CN109265560B - Molecular switch, anti-HIV-1 chimeric antigen receptor with molecular switch, preparation method, NK cell and application - Google Patents
Molecular switch, anti-HIV-1 chimeric antigen receptor with molecular switch, preparation method, NK cell and application Download PDFInfo
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Abstract
The molecular switch, the anti-HIV-1 chimeric antigen receptor with the molecular switch, the preparation method, the NK cell and the application are disclosed in the invention, the molecular switch is named as RQR8, and the molecular switch has a nucleic acid sequence shown in SEQ ID NO. 1. The chimeric antigen receptor provided by the invention is added with a molecular switch RQR8 to prevent and treat the adverse reaction of CD4-17bscFv-bicAR-NK, and the safety is improved.
Description
Technical Field
The invention relates to the technical field of genes, in particular to a molecular switch, an anti-HIV-1 chimeric antigen receptor with the molecular switch, a preparation method, NK cells and application.
Background
The clinical treatment of aids is mainly high-level anti-retroviral therapy (HAART), the use of HAART has led to a mortality rate of almost 100% within five years to a survival rate of 91% within five years. Adults infected with HIV-1 in developed countries have an estimated average life span of more than 40 years from the time of diagnosis. In developing countries, although the average life span of adults infected with HIV-1 is shorter, their survival rate has also increased. Despite the significant achievements achieved with the use of ART, we have to face a series of problems with the use of ART. For example, ART has caused HIV-1 infection to progress to a chronic disease that is not easily cured. In addition, the use of ART has many disadvantages and limitations. ART is a lifelong therapy and toxic. During the course of therapy, we are also faced with the problem of resistance to HIV-1 drugs. This therapy also does not eliminate latent HIV-1 in the viral pool. The expensive cost of treatment also leaves many HIV-1 infected patients untreated in countries with limited resources. Therefore, there is an urgent need to find new methods for treating HIV-1 infection.
T cell therapies against HIV-1 are disclosed in patents CN95192559.8, CN95195183.1, CN201710801919.4, cn201711239809.x and CN 201711239808.5. This therapy shows that genetically engineered T cells can effectively control HIV-1 virus without developing HAART therapy. The mechanism of action is mainly that linking the variable region (scFv) of an HIV-1 specific single chain antibody or the native CD4 molecule to the intracellular T cell activation region of the CD8+ T lymphocyte receptor generates HIV-1 specific single-or dual-target CAR-T cells that can kill cells expressing the HIV-1 envelope protein. However, CAR-T cells are excessively or uncontrollable in the body during transfusion to cause certain toxic and side effects, which may cause safety problems in HIV-1 therapeutic application, and besides, CAR-T has strong individuation characteristics, so that the industrialization of CAR-T cells also becomes a bottleneck in HIV-1 therapeutic application.
Therefore, the development of a molecular switch, an anti-HIV-1 chimeric antigen receptor with the molecular switch, a preparation method, NK cells and application not only have urgent research values, but also have good economic benefits and industrial application potentials, which is the basis and the motivation for the completion of the invention.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a molecular switch, an anti-HIV-1 chimeric antigen receptor with the molecular switch, a preparation method, NK cells and application, so as to improve the safety of the chimeric antigen receptor in killing a gp120 cell line.
In a first aspect, the present invention provides a molecular switch, designated RQR8, having a nucleic acid sequence as set forth in SEQ ID No. 1.
In a second aspect, the invention provides an anti-HIV-1 chimeric antigen receptor with the molecular switch, wherein the nucleic acid sequence of the anti-HIV-1 chimeric antigen receptor necessarily comprises a nucleic acid sequence of the molecular switch RQR 8.
In the present invention, as a preferred embodiment, the anti-HIV-1 chimeric antigen receptor comprises a CD4 antigen nucleic acid artificial sequence, a Linker nucleic acid artificial sequence, a 17B single-chain antibody nucleic acid artificial sequence, a CD8 α hinge region nucleic acid artificial sequence, a NKG2D transmembrane region nucleic acid artificial sequence, a 2B4 costimulatory region nucleic acid artificial sequence, a CD3 zeta signaling region nucleic acid artificial sequence, a T2A self-splicing region nucleic acid artificial sequence, and a RQR8 molecular switch region nucleic acid artificial sequence.
In the present invention, as a preferred embodiment, the anti-HIV-1 chimeric antigen receptor comprises sequentially linked
The CD4 antigen nucleic acid artificial sequence as shown in SEQ ID NO. 7;
the Linker nucleic acid artificial sequence as shown in SEQ ID NO. 8;
the 17b single-chain antibody nucleic acid artificial sequence as shown in SEQ ID NO. 9;
a CD8 a hinge region nucleic acid artificial sequence as set forth in SEQ ID No. 10;
the NKG2D transmembrane region nucleic acid artificial sequence as shown in SEQ ID NO. 11;
2B4 costimulatory region nucleic acid artificial sequence as set forth in SEQ ID No. 12;
the CD3 zeta signaling region nucleic acid artificial sequence as set forth in SEQ ID No. 13;
the T2A self-splicing region nucleic acid artificial sequence as shown in SEQ ID NO. 14;
the RQR8 molecular switch region nucleic acid artificial sequence as shown in SEQ ID NO. 1.
In the invention, as a preferable technical scheme, the nucleic acid sequence of the anti-HIV-1 chimeric antigen receptor is shown in SEQ ID NO. 6.
