CN101307327A - A kind of CEA recombinant adeno-associated virus vector and its construction method and application - Google Patents

Abstract

A CEA recombinant adeno-associated virus vector and its construction method and application. The recombinant adeno-associated virus vector is a recombinant adeno-associated virus vector obtained by replacing the adeno-associated virus structural gene in the adeno-associated virus vector with the carcinoembryonic antigen CEA gene or its mutant gene. The CEA recombinant adeno-associated virus vector of the present invention can deliver the wild-type or mutant carcinoembryonic antigen gene carried by it into the monocyte-macrophage-dendritic cell line, and the cells carrying these specific antigen genes Used to stimulate effector cells of the immune system. Experiments have proved that the CTL induced by DC infected by rAAV of the present invention can effectively inhibit the growth of malignant tumor cells or kill tumor cells in patients. Therefore, the CEA recombinant adeno-associated virus vector of the present invention or the recombinant adeno-associated virus Products related to related viral vectors can be used to prepare drugs for treating breast cancer, colon cancer, gastric cancer and lung adenocarcinoma.

Description

A kind of CEA recombinant adeno-associated virus vector and its construction method and application

technical field

The present invention relates to a vector and its application, in particular to a recombinant adeno-associated virus vector and its construction method and its application in the preparation of antitumor drugs.

Background technique

The genetic structure of adeno-associated virus (AAV) has been identified. In 1983, Samulski et al. described the terminal repeat segment of AAV (upstream 5' end segment, downstream 3' end segment) (Samulski RJ, Srivastava A, Berns KI, Muzyczka N. Rescue of adeno-associated virus from recombinant plasmamids: gene correction within the terminal repeats of AAV. Cell. 33:135-143.). In 1984, Hermonat et al. described the low infection particle (lip) gene and envelope (cap) gene of AAV (Hermonat PL, Labow MA, Wright R, Berns KI, Muzyczka N. Genetics ofadeno-associated virus: isolation and preliminary characterization of adeno-associated virus type 2 mutants. J Virol. 51: 329-339. Hermonat, P.L., and Muzyczka, N. Use of adeno-associated virus as a mammalian DNA cloning vector: transduction of neomycin resistance into mammalian tissue Proculture.culture. . Acad. Sci. U.S.A. 81:6466-6470.). In 1986, Labow et al. identified the p5 promoter located between the upstream 5' end fragment and the rep gene (Labow MA, Hermonat PL, Berns KI. Positive and negative autoregulation of the adeno-associated virus type 2 genome. J Virol. 160: 251-258.).

In 1984, Professor Paul L. Hermonat, one of the main technical directors of Broadway Gene International Co., Ltd., took the lead in proving that AAV vectors could be used for gene therapy of human diseases (Hermonat, P.L., and Muzyczka, N.Use of adeno-associated virus as a mammalian DNA cloning vector: transduction of neomycin resistance into mammalian tissue culture cells. Proc. Natl. Acad. Sci. U.S.A. 81: 6466-6470.). At present, mainly European and American countries are conducting clinical trials of AAV-based gene therapy for human diseases. According to the statistics of the US Food and Drug Administration, more than ten AAV-based gene therapy clinical trials are currently underway, mainly injecting AAV viruses carrying therapeutic genes into patients so that they can express therapeutic genes in vivo, so as to achieve therapeutic effect. disease purpose. The diseases mainly targeted for treatment include non-neoplastic diseases such as Parkinson's syndrome, rheumatoid arthritis, hemophilia, heart failure, progressive muscular atrophy and Alzheimer's syndrome. However, there are still some problems in the AAV virus used in clinical treatment, such as the obvious limitation of the size of the therapeutic gene carried, the instability of the virus itself, resulting in unstable expression of the therapeutic gene, and inconsistent efficacy. Although the immunogenicity of the AAV virus itself is very weak, the expressed therapeutic gene can easily induce autoimmune reactions in patients, and even lead to severe toxic side effects.

AAV is a non-pathogenic, defective virus that requires the assistance of gene products from other viruses (such as adenovirus) to assemble into infectious virus particles. The total length of the AAV genome is about 4700 base pairs (bp), with repeated terminal fragments (TR) at both ends, and viral structural genes in the middle, including the Rep gene and the viral capsid Cap gene related to viral replication. Due to the instability of the AAV virus itself and the limited length of the exogenous gene (therapeutic gene) it carries, it is necessary to recombine it to form a recombinant adeno-associated virus (rAAV). A large number of existing studies have shown that deleting the structural genes in the AAV genome can significantly increase the capacity of exogenous genes. In addition, exogenous genes with therapeutic effects are inserted into rAAV to prepare infectious rAAV virus particles, which are injected into patients to infect cells in the body, and then express therapeutic genes, so as to achieve the effect of treating diseases. At present, rAAV is mainly used in the treatment of non-tumor diseases such as Parkinson's syndrome, rheumatoid arthritis, hemophilia, heart failure, progressive muscular atrophy and Alzheimer's syndrome. However, rAAV still has some shortcomings, such as the instability of recombinant virus, low virus titer, and limited capacity for accepting therapeutic genes (generally, the maximum exogenous gene fragment that can be inserted is about 2000 base pairs (bp), otherwise the stability of rAAV sex will be destroyed). Therefore, it is necessary to design a more reasonable recombinant adeno-associated virus (rAAV) vector to meet the needs of practical applications.

Contents of the invention

The object of the present invention is to provide a recombinant adeno-associated virus (rAAV) vector with high stability and large capacity carrying exogenous gene (CEA).

The CEA recombinant adeno-associated virus vector provided by the present invention is a recombinant adeno-associated virus obtained by replacing the adeno-associated virus structural gene in the adeno-associated virus (AAV) vector with the carcinoembryonic antigen CEA (chorioembryonic antigen) gene or its mutant gene carrier.

