Rapid packaging method for lentivirus meeting IIT clinical quality inspection standard and application

Abstract

The invention discloses a slow virus rapid packaging method conforming to IIT clinical quality inspection standards and application, and belongs to the technical field of biological medicine. Comprising the following steps: 1) laying 293T cells into a cell culture dish for incubation to prepare a 293T cell culture dish; 2) preparing a target plasmid and a packaging helper plasmid according to a ratio, adding the target plasmid and the packaging helper plasmid into a transfection reagent to prepare a plasmid and transfection reagent compound, adding the plasmid and transfection reagent compound into a 293T cell culture dish for culture, replacing a serum-free culture medium for continuous culture, and collecting lentivirus supernatant; and (3) centrifuging and filtering the lentivirus supernatant, adding all-purpose nuclease for incubation, then adding a PEG8000 solution, uniformly mixing, standing overnight, centrifuging, collecting lentivirus precipitate, dissolving the lentivirus precipitate by adopting a resuspension, standing, blowing away the precipitate, centrifuging, and collecting the supernatant to obtain the target lentivirus. According to the packaging method, expensive instruments or consumables are not needed, the steps are simple, operation is convenient, and a large number of slow viruses meeting the IIT level can be obtained within a short time.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a lentivirus rapid packaging method and application that meets IIT clinical quality inspection standards. Background Art

[0002] Chimeric antigen receptor T cell (CAR-T cell) therapy is a new type of immunotherapy technology for precise targeted treatment of tumor cells. The production process involved greatly affects the cell quality and treatment effect. The most critical part of the production process of CAR-T therapy is the production of lentiviral packaging (Kim DW, Biomolecules, 2020: 10(2): 263; McCarron A, J Biotechnol, 2016: 240: 23-30).

[0003] Lentivirus is a type of retrovirus, but its structure and genome are much more complex than other retroviruses. This also makes lentivirus have a unique integration mechanism and viral infection persistence. The lentiviral vectors currently used in cell therapy are mainly derived from HIV-1. They are used as vectors for cell therapy because of their more effective and extensive infection of dividing and non-dividing cells and tissues, larger packaging capacity, lower cytotoxicity and immunogenicity, better expression effect, and their tendency to integrate into the inactive transcription region of the host genome away from the transcription start point (L E Ailles, Curr Top Microbiol Immunol, 2002: 261: 31-52).

[0004] However, there are still many problems in the packaging system and application of lentivirus. For example, general-level laboratories cannot package and obtain lentiviruses with qualified quality inspection and high titer by themselves, and relying on professional institutions for packaging is expensive and has a long cycle, which affects the progress of the experiment; for example, the current lentivirus packaging process is cumbersome, from the status of the tool cell 293T, the culture medium used, the selection of liposomes to the collection of lentivirus supernatant, concentration and purification, each step is critical and will affect the titer and quality inspection results of the final lentivirus; for example, after the current simplified steps, the small-scale packaged lentivirus can only meet the standards of cell or animal experiments. If you want to obtain lentiviruses above the level of clinical research (Investigator-Initiated Trials, IIT), you have to use cell factories under GMP conditions to obtain a large amount of lentivirus supernatant, and then purify it through complex and high-cost downstream processes, and finally filter it through a 0.22μm filter membrane to obtain the final product. Therefore, the lentiviruses used in most CAR-T cell-related clinical trials are currently cooperated or purchased through professional institutions, with a long cycle and high price, which is a major obstacle for many researchers with limited funds who want to conduct clinical trials quickly. From a technical perspective, the reason why lentiviruses used in clinical trials are relatively expensive is that the existing technology for lentivirus packaging uses a very complicated downstream purification step, which results in a large amount of virus loss during the purification process. Therefore, it cannot be carried out in a general laboratory and must be carried out in a cell factory (a microbial production system based on synthetic biology and genetic engineering technology). Nucleases are used in this process, and then they must be removed during the purification step. The operation is complicated, time-consuming and costly. Summary of the invention

[0005] In order to overcome the technical problems of the above-mentioned prior art, such as complicated operation, time-consuming and complex purification steps, the purpose of the present invention is to provide a method for rapid packaging of lentivirus that meets the IIT clinical quality inspection standards, so as to achieve packaging of lentivirus that meets the IIT quality inspection standards in the laboratory with simple operation steps, so that it can be applied to the preparation of CAR-T cells.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The present invention discloses a method for rapid packaging of lentivirus that meets the IIT clinical quality inspection standard, comprising the following steps:

[0008] 1) Tool cell plating: Plating 293T cells into a cell culture dish and incubating them to prepare a 293T cell culture dish;

[0009] 2) Cell transfection: After the target plasmid, packaging auxiliary plasmid pSPAX2 and packaging auxiliary plasmid pMD2G are prepared in proportion, the transfection reagent Higene is added to obtain a plasmid and transfection reagent complex, which is then added to the 293T cell culture dish prepared in step 1) for culture, and then the supernatant is discarded, and the serum-free medium is replaced for continued culture, and the lentiviral supernatant is collected;

[0010] 3) Lentivirus collection and concentration: The lentivirus supernatant is centrifuged and filtered, and the universal nuclease is added for incubation. Then, the PEG8000 solution is added, mixed and allowed to stand overnight, centrifuged, and the lentivirus precipitate is collected. The lentivirus precipitate is dissolved with a resuspension solution, and the precipitate is blown away after standing, centrifuged, and the supernatant is collected to obtain the target lentivirus.

