CN107987173A - Double target spot Chimeric antigen receptors, its encoding gene, have the gene plasmid, immune T effector cell and HIV-1 applications - Google Patents
Double target spot Chimeric antigen receptors, its encoding gene, have the gene plasmid, immune T effector cell and HIV-1 applications Download PDFInfo
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- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70503—Immunoglobulin superfamily
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Abstract
The invention discloses double target spot Chimeric antigen receptors, its encoding gene, there is the gene plasmid, expression vector, immune T effector cell and application, the Chimeric antigen receptor to include T4 antigen area (including Leader areas), Linker areas, 17b single-chain antibodies area (17bscFv), hinge area, transmembrane domain, costimulation domain and signal transduction domain.CD4 17bscFv CAR of the present invention simulate the calmodulin binding domain CaM of gp120, its validity and safe in the single target spot CD4 ζ CAR reported in the early time.
Description
Technical field
The present invention relates to biomedicine technical field, more particularly to a kind of double target spot Chimeric antigen receptors for HIV-1,
Its encoding gene, have the gene plasmid, expression vector, immune T effector cell and applied in terms of HIV-1.
Background technology
Human immunodeficiency virus (human immunodeficiency virus Type 1, HIV-1), belongs to reverse transcription
One kind of virus, invades CD4+T cells, body acquired immunity function is badly damaged, ultimately result in AIDS AIDS
(Acquired immunodeficiency syndrome).The Chinese Center for Disease Control and Prevention of in August, 2017 discloses the whole nation
AIDS Epidemic, the number of the infected of the domestic HIV-1 in China (human immunodeficiency virus) have reached 718270
People, wherein thering are 299169 people to suffer from AIDS, because the number of AIDS death has reached 221628 people.In addition HIV- is increased newly every year
1 the infected/patient AIDS is up to more than 140000 people.These numerals show the HIV infection situation very severe in China.
At present, AIDS clinical treatment is mainly highly active antiretroviral therapy (Highly active
Antiretroviral therapy, HAART), the use of HAART already lead in 5 years almost 100% lethality to five
91% survival rate in year.It it is more than 40 years from their rear average life span estimation is made a definite diagnosis in the adult of developed country's infected by HIV.
In developing country, although adult's average life span of infected by HIV is shorter, their survival rate also improves.No matter ART's
Using acquired prominent achievement, a series of problems caused by the use that we have in face of ART.For example, ART has been led
HIV infection is caused to develop into the chronic disease for being not easy to cure.In addition, the use of ART also has many inferior positions and limitation.ART is lifelong
Therapy and be virose.In therapeutic process, we will also face the drug resistance problems of HIV medicines.This therapy can not
Eliminate the HIV to hide in virus base.Expensive medical expense also makes many patients of the limited national infected by HIV of resource obtain not
To treatment.Therefore, people are badly in need of finding the new method for the treatment of HIV-1 infection.
The cytolysis patent of HIV-1 infection cells is directed in the cell by carrying chimeric CD4 acceptors
It is all public in CN95192559.8 and cell and correlation molecule and process patent CN95195183.1 with CD4 decoy acceptors
Open the T cell therapy for HIV-1.The therapy shows in the case of HAART treatments are not carried out, thin by the T of genetic modification
Born of the same parents can efficiently control HIV-1 viruses.Its mechanism of action is mainly, by CD4 extracellular domains and intracellular CD3- ζ signal nodes
The be connected CD4- ζ Chimeric antigen receptors (CAR) of composition of structure domain are transfected into human T-cell by viral vector, since CD4 is HIV-1
The major receptors of infection cell, the CD4 extracellular domains of CD4- ζ CAR are by combining outside the HIV-1 on infection cell surface
Memebrane protein gp120 simulates immunological synapse, causes CD4- ζ CAR-T cell activations, and then cracks HIV-1 infection cells, reduces and suffers from
HIV-1 virus loads in person's body.But CD4- ζ CAR-T cells combine gp120 antigens after, although T cell can be activated
The HIV-1 infection cells combined are killed, but the T cell activated is difficult further to breed, and can only play temporal effect, and CD4-
ζ CAR-T cells are easily hiv-1-infected, become new host.This method is unsatisfactory in later phase clinical test effect, so far for
Only, it is not widely used also.
Therefore, a kind of new safe efficient type Chimeric antigen receptor for being used for HIV-1 treatments is developed, is not only had urgent
Researching value, it may have good economic benefit and extensive medical applications potentiality, this be exactly the present invention be accomplished it is dynamic
Where power and basis.
The content of the invention
The defects of in order to overcome the prior art as indicated above, the present inventor has made intensive studies this, is paying
After a large amount of creative works, so as to complete the present invention.
Specifically, the technical problems to be solved by the invention are：Double target spot Chimeric antigen receptors, its coding base are provided
Cause, have the gene plasmid, expression vector, immune T effector cell and the application in terms of HIV-1.
In order to solve the above technical problems, the technical scheme is that：
In a first aspect, the present invention provides a kind of double target spot Chimeric antigen receptors, the Chimeric antigen receptor resists comprising CD4
Former area's (including CD4Leader areas), Linker areas, 17b single-chain antibodies area (17bscFv), hinge area, altogether transmembrane domain, thorn
Swash domain and signal transduction domain.
Wherein, T4 antigen area is the 1-182 amino acid of CD4.
