[go: up one dir, main page]

WO2019174610A1 - Virus oncolytique, séquence d'adn synthétique et utilisation associée - Google Patents

Virus oncolytique, séquence d'adn synthétique et utilisation associée Download PDF

Info

Publication number
WO2019174610A1
WO2019174610A1 PCT/CN2019/078117 CN2019078117W WO2019174610A1 WO 2019174610 A1 WO2019174610 A1 WO 2019174610A1 CN 2019078117 W CN2019078117 W CN 2019078117W WO 2019174610 A1 WO2019174610 A1 WO 2019174610A1
Authority
WO
WIPO (PCT)
Prior art keywords
lys
arg
oncolytic virus
trp
cys
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2019/078117
Other languages
English (en)
Chinese (zh)
Inventor
蔡立刚
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Individual
Original Assignee
Individual
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Individual filed Critical Individual
Priority to CN201980003419.3A priority Critical patent/CN111094324B/zh
Publication of WO2019174610A1 publication Critical patent/WO2019174610A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/08—RNA viruses
    • C07K14/145—Rhabdoviridae, e.g. rabies virus, Duvenhage virus, Mokola virus or vesicular stomatitis virus
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention belongs to the field of biomedicine, and more particularly to an oncolytic virus, a basic peptide segment and an application thereof.
  • the oncolytic virus has the property of infecting and proliferating in tumor cells, thereby destroying the tumor tissue.
  • the safety and therapeutic efficacy of a variety of viruses with oncolytic properties for the treatment of different cancers has been reported and confirmed.
  • the current working principles of oncolytic viruses include:
  • Oncolytic viruses can selectively infect tumor cells by inactivation or deletion of specific genes in target cells, and ultimately destroy tumor cells.
  • adenovirus, Newcastle virus, Coxsackie virus is a single-stranded RNA virus, and RNA replication is carried out in the cytoplasm without causing insertional gene mutation of the host cell.
  • Coxsackie virus is divided into two subtypes A and B based on its antigenicity in mice. In most cases, patients infected with wild-type Coxsackie virus will only have mild "cold" symptoms. Currently, both subtypes of Coxsackie virus have been tried alone or in combination for tumor therapy.
  • the oncolytic virus for the purpose of "killing" tumor cells is highly toxic, and when the body is infected with the wild type virus, pathological reactions such as fever and chills may occur. With the increase of serum AMY, CK, AST, ALT and other indicators, the organs of the body may be damaged. For example, pancreatic exocrine tissue damage, myocardial tissue and liver inflammation.
  • the present invention provides an oncolytic virus, a synthetic DNA sequence and an application thereof, which aims to inhibit tumor cells by providing an oncolytic virus capable of changing the tumor microenvironment. This solves the technical problem that the existing oncolytic virus tumor suppression effect is poor or the toxicity is too strong.
  • an oncolytic virus characterized in that an expression sequence of an exogenous basic peptide is inserted into a genome, and the basic peptide is expressed in a physiological process.
  • the pH of the host environment in which it is infected is increased by about 0.4 to about 0.6.
  • the oncolytic virus is herpes virus, coxsackie virus, adenovirus, vaccinia virus, measles virus, poliovirus, retrovirus, reovirus, respiratory tract Cytovirus, parvovirus H1, vesicular stomatitis virus, or Newcastle disease virus, preferably adenovirus, Newcastle virus or Coxsackie virus.
  • the oncolytic virus wherein the exogenous basic peptide is a 4 peptide to 10 peptide.
  • the oncolytic virus has a basic amino acid content of more than 60% in the exogenous basic peptide.
  • the oncolytic virus has a basic amino acid content of more than 80% in the exogenous basic peptide.
  • the oncolytic virus wherein the basic amino acid is selected from the group consisting of: arginine, lysine or histidine.
  • the oncolytic virus wherein the basic amino acid is selected from the group consisting of: arginine or lysine.
  • the oncolytic virus has an N-terminal amino acid of the basic peptide segment which is lysine.
  • the oncolytic virus wherein the exogenous basic peptide is selected from the group consisting of:
  • the oncolytic virus wherein the oncolytic virus is a Coxsackievirus B3 strain.
  • the oncolytic virus wherein the exogenous basic peptide is selected from the group consisting of:
  • the oncolytic virus wherein the oncolytic virus is a variant attenuated strain Coxsackie virus B3 strain, comprises the following base mutation sites: T96C, G1180A, T1654C, T1756C, G2276A, A2685C, G2690A, C3120A , A3231G, G4327A, T5088C, A5270G, C7026T, and/or G7192A.
  • the oncolytic virus, the coding sequence of the exogenous basic peptide fragment is inserted on the pVAX1 vector.
  • the oncolytic virus wherein the exogenous basic peptide is selected from the group consisting of Lys-Arg-Trp-His-Lys-Met-Arg-Lys-His.
  • the use of the oncolytic virus is provided, and the oncolytic virus provided by the present invention is applied to the preparation of an antitumor drug.
  • the use of the oncolytic virus provided by the present invention is applied to the preparation of an anti-solid tumor drug.
