CN116731974B

CN116731974B - Virus preparation and application

Info

Publication number: CN116731974B
Application number: CN202310606233.5A
Authority: CN
Inventors: 叶倩; 胡艳平; 李娜
Original assignee: Hangzhou Ronggu Biotechnology Co ltd
Current assignee: Hangzhou Ronggu Biotechnology Co ltd
Priority date: 2023-05-23
Filing date: 2023-05-23
Publication date: 2024-11-29
Anticipated expiration: 2043-05-23
Also published as: CN116731974A

Abstract

The invention relates to a preparation method and application of viruses. In particular, a cell is provided in which expression or activity of ASCT2 is inhibited. The infection efficiency and active titer of viruses prepared using the cells are significantly higher than viruses prepared using wild-type cells.

Description

Preparation method and application of virus

Technical Field

The invention relates to the field of biotechnology, in particular to a preparation method and application of viruses

Background

CAR-T cells have achieved great success in early clinical trials of recurrent or refractory B cell malignancies, and currently 7 CAR-T therapies have been commercially applied worldwide, targeting CD19 positive leukemias and lymphomasRelmacabtagene) Or B Cell Maturation Antigen (BCMA) positive multiple myelomaHowever, to avoid allograft versus host disease (GvHD), the preparation of CAR-T cells requires in vitro engineering by patient autologous peripheral blood T cells, which results in a very expensive and lengthy process flow. Furthermore, a number of clinical trials suggest that existing CAR-T cell therapies may induce serious adverse events, such as life threatening fine factor release syndrome (CRS) or neurotoxicity.

Natural killer cells (NK cells) are effector cells of the innate immune system that recognize and lyse target cells in a non-antigen specific manner, allowing them to effectively monitor and eliminate tumor cells. Notably, NK cells do not cause acute or chronic GvHD. NK cells are abundant in sources including peripheral blood, umbilical cord blood, CD34+ hematopoietic precursor cells, induced pluripotent stem cells and the like of healthy people, and allow preparation of prefabricated products in 'stock' form, which can greatly reduce cost and process cycle. Importantly, phase I/II clinical trials (accession number NCT 03056339) of recurrent or refractory B-cell tumors using CD 19-targeted CAR-NK cell therapy of umbilical cord blood sources reported encouraging results with complete remission in 7 out of 11 patients and no treatment-related CRS, neurotoxicity, and GvHD occurred. Thus, the use of CAR-NK cells of allogeneic origin against a particular antigen has better safety and efficacy, and is likely to be used as a "off-the-shelf" product, hopefully supplementing or even replacing existing CAR-T therapies.

The development of CAR-NK cell therapies is significantly retarded compared to the extensive clinical studies of existing CAR-T cell therapies. One important reason for this is the lack of a simple and effective gene delivery method for primary NK cells. Exogenous genes can be delivered to NK cells using transfection methods such as electrotransfection or lipofection, however in these cases the exogenous DNA is not normally integrated into the NK cell genome and thus expression of the transgene is transient and is greatly reduced about 3-5 days after transfection. Exogenous DNA can be integrated into the cell genome based on the PiggyBac transposon system, but two independent clinical trials have recently reported that CAR-T cells prepared using this technology are at risk for transformation into T cell tumors (Bishop DC, et al blood 2021,138 (16): 1504-1509, MICKLETHWAITE KP, et al blood 2021,138 (16): 1391-1405). In contrast, lentiviral vectors have lower genotoxicity and insertion mutation, and are safer methods for gene transduction. However, conventional lentiviral techniques for transduction of the CAR gene into primary human NK cells have encountered a technical bottleneck in that the CAR gene of CAR-T cells is mainly delivered as a pseudotyped lentiviral vector using vesicular stomatitis virus glycoprotein G gene (VSV-G) as an envelope glycoprotein, which can efficiently transduce human T cells, but has little effect when applied to NK cells.

The infection efficiency and titer of the virus particles obtained by the existing lentivirus production method are low, and the application of lentivirus technology in transfection of immune cells is severely limited.

Disclosure of Invention

In a first aspect the invention provides a cell in which expression or activity of ASCT2 is inhibited.

In one or more embodiments, the cell is a cell that can be used to produce a virus.

In one or more embodiments, the cells are HEK293 cells, including one or more of HEK293T、HEK293H、HEK293F、HEK293S、HEK293T/17、HEK293T/17SF、HEK293FT、HEK293SG、HEK293E、HEK293-6E、HEK293FTM、HEK293SGGD cells.

In one or more embodiments, the virus is a natural virus or a pseudotyped virus.

In one or more embodiments, the virus is a retrovirus or pseudotyped virus thereof, such as a lentivirus or pseudotyped virus thereof.

In one or more embodiments, the pseudovirus comprises an envelope protein of one or more viruses selected from the group consisting of baboon endogenous retrovirus (BaEV), feline leukemia virus (RD 114), vesicular stomatitis virus (VSV-G), or variants thereof.

In one or more embodiments, the pseudovirus comprises one or more envelope proteins or variants thereof selected from VSV-G, baEV and RD114. Preferably, the pseudovirus comprises RD114-TR, baEV-LV or BaEV-RLess envelope protein.

In one or more embodiments, expression or activity of the ASCT2 is inhibited by a gene editing technique, an RNA inhibition technique, a homologous recombination technique, or an immunological technique.

In one or more embodiments, the gene editing technique introduces mutations in the gene encoding ASCT2 of the cell genome that result in reduced expression levels or reduced activity of the expressed ASCT2 via a gene editing vector.

In one or more embodiments, the gene editing techniques include CRISPR, zinc finger protein, TALEN-based gene editing techniques.

In one or more embodiments, the RNA inhibition technique results in reduced expression levels or reduced activity of the expressed ASCT2 by siRNA, antisense RNA and/or ribozymes targeting the gene or transcript encoding ASCT 2.

In one or more embodiments, the homologous recombination technique knocks out the gene encoding ASCT2 in the genome by a homologous recombination vector, or homologous recombines a nucleic acid sequence encoding an inactive or reduced activity ASCT2 variant into the genome to replace the wild-type ASCT2 coding sequence.

In one or more embodiments, the immunological technique reduces the activity of ASCT2 by allowing cells to express, secrete, or contact an antibody or antigen binding fragment thereof of ASCT 2.

In one or more embodiments, the cell comprises, expresses, secretes, or is contacted with an agent, such as an antibody, nucleic acid, or small molecule, that down-regulates the expression level or activity of ASCT 2. The agent that down-regulates ASCT2 activity is an antagonist of ASCT2 activity, the agent that down-regulates the expression level of ASCT2 is selected from (a) an siRNA, an antisense RNA, and/or a ribozyme that targets the ASCT2, (b) a gene editing vector that introduces a mutation in the encoding gene of ASCT2 in the genome of a subject cell that results in the loss or attenuation of the expressed ASCT2 activity, and (c) a homologous recombination vector that is used to knock-out the gene of the RNA in the genome or to homologous recombine a nucleic acid sequence encoding an inactive or reduced activity ASCT2 mutant into the genome to replace the wild-type ASCT2 encoding sequence.

In one or more embodiments, the gene encoding the ASCT2 protein (e.g., the SLC1A5 gene) of the cell has a mutation that reduces expression or activity of ASCT 2. Preferably, the ASCT2 protein coding sequence of the cell has a mutation that reduces expression or activity of ASCT 2. Preferably, the SLC1A5 gene of said cell has a mutation at residues 630 to 1195 or a fragment thereof that reduces expression or activity of ASCT 2. The mutation includes an insertion, deletion or substitution mutation.

In one or more embodiments, the cells have sgrnas transferred therein. Preferably, the sgRNA is as shown in SEQ ID NO. 4 and/or 5. In one or more embodiments, the cell also has a Cas protein or CRISPR/Cas complex transferred into it.

In one or more embodiments, the cell has an ASCT2 protein whose activity is inhibited.

In one or more embodiments, the cells comprise an antibody, or antigen-binding fragment thereof, to an ASCT2 protein that inhibits the activity of the ASCT2 protein.

The present invention provides a method of preparing a cell according to the first aspect of the invention comprising the step of inhibiting the expression or activity of ASCT2 in the cell.

In one or more embodiments, the expression or activity of ASCT2 is inhibited by gene editing techniques, homologous recombination techniques, RNA inhibition techniques, or immunological techniques.

In one or more embodiments, the gene editing technique introduces mutations in the gene encoding ASCT2 of the cell genome that result in reduced expression levels or reduced activity of the expressed ASCT2 via a gene editing vector. In one or more embodiments, the gene editing techniques include CRISPR, zinc finger protein, TALEN-based gene editing techniques.

