CN105950645A - Humanized fusion gene segment of CAR-CD19 antigen receptor, construction method and application thereof - Google Patents
Humanized fusion gene segment of CAR-CD19 antigen receptor, construction method and application thereof Download PDFInfo
- Publication number
- CN105950645A CN105950645A CN201610014730.6A CN201610014730A CN105950645A CN 105950645 A CN105950645 A CN 105950645A CN 201610014730 A CN201610014730 A CN 201610014730A CN 105950645 A CN105950645 A CN 105950645A
- Authority
- CN
- China
- Prior art keywords
- car
- fusion gene
- carrier
- hcd19
- sequence
- 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
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—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
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70503—Immunoglobulin superfamily
- C07K14/7051—T-cell receptor (TcR)-CD3 complex
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70503—Immunoglobulin superfamily
- C07K14/70521—CD28, CD152
-
- 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
- C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
-
- 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
- C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/32—Fusion polypeptide fusions with soluble part of a cell surface receptor, "decoy receptors"
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
-
- 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/106—Plasmid DNA for vertebrates
- C12N2800/107—Plasmid DNA for vertebrates for mammalian
Abstract
The invention discloses a humanized fusion gene segment of a CAR-CD19 antigen receptor, a construction method and an application thereof, and belongs to the technical fields of biotechnology and cellular therapy. The humanized fusion gene segment of the CAR-CD19 antigen receptor is a nucleic acid sequence represented as the sequence table SEQ ID No.1. The humanized fusion gene segment can effectively avoid generation of human-anti-mouse antibody due to mouse-sourced CD19 gene. A T lymphocyte, which is modified by a CAR-hCD19 chimeric antigen receptor, can effectively kill tumor cells in a short time after specific identification of the CD19-positive tumor cells. The humanized fusion gene segment can be used for treat CD19-positive B lymphocyte leukemia or B lymphocytoma without obvious toxic and side effects.
Description
Technical field
The invention discloses the humanization fusion gene fragment of a kind of CAR-CD19 antigen receptor, its construction method and answer
With, belong to biotechnology and cell therapy technology field.
Background technology
Leukemia, is commonly called as " leukemia ", is the malignant disease of a kind of hemopoietic tissue, and its main cause is certain of hematopoietic cell
A series of or a certain leukocyte series precursor loses differentiation and maturation ability, in bone marrow and other hemopoietic groups
In malignant clone hypertrophy, accumulation in knitting, and invade other human organs, such as: liver, spleen, lymph node, finally
Cause the destruction of body tissue, organ, make normal hematopoiesis function be suppressed.With solid tumor except that, in vain
Disorders of blood is not to be grown in certain local organs, and is as blood system and is distributed in whole body, therefore influences whether that human body is each
The malignant diseases of individual system, organ and tissue.Leukemia accounts for about the 3% of tumor total incidence, is that China ten is the most occurred frequently
One of malignant tumor, wherein male is more than women.In child and youth most common, be pediatric malignancies sickness rate
In rank the first.Leukemia treating is mainly based on chemotherapy in early days, for middle and advanced stage leukaemic, main treatment
Method has chemotherapy, bone marrow transplantation.Although bone marrow transplantation is the Therapeutic Method that middle and advanced stage leukemia is maximally efficient, but it is also
There is shortcoming expensive, that bone marrow is few.
Chimeric antigen receptor (chimeric antigen receptor, CAR) T cell technology is that development in recent years is the most fast
A kind of cell therapy technology of speed.By genetic modification technology, the targeting of effector T cell, killing activity and persistency
The immunocyte of the most more conventional application is high, and can overcome tumor by local immunosuppressant microenvironment and break host immune tolerance
State.