  • the application, the oncolytic virus provided by the present invention is applied to the preparation of a medicament against an anti-respiratory system tumor, a digestive tract system tumor, an endocrine system tumor, or a gynecological tumor.
  • an antitumor drug comprising the oncolytic virus provided by the present invention is provided.
  • the anti-tumor drug further comprises a checkpoint inhibitor.
  • a method of treating a malignant tumor which comprises administering the antitumor drug of the present invention intravenously or topically to a lesion.
  • the method of treatment wherein the malignant tumor is a solid tumor.
  • the method of treatment wherein the malignant tumor is a respiratory system tumor, a digestive system tumor, an endocrine system tumor, or a gynecological tumor.
  • a synthetic DNA sequence for expressing a basic peptide having a basic amino acid content of more than 60% is provided.
  • the synthetic DNA sequence has a basic amino acid content of more than 80% in the basic peptide segment.
  • the synthetic DNA sequence wherein the basic amino acid is selected from the group consisting of: arginine, lysine or histidine.
  • the synthetic DNA sequence wherein the basic amino acid is selected from arginine or lysine.
  • the synthetic DNA sequence, wherein the N-terminal amino acid of the basic peptide segment is lysine.
  • the synthetic DNA sequence, wherein the exogenous basic peptide is selected from the group consisting of:
  • the synthetic DNA sequence, wherein the basic peptide sequence is:
  • the oncolytic virus provided by the invention can significantly change the pH value of the interstitial cells of the tumor lesion, thereby affecting the microenvironment of tumor cell growth, and finally inhibiting the growth of the tumor, and has a relatively broad broad-spectrum resistance.
  • Tumor effect applied to the preparation of anti-tumor drugs, has a good application prospect.
  • the oncolytic virus provided by the present invention is a microenvironment for tumor growth, not a tumor cell itself, and thus tumor cells are not easily resistant to mutation. What is even more striking is that since the oncolytic virus proliferates with the proliferation of malignant tumor cells and continues to express the basic polypeptide, the anti-tumor effect has a sustained cumulative effect, and automatically adapts to the degree of tumor development without excessive treatment.
  • the oncolytic virus provided by the present invention may be various types of oncolytic viruses currently known, and the expression of the basic peptide segment acts on the cell microenvironment, and kills the tumor cell or inhibits the expression of a specific gene of the tumor cell.
  • the principle of action of oncolytic viruses does not conflict, and the effects can be superimposed on each other to further inhibit tumor growth and have an anti-tumor effect.
  • Coxsackie CVB 3 and the N-terminal lysine-like 4 peptide and 9-peptide expression gene which can obviously change the acid-base environment of the tumor cell interstitial, and has excellent anti-solid tumor effect. And its toxicity is low, the side effects are small, and only a slight fever reaction is caused.
  • Coxsackie virus is an RNA virus that does not integrate with host cells and has no risk of transcription.
  • the tumor treatment method provided by the present invention is suitable for intravenous administration because of the excellent safety, targeting, specificity, and low toxic side effects of the oncolytic virus provided by the present invention. In particular, it has a good therapeutic effect on malignant tumors that do not have surgical conditions, such as metastatic type, diffusivity, and early stage tumors. In addition, the tumor treatment method provided by the invention has a certain tumor prevention effect.
  • the expression gene for encoding a basic peptide segment provided by the present invention can express a basic peptide segment having a pH value which changes the microenvironment of the cell, thereby affecting the microenvironment of the tumor cell lesion region and inhibiting the growth of the tumor cell.
  • FIG. 1 is a schematic diagram showing the structure of a Coxsackie virus gene carrying the eukaryotic expression vector pVAX1 according to an embodiment of the present invention
  • FIG. 2 is a schematic diagram showing the structure of a Coxsackie virus gene carrying the eukaryotic expression vector pVAX1 inserted into a synthetic DNA sequence according to an embodiment of the present invention
  • FIG. 3 is a schematic flow chart of a virus purification process provided by an embodiment of the present invention.
  • Figure 5 is a comparison view of a tissue section provided in Example 20 of the present invention.
  • Figure 6 is a graph showing a tumor volume curve according to Embodiment 21 of the present invention.
  • Figure 7 is a graph showing a tumor volume curve according to Embodiment 22 of the present invention.
  • Figure 8 is a graph showing a tumor volume curve according to Embodiment 23 of the present invention.
  • Figure 9 is a graph showing the inhibitory effect of tumor cells in vitro according to Example 24 of the present invention.
