WO2021062961A1 - 培干扰素和原癌基因产物靶向抑制剂在协同抑制肿瘤中的应用 - Google Patents

培干扰素和原癌基因产物靶向抑制剂在协同抑制肿瘤中的应用 Download PDF

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WO2021062961A1
WO2021062961A1 PCT/CN2019/130749 CN2019130749W WO2021062961A1 WO 2021062961 A1 WO2021062961 A1 WO 2021062961A1 CN 2019130749 W CN2019130749 W CN 2019130749W WO 2021062961 A1 WO2021062961 A1 WO 2021062961A1
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interferon
proto
combination
tumor
peg
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French (fr)
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杨宇峰
何成
楼觉人
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上海生物制品研究所有限责任公司
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Priority to EP19947849.6A priority Critical patent/EP4043028A4/en
Priority to JP2022544871A priority patent/JP7412577B2/ja
Priority to US17/754,351 priority patent/US20220370565A1/en
Publication of WO2021062961A1 publication Critical patent/WO2021062961A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A61K38/212—IFN-alpha
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • 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
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered

Definitions

  • the present invention relates to the technical field of anti-tumor drugs, and more specifically to the application of interferon and proto-oncogene product targeting inhibitors in synergistically inhibiting tumors and related drugs.
  • Tumor is a systemic and systemic disease with multi-factor participation and multi-step development. Tumors have the biological characteristics of high complexity, variability and diversity. The abnormality of cancer-related genes can cause the stability of tumor genome to be lost, which in turn leads to abnormal biological behaviors of tumor cells, such as avoidance of apoptosis, the potential for unlimited replication, angiogenesis, invasion and metastasis, and immune escape.
  • Surgery and radiotherapy are local treatment methods, and drug treatment is a method of systemic effect, which takes more into account the characteristics of malignant tumors that can spread and metastasize.
  • tumor immunotherapy has significantly improved the quality of life of patients compared with traditional tumor treatment methods.
  • the purpose of the present invention is to provide an effective and low toxic and side effect treatment method and drug for the treatment of tumors.
  • the present invention provides the use of PEG interferon and a targeted inhibitor of proto-oncogene product in the synergistic treatment of tumors. .
  • an active ingredient combination comprising the first active ingredient perinterferon and the second active ingredient proto-oncogene product targeting inhibitor, and the combination is used For the preparation of pharmaceutical compositions or kits for synergistic treatment of tumors.
  • the proto-oncogene product targeting inhibitor is an antibody.
  • the peginterferon is peginterferon ⁇ 1b.
  • the said interferon includes recombinant human interferon ⁇ 1b modified with PEG with a molecular weight of 10,000 to 100,000, preferably recombinant human interferon ⁇ 1b modified with PEG with a molecular weight of 10,000 to 100,000.
  • the mass ratio of the interferon and the targeted inhibitor of the proto-oncogene product is 1:100 to 500:1, preferably 1:20 to 200:1, more preferably 1: 10 to 100:1 or 1:5 to 50:1.
  • the ratio (IU/mg) of the said interferon and the targeted inhibitor of the proto-oncogene product is 1000 IU/mg to 100 million IU/mg, preferably 10000 IU/mg to 10 million IU/mg, more preferably 100,000 IU/mg to 5 million IU/mg or 200,000 IU/mg to 3 million IU/mg.
  • the pharmaceutical composition or kit is used for treating or administering to a mammal, and more preferably, the mammal is a rodent (such as a mouse, a rat) or a human.
  • the tumor is selected from the group consisting of lung cancer, gastric cancer, breast cancer, liver cancer, bowel cancer, ovarian cancer, cervical cancer, or a combination thereof.
  • the tumor is selected from the following group: lung cancer, gastric cancer, breast cancer, or a combination thereof.
  • the proto-oncogene product targeting inhibitor is selected from the following group: EGFR inhibitor, Her2 inhibitor, or a combination thereof.
  • the proto-oncogene product targeting inhibitor is selected from the following group: EGFR inhibitor, Her2 inhibitor, or a combination thereof.
  • the proto-oncogene product targeting inhibitor is selected from the group consisting of Avastin, Herceptin, Pertuzumab, or a combination thereof.
  • a pharmaceutical composition in the second aspect of the present invention, includes a perferon, a proto-oncogene product targeting inhibitor, and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition further includes additional therapeutic drugs (such as anti-tumor agents).
  • the proto-oncogene product targeting inhibitor is selected from the following group: EGFR inhibitor, Her2 inhibitor, or a combination thereof.
  • the proto-oncogene product targeting inhibitor is selected from the group consisting of Avastin, Herceptin, Pertuzumab, or a combination thereof.
  • the pharmaceutical composition further includes a chemotherapeutic agent, a checkpoint inhibitor, CAR-T cells, or a combination thereof.
  • the chemotherapeutic agent includes cisplatin and paclitaxel.
  • a combination of active ingredients which is composed of perinterferon and a proto-oncogene product targeting inhibitor.
  • the proto-oncogene product targeting inhibitor is selected from the following group: EGFR inhibitor, Her2 inhibitor, or a combination thereof.
  • a medicine kit which comprises:
  • a first preparation which contains interferon and a pharmaceutically acceptable carrier
  • first formulation and the second formulation are independent of each other.
  • the first preparation and the second preparation are freeze-dried preparations or liquid preparations.
  • the first preparation and the second preparation are injections.
  • the first formulation is applied before, during or after the second formulation.
  • the proto-oncogene product targeting inhibitor is selected from the following group: EGFR inhibitor, Her2 inhibitor, or a combination thereof.
  • the proto-oncogene product targeting inhibitor is selected from the group consisting of Avastin, Herceptin, Pertuzumab, or a combination thereof.
  • a non-therapeutic in vitro method for synergistically inhibiting the growth of tumor cells including the steps of: adding the active ingredient combination and/or third aspect of the second aspect of the present invention to a tumor cell culture system.
  • the pharmaceutical composition of the aspect thereby synergistically inhibiting the growth of tumor cells.
  • the method includes: mixing and culturing tumor cells with interferon and a proto-oncogene product targeting inhibitor.
  • the tumor cells include tumor cells in the logarithmic growth phase.
  • the number of said tumor cells is 10 3 -10 8 cells/ml.
  • the culture time is 0.1-120 hours, preferably 1-96 hours.
  • the final concentration of interferon in the mixed culture solution is 100-1000 ⁇ g/ml, preferably 200-500 ⁇ g/ml, more preferably about 300 ⁇ g/ml.
  • the final concentration of the proto-oncogene product targeting inhibitor in the mixed culture medium is 0.5-50 ⁇ g/ml, preferably 1-25 ⁇ g/ml, more preferably 2-15 ⁇ g/ml, such as about 3 ⁇ g/ml .
  • the mass ratio of the interferon and the targeted inhibitor of the proto-oncogene product is 1:100 to 500:1, preferably 1:20 to 200:1, more preferably 1: 10 to 100:1 or 1:5 to 50:1.
  • the ratio (IU/mg) of the said interferon and the targeted inhibitor of the proto-oncogene product is 1000 IU/mg to 100 million IU/mg, preferably 10000 IU/mg to 10 million IU/mg, more preferably 100,000 IU/mg to 5 million IU/mg or 200,000 IU/mg to 3 million IU/mg.
  • a method for treating tumors comprising the steps of: administering the active ingredient combination according to the second aspect of the present invention and/or the drug combination according to the third aspect to a subject (such as a human) in need To treat tumor cells.
  • a method for reducing the toxicity of a targeted inhibitor of proto-oncogene products comprising the steps of: administering PEG interferon to a subject (such as a human) in need, wherein PEG interferon is administered ) Interferon is performed before, during, and/or after the subject is administered the targeted inhibitor of the proto-oncogene product.
  • the toxic and side effects are selected from the following group: weight loss, gastrointestinal reactions (e.g., nausea, vomiting, loss of appetite), fatigue, skin rash, erythema.
  • Figure 1 shows the efficacy of PEG interferon, Avastin, or both, on subcutaneously transplanted tumors in nude mice with human gastric cancer NCI-N87.
  • Figure 2 shows the effect of PEG interferon, Avastin alone or both on the body weight of NCI-N87 tumor-bearing nude mice
  • Figure 3 shows the efficacy of PEG interferon, Avastin, or both, on subcutaneously transplanted tumors in nude mice with human gastric cancer NCI-N87.
  • Figure 4 shows the efficacy of PEG interferon, Avastin alone or a combination of the two on human liver cancer Huh-7 subcutaneously transplanted tumors in nude mice
  • FIG. 5 shows the effect of PEG interferon, Avastin alone or both on the body weight of Huh-7 tumor-bearing nude mice
  • Figure 6 shows the efficacy of PEG interferon, Avastin, or both, on subcutaneously transplanted tumors of human liver cancer Huh-7 in nude mice.
  • Figure 7 shows the effect of PEG interferon, Herceptin alone or both on subcutaneous transplanted tumors of human breast cancer BT-474 in nude mice
  • Figure 8 shows the effect of PEG interferon, Herceptin alone or both on the body weight of BT-474 tumor-bearing nude mice.
  • Figure 9 shows the efficacy of PEG interferon, Herceptin alone or both on subcutaneous transplanted tumors of human breast cancer BT-474 in nude mice. Top photo: D20; bottom photo: D60.
  • Figure 10 shows that the combination of interferon and Herceptin has a synergistic inhibitory activity on the proliferation of human breast cancer BT-474 cells cultured in vitro, and the inhibitory effect is significantly improved.
  • the combination index (CI value) is 0.31, indicating a synergistic effect effect.
  • PEG interferon and proto-oncogene product targeted inhibitors have a good synergistic effect in the treatment of tumors (especially breast cancer and gastric cancer).
  • the combined use of PEG interferon and proto-oncogene product targeted inhibitors can effectively inhibit tumor growth, and its effect is far superior to the additive effect of the two, and can significantly reduce the side effects of using interferon (especially high-dose Side effects caused by prolonged use of interferon drugs).
  • the present invention has been completed.
  • first active ingredient refers to PEG interferon, or PEG interferon. It should be understood that the term includes derivatives with the same function as PEG interferon.
  • the term "second active ingredient” refers to proto-oncogene product targeted inhibitors, especially small molecule targeted inhibitors and antibody-based targeted inhibitors.
  • the proto-oncogene product is a protein, such as EGFR protein, Her2 protein and the like.
  • active ingredient combination of the present invention As used herein, the terms “active ingredient combination of the present invention”, “material combination of the present invention”, and “pharmaceutical ingredient combination of the present invention” are used interchangeably and refer to the combination of the above-mentioned first active ingredient and second active ingredient.
  • the term "pharmaceutical composition of the present invention” refers to a composition comprising or containing a first active ingredient (or a first preparation containing the first active ingredient) and a second active ingredient (or a preparation containing the second active ingredient)
  • the composition of the second formulation wherein the first formulation and the second formulation may be the same or different formulations.
  • the first formulation and the second formulation may be the same formulation, or separate formulations.
  • Interferon Interferon, IFN
  • IFN Common interferon
  • Common interferon alpha was approved in 1986 as the first biological agent for the treatment of malignant tumors, and currently has a wide range of indications, including hairy cell leukemia, chronic leukemia, non-Hodg’s lymphoma, myeloma, bladder cancer, Ovarian cancer, advanced metastatic kidney cancer and pancreatic malignant endocrine tumors, melanoma, laryngeal papilloma, basal cell carcinoma and Kaposi sarcoma, etc.
  • rhIFN ⁇ 1b Common interferon ⁇ 1b
  • rhIFN ⁇ 1b is composed of 166 amino acids and has a relative molecular weight of 19.382kDa. It has broad-spectrum antiviral, anti-tumor and immunomodulatory functions. The results of clinical research conducted in the 1990s, as well as decades of clinical research and clinical actual use effects have proved that it has a certain therapeutic effect on certain tumors. However, treatment with interferon alone requires high doses and has greater side effects, and patient compliance is poor.
  • HuIFN-1b and HuIFN-2a 81%
  • Polyethylene Glycol is a class of linear or branched ethylene glycol polymers with an average molecular weight of more than 200Da. It has a large molecular weight, safety, non-toxicity, amphipathic, immunological inertness and structural stability. And other characteristics. Polyethylene glycol chemical modification of protein can prolong its half-life in vivo, improve stability, reduce immunogenicity and antigenicity, etc., without changing the amino acid composition of the protein. It is a simple and effective way to improve the pharmacodynamic properties of protein drugs. means.
  • a preferred PEG interferon ⁇ 1b is a recombinant human interferon ⁇ 1b modified with PEG of about 10,000-100,000 Daltons (e.g., about 20,000 daltons). Particularly preferred is recombinant human interferon ⁇ 1b modified with PEG with a molecular weight of about 20,000 Daltons, which is a new type of long-acting interferon ⁇ 1b drug (see Chinese Patent ZL200410067652.3).
  • PEG interferon ⁇ 1b can inhibit the proliferation of tumor cells.
  • PEG interferon ⁇ 1b can have a significant effect on subcutaneous transplanted tumors in mice.
  • PEG interferon ⁇ 1b can inhibit tumor cell proliferation and exert its anti-tumor effect by activating the JAK/STAT signal pathway. It can inhibit the growth of vascular endothelial cells and promote their apoptosis by inhibiting the expression of tumor angiogenic factors.
  • the combination of PEG interferon ⁇ 1b and the monoclonal antibody drug Avastin can significantly increase the tumor inhibition rate against human gastric cancer NCI-N87 and other tumor cells, indicating that PEG interferon ⁇ 1b can treat Avastin Human gastric cancer subcutaneously transplanted tumors in nude mice have a synergistic effect.
  • the combined use of PEG interferon ⁇ 1b and the monoclonal antibody drug Herceptin can significantly improve the tumor inhibition rate of human breast cancer BT-474 and other tumor cells, indicating that PEG interferon ⁇ 1b has an effect on Herceptin.
  • the subcutaneous transplanted tumors in nude mice for the treatment of human breast cancer have a synergistic effect.
  • first active ingredient PEG interferon and the second active ingredient proto-oncogene product targeted inhibitors such as Avastin or Herceptin, etc.
