Virus-like plasmid and application thereof

Abstract

The invention discloses a virus-like plasmid and application thereof. The virus-like plasmid is characterized in that mIL-21 and 4-1BBL coding genes are inserted into a lentivirus plasmid; the amino acid sequences of the coding genes of the mIL-21 and the 4-1BBL are respectively as shown in SEQ ID NO. 1 and SEQ ID NO. 2. Virus-like particles (VLPs) prepared from the virus-like plasmids are added into an NK cell culture solution, and the VLPs can synergistically stimulate the NK cells, promote the rapid growth of the NK cells (the amplification multiple reaches 1850 times after 18 days of culture), and can improve the purity (about 92%) and the killing rate (when the efficiency-target ratio is 2: 1, up to 80%) of the NK cells.

Description

Technical Field

[0001] The present invention relates to the field of virus-like plasmid culture and cell preparation, and in particular to a virus-like plasmid, and the use of the virus-like plasmid to construct virus-like particles and then be used for in vitro amplification and culture of NK cells. Background Art

[0002] Natural killer (NK) cells are part of the innate immune system and are different from the human body's T cells and B cells. They are widely recognized for their strong cytotoxicity, ability to kill target cells without antigen sensitization, lack of major histocompatibility complex (MHC) restriction, and fast response speed. In addition, a large amount of clinical experimental data shows that NK cells are targeted to cancers such as osteosarcoma, leukemia, lymphoma, ovarian cancer, and breast cancer. These advantages make them a new choice for the treatment of chronic or drug-resistant cancers.

[0003] NK cell-based adoptive immunotherapy is a cell biological therapy method that returns NK cells induced and cultured in vitro to tumor patients, allowing them to directly or indirectly kill tumor cells in the body, thereby achieving the purpose of treating tumors. NK cells in the human body are differentiated and developed from hematopoietic stem cells and are mainly distributed in peripheral blood, accounting for 5-10% of peripheral blood mononuclear cells (PBMC). NK cells returned to the body will not proliferate again, so a large number of NK cells need to be returned to the patient to achieve a relatively good treatment effect.

[0004] At present, how to obtain a large number of high-purity and high-activity NK cells in vitro has always been the primary prerequisite for the large-scale clinical application of NK cells.

[0005] There are two main methods reported for in vitro expansion of NK cells: pure factor cell culture method and trophoblast method. The pure factor method is known for its high safety. The cytokines used are all existing in the body environment. By adding various cytokines to stimulate mononuclear cells, they are induced in the direction of NK cells. However, there are problems such as low expansion multiples, low purity and poor activity of the expanded NK cells, and poor reproducibility between different batches. It is difficult to achieve true NK cell activation and proliferation; the trophoblast method is to add trophoblast cells to activate and expand NK cells in peripheral blood in a targeted manner, but the added trophoblast cells belong to cancer cell lines, and there may be a risk of carcinogenesis in NK cell transfusion therapy. Summary of the invention

[0006] In view of the above problems, the present invention provides a virus-like plasmid, which is used to construct virus-like particles (VLPs), and then VLPs are used to activate the in vitro expansion culture of NK cells; because VLPs are obtained by packaging the cell line 293T that overexpresses mIL-21 and 4-1BBL, and then collected and purified and added to NK cell culture, the safety risk of using NK cells is reduced, and NK cells are efficiently expanded and the cell purity is greatly improved.

[0007] The technical solution of the present invention is as follows:

[0008] A virus-like plasmid, which is a lentiviral plasmid into which mIL-21 and 4-1BBL encoding genes are inserted; wherein the amino acid sequences of the mIL-21 and 4-1BBL encoding genes are shown as SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0009] The present invention also provides a method for preparing a virus-like plasmid, the steps of which are as follows:

[0010] IL-21 was linked to the transmembrane region of CD8a to obtain the chimeric gene mIL-21;

[0011] The chimeric gene 4-1BBL was obtained by connecting the cleaved peptide to 4-1BBL;

[0012] The chimeric gene mIL-21 and the chimeric gene 4-1BBL were constructed into the same lentiviral-like plasmid.

[0013] The present invention also provides a method for preparing 293T cells containing mIL-21 and 4-1BBL, comprising the following steps:

[0014] Obtaining the above virus-like plasmid;

[0015] The virus-like plasmid, packaging plasmids pMD-gag / po l, pMD-rev, auxiliary plasmid pMD-vsvg and transfection agent are added to a 293T cell solution for cultivation, the supernatant in the culture solution is collected and centrifuged, and the centrifuged supernatant is filtered and concentrated to obtain a lentiviral concentrate;

[0016] 293T cells, DMEM complete medium and the lentivirus concentrate were mixed and cultured to obtain 293T cells containing mIL-21 and 4-1BBL.

