METHOD FOR EFFICIENTLY INFECTING HUMAN NATURAL KILLER CELLS AND OTHER IMMUNE CELLS WITH PSEUDOVIRUS

Abstract

The present disclosure belongs to the field of biotechnology, and specifically relates to a method for efficiently infecting human natural killer (NK) cells and other immune cells with a pseudovirus. Specifically, a viral transfection system provided in the present disclosure has an envelope plasmid with a protein having an XYZ structure. The X is an extracellular (ex) structure of a gibbon ape leukemia virus (GALV) envelope glycoprotein, the Y is a transmembrane (TM) structure of the GALV envelope glycoprotein, and the Z is an intracellular segment portion of a murine virus gene.

Claims

1. A method for preparing natural killer (NK) cells expressing a target gene, comprising the steps of transfecting cells with a vector composition containing an envelope plasmid, collecting a viral liquid, and contacting the viral liquid with NK cells, the envelope plasmid containing a fusion protein having an XYZ structure, the X having an amino acid sequence as shown in SEQ ID NO.1, the Y having an amino acid sequence as shown in SEQ ID NO.2, and the Z having an amino acid sequence as shown in SEQ ID NO.3.

2. The method according to claim 1, wherein the vector composition further contains the following plasmids: 1) an expression plasmid, containing a coding sequence of a target gene or a protein expressing the target gene, 2) a packaging plasmid 1, containing coding sequences of group specific antigen (GAG) and polymerase (POL) or expressing GAG and POL, and 3) a packaging plasmid 2, containing a coding sequence of regulator of expression of virion protein (REV) or expressing REV.

3. The method according to claim 2, wherein the expression plasmid, the packaging plasmid 1, the packaging plasmid 2 and the envelope plasmid are in a mass ratio of 2:1:1:1.

4. The method according to claim 2, wherein the packaging plasmid 1, the packaging plasmid 2, and the envelope plasmid each independently comprises a cytomegalovirus (CMV) promoter.

5. The method according to claim 1, further comprising steps for separating and/or purifying the viral liquid.

6. NK cells prepared by a method according to claim 1.

7. An application of NK cells according to claim 6 in preparing a drug.

8. An application of an envelope plasmid or a composition thereof in preparing NK cells expressing a target gene, an envelope plasmid containing a fusion protein having an XYZ structure, the X having an amino acid sequence as shown in SEQ ID NO.1, the Y having an amino acid sequence as shown in SEQ ID NO.2, and the Z having an amino acid sequence as shown in SEQ ID NO.3.

9. The application according to claim 8, wherein the NK cells are human cells.

10. The application according to claim 8, wherein the target gene comprises an antibody, a chimeric antigen receptor (CAR) or a functional protein.

11. The application according to claim 10, wherein the antibody and the CAR target any one or more of the following sites: BCMA, CD19, CD20, CD123, CD22, CD3D, CD3E, CD7CLEC12AGPRC5D, CD138, CD30, CD33, CD38, CD3E, CD79BSLAMF7, CD10, CD117, CD37, CD4, CD5, CD56, CD72, CD79A, CD99, Flt-3, LILRA3, LILRB4, SLAMF3, Her2, MSLN, B7-H3, CLDN18, EGFR, GPC3, KRAS, CA9, CEA, EGFRvIII, EphA2, ERBB3, ERBB4, FAP, GUCY2C, IL13RA2, MUC1, PD-1, PSMA, VEGFR2, AFP, AXL, CD133, CD147, CD171, CD80, CD86, c-Met, DLL4, EpCAM, Nectin-4, Podoplanin, ROBO1, ROR2, SSTR2, FOLR1, ROR1, CD70, NKG2D, PD-L1, and SIRP alpha.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0119] FIG. 1 is a graph showing the results of the analysis of the expression of SLC20A1 in blood cells.

[0120] FIG. 2 is a graph showing the results of the analysis of the expression of LDLR in blood cells.

