CN107987173B - Double-target-point chimeric antigen receptor, coding gene thereof, plasmid with gene, immune T effector cell and HIV-1 application - Google Patents
Double-target-point chimeric antigen receptor, coding gene thereof, plasmid with gene, immune T effector cell and HIV-1 application Download PDFInfo
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Abstract
The invention discloses a double-target chimeric antigen receptor, a coding gene thereof, a plasmid with the gene, an expression vector, an immune T-effect cell and application thereof, wherein the chimeric antigen receptor comprises a CD4 antigen region (comprising a Leader region), a Linker region, a 17b single-chain antibody region (17bscFv), a hinge region, a transmembrane domain, a co-stimulation domain and a signal transduction domain. The CD4-17bscFv-CAR mimics the binding region of gp120 and has higher effectiveness and safety than the single-target CD 4-zeta CAR reported earlier.
Description
Technical Field
The invention relates to the technical field of biomedicine, in particular to a double-target chimeric antigen receptor aiming at HIV-1, a coding gene thereof, a plasmid with the gene, an expression vector, an immune T-effector cell and application in the aspect of HIV-1.
Background
Human immunodeficiency virus Type 1 (HIV-1), a Type of retrovirus, invades CD4+ T cells, severely impairs the acquired immune function of the body, and finally causes aids (acquired immunodeficiency syndrome). The disease prevention and control center in China in 8 months in 2017 publishes the epidemic situation of AIDS in China, the number of people infected with HIV-1(human immunodeficiency virus) in China reaches 718270, 299169 people suffer from AIDS, and the number of people dead due to the AIDS reaches 221628. In addition, more than 140000 HIV-1 infected persons/AIDS patients are newly increased every year. These figures indicate that the AIDS infection in our country is very severe.
Currently, the clinical treatment for aids is mainly high-potency antiretroviral therapy (HAART), the use of HAART has resulted in a mortality rate of almost 100% within five years to a survival rate of 91% within five years. Adults infected with HIV 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 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 led to the development of HIV infection into 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 also face the problem of resistance to HIV drugs. This therapy also does not eliminate latent HIV in the viral pool. The expensive cost of treatment also leaves many HIV-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 therapy against HIV-1 is disclosed in both the cytolysis patent CN95192559.8 and cells bearing the CD4 decoy receptor, as well as related molecular and method patent CN95195183.1, directed to HIV-1 infected cells by cells bearing the chimeric CD4 receptor. This therapy shows that genetically engineered T cells can effectively control HIV-1 virus without developing HAART therapy. The action mechanism is mainly that a CD 4-zeta Chimeric Antigen Receptor (CAR) consisting of a CD4 extracellular domain connected with an intracellular CD 3-zeta signaling domain is transfected into a human T cell by a viral vector, and as CD4 is a main receptor of an HIV-1 infected cell, a CD4 extracellular domain of the CD 4-zeta CAR simulates immune synapse by binding with an HIV-1 outer membrane protein gp120 positioned on the surface of the infected cell, so that the CD 4-zeta CAR-T cell is activated, and then the HIV-1 infected cell is cracked, and the HIV-1 viral load in a patient is reduced. However, although CD 4-zeta CAR-T cells can activate T cells to kill bound HIV-1 infected cells after binding to gp120 antigen, activated T cells are difficult to further proliferate and only exert transient effects, and CD 4-zeta CAR-T cells are susceptible to HIV-1 infection and become new hosts. The method has not ideal effect in later clinical tests, and has not been widely applied so far.
Therefore, the development of a novel safe and efficient chimeric antigen receptor for HIV-1 treatment not only has urgent research value, but also has good economic benefit and large-scale medical application potential, which is the motivation and the foundation for the completion of the invention.
Disclosure of Invention
The present inventors have conducted intensive studies to overcome the above-identified drawbacks of the prior art, and as a result, have completed the present invention after having made a great deal of creative efforts.
Specifically, the technical problems to be solved by the present invention are: provides a double-target chimeric antigen receptor, a coding gene thereof, a plasmid with the gene, an expression vector, an immune T effector cell and application in the aspect of HIV-1.