Wherein, Linker areas are selected from connection peptide (G4S) 2, (G4S) 4, (G4S) 6, (G4S) 8 and (G4S) of different length
10, the present invention preferably (G4S) 8, the connection peptide being made of 40 amino acid.
Wherein, G4S is made of Gly Gly Gly Gly 5 amino acid of Ser, (G4S) 2, (G4S) 4, (G4S)
6th, (G4S) 8 and the number of repetition that (G4S) 10 is G4S.
Wherein, 17b single-chain antibodies area is the single-chain antibody 17bscFv for HIV-1 17b epitopes.
Wherein, hinge area is CD8 α hinge areas.
Wherein, transmembrane domain is CD8 α transmembrane regions.
Wherein, costimulation domain is the functional domain of CD137.
Wherein, signal transduction domain includes the functional domain of CD3 ζ.
Second aspect, the present invention provides the gene for encoding double target spot Chimeric antigen receptors, which is CD4-
17bscFv-CD8 α-CD137-CD3 ζ (also known as CD4-17bscFv-CAR DNA fragmentations).
The gene is composed in series by following nucleic acid artificial sequence：
(1) T4 antigen nucleic acid artificial sequence, such as SEQ ID NO.2；
(2) Linker nucleic acid artificial sequence, such as SEQ ID NO.3；
(3) 17b single-chain antibodies nucleic acid artificial sequence, such as SEQ ID NO.4；
(4) CD8 α hinge areas nucleic acid artificial sequence, such as SEQ ID NO.5；
(5) CD8 α transmembrane regions nucleic acid artificial sequence, such as SEQ ID NO.6；
(6) CD137 costimulations area nucleic acid artificial sequence, such as SEQ ID NO.7；
(7) CD3 ζ signal transductions area nucleic acid artificial sequence, such as SEQ ID NO.8.
In the present invention, as a kind of perferred technical scheme, the nucleotide sequence such as SEQ ID NO.1 of the gene.
The third aspect, the present invention provides recombinant plasmid, the recombinant plasmid includes gene as described above.
In the present invention, as a kind of perferred technical scheme, the recombinant plasmid is by the CD4-17bscFv-CD8
α-CD137-CD3 ζ genetic fragments are inserted into pLent-CD4- obtained from the pLent-C-GFP DNA fragmentations of linearisation
17bscFv-CAR plasmids.
Fourth aspect, the present invention provides expression vector, gene as described above is included in the expression vector.
In the present invention, as a kind of perferred technical scheme, the expression vector uses slow virus package cell line.
5th aspect, the present invention provides immune T effector cell, the immune T effector cell includes base as described above
Cause.
In the present invention, as a kind of perferred technical scheme, the immune T effector cell includes CD8+T cells.
6th aspect, the present invention provides the application in terms of the medicine for the treatment of HIV-1 is prepared.
In the present invention, as a kind of perferred technical scheme, the form of the medicine includes kit.
In the present invention, as a kind of perferred technical scheme, the kit includes：
(1) expression vector as described above for stablizing expression CD4-17bscFv-CAR is obtained；
(2) carrier diluent.
After employing above-mentioned technical proposal, the beneficial effects of the invention are as follows：
The present invention is in pairs by the single-chain antibody 17b that connected after the extracellular domain of the CD4- ζ CAR reported in the early time, development
Target spot CAR targets CD4 binding sites and 17b epitopes (also known as CD4 induced epitopes), and this patent is named as CD4-17bscFv-
CAR (see Fig. 1).CD4 bound sites on the first surface protein gp120 with HIV-1 infection cells of the CD4 of CD4-17bscFv-CAR
Point combines, and the conformation of gp120 is changed, exposes the 17b epitopes combined with 17b scFv, can pass through after the two combination
The intracellular region of CD3 makes T cell activation play effector function.It follows that the activation signals of CD4-17bscFv-CAR-T needs two
The combination in a site.Therefore, CD4-17bscFv-CAR can effectively prevent HIV-1 cell entry CAR-T cells, its safety
Property higher than single target spot CD4- ζ CAR for reporting in the early time.After present invention optimization linker, it is attached to CD4 and 17b scFv same
On gp120.Therefore, CD4-17bscFv-CAR of the present invention simulates the calmodulin binding domain CaM of gp120, enhances both affinity, its
Validity is also above the single target spot CD4- ζ CAR reported in the early time.
The present invention also transforms remaining structure of CAR, thus develop it is a kind of it is brand-new, that MHC is non-dependent is anti-
T cell activation signal can be further amplified in HIV-1CAR molecules, CD4-17bscFv-CAR, improve internal amplification ability and production
Raw cell factor produces ability.CD4-17bscFv-CAR-T cells and the cell line row of expression gp120 are co-cultured, Ke Yiqiang
The effectively cell line of killing expression gp120.And fragmentation effect is substantially better than compared to the CD4- ζ CAR-T cells reported in the early time.
For more detailed, the present invention uses CD8 α hinge areas, confirms that the homology of CD4 can be reduced in practice, prevents CAR-T quilts
HIV-1 infects.CAR-T can further be activated using 4-1BB, CAR-T is largely expanded, increased to HIV-1 infection cells
Lethality.
Brief description of the drawings
Fig. 1 is Chimeric antigen receptor CD4-17bscFv-CD8 α-CD137-CD3 ζ principle design drawings of the present invention.
