CN107974460B - Chimeric antigen receptor gene aiming at HIV-1, plasmid, T cell, kit and application with gene - Google Patents
Chimeric antigen receptor gene aiming at HIV-1, plasmid, T cell, kit and application with gene Download PDFInfo
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Abstract
The invention discloses a chimeric antigen receptor gene aiming at HIV-1, which comprises a leader region, a structural domain combined with a CD4 binding site of gp120, a hinge region, a transmembrane domain, a costimulatory structural domain, a signaling structural domain and a suicide gene system region. When the T cell provided by the invention is applied, the infection of HIV-1 can be prevented, and the lethality of HIV-1 infected cells is increased. Once an adverse reaction occurs after the HIV-1-CAR-T cells of the present invention are infused, the cells in the body can be cleared immediately by the suicide gene system to achieve prophylactic effects.
Description
Technical Field
The invention relates to the technical field of genes, in particular to a chimeric antigen receptor gene aiming at HIV-1, a plasmid, a T cell, a kit and application with the gene.
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), which not only effectively controls HIV-1 replication, but also reconstructs the immunity of AIDS patients (Michaels, S.H.; Clark, R.; Kissinger, P.decting morbidative and mortality amplitude patients with advanced human immunodeficiency virus infection. N.Engl. J.Med., 1998.339: 405-). However, since the virus is integrated in the infected cells and forms a stable reservoir of latent infection, the viral load may rebound to the level prior to treatment once the patient ceases drug treatment (David, D.Ho. Toward HIV-1 operation or administration: the tasks ahead. science,1998.280: 1866-. Thus, this therapy does not completely eliminate HIV-1 from the patient. In order to maintain efficacy, patients must take their medications year after year, which is costly (1.2-5.0 ten thousand dollars per year), and can produce serious toxic side effects (damage to the kidneys and liver, resulting in heart disease, diabetes, weakening bones, causing weight gain and psychiatric problems, such as insomnia and depression). In response to the above problems, the prior art has generally developed highly potent, low-toxicity, drug-resistant HIV-1 inhibitors acting on new targets and having new structural types (U.S. Pat. Nos. 5,849,911, 6,166,004 and 6,087,383, etc.; Chinese patents CN200480010780.2 and CN03814963.X, etc.). However, the HIV-1 gene has high variability, and mutation is easy to occur under the drug selection pressure of the HIV-1 inhibitor, so that drug-resistant strains are generated, and the effectiveness of HIV-1 inhibitor drug treatment is reduced. And the prior technical scheme does not completely get rid of the situation of long-term medication. 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) formed by connecting a CD4 extracellular domain with an intracellular CD 3-zeta signaling domain is transfected into a human T cell through a viral vector, and as CD4 is a main receptor of an HIV-1 infected cell, a CD4 extracellular domain of 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, CD 4-zeta CAR-T cell is activated, thus 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 become activated T cells to kill bound HIV-1 infected cells after binding to gp120 antigen, it is difficult for activated T cells to proliferate further and exert only transient effects, and CD 4-zeta CAR-T cells are susceptible to HIV-1 infection and become new hosts. Therefore, the effect of the traditional Chinese medicine composition in later clinical tests is not ideal, and the traditional Chinese medicine composition has not been widely applied to the present.
Therefore, the development of a novel safe and efficient chimeric antigen receptor aiming at HIV-1 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 chimeric antigen receptor gene aiming at HIV-1, a plasmid, a T cell, a kit and application with the gene.
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 chimeric antigen receptor gene for HIV-1 comprising a leader region, a domain that binds to the CD4 binding site of gp120, a hinge region, a transmembrane domain, a costimulatory domain, a signaling domain, and a suicide gene system region.
In the present invention, as a preferable embodiment, the leader region is a CD8leader region.
In the present invention, as a preferred embodiment, the domain binding to the CD4 binding site of gp120 is any one of CD4 antigen, VRC01, VRC01b, VRC01-C, 3BNC60, and NIH 45-46.
