A method for preparing CAR-NK cells based on recombinant adenovirus and its application

Abstract

The present invention discloses a method for preparing CAR-NK cells based on recombinant adenovirus and its application. The present invention provides an efficient CAR-NK cell preparation scheme, which can prepare CAR-NK cells with efficient expression of CAR molecules, solve the problem of low transduction efficiency of NK cells, and improve the efficacy of CAR-NK cells. The present invention adds a transduction-promoting reagent (0.5-1μM BX795) to enhance the transduction efficiency, and the transduction efficiency can reach more than 70%. The present invention uses recombinant adenovirus transduction without affecting the phenotype and amplification of NK cells. Based on recombinant adenovirus CAR-NK cells, it has significant in vitro killing efficiency. Recombinant adenovirus CAR-NK cells have significant in vivo anti-tumor (Raji transplant tumor model) effects.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to a method for preparing CAR-NK cells based on recombinant adenovirus and an application thereof. Background Art

[0002] Natural killer (NK) cells are important effector cells of the innate immune system, accounting for 5% to 15% of mononuclear cells in the blood and lymphoid organs. NK cells are the body's first line of defense against viral infections and malignant cells. They can directly kill cancer or infected cells, or indirectly improve antibody and T cell-mediated responses. In addition, NK cells also have immunomodulatory functions, regulating dendritic cells, macrophages and neutrophils, as well as antigen-specific T cells and B cell responses.

[0003] Chimeric antigen receptor (CAR) is a receptor protein that can specifically recognize target proteins and induce secondary signal transduction. CAR is mainly composed of three domains: extracellular region, transmembrane region and intracellular region. The extracellular domain contains a single chain variable fragment (scFv), which is usually derived from an antibody that can specifically recognize tumor antigens expressed on cancer cells; the transmembrane domain anchors the CAR structure to the effector cell membrane; the intracellular activation domain activates downstream signals and promotes the killing of target cells.

[0004] Compared with natural NK cells, CAR-NK cells expressing CAR receptors have stronger targeting and anti-tumor response. The methods for delivering genes to NK cells mainly include viral and non-viral methods, among which viral methods are represented by retroviruses and lentiviruses; and non-viral methods include mRNA electroporation, transposon and CRISPR / Cas9-mediated methods. The sensitivity and defense mechanism of NK cells to foreign genetic material usually lead to low levels of transduction and induce cell apoptosis. Therefore, compared with T cells, the transduction efficiency of NK cells is relatively low.

[0005] At present, the preparation of CAR-NK cells is mainly transduced by retrovirus and lentivirus. Among them, the transduction efficiency of recombinant lentivirus on NK cells is low, ranging from 10% to 30%. This is because the VSV-G envelope of the lentivirus has a low binding ability to the low-density lipid receptor LDL-R, which is a receptor that promotes viral entry, but this receptor is expressed at a low level on activated NK cells; and retroviruses can be randomly integrated into the genome, with the potential risk of insertion mutations. In addition, the non-viral transduction system is still unstable: nucleic acids are introduced into NK cells by electroporation, which is a simple and cost-effective method, but the permeabilization of the cell membrane by electric pulses can easily lead to a high cell mortality rate through unregulated exchange of internal or external cell components or permanent membrane leakage. Summary of the invention

[0006] Adenovirus vectors have the characteristics of high transfection efficiency, low pathogenicity, high titer, and no integration into host cell chromosomes in vivo. They are one of the commonly used vectors in gene therapy clinical trials and vaccine development. However, the most widely used adenovirus type 5 has low gene transduction efficiency for NK cells. There are no reports on the use of modified recombinant adenoviruses for the preparation of CAR-NK cells. In order to solve the problems existing in the prior art, the present invention will provide an efficient and stable CAR-NK cell preparation scheme.

[0007] In a first aspect, the present invention provides a CAR targeting CD19, wherein the CAR comprises a ScFv targeting CD19, and the CAR further comprises one or more of a signal peptide, a hinge region, a transmembrane region, a co-stimulatory region, or a signal transduction region.

[0008] Furthermore, the CAR includes a signal peptide, ScFv, a hinge region, a transmembrane region, a co-stimulatory region, and a signal transduction region connected in sequence.

[0009] Furthermore, the CAR includes a signal peptide, ScFv, a hinge region, and a transmembrane region connected in sequence.

[0010] Furthermore, the signal peptide is CD8α, the hinge region includes IgG4, IgG4(L), or CD8α, the transmembrane region includes CD28 or CD8α, the co-stimulatory region includes CD28, 4-1BB, or OX40, and the signal transduction domain is CD3ζ.

[0011] Furthermore, the CAR includes a CD8α signal peptide, a ScFv targeting CD19, an IgG4 hinge region, a CD28 transmembrane region, an OX40 co-stimulatory region, and a CD3ζ signal transduction region connected in sequence.

[0012] Furthermore, the CAR includes a CD8α signal peptide, a ScFv targeting CD19, an IgG4 hinge region, and a CD28 transmembrane region connected in sequence.

[0013] Furthermore, the CAR includes a CD8α signal peptide, a ScFv targeting CD19, an IgG4(L) hinge region, a CD28 transmembrane region, a 4-1BB co-stimulatory region, and a CD3ζ signal transduction region connected in sequence.

