CN106317228A - Chimeric antigen receptor molecule and application thereof - Google Patents

Chimeric antigen receptor molecule and application thereof Download PDF

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Publication number
CN106317228A
CN106317228A CN201610855798.7A CN201610855798A CN106317228A CN 106317228 A CN106317228 A CN 106317228A CN 201610855798 A CN201610855798 A CN 201610855798A CN 106317228 A CN106317228 A CN 106317228A
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cell
peptide fragment
chimeric antigen
antigen receptor
receptor molecule
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李华顺
韩昆昆
薛亚男
王保垒
任宝永
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Priority to PCT/CN2016/100796 priority patent/WO2018058431A1/en
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    • 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
    • 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
    • C07K14/70517—CD8
    • 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
    • C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
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    • 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
    • C12N15/86—Viral vectors
    • 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
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2510/00—Genetically modified cells
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Abstract

The invention relates to the field of molecular biology, and in particular relates to a chimeric antigen receptor molecule and application thereof. The invention provides the chimeric antigen receptor molecule. The chimeric antigen receptor molecule comprises an extracellular region peptide fragment, a membrane-spanning region peptide fragment and an intracellular structure domain peptide fragment which are sequentially and serially connected, wherein the extracellular region peptide fragment is an HAC (Human Artificial Chromosome) peptide fragment, the sequence of the HAC peptide fragment is shown in SEQ ID NO:1; meanwhile, the invention also provides nucleotide, a recombinant vector and reconstitution cells of the chimeric antigen receptor molecule. The chimeric antigen receptor molecule provided by the invention has higher affinity with PDL-1 molecules on the surface of tumor.

Description

A kind of Chimeric antigen receptor molecule and application thereof
Technical field
The present invention relates to biology field, particularly relate to a kind of Chimeric antigen receptor molecule and application thereof.
Background technology
Malignant tumor is the disease of a kind of serious threat human life.The pathogeny of malignant tumor or cancer is multiple many Sample, its common performance is that the tumor cell of variation is not removed by body immune system, it is possible to unconfined breeding and expansion Dissipate, destroy normal cell and the function of surrounding tissue.For a long time, medicine sector is attempting curing and controlling the disease of tumor disease Journey aspect has done substantial amounts of effort, but produces little effect.At present, in the treatment of malignant tumor, in addition to radical surgery, mainstream health care The clinical auxiliary treatment means on boundary remains radiation cure, chemotherapy and Antybody therapy.
1985, American scientist attempt separate patient mononuclearcell, and in vitro with various cytokine inductions, Activate, produce killer T cell (Cytokine-induced killer cells, CIK), then find that these cells pass through After intravenous drip feeds back to patient, the growth to tumor has lethal effect.Develop through the clinical practices of nearly 30 years and technology, The 4th kind of generally acknowledged malignant tumor auxiliary treatment means is had become as by immunologic cytotoxicity cell therapy tumor.It is commonly used in clinic The immunologic cytotoxicity cell for the treatment of: natural killer cell (Natural killer cells, NK), gamma delta T cells, cytotoxic T drench Bar cell (Cytotoxic T lymphocytes, CTL), NK sample T lymphocyte (Natural killer-like T Cells, NKT), Th1 effector lymphocyte (Effector cells) etc..One of antigen presenting cell dendritic cell (Dentritic Cells, DC) it is also commonly used for the application of immune cell therapy, DC can improve the specificity of immunologic cytotoxicity cell, booster immunization kills Hinder the vigor of cell.
Immunologically competent cell, to the identification of tumor cell and fragmentation effect, depends on the acceptor molecule on tumor cell membrane surface Expressing, at least both sides factor causes internal T lymphocyte can not identify cancerous cell well: (1) cancerous cell is lowered The expression of antigen presenting molecules, the antigen that (2) are rendered is the most weak with φt cell receptor affinity.Although existing in cancer patient's body The T lymphocyte of cancerous cell high degree of specificity, but quantity does not has the effect for the treatment of cancer very little.In order to overcome immunologic cytotoxicity The shortcoming that cell-specific is low, a kind of transgene method of American scientist invention: tumor surface specific antigen will be identified Monoclonal antibody hypervariable region sequence recombinate in vitro, sub-clone is single chain antibody fragments (Single chain antibody Fragment of variable regions, scFv), then merge with the transmembrane protein fragment of other genes and intracellular signal peptide Form artificial Chimeric antigen receptor (Chimeric antigen receptor, CAR), transfect to T cell formation chimeric Antigen receptor T cell (Chimeric antigen receptor, CAR-T).Chimeric antigen receptor is mainly made up of two parts, One end be positioned at extracellular can a certain antigen of specific recognition cancer cell surfaces, the other end is positioned at intracellular and contains signal activation unit Part (such as the Zeta chain of φt cell receptor), plays the effect of transmission signal activation T cell.