In the present invention, as a preferred embodiment, the domain that binds to the CD4 binding site of gp120 is CD4 antigen.
In the present invention, as a preferred embodiment, the hinge region of the chimeric antigen receptor is a CD8 hinge region.
In the present invention, as a preferred technical solution, the transmembrane domain of the chimeric antigen receptor is the CD8 transmembrane domain.
In the present invention, as a preferred embodiment, the co-stimulatory domain of the chimeric antigen receptor is a functional domain of 4-1 BB.
In a preferred embodiment of the present invention, the signaling domain of the chimeric antigen receptor is a functional domain of CD3 ζ.
In the present invention, as a preferred embodiment, the suicide gene system of the chimeric antigen receptor is iCasp9 suicide gene system.
Correspondingly, the nucleotide sequence of the chimeric antigen receptor gene aiming at the HIV-1 is shown as SEQ ID NO.1, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13 and SEQ ID NO. 14.
In the present invention, as a preferred embodiment, the chimeric antigen receptor gene against HIV-1 is preferably obtained by connecting the following modules in series:
CD8leader region nucleic acid artificial sequence as shown in SEQ ID NO. 2;
CD4 antigen nucleic acid artificial sequence shown in SEQ ID NO. 3;
a CD8 hinge region nucleic acid artificial sequence as shown in SEQ ID NO. 4;
CD8 transmembrane region nucleic acid artificial sequence as shown in SEQ ID NO. 5;
4-1BB costimulatory region nucleic acid artificial sequence as shown in SEQ ID NO. 6;
CD3 zeta signaling region nucleic acid artificial sequence as shown in SEQ ID NO. 7;
the self-cleavage polypeptide T2A nucleic acid artificial sequence is shown as SEQ ID NO. 8;
iCasp9 suicide gene region nucleic acid artificial sequence, SEQ ID NO. 9.
The chimeric antigen receptor gene aiming at HIV-1 is obtained by adopting a preparation method comprising the following steps:
(1) synthesizing the whole expression frame of the sequence according to a CD8leader region nucleic acid artificial sequence, a CD4 antigen nucleic acid artificial sequence, a CD8 hinge region nucleic acid artificial sequence, a CD8 transmembrane region nucleic acid artificial sequence, a 4-1BB costimulatory region nucleic acid artificial sequence, a CD3 zeta signaling region nucleic acid artificial sequence, a self-cleaving polypeptide T2A nucleic acid artificial sequence and an iCasp9 suicide gene region nucleic acid artificial sequence respectively, and inserting the sequences into a standard vector pUC57 to obtain pUC-HIV-1-CD 4-CAR;
(2) carrying out double enzyme digestion on pUC-HIV-1-CD4-CAR, cutting an agar part containing HIV-1-CD4-CAR DNA fragments by using agar electrophoresis, treating by using DNA extraction kit sol solution, passing through a DF column, discarding filtrate, rinsing the DF column, carrying out air separation, eluting the DF column, and collecting a centrifuge to obtain purified HIV-1-CD4-CAR DNA fragments, namely the chimeric antigen receptor gene aiming at HIV-1.
In a second aspect, the present invention provides a plasmid comprising a chimeric antigen receptor gene for HIV-1 as described above.
In the present invention, as a preferred embodiment, the plasmid is obtained by inserting the fusion gene fragment CD8leader-CD4-CD8-4-1BB-CD3 ζ -T2A-iCasp9 into a lentiviral expression vector pLent-C-GFP.
The plasmid is prepared by the following preparation method: the purified HIV-1-CD4-CAR DNA fragment and the linearized pLent-C-GFP DNA fragment were ligated in the ligation system: 10 × buffer: 1 mul; t4 ligase: 1 mul; HIV-1-CD4-CAR DNA: 4 mu l of the solution; linearized pLent-C-GFP DNA: mu.l were ligated overnight under conditions to form the pLent-HIV-1-CD4-CAR plasmid.
