CN111944018A - Antigen polypeptide for inducing liver cancer specific cytotoxic T lymphocyte and application thereof - Google Patents
Antigen polypeptide for inducing liver cancer specific cytotoxic T lymphocyte and application thereof Download PDFInfo
- Publication number
- CN111944018A CN111944018A CN202010889660.5A CN202010889660A CN111944018A CN 111944018 A CN111944018 A CN 111944018A CN 202010889660 A CN202010889660 A CN 202010889660A CN 111944018 A CN111944018 A CN 111944018A
- Authority
- CN
- China
- Prior art keywords
- cell
- liver cancer
- antigen
- specific cytotoxic
- lymphocyte
- 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.)
- Granted
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/14—Blood; Artificial blood
- A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
-
- 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
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
-
- 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
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0636—T lymphocytes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0636—T lymphocytes
- C12N5/0638—Cytotoxic T lymphocytes [CTL] or lymphokine activated killer cells [LAK]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0639—Dendritic cells, e.g. Langherhans cells in the epidermis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
- G01N33/5047—Cells of the immune system
- G01N33/505—Cells of the immune system involving T-cells
-
- 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
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/515—Animal cells
- A61K2039/5154—Antigen presenting cells [APCs], e.g. dendritic cells or macrophages
-
- 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
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/515—Animal cells
- A61K2039/5156—Animal cells expressing foreign proteins
-
- 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
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/515—Animal cells
- A61K2039/5158—Antigen-pulsed cells, e.g. T-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
- C12N2503/00—Use of cells in diagnostics
- C12N2503/02—Drug screening
-
- 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
- C12N2510/00—Genetically modified cells
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
- G01N2500/10—Screening for compounds of potential therapeutic value involving cells
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Hematology (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Medicinal Chemistry (AREA)
- Wood Science & Technology (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Pharmacology & Pharmacy (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- General Engineering & Computer Science (AREA)
- Tropical Medicine & Parasitology (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Toxicology (AREA)
- General Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Food Science & Technology (AREA)
- Epidemiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Pathology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Mycology (AREA)
- Oncology (AREA)
Abstract
The application discloses an antigen polypeptide for inducing liver cancer specific cytotoxic T lymphocytes and application thereof. The amino acid sequence of the antigen polypeptide for inducing the liver cancer specific cytotoxic T lymphocyte comprises a sequence shown in Seq ID No. 1. The antigen polypeptide can specifically and efficiently induce the hepatoma carcinoma cell cytotoxic T lymphocyte, and the induced hepatoma carcinoma specific cell cytotoxic T lymphocyte is superior to CTL induced by other existing antigen polypeptides in immunogenicity, positive rate of antigen-loaded cell cytotoxic T lymphocyte and killing efficiency of the cell in vitro experiments. The antigen polypeptide is adopted to induce the cytotoxic T lymphocyte which can efficiently and specifically kill the liver cancer tumor cells, the preparation method is efficient and convenient, compared with other immune cell therapies, the antigen polypeptide has the characteristics of low cost, high yield and simple process flow, and a new scheme and a new way are provided for clinical application of liver cancer immune cell therapy.
Description
Technical Field
The application relates to the field of liver cancer immune cell therapy, in particular to an antigen polypeptide for inducing liver cancer specific cytotoxic T lymphocytes and application thereof.
Background
Immune cell therapy is the only method which has the possibility of completely eliminating cancer cells in the prior art, makes up the defects of the traditional therapy, and is considered as a treatment means with the most promising development prospect in the twenty-first century tumor comprehensive treatment mode.
Cytotoxic T lymphocytes (abbreviated CTLs) are the most important effector cells of the body against tumors and are widely used in immune cell therapy. Antigen loading is an essential process in the preparation process of CTL, and in CTL industrialization and clinical application, the problems of specificity and effectiveness of loaded antigen polypeptide must be solved.
In the development of the CTL therapy aiming at liver cancer at present, although relevant documents report that the liver cancer is associated with positive polypeptide sequences of viruses (HBV and HCV); and the antigen polypeptide sequence with high liver cancer expression. However, there are no reports on the strong and weak immunogenicity of these antigens, and even more, there are no reports on whether these antigens can be loaded by CTLs via antigen-presenting cells such as DC, and the strong and weak killing ability of CTLs loaded with these polypeptides of liver cancer antigens.
Disclosure of Invention
The application aims to provide a novel antigen polypeptide for inducing liver cancer specific cytotoxic T lymphocytes and application thereof.
The following technical scheme is adopted in the application:
the first aspect of the application discloses an antigen polypeptide for inducing liver cancer specific cytotoxic T lymphocyte, the amino acid sequence of the antigen polypeptide comprises the sequence shown in Seq ID No.1,
Seq ID No.1：FLLXVDATV
wherein, X is selected from any one of alanine, leucine, isoleucine, methionine, cysteine and threonine.
It is noted that the antigenic polypeptide of the present application can efficiently induce liver cancer specific CTLs, and the obtained antigen-loaded liver cancer specific CTLs have strong immunogenicity, high positive rate, and strong in vitro killing ability and efficiency. In particular, in one implementation of the present application, X is leucine, such that the antigenic polypeptide has strong immunogenicity, high positive rate, strong in vitro killing ability and efficiency. As regards X as alanine, isoleucine, methionine, cysteine or threonine, the corresponding antigenic polypeptide has, in the experimental analysis, the same specificity and function as the antigenic polypeptide in which X is leucine.
It should be noted that the key point of the present application is the antigen polypeptide with sequence shown in Seq ID No.1, and it is understood that on the basis of not affecting the performance of the antigen polypeptide in inducing liver cancer specific CTL and induced CTL, amino acid sequences with other functions or functions may be added at both ends of the antigen polypeptide with sequence shown in Seq ID No.1, and are not specifically limited herein.
In a second aspect of the present application, there is disclosed a nucleic acid sequence, which may be DNA or RNA, encoding an antigenic polypeptide of the present application.
It is noted that the nucleic acid sequences of the present application are capable of encoding the antigenic polypeptides of the present application; it is understood that the specific nucleic acid sequence can be determined by referring to the amino acid sequence of the sequence shown in Seq ID No.1 with reference to the genetic code, and is not particularly limited herein.
In one embodiment of the application, the nucleic acid sequence comprises a sequence which is degenerate as any one or as any one of the sequences shown in Seq ID No.2 to Seq ID No.7,
Seq ID No.2：5’-TTCCTGGCACTAGTCGATGCAACAGTA-3’
Seq ID No.3：5’-TTCCTGCTGCTAGTCGATGCAACAGTA-3’
Seq ID No.4：5’-TTCCTGATCCTAGTCGATGCAACAGTA-3’
Seq ID No.5：5’-TTCCTGATGCTAGTCGATGCAACAGTA-3’
Seq ID No.6：5’-TTCCTGTGTCTAGTCGATGCAACAGTA-3’
Seq ID No.7：5’-TTCCTGACACTAGTCGATGCAACAGTA-3’。
wherein, the sequences shown in Seq ID No.2 to Seq ID No.7 correspond to the antigen polypeptide which codes the application and X is alanine, leucine, isoleucine, methionine, cysteine, threonine.
It will be understood that degenerate sequences in accordance with the present application are nucleic acid sequences which, by virtue of the degeneracy of the degenerate codons, are likewise capable of encoding the amino acid sequence shown in Seq ID No.1 but differ from the sequences shown in Seq ID No.2 to Seq ID No. 7.
It is to be noted that the nucleic acid sequences of the present application are critical to be capable of encoding the antigenic polypeptides of the present application; it is understood that other functional or functional sequences may be added to the nucleic acid sequences of the present application without affecting the encoding or expression of the antigenic polypeptides of the present application, and are not specifically limited herein.
In a third aspect of the present application, a gene expression vector is disclosed, wherein the gene expression vector has an inserted foreign gene capable of encoding and expressing the antigenic polypeptide of the present application.
It is understood that the key of the gene expression vector of the present application is that the foreign gene can encode and express the antigen polypeptide of the present application, and as for the framework structure of the gene expression vector, such as promoter, terminator and the like, reference can be made to the existing gene expression vector, which is not specifically limited herein.
In one implementation of the present application, the exogenous gene of the gene expression vector comprises a nucleic acid sequence of the present application.
In a fourth aspect of the present application, a recombinant cell comprising the gene expression vector of the present application is disclosed.