  • they can not only effectively inhibit tumors, but also significantly Reduce toxic and side effects, especially one or more adverse reactions selected from the following group: influenza-like syndrome, gastrointestinal reactions (such as nausea, vomiting, loss of appetite), fatigue, skin rash (including non-specific pruritus Skin rash), erythema, etc., weight loss.
  • the research of the present invention also shows that when PEG interferon ⁇ 1b is administered to tumor cells, it can inhibit tumor cell growth and/or migration.
  • proto-oncogene product targeting inhibitor and “proto-oncoprotein targeting inhibitor” are used interchangeably and refer to inhibitors that specifically target the encoded product (especially protein) of the proto-oncogene.
  • proto-oncoprotein refers to a protein encoded by a proto-oncogene, especially a membrane protein.
  • the proto-oncoprotein inhibitor can act by reducing the amount of proto-oncoprotein, inhibiting or blocking the activation of proto-oncoprotein, or inhibiting other molecules in the signal transduction pathway.
  • Non-limiting examples of targeted inhibitors of proto-oncogene products include (but are not limited to): inhibitors targeting EGFR, inhibitors targeting Her2, or a combination thereof.
  • proto-oncoprotein activity refers to the ability of the proto-oncoprotein to promote the formation, growth, proliferation, migration and/or invasion of cancer cells.
  • proto-oncoprotein expression refers to the formation of proto-oncoprotein gene products measured by any method known in the art, including (but not limited to): nucleic acid hybridization method, Northern blot, proto-oncoprotein Position hybridization, polymerase chain reaction amplification, reporter gene expression, etc.
  • the formation of proto-oncoprotein gene products can also be measured by any antibody-based technique, including immunohistochemistry, immunofluorescence, Western blot and ELISA.
  • targeted inhibitors include (but are not limited to): antibodies, small molecule drugs, nucleic acid drugs, CAR-T cells, and the like.
  • EGFR Epidermal growth factor receptor
  • NM_201282 transmembrane protein
  • EGF ErbB receptor
  • TGF- ⁇ ligand
  • EGFR gene is overexpressed or abnormally activated in a variety of tumor tissues, thereby enhancing tumor cell proliferation, invasion and metastasis.
  • EGFR has become a clinically proven therapeutic target for many types of tumors.
  • mAb monoclonal antibodies
  • a representative EGFR inhibitor is an anti-EGFR antibody, such as bevacizumab (avastin) or its biological analogue or its ADC.
  • Bevacizumab is an anti-EGFR monoclonal antibody.
  • the HER family of receptor tyrosine kinases are important mediators of cell growth, differentiation and survival.
  • the receptor family includes four different members: HER1 (EGFR or ErbB1), Her2 (ErbB2 or p185neu), HER3 (ErbB3) and HER4 (ErbB4 or tyro2).
  • Her2 cannot directly interact with HER activating ligands, it is quite certain that its kinase can signal Her2-containing heterodimers and increase the ligand's affinity for binding to EGFR or HER3.
  • the formation of Her2 dimers triggers a series of processes in cells, which ultimately leads to an increase in cell motility, cell survival and proliferation, and anti-apoptotic activity.
  • antibodies or other targeted inhibitors can be used to target the Her2 antigen on the surface of tumor cells to kill or inhibit cancer cells.
  • a representative Her2 inhibitor is Trastuzumab (Trastuzumab, Herceptin) or its biological analogue or its ADC.
  • Trastuzumab is a humanized anti-Her2 monoclonal antibody that binds to area IV of the surface area of Her2 cells.
  • Pertuzumab In addition to trastuzumab, Pertuzumab (Perjeta) is also a humanized Her2 specific monoclonal antibody. Unlike trastuzumab, Pertuzumab binds to area II of the Her2 cell surface area, thereby preventing Her2 from binding to other members of the HER family. Pertuzumab has clinically shown anti-ovarian cancer, non-small cell lung cancer, prostate cancer, breast cancer and gastric cancer effects.
  • RNAi siRNA
  • nucleic acid drugs targeting proto-oncogenes can also be used to target and inhibit the proto-oncogenes (such as EGFR or Her2).
  • nucleic acid drugs include (but are not limited to): RNAi, siRNA, shRNA, and their precursors, or expression vectors.
  • siRNA Small interfering RNA, siRNA refers to a small RNA molecule (approximately 21-25 nucleotides), which can be determined by Dicer (an enzyme in the RNase III family that is specific for double-stranded RNA). ) Is processed from its precursors (such as dsRNA, shRNA, etc.), and can also be synthesized by chemical methods or processed by other proteins.
  • siRNA is the main member of siRISC, which stimulates the target mRNA complementary to it to be rapidly cleaved and degraded, leading to the silencing of the target gene, thus becoming a key functional molecule in RNAi.
  • RNAi precursor refers to an RNA molecule that can be processed in mammalian cells to produce siRNA, specifically, it is selectively processed by Dicer, Ago2 or other similar proteins to produce mature siRNA, and then implement RNAi.
  • shRNA used herein is an abbreviation of short hairpin RNA, that is, “short hairpin RNA”.
  • shRNA consists of two short reverse complementary sequences, separated by a top loop sequence in the middle, forming a hairpin structure.
  • the endogenous RNA polymerase III (RNA polymerase III) promoter in the cell controls transcription, and the shRNA sequence ends Connect 5-6 Ts as the transcription terminator of RNA polymerase III.
  • siRNA small interfering RNA
  • plasmid vector When delivered to an animal, the hairpin sequence is expressed to form a "double-stranded RNA" (shRNA) with a top loop structure, which is recognized and processed by proteins such as Dicer and Ago2 in the cell to produce a functional siRNA.
  • miRNA is a type of non-coding single-stranded RNA molecule with a length of about 20-24 nucleotides encoded by endogenous genes, which participates in the regulation of the expression of a large number of genes in plants and animals. So far, more than 4,000 miRNA molecules have been found in animals, plants and viruses. Most miRNA genes exist in the genome in the form of single copy, multiple copies or gene clusters. Each miRNA can regulate multiple target genes, and several miRNAs can also participate in the regulation of the same gene, forming a complex regulatory network. It is speculated that miRNAs regulate the expression of more than half of human genes.
  • miRNA There are many forms of miRNA, the most primitive is pri-miRNA; after pri-miRNA is processed by Drosha, it becomes pre-miRNA, that is, miRNA precursor, with a length of about 50-90 nucleotides; pre-miRNA is then subjected to Dicer enzyme After digestion, it becomes a mature miRNA with a length of about 20-24 nucleotides. miRNA mainly inhibits the expression of target genes by inhibiting translation and accelerating the deadenylation of mRNA, and its mechanism is different from siRNA-mediated mRNA degradation.
  • the term "expression vector” refers to a vector capable of transferring a polynucleotide sequence of interest to a target cell.
  • Such vectors can self-replicate or integrate into host cell chromosomes (host cells include, for example, prokaryotic cells, yeast, animal cells, plant cells, insect cells, animal individuals, and plant individuals, etc.), and can be used in the multi-nuclear cells of the present invention.
  • the site of nucleotide transcription contains a promoter.
  • the expression vector may contain a structural gene and a promoter that regulates its expression, in addition, there are various regulatory elements that can function in the host cell. It is well known in the art that the type of expression vector for living organisms (such as animals) and the types of regulatory elements used can vary according to the type of host cell used.
  • the viral vector that can be used in the present invention is not particularly limited, and can be any viral vector that can take advantage of the characteristics of the virus to transmit its genome and bring genetic material into other cells for infection. It can occur in whole living organisms or in cell culture. Including lentivirus vectors, adenovirus vectors, herpes virus vectors, and poxvirus vectors.
  • a preferred expression vector is a lentiviral vector.
  • the present invention also provides a composition that can be used for synergistic treatment of tumors, which can be used to inhibit tumor growth and/or metastasis.
  • the pharmaceutical composition of the present invention includes: an effective amount of PEG interferon, an effective amount of a proto-oncogene product targeting inhibitor, and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition of the present invention may also include optional solid tumor chemotherapeutics.
  • the chemotherapeutic agents include (but are not limited to) cisplatin, paclitaxel, doxorubicin and the like.
  • the PEG interferon or proto-oncogene product targeting inhibitor of the present invention can be formulated in a non-toxic, inert and pharmaceutically acceptable carrier medium, where the pH is usually about 5-8, Preferably, the pH is about 6-8.
  • pharmaceutically acceptable carrier refers to a carrier used for the administration of a therapeutic agent, and includes various excipients and diluents.
  • the term refers to such pharmaceutical carriers: they themselves are not essential active ingredients, and there is no excessive toxicity after administration. Suitable carriers are well known to those of ordinary skill in the art.
  • the pharmaceutically acceptable carrier in the composition may contain liquid, such as water, saline, and buffer. In addition, these carriers may also contain auxiliary substances, such as fillers, lubricants, glidants, wetting or emulsifying agents, and pH buffer substances.
  • the vector may also contain a cell transfection reagent.
  • an effective amount or “effective dose” refers to an amount that can produce function or activity on humans and/or animals and/or cells and can be accepted by humans and/or animals.
  • pharmaceutically acceptable ingredients are substances that are suitable for humans and/or mammals without excessive side effects (such as toxicity, irritation, and allergic reactions), that is, substances that have a reasonable benefit/risk ratio.
  • pharmaceutically acceptable carrier refers to a carrier used for the administration of a therapeutic agent, and includes various excipients and diluents. Such carriers include (but are not limited to): saline, buffer, dextrose, water, glycerol, polysorbate, ethanol, and combinations thereof.
  • the pharmaceutical preparation should match the mode of administration, and the pharmaceutical composition of the present invention can be made into an injection form, for example, with physiological saline or an aqueous solution containing glucose and other adjuvants for preparation by conventional methods.
  • the pharmaceutical composition should be manufactured under aseptic conditions.
  • the amount of active ingredient administered is a therapeutically effective amount.
  • the pharmaceutical preparation of the present invention can also be made into a sustained-release preparation.
  • active ingredient combination of the present invention can also be used together with other therapeutic agents (such as antitumor agents or immunomodulators).
  • a safe and effective amount of active ingredient combination (including the first active ingredient (or its preparation) and/or the second active ingredient (or its preparation)) is administered to the mammal.
  • the effective amount of the first active ingredient (or its preparation) and/or the second active ingredient (or its preparation) in the active ingredient combination of the present invention may vary with the mode of administration and the severity of the tumor. .
  • the selection of the preferred effective amount can be determined by a person of ordinary skill in the art according to various factors (for example, through clinical trials). The factors mentioned include but are not limited to: pharmacokinetic parameters such as bioavailability, metabolism, half-life, etc.; tumor severity, patient's weight, patient's immune status, route of administration, etc.
  • the second active ingredient its safe and effective amount is usually at least about 10 ng/kg body weight, and in most cases not exceeding about 50 mg/kg body weight, preferably the dose is about 50 ng/kg body weight-about 10 mg/kg body weight.
  • the specific dosage should also consider factors such as the route of administration and the patient's health status, which are all within the skill range of a skilled physician.
  • the administration mode of the pharmaceutical composition of the present invention is not particularly limited, and representative examples include (but not limited to): intravenous injection, subcutaneous injection, intramuscular injection, and the like.
  • the present invention provides a medicine box, which comprises:
  • Component (1) a preparation containing PEG interferon
  • Component (2) preparations containing targeted inhibitors of proto-oncogene products
  • Component (3) Instructions.
  • the preparations containing PEG interferon include (but are not limited to): lyophilized agents, liquid preparations or intravenous injections.
  • the formulations containing targeted inhibitors of proto-oncogene products include (but are not limited to): freeze-dried agents, liquid formulations, tablets, capsules, suppositories, or intravenous injections.
  • protein preparations are usually freeze-dried preparations or injections.
  • the kit contains one or more (such as at least two) unit dosage forms containing PEG interferon and one or more (such as at least two) units containing proto-oncogene product targeting inhibitors Dosage form; preferably 4-10 each.
  • unit dosage form refers to the preparation of the composition into a dosage form required for single administration for the convenience of taking, including but not limited to various solid dosage forms (such as tablets), liquid dosage forms, capsules, and sustained-release dosage forms. Agent.
  • the instructions provided by the present invention may have the following description:
  • the method of using the kit is to use both a unit dosage form containing PEG interferon and a unit dosage form containing a proto-oncogene product targeting inhibitor.
  • the kit provided by the present invention is prepared by the following steps: a preparation containing PEG interferon and a preparation containing a proto-oncogene product targeting inhibitor are placed together with instructions to form a kit.
  • the preparation containing PEG interferon preferably contains a unit dosage form of PEG interferon
  • the preparation containing a proto-oncogene product targeting inhibitor preferably contains a unit dosage form of a proto-oncogene product targeting inhibitor.
  • At least one unit dosage form containing PEG interferon and at least one unit dosage form containing a proto-oncogene product targeting inhibitor are placed together with instructions to form a kit.
  • Pediatric (PEG) interferon and proto-oncogene product targeted inhibitors can synergistically act in anti-tumor therapy, thereby more significantly inhibiting tumor growth.
  • PEG interferon and targeted inhibitors of proto-oncogene products can significantly reduce the amount of PEG interferon and show faster It has a stronger anti-tumor therapeutic effect, so it can greatly reduce the side effects caused by long-term and/or high-dose interferon use.
  • PEG interferon and proto-oncogene product targeted inhibitors such as Herceptin
  • PEG interferon ⁇ 1b (Shanghai Institute of Biological Products Co., Ltd.), white freeze-dried powder injection, specification 100 ⁇ g/bottle (0.5ml), 100,000 IU (ie 1000IU/ ⁇ g).
  • Avastin (bevacizumab injection) (avastin) is a commercially available product.
  • Herceptin is a commercially available product.
  • the use and welfare of laboratory animals shall comply with the regulations of the International Laboratory Animal Evaluation and Accreditation Committee (AAALAC).
  • AALAC International Laboratory Animal Evaluation and Accreditation Committee
  • the health and death of animals are monitored daily. Routine inspections include observing the effects of test substances and drugs on the daily behavior of animals, such as behavioral activities, weight changes, physical signs, etc.