  • Figure 10 is a microscopic examination result of myocardial cytotoxicity provided in Example 25 of the present invention.
  • Figure 11 is a photograph of a mouse after 6 days of toxicity test on BALB/C mice provided in Example 25 of the present invention.
  • Figure 12 is a photomicrograph of a tissue section of a mouse myocardial tissue after 6 days of toxicity test in BALB/C mice according to Example 25 of the present invention
  • Figure 13 is a photograph showing the experimental observation of toxicity of suckling mice provided in Example 25 of the present invention.
  • Figure 14 is a diagram showing the results of a microscopic examination provided in Embodiment 26 of the present invention.
  • Figure 15 is a comparison diagram of pH detection provided in Example 24 of the present invention.
  • Figure 16 is a graph showing the in vitro inhibitory effect on different types of tumor cells provided in Example 27 of the present invention.
  • the oncolytic virus provided by the invention has an expression sequence of an exogenous basic peptide inserted in the genome, and expresses the basic peptide in a physiological process, so that the pH of the infected host environment increases, and the increase is about 0.4. To 0.6. After the infection of the oncolytic virus of the exogenous peptide gene, the large expression of the exogenous alkaline peptide can make the microenvironment of the tumor tissue alkaline, so that the tumor tissue can be better inhibited and eliminated.
  • the oncolytic virus is herpes virus, coxsackie virus, adenovirus, vaccinia virus, measles virus, poliovirus, retrovirus, reovirus, respiratory syncytial virus, parvovirus H1, vesicular mouth Inflammatory virus, or Newcastle disease virus.
  • the oncolytic virus is preferably an oncolytic virus such as an adenovirus, a Newcastle virus or a Coxsackie virus that inactivates or deletes a specific gene in a target cell.
  • the exogenous basic peptide is a 4 peptide to 10 peptide; wherein the basic amino acid content exceeds 60%, preferably exceeds 80%; the basic amino acid is selected from: arginine, lysine or histidine, preferably The basic amino acid is selected from the group consisting of: arginine or lysine; the N-terminal amino acid of the exogenous basic peptide segment is preferably lysine.
  • exogenous basic peptide is selected from the following peptides:
  • the coxsackie virus specifically the coxsackie virus attenuating variant, is used to insert the following exogenous basic peptide on the pVAX1 vector in which the viral genome is constructed:
  • the exogenous basic peptide is selected from the group consisting of:
  • the exogenous peptide segment (14) Lys-Arg-Trp-His-Lys-Met-Arg-Lys-His exhibits excellent tumor suppressing effect and Good security.
  • the Coxsackie virus CVB 3 virus preferably adopts the mutant attenuated strain Coxsackie virus B3 strain, and includes the following base mutation sites: T96C, G1180A, T1654C, T1756C, G2276A, A2685C, G2690A, C3120A, A3231G, G4327A. , T5088C, A5270G, C7026T, and / or G7192A.
  • the coding sequence of the exogenous basic peptide fragment was inserted on the pVAX1 vector.
  • the foreign basic peptide DNA sequence is inserted between the 5'UTR and VP4 fragments of the recombinant vector
  • the oncolytic virus provided by the invention is applied to prepare antitumor drugs, especially anti-solid tumor drugs, for example, drugs for preparing anti-respiratory system tumors, digestive system tumors, endocrine system tumors, or gynecological tumors.
  • antitumor drugs especially anti-solid tumor drugs, for example, drugs for preparing anti-respiratory system tumors, digestive system tumors, endocrine system tumors, or gynecological tumors.
  • the oncolytic virus provided by the invention after reaching the lesion area, utilizes the targeting and replication ability of the virus to express the basic peptide segment according to the degree of tumor. The experiment confirmed that the basic peptide segment changed the pH of the microenvironment of the tumor cells in the tumor cells and the intercellular environment. This change brings a series of combined effects to the metabolism of tumor cells, which ultimately leads to significant tumor suppression.
  • the current oncolytic virus can theoretically carry the expression gene of the exogenous basic peptide by genetic modification, thereby changing the microscopic presence of the tumor cell in addition to the original effect of inhibiting or killing the tumor cell.
  • the environment produces an inhibitory effect, and the two act synergistically to inhibit the tumor more effectively. Since the oncolytic virus provided by the present invention exerts an anti-tumor effect by affecting the microenvironment in which the tumor cells are located, it has a more pronounced inhibitory effect on the solid tumor in which the tumor cells are concentrated.
  • the present invention provides an antitumor drug comprising the oncolytic virus provided by the present invention.
  • an immunoassay point inhibitor is also included.
  • the drug can exert a good tumor suppressing effect by intravenous administration or topical administration to the lesion.
  • Immunological checkpoint inhibitors PD-1, PD-L1, CTLA4
  • the response rate to solid tumors is not high (except melanoma)
  • the stimulation of the immune system is insufficient.