It is noted that the recombinant cells of the present application are capable of encoding and expressing the antigenic polypeptides of the present application by transduction or transfection of the gene expression vectors of the present application; the antigen polypeptide of the application can be obtained by culturing the recombinant cells and extracting and purifying protein. It is understood that the key to the recombinant cell of the present application is that it contains the gene expression vector of the present application, and the specific cell to be used is not particularly limited, depending on the framework structure of the gene expression vector.
In a fifth aspect, the present application discloses an Antigen Presenting Cell (APC) for inducing hepatoma-specific cytotoxic T lymphocytes, which is capable of presenting the antigenic polypeptide of the present application to CD 8-positive T cells and activating the production of corresponding specific cytotoxic T lymphocytes.
In one implementation of the present application, the antigen presenting cells that induce hepatoma-specific cytotoxic T lymphocytes are obtained by stimulating the antigen presenting cells with the antigen polypeptide of the present application, or the nucleic acid sequence of the present application is introduced into the antigen presenting cells so that the antigen presenting cells can express the antigen polypeptide of the present application, thereby obtaining the ability to induce hepatoma-specific cytotoxic T lymphocytes.
In general, an APC having the ability to induce CTL is obtained by directly stimulating culture with an antigenic polypeptide, or an APC may be formed by introducing a nucleic acid sequence expressing the antigenic polypeptide into the APC and expressing the antigenic polypeptide by the APC. In one embodiment of the present application, the antigen presenting cells obtained by direct stimulation culture of the synthesized antigen polypeptide are used for inducing hepatoma-specific cytotoxic T lymphocytes.
In a sixth aspect of the present application, there is disclosed an antigenic polypeptide of the present application or a liver cancer-specific cytotoxic T lymphocyte induced by an antigen presenting cell of the present application which induces a liver cancer-specific cytotoxic T lymphocyte.
It can be understood that the liver cancer specific CTL is obtained by induction of the antigen polypeptide or APC, so that the liver cancer specific CTL has the advantages of strong immunogenicity, high positive rate, high in-vitro killing capacity and efficiency and the like, and can better meet the clinical application of treatment, prevention or detection of liver cancer.
A seventh aspect of the present application discloses a liver cancer specific T cell antigen receptor T cell (TCR-T) comprising a liver cancer antigen TCR sequence obtained by single cell TCR full length sequencing of the liver cancer specific cytotoxic T lymphocytes of the present application.
In one implementation of the present application, the liver cancer-specific T cell antigen receptor T cell is formed by transducing a liver cancer antigen TCR sequence to a T cell via a gene transduction vector.
It can be understood that after obtaining the TCR sequence of the liver cancer antigen by the single cell TCR full-length sequencing, the PCR amplification is generally performed by using a TCR specific primer to obtain the TCR sequence of the liver cancer antigen, and then the PCR amplification product, i.e., the TCR sequence of the liver cancer antigen, is transduced into T cells by a gene transduction vector. Wherein, the TCR specific primer or the primer design can refer to the existing PCR amplification technology; the gene transduction vector may also refer to the existing TCR-T technology, such as a lentiviral vector, etc., and is not particularly limited herein.
The eighth aspect of the present application discloses an application of the antigenic polypeptide of the present application, or the nucleic acid sequence of the present application, or the gene expression vector of the present application, or the recombinant cell of the present application, or the antigen presenting cell of the present application that induces the liver cancer specific cytotoxic T lymphocyte, or the liver cancer specific cytotoxic T lymphocyte of the present application, or the liver cancer specific T cell antigen receptor T cell of the present application, in the preparation of a medicament for treating or preventing liver cancer, or in the preparation of a reagent for detecting or screening liver cancer.
It is understood that the antigenic polypeptides, nucleic acid sequences, APC, CTL and TCR-T of the present application all have liver cancer specificity, and therefore, in addition to being used for preparing a medicament for treating or preventing liver cancer, the specificity detection or screening of liver cancer can be realized through the specificity of the antigenic polypeptides, nucleic acid sequences, APC, CTL and TCR-T. As for the gene expression vector and the recombinant cell, the gene expression vector and the recombinant cell can be used for preparing the antigen polypeptide of the application, and therefore, the gene expression vector and the recombinant cell can also be indirectly used for preparing a medicament for treating or preventing liver cancer.
The ninth aspect of the present application discloses a kit for preparing liver cancer-specific cytotoxic T lymphocytes, which contains at least one component of components (a) to (f);
(a) an antigenic polypeptide of the present application;
(b) a nucleic acid sequence of the present application;
(c) a gene expression vector of the present application;
(d) a recombinant cell of the present application;
(e) antigen presenting cells of the present application that induce hepatoma-specific cytotoxic T lymphocytes;
(f) exosomes capable of presenting the antigenic polypeptides of the present application to CD8 positive T cells and activating the production of corresponding specific cytotoxic T lymphocytes.
The kit for preparing the liver cancer specific CTL can prepare the liver cancer specific CTL with strong immunogenicity, high positive rate, high in-vitro killing capacity and high efficiency, and can better meet the use requirements of liver cancer research and clinical application. It is understood that the kit for preparing liver cancer-specific CTLs of the present application may optionally contain other key reagents for preparing liver cancer-specific CTLs, and of course, these reagents may also be obtained directly by commercial purchase, and are not specifically limited herein.
It is further to be noted that exosomes as defined herein refer to, for example, intracellular vesicles or similar functional or structural substances which present the antigenic polypeptides of the present application on their surface, thereby activating the production of corresponding specific cytotoxic T lymphocytes. The exosomes of the present application may be prepared by, for example, the methods described in JPH11-510507 and WO 99/03499. The exosomes of the present application are capable of presenting the antigenic polypeptides of the present application and, therefore, can also be used to prepare the liver cancer-specific CTLs of the present application.
A tenth aspect of the present application discloses a pharmaceutical composition for treating or preventing liver cancer, comprising a pharmaceutically acceptable carrier and at least one component of components (a) to (h);
(a) an antigenic polypeptide of the present application;
(b) a nucleic acid sequence of the present application;
(c) a gene expression vector of the present application;
(d) a recombinant cell of the present application;
(e) antigen presenting cells of the present application that induce hepatoma-specific cytotoxic T lymphocytes;
(f) exosomes capable of presenting the antigenic polypeptides of the present application to CD8 positive T cells and activating the production of corresponding specific cytotoxic T lymphocytes;
(g) the liver cancer-specific cytotoxic T lymphocytes of the present application;
(h) the liver cancer specific T cell antigen receptor T cells of the present application.
The pharmaceutically acceptable carrier includes, but is not limited to, a solution of the antigenic polypeptide, the nucleic acid sequence, the APC, exosomes, TCR-T or CTLs, and diluents, excipients, solvents, encapsulating materials, and the like of the pharmaceutical composition.
The application of the pharmaceutical composition for treating or preventing liver cancer, wherein the liver cancer specific cytotoxic T lymphocytes have the advantages of strong immunogenicity, high positive rate, high in-vitro killing capacity and efficiency and the like, and have the effect of treating or preventing liver cancer. The TCR-T can be used for target development in liver cancer immune cell therapy. The antigenic polypeptides, nucleic acid sequences, exosomes and antigen presenting cells of the application can induce immune response against liver cancer, thereby having the effect of treating or preventing liver cancer. The gene expression vector and the recombinant cell can express the antigen polypeptide, and therefore, under the condition of expressing the antigen polypeptide, the gene expression vector and the recombinant cell have similar functions and effects of the antigen polypeptide.
An eleventh aspect of the present application discloses a kit for inducing an immune response against liver cancer, which contains at least one component of components (a) to (h);
(a) an antigenic polypeptide of the present application;
(b) a nucleic acid sequence of the present application;
(c) a gene expression vector of the present application;
(d) a recombinant cell of the present application;
(e) antigen presenting cells of the present application that induce hepatoma-specific cytotoxic T lymphocytes;
(f) exosomes capable of presenting the antigenic polypeptides of the present application to CD8 positive T cells and activating the production of corresponding specific cytotoxic T lymphocytes;
(g) the liver cancer-specific cytotoxic T lymphocytes of the present application;
(h) the liver cancer specific T cell antigen receptor T cells of the present application.
It is noted that the antigenic polypeptides, nucleic acid sequences, antigen presenting cells, exosomes, liver cancer specific cytotoxic T lymphocytes and TCR-ts of the present application are capable of specifically eliciting an immune response against liver cancer; therefore, it can be used as a corresponding agent, such as "vaccine" and the like. The above components may be used alone or in combination, and are not particularly limited herein.