In order to solve the technical problems, the technical scheme of the invention is as follows:
in a first aspect, the present invention provides a dual-target chimeric antigen receptor comprising a CD4 antigen region (comprising a CD4Leader region), a Linker region, a 17b single-chain antibody region (17 bfscfv), a hinge region, a transmembrane domain, a costimulatory domain, and a signaling domain.
Wherein, the CD4 antigen region is 1-182 amino acids of CD 4.
Wherein the Linker region is selected from Linker peptides of different lengths (G4S)2, (G4S)4, (G4S)6, (G4S)8 and (G4S)10, and (G4S)8, a Linker peptide consisting of 40 amino acids is preferred in the present invention.
Wherein G4S consists of Gly Gly Gly Gly Ser 5 amino acids, and the (G4S)2, (G4S)4, (G4S)6, (G4S)8 and (G4S)10 are the repetition times of G4S.
Wherein, the 17b single-chain antibody region is a single-chain antibody 17bscFv aiming at HIV-117 b epitope.
Wherein the hinge region is a CD8 a hinge region.
Wherein the transmembrane domain is a CD8 alpha transmembrane region.
Wherein the costimulatory domain is a functional domain of CD 137.
Wherein the signaling domain comprises a functional domain of CD3 ζ.
In a second aspect, the invention provides a gene encoding the dual-target chimeric antigen receptor, which is CD4-17bscFv-CD8 α -CD137-CD3 ζ (also referred to as CD4-17bscFv-CAR DNA fragment).
The gene is composed of the following nucleic acid artificial sequences in tandem:
(1) CD4 antigen nucleic acid artificial sequence shown in SEQ ID NO. 2;
(2) linker nucleic acid artificial sequence as SEQ ID NO. 3;
(3)17b single-chain antibody nucleic acid artificial sequence shown in SEQ ID NO. 4;
(4) CD8 alpha hinge region nucleic acid artificial sequence as shown in SEQ ID NO. 5;
(5) CD8 alpha transmembrane region nucleic acid artificial sequence as shown in SEQ ID NO. 6;
(6) CD137 costimulatory region nucleic acid artificial sequence as shown in SEQ ID NO. 7;
(7) CD3 zeta signaling region nucleic acid artificial sequence shown in SEQ ID NO. 8.
In the invention, as a preferable technical scheme, the nucleic acid sequence of the gene is shown as SEQ ID NO. 1.
In a third aspect, the present invention provides a recombinant plasmid comprising a gene as described above.
In the present invention, as a preferred embodiment, the recombinant plasmid is a pLent-CD4-17bscFv-CAR plasmid obtained by inserting the CD4-17bscFv-CD8 α -CD137-CD3 ζ gene fragment into a linearized pLent-C-GFP DNA fragment.
In a fourth aspect, the present invention provides an expression vector comprising a gene as described above.
In the present invention, as a preferred embodiment, the expression vector is a lentiviral packaging cell line.
In a fifth aspect, the invention provides an immune T effector cell comprising a gene as described above.
In the present invention, as a preferred embodiment, the immune T effector cell includes a CD8+ T cell.
In a sixth aspect, the invention provides the use in the manufacture of a medicament for the treatment of HIV-1.
In the present invention, as a preferred technical scheme, the form of the medicament comprises a kit.
In the present invention, as a preferred embodiment, the kit comprises:
(1) obtaining an expression vector stably expressing CD4-17 bsfv-CAR as described above;
(2) a carrier diluent.