Chimeric antigen receptor (chimeric antigen receptor, CAR) is (such as strand by the receptor of antigenic specificity
Antibody scFv), intervening sequence (spacer), cross-film sequence (TM Domain) and intracellular costimulatory signal molecular composition.Between
Can be hlgGl, hIgG4, hlgD, CD7 or CD8 etc. every sequence, cross-film sequence can be CD3 ζ, CD4,
CD7, CD8, Fe ε RI Y, H2-Kb etc..Intracellular costimulatory signal molecule can be CD28, CD134, CD137,
CD244, Fe ε RI, (3) 3 ζ etc..The difference of the composition according to intracellular costimulatory signal molecule, CAR is divided into three
Generation.First generation CAR contains only the signaling molecule of an intracellular, and the second filial generation contains two costimulatory signal molecules, and the 3rd
In generation, is containing three costimulatory signal molecules.The T lymphocyte that CAR modifies in vivo can be special by its single-chain antibody
Identification related neoplasms surface antigen, then by intracellular costimulatory signal molecule, the signal of identification is delivered to intracellular,
Activated T lymphocytes lethal, killing tumor cell.
Bone-marrow-derived lymphocyte antigens c D19, also known as CD19, be a kind of Ig of belonging to superfamily member wear membrane glycoprotein, its
It is distributed widely in different times B cell surface, including pre B lymphocyte, immature B cell, mature B cell, activation
B cell.It addition, CD19 by major part B cell malignant tumor (such as, chronic lymphatic leukemia, acute lymphoblastic
Property B leukemia and diffusivity large B cell lymphatic cancer etc.) expressed, but be not distributed in bone marrow on stem cell, because of
The preferable tumor targets of this B cell malignant tumor.CD19 antigen receptor applied in CAR-T technology is to adopt at present
With Mus source gene, such as Mus resource monoclonal antibody FMC63, but this type of Mus source genetic fragment is in treatment human diseases mistake
Cheng Zhongyou causes the probability of the generation of human antimouse antibody (HAMA, Human anti-mouse antibodies), because of
The whole genetic fragment of this humanization just seems extremely important.
Summary of the invention
First technical problem that the invention solves the problems that is to provide the humanization of a kind of CAR-CD19 antigen receptor and merges base
Because of fragment.
Second technical problem the invention solves the problems that is to provide above-mentioned fusion gene fragment and the carrier containing this fragment
Construction method.
The 3rd technical problem the invention solves the problems that is to provide above-mentioned fusion gene fragment and the carrier containing this fragment
Purposes.
For achieving the above object, the present invention is by the following technical solutions:
The humanization fusion gene fragment of a kind of CAR-antigen receptor, is sequentially spliced humanized antigen by nitrogen end to carbon teminal
ScFv sequence, CD28 link zone sequence, CD28 transmembrane protein region sequence and signal structure territory sequence.
" antigen " of described antigen scFv sequence includes but not limited to CD10, CD19, CD20, CD22,
CD24, CD30, CD33, CD38, CD39, CD133, CD138, the preferred CD19 of the present invention.
Described " signal structure territory sequence " includes but not limited to CD3-zeta sequence, FC ε RI γ, CD28,
4-1BB (CD137), one or more in 0X-40 (CD134), safe suicide gene, such as FKBPCasp9 etc..This letter
Number domain sequence can be in any combination.The present invention selects CD3-zeta sequence.
The humanization fusion gene fragment of a kind of CAR-CD19 antigen receptor, is sequentially spliced humanization by nitrogen end to carbon teminal
CD19scFv sequence, CD28 link zone sequence, CD28 transmembrane protein region sequence, protein region in CD28 Cytoplasm
Sequence and CD3-zeta sequence.
The humanization fusion gene fragment (i.e. CAR-hCD19 fusion gene fragment) of described CAR-CD19 antigen receptor is
Nucleotide sequence shown in sequence table SEQ ID No.1.
The present invention is in order to reduce the size of vector plasmid, and then increases transfection and the production efficiency of virus, and humanization merges
Some site in genetic fragment is deleted by artificial, the most effectively shortens the sequence of whole gene,
Maintain two grades and tertiary structure of complete fusion protein, remain its effective antigen recognition, carry simultaneously
High cytotoxicity.
Contain and the carrier of humanization fusion gene fragment of CAR-CD19 antigen receptor described in expressing.Described carrier is
Finger both can include permanent being integrated into cell entrained genetic fragment at the various viruses of cell inner expression and plasmid
In genome, be also included within intracellular can only the carrier of transient expression.Described carrier includes but not limited to virus, matter
Grain or RNA；Described virus is slow virus, adenovirus, adeno-associated virus or retrovirus retrovirus or other diseases commonly used in the art
Poisonous carrier.