The beneficial effect of this application lies in:
the antigen polypeptide can specifically and efficiently induce the hepatoma carcinoma cell cytotoxic T lymphocyte, and the induced hepatoma carcinoma specific cell cytotoxic T lymphocyte is superior to CTL induced by other existing antigen polypeptides in immunogenicity, positive rate of antigen-loaded cell cytotoxic T lymphocyte and killing efficiency of the cell in vitro experiments. The antigen polypeptide is adopted to efficiently induce the cytotoxic T lymphocyte which specifically kills the liver cancer tumor cells, the preparation method is efficient and convenient, compared with other immune cell therapies, the antigen polypeptide has the characteristics of low cost, high yield and simple process flow, and a new scheme and a new way are provided for clinical application of liver cancer immune cell therapy.
Drawings
FIG. 1 is a schematic diagram showing the induction of liver cancer-specific CTL by the antigenic polypeptide in the present example;
FIG. 2 is a graph showing the results of the immunogenicity test of the antigenic polypeptides in the examples of the present application;
FIG. 3 is a graph showing the flow assay fluorescence results of the antigen-loaded CTL positivity test in the examples of the present application;
FIG. 4 is a graph showing the standard displacement efficiency of the antigen-loaded CTL positive rate test in the examples of the present application;
FIG. 5 is a graph showing the results of flow cytometry analysis of CTL positive cell rates induced by antigenic polypeptides in the examples of the present application;
FIG. 6 shows the results of the test for the killing rate of CTL induced by the antigenic polypeptide in the examples of the present application.
Detailed Description
At present, although there are related researches and reports on antigenic polypeptides of liver cancer, the existing researches and reports are basically limited to screening and expression performance research of positive polypeptide sequences; the immunogenicity of antigenic polypeptides has not been investigated, nor has the ability of CTLs to be loaded with such antigenic polypeptides been investigated or reported, as well as the killing ability of CTLs loaded with such antigenic polypeptides.
The research of the application considers that although the existing research has screened positive antigen polypeptide, the positive antigen polypeptide has high expression performance; however, if the immunogenicity is weak, or the CTL cannot be loaded, or the killing ability of the CTL loaded with the antigen polypeptide is weak, these factors directly influence the CTL treatment effect and cost of the liver cancer.
Based on the research and the recognition, the application develops a new antigen polypeptide capable of efficiently inducing the liver cancer specific CTL, and the liver cancer specific CTL induced by the antigen polypeptide is superior to the CTL induced by other existing antigen polypeptides in immunogenicity, positive rate of antigen-loaded cytotoxic T lymphocyte cells and killing efficiency of the antigen-loaded cytotoxic T lymphocyte cells in-vitro experiments.
The antigen polypeptide is adopted to efficiently induce the cytotoxic T lymphocyte with specificity for killing the liver cancer tumor cells, and the preparation method is efficient and convenient. Compared with other immune cell therapies, the method for preparing the liver cancer specific CTL by adopting the antigen polypeptide has lower cost than that of using a plasmid/virus vector to express the transfection antigen to cells; in addition, the CTL has the performance of efficiently killing the liver cancer tumor cells, so that the return dose of the CTL can be reduced, and the CTL culture cost is reduced. The process flow for preparing the liver cancer specific CTL is simple, and particularly, the process flow for preparing the CTL is more simple compared with the process flow for constructing a plasmid library and a virus cell library required by a TCR-T, CAR-T cell treatment method and the like.
In addition, CTL carrying the liver cancer antigen polypeptide screened by the invention is screened out positively by a flow cytometer, after collection, the TCR sequence corresponding to the liver cancer antigen is easily obtained only by high-throughput single-cell TCR full-length sequencing and TCR specific primer amplification, and the TCR sequence has corresponding guiding significance for the subsequent preparation of TCR-T and can promote the target development of the TCR-T in liver cancer immune cell therapy.
The present application will be described in further detail with reference to specific examples. The following examples are intended to be illustrative of the present application only and should not be construed as limiting the present application. In the following description, numerous details are set forth in order to provide a better understanding of the present application. However, one skilled in the art will readily recognize that some of the features may be omitted in different instances or may be replaced by other kits, materials, methods. In some instances, certain operations related to the present application have not been shown or described in detail in this specification in order to avoid obscuring the core of the present application from excessive description, and it is not necessary for those skilled in the art to describe these operations in detail, so that the related operations will be fully understood from the description in the specification and the general knowledge of the art. In the following examples, the reagents or instruments used are not indicated by manufacturers, but are all conventional products available on the market.
The numbers in the present application, for example, "Seq ID No.", "first aspect", "second aspect", "third aspect", "(a)", "(b)", "(c)" and the like, are used only for distinguishing the described objects, and do not have any sequential or technical meaning.
Examples
This example develops a new antigen polypeptide inducing liver cancer specific cytotoxic T lymphocyte, the development process is shown in FIG. 1, which comprises obtaining Whole exon sequencing from TCGA database of liver cancer patient, performing tumor neoantigen and polypeptide prediction (Turneoantigen), antigen and MHC class I molecule binding affinity analysis (affinity), screening out the new objective polypeptide with stronger affinity; then, antigen presenting cells are stimulated, APC cells stimulated by cytokines (IL-4, GM-CSF, IFN-gamma and LPS) present antigens to CD8+ T cells (antigen presenting), and after maturation is stimulated by corresponding cytokines (IL-21, IL-2, IL-7 and IL-15), CTLs secrete cytokines (IFN-gamma) to kill target cells or tumor cells to treat tumor patients.
The antigen polypeptide and CTL induced by the antigen polypeptide in the embodiment have immunogenicity, positive rate of antigen-loaded cytotoxic T lymphocyte cells and killing efficiency in vitro cell experiments, which are superior to CTL induced by other antigen polypeptides in the prior art, and detailed experiments are as follows:
1. tumor antigen polypeptide screening
1.1 screening of tumor neoantigen Polypeptides
According to six bioinformatics software of PSSMHCpan, NetMHC-4.0, NetMHCpan-3.0, NetMHCpan-4.0, PickPocket and SMM, a plurality of human high-frequency mutation sites in the HLA-A0201-typed liver cancer population are predicted, and polypeptide-MHC affinity prediction is carried out on the mutation sites corresponding to the several sites. In this example 364 cases of somatic mutation data for the sequencing results of the whole exon of liver cancer were downloaded from the TCGA database (https:// portal.gdc.cander.gov) (version 20170929). Screening missense SNV sites reproduced on at least 2 patients to obtain the highest frequency 10 mutation sites in a liver cancer patient population taking HLA-A0201 as an example, and intercepting an amino acid sequence into a short peptide with the length of 9 mers and containing variant amino acids according to variant information.
And generating a short peptide sequence which contains variant amino acids and is 9 mers in length according to the variation information from the 10 high-frequency mutation sites obtained by screening. In order to identify neoantigens that can be used as candidate markers for developing anti-tumor vaccines and to optimize prediction accuracy, this example incorporates six software items PSSMHCpan, NetMHC-4.0, NetMHCpan-3.0, NetMHCpan-4.0, PickPocket and SMM into an affinity prediction protocol for detection of candidate neoantigens in TCGA liver cancer samples. Finally, candidate neoantigens meeting the following conditions were selected: the conjugate IC50<500nM was predicted by at least 3 softwares and the minimum of the corresponding IC50 values of several softwares predicted to be conjugate was taken as the final prediction and IC50<50 nM.
Six software combinations are used for predicting the binding capacity of the generated short peptide and MHC, IC50<500nM indicates affinity, IC50<50nM indicates high affinity, and the lower the IC50 score, the higher the affinity. According to the sorting from low to high of IC50 score, the short peptide with IC50 score of less than 50nM is selected in the example, and finally 2 high-frequency mutant polypeptides with the strongest affinity are obtained and synthesized for subsequent experiments. The details of the 2 candidate neoantigen polypeptides synthesized in this example are shown in Table 1.
