CN112646839A

CN112646839A - Modified adeno-associated virus

Info

Publication number: CN112646839A
Application number: CN201910957102.5A
Authority: CN
Inventors: 梁亚龙
Original assignee: Individual
Current assignee: Individual
Priority date: 2019-10-10
Filing date: 2019-10-10
Publication date: 2021-04-13

Abstract

The invention belongs to the technical field of biology, and relates to a modified adeno-associated virus. The invention relates to a modified adeno-associated virus, which can realize the secretory expression of chimeric peptide or chimeric protein containing innate immune stimulating factors, immune check point inhibitors and targeted tumor apoptosis active peptide, and can be effectively combined by activating an innate immune system in vivo, blocking immune check points and using the targeted apoptosis active peptide according to tumor types, thereby being applied to the preparation of tumor medicaments for treating or preventing unlimited cancer. Solves the problems of poor clinical effect, low effective rate, short half-life period, repeated administration and the like of the existing tumor immunotherapy products.

Description

Modified adeno-associated virus

Technical Field

The invention belongs to the technical field of biology, and relates to a modified adeno-associated virus. Specifically, the invention relates to a modified adeno-associated virus, which can realize the secretory expression of chimeric peptide or chimeric protein containing an inherent immune stimulating factor and an immune checkpoint inhibitor and targeting tumor apoptosis active peptide, and can be applied to the preparation of tumor drugs for treating or preventing unlimited cancers.

Background

The development of modern anticancer drugs has so far been revolutionized three times:

the first chemotherapy drugs appeared after 1940, and most of the chemotherapy drugs used clinically at present belong to this class. However, the most difficult problem to be solved by chemotherapeutic drugs is that they cannot distinguish malignant cells from normal cells, so that the chemotherapeutic drugs kill cancer cells and normal cells of human body, causing irreversible damage to patients, and leading most patients to die due to toxicity of the chemotherapeutic drugs.

The second time, the targeted therapy started after 2000 years, and is typically gleevec marketed in 2001 for the treatment of BCL-ABL mutant gene chronic leukemia. The medicine can enable the five-year survival rate of patients to be increased from 30% to 89%. But targeting is ineffective due to genomic instability and mutation of the tumor, as well as tumor heterogeneity, which ultimately leads to patient resistance; the targeted drug resistance has become a difficult problem in the world, and the clinical drug resistance phenomenon appears in some patients after 8-10 months. It is better than our approach to block cancer growth with drugs, however cancer cells are also evolving and it develops a new approach to continue growing, so targeting drugs may introduce another profound future to patients.

The third time is immunotherapy. The target of "immunotherapy" is normal immune cells, with the goal of activating the body's own immune system to treat cancer. The immunotherapy of tumor is divided into six types, namely T cell targeted immunomodulator, immunomodulator targeted to other immune cells, tumor vaccine, cell therapy, oncolytic virus and bispecific antibody targeted to CD 3. In the aspect of medical application, an immune checkpoint inhibitor PD1 in immunotherapy is an industrially recognized psychotropic drug, and in 2013, two new drugs which are proposed by Shilubao and Shishadong and act on the same target point PD-1 have clinical effects that the two drugs reduce or even disappear more than 60 percent of patients with tumors in advanced melanoma patients (most of cancers are already metastasized) with failed treatment schemes for more than 2 years. But current immunotherapy still has the fatal defect that the effective rate is low, and in clinic, the PD-1 inhibitor is only effective for 20 percent of patients; patients gradually develop immunogenicity using PD-1 antibodies, produce anti-PD 1 antibodies in vivo, and fail the drug; and because the half-life period in vivo is very short, continuous repeated administration is required clinically, so that the treatment cost is huge and a huge workload is brought to medical treatment. This is a concern in the industry and is not expected to be a problem of coexistence of positive and negative effects, and no channel has been solved so far. Recent oncolytic virus-related immunotherapy has gradually attracted considerable attention, with only the first half of 2018 funding for two large oncolytic viruses, the first being the 3.96 million dollar acquisition of Vialytic in Musandong, and the second being the 10.4 million dollar total investment by the Johnson company under the vigorous flag to the T-Stealth oncolytic virus platform of BeneVirBiopharm. Indeed, as early as 2003 and 2005, CFDA approved two oncolytic virus products on the market, including "this-day" (P53 adenovirus injection) and echol (recombinant human adenovirus type 5 injection). However, the clinical response effect of the two products is not good, and the curative effect is not approved by international and the same industries.

Since the recombinant adeno-associated virus (rAAV) is derived from non-pathogenic wild adeno-associated virus, the recombinant rAAV is considered to be one of the most promising gene transfer vectors due to the characteristics of good safety, wide host cell range (divided and non-divided cells), low immunogenicity, long time for expressing foreign genes in vivo and the like, and is widely applied to gene therapy and vaccine research worldwide. Therefore, by utilizing the mechanism of immunotherapy, a new adeno-associated virus capable of expressing and treating broad-spectrum tumors (solid tumors and non-solid tumors) is developed, and the problems of poor clinical effect, low effective rate, short half-life period, repeated administration and the like of the existing immunotherapy products are solved.

Disclosure of Invention

To achieve the above object, the present invention constructs a novel modified adeno-associated virus (AAV).

The invention selects a virus vector (AAV) which is commonly used in clinic, preferably AAV2, and the vector can realize the secretion expression of the chimeric peptide or the chimeric protein of the anti-tumor cell through the genetic engineering construction.

