WO2022151078A1 - Virus oncolytique et son application - Google Patents

Virus oncolytique et son application Download PDF

Info

Publication number
WO2022151078A1
WO2022151078A1 PCT/CN2021/071563 CN2021071563W WO2022151078A1 WO 2022151078 A1 WO2022151078 A1 WO 2022151078A1 CN 2021071563 W CN2021071563 W CN 2021071563W WO 2022151078 A1 WO2022151078 A1 WO 2022151078A1
Authority
WO
WIPO (PCT)
Prior art keywords
cancer
nucleic acid
oncolytic virus
acid fragment
virus
Prior art date
Application number
PCT/CN2021/071563
Other languages
English (en)
Chinese (zh)
Inventor
巩子英
张道允
孙永华
王毅
Original Assignee
嘉兴允英医学检验有限公司
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 嘉兴允英医学检验有限公司 filed Critical 嘉兴允英医学检验有限公司
Priority to CN202180000514.5A priority Critical patent/CN112840020B/zh
Priority to PCT/CN2021/071563 priority patent/WO2022151078A1/fr
Publication of WO2022151078A1 publication Critical patent/WO2022151078A1/fr

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A61K35/763—Herpes virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A61K35/768—Oncolytic viruses not provided for in groups A61K35/761 - A61K35/766
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70521—CD28, CD152
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
    • C12N2710/16621—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
    • C12N2710/16633—Use of viral protein as therapeutic agent other than vaccine, e.g. apoptosis inducing or anti-inflammatory

