WO2022016844A1 - 一种基于干扰素的癌症治疗方法和药物组合 - Google Patents

一种基于干扰素的癌症治疗方法和药物组合 Download PDF

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WO2022016844A1
WO2022016844A1 PCT/CN2021/073191 CN2021073191W WO2022016844A1 WO 2022016844 A1 WO2022016844 A1 WO 2022016844A1 CN 2021073191 W CN2021073191 W CN 2021073191W WO 2022016844 A1 WO2022016844 A1 WO 2022016844A1
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interferon
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administration
treatment
mifn
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PCT/CN2021/073191
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French (fr)
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孙黎
周卫东
廖小金
庄露
何叒一
周婷
曾玲英
杨美花
王世媛
郑杰华
张林忠
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厦门特宝生物工程股份有限公司
厦门伯赛基因转录技术有限公司
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Priority to EP21847380.9A priority Critical patent/EP4186519A4/en
Priority to KR1020237006195A priority patent/KR20230065977A/ko
Priority to CA3189942A priority patent/CA3189942A1/en
Priority to JP2023504317A priority patent/JP2023534072A/ja
Priority to AU2021314116A priority patent/AU2021314116A1/en
Publication of WO2022016844A1 publication Critical patent/WO2022016844A1/zh
Priority to US18/099,664 priority patent/US20230270824A1/en

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Definitions

  • the invention relates to the field of biomedicine, and discloses an interferon-based cancer treatment method and a drug combination.
  • the present invention relates to a method of interferon-based cancer treatment comprising i) intermittently administering to a subject an interferon-based therapeutic agent, and ii) administering an additional anticancer agent, preferably gemcitabine.
  • the present invention also relates to pharmaceutical combinations for use in the methods.
  • Interferons are a class of active proteins with multiple functions and are cytokines produced by monocytes and lymphocytes. They have broad-spectrum anti-virus, influence cell growth, differentiation, regulate immune function and other biological activities, are currently the most important anti-viral infection and anti-tumor biological products.
  • Type I interferons include IFN- ⁇ and IFN- ⁇ .
  • IFN- ⁇ is mainly produced by monocytes-macrophages, and B cells and fibroblasts can also synthesize IFN- ⁇ ; IFN- ⁇ is mainly produced by fibroblasts.
  • IFN- ⁇ / ⁇ both bind to the same receptor and are widely distributed, including monocytes-macrophages, polymorphonuclear leukocytes, B cells, T cells, platelets, epithelial cells, endothelial cells and tumor cells. More than 23 subtypes of IFN ⁇ are known. There is only one subtype of IFN ⁇ .
  • Type II interferon gamma interferon
  • gamma interferon is mainly produced by activated T cells (including Th0, Th1 cells and almost all CD8+ T cells) and NK cells, and is a kind of so-called lymphokine.
  • IFN- ⁇ can exist in the form of extracellular matrix, so it controls cell growth through the adjacent mode, and it can be distributed on the surface of almost all cells except mature erythrocytes. There is only one subtype of IFN- ⁇ .
  • Type III interferon mainly refers to interferon lambda.
  • Interferons, interferon mutants and interferon derivatives have been widely approved for various types of therapy.
  • Interferons and mutants that have been approved for human clinical treatment include interferon alpha 2a, interferon alpha 2b, interferon alpha 1b, compound interferon (Ganfujin), interferon mutants (such as Lefu Neng), interferon ⁇ , interferon ⁇ (1b), etc.
  • Interferon derivatives include peginterferon alfa 2a, peginterferon alfa 2b, integrated interferon and the like.
  • Type I IFNs are the first immunotherapy drugs approved by the FDA for clinical use in cancer. Among them, hematological tumors have the best curative effect, followed by lymphatic system tumors, and solid tumors are the worst. For example, malignant melanoma, ovarian tumor, and colorectal cancer have the highest curative effect of no more than 20%.
  • interferon anti-tumor such as interferon combined with 5-fluorouracil in the treatment of liver cancer, colorectal cancer, interferon combined with cisplatin, carboplatin and other treatments
  • these studies have achieved some results in lung cancer, the overall effect is average and the prognosis is poor.
  • interferon is generally administered in a large dose and continuously in clinical practice, which inevitably brings about problems such as high drug-related toxicity, poor patient compliance and tolerance.
  • the present invention provides a method of treating cancer in a subject, comprising administering to the subject
  • the interferon-based therapeutic comprises an interferon or a mutant or derivative thereof, or a nucleic acid molecule encoding an interferon or a mutant or derivative thereof, or an agent that promotes the production of endogenous interferon substance.
  • the interferon is a type I, type II or type III interferon, eg, interferon alpha, interferon beta, interferon gamma or interferon lambda, preferably interferon alpha.
  • the interferon-based therapeutic comprises interferon alpha 2a, interferon alpha 2b, interferon alpha 1b, interferon lambda, or a mutant or derivative thereof.
  • the interferon or a mutant or derivative thereof is PEGylated.
  • the interferon-based therapeutic agent is selected from the group consisting of P1101, Peggabine, Pyloxine, PegIntron, Ganfujin, LeFuneng, Ganneneng, Rodine, Yundesu, Derived probiotics and peginterferon lambda.
  • the interferon-based therapeutics include agonists of TLRs, RLRs, and STINGs signaling pathways.
  • the interferon-based therapeutic is selected from the group consisting of GS-9620, GS-9688, RO7020531, RO6864018, TQ-A3334, JNJ-4964, SB9200, MIW815, DMXAA, MK-1454, and diABZI.
