CN115028741A - Tumor antigen-antibody complex, preparation method and application thereof - Google Patents
Tumor antigen-antibody complex, preparation method and application thereof Download PDFInfo
- Publication number
- CN115028741A CN115028741A CN202210703064.2A CN202210703064A CN115028741A CN 115028741 A CN115028741 A CN 115028741A CN 202210703064 A CN202210703064 A CN 202210703064A CN 115028741 A CN115028741 A CN 115028741A
- Authority
- CN
- China
- Prior art keywords
- antibody
- tumor antigen
- eso
- fusion protein
- intn
- Prior art date
- Legal status (The legal status 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 status listed.)
- Pending
Links
- 206010028980 Neoplasm Diseases 0.000 title claims abstract description 73
- 238000002360 preparation method Methods 0.000 title claims abstract description 21
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 55
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 48
- 239000000427 antigen Substances 0.000 claims abstract description 33
- 102000036639 antigens Human genes 0.000 claims abstract description 33
- 108091007433 antigens Proteins 0.000 claims abstract description 33
- 238000000034 method Methods 0.000 claims abstract description 29
- 238000006243 chemical reaction Methods 0.000 claims abstract description 18
- 230000017730 intein-mediated protein splicing Effects 0.000 claims abstract description 16
- 230000008878 coupling Effects 0.000 claims abstract description 11
- 238000010168 coupling process Methods 0.000 claims abstract description 11
- 238000005859 coupling reaction Methods 0.000 claims abstract description 11
- 229960005486 vaccine Drugs 0.000 claims abstract description 7
- 102000037865 fusion proteins Human genes 0.000 claims description 54
- 108020001507 fusion proteins Proteins 0.000 claims description 54
- 239000013604 expression vector Substances 0.000 claims description 31
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 25
- 239000012634 fragment Substances 0.000 claims description 12
- 239000013612 plasmid Substances 0.000 claims description 12
- 238000010276 construction Methods 0.000 claims description 9
- 102100025570 Cancer/testis antigen 1 Human genes 0.000 claims description 8
- 101000856237 Homo sapiens Cancer/testis antigen 1 Proteins 0.000 claims description 8
- 239000013598 vector Substances 0.000 claims description 7
- 125000000539 amino acid group Chemical group 0.000 claims description 6
- 102000003960 Ligases Human genes 0.000 claims description 3
- 108090000364 Ligases Proteins 0.000 claims description 3
- 238000010367 cloning Methods 0.000 claims description 3
- 238000001976 enzyme digestion Methods 0.000 claims description 2
- 238000002156 mixing Methods 0.000 claims description 2
- 238000012412 chemical coupling Methods 0.000 abstract description 4
- 230000008569 process Effects 0.000 abstract description 4
- 241001391944 Commicarpus scandens Species 0.000 abstract description 2
- 239000006227 byproduct Substances 0.000 abstract description 2
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 2
- 238000012827 research and development Methods 0.000 abstract description 2
- 230000009870 specific binding Effects 0.000 abstract description 2
- 239000000126 substance Substances 0.000 abstract description 2
- 231100000331 toxic Toxicity 0.000 abstract description 2
- 230000002588 toxic effect Effects 0.000 abstract description 2
- 230000001988 toxicity Effects 0.000 abstract description 2
- 231100000419 toxicity Toxicity 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 18
- 210000003000 inclusion body Anatomy 0.000 description 11
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 9
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- 238000009835 boiling Methods 0.000 description 6
- 238000011033 desalting Methods 0.000 description 6
- 238000010186 staining Methods 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 239000007788 liquid Substances 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 239000012096 transfection reagent Substances 0.000 description 5
- 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 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 230000006698 induction Effects 0.000 description 4
- 108090000765 processed proteins & peptides Proteins 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 3
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- 230000014509 gene expression Effects 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 3
- 239000000523 sample Substances 0.000 description 3
- 239000011534 wash buffer Substances 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- 108020004705 Codon Proteins 0.000 description 2
- 210000001744 T-lymphocyte Anatomy 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 210000004899 c-terminal region Anatomy 0.000 description 2
- 230000003197 catalytic effect Effects 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 description 2
- 210000004443 dendritic cell Anatomy 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 230000028993 immune response Effects 0.000 description 2
- 238000009169 immunotherapy Methods 0.000 description 2
- 239000012160 loading buffer Substances 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000012192 staining solution Substances 0.000 description 2
- 238000002604 ultrasonography Methods 0.000 description 2
- 101710145634 Antigen 1 Proteins 0.000 description 1
- 230000004568 DNA-binding Effects 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 108010001515 Galectin 4 Proteins 0.000 description 1
- 102100039556 Galectin-4 Human genes 0.000 description 1
- 229940076838 Immune checkpoint inhibitor Drugs 0.000 description 1
- 238000012404 In vitro experiment Methods 0.000 description 1
- 239000012880 LB liquid culture medium Substances 0.000 description 1
- 102100033486 Lymphocyte antigen 75 Human genes 0.000 description 1
- 101710157884 Lymphocyte antigen 75 Proteins 0.000 description 1
- 102000016943 Muramidase Human genes 0.000 description 1
- 108010014251 Muramidase Proteins 0.000 description 1
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 1
- 238000009004 PCR Kit Methods 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 108700005077 Viral Genes Proteins 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 230000030741 antigen processing and presentation Effects 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000002457 bidirectional effect Effects 0.000 description 1
- 210000004900 c-terminal fragment Anatomy 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000006037 cell lysis Effects 0.000 description 1
- 238000002659 cell therapy Methods 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000004891 communication Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000004042 decolorization Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000005014 ectopic expression Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 229960000789 guanidine hydrochloride Drugs 0.000 description 1