[0017] In one embodiment, in the method for preparing 293T cells, the preparation of the virus concentrate specifically comprises the following steps:

[0018] The virus-like plasmid expressing mIL-21 and 4-1BBL, the packaging plasmids pMD-gag / po l, pMD-rev, and the auxiliary plasmid pMD-vsvg were mixed at a mass ratio of 4:3:2:1 to obtain a plasmid mixture;

[0019] According to the ratio of mass to volume of 1:2, the plasmid mixture and the transfection reagent PE I were added to the 293T cell solution, and the four plasmids were transferred into the 293T cells through the transfection agent, and the cells were cultured in a 37°C, 5% CO2 incubator, and the supernatant of the cell culture solution was collected twice at 24 hours and 48 hours of culture respectively;

[0020] The supernatants collected twice were mixed and centrifuged at 3800 rpm for 5 min. The centrifuged supernatant was collected and filtered with a 0.45 μm filter membrane, and then transferred to an ultrafiltration tube for concentration to obtain a lentiviral concentrate.

[0021] In one embodiment, in the method for preparing 293T cells, 293T cells containing mIL-21 and 4-1BBL are prepared, and the specific steps are as follows:

[0022] The 293T cells were resuspended in DMEM complete medium and the cell density was controlled to 1×10 5 / ml, and add 1ml of 293T cell suspension to each well, inoculate in 2 wells of a 24-well plate, and culture in a 37°C, 5% CO2 incubator for 24h;

[0023] After 24 h, the DMEM complete medium in the two wells inoculated with cells on the 24-well plate was discarded, and 300 μl of DMEM basal medium was added to each well, and incubated in a 37°C, 5% CO2 incubator for 1 h;

[0024] After 1 hour, according to MOI=5, the lentiviral concentrate was added to one of the two wells inoculated with cells on a 24-well plate, and incubated for 6 hours;

[0025] After 6 h, add DMEM complete medium to 1 ml in each of the two wells inoculated with cells on the 24-well plate and continue culturing for 36 h;

[0026] After 36 hours, 1 μg of puromycin was added to each of the two wells inoculated with cells on the 24-well plate for cell screening and culture; monoclonal clones with high expression of mIL-21 and 4-1BBL were selected and expanded to obtain 293T cells containing mIL-21 and 4-1BBL.

[0027] The present invention also provides a method for preparing virus-like particles, the steps of which are as follows:

[0028] The 293T cells containing mIL-21 and 4-1BBL prepared above were obtained and mixed with the packaging plasmid pMD-gag / pol, and the supernatant of the culture solution was collected;

[0029] The collected supernatant was centrifuged at 3800 rpm for 5 min, the supernatant was collected and filtered through a 0.45 μm filter membrane, and then transferred to an ultrafiltration tube for concentration to obtain virus-like particles.

[0030] The present invention also provides an in vitro amplification and culture method for activating NK cells using virus-like particles, comprising the following steps:

[0031] On day 0, resuspend NK cells in 20 ml NK cell expansion medium and control the cell density to 1.0-2.0×10 6 / ml; inoculating the NK cell suspension into the coated culture bottle, and adding 20-2000 μg of the virus-like particles prepared above, and culturing in a 37°C, 5% CO2 incubator;

[0032] From the 3rd to the 18th day of culture, NK cell expansion medium was added every 2 to 3 days, and the cell density after replenishment was controlled to be 0.8 to 1.5 × 10 6 / ml until the 18th day, when the culture is completed and the cultured NK cells are collected; wherein, 20 to 2000 μg and 40 to 4000 μg of the virus-like particles prepared above need to be supplemented on the 3rd day and the 5th day, respectively.

[0033] In one embodiment, in the above-mentioned in vitro expansion and culture method of NK cells, the coating solution components in the culture bottle are: anti-CD20 antibody with a final concentration of 0.5-10 μg / ml, anti-CD137 antibody with a final concentration of 0.5-10 μg / ml, and 5-20 ml of DPBS solution.

[0034] In one embodiment, in the above-mentioned in vitro expansion and culture method of NK cells, the NK cell expansion culture medium includes KBM581 culture medium, which contains IL-2 with a final concentration of 100 to 2000 IU / ml, Nicotinamide with a final concentration of 10 to 200 μg / ml, and inactivated autologous plasma with a final concentration of 1 to 20 v / v%.

[0035] Compared with the prior art, the present invention has the following advantages:

[0036] (1) In the in vitro culture of NK cells, the addition of VLPs increases the synergistic stimulation of VLPs on NK cells, making the purity, proliferation and activity of the cultured NK cells better. On the 18th day of culture, the NK cell expansion multiple can reach 1850 times, and the NK cell CD3-C56+ purity is as high as about 92%. In addition, the experimental repeatability of NK seed cells from different sources is good, avoiding the problems of poor proliferation ability and low purity when culturing NK cells by pure factor method;

[0037] (2) Compared with the trophoblast method, the biosafety risk caused by the direct addition of trophoblasts (tumor cells) is reduced in the in vitro culture of NK cells;

[0038] (3) NK cells obtained by adding VLPs to culture have high tumor killing activity. When the effector-target ratio is 2:1, the killing rate can reach more than 80%. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 The purity flow cytometry of NK cells (CD3-C56+) on day 0 in Example 4 and Example 5 is shown;

[0040] Figure 2 The purity flow cytometry of NK cells CD3-C56+ on day 7, day 10, day 12, day 14, day 16 and day 18 in Example 4 is shown;

[0041] Figure 3 The purity flow cytometry graphs of NK cells CD3-C56+ on day 7, day 10, day 12, day 14, day 16, and day 18 in Example 5 are as follows;

[0042] Figure 4 The figure is a graph showing the NK cell expansion multiple in Example 4 and Example 5;

[0043] Figure 5 This is a graph showing the NK cell killing rate in Example 4 and Example 5. DETAILED DESCRIPTION

[0044] The preferred embodiments of the present invention are further described in detail below in conjunction with the accompanying drawings.