  • the synergistic effect of the oncolytic virus immunoassay inhibitor provided by the invention significantly improves the killing effect of the immune system on the solid tumor, enhances the permeability of the local immune cells of the tumor, and up-regulates the PD-L1.
  • CVB 3 virus Especially for recombinant CVB 3 virus, it induces local specific and non-specific immune responses in tumors, leading to some immune changes such as: calreticulin (CRT) exposure, ATP valgus, HGMB1 (Extracellular High Mobility Group Box1) Translocation within the cell.
  • CRT calreticulin
  • ATP ATP valgus
  • HGMB1 Extracellular High Mobility Group Box1
  • Translocation within the cell.
  • the oncolytic virus proliferates, by inducing IFN or/and cytokine production to activate NK cells and DC cells, various mature DCs and cytotoxic CD107a+ NK cells are promoted into the tumor site, resulting in an immune cell spectrum in the tumor microenvironment. Changes, while restoring the body's inherent anti-tumor immunity.
  • the synergistic effect of the recombinant Coxsackie virus provided by the present invention and an immunoassay inhibitor is particularly remarkable.
  • the present invention also provides an expression gene of a basic peptide fragment for expressing a basic peptide having a basic amino acid content of more than 60%, preferably more than 80%;
  • the basic amino acid is selected from the group consisting of arginine, lysine or histidine, preferably the basic amino acid is selected from arginine or lysine.
  • the N-terminal amino acid of the basic peptide segment is preferably lysine.
  • the synthetic DNA sequence encoding the exogenous basic peptide is selected from the following peptides:
  • the basic peptide sequence is preferably:
  • the full-length sequence of the Coxsackie B3 nancy strain is shown in GeneBank ID: JX312064.1 (presented by Tongji Medical College).
  • the recombinant Coxsackie virus strain (rCVB3) used in the examples contained the following base mutation sites: T96C, G1180A, T1654C, T1756C, G2276A, A2685C, G2690A, C3120A, A3231G, G4327A, T5088C, A5270G, C7026T, G7192A.
  • the complete cDNA sequence of the recombinant Coxsackie virus strain was synthesized by Wuhan Boweed Biotechnology Co., Ltd. and constructed by molecular biology to the eukaryotic expression vector pVAX1, as shown in Figure 1:
  • the oncolytic virus provided in the present example was inserted into the exogenous basic peptide gene sequence by reverse genetics between the 5' UTR and VP4 fragments of the above constructed recombinant vector.
  • a 15 bp (SEQ NO. 1) and 24 bp (SEQ NO. 2) DNA sequences were used at the 5' and 3' ends of the sequence for recognition and cleavage of protease C, as shown in Figure 2.
  • Example Peptide name Polypeptide sequence Gene name gene sequence 1 Polypeptide 1 SEQ NO.3 Nucleotide seque 1 SEQ NO.4 2 Polypeptide 2 SEQ NO.5 Nucleotide seque 2 SEQ NO.6 3 Polypeptide 3 SEQ NO.7 Nucleotide seque 3 SEQ NO.8 4 Polypeptide 4 SEQ NO.9 Nucleotide seque 4 SEQ NO.10 5 Polypeptide 5 SEQ NO.11 Nucleotide seque 5 SEQ NO.12 6 Polypeptide 6 SEQ NO.13 Nucleotide seque 6 SEQ NO.14 7 Polypeptide 7 SEQ NO.15 Nucleotide seque 7 SEQ NO.16 8 Polypeptide 8 SEQ NO.17 Nucleotide seque 8 SEQ NO.18 9 Polypeptide 9 SEQ NO.19 Nucleotide seque 9 SEQ NO.20 10 Polypeptide 10 SEQ NO.21 Nucleotide seque 10 SEQ NO.22 11 Polypeptide 11 SEQ NO.23 Nu
  • the insertion method is specifically as follows: the DNA sequence carrying the above-mentioned exogenous basic peptide is inserted between the 5'UTR and VP4 fragments of the recombinant vector, and the positive clone is screened and identified, and the plasmid is extracted and obtained for virus. The complete cDNA of the package.
  • the plasmid containing pVAX1-SalI and pUC19 was extracted using a kit from Axygen.
  • the plasmid pVAX1 (ApaI ⁇ SalI) and plasmid pUC57-CVB3-Am were double digested with NotI and SalI. After the reaction, 1% agarose gel electrophoresis was carried out, and 2999 bp vector and about 7500 bp CVB3-Am fragment were separately collected and gelatinized. Recovery, purification of the digested product according to the specific steps of Axygen's gel recovery kit.
  • CVB3-Am fragment digested with NotI and SalI was ligated to the vector pVAX1 (ApaI ⁇ SalI) in a certain ratio using TKDNA ligase of TAKARA, and transformed into Stbl3.
  • the pVAX1 vector carrying the complete cDNA sequence of the recombinant Coxsackie virus was transfected into Cos7 packaging cells, and the recombinant virus solution having infectious ability was cultured and obtained.
  • a PolyA sequence is inserted after the 3' UTR, and the length is between 20 and 100, preferably between 30 and 80, which can effectively ensure the stability of the gene encoding the exogenous alkaline peptide, thereby ensuring the expression effect.
  • the virus can be stored at -20 ° C for more than one year, can be placed at room temperature for 2 days without dropping the titer, and has strong stability, which is convenient for storage and transportation.
  • Comparative Example 1 Insertion of the above synthetic DNA sequence between the pVAX1 vector VP1 and 2A elements to obtain a virus, the expression of the basic peptide was not stably expressed, and the inhibitory effect on cancer cells was limited.