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 239000012274 immune-checkpoint protein inhibitor Substances 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 238000005304 joining Methods 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- 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 1
- 229960000318 kanamycin Drugs 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 229960000274 lysozyme Drugs 0.000 description 1
- 239000004325 lysozyme Substances 0.000 description 1
- 235000010335 lysozyme Nutrition 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 210000004898 n-terminal fragment Anatomy 0.000 description 1
- 102000007863 pattern recognition receptors Human genes 0.000 description 1
- 108010089193 pattern recognition receptors Proteins 0.000 description 1
- 238000003752 polymerase chain reaction Methods 0.000 description 1
- 239000012474 protein marker Substances 0.000 description 1
- 239000011535 reaction buffer Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000012723 sample buffer Substances 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000011537 solubilization buffer Substances 0.000 description 1
- 230000003381 solubilizing effect Effects 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 206010041823 squamous cell carcinoma Diseases 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 238000011426 transformation method Methods 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
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
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
-
- 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
- A61K39/0005—Vertebrate antigens
- A61K39/0011—Cancer antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
- A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
-
- 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
- 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
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
-
- 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
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/51—Complete heavy chain or Fd fragment, i.e. VH + CH1
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/515—Complete light chain, i.e. VL + CL
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/90—Fusion polypeptide containing a motif for post-translational modification
- C07K2319/92—Fusion polypeptide containing a motif for post-translational modification containing an intein ("protein splicing")domain
Abstract
The invention discloses a tumor antigen-antibody complex and a preparation method and application thereof, wherein the tumor antigen-antibody complex comprises a tumor antigen and an antibody, and the tumor antigen-antibody complex is obtained by combining the tumor antigen and the antibody and is antibody coupling protein 3G 9-NY-ESO-1. According to the invention, the tumor antigen and the antibody are connected by a trans-splicing method of the broken intein GP41-1, the tumor antigen and the antibody are connected by peptide bonds, the stability is higher, the specific binding is stronger, the reaction condition is mild, the efficiency is high, the process is simple, no toxic or harmful substance is added, no by-product is generated, the safety is high, the problems that a chemical reagent is required to be added when the antigen and the antibody are connected by a chemical coupling method and the like in the prior art, certain toxicity exists, the chemical coupling is unstable, the coupling bond is easy to break and the like are solved, and the tumor antigen-antibody complex can be used as a novel tumor vaccine, and provides technical support for the research and development of the novel tumor vaccine.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a tumor antigen-antibody complex, and a preparation method and application thereof.
Background
Tumor antigens mainly include tumor specific antigens and tumor associated antigens. Possible mechanisms of body production of tumor antigens are: mutation of a gene; the gene that the cell does not originally express is activated; abnormal, ectopic expression of embryonic stage antigens or differentiation antigens; exogenous gene (e.g., viral gene) expression, and the like. New York esophageal squamous carcinoma antigen 1(NY-ESO-1) is one of tumor-testis antigen (CTA) subfamilies, NY-ESO-1 is processed in cells to generate antigenic peptide, and is combined with leukocyte antigen (HLA) molecules to form a complex, and the complex is recognized by CD4+ T lymphocytes and Cytotoxic T Lymphocytes (CTL) to induce organisms to generate stronger specific immune responses against corresponding tumor cells (International journal of laboratory medicine 36(21),3138-3141, 2015).
Inteins are internal protein elements whose host proteins self-cleave and catalyze the joining of flanking sequences by peptide bonds. The split intein is split at a specific site in the middle of a protein intron to form an N-terminal fragment and a C-terminal fragment, which are respectively encoded by two genes with a longer distance, and the two fragments are mutually recognized through non-covalent bonds to reconstruct a catalytic active center and mediate protein trans-splicing (Chinese biochemistry and molecular biology reports 24(6),512-521, 2008). The split intein GP41-1 was the most stable and the most rapid splicing reported in the literature (J Biol chem.287,28686-28696,2012). Wang Han et al successfully coupled the DNA Binding Domain (DBD) of a low temperature yeast strain to the synthetic VP64 activation domain using the split intein GP41-1 to construct a GAL 4-based bidirectional expression system (Pnas.115(15), 3900-. Yao Zhong et al developed a living cell method of split intein-mediated protein ligation (SINPL) using split intein GP41-1 and was applied to mammalian cell lines (Nature communications.11:2440,2020).
3G9 is an antibody against DEC205 on human dendritic cells (DC cells) as provided in patent CN 101888856B. DEC205 is used as a pattern recognition receptor on the surface of DC cells, can specifically capture antigen molecules, and is processed and presented to the immune system of a body to induce immune response. The anti-DEC 205 monoclonal antibody can be used as a targeting vector to specifically deliver drugs to DC cells, and can effectively enhance the process. In vitro experiments show that the antigen presentation efficiency assisted by the DEC205 targeted antibody can be enhanced by 1000 times (Zhang top. preparation of DEC-205 targeted antibody and humanization modification thereof [ D ]. Beijing coordination medical college (department of medicine of Qinghua university) & Chinese medical academy of sciences, 2013).
