CN105753991A - Chimeric antigen receptor resistant to placenta chondroitin sulfate and application of chimeric antigen receptor - Google Patents
Chimeric antigen receptor resistant to placenta chondroitin sulfate and application of chimeric antigen receptor Download PDFInfo
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- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract
The invention relates to the field of cellular immunotherapy of tumors, in particular to a chimeric antigen receptor resistant to placenta chondroitin sulfate (p1-CS) and application of the chimeric antigen receptor.The chimeric antigen receptor is mainly formed by serially connecting an antigen recognition area resistant to pl-CS, a hinge area, a transmembrane area and an intracellular area, the antigen recognition area resistant to p1-CS is one of plasmodium protein VAR2CSA, part of peptide segment of the plasmodium protein VAR2CSA or p1-CS antibody, and the peptide segment of the plasmodium protein VAR2CSA is a peptide segment with number of amino acid in the plasmodium protein VAR2CSA greater than 500.T cells expressing the chimeric antigen receptor can kill tumor cells to maximum extent, are quite low in degree of damage to normal tissue, can break tumor immune to inhibit microenvironment so as to have better treatment effect on solid tumors and can almost treat tumors of various types.
Description
Technical field
The present invention relates to the cellular immunotherapy field of tumor, particularly relate to a kind of anti-Placenta Hominis sample chondroitin sulfate
Chimeric antigen receptor and application, be specially chimeric antigen based on tomour specific target spot pl-CS and be subject to
The construction method of body T (CAR-T) cell technology and the application in antineoplaston thereof.
Background technology
Chimeric antigen receptor T cell (CAR-T) technology is to carry out the novel of clinical trial at present
Cell therapy.The treatment of acute leukemia and non-Hodgkin lymphoma has significant curative effect, is considered
It it is one of the most promising oncotherapy mode.Its basic fundamental is by genetic engineering means, by T cell
It is transformed into the mode identification tumor antigen being independent of HLA so that CAR-T cell is compared to nave T cell
There is the ability of higher identification and killing tumor cell.The core component of CAR-T is CAR, CAR mono-
As include a tumor associated antigen land, be typically derived from monoclonal antibody scFV of tumor associated antigen
Section；One outer hinge region of born of the same parents；One cross-film district and an intracellular stimulus signal district.
CAR-T technology is successfully it is crucial that the tumor antigen i.e. selection of target spot.Optimal antigen is in tumor
Cell surface high expressed, at the antigen that normal cell is not expressed.Only select such antigen, just can make
Combination that CAR-T cell is single-minded killing tumor cell, and normal cell is not combined, will not be to normal group
It is woven into injury.But so preferable antigen is the most few, most of antigens be in tumor tissues high expressed or
Person's overexpression, the lowest expression.At present, CAR-T in the treatment of entity tumor far away from vain
The therapeutic effect of disorders of blood, one of its reason is exactly the most not find ideal target in each solid tumor.
CAR-T technology is that tumour immunity suppresses micro-loop for the Another reason that the effect treating solid tumor is bad
The existence in border.Tumour immunity suppression microenvironment is the interior environment formed during tumor development, by tumor
Cell, Interstitial cell, immunity regulatory cell, blood capillary, tissue fluid, cytokine, extracellular matrix etc.
Composition.Due to immunity microenvironment existence, immunologic cytotoxicity cell cannot effectively arrive tumor cell position or
Tumor cell effectively cannot be killed after arriving target site.Solid tumor to be carried out by CAR-T cell
Effective treatment, it is necessary to break through tumour immunity microenvironment and limit, enters solid tumor and realizes killing tumor cell
Wound.
CN 104788573 A disclose a kind of Chimeric antigen receptor hCD19scFv-CD8 α-CD28-CD3 ζ and
Its purposes, this Chimeric antigen receptor is by anti human CD 19 monoclonal antibody HI19a light chain and variable region of heavy chain
(hCD19scFv), people CD8 α hinge region, people CD28 cross-film district and intracellular region and people's CD3 ζ intracellular
Plot structure is in series.This Chimeric antigen receptor is used for modifying T lymphocyte, the T cell after modification
(CAR-T cell) can be used for the treatment of the positive tumor of surface C D19.The Chimeric antigen receptor of this patent is only
It is directed to the tumor that surface C D19 is positive, and recognition effect is bad, therefore, screens suitable tumor target
Mark, can also break through tumor microenvironment simultaneously, is the key of CAR-T technology treatment solid tumor.
Chondroitin sulfate (CS) is the covalently bound class glycosaminoglycans forming Dan Baiduotang proteoglycan PG on protein.
Chondroitin sulfate is distributed widely in extracellular matrix and the cell surface of animal tissue, and sugar chain is by Fructus Vitis viniferae alternately
Alduronic acid and N-acetylgalactosamine (also known as N-acetylamino galactosamine) disaccharide unit composition, by one
It is connected on the serine residue of core protein like sugar link zone.Chondroitin sulfate is present in and removes from nematicide to people
In exophytic all biologies, play many important physiological functions.Although the backbone structure of polysaccharide is not
Complexity, but with regard to degree, sulfate and two species diversity to isomerized sugar aldehydic acid for the distribution in chain,
Present the inhomogeneity of height.The fine structure of chondroitin sulfate decide function specificity and with many hatching egg
The interaction of white matter molecule.