We have discovered, through extensive studies, that the addition of GM-SCF, a factor encoding the innate immune stimulator, can be directed to induce a transition from "cold" tumors to "hot" tumors, which are more sensitive to immune checkpoint inhibitors, and whether the tumor is "cold-hot" determines its response to immune checkpoint inhibitors. Particularly, through research, the single-chain antibody immune checkpoint inhibitor which simultaneously expresses intrinsic immune stimulating factor (GM-SCF) and any one of PD1, TIM3 and LAG3, particularly preferably any one of PD1scFv, TIM3scFv and LAG3scFv is found to solve the problems of poor clinical effect and low efficiency of a single immune checkpoint inhibitor.

In addition, a large number of clinical experiences and published literature have confirmed in recent years: more than 50% of a large number of solid tumors are related to P53 mutation or deletion, P53 gene mutation can be sensitive to immune drugs (dong et al, 2017; Miao et al, 2018), under the condition, the active peptide of targeted tumor apoptosis of a P53 mechanism can be adopted to play an anti-tumor role, the research of the applicant finds that the anti-cancer effect of the N-terminal 15 peptide, the C-terminal 22 peptide and the P53N-terminal 37 peptide (P73 inhibitory peptide) of the P53 is superior to that of the P53, and the P53 mutation does not exist in normal diploid cells, the P53N-terminal 15 peptide, the C-terminal 22 peptide and the P53N-terminal 37 peptide (P73 inhibitory peptide) cannot kill normal tissues, and are nontoxic to the normal tissues, so the tumor targeting effect is realized; however, in addition to the fact that 50% of tumor patients had no abnormal or clinically insignificant mutation in their genes and immunohistochemical assays for P53, the development and progression of tumors in these patients may be P53 independent, in which case anti-tumor effects were achieved using apoptosis-targeting active peptides other than the P53 mechanism, such as Apoptin, ORF4, Shepherdin, Par-4SAC, with the ability to specifically induce apoptosis and necrosis of transformed and tumor cells, without damaging diploid cells of normal humans.

In view of the above studies, applicants have prepared a modified AAV by the present invention, which can realize the secretory expression of a chimeric peptide or chimeric protein comprising an innate immune stimulator and an immune checkpoint inhibitor and a targeted apoptosis active peptide against tumor cells. The combination can realize the simultaneous expression of the inherent immune stimulating factor, the immune check point inhibitor and the targeted tumor apoptosis active peptide in vivo, has stronger synergistic effect, can greatly improve the clinical effectiveness and the effective rate of immunotherapy, can also target and promote apoptosis to tumor cells, and is non-toxic and harmless to human bodies.

Further, said one modified AAV is preferably AAV 2.

Further, the AAV carries a protein capsid that is nearly identical to wild-type AAV, whereas the portion of the genome within the capsid that encodes the viral proteins is completely deleted, the only remaining portion being ITRs (inverted terminal repeats).

Further, in the chimeric peptide or the chimeric protein capable of realizing secretory expression of the anti-tumor cells, the intrinsic immune stimulating factor used is granulocyte colony stimulating biological factor (GM-CSF).

Further, in the chimeric peptide or the chimeric protein capable of realizing secretory expression of the anti-tumor cells, the immune checkpoint inhibitor is a single-chain antibody selected from any one of PD1, TIM3 and LAG 3;

preferably any one of PD1scFv, TIM3scFv and LAG3 scFv;

further preferably PD1 scFv.

Furthermore, in the chimeric peptide or the chimeric protein capable of realizing secretory expression of the antitumor cell, the apoptosis-targeting active peptide is selected from any one of a P53N terminal 15 peptide, a P53C terminal 22 peptide and a P53N terminal 37 peptide (P73 inhibiting peptide), or any one of Apoptin, ORF4, Sphervin and par-4 SAC.

Furthermore, the selection of the active peptide targeting tumor apoptosis is determined by the gene detection of tumor cells, if the detection result is related to P53 defect or mutation, the active peptide targeting tumor apoptosis is selected from any one of P53N terminal 15 peptide, P53C terminal 22 peptide and P53N terminal 37 peptide (P73 inhibitory peptide); if the detection result is not related to P53 deficiency or mutation, the active peptide targeting tumor apoptosis is selected from any one of Apoptin, ORF4, Sphervin and par-4 SAC.

A modified AAV described herein comprises a combination of nucleic acid sequences that effect secretory expression of a chimeric peptide or chimeric protein of an anti-tumor cell.

Further, the nucleic acid sequence combination capable of realizing the secretion expression of the chimeric peptide or the chimeric protein of the anti-tumor cell can code an inherent immune stimulating factor, an immune checkpoint inhibitor and a targeted tumor apoptosis active peptide.

Further, the nucleic acid sequence combination capable of realizing the secretory expression of the chimeric peptide or the chimeric protein of the anti-tumor cell comprises any one of an innate immune stimulating factor GM-CSF and an immune checkpoint inhibitor PD1scFv or TIM3scFv or LAG3scFv and any one of apoptosis-targeting active peptides P53N terminal 15 peptide, P53C terminal 22 peptide, P53N terminal 37 peptide (P73 inhibiting peptide) or any one of Apoptin, ORF4, Shepherdin and par-4 SAC;

wherein, the selection of the active peptide nucleic acid sequence targeting tumor apoptosis is determined by the gene detection of tumor cells, if the detection result is related to P53 defect or mutation, the active peptide nucleic acid sequence targeting tumor apoptosis is selected from any one of P53N terminal 15 peptide, P53C terminal 22 peptide and P53N terminal 37 peptide (P73 inhibitory peptide); if the detection result is not related to P53 deficiency or mutation, the active peptide nucleic acid sequence targeting tumor apoptosis is selected from any one of Apoptin, ORF4, Shepherdin and par-4 SAC.