  • the infectious virus-containing recombinant virus solution described in Examples 1 to 18 was inoculated into the expanded and cultured Vero cells, and a virus purification liquid was obtained as a test product by a production and purification process.
  • the virus purification process diagram is shown in Figure 3:
  • the virus purification solution is tested and should meet the following criteria:
  • test article used in this example was prepared and tested according to the protocol described in Example 19.
  • the recombinant Coxsackie viruses of Examples 1 to 18 were used as test articles, which were prepared in Example 5, Example 14, and Example 17, respectively.
  • the above virus was prepared into the test sample according to the method described in Example 19.
  • the grouping, dosage and method of administration are as follows:
  • Group 2 (cisplatin) was administered once a week for 4 weeks, and for 1 week, D41 animals were euthanized; physiological saline was administered once a day for 6 weeks, and D41 animals were euthanized. Observe 2 times a day during the administration, observe the general clinical symptoms of the animals, and measure the body weight and tumor diameter twice a week.
  • test group prepared in the right example 5 has obvious tumor cell damage and deeper eosin staining than the left negative control group.
  • test article used in this example was prepared and tested according to the protocol described in Example 19.
  • Example 1 recombinant coxsackie virus in which three kinds of genomes were inserted with a basic peptide segment was used as a test article, which were prepared in Example 1, Example 2, Example 4, and Example 5, respectively.
  • the above virus was prepared into the test sample according to the method described in Example 19.
  • the grouping, dosage and method of administration are as follows:
  • Group 2 (cisplatin) was administered once a week for 4 weeks, and for 1 week, D48 animals were euthanized; physiological saline was administered once a day for 7 weeks, and D48 animals were euthanized. The drug was observed twice a day during the administration, and the general clinical symptoms of the animals were observed, and the body weight and tumor diameter were measured twice a week.
  • Example 1 the test articles prepared in Example 1, Example 2, Example 4, and Example 5 all have an anti-tumor effect, wherein the test sample of Example 5 has a significant inhibitory effect on tumor growth.
  • test article used in this example was prepared and tested according to the protocol described in Example 19.
  • the above virus was prepared into the test sample according to the method described in Example 19.
  • A549 cells established subcutaneous tumor nude mouse model of lung cancer, 20 tumor-bearing animals screened into homogeneous tumor volume, tumor volume 62-92mm 20 to select animals are divided into 3 groups of 1 to 4, the mean tumor volume of about 79mm 3, a fully Randomized grouping, each group of animals was given a random number by Excel software, and the random numbers were sorted in ascending order. They were divided into 4 groups of 5 animals each.
  • the grouping, dosage and method of administration are as follows:
  • Group 2 (cisplatin) was administered once a week for 4 weeks, and for 1 week, D42 animals were euthanized; physiological saline was administered once a day for 6 weeks, and D42 animals were euthanized. Observe 2 times a day during the administration, observe the general clinical symptoms of the animals, and measure the body weight and tumor diameter twice a week.
  • test samples prepared in Example 18 and Example 13 all have certain anti-tumor effects.
  • test article used in this example was prepared and tested according to the protocol described in Example 19.
  • Example 8 recombinant coxsackie virus in which three kinds of genomes were inserted with a basic peptide segment was used as a test article, which were prepared in Example 8, Example 9, and Example 10, respectively.
  • the above virus was prepared into the test sample according to the method described in Example 19.
  • tumor screening animal tumor volume 25 uniform, first select tumor volume 65-90mm 25 animals were divided into 3 groups of 1 to 5, the mean tumor volume of about 79mm 3, a fully Randomized grouping, each group of animals was given a random number by Excel software, and the random numbers were sorted in ascending order. They were divided into 5 groups of 5 animals each.
  • the grouping, dosage and method of administration are as follows:
  • Group 2 (cisplatin) was administered once a week for 4 weeks, and for 1 week, D33 animals were euthanized; physiological saline was administered once a day for 5 weeks, and D33 animals were euthanized. Observe 2 times a day during the administration, observe the general clinical symptoms of the animals, and measure the body weight and tumor diameter twice a week.
  • test samples prepared in Example 8, Example 9, and Example 10 all have certain anti-tumor effects.
  • test articles prepared in Examples 1 to 18 all had antitumor effects, and the test articles prepared in Example 5 and the test samples prepared in Example 14 had a significant inhibitory effect on tumor growth.
  • the human lung cancer cell line A549 was assayed for 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl 2H-tetrazolium bromide (MTT). Cells were seeded in 96-well plates 24 hours prior to treatment and grown to approximately 80% confluence.
  • MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl 2H-tetrazolium bromide
  • Recombinant CVB3 with different concentrations (1 PFU/mL, 1 x 101 PFU/mL, 1 x 102 PFU/mL, 1 x 103 PFU/mL, 1 x 104 PFU/mL, 1 x 105 PFU/mL, 1 x 106 PFU/mL, 1 x 107 PFU/mL or 1 x 108 PFU/mL), and Example 5, implementation Example 14, Example 17 Infected cells, or normal saline (NS) was used as a negative control, and cisplatin was used as a positive control. After 72 hours, the MTT assay was performed according to the manufacturer's protocol (VWR Life Sciences Amresco, Radnor, PA, USA).
  • Example 5 and Example 14 (peptide amino acid over 60%) inhibited tumor cells by more than 95% at a viral concentration of 10 7 ; In the peptide example of Example 5, the inhibitory effect was particularly remarkable.
  • Example 5 The oncolytic viruses of Example 5 (rCVB3-4pep5) and Example 14 (rCVB3-9pep) were evaluated using rCVB3 and CVB3Nancy strains as positive controls.
  • Cardiomyocyte toxicity test The viruses of Example 5, Example 14 and the positive control group were respectively infected with human cardiomyocytes (purchased from Suzhou Bei Na Chuanglian Biotechnology Co., Ltd.), and the final concentration of the virus was 10 7 PFU/ml. The group used saline. Microscopic examination after 72 hours. As a result, as shown in Fig. 10, the CVB3Nancy strain caused cardiomyocyte lesions, while the rCVB3, Example 5 and Example 14 administration groups had no lesions.
  • Example 5 The viruses of Example 5, Example 14 and the positive control group were injected intraperitoneally into BALB/C mice (license No. 42000600028329) in a dosage form of 10 8 PFU/ml, 0.3 ml per day.
  • the negative control group used physiological saline. Observation was performed every day, and after 6 days, as shown in Fig. 11, mouse myocardial tissue was taken for tissue sectioning, and the results are shown in Fig. 12.
  • the experimental results showed that the mice in the CVB3Nancy strain administration group were in a bad state. Myocardial tissue section results showed that the CVB3Nancy administration group caused significant myocardial damage, while the recombinant CVB3 administration group was normal.
  • Toxicity test on suckling rats The viruses of Example 5, Example 14 and the positive control group were injected intraperitoneally into the suckling mice (license No. 42816300002647) at a dose of 10 8 PFU/ml, each 0.1ml.
  • the negative control group used physiological saline. Observe every day. As a result, as shown in Fig. 13, the CVB3Nancy strain administration group died on the sixth day, and the rCVB3, Example 5, and Example 14 administration groups were normal.
  • test samples two kinds of recombinant Coxsackie viruses in which the basic peptide was inserted into the genome were used as the test samples, which were Example 5 and Example 14, respectively.
  • Example 5 and Example 14 were designated as 4p5 group and 9pep group, respectively.
  • Another group of cells served as a negative control.
  • Each group of cells was divided into two parts, cultured under the same conditions and subjected to test operations.
  • One of each group of cells was stained with eosin and microscopically after 3 hours of infection. The result is shown in Figure 14.
  • the two groups of cells infected with the recombinant Coxsackie virus cDNA showed significant lesions. From the staining results, the infected group was more deeply stained than the negative control group, indicating that the cytoplasm and interstitial cells were more eosinophilic.
  • Example 20 Each of the experimental groups in Example 20 was randomly selected for 3 samples, and the live pH measurement of the tumor site was performed on a D41 using a CL-9D02 benchtop pH/mV instrument. The measurement results of each group were taken as arithmetic mean values. As shown in Fig. 10, the pH values measured by the sampling of each of the examples were improved, and the lift value was 0.4 to 0.6, as shown in Fig. 15.
  • Example 27 In vitro inhibitory effect of different kinds of tumor cells in vitro
  • human lung cancer cell lines A549, GLC-82, NCI-H460, NCI-H1299, liver cancer SNU-398, and human lung fibroblasts were 3-(4,5- Determination of dimethylthiazol-2-yl)-2,5-diphenyl 2H-tetrazole ammonium bromide (MTT). Cells were seeded in 96-well plates 24 hours prior to treatment and grown to approximately 80% confluence.
  • Infect cells with recombinant CVB3 at different concentrations (1 PFU/mL, 1 x 101 PFU/mL, 1 x 102 PFU/mL, 1 x 103 PFU/mL, 1 x 104 PFU/mL, 1 x 105 PFU/mL, 1 x 106 PFU/mL, 1 x 107 PFU/mL or 1 x 108 PFU/mL), or with saline (NS)
  • a negative control cisplatin was used as a positive control. After 72 hours, the MTT assay was performed according to the manufacturer's protocol (VWR Life Sciences Amresco, Radnor, PA, USA).
  • Example 27 have broad-spectrum inhibitory effects on different types of lung cancer, liver cancer cells, and have little lethality against normal cells.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Virology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Epidemiology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Immunology (AREA)
  • Biophysics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