With the development of the field of immunotherapy, the limitations of NY-ESO-1 adoptive T cell therapy, combined check point inhibitor therapy and other methods are highlighted, and the DEC205 monoclonal antibody as a carrier has huge application prospects in tumor immunotherapy.
Disclosure of Invention
In order to solve the technical problems in the prior art, the invention aims to provide a tumor antigen-antibody complex, and a preparation method and application thereof.
In order to achieve the purpose and achieve the technical effect, the invention adopts the technical scheme that:
a tumor antigen-antibody complex comprises a tumor antigen and an antibody, wherein the heavy chain amino acid sequence of the tumor antigen-antibody complex is shown as SEQ ID NO. 10, the light chain amino acid sequence is shown as SEQ ID NO. 6, and the light chain does not participate in the reaction.
Furthermore, the tumor antigen-antibody complex is obtained by combining a tumor antigen with an antibody, the tumor antigen-antibody complex is antibody coupling protein 3G9-NY-ESO-1, the tumor antigen is NY-ESO-1, and the antibody is monoclonal antibody 3G9 of DEC 205.
Further, the tumor antigen-antibody complex is obtained by combining the tumor antigen with the antibody by splitting the intein GP41-1 in a trans-splicing mode.
A preparation method of a tumor antigen-antibody complex comprises the following steps:
step (1): construction of antibody fusion protein 3G9-IntN heavy chain expression vector
Step (2): preparation of antibody fusion protein 3G9-IntN
And (3): construction of fusion protein GB1-IntC-NY-ESO-1 expression vector
And (4): preparation of fusion protein GB1-IntC-NY-ESO-1
And (5): preparing antibody coupling protein 3G 9-NY-ESO-1.
Further, in step (1), the construction of the antibody fusion protein 3G9-IntN heavy chain expression vector comprises the following steps: the plasmid pUC57-Kan-GP41IN is used as a template, Fc-IntN-F with a base sequence shown as SEQ ID NO. 3 and Fc-IntN-R with a base sequence shown as SEQ ID NO. 4 are designed, an Fc-IntN gene fragment is amplified through PCR, then a corresponding fragment is recovered through gel and cloned between multiple cloning sites BamH I and Hind III on a phIgG1-3G9 vector to construct a heavy chain expression vector phIgG1-3G9-IntN capable of expressing the antibody fusion protein 3G 9-IntN.
Further, in the step (2), the preparation of the antibody fusion protein 3G9-IntN comprises the following steps: co-transfecting the antibody fusion protein 3G9-IntN heavy chain expression vector constructed in the step (1) and the antibody 3G9 light chain expression vector phIgK-3G9 into 293T cells according to the mass ratio of 1:1, and expressing the antibody fusion protein 3G9-IntN, wherein the amino acid sequence of the protein heavy chain is shown as SEQ ID NO:5, and the amino acid sequence of the light chain is shown as SEQ ID NO: 6.
Further, in the step (3), the construction of the expression vector of the fusion protein GB1-IntC-NY-ESO-1 comprises the following steps:
by using BamH I and NdeI double enzyme digestion plasmid pUC57-Kan-GB1-GP41IC and pET-30a (+) -NY-ESO-1, GB1-intC fragment and linearized vector pET-30a (+) -NY-ESO-1 are respectively obtained, and then fusion protein GB1-intC-NY-ESO-1 expression vector pET-30a (+) -GB1-intC-NY-ESO-1 is constructed by using T4 ligase and using T4 ligation method.
Further, in step (4), the preparation of the fusion protein GB1-IntC-NY-ESO-1 comprises the following steps:
the fusion protein GB1-IntC-NY-ESO-1 expression vector pET-30a (+) -GB1-IntC-NY-ESO-1 constructed in the step (3) is transformed into BL21(DE3) competent cells, the fusion protein GB1-IntC-NY-ESO-1 is prepared according to pET systems handbook, the protein amino acid sequence is shown as SEQ ID NO:9, and HisTrpTM HP is used for purifying the target protein.
Further, in the step (5), the preparation of the antibody-conjugated protein 3G9-NY-ESO-1 comprises the following steps:
and (3) mixing the antibody fusion protein 3G9-IntN obtained in the step (2) and the fusion protein GB1-IntC-NY-ESO-1 obtained in the step (4) according to a certain molar ratio, adding DTT with the final concentration of 10 micromole per liter to induce and catalyze the trans-splicing reaction of the broken intein GP41-1, and after the splicing reaction, connecting five residual amino acid residues of the IntN TRSGY and five amino acid residues SSSDV of the IntC by peptide bonds to obtain the required antibody coupling protein 3G9-NY-ESO-1, wherein the amino acid sequence of a heavy chain of the protein is shown as SEQ ID NO. 10, the amino acid sequence of a light chain is shown as SEQ ID NO. 6, and the light chain does not participate in the reaction.
An application of tumor antigen-antibody complex in preparing tumor vaccine.