A kind of construction method of the carrier of the humanization fusion gene fragment containing CAR-CD19 antigen receptor, including with
Lower step:
(1) utilize PCR and overlap extension technology design packet respectively containing BsmI and NotI restriction enzyme site and at least two with
The primer of machine base, introduces BsmI and NotI the two ends of hCD19 antibody human source code gene, and will comprise hCD19
PCR fragment product cloning enter pGEM-T easy carrier, obtain pGEM-T-hCD19；
(2) with the method that step (1) is same expand respectively CD28 link zone sequence, CD28 transmembrane protein region sequence,
Protein region sequence and CD3zeta fragment in CD28 Cytoplasm, be integrated in pGEM-T-hCD19 carrier in order,
To the carrier comprising CAR-hCD19 fusion gene fragment；
(3) by BsmI and SalI, whole CAR-hCD19 fusion gene fragment is inserted into pHIV-EGFP eucaryon table
Reach in carrier, obtain pcHIV-CAR-hCD19 expression vector.
Primer in described step (1) is sequence table SEQ ID No.2 and the core shown in sequence table SEQ ID No.3
Nucleotide sequence；In step (2), CAR-hCD19 fusion gene fragment is the nucleoside shown in sequence table SEQ ID No.1
Acid sequence.
The host cell of the humanization fusion gene fragment containing above-mentioned CAR-hCD19 antigen receptor.
The host cell of the carrier of the humanization fusion gene fragment containing CAR-hCD19 antigen receptor.
A kind of medicine treating cancer, the carrier of the humanization fusion gene fragment containing CAR-hCD19 antigen receptor
Host cell, be prepared from as active component.
Described host cell is lymphocyte.The present invention carries the carrier of fusion gene, whole by its distinctive mode
Closing in the genome of T lymphocyte, fusion gene is correctly expressed, and is formed and has expressed by identification cancer cell
The scFv part of specific antigen, transmembrane protein part and two synergistic signal domains.Now, T lymphocyte
It is configured to CAR-T lymphocyte, therefore there is specific killing and express the cancer cell of certain specific antigen.
The humanization fusion gene fragment of described CAR-CD19 antigen receptor is for preparing the use of the medicine for the treatment of cancer
On the way.
The carrier of the humanization fusion gene fragment containing CAR-CD19 antigen receptor is for preparing the medicine for the treatment of cancer
Purposes.
The host cell of the carrier of the humanization fusion gene fragment containing CAR-CD19 antigen receptor is used for preparing treatment
The purposes of the medicine of cancer.
Described cancer is bone-marrow-derived lymphocyte leukemia positive for CD19 or bone-marrow-derived lymphocyte tumor.
The present invention can be used for treating leukemia positive for CD19 and bone-marrow-derived lymphocyte tumor, it is provided that a kind of novel CAR-T
(Chimeric Antigen Receptor T-Cell Immunotherapy, Chimeric antigen receptor T cell immunity is treated
Method) cell therapeutic approach.
Cell experiment shows, the carrier for expression of eukaryon containing CAD-hCD19 fusion gene that the present invention builds, and is cultivating
Under the conditions of quickly rise in value, in retrovirus is incorporated into the genome of T lymphocyte, enter at T lymphocytic cell surface
Row is expressed, the CD19 albumen expressed by specific recognition bone-marrow-derived lymphocyte surface, and then effectively kills bone-marrow-derived lymphocyte.
After 7-10 days cultivate, the quantity of CAR-T lymphocyte is by hundreds and thousands of times of ground propagation, and cytotoxicity reaches
High.From culture environment, the isolated and purified CAR-hCD19 T lymphocyte obtained can be frozen, on suitable opportunity
Under feed back in the patient.
