WO2021244628A1 - 一种酶和病毒的药物组合及其应用 - Google Patents

一种酶和病毒的药物组合及其应用 Download PDF

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WO2021244628A1
WO2021244628A1 PCT/CN2021/098263 CN2021098263W WO2021244628A1 WO 2021244628 A1 WO2021244628 A1 WO 2021244628A1 CN 2021098263 W CN2021098263 W CN 2021098263W WO 2021244628 A1 WO2021244628 A1 WO 2021244628A1
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virus
cancer
hours
drug
viral vector
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French (fr)
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刘彦君
王征
徐云霞
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Shanghai Bao Pharmaceuticals Co Ltd
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Shanghai Bao Pharmaceuticals Co Ltd
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Priority to US18/000,516 priority Critical patent/US20230277633A1/en
Priority to EP21816711.2A priority patent/EP4162951A4/en
Priority to CN202180013742.6A priority patent/CN115066261A/zh
Priority to JP2022574838A priority patent/JP7795790B2/ja
Publication of WO2021244628A1 publication Critical patent/WO2021244628A1/zh
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    • A61K38/46—Hydrolases (3)
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    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
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    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
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    • C12N9/6489—Metalloendopeptidases (3.4.24)
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    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
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    • C12R2001/46—Streptococcus ; Enterococcus; Lactococcus
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    • 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
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Definitions

  • the invention relates to the field of biomedicine, in particular to a drug combination of enzyme and virus and its application.
  • Viruses are now commonly used therapeutic vectors, such as oncolytic viruses, viral vaccines, and gene therapy viruses.
  • Commonly used viruses include HSV-1, adenovirus and so on.
  • HSV-1 adenovirus
  • a major problem with the application of these viruses to treatment is that most of these viruses have been infected and are immunogenic, and neutralizing antibodies are widely present in the population. The positive rate of HSV-1 pre-existing neutralizing antibody is considerable.
  • These neutralizing antibodies can block the virus from exerting its effect, which is one of the main limitations of viral vector therapy.
  • the titer of neutralizing antibodies will increase and the drug can no longer be administered within one year. This is another major obstacle to limiting viral vector therapy.
  • immunosuppressive agents are mainly used to solve this problem, but such methods have poor therapeutic effect.
  • Oncolytic virus is a type of virus that preferentially infects and kills tumor cells. In the early stage, some tumor cells were specifically infected and destroyed by oncolytic viruses. Subsequently, the oncolytic virus replicates and multiplies in tumor cells, releasing new infectious virus particles to infect and destroy other tumor cells. Oncolytic viruses exert oncolytic effects by directly lysing tumor cells or stimulating the host to produce an anti-tumor immune response.
  • intratumoral injection has become the current mainstream drug delivery method because it is not easy to resist drug resistance, can also stimulate tumor microenvironmental immune response, and is not easy to produce cytokine storm.
  • intratumoral injection cannot achieve the effect of eliminating metastatic tumors. Therefore, there are more and more researches on intravenous injection of oncolytic virus compared to intratumoral injection.
  • the problem of neutralizing antibody and the safety of system injection are the main obstacles to intravenous injection of oncolytic virus. (Russell, S., Peng, K. & Bell, J.
  • the pre-existing neutralizing antibodies in the human body are also one of the treatment obstacles encountered by viral vector vaccines. Academician Chen Wei pointed out in his newly published article on the clinical trial of a new coronavirus vaccine Ad5-nCoV with adenovirus as a carrier, that pre-existing Ad5 immunity can slow down the rapid immune response to SARS-CoV-2 and reduce the peak of the response level. In addition, the high level of Ad5 immunity may also have a negative impact on the persistence of the immune response caused by the vaccine. (Zhu Feng-Cai, Li Yu-Hua, Guan Xu-Hua et al.
  • WO2020102740A2 discloses a method of gene therapy, which is a therapeutic method of using IdeS or EndoS to cut IgG or sugar chains contained in IgG in the serum of AAV treated subjects.
  • IdeS or EndoS to cut IgG or sugar chains contained in IgG in the serum of AAV treated subjects.
  • an enzyme that can effectively degrade the Fc part of antibodies or sugar chains contained in IgG.
  • the purpose of the present invention is to provide a reagent for reducing the interference of blood IgG on viral therapy, including a drug combination of immunoglobulin degrading enzyme or endoglycosidase and viral vector drug and its application.
  • Drugs include oncolytic viruses and viral vaccines.
  • oncolytic viruses the main obstacles to intravenous administration of oncolytic viruses are eliminated by pre-clearing the virus vector drug-related neutralizing antibody in the human body and/or removing the neutralizing antibody produced after the administration of the virus vector.
  • virus vaccines the virus carrier drug neutralizing antibody is removed in advance to eliminate the influence of the neutralizing antibody.
  • the present invention relates to the following items:
  • a pharmaceutical combination characterized in that the pharmaceutical combination comprises: 1) a reagent for reducing the binding of Fc receptors to endogenous serum antibodies, wherein the reagent includes an immunoglobulin degrading enzyme or an endoglycosidase, And 2) Viral vector drugs, wherein the viral vector drugs are selected from oncolytic viruses, viral vaccines; and wherein the drug combination allows the separate administration of the viral vector drugs and the agents.
  • the immunoglobulin degrading enzyme is an IgG degrading enzyme
  • the IgG degrading enzyme is selected from the IgG cysteine protease of Streptococcus pyogenes or a variant thereof or Fragment or human-derived MMP protease or a variant or fragment thereof, the variant or fragment retains the activity of cleaving IgG; preferably, the IgG degrading enzyme is selected from IdeS, MAC2, IdeZ, IdeZ2, IdeE, IdeE2, IdeP , MMP.
  • the endoglycosidase is an IgG endoglycosidase
  • the IgG endoglycosidase is selected from streptococcus, Corynebacterium pseudotuberculosis, Enterococcus faecalis or meninges IgG endoglycosidase of septic bacteria or variants or fragments thereof, the variants or fragments retain the activity of IgG endoglycosidase
  • the streptococcus is, for example, Streptococcus pyogenes, Streptococcus equi, or Streptococcus zooepidemicus ;
  • the IgG endoglycosidase is EndoS, CP40, EndoE or EndoF2.
  • the virus used in the viral vector drug is selected from the group consisting of ssDNA virus, dsDNA virus, ssRNA virus or dsRNA virus; and/ Or, the virus used in the viral vector medicine is selected from a wild-type virus strain or a naturally attenuated strain, a genetically engineered selective attenuated strain, a gene-loaded virus strain, and a gene transcription targeted virus strain.
  • wild-type virus strain or naturally attenuated strain is selected from the group consisting of Newcastle disease virus, reovirus, mumps virus, West Nile virus, adenovirus, Vaccinia virus etc.
  • GM-CSF granulocyte macrophage colony stimulating factor
  • the gene-loaded virus strain is, for example, JX-594 or T-VEC.
  • the drug combination according to item 6 characterized in that the gene transcription targeting virus strain, that is, inserting a tissue or tumor-specific promoter before essential viral genes to control the replication of oncolytic viruses in tumor cells,
  • the gene transcription targeting virus strain is, for example, G92A.
  • ssDNA-like virus is selected from parvovirus, and the parvovirus is, for example, H-1PV virus.
  • the dsDNA virus is selected from herpes simplex virus, adenovirus, and poxvirus; preferably, the adenovirus is selected from Enadenotucirev , DNX-2401, C-REV, NG-348, ProsAtak, CG0070, ADV-TK, EDS01, KH901, H101, H103, VCN-01, Telomelysin (OBP-301), the herpes simplex virus is preferably type I simplex virus Herpes virus HSV-1; the herpes simplex virus is selected from R3616, T-VEC, HF10, G207, NV1020, OrienX010, and the poxvirus is selected from Pexa-Vec (vaccinia virus), JX-594 (vaccinia virus), GL- ONC1, Myxoma.
  • the adenovirus is selected from Enadenotucirev , DNX-2401, C-REV, NG-348, ProsAtak,
  • the ssRNA virus is selected from Piconavirus, alphavirus, Retroviruses, Paramyxoviruses, Rhabdoviruses; preferably, the Piconavirus is selected from CAVATAK, PVS-RIPO, CVA21 (enterovirus) RIGVIR, the alphavirus is selected from M1, Sindbis AR339, Semliki Forest virus, the Retroviruses is selected from Toca511, the Paramyxoviruses is selected from MV-NIS, PV701 (Newcastle disease virus), and the Rhabdoviruses are selected from VSV-IFN ⁇ , MG1 -MAGEA3, VSV-GP.
  • the Piconavirus is selected from CAVATAK, PVS-RIPO, CVA21 (enterovirus) RIGVIR
  • the alphavirus is selected from M1, Sindbis AR339, Semliki Forest virus
  • the Retroviruses is selected from Toca511
  • the Paramyxoviruses is selected from
  • the dsRNA virus is selected from Reoviruses; preferably, the Reoviruses are selected from Pelareorep, Reolysin, vaccinia virus, and parotid gland Inflammatory virus, human immunodeficiency virus (human immunodeficiency virus, HIV).
  • ssRNA virus is selected from the group consisting of reovirus, coxsackievirus, poliovirus, and porcine sackie virus.
  • Kagu virus seeneca valley virus
  • measles virus measles virus
  • newcastle disease virus newcastle disease virus
  • VSV vesicular stomatitis virus
  • the oncolytic virus expresses exogenous genes, and the exogenous genes are selected from the group consisting of Bispecific T cell engagers (BiTE), GM- CSF, interleukin-2 (IL-2), interleukin-12 (IL-12), interferon (IFN), tumor necrosis factor (TNF), soluble CD80, CCL3.
  • BiTE Bispecific T cell engagers
  • GM- CSF interleukin-2
  • IL-12 interleukin-12
  • IFN interferon
  • TNF tumor necrosis factor
  • soluble CD80 soluble CD80
  • the drug combination further comprises a targeted drug or a chemotherapeutic drug or an immune checkpoint blocker
  • the targeted drug is selected from epigenetic Chemical drugs, inhibitors targeting PI3K/Akt/mTOR signaling pathways and tyrosine kinase inhibitors
  • the chemotherapeutic drugs are selected from immunosuppressants and proteasome inhibitors
  • the immune checkpoint blockers are selected from anti-CTLA -4 antibody, anti-PD-1 antibody and anti-TIM-3 antibody
  • the epigenetic drugs such as histone deacetylase inhibitors, the inhibitors targeting the PI3K/Akt/mTOR signaling pathway such as Tricibine
  • the tyrosine kinase inhibitor is for example sunitinib
  • the immunosuppressant is for example cyclophosphamide, gemcitabine, temozolomide, mitoxantrone, and bortezomib.
  • the agent is administered to the subject by intravenous infusion or subcutaneous injection, and/or the administered
  • the amount of the reagent is 0.01 mg/kg body weight to 2 mg/kg body weight, 0.04 to 2 mg/kg body weight, 0.12 mg/kg body weight to 2 mg/kg body weight, 0.24 mg/kg body weight to 2 mg/kg body weight or 1 mg/kg body weight To 2 mg/kg body weight; preferably, the disease is cancer, viral infection, bacterial infection or fungal infection.
  • the administration time interval of the agent and the viral vector drug is at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 4 hours, At least 5 hours or at least 6 hours; and up to 35 days, up to 28 days, up to 21 days, up to 18 days, up to 14 days, up to 13 days, up to 12 days, up to 11 days, up to 10 days, up to 9 days, up to 8 days, up to 7 days, up to 6 days, up to 5 days, up to 4 days, up to 3 days, up to 2 days, up to 24 hours, up to 18 hours, up to 12 hours, up to 10 hours, up to 8 hours, up to 7 hours Or up to 6 hours.
  • time interval between the reagent and the viral vector drug is 30 minutes to 1 hour, 30 minutes to 2 hours, 30 minutes to 3 hours, 30 minutes to 4 hours, 30 minutes To 5 hours, 30 minutes to 6 hours, 1 to 2 hours, 1 to 3 hours, 1 to 4 hours, 1 to 5 hours, 1 to 6 hours, 2 to 3 hours, 2 to 4 hours, 2 to 5 hours, 2 to 6 hours, 3 to 4 hours, 3 to 5 hours, 3 to 6 hours, 4 to 5 hours, 4 to 6 hours or 5 to 6 hours.
  • a method of administering the drug combination according to any one of items 1-17 to a subject to treat or prevent cancer or infection wherein the method results in a 20-50% reduction in the viral vector drug binding, 50 -75%, 75-90%, 90-95% or 95% or more antibodies; the infection is preferably a viral infection, a bacterial infection or a fungal infection.
  • the viral vector drug is an oncolytic virus; preferably, the cancer is selected from prostate cancer, breast cancer, bladder cancer, colon cancer, rectal cancer, pancreatic cancer, ovarian cancer, Lung cancer, cervical cancer, endometrial cancer, kidney (kidney cell) cancer, esophageal cancer, thyroid cancer, lymphoma, skin cancer, melanoma and leukemia.
  • the cancer is selected from prostate cancer, breast cancer, bladder cancer, colon cancer, rectal cancer, pancreatic cancer, ovarian cancer, Lung cancer, cervical cancer, endometrial cancer, kidney (kidney cell) cancer, esophageal cancer, thyroid cancer, lymphoma, skin cancer, melanoma and leukemia.
  • the viral vector drug is a viral vaccine; preferably, the viral vaccine is used to target or treat prostate cancer, breast cancer, bladder cancer, colon cancer, rectal cancer, and pancreatic cancer , Ovarian cancer, lung cancer, cervical cancer, endometrial cancer, kidney (kidney cell) cancer, esophageal cancer, thyroid cancer, lymphoma, skin cancer, melanoma, leukemia or diseases caused by coronavirus, new coronavirus.
  • Figure 1 is the electrophoresis diagram of IdeS and IdeE expression. 1 is the molecular weight marker, 2 is before induction, 3 is IdeS, and 4 is IdeE.
  • Figures 2 and 3 are the electrophoresis diagrams of IgG1 digested by IdeS and IdeE in vitro, respectively.
  • the arrows on the right side of the figure indicate different cleavage products from IgG.
  • Arrow 1 intact IgG1
  • Arrow 2 scIgG1 (single-cut IgG1-produced by the cleavage of the first IgG heavy chain)
  • Arrow 3 F(ab')2 fragment (produced by the cleavage of the second IgG heavy chain).
  • FIGS 4 and 5 are respectively IdeS and IdeE digestion IVIg electrophoresis diagrams.
  • Figure 6 shows the virus infection of mice in different groups.
  • Figure 7 shows the detection of neutralizing antibodies in mouse serum.
  • Figure 8 shows the tumor volume growth curves in different groups of mice.
  • the immunoglobulin degrading enzyme provided by the present invention can effectively cleave the immunoglobulin in the blood, and there is no antibody against the enzyme in the human body, and is safe to use.
  • the following is the technical solution of the present invention:
  • a pharmaceutical combination comprising: 1) an agent that reduces the binding of Fc receptors to endogenous serum antibodies, wherein the agent includes an immunoglobulin degrading enzyme or an endoglycosidase , And 2) Viral vector drugs, wherein the viral vector drugs are selected from oncolytic viruses, viral vaccines; and wherein the drug combination allows separate administration of the antibody and the agent.