[0011] Preferably, in step 1), 293T cells that have been passaged to the logarithmic phase are cultured at 6×10 6 The counting amount of cells / dish was plated into cell culture dishes for incubation, and the number of plates was 20 dishes.

[0012] Preferably, in step 1), the culture medium used for 293T cell expansion and plating is DMEM + 10% FBS.

[0013] Preferably, in step 2), the mass ratio of the target plasmid, the packaging helper plasmid pSPAX2 and the packaging helper plasmid pMD2G is 4:3:1, and the total mass of the three plasmids is 18 μg / dish.

[0014] Preferably, in step 2), the amount of transfection reagent Higene used is 43.2 μL / dish.

[0015] Preferably, in step 2), OptiPRO TM SFM serum-free medium; lentivirus collection time is 6 hours after adding plasmid and transfection reagent complex, and all the medium is replaced with OptiPRO TM The cells were cultured in SFM serum-free medium and then the supernatant was collected after culturing for 48 h.

[0016] Preferably, in step 3), the incubation time with the universal nuclease is 0.5 h, and the working concentration is 2.5 U / mL.

[0017] Preferably, in step 3), filter with a 0.45 μm filter, add a PEG8000 solution with a volume of 1 / 4 of the lentiviral solution, mix well and let stand overnight.

[0018] Preferably, in step 3), the lentiviral precipitate obtained from every 20 10 cm cell culture dishes is dissolved with 1 mL of lentiviral resuspension solution.

[0019] The present invention also discloses the use of a lentivirus that meets the IIT clinical quality inspection standard and is prepared by the above-mentioned lentivirus rapid packaging method in the preparation of CAR-T cells.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The rapid packaging method of lentivirus that meets the IIT clinical quality inspection standard disclosed in the present invention simplifies the steps of lentivirus concentration and purification, and clearly screens and confirms the key conditions in the packaging process, such as the special selection of Higene as a transfection reagent during cell transfection to obtain a higher lentivirus titer; for example, a very critical operation of the present invention is the introduction of a universal nuclease during the lentivirus concentration process, which is the same as the addition of a universal nuclease before the purification of the lentivirus by a professional institution at present, but the invention of the present invention is that after the universal nuclease fully exerts the effect of removing residual nucleic acids in the lentivirus, the added PEG8000 solution can cause the inactivation of the universal nuclease, which means that the present invention does not need to use a complex downstream purification process to remove the universal nuclease, or other proteins, nucleic acid impurities. After testing, the clinical indicators such as titer, safety, purity and impurities of the lentivirus obtained by the rapid packaging method of the present invention meet the standard requirements. The packaging method does not require expensive instruments or consumables, has simple steps, is easy to operate, and can obtain a large number of IIT-level lentiviruses in a short time. The lentivirus packaging, lentivirus titer detection and lentivirus quality inspection process of the present invention only takes about 10 days, and no expensive cell factory equipment and purification equipment are used. The entire packaging process can effectively control costs and is easy for operators to master. It is very suitable for rapid packaging and preparation of lentivirus that meets IIT clinical quality inspection standards in the laboratory.

[0022] Furthermore, when 293T cells were plated into the cell culture dish, a cell wall adhesive was introduced, which greatly reduced the residual host cell protein in the final lentiviral solution.

[0023] Furthermore, during the lentivirus packaging process, OptiPROTM SFM serum-free culture medium was introduced, which not only increased the lentivirus titer but also significantly reduced the residual bovine serum albumin. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a comparison of the host cell protein residues in the lentivirus collected at 48h and 72h;

[0025] Figure 2 The CAR positive rate after lentivirus infected T cells at different MOI values; A is the CAR positive rate of patients when the MOI value was 1 or 2 as detected by flow cytometry; B is the statistical chart of CAR positive rate of 4 patients;

[0026] Figure 3 These are the results of the proportions of CD3+T, CD3+CD4+T and CD3+CD8+T cells in CAR-T cells; A is the proportion of each immune cell in CAR-T cells detected by flow cytometry; B is the statistical proportion of each immune cell in CAR-T cells of 4 patients. DETAILED DESCRIPTION

[0027] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0028] It should be noted that the terms "first", "second", etc. in the specification and claims of the present invention and the above-mentioned drawings are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequence. It should be understood that the data used in this way can be interchanged where appropriate, so that the embodiments of the present invention described herein can be implemented in an order other than those illustrated or described herein. In addition, the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusions, for example, a process, method, system, product or device that includes a series of steps or units is not necessarily limited to those steps or units that are clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.