Definitions

  • the present application relates to the field of biotechnology, in particular to oncolytic viruses containing recombinant nucleic acids and applications thereof.
  • Oncolytic viruses refer to a class of viruses that can effectively infect and destroy cancer cells. Oncolytic viruses can replicate and proliferate in cancer cells, release new infectious virus particles to infect and destroy other cancer cells, or express proteins that have effects on cancer cells to affect the tumor microenvironment and stimulate host anti-tumor immune responses Or directly dissolve the tumor. Due to the properties of oncolytic viruses, this therapy can be administered both systemically and locally to treat primary and metastatic tumors. When cancer cells burst and die under the infection of an oncolytic virus, newly generated viral particles are released to further infect surrounding cancer cells.
  • Oncolytic viruses can not only directly kill tumors, but also are expected to stimulate the body's immune response and enhance anti-tumor effects.
  • oncolytic viruses can also be administered in combination with other anticancer drugs, and in addition, oncolytic viruses can be recombined with foreign genes that are beneficial for cancer treatment. In this way, on the one hand, the oncolytic effect of the oncolytic protein can be exerted, and on the other hand, the anticancer effect of other drugs can be exerted. Therefore, in order to achieve better therapeutic effects against cancer, there has always been a need for oncolytic viruses containing recombinant nucleic acids.
  • an oncolytic virus containing a recombinant nucleic acid is provided.
  • the recombinant nucleic acid may comprise: (i) a first nucleic acid fragment encoding a soluble PD-1 molecule; (ii) a second nucleic acid fragment encoding a VAR2CSA protein; and (iii) a third nucleic acid fragment encoding an antibody to the CD3 molecule.
  • the similarity between the first nucleic acid fragment and the sequence shown in SEQ ID NO: 1 may be greater than or equal to 90%.
  • the similarity between the second nucleic acid fragment and the sequence shown in SEQ ID NO: 2 may be greater than or equal to 90%.
  • the similarity between the third nucleic acid fragment and the sequence shown in SEQ ID NO: 3 may be greater than or equal to 90%.
  • the recombinant nucleic acid may further comprise (iv) a fourth nucleic acid fragment encoding the US11 protein.
  • the similarity between the fourth nucleic acid fragment and the sequence shown in SEQ ID NO: 4 may be greater than or equal to 90%.
  • the oncolytic virus can be of the genus Herpes simplex.
  • the oncolytic virus can be HSV-1 virus, and the similarity of the recombinant nucleic acid to the sequence shown in SEQ ID NO:5 can be greater than or equal to 80%.
  • the recombinant sequence may also include at least one of the following nucleic acid fragments: a nucleic acid fragment encoding a cytokine, a nucleic acid fragment encoding a costimulatory molecule, a nucleic acid fragment encoding an anti-angiogenic factor, and a matrix metalloproteinase encoding nucleic acid fragments.
  • an oncolytic virus containing a recombinant nucleic acid may comprise: (i) a first nucleic acid fragment encoding a soluble PD-1 molecule; (ii) a second nucleic acid fragment encoding a VAR2CSA protein; (iii) a third nucleic acid fragment encoding an antibody to the CD3 molecule; and ( iv) a fourth nucleic acid fragment encoding the US11 protein.
  • the oncolytic virus can be an HSV-1 or HSV-2 virus
  • the fourth nucleic acid fragment can comprise an exogenous nucleic acid fragment inserted into the recombinant nucleic acid.
  • the oncolytic virus can be HSV-1 virus, and the similarity of the recombinant nucleic acid to the sequence shown in SEQ ID NO:5 can be greater than or equal to 80%.
  • the oncolytic virus when the oncolytic virus acts on human non-small cell lung cancer cells, human liver cancer cells, human breast cancer cells, or human pancreatic cancer cells at a multiplicity of infection of 1 in a culture environment, the oncolytic virus is capable of, respectively, within 48 hours. Correspondingly results in the death of at least 40%, 80%, 40%, 50% of cancer cells.
  • the oncolytic virus when it acts on human liver cancer cells, human breast cancer cells, or human pancreatic cancer cells at a multiplicity of infection of 0.1 in a culture environment, it can cause about 80%, 40%, 30% of cancer cells die.
  • the oncolytic virus is capable of causing at least 70% or 80%, respectively, within 32 days when the oncolytic virus is administered to a human pancreatic cancer tumor in a single injection of 2x106 pfu or 3 injections of 1x106 pfu, respectively. % tumor volume reduction.
  • the oncolytic virus when administered to a human non-small cell lung cancer tumor in 3 injections of 2x106 pfu or 3 injections of 4x106 pfu, respectively, results in at least 70% of the corresponding results within 32 days. or 90% tumor volume reduction.
  • compositions for treating cancer may include the oncolytic virus of any one of the above and a pharmacologically acceptable carrier or excipient.
  • the cancer can be melanoma, lung cancer, leukemia, gastric cancer, ovarian cancer, pancreatic cancer, breast cancer, prostate cancer, bladder cancer, colon cancer, rectal cancer, liver cancer, cervical cancer, or osteosarcoma.
  • the cancer can be lung cancer, liver cancer, breast cancer, or pancreatic cancer.
  • compositions for treating pancreatic cancer may include the oncolytic virus of any one of the above and a pharmacologically acceptable carrier or excipient.
  • an application of the above oncolytic virus in the preparation of a medicament for treating cancer is provided.
  • the cancer can be melanoma, lung cancer, leukemia, gastric cancer, ovarian cancer, pancreatic cancer, breast cancer, prostate cancer, bladder cancer, colon cancer, rectal cancer, liver cancer, cervical cancer, or osteosarcoma.
  • the cancer can be lung cancer, liver cancer, breast cancer, or pancreatic cancer.
  • an oncolytic virus in the preparation of a medicament for treating pancreatic cancer.
  • a method for treating cancer is provided.
  • the method can include administering to a subject suffering from cancer an effective amount of the above-described composition.
  • the cancer may be melanoma, lung cancer, leukemia, gastric cancer, ovarian cancer, pancreatic cancer, breast cancer, prostate cancer, bladder cancer, colon cancer, rectal cancer, liver cancer, cervical cancer, or osteosarcoma.
  • the cancer can be lung cancer, liver cancer, breast cancer, or pancreatic cancer.
  • the subject may be a mammal.
  • the ratio of the amount of the oncolytic virus contained in the effective dose of the composition to the subject's body weight may range from 2.5 x 10 4 pfu/g to 5 x 10 5 pfu/g .
  • the ratio of the amount of the oncolytic virus contained in the effective dose of the composition to the subject's body weight can range from 2.5 x 10 4 pfu/g to 5 x 10 6 pfu/g .
  • the administering to a subject with cancer an effective dose of the composition may comprise administering the composition to the subject by injection.
  • the administering the composition to the subject by injection may comprise injecting the composition into the subject's tumor or at a site near the tumor.
  • FIG. 1 is a schematic diagram of the vector plasmid of the recombinant oncolytic virus constructed according to some embodiments of the present application;
  • FIG. 2 is a schematic diagram of human non-small cell lung cancer cells A549 transfected with recombinant oncolytic virus according to some embodiments of the present application after staining;
  • FIG. 3 is a schematic diagram of HepG2 staining of human hepatoma cells transfected with recombinant oncolytic virus according to some embodiments of the present application;
  • Figure 4 is a schematic diagram of human breast cancer cells transfected with recombinant oncolytic virus MCF-7 stained according to some embodiments of the present application;
  • FIG. 5 is a schematic diagram of human pancreatic cancer cells SW1990 transfected with recombinant oncolytic virus according to some embodiments of the present application after staining;
  • Figure 6 shows the cell viability of human non-small cell lung cancer cell A549, human liver cancer cell HepG2, human breast cancer cell MCF-7 and human pancreatic cancer cell SW1990 transfected with recombinant oncolytic virus according to some embodiments of the present application Schematic diagram;
  • FIG. 7 is a schematic diagram of SW1990 and A549 subcutaneous tumor models constructed by treating immunodeficient nude mice with recombinant oncolytic virus according to some embodiments of the present application;
  • Figure 8 is a schematic diagram of the time and tumor volume of SW1990 subcutaneous tumor models treated with different doses of recombinant oncolytic virus for different times according to some embodiments of the present application;