The recombinant adeno-associated virus vector of the present invention is modified on the basis of known adeno-associated virus vectors, the structural genes of the adeno-associated virus are Rep and Cap/Lip genes, and the carcinoembryonic antigen (CEA) gene is a Tumor antigen gene, the mutant gene is a carcinoembryonic antigen gene fragment with the same function.

Known adeno-associated virus vectors have a p5 promoter, in order to increase the transcription level of the target gene, the p5 promoter in the recombinant adeno-associated virus vector can be further replaced with a macrophage virus (cytomegalovirus, CMV) promoter, One or more of the beta-actin promoter and the SV40 viral promoter.

The second object of the present invention is to provide a method for constructing the above-mentioned CEA recombinant adeno-associated virus vector.

The construction method provided by the present invention uses a conventional gene recombination method to first delete the adeno-associated virus structural gene in the adeno-associated virus vector, and then replace the deleted gene with the carcinoembryonic antigen CEA gene or its mutant gene to obtain CEA recombinant adeno-associated virus vector.

In the construction method of the above-mentioned CEA recombinant adeno-associated virus vector, in order to improve the transcription level of the target gene, the p5 promoter in the recombinant adeno-associated virus vector can be further replaced with a macrophage virus promoter, beta-actin promoter and one or more of the SV40 viral promoters.

Products related to the CEA recombinant adeno-associated virus vector of the present invention include recombinant adeno-associated virus plasmids, recombinant adeno-associated virus particles and cell lines infected or transfected by the recombinant adeno-associated virus vector of the present invention, such as monocytes-macrophages - dendritic cell lines, T lymphocyte lines, etc. In cell lines such as T lymphocytes, the expression is obtained under the action of a transcriptional promoter), etc., etc., all belong to the protection scope of the present invention.

In terms of medical use, another object of the present invention is to provide an antitumor drug.

The active ingredient of the antitumor drug provided by the present invention is the above-mentioned CEA recombinant adeno-associated virus vector or products related to the recombinant adeno-associated virus vector of the present invention.

For example, using the CEA recombinant adeno-associated virus of the present invention as a carrier, the tumor antigen-wild type and/or mutant carcinoembryonic antigen CEA is introduced into the monocyte-macrophage-dendritic cell line, and the dendritic cells are induced , in order to achieve the purpose of immunostimulation in vitro and in vivo, to treat certain tumors, or to treat certain tumors with cytotoxic T lymphocytes (such as but not limited to T lymphocytes and B lymphocytes) stimulated by the dendritic cells some tumors.

The antitumor drug can be used for treating CEA antigen-positive malignant tumors such as breast cancer, colon cancer, gastric cancer and lung adenocarcinoma.

The medicine of the present invention can adopt dosage forms such as solvent or powder.

The choice of the solvent is various, such as cell culture medium (base), physiological saline or phosphate buffer saline, etc. are all available.

When necessary, one or more pharmaceutically acceptable carriers can also be added to the above drugs. The carrier includes conventional diluents, absorption enhancers, surfactants and the like in the field of pharmacy.

The way of administration can be to isolate the mononuclear cells in the body of the tumor patient first, and then infect or transfect the mononuclear cells of the patient with this drug. Or reinfuse the cytotoxic T lymphocytes stimulated by the mature dendritic cells transformed with the wild-type and/or mutant carcinoembryonic antigen CEA gene into the tumor patient.

The dosage of the above drugs is generally 100 μl/5×10 ⁶ /each time, twice a month, and the course of treatment is usually 6 months. Both the dose and the course of treatment can be adjusted according to the actual situation.

In order to improve the curative effect, the medicine of the present invention can also be combined with antibiotics, immune stimulants and tumor targeting drugs.

The invention also provides a method for killing tumors in vitro.

The method for killing tumor provided by the present invention may comprise the following steps:

1) The monocyte-macrophage-dendritic cell or T lymphocyte naturally produced in the tumor system (this system can be produced by artificial simulation) is recombined with the wild-type carcinoembryonic antigen CEA gene of the present invention The adeno-associated virus vector and/or the recombinant adeno-associated virus vector carrying the mutant carcinoembryonic antigen CEA gene is infected or transfected, or is treated with a product related to the CEA recombinant adeno-associated virus vector of the present invention, and the treated cells are obtained respectively;

2) adding the monocyte-macrophage-dendritic cells treated in step 1) into the system where the tumor is located to kill the tumor; or combining untreated T lymphocytes with the treated monocyte- Macrophage-dendritic cell mixed culture to form antigen-specific cytotoxic T lymphocytes, and then add the antigen-specific cytotoxic T lymphocytes to the system where the tumor is located to kill the tumor; or combine the treated T lymphocytes with untreated T lymphocytes The treated monocytes-macrophages-dendritic cells are added to the system where the tumor is located to kill the tumor.

The method for killing tumors described in the present invention can be specifically applied to tumor treatment, including giving a tumor patient a reinfusion of antigen-specific cytotoxic T lymphocytes, which are composed of naturally occurring T lymphocytes derived from the patient and T lymphocytes derived from the patient. The patient's monocyte-macrophage-dendritic cell mixed culture was produced. Before the mixed culture, these monocyte-macrophage-dendritic cells have been infected by the recombinant adeno-associated virus carrying the wild-type carcinoembryonic antigen CEA gene and/or the recombinant adeno-associated virus carrying the mutant carcinoembryonic antigen CEA gene of the present invention. Infection or transfection with related viral vectors, or treatment with products related to the CEA recombinant adeno-associated viral vector of the present invention;

Alternatively, a tumor patient is given a reinfusion of monocyte-macrophage-dendritic cells derived from the patient. Before reinfusion, these monocyte-macrophage-dendritic cells have been infected by the recombinant adeno-associated virus carrying the wild-type carcinoembryonic antigen CEA gene and/or the recombinant adeno-associated virus carrying the mutant carcinoembryonic antigen CEA gene of the present invention. Infection or transfection with related viral vectors, or treatment with products related to the CEA recombinant adeno-associated viral vector of the present invention;

Alternatively, a tumor patient is given a reinfusion of the above-mentioned T lymphocytes derived from the patient and the naturally occurring monocyte-macrophage-dendritic cells derived from the patient. Before reinfusion, these T lymphocytes have been infected or transfected by the recombinant adeno-associated virus carrying the wild-type carcinoembryonic antigen CEA gene and/or the recombinant adeno-associated virus vector carrying the mutant carcinoembryonic antigen CEA gene of the present invention, or by Product handling related to the CEA recombinant adeno-associated virus vector of the present invention.

