CN117089578A - Preparation method of CAR-NK from peripheral blood - Google Patents

Abstract

The invention provides a preparation method of EGFR and EGFRvIII targeted CAR-NK cells. In particular, the invention provides a viral packaging system and methods for preparing EGFR and egfrvlll targeted CAR-NK cells thereof. The transfection efficiency of the virus vector prepared by the virus packaging system on NK cells is higher, and the preparation efficiency of the NK cells can be increased. The invention also provides the CAR-NK cells prepared by the method and application thereof.

Description

Preparation method of CAR-NK from peripheral blood

Technical Field

The invention relates to the field of biotechnology, in particular to a preparation method of CAR-NK cells from peripheral blood.

Background

Natural killer cells (natural killer cell, NK) are an important class of immune cells in the body, are an important component of the innate immune system (Innate immune system), and are one of the important components of the first line of defense against pathogenic invasion and malignant tumors in the human body. NK cell killing target cells are antigen non-specific, without Major Histocompatibility Complex (MHC) restriction, allogeneic NK cell transplantation does not elicit an anti-host response (GVHD). NK cells are widely available, such as NK tumor cell lines (NK-92, NKL, etc.), peripheral blood NK cells (PB-NK), umbilical cord blood NK cells (UCB-NK), stem cell-induced differentiation NK cells (hematopoietic stem cells, induced pluripotent stem cells). In normal human peripheral blood, NK cells account for about 5% -15% of circulating lymphocytes. NK cells from peripheral blood can be prepared in a large quantity after being cultured and amplified, so that the clinical application of large dosage is satisfied.

Chimeric antigen receptor (Chimeric antigen receptor, CAR) T cells have made breakthrough progress in the treatment of drug-resistant, relapsed, refractory B-cell tumors, such as leukemia and lymphoma, but the therapeutic effect of CAR-T therapy in solid tumors is not as pronounced as in the treatment of hematological tumors. Based on the tumor killing capability of NK cells, the tumor targeting and cracking killing capability of NK cells can be further enhanced by utilizing a mature CAR technology to modify the NK cells, and the research of the CAR-NK has great clinical significance.

Epidermal growth factor receptor (Epidermal Growth Factor Receptor, EGFR) is a member of the subfamily of tyrosine kinases, which induces EGFR homodimerization or heterodimerization by binding to its ligands, promoting autophosphorylation of its tyrosine kinase domain, further activating downstream signaling pathways (e.g., PI3K/AKT signaling pathway, ras/Raf/MEK/ERK signaling pathway), and performing its biological functions. EGFR is overexpressed in many tumors, including bladder, brain, head and neck, pancreas, lung, breast, ovary, colon, prostate, and kidney cancers, and EGFR is also expressed in normal tissues, with higher levels of expression in skin, liver, and gastrointestinal epithelial tissues, but at far lower levels than in tumor tissues. Approximately 30% of glioblastomas carry epidermal growth factor receptor mutations (Epidermal growth factor receptor variant III, EGFRvIII), which are tumor-specific mutations resulting from mutations in the wild-type EGFR (Wildtype EGF, EGFR or wtEGFR) gene, which are tumor neoantigens (neo-antigens), which are not only very immunogenic and prone to activate immune responses, but also are not expressed by normal cells and tissues. Therefore, the development of the CAR-NK cell capable of simultaneously targeting EGFR and EGFRvIII has a very deep clinical application prospect in the field of treatment of related solid tumors.

The current clinical test of the CAR-NK cells mostly uses an NK92 cell line, can be continuously subcultured and is easy to transfect. However, NK92 cells as a tumor cell line have inherent drawbacks including potential tumorigenic risk, etc., and are therefore not optimal cell sources.

PBMC are an important source of primary NK cells, with strong cytotoxicity, but weaker proliferation capacity and harder transfection.

For NK cells, especially peripheral blood derived NK cells, the preparation efficiency of CAR-NK is far lower than that of CAR-T due to the characteristic of difficult transfection, which also limits the clinical application of CAR-NK. Therefore, the development of the efficient preparation method of the CAR-NK cells has higher clinical value.

Thus, there is an urgent need in the art to develop new methods of preparing EGFR-targeted peripheral blood-derived CAR-NK cells.

Disclosure of Invention

The invention aims to provide a preparation method of EGFR-targeting CAR-NK cells.

Specifically, the invention provides a CAR-NK cell virus packaging system and a recombinant virus vector for preparing EGFR-targeting, and a method for preparing the CAR-NK cell targeting the EGFR.

In a first aspect of the present invention, there is provided a viral packaging system comprising a gene expression plasmid of interest, a viral envelope protein plasmid and a helper packaging plasmid,

Wherein the target gene expression plasmid is selected from the group consisting of: pMSCV, pMX, pMY, pLenti, or a combination thereof, and the gene expression plasmid of interest expresses a Chimeric Antigen Receptor (CAR) targeting EGFR/egfrvlll or a fusion protein containing the chimeric antigen receptor.

In another preferred embodiment, the EGFR/EGFRvIII targeting CAR has the amino acid sequence shown in SEQ ID NO. 1.

In another preferred embodiment, the chimeric antigen receptor-containing fusion protein further comprises a fusion protein (mb 15) encoding the cytokine IL-15 and its receptor IL-15 Ra.

In another preferred embodiment, the amino acid sequence of mb15 is shown in SEQ ID NO. 4.

In another preferred embodiment, the gene expression plasmid of interest expresses a fusion protein of CAR and mb15 targeting EGFR/egfrvlll, preferably wherein CAR and mb15 are linked by a T2A short peptide.

In another preferred embodiment, the EGFR/EGFRvIII targeting CAR and mb15 fusion protein has the amino acid sequence shown in SEQ ID NO. 5.

In another preferred embodiment, the expression plasmid of the target gene is a 5' LTR promoter self-inactivating pLenti, and it contains a promoter driving the expression of the target gene.

In another preferred embodiment, the promoter is selected from the group consisting of: enCMV, short EF1a, mPGK, MSCV, UCOE-SFFV, or combinations thereof.

In another preferred embodiment, the viral envelope protein plasmid is selected from the group consisting of: pCMV-Vsg, pCMV-RD114A, pCMV-BaEV-TR, pCMV-Ampho, pCMV-10A1, or combinations thereof.

In another preferred embodiment, the helper packaging plasmid is selected from the group consisting of: psPAX2, pCMV-gag-pol, or a combination thereof.

In another preferred embodiment, the viral packaging system is a lentiviral packaging system or a retroviral packaging system.

In another preferred embodiment, the viral packaging system is a lentiviral packaging system, and the gene expression plasmid of interest is a 5' LTR promoter self-inactivating pLenti; the auxiliary packaging plasmid is psPAX2.

In another preferred embodiment, said pLenti comprises a promoter driving expression of said nucleic acid sequence, preferably said promoter is EnCMV.

In another preferred embodiment, the viral packaging system is a retroviral packaging system and the gene expression plasmid of interest is selected from the group consisting of: pMX, pMY, pMSCV, pBabe, or a combination thereof; the auxiliary packaging plasmid is pCMV-gag-pol.

In another preferred embodiment, the viral packaging system is a retroviral packaging system and the gene expression plasmid of interest is selected from the group consisting of: pMSCV, pMX, or a combination thereof;

The auxiliary packaging plasmid is pCMV-gag-pol;

the viral envelope protein plasmid is selected from the group consisting of: pCMV-RD114A, pCMV-BaEV-TR, or a combination thereof.

In another preferred embodiment, in the virus packaging system, the gene expression plasmid of interest: viral envelope protein plasmid: the ratio of the auxiliary packaging plasmids is 1-10:1-10:1:10, preferably 1-5:1-5, more preferably 1-2:1-2, and most preferably 1:1:1.

In another preferred embodiment, the viral packaging system is used for packaging recombinant viral vectors for transfection of NK cells.

In another preferred embodiment, the NK cells are peripheral blood derived NK cells.

In another preferred embodiment, the NK cells are NK cells derived from peripheral blood of healthy people.

In a second aspect of the present invention, there is provided a method for preparing a recombinant viral vector comprising the steps of:

(a) Providing a packaging cell;

(b) Transfecting said packaging cell with a viral packaging system according to the first aspect of the invention;

(c) Culturing the transfected packaging cells, thereby collecting the recombinant viral vector.

In another preferred embodiment, the packaging cell is selected from the group consisting of: 293T cells, 293F cells, 293FT cells, HEK293 cells.

In another preferred embodiment, in step (b) the packaging cells are transfected with a combination of transfection reagents, preferably PEI-MAX, more preferably in an amount of 1-10 times (preferably 1-5 times, most preferably 3 times) the total mass of plasmids in the viral packaging system.

In another preferred embodiment, in step (c), the recombinant viral vector is obtained by culturing the transfected packaging cells 48 hours later and collecting the viral supernatant for the first time and 72 hours later and collecting the viral supernatant for the second time.

In another preferred embodiment, step (c) further comprises the step of concentrating the collected viral supernatant by filtration.

In a third aspect of the invention there is provided a recombinant viral vector expressing an EGFR/egfrvlll targeted CAR, said recombinant viral vector being prepared using the method of the second aspect of the invention.

In another preferred embodiment, the recombinant viral vector is used to prepare EGFR/EGFRvIII targeted CAR-NK cells.

In another preferred embodiment, the NK cells are peripheral blood derived NK cells.

In another preferred embodiment, the NK cells are NK cells derived from peripheral blood of healthy people.

In a fourth aspect of the invention, there is provided a method of preparing EGFR/egfrvlll-targeted CAR-NK cells comprising the steps of:

(S1) providing NK cells to be transfected;

(S2) transfecting said NK cells with the recombinant viral vector according to the third aspect of the present invention, thereby obtaining said EGFR/egfrvlll targeted CAR-NK cells.

In another preferred embodiment, in step (S2), the multiplicity of viral infection (MOI) is 1 to 20, preferably 5 to 15, more preferably 8 to 12, most preferably moi=10.

In another preferred embodiment, the CAR-NK cells prepared by the method have a CAR expression rate of 50% or more, preferably 60% or more, more preferably 70% or more.

In a fifth aspect of the invention there is provided a CAR-NK cell prepared according to the method of the fourth aspect of the invention.

In another preferred embodiment, the CAR-NK cells express a CAR043-mb15 fusion protein, the amino acid sequence of which is shown in SEQ ID NO. 5.

In another preferred embodiment, the NK cells are peripheral blood derived NK cells.

In another preferred embodiment, the NK cells are NK cells derived from peripheral blood of healthy people.

In a sixth aspect of the invention, there is provided a formulation comprising a CAR-NK cell according to the fifth aspect of the invention, and a pharmaceutically acceptable carrier.

In a seventh aspect of the invention there is provided the use of a CAR-NK cell as described in the fifth aspect of the invention, or a formulation as described in the sixth aspect of the invention, for the manufacture of a medicament or formulation for the prophylaxis and/or treatment of cancer or tumour.

In another preferred embodiment, the tumor is selected from the group consisting of: hematological tumors, solid tumors, or combinations thereof.

In another preferred embodiment, the hematological neoplasm is selected from the group consisting of: acute Myelogenous Leukemia (AML), multiple Myeloma (MM), chronic Lymphocytic Leukemia (CLL), acute Lymphoblastic Leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), or combinations thereof.

In another preferred embodiment, the solid tumor is selected from the group consisting of: bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, colon cancer, prostate cancer, kidney cancer, stomach cancer, peritoneal metastasis of stomach cancer, liver cancer, small intestine cancer, bone cancer, rectal cancer, large intestine cancer, cervical cancer, lymphatic cancer, nasopharyngeal cancer, adrenal tumor, brain glioma, endometrial cancer, or a combination thereof.

In another preferred embodiment, the tumor is a tumor that highly expresses EGFR and/or EGFRvIII.

In another preferred embodiment, the high expression means that the ratio of EGFR and/or EGFRvIII expression level (F1) of the tumor cells to physiological state expression level (F0) of the healthy subject (i.e. F1/F0) is not less than 1.5, preferably not less than 2, more preferably not less than 2.5.

In another preferred embodiment, the medicament or formulation is administered to a subject in need thereof, preferably a mammal, more preferably a human.

In an eighth aspect of the invention there is provided a method of treating a disease comprising administering to a subject in need thereof an amount of a vector according to the third aspect of the invention, a CAR-NK cell according to the fifth aspect of the invention, or a formulation according to the sixth aspect of the invention, or a medicament containing the same.

In another preferred embodiment, the disease is cancer or tumor.

In another preferred embodiment, the CAR-NK cells have an uptake of (0.5-5). Times.10 ⁹ Cells/time.

In another preferred embodiment, the method of ingestion of the CAR-NK cell-containing drug is intratumoral injection, intravenous injection, intrathoracic injection, or local intervention.

In a specific embodiment of the invention, the method of ingestion of the CAR-NK cell-containing drug is intratumoral injection.

In another preferred embodiment, the cancer is a tumor and related diseases that highly express EGFR and/or EGFRvIII.

In another preferred embodiment, the cancer is bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, colon cancer, prostate cancer, and kidney cancer; preferably, the cancer is glioblastoma.

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

FIG. 1a shows the results of a flow assay for NK cell expansion using peripheral blood derived mononuclear cells (PBMC);

FIG. 1b is a schematic diagram showing a process for preparing CAR-NK using virus infected peripheral blood derived NK cells;

FIG. 2a is a schematic diagram of a lentiviral vector expressing a CAR gene using different promoters;

FIG. 2b is a schematic diagram of several retroviral vectors differing in the 5' LTR promoter region;

FIG. 3 shows the results of a flow assay for preparing CAR-NK using lentiviral vectors containing different promoters and pMSCV retroviral vectors;

FIG. 4 shows the results of a flow assay for preparing CAR-NK using retrovirus packaged with different envelope proteins;

FIG. 5 shows the results of a flow assay for preparing CAR-NK using viruses packaged with different retroviral vectors;

FIG. 6a is a schematic representation of a retroviral vector and CAR-NK simultaneously expressing EGFR/EGFRvIII targeting chimeric antigen receptor and IL-15/IL-15Ra fusion protein;

FIG. 6b shows the results of flow assays for NK cell surface CAR and IL-15 at various time points;

FIG. 7 shows the results of the detection of the killing ability of CAR-NK expressing EGFR/EGFRvIII targeting chimeric antigen receptor and IL-15/IL-15Ra fusion protein simultaneously to different tumor cells.

Detailed Description

The present inventors have studied extensively and intensively, and have unexpectedly found a CAR-NK cell virus packaging system for preparing EGFR-targeting. Specifically, the optimized virus packaging system is obtained through screening, a specific three-plasmid combination system is used, and the recombinant virus vector obtained through packaging can be used for efficiently transfecting NK cells from peripheral blood and stably expressing the CAR in the NK cells for a long time. The present application has been completed on the basis of this finding.

Terminology

In order that the present disclosure may be more readily understood, certain terms are first defined. As used in the present application, each of the following terms shall have the meanings given below, unless explicitly specified otherwise herein. Other definitions are set forth throughout the application.

The term "about" may refer to a value or composition that is within an acceptable error of a particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or measured.