  • Figure 9 is a schematic diagram of the time and tumor volume of A549 subcutaneous tumor models treated with different doses of recombinant oncolytic virus for different times according to some embodiments of the present application.
  • Figure 10 is a graph showing the time and mouse survival rate of CT26 abdominal tumor model treated with different doses of recombinant oncolytic virus according to some examples of the present application.
  • a "subject” (may also be referred to as an "individual", “subject”) is an individual who is treated with an oncolytic virus or composition of the present application.
  • the subject may be a vertebrate.
  • Vertebrates can include fish (eg, sharks), amphibians (eg, frogs, toads, salamanders), reptiles (eg, turtles, snakes, lizards), birds (eg, ostriches), mammals, and the like.
  • the vertebrate is a mammal. Mammals include, but are not limited to, primates (including humans and non-human primates) and rodents (eg, mice and rats).
  • the mammal can be a human.
  • the subject may have cancer and has received other treatment (eg, chemotherapy) or has not yet received treatment.
  • treating refers to ameliorating or curing a disease (eg, cancer) in a subject.
  • treatment can include reducing, delaying, alleviating the severity of symptoms of cancer (eg, reduction in tumor volume), reducing the frequency of symptoms of cancer (eg, pain, etc.), prolonging survival of a subject with cancer time, increased survival rate, decreased cancer cell survival ability, or killed cancer cells, etc.
  • the term "effective dose” refers to an amount of a composition (eg, an amount sufficient to treat a disease) that is sufficient to provide a useful or otherwise reduce detrimental unbeneficial event.
  • the composition includes an oncolytic virus.
  • the amount of oncolytic virus contained in an effective dose of the composition depends on a variety of factors including, but not limited to, the purpose of treatment, the subject's weight, sex, age and general health, the route of administration, the time of administration, and the disease to be treated nature.
  • herpes simplex virus is an enveloped, neurotropic, double-stranded DNA virus.
  • viruses can be divided into herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2).
  • immune checkpoint inhibitor refers to an antibody that inhibits or blocks inhibitory immune checkpoint molecules.
  • Immune checkpoints are regulators and regulators of the immune system, which are essential for self-tolerance by preventing the immune system from indiscriminately attacking cells.
  • the present application provides an oncolytic virus containing recombinant nucleic acid.
  • the oncolytic virus may be of the herpes simplex virus genus, eg, HSV-1 or HSV-2 virus.
  • the recombinant nucleic acid may include a first nucleic acid fragment encoding a soluble PD-1 (soluble programmed death-1, sPD-1) molecule, a second nucleic acid fragment encoding a VAR2CSA protein, and an antibody encoding a CD3 (cluster of differentiation 3, CD3) molecule
  • the third nucleic acid fragment of , and the fourth nucleic acid fragment encoding the US11 protein.
  • the recombinant nucleic acid may also include nucleic acid fragments encoding cytokines, nucleic acid fragments encoding costimulatory molecules, nucleic acid fragments encoding anti-angiogenic factors, nucleic acid fragments encoding matrix metalloproteinases, and the like or combinations thereof.
  • the application also provides a composition for treating cancer.
  • the composition may include the recombinant nucleic acid-containing oncolytic virus described above and a pharmacologically acceptable carrier or excipient.
  • Cancers that can be treated by the composition may include, but are not limited to, melanoma, lung cancer, leukemia, gastric cancer, ovarian cancer, pancreatic cancer, breast cancer, prostate cancer, bladder cancer, colon cancer, rectal cancer, liver cancer, cervical cancer, osteosarcoma, etc. .
  • the present application also provides an application of the above oncolytic virus in the preparation of a medicament for treating cancer. Specifically, the present application also provides an application of the above oncolytic virus in the preparation of a medicament for treating pancreatic cancer.
  • the application further provides a method for treating cancer.
  • the method may comprise administering to a subject suffering from cancer an effective amount of the above-described composition.
  • the composition can be administered to a subject by injection, for example, into a subject's tumor or at a site near the tumor.
  • the composition may be administered to the subject in combination with other drugs (eg, anticancer drugs).
  • the oncolytic virus disclosed in the present application and the composition thereof can inhibit and kill cancer cells, effectively reduce the survival rate of cancer cells in the administered object, effectively improve or slow down the symptoms of cancer (eg, reduce tumor volume) and improve the survival ability of the object .
  • an oncolytic virus (also referred to as a "recombinant oncolytic virus") containing a recombinant nucleic acid is provided.
  • oncolytic viruses can be divided into DNA-based oncolytic viruses and RNA-based oncolytic viruses.
  • Exemplary DNA-like oncolytic viruses can include, but are not limited to, oncolytic adenovirus (adenovirus), vaccinia virus (vaccinia virus), parvovirus (parvovirus), herpes simplex virus (HSV), and the like.
  • Exemplary RNA-based oncolytic viruses can include, but are not limited to, reovirus, polio virus, seneca valley virus, and the like.
  • the oncolytic virus may be HSV, which belongs to the family Herpesviridae, the genus Herpes simplex. HSV can include HSV-1 and HSV-2.
  • the oncolytic virus can be an engineered oncolytic virus.
  • the oncolytic virus encoding the neurotropic ICP34.5 (or ⁇ -34.5) gene is removed in wild-type HSV-1 virus, thereby rendering it devoid of neurovirulence, ie, the resulting oncolytic virus is non-pathogenic Sexual/non-neurotoxic and oncolytic.
  • HSV-1 is one of the oncolytic viruses that can be used to selectively attack cancer cells due to its ease of manipulation and relative harmlessness in its native state.
  • oncolytic viruses eg, HSV-1
  • HSV-1 By modifying the genes of oncolytic viruses (eg, HSV-1), such as inserting other gene segments that inhibit and kill cancer cells, the ability of oncolytic viruses to target and infect cancer cells and/or oncolytic viruses can be improved The ability to kill cancer cells, so that the oncolytic virus has a better anti-tumor therapeutic effect.
  • a recombinant nucleic acid can include one or more exogenous nucleic acid fragments.
  • Exogenous nucleic acid fragments may include, but are not limited to, nucleic acid fragments encoding immune checkpoint inhibitors, nucleic acid fragments encoding molecules that facilitate the targeting of oncolytic viruses to infect cancer cells, nucleic acid fragments encoding antibodies to effector cell surface antigens, nucleic acid fragments encoding antibodies against effector cell surface antigens, Nucleic acid fragments that facilitate evasion or resistance of host immune responses by oncolytic viruses, nucleic acid fragments encoding cytokines, nucleic acid fragments encoding costimulatory molecules, nucleic acid fragments encoding anti-angiogenic factors, nucleic acid fragments encoding matrix metalloproteinases, blocking or down-regulation Antisense RNAs or small RNAs of proto-oncogenes and metabolic genes overexpressed by tumors, prodrug converting enzymes, etc., or
  • nucleic acid fragments encoding immune checkpoint inhibitors may include, but are not limited to, nucleic acid fragments encoding soluble PD-1 molecules (ie, first nucleic acid fragments), nucleic acid fragments encoding PD-1 inhibitors, PD-1 - Nucleic acid fragments encoding L1 (or B7-H1, CD274) inhibitors, nucleic acid fragments encoding PD-L2 (or B7-DC, CD273) inhibitors, nucleic acid fragments encoding CTLA-4 inhibitors, encoding LAG-3 inhibitors
  • nucleic acid fragments encoding molecules that facilitate targeting of oncolytic viruses to infect cancer cells may include, but are not limited to, nucleic acid fragments encoding VAR2CSA protein (ie, second nucleic acid fragments), and the like.
  • nucleic acid fragments encoding antibodies to effector cell surface antigens can include nucleic acid fragments encoding antibodies to T cell surface antigens and nucleic acid fragments encoding antibodies to B cell surface antigens, such as nucleic acid fragments encoding antibodies to CD3 molecules ( Namely, the third nucleic acid fragment), the nucleic acid fragment of the antibody encoding CD4 molecule, the nucleic acid fragment of the antibody encoding the CD5 molecule, the nucleic acid fragment of the antibody encoding the CD8 molecule, the nucleic acid fragment of the antibody encoding the CD45RO molecule, the nucleic acid fragment of the antibody encoding the CD20 molecule Nucleic acid fragments, nucleic acid fragments encoding antibodies to CD21 molecules, nucleic acid fragments encoding antibodies to CD45RA molecules, and the like, or combinations thereof.