The invention provides a recombinant adeno-associated virus (rAAV) vector with high stability and large capacity of exogenous gene (CEA). In the rAAV vector of the present invention, the AAV structural genes Rep and Cap/Lip genes are replaced by wild-type or mutant carcinoembryonic antigen (chorioembryonic antigen, CEA) genes. The recombinant adeno-associated virus vector of the present invention can deliver the wild-type or mutant carcinoembryonic antigen CEA gene carried by it into the monocyte-macrophage-dendritic cell line, and the cells carrying these specific antigen genes Used to stimulate effector cells of the immune system (not limited to T and B lymphocytes). Experiments have proved that the CTL induced by DC infected by rAAV of the present invention can effectively inhibit the growth of malignant tumor cells or kill tumor cells in patients. Therefore, the CEA recombinant adeno-associated virus vector of the present invention or recombinant Products related to adeno-associated virus vectors can be used to prepare antitumor drugs. The invention has important theoretical and practical significance in the clinical application of CEA antigen-positive malignant tumors such as breast cancer, colon cancer, gastric cancer and lung adenocarcinoma, and has broad application prospects.

The present invention will be described in further detail below in conjunction with specific embodiments.

Description of drawings

Figure 1 is a schematic diagram of the structure of the recombinant adeno-associated virus vector

Figure 2A is the result of enzyme digestion detection of recombinant adeno-associated virus vector rAAV/CEA

Figure 2B is the PCR detection result of recombinant adeno-associated virus vector rAAV/CEA

Figure 3 is a flow chart for the preparation of recombinant adeno-associated virus rAAV

Figure 4 is the virus titer detection result of recombinant adeno-associated virus rAAV/CEA

Figure 5 is the experimental process for killing tumors based on monocytes of tumor patients infected by one or more rAAV viruses carrying tumor antigen genes

Figure 6 is the detection result of the efficiency of recombinant adeno-associated virus rAAV/CEA infecting peripheral blood mononuclear cells

Figure 7 shows the detection results of CD80, CD83 and CD86 levels in DCs infected with recombinant adeno-associated virus rAAV/CEA

Figure 8 is the detection result of IFN-γ expression level of CTL induced by rAAV/CEA-infected DC

Figure 9 shows the killing of tumor cells by CTLs induced by rAAV/CEA-infected DCs and the detection results of killing specificity

Figure 10 shows the imaging observation results of the changes of metastatic lesions before and after treatment with rAAV/CEA-infected DCs in a patient with colon cancer

Figure 11 shows the changes of CTL tumor antigen levels in the serum of four patients with metastatic colorectal cancer before and after treatment with rAAV/CEA-infected DCs

Detailed ways

The methods used in the following examples are conventional methods unless otherwise specified, and the specific steps can be found in: "Molecular Cloning: A Laboratory Manual" (Sambrook, J., Russell, David W., Molecular Cloning: A Laboratory Manual, 3rd edition , 2001, NY, Cold Spring Harbor).

The percentage concentrations are mass/volume (W/V) percentage concentrations or volume/volume (V/V) percentage concentrations unless otherwise specified.

The primers used, DNA sequence synthesis and DNA sequence determination were all completed by Invitrogen Company of the United States.

Example 1. Construction and detection of recombinant adeno-associated virus vectors rAAV/CEA and rAAV/mCEA

Materials and their sources:

A. The pBR322 plasmid (named pBR-AAV2) carrying the whole genome DNA of AAV type 2: prepared by Professor Paul L. Hermonat, one of the main technical directors of the United States Broadway Gene International Co., Ltd. (Hermonat, P.L., and Muzyczka, N. Use of adeno-associated virus as a mammalian DNA cloning vector: transduction of neomycin resistance into mammalian tissue culture cells. Proc. Natl. Acad. Sci. U.S.A. 81: 6466-6470.).

B. Human primary colon cancer cells: isolated from cancer tissues of colon cancer patients or obtained from commercial sources, immunohistochemically confirmed positive for carcinoembryonic antigen (CEA).

C. The pCI-neo plasmid carrying the CMV promoter was purchased from Promega Corporation in the United States, and the plasmid pSG424 carrying the SV40 early promoter was purchased from Clonitic Corporation in the United States.

D. Nucleotide primers for gene amplification: designed by our company based on the published carcinoembryonic antigen (CEA) gene sequence in the American Gene Bank (US NCI Gene Bank: M29540).

Construct the recombinant adeno-associated virus vector (as shown in Figure 1) of the present invention carrying carcinoembryonic antigen CEA (chorioembryonic antigen) gene or its mutant gene (as shown in Figure 1) with following method, concrete process comprises the following steps:

1. Construction of recombinant adeno-associated virus vector

A. The transformation of the pBR-AAV2 plasmid, the specific method is: first use the restriction endonuclease Bst98I and HpaI (purchased from the U.S. Promega company) to make the structural gene Rep and Lip/Cap of the adeno-associated virus AAV genome in the pBR-AAV2 plasmid The gene was completely excised, and the reaction system was: 1 μg pBR-AAV2, 10U Bst98I, 10U Hpa I, 2.5 μl 10× buffer D and 19.5 μl deionized water; the reaction conditions were: water bath at 37°C for 4 hours. Then, insert the nucleotide sequence (CGAATTCATGCGATATCGTT) containing restriction endonuclease EcoR I and EcoR V restriction sites into the plasmid, the reaction system is: 500ng plasmid, 300ng EcoR I and the nucleotide sequence of EcoR V, 10IU T ₄ DNA ligase (purchased from Promega, USA), 1.5 μl of 10×T ₄ DNA ligation buffer and 11.5 μl of deionized water; the reaction conditions were: water bath at 4° C. for 8 hours. Then, retain the complete TR sequence at both ends or insert a fragment consisting of 9 nucleotides at the 75th nucleotide sequence of the TR at both ends of the AAV genome: CTGCGCTGG, the purpose is to improve the stability of the rAAV virus and improve The replication efficiency of the virus, the method is: first cut the TR at both ends with the restriction endonuclease Ban I (purchased from Promega, USA), and the reaction system is: 1 μg of the plasmid prepared above, 10U Ban I, 1.5 μl 10× buffer G and 11.5 μl of deionized water; the reaction conditions are: in a water bath at 37°C for 4 hours, and then insert 9 nucleotide fragments into the plasmid, the reaction system is: 500ng plasmid, 300ng 9 nucleotide sequences, 10IU T ₄ DNA Ligase (purchased from Promega, USA), 1.5 μl of 10×T ₄ DNA ligation buffer and 11.5 μl of deionized water; reaction conditions: water bath at 4° C. for 8 hours.

B. Using gene amplification technology (polymerase chain reaction, PCR) to amplify the CMV promoter and SV40 early promoter. The specific method is: first use the pCI-neo plasmid (purchased from Promega, USA) as a template, and PCR amplify the CMV promoter under the guidance of primer 1: AGATCTTCAATATTGGCCAT and primer 2: TGTCAGAAGCACTGACTGC, the PCR amplification conditions are: first 94 ° C 4 Minutes; then 94°C for 30 seconds, 60°C for 35 seconds, 72°C for 1 minute, a total of 30 cycles; finally 72°C for 8 minutes, after the reaction, the PCR product was detected by 1.2% agarose gel electrophoresis, and it appeared at 740bp An expected specific band, the target band was recovered and purified to obtain the CMV promoter. Using the pSG424 plasmid (purchased from Clonitic, USA) as a template, under the guidance of primer 3: GAACCAGCTGTGGAATGTGTC and primer 4: TCAGGAAGCTTAGATCTAGC, the SV40 early promoter was amplified by PCR. The PCR amplification conditions were: first 94°C for 4 minutes; then 94°C 30 seconds at 60°C, 35 seconds at 72°C, 40 seconds at 72°C, a total of 30 cycles; the last 8 minutes at 72°C, after the reaction, the PCR product was detected by 1.2% agarose gel electrophoresis, and an expected specificity appeared at 359bp The target band was recovered and purified to obtain the SV40 early promoter.

C.PCR amplification of beta actin promoter, full-length CEA cDNA and partial CEA cDNA fragments (respectively named A (from 5' end 115-717 bases), B (from 5' end 718-1353 base), C (base 1354-2223 from the 5' end), this embodiment takes the above fragment as an example but is not limited to the above fragment, other CEA cDNA fragments with the same function as CEA cDNA can be used to construct this Invented recombinant adeno-associated virus vector), the specific method is: use nucleic acid isolation technology to isolate total DNA and mRNA from human primary colon cancer cells (the total DNA and mRNA can also be artificially synthesized or obtained from commercial channels), and then Using the total DNA as a template, under the guidance of primer 5: CCCGGGCCCAGCACCCCAAG and primer 6: CATCCATGGTGAGCTGCG, the beta-actin promoter was amplified by PCR. The PCR amplification conditions were as follows: first 94°C for 4 minutes; then 94°C for 30 seconds; second, 72°C for 1 minute and 20 seconds, a total of 30 cycles; the last 72°C for 8 minutes, after the reaction, the PCR product was detected by 1.2% agarose gel electrophoresis, and an expected specific band appeared at 1176bp. The target band is recovered and purified to obtain the beta actin promoter. Then reverse-transcribe the mRNA to synthesize its cDNA and use it as a template. Under the guidance of primer 7: CATGGAGTCTCCCTCGGC and primer 8: TGCTATATCAGAGCGCAACC, the full-length CEA cDNA is amplified by PCR. The PCR amplification conditions are: first 94°C for 4 minutes; 30 seconds at ℃, 35 seconds at 64℃, 2 minutes and 30 seconds at 72℃, a total of 30 cycles; the last 8 minutes at 72℃, after the reaction, the PCR product was detected by 1.2% agarose gel electrophoresis, and an expected line appeared at 2120bp The specific band of the target band was recovered and purified to obtain the full-length CEA cDNA, and the same method as above was used to obtain CEA cDNA fragment A (primer 7 and primer 9: AGTGAGGGTCCTGTTGCCATTG), CEA cDNA fragment B (primer 10: CTATTCAATGTCACAAGAAATG and Primer 11: ATAGAGGACATTCAGGATG), CEA cDNA Fragment C (Primer 12: GGCCCAGACGACCCCAC and Primer 8).

D. Using DNA ligation technology, insert the above-mentioned amplified CMV promoter, SV40 early promoter, beta-actin promoter, full-length CEA cDNA or partial CEA cDNA fragments into the modified pBR-AAV2 vector of step A in sequence, For inserting the promoter, first carry out enzyme digestion reaction, then carry out ligation reaction, wherein, enzyme digestion reaction system is: 1 μ g plasmid; 10 U restriction endonucleases BamH I and Sal I (purchased from American Promega Company), 2.5 μ l 10× Buffer C and 19.5 μl deionized water; the reaction conditions are: water bath at 37°C for 4 hours, the ligation reaction system is: 500ng digested plasmid, 300ng promoter DNA, 10IU T ₄ DNA ligase (purchased from Promega company), 1.5 μl 10×T ₄ DNA ligation buffer and 11.5 μl deionized water; the reaction conditions were: water bath at 4° C. for 8 hours. Then, the plasmid carrying the promoter and the full-length PSA cDNA were digested with restriction endonucleases EcoR I and EcoR V, respectively. The system and conditions for enzyme cleavage reaction and ligation reaction are the same as above. Finally, recombinant adeno-associated virus vectors (named rAAV/CEA) carrying CMV promoter, SV40 early promoter, beta actin promoter and full-length CEA cDNA, and carrying CMV promoter, SV40 early promoter were respectively obtained. , beta-actin promoter and A or B or C different CEA cDNA fragments (mutant) recombinant adeno-associated virus vectors (respectively named rAAV/AmCEA, rAAV/BmCEA and rAAV/CmCEA, collectively named rAAV/mCEA) .