Fig. 2 is CD4-17bscFv-CAR DNA fragmentations (left side) and the pLent-C-GFP DNA (right side) of linearisation in the present invention
Fragment electrophoretic figure.
Fig. 3 is the schematic diagram of slow virus CD4-17bscFv-CD8 α-CD137-CD3 ζ expression plasmids of the present invention.
The efficiency that Fig. 4 is the expression CD4-17bscFv-CAR of CD4-17bscFv-CAR-T cells of the present invention is 72.0%
(right side is streaming peak figure, and a left side is scatter diagram).
Fig. 5 is experiment knots of the Linker to the fragmentation effects of CD4-17bscFv-CAR-T cells of different length of the present invention
Fruit is schemed.In E：T is 1：When 1, CD4-linker (G4S) 8-17bscFv-CAR-T are to expressing HIV-1NL4-3Envelope glycoprotein cell
The killing-efficiency of strain Jurkat is 92.57 ± 7.43% apparently higher than CD4- ζ CAR-T and does not transform CD8+T cells
Fig. 6 is ELIspot of the present invention experiment detection figures.Left figure is the situation of CD4-17bscFv-CAR-T secretion of gamma-IFN；
Middle figure is the situation of CD4- ζ CAR-T secretion of gamma-IFN；Right figure is not transform the situation of CD8+T cell secretion of gamma-IFN；Expression
HIV-1NL4-3The secretion energy of the CD4-17bscFv-CAR-T cell IFN-γ of envelope glycoprotein cell line Jurkat mixed culture
Power is significantly higher than CD4- ζ CAR-T and does not transform CD8+T cells.
Embodiment
With reference to specific embodiment, the present invention is further described.But the purposes and mesh of these exemplary embodiments
Be only used for enumerate the present invention, any type of any restriction not is formed to the real protection scope of the present invention, it is more non-to incite somebody to action this
The protection domain of invention is confined to this.
Embodiment 1
A kind of double target spot Chimeric antigen receptors for HIV-1, the Chimeric antigen receptor (are included comprising T4 antigen area
CD4Leader areas), Linker areas, 17b single-chain antibodies area (17bscFv), hinge area, transmembrane domain, costimulation domain,
Signal transduction domain and suicide gene system area.
Wherein, T4 antigen area is the 1-182 amino acid of CD4；Connection peptide (G4S) 2 of the Linker areas selected from different length,
(G4S) 4, (G4S) 6, (G4S) 8 and (G4S) 10, the present invention preferably (G4S) 8, the connection peptide being made of 40 amino acid；17b is mono-
Chain antibody area is the single-chain antibody 17bscFv for HIV-1 17b epitopes；Hinge area is CD8 α hinge areas；Transmembrane domain is
CD8 α transmembrane regions；Costimulation domain is the functional domain of CD137；Signal transduction domain includes the functional structure of CD3 ζ
Domain.
Embodiment 2
The gene of the Chimeric antigen receptor is encoded, which (is also known as CD4-17bscFv-CD8 α-CD137-CD3 ζ
CD4-17bscFv-CAR DNA fragmentations).
The structure of CD4-17bscFv-CD8 α-CD137-CD3 ζ
By fusion fragment CD4-17bscFv-CD8 α-CD137-CD3 ζ insertion Lentivirals pLent-C-
GFP。
The signal of CD4-17bscFv-CD8 α-CD137-CD3 ζ modules is shown in that (complete nucleic-acid sequences are shown in annex SEQ ID to Fig. 1
NO.1)。
Each sequence of modules of CD4-17bscFv-CD8 α-CD137-CD3 ζ
(1) T4 antigen nucleic acid artificial sequence (SEQ ID NO.2)
(2) Linker nucleic acid artificial sequence (SEQ ID NO.3)
(3) 17b single-chain antibodies nucleic acid artificial sequence (SEQ ID NO.4)
(4) CD8 α hinge areas nucleic acid artificial sequence (SEQ ID NO.5)
(5) CD8 α transmembrane regions nucleic acid artificial sequence (SEQ ID NO.6)
(6) CD137 costimulations area nucleic acid artificial sequence (SEQ ID NO.7)
(7) CD3 ζ signal transductions area nucleic acid artificial sequence (SEQ ID NO.8)
Respectively by T4 antigen nucleic acid artificial sequence, Linker nucleic acid artificial sequence, 17b single-chain antibody nucleic acid artificial sequence,
CD8 α hinge area nucleic acid artificial sequence, CD8 α transmembrane region nucleic acid artificial sequence, CD137 costimulations area nucleic acid artificial sequence, CD3 ζ
Signal transduction area nucleic acid artificial sequence entrusts Sangon Biotech (Shanghai) Co., Ltd. to synthesize its whole expression cassette and is inserted into mark
On quasi- carrier pUC57, therefore pUC-CD4-17bscFv-CAR is named as, pUC-CD4-17bscFv-CAR is subjected to Fast
Digest AsiSI (being purchased from ThermoFisher companies) and Fast Digest NotI (being purchased from ThermoFisher companies) are double
Digestion, 37 DEG C, digestion 20min.100 μ l digestion systems are：10×buffer：10μl；DNA 6μg；AsiSI enzymes： 3μl；
NotI enzymes：3μl；Deionized water supplies volume.Will be containing HIV-1-CD4-CAR DNA fragmentation agar-agars position using agar-agar electrophoresis
Cut, be placed in two centrifuge tubes.Using DNA extraction kit (being purchased from ThermoFisher companies) by DNA from fine jade
Dissolution and concentrated in glue, add 500 μ l DF buffer toward above-mentioned centrifuge tube first, 55 DEG C act on 10 minutes, per shaking within 2-3 minutes
Shake once, until agar-agar is completely dissolved.Again by agar-agar solution all suction DF Column, and put on Collection Tube
(collection filtered fluid).8000rpm is centrifuged 1 minute, and filtered fluid is outwelled.Add 500 μ l Wash Buffer, 8000rpm from
The heart 1 minute, filtered fluid is outwelled.12000rpm, which is centrifuged 2 minutes, ensures that ethanol is removed.Finally DF Column are transferred to another
Clean microcentrifugal tube, adds 25 μ l Elution Buffer, and after being stored at room temperature 2 minutes, 14000rpm is centrifuged 2 minutes,
Liquid in microcentrifugal tube is the CD4-17bscFv-CAR DNA fragmentations purified (see Fig. 2).