After the technical scheme is adopted, the invention has the beneficial effects that:
the present invention will develop a dual target CAR, targeting both the CD4 binding site and the 17b epitope (also known as CD 4-inducing epitope), with a single chain antibody 17b in tandem behind the extracellular domain of the earlier reported CD 4-zeta CAR, named CD4-17 bsfv-CAR (see figure 1). CD4 of CD4-17bscFv-CAR binds to the binding site of CD4 on gp120, the surface protein of HIV-1 infected cells, changes the conformation of gp120, exposes the 17b epitope bound by 17b scFv, and after binding, activates T cells to perform effector functions via the intracellular domain of CD 3. Thus, the activation signal of CD4-17bscFv-CAR-T requires two sites of binding. Thus, CD4-17bscFv-CAR can effectively prevent HIV-1 virus from entering CAR-T cells with safety higher than the single-target CD 4-zeta CAR reported earlier. After the invention optimizes the linker, the CD4 and 17b scFv are combined on the same gp 120. Thus, the CD4-17bscFv-CAR of the invention mimics the binding region of gp120, enhancing the affinity of both, and is also more effective than the single-target CD 4-zeta CAR previously reported.
The invention also modifies the rest of the structure of the CAR, so that a novel MHC independent anti-HIV-1 CAR molecule is developed, and the CD4-17bscFv-CAR can further amplify T cell activation signals, and improve the in vivo amplification capacity and cytokine production capacity. Co-culturing CD4-17bscFv-CAR-T cells with a gp 120-expressing cell line can potently kill gp 120-expressing cell lines. And the killing effect is obviously better than that of CD 4-zeta CAR-T cells reported earlier. In more detail, the present invention, which employs the CD8 α hinge region, has been shown in practice to reduce the homology of CD4 and prevent CAR-T from being infected with HIV-1. CAR-T can be further activated by using 4-1BB, which allows substantial expansion of CAR-T and increases lethality to HIV-1 infected cells.
Drawings
FIG. 1 is a schematic design diagram of the chimeric antigen receptor CD4-17bscFv-CD8 alpha-CD 137-CD3 zeta.
FIG. 2 is an electrophoretogram of the CD4-17bscFv-CAR DNA fragment (left) and the linearized pLent-C-GFP DNA fragment (right) of the present invention.
FIG. 3 is a schematic diagram of lentivirus CD4-17bscFv-CD8 alpha-CD 137-CD3 zeta expression plasmid.
FIG. 4 shows that the CD4-17bscFv-CAR-T cells of the invention express CD4-17bscFv-CAR with an efficiency of 72.0% (flow peak on the right, scatter plot on the left).
FIG. 5 is a graph showing the results of experiments on the killing effect of Linker of different lengths on CD4-17bscFv-CAR-T cells. At E: t is 1:1, CD4-linker (G4S)8-17bscFv-CAR-T pairs express HIV-1NL4-3The killing efficiency of the envelope glycoprotein cell line Jurkat is 92.57 +/-7.43 percent and is obviously higher than that of CD 4-zeta CAR-T and unmodified CD8+ T cells
FIG. 6 is an ELIspot assay of the present invention. The left panel shows the IFN-gamma secretion of CD4-17 bscFv-CAR-T; the middle panel shows the IFN-gamma secretion of CD 4-zeta CAR-T; the right panel shows IFN-gamma secretion from unmodified CD8+ T cells; expression of HIV-1NL4-3The secretory capacity of IFN-gamma of CD4-17bscFv-CAR-T cells cultured by the envelope glycoprotein cell strain Jurkat mixed culture is remarkably higher than that of CD 4-zeta CAR-T cells and non-modified CD8+ T cells.
Detailed Description
The invention is further illustrated by the following specific examples. The use and purpose of these exemplary embodiments are to illustrate the present invention, not to limit the actual scope of the present invention in any way, and not to limit the scope of the present invention in any way.
Example 1
A dual-target chimeric antigen receptor to HIV-1 comprising a CD4 antigen region (comprising a CD4Leader region), a Linker region, a 17b single-chain antibody region (17 bfscfv), a hinge region, a transmembrane domain, a costimulatory domain, a signaling domain, and a suicide gene system region.