L'invention concerne un virus oncolytique, une séquence d'ADN synthétique et une utilisation associée. La séquence d'expression d'un peptide de base exogène est insérée dans le génome du virus oncolytique, et le peptide basique est exprimé dans un processus physiologique, de telle sorte que la valeur du pH du milieu hôte infecté par celui-ci est augmentée. La séquence d'ADN synthétique est utilisée pour exprimer un peptide basique, la teneur en acide aminé basique dans le peptide basique dépassant 60 %. Le virus oncolytique et la séquence d'ADN synthétique fournis par l'invention sont appliqués dans la préparation d'un médicament antitumoral et ont un bon effet antitumoral.
PCT/CN2019/078117 2018-03-14 2019-03-14 Virus oncolytique, séquence d'adn synthétique et utilisation associée Ceased WO2019174610A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201980003419.3A CN111094324B (zh) 2018-03-14 2019-03-14 一种溶瘤病毒、合成dna序列及其应用

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201862643166P 2018-03-14 2018-03-14
US62/643,166 2018-03-14

Publications (1)

Publication Number Publication Date
WO2019174610A1 true WO2019174610A1 (fr) 2019-09-19

Family

ID=67908669

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2019/078117 Ceased WO2019174610A1 (fr) 2018-03-14 2019-03-14 Virus oncolytique, séquence d'adn synthétique et utilisation associée

Country Status (2)

Country Link
CN (1) CN111094324B (fr)
WO (1) WO2019174610A1 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112538466A (zh) * 2020-12-10 2021-03-23 武汉博威德生物技术有限公司 一种柯萨奇病毒及其在制备抗肝癌药物中的应用

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005074968A2 (fr) * 2004-02-10 2005-08-18 Universiteit Maastricht Utilisation medicale de peptides basiques
WO2011100688A1 (fr) * 2010-02-12 2011-08-18 Dana-Farber Cancer Institute, Inc. Antagonistes améliorés du muc1
WO2013110120A1 (fr) * 2012-01-24 2013-08-01 Inter-K Pty Limited Agents peptidiques utilisés en thérapie anticancéreuse
US20160008480A1 (en) * 2014-07-08 2016-01-14 Samsung Electronics Co., Ltd. Fusion protein comprising targeting moiety, cleavage site, and cell membrane penetrating domain, and use thereof
WO2018027316A1 (fr) * 2016-08-09 2018-02-15 Alkayyal Almohanad Rhabdovirus oncolytique exprimant il12