  • Antigens encoding nucleic acid fragments that facilitate evasion or resistance to host immune responses by oncolytic viruses include, but are not limited to, nucleic acid fragments encoding US11 (ie, the fourth sequence), nucleic acid fragments encoding UL82, and the like, or combinations thereof.
  • Nucleic acid fragments encoding cytokines may include, but are not limited to, nucleic acid fragments encoding GM-CSF, nucleic acid fragments encoding G-CSF, nucleic acid fragments encoding M-CSF, nucleic acid fragments encoding IL-1, nucleic acid fragments encoding IL-2 , nucleic acid fragments encoding IL-3, nucleic acid fragments encoding IL-4, nucleic acid fragments encoding IL-5, nucleic acid fragments encoding IL-6, nucleic acid fragments encoding IL7, nucleic acid fragments encoding IL-8, IL- Nucleic acid fragments of 10, nucleic acid fragments encoding IL-12, nucleic acid fragments encoding IL-13, nucleic acid fragments encoding IL-15, nucleic acid fragments encoding IL-18, nucleic acid fragments encoding IL-21, nucleic acid fragments en
  • Nucleic acid fragments encoding costimulatory molecules may include, but are not limited to, nucleic acid fragments encoding CD27, nucleic acid fragments encoding CD28, nucleic acid fragments encoding CD70, nucleic acid fragments encoding CD80, nucleic acid fragments encoding CD83, nucleic acid fragments encoding CD86, nucleic acid fragments encoding CD134 (or OX-40) nucleic acid fragments, nucleic acid fragments encoding CD134L (or OK-40L), nucleic acid fragments encoding CD137 (41BB), nucleic acid fragments encoding CD137L (or 41BBL), nucleic acid fragments encoding CD224, nucleic acid fragments encoding GITR Nucleic acid fragments, nucleic acid fragments encoding ICOS, and the like, or combinations thereof.
  • Nucleic acid fragments encoding anti-angiogenic factors can include, but are not limited to, those encoding disrupting cell types (eg, endothelial cells (EC) and circulating endothelial progenitor cells, pericytes, vascular smooth muscle cells, mesenchymal cells, including stem cells and parenchymal cells). Nucleic acid fragments of one or more interacting polypeptides, disrupting one or more interactions of secreted factors (eg, VEGF, fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), or angiopoietin) acting polypeptides, etc., or a combination thereof.
  • disrupting cell types eg, endothelial cells (EC) and circulating endothelial progenitor cells, pericytes, vascular smooth muscle cells, mesenchymal cells, including stem cells and parenchymal cells.
  • Nucleic acid fragments encoding MMPs may include, but are not limited to, MMP1, MMP2, MMP3, MMP7, MMP9 ), matrix metalloproteinase 12 (MMP12).
  • the recombinant nucleic acid of an oncolytic virus can include nucleic acid fragments encoding immune checkpoint inhibitors, nucleic acid fragments encoding molecules that facilitate targeting of oncolytic viruses to infect cancer cells, nucleic acid fragments encoding antibodies to effector cell surface antigens Nucleic acid fragments and nucleic acid fragments encoding nucleic acid fragments that facilitate oncolytic virus evasion or resistance to host immune responses.
  • the recombinant nucleic acid may include a first nucleic acid fragment encoding a soluble PD-1 molecule, a second nucleic acid fragment encoding a VAR2CSA protein, and a third nucleic acid fragment encoding an antibody to the CD3 molecule. Additionally or alternatively, the recombinant nucleic acid may include a fourth nucleic acid fragment encoding the US11 protein.
  • Soluble PD-1 molecule is the extracellular domain of PD-1 immunosuppressive molecule, which can competitively bind to the ligand PD-L1 expressed by cancer cells, thereby releasing the inhibitory effect of PD-1 and PD-L1 on T cells.
  • Oncolytic viruses reconstituted with soluble PD-1 molecules can effectively promote immune response.
  • the CD3 molecule is an important marker on the surface of T cells and consists of five polypeptide chains of ⁇ , ⁇ , ⁇ , ⁇ and ⁇ . Antibodies against CD3 molecules can specifically recruit T cells expressing CD3 molecules. Therefore, inserting a gene fragment capable of expressing an antibody of CD3 molecule into the nucleic acid of an oncolytic virus can promote the activation and/or proliferation of T cells and enhance the anti-tumor effect of the oncolytic virus.
  • Exemplary nucleic acid fragments encoding antibodies to CD3 molecules can include nucleic acid fragments encoding OKT3, nucleic acid fragments encoding L2K, nucleic acid fragments encoding UCHT1, and the like, or combinations thereof.
  • the third nucleic acid fragment can be a nucleic acid fragment of OKT3.
  • the VAR2CAS protein is a parasite-driven antigen that mediates the binding of malaria-infected erythrocytes to chondroitin sulfate A (CSA) in the placenta.
  • the VAR2CAS protein can specifically bind to cancer cells, including liver cancer cells, lung cancer cells, prostate cancer cells, etc., but not to other normal tissue cells except the placenta. Insertion of nucleic acid fragments of the VAR2CAS protein into oncolytic viruses facilitates targeting of cancer cells.
  • the US11 protein inhibits the RLR-mediated activation of IRF3, a downstream signaling pathway of innate immunity, and prevents interferon-beta by interacting with the endogenous pattern recognition receptors RIG-I and MDA-5, interfering with their interaction with the adaptor protein MAVS production.
  • the additional addition of a nucleic acid fragment encoding the US11 protein ie, the fourth nucleic acid fragment
  • the fourth nucleic acid fragment can be used to enhance the ability of the oncolytic virus to evade the host's innate immune defense and prolong the persistence time of the oncolytic virus in the body.
  • the fourth nucleic acid fragment may comprise an exogenous nucleic acid fragment inserted into the recombinant nucleic acid.
  • the exogenous nucleic acid fragment can be a nucleic acid fragment encoding an exogenous US11 protein (eg, human, animal).
  • the fourth nucleic acid fragment may comprise a non-exogenous nucleic acid fragment inserted into the recombinant nucleic acid, eg, a nucleic acid fragment from a US11 protein encoding an oncolytic virus.
  • the similarity between the first nucleic acid fragment and the sequence shown in SEQ ID NO: 1 may be greater than or equal to 95%, 90%, 85%, 80%, etc.
  • the similarity between the second nucleic acid fragment and the sequence shown in SEQ ID NO: 2 may be greater than or equal to 95%, 90%, 85%, 80%, etc.
  • the similarity between the third nucleic acid fragment and the sequence shown in SEQ ID NO: 3 may be greater than or equal to 95%, 90%, 85%, 80%, etc.
  • the similarity between the fourth nucleic acid fragment and the sequence shown in SEQ ID NO:4 may be greater than or equal to 95%, 90%, 85%, 80%, etc.
  • the similarity between the recombinant nucleic acid of the oncolytic virus and the sequence shown in SEQ ID NO: 5 can be greater than or equal to 95%, 90%, 85%, 80%, etc.
  • the oncolytic virus further contains a fifth nucleic acid fragment encoding a recombinant oncolytic virus for screening, eg, enhanced yellow fluorescent protein (EYFP).
  • EYFP enhanced yellow fluorescent protein
  • one or more of the first nucleic acid segment, the second nucleic acid segment, the third nucleic acid segment, the fourth nucleic acid segment, and the fifth nucleic acid segment can be linked to one or more expression control sequences.
  • the one or more expression control sequences may include promoters, enhancers, polynucleotides (eg, terminators), the like, or combinations thereof.
  • Exemplary promoters can include SV40 promoter, CMV promoter, MSV promoter, EF1 promoter, MMLV promoter, U6 promoter, H1 promoter, and the like.
  • Exemplary enhancers can include SV40 enhancers, CMV enhancers, and the like.
  • Terminators can include SV40PolyA, TK PolyA, BGH PolyA, and the like.
  • the first nucleic acid fragment can be operably linked to a promoter.
  • the fifth nucleic acid fragment can be operably linked to a CMV promoter, a CMV enhancer, BGH PolyA.
  • each exogenous nucleic acid fragment can be inserted into the nucleic acid of an oncolytic virus by one or more methods common in the art to obtain the above-mentioned recombinant nucleic acid, which is not limited in this application.
  • one or more exogenous nucleic acid fragments can be inserted into the vector using ligase, using fusion PCR techniques, the like, or a combination thereof.
  • a ligase can be used in sequence to ligate the digested vector and the gene fragment to be inserted.
  • fusion PCR can be used to ligate the fragments together in sequence, and then fused to the vector.