TABLE 1 sequence information of the screened antigenic polypeptides
| Serial number | HLA typing | Name of Gene | Amino acid sequence | Length of polypeptide | SeqIDNo. |
| 1 | HLA-A0201 | TGIF2LX | FLLLVDATV | 9 | 8 |
| 2 | HLA-A0201 | OR2G2 | ILLGFSDYLQL | 11 | 9 |
Wherein, the nucleic acid sequence of the TGIF2LX antigen polypeptide is the sequence shown in Seq ID No.3,
Seq ID No.3：5’-TTCCTGCTGCTAGTCGATGCAACAGTA-3’。
1.2 Virus-associated antigen screening
In order to compare the immunogenicity of the predicted neoantigenic polypeptide with the viral-associated antigenic polypeptide, positive HBV-associated polypeptide sequences were obtained according to the reference, and the polypeptide information is shown in Table 2.
TABLE 2 information on the synthesis of HBV, HCV-related viral polypeptides
| Serial number | Name (R) | Amino acid sequence | Length of polypeptide | SeqIDNo. |
| 1 | HBV_01 | FLLTRILT | 8 | 10 |
1.3 tumor-associated antigen screening
Meanwhile, in order to compare the immunogenicity of the predicted new antigen polypeptide with the immunogenicity of the related antigen polypeptide with high tumor expression, the present example also obtains related polypeptide sequences with high liver cancer expression according to the reference literature, in the present example, 6 related polypeptide sequences with high tumor expression are obtained in total, the polypeptide sequence with the best effect is selected as the control group test of the present example, and the polypeptide information is shown in table 3.
TABLE 3 Synthesis of information on liver cancer highly expressed related Polypeptides
| Serial number | Name (R) | Amino acid sequence | Length of polypeptide | SeqIDNo. |
| 1 | VEGFR2-775 | AMFFWLLLV | 9 | 11 |
Tumor-associated antigen screening references are as follows:
Tada Y,Yoshikawa T,Shimomura M,Sawada Y,Sakai M,Shirakawa H,Nobuoka D,Nakatsura T:Analysis of cytotoxic T lymphocytes from a patient with hepatocellular carcinoma who showed a clinical response to vaccination with a glypican3derivedpeptide.Int J Oncol 2013,43(4):1019-1026.
Kan Z,Zheng H,Liu X,Li S,Barber TD,Gong Z,Gao H,Hao K,Willard MD,Xu J et al:Whole-genome sequencing identifies recurrent mutations in hepatocellular carcinoma.Genome Res 2013,23(9):1422-1433.
LofflerMW,Mohr C,Bichmann L,Freudenmann LK,WalzerM,Schroeder CM,Trautwein N,Hilke FJ,Zinser RS,Muhlenbruch L et al:Multi-omics discovery of exome-derived neoantigens in hepatocellular carcinoma.Genome Med 2019,11(1):28.
Schulze K,Nault JC,Villanueva A:Genetic profiling of hepatocellular carcinoma using next-generation sequencing.J Hepatol 2016,65(5):1031-1042.
Meyerson M,Gabriel S,Getz G:Advances in understanding cancer genomes through second-generation sequencing.Nat Rev Genet 2010,11(10):685-696.
2. immunogenicity assays
After synthesis of polypeptides having the sequences shown in tables 1, 2 and 3, by entrustochem bioengineering (shanghai) gmbh PBMC cells were purchased, in this example from mitsunotong biotechnology limited, product number: PER-F, and sorting CD8 positive T cells and DC cells with specific magnetic beads and MS columns. Wherein, the specificity magnetic bead specifically is, the magnetic bead and the magnetic force frame of thermo fisher, the magnetic bead name: dynabeadsTMCD8, cat number: 11147D; magnetic bead name: dynabeadsTMFlowCompTMHuman CD14 Kit, cat No.: 11367D; magnetic bead separator, name: DynaMagTM-2Magnet, cat #: 12321D; specificity refers to magnetic beads positive for the CD8 target and magnetic beads positive for DC cells (CD14+ magnetic beads). After sorting addIL-4 and GM-CSF cytokines were added to culture DC cells first, and the corresponding cytokines IL-4, GM-CSF, LPS and IFN- γ were added to stimulate maturation for 24h, followed by stimulation with the above synthesized polypeptides of interest, respectively. DC cells were co-cultured with CD8 positive T cells for one week, specifically, IL-21 was cultured for 72h, followed by culture with IL-2, IL-7 and IL-15. CD8 positive T cells are collected after stimulation and maturation and are subjected to EliSpot assay, whether the polypeptide has immunogenicity is observed through the condition that secreted IFN-gamma generates purple spots, and then the EliSpot plate is subjected to AID reading. The specific experimental steps are as follows:
2.1 sorting of DCs and T cells
(1) Collecting 50-100mL of HLA-A0201 typed healthy human Peripheral blood, resuspending with PBS, adding Ficoll separating medium with corresponding volume, centrifuging with density gradient, sucking out mononuclear cell layer with a pipette, transferring to a centrifuge tube, resuspending with sterile PBS, centrifuging, washing repeatedly for one time, removing redundant Ficoll separating medium and platelets to obtain Peripheral Blood Mononuclear Cells (PBMCs), and freezing for later use;
(2) take 50X 106After the peripheral blood mononuclear cells are recovered, washing the peripheral blood mononuclear cells once by using a 1640 culture medium, centrifuging the peripheral blood mononuclear cells for 5min at the rotating speed of 300g, counting the cells, and incubating the cells for 6-12h at 37 ℃ by using the 1640 culture medium and 10% FBS;
(3) after incubation, the cells were centrifuged at 300g for 5min and resuspended in a MACS buffer and counted. Adding corresponding CD14 magnetic beads and MACS buffer according to the quantity, incubating for 15min at 4 ℃, and shaking once every 5 min; wherein the addition amount of the magnetic beads is 20 μ L/107cells, MACS buffer was added in an amount of 80. mu.L/107cells；
(4) Taking out, washing with 20mL of MACS buffer, centrifuging at 300g for 10min, resuspending with MACS, passing through MC column, washing MC column with 1mL of MACS, filtering to obtain CD14 negative cells, washing the old tube with 1mL of MACS after cell suspension filtration, washing the column with 1mL of MACS twice, removing MC column, pushing out CD14 positive cells (DC cells) with piston, and counting 1.722 × 107Half of the cells were cultured in CG-DC + 5% FBS +10ng/mL IL-4+86ng/mL GM-CSF medium, and half of the cells were cryopreserved in CS10 medium；
(5) Counting CD14 negative cells, adding corresponding volumes of CD8 magnetic beads and MACS buffer, incubating for 15min at 4 ℃, and shaking once every 5 min; wherein the amount of CD8 magnetic beads is 20 μ L/107cells, MACS buffer was added in an amount of 80. mu.L/107cells；
(6) Taking out, washing with 20mL of MACS buffer, centrifuging at 300g for 10min, resuspending with MACS, passing through MC column, washing MC column with 1mL of MACS, filtering to obtain CD8 negative cells, washing the old tube with 1mL of MACS after cell suspension filtration, washing the column with 1mL of MACS twice, thrusting out CD8 positive cells (T cells) with piston, and counting 3.42 × 106And freezing and storing.
2.2 culture of DCs and T cells
(1) D1, DC cell count 1.722X 10 after magnetic bead sorting7Half of the cells were frozen, half of the cells were cultured, resuspended in CG-DC + 5% FBS +10ng/mL of IL-4+86ng/mL of GM-CSF medium, 1 mL/well, and cultured in 12-well plates for a total of 12 wells, with the number of cells per well being 5X 105Culturing for 48h per well;
(2) d3, each well supplemented with CG-DC + 5% FBS +20ng/mL IL-4+176ng/mL GM-CSF 500 μ L, i.e., supplemented with 1/2 in original volume, and doubling cytokine concentration of medium; continuously culturing for 48 h;
(3) d5, preparing CG-DC + 5% FBS +10ng/mL IL-4+86ng/mL GM-CSF +10ng/mL LPS +10ng/mL IFN-gamma culture medium, and changing the culture solution in full; specifically, centrifuging at a rotating speed of 300g for 10min, removing the original culture medium, resuspending by using the prepared culture medium, and performing mature culture for 24 h; simultaneously recovering T cells, and using CG-DC + 5% FBS +10ng/mL IL-7 culture medium for resuspension;
(4) d6, loading the DC cells with the corresponding target polypeptide, in this case 10. mu.g/mL, 37 ℃ CO2Culturing in an incubator for 16 h;
2.3 Co-culture of DCs and T cells
(5) D7, after completion of polypeptide loading, co-culturing DC cells and T cells in amounts greater than the ratio of DC: t is 1: 4-1: 8, and the volume ratio is 1:1, the culture density is 1.0-1.5 × 106cells/cm2(ii) a At this point, 30ng/mL IL-21 was added,continuously culturing for 72 h;
(6) d10, adding 10ng/mL IL-2, 10ng/mL IL-7(10ng/mL) and 10ng/mL IL-15 after 3 days, and culturing for 48-72 h;
(7) d12 or D13, half the volume of the solution was changed, and the solution was supplemented with cytokines IL-2 at 10ng/mL, IL-7 at 10ng/mL and IL-15 at 10ng/mL to ensure the cell density at 1X 106about/mL, culturing for 48-72 h;
(8) d14 or D16, total volume exchange solution, supplemented with cytokines IL-2 at 10ng/mL, IL-7 at 10ng/mL and IL-15 at 10 ng/mL.