Embodiment 3
Table recombinant plasmid, the recombinant plasmid include gene as described above.The recombinant plasmid is by the CD4-
17bscFv-CD8 α-CD137-CD3 ζ genetic fragments are inserted into obtained from the pLent-C-GFP DNA fragmentations of linearisation
PLent-CD4-17bscFv-CAR plasmids.
It is the preparation method of table recombinant plasmid below：
Respectively by T4 antigen nucleic acid artificial sequence, Linker nucleic acid artificial sequence, 17b single-chain antibody nucleic acid artificial sequence,
CD8 hinge area nucleic acid artificial sequence, CD8 transmembrane region nucleic acid artificial sequence, CD137 costimulations area nucleic acid artificial sequence, CD3 ζ letters
Number conducting region nucleic acid artificial sequence entrusts Sangon Biotech (Shanghai) Co., Ltd. to synthesize its whole expression cassette and is inserted into standard
On carrier pUC57, therefore pUC-CD4-17bscFv-CAR is named as, while by pUC-CD4-17bscFv-CAR and pLent-
C-GFP carriers carry out Fast Digest AsiSI (being purchased from ThermoFisher companies) and Fast Digest NotI (are purchased from
ThermoFisher companies) double digestion, 37 DEG C, digestion 20min.100 μ l digestion systems are：10×buffer： 10μl；DNA 6
μg；AsiSI enzymes：3μl；NotI enzymes：3μl；Deionized water supplies volume.Will be respectively containing HIV-1-CD4- using agar-agar electrophoresis
The agar-agar position of CAR DNA fragmentations and the pLent-C-GFP DNA fragmentations of linearisation is cut, and is placed in two centrifuge tubes.Using
DNA extraction kit (being purchased from ThermoFisher companies) dissolution and concentrate DNA from agar-agar, first toward it is above-mentioned from
Heart pipe adds 500 μ l DF buffer, and 55 DEG C act on 10 minutes, per rocking once within 2-3 minutes, until agar-agar is completely dissolved.Again
By agar-agar solution all suction DF Column, and put on Collection Tube (collection filtered fluid).8000rpm centrifuges 1 point
Clock, filtered fluid is outwelled.500 μ l Wash Buffer, 8000rpm centrifugation 1 minute is added, filtered fluid is outwelled.12000rpm
Centrifugation ensures that ethanol is removed in 2 minutes.DF Column are finally transferred to another clean microcentrifugal tube, add 25 μ l
Elution Buffer, after being stored at room temperature 2 minutes, 14000rpm is centrifuged 2 minutes, and the liquid in microcentrifugal tube is to purify
CD4-17bscFv-CAR DNA fragmentations (see Fig. 2) and linearisation pLent-C-GFP DNA (see Fig. 2) fragment.
Above two DNA fragmentation is subjected to overnight connection at 16 DEG C and forms pLent-CD4-17bscFv-CAR (see Fig. 3)
Plasmid.Linked system is：10×buffer：1μl；T4 ligases：1μl；CD4-17bscFv-CAR DNA：4μl；Linearisation
pLent-C-GFP DNA：4μl.
Above-mentioned pLent-CD4-17bscFv-CAR is transformed into E.coli (DH5 α).Comprise the following steps that：By plasmid and sense
Mixed by state cell and be incubated half an hour on ice, then 42 degree heat shocks 90 seconds, then 2min is placed on ice, finally add LB liquid medium
It is slow to shake or so 1 hour 3000rpm centrifuges 5min again, 100 μ l bacterium solutions are coated on containing ammonia benzyl LB solid plates.Next day picking
Single bacterium colony is incubated overnight, and pLent-CD4- is extracted using plasmid extraction purification kit (being purchased from Qiagen companies)
17bscFv-CAR plasmids, comprise the following steps that：(1) 10000 × g of 1.5ml bacterium solution room temperatures is taken to centrifuge 1min.(2) supernatant is removed, is added
250 μ l solution I (A containing RNase), vortex oscillator is shaken to thalline to suspend completely.(3) 250 μ l solution II, gentle top are added
Centrifuge tube 4~6 times, obtain the lysate of clarification.Preferably it is incubated at room temperature 2min.(4) plus 350 μ l solution III, gentle overturn count
Secondary mixing, to there is white flock precipitate, 10000 × g of room temperature centrifuges 10min.(5) especially careful absorption supernatant, moves to cleaning
The absorbing column for assembling volume 2ml centrifuge tubes in.Ensure not suck precipitation and cell fragment.10000 × g of room temperature is centrifuged
1min, passes through absorbing column completely to lysate.(6) filtered solution is abandoned, adds 500 μ l Buffer HBC, 10000 × g centrifugation 1min,
Absorbing column is cleaned, removes the purity that residual protein ensures DNA.(7) filtered solution is abandoned, then with the diluted 750 μ l of 100% ethanol
Wash Buffer clean absorbing column, 10000 × g centrifugations 1min.(8) filtered solution is abandoned, then adds 750 μ l Wash Buffer cleanings
Absorbing column.(9) 10000 × g of absorbing column must be centrifuged 2min ensures that ethanol is removed.(10) absorbing column is put into totally
1.5ml centrifuge tubes, add 50-100 μ l (final concentration for depending on needs) aseptic deionized waters or TE buffer solutions on filter membrane,
10000 × g centrifuges 5min, collects Plasmid DNA.(11) and concentration known DNA sample (Marker) does Ago-Gel electricity together
Swimming, comparing result, it is 376ng/ μ l to draw pLent-CD4-17bscFv-CAR plasmid concentrations.