In a third aspect, the present invention provides a method for preparing an anti-HIV-1 chimeric antigen receptor, comprising the steps of:
(1) synthesizing the whole expression frame according to a CD4 antigen nucleic acid artificial sequence, a Linker nucleic acid artificial sequence, a 17B single-chain antibody nucleic acid artificial sequence, a CD8 alpha hinge region nucleic acid artificial sequence, a NKG2D transmembrane region nucleic acid artificial sequence, a 2B4 costimulatory region nucleic acid artificial sequence, a CD3 zeta signaling region nucleic acid artificial sequence, a T2A self-cutting region nucleic acid artificial sequence and a RQR8 molecular switch region nucleic acid artificial sequence, and inserting the sequences into a standard vector pUC to obtain pUC-CD4-17 bscFv-biscar;
(2) performing double enzyme digestion on pUC-CD4-17 bscFv-biscar, cutting off the agar part of the CD4-17 bscFv-biscar DNA fragment by using agar electrophoresis, treating by using a DNA extraction kit sol solution, passing through a DF column, discarding filtrate, rinsing the DF column, performing air separation, eluting the DF column, and collecting a centrifuge to obtain a purified CD4-17 bscFv-biscar DNA fragment, namely the chimeric antigen receptor.
In a fourth aspect, the present invention provides an NK cell containing the anti-HIV-1 chimeric antigen receptor as described above.
In the present invention, as a preferred embodiment, the NK cells are prepared by a method comprising the steps of:
the pLent-CD4-17bscFv-bicAR plasmid was first lentivirally packaged and then immune cells were infected with recombinant lentiviruses.
In the present invention, as a preferred embodiment, the plext-CD 4-17 bsfv-biCAR plasmid is a plex-CD 4-17 bsfv-biCAR plasmid obtained by inserting a fusion gene fragment CD4-17 bsfv-biCAR DNA fragment into a lentiviral expression vector plex-C-GFP.
In the invention, as a preferable technical scheme, the pLent-CD4-17bscFv-BICAR plasmid is obtained by a preparation method comprising the following steps: ligating the purified CD4-17 bscFv-biscar DNA fragment and the linearized pLent-C-GFP DNA fragment overnight at 16 ℃ to form a pLent-CD4-17 bscFv-biscar plasmid; the connecting system is as follows: 10 × buffer: 1 mul; t4 ligase: 1 mul; CD4-17 bsfv-biCAR DNA: 4 mu l of the solution; linearized pLent-C-GFP DNA: 4 μ l.
In a fifth aspect, the invention provides the use of an anti-HIV-1 chimeric antigen receptor in the manufacture of a medicament for the treatment of HIV-1.
The pharmaceutical form includes, but is not limited to, a kit.
In the present invention, as a preferred embodiment, the kit comprises
(1) Obtaining a vector stably expressing CD4-17 bsfv-CAR as described above;
(2) a carrier diluent.
Due to the adoption of the technical scheme, the invention has the following beneficial effects:
the invention reserves double targets, makes the double targets simulate the binding region of HIV-1gp120 tripolymer, enhances the affinity of the double targets, and reduces the risk of virus entering CAR-NK cells.
The invention adopts NK cells, Natural Killer (NK) cells as an indispensable component in the Natural immune system of an organism, is the first Natural defense line of the organism for resisting tumor and virus infection cells, and is also a bridge for connecting Natural immunity and acquired immunity. NK cells can perform a killing function by binding activating receptors and inhibitory receptors on the surface to target cells, and regulate the killing effect by secreting granzyme, perforin, TNF and Fas-L, TRAIL expressed by the TNF, and the like, independently of specific antigens presented by MHC. Compared with T cells, the NK cells modified by the CD4-17bscFv-bicAR genes cannot have serious toxic and side effects such as cell storm, neurotoxicity and the like caused by excessive activity or uncontrolled activity, and the safety is high. And the CD4-17 bscFv-biscar gene-modified NK cells of the invention have low graft versus host response (GVHD), which means that the CD4-17 bscFv-biscar gene-modified NK cells of the invention can play a role in many patients. Thus, the invention overcomes the difficult problem of CAR-T technology that can not be industrialized.
The CD4-17bscFv-bicAR transmembrane region and the co-stimulation region adopt an NK cell activation signal NKG2D transmembrane region and an intracellular signal region of 2B4, and compared with CD4-17bscFv-CAR, the proliferation and the survival rate of NK cells are improved, so that the killing effect on a gp 120-expressing cell line is improved.
The CAR structure of the CD4-17bscFv-BICAR prevents and treats the adverse reaction of CD4-17bscFv-BICAR-NK by adding a molecular switch RQR8, and both rituximab and QBEND10 mab can directly induce CD4-17bscFv-BICAR-NK cell apoptosis through ADCC and CDC of antibodies. Compared with the traditional suicide gene, the molecular weight of RQR8 is small, which is beneficial to the expression of transfected NK cells. Secondly, the apoptosis can be efficiently and rapidly induced, and the apoptosis rate of the rituximab can reach 99% when the rituximab acts for 15min (the HSV-TK suicide gene system can achieve the effect only after lasting for 3 days under the action of ganciclovir). Of course, this molecular switch RQR8 can be applied to a variety of CAR structures to play its role, for example, the CAR structure of Anti-EGFRvIII CAR-NK cells.
In conclusion, the molecular switch RQR8 is added into the chimeric antigen receptor provided by the invention to prevent and treat the adverse reaction of CD4-17bscFv-bicAR-NK, so that the safety is improved.
Drawings
FIG. 1 shows the design of the chimeric antigen receptor CD4-17bscFv-CD8 alpha-NKG 2D-2B4-CD3 zeta-T2A-RQR 8.
FIG. 2 shows that the CAR-expressing efficiency of the CD4-17 bscFv-biscar-NK cells of the invention was 51.8%.
FIG. 3 is a graph showing the results of experiments on the killing effect of CD4-17bscFv-bicAR-NK cells of the present invention. The killing efficiency of CD4-17 bscFv-biscar-NK on cell strain Jurkat expressing HIV-1NL4-3 envelope glycoprotein is 94.42 +/-8.97 percent, which is obviously higher than that of CD4-17bscFv-CAR-NK and NK cells and is consistent with that of CD4-17 bscFv-CAR-T.
