WO2022262100A1 - 具有增强的adcp效应的抗cd70抗体及其应用 - Google Patents
具有增强的adcp效应的抗cd70抗体及其应用 Download PDFInfo
- Publication number
- WO2022262100A1 WO2022262100A1 PCT/CN2021/112518 CN2021112518W WO2022262100A1 WO 2022262100 A1 WO2022262100 A1 WO 2022262100A1 CN 2021112518 W CN2021112518 W CN 2021112518W WO 2022262100 A1 WO2022262100 A1 WO 2022262100A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- seq
- acid sequence
- amino acid
- cdr1
- cdr2
- Prior art date
Links
- 230000000694 effects Effects 0.000 title claims abstract description 23
- 101000934356 Homo sapiens CD70 antigen Proteins 0.000 claims abstract description 43
- 230000005888 antibody-dependent cellular phagocytosis Effects 0.000 claims abstract description 34
- 102100025221 CD70 antigen Human genes 0.000 claims abstract description 31
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 10
- 239000003814 drug Substances 0.000 claims abstract description 4
- 150000001413 amino acids Chemical class 0.000 claims description 80
- 238000002360 preparation method Methods 0.000 claims description 3
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical group C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims description 2
- 108020004414 DNA Proteins 0.000 claims description 2
- 102000053602 DNA Human genes 0.000 claims description 2
- 239000002773 nucleotide Substances 0.000 claims description 2
- 125000003729 nucleotide group Chemical group 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 75
- 210000004027 cell Anatomy 0.000 abstract description 47
- 241000282567 Macaca fascicularis Species 0.000 abstract description 9
- 108020001507 fusion proteins Proteins 0.000 abstract description 4
- 102000037865 fusion proteins Human genes 0.000 abstract description 4
- 230000019491 signal transduction Effects 0.000 abstract description 4
- 229940079593 drug Drugs 0.000 abstract description 3
- 238000009169 immunotherapy Methods 0.000 abstract description 3
- 238000001514 detection method Methods 0.000 description 17
- 102000053826 human CD70 Human genes 0.000 description 12
- 239000006228 supernatant Substances 0.000 description 12
- 239000007788 liquid Substances 0.000 description 11
- 239000011324 bead Substances 0.000 description 10
- 108700008625 Reporter Genes Proteins 0.000 description 9
- 210000002540 macrophage Anatomy 0.000 description 8
- 239000000203 mixture Substances 0.000 description 7
- 238000004091 panning Methods 0.000 description 7
- 108090000623 proteins and genes Proteins 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 6
- 239000001963 growth medium Substances 0.000 description 6
- 239000002609 medium Substances 0.000 description 6
- 238000002965 ELISA Methods 0.000 description 5
- 230000004913 activation Effects 0.000 description 5
- 239000000427 antigen Substances 0.000 description 5
- 108091007433 antigens Proteins 0.000 description 5
- 102000036639 antigens Human genes 0.000 description 5
- 238000000684 flow cytometry Methods 0.000 description 5
- 150000007523 nucleic acids Chemical group 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 5
- 108091028043 Nucleic acid sequence Proteins 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
- 239000012636 effector Substances 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 238000000034 method Methods 0.000 description 4
- 239000013642 negative control Substances 0.000 description 4
- 239000002245 particle Substances 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 3
- 108060001084 Luciferase Proteins 0.000 description 3
- 239000005089 Luciferase Substances 0.000 description 3
- 239000012980 RPMI-1640 medium Substances 0.000 description 3
- 108010090804 Streptavidin Proteins 0.000 description 3
- 229960000723 ampicillin Drugs 0.000 description 3
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 3
- 229960002685 biotin Drugs 0.000 description 3
- 235000020958 biotin Nutrition 0.000 description 3
- 239000011616 biotin Substances 0.000 description 3
- 239000002981 blocking agent Substances 0.000 description 3
- 230000000903 blocking effect Effects 0.000 description 3
- 239000006285 cell suspension Substances 0.000 description 3
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000013604 expression vector Substances 0.000 description 3
- 238000002823 phage display Methods 0.000 description 3
- 239000013612 plasmid Substances 0.000 description 3
- 239000013641 positive control Substances 0.000 description 3
- 238000002560 therapeutic procedure Methods 0.000 description 3
- 102000007469 Actins Human genes 0.000 description 2
- 108010085238 Actins Proteins 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 2
- 108010021468 Fc gamma receptor IIA Proteins 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 102100029204 Low affinity immunoglobulin gamma Fc region receptor II-a Human genes 0.000 description 2
- 229920001213 Polysorbate 20 Polymers 0.000 description 2
- 108010076504 Protein Sorting Signals Proteins 0.000 description 2
- 108010003723 Single-Domain Antibodies Proteins 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 102000004142 Trypsin Human genes 0.000 description 2
- 108090000631 Trypsin Proteins 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 239000003085 diluting agent Substances 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 2
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 229930027917 kanamycin Natural products 0.000 description 2
- 229960000318 kanamycin Drugs 0.000 description 2
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 2
- 229930182823 kanamycin A Natural products 0.000 description 2
- 239000010413 mother solution Substances 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 230000000242 pagocytic effect Effects 0.000 description 2
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 2
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 235000020183 skimmed milk Nutrition 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 239000012588 trypsin Substances 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- IVLXQGJVBGMLRR-UHFFFAOYSA-N 2-aminoacetic acid;hydron;chloride Chemical compound Cl.NCC(O)=O IVLXQGJVBGMLRR-UHFFFAOYSA-N 0.000 description 1
- 102100027207 CD27 antigen Human genes 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 101000914511 Homo sapiens CD27 antigen Proteins 0.000 description 1
- 206010057249 Phagocytosis Diseases 0.000 description 1
- 229920002594 Polyethylene Glycol 8000 Polymers 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- 229930003451 Vitamin B1 Natural products 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 229940125644 antibody drug Drugs 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000007969 cellular immunity Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 230000007012 clinical effect Effects 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 230000003750 conditioning effect Effects 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000037406 food intake Effects 0.000 description 1
- 230000005965 immune activity Effects 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 230000003832 immune regulation Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 238000003670 luciferase enzyme activity assay Methods 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 210000000822 natural killer cell Anatomy 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 230000003472 neutralizing effect Effects 0.000 description 1
- 210000000440 neutrophil Anatomy 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 230000008782 phagocytosis Effects 0.000 description 1
- 230000006461 physiological response Effects 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 210000001243 pseudopodia Anatomy 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 230000008054 signal transmission Effects 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 230000007781 signaling event Effects 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 238000002626 targeted therapy Methods 0.000 description 1
- 229960003495 thiamine Drugs 0.000 description 1
- DPJRMOMPQZCRJU-UHFFFAOYSA-M thiamine hydrochloride Chemical compound Cl.[Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N DPJRMOMPQZCRJU-UHFFFAOYSA-M 0.000 description 1
- 239000012096 transfection reagent Substances 0.000 description 1
- 239000011691 vitamin B1 Substances 0.000 description 1
- 235000010374 vitamin B1 Nutrition 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2875—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF/TNF superfamily, e.g. CD70, CD95L, CD153, CD154
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
-
- 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
- G01N2333/70575—NGF/TNF-superfamily, e.g. CD70, CD95L, CD153 or CD154
-
- 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
- This application belongs to biomedical technology, and in particular relates to an anti-CD70 antibody with enhanced ADCP effect and its application.