In a third aspect, the present invention provides a T cell comprising a chimeric antigen receptor gene for HIV-1 as described above.
In the present invention, as a preferred technical scheme, the T cell is prepared by the following preparation method: the pLent-HIV-1-CD4-CAR plasmid described above was first lentivirally packaged and the T cells were then infected with recombinant lentiviruses.
In a fourth aspect, the present invention provides a kit comprising
Obtaining a vector stably expressing HIV-1-CD4-CAR as described above;
and a carrier diluent.
In a fifth aspect, the invention provides application of the gene, which means that the gene can be applied to preparing medicines for treating HIV-1. The form of the medicament includes, but is not limited to, a kit.
After the technical scheme is adopted, the invention has the beneficial effects that:
the present invention combines a structure that binds to the CD4 binding site of gp120 with a CAR motif as the extra-and intra-membrane domains of an anti-HIV-1 CAR molecule, respectively, and modifies the design of the CAR, thus developing a completely novel, MHC independent anti-HIV-1 CAR molecule, named HIV-1-CAR (see figure 1). HIV-1-CAR, on the one hand, reduces the homology of HIV cell receptors, protecting HIV-1-CAR-T cells from HIV infection; on the other hand, HIV-1-CAR can further amplify T cell activation signals, and improve the in vivo amplification capacity and cytokine production capacity. HIV-1-CAR-T can kill gp 120-expressing cell lines strongly by co-culturing with 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, using the CD8leader region and hinge region, has been shown in practice to reduce the homology of CD4 and prevent infection of the modified CAR-T by HIV-1. The use of 4-1BB can further activate the modified CAR-T, allowing substantial expansion of the CAR-T, increasing lethality to HIV-1 infected cells.
The HIV-1-CAR-T provided by the invention is provided with an iCasp9 suicide gene system, and once an adverse reaction occurs after the HIV-1-CAR-T provided by the invention is infused, cells in a body can be immediately cleared through the suicide gene system so as to achieve a preventive effect.
Drawings
FIG. 1 is a schematic design diagram of the safe and highly efficient chimeric antigen receptor of the present invention.
FIG. 2 is an electrophoretogram of the DNA fragment of HIV-1-CD4-CAR (right) and the linearized pLent-C-GFP DNA (left) of the present invention.
FIG. 3 is a schematic diagram of the lentiviral CD8leader-CD4-CD8-4-1BB-CD3 zeta-T2A-iCasp 9 expression plasmid of the present invention.
FIG. 4 shows that the HIV-1-CD4-CAR-T cells of the present invention express HIV-1-CD4-CAR with an efficiency of 81.8%.
FIG. 5 is an ELIspot assay of the invention, the left panel is the IFN-gamma secretion of HIV-1-CD 4-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; the secretion capacity of IFN-gamma of HIV-1-CD4-CAR-T cells cultured in mixed culture of a cell strain Jurkat expressing HIV-1NL4-3 envelope glycoprotein is significantly higher than that of CD 4-zeta CAR-T and unmodified CD8+ T cells.
FIG. 6 is a graph showing the results of the killing experiment of HIV-1-CD4-CAR-T cells of the present invention. At E: t is 1:1, the killing efficiency of HIV-1-CD4-CAR-T on cell line Jurkat expressing HIV-1NL4-3 envelope glycoprotein is 90.56 +/-8.23% which is obviously higher than that of CD 4-zeta CAR-T and non-modified CD8+ T cells;
FIG. 7 is a graph showing the results of the killing experiment of HIV-1-CAR-T cells of the present invention. At E: t is 1:1, the killing efficiency of HIV-1-CAR-T on cell line Jurkat expressing HIV-1NL4-3 envelope glycoprotein reaches over 85 percent, which is obviously higher than that of CD 4-zeta CAR-T and unmodified 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 chimeric antigen receptor gene for HIV-1 comprising a leader region, a domain that binds to the CD4 binding site of gp120, a hinge region, a transmembrane domain, a costimulatory domain, a signaling domain, and a suicide gene system region. Wherein the domain that binds to the CD4 binding site of gp120 employs any one of the CD4 antigen, VRC01, VRC01b, VRC01-C, 3BNC60, NIH 45-46.