FIG. 4 shows the cytokine secretion of CD4-17bscFv-bicAR-NK cells of the present invention. The comparative trend of IFN- γ cytokine content in CD4-17 bscFv-biscar-NK, CD4-17bscFv-CAR-NK and CD4-17bscFv-CAR-T is consistent with the killing results, while the ability of CD4-17 bscFv-biscar-NK to secrete IL6 and INF- α is lower than that of CD4-17bscFv-CAR-NK, and more significantly lower than that of CD4-17 bscFv-CAR-T.
Detailed Description
The technical solution of the present invention will be described in detail with reference to specific examples. The following examples are only for illustrating the technical solutions of the present invention more clearly, and therefore are only examples, and the protection scope of the present invention is not limited thereby.
Example 1
A molecular switch, named RQR8, having a nucleic acid sequence as set forth in SEQ ID No. 1. The RQR8 is formed by connecting a signal peptide nucleic acid sequence (SEQ ID NO.2), a mimic epitope nucleic acid sequence (SEQ ID NO.3) of CD20, a mimic epitope nucleic acid sequence (SEQ ID NO.4) of CD34, a mimic epitope nucleic acid sequence (SEQ ID NO.3) of CD20 and a nucleic acid sequence (SEQ ID NO.5) of a CD8 hinge transmembrane region in series in sequence, and the complete nucleic acid sequence is shown in SEQ ID NO. 1. The nucleotide sequence was synthesized by the firm of Venezetian Bioengineering (Shanghai) Co.Ltd.
Example 2
The anti-HIV-1 chimeric antigen receptor must have a nucleic acid sequence comprising a molecular switch RQR8 nucleic acid sequence. In this embodiment, the anti-HIV-1 chimeric antigen receptor comprises a sequentially linked artificial sequence of the CD4 antigen nucleic acid as set forth in SEQ ID No. 7; the Linker nucleic acid artificial sequence as shown in SEQ ID NO. 8; the 17b single-chain antibody nucleic acid artificial sequence as shown in SEQ ID NO. 9; a CD8 a hinge region nucleic acid artificial sequence as set forth in SEQ ID No. 10; the NKG2D transmembrane region nucleic acid artificial sequence as shown in SEQ ID NO. 11; 2B4 costimulatory region nucleic acid artificial sequence as set forth in SEQ ID No. 12; the CD3 zeta signaling region nucleic acid artificial sequence as set forth in SEQ ID No. 13; the T2A self-splicing region nucleic acid artificial sequence as shown in SEQ ID NO. 14; the RQR8 molecular switch region nucleic acid artificial sequence as shown in SEQ ID NO. 1.
Namely: as shown in figure 1, the nucleic acid sequence of the anti-HIV-1 chimeric antigen receptor is shown in SEQ ID NO. 6.
Example 3
A method for preparing a chimeric antigen receptor comprising the steps of: (1) synthesizing the whole expression frame according to a CD4 antigen nucleic acid artificial sequence, a Linker nucleic acid artificial sequence, a 17B single-chain antibody nucleic acid artificial sequence, a CD8 alpha hinge region nucleic acid artificial sequence, a NKG2D transmembrane region nucleic acid artificial sequence, a 2B4 costimulatory region nucleic acid artificial sequence, a CD3 zeta signaling region nucleic acid artificial sequence, a T2A self-cutting region nucleic acid artificial sequence and a RQR8 molecular switch region nucleic acid artificial sequence, and inserting the sequences into a standard vector pUC to obtain pUC-CD4-17 bscFv-biscar; (2) performing double enzyme digestion on pUC-CD4-17 bscFv-biscar, cutting off the agar part of the CD4-17 bscFv-biscar DNA fragment by using agar electrophoresis, treating by using a DNA extraction kit sol solution, passing through a DF column, discarding filtrate, rinsing the DF column, performing air separation, eluting the DF column, and collecting a centrifuge to obtain a purified CD4-17 bscFv-biscar DNA fragment, namely the chimeric antigen receptor.
In this example, the chimeric antigen receptor was prepared as follows:
the whole expression cassette was synthesized by committing Biotechnology (Shanghai) limited and inserted into a standard vector pUC according to the nucleic acid artificial sequence of CD4 antigen, the nucleic acid artificial sequence of Linker, the nucleic acid artificial sequence of 17B single-chain antibody, the nucleic acid artificial sequence of CD8 alpha hinge region, the nucleic acid artificial sequence of NKG2D transmembrane region, the nucleic acid artificial sequence of 2B4 costimulatory region, the nucleic acid artificial sequence of CD3 zeta signaling region, the nucleic acid artificial sequence of T2A self-cleavage region and the nucleic acid artificial sequence of RQR8 molecular switch region, respectively, and thus named as pUC-CD4-17 bscFv-bicaCAR, and pUC-CD4-17 bscFv-bicaCAR was subjected to a Fast Digest AsiSI (available from ThermoFisher) and a Fast Digest NotI (available from ThermoFisher) double enzyme digestion at 37 ℃ for 20 min. The 100. mu.l enzyme system is: 10 × buffer: 10 mu l of the mixture; 6 mu g of DNA; AsiSI enzyme: 3 mu l of the solution; NotI enzyme: 3 mu l of the solution; deionized water to make up the volume. The agar sites containing the CD4-17 bscFv-biscar DNA fragments were excised using agar electrophoresis and placed in two centrifuge tubes. The DNA was dissolved from the agar using a DNA extraction kit (available from ThermoFisher Co.) and concentrated by first adding 500. mu.l DF buffer to the centrifuge tube and allowing to act at 55 ℃ for 10 minutes, shaking every 2-3 minutes until the agar was completely dissolved. The agar solution was then aspirated into the DF Column and covered with the Collection Tube (Collection of filtrate). Centrifuge at 8000rpm for 1 minute and pour off the filtrate. Then 500. mu.l of Wash Buffer was added and centrifuged at 8000rpm for 1 minute, and the filtrate was decanted off. Centrifugation at 12000rpm for 2 minutes ensured that ethanol was removed. And finally transferring the DF Column to another clean micro-centrifuge tube, adding 25 mu l of Elution Buffer, standing for 2 minutes at room temperature, and centrifuging for 2 minutes at 14000rpm, wherein the liquid in the micro-centrifuge tube is the purified CD4-17bscFv-bicAR DNA fragment.