[0045] The present invention provides an in vitro expansion and culture method for activating NK cells using VLPs. The VLPs are obtained by packaging a cell line 293T that overexpresses mIL-21 and 4-1BBL, and then collected and purified, and added to NK cell culture, thereby avoiding the risks caused by direct use of trophoblast cells, and also solving the problems of limited expansion capacity and low cell purity when culturing NK cells by the pure factor method.

[0046] The technical solution of the present invention is as follows:

[0047] An in vitro amplification and culture method for activating NK cells using virus-like particles comprises the following steps:

[0048] 1) Preparing a virus-like plasmid overexpressing mIL-21 and 4-1BBL;

[0049] 2) preparing a lentiviral concentrate of a virus-like plasmid overexpressing mIL-21 and 4-1BBL;

[0050] 3) Using virus-like plasmids to prepare the overexpression cell line 293T-mIL-21-4-1BBL;

[0051] 4) The expression cell line 293T-mIL-21-4-1BBL was co-cultured with the packaging plasmid pMD-gag / pol to prepare virus-like particles (VLPs) overexpressing mIL-21 and 4-1BBL;

[0052] 5) Adding NK cells to the mixture of NK culture medium and VLPs to activate and expand NK cells.

[0053] In the present invention, mononuclear cells (PBMC) separated from peripheral blood are used for NK cell expansion and culture; the specific preparation process is as follows.

[0054] 1. Mononuclear Cell Isolation

[0055] Fresh peripheral blood drawn from a healthy person is slowly transferred to a 50 ml centrifuge tube containing Ficoll solution. The ratio of peripheral blood to Ficoll solution is 1:2. Try not to destroy the boundary layer between the two liquids during the operation.

[0056] Transfer the centrifuge tube to the centrifuge, balance it, and centrifuge it at 800g for 20 min at room temperature, setting the speed to 5 for ascending and 0 for descending;

[0057] After centrifugation, transfer the mononuclear cells of PBMC in the middle buffy coat layer to a new 50 ml centrifuge tube, add DPBS solution for washing, centrifuge the washed cells at 500 g for 10 min in room temperature, repeat washing and centrifugation 2 to 3 times, collect the mononuclear cells (PBMC) and set aside.

[0058] 2. Preparation of virus-like plasmids overexpressing mIL-21 and 4-1BBL

[0059] Design or obtain the coding genes of IL-21 and 4-1BBL;

[0060] IL-21 was linked to the transmembrane region of CD8a to obtain the chimeric gene mIL-21;

[0061] The chimeric gene 4-1BBL was obtained by connecting the cleaved peptide to 4-1BBL;

[0062] The chimeric gene mIL-21 and the chimeric gene 4-1BBL were constructed into the same lentiviral plasmid to obtain a lentiviral-like plasmid overexpressing mIL-21 and 4-1BBL;

[0063] Among them, the amino acid sequences corresponding to the coding genes of mIL-21 and 4-1BBL are as follows:

[0064] The amino acid sequence of mIL-21 is shown in SEQ ID NO.1:

[0065] MPLTSQVTSSSHAAKPDFYIYAQLKSANTGNNERI INVS INCAADSPCFSCMSACLDFSMALL TYAHSLLRPEACRPNCEWSNYVQGKNYLPLAPT IACDSYEKKPGLDFACDPALSDASTTTYASDHNSHAG IYCSAPLLLFGCPSCDSYEAQLKSAYCSTNAGLIDEFFKSPEACRPRQKHRLTLSLVITLAVHTAFSCFQKAQLKSANTHLSSRSIKKGRQIDYC;

[0066] The amino acid sequence of 4-1BBL is shown in SEQ ID NO.2:

[0067] MLPFRVTPEI GPELSPFFFSKADASLACPWANKNSSAPDSMPDLKKKADDDASDWGHQDSIDGPLSPGSNKPLASDAQGMFAQPSNHNDSAKCSHAPHPSLVGSVSLGPLSALVAKAGVHDCNPCAPASLPWALVDTEARARHAWQLSAIWNLALHLQPLRSCWVVAKAGVYYYSE IPACCCAD IWLADNSLARHVYYVAWANS ICHYSDCKAYHCCKCHHHHLNCDSVADHAHNPAGLPSPLRSEE.