Placenta Hominis also exists a kind of special chondroitin sulfate, its glycosylation pattern and conventional chondroitin sulfate
, in plasmodium, there is a kind of entitled VAR2CSA albumen, the sulphuric acid in combination Placenta Hominis that can be special in difference
Chrondroitin, and on the surface of tumor cell, there is Placenta Hominis sample chondroitin sulfate (pl-CS), therefore,
Pl-CS is the promising target of CAR-T technology treatment tumor.
Summary of the invention
Select the most very good for current CAR-T technology treatment tumor point of impact on target, and tumor microenvironment shadow
Ringing the situation of CAR-T technology therapeutic effect, the present invention provides the inosculating antibody of a kind of anti-Placenta Hominis sample chondroitin sulfate
Original receptor and application thereof, described Chimeric antigen receptor, tumor cell that can be more special, micro-to tumor
Environment has more preferable targeting.
For reaching this purpose, the present invention by the following technical solutions:
On the one hand, the present invention provides the Chimeric antigen receptor of a kind of anti-Placenta Hominis sample chondroitin sulfate, described chimeric
Antigen receptor mainly includes the antigen recognition district of anti-pl-CS, hinge region, cross-film district and intracellular region series connection structure
Become；
Wherein, the antigen recognition district of described anti-pl-CS is plasmodium albumen VAR2CSA, plasmodium albumen
Any one in the part peptide fragment of VAR2CSA or pl-CS antibody；
The part peptide fragment of described plasmodium albumen VAR2CSA is the aminoacid in plasmodium albumen VAR2CSA
The quantity peptide fragment more than 500.
In the present invention, the part peptide fragment of described plasmodium albumen VAR2CSA refers to plasmodium albumen
The aminoacid quantity in the VAR2CSA peptide fragment more than 500, can be in plasmodium albumen VAR2CSA
Peptide fragments more than any 500 aminoacid or 500 aminoacid is all feasible.In the present invention, malaria is former
Worm albumen VAR2CSA, the part peptide fragment ID1-ID2a of plasmodium albumen VAR2CSA and pl-CS antibody are equal
Can be with specific binding pl-CS, pl-CS is almost present in all types of tumor cell surface, and the thinnest
There is not pl-CS in the surface of born of the same parents；Selecting pl-CS is the specificity target spot of tumor, using VAR2CSA as
CAR identifies that domain identifies the pl-CS of tumor, by the way of VAR2CSA identifies pl-CS, and can
So that the tumor cell of CAR-T cell-specific, it is easier to it is targeted to tumor microenvironment, enters into tumor
Organization internal, without damaging normal cell and tissue；Additionally, pl-CS or tumor cell
The ingredient of epimatrix, is the material base forming tumor microenvironment, and selection pl-CS is target spot, favorably
Tumour immunity suppression microenvironment is broken through in CAR-T cell.
Preferably, described plasmodium albumen VAR2CSA be plasmodium pf 3D7 VAR2CSA albumen and/
Or the VAR2CSA albumen of plasmodium pf FCR3.
Preferably, the aminoacid sequence of the VAR2CSA albumen of described plasmodium pf 3D7 is SEQ ID
NO.3, nucleotide sequence is SEQ ID NO.4.
Preferably, the aminoacid sequence of the VAR2CSA albumen of described plasmodium pf FCR3 is SEQ ID
NO.5, nucleotide sequence is SEQ ID NO.6.
Preferably, the part peptide fragment of described plasmodium albumen VAR2CSA is ID1-ID2a, described
The aminoacid sequence of ID1-ID2a is SEQ ID NO.7, and nucleotide sequence is SEQ ID NO.8.
In the present invention, described ID1-ID2a is that in this part peptide fragment, length is smaller, is that effect is best
One section.
Preferably, described hinge region behaviour CD8 α hinge region.
Preferably, the aminoacid sequence of described people CD8 α hinge region is SEQ ID NO.9, and nucleotide sequence is
SEQ ID NO.10。
Preferably, described cross-film district behaviour CD28 cross-film district.
Preferably, the aminoacid sequence in described people's CD28 cross-film district is SEQ ID NO.11, and nucleotide sequence is
SEQ ID NO.12。
As optimal technical scheme, described intracellular region behaviour CD3 ζ intracellular region, people's CD28 intracellular region or people
Any one or the combination of at least two in 4-1BB intracellular region, such as, can be CD3 ζ intracellular region, people
The connecting of CD28 intracellular region, people's 4-1BB intracellular region, people's CD28 intracellular region and people's CD3 ζ intracellular region, people
The connecting of 4-1BB intracellular region and people's CD3 ζ intracellular region, the string of people's CD3 ζ intracellular region and people's 4-1BB intracellular region
Connection or people's CD28 intracellular region, people's 4-1BB intracellular region are connected with people's CD3 ζ intracellular region, preferably CD3 ζ
The connecting of intracellular region, people's CD28 intracellular region and people's CD3 ζ intracellular region, people's 4-1BB intracellular region and people CD3 ζ
The series connection of intracellular region or people's CD28 intracellular region, people's 4-1BB intracellular region are connected with people's CD3 ζ intracellular region.