Example 4
preparation of pLent-CD4-17bscFv-bicAR plasmid.
The whole expression cassette was synthesized by the CD4 antigen nucleic acid artificial sequence, Linker nucleic acid artificial sequence, 17B single-chain antibody nucleic acid artificial sequence, CD8 alpha hinge region nucleic acid artificial sequence, NKG2D transmembrane region nucleic acid artificial sequence, 2B4 costimulatory region nucleic acid artificial sequence, CD3 zeta signaling region nucleic acid artificial sequence, T2A self-cleavage region nucleic acid artificial sequence and RQR8 molecular switch region nucleic acid artificial sequence committing the Bioengineering (Shanghai) Limited company and inserted into the standard vector pUC, thus named pUC-CD4-17 bscFv-bicaCAR, while the pUC-CD4-17 bscFv-bicaCAR and pLent-C-GFP vectors were subjected to Fast Digest AsiSI (from ThermoFisher) and Fast Digest I (from ThermoFisher) double digestion at 37 ℃ for 20min, respectively. The 100. mu.l enzyme system is: 10 × buffer: 10 mu l of the mixture; 6 mu g of DNA; AsiSI enzyme: 3 mu l of the solution; NotI enzyme: 3 mu l of the solution; deionized water to make up the volume. The agar sites containing the CD4-17 bscFv-biscar DNA fragment and the linearized pLent-C-GFP DNA fragment were excised by agarose electrophoresis and placed in two centrifuge tubes. The DNA was dissolved from the agar using a DNA extraction kit (available from ThermoFisher Co.) and concentrated by first adding 500. mu.l DF buffer to the centrifuge tube and allowing to act at 55 ℃ for 10 minutes, shaking every 2-3 minutes until the agar was completely dissolved. The agar solution was then aspirated into the DF Column and covered with the Collection Tube (Collection of filtrate). Centrifuge at 8000rpm for 1 minute and pour off the filtrate. Then 500. mu.l of Wash Buffer was added and centrifuged at 8000rpm for 1 minute, and the filtrate was decanted off. Centrifugation at 12000rpm for 2 minutes ensured that ethanol was removed. And finally transferring the DF Column to another clean micro-centrifuge tube, adding 25 mu l of Elution Buffer, standing for 2 minutes at room temperature, centrifuging for 2 minutes at 14000rpm, wherein the liquid in the micro-centrifuge tube is the purified CD4-17 bscFv-biscAR DNA fragment and the linearized pLent-C-GFP DNA fragment.
The two DNA fragments were ligated overnight at 16 ℃ to form the pLent-CD4-17bscFv-bicAR plasmid. The connecting system is as follows: 10 × buffer: 1 mul; t4 ligase: 1 mul; CD4-17 bsfv-biCAR DNA: 4 mu l of the solution; linearized pLent-C-GFP DNA: 4 μ l.
Coli (DH 5. alpha.) was transformed with pLent-CD4-17bscFv-bicAR as described above. The method comprises the following specific steps: the plasmid and the competent cells are evenly mixed and incubated on ice for half an hour, then heat shock is carried out at 42 ℃ for 90 seconds, then the mixture is placed on ice for 2min, finally liquid LB culture medium is added and slowly shaken for about 1 hour, then centrifugation is carried out at 3000rpm for 5min, and 100 mul of bacterial liquid is coated on a solid plate containing ampicillin LB.
Example 5
And (5) purifying the plasmid.
The next day, a single colony was picked for overnight culture, and the pLent-CD4-17bscFv-BICAR plasmid was extracted using a plasmid extraction and purification kit (purchased from Qiagen), comprising the following specific steps: (1) 1.5ml of the bacterial solution was centrifuged at room temperature at 10000 Xg for 1 min. (2) The supernatant was removed, 250. mu.l of solution I (containing RNase A) was added, and the cells were shaken by a vortex shaker until they were completely suspended. (3) Add 250. mu.l of solution II and gently invert the tube 4-6 times to obtain a clear lysate. Preferably, the incubation is carried out at room temperature for 2 min. (4) Add 350. mu.l of solution III, mix gently by inversion several times until white flocculent precipitate appears, centrifuge at room temperature 10000 Xg for 10 min. (5) The supernatant was aspirated with special care and transferred to a clean adsorption column equipped with 2ml centrifuge tubes. It is ensured that there are no aspiration deposits and cell debris. Centrifugation was carried out at room temperature at 10000 Xg for 1min until the lysate was completely passed through the column. (6) The filtrate was discarded, 500. mu.l Buffer HBC was added, 10000 Xg was centrifuged for 1min, and the column was washed to remove residual protein to ensure the purity of DNA. (7) The filtrate was discarded, and the column was washed with 750. mu.l of Wash Buffer diluted with 100% ethanol and centrifuged at 10000 Xg for 1 min. (8) The column was washed with 750. mu.l of Wash Buffer. (9) The column must be centrifuged at 10000 Xg for 2min to ensure that the ethanol is removed. (10) The column was placed into a clean 1.5ml centrifuge tube, 100. mu.l of sterile deionized water or TE buffer was added to the filter, 10000 Xg was centrifuged for 5min, and plasmid DNA was collected. (11) Agarose gel electrophoresis was performed with a DNA sample (Marker) of a predetermined concentration, and the results were compared to obtain pLent-CD4-17 bscFv-biscAR plasmid at a concentration of 539 ng/. mu.l.
