kellner et al. made MICA and anti-tumor surface antigen antibodies into fusion proteins, which can activate NK cells to kill tumor cells.
  • a truncated MICA i.e., DMIC molecule
  • Another object of the present invention is to provide a process for the preparation and use of the high stability and high affinity DMIC molecules.
  • a truncated MICA comprising an alpha 1 alpha 2 domain of a mutated MICA, said "mutation" comprising one or more of the amino acid sequences of the alpha 1 alpha 2 domain of native MICA
  • the hydrophobic amino acid residues exposed to the surface are mutated to hydrophilic amino acid residues.
  • amino acid sequence of the ⁇ 1 ⁇ 2 domain of the native MICA is set forth in SEQ ID NO.
  • the "surface-exposed hydrophobic amino acid residue” is selected from the group consisting of 13L, 15W, 24L, 51A, 54V, 88L, 94I, 96V, 130M, 141M, and 179L, wherein the amino acid
  • the residue numbering is given by the number shown in SEQ ID NO.
  • the truncated MICA has a higher stability than the native MICA, and preferably the truncated MICA has a Tm value greater than or equal to 36 °C.
  • the affinity of the truncated MICA to NKG2D is at least 2 times the affinity of the native MICA molecule to NKG2D; preferably at least 3 fold; more preferably at least 5 fold; most preferably at least 10 fold.
  • the truncated MICA comprises one or more mutated amino acid residues selected from the group consisting of: 13S, 15Q, 24Q, 51G, 54T, 88S, 94T, 96S, 130V, 141Q and 179P, wherein the amino acid residue numbering is the number shown in SEQ ID NO.
  • the truncated MICA has at least 80% homology, preferably at least 90% homology, to the amino acid sequence set forth in SEQ ID NO.
  • the truncated MICA has at least 85% homology, preferably at least 90% homology, more preferably has at least 85% homology to the amino acid sequence set forth in SEQ ID NO. At least 95% homology, most preferably at least 98% homology.
  • the "mutation" further comprises mutation of an amino acid residue in the hydrophobic core of one or more (preferably 2-30, more preferably 2-25) ⁇ 1 ⁇ 2 domains of native MICA. .
  • the "amino acid residue in the hydrophobic core of the ⁇ 1 ⁇ 2 domain of native MICA” is selected from the group consisting of 9N, 10L, 14S, 16D, 25T, 35L, 39R, 49Q, 51A, 52E, 54T, 70N, 109Q, 121Q, 128W, 147L, 150D, 153K, 155K, 158Y, 159H, 162H, and 178V, wherein the amino acid residue numbering is the number shown in SEQ ID NO.
  • amino acid residue in the hydrophobic core of the ⁇ 1 ⁇ 2 domain of native MICA includes 178V, and/or 162H, wherein the amino acid residue numbering is represented by the number shown in SEQ ID NO.
  • the "amino acid residue in the hydrophobic core of the ⁇ 1 ⁇ 2 domain of native MICA" includes 158Y, and/or 147L, wherein the amino acid residue numbering is represented by the number shown in SEQ ID NO.
  • amino acid residue in the hydrophobic core of the ⁇ 1 ⁇ 2 domain of native MICA further includes 121Q, and/or 109Q, wherein the amino acid residue numbering is represented by the number shown in SEQ ID NO.
  • amino acid residue in the hydrophobic core of the ⁇ 1 ⁇ 2 domain of native MICA further includes 128W, wherein the amino acid residue numbering is represented by the number shown in SEQ ID NO.
  • the "amino acid residue in the hydrophobic core of the ⁇ 1 ⁇ 2 domain of native MICA" further includes 35L, 10L, and/or 9N, wherein the amino acid residue numbering is represented by SEQ ID NO. Numbering.
  • the "amino acid residue in the hydrophobic core of the ⁇ 1 ⁇ 2 domain of native MICA" further includes 14S, 25T, 49Q, 51A, 52E, and/or 54T, wherein the amino acid residue numbering uses SEQ ID The number shown in NO.1.
  • the truncated MICA further comprises one or more mutated amino acid residues selected from the group consisting of 9D, 10F, 14M or 14I, 16N, 25A, 35I, 39T, 49D, 51E , 52N, 54Q, 70W, 109I or 109H, 121G, 121M, 121W or 121N, 128F, 147W, 150V, 153E, 155D, 158W or 158F, 159D, 162R, 178I or 178M, wherein the amino acid residue numbering uses SEQ ID NO The number shown in .1.
  • the truncated MICA further comprises a mutated amino acid residue: 162R, and/or 178I or 178M, wherein the amino acid residue numbering is the number shown in SEQ ID NO.
  • the truncated MICA further comprises a mutated amino acid residue: 158W or 158F, and/or 147W, wherein the amino acid residue numbering is the number shown in SEQ ID NO.
  • the truncated MICA further comprises a mutated amino acid residue: 121G, 121M, 121W or 121N, and/or 109I or 109H, wherein the amino acid residue numbering is represented by SEQ ID NO. The number.
  • the truncated MICA further comprises a mutated amino acid residue: 128F, wherein the amino acid residue numbering is the number shown in SEQ ID NO.
  • the truncated MICA further comprises a mutated amino acid residue: 35I, 10F, and/or 9D, wherein the amino acid residue numbering is the number shown in SEQ ID NO.
  • the truncated MICA further comprises a mutated amino acid residue: 14M or 14I, 25A, 49D, 51E, 52N, and/or 54Q, wherein the amino acid residue numbering is SEQ ID NO. The number shown.
  • the truncated MICA further comprises a mutated amino acid residue: 16N, 39T, 70W, 150V, 153E, 155D, and/or 159D, wherein the amino acid residue numbering is SEQ ID NO. The number shown.
  • the truncated MICA has an amino acid sequence selected from the group consisting of SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11. SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21, SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. SEQ ID NO. 29 and SEQ ID NO.
  • the truncated MICA is soluble.
  • a fusion protein having the truncated MICA of the first aspect of the invention.
  • the fusion protein has an optional tag sequence that facilitates expression and/or purification.
  • the fusion protein further comprises an antibody sequence and/or a TCR sequence.
  • a nucleic acid molecule comprising a polynucleotide sequence encoding the truncated MICA of the first aspect of the invention or a complement thereof is provided.
  • a vector comprising the nucleic acid molecule of the third aspect of the invention is provided.
  • a host cell comprising the vector of the fourth aspect of the invention or the nucleic acid molecule of the third aspect of the invention integrated with exogenous in the chromosome is contained.
  • a conjugate comprising
  • a coupling moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.
  • the coupling moiety is selected from the group consisting of a fluorescent or luminescent label, a radioactive label, an MRI (magnetic resonance imaging) or CT (computer tomography) contrast agent, or is capable of producing a detectable agent
  • Product enzymes radionuclides, biotoxins, cytokines (such as IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles/nanorods, viral particles, liposomes, nanomagnetic particles, pre- Drug activating enzyme (eg, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agent (eg, cisplatin) or any form of nanoparticles, etc.
  • DTD DT-diaphorase
  • BPHL biphenyl hydrolase-like protein
  • a seventh aspect of the invention there is provided a method of preparing the truncated MICA of the first aspect of the invention, comprising the steps of:
  • the truncated MICA of the first aspect of the invention is isolated or purified.
  • the medicament is for treating a tumor or a viral infection
  • the reagent is used to detect NKG2D.
  • Figure 1 shows a schematic diagram of the hydrophobic core region structure of a DMIC molecule.
  • Figures 2a and 2b are the amino acid sequence and nucleotide sequence corresponding to the ⁇ 1 ⁇ 2 domain of the MICA molecule, respectively (SEQ ID NOS. 1 and 2).
  • Figures 3a and 3b are the amino acid sequence and nucleotide sequence (SEQ ID NOS. 3 and 4) of the wild-type DMIC, respectively.
  • the hydrophobic amino acid residues exposed on the surface are mutated with respect to the sequence corresponding to the ⁇ 1 ⁇ 2 domain of the MICA molecule.
  • the mutated amino acid residues are shown in boldface and underline.
  • Figures 4a and 4b are the amino acid sequence and nucleotide sequence (SEQ ID NOS. 5 and 6), respectively, of the DMIC mutant B7.
  • the mutated amino acid residues are shown in boldface and underlined relative to the wild-type DMIC.
  • Figures 5a and 5b are the amino acid sequence and nucleotide sequence (SEQ ID NOS. 7 and 8), respectively, of the DMIC mutant strain C3, and the mutated amino acid residues are shown in boldface and underlined relative to the wild-type DMIC.
  • Figures 6a and 6b are the amino acid sequence and nucleotide sequence (SEQ ID NOS. 9 and 10) of the DMIC mutant strain C5, respectively, and the mutated amino acid residues are shown in boldface and underlined relative to the wild-type DMIC.
  • Figures 7a and 7b are the amino acid sequence and nucleotide sequence (SEQ ID NOS. 11 and 12) of the DMIC mutant strain D16, respectively, and the mutated amino acid residues are shown in boldface and underlined relative to the wild-type DMIC.
  • Figures 8a and 8b are the amino acid sequence and nucleotide sequence (SEQ ID NOS. 13 and 14), respectively, of the DMIC mutant strain M1, and the mutated amino acid residues are shown in boldface and underlined relative to the wild-type DMIC.
  • Figures 9a and 9b are the amino acid sequence and nucleotide sequence (SEQ ID NOS. 15 and 16), respectively, of the DMIC mutant strain M1a, and the mutated amino acid residues are shown in boldface and underlined relative to the wild-type DMIC.
  • Figures 10a and 10b are the amino acid sequence and nucleotide sequence (SEQ ID NOS. 17 and 18), respectively, of the DMIC mutant strain M2a, and the mutated amino acid residues are shown in boldface and underlined relative to the wild-type DMIC.
  • Figures 11a and 11b are the amino acid sequence and nucleotide sequence (SEQ ID NO. 19 and 20), respectively, of the DMIC mutant strain M2b, and the mutated amino acid residues are shown in boldface and underlined relative to the wild-type DMIC.
  • Figures 12a and 12b are the amino acid sequence and nucleotide sequence (SEQ ID NOS. 21 and 22) of the DMIC mutant strain C3H7, respectively, and the mutated amino acid residues are shown in boldface and underlined relative to the wild-type DMIC.
  • Figures 13a and 13b are the amino acid sequence and nucleotide sequence (SEQ ID NOS. 23 and 24) of the DMIC mutant strain C5H7, respectively, and the mutated amino acid residues are shown in boldface and underlined relative to the wild-type DMIC.
  • Figures 14a and 14b are the amino acid sequence and nucleotide sequence (SEQ ID NOS. 25 and 26) of the DMIC mutant strain D16H7, respectively, and the mutated amino acid residues are shown in boldface and underlined relative to the wild-type DMIC.
  • Figures 15a and 15b are the amino acid sequence and nucleotide sequence (SEQ ID NOS. 27 and 28), respectively, of the DMIC mutant strain M1aH7, and the mutated amino acid residues are shown in boldface and underlined relative to the wild-type DMIC.
  • Figures 16a and 16b are the amino acid sequence and nucleotide sequence (SEQ ID NOS. 29 and 30), respectively, of the DMIC mutant strain M2aH7, and the mutated amino acid residues are shown in boldface and underlined relative to the wild-type DMIC.
  • Figures 17a and 17b are the amino acid sequence and nucleotide sequence (SEQ ID NOS. 31 and 32), respectively, of the DMIC mutant strain M2bH7, and the mutated amino acid residues are shown in boldface and underlined relative to the wild-type DMIC.
  • Figure 18 is a graph showing the elution peak of protein purification.
  • Figure 19 is a Non-reducing SDS-PAGE gel of the purified protein.
  • Lane 1 molecular weight marker
  • lane 2 wild-type DMIC
  • lane 3 mutant B7
  • lane 4 mutant C3, lane 5: mutant C5,
  • lane 6 mutant D16
  • lane 7 mutant M1
  • lane 8 mutant M1a
  • lane 9 H7
  • lane 10 mutant C3H7
  • lane 11 mutant C5H7
  • lane 12 mutant D16H7
  • lane 13 mutant M1aH7
  • lane 14 molecular weight marker
  • lane 15 mutant M2a
  • Lane 16 Mutant M2b
  • Lane 17 Mutant M2aH7
  • Lane 18 Mutant M2bH7
  • Lane 19 MICA
  • Lane 20 Molecular Weight Markers.
  • Figure 20 is a graph showing the interaction of purified MICA protein and DMIC protein with NKG2D by Biacore SPR.
  • Figure 21 is a map showing the interaction of purified MICA protein, DMIC protein and NKG2D by Biacore SPR.
  • MICA MICA
  • H7 wild-type DMIC
  • mutant B7 mutant B7
  • mutant C3H7 mutant C5H7
  • mutant D16H7 mutant D16H7
  • mutant M1aH7 mutant M1aH7
  • mutant M2aH7 mutant M2aH7
  • mutant M2bH7 mutant M2bH7.
  • Figure 22 shows the Tm value and dH value of the purified protein after DSC detection.
  • Figure 23 is a DSC raw graph of MICA and wild-type DMIC.
  • Figure 24 is a DSC fit plot of DMIC high stability and high affinity mutants and H7.
  • Figure 25 shows the flow detection of biotinylated MICA protein, wild-type DMIC protein, high-affinity DMIC proteins B7, C3, C5, D16, M1, M1a and peripheral blood mononuclear cell (PBMC) surface. The combination of NKG2D.
  • the inventors have conducted extensive and intensive research and found that the present invention has been completed on the basis of a targeted mutation of a hydrophobic core region of a protein to obtain a mutant having high stability and high affinity.
  • the present inventors removed the ⁇ 3 domain of MICA (amino acids 182-275), resulting in a new molecular DMIC containing only the ⁇ 1 ⁇ 2 domain of MICA (amino acids 1-181) (SEQ ID NOS. 1 and 2, That is, truncated MICA).
  • wild-type DMICs DMIC-WT, SEQ ID NOS. 3 and 4
  • Mutants that were more stable than DMIC-WT were isolated by directed molecular evolution and phage display techniques by introducing random mutations into the hydrophobic core of DMIC-WT.
  • the mutant obtained by the present invention has a binding affinity with NKG2D which is 10-2600 times more than that of DMIC-WT and NKG2D, except that the stability is greatly improved compared with DMIC-WT.
  • the mutation sites on the stability mutant were separately transplanted into the DMIC mutant H7 (the mutation sites include 16N, 39T, 70W, 150V, 153E, 155D, 159D, wherein the amino acid residue numbering is represented by SEQ ID NO. No.), the stability of the mutant transplanted with the stable mutation site was further enhanced as compared with H7.
  • MICA protein crystal structure
  • NKG2D complex MICA (NCBI Accession Number: 1HYR_C) contains three domains, namely ⁇ 1, ⁇ 2, and ⁇ 3, and ⁇ 3, respectively. Does not participate in the combination with NKG2D.
  • the inventors removed the ⁇ 3 domain of MICA (amino acids 182-275), forming a truncated MICA, and further The hydrophobic amino acid residue exposed to the surface was replaced with a hydrophilic amino acid residue (L13S; W15Q; L24Q; A51G; V54T; L88S; I94T; V96S; M130V; M141Q; L179P), resulting in an ⁇ 1 ⁇ 2 structure containing only MICA.
  • the new molecule of the domain (amino acids 1-181), the wild type DMIC (DMIC-WT, SEQ ID NOS. 3 and 4).
  • a common feature of protein structure is that there are hydrophobic cores inside the molecule, which are composed of closely packed hydrophobic side chains.
  • the hydrophobic side chains are buried in the water molecules, which is the original driving force for the folding of the polypeptide chains. It is the main factor that stabilizes the three-dimensional structure of proteins.
  • the inventors introduced random mutations in the hydrophobic core region of DMIC-WT to construct a phage library, and used the phage display technology to screen the library for stability. After obtaining the mutant strain, it was found that the obtained mutant not only greatly improved the stability, but also improved the affinity by 10-2600 times.
  • the inventors introduced three mutation sites (D16N, R39T, N70W, D150V, K153E, K155D, H159D) using DMIC-WT as a template, and a new DMIC mutant was generated, which was named H7. Then, the mutation sites of the stable mutants screened from the phage library were transplanted into H7, respectively, and the results showed that the stability of the mutant transplanted with the stable mutation site was further improved as compared with H7.
  • the present invention provides a high stability, high affinity DMIC molecule, which provides a solid foundation for further research on the function of DMIC molecules, such as fusion proteins.
  • truncated MICA and “DMIC” are used interchangeably and refer to a protein derived from MICA including the ⁇ 1 ⁇ 2 domain of MICA, and the protein contains a mutation that increases affinity and stability.
  • the invention also provides a fusion protein comprising the truncated MICA, such as an isolated fusion protein.
  • the protein of the invention may be a monomer or a multimer (e.g., a dimer) formed from a monomer.
  • the term also encompasses active fragments and derivatives of DMIC or its fusion proteins.
  • isolated means that the substance is separated from its original environment (if it is a natural substance, the original environment is the natural environment).
  • the polynucleotides and polypeptides in the natural state in living cells are not isolated and purified, but the same polynucleotide or polypeptide is isolated and purified, as separated from other substances present in the natural state.
  • isolated fusion protein means that the fusion protein is substantially free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated. Those skilled in the art can purify the fusion protein using standard protein purification techniques. Substantially pure proteins produce a single major band on a non-reducing polyacrylamide gel.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • the DNA can be a coding strand or a non-coding strand.
  • the present invention also relates to variants of the above polynucleotides which encode protein fragments, analogs and derivatives having the same amino acid sequence as the present invention.
  • Variants of this polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide that may be a substitution, deletion or insertion of one or more nucleotides, but does not substantially alter the function of the polypeptide encoded thereby.
  • the term "primer” refers to a generic term for oligonucleotides that are paired with a template and which are capable of synthesizing a DNA strand complementary to a template starting from the action of a DNA polymerase.
  • the primer may be native RNA, DNA, or any form of natural nucleotide.
  • the primer may even be a non-natural nucleotide such as LNA or ZNA.
  • the primer is “substantially” (or “substantially") complementary to a particular sequence on a strand on the template.
  • the primer must be sufficiently complementary to a strand on the template to initiate extension, but the sequence of the primer need not be fully complementary to the sequence of the template.
  • a sequence that is not complementary to the template is added to the 5' end of the primer complementary to the template at the 3' end, and such primer is still substantially complementary to the template.
  • the non-fully complementary primers can also form a primer-template complex with the template for amplification.
  • the full-length nucleotide sequence of the protein of the present invention or a fusion protein thereof or a fragment thereof can be usually obtained by a PCR amplification method, a recombinant method or a synthetic method.
  • primers can be designed according to published nucleotide sequences, particularly open reading frame sequences, and used as commercially available cDNA libraries or cDNA libraries prepared by conventional methods known to those skilled in the art.
  • the template is amplified to obtain the relevant sequence. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then the amplified fragments are spliced together in the correct order.
  • the recombinant sequence can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it to a cell, and then separating it from the proliferated host cell by conventional methods. sequence.
  • synthetic sequences can be used to synthesize related sequences, especially when the fragment length is short.
  • a long sequence of fragments can be obtained by first synthesizing a plurality of small fragments and then performing the ligation.
  • a method of amplifying DNA/RNA using PCR technology is preferably used to obtain the gene of the present invention.
  • the primers for PCR can be appropriately selected according to the sequence information of the present invention disclosed herein, and can be synthesized by a conventional method.
  • the amplified DNA/RNA fragment can be isolated and purified by conventional methods such as by gel electrophoresis.
  • the invention also relates to vectors comprising the polynucleotides of the invention, as well as host cells genetically engineered using the vector or fusion protein coding sequences of the invention, and methods of producing the proteins of the invention by recombinant techniques.
  • polynucleotide sequences of the present invention can be utilized to express or produce recombinant proteins by conventional recombinant DNA techniques. Generally there are the following steps:
  • Methods well known to those skilled in the art can be used to construct expression vectors containing the DNA sequences of the proteins of the invention and suitable transcription/translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like.
  • the DNA sequence can be operably linked to an appropriate promoter in an expression vector to direct mRNA synthesis.
  • the expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.
  • the expression vector preferably comprises one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
  • selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
  • Vectors comprising the appropriate DNA sequences described above, as well as appropriate promoters or control sequences, can be used to transform appropriate host cells to enable expression of the protein.
  • the host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • a prokaryotic cell such as a bacterial cell
  • a lower eukaryotic cell such as a yeast cell
  • a higher eukaryotic cell such as a mammalian cell.
  • Representative examples are: Escherichia coli, bacterial cells of the genus Streptomyces; fungal cells such as yeast; plant cells; insect cells of Drosophila S2 or Sf9; animal cells of CHO, NS0, COS7, or 293 cells, and the like.
  • Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as E. coli
  • competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated by the CaCl 2 method, and the procedures used are well known in the art.
  • Another method is to use MgCl 2 .
  • Conversion can also be carried out by electroporation if desired.
  • the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, and the like.
  • the obtained transformant can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention.
  • the medium used in the culture may be selected from various conventional media depending on the host cell used.
  • the cultivation is carried out under conditions suitable for the growth of the host cell.
  • the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction) and the cells are cultured for a further period of time.
  • the protein in the above method can be expressed intracellularly, or on the cell membrane, or secreted outside the cell. If desired, the protein can be isolated and purified by various separation methods using its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting method), centrifugation, osmotic sterilizing, super treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • conventional renaturation treatment treatment with a protein precipitant (salting method), centrifugation, osmotic sterilizing, super treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and
  • the term "truncated MICA” also includes variant forms having the above activities. These variants include, but are not limited to, 1-3 (usually 1-2, more preferably 1) amino acid deletions, insertions and/or substitutions, and additions at the C-terminus and/or N-terminus or One or several (usually 3 or less, preferably 2 or less, more preferably 1 or less) amino acids are deleted. For example, in the art, similar or similar performance When an amino acid is substituted, it usually does not change the function of the protein. As another example, the addition or deletion of one or more amino acids at the C-terminus and/or N-terminus will generally not alter the structure and function of the protein. Furthermore, the term also encompasses polypeptides of the invention in both monomeric and multimeric forms. The term also includes both linear as well as non-linear polypeptides (such as cyclic peptides).
  • the invention also includes active fragments, derivatives and analogs of the above proteins.
  • fragment refers to a polypeptide that substantially retains the function or activity of a fusion protein of the invention.
  • a polypeptide fragment, derivative or analog of the invention may be (i) a polypeptide having one or several conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, or (ii) at one or more a polypeptide having a substituent group in one amino acid residue, or (iii) a polypeptide formed by fusing an antigen peptide with another compound (such as a compound that extends half-life of the polypeptide, such as polyethylene glycol), or (iv) an additional amino acid sequence
  • a polypeptide formed by fusion of the polypeptide sequence a fusion protein formed by fusion with a leader sequence, a secretory sequence or a tag sequence such as 6His).
  • a preferred class of reactive derivatives means that up to 3, preferably up to 2, and more preferably up to 1 amino acid are replaced by amino acids of similar or similar nature to the amino acid sequence of Formula Ia or Formula Ib. Peptide. These conservative variant polypeptides are preferably produced by amino acid substitution according to Table A.
  • the invention also provides analogs of the truncated MICA proteins of the invention.
  • the difference between these analogs and the polypeptide represented by the specific sequence of the present invention may be a difference in amino acid sequence, a difference in a modified form which does not affect the sequence, or a combination thereof.
  • Analogs also include analogs having residues other than the native L-amino acid (such as D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (such as beta, gamma-amino acids). It is to be understood that the polypeptide of the present invention is not limited to the representative polypeptides exemplified above.
  • Modifications include chemically derived forms of the polypeptide, such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those produced by glycosylation modifications in the synthesis and processing of the polypeptide or in further processing steps. Such modification can be accomplished by exposing the polypeptide to an enzyme that performs glycosylation, such as a mammalian glycosylation enzyme or a deglycosylation enzyme. Modified forms also include phosphorylated amino acid residues The sequence of (such as phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides modified to increase their resistance to proteolytic properties or to optimize solubility properties.
  • the polypeptide (fusion protein) of the present invention can also be used in the form of a salt derived from a pharmaceutically or physiologically acceptable acid or base.
  • These salts include, but are not limited to, salts formed with hydrochloric acid, hydrobromic acid, sulfuric acid, citric acid, tartaric acid, phosphoric acid, lactic acid, pyruvic acid, acetic acid, succinic acid, oxalic acid, fumaric acid, Malay. Acid, oxaloacetic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, or isethionic acid.
  • Other salts include those formed with alkali or alkaline earth metals such as sodium, potassium, calcium or magnesium, as well as esters, carbamates or other conventional "prodrugs".
  • the present invention provides fusion proteins which optionally contain a peptide linker to which a truncated MICA and other polypeptide elements (e.g., antibodies, TCRs, etc.) according to the present invention are linked by peptide bonds or peptide linkers.
  • the size and complexity of the peptide linker may affect the activity of the protein.
  • the peptide linker should be of sufficient length and flexibility to ensure that the two proteins attached have sufficient freedom in space to perform their function. At the same time, the effect of the formation of an alpha helix or a beta sheet in the peptide linker on the stability of the fusion protein is avoided.
  • the length of the linker peptide is generally from 0 to 10 amino acids, preferably from 1 to 5 amino acids.
  • the invention also provides a composition comprising: (i) a truncated MICA protein of the invention or a polynucleotide encoding a truncated MICA protein of the invention, and (ii) pharmaceutically acceptable Carrier, or excipient.
  • the term "containing” means that the various ingredients may be applied together or in the composition of the present invention. Therefore, the terms “consisting essentially of” and “consisting of” are encompassed by the term “contains.”
  • compositions of the invention include pharmaceutical compositions.
  • compositions of the present invention can be prepared in a variety of conventional dosage forms including, but not limited to, injections, granules, tablets, pills, suppositories, capsules, suspensions, sprays and the like.
  • the fusion proteins of the invention may be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium wherein the pH is usually from about 5 to about 8, preferably from about 6 to about 8.
  • the term "effective amount” or “effective amount” refers to an amount that is functional or active to a human and/or animal and that is acceptable to humans and/or animals, such as from 0.001 to 99% by weight; preferably 0.01-95 wt%; more preferably, 0.1-90 wt%.
  • a "pharmaceutically acceptable” ingredient is one that is suitable for use in humans and/or mammals without excessive adverse side effects (eg, toxicity, irritation, and allergies), ie, having a reasonable benefit/risk ratio.
  • pharmaceutically acceptable carrier refers to a carrier for the administration of a therapeutic agent, including various excipients and diluents.
  • compositions of the present invention comprise a safe and effective amount of a fusion protein of the invention and a pharmaceutically acceptable carrier.
  • Such carriers include, but are not limited to, saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should be matched to the mode of administration, and the pharmaceutical composition of the present invention can be prepared into an injection form, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants.
  • the pharmaceutical composition is preferably manufactured under sterile conditions.
  • the amount of active ingredient administered is a therapeutically effective amount.
  • the pharmaceutical preparation of the present invention can also be formulated into a sustained release preparation.
  • the effective amount of the truncated MICA protein of the present invention may vary depending on the mode of administration and the severity of the disease to be treated and the like. The selection of a preferred effective amount can be determined by one of ordinary skill in the art based on various factors (e.g., by clinical trials). The factors include, but are not limited to, pharmacokinetic parameters of the fusion protein of the invention such as bioavailability, metabolism, half-life, etc.; severity of the disease to be treated by the patient, body weight of the patient, immune status of the patient, administration Ways, etc.
  • the fusion protein of the present invention is administered at a dose of about 0.5 mg to 5 mg/kg of animal body weight per day (preferably 2 mg to 4 mg/kg of animal body weight), a satisfactory effect can be obtained.
  • a dose of about 0.5 mg to 5 mg/kg of animal body weight per day preferably 2 mg to 4 mg/kg of animal body weight
  • several separate doses may be administered per day, or the dose may be proportionally reduced, as is critical to the condition of the treatment.
  • One method of producing a DMIC molecule of the invention is to select a high stability, high affinity DMIC mutant from a diverse library of phage particles displaying this DMIC molecule.
  • Mutations can be carried out by any suitable method including, but not limited to, polymerase chain reaction (PCR), restriction enzyme based cloning or ligation-independent cloning (LIC) methods.
  • PCR polymerase chain reaction
  • LIC ligation-independent cloning
  • the DMIC molecules of the invention may be chemically synthesized or recombinant. Accordingly, the DMIC molecules of the present invention can be artificially synthesized by a conventional method or can be produced by a recombinant method.
  • the polynucleotide of the present invention can be utilized to express or produce a recombinant DMIC by conventional recombinant DNA techniques. Generally there are the following steps:
  • the DMIC of the present invention is isolated and purified from the culture medium or cells.
  • DMIC can be expressed intracellularly, or on the cell membrane, or secreted outside the cell.
  • the recombinant protein can be isolated and purified by various separation methods using its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting method), centrifugation, osmotic sterilizing, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • the high stability, high affinity DMIC molecular uses of the present invention include the following:
  • TCR T cell receptor
  • the DMIC molecule of the present invention has high stability
  • the DMIC molecule of the present invention has high affinity
  • the hydrophobic amino acid residues exposed to the surface are replaced with hydrophilic residues, and the substitution conditions are L13S, W15Q, L24Q, A51G, V54T, L88S, I94T, V96S, M130V, M141Q, L179P, thereby obtaining wild Type DMIC (DMIC-WT, SEQ ID NOS. 3 and 4).
  • the amino acid sequence of DMIC-WT was optimized to a nucleotide sequence suitable for expression in E. coli, and the gene was synthesized and cloned into a PUC57 vector (Genscript).
  • DMIC-WT was cloned into the pET-28a expression plasmid (purchased from Novagen) by standard methods described in the Molecular Cloning a Laboratory Manual (3rd edition, Sambrook and Russell).
  • NcoI and NotI-cleaved DNA sequences encoding DMIC-WT were ligated into the pET-28a vector cleaved with NcoI and NotI.
  • the ligated plasmid was transformed into a conventional competent Escherichia coli strain BL21 (DE3) cell (purchased from Vazyme) and plated on an LB agar plate containing 50 ⁇ g/mL kanamycin.
  • the E. coli strain BL21 (DE3) containing the DMIC-WT expression plasmid obtained in Example 2 was inoculated into 5 ml of LB medium containing 50 ⁇ g/mL kanamycin, and cultured overnight at 37 ° C with shaking at 250 rpm. On the second day, transfer to a 400 ml LB medium containing 50 ⁇ g/mL kanamycin at a volume ratio of 1:100, and incubate at 37 ° C at 250 rpm to an OD600 of 0.4-0.6 (about 2-3 h), and add a final concentration of 0.7. Expression was induced by mM IPTG for 4 hours. Cells were harvested by centrifugation at 5,000 rpm for 20 min.
  • the cell pellet was lysed with Bugbuster MasterMix (Merck) and the inclusion body pellet was recovered by centrifugation at 6,000 g for 15 min. The inclusion bodies were then washed three times with a 10-fold diluted Bugbuster solution, and finally the inclusion bodies were dissolved in 6 M guanidine hydrochloride buffer. After quantification by the BCA method, 12 mg per tube was dispensed and stored frozen at -80 °C.
  • the dialyzed refolding protein solution DMIC-WT was centrifuged and the anion exchange column QFF (5 ml, GE) was eluted by a GE AKTA protein purification system using a linear gradient of 1 M NaCl prepared with 10 mM Tris.Cl pH 8.5. Peak, run SDS-PAGE gel analysis.
  • the fraction containing DMIC-WT was diluted 10-fold with 10 mM Tris.Cl pH 8.5, and then again anion exchange column QFF was eluted with a linear gradient of 1 M NaCl prepared with 10 mM Tris.Cl pH 8.5 (Fig. 18(b)). Identification by SDS-PAGE gel (relative molecular mass about 22 kD, Figure 19).
  • Random mutations were introduced into the DMIC-WT hydrophobic core to construct four phage mutant libraries, and the library was panned using phage display technology.
  • the above hydrophobic core library construction and screening method can be referred to the construction and screening method of the high affinity TCR phage library described by Li et al. ((2005) Nature Biotech 23(3): 349-354).
  • the designed primers are shown in the following table (Table 1-4).
  • B C or G or T
  • D A or G or T
  • H A or C or T
  • K G or T
  • M A or C
  • N A or C or G or T
  • R A or G
  • S C or G
  • V A or C or G
  • W A or T
  • Y C or T.
  • the inventors first displayed DMIC-WT on the surface of filamentous phage, and detected the binding of DMIC-WT to NKG2D by phage ELISA (enzyme-linked immunosorbent assay) and obtained the interaction strength.
  • DMIC-WT under the following 12 conditions: 26 ° C (IPTG concentration of 0 mM, 0.2 mM, 0.5 mM, 1 mM, respectively), 30 ° C (IPTG concentrations were respectively 0 mM, 0.2 mM, 0.5 mM, 1 mM), 37 ° C (IPTG concentration of 0 mM, 0.2 mM, 0.5 mM, 1 mM, respectively), and then the binding of the displayed DMIC-WT to NKG2D was detected by phage Elisa, and the result was at 37 ° C, 1 mM. DMIC-WT showed the worst performance in the presence of IPTG.
  • the inventors In order to allow those mutants whose stability is comparable to DMIC-WT or even worse than DMIC-WT to be directly eliminated during expression, retaining a mutant having better stability than DMIC-WT, that is, in order to reduce background expression, the inventors The conditions in which 37 mM, 1 mM IPTG was present were selected as the expression conditions for the subsequent mutant library. In order to further reduce the screening background, the DMIC mutant library displayed on the surface of the phage was first treated in the presence of 0.02% SDS, treated with a water bath at 65 ° C for 1 h, cooled on ice for 15 min, and then combined with NKG2D for screening.
  • the hydrophobic cores of more than 50 DMIC mutant strains screened by the above method were all mutated. Then, it was identified by phage Elisa, the combination of mutation sites, and finally 8 mutant strains were selected for further identification.
  • the 8 mutant strains were B7, C3, C5, D16, M1, M1a, M2a, M2b. Among them, B7, C3, C5, and D16 are directly selected mutant strains, and M1, M1a, M2a, and M2b are mutant strains after combination of mutation sites.
  • DMIC-WT and each mutant were inoculated separately to 5 mL 2xTY (100 ⁇ g/mL ampicillin, 2% glucose), 250 rpm/min, and cultured overnight at 37 °C.
  • the supernatant of the culture supernatant was collected by centrifugation, and the phage in the supernatant was precipitated with 1/4 volume ratio of PEG/NaCl, placed on ice for 1 hour, and the pellet was collected by centrifugation and resuspended in 3 mL of PBS.
  • the wild type MICA, DMIC-WT and mutants B7, C3, C5, D16, M1, M1a, M2a, M2b were expressed, renatured and purified by the method described in Example 3, and then run SDS-PAGE gel.
  • the binding activity of MICA, DMIC-WT and mutants B7, C3, C5, D16, M1, M1a, M2a, M2b and NKG2D was detected using a BIAcore T200 real-time analysis system.
  • the anti-streptavidin antibody was added to a coupling buffer (10 mM sodium acetate buffer, pH 4.77), and then the antibody was passed through a CM5 chip previously activated with EDC and NHS to immobilize the antibody on the surface of the chip.
  • a solution of ethanolamine in hydrochloric acid blocks the unreacted activated surface, completing the coupling process at a coupling level of about 15,000 RU.
  • a low concentration of streptavidin is passed over the surface of the coated antibody chip, then biotinylated NKG2D is passed through the detection channel, and the other channel is used as a reference channel, and 0.05 mM biotin is again at 10 ⁇ L/min. Flow rate Flow through the chip for 2 min, blocking the remaining binding sites of streptavidin. The affinity was determined by single-cycle kinetic analysis.
  • HEPES-EP buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% P20, pH 7.4) was diluted into several different concentrations, and flowed through the surface of the chip at a flow rate of 30 ⁇ L/min.
  • the binding time of each injection was 120 s, and it was dissociated for 600 s after the last injection.
  • the chip was regenerated with 10 mM Gly-HCl, pH 1.75, after each round of assay.
  • Kinetic parameters were calculated using BIAcore Evaluation software.
  • the purified NKG2D protein was concentrated using a Millipore ultrafiltration tube while the buffer was replaced with 10 mM Tris.Cl pH 8.0, followed by biotinylation reagent 0.05M Bicine pH 8.3, 10 mM ATP, 10 mM MgOAc, 50 ⁇ M D-Biotin, 100 ⁇ g/
  • the ml BirA enzyme (GST-BirA) was incubated overnight at 20 ° C with shaking at 80 rpm, and the biotinylation was determined by SDS-PAGE.
  • the biotinylated labeled NKG2D protein was concentrated to 0.5 ml using a Millipore ultrafiltration tube, and biotinylated NKG2D was purified by gel filtration chromatography, and the Superdex 75 16/20 column was equilibrated with PBS using an AKTA Protein Purification System (GE). GE), loaded with 0.5 ml of concentrated biotinylated NKG2D protein and continued to equilibrate with PBS at a flow rate of 0.5 ml/min. The biotinylated NKG2D protein appeared as a single peak elution at about 10 ml. The protein-containing fractions were pooled, concentrated using a Millipore ultrafiltration tube, protein concentration was determined by BCA method, and biotinylated NKG2D protein was dispensed at -80 °C.
  • the KD value of MICA binding to NKG2D protein was 5.62E-07M by the process described in this example; the KD value of DMIC-WT binding to NKG2D protein was 1.479E-07M; the KD value of binding of B7 to NKG2D protein was 1.177E.
  • the Tm value and the dH value of the purified protein MICA, DMIC-WT and each mutant were measured by a differential scanning calorimeter (Nano DSC) of TA (waters), USA.
  • the scanning range is 10-100 ° C
  • the heating rate is 1 ° C / min
  • the sample concentration is 0.5 mg / mL
  • the sample loading is 900 ⁇ L.
  • the Tm value is obtained by fitting the fitting model TwostateScaled of the analysis software Nanoanalyze.
  • Figure 19 is a SDS-PAGE gel of the protein MICA, DMIC-WT and each mutant obtained after purification by two anion exchange columns as described in Example 3.
  • Figure 22 is the DSC data of the proteins MICA, DMIC-WT and each mutant. The results showed that MICA and DMIC-WT had minimal protein content in the correct conformation after purification, and there was no obvious protein unfolding endothermic peak. The Tm value and dH value could not be obtained by the analysis software Nanoanalyze, and the hydrophobic core mutation was obtained.
  • the Tm value and dH value of the subsequent mutant are as follows: B7 has a Tm value of 38.62 ° C, dH is 430.34 kJ/mol; C3 has a Tm value of 45.78 ° C, dH is 449.20 kJ/mol; C5 has a Tm value of 44.38 ° C, dH is 484.33 kJ/mol; D16 appears double peak, Tm value is 43.50 ° C and 61.16 ° C, dH is 596.70 kJ / mol and 375.40 kJ / mol; M1 Tm value 40.81 ° C, dH is 382.62 kJ / mol; M1a Tm value is 41.60 ° C, dH is 415.10kJ / mol; M2a Tm value is 43.48 ° C, dH is 324.74kJ / mol; M2b Tm value is 43.61
  • Figure 23 is a DSC curve of MICA and DMIC-WT
  • Figure 24 is a DSC fit curve of each mutant. It is indicated that the mutants B7, C3, C5, D16, M1, M1a, M2a, M2b are more resistant to unfolding than DMIC-WT, more resistant to inappropriate or undesired folding and a significant increase in thermal stability.
  • the mutation sites of the high stability mutants C3, C5, D16, M1a, M2a, M2b screened in Example 4 were transplanted to the DMIC mutant H7 (containing 7 mutation sites compared to DMIC-WT: D16N, R39T, N70W, D150V, K153E, K155D, H159D), six new mutants were constructed, namely C3H7, C5H7, D16H7, M1aH7, M2aH7, M2bH7, and protein expression and renaturation of 6 molecules. Purification, Biacore SPR affinity assay, DSC stability assay.
  • H7, C3H7, C5H7, D16H7, M1aH7, M2aH7, M2bH7 were expressed, renatured, purified (Fig. 18) and run SDS-PAGE gel (Fig. 19) by the method described in Example 3.
  • the expression amount and protein refolding yield were calculated.
  • the expression amount was 1 L of E. coli induced expression and purification of the inclusion body.
  • the expression amount and protein refolding yield referred to in the present invention are calculated according to the above calculation methods, unless otherwise specified.
  • the following table (Table 5) is the protein expression amount and protein refolding yield before and after the introduction of the stable hydrophobic core by H7.
  • Protein name Expression amount (mg/L) Protein refolding yield (%) H7 406 14.7 C3H7 607 41 C5H7 476 34 D16H7 420 28.6 M1aH7 657 28.5 M2aH7 443 17.5 M2bH7 473 16.6
  • the binding activity of H7, C3H7, C5H7, D16H7, M1aH7, M2aH7, M2bH7 and NKG2D was detected by Biacore SPR using the method described in Example 5.
  • the KD value of H7 binding to NKG2D protein is 3.556E-09M; the KD value of C3H7 binding to NKG2D protein is 2.872E-09M; the KD value of C5H7 binding to NKG2D protein is 1.736E-09M; the KD value of binding of D16H7 to NKG2D protein It is 1.478E-08M; the KD value of M1aH7 binding to NKG2D protein is 4.264E-09M; the KD value of M2aH7 binding to NKG2D protein is 1.967E-09M; the KD value of M2bH7 binding to NKG2D protein is 5.121E-09M (Fig.
  • Figure 21 is a binding curve of each protein detected by Biacore SPR to NKG2D.
  • the above data indicate that, except that the affinity of D16H7 to NKG2D is lower than that of H7 and NKG2D, the affinity of C3H7, C5H7, M1aH7, M2aH7, M2bH7 and NKG2D is substantially equivalent to that of H7 and NKG2D.
  • the thermal stability of H7, C3H7, C5H7, D16H7, M1aH7, M2aH7, M2bH7 was determined by DSC using the method described in Example 6.
  • the Tm value of H7 is 48.40 ° C, dH is 335 kJ / mol; the Tm value of C3H7 is 55.10 ° C, dH is 270 kJ / mol; the Tm value of C5H7 is 53.90 ° C, dH is 233 kJ / mol; Tm value of D16H7 50.2 ° C, dH is 304 kJ / mol; M1aH7 Tm value is 50.4 ° C, dH is 367kJ / mol; M2aH7 has double peaks, Tm values are 55.57 ° C and 84.84 ° C, dH is 216.39kJ / mol and 269.92kJ
  • Figure 24 is a DSC fit curve for each protein of DMIC. The above data indicate that the Tm values of C3H7, C5H7, D16H7, M1aH7, M2aH7, and M2bH7 are higher than those of H7.
  • the stability and affinity of the DMIC mutants screened by the present invention were significantly higher than DMIC-WT. At the same time, the transplantation of the mutation sites of the DMIC mutants to H7 can also enhance the stability.
  • Example 8 Flow cytometry detection of MICA, binding of wild-type DMIC and high-affinity DMIC proteins to NKG2D on the surface of PBMC
  • PBMCs were stained with biotinylated MICA protein, wild-type DMIC protein and high-affinity DMIC proteins B7, C3, C5, D16, M1, M1a (Figure 25). The experiment is divided into direct staining method and indirect staining method.
  • Indirect method was to compare 1 ug of high affinity DMIC protein C5 and 1 uL of PE mouse anti-human NKG2D to PBMC for 30 min and compare with PBMC samples stained with PE mouse anti-human NKG2D alone.
  • the results showed that high-affinity DMIC C5 inhibited the binding of anti-NKG2D antibody to NKG2D on the surface of PBMC, resulting in a decrease in the positive rate of anti-NKG2D antibody binding to PBMC from 55.9% to 2.18% (Fig. 25A). It is indicated that high affinity DMIC C5 can bind well to NKG2D on the surface of PBMC.
  • the direct method is to first stain the biotinylated MICA protein, the wild-type DMIC protein and the high-affinity DMIC proteins B7, C3, C5, D16, M1, M1a, respectively, and wash the PBMC three times, then add 0.5 respectively.
  • uL SA-PE was stained and finally detected by flow cytometry. The results showed that the detected protein could bind well to PBMC, and the positive rate increased with the increase of protein concentration. The positive rate increased with the increase of protein affinity.
  • the affinity of 10 -11 M to reach the level of high-affinity DMIC D16, M1, C5 positive rates of anti-NKG2D antibody staining with comparable (FIG. 25B).
  • the highly stable, high-affinity DMIC mutants screened by the present invention are for the subsequent study of the function of DMIC (such as studying the effect of DMIC and NKG2D affinity on activating NK cells or T cells), and developing DMIC-related drugs (such as with antibodies or TCR provides a firm basis for making fusion proteins, preparing drugs for treating tumors or viral infections, and the like.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Organic Chemistry (AREA)
  • Biochemistry (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Zoology (AREA)
  • Epidemiology (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • Physics & Mathematics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Microbiology (AREA)
  • Analytical Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Biophysics (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Wood Science & Technology (AREA)
  • Oncology (AREA)
  • General Chemical & Material Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Optics & Photonics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Hospice & Palliative Care (AREA)

Abstract

本发明提供了一种截短了的高稳定性和高亲和性的MICA(MHC classⅠ-related chain A)，即DMIC分子及其制法与用途。本发明的DMIC分子的疏水芯区域发生了突变，引起了其稳定性和亲和力的提高。

Description

高稳定性和高亲和力的DMIC及其制法 技术领域
本发明涉及生物技术领域，更具体地涉及疏水芯区域突变的、高稳定性的、高亲和力的可溶性DMIC。本发明还涉及所述DMIC的制备方法及用途。
背景技术
NKG2D(Natural Killer Group 2D)是一种活化性受体，主要表达在NK细胞、CD8+αβT细胞、γδ+T细胞和部分CD4+T细胞表面。MICA(MHC classⅠ-related chain A)是人NKG2D的配体之一，在多种肿瘤表面高表达，如黑色素瘤，胶质瘤，以及结肠癌、肾癌、肺癌、卵巢癌、乳腺癌、前列腺癌等多种癌细胞，而在正常细胞和成熟组织中不表达或低表达。研究表明，NKG2D与MICA的相互作用在肿瘤免疫监督中起到了重要的作用。Bauer的研究表明，NKG2D与MICA的相互作用可以直接激活NK细胞杀伤肿瘤细胞，同时，做为TCR的共刺激分子，激活CD8+αβT细胞和γδ+T细胞。过表达MICA的胶质瘤细胞在体外可以增强NK细胞和T细胞的活性，同时，过表达MICA的胶质瘤在裸鼠和VMDK鼠体内的生长也被明显延迟。NKG2D介导的NK细胞的细胞毒性与肿瘤表面表达的MICA的密度息息相关。
肿瘤表面的MICA即膜上的MICA发挥着肿瘤免疫监督和抗肿瘤的作用，而游离的、可溶性的MICA(sMICA)则会损伤机体的免疫反应，促进肿瘤的免疫逃逸。研究表明，肿瘤表面的MICA会在内质网蛋白酶5(Erp5)和金属蛋白酶(MPs)的作用下，从肿瘤表面脱落，形成游离的sMICA。sMICA会下调CD8+αβT细胞，NK细胞，γδ+T细胞和CD3+CD56+NKT细胞表面NKG2D的表达，进而实现肿瘤的免疫逃逸。由于MICA既参与了肿瘤的免疫监督，又参与了肿瘤的免疫逃逸，双重的身份，使得MICA成为了免疫治疗的潜在靶点。对MICA进行深入研究，可以更好的发挥其抗肿瘤的功效，Kellner等将MICA与抗肿瘤表面抗原的抗体做成融合蛋白，可以激活NK细胞杀伤肿瘤细胞。
发明内容
本发明的目的是提供一种截短了的高稳定性和高亲和性的MICA(即DMIC分子)。
本发明的另一目的是提供所述高稳定性和高亲和性DMIC分子的制备方法及用途。
本发明的第一方面，提供了一种截短的MICA，所述截短的MICA包括突变的MICA的α1α2结构域，所述“突变”包括天然MICA的α1α2结构域的氨基酸序列中一个或多个暴露于表面的疏水性氨基酸残基突变为亲水性氨基酸残基。
在另一优选例中，所述天然MICA的α1α2结构域的氨基酸序列如SEQ ID NO.1所示。
在另一优选例中，所述“暴露于表面的疏水性氨基酸残基”选自下组：13L、15W、24L、51A、54V、88L、94I、96V、130M、141M、和179L，其中氨基酸残基编号采用SEQ ID NO.1所示的编号。
在另一优选例中，所述截短的MICA的稳定性高于天然MICA，优选地，所述截短的MICA的Tm值大于或等于36℃。
在另一优选例中，所述截短的MICA与NKG2D的亲和力是天然MICA分子与NKG2D的亲和力的至少2倍；优选地至少3倍；更优选地至少5倍；最优选地至少10倍。
在另一优选例中，所述截短的MICA包含选自下组的一个或多个突变后的氨基酸残基：13S、15Q、24Q、51G、54T、88S、94T、96S、130V、141Q和179P，其中氨基酸残基编号采用SEQ ID NO.1所示的编号。
在另一优选例中，所述截短的MICA与SEQ ID NO.1所示的氨基酸序列相比具有至少80％的同源性，优选地具有至少90％的同源性。
在另一优选例中，所述截短的MICA与SEQ ID NO.3所示的氨基酸序列相比具有至少85％的同源性，优选地具有至少90％的同源性，更优选地具有至少95％的同源性，最优选地具有至少98％的同源性。
在另一优选例中，所述“突变”还包括一个或多个(优选为2-30个，更优选为2-25个)天然MICA的α1α2结构域的疏水芯中的氨基酸残基发生突变。
在另一优选例中，所述“天然MICA的α1α2结构域的疏水芯中的氨基酸残基”选自下组：9N、10L、14S、16D、25T、35L、39R、49Q、51A、52E、54T、70N、109Q、121Q、128W、147L、150D、153K、155K、158Y、159H、162H、和178V，其中氨基酸残基编号采用SEQ ID NO.1所示的编号。
在另一优选例中，所述“天然MICA的α1α2结构域的疏水芯中的氨基酸残基”包括178V、和/或162H，其中氨基酸残基编号采用SEQ ID NO.1所示的编号。
在另一优选例中，所述“天然MICA的α1α2结构域的疏水芯中的氨基酸残基”包括158Y、和/或147L，其中氨基酸残基编号采用SEQ ID NO.1所示的编号。
在另一优选例中，所述“天然MICA的α1α2结构域的疏水芯中的氨基酸残基”还包括121Q、和/或109Q，其中氨基酸残基编号采用SEQ ID NO.1所示的编号。
在另一优选例中，所述“天然MICA的α1α2结构域的疏水芯中的氨基酸残基”还包括128W，其中氨基酸残基编号采用SEQ ID NO.1所示的编号。
在另一优选例中，所述“天然MICA的α1α2结构域的疏水芯中的氨基酸残基”还包括35L、10L、和/或9N，其中氨基酸残基编号采用SEQ ID NO.1所示的编号。
在另一优选例中，所述“天然MICA的α1α2结构域的疏水芯中的氨基酸残基”还包括14S、25T、49Q、51A、52E、和/或54T，其中氨基酸残基编号采用SEQ ID NO.1所示的编号。
在另一优选例中，所述截短的MICA还包含选自下组的一个或多个突变后的氨基酸残基：9D，10F，14M或14I，16N，25A，35I，39T，49D，51E，52N，54Q，70W，109I或109H，121G、121M、121W或121N，128F，147W，150V，153E，155D，158W或158F，159D，162R，178I或178M，其中氨基酸残基编号采用SEQ ID NO.1所示的编号。
在另一优选例中，所述截短的MICA还包含突变后的氨基酸残基：162R，和/或178I或178M，其中氨基酸残基编号采用SEQ ID NO.1所示的编号。
在另一优选例中，所述截短的MICA还包含突变后的氨基酸残基：158W或158F，和/或147W，其中氨基酸残基编号采用SEQ ID NO.1所示的编号。
在另一优选例中，所述截短的MICA还包含突变后的氨基酸残基：121G、121M、121W或121N，和/或109I或109H，其中氨基酸残基编号采用SEQ ID NO.1所示的编号。
在另一优选例中，所述截短的MICA还包含突变后的氨基酸残基：128F，其中氨基酸残基编号采用SEQ ID NO.1所示的编号。
在另一优选例中，所述截短的MICA还包含突变后的氨基酸残基：35I、10F、和/或9D，其中氨基酸残基编号采用SEQ ID NO.1所示的编号。
在另一优选例中，所述截短的MICA还包含突变后的氨基酸残基：14M或14I、25A、49D、51E、52N、和/或54Q，其中氨基酸残基编号采用SEQ ID NO.1所示的编号。
在另一优选例中，所述截短的MICA还包含突变后的氨基酸残基：16N、39T、70W、150V、153E、155D、和/或159D，其中氨基酸残基编号采用SEQ ID NO.1所示的编号。
在另一优选例中，所述截短的MICA具有选自下组的氨基酸序列：SEQ ID NO.3、SEQ ID NO.5、SEQ ID NO.7、SEQ ID NO.9、SEQ ID NO.11、SEQ ID NO.13、SEQ ID NO.15、SEQ ID NO.17、SEQ ID NO.19、SEQ ID NO.21、SEQ ID NO.23、SEQ ID NO.25、SEQ ID NO.27、SEQ ID NO.29和SEQ ID NO.31。
在另一优选例中，所述截短的MICA是可溶的。
本发明的第二方面，提供了一种融合蛋白，所述融合蛋白具有本发明第一方面所述的截短的MICA。
在另一优选例中，所述融合蛋白具有任选的协助表达和/或纯化的标签序列。
在另一优选例中，所述融合蛋白还包括抗体序列和/或TCR序列。
本发明的第三方面，提供了一种核酸分子，所述核酸分子包含编码本发明第一方面所述的截短的MICA的多核苷酸序列或其互补序列。
本发明的第四方面，提供了一种载体，所述的载体含有本发明第三方面所述的核酸分子。
本发明的第五方面，提供了一种宿主细胞，所述的宿主细胞含有本发明第四方面所述的载体或染色体中整合有外源的本发明第三方面所述的核酸分子。
本发明的第六方面，提供了一种偶联物，所述偶联物包括
(a)如权利要求1所述的截短的MICA；和
(b)选自下组的偶联部分：可检测标记物、药物、毒素、细胞因子、放射性核素、或酶。
在另一优选例中，所述偶联部分选自：荧光或发光标记物、放射性标记物、MRI(磁共振成像)或CT(电子计算机X射线断层扫描技术)造影剂、或能够产生可检测产物的酶、放射性核素、生物毒素、细胞因子(如IL-2等)、抗体、抗体Fc片段、抗体scFv片段、金纳米颗粒/纳米棒、病毒颗粒、脂质体、纳米磁粒、前药激活酶(例如，DT-心肌黄酶(DTD)或联苯基水解酶-样蛋白质(BPHL))、化疗剂(例如，顺铂)或任何形式的纳米颗粒等
本发明的第七方面，提供了一种制备本发明第一方面所述的截短的MICA的方法，其特征在于，包括步骤：
1)培养本发明第五方面所述的宿主细胞，从而表达本发明第一方面所述的截短的MICA；
2)分离或纯化出本发明第一方面所述的截短的MICA。
本发明的第八方面，提供了本发明第一方面所述的截短的MICA、本发明第二方面所述的融合蛋白、本发明第六方面所述的偶联物的用途，用于制备药物或试剂；
所述药物用于治疗肿瘤或病毒感染；
所述试剂用于检测NKG2D。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇 幅，在此不再一一累述。
附图说明
图1显示了DMIC分子的疏水芯区域结构示意图。
图2a和2b分别为MICA分子的α1α2结构域对应的氨基酸序列与核苷酸序列(SEQ ID NO.1和2)。
图3a和3b分别为野生型DMIC的氨基酸序列与核苷酸序列(SEQ ID NO.3和4)，相对于MICA分子α1α2结构域对应的序列，暴露于表面的疏水性氨基酸残基发生了突变，突变后的氨基酸残基以黑体字和下划线显示。
图4a和4b分别为DMIC突变株B7的氨基酸序列与核苷酸序列(SEQ ID NO.5和6)，相对于野生型DMIC，突变后的氨基酸残基以黑体字和下划线显示。
图5a和5b分别为DMIC突变株C3的氨基酸序列与核苷酸序列(SEQ ID NO.7和8)，相对于野生型DMIC，突变后的氨基酸残基以黑体字和下划线显示。
图6a和6b分别为DMIC突变株C5的氨基酸序列与核苷酸序列(SEQ ID NO.9和10)，相对于野生型DMIC，突变后的氨基酸残基以黑体字和下划线显示。
图7a和7b分别为DMIC突变株D16的氨基酸序列与核苷酸序列(SEQ ID NO.11和12)，相对于野生型DMIC，突变后的氨基酸残基以黑体字和下划线显示。
图8a和8b分别为DMIC突变株M1的氨基酸序列与核苷酸序列(SEQ ID NO.13和14)，相对于野生型DMIC，突变后的氨基酸残基以黑体字和下划线显示。
图9a和9b分别为DMIC突变株M1a的氨基酸序列与核苷酸序列(SEQ ID NO.15和16)，相对于野生型DMIC，突变后的氨基酸残基以黑体字和下划线显示。
图10a和10b分别为DMIC突变株M2a的氨基酸序列与核苷酸序列(SEQ ID NO.17和18)，相对于野生型DMIC，突变后的氨基酸残基以黑体字和下划线显示。
图11a和11b分别为DMIC突变株M2b的氨基酸序列与核苷酸序列(SEQ ID NO.19和20)，相对于野生型DMIC，突变后的氨基酸残基以黑体字和下划线显示。
图12a和12b分别为DMIC突变株C3H7的氨基酸序列与核苷酸序列(SEQ ID NO.21和22)，相对于野生型DMIC，突变后的氨基酸残基以黑体字和下划线显示。
图13a和13b分别为DMIC突变株C5H7的氨基酸序列与核苷酸序列(SEQ ID NO.23和24)，相对于野生型DMIC，突变后的氨基酸残基以黑体字和下划线显示。
图14a和14b分别为DMIC突变株D16H7的氨基酸序列与核苷酸序列(SEQ ID NO.25和26)，相对于野生型DMIC，突变后的氨基酸残基以黑体字和下划线显示。
图15a和15b分别为DMIC突变株M1aH7的氨基酸序列与核苷酸序列(SEQ ID NO.27和28)，相对于野生型DMIC，突变后的氨基酸残基以黑体字和下划线显示。
图16a和16b分别为DMIC突变株M2aH7的氨基酸序列与核苷酸序列(SEQ ID NO.29和30)，相对于野生型DMIC，突变后的氨基酸残基以黑体字和下划线显示。
图17a和17b分别为DMIC突变株M2bH7的氨基酸序列与核苷酸序列(SEQ ID NO.31和32)，相对于野生型DMIC，突变后的氨基酸残基以黑体字和下划线显示。
图18为蛋白纯化的洗脱峰图。(a)MICA，(b)野生型DMIC，(c)H7，(d)突变体B7，(e)突变体C3，(f)突变体C5，(g)突变体D16，(h)突变体M1，(i)突变体M1a，(j)突变体M2a，(k)突变体M2b，(l)突变体C3H7，(m)突变体C5H7，(n)突变体D16H7，(o)突变体M1aH7，(p)突变体M2aH7，(q)突变体M2bH7。
图19为纯化后的蛋白的Non-reducing SDS-PAGE胶图。泳道1：分子量标记，泳道2：野生型DMIC，泳道3：突变体B7，泳道4：突变体C3，泳道5：突变体C5，泳道6：突变体D16，泳道7：突变体M1，泳道8：突变体M1a，泳道9：H7，泳道10：突变体C3H7，泳道11：突变体C5H7，泳道12：突变体D16H7，泳道13：突变体M1aH7，泳道14：分子量标记，泳道15：突变体M2a，泳道16：突变体M2b， 泳道17：突变体M2aH7，泳道18：突变体M2bH7，泳道19：MICA，泳道20：分子量标记。
图20为用Biacore SPR检测纯化后的MICA蛋白、DMIC蛋白与NKG2D相互作用的数据。