Wherein the CD4 antigen region is 1-182 amino acids of CD 4; the Linker region is selected from Linker peptides of different lengths (G4S)2, (G4S)4, (G4S)6, (G4S)8 and (G4S)10, with (G4S)8, a Linker peptide consisting of 40 amino acids being preferred in the present invention; the 17b single-chain antibody region is a single-chain antibody 17bscFv aiming at HIV-117 b epitope; the hinge region is a CD8 a hinge region; the transmembrane domain is a CD8 alpha transmembrane region; the costimulatory domain is a functional domain of CD 137; the signaling domain comprises a functional domain of CD3 ζ.
Example 2
The gene for coding the chimeric antigen receptor is CD4-17bscFv-CD8 alpha-CD 137-CD3 zeta (also named CD4-17bscFv-CAR DNA fragment).
Construction of CD4-17bscFv-CD8 alpha-CD 137-CD3 zeta
The fusion gene fragment CD4-17bscFv-CD8 alpha-CD 137-CD3 zeta is inserted into a lentivirus expression vector pLent-C-GFP.
The CD4-17bscFv-CD8 α -CD137-CD3 ζ module is schematically shown in FIG. 1 (see appendix SEQ ID NO.1 for the complete nucleic acid sequence).
Module sequences of CD4-17bscFv-CD8 alpha-CD 137-CD3 zeta
(1) CD4 antigen nucleic acid artificial sequence (SEQ ID NO.2)
(2) Linker nucleic acid artificial sequence (SEQ ID NO.3)
(3)17b Single-chain antibody nucleic acid Artificial sequence (SEQ ID NO.4)
(4) CD8 alpha hinge region nucleic acid artificial sequence (SEQ ID NO.5)
(5) CD8 alpha transmembrane region nucleic acid artificial sequence (SEQ ID NO.6)
(6) CD137 costimulatory region nucleic acid artificial sequence (SEQ ID NO.7)
(7) CD3 zeta signaling region nucleic acid artificial sequence (SEQ ID NO.8)
The whole expression cassette was synthesized by committing to bioengineering (Shanghai) Limited according to 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, CD8 alpha transmembrane region nucleic acid artificial sequence, CD137 co-stimulatory region nucleic acid artificial sequence, CD3 zeta signaling region nucleic acid artificial sequence, and inserted into the standard vector pUC57, thus named pUC-CD4-17bscFv-CAR, and the pUC-CD4-17bscFv-CAR was subjected to Fast Digest AsiSI (purchased from ThermoFisher) and Fast Digest NotI (purchased from ThermoFisher) double enzyme 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 HIV-1-CD4-CAR DNA fragment 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 microcentrifuge tube, adding 25. mu.l of Elution Buffer, standing at room temperature for 2 minutes, and centrifuging at 14000rpm for 2 minutes, wherein the liquid in the microcentrifuge tube is the purified CD4-17bscFv-CAR DNA fragment (shown in figure 2).
Example 3
Recombinant plasmids comprising the genes as described above. The recombinant plasmid is a pLent-CD4-17bscFv-CAR plasmid which is obtained by inserting the CD4-17bscFv-CD8 alpha-CD 137-CD3 zeta gene fragment into a linearized pLent-C-GFP DNA fragment.
The following is a preparation method of the recombinant plasmid:
the whole expression cassette was synthesized by CD4 antigen nucleic acid artificial sequence, Linker nucleic acid artificial sequence, 17b single-chain antibody nucleic acid artificial sequence, CD8 hinge region nucleic acid artificial sequence, CD8 transmembrane region nucleic acid artificial sequence, CD137 costimulatory region nucleic acid artificial sequence, CD3 zeta signaling region nucleic acid artificial sequence, committee for Biotechnology (Shanghai) and inserted into a standard vector pUC57, thus named pUC-CD4-17bscFv-CAR, while pUC-CD4-17bscFv-CAR and pLent-C-GFP vectors were subjected to a double enzyme digestion of Fast Digest AsiSI (available from ThermoFisher) and Fast Digest NotI (available from ThermoFisher) 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 HIV-1-CD4-CAR 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 microcentrifuge 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 microcentrifuge tube is the purified CD4-17bscFv-CAR DNA fragment (shown in figure 2) and the linearized pLent-C-GFP DNA fragment (shown in figure 2).