[0029] The present invention is further described in detail below in conjunction with the accompanying drawings:

[0030] In order to prepare a lentivirus that meets the IIT clinical quality inspection standard, the present invention strictly screened the key condition parameters that may be involved in the experimental process, and finally obtained a rapid packaging method, including the following steps:

[0031] 1) Tool cell plating: Plating 293T cells into a cell culture dish and incubating them to prepare a 293T cell culture dish;

[0032] 2) Cell transfection: After the target plasmid, packaging auxiliary plasmid pSPAX2 and packaging auxiliary plasmid pMD2G are prepared in proportion, a transfection reagent is added to prepare a plasmid and transfection reagent complex, which is then added to the 293T cell culture dish prepared in step 1) for culture, and then the supernatant is discarded, and the serum-free medium is replaced for continued culture, and the lentiviral supernatant is collected;

[0033] 3) Lentivirus collection and concentration: The lentivirus supernatant is centrifuged and filtered, and the universal nuclease is added for incubation. Then, the PEG8000 solution is added, mixed and allowed to stand overnight, centrifuged, and the lentivirus precipitate is collected. The lentivirus precipitate is dissolved with a resuspension solution, and the precipitate is blown away after standing, centrifuged, and the supernatant is collected to obtain the target lentivirus.

[0034] The target lentivirus obtained by packaging according to the above method can be used in the preparation of CAR-T cells.

[0035] The "lentivirus" described in the present invention is a symmetrical spherical enveloped virus with a diameter of about 80 to 120 nm, which is composed of an envelope protein, an envelope, a matrix protein, a capsid, two single-stranded RNAs, a reverse transcriptase, an integrase and a protease.

[0036] The "293T cells" described in the present invention are human embryonic kidney cells, also known as Hek293T, HEK-293T, etc. 293T cells have weak wall adhesion ability, are eosinophilic, and aggregate in islands when growing. 293T cells are very easy to transfect, and the efficiency can reach more than 90% when transfected using methods such as calcium phosphate, polyethyleneimine (PEI) or liposomes.

[0037] The "target plasmid" described in the present invention is a special plasmid that is artificially designed or modified to carry and express specific target genes. These genes may be selected for the purpose of studying gene function, producing proteins, performing gene therapy or vaccine development. The target plasmid usually has the ability to replicate autonomously and can be amplified in large quantities in host cells. The target plasmid usually includes elements such as a replication origin, a selection marker, a multiple cloning site, a promoter and a terminator.

[0038] The "packaging helper plasmid" described in the present invention refers to a plasmid used to support the packaging and expression of exogenous DNA in a three-plasmid packaging system, and is usually transformed into a host cell together with an infectious bacteriophage (or viral vector) and a target plasmid. In particular, in the process of supporting the packaging of the target plasmid, the proteins provided by the helper plasmid participate in the packaging process of the target plasmid. They help the bacteriophage (or virus) to encapsulate the DNA of the target plasmid in its head structure, thereby ensuring that the target plasmid can be successfully delivered into the host cell.

[0039] The "PEG8000" described in the present invention is a high molecular polymer with high hydrophilicity. It plays an important role in the preparation process of lentivirus, especially in the purification and concentration stage of lentivirus. PEG8000 absorbs a large amount of water in the solution. This characteristic reduces the distance between virus particles, making it easy to aggregate together. Through the action of PEG8000, the relative concentration of the virus is improved, and the purpose of precipitation and concentration is finally achieved. In this process, the amount of PEG8000 has a great influence on the results, which is mainly reflected in three aspects: 1) precipitation effect. If the amount of PEG8000 is insufficient, the virus may not be fully concentrated, resulting in poor precipitation effect. On the contrary, if the amount of PEG8000 is too much, the solution may be too viscous, which is not conducive to the precipitation of virus particles and subsequent separation operations; 2) virus recovery rate. If the amount of PEG8000 is insufficient or excessive, it may lead to a decrease in virus recovery rate; 3) virus titer. An appropriate amount of PEG8000 can maintain the activity of the virus, thereby improving the virus titer. If the dosage of PEG8000 is inappropriate, it will cause aggregation or degradation of viral particles, thereby reducing the viral titer.

[0040] The "CAR-T cells" described in the present invention are called chimeric antigen receptor T cells (Chimeric Antigen Receptor T-Cell), which are genetically engineered T cells commonly used in cancer treatment. The preparation of CAR-T cells usually includes T cell collection, gene transduction, cell expansion, quality control and reinfusion operations.

