CN116103242A - ESAT6-CAR-T for targeted treatment of tuberculosis, and preparation method and application thereof - Google Patents
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Abstract
The invention belongs to the field of tuberculosis immunotherapy, and in particular relates to ESAT6-CAR-T for targeted therapy of tuberculosis, and a preparation method and application thereof. The preservation number of ESAT6-CAR-T is CCTCC NO: C2022374, and the preparation method comprises the following steps: step 1: cloning, expressing, purifying and immunizing the Mtb specific secretion antigen ESAT 6; step 2: development and screening of an anti-ESAT 6 protein specific single-chain antibody amino acid coding sequence; step 3: preparation of lentivirus and retrovirus carrying an anti-ESAT 6 protein single-chain antibody sequence; step 4: preparation of ESAT6-CAR-T lymphocytes. Application of ESAT6-CAR-T in preparing medicine, vaccine and detection reagent for treating tuberculosis. Compared with the traditional tuberculosis medicines, the invention has higher safety; the treatment effect is good; has potential value for pressure-resistant tuberculosis and Mtb detention bacteria infection; no off-target effect.
Description
Technical Field
The invention belongs to the field of tuberculosis immunotherapy, and in particular relates to ESAT6-CAR-T for targeted therapy of tuberculosis, and a preparation method and application thereof.
Background
Tuberculosis (tuberculosis) is a highly lethal infectious disease caused by mycobacterium tuberculosis (Mycobacterium tuberculosis, mtb), and has so far been the single infectious disease responsible for the greatest number of deaths. In recent years, the incidence rate is continuously increased, the new incidence rate is approximately 900 ten thousand worldwide, the death cases are approximately 200 ten thousand worldwide, and although China achieves a certain achievement in pulmonary tuberculosis prevention, control and diagnosis, china is still one of the countries with high tuberculosis burden.
The treatment of tuberculosis is still mainly based on traditional chemical drug treatment, but tuberculosis drugs expose a plurality of short plates in clinical application, for example: the existing tuberculosis drug has long treatment period and is easy to cause bacterial drug resistance; the tuberculosis medicine (isoniazid, rifampicin, etc.) has obvious toxic and side effects and weakens the immunity of the patient to a certain extent. In addition, mtb, an ancient bacterial parasite, is dormant when it encounters a special environment (nutrient deficiency, extreme pH) within the host cell, which further reduces the sensitivity and killing ability of traditional drugs. Under the catalysis of the above elements, the body of a patient cannot generate enough immune response and specific immune protection, and the cure and the prognosis of tuberculosis depend greatly on the autoimmune state of the patient.
The long-term use of tuberculosis drugs causes the drug resistance problem to be serious, causes different types of drug resistance (single drug resistance, multiple drug resistance, wide drug resistance and the like) and brings great challenges to the clinical treatment of tuberculosis. The current coping mode is still traditional chemotherapy, and the treatment effect is extremely limited, and meanwhile, the method has extremely great toxic and side effects. The immunity of the organism of the patient is impaired to different degrees. The autoimmune power of patients affects the occurrence, development and prognosis of tuberculosis. The research view angle at home and abroad is dynamically transferred to the immunotherapy of tuberculosis, and aims to correct inflammatory response, shorten treatment process and improve curative effect. Several studies have shown broad prospects in the treatment of tuberculosis. However, the existing immune preparations are various and mostly exogenous preparations, and have certain biological safety and hidden danger. Therefore, avoiding traditional medicines and correcting the immune state of a patient, and developing a new immunotherapy scheme is important for solving the Mtb infection.
Chimeric antigen receptor T cells (Chimeric antigen receptor T cell, CAR-T) are novel and efficient genetically engineered immune cell therapeutic preparations, and the Chimeric Antigen Receptor (CAR) for recognizing specific antigen proteins is expressed and anchored on cell membranes by virtue of virus vectors, so that the chimeric antigen receptor T cells play a role in targeted recognition and specific killing of target cells. The method is initially applied to tumor treatment, and has great success in the treatment of blood tumors, and has good targeting and specificity in clinical experiments and various researches. Mycobacterium tuberculosis as intracellular parasitic bacteria, and in vivo clearance requires cooperation of innate immunity and acquired immunity. Antigen presenting cells infected with Mtb present a variety of unique antigens, suggesting that CAR-T cell therapies have good application value for Mtb infection.
The bacterial components of Mtb, secreted proteins and other components can excite the immune function of the organism of a patient, and a plurality of ideal target antigens (Ag 85 and CFP-10) exist. Wherein ESAT6 is enriched in T cell epitopes, is a putative specific target protein and is non-cross-pathogenic with other mycobacteria. Moreover, a plurality of researches develop the antagonistic drugs aiming at the target spot, and the method has higher application value.
Disclosure of Invention
Aiming at the defects existing in the prior art, the invention provides ESAT6-CAR-T for targeted treatment of tuberculosis, a preparation method and application thereof, ESAT6 is used as a target antigen, and a high-affinity specific single-chain antibody (scFv) is obtained through a hybridoma technology and a sequencing technology, and CAR-T cells are further designed. By recognizing ESAT6 as a target antigen, a cooperative mechanism of CAR-T cells and Mtb infected cells is utilized to bypass a chemotherapy drug resistance mechanism, so that the autoimmune system of a patient is stimulated to the maximum extent, the problems of low immunity and intracellular latency are solved, and the purpose of tuberculosis treatment is realized by killing Mtb through the cell cooperative mechanism.
In order to achieve the above purpose, the present invention adopts the following technical scheme: ESAT6-CAR-T for targeted treatment of tuberculosis, wherein the preservation number of the ESAT6-CAR-T is CCTCC NO: C2022374, and the ESAT6-CAR-T is classified and named: human ESAT6-CAR-T lymphocytes 2022H-ECT1. Preservation unit: china center for type culture Collection, address: the preservation date of the university of Wuhan in China is 2022, 12 months and 08 days.
Further, the ESAT6-CAR-T specifically targets Mtb-ESAT6 antigen using CD4 + And CD8 + In combination, recognizes and kills Mtb host cells.
A method of preparing ESAT6-CAR-T for targeted treatment of tuberculosis, the method comprising:
step 1: cloning, expressing, purifying and immunizing the Mtb specific secretion antigen ESAT 6;
step 2: development and screening of an anti-ESAT 6 protein specific single-chain antibody amino acid coding sequence;
step 3: preparation of lentivirus and retrovirus carrying an anti-ESAT 6 protein single-chain antibody sequence;
step 4: preparation of ESAT6-CAR-T lymphocytes.
Further, the step 1 specifically includes:
(1) Optimizing codons and designing primers according to the amino acid sequence of ESAT6 protein of Mtb and the codon preference of escherichia coli, amplifying ESAT6 full-length fragments and inserting a vector pET-22b to construct a recombinant plasmid pET22b-ESAT6, and identifying by double enzyme digestion and sequencing;
(2) Transforming the identified correct recombinant plasmid pET22b-ESAT6 into BL21 competent cells, performing IPTG induction expression, collecting induced thalli, performing ultrasonic disruption, preparing a protein sample, performing SDS-PAGE electrophoresis analysis on protein expression and expression form, performing Ni column affinity chromatography purification on the prepared sample after optimizing induction conditions, performing SDS-PAGE electrophoresis separation on the purified sample, transferring a membrane, incubating a His tag antibody, performing membrane washing, incubating an HRP-marked secondary antibody, and performing color development and exposure;
(3) The ESAT6 recombinant protein identified correctly was mixed and emulsified with an adjuvant, balb/c mice were immunized by subcutaneous multipoint injection at a dose of 100. Mu.g/mouse, boosted once for 2-3 weeks, for a total of 5 immunizations, and antibody titers were detected by ELISA after rat tail blood collection.
Further, the step 2 specifically includes:
the immunized Balb/c mice are subjected to aseptic spleen taking, single cell suspension is prepared, cell fusion is carried out by utilizing a semi-solid and liquid phase combination mode, hybridoma cells are screened, positive hybridoma cells secreting antibodies are identified and screened through ELISA, the hybridoma cells meeting expectations are subjected to expanded culture and cryopreservation, the monoclonal antibodies are subjected to subtype identification, the hybridoma cells are inoculated into the abdominal cavity of the mice after identification, a large amount of monoclonal antibodies are prepared, then ascites of the mice are collected, antibodies are obtained through protein G affinity chromatography, and the corresponding hybridoma cells are sequenced.
Further, the step 3 specifically includes:
connecting the sequences obtained by screening with viral vector plasmids, transforming into host bacteria, selecting positive clones, performing target sequence PCR and sequencing, co-transfecting 293T cells with the identified correct viral recombinant vectors and auxiliary plasmids, collecting supernatant of the transfected 293T cells according to the growth state of the cells, and concentrating and storing the supernatant containing the viruses for subsequent experiments.