The pLent-CD4-17bscFv-bicAR plasmid described above was sequenced by committee Biotechnology engineering (Shanghai) Co. Sequencing is carried out correctly for later use.
Example 6
The invention provides NK cells containing the anti-HIV-1 chimeric antigen receptor. The preparation method comprises the following steps: the pLent-CD4-17bscFv-bicAR plasmid was first lentivirally packaged and then immune cells were infected with recombinant lentiviruses.
Lentivirus packaging and titer detection
A Lentiviral Packaging Kit is adopted, and the specific method comprises the following steps: the lentivirus packaging cell line 293T is inoculated in a 10cm culture dish containing DMEM and 10% FBS, cultured at 37 ℃ under the condition of 5% CO2, and prepared for transfection when the anchorage rate is 70% -80%. A sterile 1.5ml EP tube or 15ml centrifuge tube was used to prepare the reaction system as follows: serum-free DMEM: 4 ml; pLent-CD4-17bscFv-bicAR plasmid: 10 mu g of the mixture; GM easy (TM) Lentiviral Mix: 10 μ l (10 μ g); HG Transgene Reagent: 60 μ l. After mixing, the mixture was left at room temperature for 20min, and then dropped into a dish containing 293T cells, and then cultured in a CO2 incubator. After transfection 24, the cell culture solution was carefully aspirated off and discarded in a waste liquid cup containing a disinfectant solution, and then 15ml of fresh culture medium containing 10% serum was added to continue the culture. After 48h of liquid change, the cell supernatant was aspirated into a 50ml centrifuge tube, centrifuged at 500g for 5min at 4 ℃, filtered through a 0.45 μm filter and transferred to a new centrifuge tube. The virus particles in the supernatant can then be directly assayed for titer. The titer of 100. mu.l of the virus solution was determined using a lentiviral vector (HIV P24) rapid test card, and the titer of the recombinant lentivirus was 1.25X 107 TU/ml.
Preparation of NK cells
1) Extraction of plasma
Taking 50ml of cord blood, evenly distributing into 250 ml centrifuge tubes, centrifuging for 15min at room temperature at 650g, taking the upper layer of faint yellow plasma into a new 50ml centrifuge tube (the lower layer of red liquid is used for extracting mononuclear cells), inactivating in a 56 ℃ water bath for 30min, centrifuging for 10min at 900g, taking the supernatant, placing at-20 ℃ for 15min, centrifuging again, taking 900g, 10min, taking the supernatant, and placing at 4 ℃ for storage. (centrifuge adjustment speed 1, speed 1)
2) Isolation of monocytes
A. And (3) diluting the lower layer red liquid obtained in the last step of plasma extraction with physiological saline in equal volume, wherein the total volume is about 20ml, and uniformly mixing the lower layer red liquid and the physiological saline in a reversed way for later use. B. Another 50ml centrifuge tube was prepared by adding 20ml of lymphocyte separation medium to each tube. C. Carefully add 20ml of diluted blood to the centrifuge tube containing 20ml of lymphocyte separation medium to form a distinct layer between the blood and lymphocyte separation medium, take care not to mix the diluted blood into the lymphocyte separation medium, and centrifuge at 650g for 30min at room temperature. C. Gently aspirate the monocyte (buffy coat) and its lower half of the lymphocyte isolate and transfer to a new 50ml centrifuge tube; an equal volume of physiological saline was added and centrifuged at 650g for 10min at room temperature. The supernatant was discarded. And repeating the washing step, resuspending the cells by using normal saline, simultaneously taking a small amount of cell suspension trypan blue for staining and counting, centrifuging for 10min at 260g after counting, and discarding the supernatant for later use.
3) Seeding and induced activation of monocytes
NK cell infected by lentivirus and amplification culture of infected NK cell
NK cells were infected with the recombinant lentivirus described above at MOI ═ 5. Infected cells at 37 ℃ with 5% CO2After 12 hours of incubation in the incubator, the cell supernatants were collected and discarded, and equal amounts of virus solution, Polybrene (8. mu.g/ml) and cell culture medium were added again at 37 ℃ with 5% CO2Continuously culturing in the incubator, sucking and removing culture supernatant after 12h, adding fresh CORNING culture medium again, continuously performing amplification culture, and culturing for 17 days until the cells are amplified to a sufficient amount. Chimeric antigen receptor expression was detected by FC500 flow cytometer (purchased from BECKMAN corporation) FL1 channel (fig. 2). The positive rate of the recombinant lentivirus infected NK cells is 51.8 percent by taking uninfected NK lymphocytes as negative control.
Example 7
Analysis of control effectiveness of molecular switch on CD4-17bscFv-bicAR-NK cells
CD4-17bscFv-bicAR-NK cells at a density of 1X 105Each/ml was inoculated into 96-well plates, 100ul per well, in 5% CO2Culturing in an incubator at 37 ℃ for 24 hours; adding 10nM rituximab (product of Roche), adding 20. mu.L CCK-8 (product of MCE) into each well after 15min, incubating for 2h, detecting with microplate reader, and reading OD value at 450nM wavelength. CD4-17bscFv-bicAR-NK cell control group without rituximab and blank control group without rituximab only added were set.