The two DNA fragments were ligated overnight at 16 ℃ to form the pLent-CD4-17bscFv-CAR (see FIG. 3) plasmid. The connecting system is as follows: 10 × buffer: 1 mul; t4 ligase: 1 mul; CD4-17 bsfv-CAR 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-CAR 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. The next day, a single colony was picked for overnight culture, and the pLent-CD4-17bscFv-CAR 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) Adding 250 mu l of solution II, and gently inverting the centrifuge tube for 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 filtrate was discarded, and 750. mu.l of Wash Buffer was added to Wash the column. (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, 50-100. mu.l (depending on the desired final concentration) sterile deionized water or TE buffer was added to the filter, and the plasmid DNA was collected by centrifugation at 10000 Xg for 5 min. (11) Agarose gel electrophoresis was performed with DNA samples (Marker) of known concentration, and comparison of the results gave a pLent-CD4-17bscFv-CAR plasmid concentration of 376 ng/. mu.l.
The pLent-CD4-17bscFv-CAR plasmid described above was sequenced by committee Biotechnology engineering (Shanghai) Co., Ltd. Sequencing is carried out correctly for later use.
Example 4
An expression vector comprising the gene as described above. The expression vector adopts a lentivirus packaging cell line.
The expression vector was prepared as follows.
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: 3 ml; pLent-HIV-1-CD4-CAR plasmid: 10 mu g of the mixture; GM easy (TM) Lentiviral Mix: 10 μ l (10 μ g); HG Transgene Reagent: 60 μ l. Mixing, standing at room temperature for 20min, adding into 293T cell culture dish, and placing in CO2Culturing in an incubator. After 24h of transfection, 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 above viruses were titrated with TCID50, and 293T cells in logarithmic growth phase were grown at 1X 104The amount of Cells/well was seeded in 96-well cell culture plates, samples were diluted 10-fold in serial concentrations with 5% FBS DMEM, loaded in 96-well plates, 10 wells per concentration,set 2 well blank. The cells are cultured at 37 ℃ and 5% CO2, the virus spot condition of the cells is observed day by day, generally, 5-7 days are needed to be observed, and the TCID50 result of the samples is calculated according to the concentration of the virus spot and the number of the holes. The results showed that the titer of the recombinant lentivirus was 6.72X 106TCID50/ml。
Example 5
Immune T effector cells comprising a gene as described above, including CD8+ T cells. Immune T effector cells were prepared as follows.
Preparation of PBMC cells
Fresh peripheral blood from 75ml healthy donors was taken and used with TBD sample density separation (purchased from tianjin primary opacific organisms) to isolate peripheral blood mononuclear cells PBMCs as follows:
(1) peripheral blood 75ml was diluted with physiological saline at a ratio of 1: 1. The diluted blood was carefully added to the same volume of lymphocyte separation medium to form a distinct layer, and the layer was centrifuged horizontally at 800rpm/min for 20min at room temperature. At the moment, 4 layers are formed in the centrifugal tube from top to bottom; serum, a white membrane layer composed of PBMCs, a lymphocyte separation liquid layer, and a lowest erythrocyte sedimentation layer.
(2) The buffy coat was carefully aspirated with a pipette, and the PBMC aspirated as completely as possible. Adding 2 times of physiological saline, washing cells for 2 times, mixing uniformly each time, centrifuging at 800rpm/min for 10 min. The low-speed centrifugation is favorable for removing platelet and lymphocyte separation liquid remained in the cell suspension, the supernatant is discarded after the centrifugation, and PBMC cells are collected.