The present invention provides a method for increasing the efficiency and/or titer of viral infection, or a method for preparing a virus, comprising the step of producing a virus using cells in which expression or activity of ASCT2 is inhibited.

In one or more embodiments, the cell is as described in any one of the embodiments of the first aspect of the invention.

In one or more embodiments, the virus is as described in any one of the embodiments of the first aspect of the invention.

In one or more embodiments, the method includes the steps of:

(1) Transfecting the cells with a viral vector or virus,

(2) Harvesting the virus produced by the cells.

In one or more embodiments, the virus harvested in (2) is located in the cell culture supernatant, and/or in the cells.

In one or more embodiments, step (2) comprises harvesting the virus 48 hours or more after transfection of the cells.

In one or more embodiments, in (1), the viral vector comprises a coding sequence for an envelope protein or variant thereof of one or more viruses selected from the group consisting of baboon endogenous retrovirus, feline endogenous retrovirus, vesicular stomatitis virus, or the virus comprises an envelope protein or variant thereof of one or more viruses selected from the group consisting of baboon endogenous retrovirus, feline endogenous retrovirus, vesicular stomatitis virus.

In one or more embodiments, in (1), the viral vector comprises a coding sequence for one or more envelope proteins or variants thereof selected from the group consisting of VSV-G, baEV and RD114, or the virus comprises one or more envelope proteins or variants thereof selected from the group consisting of VSV-G, baEV and RD114. Preferably, the viral vector comprises a coding sequence for one or more envelope proteins selected from RD114-TR, baEV-LV or BaEV-RLess envelope proteins, or the virus comprises one or more envelope proteins selected from RD114-TR, baEV-LV or BaEV-RLess envelope proteins.

In one or more embodiments, the viral vector is a lentiviral packaging plasmid, such as a three-plasmid system or a four-plasmid system lentiviral packaging plasmid. Preferably, the viral vector is selected from one or more or all of pLP1, pLP2, pENV and plenti. pENV comprises the coding sequence of the envelope protein.

In one or more embodiments, the method further comprises the step of inhibiting expression or activity of ASCT2 in the cell. In one or more embodiments, the expression or activity of ASCT2 is inhibited by gene editing techniques, homologous recombination techniques, RNA inhibition techniques, or immunological techniques.

The invention also provides viruses produced by the methods of producing viruses of any of the embodiments of the invention.

The second aspect of the present invention also provides a method for preparing an immune effector cell, comprising:

1) Viruses are produced using the methods of producing viruses according to any of the embodiments of the present invention,

2) Transfecting immune effector cells with the virus obtained in 1),

3) Transfected immune effector cells were harvested.

In one or more embodiments, the virus in step 2) is the virus harvested in the method of step 1) after 48 hours or more (e.g., at least 72, at least 96, at least 120 hours) transfection of cells in which expression or activity of ASCT2 is inhibited.

In one or more embodiments, the virus in step 2) is diluted, e.g., 1:50, 1:100, 1:200, or 1:500.

In one or more embodiments, the virus contains a nucleic acid sequence of interest.

In one or more embodiments, the nucleic acid sequence includes a coding sequence for a Chimeric Antigen Receptor (CAR), optionally further including a coding sequence for a cytokine.

In one or more embodiments, the CAR includes an extracellular antigen-recognition region, a hinge region, a transmembrane region, and an intracellular region.

In one or more embodiments, the extracellular antigen-recognition region targets a tumor antigen. Such antigens include, but are not limited to CD19,CD5、CD20、CD22、CD23、CD27、CD30、CD33、CD34、CD37、CD38、CD43、CD72a、CD78、CD79a、CD79b、CD86、CD134、CD137、CD138、CD319、GPC3、CD32b、CD171、CS-1、CLL-1、BCMA、GD2、GD3、PSMA、ROR1、FLT3、FAP、CD44v6、CEA、EPCAM、B7H3、IL-13Ra2、Mesothelin、Her2、MUC1、EGFR、CLDN6、CLDN18.2、DLL3、NY-ESO-1、WT1、Nectin-4、GPC3、PDL1、NKG2DL, any combination of the above antigens.

In one or more embodiments, the extracellular antigen-recognition region is an antibody or antigen-binding fragment thereof, such as a single chain antibody or nanobody.

In one or more embodiments, the hinge region of the CAR comprises any one or more of a CD8 hinge region, a CD4 hinge region, an IgD hinge region, an IgG hinge region.

In one or more embodiments, the transmembrane region of the CAR includes any one or more of the transmembrane regions from CD8, NKG2D, 2B4, DAP10, DAP12, CD28, CD134 (OX 40), CD137 (4-1 BB), CD27, and CD 40.

In one or more embodiments, the intracellular region comprises an intracellular co-stimulatory domain and/or an intracellular signaling domain. The intracellular region includes the intracellular domains of CD28, OX40 and/or 4-1BB, CD3 ζ, fcεRIγ, CD27, CD28, CD134, ICOS, GITR, 2B4, DAP10, DAP12, DNAM-1, NKp30, NKp44, NKp 46.

In one or more embodiments, the CAR further comprises a signal peptide, such as a CD8 signal peptide, a CD28 signal peptide, a CD4 signal peptide, or a light chain signal peptide.

In one or more embodiments, the cytokines include interleukins and chemokines. The interleukin-like factors include one or more of IL-1, IL-2, IL-7, IL-9, IL-10, IL-12, IL-15, IL-18, and IL-21. The chemokines include one or more of CCL1、CCL2、CCL3、CCL3L1、CCL4、CCL4L1、CCL5、CCL7、CCL8、CCL11、CCL13、CCL14、CCL15、CCL16、CCL17、CCL18、CCL19、CCL20、CCL21、CCL22、CCL23、CCL24、CCL25、CCL26、CCL27、CCL28、CXCL1、CXCL2、CXCL3、CXCL4、CXCL4L1、CXCL5、CXCL6、CXCL7、CXCL8、CXCL9、CXCL10、CXCL11、CXCL12、CXCL13、CXCL14、CXCL16、CXCL17、XCL1、XCL2 and CX3CL 1.

In one or more embodiments, the coding sequence for the chimeric antigen receptor and the coding sequence for the cytokine are linked by a coding sequence derived from 2A. The 2A comprises P2A, T A or F2A.

In one or more embodiments, the nucleic acid sequence encodes a sequence set forth in SEQ ID NO. 7.

In one or more embodiments, the immune effector cells include T cells, TCR-T, CAR-T, TIL, DC-CIK, NK cells. Preferably, the immune effector cells are derived from human umbilical cord blood or peripheral blood.

The invention also provides an immune effector cell obtained by the method of preparing an immune effector cell described in any of the embodiments herein.

The invention also provides a pharmaceutical composition comprising an immune effector cell obtained by the method of preparing an immune effector cell described in any of the embodiments herein, and a pharmaceutically acceptable adjuvant.

The invention also provides a cell with the ASCT2 expression or activity inhibited, and application of the virus in preparing a reagent containing immune effector cells or preparing a medicament for preventing or treating diseases.

In one or more embodiments, the cells in which expression or activity of ASCT2 is inhibited are as described in any of the embodiments of the first aspect herein.

In one or more embodiments, the immune effector cell is as described in any one of the embodiments of the second aspect herein.

In one or more embodiments, the nucleic acid sequence comprises a coding sequence for a Chimeric Antigen Receptor (CAR) or a coding sequence for a T Cell Receptor (TCR), optionally further comprising a coding sequence for a cytokine.

In one or more embodiments, the CAR is as described in any one of the embodiments of the second aspect herein.

In one or more embodiments, the coding sequence for the chimeric antigen receptor and the coding sequence for the cytokine are linked by a coding sequence derived from 2A. The 2A comprises T2A or F2A.

In one or more embodiments, the gene encodes a sequence set forth in SEQ ID NO. 7.

The invention also provides the use of an immune effector cell or a pharmaceutical composition comprising the same as described in any of the embodiments herein in the preparation of a medicament for treating or preventing a disease.

Drawings

FIG. 1 ASCT2 knockout of lentiviral vector particle producer cells HEK-293. A, flow cytometry analysis of sgRNA targeting. B, sequence analysis of sgRNA targeting. Flow cytometry analysis of ASCT2 protein expression.

FIG. 2. Plasmid construction of lentivirus-expressed fluorescent reporter protein BFP. A, plasmid map. And B, designing a carrier of the fluorescent protein BFP.

FIG. 3 microscopic fields of HEK-293 cells at various time points in lentiviral packaging.

FIG. 4. Collection of lentiviral vector particles at different time points, after transduction of NK92 cells, flow cytometry examined BFP fluorescent expression. A, collecting lentiviral vector particles 48 hours after plasmid transfection, purifying and concentrating 200 times. After infection of NK92 cells at different dilution ratios, flow cytometry examined BFP expression. And B, a statistical graph of results of 3 repeated holes of the experiment of the graph A. The lentiviral vector particles were collected 72 hours after plasmid transfection, purified and concentrated 200-fold. After infection of NK92 cells at different dilution ratios, flow cytometry examined BFP expression. D, 3 duplicate well results statistics for C plot experiments. E, collecting lentiviral vector particles 96 hours after plasmid transfection, purifying and concentrating 200 times. After infection of NK92 cells at different dilution ratios, flow cytometry examined BFP expression. F: statistical plot of results from 3 replicate wells of the E plot experiment. The lentiviral vector particles were collected 120 hours after plasmid transfection, purified and concentrated 200-fold. After infection of NK92 cells at different dilution ratios, flow cytometry examined BFP expression. H G figure 3 replicate well results statistics for the experiment.

FIG. 5 functional titre comparison of wild type and ASCT2 knockout HEK-293 cell packaging lentiviral vector particles.

FIG. 6 preparation of CD 19-targeting CAR-NK cells. Design of CAR expression vector. And B, detecting the CAR molecules after the NK cells of different donors are prepared into the CAR-NK cells by flow cytometry. ELISA method for detecting secreted IL-15 by CAR-NK cells from multiple donors. Amplification of CAR-NK cells.

Figure 7 killing of tumor cells in vitro and in vivo by CD19 targeted CAR-NK cells. Killing ability of CAR-NK cells on Raji. And B, killing capacity of the CAR-NK cells to JeKo-1. And C, constructing a lymphoma model by using an immunodeficient mouse, and observing tumor loads at different time points by using a living body bioluminescence imaging technology after NK or CAR-NK cells are input into the model by veins. And D, tumor load dynamic change of each mouse. And E, survival statistics of two groups of mice.

Detailed Description

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. These techniques are well explained in the literature, such as Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al, 1989), oligonucleotide Synthesis (M.J. Gait edit, 1984), ANIMAL CELL Culture (R.I. Freshney edit ,1987);Methods in Enzymology(Academic Press,Inc.);Current Protocols in Molecular Biology(F.M.Ausubel, et al, 1987 edition and periodically updated versions thereof), PCR: the Polymerase Chain Reaction (Mullis et al, edit ,1994);A Practical Guide to Molecular Cloning(Perbal Bernard V.,1988);Phage Display：A Laboratory Manual(Barbas, et al, 2001).

During the preparation of the BaEV or RD114 envelope protein pseudotyped lentiviral vector particles, the HEK-293 cells of the packaging cells can form huge syncytia (adjacent 293 cells are subjected to cell fusion to form a large cell and have a plurality of cell nuclei), and the BaEV-RLess is the most obvious. This resulted in massive death of HEK-293 cells, severely affecting the productivity of lentiviral vector particles. In addition, fragments formed by cell death further increase the difficulty in subsequent purification of lentiviral vector particles. This feature severely limits the potential for use of the pseudotyped lentiviral vector, especially not applicable to process development of GMP-level large-scale lentiviral products, failing to meet the urgent need of current CAR-NK cell therapy development.

The inventors found that lentiviruses prepared using cells with down-regulated ASCT2 expression or activity were significantly more efficient in infection and active titers than lentiviruses prepared using wild-type cells. Accordingly, the present invention provides cells in which expression or activity of ASCT2 is inhibited, methods of preparing the same, methods of preparing viruses, and methods of preparing immune effector cells therefrom.

The term "envelope protein" or "envelope glycoprotein" is a layer of envelope that some viruses in nature contain, which is enclosed by a protein capsid, often from the host cell membrane, which contains the glycoprotein characteristic of the virus itself. These glycoproteins are used to recognize and bind to surface receptors of host cells, then the viral envelope binds to the host cell membrane, and finally the viral capsid and genome enter the host, completing the infection process.

Cells

The invention firstly provides a cell with the expression or activity of ASCT2 inhibited and a preparation method thereof. Any host cell that can be used to produce or be transfected with a virus can be used in the present invention. The host cell may be a mammalian cell or a non-mammalian cell. Host mammalian cell lines useful as virus production are well known in the art and include, but are not limited to, a variety of immortalized cell lines available from the American Type Culture Collection (ATCC), including, but not limited to, chinese Hamster Ovary (CHO) cells, heLa cells, baby Hamster Kidney (BHK) cells, monkey kidney Cells (COS), human hepatocellular carcinoma cells (e.g., hepG 2), HEK293 cells, and the like. The cells may be adherent cells or suspension cells. The HEK293 cells include adherent HEK293 cells or cell lines thereof subjected to suspension domestication, such as HEK293T、HEK293H、HEK293F、HEK293S、HEK293T/17、HEK293T/17SF、HEK293FT、HEK293SG、HEK293E、HEK293-6E、HEK293FTM、HEK293SGGD cells.

Herein, viruses include natural viruses or pseudotyped viruses. In exemplary embodiments, the virus is a retrovirus or pseudotyped virus thereof, such as a lentivirus or pseudotyped virus thereof. The term "pseudotyped virus", "pseudovirus" or "pseudotyped virus" refers to a virus formed by the integration of the envelope glycoprotein of one recombinant virus with another virus. The pseudotyped virus has a similar phenotype but has the cell tropism of another virus relative to the wild-type virus, is easier to produce and safer. In the case of lentiviruses, lentiviral vectors were derived from HIV-1 virus. Pseudotyped lentiviral envelope proteins can be derived from glycoproteins of a variety of heterologous viruses, thereby expanding target cell tropism. For example, the transmembrane and extracellular domains of the baboon endogenous retrovirus (BaEV) envelope glycoprotein are expressed in fusion with the cytoplasmic tail domain of the amphotropic Murine Leukemia Virus (MLV) envelope glycoprotein (BaEV-TR), or the BaEV envelope glycoprotein is depleted of fusion inhibitory R peptide (BaEV-RLess). Pseudotyped lentiviral vectors based on BaEV-TR or BaEV-RLess can deliver genes to hematopoietic stem cells as well as resting T cells and B cells with high efficiency. In addition, fusion expression of the extracellular and transmembrane domains of the envelope glycoprotein of feline leukemia virus (RD 114) with the cytoplasmic tail domain of the amphotropic Murine Leukemia Virus (MLV) envelope glycoprotein (RD 114-TR) is also an alternative envelope. Pseudotyped lentiviral vectors can also be used to transduce NK cells. In addition, VSV-G derived from vesicular stomatitis virus is also a common envelope protein.

Herein, "inhibit," "reduce," or "down-regulate" includes as low as 0.

The cells may be prepared by reducing the expression or activity of ASCT2 in the cells using techniques known in the art. These techniques include, but are not limited to, gene editing techniques, RNA inhibition techniques, homologous recombination techniques, or immunological techniques.

In some embodiments, the gene encoding the ASCT2 protein of the cell SLC1A5 gene is provided with a mutation, provided that the mutation reduces expression or activity of ASCT 2. The mutation may be in the ASCT2 protein coding sequence or non-coding sequence. Illustratively, the mutation is at residues 630 to 1195 of the SLC1A5 gene or a fragment thereof. The mutation includes an insertion, deletion or substitution mutation.

Herein, the gene editing technique includes introducing a mutation, which results in a decrease in the expression level or activity of expressed ASCT2, into a gene encoding ASCT2 in the genome of a cell by a gene editing vector. Illustratively, the gene editing techniques include CRISPR, zinc finger protein, TALEN based gene editing techniques. Thus, in some embodiments, the cells have sgrnas transferred therein. The sgRNA is shown as SEQ ID NO. 4 and/or 5. The cells may also have a Cas protein or CRISPR/Cas complex transferred into them.

RNA inhibition techniques herein include reduced expression levels or reduced activity of expressed ASCT2 by siRNA, antisense RNA and/or ribozymes targeting the gene or transcript encoding ASCT 2. Thus, in some embodiments, the activity of the transcript of ASCT2 protein of said cell is inhibited.

Homologous recombination techniques herein include knocking out the gene encoding ASCT2 in the genome by a homologous recombination vector or homologous recombination of a nucleic acid sequence encoding an inactive or reduced activity ASCT2 variant into the genome to replace the wild-type ASCT2 coding sequence.

