Disclosure of Invention
In view of the above, it is an object of the present invention to provide a virus-like particle expressing mIL21 and 4-1BBL and its use in amplifying NK cells in vitro for the art.
Virus-like particles (VLPs) are highly structured protein particles assembled from viral single or multiple structural proteins, similar in morphology and structure to natural viral particles. VLPs lack regulatory proteins and infectious nucleic acids, have no replication ability, and have high safety. The present invention aims at using VLPs to express mll 21, 4-1BBL to achieve NK cell expansion and circumvent the tumorigenic risk of K562 cells.
The invention adopts the following technical scheme to realize the aim of the invention:
in a first aspect the invention provides a virus-like particle B21-VLP expressing mIL21 and 4-1 BBL.
Further, the B21-VLP is formed by sequentially connecting operably linked elements such as mIL21, T2A, 4-1BBL and T2A, VSV-G;
The amino acid sequence of mIL21 is shown as SEQ ID NO.16, and the amino acid sequence of 4-1BBL is shown as SEQ ID NO. 17.
Further, the amino acid sequence of the VSV-G is shown as SEQ ID NO. 18;
Optionally, the base sequence of the mIL21 is shown as SEQ ID NO.20, and the base sequence of the 4-1BBL is shown as SEQ ID NO. 21;
Alternatively, the base sequence of VSV-G is shown as SEQ ID NO. 22.
In the invention, the mIL21 and the mbIL21 are obtained by molecular modification on the basis of mouse IL 21. It generally comprises the functional domains of IL21, which are critical for its binding to the receptor and for its biological activity. It has immunoregulatory, anti-tumor and antiviral effects. In a specific embodiment of the invention, the amino acid sequence of the mIL21 is shown in SEQ ID NO. 16.
In the present invention, the 4-1BBL refers to a 4-1BB ligand, also called CD137L, which is one of the members of the tumor necrosis factor superfamily and plays an important role in the immune system. 4-1BBL is a type II transmembrane protein, consisting of 306 amino acids. The extracellular domain contains 171 amino acids and can be hydrolyzed by protease to form soluble 4-1BBL. It has typical structural characteristics of the tumor necrosis factor superfamily, including a beta-sheet structure and a plurality of alpha-helices, which are critical for its binding to receptors and signaling. In a specific embodiment of the present invention, the amino acid sequence of the 4-1BBL is shown as SEQ ID NO. 17.
In the present invention, the T2A is a 2A peptide sequence from Foot and Mouth Disease Virus (FMDV). The 2A peptide is a short peptide, which can make virus polyprotein self-cut after translation during virus infection, and generate a plurality of independent functional proteins. T2A peptides typically consist of about 20 amino acids, with a specific amino acid sequence and spatial structure. T2A can be connected with different types of genes, and can effectively realize co-expression whether the genes code structural proteins or regulatory proteins and the like. In a specific embodiment of the invention, the amino acid sequence of T2A is shown in SEQ ID NO. 19.
In the present invention, the VSV-G refers to vesicular stomatitis virus glycoprotein (Vesicular Stomatitis Virus Glycoprotein), which is a transmembrane glycoprotein consisting of 511 amino acids and having a molecular weight of about 67kDa. In a specific embodiment of the present invention, the amino acid sequence of VSV-G is shown as SEQ ID NO. 18.
In a second aspect, the invention provides a method of constructing a virus-like particle B21-VLP expressing mIL21 and 4-1 BBL.
Further, the construction method comprises the following steps:
(1) Construction of B21-VSVG envelope plasmid:
(2) B21-VLPs were constructed by transfecting host cells with B21-VSVG envelope plasmids.
Further, the construction of the B21-VSVG envelope plasmid comprises the following steps:
(1) Obtaining mIL21 and 4-1BBL fragments through gene synthesis, wherein the amino acid sequence of the mIL21 is shown as SEQ ID NO.16, and the amino acid sequence of the 4-1BBL is shown as SEQ ID NO.17 as fragment I;
(2) Using pMD2.G as a plasmid skeleton, and performing restriction enzyme digestion with EcoRI restriction enzyme to obtain a fragment II;
(3) The pMD2.G is used as a plasmid template, a fragment III is obtained by PCR, a primer VSVG-F1 is designed, the sequence from the 5 'end to the 3' end is shown as SEQ ID NO.1, a homologous arm is designed by using a homologous recombination method and added to the 5 'end, the sequence from the amino end is shown as SEQ ID NO.2, a 3' end primer VSVG-R1 is designed, and the sequence from the 5 'end to the 3' end is shown as SEQ ID NO. 3;
(4) And connecting the obtained fragments I, II and III by using homologous recombination enzyme to obtain the complete plasmid, namely the B21-VSVG envelope plasmid.
Further, the construction of the B21-VLP using B21-VSVG envelope plasmid transfection host cells comprises the steps of:
(1) Mixing envelope plasmids B21-VSVG, auxiliary plasmids pMDLg and pRSV as solution A, mixing PEI with a culture medium as solution B, mixing A, B to obtain AB mixed solution, adding the AB mixed solution into a host cell culture medium, and culturing host cells;
(2) 3-5h later, executing liquid changing operation;
(3) Feeding after 20-22 h;
(4) Harvesting 48h after packaging to obtain the B21-VLP.
In some embodiments, the host cells are cultured under conditions of 37 ℃ in a 5% CO2 incubator at 125 rpm.
Further, the dosage of the envelope plasmid and the auxiliary plasmid is respectively B21-VSVG (1-10) mug, pMDLg (5-50) mug and pRSV (1-10) mug;
Alternatively, the dosage of the envelope plasmid and the auxiliary plasmid is respectively B21-VSVG 5 mug, pMDLg 20 mug and pRSV 5 mug;
Alternatively, the host cell is a 293T cell, 293 cell, HEK293F cell, CHO cell, vero cell or HeLa cell;
alternatively, the host cell is a 293T cell;
optionally, the culture medium isTransient medium, emCD HEK293 Plus medium, CELL-WISE 293 medium CW001, complete medium M293TI, glutamine-free medium M293TIS, glutamine-free phenol red-free medium M293TINPR or union293 medium;
optionally, the culture medium isTransient medium;
Optionally, the culture medium is used in an amount of 0.5-5mL when preparing the solution a;
optionally, in preparing the solution a, the culture medium is used in an amount of 1mL;
optionally, the PEI and the culture medium are used in an amount of 25-125 mu L and 0.5-5mL respectively when the solution B is prepared;
optionally, in preparing the solution B, the PEI and the culture medium are used in an amount of 75 mu L and 1mL respectively;
Optionally, the feeding comprises glucose and glutamine supplementation.
In some embodiments, the pipetting comprises centrifugation of the host cells at 1000rpm for 5min followed by 20mLTransient medium resuspended cells and transferred to 125mL cell culture shake flasks. Placed in a 37℃5% CO2 incubator, and shake cultured at 125 rpm.
In some embodiments, the feed comprises the steps of adding 80. Mu.L of 50% glucose injection, 800. Mu. L L-glutamine to a 125mL cell culture shake flask, shaking slowly and mixing, and shaking culture at 125rpm in a 37℃5% CO2 incubator.