2.4 Enzyme-linked Immunospot Assay (ELISPOT, Enzyme-linked Immunospot Assay)
(1) D20, recovering HLA-0201 typed T2 cells (purchased from ATCC) -T02-0201 cells (IMDM + 10% FBS), centrifuging at 300g rotation speed for 5min, and counting;
(2) d22, collecting T02-0201 cells, centrifuging for 5min at the rotating speed of 300g, counting the cells, resuspending the cells by serum-free IMDM, adding 1.5mL of EP tube, respectively loading corresponding target polypeptides, wherein the concentration of the polypeptides is 10 mu g/mL, and taking the polypeptide as an experimental group; setting a group as a negative control group independently, namely adding DMSO into T02-0201 cells, wherein the volume of DMSO is consistent with that of polypeptide, and the temperature is 37 ℃ for 4 h;
(3) after 4h, the peptide-loaded T02-0201 cells were harvested and resuspended in CG-DC + 2% FBS medium at a cell concentration of 5X 104Per mL;
(4) washing the elispot plate 5 times with PBS, 150 μ L/well, every 5min, then adding IMDM + 10% FBS, 100 μ L/well, 37 deg.C, 30 min;
(5) taking a T cell sample to be detected, re-suspending the T cell sample by using CG-DC + 2% FBS culture medium, and adjusting the cell concentration to be 2 x 105Per mL;
(6) taking out an elispot plate, beating liquid on a clean paper towel, adding T02 and T cells into a hole of 100 mu L/hole respectively according to the plate paving sequence, simultaneously adding a positive control group OKT3 and a negative control group, and carrying out heating at 37 ℃ for 16-20 h;
(7) d23, removing cells on the plate, washing the elispot plate with PBS 5 times at 150 μ L/well, washing once every 5min, adding the prepared 7-b6-1-ALP antibody at 100 μ L/well, incubating for 2h at 37 ℃, preparing PBS + 0.5% FBS, adding the antibody at a ratio of 1:200, and filtering at 0.22 μm; among them, the 7-b6-1-ALP antibody kit was purchased from mabtech, under the name: human IFN-. gamma.ELISpot kit (ALP), cat #: 3420-2APW-2, antibody concentration consistent with kit instructions, 1:200 refers to the volume ratio of 7-b6-1-ALP antibody to PBS + 0.5% FBS, used to formulate the antibody;
(8) removing primary antibody, washing the elispot plate with PBS for 5 times at 150 μ L/well every 5min, adding NBT/BCIP, and developing in dark for 2-8min at 100 μ L/well;
(9) observation, washing with a large amount of tap water.
(10) And (4) drying by using an oven, counting the purple spots of each pore plate by using an AID (AID-reading) instrument, and finishing and counting.
In the above test procedures, "D" represents the number of days, for example, "D1" represents the first day, and is 1.722X 10 in terms of DC cell count7Half of the total number was used for the number of days counted from the start of the culture.
The AID reading plate results are shown in fig. 2, the first column is TGIF2LX, the second column is OR2G2, the third column is HBV _01, and the fourth column is VEGFR 2-775; rows 1 to 3 are 3 replicates of each experimental group, and rows 4 to 6 are 3 replicates of the negative control group.
The results in fig. 2 show that more purple spots were observed in the neoantigen polypeptide screening group TGIF2LX, indicating that the antigen polypeptide has higher immunogenicity and is statistically significant.
3. Antigen-loaded polypeptide CTL positive rate test
In order to evaluate the ability of the selected neo-antigenic polypeptides to pass antigen presenting cells such as DC, present antigenic peptides to CD8 positive T cells, and activate and produce corresponding specific CTL cells, the positive rate test of detecting antigen-specific CTL cells by tetramer staining was performed. In this example, a tetramer displacement kit (QuickSwitchTM, PE staining) from Beijing Boelmei Biotechnology Inc. (MBL Biotech) was purchased and tested, and the specific steps were as follows:
3.1 tetramer Displacement experiments
(1) A2 mg/mL (2mM) solution of the target polypeptide for replacement and a positive polypeptide standard were prepared in DMSO:
(2) add 50. mu.L of tetramer to round bottom 96-well plate;
(3) adding 1 mu L of the polypeptide solution prepared in the step (1), and uniformly mixing by using a pipettor;
(4) adding 1 mu L of polypeptide replacement factor, and uniformly mixing by using a pipettor;
(5) incubating for 4h at room temperature in the dark;
(6) then stored at 4 ℃ in the dark.
3.2 detection of tetramer displacement efficiency:
(1) 20 mu L of capture magnetic beads are added into a hole 1-5 of a 96-hole plate with a conical bottom;
(2) add 5. mu.L of 1 × detection buffer to well 2, 5. mu.L of the non-displaced tetramer to wells 1 and 3, and the displaced tetramer to wells 4 and 5;
(3) wrapping the 96-well plate with tinfoil, placing the 96-well plate on a flat plate shaker, and oscillating at 550rpm for 45min at room temperature;
(4) after oscillation, adding 1 Xdetection buffer solution into the holes 1-5, washing 150 μ L per hole, planting the 96-well plate on a magnetic plate, standing for 5min, discarding the supernatant after standing, keeping the 96-well plate on the magnetic plate, stirring for 2 seconds by using a vortex mixer, and then taking the 96-well plate off the magnetic plate;
(5) add 1 Xantibody-FITC (antibody specific for the polypeptide in the tetramer of the kit) 25. mu.L each into well 2-5, add 1 Xdetection buffer into well 1;
(6) wrapping the 96-well plate with tinfoil, placing the 96-well plate on a flat plate shaker, and oscillating at 550rpm for 45min at room temperature;
(7) after oscillation, adding 1 Xdetection buffer solution into the holes 1-5, washing 150 μ L per hole, planting the 96-well plate on a magnetic plate, standing for 5min, discarding the supernatant after standing, keeping the 96-well plate on the magnetic plate, stirring for 2 seconds by using a vortex mixer, and then taking the 96-well plate off the magnetic plate;
(8) adding 1 Xdetection buffer solution into the hole 6, adding 5 μ L of capture magnetic beads, and mixing well;
(9) all the wells were transferred to a flow tube and placed on ice, and then the fluorescent intensity of the polypeptide in the tetramer of the kit was detected by an up-flow cytometer, and the substitution efficiency of the polypeptide was calculated, and the results are shown in fig. 3, fig. 4 and table 4.
TABLE 4 replacement efficiency test results
In Table 4, FITC is the mean fluorescence intensity of the displaced prototetramer. FIG. 3 is a graph showing the results of flow-through fluorescence, wherein the first column shows the results of the unsubstituted polypeptide, the second column shows the results of the positive polypeptide, and the third column shows the results of the nascent polypeptide TGIF2 LX. Fig. 4 is a standard curve of the displacement efficiency, wherein the left graph is a specific value and the right graph is a standard curve.
The results in FIG. 3, FIG. 4 and Table 4 show that the substitution efficiency of the nascent polypeptide TGIF2LX of this example is about 95.42%; according to the standard of the kit, if the replacement rate is more than 75%, the result is positive; therefore, the substitution of the nascent polypeptide TGIF2LX was successful and the tetramer was used for subsequent detection.