The term "optionally" or "optionally" means that the subsequently described event or circumstance may occur, but need not occur.

The term "comprising" or "includes" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of …" or "consisting of …".

The terms "EGFR/egfrvlll-targeting CAR", "CAR043" are used interchangeably, and each refer to a chimeric antigen receptor capable of specifically binding both EGFR and egfrvlll. In a preferred embodiment of the invention, the sequence of the CAR is shown in SEQ ID No. 1.

"transduction," "transfection," "transformation," or other similar terms as used herein refer to the process of transferring an exogenous polynucleotide into a host cell, and transcription and translation to produce a polypeptide product, including the use of plasmid molecules to introduce the exogenous polynucleotide into the host cell (e.g., E.coli).

"Gene expression" or "expression" refers to the process by which a gene is transcribed, translated, and post-translationally modified to produce an RNA or protein product of the gene.

The term "administering" refers to physically introducing a product of the invention into a subject using any of a variety of methods and delivery systems known to those of skill in the art, including intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, e.g., by injection or infusion.

It should be understood that, in this document, the amino acid names are identified by international single english letters, and the amino acid names corresponding to the amino acid names are abbreviated as "three english letters: ala (A), arg (R), asn (N), asp (D), cys (C), gln (Q), glu (E), gly (G), his (H), I1E (I), leu (L), lys (K), met (M), phe (F), pro (P), ser (S), thr (T), trp (W), tyr (Y), val (V).

It should be understood that for any method described herein that includes more than one step, the order of the steps is not necessarily limited to the order described in these embodiments.

Chimeric Antigen Receptor (CAR) and CAR-NK cells

In the present invention, "chimeric antigen receptor targeting EGFR/egfrvlll", "EGFR/egfrvlll-targeting CAR", "CAR of the invention" or similar terms are used interchangeably, all referring to chimeric antigen receptor capable of specifically binding EGFR and egfrvlll.

Chimeric immune antigen receptor (Chimeric antigen receptor, CAR) consists of extracellular antigen recognition region, transmembrane region and intracellular co-stimulatory signaling region.

CAR-immune cell therapy achieves a very high clinical response rate in hematological malignancy therapy, which is not achieved by any previous therapeutic means. Based on the tumor killing capability of NK cells, the tumor targeting and cracking killing capability of NK cells can be further enhanced by utilizing a mature CAR technology to modify the NK cells.

The Chimeric Antigen Receptor (CAR) of the invention includes an extracellular domain, a transmembrane domain, and an intracellular domain. Extracellular domains include target-specific binding elements (also referred to as antigen binding domains). The intracellular domain includes a costimulatory signaling region and a zeta chain moiety. A costimulatory signaling region refers to a portion of an intracellular domain that comprises a costimulatory molecule. Costimulatory molecules are cell surface molecules that are required for the efficient response of lymphocytes to antigens, rather than antigen receptors or their ligands.

In a preferred embodiment of the invention, the extracellular domain of the CAR provided by the invention comprises an antigen binding domain that targets EGFR and egfrvlll simultaneously. The CARs of the invention, when expressed in NK cells, are capable of antigen recognition based on antigen binding specificity. When it binds to its cognate antigen, affects tumor cells, causes tumor cells to not grow, to be caused to die or to be otherwise affected, and causes the patient's tumor burden to shrink or eliminate. The antigen binding domain is preferably fused to an intracellular domain from one or more of the costimulatory molecule and zeta chain. Preferably, the antigen binding domain is fused to an intracellular domain of a combination of a co-stimulatory signaling molecule of CD28 and/or 4-1BB origin, and a cd3ζ signaling domain.

As used herein, "antigen binding domain" refers to Fab fragments, fab 'fragments, F (ab') ₂ Fragments, or single Fv fragments. In a preferred embodiment of the invention, the antigen binding domain comprises an scFv that specifically recognizes EGFR and egfrvlll.

For hinge and transmembrane regions (transmembrane domains), the CAR may be designed to include a transmembrane domain fused to the extracellular domain of the CAR. In one embodiment, a transmembrane domain is used that naturally associates with one of the domains in the CAR. In some examples, the transmembrane domain may be selected, or modified by amino acid substitutions, to avoid binding such domain to the transmembrane domain of the same or a different surface membrane protein, thereby minimizing interactions with other members of the receptor complex.

The invention also provides EGFR and EGFRvIII targeted CAR-NK cells.

Cytokine IL-15

IL-15 is a pleiotropic cytokine that plays a central role in NK, T and B cell development, survival and activation. IL-15 not only increases NK cell and T cell numbers, but also activates NK cells and cytotoxic CD8+ T cells, thereby killing tumors and other pathogens.

Cytokine IL-15 receptor IL-15Ra (CD 215)

IL-15 receptor comprises three subunits, alpha (CD 215), beta (CD 122), gamma (CD 132), wherein the alpha subunit is independent of the beta, gamma subunits. IL-15 binds to a high affinity alpha receptor expressed on antigen presenting cells, and IL-15 is presented to beta/gamma dimers to form quaternary complexes, which activate JAK and STAT model pathways, and regulate proliferation and activation of immune cells such as T, NK.

Carrier body

Nucleic acid sequences encoding a desired molecule can be obtained using recombinant methods known in the art, such as, for example, by screening libraries from cells expressing the gene, by obtaining the gene from vectors known to include the gene, or by direct isolation from cells and tissues containing the gene using standard techniques. Alternatively, the gene of interest may be produced synthetically.

The present invention provides viral plasmid vectors for use in gene therapy. Retroviral and/or lentiviral derived vectors are suitable tools for achieving long term gene transfer because they allow long term, stable integration of the transgene and its proliferation in daughter cells.

In the present invention, the term "lentivirus" refers to a gene therapy vector developed based on HIV-1 (human immunodeficiency type I virus). A distinction is made between a general retrovirus, which has the ability to infect both dividing cells and non-dividing cells.

In the present invention, the term "retrovirus" refers to a virus packaged using a retroviral vector, which transfers a gene of interest to a target cell with high efficiency using a unique life cycle of the retrovirus. Retroviruses generally infect only dividing cells.

In brief summary, a CAR of the invention or a nucleic acid sequence thereof is typically operably linked to a promoter and incorporated into an expression vector. The vector is suitable for replication and integration of eukaryotic cells. Typical cloning vectors contain transcriptional and translational terminators, initiation sequences, and promoters useful for regulating expression of the desired nucleic acid sequence.

Further, the expression vector may be provided to the cell in the form of a viral vector. Viral vector techniques are well known in the art and are described, for example, in Sambrook et al (2001,Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Laboratory,New York) and other virology and molecular biology manuals. Viruses that may be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. In general, suitable vectors include an origin of replication, a promoter sequence, a convenient restriction enzyme site, and one or more selectable markers that function in at least one organism (e.g., WO01/96584; WO01/29058; and U.S. Pat. No. 6,326,193).

Many virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. Selected genes can be inserted into vectors and packaged into retroviral particles using techniques known in the art. The recombinant virus may then be isolated and delivered to a subject cell in vivo or ex vivo. Many retroviral systems are known in the art. In some embodiments, an adenovirus vector is used. Many adenoviral vectors are known in the art. In one embodiment, a lentiviral vector is used.

Additional promoter elements, such as enhancers, may regulate the frequency of transcription initiation. Typically, these are located in the 30-110bp region upstream of the start site, although many promoters have recently been shown to also contain functional elements downstream of the start site. The spacing between promoter elements is often flexible so as to maintain promoter function when the elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased by 50bp before the activity begins to decrease. Depending on the promoter, it appears that individual elements may act cooperatively or independently to initiate transcription.

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 ebustan-balr (Epstein-Barr) virus immediate early promoter, the ruses sarcoma virus promoter, and human gene promoters such as but not limited to the actin promoter, myosin promoter, heme promoter, and creatine kinase promoter. Further, the invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present invention. 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 such expression is desired, or switching off expression when expression is undesired. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

To assess expression of the CAR polypeptide or portion thereof, the expression vector introduced into the cell may also comprise either or both a selectable marker gene or a reporter gene to facilitate identification and selection of the expressing cell from a population of cells sought to be transfected or infected by the viral vector. In other aspects, the selectable marker may be carried on a single piece of DNA and used in a co-transfection procedure. Both the selectable marker and the reporter gene may be flanked by appropriate regulatory sequences to enable expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes, such as neo and the like.

The reporter gene is used to identify potentially transfected cells and to evaluate the functionality of the regulatory sequences. Typically, the reporter gene is the following gene: which is not present in or expressed by the recipient organism or tissue and which encodes a polypeptide whose expression is clearly indicated by some readily detectable property, such as enzymatic activity. After the DNA has been introduced into the recipient cell, the expression of the reporter gene is assayed at the appropriate time. Suitable reporter genes may include genes encoding luciferases, beta-galactosidases, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or green fluorescent protein (e.g., ui-Tei et al 2000FEBS Letters479:79-82). Suitable expression systems are well known and can be prepared using known techniques or commercially available. Typically, constructs with a minimum of 5 flanking regions that show the highest level of reporter gene expression are identified as promoters. Such promoter regions can be linked to reporter genes and used to evaluate agents for their ability to regulate promoter-driven transcription.

Methods for introducing genes into cells and expressing genes into cells are known in the art. In the context of expression vectors, the vector may be readily introduced into a host cell, e.g., a mammalian, bacterial, yeast or insect cell, by any method known in the art. For example, the expression vector may be transferred into the host cell by physical, chemical or biological means.

In a preferred embodiment of the invention, the vector is a retroviral vector.

Virus packaging system

The present invention provides a viral packaging system for use in preparing a recombinant viral vector expressing an EGFR/egfrvlll targeted CAR.

The virus packaging system of the invention is a three-plasmid virus packaging system, comprising target gene expression plasmid, auxiliary packaging plasmid and virus envelope protein plasmid. The target gene expression plasmid is a transfer plasmid encoding a target protein or transcribing a target gene, both sides of which contain long terminal repeats (LTR, long terminal repeat), which assist in the insertion of the target gene into the genome of the host cell. The helper packaging plasmid encodes genes for structural proteins associated with helper virus packaging, such as gag, pol, etc. Viral envelope protein plasmids encode the genes of the viral protein coat, and different viruses have different infectivity due to different envelope proteins.

The viral packaging system of the present invention is preferably a lentiviral packaging system or a retroviral packaging system.

The third generation lentiviral packaging vector is optimized, and the promoter of the 5' LTR is self-inactivated, so that the expression of the exogenous gene can be flexibly driven by different promoters. The different promoters have different capacities for controlling gene expression, and the length of the sequence of the promoter can influence the size of a viral vector, thereby influencing the efficiency of viral packaging.

In a preferred embodiment of the present invention, the lentiviral packaging vector (i.e., the gene expression plasmid of interest) used is a pLenti vector whose promoter of the 5' LTR has been self-inactivated. Preferably, the lentiviral packaging vector has a promoter introduced to drive gene expression of the CAR of the invention, the promoter being selected from the group consisting of EnCMV, short EF1a, mPGK, MSCV and UCOE-SFFV, preferably the promoter is EnCMV.

A transcription virus gene expression system is another very efficient system that can stably integrate exogenous genes into the genome of mammalian cells. The 5' LTR of a retroviral vector contains a very strong promoter that drives high levels of expression of the gene of interest. In retroviral expression systems, expression of the gene of interest is dependent on the promoter region of the 5' LTR, and the efficiency of gene transduction mediated by different retroviral expression vectors varies. For example, pMX, pBabe vectors based on Moloney Murine Leukemia Virus (MMLV) have high-efficiency gene expression capacity, but can be silenced in immature cells, and further engineered pMY vectors are more suitable for exogenous gene expression in hematopoietic stem cells; pMSCV vectors based on Murine Stem Cell Virus (MSCV) are capable of expressing genes of interest in large amounts in embryonic stem cells, embryonic cancer cells, hematopoietic stem cells and other mammalian cell lines.

In a preferred embodiment of the present invention, the gene expression plasmid of interest used is a retroviral pMX, pMY, or pMSCV vector. Preferably, the target gene expression plasmid is pMX or pMSCV.

When a retrovirus infects a host cell, the interaction of envelope proteins on the surface of the virus and corresponding receptors expressed by host cell membranes is mainly relied on to mediate the fusion of the viral envelope and the host cell membranes, so that the tropism of the packaged retrovirus, namely the preference of the infection of different host cells, can be changed by using different envelope proteins in a retrovirus packaging system.

In a preferred embodiment of the invention, the viral envelope protein plasmid used is a pCMV-BaEV-TR vector or a pCMV-RD114A vector.

Formulations

The invention provides a preparation containing the CAR-NK cells and a pharmaceutically acceptable carrier. In one embodiment, the formulation is a liquid formulation. Preferably, the formulation is an injection. Preferably, the concentration of said CAR-NK cells in said formulation is 1 x 10 ³ -1×10 ⁸ Individual cells/ml, more preferably 1X 10 ⁴ -1×10 ⁷ Individual cells/ml.

In one embodiment, the formulation may include a buffer such as neutral buffered saline, sulfate buffered saline, or the like; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; a protein; polypeptides or amino acids such as glycine; an antioxidant; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and a preservative. The formulations of the present invention are preferably formulated for intravenous administration.

Therapeutic applications

The invention includes therapeutic applications of NK cells (e.g., peripheral blood derived NK cells) transduced with recombinant viral vectors packaged using the viral packaging systems of the invention. The transduced NK cells can target the tumor cell markers EGFR/EGFRvIII, synergistically activate the NK cells and cause the NK cell immune response, thereby obviously improving the killing efficiency of the NK cells on the tumor cells.

Accordingly, the present invention also provides a method of stimulating an NK cell-mediated immune response to a target cell population or tissue of a mammal comprising the steps of: administering the CAR-NK cells of the invention to a mammal.

In one embodiment, the invention includes a class of cell therapies in which autologous NK cells (or heterologous donors) from a patient are isolated, activated and genetically engineered to produce CAR-NK cells, and subsequently injected into the same patient. This way the probability of graft versus host disease is very low and the antigen is recognized by NK cells in a non-MHC restricted manner. Furthermore, one CAR-NK can treat all cancers that express this antigen. Unlike antibody therapies, CAR-NK cells are able to replicate in vivo, producing long-term persistence that can lead to persistent tumor control.

In one embodiment, the CAR-NK cells of the invention can undergo robust in vivo NK cell expansion and can last for an extended amount of time. Additionally, the CAR-mediated immune response can be part of an adoptive immunotherapy step in which the CAR-modified NK cells induce an immune response specific for the antigen binding domain in the CAR. For example, CAR-NK cells against EGFR/egfrvlll elicit a specific immune response against EGFR/egfrvlll expressing cells.

Treatable cancers include tumors that are not vascularized or have not been substantially vascularized, as well as vascularized tumors. Cancers may include non-solid tumors (such as hematological tumors, e.g., leukemia and lymphoma) or may include solid tumors. Types of cancers treated with the CAR-NK of the invention include, but are not limited to, carcinomas, blastomas and sarcomas, and certain leukemia or lymphoid malignancies, benign and malignant tumors, such as sarcomas, carcinomas and melanomas. Adult tumors/cancers and pediatric tumors/cancers are also included.

Hematological cancers are cancers of the blood or bone marrow. Examples of hematologic (or hematogenic) cancers include leukemias, including acute leukemias (such as acute lymphoblastic leukemia, acute myelogenous leukemia and myeloblastic, promyelocytic, granulo-monocytic, monocytic and erythroleukemia), chronic leukemias (such as chronic myelogenous (myelogenous) leukemia, chronic myelogenous leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphomas, hodgkin's disease, non-hodgkin's lymphomas (indolent and high grade forms), multiple myelomas, waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia.

Solid tumors are abnormal masses of tissue that do not normally contain cysts or fluid areas. Solid tumors may be benign or malignant. Different types of solid tumors are named for the cell type that they are formed of (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumors such as sarcomas and carcinomas include fibrosarcoma, myxosarcoma, liposarcoma mesothelioma, lymphoid malignancies, pancreatic cancer, ovarian cancer.

The CAR-modified NK cells of the invention can also be used as a vaccine type for ex vivo immunization and/or in vivo therapy of mammals. Preferably, the mammal is a human.

For ex vivo immunization, at least one of the following occurs in vitro prior to administration of the cells into a mammal: i) Expanding the cells, ii) introducing nucleic acid encoding the CAR into the cells, and/or iii) cryopreserving the cells.

Ex vivo procedures are well known in the art and are discussed more fully below. Briefly, cells are isolated from a mammal (preferably a human) and genetically modified (i.e., transduced or transfected in vitro) with vectors expressing the CARs disclosed herein. The CAR-modified cells can be administered to a mammalian recipient to provide a therapeutic benefit. The mammalian recipient can be a human, and the CAR-modified cells can be autologous with respect to the recipient. Alternatively, the cell may be allogeneic, syngeneic (syngeneic) or xenogeneic with respect to the recipient.

In addition to the use of cell-based vaccines for ex vivo immunization, the present invention also provides compositions and methods for in vivo immunization to elicit an immune response against an antigen in a patient.

The invention provides a method of treating a tumor comprising administering to a subject in need thereof a therapeutically effective amount of a CAR-modified NK cell of the invention.

The CAR-modified NK cells of the invention can be administered alone or as a pharmaceutical composition in combination with diluents and/or with other components such as IL-2, IL-17 or other cytokines or cell populations. Briefly, the pharmaceutical compositions of the invention may comprise a target cell population as described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may include buffers such as neutral buffered saline, sulfate buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; a protein; polypeptides or amino acids such as glycine; an antioxidant; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and a preservative. The compositions of the present invention are preferably formulated for intravenous administration.

The pharmaceutical composition of the present invention may be administered in a manner suitable for the disease to be treated (or prevented). The number and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient's disease-although the appropriate dosage may be determined by clinical trials.

When it is indicated that "immunologically there isThe precise amount of the composition of the present invention to be administered, when an effective amount "," an antitumor effective amount "," a tumor-inhibiting effective amount "or" therapeutic amount "is determined by a physician, taking into account the age, weight, tumor size, degree of infection or metastasis and individual differences of the condition of the patient (subject). It can be generally stated that: pharmaceutical compositions comprising NK cells described herein can be 10 ⁴ To 10 ¹⁰ A dose of individual cells/kg body weight, preferably 10 ⁵ To 10 ⁶ Individual cells/kg body weight doses (including all integer values within those ranges) are administered. NK cell compositions can 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.

Administration of the subject compositions may be performed in any convenient manner, including by spraying, injection, swallowing, infusion, implantation, or transplantation. The compositions described herein may be administered to a patient subcutaneously, intradermally, intratumorally, intradesmally, intraspinal, intramuscularly, by intravenous (i.v.) injection or intraperitoneally. In one embodiment, the NK cell composition of the invention is administered to a patient by intradermal or subcutaneous injection. In another embodiment, the NK cell composition of the invention is preferably administered by i.v. injection. The composition of NK cells can be injected directly into tumors, lymph nodes or the site of infection.

In certain embodiments of the invention, cells activated and expanded using the methods described herein or other methods known in the art for expanding NK cells to therapeutic levels are administered to a patient in combination (e.g., before, simultaneously with, or after) any number of relevant therapeutic modalities, including, but not limited to, treatment with: such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C) or natalizumab therapy for MS patients or ertapelizumab therapy for psoriasis patients or other therapy for PML patients. In a further embodiment, the NK cells of the invention may be used in combination with: chemotherapy, radiation, immunosuppressives such as cyclosporine, azathioprine, methotrexate, mycophenolate and FK506, antibodies or other immunotherapeutic agents. In further embodiments, the cell compositions of the invention are administered to a patient in combination (e.g., before, simultaneously or after) with bone marrow transplantation, using a chemotherapeutic agent such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide. For example, in one embodiment, the subject may undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation. In some embodiments, the subject receives injection of expanded immune cells of the invention after transplantation. In an additional embodiment, the expanded cells are administered pre-operatively or post-operatively.

The dose of the above treatments administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment. The dosage ratio administered to humans may be carried out according to accepted practices in the art. Typically, 1X 10 will be administered per treatment or per course of treatment ⁶ Up to 1X 10 ¹⁰ The modified NK cells (e.g., CAR-NK cells) of the present invention are administered to patients by means such as intravenous infusion.

The main advantages of the invention include:

(1) The invention establishes a preparation method of CAR-NK cells from peripheral blood for the first time. The method comprises the amplification of NK cells and the virus efficient transfection of the CAR genes. The CAR-NK cell prepared by the invention can obviously enhance the cracking capability of the NK cell to target cells, and simultaneously, the exogenous gene can be stably expressed in the NK cell for a long time.

(2) The invention discovers for the first time that the gene transfection efficiency of retrovirus in CAR-NK preparation is higher than that of lentivirus, the virus-mediated gene transduction efficiency of different retrovirus expression vector packages is different, and the gene transduction efficiency of pMX and pMSCV vectors is highest. The different envelope proteins are packaged with different gene transduction efficiencies, and the retroviral packaging system using envelope proteins BaEV and RD114 is the most efficient for NK cells.

(3) The invention designs a CAR-NK cell for the first time, and the NK cell expresses a chimeric antigen receptor (CAR 043) targeting EGFR and EGFRvIII and a fusion protein (mb 15) of IL-15 and a receptor IL-15Ra thereof. Compared with non-modified NK cells, the CAR-NK cells have higher tumor killing capacity, and are particularly obvious when the NK cells are not sensitive to tumor cells.

The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental methods, in which specific conditions are not noted in the following examples, are generally conducted under conventional conditions or under conditions recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated.

Example 1 expansion of NK cells Using peripheral blood derived mononuclear cells (PBMC)

(1) Isolation of peripheral blood mononuclear cells

Peripheral blood of a healthy person is collected, and peripheral blood mononuclear cells and plasma are separated by using lymphocyte separation liquid or a medical cell separation tube. After centrifugation, the blood sample was separated into 4 layers, a pale yellow plasma layer, a white annular PBMC layer (buffy coat layer), a separated liquid layer and a red blood cell layer from bottom to bottom. The PBMC layer was pipetted into another centrifuge tube and the cells were washed 2 times with isotonic solution (PBS, physiological saline) (room temperature, horizontal rotor 300g, centrifugation 10 min) to obtain peripheral blood mononuclear cell PBMC, which were counted.