In some embodiments, the harvesting comprises the steps of transferring the host cell suspension into a centrifuge tube, centrifuging at 2000rpm for 10min, collecting the supernatant as a virus harvest, filtering the virus harvest using a 0.45 μm filter, transferring the virus harvest into the centrifuge tube, centrifuging at 4 ℃ for 2h with 9 drops, 18300g, and resuspending the virus pellet using DPBS containing 2% HSA to obtain a B21-VLP virus concentrate.
A third aspect of the invention provides a composition comprising a B21-VLP of the first aspect of the invention.
In a fourth aspect, the invention provides a culture of expanded NK cells.
Further, the culture comprises the B21-VLP of the first aspect of the invention or the composition of the third aspect of the invention.
In a fourth aspect, the invention provides a method of expanding NK cells in vitro, culturing in vitro or stimulating NK cells.
Further, the method comprises contacting NK cells with the B21-VLP of the first aspect of the invention, the composition of the third aspect of the invention or the culture of the fourth aspect of the invention;
alternatively, the NK cells are present in a population of cord blood mononuclear cells.
In some embodiments, cord blood is enriched for hematopoietic stem cells and a variety of immune cells, including NK cells. NK cells in cord blood have some unique characteristics and advantages such as low immunogenicity, strong proliferation capacity, antitumor activity and immunoregulatory effect.
A fifth aspect of the invention provides any one of the following applications:
(1) Use of a B21-VLP of the first aspect of the invention, a composition of the third aspect of the invention or a culture of the fourth aspect of the invention for in vitro expansion of NK cells, in vitro culture or stimulation of NK cells;
(2) Use of NK cells cultured from the B21-VLP of the first aspect of the invention, the composition of the third aspect of the invention or the culture of the fourth aspect of the invention for the preparation of an anti-tumor drug.
In some embodiments, the present invention is not particularly limited to the specific source of the NK cells including but not limited to bone marrow-derived NK cells, peripheral blood-derived NK cells, peripheral lymphoid tissue-derived NK cells, thymus-derived NK cells, liver, lung, intestinal tract, and other non-lymphoid tissue-derived NK cells.
In some embodiments, the tumor includes, but is not limited to, hematological malignancies, solid tumors. Wherein the hematological malignancy includes, but is not limited to, acute myelogenous leukemia, non-hodgkin lymphoma, acute lymphoblastic leukemia, chronic myelogenous leukemia, hodgkin lymphoma, multiple myeloma. The solid tumors include, but are not limited to, melanoma, renal cell carcinoma, non-small cell lung cancer, brain glioma, meningioma, nasopharyngeal carcinoma, oral cancer, breast cancer, esophageal cancer, mediastinal tumor, gastric cancer, liver cancer, colorectal cancer, pancreatic cancer, bladder cancer, prostate cancer, ovarian cancer, cervical cancer, osteosarcoma, ewing's sarcoma, soft tissue sarcoma, skin cancer, thyroid cancer.
Compared with the prior art, the invention has the following beneficial effects:
The invention creatively constructs a brand new virus-like particle (B21-VLP) for expressing mIL21 and 4-1BBL for the first time, realizes high-efficiency expansion of NK cells and avoids the tumorigenic risk of K562 cells, in addition, the B21-VLP can enable NK cells to be selectively expanded when stimulating CBMC, the NK cells account for more than 90% of the total cell content in 21 days of culture, and the NK cells cultured by adopting the B21-VLP have stronger killing capacity to tumor cells, and have wide application prospect in the technical field of in-vitro expansion of NK cells.
Detailed Description
The invention is further illustrated below in conjunction with specific examples, which are provided solely to illustrate the invention and are not to be construed as limiting the invention. It will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations may be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the claims and their equivalents.
The reagents and raw materials used in the present invention are readily available to those of ordinary skill in the art, and unless otherwise indicated, are commercially available, and the experimental methods of the present invention without specifying the specific conditions are generally carried out according to conventional conditions or according to conditions suggested by the manufacturer, and in particular, the following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the present invention in any way. It should be noted that the experimental conditions and the results thereof described in the following examples are only for illustrating the present invention and should not limit the present invention described in detail in the claims.
EXAMPLE 1 construction of B21-VSVG envelope plasmid
1. Experimental materials
Primers were all synthesized by Jin Weizhi biosciences, inc. In su;
PCR MIX enzyme, jinsha organism, with the product number SF212;
AgeI restriction endonuclease: biolabs, cat# R3552SVIAL;
XholI restriction enzyme Biolabs, cat# R0146VVIAL;
EcoRI restriction endonuclease: biolabs, cat# R0101VVIAL;
homologous recombinase, jinsha organism, with the product number of SC612;
Agarose gel DNA recovery kit, tiangen biochemical technology, with the product number of DP209-02;
a small amount of DNA extraction kit is Jinsha organism, and the product number is PE707-50.
2. Experimental method
(1) Construction of B21-VSVG envelope plasmid
1) Obtaining mbIL21 and 4-1BBL (CD 137L) fragments by gene synthesis as fragment one;
2) Using pMD2.G as a plasmid skeleton, and performing restriction enzyme digestion with EcoRI restriction enzyme to obtain a fragment II;
3) pMD2.G was used as the plasmid template, PCR gave fragment three. The primer VSVG-F1 is designed, the base sequence from the 5' end to the 3' end is GAATTCTGACACTATGAAGTGCCTTT (SEQ ID NO. 1), and a homologous arm is designed by using a homologous recombination method and added to the 5' end, and the base sequence is GAGAACCCCGGCCCC (SEQ ID NO. 2). Designing a 3' end primer VSVG-R1, wherein the base sequence from the 5' end to the 3' end is TGTGCAGGATTTGAGTTACTTTCCAAGT (SEQ ID NO. 3);
4) The obtained fragments I, II and III are connected by homologous recombination enzymes to obtain complete plasmids, the complete plasmids are named as B21-VSVG, the B21-VLP is formed by sequentially connecting operable connecting elements, namely mbiL21, T2A, 4-1BBL and T2A, VSV-G, wherein the amino acid sequences of the mbiL21, 4-1BBL and VSV-G, T A are respectively shown as SEQ ID NO.16-19, and the base sequences of the mbiL21, 4-1BBL and VSV-G, T A are respectively shown as SEQ ID NO. 20-23.
5) Transforming plasmid by using competent cells of escherichia coli, coating overnight for growing bacteria, and sequencing to verify the correctness of the plasmid;
6) Plasmid colonies that were verified to be correct were grown up and plasmid DNA was extracted using a DNA extraction kit.