3.3 detection of Positive Rate of antigen-specific cytotoxic T cells:
(1) collection of mature effector cells 2X 106Centrifuging the mixture in a 1.5mL centrifuge tube at 400g for 5 min;
(2) discard the supernatant and resuspend the cells using PBS (0.5% FBS), 200 μ Ι _ per tube;
(3) adding 2 μ L of tetramer-PE and CD8 surface antibody-FITC (1:1000) into each tube, staining, incubating at 4 deg.C for 30min, and preparing cells with one double negative, one single PE staining and one single FITC staining;
(4) after the incubation was completed, 400. mu.L each of PBS (0.5% FBS) was added to each tube for washing, and 400g was centrifuged for 5 min;
(5) adding PBS (0.5% FBS) into each tube, washing with 400 μ L, centrifuging at 400g for 5 min;
(6) 300 μ L of PBS (0.5% FBS) was added to each tube for resuspension, transferred to a flow tube, placed on ice, and then the fluorescence intensity of the cells was measured by an up-flow cytometry, and the Q2 quadrant was positive cells of antigen-specific cytotoxic T cells, and the results are shown in fig. 5.
FIG. 5 is a graph showing the results of analysis of the CTL positive cell rate induced by detecting neoantigen peptide by flow cytometry, wherein the left graph "Control" is a Control group, and the right graph is a test group of the neoantigen peptide TGIF2 LX. The results in fig. 5 show that T cells stimulated with TGIF2LX antigenic polypeptide antigen in this example detected 1.21% of positive tumor-specific T cells with tetramers, and that the Q2 quadrant was an in-frame positive population. Correspondingly, the control group was only 0.075%.
The CTL positive rate of the existing liver cancer antigen polypeptide after induction is lower than 0.5 percent, and even CTL which can be detected by flow cytometry cannot be induced. The results in FIG. 5 show that the liver cancer-specific cytotoxic T lymphocytes induced by the TGIF2LX antigenic polypeptide of the present example have a significantly higher positive rate than CTLs induced by the existing liver cancer antigenic polypeptide.
4. Test of killing ability of liver cancer specific CTL in vitro cell
In order to evaluate the in vitro cell-killing ability of the selected neoantigen peptides of this example to induce CTLs, the following experimental procedures were performed.
Selecting the newborn antigen polypeptide with immunogenicity screened in the EliSpot assay experiment, and adopting a lactate dehydrogenase detection kit (LDHAssaykit) to perform target cell killing performance test. Lactate Dehydrogenase (LDH) is one of the cytosolic enzymes of living cells and is normally impermeable to the cell membrane. When target cells T2 are attacked and damaged by effector cells CTL, the permeability of cell membranes is changed, LDH can be released into a medium, the released LDH enables oxidized coenzyme I (NAD +) to be changed into reduced coenzyme I (NADH2) in the process of catalyzing lactic acid to generate pyruvic acid, the latter reduces iodonitronitrocloazodicarbonyl blue (INT) or Nitrocloazodicarbonyl Blue (NBT) through hydrogen donor-phenazine dimethyl sulfate (PMS) to form a colored formazan compound, a high absorption peak exists at 490nm wavelength, the OD value read by an enzyme labeling instrument is used, and the activity of the target cells can be obtained through calculation, so that the killing performance of antigen polypeptide can be detected. The specific operation steps are as follows:
(1) taking T2 cells, counting, and carrying out resuspension by serum-free IMDM, wherein an experimental group is used for loading screened newborn antigen polypeptide, a control group is used for adding DMSO with the same volume, the concentration of the polypeptide is 10 mu g/mL, the polypeptide is loaded for 4 hours at 37 ℃;
(2) after 4h, the T2 cells were centrifuged and resuspended T2 in CG-DC + 5% FBS medium and adjusted to a concentration of 2X 105Per mL; wherein, CG-DC is purchased from CellGenix, name:
GMP DC Medium, good number: 20801-;
(3) effector cells were taken and diluted in culture according to the plating protocol indicated in table 5 with an effective target ratio of 5:1(E: T ═ 5: 1); the culture medium is CG-DC + 5% FBS;
TABLE 5 LDH plating Condition
In Table 5, ` r ` indicates the experimental group (50. mu.L effector cells + 50. mu.L target cells), ` c ` indicates the effector cells released naturally (50. mu.L effector cells + 50. mu.L medium), ` c ` indicates the target cells released naturally (50. mu.L target cells + 50. mu.L medium), ` c ` indicates the maximum release of the target cells (50. mu.L target cells + 50. mu.L medium + 50. mu.L lysate), ` c ` indicates the volume correction (110. mu.L), ` c ` indicates the culture medium control (100. mu.L).
(4) Plating with 96-well plate with pointed bottom or round bottom, and final volume of all wells is 100 μ L;
(5)96 well plates at 37 ℃ 5% CO2Culturing for 4 hours in an incubator;
(6) adding 10 mu L of lysis buffer to the maximum release group and the volume correction group of the target cells when culturing for 3.5 h;
(7) centrifuging a 96-well plate after incubation is finished, and centrifuging for 4min at 250 g;
(8) taking 50 mu L/well of the supernatant to a flat-bottom 96-well plate for detection, and avoiding bubbles;
(9) adding 50 mu L/hole of Substrate Mix, and preserving at-20 ℃ after the use;
(10) reacting at room temperature in dark for 30 min;
(11) adding 50 mu L/hole Stop solution, and lightly beating and uniformly mixing to avoid bubbles;
(12) in the inspection hole, the bubble is punctured by a syringe, and the absorbed light is read on a full-wavelength reading instrument at 490nm to derive the original data.
Calculated% kill:
(13) all experimental and control groups should be subtracted with the background mean.
(14) Corrected values were used for the calculation of killing efficiency, cell killing rate (%) × (experimental group release-effector cell spontaneous release-target cell spontaneous release)/(target cell maximal release-target cell spontaneous release) x 100%.
The results of killing rate are shown in FIG. 6, the abscissa "TGIF 2 LX" represents CTL induced by TGIF2LX antigenic polypeptide, and "DMSO" represents that DMSO is equally substituted for CTL induced by TGIF2LX antigenic polypeptide in the negative control; the ordinate represents cytotoxicity, i.e. killing rate.
The results in FIG. 6 show that CTL cells induced and stimulated by the selected neoantigen polypeptide TGIF2LX have in vitro cell killing ability, and the killing efficiency is 76% when the ratio of E to T is 5: 1; the background killing rate of general T cells is lower than 10%, the DMSO group is a negative control group, and the killing rate of the T cell background group without loaded polypeptide is lower than 5%. As can be seen, the liver cancer specific CTL induced by TGIF2LX has strong killing ability in vitro.
The test results show that the TGIF2LX antigen polypeptide with the amino acid sequence shown in Seq ID No.8 obtained in the embodiment can efficiently induce the liver cancer specific cytotoxic T lymphocyte, and the positive rate of the induced liver cancer specific cytotoxic T lymphocyte and the killing rate of the induced liver cancer specific cytotoxic T lymphocyte in immunogenicity, antigen-loaded cytotoxic T lymphocyte cell and in vitro experiments of the cell are superior to those of the existing antigen polypeptide and CTL induced by the antigen polypeptide, and the clinical application value is very high.
The results of the specificity and function prediction and analysis of the TGIF2LX antigen polypeptide of the present example by using protein analysis software and antigen polypeptide prediction software show that the antigen polypeptides of the sequence shown in Seq ID No.1 all have the same specificity and function as the TGIF2LX antigen polypeptide of the present example, namely, can efficiently induce liver cancer specific cytotoxic T lymphocytes, and the induced liver cancer specific cytotoxic T lymphocytes are superior to the existing antigen polypeptides and CTL induced by the same in immunogenicity, positive rate of antigen-loaded cytotoxic T lymphocytes and killing rate of the cells in vitro experiments.
Seq ID No.1：FLLXVDATV
Wherein, X is selected from any one of alanine, leucine, isoleucine, methionine, cysteine and threonine. When X is leucine, the TGIF2LX antigen polypeptide of the present example.