  • the administration of the interferon-based therapeutic agent in the consecutive courses of treatment is such that the concentration of neopterin in the subject is higher than the neopterin concentration prior to the first administration during substantially the entire course of treatment, eg is about 110%, about 120%, about 130%, about 140%, about 150%, about 200%, about 250% or more of the pre-first administration neopterin concentration.
  • the length of the continuous course of treatment is the time from the first administration to the last administration of the course of treatment, plus about 5 in vivo half-lives of the therapeutic agent.
  • each of the plurality of consecutive courses of treatment has a length of about 1 week to about 24 weeks, preferably about 1 week to about 12 weeks, more preferably about 1 week to about 8 weeks, more preferably about 2 weeks to about 2 weeks 6 weeks.
  • each consecutive course of treatment is about 1 week to about 12 weeks in length, and wherein each course of treatment is separated by about 1 week to about 12 weeks in length.
  • each successive course of treatment is separated by about 1 week to about 24 weeks, preferably about 1 week to about 12 weeks, more preferably about 1 week to about 8 weeks, more preferably about 2 weeks to about 6 weeks.
  • each successive course of treatment is about 1 week to about 8 weeks in length, and wherein each course of treatment is separated by about 1 week to about 8 weeks.
  • each successive course of treatment is about 2 weeks to about 6 weeks in length, and wherein each treatment course is separated by about 2 weeks to about 6 weeks.
  • the interferon-based therapeutic is administered for 2-25 or more consecutive courses of treatment.
  • the plurality of consecutive courses of treatment are substantially the same length.
  • the sessions have substantially the same time interval between sessions.
  • the cancer is selected from leukemia (eg, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), chronic lymphocytic leukemia, polycapillary leukemia), liver cancer , lung cancer, colorectal cancer, skin cancer, stomach cancer, breast cancer, prostate cancer, non-Hodgkin's lymphoma, melanoma, multiple myeloma, laryngeal papilloma, follicular lymphoma, AIDS-related Kaposi's sarcoma and renal cell carcinoma, preferably liver, lung, breast, colorectal or melanoma.
  • ALL acute lymphoblastic leukemia
  • AML acute myeloid leukemia
  • CML chronic myeloid leukemia
  • chronic lymphocytic leukemia polycapillary leukemia
  • liver cancer eg, lung cancer, colorectal cancer, skin cancer, stomach cancer, breast cancer, prostate cancer, non
  • the administration of the interferon-based therapeutic does not overlap with the administration of the additional anticancer agent.
  • the additional agent is administered between the plurality of consecutive courses of treatment.
  • the administration of the "interferon-based therapeutic" overlaps the administration of the additional agent.
  • the additional agent is administered during and between the plurality of consecutive courses of treatment.
  • the additional anticancer agent is administered according to its conventional regimen.
  • the anticancer agent is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-oxidedoxedoxedoxedoxethyl
  • chemotherapeutic agents such as alkylating agents: nimustine, carmustine, lomustine, cyclophosphamide, ifosfamide, mustard, semustine; antimetabolites: desoxy Fluoroguanosine, Doxefluoroguanidine, 5-Fluorouracil, Mercaptopurine, Thioguanine, Cytarabine, Fluoroguanosine, Tegafur, Gemcitabine, Carmofur, Hydroxyurea, Methotrexate, Euphor Antitumor antibiotics: actinomycin D, doxorubicin, daunorubicin, epirubicin, mitomycin, pelomycin, pingyangmycin, Pirarubicin; anti-tumor animal and plant ingredients: irinotecan, harringtonine, hydroxycamptothecin, vinorelbine, paclitaxel, nab-paclitaxel, taxotere, topotecan, vincristine
  • Immune checkpoint inhibitors eg inhibitors of PD-1, PD-L1, CTLA4, eg antibodies selected from the group consisting of Nivolumab, Pembrolizumab, Atezolizumab, Durvalumab, Avelumab; or
  • iii) Small molecule targeted drugs such as Imatinib, Gefitinib, Bortezomib, Erlotinib, Sorafenib, Lenalidomide, Sunitinib, Dasatinib, Nilo tinib, lapatinib, pazopanib, everolimus, vandetanib, crizotinib, vemurafenib, ruxolitinib, axitinib, vismodegib, carfilzomib, regora Nitrate, bosutinib, tofacitinib, cabozantinib, ponatinib, pomalidomide, trametinib, dabrafenib, afatinib, icotinib, ibrutinib Ceritinib, Ceritinib, Idelaris, Apatinib, Palbociclib, Lenvatin
  • the anticancer agent is selected from the group consisting of oxaliplatin, epirubicin, paclitaxel and gemcitabine, more preferably gemcitabine.
  • the present invention provides a pharmaceutical combination for treating cancer in a subject comprising an interferon-based therapeutic agent and an anticancer agent.
  • the interferon-based therapeutic comprises an interferon or a mutant or derivative thereof, or a nucleic acid molecule encoding an interferon or a mutant or derivative thereof, or an agent that promotes the production of endogenous interferon substance.
  • the interferon is a type I, type II or type III interferon, eg, interferon alpha, interferon beta, interferon gamma or interferon lambda, preferably interferon alpha.
  • the interferon-based therapeutic comprises interferon alpha 2a, interferon alpha 2b, interferon alpha 1b, interferon lambda, or a mutant or derivative thereof.
  • the interferon or a mutant or derivative thereof is PEGylated.