The mortality rate of NK cells ═ 100% x [1- (plus rituximab group OD value-blank OD value)/(not plus rituximab group OD value-blank OD value) ]. The death rate of NK cells is 51.36%, and the control rate of rituximab on the activity of CAR-NK cells is 99.15%; the result shows that the activity of the CD4-17bscFv-BICAR-NK cell designed by the invention is controlled by rituximab, and the rituximab can induce over 99 percent of apoptosis within 15min, thereby greatly improving the clinical safety of CAR technology.
Example 8
Effect of killing activity of CD4-17 bsfv-biCAR-NK cells.
The cell line Jurkat expressing HIV-1NL4-3 envelope glycoprotein is used as a target cell, and the effector cells are CD4-17bscFv-bicAR-NK, CD4-17bscFv-CAR-NK, CD4-17bscFv-CAR-T and NK cells respectively.
According to E: t is 1: 1, adding 1 × 106 Jurkat cells, collecting CD4-17 bscFv-biscar-NK, CD4-17bscFv-CAR-NK and NK cell cells after the cells are completely attached to the wall, and respectively adjusting the cell concentration to be 1 × 107Per ml, 100. mu.L of 5% CO at 37 ℃ per well2Culturing under the condition for 12 h. 20. mu.L of diluted CCK8 (from MCE) was added to the supernatant, incubated for 4-6 hours, and the absorbance of OD450 was measured by a microplate reader. Killing rate ═ 1- (effector cell + target cell well OD value-OD value of effector cell alone)/OD value of target cell alone]×100％。
The killing efficiency of CD4-17 bscFv-biscAR-NK on Jurkat cells is 94.42 +/-8.97% which is obviously higher than that of the CD4-17bscFv-CAR-NK and NK cell groups (figure 3), and is consistent with the killing effect of CD4-17 bscFv-CAR-T.
Example 9
CD4-17 bscFv-biscar-NK, CD4-17bscFv-CAR-NK and CD4-17bscFv-CAR-T cell cytokine secretion capacity
CD4-17 bsfc-biCAR-NK, CD4-17 bsfc-CAR-NK and CD4-17 bsfc-CAR-T cells were differentiated as follows E: t (effector cell to target cell ratio) is 1: 1 was cultured in admixture with a cell line Jurkat expressing the HIV-1NL4-3 envelope glycoprotein for 20 hours, and cytokine secretion by CD4-17 bscFv-biscar-NK, CD4-17bscFv-CAR-NK and CD4-17bscFv-CAR-T cells were examined by ELISA assay, respectively.
The secretion of the cytokine IFN-. gamma.was measured using a Human IFN-gamma Quantikine ELISA Kit (purchased from R & D Systems). Cytokine IL-6 secretion was determined using an IL-6Human ELISA Kit (available from Thermo Fisher). The secretion of the cytokine TNF-. alpha.was measured using a Human TNF-alpha Quantikine ELISA Kit (purchased from R & D Systems). The specific operation steps are shown in the specification.
As can be seen from FIG. 4, the comparative trend of IFN- γ cytokine content in CD4-17 bscFv-biscar-NK, CD4-17bscFv-CAR-NK and CD4-17bscFv-CAR-T is consistent with the killing results, while the ability of CD4-17 bscFv-biscar-NK to secrete IL6 and INF- α is lower than that of CD4-17bscFv-CAR-NK, more significantly lower than that of CD4-17bscFv-CAR-T, and does not cause cell storm. Therefore, the effectiveness and safety of the CD4-17 bscFv-bicOR-NK are higher than those of the CD4-17 bscFv-CAR-T. The result shows that the CD4-17bscFv-bicAR-NK molecule is reasonable in design, safe and effective, and lays a foundation for treating HIV-1.
Example 11
Kit for preparing CD4-17bscFv-bicAR-NK cells
(1) Obtaining a vector stably expressing CD4-17 bsfv-CAR as described above;
(2) a carrier diluent;
(3) instructions for use;
wherein the instructions for use comprise the methods of examples 3-5 above.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; such modifications and substitutions do not depart from the spirit and scope of the present invention, and they should be construed as being included in the following claims and description.
Sequence listing
<110> Shandong Xingyi Biotechnology Ltd
<120> molecular switch, anti-HIV-1 chimeric antigen receptor with molecular switch, preparation method, NK cell and application
<130> 2018
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atagtggcac ctggacatgc actgtcttgc agaaccagaa gaaggtggag ttcaaaatag 600
acatcgtggt gctagctttc cagaaggcct ccagcatagg aggaggagga agcggaggag 660
gaggaagcgg aggaggagga agcggtggtg gtggtagtgg tggtggtggt agtggtggtg 720
gtggtagtgg tggtggtggt agtggtggtg gtggtagtat ggccctgcct gtgacagccc 780
tgctgctgcc tctggctctg ctgctgcatg ccgctagacc ccaagtgcag ctgcttgagt 840
ctggggctga ggtgaagaag cctgggtcct cggtgaaggt ctcctgcaag gcctctggag 900
acaccttcat cagatatagt tttacctggg tgcgacaggc ccctggacaa ggccttgagt 960
ggatgggaag gatcatcact atccttgatg tagcacacta cgcaccgcac ctccagggca 1020
gagtcacgat taccgcggac aagtccacga gcacagtcta cctggagctg cggaatctaa 1080
gatctgacga tacggccgta tatttctgtg cgggagtgta cgagggagag gcggacgagg 1140