图21为用Biacore SPR检测纯化后的MICA蛋白、DMIC蛋白与NKG2D相互作用的图谱。(a)MICA，(b)野生型DMIC，(c)H7，(d)突变体B7，(e)突变体C3，(f)突变体C5，(g)突变体D16，(h)突变体M1，(i)突变体M1a，(j)突变体M2a，(k)突变体M2b，(l)突变体C3H7，(m)突变体C5H7，(n)突变体D16H7，(o)突变体M1aH7，(p)突变体M2aH7，(q)突变体M2bH7。
图22为纯化后的蛋白经过DSC检测，得到的Tm值和dH值。
图23为MICA和野生型DMIC的DSC原始曲线图。
图24为DMIC高稳定性和高亲和性突变体及H7的DSC拟合曲线图。
图25为流式检测生物素化的MICA蛋白，野生型DMIC蛋白，高亲和性DMIC蛋白B7，C3，C5，D16，M1，M1a与外周血单个核细胞(peripheral blood mononuclear cell，PBMC)表面NKG2D的结合。
具体实施方式
本发明人经过广泛而深入的研究发现，通过有针对性地对蛋白的疏水芯区域进行突变，可获得高稳定性和高亲和性的突变体，在此基础上完成了本发明。
本发明人将MICA的α3结构域(第182-275位氨基酸)去掉，产生了一个只含有MICA的α1α2结构域(第1-181位氨基酸)的新分子DMIC(SEQ ID NO.1和2，即截短的MICA)。将DMIC中暴露于表面的疏水性氨基酸替换成亲水性氨基酸后，便形成了野生型的DMIC(DMIC-WT，SEQ ID NO.3和4)。通过在DMIC-WT的疏水芯引入随机突变，用定向分子进化法及噬菌体展示技术，分离出了比DMIC-WT更稳定的突变体。本发明获得的突变体，除了稳定性比DMIC-WT有很大提高外，与NKG2D的结合亲和力也比DMIC-WT与NKG2D的结合亲和力提高了10-2600倍。将稳定性突变体上的突变位点分别移植入DMIC突变体H7(突变位点包括16N、39T、70W、150V、153E、155D、159D，其中氨基酸残基编号采用SEQ ID NO.3所示的编号)，与H7相比，移植了稳定性突变位点的突变体的稳定性进一步增强。
具体地，本发明人从MICA与NKG2D复合物的蛋白晶体结构(PDB ID:1HYR)分析得知，MICA(NCBI Accession Number:1HYR_C)包含3个结构域，分别是α1、α2和α3，且α3不参与跟NKG2D的结合。考虑到分子量较小的蛋白有利于后续的研究开发(如做成融合蛋白等)，本发明人将MICA的α3结构域(第182-275位氨基酸)去掉，形成了截短的MICA，进一步将暴露于表面的疏水性氨基酸残基替换为亲水性氨基酸残基(L13S；W15Q；L24Q；A51G；V54T；L88S；I94T；V96S；M130V；M141Q；L179P)，产生了一个只含有MICA的α1α2结构域(第1-181位氨基酸)的新分子，即野生型DMIC(DMIC-WT，SEQ ID NO.3和4)。
经过包涵体表达和蛋白体外复性，用Elisa检测不到DMIC-WT与NKG2D结合的信号，而用灵敏度较高的Biacore SPR则可以检测到DMIC-WT与NKG2D结合的信号，这一现象表明DMIC-WT在体外复性时，折叠成正确构象的蛋白比例较少，所以用Elisa检测不到其与NKG2D结合的信号。这很可能由于DMIC-WT的热稳定性不好，导致其体外复性效率较差。稳定性不好，复性效率差，势必影响到蛋白的后续研究，特别是功能上的研究，如与抗体或者TCR做成融合蛋白后，复性将会更加困难。因此，对DMIC进行稳定性优化，提高其表达和复性效率，显得至关重要。
蛋白质结构具有一个共同的特征，那就是分子内部都有疏水芯，是由紧密堆积的疏水侧链构成，疏水侧链的内埋使其屏蔽于水分子，是多肽链折叠的原始驱动力，也 是稳定蛋白质三维结构的主要因素。为了提高DMIC蛋白的热稳定性，改善其体外复性效率，本发明人在DMIC-WT的疏水芯区域引入随机突变，构建噬菌体文库，利用噬菌体展示技术对文库进行稳定性筛选。获得突变株后，发现得到的突变体不仅稳定性有很大提高，而且亲和力也提高了10-2600倍。进一步地，本发明人以DMIC-WT为模板，引入7个突变位点(D16N，R39T，N70W，D150V，K153E，K155D，H159D)，产生了新的DMIC突变体，命名为H7。然后将从噬菌体库筛选得到的稳定性突变体的突变位点分别移植入H7，结果表明，与H7相比，移植了稳定性突变位点的突变体的稳定性进一步提高。本发明提供了一种高稳定性、高亲和力的DMIC分子，为后续进一步研究DMIC分子的功能(如融合蛋白)提供了坚实的基础。
蛋白及其制备
在本发明中，“截短的MICA”和“DMIC”可互换使用，指衍生自MICA的包括MICA的α1α2结构域的蛋白，且蛋白中包含提高亲和力和稳定性的突变。本发明还提供了包括所述截短的MICA的融合蛋白，如分离的融合蛋白。本发明蛋白可以是单体或由单体形成的多聚体(如二聚体)。此外，应理解，所述术语还包括DMIC或其融合蛋白的活性片段和衍生物。
如本文所用，“分离的”是指物质从其原始环境中分离出来(如果是天然的物质，原始环境即是天然环境)。如活体细胞内的天然状态下的多核苷酸和多肽是没有分离纯化的，但同样的多核苷酸或多肽如从天然状态中同存在的其他物质中分开，则为分离纯化的。
如本文所用，“分离的融合蛋白”是指融合蛋白基本上不含天然与其相关的其它蛋白、脂类、糖类或其它物质。本领域的技术人员能用标准的蛋白质纯化技术纯化融合蛋白。基本上纯的蛋白在非还原聚丙烯酰胺凝胶上能产生单一的主带。
本发明的多核苷酸可以是DNA形式或RNA形式。DNA形式包括cDNA、基因组DNA或人工合成的DNA。DNA可以是单链的或是双链的。DNA可以是编码链或非编码链。
本发明还涉及上述多核苷酸的变异体，其编码与本发明有相同的氨基酸序列的蛋白质片段、类似物和衍生物。此多核苷酸的变异体可以是天然发生的等位变异体或非天然发生的变异体。这些核苷酸变异体包括取代变异体、缺失变异体和插入变异体。如本领域所知的，等位变异体是一个多核苷酸的替换形式，它可能是一个或多个核苷酸的取代、缺失或插入，但不会从实质上改变其编码多肽的功能。
如本文所用，术语“引物”指的是在与模板配对，在DNA聚合酶的作用下能以其为起点进行合成与模板互补的DNA链的寡核苷酸的总称。引物可以是天然的RNA、DNA，也可以是任何形式的天然核苷酸。引物甚至可以是非天然的核苷酸如LNA或ZNA等。引物“大致上”(或“基本上”)与模板上一条链上的一个特殊的序列互补。引物必须与模板上的一条链充分互补才能开始延伸，但引物的序列不必与模板的序列完全互补。比如，在一个3'端与模板互补的引物的5'端加上一段与模板不互补的序列，这样的引物仍大致上与模板互补。只要有足够长的引物能与模板充分的结合，非完全互补的引物也可以与模板形成引物-模板复合物，从而进行扩增。
本发明蛋白或其融合蛋白的核苷酸全长序列或其片段通常可以用PCR扩增法、重组法或人工合成的方法获得。对于PCR扩增法，可根据已公开的有关核苷酸序列，尤其是开放阅读框序列来设计引物，并用市售的cDNA库或按本领域技术人员已知的常规方法所制备的cDNA库作为模板，扩增而得到有关序列。当序列较长时，常常需要进行两次或多次PCR扩增，然后再将各次扩增出的片段按正确次序拼接在一起。
一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关 序列。
此外，还可用人工合成的方法来合成有关序列，尤其是片段长度较短时。通常，通过先合成多个小片段，然后再进行连接可获得序列很长的片段。
应用PCR技术扩增DNA/RNA的方法被优选用于获得本发明的基因。用于PCR的引物可根据本文所公开的本发明的序列信息适当地选择，并可用常规方法合成。可用常规方法如通过凝胶电泳分离和纯化扩增的DNA/RNA片段。
本发明也涉及包含本发明的多核苷酸的载体，以及用本发明的载体或融合蛋白编码序列经基因工程产生的宿主细胞，以及经重组技术产生本发明所述蛋白质的方法。
通过常规的重组DNA技术，可利用本发明的多核苷酸序列可用来表达或生产重组蛋白。一般来说有以下步骤：
(1)用本发明的编码本发明蛋白的多核苷酸(或变异体)，或用含有该多核苷酸的重组表达载体转化或转导合适的宿主细胞；
(2)在合适的培养基中培养的宿主细胞；
(3)从培养基或细胞中分离、纯化蛋白质。
本领域的技术人员熟知的方法能用于构建含本发明蛋白的编码DNA序列和合适的转录/翻译控制信号的表达载体。这些方法包括体外重组DNA技术、DNA合成技术、体内重组技术等。所述的DNA序列可有效连接到表达载体中的适当启动子上，以指导mRNA合成。表达载体还包括翻译起始用的核糖体结合位点和转录终止子。
此外，表达载体优选地包含一个或多个选择性标记基因，以提供用于选择转化的宿主细胞的表型性状，如真核细胞培养用的二氢叶酸还原酶、新霉素抗性以及绿色荧光蛋白(GFP)，或用于大肠杆菌的四环素或氨苄青霉素抗性。
包含上述的适当DNA序列以及适当启动子或者控制序列的载体，可以用于转化适当的宿主细胞，以使其能够表达蛋白质。
宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞。代表性例子有：大肠杆菌，链霉菌属的细菌细胞；真菌细胞如酵母；植物细胞；果蝇S2或Sf9的昆虫细胞；CHO、NS0、COS7、或293细胞的动物细胞等。
用重组DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。当宿主为原核生物如大肠杆菌时，能吸收DNA的感受态细胞可在指数生长期后收获，用CaCl2法处理，所用的步骤在本领域众所周知。另一种方法是使用MgCl2。如果需要，转化也可用电穿孔的方法进行。当宿主是真核生物，可选用如下的DNA转染方法：磷酸钙共沉淀法，常规机械方法如显微注射、电穿孔、脂质体包装等。
获得的转化子可以用常规方法培养，表达本发明的基因所编码的多肽。根据所用的宿主细胞，培养中所用的培养基可选自各种常规培养基。在适于宿主细胞生长的条件下进行培养。当宿主细胞生长到适当的细胞密度后，用合适的方法(如温度转换或化学诱导)诱导选择的启动子，将细胞再培养一段时间。
在上面的方法中的蛋白质可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。
如本文所用，术语“截短的MICA”还包括具有上述活性的变异形式。这些变异形式包括(但并不限于)：1-3个(通常为1-2个，更佳地1个)氨基酸的缺失、插入和/或取代，以及在C末端和/或N末端添加或缺失一个或数个(通常为3个以内，较佳地为2个以内，更佳地为1个以内)氨基酸。例如，在本领域中，用性能相近或相似的 氨基酸进行取代时，通常不会改变蛋白质的功能。又比如，在C末端和/或N末端添加或缺失一个或数个氨基酸通常也不会改变蛋白质的结构和功能。此外，所述术语还包括单体和多聚体形式的本发明多肽。该术语还包括线性以及非线性的多肽(如环肽)。
本发明还包括上述蛋白的活性片段、衍生物和类似物。如本文所用，术语“片段”、“衍生物”和“类似物”是指基本上保持本发明融合蛋白的功能或活性的多肽。本发明的多肽片段、衍生物或类似物可以是(i)有一个或几个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的多肽，或(ii)在一个或多个氨基酸残基中具有取代基团的多肽，或(iii)抗原肽与另一个化合物(比如延长多肽半衰期的化合物，例如聚乙二醇)融合所形成的多肽，或(iv)附加的氨基酸序列融合于此多肽序列而形成的多肽(与前导序列、分泌序列或6His等标签序列融合而形成的融合蛋白)。根据本文的教导，这些片段、衍生物和类似物属于本领域熟练技术人员公知的范围。
一类优选的活性衍生物指与式Ia或式Ib的氨基酸序列相比，有至多3个，较佳地至多2个，更佳地至多1个氨基酸被性质相似或相近的氨基酸所替换而形成多肽。这些保守性变异多肽最好根据表A进行氨基酸替换而产生。
表A
最初的残基 代表性的取代 优选的取代
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
本发明还提供本发明截短的MICA蛋白的类似物。这些类似物与本发明具体序列所示的多肽的差别可以是氨基酸序列上的差异，也可以是不影响序列的修饰形式上的差异，或者兼而有之。类似物还包括具有不同于天然L-氨基酸的残基(如D-氨基酸)的类似物，以及具有非天然存在的或合成的氨基酸(如β、γ-氨基酸)的类似物。应理解，本发明的多肽并不限于上述例举的代表性的多肽。
修饰(通常不改变一级结构)形式包括：体内或体外的多肽的化学衍生形式如乙酰化或羧基化。修饰还包括糖基化，如那些在多肽的合成和加工中或进一步加工步骤中进行糖基化修饰而产生的多肽。这种修饰可以通过将多肽暴露于进行糖基化的酶(如哺乳动物的糖基化酶或去糖基化酶)而完成。修饰形式还包括具有磷酸化氨基酸残基 (如磷酸酪氨酸，磷酸丝氨酸，磷酸苏氨酸)的序列。还包括被修饰从而提高了其抗蛋白水解性能或优化了溶解性能的多肽。
本发明多肽(融合蛋白)还可以以由药学上或生理学可接受的酸或碱衍生的盐形式使用。这些盐包括(但不限于)与如下酸形成的盐：氢氯酸、氢溴酸、硫酸、柠檬酸、酒石酸、磷酸、乳酸、丙酮酸、乙酸、琥珀酸、草酸、富马酸、马来酸、草酰乙酸、甲磺酸、乙磺酸、苯磺酸、或羟乙磺酸。其他盐包括：与碱金属或碱土金属(如钠、钾、钙或镁)形成的盐，以及以酯、氨基甲酸酯或其他常规的“前体药物”的形式。
本发明提供了的融合蛋白，它可任选地含有肽接头，通过肽键或肽接头连接根据本发明的截短的MICA和其他多肽元件(如抗体、TCR等)。肽接头大小和复杂性可能会影响蛋白的活性。通常，肽接头应当具有足够的长度和柔韧性，以保证连接的两个蛋白在空间上有足够的自由度以发挥其功能。同时避免肽接头中形成α螺旋或β折叠等对融合蛋白的稳定性的影响。
连接肽的长度一般为0-10个氨基酸，较佳地1-5个氨基酸。
药物组合物及施用方法
本发明还提供了一种组合物，它含有：(i)本发明的截短的MICA蛋白或可编码本发明的截短的MICA蛋白的多核苷酸，以及(ii)药学上上可接受的载体、或赋形剂。
本发明中，术语“含有”表示各种成分可一起应用于或存在于本发明的组合物中。因此，术语“主要由...组成”和“由...组成”包含在术语“含有”中。
本发明的组合物包括药物组合物。
本发明的药物组合物可制备成各种常规剂型，其中包括(但并不限于)：注射剂、粒剂、片剂、丸剂、栓剂、胶囊、悬浮液、喷雾剂等。
通常，可将本发明的融合蛋白配制于无毒的、惰性的和药学上可接受的水性载体介质中，其中pH通常约为5-8，较佳地，pH约为6-8。
如本文所用，术语“有效量”或“有效剂量”是指可对人和/或动物产生功能或活性的且可被人和/或动物所接受的量，如0.001-99wt％；较佳的0.01-95wt％；更佳的，0.1-90wt％。