E. Introduce the ligated DNA-rAAV/CEA and rAAV/mCEA into genetically engineered Escherichia coli (E.coli) DH5α competent cells (Invitrogen, USA), and use LB plates containing 100 μg/mL ampicillin for resistance screening , pick a single white colony, extract the plasmid and purify it to obtain rAAV/CEA plasmid and rAAV/mCEA plasmid.

2. Detection of recombinant adeno-associated virus vector

First, the purified rAAV/CEA plasmid and rAAV/mCEA plasmid obtained in step 1 were digested with restriction enzymes (EcoR I&BamH I, Not I, BamH I&Nsi I in turn), and the restriction enzymes used were purchased from From the United States Promega company. At the same time, the rAAV vector without the CEA gene was used as a negative control (the CMV promoter, the SV40 early promoter, and the beta-actin promoter were inserted into the modified pBR-AAV2 vector in step A in sequence, and the vector was treated with a restriction endonuclease Pst I enzyme digestion), after the reaction finished, the enzyme digestion product was carried out to 1.2% agarose gel electrophoresis detection, wherein the detection result of rAAV/CEA plasmid is as shown in Figure 2A (1.DNA molecular weight standard. 2.No CEA gene rAAV vector (Pst I endonuclease). 3.rAAV/CEA (EcoR I and BamH I endonuclease). 4.rAAV/CEA (Not I endonuclease). 5.rAAV/CEA (BamH I and Nsi I endonuclease).), a specific band of 2115bp was obtained by digestion with EcoR I and BamHI, a specific band of 4kb was obtained by digestion with Not I, and a specific band of 1.3kb and The specific band of 2.1kb is consistent with the expected result. The enzyme digestion results of rAAV/mCEA plasmids were also consistent with the expected results. The rAAV/CEA plasmid and the rAAV/mCEA plasmid were further detected by gene amplification (PCR), wherein the detection results of the rAAV/CEA plasmid were as shown in Figure 2B (1. DNA molecular weight standard. 2. Negative control. 3. The PCR amplified product of CEA cDNA.), obtained the expected specificity band of 2115bp through amplification. The PCR detection results of the rAAV/mCEA plasmid were also consistent with the expected results (the size of the amplified product was rAAV/AmPSA: 603bp, rAAV/BmPSA: 636bp, rAAV/CmPSA: 870bp). The above detection results show that the recombinant adeno-associated virus vector rAAV/CEA carrying the carcinoembryonic antigen (CEA) gene and the recombinant adeno-associated virus vector rAAV/CEA carrying the carcinoembryonic antigen (CEA) mutant gene with the correct insertion position and sequence have been obtained. mCEA.

Embodiment 2, preparation of recombinant adeno-associated virus (rAAV) and virus titer determination

Materials and their sources:

A. The recombinant adeno-associated virus vector rAAV/CEA carrying the carcinoembryonic antigen (CEA) gene constructed in Example 1 and the recombinant adeno-associated virus vector rAAV/mCEA (rAAV/AmCEA, rAAV/BmCEA and rAAV/CmCEA).

B. Auxiliary plasmid pHelper containing AAV Rep gene and Lip/Cap gene: It was constructed by Professor Liu Yong from the Gene Therapy Center of the Affiliated Hospital of the University of Arkansas School of Medicine. (Liu, Y., Chiriva-Internati, M., Grizzi, F. Salati, E., Roman, J.J., Lim S., and Hermonat, P.L. Rapid induction of cytotoxic T cell response against cervical cancer cells by humanpapillomavirus type 16 E6 antigen gene delivery into human dendritic cellsbv an adeno-associated virus vector. Cancer Gene Therapy 8:948-957.).

C. AAV-HEK293 cells containing adenovirus genes (E1, E2A, E4, VAI, and VAII genes) integrated in the cell chromosome and expressed: established by the Gene Therapy Center of the Affiliated Hospital of the University of Arkansas School of Medicine in the United States. (Liu, Y., Chiriva-Internati, M., Grizzi, F. Salati, E., Roman, J.J., Lim S., and Hermonat, P.L. Rapid induction of cytotoxic T cell response against cervical cancer cellsbv human papillomavirus type 16 E6 Antigen gene delivery into human dendritic cells bv an adeno-associated virus vector. Cancer Gene Therapy 8:948-957.).

D. Lipofectin, a liposome transfection reagent: purchased from Invotrogen, USA.

E. DMEM medium and fetal bovine serum (or calf serum): purchased from Cellgro, USA.

F. PCR DIG labeling kit and DIG hybridization detection kit: purchased from Roche, Switzerland.

G. DNA copy number standards: 10 ¹² copies (copies)/μl to 10 ⁹ (copies)/μl respectively, purchased from Promega, USA.

1. Preparation of recombinant adeno-associated virus (rAAV)

Referring to Figure 3, the following method is used to prepare recombinant adeno-associated virus (rAAV). Take the preparation of a 10.0 cm cell culture dish as an example. %, proceed as follows:

A. Follow the instructions of Lipofectin: Mix 1.0 μg rAAV vector (rAAV/CEA or rAAV/mCEA), 1.0 μg pHelper plasmid, 4.0 μl Lipofectin and 50.0 μl DMEM containing 10% fetal calf serum (or calf serum) The culture medium was mixed and allowed to stand at room temperature for 20 minutes.