  • first and second vectors e.g., plasmids
  • part of the gene fragments e.g., 3 gene fragments
  • other genes can be sequentially ligated using ligase.
  • the fragment is ligated with the second vector, and then PCR is used to obtain the remaining gene fragments that are ligated together, so as to be ligated with the first vector.
  • the vector can be the nucleic acid of a wild-type oncolytic virus.
  • the vector can be the nucleic acid of wild-type HSV-1 virus.
  • the vector can be the nucleic acid of an oncolytic virus in which one or more encoding genes (eg, ICP34.5) are deleted.
  • the present application does not limit the order of the individual nucleic acid fragments in the recombinant nucleic acid.
  • the first, second, third, and fourth nucleic acid segments can be inserted into different sites of the oncolytic viral nucleic acid, respectively.
  • one or more of the first, second, third, and fourth nucleic acid segments can be inserted into the same site of an oncolytic viral nucleic acid.
  • the insertion site of the above-mentioned nucleic acid fragment can be any suitable site in the coding region of the oncolytic virus nucleic acid.
  • the insertion site of the above-mentioned nucleic acid fragment may be the position in the HSV-1 virus where one or more coding genes (eg, ICP34.5) are deleted.
  • exogenous nucleic acid fragments can be sequentially inserted into the same site or different sites of the oncolytic viral nucleic acid.
  • the sequence of the exogenous nucleic acid fragments in the recombinant nucleic acid can be arbitrary.
  • the sequence (5' end to 3' end) of the nucleic acid fragments in the recombinant nucleic acid of the oncolytic virus can be the fourth nucleic acid fragment, the fifth nucleic acid fragment, the first nucleic acid fragment, the second nucleic acid fragment and the first nucleic acid fragment Three nucleic acid fragments.
  • compositions for treating cancer may include the above-described oncolytic virus and a pharmacologically acceptable carrier or excipient.
  • Pharmacologically acceptable carriers can include coatings, capsules, microcapsules, nanocapsules, etc., or any combination thereof. It should be noted that the carrier needs to be non-toxic and capable of supporting key components in the composition (eg, the above-mentioned oncolytic viruses, anti-cancer-promoting molecules expressed by oncolytic viruses, such as soluble PD-1 molecules) Activity was not significantly affected.
  • the carrier can protect the key components in the composition and reduce or avoid the inactivation or decomposition of the key components under some adverse conditions (eg, oxidation, denaturation caused by strong acid or strong base, etc.). For example, enzymes or relatively low pH in gastric juices can cause key components to break down or be inactivated. Carriers can help maintain or enhance the efficacy of a pharmaceutical composition by protecting key ingredients in the composition.
  • carriers can be used for controlled release of key components (eg, oncolytic viruses).
  • Controlled release can include, but is not limited to, slow release, sustained release, targeted release, and the like.
  • the carrier may include hydrogel capsules, microcapsules or nanocapsules made of collagen, gelatin, chitosan, alginate, polyvinyl alcohol, polyethylene oxide, starch, cross-linked starch, etc., or any combination thereof capsule.
  • pharmaceutically acceptable carriers can include dispersion media (eg, solvents), coatings, buffers, stabilizing agents, isotonic and absorption delaying agents, and the like.
  • exemplary pharmacologically acceptable carriers can include phosphate buffered saline solutions, water, emulsions (eg, oil/water emulsions), various types of wetting agents, sterile solutions, gels, bioabsorbable matrix materials, and the like, or Other suitable materials, or any combination thereof.
  • excipients can include, but are not limited to, water, saline, polyethylene glycol, hyaluronic acid, ethanol, pharmaceutically acceptable salts, eg, salts of inorganic acids (eg, hydrochloric acid, hydrobromic acid, phosphoric acid) salts, sulfates, etc.) and salts of organic acids (eg acetate, propionate, benzoate, etc.).
  • inorganic acids eg, hydrochloric acid, hydrobromic acid, phosphoric acid
  • salts of organic acids eg acetate, propionate, benzoate, etc.
  • cancers that can be treated by the composition can include, but are not limited to, glioma, melanoma, liver cancer, lung cancer, colon cancer, rectal cancer, head and neck tumors, breast cancer, renal cell carcinoma, ovarian cancer cancer, prostate cancer, gastric cancer, lymphoma, pancreatic cancer, bladder cancer, breast cancer, endometrial cancer, lymphoma, cervical cancer, sarcoma (eg, soft tissue sarcoma and osteosarcoma), and the like.
  • glioma, melanoma liver cancer, lung cancer, colon cancer, rectal cancer, head and neck tumors
  • breast cancer renal cell carcinoma
  • ovarian cancer cancer prostate cancer
  • gastric cancer lymphoma
  • pancreatic cancer bladder cancer
  • breast cancer endometrial cancer
  • lymphoma cervical cancer
  • sarcoma eg, soft tissue sarcoma and osteosarcoma
  • compositions suitable for oral administration may include, but are not limited to, tablets, liposomal formulations, sustained release capsules, microparticles, microspheres, or any other suitable form.
  • compositions suitable for injectable means may include, but are not limited to, sterile aqueous or oily formulations and the like.
  • compositions suitable for topical administration may include, but are not limited to, sterile aqueous or non-aqueous solutions, suspensions, emulsions.
  • the form of the composition may include aerosols, mists, powders, solutions, suspensions, gels, and the like.
  • the composition can be stored at a suitable temperature, which can include room temperature (about 20°C), 4°C, -20°C, -80°C, and the like.
  • the compositions can also be presented in various forms convenient for storage and transport, such as powders. Powders can be sterile powders to which a solvent can be added and mixed well before use to prepare solutions for oral, injectable, or topical administration.
  • the composition may also include components that have antibacterial effects but do not significantly negatively affect the survival of oncolytic viruses, such that the composition is stable under certain storage conditions (eg, refrigeration and freezing) and can prevent Contamination by microorganisms such as bacteria and fungi.
  • the multiplicity of infection of the oncolytic virus on cancer cells in a culture environment can be 0.1, 0.2, 0.5, 0.8, 1.0, and the like.
  • the oncolytic virus is capable of correspondingly causing at least 30% of the cancer cells to die within 48 hours in a culture environment at a multiplicity of infection of 1 on human non-small cell lung cancer cells. In some embodiments, the oncolytic virus is capable of correspondingly causing at least 40% of the cancer cells to die within 48 hours in a culture environment at a multiplicity of infection of 1 on human non-small cell lung cancer cells. In some embodiments, the oncolytic virus is capable of correspondingly causing at least 50% of the cancer cells to die within 48 hours in a culture environment at a multiplicity of infection of 1 on human non-small cell lung cancer cells.
  • the oncolytic virus is capable of correspondingly causing at least 60% of the cancer cells to die within 48 hours when acting on human hepatoma cells at a multiplicity of infection of 1 in a culture environment. In some embodiments, the oncolytic virus is capable of correspondingly causing at least 70% of the cancer cells to die within 48 hours when acting on human hepatoma cells at a multiplicity of infection of 1 in a culture environment. In some embodiments, the oncolytic virus is capable of correspondingly causing at least 80% of the cancer cells to die within 48 hours when acting on human hepatoma cells at a multiplicity of infection of 1 in a culture environment. In some embodiments, the oncolytic virus is capable of correspondingly causing at least 90% of the cancer cells to die within 48 hours when acting on human hepatoma cells at a multiplicity of infection of 1 in a culture environment.
  • the oncolytic virus is capable of correspondingly causing at least 30% of the cancer cells to die within 48 hours when acting on human breast cancer cells at a multiplicity of infection of 1 in a culture environment. In some embodiments, the oncolytic virus is capable of correspondingly causing at least 40% of the cancer cells to die within 48 hours when acted on human breast cancer cells at a multiplicity of infection of 1 in a culture environment. In some embodiments, the oncolytic virus is capable of correspondingly causing at least 50% of the cancer cells to die within 48 hours when acting on human breast cancer cells at a multiplicity of infection of 1 in a culture environment.