[0014] Furthermore, the CAR includes a CD8α signal peptide, a ScFv targeting CD19, a CD8α hinge region, a CD8α transmembrane region, a 4-1BB co-stimulatory region, and a CD3ζ signal transduction region, which are connected in sequence.

[0015] Further, the CAR comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO:6.

[0016] Further, the CAR comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO:8.

[0017] Furthermore, the CAR comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:10.

[0018] Further, the CAR comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO:12.

[0019] The second aspect of the present invention provides a polynucleotide molecule or a vector containing the same, wherein the polynucleotide molecule includes a nucleotide sequence encoding the CAR described above.

[0020] Furthermore, the vector is an adenovirus vector.

[0021] Furthermore, the adenoviral vector is a pAd.NK adenoviral vector.

[0022] In the present invention, the pKAd.NK adenovirus vector refers to the invention patent derived from application number 202110677612.4, the principle of which is that the HAdV-5 fiber gene is modified into a human adenovirus type 5 with an artificially modified f11p228R gene, wherein the amino acid sequence of the f11p228R gene is as shown in SEQ ID Shown in NO:19 (i.e., MKRARPSEDTFNPVYPYDTETGPPTVPFLTPPFVSPNGFQESPPGVLTLKCLTPLTTTGGSLQLKVGGGLTVDDTNGFLKENISATTPLVKTGHSIGLPLGAGLGTNENKLCIKLGQGLTFNSNNICIDDNINTLWTGVNPTEANCQIMNSSESNDCKLILTL VKTGALVTAFVYVIGVSNNFNMLTTHRNINFTAELFFDSTGNLLTRLSSLKTPLNHKSGQNMATGCDCRGDCFCAITNAKGFMPSTTAYPFNDNSREKENYIYGTCYYTASDRTAFPIDISVMLNRRAINDETSYCIRITWSWNTGDAPEVQTSATTLVTSPFTFYYIREDD).

[0023] As used herein, the term "vector" refers to an agent that can transduce, transfect, transform or infect cells, thereby causing the cells to express nucleic acids and / or proteins other than those that are natural to the cells, or to express them in a manner that is not natural to the cells. When nucleic acids are translocated from the extracellular environment into cells, the cells are "transduced" by nucleic acids. Any method for transferring nucleic acids into cells can be used; unless otherwise specified, the term does not imply any specific method for delivering nucleic acids to cells. When nucleic acids are transduced into cells and stably replicated, cells are "transformed" by nucleic acids. The vector includes nucleic acids (usually RNA or DNA) to be expressed by the cell. In a specific embodiment of the present invention, the specific type of the vector is an adenovirus vector, specifically a modified pAd.NK adenovirus vector.

[0024] Furthermore, the polynucleotide molecule comprises a nucleotide sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, or SEQ ID NO:11.

[0025] The term "identity" refers to the sequence similarity between two polypeptide molecules or between two nucleic acid molecules. When a position in two comparison sequences is occupied by the same base or the same amino acid residue, the corresponding molecules are identical at that position. The percent identity between two sequences corresponds to the number of matching positions shared by the two sequences divided by the number of positions compared and multiplied by 100. Typically, two sequences are compared when aligned to obtain maximum identity. Identity can be calculated by alignment using, for example, GCG (Genetics Computer Group, Program Manual for the GCG Package, 7th Edition, Madison, Wisconsin) stacking programs or any sequence comparison algorithm (such as BLAST, FASTA or CLUSTALW).

[0026] The sequence homology or identity of conserved polynucleotides or polypeptides can be determined by standard alignment algorithms. Basically, homologous or identical sequences are generally expected to hybridize to all or at least about 50%, 60%, 70%, 80% or 90% or more of the sequence length under moderate or high stringency conditions. Polynucleotides containing degenerate codons rather than codons in the hybridizing polynucleotides are also contemplated.

[0027] Furthermore, the polynucleotide molecule also includes a nucleotide sequence encoding a sIL-15 molecule, and the amino acid sequence of the sIL-15 molecule is shown in SEQ ID NO:14.

[0028] Furthermore, the nucleotide sequence encoding the sIL-15 molecule is shown in SEQ ID NO:13.

[0029] Furthermore, the polynucleotide molecule also includes a nucleotide sequence as shown in SEQ ID NO: 15 encoding EF1α.

[0030] Furthermore, the polynucleotide molecule also includes a nucleotide sequence as shown in SEQ ID NO: 16 encoding BGHpa.

[0031] Furthermore, the polynucleotide molecule also includes a nucleotide sequence as shown in SEQ ID NO: 17 encoding hPGKp.

[0032] Furthermore, the polynucleotide molecule also includes a nucleotide sequence as shown in SEQ ID NO: 18 encoding SV40 PA.

[0033] Furthermore, the nucleotide sequences encoding EF1α, the aforementioned CAR, BGHpa, hPGKp, sIL-15, and SV40 PA in the polynucleotide molecule are sequentially connected.

[0034] Furthermore, the nucleotide sequences encoding EF1α, the aforementioned CAR, BGHpa, hPGKp, sIL-15, and SV40 PA in the polynucleotide molecule are connected by P2A, T2A or E2A.

[0035] The term "connection" refers to the functional connection between the nucleic acid expression regulatory sequence and the nucleic acid sequence encoding the target protein so as to perform the overall function. The effective connection with the recombinant vector can be prepared using gene recombination techniques well known in the art, and site-specific DNA cutting and connection can be performed using enzymes well known in the art.