Compared with the prior art, the invention has the following beneficial effects:
the invention discloses a tumor antigen-antibody complex, a preparation method and an application thereof, wherein the tumor antigen-antibody complex comprises a tumor antigen and an antibody, the tumor antigen is NY-ESO-1, the antibody is a monoclonal antibody 3G9 of DEC205, the tumor antigen-antibody complex is obtained by combining the tumor antigen and the antibody, the tumor antigen-antibody complex is an antibody coupling protein 3G9-NY-ESO-1, a heavy chain amino acid sequence is shown as SEQ ID NO. 10, a light chain amino acid sequence is shown as SEQ ID NO. 6, and a light chain does not participate in reaction. According to the invention, the tumor antigen and the antibody are connected by a trans-splicing method of the broken intein GP41-1, the tumor antigen and the antibody are connected by peptide bonds, the stability is higher, the specific binding is stronger, the reaction condition is mild, the efficiency is high, the process is simple, no toxic or harmful substance is added, no by-product is generated, the safety is high, the problems that a chemical reagent is required to be added when the antigen and the antibody are connected by a chemical coupling method and the like in the prior art, certain toxicity exists, the chemical coupling is unstable, the coupling bond is easy to break and the like are solved, the tumor antigen-antibody complex can be used as a novel tumor vaccine, and a technical support and a new thought are provided for the research and development of the novel tumor vaccine.
Drawings
FIG. 1 is a plasmid map of the heavy chain expression vector phIgG1-3G9-IntN of the antibody fusion protein 3G9-IntN of the present invention;
FIG. 2 is an SDS-PAGE staining identification of the antibody fusion protein 3G9-IntN of the present invention;
FIG. 3 is a plasmid map of expression vector pET-30a (+) -GB1-IntC-NY-ESO-1 of fusion protein GB1-IntC-NY-ESO-1 of the present invention;
FIG. 4 is an SDS-PAGE staining identification of the fusion protein GB1-IntC-NY-ESO-1 of the present invention;
FIG. 5 is a photograph of the SDS-PAGE staining identification of the split intein trans-splicing of the present invention to prepare antibody-coupled protein 3G 9-NY-ESO-1.
Detailed Description
The present invention is described in detail below so that the advantages and features of the present invention can be more easily understood by those skilled in the art, and thus the scope of the present invention can be clearly and clearly defined.
The following presents a simplified summary of one or more aspects in order to provide a basic understanding of such aspects. This summary is not an extensive overview of all contemplated aspects, and is intended to neither identify key or critical elements of all aspects nor delineate the scope of any or all aspects. Its sole purpose is to present some concepts of one or more aspects in a simplified form as a prelude to the more detailed description that is presented later.
A tumor antigen-antibody complex comprises a tumor antigen NY-ESO-1 and an antibody, wherein the tumor antigen is a monoclonal antibody 3G9 of DEC205 on a human dendritic cell (DC cell) provided in Chinese patent publication No. CN 101888856B, the tumor antigen-antibody complex is obtained by combining the tumor antigen and the antibody through the reverse splicing of a broken intein GP41-1, the tumor antigen-antibody complex is an antibody coupling protein 3G9-NY-ESO-1, the heavy chain amino acid sequence of the tumor antigen-antibody complex is shown as SEQ ID NO. 10, the light chain amino acid sequence is shown as SEQ ID NO. 6, and a light chain does not participate in reaction.
A preparation method of a tumor antigen-antibody complex comprises the following steps:
preparing a fusion protein GB1-IntC-NY-ESO-1 by fusing and expressing a tumor antigen NY-ESO-1 and GB1-GP41-1-InteinC (C-terminal exopeptide), wherein GB1 is a common protein solubilizing label and can be cut off together with IntC after completing splicing reaction;
antibody 3G9 and GP41-1-InteinN (C-terminal exon peptide) are fused and expressed to prepare antibody fusion protein 3G 9-IntN;
the fusion protein GB1-IntC-NY-ESO-1 is mixed with the antibody fusion protein 3G9-IntN, and the antibody coupling protein 3G9-NY-ESO-1 is generated by the trans-splicing of the split intein GP 41-1. The preparation steps of the fusion protein GB1-IntC-NY-ESO-1 and the antibody fusion protein 3G9-IntN are not in mutual sequence.
Example 1
Construction of antibody fusion protein 3G9-IntN heavy chain expression vector
The mammalian codon optimized base sequence (SEQ ID NO:2) for the amino acid sequence of GP41-1-InteinN polypeptide fragment (SEQ ID NO:1) was obtained from plasmid pUC57-Kan-GP41IN (provided by Sovik institute of Industrial park Tokyo Biotech, Inc.), and antibody 3G9 heavy chain expression vector phIgG1-3G9 and light chain expression vector phIgK-3G9 are provided by Sovik institute of Industrial park Tokyo Biotech, Inc. The construction method comprises the following steps:
plasmid pUC57-Kan-GP41IN (provided by Soochow Industrial park Tokyo Biotechnology Co., Ltd.) was used as a template, primers in Table 1 were designed, Fc-IntN gene fragments were amplified by PCR, and then corresponding fragments were recovered by gel (DNA gel recovery kit, Byuntian Biotechnology research institute, cat # D0056) and cloned between BamH I and HindIII, which were multiple cloning sites on a phIgG1-3G9 vector, to construct a heavy chain expression vector IgG phG 1-3G9-IntN that can express antibody fusion protein 3G9-IntN, and the plasmid map is shown in FIG. 1, and was sequenced, identified correctly and then stored.
The PCR amplification conditions were as follows:
PCR kit:
FastPfu DNA Polymerase (containing 2.5mM dNTPs, catalog number AP 221-11);
PCR amplification procedure: the PCR product was run on a 1% agarose gel at 98 ℃ for 10min, 98 ℃ for 40s, 56 ℃ for 30s, 72 ℃ for 1min, 72 ℃ for 10min, 10 ℃ for 10 min.