BD magnetic bead sorting and culturing of CD8+ T lymphocytes and preparation of CD4-17bscFv-CAR-T
1) The isolated PBMCs were washed 2 times with PBS. 2) The cells were pelleted by centrifugation at 300 Xg for 10 min. 3) Cells were resuspended and biotin conjugated negative selection antibody (purchased from BD) was added. 4) Incubate for 20 minutes at room temperature. The cells were then washed with PBS buffer and pelleted by centrifugation at 300 Xg for 10 minutes. 5) Avidin-coupled magnetic beads (purchased from BD) were added. 6) Incubate for 30 minutes at room temperature. 7) The cells were resuspended in PBS buffer and added to flow tubes, no more than 3ml per tube. 8) The flow tube was placed on a cell sorting magnetic rack and allowed to stand for 8 minutes to allow non-CD 8+ T cells with bound beads to adhere to the side tube wall. 9) The supernatant not enriched by the magnetic beads was collected and centrifuged at 300 Xg for 10 minutes to obtain CD8+ T cells.
Resuspend CD8+ T cells in RPMI1640 complete Medium, then 1X 106Cell concentration per ml, plated evenly onto cell culture plates. Then, the cells were stimulated with anti-CD3 (purchased from BD Co.), anti-CD28 (purchased from BD Co.) and IL2 (purchased from BD Co.) and harvested after 48 hours. And infecting the recombinant lentivirus with CD8+ T at the MOI of 10, replacing fresh culture medium after infecting for 24h, and continuously carrying out expansion culture until the amount is enough. Chimeric antigen receptor expression was detected by FC500 flow cytometer (purchased from BECKMAN corporation) FL1 channel (fig. 4). By taking an unmodified CD8+ T cell as a negative control, the positive rate of CD4-17bscFv-CAR-T is 72.0%.
Example 6
Effect of Linker of varying lengths on the killing Activity of CD4-17bscFv-CAR-T cells
Expression of HIV-1NL4-3The envelope glycoprotein cell line Jurkat is used as a target cell, and the effector cells are CD4-17bscFv-CAR-T, CD 4-zeta CAR-T cells containing linkers with different lengths and CD8+ T cells without modification respectively. According to E: t is 1:1, adding 1X 106Collecting CD4-17bscFv-CAR-T, CD 4-zeta CAR-T cells and non-modified CD8+ T cells containing different lengths of Linker after the cells are completely attached to the wall, and respectively adjusting the cell concentration to be 1 x 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 with 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-Linker (G4S)8-17bscFv-CAR-T on Jurkat cells was 92.57 + -7.43% higher than that of the CD4-17bscFv-CAR-T group of other linkers of different lengths (FIG. 5). The fact that the Linker is 40 amino acids, the killing effect of the CD4-17bscFv-CAR-T is optimal, and the killing effect of the CD4-Linker (G4S)8-17bscFv-CAR-T is obviously higher than that of CD 4-zeta CAR-T and non-modified CD8+ T cells.
Example 7
Detection of susceptibility of CD4-17bscFv-CAR-T cells to HIV-1
Wild type HIV-1NL4-3CD4+ T lymphocytes, unmodified CD8+ T lymphocytes, transduced empty control CD8+ T cells, CD 4-zeta CAR-T and CD4-17bscFv-CAR-T cells were infected separately, with CD4+ T lymphocytes as experimental positive control. The 5 experimental groups were then analyzed for intracellular HIV-1 antigen p24 content by HIV-1p24 antigen ELISA detection kit (purchased from Thermo Fisher Scientific Co.) over 10 days of culture. The ELISA detection method comprises the following specific steps:
A. addition of conjugate 1:1, 25. mu.L of conjugate was added to each well. B. Sample adding: the sample, negative control, p24 antigen positive control 75 μ L were added sequentially. C. And (3) incubation: the plate was sealed with a sealing membrane and incubated at 37 ℃ for 60 minutes. D. Washing the plate: discard the liquid in the reaction well of the microplate, add 350. mu.L of working concentration washing solution to each well (containing blank control well), and then discard the washing solution. The plate washing was repeated 5 times and finally patted dry. E. Adding an enzyme: conjugate 2 was added to each well (blank control wells not), the plate was sealed with a sealing plate membrane and incubated at 37 ℃ for 30 minutes. F. Washing the plate: discard the liquid in the reaction well of the microplate, add 350. mu.L of working concentration washing solution to each well (containing blank control well), and then discard the washing solution. The plate washing was repeated 5 times and finally patted dry. G. Adding a substrate solution: add 50. mu.L of substrate buffer and developer to each well (containing blank control wells), seal the plate with a sealing membrane, and incubate in the dark at room temperature (18-30 ℃) for 30 minutes. H, adding a stop solution: add 50. mu.L of stop solution (containing blank control well) to each well, and gently shake the plate to mix the contents well. I. And (3) detection: within 1 hour after the reaction is stopped, the absorbance value of each well is measured by using an enzyme-labeling instrument at the wavelength of 450nm, and whether the sample contains the HIV-1p24 antigen or not is judged according to the critical value.