  • the mouse tumor inhibition experiment index is to investigate the effect of drugs on tumor growth, and the specific index is T/C% or tumor inhibition rate TGI (%).
  • the tumor diameter was measured with a vernier caliper twice a week.
  • T/C(%) (TT 0 )/(CC 0 ) ⁇ 100
  • T, C are the tumor volume at the end of the experiment; T 0 , C 0 are the tumor volume at the beginning of the experiment.
  • TGI Tumor inhibition rate
  • TGI tumor inhibition rate
  • the tumor is smaller than the initial volume, that is, when T ⁇ T 0 or C ⁇ C 0 , it is defined as partial tumor regression (PR); if the tumor disappears completely, it is defined as complete tumor regression (CR).
  • PR partial tumor regression
  • CR complete tumor regression
  • the end of the experiment (such as D21) the end of the experiment was reached, or the tumor volume reached 1500 mm 3 , the animals were killed under CO 2 anesthesia, and then the tumors were dissected and photographed.
  • PEG interferon 0.1, 1, 10mg/kg, SC, 2 times a week, 5 times in total
  • PEG interferon ⁇ 1b for injection (abbreviated as "PEG interferon"): white freeze-dried powder injection, specification 100 ⁇ g/bottle (0.5ml), batch number S20170908; stored at 2-8°C and protected from light. Produced and provided by Shanghai Institute of Biological Products Co., Ltd.
  • Anvastin (bevacizumab injection): colorless and clear liquid, 100mg (4ml)/bottle; batch number H0182B03; stored at 2-8°C and protected from light, purchased from Roche Pharmaceutical Company.
  • the concentration is 1mg/ml, prepare before use, and dilute with normal saline; the concentration of Avastin is 25mg/ml, directly dilute with normal saline to the required concentration.
  • NCI-N87 cells Human gastric cancer NCI-N87 cells were purchased from American Type Culture Collection. NCI-N87 cells are cultured adherently in a 10-cm petri dish. The culture conditions are RPMI 1640 medium with 10% fetal bovine serum, penicillin and streptomycin, and culture in an incubator at 37°C and 5% CO 2 air. . Passage 2-3 times a week, when the cells are in the exponential growth phase, trypsinize, collect the cells, count, and inoculate.
  • BALB/c-Foxn1 nu /Gpt, 4-8 weeks, ⁇ purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. Production license number: SCXK (Su) 2018-0008; animal certificate number 201900949. Feeding environment: SPF level.
  • Nude mice were inoculated subcutaneously (SC) with 6 ⁇ 10 6 human gastric cancer NCI-N87 cells. After the tumor grew to 100-150 mm 3 , the animals were divided into groups according to the tumor volume (D0). Mice were administered by subcutaneous injection (SC) or intravenous injection (IV) twice a week (BIW); the administration volume was 10 mL/kg; the solvent group was given the same volume of "solvent" (physiological saline); the specific dosage and The dosage regimen is shown in Table 1. The tumor volume was measured twice a week, the mice were weighed, and the data was recorded.
  • SC subcutaneous injection
  • IV intravenous injection
  • Table 1 The tumor volume was measured twice a week, the mice were weighed, and the data was recorded.
  • D0 Time of first administration; doubled the first dose of Avastin; SC: subcutaneous injection; IV: intravenous injection; P value refers to the comparison with solvent; *P ⁇ 0.05, **P ⁇ 0.01, and Pei (PEG) Comparison of interferon 1mg/kg + Avastin 10mg/kg dose group.
  • PEG interferon (0.1, 1, 10mg/kg, SC, 2 times a week, 6 times in total) dose-dependently inhibited the growth of human gastric cancer NCI-N87 subcutaneously transplanted tumors in nude mice, and the tumor inhibition rates were respectively 19%, 32% and 55%; (10mg/kg, IV, 2 times a week, 6 times in total)
  • the tumor inhibition rate of NCI-N87 subcutaneous transplantation tumor is 49%.
  • the tumor-bearing mice showed good tolerance to the drug when used in combination, and no symptoms such as weight loss occurred.
  • PEG interferon ⁇ 1b (1 mg/kg, SC, 2 times a week, 6 times in total) is used in combination with Avastin (10 mg/kg, IV, 2 times a week, 6 times in total)
  • Avastin 10 mg/kg, IV, 2 times a week, 6 times in total
  • the body weight of the mice in the group was basically the same as that in the perinterferon group (1 mg/kg) and the Avastin group (10 mg/kg).
  • PEG interferon 0.1, 1, 10 mg/kg, SC, 2 times a week, 6 times in total
  • dose-dependently inhibited human gastric cancer NCI-N87 nude mice subcutaneously
  • the growth of transplanted tumors; (10mg/kg, IV, 2 times a week, 6 times in total) is also effective for NCI-N87 subcutaneous transplantation tumor.
  • PEG interferon When used in combination, PEG interferon has a significant synergistic effect on Avastin in the treatment of NCI-N87 nude mice subcutaneously transplanted tumors (P ⁇ 0.05, compared with PEG interferon or Avastin monotherapy) . In addition, when used in combination, the toxicity did not increase, and the tumor-bearing mice showed good tolerance to the drug.
  • PEG interferon 0.1, 1, 10mg/kg, SC, 2 times a week, 5 times in total
  • PEG interferon ⁇ 1b for injection (referred to as "PEG interferon"): the same as in Example 1.
  • Anvastin (bevacizumab injection): Same as Example 1.
  • the concentration is 1mg/ml, prepare before use, and dilute with normal saline; the concentration of Avastin is 25mg/ml, directly dilute with normal saline to the required concentration.
  • Huh-7 cells Human liver cancer Huh-7 cells were purchased from the Cell Bank of the Chinese Academy of Sciences. Huh-7 cells are cultured adherently in a 10-cm petri dish. The culture conditions are RPMI 1640 medium plus 10% fetal bovine serum, penicillin and streptomycin, and culture in an incubator at 37°C and 5% CO 2 air. . Passage 2-3 times a week, when the cells are in the exponential growth phase, trypsinize, collect the cells, count, and inoculate.
  • BALB/c-Foxn1nu/Nju, 6-7 weeks, ⁇ purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. Production license number: SCXK (Su) 2018-0008; animal certificate number 201900949. Feeding environment: SPF level.
  • mice were subcutaneously inoculated with 6 ⁇ 10 6 human liver cancer Huh-7 cells. After the tumor grew to 100-150 mm 3 , the animals were divided into groups according to the tumor volume (D0). Mice were administered by subcutaneous injection (SC) or intravenous injection (IV) twice a week (BIW); the administration volume was 10 mL/kg; the solvent group was given the same volume of "solvent" (physiological saline); the specific dosage and The dosage regimen is shown in Table 2. The tumor volume was measured twice a week, the mice were weighed, and the data was recorded.
  • SC subcutaneous injection
  • IV intravenous injection
  • Table 2 The tumor volume was measured twice a week, the mice were weighed, and the data was recorded.
  • D0 time of first administration; doubled the first dose of Avastin; SC: subcutaneous injection; IV: intravenous injection; P value refers to the comparison with solvent; *P ⁇ 0.05, compared with PEG interferon 1mg/kg+A Vitin 10mg/kg dose group comparison.
  • PEG interferon (0.1, 1, 10 mg/kg, SC, 2 times a week, 5 times in total) has no significant inhibitory effect on the growth of human liver cancer Huh-7 subcutaneous xenograft tumors in nude mice.
  • the tumor inhibition rates are- 10%, -7% and 9%; (10mg/kg, IV, 2 times a week, 5 times in total)
  • the tumor inhibition rate of Huh-7 subcutaneous transplantation tumor is 40%.
  • PEG interferon (1mg/kg, SC, 2 times a week, 5 times in total) and (10mg/kg, IV, 2 times a week, 5 times in total) when combined, the tumor inhibition rate of Huh-7 increased to 49%.
  • Pei (PEG) interferon has a certain synergistic effect on Avastin in the treatment of human liver cancer Huh-7 nude mice subcutaneously transplanted tumors.
  • the tumor-bearing mice showed good tolerance to the drug when used in combination, and no symptoms such as weight loss occurred.
  • PEG interferon ⁇ 1b (1 mg/kg, SC, 2 times a week, 6 times in total) is used in combination with Avastin (10 mg/kg, IV, 2 times a week, 6 times in total)
  • Avastin 10 mg/kg, IV, 2 times a week, 6 times in total
  • the body weight of the mice in the group was not significantly different from the body weight of the mice in the perinterferon group (1 mg/kg) and the Avastin group (10 mg/kg).
  • the body weight of the combination group was higher than that of Avastin alone (10 mg/kg), which suggests that the combined use can further reduce the side effects of Avastin administration.
  • PEG interferon 0.1, 1, 10 mg/kg, SC, 2 times a week, 5 times in total
  • Huh-7 subcutaneous transplantation tumors Obviously inhibiting effect; (10mg/kg, IV, 2 times a week, 5 times in total) is effective for Huh-7 subcutaneous transplantation tumor.
  • Pei (PEG) interferon When used in combination; Pei (PEG) interferon has a synergistic effect on the treatment of human liver cancer Huh-7 subcutaneous transplanted tumors in nude mice with Avastin. In addition, when used in combination, the toxicity did not increase, and the tumor-bearing mice showed good tolerance to the drug.
  • PEG interferon PEG interferon ⁇ 1b
  • Herceptin Herceptin on human breast cancer BT-474 nude mice subcutaneously transplanted tumors.
  • PEG interferon 0.1, 1, 10mg/kg, SC, 2 times a week, 6 times in total
  • 5mg/kg, IV, 2 times a week, 6 times in total or PEG interferon (1mg/kg, SC, 2 times a week, 6 times in total) and (5mg/kg, IV, 2 times a week, 6 times in total) combined use.
  • PEG interferon ⁇ 1b for injection (referred to as "PEG interferon"): the same as in Example 1.
  • Herceptin (trastuzumab for injection), white to light yellow loose lumps, specification 440mg (20ml)/bottle, batch number/diluent batch number N3886/B3083; production date 20170220(product)/20171118 (Dilution solution), valid until 20200219. Store at 2-8°C in the dark.
  • PEG interferon was provided by Shanghai Institute of Biological Products Co., Ltd.; Herceptin was purchased from Roche Pharmaceuticals.
  • PEG interferon add 120 ⁇ l of sterile water for injection to each bottle of PEG interferon, the concentration is 1mg/ml, prepare before use; dilute with normal saline;
  • Herceptin first reconstituted with the solvent provided by the original research manufacturer, after dissolution, it is colorless to light yellow, clear to slightly opalescent solution, and the concentration after reconstitution is 21mg/ml; immediately aliquot at -70 ⁇ -80°C Frozen. Dilute with normal saline to the required concentration before use.
  • BT-474 cells Human breast cancer BT-474 cells were purchased from American Type Culture Collection. BT-474 cells were cultured adherently in a 10-cm petri dish. The culture conditions were DMEM medium (Gibco) with 10% fetal bovine serum (Gibco), penicillin and streptomycin, at 37°C, 5% CO 2 air In the incubator (Thermo). Passage 2-3 times a week, when the cells are in the exponential growth phase, trypsinize, collect the cells, count, and inoculate.
  • BALB/cJGpt-Foxn1nu/Gpt, 5 weeks, ⁇ purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. Production License Number: SCXK (Su) 2018-0008 Animal Certificate Number 201901062. Feeding environment: SPF level.
  • mice were subcutaneously inoculated with 1 ⁇ 10 7 human breast cancer BT-474 cells. After the tumor grew to 100-150 mm 3 , the animals were divided into groups according to the tumor volume (D0). Mice were administered by subcutaneous injection (SC) or intravenous injection (IV) twice a week (BIW); the administration volume was 10 mL/kg; the solvent group was given the same volume of "solvent" (physiological saline); the specific dosage and The dosage regimen is shown in Table 3. The tumor volume was measured twice a week, the mice were weighed, and the data was recorded.
  • SC subcutaneous injection
  • IV intravenous injection
  • Table 3 The tumor volume was measured twice a week, the mice were weighed, and the data was recorded.
  • D0 time of first administration; double the first dose of Herceptin; SC: subcutaneous injection; IV: intravenous injection; P value refers to the comparison with solvent; **P ⁇ 0.01, 1mg/kg+ with PEG interferon Comparison of Herceptin 5mg/kg dose group.
  • PEG interferon (0.1, 1, 10 mg/kg, SC, 2 times a week, 6 times in total) dose-dependently inhibited the growth of human breast cancer BT-474 subcutaneously transplanted tumors in nude mice, and the tumor inhibition rates were respectively 66%, 98% and 156%, 1mg/kg group had 3/6 tumor partial regression, 10mg/kg group had 4/6 tumor partial regression and 2/6 tumor complete regression (D20), until the end of the experiment (D60 ), 4/6 tumors in the 10mg/kg group still partially regressed; (5mg/kg, IV, 2 times a week, 6 times in total), the tumor inhibition rate of BT-474 subcutaneously transplanted tumor is 57% (D20).
  • PEG interferon (1mg/kg, SC, 2 times a week, 6 times in total) and (5mg/kg, IV, 2 times a week, 6 times in total)
  • the tumor inhibition rate of BT-474 was significantly increased to 170% (P ⁇ 0.01, compared with single agent), and 3/6 tumors partially resolved And 3/6 tumors completely resolved (D20); to the end of the experiment (D60), there were still 3/6 tumors partially resolved and 3/6 tumors completely resolved; this shows that PEG interferon combined with Herceptin treatment of human breast
  • the tumor BT-474 subcutaneously transplanted in nude mice has a very significant synergistic effect, and even achieves a partial or complete treatment effect.
  • the tumor-bearing mice showed good tolerance to the drug when used in combination, and no symptoms such as weight loss occurred.
  • Pei (PEG) interferon ⁇ 1b (1mg/kg, SC, 2 times a week, 6 times in total) and (5mg/kg, IV, 2 times a week, 6 times in total)
  • the body weight of the mice in the combination group was not different from the body weight of the mice in the Peinterferon alone group and the Herceptin alone group.