- ADCP Antibody-dependent cellular phagocytosis
- macrophages and neutrophils have this function, among which the phagocytic activity of macrophages is relatively high. Strong, is the main effector cells of ADCP.
- macrophages themselves have natural immune activity, and after activation, they can play an immune conditioning role and enhance the overall immune function.
- solid tumors themselves have cell and tissue differentiation and compact structure, so it is difficult for CART cells to infiltrate, and the phagocytic function of macrophages can play a role in dissolving its structure. Therefore, ADCP has more special practical significance for the treatment of solid tumors. In fact, due to the bottleneck encountered in the application of CART therapy in solid tumors, the use of this characteristic of macrophages for targeted therapy has attracted the attention of researchers.
- the antigen binds to the antibody, and then the crystallizable fragment (fragment crystallizable, Fc) of the antibody mainly binds to Fc ⁇ IIA (Fc gamma receptorIIA) on the surface of the macrophage, and the antigen (particle) binds to the surface of the macrophage, causing actin at the ingestion site Polymerization and pseudopodia wrap particles, and actin depolymerization after phagocytosis is complete. During this process, the NFAT signaling pathway in macrophages is activated. Using this early onset signaling event, ADCP effects can be detected.
- ADCP has aroused great interest in tumor therapy
- the ADCP effect is generally weak in the existing antibody drugs.
- the regulation of CD70 will inevitably cause a series of signal transmission and physiological responses of lymphocytes such as T cells, even B cells and NK cells, which is an ideal immunotherapy target.
- lymphocytes such as T cells, even B cells and NK cells, which is an ideal immunotherapy target.
- Proper application has the potential to mobilize a variety of cellular immunity. This application targets human CD70 to develop an antibody with strong ADCP effect.
- the current CD70-targeting therapies include CART and all-antibody, but the clinical effect is limited, especially for solid tumors, these two methods are almost ineffective.
- increasing the type of antibody mechanism is a way to improve the therapeutic effect of antibodies.
- the ADCP effect of antibodies in clinical trials is still weak, and it is still insufficient to enhance the therapeutic effect.
- the amino acid sequence of CDR1 of the heavy chain variable region is selected from SEQ ID NO: 1-24
- the amino acid sequence of CDR2 is selected from SEQ ID NO : 25-48
- the amino acid sequence of CDR3 is selected from SEQ ID NO: 49-72.
- the anti-CD70 Nanobody with enhanced ADCP effect comprises any of the following heavy chain variable regions:
- the amino acid sequence of CDR1 is selected from SEQ ID NO: 1
- the amino acid sequence of CDR2 is selected from SEQ ID NO: 25
- the amino acid sequence of CDR3 is selected from SEQ ID NO: 49;
- the amino acid sequence of CDR1 is selected from SEQ ID NO: 2
- the amino acid sequence of CDR2 is selected from SEQ ID NO: 26
- the amino acid sequence of CDR3 is selected from SEQ ID NO: 50;
- the amino acid sequence of CDR1 is selected from SEQ ID NO: 3
- the amino acid sequence of CDR2 is selected from SEQ ID NO: 27
- the amino acid sequence of CDR3 is selected from SEQ ID NO: 51;
- the amino acid sequence of CDR1 is selected from SEQ ID NO: 4
- the amino acid sequence of CDR2 is selected from SEQ ID NO: 28
- the amino acid sequence of CDR3 is selected from SEQ ID NO: 52;
- amino acid sequence of CDR1 is selected from SEQ ID NO:5
- amino acid sequence of CDR2 is selected from SEQ ID NO:29
- amino acid sequence of CDR3 is selected from SEQ ID NO:53;
- the amino acid sequence of CDR1 is selected from SEQ ID NO: 6, the amino acid sequence of CDR2 is selected from SEQ ID NO: 30, and the amino acid sequence of CDR3 is selected from SEQ ID NO: 54;
- the amino acid sequence of CDR1 is selected from SEQ ID NO: 7
- the amino acid sequence of CDR2 is selected from SEQ ID NO: 31
- the amino acid sequence of CDR3 is selected from SEQ ID NO: 55;
- the amino acid sequence of CDR1 is selected from SEQ ID NO: 8
- the amino acid sequence of CDR2 is selected from SEQ ID NO: 32
- the amino acid sequence of CDR3 is selected from SEQ ID NO: 56;
- the amino acid sequence of CDR1 is selected from SEQ ID NO: 9
- the amino acid sequence of CDR2 is selected from SEQ ID NO: 33
- the amino acid sequence of CDR3 is selected from SEQ ID NO: 57;
- the amino acid sequence of CDR1 is selected from SEQ ID NO: 10
- the amino acid sequence of CDR2 is selected from SEQ ID NO: 34
- the amino acid sequence of CDR3 is selected from SEQ ID NO: 58;
- the amino acid sequence of CDR1 is selected from SEQ ID NO: 11
- the amino acid sequence of CDR2 is selected from SEQ ID NO: 35
- the amino acid sequence of CDR3 is selected from SEQ ID NO: 59;
- the amino acid sequence of CDR1 is selected from SEQ ID NO: 12
- the amino acid sequence of CDR2 is selected from SEQ ID NO: 36
- the amino acid sequence of CDR3 is selected from SEQ ID NO: 60;
- the amino acid sequence of CDR1 is selected from SEQ ID NO: 13
- the amino acid sequence of CDR2 is selected from SEQ ID NO: 37
- the amino acid sequence of CDR3 is selected from SEQ ID NO: 61;
- the amino acid sequence of CDR1 is selected from SEQ ID NO: 14, the amino acid sequence of CDR2 is selected from SEQ ID NO: 38, and the amino acid sequence of CDR3 is selected from SEQ ID NO: 62;
- the amino acid sequence of CDR1 is selected from SEQ ID NO: 15, the amino acid sequence of CDR2 is selected from SEQ ID NO: 39, and the amino acid sequence of CDR3 is selected from SEQ ID NO: 63;
- the amino acid sequence of CDR1 is selected from SEQ ID NO: 16
- the amino acid sequence of CDR2 is selected from SEQ ID NO: 40
- the amino acid sequence of CDR3 is selected from SEQ ID NO: 64;
- the amino acid sequence of CDR1 is selected from SEQ ID NO: 17
- the amino acid sequence of CDR2 is selected from SEQ ID NO: 41
- the amino acid sequence of CDR3 is selected from SEQ ID NO: 65;
- the amino acid sequence of CDR1 is selected from SEQ ID NO: 18, the amino acid sequence of CDR2 is selected from SEQ ID NO: 42, and the amino acid sequence of CDR3 is selected from SEQ ID NO: 66;
- the amino acid sequence of CDR1 is selected from SEQ ID NO: 19
- the amino acid sequence of CDR2 is selected from SEQ ID NO: 43
- the amino acid sequence of CDR3 is selected from SEQ ID NO: 67;
- the amino acid sequence of CDR1 is selected from SEQ ID NO: 20
- the amino acid sequence of CDR2 is selected from SEQ ID NO: 44
- the amino acid sequence of CDR3 is selected from SEQ ID NO: 68;
- the amino acid sequence of CDR1 is selected from SEQ ID NO: 21, the amino acid sequence of CDR2 is selected from SEQ ID NO: 45, and the amino acid sequence of CDR3 is selected from SEQ ID NO: 69;
- the amino acid sequence of CDR1 is selected from SEQ ID NO: 22
- the amino acid sequence of CDR2 is selected from SEQ ID NO: 46
- the amino acid sequence of CDR3 is selected from SEQ ID NO: 70;
- the amino acid sequence of CDR1 is selected from SEQ ID NO: 23
- the amino acid sequence of CDR2 is selected from SEQ ID NO: 47
- the amino acid sequence of CDR3 is selected from SEQ ID NO: 71;
- the amino acid sequence of CDR1 is selected from SEQ ID NO:24
- the amino acid sequence of CDR2 is selected from SEQ ID NO:48
- the amino acid sequence of CDR3 is selected from SEQ ID NO:72.
- SEQ ID NO: 1-72 is shown in the following table:
- the present application also discloses a DNA molecule encoding the above-mentioned anti-CD70 nanobody with enhanced ADCP effect, the coding nucleotide sequence corresponding to the amino acids described in SEQ ID NO: 1-72 of the heavy chain variable region is as follows in sequence SEQ ID NO: 73-144.
- SEQ ID NO: 73-144 are shown in the table below:
- the antibodies described herein target the human or cynomolgus CD70 molecule.
- the present application also discloses the application of the above-mentioned anti-CD70 nanobody with enhanced ADCP effect for detecting CD70 molecules.
- the application of the above-mentioned anti-CD70 nanobody with enhanced ADCP effect in the preparation of drugs for treating tumors is also within the protection scope of the present application.
- This application uses commercialized recombinant human CD70 biotin-tagged protein and phage display antibody library as starting materials.
- the CD70 biotin-labeled protein was co-incubated with streptavidin-coated magnetic beads, CD70 was immobilized on the magnetic beads through the interaction between streptavidin and biotin, incubated with the antibody library, and washed away Bind/weakly bind phages, elute phages bound to CD70 protein.
- the coding sequences of the signal peptide, anti-CD70 antibody and human IgG1Fc are linked and read in-frame to construct a mammalian expression vector.
- the 293F cells were transfected, and the supernatant was harvested after shaking for 5 days, and the fusion protein was purified from the supernatant by protein A magnetic bead affinity chromatography.
- Cultivate target cells add luciferase reporter gene cells that can display ADCP signal transduction in a certain proportion, and co-incubate with a series of gradient concentrations of antibodies, and the reporter gene is activated to varying degrees.
- the final ADCP intensity is expressed by reporter gene activity.
- the present application successfully prepared an antibody (fusion protein) capable of binding to human CD70 molecules.
- the antibody has good specificity and high affinity, and binds to human CD70 expressed on the cell surface. Binding of CD70 successfully activates signaling required for the ADCP effect in reporter cells.
- the antibody can also effectively bind to cynomolgus monkey CD70.
- the anti-human CD70 monoclonal antibody is a potential drug for tumor immunotherapy.
- Figure 1 is the ELISA detection result of the combination of phage crude culture solution and human CD70;
- Figure 2 is the FACS detection result of the combination of phage crude culture solution and human CD70
- Figure 3 is a schematic diagram of the CD70 antibody expression vector structure, MCS is a multiple cloning site, and the target gene is inserted into this position;
- Figure 4 is the SDS-PAGE electrophoresis of the purified antibody
- Figure 5 is the detection of the binding performance of the purified antibody to CD70 on the cell surface
- Figure 6 is a purified antibody ADCP reporter gene activation detection
- Figure 7 is the detection of the binding of the purified antibody to CD70 in cynomolgus monkeys.
- Activation of host bacteria TG1 prepare a mini agar medium plate [1 ⁇ M9 salt, 2% glucose, 2mM MgSO 4 , 0.1mM CaCl 2 , 1mM vitamin B1], culture TG1 overnight in a 37°C incubator by streaking.
- Phage binding place the magnetic beads on the magnetic stand and remove the liquid. Add the blocked library to magnetic beads, resuspend and incubate with rotation for 1 hour, and remove the liquid.
- Phage precipitation centrifuge at 10,000 rpm for 15 minutes to remove bacteria, add 1/5 volume of 2.5M NaCl/20% PEG8000 to the supernatant, and ice-bath for 2 hours. Centrifuge at 10,000rpm for 10 minutes to obtain phage precipitate, remove the residual liquid, add 0.2ml 0.01M PBS (pH7.4) to resuspend the precipitate, and measure the titer as above.
- Table 1 ELISA detection results of the combination of phage crude culture medium and human CD70
- the full-length amino acid sequence of the variable region of the antibody number 1 is shown in SEQ ID NO: 145, and the nucleic acid sequence is shown in SEQ ID NO: 146; the full-length amino acid sequence of the variable region of the antibody number 2 is shown in SEQ ID NO: 147 As shown, the nucleic acid sequence is shown in SEQ ID NO: 148; the full-length amino acid sequence of the variable region of the antibody number 3 is shown in SEQ ID NO: 149, and the nucleic acid sequence is shown in SEQ ID NO: 150.
- step (3) 4 Add 100 ⁇ l of the phage supernatant in step (2) 2) or 100 ul concentration gradient [see Figure 5 or Table 3], and the purified antibody in step (3) 8), mix well, and incubate at room temperature for 30 minutes to 1 hour.
- phage detection add 100ul of anti-M13 phage antibody (purchased from Beijing Sino Biological) diluted 1000 times with PBS, incubate for 30min, wash once with PBS, add 100ul of fluorescent marker (such as FITC, APC) diluted 100 times with PBS
- fluorescent marker such as FITC, APC
- ADCP reporter gene cells (ADCP reporter gene cells)
- ADCP Fc ⁇ RIIa Jurkat Effector Cell Line (purchased from Jiman Biotech).
- the antibody was diluted with 1% FBS+RPMI-1640 culture medium to make a series of mother solutions with a working concentration of 5 times, and 10ul/well was added to the cells.
- the working concentration of ARGX-110, negative control IgG1 and detection antibody starts from 10ug/ml, and decreases by 4 times in 8 gradients.
- step 6) Repeat step 4) to wash the cells.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Medicinal Chemistry (AREA)
- Hematology (AREA)
- Biochemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Cell Biology (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- General Physics & Mathematics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Food Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Genetics & Genomics (AREA)
- Pathology (AREA)
- Biotechnology (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Peptides Or Proteins (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明公开了一种具有增强的ADCP效应的抗CD70抗体及其应用。本申请成功制备了可结合人源CD70分子的抗体(融合蛋白)，该抗体特异性好，亲合力较高，结合细胞表面表达的人源CD70。结合CD70后成功激活了报告细胞中ADCP效应所需信号转导。该抗体亦可以与食蟹猴CD70有效结合。该抗人CD70单克隆抗体是肿瘤免疫治疗的潜在药物。
Description
本申请属于生物医药技术，特别是涉及一种具有增强的ADCP效应的抗CD70抗体及其应用。
抗体依赖性细胞介导的吞噬作用(antibody-dependent cellular phagocytosis,ADCP)是抗体的重要特性，巨噬细胞(macrophage)和中性粒细胞(neutrophil)等具有此功能，其中巨噬细胞吞噬活性较强，是ADCP的主要效应细胞。同时巨噬细胞本身具有天然免疫活性，其激活后可以发挥免疫调理作用，起到增强整体免疫功能的作用。特别是实体瘤本身具有细胞、组织分化，结构紧密，因而CART细胞难以浸润，而巨噬细胞的吞噬功能可起到消解其结构的作用，因此ADCP对于实体瘤的治疗具有更特殊的实际意义。实际上，因CART治疗在实体瘤中应用遇到的瓶颈，利用巨噬细胞的这种特点进行靶向治疗的已引起研究者的关注。
抗原与抗体结合，进而抗体的可结晶片段(fragment crystallizable,Fc)主要与巨噬细胞表面的FcγⅡA(Fc gamma receptorⅡA)结合，抗原(颗粒)结合到巨噬细胞表面，引起摄入部位肌动蛋白聚合及伪足包裹颗粒，吞噬完成后肌动蛋白解聚。在此过程中，巨噬细胞中的NFAT信号转导通路被激活。利用该早期发生的信号转导事件，可以检测ADCP效应。
虽然ADCP在肿瘤治疗中引起人们的极大兴趣，ADCP效应却在已有的抗体药中普遍较弱。鉴于CD70/CD27分子对重要免疫调节功能，对CD70进行调控必然会引起一系列的T细胞、甚至B细胞及NK细胞等淋巴细胞的信号传递及生理反应，是一个很理想的免疫治疗靶点，合理应用有可能调动多种细胞免疫。本申请以人源CD70为靶点，开发具有强烈ADCP效应的抗体。
目前靶向CD70的疗法有CART及全抗，临床效果有限，特别是对实体瘤来说，这两种方法几乎无效。从ADCP效应入手，增加抗体作用机制的类型，是提高抗体的治疗效果一种方式。目前，临床实验中的抗体的ADCP效应仍较弱，对于增强治疗效果仍有欠缺。
发明内容
发明目的：针对上述现有技术中存在的技术问题，本申请提供了一组具有增强的ADCP效应的抗CD70纳米抗体及其应用。
技术方案：本申请所述的一组具有增强的ADCP效应的抗CD70纳米抗体，其重链可变区CDR1的氨基酸序列选自SEQ ID NO：1-24，CDR2的氨基酸序列选自SEQ ID NO：25-48，CDR3的氨基酸序列选自SEQ ID NO：49-72。
优选的，所述的具有增强的ADCP效应的抗CD70纳米抗体，包括以下任一所示重链可变区：
CDR1的氨基酸序列选自SEQ ID NO：1，CDR2的氨基酸序列选自SEQ ID NO：25，CDR3的氨基酸序列选自SEQ ID NO：49；
CDR1的氨基酸序列选自SEQ ID NO：2，CDR2的氨基酸序列选自SEQ ID NO：26，CDR3的氨基酸序列选自SEQ ID NO：50；
CDR1的氨基酸序列选自SEQ ID NO：3，CDR2的氨基酸序列选自SEQ ID NO：27，CDR3的氨基酸序列选自SEQ ID NO：51；
CDR1的氨基酸序列选自SEQ ID NO：4，CDR2的氨基酸序列选自SEQ ID NO：28，CDR3的氨基酸序列选自SEQ ID NO：52；
CDR1的氨基酸序列选自SEQ ID NO：5，CDR2的氨基酸序列选自SEQ ID NO：29，CDR3的氨基酸序列选自SEQ ID NO：53；
CDR1的氨基酸序列选自SEQ ID NO：6，CDR2的氨基酸序列选自SEQ ID NO：30，CDR3的氨基酸序列选自SEQ ID NO：54；
CDR1的氨基酸序列选自SEQ ID NO：7，CDR2的氨基酸序列选自SEQ ID NO：31，CDR3的氨基酸序列选自SEQ ID NO：55；
CDR1的氨基酸序列选自SEQ ID NO：8，CDR2的氨基酸序列选自SEQ ID NO：32，CDR3的氨基酸序列选自SEQ ID NO：56；
CDR1的氨基酸序列选自SEQ ID NO：9，CDR2的氨基酸序列选自SEQ ID NO：33，CDR3的氨基酸序列选自SEQ ID NO：57；
CDR1的氨基酸序列选自SEQ ID NO：10，CDR2的氨基酸序列选自SEQ ID NO：34，CDR3的氨基酸序列选自SEQ ID NO：58；
CDR1的氨基酸序列选自SEQ ID NO：11，CDR2的氨基酸序列选自SEQ ID NO：35，CDR3的氨基酸序列选自SEQ ID NO：59；
CDR1的氨基酸序列选自SEQ ID NO：12，CDR2的氨基酸序列选自SEQ ID NO：36，CDR3的氨基酸序列选自SEQ ID NO：60；
CDR1的氨基酸序列选自SEQ ID NO：13，CDR2的氨基酸序列选自SEQ ID NO：37，CDR3的氨基酸序列选自SEQ ID NO：61；
CDR1的氨基酸序列选自SEQ ID NO：14，CDR2的氨基酸序列选自SEQ ID NO：38，CDR3的氨基酸序列选自SEQ ID NO：62；
CDR1的氨基酸序列选自SEQ ID NO：15，CDR2的氨基酸序列选自SEQ ID NO：39，CDR3的氨基酸序列选自SEQ ID NO：63；
CDR1的氨基酸序列选自SEQ ID NO：16，CDR2的氨基酸序列选自SEQ ID NO：40，CDR3的氨基酸序列选自SEQ ID NO：64；
CDR1的氨基酸序列选自SEQ ID NO：17，CDR2的氨基酸序列选自SEQ ID NO：41，CDR3的氨基酸序列选自SEQ ID NO：65；
CDR1的氨基酸序列选自SEQ ID NO：18，CDR2的氨基酸序列选自SEQ ID NO：42，CDR3的氨基酸序列选自SEQ ID NO：66；
CDR1的氨基酸序列选自SEQ ID NO：19，CDR2的氨基酸序列选自SEQ ID NO：43，CDR3的氨基酸序列选自SEQ ID NO：67；
CDR1的氨基酸序列选自SEQ ID NO：20，CDR2的氨基酸序列选自SEQ ID NO：44，CDR3的氨基酸序列选自SEQ ID NO：68；