TABLE 1
Example 2
Preparation of antibody fusion protein 3G9-IntN
The antibody fusion protein 3G9-IntN heavy chain expression vector constructed in example 1 and the antibody 3G9 (anti-human DEC205 monoclonal antibody) light chain expression vector phIgK-3G9 (provided by Sozhou Industrial park Tokyo Biotech Co., Ltd.) were co-transfected into 293T cells at a mass ratio of 1:1 to express 3G9-IntN protein (protein heavy chain amino acid sequence shown in SEQ ID NO:5 and light chain amino acid sequence shown in SEQ ID NO: 6). The specific method comprises the following steps:
Example 3
Construction of fusion protein GB1-IntC-NY-ESO-1 expression vector
The E.coli codon optimized base sequence (SEQ ID NO:8) of the amino acid sequence (SEQ ID NO:7) of the GB1-GP41-1-InteinC polypeptide fragment was obtained from the plasmid pUC57-Kan-GB1-GP41IC (provided by Sozhou Industrial park Tokyo Biotech, Inc.).
By using BamH I (Baobioengineering (Dalian) Co., Ltd., product No. 1010A) and NdeI (Baobioengineering (Dalian) Co., Ltd., product No. 1161A) double digestion plasmids pUC57-Kan-GB1-GP41IC and pET-30A (+) -NY-ESO-1 (provided by Sovizhou Industrial park Tokyo Biotech Co., Ltd.), respectively, GB1-IntC fragment and linearized vector pET-30A (+) -NY-ESO-1 are obtained, and then using T4 ligase (Baobioengineering (Dalian) Co., Ltd., product No. 2050A), fusion protein GB1-IntC-NY-ESO-1 expression vector pET-30A (+) -1-IntC-NY-ESO-1 is constructed by using T4 ligation method well known in the art, and the plasmid map is shown in FIG. 3, and (5) warehousing after sequencing and identification are correct.
Example 4
Preparation of fusion protein GB1-IntC-NY-ESO-1
The fusion protein GB1-IntC-NY-ESO-1 expression vector pET-30a (+) -GB1-IntC-NY-ESO-1 constructed in example 3 is transformed into BL21(DE3) competent cells (Kangji, CW0809S), and the transformation method can be referred to the specification of the competent cells. The fusion protein GB1-IntC-NY-ESO-1 (amino acid sequence of the protein is shown in SEQ ID NO:9) was prepared according to the handbook of pET systems (TB 0558 th Edition02/99, Novagen). The specific method comprises the following steps:
BL21(DE3) competent cells were transformed, plated with kanamycin-resistant plates, and the colonies were picked up to 1 ml of LB liquid medium (available from Sovium Industrial park, Tokyo Biotech Co., Ltd.) and cultured at 37 ℃ and 200rpm for 8 hours to preserve glycerol. 10 μ l of glycerol was inoculated into 5 ml of LB liquid medium and cultured overnight at 37 ℃ and 200 rpm. Then 5 ml of the bacterial liquid is expanded and cultured into 500 ml of LB liquid culture medium, the culture is carried out for 2-3h at 37 ℃ and 200rpm, the bacterial liquid OD600 is about 0.8h, and IPTG (Solarbio, product number I8070) with the final concentration of 1 millimole per liter is added for induction expression, and the induction conditions are as follows: induction was carried out at 25 ℃ and 180rpm for 20 h. After induction, the cells were collected by centrifugation at 10000g for 5min, resuspended in 60 ml of inclusion body wash buffer (supplied by Soviea Industrial park Weida Biotech Co., Ltd.), incubated at 30 ℃ for 15min with lysozyme (Sigma-Aldrich, cat # L6876) at a final concentration of 100. mu.g per ml, and then sonicated on ice using an ultrasonic cell disruptor under the following conditions: 400W, 5 seconds of ultrasound/5 seconds of interval, 30min totally, the result of ultrasound is that the bacterial liquid is not sticky. After cell lysis, 10000g are centrifuged for 10min, and inclusion bodies are collected. Removing supernatant, washing the inclusion body precipitate by using 100 ml of inclusion body washing buffer solution for heavy suspension washing, and repeatedly washing the inclusion body for at least 3 times until the inclusion body is cleaned. The inclusion bodies were dissolved by adding 50 ml of inclusion body dissolving buffer (supplied by Soviea Industrial park Tokyo Biotech Co., Ltd.), and dissolved on a magnetic stirrer at room temperature for 2-3 hours, and the inclusion bodies were substantially completely dissolved. 20000g, centrifugation at 4 ℃ for 30min, discarding the precipitate, filtering the supernatant with 0.22 μm, desalting with desalting column (GE Healthcare, 17-5087-01), the desalting method can be found in the specification of desalting column. After the buffer solution was replaced with a desalting buffer (supplied by Soviea Industrial park, Tokyo Biotech Co., Ltd.), the target protein was purified using HisTraptM HP (GE Healthcare, 17-5248-01), and the purification method was described in the specification of the purification column. The concentration of the prepared recombinant protein is detected by a BCA method (Biyuntian biotechnology institute, Cat number P0009), and the detection method refers to the specification of a detection kit. The prepared protein is subpackaged and stored at-80 ℃. 80 microliters of GB1-IntC-NY-ESO-1 protein was added with 20 microliters of 5 XSDS-PAGE loading buffer (provided by Soochow Industrial park, Tech-Biotech Co., Ltd.), and the sample was prepared by boiling water bath for 10 minutes, and SDS-PAGE was followed by examination and identification of the protein. The method of SDS-PAGE is well known to those skilled in the art and is briefly described below. Protein tags (Thermo, cat # 26616) and GB1-IntC-NY-ESO-1 proteins were sequentially spotted in this order, after electrophoresis for 80V for 30 minutes, the voltage was adjusted to 120V until the indicator reached the end, and after staining with Coomassie brilliant blue staining solution (available from Soviet industries, Inc.) for 1 hour at room temperature, the staining was carried out in a boiling water bath for 30 minutes to decolorize, and the results are shown in FIG. 4.