The CAR-T cell therapy developed with CD19 for target spot at present, at a phase and second phase hematological system tumor clinical treatment On all achieve obvious effect, but CAR-T cell therapy making slow progress in the research of solid tumor, there is presently no aobvious The breakthrough of work property.One of reason is 95% all to express B cell antigen CD19 in lymphoid leukemia cancerous cell, and other solid tumors The specific antigen of cell is expressed between 40-70%；So in the treatment of solid tumor, single monoclonal antibody CAR-T cell is not The cancerous cell expressing other tumor specific antigens may be killed.The two of reason are that immunologic cytotoxicity cell is by blood/lymph fluid Speed and the number of entity tumor is arrived in circulation infiltration.The three of reason are that the negative regulator of immunologically competent cell is made by tumor tissue cell With.
Under normal circumstances, the blood circulation of human body exists a small amount of activity and kills cell, such as NK, gamma delta T cells etc., Its role is to remove old and feeble, the histiocyte of variation or resist poisoning intrusion.The activity of immunocyte is mainly thin by T regulation The control of born of the same parents (Regulatory T cells, Treg), when the control of T regulation cell weakens, can cause immunologic function hyperfunction Or generation autoimmune disease；When controlling to strengthen, immunologic hypofunction can be made, grow tumor or other viral skins Sick.The protein factor of the participation negative regulator confirmed at present has Cytotoxic T lymphocyte associated antigen-4 (Cytotoxic T Lymphocyte-associated antigen-4, CTLA4) and death protein 1/ death protein 1 aglucon 1 (Programmed cell death protein 1/Programmed cell death protein 1 ligand 1, PD- 1/PD-L1) equimolecular.Inhibition T regulation cell expresses CTLA4, can be with the B7 race albumen on immunocyte DC and T cell surface Subunit interacts, and suppresses immune cell function.B7 protein family member has B7-H1 (PD-L1), B7-H2 (PD-1L2) etc.. B7-H1 (PD-L1) can also pass through bind lymphocytes PD-1 molecule, suppresses the active function of target cell further.PD-1 and PD-L1 interaction is a major obstacle of immune cell therapy tumor to the suppression of T cell function.At most of solid tumors In tissue, cancerous cell is expressed the level of PD-L1/PD-1 and is increased, and produces the activated lymphocyte of infiltration to cancerous tissue directly Suppression, this is also one of the mechanism of tumor escape immunity of organism supervision；The expression of activating T cell surface PD-1 increases, it is easier to Suppressed by negative regulator.It is pernicious swollen that up-to-date antibody drug keytruda and opdivo has been approved by the FDA in the United States clinical treatment Tumor, its action principle is to block be combineding with each other of CTLA4/B7 and PD-1/PD-L1, thus releases internal T regulation cell or cancer Cell kills the suppression of cell to activity T, allows vivo immunization competent cell kill tumor cell, to remove.The present invention is led to Cross the PD-1 molecule of change Chimeric antigen receptor molecule (CAR) extracellular fragment, select higher point of the affinity than PD-1 Yu PDL1 Son, prepares specificity and lethal higher high-affinity Chimeric antigen receptor molecule.
Summary of the invention
For solving above-mentioned technical problem, it is an object of the invention to provide one and have higher with tumor surface PDL-1 molecule The Chimeric antigen receptor molecule of affinity and application thereof.
First aspect present invention provides a kind of Chimeric antigen receptor molecule, including the extracellular region peptide fragment being sequentially connected in series, cross-film District's peptide fragment and intracellular domain peptide fragment, described extracellular region peptide fragment includes HAC peptide fragment, and its sequence is as shown in SEQ ID NO:1.According to 《Engineering high-affinity PD-1 variants for optimized immunotherapy and Immuno-PET imaging " article report, the mutant HAC of PD-1 has and tumor surface PDL-1 molecule more high-affinity.