Preferably, the aminoacid sequence of described people's CD3 ζ intracellular region is SEQ ID NO.13, and nucleotide sequence is
SEQ ID NO.14。
Preferably, described people's CD28 intracellular region is SEQ with the aminoacid sequence connected of people's CD3 ζ intracellular region
ID NO.15, nucleotide sequence is SEQ ID NO.16.
Preferably, described people's 4-1BB intracellular region with the aminoacid sequence connected of people's CD3 ζ intracellular region is
SEQ ID NO.17, nucleotide sequence is SEQ ID NO.18.
Preferably, described people's CD28 intracellular region, the connecting of people's 4-1BB intracellular region and people's CD3 ζ intracellular region
Aminoacid sequence is SEQ ID NO.19, and nucleotide sequence is SEQ ID NO.20.
Preferably, the amino terminal of described Chimeric antigen receptor contains people's CD8 alpha signal peptide.
Preferably, the aminoacid sequence of described people's CD8 alpha signal peptide is SEQ ID NO.21, and nucleotide sequence is
SEQ ID NO.22。
As optimal technical scheme, described Chimeric antigen receptor is by people's CD8 alpha signal peptide, VAR2CSA, people
CD8 α hinge region, people CD28 cross-film district and intracellular region, people's 4-1BB intracellular region and people's CD3 ζ intracellular region
In series.
Preferably, the aminoacid sequence of described Chimeric antigen receptor is SEQ ID NO.1, and nucleotide sequence is
SEQ ID NO.2。
Preferably, described Chimeric antigen receptor is transfected into table in T cell by its nucleotide sequence encoded
Reach.
Preferably, appointing during the mode of described transfection is viral vector, eukaryon expression plasmid or mRNA sequence
Anticipate a kind of or combination of at least two.
Preferably, during described viral vector is slow virus carrier, adenovirus vector or retroviral vector
Any one or the combination of at least two.
Second aspect, the present invention provides a kind of compositions, and described compositions includes being fitted together to described in first aspect
Antigen receptor.
The third aspect, the present invention provides Chimeric antigen receptor as described in relation to the first aspect and/or such as second aspect institute
The compositions stated is in preparation Chimeric antigen receptor T cell and the application in anti-tumor medicine thereof.
Compared with prior art, there is advantages that
(1) Chimeric antigen receptor of the present invention, by the way of VAR2CSA identifies pl-CS, can make
The tumor cell of CAR-T cell-specific, it is easier to be targeted to tumor microenvironment, enters into tumor tissues
Inside, tumor cell that can be more special, tumor microenvironment is had more preferable targeting；
(2) present invention expresses the T cell energy farthest killing tumor cell of this Chimeric antigen receptor, right
The extent of injury of normal structure is the least, can break through tumour immunity suppression microenvironment, thus have solid tumor more
Good curative effect, and almost can treat various types of tumor.
Accompanying drawing explanation
Fig. 1 is the CAR Lentiviral schematic diagram of targeting pl-CS；
Fig. 2 (A) is that the green fluorescence (GFP) of T cell is expressed after fluorescence microscopy Microscopic observation slow virus infection 96h
Situation, Fig. 2 (B) is the GFP positive rate of T cell after FCM results show slow virus infection 96h；
Fig. 3 (A) is that in experimental group, negative control group and wild-type T cells group supernatant, IL-2 concentration compares, figure
3 (B) are that in experimental group, negative control group and wild-type T cells group supernatant, IFN-γ concentration compares, Fig. 3 (C)
Compare for TNF-α concentration in experimental group, negative control group and wild-type T cells group supernatant；
Fig. 4 (A) is in the case of difference effect target ratio, experimental group, negative control group and wild-type T cells group
Comparing the cytotoxicity of lung cancer cell line A549, Fig. 4 (B) is in the case of difference effect target ratio, experimental group,
The cytotoxicity of hepatoma cell line Huh-7 is compared by negative control group and wild-type T cells group, and Fig. 4 (C) is
In the case of difference effect target ratio, experimental group, negative control group and wild-type T cells group are to human osteosarcoma cell
The cytotoxicity of cell line MG63 compares, and Fig. 4 (D) is in the case of difference effect target ratio, experimental group, the moon
The cytotoxicity of CCL188 Colo205 is compared by property matched group and wild-type T cells group.
Detailed description of the invention
By further illustrating the technological means and effect thereof that the present invention taked, below in conjunction with accompanying drawing and pass through
Detailed description of the invention further illustrates technical scheme, but the present invention is not limited to embodiment model
In enclosing.
Embodiment 1: the determination of the CAR gene order of targeting pl-CS
Plasmodium VAR2CSA gene, people CD8 α letter is searched from GenBank data base and document
Number peptide gene, people CD8 α hinge region gene, people CD28 cross-film district and intracellular region gene, people 4-1BB born of the same parents
Inner region gene and people's CD3 ζ intracellular region gene order.Carried out codon optimized, it is ensured that at coding amino
It is more suitable for human cell in the case of acid sequence is constant to express.
Each gene sequence information is shown in SEQUENCE LISTING (sequence table SEQ ID NO.1-22).