Family Cites Families (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008140621A2 (fr) * 2006-12-21 2008-11-20 Mount Sinai School Of Medicine Of New York University Virus oncolytiques transgéniques et leurs utilisations
KR100931863B1 (ko) * 2007-09-04 2009-12-15 가톨릭대학교 산학협력단 약독화된 콕사키바이러스게놈을 이용한 외래 유전자 발현시스템
EP2085092A1 (fr) * 2008-01-29 2009-08-05 Bayer Schering Pharma Aktiengesellschaft Paramyxovirus oncolytiques atténués encodant des cytokines aviaires
ES2385251B1 (es) * 2009-05-06 2013-05-06 Fundació Privada Institut D'investigació Biomèdica De Bellvitge Adenovirus oncolíticos para el tratamiento del cáncer.
EP2619224A1 (fr) * 2010-09-24 2013-07-31 Oncos Therapeutics Oy Vecteurs adénoviraux oncolytiques, et procédés et utilisations associés
CN103981152B (zh) * 2014-04-16 2015-01-21 武汉博威德生物技术有限公司 一种柯萨奇病毒及其制备抗肿瘤药物之应用
CN104151402B (zh) * 2014-08-06 2017-01-18 苏州大学 病毒性心肌炎环肽疫苗及其制备方法
CN106632613B (zh) * 2017-01-17 2020-07-31 中国药科大学 与柯萨奇病毒腺病毒受体相关的亲和肽
CN107164338A (zh) * 2017-06-27 2017-09-15 镇江市卫克生物科技有限公司 一种重组溶瘤病毒及其应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005074968A2 (fr) * 2004-02-10 2005-08-18 Universiteit Maastricht Utilisation medicale de peptides basiques
WO2011100688A1 (fr) * 2010-02-12 2011-08-18 Dana-Farber Cancer Institute, Inc. Antagonistes améliorés du muc1
WO2013110120A1 (fr) * 2012-01-24 2013-08-01 Inter-K Pty Limited Agents peptidiques utilisés en thérapie anticancéreuse
US20160008480A1 (en) * 2014-07-08 2016-01-14 Samsung Electronics Co., Ltd. Fusion protein comprising targeting moiety, cleavage site, and cell membrane penetrating domain, and use thereof
WO2018027316A1 (fr) * 2016-08-09 2018-02-15 Alkayyal Almohanad Rhabdovirus oncolytique exprimant il12

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
POST, D. E.: "Cancer Therapy with a Replicating Oncolytic Adenovirus Targeting the Hypoxic Microenvironment of Tumors", CLINICAL CANCER RESEARCH, vol. 10, no. 24, 15 December 2004 (2004-12-15), pages 8603 - 8612, XP055637037 *

Also Published As

Publication number Publication date
CN111094324B (zh) 2023-10-10
CN111094324A (zh) 2020-05-01

Similar Documents

Publication Publication Date Title
ES2878549T3 (es) Adenovirus oncolítico para su uso en el tratamiento del cáncer cerebral
US20220288142A1 (en) Recombinant oncolytic virus, synthetic dna sequence, and application thereof
JP5807788B2 (ja) ニューカッスル病ウイルスの新規クローン、その作製及び癌治療への応用
Carter et al. Clinical trials of oncolytic viruses in breast cancer
CN109414487B (zh) 利用stat1/3抑制剂与溶瘤型疱疹病毒的组合物和方法
CN119709652A (zh) 溶瘤病毒及其应用
Deng et al. Oncolytic cancer therapy with a vaccinia virus strain
JP2002515442A (ja) 癌を処置するためのアデノウィルス−化学治療剤のコンビネーション
WO2019174610A1 (fr) Virus oncolytique, séquence d'adn synthétique et utilisation associée
WO2019201192A1 (fr) Virus coxsackie b pour le traitement de tumeurs
Rasoul The Anti-cancer Impact of Genetically Engineered Newcastle Disease Virus Expressing GFP Gene Against U87-MG Cell Line
CN111939262B (zh) 一种治疗肿瘤或癌症的药物组合物和其应用
EP3624825A1 (fr) Virus oncolytique recombinant
Chen et al. Advances in the therapeutic study of oncolytic virus in colorectal cancer
CN108495934B (zh) 将腺病毒和化学治疗剂组合用于治疗癌症
CN117660368A (zh) 表达趋化因子ccl19的重组溶瘤流感病毒及应用
US20240318147A1 (en) Recombinant oncolytic virus, and construction method therefor and use thereof
WO2022033467A1 (fr) Procédé de construction d'un virus oncolytique
JP2011520994A (ja) 腫瘍崩解性レオウイルス療法中の炎症性サイトカイン産生の排除
CN106591248B (zh) 重组溶瘤流感病毒制备方法和应用
JP2022543610A (ja) 遺伝子改変エンテロウイルスベクター
US20190284538A1 (en) Recombinant oncolytic virus, synthetic dna sequence, and application thereof
CN118593493B (zh) Anti-inflammatory agent 35在制备预防或治疗非洲猪瘟药物中的应用
WO2019144874A1 (fr) Application d'une souche atténuée de virus zika (zikv) dans le traitement d'un gliome cérébral
Frendéus et al. Vector-based Cancer Immunotherapy

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19766512

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19766512

Country of ref document: EP

Kind code of ref document: A1