It should be noted that, described cross-film district peptide fragment is the region crossing over cell membrane in protein sequence, usually α-spiral shell Rotation structure, about 20～25 amino acid residues, its aminoacid major part is hydrophobic amino acid；Wherein, described cross-film district peptide fragment Include but not limited to CD8 cross-film district's peptide fragment or PD-1 cross-film district peptide fragment, when cross-film district peptide fragment is CD8 cross-film district's peptide fragment, described HAC peptide fragment is connected also by the hinge district peptide fragment of CD8 with CD8 cross-film district peptide fragment.The sequence such as SEQ ID of CD8 cross-film district peptide fragment Shown in NO:2, the sequence of the hinge district peptide fragment of CD8 as shown in SEQ ID NO:3, the sequence such as SEQ ID of PD-1 cross-film district peptide fragment Shown in NO:4.
Further, described intracellular domain peptide fragment is costimulatory signal molecule, selected from 4-1BB (also known as CD137), One or more in the intracellular domain peptide fragment of CD28, CD3 ζ.Preferably, described intracellular domain peptide fragment is selected from being connected with each other 4-1BB and CD3 ζ intracellular domain peptide fragment, its sequence is respectively as shown in SEQ ID NO:5 and 6.
Further, described Chimeric antigen receptor molecule also includes signal peptide.Signal peptide can improve Chimeric antigen receptor The effect of this fusion protein of molecule secretion, after being expressed together with other aminoacid sequence of signal peptide and fusion protein, finally Removed by proteolytic cleavage.Protease has certain recognition sequence, and signal peptide constitutes new ammonia after merging with peptide fragment behind Base acid sequence, if so the signal peptide selected is improper, may result in cutting of protease, protein inactivation by mistake.Signal peptide is optional From signal peptide or the signal peptide of PD-1 protein excretion of light chain immunoglobulin, its sequence is respectively such as SEQ ID NO:7 and 8 Shown in.
Preferably, the Chimeric antigen receptor molecule of the present invention, by the signal peptide of light chain immunoglobulin, HAC peptide fragment, CD8 Hinge district peptide fragment, the cross-film district peptide fragment of CD8,4-1BB intracellular domain peptide fragment and CD3 ζ intracellular domain peptide fragment be sequentially connected with Forming, its sequence is as shown in SEQ ID NO:9.
Second aspect present invention provides a kind of nucleotide, and the Chimeric antigen receptor that its coding first aspect present invention provides is divided Son.
Further, comprise or for the nucleotide sequence shown in SEQ ID NO:10.
Third aspect present invention provides a kind of recombinant vector, and it comprises the nucleotide that second aspect present invention provides.
Further, described carrier is slow virus carrier, and the shRNA of exogenous gene or external source can be integrated by effectively On host chromosome, thus reach persistency and express the effect of aim sequence.God can be effectively infected in terms of infection ability Through polytype cells such as unit cell, hepatocyte, myocardial cell, tumor cell, endotheliocyte, stem cell, thus reach good Good gene therapy effect.For the cell of some more difficult transfections, such as primary cell, stem cell, undifferentiated cell etc., make With slow virus carrier, the transduction efficiency of genes of interest or purpose shRNA can be greatly improved, and genes of interest or purpose shRNA whole Close the probability of host cell gene group to be greatly increased, it is possible to more convenient genes of interest or purpose shRNA of realizing quickly For a long time, stable expression.
It should be noted that, slow virus carrier used in the present invention, it should include but not limited to pRRSLIN slow virus table Reach carrier and pLVX carrier, preferably pRRSLIN Lentiviral.
Fourth aspect present invention provides a kind of reconstitution cell, and it comprises the recombinant vector that third aspect present invention provides.Institute State reconstitution cell and be preferably T cell or NK cell.The encoding gene of this Chimeric antigen receptor can be transferred by aforementioned bearer Intracellular to T cell or NK, it is used for modifying T cell or NK cell, becomes CAR-T or CAR-NK cell；Utilize this chimeric antigen The T cell of receptor modification or NK cell, it is possible to by the tissue factor on tumor cell surface, kill tumor cell, carry out Oncotherapy.