Embodiment 2: the structure of Lentiviral pLHlentiCAR004
By said gene sequence successively by people's CD8 alpha signal peptide gene, VAR2CSA, people CD8 α hinge region
Gene, people CD28 cross-film district and intracellular region gene, people's 4-1BB intracellular region gene and people's CD3 ζ intracellular region
Gene order is attached, and forms Chimeric antigen receptor, the nucleotide sequence of described antigen receptor such as SEQ ID
Shown in NO.2, introduce suitable restriction enzyme site, carry out artificial full genome synthesis, be cloned into slow virus and express load
Body obtains pLHlentiCAR004 plasmid.Fig. 1 is shown in by plasmid schematic diagram.
Recombiant plasmid is served Hai Ying fine horse Bioisystech Co., Ltd check order, sequencing result with fit to
Sequence alignment is to confirm that sequence is the most correct.
Embodiment 3: Lentiviral and a large amount of extractions of package carrier
By the stbl3 large intestine bar of pLHlentiCAR004 plasmid and psPAX2 and pMD2.G packaging plasmid
Bacterium is mass propgation in LB culture fluid, and with alkaline lysis large quantity extracting plasmid, in case transfection, detailed process is:
1) respectively the stbl3 escherichia coli kind with pLHlentiCAR004, psPAX2, pMD2.G is connect
Planting in the culture bottle containing 500mL LB/ antibiotic culture fluid, 37 DEG C of shaking tables are cultivated 12-16 hour；
2) 3000*g is centrifuged 10min, collects thalline, outwell culture medium left-hand thread in absorbent paper pat gently with
Exhaust residual liquid.The thalline collected uses Mei Ji bio tech ltd, Guangzhou MaxPure Plasmid Maxi
Kit big upgrading grain, concretely comprising the following steps of extraction plasmid:
(1) adding 20mL BufferP1/RNase A in thalline, the resuspended antibacterial of vortex (guarantees that antibacterial is complete
Full weight hang, resuspended after solution there is no cell mass)；20mL Buffer P2 is added in re-suspension liquid, reverse
Mix 10-15 time, stand 1-2min (must mix gently, notice that the operating time not can exceed that 4 minutes)；
(2) in lysate, 20mL Buffer E3 is added, immediately reverse mixing 15-20 time, blending process
Wanting soft and abundant, neutralizing sufficient standard is: solution becomes less viscous thick, precipitate dispersion whitening color；3000*g
Centrifugal 10min, then transfer supernatant is in new centrifuge tube, adds 1/3 times of volume Buffer E4 supreme
In clear liquid, vortex mixes；
(3) being enclosed within 50mL centrifuge tube by MaxPure Micro Column, transfer 15mL mixed liquor is extremely
In pillar, 3000*g is centrifuged 10min；Outwell effluent, pillar is recovered in collecting pipe, continue transfer surplus
Under solution in pillar, 3000*g is centrifuged 10 minutes；Outwell effluent, pillar recovered in collecting pipe,
20mL Buffer E5 is to pillar in addition, and 3000*g is centrifuged 10 minutes；Outwell filtrate and pillar is recovered collecting pipe
In；
(4) addition 20mL Buffer PW2 (diluting with dehydrated alcohol) is to pillar, and 3000*g is centrifuged 10
Minute；Outwell filtrate and pearl is recovered in collecting pipe, add 20mL Buffer PW2 (dilute with dehydrated alcohol
Release) to pillar, 3000*g is centrifuged 10 minutes；Abandon filtrate and pillar recovered in collecting pipe, 4000*g from
The heart 10 minutes；
(5) pillar is enclosed within the 50mL centrifuge tube of sterilizing, adds 30-200mL aquesterilisa to pillar
Film central authorities, stand 2 minutes, and 10000*g is centrifuged 5min, collect and separate liquid；
(6) further endotoxin is removed in extracting, with aquesterilisa adjust plasmid concentration 0.1g-0.6mg/mL it
Between；
(7) 0.1 times of volume Buffer ER1 and 0.1 times of volume Buffer ER2 are added in plasmid solution,
Reverse mixing；Placing 20min on ice, period reverse mixing is repeatedly；After 42 DEG C of water-bath 5min, room temperature is at 10000*g
Centrifugal 3min under speed；
(8) take out sample gently, define one layer of red solution bottom discovery, carefully supernatant is shifted
To centrifugal Herba potentillae fulgentis, in the isopropanol of 0.7 times of volume of addition to supernatant, reverse mixing is repeatedly；Under room temperature,
Under 10000*g speed, centrifugal 10min, outwells supernatant, adds in the 70% ethanol extremely precipitation of 1 times of volume.
Vortex mixing 15-30s；Under room temperature, under 10000*g speed, centrifugal 3min, carefully outwells supernatant；Room
Under temperature, under 10000*g speed, centrifugal 1min collects the drop on tube wall, is not drawn onto precipitation, at air
In be dried 10min；
(9) adding appropriate amounts of sterilized water in plasmid, vortex 10-20s mixes, and room temperature stands 30min and allows plasmid
Fully dissolving, period reverse mixing is repeatedly；
Nanodrop ultraviolet spectrophotometer is measured plasmid concentration, then plasmid is saved in-20 DEG C.
Embodiment 4: the packaging of slow virus, concentrate and titrate
1) packaging of slow virus
(1) the pre-plate of cell: 24h before transfection, takes the 293T cell covered with and carries out pre-plate, with second day energy
Reach 70% degree of converging and be advisable (pre-plate 5,000,000 in 75mm culture dish).