Accordingly, the nucleic acid sequences encoding the antigenic polypeptides in which X is alanine, leucine, isoleucine, methionine, cysteine or threonine are the sequences shown in Seq ID No.2 to Seq ID No.7,
Seq ID No.2：5’-TTCCTGGCACTAGTCGATGCAACAGTA-3’
Seq ID No.3：5’-TTCCTGCTGCTAGTCGATGCAACAGTA-3’
Seq ID No.4：5’-TTCCTGATCCTAGTCGATGCAACAGTA-3’
Seq ID No.5：5’-TTCCTGATGCTAGTCGATGCAACAGTA-3’
Seq ID No.6：5’-TTCCTGTGTCTAGTCGATGCAACAGTA-3’
Seq ID No.7：5’-TTCCTGACACTAGTCGATGCAACAGTA-3’。
the antigen polypeptide used in this example is an antigen polypeptide synthesized by entrusted organism company, and it is understood that, according to the conventional genetic engineering technique, the nucleic acid sequences of the sequences shown in Seq ID No.2 to Seq ID No.7 can be completely inserted into a plasmid vector having an expression function to form a gene expression vector, and then the gene expression vector is introduced into a corresponding host cell to form a recombinant cell; and culturing the recombinant cell to express the nucleic acid sequence to the antigen polypeptide of the embodiment, and extracting and purifying the polypeptide to obtain the antigen polypeptide of the embodiment.
In addition, on the basis of the liver cancer specific cytotoxic T lymphocyte prepared in the embodiment, the sequence information of a liver cancer specific TCR sequence can be obtained through high-throughput single-cell TCR full-length sequencing, a complete TCR sequence is obtained through TCR specific primer amplification, the TCR sequence is inserted into a gene transduction vector, such as lentivirus, and the TCR sequence is transduced into a T cell through a gene transduction technology, so that a liver cancer specific T cell antigen receptor T cell, namely a liver cancer specific TCR-T cell, can be obtained.
The foregoing is a more detailed description of the present application in connection with specific embodiments thereof, and it is not intended that the present application be limited to the specific embodiments thereof. It will be apparent to those skilled in the art from this disclosure that many more simple derivations or substitutions can be made without departing from the spirit of the disclosure.
SEQUENCE LISTING
<110> Shenzhen Letu biomedicine Limited
<120> antigenic polypeptide for inducing liver cancer specific cytotoxic T lymphocyte and application thereof
<130> 20I30206
<160> 11
<170> PatentIn version 3.3
<210> 1
<211> 9
<212> PRT
<213> Artificial sequence
<220>
<221> X
<222> (4)..(4)
<223> A or L or I or M or C or T
<400> 1
Phe Leu Leu Xaa Val Asp Ala Thr Val
1 5
<210> 2
<211> 27
<212> DNA
<213> Artificial sequence
<400> 2
ttcctggcac tagtcgatgc aacagta 27
<210> 3
<211> 27
<212> DNA
<213> Artificial sequence
<400> 3
ttcctgctgc tagtcgatgc aacagta 27
<210> 4
<211> 27
<212> DNA
<213> Artificial sequence
<400> 4
ttcctgatcc tagtcgatgc aacagta 27
<210> 5
<211> 27
<212> DNA
<213> Artificial sequence
<400> 5
ttcctgatgc tagtcgatgc aacagta 27
<210> 6
<211> 27
<212> DNA
<213> Artificial sequence
<400> 6
ttcctgtgtc tagtcgatgc aacagta 27
<210> 7
<211> 27
<212> DNA
<213> Artificial sequence
<400> 7
ttcctgacac tagtcgatgc aacagta 27
<210> 8
<211> 9
<212> PRT
<213> Artificial sequence
<400> 8
Phe Leu Leu Leu Val Asp Ala Thr Val
1 5
<210> 9
<211> 11
<212> PRT
<213> Artificial sequence
<400> 9
Ile Leu Leu Gly Phe Ser Asp Tyr Leu Gln Leu
1 5 10
<210> 10
<211> 8
<212> PRT
<213> Artificial sequence
<400> 10
Phe Leu Leu Thr Arg Ile Leu Thr
1 5
<210> 11
<211> 9
<212> PRT
<213> Artificial sequence
<400> 11
Ala Met Phe Phe Trp Leu Leu Leu Val
1 5
Claims (13)
1. An antigenic polypeptide for inducing hepatoma-specific cytotoxic T lymphocytes, characterized in that: the amino acid sequence of the antigen polypeptide comprises a sequence shown in Seq ID No.1,
Seq ID No.1：FLLXVDATV
wherein, X is selected from any one of alanine, leucine, isoleucine, methionine, cysteine and threonine.
2. A nucleic acid sequence encoding the antigenic polypeptide of claim 1.
3. The nucleic acid sequence of claim 2, wherein: the nucleic acid sequence comprises any one of the sequences shown in Seq ID No.2 to Seq ID No.7 or a degenerate sequence of any one of the sequences,
Seq ID No.2：5’-TTCCTGGCACTAGTCGATGCAACAGTA-3’
Seq ID No.3：5’-TTCCTGCTGCTAGTCGATGCAACAGTA-3’
Seq ID No.4：5’-TTCCTGATCCTAGTCGATGCAACAGTA-3’
Seq ID No.5：5’-TTCCTGATGCTAGTCGATGCAACAGTA-3’
Seq ID No.6：5’-TTCCTGTGTCTAGTCGATGCAACAGTA-3’
Seq ID No.7：5’-TTCCTGACACTAGTCGATGCAACAGTA-3’。
4. a gene expression vector characterized by: the gene expression vector is inserted with a foreign gene capable of encoding and expressing the antigenic polypeptide of claim 1;
preferably, the exogenous gene comprises the nucleic acid sequence of claim 2 or 3.
5. A recombinant cell, wherein: the recombinant cell contains the gene expression vector of claim 4.
6. An antigen presenting cell that induces hepatoma-specific cytotoxic T lymphocytes, comprising: the antigen presenting cell for inducing liver cancer specific cytotoxic T lymphocyte can present the antigen polypeptide of claim 1 to CD8 positive T cell and activate to generate corresponding specific cytotoxic T lymphocyte.
7. The antigen presenting cell for inducing hepatoma-specific cytotoxic T lymphocytes according to claim 6, characterized in that: the antigen presenting cell for inducing the hepatoma-specific cytotoxic T lymphocyte is obtained by stimulating the antigen presenting cell with the antigenic polypeptide of claim 1, or introducing the nucleic acid sequence of claim 2 or 3 into the antigen presenting cell so that the antigen presenting cell can express the antigenic polypeptide of claim 1, thereby obtaining the capacity of inducing the hepatoma-specific cytotoxic T lymphocyte.
8. The antigen polypeptide of claim 1 or the liver cancer-specific cytotoxic T lymphocyte induced by the antigen presenting cell of claim 6 or 7 which induces a liver cancer-specific cytotoxic T lymphocyte.
9. A liver cancer specific T cell antigen receptor T cell, which is characterized in that: contains a hepatoma antigen TCR sequence obtained by single cell TCR full-length sequencing of hepatoma specific cytotoxic T lymphocytes according to claim 8;
preferably, the liver cancer specific T cell antigen receptor T cell is formed by transducing the liver cancer antigen TCR sequence to a T cell via a gene transduction vector.
10. The antigenic polypeptide of claim 1, or the nucleic acid sequence of claim 2 or 3, or the gene expression vector of claim 4, or the recombinant cell of claim 5, or the antigen presenting cell inducing liver cancer-specific cytotoxic T lymphocyte of claim 6 or 7, or the liver cancer-specific cytotoxic T lymphocyte of claim 8, or the liver cancer-specific T cell antigen receptor T cell of claim 9, for use in the preparation of a medicament for treating or preventing liver cancer, or for use in the preparation of a reagent for detecting or screening liver cancer.
11. A kit for preparing liver cancer specific cytotoxic T lymphocytes is characterized in that: the kit comprises at least one component of the components (a) to (f);
(a) the antigenic polypeptide of claim 1;
(b) the nucleic acid sequence of claim 2 or 3;
(c) the gene expression vector of claim 4;
(d) the recombinant cell of claim 5;
(e) the antigen presenting cell of claim 6 or 7 which induces hepatoma-specific cytotoxic T lymphocytes;
(f) exosomes capable of presenting the antigenic polypeptide of claim 1 to CD8 positive T cells and activating the production of corresponding specific cytotoxic T lymphocytes.
12. A pharmaceutical composition for treating or preventing liver cancer, characterized in that: the pharmaceutical composition comprises a pharmaceutically acceptable carrier and at least one ingredient of components (a) to (h);
(a) the antigenic polypeptide of claim 1;
(b) the nucleic acid sequence of claim 2 or 3;
(c) the gene expression vector of claim 4;
(d) the recombinant cell of claim 5;
(e) the antigen presenting cell of claim 6 or 7 which induces hepatoma-specific cytotoxic T lymphocytes;
(f) exosomes capable of presenting the antigenic polypeptide of claim 1 to CD8 positive T cells and activating production of corresponding specific cytotoxic T lymphocytes;
(g) the liver cancer-specific cytotoxic T lymphocyte of claim 8;
(h) the liver cancer-specific T cell antigen receptor T cell of claim 9.