gggaatatga taataatggg tttctgaaac attggggcca gggaaccctg gtcaccgtca 1200
cctcaggagg aggaggaagc ggaggaggag gaagcggagg aggaggaagc gaacttgaac 1260
tgacgcagtc tccagccacc ctgtctgtgt ctccagggga aagagccacc ctctcctgca 1320
gggccagtga aagtgttagc agcgacttag cctggtacca gcagaaacct ggccaggctc 1380
ccaggctcct catctatggt gcatccacca gggccactgg tgttccagcc aggttcagtg 1440
gcagtgggtc tggggcagag ttcactctca ccatcagcag cctgcagtct gaagattttg 1500
cagtttatta ctgtcagcag tataataact ggcctccgag atatacgttc ggccaaggga 1560
ccagactgga aatcaaaacg cgtaccacga cgccagcgcc gcgaccacca acaccggcgc 1620
ccaccatcgc gtcgcagccc ctgtccctgc gcccagaggc gtgccggcca gcggcggggg 1680
gcgcagtgca cacgaggggg ctggacttcg cctgtgatcc attttttttc tgctgcttca 1740
tcgctgtagc catgggaatc cgtttcatta ttatggtagc atggaggaga aagaggaagg 1800
agaagcagtc agagaccagt cccaaggaat ttttgacaat ttacgaagat gtcaaggatc 1860
tgaaaaccag gagaaatcac gagcaggagc agacttttcc tggagggggg agcaccatct 1920
actctatgat ccagtcccag tcttctgctc ccacgtcaca agaacctgca tatacattat 1980
attcattaat tcagccttcc aggaagtctg gatccaggaa gaggaaccac agcccttcct 2040
tcaatagcac tatctatgaa gtgattggaa agagtcaacc taaagcccag aaccctgctc 2100
gattgagccg caaagagctg gagaactttg atgtttattc cagagtgaag ttcagcagga 2160
gcgcagacgc ccccgcgtac aagcagggcc agaaccagct ctataacgag ctcaatctag 2220
gacgaagaga ggagtacgat gttttggaca agagacgtgg ccgggaccct gagatggggg 2280
gaaagccgag aaggaagaac cctcaggaag gcctgtacaa tgaactgcag aaagataaga 2340
tggcggaggc ctacagtgag attgggatga aaggcgagcg ccggaggggc aaggggcacg 2400
atggccttta ccagggtctc agtacagcca ccaaggacac ctacgacgcc cttcacatgc 2460
aggccctgcc ccctcgcgaa ggccgaggga gcctgctgac atgtggcgat gtggaggaaa 2520
acccaggacc aatgggcacc agcctcctct gctggatggc cctgtgtctc ctgggggcag 2580
atcacgcaga tgcttgtcct tactctaacc cctctctctg ttctggaggt ggaggatctg 2640
agttacctac ccagggaaca ttttcaaatg tttctacaaa tgtatcccct gctaagccta 2700
caacaactgc atgtccttac tctaacccct ctctctgttc tggaggtgga ggatctcctg 2760
ctcctcgtcc tcctacccct gctcctacta tcgcgtcgca gcccctgtcc ctgcgcccag 2820
aggcgtgccg gccagcggcg ggtggcgcag tgcacacgag gggtctggac ttcgcctgtg 2880
atatctacat ctgggcgccc ttggccggga cttgtggggt ccttctcctg tcactggtta 2940
tcacccttta ctgcaaccac agaaatagga gaagagtttg caagtgtcct agacctgttg 3000
tttaggcggc cgc 3013
<210> 7
<211> 630
<212> DNA
<213> ethnic species (Homo sapiens)
<400> 7
atgaaccggg gagtcccttt taggcacttg cttctggtgc tgcaactggc gctcctccca 60
gcagccactc agggaaagaa agtggtgctg ggcaaaaaag gggatacagt ggaactgacc 120
tgtacagctt cccagaagaa gagcatacaa ttccactgga aaaactccaa ccagataaag 180
attctgggaa atcagggctc cttcttaact aaaggtccat ccaagctgaa tgatcgcgct 240
gactcaagaa gaagcctttg ggaccaagga aactttcccc tgatcatcaa gaatcttaag 300
atagaagact cagatactta catctgtgaa gtggaggacc agaaggagga ggtgcaattg 360
ctagtgttcg gattgactgc caactctgac acccacctgc ttcaggggca gagcctgacc 420
ctgaccttgg agagcccccc tggtagtagc ccctcagtgc aatgtaggag tccaaggggt 480
aaaaacatac agggggggaa gaccctctcc gtgtctcagc tggagctcca ggatagtggc 540
acctggacat gcactgtctt gcagaaccag aagaaggtgg agttcaaaat agacatcgtg 600
gtgctagctt tccagaaggc ctccagcata 630
<210> 8
<211> 756
<212> DNA
<213> ethnic species (Homo sapiens)
<400> 8
ggaggaggag gaagcggagg aggaggaagc ggaggaggag gaagcggtgg tggtggtagt 60
ggtggtggtg gtagtggtgg tggtggtagt ggtggtggtg gtagtggtgg tggtggtagt 120
<210> 9
<211> 756
<212> DNA
<213> ethnic species (Homo sapiens)
<400> 9
caagtgcagc tgcttgagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc 60
tcctgcaagg cctctggaga caccttcatc agatatagtt ttacctgggt gcgacaggcc 120
cctggacaag gccttgagtg gatgggaagg atcatcacta tccttgatgt agcacactac 180
gcaccgcacc tccagggcag agtcacgatt accgcggaca agtccacgag cacagtctac 240
ctggagctgc ggaatctaag atctgacgat acggccgtat atttctgtgc gggagtgtac 300
gagggagagg cggacgaggg ggaatatgat aataatgggt ttctgaaaca ttggggccag 360
ggaaccctgg tcaccgtcac ctcaggagga ggaggaagcg gaggaggagg aagcggagga 420
ggaggaagcg aacttgaact gacgcagtct ccagccaccc tgtctgtgtc tccaggggaa 480
agagccaccc tctcctgcag ggccagtgaa agtgttagca gcgacttagc ctggtaccag 540
cagaaacctg gccaggctcc caggctcctc atctatggtg catccaccag ggccactggt 600
gttccagcca ggttcagtgg cagtgggtct ggggcagagt tcactctcac catcagcagc 660
ctgcagtctg aagattttgc agtttattac tgtcagcagt ataataactg gcctccgaga 720
tatacgttcg gccaagggac cagactggaa atcaaa 756
<210> 10
<211> 135
<212> DNA
<213> ethnic species (Homo sapiens)
<400> 10
accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60
tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120
gacttcgcct gtgat 135
<210> 11
<211> 63
<212> DNA
<213> ethnic species (Homo sapiens)
<400> 11
ccattttttt tctgctgctt catcgctgta gccatgggaa tccgtttcat tattatggta 60
gca 63
<210> 12
<211> 360
<212> DNA
<213> ethnic species (Homo sapiens)