[0036] The third aspect of the present invention provides a cell, which includes the CAR described above, or the polynucleotide molecule described above, or a vector containing the same.

[0037] Furthermore, the cell includes a eukaryotic cell or a prokaryotic cell.

[0038] Furthermore, the cells include lymphocytes.

[0039] Furthermore, the cells include T cells, B cells, or NK cells.

[0040] As used herein, the term "lymphocyte" refers to a cell of the immune system, which is a type of white blood cell. Lymphocytes include, but are not limited to, T cells (cytotoxic and helper T cells), B cells, and natural killer cells (NK cells).

[0041] As used herein, the term "T cell" or "T lymphocyte" is recognized in the art and is intended to include thymocytes, initial T lymphocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes or activated T lymphocytes. The term "T cell" includes natural T cells (e.g., separated from an organism, such as a mammal, e.g., a human, e.g., a subject), T cells grown in vitro, and genetically engineered T cells within its scope. The term T cell also includes T cells comprising T cell receptors (e.g., natural TCRs, or recombinant TCRs) and T cells comprising artificial T cell receptors (e.g., CAR-T cells).

[0042] As used herein, the term "NK cells" refers to natural killer cells, which are important immune cells in the body and are mainly distributed in the bone marrow, peripheral blood, liver, spleen, lungs and lymph nodes.

[0043] A fourth aspect of the present invention provides a method for preparing a CAR cell targeting CD19, the method comprising transducing the aforementioned CAR, or the aforementioned polynucleotide molecule or a vector containing the same into the cell via adenovirus.

[0044] The term "transduction" when used to produce recombinant antigen receptor cells or chimeric antigen receptor cells refers to the process of introducing exogenous nucleotide sequences into cells. In some embodiments, the transduction is performed by a vector.

[0045] Furthermore, the method specifically comprises the steps of:

[0046] 1) synthesizing the aforementioned signal peptide, ScFv, hinge region, transmembrane region, co-stimulatory region, signal transduction region and the aforementioned nucleotide fragments of BGHpa, hPGKp, sIL15, and SV40pa by molecular biology techniques;

[0047] 2) connecting the molecules in step 1) in the order of the nucleotide sequences in the second aspect of the present invention, and cloning the complete nucleotide sequence into an adenovirus vector;

[0048] 3) Select mononuclear cells from the healthy lymphatic system and inoculate them into NK cell activation medium and NK cell expansion medium for 10-15 days;

[0049] 4) The cultured cells were inoculated into NK cell basal medium, the cells were infected with adenovirus vectors, BX795 transduction promoter was added, transduction was performed for 4 hours, an equal volume of NK cell complete medium containing double factors was added, transduction was performed for 24 hours, the medium was replaced by centrifugation, and the cells were cultured continuously with NK cell expansion medium, and transduction was performed for 3 days to obtain CAR cells.

[0050] Furthermore, the NK cell activation medium is NK MACS containing 5% autologous plasma, 1000 IU / mL IL-2, and 500 IU / mL IL-15, and its commercial source is Miltenyi.

[0051] Furthermore, the NK cell expansion medium is NK MACS containing 5% serum replacement, 1000 IU / mL IL-2, and 500 IU / mL IL-15, and its commercial source is Miltenyi.

[0052] Furthermore, the NK cell basal medium is NK MACS, and its commercial source is Miltenyi.

[0053] Furthermore, the NK cell complete culture medium is NKMACS containing 2000 IU / mL IL-2 and 1000 IU / mL IL-15, and its commercial source is Miltenyi.

[0054] Furthermore, the molecular biology techniques include PCR, restriction enzyme digestion, and homologous recombination.

[0055] Furthermore, the adenovirus vector is a pAd.NK adenovirus vector.

[0056] Furthermore, in step 2), the connection method is forward insertion, and the connection site is PmeI.

[0057] Furthermore, the cells in step 3) are NK cells.

[0058] Furthermore, in the step 4), the MOI of the adenovirus vector to infect cells is 2500 VPs / cell.

[0059] The fifth aspect of the present invention provides a cell therapy composition for treating or preventing cancer, wherein the cell therapy composition comprises the cells described in the third aspect of the present invention, or the cells prepared by the aforementioned method as an active ingredient.

[0060] As used herein, the terms "cancer," "hyperproliferative," and "neoplastic" refer to cells with the capacity for spontaneous growth, i.e., an abnormal state or condition characterized by rapidly proliferating cell growth. Hyperproliferative and neoplastic disease states may be classified as pathological, i.e., characterizing or constituting a disease state, or as nonpathological, i.e., a deviation from normal but unrelated to a disease state. The term is intended to include all types of cancerous growths or oncogenic processes, metastatic tissues, or malignantly transformed cells, tissues, or organs, regardless of histopathological type or stage of invasiveness.

[0061] As used herein, the term "treatment" refers to a clinical intervention designed to change the natural course of an individual or cell to be treated during clinical pathology. The desired therapeutic effect includes reducing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving prognosis. For example, one or more conditions associated with the disease to which the present invention relates are reduced or eliminated, including but not limited to reducing (or destroying) the proliferation of cancer cells, reducing pathogen infection, reducing symptoms caused by the disease, improving the quality of life of those suffering from the disease, reducing the dosage of other drugs required for treating the disease, and / or prolonging the survival of the individual, then the individual is successfully "treated".