Sangon Biotech (Shanghai) Co., Ltd. is entrusted to be surveyed above-mentioned pLent-CD4-17bscFv-CAR plasmids
Sequence.It is spare after being sequenced correctly.
Embodiment 4
Expression vector, includes gene as described above in the expression vector.The expression vector is thin using slow virus packaging
Born of the same parents system.
Expression vector uses following preparation method.
Slow virus is packed, titre detection
It is as follows using Lentiviral Packaging Kit slow virus package kits, specific method：By slow virus bag
Dress cell line 293T is inoculated in containing in DMEM+10%FBS 10cm culture dishes, 37 DEG C, is cultivated under the conditions of 5% CO2, adherent
Prepare transfection when rate is 70%-80%.Sterile 1.5ml EP pipes or 15ml centrifuge tubes are taken, reactant is prepared by following component
System：Serum-free DMEM：3ml；PLent-HIV-1-CD4-CAR plasmids：10μg；GM easyTM Lentiviral Mix：10μl
(10μg)；HG TransgeneTM Reagent： 60μl.After mixing, after room temperature places 20min, drop evenly containing 293T
In Tissue Culture Dish, CO is placed in2Cultivated in incubator.After transfecting 24h, carefully sop up cell culture fluid and abandon in filling thimerosal
Waste liquid cup in, then plus 15ml contains the fresh culture medium of 10% serum and continues to cultivate.After changing liquid 48h, cell conditioned medium is drawn
Liquid is in 50ml centrifuge tubes, and 4 DEG C, 500g centrifugation 5min, supernatant is transferred in new centrifuge tube after being filtered with 0.45 μm of filter.This
When supernatant in virion can directly go detection titre.
By above-mentioned virus using TCID50 measure titres, by the 293T cells in exponential phase with 1 × 104
The amount in Cells/ holes is inoculated in 96 porocyte culture plates, and sample presses 10 times of double dilution series concentration with 5%FBS DMEM, sample-adding
In 96 orifice plates, each concentration is loaded 10 holes, if 2 hole blank controls.Cultivated in 37 DEG C, 5%CO2, observe cell day by day and occur
Malicious spot situation, generally requires observation 5-7 days, and the TCID50 results of sample are calculated according to concentration and hole count that malicious spot occur.As a result table
It is bright, the titre 6.72 × 10 of recombinant slow virus6 TCID50/ml。
Embodiment 5
Immune T effector cell, the immune T effector cell include gene as described above, the immune T effector cell
Including CD8+T cells.Immune T effector cell uses following preparation method.
The preparation of PBMC cells
The fresh peripheral blood of 75ml health contributors is taken, (is given birth to TBD sample rates separating liquid purchased from Tianjin Hao oceans China Tech
Thing), separating peripheral blood mononuclear cells PBMC methods are as follows：
(1) peripheral blood 75ml and physiological saline are pressed 1:1 dilution proportion.Blood after dilution is carefully added on an equal basis
On volume lymphocyte separation medium, obvious layering, room temperature horizontal centrifugal 800rpm/min, 20min are formed.At this time in centrifuge tube
4 layers are formed from top to bottom；Serum, the tunica albuginea layer being made of PBMC, lymphocyte separation medium layer and nethermost erythroprecipitin
Layer.
(2) tunica albuginea layer is carefully drawn with suction pipe, all suctions out PBMC as far as possible.Add 2 times of amount physiological saline, wash cell 2 times,
Centrifugation, 800rpm/min, 10min after mixing every time.Low-speed centrifugal is conducive to remove the blood platelet retained in cell suspension and leaching
Bar cell separating liquid, supernatant discarding after centrifugation, collects PBMC cells.
BD magnetic bead sortings, culture and the preparation of CD4-17bscFv-CAR-T of CD8+T lymphocytes
1) isolated PBMC is washed 2 times with PBS.2) 300 × g of centrifugation cell, 10 minutes.3) cell is resuspended,
The moon for adding biotin coupling selects antibody (being purchased from BD companies).4) at room temperature, it is incubated 20 minutes.Then washed with PBS buffer
Cell, centrifugation cell 300 × g, 10 minutes.5) Avidin coupled bead (being purchased from BD companies) is added.6) at room temperature, it is incubated
30 minutes.7) cell is resuspended with PBS buffer, adds in streaming pipe, often pipe is no more than 3ml.8) streaming pipe is placed on cell point
Select on magnet stand, quiet 8 minutes, allow the non-CD8+T cells for being combined with magnetic bead to be adsorbed in side wall.9) collect not by enrichment with magnetic bead
Supernatant, centrifuges 300 × g, 10 minutes, what is obtained was CD8+T cells.