This example shows, as a preferred embodiment, the nucleotide sequence of the chimeric antigen receptor gene for HIV-1 is shown in SEQ ID NO. 1. Namely: the following modules are connected in series to obtain: CD8leader region nucleic acid artificial sequence as shown in SEQ ID NO. 2; CD4 antigen nucleic acid artificial sequence shown in SEQ ID NO. 3; a CD8 hinge region nucleic acid artificial sequence as shown in SEQ ID NO. 4; CD8 transmembrane region nucleic acid artificial sequence as shown in SEQ ID NO. 5; 4-1BB costimulatory region nucleic acid artificial sequence as shown in SEQ ID NO. 6; CD3 zeta signaling region nucleic acid artificial sequence as shown in SEQ ID NO. 7; the self-cleavage polypeptide T2A nucleic acid artificial sequence is shown as SEQ ID NO. 8; an iCasp9 suicide gene region nucleic acid artificial sequence shown as SEQ ID NO. 9.
Example 2
Preparation example of chimeric antigen receptor gene against HIV-1.
This example also illustrates the case where the domain binding to the CD4 binding site of gp120 is exemplified by CD4 antigen, and when any of VRC01(SEQ ID NO.10), VRC01b (SEQ ID NO.11), VRC01-C (SEQ ID NO.12), 3BNC60(SEQ ID NO.13), and NIH45-46(SEQ ID NO.14) is used, the preparation method is substantially the same, and no redundant description is given here.
The preparation method of the chimeric antigen receptor gene aiming at HIV-1 comprises the following steps:
(1) synthesizing the whole expression frame of the sequence according to a CD8leader region nucleic acid artificial sequence, a CD4 antigen nucleic acid artificial sequence, a CD8 hinge region nucleic acid artificial sequence, a CD8 transmembrane region nucleic acid artificial sequence, a 4-1BB costimulatory region nucleic acid artificial sequence, a CD3 zeta signaling region nucleic acid artificial sequence, a self-cleaving polypeptide T2A nucleic acid artificial sequence and an iCasp9 suicide gene region nucleic acid artificial sequence respectively, and inserting the sequences into a standard vector pUC57 to obtain pUC-HIV-1-CD 4-CAR;
(2) carrying out double enzyme digestion on pUC-HIV-1-CD4-CAR, cutting an agar part containing HIV-1-CD4-CAR DNA fragments by using agar electrophoresis, treating by using DNA extraction kit sol solution, passing through a DF column, discarding filtrate, rinsing the DF column, carrying out air separation, eluting the DF column, and collecting a centrifuge to obtain purified HIV-1-CD4-CAR DNA fragments, namely the chimeric antigen receptor gene aiming at HIV-1.
In more detail, the preparation method of the chimeric antigen receptor gene for HIV-1 provided in this embodiment includes the following steps:
the expression cassette was synthesized by CD8leader region nucleic acid artificial sequence, CD4 antigen nucleic acid artificial sequence, CD8 hinge region nucleic acid artificial sequence, CD8 transmembrane region nucleic acid artificial sequence, 4-1BB costimulatory region nucleic acid artificial sequence, CD3 zeta signaling region nucleic acid artificial sequence, self-cleaving polypeptide T2A nucleic acid artificial sequence, iCasp9 suicide gene region nucleic acid artificial sequence, committee bioengineering (Shanghai) Limited, and inserted into a standard vector pUC57, respectively, thus being named pUC-HIV-1-CD4-CAR,
pUC-HIV-1-CD4-CAR was subjected to double digestion with Fast Digest AsiSI (available from ThermoFisher) and Fast Digest NotI (available from ThermoFisher) 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 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 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 HIV-1-CD4-CAR DNA fragment (shown in figure 2).