[0062] The sixth aspect of the present invention provides the use of the aforementioned CAR, the aforementioned polynucleotide molecule or a vector comprising the same, or the aforementioned cell in the preparation of a pharmaceutical composition for treating CD19-positive cancer.

[0063] Further, the CD19-positive cancer includes myelodysplasia, myelodysplastic syndrome, preleukemia, blood cancer, acute leukemia, B-cell acute lymphoblastic leukemia, T-cell acute lymphoblastic leukemia, small lymphocytic leukemia, acute lymphoblastic leukemia, chronic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, non-Hodgkin's lymphoma, lymphoma, myeloma, pancreatic cancer, biliary tract cancer, lung cancer, ovarian cancer, breast cancer, uterine cancer, rectal cancer, colorectal cancer, colon cancer, bone marrow cancer, liver cancer, brain cancer, prostate cancer, gastric cancer, glioma, melanoma, squamous cell carcinoma, head and neck cancer, renal cell carcinoma, glioblastoma, or medulloblastoma.

[0064] The seventh aspect of the present invention provides the use of the aforementioned CAR, the aforementioned polynucleotide molecule or a vector comprising the same in the preparation of NK cells that express CAR with high targeting CD19 and low expression of sIL-15.

[0065] The eighth aspect of the present invention provides a pharmaceutical composition, which comprises the cells described above.

[0066] Furthermore, the pharmaceutical composition includes pharmaceutically acceptable excipients.

[0067] Furthermore, the pharmaceutical composition is delivered via a pharmaceutically acceptable carrier.

[0068] The term "pharmaceutical composition" refers to a preparation in a form that allows the biological activity of the active ingredient to be effective and does not contain other components that are unacceptably toxic to the subject to which the composition or preparation is administered. Such preparations are sterile. "Pharmaceutically acceptable" excipients (media, additives) are those that can be reasonably administered to a subject mammal to provide an effective dose of the active ingredient used.

[0069] "Pharmaceutically acceptable" ingredients are suitable for use in humans and / or mammals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), that is, substances with a reasonable benefit / risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier used for the administration of therapeutic agents, including various excipients and diluents. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, polysorbate, ethanol, and combinations thereof. Generally, drug preparations should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. The pharmaceutical composition is preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount.

[0070] As used herein, the term "about" or "approximately" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by up to 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% relative to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In specific embodiments, when preceding a numerical value, the term "about" or "approximately" means that value plus or minus a range of 15%, 10%, 5%, or 1%.

[0071] Advantages and beneficial effects of the present invention:

[0072] The present invention provides an efficient CAR-NK cell preparation scheme, which can prepare CAR-NK cells with efficient expression of CAR molecules, solve the problem of low NK cell transduction efficiency, and improve the therapeutic effect of CAR-NK cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Figure 1 It is a schematic diagram of the CAR structure;

[0074] Figure 2 is a schematic diagram of the recombinant adenovirus structure;

[0075] Figure 3 This is the result of flow cytometry detection of CAR expression efficiency after transduction at different MOIs;

[0076] Figure 4 This is a graph showing the effect of different concentrations of transduction-promoting reagents on CAR transduction efficiency;

[0077] Figure 5 This is a comparison chart of the efficiency of NK cell transduction by recombinant adenovirus and recombinant lentivirus;

[0078] Figure 6 This is a statistical chart of the killing efficiency of NK cells after transduction of recombinant adenovirus and recombinant lentivirus;

[0079] Figure 7 It is a graph showing the killing efficiency of CAR19-NK cells against CD19+ tumor cell lines Raji, Daudi, Nalm-6, and H929;

[0080] Figure 8 This is the in vivo imaging result at different time points after CAR19-NK cells were used to treat Raji transplanted tumors;

[0081] Fig. 9 This is a statistical graph of in vivo imaging results at different time points after CAR19-NK cells were used to treat Raji transplanted tumors;

[0082] Fig.10 This is the test result of mouse body weight after CAR19-NK cells were used to treat Raji transplanted tumor;

[0083] Fig.11 This is the survival curve of mice after CAR19-NK cell treatment of Raji transplanted tumor. DETAILED DESCRIPTION

[0084] The present invention will be described in detail below in conjunction with the accompanying drawings and embodiments, so that those skilled in the art can understand and implement the present invention and further recognize the advantages of the present invention.

[0085] Unless otherwise defined in the specification of the present invention, all technical terms herein are used according to the customary definitions commonly used and understood by those skilled in the art. The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0086] Example 1 Design of different CAR structures and preparation of recombinant adenovirus

[0087] 1. Experimental methods

[0088] 1) The present invention is designed as follows Figure 1 The CAR structure shown in FIG. 1 and its specific nucleotide and amino acid sequences are shown in Table 1.

[0089] 2) Preparation steps of recombinant adenovirus

[0090] Synthesize CD8α signal peptide, FMC63 (ScFv targeting CD19), IgG4 hinge region, CD8α hinge region, CD28 transmembrane region, CD8α transmembrane region, OX40 co-stimulatory signal region, 4-1BB co-stimulatory signal region, CD28 co-stimulatory signal region and CD3ζ signal transduction region, BGHpa, hPGKp, sIL15, SV40pa and other DNA fragments, and synthesize the above CAR molecules by molecular biology techniques such as PCR, restriction enzyme digestion, and homologous recombination. The above CAR molecules obtained are forward inserted into the PmeI site of the recombinant adenovirus vector pAd.NK by homologous recombination to obtain recombinant adenovirus plasmids JD-A-CAR1, JD-A-CAR2, JD-A-CAR8, JD-A-CAR13, JD-A-CAR14 and JD-A-CAR16 with CD19 CAR gene targeting, and their basic structures are as follows Figure 2 As shown, CARs represents different CAR molecules, and the Ad5 genome is a type 5 adenovirus genome with E1 and E3 regions deleted (wherein the fiber top ball is transformed with F11p228RGD).