[0068] 3. Preparation of Lentivirus Concentrate Overexpressing mIL-21 and 4-1BBL

[0069] The lentiviral plasmid overexpressing mIL-21 and 4-1BBL prepared in step 2, the packaging plasmids pMD-gag / pol, pMD-rev, and the auxiliary plasmid pMD-vsvg were mixed at a mass ratio of 4:3:2:1 to obtain a plasmid mixture;

[0070] According to the ratio of mass to volume of 1:2, the plasmid mixture and the transfection reagent PEI were added to the 293T cell solution, and the four plasmids were transferred into the 293T cells through the transfection agent, and cultured in a 37°C, 5% CO2 incubator for 48 hours, and the supernatant of the cell culture solution was collected at 24 hours and 48 hours respectively;

[0071] The supernatants collected twice were mixed and centrifuged at 3800 rpm for 5 min. The centrifuged supernatant was collected and filtered with a 0.45 μm filter membrane, and then transferred to an ultrafiltration tube (Amicon@Ultra-15, 10 kDa) for concentration to obtain a lentiviral concentrate containing mIL-21 and 4-1BBL.

[0072] 4. Preparation of overexpression cell line 293T-mIL-21-4-1BBL

[0073] The 293T cells were resuspended in DMEM complete medium and the cell density was controlled to 1×10 5 1 ml of 293T cell suspension was added to each well, and the 293T cell suspension was inoculated into two wells of a 24-well plate and cultured in a 37°C, 5% CO2 incubator for 24 h.

[0074] After 24 h, the DMEM complete medium in the two wells inoculated with cells on the 24-well plate was discarded, and 300 μl of DMEM basal medium was added, and incubated in a 37°C, 5% CO2 incubator for 1 h;

[0075] After 1 hour, add the lentiviral concentrate prepared in step 3 to one of the two wells inoculated with cells on a 24-well plate at an MOI of 5, and incubate for 6 hours;

[0076] After 6 h, add DMEM complete medium to 1 ml in each of the two wells inoculated with cells on the 24-well plate and continue culturing for 36 h;

[0077] After 36 hours, 1 μg of puromycin was added to each of the two wells inoculated with cells on the 24-well plate for cell screening and culture; monoclonal clones with high expression of mIL-21 and 4-1BBL were selected and expanded to obtain 293T cells containing mIL-21 and 4-1BBL.

[0078] 5. Preparation of virus-like particles (VLPs) overexpressing mIL-21 and 4-1BBL

[0079] The 293T cells containing mIL-21 and 4-1BBL prepared in step 4 were taken and mixed with the packaging plasmid pMD-gag / pol, and the supernatant of the culture solution was collected;

[0080] The collected supernatant was centrifuged at 3800 rpm for 5 min at 4°C. The supernatant was collected and filtered through a 0.45 μm filter membrane, then transferred to an ultrafiltration tube (Amicon@Ultra-15, 10 kDa) for concentration and ultrafiltration purification to obtain virus-like particles (VLPs).

[0081] After completing the above preparations, the additional components required for preparing NK cell expansion culture, namely virus-like particles (VLPs) and 293T cells containing mIL-21 and 4-1BBL, are obtained; the next step is to use the PBMCs isolated in step 1 to carry out NK cell expansion culture.

[0082] 6. NK cell culture

[0083] 1. Preparation

[0084] 1. Prepare coating solution: final concentration of 0.5-10 μg / ml anti-CD20 antibody, final concentration of 0.5-10 μg / ml anti-CD137 antibody and 5-20 ml DPBS solution.

[0085] Preferably, in the coating solution components, the final concentration of anti-CD20 antibody is 1-5 μg / ml, the final concentration of anti-CD137 antibody is 1-5 μg / ml and 5-20 ml of DPBS solution.

[0086] 2. Prepare NK cell expansion medium: Use KBM581 medium as the basal medium, and contain IL-2 with a final concentration of 100-2000 IU / ml, Nicotinamide with a final concentration of 10-200 μg / ml, and inactivated autologous plasma with a final concentration of 1-20 v / v%.

[0087] Preferably, the NK cell expansion medium is based on KBM581 medium, which contains IL-2 at a final concentration of 200 to 1000 IU / ml, Nicotinamide at a final concentration of 20 to 100 μg / ml, and inactivated autologous plasma at a final concentration of 5 to 10 v / v%.

[0088] (II) NK cell culture process

[0089] On day 0, add 20 l of NK cell culture coating solution to the TC-treated T75 culture flask; resuspend the PBMC cells isolated in step 1 with 20 ml of NK cell expansion medium and control the cell density to 1.0-2.0×10 6 / ml, to obtain a cell suspension; inoculate the cell suspension into the coated TC culture bottle, and add 20-2000 μg of the virus-like particles (VLPs) prepared in step 5, and culture in a 37°C, 5% CO2 incubator for 18 days;

[0090] On the third and fifth days, NK cell expansion medium was added respectively; at the same time, 20-2000 μg and 40-4000 μg of virus-like particles (VLPs) prepared in step 5 were added respectively, and the cell density after rehydration was controlled to be 0.8-1.5×10 6 Pieces / ml;

[0091] On the 7th day, the cells were transferred to the culture bag and NK cell expansion medium was added. The NK cell expansion medium was replenished every 2 to 3 days, and the cell density after replenishment was controlled to be 0.8 to 1.5 × 10 6 / ml until the 18th day of culture;

[0092] On the 18th day, the culture was completed, and the NK cells were collected and transferred to a centrifuge tube, centrifuged at 700g for 10 minutes, and the supernatant was removed; the NK cell pellet was resuspended with freezing solution, and the obtained NK cell suspension was divided into freezing tubes, transferred to liquid nitrogen for standby use; wherein the freezing solution was FBS freezing solution containing 10% DMSO.

[0093] Preferably, during the NK cell culture process, the amount of VLPs added is preferably 100 to 1000 μg.

[0094] In order to better understand the present invention, the technical scheme of the present invention is further described in detail below through specific embodiments and comparative examples.

[0095] Example 1 Preparation of 293T-mIL-21-4-1BBL overexpression cell line

[0096] 1.1 Construction of lentiviral plasmid pMD-mIL-21 / 4-1BBL

[0097] IL-21 was connected to the CD8a transmembrane region to obtain the chimeric gene mIL-21, which was then connected to 4-1BBL through cleavage peptide to obtain a chimeric sequence. Finally, the co-expression of mIL-21 and 4-1BBL was constructed into the same lentiviral vector to obtain the lentiviral-like plasmid pMD-mIL-21 / 4-1BBL; wherein the amino acid sequences of mIL-21 and 4-1BBL are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0098] 1.2 Packaging of recombinant lentivirus LV-mIL-21 / 4-1BBL

[0099] 1.2.1. Take 293T cells in the logarithmic growth phase and inoculate them into T175 cell culture flasks. When the cell confluence reaches more than 90%, remove the culture medium from the cell culture flask and add 17.5 ml of preheated fresh DMEM basal medium. Transfer to a 37°C, 5% CO2 incubator and incubate for 1 hour.

[0100] 1.2.2. According to the mass ratio of 4:3:2:1, 37.3 μg of virus-like plasmid pMD-mIL-21 / 4-1BBL, 28.0 μg of packaging plasmid pMD-gag / pol, 18.7 μg of packaging plasmid pMD-rev, and 9.3 μg of auxiliary plasmid pMD-vsvg were respectively added to a 6-well plate to obtain a total of 93.3 μg of four plasmids. The four plasmids were added to 2 ml of GIBCO OPTI-MEM (1X) medium and mixed to prepare a plasmid mixture;

[0101] 1.2.3, take 186.6μl PEI, add 2ml GIBCO OPTI-MEM (1X) medium to mix, prepare PEI mixture, mix 93.3ug plasmid mixture and 186.6μl PEI mixture to form a plasmid-PEI mixture, incubate at room temperature for 20min, add to the 293T cells incubated in step 1.2.1, and replace with 35ml fresh DMEM basal medium containing 2% FBS after 6h;

[0102] 1.2.4. Collect the 24h and 48h lentiviral supernatants separately, mix them, centrifuge the mixed supernatants at 3800rpm, 5min, and 4°C, collect the centrifuged supernatants, filter them with a 0.45μm filter membrane, transfer them to an ultrafiltration tube (Amicon@Ultra-15, 100kDa) for concentration, and obtain the recombinant lentivirus LV-mIL-21-4-1BBL (i.e., lentiviral concentrate), and store them in aliquots at -80°C for later use;

[0103] 1.2.5. After infecting 293T cells with the prepared recombinant lentivirus LV-mIL-21-4-1BBL for 48 hours, the infection positive rate was detected by flow cytometry, and the titer was detected according to the following formula. The volume of lentivirus concentrate (i.e., recombinant lentivirus) added in the subsequent steps was controlled according to the calculated cell titer value;

[0104] The virus titer was calculated according to the following formula:

[0105] Virus titer (TU / ml) = {(transduction positive rate - blank positive rate) × number of cells plated for titer detection} / virus loading volume (ml).

[0106] 1.3 Preparation of 293T-mIL-21-4-1BBL overexpression cell line

[0107] 1.3.1. Resuspend the collected 293T cells in DMEM complete medium and adjust the cell density to 1×10 5 After the cells / ml were inoculated into 2 wells of a 24-well plate, 1 ml / well, the plate was placed in a 37°C, 5% CO2 incubator for 24 h;

[0108] 1.3.2. 24 hours after inoculation, discard the DMEM complete medium in the two wells of the 24-well plate where the cells were inoculated, add 300 μl of DMEM basal medium, and incubate in a 37°C, 5% CO2 incubator for 1 hour;

[0109] 1.3.3. After the incubation is completed, according to the MOI value of recombinant lentivirus infection of 5, recombinant lentivirus LV-mIL-21-4-1BBL is added to one of the wells to infect 293T cells. After 6 hours, DMEM complete medium is added to 1 ml and placed in a 37°C, 5% CO2 incubator for overnight culture;

[0110] 1.3.4. After culturing for 36 hours, add cell culture medium and 1 μg of puromycin to the two wells on the 24-well plate where cells were inoculated, respectively, for cell screening and culture, and then select monoclonal clones, and select monoclonal clones with high expression of 4-1BBL by flow cytometry. After expanded culture, the engineered cell line 293T-mIL-21-4-1BBL that stably expresses mIL-21 and 4-1BBL molecules is obtained and set aside.