[0121] FIG. 3 is a graph showing the results of the analysis of the expression of SLC1A5 in blood cells.

[0122] FIG. 4 is a graph showing the expression of LDLR, SLC1A5 and SLC20A1 on NK cells.

[0123] FIG. 5 is a schematic diagram comparing a viral system of the present disclosure with a conventional third-generation lentiviral packaging system.

[0124] FIG. 6 is a schematic structural diagram of a CAR expressing BCMA antibodies.

[0125] FIG. 7A shows the results of the CAR+ positive rates detected by a flow cytometry on days 3, 6, 9, and 15.

[0126] FIG. 7B presents the change curves for the CAR+ positive rates on days 3, 6, 9, and 15. It can be seen that the positive rate for cells infected with QMV virus is above 80% on day 3, drops to around 65% on day 6, and stabilizes at over 50% after day 9. In contrast, the positive rate for cells infected with VSVG virus declines rapidly, with 40% on day 3, dropping to 20% on day 6, 10% on day 9, and reaching 5% on day 15.

[0127] FIG. 7C displays the cell viability on days 3, 6, 9, and 15, showing no significant differences in cell viability between cells infected with QMV and VSVG at the aforementioned time points.

[0128] FIG. 8 is a graph showing detection results for positive rates after transduction of CAR to NK cells or T cells using the viral system of the present disclosure versus the conventional third-generation lentiviral packaging system.

[0129] FIG. 9 is a graph showing assay results for the positive rates after transduction of green fluorescent protein (GFP) into NK cells or T cells using the viral system of the present disclosure versus the conventional third-generation lentiviral packaging system.

DETAILED DESCRIPTION

[0130] The present disclosure is further described below with reference to specific embodiments. The embodiments described below are merely preferred embodiments of the present disclosure and are not intended to limit the present disclosure in any other form. Any person skilled in the art may make equivalent embodiments by modifying the disclosed technical content in the same manner. Any simple modification or equivalent change made to the following embodiments based on the technical essence of the present disclosure without departing from the scope of the present disclosure is within the scope of protection of the present disclosure.

Universal Method-Steps for Preparation of Viral Liquid

[0131] 1. HEK293T cells were prepared and cultured in a Dulbecco's modified eagle's medium (DMEM) with 10% serum until a convergence rate reached 80%. [0132] 2. A PEI solution (Shanghai Liji Biotechnology Co., Ltd., Cat: AC04L092) was used to mix with DNA at a volume-to-mass ratio of 3:1 to form a transfection complex. Four plasmids included an expression plasmid (containing a target gene intended for expression in the present disclosure), a packaging plasmid 1, a packaging plasmid 2, and an envelope plasmid, with a mass ratio of 2:1:1:1. The packaging plasmid 1 and the packaging plasmid 2 were derived from a Cell Biolabs VPK-206 viral transduction product. [0133] 3. After being placed at room temperature for 10-20 minutes, the transfection complex was added to HEK293T cells. After 12 hours, the transfection complex was removed, a DMEM with 5% serum was replaced with, followed by continuing to culture for 52 hours. Finally, a viral liquid was obtained and concentrated to measure biological titer, and then was store at 80 degrees.

Embodiment 1: Comparison of a Transduction System of the Present Disclosure with a Third-Generation Lentiviral Packaging System

[0134] Using data from the proteinatlas.org database, the receptor expression of NK cells was analyzed, with the results shown in FIGS. 1-4. SLC20A1 was a receptor for GALV, LDLR was a receptor for a VSV-G envelope, and SLC1A5 was a Baboon retroviral (BaEV) receptor. The data in FIGS. 1-4 presented RNA-seq data and three separate multi-sample sequencing results. In the first row, which was derived from the sequencing of 6 samples, the RNA expression of SLC20A1 in NK cells was found to be at an average of 50.2 nTPM, the RNA expression of LDLR averaged 2.5 nTPM, and the RNA expression of SLC1A5 averaged 21 nTPM. The second row, derived from the sequencing of 4 samples, showed that the RNA expression of SLC20A1 in NK cells averaged 295 nTPM, the RNA expression of LDLR averaged 39.7 nTPM, and the RNA expression of SLC1A5 averaged 53 nTPM. The third row, derived from the sequencing of 105 samples, indicated that the RNA expression of SLC20A1 in NK cells averaged 175 nTPM, the RNA expression of LDLR averaged 23.7 nTPM, and the RNA expression of SLC1A5 averaged 18 nTPM.