(2) second days: transfection
A () observes the growing state of pre-plate cell, be advisable about 90% with the degree of converging of cell, suck training
Support base, add the complete medium (15mL in 75mm culture dish) of fresh preheating；
By the PEI needed for transfection, plasmid, Opti-MEM I Reduced Serum Medium before (b) transfection
Deng placement equilibrium at room temperature；
C () prepares the mixed liquor of plasmid: this packaging totally 10 ware 293T cell, each ware cell prepares mixed
Close liquid 500 μ L, therefore need to prepare mixed liquor cumulative volume is 5mL；First add three kinds of matter according to various plasmid consumptions
Grain, totally 420 μ g (pLHlentiCAR004:psPAX2:pMD2.G=3:2:1), add Opti-MEM I
Reduced Serum Medium adjustment volume is to 2.5mL, and after piping and druming mixing, room temperature places 10min；Take again
PEI and the Opti-MEM I Reduced each 1.25mL of Serum Medium, after mixing, room temperature places 10min,
Finally being mixed by both the above solution, after mixing, room temperature places 10min, is the mixed liquor of transfection；
D () mixed liquor is added dropwise in culture dish, every ware 500 μ L；
(3), after transfection 6h, every ware culture medium need to be changed into the training of 17mL UltraCLUTURE serum-free
Support base；
(4) the three to five days, the collection of virus: transfection 48h-72h, collect culture supernatant respectively, collect
Viral supernatants be temporarily stored into 4 DEG C.
2) slow virus concentrates
By (corresponding with the rotor 25000rpm of SW-32Ti after the membrane filtration of culture supernatant 0.45um collected
Centrifugal force is 106750g), 4 DEG C of centrifugal 2h, after removing supernatant, resuspended with the PBS of 200uL, it is stored in-80 DEG C.
3) titration of slow virus
Pre-bed board 293T cell in (1) 6 orifice plate, 3 × 105Cells/well, 37 DEG C of 5%CO2In incubator
Overnight incubation (about 18-20h)；
(2) preparation polybrene mother solution: sterilizing pure water, 8mg/mL, 0.22 μm filter filters, subpackage,
Be stored in-20 DEG C standby；
(3) preparation DMEM culture medium containing polybrene: 10%FBS, 1% is dual anti-, 8 μ g/mL
Polybrene (one thousandth of culture volume)；
(4) melt virus: take out virus frozen stock solution from-80 DEG C, melt in room temperature, melt to be placed on and take on ice
Enter iuntercellular standby；
(5) virus dilution: with the 5%FBS DMEM complete medium containing polybrene prepared by disease
Toxogen liquid carries out 10 times of dilutions, obtains 10-1-10-4Viral dilution liquid, notice that draw virus liquid is carried out every time
To mix before next step dilution；
(6) discard old culture medium, the first hole adds the 1mL culture medium without virus as negative control.
Remaining every hole adds viral dilution liquid corresponding to 1mL, cultivates 18-20h for 37 DEG C；
(7) remove the culture medium containing virus the next morning, replace the 5%FBS not containing polybrene with 2mL
DMEM culture medium；Basis of microscopic observation cell has expression GFP；
(8) suck culture medium, then use trypsin digestion cell；Cell is blown out individual cells, adds appropriate
Complete medium terminates pancreatin reaction, and 300g is centrifuged 3min and collects cell；
(9) remove supernatant, with appropriate pre-cooling PBS resuspended after, 300g is centrifuged 3min, collects cell, then
It is repeated once；
(10) finally resuspended with 1mL pre-cooling PBS, it is placed on ice, for flow cytometer showed.
(11) selecting GFP positive rate in the hole of 1%-20%, each hole calculates virus titer respectively, finally takes
Meansigma methods；
Computing formula: titer={ (F × Cn)/V} × DF, unit is TU/mL；
Wherein, F:GFP positive cell rate；Cn: the cell quantity of every hole bed board；V: added virus dilute
Release liquid to amass；DF: viral dilution multiple
Being calculated virus titer is: 1.6 × 108-1×1010Between.
The separation of embodiment 5:T cell, cultivates and infects
1) separation of PBMC cell
(1) blood sampling 50mL, 800g is centrifuged 10min (acceleration is set to 3, and reduction of speed is set to 4)；
(2) take leukocytic cream, be diluted to 8mL with 2%FBS；
(3) draw 4mL LymphoPrep to add in 15mL centrifuge tube, then by careful for dilute blood along pipe
Wall adds to be layered on liquid, keeps both interfaces clear；
(4) 800g is centrifuged 20min (acceleration is set to 6, and reduction of speed is set to 1)；
(5) with the pasteur pipet linen mononuclearcell of sucking-off gently, add another and contained 10mL
In the centrifuge tube of RF-10, mixing；
(6) it is centrifuged 5min with 500g, abandons supernatant；
(7) adding 10mL RF-10 re-suspended cell, trypan blue counts, and 350g is centrifuged 10min, abandons
Clearly.