Immunological techniques herein include reducing the activity of ASCT2 by expressing, secreting, or contacting cells with an antibody or antigen binding fragment thereof of ASCT 2. Thus, in some embodiments, the cell comprises an antibody, or antigen-binding fragment thereof, to an ASCT2 protein, which inhibits the activity of the ASCT2 protein.

In the above techniques, the cells comprise, express, secrete, or are contacted with an agent, such as an antibody, nucleic acid, or small molecule, that down-regulates the expression level or activity of ASCT 2. Agents that down-regulate ASCT2 activity may be antagonists of ASCT2 activity. The agent that down-regulates the expression level of ASCT2 may be selected from (a) siRNA, antisense RNA and/or ribozyme targeting said ASCT2, (b) a gene editing vector that introduces a mutation in the encoding gene of ASCT2 in the genome of a subject cell that results in the loss or attenuation of the expressed ASCT2 activity, and (c) a homologous recombination vector for knocking out the gene of said RNA in the genome or homologous recombination of a nucleic acid sequence encoding an inactive or reduced activity ASCT2 mutant into the genome to replace the wild-type ASCT2 encoding sequence.

Method for preparing virus

Based on the above cells, the present invention provides a method for preparing a virus comprising the step of producing a virus using a cell in which expression or activity of ASCT2 is inhibited. The invention also provides a method for increasing the efficiency and/or titer of viral infection.

In one or more embodiments, the method comprises the steps of (1) transfecting the cell with a viral vector or virus, and (2) generating virus from the cell. Viruses produced by cells may be located in cell culture supernatants or in cells. The invention also includes viruses obtained by the method.

In exemplary embodiments, the viral vector comprises a coding sequence for an envelope protein or variant thereof described herein, and the virus comprises an envelope protein or variant thereof described herein, e.g., a VSV-G, baEV, RD114, RD114-TR, baEV-LV or BaEV-RLess envelope protein.

When producing lentiviruses or pseudoviruses thereof, the viral vector is a lentivirus packaging plasmid, such as a three-plasmid system or a four-plasmid system lentivirus packaging plasmid. Exemplary four plasmid cells contain pLP1, pLP2, pENV, and plenti. The pLP1 plasmid contains the gag and pol genes necessary for the preparation of lentiviral vector particles, while the Rev proteins that the pLP2 plasmid uses to express can co-act with the response elements on pLP1 to induce gag and pol expression and direct nuclear transport of viral RNA. pENV comprises the coding sequence of the envelope protein. The pLenti is a lentiviral expression plasmid into which a nucleotide sequence of interest or a marker gene sequence for expression of the generation may be inserted. After transfection of immune effector cells with the produced lentivirus, the expression product of the marker gene can be detected by techniques conventional in the art such as flow cytometry.

Expression plasmids may contain expression cassettes, which generally contain sequences for cloning or expressing exogenous nucleotide sequences. Such sequences (referred to in certain embodiments as "flanking sequences") typically include one or more nucleotide sequences including a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence containing donor and acceptor splice sites, a sequence encoding a leader sequence for secretion of the polypeptide, a ribosome binding site, a polyadenylation sequence, a polylinker region for insertion of nucleic acid encoding an antibody to be expressed, and optional marker elements. See, for example, WO 01/96584, WO01/29058, and U.S. Pat. No. 6,326,193.

One example of a suitable promoter is the immediate early Cytomegalovirus (CMV) promoter sequence. The promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is extended growth factor-1α (EF-1α). However, other constitutive promoter sequences may also be used, including but not limited to the simian virus 40 (SV 40) early promoter, the mouse mammary carcinoma virus (MMTV), the Human Immunodeficiency Virus (HIV) Long Terminal Repeat (LTR) promoter, the MoMuLV promoter, the avian leukemia virus promoter, the epstein barr virus immediate early promoter, the ruses sarcoma virus promoter, and human gene promoters such as but not limited to the actin promoter, the myosin promoter, the heme promoter, and the creatine kinase promoter. Further, the use of inducible promoters is also contemplated. The use of an inducible promoter provides a molecular switch that is capable of switching on expression of a polynucleotide sequence operably linked to the inducible promoter when expressed for a period of time and switching off expression when expression is undesirable. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

The expression plasmid may also be an integrated expression plasmid, such as a transposable vector or a recombinant vector based on gene editing technology, comprising sequences (e.g., transposon 5 'and 3' inverted terminal repeats, donor DNA) recognized by an integration element (e.g., a transposase or CRISPR-associated transposase) and mediating integration.

Methods for constructing plasmids containing nucleic acid sequences of interest are well known in the art. In general, the full-length sequence of the nucleic acid sequence of interest, or a fragment thereof, can be obtained, in general, by PCR amplification, recombinant methods or synthetic methods. Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods. The host cell used to produce the nucleic acid or protein may be a prokaryotic cell, such as a bacterial cell, or a lower eukaryotic cell, such as a yeast cell, or a higher eukaryotic cell, such as a mammalian cell. Representative examples are E.coli, streptomyces, salmonella typhimurium, fungal cells such as yeast, drosophila S2 or Sf9 insect cells, CHO, COS7, 293 cell animal cells, and the like. Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art.

In exemplary embodiments, the nucleic acid sequence of interest encodes a chimeric antigen receptor and/or a cytokine. The term "Chimeric Antigen Receptor (CAR)" is an artificially designed protein fragment, comprising an extracellular fragment, a transmembrane region and an intracellular fragment. Extracellular fragments can recognize specific membrane proteins of tumor cells, and intracellular fragments can provide co-stimulatory and activating signals to enhance the killing capacity of the cells. In particular, the CAR contains an optional signal peptide sequence, an extracellular target recognition region (antigen binding domain), a hinge region, a transmembrane region, an intracellular co-stimulatory domain, and an intracellular signal domain. The extracellular recognition region comprises an antibody that targets an antigen (e.g., a tumor antigen), such as a full-length antibody, an antigen-binding fragment, a single domain antibody (nanobody), a single chain variable fragment (scFv).

The targeting antigen of the chimeric antigen receptor is not limited, including but not limited to ：CD19,CD5、CD20、CD22、CD23、CD27、CD30、CD33、CD34、CD37、CD38、CD43、CD72a、CD78、CD79a、CD79b、CD86、CD134、CD137、CD138、CD319、GPC3、CD32b、CD171、CS-1、CLL-1、BCMA、GD2、GD3、PSMA、ROR1、FLT3、FAP、CD44v6、CEA、EPCAM、B7H3、IL-13Ra2、Mesothelin、Her2、MUC1、EGFR、CLDN6、CLDN18.2、DLL3、NY-ESO-1、WT1、Nectin-4、GPC3、PDL1、NKG2DL and any combination of the above antigens.

The variable region of CD19 single chain antibody (FMC 63, genebank: HM 852952.1), CD8 outer membrane (hinge region), transmembrane segment, 4-1BB intracellular segment and CD3zeta intracellular segment, and human IL-15 is expressed in series by T2A polypeptide structure

The optional signal peptide on the CAR may be selected as desired. Such as a CD8 signal peptide, a CD28 signal peptide, a CD4 signal peptide or a light chain signal peptide, the sequences of which are within the knowledge of a person skilled in the art.

The hinge region of the CAR may be selected from a CD8 hinge region, an IgD hinge region, an IgG1 Fc CH2CH3 hinge region, or an IgG4 Fc CH2CH3 hinge region, the sequence of which is within the knowledge of one skilled in the art. In an exemplary embodiment, the hinge region is a CD8 hinge region.

The transmembrane region of the CAR is selected from the group consisting of the CD28 transmembrane region, the CD8 transmembrane region, the cd3ζ transmembrane region, the CD134 transmembrane region, the CD137 transmembrane region, the ICOS transmembrane region, and the DAP10 transmembrane region, the sequences of which are within the knowledge of those skilled in the art. In an exemplary embodiment, the transmembrane region is a CD8 transmembrane region.

Suitable intracellular co-stimulatory domains may be selected as desired, including those with co-stimulatory signaling molecules, such as the intracellular domains of CD28, CD134/OX40, CD137/4-1BB, lymphocyte-specific protein tyrosine kinase, inducible T cell co-stimulatory factor (ICOS) and DNAX activator protein 10. In an exemplary embodiment, the intracellular co-stimulatory domain is a 4-1BB co-stimulatory domain.