CD8+T cells are resuspended with RPMI1640 complete mediums, then by 1 × 106The cell concentration of/ml, uniformly spreads extremely
In Tissue Culture Plate.Then (it is purchased from using anti-CD3 (being purchased from BD companies), anti-CD28 (being purchased from BD companies) antibody and IL2
BD companies) cell is stimulated, effect 48 it is small when after, collect cell.Recombinant slow virus is infected in the ratio of MOI=10
CD8+T, fresh culture is changed after infecting 24h, continues to expand culture to enough dosages.(it is purchased from by FC500 flow cytometers
BECKMAN companies) FL1 Air conduct measurements Chimeric antigen receptor expression (Fig. 4).Using the CD8+T cells do not transformed as negative right
According to CD4-17bscFv-CAR-T positive rates 72.0%.
Embodiment 6
Influences of the Linker of different length to the killing activity of CD4-17bscFv-CAR-T cells
Express HIV-1NL4-3For envelope glycoprotein cell line Jurkat as target cell, effector cell is respectively to contain different length
Spend CD4-17bscFv-CAR-T, the CD4- ζ CAR-T of Linker and do not transform CD8+T cells.By E：T is 1：1, add 1 × 106
A Jurkat cell, after cell is completely adherent, collects CD4-17bscFv-CAR-T, CD4- ζ containing different length Linker
CAR-T and CD8+T cells are not transformed, it is 1 × 10 to adjust cell concentration respectively7/ ml, adds 100 μ L per hole, 37 DEG C, 5% CO2
Under the conditions of cultivate 12h.Abandon supernatant and add the 20 diluted CCK8 of μ L (being purchased from MCE companies), when incubation 4~6 is small, microplate reader detection
The light absorption value of OD450.Killing rate=[1- (the OD values of effector cell+Target cell wells OD values-individual effect cell)/independent target is thin
The OD values of born of the same parents] × 100%.
CD4-linker (G4S) 8-17bscFv-CAR-T are high for 92.57 ± 7.43% to the killing-efficiency of Jurkat cell
In the CD4-17bscFv-CAR-T groups (Fig. 5) of other different length Linker.Illustrate that Linker is 40 amino acid, its CD4-
The fragmentation effect of 17bscFv-CAR-T is optimal, and the fragmentation effect of CD4-linker (G4S) 8-17bscFv-CAR-T is obvious
CD8+T cells are not transformed higher than CD4- ζ CAR-T and.
Embodiment 7
CD4-17bscFv-CAR-T cells detect the neurological susceptibility of HIV-1
Wild type HIV-1NL4-3Pair unloaded to CD4+T lymphocytes, the CD8+T lymphocytes do not transformed, transduction respectively
Infected according to CD8+T cells, CD4- ζ CAR-T and CD4-17bscFv-CAR-T cells, wherein CD4+T lymphocytes are as real
Test positive control.Then pass through the culture of 10 days, Thermo (is purchased from by HIV-1p24 antigen ELISA detecting kits
Fisher Scientific companies) analysis 5 experimental groups intracellular HIV-1 antigen p24 contents.The specific step of ELISA detections
Suddenly：
Plus conjugate 1 A.：1,25 μ L of conjugate are added per hole.B. it is loaded：Sequentially add sample, negative control, p24 antigens
75 μ L of positive control.C. incubate：Microwell plate is sealed with sealing plate film, 37 DEG C is put and incubates 60 minutes.D. board-washing：Discard microwell plate
Liquid in reacting hole, 350 μ L working concentration cleaning solutions are added per hole (containing blank control wells), then discard cleaning solution.Repeat
Board-washing totally 5 times, finally pats dry.E. it is enzyme：Add conjugate 2 (blank control wells are not added with) per hole, sealed microwell plate with sealing plate film,
37 DEG C are put to incubate 30 minutes.F. board-washing：Liquid in microwell plate reacting hole is discarded, 350 μ L are added per hole (containing blank control wells)
Working concentration cleaning solution, then discards cleaning solution.Repeat board-washing totally 5 times, finally pat dry.Plus substrate solution G.：Add substrate per hole
Buffer solution and each 50 μ L of color developing agent (containing blank control wells), are sealed microwell plate with sealing plate film, put room temperature (18~30 DEG C) lucifuge
Incubate 30 minutes.H adds terminate liquid：Add 50 μ L of terminate liquid (containing blank control wells) per hole, gently vibrate microwell plate, make content
Fully mix.I. detect：It is interior when 1 is small after termination reaction, with microplate reader at 450nm wavelength, each hole absorbance is measured, according to
Whether contain HIV-1p24 antigens in critical value judgement sample.
The CD4+T lymphocytes that the results show has more than half are positive for p24, and the CD4- ζ CAR-T cells for having 10% are
P24 is positive, and p24 positive cells ratio in CD4-17bscFv-CAR-T cells, with do not transform CD8+T lymphocytes, transduction
Unloaded control CD8+T cells are suitable, are p24 feminine genders.Therefore, CD4-17bscFv-CAR-T cells can be avoided HIV-
1 infects.