[0111] Example 2 Packaging and purification of VLPs

[0112] 2.1. Take the 293T-mIL-21-4-1BBL cells prepared in Example 1 and inoculate them into a T175 cell culture flask. After the cell confluence reaches more than 90%, remove the culture medium from the cell culture flask, add 17.5 ml of preheated fresh DMEM basal medium, transfer to a 37°C, 5% CO2 incubator and incubate for 1 hour for later use;

[0113] 2.2. Take 56.0 μg of auxiliary plasmid pMD-gag / pol and add it to a 12-well plate. Add 1 ml of GIBCOOPTI-MEM (1X) culture medium to each well to prepare a plasmid mixture.

[0114] 2.3. Take 112.0 μl of PEI, add 1 ml of GIBCO OPTI-MEM (1X) medium to prepare a PEI mixture, then mix 56.0 μg of the plasmid mixture and 112.0 μl of the PEI mixture to form a plasmid-PEI mixture, incubate at room temperature for 20 min, add it to the 293T-mIL-21-4-1BBL cells incubated in step 2.1, and replace it with 35 ml of fresh DMEM basal medium after 6 hours;

[0115] 2.4. Collect the VLPs supernatant of 24h and 48h respectively, centrifuge at 3800rpm, 5min, 4℃, collect the centrifugal supernatant, filter with 0.45μm filter membrane, transfer to ultrafiltration tube (Amicon@Ultra-15, 10kDa) for concentration, and obtain VLPs, which are then packaged and frozen in a -20℃ refrigerator for later use;

[0116] 2.5. Detect the protein concentration of the prepared VLPs. In subsequent experiments, the corresponding volume will be added according to this protein concentration value.

[0117] Example 3 Extraction of PBMC cells

[0118] 3.1. Collect 50 ml of peripheral blood from healthy volunteers and transfer it to a 50 ml centrifuge tube containing Ficoll solution. The ratio of peripheral blood to Ficoll solution is 1:2. Try not to destroy the boundary layer between the two liquids during the operation.

[0119] 3.2. Transfer the centrifuge tube to the centrifuge, balance it, and centrifuge it at 800g for 20 minutes in the greenhouse, setting the speed to 5 for ascending and 0 for descending;

[0120] 3.2. After centrifugation, pipette the PBMC cells in the middle buffy coat layer into a new 50 ml centrifuge tube, add DPBS solution to wash, centrifuge at 500 g for 10 min, repeat 2 to 3 times, collect the mononuclear cells (PBMC) and set aside.

[0121] Example 4 Activation and expansion of NK cells

[0122] 4.1 Preparation of NK cell culture coating solution and NK cell expansion medium

[0123] Prepare 15 ml of coating solution: measure 15 ml of DPBS solution containing anti-CD20 antibody at a final concentration of 2 μg / ml and anti-CD137 antibody at a final concentration of 2 μg / ml in the DPBS solution;

[0124] Prepare 2000 ml of NK expansion medium: use KBM581 medium as the basal medium, and the KBM581 medium contains IL-2 with a final concentration of 200 IU / ml, Nicotinamide with a final concentration of 50 μg / ml, and inactivated autologous plasma with a final concentration of 5 v / v%.

[0125] 4.2NK cell activation and expansion

[0126] On day 0, add NK cell culture coating solution to the TC-treated T75 culture flask and place it horizontally in a 37°C, 5% CO2 incubator for 2-4 hours. After the coating is completed, discard the coating solution and resuspend the PBMC cells in 20 ml of NK cell expansion medium at a concentration of 1.8×10 6 The cells were inoculated into a coated T75 culture flask at a density of 10 / ml, and 50 μg / ml of VLPs prepared in Example 2 were added simultaneously; the proportion of NK cells in PBMC was about 8%; therefore, on day 0, the number of NK cells was about: 1.8×10 6 / ml×20ml×8%=2.88×10 6 indivual;

[0127] On the third day, 20 ml of NK cell expansion medium was added to the T75 culture flask, and 50 μg / ml of VLPs prepared in Example 2 were added simultaneously, and the culture was continued;

[0128] On the 5th day, 40 ml of NK cell expansion medium was added to the T75 culture flask, and 50 μg / ml of VLPs prepared in Example 2 were added simultaneously, and the culture was continued;

[0129] On the 7th day, all cells in the T75 culture flask were transferred to a cell culture bag, 80 ml of NK cell expansion medium was added to the cell culture bag, and the culture was continued;

[0130] On the 10th day, add 160 ml of NK cell expansion medium to the cell culture bag and continue culturing;

[0131] On day 12, add 320 ml of NK cell expansion medium to the cell culture bag and continue culturing;

[0132] On day 14, add 640 ml of NK cell expansion medium to the cell culture bag and continue culturing;

[0133] On day 16, add 720 ml of NK cell expansion medium to the cell culture bag and continue culturing;

[0134] On day 18, cell culture was stopped and NK cells were collected.

[0135] Example 5 (Comparative Example 1, pure factor method for culturing NK cells)

[0136] 5.1. NK cell culture test

[0137] In this example, no VLPs need to be added during the NK cell culture process, and the other processes for preparing NK cells are the same as those in Example 4.