[0135] Based on the data in FIGS. 1-4, it could be seen that the expression of GALV receptor (SLC20A1) was significantly higher than that of LDLR (VSV-G receptor), and significantly higher than that of BaEV receptor (SLC1A5), in all kinds of blood cells.

[0136] Therefore, SEQ ID NO.1-3 were sequentially ligated and constructed into the vector as the envelope plasmid of the viral transduction system of the present disclosure. A plasmid structure of the viral transduction system is shown in FIG. 5.

TABLE-US-00001 TABLE1 SequenceoftheproteinQMVofthepresentdisclosure Site Name Specificsequence SEQID Full Proteinof MVLLPGSMLLTSNLHHLRHQMSPGSWKRLIILL length thepresent SCVFGGGGTSLQNKNPHQPMTLTWQVLSQTGD disclosure VVWDTKAVQPPWTWWPTLKPDVCALAASLES (GalVex- WDIPGTDVSSSKRVRPPDSDYTAAYKQITWGAI GTM-MEVc) GCSYPRARTRMASSTFYVCPRDGRTLSEARRCG GLESLYCKEWDCETTGTGYWLSKSSKDLITVK WDQNSEWTQKFQQCHQTGWCNPLKIDFTDKGK LSKDWITGKTWGLRFYVSGHPGVQFTIRLKITN MPAVAVGPDLVLVEQGPPRTSLALPPPLPPREAP PPSLPDSNSTALATSAQTPTVRKTIVTLNTPPPTT GDRLFDLVQGAFLTLNATNPGATESCWLCLAM GPPYYEAIASSGEVAYSTDLDRCRWGTQGKLTL TEVSGHGLCIGKVPFTHQHLCNQTLSINSSGDHQ 1 YLLPSNHSWWACSTGLTPCLSTSVFNQTRDFCIQ VQLIPRIYYYPEEVLLQAYDNSHPRTKREAVSLT LAVLLGLGITAGIGTGSTALIKGPIDLQQGLTSLQ IAIDADLRALQDSVSKLEDSLTSLSEVVLQNRRG LDLLFLKEGGLCAALKEECCFYIDHSGAVRDSM KKLKEKLDKRQLERQKSQNWYEGWENNSPWFT TLLSTIAGPLLLLLLLLILGPCIINRLVQFVKDRIS VVQAL 1-632 Exstructure MVLLPGSMLLTSNLHHLRHQMSPGSWKRLIILL ofGALV SCVFGGGGTSLQNKNPHQPMTLTWQVLSQTGD envelope VVWDTKAVQPPWTWWPTLKPDVCALAASLES glycoprotein WDIPGTDVSSSKRVRPPDSDYTAAYKQITWGAI GCSYPRARTRMASSTFYVCPRDGRTLSEARRCG GLESLYCKEWDCETTGTGYWLSKSSKDLITVK WDQNSEWTQKFQQCHQTGWCNPLKIDFTDKGK LSKDWITGKTWGLRFYVSGHPGVQFTIRLKITN MPAVAVGPDLVLVEQGPPRTSLALPPPLPPREAP PPSLPDSNSTALATSAQTPTVRKTIVTLNTPPPTT GDRLFDLVQGAFLTLNATNPGATESCWLCLAM GPPYYEAIASSGEVAYSTDLDRCRWGTQGKLTL TEVSGHGLCIGKVPFTHQHLCNQTLSINSSGDHQ YLLPSNHSWWACSTGLTPCLSTSVFNQTRDFCIQ VQLIPRIYYYPEEVLLQAYDNSHPRTKREAVSLT LAVLLGLGITAGIGTGSTALIKGPIDLQQGLTSLQ IAIDADLRALQDSVSKLEDSLTSLSEVVLQNRRG LDLLFLKEGGLCAALKEECCFYIDHSGAVRDSM KKLKEKLDKRQLERQKSQNWYEGWENNSPWFT TLL 633-653 TMstructure STIAGLLLLLLLLILGPCII 2 ofGALV 654-670 MEVc NRLVQFVKDRISVVQAL 3