(2) Construction of IL21-VSVG envelope plasmid
1) Taking the B21-VSVG plasmid as a plasmid skeleton, and performing restriction enzyme digestion by using AgeI and XholI to obtain a fragment I;
2) The B21-VSVG plasmid was used as a template to obtain fragment two by PCR. The 5' -end primer IL21-VSVG-F1 was designed, and the base sequence from the 5' -end to the 3' -end was TCAGCATCTGTCCTCGAGAACAC (SEQ ID NO. 4). Designing a 3 'end primer, wherein the base sequence from the 5' end to the 3 'end is TGGTCCTGGATTTTCCTCCACG (SEQ ID NO. 5), designing a homology arm by using a homologous recombination method, adding the homology arm to the 5' end primer, and the base sequence from the 5 'end to the 3' end is GTACAGCAGGCACT (SEQ ID NO. 6);
3) Fragment three was obtained by PCR using the B21-VSVG plasmid as template. The 5 'primer IL21-VSVG-F2 was designed, the base sequence from 5' to 3 'was CCAAGTGCCTGCTGTACCTGG (SEQ ID NO. 7), and the homology arm was designed to 5' by homologous recombination, and the base sequence was GGAAAATCCAGGA (SEQ ID NO. 8). Designing a 3' end primer IL21-VSVG-R2, wherein the base sequence from the 5' end to the 3' end is TTTATGGTGAAAGCAGGACCGGT (SEQ ID NO. 9);
4) Electrophoresis of the fragments I, II and III on agarose gel, and extraction and quantification of corresponding plasmid fragment bands by using agarose gel DNA recovery kit;
5) Ligating the obtained fragments I, II and III by homologous recombination enzymes to obtain a complete plasmid which is named IL21-VSVG;
6) Transforming plasmid by using competent cells of escherichia coli, coating overnight for growing bacteria, and sequencing to verify the correctness of the plasmid;
7) Plasmid colonies that were verified to be correct were grown up and plasmid DNA was extracted using a DNA extraction kit.
(3) Construction of B-VSVG envelope plasmid
1) Taking the B21-VSVG plasmid as a plasmid skeleton, and carrying out enzyme digestion by EcoRI restriction enzyme to obtain a fragment I;
2) The B21-VSVG plasmid is used as a plasmid template, and the fragment II is obtained by PCR. The 5 'primer B-VSVG-F1 was designed, the base sequence from the 5' end to the 3 'end was GAATACGCCTCTGACGCTTCAC (SEQ ID NO. 10), and the homology arm was designed to the 5' end by using the method of homologous recombination, and the base sequence was AAGCACGTGAGATCTGCCACCATG (SEQ ID NO. 11). Designing a 3 'end primer B-VSVG-R1, wherein the base sequence of the 3' end primer B-VSVG-R1 is CTGCACTGGTGGGGTGAATTC (SEQ ID NO. 12);
3) Electrophoresis of the first and second fragments on agarose gel, and extraction and quantification of the corresponding plasmid fragment bands by using agarose gel DNA recovery kit;
4) Connecting the obtained fragment I and the fragment II by utilizing homologous recombinant enzyme to obtain a complete plasmid which is named as B-VSVG;
5) Transforming plasmid by using competent cells of escherichia coli, coating overnight for growing bacteria, and sequencing to verify the correctness of the plasmid;
6) Plasmid colonies that were verified to be correct were grown up and plasmid DNA was extracted using a small DNA extraction kit.
(4) Construction of B21-noVSVG envelope plasmid
1) Taking the B21-VSVG plasmid as a plasmid skeleton, and carrying out enzyme digestion by EcoRI restriction enzyme to obtain a fragment I;
2) The B21-VSVG plasmid is used as a plasmid template, and the fragment II is obtained by PCR. The 5' -end primer B21-F1 was designed, and the 5' -end to 3' -end base sequence was AAGCACGTGAGATCTGAATTCG (SEQ ID NO. 13). Designing a 3 'end primer TTCCGACCTCGGTGAAGGGA (SEQ ID NO. 14), designing a homology arm by using a homologous recombination method, adding the homology arm to the 5' end, and setting the base sequence as CTGCACTGGTGGGGTTCTAGA (SEQ ID NO. 15);
Electrophoresis of the first and second fragments on agarose gel, and extraction and quantification of the corresponding plasmid fragment bands by using agarose gel DNA recovery kit;
3) Ligating the obtained fragment I and the fragment II by homologous recombination enzyme to obtain a complete plasmid which is named as B21-noVSVG;
4) Transforming plasmid by using competent cells of escherichia coli, coating overnight for growing bacteria, and sequencing to verify the correctness of the plasmid;
5) Plasmid colonies that were verified to be correct were grown up and plasmid DNA was extracted using a small DNA extraction kit.
3. Experimental results
The constructed B21-VSVG envelope plasmid map is shown in figure 1, the schematic diagram of the amplification of NK cells by B21-VLP in vitro is shown in figure 2, and the schematic diagram of the structures of B21-VLP, B-VLP, 21-VLP and no VSV-B21-VLP is shown in figure 3.
EXAMPLE 2 construction of virus-like particle B21-VLP expressing mIL21 and 4-1BBL
1. Experimental materials
The transient culture medium is Kang organisms, and the product number is A21501;
KBM581 Medium, corning, cat# 88-591-CM;
PEI is Polyplus, the commodity number is 101000026;
50% glucose injection, henan Kolun pharmaceutical industry;
l-glutamine Solarbio, cat# G0200;
human Serum Albumin (HSA) Pis fei Ke biopharmaceutical;
Dulbecco's Phosphate Buffer Solution (DPBS) Gibco, cat# 14190250;
Recombinant human interleukin-2 (IL 2) for injection is available in the pharmaceutical industry;
P24 ELISA detection kit, namely the next holy organism, with the product number of 99301ES24;
FITC Anti-Human CD3, kuang Bo organism, cat# 6610004;
PE anti-human CD56, kuang Bo organism, cat No. A6803;
PE/Cyanine7 Anti-Human CD4 Anti-body: elabscience, cat# E-AB-F1109H;
PERCP ANTI-Human CD8 Antibody Elabscience, accession number AN00427F.
2. Experimental method
(1) Transfection of 293T cell expression Processes Using constructed envelope plasmids
1) Two VLPs were prepared using B21-VSVG, B21-noVSVG envelope plasmids, respectively. First, 293T cells were inoculated, 5X 106 cells were taken, and 20mL was usedThe transient medium was resuspended in 125mL cell culture shake flasks and incubated in 37℃5% CO2 incubator at 125rpm with shaking. Two envelope plasmids (5. Mu.g) were mixed with helper plasmids (pMDLg 20. Mu.g, pRSV 5. Mu.g) as solution A, and 75. Mu.L of PEI was mixed with 1mL of medium as solution B. And (3) gently mixing A, B liquids, avoiding generating bubbles, and standing for 5 minutes at room temperature after uniformly mixing. And adding the solution B into the solution A, gently mixing, and standing at room temperature for 20min after mixing. The AB mixture was slowly added to the cell culture flask, during which time it was slowly shaken and mixed. The cell culture flask was placed in a 37℃5% CO2 incubator and shake cultured at 125 rpm.
2) And executing liquid change operation after 3-5 h. After centrifugation of the cells at 1000rpm for 5min, 20mL was usedTransient medium resuspended cells and transferred to 125mL cell culture shake flasks. Placed in a 37℃5% CO2 incubator, and shake cultured at 125 rpm.
3) And (5) feeding for 20-22 hours. 80. Mu.L of 50% glucose injection, 800. Mu. L L-glutamine, was added to each 125mL cell culture flask, and mixed by slow shaking. Placed in a 37℃5% CO2 incubator, and shake cultured at 125 rpm.
4) Harvesting 48h after packaging. The cell suspension was transferred to a centrifuge tube, centrifuged at 2000rpm for 10min, and the supernatant was collected as a virus harvest, which was filtered using a 0.45 μm filter. The virus harvest is transferred into a centrifuge tube, and the virus harvest is lifted by 9 to 0,18300g and centrifuged for 2 hours under the environment of 4 ℃. The viral pellet was resuspended using DPBS containing 2% HSA to give B21-VLP, B-VLP, 21-VLP, B21-noVSVG-VLP virus concentrates, respectively.