  • the drug combination comprises a therapeutically effective amount of the immunoglobulin degrading enzyme and a viral vector drug.
  • the pharmaceutical combination is a pharmaceutical composition, and further comprises a pharmaceutically acceptable carrier or diluent.
  • the immunoglobulin degrading enzyme is an IgG degrading enzyme
  • the IgG degrading enzyme is selected from the IgG cysteine protease of Streptococcus pyogenes or its variants or fragments or human Source MMP protease or a variant or fragment thereof, the variant or fragment retains the activity of cleaving IgG; preferably, the IgG degrading enzyme is selected from IdeS, MAC2, IdeZ, IdeZ2, IdeE, IdeE2, IdeP, MMP.
  • the pharmaceutical combination as described above wherein the endoglycosidase is an IgG endoglycosidase, and the IgG endoglycosidase is selected from Streptococcus, Corynebacterium pseudotuberculosis, Enterococcus faecalis or Meningococcus
  • the IgG endoglycosidase or a variant or fragment thereof, the variant or fragment retains the activity of the IgG endoglycosidase
  • the streptococcus is, for example, Streptococcus pyogenes, Streptococcus equi or Streptococcus zooepidemicus; preferably
  • the IgG endoglycosidase is EndoS, CP40, EndoE or EndoF2.
  • the IgG degrading enzyme comprises the amino acid sequence of SEQ ID NO: 1 to 41 or a variant with the same function or a protein composed of the amino acid sequence or a fragment thereof.
  • the IgG degrading enzyme includes the amino acid sequence of SEQ ID NO: 1 to 41, and may further include: 1) the amino acid sequence of SEQ ID NO: 1 to 41; 2) a variant thereof, and the variant and amino acid Sequence SEQ ID NO: 1 to 41 have at least 50% identity and have IgG degrading enzyme; or 1) and 2) fragments with IgG degrading enzyme activity.
  • SEQ ID NOs: 7-41 are IgG degrading enzyme mutants shown in CN107532156A and CN107532158A.
  • the IgG endoglycosidase comprises the amino acid sequence of SEQ ID NO: 42-45 or a variant with the same function or a protein composed of the amino acid sequence.
  • the IgG endoglycosidase comprises: 1) the amino acid sequence of SEQ ID NO: 42 to 45; 2) a variant thereof, which has at least 50% of the amino acid sequence of SEQ ID NO: 42 to 45 1) and 2) fragments with IgG endoglycosidase activity.
  • the fourth aspect of the present invention provides a composition, especially a pharmaceutical composition or a combination of drugs, including the mutant or protein as described above, and a therapeutic agent;
  • the therapeutic agent can be any drug that is in the body after administration.
  • Therapeutic agents that have produced anti-drug antibodies or are prone to produce anti-drug antibodies including but not limited to antibody drugs, fusion proteins, small molecule drugs, nucleic acid drugs, antibody-conjugated drugs, or viral carrier drugs; the pharmaceutical composition also includes pharmaceutically Acceptable carrier or excipient.
  • the virus used in the viral vector drug is selected from the group consisting of ssDNA virus, dsDNA virus, ssRNA virus or dsRNA virus; and/or,
  • the virus used in the viral vector medicine is selected from a wild-type virus strain or a naturally attenuated strain, a genetically engineered selective attenuated strain, a gene-loaded virus strain, and a gene transcription-targeted virus strain.
  • the wild-type virus strain or naturally attenuated strain is selected from Newcastle disease virus, reovirus, mumps virus, West Nile virus, adenovirus, vaccinia virus and the like.
  • the genetically engineered selective attenuated strain achieves tumor selectivity of virus replication by manually deleting key genes, such as thymidine kinase (Thymidine kinase, TK) knockout genetically modified human herpes simplex virus I (HSV-1) ),
  • the genetically engineered selectively attenuated strains are, for example, ONYX-015, G207.
  • ONYX-015 deleted 827bp in the E1b region, and carried out a point mutation in the gene for E1B55K protein, so that the expression gene was terminated prematurely, and the E1B55K protein could not be expressed.
  • G207 deleted the ⁇ 34.5 gene, which is the determinant of HSV-1 neurotoxicity.
  • the gene-loaded virus strain is loaded with a foreign gene
  • the foreign gene is, for example, granulocyte macrophage colony stimulating factor (GM-CSF)
  • GM-CSF granulocyte macrophage colony stimulating factor
  • the gene-loaded virus strain is, for example, JX-594 or JX-594. T-VEC.
  • the gene transcription-targeted virus strain that is, a tissue or tumor-specific promoter is inserted in front of the virus essential genes to control the replication of oncolytic virus in tumor cells
  • the gene transcription-targeted virus strain is, for example, G92A .
  • the dsDNA virus is selected from the group consisting of herpes simplex virus, adenovirus, and poxvirus; more preferably, the herpes simplex virus is preferably type I herpes simplex virus HSV-1, for example, R3616, T-VEC, HF10, G207, NV1020, OrienX010, the poxvirus is selected from Pexa-Vec (vaccinia viruse), JX-594 (vaccinia viruse), GL-ONC1, Myxoma; the adenovirus is selected from Enadenotucirev, DNX -2401, C-REV, NG-348, ProsAtak, CG0070, ADV-TK, EDS01, KH901, H101, H103, VCN-01, Telomelysin (OBP-301).
  • the herpes simplex virus is preferably type I herpes simplex virus HSV-1, for example, R3616, T-VEC, HF10, G207,
  • the ssRNA virus is selected from Piconavirus, alphavirus, Retroviruses, Paramyxoviruses, Rhabdoviruses; preferably, the Piconavirus is selected from CAVATAK, PVS-RIPO, CVA21 (enterovirus), RIGVIR, and the alphavirus is selected from M1, Sindbis AR339 , Semliki Forest virus, the Retroviruses are selected from Toca511, the Paramyxoviruses are selected from MV-NIS, PV701 (Newcastle disease virus), and the Rhabdoviruses are selected from VSV-IFN ⁇ , MG1-MAGEA3, VSV-GP.
  • the Piconavirus is selected from CAVATAK, PVS-RIPO, CVA21 (enterovirus), RIGVIR
  • the alphavirus is selected from M1, Sindbis AR339 , Semliki Forest virus
  • the Retroviruses are selected from Toca511
  • the Paramyxoviruses are selected
  • the dsRNA virus is selected from Reoviruses; preferably, the Reoviruses are selected from Pelareorep, Reolysin, vaccinia virus, mumps virus, human immunodeficiency virus, HIV);
  • the RNA virus is selected from the group consisting of reovirus, coxsackievirus, polio virus, seneca valley virus, Measles virus (measles virus), Newcastle disease virus (newcastle disease virus), vesicular stomatitis virus (vesicular stomatitis virus), influenza virus.
  • the BiTE can bind to CD3 and other molecules that activate T cells, and at the same time can bind to antigen targets on the surface of cancer cells; the scFv targets immune checkpoints; the immune checkpoints include CTLA-4, PD-1, and TIM-3 , LAG3, Siglec15, 4-1BB, GITR, OX40, CD40L, CD28, TIGIT, VISTA.
  • the cytokines, chemokines such as GM-CSF, interleukin-2 (IL-2), interleukin-12 (IL-12), interferon (IFN), tumor necrosis factor (TNF), soluble CD80 , CCL3.
  • the drug combination as described above, wherein the target of the drug can be a cell surface protein, including but not limited to: AFP, ⁇ v integrin, ⁇ 4 ⁇ 7 integrin, BCMA, CD2, CD3, CD19, CD20, CD22, CD25, CD30, CD32, CD33, CD36, CD40, CD46, CD52, CD56, CD64, CD70, CD74, CD79, CD80, CD86, CD105, CD121, CD123, CD133, CD138, CD174, CD205, CD227, CD326, CD340, CEA, c-Met, Cripto, CA1X, Claudin18.2, ED-B, EGFR, EpCAM, EphA2, EphB2, FAP, FOLR1, GD2, Globo H, GPC3, GPNMB, HER-1, HER -2, HER-3, MAGE-A3, Mesothelin, MUC16, GPNMB, PSMA, TMEFF2, TAG-72, 5T4, ROR
  • the target of the antibody drug can be a cytokine, including but not limited to: interleukins IL-1 to IL-13, tumor necrosis factor ⁇ and ⁇ , interferon ⁇ , ⁇ and ⁇ , tumor growth factor ⁇ (TGF- ⁇ ), colony stimulating factor (CSF) or granulocyte monocyte colony stimulating factor (GM-CSF).
  • cytokine including but not limited to: interleukins IL-1 to IL-13, tumor necrosis factor ⁇ and ⁇ , interferon ⁇ , ⁇ and ⁇ , tumor growth factor ⁇ (TGF- ⁇ ), colony stimulating factor (CSF) or granulocyte monocyte colony stimulating factor (GM-CSF).
  • the target of the antibody drug can be hormones, enzymes, intracellular and intercellular messengers, such as adenosine cyclase, guanosine cyclase or phospholipase C.
  • the target of the antibody drug may be an immune checkpoint, and the immune checkpoint includes: CTLA-4, PD-1, PD-L1, TIM-3, LAG3, Siglec15, 4-1BB, GITR, OX40, CD40L, CD28, TIGIT, VISTA.
  • the drug combination as described above, wherein the drug combination further includes a targeted drug or a chemotherapy drug or an immune checkpoint blocker, and the targeted drug is selected from the group consisting of epigenetic drugs, targeting PI3K/ Inhibitors of Akt/mTOR signaling pathway and tyrosine kinase inhibitors, the chemotherapeutic drugs are selected from immunosuppressive agents, proteasome inhibitors, cytotoxic drugs and cell cycle non-specific drugs, and the immune checkpoint blockers are selected From anti-CTLA-4 antibody, anti-PD-1 antibody, anti-TIM-3 antibody, anti-LAG3 antibody, anti-Siglec15 antibody, anti-4-1BB antibody, anti-GITR antibody, anti-OX40 antibody, anti-CD40L antibody, anti-CD28 antibody, anti- TIGIT antibody, anti-VISTA antibody; the epigenetic drugs such as histone deacetylase inhibitors, the inhibitors targeting the PI3K/Akt/mTOR signaling pathway such as Tricibine, and the tyrosine
  • the present invention also provides an application of any one of the drug combinations in the preparation of drugs for treating or preventing diseases.
  • the disease is cancer or infectious disease.
  • infectious diseases include viral infections, bacterial infections or fungal infections.
  • the cancer is selected from the group consisting of the following cancers: acute lymphoblastic leukemia, acute medullary leukemia, adrenocortical carcinoma, AIDS-related cancer, AIDS-related lymphoma, anal cancer, appendix cancer, astrocytoma, childhood cerebellum or Brain cancer, basal cell carcinoma, extrahepatic cholangiocarcinoma, bladder cancer, bone cancer, osteosarcoma/malignant fibrous histiocytoma, brain stem glioma, brain cancer, brain tumor-cerebellar astrocytoma, brain tumor-brain star Form cell tumor/malignant glioma, brain tumor-ependymoma, brain tumor-medulloblastoma, brain tumor-supratentorial primitive neuroectodermal tumor, brain tumor-visual pathway and hypothalamic glioma, breast cancer , Bronchial Adenoma/Carcinoid, Burkitt Lymphoma, Carcinoid Tu
  • the cancer or infectious disease can be a veterinary disease or a human disease. Examples are as follows:
  • the agent and the viral vector drug are present as a combined preparation for simultaneous, separate or sequential use.
  • the method includes the following steps: 1) administering the agent to the subject; subsequently, 2) administering the viral vector drug to the subject.
  • the reagent is an immunoglobulin degrading enzyme, and there is a time interval between the administration of the immunoglobulin degrading enzyme and the viral vector drug.
  • the method includes the following steps: 1) administering the viral vector drug to the subject; subsequently, 2) administering the agent to the subject.
  • the reagent is an immunoglobulin degrading enzyme, and there is a time interval between the administration of the immunoglobulin degrading enzyme and the viral vector drug.
  • the dosage and time interval of the mutant as described above are sufficient to reduce the immunoglobulin level in the subject to 60% of the initial level. More preferably, the agent administration amount and time interval are sufficient to reduce the immunoglobulin level binding in the subject to less than 50%, 40%, 30%, 20%, or 10% of the initial level in the patient.
  • the agent can be administered at a single time point or within a set time.
  • the mutant as described above is administered by intravenous infusion, intraperitoneal injection, intramuscular injection, joint injection, intradermal injection or subcutaneous injection, and the preferred injection method is intravenous infusion.
  • the amount of the mutant as described above administered is 0.01 mg/kg body weight to 2 mg/kg body weight, 0.04 to 2 mg/kg body weight, 0.12 mg/kg body weight to 2 mg/kg body weight, 0.24 mg/kg body weight to 2mg/kg body weight or 1mg/kg body weight to 2mg/kg body weight.
  • the interval between the administration of the mutant as described above and the therapeutic agent is at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 4 hours, at least 5 hours or at least 6 hours; and up to 35 days, up to 28 days, up to 21 days, up to 18 days, up to 14 days, up to 13 days, up to 12 days, up to 11 days, up to 10 days, up to 9 days, up to 8 days, up to 7 Days, up to 6 days, up to 5 days, up to 4 days, up to 3 days, up to 2 days, up to 24 hours, up to 18 hours, up to 12 hours, up to 10 hours, up to 8 hours, up to 7 hours, or up to 6 hours.
  • the time interval between the mutant as described above and the therapeutic agent is 30 minutes to 1 hour, 30 minutes to 2 hours, 30 minutes to 3 hours, 30 minutes to 4 hours, 30 minutes to 5 hours, 30 minutes to 6 hours, 1 to 2 hours, 1 to 3 hours, 1 to 4 hours, 1 to 5 hours, 1 to 6 hours, 2 to 3 hours, 2 to 4 hours, 2 to 5 hours, 2 to 6 hours , 3 to 4 hours, 3 to 5 hours, 3 to 6 hours, 4 to 5 hours, 4 to 6 hours or 5 to 6 hours.
  • the method includes the following steps: 1) using the mutant as described above to treat blood from the subject ex vivo; 2) returning the blood to the subject; 3) The therapeutic agent is administered to the subject.
  • the method includes the following steps: 1) administering the therapeutic agent to the subject; 2) treating the blood from the subject ex vivo with the mutant as described above ; 3) Return the blood to the subject.
  • mutants and therapeutic agents described above are used to prevent and/or treat cancer.
  • mutants and therapeutic agents described above are used to prevent and/or treat viral infections.
  • mutants and therapeutic agents described above are used to prevent and/or treat bacterial infections.
  • mutants and therapeutic agents described above are used to prevent and/or treat fungal infections.
  • the present invention also provides a method for administering the drug combination to a subject to treat or prevent cancer or infection.
  • the method results in a reduction of 20-50%, 50-75%, 75-90%, 90-95% or 95% or more of antibodies in the viral vector binding antibody; wherein the method causes the pathogenicity Sex IgG antibody is reduced by 20-50%, 50-75%, 75-90%, 90-95% or 95% or more than 95%.
  • the drug is used in a method of treating cancer, preventing cancer, and preventing infection.
  • the infection is preferably a viral infection, a bacterial infection or a fungal infection.