[0091] 2. Experimental results

[0092] After molecular sequencing, the EF1α nucleotide sequence, BGHpa nucleotide sequence, hPGKp nucleotide sequence, and SV40 PA nucleotide sequence were successfully constructed, and their sequences are shown in Table 2; and the recombinant adenovirus plasmid constructed by the present invention was successfully constructed, and its CARs molecule was consistent with the sequence in Table 1, and the sIL-15 nucleotide sequence was successfully constructed, and its amino acid and nucleotide sequences are shown in Table 1.

[0093] Table 1

[0094]

[0095]

[0096]

[0097]

[0098]

[0099] Table 2

[0100]

[0101]

[0102] Example 2 Effects of different CAR structures on virus yield and activity

[0103] A. Experimental Methods

[0104] 1. Packaging of recombinant adenovirus:

[0105] 1.1 Add 400 μl Opti-MEM, 10 μl P3000 and 10 μg plasmid to tube 1, and add 400 μl Opti-MEM and 15 μl Lip3000 to tube 2. Mix tubes 1 and 2 evenly and let stand at room temperature for 20 min.

[0106] 1.2 Add the above mixed plasmids to HEK293 cells with a cell confluence of 80%, and then replenish the solution once every 3-4 days to observe the cell lesions until 80% of the lesions appear, then collect the cells and supernatant into a centrifuge tube.

[0107] 2. Amplification of recombinant adenovirus:

[0108] 2.1 First round of amplification: The above collected cells and supernatant were repeatedly frozen and thawed three times, centrifuged at 500g for 10 min, and 5 mL of supernatant was taken to infect two 15 cm dishes. The cell pathological changes were observed. When 80% of the lesions appeared in about 3 days, the cells and supernatant were collected into centrifuge tubes.

[0109] 2.2 Second round of amplification: The above collected cells and supernatant were repeatedly frozen and thawed three times, centrifuged at 500g for 10 min, and 5 mL of supernatant was taken to infect 10 15 cm dishes. The cell pathological changes were observed. When 80% of the lesions appeared in about 3 days, the cells and supernatant were collected into centrifuge tubes.

[0110] 2.3 The third round of amplification: The above collected cells and supernatant were repeatedly frozen and thawed three times, centrifuged at 500g for 10 min, and 5 mL of supernatant was taken to infect 20 15 cm dishes. The cell pathological changes were observed. When 80% of the pathological changes appeared in about 3 days, the cells and supernatant were collected into centrifuge tubes.

[0111] 3. Purification of recombinant adenovirus:

[0112] The collected cells were resuspended in PBS, frozen and thawed three times, and the virus was purified by cesium chloride density gradient centrifugation. Then, the virus was transferred into a dialysis bag and dialyzed at 4°C overnight and then packaged to obtain recombinant adenoviruses AD.CAR1, AD.CAR2, AD.CAR8, AD.CAR13, AD.CAR14 and AD.CAR16 with different CAR structures.

[0113] 4. Determination of recombinant adenovirus titer:

[0114] 4.1 Infection titer determination: K562 cells were plated at 2×10 5 cells / well were inoculated in a 24-well plate, and 0.00001μl, 0.0001μl, 0.001μl, 0.01μl, 0.1μl and 1μl of purified virus were added to K562 cells. After 48h of infection, the cells were collected and subjected to flow cytometry detection. The infection titer was calculated based on the positive rate of flow cytometry detection.

[0115] 4.2 Particle titer determination: The purified virus was diluted 5-fold, 10-fold, and 20-fold, respectively, and 1% SDS was added. The mixture was incubated at 55°C for 10 min, and the supernatant was collected by centrifugation. The OD260 value was determined using Nanodrop 2000 to calculate the particle titer.

[0116] B. Experimental Results

[0117] The results are shown in Table 3. There is no significant difference in the virus yields of different CAR structures, but the virus activity of AD.CAR1 and AD.CAR2 is poor, with a specific activity lower than 1 / 1000, indicating that the CAR structure has a greater impact on the virus yield. The infection titers of other viruses, including AD.CAR8, AD.CAR13, AD.CAR14, and AD.CAR16, were all greater than 1.0×10 10 / ml, and the specific activity was greater than 1 / 100IU, which met the quality standards of recombinant adenovirus.

[0118] Table 3 Statistics of recombinant adenovirus volume and titer

[0119]

[0120]

[0121] Example 3 Optimization of recombinant adenovirus transduction system

[0122] 1. Effect of different MOI of recombinant adenovirus on transduction efficiency.

[0123] A. Experimental Methods

[0124] 1.1 NK cell plating: Take the suspension of NK cells KA062 cultured to day 14 and place it in a centrifuge tube. Centrifuge at 1500 rpm for 8 min, discard the supernatant, resuspend in NK cell basal medium, and plate at 1×10 6 cells / 0.5mL / well system was inoculated in a 24-well plate, maintaining 2×10 6 The plating density is 2.5~3.0 mL.