Example 3
A plasmid comprising a chimeric antigen receptor gene for HIV-1 as described above. The plasmid of the embodiment is obtained by inserting a fusion gene fragment CD8leader-CD4-CD8-4-1BB-CD3 zeta-T2A-iCasp 9 into a lentiviral expression vector pLent-C-GFP.
Example 4
Examples of plasmid preparation.
The plasmid is prepared by the following preparation method: the purified HIV-1-CD4-CAR DNA fragment and the linearized pLent-C-GFP DNA fragment were ligated in the ligation system: 10 × buffer: 1 mul; t4 ligase: 1 mul; HIV-1-CD4-CAR DNA: 4 mu l of the solution; linearized pLent-C-GFP DNA: mu.l were ligated overnight under conditions to form the pLent-HIV-1-CD4-CAR plasmid.
More specifically, the plasmid preparation method adopted in this example includes the following steps:
the entire expression cassette was synthesized by the company named pUC-HIV-1-CD4-CAR from the CD8leader region nucleic acid artificial sequence, CD4 antigen nucleic acid artificial sequence, CD8 hinge region nucleic acid artificial sequence, CD8 transmembrane region nucleic acid artificial sequence, 4-1BB costimulatory region nucleic acid artificial sequence, CD3 zeta signaling region nucleic acid artificial sequence, self-cleaving polypeptide T2A nucleic acid artificial sequence, iCasp9 suicide gene region nucleic acid artificial sequence, Consortium Biotech (Shanghai) and inserted into the standard vector pUC, respectively, and the pUC-HIV-1-CD4-CAR and pLent-C-GFP vectors were subjected to the double digestion of Fast Digest AsiSI (from ThermoFisher) and Fast Digest NotI (from ThermoFisher) 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 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 HIV-1-CD4-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-HIV-1-CD4-CAR (see FIG. 3) plasmid. The connecting system is as follows: 10 × buffer: 1 mul; t4 ligase: 1 mul; HIV-1-CD4-CAR DNA: 4 mu l of the solution; linearized pLent-C-GFP DNA: 4 μ l.
Example 5
Example of purification of pLent-HIV-1-CD4-CAR plasmid.
Coli (DH5 α) was transformed with the above-described pnent-HIV-1-CD 4-CAR. 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-HIV-1-CD4-CAR plasmid was extracted using a plasmid extraction and purification kit (purchased from Qiagen) by 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 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, 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 a DNA sample (Marker) of a predetermined concentration, and the result was compared to obtain pLent-HIV-1-CD4-CAR plasmid at a concentration of 332 ng/. mu.l.
The pLent-HIV-1-CD4-CAR plasmid described above was sequenced by committee Biotechnology (Shanghai) Co., Ltd. Sequencing is carried out correctly for later use.
Example 6
A T cell comprising a chimeric antigen receptor gene for HIV-1 as described above. The T cells of this example were prepared as follows: the pLent-HIV-1-CD4-CAR plasmid described above was first lentivirally packaged and the T cells were then infected with recombinant lentiviruses.
The following examples are methods of lentivirus packaging, preparation of PBMC cells, recombinant lentivirus infection of T cells.
Example 7
Lentivirus packaging example.
Slow Using Lentiviral Packaging KitThe virus packaging kit comprises the following specific 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-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 plated on 96-well cell culture plates, and samples were loaded in 96-well Cells using 5% FBS DMEM diluted in series at 10 fold ratios, with 10 wells for each concentration, and 2-well blanks were set. 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 6.5X 106TCID50/ml。
Example 8
Preparation example 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; plasma, a buffy coat 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.