  • the drug is used in the treatment method of cancer.
  • the therapeutic agent is a viral vector drug; preferably, the viral vector drug is an oncolytic virus or a viral vaccine.
  • the components of the drug combination are administered separately, or the components of the drug combination are administered simultaneously.
  • the present invention also provides a product that contains reagents for reducing blood IgG levels, including IgG degrading enzymes and viral carrier drugs, as a combined preparation for simultaneous, separate or sequential application in cancer treatment, cancer and/or infection prevention.
  • the combined preparation is used in a method of treating cancer.
  • the cancer is selected from the group consisting of: acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancer, AIDS-related lymphoma, anal cancer, appendix cancer, astrocytoma , Cerebellar or brain cancer in children, basal cell carcinoma, extrahepatic cholangiocarcinoma, bladder cancer, bone cancer, osteosarcoma/malignant fibrous histiocytoma, brain stem glioma, brain cancer, brain tumor-cerebellar astrocytoma, brain Tumor-Cerebral Astrocytoma/Malignant Glioma, Brain Tumor-Ependymoma, Brain Tumor-Medulloblastoma, Brain Tumor-Supratentorial Primitive Neuroectodermal Tumor, Brain Tumor-Visual Pathway and Hypothalamic Glia Tumor, breast cancer, bronchial adenoma
  • the present invention also provides a pharmaceutical composition or therapeutic agent for cancer treatment, cancer and/or infection prevention method, said pharmaceutical composition or therapeutic agent comprising: 1) a therapeutically effective amount of an agent for reducing blood IgG levels It includes an IgG degrading enzyme; and 2) a therapeutically effective amount of a viral vector drug, preferably an oncolytic virus; and 3) a pharmaceutically acceptable carrier or diluent.
  • the pharmaceutical composition of the present invention can be used in combination with targeted drugs.
  • targeted drugs are selected from epigenetic drugs, inhibitors targeting PI3K/Akt/mTOR signaling pathway, and receptor tyrosine kinase inhibitors.
  • Histone deacetylase inhibitor is a widely studied epigenetic drug. HDACi can not only promote tumor cell differentiation and apoptosis by inhibiting tumor cell proliferation and inducing cell cycle arrest, but also The antiviral immune response of the body can be reduced by inhibiting the interferon signal pathway.
  • the histone deacetylase inhibitor HDAC6 has been proven to significantly increase the replication level of HSV-1 oncolytic virus in glioma cells, and can cooperate with HSV-1 to kill tumors.
  • PI3K/Akt signaling pathway is an important signaling pathway that regulates cell proliferation and apoptosis under stress conditions.
  • the Akt inhibitor Tricibine can cooperate with the oncolytic virus MG18L to induce apoptosis in glioma cells. The two are combined to treat mouse gliomas The curative effect is significantly better than single-agent treatment.
  • Rapamycin is an inhibitor of the mTOR signaling pathway, which can cooperate with adenovirus and HSV-1 to kill tumor cells that are not susceptible.
  • Protein tyrosine kinases (PTKs) inhibitors have multiple effects of inhibiting tumor angiogenesis and anti-tumor cell growth.
  • Sunitinib is a small molecule receptor tyrosine kinase inhibitor, which can increase the replication of VSV oncolytic virus in tumor cells by inhibiting the activity of intracellular PTK, and it can also destroy tumor angiogenesis by inhibiting the VEGFR signaling pathway To enhance the infection ability of the oncolytic virus in the tumor, thereby significantly enhancing the therapeutic effect of the oncolytic virus.
  • the pharmaceutical composition of the present invention can be used in combination with chemotherapy drugs.
  • the combined use can immunogenic cell death, enhance tumor cell antigenicity or susceptibility to immune cells, suppress negatively regulated Treg cells and myeloid-derived suppressor cells (MDSC).
  • the chemotherapeutic drugs are selected from immunosuppressive agents, proteasome inhibitors, cytotoxic drugs and cell cycle non-specific drugs, the immunosuppressive agents are for example cyclophosphamide, the proteasome inhibitors are for example bortezomib, and the immunosuppressive agents are Agents such as thalidomide and pomalidomide, the cytotoxic drugs such as gemcitabine, temozolomide, and the cell cycle non-specific drugs such as mitoxantrone.
  • the combined use is a combination of oncolytic reovirus (RV) and bortezomib (BTZ).
  • the pharmaceutical composition of the present invention can be used in combination with an immune checkpoint blocker.
  • the immune checkpoint is selected from CTLA-4, PD-1, TIM-3, LAG3, Siglec15, 4-1BB, GITR, OX40, CD40L, CD28, TIGIT, VISTA;
  • the immune checkpoint blocker is selected from anti- CTLA-4 antibody, anti-PD-1 antibody, anti-TIM-3 antibody, anti-LAG3 antibody, anti-Siglec15 antibody, anti-4-1BB antibody, anti-GITR antibody, anti-OX40 antibody, anti-CD40L antibody, anti-CD28 antibody, anti-TIGIT antibody , Anti-VISTA antibody.
  • the present invention also provides a kit or kit for preventing or treating cancer or infection, the kit comprising: 1) a therapeutically effective amount of a drug containing the mutant as described above; and 2) a therapeutically effective amount
  • the therapeutic agent is selected from the group consisting of viral vector drugs, antibodies, and polypeptide drugs that can reduce blood IgG levels; the viral vector drugs are preferably oncolytic viruses and gene therapy viruses.
  • the kit may also include 3) targeted drugs or chemotherapeutic drugs or immune checkpoint blockers.
  • the targeted drugs are selected from epigenetic drugs, inhibitors targeting the PI3K/Akt/mTOR signaling pathway, and tyrosine kinase inhibitors, and the chemotherapeutic drugs are selected from immunosuppressants, proteasome inhibitors, and cytotoxic drugs.
  • the immune checkpoint blocker is selected from the group consisting of anti-CTLA-4 antibody, anti-PD-1 antibody, anti-TIM-3 antibody, anti-LAG3 antibody, anti-Siglec15 antibody, anti-4-1BB antibody, Anti-GITR antibody, anti-OX40 antibody, anti-CD40L antibody, anti-CD28 antibody, anti-TIGIT antibody, anti-VISTA antibody; the epigenetic drugs such as histone deacetylase inhibitors, the targeting PI3K/Akt/mTOR Signal pathway inhibitors such as Tricibine, the tyrosine kinase inhibitors such as sunitinib, the immunosuppressants such as cyclophosphamide, the proteasome inhibitors such as bortezomib, the immunosuppressants such as sa Lidamide, pomalidomide, the cytotoxic drugs such as gemcitabine, temozolomide, and the cell cycle non-specific drugs such as mitoxantrone.
  • the epigenetic drugs such as histone deace
  • the kit or kit includes a kit A and a kit B, the kit A includes a therapeutically effective amount of a drug for reducing blood immunoglobulin levels including IgG degrading enzymes, and the kit B includes a therapeutically effective amount of Therapeutic agent; the therapeutic agent is selected from viral carrier drugs, antibodies, and polypeptide drugs that can reduce blood IgG levels; the viral carrier drugs are preferably oncolytic viruses.
  • the kit medicine box may also include a medicine box C.
  • the kit C includes targeted drugs or chemotherapeutic drugs or immune checkpoint blockers.
  • the targeted drugs are selected from epigenetic drugs, inhibitors targeting the PI3K/Akt/mTOR signaling pathway, and tyrosine kinase inhibitors, and the chemotherapeutic drugs are selected from immunosuppressants, proteasome inhibitors, and cytotoxic drugs.
  • the immune checkpoint blocker is selected from the group consisting of anti-CTLA-4 antibody, anti-PD-1 antibody, anti-TIM-3 antibody, anti-LAG3 antibody, anti-Siglec15 antibody, anti-4-1BB antibody, Anti-GITR antibody, anti-OX40 antibody, anti-CD40L antibody, anti-CD28 antibody, anti-TIGIT antibody, anti-VISTA antibody; the epigenetic drugs such as histone deacetylase inhibitors, the targeting PI3K/Akt/mTOR Signal pathway inhibitors such as Tricibine, the tyrosine kinase inhibitors such as sunitinib, the immunosuppressants such as cyclophosphamide, the proteasome inhibitors such as bortezomib, the immunosuppressants such as sa Lidamide, pomalidomide, the cytotoxic drugs such as gemcitabine, temozolomide, and the cell cycle non-specific drugs such as mitoxantrone.
  • the epigenetic drugs such as histone deace
  • the kit may include instructions concerning the administration of a therapeutically effective amount of a drug for lowering blood immunoglobulin levels and a therapeutically effective amount of the therapeutic agent (for example, dosage information, administration time interval information).
  • the therapeutic agent is selected from viral vector drugs, antibodies, and polypeptide drugs capable of reducing blood IgG levels; the viral vector drugs are preferably oncolytic viruses.
  • the above-mentioned drugs for reducing blood immunoglobulin levels and therapeutically effective amounts of therapeutic agents can be administered simultaneously, separately or sequentially, for example, for cancer treatment, cancer and/or infection prevention and / Or treatment.
  • the mutant or protein and viral vector drug as described above can be provided as separate formulations or as a combined formulation.
  • drug (or agent) that reduces blood immunoglobulin levels preferably refers to a drug or agent that reduces blood immunoglobulin levels to 60% or less of the original level.
  • the drug or agent reduces blood immunoglobulin to at most 60% of the original level, 50% at the original level, 40% at the original level, 30% at the original level, 20% at the original level, and 10% at the original level. Or up to 0% of the original level. More preferably, the drug or agent reduces blood immunoglobulin to at most 20% of the original level, at most 10% of the original level, or at most 0% of the original level.
  • a mature and complete expression system can be used to manufacture viral vector drugs.
  • Some examples of methods include the use of mammalian cell expression systems to produce viral particles, such as the use of HEK293 cells to produce adenovirus-like virus vector drugs (Freedman Joshua D, Duffy Margaret R, Lei-Rossmann Janet, etc., An Oncolytic Virus Expressing a T-cell Engager Simultaneously Targets Cancer and Immunosuppressive Stromal Cells.[J].Cancer Res.,2018,78:6852-6865.).
  • the pharmaceutical carrier may be liquid, and the pharmaceutical composition may be in the form of a solution.
  • Liquid carriers are used to prepare solutions, suspensions, emulsions, syrups, elixirs and pressurized compositions.
  • the active ingredient can be dissolved or suspended in a pharmaceutically acceptable liquid carrier, such as water, an organic solvent, a mixture of the two, or a pharmaceutically acceptable oil or fat.
  • the pharmaceutical composition for parenteral administration is sterile, substantially isotonic, pyrogen-free, and prepared in accordance with the GMP of the FDA or similar agencies.
  • Viral vector drugs can be administered as an injectable form of a solution or suspension of the substance, where the substance is in a physiologically acceptable diluent and drug carrier (which can be a sterile liquid, such as water, oil, saline, glycerin) Or ethanol).
  • auxiliary substances such as wetting or emulsifying agents, surfactants, and pH buffering substances may be present in the composition.
  • Other components of the pharmaceutical composition are those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil and mineral oil.
  • glycols such as propylene glycol or polyethylene glycol are the preferred liquid carriers, especially for injectable solutions.
  • Viral vector drugs can be administered in the form of depot injections or implant preparations, which can be formulated to allow sustained release of the active ingredient.
  • the composition is prepared as an injectable, that is, a liquid solution or suspension; it can also be prepared in a solid form suitable for dissolution or suspension in a liquid carrier before injection.
  • the agent and oncolytic virus or viral vaccine can be administered by any suitable route.
  • both the agent and the oncolytic virus are administered intravenously (i.v.).
  • both the reagent and the viral vaccine are administered intravenously (i.v.).
  • the agent is administered intravenously (i.v.) and the viral vaccine is administered intramuscularly.
  • polypeptide and protein are used interchangeably herein to mean a polymer of amino acid residues. That is, the description for polypeptides is equally applicable to describing peptides and describing proteins, and vice versa.
  • the term applies to naturally occurring amino acid polymers and amino acid polymers in which one or more amino acid residues are non-naturally encoded amino acids.
  • the term encompasses amino acid chains of any length, including full-length proteins (ie, antigens) in which the amino acid residues are linked via covalent peptide bonds.
  • the reagents and raw materials used in the present invention are all commercially available.
  • the beneficial effects of the present invention are at least: by combining immunoglobulin degrading enzymes or endoglycosidase with oncolytic viruses, on the one hand, there is no interference of oncolytic virus neutralizing antibodies, and on the other hand, intravenous injection of oncolytic viruses is also eliminated.
  • the mediated cytokine storm and other side effects make intravenous injection of oncolytic virus no longer an obstacle to its administration, making it possible to treat oncolytic virus in migrating tumors.
  • the IdeS and IdeE protease nucleotide sequences were obtained by gene synthesis technology, and the IdeS and IdeE protease nucleotide sequences were recombined into the expression vector pET32a to construct a recombinant expression vector.
  • the recombinant expression vector proved to be correct by sequencing is transformed into the expression bacteria BL21 Escherichia coli to obtain positive expression bacteria containing the IdeS and IdeE protease gene sequences.
  • the synthesized sequence encoding IdeS is as follows (SEQ ID NO: 46):
  • the synthesized sequence encoding IdeE is as follows (SEQ ID NO: 47):
  • trastuzumab was selected as the substrate. Dilute IdeS and IdeE to 0.1mg/mL, 0.05mg/mL, 0.025mg/mL and 0.0125mg/mL. Separately take 1ul of different concentrations of enzyme and add 9ul of the reaction system containing 1mg/ml trastuzumab to start the cleavage reaction, and the reaction system is placed at 37°C for 30 minutes. After mixing the sample with the same volume of 2 ⁇ SDS loading buffer, the cleavage product was detected by SDS-PAGE in a water bath at 75°C for 5 min. IdeS and IdeE have obvious cleavage effects on human IgG1 ( Figure 2-3).
  • IdeE, IdeS, and IdeZ The in vitro cleavage activity of IdeE, IdeS, and IdeZ on human IVIg immunoglobulin was evaluated by detecting the amount of intact or single-cutting IVIg by adding IdeE, IdeS, and IdeZ proteases.
  • IdeE Dilute 5ul IdeE, IdeS, IdeZ (Genovis) protease separately and add 45ul reaction system containing IVIg to start the cleavage reaction.
  • the mass ratio of enzyme and IVIg is 1:200.
  • IdeZ cuts 10 ⁇ g IVIg according to 1Unit for experiment.
  • the reaction system was placed at 37°C for 1 hr. After mixing the sample with the same volume of 2 ⁇ SDS loading buffer, the cleavage product was detected by SDS-PAGE in a water bath at 75°C for 5 min.
  • Figure 4 shows the cleavage effect of three enzymes to cut human IVIg in vitro. The results of electrophoresis showed that all three proteases can effectively cleave human IVIg.
  • mice Under aseptic conditions, 8 mice were injected intraperitoneally with human IVIg (intravenous human immunoglobulin), 2 mice in each group (two mice are parallel experiments, mouse No. 1 and No. 2), the injection dose is 1g/ kg. 24 hours after the injection of human IVIg, IgG degrading enzymes IdeS and IdeE were grouped into mice at a dose of 5 mg/kg and IdeZ at a dose of 1000 unit/kg. One group was a blank control (normal saline). Two mice in each group were given blood and serum samples 24 hours after injection of IgG degrading enzyme. Take serum samples for non-reducing SDS-PAGE detection. The results showed that IdeS, IdeZ, and IdeE were effective in cutting IVIg in mice, and IVIg can be completely digested within 24 hours (Figure 5).