  • PEG interferon 0.1, 1, 10 mg/kg, SC, 2 times a week, 6 times in total
  • PEG interferon (1mg/kg, SC, 2 times a week, 6 times in total) and (5mg/kg, IV, 2 times a week, 6 times in total) has a significant synergistic effect on the curative effect of BT-474 tumor (P ⁇ 0.05, compared with single agent).
  • the toxicity did not increase, and the tumor-bearing mice showed good tolerance to the drug.
  • SRB method The sulforhodamine B protein staining method (SRB method) was used to evaluate the effect of interferon alone or in combination with other drugs on the proliferation of cultured tumor cells in vitro.
  • Inhibition rate (OD value control hole-OD value administration hole)/OD value control Hole ⁇ 100%
  • IFN- ⁇ 1b in 600 ⁇ g interferon is 300 ⁇ g (about 300,000 IU), and so on.
  • the concentration of interferon when used in combination, when the concentration of interferon is about 9.38-600 ⁇ g/ml and the concentration of Herceptin is 0.05-3 ⁇ g/ml, a significant synergistic inhibitory effect can be observed.
  • the inhibition rate of 150 ⁇ g/ml interferon + 0.75 ⁇ g/ml Herceptin in combination is significantly higher than that of 300 ⁇ g/ml alone or 1.5 ⁇ g/ml Herceptin.
  • Interferon is a class of highly active protein molecules with broad-spectrum antiviral proliferation, anti-tumor, immune regulation and other activities.
  • common interferon has been approved by the US FDA and European EMA for the treatment of hairy cell leukemia, chronic myelogenous leukemia, kidney cancer, Carpos sarcoma, melanoma, follicular lymphoma, multiple myeloma, and cervical cancer.
  • Intraepithelial tumor (Elena Garcia-Martinez, etc. Trial Watch: Immunostimulation with recombinant cytokines for cancer therapy, ONCOIMMUNOLOGY, 2018, VOL. 7, NO.
  • interferon Although ordinary interferon is a safe and effective preparation, there are still some problems, especially serious side effects.
  • the common interferon currently used clinically has a small molecular weight (below 20kDa) and is easily filtered by the glomerulus, resulting in a half-life of only 1.5-2 hours in the human body.
  • common clinical interferon treatment is generally used.
  • the use of high-dose, multiple injections, the patient's compliance is poor, and it is easy to cause side effects.
  • the adverse reactions of common interferon alpha in the human body are systemic (Cao Xuelin, Adverse Reaction Mechanisms of Interferon, Biomedical Engineering and Clinics, 2010, Volume 14, Issue 4: 340-342): In the early stage of treatment, most patients will appear Influenza-like syndrome, the clinical symptoms include fever, chills, headache, myalgia, gastrointestinal pain, etc. Nausea, vomiting, and loss of appetite are common gastrointestinal reactions in the early stage of ordinary interferon alpha treatment. In ordinary interferon alpha treatment, more than 70% of patients will experience fatigue. The main adverse reactions of the neuropsychiatric system are mental disorders, depression, trance, anxiety, irritability and so on.
  • Approximately 20% of patients receiving common interferon alpha treatment may have mild to moderate reductions in the total number of white blood cells, neutrophils, and platelets.
  • Skin and mucosal lesions caused by common interferon alpha are the most common skin and mucosal lesions, which are often non-specific rashes with itching; other manifestations include erythema at the injection site, skin ulcers, oral mucosal ulcers, and lip inflammation.
  • Common interferon alpha therapy can cause thyroid dysfunction through autoimmune and non-autoimmune mechanisms, and hypothyroidism is the most common thyroid dysfunction.
  • Common interferon alpha can also induce autoimmune damage to pancreatic beta cells and induce diabetes. Other relatively rare adverse reactions include interstitial pneumonia, retinopathy, hearing loss and tinnitus, hair loss, diarrhea, and weight loss.
  • Human interferon ⁇ 1b is a protein composed of 166 amino acids produced by human white blood cells, and its relative molecular weight is 19.382kDa. Studies have shown that after the Chinese leukocytes are attacked by the virus, the interferon ⁇ 1b is the main type of interferon produced.
  • ordinary interferon ⁇ 1 Compared with ordinary interferon ⁇ 2 products, ordinary interferon ⁇ 1 has relatively low in vitro biological activity, but it has a stronger effect in the body and has a wide range and small side effects.
  • the specific activity of common interferon ⁇ 2a and 2b in vitro is 1 ⁇ 10 8 IU/mg, while the specific activity of common interferon ⁇ 1b is only 1 ⁇ 10 7 IU/mg, which is 10% of the former two.
  • phase I and phase II clinical trials conducted in the United States showed that high-dose (500 ⁇ g/day) common interferon alpha 1b injection has a certain curative effect in the treatment of patients with metastatic renal cell carcinoma and multiple myeloma, and the adverse reactions are similar to other common ones.
  • Interferon has milder adverse reactions than interferon (P.Masci, etc. Gene Modulatory Effects, Pharmacokinetics, and Cl inical Tolerance of Interferon ⁇ -1b: A Second Member of the Interferon ⁇ Family.Clinical Pharmacology&Therapeutics.2007.Vol.81, No.3 :354-361).
  • Common interferon ⁇ 1b like other common interferons, also has the defect that it has a small molecular weight, is easily filtered by the glomerulus, and has a short half-life in the human body.
  • the second-generation polyethylene glycol modifier was used to modify the common interferon ⁇ 1b, and a single PEG-modified interferon ⁇ 1b was obtained, which was named PEG interferon ⁇ 1b.
  • PEG interferon ⁇ 1b is used as a single agent or in combination with other drugs for preclinical in vitro and in vivo pharmacodynamic studies.
  • PEG interferon alpha 1b has a certain inhibitory effect on breast cancer, lung cancer, liver cancer and gastric cancer cells (19.6%-58.4% inhibition rate).
  • PEG interferon ⁇ 1b When PEG interferon ⁇ 1b is used in combination with Avastin, the therapeutic effect is obviously enhanced in the subcutaneous transplanted tumor model of human gastric cancer NCI-N87 nude mice. At the same time, it also shows that the synergistic effect produced by the combination with monoclonal antibodies can greatly reduce the dosage of PEG interferon ⁇ 1b (a 10-fold reduction), which provides a better way for the combination of PEG interferon ⁇ 1b and monoclonal antibodies and other anti-tumor drugs. Large range of dosage options.