The results showed that more than half of the CD4+ T lymphocytes were p24 positive, 10% of the CD 4-zeta CAR-T cells were p24 positive, and the proportion of p24 positive cells in CD4-17 bsfv-CAR-T cells was p24 negative, comparable to the unmodified CD8+ T lymphocytes, transduced empty control CD8+ T cells. Thus, CD4-17bscFv-CAR-T cells can avoid HIV-1 infection.
Example 8
CD4-17bscFv-CAR-T cells stimulated by HIV-1 envelope protein can efficiently secrete antiviral cytokines
CD4-17 bsfv-CAR-T, CD 4-zeta CAR-T and non-engineered CD8+ T were each as follows: t (effector cell to target cell ratio) is 1:1 and expression of HIV-1NL4-3The envelope glycoprotein cell line Jurkat was cultured for 20h in mixed culture, and secretion of IFN-. gamma.by CD4-17bscFv-CAR-T, CD 4-zeta CAR-T and unmodified CD8+ T cells were examined by ELIspot assay (purchased from eBioscience, Inc.), respectively. The ELIspot specific steps are as follows:
day 1: cell culture (sterile procedure) 1. pre-coated plate activation: adding RPMI-1640 culture medium into each well, standing at room temperature for 5-10 min, and removing the culture medium by plate beating. 2. Adding a cell suspension: adding the cell suspension with the adjusted concentration into each experimental hole, wherein each experimental hole is 100 mu L; positive control wells: cell concentration 1X 105Well, add cell culture medium and PHA; negative control wells: cell concentration 1X 105Per well, add equal volume of cell culture medium; background negative control: RPMI-1640 medium containing fetal bovine serum was added. 3. And (3) incubation: put at 37 ℃ with 5% CO2The incubator is used for 16-20 hours.
Day 2: post-culture manipulation (sterile manipulation is no longer required) 4 lysis of cells: pour the cells and media from the wells. The cells were hypotonic lysed by adding ice-washed deionized water at 200. mu.L/well and placing in a freezer at 4 ℃ for 10 minutes. 5. Washing the plate: the well contents were poured, 1 × Washing buffer, 200 μ L/well, and washed 5-7 times. Standing for 30-60s each time. And finally, patting the water-absorbing paper to remove the liquid. 6. And (3) incubation of the detection antibody: biotin-labeled antibody solution was added to the wells at 100. mu.L/well. Incubate at 37 ℃ for 1 hour. 7. Washing the plate: the well contents were poured, 1 × Washing buffer, 200 μ L/well, and washed 5 times. Standing for 30-60 seconds each time. And finally, patting the water-absorbing paper to remove the liquid. 8. Incubation with enzyme-linked avidin: the diluted enzyme-labeled avidin solution was added to the plate wells at 100. mu.L/well. Incubate at 37 ℃ for 1 hour. 9. Washing the plate: the well contents were poured, 1 × Washing buffer, 200 μ L/well, and washed 5 times. Standing for 30-60 seconds each time. And finally, patting the water-absorbing paper to remove the liquid. 10. Color development: add the AEC color developing solution prepared at present into each plate well at 100. mu.L/well. Standing at room temperature in dark place for color development for 25 minutes, and 11. terminating the color development: the liquid in the wells was poured out, the plate base was uncovered and washed 3-5 times with deionized water to stop the development. The plate is placed in a cool place at room temperature, and the base is closed after the plate is naturally dried. Spot photograph of ELISPOT plate and analysis.