  • the interferon-based therapeutic agent is selected from the group consisting of P1101, Peggabine, Pyloxine, PegIntron, Ganfujin, LeFuneng, Ganneneng, Rodine, Yundesu, Derived probiotics and peginterferon lambda.
  • the interferon-based therapeutics include agonists of TLRs, RLRs, and STINGs signaling pathways.
  • the interferon-based therapeutic is selected from the group consisting of GS-9620, GS-9688, RO7020531, RO6864018, TQ-A3334, JNJ-4964, SB9200, MIW815, DMXAA, MK-1454, and diABZI.
  • the anticancer agent is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-oxidedoxedoxedoxedoxethyl
  • chemotherapeutic agents such as alkylating agents: nimustine, carmustine, lomustine, cyclophosphamide, ifosfamide, mustard, semustine; antimetabolites: desoxy Fluoroguanosine, Doxefluoroguanidine, 5-Fluorouracil, Mercaptopurine, Thioguanine, Cytarabine, Fluoroguanosine, Tegafur, Gemcitabine, Carmofur, Hydroxyurea, Methotrexate, Euphor Antitumor antibiotics: actinomycin D, doxorubicin, daunorubicin, epirubicin, mitomycin, pelomycin, pingyangmycin, Pirarubicin; anti-tumor animal and plant ingredients: irinotecan, harringtonine, hydroxycamptothecin, vinorelbine, paclitaxel, nab-paclitaxel, taxotere, topotecan, vincristine
  • Immune checkpoint inhibitors eg inhibitors of PD-1, PD-L1, CTLA4, eg antibodies selected from the group consisting of Nivolumab, Pembrolizumab, Atezolizumab, Durvalumab, Avelumab; or
  • iii) Small molecule targeted drugs such as Imatinib, Gefitinib, Bortezomib, Erlotinib, Sorafenib, Lenalidomide, Sunitinib, Dasatinib, Nilo tinib, lapatinib, pazopanib, everolimus, vandetanib, crizotinib, vemurafenib, ruxolitinib, axitinib, vismodegib, carfilzomib, regora Nitrate, bosutinib, tofacitinib, cabozantinib, ponatinib, pomalidomide, trametinib, dabrafenib, afatinib, icotinib, ibrutinib Ceritinib, Ceritinib, Idelaris, Apatinib, Palbociclib, Lenvatin
  • the anticancer agent is selected from the group consisting of oxaliplatin, epirubicin, paclitaxel and gemcitabine, more preferably gemcitabine.
  • the pharmaceutical combination is used to treat cancer in a subject by the methods of the present invention.
  • Figure 1 The elution profile of mIFN- ⁇ 4 fermentation supernatant SP Sepharose Fast Flow.
  • Figure 7 The plasma concentration-time curve of a single subcutaneous injection of 1 ⁇ g/mice of PEG-mIFN- ⁇ 4 in BALB/c mice.
  • Figure 8 Survival curve of the experimental comparison of the therapeutic effects of PEG-mIFN- ⁇ 4 continuous and intermittent administration in the treatment of transplanted liver cancer H 22 in mice.
  • FIG. 1 Survival curve (A) and mortality curve (B) of PEG-mIFN- ⁇ 4 continuous administration, intermittent administration and combined administration of gemcitabine in the treatment of mouse transplanted liver cancer H22.
  • Figure 10 The experimental mortality curve comparing the therapeutic effects of PEG-mIFN- ⁇ 4 continuous administration, intermittent administration, and gemcitabine administration in the treatment of mouse transplanted lung cancer LLC.
  • Figure 13 The mortality curve of PEG-mIFN- ⁇ 4 in the treatment of breast cancer 4T1 by continuous administration, intermittent administration, and combined administration of gemcitabine.
  • Figure 14 Survival curve of intermittent administration of interferon combined with anticancer agent epirubicin in the treatment of transplanted liver cancer H22 in mice.
  • Figure 15 Survival curve of intermittent administration of interferon combined with anticancer agent oxaliplatin in the treatment of transplanted liver cancer H22 in mice.
  • Figure 16 Survival curve of intermittent administration of interferon combined with anticancer agent paclitaxel in the treatment of transplanted liver cancer H22 in mice.
  • the inventors have unexpectedly discovered that a significantly better therapeutic effect can be achieved by intermittently administering interferon over multiple courses of treatment, eg at substantially the same dose, administering substantially the same amount of The same number of times, or in substantially the same treatment time to achieve significantly better treatment effect.
  • intermittently administering interferon over multiple courses of treatment eg at substantially the same dose, administering substantially the same amount of The same number of times, or in substantially the same treatment time to achieve significantly better treatment effect.
  • continuous administration of interferon over a long period of time will result in depletion of immune cells and difficult recovery.
  • the efficacy of interferon depends on the immune system, so interferon-based therapy can achieve good therapeutic effects such as high tumor suppression in the early stage of treatment when immune cells are still sufficient. With cell depletion, the efficacy of the treatment will decrease substantially, and even continued administration of interferon may not improve the efficacy of the treatment.
  • interferon By intermittently administering interferon, this problem can be avoided. For example, after administering interferon for a period of time to achieve a certain therapeutic effect, stop the administration of interferon for a period of time before partial immune suppression and depletion of immune cells, so that immune cells can recover as soon as possible, and then re-administration of interferon can still achieve better results. therapeutic efficacy. On the basis of intermittent administration of interferon, the combined use of anticancer agents can further improve the therapeutic efficacy.