13. A kit for inducing an immune response against liver cancer, characterized by: the kit comprises at least one component of the components (a) to (h);
(a) the antigenic polypeptide of claim 1;
(b) the nucleic acid sequence of claim 2 or 3;
(c) the gene expression vector of claim 4;
(d) the recombinant cell of claim 5;
(e) the antigen presenting cell of claim 4 or 5 which induces hepatoma-specific cytotoxic T lymphocytes;
(f) exosomes capable of presenting the antigenic polypeptide of claim 1 to CD8 positive T cells and activating production of corresponding specific cytotoxic T lymphocytes;
(g) the liver cancer-specific cytotoxic T lymphocyte of claim 6;
(h) the liver cancer-specific T cell antigen receptor T cell of claim 9.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010889660.5A CN111944018B (en) | 2020-08-28 | 2020-08-28 | Antigen polypeptide for inducing liver cancer specific cytotoxic T lymphocyte and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010889660.5A CN111944018B (en) | 2020-08-28 | 2020-08-28 | Antigen polypeptide for inducing liver cancer specific cytotoxic T lymphocyte and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111944018A true CN111944018A (en) | 2020-11-17 |
| CN111944018B CN111944018B (en) | 2022-12-23 |
Family
ID=73368128
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010889660.5A Active CN111944018B (en) | 2020-08-28 | 2020-08-28 | Antigen polypeptide for inducing liver cancer specific cytotoxic T lymphocyte and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111944018B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112666358A (en) * | 2021-01-14 | 2021-04-16 | 深圳裕康医学检验实验室 | Method for detecting antigen immunogenicity by using peripheral blood |
| CN113943347A (en) * | 2021-10-26 | 2022-01-18 | 维肽瀛(上海)生物技术有限公司 | Polypeptide composition, T lymphocyte and application of T lymphocyte in resisting tumor cells |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030170212A1 (en) * | 2001-05-15 | 2003-09-11 | Zeling Cai | Ex-vivo priming for generating cytotoxic T lymphocytes specific for non-tumor antigens to treat autoimmune and allergic disease |
| US20050048646A1 (en) * | 2003-08-25 | 2005-03-03 | Medinet Co., Ltd. | Method for inducing cytotoxic T lymphocyte |
| CN102168066A (en) * | 2011-01-31 | 2011-08-31 | 浙江大学 | Method for in vitro induction of specific cytotoxic T lymphocytes of hepatitis B virus (HBV) |
| CA2792910A1 (en) * | 2010-03-11 | 2011-09-15 | Yusuke Nakamura | Hjurp peptides and vaccines including the same |
| CN107488235A (en) * | 2016-10-21 | 2017-12-19 | 闾民 | A kind of preparation and application of new enhanced antigen combined polypeptid induction liver cancer-specific CTL cells |
| CN109485721A (en) * | 2018-11-23 | 2019-03-19 | 杜学明 | A method of obtaining tumor specific T cells receptor |
| CN111978375A (en) * | 2020-08-28 | 2020-11-24 | 深圳市乐土生物医药有限公司 | Protein or polypeptide having cytotoxic T cell-inducing ability |
-
2020
- 2020-08-28 CN CN202010889660.5A patent/CN111944018B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030170212A1 (en) * | 2001-05-15 | 2003-09-11 | Zeling Cai | Ex-vivo priming for generating cytotoxic T lymphocytes specific for non-tumor antigens to treat autoimmune and allergic disease |
| US20050048646A1 (en) * | 2003-08-25 | 2005-03-03 | Medinet Co., Ltd. | Method for inducing cytotoxic T lymphocyte |
| CA2792910A1 (en) * | 2010-03-11 | 2011-09-15 | Yusuke Nakamura | Hjurp peptides and vaccines including the same |
| CN102168066A (en) * | 2011-01-31 | 2011-08-31 | 浙江大学 | Method for in vitro induction of specific cytotoxic T lymphocytes of hepatitis B virus (HBV) |
| CN107488235A (en) * | 2016-10-21 | 2017-12-19 | 闾民 | A kind of preparation and application of new enhanced antigen combined polypeptid induction liver cancer-specific CTL cells |
| CN109485721A (en) * | 2018-11-23 | 2019-03-19 | 杜学明 | A method of obtaining tumor specific T cells receptor |
| CN111978375A (en) * | 2020-08-28 | 2020-11-24 | 深圳市乐土生物医药有限公司 | Protein or polypeptide having cytotoxic T cell-inducing ability |
Non-Patent Citations (3)
| Title |
|---|
| EMBL: "UniProtKB - Q8IUE1 (TF2LX_HUMAN)", 《UNIPROT》 * |
| TOSHIE MASHIBA等: "Identification of CTL epitopes in hepatitis C virus by a genome-wide computational scanning and a rational design of peptide vaccine", 《IMMUNOGENETICS》 * |
| 吕建锋等: "应用MAGE-1抗原肽体外诱导特异性细胞毒T淋巴细胞形成", 《中华普通外科杂志》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112666358A (en) * | 2021-01-14 | 2021-04-16 | 深圳裕康医学检验实验室 | Method for detecting antigen immunogenicity by using peripheral blood |
| CN113943347A (en) * | 2021-10-26 | 2022-01-18 | 维肽瀛(上海)生物技术有限公司 | Polypeptide composition, T lymphocyte and application of T lymphocyte in resisting tumor cells |
| CN113943347B (en) * | 2021-10-26 | 2024-01-05 | 维肽瀛(上海)生物技术有限公司 | Polypeptide composition, T lymphocyte and application thereof in anti-tumor cells |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111944018B (en) | 2022-12-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108546679B (en) | Method for amplifying human mature high-activity dendritic cells in large quantity in vitro and application thereof | |
| CN112048001B (en) | Tumor neogenesis antigen polypeptide and application thereof | |
| CN104910278B (en) | A kind of slow virus with high-efficiency transfection ability and biological activity for being used to prepare CART cells | |
| CN111944018B (en) | Antigen polypeptide for inducing liver cancer specific cytotoxic T lymphocyte and application thereof | |
| JP5840857B2 (en) | Composition for inducing cytotoxic T cells | |
| WO2009139413A1 (en) | Method for production of cell mass containing cytokine-induced killer cell | |
| CN110551198B (en) | Lung cancer antigen composition, application thereof and cytotoxic T lymphocyte | |
| CN114230658A (en) | Novel coronavirus-specific T cell receptor and uses thereof | |
| CN111978375B (en) | Protein or polypeptide having cytotoxic T cell-inducing ability | |
| CN106366167B (en) | Epitope peptide and application thereof | |
| CN114729320B (en) | Compositions, methods and uses for reprogramming cells to dendritic cell type 2 capable of presenting antigen | |
| CN112940105B (en) | HLA-A11 restrictive hepatitis B virus HBc 141-151 T cell receptor of epitope peptide and application thereof | |
| CN113913386B (en) | Trophoblast cell and application thereof in amplifying human NK cells | |
| CN1686538A (en) | Dendron shaped cell tumour vaccine for loading withered heat shock tumour cell, its preparation method and application | |
| CN112694519B (en) | Antigens, nucleic acids, expression vectors, antigen presenting cells and cytotoxic T cells and uses thereof | |
| CN114134182A (en) | Preparation method and application of novel immune cells | |
| CN113355283A (en) | Preparation method and application of antigen reactive T cells based on RNA vaccine | |
| CN109294997B (en) | LRFFT1 cell | |
| WO2018032619A1 (en) | Applications of soluble protein baff in b cell in-vitro culture and proliferation | |
| CN116836231B (en) | New antigen peptide of t (8; 21) AML and application thereof | |
| CN117624340B (en) | T Cell Receptor (TCR) recognizing human Hepatitis B Virus (HBV) antigen and use thereof | |
| CN111363009B (en) | Rectal cancer target antigen, CTL cell stimulated and cultured by rectal cancer target antigen and application of CTL cell | |
| WO2020133050A1 (en) | Ebv epitope high affinity t cell receptor | |
| CN110093371B (en) | Construction method of LRFF cell | |
| WO2023179795A1 (en) | Method for rapidly, simply and conveniently obtaining correctly paired tcrs, and obtained tcrs |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |