Preparation and application of HER2-targeted CAR-NK cells expressing mIL-15

Abstract

The present invention belongs to the field of biomedicine, and specifically relates to the preparation and application of HER2-targeted CAR-NK cells expressing mIL-15. Specifically, the present invention provides a membrane-bound IL-15, wherein the membrane-bound IL-15 contains an IL-15 core sequence, a hinge region, and a transmembrane region. The membrane-bound IL-15 can better bind to the cell's own IL-15 receptor, thereby activating the IL-15 receptor downstream signaling pathway, promoting the self-proliferation of CAR-NK cells, and avoiding the cytokine toxicity caused by secretory IL-15.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to the preparation and application of HER2-targeted CAR-NK cells expressing mIL-15. Background Art

[0002] Natural killer (NK) cells are effector cells that play an important role in the human immune system. They are named because they can exert cell killing effects without antigen pre-sensitization. NK cells account for about 10% of the total peripheral blood mononuclear cells and are mainly distributed in lymph nodes, bone marrow, spleen, liver, intestinal epithelium and lung epithelium. NK cells mainly exert cytotoxic and immunomodulatory effects by releasing perforin and granzymes, and secreting cytokines IFN-γ, TNF-α, and GM-CSF. At present, the main genetic modifications of NK cells are cytokine modification and CAR gene modification, among which CAR gene modification can enhance the effect of NK cells in targeting and killing tumors. Chimeric antigen receptor (CAR) technology refers to an emerging cell therapy technology that uses genetic engineering to express an artificially constructed antigen that can recognize specific tumor cell surface antigens on the surface of immune effector cells, and can activate immune cells through its intracellular signal transduction activation domain to exert immune cell killing effects.

[0003] Human Epidermal Growth Factor Receptor 2 (HER2) is a tyrosine kinase receptor membrane glycoprotein encoded by the HER2 gene and is a member of the epidermal growth factor receptor family. Studies have found that the occurrence of many human tumors is related to HER2 proto-oncogene amplification and HER2 protein overexpression, such as breast cancer, gastric cancer, ovarian cancer, prostate cancer, lung cancer, etc., among which about 20-30% of breast cancer patients and 10-30% of gastric cancer patients overexpress the HER2 gene, so the use of targeted drugs for treatment can achieve good therapeutic effects. At present, there are many HER2 targeted drugs, mainly including monoclonal antibody drugs (trastuzumab and pertuzumab), ADC drugs developed on monoclonal antibodies (enmetazocortuzumab), small molecule tyrosine kinase inhibitor drugs (lapatinib and neratinib), etc. Although these drugs have significantly improved the prognosis of patients with HER2-positive diseases, they will produce acquired drug resistance, and the tumor will recur again and further deteriorate.

[0004] Therefore, it is necessary to develop other anti-tumor strategies targeting HER2. Currently, the genetic modification of NK cells mainly includes cytokine modification and CAR gene modification. Among them, CAR gene modification can enhance the effect of NK cells in targeting and killing tumors, which will provide a better solution for the treatment of cancer patients. Summary of the invention

[0005] The main purpose of the present invention is to provide a membrane-bound IL-15 (mIL-15), which can better bind to the cell's own IL-15 receptor (IL15Rβγ), thereby activating the IL-15 receptor downstream signaling pathway, promoting the self-proliferation of CAR-NK cells, and avoiding the cytokine toxicity caused by secretory IL-15.

[0006] Specifically, the present invention provides the following technical solutions:

[0007] In a first aspect, the present invention provides a membrane-bound IL-15 (mIL-15), wherein the membrane-bound IL-15 comprises an IL-15 core sequence, a hinge region, and a transmembrane region.

[0008] Preferably, the amino acid sequence of the hinge region is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the sequence shown in SEQ ID NO. 15. Or there are 1, 2, 3, 4, 5 or more amino acid substitutions relative to SEQ ID NO. 15, such as conservative substitutions.

[0009] Preferably, the amino acid sequence of the hinge region is as shown in SEQ ID NO.15.

[0010] Preferably, the amino acid sequence of the transmembrane region (TM) has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO. 17. Or there are 1, 2, 3, 4, 5 or more amino acid substitutions relative to SEQ ID NO. 17, such as conservative substitutions.

[0011] Preferably, the amino acid sequence of the transmembrane region is as shown in SEQ ID NO.17.

[0012] Preferably, the IL-15 is IL-15 from any source, preferably IL-15 from the human genome.

[0013] Preferably, the IL-15 refers to IL-15 in the human genome.

[0014] Preferably, the amino acid sequence of the IL-15 core sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO. 13. Or there are 1, 2, 3, 4, 5 or more amino acid substitutions relative to SEQ ID NO. 13, such as conservative substitutions.

[0015] Preferably, the amino acid sequence of the IL-15 core sequence is shown as SEQ ID NO.13.

[0016] That is, the complete structure of the membrane-bound IL-15 has the amino acid sequence shown in SEQ ID NO. 13-15-17.

[0017] On the other hand, the present invention provides a fusion protein comprising CAR and the above-mentioned membrane-bound IL-15 (mIL-15).

[0018] The fusion protein described in the present invention is also referred to as CAR-mIL15, and in a specific embodiment, is also referred to as CAR27, which specifically refers to a fusion protein formed by connecting CAR and mIL-15.

[0019] Specifically, in the fusion protein, the membrane-bound IL-15 can be connected to the N-terminus or C-terminus of any CAR, and can be connected through any sequence.

[0020] Preferably, the structure of the fusion protein is composed of the structures of the first signal peptide-CAR-self-cleavage sequence-second signal peptide-membrane-bound IL-15 connected in sequence.

[0021] Preferably, the first signal peptide is identical or different from the second signal peptide.

[0022] Preferably, the first signal peptide and the second signal peptide are each independently selected from 5 to 30 amino acids at the N-terminus of any secretory protein.

[0023] Preferably, the amino acid sequence of the first signal peptide is as shown in SEQ ID NO.1.

[0024] Preferably, the amino acid sequence of the second signal peptide is as shown in SEQ ID NO.1.

[0025] Preferably, the self-cleavage sequence is, for example, self-cleavage polypeptide 2A, more specifically including P2A, E2A, F2A, T2A or peptides from other sources with spontaneous cleavage function, more preferably, the amino acid sequence of the self-cleavage sequence is shown in SEQ ID NO.11.

[0026] In the present invention, the term "chimeric antigen receptor (CAR)" is a genetically engineered structure comprising an extracellular domain capable of binding to an antigen, a transmembrane domain derived from a different polypeptide from the extracellular domain, and at least one intracellular domain. "Chimeric Antigen Receptor (CAR)" is also called "chimeric receptor" or "chimeric immune receptor (CIR)". The "extracellular domain capable of binding to an antigen" refers to any oligopeptide or polypeptide capable of binding to an antigen, such as any type of antibody or fragment thereof. "Intracellular domain" refers to any oligopeptide or polypeptide known to act as a domain that transmits signals to activate or inhibit intracellular biological processes. The hinge region and transmembrane region in the CAR may be the same or different from those in mIL-15.

[0027] Preferably, the fusion protein CAR includes the first, second and / or third generation CAR. In some aspects, the first generation CAR is a CAR that only provides a CD3 chain induction signal when the antigen binds; in some aspects, the second generation CAR is a CAR that provides this signal and a costimulatory signal, such as a CAR including an intracellular signaling domain from a costimulatory receptor (e.g., CD28 or CD137); in some aspects, the third generation CAR is a CAR including multiple costimulatory domains of different costimulatory receptors.

[0028] Most preferably, the fusion protein has the amino acid sequence shown in SEQ ID NO.19.

[0029] Specifically, in the specific embodiments, the fusion protein is also called CAR27.

[0030] On the other hand, the present invention provides a chimeric antigen receptor, wherein the chimeric antigen receptor contains an antibody specifically targeting HER2, and the antibody specifically targeting HER2 has an amino acid sequence shown in SEQ ID NO.3.

[0031] Preferably, the chimeric antigen receptor further contains one or more of the following structures:

[0032] 1) CD8α hinge region and transmembrane region, having the amino acid sequence shown in SEQ ID NO.5;

[0033] 2) 4-1BB co-stimulatory signal domain, having the amino acid sequence shown in SEQ ID NO.7;

[0034] 3) CD3ζ signaling domain, having the amino acid sequence shown in SEQ ID NO.9.

[0035] On the other hand, the present invention provides a nucleic acid molecule, which comprises a nucleic acid sequence encoding the aforementioned membrane-bound IL-15 or fusion protein or chimeric antigen receptor, or the nucleic acid molecule encodes the aforementioned membrane-bound IL-15 or fusion protein or chimeric antigen receptor.

[0036] In the present invention, "nucleic acid molecule" or "nucleic acid" are used interchangeably and refer to a nucleotide chain of any length, and include DNA or RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into the chain by DNA or RNA polymerase.

[0037] Preferably, the coding sequence of the aforementioned membrane-bound IL-15 and the sequence formed by connecting SEQ ID NO.14-16-18 have 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology, and codon optimization can be performed according to the preferences of different species depending on the usage environment.

[0038] Preferably, the coding sequence of the membrane-bound IL-15 is composed of SEQ ID NOs. 14-16-18 connected together.

[0039] In another aspect, the present invention provides a vector, comprising the aforementioned nucleic acid molecule, or the vector expresses the aforementioned membrane-bound IL-15 or fusion protein or chimeric antigen receptor.

[0040] Preferably, the vector comprises a bacterial plasmid vector, a bacteriophage vector, a yeast plasmid vector, an adenoviral vector, a retroviral vector or a lentiviral vector.

[0041] Preferably, the vector is an adenoviral vector.

[0042] In some embodiments, the vector of the present invention contains regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal ribosome entry sites (IRES) and other expression control elements (such as transcription termination signals, or polyadenylation signals and poly-U sequences, etc.).

[0043] The vector or nucleic acid molecule of the present invention can be used to transform or transfect a host cell or enter a host cell in any manner for purposes such as storing or expressing antibodies.

[0044] In another aspect, the present invention provides a host cell, wherein the host cell expresses the aforementioned membrane-bound IL-15 or fusion protein or chimeric antigen receptor, or contains the aforementioned nucleic acid molecule or the aforementioned vector.

[0045] Preferably, the host cell comprises a human immune cell.

[0046] Preferably, the immune cells are, for example, CD4+T cells and / or CD8+T cells, NK cells (natural killer cells), NKT cells (invariant natural killer T cells), γδT cells (gamma delta T cells) or macrophages.

[0047] Preferably, the immune cells are NK cells.

[0048] Preferably, the host cell can be separated from human tissues, cell populations, or obtained by in vitro culture, such as obtained by induced differentiation of stem cells, or the host cell can also be a mature commercial cell line product.

[0049] On the other hand, the present invention provides a pharmaceutical composition, comprising at least one of the aforementioned membrane-bound IL-15, fusion protein, chimeric antigen receptor, nucleic acid molecule, vector, and host cell.

[0050] In the present invention, "pharmaceutical composition" may refer to a composition used for the treatment of a disease, and may also be used for in vitro culture experiments of cells. When used for the treatment of a disease, the term "pharmaceutical composition" generally refers to a unit dosage form, and may be prepared by any of the methods well known in the pharmaceutical field. All methods include the step of combining the active ingredient with an excipient constituting one or more accessory ingredients. Typically, the composition is prepared by uniformly and fully combining the active compound with a liquid excipient, a finely divided solid excipient, or both.

[0051] Preferably, the pharmaceutical composition further comprises: a pharmaceutically acceptable excipient.

[0052] In the present invention, the term "pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier" may include any solvent, solid excipient, diluent or other liquid excipient, etc., suitable for a specific target dosage form.

[0053] Preferably, the pharmaceutical composition can be a tablet, pill, powder, granule, capsule, lozenge, syrup, liquid, emulsion, suspension, controlled release preparation, aerosol, film, injection, intravenous drip, transdermal absorption preparation, ointment, lotion, adhesive preparation or suppository.

[0054] Preferably, the pharmaceutical composition is prepared as an injection for administration, and the injection includes injection at any site, such as intradermal injection, subcutaneous injection, intramuscular injection, and intravenous injection.

[0055] In another aspect, the present invention provides use of the aforementioned membrane-bound IL-15 or fusion protein or chimeric antigen receptor in preparing cells.

[0056] Specifically, CAR and / or mIL15 are expressed on the cells.

[0057] Preferably, the cell is an immune cell.

[0058] Preferably, the cells comprise human immune cells.

[0059] Preferably, the immune cells include CD4+T cells, CD8+T cells, NK cells, iNKT cells, γδT cells, and macrophages.

[0060] Preferably, the immune cells are NK cells.

[0061] When the cell expresses CAR, it can be called a CAR cell, and when the cell is a NK cell, it is a CAR-NK cell. In a specific embodiment, the CAR structure introduced during the preparation of the CAR cell is also connected to the membrane-bound IL-15 of the present invention, and the cell obtained at this time is still called a CAR cell.

[0062] Preferably, after the membrane-bound IL-15 is connected to the CAR structure, the therapeutic efficacy of the CAR cells can be enhanced, specifically including promoting the proliferation of CAR cells and reducing cytotoxicity.

[0063] Most preferably, the CAR cell is a CAR-NK cell.

[0064] Preferably, the cells are used in adoptive cell therapy.

[0065] On the other hand, the present invention provides a method for preparing highly therapeutic cells, the method comprising the step of introducing the aforementioned membrane-bound IL-15 or fusion protein or chimeric antigen receptor into cells.

[0066] More specifically, the method comprises the step of inducing expression of the fusion protein.

[0067] Various methods for introducing genetically engineered components (e.g., antigen receptors, e.g., CAR or TCR) are well known and can be used for the methods and compositions provided herein. Exemplary methods include methods for transferring nucleic acids encoding receptors, including via viral vectors, such as retroviruses or lentiviruses, non-viral vectors or transposons, such as the Sleeping Beauty transposon system. The method of gene transfer can include transduction, electroporation or other methods that result in gene transfer to cells.

[0068] On the other hand, the present invention provides a method for improving the therapeutic effect of CAR cells, comprising the step of connecting the aforementioned membrane-bound IL-15 after the CAR structure.

[0069] More specifically, the method comprises the step of introducing the aforementioned fusion protein into cells.

[0070] On the other hand, the present invention provides a method for treating a disease, comprising administering cells expressing a fusion protein or a chimeric antigen receptor to a subject, wherein the disease is a disease that can be treated by CAR cell therapy.

[0071] The cell therapy may also be referred to as adoptive cell therapy. With respect to the subject to be treated, the cells may be allogeneic and / or autologous.

[0072] The diseases described in the present invention include cancer, autoimmune diseases, and metabolic diseases.

[0073] The cancer of the present invention may be a blood cancer or a cancer with a solid tumor. Preferably, the cancer includes cervical cancer, seminoma, testicular lymphoma, prostate cancer, ovarian cancer, lung cancer, rectal cancer, breast cancer, skin squamous cell carcinoma, colon cancer, liver cancer, pancreatic cancer, gastric cancer, esophageal cancer, thyroid cancer, bladder transitional cell carcinoma, leukemia, brain tumor, gastric cancer, peritoneal cancer, head and neck cancer, endometrial cancer, kidney cancer, female reproductive tract cancer, carcinoma in situ, neurofibroma, bone cancer, skin cancer, gastrointestinal stromal tumor, mast cell tumor, multiple myeloma, melanoma, glioma;

[0074] Exemplary autoimmune diseases described in the present invention include achalasia; Addison's disease; adult-onset Still's disease; agammaglobulinemia; alopecia areata; amyloidosis; ankylosing spondylitis; anti-GBM / anti-TBM nephritis; antiphospholipid syndrome; autoimmune angioedema; autoimmune autonomic dysfunction; autoimmune encephalomyelitis; autoimmune hepatitis; autoimmune inner ear disease (AIED); autoimmune myocarditis; autoimmune oophoritis; autoimmune orchitis; autoimmune pancreatitis; autoimmune retinopathy; and autoimmune urticaria.

[0075] The metabolic diseases of the present invention include, for example, diabetes, diabetic ketoacidosis, hyperglycemic hyperosmolar syndrome, hypoglycemia, gout, protein-energy malnutrition, vitamin A deficiency, scurvy, vitamin D deficiency, and osteoporosis. Metabolic diseases well known to those skilled in the art are diseases caused by metabolic problems, including metabolic disorders and hypermetabolism.

[0076] Preferably, the disease is a HER2-positive cancer.

[0077] As used herein, the term "treat" (and grammatical variations such as "therapeutic" and "therapeutic") refers to the complete or partial alleviation or reduction of a disease or condition or disorder or symptoms, adverse effects or consequences or phenotypes associated therewith. Desired therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, ameliorating or alleviating the disease state, and alleviating or improving prognosis. The term does not imply complete cure of the disease or complete elimination of any symptom or effect or all symptoms or consequences.

[0078] The term "therapeutic effect" refers to a biological effect that can be manifested by a variety of means, including but not limited to, for example, a reduction in tumor volume, a reduction in the number of cancer cells, a reduction in the number of metastases, an increase in life expectancy, a reduction in cancer cell proliferation, a reduction in cancer cell survival, or an improvement in various physiological symptoms associated with a cancerous condition.

[0079] The CAR cell of the present invention refers to a cell containing CAR, more specifically, a cell expressing CAR, and the CAR-NK cell refers to a NK cell containing or expressing CAR.

[0080] The target (antigen) of the antigen binding domain of the present invention can be a therapeutic target for any disease, such as CD16a, CD33, CD20, CD19, CD22, CD123, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFα, CD26, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, UL16 binding protein, H LA-DR, DLL4, TYRO3, AXL, MER, CD122, CD155, PDGF-DD, ligand of TGF-β receptor II (TGF-βRII), ligand of TGF-βRIII, ligand of DNAM1, ligand of NKp46, ligand of NKp44, ligand of NKG2D, ligand of NKp30, ligand of scMHCI, ligand of scMHCII, ligand of scTCR, receptor for PDGF-DD, receptor for stem cell factor (SCF), receptor for stem cell-like tyrosine kinase 3 ligand (FLT3L), receptor for MICA, receptor for MICB, receptor for ULP16 binding protein, receptor for CD155 and receptor for CD122. BRIEF DESCRIPTION OF THE DRAWINGS

[0081] Figure 1 This is a schematic diagram of the CAR27 structure.

[0082] Figure 2 is the transduction efficiency of recombinant adenovirus CAR27 on primary NK cells.

[0083] Figure 3 It is the expression efficiency of HER2 on HER2-positive target cells SKBR3 and NCI-N87.

[0084] Figure 4 It is the killing efficiency of HER2 CAR NK on target cells SKBR3 and NCI-N87.

[0085] Figure 5 It is the cytokine secretion ability of HER2 CAR NK after co-culture with target cells SKBR3 and NCI-N87.

[0086] Figure 6 It is the tumor inhibitory effect of HER2 CAR NK cells in treating NCI-N87 transplanted tumors.

[0087] Figure 7This is the result of the body weight test of mice after HER2 CAR-NK cell treatment of NCI-N87 transplanted tumor. DETAILED DESCRIPTION

[0088] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto, and any technician familiar with the technical field within the technical scope disclosed by the present invention, according to the technical solution and inventive concept of the present invention, shall be equivalently replaced or changed, and all shall be covered within the protection scope of the present invention. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial channels.

[0089] General method:

[0090] 1. Design of CAR and fusion protein (CAR+mIL-15)

[0091] This example takes the design of a fusion protein of a HER2 chimeric antigen receptor and IL-15 structure having an antigen recognition domain, a costimulatory signal domain, and a CD3ξ signal domain as an example. The specific structure is as follows Figure 1 shown.

[0092] Antigen recognition domain design: Without changing the properties and functions of HER2 scFv, the antibody framework sequence of scFv is replaced with the human germline antibody framework sequence to reduce immunogenicity, improve the affinity of scFv to the HER2 target, reduce the possibility of CAR-NK cell self-activation, increase the efficiency of signal transmission into the cell, inhibit the terminal differentiation of CAR-NK, prolong the survival time of CAR-NK cells in the body, and increase the anti-tumor performance of CAR-NK cells.

[0093] Costimulatory signal domain design: Without changing the properties and functions of the 4-1BB costimulatory domain, it reduces the possibility of CAR-NK cell self-activation, increases the efficiency of 4-1BB signal transmission into the cell, prolongs the survival time of CAR-NK cells in the body, and increases the anti-tumor performance of CAR-NK cells.

[0094] CD3ζ signal domain design: The CD3ζ intracellular signal region modifies the amino acid sites related to signal transduction, ultimately improving the intracellular signal transduction pathway of CAR-NK cells, enhancing the proliferation and sustainability of CAR-NK cells in the body, improving cytokine secretion, and thereby enhancing the anti-tumor performance of CAR-NK cells.

[0095] mIL-15 structural design: The structure of membrane-bound IL-15 includes IL-15-CD8 hinge-CD8 TM. This type of membrane-bound IL-15 has a longer hinge region and a large activity space, and can easily bind to the IL-15 receptor (IL15Rβγ) of NK cells themselves, thereby activating the downstream signaling pathway of the IL-15 receptor and promoting the self-proliferation of CAR-NK cells, while avoiding the cytokine toxicity caused by secretory IL-15.

[0096] 2. Preparation of recombinant adenovirus (Ad.NK.HER2) expressing fusion protein targeting HER2 (CAR27 / CAR+mIL-15)

[0097] 1. According to the amino acid sequence shown in Table 1, the corresponding nucleic acid sequence was synthesized. Figure 1 CAR27 was synthesized by using the structure of , and its amino acid sequence is shown in SEQ ID NO.19.

[0098] Table 1. Amino acid sequence

[0099] name SEQ ID NO. length CD8α signal peptide 1 21 HER2 scFv 3 242 CD8 hinge region + transmembrane region 5 68 4-1BB co-stimulatory signaling domain 7 42 CD3ζ signaling domain 9 112 2A 11 18 mIL-15 13 114 Hinge 15 55 TM 17 21

[0100] 2. The pKAd.NK plasmid was digested with PmeI, the Ad.NK fragment after digestion was dephosphorylated, and the vector was recovered; the complete CAR27 nucleic acid fragment was inserted into the adenovirus vector Ad.NK through terminal homologous recombination to obtain the adenovirus plasmid pAd.NK.CAR27 carrying the HER2-targeted CAR gene.

[0101] 3. The adenovirus plasmid pAd.NK.CAR27 linearized with PacI was transfected into HEK293 cells of passage P20-P30.

[0102] 4. Plaques will appear about 7 to 10 days after transfection. After the disease is completely formed, centrifuge at 1800 rpm for 5 minutes, collect the cells and supernatant, mark the passage (P0), and store at -80℃.

[0103] 5. Freeze and thaw the P0 virus three times, centrifuge at 1800 rpm for 5 minutes, take the supernatant to infect 293A cells, and amplify the virus. After the cells are completely diseased, collect the virus, mark the name, generation, and batch number, and store it at -80°C.

[0104] 6. Purify the collected viruses by cesium chloride density gradient centrifugation and store at -80°C.

[0105] 7. The purified virus was used to infect K562 cells for titer determination.

[0106] Example 1. Preparation of CAR27-NK cells

[0107] 1. Activation and expansion of NK cells

[0108] Peripheral blood mononuclear cells (PBMCs) were separated by Ficoll density gradient centrifugation, and the separated PBMCs were activated and cultured in vitro until day 12 using NK cell activation medium (NK MACS containing 5% autologous plasma, 1000 IU / mL IL-2, and 500 IU / mL IL-15, Miltenyi) and NK cell expansion medium (NK MACS containing 5% serum replacement, 1000 IU / mL IL-2, and 500 IU / mL IL-15, Miltenyi).

[0109] 2. Cell Transduction

[0110] (1) NK cell plating:

[0111] NK cells on day 12 were collected by centrifugation at 1500 rpm for 10 min, resuspended in NK cell culture medium, and counted at 2×10 6 The cells were seeded at a density of 1.54 × 10 cells / mL in a T75 cell culture flask.

[0112] (2) Infection of NK cells with recombinant adenovirus: Take the melted and mixed recombinant adenovirus and infect NK cells at an infection dose of 2500VP.

[0113] (3) Replace the medium: After 24 hours, centrifuge and replace the medium at 1x10 6 NK cell expansion medium was added to a cell density of 1:1 / mL and cultured in an incubator (37°C, CO2 concentration of 5%).

[0114] (4) Detect HER2 CAR expression.

[0115] On the third day after transduction, 1×10 6 Flow cytometry was used to detect HER2 CAR expression. Figure 2 As shown, the CAR27-NK expression efficiency of the three samples was greater than 60%.

[0116] Example 2: Killing efficiency of HER2 CAR27-NK on target cells

[0117] This example takes the detection of the killing efficiency of CAR27-NK cells (the preparation method is shown in Example 1) on target cells expressing HER2 as an example.

[0118] 1. Target cell preparation: Take sufficient target cells (SKBR3 and NCI-N87, such as Figure 3 As shown, HER2 is highly expressed on its surface), centrifuge at 500 g for 10 min, discard the supernatant, and resuspend and count with 1640 basal medium. According to the counting density, the target cell density is adjusted to 1×106 Cells / mL, add 1mM Calcein-AM solution (10uL / 1×10 6 cells / mL) and stained for 30 min. After staining, wash twice with PBS and resuspend in 1640 basal medium for counting. According to the counting density, the density of target cells was adjusted to 2×10 5 Cells / mL, reserve for use.

[0119] 2. Preparation of effector cells: Take enough CAR27-NK cells, centrifuge at 500g for 10 minutes, discard the supernatant, and resuspend and count in 1640 basal medium. Dilute the effector cells to the corresponding concentration according to the effector-target ratio. Effector-target ratio: 0.5:1, 1:1, 2:1, 5:1, 10:1

[0120] 3. Plating: Set up 3 duplicate wells, spontaneous well (100 μL culture medium + 100 μL target cells), maximum well (100 μL target cells + 50 μL maximum release well + 50 μL basal culture medium), experimental well (100 μL target cells + 100 μL effector cells), and incubate in a CO2 incubator in the dark for 4 h.

[0121] 4. On-line detection: Add 50 μL of Triton-X-100 with a final concentration of 0.1% to the maximum release well, centrifuge at 400 g for 5 min, transfer 150 μL of the supernatant to a light-proof ELISA plate, and detect on the instrument (excitation wavelength: 488 nm, emission wavelength: 520 nm).

[0122] like Figure 4 As shown, CAR27-NK cells can recognize HER2 target protein and effectively kill SKBR3 and NCI-N87 cells, indicating that within a short period of incubation with target cells, CAR27-NK cells can be activated and lyse target cells, and have stronger killing ability than NK cells.

[0123] Example 3: Cytokine release experiment after co-culture of HER2 CAR27-NK and target cells

[0124] This example takes the detection of the secretion of IFN-γ and granzyme (GZMB) after HER2 CAR27-NK cells were co-incubated with target cells as an example.

[0125] 1. Preparation of target cells and effector cells

[0126] (1) Take enough target cells (SKBR3 and NCI-N87), centrifuge at 500g for 10 min, discard the supernatant, resuspend and count in 1640 basal medium. Adjust the cell density and set aside.

[0127] (2) Take a sufficient amount of effector cells, collect the cells by centrifugation, discard the supernatant, resuspend the cells in 1640 basal medium, and dilute the effector cells to the corresponding concentration according to the effector-target ratio (0.25:1 or 0.5:1).

[0128] 2. Co-incubation of target cells and effector cells

[0129] Take enough CAR27-NK, centrifuge at 500g for 10min, discard the supernatant, and resuspend and count in 1640 basal medium. Plate the effector cells and target cells in a 96-well plate at a ratio of 1:1, and the control group is NK cells that are not infected with the virus. Set up the background well (only effector cells) at the same time. Incubate at 37℃, 5% CO2 for 4 hours.

[0130] 3. Sample collection: After incubation, centrifuge and place the supernatant in an EP tube for later use.

[0131] 4. Cytokine detection: Detect the content of IFN-γ and Granzyme B according to the instructions of the multi-factor flow cytometry kit.

[0132] like Figure 5 As shown, the ability of CAR27-NK cells to secrete IFN-γ and Granzyme B was significantly enhanced compared with NK cells not infected with the virus.

[0133] Example 4: Anti-tumor ability of CAR27-NK cells in tumor-bearing mice

[0134] This example takes the anti-tumor ability of a chimeric antigen receptor CAR (CAR27) formed by connecting HER2 scFv, a natural 4-1BB co-stimulatory signal domain, a natural CD3ζ signal domain and an mIL-15 domain in tumor-bearing mice as an example.

[0135] 1. The NK cells activated and expanded to day 12 were infected with HER2-CAR adenovirus to prepare HER2-CAR27-NK cells (NK cells not infected with adenovirus were cultured at the same time for control experiments).

[0136] 2. 24 hours after virus infection, the cells were centrifuged and the medium was changed. The cells were counted and the number of cells was calculated as 1.0×10 6 The cells / mL were inoculated and cultured in NK cell expansion medium (NK MACS, Miltenyi containing 5% serum replacement, 1000 IU / mL IL-2, and 500 IU / mL IL-15).

[0137] 3. On the third day of culture, harvest the cells and count them, and keep the corresponding cell samples for flow cytometry analysis of CAR expression rate. Cryopreserved cells are stored in liquid nitrogen for later use.

[0138] 4. A total of 24 6-8 week old NCG mice (Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) were divided into 3 groups of 8 mice each. Each mouse was subcutaneously inoculated with 1.0×10 6 NCI-N87 cells (Nanjing Kebai Biotechnology Co., Ltd.).

[0139] 5. After the mice were successfully modeled, they were injected with drugs on days 7, 14, 21, and 28. The mice in the CAR27-NK group were injected with CAR27-NK cells (1×10 7 At the same time, the other two groups of mice were injected with the corresponding number of NK cells and the corresponding volume of normal saline as controls.

[0140] 6. The tumor volume of mice was measured and the weight of mice was weighed every 3 days after CAR27-NK cell injection.

[0141] like Figure 6 As shown in the figure, CAR27-NK cells can significantly inhibit tumor growth compared with the tumor-bearing mice injected with NK cells. Figure 7 As shown, CAR27-NK treatment had no significant effect on the body weight of mice, indicating no significant toxic side effects.

[0142] The above contents are further detailed descriptions of the present invention in combination with specific preferred embodiments, and it cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, several simple deductions or substitutions can be made without departing from the concept of the present invention, which should be regarded as falling within the protection scope of the present invention.

Claims

1. A fusion protein, wherein the fusion protein is composed of CAR and membrane-bound IL-15 connected together, and has an amino acid sequence shown in SEQ ID NO.

19.

2. A nucleic acid molecule comprising a nucleic acid sequence encoding the fusion protein of claim 1.

3. A vector, comprising the nucleic acid molecule of claim 2, or expressing the fusion protein of claim 1.

4. The vector of claim 3, wherein the vector comprises a bacterial plasmid vector, a phage vector, a yeast plasmid vector, an adenovirus vector, a retrovirus vector or a lentivirus vector. The vector according to claim 3 , which is an adenovirus vector.

6. A host cell, wherein the fusion protein of claim 1 is expressed in the host cell, or the host cell contains the nucleic acid molecule of claim 2 or the vector of any one of claims 3 to 5.

7. The host cell of claim 6, comprising a human immune cell.

8. The host cell according to claim 7, wherein the immune cells include CD4+ T cells, CD8+ T cells, NK cells, iNKT cells, γδ T cells, and macrophages.

9. The host cell of claim 7, wherein the immune cell is a NK cell.

10. The host cell according to claim 6, wherein the host cell comprises a cell isolated from a human tissue or cell population, a cell obtained by in vitro culture, or a mature commercial cell line product.

11. A pharmaceutical composition comprising one or more of the following: 1) The fusion protein according to claim 1, 2) The nucleic acid molecule according to claim 2, 3) The vector according to any one of claims 3 to 5, 4) The host cell according to any one of claims 6 to 10.

12. The pharmaceutical composition according to claim 11, further comprising: Pharmaceutically acceptable excipients.

13. The pharmaceutical composition of claim 11, wherein the dosage form of the pharmaceutical composition comprises tablets, pills, powders, granules, capsules, lozenges, syrups, liquids, emulsions, suspensions, controlled release preparations, aerosols, films, injections, intravenous drips, transdermal absorption preparations, ointments, lotions, adhesive preparations or suppositories.

14. Use of the fusion protein according to claim 1 in preparing cells.

15. The use according to claim 14, wherein the cells express CAR and / or mIL15.

16. The use according to claim 14, wherein the cells comprise immune cells.

17. The use according to claim 14, wherein the cells comprise human immune cells.

18. The use according to claim 16, wherein the immune cells include CD4+ T cells, CD8+ T cells, NK cells, iNKT cells, γδ T cells, and macrophages.

19. The use according to claim 16, wherein the immune cells are NK cells.

20. The use as claimed in claim 16, wherein the membrane-bound IL-15 is connected to the CAR structure and then introduced into the cell to obtain a CAR cell with improved therapeutic effect.

21. The use according to claim 20, wherein the improved therapeutic effect comprises promoting cell proliferation and reducing cytotoxicity.

22. The use according to claim 14, wherein the cell is a CAR-NK cell.

23. The use according to claim 14, wherein adoptive cell therapy using the cells has a therapeutic effect on cancer, autoimmune diseases, and metabolic diseases.

24. The use of claim 23, wherein the cancer comprises cervical cancer, seminoma, testicular lymphoma, prostate cancer, ovarian cancer, lung cancer, rectal cancer, breast cancer, skin squamous cell carcinoma, colon cancer, liver cancer, pancreatic cancer, gastric cancer, esophageal cancer, thyroid cancer, bladder transitional cell carcinoma, leukemia, brain tumor, gastric cancer, peritoneal cancer, head and neck cancer, endometrial cancer, kidney cancer, female reproductive tract cancer, carcinoma in situ, neurofibroma, bone cancer, skin cancer, gastrointestinal stromal tumor, mast cell tumor, multiple myeloma, melanoma, glioma.

25. The use of claim 23, wherein the autoimmune disease comprises achalasia, Addison's disease, adult Still's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM / anti-TBM nephritis, antiphospholipid syndrome, autoimmune angioedema, autoimmune dysautonomia, autoimmune encephalomyelitis, autoimmune hepatitis, autoimmune inner ear disease, autoimmune myocarditis, autoimmune oophoritis, autoimmune orchitis, autoimmune pancreatitis, autoimmune retinopathy, and autoimmune urticaria.

26. The use according to claim 23, wherein the metabolic diseases include diabetes, diabetic ketoacidosis, hyperglycemic hyperosmolar syndrome, hypoglycemia, gout, protein-energy malnutrition, vitamin A deficiency, scurvy, vitamin D deficiency, and osteoporosis.

27. The use according to claim 23, wherein the cancer is HER2-positive breast cancer or gastric cancer.

28. A method for preparing cells with high therapeutic efficacy, the method comprising the step of introducing the fusion protein of claim 1 into cells.

29. The preparation method according to claim 28, comprising the step of inducing expression.

30. The preparation method according to claim 28, wherein the high therapeutic effect includes improved therapeutic effects on cancer, autoimmune diseases, and metabolic diseases.

31. The preparation method of claim 30, wherein the cancer comprises cervical cancer, seminoma, testicular lymphoma, prostate cancer, ovarian cancer, lung cancer, rectal cancer, breast cancer, skin squamous cell carcinoma, colon cancer, liver cancer, pancreatic cancer, gastric cancer, esophageal cancer, thyroid cancer, bladder transitional cell carcinoma, leukemia, brain tumor, gastric cancer, peritoneal cancer, head and neck cancer, endometrial cancer, kidney cancer, female reproductive tract cancer, carcinoma in situ, neurofibroma, bone cancer, skin cancer, gastrointestinal stromal tumor, mast cell tumor, multiple myeloma, melanoma, glioma.

32. The preparation method of claim 30, wherein the autoimmune disease comprises achalasia, Addison's disease, adult Still's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM / anti-TBM nephritis, antiphospholipid syndrome, autoimmune angioedema, autoimmune dysautonomia, autoimmune encephalomyelitis, autoimmune hepatitis, autoimmune inner ear disease, autoimmune myocarditis, autoimmune oophoritis, autoimmune orchitis, autoimmune pancreatitis, autoimmune retinopathy, and autoimmune urticaria.

33. The preparation method according to claim 30, wherein the metabolic diseases include diabetes, diabetic ketoacidosis, hyperglycemic hyperosmolar syndrome, hypoglycemia, gout, protein-energy malnutrition, vitamin A deficiency, scurvy, vitamin D deficiency, and osteoporosis.

34. The preparation method according to claim 30, wherein the cancer is HER2-positive breast cancer or gastric cancer.

35. The preparation method according to claim 28, wherein the cells comprise immune cells.

36. The preparation method according to claim 28, wherein the cells comprise human immune cells.

37. The preparation method according to claim 35, wherein the immune cells include CD4+ T cells, CD8+ T cells, NK cells, iNKT cells, γδ T cells, and macrophages.

38. The preparation method according to claim 35, wherein the immune cells are NK cells.

39. The preparation method according to claim 28, wherein the cell is a CAR-NK cell.

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