Further, the step 4 specifically includes:
PBMC cells were isolated from peripheral blood of healthy adults and purified CD3 by magnetic bead anion separation + T cell final concentration of 25
g/mL ConA stimulated CD3 + And (3) activating the T cells for 24 hours, and then adding slow viruses to perform transfection to obtain ESAT6-CAR-T cells.
Further, the step 4 specifically includes:
1. human peripheral blood CD3 + T cell sorting and in vitro activation:
(1) Collecting fresh EDTA anticoagulated whole blood, and mixing the whole blood reversely;
(2) Diluting the freshly collected anticoagulated whole blood with PBS according to the volume ratio of 1:1 and fully and uniformly mixing;
(3) Adding the Ficoll separating liquid which is balanced to the room temperature in advance into a 50mL centrifuge tube;
(4) Slowly adding diluted whole blood above the liquid surface of Ficoll separating liquid, wherein the boundary between blood and separating liquid cannot be destroyed;
(5) After sample addition, carefully placing the centrifuge tube into a horizontal centrifuge, centrifuging at room temperature for 800 Xg and 20min, and slowly lifting; the liquid after centrifugation is divided into four layers, and the four layers are sequentially from top to bottom: a plasma layer, a white membrane layer, an outer blood mononuclear cell, a Ficoll separating liquid layer and a red blood cell layer;
(6) Discarding part of upper plasma, collecting cells of the tunica media into a new centrifuge tube, adding a proper amount of PBS, blowing and mixing uniformly by using a sample adding gun, and then carrying out 120 Xg for 10min;
(7) Discarding the supernatant, continuing to resuspend the cells with PBS, 120 Xg, 10min, repeating this step 2 times, with the aim of removing platelets;
(8) Counting the obtained PBMC, and adjusting the cell concentration to 5X 107cells/mL by using PBS, wherein the volume range of the cell suspension is between 0.25 and 2 mL;
(9) The conditioned cell suspension was added to a polystyrene underflow tube to which was added 50L/mL EasySep TM Human T Cell Isolation Cocktail；
(10) Mixing gently with a sample adding gun, and incubating at room temperature for 5min;
(11) EasySep with an oscillator TM Dextran RapidSpheres TM Vortex for 30s, fully mix the magnetic beads;
(12) EasySep TM Dextran RapidSpheres TM Adding the mixture into a sample to 40L/mL, and uniformly mixing;
(13) Supplementing the sample volume to 2.5mL by using PBS, and lightly mixing for 2-3 times by using a sample adding gun;
(14) Put the flow tube into easy Sep TM Placing the magnet at room temperature for 3min;
(15) Taking up the magnet, pouring the liquid in the tube into a new tube, and separating CD3+ T cells from the liquid;
(16) Culturing CD3 with RPMI 1640 complete Medium + T cells, added with Con A protein to a final concentration of 25
g/mL, adding human IL-2 to a final concentration of 20ng/mL, and adding 5% CO 2 Culturing in a cell incubator at 37 ℃ for 24 hours;
2. human CD3 + T cell lentiviral infection:
taking out the virus from the refrigerator at the temperature of-80 ℃, melting in an ice bath in a biosafety cabinet, and storing at the temperature of 4 ℃; human CD3 + The T cells were activated for 24h and then subjected to lentiviral infection,
regulating cell concentration, calculating virus dosage, adding 1×HiTransG A and 1×HiTransG P infection promoting agent, mixing, and adding 5% CO 2 After static culture for 12 hours in a 37 ℃ incubator, the culture medium is replaced by a new RPMI1640 complete culture medium containing human IL-2, and liquid supplementing, passage and harvesting are carried out according to requirements.
Use of ESAT6-CAR-T for targeted treatment of tuberculosis: the ESAT6-CAR-T is used for preparing therapeutic drugs, vaccines and detection reagents for tuberculosis.
Further, scraping a well-grown Mtb colony on a Roche medium into an EP tube containing 0.05% Tween-80 physiological saline, blowing the colony into a single bacterial suspension by using a sterile syringe, standing overnight, transferring the supernatant into a new sterile EP tube, and measuring OD 600 The method comprises the steps of carrying out a first treatment on the surface of the Acquisition ofEnriching mononuclear cells from peripheral blood of healthy adult according to STEM CELL RosetteSepTM Human Monocyte Enrichment Cocktail operation instructions, culturing in vitro with RPMI1640 medium containing GM-CSF, adding the bacterial suspension dropwise after cell adhesion to prepare Mtb macrophage infection model,
ESAT6-CAR-T cells and Mtb-infected macrophages were according to E: t=0.5:1; an effective target ratio of 1:1 or 2:1 to 6h;12h; co-culture was performed for 24h or 48h of time, after cell lysis, roche medium was plated and CFU was counted to evaluate the killing capacity of human ESAT6-CAR-T cells against Mtb.
The ESAT6-CAR-T for targeted treatment of tuberculosis and the preparation method and application thereof have the advantages that:
1. compared with the traditional tuberculosis medicines, the medicine has higher safety: most of tuberculosis treatment medicines have strong toxic and side effects, and have great harm to organs such as liver, kidney and the like; and the clinical treatment cycle of tuberculosis is long, and patients take tuberculosis medicines for a long time, so that the autoimmunity is greatly weakened, and the prognosis and prognosis of tuberculosis are not facilitated. The invention starts from exciting the autoimmune force, skillfully avoids the traditional chemical medicines in the treatment process, and achieves the aim of treating tuberculosis by killing Mtb through intercellular interaction.
2. The treatment effect is good: at a certain dosage, the performance of the invention in an in vitro killing Mtb experiment is superior to the combined treatment of isoniazid and rifampicin which are traditional tuberculosis medicines. The CAR-T cells are suggested to have wide application prospect for tuberculosis, and a certain degree of reference and reference are provided for developing more immunotherapy.
3. Has potential value for pressure-resistant tuberculosis and Mtb detention bacteria infection: traditional tuberculosis medicines are easy to cause bacterial drug resistance in the treatment of tuberculosis; or the Mtb is taken off to form L-shaped bacteria, so that the drug sensitivity and the killing effect are further reduced. Not only has no treatment effect, but also is easy to cause repeated infection. The invention avoids the traditional chemotherapy drugs, utilizes CAR-T cells to treat tuberculosis, and CD4 on the premise of not causing bacterial drug resistance + CD8 + T cells cooperate and cooperate to correct unbalance of cellular immunity and humoral immunity, and excite autoimmune power, so as to treat tuberculosis.
4. No off-target effect: when the CAR-T cells recognize the killer tumor cells, the CAR-T cells recognize similar antigens which are underexpressed in normal cells, thereby causing damage to the normal cells. The invention adopts ESAT6 of Mtb as target antigen, and the host normal cells have no ESAT6 and homologous protein expression, thus avoiding off-target effect.
Drawings
FIG. 1 is a diagram showing the double restriction enzyme identification of recombinant plasmid pET22b-ESAT6 according to the embodiment of the invention;
FIG. 2 is a diagram showing sequencing and identification of recombinant plasmid pET22b-ESAT6 according to the embodiment of the invention;
FIG. 3 is a diagram showing SDS-PAGE results of affinity purification of Ni columns according to the embodiment of the present invention;
FIG. 4 is a diagram of a western blot identification purified protein according to an embodiment of the present invention;
FIG. 5A is a schematic illustration of the subtype identification of a monoclonal antibody according to an embodiment of the present invention;
FIG. 5B is a hybridoma cell sequencing display according to an embodiment of the invention;
FIG. 6A shows the results of ascites antibody purification in accordance with an embodiment of the present invention;
FIG. 6B shows the results of antibody titer detection according to the embodiments of the present invention;
FIG. 7A is a human CAR-T cell expressing EGFP under a fluorescence microscope according to an embodiment of the present invention;
FIG. 7B is a mouse CAR-T cell expressing EGFP under a fluorescence microscope according to an embodiment of the present invention;
FIG. 7C is a flow cytometry determination of lentivirus infection efficiency according to an embodiment of the present invention;
FIG. 7D is a graph showing the efficiency of MSCV virus infection as determined by cytometry in accordance with an embodiment of the present invention;
FIG. 8A is a diagram of a human monocyte-macrophage infected with Mtb according to an embodiment of the invention;
FIG. 8B is a BMDM of Mtb-infected mice according to an embodiment of the invention;
FIG. 9 is a graph showing bacterial culture colony counts for in vitro treatment of different potency target versus humanized ESAT6-CAR-T cells in accordance with an embodiment of the present invention;
FIG. 10 is a graph showing bacterial culture colony counts for in vitro treatment of human ESAT6-CAR-T cells at various time points in accordance with an embodiment of the present invention;
FIG. 11 is a graph showing bacterial culture colony counts following in vitro treatment of murine ESAT-6-CAR-T cells at different doses in accordance with an embodiment of the present invention;
FIG. 12 is a bacterial culture colony count following in vitro treatment of murine ESAT-6-CAR-T cells at various time points in accordance with an embodiment of the present invention;
FIG. 13 is an illustration of the identification of a model of infection in animals with lung tissue in accordance with an embodiment of the present invention;
FIG. 14A is a colony count of lungs after 28d of in vivo treatment according to an embodiment of the invention;
FIG. 14B is a colony count of spleen after 28d in vivo treatment according to an embodiment of the present invention.