CDR1的氨基酸序列选自SEQ ID NO：21，CDR2的氨基酸序列选自SEQ ID NO：45，CDR3的氨基酸序列选自SEQ ID NO：69；
CDR1的氨基酸序列选自SEQ ID NO：22，CDR2的氨基酸序列选自SEQ ID NO：46，CDR3的氨基酸序列选自SEQ ID NO：70；
CDR1的氨基酸序列选自SEQ ID NO：23，CDR2的氨基酸序列选自SEQ ID NO：47，CDR3的氨基酸序列选自SEQ ID NO：71；
CDR1的氨基酸序列选自SEQ ID NO：24，CDR2的氨基酸序列选自SEQ ID NO：48，CDR3的氨基酸序列选自SEQ ID NO：72。
SEQ ID NO：1-72如下表所示：
本申请还公开了编码上述具有增强的ADCP效应的抗CD70纳米抗体的DNA分子，其重链可变区SEQ ID NO：1-72所述氨基酸对应的编码核苷酸序列依次如SEQ ID NO：73-144所示。
SEQ ID NO：73-144如下表所示：
本申请所述抗体靶向人或食蟹猴CD70分子。
本申请还公开了上述具有增强的ADCP效应的抗CD70纳米抗体用于检测CD70分子的应用。
进一步的，上述具有增强的ADCP效应的抗CD70纳米抗体在制备治疗肿瘤的药物中的应用也在本申请的保护范围内。本申请以商品化的重组人CD70生物素标签蛋白和噬菌体展示抗体库为起始材料。将CD70生物素标签蛋白与链霉亲和素包被磁珠共孵育，通过链霉亲和素与生物素之间的互作将CD70固定在磁珠上，用抗体库与其孵育，洗去未结合/结合弱的噬菌体，将与CD70蛋白结合的噬菌体洗脱下来。如此反复3次，每次改变CD70生物素标签蛋白的使用量和洗涤条件，逐次淘汰掉结合弱的噬菌体并尽可能保留更多的结合强的噬菌体。用强结合的噬菌体侵染宿主菌，涂板并过夜培养，获得单克隆菌落，即将可结合CD70的噬菌体抗体进行单克隆化。利用单克隆培养上清进行ELISA筛选，并对单克隆进行核酸序列测定。
将信号肽、抗CD70抗体、人源IgG1Fc的编码序列连接，同框阅读，构建哺乳动物表达载体。转染293F细胞，振荡培养5天后收获上清，利用蛋白A磁珠亲和层析从上清中纯化获得融合蛋白。
将不同浓度的纯化抗体与表达人源CD70的癌细胞系或食蟹猴CD70超表达细胞系进行孵育，利用荧光标记的二抗检测结合到细胞表面的抗体，测定其与细胞表面的人源或食蟹猴CD70结合能力。
培养靶细胞，按一定比例加入可显示ADCP信号转导的荧光素酶报告基因的细胞，与一系列梯度浓度的抗体共孵育，报告基因不同程度被激活。最终ADCP强度通过报告基因活性表现出来。
有益效果：本申请成功制备了可结合人源CD70分子的抗体(融合蛋白)，该抗体特异性好，亲合力较高，结合细胞表面表达的人源CD70。结合CD70后成功激活了报告细胞中ADCP效应所需信号转导。该抗体亦可以与食蟹猴CD70有效结合。该抗人CD70单克隆抗体是肿瘤免疫治疗的潜在药物。
图1是phage粗培液与人源CD70结合ELISA检测结果；
图2是phage粗培液与人源CD70结合FACS检测结果；
图3是CD70抗体表达载体结构示意图，MCS为多克隆位点，靶基因插入此位置；
图4是纯化抗体SDS-PAGE电泳；
图5是纯化抗体与细胞表面CD70结合性能检测；
图6是纯化抗体ADCP报告基因激活检测；
图7是纯化抗体与食蟹猴CD70结合检测。
下面结合实施例对本申请作出详细说明。
(1)噬菌体展示抗体文库淘选
1)宿主菌TG1活化：制备mini agar培养基平板[1×M9盐，2％葡萄糖，2mM MgSO
4，0.1mM CaCl
2，1mM维生素B1]，划线法过夜培养TG1于37℃培养箱。
2)磁珠洗涤及封闭：吸取50ul磁珠(购自Invitrogen)，置于磁力架上，吸附后吸去液体，1ml PBS重悬、洗涤两次，用1ml 1.5％脱脂奶粉+1.5％BSA的封闭剂(第二轮、第三轮淘选时封闭剂浓度逐渐增加)封闭1小时，去除液体。
3)抗原结合：按16ug/ml的浓度将人源CD70蛋白(购自Acro Biosystem，以后各轮淘选时抗原浓度逐渐降低)用PBS中(pH7.2-7.4)稀释至1ml，重悬磁珠，旋转孵育1小时。
4)库封闭：与抗原结合至磁珠同步，取10
11pfu噬菌体病毒颗粒(来自于原始抗体库或淘选扩增产物)，用1ml 1.0％脱脂奶粉+1.0％BSA的封闭剂旋转孵育1小时。
5)噬菌体结合：将磁珠置于磁力架上，去除液体。将封闭后的库加入磁珠，重悬并旋转孵育1小时，去除液体。
6)洗涤：用1ml PBST[0.01M PBS(pH7.4),0.1％Tween-20(第二、三轮Tween-20浓度分别用0.2％、0.3％)]洗涤，再用0.01M PBS(pH7.4)洗涤。
7)洗脱：吸去液体，用300ul 0.2M甘氨酸-盐酸(pH2.2)洗脱10分钟，加入20ul中和液[1M Tris-Cl(pH9.0)]混匀，暂时保存于4℃。
8)测滴度：取2ul、0.2ul(原液用2×YT培养基稀释10倍后取2ul)、0.02ul(原液用2×YT培养基稀释100倍后取2ul)洗脱液，与0.2ml对数中期(OD600＝0.5)的TG1混匀，室温孵育30分钟，均匀涂于2×YT-GA100[含2％葡萄糖，100ug/ml氨苄青霉素]平板上，37℃过夜培养，计数约50个克隆的平板上的克隆数，根据稀释倍数计算滴度。
9)噬菌体扩增：在淘选进行的同时，挑取mini agar平板上的TG1单克隆，接种于10ml 2×YT培养液中，37℃下，250rpm振荡培养至对数中期(OD600＝0.5)。加入200ul淘选获得的洗脱产物，37℃孵育30分钟。加入辅助噬菌体M13KO7，37℃再孵育30分钟，37℃下，250rpm振荡培养1小时。离心去上清，用20ml含工作浓度100ug/ml氨苄青霉素和工作浓度50ug/ml卡那霉素的2×YT重悬，30℃，220rpm振荡培养过夜。
10)噬菌体沉淀：10000rpm离心15分钟去除菌体，上清中加入1/5体积的2.5M NaCl/20％PEG8000，冰浴2小时。10000rpm离心10分钟得到噬菌体沉淀，将残液去除干净，加入0.2ml 0.01M PBS(pH7.4)液重悬沉淀，如上文测滴度。
11)重复步骤2)-10)两或三次，获得结合力强的噬菌体展示抗体。
(2)单克隆ELISA
1)0.3ug/ml链霉亲和素4℃过夜包被酶标板，2％BSA/PBS封闭液处理2h，并用PBS洗涤3次。
2)从2×YT-GA100平板上挑取的单克隆菌落，振荡培养至对数中期，加入辅助噬菌体M13KO7，37℃孵育30分钟。37℃，220rpm振荡培养1小时，4000rpm离心15分钟。用400ul含工作浓度100ug/ml氨苄青霉素和工作浓度50ug/ml卡那霉素的2×YT重悬，30℃，220rpm振荡培养过夜。4000rpm离心15分钟，沉淀菌体。
3)取50ul 4％BSA/PBS加入到酶标板中，同时取50ul噬菌体上清，混匀，孵育1小时。
4)去除液体，0.1％PBST洗5次，PBS洗3次，去除液体。
5)将HRP标记抗M13噬菌体抗体(购自北京义翘神州)用2％BSA稀释3000倍，取100ul加入到酶标板中，孵育1小时，去除液体，0.1％PBST洗3次，拍干残液。
6)加入100ul TMB显色液，37℃孵育10min或至蓝色充分显现，加入100ul 1M硫酸终止反应，在酶标仪上读取OD450。结果如图1所示，在噬菌体淘选过程中，用含有phage病毒颗粒的2×YT培养基50ul做检测。其原始数据如表1所示，其中数值为450nm处的吸光值及其比值。
表1：phage粗培液与人源CD70结合ELISA检测结果
| No.ID | 空白组 | 实验组 | 实验/空白 | No.ID | 空白组 | 实验组 | 实验/空白 |