5) Physical titers were detected after sampling using P24 ELISA. And (5) after the rest is packaged, placing the mixture at-80 ℃ for standby.
6) The physical titres of B21-VSVG and B21-noVSVG were compared.
(2) Isolation and stimulation of cord blood mononuclear cells
1) Isolation of mononuclear cells
Separating 200mL of cord blood into a centrifuge tube, centrifuging at 2000rpm and room temperature for 20min, removing upper pale yellow blood plasma, adding physiological saline with the same volume as the upper pale yellow blood plasma to obtain diluted cord blood, taking the centrifuge tube, adding lymphocyte separation liquid into the centrifuge tube, adding the diluted cord blood into the lymphocyte separation liquid, layering the diluted cord blood and the lymphocyte separation liquid, centrifuging at 2000rpm and room temperature for 30min, removing part of supernatant, sucking the middle white membrane layer into the centrifuge tube, adding the same volume of physiological saline, centrifuging at 2000rpm for 10min at room temperature, removing supernatant, washing for three times, and counting, wherein the volume ratio of the lymphocyte separation liquid to the diluted cord blood is 15:45-50.
2) Inoculation and stimulation of mononuclear cells
The sediment after the supernatant is removed in the last step is inoculated into KBM 581 culture medium containing IL-2 with the concentration of 200IU/mL according to the cell density of 5.0X10-6/mL to form a mixed solution, and the mixed solution is placed into a T25 culture flask after being coated and then is cultured in an incubator with the saturated humidity of CO2 with the temperature of 37 ℃ and the volume percentage content of 5%.
3) In vitro expansion of cord blood NK cells
After the single nucleus cell separation of the umbilical cord blood in the previous step, 2 bottles are placed in T25 bottles, and each bottle contains 5.0X106 cells and has a volume of 5mL. B21-VLP, B21-noVSVG-VLP stimulations, 5% autologous serum, with a mass of 500ng, were added to the coated T25 flask, day 0 of inoculation.
On day 2, the same volume of KBM 581 medium containing IL-2 at a concentration of 200IU/mL was added.
On days 4-6, cells were observed daily, KBM 581 medium containing IL-2 at a concentration of 200IU/mL was added according to the color or cell volume of the cell suspension, and the volume per addition should not exceed one time the existing volume.
Day 7 counts, cell densities were adjusted to 0.8-1.0X106 cells/mL depending on cell suspension color and cell number, and NK phenotypes were examined by flow cytometry. From day 7, cells were counted at 2-day intervals and KBM 581 medium containing IL-2 at 200IU/mL was supplemented to maintain the cell concentration at 8-1.0X106 cells/mL. Co-culturing for 21 days.
3. Experimental results
VSV-G (lentiviral packaging plasmid), which embeds the vesicular stomatitis virus G protein (VSV-G) gene sequence, plays a helper role in the lentiviral packaging process. The gene replaces the encoding gene of the viral envelope protein in the provirus, and remarkably enhances the infection range of host cells of the virus. VLPs were packaged using a plasmid containing VSV-G and a plasmid without VSV-G, respectively, and the P24 content of the VLPs was measured as its physical titer using ELISA, and experiments demonstrated that the presence or absence of the VSV-G structure had a greater effect on the physical titer of the VLPs, and that the physical titer of VLPs with VSV-G was significantly higher than that of VLPs without VSV-G (FIG. 4).
Isolation of mononuclear cells (CBMC) from cord blood. After measuring the physical titer of VLPs, CBMC was amplified with B21-VLPs and B21-noVSVG-VLPs of the same mass of 500ng, 5% autologous serum was added, cells were counted every 2-3 days from 7 days of culture, and after 22 days of culture, the results showed that VLPs with or without VSV-G structure had an amplifying effect on cells, but B21-VLPs with VSV-G had a better amplifying effect (FIG. 5).
Example 3B21-VLP has good amplification effect on NK cells in CBMC
1. Experimental materials
The transient culture medium is Kang organisms, and the product number is A21501;
KBM581 Medium, corning, cat# 88-591-CM;
PEI is Polyplus, the commodity number is 101000026;
50% glucose injection, henan Kolun pharmaceutical industry;
l-glutamine Solarbio, cat# G0200;
human Serum Albumin (HSA) Pis fei Ke biopharmaceutical;
Dulbecco's Phosphate Buffer Solution (DPBS) Gibco, cat# 14190250;
Recombinant human interleukin-2 (IL 2) for injection is available in the pharmaceutical industry;
P24 ELISA detection kit, namely the next holy organism, with the product number of 99301ES24;
FITC Anti-Human CD3, kuang Bo organism, cat# 6610004;
PE anti-human CD56, kuang Bo organism, cat No. A6803;
PE/Cyanine7 Anti-Human CD4 Anti-body: elabscience, cat# E-AB-F1109H;
PERCP ANTI-Human CD8 Antibody Elabscience, accession number AN00427F.
2. Experimental method
(1) Transfection of 293T cell expression Processes Using constructed envelope plasmids
1) B21-VLP was prepared using envelope plasmid B21-VSVG. 293T cells were first seeded, 5X 106 cells were resuspended in 125mL cell culture shake flasks using 20mL 293 medium and incubated in 37℃5% CO2 incubator with shaking at 125 rpm. The envelope plasmid B21-VSVG (5. Mu.g) and helper plasmid (pMDLg 20. Mu.g, pRSV 5. Mu.g) were mixed as solution A, and 75. Mu.L of PEI was mixed with 1mL of medium as solution B. And (3) gently mixing A, B liquids, avoiding generating bubbles, and standing for 5 minutes at room temperature after uniformly mixing. And adding the solution B into the solution A, gently mixing, and standing at room temperature for 20min after mixing. The AB mixture was slowly added to the cell culture flask, during which time it was slowly shaken and mixed. The cell culture flask was placed in a 37℃5% CO2 incubator and shake cultured at 125 rpm.
2) And executing liquid change operation after 3-5 h. After centrifugation at 1000rpm for 5min, cells were resuspended using 20mL 293 medium and transferred to 125mL cell culture shake flasks. Placed in a 37℃5% CO2 incubator, and shake cultured at 125 rpm.
3) And (5) feeding for 20-22 hours. 80. Mu.L of 50% glucose injection and 800. Mu.L of glutamine were added to each 125mL cell culture flask, and mixed by slow shaking. Placed in a 37℃5% CO2 incubator, and shake cultured at 125 rpm.
4) Harvesting 48h after packaging.
5) Transferring the cell suspension into a centrifuge tube, centrifuging at 2000rpm for 10min, and taking the supernatant as a virus harvest liquid. The virus harvest was filtered using a 0.45 μm filter. The virus harvest is transferred into a centrifuge tube, and the virus harvest is lifted by 9 to 0,18300g and centrifuged for 2 hours under the environment of 4 ℃. The viral pellet was resuspended with PBS containing 2% HSA to give a B21-VLP virus concentrate.
6) Physical titers were detected after sampling using P24 ELISA. And (5) after the rest is packaged, placing the mixture at-80 ℃ for standby.
(2) Isolation and stimulation of cord blood mononuclear cells
1) Isolation of mononuclear cells
Separating 200mL of cord blood into centrifuge tubes, centrifuging at 2000rpm at room temperature for 20min, removing upper pale yellow blood plasma, adding physiological saline with the same volume as the upper pale yellow blood plasma to obtain diluted cord blood, taking the centrifuge tubes, adding lymphocyte separation liquid into the centrifuge tubes, adding the diluted cord blood into the lymphocyte separation liquid to separate the diluted cord blood from the lymphocyte separation liquid, centrifuging at 2000rpm at room temperature for 30min, removing part of supernatant, sucking the middle white membrane layer into the centrifuge tubes, adding the same volume of physiological saline, centrifuging at 2000rpm at room temperature for 10min, removing supernatant, washing for three times, and counting, wherein the volume ratio of the lymphocyte separation liquid to the diluted cord blood is 15:45-50.
2) Inoculation and stimulation of mononuclear cells
Inoculating the sediment after removing the supernatant in the previous step into a K581 serum-free culture medium according to the cell density of 5.0X10-6/mL, adding IL2 factor with the concentration of 200IU/mL to form a mixed solution, placing the mixed solution into a T25 culture flask after coating, and culturing in a CO2 saturated humidity incubator with the temperature of 37 ℃ and the volume percentage content of 5%.
3) In vitro expansion of cord blood NK cells
After the single nuclear cell separation of the umbilical cord blood in the previous step, 3 bottles are placed in T25 bottles, and 5.0X106 cells per bottle and 5mL of the volume are filled. B21-VLPs with mass of 1500ng, 500ng, 150ng were added to each of the coated T25 flasks, stimulated, 5% autologous serum, day 0 of inoculation. The fluid replacement in this step was performed using K581 medium containing IL-2 at a concentration of 200 IU/mL.
And supplementing liquid on the 2 nd day, namely adding the culture medium with the same volume amount.
And 4-6 days of fluid infusion, namely observing cells every day, adding K581 complete medium according to the color or the cell quantity of the cell suspension, wherein the volume of each addition is not more than one time of the existing volume.
Day 7 counts, cell densities were adjusted to 0.8-1.0X106 cells/mL depending on cell suspension color and cell number, and NK phenotypes were examined by flow cytometry. From day 7, cells were counted at 2 intervals and the cell concentration was maintained at 8-1.0X106 cells/mL with the addition of expansion medium, and the CD3+CD56-cell fraction was detected by flow cytometry when cultured for ten days. On days 10, 13, 19, 22 of culture, respectively, CBMC were counted and their fold increase calculated.
3. Experimental results
Isolation of mononuclear cells (CBMC) from cord blood. After measuring physical titers of VLPs, CBMC was stimulated with B21-VLPs of mass 1500ng, 500ng, 150ng, respectively, and after ten days of culture, CD3+CD56-cell ratios were detected using a flow cytometer, which showed that NK cell ratios in the cultured CBMC cells increased with increasing B21-VLP concentration (FIG. 6).
On days 10, 13, 19, 22 of culture, the cultured CBMC were counted and their fold increase calculated, respectively, and the results showed that the difference in cell proliferation was not large when the B21-VLP mass was 1500ng and 500 ng. Therefore, we considered that using B21-VLP of 500ng mass had good expansion effect on NK cells in CBMC (fig. 7).
EXAMPLE 4 comparison of the stimulating effects of B21-VLP, B-VLP, 21-VLP on CBMC
1. Experimental materials
The transient culture medium is Kang organisms, and the product number is A21501;
KBM581 Medium, corning, cat# 88-591-CM;
PEI is Polyplus, the commodity number is 101000026;
50% glucose injection, henan Kolun pharmaceutical industry;
l-glutamine Solarbio, cat# G0200;
human Serum Albumin (HSA) Pis fei Ke biopharmaceutical;
Dulbecco's Phosphate Buffer Solution (DPBS) Gibco, cat# 14190250;
Recombinant human interleukin-2 (IL 2) for injection is available in the pharmaceutical industry;
P24 ELISA detection kit, namely the next holy organism, with the product number of 99301ES24;
FITC Anti-Human CD3, kuang Bo organism, cat# 6610004;
PE anti-human CD56, kuang Bo organism, cat No. A6803;
PE/Cyanine7 Anti-Human CD4 Anti-body: elabscience, cat# E-AB-F1109H;
PERCP ANTI-Human CD8 Antibody Elabscience, accession number AN00427F.
2. Experimental method
(1) Transfection of 293T cell expression Processes Using constructed envelope plasmids
1) VLPs were prepared using plasmids B21-VSVG, B-VSVG, 21-VSVG, respectively. First, 293T cells were inoculated, 5X 106 cells were taken, and 20mL was usedThe transient medium was resuspended in 125mL cell culture shake flasks and incubated in 37℃5% CO2 incubator at 125rpm with shaking. The envelope plasmid B21-VSVG (5. Mu.g) and helper plasmid (pMDLg 20. Mu.g, pRSV 5. Mu.g) were mixed as solution A, and 75. Mu.L of PEI was mixed with 1mL of medium as solution B. And (3) gently mixing A, B liquids, avoiding generating bubbles, and standing for 5 minutes at room temperature after uniformly mixing. And adding the solution B into the solution A, gently mixing, and standing at room temperature for 20min after mixing. The AB mixture was slowly added to the cell culture flask, during which time it was slowly shaken and mixed. The cell culture flask was placed in a 37℃5% CO2 incubator and shake cultured at 125 rpm.
2) And executing liquid change operation after 3-5 h. After centrifugation of the cells at 1000rpm for 5min, 20mL was usedTransient medium resuspended cells and transferred to 125mL cell culture shake flasks. Placed in a 37℃5% CO2 incubator, and shake cultured at 125 rpm.
3) And (5) feeding for 20-22 hours. 80. Mu.L of 50% glucose injection and 800. Mu.L of glutamine were added to each 125mL cell culture flask, and mixed by slow shaking. Placed in a 37℃5% CO2 incubator, and shake cultured at 125 rpm.
4) Harvesting 48h after packaging.
5) Transferring the cell suspension into a centrifuge tube, centrifuging at 2000rpm for 10min, and taking the supernatant as a virus harvest liquid. The virus harvest was filtered using a 0.45 μm filter. The virus harvest is transferred into a centrifuge tube, and the virus harvest is lifted by 9 to 0,18300g and centrifuged for 2 hours under the environment of 4 ℃. The viral pellet was resuspended using PBS containing 2% HSA to give B21-VLP, B-VLP, 21-VLP virus concentrate.
6) Physical titers were detected after sampling using P24 ELISA. And (5) after the rest is packaged, placing the mixture at-80 ℃ for standby.
(2) Isolation and stimulation of cord blood mononuclear cells
1) Isolation of mononuclear cells
Separating 200mL of cord blood into centrifuge tubes, centrifuging at 2000rpm at room temperature for 20min, removing upper pale yellow blood plasma, adding physiological saline with the same volume as the upper pale yellow blood plasma to obtain diluted cord blood, taking the centrifuge tubes, adding lymphocyte separation liquid into the centrifuge tubes, adding the diluted cord blood into the lymphocyte separation liquid to separate the diluted cord blood from the lymphocyte separation liquid, centrifuging at 2000rpm at room temperature for 30min, removing part of supernatant, sucking the middle white membrane layer into the centrifuge tubes, adding the same volume of physiological saline, centrifuging at 2000rpm at room temperature for 10min, removing supernatant, washing for three times, and counting, wherein the volume ratio of the lymphocyte separation liquid to the diluted cord blood is 15:45-50.