The invention have the advantage that the humanized CAD-hCD19 fusion gene sequence that the present invention designs, effectively prevent
The generation of the human antimouse antibody that Mus source CD19 gene causes.This Chimeric antigen receptor is proceeded to expression vector, it is achieved that
The high efficient expression on human lymphocyte surface, by nonspecific lymphocyte directional transformation for being capable of identify that people CD19 thus
Tumor cell positive for mediation CD19 carries out the specific lymphocyte of target killing.The most particularly preferred fusion gene
Sequence makes mosaic antigen receptor preferably play a role.The T lymph that CAR-hCD19 Chimeric antigen receptor is modified is thin
Born of the same parents, after the tumor cell (bone-marrow-derived lymphocyte) that special knowledge CD19 is positive, can the most effectively kill tumor cell,
And exist the most for a long time, can effectively prevent the recurrence of the state of an illness, can be used for treating B lymph positive for CD19 thin
Born of the same parents' leukemia or bone-marrow-derived lymphocyte tumor, and there is no obvious toxic and side effects.
Below in conjunction with the drawings and specific embodiments, the present invention will be further described, not limitation of the present invention.Root
According to general knowledge known in this field, the implementation of the present invention is not limited in embodiment disclosure of that, the most all according to
The equivalent of this area that the technology of the present invention enlightenment is carried out, belongs to protection scope of the present invention.
Accompanying drawing explanation
Fig. 1 is principle of the invention figure
Fig. 2 is CAR-hCD19 fusion gene fragment design drawing
Fig. 3 is the CAR-hCD19 fusion gene expression spirogram at T lymphocytic cell surface
Fig. 4 is the killing experiments result figure of the T lymphocyte expressing CAR-hCD19 fusion gene
Detailed description of the invention
Embodiment 1: plamid vector construction
Hereinafter the DNA genetic fragment in experiment is synthesized by amplification in vitro, and is determined its correct sequence by gene sequencing.
1, utilize PCR and overlap extension technology design packet respectively containing BsmI and NotI restriction enzyme site and at least two with
The primer 5 of machine base '-GGGCATTCCTCCTGATCCAGACATCCAG (SEQ ID No.2) and
5’-AGTCAGTGGCAGAGGAGTCGCCGGCGTGG(SEQ ID No.3).BsmI and NotI is introduced hCD19
The two ends of antibody human source code gene, and the PCR fragment product cloning comprising hCD19 is entered pGEM-T easy carrier
(carrier is purchased from Promega company), obtains pGEM-T-hCD19；
2, CD28 link zone sequence, CD28 transmembrane protein region sequence, CD28 are expanded respectively by the method for step (1)
In Cytoplasm, protein region sequence and CD3zeta fragment, be integrated in order in pGEM-T-hCD19 carrier, wrapped
The carrier of the humanization fusion gene fragment containing CAR-hCD19 antigen receptor；
3, by BsmI and SalI restriction enzyme site by the humanization fusion gene sheet of whole CAR-hCD19 antigen receptor
Section is inserted into slow virus plasmid vector pHIV-EGFP (https: //www.addgene.org/21373/, addgene company
Product, Plasmid#21373) EF-1 α promoter after, obtain pcHIV-CAR-hCD19 expression vector.
The CAR-hCD19 core gene fragment obtained by humanized CD19scFv, immediately after be CD28
In link zone, CD28 transmembrane protein district, CD28 Cytoplasm, protein region and the firsts and seconds being made up of CD3-zeta are believed
Number domain (see Fig. 2), CAR-hCD19 core gene fragment is the nucleotides sequence shown in sequence table SEQ ID No.1
Row.
Embodiment 2: prepared by slow virus
The vector plasmid that embodiment 1 obtains with other slow virus packaging plasmids, as packaging plasmid pMDLg/pRRE,
PRSV-Rev and envelope protein plasmid pMD2.G, according to the ratio of 1: 1: 1: 0.5, uses transfection reagent, such as calcium phosphate
Or Lipofectamine (Thermo Fisher) etc., illustrate that common transfection densities reaches according to concrete reagent operation
The 293T cell strain of 60%-70%.