  • the oncolytic virus is capable of correspondingly causing at least 30% of the cancer cells to die within 48 hours at a multiplicity of infection of 1 on human pancreatic cancer cells in a culture environment. In some embodiments, the oncolytic virus is capable of correspondingly causing at least 50% of the cancer cells to die within 48 hours when acting on human pancreatic cancer cells at a multiplicity of infection of 1 in a culture environment. In some embodiments, the oncolytic virus is capable of correspondingly causing at least 60% of the cancer cells to die within 48 hours when acting on human breast cancer cells at a multiplicity of infection of 1 in a culture environment.
  • the oncolytic virus when it acts on human liver cancer cells, human breast cancer cells, or human pancreatic cancer cells at a multiplicity of infection of 0.1 in a culture environment, it can cause about 80%, 40%, 30% of cancer cells die.
  • the oncolytic virus is capable of causing at least 70% or 80%, respectively, within 32 days when the oncolytic virus is administered to a human pancreatic cancer tumor in a single injection of 2x106 pfu or 3 injections of 1x106 pfu, respectively. % tumor volume reduction.
  • the oncolytic virus when administered to a human non-small cell lung cancer tumor in 3 injections of 2x106 pfu or 3 injections of 4x106 pfu, respectively, results in at least 70% of the corresponding results within 32 days. or 90% tumor volume reduction.
  • an application of an oncolytic virus in the preparation of a medicament for treating cancer can be used to treat a subject with cancer, eg, a mammal.
  • Exemplary cancers can include melanoma, lung cancer, leukemia, gastric cancer, ovarian cancer, pancreatic cancer, breast cancer, prostate cancer, bladder cancer, colon cancer, rectal cancer, liver cancer, cervical cancer, or osteosarcoma.
  • the cancer may include lung cancer, liver cancer, breast cancer, or pancreatic cancer.
  • a method for treating cancer may comprise administering to a subject suffering from cancer an effective amount of the above-described composition.
  • the subject can be a mammal, eg, a human.
  • an effective dose of the composition can be administered to a subject with cancer.
  • an effective dose can be determined based on the characteristics of the subject to be treated, the route of administration, and/or the characteristics of the cancer (eg, the type of cancer, progression, etc.).
  • the characteristics of the subject may include, but are not limited to, age, gender, height, weight, health status, and the like. Therefore, the effective doses described in the embodiments of the present application are exemplary contents, which can be modified by those skilled in the art according to specific conditions.
  • the ratio of the amount of oncolytic virus contained in the composition to the subject's body weight can range from 1 x 10 2 pfu/g to 1 x 10 8 pfu/g, 1 x 10 3 pfu/g to 1 x 10 7 pfu/g, 5 ⁇ 10 3 pfu/g-5 ⁇ 10 6 pfu/g, 2.5 ⁇ 10 4 pfu/g-5 ⁇ 10 6 pfu/g, 2.5 ⁇ 10 4 pfu/g-5 ⁇ 10 5 pfu/g, 2.5 ⁇ 10 4 pfu/g-4 ⁇ 10 5 pfu/g, 2.5 ⁇ 10 4 pfu/g-2 ⁇ 10 5 pfu/g, 2.5 ⁇ 10 4 pfu/g-1 ⁇ 10 5 pfu/ g, 5 ⁇ 10 4 pfu/g-5 ⁇ 10 4 pfu/g, etc.
  • the compositions can be administered to a subject by a variety of modes of administration. Administration may include, but is not limited to, oral, injection, or topical. In some embodiments, the composition can be administered to a subject by injection. Exemplary injection modes may include subcutaneous injection, intramuscular injection, intravenous injection, and the like. In some embodiments, the mode of injection can include injecting the composition into the subject's tumor at or near the tumor. In some embodiments, the mode of injection can include injecting the composition into a tissue or organ of the subject, such as the kidney, liver, heart, thyroid, or joint. In some embodiments, topical administration can include applying the composition to the skin to alleviate cancers such as skin cancer, lymphoma, and the like.
  • topical administration can include vaginal administration, rectal administration, nasal administration, auricular administration, intramedullary administration, intraarticular administration, intrapleural administration, etc., or any combination thereof.
  • the composition can be administered to a subject by a combination of different modes of administration.
  • the method can include administering to the subject three times a day, twice a day, once a day, every two days, and the like.
  • compositions of the present application can be used before or after administration of other pharmaceutical compositions for the treatment of cancer.
  • the compositions disclosed in this application can be combined with other treatment modalities to treat cancer in a subject.
  • other treatment modalities include, but are not limited to, administering to the subject other pharmaceutical compositions that can treat cancer, surgical removal of the subject's tumor, radiation therapy, and the like.
  • pharmaceutical compositions that can be used to treat cancer include, but are not limited to, cytotoxic anticancer drugs and non-cytotoxic anticancer drugs.
  • Non-cytotoxic anticancer drugs may include hormonal drugs (eg, tamoxifen, exemestane), targeted drugs (eg, bevacizumab), and immunotherapy drugs (eg, monoclonal antibodies, tumor vaccine) etc.
  • test materials in the following examples are conventional methods unless otherwise specified.
  • the test materials used in the following examples were purchased from conventional biochemical reagent companies unless otherwise specified.
  • Example 1 Vector for constructing recombinant oncolytic virus (ie, oncolytic virus containing recombinant nucleic acid) strains
  • FIG. 1 is a schematic diagram of the vector plasmid of recombinant oncolytic virus constructed according to some embodiments of the present application. The ligation sequence and positions of the individual fragments are shown in Figure 1.
  • the wild-type HSV-1 virus and R149 plasmid were co-transfected into green monkey kidney cells (vero cells), and the cell debris was removed after harvesting. Save for backup. Vero cells were purchased from the American Type Culture Collection (ATCC).
  • the oncolytic virus containing the recombinant nucleic acid can be a recombinant HSV-1 virus, called HSV1-R149, which has been deposited in the General Microbiology Center (CGMCC) of the China Microorganism Culture Collection Management Committee (CGMCC) with a preservation date of September 24, 2020,
  • CGMCC General Microbiology Center
  • CGMCC China Microorganism Culture Collection Management Committee
  • the registration number of the deposit center is CGMCC No. 20707, and the address of the deposit unit is No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.
  • Human non-small cell lung cancer cells A549, human liver cancer cells HepG2, human breast cancer cells MCF-7 and human pancreatic cancer cells SW1990 were inoculated in 12-well plates according to the appropriate inoculation amount, and cultured for 24 hours, and the cells basically covered a monolayer.
  • the original culture medium in the 12-well plate was aspirated, washed twice with DPBS or serum-free DMEMP medium, and then added 300 ⁇ l of serum-free DMEM medium.
  • the amount of virus added to each well was calculated and divided into the following 4 groups: the multiplicity of infection (MOI) of the control group was 0; the MOI of the low-dose group was 0.1; The MOI in the middle-dose group was 1.0; the MOI in the high-dose group was 2.0.
  • MOI multiplicity of infection
  • FIG. 2 is a schematic diagram of human non-small cell lung cancer cells A549 transfected with recombinant oncolytic virus according to some embodiments of the present application after staining.
  • 3 is a schematic diagram of HepG2 staining of human hepatoma cells transfected with recombinant oncolytic virus according to some embodiments of the present application.
  • FIG. 4 is a schematic diagram of human breast cancer cells transfected with recombinant oncolytic virus MCF-7 stained according to some embodiments of the present application.
  • Figure 5 is a schematic diagram of human pancreatic cancer cells SW1990 transfected with recombinant oncolytic virus according to some embodiments of the present application after staining.
  • Figure 6 shows the cell viability of human non-small cell lung cancer cell A549, human liver cancer cell HepG2, human breast cancer cell MCF-7 and human pancreatic cancer cell SW1990 transfected with recombinant oncolytic virus according to some embodiments of the present application schematic diagram.
  • the four groups of cancer cells A549, HepG2, MCF-7 and SW1990 were cultured for 24 hours and 48 hours, and the cell survival rates were all above 90%.
  • medium dose (MOI of 1) and high dose (MOI of 2) can effectively kill these four cancer cells.
  • the killing effect of the virus action time of 48h was significantly higher than that of the action time of 24h.
