CN112831525A - Simple and efficient lentivirus cryopreservation liquid and application thereof - Google Patents
Simple and efficient lentivirus cryopreservation liquid and application thereof Download PDFInfo
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Abstract
The invention provides a simple and efficient lentivirus cryopreservation solution and application thereof, belonging to the field of immune cell therapy. The formula of the lentivirus freezing solution comprises: human plasma albumin (HSA) injection, 0.9% normal saline injection; wherein the concentration of HAS comprises 10%, 20%, 30%, 40%.
Description
Technical Field
The invention relates to the field of immune cell therapy, in particular to a chimeric antigen receptor (CAR-T) lentivirus cryopreservation solution and application thereof.
Background
Chimeric Antigen Receptor T Cells (CART) are produced by expressing an artificially synthesized CAR molecule (Chimeric Antigen Receptor) on the T cell membrane by means of genetic modification, so that the T cells recognize and kill tumor cells in an Antigen-antibody binding manner. CAR molecules include an extracellular binding region (a monoclonal antibody-derived single chain antibody (scFv) that specifically recognizes a target antigen), a hinge region, a transmembrane region, and an intracellular signal segment. This technique has several advantages, for example, the recognition of TAA (tumor associated antigen) on the tumor surface by CAR proteins is performed in an MHC (major histocompatibility complex) -independent manner, so that CART cells can overcome the immune attack of tumor cells by down-regulating MHC molecules, and CAR recognition is not MHC-restricted, and the same CAR can be applied to different patients. In addition, CAR proteins can recognize any type of antigen on the cell surface, including proteins, carbohydrates, glycolipids, such that the CAR greatly increases the range of tumor surface markers that T cells can recognize relative to TCR which can only recognize MHC-peptide fragments.
At present, the CAR-T treatment mainly adopts lentivirus carrying CAR specifically aiming at tumor antigens as a gene delivery carrier, but one bottleneck at present is that after the lentivirus is subjected to super-separation concentration or concentration purification and is re-dissolved by PBS or normal saline, the T cell infection efficiency of fresh lentivirus is very high, but the lentivirus is preserved at minus 80 ℃, and the infection efficiency of the lentivirus after freeze thawing is about 40% -60%.
The invention takes CD19 CAR-T with mature technology at home and abroad as an example at present, and verifies the protection efficiency of the developed lentivirus frozen stock solution on lentivirus particles, wherein CD19 scFv adopts a classical FMC63 antibody, and the optimal effective concentration component is finally determined to be 30-40% by screening the comparison of infection efficiency of HSA with different concentrations on the lentivirus after freeze thawing, so that the lentivirus frozen stock solution meeting the requirements of IND and NDA is provided for the industrial development of CAR-T treatment.
Disclosure of Invention
The invention aims to solve the problems that in the current gene therapy, lentivirus serving as a gene delivery vector is unstable in low-temperature storage, and the infection efficiency is obviously reduced after freeze thawing, so that the treatment effect is unstable and even fails, and the invention provides the following technical scheme:
in a first aspect, the invention provides a formula of a lentivirus freezing medium, which comprises the following components in volume fraction: 10-40% of human plasma albumin (HSA) and 60-90% of normal saline injection.
In the second aspect, the invention provides a method for optimizing the formula of the slow virus frozen stock solution, and the method has the advantages of single formula component, simple and easy operation, obvious effect, low preparation cost and easy industrial production.
In a third aspect, the invention provides a use of the lentiviral cryopreservation solution of the first aspect in the preparation of CART cells.
In a fourth aspect, the invention provides a use of the above-mentioned lentivirus frozen stock solution in the preparation of a medicine for treating tumors by using CART cells.
In the fifth aspect, the formula of the lentivirus frozen stock solution provided by the invention is not limited to CAR-T treatment methods, but can also be used for all lentivirus-based gene treatment methods.
Advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of embodiments of the invention.
Drawings
FIG. 1 is a comparison of cryoS 001-20% versus two commercial lentivirus stocks after three freeze-thaw cycles
FIG. 2 is a comparison of CAR positivity after three freeze-thaw of cryoS 001-20% and two commercial lentivirus cryopreserved fluids
FIG. 3 is a comparison of flow assays of three freeze-thaw cycles of four lentivirus stocks of different concentrations
FIG. 4 is a comparison of infection efficiency of three freeze-thaw cycles of lentiviruses for four different concentration cryopreservation solution formulations
FIG. 5 is a graph showing the stability comparison of three freeze thawing of four different concentrations of lentivirus frozen stock solutions
FIG. 6 comparison of infection efficiency of three freeze-thaw cycles of lentiviruses for four different concentration cryopreservation solution formulations
Detailed Description
While the following is a description of the preferred embodiments of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention. And the instruments, reagents, materials and the like referred to in the following examples are conventional instruments, reagents, materials and the like in the prior art and are commercially available in a normal way unless otherwise specified. Unless otherwise specified, the experimental methods, detection methods, and the like in the following examples are conventional experimental methods, detection methods, and the like in the prior art.
The following will further describe the present invention with reference to specific embodiments.
Example 1: CD19 CAR lentiviral vector construction
FMC63 scFv fragment SEQ ID NO: 1, the hinge, transmembrane and intracellular regions of the corresponding secondary CAR after the signal peptide SEQ ID NO: 2, handed over to Huada Gene Co. After two sections of synthetic genes are obtained respectively, the vector construction of CD19 CAR is carried out, firstly PCR amplification is carried out, enzyme cutting sites are added at the head and the tail of the two sections of sequences respectively, and then the two sections of sequences are subjected to enzyme cutting and enzyme linkage to obtain the sequence shown in SEQ ID NO: 3, mixing a lentiviral vector pCDH-EF1a-T2A-puro and SEQ ID NO: 3, respectively carrying out double enzyme digestion and T4DNA ligase connection, transforming competent cells, selecting single clone, carrying out bacterial liquid sequencing, and greatly extracting plasmid to obtain the lentiviral vector pCDH-EF1a-CD19 CAR plasmid with correct sequence.
Example 2: CD19 CAR lentivirus preparation
After mixing the lentiviral packaging plasmid mixture (including PSPAX2 and VSVG) with the pCDH-EF1a-CD19 CAR plasmid in pre-optimized ratios, the co-transfection reagent was added and incubated for 30 minutes at room temperature. The transfection mixture was then slowly added to 293T cells (purchased from ATCC). Collecting culture medium supernatant after 1-3 days to obtain crude lentivirus solution. After centrifugation at 500g for 10min at 4 ℃ the supernatant was collected for a total of 180 ml. 180 ml of the sample was divided into 6 tubes on average, each 30 ml tube was placed in an ultracentrifuge tube, and after trimming, the lentivirus was concentrated by ultracentrifugation at 20000g for 2 hours.
Example 3: pre-experiment of lentivirus frozen stock solution
In this example, the protective efficiency of a lentivirus of CD19 CAR was compared with that of a commercial lentivirus stock solution of type 2 (brand 1, Yumeibo, cat # UR40202, cat # UB, brand 2, Andefuno, cat # D022, cat # ABP) to confirm the effectiveness of the lentivirus stock solution.
After the end of the dispensed lentivirus superisolation in example 2 above, the supernatant was carefully discarded and the lentiviral particles were resuspended in a pre-cooled lentivirus stock: concentrating 60 ml of lentivirus stock solution in two super-dissociation tubes, removing supernatant, re-suspending each tube with 0.5ml of UB or ABP or cryoS001, namely dividing the super-concentrated 180 ml of lentivirus stock solution into three equal parts, each 60 ml, re-suspending each part with 1ml of corresponding lentivirus frozen stock solution after concentration, lightly blowing, uniformly mixing, sterilizing and filtering with a 0.22 mu m filter membrane, subpackaging into 100 mu L/tube, and storing in a refrigerator at-80 ℃.
After 24 hours of cryopreservation, the infection efficiency of lentiviruses preserved in the three lentivirus cryopreservation solutions was tested:
1) the first day, 1E +05 293T cells were seeded in 24-well plates in 1ml DMEM/FBS medium containing 10% Fetal Bovine Serum (FBS) per well.
2) The following day, 3ml of DMEM/FBS +1/1000 polyclonal mixture was prepared, vortexed and after yesterday inoculation of 293T cells in each well was aspirated, 0.5ml of DMEM/FBS +1/1000 polyclonal mixture was added to each well. Freezing and thawing the three kinds of lentiviruses on ice, adding 1-10 mu L of each lentivirus into each well, infecting cells of 2 wells with each lentivirus, and freezing and storing the rest lentiviruses in an ultra-low temperature refrigerator at-80 ℃.
3) On the third day, each well was supplemented with 1ml of DMEM/FBS medium and the culture was continued.
4) On the fifth day, cells were digested from 24-well plates with 2.5% pancreatin, 1E +06 cells were counted in one well and subjected to flow assay, and DNA was extracted from all cells in the other well and frozen at-80 ℃.
5) The stained 293T cells were flow-tested for CAR positivity of 293T cells 72 hours after infection to test the infection efficiency of lentiviruses.
6) Repeating the steps 1-5, putting the lentivirus subjected to freeze thawing once into an ultralow temperature refrigerator at minus 80 ℃ for 24 hours, then carrying out secondary infection, and putting the rest lentivirus tubes into a refrigerator at minus 80 ℃ for storage after the experiment is finished.
7) Repeating the steps 1-5, putting the lentivirus subjected to freeze thawing twice into an ultralow-temperature refrigerator at-80 ℃ for 24 hours, then carrying out infection for the third time, and after the experiment is finished, putting the rest lentivirus tubes into a 0.6% sodium hypochlorite solution, soaking for 1 hour, and then treating according to the biological waste.
Results are shown in fig. 1, and results of three freeze-thaw experiments are shown in fig. 2 by bar graph representation. The results show that the efficiency of each 293T infection of the lentivirus frozen stock solution is higher than that of 2 commercial lentivirus frozen stock solutions after repeated freeze thawing.
Example 4: optimization experiment of slow virus frozen stock solution formula
1) A batch of CD19 CAR lentivirus was reconstituted as in example 2 above, with a total lentivirus stock volume of 120 ml, 30 ml virus stock in one tube for 4 tubes, and lentivirus concentration was performed by ultracentrifugation at 20000g for 2 hours.
2) Preparing slow virus freezing solutions with different HSA concentrations:
0.1mL +0.9mL of physiological saline for cryoS 001-10%, 0.2mL +0.8mL of physiological saline for cryoS 001-20%, 0.3mL +0.7mL of physiological saline for cryoS 001-30%, and 0.4mL +0.6mL of physiological saline for cryoS 001-40%, mixing the above lentivirus frozen stock solution uniformly, and pre-cooling at 4 ℃.
3) After the completion of lentivirus superisolation, the supernatant was carefully discarded, and the lentivirus particles were resuspended in 0.5mL of each of the above-mentioned precooled lentivirus stocks. Namely, 240 mL of the stock solution of the lentivirus subjected to the super-separation concentration is divided into four parts, each part is 60 mL, the four parts are respectively resuspended by 1mL of the cryoS 001-10%/20%/30%/40%, the mixture is lightly blown, sterilized and filtered by a 0.22 mu m filter membrane, and the mixture is stored in an ultra-low temperature refrigerator at minus 80 ℃ after being subpackaged into 100 mu L/tube.
4) After the lentiviruses are frozen and stored for 24 hours, the infection efficiency of the lentiviruses stored in four lentivirus frozen stock solutions is detected:
5) the steps 1-5 in the example 3 are repeated, and the lentivirus after freeze thawing once is stored in an ultra-low temperature refrigerator at-80 ℃.
6) Repeating the steps 1-5 in the embodiment 3, placing the lentivirus subjected to freeze thawing once into an ultra-low temperature refrigerator at minus 80 ℃ for 24 hours, then carrying out secondary infection, placing the rest lentivirus tubes into the ultra-low temperature refrigerator at minus 80 ℃ for storage after the experiment is finished,
7) repeating the steps 1-5 in the embodiment 3, placing the lentivirus subjected to freeze thawing twice into an ultralow temperature refrigerator at-80 ℃ for 24 hours, then carrying out infection for the third time, placing the rest lentivirus tubes into a 0.6% sodium hypochlorite solution after the experiment is finished, soaking for 1 hour, and then disposing according to biohazard garbage.
The results are shown in FIG. 3 and FIG. 4. The result shows that the lentivirus frozen stock solution with the cryoS 001-40% formula has the best stability of the lentivirus stored in the lentivirus frozen stock solution after repeated freeze thawing, but the lentivirus frozen stock solution with the cryoS 001-30% formula can meet the requirements based on that the lentivirus is frozen and thawed once for use in the prior art.
In fig. 3, the virus titers of each reagent used in three freeze-thaw cycles are shown in the following table:
example 5: stability study of Lentiviral cryopreservation solution
After the lentiviruses preserved in the four lentivirus frozen stock solutions prepared by the method in the embodiment 4 are placed in an ultra-low temperature refrigerator with the temperature of minus 80 ℃ for 6 months, one lentivirus preserved for 6 months is taken to perform freeze thawing and 293T infection experiments again for three times, the protective effect of the lentivirus frozen stock solutions with different concentrations of HSA formulas on the lentiviruses is verified, the steps are the same as those in the embodiment 4, and the results are shown in a figure 5 and a figure 6. The result shows that the lentivirus preserved by the lentivirus freezing solution disclosed by the invention is preserved for 6 months at the temperature of-80 ℃, the infection efficiency of the lentivirus of each formula is basically unchanged, after repeated freezing and thawing, the stability of the lentivirus preserved by the lentivirus freezing solution with the cryoS 001-40% formula is optimal, but the lentivirus freezing solution with the cryoS 001-30% formula can meet the requirements based on the fact that the existing lentivirus in the industry is frozen and thawed once for use.
In fig. 5, the virus titers of each reagent used in three freeze-thaw cycles are shown in the following table:
Claims (5)
1. a simple and efficient lentivirus freezing medium and application thereof are characterized in that: is prepared from human plasma albumin (HSA) 10-40% and physiological saline 60-90%.
2. The simple and highly effective lentiviral cryopreservation solution and use thereof on claim 1, wherein the concentration of HAS is 10%, 20%, 30%, 40%.
3. A simple and highly effective lentiviral cryopreservation solution according to claim 1 as well as the use thereof in any one of claims 1 to 2, comprising:
and (3) precooling the lentivirus frozen stock solution, adding the precooled lentivirus frozen stock solution into the lentivirus particles for resuspension, lightly blowing, uniformly mixing, sterilizing and filtering by using a filter membrane of 0.22 mu m, and storing in an ultra-low temperature refrigerator at-80 ℃.
4. A simple and highly potent frozen stock solution of lentivirus according to claim 1 as defined in any of claims 1 to 3 and its use for the preparation of CART cells and lentivirus-based gene therapy.
5. The use of claim 4, wherein the CART cells prepared are for use in a medicament for treating a tumor.
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