Further, the nucleic acid sequence combination capable of realizing the secretory expression of the chimeric peptide or the chimeric protein of the anti-tumor cell contains a signal peptide and a cell penetrating peptide sequence.

Further, the nucleic acid sequence combination capable of realizing the secretory expression of the chimeric peptide or the chimeric protein of the anti-tumor cell contains an intron sequence.

Applicants prepared a modified AAV prepared by the present invention, administered by intramuscular injection at a dose of 5x10⁷-5*10¹²vp, through "in vivo administration," modified AAV can survive in vivo for more than one year, ensuring a once-administered, effective treatment regimen within one year. Solves the problems of short half-life in vivo and repeated clinical administration of a single immune checkpoint inhibitor (such as a PD1 antibody) or a combination of GM-CSF and the immune checkpoint inhibitor.

In addition, the modified AAV provided by the invention can promote receptor autoimmunity by stimulating innate immune factors in tumor treatment, efficiently identify tumor cells by an immune checkpoint inhibitor, and accurately identify tumor types according to gene detection, thereby increasing targeted tumor apoptosis bioactive peptides, further exerting unique advantages of unlimited cancer treatment (including lung cancer, melanoma cancer, head and neck cancer, liver cancer, lymph cancer, stomach cancer, kidney cancer, prostate cancer, breast cancer, bladder cancer, colorectal cancer, cervical cancer, ovarian cancer, pancreatic cancer, gallbladder cancer and the like), remarkably improving anti-tumor effect and tumor response rate, and realizing the expected purpose and beneficial effect of the invention.

The invention also relates to the application of any nucleic acid sequence of any chimeric peptide or chimeric protein for realizing secretory expression of anti-tumor cells and/or any vector and/or any virus in preparing tumor drug species for treating or preventing unlimited cancer species.

Drawings

FIG. 1 is a schematic diagram of the expression cassette structure of the modified AVV constructed in accordance with FIG. 1 and the expression cassette structure of the control AVV

FIG. 2 graph of HCT116 tumor cell volume change and drug administration time in nude mice

FIG. 3 is a graph showing the volume change of Caco-2 tumor cells in nude mice and the administration time

FIG. 4 graph of SiHa tumor cell volume change and dosing time in nude mice

FIG. 5 graph of the volume change of MCF7 tumor cells in nude mice versus the time of administration

Detailed Description

Example 1: construction of modified adeno-associated viruses (AAV)

The invention provides a modified adeno-associated virus (AAV), wherein the chimeric peptide or chimeric protein capable of realizing secretory expression of anti-tumor cells comprises an immune intrinsic factor, an immune checkpoint inhibitor and a targeted tumor apoptosis active peptide, and a recombinant adeno-associated virus capable of realizing secretory expression of three chimeric peptides andor chimeric proteins is constructed. The specific construction method is as follows: the CMV promoter is spliced with the first chimeric peptide or chimeric protein by adding a signal peptide (NT4 or IgG) and an artificial intron, the first chimeric peptide/chimeric protein and the second chimeric peptide/chimeric protein sequence are spliced by adding 2A (self-splicing polypeptide sequence), and the second chimeric peptide/chimeric protein and the third chimeric peptide/chimeric protein sequence are spliced by adding 2A (self-splicing polypeptide sequence).

Furin-2A is one of the 2A self-splicing polypeptide elements (2A self-cleavage peptide). As a splicing sequence, 2A peptide segments are best researched by foot-and-mouth disease virus (FMDV)2A (F2A), the self-shearing efficiency is high, the expression balance of an upper gene and a lower gene is good, the structure is short (20-22 amino acids), and the splicing sequence is a good connecting tool in the construction process of a vector. The conserved sequence of the 2A peptide is the terminal-NPGP, the cleavage site is between G and P. Adding a Furin protease enzyme cutting site RAKR in front of F2A, and adding a-GSG-to enhance the shearing capability of 2A peptide, and removing the sequence remained at the carboxyl terminal of the front protein after 2A self-shearing. Therefore, Furin-2A is preferred in the selection of the splicing sequence.

The invention adds Cell Penetrating Peptides (CPPs) at the carboxyl terminal of the target apoptosis active peptide to help the target apoptosis active peptide to better enter tumor cells, wherein the CPPs comprises TAT cell penetrating peptide or ANTP cell penetrating peptide or a cyclic tumor cell penetrating peptide (CRGDKGPDC) with 9 amino acids.

The invention constructs 7 modified adeno-associated virus, CMV and polyA, namely expression cassettes of different adeno-associated virus vectors, which are respectively (the components are shown in detail as a, b, c, d, e, f and g in the attached figure 1):

AAV2-GM-CSF-PD1scFv-P53(C22)，

AAV2-GM-CSF-PD1scFv-P53(N15),

AAV2-GM-CSF-PD1scFv-P53(N37),

AAV2-GM-CSF-PD1scFv-Apoptin，

AAV2-GM-CSF-PD1scFv-Shepherdin，

AAV2-GM-CSF-PD1scFv-ORF4,

AAV2-GM-CSF-PD1scFv-Par4SAC，

and 7 control adeno-associated viruses were constructed, the expression cassettes of which are respectively (see h, i, j, k, l, m, n in FIG. 1 for details):

AAV2-GM-CSF-PD1scFv，

AAV2--PD1scFv-P53(N15)，

AAV2-GM-CSF-P53(N15)，

AAV2-GM-CSF-apoptin，

AAV2-PD1scFv-apoptin，

AAV2-GM-CSF，

AAV2-PD1scFv。

all the expression cassettes are obtained by adopting a whole gene synthesis mode, and then inserted into a pAAV-MCS vector to form a virus vector containing the inserted target gene.

Example 2: identification of modified adeno-associated viruses

And constructing the fused gene in pAAV-MCS, transforming the gene into DH5 alpha competent cells, coating the cell on an LB solid culture plate, placing the cell on a 37 ℃ incubator for inverted culture overnight, picking out colonies after the colonies grow out for colony PCR, carrying out amplification culture on the colonies with the PCR result being positive, and carrying out sequence identification without errors after the plasmids are extracted slightly.

The correct monoclonal colonies were grown up and plasmids were extracted using Qiagen endotoxin removal plasmid Production kit for subsequent testing.

Example 3: packaging and purification of modified adeno-associated virus

The packaging and purification of the recombinant adeno-associated virus according to the present invention will be described below by taking as an example only the packaging and purification of the recombinant adeno-associated virus AAV2-GM-CSF-PD1scFv-P53(N15) represented by the b expression cassette in fig. 1, which is shown in fig. 1.

(1) AAV293 cell culture

The cells are cultured in DMEM high-glucose medium containing 10% FBS at 37 ℃ and 5% CO₂Culturing in incubator with 75cm²The cell culture flask of (1). When the cells grow to 90% confluence, subculturing and removing the culture medium, adding 1ml of 0.25% trypsin, digesting at 37 ℃ for about 3min, observing the cells under a microscope, adding 9ml of DMEM medium containing 10% FBS when the cells shrink and become round, blowing and beating into a single cell suspension for cell counting, and counting the cells according to the ratio of 1: 3 for passage.

(2) Virus package

AAV293 cells were cell passaged and then 5% CO at 37 ℃₂The cells were cultured in an incubator and when the cells grew to 60% -70% confluence, transfection was performed. And (2) transfecting the pAAV-mcs-GM-CSF-PD1scFv-P53(N15), the pAAV-RC and pHelper to AAV-293 cells together by using a three-plasmid transfection system, specifically transfecting the pAAV-mcs-GM-CSF-PD1scFv-P53(N15), the pAAV-RC and pHelper in a molar mass ratio of 1: 1: 1, total plasmid amount 400ug per bottle for transfection.

400ug of plasmid, ultrapure water and 2.5mol/L CaCl₂Mix well in a 15ml centrifuge tube and add 2XBBS (50mmol/L BES, 280mol/L NaCl, 1.5mmol/L Na) dropwise₂HPO₄Ph6.95) with addition and blowing to form smaller calcium phosphorusAnd (3) granules. After addition was complete, incubation was carried out at room temperature for 20 min.

Taking out cells, removing culture medium by suction, washing with PBS once, adding calcium phosphate-DNA solution into AAV-293 cells grown to 60% -70% confluence, and culturing at 37 deg.C and 5% CO₂And (4) continuing culturing in the incubator, and after 18h, replacing the culture medium with a DMEM medium and continuing culturing. After 72h, the medium was transferred to a 15ml centrifuge tube, the cells were washed once with PBS, a small amount of PBS was added, and 293 cells in the flask were scraped with a cell scraper. If not used immediately, the cell suspension was transferred to a cryopreservation tube and stored at-80 ℃.

(3) Harvesting

The virus-infected AVV293 cells were collected together with the culture medium in a 15ml centrifuge tube, centrifuged at 1500g/min for 3 minutes, the cells and supernatant were separated, the supernatant was stored separately, and the cells were resuspended in 1ml PBS. The cell suspension was repeatedly transferred in liquid nitrogen and 37 ℃ water bath, frozen and thawed four times, each time for about 10 min. After each melting, the mixture is shaken and mixed evenly.

(4) Concentration of virus

The cells were then centrifuged at 8000g for 15min at 4 ℃ with repeated freeze thawing, and the supernatant was removed to a new 15ml centrifuge tube. The previous culture medium supernatant was also centrifuged at 8000g for 15min at 4 ℃ and the two centrifuged supernatants were transferred to the same centrifuge tube and mixed well. The impurities were removed by filtration through a 0.45um filter. 1/2 volumes of 1M NaCl, 10% PEG8000 solution were added and mixed well overnight at 4 ℃.

The overnight supernatant solution was centrifuged at 12,000rpm for 2h at 4 ℃. The supernatant was discarded, the viral pellet was dissolved in an appropriate amount of PBS solution, and after complete dissolution, the pellet was sterilized by filtration through a 0.22um filter. The residual plasmid DNA (final concentration of 50U/ml) was removed by digestion with nuclease. Close the tube lid and invert several times to mix well. Incubate at 37 ℃ for 30 minutes. Filtering with 0.22um filter head, and collecting filtrate to obtain concentrated AAV.

(5) Determination of viral titre (Q-PCR method)

20ul of the concentrated virus solution was added with 1ul of RNAse-free DNAse, mixed well, and reacted in a water bath at 37 ℃ for 30 min. Then, the mixture was centrifuged at 12000rpm/min at 4 ℃ for 10min, and 10. mu.l of the supernatant was taken in a 1.5ml EP tube. 90ul Dilution Buffer (1mM Tris-HCl, pH 8.0, 0.1mM EDTA, 150mM NaCl) was added thereto, mixed well, and subjected to water bath at 37 ℃ for 30 min. Naturally cooling the EP tube to room temperature, adding 1ul of proteinase K, reacting in water bath at 65 ℃ for 1h, and removing the protein shell of the virus. Then, the mixture is boiled and bathed for 10min at 100 ℃ and naturally cooled to the room temperature. Then Q-PCR was performed to detect the titer.