  • Human hepatoma cells HepG2 were treated with recombinant oncolytic virus for 24 hours, and the cell survival rate was about 30% in the middle and high dose groups. After prolonging the treatment time to 48 hours, the cell survival rate of the low dose group was only 20.25%, the cell survival rate of the middle dose group was 11.37%, and the cell survival rate of the high dose group was 9.46%. It shows that prolonging the treatment time of recombinant oncolytic virus or increasing the dose of recombinant oncolytic virus can greatly increase the mortality rate of human hepatoma cells HepG2, and reduce the survival ability of human hepatoma cells HepG2.
  • the cell survival rates in the middle and high dose groups were 54.22% and 46.06%, respectively. From the above results, it can be seen that the recombinant oncolytic virus can quickly kill A549, MCF-7, SW1900 cancer cells in a relatively short period of time (for example, 48 hours), and the higher the dose of recombinant oncolytic virus, the better the cell survival rate of cancer cells. lower.
  • the recombinant oncolytic virus can quickly and effectively kill four cancer cells A549, HepG2, MCF-7 and SW1990, and has obvious inhibitory effects on lung cancer, liver cancer, breast cancer, and pancreatic cancer. Prolonging the duration of viral action or increasing the dose of the virus drastically reduces the cell viability of cancer cells.
  • a subcutaneous (SQ) tumor model in nude mice was established. 4-6 week old female BALB/c nude mice were injected with 100 ul of 1.3 x 106 A549 or 1.3 x 106 SW1990 diluted in sterile PBS into the right armpit. Treatment was initiated when the tumor volume had grown to approximately 50-200 mm3 . Intratumoral injections were performed every two days for a total of three injections. The control group was injected with sterile PBS solution, and the experimental group was injected with recombinant oncolytic virus solution at high, medium and low doses.
  • the recombinant oncolytic virus solution was obtained by dissolving the recombinant oncolytic virus in sterile 100ul PBS solution.
  • the high dose was 2 ⁇ 10 6 pfu
  • the medium dose was 1 ⁇ 10 6 pfu
  • the low dose was 5 ⁇ 10 5 pfu.
  • the physical condition of the mice was observed daily, and the body weight of the mice was monitored every two days. Tumor diameters were monitored using vernier calipers and tumor volumes were calculated, and mice were euthanized when they survived 32 days of treatment or when tumor volume reached 4000 mm.
  • FIG. 7 is a schematic diagram of SW1990 and A549 subcutaneous tumor models constructed according to the treatment of immunodeficient nude mice with recombinant oncolytic virus shown in some examples of the present application.
  • the control group represents the mice before the injection of the recombinant oncolytic virus solution.
  • high-dose recombinant oncolytic virus solution injection was started when the tumor reached 50-200 mm 3 . That is, the tumor size of the mice in the control group was 50-200 mm 3 .
  • the mice in the control group injected with human pancreatic cancer cells SW1990 developed rapidly and grew rapidly.
  • the tumor volume of both tumors was significantly reduced relative to the control group.
  • Figure 8 is a graph showing the time and tumor volume of the SW1990 subcutaneous tumor model treated with different doses of recombinant oncolytic virus for different times according to some embodiments of the present application.
  • the tumors in the control group injected with PBS continued to grow rapidly, while the tumor volume in the experimental group injected with recombinant oncolytic virus solution was significantly reduced whether it was a single injection or multiple consecutive injections.
  • the tumor volume in the control group increased by 3.7 times, while the tumor volume in the experimental group increased by 60% in the low-dose group, and decreased by 73% and 87% in the medium- and high-dose groups, respectively.
  • a medium dose of recombinant oncolytic virus requires three consecutive injections to have a good oncolytic effect, but when the virus amount is increased to a high dose, that is, 2 ⁇ 10 6 pfu, only one injection can achieve three consecutive injections with a medium dose.
  • Oncolytic effect of injection is
  • Figure 9 is a graph showing the time and tumor volume of A549 subcutaneous tumor models treated with different doses of recombinant oncolytic virus for different times according to some embodiments of the present application.
  • the recombinant oncolytic virus has better oncolytic effect in the tumor model of A549 immunodeficient nude mice.
  • tumor volume increased 2.6-fold 32 days after injection.
  • a single injection of 8 ⁇ 10 6 pfu virus reduced tumor volume by 58%, and three consecutive injections of 2 ⁇ 10 6 pfu virus reduced tumor volume by 73%.
  • the virus dose was increased to (4-8) ⁇ 10 6 pfu, the tumor basically disappeared after three consecutive injections.
  • this example shows that the recombinant oncolytic virus provided in this application has a good therapeutic effect on lung cancer and pancreatic cancer.
  • IP intraperitoneal
  • mice were intraperitoneally injected with recombinant oncolytic virus (dissolved in sterile 100 ul PBS solution), once every two days, for a total of three injections.
  • the control group was injected with sterile PBS solution, and the experimental group was injected with high and low doses of recombinant oncolytic virus.
  • the low dose was 10 6 pfu and the high dose was 10 7 pfu.
  • Mice body weights were monitored before and after virus injection. The physical condition and survival status of the mice were observed every day.
  • Figure 10 is a graph showing the time and mouse survival rate of CT26 abdominal tumor model treated with different doses of recombinant oncolytic virus according to some examples of the present application.
  • the survival rate of mice was 10% and 90% of the mice died after 19 days after injection, while the mice in the experimental group injected with 10 7 pfu and 10 6 pfu virus amount survived The rates were 100% and 87.5%, respectively.
  • mice in the experimental groups with 10 7 pfu and 10 6 pfu viral loads still had higher survival rates, 60% and 50%, respectively.
  • the recombinant oncolytic virus significantly improved the viability of tumor-bearing mice. After three injections of 10 7 pfu of virus, 60% of mice with abdominal tumors survived long-term.
  • a recombinant oncolytic virus disclosed in this application and a method for its application may bring about beneficial effects including but not limited to: (1) The nucleic acid fragments containing soluble PD-1 molecules in the recombinant oncolytic virus can relieve or reduce the The inhibitory effect of T cells on oncolytic viruses, thereby enhancing the survival and proliferation of oncolytic viruses in host cells; (2) The nucleic acid fragments of antibodies containing CD3 molecules in recombinant oncolytic viruses can activate T cell proliferation and activation, Enhance the anti-tumor effect of the immune system; (3) The nucleic acid fragment containing the VAR2CAS protein in the recombinant oncolytic virus is beneficial to target cancer cells; (4) The nucleic acid fragment containing the US11 protein in the recombinant oncolytic virus enhances the escape of the oncolytic virus The ability of the host's natural immune defense to prolong the persistence of oncolytic viruses in the body, thereby enhancing the targeted infection and killing of cancer cells.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Genetics & Genomics (AREA)
  • Animal Behavior & Ethology (AREA)
  • Virology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Biochemistry (AREA)
  • Zoology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Epidemiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Wood Science & Technology (AREA)
  • Toxicology (AREA)
  • Oncology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Mycology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Cell Biology (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Dermatology (AREA)
  • General Engineering & Computer Science (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne un virus oncolytique et son application. Le virus oncolytique contient un acide nucléique recombinant. L'acide nucléique recombinant comprend : (i) un premier fragment d'acide nucléique pour coder une molécule PD-1 soluble ; (ii) un deuxième fragment d'acide nucléique pour coder une protéine VAR2CSA ; et (iii) un troisième fragment d'acide nucléique pour coder un anticorps d'une molécule CD3.
PCT/CN2021/071563 2021-01-13 2021-01-13 Virus oncolytique et son application WO2022151078A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CN202180000514.5A CN112840020B (zh) 2021-01-13 2021-01-13 溶瘤病毒及其应用
PCT/CN2021/071563 WO2022151078A1 (fr) 2021-01-13 2021-01-13 Virus oncolytique et son application