293T cell is after transfection, within 24-72 hour, starts to collect by the form collecting cell supernatant
Slow virus carrier, process is centrifuged afterwards, the mode of filtration concentrates, purification slow virus carrier, finally under the conditions of-80 DEG C
Long-term frozen preserves.
The preparation of embodiment 3:CAR-T cell and model test
The peripheral blood cells product obtained by leukopheresis, uses the mode separation peripheral blood of density gradient centrifugation
Mononuclearcell (mainly comprise the T lymphocyte of CD3+, the bone-marrow-derived lymphocyte of CD19+ and a small amount of dendritic cell,
Macrophage etc.), cultivate without in the cell culture medium of Ox blood serum.
T lymphocyte activate through CD3/CD28 Dynabeads (be purchased from Thermo Fisher company) in vitro and
After slow virus (the conversion concentration of 1: 1-1: 50) transduction, the most quickly rise in value.Take different time
The cell of section, diluted concentration to 1E6 cell/100 μ l PBS, use antibody CD3-FITC, CD19-PE (is purchased from Becton
Dickinson) according to the concentration of producer's suggestion for operation, dye on ice 30 minutes, in concentration more than 0.5% after cleaning
Eddy diffusion in polyformaldehyde solution, is finally carried out quantitatively on Accuri C6 flow cytometer (being purchased from BD company)
Analyze.To be integrated into T lymph thin by permanent after reverse transcription for CAR-hCD19 fusion gene entrained by slow virus
In the genome of born of the same parents, and carry out expressing (see Fig. 3) at T lymphocytic cell surface, and can specific recognition bone-marrow-derived lymphocyte
CD19 albumen expressed by surface, and then kill bone-marrow-derived lymphocyte (see Fig. 4).
Fig. 3 shows, takes the cell before the 5th day and cell harvesting, according to the method described above, staining cell respectively, and
Being analyzed on flow cytometer, display CAR-hCD19 expresses at T lymphocytic cell surface, and express cell contains
Measure and increase along with incubation time and increase.
Fig. 4 shows, after using the mode separating peripheral blood mononuclear cells of density gradient centrifugation, at the 0th day, and the 3rd
My god, the 6th day, take cell respectively, according to the method described above, CD3+T cell and CD19+B cell are carried out flow cytometer showed.
Wherein, Mock group is matched group (being not added with virus to transform, do not express CAR), and Vector group is experimental group (warp
Cross slow virus transformation, express CAR+), 1 and 2 is the sample group of two different people.Matched group 1-Mock and 2-Mock
Display, the T cell transforming, not expressing CAR+ without slow virus does not have the function killing CD19+B cell, from
The B cell that always there is CD19+ in 0th day to the 6th day；And experimental group 1-Vector and 2-Vector display is passed through
Slow virus transformation, the T cell of expression CAR+ have the function killing CD19+B cell, start to detect not from the 3rd day
B cell to CD19+.
After the cultivation of 7-10 days, the quantity of T lymphocyte is by the increment of hundreds and thousands of times, and cytotoxicity reaches
To the highest.Finally, the CD3/CD28 Dynabeads in mixed culture is separated, CAR-hCD19 T after purification
Lymphocyte is frozen, and is fed back in patient body on suitable opportunity.
The present invention carries the carrier of fusion gene, is integrated in the genome of T lymphocyte by its distinctive mode,
Fusion gene is correctly expressed, formed have identify specific antigen expressed by cancer cell scFv part, across
Memebrane protein part and two synergistic signal domains.Now, T lymphocyte is configured to CAR-T lymphocyte, because of
This has specific killing and expresses the cancer cell of certain specific antigen.
Claims (10)
1. the humanization fusion gene fragment of a CAR-CD19 antigen receptor, it is characterised in that: by nitrogen end to carbon teminal
Sequentially splice humanized CD19 scFv sequence, CD28 link zone sequence, CD28 transmembrane protein region sequence, CD28
Protein region sequence and CD3-zeta sequence in Cytoplasm.
Humanization fusion gene fragment the most according to claim 1, it is characterised in that: described genetic fragment is
Nucleotide sequence shown in sequence table SEQ ID NO.1.