Obtaining recombinant adeno-associated virus AAV2-GM-CSF-PD1scFv-P53(N15) co-expressing human GM-CSF, PD1svFc and P53(N15) with titer of 10X10⁹pfu/L.

Example 4: modified adeno-associated virus for detection of killing activity of different types of tumor cells

Respectively mixing Hela299 (cervical cancer cells), Eca-109 (esophageal cancer cells), A549 (lung cancer cells), MCF7 (breast cancer cells), GBC-SD (gallbladder cancer cells), Caco-2 (colon adenocarcinoma cells), U251 (glial cancer cells), PC-3 (prostate cancer cells), HCT116 (colon cancer cells), SiHa (cervical cancer cells), MG-63 (osteosarcoma cells) and A3T (lymphocytic leukemia cells) into 5x10³AAV2-GM-CSF-PD1scFv-P53(N15) was diluted to infect the above tumor cells at 1-fold MOI (ratio of the number of Mulitliicitoffectin viruses to the number of cells) 24 hours after inoculation, the viral residual was discarded after 7 days, and the killing effect of AAV2-GM-CSF-PD1scFv-P53(N15) on various cells was examined by the MTT method. OD570 was read in a microplate reader at a reference wavelength of 630 nm. Duplicate wells were made in 8 independent replicates three times.

Killing rate ═ 1- [ (test well mean OD value-reagent blank mean OD value)/(negative well mean OD value-reagent blank mean OD value)]Computing IC₅₀Value (MOI IC)₅₀)。

The killing activity of AAV2-GM-CSF-PD1 scFv-apaptin on the above tumor cells was tested by the same method, and the results are shown in the following table:

note: as the result of prophase cell experiments shows that the results of AAV2-GM-CSF-PD1scFv-P53(N15), AAV2-GM-CSF-PD1scFv-P53(C22) and AAV2-GM-CSF-PD1scFv-P53(N37) are equivalent and have no significant difference, AAV2-GM-CSF-PD1scFv-P53(N15) is taken as a representative of P53 active peptide for targeting tumor apoptosis; AAV2-GM-CSF-PD1scFv-apoptin, AAV2-GM-CSF-PD1scFv-Shepherdin, AAV2-GM-CSF-PD1scFv-ORF4 and AAV2-GM-CSF-PD1scFv-Par4SAC have equivalent results and no significant difference, so that AAV2-GM-CSF-PD1scFv-apoptin is taken as a representative of non-P53 targeting tumor apoptosis active peptide.

And (3) knotting: AAV2-GM-CSF-PD1scFv-P53(N15) has obvious killing capacity on the infected tumor cells, especially has strong killing capacity on Hela299, Eca-109, Caco-2, PC-3, HCT116 and MG-63, wherein the killing capacity on the tumor cells (marked as P53-) Caco-2, PC-3, HCT116 and MG-63 which do not express P53 or express loss-activity mutant P53 or P53 segment is higher, and the killing capacity of AAV2-GM-CSF-PD1scFv-P53(N15) on the Caco-2, PC-3, HCT116 and MG-63 tumor cells is obviously better than that of AAV2-GM-CSF-PD 1-apoptin;

AAV2-GM-CSF-PD1 scFv-apaptin has obvious killing capacity on the infected tumor cells, especially has strong killing capacity on Hela299, Eca-109, A549, MCF7(P53+), SiHa (P53+), wherein the killing capacity on tumor cells expressing P53 or P53 fragments (marked as P53+) MCF7 and SiHa is higher, and the killing capacity of AAV2-GM-CSF-PD1 scFv-apaptin on MCF7 and SiHa tumor cells is obviously better than that of AAV2-GM-CSF-PD1scFv-P53 (N15);

example 5: modified adeno-associated virus nude mice in vivo experiments:

SiHa (cervical cancer cells), Caco-2 (colon adenocarcinoma cells), MCF7 (breast cancer cells) and HCT116 (colon cancer cells) in logarithmic phase are respectively inoculated into BALB/c nude mice, and the inhibition effect of the AAV constructed in the invention on the growth of transplanted tumors is detected, and the results are shown in figures 2-5. Taking the inhibiting effect on breast cancer cells (MCF7) as an example, the specific operation is as follows:

taking MCF7 cells in logarithmic growth phase and diluting the cells to 1X10 by using physiological saline⁸The injection is injected into the lateral abdomen of a nude mouse under the skin of the near axillary part at 100 uL/mL. The conditions of the nude mice were continuously observed, aboutAfter 5 weeks, hard nodules of rice grain size appeared subcutaneously in the inoculated area. Nude mice were randomly divided into 9 groups (AAV2-GM-CSF-PD1scFv-P53(N15), AAV2-GM-CSF-PD1scFv-Apoptin, AAV2-GM-CSF-PD1scFv, AAV2-PD1scFv-P53(N15), AAV2-GM-CSF-P53(N15), AAV2-GM-CSF-Apoptin, AAV2-PD1scFv-Apoptin, AAV2-GM-CSF, AAV2-PD1scFv), 5 mice per group. With a catalyst containing 2X10⁸100uL of recombinant adeno-associated virus solution with pfu virus amount is injected into tumor at multiple points, once every other day, for 5 times; the blank control group was injected 100uLX5 times with physiological saline instead of recombinant virus. Tumor growth was measured before and after injection at 3 rd, 7 th, 14 th, 21 th, 28 th, 35 th, 42 th, 49 th d, and tumor size was measured with a vernier caliper, and tumor volume was calculated using the formula "axb2x0.5" (a: maximum diameter, b: minimum diameter).

While FIGS. 2-5 show that all of the nine modified adeno-associated viruses reduced tumor volume, AAV2-GM-CSF-PD1scFv-P53(N15) and AAV2-GM-CSF-PD1scFv-Apoptin showed the best effect in reducing tumor volume, AAV2-GM-CSF-PD1scFv, AAV2-PD1scFv-P53(N15), AAV2-GM-CSF-P53(N15), AAV2-GM-CSF-Apoptin, AAV2-PD1scFv-Apoptin showed the next least effect, AAV2-GM-CSF, AAV2-PD1scv showed the weaker effect.

Wherein the effect of AAV2-GM-CSF-PD1scFv-Apoptin on SiHa and MCF7 transplants of P53+ is better than that of AAV2-GM-CSF-PD1scFv-P53(N15), and the effect of AAV2-GM-CSF-Apoptin and AAV2-PD1scFv-Apoptin on SiHa and MCF7 transplants of P53+ is better than that of AAV2-GM-CSF-P53(N15) and AAV2-PD1scFv-P53 (N15).

Wherein the effect of AAV2-GM-CSF-PD1scFv-P53(N15) on HCT116 and Caco-2 transplants of P53-is better than that of AAV2-GM-CSF-PD1scFv-Apoptin, and the effect of AAV2-GM-CSF-P53(N15) and AAV2-PD1scFv-P53(N15) on HCT116 and Caco-2 transplants of P53-is better than that of AAV2-GM-CSF-Apoptin and AAV2-PD1 scFv-Apoptin.

Example 6: modified adeno-associated virus ascites tumor model in vivo assay

Hela299 (cervical cancer cells) in log phase was digested with pancreatin to a concentration of 2.0 x10⁶The cell suspension of the/mL is inoculated into BALB/c nude mice of 3-5 weeks old by means of intraperitoneal injection, and the injection volume is 200 mu L. SmallAscites tumor appears 21 days after MCF7 cells are injected into the mice, the tumor formation rate is 90%, the disease condition is slow after tumor formation, and the survival time after the ascites tumor is 28-35 days.

BALB/c nude mice of 3-5 weeks of age were randomly divided into 3 groups of 20 mice each, control group, treatment group 1 (AAV2-GM-CSF-PD1scFv-P53(N15)) treatment group 2(AAV2-GM-CSF-PD1 scFv-aproptin), respectively. After the nude mouse is inoculated with Hela299 (cervical carcinoma cells) and has ascites tumor, the virus is injected into the abdominal cavity, and the injection amount is 4 x10⁵pfu/stick; in the control group, 20 mice died sequentially from week 4 and all died at week 6. Ascites disappeared 7-10 days after treatment in 20 mice in group 1, 19 of the 20 mice survived with a survival period of more than 12 weeks and no ascites tumor recurrence. Ascites disappeared 7-10 days after treatment in 20 mice in group 2, 18 of 20 mice survived with a survival period of more than 12 weeks and no ascites tumor recurred. AAV2-GM-CSF-PD1 scFv-apaptin and AAV2-GM-CSF-PD1scFv-P53(N15) have been shown to have very effective therapeutic effects.

Example 7: clinical examples of modified adeno-associated viruses

Patient information: the clinical diagnosis of the gallbladder medium-low differentiation adenocarcinoma is 64 years old for women, the PD-L1 immunohistochemical monitoring shows that the expression of PD-L1 TPS is negative, the expression of TPS is less than 1 percent, the expression of PD-L1 CPS is positive, and the CPS is more than or equal to 1. The PD1 monoclonal antibody is clinically ineffective in treatment and chemotherapy is ineffective, and the survival period is less than three months. The gene was detected as P53 without mutation.

The treatment scheme comprises the following steps: administering AAV-GM-SCF-PD1scFv-Apoptin by intramuscular injection of 1x10¹⁰vp。

The effect is as follows: after one week, the ascites is less, the pleural fluid is absorbed, the lung shadow is less, the symptom is relieved, the pain is relieved, the tumor antigens such as the alpha-embryonic protein, the carcinoembryonic antigen, the C125 determination and the like are checked, the obvious reduction is realized, the continuous state of the patient is excellent at present, and the survival cycle is more than 1 year.

Example 8: clinical examples of modified adeno-associated viruses

Patient information: male, 69 years old, lung cancer, right lower lung cancer, both lung metastasis mediastinal lymph node metastasis lumbar metastatic cancer TNM staging: the T4N2M1b gene was diagnosed with the P53 mutation.

The treatment scheme comprises the following steps: administration of AAV-GMSCF-PD1scFv-P53(N15), hip intramuscular injection 1x10¹⁰vp。

The effect is as follows: after one week, the abdominal carcinoembryonic antigen and the carbohydrate antigen 125 are obviously reduced, and the carcinoembryonic antigen is reduced by 40%. The sugar chain antigen 125 is reduced by 32 percent, and the subjective symptoms and the survival state of the patient are obviously improved.

Example 9: clinical examples of modified adeno-associated viruses

Patient information: for men, 75 years old, lung cancer in the lower right, metastatic mediastinal lymph node of both lungs metastasizing lumbar metastatic cancer TNM stage: the T4N2M1b gene was diagnosed with the P53 mutation.

The treatment scheme comprises the following steps: administering AAV-GM-SCF-PD1scFv-P53(C22) by hip intramuscular injection of 1x10¹⁰vp。

The effect is as follows: after one week, the abdominal carcinoembryonic antigen and the carbohydrate antigen 125 are obviously reduced, and the carcinoembryonic antigen is reduced by 37%. The sugar chain antigen 125 is reduced by 30 percent, and the subjective symptoms and the survival state of the patient are obviously improved.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, simple combination, equivalent modification and the like within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

SEQUENCE LISTING

<110> Liangya Longipedun

<120> a modified adeno-associated virus

<160> 12

<170> PatentIn version 3.3

<210> 1

<211> 435

<212> DNA

<213> Artificial

<220>

<223> human GM-CSF sequence

<400> 1

atgtggctgc agagcctgct gctcttgggc actgtggcct gcagcatctc tgcacccgcc 60

cgctcgccca gccccagcac acagccctgg gagcatgtga atgccatcca ggaggcccgg 120

cgtctcctga acctgagtag agacactgct gctgagatga atgaaacagt agaagtcatc 180

tcagaaatgt ttgacctcca ggagccgacc tgcctacaga cccgcctgga gctgtacaag 240

cagggcctgc ggggcagcct caccaagctc aagggcccct tgaccatgat ggccagccac 300

tacaaacagc actgccctcc aaccccggaa acttcctgtg caacccagat tatcaccttt 360

gaaagtttca aagagaacct gaaggacttt ctgcttgtca tcccctttga ctgctgggag 420

ccagtccagg agtga 435

<210> 2

<211> 798

<212> DNA

<213> Artificial

<220>

<223> anti-PD-1 scFv sequence

<400> 2

atgggatggt catgtatcat cctttttcta gtagcaactg caaccggcgc gcactccgag 60

gtgcagctgg tgcagtctgg gggaggcgtg gttcagcctg ggaggtccct gagactctcc 120

tgtgcagcgt ctggattcac ctttagtagc tattggatga gctgggtccg ccaggctcca 180

gggaaggggc tggagtgggt ctcagctatt agtggtagtg gtggtagcac atactacgca 240

gactccgtga agggccggtt caccatctcc agagacaatt ccaagaacac gctgtatctg 300

caaatgaaca gcctaagagc cgaggacacg gccgtatatt actgtgcgaa agagaactgg 360

ggatcgtact tcgatctctg ggggcaaggg accacggtca ccgtctcctc aggtggcgga 420

gggtcaggtg gcggagggtc aggtggcgga gggtcaggcg tgcactccga catcgtgatg 480

acccagtctc cttccaccct gtctgcatct gtaggagaca gagtcaccat cacttgccgg 540

gccagtcagg gtattagtag ctggttggcc tggtatcagc agaaaccagg gagagcccct 600

aaggtcttga tctataaggc atctacttta gaaagtgggg tcccatcaag gttcagcggc 660

agtggatctg ggacagattt cactctcacc atcagcagtc tgcaacctga agattttgca 720

acttactact gtcaacagag ttacagtacc ccgtggacgt tcggccaggg gaccaagctg 780

gaaatcaaga gatgataa 798

<210> 3

<211> 45

<212> DNA

<213> Artificial

<220>

<223> P53(N15) sequence

<400> 3

ccccctctga gtcaggaaac attttcagac ctatggaaac tactt 45

<210> 4

<211> 108

<212> DNA

<213> Artificial

<220>

<223> p53(N37) sequence

<400> 4

acagccaagt ctgtgacttg cacgtactcc cctgccctca acaagatgtt ttgccaactg 60

gccaagacct gccctgaggt tgtgaggcgc tgcccccacc atgagcgc 108

<210> 5

<211> 140

<212> DNA

<213> Artificial

<220>

<223> p53(C22) sequence

<400> 5

gggccggcgt gggagcaggg ctcactccag ccacctgaag tccaagaagg gtcagtctac 60

ctcccgccat aagaagctta agaagtggaa gatgcgccgg aatcagttct gggtaaaggt 120

acagcgcgga tgaggatccc 140

<210> 6

<211> 366

<212> DNA

<213> Artificial

<220>

<223> Apoptin sequence

<400> 6

atgaacgctc tccaagaaga tactccaccc ggaccatcaa cggtgttcag gccaccaaca 60

agttcacggc cgttggaaac ccctcactgc agagagatcc ggattggtat cgctggaatt 120

acaatcactc tatcgctgtg tggctgcgcg aatgctcgcg ctcccacgct aagatctgca 180

actgcggaca attcagaaag cactggtttc aagaatgtgc cggacttgag gaccgatcaa 240

cccaagcctc cctcgaagaa gcgatcctgc gacccctccg agtacagggt aagcgagcta 300

aaagaaagct tgattaccac tactcccagc cgaccccgaa ccgcaagaag gcgtataaga 360

ctgtaa 366

<210> 7

<211> 369

<212> DNA

<213> Artificial

<220>

<223> ORF4 sequences

<400> 7

atggttcttc ctgttcttcc ctcccccgcc gttatcgaaa cccaacaaaa ttgcatcatc 60

tggctgggtc tggcccattc caccgtggtc gatgtgatta gagccattag ggcaaatggg 120

attttcatca cccaagaggc tgaggagata ctgcacgttt taagggagtg gctgttctat 180

aacttcaaca tcgagcgctc aaagcgccga gaccgtcgcc gacgagcggt gtgcagtgcc 240

cggaccaggt tctgcttcgt aaaatacgaa aatgtccgga aacagctcca tcatgacacg 300

atccagaaca cgattagcgt tatcttgcca tcatccgtac caaccgccgg ccaccttacc 360

tcgctgtga 369

<210> 8

<211> 27

<212> DNA

<213> Artificial

<220>

<223> Shepherdin sequence

<400> 8

aaacactcgt cgggctgcgc attcctg 27

<210> 9

<211> 177

<212> DNA

<213> Artificial

<220>

<223> Par-4SAC sequence

<400> 9

ggcaagagct cgggccccag tgccaggaaa ggcaaggggc agatcgagaa gaggaagctg 60

cgggagaagc ggcgctccac cggcgtggtc aacatccctg ccgcagagtg cttagatgag 120

tacgaagatg atgaagcagg gcagaaagag cggaaacgag aagatgcaat tacacaa 177

<210> 10

<211> 240

<212> DNA

<213> Artificial

<220>

<223> NT4 Signal peptide sequence

<400> 10

atgctccctc tcccctcatg ctccctcccc atcctcctcc ttttcctcct ccccagtgtg 60

ccaattgagt cccaaccccc accctcaaca ttgccccctt ttctggcccc tgagtgggac 120

cttctctccc cccgagtagt cctgtctagg ggtgcccctg ctgggccccc tctgctcttc 180

ctgctggagg ctggggcctt tcgggagtca gcaggtgccc cggccaaccg cagccggcgt 240

<210> 11

<211> 9

<212> DNA

<213> Artificial

<220>

<223> (GSG)

<400> 11

ggcagcggt 9

<210> 12

<211> 84

<212> DNA

<213> Artificial

<220>

<223> FURIN2A

<400> 12

agagctaaga gggctccagt aaagcagact ctaaacttcg atcttctcaa gctcgctgga 60

gatgttgaga gcaacccagg tcca 84

Claims

1. A modified adeno-associated virus (AAV) which is capable of effecting the secretory expression of a chimeric peptide or chimeric protein of an anti-tumor cell.

2. The modified AAV according to claim 1, wherein said AAV is preferably AAV 2.

3. The modified AAV according to any of claims 1-2, wherein said chimeric peptide or protein effecting secretory expression of anti-tumor cells comprises an innate immune stimulator and an immune checkpoint inhibitor and a peptide active targeting tumor apoptosis.

4. The modified AAV according to claim 3, wherein the innate immune stimulating factor for use in a chimeric peptide or protein for secretory expression of anti-tumor cells is granulocyte colony-stimulating biological factor (GM-CSF).

5. The modified AAV according to claim 3, wherein the immune checkpoint inhibitor is a single chain antibody selected from any one of PD1, TIM3 and LAG3 in a chimeric peptide or protein capable of effecting secretory expression of an anti-tumor cell.

6. The modified AAV according to claim 5, wherein the immune checkpoint inhibitor is preferably any one of PD1scFv, TIM3scFv, LAG3 scFv;

further preferably, the immune checkpoint inhibitor is PD1 scFv.

7. The modified AAV according to claim 3, wherein the apoptosis-targeting active peptide of the chimeric peptide or protein capable of effecting secretory expression of anti-tumor cells is selected from the group consisting of the N-terminal 15 peptide of P53, the C-terminal 22 peptide of P53, the C-terminal 37 peptide of P53N (P73 inhibitory peptide), and any one of Apoptin, ORF4, Shepherdin, and par-4 SAC.

Furthermore, the selection of the active peptide targeting tumor apoptosis is determined by the gene detection of tumor cells, if the detection result is related to P53 defect or mutation, the active peptide targeting tumor apoptosis is selected from any one of P53N-terminal 15 peptide, P53C-terminal 22 peptide and P53N-terminal 37 peptide (P73 inhibitory peptide); if the detection result is not related to P53 deficiency or mutation, the active peptide targeting tumor apoptosis is selected from any one of Apoptin, ORF4, Shepherdin, par-4 SAC.

8. A modified AAV according to claim 1, wherein said modified AAV comprises a combination of nucleic acid sequences which effect secretory expression of a chimeric peptide or chimeric protein to a tumor cell.

9. The modified AAV of claim 8, wherein said combination of nucleic acid sequences effecting secretory expression of a chimeric peptide or protein to a tumor cell encodes an innate immune stimulator and an immune checkpoint inhibitor and a peptide targeting apoptosis.

10. The modified AAV according to any of claims 8-9, wherein said combination of nucleic acid sequences capable of effecting secretory expression of a chimeric peptide or protein against a tumor cell comprises GM-CSF and any one of the immune checkpoint inhibitors PD1scFv or TIM3scFv or LAG3scFv and any one of the apoptosis-targeting active peptides P53N-terminal 15 peptide or P53C-terminal 22 peptide or P53N terminal 37 peptide (P73 inhibitory peptide).

11. The modified AAV according to any one of claims 8 to 9, wherein the combination of nucleic acid sequences capable of effecting secretory expression of the chimeric peptide or protein against a tumor cell comprises an innate immune stimulator GM-CSF and any one of the immune checkpoint inhibitors PD1scFv or TIM3scFv or LAG3scFv and any one of the apoptosis-targeting active peptides Apoptin or ORF4 or Shepherdin or par-4 SAC.

12. The modified AAV according to any of claims 8 to 11, wherein said combination of nucleic acid sequences effecting secretory expression of a chimeric peptide or chimeric protein from an anti-tumor cell comprises a signal peptide and a cell-penetrating peptide sequence.

13. The modified AAV according to any one of claims 8-11, wherein the nucleic acid sequence capable of effecting secretory expression of the chimeric peptide or chimeric protein of an anti-tumor cell comprises an intron sequence.

14. The modified AAV according to any of claims 1 to 13, wherein said nucleic acid sequence is any that effects secretory expression of a chimeric peptide or chimeric protein against a tumor cell and/or any vector and/or any virus for use in the preparation of a medicament for the treatment or prevention of a tumor species, not limited to cancer species.