| 1 | 0.3393 | 1.5412 | 4.5423 | 13 | 0.7909 | 2.7965 | 3.5358 |
| 2 | 0.3801 | 0.6504 | 1.7111 | 14 | 0.5959 | 2.3806 | 3.9949 |
| 3 | 0.9456 | 1.8750 | 1.9829 | 15 | 0.5943 | 2.4666 | 4.1504 |
| 4 | 1.2482 | 2.9280 | 2.3458 | 16 | 0.8776 | 2.5020 | 2.8510 |
| 5 | 1.1651 | 2.1283 | 1.8267 | 17 | 0.8653 | 2.2645 | 2.6170 |
| 6 | 1.1849 | 2.2273 | 1.8797 | 18 | 0.6055 | 1.5305 | 2.5277 |
| 7 | 0.7467 | 1.5409 | 2.0636 | 19 | 0.9782 | 2.6730 | 2.7326 |
| 8 | 1.1179 | 2.0062 | 1.7946 | 20 | 1.0186 | 2.0023 | 1.9657 |
| 9 | 0.9789 | 1.9353 | 1.9770 | 21 | 0.5214 | 2.2278 | 4.2727 |
| 10 | 1.1326 | 2.8815 | 2.5441 | 22 | 0.5376 | 2.1932 | 4.0796 |
| 11 | 0.8119 | 1.6906 | 2.0823 | 23 | 1.0740 | 2.2402 | 2.0858 |
| 12 | 0.5712 | 2.3354 | 4.0886 | 24 | 0.5522 | 1.7882 | 3.2383 |
(3)人源CD70单抗制备及流式检测
1)从2×YT-GA100平板上挑取的单克隆菌落，液体振荡培养过夜，按质粒提取法提取噬菌粒(phagemid)。
2)合成引物，PCR扩增噬菌体所展示的抗体基因编码区。
3)将以上核酸片段按序插入到真核表达载体Abexp-uIgG1的MCS区(图3)，以编码N端为信号肽、中段为抗体、C端为Fc标签的融合蛋白。
4)制备无菌无内毒素的质粒。取23ug，用0.75mL的稀释液(如OPM-293CD05培养基)稀释，同时取70μL的转染试剂(如PEI溶液)加入到0.75mL的稀释液中(如OPM-293CD05培养基)轻柔混匀。将PEI稀释液加入质粒稀释液中，立即用枪轻柔混匀，室温静置15min，避免扰动。
5)加入到25ml 293F细胞及其培养液中，80rpm，37℃，5％CO
2条件下培养24小时，再加入25mL新鲜生长培养基(如OPM-293CD05)，80rpm，37℃，5％CO
2继续培养72小时。
6)10000rpm离心10分钟，取上清。与平衡后的proteinA亲和磁珠旋转孵育1小时，置于磁力架上，去除上清。
7)用30ml PBS洗杂3次，加入5ml 0.1M甘氨酸(pH 3.0)洗脱10分钟，置于磁力架上，吸取上清立即用1M Tris-HCl缓冲液(pH 8.5)至中性，即获得纯化的抗体。
8)SDS-PAGE检测纯化抗体，同时进行浓度测定。采用还原电泳，抗体分子中的二硫键被打开，分子以伸展的单肽链状态进行电泳迁移，结果如图4所示。
对筛选所得纯化抗体测序，得到编号下表所示的编号1-24的抗体：
其中，编号1抗体的可变区全长氨基酸序列如SEQ ID NO：145所示，核酸 序列如SEQ ID NO：146所示；编号2抗体的可变区全长氨基酸序列如SEQ ID NO：147所示，核酸序列如SEQ ID NO：148所示；编号3抗体的可变区全长氨基酸序列如SEQ ID NO：149所示，核酸序列如SEQ ID NO：150所示。
SEQ ID NO：145
SEQ ID NO：146
SEQ ID NO：147
SEQ ID NO：148
SEQ ID NO：149
SEQ ID NO：150
9)流式细胞术检测纯化抗体的细胞结合能力
①将表达CD70的细胞株(如786-0)用0.25％的胰酶充分消化，血清终止消化，离心收集细胞，PBS轻轻吹打制备单细胞悬液。
②10ml PBS洗涤细胞1次，1000rpm离心5min,再用1ml PBS悬浮细胞，细胞计数。
③取2.5×10
5个细胞于96孔细胞培养板中，离心收集细胞。
④加入100μl步骤(2)2)中的phage上清或100ul浓度梯度的[见图5或表3]、步骤(3)8)中的纯化抗体，混匀，室温孵育30分钟至1小时。
⑤离心收集细胞，用300ul PBS洗涤细胞1次。
⑥若为phage检测，加入100ul用PBS稀释1000倍的抗M13噬菌体抗体(购自北京义翘神州)，孵育30min，PBS洗一次，加入100ul用PBS稀释100倍的荧光标记(如FITC、APC)抗体，室温下避光反应20min；若为纯化抗体检测，加入100μl用PBS稀释200倍的荧光标记(如FITC、APC)抗体，室温下避光反应20min。
⑦用1ml PBS洗涤细胞1次，1000rpm离心8min，去除上清液。
⑧加入100μl PBS重悬成单细胞悬液，用流式细胞仪上机检测。phage流式如图2所示，图中左侧、右侧柱形图分别代表与野生型CHO-K1、CD70超表达CHO-K1细胞株结合信号。其原始数据如表2所示，其中数值为荧光强度中值(MFI)及其比值。
表2：phage粗培液与人源CD70结合FACS检测结果
| No ID | 空白组 | 实验组 | 实验/空白 | No ID | 空白组 | 实验组 | 实验/空白 |
| 1 | 961.3 | 9311.2 | 9.69 | 13 | 541.0 | 8218.2 | 15.19 |
| 2 | 822.3 | 2162.4 | 2.63 | 14 | 545.0 | 6999.0 | 12.84 |
| 3 | 566.7 | 5450.0 | 9.62 | 15 | 553.5 | 10431.1 | 18.85 |
| 4 | 539.4 | 7917.5 | 14.68 | 16 | 476.9 | 7858.0 | 16.48 |
| 5 | 537.8 | 6657.0 | 12.38 | 17 | 542.0 | 9965.4 | 18.39 |
| 6 | 546.0 | 6216.5 | 11.39 | 18 | 569.4 | 5880.3 | 10.33 |
| 7 | 544.2 | 7686.4 | 14.12 | 19 | 554.0 | 11803.8 | 21.31 |
| 8 | 537.4 | 8517.8 | 15.85 | 20 | 611.1 | 7568.3 | 12.38 |
| 9 | 525.50 | 6962.40 | 13.25 | 21 | 575.80 | 9114.20 | 15.83 |
| 10 | 537.30 | 7651.50 | 14.24 | 22 | 548.70 | 8939.80 | 16.29 |
| 11 | 540.00 | 5013.90 | 9.29 | 23 | 550.20 | 9450.70 | 17.18 |
| 12 | 538.50 | 5671.00 | 10.53 | 24 | 547.50 | 4370.20 | 7.98 |
纯化抗体流式如图5所示，流式细胞术运用于该检测，其中ARGX-110为阳性对照，hIgG1为阴性对照。其原始数据如表3所示，其中的数值为MFI。
表3：纯化抗体与细胞表面CD70结合性能检测
(4)纯化抗体ADCP报告基因激活检测
1)培养靶细胞raji和效应细胞(ADCP报告基因细胞)ADCP FcγRIIa Jurkat Effector Cell Line(购自吉满生物)。