In the present invention:
Term " costimulatory signal molecule " (Co-stimulating molecule) refer to immunocyte surface some glue Attached molecule, such as CD28, CD134/OX40, CD137/4-1BB, CD40 etc., by being combined with its part, the of immune cell activated Binary signal, strengthens multiplication capacity and the secretory function of cytokine of immunocyte, extends the time-to-live of activating immune cell.
Term " extracellular region " refers to that memebrane protein is positioned at extracellular section.
Term " domain " refers to the region in protein bio macromole with specific structure and standalone feature, common knot The total number of atnino acid in structure territory is between 100～400, and minimum domain only has 40～50 amino acid residues, big knot Structure territory can be more than 400 amino acid residues.
Term " PD-1 " refers to mankind's programmed death factor 1 (programmed cell death protein1), base Because of title PDCD1_HUMAN, corresponding protein sequence numbering has UniProtKB-Q15116, is T cell immunosuppression molecule, its born of the same parents Outer region domain is similar to the variable region (V-section) of immunoglobulin, has specific binding its part PD-L1 and PD-L2 The characteristic of (Programmed cell death protein 1ligand 1/2).PD-1 is generally in the T lymphocyte of activation Express, Several Kinds of Malignancy cell also has expression.
Term " PD-L1 ", " PD-L2 " refer to the mankind's programmed death factor 1 aglucon 1 and the aglucon 2 having now been found that (programmed cell death protein 1ligand 1/2).Its extracellular region structural domain has similar immunoglobulin V and C1 district, combine (4zqk Structure 23 2341-2348,2015) with the V district of PD-1 by V district.Generally in tree Prominent shape cell DC, T regulation cell and Th cell, macrophage, Mast cell and bone marrow are expressed, on a small quantity at Several Kinds of Malignancy Cell also has expression.
By such scheme, the present invention at least has the advantage that the Chimeric antigen receptor molecule of the present invention, has with swollen The HAC molecule of tumor surface PDL-1 molecule more high-affinity, is assembled in CAR-T cell extracellular fragment, is used for identifying that tumor is thin Born of the same parents, have higher affinity and killing activity.
Described above is only the general introduction of technical solution of the present invention, in order to better understand the technological means of the present invention, And can be practiced according to the content of description, below with presently preferred embodiments of the present invention and coordinate accompanying drawing describe in detail as after.
Accompanying drawing explanation
Fig. 1 is that Lentiviral builds schematic diagram；
Fig. 2 is the streaming result figure that pRRSLIN-HAC infects 3 days；
Fig. 3 is the CAR-HAC Mortaility results figure to target cell the most of the same race；
Fig. 4 is the CAR-HAC testing result figure to the in-vitro multiplication of target cell the most of the same race；
Fig. 5 is the CAR-HAC testing result figure to the cytokine of target cell the most of the same race；
Fig. 6 is the CAR-HAC testing result figure to 4 kinds of cell strains of MCF7/MCF7-PDL1/HeLa/SMC7721；
Fig. 7 is the fragmentation effect comparison diagram for different cell strain CAR-PD-1 and CAR-HAC.
Detailed description of the invention
Below in conjunction with the accompanying drawings and embodiment, the detailed description of the invention of the present invention is described in further detail.Hereinafter implement Example is used for illustrating the present invention, but is not limited to the scope of the present invention.
Prepared by embodiment 1 Lentiviral
The present invention provides the preparation method of a kind of Lentiviral for expressing Chimeric antigen receptor molecule, including Following steps:
S1, basis " Engineering high-affinity PD-1 variants for optimized Immunotherapy and immuno-PET imaging " article report PD-1 molecular mutation body HAC sequence information, synthesis HAC genetic fragment, searches known people's CD8 cross-film district gene order, people's 4-1BB intracellular region gene from GenBank data base Sequence and CD3 ζ intracellular region gene order；
S2, by said gene sequence successively by people's HAC gene, CD8 Envelope region, people's 4-1BB intracellular region gene and CD3 ζ Intracellular region gene is attached, and introduces different restriction enzyme sites in each sequence junction, forms complete HAC-CD8-4-1BB- CD3 ζ gene sequence information,
S3, by the gene order of HAC-CD8-4-1BB-CD3 ζ by enzyme action convert be connected in pRRSLIN carrier, gene Upstream is EP-1 α promoter.After vector to Stbl3 coli strain, transferred species is to the solid containing ampicillin Breeding in culture medium, screening, it is thus achieved that positive colony, extract plasmid, enzyme action identifies clone, confirms vector construction by order-checking Success, it is thus achieved that pRRSLIN-HAC Lentiviral, Lentiviral builds schematic diagram as shown in Figure 1.