2) magnetic bead sorting CD4+T (CD8+T) cell
(1) vortex mixing magnetic bead, takes 25 μ L magnetic beads in test tube, adds 1mL Buffer 1 and mixes, will
Test tube is placed on 1min on magnetic frame, removes supernatant.Add the 25 μ resuspended magnetic beads of L Bffer1 standby；
(2) PBMC Buffer 1 resuspended to density be 107/mL；
(3) in the PBMC of 1mL, add the magnetic bead of 25 μ L washings, hatch 20min for 2-8 DEG C, be placed on
Sloped rotating on shaking table；
(4) test tube is placed on 2min on Magnet, collects supernatant；
(5) remove test tube, add 1mL Buffer 1, piping and druming mixing, be placed on 2min on magnetic bead, collect supernatant,
It is repeated once；
(6) add 100 μ L Buffer 2 re-suspended cells, add 10 μ L DETCHaBEAD, incubated at room temperature 45min
Cell is made to discharge from magnetic bead；
(7) test tube being placed on 1min on magnetic frame, the supernatant containing cell is transferred to, in new test tube, add
500 μ L Buffer 2 wash magnetic bead 2-3 time, collect supernatant；
(8) adding 4mL Buffer 2,350g is centrifuged 5min, removes supernatant, with Buffer 2 re-suspended cell；
(9) supernatant collected in sorting CD4+T cell processes is pressed test kit operation and is collected CD8+T cell.
3) cultivation of T cell
(1) CD4+T (CD8+T) the cell 350g after magnetic bead sorting is centrifuged 10min；
(2) the resuspended counting of RF-10；
(3) cultivation, cell density in CD4+T and CD8+T Tissue Culture Plate are added according to the ratio of 1:1
It is 5 × 105Individual/mL；
(4) T cell special culture media add CD3/CD28 antibody magnetic bead, add magnetic bead amount by with carefully
Ratio 1:1 of born of the same parents adds；
(5) add rhIL-2, make final concentration of 10ng/mL；
(6) counting 2-3 time weekly, the proliferative conditions of record cell.
4) slow-virus transfection of T cell.
(1) CD4+T (CD8+T) the cell 350g after magnetic bead sorting is centrifuged 10min, adds and cultivates completely
The outstanding counting of basic weight；
(2) adding CD4+T and CD8+T cell according to the ratio of 1:1 to cultivate in 96 orifice plates, cell is close
Degree is 5 × 105Individual/mL, every porocyte number is 1 × 105Individual；
(3) washing magnetic bead, adds the magnetic bead with cell 1:1 ratio in culture dish；
(4) add rhIL-2, make final concentration of 10 μ g/L；
(5) after stimulating 24h, infection cell, add polybrene, make final concentration of 6 μ g/mL, mixing；
(6) liquid is changed after 16-24h；
Carry out fluorescence microscope after (7) 3 days to take pictures.
The result figure such as Fig. 2 (A) of fluorescence microscope, shown in flow cytometer detection efficiency of infection such as Fig. 2 (B), it is seen then that
The efficiency of infection of flow cytometer detection is about 50%, can efficiently prepare CAR-T cell.
Embodiment 6:CAR-T cells in vitro release of cytokines and detection
In (1) 48 orifice plate, every hole CAR-T cell 1.5 × 105Individual with Huh7 cell per well 1.5 × 105Individual
Co-culture 24h, add 500 μ L RPMI-1640 culture medium (without serum and rhIL-2)；
(2) sample collection and storage: centrifugal supernatant of collecting, directly starts experiment or subpackage is stored in-20 DEG C
(100 μ L often manage)；
(3) reagent prepares: is taken out by all reagent before using and recovers to room temperature, with deionized water by Wash
Buffer concentrated solution is diluted to 500mL；Substrate nitrite ion, mixes A with B equal-volume, uses in 15min
Complete, every hole 200 μ L mixed liquor；Cytokine standards product, go 500 μ L to store liquid, gradient concentration dilution 6
Group；
(4) standard substance that sample and Concentraton gradient dilute are added in the ELISA orifice plate of coated antibody,
The 100 every holes of μ L, remove bubble incubated at room 2h (sample often group sets three multiple holes)；
(5) removing liquid in hole, every hole adds 300 μ LWB washings, washs three times, blots WB for the last time；
(6) 200 detection antibody corresponding for μ L, incubated at room 2h are added；
(7) removing liquid in hole, every hole adds 300 μ LWB washings, washs three times, blots WB for the last time；
(8) 200 μ L substrate solutions, incubated at room 20min (lucifuge) are added；
(9) adding 50 μ L stop buffers, solution is become yellow from blueness, if color for green or does not change
Become color, pat and make it mix；
(10) absorbance at 450nm is detected in 30min；
(11) each cytokine concentrations is counted according to standard curve,
Shown in result such as Fig. 3 (A), 3 (B) and 3 (C), it is seen then that CAR-T cell co-cultures with tumor cell and releases
Put substantial amounts of cytokine, shown that CAR-T cells against tumor cells creates strong immunoreation.