Detailed Description
The following description of the embodiments is provided in conjunction with the accompanying drawings.
The reagents and apparatus in the examples below were conventional experimental reagents and apparatus.
Example 1:
ESAT6-CAR-T for targeted treatment of tuberculosis has a preservation number of CCTCCNO: C2022374. The ESAT6-CAR-T specifically targets Mtb-ESAT6 antigen and uses CD4 + And CD8 + In combination, recognizes and kills Mtb host cells.
A method of preparing ESAT6-CAR-T for targeted treatment of tuberculosis, the method comprising:
step 1: cloning, expressing, purifying and immunizing the Mtb specific secretion antigen ESAT 6;
step 2: development and screening of an anti-ESAT 6 protein specific single-chain antibody amino acid coding sequence;
step 3: preparation of lentivirus and retrovirus carrying an anti-ESAT 6 protein single-chain antibody sequence;
step 4: preparation of ESAT6-CAR-T lymphocytes.
The step 1 specifically includes:
(1) Optimizing codons and designing primers according to the amino acid sequence of ESAT6 protein of Mtb and the codon preference of escherichia coli, amplifying ESAT6 full-length fragments and inserting a vector pET-22b to construct a recombinant plasmid pET22b-ESAT6, and identifying by double enzyme digestion and sequencing;
(2) Transforming the identified correct recombinant plasmid pET22b-ESAT6 into BL21 competent cells, performing IPTG induction expression, collecting induced thalli, performing ultrasonic disruption, preparing a protein sample, performing SDS-PAGE electrophoresis analysis on protein expression and expression form, performing Ni column affinity chromatography purification on the prepared sample after optimizing induction conditions, performing SDS-PAGE electrophoresis separation on the purified sample, transferring a membrane, incubating a His tag antibody, performing membrane washing, incubating an HRP-marked secondary antibody, and performing color development and exposure;
(3) The ESAT6 recombinant protein identified correctly was mixed and emulsified with an adjuvant, balb/c mice were immunized by subcutaneous multipoint injection at a dose of 100. Mu.g/mouse, boosted once for 2-3 weeks, for a total of 5 immunizations, and antibody titers were detected by ELISA after rat tail blood collection.
The step 2 specifically includes:
the immunized Balb/c mice are subjected to aseptic spleen taking, single cell suspension is prepared, cell fusion is carried out by utilizing a semi-solid and liquid phase combination mode, hybridoma cells are screened, positive hybridoma cells secreting antibodies are identified and screened through ELISA, the hybridoma cells meeting expectations are subjected to expanded culture and cryopreservation, the monoclonal antibodies are subjected to subtype identification, the hybridoma cells are inoculated into the abdominal cavity of the mice after identification, a large amount of monoclonal antibodies are prepared, then ascites of the mice are collected, antibodies are obtained through protein G affinity chromatography, and the corresponding hybridoma cells are sequenced.
The step 3 specifically includes:
connecting the sequences obtained by screening with viral vector plasmids, transforming into host bacteria, selecting positive clones, performing target sequence PCR and sequencing, co-transfecting 293T cells with the identified correct viral recombinant vectors and auxiliary plasmids, collecting supernatant of the transfected 293T cells according to the growth state of the cells, and concentrating and storing the supernatant containing the viruses for subsequent experiments.
The step 4 specifically includes:
PBMC cells were isolated from peripheral blood of healthy adults and purified CD3 by magnetic bead anion separation + T cells, conA stimulated CD3 at a final concentration of 25 g/mL + T cells are activated for 24 hours and then added with slow virus to be transfected to obtain ESAT6-CAR-T cells.
The step 4 specifically includes:
1. human peripheral blood CD3 + T cell sorting and in vitro activation:
(1) Collecting fresh EDTA anticoagulated whole blood, and mixing the whole blood reversely;
(2) Diluting the freshly collected anticoagulated whole blood with PBS according to the volume ratio of 1:1 and fully and uniformly mixing;
(3) Adding the Ficoll separating liquid which is balanced to the room temperature in advance into a 50mL centrifuge tube;
(4) Slowly adding diluted whole blood above the liquid surface of Ficoll separating liquid, wherein the boundary between blood and separating liquid cannot be destroyed;
(5) After sample addition, carefully placing the centrifuge tube into a horizontal centrifuge, centrifuging at room temperature for 800 Xg and 20min, and slowly lifting; the liquid after centrifugation is divided into four layers, and the four layers are sequentially from top to bottom: a plasma layer, a white membrane layer, an outer blood mononuclear cell, a Ficoll separating liquid layer and a red blood cell layer;
(6) Discarding part of upper plasma, collecting cells of the tunica media into a new centrifuge tube, adding a proper amount of PBS, blowing and mixing uniformly by using a sample adding gun, and then carrying out 120 Xg for 10min;
(7) Discarding the supernatant, continuing to resuspend the cells with PBS, 120 Xg, 10min, repeating this step 2 times, with the aim of removing platelets;
(8) Counting the obtained PBMC, and adjusting the cell concentration to 5X 107cells/mL by using PBS, wherein the volume range of the cell suspension is between 0.25 and 2 mL;
(9) The conditioned cell suspension was added to a polystyrene underflow tube to which was added 50L/mL EasySep TM Human T Cell Isolation Cocktail；
(10) Mixing gently with a sample adding gun, and incubating at room temperature for 5min;
(11) EasySep with an oscillator TM Dextran RapidSpheres TM Vortex for 30s, fully mix the magnetic beads;
(12) EasySep TM Dextran RapidSpheres TM Adding the mixture into a sample to 40L/mL, and uniformly mixing;
(13) Supplementing the sample volume to 2.5mL by using PBS, and lightly mixing for 2-3 times by using a sample adding gun;
(14) Put the flow tube into easy Sep TM Placing the magnet at room temperature for 3min;
(15) Taking up the magnet, pouring the liquid in the tube into a new tube, and separating CD3+ T cells from the liquid;
(16) Culturing CD3 with RPMI 1640 complete Medium + T cells, added with Con A protein to a final concentration of 25
g/mL, adding human IL-2 to a final concentration of 20ng/mL, and adding 5% CO 2 Culturing in a cell incubator at 37 ℃ for 24 hours;
2. human CD3 + T cell lentiviral infection:
taking out the virus from the refrigerator at the temperature of-80 ℃, melting in an ice bath in a biosafety cabinet, and storing at the temperature of 4 ℃; human CD3 + The T cells were activated for 24h and then subjected to lentiviral infection,
regulating cell concentration, calculating virus dosage, adding 1×HiTransG A and 1×HiTransG P infection promoting agent, mixing, and adding 5% CO 2 After static culture for 12 hours in a 37 ℃ incubator, the culture medium is replaced by a new RPMI1640 complete culture medium containing human IL-2, and liquid supplementing, passage and harvesting are carried out according to requirements.
Use of ESAT6-CAR-T for targeted treatment of tuberculosis: the ESAT6-CAR-T is used for preparing therapeutic drugs, vaccines and detection reagents for tuberculosis.
Scraping Mtb colony with good growth state on Roche medium into an EP tube containing 0.05% Tween-80 physiological saline, blowing to single bacterial suspension by a sterile syringe, standing overnight, transferring the supernatant into a new sterile EP tube, and measuring OD 600 The method comprises the steps of carrying out a first treatment on the surface of the Collecting peripheral blood of healthy adult, enriching mononuclear cells according to STEM CELL RosetteSepTM Human Monocyte Enrichment Cocktail operation instruction, culturing in vitro with RPMI1640 medium containing GM-CSF, adding the bacterial suspension dropwise after cell adhesion to prepare Mtb macrophage infection model,
ESAT6-CAR-T cells and Mtb-infected macrophages were according to E: t=0.5:1; an effective target ratio of 1:1 or 2:1 to 6h;12h; co-culture was performed for 24h or 48h of time, after cell lysis, roche medium was plated and CFU was counted to evaluate the killing capacity of human ESAT6-CAR-T cells against Mtb.
Example 2:
a preparation method of ESAT6-CAR-T for targeted treatment of tuberculosis,
step 1: cloning, expression, purification and immunization of mycobacterium tuberculosis specific secretion antigen ESAT6
1.1 cloning, expression and purification of Mycobacterium tuberculosis specific secretion antigen ESAT6
(1) According to the codon preference of the host bacteria, optimizing the codon of ESAT6 protein, inserting the ESAT6 full-length fragment into an expression vector pET-22b to construct a recombinant plasmid, and carrying out enzyme digestion identification and gene sequencing verification.