Embodiment 2 is prepared by slow virus
The present invention provides the method that in embodiment 1, Lentiviral expresses preparation slow virus, comprises the following steps:
S1, transfect first 24 hours, with every ware about 8 × 106293T cell is seeded in 15cm culture dish.When guaranteeing transfection Cell about 80% degree of converging and be uniformly distributed in culture dish.
S2, prep solution A and solution B
Solution A: 6.25mL 2 × HEPES buffer buffer
Solution B: be separately added into the mixture of following plasmid: 112.5 μ g pRRLSIN-HAC (target plasmid)； 39.5μg pMD2.G(VSV-G envelop)；73 μ g pCMVR8.74 (gag, pol, tat, rev)；625 μ L 2M calcium ions are molten Liquid.
S3, fully mix solution B, gently while vortex solution A, be added dropwise over solution B, stand 2-3 minute.Whirlpool gently Screwing on the mixed solution stating A and B, be added dropwise in the culture dish containing 293T cell, front and back rocking culture dish makes DNA and calcium gently The mixture of ion is uniformly distributed.It is positioned in incubator cultivation 16-18 hour.Change fresh culture, continue to cultivate, turning Speed 500g, centrifugal 10min at temperature 25 DEG C, use PES film (0.45 μm) to filter；With 70% ethanol disinfection centrifuge tube, it is placed in Sterilize under uviol lamp 30min；The filtered supernatant containing slow virus is transferred in centrifuge tube, carefully spreads bottom centrifuge tube Last layer 20% sucrose (every 8mL supernatant adds 1mL sucrose), with PBS equilibrium centrifugation pipe, at rotating speed 25000rpm, temperature 4 DEG C Centrifugal 2h；Carefully take out centrifuge tube, outwell supernatant, be inverted centrifuge tube and remove residual liquid；Add 100 μ L PBS, seal from Heart pipe, places 2h, every 20min vortex gently once at 4 DEG C, and 500g is centrifuged 1min (25 DEG C), collects viral supernatants；Cooled on ice After, it is placed in-80 DEG C of preservations.
Prepared by embodiment 3 CAR-T cell
The present invention provides the method that in embodiment 2, slow virus infected cell prepares CAR-T cell, comprises the following steps:
S1, take 0.5mL blood and carry out quick the pathogenic microorganism examination, get rid of HBV, HCV, HDV and HEV, HIV-1/2, prunus mume (sieb.) sieb.et zucc. The poison microorganism such as spirillum and parasite is infected；Under aseptic condition, with heparin bottle blood sampling 50mL (anticoagulant heparin), immediately (4 DEG C, In 24 hours) to deliver to cell and prepare laboratory, it is ensured that this process is without microbiological contamination.After obtaining blood samples of patients, in GMP system Standby room, puts into Biohazard Safety Equipment after carrying out disinfection with cotton ball soaked in alcohol wiping heparin bottle surface.
S2, open 2 50mL centrifuge tubes in advance, proceed to blood, in two 50mL centrifuge tubes, screw；Blood is installed by above-mentioned Two 50mL centrifuge tubes of liquid put into centrifuge, and 2000rpm is centrifuged 10min, room temperature collected after centrifugation upper plasma, stays The beds of precipitation；The autologous plasma collected is through 56 DEG C, and 30min inactivates, after 4 DEG C are placed 15min, and 900g, centrifugal 30min (4 DEG C), take The most standby.
S3, by the hemocyte normal saline dilution of above-mentioned enrichment to 30mL/ manage, open 2 new 50mL centrifuge tubes, often Individual centrifuge tube is separately added into 15mL human lymphocyte separation liquid, with pipet, the hemocyte liquid after dilution is added slowly to fill In the centrifuge tube of people's lymph separation liquid, screw.Note the upper strata of blood lymph to be added to separation liquid, do not break people's lymph separation liquid Interface.The hemocyte liquid added being put into centrifuge, is adjusted to the elevation rate of minimum, room temperature 2000rpm is centrifuged 20min.Receive Collecting the middle level leukocytic cream of two pipes in a 15mL sterile centrifugation tube, add 5mL normal saline, (2000rpm is centrifuged to wash twice 10min), peripheral blood lymphocytes (PBMC) is obtained.