Similarly, the intracellular signaling domain of the CAR may be selected as desired, including but not limited to the CD3zeta intracellular signaling domain or fceriy intracellular signaling domain. In an exemplary embodiment, the intracellular signaling domain is a CD3zeta intracellular signaling domain.

The above-described parts forming the chimeric antigen receptor of the invention may be directly linked to each other or may be linked by a linker sequence. The linker sequences may be linker sequences suitable for antibodies as known in the art, such as G and S containing linker sequences. Typically, a linker contains one or more motifs that repeat back and forth. Preferably, the motifs are contiguous in the linker sequence with no amino acid residues inserted between the repeats. The linker sequence may comprise 1,2, 3,4 or 5 repeat motif compositions. The length of the linker may be 3 to 25 amino acid residues, e.g. 3 to 15, 5 to 15, 10 to 20 amino acid residues. In certain embodiments, the linker sequence is a glycine linker sequence. The number of glycine in the linker sequence is not particularly limited, and is usually 2 to 20, for example, 2 to 15, 2 to 10, 2 to 8. In addition to glycine and serine, other known amino acid residues may be contained in the linker, such as alanine (A), leucine (L), threonine (T), glutamic acid (E), phenylalanine (F), arginine (R), glutamine (Q), etc. In certain embodiments, the linker sequence is a (GGGGS) n linkage, where n is an integer from 1 to 5.

It will be appreciated that in gene cloning operations, it is often necessary to design suitable cleavage sites, which tend to introduce one or more unrelated residues at the end of the expressed amino acid sequence, without affecting the activity of the sequence of interest. To construct fusion proteins, facilitate expression of recombinant proteins, obtain recombinant proteins that are automatically secreted outside of the host cell, or facilitate purification of recombinant proteins, it is often desirable to add some amino acid to the N-terminus, C-terminus, or other suitable region within the recombinant protein, including, for example, but not limited to, suitable linker peptides, signal peptides, leader peptides, terminal extensions, and the like. Thus, the amino-or carboxy-terminus of a CAR of the invention may also contain one or more polypeptide fragments as protein tags. Any suitable label may be used herein. For example, the tag may be FLAG, HA, HA1, c-Myc, poly-His, poly-Arg, strep-TagII, AU1, EE, T7,4A6, B, gE, and Ty1. These tags can be used to purify proteins.

The invention also includes mutants of the CAR of any of the embodiments. These mutants include amino acid sequences that have at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 97% sequence identity to the CAR and retain the biological activity of the CAR (e.g., activating T cells). Sequence identity between two aligned sequences can be calculated using BLASTp, e.g., NCBI.

The nucleic acid sequence of interest may comprise a coding sequence for a cytokine, including interleukins and chemokines. The interleukin-like factors include one or more of IL-1, IL-2, IL-7, IL-9, IL-10, IL-12, IL-15, IL-18, and IL-21. The chemokines include one or more of CCL1、CCL2、CCL3、CCL3L1、CCL4、CCL4L1、CCL5、CCL7、CCL8、CCL11、CCL13、CCL14、CCL15、CCL16、CCL17、CCL18、CCL19、CCL20、CCL21、CCL22、CCL23、CCL24、CCL25、CCL26、CCL27、CCL28、CXCL1、CXCL2、CXCL3、CXCL4、CXCL4L1、CXCL5、CXCL6、CXCL7、CXCL8、CXCL9、CXCL10、CXCL11、CXCL12、CXCL13、CXCL14、CXCL16、CXCL17、XCL1、XCL2 and CX3CL 1.

The coding sequences for the chimeric antigen receptor and the cytokine may be in the same expression cassette. At this time, the coding sequence of the chimeric antigen receptor and the coding sequence of the cytokine are linked by a coding sequence derived from 2A (P2A, T a or F2A) or an IRES sequence. Alternatively, the coding sequences for the chimeric antigen receptor and the cytokine are in two expression cassettes.

Methods for transfecting host cells (e.g., host cells described herein) with viruses or viral vectors are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of polynucleotides in liposomes, and direct microinjection of DNA into the nucleus, etc.

Immune effector cells

The present invention also provides a method for preparing immune effector cells, comprising 1) preparing a virus using the method for preparing a virus according to the present invention, 2) transfecting immune effector cells with the virus obtained in 1), and 3) harvesting the transfected immune effector cells. Step 2) the virus transfected into immune effector cells is the virus harvested in the method of step 1) after 48 hours or more (e.g., at least 72, at least 96, at least 120 hours) of transfection of cells in which expression or activity of ASCT2 is inhibited. The virus obtained in step 1) may be diluted for step 2), for example 1:50, 1:100, 1:200 or 1:500 dilution.

As used herein, "immune effector cells" include T cells, tumor Infiltrating Lymphocytes (TIL) cells, natural Killer (NK) cells, or Natural Killer T (NKT) cells. NK cells suitable for use in the present invention may be of various types of NL cells of various origins. For example, NK cells may be derived from human umbilical cord blood or peripheral blood.

The invention also includes immune effector cells obtained by the method for preparing immune effector cells. The effector cells express a nucleic acid sequence of interest described herein, e.g., a coding sequence for a Chimeric Antigen Receptor (CAR) and/or a coding sequence for a cytokine.

Pharmaceutical composition

The invention also provides a pharmaceutical composition comprising an immune effector cell obtained by the method of preparing an immune effector cell described herein, and a pharmaceutically acceptable adjuvant. The invention also provides the use of an immune effector cell or a pharmaceutical composition comprising the same as described herein in the manufacture of a medicament for the treatment or prophylaxis of a disease. The present invention also provides a method of treating or preventing a disease comprising administering to a patient in need thereof a therapeutically effective amount of an immune effector cell or pharmaceutical composition described herein. The disease is a disease suitable for cellular immunotherapy, such as autoimmune disease or cancer. One skilled in the art can determine the appropriate disease based on the antigen recognized by the extracellular region of the CAR and the type of immune effector cells.

The immune effector cells of the invention may be administered alone or as a pharmaceutical composition in combination with diluents and/or with other components such as associated cytokines or cell populations. Briefly, the pharmaceutical compositions herein contain the immune cells described herein, and pharmaceutically acceptable excipients, including but not limited to diluents, carriers, solubilizers, emulsifiers, preservatives and/or adjuvants. The adjuvant is preferably non-toxic to the recipient at the dosage and concentration employed. Such excipients include, but are not limited to, saline, buffers, dextrose, water, glycerol, ethanol, and combinations thereof. In certain embodiments, the pharmaceutical composition may contain substances for improving, maintaining or retaining, for example, pH, permeability, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, absorption or permeation of the composition. These substances are known from the prior art. The optimal pharmaceutical composition can be determined depending on the intended route of administration, the mode of delivery and the dosage required.

Pharmaceutical compositions for in vivo administration are generally provided in the form of sterile formulations. Sterilization is achieved by filtration through sterile filtration membranes. In the case of lyophilization of a composition, this method may be used to sterilize the composition either before or after lyophilization and reconstitution. The pharmaceutical compositions of the present invention may be selected for parenteral delivery. Compositions for parenteral administration may be stored in lyophilized form or in solution. For example, by using physiological saline or an aqueous solution containing glucose and other auxiliary agents by conventional methods. Parenteral compositions are typically placed in a container having a sterile access port, such as an intravenous solution tape or vial having a stopper pierceable by a hypodermic injection needle. Or alternatively the composition may be for inhalation or delivery through the digestive tract (such as orally). The preparation of such pharmaceutically acceptable compositions is within the skill of the art. Other pharmaceutical compositions will be apparent to those skilled in the art, including formulations comprising antibodies in sustained or controlled release delivery formulations. Techniques for formulating a variety of other sustained or controlled delivery means, such as liposome carriers, bioerodible particles or porous beads, and depot injections, are also known to those skilled in the art.

Once formulated, the pharmaceutical compositions are stored in sterile vials as solutions, suspensions, gels, emulsions, solids, crystals, or as dehydrated or lyophilized powders. The formulation may be stored in a ready-to-use form or reconstituted (e.g., lyophilized) prior to administration. The invention also provides kits for producing single dose administration units. Kits of the invention may each contain a first container having a dried protein and a second container having an aqueous formulation. In certain embodiments of the invention, kits are provided that contain single and multi-chamber prefilled syringes (e.g., liquid syringes and lyophilized syringes).