  • Adenovirus neutralizing antibody titer detection HEK293 cells 5 ⁇ 10 4 cells per well are seeded into a 96-well plate, and cultured at 37°C and 5% CO 2 for about 7 hours. After diluting the mouse serum 10 times, 10 gradients were diluted according to the 2-fold ratio, and then mixed with Ad5-Luc virus (2 ⁇ 10 4 TU/well) in equal volume, and incubated for 1 hour at room temperature. There are 3 replicate wells for each dilution, and no serum and virus are added to the negative control. After the incubation was completed, it was added to the cell culture plate and incubated at 37°C and 5% CO 2 for 24 hours. After the culture is completed, add the Bio-Bright TM One-Step Firefly Luciferase Assay Kit reagents, operate according to the instructions, and detect the chemiluminescence signal.
  • HSV-1 virus neutralizing antibody titer detection U-2OS cells are seeded into 96-well plates according to 2 ⁇ 10 4 cells per well, and cultured at 37°C and 5% CO 2 for about 7 hours. After diluting mouse serum 10 times, 10 gradients were diluted according to a 2-fold ratio, mixed with HSV-1 virus (1 ⁇ 10 4 PFU/well) in equal volume, and incubated for 1 hour at room temperature. There are 3 replicate wells for each dilution, and no serum and virus are added to the negative control. After the incubation, the mixture was added to the cell culture plate and cultured for 16 hours.
  • the cells are fixed with 1% paraformaldehyde, and then 0.1% Triton X-100 is added to incubate at room temperature for 5 minutes. Subsequently, HSV1 antibody (10 ⁇ g/ml) was added and incubated for 1 hour. Then incubate with anti-mouse IgG-HRP secondary antibody (diluted 1:2000) for 30 minutes. Add TMB substrate and incubate for 15 min. After drying the well plate, use the CTL enzyme-linked spot analyzer to read and analyze the data.
  • Example 5 The effect of IgG degrading enzyme on Adv5 infection in vivo
  • C57BL/6 mice were used to evaluate the effect of IgG degrading enzyme on Adv5-Luc infection in vivo.
  • C57BL/6 mice were injected intraperitoneally with human IVIg, 6 mice in each group.
  • human IVIg was injected intraperitoneally at a dose of 1 g/kg.
  • 30 minutes after the injection of human IVIg, IdeS and IdeE protease were administered intravenously at a dose of 5 mg/kg, and IdeZ was intravenously administered at a dose of 1000 units/kg to mice.
  • Adv5-Luc was administered at 5 ⁇ 10 10 vg/mouse on day 0, and fluorescence of mice was detected on day 6 and 10 during the process.
  • the experimental design is shown in Table 2.
  • the experimental results are shown in Figure 6. All three IgG degrading enzymes can reduce the effect of IVIg on Adv5-Luc virus infection.
  • mice Female athymic mice aged 6-8 weeks were subcutaneously inoculated with A549 tumor cells (5 ⁇ 10 6 ) in 0.1 ml PBS in the right lower limb area for tumorigenesis. When the tumor reached ⁇ 150mm 3 , the mice were randomly divided into 3 groups with 6 mice in each group. On day -1, blood was collected, and human IVIg was intraperitoneally injected after blood collection. 30 minutes after the injection of human IVIg, the protease was administered intravenously at a dose of 5 mg/kg.
  • Neutralizing antibody detection After diluting mouse serum 10 times, dilute 10 gradients according to 2 times ratio, mix with Ad5-Luc adenovirus (2 ⁇ 10 4 TU/well), add to 96-well plate, and incubate at room temperature 1 hour. There are 2 replicate wells for each dilution, and no serum and virus are added to the negative control. After the incubation was completed, 5 ⁇ 10 4 HEK293 cells were added to each well, and cultured at 37°C and 5% CO 2 for 24 hours. After the culture is completed, add the Bio-Bright TM One-Step Firefly Luciferase Assay Kit reagents, operate according to the instructions, and detect the chemiluminescence signal.
  • Figure 7 shows that IVIg contains adenovirus neutralizing antibodies, which can be degraded using IdeS or IdeE.

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Abstract

本发明公开了一种药物组合，其包含：1)降低Fc受体与内源性血清抗体结合的试剂，其中，所述试剂包括免疫球蛋白降解酶或者糖苷内切酶，和2)病毒载体药物，其中，所述病毒载体药物选自溶瘤病毒、病毒疫苗；并且其中所述药物组合允许所述病毒载体药物和所述试剂的单独给药。本发明还公开了所述药物组合在制备治疗或预防疾病的药物中的应用，和将药物组合施用于受试者来治疗或预防癌症或感染的方法。

Description

一种酶和病毒的药物组合及其应用 技术领域
本发明涉及生物医药领域，具体涉及一种酶和病毒的药物组合及其应用。
背景技术
病毒是现在常用的治疗载体，比如溶瘤病毒、病毒疫苗、基因治疗病毒。常用的病毒包括HSV-1、腺病毒等。这些病毒应用于治疗的一大问题是这类病毒绝大多数人都感染过且具有免疫原性，其中和抗体广泛存在于人群中。HSV-1预存的中和抗体阳性率可观。这些中和抗体会阻断病毒发挥效果，是病毒载体治疗的主要限制之一。除此之外，病毒载体类治疗完成一次给药后，中和抗体的滴度会增高并且一年之内无法再进行给药，此为限制病毒载体治疗的另一个主要障碍。目前主要采用免疫抑制剂来解决此问题，但这类方法治疗效果不佳。
溶瘤病毒(Oncolytic virus)是优先感染并杀死肿瘤细胞的一类病毒。初期，部分肿瘤细胞被溶瘤病毒特异性感染和破坏。随后，溶瘤病毒在肿瘤细胞进行复制及增殖，释放出新的感染性病毒颗粒感染和破坏其他肿瘤细胞。溶瘤病毒通过直接溶解肿瘤细胞或者刺激宿主产生抗肿瘤免疫反应来发挥溶瘤功效。
溶瘤病毒的给药方式有瘤内注射和静脉注射两种。其中瘤内注射因其不容易耐药、还可激发肿瘤微环境免疫应答、不容易产生细胞因子风暴等特点成为目前的主流给药方式。但对于晚期、已经转移到全身的肿瘤，瘤内注射给药方式的效果有限，瘤内注射无法达到消除已转移肿瘤的效果。因此溶瘤病毒静脉注射的研发相对于瘤内注射的研究越来越多，但中和抗体问题以及系统注射带来安全性等问题，是目前溶瘤病毒静脉注射的主要障碍。(Russell,S.,Peng,K.& Bell,J.Oncolytic virotherapy.Nat Biotechnol 30,658–670,2012.https://doi.org/10.1038/nbt.2287)。因此亟需一种能够减缓或者消除溶瘤病毒静脉注射障碍的治疗药物。
人体内预先存在的中和抗体也是病毒载体疫苗遇到的治疗障碍之一。陈 薇院士在其最新发表的以腺病毒为载体的新冠病毒疫苗Ad5-nCoV临床实验文章中指出，预先存在的Ad5免疫可以减慢对SARS-CoV-2的快速免疫反应，并降低反应的峰值水平。此外，高度存在的Ad5免疫力也可能会对疫苗引起的免疫反应的持续性产生负面影响。(Zhu Feng-Cai,Li Yu-Hua,Guan Xu-Hua et al.Safety,tolerability,and immunogenicity of a recombinant adenovirus type-5 vectored COVID-19 vaccine:a dose-escalation,open-label,non-randomised,first-in-human trial.[J].Lancet,2020.)因此，亟需一种能安全有效降解IgG的酶，用于酶切Ad5-nCoV疫苗治疗对象血清中IgG。
WO2020102740A2公开了一种基因治疗的方法，所述方法为采用IdeS或者EndoS用于酶切AAV治疗对象血清中IgG或者IgG所含糖链的治疗方法。但在溶瘤病毒和病毒疫苗领域急缺一种有效降解抗体Fc部分或IgG所含糖链的酶。
发明内容
针对现有技术的不足，本发明的目的在于提供一种降低血液IgG对病毒治疗干扰的试剂包括免疫球蛋白降解酶或糖苷内切酶与病毒载体药物的药物组合及其应用，所述病毒载体药物包括溶瘤病毒以及病毒疫苗。针对溶瘤病毒，通过对人体中病毒载体药物相关中和抗体的预先清除、和/或清除病毒载体给药后产生的中和抗体，扫除溶瘤病毒静脉给药的主要障碍。针对病毒疫苗，预先清除病毒载体药物中和抗体，消除中和抗体的影响。
具体地，本发明涉及如下各项：
1.一种药物组合，其特征在于，所述药物组合包含：1)降低Fc受体与内源性血清抗体结合的试剂，其中，所述试剂包括免疫球蛋白降解酶或者糖苷内切酶，和2)病毒载体药物，其中，所述病毒载体药物选自溶瘤病毒、病毒疫苗；并且其中所述药物组合允许所述病毒载体药物和所述试剂的单独给药。
2.如项1所述的药物组合，其特征在于，所述免疫球蛋白降解酶为IgG降解酶，所述IgG降解酶选自化脓性链球菌的IgG半胱氨酸蛋白酶或其变体或片段或者人来源MMP蛋白酶或其变体或片段，所述变体或片段保持了酶切IgG的活性；优选地，所述IgG降解酶选自IdeS、MAC2、IdeZ、IdeZ2、IdeE、IdeE2、IdeP、MMP。
3.如项1所述的药物组合，其特征在于，所述糖苷内切酶为IgG糖苷内 切酶，所述IgG糖苷内切酶选自链球菌、假结核棒状杆菌、粪肠球菌或脑膜脓毒性菌的IgG内切糖苷酶或其变体或片段，所述变体或片段保持了IgG糖苷内切酶的活性，所述链球菌例如为化脓性链球菌、马链球菌或兽疫链球菌；优选地，所述IgG内切糖苷酶为EndoS、CP40、EndoE或EndoF2。