2) Inoculation and stimulation of mononuclear cells
Inoculating the sediment after removing the supernatant in the previous step into a K581 serum-free culture medium according to the cell density of 5.0X10-6/mL, adding IL2 factor with the concentration of 200IU/mL to form a mixed solution, placing the mixed solution into a T25 culture flask after coating, and culturing in a CO2 saturated humidity incubator with the temperature of 37 ℃ and the volume percentage content of 5%.
3) In vitro expansion of cord blood NK cells
After the single nuclear cell separation of the umbilical cord blood in the previous step, 3 bottles are placed in T25 bottles, and 5.0X106 cells per bottle and 5mL of the volume are filled. B-VLP, 21-VLP, B21-VLP stimulations of 500ng in mass, 5% autologous serum, day 0 of inoculation, were added to the coated T25 flasks, respectively. The fluid replacement described in this step was KBM 581 medium containing IL-2 at a concentration of 200 IU/mL.
And (2) supplementing the liquid on the 2 nd day, namely adding the same volume amount of KBM 581 complete medium.
And 4-6 days of fluid infusion, namely observing cells every day, adding KBM 581 complete medium according to the color or the cell quantity of the cell suspension, wherein the volume of each addition is not more than one time of the existing volume.
Day 7 counts, cell densities were adjusted to 0.8-1.0X106 cells/mL depending on cell suspension color and cell number, and NK phenotypes were examined by flow cytometry. From day 7, cells were counted at 2-day intervals and the cell concentration was maintained at 8-1.0X106 cells/mL with the addition of the expansion KBM 581 complete medium. At 22 days of culture, CBMC were counted and fold increase was calculated for the culture.
3. Experimental results
Cord blood CBMC was isolated. Physical titers of B21-VLP, B-VLP, 21-VLP were determined, CBMC were stimulated at a mass of 500ng, and at 22 days of culture, CBMC were counted and fold increase calculated. The experimental results show that the best stimulation was achieved with B21-VLP, and that the CBMC fold increase of B-VLP, 21-VLP, was almost 0, whereas the CBMC fold increase of B21-VLP was 250, and that mIL21 and 4-1BBL had a synergistic effect on cell expansion (FIG. 8), which was an unexpected technical effect based on the prior art by the person skilled in the art.
EXAMPLE 5 comparison of amplification effects of B21-VLP and K562-41BBL-mbiL21 on NK cells
1. Experimental materials
The transient culture medium is Kang organisms, and the product number is A21501;
KBM581 Medium, corning, cat# 88-591-CM;
PEI is Polyplus, the commodity number is 101000026;
50% glucose injection, henan Kolun pharmaceutical industry;
l-glutamine Solarbio, cat# G0200;
human Serum Albumin (HSA) Pis fei Ke biopharmaceutical;
Dulbecco's Phosphate Buffer Solution (DPBS) Gibco, cat# 14190250;
Recombinant human interleukin-2 (IL 2) for injection is available in the pharmaceutical industry;
P24 ELISA detection kit, namely the next holy organism, with the product number of 99301ES24;
FITC Anti-Human CD3, kuang Bo organism, cat# 6610004;
PE anti-human CD56, kuang Bo organism, cat No. A6803;
PE/Cyanine7 Anti-Human CD4 Anti-body: elabscience, cat# E-AB-F1109H;
PERCP ANTI-Human CD8 Antibody Elabscience, accession number AN00427F.
The K562-41BBL-mbiL21 is derived from literature ：Shman TV,Vashkevich KP,Migas AA,Matveyenka MA,Lasiukov YA,Mukhametshyna NS,Horbach KI,Aleinikova OV.Phenotypic and functional characterisation of locally produced natural killer cells ex vivo expanded with the K562-41BBL-mbIL21 cell line.Clin Exp Med.2023Oct;23(6):2551-2560.doi:10.1007/s10238-022-00974-2.Epub 2022Dec 17.PMID:36527513.
2. Experimental method
(1) Transfection of 293T cell expression Processes Using constructed envelope plasmids
1) B21-VLP was prepared using envelope plasmid B21-VSVG. First, 293T cells were seeded to obtain 5X 106 cells using 20mLThe transient medium was resuspended in 125mL cell culture shake flasks and incubated in 37℃5% CO2 incubator at 125rpm with shaking. The envelope plasmid B21-VSVG (5. Mu.g) and helper plasmid (pMDLg 20. Mu.g, pRSV 5. Mu.g) were combined with 1mLMixing transient culture medium as solution A, and mixing 75 μl PEI with 1mLThe transient medium was mixed as solution B. And (3) gently mixing A, B liquids, avoiding generating bubbles, and standing for 5 minutes at room temperature after uniformly mixing. And adding the solution B into the solution A, gently mixing, and standing at room temperature for 20min after mixing. The AB mixture was slowly added to the cell culture flask, during which time it was slowly shaken and mixed. The cell culture flask was placed in a 37℃5% CO2 incubator and shake cultured at 125 rpm.
2) And executing liquid change operation after 3-5 h. After centrifugation of the cells at 1000rpm for 5min, 20mL was usedTransient medium resuspended cells and transferred to 125mL cell culture shake flasks. Placed in a 37℃5% CO2 incubator, and shake cultured at 125 rpm.
3) And (5) feeding for 20-22 hours. 80. Mu.L of 50% glucose injection and 800. Mu.L of glutamine were added to each 125mL cell culture flask, and mixed by slow shaking. Placed in a 37℃5% CO2 incubator, and shake cultured at 125 rpm.
4) Harvesting 48h after packaging.
5) Transferring the cell suspension into a centrifuge tube, centrifuging at 2000rpm for 10min, and taking the supernatant as a virus harvest liquid. The virus harvest was filtered using a 0.45 μm filter. The virus harvest is transferred into a centrifuge tube, and the virus harvest is lifted by 9 to 0,18300g and centrifuged for 2 hours under the environment of 4 ℃. The viral pellet was resuspended with PBS containing 2% HSA to give a B21-VLP virus concentrate.
6) Physical titers were detected after sampling using P24 ELISA. And (5) after the rest is packaged, placing the mixture at-80 ℃ for standby.
(2) Isolation and stimulation of cord blood mononuclear cells
1) Isolation of mononuclear cells
Separating 200mL of cord blood into a centrifuge tube, centrifuging at 2000rpm and room temperature for 20min, removing upper pale yellow blood plasma, adding physiological saline with the same volume as the upper pale yellow blood plasma to obtain diluted cord blood, taking the centrifuge tube, adding lymphocyte separation liquid into the centrifuge tube, adding the diluted cord blood into the lymphocyte separation liquid to separate the diluted cord blood from the lymphocyte separation liquid, centrifuging at 2000rpm and room temperature for 30min, removing part of supernatant, sucking the middle white membrane layer into the centrifuge tube, adding the same volume of physiological saline, centrifuging at 2000rpm for 10min at room temperature, removing supernatant, washing for three times, and counting, wherein the volume ratio of the lymphocyte separation liquid to the diluted cord blood is 15:45-50.