S4, configuring complete growth medium, it is 5% that V-VIVO15 adds autologous AB (FBS) concentration, interleukin II (IL-2) concentration is 40ng/mL, and the PBMC culture medium of isolated is diluted to 2 × 106/ mL, takes 50 μ L flow cytometer detections The purity of T cell in PBMC.
S5, Day 0, configuration buffer (adds the hyclone (FBS) of 1% in PBS), selects microballon conduct Cell culture vector, vibrate 30s or manually shake up 5min up and down by microballon, is that 3:1 takes according to the amount ratio of microballon with T cell CD3/CD28 microballon is placed in 1.5mL EP pipe, adds 1mL buffer solution for cleaning microballon, uses Magnet outwards to inhale micro-from EP pipe afterwards Pearl 1min, abandons washing liquid, is repeated twice, and re-uses culture medium and microballon is resuspended to original volume, after cell and microballon are mixed by 2 × 106PBMC/mL is added in suitable culture bottle.
Cell density is adjusted to 3-5 × 10 by S6, Day 26/ mL, is that 1:5 adds in fact in the ratio of viral vector Yu cell Execute the pRRSLIN-HAC Lentiviral that example 1 prepares, add simultaneously polybrene (polybrene) 4 μ g/mL and 40ng/mL IL-2.After 4h, add fresh complete medium and cell density is adjusted to 1 × 106/ mL continues to cultivate.Will All of cell centrifugation, adds fresh culture medium, continues to cultivate.
S7, carry out half amount every 2-3 days and change liquid, maintain cell density in 0.5-1 × 106/mL。
S8, Day 10-12, cell quantity reaches 109Rank, under 400g, centrifugal 5min obtains immunocyte, then with pre- Cold PBS washs twice.
S9, with blood counting chamber count, flow cytomery cell population, CAR-T cell proportion.Observe every day and cultivate The color change of base, cell density, cellular morphology also make respective record.Progressively during amplification culture, needed for adding cumulative volume Interleukin II.
Embodiment 4 CAR-T cell flow cytometer showed
The CAR-T cell of embodiment 3 preparation is carried out flow cytometer showed, and it specifically comprises the following steps that
S1, take 5 × 104Cell (including T cell, CAR-T cell) is used for dyeing；
S2, cell hatch 45min with HAC molecular recognition altogether with antibody (antibody can be combined, coupling FITC fluorescence molecule), and 50 μ l, is placed on ice；
S3, PBS eluting twice；
S4, with 120 μ l FACS reagent re-suspended cells；
FITC fluorescence signal measured by S5, fluidic cell instrument, if contrasted with compareing T cell, CAR cell FITC fluorescence is believed Number strengthen, surface C AR cell construction success.
CAR-T cell streaming contaminate effect as in figure 2 it is shown, in figure, vertical coordinate is streaming SSC-H side scatter signal, Abscissa is FITC fluorescence signal, and this signal value is the strongest, shows that HAC molecule is expressed on film the most, and CAR-T cell transfecting becomes The ratio of merit is the highest.A figure and B figure are matched group, for not infecting the T cell of virus；The antibody of the detection CAR molecule of FITC coupling Can't detect CAR developed by molecule；C figure and D scheme, and for transfecting the T cell of PRRSLIN-HAC slow virus, through flow cytometer detection, scheme with A With B figure contrast, there is cell Successful transfection；After virus infection T cell, after flow cytometer detection infects 3 days, infecting efficiency can reach 53.26%, illustrate successfully to prepare HAC-CAR-T cell.
Embodiment 5 HAC-CAR-T cells in vitro Activity determination
Use LDH method for releasing detection HAC-CAR-T cell that engineering cell strain MCF-1/PDL1 and high expressed PDL1 is swollen The lethal effect of oncocyte, is discharged by ELISA method detection LDH, comprises the following steps:
S1, with the RPMI-1640 culture fluid containing 5% calf serum, target cell is adjusted to 5 × 104/mL。
S2, in 96 porocyte culture plates add target cell, every hole adds 100 μ L.Take 3 hole action effect cell (HAC- CAR-T cell) Spontaneous release control wells, it is not added with target cell, only adds 100 μ L culture fluid.