Embodiment 7:CAR-T cells in vitro killing tumor cell
1) optimal plating cells concentration is determined
(1) collecting each tumor cell, with analyzing culture medium washing, adjusting cell concentration is 1 × 105Individual/mL；
(2) in 96 orifice plates, add 100 μ L analysis culture medium, be paved with；
(3) add 100 μ L cell suspension in 96 orifice plates, dilute successively, last Concentraton gradient sucking-off
100 μ L cell suspension.Arranging six multiple holes, three holes are High control group, and three holes are Low control
Group, and the three acellular analysis in hole culture medium are set as Background control (range, 500-50000)；
(4) incubator hatches 18h；
(5) the every hole of HC group adds 5 μ L cytolysates, hatches 15min, and vibrations are accelerated cell and dissolved；
(6) every hole adds the reactant liquor that 100 μ L now join, and hatches 30min for 15 DEG C-25 DEG C and (notes 96 orifice plates
Lucifuge)；
(7) every hole adds 50 μ L stop buffers, shakes 10s；
(8) 490nm surveys absorbance；
(9) analytical data, determines optimum cell concentration (maximum difference of HC Yu LC).
2) cell-mediated toxicity detection
(1) CAR-T cell is collected, with analyzing culture medium washed cell；
(2) according to different effect target than and 1) in the optium concentration of result determine and be laid in 96 orifice plates
CAR-T cell number；
(3) the every pore volume of corresponding cell suspension at 96 orifice plate middle berths is adjusted to 50 μ L；
(4) collecting each tumor cell, adjustment cell concentration is 2 times of optium concentration, adds 50 μ L cells and hangs
Liquid is in T cell；
(5) different matched group is set；
(6) 37 degree of incubators hatch 18h；
(7) every hole adds 5 μ L cytolysates, hatches 15min, and vibrations are accelerated cell and dissolved；
(8) every hole adds the reactant liquor that 100 μ L now join, and hatches 30min for 15 DEG C-25 DEG C and (notes 96 orifice plates
Lucifuge)；
(9) every hole adds 50 μ L stop buffers, shakes 10s；
(10) 490nm surveys absorbance；
(11) percentage ratio of each sample cell toxicity is calculated.
Shown in result such as Fig. 4 (A)-4 (D), it is seen then that CAR-T cell has the toxicity to tumor cell, can be high
The killing tumor cell of effect.
In sum, the present invention expresses the T cell of this Chimeric antigen receptor farthest to kill tumor thin
Born of the same parents, the injury of normal tissue is the least, can break through tumour immunity suppression microenvironment, thus have solid tumor
Preferably curative effect, and almost can treat various types of tumor.
Applicant states, the present invention illustrates the method detailed of the present invention by above-described embodiment, but the present invention
It is not limited to above-mentioned method detailed, does not i.e. mean that the present invention has to rely on above-mentioned method detailed ability real
Execute.Person of ordinary skill in the field is it will be clearly understood that any improvement in the present invention, to product of the present invention
The equivalence of each raw material is replaced and the interpolation of auxiliary element, concrete way choice etc., all falls within the guarantor of the present invention
Within the scope of protecting scope and disclosure.
Claims (10)
1. the Chimeric antigen receptor of an anti-Placenta Hominis sample chondroitin sulfate, it is characterised in that described inosculating antibody
Original receptor mainly includes that the antigen recognition district of anti-pl-CS, hinge region, cross-film district and intracellular region are in series；
Wherein, the antigen recognition district of described anti-pl-CS is plasmodium albumen VAR2CSA, plasmodium albumen
Any one in the part peptide fragment of VAR2CSA or pl-CS antibody；
The part peptide fragment of described plasmodium albumen VAR2CSA is the amino in plasmodium albumen VAR2CSA
The acid number amount peptide fragment more than 500.
Chimeric antigen receptor the most according to claim 1, it is characterised in that described plasmodium albumen
VAR2CSA is VAR2CSA albumen and/or the plasmodium strain pf FCR3 of plasmodium strain pf 3D7
VAR2CSA albumen；
Preferably, the aminoacid sequence of the VAR2CSA albumen of described plasmodium strain pf 3D7 is SEQ ID
NO.3, nucleotide sequence is SEQ ID NO.4；
Preferably, the aminoacid sequence of the VAR2CSA albumen of described plasmodium strain pf FCR3 is SEQ ID
NO.5, nucleotide sequence is SEQ ID NO.6；
Preferably, the part peptide fragment of described plasmodium albumen VAR2CSA is ID1-ID2a, described
The aminoacid sequence of ID1-ID2a is SEQ ID NO.7, and nucleotide sequence is SEQ ID NO.8.