3. contain and express the humanization fusion gene fragment of CAR-CD19 antigen receptor described in claim 1 or 2
Carrier.
Carrier the most according to claim 3, it is characterised in that: described carrier is virus, plasmid or RNA；Institute
State virus for slow virus, adenovirus, adeno-associated virus or retrovirus retrovirus.
5. a construction method for the carrier of the humanization fusion gene fragment containing CAR-CD19 antigen receptor, it is special
Levy and be to comprise the following steps:
(1) utilize PCR and overlap extension technology design packet respectively containing BsmI and NotI restriction enzyme site and at least two with
The primer of machine base, introduces BsmI and NotI the two ends of hCD19 antibody human source code gene, and will comprise hCD19
PCR fragment product cloning enter pGEM-T easy carrier, obtain pGEM-T-hCD19；
(2) CD28 link zone sequence, CD28 transmembrane protein region sequence, CD28 are expanded respectively by the method for step (1)
In Cytoplasm, protein region sequence and CD3zeta fragment, be integrated in order in pGEM-T-hCD19 carrier, wrapped
Carrier containing CAR-hCD19 fusion gene fragment；
(3) by BsmI and SalI, whole CAR-hCD19 fusion gene fragment is inserted into pHIV-EGFP eucaryon table
Reach in carrier, obtain pcHIV-CAR-hCD19 expression vector.
A kind of humanization fusion gene fragment containing CAR-CD19 antigen receptor the most according to claim 5
The construction method of carrier, it is characterised in that: the primer in described step (1) be sequence table SEQ ID NO.2 and
Nucleotide sequence shown in sequence table SEQ ID NO.3；In step (2), CAR-hCD19 fusion gene fragment is sequence
Nucleotide sequence shown in list SEQ ID NO.1.
7. contain CAR-CD19 antigen receptor described in claim 1 or 2 humanization fusion gene fragment and/or
The host cell of the carrier described in right 3 or 4；Described host cell is lymphocyte.
8. the medicine treating cancer, it is characterised in that: containing the fusion gene sheet described in claim 1 or 2
Section or containing the carrier described in claim 3 or 4 or the host cell described in claim 7.
9. the humanization fusion gene fragment of the CAR-CD19 antigen receptor described in claim 1 or 2 and/or right
Carrier described in 3 or 4 and/or the host cell described in claim 7 are for preparing the purposes of the medicine for the treatment of cancer.
The purposes of the medicine of preparation treatment cancer the most according to claim 9, it is characterised in that: described cancer
Disease is bone-marrow-derived lymphocyte leukemia positive for CD19 or bone-marrow-derived lymphocyte tumor.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610014730.6A CN105950645A (en) | 2016-01-11 | 2016-01-11 | Humanized fusion gene segment of CAR-CD19 antigen receptor, construction method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610014730.6A CN105950645A (en) | 2016-01-11 | 2016-01-11 | Humanized fusion gene segment of CAR-CD19 antigen receptor, construction method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105950645A true CN105950645A (en) | 2016-09-21 |
Family
ID=56917286
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610014730.6A Pending CN105950645A (en) | 2016-01-11 | 2016-01-11 | Humanized fusion gene segment of CAR-CD19 antigen receptor, construction method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105950645A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108508200A (en) * | 2018-04-18 | 2018-09-07 | 上海尚珞生物医药科技有限公司 | Detect the method and its application of the cell of CD19 CAR |
| CN111253487A (en) * | 2018-12-03 | 2020-06-09 | 广东东阳光药业有限公司 | CD19 antibodies and uses thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103492406A (en) * | 2010-12-09 | 2014-01-01 | 宾夕法尼亚大学董事会 | Use of chimeric antigen receptor-modified t cells to treat cancer |
| WO2014153270A1 (en) * | 2013-03-16 | 2014-09-25 | Novartis Ag | Treatment of cancer using humanized anti-cd19 chimeric antigen receptor |
-
2016