  • the present invention provides a method of treating cancer in a subject comprising administering to the subject
  • interferon may be a human interferon, eg may be a type I, II or III interferon, such as interferon alpha, interferon beta, interferon gamma or interferon lambda, preferably interferon alpha.
  • interferon-based therapeutic agent refers to a therapeutic agent capable of producing at least part of the effects of natural interferon.
  • an "interferon-based therapeutic” may include naturally isolated or recombinantly produced interferons, such as type I interferons, preferably interferon alpha.
  • Suitable interferon alphas include, but are not limited to, interferon alpha 2a, interferon alpha 2b, or interferon alpha 1b.
  • Interferon-based therapeutics may also include interferon mutants, such as Ganfujin (recombinant integrated interferon, Infergen).
  • Interferon-based therapeutics may also include interferon derivatives, such as PEGylated interferons or mutants thereof, albuminated interferons or mutants thereof, and other protein and organic modified interferons and their mutants etc.
  • PEGylated interferons or mutants thereof include, but are not limited to, pegylated interferon alfa 2a (e.g., pegylated interferon alfa 2a (e.g.
  • PegIntron, 40Kd bibranched UPEG-NHS modified Peginterferon alfa 2b (such as PegIntron , 12Kd linear PEG-SC modification), pegylated interferon ⁇ 2b (such as pegbine, Y-type 40Kd PEG modified), cultured interferon ⁇ -2 (such as derived prebiotics), pegylated interferon ⁇ , P1101, etc.
  • pegIntron alfa 2b such as PegIntron , 12Kd linear PEG-SC modification
  • pegylated interferon ⁇ 2b such as pegbine, Y-type 40Kd PEG modified
  • cultured interferon ⁇ -2 such as derived prebiotics
  • the "interferon-based therapeutic” comprises a long-acting interferon, eg, a PEGylated interferon or a mutant thereof. In some embodiments, the "interferon-based therapeutic” comprises a short-acting interferon.
  • the "interferon-based therapeutic” comprises a plurality of different types of interferons or mutants or derivatives thereof.
  • interferon-based therapeutic agent encompasses various therapeutic agents that have been approved for marketing comprising interferon or a mutant or derivative thereof.
  • the "interferon-based therapeutic” is pegbine (Y-type 40Kd PEG-modified, peginterferon alfa 2b, Tebao Bio).
  • the "interferon-based therapeutic” is Pegasys (pegylated interferon alfa 2a, Roche).
  • the "interferon-based therapeutic” is PegIntron (pegylated interferon alfa 2b injection, Schering-Plough).
  • the "interferon-based therapeutic agent” is dryfujin (Infergen, recombinant integrated interferon, Amgen, USA).
  • the "interferon-based therapeutic” is Glycin (recombinant human interferon alpha 2b, Schering-Plough).
  • the "interferon-based therapeutic” is rosin (interferon alpha 2a, Roche).
  • the "interferon-based therapeutic agent” is Yundesu (recombinant human interferon alpha 1b, Beijing Sanyuan Genetic Engineering Co., Ltd.).
  • the "interferon-based therapeutic agent” is Peiyisheng (Peicheng Interferon Alpha-2 Injection, Beijing Kain Technology Co., Ltd.). In some embodiments, the "interferon-based therapeutic” is peginterferon lambda (Nanogen Pharmaceutical biotechbology).
  • the interferon-based therapeutic comprises interferon alpha 2b. In some preferred embodiments, the interferon-based therapeutic comprises polyethylene glycol-modified interferon alpha 2b. In some preferred embodiments, the interferon-based therapeutic is pegbine.
  • the "interferon-based therapeutic” comprises an interferon, or a mutant or derivative thereof, comprising any one of SEQ ID NOs: 1-5
  • the amino acid sequence of , or said interferon or a mutant or derivative thereof comprises at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, and Amino acid sequences of at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity.
  • the "interferon-based therapeutic” also includes a nucleic acid molecule, eg, a recombinant nucleic acid expression vector, that encodes an interferon or a mutant or derivative thereof.
  • a nucleic acid molecule eg, a recombinant nucleic acid expression vector, that encodes an interferon or a mutant or derivative thereof.
  • Suitable expression vectors, particularly for therapeutic applications, can be readily determined by those skilled in the art.
  • Interferon-based therapeutics may also include substances capable of promoting endogenous interferon production, such as agonists of TLRs, RLRs, and STINGs signaling pathways.
  • substances that promote endogenous interferon production include, but are not limited to, GS-9620, GS-9688, RO7020531, RO6864018, TQ-A3334, JNJ-4964, SB9200, MIW815, DMXAA, MK-1454, diABZI, etc. [1]- [34] .
  • a "continuous course of treatment” refers to a course of treatment in which the therapeutic agent is administered such that an effective concentration (eg, effective blood concentration) of an interferon (exogenous or endogenous) in a patient is continuously maintained or maintained
  • an effective concentration eg, effective blood concentration
  • an interferon exogenous or endogenous
  • In vivo concentrations eg, blood concentrations
  • neopterin the primary pharmacodynamic marker of steroids
  • a "continuous course of treatment” can be defined as a course of treatment in which one or more interferon-based therapeutics are administered such that, substantially throughout the course of treatment, the concentration of neopterin in the subject is higher than the neopterin level prior to the first administration
  • concentration of neopterin baseline concentration
  • concentration of neopterin concentration in vivo can be determined by methods well known in the art.
  • the administration regimen of the interferon-based therapeutic will generally depend on the properties of the selected therapeutic itself, such as its in vivo half-life.