S3, add 100 μ L effector lymphocytes to each hole, ratio 10:1 of effector lymphocyte and target cell；5:1；1:1.Spontaneous release Hole is not added with effector lymphocyte and only adds 100 μ L culture fluid, and effector lymphocyte and target cell hatch 6 hours altogether, and three multiple holes are put in each experiment.
In S4, maximum release aperture, (positive control) adds 10 μ L Lysis Solution (10 ×), hatches 45min-60min, Three multiple holes are put in each experiment.
S5, take testing sample and each 50 μ L of control sample in above-mentioned 3 and 4, add in 96 fresh hole ELISA Plate, add Reactant liquor and substrate, lucifuge 30min.
S6, add 50 μ L stop buffers.
S7, measuring the optical density (OD value) in each hole on enzyme connection detector, detection wavelength 490nm or 492nm, at 1 hour Interior survey is complete.
S8, specific killing efficiency calculation
Killing rate=experimental group LDH (OD)/maximum LDH release group (OD).
Computing formula: killing-efficiency=(experimental group-effect Spontaneous release-target Spontaneous release)/(target maximum release-target is certainly So release) × 100%.
S9, by CBA kit measurement cytokine secretion profile, calculate the propagation feelings in each group of CAR-T cell simultaneously Condition, and utilize CD3 and CD8 antibody staining, confirm the ratio of T cell positive for CD8 in the T cell of propagation.
As it is shown on figure 3, HAC CAR-T can significantly kill SMCCC7721 tumor cell, the MCF-7 to high expressed PDL-1 The fragmentation effect of cell is better than MCF-7 Common tumors cell, and in figure, abscissa represents that CAR-T cell is different from tumor cell Effect target ratio, vertical coordinate represents killing-efficiency, different types of histogram graph representation difference tumor cell.Accordingly, as shown in Figure 4, Abscissa represents the effect target ratio of T cell and tumor or CAR-T cell and tumor, and vertical coordinate represents that cell number, T represent T cell, HAC represents CAR-T cell, it is seen that compared with general T cell, and CAR-T cell, after touching tumor cell stimulation, can occur spy The opposite sex activates and propagation, and the most notable in the case of efficient target ratio.
In Fig. 5 killing experiments, abscissa represents the effect target ratio that CAR-T cell is different from tumor cell, and vertical coordinate represents Cytokine content, the cytokine in detection culture supernatant, find CAR-T killing experiments group IL-2 (Fig. 5 A) and TNF-α (figure Secretion 5B) significantly raises.As shown in Figure 6, for CD3/CD8 streaming antibody test CAR-T to MCF7/MCF7-PDL1/HeLa/ The ratio of cd8 t cell after 4 kinds of cell strains killings of SMC7721, vertical coordinate PE signal represents specific detection CD3 developed by molecule, Abscissa FITC signal represents detection CD8 developed by molecule, by flow cytomery, after HAC CAR-T cell-stimulating, mainly It it is cd8 t cell generation proliferated specifically.
As it is shown in fig. 7, to different cell MCF7 (Fig. 7 A), MCF7-PD-L1 (Fig. 7 B) and SMC7721 (Fig. 7 C), CAR- PD-1 represents that the extracellular fragment of CAR molecule is common PD-1 molecule, and CAR-PD-1-HAC represents that the HAC-CAR that the present invention builds is fitted together to Antigen receptor molecule, compared with PD-1CAR-T, HAC CAR-T effect is more preferable.
The above results proves, HAC-CART cell, after contact tumor cell, specific can occur activation and propagation, The release cells factor, killing tumor cell, wherein CD8 positive T cell plays Main Function.Compare simultaneously PD-1CAR-T and The HAC CAR-T fragmentation effect to tumor, result display HAC-CAR-T is substantially better than PD-1CAR-T.
The above is only the preferred embodiment of the present invention, is not limited to the present invention, it is noted that for this skill For the those of ordinary skill in art field, on the premise of without departing from the technology of the present invention principle, it is also possible to make some improvement and Modification, these improve and modification also should be regarded as protection scope of the present invention.