[0041] Specifically, the screening process of some key links in the packaging method of the present invention is described in detail below:

[0042] 1. Determination of 293T cell status and quantity

[0043] As a commonly used packaging host, 293T cells, their health status and quantity are key factors affecting virus yield, and are also related to the residual amount of host cell protein in the lentivirus quality inspection standard. The 293T cells used in the present invention were purchased from the Chinese Academy of Sciences Cell Bank (Order No. 275316, Order Date January 18, 2024), and the number of passages is not high and the cell growth state is good. Since the 293T cell wall adhesion is not very good, a cell wall adhesion agent is used to increase the wall adhesion of 293T cells, which can also prevent 293T cells from easily falling off during the fluid exchange process.

[0044] The cell wall adhesion agent C1010, 200-500X used in the embodiment of the present invention was purchased from Beijing Prilai Gene Technology Co., Ltd., with a specification of 1 mL, 200-500X concentrate, which was dissolved in filtered sterilized physiological saline for use.

[0045] The number of 293T cells plated in a 10 cm cell culture dish varies widely, ranging from 1×10 5 to 1×10 7 The present invention compares the titer of lentivirus obtained by packaging three cell numbers and the residual host cell protein based on the plating rate. The transfection reagent Higene, the ratio of three plasmids (target plasmid: pSPAX2: pMD2G = 2:1:1), the medium used for lentivirus packaging (DMEM medium + 10% FBS) and the lentivirus collection time (adding plasmid and transfection reagent complex 6h after half-volume liquid replacement, 48h and 72h after collecting the supernatant) are all consistent. Each group has 10 culture dishes, and the volume of the concentrated lentivirus precipitate resuspended is 1mL. The test results show that the number of 293T cells plated is 6×10 6 / dish, the lentivirus titer is high and the host cell protein residue is minimal.

[0046] Table 1. Determination of the number of 293T cells plated in a 10 cm cell culture dish

[0047]

[0048] 2. Ratio and total amount of three plasmid systems

[0049] The ratio and total amount of the target plasmid and the auxiliary plasmids pSPAX2 and pMD2G will also affect the titer of the final lentivirus. Therefore, the present invention compares the lentivirus titers obtained under four ratios. 293T cell plating number 6×10 6 , transfection reagent Higene, total amount of three plasmids, medium for lentivirus packaging (DMEM medium + 10% FBS) and lentivirus collection time (add plasmid and transfection reagent complex 6h after half-volume medium replacement, collect supernatant 48h, 72h) were all kept consistent, 10 culture dishes per group, and the volume of lentivirus precipitation resuspended after concentration was 1mL. The results showed that the highest lentivirus titer was obtained when the ratio of the three plasmids was 4:3:1.

[0050] Table 2. Determination of the ratio of target plasmid, pSPAX2 and pMD2G in 10 cm cell culture dishes

[0051]

[0052] We then compared the lentiviral titers at four different total plasmid concentrations. 6, the ratio of the three plasmids (target plasmid: pSPAX2: pMD2G = 4:3:1), the medium used for lentivirus packaging (DMEM medium + 10% FBS) and the time for collecting lentivirus (adding the plasmid and transfection reagent complex for 6 hours, half-volume medium replacement, 48h and 72h supernatant collection) were all kept consistent, with 10 culture dishes per group, and the volume of the concentrated lentivirus precipitate resuspended was 1mL. The results showed that the highest lentivirus titer was obtained when the total amount of the three plasmids was 18μg.

[0053] Table 3. Determination of the total amount of three plasmids in a 10 cm cell culture dish

[0054]

[0055] Note: The volume calculation formula of Higene added is: Higene = 0.4 × 6 × the amount of plasmid (It should be noted here that the amount of plasmid affects the titer. The transfection reagent will vary with the amount of plasmid, but the coefficient of the transfection reagent is the same, which is 6.)

[0056] 3. Selection of transfection reagents

[0057] There are many types of transfection reagents at present, with different prices and effects. We compared three domestic transfection reagents with low prices. 293T cell plating number 6×10 6 , the ratio of the three plasmids (target plasmid: pSPAX2: pMD2G = 4:3:1), the total amount of the three plasmids was 18μg, the medium used for lentivirus packaging (DMEM medium + 10% FBS) and the lentivirus collection time (adding plasmid and transfection reagent complex 6h after half-volume medium replacement, 48h and 72h after supernatant collection) were all kept consistent, 10 culture dishes per group, and the volume of the concentrated lentivirus precipitate resuspended was 1mL. The results showed that the lentivirus titer was the highest when the transfection reagent was Higene.

[0058] Table 4. Determination of transfection reagents

[0059] Transfection reagents Transfection reagent volume (μL) Lentivirus titer (TU / mL) Higene 43.2 <![CDATA[3.82×10 8 ]]> TF 54 <![CDATA[2.44×10 8 ]]> EZ Trans 54 <![CDATA[1.56×10 8 ]]>

[0060] The effects of different Higene volumes on the lentiviral titer were then compared. The results showed that the lentiviral titer was highest when the volume of transfection reagent Higene added to a 10 cm cell culture dish was 43.2 μL.

[0061] Table 5. Determination of the volume of transfection reagent Higene

[0062]

[0063] 4. Comparison of fluid change time and volume during lentivirus packaging

[0064] The time and volume of medium replacement may have a certain effect on the lentiviral titer. We compared the lentiviral titer under different medium replacement time or medium replacement volume conditions. 293T cell plating number 6×10 6 , the ratio of the three plasmids (target plasmid: pSPAX2: pMD2G = 4:3:1), the total amount of the three plasmids was 18 μg, the transfection reagent Higene was 43.2 μL, and the culture medium used for lentivirus packaging (DMEM culture medium + 10% FBS) were all kept consistent. There were 10 culture dishes in each group, and the volume of the resuspended lentivirus precipitate after concentration was 1 mL.

[0065] Table 6. Determination of half-volume liquid replacement time after adding plasmid and transfection reagent complex

[0066] Half-volume medium replacement time (h) Lentivirus titer (TU / mL) 6 <![CDATA[2.34×10 8 ]]> 18 <![CDATA[2.01×10 8 ]]> 24 <![CDATA[1.98×10 8 ]]>

[0067] After half-volume liquid exchange at the corresponding time points, the above groups collected the lentiviral supernatant at 48h and 72h, respectively. The results showed that half-volume liquid exchange 6h after the addition of the plasmid and transfection reagent complex resulted in a high titer of the lentiviral collected. Subsequently, we compared the titers of the lentiviral collected when half-volume or full liquid exchange was performed 6h after the addition of the plasmid and transfection reagent complex. The results showed that the lentiviral titer was high when the full liquid exchange was performed 6h after the addition of the plasmid and transfection reagent complex.

[0068] Table 7. Determination of the volume of liquid replacement 6 hours after adding plasmid and transfection reagent complex

[0069] Fluid replacement volume (mL) Lentivirus titer (TU / mL) 5 <![CDATA[2.65×10 8 ]]> 10 <![CDATA[2.97×10 8 ]]>

[0070] 5. Comparison of culture medium types during lentiviral packaging

[0071] There are many types of culture media currently used for lentiviral packaging. We compared DMEM complete medium, Opti-MEM TM I Reduced Serum Medium and OptiPRO TM Effects of SFM serum-free medium on lentivirus titer, residual bovine serum albumin and residual plasmid DNA.

[0072] 293T cells plated 6×10 6 , the ratio of the three plasmids (target plasmid: pSPAX2: pMD2G = 4:3:1), the total amount of the three plasmids was 18μg, the transfection reagent Higene was 43.2μL, and the lentivirus collection time (adding plasmid and transfection reagent complex for 6 hours and replacing all the liquids, collecting the supernatant 48h and 72h after), each group of 10 culture dishes, the volume of the concentrated lentivirus precipitate resuspended was 1mL. The results showed that the residual bovine serum albumin in the lentivirus obtained by DMEM medium containing 10% FBS significantly exceeded the clinical standard (≤500ng / mL), Opti-MEM TMI Reduced Serum Medium and OptiPRO TM The lentivirus titers obtained from SFM serum-free medium were not much different, and the residual bovine serum albumin met the clinical standards. However, compared with the two, OptiPRO TM The residual bovine serum albumin in the lentivirus obtained by SFM serum-free medium was the least. The residual plasmid DNA in the lentivirus obtained under the three culture medium conditions was higher than the clinical standard (≤50ng / mL), and it was necessary to introduce a universal nuclease to treat the lentiviral supernatant.

[0073] Table 8. Determination of the type of culture medium used for medium replacement during lentiviral packaging

[0074]

[0075] We then compared the effects of the time of omnipotent nuclease treatment of lentiviral supernatant and the amount of omnipotent nuclease added on lentiviral titer, residual nuclease and residual plasmid DNA.

[0076] 293T cells plated 6×10 6 , the ratio of the three plasmids (target plasmid: pSPAX2: pMD2G = 4:3:1), the total amount of the three plasmids was 18 μg, the transfection reagent Higene was 43.2 μL, and the lentivirus collection time (add plasmid and transfection reagent complex for 6 hours, and replace all the liquid with OptiPRO TM SFM serum-free medium was used to collect the supernatant at 48h and 72h respectively) and each group had 10 culture dishes. The volume of the lentivirus precipitate resuspended after concentration was 1mL. The results showed that when the lentivirus supernatant was treated with the lowest recommended working concentration in the instructions, i.e. 0.25U / mL, the final lentivirus titer, nuclease residue and plasmid DNA residue decreased with the increase of the universal nuclease treatment time. Among them, when the treatment time was 0.5h, although the lentivirus titer was high and the nuclease residue met the clinical standard (≤50ng / mL), the plasmid DNA residue was higher than the clinical standard (≤50ng / mL).

[0077] Table 9. Determination of the time for treating lentiviral supernatant with universal nuclease

[0078]

[0079] To ensure that the lentivirus titer remained at a high level after the addition of the universal nuclease, we then compared the effects of the universal nuclease working time of 0.5 h on the lentivirus titer, residual nuclease, and residual plasmid DNA at different working concentrations.

[0080] The results showed that when the working time was 0.5 h and the working concentration of universal nuclease was 2.5 U / mL or 25 U / mL, the residual nuclease and plasmid DNA in the lentivirus met the clinical standards, but the lentivirus titer obtained at the working concentration of 2.5 U / mL was higher than that at 25 U / mL and met the standard of ≥1×10 8 Clinical standard. Therefore, the working time of the universal nuclease was finally determined to be 0.5h and the working concentration was 2.5U / mL.

[0081] Table 10. Determination of the working concentration of universal nuclease

[0082]

[0083] “-” means no nuclease was detected by the kit.

[0084] 6. Comparison of lentivirus purification methods

[0085] Currently, there are a variety of lentivirus purification kits on the market. We compared the titer and host cell protein residues of lentivirus resuspended in PEG8000 concentrated lentivirus resuspension (without further purification) and lentivirus treated with BioMedical lentivirus purification kit, Nereus LentiHERO lentivirus purification column or Sartorius ultrafiltration tube (100kDa).

[0086] 293T cells plated 6×10 6 , the ratio of the three plasmids (target plasmid: pSPAX2: pMD2G = 4:3:1), the total amount of the three plasmids was 18 μg, the transfection reagent Higene was 43.2 μL, and the lentivirus collection time (add plasmid and transfection reagent complex for 6 hours, and replace all the liquid with OptiPRO TM SFM serum-free medium was used to collect the supernatant at 48h and 72h respectively), the working concentration of universal nuclease (2.5U / mL) and the working time (0.5h) were kept consistent, with 10 culture dishes in each group, and the volume of the concentrated lentiviral precipitate resuspended was 1mL.

[0087] The results showed that the highest titer of lentivirus obtained under the four conditions was obtained under the PEG8000 concentration condition, but the residual host cell protein in the lentivirus obtained under this condition still exceeded the clinical standard (≤4μg / 1×10 8 TU), further optimization of conditions is needed.

[0088] Table 11. Comparison of lentiviral purification conditions

[0089]

[0090]

[0091] Subsequently, we compared the host cell protein residues in the lentivirus collected at 48h and 72h. The results showed that the host cell protein residues in the lentivirus collected at 72h were significantly higher than those at 48h ( Figure 1 ).

[0092] To further increase the lentivirus titer, we then collected lentivirus supernatants from 20 10 cm cell culture dishes at 48 h, concentrated them with PEG8000, resuspended them in 1 mL of lentivirus resuspension solution, and tested the titer, safety, purity, and clinical indicators of impurities of the obtained lentivirus.

[0093] The results showed that when the lentivirus packaging was carried out according to the following conditions, the obtained lentivirus met the IIT clinical use standards. Determined conditions: 293T cell plating number 6×10 6 / dish, 20 dishes were plated; the ratio of three plasmids (target plasmid: pSPAX2: pMD2G = 4:3:1), the total amount of three plasmids was 18μg / dish, and the transfection reagent Higene was 43.2μL / dish; the lentivirus collection time was 6 hours after the addition of plasmid and transfection reagent complex, and all the liquid was replaced with OptiPRO TM SFM serum-free medium, and then collect the supernatant after 48 hours; the working concentration of universal nuclease is 2.5U / mL and the working time is 0.5h; after filtering with a 0.45μm filter, add 1 / 4 volume of PEG8000 solution of the lentivirus solution and mix overnight; after centrifugation, resuspend the lentivirus precipitate with 1mL lentivirus resuspension solution.

[0094] Table 12. Results of various lentivirus index tests

[0095]

[0096] Example 1

[0097] 1. Plating of tool cells 293T

[0098] (1) Dilute the cell wall adhesive (Beijing Prilai Gene Technology Co., Ltd., C1010) in PBS buffer at a ratio of 1:500 to prepare a working solution. Take 4 mL of the working solution and add it to a 10 cm cell culture dish. Shake well and place in a 37°C incubator for 30 min. Then discard the working solution in the dish and add 2 mL of PBS buffer to wash once;

[0099] (2) The expanded and passaged logarithmic phase 293T cells were collected and counted, and then 6×10 6 cells / dish, shake well and incubate in a 37°C, 5% CO2 incubator for 24 hours. The culture medium used for 293T cell expansion and plating was DMEM + 10% FBS;

[0100] 2. Cell transfection

[0101] (3) Preparation of plasmid and transfection reagent complex: The ratio of the target plasmid to the packaging auxiliary plasmids pSPAX2 and pMD2G in the three-plasmid system is 4:3:1. Specifically, the amounts of the three plasmids added to a cell culture dish are 9μg, 6.75μg, and 2.25μg, respectively. Calculate the volume of plasmids to be added according to the added amount and the concentration of each plasmid. Then take a test tube A and add the three plasmids to the PBS buffer in turn, with a final volume of 500μL. At the same time, in another test tube B, add 43.2μL of transfection reagent Higene (Beijing Pulilai Gene Technology Co., Ltd., C1506) to the PBS buffer, with a final volume of 500μL. Gently drip the solution in tube A into tube B, mix gently, and let stand at room temperature for 15 minutes;

[0102] (4) Remove the 293T cell culture dish, discard the supernatant, add 9 mL of serum-free DMEM medium, and then add 1 mL of a mixture of plasmid and transfection reagent complex;

[0103] (5) After incubation at 37°C, 5% CO2 for 6-8 h, the supernatant was discarded and replaced with 10 mL of serum-free OptiPROTM medium (Gibco, catalog number 12309019);

[0104] 3. Lentivirus collection and concentration

[0105] (6) After changing the medium, the culture was continued for 48 h, and the lentiviral supernatant was collected and centrifuged at 4000 g for 5 min, and then filtered using a 0.45 μm cellulose acetate CA membrane (Sartorius, S7598-FXOSK);

[0106] (7) Add 2.5 U / mL of GMP-grade universal nuclease (YEASEN, catalog number 20157ES25) to the filtered lentiviral solution and incubate in a 37°C, 5% CO2 incubator for 30 min;

[0107] (8) Concentration: Add 1 / 4 volume of the prepared autoclaved PEG8000 solution (5×PEG8000 solution is prepared by dissolving 50 g of PEG8000 and 8.766 g of NaCl in 200 mL of Milli-Q pure water) to the lentivirus solution, mix well, and place at 4°C. Mix well every 30 min for three times, and place overnight; centrifuge at 4°C, 4000 g for 30 min, and discard the supernatant (the tube can be inverted to remove residual liquid);

[0108] (9) Dissolve the lentivirus precipitate with lentivirus resuspension solution (Biyuntian, C2901M-2), let it stand for 10 minutes, then gently blow off the precipitate, then centrifuge at 4°C, 12000g for 5 minutes, and take the clear supernatant as the final lentivirus.

[0109] Note: In the present invention, the lentivirus precipitate obtained from every 20 10 cm cell culture dishes was dissolved with 1 mL of lentivirus resuspension solution, and the obtained lentivirus met the IIT clinical quality inspection standards.

[0110] The lentivirus quality inspection standards used in the present invention are as shown in Table 13:

[0111] Table.13 Lentivirus quality inspection standards

[0112]

[0113]

[0114] 4. T cell infection and CAR-T cell quality control

[0115] The qualified lentivirus prepared by the present invention is used to infect human T cells, and the quality inspection of bacteria, fungi, mycoplasma and endotoxin is carried out on the 6th day of culture, and the CAR positive rate, CD3+CD4+T cell and CD3+CD8+T cell ratio are detected to evaluate the quality of CAR-T cell products. Specifically, the following steps are included:

[0116] (1) Isolate human PBMCs, obtain CD3+ T cells using a human CD3 T cell isolation kit (BD, catalog number 480131), resuspend in X-VIVO 15 medium, and add CD3CD28 dual anti-magnetic beads and IL-2; on the second day, add polybrene and lentivirus that has passed quality control;

[0117] (2) On day 3, the cell pellet was resuspended in medium X-VIVO 15 + 10% human serum + IL-2 and plated;

[0118] (3) On the 6th day, CAR positivity, bacteria, fungi, mycoplasma, and endotoxin were tested.

[0119] The safety test results are as follows (Table 14), CAR positive rate ( Figure 2 ) and the ratio of CD3+CD4+T cells and CD3+CD8+T cells are as follows ( Figure 3 ), the results showed that the lentivirus packaged by the present invention infected T cells, and the CAR-T cells finally produced met the quality inspection requirements and standards for clinical transfusion. All test results show that the present invention can quickly package high-quality lentivirus that meets IIT standards in the laboratory with low cost and simplified steps.

[0120] Table 14. Test results of CAR-T cell safety indicators

[0121] bacteria Negative Fungi Negative Mycoplasma Negative endotoxin Negative

[0122] In summary, the present invention successfully prepared a lentivirus that meets the IIT clinical quality inspection standards, and the method of the present invention has significant advantages, which are mainly reflected in the following aspects:

[0123] First, cost saving: A rough calculation of the amount spent on lentivirus packaging, lentivirus titer detection, lentivirus quality inspection, and final CAR-T cell preparation and quality control shows that the cost is about RMB 7,000 per person per time (Table 15). However, some professional institutions in the industry (such as GenScript, Heyuan Bio, etc.) currently only provide lentiviruses, and the price required is more than RMB 20,000 per person per time, which does not include the subsequent preparation and quality control of CAR-T cells.

[0124] Table 15. Cost accounting details of the present invention

[0125]

[0126] Second, it is time-saving: the rapid packaging method of lentivirus disclosed in the present invention can prepare target substances that meet the IIT clinical quality inspection standards without the need for expensive instruments or consumables (such as expensive cell factory equipment and purification equipment) through the screening and examination of reagent materials used in key operation processes. The whole method has simple steps and is easy to operate. A large amount of lentivirus that meets the IIT level can be obtained in a short time (the lentivirus packaging process, lentivirus titer detection and lentivirus quality inspection only take about 10 days).

[0127] Third, quality advantage: This paper compares two lentivirus purification kits on the market, namely, ViraTrap from Hangzhou Bio-Medical Technology Co., Ltd. TM The limitations of the large-scale purification kit for lentivirus (V1270) and Astrea's Nereus LentiHERO purification column are that the price is high, the volume of lentivirus supernatant that can be processed is limited, and the final lentivirus titer is not high and cannot meet the lentivirus quality inspection standards (results shown in Table 11). The PEG8000 solution used in the present invention is extremely low in cost, has no limit on the volume of lentivirus supernatant, and the lentivirus obtained not only has a high titer, but also meets the lentivirus quality inspection standards. In addition, in the subsequent lentivirus applications obtained by the present invention, the CAR positive rate is high, and the prepared CAR-T cells meet the quality inspection standards for clinical patient cell transfusion.

[0128] The above contents are only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. A method for rapid packaging of lentivirus that meets IIT clinical quality inspection standards, characterized in that: The following steps are involved: 1) Tool cell plating: Plating 293T cells into a cell culture dish and incubating them to prepare a 293T cell culture dish; 2) Cell transfection: After the target plasmid, packaging auxiliary plasmid pSPAX2 and packaging auxiliary plasmid pMD2G are prepared in proportion, the transfection reagent Higene is added to obtain a plasmid and transfection reagent complex, which is then added to the 293T cell culture dish prepared in step 1) for culture, and then the supernatant is discarded, and the serum-free medium is replaced for continued culture, and the lentiviral supernatant is collected; 3) Lentivirus collection and concentration: The lentivirus supernatant is centrifuged and filtered, and the universal nuclease is added for incubation. Then, the PEG8000 solution is added, mixed and allowed to stand overnight, centrifuged, and the lentivirus precipitate is collected. The lentivirus precipitate is dissolved with a resuspension solution, and the precipitate is blown away after standing, centrifuged, and the supernatant is collected to obtain the target lentivirus.

2. The method for rapid packaging of lentivirus according to claim 1, characterized in that: In step 1), when culturing on the plate, a cell wall adhesive is added to the cell culture dish; when culturing on the plate, 293T cells that have been passaged to the logarithmic phase are plated at 6×10 6 The counting amount of cells / dish was plated into cell culture dishes for incubation, and the number of plates was 20 dishes.

3. The method for rapid packaging of lentivirus according to claim 1, characterized in that: In step 1), the culture medium used for 293T cell expansion and plating was DMEM+10% FBS.

4. The method for rapid packaging of lentivirus according to claim 1, characterized in that: In step 2), the mass ratio of the target plasmid, the packaging helper plasmid pSPAX2 and the packaging helper plasmid pMD2G was 4:3:1, and the total mass of the three plasmids was 18 μg / dish.

5. The method for rapid packaging of lentivirus that complies with IIT clinical quality inspection standards according to claim 1, characterized in that: In step 2), the amount of transfection reagent Higene used is 43.2 μL / dish.

6. The method for rapid packaging of lentivirus that complies with IIT clinical quality inspection standards according to claim 1, characterized in that: In step 2), use OptiPRO TM SFM serum-free medium; lentivirus collection time is 6 hours after adding plasmid and transfection reagent complex, and all the medium is replaced with OptiPRO TM The cells were cultured in SFM serum-free medium and then the supernatant was collected after culturing for 48 h.

7. The method for rapid packaging of lentivirus that complies with IIT clinical quality inspection standards according to claim 1, characterized in that: In step 3), the incubation time with universal nuclease is 0.5 h, and the working concentration is 2.5 U / mL.

8. The method for rapid packaging of lentivirus that complies with IIT clinical quality inspection standards according to claim 1, characterized in that: In step 3), filter with a 0.45 μm filter, add PEG8000 solution with 1 / 4 volume of the lentivirus solution, mix well and let stand overnight.

9. The method for rapid packaging of lentivirus that complies with IIT clinical quality inspection standards according to claim 1, characterized in that: In step 3), the lentiviral precipitate obtained from each of 20 10 cm cell culture dishes was dissolved with 1 mL of lentiviral resuspension solution.

10. Use of a lentivirus that meets the IIT clinical quality inspection standard and is prepared by the lentivirus rapid packaging method according to any one of claims 1 to 9 in the preparation of CAR-T cells.

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