Inclusion body wash buffer: 20mM Tris-HCl pH 7.5, 10mM EDTA, 1% Triton X-100.
Inclusion body solubilization buffer: 20mM Tris-HCl pH 8.5, 6M guanidine hydrochloride.
Desalting buffer solution: 20mM Tris-HCl pH 8.5, 150mM sodium chloride.
Example 5
Preparation of antibody-conjugated protein 3G9-NY-ESO-1
The antibody fusion protein 3G9-IntN prepared in example 2 and the fusion protein GB1-IntC-NY-ESO-1 prepared in example 4 were mixed at different molar ratios (the molar ratios of 3G 9-IntN: GB1-IntC-NY-ESO-1 were 3:1, 1.5:1, 1:1.5 and 1:3, respectively), DTT (Solarbio, D8220-25G) was added at a final concentration of 10. mu. mol per liter to induce catalytic cleavage of the intein GP41-1 trans-splicing reaction, after the splicing reaction, the five remaining amino acid residues of the IntN TRSGY are connected with the five amino acid residues SSSDV of the IntC by peptide bonds to prepare the tumor vaccine 3G9-NY-ESO-1 protein (the amino acid sequence of the heavy chain of the protein is shown in SEQ ID NO:10, the amino acid sequence of the light chain is shown in SEQ ID NO:6, and the light chain does not participate in the reaction). Reaction buffer: pH 8.5, 20 mmol/l Tris hcl, 150 mmol/l sodium chloride (provided by soyama industrial park reachability biotechnology limited), reaction conditions: the reaction was carried out at 37 ℃ for 2 hours. 80 microliters of the reaction product was added to 20 microliters of 5 xSDS-PAGE sample buffer (supplied by Soviet Industrial park, Tech Biotech Co., Ltd.), and subjected to boiling water bath for 10 minutes to prepare a sample, SDS-PAGE was performed, followed by examination and protein identification. The method of SDS-PAGE is well known to those skilled in the art and is briefly described below. According to protein markers (Thermo, cat # 26616), 3G9-IntN protein, GB1-IntC-NY-ESO-1 protein, 3G 9-IntN: GB1-IntC-NY-ESO-1 is sequentially spotted in the order of 3:1, 1.5:1, 1:1.5 and 1:3, the electrophoresis is carried out for 80V and 30 minutes, then the voltage is regulated to 120V until the indicator is bottom, Coomassie brilliant blue staining solution (provided by Soviet Biotech limited, Suzhou industrial park) is used for staining for 1 hour at room temperature, boiling water bath is carried out for 30 minutes for decolorization, and the result is shown in FIG. 5, and 3G9-IntN is determined: the optimal molar ratio of GB1-IntC-NY-ESO-1 is 1.5: 1.
The parts or structures of the invention not specifically described may be any parts or structures of the prior art or the prior art, and are not described herein again.
The above description is only an embodiment of the present invention, and is not intended to limit the scope of the present invention, and all equivalent structures or equivalent processes performed by the present invention or directly or indirectly applied to other related technical fields are also included in the scope of the present invention.
Claims (10)
1. A tumor antigen-antibody complex is characterized by comprising a tumor antigen and an antibody, wherein the heavy chain amino acid sequence of the tumor antigen-antibody complex is shown as SEQ ID NO. 10, the light chain amino acid sequence is shown as SEQ ID NO. 6, and the light chain does not participate in reaction.
2. The tumor antigen-antibody complex of claim 1, wherein the tumor antigen-antibody complex is obtained by combining a tumor antigen with an antibody, the tumor antigen-antibody complex is antibody-conjugated protein 3G9-NY-ESO-1, the tumor antigen is NY-ESO-1, and the antibody is monoclonal antibody 3G9 of DEC 205.
3. The tumor antigen-antibody complex of claim 2, wherein said tumor antigen-antibody complex is obtained by binding tumor antigen to antibody by cleaving the intein GP41-1 by trans-splicing.
4. The method for preparing a tumor antigen-antibody complex according to any one of claims 1 to 3, comprising the steps of:
step (1): construction of antibody fusion protein 3G9-IntN heavy chain expression vector
Step (2): preparation of antibody fusion protein 3G9-IntN
And (3): construction of fusion protein GB1-IntC-NY-ESO-1 expression vector
And (4): preparation of fusion protein GB1-IntC-NY-ESO-1
And (5): preparing antibody coupling protein 3G 9-NY-ESO-1.