4.如项2所述的药物组合，其特征在于，所述IgG降解酶包含如SEQ ID NO:1～41所示的氨基酸序列或为由所述氨基酸序列组成的蛋白质。
5.如项3所述的药物组合，其特征在于，所述IgG内切糖苷酶包含如SEQ ID NO:42～45所示的氨基酸序列或为由所述氨基酸序列组成的蛋白质。
6.如项1所述的药物组合，其特征在于，所述病毒载体药物中，所述病毒载体药物所用的病毒选自ssDNA类病毒、dsDNA类病毒、ssRNA类病毒或dsRNA类病毒；和/或，所述病毒载体药物所用的病毒选自野生型病毒株或自然减毒株、基因工程选择性减毒株、基因加载型病毒株、基因转录靶向型病毒株。
7.如项6所述的药物组合，其特征在于，所述野生型病毒株或自然减毒株选自新城疫病毒、呼肠孤病毒、流行性腮腺炎病毒、西尼罗河病毒、腺病毒、牛痘病毒等。
8.如项6所述的药物组合，其特征在于，所述基因工程选择性减毒株通过人工方式删除关键基因而实现病毒复制的肿瘤选择性，所述基因工程选择性减毒株例如ONYX-015、G207。
9.如项6所述的药物组合，其特征在于，所述基因加载型病毒株加载了外源基因，所述外源基因例如为粒细胞巨噬细胞集落刺激因子(GM-CSF)，所述基因加载型病毒株例如为JX-594或T-VEC。
10.如项6所述的药物组合，其特征在于，所述基因转录靶向型病毒株，即在病毒必需基因前插入组织或肿瘤特异性启动子来控制溶瘤病毒在肿瘤细胞内复制，所述基因转录靶向型病毒株例如为G92A。
11.如项6所述的药物组合，其特征在于，所述ssDNA类病毒选自细小病毒(parvovirus)，所述细小病毒例如为H-1PV病毒。
12.如项6所述的药物组合，其特征在于，所述dsDNA类病毒选自单纯疱疹病毒(herpes simplex virus)、腺病毒(adeno virus)、poxvirus；优选地，所述腺病毒选自Enadenotucirev、DNX-2401、C-REV、NG-348、ProsAtak、CG0070、ADV-TK、EDS01、KH901、H101、H103、VCN-01、Telomelysin (OBP-301)，所述单纯疱疹病毒优选为I型单纯疱疹病毒HSV-1；所述单纯疱疹病毒选自R3616、T-VEC、HF10、G207、NV1020、OrienX010，所述poxvirus选自Pexa-Vec(vaccinia virus)、JX-594(vaccinia virus)、GL-ONC1、Myxoma。
13.如项6所述的药物组合，其特征在于，所述ssRNA类病毒选自Picornavirus、alphavirus、Retroviruses、Paramyxoviruses、Rhabdoviruses；优选地，所述Picornavirus选自CAVATAK、PVS-RIPO、CVA21(enterovirus)、RIGVIR，所述alphavirus选自M1、Sindbis AR339、Semliki Forest virus，所述Retroviruses选自Toca511，所述Paramyxoviruses选自MV-NIS、PV701(Newcastle disease virus)，所述Rhabdoviruses选自VSV-IFNβ、MG1-MAGEA3、VSV-GP。
14.如项6所述的药物组合，其特征在于，所述dsRNA类病毒选自Reoviruses；优选地，所述Reoviruses选自Pelareorep、呼肠孤病毒(Reolysin)、牛痘病毒(vaccinia virus)、腮腺炎病毒、人类免疫缺陷病毒(human immunodeficiency virus，HIV)。
15.如项6所述的药物组合，其特征在于，所述ssRNA类病毒选自呼肠孤病毒(reovirus)、柯萨奇病毒(coxsackievirus)、脊髓灰质炎病毒(polio virus)、猪塞内加谷病毒(seneca valley virus)、麻疹病毒(measles virus)、新城疫病毒(newcastle disease virus)、水泡性口炎病毒(vesicular stomatitis virus,VSV)、流感病毒。
16.如项1所述的药物组合，其特征在于，所述溶瘤病毒表达外源基因，所述外源基因选自双特异性T细胞结合子(Bispecific T cell engagers，BiTE)、GM-CSF、白细胞介素-2(IL-2)、白细胞介素-12(IL-12)、干扰素(IFN)、肿瘤坏死因子(TNF)、可溶性CD80、CCL3。
17.如项1-16中任一项所述药物组合，其特征在于，所述药物组合还包括靶向药物或化疗药物或免疫检查点阻断剂，所述靶向药物选自表观遗传学药物、靶向PI3K/Akt/mTOR信号通路的抑制剂和酪氨酸激酶抑制剂，所述化疗药物选自免疫抑制剂和蛋白酶体抑制剂，所述免疫检查点阻断剂选自抗CTLA-4抗体、抗PD-1抗体和抗TIM-3抗体；所述表观遗传学药物例如组蛋白去乙酰化酶抑制剂，所述靶向PI3K/Akt/mTOR信号通路的抑制剂例如Tricibine，所述酪氨酸激酶抑制剂例如舒尼替尼，所述免疫抑制剂例如环磷酰胺、吉西他滨、替莫唑胺、米托蒽醌和硼替佐米。
18.如项1-17中任一项所述药物组合在制备治疗或预防疾病的药物中的应用，其中所述试剂通过静脉输液或者皮下注射施用于受试者，和/或，所施用的所述试剂的量为0.01mg/kg体重至2mg/kg体重、0.04至2mg/kg体重、0.12mg/kg体重至2mg/kg体重、0.24mg/kg体重至2mg/kg体重或1mg/kg体重至2mg/kg体重；优选地，所述疾病为癌症、病毒感染、细菌感染或真菌感染。
19.如项18所述的应用，其中所述试剂和所述病毒载体药物的给药时间间隔为至少30分钟、至少1小时、至少2小时、至少3小时、至少4小时、至少4小时、至少5小时或至少6小时；并且最多35天、最多28天、最多21天、最多18天、最多14天、最多13天、最多12天、最多11天、最多10天、最多9天、最多8天、最多7天、最多6天、最多5天、最多4天、最多3天、最多2天、最多24小时、最多18小时、最多12小时、最多10小时、最多8小时、最多7小时或最多6小时。
20.如项18所述的应用，其中所述试剂和所述病毒载体药物的时间间隔为30分钟至1小时、30分钟至2小时、30分钟至3小时、30分钟至4小时、30分钟至5小时、30分钟至6小时、1至2小时、1至3小时、1至4小时、1至5小时、1至6小时、2至3小时、2至4小时、2至5小时、2至6小时、3至4小时、3至5小时、3至6小时、4至5小时、4至6小时或5至6小时。
21.如项18所述的应用，其中所述试剂在所述病毒载体药物给药前施用，或者所述试剂在所述病毒载体药物给药后施用。
22.如项19所述的应用，其中所述试剂在所述病毒载体药物给药前施用时，所述试剂给药前、所述试剂给药后且所述病毒载体药物给药前、所述病毒载体药物给药后，定量检测所述受试者血液中结合病毒载体的抗体、对其进行抗体介导的效应功能的确认。
23.如项20所述的应用，其中所述试剂在所述病毒载体药物给药后施用时，所述述病毒载体药物给药前、所述病毒载体药物给药后且所述试剂给药前、所述试剂给药后，定量检测所述受试者血液中结合病毒载体的抗体、对其进行抗体介导的效应功能的确认。
24.将如项1-17中任一项所述的药物组合施用于受试者来治疗或预防癌症或感染的方法，其中所述方法导致所述病毒载体药物结合减少20-50％，50-75％，75-90％，90-95％或95％或更多抗体；所述感染优选为病毒感染、细 菌感染或真菌感染。
25.如项24所述的方法，其中所述病毒载体药物为溶瘤病毒；优选地，所述癌症选自前列腺癌、乳腺癌、膀胱癌、结肠癌、直肠癌、胰腺癌、卵巢癌、肺癌、宫颈癌、子宫内膜癌、肾(肾细胞)癌、食管癌、甲状腺癌、淋巴瘤、皮肤癌，黑色素瘤和白血病。
26.如项24所述的方法，其中所述病毒载体药物为病毒疫苗；优选地，所述病毒疫苗用于靶向或治疗前列腺癌、乳腺癌、膀胱癌、结肠癌、直肠癌、胰腺癌、卵巢癌、肺癌、宫颈癌、子宫内膜癌、肾(肾细胞)癌、食管癌、甲状腺癌、淋巴瘤、皮肤癌，黑色素瘤、白血病或冠状病毒、新型冠状病毒导致的疾病。
27.如项24-26中任一项所述的方法，其中所述药物组合的成分单独给药。
28.如项24-26中任一项所述的方法，其中所述药物组合的成分同时给药。
附图说明
图1为IdeS、IdeE表达电泳图，1为分子量marker，2为诱导前，3为IdeS、4为IdeE。
图2、3分别为IdeS、IdeE体外酶切IgG1电泳图。图中右侧的箭头表示来自IgG的不同切割产物。箭头1：完整的IgG1；箭头2：scIgG1(单切割的IgG1-由第一IgG重链切割产生)；箭头3：F(ab’)2片段(由第二IgG重链的切割产生)。
图4、5分别为IdeS、IdeE体外酶切IVIg电泳图。
图6为不同组别小鼠病毒感染情况。
图7为小鼠血清中和抗体检测。
图8为不同组别小鼠体内肿瘤体积生长曲线。
发明详述
本发明提供的免疫球蛋白降解酶可有效酶切血液中的免疫球蛋白，并且人体中不存在针对该酶的抗体，使用安全。为解决上述技术问题，以下为本发明的技术方案：
I.药物组合
第一方面，提供一种药物组合，其中，所述药物组合包含：1)降低Fc受 体与内源性血清抗体结合的试剂，其中，所述试剂包括免疫球蛋白降解酶或者糖苷内切酶，和2)病毒载体药物，其中，所述病毒载体药物选自溶瘤病毒、病毒疫苗；并且其中所述药物组合允许所述抗体和所述试剂的单独给药。优选地，所述药物组合包含治疗有效量的所述免疫球蛋白降解酶和病毒载体药物。优选地，所述药物组合是药物组合物，并且还包含药学上可接受的载剂或稀释剂。
1.1免疫球蛋白降解酶或者糖苷内切酶
优选地，如上所述的药物组合，其中，所述免疫球蛋白降解酶为IgG降解酶，所述IgG降解酶选自化脓性链球菌的IgG半胱氨酸蛋白酶或其变体或片段或者人来源MMP蛋白酶或其变体或片段，所述变体或片段保持了酶切IgG的活性；优选地，所述IgG降解酶选自IdeS、MAC2、IdeZ、IdeZ2、IdeE、IdeE2、IdeP、MMP。
优选地，如上所述的药物组合，其中，所述糖苷内切酶为IgG糖苷内切酶，所述IgG糖苷内切酶选自链球菌、假结核棒状杆菌、粪肠球菌或脑膜脓毒性菌的IgG内切糖苷酶或其变体或片段，所述变体或片段保持了IgG糖苷内切酶的活性，所述链球菌例如为化脓性链球菌、马链球菌或兽疫链球菌；优选地，所述IgG内切糖苷酶为EndoS、CP40、EndoE或EndoF2。
优选地，所述IgG降解酶包含SEQ ID NO:1～41的氨基酸序列或其具有相同功能的变体或为由所述氨基酸序列组成的蛋白质或其片段。在一个实施方式中，所述IgG降解酶包含SEQ ID NO:1～41的氨基酸序列，还可包括：1)氨基酸序列SEQ ID NO:1～41；2)其变体，该变体与氨基酸序列SEQ ID NO:1～41具有至少50％的同一性并具有IgG降解酶；或1)和2)的具有IgG降解酶活性的片段。其中SEQ ID NOs:7-41是CN107532156A、CN107532158A中所示的IgG降解酶突变体。
优选地，所述IgG内切糖苷酶包含SEQ ID NO:42～45的氨基酸序列或其具有相同功能的变体或为由所述氨基酸序列组成的蛋白质。在一个实施方式中，所述IgG内切糖苷酶包含：1)氨基酸序列SEQ ID NO:42～45；2)其变体，该变体与氨基酸序列SEQ ID NO:42～45具有至少50％的同一性并具有IgG内切糖苷酶活性；或1)和2)的具有IgG内切糖苷酶活性的片段。
本发明的第四方面，提供了一种组合物，特别是药物组合物或者药物组合，包括如上所述的突变体或者蛋白，以及治疗剂；所述的治疗剂可为任何 用药后在机体内已产生抗药抗体或易产生抗药抗体的治疗剂，包括但不限于抗体药物、融合蛋白、小分子药物、核酸药物、抗体偶联药物或病毒载体药物；所述药物组合物还包括药学上可接受的载体或赋形剂。
1.2病毒载体药物
优选地，如上所述的药物组合，其中，所述病毒载体药物中，所述病毒载体药物所用的病毒选自ssDNA类病毒、dsDNA类病毒、ssRNA类病毒或dsRNA类病毒；和/或，所述病毒载体药物所用的病毒选自野生型病毒株或自然减毒株、基因工程选择性减毒株、基因加载型病毒株、基因转录靶向型病毒株。
优选地，所述野生型病毒株或自然减毒株选自新城疫病毒、呼肠孤病毒、流行性腮腺炎病毒、西尼罗河病毒、腺病毒、牛痘病毒等。
优选地，所述基因工程选择性减毒株通过人工方式删除关键基因而实现病毒复制的肿瘤选择性，例如胸苷激酶(Thymidinekinase,TK)敲除的基因改造人单纯疱疹病毒I(HSV-1)，所述基因工程选择性减毒株例如ONYX-015、G207。ONYX-015在E1b区域删除了827bp，并且在针对E1B55K蛋白的基因进行点突变，使其表达基因提前终止，无法表达E1B55K蛋白。G207删除了γ34.5基因，该基因为HSV-1的神经毒性决定因素。
优选地，所述基因加载型病毒株加载了外源基因，所述外源基因例如为粒细胞巨噬细胞集落刺激因子(GM-CSF)，所述基因加载型病毒株例如为JX-594或T-VEC。
优选地，所述基因转录靶向型病毒株，即在病毒必需基因前插入组织或肿瘤特异性启动子来控制溶瘤病毒在肿瘤细胞内复制，所述基因转录靶向型病毒株例如为G92A。
优选地，如上所述的药物组合，其中，所述ssDNA类病毒选自细小病毒(parvovirus)，优选所述细小病毒为H-1PV病毒。
优选地，所述dsDNA类病毒选自单纯疱疹病毒(herpes simplex virus)、腺病毒(adeno virus)、poxvirus；更优选地，所述单纯疱疹病毒优选为I型单纯疱疹病毒HSV-1，例如为R3616、T-VEC、HF10、G207、NV1020、OrienX010，所述poxvirus选自Pexa-Vec(vaccinia viruse)、JX-594(vaccinia viruse)、GL-ONC1、Myxoma；所述腺病毒选自Enadenotucirev、DNX-2401、C-REV、NG-348、ProsAtak、CG0070、ADV-TK、EDS01、KH901、H101、H103、VCN-01、Telomelysin(OBP-301)。