(2) BL21 (DE 3) competent cells were transformed with the recombinant plasmid, plated and cultured upside down at 37℃overnight. Selecting a single positive colony, culturing the single positive colony in LB culture solution at 37 ℃ until the OD600 of the thalli is 0.6-0.8, adding IPTG to a final concentration of 0.2mM, and collecting the bacteria after induction for 16 hours at 15 ℃; centrifuging to collect bacteria, re-suspending the bacteria, and performing ultrasonic crushing; centrifuging, subjecting the supernatant to Ni column affinity purification, and identifying the purified protein by Western Blot.
Expression, purification and Western-blot identification of mycobacterium tuberculosis specific secretion protein ESAT 6:
the cloning of the specific secretory protein ESAT6 gene of the mycobacterium tuberculosis is successfully completed, the recombinant plasmid pET22b-ESAT6 is successfully constructed, and the double enzyme digestion identification and sequencing are correct and meet the expectations (figures 1 and 2); transforming the plasmid into BL21 (DE 3) expression strain, performing amplification culture by inducing with 0.2mM IPTG at 15 ℃, purifying and dialyzing by Ni column to obtain target Protein, wherein the band is single (figure 3: M: protein Mark; P: precipitation after crushing; S: supernatant after crushing; FT: effluent; 1: cleaning sample 20;2: cleaning sample 50;3: eluting sample); western-blot showed that the purified protein could specifically bind to the corresponding antibody, in line with expectations (FIG. 4).
Step 2: development and screening of anti-ESAT 6 protein specific single-chain antibody amino acid coding sequence
1.1 preparation of anti-ESAT 6 protein monoclonal antibodies
1.1.1 animal immunization and serum detection:
(1) Immunization of animals
TABLE 1 animal immunization protocol
(2) Antiserum potency detection and screening
TABLE 2 antiserum titers detection and screening procedure
1.2 monoclonal antibody screening
1.2.1 preparation of hybridoma cells
TABLE 3 hybridoma cell preparation procedure
1.2.2 ascites purification
TABLE 4 purification scheme for ascites
2.1 immunization of animals:
the potency detection shows that the immune effect of the mice meets the expectations, and the next experiment is arranged
2.2 monoclonal antibody detection:
(1) 8 strains of better hybridoma cell fixed strains are selected, sequencing is carried out (figure 5A) and subtype identification is carried out (figure 5B), and 3 strains are selected to prepare ascites.
(2) The purity of the antibody obtained by ascites purification is over 90 percent through SDS-PAGE verification. Purified antibodies were identified and titers were measured as shown in fig. 6.
Step 3: preparation of lentivirus for expressing ESAT6 protein single-chain antibody
1.1 selection of Single chain antibody expression sequences
And (3) synthesizing the early data and comparing the antibody sequences, and selecting 1 clone to construct a subsequent recombinant virus vector, wherein the sequence is as follows:
heavy chain:
base sequence
caggtgcagctgaaggagtctggagctgaactggtaaggcctgggacttcagtgacgatatcctgcaaggcttctggatacgctttcactaactactggctagtttgggtaaagcagaggcctggacatggacttgagtggattggagatatttaccctggaagtgatagtacttactacaatgagaagttcaagggcaaagccacactgactgcagacaaatcctcgagcacagcctatatgcagctcagtagcctgacatctgaggactctgctgtctatttctgtgcaagcggagactactggggccaaggcaccactctcaccgtctcctca
Amino acid sequence
QVQLKESGAELVRPGTSVTISCKASGYAFTNYWLVWVKQRPGHGLEWIGDIYPGSDSTYYNEKFKGKATLTADKSSSLTSEDSAVYFCASGDYWGQGTTLTVSS
Light chain:
base sequence
gatgttttgatgacccaaactccactcactttgtcggttactattggacaaccagcctccatctcttgcaagtcaagtcagagcctcttaggtagtgatggaaagacatatttgaattggttgttacagaggccaggccagtctccaaagcgcctaatctatctggtgtctaaactggactctggagtccctgacaggttcactggcagtggatcagggacagatttcacactgaaaatcagcagagtggaggctgaggatttgggagtttattattgctggcaaggtacacattttcctcagacgttcggtggaggcaccaagctggaaataaaa
Amino acid sequence
DVLMTQTPLTLSVTIGQPASISCKSSQSLLGSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSG
VPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPQTFGGGTKLEIK
1.2 construction of viral expression vectors
1.2.1 plasmid transfection and lentiviral harvesting
(1) 24h before transfection, digesting 293T cells in logarithmic growth phase with trypsin, adjusting cell density to about 5×106 cells/15 ml with 10% serum-containing medium, re-inoculating to 10cm cell culture dish, 37 ℃ and 5% CO 2 Culturing in an incubator. The cell can be used for transfection after 24 hours until the cell density reaches 70% -80%;
(2) 2h before transfection, changing to serum-free culture medium;
(3) Adding each prepared DNA solution (target vector plasmid 20 mug, pHelper 1.0 vector plasmid 15 mug, pHelper 2.0 vector plasmid 10 mug) into a sterilizing centrifuge tube, uniformly mixing with the corresponding volume of transfection reagent, adjusting the total volume to 1ml, and incubating for 15min at room temperature;
(4) Slowly dripping the mixed solution into 293T cell culture solution, mixing, and adding 5% CO at 37deg.C 2 Culturing in a cell incubator;
note that: the addition must be uniform and as little as possible to blow up the cells.
(5) After 6h of culture, the culture medium containing the transfection mixture is discarded, 10ml of PBS solution is added for cleaning once, and the culture dish is gently rocked to wash the residual transfection mixture and then discarded;
(6) Slowly adding 20ml cell culture medium containing 10% serum at 37deg.C and 5% CO 2 Culturing in the incubator for 48-72 h.
1.2.2 lentivirus concentration and purification
(1) Collecting supernatant of 293T cells 48h after transfection (0 h after transfection) according to the cell state;
(2) Centrifuging at 4deg.C for 10min at 4000g to remove cell debris;
(3) Filtering the supernatant with a 0.45 μm filter in a 40ml ultracentrifuge tube;
(4) Balancing samples, putting the ultracentrifuge tubes with virus supernatant into a Beckman ultracentrifuge one by one, setting the centrifugation parameters to 25000rpm, and controlling the centrifugation temperature to 4 ℃ for 2 hours;
(5) Removing the supernatant after centrifugation, removing the liquid remained on the tube wall as much as possible, adding virus preservation liquid (PBS or cell culture medium can be used for substitution), and lightly and repeatedly blowing to resuspension;
(6) After fully dissolving, centrifuging at a high speed of 10000rpm for 5min, sterilizing and filtering by using a filter membrane, and then sub-packaging the supernatant according to the requirement; preparing a sample to be detected.
1.2.3 lentiviral titer detection:
the day before the assay, 293T adherent cells were plated, 96 well plates, 4X 104 cells per well, 100 μl in volume; according to the expected titer of the virus, 7-10 sterile EP tubes were prepared, each tube being added with 90 μl of serum-free medium; adding 10 mu L of virus stock solution to be measured into a first tube, uniformly mixing, adding 10 mu L of virus stock solution into a second tube, and continuing the same operation until the last tube; selecting required cell holes, discarding 90 mu L of culture medium, adding 90 mu L of released virus solution, culturing in an incubator for 24 hours, adding 100 mu L of complete culture medium, and carefully operating without blowing up cells; after 4 days, the fluorescent expression was observed, and the number of fluorescent cells decreased with the increase of dilution factor.
1.3 retroviral vector construction
1.3.1 plasmid transfection and retroviral harvesting
(1) Inoculating HEK293T cells into 100mm culture dish one day before transfection, adding DMEM medium containing 10% FBS, 37deg.C, 5% CO 2 Culturing for 24 hours, wherein the cell fusion degree is about 80-90% before transfection;
(2) 1 hour before transfection, add
Continuously culturing the culture solution;
(3) Mixing A, B solutions: wherein, the solution A is 1.5mL of Opti-MEM and 4 mug of DNA (respectively packaging plasmids SL3, SL4, SL5 and target gene plasmid), the plasmids are added into the Opti-MEM and then blown and evenly mixed by a gun head; the solution B is 1.5mL of Opti-MEM and 40 mu L of Lipofectamine 2000, and the mixture is blown and mixed uniformly by a gun head, and the mixture is kept stand at room temperature for 5min for incubation; adding the solution A into the solution B, blowing and mixing uniformly by using a gun head, standing at room temperature, and incubating for 20min;
(4) Slowly and dropwise adding the incubated transfection complex into a 100mm culture dish for culturing cells, and waterThe mixture is evenly distributed by slight shaking at 37 ℃ and 5 percent of CO 2 Culturing, changing the culture medium to DMEM medium containing 10% FBS after transfection for 6 hr, continuing 37 deg.C, and 5% CO 2 Culturing;
(5) Collecting culture medium supernatant after 48h of transfection, storing at 4 ℃, adding fresh DMEM culture medium containing 10% FBS in the same volume for continuous culture, and collecting the culture medium supernatant once again after 24 h; concentrating the culture medium supernatants collected twice, centrifuging at 4deg.C for 30min at 2000g, and filtering the supernatant with 0.45 μm filter to remove cell debris;
(6) Adding the filtrate into a centrifuge tube, centrifuging at 4deg.C at 50,000g for 2 hr, discarding supernatant, re-suspending retroviral particles with 200 μl HBSS buffer per tube of sediment, packaging, and storing at-80deg.C to obtain virus for transfecting cells.