如本文所用，“药学上可接受的”的成分是适用于人和/或哺乳动物而无过度不良副反应(如毒性、刺激和变态反应)的，即具有合理的效益/风险比的物质。术语“药学上可接受的载体”指用于治疗剂给药的载体，包括各种赋形剂和稀释剂。
本发明的药物组合物含有安全有效量的本发明的融合蛋白以及药学上可接受的载体。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。通常药物制剂应与给药方式相匹配，本发明的药物组合物可以被制成针剂形式，例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。所述的药物组合物宜在无菌条件下制造。活性成分的给药量是治疗有效量。本发明的药物制剂还可制成缓释制剂。
本发明截短的MICA蛋白的有效量可随给药的模式和待治疗的疾病的严重程度等而变化。优选的有效量的选择可以由本领域普通技术人员根据各种因素来确定(例如通过临床试验)。所述的因素包括但不限于：本发明融合蛋白的药代动力学参数例如生物利用率、代谢、半衰期等；患者所要治疗的疾病的严重程度、患者的体重、患者的免疫状况、给药的途径等。针对结核病患者，通常，当本发明的融合蛋白每天以约0.5mg-5mg/kg动物体重(较佳的2mg-4mg/kg动物体重)的剂量给予，能得到令人满意的效果。例如，由治疗状况的迫切要求，可每天给予若干次分开的剂量，或将剂量按比例地减少。
制备方法
产生本发明的DMIC分子的一种方法是从展示此DMIC分子的噬菌体颗粒的多样性文库中选择出高稳定性、高亲和性的DMIC突变体。
可采用任何合适的方法进行突变，包括但不限于依据聚合酶链式反应(PCR)、依据限制性酶的克隆或不依赖连接的克隆(LIC)方法。许多标准分子生物学教材详述了这些方法。聚合酶链式反应(PCR)诱变和依据限制性酶的克隆的更多细节可参见Sambrook和Russell,(2001)分子克隆-实验室手册(Molecular Cloning-A Laboratory Manual)(第三版)CSHL出版社。LIC方法的更多信息可见(Rashtchian,(1995)Curr Opin Biotechnol 6(1):30-6)。
本发明的DMIC分子可以是化学合成的，或重组的。相应地，本发明DMIC分子可用常规方法人工合成，也可用重组方法生产。
通过常规的重组DNA技术，可利用本发明的多核苷酸来表达或生产重组的DMIC。一般来说有以下步骤：
1)用编码本发明DMIC的多核苷酸(或变异体)，或用含有该多核苷酸的重组表达载体转化或转导合适的宿主细胞；
2)在合适的培养基中培养宿主细胞；
3)从培养基或细胞中分离、纯化出本发明的DMIC。
DMIC可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超声处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。
本发明DMIC的用途
本发明的高稳定性、高亲和力DMIC分子用途包括以下几点：
1)用于研究DMIC分子与NKG2D之间的相互作用，如DMIC分子与NKG2D的亲和力对激活NK细胞或T细胞的影响；
2)与抗体做成融合蛋白，制备治疗肿瘤或病毒感染的药物；
3)与T细胞受体(TCR)做成融合蛋白，制备治疗肿瘤或病毒感染的药物；
4)可用于免疫动物，制备抗DMIC或抗MICA的抗体；
5)可与荧光素偶联，用流式细胞仪检测细胞表面NKG2D的表达。本发明的主要优点包括：
1)本发明的DMIC分子稳定性高；
2)本发明的DMIC分子亲和力高；
3)可以高效、简便地筛选获得高稳定性、高亲和性的DMIC分子。
下面的具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook和Russell等人，分子克隆：实验室手册(Molecular Cloning-A Laboratory Manual)(第三版)(2001)CSHL出版社中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
实施例1野生型DMIC的构建和序列的优化
根据MICA与NKG2D复合物的晶体结构(PDB ID:1HYR)(Li等，(2001)，自然免疫学(Nat Immunol)2(5):443-451)，将MICA(NCBI Accession Number:1HYR_C)的α3结构域(第182-275位氨基酸)去掉，保留α1α2结构域(第1-181位氨基酸)，形成 初级DMIC分子(SEQ ID NO.1和2)。再根据晶体结构，将暴露于表面的疏水性氨基酸残基替换成亲水残基，替换情况为L13S，W15Q，L24Q，A51G，V54T，L88S，I94T，V96S，M130V，M141Q，L179P，从而得到野生型DMIC(DMIC-WT，SEQ ID NO.3和4)。将DMIC-WT的氨基酸序列优化成适于在大肠杆菌表达的核苷酸序列，基因合成后克隆到PUC57载体(Genscript)。
实施例2将DMIC-WT序列克隆入基于pET-28a的表达质粒
通过《分子克隆实验室手册》(Molecular Cloning a Laboratory Manual)(第三版，Sambrook和Russell)中描述的标准方法将DMIC-WT克隆入pET-28a表达质粒(购自Novagen)。将NcoI和NotI切割的编码DMIC-WT的DNA序列连接入用NcoI和NotI切割的pET-28a载体。将连接的质粒转化入常规的感受态大肠杆菌(Escherichia coli)菌株BL21(DE3)细胞(购自Vazyme),接种在含有50μg/mL卡那霉素的LB琼脂板上。37℃培养过夜后，挑取单个菌落，在含有50μg/mL卡那霉素的5ml LB培养基中37℃250rpm震荡培养过夜。将过夜培养菌液提质粒后测序。
实施例3 DMIC-WT的表达、复性和纯化
将实施例2中得到的含有DMIC-WT表达质粒的大肠杆菌菌株BL21(DE3)接种在含有50μg/mL卡那霉素的5ml LB培养基中，37℃250rpm震荡培养过夜。第2天，按体积比1:100转接到含有50μg/mL卡那霉素的400ml LB培养基中，37℃250rpm震荡培养至OD600为0.4-0.6(约2-3h)，加入终浓度0.7mM IPTG诱导表达4小时。5,000rpm离心20min收获细胞。用Bugbuster MasterMix(Merck)裂解细胞沉淀物，6,000g离心15min回收包涵体沉淀物。之后用10倍稀释的Bugbuster溶液三次洗涤包涵体，最后将包涵体溶解在6M盐酸胍缓冲液中。用BCA法定量后分装为12mg每管，在-80℃冷冻保存。
解冻12mg DMIC-WT包涵体，缓慢滴到200ml复性缓冲液中：100mM Tris.Cl，pH 8.5,400mM L-精氨酸，2mM EDTA，氧化还原对终浓度分别为1mM和10mM。溶液在4℃缓慢搅拌10分钟，装进分子截留量为3.5kD的透析袋，用5L纯水4-8℃缓慢搅拌透析复性过夜。第2天，用10mM Tris.Cl pH 8.5的缓冲液透析复性8小时，再更换相同的新鲜缓冲液透析过夜1次。
将透析完的复性蛋白液DMIC-WT离心后上阴离子交换柱QFF(5ml，GE)，利用GE AKTA蛋白纯化系统，用10mM Tris.Cl pH 8.5配制的1M NaCl线性梯度洗脱，收集洗脱峰，跑SDS-PAGE胶分析。包含DMIC-WT的组分用10mM Tris.Cl pH 8.5稀释10倍后，再次上阴离子交换柱QFF，用10mM Tris.Cl pH 8.5配制的1M NaCl线性梯度洗脱(图18(b))，跑SDS-PAGE胶分析鉴定(相对分子质量约22kD，图19)。
实施例4产生高稳定性和高亲和性DMIC突变体
在DMIC-WT疏水芯引入随机突变，构建4个噬菌体突变文库，用噬菌体展示技术对文库进行淘选。上述疏水芯文库构建及筛选方法可以参照Li等在((2005)Nature Biotech 23(3):349-354)中描述的高亲和性TCR噬菌体文库的构建及筛选方法。构建4个DMIC疏水芯突变文库时，设计的引物如下表(表1-4)所示。
表1构建DMIC疏水芯突变体文库1时所设计的引物
引物名称 引物序列(5'-3') SEQ ID NO
dmLA-F catgccatgGCCGATTACAAAGACGAAC 33
dmLA-F1 catgccatggccgattacaaagacgaaccgcatagcNtKcgttataatctgacc 34
dmLA-R1 ctgacgatcacaacgcagMaNcggctgaccatcMaNatgaacttcggtctg 35
dmLA-F2 ctgcgttgtgatcgtcag 36
dmLA-R2 cattaccggtcagatcacgggtttcacgatcMHWggttttattaccc 37
dmLA-F3 gtgatctgaccggtaatg 38
dmLA-R3 gctcagaaacagttcaccatcataatagaaatgMHDactgctacgggtgc 39
dmLA-F4 ggtgaactgtttctgagcHDKaatctggaaaccaaag 40
dmLA-R4 ctgcatgcattgcatgMHWatgggttttggttttc 41
dmLA-F5 catgcaatgcatgcagattg 42
dmLA-R5 cattttcagggatagcaagc 43
表2构建DMIC疏水芯突变体文库2时所设计的引物
Figure PCTCN2017107145-appb-000001
表3构建DMIC疏水芯突变体文库3时所设计的引物
Figure PCTCN2017107145-appb-000002
表4构建DMIC疏水芯突变体文库4时所设计的引物
Figure PCTCN2017107145-appb-000003
本发明中所用的简并碱基，如本领域技术人员熟知的那样，可以分别代表的碱基类型如下：B＝C或G或T；D＝A或G或T；H＝A或C或T；K＝G或T；M＝A或C；N＝A或C或G或T；R＝A或G；S＝C或G；V＝A或C或G；W＝A或T；Y＝C或T。
筛选条件优化：本发明人首先将DMIC-WT展示在丝状噬菌体的表面，通过噬菌体ELISA(酶联免疫吸附测定法)进行检测DMIC-WT与NKG2D的结合并得到其相互作用强度。为了得到最优的筛选条件，本发明人将DMIC-WT分别在以下12种条件下展示：26℃(IPTG浓度分别为0mM,0.2mM,0.5mM,1mM)，30℃(IPTG浓度分别为 0mM,0.2mM,0.5mM,1mM)，37℃(IPTG浓度分别为0mM,0.2mM,0.5mM,1mM)，然后通过噬菌体Elisa检测展示的DMIC-WT与NKG2D的结合，结果在37℃，1mM IPTG存在的条件下，DMIC-WT展示效果最差。为了让那些稳定性与DMIC-WT相当甚至比DMIC-WT差的突变体在表达过程中被直接淘汰，保留稳定性比DMIC-WT更好的突变体，即为了降低背景的表达，本发明人选择用37℃，1mM IPTG存在的条件做为后续突变文库的表达条件。为了进一步降低筛选背景，展示在噬菌体表面的DMIC突变体文库先在0.02％SDS存在的条件下，65℃水浴处理1h，冰上冷却15min后，再与NKG2D结合进行筛选。所以，为了让稳定性差的突变株在噬菌体展示过程中被淘汰，更稳定的突变株被筛选到，将筛选条件确定如下：1.在37℃展示DMIC突变文库；2.展示DMIC文库时加入1mM IPTG；3.筛选前将展示DMIC突变体的噬菌体在0.02％SDS存在的条件下，60-65℃孵育60min。
经测序鉴定，利用上述方法筛选到的50多个DMIC突变株的疏水芯均发生了突变。然后经过噬菌体Elisa鉴定，突变位点的组合，最终选定8个突变株做进一步鉴定，8个突变株分别为B7，C3，C5，D16，M1，M1a，M2a，M2b。其中B7，C3，C5，D16是直接筛选出来的突变株，M1，M1a，M2a，M2b是突变位点组合后的突变株。
噬菌体ELISA实验步骤：
1.分别接种DMIC-WT及各突变体至5mL 2xTY(100μg/mL氨苄青霉素，2％葡萄糖)，250rpm/min，37℃培养过夜。
2.分别转接培养过夜的菌液50μL至新鲜的5mL 2xTY(100μg/mL氨苄青霉素，2％葡萄糖)，250rpm/min 37℃培养至OD600＝0.4；用5μL(6.5x 1010)KM13辅助噬菌体感染，37℃静止30min后，250rpm/min 37℃振荡30min，离心后将沉淀重悬于30mL 2xTY(100μg/mL氨苄青霉素，50μg/mL卡那霉素，0.1％葡萄糖，1mM IPTG),250rpm/min 37℃培养过夜。
3.用10μg/mL链霉亲和素(PBS,pH＝7.4)包被免疫吸附板(NUNC),100μL每孔,4℃过夜。
4.离心收集过夜培养的菌液上清，用1/4体积比的PEG/NaCl沉淀上清液中的噬菌体，冰上放置1h，离心收集沉淀，重悬于3mL PBS。
5.用0.1％PBST洗板3次后，每孔加300μL 3％Marvel-PBS，37℃封闭1h；PBST洗板3次，每孔加100μL 10ug/mL的生物素化的NKG2D，室温放置1h；洗板3次，每孔加100μL噬菌体样品(10μL PEG沉淀的样品与3％的Marvel-PBS室温孵育1h)，室温放置1h；洗板3次后，每孔加100uL，anti-M13-HRP conjugate(GE Healthcare)(1:5000稀释于3％的Marvel-PBS中)，室温放置1h；洗板5次，每孔加100μL TMB，放置5min后，每孔加100μL 1M硫酸终止。用酶标仪读取OD 450nm的值。
实施例5 DMIC-WT及各突变体蛋白与NKG2D的结合活性分析
采用实施例3中所述的方法对野生型MICA，DMIC-WT及突变体B7，C3，C5，D16，M1，M1a，M2a，M2b进行表达、复性、纯化后跑SDS-PAGE胶。
使用BIAcore T200实时分析系统检测MICA，DMIC-WT及突变体B7，C3，C5，D16，M1，M1a，M2a，M2b与NKG2D的结合活性。将抗链霉亲和素的抗体加入偶联缓冲液(10mM醋酸钠缓冲液，pH 4.77)，然后将抗体流过预先用EDC和NHS活化过的CM5芯片，使抗体固定在芯片表面，最后用乙醇胺的盐酸溶液封闭未反应的活化表面，完成偶联过程，偶联水平约为15,000RU。