5. The method for preparing the tumor antigen-antibody complex of claim 4, wherein the step (1) of constructing the antibody fusion protein 3G9-IntN heavy chain expression vector comprises the steps of: the plasmid pUC57-Kan-GP41IN is used as a template, Fc-IntN-F with a base sequence shown as SEQ ID NO. 3 and Fc-IntN-R with a base sequence shown as SEQ ID NO. 4 are designed, Fc-IntN gene fragments are amplified through PCR, corresponding fragments are recovered through gel, and are cloned between a multiple cloning site BamH I and Hind III on a phIgG1-3G9 vector to construct a heavy chain expression vector phIgG1-3G9-IntN capable of expressing the antibody fusion protein 3G 9-IntN.
6. The method for preparing a tumor antigen-antibody complex according to claim 4, wherein the step of preparing the antibody fusion protein 3G9-IntN in step (2) comprises: co-transfecting the antibody fusion protein 3G9-IntN heavy chain expression vector constructed in the step (1) and the antibody 3G9 light chain expression vector phIgK-3G9 into 293T cells according to the mass ratio of 1:1, and expressing the antibody fusion protein 3G9-IntN, wherein the amino acid sequence of the protein heavy chain is shown as SEQ ID NO:5, and the amino acid sequence of the light chain is shown as SEQ ID NO: 6.
7. The method for preparing a tumor antigen-antibody complex according to claim 4, wherein in step (3), the step of constructing the expression vector of fusion protein GB1-IntC-NY-ESO-1 comprises:
by using BamH I and NdeI double enzyme digestion plasmid pUC57-Kan-GB1-GP41IC and pET-30a (+) -NY-ESO-1, GB1-intC fragment and linearized vector pET-30a (+) -NY-ESO-1 are respectively obtained, and then fusion protein GB1-intC-NY-ESO-1 expression vector pET-30a (+) -GB1-intC-NY-ESO-1 is constructed by using T4 ligase and using T4 ligation method.
8. The method for preparing a tumor antigen-antibody complex according to claim 4, wherein the step of preparing fusion protein GB1-IntC-NY-ESO-1 in step (4) comprises:
the fusion protein GB1-IntC-NY-ESO-1 expression vector pET-30a (+) -GB1-IntC-NY-ESO-1 constructed in the step (3) is transformed into BL21(DE3) competent cells, the fusion protein GB1-IntC-NY-ESO-1 is prepared according to pET systems handbook, the protein amino acid sequence is shown as SEQ ID NO:9, and HisTrpTM HP is used for purifying the target protein.
9. The method for preparing a tumor antigen-antibody complex according to claim 4, wherein in step (5), the step of preparing antibody-conjugated protein 3G9-NY-ESO-1 comprises:
and (3) mixing the antibody fusion protein 3G9-IntN obtained in the step (2) and the fusion protein GB1-IntC-NY-ESO-1 obtained in the step (4) according to a certain molar ratio, adding DTT with the final concentration of 10 micromole per liter to induce and catalyze the trans-splicing reaction of the broken intein GP41-1, after the splicing reaction, connecting five residual amino acid residues of IntN TRSGY and five amino acid residues SSSDV of IntC by peptide bonds to obtain the required antibody coupling protein 3G9-NY-ESO-1, wherein the amino acid sequence of a heavy chain of the protein is shown as SEQ ID NO:10, the amino acid sequence of a light chain is shown as SEQ ID NO:6, and the light chain does not participate in the reaction.
10. Use of a tumor antigen antibody complex according to any one of claims 1-4 in the preparation of a tumor vaccine.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210703064.2A CN115028741A (en) | 2022-06-21 | 2022-06-21 | Tumor antigen-antibody complex, preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210703064.2A CN115028741A (en) | 2022-06-21 | 2022-06-21 | Tumor antigen-antibody complex, preparation method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN115028741A true CN115028741A (en) | 2022-09-09 |
Family
ID=83124898
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210703064.2A Pending CN115028741A (en) | 2022-06-21 | 2022-06-21 | Tumor antigen-antibody complex, preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115028741A (en) |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040077842A1 (en) * | 2000-11-01 | 2004-04-22 | Jeff Himawan | Method of producing biospecific molecules by protein trans-splicing |
| CN101888856A (en) * | 2007-11-07 | 2010-11-17 | 塞尔德克斯医疗公司 | Antibodies that bind human dendritic and epithelial cell 205 (DEC-205) |
| CN103409451A (en) * | 2013-06-28 | 2013-11-27 | 扬州维克斯生物科技有限公司 | Method for loading tumor antigen peptide to dendritic cell (DC) in targeting manner |
| CN107922471A (en) * | 2015-06-24 | 2018-04-17 | 优瑞科生物技术公司 | Target construct of 1 peptides of NY ESO/MHC compounds and application thereof |
| CN110770345A (en) * | 2017-04-14 | 2020-02-07 | 托尔奈公司 | Immunomodulatory polynucleotides, antibody conjugates, and methods of use thereof |
| WO2021099607A1 (en) * | 2019-11-22 | 2021-05-27 | Cytiva Bioprocess R&D Ab | Protein purification using a split intein system |