1.3.2 retrovirus concentration and purification
(1) Adding 200 mu L of virus concentrate into a centrifuge tube, adding 1.5mL of 20% sucrose solution into the upper layer, and centrifuging at 4 ℃ for 2h at 50,000g after balancing;
(2) Centrifuging to remove supernatant, collecting precipitated virus particles, re-suspending each tube of precipitate with 200 μl HBSS buffer, sub-packaging, freezing at-80deg.C, and taking one tube for measuring virus titer.
1.3.3 retrovirus titre assay
(1) Inoculating HEK293T cells into 96-well plates, and inoculating 100 μl of cell suspension (104 cells/well) per well, DMEM medium containing 10% FBS, 5% CO 2 Culturing overnight at 37 ℃;
(2) Carrying out gradient dilution on the virus stock solution by using a DMEM culture medium containing 10% FBS (FBS) according to a limiting dilution method in the next day, sucking off the culture solution in the cell holes from 10 < -1 > to 10 < -10 >, respectively adding the virus solution with the dilution degree of 10 < -4 > to 10 < -10 >, adding 3 parallel holes for each dilution degree, and diluting the suspension with 100 mu L of virus per hole;
(3) After 16h of transduction, the original DMEM medium containing virus particles was replaced with fresh DMEM medium containing 10% FBS, and the incubation was continued at 37℃with 5% CO 2 Culturing;
(4) And observing the fluorescent expression condition under a fluorescent microscope after 48-72 hours. The titer calculation was performed by selecting wells with fluorescence of 1 to 20% according to the following formula.
Viral titer = number of fluorescent cells x dilution x 1000
Experimental results:
lentiviral and retrovirus strains carrying ESAT6-CAR expression sequences are successfully synthesized and can be used for subsequent experiments.
Step 4: preparation of ESAT6-CAR-T lymphocyte and preclinical sterilization effect study
Preparation of 1.1ESAT6-CAR-T lymphocytes
1.1.1CD3 + T cell sorting
1.1.1.1 human peripheral blood CD3 + T cell sorting and in vitro activation
Peripheral blood as used in this application was derived from PPD-negative healthy volunteers.
(1) Collecting fresh EDTA anticoagulated whole blood, and mixing the whole blood reversely;
(2) Diluting the freshly collected anticoagulated whole blood with PBS according to the volume ratio of 1:1 and fully and uniformly mixing;
(3) Adding the Ficoll separating liquid which is balanced to the room temperature in advance into a 50mL centrifuge tube;
(4) Slowly adding diluted whole blood above the liquid surface of Ficoll separating liquid, wherein the boundary between blood and separating liquid cannot be destroyed;
(5) After sample addition, carefully placing the centrifuge tube into a horizontal centrifuge, centrifuging at room temperature for 800 Xg and 20min, and slowly lifting; the liquid after centrifugation is divided into four layers, and the four layers are sequentially from top to bottom: a plasma layer, a white membrane layer, peripheral blood mononuclear cells (Peripheral blood mononuclear cell, PBMC), a Ficoll separation liquid layer, and a red blood cell layer;
(6) Discarding part of upper plasma, collecting cells of a tunica media layer into a new centrifuge tube, adding a proper amount of PBS (diluting redundant Ficoll separating liquid as much as possible), blowing and mixing uniformly by a sample adding gun, and then carrying out 120 Xg for 10min;
(7) Discarding the supernatant, continuing to resuspend the cells with PBS, 120 Xg, 10min, repeating this step 2 times, with the aim of removing platelets;
(8) The obtained PBMC were counted and their cell concentration was adjusted to 5X 10 with PBS 7 cells/mL, the volume range of the cell suspension is between 0.25 and 2 mL;
(9) The conditioned cell suspension was added to a polystyrene underflow tube to which easy Sep was added TM Human T Cell Isolation Cocktail(50μL/mL)；
(10) Mixing gently with a sample adding gun, and incubating at room temperature for 5min;
(11) EasySep with an oscillator TM Dextran RapidSpheres TM Vortex for 30s, fully mix the magnetic beads;
(12) EasySep TM Dextran RapidSpheres TM Adding into a sample (40 mu L/mL), and uniformly mixing;
(13) Supplementing the sample volume to 2.5mL by using PBS, and lightly mixing for 2-3 times by using a sample adding gun;
(14) Put the flow tube into easy Sep TM Placing the magnet at room temperature for 3min;
(15) Picking up the magnet and pouring the liquid in the tube into a new tube, wherein the discharged liquid contains the sorted CD3 + T cells;
(16) Culturing CD3 with RPMI 1640 complete Medium + T cells, con A protein was added to a final concentration of 25. Mu.g/mL, human IL-2 was added to a final concentration of 20ng/mL, and 5% CO was added 2 The cells were cultured in a 37℃incubator for 24 hours.
1.1.1.2 mice spleen CD3 + T cell sorting and in vitro activation
(1) Spleen is taken: selecting healthy 6-7 w female C57BL/6 mice, killing cervical dislocation, and soaking in 75% alcohol for 5min;
(2) Fixing the mouse on a foam plate with the ventral surface facing upwards, cutting a small opening by using ophthalmic scissors, tearing open skin, exposing abdominal cavity, and enabling spleen to be red long;
(3) Taking out spleen with forceps, removing connective tissue or fat on spleen, noticing the presence or absence of necrotic area, and checking whether spleen has tumor, discoloration or lesions, recording the initial state of sample is important for later evaluation of cell quality. The spleen was rinsed 1 time in PBS;
(4) Transferring the spleen to a cell sieve, grinding the spleen by using a syringe needle plug in a gentle circle drawing mode, and releasing spleen cells;
(5) Washing the cell sieve with an appropriate amount of PBS;
(6) Collecting single cell suspension obtained after grinding into a centrifuge tube, 300 Xg, 5min, and collecting cells;
(7) Discarding the supernatant, re-suspending the cells with PBS containing 1mM EDTA, counting the cells, and adjusting the concentration to 1X 108cells/mL, wherein the volume of the cell suspension is between 0.1 and 2 mL;
(8) Adding Rat Serum (50. Mu.L/mL) to the cell suspension;
(9) The sample was added to a 5mL polystyrene underflow tube;
(10) Addition of EasySep to sample TM Mouse T Cell Isolation Cocktail(50μL/mL)；
(11) Mixing, and incubating for 10min at room temperature;
(12) EasySep with an oscillator TM Streptavidin RapidSpheres TM 50001 vortex for 30s, and fully mixing the magnetic beads;
(13) To rapidsphere TM Added to the sample (75. Mu.L/mL);
(14) After mixing evenly, incubating for 2.5min at room temperature;
(15) Supplementing the sample volume to 2.5mL by using PBS containing 1mM EDTA, and lightly mixing for 2-3 times by using a sample adding gun;
(16) Put the flow tube into easy Sep TM Placing in a magnet at room temperature for 2.5min;
(17) The magnet is taken up, and the liquid is poured into a sterile 15ml centrifuge tube; namely CD3 + T cell
(18) Culturing mouse CD3 with RPMI 1640 complete Medium + T cells, con A protein was added to a final concentration of 5. Mu.g/mL, mouse IL-2 was added to a final concentration of 20ng/mL, and 5% CO was added 2 Culturing in an incubator at 37 ℃ for 24 hours.
1.1.2 viral infection
1.1.2.1 human CD3 + T cell lentiviral infection
The virus was taken out of the freezer at-80℃and thawed in an ice bath in a biosafety cabinet and stored at 4℃under strict operating instructions.
(1) Human CD3 + T cells are activated for 24 hours and then are infected by slow viruses, the slow viruses are suicide defective viruses, toxic genes in the viruses are removed and inserted and replaced by exogenous target genes, and the slow viruses belong to pseudotyped viruses. After infection of the target lymphocytes, other cells will not be infected again, and the host cells will not be used to produce new viral particles.