使低浓度的链霉亲和素流过已包被抗体的芯片表面，然后将生物素化的NKG2D流过检测通道，另一通道作为参比通道，再将0.05mM的生物素以10μL/min的流速 流过芯片2min，封闭链霉亲和素剩余的结合位点。采用单循环动力学分析方法测定其亲和力，将MICA，DMIC-WT及突变体B7，C3，C5，D16，M1，M1a，M2a，M2b用HEPES-EP缓冲液(10mM HEPES,150mMNaCl，3mM EDTA,0.005％P20，pH 7.4)稀释成几个不同的浓度，以30μL/min的流速，依次流过芯片表面，每次进样的结合时间为120s，最后一次进样结束后让其解离600s。每一轮测定结束后用pH1.75的10mM Gly-HCl再生芯片。利用BIAcore Evaluation软件计算动力学参数。
本实施例中所用的NKG2D，其表达、复性和纯化过程与实施例3中DMIC-WT的表达、复性和纯化过程相同。其生物素化的过程如下：
a.生物素化
用Millipore超滤管将纯化的NKG2D蛋白浓缩，同时将缓冲液置换为10mM Tris.Cl pH 8.0，然后加入生物素化试剂0.05M Bicine pH 8.3、10mM ATP、10mM MgOAc、50μM D-Biotin、100μg/ml BirA酶(GST-BirA)，20℃80rpm振荡孵育混合物过夜，SDS-PAGE检测生物素化是否完全。
b.纯化生物素化后的复合物
用Millipore超滤管将生物素化标记后的NKG2D蛋白浓缩至0.5ml，采用凝胶过滤层析纯化生物素化的NKG2D，利用AKTA蛋白纯化系统(GE)，用PBS平衡Superdex75 16/20柱(GE)，加载0.5ml浓缩过的生物素化NKG2D蛋白，继续用PBS以0.5ml/min流速平衡。生物素化的NKG2D蛋白在约10ml时作为单峰洗脱出现。合并含有蛋白质的组分，用Millipore超滤管浓缩，BCA法测定蛋白质浓度，将生物素化的NKG2D蛋白分装保存在-80℃。
通过本实施例所述过程检测到MICA与NKG2D蛋白结合的KD值为5.62E-07M；DMIC-WT与NKG2D蛋白结合的KD值为1.479E-07M；B7与NKG2D蛋白结合的KD值为1.177E-09M；C3与NKG2D蛋白结合的KD值为1.056E-10M；C5与NKG2D蛋白结合的KD值为5.489E-11M；D16与NKG2D蛋白结合的KD值为6.840E-11M；M1与NKG2D蛋白结合的KD值为6.740E-11M；M1a与NKG2D蛋白结合的KD值为1.751E-10M；M2a与NKG2D蛋白结合的KD值为2.137E-10M；M2b与NKG2D蛋白结合的KD值为4.435E-10M(图20)。图21为用Biacore SPR检测的各蛋白与NKG2D结合的曲线。结果表明，与DMIC-WT相比，各突变体与NKG2D的亲和力提高了10-2600倍。
实施例6 DMIC-WT及各突变体蛋白的稳定性测试
采用实施例3中所述的方法对野生型MICA，DMIC-WT及突变体B7，C3，C5，D16，M1，M1a，M2a，M2b进行表达、复性、纯化(图18)、跑SDS-PAGE胶鉴定(图19)。
利用美国TA(waters)公司的差示扫描量热仪(Nano DSC)测定上述纯化后的蛋白MICA，DMIC-WT与各突变体的Tm值和dH值。其扫描范围为10-100℃，升温速率为1℃/min，样品浓度为0.5mg/mL，上样量为900μL。其中，Tm值是通过分析软件Nanoanalyze的拟合模型TwostateScaled拟合而得到。
图19为按实施例3中所述经过2次阴离子交换柱纯化后得到的蛋白MICA，DMIC-WT及各突变体的SDS-PAGE胶图。
图22为蛋白MICA，DMIC-WT及各突变体的DSC数据。结果显示，MICA和DMIC-WT复性纯化后得到正确构象的蛋白含量极少，没有明显的蛋白去折叠吸热峰，利用分析软件Nanoanalyze得不到其Tm值和dH值，而经疏水芯突变后的突变体的Tm值和dH值如下：B7的Tm值为38.62℃，dH为430.34kJ/mol；C3的Tm值为45.78℃，dH为449.20kJ/mol；C5的Tm值为44.38℃，dH为484.33kJ/mol；D16出现了双峰，Tm值为43.50℃和61.16℃，dH为596.70kJ/mol和375.40kJ/mol；M1的Tm值为 40.81℃，dH为382.62kJ/mol；M1a的Tm值为41.60℃，dH为415.10kJ/mol；M2a的Tm值为43.48℃，dH为324.74kJ/mol；M2b的Tm值为43.61℃，dH为354.87kJ/mol。图23为MICA与DMIC-WT的DSC曲线，图24为各突变体的DSC拟合曲线。说明突变体B7，C3，C5，D16，M1，M1a，M2a，M2b相较于DMIC-WT更抗解折叠、更抗不适当或不希望的折叠以及热稳定性有显著提高。
实施例7 DMIC突变位点的移植
将实施例4中筛选到的高稳定性突变体C3，C5，D16，M1a，M2a，M2b的突变位点分别移植到DMIC突变体H7(与DMIC-WT相比，含7个突变位点：D16N，R39T，N70W，D150V，K153E，K155D，H159D)，构建了6个新的突变体，分别是C3H7，C5H7，D16H7，M1aH7，M2aH7，M2bH7，并对6个分子进行蛋白表达，复性，纯化，Biacore SPR亲和力检测，DSC稳定性测定。
采用实施例3中所述的方法对H7，C3H7，C5H7，D16H7，M1aH7，M2aH7，M2bH7，进行表达、复性、纯化(图18)、跑SDS-PAGE胶(图19)鉴定。同时计算其表达量和蛋白复性收率。其中，表达量为1L大肠杆菌诱导表达并提纯后的包涵体的产量。蛋白复性收率的计算式如下：蛋白复性收率(％)＝100*纯化后所得蛋白量(mg)/复性所用包涵体的量(mg)。在本发明中提及的表达量和蛋白复性收率均按上述计算方法进行计算，有特别说明的除外。下表(表5)为H7引入稳定性疏水芯前后的蛋白表达量和蛋白复性收率。
表5DMIC蛋白表达量及复性收率
蛋白名称 表达量(mg/L) 蛋白复性收率(％)
H7 406 14.7
C3H7 607 41
C5H7 476 34
D16H7 420 28.6
M1aH7 657 28.5
M2aH7 443 17.5
M2bH7 473 16.6
由上表数据可知，从蛋白表达量看，C3H7，C5H7，D16H7，M1aH7，M2aH7，M2bH7的表达量都比H7高。从复性收率看，M2aH7和M2bH7的复性得率比H7略高，而C3H7，C5H7，D16H7，M1aH7的复性收率较H7有很大的提高。
采用实施例5中所述的方法，用Biacore SPR检测H7，C3H7，C5H7，D16H7，M1aH7，M2aH7，M2bH7与NKG2D的结合活性。H7与NKG2D蛋白结合的KD值为3.556E-09M；C3H7与NKG2D蛋白结合的KD值为2.872E-09M；C5H7与NKG2D蛋白结合的KD值为1.736E-09M；D16H7与NKG2D蛋白结合的KD值为1.478E-08M；M1aH7与NKG2D蛋白结合的KD值为4.264E-09M；M2aH7与NKG2D蛋白结合的KD值为1.967E-09M；M2bH7与NKG2D蛋白结合的KD值为5.121E-09M(图20)。图21为用Biacore SPR检测的各蛋白与NKG2D的结合曲线。以上数据表明，除了D16H7与NKG2D的亲和力比H7与NKG2D的亲和力低外，C3H7，C5H7，M1aH7，M2aH7，M2bH7与NKG2D的亲和力跟H7与NKG2D的亲和力基本相当。
采用实施例6中所述的方法，用DSC检测H7，C3H7，C5H7，D16H7，M1aH7，M2aH7，M2bH7的热稳定性。如图22，H7的Tm值为48.40℃，dH为335kJ/mol；C3H7的Tm值为55.10℃，dH为270kJ/mol；C5H7的Tm值为53.90℃，dH为233kJ/mol；D16H7的Tm值为50.2℃，dH为304kJ/mol；M1aH7的Tm值为50.4℃，dH为367kJ/mol；M2aH7有双峰，Tm值分别为55.57℃和84.84℃，dH为216.39kJ/mol和269.92kJ/mol；M2bH7有双峰，Tm值分别为57.04℃和85.49℃，dH为223.28kJ/mol和252.67kJ/mol。 图24为DMIC各蛋白的DSC拟合曲线。以上数据表明，C3H7，C5H7，D16H7，M1aH7，M2aH7，M2bH7的Tm值都较H7高。
通过蛋白表达量、蛋白复性收率及Tm值的对比分析可知，经过疏水芯优化的C3H7，C5H7，D16H7，M1aH7，M2aH7，M2bH7相较于疏水芯未经改造的H7，蛋白表达量更高，复性能力更强、更抗解折叠、更抗不适当或不希望的折叠、蛋白复性收率更高以及热稳定性有显著提高。因此，C3H7，C5H7，D16H7，M1aH7，M2aH7，M2bH7相较于H7稳定性有显著提高。
本发明筛选到的DMIC突变体稳定性和亲和力都显著高于DMIC-WT。同时，将DMIC突变体的突变位点分别移植到H7，也都能够起到增强稳定性的作用。
实施例8流式检测MICA，野生型DMIC和高亲和性DMIC蛋白与PBMC表面NKG2D的结合
使用生物素化的MICA蛋白，野生型DMIC蛋白和高亲和性DMIC蛋白B7，C3，C5，D16，M1，M1a对PBMC进行染色(图25)。实验分为直接染色法和间接染色法。
间接法是将1ug高亲和性DMIC蛋白C5和1uL PE mouse anti-human NKG2D同时对PBMC染色30min并检测，与单独染PE mouse anti-human NKG2D的PBMC样品进行对比。结果表明，高亲和性DMIC C5可以抑制anti-NKG2D抗体与PBMC表面的NKG2D结合，导致anti-NKG2D抗体与PBMC结合的阳性率从55.9％下降到2.18％(图25A)。说明高亲和性DMIC C5可以与PBMC表面的NKG2D很好的结合。
直接法是先将生物素化的MICA蛋白，野生型DMIC蛋白和高亲和性DMIC蛋白B7，C3，C5，D16，M1，M1a分别对PBMC进行染色，PBS清洗3次后，再分别加入0.5uL的SA-PE进行染色，最后用流式细胞仪检测。结果表明，检测的蛋白都能很好的与PBMC结合，且随着蛋白浓度的增加，阳性率增加；随着蛋白亲和力的增加，阳性率也增加。其中亲和力达到10-11M级别的高亲和性DMIC D16，M1，C5的阳性率与anti-NKG2D抗体染色的阳性率相当(图25B)。
本发明筛选到的高稳定性、高亲和力DMIC突变体，为后续研究DMIC的功能(如研究DMIC与NKG2D的亲和力对激活NK细胞或T细胞的影响)，开发DMIC相关的药物(如与抗体或TCR做成融合蛋白，制备治疗肿瘤或病毒感染的药物等)或试剂，提供了坚定的基础。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种截短的MICA，其特征在于，所述截短的MICA包括突变的MICA的α1α2结构域，所述“突变”包括天然MICA的α1α2结构域的氨基酸序列中一个或多个暴露于表面的疏水性氨基酸残基突变为亲水性氨基酸残基；
    优选地，所述“暴露于表面的疏水性氨基酸残基”选自下组：13L、15W、24L、51A、54V、88L、94I、96V、130M、141M、和179L，其中氨基酸残基编号采用SEQ ID NO.1所示的编号。
  2. 如权利要求1所述的截短的MICA，其特征在于，所述“突变”还包括一个或多个(优选为2-30个，更优选为2-25个)天然MICA的α1α2结构域的疏水芯中的氨基酸残基发生突变；
    优选地，所述“天然MICA的α1α2结构域的疏水芯中的氨基酸残基”选自下组：9N、10L、14S、16D、25T、35L、39R、49Q、51A、52E、54T、70N、109Q、121Q、128W、147L、150D、153K、155K、158Y、159H、162H、和178V，其中氨基酸残基编号采用SEQ ID NO.1所示的编号。
  3. 如权利要求2所述的截短的MICA，其特征在于，所述“天然MICA的α1α2结构域的疏水芯中的氨基酸残基”包括178V、和/或162H，其中氨基酸残基编号采用SEQ ID NO.1所示的编号；和/或
    所述“天然MICA的α1α2结构域的疏水芯中的氨基酸残基”包括158Y、和/或147L，其中氨基酸残基编号采用SEQ ID NO.1所示的编号；
    优选地，所述“天然MICA的α1α2结构域的疏水芯中的氨基酸残基”还包括121Q、和/或109Q，其中氨基酸残基编号采用SEQ ID NO.1所示的编号；
    最优选地，所述截短的MICA具有选自下组的氨基酸序列：SEQ ID NO.3、SEQ ID NO.5、SEQ ID NO.7、SEQ ID NO.9、SEQ ID NO.11、SEQ ID NO.13、SEQ ID NO.15、SEQ ID NO.17、SEQ ID NO.19、SEQ ID NO.21、SEQ ID NO.23、SEQ ID NO.25、SEQ ID NO.27、SEQ ID NO.29和SEQ ID NO.31。
  4. 一种融合蛋白，其特征在于，所述融合蛋白具有权利要求1所述的截短的MICA。
  5. 一种核酸分子，其特征在于，所述核酸分子包含编码权利要求1中所述的截短的MICA的多核苷酸序列或其互补序列。
  6. 一种载体，其特征在于，所述的载体含有权利要求5所述的核酸分子。
  7. 一种宿主细胞，其特征在于，所述的宿主细胞含有权利要求6所述的载体或染色体中整合有外源的权利要求5所述的核酸分子。
  8. 一种偶联物，其特征在于，所述偶联物包括
    (a)如权利要求1所述的截短的MICA；和
    (b)选自下组的偶联部分：可检测标记物、药物、毒素、细胞因子、放射性核素、或酶。
  9. 一种制备权利要求1所述的截短的MICA的方法，其特征在于，包括步骤：
    1)培养权利要求7所述的宿主细胞，从而表达权利要求1所述的截短的MICA；
    2)分离或纯化出权利要求1所述的截短的MICA。
  10. 权利要求1所述的截短的MICA、权利要求4所述的融合蛋白、权利要求8所述的偶联物的用途，其特征在于，用于制备药物或试剂；
    所述药物用于治疗肿瘤或病毒感染；
    所述试剂用于检测NKG2D。
PCT/CN2017/107145 2016-10-20 2017-10-20 高稳定性和高亲和力的dmic及其制法 WO2018072751A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201780065101.9A CN109890837B (zh) 2016-10-20 2017-10-20 高稳定性和高亲和力的dmic及其制法