[0125] 1.2 Adding recombinant adenovirus: Take out the recombinant adenovirus from the -80°C refrigerator and place it on an ice box to melt. After mixing, add the corresponding virus volume according to MOI=2500, MOI=5000 and MOI=10000 (VPs / cell).

[0126] 1.3 Rehydration: 4 hours after transduction, add 0.5 mL of NK cell complete medium containing double factors to each well.

[0127] 1.4 Centrifugation and medium replacement: 24 hours after transduction, take the transduced cell suspension and place it in a centrifuge tube. Centrifuge at 1500 rpm for 8 minutes, discard the supernatant, and resuspend the cells in NK cell expansion medium. The inoculation density is 1×10 6 / mL was inoculated into 24-well culture plates.

[0128] B. Experimental Results

[0129] 72 hours after transduction, appropriate amount of cells were taken for counting and flow cytometry detection of CAR19 expression, as shown in Table 4 and Figure 3 As shown in the figure, as the MOI increases, the transduction efficiency also increases. When MOI = 10000, the transduction efficiency is 77%, and the cell viability does not change significantly. However, the cell growth rate of the 5000VPs / cell group and the 10000VPs / cell group is significantly slower than that of the 2500VPs / cell group. Therefore, 2500VPs / cell is considered to be selected as the virus infection intensity, and at the same time, the transduction efficiency is improved by adding a transduction-promoting agent.

[0130] Table 4 Statistics of cell proliferation, cell viability and transduction efficiency after transduction at different MOIs

[0131] Group Cell expansion times Survival rate (%) Transduction efficiency (%) Comparison 2.43 94.59 0.07 2500VPs / cell 2.06 92.35 49.09 5000VPs / cell 1.78 93.89 64.95 10000VPs / cell 1.35 93.75 77.34

[0132] 2. Effect of adding transduction-promoting reagents on transduction efficiency

[0133] A. Experimental Methods

[0134] 2.1 NK cell plating: Take the suspension of NK cells WM005 cultured to day 14 and place it in a centrifuge tube. Centrifuge at 1500 rpm for 8 min, discard the supernatant, resuspend in NK cell basal medium, and plate at 1×10 6 cells / 0.5mL / well system was inoculated in a 24-well plate, maintaining 2×10 6 The plating density is 2.5~3.0 mL.

[0135] 2.2 Adding recombinant adenovirus: Take out the recombinant adenovirus from the -80°C refrigerator and place it on an ice box to melt. After mixing, add the corresponding volume of virus according to MOI = 2500.

[0136] 2.3 Add different concentrations of transduction-promoting reagents: 10 mM BX795 diluted 100 times and then add 2.5 μl, i.e. the final concentration is 0.5 μM; 10 mM BX795 diluted 100 times and then add 5 μl, i.e. the final concentration is 1 μM.

[0137] 2.4 Centrifugation and medium replacement: 24 hours after transduction, take the transduced cell suspension and place it in a centrifuge tube. Centrifuge at 1500 rpm for 8 minutes, discard the supernatant, and resuspend the cells in NK cell expansion medium. The inoculation density is 1×10 6 / mL was inoculated into 24-well culture plates.

[0138] B. Experimental Results

[0139] 72 hours after transduction, appropriate amount of cells were taken for counting and flow cytometry detection of CD19 scFv expression, as shown in Table 5 and Figure 4 As shown in the figure, under the same MOI conditions, adding a transduction-promoting reagent can greatly improve the transduction efficiency, which is more than 1 times higher, but has little effect on the growth of NK cells. Therefore, 0.5-1 μM is selected as the added concentration.

[0140] Table 5 Statistics of cell concentration, cell viability and transduction efficiency after transduction with different concentrations of transduction-promoting reagents

[0141] name Cell expansion times Survival rate (%) Transduction efficiency (%) Comparison 3.71 89.83 0.09 0μM BX795 3.54 88.25 33.64 0.5 μM BX795 3.55 88.31 70.42 1μM BX795 3.33 87.47 79.17

[0142] Example 4 Comparison of the functions of CAR-NK cells based on recombinant lentivirus and CAR-NK cells based on recombinant adenovirus

[0143] A. Experimental Methods

[0144] 1. Preparation of CAR19-NK cells based on recombinant adenovirus:

[0145] 1.1 NK cell plating: Take the suspension of NK cells WM005 cultured to day 14 and place it in a centrifuge tube. Centrifuge at 1500 rpm for 8 min, discard the supernatant, resuspend in NK cell basal medium, and plate at 1×10 6 cells / 0.5mL / well system was inoculated in a 24-well plate, maintaining 2×10 6 The plating density is 2.5~3.0 mL.

[0146] 1.2 Adding recombinant adenovirus: Take out the recombinant adenovirus from the -80°C refrigerator and place it on an ice box to melt. After mixing, add the corresponding volume of virus according to MOI = 2500.

[0147] 1.3 Add different concentrations of transduction-promoting reagents: 10 mM BX795 diluted 100 times and then add 2.5 μl, i.e. the final concentration is 0.5 μM; 10 mM BX795 diluted 100 times and then add 5 μl, i.e. the final concentration is 1 μM.