Embodiment 8
By HIV-1 envelope proteins stimulate CD4-17bscFv-CAR-T cells can with the disease-resistant poison cell of efficient secretion because
Son
CD8+T is not transformed CD4-17bscFv-CAR-T, CD4- ζ CAR-T and respectively by E：T (effector cell and target cell
Than) it is 1：1 with expressing HIV-1NL4-3When envelope glycoprotein cell line Jurkat is mixed 20h, and (purchase is tested with ELIspot
From eBioscience companies) detect CD4-17bscFv-CAR-T respectively, CD4- ζ CAR-T and CD8+T cells IFN- is not transformed
The secretion of γ.ELIspot specific steps：
1st day：1. pre-coated plate of cell culture (sterile working) activates：RPMI-1640 culture mediums are added per hole, room temperature is quiet
Clappers remove culture medium after putting 5-10 minutes.2. add cell suspension：The cell suspension for adjusting concentration is added into each experimental port,
100 μ L/ holes；Positive control wells：Cell concentration 1 × 105/ hole, adds cell culture medium and PHA；Negative control hole：Cell concentration
1×105/ hole, adds isometric cell culture medium；Background negative control：Add the RPMI-1640 culture mediums containing hyclone.3.
It is incubated：37 DEG C are put into, 5% CO2When incubator culture 16-20 is small.
2nd day：(no longer needing sterile working) 4. cell lysis is operated after culture：Pouring aperture inner cell and culture medium.Add
The deionized water that ice bath is crossed, 200 μ L/ holes, 4 DEG C of refrigerators place 10 minutes hypotonic lysis cells.5. board-washing：Liquid in pouring aperture, 1
× Washing buffer, 200 μ L/ holes, are washed 5-7 times.30-60s is stood every time.For the last time, have the final say and go on blotting paper
Net liquid body.6. detect antibody incubation：The antibody-solutions of biotin labeling are added into plate hole, 100 μ L/ holes.When 37 DEG C of incubations 1 are small.
7. board-washing：Liquid in pouring aperture, 1 × Washing buffer, 200 μ L/ holes, are washed 5 times.Stand 30-60 seconds every time.Finally
Once, clappers remove liquid on blotting paper.8. enzyme-linked Avidin is incubated：The enzyme mark avidin solution diluted is added into plate
Hole, 100 μ L/ holes.When 37 DEG C of incubations 1 are small.9. board-washing：Liquid in pouring aperture, 1 × Washing buffer, 200 μ L/ holes, are washed
Wash 5 times.Stand 30-60 seconds every time.For the last time, clappers remove liquid on blotting paper.10. colour developing：The AEC now matched somebody with somebody is shown
Color liquid adds each plate hole, 100 μ L/ holes.Room temperature lucifuge stands colour developing 25 minutes, 11. color development stoppings：Liquid in pouring aperture, is opened
Plate base, is washed with deionized 3-5 times, color development stopping.Plate is placed on room temperature shady place, closes bottom after its naturally dry
Seat.12.ELISPOT plate spots are taken pictures and are analyzed.
The result shows that：With expressing HIV-1NL4-3The CD4-17bscFv- of envelope glycoprotein cell line Jurkat mixed culture
The secretion capacity of CAR-T (left side) cell IFN-γ be significantly higher than CD4- ζ CAR-T (in) and do not transform CD8+T (right side) cell (figure
6)。
From the above results, CD4-17bscFv-CAR-T molecules of the present invention, design is reasonable, safely and effectively, for treatment
HIV-1 lays the foundation.
Embodiment 9
Prepare the kit of CD4-17bscFv-CAR-T cells
(1) carrier as described above for stablizing expression CD4-17bscFv-CAR is obtained；
(2) carrier diluent；
(3) operation instructions；
In terms of the kit is applied to HIV-1 treatments, operation instructions include the method as described in embodiment 4-6.
It should be appreciated that the purposes of these embodiments is merely to illustrate the present invention and is not intended to the protection model of the limitation present invention
Enclose.In addition, it should also be understood that, after the technology contents of the present invention have been read, those skilled in the art can make the present invention each
Kind change, modification and/or variation, all these equivalent forms equally fall within the guarantor that the application the appended claims are limited
Within the scope of shield.
Claims (10)
1. pair target spot Chimeric antigen receptor, it is characterised in that：The Chimeric antigen receptor (is included comprising T4 antigen area
CD4Leader areas), Linker areas, 17b single-chain antibodies area, hinge area, transmembrane domain, costimulation domain and signal transduction
Domain.
2. double target spot Chimeric antigen receptors as claimed in claim 1, it is characterised in that：Linker areas are selected from the company of different length
Meet peptide (G4S) 2, (G4S) 4, (G4S) 6, (G4S) 8 and (G4S) 10.
3. double target spot Chimeric antigen receptors as claimed in claim 2, it is characterised in that：Linker areas select (G4S) 8, by 40
The connection peptide of amino acid composition.