  • a long-acting interferon typically 30-120 hours in vivo half-life
  • it may be administered about once a week, about every two weeks in the continuous course of treatment, or monthly or even longer at elevated doses
  • short-acting interferons usually 2-5 hours in vivo half-life
  • it can be administered once a day or every other day or three times a week, or at elevated doses (eg, 9-36 MIU or higher) , may be administered once a week or, in the case of reduced doses, multiple times a day.
  • the number of administrations of the interferon-based therapeutic agent is not particularly limited as long as it satisfies the above-mentioned definition of a continuous course of treatment.
  • One of skill in the art can determine the continuous course of treatment based on in vivo concentrations (eg, blood concentrations) of pharmacodynamic markers of the interferon-based therapeutic, eg, neopterin.
  • the "interferon-based therapeutic” may be administered on its conventional schedule in the "continuous course of treatment.”
  • dry Fujin synthetic integrated interferon, Infergen
  • interferon alpha 2b such as Ganneng
  • interferon alpha 2a such as rosin
  • interferon alpha 1b such as Yundesu
  • peginterferon alfa 2a such as Peginterferon
  • peginterferon alfa 2b such as PegIntron or Interferon alfa-2 (eg, Peginterferon lambda) or peginterferon lambda
  • P1101 was administered at approximately 400 ⁇ g every two weeks.
  • Albuminylated interferon alfa 2b can be administered from about 900 to about 1800 ⁇ g every two weeks or about 1200
  • the lengths of each of the multiple consecutive courses of treatment should allow the therapeutic agent to achieve some therapeutic effect, but should avoid excessive depletion of immune cells.
  • Immune cell depletion during a course of treatment can often be identified by changes in relevant therapeutic indicators. For example, when the relevant therapeutic index shows that the therapeutic effect of the therapeutic agent is getting worse, it means that the immune cells may have been over-consumed.
  • each of the plurality of consecutive courses of treatment is at least about 1 week in length.
  • each of the plurality of consecutive courses of treatment is up to about 24 weeks in length.
  • each of the plurality of consecutive courses of treatment is about 1 week to about 24 weeks in length, eg, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 13 weeks, about 14 weeks, about 15 weeks, about 16 weeks, about 17 weeks, about 18 weeks, about 19 weeks , about 20 weeks, about 21 weeks, about 22 weeks, about 23 weeks, about 24 weeks.
  • each of the plurality of consecutive courses of treatment is about 1 week to about 12 weeks in length. In some more preferred embodiments, each of the plurality of consecutive courses of treatment is about 1 week to about 8 weeks in length. In some further preferred embodiments, each of the plurality of consecutive courses of treatment is about 2 weeks to about 6 weeks in length. In some further preferred embodiments, each of the plurality of consecutive courses of treatment is about 2 weeks in length.
  • the endpoint of each successive course of therapy may be the last administration of the interferon-based therapeutic plus about 5 of the therapeutic agent in the successive course of treatment
  • the length of the continuous course of treatment is the time from the first administration to the last administration, plus about 5 in vivo half-lives of the therapeutic agent. It is believed that after 5 half-lives of administration of the therapeutic agent, there will no longer be a substantial therapeutic effect.
  • the time interval between the plurality of consecutive courses of treatment may depend on the regeneration cycle of the immune cells.
  • the length of the interval should allow for the recovery of immune cells in the patient that have been reduced as a result of the treatment to a level that is effective for the treatment. It is generally believed that about 1-2 weeks are required for immune cell proliferation, therefore, the interval between the multiple consecutive courses of treatment may be as short as about 1 week.
  • each successive course of treatment is separated by at least about 1 week.
  • the consecutive courses of treatment are separated by at most about 24 weeks.
  • each successive course of treatment is separated by about 1 week to about 24 weeks, eg, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 13 weeks, about 14 weeks, about 15 weeks, about 16 weeks, about 17 weeks, about 18 weeks, about 19 weeks, about 20 weeks , about 21 weeks, about 22 weeks, about 23 weeks, about 24 weeks.
  • each successive course of treatment is separated by about 1 week to about 12 weeks. In some more preferred embodiments, each successive course of treatment is separated by about 1 week to about 8 weeks. In some further preferred embodiments, each successive course of treatment is separated by about 2 weeks to about 6 weeks.
  • each successive course of treatment is about 1 week to about 24 weeks in length, and wherein each course of treatment is separated by about 1 week to about 24 weeks.
  • each successive course of treatment is about 1 week to about 12 weeks in length, and wherein each course of treatment is separated by about 1 week to about 12 weeks.
  • each successive course of treatment is about 1 week to about 8 weeks in length, and wherein each course of treatment is separated by about 1 week to about 8 weeks.
  • each successive course of treatment is about 2 weeks to about 6 weeks in length, and wherein each course of treatment is separated by about 2 weeks to about 6 weeks.
  • each consecutive course of treatment is about 2 weeks in length, and wherein each course of treatment is separated by about 2 weeks.
  • the "interferon-based therapeutic" is administered at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25 or more consecutive courses.
  • the sessions have substantially the same time interval between sessions.
  • the interferon-based therapeutic is pegbine, and each successive course of treatment is about 5 weeks to about 24 weeks in length, with about 2 to 8 weeks between courses of treatment.
  • the cancer includes, but is not limited to, leukemia (eg, acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), chronic lymphocytic leukemia, polycapillary leukemia) , liver cancer, lung cancer, colorectal cancer, skin cancer, stomach cancer, breast cancer, prostate cancer, non-Hodgkin's lymphoma, melanoma, multiple myeloma, laryngeal papilloma, follicular lymphoma, AIDS-related Kapo sarcoma, renal cell carcinoma, etc.