The results show that: and expression of HIV-1NL4-3The secretory capacity of IFN-gamma of CD4-17bscFv-CAR-T (left) cells cultured by the envelope glycoprotein cell line Jurkat mixed culture is remarkably higher than that of CD 4-zeta CAR-T (middle) and CD8+ T (right) cells which are not modified (FIG. 6).
The results show that the CD4-17bscFv-CAR-T molecule is reasonable in design, safe and effective, and lays a foundation for treating HIV-1.
Example 9
Kit for preparing CD4-17bscFv-CAR-T cells
(1) Obtaining a vector stably expressing CD4-17 bsfv-CAR as described above;
(2) a carrier diluent;
(3) instructions for use;
the kit is used in the treatment of HIV-1 and the instructions for use include the methods described in examples 4-6.
It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should also be understood that various alterations, modifications and/or variations can be made to the present invention by those skilled in the art after reading the technical content of the present invention, and all such equivalents fall within the protective scope defined by the claims of the present application.
Claims (8)
1. A dual-target chimeric antigen receptor characterized by: the chimeric antigen receptor comprises a CD4 antigen region, a Linker region, a 17b single-chain antibody region, a hinge region, a transmembrane domain, a costimulatory domain, and a signaling domain;
the Linker region is (G4S)8, and is a connecting peptide consisting of 40 amino acids;
the CD4 antigen nucleic acid artificial sequence is shown as SEQ ID NO. 2;
the nucleic acid artificial sequence of the 17b single-chain antibody is shown in SEQ ID NO. 4;
the hinge region is a CD8 a hinge region;
the nucleic acid artificial sequence of the CD8 alpha hinge region is shown as SEQ ID NO. 5;
the transmembrane domain is a CD8 alpha transmembrane region;
the nucleic acid artificial sequence of the CD8 alpha transmembrane region is shown as SEQ ID NO. 6;
the co-stimulatory domain is a CD137 co-stimulatory region;
the nucleic acid artificial sequence of the CD137 co-stimulation area is shown as SEQ ID NO. 7;
the signaling domain is a CD3 zeta signaling region;
the nucleic acid artificial sequence of the CD3 zeta signaling region is shown as SEQ ID NO. 8.
2. A gene encoding the dual-target chimeric antigen receptor of claim 1, wherein: the gene is CD4-17bscFv-CD8 alpha-CD 137-CD3 zeta;
and the gene is composed of the following nucleic acid artificial sequences in tandem:
(1) CD4 antigen nucleic acid artificial sequence shown in SEQ ID NO. 2;
(2) linker nucleic acid artificial sequence as SEQ ID NO. 3;
(3)17b single-chain antibody nucleic acid artificial sequence shown in SEQ ID NO. 4;
(4) CD8 alpha hinge region nucleic acid artificial sequence as shown in SEQ ID NO. 5;
(5) CD8 alpha transmembrane region nucleic acid artificial sequence as shown in SEQ ID NO. 6;
(6) CD137 costimulatory region nucleic acid artificial sequence as shown in SEQ ID NO. 7;
(7) CD3 zeta signaling region nucleic acid artificial sequence shown in SEQ ID NO. 8.
3. The coding gene of claim 2, wherein: the nucleic acid sequence of the gene is shown as SEQ ID NO. 1.
4. A recombinant plasmid characterized by: the recombinant plasmid comprises the gene of claim 2 or 3.
5. The recombinant plasmid of claim 4, wherein: the recombinant plasmid is a pLent-CD4-17bscFv-CAR plasmid which is obtained by inserting the CD4-17bscFv-CD8 alpha-CD 137-CD3 zeta gene fragment into a linearized pLent-C-GFP DNA fragment.
6. An expression vector characterized by: the expression vector comprising the gene of claim 2 or 3.
7. An immune T effector cell characterized by: the immune T effector cell comprises the gene of claim 2 or 3.
8. Use of a gene according to claim 2 or 3 for the preparation of a medicament for the treatment of HIV-1.
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