[0148] 1.4 Centrifugation and medium replacement: 24 hours after transduction, take the transduced cell suspension and place it in a centrifuge tube. Centrifuge at 1500 rpm for 8 minutes, discard the supernatant, and resuspend the cells in NK cell expansion medium. The inoculation density is 1×10 6 / mL was inoculated into 24-well culture plates and cultured for 2 days.

[0149] 1.5 Collect cells and detect transduction efficiency and killing activity.

[0150] 2. Preparation of CAR19-NK cells based on recombinant lentivirus:

[0151] 2.1 Culture to day 9, centrifuge at room temperature at 1800 rpm for 10 min to collect activated NK cells, resuspend the cells in complete medium, add the transduction reagent BX795 (0.1 μM), inoculate into 6-well plates at 2.0 ml / well, add recombinant lentivirus expressing CAR19, and the CAR structure is the same as CAR16.

[0152] 2.2 After mixing, place in an incubator and culture for 4 hours. Then add 2 ml of fresh complete medium and continue culturing.

[0153] 2.3 After 24 hours of culture, take out the cells and centrifuge at 1800 rpm for 10 minutes at room temperature to collect the cells. Harvest the precipitate by centrifugation. Resuspend the cells in expansion medium and add 1×10 6 The cells were expanded at a density of 10 cells / ml.

[0154] 2.4 Culture until the 7th day after transduction, collect cells, and detect transduction efficiency and killing effect.

[0155] B. Experimental Results

[0156] like Figure 5 As shown in Figure 2, the gene transduction efficiency of recombinant adenovirus is significantly higher than that of recombinant lentivirus; in addition, under the same effector-target ratio, the killing activity of NK cells based on recombinant adenovirus is significantly higher than that of CAR-NK cells based on recombinant lentivirus (such as Figure 6 Therefore, recombinant adenovirus has great advantages in preparing CAR-NK cells.

[0157] Example 5 Functional evaluation of CAR-NK cells based on recombinant adenovirus

[0158] 1. Preparation of CAR19-expressing NK cells

[0159] 1.1 NK cell plating: Take the NK cell suspension cultured to day 12 and place it in a centrifuge tube. Centrifuge at 1500 rpm for 8 min, discard the supernatant, resuspend it with NK cell basal medium, and plate at 4×10 8 cells / 200mL / bottle system was inoculated in a T225 culture flask and maintained at 2×10 6 The plating density is 2.5~3.0 mL.

[0160] 1.2 Add transduction-promoting reagents: Add 20 μl of 10 mM BX795, i.e. the final concentration is 1 μM.

[0161] 1.3 Adding recombinant adenovirus: Take out the recombinant adenovirus from the -80°C refrigerator and place it on an ice box to melt. After mixing, add the corresponding volume of virus according to MOI = 2500 (VPs / cell).

[0162] 1.4 Fluid replenishment: 4 hours after transduction, replenish each bottle with 200 mL of NK cell complete medium containing double factors.

[0163] 1.5 Centrifugation and medium replacement: 24 hours after transduction, take the transduced cell suspension and place it in a centrifuge tube. Centrifuge at 1500 rpm for 8 minutes, discard the supernatant, and resuspend the cells in NK cell expansion medium. The inoculation density is 1×10 6 / mL was inoculated into T225 culture flask.

[0164] 2. Evaluation of the killing function of CAR19-NK cells in vitro

[0165] A. Experimental Methods

[0166] 2.1 Target cells Raji, Daudi, Nalm-6 and H929 were stained with Calcein-AM and plated in 96-well plates at 20,000 cells / 100 μl;

[0167] 2.2 CAR19-NK cells transduced for 72 hours were added at effector-target ratios of 0.5:1, 1:1, and 2:1, respectively, and incubated in the dark for 4 hours;

[0168] 2.3 Use a multifunctional microplate reader to detect the OD value and calculate the killing efficiency of each group.

[0169] B. Experimental Results

[0170] like Figure 7 As shown, the killing efficiency of transduced CAR-NK cells on tumor cells is much greater than that of the NK cell group, and there is not much difference between the CAR-NK groups.

[0171] 3. Evaluation of the anti-tumor effect of CAR19-NK cells in vivo

[0172] A. Experimental Methods

[0173] 3.1 Raji cells, a human Burkkit lymphoma cell line in the logarithmic growth phase, were prepared into a single cell suspension and injected into the tail vein of mice;

[0174] 3.2 The mice were randomly divided into 6 groups: control group (Vehicle group and NK cell group) and experimental group CAR-NK (CAR13 and CAR16 group). Each group had 6 tumor-bearing mice, and each group was injected with 1.0×10 7 cell / 100 μl, administered once every 5 days, for a total of 4 times, the schedule is shown in Table 6;

[0175] 3.3 The tumor burden of each mouse was detected by in vivo imaging twice a week, and the body weight of each mouse was measured twice a week.