The CAR-T lentivirus is a self-inactivating lentivirus vector, and is coated by VSVG membrane protein. After successful infection of the T cells, the T cells are allowed to express the desired single chain antibody (scFv-chain variable fragment) and intracellular signaling region. According to the currently mainstream dividing method, the two-generation structure carries one costimulatory molecule except for CD3 zeta, and the two-generation structure carries two costimulatory molecules. The intracellular segments of all CAR frames have a structure with self-cleaving polypeptide 2A (self-cleaving 2A peptide) linked to EGFP green fluorescent tag for ease of identification and selection.
Adjusting cell concentration, calculating virus dosage, adding 1×HiTransG A (stock solution 25×) and 1×HiTransG P infection promoter (stock solution 25×), mixing, adding 5% CO 2 After the culture is kept for 12 hours in a 37 ℃ incubator, the culture medium is replaced by a new RPMI 1640 complete culture medium containing human IL-2, the cell state is closely observed during the period, and if the state is not good, the liquid is replaced in advance. According to the experimental requirements, a culture vessel with a larger bottom area is inoculated, from which fluid replacement, passaging and harvesting are carried out as required (whether the medium turns yellow or not).
1.1.2.2 murine CD3 + T cell MSCV Virus infection
Mouse CD3 + Activating T cells for 24 hr, performing MSCV virus infection, regulating cell concentration, calculating virus dosage, adding 1×HiTransG A (stock solution 25×) and 1×HiTransG P infection promoting agent (stock solution 25×), mixing, adding 5% CO 2 After the culture is kept for 12 hours in a 37 ℃ incubator, the culture medium is replaced by a new RPMI 1640 complete culture medium containing mouse IL-2, the cell state is observed during the period, and if the state is not good, the liquid is replaced in advance. According to the experimental requirements, a culture vessel with a larger bottom area is inoculated, from which fluid replacement, passaging and harvesting are carried out as required (whether the medium turns yellow or not).
1.2 in vitro bactericidal effect study of CAR-T cells
1.2.1 Mtb infection model establishment of macrophages
Mononuclear-macrophage induction culture of 1.2.1.1 human peripheral blood source
(1) Fresh whole blood (with CD 3) + T from the same volunteer), mixed EDTA for anticoagulation;
(2) EDTA was added to whole blood to a final concentration of 1mM;
(3) Adding RosetteSep to sample TM Human Monocyte Enrichment Cocktail (50. Mu.L/mL), and incubating at room temperature for 20min after mixing;
(4) Adding PBS with the same volume into the sample, and gently mixing;
(5) Adding a proper amount of Ficoll separating liquid which is restored to the room temperature in advance into a 50mL centrifuge tube;
(6) Slowly adding diluted blood above the liquid level of the Ficoll separating liquid, wherein the boundary between the sample and the separating liquid cannot be destroyed;
(7) After sample addition, carefully placing the centrifuge tube into a horizontal centrifuge, centrifuging at room temperature for 1200 Xg and 20min, and slowly lifting; after centrifugation, the liquid is divided into four layers, which are sequentially from top to bottom: plasma layer, buffy coat (enriched monocytes), ficoll separating layer and other cell layers containing erythrocytes;
(8) Discarding part of upper plasma, collecting cells of the tunica media into a new centrifuge tube, adding a proper amount of PBS, blowing and mixing uniformly by using a sample adding gun, and then carrying out 300 Xg for 10min;
(9) Discarding the supernatant;
(10) Repeating the steps (8) (9);
(11) The cells obtained finally are monocytes;
(12) Monocyte induction system: RPMI 1640 complete medium, final concentration 75ng/mL GM-CSF, completely changed to RPMI 1640 complete medium containing final concentration 75ng/mL GM-CSF at 3d, the induction period was concerned about the cell state, after induction for 5d in vitro, the cell volume was increased, and the suspension cells were changed to adherent cells, and the subsequent infection experiment was performed.
1.2.1.2 induced culture of bone marrow-derived macrophages in mice
(1) Healthy 6-7 w female C57BL/6 mice are selected, the cervical vertebra dislocation is killed, and the mice are soaked in 75% alcohol for 5min;
(2) Fixing the mouse, carefully shaving the legs upward along the feet, opening a small opening at the ankle of the mouse, cutting longitudinally upward, fixing the skin on both sides of the legs, and treating the other side in the same manner;
(3) Trimming the muscles around the femur, and transversely shearing the muscles below the femur along the medial diaphysis of the tibia;
(4) The femur and tibia were separated with sterile gauze and the remaining muscles were stripped, then placed in a 1.5mL Ep tube with RPMI 1640 complete medium, and placed on ice;
(5) Carefully isolating the upper and lower ends of the femur and tibia, rinsing the bone marrow cavity with sterilized PBS, and collecting bone marrow cells in a sterile plate;
(6) The pellets were carefully blown using a 1mL syringe and collected into a centrifuge tube at 800rpm/min for 5min;
(7) A subsequent infection experiment was performed using complete RPMI 1640 medium containing 30ng/mL M-CSF, supplemented with complete RPMI 1640 medium containing 30ng/mL M-CSF for 3d, during which time the cellular status was closely focused and induced to macrophages after 5 d.
1.2.1.3 preparation of Mtb bacterial suspension and construction of macrophage infection model
(1) Preparation of Mtb bacterial suspension
1mL of sterile 0.05% Tween-80 is taken out in a sterilized 1.5mL Ep tube, a bacterial colony growing well on the Roche medium is picked up by a burnt bacterial-taking ring to the 1.5mL Ep tube, firstly, the bacterial colony is repeatedly sucked by a 1mL syringe until no obvious agglomerate exists, and then the bacterial colony is stood at the temperature of 4 ℃ for overnight. The supernatant was transferred to a fresh sterilized 1.5mL Ep tube, and the absorbance (OD) of the bacterial liquid at 600nm was measured by an ultraviolet spectrophotometer 600 ) Smears were acid fast stained to determine if the bacteria were in a single uniform distribution. Bacterial count/mL = OD 600 X 108 x dilution.
(2) Macrophage infection model construction
Calculating the volume of the required bacterial suspension according to MOI and cell number Discarding cell culture supernatant, adding bacterial suspension, 5% CO 2 Placing in a 37 ℃ incubator for 6 hours, and fishing out the climbing slices to carry out acid-resistant staining. The culture was continued after 6h by changing the new RPMI 1640 medium.
1.2.2 CD8 carrying ESAT6-CAR + 、CD4 + T lymphocyte combined sterilization experiment
A ESAT6-CAR-T cell dose gradient:
experimental grouping: infection group, empty T cell group infected with control virus, effective target ratio 0.5:1, effective target ratio 1:1, effective target ratio 2:1, rfp+inh combined treatment group;
b ESAT6-CAR-T cell time gradient:
experimental grouping: infection group, 6h,12h,24h,48h;
1.3 in vivo Sterilization Effect study of CAR-T cells
1.3.1 establishment of a model of Mtb infected mice
(1) Preparing a bacterial suspension: preparing bacterial suspension by using the re-strain, taking 1mL of sterile 0.05% Tween-80 in a sterilized 1.5mL Ep tube, picking a good colony growing on a Roche medium by using a burnt fungus-taking ring to the Ep tube, repeatedly sucking the colony with a 1mL syringe until no obvious agglomerate exists, and standing at 4 ℃ overnight. The supernatant was transferred to a fresh sterilized 1.5mL Ep tube, and the absorbance (OD) of the bacterial liquid at 600nm was measured by an ultraviolet spectrophotometer 600 ) Diluting the suspension to 107CFU/mL;
(2) Infected mice: infection was performed by nasal drip, 50 μl per mouse, 3 per group;
(3) Identification of infection model: mice were sacrificed after 4w cervical fracture, lungs and spleens were taken and placed in a homogenizer, 1mL of physiological saline was added for homogenization, the homogenate was diluted in a proper ratio, 100 μl of coated rogowski medium was taken, and the mixture was placed in a 37 ℃ incubator for 20d for observation. Bacterial colonies are taken to prepare bacterial suspension, and acid-fast staining is carried out.
1.3.2CAR-T cell mice in vivo therapeutic experiments
Mice were sacrificed after 28d following experimental grouping (normal, infected, control virus infected T cell group, ESAT6-CAR-T cell treated group, rfp+inh combined treatment group) by CAR-T cell reinfusion. Lung and spleen tissue homogenate is then coated with Roche medium, and the change of tissue load after treatment of mice is observed.