Herein, the term "disease and/or condition" refers to a physical state of a subject associated with the diseases and/or conditions described herein. The terms "patient," "subject," "individual," "subject," and "subjects" are used interchangeably herein to refer to any organism, preferably an animal, more preferably a mammal, such as a rat, mouse, dog, cat, rabbit, monkey, cow, horse, human, etc., that receives a pharmaceutical composition of the invention to treat, prevent, ameliorate, and/or ameliorate a disease or condition of the invention. "preventing" refers to the use of a therapeutic regimen described herein by a subject to achieve an effect of preventing or reducing the adverse effects of a disease or disorder. "treating" refers to a subject employing a treatment regimen described herein to achieve at least one positive therapeutic effect (e.g., reduced number of cancer cells, reduced tumor volume, reduced rate of infiltration of cancer cells into peripheral organs, or reduced rate of tumor metastasis or tumor growth). The treatment regimen effective to treat a patient can vary depending on a variety of factors, such as the disease state, age, weight, and ability of the patient to elicit an anti-cancer response in the subject by therapy.

When referring to a "therapeutically effective amount," the precise amount of the composition of the invention to be administered can be determined by a physician and will vary depending in part on the degree of treatment, the target, the molecule delivered, the indication, the route of administration, the age of the patient, body weight, body surface or organ size, general health, tumor size, the degree of infection or metastasis, and the individual variability of the condition. In certain embodiments, the clinician may titrate the dose and alter the route of administration to obtain the optimal therapeutic effect. It may be generally pointed out that the pharmaceutical composition comprising the immune cells described herein may be administered in a dose of 10 ⁴ to 10 ⁹ cells/kg body weight, preferably in a dose of 10 ⁵ to 10 ⁶ cells/kg body weight. The immune cell composition may also be administered multiple times at these doses. Cells can be administered by using injection techniques well known in immunotherapy (see, e.g., rosenberg et al, new Eng. J. Of Med.319:1676,1988). Optimal dosages and treatment regimens for a particular patient can be readily determined by one skilled in the medical arts by monitoring the patient for signs of disease and adjusting the treatment accordingly.

The frequency of administration will depend on the pharmacokinetic parameters of the binding molecule in the formulation used. The clinician typically administers the composition until a dose is reached that achieves the desired effect. The composition may thus be administered as a single dose, or over time as two or more doses (which may or may not contain the same amount of the desired molecule), or as a continuous infusion through an implanted device or catheter.

Administration of the subject compositions may be performed in any convenient manner, including by spraying, injection, swallowing, infusion, implantation, or transplantation. The routes of administration of the compositions described herein are known methods, such as injection orally, by intravenous, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, portal, or intralesional routes, by sustained release systems, or by implanted devices. In one embodiment, the immune cell composition of the invention is administered to a patient by intradermal or subcutaneous injection. In another embodiment, the immune cell composition of the invention is preferably administered by intravenous injection. The composition of immune cells may be injected directly into the tumor, lymph node or site of infection.

In some embodiments of the invention, the immune effector cells of the invention or compositions thereof may be combined with other therapies known in the art. Such therapies include, but are not limited to, chemotherapy, radiation therapy, and immunosuppressants. For example, treatment may be in combination with radiation or chemotherapy agents known in the art for the treatment of mesothelin-mediated diseases.

Embodiments of the present invention will be described in detail below with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The specific techniques or conditions are not noted in the examples, and are carried out according to techniques or conditions described in the literature in the art (for example, refer to J. Sam Brookfield et al, edited by Huang Peitang et al, molecular cloning Experimental guidelines, third edition, scientific Press) or according to the product specifications. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

Examples

Example 1 ASCT2 in HEK-293 cell producing cell based on CRISPR/Cas9 knockout lentiviral vector particle

1. Design and acquisition of sgRNA

Human ASCT2 was encoded by the SLC1A5 gene, located in chromosome 19 at 13.3 in the q-arm, the full length 13712bp of the transcription unit, and the sequence of segment 1 of the protein code was 630..1195 (SEQ ID No. 1) by genome browser query (https:// genome-asia. Ucsc. Edu) from Style, university California.

Two sgRNA guide sequences were designed as follows:

sgRNA1 guide DNA:5’-CGCGCCTTGGTCCTCGATGG-3’(SEQ ID No.2)

sgRNA2 guide DNA:5’-GCGAGCCCCTTGGAGTCTCG-3’(SEQ ID NO.3)

The sequence of the sgRNA synthesized by the entrusted Kirschner biosciences Inc., 3 thio and oxymethyl modifications at the 5 'end and 3' end are shown as SEQ ID NO. 4 (sgRNA 1) and SEQ ID NO. 5 (sgRNA 2).

2. Electrotransport CRISPR/Cas9 ribonucleoprotein complex (Cas 9 RNP)

HEK-293 cells (ATCC, cat# CRL-1573) were cultured, plated into DMEM high-sugar medium (ThermoFisher, cat# 11965118) containing 10% FBS,2mM L-glutamine, 10mM HEPES and 1.0mM sodium pyruvate and placed in a 37℃incubator.

60Pmol of CRISPR/Cas9 Protein TrueCut ^TM Cas9 Protein v2 (Thermo Fisher, cat. No. A36499) was added to each of the two sgRNAs (150 pmol) in PBS to form a total of 10uL of Cas9 RNP mix, which was mixed and allowed to stand at room temperature for about 10 minutes.

10 ⁶ 293 Cells were collected, centrifuged at 90g at room temperature for 10 minutes, resuspended in 100uL SF 4D-Nucleofector ^TM X solution and the RNP mixture was added. Transfer to an electric rotating cup, use Lonza 4D-Nucleofector electric, program code CM-130.

After electrotransformation, the cells were placed in a 37℃incubator for continuous culture

3. Genomic target region sequence analysis

On day 5 after electrotransformation, 293 cells were incubated with ASCT2 antibody (CELL SIGNALING, cat# 5345) and Alexa was used647 Fluorescent-labeled anti-rabbit IgG antibody (Biolegend, cat. 406414) was stained and analyzed by flow cytometry using a ACEA NovoCyte flow cytometer. As shown in FIG. 1, A, the ASCT 2-negative cell ratio after sgRNA1 targeting was relatively low, and we used the limiting dilution method to obtain a 96-well monoclonal cell culture.

After 2 weeks of further culture, genomic DNA of each monoclonal cell line was prepared using QuickExtract DNA extraction kit (Lucigen, cat# QE 09050). PCR amplification of the target region was then performed using PrimSTAR GXL DNA polymerase (Takara, cat. No. R050A).

The PCR primers are shown in SEQ ID NO. 8 (forward) and SEQ ID NO. 9 (reverse).

The PCR scheme comprises steps of 1:98 ℃ and 10s, 2:60 ℃ and 15s, 3:68 ℃ and 20s, and 4, repeating the steps of 1-3 for 30 cycles.

The PCR products were electrophoresed on a 1% agarose gel, and bands of the PCR products were excised from the gel at a position about 400bp in size, purified and sent to Sanger sequencing (commissioned Beijing, biotechnology Co., ltd.).

The sequencing primer 5'-ACACTCCAGCTTCCAAGAGC-3' (SEQ ID NO: 6).

Sequence analysis confirmed that there were multiple cloned insertion or deletion mutations in the sgRNA 1-targeted DNA (shown bolded in font), base substitutions were indicated by "underlines", base deletions were indicated by "-", and base insertions were indicated by "strikethrough" (fig. 1, b).

4. Identification of ASCT2 stable knockout HEK-293 cells

Further analysis of ASCT2 expression was performed on HEK-293 cells at the protein expression level, confirming successful knockout of ASCT2 by flow cytometry analysis of #30 monoclonal cells (FIG. 1, C).

EXAMPLE 2 suspension acclimation of ASCT2 knockout HEK-293 cells to serum-free culture

HEK-293 wild-type WT and ASCT2 knocked out #30 were first cultured with 25% serum-free medium OPM-293CD05 (brand: om Pu Mai, cat# 81075-001) +75% stock culture (DMEM+10% FBS) and subcultured for two generations at 3M cells/10cm dish. The cells grow into a compact monolayer within 48-72 h, and the cell viability is 89% -99%. And then increasing the proportion of the serum-free culture medium in sequence according to the step, reducing the proportion of the original culture medium, finally completely culturing HEK-293 in the serum-free culture medium, culturing (37 ℃ C., 5% CO ₂, 90 rpm) according to 10x10 ⁶ cells/20mL (2 shaking flasks) on a shaking table, and carrying out passage when the density reaches 2x10 ⁶ cells/mL, wherein the cell viability is more than or equal to 90% until the HEK-293 cell line HEK-293/SC adapting to serum-free culture is obtained after suspension domestication.