2)将靶细胞用1％FBS+RPMI-1640培养基悬浮，取20ul/孔，并确保取20000个/孔，加入到384孔板中。
3)将效应细胞用1％FBS+RPMI-1640培养基悬浮，取20ul/孔，并确保取50000个/孔，加入到384孔板中。
3)抗体用1％FBS+RPMI-1640培养基培养基稀释成一系列工作浓度5倍的母液，取10ul/孔加入到细胞中。ARGX-110、阴性对照IgG1和检测抗体工作浓度从10ug/ml开始，4倍降低8个梯度。
4)置于5％CO
2培养箱中，37℃孵育6小时。
5)将One-Lite Luciferase Assay System(购自诺唯赞)提前恢复至室温，按按30ul/孔加入。
6)反应3分钟，用酶标仪读数(15分钟内完成)。结果如图6所示，运用荧光素酶报告基因系统，若抗体激活了ADCP信号转导，报告基因转录激活，表达出荧光素酶，其中ARGX-110为阳性对照，hIgG1为阴性对照。其原始数据如表4所示，其中的数值为MFI。
表4：纯化抗体ADCP报告基因激活检测
(5)纯化抗体与食蟹猴CD70结合检测
1)培养食蟹猴CD70超表达的CHO-K1细胞，用0.25％的胰酶充分消化，血清终止消化，离心收集细胞，PBS轻轻吹打制备单细胞悬液。
2)按200000个细胞每孔加入到96孔板中，并保证每孔加入50ul。
3)将阴性、阳性及对照抗体按工作浓度2倍配制成母液(例如，10、1、0.1ug/ml)，取50ul加入细胞中，混匀，4℃下孵育30分钟。
4)加入100ul PBS，轻缓摇动，500g离心5分钟，去除上清，再加入200ul PBS轻缓摇动，500g离心5分钟，去除上清。
5)加入100ul稀释的荧光二抗，摇动96孔板悬浮细胞，4℃孵育30分钟。
6)重复步骤4)，以洗涤细胞。
7)用80ul PBS重悬细胞，流式细胞仪检测荧光。结果如图7所示，抗体与可于细胞表面的超表达食蟹猴CD70的细胞共孵育，荧光标记二抗检测细胞结合的抗体。其中hIgG1为阴性对照，ARGX-110为阳性对照。其原始数据如表5所示，其中的数值为MFI。
表5：纯化抗体与食蟹猴CD70结合检测
Claims (6)
- 一组具有增强的ADCP效应的抗CD70抗体，其特征在于，其重链可变区CDR1的氨基酸序列选自SEQ ID NO：1-24，CDR2的氨基酸序列选自SEQ ID NO：25-48，CDR3的氨基酸序列选自SEQ ID NO：49-72。
- 根据权利要求1所述的具有增强的ADCP效应的抗CD70抗体，其特征在于，包括以下任一所示重链可变区：CDR1的氨基酸序列选自SEQ ID NO：1，CDR2的氨基酸序列选自SEQ ID NO：25，CDR3的氨基酸序列选自SEQ ID NO：49；CDR1的氨基酸序列选自SEQ ID NO：2，CDR2的氨基酸序列选自SEQ ID NO：26，CDR3的氨基酸序列选自SEQ ID NO：50；CDR1的氨基酸序列选自SEQ ID NO：3，CDR2的氨基酸序列选自SEQ ID NO：27，CDR3的氨基酸序列选自SEQ ID NO：51；CDR1的氨基酸序列选自SEQ ID NO：4，CDR2的氨基酸序列选自SEQ ID NO：28，CDR3的氨基酸序列选自SEQ ID NO：52；CDR1的氨基酸序列选自SEQ ID NO：5，CDR2的氨基酸序列选自SEQ ID NO：29，CDR3的氨基酸序列选自SEQ ID NO：53；CDR1的氨基酸序列选自SEQ ID NO：6，CDR2的氨基酸序列选自SEQ ID NO：30，CDR3的氨基酸序列选自SEQ ID NO：54；CDR1的氨基酸序列选自SEQ ID NO：7，CDR2的氨基酸序列选自SEQ ID NO：31，CDR3的氨基酸序列选自SEQ ID NO：55；CDR1的氨基酸序列选自SEQ ID NO：8，CDR2的氨基酸序列选自SEQ ID NO：32，CDR3的氨基酸序列选自SEQ ID NO：56；CDR1的氨基酸序列选自SEQ ID NO：9，CDR2的氨基酸序列选自SEQ ID NO：33，CDR3的氨基酸序列选自SEQ ID NO：57；CDR1的氨基酸序列选自SEQ ID NO：10，CDR2的氨基酸序列选自SEQ ID NO：34，CDR3的氨基酸序列选自SEQ ID NO：58；CDR1的氨基酸序列选自SEQ ID NO：11，CDR2的氨基酸序列选自SEQ ID NO：35，CDR3的氨基酸序列选自SEQ ID NO：59；CDR1的氨基酸序列选自SEQ ID NO：12，CDR2的氨基酸序列选自SEQ ID NO：36，CDR3的氨基酸序列选自SEQ ID NO：60；CDR1的氨基酸序列选自SEQ ID NO：13，CDR2的氨基酸序列选自SEQ ID NO：37，CDR3的氨基酸序列选自SEQ ID NO：61；CDR1的氨基酸序列选自SEQ ID NO：14，CDR2的氨基酸序列选自SEQ ID NO：38，CDR3的氨基酸序列选自SEQ ID NO：62；CDR1的氨基酸序列选自SEQ ID NO：15，CDR2的氨基酸序列选自SEQ ID NO：39，CDR3的氨基酸序列选自SEQ ID NO：63；CDR1的氨基酸序列选自SEQ ID NO：16，CDR2的氨基酸序列选自SEQ ID NO：40，CDR3的氨基酸序列选自SEQ ID NO：64；CDR1的氨基酸序列选自SEQ ID NO：17，CDR2的氨基酸序列选自SEQ ID NO：41，CDR3的氨基酸序列选自SEQ ID NO：65；CDR1的氨基酸序列选自SEQ ID NO：18，CDR2的氨基酸序列选自SEQ ID NO：42，CDR3的氨基酸序列选自SEQ ID NO：66；CDR1的氨基酸序列选自SEQ ID NO：19，CDR2的氨基酸序列选自SEQ ID NO：43，CDR3的氨基酸序列选自SEQ ID NO：67；CDR1的氨基酸序列选自SEQ ID NO：20，CDR2的氨基酸序列选自SEQ ID NO：44，CDR3的氨基酸序列选自SEQ ID NO：68；CDR1的氨基酸序列选自SEQ ID NO：21，CDR2的氨基酸序列选自SEQ ID NO：45，CDR3的氨基酸序列选自SEQ ID NO：69；CDR1的氨基酸序列选自SEQ ID NO：22，CDR2的氨基酸序列选自SEQ ID NO：46，CDR3的氨基酸序列选自SEQ ID NO：70；CDR1的氨基酸序列选自SEQ ID NO：23，CDR2的氨基酸序列选自SEQ ID NO：47，CDR3的氨基酸序列选自SEQ ID NO：71；CDR1的氨基酸序列选自SEQ ID NO：24，CDR2的氨基酸序列选自SEQ ID NO：48，CDR3的氨基酸序列选自SEQ ID NO：72。
- 编码权利要求1所述具有增强的ADCP效应的抗CD70抗体的DNA分子，其特征在于，其重链可变区SEQ ID NO：1-72所述氨基酸对应的编码核苷酸序列依次如SEQ ID NO：73-144所示。
- 根据权利要求1或2所述的具有增强的ADCP效应的抗CD70抗体，其特征在于，所述抗体靶向人或食蟹猴CD70分子。
- 权利要求1或2所述具有增强的ADCP效应的抗CD70抗体用于检测CD70分子的应用。
- 权利要求1或2所述具有增强的ADCP效应的抗CD70抗体在制备治疗肿瘤的药物中的应用。
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110673784.4 | 2021-06-17 | ||
| CN202110673784.4A CN113292653B (zh) | 2021-06-17 | 2021-06-17 | 具有增强的adcp效应的抗cd70抗体及其应用 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2022262100A1 true WO2022262100A1 (zh) | 2022-12-22 |