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201610918179.8A CN107964041B (zh) 2016-10-20 2016-10-20 高稳定性和高亲和力的dmic及其制法
CN201610918179.8 2016-10-20

Publications (1)

Publication Number Publication Date
WO2018072751A1 true WO2018072751A1 (zh) 2018-04-26

Family

ID=61996493

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2017/107145 WO2018072751A1 (zh) 2016-10-20 2017-10-20 高稳定性和高亲和力的dmic及其制法

Country Status (2)

Country Link
CN (2) CN107964041B (zh)
WO (1) WO2018072751A1 (zh)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109557064A (zh) * 2018-12-30 2019-04-02 长春中医药大学 一种通过荧光金纳米簇探针检测丙酮酸及其浓度的方法、检测丙酮酸氧化酶及其浓度的方法
CN111471099B (zh) * 2020-03-31 2022-04-12 长沙学院 MICA胞外区突变体及其筛选的方法、scFv-MICA融合抗体及其制备方法和应用

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016090278A2 (en) * 2014-12-05 2016-06-09 Avidbiotics Corp. Insertable variable fragments of antibodies and modified a1-a2 domains of nkg2d ligands

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB0403491D0 (en) * 2004-02-17 2004-03-24 Univ Cambridge Tech Polypeptides, methods and means
US8753640B2 (en) * 2011-05-31 2014-06-17 University Of Washington Through Its Center For Commercialization MIC-binding antibodies and methods of use thereof
WO2013117647A1 (en) * 2012-02-07 2013-08-15 Innate Pharma Mica binding agents
JP6518199B6 (ja) * 2013-03-15 2019-06-12 ノヴェロジックス・バイオテクノロジー，インコーポレーテッド Ｍｉｃａおよびｍｉｃｂタンパク質に対する抗体
NZ715815A (en) * 2013-06-26 2017-02-24 Guangzhou Xiangxue Pharmaceutical Co Ltd High-stability t-cell receptor and preparation method and application thereof

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016090278A2 (en) * 2014-12-05 2016-06-09 Avidbiotics Corp. Insertable variable fragments of antibodies and modified a1-a2 domains of nkg2d ligands

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LENGYEL, C.S.E. ET AL.: "Mutations Designed to Destabilize the Receptor-Bound Conformation Increase MICA-NKG2D Association Rate and Affinity", J. BIOL. CHEM., vol. 282, no. 42, 19 October 2007 (2007-10-19), XP055432102 *

Also Published As

Publication number Publication date
CN107964041A (zh) 2018-04-27
CN109890837A (zh) 2019-06-14
CN107964041B (zh) 2023-08-01
CN109890837B (zh) 2023-09-08

Similar Documents

Publication Publication Date Title
RU2645256C2 (ru) Высокостабильный т-клеточный рецептор и способ его получения и применения
EP1012280B1 (en) Trimerising module
JP6346159B2 (ja) 所定の標的に対して親和性を有するヒトリポカリン２（Ｌｃｎ２、ｈＮＧＡＬ）の突然変異タンパク質
WO2021083363A1 (zh) 一种识别Kras G12V的高亲和力TCR
WO2018072751A1 (zh) 高稳定性和高亲和力的dmic及其制法
WO2016058547A1 (zh) 高活性的肿瘤抑制剂及其制法和应用
WO2015055148A1 (zh) Yap蛋白抑制多肽及其应用
WO2020182082A1 (zh) 一种识别afp抗原的高亲和力tcr
WO2020098717A1 (zh) 一种识别afp的高亲和力tcr
CN107223133B (zh) 一种可溶的异质二聚t细胞受体及其制法和应用
CN105985444B (zh) 一种嵌合抗原受体、以及快速构建嵌合抗原受体的方法及应用
WO2023179768A1 (zh) 一种识别mage-a4抗原的高亲和力tcr及其序列和应用
CN112480262B (zh) 一种融合蛋白及其制备与应用
WO2023221959A1 (zh) 一种识别mage的高亲和力t细胞受体及其应用
JPH11508453A (ja) Ｉｌ―１６活性を有するプロセスされたポリペプチド、それらの製法、及びそれらの使用
JP2776480B2 (ja) 組み換え型ヒトリンホトキシン
KR20220087511A (ko) 특이적 결합 분자

Legal Events

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

Ref document number: 17863213

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 17863213

Country of ref document: EP

Kind code of ref document: A1