Example 9
BD magnetic bead sorting and culture of CD8+ T lymphocytes and preparation examples of HIV-1-CD 4-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 HIV-1-CD4-CAR-T is 81.8 percent.
Example 10
Detection of susceptibility of HIV-1-CD4-CAR-T cells to HIV-1
Wild type HIV-1NL4-3Respectively to CD4+ T lymphocytes and unmodified CD8+ T lymphocytesCells, transduced unloaded control CD8+ T cells, CD 4-zeta CAR-T and HIV-1-CD4-CAR-T cells were infected 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 HIV-1-CD4-CAR-T cells was p24 negative, comparable to the unmodified CD8+ T lymphocytes, transduced empty control CD8+ T cells. Thus, HIV-1-CD4-CAR-T cells can be protected from HIV-1 infection.
Example 11
HIV-1-CD4-CAR-T cells stimulated by HIV-1 envelope protein can efficiently secrete antiviral cytokines
HIV-1-CD4-CAR-T, CD 4-zeta CAR-T andtransforming the CD8+ T according to the following steps of E: t (effector cell to target cell ratio) is 1:1 and expression of HIV-1NL4-3The envelope glycoprotein cell line Jurkat was cultured in a mixed manner and tested for secretion of HIV-1-CD4-CAR-T, CD 4-zeta CAR-T and unmodified CD8+ T cell IFN-. gamma.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 cell suspension (mixed cell suspension of effector cells and target cells) with adjusted concentration into each experimental hole, wherein the concentration is 100 mu L/hole; 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 was incubated for 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 IFN-gamma secretion capacity of HIV-1-CD4-CAR-T (left) cells cultured in the envelope glycoprotein cell line Jurkat mixture was significantly higher than that of CD 4-zeta CAR-T (middle) and unmodified CD8+ T (right) cells (FIG. 5).
Example 12
Killing activity assay for HIV-1-CD4-CAR-T cells
Expression of HIV-1NL4-3Envelope glycoprotein cell line Jurkat as target cell, effector cells are HIV-1-CD4-CAR-T, CD 4-zeta CAR-T and non-engineered CD8+ T cells, respectively.
According to E: t is 1:1, adding 1X 106Collecting HIV-1-CD4-CAR-T, CD 4-zeta CAR-T and unmodified CD8+ T cells after the cells are completely attached to the wall, and respectively adjusting the cell concentration to 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 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]X 100%. The killing efficiency of HIV-1-CD4-CAR-T on Jurkat cells was 90.56. + -. 8.23% significantly higher than that of CD 4-zeta CAR-T and unmodified CD8+ T cells (FIG. 6).
Example 13
Assay of AP1903 control of HIV-1-CD4-CAR-T cell Activity
The results show that the HIV-1-CD4-CAR molecule is reasonable in design, safe and effective, and lays a foundation for treating HIV-1.
Example 14
Killing activity assay for HIV-1-VRC01-CAR-T, HIV-1-VRC01b-CAR-T, HIV-1-VRC01-C-CAR-T, HIV-1-3BNC60-CAR-T and HIV-1-NIH45-46-CAR-T cells
Expression of HIV-1NL4-3Envelope glycoprotein cell line Jurkat as target cell, effector cell HIV-1-VRC01-CAR-T, HIV-1-VRC01b-CAR-T, HIV-1-VRC01-C-CAR-T, HIV-1-3BNC60-CAR-T, HIV-1-NIH45-46-CAR-T, CD 4-zeta CAR-T and non-engineered CD8+ T cell respectively.
According to E: t is 1:1, adding 1X 106Collecting HIV-1-VRC01-CAR-T, HIV-1-VRC01b-CAR-T, HIV-1-VRC01-C-CAR-T, HIV-1-3BNC60-CAR-T, HIV-1-NIH45-46-CAR-T, CD 4-zeta CAR-T and non-modified CD8+ T cells 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]X 100%. The killing efficiency of HIV-1-CAR-T on Jurkat cells reached over 85%, which was significantly higher than that of CD 4-zeta CAR-T and unmodified CD8+ T cells (FIG. 7).