2) Inoculation and stimulation of mononuclear cells
Inoculating the sediment after removing the supernatant in the previous step into a K581 serum-free culture medium according to the cell density of 5.0X10-6/mL, adding IL2 factor with the concentration of 200IU/mL to form a mixed solution, placing the mixed solution into a T25 culture flask after coating, and culturing in a CO2 saturated humidity incubator with the temperature of 37 ℃ and the volume percentage content of 5%.
3) In vitro expansion of cord blood NK cells
After the single nucleus cell separation of the umbilical cord blood in the previous step, 2 bottles are placed in T25 bottles, and each bottle contains 5.0X106 cells and has a volume of 5mL. One of the bottles coated with the above-mentioned solution was stimulated with 500ng of B21-VLP, and the other bottle was stimulated with K562-41BBL-mbiL21 cells, 1:1,5% autologous serum with CBMC, on day 0. The fluid replacement in this step was performed using K581 medium containing IL-2 at a concentration of 200 IU/mL.
And supplementing liquid on the 2 nd day, namely adding the culture medium with the same volume amount.
And 4-6 days of fluid infusion, namely observing cells every day, adding K581 complete medium according to the color or the cell quantity of the cell suspension, wherein the volume of each addition is not more than one time of the existing volume.
Day 7 counts, cell densities were adjusted to 0.8-1.0X106 cells/mL depending on cell suspension color and cell number, and NK phenotypes were examined by flow cytometry. From day 7, cells were counted at 2-3 days intervals and the cell concentration was maintained at 8-1.0X106 cells/mL with the addition of expansion medium, and the CD3-CD56+ NK cell fraction was detected on day 22 of culture using a flow cytometer.
3. Experimental results
Experiments show that the amplification capability of B21-VLP on NK cells can achieve the effect similar to that of K562-41BBL-mbiL 21. The proportion of NK cells in total cells can reach more than 90% (FIGS. 9-10).
The CBMC was isolated and the physical titer of B21-VLPs was determined, and CBMC was stimulated with B21-VLPs and K562-41BBL-mbiL21 of 500ng mass, respectively, which showed that the flow assay showed a significant reduction in cell debris when B21-VLPs stimulated CBMC, and therefore, the stimulation with B21-VLPs constructed by the present invention was more advantageous (FIG. 11).
Example 6B21-VLP allows NK cells to selectively expand when CBMC is stimulated
1. Experimental materials
The transient culture medium is Kang organisms, and the product number is A21501;
KBM581 Medium, corning, cat# 88-591-CM;
PEI is Polyplus, the commodity number is 101000026;
50% glucose injection, henan Kolun pharmaceutical industry;
l-glutamine Solarbio, cat# G0200;
human Serum Albumin (HSA) Pis fei Ke biopharmaceutical;
Dulbecco's Phosphate Buffer Solution (DPBS) Gibco, cat# 14190250;
Recombinant human interleukin-2 (IL 2) for injection is available in the pharmaceutical industry;
P24 ELISA detection kit, namely the next holy organism, with the product number of 99301ES24;
FITC Anti-Human CD3, kuang Bo organism, cat# 6610004;
PE anti-human CD56, kuang Bo organism, cat No. A6803;
PE/Cyanine7 Anti-Human CD4 Anti-body: elabscience, cat# E-AB-F1109H;
PERCP ANTI-Human CD8 Antibody Elabscience, accession number AN00427F.
2. Experimental method
(1) Transfection of 293T cell expression Processes Using constructed envelope plasmids
1) B21-VLP was prepared using envelope plasmid B21-VSVG. First, 293T cells were seeded to obtain 5X 106 cells using 20mLThe transient medium was resuspended in 125mL cell culture shake flasks and incubated in 37℃5% CO2 incubator at 125rpm with shaking. The envelope plasmid B21-VSVG (5. Mu.g) and helper plasmid (pMDLg 20. Mu.g, pRSV 5. Mu.g) were combined with 1mLMixing transient culture medium as solution A, and mixing 75 μl PEI with 1mLThe transient medium was mixed as solution B. And (3) gently mixing A, B liquids, avoiding generating bubbles, and standing for 5 minutes at room temperature after uniformly mixing. And adding the solution B into the solution A, gently mixing, and standing at room temperature for 20min after mixing. The AB mixture was slowly added to the cell culture flask, during which time it was slowly shaken and mixed. The cell culture flask was placed in a 37℃5% CO2 incubator and shake cultured at 125 rpm.
2) And executing liquid change operation after 3-5 h. After centrifugation of the cells at 1000rpm for 5min, 20mL was usedTransient medium resuspended cells and transferred to 125mL cell culture shake flasks. Placed in a 37℃5% CO2 incubator, and shake cultured at 125 rpm.
3) And (5) feeding for 20-22 hours. 80. Mu.L of 50% glucose injection and 800. Mu.L of glutamine were added to each 125mL cell culture flask, and mixed by slow shaking. Placed in a 37℃5% CO2 incubator, and shake cultured at 125 rpm.
4) Harvesting 48h after packaging.
5) Transferring the cell suspension into a centrifuge tube, centrifuging at 2000rpm for 10min, and taking the supernatant as a virus harvest liquid. The virus harvest was filtered using a 0.45 μm filter. The virus harvest is transferred into a centrifuge tube, and the virus harvest is lifted by 9 to 0,18300g and centrifuged for 2 hours under the environment of 4 ℃. The viral pellet was resuspended with PBS containing 2% HSA to give a B21-VLP virus concentrate.
6) Physical titers were detected after sampling using P24 ELISA. And (5) after the rest is packaged, placing the mixture at-80 ℃ for standby.
(2) Isolation and stimulation of cord blood mononuclear cells
1) Isolation of mononuclear cells
Separating 200mL of cord blood into a centrifuge tube, centrifuging at 2000rpm and room temperature for 20min, removing upper pale yellow blood plasma, adding physiological saline with the same volume as the upper pale yellow blood plasma to obtain diluted cord blood, taking the centrifuge tube, adding lymphocyte separation liquid into the centrifuge tube, adding the diluted cord blood into the lymphocyte separation liquid to separate the diluted cord blood from the lymphocyte separation liquid, centrifuging at 2000rpm and room temperature for 30min, removing part of supernatant, sucking the middle white membrane layer into the centrifuge tube, adding the same volume of physiological saline, centrifuging at 2000rpm for 10min at room temperature, removing supernatant, washing for three times, and counting, wherein the volume ratio of the lymphocyte separation liquid to the diluted cord blood is 15:45-50.
2) Inoculation and stimulation of mononuclear cells
Inoculating the sediment after removing the supernatant in the previous step into a K581 serum-free culture medium according to the cell density of 5.0X10-6/mL, adding IL2 factor with the concentration of 200IU/mL to form a mixed solution, placing the mixed solution into a T25 culture flask after coating, and culturing in a CO2 saturated humidity incubator with the temperature of 37 ℃ and the volume percentage content of 5%.
3) In vitro expansion of cord blood NK cells
After the single nucleus cell separation of the umbilical cord blood in the previous step, 2 bottles are placed in T25 bottles, and each bottle contains 5.0X106 cells and has a volume of 5mL. One of the bottles coated with the above-mentioned solution was stimulated with 500ng of B21-VLP, and the other bottle was stimulated with K562-41BBL-mbiL21 cells, 1:1,5% autologous serum with CBMC, on day 0. The fluid replacement in this step was performed using K581 medium containing IL-2 at a concentration of 200 IU/mL.