B. Add the mixed solution into the cell culture dish, and continue to culture in the carbon dioxide cell incubator.

C. After 72 hours, harvest all the cells and culture medium in the culture dish.

D. After vigorously shaking for 1 minute, centrifuge to save the supernatant, that is, the rAAV virus liquid.

E. Sterilize the collected rAAV virus liquid by filtration. The obtained rAAV viruses carrying tumor-associated antigen gene-carcinoembryonic antigen gene full-length CEA cDNA or partial CEA cDNA fragments (A, B, C mutant genes) were named rAAV/CEA, rAAV/AmCEA, rAAV/BmCEA, rAAV/BmCEA, respectively. rAAV/CmCEA.

2. Determination of virus titer of recombinant adeno-associated virus (rAAV)

The various rAAV viruses (rAAV/CEA, rAAV/AmCEA, rAAV/BmCEA, rAAV/CmCEA) obtained in step 1 were subjected to virus titer determination by conventional dot blot method. The specific method included the following steps: Needles are specific probes for tumor antigen genes.

A. Using the conventional DNA phenol/chloroform extraction method, rAAV virus particle DNA was extracted.

B. Place the nylon membrane in a dot blot apparatus, add alkali-denatured rAAV virus particle DNA, and add a DNA copy number standard, and then vacuumize.

C. After taking out the nylon film and drying it, fix it with UV light.

D. Use the PCR DIG labeling kit and refer to the kit instructions to prepare DIG-labeled specific probes. The probes are the CEA cDNA obtained in step C of Example 1. After the PCR amplification, the PCR amplification products were subjected to 1.2% agarose gel electrophoresis, and the PCR amplification products were detected under ultraviolet light, and positive bands appeared in all the results, indicating that the probes were successfully labeled.

E. Using the DIG hybridization detection kit and referring to the kit instructions, carry out DNA hybridization on various rAAV virus particle DNAs in a hybridization oven.

Among them, the detection results of rAAV/CEA are shown in Figure 4, the virus titer of rAAV/CEA is 10 ¹² copies/mL, and the virus titer of rAAV/mCEA is 10 ¹¹ -10 ¹² copies/mL.

Example 3. Tumor killing experiment of introducing tumor antigen into monocyte-macrophage-dendritic cell line

Materials and their sources:

A. rAAV virus: rAAV/CEA and rAAV/mCEA (rAAV/AmCEA, rAAV/BmCEA, rAAV/CmCEA,).

B. AIM-V cell culture medium: purchased from Invitrogen, USA.

C. Cytokines: colony cell stimulating factor (GM-CSF), interleukins 2, 4, 7 (IL-2, 4, 7) and tumor necrosis factor (TNF-α) were purchased from American R&D Corporation.

1. Tumor killing experiment

As shown in Figure 5, the whole process of the tumor killing experiment based on one or more rAAV viruses carrying tumor antigen genes (carcinoembryonic antigen CEA gene and mutant gene thereof) of the present invention infecting tumor patient's monocytes includes The following steps:

A. Take 50-150 ml of peripheral blood from a tumor patient, use a blood cell separator (or lymphocyte separation medium) to obtain peripheral blood mononuclear cells (PBMC) according to conventional methods, mix with AIM-V medium, and add to a cell culture bottle , placed in a constant temperature carbon dioxide incubator for 2 hours.

B. Remove the suspension cells and keep the adherent cells (monocyte, Mo). Suspension cells are peripheral blood lymphocytes, which are mixed with AIM-V medium and then cultured for further use.

C. Add one (or more, better effect) rAAV virus obtained in Example 2 of the present invention, the addition amount is about 100-1000MOI, and then add GM-CSF (800IU/mL) at the same time, and continue to cultivate for 4 hours.

D. Remove the old medium, supplement AIM-V medium containing GM-CSF, IL-4 (800IU/mL) and TNF-α (20IU/mL), and continue culturing.

E. After culturing for 5 days, harvest the mature dendritic cells (DC), mix them with the cultured peripheral blood lymphocytes, add IL-2 (20IU/mL) and IL-7 (500IU /mL), continue to culture.

F. After culturing for 7-9 days, harvest activated cytotoxic T lymphocytes (CTL) for detection.

2. Detection of dendritic cells (DC) and cytotoxic T lymphocytes (CTL)

A. Detection of the efficiency of rAAV infecting peripheral blood mononuclear cells

Using conventional fluorescent antibody labeling and staining method, the mononuclear cells or non-nuclear cells infected by rAAV of the present invention obtained in step 1 were labeled with specific fluorescent antibodies against tumor antigen-carcinoembryonic antigen CEA and mutants thereof (purchased from BD Company of the United States). Mature DCs were then subjected to flow cytometry to detect the number of positive cells. Among them, the test results of the efficiency of recombinant adeno-associated virus rAAV/CEA infecting peripheral blood mononuclear cells are shown in Figure 6. The efficiency of rAAV/CEA infecting peripheral blood mononuclear cells was 92.8%. The efficiency of rAAV (rAAV/AmCEA, rAAV/BmCEA, rAAV/CmCEA) infecting peripheral blood mononuclear cells is about 90%, that is, about 90% of peripheral blood mononuclear cells can be infected by rAAV virus, which proves that The invented rAAV has higher infection efficiency.

B. Detection of CD Molecular Levels in Dendritic Cells (DC)

The levels of CD80, CD83 and CD86 expressed by DC were positively correlated with the function of DC. Use the same detection method as in step A, that is, use fluorescently labeled antibodies against these three CD molecules (purchased from BD, USA) to detect the expression levels of CD80, CD83, and CD86 in DCs obtained in step 1. DCs were used as controls. Among them, the detection results of CD80, CD83 and CD86 levels in DCs infected with recombinant adeno-associated virus rAAV/CEA are shown in Figure 7. CmCEA)-infected DCs expressed significantly higher levels of CD molecules than the control, proving that the constructed and prepared rAAV carrying the tumor antigen-carcinoembryonic antigen CEA and its mutants infected peripheral blood mononuclear cells, and the induced DCs had powerful functions. .