[0138] Example 6 NK cell functional test detection

[0139] 6.1. NK cell purity flow cytometry detection

[0140] On the 7th, 10th, 12th, 14th, 16th and 18th days during the NK cell culture process of Example 4 and Example 5, 0.8×10 NK cells were collected respectively. 6 100 μl PBS buffer was used to wash the NK cells twice; 200 μl PBS buffer containing 2% FBS was used to resuspend the washed NK cells; 2 μl anti-human CD56-APC and CD3-PE antibodies were added to each NK cell suspension, and the mixture was gently blown and incubated at room temperature and away from light for 15 min; after the incubation, the 2 cells were washed twice with 1 ml PBS buffer containing 2% FBS; the washed NK cells were resuspended again with 200 μl PBS buffer containing 2% FBS; the purity ratio of NK cells (CD3-C56+) was detected by flow cytometry, and the detection results of the ratio of NK cells (CD3-C56+) are shown in Table 1. Figure 1 , Figure 2 and Figure 3 shown.

[0141] Table 1 Detection ratio of NK cells (CD3-C56+)

[0142]

[0143] From Table 1, Figure 1 , Figure 2 and Figure 3 It can be seen that on the 18th day of NK cell culture, the proportion of NK cells (CD3-C56+) cultured in Example 4 was 91.24%, and the proportion of NK cells (CD3-C56+) in Comparative Example 1 was 74.71%, that is, the purity of NK cells in Example 4 was much greater than that in Comparative Example 1. This is because, during the NK cell culture process of Example 4, VLPs were added on the 0th day, the 3rd day, and the 5th day, respectively, and VLPs can synergistically stimulate NK cells and promote the rapid proliferation and growth of NK cells.

[0144] 6.2. NK cell expansion multiple detection

[0145] 6.2.1. Total cell growth assay

[0146] During the cell culture process of Example 4 and Example 5, samples were taken on day 0, day 7, day 10, day 12, day 14, day 16, and day 18, and the number of cells was recorded using a Countstar cell counter. The total cell count results are shown in Table 2.

[0147] Table 2 Total cell count results

[0148]

[0149] 6.2.2 Calculation of NK cell expansion

[0150] Since Examples 4 and 5 use PBMC seed cells for NK cell culture, and the initial content of NK cells in the PBMC seed cells is about 8%; during the culture process, the number of NK cells at each time point = the total number of PBMC cells * NK cell purity; the NK cell expansion multiples calculated from Tables 1 and 2 are shown in Table 3; Figure 4 It is a cell expansion multiple curve chart drawn according to the data in Table 3.

[0151] Table 3 NK cell expansion multiples

[0152]

[0153] From Table 3 and Figure 4 It can be seen that on the 18th day of NK cell culture, the expansion of NK cells in Example 4 was 1849.8 times, which was much greater than the expansion of NK cells in Comparative Example 1 by 814.8 times. This is because, during the NK cell culture process of Example 4, VLPs were added on the 0th day, the 3rd day, and the 5th day, respectively, and VLPs can synergistically stimulate NK cells and promote the rapid expansion and growth of NK cells.

[0154] 6.3 Evaluation of NK cell killing assay in vitro

[0155] K562 cells stably expressing Luciferase (luciferase) were used as target cells, and NK cells harvested on the 18th day in Example 4 and Example 5 (Comparative Example 1) were used as effector cells. 50 μl of NK cells and 50 μl of K562-Luciferase cells were inoculated into 96-well plates according to the effector-target ratio E:T=0:1, 1:1, 2:1, 5:1, and 10:1, respectively. After incubation for 6 hours at 37°C and 5% CO2 incubator, the release level of target cell Luciferase was determined using the GMOne-Step Luciferase Reporter Gene Assay Kit (purchased from Genomeditech) to determine the killing ability of NK cells. The NK cell killing rate was calculated using the following formula:

[0156] NK cell cytotoxicity (%) = {(target cell maximum release group only - experimental group) / target cell maximum release group only} × 100%.

[0157] The calculation results are shown in Table 4. The curve graph drawn from the data in Table 4 is shown in Figure 5 shown.

[0158] Table 4 NK cell killing rate

[0159] Effector-target ratio (E:T) Example 4 Example 5 (Comparative Example 1) 0：1 0 0 1：1 44.50％ 40.20％ 2：1 80.70％ 65.50％ 5：1 91.20％ 85.00％ 10：1 96.90％ 94.40％

[0160] From Table 4 and Figure 5 It can be seen that when the effector-target ratio is 2:1, the tumor killing rate of NK cells cultured in Example 4 is 80.7%, which is significantly higher than the tumor killing rate of 61.5% of NK cells cultured in Comparative Example 1. This is because VLPs are added in Example 4, which can synergistically stimulate NK cells, promote the rapid proliferation and growth of NK cells, and improve the purity of NK cells, which greatly improves the tumor killing rate of NK cells.

[0161] It should be understood that the above description of the preferred embodiments of the present invention is relatively detailed and cannot be regarded as limiting the scope of patent protection of the present invention. The scope of patent protection of the present invention shall be based on the attached claims.

Claims

1. A virus-like plasmid, characterized in that The virus-like plasmid is a lentiviral plasmid into which mIL-21 and 4-1BBL coding genes are inserted.