优选地，所述ssRNA类病毒选自Picornavirus、alphavirus、Retroviruses、Paramyxoviruses、Rhabdoviruses；优选地，所述Picornavirus选自CAVATAK、PVS-RIPO、CVA21(enterovirus)、RIGVIR，所述alphavirus选自M1、Sindbis AR339、Semliki Forest virus，所述Retroviruses选自Toca511，所述Paramyxoviruses选自MV-NIS、PV701(Newcastle disease virus)，所述Rhabdoviruses选自VSV-IFNβ、MG1-MAGEA3、VSV-GP。
优选地，所述dsRNA类病毒选自Reoviruses；优选地，所述Reoviruses选自Pelareorep、呼肠孤病毒(Reolysin)、牛痘病毒(vaccinia virus)、腮腺炎病毒、人类免疫缺陷病毒(human immunodeficiency virus，HIV)；优选的，所述RNA类病毒选自呼肠孤病毒(reovirus)、柯萨奇病毒(coxsackievirus)、脊髓灰质炎病毒(polio virus)、猪塞内加谷病毒(seneca valley virus)、麻疹病毒(measles virus)、新城疫病毒(newcastle disease virus)、水泡性口炎病毒(vesicular stomatitis virus)、流感病毒。
优选地，如上所述的药物组合，其中，所述溶瘤病毒表达外源基因，所述外源基因优选双特异性T细胞结合子(Bispecific T cell engagers，BiTE)、scFv片段、细胞因子、趋化因子。所述BiTE能结合CD3等激活T细胞的分子，同时能结合癌细胞表面的抗原靶点；所述scFv靶向免疫检查点；所述免疫检查点包括CTLA-4、PD-1、TIM-3、LAG3、Siglec15、4-1BB、GITR、OX40、CD40L、CD28、TIGIT、VISTA。所述细胞因子、趋化因子例如GM-CSF、白细胞介素-2(IL-2)、白细胞介素-12(IL-12)、干扰素(IFN)、肿瘤坏死因子(TNF)、可溶性CD80、CCL3。
1.3药物的靶点
优选地，如上所述的药物组合，其中，所述药物的靶点可为细胞表面蛋白，包括但不限于：AFP、αv整联蛋白(integrin)、α4β7整联蛋白、BCMA、CD2、CD3、CD19、CD20、CD22、CD25、CD30、CD32、CD33、CD36、CD40、CD46、CD52、CD56、CD64、CD70、CD74、CD79、CD80、CD86、CD105、CD121、CD123、CD133、CD138、CD174、CD205、CD227、CD326、CD340、CEA、c-Met、Cripto、CA1X、Claudin18.2、ED-B、EGFR、EpCAM、EphA2、EphB2、FAP、FOLR1、GD2、Globo H、GPC3、GPNMB、HER-1、HER-2、HER-3、MAGE-A3、Mesothelin、MUC16、GPNMB、PSMA、TMEFF2、TAG-72、5T4、ROR-1、Sca-1、SP、VEGF或WT1。
所述的抗体药物的靶点可为细胞因子，包括但不限于：白介素IL-1至IL-13、肿瘤坏死因子α和β、干扰素α、β和γ、肿瘤生长因子β(TGF-β)、集落刺激因子(CSF)或粒细胞单核细胞集落刺激因子(GM-CSF)。见HumanCytokines:Handbook for Basic & Clinical Research(Aggrawal等编,Blackwell Scientific,Boston,MA 1991)。
所述的抗体药物的靶点可为激素、酶、胞内和胞间信使，例如：腺苷环化酶、鸟苷环化酶或磷脂酶C。
所述的抗体药物的靶点可为免疫检查点，所述的免疫检查点包括：CTLA-4、PD-1、PD-L1、TIM-3、LAG3、Siglec15、4-1BB、GITR、OX40、CD40L、CD28、TIGIT、VISTA。
1.4其他药物
优选地，如上任一所述药物组合，其中，所述药物组合还包括靶向药物或化疗药物或免疫检查点阻断剂，所述靶向药物选自表观遗传学药物、靶向PI3K/Akt/mTOR信号通路的抑制剂和酪氨酸激酶抑制剂，所述化疗药物选自免疫抑制剂、蛋白酶体抑制剂、细胞毒药物和细胞周期非特异性药物，所述免疫检查点阻断剂选自抗CTLA-4抗体、抗PD-1抗体、抗TIM-3抗体、抗LAG3抗体、抗Siglec15抗体、抗4-1BB抗体、抗GITR抗体、抗OX40抗体、抗CD40L抗体、抗CD28抗体、抗TIGIT抗体、抗VISTA抗体；所述表观遗传学药物例如组蛋白去乙酰化酶抑制剂，所述靶向PI3K/Akt/mTOR信号通路的抑制剂例如Tricibine，所述酪氨酸激酶抑制剂例如舒尼替尼，所述免疫抑制剂例如环磷酰胺，所述蛋白酶体抑制剂例如硼替佐米，所述免疫抑制剂例如沙利度胺、泊马度胺，所述细胞毒药物例如吉西他滨、替莫唑胺，所述细胞周期非特异性药物例如米托蒽醌。
II.药物组合的应用
本发明还提供了一种任一项所述药物组合在制备治疗或预防疾病的药物中的应用。优选地，所述疾病为癌症、传染性疾病。所述传染性疾病包括病毒感染、细菌感染或真菌感染。
2.1癌症
所述癌症选自由以下癌症组成的组：急性淋巴母细胞白血病、急性髓样白血病、肾上腺皮质癌、AIDS相关的癌症、AIDS相关淋巴瘤、肛门癌、阑 尾癌、星形细胞瘤、儿童小脑或大脑癌、基细胞癌、肝外胆管癌、膀胱癌、骨癌、骨肉瘤/恶性纤维组织细胞瘤、脑干胶质瘤、脑癌、脑瘤-小脑星形细胞瘤、脑瘤-大脑星形细胞瘤/恶性胶质瘤、脑瘤-室管膜瘤、脑瘤-髓母细胞瘤、脑瘤-幕上原始神经外胚层瘤、脑瘤-视觉通路和下丘脑胶质瘤、乳腺癌、支气管腺瘤/类癌、Burkitt淋巴瘤、类癌肿瘤、胃肠道类癌肿瘤、不明原发癌、中枢神经系统淋巴瘤、小脑星形细胞瘤、大脑星形细胞瘤/恶性神经胶质瘤、宫颈癌、慢性淋巴细胞性白血病、慢性髓性白血病慢性骨髓增殖性疾病、结肠癌、皮肤T细胞淋巴瘤、结缔组织增生性小圆细胞肿瘤、子宫内膜癌、室管膜瘤、食管癌、尤因肿瘤家族中的尤因氏肉瘤、颅外生殖细胞肿瘤、儿童性腺外生殖细胞瘤、肝外胆道癌、眼癌-眼内黑色素瘤、眼癌-视网膜母细胞瘤、胆囊癌、胃癌、胃肠道类癌肿瘤、胃肠道间质瘤(GIST)、颅外、性腺外、或卵巢生殖细胞瘤、妊娠滋养细胞瘤、脑干胶质瘤、童年大脑星形细胞瘤神经胶质瘤、儿童视觉通路和下丘脑神经胶质瘤、胃类癌、毛细胞白血病、头颈癌、心脏癌、肝细胞(肝)癌、霍奇金淋巴瘤、下咽癌、下丘脑和视觉通路胶质瘤、眼内黑色素瘤、胰岛细胞癌(内分泌胰腺)、卡波西肉瘤、肾癌(肾细胞癌)、喉头癌、白血病、急性成淋巴细胞白血病(也称为急性淋巴细胞性白血病)、急性髓样白血病(也称为急性髓细胞性白血病)、慢性淋巴细胞白血病(也称为慢性淋巴细胞性白血病)、慢性髓细胞性白血病(也称为慢性髓样白血病)、毛细胞白血病、唇和口腔癌、脂肪肉瘤、肝癌(原发性)、非小细胞肺癌、小细胞肺癌、淋巴瘤、AIDS相关淋巴瘤、Burkitt淋巴瘤、皮肤T细胞淋巴瘤、霍奇金淋巴瘤、非霍奇金淋巴瘤(旧分类：除了霍奇金氏淋巴瘤之外的其他全部淋巴瘤)、原发性中枢神经系统淋巴瘤、巨球蛋白血症、骨恶性纤维组织细胞瘤/骨肉瘤、髓母细胞瘤、黑色素瘤、眼内(眼)黑色素瘤、Merkel细胞癌、间皮瘤、成人恶性间皮瘤、原发性隐性转移性鳞状颈部癌、口腔癌、多发性内分泌腺瘤综合征、多发性骨髓瘤/浆细胞性肿瘤、蕈样肉芽肿、骨髓增生异常综合征、骨髓增生异常/骨髓增生性疾病、慢性骨髓性白血病、成人急性髓样白血病、儿童急性髓样白血病、多发性骨髓瘤(骨髓癌症)、骨髓增生病、鼻腔和鼻旁窦癌、鼻咽癌、神经母细胞瘤、非霍奇金淋巴瘤、非小细胞肺癌、口腔癌、口咽癌、骨肉瘤/骨恶性纤维组织细胞瘤、卵巢癌、卵巢上皮癌(表面上皮-间质细胞瘤)、卵巢生殖细胞肿瘤、卵巢低度恶性潜在肿瘤、胰腺癌、胰岛细胞胰腺癌、鼻旁窦和鼻腔癌、甲状旁腺癌、 阴茎癌、咽癌、嗜铬细胞瘤、松果腺星形细胞瘤、松果腺生殖细胞瘤、成松果腺细胞瘤和幕上原始神经外胚层肿瘤、脑垂体瘤、浆细胞瘤/多发性骨髓瘤、胸膜肺母细胞瘤、原发性中枢神经系统淋巴瘤、前列腺癌、直肠癌、肾盂和输尿管肾细胞癌(肾癌)、移行细胞癌、视网膜母细胞瘤、横纹肌肉瘤、唾液腺癌、尤因家族肿瘤肉瘤、卡波济肉瘤、软组织肉瘤、子宫肉瘤、塞扎里(Sézary)综合征、皮肤癌(非黑色素瘤)、皮肤癌(黑色素瘤)、Merkel细胞皮肤癌、小细胞肺癌、小肠癌、软组织肉瘤、鳞状细胞癌、原发性隐性鳞状颈部癌、转移性胃癌、幕上原始神经外胚层瘤、皮肤T细胞淋巴瘤(见蕈样肉芽肿和塞扎里综合征)、睾丸癌、咽喉癌、胸腺瘤、胸腺瘤和胸腺癌、甲状腺癌、甲状腺癌、肾盂和输尿管移行细胞癌、输尿管和肾盂滋养细胞瘤、移行细胞癌尿道癌、子宫内膜子宫癌、子宫肉瘤、阴道癌、视觉通路和下丘脑胶质瘤、外阴癌、巨球蛋白血症和肾母细胞瘤(肾癌)。
2.2癌症或传染性疾病
所述癌症或传染性疾病可以为兽类疾病、也可以为人类疾病，举例如下：
Figure PCTCN2021098263-appb-000001
Figure PCTCN2021098263-appb-000002
III.药物组合物的治疗方法
在本发明的应用中，所述试剂和病毒载体药物作为用于同时、分开或依次使用的联合制剂存在。
在一些实施方式中，所述方法包括以下步骤：1)向受试者施用所述试剂；随后，2)向所述受试者施用所述病毒载体药物。优选地，所述试剂为免疫球蛋白降解酶，所述免疫球蛋白降解酶和所述病毒载体药物的施用存在时间间隔。
在一些实施方式中，所述方法包括以下步骤：1)向所述受试者施用所述病毒载体药物；随后，2)向受试者施用所述试剂。优选地，所述试剂为免疫球蛋白降解酶，所述免疫球蛋白降解酶和所述病毒载体药物的施用存在时间间隔。
优选的是，如上所述的突变体的施用量和时间间隔足以将受试者体内的免疫球蛋白水平降至起始水平的60％。更优选的是，试剂施用量和时间间隔足以将受试者体内的免疫球蛋白水平结合降低至低于该患者体内起始水平的50％、40％、30％、20％或10％。该试剂可以在一个单一时间点施用或在设定的时间内施用。
优选的是，其中如上所述的突变体通过静脉输液、腹腔注射、肌肉注射、关节注射、皮内注射或者皮下注射施用，优选的注射方式为静脉输液方式。并且/或者所施用的如上所述的突变体的量为0.01mg/kg体重至2mg/kg体重、0.04至2mg/kg体重、0.12mg/kg体重至2mg/kg体重、0.24mg/kg体重至2mg/kg体重或1mg/kg体重至2mg/kg体重。
优选的是，其中如上所述的突变体和所述治疗剂的给药时间间隔至少30分钟、至少1小时、至少2小时、至少3小时、至少4小时、至少4小时、至少5小时或至少6小时；并且最多35天、最多28天、最多21天、最多18天、最多14天、最多13天、最多12天、最多11天、最多10天、最多9天、最多8天、最多7天、最多6天、最多5天、最多4天、最多3天、最多2天、最多24小时、最多18小时、最多12小时、最多10小时、最多8小时、最多7小时或最多6小时。
优选的是，其中如上所述的突变体和所述治疗剂的时间间隔为30分钟至1小时、30分钟至2小时、30分钟至3小时、30分钟至4小时、30分钟至5 小时、30分钟至6小时、1至2小时、1至3小时、1至4小时、1至5小时、1至6小时、2至3小时、2至4小时、2至5小时、2至6小时、3至4小时、3至5小时、3至6小时、4至5小时、4至6小时或5至6小时。
在又一实施方式中，所述方法包括以下步骤：1)用如上所述的突变体对来自所述受试者的血液进行离体处理；2)将血液返回所述受试者；3)向所述受试者施用所述治疗剂。
在又一实施方式中，所述方法包括以下步骤：1)向所述受试者施用所述治疗剂；2)用如上所述的突变体对来自所述受试者的血液进行离体处理；3)将血液返回所述受试者。
在优选的实施方式中，如上所述的突变体与治疗剂用于预防和/或治疗癌症。
在优选的实施方式中，如上所述的突变体与治疗剂用于预防和/或治疗病毒感染。
在优选的实施方式中，如上所述的突变体与治疗剂用于预防和/或治疗细菌感染。
在优选的实施方式中，如上所述的突变体与治疗剂用于预防和/或治疗真菌感染。
本发明还提供了所述药物组合施用于受试者来治疗或预防癌症或感染的方法。其中所述方法导致所述病毒载体结合抗体减少20-50％，50-75％，75-90％，90-95％或95％或95％以上的抗体；其中所述方法导致所述致病性IgG抗体减少20-50％，50-75％，75-90％，90-95％或95％或95％以上。优选的是，所述药物用于治疗癌症、预防癌症、预防感染的方法。所述感染优选为病毒感染、细菌感染或真菌感染。优选的是，所述药物用于癌症的治疗方法。
优选地，其中所述治疗剂为病毒载体药物；优选地，所述病毒载体药物为溶瘤病毒、病毒疫苗。
优选的，其中所述药物组合的成分单独给药，或所述药物组合的成分同时给药。
IV.产品
本发明还提供了一种产品，所述产品含有降低血液IgG水平的试剂包括IgG降解酶和病毒载体药物，作为联合制剂同时、分开或依次应用于癌症治疗、癌症和/或感染的预防中。优选地，所述联合制剂应用于治疗癌症的方法中。
在一些实施方式中，癌症选自由以下癌症组成的组：急性淋巴母细胞白血病、急性髓样白血病、肾上腺皮质癌、AIDS相关的癌症、AIDS相关淋巴瘤、肛门癌、阑尾癌、星形细胞瘤、儿童小脑或大脑癌、基细胞癌、肝外胆管癌、膀胱癌、骨癌、骨肉瘤/恶性纤维组织细胞瘤、脑干胶质瘤、脑癌、脑瘤-小脑星形细胞瘤、脑瘤-大脑星形细胞瘤/恶性胶质瘤、脑瘤-室管膜瘤、脑瘤-髓母细胞瘤、脑瘤-幕上原始神经外胚层瘤、脑瘤-视觉通路和下丘脑胶质瘤、乳腺癌、支气管腺瘤/类癌、Burkitt淋巴瘤、类癌肿瘤、胃肠道类癌肿瘤、不明原发癌、中枢神经系统淋巴瘤、小脑星形细胞瘤、大脑星形细胞瘤/恶性神经胶质瘤、宫颈癌、慢性淋巴细胞性白血病、慢性髓性白血病慢性骨髓增殖性疾病、结肠癌、皮肤T细胞淋巴瘤、结缔组织增生性小圆细胞肿瘤、子宫内膜癌、室管膜瘤、食管癌、尤因肿瘤家族中的尤因氏肉瘤、颅外生殖细胞肿瘤、儿童性腺外生殖细胞瘤、肝外胆道癌、眼癌-眼内黑色素瘤、眼癌-视网膜母细胞瘤、胆囊癌、胃癌、胃肠道类癌肿瘤、胃肠道间质瘤(GIST)、颅外、性腺外、或卵巢生殖细胞瘤、妊娠滋养细胞瘤、脑干胶质瘤、童年大脑星形细胞瘤神经胶质瘤、儿童视觉通路和下丘脑神经胶质瘤、胃类癌、毛细胞白血病、头颈癌、心脏癌、肝细胞(肝)癌、霍奇金淋巴瘤、下咽癌、下丘脑和视觉通路胶质瘤、眼内黑色素瘤、胰岛细胞癌(内分泌胰腺)、卡波西肉瘤、肾癌(肾细胞癌)、喉头癌、白血病、急性成淋巴细胞白血病(也称为急性淋巴细胞性白血病)、急性髓样白血病(也称为急性髓细胞性白血病)、慢性淋巴细胞白血病(也称为慢性淋巴细胞性白血病)、慢性髓细胞性白血病(也称为慢性髓样白血病)、毛细胞白血病、唇和口腔癌、脂肪肉瘤、肝癌(原发性)、非小细胞肺癌、小细胞肺癌、淋巴瘤、AIDS相关淋巴瘤、Burkitt淋巴瘤、皮肤T细胞淋巴瘤、霍奇金淋巴瘤、非霍奇金淋巴瘤(旧分类：除了霍奇金氏淋巴瘤之外的其他全部淋巴瘤)、原发性中枢神经系统淋巴瘤、巨球蛋白血症、骨恶性纤维组织细胞瘤/骨肉瘤、髓母细胞瘤、黑色素瘤、眼内(眼)黑色素瘤、Merkel细胞癌、间皮瘤、成人恶性间皮瘤、原发性隐性转移性鳞状颈部癌、口腔癌、多发性内分泌腺瘤综合征、多发性骨髓瘤/浆细胞性肿瘤、蕈样肉芽肿、骨髓增生异常综合征、骨髓增生异常/骨髓增生性疾病、慢性骨髓性白血病、成人急性髓样白血病、儿童急性髓样白血病、多发性骨髓瘤(骨髓癌症)、骨髓增生病、鼻腔和鼻旁窦癌、鼻咽癌、神经母细胞瘤、非霍奇金淋巴瘤、非小细胞肺癌、口腔癌、口咽癌、骨肉瘤/骨恶性纤维 组织细胞瘤、卵巢癌、卵巢上皮癌(表面上皮-间质细胞瘤)、卵巢生殖细胞肿瘤、卵巢低度恶性潜在肿瘤、胰腺癌、胰岛细胞胰腺癌、鼻旁窦和鼻腔癌、甲状旁腺癌、阴茎癌、咽癌、嗜铬细胞瘤、松果腺星形细胞瘤、松果腺生殖细胞瘤、成松果腺细胞瘤和幕上原始神经外胚层肿瘤、脑垂体瘤、浆细胞瘤/多发性骨髓瘤、胸膜肺母细胞瘤、原发性中枢神经系统淋巴瘤、前列腺癌、直肠癌、肾盂和输尿管肾细胞癌(肾癌)、移行细胞癌、视网膜母细胞瘤、横纹肌肉瘤、唾液腺癌、尤因家族肿瘤肉瘤、卡波济肉瘤、软组织肉瘤、子宫肉瘤、塞扎里(Sézary)综合征、皮肤癌(非黑色素瘤)、皮肤癌(黑色素瘤)、Merkel细胞皮肤癌、小细胞肺癌、小肠癌、软组织肉瘤、鳞状细胞癌、原发性隐性鳞状颈部癌、转移性胃癌、幕上原始神经外胚层瘤、皮肤T细胞淋巴瘤(见蕈样肉芽肿和塞扎里综合征)、睾丸癌、咽喉癌、胸腺瘤、胸腺瘤和胸腺癌、甲状腺癌、甲状腺癌、肾盂和输尿管移行细胞癌、输尿管和肾盂滋养细胞瘤、移行细胞癌尿道癌、子宫内膜子宫癌、子宫肉瘤、阴道癌、视觉通路和下丘脑胶质瘤、外阴癌、巨球蛋白血症和肾母细胞瘤(肾癌)。
本发明还提供了一种用于癌症治疗、癌症和/或感染的预防方法的药物组合物或治疗剂，所述药物组合物或治疗剂包含：1)治疗有效量的降低血液IgG水平的试剂包括IgG降解酶；和2)治疗有效量的病毒载体药物，优选溶瘤病毒；和3)药学上可接受的载剂或稀释剂。
本发明的药物组合物可以与靶向药物联合使用。所述靶向药物选自表观遗传学药物、靶向PI3K/Akt/mTOR信号通路的抑制剂和受体酪氨酸激酶抑制剂。
组蛋白去乙酰化酶抑制剂(HDACi)是一种被广泛研究的表观遗传学药物，HDACi不仅可通过抑制肿瘤细胞增殖和诱导细胞周期阻滞等方式促进肿瘤细胞的分化和凋亡，还可以通过抑制干扰素信号途径来降低机体的抗病毒免疫应答。组蛋白去乙酰化酶抑制剂HDAC6被证实能够显著提高HSV-1溶瘤病毒在神经胶质瘤细胞中的复制水平，可与HSV-1协同杀伤肿瘤。
PI3K/Akt信号通路是应激条件下调节细胞增殖和凋亡的重要信号通路，Akt抑制剂Tricibine能够协同溶瘤病毒MG18L诱导胶质瘤细胞发生凋亡，两者联合治疗小鼠神经胶质瘤的疗效明显优于单药治疗。雷帕霉素是mTOR信号通路的抑制剂，可协同腺病毒和HSV-1杀伤不易感的肿瘤细胞。
蛋白酪氨酸激酶(PTKs)抑制剂具有抑制肿瘤血管生成和抗肿瘤细胞生 长的多重作用。舒尼替尼是一种小分子受体酪氨酸激酶抑制剂，能通过抑制细胞内PTK的活性来提高VSV溶瘤病毒在肿瘤细胞中的复制，也能通过抑制VEGFR信号通路破坏肿瘤血管生成来增强溶瘤病毒在瘤内的感染能力，从而显著增强溶瘤病毒的治疗效果。
本发明的药物组合物可以与化疗药物联合使用。所述联合使用能免疫原性细胞死亡、增强肿瘤细胞抗原性或对免疫细胞的易感性、抑制具有负调控的Treg细胞和髓样来源的抑制细胞(MDSC)。所述化疗药物选自免疫抑制剂、蛋白酶体抑制剂、细胞毒药物和细胞周期非特异性药物，所述免疫抑制剂例如环磷酰胺，所述蛋白酶体抑制剂例如硼替佐米，所述免疫抑制剂例如沙利度胺、泊马度胺，所述细胞毒药物例如吉西他滨、替莫唑胺，所述细胞周期非特异性药物例如米托蒽醌。优选的，所述联合使用为溶瘤呼肠孤病毒(RV)和硼替佐米(BTZ)联合用药。
本发明的药物组合物可以与免疫检查点阻断剂联合使用。所述免疫检查点选自CTLA-4、PD-1、TIM-3、LAG3、Siglec15、4-1BB、GITR、OX40、CD40L、CD28、TIGIT、VISTA；所述免疫检查点阻断剂选自抗CTLA-4抗体、抗PD-1抗体、抗TIM-3抗体、抗LAG3抗体、抗Siglec15抗体、抗4-1BB抗体、抗GITR抗体、抗OX40抗体、抗CD40L抗体、抗CD28抗体、抗TIGIT抗体、抗VISTA抗体。
本发明还提供了一种用于预防或治疗癌症或感染的试剂盒或套装药盒，所述试剂盒包含：1)治疗有效量的包含如上所述的突变体的药物；和2)治疗有效量的治疗剂；所述治疗剂选自病毒载体药物、抗体、能降低血液IgG水平的多肽药物；所述病毒载体药物优选溶瘤病毒、基因治疗病毒。所述试剂盒还可以包括3)靶向药物或化疗药物或免疫检查点阻断剂。
所述靶向药物选自表观遗传学药物、靶向PI3K/Akt/mTOR信号通路的抑制剂和酪氨酸激酶抑制剂，所述化疗药物选自免疫抑制剂、蛋白酶体抑制剂、细胞毒药物和细胞周期非特异性药物，所述免疫检查点阻断剂选自抗CTLA-4抗体、抗PD-1抗体、抗TIM-3抗体、抗LAG3抗体、抗Siglec15抗体、抗4-1BB抗体、抗GITR抗体、抗OX40抗体、抗CD40L抗体、抗CD28抗体、抗TIGIT抗体、抗VISTA抗体；所述表观遗传学药物例如组蛋白去乙酰化酶抑制剂，所述靶向PI3K/Akt/mTOR信号通路的抑制剂例如Tricibine，所述酪氨酸激酶抑制剂例如舒尼替尼，所述免疫抑制剂例如环磷酰胺，所述蛋白酶 体抑制剂例如硼替佐米，所述免疫抑制剂例如沙利度胺、泊马度胺，所述细胞毒药物例如吉西他滨、替莫唑胺，所述细胞周期非特异性药物例如米托蒽醌。
所述试剂盒或套装药盒包括药盒A和药盒B，所述药盒A包括治疗有效量的降低血液免疫球蛋白水平的药物包括IgG降解酶，所述药盒B包括治疗有效量的治疗剂；所述治疗剂选自病毒载体药物、抗体、能降低血液IgG水平的多肽药物；所述病毒载体药物优选溶瘤病毒。所述套装药盒还可以包括药盒C。所述药盒C包括靶向药物或化疗药物或免疫检查点阻断剂。所述靶向药物选自表观遗传学药物、靶向PI3K/Akt/mTOR信号通路的抑制剂和酪氨酸激酶抑制剂，所述化疗药物选自免疫抑制剂、蛋白酶体抑制剂、细胞毒药物和细胞周期非特异性药物，所述免疫检查点阻断剂选自抗CTLA-4抗体、抗PD-1抗体、抗TIM-3抗体、抗LAG3抗体、抗Siglec15抗体、抗4-1BB抗体、抗GITR抗体、抗OX40抗体、抗CD40L抗体、抗CD28抗体、抗TIGIT抗体、抗VISTA抗体；所述表观遗传学药物例如组蛋白去乙酰化酶抑制剂，所述靶向PI3K/Akt/mTOR信号通路的抑制剂例如Tricibine，所述酪氨酸激酶抑制剂例如舒尼替尼，所述免疫抑制剂例如环磷酰胺，所述蛋白酶体抑制剂例如硼替佐米，所述免疫抑制剂例如沙利度胺、泊马度胺，所述细胞毒药物例如吉西他滨、替莫唑胺，所述细胞周期非特异性药物例如米托蒽醌。
该试剂盒可以包含涉及治疗有效量的降低血液免疫球蛋白水平的药物和治疗有效量的治疗剂施用(例如，剂量信息、给药时间间隔信息)的说明书。所述治疗剂选自病毒载体药物、抗体、能降低血液IgG水平的多肽药物；所述病毒载体药物优选溶瘤病毒。
在本发明的任何方面，如上所述的降低血液免疫球蛋白水平的药物和治疗有效量的治疗剂施用均可以同时、分开或依次施用，例如用于癌症治疗、癌症和/或感染的预防和/或治疗。如上所述的突变体或者蛋白和病毒载体药物可以作为分开的制剂或作为联合制剂提供。
本文所用的术语“降低血液免疫球蛋白水平的药物(或试剂)”优选是指将血液免疫球蛋白水平降至原水平60％或以下的药物或试剂。优选的是，该药物或试剂将血液免疫球蛋白降到至多原水平60％、至多原水平50％、至多原水平40％、至多原水平30％、至多原水平20％、至多原水平10％或至多原水平0％。更优选的是，该药物或试剂将血液免疫球蛋白降到至多原水平20％、至多原水平10％或至多原水平0％。
可利用成熟完善的表达系统来制造病毒载体药物。一些方法示例包括利用哺乳动物细胞表达系统来产生病毒颗粒，例如使用HEK293细胞生产腺病毒类病毒载体药物(Freedman Joshua D,Duffy Margaret R,Lei-Rossmann Janet等,An Oncolytic Virus Expressing a T-cell Engager Simultaneously Targets Cancer and Immunosuppressive Stromal Cells.[J].Cancer Res.,2018,78:6852-6865.)。
药物载剂可以是液体，并且药物组合物可以为溶液形式。液体载剂用于制备溶液、悬浮液、乳液、糖浆、酏剂和加压组合物。活性成分可以溶解或悬浮在药学上可接受的液体载剂中，例如水、有机溶剂、二者的混合物或药学上可接受的油或脂肪。
用于肠胃外施用的药物组合物是无菌的、基本上等渗的、无热原的，并根据FDA或类似机构的GMP来制备。病毒载体药物可以作为该物质的溶液或悬浮液的可注射剂型施用，其中，该物质处在生理上可接受的稀释剂和药物载剂(可以是无菌液体，例如水、油、盐水、甘油或乙醇)中。另外，组合物中可存在辅助物质，例如润湿剂或乳化剂、表面活性剂和pH缓冲物质等。药物组合物的其他组分有石油、动物、植物或合成来源的组分，例如花生油、大豆油和矿物油。通常，例如丙二醇或聚乙二醇等二醇是优选的液体载剂，对于可注射溶液尤其如此。病毒载体药物可以以积存注射剂或植入制剂的形式施用，这些形式能够被配制成允许活性成分持续释放。通常，将组合物制备成可注射物，即液体溶液或悬浮液；也可以制备成适合于在注射前溶解或悬浮在液体载质中的固体形式。
所述试剂和溶瘤病毒或者病毒疫苗可以通过任何适合的途径施用。优选的是，所述试剂和溶瘤病毒均为静脉内(i.v.)施用。选的是，所述试剂和病毒疫苗均为静脉内(i.v.)施用。优选的是，所述试剂为静脉内(i.v.)施用，所述病毒疫苗为肌肉内施用。
除非另外定义，否则本文所用的所有技术及科学术语都具有与本发明所属领域的普通技术人员通常所了解相同的含义。虽然在本发明的实践或测试中可使用与本文所述者类似或等效的任何方法、装置和材料，但现在描述优选方法、装置和材料。
术语“多肽”和“蛋白”在本文中互换使用以意指氨基酸残基的聚合物。即，针对多肽的描述同样适用于描述肽和描述蛋白，且反之亦然。所述术语适用于天然产生氨基酸聚合物以及其中一个或一个以上氨基酸残基为非天然编 码氨基酸的氨基酸聚合物。如本文中所使用，所述术语涵盖任何长度的氨基酸链，其包括全长蛋白(即抗原)，其中氨基酸残基经由共价肽键连接。
在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明的有益效果至少在于：通过将免疫球蛋白降解酶或者糖苷内切酶与溶瘤病毒联用，一方面没有溶瘤病毒中和抗体的干扰，另一方面也排除了溶瘤病毒静脉注射介导的细胞因子风暴等副作用，使得溶瘤病毒的静脉注射不再是其施用的障碍，让溶瘤病毒在迁移型肿瘤中的治疗成为可能。通过将免疫球蛋白降解酶或者糖苷内切酶与病毒疫苗联用，一方面没有针对疫苗病毒载体的中和抗体的干扰，另一方面也排除了病毒疫苗静脉注射介导的细胞因子风暴等副作用，使得病毒疫苗安全性大大提高。
具体实施方式
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。
实施例1.样品制备
通过基因合成技术获得IdeS、IdeE蛋白酶核苷酸序列，将IdeS、IdeE蛋白酶核苷酸序列重组到表达载体pET32a中，构建重组表达载体。将所述测序证明正确的重组表达载体转化到表达菌BL21大肠杆菌中，得到含有所述IdeS、IdeE蛋白酶基因序列的阳性表达菌。
所合成的编码IdeS的序列如下(SEQ ID NO:46)：
Figure PCTCN2021098263-appb-000003
Figure PCTCN2021098263-appb-000004
所合成的编码IdeE的序列如下(SEQ ID NO:47)：
Figure PCTCN2021098263-appb-000005
具体地，挑取6个单菌落37℃过夜培养。将过夜培养的菌液以1:100的比例加入5mL Amp+LB培养基中，37℃220rpm培养2-3h。取出1mL菌液作为未诱导对照，其余按照体积加入IPTG至终浓度为0.5mM，37℃180rmp培养4h。表达产物用12％SDS-PAGE电泳检测(图1)。收集表达菌体，采用常规方法破胞，通过镍柱纯化得到IdeS、IdeE蛋白酶，去除内毒素并除菌过滤后备用。
实施例2.对人IgG1切割活性的评估
测试对人IgG1的切割活性，选择曲妥珠单抗作为底物。将IdeS、IdeE稀释至0.1mg/mL、0.05mg/mL、0.025mg/mL和0.0125mg/mL。分别取1ul不同浓度的酶加入9ul含有1mg/ml曲妥珠单抗的反应体系中开始切割反应，反应体系置于37℃反应30min。将样品与同等体积的2×SDS上样缓冲液混合后75℃水浴5min，SDS-PAGE检测切割产物。IdeS、IdeE对人IgG1都有明显的切割作用(图2-3)。
实施例3.IgG降解酶体外切割IVIg
通过检测加入IdeE、IdeS、IdeZ蛋白酶完整或单切割IVIg的量来评价 IdeE、IdeS、IdeZ对人IVIg免疫球蛋白的体外切割活性。
分别稀释取5ul IdeE、IdeS、IdeZ(Genovis)蛋白酶加入45ul含有IVIg的反应体系中开始切割反应，酶和IVIg质量比为1:200，IdeZ按照1Unit切10μg IVIg进行实验。反应体系置于37℃反应1hr。将样品与同等体积的2×SDS上样缓冲液混合后75℃水浴5min，SDS-PAGE检测切割产物。
图4展示了3种酶体外切割人IVIg的酶切效果。电泳结果显示3种蛋白酶均能有效切割人IVIg。
实施例4.IgG降解酶体内切割IVIg
在无菌条件下用人IVIg(静注人免疫球蛋白)腹腔注射8只小鼠，每组2只(两只小鼠为平行实验，小鼠编号1号和2号)，注射剂量为1g/kg。注射人IVIg 24h后，再将IgG降解酶IdeS和IdeE以5mg/kg、IdeZ以1000unit/kg的剂量分组静脉注射小鼠，一组为空白对照(生理盐水)。每组2只小鼠都分别在注射IgG降解酶后24hr采血并收集血清样品。取血清样品进行非还原SDS-PAGE检测。结果显示，IdeS、IdeZ、IdeE在小鼠体内切割IVIg效果明显，24hr基本可完全酶切IVIg(图5)。
腺病毒中和抗体滴度检测：HEK293细胞5×10 4个每孔种至96孔板，37℃、5％CO 2培养约7hr。取小鼠血清稀释10倍后，按照2倍比稀释10个梯度，分别和Ad5-Luc病毒(2×10 4TU/孔)等体积混合，室温孵育1小时。每个稀释3复孔，阴性对照不加血清和病毒。孵育完成后，加入至细胞培养板中，37℃、5％CO 2培养24hr。培养完成后，加入Bio-Bright TM One-Step萤火虫荧光素酶测定试剂盒试剂，按照说明书操作，检测化学发光信号。
HSV-1病毒中和抗体滴度检测：U-2OS细胞按照2×10 4个每孔种至96孔板，37℃、5％CO 2培养约7hr。取小鼠血清稀释10倍后，按照2倍比稀释10个梯度，分别和HSV-1病毒(1×10 4PFU/孔)等体积混合，室温孵育1小时。每个稀释3复孔，阴性对照不加血清和病毒。孵育后的混合物，加入至细胞培养板中，培养16hr。培养完成后，细胞采用1％多聚甲醛固定，后加入0.1％Triton X-100室温孵育5分钟。随后，加入HSV1抗体(10μg/ml)孵育1小时。随后采用抗鼠IgG-HRP二抗(1:2000稀释)孵育30分钟。加入TMB底物孵育15min。干燥孔板后，使用CTL酶联斑点分析仪读数并分析数据。
表1.酶对中和抗体滴度的影响
Figure PCTCN2021098263-appb-000006
中和抗体滴度检测实验显示，IVIg中具有HSV1和Adv的中和抗体，而IdeS、IdeZ、IdeE可在小鼠体内有效切割中和抗体。
实施例5.IgG降解酶对Adv5体内感染的影响
采用C57BL/6小鼠评价IgG降解酶对Adv5-Luc体内感染的影响。人IVIg腹腔注射C57BL/6小鼠，每组6只。第-1天腹腔注射人IVIg，剂量为1g/kg，注射人IVIg后30min，将IdeS和IdeE蛋白酶以5mg/kg剂量静脉施用，IdeZ以1000单位/kg的剂量静脉注射小鼠。第0天将Adv5-Luc以5×10 10vg/只施用，过程中第6、10天检测小鼠荧光。实验设计如表2。实验结果如图6所示，3种IgG降解酶均可降低IVIg对Adv5-Luc病毒感染的影响。
表2.Adv5-Luc动物模型设计
Figure PCTCN2021098263-appb-000007
实施例6.小鼠肿瘤模型
采用A549无胸腺裸鼠肿瘤模型，IdeE与KJ-Adv5腺病毒联合用药与单独KJ-Adv5病毒载体药物进行比较。6-8周龄的雌性无胸腺小鼠在右下肢区域中用0.1ml PBS中的A549肿瘤细胞(5×10 6)皮下接种，用于肿瘤发生。 当肿瘤达到～150mm 3时，将小鼠随机分3组，6只每组。第-1天，取血，取血后腹腔注射人IVIg。注射人IVIg后30min，将蛋白酶以5mg/kg剂量静脉施用。第0天取血，用于中和抗体检测。取血后静脉注射KJ-Adv5，5×10 10vp/只。每周2次测量肿瘤体积，并称重，第28天处死小鼠。实验设计如表3。
表3.不同小组给与IVIg、酶、KJ-Adv5剂量
Figure PCTCN2021098263-appb-000008
中和抗体检测：取小鼠血清稀释10倍后，按照2倍比稀释10个梯度，分别和Ad5-Luc腺病毒(2×10 4TU/孔)混合，加入到96孔板中，室温孵育1小时。每个稀释2复孔，阴性对照不加血清和病毒。孵育完成后，加入5×10 4个HEK293细胞每孔，37℃、5％CO 2培养24hr。培养完成后，加入Bio-Bright TM One-Step萤火虫荧光素酶测定试剂盒试剂，按照说明书操作，检测化学发光信号。图7显示IVIg中含有腺病毒中和抗体，采用IdeS或者IdeE可降解中和抗体。
实验结果显示IdeS和IdeE均可排除IVIg对病毒静脉给药治疗效果的负面影响(图8)。
申请人声明，本发明通过上述实施例来说明本发明的详细方法，但本发明并不局限于上述详细方法，即不意味着本发明必须依赖上述详细方法才能实施。所属技术领域的技术人员应该明了，对本发明的任何改进，对本发明产品各原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本发明的保护范围和公开范围之内。

Claims (28)

  1. 一种药物组合，其特征在于，所述药物组合包含：1)降低Fc受体与内源性血清抗体结合的试剂，其中，所述试剂包括免疫球蛋白降解酶或者糖苷内切酶，和2)病毒载体药物，其中，所述病毒载体药物选自溶瘤病毒、病毒疫苗；并且其中所述药物组合允许所述病毒载体药物和所述试剂的单独给药。
  2. 如权利要求1所述的药物组合，其特征在于，所述免疫球蛋白降解酶为IgG降解酶，所述IgG降解酶选自化脓性链球菌的IgG半胱氨酸蛋白酶或其变体或片段或者人来源MMP蛋白酶或其变体或片段，所述变体或片段保持了酶切IgG的活性；优选地，所述IgG降解酶选自IdeS、MAC2、IdeZ、IdeZ2、IdeE、IdeE2、IdeP、MMP。
  3. 如权利要求1所述的药物组合，其特征在于，所述糖苷内切酶为IgG糖苷内切酶，所述IgG糖苷内切酶选自链球菌、假结核棒状杆菌、粪肠球菌或脑膜脓毒性菌的IgG内切糖苷酶或其变体或片段，所述变体或片段保持了IgG糖苷内切酶的活性，所述链球菌例如为化脓性链球菌、马链球菌或兽疫链球菌；优选地，所述IgG内切糖苷酶为EndoS、CP40、EndoE或EndoF2。
  4. 如权利要求2所述的药物组合，其特征在于，所述IgG降解酶包含如SEQ ID NO:1～41所示的氨基酸序列或为由所述氨基酸序列组成的蛋白质。
  5. 如权利要求3所述的药物组合，其特征在于，所述IgG内切糖苷酶包含如SEQ ID NO:42～45所示的氨基酸序列或为由所述氨基酸序列组成的蛋白质。
  6. 如权利要求1所述的药物组合，其特征在于，所述病毒载体药物中，所述病毒载体药物所用的病毒选自ssDNA类病毒、dsDNA类病毒、ssRNA类病毒或dsRNA类病毒；和/或，所述病毒载体药物所用的病毒选自野生型病毒株或自然减毒株、基因工程选择性减毒株、基因加载型病毒株、基因转录靶向型病毒株。
  7. 如权利要求6所述的药物组合，其特征在于，所述野生型病毒株或自然减毒株选自新城疫病毒、呼肠孤病毒、流行性腮腺炎病毒、西尼罗河病毒、腺病毒、牛痘病毒等。
  8. 如权利要求6所述的药物组合，其特征在于，所述基因工程选择性减 毒株通过人工方式删除关键基因而实现病毒复制的肿瘤选择性，所述基因工程选择性减毒株例如ONYX-015、G207。
  9. 如权利要求6所述的药物组合，其特征在于，所述基因加载型病毒株加载了外源基因，所述外源基因例如为粒细胞巨噬细胞集落刺激因子(GM-CSF)，所述基因加载型病毒株例如为JX-594或T-VEC。
  10. 如权利要求6所述的药物组合，其特征在于，所述基因转录靶向型病毒株，即在病毒必需基因前插入组织或肿瘤特异性启动子来控制溶瘤病毒在肿瘤细胞内复制，所述基因转录靶向型病毒株例如为G92A。
  11. 如权利要求6所述的药物组合，其特征在于，所述ssDNA类病毒选自细小病毒(parvovirus)，所述细小病毒例如为H-1PV病毒。
  12. 如权利要求6所述的药物组合，其特征在于，所述dsDNA类病毒选自单纯疱疹病毒(herpes simplex virus)、腺病毒(adeno virus)、poxvirus；优选地，所述腺病毒选自Enadenotucirev、DNX-2401、C-REV、NG-348、ProsAtak、CG0070、ADV-TK、EDS01、KH901、H101、H103、VCN-01、Telomelysin(OBP-301)，所述单纯疱疹病毒优选为I型单纯疱疹病毒HSV-1；所述单纯疱疹病毒选自R3616、T-VEC、HF10、G207、NV1020、OrienX010，所述poxvirus选自Pexa-Vec(vaccinia virus)、JX-594(vaccinia virus)、GL-ONC1、Myxoma。
  13. 如权利要求6所述的药物组合，其特征在于，所述ssRNA类病毒选自Picornavirus、alphavirus、Retroviruses、Paramyxoviruses、Rhabdoviruses；优选地，所述Picornavirus选自CAVATAK、PVS-RIPO、CVA21(enterovirus)、RIGVIR，所述alphavirus选自M1、Sindbis AR339、Semliki Forest virus，所述Retroviruses选自Toca511，所述Paramyxoviruses选自MV-NIS、PV701(Newcastle disease virus)，所述Rhabdoviruses选自VSV-IFNβ、MG1-MAGEA3、VSV-GP。
  14. 如权利要求6所述的药物组合，其特征在于，所述dsRNA类病毒选自Reoviruses；优选地，所述Reoviruses选自Pelareorep、呼肠孤病毒(Reolysin)、牛痘病毒(vaccinia virus)、腮腺炎病毒、人类免疫缺陷病毒(human immunodeficiency virus，HIV)。
  15. 如权利要求6所述的药物组合，其特征在于，所述ssRNA类病毒选自呼肠孤病毒(reovirus)、柯萨奇病毒(coxsackievirus)、脊髓灰质炎病毒(polio virus)、猪塞内加谷病毒(seneca valley virus)、麻疹病毒(measles virus)、新 城疫病毒(newcastle disease virus)、水泡性口炎病毒(vesicular stomatitis virus,VSV)、流感病毒。
  16. 如权利要求1所述的药物组合，其特征在于，所述溶瘤病毒表达外源基因，所述外源基因选自双特异性T细胞结合子(Bispecific T cell engagers，BiTE)、GM-CSF、白细胞介素-2(IL-2)、白细胞介素-12(IL-12)、干扰素(IFN)、肿瘤坏死因子(TNF)、可溶性CD80、CCL3。
  17. 如权利要求1-16中任一项所述药物组合，其特征在于，所述药物组合还包括靶向药物或化疗药物或免疫检查点阻断剂，所述靶向药物选自表观遗传学药物、靶向PI3K/Akt/mTOR信号通路的抑制剂和酪氨酸激酶抑制剂，所述化疗药物选自免疫抑制剂和蛋白酶体抑制剂，所述免疫检查点阻断剂选自抗CTLA-4抗体、抗PD-1抗体和抗TIM-3抗体；所述表观遗传学药物例如组蛋白去乙酰化酶抑制剂，所述靶向PI3K/Akt/mTOR信号通路的抑制剂例如Tricibine，所述酪氨酸激酶抑制剂例如舒尼替尼，所述免疫抑制剂例如环磷酰胺、吉西他滨、替莫唑胺、米托蒽醌和硼替佐米。
  18. 如权利要求1-17中任一项所述药物组合在制备治疗或预防疾病的药物中的应用，其中所述试剂通过静脉输液或者皮下注射施用于受试者，和/或，所施用的所述试剂的量为0.01mg/kg体重至2mg/kg体重、0.04至2mg/kg体重、0.12mg/kg体重至2mg/kg体重、0.24mg/kg体重至2mg/kg体重或1mg/kg体重至2mg/kg体重；优选地，所述疾病为癌症、病毒感染、细菌感染或真菌感染。
  19. 如权利要求18所述的应用，其中所述试剂和所述病毒载体药物的给药时间间隔为至少30分钟、至少1小时、至少2小时、至少3小时、至少4小时、至少4小时、至少5小时或至少6小时；并且最多35天、最多28天、最多21天、最多18天、最多14天、最多13天、最多12天、最多11天、最多10天、最多9天、最多8天、最多7天、最多6天、最多5天、最多4天、最多3天、最多2天、最多24小时、最多18小时、最多12小时、最多10小时、最多8小时、最多7小时或最多6小时。
  20. 如权利要求18所述的应用，其中所述试剂和所述病毒载体药物的时间间隔为30分钟至1小时、30分钟至2小时、30分钟至3小时、30分钟至4小时、30分钟至5小时、30分钟至6小时、1至2小时、1至3小时、1至4小时、1至5小时、1至6小时、2至3小时、2至4小时、2至5小时、 2至6小时、3至4小时、3至5小时、3至6小时、4至5小时、4至6小时或5至6小时。
  21. 如权利要求18所述的应用，其中所述试剂在所述病毒载体药物给药前施用，或者所述试剂在所述病毒载体药物给药后施用。
  22. 如权利要求19所述的应用，其中所述试剂在所述病毒载体药物给药前施用时，所述试剂给药前、所述试剂给药后且所述病毒载体药物给药前、所述病毒载体药物给药后，定量检测所述受试者血液中结合病毒载体的抗体、对其进行抗体介导的效应功能的确认。
  23. 如权利要求20所述的应用，其中所述试剂在所述病毒载体药物给药后施用时，所述述病毒载体药物给药前、所述病毒载体药物给药后且所述试剂给药前、所述试剂给药后，定量检测所述受试者血液中结合病毒载体的抗体、对其进行抗体介导的效应功能的确认。
  24. 将如权利要求1-17中任一项所述的药物组合施用于受试者来治疗或预防癌症或感染的方法，其中所述方法导致所述病毒载体药物结合减少20-50％，50-75％，75-90％，90-95％或95％或更多抗体；所述感染优选为病毒感染、细菌感染或真菌感染。
  25. 如权利要求24所述的方法，其中所述病毒载体药物为溶瘤病毒；优选地，所述癌症选自前列腺癌、乳腺癌、膀胱癌、结肠癌、直肠癌、胰腺癌、卵巢癌、肺癌、宫颈癌、子宫内膜癌、肾(肾细胞)癌、食管癌、甲状腺癌、淋巴瘤、皮肤癌，黑色素瘤和白血病。
  26. 如权利要求24所述的方法，其中所述病毒载体药物为病毒疫苗；优选地，所述病毒疫苗用于靶向或治疗前列腺癌、乳腺癌、膀胱癌、结肠癌、直肠癌、胰腺癌、卵巢癌、肺癌、宫颈癌、子宫内膜癌、肾(肾细胞)癌、食管癌、甲状腺癌、淋巴瘤、皮肤癌，黑色素瘤、白血病或冠状病毒、新型冠状病毒导致的疾病。
  27. 如权利要求24-26中任一项所述的方法，其中所述药物组合的成分单独给药。
  28. 如权利要求24-26中任一项所述的方法，其中所述药物组合的成分同时给药。
PCT/CN2021/098263 2020-06-05 2021-06-04 一种酶和病毒的药物组合及其应用 Ceased WO2021244628A1 (zh)

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