  • the PEG interferon ⁇ 1b single-use group did not show obvious anti-tumor effect. It can be seen that PEG interferon ⁇ 1b has anti-tumor activity, but for different tumors. There are still some or greater differences in its efficacy. However, when peginterferon is used in combination with Avastin, it shows a certain synergistic effect.
  • PEG interferon ⁇ 2a and ⁇ 2b have obvious advantages over common interferon in chronic hepatitis B and C, they have achieved great success. But in the field of tumor treatment, it is completely different.
  • the study of the present invention shows that the combination of PEG interferon ⁇ 1b and Avastin or Herceptin can synergistically inhibit tumors at the same time, It also significantly reduces the incidence or degree of respective side effects (such as weight loss, fever, adverse gastrointestinal reactions, skin rash, etc.).

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Abstract

培干扰素和原癌基因产物靶向抑制剂在协同治疗肿瘤中的应用。具体地，提供了一种活性成分组合的用途，所述活性成分组合包括培（PEG）干扰素和原癌基因产物靶向抑制剂，并且所述组合用于制备协同治疗肿瘤的药物组合物。该活性成分组合能够有效地协同抑制肿瘤（尤其是肺癌、胃癌、乳腺癌、肝癌）的生长，并且可显著降低毒副作用，因此可在靶向治疗肿瘤中得到广泛应用。

Description

培干扰素和原癌基因产物靶向抑制剂在协同抑制肿瘤中的应用 技术领域
本发明涉及抗肿瘤药物技术领域，更具体地涉及培干扰素和原癌基因产物靶向抑制剂在协同抑制肿瘤中的应用及其相关药物。
背景技术
肿瘤是一种多因素参与、多步骤发展的全身性、系统性疾病。肿瘤具有高度复杂性、可变性和多样性的生物学特征。癌症相关基因的异常可以引起肿瘤基因组稳定性丧失，进而导致肿瘤细胞出现异常的生物学行为，如逃避凋亡、无限复制的潜能、血管生成、侵袭及转移和免疫逃逸等。
目前，外科手术、放疗、化疗是治疗恶性肿瘤的主要方法。外科手术和放射治疗是局部治疗的方法，药物治疗属于全身效应的方法，更多地考虑到了恶性肿瘤能够扩散和转移的特质。
传统的肿瘤治疗方式包括外科手术、放疗、化疗，显示出较好疗效但均存在诸多弊端。近年来兴起的肿瘤免疫疗法较传统肿瘤治疗手段明显提高患者的生存质量。
然而，临床治疗效果不佳以及耐药性的频繁出现，促使人们需要开发更有效的治疗方案。此外，一些抗肿瘤药物的毒副作用很大，导致病人不得不放弃治疗。
因此，本领域迫切需要开发新的有效且毒副作用低的治疗肿瘤的治疗方法和药物。
发明内容
本发明的目的就是提供了一种有效且毒副作用低的治疗肿瘤的治疗方法和药物，具体地本发明提供了培(PEG)干扰素与原癌基因产物靶向抑制剂在协同治疗肿瘤的用途。
在本发明的第一方面，提供了一种活性成分组合的用途，所述活性成分组合包括第一活性成分培干扰素和第二活性成分原癌基因产物靶向抑制剂，并且所述组合用于制备协同治疗肿瘤的药物组合物或药盒。
在另一优选例中，所述的原癌基因产物靶向抑制剂为抗体。
在另一优选例中，所述的培干扰素为培干扰素α1b。
在另一优选例中，所述的培干扰素包括1-10万分子量PEG修饰的重组人干扰素α1b，较佳地用1-5万分子量的PEG修饰的重组人干扰素α1b。
在另一优选例中，所述的培干扰素和原癌基因产物靶向抑制剂的质量比为1:100至500:1，较佳地1:20至200:1，更佳地1:10至100:1或1:5至50:1。
在另一优选例中，所述的培干扰素和原癌基因产物靶向抑制剂的之比(IU/mg)为1000IU/mg至10000万IU/mg，较佳地10000IU/mg至1000万IU/mg，更佳地10万IU/mg至500万IU/mg或20万IU/mg至300万IU/mg。
在另一优选例中，所述的药物组合物或药盒用于治疗哺乳动物或施用于哺乳动物，更佳地所述哺乳动物为啮齿类动物(如小鼠、大鼠)或人。
在另一优选例中，所述的肿瘤选自下组：肺癌、胃癌、乳腺癌、肝癌、肠癌、卵巢癌、子宫颈癌、或其组合。
在另一优选例中，所述的肿瘤选自下组：肺癌、胃癌、乳腺癌、或其组合。
在另一优选例中，所述的原癌基因产物靶向抑制剂选自下组：EGFR抑制剂、Her2抑制剂、或其组合。
在另一优选例中，所述的原癌基因产物靶向抑制剂选自下组：EGFR抑制剂、Her2抑制剂、或其组合。
在另一优选例中，所述的原癌基因产物靶向抑制剂选自下组：安维汀、赫赛汀、帕妥珠单抗、或其组合。
在本发明的第二方面，提供了一种药物组合物，所述的药物组合物包括培干扰素、原癌基因产物靶向抑制剂和药学上可接受的载体。
在另一优选例中，所述的药物组合物还包括额外的治疗药物(如抗肿瘤剂)。
在另一优选例中，所述的原癌基因产物靶向抑制剂选自下组：EGFR抑制剂、Her2抑制剂、或其组合。
在另一优选例中，所述的原癌基因产物靶向抑制剂选自下组：安维汀、赫赛汀、帕妥珠单抗、或其组合。
在另一优选例中，所述的药物组合物还包括化疗剂、检测点抑制剂、CAR-T细胞、或其组合。
在另一优选例中，所述的化疗剂包括顺铂、紫杉醇。
在本发明的第三方面，提供了一种活性成分组合，所述组合由培干扰素和原癌基因产物靶向抑制剂构成。
在另一优选例中，所述的原癌基因产物靶向抑制剂选自下组：EGFR抑制剂、Her2抑制剂、或其组合。
在本发明的第四方面，提供了一种药盒，所述药盒包括：
(a)第一制剂，所述第一制剂含有培干扰素以及药学上可接受的载体；
(b)第二制剂，所述第二制剂含有原癌基因产物靶向抑制剂以及药学上可接受的载体；
(c)说明书，所述说明书描述了将培干扰素和原癌基因产物靶向抑制剂联用 以治疗肿瘤的方法。
在另一优选例中，所述的第一制剂和第二制剂是各自独立的。
在另一优选例中，所述的第一制剂和第二制剂为冻干制剂或液态制剂。
在另一优选例中，所述的第一制剂和第二制剂是注射剂。
在另一优选例中，所述的第一制剂在施用第二制剂之前、之中或之后被施用。
在另一优选例中，所述的原癌基因产物靶向抑制剂选自下组：EGFR抑制剂、Her2抑制剂、或其组合。
在另一优选例中，所述的原癌基因产物靶向抑制剂选自下组：安维汀、赫赛汀、帕妥珠单抗、或其组合。
在本发明的第五方面，提供了一种体外非治疗性的协同抑制肿瘤细胞生长的方法，包括步骤：向肿瘤细胞培养体系中加入本发明第二方面所述活性成分组合和/或第三方面所述的药物组合物，从而协同抑制肿瘤细胞生长。
在另一优选例中，所述方法包括：将肿瘤细胞和培干扰素及原癌基因产物靶向抑制剂一起混合培养。
在另一优选例中，所述的肿瘤细胞包括处于对数生长期的肿瘤细胞。
在另一优选例中，所述的肿瘤细胞的数量为10 3-10 8个/ml。
在另一优选例中，所述的培养时间为0.1-120小时，较佳地1-96小时。
在另一优选例中，混合培养液中培干扰素的终浓度为100-1000μg/ml，较佳地200-500μg/ml，更佳地约300μg/ml。
在另一优选例中，混合培养液中原癌基因产物靶向抑制剂的终浓度为0.5-50μg/ml，较佳地1-25μg/ml，更佳地2-15μg/ml，如约3μg/ml。
在另一优选例中，所述的培干扰素和原癌基因产物靶向抑制剂的质量比为1:100至500:1，较佳地1:20至200:1，更佳地1:10至100:1或1:5至50:1。
在另一优选例中，所述的培干扰素和原癌基因产物靶向抑制剂的之比(IU/mg)为1000IU/mg至10000万IU/mg，较佳地10000IU/mg至1000万IU/mg，更佳地10万IU/mg至500万IU/mg或20万IU/mg至300万IU/mg。
在本发明的第六方面，提供了一种治疗肿瘤的方法，包括步骤：向需要的对象(如人)施用本发明第二方面所述活性成分组合和/或第三方面所述的药物组合物，从而治疗肿瘤细胞。
在本发明的第七方面，提供了一种降低原癌基因产物靶向抑制剂的毒性的方法，包括步骤：向需要的对象(如人)施用培(PEG)干扰素，其中施用培(PEG)干扰素在所述对象施用所述原癌基因产物靶向抑制剂的之前、之中和/或之后进行。
在另一优选例中，所述的毒副作用选自下组：体重下降、胃肠道反应(例如 恶心、呕吐、食欲不振)、疲劳、皮疹、红斑。
应理解，在本发明范围内，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了培(PEG)干扰素、安维汀单用或二者合用对人胃癌NCI-N87裸小鼠皮下移植瘤的疗效。
图2显示了培(PEG)干扰素、安维汀单用或二者合用对NCI-N87荷瘤裸小鼠体重的影响
图3显示了培(PEG)干扰素、安维汀单用或二者合用对人胃癌NCI-N87裸小鼠皮下移植瘤的疗效。
图4显示了培(PEG)干扰素、安维汀单用或二者合用对人肝癌Huh-7裸小鼠皮下移植瘤的疗效
图5显示了培(PEG)干扰素、安维汀单用或二者合用对Huh-7荷瘤裸小鼠体重的影响
图6显示了培(PEG)干扰素、安维汀单用或二者合用对人肝癌Huh-7裸小鼠皮下移植瘤的疗效。
图7显示了培(PEG)干扰素、赫赛汀单用或二者合用对人乳腺癌BT-474裸小鼠皮下移植瘤的疗效
图8显示了培(PEG)干扰素、赫赛汀单用或二者合用对BT-474荷瘤裸小鼠体重的影响。
图9显示了培(PEG)干扰素、赫赛汀单用或二者合用对人乳腺癌BT-474裸小鼠皮下移植瘤的疗效。上图：D20；下图：D60。
图10显示了培干扰素和赫赛汀(Herceptin)合用对体外培养人乳腺癌BT-474细胞增殖有协同抑制活性，抑制作用显著提高，合用指数(CI值)为0.31，说明有协同增效作用。
具体实施方式
本发明人经过广泛而深入的研究，首次意外地发现了培(PEG)干扰素与原癌基因产物靶向抑制剂在治疗肿瘤方面(尤其是乳腺癌、胃癌)有良好的协同作用。联合运用培(PEG)干扰素与原癌基因产物靶向抑制剂能够有效抑制肿瘤的生长，其效果远优于二者的加合作用，并且可以显著降低使用干扰素的副作用(尤其是高剂量长时间使用干扰素类药物所造成的副作用)。在此基础上，完成了本发明。
术语
如本文所用，术语“第一活性成分”指培(PEG)干扰素，或培干扰素。应理解，该术语包括与培(PEG)干扰素具有相同功能的衍生物。
如本文所用，术语“第二活性成分”指原癌基因产物靶向抑制剂，尤其是小分子的靶向抑制剂和抗体类靶向抑制剂。优选地，所述的原癌基因产物为蛋白，例如EGFR蛋白、Her2蛋白等。
如本文所用，术语“本发明的活性成分组合”、“本发明的物质组合”、“本发明的药物成分组合”可互换使用，指上述第一活性成分和第二活性成分的组合。
如本文所用，术语“本发明的药物组合物”指包括或含有第一活性成分(或含所述第一活性成分的第一制剂)和第二活性成分(或含所述第二活性成分的第二制剂)的组合物，其中，所述的第一制剂和第二制剂可以是相同或不同的制剂。此外，第一制剂和第二制剂可以是同一制剂，或各自独立的制剂。
普通干扰素和培(PEG)干扰素
普通干扰素(Interferon，IFN)疗法作为肿瘤免疫疗法中的重要一员，已有多年肿瘤治疗的临床经验，大量研究表明，普通干扰素对多种肿瘤的生长具有很好的抑制作用。
普通干扰素α在1986年即获批作为首个生物制剂用于恶性肿瘤的治疗，目前有较广泛的适应症范围，包括毛细胞白血病、慢性白血病、非霍淋巴瘤、骨髓瘤、膀胱癌、卵巢癌、晚期转移性肾癌及胰腺恶性内分泌肿瘤、黑色素瘤、喉乳头状瘤、基底细胞癌和Kaposi肉瘤等。
普通干扰素α1b(rhIFNα1b)是上世纪80年代从中国健康人脐血白细胞中克隆到基因并开发而成的我国第一个基因工程蛋白药物。
rhIFNα1b由166个氨基酸组成，相对分子量为19.382kDa，具有广谱抗病毒、抗肿瘤和免疫调节功能。在上世纪90年代进行的临床研究结果和几十年来的临床研究及临床实际使用效果证明了其对于某些肿瘤有一定治疗效果。然而，单用干扰素进行治疗需要使用高剂量且副作用较大，患者依从性较差。
干扰素α亚型氨基酸序列和同源性分析
HuIFNα-1b(166aa)ACCESSION:AAL35223(SEQ ID No.:1)
Figure PCTCN2019130749-appb-000001
HuIFNα-2a(165aa)ACCESSION:1ITF_A(SEQ ID No.:2)
Figure PCTCN2019130749-appb-000002
HuIFNα-2b(165aa)ACCESSION:1RH2_A(SEQ ID No.:3)
Figure PCTCN2019130749-appb-000003
同源性分析：NCBI BLAST
HuIFN-1b与HuIFN-2a：81％
Figure PCTCN2019130749-appb-000004
HuIFN-1b与HuIFN-2b：83％
Figure PCTCN2019130749-appb-000005
聚乙二醇(Polyethylene Glycol，PEG)是一类线性或分支结构，平均分子量200Da以上的乙二醇高聚物的总称，它具有分子量大、安全无毒、两亲性、免疫惰性和结构稳定等特性。蛋白质的聚乙二醇化学修饰可以在不改变蛋白质氨基酸组成的前提下延长其体内半衰期、提高稳定性、降低免疫原性和抗原性等，是改善蛋白质类药物药效学性质的简便、有效的手段。
一种优选的培(PEG)干扰素α1b是采用约1-10万道尔顿(如约2万)的PEG修饰的重组人干扰素α1b。特别优选的是用分子量约2万道尔顿PEG修饰的重组人干扰素α1b，它是一种新型的长效化干扰素α1b药物(见中国专利 ZL200410067652.3)。在体外实验中观察到培(PEG)干扰素α1b能够对肿瘤细胞增殖产生抑制作用，在体内实验中观察到培(PEG)干扰素α1b能够对小鼠皮下移植瘤有明显疗效。
体外实验结果表明，培(PEG)干扰素α1b可以通过激活JAK/STAT信号通路，抑制肿瘤细胞增殖，发挥其抗肿瘤作用。可以通过抑制肿瘤血管生成因子的表达，来抑制血管内皮细胞的生长并促进其凋亡。
优选地，当培(PEG)干扰素α1b与单抗药物安维汀合用，可显著提高对人胃癌NCI-N87等肿瘤细胞的抑瘤率，说明培(PEG)干扰素α1b对安维汀治疗人胃癌裸小鼠皮下移植瘤有协同增效作用。