In order to obtain domesticated cells with higher activity rate and proliferation rate, a limiting dilution method is adopted to pick up the monoclonal cells, and the monoclonal cells with the lowest aggregation rate and highest growth rate are obtained to be used as a domesticated cell line for library construction and preservation.

Example 3 Effect of knockout of ASCT2 on lentiviral titres and yield in HEK-293 cells or HEK-293/SC cells

1. Packaging system for lentiviral vectors

Preparation of lentiviral vector particles following transfection of HEK-293 cells or HEK-293/SC cells with a third generation lentiviral vector system, i.e.4 lentiviral packaging plasmid mixtures, secreted lentiviral vector particles were collected for subsequent purification and concentration. The 4 lentiviral packaging plasmids specifically included pLP1, pLP2, pENV and plenti. The pLP1 plasmid contains the gag and pol genes necessary for the preparation of lentiviral vector particles, while the Rev proteins used for expression by the pLP2 plasmid can co-act with the response elements on pLP1 to induce gag and pol expression and direct nuclear transport of viral RNA, all available from Thermo fisher (cat. K497500). Human NK cells can be transduced with RD114-TR, baEV-TR or BaEV-RLess envelope-pseudotyped lentiviral vector particles to construct pENV expressing one of the envelope glycoproteins described above (the following experiments are exemplified by BaEV-RLess). pLenti is a lentiviral expression plasmid, the backbone is based on pLenti-CMV-V5-LUC Blast (Addgene, cat# 21474), the CMV promoter is replaced with EF1a, and multiple restriction cloning sites are designed to facilitate subsequent plasmid construction (FIG. 2, A). Cloning the expression gene of fluorescent protein BFP to pLenti (FIG. 2, B), constructing pLenti-BFP slow virus vector particles to transduce NK cells, and detecting BFP by flow cytometry.

2. Packaging of lentiviral vector particles

(1) Preparation of packaging cells

The ability of HEK-293 cells or HEK-293/SC cells to produce lentiviral vector particles was compared with wild-type WT and ASCT2 knockout # 30. HEK-293 cells were cultured in a 15mm cell culture dish (Thermo, accession number 174888) of Nunc, and about 5×10 ⁶ cells were inoculated in DMEM high sugar medium containing 10% fbs and placed in an incubator with 5% CO2 at 37 ℃ for culturing. After inoculation, the cell confluence was observed to be about 70%, and plasmid transfection was performed.

HEK-293/SC cells were diluted to a final density of 1.5X10 ⁶ cells/mL with serum-free medium OPM-293CD05 and grown overnight in an incubator (37 ℃, 5% CO ₂, 120 rpm).

(2) Plasmid transfection

A calcium phosphate transfection system was prepared and a 15mL centrifuge tube contained 900. Mu.L of Opti-MEM medium (Thermo, cat. No. 11058-021), 4 plasmids (pLenti: pLP1: pLp: pENV plasmid molar ratio 2:1:1:0.5) packaged with lentiviruses were added first, then 100. Mu.L of 2.5MCaC1 ₂ solution was added, and finally 2mL of 2 XHBS (Sigma, cat. No. H3375) was added dropwise on a stirring shaker. The transfection mixture was left to stand at room temperature for 20 minutes. HEK-293 cells were replaced with 20mL of DMEM medium containing 2% FBS, and steam was added to a final concentration of 20-30mmol/L. The transfection mixture was added drop-wise to the medium, shaken well and placed in an incubator. After 12 hours, the cell culture broth was replaced with 20mL DMEM medium containing 2% fbs, and the culture was continued. The culture medium of HEK-293/SC cells is serum-free medium OPM-293CD05, and other operations are the same as HEK-293.

(3) Collection of culture supernatant

The under-microscope morphology of the cultured cells was observed and recorded 48, 72, 96 and 120 hours after transfection, respectively, after which the cell culture supernatant containing lentiviral vector particles was collected, after which DMEM medium containing 2% fbs was added. As shown in FIG. 3, after 48 hours, a large number of WT HEK-293 cells were detached from the culture dish, and a part of cells were in the form of large cells containing a plurality of nuclei, while #30 HEK-293 cells were still growing on the wall in the visual field, and there was no significant difference in the number of cells that were continuously expanded, and the morphology and the pre-transfection. The suspended HEK-293/SC cells, the WT cells have similar large cell morphology of a plurality of cell nuclei, while the suspended cells acclimatized by #30 are transparent and glossy, and the cell boundaries are clear.

(4) Purification and concentration of lentiviral vector particles:

The lentiviral supernatant collected at each time period was first passed through a PES filter (Jet Biofil, cat. FPE 4030) with a pore size of 0.45 μm to remove the clarified liquid recovered from the cells and cell debris. Since the HEK-293 cells of WT had a large number of cell sloughing, severely interfering with the filter filtration of the collected lentiviral supernatants, the HEK-293 group at each time point had no similar problem with lentiviral supernatants. Culture medium of HEK-293/SC cells was collected, centrifuged at 300g for 10 minutes, and the supernatant was collected.

4ML of 20% sucrose cushion was added to the bottom of a pre-sterilized ultracentrifuge tube (Beckman, cat. 344058). The recovered lentivirus clarified solution was added to sucrose pad in centrifuge tube, trimmed and carefully placed in bucket. Using SW 32Ti rotor, place in an Optima XPN-100 ultracentrifuge and centrifuge for 90 minutes at 4℃at 125000 g. After centrifugation, the supernatant was removed using a pipette, PBS was added to resuspend lentiviral pellet and split-packed for storage at-80 ℃.

3. Determination and comparison of active titres of lentiviral vectors

(1) Comparison of lentiviral vector particle transduction efficiencies at different time points

NK92MI cells (ATCC, cat. No. CRL-2408) were seeded at a density of 0.3X10 ⁶ cells per well in 96-well plates and cultured using 0.2mL of MEM alpha medium (Thermo, cat. No. 12571063) containing 12.5% FBS. After 24 hours of inoculation, different lentiviral vector particles were separately added for transduction. Different dilution ratios of lentiviruses were used for each group, specifically 1:50, 1:100, 1:200 or 1:500, and 3 replicates were established. After mixing, the mixture was placed in a horizontal-turning-head centrifuge model for 90 minutes at 32 ℃ and 1200 g. At the end, 96-well plates were incubated at 37 ℃ for 4 hours. The cells were then aspirated, centrifuged 1 time in PBS, the supernatant discarded, resuspended in MEM alpha medium, and plated into 24 well plates for further culture. After 48 hours, the BFP expression proportion of each group of NK92MI cells was examined by flow cytometry. As shown in FIGS. 4A-H, lentiviral vector particles collected at 48 hours, WT and #30, were similar in infection efficiency. However, lentiviruses collected at 72, 96 and 120 hours, lentivirus infection efficiency of #30 was significantly higher than WT.

(2) Comparison of active titres of lentiviral vectors

Titer calculation of lentiviral viral particles t= (P x N)/(D x V). Where t=titer (TU/ml), p=flow positive cell ratio (infection efficiency at different dilutions of lentivirus, calculated by selecting the group with positive ratio around 10-20%, if positive cells account for 15%, p=1.5), n=transduced cell number, d=dilution, v=transduced volume. In FIG. 5, HEK-293 cells of #30 produced lentiviral vector particles approximately 3-fold higher than HEK-293 cells of WT in total active titer. The results for HEK-293/SC cells were similar to those for HEK-293 cells.

Example 4 preparation of CD 19-targeting CAR-NK cells for treatment of lymphomas

The design of the CAR-expressing lentiviral vector is shown in fig. 6, a. Specifically, the CAR structure includes an anti-CD 19 single chain antibody variable region (FMC 63, genebank: HM 852952.1), CD8 extramembranous (hinge region), transmembrane segment, 4-1BB intracellular segment and CD3zeta intracellular segment, and human IL-15 is expressed in tandem by the T2A polypeptide structure (UniProtKB: P40933-1). The amino acid sequence is shown as SEQ ID NO. 7.

The ASCT2 knocked-out HEK-293 cell #30 or the domesticated HEK-293/SC cell is used for preparing lentiviral vector particles, so that NK cells derived from human umbilical cord blood can be transduced efficiently. The CAR expression ratios of multiple donor-derived NK cells were examined by flow cytometry, with an average of around 50% (fig. 6, b, C). Lentiviral transduction did not affect NK cell growth, and amplified more than 2 thousand-fold in vitro within 14 days (fig. 6,D). The results for HEK-293/SC cells were similar to those for HEK-293 cells.