[0176] B. Experimental Results

[0177] Compared with the control group (Vehicle group), NK cell group and CAR13-NK cell group (non-complete functional CAR-NK cells, no intracellular signaling region), CAR16-NK cell therapy can significantly inhibit tumor growth and prolong the survival of tumor-bearing mice. At the same time, CAR16-NK treatment has no significant effect on the weight of mice, indicating that there are no significant toxic side effects. (such as Figure 8-11 (shown)

[0178] Table 6 Dosage regimen

[0179] Group No. Group Name Number of animals Dosage and frequency Route of administration 1 Vehicle Group 6 - iv 2 NK Group 6 1*10^7, Q5D*4 iv 3 CAR13 group 6 1*10^7, Q5D*4 iv 4 CAR16 group 6 1*10^7, Q5D*4 iv

[0180] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. An adenovirus vector, characterized in that: The adenoviral vector comprises a polynucleotide molecule encoding a CAR targeting CD19, wherein the polynucleotide molecule encoding a CAR targeting CD19 comprises a nucleotide sequence shown in SEQ ID NO: 11, The nucleotide sequences encoding EF1α, CAR targeting CD19, BGHpa, hPGKp, sIL-15, and SV40 PA in the polynucleotide molecule are sequentially connected, The CAR includes a CD8α signal peptide, a ScFv targeting CD19, a CD8α hinge region, a CD8α transmembrane region, a 4-1BB co-stimulatory region, and a CD3ζ signal transduction region, which are connected in sequence.

2. The adenoviral vector according to claim 1, characterized in that The amino acid sequence of the sIL-15 molecule in the polynucleotide molecule is shown in SEQ ID NO:

14.

3. The adenoviral vector according to claim 2, characterized in that The nucleotide sequence encoding the sIL-15 molecule is shown in SEQ ID NO:

13.

4. The adenoviral vector according to claim 1, characterized in that The nucleotide sequence encoding EF1α in the polynucleotide molecule is shown in SEQ ID NO:

15.

5. The adenoviral vector according to claim 1, characterized in that The nucleotide sequence encoding BGHpa in the polynucleotide molecule is shown in SEQ ID NO:

16.

6. The adenoviral vector according to claim 1, characterized in that The nucleotide sequence encoding hPGKp in the polynucleotide molecule is shown in SEQ ID NO:

17.

7. The adenoviral vector according to claim 1, characterized in that The nucleotide sequence encoding SV40 PA in the polynucleotide molecule is shown as SEQ ID NO:

18.

8. The adenoviral vector according to claim 1, characterized in that The nucleotide sequences encoding EF1α, CAR, BGHpa, hPGKp, sIL-15 and SV40 PA in the polynucleotide molecule are connected by P2A, T2A or E2A.

9. A cell, characterized in that The cell comprises the adenoviral vector according to any one of claims 1 to 8.

10. The cell according to claim 9, characterized in that The cells include eukaryotic cells or prokaryotic cells.

11. The cell according to claim 9, characterized in that The cells include T cells, B cells, or NK cells.

12. A method for preparing a CAR cell targeting CD19, characterized in that: The method comprises transducing the adenoviral vector according to any one of claims 1 to 8 into cells.

13. The method according to claim 12, characterized in that The method specifically comprises the steps of: 1) synthesizing the signal peptide, ScFv, hinge region, transmembrane region, co-stimulatory region, signal transduction region and the nucleotide fragments of BGHpa, hPGKp, sIL-15, SV40PA and EF1α described in claim 4 as described in claim 1 by molecular biology techniques; 2) connecting the nucleotide fragments in step 1) in the order of the nucleotide sequence in claim 1, and cloning the complete nucleotide sequence into an adenovirus vector; 3) Select mononuclear cells from the healthy lymphatic system and inoculate them into NK cell activation medium and NK cell expansion medium for 10-15 days; 4) The cultured cells were inoculated into NK cell basal medium, the adenovirus vector was used to infect the cells, BX795 transduction promoter was added, the transduction was continued for 4 hours, an equal volume of NK cell complete medium containing double factors was added, the transduction was continued for 24 hours, the medium was replaced by centrifugation, and the NK cell expansion medium was used for further culture, and the cells were transduced for 3 days to obtain CAR cells.

14. The method according to claim 13, characterized in that The NK cell activation medium is NK MACS containing 5% autologous plasma, 1000 IU / mL IL-2, and 500 IU / mL IL-15.

15. The method according to claim 13, characterized in that The NK cell expansion medium is NK MACS containing 5% serum replacement, 1000 IU / mL IL-2, and 500 IU / mL IL-15.

16. The method according to claim 13, characterized in that The NK cell basal culture medium is NK MACS.

17. The method according to claim 13, characterized in that The NK cell complete culture medium is NK MACS containing 2000 IU / mL of IL-2 and 1000 IU / mL of IL-15.

18. The method according to claim 13, characterized in that The molecular biology techniques include PCR, restriction enzyme digestion, and homologous recombination.

19. The method according to claim 13, characterized in that The connection method in step 2) is forward insertion, and the connection site is PmeI.

20. The method according to claim 13, characterized in that The cells in step 3) are NK cells.

21. The method according to claim 13, characterized in that In the step 4), the MOI of cells infected with adenovirus vector is 2500 VPs / cell.

22. A cell therapy composition for treating or preventing cancer, characterized in that: The cell therapy composition comprises the cells described in any one of claims 9 to 11, or the cells prepared by the method described in any one of claims 12 to 21 as an active ingredient.

23. Use of the adenovirus vector according to any one of claims 1 to 8 or the cell according to any one of claims 9 to 11 in the preparation of a pharmaceutical composition for treating CD19-positive cancer, wherein the CD19-positive cancer is myelodysplasia, B-cell acute lymphoblastic leukemia, T-cell acute lymphoblastic leukemia, small lymphocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, or lymphoma.

24. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the cell according to any one of claims 9 to 11.

25. The pharmaceutical composition according to claim 24, characterized in that The pharmaceutical composition includes pharmaceutically acceptable excipients.

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