优选地，当培(PEG)干扰素α1b与单抗药物赫赛汀合用，可显著提高对人乳腺癌BT-474等肿瘤细胞的抑瘤率，说明培(PEG)干扰素α1b对赫赛汀治疗人乳腺癌的裸小鼠皮下移植瘤有协同增效作用。
此外，当第一活性成分培(PEG)干扰素和第二活性成分原癌基因产物靶向抑制剂(如安维汀或赫赛汀等)联用时，不仅可以有效抑制肿瘤，还可显著地降低毒副作用，尤其是可降低选自下组的一种或多种不良反应：流感样综合征、胃肠道反应(例如恶心、呕吐、食欲不振)、疲劳、皮疹(包括非特异伴瘙痒的皮疹)、红斑等、体重下降。
本发明的研究还表明，培(PEG)干扰素α1b施用于肿瘤细胞时，能够抑制肿瘤细胞生长和/或迁移。
原癌基因产物靶向抑制剂
如本文所用，术语“原癌基因产物靶向抑制剂”、“原癌蛋白靶向抑制剂”可互换使用，指特异性靶向原癌基因的编码产物(尤其是蛋白)的抑制剂。如本文所用，术语“原癌蛋白”指原癌基因所编码的蛋白，尤其膜蛋白。在本发明的一些实施方案中，原癌蛋白抑制剂可通过减少原癌蛋白的量、抑制或阻断原癌蛋白活化或抑制信号传导途径中的其他分子而起作用。
原癌基因产物靶向抑制剂的非限制性实例包括(但并不限于)：靶向EGFR的抑制剂、靶向Her2的抑制剂、或其组合。
如本文所用，术语“原癌蛋白活性”是指原癌蛋白表现出的促进癌细胞形成、生长、增殖、迁移和/或侵袭的能力。
如本文所用，术语“原癌蛋白表达”是指通过本领域已知的任何方法测量的原癌蛋白基因产物的形成，所述方法包括(但并不限于)：核酸杂交方法、Northern印迹、原位杂交、聚合酶链反应扩增、报告基因表达等。原癌蛋白基因产物的形成也可以通过任何基于抗体的技术测量，包括免疫组织化学、免疫荧光、Western印迹和ELISA法。
在本发明中，靶向抑制剂包括(但并不限于)：抗体、小分子药物、核酸药物、CAR-T细胞等。
EGFR及其抑制剂
表皮生长因子受体(epidermalgrowth factor receptor，EGFR)EGFR是一种跨膜蛋白质(NM_201282)，属于ErbB受体家族成员，其配体(EGF、TGF-α等)与EGFR胞外段结合使之二聚化，由此导致胞内段酪氨酸激酶活化以及一系列信号转导的级联反应，促进细胞增殖，血管生成，转移并抑制细胞凋亡，从而导致肿瘤的发生。大量研究发现EGFR基因在多种肿瘤组织中过表达或异常活化，从而使肿瘤细胞增殖、侵袭和转移能力增强。EGFR已成为经临床验证的多种类型肿瘤的治疗靶点。
在癌症靶向治疗的发展过程中，一个成功的策略是利用单克隆抗体(monoclonal antibodies，mAb)定位肿瘤细胞表面的抗原决定簇，从而靶向杀死癌细胞。因此，可以利用抗体或其他靶向抑制剂来靶向肿瘤细胞表面的EGFR来杀死或抑制癌细胞。
一种代表性的EGFR抑制剂是抗EGFR抗体，例如贝伐珠单抗(安维汀avastin)或其生物类似药或其ADC。贝伐珠单抗是一种抗EGFR单克隆抗体。
Her2及其抑制剂
受体酪氨酸激酶的HER家族是细胞生长、分化和生存的重要介体。该受体家族包括四个不同的成员：HER1(EGFR或ErbB1)、Her2(ErbB2或p185neu)、HER3(ErbB3)和HER4(ErbB4或tyro2)。
尽管Her2不能直接与HER激活性配体相互作用，但很确定的是，它的激酶能够使包含Her2的异二聚体传递信号，并且增加配体结合至EGFR或HER3的亲和性。Her2二聚体的形成触发了一系列细胞中的进程，最终导致细胞运动、细胞生存率和增殖、以及抗凋亡活性的增加。
类似地，可以利用抗体或其他靶向抑制剂来靶向肿瘤细胞表面的Her2抗原来杀死或抑制癌细胞。
一种代表性的Her2抑制剂是曲妥珠单抗(Trastuzumab，Herceptin)或其生物类似药或其ADC。曲妥珠单抗是一种人源化的抗Her2单克隆抗体，结合Her2细胞表面区域的区域Ⅳ。
除了曲妥珠单抗外，帕妥珠单抗(Pertuzumab，Perjeta)也是一种人源化的Her2特异性的单克隆抗体。与曲妥珠单抗不同的是，帕妥珠单抗结合的是Her2细胞表面区域的区域Ⅱ，从而阻止Her2与HER家族的其它成员结合。帕妥珠单抗在临床上表现出了抗卵巢癌、非小细胞肺癌、前列腺癌、乳腺癌和胃癌的 作用。
RNAi、siRNA、shRNA
在本发明中，还可以用靶向原癌基因的核酸类药物来靶向抑制所述的原癌基因(如EGFR或Her2)。
代表性的核酸类药物包括(但并不限于)：RNAi、siRNA、shRNA，及其前体、或表达载体。
如本文所用，术语“RNAi”(RNA interference，RNA干扰)是指在进化过程中高度保守的、由双链RNA(dsRNA)诱发的、高效特异性降解同源mRNA的现象。由于使用RNAi技术可以特异性剔除或关闭特定基因的表达，所以该技术已被广泛用于探索基因功能和传染性疾病及恶性肿瘤的基因治疗领域。
如本文所用，术语“siRNA”(Small interfering RNA，siRNA)是指一种小RNA分子(约21-25个核苷酸)，可由Dicer(RNA酶Ⅲ家族中对双链RNA具有特异性的酶)从其前体(比如dsRNA、shRNA等)加工而成，也可由化学方法合成或由其它蛋白加工产生。siRNA是siRISC的主要成员，激发与之互补的目标mRNA被迅速切割降解，导致目标基因的沉默，因此成为RNAi中的关键功能分子。
本文所用的术语“表达盒”是指包含本发明RNAi前体的编码序列以及与所述编码序列操作性相连的启动子和终止信号的表达盒，所述表达盒在转录后产生本发明的RNAi前体。本文所用的术语“RNAi前体”是指可以在哺乳动物细胞中加工产生siRNA的RNA分子，具体地说，是由Dicer、Ago2或其它类似蛋白选择性加工从而产生成熟的siRNA，进而实施RNAi。
本文所用的术语“shRNA”是short hairpin RNA的缩写，即，“短发夹RNA”。shRNA包括两个短反向互补序列，中间由一顶端环(loop)序列分隔的，组成发夹结构，由细胞内源的RNA聚合酶Ⅲ(RNA polymerase Ⅲ)启动子控制转录，shRNA序列的末端连接5-6个T作为RNA聚合酶Ⅲ的转录终止子。在活体中产生“小干扰RNA”(siRNA)的一种办法是，将siRNA序列作为“短发夹”的一部分克隆进质粒载体中。当送入动物体内时，该发夹序列被表达出来，形成一个带有顶端环结构的“双链RNA”(shRNA)，被细胞内的Dicer和Ago2等蛋白所识别和加工，产生有功能的siRNA。
本文所用的术语“miRNA”(microRNA)是一类由内源基因编码的长度约20-24个核苷酸的非编码单链RNA分子，在动植物中参与对大量基因的表达调控。到目前为止，在动植物以及病毒中已经发现四千多种miRNA分子。大多数miRNA基因以单拷贝、多拷贝或基因簇(cluster)的形式存在于基因组中。每种miRNA可以调控多个靶基因，而几种miRNA也可以共同参与调节同一基因，组成复杂的调节网络。据推测，miRNA调节着人类一半以上基因的表达。miRNA存 在多种形式，最原始的是pri-miRNA；pri-miRNA经过Drosha加工后，成为pre-miRNA，即miRNA前体，长度大约为50-90个核苷酸；pre-miRNA再经过Dicer酶酶切后，成为长约20-24个核苷酸的成熟miRNA。miRNA主要通过抑制翻译和加速mRNA的脱腺苷酸化抑制靶基因表达，其机制有别于siRNA介导的mRNA降解。
表达载体
如本文所用，术语“表达载体”指的是能将感兴趣的多聚核苷酸序列转移到目标细胞的载体。此类载体能自我复制或结合进宿主细胞染色体(宿主细胞有，例如原核细胞、酵母、动物细胞、植物细胞、昆虫细胞、动物个体、和植物个体等)，并可在适合本发明多聚核苷酸转录的位点含有启动子。表达载体可包含结构基因和调控其表达的启动子，此外，还有能在宿主细胞中起作用的各种调控元件。本领域熟知，生物活体(如动物)的表达载体类型及所用调控元件的种类可根据所用宿主细胞的类型而改变。
可用于本发明的病毒载体没有特别限制，可以是任何能够利用病毒具有传送其基因组的特点，将遗传物质带入其他细胞，进行感染的病毒载体。可发生于完整活体或是细胞培养中。包括慢病毒载体、腺病毒载体、疱疹病毒载体、痘病毒载体。在本发明中，一种优选的表达载体是慢病毒载体。
药物组合物以及给药方式
本发明还提供一种可用于协同治疗肿瘤的组合物，它可用于抑制肿瘤生长和/或转移。
本发明药物组合物包括：有效量的培(PEG)干扰素，和有效量的原癌基因产物靶向抑制剂，以及药学上可接受的载体。
此外，本发明药物组合物还可以包括任选的实体肿瘤化疗剂。所述的化疗剂包括(但不限于)顺铂、紫杉醇、阿霉素等。
通常，可将本发明的培(PEG)干扰素，或原癌基因产物靶向抑制剂配制于无毒的、惰性的和药学上可接受的载体介质中，其中pH通常约为5-8，较佳地，pH约为6-8。
如本文所用，“药学上可接受的载体”指用于治疗剂给药的载体，包括各种赋形剂和稀释剂。
术语指这样一些药剂载体：它们本身并不是必要的活性成分，且施用后没有过分的毒性。合适的载体是本领域普通技术人员所熟知的。在组合物中药学上可接受的载体可含有液体，如水、盐水、缓冲液。另外，这些载体中还可能存在辅助性的物质，如填充剂、润滑剂、助流剂、润湿剂或乳化剂、pH缓冲物 质等。所述的载体中还可以含有细胞转染试剂。
如本文所用，术语“有效量”或“有效剂量”是指可对人和/或动物和/或细胞产生功能或活性的且可被人和/或动物所接受的量。
如本文所用，“药学上可接受的”的成分是适用于人和/或哺乳动物而无过度不良副反应(如毒性、刺激和变态反应)的，即具有合理的效益/风险比的物质。术语“药学上可接受的载体”指用于治疗剂给药的载体，包括各种赋形剂和稀释剂。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、聚山梨酯、乙醇及其组合。通常药物制剂应与给药方式相匹配，本发明的药物组合物可以被制成针剂形式，例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。所述的药物组合物宜在无菌条件下制造。活性成分的给药量是治疗有效量。本发明的药物制剂还可制成缓释制剂。
此外，本发明的活性成分组合还可与其他治疗剂(如抗肿瘤剂或免疫调节剂)一起使用。
使用药物组合物时，是将安全有效量的活性成分组合(包括第一活性成分(或其制剂)和/或第二活性成分(或其制剂))施用于哺乳动物。
应理解，本发明所述活性成分组合中第一活性成分(或其制剂)和/或第二活性成分(或其制剂)的有效量，可随给药的模式和肿瘤的严重程度等而变化。优选的有效量的选择可以由本领域普通技术人员根据各种因素来确定(例如通过临床试验)。所述的因素包括但不限于：药代动力学参数例如生物利用率、代谢、半衰期等；肿瘤的严重程度、患者的体重、患者的免疫状况、给药的途径等。
典型地，对于第一活性成分，其安全有效量通常至少约10纳克/千克体重，而且在大多数情况下不超过约50毫克/千克体重，较佳地该剂量是约50纳克/千克体重-约10毫克/千克体重。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。
对于第二活性成分，其安全有效量通常至少约10纳克/千克体重，而且在大多数情况下不超过约50毫克/千克体重，较佳地该剂量是约50纳克/千克体重-约10毫克/千克体重。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。
本发明所述的药物组合物的给药方式没有特别限制，代表性的例子包括(但并不限于)：静脉注射、皮下注射、肌肉注射等。
药盒
本发明提供一种药盒，所述药盒包括：
组分(1)：含有培(PEG)干扰素的制剂；
组分(2)：含有原癌基因产物靶向抑制剂的制剂；
组分(3)：说明书。
所述的含有培(PEG)干扰素的制剂包括(但并不限于)：冻干剂、液体制剂或静脉注射液。
所述的含有原癌基因产物靶向抑制剂的制剂包括(但并不限于)：冻干剂、液体制剂、片剂、胶囊、栓剂、或静脉注射液。
在本发明中，蛋白类的制剂通常为冻干制剂或注射剂。
典型地，药盒中装有一个或多个(如至少两个)含有培(PEG)干扰素的单元剂型和一个或多个(如至少两个)含有原癌基因产物靶向抑制剂的单元剂型；较佳地各为4-10个。
如本文所用，术语“单元剂型”是指为了服用方便，将组合物制备成单次服用所需的剂型，包括但不限于各种固体剂(如片剂)、液体剂、胶囊剂、缓释剂。
本发明提供的说明书中可以有如下描述：所述药盒的使用方法是同时使用含有培(PEG)干扰素的单元剂型和含有原癌基因产物靶向抑制剂的单元剂型。
本发明提供的药盒通过下述步骤制备得到：将含有培(PEG)干扰素的制剂和含有原癌基因产物靶向抑制剂的制剂，以及说明书一起放置，形成药盒。
所述的含有培(PEG)干扰素的制剂优选含有培(PEG)干扰素的单元剂型，所述含有原癌基因产物靶向抑制剂的制剂优选含有原癌基因产物靶向抑制剂的单元剂型。
所述步骤优选将至少一个含有培(PEG)干扰素的单元剂型和至少一个含有原癌基因产物靶向抑制剂的单元剂型，以及说明书一起放置，形成药盒。
本发明的有益效果：
1.培(PEG)干扰素和原癌基因产物靶向抑制剂(如安维汀或赫赛汀等)能够协同作用于抗肿瘤治疗，从而更显著地抑制肿瘤生长的作用。
2.培(PEG)干扰素和原癌基因产物靶向抑制剂(如安维汀或赫赛汀等)的联用，可以显著降低培(PEG)干扰素的用量，并可更快地显现出更强的抗肿瘤治疗效果，因此可以大幅减少长期和/或高剂量使用干扰素所导致的副作用。
3.培(PEG)干扰素和原癌基因产物靶向抑制剂(如赫赛汀)的联用，在乳腺癌等特定肿瘤中表现出出乎意料的优异效果，甚至出现高比例的肿瘤组织完全消失的治疗效果，而在单用培(PEG)干扰素或单用赫赛汀时则未观察到类似现象。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方 法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press，1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
试剂
在各实施例中，培干扰素指培(PEG)干扰素α1b(上海生物制品研究所有限公司)，白色冻干粉针剂，规格100μg/瓶(0.5ml)，合10万IU(即1000IU/μg)。
安维汀(贝伐珠单抗注射液)(avastin)为市售产品。
赫赛汀(Herceptin)为市售产品。
通用方法
实验动物的使用及福利遵照“国际实验动物评估和认可委员会(AAALAC)”的规定执行。每天监测动物的健康状况及死亡情况，例行检查包括观察受试物和药物对动物日常行为表现的影响如行为活动，体重变化，外观体征等。
小鼠抑瘤实验指标为考察药物对肿瘤生长的影响，具体指标为T/C％或抑瘤率TGI(％)。
每周二次用游标卡尺测量肿瘤直径，肿瘤体积(V)计算公式为：V＝1/2×a×b 2，其中a、b分别表示长、宽。
T/C(％)＝(T-T 0)/(C-C 0)×100，
其中,T、C为实验结束时的肿瘤体积；T 0、C 0为实验开始时的肿瘤体积。
抑瘤率(TGI)(％)＝100-T/C(％)。
当肿瘤出现消退时，抑瘤率(TGI)(％)＝100-(T-T 0)/T 0×100
如果肿瘤比起始体积缩小，即T<T 0或C<C 0时，即定义为肿瘤部分消退(PR)；如果肿瘤完全消失，即定义为肿瘤完全消退(CR)。
实验结束(如D21)、达到实验终点、或肿瘤体积达到1500mm 3，CO 2麻醉处死动物，随后解剖取瘤并拍照。
实施例1
注射用培(PEG)干扰素α1b单用或与安维汀合用对人胃癌NCI-N87裸小鼠皮下移植瘤的疗效
1摘要
评价注射用培(PEG)干扰素α1b(简称“培(PEG)干扰素”)单用或与安维汀合用对人胃癌NCI-N87裸小鼠皮下移植瘤的疗效。培(PEG)干扰素(0.1、1、10mg/kg，SC，每周2次，共5次)；
Figure PCTCN2019130749-appb-000006
(10mg/kg，IV，每周2次，共5次)；或培(PEG)干扰素(1mg/kg，SC，每周2次，共5次)与
Figure PCTCN2019130749-appb-000007
(10mg/kg，IV，每周2次，共5次)合用。
2实验目的
评价培(PEG)干扰素单用或与安维汀合用对人胃癌NCI-N87裸小鼠皮下移植瘤的疗效。
3受试药物
3.1药物信息
注射用培(PEG)干扰素α1b(简称“培(PEG)干扰素”)：白色冻干粉针剂，规格100μg/瓶(0.5ml)，批号S20170908；2-8℃避光密闭保存。由上海生物制品研究所有限公司生产并提供。
安维汀(贝伐珠单抗注射液):无色澄明液体，100mg(4ml)/瓶；批号H0182B03；2-8℃避光密闭保存,购自罗氏制药公司。
3.2药物配制
每瓶培(PEG)干扰素加入120μl灭菌注射用水，浓度为1mg/ml，临用前配制，用生理盐水稀释；安维汀浓度为25mg/ml，直接用生理盐水稀释至所需浓度。
4细胞
人胃癌NCI-N87细胞购自American Type Culture Collection。NCI-N87细胞用10-cm培养皿贴壁培养，培养条件为RPMI 1640培养基中加10％胎牛血清以及青、链霉素，于37℃、含5％CO 2空气的培养箱中培养。一周2-3次传代，当细胞呈指数生长期时，胰酶消化、收集细胞，计数，接种。
5实验动物
[D000521]BALB/c-Foxn1 nu/Gpt，4-8周，♀，购自江苏集萃药康生物科技有限公司。生产许可证号：SCXK(苏)2018-0008；动物合格证号201900949。饲养环境：SPF级。
6实验步骤
裸小鼠皮下(SC)接种6×10 6人胃癌NCI-N87细胞，待肿瘤生长至100-150mm 3后，根据肿瘤体积将动物分组(D0)。小鼠皮下注射(SC)或静脉注射(IV)给药，每周2次(BIW)；给药体积10mL/kg；溶剂组给予相同体积的“溶剂”(生理盐水)；具体给药剂量和给药方案见表1。每周测2次肿瘤体积，称小鼠体重，记录数据。
7统计学分析
二组肿瘤体积之间比较采用双尾Student's t检验，P<0.05定义为有统计学显著性差异。
8结果
结果如表1和图1、图2和图3所示。
表1.培(PEG)干扰素、安维汀单用或二者合用对人胃癌NCI-N87裸小鼠皮下移植瘤的疗效。
Figure PCTCN2019130749-appb-000008
D0:第一次给药时间；安维汀首剂加倍；SC：皮下注射；IV：静脉注射；P值指与溶剂相比；*P<0.05，**P<0.01，与培(PEG)干扰素1mg/kg+安维汀10mg/kg剂量组比较。
培(PEG)干扰素(0.1、1、10mg/kg，SC，每周2次，共6次)剂量依赖性地抑制人胃癌NCI-N87裸小鼠皮下移植瘤的生长，抑瘤率分别为19％、32％和55％；
Figure PCTCN2019130749-appb-000009
(10mg/kg，IV，每周2次，共6次)对NCI-N87皮下移植瘤的抑瘤率为49％。
培(PEG)干扰素(1mg/kg，SC，每周2次，共6次)与
Figure PCTCN2019130749-appb-000010
(10mg/kg，IV，每周2次，共6次)合用时，对NCI-N87的抑瘤率显著提高到67％(P<0.05，与单药比较)，显示出协同抑制作用。
此外，联用时荷瘤小鼠对药物表现出很好耐受，没有体重减轻等症状发生。如图2所示，培(PEG)干扰素α1b(1mg/kg，SC，每周2次，共6次)与安维汀(10mg/kg，IV，每周2次，共6次)合用组的小鼠体重与单用培干扰素组(1mg/kg)以及单用安维汀组(10mg/kg)的小鼠体重基本无区别。
上述实验结果表明：培(PEG)干扰素α1b联用安维汀，对人胃癌NCI-N87裸小鼠皮下移植瘤有显著协同增效的抑制作用，且荷瘤小鼠对药物耐受性好，副作用下降，没有体重减轻等症状发生。
9结论
如图1、2和3所示，培(PEG)干扰素(0.1、1、10mg/kg，SC，每周2次，共6次)剂量依赖性地抑制人胃癌NCI-N87裸小鼠皮下移植瘤的生长；
Figure PCTCN2019130749-appb-000011
Figure PCTCN2019130749-appb-000012
(10mg/kg，IV，每周2次，共6次)对NCI-N87皮下移植瘤同样有效。
联用时，培(PEG)干扰素对安维汀治疗NCI-N87裸小鼠皮下移植瘤有显著的协同增效作用(P<0.05，与培(PEG)干扰素或安维汀单药比较)。此外，联用时，而毒性没有增加，荷瘤小鼠对药物表现出良好的耐受性。
实施例2
注射用培(PEG)干扰素α1b单用或与安维汀合用对人肝癌Huh-7裸小鼠皮下移植瘤的疗效
1摘要
评价注射用培(PEG)干扰素α1b(简称“培(PEG)干扰素”)单用或与安维汀合用对人肝癌Huh-7裸小鼠皮下移植瘤的疗效。培(PEG)干扰素(0.1、1、10mg/kg，SC，每周2次，共5次)；；
Figure PCTCN2019130749-appb-000013
(10mg/kg，IV，每周2次，共5次)；或培(PEG)干扰素(1mg/kg，SC，每周2次，共5次)与
Figure PCTCN2019130749-appb-000014
(10mg/kg，IV，每周2次，共5次)合用。
2实验目的
评价培(PEG)干扰素单用或与安维汀合用对人肝癌Huh-7裸小鼠皮下移植瘤的疗效。
3受试药物
3.1药物信息
注射用培(PEG)干扰素α1b(简称“培(PEG)干扰素”)：同实施例1。
安维汀(贝伐珠单抗注射液):同实施例1。
3.2药物配制
每瓶培(PEG)干扰素加入120μl灭菌注射用水，浓度为1mg/ml，临用前配制，用生理盐水稀释；安维汀浓度为25mg/ml，直接用生理盐水稀释至所需浓度。
4细胞
人肝癌Huh-7细胞购自中国科学院细胞库。Huh-7细胞用10-cm培养皿贴壁培养，培养条件为RPMI 1640培养基中加10％胎牛血清以及青、链霉素，于37℃、含5％CO 2空气的培养箱中培养。一周2-3次传代，当细胞呈指数生长期时，胰酶消化、收集细胞，计数，接种。
5实验动物
[D000521]BALB/c-Foxn1nu/Nju，6-7周，♀，购自江苏集萃药康生物科技 有限公司。生产许可证号：SCXK(苏)2018-0008；动物合格证号201900949。饲养环境：SPF级。
6实验步骤
裸小鼠皮下接种6×10 6人肝癌Huh-7细胞，待肿瘤生长至100-150mm 3后，根据肿瘤体积将动物分组(D0)。小鼠皮下注射(SC)或静脉注射(IV)给药，每周2次(BIW)；给药体积10mL/kg；溶剂组给予相同体积的“溶剂”(生理盐水)；具体给药剂量和给药方案见表2。每周测2次肿瘤体积，称小鼠体重，记录数据。
7统计学分析
二组肿瘤体积之间比较采用双尾Student's t检验，P<0.05定义为有统计学显著性差异。
8结果
结果如表2和图4、图5和图6所示。
表2.培(PEG)干扰素、安维汀单用或二者合用对人肝癌Huh-7裸小鼠皮下移植瘤的疗效。
Figure PCTCN2019130749-appb-000015
D0:第一次给药时间；安维汀首剂加倍；SC：皮下注射；IV：静脉注射；P值指与溶剂相比；*P<0.05，与培(PEG)干扰素1mg/kg+安维汀10mg/kg剂量组比较。
培(PEG)干扰素(0.1、1、10mg/kg，SC，每周2次，共5次)对人肝癌Huh-7裸小鼠皮下移植瘤生长没有明显抑制作用，抑瘤率分别为-10％、-7％和9％；
Figure PCTCN2019130749-appb-000016
Figure PCTCN2019130749-appb-000017
(10mg/kg，IV，每周2次，共5次)对Huh-7皮下移植瘤的抑瘤率为40％。
培(PEG)干扰素(1mg/kg，SC，每周2次，共5次)与
Figure PCTCN2019130749-appb-000018
(10mg/kg， IV，每周2次，共5次)合用时，对Huh-7的抑瘤率提高到49％。培(PEG)干扰素对安维汀治疗人肝癌Huh-7裸小鼠皮下移植瘤有一定的协同增效作用。
此外，联用时荷瘤小鼠对药物表现出很好耐受，没有体重减轻等症状发生。如图5所示，培(PEG)干扰素α1b(1mg/kg，SC，每周2次，共6次)与安维汀(10mg/kg，IV，每周2次，共6次)合用组的小鼠体重与单用培干扰素组(1mg/kg)以及单用安维汀组(10mg/kg)的小鼠体重无明显区别。此外，联用组的体重高于单用安维汀(10mg/kg)的体重，这提示，联用时可进一步降低安维汀给药的副作用。
9结论
如图4、5和6所示，培(PEG)干扰素(0.1、1、10mg/kg，SC，每周2次，共5次)对人肝癌Huh-7裸小鼠皮下移植瘤生长没有明显抑制作用；
Figure PCTCN2019130749-appb-000019
(10mg/kg，IV，每周2次，共5次)对Huh-7皮下移植瘤有效。
联用时；培(PEG)干扰素对安维汀治疗人肝癌Huh-7裸小鼠皮下移植瘤有协同增效作用。此外，联用时，而毒性没有增加，荷瘤小鼠对药物表现出良好的耐受性。
实施例3
注射用培(PEG)干扰素α1b单用或与赫赛汀合用对人乳腺癌BT-474裸小鼠皮下移植瘤的疗效
1摘要
评价注射用培(PEG)干扰素α1b(简称“培(PEG)干扰素”)单用或与赫赛汀合用对人乳腺癌BT-474裸小鼠皮下移植瘤的疗效。培(PEG)干扰素(0.1、1、10mg/kg，SC，每周2次，共6次)；
Figure PCTCN2019130749-appb-000020
(5mg/kg，IV，每周2次，共6次)；或培(PEG)干扰素(1mg/kg，SC，每周2次，共6次)与
Figure PCTCN2019130749-appb-000021
(5mg/kg，IV，每周2次，共6次)合用。
2实验目的
评价培(PEG)干扰素单用或与赫赛汀合用对人乳腺癌BT-474裸小鼠皮下移植瘤的疗效。
3受试药物
3.1药物信息
注射用培(PEG)干扰素α1b(简称“培(PEG)干扰素”)：同实施例1。
赫赛汀(注射用曲妥珠单抗)，白色至淡黄色疏松块状物，规格440毫克(20毫升)/瓶，产品批号/稀释液批号N3886/B3083；生产日期20170220(产品)/20161118(稀释液)，有效期至20200219。2-8℃避光保存。
3.2提供单位
培(PEG)干扰素由上海生物制品研究所有限公司提供；赫赛汀购自罗氏制药公司。
3.3药物配制
培(PEG)干扰素：每瓶培(PEG)干扰素加入120μl灭菌注射用水，浓度为1mg/ml，临用前配制；用生理盐水稀释；
赫赛汀：先用原研厂家提供的溶剂复溶，溶解后为无色至淡黄色，澄清至微乳光溶液，复溶后浓度为21mg/ml；立刻分装在-70～-80℃条件下冻存。临用前用生理盐水稀释至所需浓度。
4细胞
人乳腺癌BT-474细胞购自American Type Culture Collection。BT-474细胞用10-cm培养皿贴壁培养，培养条件为DMEM培养基(Gibco)中加10％胎牛血清(Gibco)以及青、链霉素，于37℃、含5％CO 2空气的培养箱(Thermo)中培养。一周2-3次传代，当细胞呈指数生长期时，胰酶消化、收集细胞，计数，接种。
5实验动物
[D000521]BALB/cJGpt-Foxn1nu/Gpt，5周，♀，购自江苏集萃药康生物科技有限公司。生产许可证号：SCXK(苏)2018-0008动物合格证号201901062。饲养环境：SPF级。
6实验步骤
裸小鼠皮下接种1×10 7人乳腺癌BT-474细胞，待肿瘤生长至100-150mm 3后，根据肿瘤体积将动物分组(D0)。小鼠皮下注射(SC)或静脉注射(IV)给药，每周2次(BIW)；给药体积10mL/kg；溶剂组给予相同体积的“溶剂”(生理盐水)；具体给药剂量和给药方案见表3。每周测2次肿瘤体积，称小鼠体重，记录数据。
7统计学分析
二组肿瘤体积之间比较采用双尾Student's t检验，P<0.05定义为有统计学显著性差异。
8结果
结果如表3和图7、图8和图9所示。
表3.培(PEG)干扰素、赫赛汀单用或二者合用对人乳腺癌BT-474裸小鼠皮下移植瘤的疗效。
Figure PCTCN2019130749-appb-000022
Figure PCTCN2019130749-appb-000023
D0:第一次给药时间；赫赛汀首剂加倍；SC：皮下注射；IV：静脉注射；P值指与溶剂相比；**P<0.01，与培(PEG)干扰素1mg/kg+赫赛汀5mg/kg剂量组比较。
培(PEG)干扰素(0.1、1、10mg/kg，SC，每周2次，共6次)剂量依赖性地抑制人乳腺癌BT-474裸小鼠皮下移植瘤的生长，抑瘤率分别为66％、98％和156％，1mg/kg组有3/6肿瘤部分消退，10mg/kg组有4/6肿瘤部分消退和2/6肿瘤完全消退(D20)，至实验结束时(D60)，10mg/kg组仍有4/6肿瘤部分消退；
Figure PCTCN2019130749-appb-000024
Figure PCTCN2019130749-appb-000025
(5mg/kg，IV，每周2次，共6次)对BT-474皮下移植瘤的抑瘤率为57％(D20)。
培(PEG)干扰素(1mg/kg，SC，每周2次，共6次)与
Figure PCTCN2019130749-appb-000026
(5mg/kg，IV，每周2次，共6次)合用时，对BT-474的抑瘤率显著提高到170％(P<0.01，与单药比较)，有3/6肿瘤部分消退和3/6肿瘤完全消退(D20)；至实验结束(D60)，仍有3/6肿瘤部分消退和3/6肿瘤完全消退；这说明，培(PEG)干扰素联合赫赛汀治疗人乳腺癌BT-474裸小鼠皮下移植瘤有非常显著的协同增效作用，甚至实现了部分消退或完全消退的治疗效果。
此外，联用时荷瘤小鼠对药物表现出很好耐受，没有体重减轻等症状发生。尤其是如图8所示，培(PEG)干扰素α1b(1mg/kg，SC，每周2次，共6次)与
Figure PCTCN2019130749-appb-000027
(5mg/kg，IV，每周2次，共6次)合用组的小鼠体重与单用培干扰素组以及单用赫赛汀组的小鼠体重没有区别。
9结论
如图7、8和9所示，培(PEG)干扰素(0.1、1、10mg/kg，SC，每周2次，共6次)剂量依赖性地抑制人乳腺癌BT-474裸小鼠皮下移植瘤的生长，引起肿瘤部分或完全消退；
Figure PCTCN2019130749-appb-000028
(5mg/kg，IV，每周2次，共6次)对BT-474皮下移植瘤同样有效。
联用时，培(PEG)干扰素(1mg/kg，SC，每周2次，共6次)和
Figure PCTCN2019130749-appb-000029
(5mg/kg， IV，每周2次，共6次)对BT-474肿瘤的疗效有显著的协同增效作用(P<0.05，与单药比较)。此外，联用时，而毒性没有增加，荷瘤小鼠对药物表现出良好的耐受性。
实施例4
培(PEG)干扰素对肿瘤细胞株的体外抑制作用
应用磺酰罗丹明B蛋白染色法(SRB法)评价陪干扰素单用或与其他药物合用对体外培养肿瘤细胞增殖影响。
接种一定数量的对数生长期细胞于96孔培养板。贴壁生长24小时后，加入不同浓度(终浓度为1，3，10，30，100，300，1000，3000，10000nM)的药物。药物作用120小时后，用三氯乙酸固定细胞。然后SRB溶液染色；最后加入Tris溶液溶解SRB，酶标仪510nm波长下测定OD值，以下列公式计算细胞生长抑制率：抑制率＝(OD值对照孔-OD值给药孔)/OD值对照孔×100％
结果如表4所示。
表4 培(PEG)干扰素对不同肿瘤细胞株的体外抑制率
Figure PCTCN2019130749-appb-000030
实施例5
培(PEG)干扰素和赫赛汀对肿瘤细胞株的体外协同抑制作用
重复实施例4的方法，不同点在于，采用表5所示的不同剂量的培干扰素(单用组1)、赫赛汀(单用组2)、以及不同剂量的培干扰素+赫赛汀(合用组)，采用乳腺癌细胞BT474，并计算相应的抑制率(％)。
表5 药物用量
Figure PCTCN2019130749-appb-000031
Figure PCTCN2019130749-appb-000032
注：培干扰素中，IFN-α1b与PEG的重量比为约1:1，因此，600μg培干扰素中的IFN-α1b为300μg(约合30万IU)，依此类推。
如图10所示。结果表明，培干扰素和赫赛汀(Herceptin)合用对体外培养人乳腺癌BT-474细胞增殖有协同抑制活性，抑制作用显著提高，合用指数(CI值)为0.31，表明有协同增效作用。
具体地，合用时，当培干扰素浓度为约9.38-600μg/ml而赫赛汀浓度为0.05-3μg/ml时，均可观察到明显的协同抑制作用。例如，150μg/ml培干扰素+0.75μg/ml赫赛汀合用时的抑制率，显著高于单用300μg/ml培干扰素或单用1.5μg/ml赫赛汀时的抑制率。
讨论
干扰素是一类具有广谱抗病毒增殖、抗肿瘤、免疫调节等多种活性的高活性蛋白质分子。在肿瘤治疗领域，普通干扰素已被美国FDA和欧洲EMA批准用于治疗毛细胞白血病、慢性粒细胞白血病、肾癌、卡波斯肉瘤、黑色素瘤、滤泡性淋巴瘤、多发性骨髓瘤、宫颈上皮内瘤(Elena Garcia-Martinez,etc.Trial Watch:Immunostimulation with recombinant cytokines for cancer therapy，ONCOIMMUNOLOGY，2018，VOL.7，NO.6:e1433982,16pages；陈翔等，肾癌免疫治疗的临床研究进展，中华泌尿外科杂志，2017卷38第9期：717-720)。另外，在肺癌、消化道癌、前列腺癌、头颈部鳞癌等肿瘤治疗中也有一定疗效。
虽然普通干扰素是一种安全有效的制剂，但仍存在一些问题，尤其是严重的副作用。目前临床上使用的普通干扰素由于分子量较小(20kDa以下)，易被肾小球滤过，导致在人体内半衰期仅为1.5-2小时，为达到理想的疗效，一般临床上普通干扰素治疗采用大剂量、多次数注射用药，患者依从性较差，也易引起副作用的发生。
以黑色素瘤为例，需要先皮下注射普通干扰素2000万IU/m 2(约等于400μg/人)给药5天/周，1个月后再给药1000万IU/m 2(约等于200μg/人)，每周3次维持治疗48周，可延长患者存活期，但大剂量给药普通干扰素的不良反应十分明显，至少50％的患者需要推迟或减少给药剂量，因此只能用于高危患者。
普通干扰素α在人体中的不良反应是全身性的(曹学琳，干扰素不良反应机制,生物医学工程与临床，2010，第14卷第4期：340-342)：治疗初期，多数患者会出现流感样综合征，临床症状有发热、寒战、头痛、肌痛、胃肠痛等。 恶心、呕吐、食欲不振是普通干扰素α治疗早期常见的胃肠道反应。在普通干扰素α治疗中超过70％的患者会产生疲劳。神经精神系统不良反应主要为精神紊乱、抑郁、精神恍惚、焦虑、烦躁等。接受普通干扰素α治疗过程中约20％的患者可能出现白细胞总数、中性粒细胞及血小板计数轻到中度减少。普通干扰素α所致的皮肤黏膜病变以皮疹最为常见，常表现为非特异伴瘙痒的皮疹；其他表现有注射部位红斑、皮肤溃烂、口腔黏膜溃烂和口唇炎等。普通干扰素α治疗可通过自身免疫和非自身免疫机制导致甲状腺功能异常，甲状腺功能异常以甲状腺功能低下最为常见。普通干扰素α还可诱导对胰岛β细胞的自身免疫性损伤而诱发糖尿病。其它较为少见的不良反应有间质性肺炎、视网膜病变、听力受损和耳鸣、脱发、腹泻、体重减轻。
为了提高普通干扰素α治疗肿瘤的疗效，降低副作用，对普通干扰素α与其它肿瘤药物联用的方案进行了有益的探索(郭军，转移性肾癌的内科治疗进展，第二届中国肿瘤内科大会，2008：33-36)。其中贝伐珠单抗联合普通干扰素α治疗肾癌晚期的方案最为成功，2项独立的临床试验均证实贝伐珠单抗联合普通干扰素α治疗肾癌较单用普通干扰素可显著延长患者PFS(10.2vs 5.4个月、8.5vs5.2个月)。基于以上结果，2009年美国FDA批准了贝伐珠单抗联合普通干扰素治疗肾癌晚期。但3级及以上不良反应发生率最高可达80％(Brian I.Rini，etc.Phase III Trial of Bevacizumab Plus Interferon Alfa Versus Interferon Alfa Monotherapy in Patients With Metastatic Renal Cell Carcinoma:Final Results of CALGB 90206，JOURNAL OF CLINICAL ONCOLOGY，2010，VOL28，No.13:2137-2143)，因此患者依从性不高，限制了该方案的应用。
人干扰素α1b是由人白细胞产生的一种由166个氨基酸组成的蛋白质，其相对分子量为19.382kDa。研究表明，中国人的白细胞在受到病毒攻击后，产生的多种干扰素中以α1b型干扰素为主。
八十年代初，医科院病毒学研究所侯云德等首次克隆到α1b干扰素基因，由病毒所和上海生物制品研究所联合开发成功我国第一个基因工程蛋白药物----重组人干扰素α1b，是我国少数具有自主知识产权的一类新药。
与普通干扰素α 2产品相比，普通干扰素α 1体外生物学活性相对较低，但在体内作用较强而且范围广、副作用小。如在体外普通干扰素α2a和2b的比活性均为1×10 8IU/mg，而普通干扰素α 1b的比活性仅为1×10 7IU/mg，是前两者的10％。在临床应用中，使用同样重量的普通干扰素α 1b与普通干扰素α2a、2b疗效相当，而副作用明显小于后两者(童葵塘，干扰素α治疗黑色素瘤，第17次全国干扰素及细胞因子学术会议，2011:2-6)。在普通干扰素α 1b治疗毛细胞白血病的临床试验中，有效率为63.64％，缓解率达36.36％，而不良反应仅有短暂的低热，不需服药可自行消退，其他不良反应也不明显，患者均能耐受，无一例患者停药。
在美国进行的Ⅰ、Ⅱ期临床试验结果表明，采用大剂量(500μg/天)普通干扰素α 1b注射治疗转移性肾癌患者和多发性骨髓瘤患者具有一定的疗效，而不良反应与其它普通干扰素相比不良反应更为轻微(P.Masci,etc.Gene Modulatory Effects,Pharmacokinetics,and Cl inical Tolerance of Interferonα-1b：A Second Member of the InterferonαFamily.Clinical Pharmacology&Therapeutics.2007.Vol.81，No.3:354-361)。
普通干扰素α1b与其它普通干扰素一样，也存在着分子量较小，易被肾小球滤过，在人体内半衰期短的缺陷。为延长半衰期，提高其疗效，采用第二代聚乙二醇修饰剂对普通干扰素α1b进行修饰，获得了单个PEG修饰的干扰素α1b，命名为培(PEG)干扰素α1b。研究表明其分子量增大，半衰期较未修饰的普通干扰素明显延长：在大鼠体内培(PEG)干扰素α1b半衰期为13.98小时，而普通干扰素α1b为1.84小时；在食蟹猴体内培(PEG)干扰素α1b半衰期为22.03±3.32小时，而普通干扰素α1b为3.27±1.48h。根据已上市PEG修饰类重组治疗性蛋白药物的相关研究报道，此类药物的分子量增大与其临床应用上的疗效提高成正相关。
在本发明中，将培(PEG)干扰素α1b单药或与其它药物联用进行临床前体外和体内药效学研究。
研究结果表明，培(PEG)干扰素α1b与安维汀、赫赛汀等某些抗肿瘤药物联合使用，不仅在疗效方面有协同作用，而且还协同地显著降低各种不同的副作用。
在本发明的依据中，在体外肿瘤细胞模型上，观察到了培(PEG)干扰素α1b对乳腺癌、肺癌、肝癌和胃癌细胞均具有一定的抑制作用(19.6％～58.4％抑制率)。
在体内裸鼠乳腺癌BT-474肿瘤模型中，中剂量培(PEG)干扰素α1b(1mg/kg)与赫赛汀联用组的疗效增强效果明显(联用组170％抑制率vs单用中剂量培(PEG)干扰素α1b组98％抑制率vs单用赫赛汀组57％抑制率vs单用高剂量培(PEG)干扰素α1b组156％抑制率)，甚至观察到了50％的裸鼠肿瘤完全消退。这些结果不但证实了培(PEG)干扰素α1b确实具有明显抗肿瘤效应，为其用于临床肿瘤治疗提供了坚实的基础。同时也表明与单抗联用产生的协同作用可以大幅降低培(PEG)干扰素α1b的用量(降低10倍)，为培(PEG)干扰素α1b与单抗等抗肿瘤药物联用提供了更大的剂量选择范围。
在培(PEG)干扰素α1b与安维汀合用时，对人胃癌NCI-N87裸小鼠皮下移植瘤模型，观察到疗效增强效果明显。同时也表明与单抗联用产生的协同作用可以大幅降低培(PEG)干扰素α1b的用量(降低10倍)，为培(PEG)干扰素α1b与单抗等抗肿瘤药物联用提供了更大的剂量选择范围。
在肝癌Huh-7裸鼠模型上，培(PEG)干扰素α1b单用组未表现出明显的抑瘤效果，由此可见培(PEG)干扰素α1b尽管具有抗肿瘤活性，但对于不同的肿 瘤其疗效仍有一定或较大的的差异。然而，培干扰素与安维汀联用时，却表现出一定的协同增效作用。
尽管培(PEG)干扰素α2a和α2b在慢性乙型肝炎和丙型肝炎上疗效较普通干扰素具有明显的优势，取得了巨大的成功。但在肿瘤治疗领域则完全不同，
在一项三期临床研究中(T.K.Eigentler,etc.Adjuvant treatment with pegylated interferon α-2a versus low-dose interferon α-2a in patients with high risk melanoma:a randomized phase III DeCOG trial，Annals of Oncology，2016，27:1625–1632)，比较了培(PEG)干扰素α2a与普通干扰素α2a高风险黑素瘤患者的疗效与安全性，结果发现培(PEG)干扰素α2a的疗效并不优于普通干扰素α2a，而副作用却更高。
在另一项三期临床研究中(THOMAS M.H,etc.U.S.Food and Drug Administration Approval:Peginterferon-alfa-2b for the Adjuvant Treatment of Patients with Melanoma，The oncologist，2012；17:1323–1328)，培(PEG)干扰素α2b辅助治疗黑色素瘤患者的平均无复发生存期为34.8个月，较观察组的25.5个月明显延长，但对总体生存期无显著性改善。尽管治疗组的严重不良反应发生率几乎达到了观察组的2倍(33％vs15％)，2011年美国FDA还是批准了培(PEG)干扰素α2b用于辅助治疗区域淋巴结受累的恶性黑色素瘤患者，每周皮下给药6μg/kg，连续8针后维持每周3μg/kg给药(NCCN肿瘤学临床实践指南-恶性黑色瘤，2018年第2版：MS-20)。目前培(PEG)干扰素类药物用于肿瘤适应症的批准方案仅有这一个，表明在肿瘤治疗领域不同培(PEG)干扰素亚型和不同肿瘤类型对治疗效果的影响有明显的差异，不能简单的进行同理类推。
出乎意料的是，与培(PEG)干扰素α2a不同，本发明的研究表明，培(PEG)干扰素α1b与安维汀或赫赛汀的联用在协同增效地抑制肿瘤的同时，还显著降低各自相应的副作用(如体重下降、发热、不良胃肠道反应、皮疹等)的发生率或程度。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种活性成分组合的用途，其特征在于，所述活性成分组合包括第一活性成分培干扰素和第二活性成分原癌基因产物靶向抑制剂，并且所述组合用于制备协同治疗肿瘤的药物组合物或药盒。
  2. 如权利要求1所述的用途，其特征在于，所述的培干扰素为培干扰素α1b。
  3. 如权利要求1所述的用途，其特征在于，所述的培干扰素和原癌基因产物靶向抑制剂的质量比为1:100至500:1；
    或者，所述的培干扰素和原癌基因产物靶向抑制剂的之比(IU/mg)为1000IU/mg至10000万IU/mg，较佳地10000IU/mg至1000万IU/mg，更佳地10万IU/mg至500万IU/mg或20万IU/mg至300万IU/mg。
  4. 如权利要求1所述的用途，其特征在于，所述的肿瘤选自下组：肺癌、胃癌、乳腺癌、肝癌、肠癌、卵巢癌、子宫颈癌、或其组合。
  5. 如权利要求1所述的用途，其特征在于，所述的原癌基因产物靶向抑制剂选自下组：EGFR抑制剂、Her2抑制剂、或其组合。
  6. 如权利要求1所述的用途，其特征在于，所述的原癌基因产物靶向抑制剂选自下组：安维汀、赫赛汀、帕妥珠单抗、或其组合。
  7. 一种药物组合物，其特征在于，所述的药物组合物包括培干扰素、原癌基因产物靶向抑制剂和药学上可接受的载体。
  8. 一种活性成分组合，其特征在于，所述组合由培干扰素和原癌基因产物靶向抑制剂构成。
  9. 一种药盒，其特征在于，所述药盒包括：
    (a)第一制剂，所述第一制剂含有培干扰素以及药学上可接受的载体；
    (b)第二制剂，所述第二制剂含有原癌基因产物靶向抑制剂以及药学上可接受的载体；
    (c)说明书，所述说明书描述了将培干扰素和原癌基因产物靶向抑制剂联用以治疗肿瘤的方法。
  10. 一种体外非治疗性的协同抑制肿瘤细胞生长的方法，其特征在于，包括步骤：向肿瘤细胞培养体系中加入如权利要求8所述活性成分组合，从而协同抑制肿瘤细胞生长。
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