4. the gene of coding double target spot Chimeric antigen receptors as claimed in claim 3, it is characterised in that：The gene is CD4-
17bscFv-CD8α-CD137-CD3ζ；
And the gene is composed in series by following nucleic acid artificial sequence：
(1) T4 antigen nucleic acid artificial sequence, such as SEQ ID NO.2；
(2) Linker nucleic acid artificial sequence, such as SEQ ID NO.3；
(3) 17b single-chain antibodies nucleic acid artificial sequence, such as SEQ ID NO.4；
(4) CD8 α hinge areas nucleic acid artificial sequence, such as SEQ ID NO.5；
(5) CD8 α transmembrane regions nucleic acid artificial sequence, such as SEQ ID NO.6；
(6) CD137 costimulations area nucleic acid artificial sequence, such as SEQ ID NO.7；
(7) CD3 ζ signal transductions area nucleic acid artificial sequence, such as SEQ ID NO.8.
5. encoding gene as claimed in claim 4, it is characterised in that：The nucleotide sequence of the gene such as SEQ ID NO.1.
6. recombinant plasmid, it is characterised in that：The recombinant plasmid includes gene as described in claim 4 or 5.
7. recombinant plasmid as claimed in claim 6, it is characterised in that：The recombinant plasmid is by the CD4-17bscFv-
CD8 α-CD137-CD3 ζ genetic fragments are inserted into pLent-CD4- obtained from the pLent-C-GFP DNA fragmentations of linearisation
17bscFv-CAR plasmids.
8. expression vector, it is characterised in that：Gene as described in claim 4 or 5 is included in the expression vector.
9. immune T effector cell, it is characterised in that：The immune T effector cell includes gene as described above.
10. application of the gene in terms of the medicine for the treatment of HIV-1 is prepared as described in 4 or 5.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109694875A (en) * | 2018-12-27 | 2019-04-30 | 山东兴瑞生物科技有限公司 | Anti- CII Chimeric antigen receptor encoding gene, slow virus plasmid, Treg immunocyte and its application |
| WO2020061796A1 (en) * | 2018-09-26 | 2020-04-02 | Hrain Biotechnology Co., Ltd. | Bcma-and-cd19-targeting chimeric antigen receptor and uses thereof |
| CN111234032A (en) * | 2019-12-12 | 2020-06-05 | 首都医科大学附属北京妇产医院 | Double-target chimeric antigen receptor for treating ovarian cancer and preparation method and application thereof |
| CN113943709A (en) * | 2021-09-14 | 2022-01-18 | 复旦大学 | Multifunctional anti-HIV-1 CAR-T cell and construction method and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015077789A2 (en) * | 2013-11-25 | 2015-05-28 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Chimeric antigen receptors to control hiv infection |
| CN107106665A (en) * | 2014-06-06 | 2017-08-29 | 纪念斯隆-凯特琳癌症中心 | Target Chimeric antigen receptor of mesothelin and application thereof |
| CN107098969A (en) * | 2017-06-28 | 2017-08-29 | 武汉云谷生物医药科技有限公司 | A kind of recombination of Chimeric antigen receptor for treating HIV builds and its applied |
-
2017
- 2017-11-30 CN CN201711239808.5A patent/CN107987173B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015077789A2 (en) * | 2013-11-25 | 2015-05-28 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Chimeric antigen receptors to control hiv infection |
| CN107106665A (en) * | 2014-06-06 | 2017-08-29 | 纪念斯隆-凯特琳癌症中心 | Target Chimeric antigen receptor of mesothelin and application thereof |
| CN107098969A (en) * | 2017-06-28 | 2017-08-29 | 武汉云谷生物医药科技有限公司 | A kind of recombination of Chimeric antigen receptor for treating HIV builds and its applied |
Non-Patent Citations (4)
| Title |
|---|
| GENBANK: AB590293.1: "Synthetic construct DNA, clone: pFN21AE2271, Homo sapiens CD4 gene for CD4 molecule,without stop codon, in Flexi system", 《NCBI BLAST》 * |
| PDB: 1RZ8_A: "Chain A, Fab 17b Light Chain", 《NCBI BLAST》 * |
| PDB: 1RZ8_B: "Chain B, Fab 17b Heavy Chain", 《NCBI BLAST》 * |
| 陈骋等: "嵌合抗原受体修饰的T细胞的临床应用及研究进展", 《肿瘤》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020061796A1 (en) * | 2018-09-26 | 2020-04-02 | Hrain Biotechnology Co., Ltd. | Bcma-and-cd19-targeting chimeric antigen receptor and uses thereof |
| CN109694875A (en) * | 2018-12-27 | 2019-04-30 | 山东兴瑞生物科技有限公司 | Anti- CII Chimeric antigen receptor encoding gene, slow virus plasmid, Treg immunocyte and its application |
| CN109694875B (en) * | 2018-12-27 | 2022-04-29 | 山东兴瑞生物科技有限公司 | anti-CII chimeric antigen receptor coding gene, lentiviral plasmid, Treg immune cell and application thereof |
| CN111234032A (en) * | 2019-12-12 | 2020-06-05 | 首都医科大学附属北京妇产医院 | Double-target chimeric antigen receptor for treating ovarian cancer and preparation method and application thereof |
| CN111234032B (en) * | 2019-12-12 | 2021-07-23 | 首都医科大学附属北京妇产医院 | Double-target chimeric antigen receptor for treating ovarian cancer and preparation method and application thereof |
| CN113943709A (en) * | 2021-09-14 | 2022-01-18 | 复旦大学 | Multifunctional anti-HIV-1 CAR-T cell and construction method and application thereof |
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| CN107987173B (en) | 2021-07-13 |
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