2. The virus-like plasmid according to claim 1, characterized in that The amino acid sequences of the mIL-21 and 4-1BBL encoding genes are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

3. The method for preparing a virus-like plasmid according to claim 1 or 2, characterized in that: The steps include: IL-21 was linked to the transmembrane region of CD8a to obtain the chimeric gene mIL-21; The chimeric gene 4-1BBL was obtained by connecting the cleaved peptide to 4-1BBL; The chimeric gene mIL-21 and the chimeric gene 4-1BBL were constructed into the same virus-like plasmid.

4. A method for preparing 293T cells containing mIL-21 and 4-1BBL, characterized in that: The steps include: Take the virus-like plasmid as claimed in claim 1; The virus-like plasmid, packaging plasmids pMD-gag / pol, pMD-rev, auxiliary plasmid pMD-vsvg and transfection agent are added to a 293T cell solution for cultivation, the supernatant in the culture solution is collected and centrifuged, and the centrifuged supernatant is filtered and concentrated to obtain a lentiviral concentrate; 293T cells, DMEM complete medium and the lentivirus concentrate were mixed and cultured to obtain 293T cells containing mIL-21 and 4-1BBL.

5. The preparation method according to claim 4, characterized in that: The preparation of the virus concentrate specifically includes the following steps: The virus-like plasmid expressing mIL-21 and 4-1BBL, the packaging plasmids pMD-gag / pol, pMD-rev, and the auxiliary plasmid pMD-vsvg were mixed at a mass ratio of 4:3:2:1 to obtain a plasmid mixture; According to the ratio of mass to volume of 1:2, the plasmid mixture and the transfection reagent PEI were added to the 293T cell solution, and the four plasmids were transferred into the 293T cells through the transfection reagent, and the cells were cultured in a 37° C., 5% CO2 incubator, and the supernatant of the cell culture solution was collected at 24 hours and 48 hours of culture respectively; The supernatants collected twice were mixed and centrifuged at 3800 rpm for 5 min. The centrifuged supernatant was collected and filtered with a 0.45 μm filter membrane, and then transferred to an ultrafiltration tube for concentration to obtain a virus concentrate.

6. The preparation method according to claim 4, characterized in that: Prepare 293T cells containing mIL-21 and 4-1BBL. The specific steps are as follows: The 293T cells were resuspended in DMEM complete medium and the cell density was controlled to 1×10 5 / ml, and add 1ml of 293T cell suspension to each well, inoculate into 2 wells of a 24-well plate, and culture in a 37°C, 5% CO2 incubator for 24h; After 24 h, the DMEM complete medium in the two wells inoculated with cells on the 24-well plate was discarded, and 300 μl of DMEM basal medium was added to each well, and incubated in a 37°C, 5% CO2 incubator for 1 h; After 1 hour, according to MOI=5, the lentiviral concentrate was added to one of the two wells inoculated with cells on a 24-well plate, and incubated for 6 hours; After 6 h, add DMEM complete medium to 1 ml in each of the two wells inoculated with cells on the 24-well plate and continue culturing for 36 h; After 36 hours, 1 μg of puromycin was added to each of the two wells inoculated with cells on the 24-well plate for cell screening and culture; monoclonal clones with high expression of mIL-21 and 4-1BBL were selected and expanded to obtain 293T cells containing mIL-21 and 4-1BBL.

7. A method for preparing virus-like particles, characterized in that: Here are the steps: The 293T cells containing mIL-21 and 4-1BBL prepared by the preparation method as claimed in claim 4 were mixed and cultured with the packaging plasmid pMD-gag / pol, and the supernatant of the culture solution was collected; The collected supernatant was centrifuged at 3800 rpm for 5 min, the centrifuged supernatant was collected and filtered with a 0.45 μm filter membrane, and then transferred to an ultrafiltration tube for concentration to obtain virus-like particles.

8. A method for in vitro amplification and culture of NK cells activated by virus-like particles, characterized in that: The steps include: On day 0, resuspend NK cells in 20 ml of NK cell expansion medium and control the cell density to 1.0-2.0×10 6 / ml; inoculating the NK cell suspension into the coated culture bottle, and adding 20-2000 μg of the virus-like particles prepared by the preparation method according to claim 7, and culturing in a 37°C, 5% CO2 incubator; From the 3rd to the 18th day of culture, NK cell expansion medium was added every 2 to 3 days, and the cell density after replenishment was controlled to be 0.8 to 1.5 × 10 6 / ml until the 18th day, when the culture is completed and the cultured NK cells are collected; wherein, on the 3rd day and the 5th day, 20-2000 μg and 40-4000 μg of the virus-like particles prepared by the preparation method according to claim 7 need to be supplemented respectively.

9. The in vitro expansion and culture method according to claim 8, characterized in that: The coating solution components in the culture flask are: anti-CD20 antibody with a final concentration of 0.5-10 μg / ml, anti-CD137 antibody with a final concentration of 0.5-10 μg / ml, and 5-20 ml of DPBS solution.

10. The in vitro expansion and culture method according to claim 8, characterized in that: The NK cell expansion culture medium comprises KBM581 culture medium, which contains IL-2 with a final concentration of 100-2000 IU / ml, Nicotinamide with a final concentration of 10-200 μg / ml, and inactivated autologous plasma with a final concentration of 1-20 v / v%.

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