Claims (10)

1. a Chimeric antigen receptor molecule, it is characterised in that: include extracellular region peptide fragment, cross-film district peptide fragment and the born of the same parents being sequentially connected in series Intracellular domain peptide fragment, described extracellular region peptide fragment includes HAC peptide fragment, and its sequence is as shown in SEQ ID NO:1.
Chimeric antigen receptor molecule the most according to claim 1, it is characterised in that: described cross-film district peptide fragment includes but does not limits In CD8 cross-film district's peptide fragment or PD-1 cross-film district peptide fragment, when cross-film district peptide fragment is CD8 cross-film district's peptide fragment, described HAC peptide fragment with CD8 cross-film district peptide fragment is connected also by the hinge district peptide fragment of CD8.
Chimeric antigen receptor molecule the most according to claim 1, it is characterised in that: described intracellular domain peptide fragment is selected from 4- One or more in the intracellular domain peptide fragment of 1BB, CD28, CD3 ζ.
Chimeric antigen receptor molecule the most according to claim 1, it is characterised in that: described Chimeric antigen receptor molecule also wraps Include signal peptide.
5. a nucleotide, it is characterised in that: its coding divides according to the Chimeric antigen receptor described in any one of Claims 1-4 Son.
Nucleotide the most according to claim 5, it is characterised in that: comprise or for the nucleotides sequence shown in SEQ ID NO:10 Row.
7. a recombinant vector, it is characterised in that: its comprise according to described in claim 5 or 6 nucleotide.
Recombinant vector the most according to claim 7, it is characterised in that: described recombinant vector is that pRRSLIN slow virus is expressed Carrier.
9. a reconstitution cell, it is characterised in that: it comprises according to the recombinant vector described in any one of claim 7 or 8.
Reconstitution cell the most according to claim 9, it is characterised in that: described reconstitution cell is preferably T cell or NK is thin Born of the same parents.
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107082813A (en) * 2017-05-17 2017-08-22 董克海 Target PD 1 Chimeric antigen receptor molecule and its application
CN108728416A (en) * 2017-04-17 2018-11-02 沈阳美达博生物科技有限公司 A kind of CAR-T cell culture flow
WO2018218710A1 (en) * 2017-06-02 2018-12-06 阿思科力(苏州)生物科技有限公司 Chimeric antigen receptor cells targeting robo1, preparation and application thereof
CN110028589A (en) * 2018-02-07 2019-07-19 阿思科力(苏州)生物科技有限公司 Chimeric antigen receptor, the NKG2D CAR-NK cell for expressing the Chimeric antigen receptor and its preparation method and application
CN112638950A (en) * 2018-10-22 2021-04-09 上海一宸医药科技有限公司 Bispecific antibody

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109401969B (en) * 2018-12-13 2024-02-02 珠海西格膜生物技术有限公司 Pipeline connection system of cell factory and application method thereof
CN110964697B (en) * 2019-12-19 2023-07-18 中国海洋大学 Anti-tumor NK cell and preparation method and application thereof
CA3204041A1 (en) * 2021-01-04 2022-07-07 Neomics Pharmaceuticals Llc Chimeric co-stimulatory proteins comprising mutant intracellular domains with increased expression

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015142675A2 (en) * 2014-03-15 2015-09-24 Novartis Ag Treatment of cancer using chimeric antigen receptor

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
TWI654206B (en) * 2013-03-16 2019-03-21 諾華公司 Treatment of cancer with a humanized anti-CD19 chimeric antigen receptor

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015142675A2 (en) * 2014-03-15 2015-09-24 Novartis Ag Treatment of cancer using chimeric antigen receptor

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ROY L. MAUTE ET AL.: "Engineering high-affinity PD-1 variants for optimized immunotherapy and immuno-PET imaging", 《PNAS》 *
付小兵等: "《生物治疗中的转化医学》", 30 November 2014 *

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CN107082813A (en) * 2017-05-17 2017-08-22 董克海 Target PD 1 Chimeric antigen receptor molecule and its application
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CN108977453A (en) * 2017-06-02 2018-12-11 阿思科力(苏州)生物科技有限公司 It is a kind of using ROBO1 as the Chimeric antigen receptor cell of target spot and its preparation and application
US11697685B2 (en) 2017-06-02 2023-07-11 Asclepius (Suzhou) Technology Company Group Co., Ltd. Chimeric antigen receptor cells targeting ROBO1, preparation method and use thereof
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