C. Detection of Interferon-γ (IFN-γ) Levels Expressed by Cytotoxic T Lymphocytes (CTL)

The function of CTL and its ability to kill tumor cells are positively correlated with the expression level of IFN-γ. Use a method similar to step A to detect the level of IFN-γ expression in CTLs induced by rAAV-infected DCs of the present invention (with the CTLs induced by non-stimulated DCs as a control), after the mixed culture of DCs and peripheral blood lymphocytes , the cells were harvested, and the traditional intracellular staining method was used for fluorescent staining and labeling of the cells. The antibody used was a fluorescently labeled antibody against IFN-γ (purchased from BD Company in the United States), and finally the results were detected by flow cytometry. Among them, the IFN-γ expression levels of CTLs induced by rAAV/CEA-infected DCs are shown in Figure 8, and those infected by rAAV/CEA and other rAAVs carrying tumor antigens (rAAV/AmCEA, rAAV/BmCEA, rAAV/CmCEA) The level of IFN-γ expression of CTLs induced by DCs is significantly higher than that of the control, which proves that the CTLs induced by DCs infected with rAAV carrying tumor antigens CEA-carcinoembryonic antigen and its mutants constructed and prepared by the present invention are powerful.

D. Cytotoxic T lymphocyte (CTL) killing tumor cell test

After the mixed culture, the cytotoxic T lymphocytes induced by DCs infected with rAAV (rAAV/CEA, rAAV/AmCEA, rAAV/BmCEA, rAAV/CmCEA) in step 1 were divided into 20:1 (lymphocytes: tumor cells) After mixed with primary lung adenocarcinoma cells, gastric adenocarcinoma cells, intestinal adenocarcinoma cells or breast cancer cells, the traditional MTT method and ⁵¹ Cr (chromium-51) killing test were used to detect the activity of CTL in killing tumor cells. The statistical results of the tumor cell killing rate of CTLs induced by rAAV/CEA-infected DCs are shown in Figure 9, and were induced by rAAV-infected DCs constructed and prepared by the present invention carrying tumor antigen-carcinoembryonic antigen and its mutant CTL can more effectively lyse (kill) tumor cells, and the killing rate can reach about 50%.

Using the CEA antigen-negative stomach, lung, intestine, and breast cells (A, B, C, and D) as controls, use the same method as above to detect cells infected by rAAV (rAAV/CEA, rAAV/AmCEA, rAAV/BmCEA, rAAV/BmCEA, The specificity of cytotoxic T lymphocytes induced by rAAV/CmCEA)-infected DCs to kill tumor cells. Among them, the tumor cell killing specific detection results of CTLs induced by rAAV/CEA-infected DCs are shown in Figure 9. The CTLs induced by DCs have no killing effect on the above-mentioned CEA antigen-negative cells, which proves that the CTLs induced by DCs infected with rAAV carrying tumor antigen-carcinoembryonic antigen CEA and its mutant type constructed and prepared by the present invention have antigen specificity, that is, No killing effect on antigen-negative cells.

The above test results show that the CTLs induced by DCs infected with rAAV carrying tumor antigen-carcinoembryonic antigen CEA and its mutant type (collectively referred to as rAAV-DC) of the present invention have positive effects on CEA antigen-positive breast cancer, colon cancer, gastric cancer and lung cancer. Adenocarcinoma and other malignant tumors have good curative effect and can be used to prepare antitumor drugs.

Embodiment 4, the clinical experiment of tumor therapy

1. Detection of curative effect and survival time

Applying recombinant adeno-associated virus-dendritic cell technology, the DC (rAAV -DC) induced CTLs were reinfused into 20 cases of breast cancer, colon cancer, gastric cancer and lung adenocarcinoma, and the infusion volume was 1×10 ⁹ -5×10 ⁹ . Treatment course: usually 6 months, 2-3 times a month, after the condition improves, it can be reduced to 1-2 times a month, and further can be reduced to once every 1-3 months. To test its anti-tumor effect in vivo. The statistical results of treatment effect (response after rAAV-DC treatment) are shown in Table 1 (B: Serum tumor markers decrease or disappear. Q: Patient’s quality of life is improved. Such as pain relief or disappearance, increased appetite, etc. C: CT or PET-CT showed that cancer lesions or metastatic lesions were significantly reduced or disappeared.), Adverse reactions: Most cases will have mild flu-like reactions within a short time after treatment, but patients can tolerate them, and the symptoms disappear in a short period of time, no observation to serious adverse reactions and toxic reactions. The statistical results of the course of treatment and survival time are shown in Table 2 (survival time after treatment: the survival time after the patient started receiving rAAV-DC treatment (calculated from the dead cases to the time of death).), none of the death cases were caused by rAAV-DC treatment Most of the patients in this group are in the terminal stage of cancer, and some patients have already caused immune function, liver and kidney failure due to excessive radiotherapy and chemotherapy. The above statistical results further prove that the CTLs induced by rAAV-infected DCs of the present invention (collectively referred to as rAAV-DCs) can exert a certain curative effect in patients, and can effectively inhibit the growth of malignant tumor cells or kill tumor cells, and It has high safety and can be used to prepare antitumor drugs.