(2) NK cell expansion

PBMC cell density was adjusted to 1E6/mL using NK cell expansion complete medium (X-VIVO 15 serum free medium containing 100IU/mL IL-2), and then trophoblast cells were added in proportion for co-culture in T75 flasks with a cell number ratio of trophoblast to PBMC cells of 2:1. the cells were observed every 2-3 days, and when the cell density was high, the complete medium was supplemented to a cell density of about 1E 6/mL. On day 7 of culture, cell counts, 2 nd round of stimulation with addition of trophoblasts, number ratio of trophoblasts to PBMC cells 1:1, transferring the cells into a T175 culture flask for culture, and adding a culture medium according to the growth condition of the cells. Cell expansion results were counted after 14 days of culture.

(3) NK cell flow assay

Culturing for 0, 7, and 14 days, taking 5×10 ⁵ Cells were washed, added with CD3 and CD56 antibodies, incubated at 4 degrees in the dark for 30 minutes, washed and checked on a machine.

Results: the result of peripheral blood-derived NK cell amplification is shown in FIG. 1a, the purity of NK cells (CD 3-CD56+) after 7 days of amplification can reach 80%, and the purity of NK cells after 14 days of amplification is more than 90%.

In examples 2-5 below, the transfection efficiency of using different methods to transfect chimeric antigen receptor targeting EGFR and egfrvlll (CAR 043) in peripheral blood derived NK cells was examined.

Example 2 preparation of CAR-NK cell experimental procedure using viral infection

(1) Virus package

24h before plasmid transfection, 293T cells are inoculated in a 10cm culture dish, the cell number is controlled, and the confluency of the 293T cells is ensured to be about 80% and evenly distributed during transfection. The cells were transfected with a three plasmid virus packaging system (gene expression plasmid of interest, helper packaging plasmid, viral envelope protein plasmid), all 10ug. When the lentivirus is packaged, the target gene expression plasmid is a pLenti vector, the auxiliary packaging plasmid is a psPAX2 vector, and the viral envelope protein plasmid is any one of pCMV-Vsg, pCMV-RD114A, pCMV-BaEV-TR, pCMV-Ampho, and pCMV-10A1 vectors. When the retrovirus is packaged, the target gene expression plasmid is any one of pMX, pBabe, pMY, pMSCV vectors, the auxiliary packaging plasmid is a pCMV-gag-pol vector, and the virus envelope protein plasmid is any one of pCMV-Vsg, pCMV-RD114A, pCMV-BaEV-TR, pCMV-Ampho and pCMV-10A1 vectors. The transfection reagent is PEI-MAX, and the usage amount is 3 times of the total mass of the plasmid. Fresh medium was changed 4-6h after transfection, and virus supernatant was collected for the first time 48h later and stored at 4 ℃. An equal volume of fresh medium was continuously added to the dish, the second virus supernatant was collected for 72 hours, the virus supernatants were mixed twice, filtered through a 0.45 μm filter, the virus supernatant was concentrated by high-speed centrifugation, and the virus was preserved at-80 degrees after measuring the virus titer.

(2) Virus infection of NK cells

NK cells expanded for 6-8 days were stimulated with trophoblast cells to receive viral infection.One day before infection of NK cells with virus, 12 well plates were coated with RetroNectin (1 mg/mL) protein, 10ug/well, overnight at 4 ℃, PBS washed once before addition of virus, concentrated virus (moi=10) was taken, 12 well plates after coating were added, 2000g were centrifuged horizontally for 2h, and virus supernatant was discarded. NK cell addition 1X10 per well ⁶ 1mL. Adding 500IU/mL IL-2, horizontally centrifuging for 10min at 400g, standing in an incubator for culturing for 24h, and replacing fresh culture medium. And taking part of cells after 72h of virus infection, and detecting the expression condition of the target protein by using a flow method. The remaining cells were further expanded to Day14 or Day21 and the time point for harvesting cells was determined according to the number of NK cells desired to be harvested. The flow sheet for virus infection to make CAR-NK is shown in figure 1 b.

Example 3 comparison of transfection efficiencies of CAR-NK prepared by different lentiviruses and retrovectors

In this example, the transfection efficiency of various target gene expression plasmids was examined.

This example compares a self-inactivating lentiviral vector incorporating different promoter sequences (including EnCMV, short EF1a, mPGK, MSCV, UCOE-SFFV) on the backbone, with a retroviral vector pMSCV expressing the CAR gene. The lentiviral vector and retroviral vector construction schemes are shown in FIGS. 2a and 2 b.

Wherein, when carrying out slow virus packaging, the auxiliary packaging plasmid is a psPAX2 vector, and the virus envelope protein plasmid is a pCMV-BaEV-TR vector.

When the retrovirus packaging is carried out, the auxiliary packaging plasmid is a pCMV-gag-pol vector, and the virus envelope protein plasmid is a pCMV-BaEV-TR vector.

Results: the results of the flow assay after NK cell infection with the virus packaged by the above-constructed different vectors are shown in FIG. 3. In lentiviral systems, the expression efficiencies of CARs mediated by different promoters are different, the gene expression efficiencies driven by the MSCV and enCMV promoters are highest, and the gene expression efficiencies of short EF1a and mPGK are weaker. It was also found that the gene transduction effect of the retroviral vector pMSCV was higher than that of all of the above-mentioned lentiviral vectors. The subsequent CAR-NK preparation options were therefore optimized on the retroviral vector system.

Example 4 comparison of the transfection efficiency of different envelope protein assembled retroviruses on NK cells

In this example, the effects of the envelope proteins of vesicular stomatitis virus (Vsvg) and moloney murine leukemia variant virus (ampho) (4070A) and 10A1, which are capable of efficiently infecting hematopoietic stem cells, on transfection efficiency were compared.

In this example, a retroviral vector system was used, and the target gene expression plasmid was pMSCV, and the helper packaging plasmid was pCMV-gag-pol vector.

Results: the results of flow assays after NK cells infection with retrovirus packaged on pMSCV vector using the above-mentioned different envelope proteins are shown in FIG. 4. The envelope proteins RD114 and BaEV packaged retrovirus have the highest gene transduction efficiency when the NK cells are infected, and the transfection efficiency mediated by the envelope proteins of amyo, 10A1 and Vsvg is obviously lower than that of RD114 and BaEV. Because of the efficient transduction efficiency of the envelope protein BaEV on NK cells, we chose retroviral vector systems using the envelope protein BaEV in the subsequent preparation of CAR-NK cells.

Example 5 comparison of the transfection efficiency of NK cells by retroviruses assembled with different retroviral expression vectors

In this example, the transfection efficiency of different reverse transcription expression vectors was examined.

In this example, different retroviral vectors expressing the CAR gene were constructed, including pMX, pMY, pBabe, pMSCV, etc. A schematic diagram of retroviral vector construction is shown in FIG. 2 b. The helper packaging plasmid used was pCMV-gag-pol vector and the viral envelope protein plasmid was pCMV-BaEV-TR vector.

Results: the results of the flow assay after infection of NK cells with the four different retroviral vector packaged viruses described above are shown in FIG. 5. The virus of pMX and pMSCV vector packages transferred the gene most efficiently, the pBabe vector packages transferred the virus least efficiently. Thus in subsequent CAR-NK cell preparation we used the gene expression vector pMSCV or the retroviral system consisting of pMX and the envelope protein BaEV.

Examples 3-5 transfection methods the results of the detection summary are shown in table 1 below:

TABLE 1 CAR043 transfection efficiencies of different transfection methods in peripheral blood derived NK cells

Example 6 preparation of EGFR/EGFRvIII targeting CAR-NK cells

The chimeric antigen receptor sequence targeting EGFR/EGFRvIII (CAR 043) was constructed onto retroviral vector pMX, while the fusion protein of cytokine IL-15 and its receptor IL-15Ra (mb 15) was added, both linked using a T2A short peptide. The schematic of the constructed retroviral expression vector and CAR-NK cells is shown in figure 6 a.

The amino acid sequence of CAR043 is shown in SEQ ID NO. 1:

MALPVTALLLPLALLLHAARPEIVLTQSPATLSLSPGERATLSCRASSSVSSSYLHWYQQKPGQAPRLLIYSTSNLAAGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQYSGYPLTFGGGTKVEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMDWVRQAPGQGLEWMGDINPNNADTIYNQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGDYDGYYPVYYAMDYWGQGTTVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

the amino acid sequence of IL-15 is shown in SEQ ID NO. 2:

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

the amino acid sequence of IL-15Ra is shown in SEQ ID NO. 3:

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYLKSRQTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHL

the amino acid sequence of mb15 is shown as SEQ ID NO. 4:

MLLLVTSLLLCELPHPAFLLIPNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYLKSRQTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHL

the amino acid sequence of the CAR043-mb15 fusion protein is shown in SEQ ID NO. 5:

MALPVTALLLPLALLLHAARPEIVLTQSPATLSLSPGERATLSCRASSSVSSSYLHWYQQKPGQAPRLLIYSTSNLAAGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQYSGYPLTFGGGTKVEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMDWVRQAPGQGLEWMGDINPNNADTIYNQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGDYDGYYPVYYAMDYWGQGTTVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPREGRGSLLTCGDVEENPGPMLLLVTSLLLCELPHPAFLLIPNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYLKSRQTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHL

And (3) detecting the expression efficiency of the CAR-NK by using the flow type on the 3 rd day, the 7 th day and the 14 th day after the NK cells are transfected by the retrovirus, and respectively detecting the protein expression condition on the surfaces of the NK cells by using fluorescent antibodies of the anti-CAR and the anti-IL-15.

Results: as shown in FIG. 6b, the retrovirus-mediated exogenous gene produced efficient expression at 3 days of transfection, and the CAR expression rate was detected to be greater than 70%, and the efficient expression was maintained after two weeks of transfection.

Example 7 evaluation of killing effect of CAR-NK cells expressing CAR043-mb15 on tumor cells

In this example, the killing effect of CAR-NK cells on tumor cell lines was detected using the luciferase reporter method. The tumor cells detected included: EGFR-negative K562, EGFR-positive glioma cells U87, U251 and skin squamous carcinoma cell a431, wherein the EGFR expression of a431 is highest. All tumor cells were labeled with firefly luciferase and the specific experimental procedure was as follows:

(1) 100uL of tumor cell suspension (5X 10) was added to a white, impermeable-bottomed 96-well plate ³ And/or holes). The culture plates were pre-incubated in an incubator for 12-24 hours.

(2) The culture supernatants of 96-well plates were discarded, 100uL of effector cells were added per well, and the ratio of effector cells to target cell number (E/T ratio) was 0.5:1, 1:1, 5:1, and 10:1, respectively. The blank wells were filled with 100ul of medium and three duplicate wells were placed for each experiment. Effector cells were incubated with target cells for 24 hours.

(3) 100ul of Steady-Glo luciferase substrate solution was added to each well, and the relative light intensity (RLU) of the luminescence was measured using a microplate reader after shaking the plate on a horizontal shaker for 5 minutes.

(4) The Tumor cell killing effect was calculated, and the killing rate = (1- (Sample-control))/(turncell-control) ×100%

Results: as shown in fig. 7, the difference in results between CAR-NK cells and NK cell groups was not apparent in EGFR-negative K562 cell killing experiments. In the killing experiments of EGFR positive tumor cells of each group, the killing effect of the CAR-NK cells is obviously better than that of unmodified NK cells. Brain glioma cells U87 and U251 are higher in sensitivity to NK cells, and the difference of killing effects of the CAR-NK cells and the NK cells is gradually leveled with the increase of the E/T ratio. Skin squamous carcinoma cell A431 belongs to NK cell insensitive cells, but the EGFR is expressed in high level, and the CAR-NK cells prepared by the invention have more obvious killing effect than NK cells, thus reflecting the advantage of stronger killing power of the CAR-NK against EGFR high expression tumor.

Discussion of the invention

The main content of the invention is that a novel virus packaging system is used for preparing a recombinant virus vector with higher transfection efficiency on NK cells derived from peripheral blood of healthy people, and in-vitro functional experiments prove that the CAR-NK cells prepared by the method have remarkable specific killing capacity on EGFR high-expression tumor cells.

All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.

Sequence listing

<110> Shanghai Bow Biotech Co., ltd

<120> method for producing peripheral blood-derived CAR-NK

<130> P2022-0525

<160> 5

<170> PatentIn version 3.5

<210> 1

<211> 491

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 1

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu

20 25 30

Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser

35 40 45

Ser Val Ser Ser Ser Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln

50 55 60

Ala Pro Arg Leu Leu Ile Tyr Ser Thr Ser Asn Leu Ala Ala Gly Ile

65 70 75 80

Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

85 90 95

Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln

100 105 110

Tyr Ser Gly Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

115 120 125

Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

130 135 140

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

145 150 155 160

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

165 170 175

Asn Met Asp Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

180 185 190

Gly Asp Ile Asn Pro Asn Asn Ala Asp Thr Ile Tyr Asn Gln Lys Phe

195 200 205

Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

210 215 220

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

225 230 235 240

Ala Arg Gly Asp Tyr Asp Gly Tyr Tyr Pro Val Tyr Tyr Ala Met Asp

245 250 255

Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Thr Thr Thr Pro

260 265 270

Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu

275 280 285

Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His

290 295 300

Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu

305 310 315 320

Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr

325 330 335

Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe

340 345 350

Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg

355 360 365

Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser

370 375 380

Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr

385 390 395 400

Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys

405 410 415

Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn

420 425 430

Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu

435 440 445

Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly

450 455 460

His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr

465 470 475 480

Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

485 490

<210> 2

<211> 114

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 2

Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile

1 5 10 15

Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His

20 25 30

Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln

35 40 45

Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu

50 55 60

Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val

65 70 75 80

Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile

85 90 95

Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn

100 105 110

Thr Ser

<210> 3

<211> 237

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 3

Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val

1 5 10 15

Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly

20 25 30

Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn

35 40 45

Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile

50 55 60

Arg Asp Pro Ala Leu Val His Gln Arg Pro Ala Pro Pro Ser Thr Val

65 70 75 80

Thr Thr Ala Gly Val Thr Pro Gln Pro Glu Ser Leu Ser Pro Ser Gly

85 90 95

Lys Glu Pro Ala Ala Ser Ser Pro Ser Ser Asn Asn Thr Ala Ala Thr

100 105 110

Thr Ala Ala Ile Val Pro Gly Ser Gln Leu Met Pro Ser Lys Ser Pro

115 120 125

Ser Thr Gly Thr Thr Glu Ile Ser Ser His Glu Ser Ser His Gly Thr

130 135 140

Pro Ser Gln Thr Thr Ala Lys Asn Trp Glu Leu Thr Ala Ser Ala Ser

145 150 155 160

His Gln Pro Pro Gly Val Tyr Pro Gln Gly His Ser Asp Thr Thr Val

165 170 175

Ala Ile Ser Thr Ser Thr Val Leu Leu Cys Gly Leu Ser Ala Val Ser

180 185 190

Leu Leu Ala Cys Tyr Leu Lys Ser Arg Gln Thr Pro Pro Leu Ala Ser

195 200 205

Val Glu Met Glu Ala Met Glu Ala Leu Pro Val Thr Trp Gly Thr Ser

210 215 220

Ser Arg Asp Glu Asp Leu Glu Asn Cys Ser His His Leu

225 230 235

<210> 4

<211> 399

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 4

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro Asn Trp Val Asn Val Ile Ser Asp Leu Lys

20 25 30

Lys Ile Glu Asp Leu Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr

35 40 45

Thr Glu Ser Asp Val His Pro Ser Cys Lys Val Thr Ala Met Lys Cys

50 55 60

Phe Leu Leu Glu Leu Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser

65 70 75 80

Ile His Asp Thr Val Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu

85 90 95

Ser Ser Asn Gly Asn Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu

100 105 110

Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile

115 120 125

Val Gln Met Phe Ile Asn Thr Ser Ser Gly Gly Gly Ser Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser

145 150 155 160

Leu Gln Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile

165 170 175

Trp Val Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn

180 185 190

Ser Gly Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val

195 200 205

Leu Asn Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys

210 215 220

Cys Ile Arg Asp Pro Ala Leu Val His Gln Arg Pro Ala Pro Pro Ser

225 230 235 240

Thr Val Thr Thr Ala Gly Val Thr Pro Gln Pro Glu Ser Leu Ser Pro

245 250 255

Ser Gly Lys Glu Pro Ala Ala Ser Ser Pro Ser Ser Asn Asn Thr Ala

260 265 270

Ala Thr Thr Ala Ala Ile Val Pro Gly Ser Gln Leu Met Pro Ser Lys

275 280 285

Ser Pro Ser Thr Gly Thr Thr Glu Ile Ser Ser His Glu Ser Ser His

290 295 300

Gly Thr Pro Ser Gln Thr Thr Ala Lys Asn Trp Glu Leu Thr Ala Ser

305 310 315 320

Ala Ser His Gln Pro Pro Gly Val Tyr Pro Gln Gly His Ser Asp Thr

325 330 335

Thr Val Ala Ile Ser Thr Ser Thr Val Leu Leu Cys Gly Leu Ser Ala

340 345 350

Val Ser Leu Leu Ala Cys Tyr Leu Lys Ser Arg Gln Thr Pro Pro Leu

355 360 365

Ala Ser Val Glu Met Glu Ala Met Glu Ala Leu Pro Val Thr Trp Gly

370 375 380

Thr Ser Ser Arg Asp Glu Asp Leu Glu Asn Cys Ser His His Leu

385 390 395

<210> 5

<211> 908

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 5

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu

20 25 30

Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser

35 40 45

Ser Val Ser Ser Ser Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln

50 55 60

Ala Pro Arg Leu Leu Ile Tyr Ser Thr Ser Asn Leu Ala Ala Gly Ile

65 70 75 80

Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

85 90 95

Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln

100 105 110

Tyr Ser Gly Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

115 120 125

Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

130 135 140

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

145 150 155 160

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

165 170 175

Asn Met Asp Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

180 185 190

Gly Asp Ile Asn Pro Asn Asn Ala Asp Thr Ile Tyr Asn Gln Lys Phe

195 200 205

Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

210 215 220

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

225 230 235 240

Ala Arg Gly Asp Tyr Asp Gly Tyr Tyr Pro Val Tyr Tyr Ala Met Asp

245 250 255

Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Thr Thr Thr Pro

260 265 270

Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu

275 280 285

Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His

290 295 300

Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu

305 310 315 320

Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr

325 330 335

Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe

340 345 350

Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg

355 360 365

Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser

370 375 380

Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr

385 390 395 400

Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys

405 410 415

Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn

420 425 430

Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu

435 440 445

Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly

450 455 460

His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr

465 470 475 480

Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Glu Gly Arg Gly Ser

485 490 495

Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro Gly Pro Met Leu Leu

500 505 510

Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro Ala Phe Leu

515 520 525

Leu Ile Pro Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu

530 535 540

Asp Leu Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser

545 550 555 560

Asp Val His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu

565 570 575

Glu Leu Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp

580 585 590

Thr Val Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn

595 600 605

Gly Asn Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu

610 615 620

Lys Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met

625 630 635 640

Phe Ile Asn Thr Ser Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

645 650 655

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Leu Gln Ile

660 665 670

Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val Lys

675 680 685

Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe

690 695 700

Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn Lys

705 710 715 720

Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg

725 730 735

Asp Pro Ala Leu Val His Gln Arg Pro Ala Pro Pro Ser Thr Val Thr

740 745 750

Thr Ala Gly Val Thr Pro Gln Pro Glu Ser Leu Ser Pro Ser Gly Lys

755 760 765

Glu Pro Ala Ala Ser Ser Pro Ser Ser Asn Asn Thr Ala Ala Thr Thr

770 775 780

Ala Ala Ile Val Pro Gly Ser Gln Leu Met Pro Ser Lys Ser Pro Ser

785 790 795 800

Thr Gly Thr Thr Glu Ile Ser Ser His Glu Ser Ser His Gly Thr Pro

805 810 815

Ser Gln Thr Thr Ala Lys Asn Trp Glu Leu Thr Ala Ser Ala Ser His

820 825 830

Gln Pro Pro Gly Val Tyr Pro Gln Gly His Ser Asp Thr Thr Val Ala

835 840 845

Ile Ser Thr Ser Thr Val Leu Leu Cys Gly Leu Ser Ala Val Ser Leu

850 855 860

Leu Ala Cys Tyr Leu Lys Ser Arg Gln Thr Pro Pro Leu Ala Ser Val

865 870 875 880

Glu Met Glu Ala Met Glu Ala Leu Pro Val Thr Trp Gly Thr Ser Ser

885 890 895

Arg Asp Glu Asp Leu Glu Asn Cys Ser His His Leu

900 905

Claims (10)

1. A virus packaging system is characterized in that the virus packaging system comprises a target gene expression plasmid, a virus envelope protein plasmid and an auxiliary packaging plasmid,

wherein the target gene expression plasmid is selected from the group consisting of: pMSCV, pMX, pMY, pLenti, or a combination thereof, and the gene expression plasmid of interest expresses a Chimeric Antigen Receptor (CAR) targeting EGFR/EGFRvII I or a fusion protein containing said chimeric antigen receptor.

2. The viral packaging system according to claim 1, wherein the viral envelope protein plasmid is selected from the group consisting of: pCMV-Vsg, pCMV-RD114A, pCMV-BaEV-TR, pCMV-Ampho, pCMV-10A1, or combinations thereof.

3. The viral packaging system according to claim 1, wherein the helper packaging plasmid is selected from the group consisting of: psPAX2, pCMV-gag-pol, or a combination thereof.

4. The viral packaging system according to claim 1, wherein the gene expression plasmid of interest is a 5' ltr promoter self-inactivating pLenti; and the helper packaging plasmid is psPAX2.

5. The viral packaging system according to claim 1, wherein the gene expression plasmid of interest is selected from the group consisting of: pMSCV, pMX, pMY, or a combination thereof; and the helper packaging plasmid is pCMV-gag-pol.

6. The viral packaging system according to claim 1, wherein said viral packaging system is a retroviral packaging system and said gene expression plasmid of interest is selected from the group consisting of: pMSCV, pMX, or a combination thereof;

the auxiliary packaging plasmid is pCMV-gag-pol;

the viral envelope protein plasmid is selected from the group consisting of: pCMV-RD114A, pCMV-BaEV-TR, or a combination thereof.

7. A method for preparing a recombinant viral vector, comprising the steps of:

(a) Providing a packaging cell;

(b) Transfecting the packaging cell with the viral packaging system of any one of claims 1-6;

(c) Culturing the transfected packaging cells, thereby collecting the recombinant viral vector.

8. A recombinant viral vector expressing an EGFR/EGFRvI II targeted CAR, said recombinant viral vector being prepared using the method of claim 7.

9. A method of making EGFR/egfrvlll-targeted CAR-NK cells comprising the steps of:

(S1) providing NK cells to be transfected;

(S2) transfecting the NK cells with the recombinant viral vector of claim 8, thereby obtaining the EGFR/egfrvlll-targeted CAR-NK cells.

10. A CAR-NK cell prepared according to the method of claim 9.