Experimental results:
2.1 viral infection and expression
After 72h of lentivirus infection, human CD3 was successfully infected with lentivirus + Green fluorescence was seen under T cell fluorescence microscopy (fig. 7A), and flow cytometry results suggested that lentiviruses infected human peripheral blood CD3 + The infection efficiency of T cells was 55.3% (fig. 7C); 72h after MSCV virus infection, mice successfully infected with the virus spleen CD3 + Green fluorescence was seen under a T cell fluorescence microscope (FIG. 7B), and the results of flow cytometry suggested that MSCV virus infected mice spleen CD3 + The T cell infection efficiency was 14.2% (FIG. 7D).
2.2 in vitro experiments to study the intracellular killing effects of ESAT6-CAR-T cells
2.2.1 successful construction of Mtb infected cell models
After primary macrophages were infected with the supernatant of Mtb bacterial liquid left standing overnight, the climbing plates were acid-fast stained, and the result showed that Mtb positive for red rod-shaped acid-fast staining was engulfed by macrophages and scattered in the cytoplasm (fig. 8).
2.2.2 in vitro killing of human ESAT6-CAR-T cells against Mtb in human monocyte-macrophages
To determine the efficacy of ESAT6-CAR-T cells, we first determined their effect on bacteria within macrophages. In vitro killing experiments ESAT6-CAR-T cells were evaluated for effectiveness in vitro against tuberculosis by comparing the toxicity of ESAT6-CAR-T cells and empty T cells infected with control virus (hereinafter referred to as empty T cells) to Mtb in macrophages. After in vitro treatment, the cells are lysed for bacterial culture, and bacterial colonies are counted after bacterial colonies are grown.
2.2.2.1 Effect of different potency targeting ratios human ESAT6-CAR-T cells on Mtb survival in human monocyte-macrophages
We judged the effect of different effective target ratios ESAT6-CAR-T cells on bacteria in macrophages by colony counting. Bacterial culture was performed on lysed cells 24h after in vitro treatment, colonies were counted after bacterial colonies developed (FIG. 9), and the results of Roche culture counts showed that ESAT6-CAR-T cells to macrophages had an effective target ratio of 0.5:1 (2.262.+ -. 0.028, P < 0.01), an effective target ratio of 1:1 (2.139.+ -. 0.017, P < 0.01) and an effective target ratio of 2:1 (0.577.+ -. 0.508, P < 0.01) compared to the infected group (2.471.+ -. 0.066), and the colony counts were significantly reduced. Wherein the effective target ratio 2:1 group had significantly decreased colony counts compared to the empty T cell group (2.073 ±0.165, p < 0.01) and the combined rif+inh treatment group (1.698±0.037, p < 0.05) infected with the control lentivirus. It is demonstrated that human CAR-T cells against ESAT6 protein have killing effect on Mtb in human mononuclear-macrophages, wherein the effect of killing intracellular Mtb is superior to the rif+inh combination therapy at an effective target ratio of 2:1.
2.2.2.2 effects of human ESAT6-CAR-T cells on Mtb survival in human monocyte-macrophages at different time points
We selected the effect of human ESAT6-CAR-T cells on Mtb survival in human monocytes-macrophages at different time points under the same effective targeting conditions. After various times of in vitro treatment, the lysed cells were subjected to bacterial culture, and the number of colonies grown was counted (FIG. 10), and the results of Roche culture showed that the colony counts were significantly decreased after 6h (2.187.+ -. 0.042, P < 0.01), 12h (2.007.+ -. 0.075, P < 0.01), 24h (1.901.+ -. 0.025, P < 0.0001), 48h (1.797.+ -. 0.053, P < 0.0001) of treatment compared to the infection group (2.368.+ -. 0.030).
2.2.3 in vitro killing of mouse ESAT6-CAR-T cells against Mtb in mouse BMDM
2.2.3.1 Effect of different targeting ratios murine ESAT6-CAR-T cells on Mtb survival in mouse BMDM
We judged the effect of different effective target ratios ESAT6-CAR-T cells on intracellular bacteria by colony counting. After 24h of in vitro treatment, cells were lysed for bacterial culture, colonies were counted after bacterial colony growth (FIG. 11), and the Roche culture results showed that the effective target ratio was 0.5:1 (2.277 + -0.038, P < 0.05), the effective target ratio was 1:1 (2.244 + -0.036, P < 0.05), the effective target ratio was 2:1 (2.171 + -0.048, P < 0.01) and the colony count was significantly reduced compared to the infected group (2.407+ -0.050). The colony counts were significantly reduced in the 1:1 (P < 0.05), 2:1 (P < 0.01) and combined RIF+INH treatment groups (1.698+ -0.037, P < 0.05) compared to the empty T cell group, but none of the CAR-T treatment groups had as much therapeutic effect as the combined RIF+INH treatment group. Taken together, it is demonstrated that mouse ESAT6-CAR-T cells have killing effects on Mtb in mouse BMDM.
2.2.3.2 Effect of mouse ESAT6-CAR-T cells on Mtb survival in mouse BMDM at different time points
The effect of mouse ESAT6-CAR-T cells on Mtb survival in mouse BMDM at different time points under the same effective targeting conditions was selected. After various times of in vitro treatment, the lysed cells were subjected to bacterial culture, and the number of colonies grown was counted (FIG. 12), and the results of Roche culture showed a significant decrease in colony counts after 12h (1.941.+ -. 0.044, P < 0.05), 24h (1.882.+ -. 0.048, P < 0.01) and 48h (1.825.+ -. 0.096, P < 0.05) compared to the infected group (2.702.+ -. 0.051).
2.3 in vivo experiments to study the Mtb killing effect of ESAT6-CAR-T cells
2.3.1 identification of animal tuberculosis infection model
To examine the in vivo therapeutic effects of ESAT6-CAR-T, we first constructed an infected mouse model. Mice were sacrificed 28d after infection, homogenized lung and spleen tissues, plated with roche medium, and after 20d incubation, granular, beige, opaque colonies were observed on the roche medium, which proved to be mycobacterium tuberculosis after acid fast staining, and elongated rod-like red bacteria were found in acid fast staining of lung tissues (fig. 13).
2.3.2 in vivo post-treatment organ colony count
In vivo treatment 28d mice were homogenized for lung and spleen and then plated with rogowski medium, and colony counts were performed after 4w, and lung colony count results (fig. 14A) showed that no-load T cell group (2.357±0.112), CAR-T group (2.039 ±0.054), rif+inh group (0±0) had a reduced number of colonies compared to the infected group (2.387 ±0.080), where the reduction in the number of colonies in CAR-T group (P < 0.01), rif+inh group (P < 0.0001) had a statistical significance, and there was no statistical difference compared to no-load T cell group; the colony count was reduced in the CAR-T group (P < 0.05) compared to the empty T cell group, and the difference was statistically significant. Spleen colony count results (fig. 14B) showed that CAR-T group (1.901±0.173) and rif+inh group (0.515±0.452) had statistically significant colony count reduction compared to the infected group (2.189±0.430), and CAR-T group reduction did not have statistically significant (P < 0.01); the CAR-T group showed a decreasing trend but no statistical significance compared to the empty T cell group (2.239 ±0.189).
The invention obtains a high-affinity single-chain antibody sequence of a targeted Mtb-ESAT6 protein: ESAT6 as specific secretory protein of mycobacterium tuberculosis plays an important role in the occurrence and development of tuberculosis, and various researches show that the ESAT6 protein can promote the occurrence of diseases by regulating and controlling the function change of T cells, influencing macrophage lipid dripping level and other various ways. But simultaneously ESAT6 is used as antigen protein which is rich in T cell expression and is also an excellent target antigen, and the invention screens high-affinity targeted single-chain antibody (scFv) by the traditional hybridoma technology aiming at the ESAT6 antigen, thereby laying a preliminary experiment foundation for developing target drugs and constructing CAR-T cells.
Obtaining Mtb-ESAT6-CAR-T cells: traditional drug therapy of tuberculosis is easy to cause bacterial drug resistance and has great toxic and side effects, and a plurality of researches and clinical experiments on tuberculosis immunotherapy exist at present. However, in most schemes, a primer exogenous immune preparation is needed to assist the organism to correct the immune state, and a certain biological safety exists. The invention selects the cell immune preparation constructed by autologous T cells, and kills the mycobacterium tuberculosis through intercellular cooperation under the condition of not introducing exogenous preparation.
CAR-T cells were first used for in vivo, in vitro treatment of tuberculosis: CAR-T cell therapy has shown significant clinical value in hematological tumor therapy, with Mtb infection resembling to some extent the occurrence and progression of tumors. The invention discovers the great clinical transformation value of the CAR-T cell therapy in tuberculosis for the first time and utilizes CD4 + And CD8 + The Mtb host cells are identified and killed in combination and the therapeutic effect on tuberculosis is verified by both in vitro and in vivo therapies.
The above embodiments are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement the same, and are not intended to limit the scope of the present invention. All equivalent changes or modifications made in accordance with the essence of the present invention are intended to be included within the scope of the present invention.
Claims (10)
1. An ESAT6-CAR-T for targeted treatment of tuberculosis, characterized by: the preservation number of ESAT6-CAR-T is CCTCC NO: C2022374.
2. ESAT6-CAR-T targeted to treat tuberculosis according to claim 1, characterized in that: the ESAT6-CAR-T specifically targets Mtb-ESAT6 antigen and uses CD4 + And CD8 + In combination, recognizes and kills Mtb host cells.
3. A method of preparing ESAT6-CAR-T for targeted treatment of tuberculosis according to any one of claims 1-2, wherein: the preparation method comprises the following steps:
step 1: cloning, expressing, purifying and immunizing the Mtb specific secretion antigen ESAT 6;
step 2: development and screening of an anti-ESAT 6 protein specific single-chain antibody amino acid coding sequence;
Step 3: preparation of lentivirus and retrovirus carrying an anti-ESAT 6 protein single-chain antibody sequence;
step 4: preparation of ESAT6-CAR-T lymphocytes.
4. The method for preparing ESAT6-CAR-T for targeted therapy of tuberculosis according to claim 3, wherein said step 1 specifically comprises:
(1) Optimizing codons and designing primers according to the amino acid sequence of ESAT6 protein of Mtb and the codon preference of escherichia coli, amplifying ESAT6 full-length fragments and inserting a vector pET-22b to construct a recombinant plasmid pET22b-ESAT6, and identifying by double enzyme digestion and sequencing;
(2) Transforming the identified correct recombinant plasmid pET22b-ESAT6 into BL21 competent cells, performing IPTG induction expression, collecting induced thalli, performing ultrasonic disruption, preparing a protein sample, performing SDS-PAGE electrophoresis analysis on protein expression and expression form, performing Ni column affinity chromatography purification on the prepared sample after optimizing induction conditions, performing SDS-PAGE electrophoresis separation on the purified sample, transferring a membrane, incubating a His tag antibody, performing membrane washing, incubating an HRP-marked secondary antibody, and performing color development and exposure;
(3) The ESAT6 recombinant protein identified correctly was mixed and emulsified with an adjuvant, balb/c mice were immunized by subcutaneous multipoint injection at a dose of 100. Mu.g/mouse, boosted once for 2-3 weeks, for a total of 5 immunizations, and antibody titers were detected by ELISA after rat tail blood collection.
5. A method of preparing ESAT6-CAR-T for targeted treatment of tuberculosis according to claim 3, wherein: the step 2 specifically includes:
the immunized Balb/c mice are subjected to aseptic spleen taking, single cell suspension is prepared, cell fusion is carried out by utilizing a semi-solid and liquid phase combination mode, hybridoma cells are screened, positive hybridoma cells secreting antibodies are identified and screened through ELISA, the hybridoma cells meeting expectations are subjected to expanded culture and cryopreservation, the monoclonal antibodies are subjected to subtype identification, the hybridoma cells are inoculated into the abdominal cavity of the mice after identification, a large amount of monoclonal antibodies are prepared, then ascites of the mice are collected, antibodies are obtained through protein G affinity chromatography, and the corresponding hybridoma cells are sequenced.
6. A method of preparing ESAT6-CAR-T for targeted treatment of tuberculosis according to claim 3, wherein: the step 3 specifically includes:
connecting the sequences obtained by screening with viral vector plasmids, transforming into host bacteria, selecting positive clones, performing target sequence PCR and sequencing, co-transfecting 293T cells with the identified correct viral recombinant vectors and auxiliary plasmids, collecting supernatant of the transfected 293T cells according to the growth state of the cells, and concentrating and storing the supernatant containing the viruses for subsequent experiments.
7. A method of preparing ESAT6-CAR-T for targeted treatment of tuberculosis according to claim 3, wherein: the step 4 specifically includes:
PBMC cells were isolated from peripheral blood of healthy adults and purified CD3 by magnetic bead anion separation + T cells, terminalConA at a concentration of 25g/mL stimulated CD3 + And (3) activating the T cells for 24 hours, and then adding slow viruses to perform transfection to obtain ESAT6-CAR-T cells.
8. A method of preparing ESAT6-CAR-T for targeted treatment of tuberculosis according to claim 3, wherein: the step 4 specifically includes:
1. human peripheral blood CD3 + T cell sorting and in vitro activation:
(1) Collecting fresh EDTA anticoagulated whole blood, and mixing the whole blood reversely;
(2) Diluting the freshly collected anticoagulated whole blood with PBS according to the volume ratio of 1:1 and fully and uniformly mixing;
(3) Adding the Ficoll separating liquid which is balanced to the room temperature in advance into a 50mL centrifuge tube;
(4) Slowly adding diluted whole blood above the liquid surface of Ficoll separating liquid, wherein the boundary between blood and separating liquid cannot be destroyed;
(5) After sample addition, carefully placing the centrifuge tube into a horizontal centrifuge, centrifuging at room temperature for 800 Xg and 20min, and slowly lifting; the liquid after centrifugation is divided into four layers, and the four layers are sequentially from top to bottom: a plasma layer, a white membrane layer, an outer blood mononuclear cell, a Ficoll separating liquid layer and a red blood cell layer;
(6) Discarding part of upper plasma, collecting cells of the tunica media into a new centrifuge tube, adding a proper amount of PBS, blowing and mixing uniformly by using a sample adding gun, and then carrying out 120 Xg for 10min;
(7) Discarding the supernatant, continuing to resuspend the cells with PBS, 120 Xg, 10min, repeating this step 2 times, with the aim of removing platelets;
(8) Counting the obtained PBMC, and adjusting the cell concentration to 5X 107cells/mL by using PBS, wherein the volume range of the cell suspension is between 0.25 and 2 mL;
(9) The conditioned cell suspension was added to a polystyrene underflow tube to which was added 50L/mL EasySep TM Human T Cell Isolation Cocktail；
(10) Mixing gently with a sample adding gun, and incubating at room temperature for 5min;
(11) EasySep with an oscillator TM Dextran RapidSpheres TM Vortex for 30s, fully mix the magnetic beads;
(12) EasySep TM Dextran RapidSpheres TM Adding the mixture into a sample to 40L/mL, and uniformly mixing;
(13) Supplementing the sample volume to 2.5mL by using PBS, and lightly mixing for 2-3 times by using a sample adding gun;
(14) Put the flow tube into easy Sep TM Placing the magnet at room temperature for 3min;
(15) Picking up the magnet and pouring the liquid in the tube into a new tube, wherein the discharged liquid contains the sorted CD3 + T cells;
(16) Culturing CD3 with RPMI 1640 complete Medium + T cells, con A protein was added to a final concentration of 25g/mL, human IL-2 was added to a final concentration of 20ng/mL, and 5% CO was added 2 Culturing in a cell incubator at 37 ℃ for 24 hours;
2. human CD3 + T cell lentiviral infection:
taking out the virus from the refrigerator at the temperature of-80 ℃, melting in an ice bath in a biosafety cabinet, and storing at the temperature of 4 ℃; human CD3 + The T cells were activated for 24h and then subjected to lentiviral infection,
regulating cell concentration, calculating virus dosage, adding 1×HiTransG A and 1×HiTransG P infection promoting agent, mixing, and adding 5% CO 2 After static culture for 12 hours in a 37 ℃ incubator, the culture medium is replaced by a new RPMI1640 complete culture medium containing human IL-2, and liquid supplementing, passage and harvesting are carried out according to requirements.
9. Use of ESAT6-CAR-T according to any one of claims 1-2 for the targeted treatment of tuberculosis, or the ESAT6-CAR-T prepared according to the method of any one of claims 3-8, characterized in that: the ESAT6-CAR-T is used for preparing therapeutic drugs, vaccines and detection reagents for tuberculosis.
10. Use of ESAT6-CAR-T for the targeted treatment of tuberculosis according to claim 9, characterized in that:
scraping well-grown Mtb colonies on Roche medium into EP tube containing 0.05% Tween-80 physiological saline withoutThe bacterial syringes were blown into a single bacterial suspension, allowed to stand overnight, the supernatant transferred to a fresh sterile EP tube and OD was measured 600 The method comprises the steps of carrying out a first treatment on the surface of the Collecting peripheral blood of healthy adult, enriching mononuclear cells according to STEM CELL RosetteSepTM Human Monocyte Enrichment Cocktail operation instruction, culturing in vitro with RPMI1640 medium containing GM-CSF, adding the bacterial suspension dropwise after cell adhesion to prepare Mtb macrophage infection model,
ESAT6-CAR-T cells and Mtb-infected macrophages were according to E: t=0.5:1; an effective target ratio of 1:1 or 2:1 to 6h;12h; co-culture was performed for 24h or 48h of time, after cell lysis, roche medium was plated and CFU was counted to evaluate the killing capacity of human ESAT6-CAR-T cells against Mtb.
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