- 2016-01-11 CN CN201610014730.6A patent/CN105950645A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103492406A (en) * | 2010-12-09 | 2014-01-01 | 宾夕法尼亚大学董事会 | Use of chimeric antigen receptor-modified t cells to treat cancer |
| WO2014153270A1 (en) * | 2013-03-16 | 2014-09-25 | Novartis Ag | Treatment of cancer using humanized anti-cd19 chimeric antigen receptor |
Non-Patent Citations (2)
| Title |
|---|
| GENBANK登录号：HM852952.1: "Synthetic construct FMC63-28Z receptor protein gene, complete cds", 《GENBANK数据库》 * |
| KOCHENDERFER JN等: "Construction and Pre-clinical Evaluation of an Anti-CD19 Chimeric Antigen Receptor", 《JOURNAL OF IMMUNOTHEREPY》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108508200A (en) * | 2018-04-18 | 2018-09-07 | 上海尚珞生物医药科技有限公司 | Detect the method and its application of the cell of CD19 CAR |
| CN108508200B (en) * | 2018-04-18 | 2023-12-22 | 上海星湾生物技术有限公司 | Method for detecting CD19 CAR-expressing cells and application thereof |
| CN111253487A (en) * | 2018-12-03 | 2020-06-09 | 广东东阳光药业有限公司 | CD19 antibodies and uses thereof |
| WO2020114358A1 (en) * | 2018-12-03 | 2020-06-11 | 广东东阳光药业有限公司 | Cd19 antibody and uses thereof |
| CN111253487B (en) * | 2018-12-03 | 2024-02-02 | 广东东阳光药业股份有限公司 | CD19 antibodies and uses thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| AU2018202976B2 (en) | Polyclonal gamma delta t cells for immunotherapy | |
| CN106146666B (en) | Target the immune effector cell and its preparation method and application of CLDN6 | |
| CN106279434B (en) | Engineered CD20 targeted NKT cell and preparation method and application thereof | |
| US10604740B2 (en) | Nucleic acids encoding chimeric antigen receptor proteins which bind epidermal growth factor receptor and T lymphocyte expressing the protein | |
| US10647997B2 (en) | Modified effector cell (or chimeric receptor) for treating disialoganglioside GD2-expressing neoplasia | |
| JP7190096B2 (en) | Gene-edited T cells and uses thereof | |
| TW201839127A (en) | Methods and compositions for transducing and expanding lymphocytes and regulating the activity thereof | |
| CN116063574A (en) | HER 2-targeting chimeric antigen receptor | |
| CN107249602A (en) | Target the Chimeric antigen receptor of hematologic malignancies（CAR）, its composition and application method | |
| CN110734931A (en) | humanized scFv chimeric antigen receptor T cells targeting CD19, and preparation method and application thereof | |
| CN107206100A (en) | The method and composition of NK | |
| CN112979828A (en) | T lymphocyte targeting CLD18A2 and preparation method and application thereof | |
| CN109721659B (en) | Novel Chimeric Antigen Receptor (CAR) targeting CD19 and application thereof | |
| JP2021531008A (en) | GD2-based chimeric antigen receptor and its utilization | |
| JP2018065805A (en) | Chimeric antigen receptor specific for tumor cells | |
| CN108103105A (en) | A kind of preparation method of CAR-T cells, CAR-T cells obtained and its application | |
| CN109055380A (en) | A kind of preparation method of universal CAR-T cell | |
| CN108368159A (en) | Chimeric antigen receptor containing catilan domain | |
| CN107033248A (en) | Recognize Chimeric antigen receptor and its application of carcinomebryonic antigen | |
| CN109021114B (en) | Bispecific chimeric antigen receptor combining two single-chain antibodies and expression vector | |
| CN110317822A (en) | TROP2 Chimeric antigen receptor, its T cell and its preparation method and application | |
| Morimoto et al. | Natural killer cell-based immunotherapy against glioblastoma | |
| CN105950645A (en) | Humanized fusion gene segment of CAR-CD19 antigen receptor, construction method and application thereof | |
| CN108753773A (en) | Interfere CD19-CAR-T cells and its application of IFN-gama expression | |
| CN111983218A (en) | Kit for detecting interaction of living cell-living cell surface receptor-ligand |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20160921 |
|
| RJ01 | Rejection of invention patent application after publication |