Family
ID=77328645
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/CN2021/112518 WO2022262100A1 (zh) | 2021-06-17 | 2021-08-13 | 具有增强的adcp效应的抗cd70抗体及其应用 |
Country Status (2)
| Country | Link |
|---|---|
| CN (2) | CN113292653B (zh) |
| WO (1) | WO2022262100A1 (zh) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114262377B (zh) * | 2021-10-28 | 2024-03-22 | 新疆优迈生物技术有限公司 | 一种阻断cd70与其配体cd27结合的抗人cd70纳米抗体的制备方法及其编码序列 |
| CN117924489A (zh) * | 2022-01-19 | 2024-04-26 | 上海恒润达生生物科技股份有限公司 | 抗cd70纳米抗体及其用途 |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101203241A (zh) * | 2005-04-19 | 2008-06-18 | 西雅图基因公司 | 人源化抗-cd70结合物和其应用 |
| CN103596979A (zh) * | 2011-03-16 | 2014-02-19 | 阿尔金-X公司 | 针对cd70的抗体 |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109293778B (zh) * | 2018-11-01 | 2021-09-17 | 浙江蓝盾药业有限公司 | 同时抗cd70和cd47的重链抗体及其制备方法和应用 |
| CN109880802B (zh) * | 2018-11-30 | 2022-12-13 | 北京美康基免生物科技有限公司 | 一种基于cd19和cd70的双重嵌合抗原受体基因修饰的免疫细胞及其应用 |
| CN110699327B (zh) * | 2019-10-31 | 2021-04-13 | 浙江蓝盾药业有限公司 | 杂交瘤细胞株6f9、抗体及其应用 |
| CN110724670B (zh) * | 2019-10-31 | 2021-04-13 | 浙江蓝盾药业有限公司 | 杂交瘤细胞株3h4、抗体及其应用 |
-
2021
- 2021-06-17 CN CN202110673784.4A patent/CN113292653B/zh active Active
- 2021-06-17 CN CN202210929990.1A patent/CN116375865A/zh active Pending
- 2021-08-13 WO PCT/CN2021/112518 patent/WO2022262100A1/zh unknown
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101203241A (zh) * | 2005-04-19 | 2008-06-18 | 西雅图基因公司 | 人源化抗-cd70结合物和其应用 |
| CN103596979A (zh) * | 2011-03-16 | 2014-02-19 | 阿尔金-X公司 | 针对cd70的抗体 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113292653B (zh) | 2022-11-11 |
| CN113292653A (zh) | 2021-08-24 |
| CN116375865A (zh) | 2023-07-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| WO2022262100A1 (zh) | 具有增强的adcp效应的抗cd70抗体及其应用 | |
| JP2023527927A (ja) | 新型コロナウイルス（ｓａｒｓ－ｃｏｖ－２）スパイクタンパク質結合分子及びその使用 | |
| CN114163523A (zh) | 一种针对新型冠状病毒的单域抗体及其应用 | |
| WO2022262101A1 (zh) | 具有增强的adcc效应的抗cd70抗体及其应用 | |
| WO2022262099A1 (zh) | 抗cd70内化的抗体、抗体偶联物及其应用 | |
| JP7236562B2 (ja) | インターロイキン－４受容体抗体及びその用途 | |
| CN107474135B (zh) | 抗PD-1的纳米抗体PD-1/Nb20及其制备方法与应用 | |
| CN111378037B (zh) | 一种抗hIL-33人源化单抗及其应用 | |
| CN114292326B (zh) | 新型冠状病毒(sars-cov-2)刺突蛋白结合分子及其应用 | |
| CN110590955B (zh) | 一种双特异性抗体 | |
| WO2023024444A1 (zh) | 抗msln单克隆内化抗体及其制备方法和应用 | |
| CN111320690B (zh) | 一种抗人Tim3单克隆抗体及其应用 | |
| CN114853895B (zh) | 一种抗糖基转移酶a亚单位的纳米抗体及其应用 | |
| Alizadeh et al. | Identification of novel single chain fragment variable antibodies against TNF-α using phage display technology | |
| CN113980127A (zh) | 抗耐甲氧西林葡萄球菌的纳米抗体及其制备方法和用途 | |
| CN110885375B (zh) | 特异性针对mmp-9蛋白锌离子结合结构域的单域抗体及产品与应用 | |
| CN110804096A (zh) | Cd123单域抗体、核苷酸序列、表达载体及试剂盒 | |
| CN117327178A (zh) | 一种抗LILRB2全人源ScFv抗体及其应用 | |
| CN116496392B (zh) | 抗新型冠状病毒n蛋白单域抗体、融合蛋白及其编码基因和应用 | |
| CN114605555B (zh) | 一种抗新型冠状病毒SARS-CoV-2的双特异中和抗体及其应用 | |
| CN111909267B (zh) | 低免疫原性抗TNF-α人源化单克隆抗体TCX063及其应用 | |
| CN112062848B (zh) | 抗cd47单克隆抗体及其应用 | |
| EP4223878A1 (en) | Sars-cov-2-binding peptide | |
| CN116789826A (zh) | 抗人lilrb2全人源单域抗体及其应用 | |
| CN116120443A (zh) | 靶向SARS-CoV-2 RBD与其受体ACE2共享表位的驼源纳米抗体 |
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: 21945681 Country of ref document: EP Kind code of ref document: A1 |
|
| NENP | Non-entry into the national phase |
Ref country code: DE |