And supplementing liquid on the 2 nd day, namely adding the culture medium with the same volume amount.
And 4-6 days of fluid infusion, namely observing cells every day, adding K581 complete medium according to the color or the cell quantity of the cell suspension, wherein the volume of each addition is not more than one time of the existing volume.
Day 7 counts, cell densities were adjusted to 0.8-1.0X106 cells/mL depending on cell suspension color and cell number, and NK phenotypes were examined by flow cytometry. From day 7, cells were counted at 2-3 days intervals and the cell concentration was maintained at 8-1.0X106 cells/mL with the addition of expansion medium, and the CD3-CD56+ NK cell fraction was detected on day 22 of culture using a flow cytometer.
3. Experimental results
CBMC was isolated and B21-VLP physical titers were determined. The B21-VLP was used to stimulate CBMC at a mass of 500ng for 21 days, and the flow cytometer was used to detect T cells, NKT cells and NK cell content every 2 days, which indicated that the B21-VLP stimulated CBMC could selectively expand NK cells, and that NK cells accounted for more than 90% of the total cell content at day 21 of culture (FIG. 12).
EXAMPLE 7B21-VLP cultured NK cells have a stronger killing ability against K562 cells
1. Experimental materials
The transient culture medium is Kang organisms, and the product number is A21501;
KBM581 Medium, corning, cat# 88-591-CM;
PEI is Polyplus, the commodity number is 101000026;
50% glucose injection, henan Kolun pharmaceutical industry;
l-glutamine Solarbio, cat# G0200;
human Serum Albumin (HSA) Pis fei Ke biopharmaceutical;
Dulbecco's Phosphate Buffer Solution (DPBS) Gibco, cat# 14190250;
Recombinant human interleukin-2 (IL 2) for injection is available in the pharmaceutical industry;
P24 ELISA detection kit, namely the next holy organism, with the product number of 99301ES24;
FITC Anti-Human CD3, kuang Bo organism, cat# 6610004;
PE anti-human CD56, kuang Bo organism, cat No. A6803;
PE/Cyanine7 Anti-Human CD4 Anti-body: elabscience, cat# E-AB-F1109H;
PERCP ANTI-Human CD8 Antibody Elabscience, accession number AN00427F.
2. Experimental method
(1) Transfection of 293T cell expression Processes Using constructed envelope plasmids
1) B21-VLP was prepared using envelope plasmid B21-VSVG. First, 293T cells were inoculated, 5X 106 cells were taken, and 20mL was usedThe transient medium was resuspended in 125mL cell culture shake flasks and incubated in 37℃5% CO2 incubator at 125rpm with shaking. The envelope plasmid B21-VSVG (5. Mu.g) and helper plasmid (pMDLg 20. Mu.g, pRSV 5. Mu.g) were combined with 1mLMixing transient culture medium as solution A, and mixing 75 μl PEI with 1mLThe transient medium was mixed as solution B. And (3) gently mixing A, B liquids, avoiding generating bubbles, and standing for 5 minutes at room temperature after uniformly mixing. And adding the solution B into the solution A, gently mixing, and standing at room temperature for 20min after mixing. The AB mixture was slowly added to the cell culture flask, during which time it was slowly shaken and mixed. The cell culture flask was placed in a 37℃5% CO2 incubator and shake cultured at 125 rpm.
2) And executing liquid change operation after 3-5h. After centrifugation of the cells at 1000rpm for 5min, 20mL and 1mL were usedTransient medium resuspended cells and transferred to 125mL cell culture shake flasks. Placed in a 37℃5% CO2 incubator, and shake cultured at 125 rpm.
3) And (5) feeding for 20-22 hours. 80. Mu.L of 50% glucose injection and 800. Mu.L of glutamine were added to each 125mL cell culture flask, and mixed by slow shaking. Placed in a 37℃5% CO2 incubator, and shake cultured at 125 rpm.
4) Harvesting 48h after packaging.
5) Transferring the cell suspension into a centrifuge tube, centrifuging at 2000rpm for 10min, and taking the supernatant as a virus harvest liquid. The virus harvest was filtered using a 0.45 μm filter. The virus harvest is transferred into a centrifuge tube, and the virus harvest is lifted by 9 to 0,18300g and centrifuged for 2 hours under the environment of 4 ℃. The viral pellet was resuspended with PBS containing 2% HSA to give a B21-VLP virus concentrate.
6) Physical titers were detected after sampling using P24 ELISA. And (5) after the rest is packaged, placing the mixture at-80 ℃ for standby.
(2) Isolation and stimulation of cord blood mononuclear cells
1) Isolation of mononuclear cells
Separating 200mL of cord blood into a centrifuge tube, centrifuging at 2000rpm and room temperature for 20min, removing upper pale yellow blood plasma, adding physiological saline with the same volume as the upper pale yellow blood plasma to obtain diluted cord blood, taking the centrifuge tube, adding lymphocyte separation liquid into the centrifuge tube, adding the diluted cord blood into the lymphocyte separation liquid, layering the diluted cord blood and the lymphocyte separation liquid, centrifuging at 2000rpm and room temperature for 30min, removing part of supernatant, sucking the middle white membrane layer into the centrifuge tube, adding the same volume of physiological saline, centrifuging at 2000rpm for 10min at room temperature, removing supernatant, washing for three times, and counting, wherein the volume ratio of the lymphocyte separation liquid to the diluted cord blood is 15:45-50.
2) Inoculation and stimulation of mononuclear cells
Inoculating the sediment after removing the supernatant in the previous step into a K581 serum-free culture medium according to the cell density of 5.0X10-6/mL, adding IL2 factor with the concentration of 200IU/mL to form a mixed solution, placing the mixed solution into a T25 culture flask after coating, and culturing in a CO2 saturated humidity incubator with the temperature of 37 ℃ and the volume percentage content of 5%.
3) In vitro expansion of cord blood NK cells
After the single nucleus cell separation of the cord blood in the previous step, 5.0X106 cells per bottle were placed in T25 bottle with a volume of 5mL. A bottle coated with the B21-VLP was stimulated with 500ng of autologous serum at a ratio of 1:1,5% to CBMC, day 0. The fluid replacement in this step was performed using K581 medium containing IL-2 at a concentration of 200 IU/mL.
4) And supplementing liquid on the 2 nd day, namely adding the culture medium with the same volume amount.
5) And 4-6 days of fluid infusion, namely observing cells every day, adding K581 complete medium according to the color or the cell quantity of the cell suspension, wherein the volume of each addition is not more than one time of the existing volume.
6) Day 7 counts, cell densities were adjusted to 0.8-1.0X106 cells/mL depending on cell suspension color and cell number, and NK phenotypes were examined by flow cytometry.
7) NK cell killing experiments
NK cells cultured with B21-VLP are taken as effector cells, and K562 tumor cells are taken as target cells. Co-culturing for 24 hours at effective target ratios of 10:1, 3:1 and 1:1 respectively, and detecting the ratio of effector cells to target cells by using a flow analyzer.
3. Experimental results
The experimental results are shown in FIG. 13, and the results show that after 24 hours, effector cells and target cells are detected, and the proportion of the target cells is observed to be less than 1%, which indicates that NK cells cultured by the B21-VLP constructed by the invention have stronger killing capacity on K562 cells.