<400> 12
tggaggagaa agaggaagga gaagcagtca gagaccagtc ccaaggaatt tttgacaatt 60
tacgaagatg tcaaggatct gaaaaccagg agaaatcacg agcaggagca gacttttcct 120
ggagggggga gcaccatcta ctctatgatc cagtcccagt cttctgctcc cacgtcacaa 180
gaacctgcat atacattata ttcattaatt cagccttcca ggaagtctgg atccaggaag 240
aggaaccaca gcccttcctt caatagcact atctatgaag tgattggaaa gagtcaacct 300
aaagcccaga accctgctcg attgagccgc aaagagctgg agaactttga tgtttattcc 360
<210> 13
<211> 336
<212> DNA
<213> ethnic species (Homo sapiens)
<400> 13
agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc 60
tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120
cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180
gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240
cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300
tacgacgccc ttcacatgca ggccctgccc cctcgc 336
<210> 14
<211> 54
<212> DNA
<213> ethnic species (Homo sapiens)
<400> 14
gaaggccgag ggagcctgct gacatgtggc gatgtggagg aaaacccagg acca 54
Claims (8)
1. An anti-HIV-1 chimeric antigen receptor characterized by:
the anti-HIV-1 chimeric antigen receptor is formed by sequentially connecting a CD4 antigen, a 17B single-chain antibody, a CD8 alpha hinge region, an NKG2D transmembrane region, a 2B4 costimulatory region, a CD3 zeta signaling region, a T2A self-cleavage region and an RQR8 molecular switch region; the nucleic acid sequence of the RQR8 molecular switch region is shown in SEQ ID NO. 1; the nucleic acid artificial sequence of the 17b single-chain antibody is shown in SEQ ID NO. 9.
2. The anti-HIV-1 chimeric antigen receptor according to claim 1, wherein: the anti-HIV-1 chimeric antigen receptor comprises sequentially linked
The CD4 antigen nucleic acid artificial sequence as shown in SEQ ID NO. 7;
the Linker nucleic acid artificial sequence as shown in SEQ ID NO. 8;
the 17b single-chain antibody nucleic acid artificial sequence as shown in SEQ ID NO. 9;
a CD8 a hinge region nucleic acid artificial sequence as set forth in SEQ ID No. 10;
the NKG2D transmembrane region nucleic acid artificial sequence as shown in SEQ ID NO. 11;
2B4 costimulatory region nucleic acid artificial sequence as set forth in SEQ ID No. 12;
the CD3 zeta signaling region nucleic acid artificial sequence as set forth in SEQ ID No. 13;
the T2A self-splicing region nucleic acid artificial sequence as shown in SEQ ID NO. 14;
the RQR8 molecular switch region nucleic acid artificial sequence as shown in SEQ ID NO. 1.
3. The anti-HIV-1 chimeric antigen receptor according to claim 2, wherein: the nucleic acid sequence of the anti-HIV-1 chimeric antigen receptor is shown in SEQ ID NO. 6.
4. The method of producing an anti-HIV-1 chimeric antigen receptor according to claim 1, wherein: the method comprises the following steps:
(1) synthesizing the whole expression frame according to a CD4 antigen nucleic acid artificial sequence, a Linker nucleic acid artificial sequence, a 17B single-chain antibody nucleic acid artificial sequence, a CD8 alpha hinge region nucleic acid artificial sequence, a NKG2D transmembrane region nucleic acid artificial sequence, a 2B4 costimulatory region nucleic acid artificial sequence, a CD3 zeta signaling region nucleic acid artificial sequence, a T2A self-cutting region nucleic acid artificial sequence and a RQR8 molecular switch region nucleic acid artificial sequence, and inserting the sequences into a standard vector pUC to obtain pUC-CD4-17 bscFv-biscar;
(2) performing double enzyme digestion on pUC-CD4-17 bscFv-biscar, cutting off the agar part of the CD4-17 bscFv-biscar DNA fragment by using agar electrophoresis, treating by using a DNA extraction kit sol solution, passing through a DF column, discarding filtrate, rinsing the DF column, performing air separation, eluting the DF column, and collecting a centrifuge to obtain a purified CD4-17 bscFv-biscar DNA fragment, namely the chimeric antigen receptor.
NK cells characterized by: the NK cell contains the anti-HIV-1 chimeric antigen receptor of claim 1.
6. The NK cell of claim 5, wherein: the NK cell is prepared by adopting a method comprising the following steps: the pLent-CD4-17bscFv-bicAR plasmid was first lentivirally packaged and then immune cells were infected with recombinant lentiviruses.
7. The NK cell of claim 6, wherein: the pLent-CD4-17bscFv-BICAR plasmid is a pLent-CD4-17bscFv-BICAR plasmid obtained by inserting a fusion gene fragment CD4-17bscFv-BICAR DNA fragment into a lentivirus expression vector pLent-C-GFP.
8. Use of the anti-HIV-1 chimeric antigen receptor of claim 1 in the manufacture of a medicament for the treatment of HIV-1.
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