  • the disease is myeloproliferative neoplasm (MPN).
  • the disease is liver cancer, lung cancer, breast cancer, colorectal cancer, or melanoma.
  • an "additional anticancer agent” may be a chemotherapeutic agent including, but not limited to, alkylating agents such as: nimustine, carmustine, lomustine, cyclophosphamide, ifosfamide, glycerol Phosphoryl mustard, semustine; such as chemotherapeutic antimetabolites: deoxyfluguanosine, doxifloguanidine, 5-fluorouracil, mercaptopurine, thioguanine, cytarabine, fluguanosine, tega Fluoride, gemcitabine, carmofluor, hydroxyurea, methotrexate, euflavin, amcitabine, capecitabine; such as chemotherapy drugs
  • Antitumor antibiotics actinomycin D, doxorubicin, daunorubicin such as chemotherapeutic drugs, anti-tumor animal and plant ingredients: irinotecan, harringtonine, hydroxycampto
  • “Additional anticancer agents” can also be small molecule targeted drugs, including but not limited to imatinib, gefitinib, bortezomib, erlotinib, sorafenib, lenalidomide, Nilotinib, dasatinib, nilotinib, lapatinib, pazopanib, everolimus, vandetanib, crizotinib, vemurafenib, ruxolitinib, axitinib , vismodegib, carfilzomib, regorafenib, bosutinib, tofacitinib, cabozantinib, ponatinib, pomalidomide, trametinib, dabrafenib, alfa Apatinib, Icotinib, Ibrutinib, Ceritinib, Idelaris, Apatinib,
  • Additional anticancer agents can also be tumor-associated antigen-specific antibodies such as Rituxan, Herceptin, and the like.
  • an “additional anticancer agent” may also be an immune checkpoint inhibitor, eg, an inhibitor of PD1, PDL1, CTLA4, etc., such as a specific antibody.
  • immune checkpoint inhibitors include, but are not limited to, Nivolumab, Pembrolizumab, Atezolizumab, Durvalumab, Avelumab, and the like.
  • the chemotherapeutic agent is oxaliplatin. In some specific embodiments, the chemotherapeutic agent is epirubicin. In some specific embodiments, the chemotherapeutic agent is paclitaxel.
  • the chemotherapeutic agent is an antimetabolite chemotherapeutic agent, such as gemcitabine, capecitabine, amcitabine. In some most preferred embodiments, the chemotherapeutic agent is gemcitabine.
  • the administration of the "interferon-based therapeutic" does not overlap the administration of the additional anticancer agent.
  • the additional anticancer agent is administered between the multiple consecutive courses of treatment.
  • the additional anticancer agent can be administered for a period of time between the administration of the "interferon-based therapeutic agent" that is less than or equal to the interval.
  • the administration of the "interferon-based therapeutic" overlaps the administration of the additional anticancer agent.
  • the additional anti-cancer agent is administered during and between the plurality of consecutive courses of treatment, i.e., the additional anti-cancer agent is administered throughout the treatment period (all overlapping).
  • the additional anticancer agent is administered according to a conventional regimen, eg, its conventional administration regimen for the particular cancer being treated.
  • the additional anticancer agent is gemcitabine
  • the administration regimen of gemcitabine may be three times every four weeks: once a week for three weeks followed by one week off; twice every three weeks: every Twice a week, followed by one week off; or once every two weeks.
  • the methods of the invention result in tumor regression or reduction in tumor volume or prolong survival in a subject.
  • the methods of the invention result in: reducing the number of cancer cells, reducing tumor volume, inhibiting (ie, slowing or stopping) infiltration of cancer cells into peripheral organs, inhibiting (ie, slowing or stopping) tumor metastasis, inhibiting tumor growth, and/or Or relieve one or more symptoms associated with cancer.
  • the present invention provides a pharmaceutical composition comprising an interferon-based therapeutic for use in the treatment of cancer in a subject by the methods of the present invention.
  • the pharmaceutical composition further includes an additional anticancer agent.
  • the interferon-based therapeutic and anticancer agents are as defined above.
  • the present invention provides the use of an interferon-based therapeutic in the manufacture of a pharmaceutical composition for treating cancer in a subject by the methods of the present invention.
  • the pharmaceutical composition further includes an additional anticancer agent.
  • the interferon-based therapeutic and anticancer agents are as defined above.
  • the present invention provides the use of an interferon-based therapeutic in the manufacture of a pharmaceutical composition for treating cancer in a subject in combination with an additional anticancer agent.
  • the interferon-based therapeutic and anticancer agents are as defined above.
  • the interferon-based therapeutic agent and/or additional anticancer agent can be administered according to the methods described herein.
  • the present invention provides the use of an interferon-based therapeutic agent in the manufacture of a pharmaceutical composition for enhancing the efficacy of additional anticancer agents.
  • the interferon-based therapeutic and anticancer agents are as defined above.
  • the interferon-based therapeutic agent and/or additional anticancer agent can be administered according to the methods described herein.
  • the present invention provides a pharmaceutical combination comprising an interferon-based therapeutic agent and an additional anticancer agent for use in the treatment of cancer in a subject by the methods of the present invention.
  • the interferon-based therapeutic and anticancer agents are as defined above.
  • the invention provides a kit comprising an interferon-based therapeutic agent, an additional anticancer agent, and instructions for use providing information on the interferon-based therapeutic agent and the additional anticancer agent Cancer agents can be administered according to the methods of the invention or according to the descriptions of the methods of the invention to treat cancer in a subject.
  • the interferon-based therapeutic and anticancer agents are as defined above.
  • Interferon is also widely used in cancer treatment, but the effect needs to be improved. To investigate whether interferon indeed results in partial immunosuppression and depletion of immune cells due to continuous administration, resulting in suboptimal subsequent treatment effects, a comparison of the efficacy of intermittent and continuous interferon administration was performed on mice.
  • the cDNA sequence of mIFN- ⁇ 4 was optimized and coded according to the preferred codons of Pichia pastoris. GenScript Biotechnology Co., Ltd. was entrusted to synthesize the cDNA.
  • the cDNA encoding mIFN- ⁇ 4 was inserted into pPIC9K plasmid by homologous recombination, transformed into TOP10 competent cells, coated with LB solid medium, and cultured at 37°C overnight.
  • Single clones were picked and inoculated in LB liquid medium, and cultured at 37°C overnight; plasmids were extracted, double digested with XhoI and NotI, and positive clones were identified by nucleic acid electrophoresis, which were further confirmed by nucleic acid sequencing.
  • the positive cloned plasmid was cut and linearized with SalI, and electrotransformed into Pichia GS115, spread on RD plates, and cultured at 28-30°C for 3 days.
  • the scale of the fermenter is 30L.
  • the induction time is about 30h; the fermentation supernatant is collected by centrifugation, and the 5kD hollow fiber membrane tube is ultrafiltered and concentrated by 3 to 5 times, and the buffer system is replaced by 20mM phosphate buffer-20mM arginine hydrochloride-50mM sodium chloride buffer solution (pH6.5); then loaded on SP Sepharose Fast Flow chromatography column (GE Healthcare, column bed ⁇ 38mm ⁇ 160mm), 20mM phosphate Buffer - 20 mM arginine hydrochloride (pH 6.5) (solution A) washes about 3 bed volumes; with solution A and 20 mM phosphate buffer - 20 mM arginine hydrochloride - 1 M sodium chloride solution (pH 6.5) (solution B) to carry out gradient elution, collect mIFN- ⁇ 4 target samples, and take samples for non-reducing SDS
  • the mIFN- ⁇ 4 SP Sepharose Fast Flow purification target sample was concentrated by ultrafiltration with a 5K ultrafiltration membrane pack and replaced the buffer with 20mM phosphate buffer-50mM arginine hydrochloride-10mM methionine-20mM sodium chloride (pH7 .0), the concentration was adjusted to about 1.0 mg/ml; glycosidase was added according to the mass ratio of mIFN- ⁇ 4 protein to enzyme of about 20:1, and the glycosyl was removed by digestion at 25°C for about 20 h.
  • the digested sample was diluted about 6 times with 5mM boric acid buffer-10mM arginine hydrochloride (pH9.0), and loaded on a Q Sepharose Fast Flow column (GE Healthcare, column bed ⁇ 50mm ⁇ 154mm), 20mM boric acid buffer - 20 mM arginine hydrochloride - 10 mM methionine (pH 9.0) (solution C) washed about 3 bed volumes; with solution C and 20 mM borate buffer - 20 mM arginine hydrochloride - 10 mM formazan Gradient elution was performed with thionine-0.3M sodium chloride (pH9.0) (solution D), and the target sample for removing glycosyl mIFN- ⁇ 4 was collected, and the pH was adjusted to about pH5.0 with 10% acetic acid; The package was concentrated by ultrafiltration, and the buffer was replaced with 5mM acetic acid/sodium acetate buffer-50mM arginine hydrochloride-100m
  • the deglycosylated mIFN- ⁇ 4 stock solution was replaced by ultrafiltration with a 5kD ultrafiltration membrane and the buffer was 5mM acetic acid/sodium acetate buffer-50mM sodium chloride (pH 5.0); about 333ml of the sample was taken (the content of deglycosylated mIFN ⁇ 4 was About 500mg), add about 22ml 0.8M boric acid/sodium hydroxide buffer (pH9.4), stir well; according to the mass ratio of protein and 40kD Y-type polyethylene glycol succinimide ester (YPEG-NHS) is about 1 :8 Add YPEG-NHS, stir rapidly, and react at room temperature for 10 minutes; add about 20 ml of 0.2M methionine to terminate the reaction, and adjust the pH to 5.0 with 10% acetic acid.
  • YPEG-NHS Y-type polyethylene glycol succinimide ester
  • the content of bacterial endotoxin determined by PEG-mIFN- ⁇ 4 stock solution Limulus Reagent method is ⁇ 15EU/mg; using commercial mIFN- ⁇ 4 (R&D, Cat. No. 12115-1) as the standard, mouse fibroblast/encephalomyocarditis virus (L929/ The specific activity determined by the EMCV) cytopathic inhibition method was 6.1 ⁇ 10 6 U/mg.
  • BALB/c mice were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.
  • the plasma concentration of PEG-mIFN- ⁇ 4 was detected by double-antibody sandwich ELISA.
  • the detection kit uses Mouse IFN alpha Platinum ELISA kit (Cat. No. BMS6027/BMS6027TEN, Thermo), but replaces its reporter antibody with anti-PEG antibody 3.3biotin (Institute of Biomedical Sciences, Academia Sinica, Taiwan) to avoid endogenous mIFN - Interference of alpha on assay results.