Example 15
Kit for preparing HIV-1-CD4-CAR-T cells
(1) Obtaining a vector stably expressing HIV-1-CD4-CAR as described above;
(2) a carrier diluent;
(3) instructions for use;
example 16
Application of kit in treatment of HIV-1
(1) Packaging the vector stably expressing the HIV-1-CD4-CAR in the kit by lentivirus: a lentivirus Packaging Kit is adopted, a lentivirus Packaging cell line 293T is inoculated in a 10cm culture dish containing DMEM and 10% FBS, the culture is carried out at 37 ℃ and under the condition of 5% CO2, and transfection is prepared when the anchorage rate is 70% -80%. Taking sterile 1.5ml EP tube or 15ml for centrifugationThe reaction system was prepared as follows: serum-free DMEM: 4 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 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.
(2) Peripheral blood mononuclear cells PBMCs were isolated from 75ml of fresh peripheral blood of healthy donors using TBD sample density separation (purchased from tianjin primary opacific organisms) as follows:
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; plasma, a buffy coat layer composed of PBMCs, a lymphocyte separation liquid layer, and a lowest erythrocyte sedimentation layer.
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.
(3) BD magnetic bead sorting, culture of CD8+ T lymphocytes and preparation of HIV-1-CD 4-CAR-T1) 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.
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 (6)
1. A chimeric antigen receptor gene against HIV-1 characterized by: the gene comprises a leader region, a domain that binds to the CD4 binding site of gp120, a hinge region, a transmembrane domain, a costimulatory domain, a signaling domain, and a suicide gene system region;
the domain binding to the CD4 binding site of gp120 employs any one of CD4 antigen, VRC01, VRC01b, VRC01-C, 3BNC60, NIH 45-46;
when the structural domain combined with the CD4 binding site of gp120 is CD4 antigen, the nucleotide sequence of the chimeric antigen receptor gene aiming at HIV-1 is obtained by connecting the nucleotide sequence of the leader region with SEQ ID NO. 1;
the nucleotide sequence of the guide region is shown as SEQ ID NO. 2;
when the domain binding to the CD4 binding site of gp120 is VRC01, VRC01b, VRC01-C, 3BNC60 or NIH45-46, the nucleotide sequence of the chimeric antigen receptor gene for HIV-1 is SEQ ID No.10, SEQ ID No.11, SEQ ID No.12, SEQ ID No.13 or SEQ ID No.14, respectively.
2. A plasmid, characterized in that: the plasmid comprising the chimeric antigen receptor gene for HIV-1 according to claim 1.
A T cell characterized by: the T cell comprises the chimeric antigen receptor gene for HIV-1 of claim 1.
4. A kit comprising
A vector stably expressing the chimeric antigen receptor gene for HIV-1 according to claim 1;
and a carrier diluent.
5. Use of the chimeric antigen receptor gene against HIV-1 according to claim 1 for the preparation of a medicament for the treatment of HIV-1.
6. The use of claim 5, wherein: the form of the medicament includes a kit.
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| CN107400168A (en) * | 2017-07-18 | 2017-11-28 | 深圳市免疫基因治疗研究院 | A kind of Chimeric antigen receptor and its application based on CD117 |
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| CN107337736A (en) * | 2017-06-06 | 2017-11-10 | 上海优卡迪生物医药科技有限公司 | The double targeting Chimeric antigen receptors of OCTS CAR, encoding gene, recombinant expression carrier and its structure and application |
| CN107400168A (en) * | 2017-07-18 | 2017-11-28 | 深圳市免疫基因治疗研究院 | A kind of Chimeric antigen receptor and its application based on CD117 |
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