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2021/071563 WO2022151078A1 (fr) 2021-01-13 2021-01-13 Virus oncolytique et son application

Publications (1)

Publication Number Publication Date
WO2022151078A1 true WO2022151078A1 (fr) 2022-07-21

Family

ID=75929866

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/071563 WO2022151078A1 (fr) 2021-01-13 2021-01-13 Virus oncolytique et son application

Country Status (2)

Country Link
CN (1) CN112840020B (fr)
WO (1) WO2022151078A1 (fr)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4332218A1 (fr) 2021-06-03 2024-03-06 Shanghai Yunying Biopharmaceutical Technology Co., Ltd. Vecteur de virus oncolytique et son utilisation
CN116836283B (zh) * 2022-05-17 2024-05-07 苏州万灏生物科技有限公司 具有多种免疫调节因子的增强型溶瘤病毒及其制备方法和应用

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018049248A1 (fr) * 2016-09-09 2018-03-15 Icellhealth Consulting Llc Virus oncolytique équipé de molécules d'engagement bispécifiques
CN109983121A (zh) * 2016-06-30 2019-07-05 昂克诺斯公司 治疗性多肽的假型化溶瘤病毒递送
CN110124045A (zh) * 2019-04-30 2019-08-16 云南大学 疟原虫var2csa蛋白在抗肿瘤药物中的应用
WO2019200316A1 (fr) * 2018-04-13 2019-10-17 Bluebird Bio, Inc. Thérapie cellulaire adoptive
CN111212656A (zh) * 2017-07-26 2020-05-29 昂克诺斯公司 溶瘤病毒载体及其用途

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109983121A (zh) * 2016-06-30 2019-07-05 昂克诺斯公司 治疗性多肽的假型化溶瘤病毒递送
WO2018049248A1 (fr) * 2016-09-09 2018-03-15 Icellhealth Consulting Llc Virus oncolytique équipé de molécules d'engagement bispécifiques
CN111212656A (zh) * 2017-07-26 2020-05-29 昂克诺斯公司 溶瘤病毒载体及其用途
WO2019200316A1 (fr) * 2018-04-13 2019-10-17 Bluebird Bio, Inc. Thérapie cellulaire adoptive
CN110124045A (zh) * 2019-04-30 2019-08-16 云南大学 疟原虫var2csa蛋白在抗肿瘤药物中的应用

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
LIU HUI-LING;HUANG CHENG-HAO: "Progress in Oncolytic Virotherapy for the Treatment of Pancreatic Cancer", CHINESE BULLETIN OF LIFE SCIENCES, vol. 32, no. 1, 29 February 2020 (2020-02-29), pages 83 - 91, XP055950443, ISSN: 1004-0374, DOI: 10.13376/j.cbls/2020011 *
SASSO EMANUELE; D’ALISE ANNA MORENA; ZAMBRANO NICOLA; SCARSELLI ELISA; FOLGORI ANTONELLA; NICOSIA ALFREDO: "New viral vectors for infectious diseases and cancer", SEMINARS IN IMMUNOLOGY, vol. 50, 1 August 2020 (2020-08-01), US , pages 1 - 26, XP086411838, ISSN: 1044-5323, DOI: 10.1016/j.smim.2020.101430 *
WU YANHONG;WANG HUIRU;DENG ZHENLING;GE KELI;ZHANG SHUREN: "Progress in Cancer Bio-immunotherapy", SCIENCE & TECHNOLOGY REVIEW, vol. 32, no. 26, 18 September 2014 (2014-09-18), pages 27 - 36, XP055950453, ISSN: 1000-7857, DOI: 10.3981/j.issn.1000-7857.2014.26.003 *

Also Published As

Publication number Publication date
CN112840020B (zh) 2023-03-17
CN112840020A (zh) 2021-05-25

Similar Documents

Publication Publication Date Title
JP6754532B2 (ja) 二重特異性抗体をコードする腫瘍溶解性アデノウイルスおよびそれに関連する方法と使用
JP7038065B2 (ja) 腫瘍溶解性ウイルス株
EP3490583B1 (fr) Vecteurs de virus de l'herpès simplex oncolytique exprimant des molécules stimulatrices du système immunitaire
US20210213079A1 (en) Treatment of brain cancer with oncolytic adenovirus
US20120207840A1 (en) Virion Derived Protein Nanoparticles For Delivering Diagnostic Or Therapeutic Agents For The Treatment Of Non-Melanoma Skin Cancer
KR20190140915A (ko) 재조합 헤르페스 심플렉스 바이러스 및 이의 용도
US20130012426A1 (en) Virion Derived Protein Nanoparticles For Delivering Diagnostic Or Therapeutic Agents For the Treatment of Psoriasis
WO2022151078A1 (fr) Virus oncolytique et son application
JP2021527694A (ja) 腫瘍溶解性ウイルスを用いた処置
WO2005086922A2 (fr) Adenovirus oncolytique dote de genes therapeutiques
WO2019062251A1 (fr) Adénovirus oncolytique recombiné isolé, composition pharmaceutique, et application de l'adénovirus oncolytique recombiné isolé dans un médicament pour le traitement d'une tumeur et/ou d'un cancer
KR20190134786A (ko) 방광암의 치료 방법
CN116042723A (zh) 表达免疫系统-刺激分子的溶瘤性单纯疱疹病毒载体
CN107614515A (zh) 用于抗hiv(人免疫缺陷病毒)疗法和/或疫苗的t20构建体
WO2023226942A1 (fr) Vecteur hsv-1 recombinant codant pour facteur immunostimulateur et anticorps anti-point de contrôle immunitaire
CN115960966A (zh) 在癌细胞中具有增强复制的hsv载体
WO2022252185A1 (fr) Vecteur de virus oncolytique et son utilisation
CN114375331B (zh) 用于脑肿瘤治疗的溶瘤性i型单纯疱疹病毒
CN113227384A (zh) 用于治疗肿瘤的药物组合物、药盒和方法
Qiu et al. Adenovirus-Mediated Dual Gene Expression of Human Interleukin-10 and Hepatic Growth Factor Exerts Protective Effect Against CCl 4-Induced Hepatocyte Injury in Rats
WO2006046243A1 (fr) Sv40 pour therapie genique de troubles renaux
CN110404077B (zh) 肿瘤ecm降解和/或抑制剂及其成套药盒和应用
CN110982841A (zh) 重组腺相关病毒载体、重组腺相关病毒aav8-pd1及其应用
NL2031110B1 (en) Recombinant adeno-associated virus vector, recombinant adeno-associated virus aav8-pd1 and use thereof
US20240041939A1 (en) Encapsulated cells expressing il-12 and uses thereof

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: 21918293

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: 21918293

Country of ref document: EP

Kind code of ref document: A1