Table 1 Using recombinant adeno-associated virus-dendritic cell technology (rAAV-DC)

Statistical results of the curative effect of treating 20 patients with breast cancer, colon cancer, stomach cancer and lung adenocarcinoma

serial number diagnosis Clinical stage Target genes carried by rAAV rAAV-DC treatment course (months) Survival time after treatment (months) treatment effect The current situation 1 breast cancer IV CEA 7 7 B, Q die 2 breast cancer IV CEA 12 28 B, Q, C 3 colon cancer IV CEA 8 8 B, Q die 4 colon cancer III/IV CEA 10 18 B, Q, C 5 colon cancer IV CEA 18 twenty two B, Q, C 6 colon cancer IV CEA+CEA mutant (fragment A) 20 30 B, Q, C 7 colon cancer IV CEA+CEA mutant (fragment A) 12 14 B, Q die 8 colon cancer IV CEA+CEA mutant (fragment B) 13 13 B, Q, C 9 colon cancer IV CEA+CEA mutant (fragment B) 13 13 B, Q, C 10 colon cancer IV CEA+CEA mutant (fragment C) 12 12 B, Q, C 11 colon cancer IV CEA+CEA mutant (fragment C) 6 6 B, Q die 12 stomach cancer IV CEA 6 6 B, Q die

13 stomach cancer IV CEA 12 20 B, Q, C 14 stomach cancer IV CEA 12 twenty two B, Q, C 15 stomach cancer IV CEA 16 16 B, Q, C 16 Lung adenocarcinoma IV CEA 12 15 B, Q, C 17 Lung adenocarcinoma IV CEA 14 14 B, Q, C 18 Lung adenocarcinoma IV CEA 6 7 Q die 19 Lung adenocarcinoma IV CEA 9 9 B, Q die 20 Lung adenocarcinoma IV CEA twenty two 28 B, Q, C Total III-IV 240 308 7

Table 2 Statistical results of the course of treatment and survival time of 20 patients with breast cancer, colon cancer, gastric cancer and lung adenocarcinoma

2. Changes in imaging and serum tumor markers of tumor patients before and after treatment

A. Changes in imaging of tumor patients before and after treatment

Imaging observations were performed on the changes of metastatic lesions in 20 patients with breast cancer, colon cancer, gastric cancer and lung adenocarcinoma before and after treatment in step 1, and the CTL induced by rAAV/CEA-infected DC in a patient with stage IV metastatic colon cancer The imaging observation results of the changes of metastatic lesions before and after treatment are shown in Figure 10 (the left figure is before the treatment, and the right figure is the situation after four months of treatment). After CTL treatment induced by one or two infected DCs (rAAV-DC) in /BmCEA and rAAV/CmCEA), the patient's metastatic lesions obviously disappeared, which further proved that the DC induced by the rAAV infection of the present invention CTL can effectively inhibit the growth of malignant tumor cells or kill tumor cells in patients, and can be used to prepare antitumor drugs.

B. Changes of serum tumor-associated antigen levels in patients before and after treatment

The changes of serum tumor marker-tumor-associated antigen CEA levels (data from the test results of the experimental hospital) in the above 20 patients with breast cancer, colon cancer, gastric cancer and lung adenocarcinoma before and after treatment were detected, and four of them were treated with rAAV/CEA. The changes of the CEA tumor-associated antigen level in the serum of patients with metastatic colorectal cancer before and after the treatment of CTL induced by infected DCs (the data comes from the hospital laboratory results) are shown in Figure 11. The results were obtained by rAAV (rAAV/CEA, rAAV/ After CTL treatment induced by one or two infected DCs (rAAV-DC) among AmCEA, rAAV/BmCEA and rAAV/CmCEA), the level of tumor-associated antigen CEA in the serum decreased significantly, indicating that the tumor burden in the patient significantly decreased (tumor cells were significantly reduced), which further proves that the CTL induced by DC infected by rAAV of the present invention can effectively inhibit the growth of malignant tumor cells or kill tumor cells in patients, and can be used to prepare antitumor drugs.

Claims (10)

1, a kind of CEA recombined glandulae correlation viral vectors is that the adeno-associated virus structure gene in the gland relevant viral vector is replaced with the recombined glandulae correlation viral vectors that carcinoembryonic antigen or its mutant carcinomebryonic antigen CEA gene obtain.

2, CEA recombined glandulae correlation viral vectors according to claim 1 is characterized in that: the p5 promotor in the described recombined glandulae correlation viral vectors is replaced with one or several promotor in scavenger cell viral promotors, beta actin promoter and the SV40 viral promotors.

3, a kind of method that makes up the described heavy CEA group gland relevant viral vector of claim 1, be earlier the adeno-associated virus structure gene in the gland relevant viral vector to be rejected, replace this rejecting gene with carcinomebryonic antigen CEA gene or its mutated genes again, obtain recombined glandulae correlation viral vectors.

4, construction process according to claim 3 is characterized in that: described method also comprises the step that the p5 promotor in the described recombined glandulae correlation viral vectors is replaced with one or several promotor in scavenger cell viral promotors, beta actin promoter and the SV40 viral promotors.

5,, comprise recombinant adeno-associated virus plasmid, recombinant adeno-associated virus particle and by recombinant gland relevant viral vector infection or cells transfected system with the relevant product of the described CEA recombined glandulae correlation viral vectors of claim 1.

6, product according to claim 5 is characterized in that: described clone comprises monocyte-scavenger cell-dendritic cell system and T lymphocyte series.

7, claim 1 or the 2 described CEA recombined glandulae correlation viral vectors application in the preparation antitumor drug.

8, claim 5 or the 6 described products application in the preparation antitumor drug.

9, according to claim 7 or 8 described application, it is characterized in that: described tumour is mammary cancer, colorectal carcinoma, cancer of the stomach or adenocarcinoma of lung.

10, a kind of method of killing in vitro tumour may further comprise the steps:

1) spontaneous monocyte-scavenger cell-dendritic cell or T lymphocyte in the system of tumour place are describedly carried the recombined glandulae correlation viral vectors of wild-type carcinomebryonic antigen CEA gene and/or the recombinant gland relevant viral vector infection or the transfection of carrying mutant carcinomebryonic antigen CEA gene by claim 1 or 2, or by claim 5 or 6 described product treatment, the cell after obtaining separately handling;

2) will kill tumour in the monocyte-scavenger cell after handling in the step 1)-dendritic cell adding tumour place system; Or T lymphocyte that will be not processed and the monocyte-scavenger cell after the described processing-dendritic cell mixed culture formation antigen-specific cytotoxic T lymphocyte, will kill tumour in the system of this antigen-specific cytotoxic T lymphocyte adding tumour place again; Or will kill tumour in processed T lymphocyte and the not processed monocyte-scavenger cell-dendritic cell adding tumour place system.

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