Chimeric antigen receptor the most according to claim 1 and 2, it is characterised in that described hinge region is
People CD8 α hinge region；
Preferably, the aminoacid sequence of described people CD8 α hinge region is SEQ ID NO.9, and nucleotide sequence is
SEQ ID NO.10。
4. according to the Chimeric antigen receptor according to any one of claim 1-3, it is characterised in that described across
Film district behaviour CD28 cross-film district；
Preferably, the aminoacid sequence in described people's CD28 cross-film district is SEQ ID NO.11, and nucleotide sequence is
SEQ ID NO.12。
5. according to the Chimeric antigen receptor according to any one of claim 1-4, it is characterised in that described born of the same parents
In inner region behaviour CD3 ζ intracellular region, people's CD28 intracellular region or people's 4-1BB intracellular region any one or extremely
The string of the combination of few two kinds, preferably CD3 ζ intracellular region, people's CD28 intracellular region and people's CD3 ζ intracellular region
Connection, people's 4-1BB intracellular region are connected or people's CD28 intracellular region, people 4-1BB born of the same parents with people's CD3 ζ intracellular region
Inner region is connected with people's CD3 ζ intracellular region；
Preferably, the aminoacid sequence of described people's CD3 ζ intracellular region is SEQ ID NO.13, and nucleotide sequence is
SEQ ID NO.14；
Preferably, described people's CD28 intracellular region is SEQ with the aminoacid sequence connected of people's CD3 ζ intracellular region
ID NO.15, nucleotide sequence is SEQ ID NO.16；
Preferably, described people's 4-1BB intracellular region with the aminoacid sequence connected of people's CD3 ζ intracellular region is
SEQ ID NO.17, nucleotide sequence is SEQ ID NO.18；
Preferably, described people's CD28 intracellular region, people's 4-1BB intracellular region are connected with people's CD3 ζ intracellular region
Aminoacid sequence be SEQ ID NO.19, nucleotide sequence is SEQ ID NO.20.
6. according to the Chimeric antigen receptor according to any one of claim 1-5, it is characterised in that described embedding
The amino terminal closing antigen receptor also comprises people's CD8 alpha signal peptide；
Preferably, the aminoacid sequence of described people's CD8 alpha signal peptide is SEQ ID NO.21, and nucleotide sequence is
SEQ ID NO.22。
7. according to the Chimeric antigen receptor according to any one of claim 1-6, it is characterised in that described embedding
Close antigen receptor by people's CD8 alpha signal peptide, VAR2CSA, people CD8 α hinge region, people's CD28 cross-film district
In series with intracellular region, people's 4-1BB intracellular region and people's CD3 ζ intracellular region；
Preferably, the aminoacid sequence of described Chimeric antigen receptor is SEQ ID NO.1, and nucleotide sequence is
SEQ ID NO.2。
8. according to the Chimeric antigen receptor according to any one of claim 1-7, it is characterised in that described
Chimeric antigen receptor is transfected in T cell expression by its nucleotide sequence encoded；
Preferably, appointing during the mode of described transfection is viral vector, eukaryon expression plasmid or mRNA sequence
Anticipate a kind of or combination of at least two；
Preferably, during described viral vector is slow virus carrier, adenovirus vector or retroviral vector
Any one or the combination of at least two.
9. a compositions, it is characterised in that described compositions includes according to any one of claim 1-8
Chimeric antigen receptor.
10. Chimeric antigen receptor as according to any one of claim 1-8 and/or as claimed in claim 9
Compositions in preparation Chimeric antigen receptor T cell and the application in anti-tumor medicine thereof.
Priority Applications (6)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510843720.9A CN105753991B (en) | 2015-11-26 | 2015-11-26 | Chimeric antigen receptor for resisting placenta-like chondroitin sulfate and application thereof |
| GB1810451.3A GB2561755A (en) | 2015-11-26 | 2016-10-26 | Anti-placenta-chondroitin-sulfate chimeric antigen receptor and application thereof |
| EP16867848.0A EP3381946A4 (en) | 2015-11-26 | 2016-10-26 | Anti-placenta-chondroitin-sulfate chimeric antigen receptor and application thereof |
| PCT/CN2016/103457 WO2017088623A1 (en) | 2015-11-26 | 2016-10-26 | Anti-placenta-chondroitin-sulfate chimeric antigen receptor and application thereof |
| CN201680001176.6A CN106459224B (en) | 2015-11-26 | 2016-10-26 | Chimeric antigen receptor for resisting placenta-like chondroitin sulfate and application thereof |
| US15/779,382 US20190153049A1 (en) | 2015-11-26 | 2016-10-26 | Anti-placenta-chondroitin-sulfate chimeric antigen receptor and application thereof |
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| CN108066361A (en) * | 2017-01-23 | 2018-05-25 | 广州中科蓝华生物科技有限公司 | A kind of excretion body, its preparation method and its purposes in anti-tumor drug or preparation is prepared |
| CN108570118A (en) * | 2017-10-17 | 2018-09-25 | 中国科学院深圳先进技术研究院 | A kind of affinity chromatographic purification process of placenta sample chondroitin sulfate A (CSA) or derivatives thereof |
| CN108570118B (en) * | 2017-10-17 | 2020-07-03 | 中国科学院深圳先进技术研究院 | Affinity chromatography purification method of placenta-like chondroitin sulfate A or derivative thereof |
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| CN109387627A (en) * | 2018-10-16 | 2019-02-26 | 中国科学院深圳先进技术研究院 | A kind of Reagent Protocol of screening for cancer and early diagnosis based on placenta sample chondroitin sulfate A (CSA) |
| CN109387627B (en) * | 2018-10-16 | 2021-09-24 | 中国科学院深圳先进技术研究院 | Reagent method for screening and early diagnosis of cancer based on placenta-like chondroitin sulfate A |
| CN110124045A (en) * | 2019-04-30 | 2019-08-16 | 云南大学 | Application of the plasmodium VAR2CSA albumen in anti-tumor drug |
| WO2023130667A1 (en) * | 2022-01-07 | 2023-07-13 | 中科蓝华(广州)生物医药技术有限公司 | Catenated var2csa recombinant protein, preparation method therefor, and use thereof |
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