The prepared CAR-NK cells and a human lymphoma cell line (Raji or JeKo-1) expressing CD19 are subjected to co-incubation culture to verify the in-vitro specific killing research on tumors, and the killing capacity of the CAR-NK cells on B cell lymphomas Raji and JeKo-1 is found to be obviously stronger than that of the NK cells under the condition of gradient progressive effective target ratio based on luciferase assay (figures 7, A and B).

Animal experiments are carried out on NSG mice after Raji cell implantation, and the results show that the CAR-NK019 has obvious killing effect on CD19 ⁺ tumor cells, can completely remove tumors and obviously prolongs the survival time of the mice (figure 7, C-E).

Sequences herein

SEQ ID NO:1

ATGGTGGCCGATCCTCCTCGAGACTCCAAGGGGCTCGCAGCGGCGGAGCCCACCGCCAACGGGGGCCTGGCGCTGGCCTCCATCGAGGACCAAGGCGCGGCAGCAGGCGGCTACTGCGGTTCCCGGGACCAGGTGCGCCGCTGCCTTCGAGCCAACCTGCTTGTGCTGCTGACAGTGGTGGCCGTGGTGGCCGGCGTGGCGCTGGGACTGGGGGTGTCGGGGGCCGGGGGTGCGCTGGCGTTGGGCCCGGAGCGCTTGAGCGCCTTCGTCTTCCCGGGCGAGCTGCTGCTGCGTCTGCTGCGGATGATCATCTTGCCGCTGGTGGTGTGCAGCTTGATCGGCGGCGCCGCCAGCCTGGACCCCGGCGCGCTCGGCCGTCTGGGCGCCTGGGCGCTGCTCTTTTTCCTGGTCACCACGCTGCTGGCGTCGGCGCTCGGAGTGGGCTTGGCGCTGGCTCTGCAGCCGGGCGCCGCCTCCGCCGCCATCAACGCCTCCGTGGGAGCCGCGGGCAGTGCCGAAAATGCCCCCAGCAAGGAGGTGCTCGATTCGTTCCTGGATCTTGCGAG

SEQ ID No.2:sgRNA1 guide DNA

CGCGCCTTGGTCCTCGATGG

SEQ ID NO.3:sgRNA2 guide DNA

GCGAGCCCCTTGGAGTCTCG

SEQ ID NO.4:sgRNA1

CGCGCCUUGGUCCUCGAUGGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU

SEQ ID NO.5:sgRNA2

GCGAGCCCCUUGGAGUCUCGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU

SEQ ID NO.6

ACACTCCAGCTTCCAAGAGC

SEQ ID NO:7

MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRARRSGSGEGRGSLLTCGDVEENPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

SEQ ID NO:8

CTGGGGAATTTAAACACTCCAGC

SEQ ID NO:9

ACACCCCCAGTCCCAGCG

Claims

1. A method of increasing the efficiency and/or titer of infection with a virus, or a method of making a virus, comprising the step of producing a virus using a cell in which expression or activity of ASCT2 is inhibited, wherein the virus is a lentivirus or a pseudotyped virus thereof, and the cell is a HEK293 cell.

2. The method of claim 1, wherein the pseudotyped virus comprises an envelope protein of one or more viruses selected from the group consisting of baboon endogenous retrovirus, feline leukemia virus, vesicular stomatitis virus.

3. The method of claim 1, wherein the pseudotyped virus comprises one or more envelope proteins selected from the group consisting of VSV-G, baEV and RD114.

4. The method of claim 3, wherein the pseudotyped virus comprises RD114-TR, baEV-LV, or BaEV-RLess envelope protein.

5. The method of claim 1, wherein expression or activity of ASCT2 is inhibited by a gene editing technique, an RNA inhibition technique, a homologous recombination technique, or an immunological technique.

6. The method of claim 5, wherein the gene editing technique introduces a mutation in the gene encoding ASCT2 of the genome of the cell that results in a reduced level of expression or reduced activity of the expressed ASCT2 via a gene editing vector.

7. The method of claim 5, wherein the RNA inhibition technique results in a reduced expression level or reduced activity of the expressed ASCT2 by siRNA, antisense RNA and/or ribozyme targeting the gene or transcript encoding ASCT 2.

8. The method of claim 5, wherein the homologous recombination technique knocks out the gene encoding ASCT2 in the genome by homologous recombination vectors or homologous recombines a nucleic acid sequence encoding an inactive or reduced activity ASCT2 variant into the genome to replace the wild-type ASCT2 coding sequence.

9. The method of claim 5, wherein the immunological technique reduces ASCT2 activity by expressing, secreting, or contacting cells with an antibody or antigen binding fragment thereof of ASCT 2.

10. The method of claim 1, wherein the cell comprises, expresses, secretes, or is contacted with an agent that down-regulates ASCT2 expression levels or activity.

11. The method of claim 1, wherein the gene encoding ASCT2 protein of the cell has a mutation that reduces expression or activity of ASCT 2.

12. The method of claim 1, wherein the cells have sgrnas transferred therein, resulting in reduced expression levels or reduced activity of expressed ASCT 2.

13. The method according to claim 1, characterized in that it comprises the steps of:

(1) Transfecting the cells with a viral vector or virus, and

(2) Harvesting the virus produced by the cells.

14. The method of claim 13, wherein in (1) the viral vector comprises a coding sequence for an envelope protein of one or more viruses selected from the group consisting of baboon endogenous retrovirus, feline endogenous retrovirus, vesicular stomatitis virus.

15. The method of claim 13, wherein in (1) the viral vector comprises a coding sequence for one or more envelope proteins selected from the group consisting of VSV-G, baEV and RD114, or the virus comprises one or more envelope proteins selected from the group consisting of VSV-G, baEV and RD114.

16. The method of claim 13, wherein the viral vector is a lentiviral packaging plasmid.

17. The method of claim 13, further comprising the step of inhibiting expression or activity of ASCT2 in said cell.

18. The method of claim 17, wherein the expression or activity of ASCT2 is inhibited by gene editing techniques, homologous recombination techniques, RNA inhibition techniques, or immunological techniques.

19. A method of making an immune effector cell comprising:

1) The method of any one of claim 1 to 18 for producing a virus,

2) Transfecting immune effector cells with the virus obtained in 1), and

3) Harvesting transfected immune effector cells, wherein the virus is a lentivirus or a pseudotyped virus thereof.

20. The method of claim 19, wherein the virus in step 2) is harvested 48 hours or more after transfection of cells in which ASCT2 expression or activity is inhibited in the method of step 1).

21. The method of claim 19, wherein the virus in step 2) is diluted.

22. The method of claim 21, wherein the dilution is a 1:50, 1:100, 1:200, or 1:500 dilution.

23. The method of claim 19, wherein the virus comprises a nucleic acid sequence of interest.

24. The method of claim 23, wherein the nucleic acid sequence comprises a coding sequence for a chimeric antigen receptor, optionally further comprising a coding sequence for a cytokine.

25. The method of claim 19, wherein the immune effector cells comprise T cells, TCR-T, CAR-T, TIL, DC-CIK, NK cells.

Use of a cell in which expression or activity of asct2 is inhibited in the manufacture of a medicament for the treatment of lymphoma, wherein the cell is a HEK293 cell.

27. The use of claim 26, wherein expression or activity of ASCT2 is inhibited by gene editing techniques, RNA inhibition techniques, homologous recombination techniques, or immunological techniques.

28. The use according to claim 27, wherein the gene editing technique introduces a mutation in the gene encoding ASCT2 of the genome of the cell that results in a reduced level of expression or reduced activity of the expressed ASCT2 by means of a gene editing vector.

29. The use according to claim 27, wherein the RNA inhibition technique results in reduced expression levels or reduced activity of expressed ASCT2 by siRNA, antisense RNA and/or ribozymes targeting the gene or transcript encoding ASCT 2.

30. Use according to claim 27, wherein the homologous recombination technique knocks out the gene encoding ASCT2 in the genome by means of a homologous recombination vector or homologous recombines a nucleic acid sequence encoding an inactive or reduced activity ASCT2 variant into the genome to replace the wild type ASCT2 coding sequence.

31. The use of claim 27, wherein the immunological technique reduces ASCT2 activity by allowing the cells to express, secrete or contact an antibody or antigen binding fragment thereof to ASCT 2.

32. The use of claim 26, wherein the cell comprises, expresses, secretes or is contacted with an agent which down-regulates ASCT2 expression levels or activity.

33. The use according to claim 26, wherein the gene encoding ASCT2 protein of the cell has a mutation that reduces expression or activity of ASCT 2.

34. The use of claim 26, wherein the cells have sgrnas transferred therein.