| WO2021158073A1 (en) * | 2020-02-06 | 2021-08-12 | 아주대학교산학협력단 | Fusion antibody for presenting antigen-derived t cell antigen epitope or peptide containing same on cell surface, and composition comprising same |
| US20210388364A1 (en) * | 2019-02-06 | 2021-12-16 | Helmholtz Zentrum Müncher - Deutsches Forschungszentrum Für Gesundheit Und Umwell (GMBH) | Method for detecting a specific splice event of a gene of interest |
| CN114450292A (en) * | 2019-09-09 | 2022-05-06 | 武汉友芝友生物制药股份有限公司 | Cleavage type intein, and method for producing recombinant polypeptide using same |
-
2022
- 2022-06-21 CN CN202210703064.2A patent/CN115028741A/en active Pending
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040077842A1 (en) * | 2000-11-01 | 2004-04-22 | Jeff Himawan | Method of producing biospecific molecules by protein trans-splicing |
| CN101888856A (en) * | 2007-11-07 | 2010-11-17 | 塞尔德克斯医疗公司 | Antibodies that bind human dendritic and epithelial cell 205 (DEC-205) |
| CN103409451A (en) * | 2013-06-28 | 2013-11-27 | 扬州维克斯生物科技有限公司 | Method for loading tumor antigen peptide to dendritic cell (DC) in targeting manner |
| CN107922471A (en) * | 2015-06-24 | 2018-04-17 | 优瑞科生物技术公司 | Target construct of 1 peptides of NY ESO/MHC compounds and application thereof |
| CN110770345A (en) * | 2017-04-14 | 2020-02-07 | 托尔奈公司 | Immunomodulatory polynucleotides, antibody conjugates, and methods of use thereof |
| US20210388364A1 (en) * | 2019-02-06 | 2021-12-16 | Helmholtz Zentrum Müncher - Deutsches Forschungszentrum Für Gesundheit Und Umwell (GMBH) | Method for detecting a specific splice event of a gene of interest |
| CN114450292A (en) * | 2019-09-09 | 2022-05-06 | 武汉友芝友生物制药股份有限公司 | Cleavage type intein, and method for producing recombinant polypeptide using same |
| WO2021099607A1 (en) * | 2019-11-22 | 2021-05-27 | Cytiva Bioprocess R&D Ab | Protein purification using a split intein system |
| WO2021158073A1 (en) * | 2020-02-06 | 2021-08-12 | 아주대학교산학협력단 | Fusion antibody for presenting antigen-derived t cell antigen epitope or peptide containing same on cell surface, and composition comprising same |
Non-Patent Citations (2)
| Title |
|---|
| ACCESSION NO.:NP_001318.1: "cancer/testis antigen 1 [Homo sapiens]", GENPEPT, 4 December 2021 (2021-12-04) * |
| TAKEMASA TSUJI: "Antibody-Targeted NY-ESO-1 to Mannose Receptor or DEC-205 In Vitro Elicits Dual Human CD8+ and CD4+ T Cell Responses with Broad Antigen Specicity", THE JOURNAL OF IMMUNOLOGY, vol. 186, no. 2, 31 December 2011 (2011-12-31), pages 1218 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| KR20140007394A (en) | Novel immunoglobulin-binding polypeptide | |
| JP6238967B2 (en) | Process for purification of recombinant P. falciparum sporozoite surrounding protein | |
| JP2015508664A (en) | Enzymatic cleavage on the column | |
| CN110835366B (en) | Tag polypeptide for promoting soluble expression of protein and application thereof | |
| JP4088584B2 (en) | A method for separating a target protein from a fusion protein. | |
| CN115028741A (en) | Tumor antigen-antibody complex, preparation method and application thereof | |
| CN114989311A (en) | 3G9-LNM antibody coupling protein and preparation method and application thereof | |
| CN114887047A (en) | NY-ESO-1 tumor antigen-based vaccine and preparation method thereof | |
| CN107629129B (en) | Method for producing and purifying polypeptides | |
| CN110540601B (en) | Recombinant PLB-hEGF fusion protein and application thereof | |
| CN114949191A (en) | Tumor vaccine and preparation method thereof | |
| JP2561122B2 (en) | Functional polypeptide | |
| RU2453604C1 (en) | Hybrid protein (versions), bacterial strain escherichia coli - hybrid protein producer (versions) and method for producing methionine-free human interferon alpha-2 | |
| US9580488B2 (en) | Fusion tags and expression vector system for the expression of human parathyroid hormone (rhPTH) | |
| RU2441072C1 (en) | FUSION PROTEIN, ESCHERICHIA COLI STRAIN BEING FUSION PROTEIN PRODUCER AND METHOD FOR PRODUCING METHIONINE-FREE HUMAN INTERFERON ALPHA-2b OF SUCH FUSION PROTEIN | |
| CN108707196B (en) | Method for preparing human tumor necrosis factor I type receptor extracellular domain protein | |
| Wang et al. | Preparation of a peptide vaccine against GnRH by a bioprocess system based on asparaginase | |
| CN114073759A (en) | Skin wound healing preparation | |
| WO2016128363A1 (en) | Method for producing a recombinant protein of interest | |
| CN112898407B (en) | Preparation method of recombinant camel-derived serum albumin | |
| WO2007096899A2 (en) | Affinity polypeptide for purification of recombinant proteins | |
| CN114292321B (en) | Soluble expression EG95 protein and preparation method and application thereof | |
| TWI712691B (en) | Dextran affinity tag and application thereof | |
| CN113528544B (en) | Gene for coding soluble HPV23L1 protein and construction and application of recombinant plasmid thereof | |
| CN114381471A (en) | Application of auxiliary protein in recombinant protein production and fusion expression system |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |