Preparation for lentivirus transduction and application thereof

Abstract

The invention relates to the field of biological medicine, in particular to a preparation for lentivirus transduction and application thereof. The preparation provided by the invention contains multiple of chlorine salt, albumin and a serum-free immune cell culture medium, and on the basis of the total volume of the preparation, the mass volume fraction of the chlorine salt is 0.24-0.96%, the volume fraction of the albumin is 1-10%, and the volume fraction of the serum-free immune cell culture medium is 10-50%. After the preparation provided by the invention is mixed with lentivirus, stable preservation of a lentivirus vector can be ensured, and after mixing, immune cells are transfected, so that the immune cells have better motility rate, amplification effect, positive rate and killing effect.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a preparation for lentiviral transduction and uses thereof. Background Art

[0002] Cell and gene therapy products have become the forefront of global medical development. Various related clinical studies have been carried out around the world, and cell therapy-related companies have also emerged.

[0003] Lentivirus is a commonly used vector in gene-modified cell therapy projects. Compared with other methods, lentivirus can efficiently transduce cyclical and non-cyclical cells and is suitable for the needs of most projects. The effect of lentiviral transduction determines the effect of gene modification. In the transduction system, the general virus volume accounts for about 5-10% of the total volume. Therefore, the preparation of the virus must ensure its stability while ensuring that the preparation will not have a significant impact on the transduction system during transduction. Summary of the invention

[0004] In view of the above-mentioned shortcomings of the prior art, an object of the present invention is to provide a preparation for lentiviral transduction and use thereof, so as to solve the problems in the prior art.

[0005] To achieve the above-mentioned object and other related objects, the present invention provides a preparation for lentiviral transduction, wherein the preparation comprises multiple types of chloride salt, albumin and serum-free immune cell culture medium. Based on the total volume of the preparation, the mass volume fraction of the chloride salt is 0.24-0.96% (g / L), the volume fraction of the albumin is 1-10%, and the volume fraction of the serum-free immune cell culture medium is 10-50%.

[0006] Preferably, the serum-free immune cell culture medium is selected from X-VIVO 15 culture medium, AIM-V culture medium, X-VIVO 10 culture medium, NK cell culture medium, dendritic cell (DC) culture medium, cytokine-induced killer cell (CIK) culture medium, cytotoxic T lymphocyte (CTL) culture medium or tumor infiltrating lymphocyte (TIL) culture medium.

[0007] The present invention also provides the use of the above-mentioned preparation in improving the killing efficiency or amplification efficiency of immune cells after in vitro lentiviral transduction.

[0008] The present invention also provides a composition comprising a lentiviral vector and the above-mentioned preparation.

[0009] The present invention also provides a method for improving the expansion multiple or killing efficiency of T cells, characterized in that the method comprises the following steps: mixing the lentiviral vector with the aforementioned preparation, and then mixing it with in vitro T cells to obtain lentiviral-transduced T cells, wherein the expansion multiple or killing efficiency of the lentiviral-transduced T cells is higher than that of T cells obtained after transduction with the lentiviral vector alone.

[0010] As described above, the preparation for lentiviral transduction and its use of the present invention have the following beneficial effects: The preparation provided by the present invention can ensure the stable preservation of the lentiviral vector after being mixed with the lentivirus, and can make the immune cells have better viability, amplification effect, positive rate and killing effect after transfection of immune cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 Shown is a flow chart of the purification process of the lentiviral vector in the present invention.

[0012] Figure 2 The results are shown for the stability of different formulations of the present invention at -80 degrees Celsius P24.

[0013] Figure 3 The results are shown for the TU stability of different formulations of the present invention at -80 degrees Celsius.

[0014] Figure 4 The results show the reduction ratio of TU at -80 degrees Celsius for different formulations of the present invention.

[0015] Figure 5 The process flow of the preparation containing lentiviral vectors infecting immune cells in the present invention is shown.

[0016] Figure 6 The results are shown as the cell expansion times of different formulas in the present invention.

[0017] Figure 7 The results are shown for cell viability after cells were infected with different formulations of the present invention.

[0018] Figure 8 The results are shown as the T cell positivity rates of cells in different formulas of the present invention.

[0019] Fig. 9 Shown are the killing ratio results of T cells after transduction with different formulations of the present invention. DETAILED DESCRIPTION

[0020] The present invention provides a preparation for lentiviral transduction, wherein the preparation comprises multiple types of chloride salt, albumin and serum-free immune cell culture medium. Based on the total volume of the preparation, the mass volume fraction of the chloride salt is 0.24-0.96% (g / L), the volume fraction of the albumin is 1-10%, and the volume fraction of the serum-free immune cell culture medium is 10-50%.

[0021] In some embodiments, the chloride salt is selected from sodium chloride, potassium chloride, calcium chloride or magnesium chloride. Preferably, the chloride salt is sodium chloride.

[0022] In some embodiments, the albumin is selected from serum albumin, lactalbumin, ovalbumin or myoalbumin. Preferably, the albumin is serum albumin. More preferably, the albumin is selected from human serum albumin or bovine serum albumin.

[0023] In some specific embodiments, the serum-free immune cell culture medium is selected from X-VIVO 15 culture medium, AIM-V culture medium, X-VIVO 10 culture medium, NK cell culture medium, dendritic cell (DC) culture medium, cytokine-induced killer cell (CIK) culture medium, cytotoxic T lymphocyte (CTL) culture medium or tumor infiltrating lymphocyte (TIL) culture medium. Preferably, the serum-free immune cell culture medium is X-VIVO 15 culture medium.

[0024] In some specific embodiments, based on the total volume of the preparation, the mass volume fraction of the chloride salt is 0.24-0.96% (g / L). Specifically, the mass volume fraction is 0.24-0.3%, 0.3-0.36%, 0.36-0.42%, 0.42-0.48%, 0.48-0.54%, 0.54-0.60%, 0.6-0.66%, 0.66-0.72%, 0.72-0.78%, 0.78-0.84%, 0.84-0.9% or 0.9-0.96%. Preferably, the mass volume fraction of the chloride salt is 0.3-0.84% ​​(g / L).

[0025] In some specific embodiments, based on the total volume of the preparation, the volume fraction of the albumin in the total volume of the preparation is 1-10%. Specifically, the volume fraction is 1-2%, 2-3%, 3-5%, 5-7%, 7-9% or 9-10%. Preferably, the volume fraction is 2-3%.

[0026] In some specific embodiments, based on the total volume of the preparation, the volume fraction of the serum-free immune cell culture medium to the total volume of the preparation is 10-50%. Specifically, the volume fraction is 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, 35-40%, 40-45% or 45-50%. Preferably, the volume fraction is 20-40%.

[0027] In some embodiments, the lentivirus may be a recombinant human immunodeficiency virus (eg, HIV-1) or vesicular stomatitis virus G (VSV-G). In addition, the lentivirus may contain one or more exogenous genes, including, for example, an exogenous gene encoding a chimeric antigen receptor (CAR).

[0028] In some embodiments, CAR each comprises an antigen binding domain, a transmembrane domain and one or more signaling domains in the N-terminal to C-terminal direction. The signaling domain may include one or more primary signaling domains (e.g., CD3-ζ stimulatory domains) and / or one or more costimulatory signaling domains (e.g., intracellular domains selected from costimulatory proteins, the costimulatory proteins selected from CD27, CD28, 4-1BB (CD137), OX40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, ICAM-1, lymphocyte function-associated antigen-1 (LFA-1), CD2, CDS, CD7, CD287, LIGHT, NKG2C, NKG2D, SLAMF7, NKp80, NKp30, NKp44, NKp46, CD160, B7-H3 and a ligand specifically bound to CD83).

[0029] In some embodiments, the lentivirus dispersed in the aforementioned formulation maintains a concentration of about 70% to about 100% relative to the concentration of the lentivirus dispersed in the aforementioned formulation prior to the freeze / thaw cycle after 3-9 freeze / thaw cycles, wherein each freeze / thaw cycle comprises freezing the aforementioned formulation containing the lentivirus and subsequently thawing the aforementioned formulation containing the lentivirus at room temperature.

[0030] The present invention also provides the use of the above-mentioned preparation in improving the killing efficiency or amplification efficiency of immune cells after in vitro lentiviral transduction.

[0031] In some embodiments, the immune cell is selected from B cells, T cells, macrophages, dendritic cells, neutrophils, eosinophils, basophils, natural killer cells, monocytes or mast cells. Preferably, the immune cell is a T cell.

[0032] The present invention also provides a composition comprising a lentiviral vector and the above-mentioned preparation.

[0033] In some embodiments, the lentiviral vector can be expressed as, for example, 1×10 7 Transduction units per milliliter (TU / mL) up to 1×10 8 TU / mL (e.g. 1x10 7 TU / mL, 2.5x10 7 TU / mL,3x10 7 TU / mL, 3.5x10 7 TU / mL, 4x 10 7 TU / mL, 4.5x 10 7 TU / mL, 5x10 7 TU / mL, 5.5x10 7 TU / mL, 6x10 7 TU / mL, 6.5x10 7 TU / mL, 7x10 7 TU / mL, 7.5x10 7 TU / mL, 8x 10 7 TU / mL, 8.5x 10 7 TU / mL, 9x 10 7 TU / mL, 9.5x 10 7 TU / mL or 1x 10 8 TU / mL) is present in the composition.

[0034] In some embodiments, the composition further comprises a pharmaceutically acceptable excipient. Specifically, the pharmaceutically acceptable excipient may be selected from sugars, such as lactose, glucose and sucrose; polyols, such as propylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol; alginic acid; emulsifiers, such as Tween; wetting agents, such as sodium lauryl sulfate; stabilizers; antioxidants; preservatives; diluents, such as pyrogen-free water; isotonic saline solution; and phosphate buffer, etc.

[0035] The present invention also provides a method for improving the expansion multiple or killing efficiency of T cells, the method comprising the following steps: mixing a lentiviral vector with the aforementioned preparation, and then mixing it with in vitro T cells to obtain lentiviral-transduced T cells, wherein the expansion multiple or killing efficiency of the lentiviral-transduced T cells is higher than that of T cells obtained after transduction with the lentiviral vector alone.

[0036] In the present invention, the term "freeze / thaw cycle" refers to exposing a liquid mixture, such as an aqueous solution or a suspension, to a temperature equal to or lower than its freezing point until the mixture is frozen, and then melting the mixture at a temperature greater than its freezing point. The freezing step can be performed by, for example, placing the mixture in an environment with a temperature from about -80°C to about -20°C. The mixture can be kept frozen before melting, for example, for a period of one or more days, weeks, months or years. The thawing step can be performed by exposing the mixture to a condition with a temperature of about 2°C to about 8°C, or by storing the mixture at room temperature (e.g., ambient temperature of a laboratory or about 25°C). Alternatively, thawing can occur by using a water bath (e.g., at 37°C).

[0037] In the present invention, the term "lentiviral vector" refers to a lentiviral particle having the ability to introduce exogenous nucleic acid molecules into a host. "Lentiviral vector" includes a viral vector comprising a sequence derived from HIV-1. The lentiviral vector carrying an exogenous gene is packaged into infectious viral particles via viral packaging with the assistance of a packaging plasmid using a specific cell line. Infectious viral particles infect cells to achieve the expression of exogenous genes. "Recombinant" viral vector refers to a viral vector constructed by genetic recombination technology. Recombinant viral vectors can be constructed using methods known in the art, such as by transducing a packaging cell line with a nucleic acid encoding a viral genome and then isolating newly packaged viral particles.

[0038] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0039] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing the specific embodiments rather than for limiting the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

[0040] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.

[0041] Example 1 Study on the stability of lentivirus in formulation

[0042] Cell recovery, amplification and lentivirus packaging are carried out upstream, and finally the crude lentivirus is harvested and dispensed into clean centrifuge bottles, which are balanced using an electronic balance. The balanced centrifuge bottles are placed in a laboratory centrifuge for centrifugation, and the supernatant is collected after centrifugation.

[0043] Place the pump feed tube into the crude supernatant and filter using a 1.0 μm capsule filter.

[0044] Add 40 units of nuclease per ml of lentiviral solution, mix well, and incubate in a 37 (36-38) °C water bath for 2 h.

[0045] Use hollow fiber column to ultrafiltration concentrate the virus and change the liquid: put the hose at the inlet and reflux end of the hollow fiber column into the virus liquid to be concentrated, and put the hose at the outlet end into the waste liquid bucket. Mix once every 15 minutes. When it is concentrated to 5 (4~6) times, add an equal volume of 0.9% NaCl to change the liquid. Change the liquid 5 times in total. After the liquid change is completed, when the sample is concentrated to 5 (4~6) times, completely open the reflux end tube clamp, close the permeate end tube clamp, circulate for 3~5 minutes, and discharge the feed liquid in the circulation pipeline into the feed liquid bottle.

[0046] The process solution 0.9% NaCl balanced the chromatography column to keep the pH, conductivity, and UV280 curves stable; After the chromatography column is balanced, the sample is loaded. After filtering with a 0.5 μm filter, the sample is loaded for one cycle. The inlet of the chromatography system is switched to the loading liquid. The UV is adjusted to zero, and the sample is loaded. The change of UV280 is observed. When the UV value rises to 30-50 mAU, the virus flow-through liquid is collected.

[0047] Place the hose at the inlet and reflux ends of the hollow fiber column into the virus solution to be concentrated, and the hose at the outlet end into the waste liquid bucket, and adjust the hose clamp at the reflux end. Mix while concentrating, and mix once every 15 minutes. When the concentration reaches 15 (15~20) times, completely open the hose clamp at the reflux end, close the hose clamp at the purging end, and circulate for 3~5 minutes. Discharge the liquid in the circulation pipeline into the liquid bottle, weigh it, record the volume, and take samples according to the sampling plan. Concentration multiple = volume of virus solution after clarification and filtration / volume of virus solution before liquid replacement after concentration.

[0048] Prepare sterilizing filters, screw-mouth syringes and other materials in advance, turn on the biosafety cabinet for UV disinfection for 30 minutes, and blow air for self-purification for 20 minutes. In the biosafety cabinet, sterilize and filter the sample of Experiment 2 (the virus concentrate of TFF2) using a 0.22 μm disc filter to obtain the stock solution of lentivirus experimental group 2.

[0049] The harvested lentivirus was cultured as described above (i.e. Figure 1 ) After the purification step, the six formulations in Table 2 were used for filling, and stored at -80°C, and p24 and TU were detected at D1, D2, D3, D5, D7, D10, and D12. The raw materials used in the formulations of this embodiment are shown in Table 1; Table 1

[0050] Table 2

[0051] The test results of the above formula are shown in Table 3. Figure 2-4 shown.

[0052] Table 3

[0053] Note: TU recovery rate = TU value at this time point / D1 TU value From the above results, it can be seen that at D10, the test results of TU were as follows: Preparation 1 decreased by 38%, Preparation 2 decreased by 37%, Preparation 3 decreased by 41%, Preparation 4 decreased by 0%, Preparation 5 decreased by 2%, and Preparation 6 decreased by 17%.

[0054] Example 2 Performance of different formulations of viruses after infection according to Figure 5 The process was carried out to infect T cells with 6 different preparations of lentivirus, and the viability, amplification, CD3+CAR+ and killing at D12 were tested during the process.

[0055] The PBMCs were taken out of liquid nitrogen and revived at 37°C. In a biosafety cabinet, the revived PBMCs were carefully transferred to preheated TexMACS medium with a pipette. After centrifugation at 400g for 10 min, the supernatant was discarded. The cells were then resuspended evenly with preheated complete medium and sampled for viable cell counting.

[0056] According to the live cell count results and CD3+ percentage, add sorting buffer according to 100μl sorting buffer / 1E+07 cells and incubate. Use LS column for sorting and use the matching piston to slowly elute the magnetically labeled CD3+T cells into a clean centrifuge tube.

[0057] The sorted T cells were mixed and sampled for counting. According to the counting results, 2E7 T cells were placed in a 15 ml centrifuge tube, centrifuged at 400 g for 10 min, and the supernatant was discarded. The cells were resuspended evenly with complete culture medium, and the T cell density was adjusted and cell activation reagent was added. After mixing, the cells were transferred to a six-well plate and activated and cultured in a cell culture incubator at 37°C and 5% CO2.

[0058] Take out each group of lentivirus and thaw in a dark environment at 2-8℃. Take out the cultured cells from the carbon dioxide incubator, take samples and count, centrifuge at 400g for 10min, discard the supernatant, add an appropriate amount of complete medium to resuspend, add lentivirus and transduce by centrifugation, and culture in a cell culture incubator at 37℃ and 5% CO2 after centrifugation.

[0059] After 24±2h of cell transduction, samples were taken and counted from each group of cells, and then transferred to 15ml centrifuge tubes and centrifuged at 400g for 10min. After discarding the supernatant, the density was adjusted to 5E5 cells / mL with complete culture medium. After resuspending evenly, the cells were transferred to a six-well plate and cultured in a cell culture incubator at 37℃ and 5% CO2.

[0060] The culture plate was removed, samples were taken from each well and counted. Based on the counting results, the density was adjusted to 5E5 cells / mL with complete culture medium. Each group of cells was cultured in a cell culture incubator at 37°C and 5% CO2.

[0061] The culture bottles were removed, samples were counted from each group, and sent for testing of CAR positive rate. VCN samples were also kept. The remaining cells were washed twice with 0.9% saline containing 1% HSA, and harvested by centrifugation at 400g for 10 minutes. Killing experiments were also performed.

[0062] The experimental groups and lentivirus information are shown in Table 4, and the specific culture parameters are shown in Table 5.

[0063] Table 4

[0064] Table 5

[0065] The above experimental results are as follows: Cell Expansion: like Figure 6 As shown: By D12, the amplification multiples of each group were all above 100 times, and those of preparations 4, 5, and 6 were above 200 times, among which preparation group 4 had the highest amplification multiple of 312 times.

[0066] Cell viability: like Figure 7 As shown: After D1 sorting, the cell viability of each group was above 90%, the cell viability decreased after viral transduction, and the cell viability began to increase after D5. By D12, the cell viability of each group was around 90%.

[0067] CD3+CAR+: like Figure 8 As shown: As of D12, the CD3+CAR+% in each group was above 65%.

[0068] Cell Killing: The cell sample killing results of D12 are as follows Fig. 9 As shown, the killing rates of preparations 4, 5, and 6 were greater than 30%.

[0069] In summary, the preparation provided by the present invention can increase the viable expansion multiple, infection positive rate and cell killing effect of transduced T cells.

[0070] The above examples are for the purpose of illustrating the embodiments disclosed by the present invention and are not to be construed as limiting the present invention. In addition, the various modifications listed herein and the variations of the methods in the invention are obvious to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been specifically described in conjunction with various specific preferred embodiments of the present invention, it should be understood that the present invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of the present invention.

Claims

1. A preparation for lentiviral transduction, characterized in that The preparation comprises multiple types of chloride salt, albumin and serum-free immune cell culture medium. Based on the total volume of the preparation, the mass volume fraction of the chloride salt is 0.24-0.96%, the volume fraction of the albumin is 1-10%, and the volume fraction of the serum-free immune cell culture medium is 10-50%; The chloride salt is selected from sodium chloride; The albumin is selected from serum albumin; The serum-free immune cell culture medium is selected from X-VIVO 15 culture medium.

2. The preparation according to claim 1, characterized in that Based on the total volume of the preparation, the mass volume fraction of the chloride salt is 0.3-0.84%.

3. The preparation according to claim 1, characterized in that Based on the total volume of the preparation, the volume fraction of the albumin is 2-3%.

4. The preparation according to claim 1, characterized in that Based on the total volume of the preparation, the volume fraction of the serum-free immune cell culture medium is 20-40%.

5. Use of the preparation according to any one of claims 1 to 4 for improving the killing efficiency or expansion efficiency of immune cells after in vitro lentiviral transduction.

6. The use according to claim 5, characterized in that The immune cells are selected from B cells, T cells, macrophages, dendritic cells, neutrophils, eosinophils, basophils, natural killer cells, monocytes or mast cells.

7. A composition, characterized in that The composition comprises a lentiviral vector and the preparation according to any one of claims 1 to 4.

8. A method for increasing T cell expansion multiple or killing efficiency, characterized in that: The method comprises the following steps: mixing a lentiviral vector with the preparation of any one of claims 1 to 4, and then mixing the mixture with in vitro T cells to obtain lentiviral-transduced T cells, wherein the expansion multiple or killing efficiency of the lentiviral-transduced T cells is higher than that of T cells obtained by transduction with the lentiviral vector alone.

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