[0137] According to the method in the universal method, viral liquids of the viral system of the present disclosure and the third-generation lentiviral packaging system were prepared, and NK cells were transduced. The target gene was a CAR structure expressing a BCMA antibody (structural diagram shown in FIG. 6).

[0138] NK cells were derived from peripheral blood monoculear cells (PBMC), and 8 g/mL of polybrene was added along with 10 MOI of a virus. After 16 hours, a fresh NK cell culture medium was added to remove a medium containing the virus.

[0139] After being infected with the virus, NK cells were taken out for detecting cell viability and positive rates on days 3, 6, 9, and 15. Specifically, 0.5 of E6NK cells were taken and washed twice with phosphate buffered saline (PBS), followed by adding 100 L of magnetic-activated cell sorting (MACS) buffer, and adding 1 L of BCMA ScFv-specific recognition reagent (Miltenyi order #: 130-126-090). After that, the E6NK cells were incubated at room temperature for 10 minutes, and then washed twice. The BCMA-positive cell ratio was detected using a flow cytometry.

Experimental Results

[0140] FIGS. 7A and 7B show that the positive rate of cells infected with QMV virus is over 80% on the third day, drops to around 65% on the sixth day, and stabilizes above 50% after the ninth day. In contrast, the positive rate of cells infected with VSV-G virus declines rapidly, with a positive rate of 40% on the third day, dropping to 20% on the sixth day, 10% on the ninth day, and 5% on the fifteenth day.

[0141] Simultaneously, cell viability is monitored on days 3, 6, 9, and 15. The cell viability of both QMV and VSV-G infected cells remains unchanged after 15 days of culture, as shown in the statistics in FIG. 7C.

Embodiment 2: Comparison of Transfection of CAR and GFP into NK Cells and T Cells

[0142] To determine whether the QMV virus was CAR-specific and NK cell-specific, further comparative experiments were conducted.

[0143] Using the method described in Embodiment 1, VSV-G and QMV viruses were used to transduce BCMA-CAR into PB-NK, NK92, and T cells. The positive rates were detected on the ninth day post-transduction, using the same detection method as in Embodiment 1. As shown in FIG. 8, the BCMA-CAR positive rates for NK92 and PB-NK cells infected with VSV-G were around 10%, while the BCMA-CAR positive rates for NK92 and PB-NK cells infected with QMV were approximately 95% and 60%, respectively. For T cells infected with VSV-G and QMV viruses, the BCMA-CAR positive rates were similar, around 83%.

[0144] Following the same method as in Embodiment 1, VSV-G and QMV viruses were used to transduce GFP into PB-NK, NK92, and T cells, with the positive rates detected on the ninth day post-transduction, using the same detection method as in Embodiment 1. As shown in FIG. 9, the GFP positive rates for NK92 and PB-NK cells infected with VSV-G were 36% and 22%, respectively, while the GFP positive rates for NK92 and PB-NK cells infected with QMV were 60% and 50%, respectively. For T cells infected with VSV-G and QMV viruses, the GFP positive rates were similar, both in the range of 96-97%.

[0145] The above results indicate that the QMV viral transduction system of the present disclosure is suitable for various immune cells, demonstrating high and stable transduction efficiency when expressing different target genes.