  • mice were subcutaneously injected with 1 ⁇ g/mice of PEG-mIFN- ⁇ 4 on the back of the neck, the plasma drug concentration peak time (T max ) was 24h, the peak concentration (C max ) was 268ng/ml, and the elimination half-life (T 1 /2 ) is 28.3h.
  • the samples whose blood drug concentration is lower than the lower limit of quantification are counted as 0 before the blood drug concentration reaches the peak, and the samples after reaching the peak are expressed as BLQ.
  • Example 4 Comparative study on the therapeutic effects of intermittent administration and continuous administration of PEG-mIFN- ⁇ 4 on transplanted liver cancer H 22 in mice.
  • BALB/c mice were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.
  • mouse liver cancer H 22 cell line was purchased from China Center for Type Culture Collection (CCTCC).
  • mice 6-8 weeks old, 18-22g SPF grade BALB/c mice were subcutaneously inoculated with H 22 cells (0.5 ⁇ 10 6 cells/mL, 0.2 ml/mice) in the axilla of the right forelimb.
  • the patients were randomly divided into groups on the day of tumor receiving.
  • the mode of administration of PEG-mIFN- ⁇ 4 was subcutaneous injection on the back of the neck, and the dose was 1 microgram per animal.
  • the PEG-mIFN- ⁇ 4 intermittent administration group was administered once every 48 hours for 3 consecutive times, and the drug was discontinued for 144 hours; 4 consecutive rounds.
  • the PEG-mIFN- ⁇ continuous administration group was administered once every 48 hours for 21 consecutive administrations.
  • the normal saline control group was given an equal volume of normal saline.
  • the survival curve is shown in Figure 8, and the statistical comparison results are shown in Table 3. To 40 days after tumor receiving, the median survival time of the normal saline control group was 19.5 days, and that of the PEG-mIFN- ⁇ 4 continuous administration group was 38 days; the survival rate of the PEG-mIFN- ⁇ 4 intermittent administration group was 93.3%, which was not yet available. Calculate the median survival time.
  • the inventors have surprisingly found that by intermittently administering PEG-mIFN- ⁇ 4 and concurrently administering gemcitabine, significantly better results can be obtained in H22 tumor therapy than either gemcitabine alone or intermittent administration of PEG-mIFN- ⁇ 4 alone treatment effect.
  • intermittent administration every 48 hours, 3 consecutive administrations, and withdrawal for 144 hours; 8 rounds
  • continuous administration administration every 48 hours, continuous administration
  • gemcitabine administration PEG-mIFN- ⁇ 4 administered once every 48 hours for 3 consecutive times, drug withdrawal for 240 hours, continued for 6 rounds; gemcitabine administered once a week
  • BALB/c mice were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd., and the mouse liver cancer H22 cell line was purchased from China Center for Type Culture Collection (CCTCC).
  • CTCC China Center for Type Culture Collection
  • mice 6-8 weeks old, 18-22g SPF grade BALB/c mice were subcutaneously inoculated with H22 cells (1 ⁇ 10 6 cells/mL, 0.2ml/mice) in the axilla of the right forelimb.
  • the patients were randomly divided into groups on the day of tumor receiving.
  • the mode of administration of PEG-mIFN- ⁇ 4 was subcutaneous injection on the back of the neck, and the dose was 1 microgram per animal.
  • Gemcitabine was administered intraperitoneally at a dose of 60 mg/kg. The administration started on the same day after the grouping; the PEG-mIFN- ⁇ 4 intermittent administration group was administered once every 48 hours for 3 consecutive administrations, and the drug was discontinued for 144 hours for 8 rounds.
  • the PEG-mIFN- ⁇ continuous administration group was administered once every 48 hours.
  • PEG-mIFN- ⁇ 4 was administered once every 48 hours for 3 consecutive times, and the drug was discontinued for 240 hours for 6 rounds; gemcitabine was administered once a week.
  • the gemcitabine monotherapy group was dosed once a week.
  • the normal saline control group was given an equal volume of normal saline.
  • the survival curve is shown in Figure 9, and the statistical comparison results are shown in Table 4.
  • the median survival time of PEG-mIFN- ⁇ 4 continuous administration group was 37.0 days, and intermittent administration group was 64.0 days, both longer than 17.5 days of normal saline control group.
  • the survival time of the PEG-mIFN- ⁇ 4 intermittent administration group was significantly longer than that of the continuous administration group.
  • intermittent administration of PEG-mIFN- ⁇ 4 showed significantly better efficacy than continuous administration.
  • the median survival time of gemcitabine alone group was 58.5 days, and the survival rate at the end of the observation period (108 days after receiving the tumor) in the PEG-mIFN- ⁇ 4 intermittent administration group combined with gemcitabine group was 75.0%, and the survival curves of the two groups were significantly different ( P ⁇ 0.0001), the survival time of PEG-mIFN- ⁇ 4 combined with gemcitabine group was significantly prolonged compared with gemcitabine alone group.
  • the median survival time of the PEG-mIFN- ⁇ 4 intermittent administration group was 64.0 days, and the survival rate at the end of the observation period (108 days after receiving the tumor) in the PEG-mIFN- ⁇ 4 intermittent administration group combined with gemcitabine was 75.0%.
  • the survival curves were significantly different (P ⁇ 0.0001), and the survival time of the PEG-mIFN- ⁇ 4 intermittent administration combined with gemcitabine group was significantly longer than that of the PEG-mIFN- ⁇ 4 intermittent administration group alone.
  • the present inventors studied the tumor suppressive effect of intermittent administration of PEG-mIFN- ⁇ 4 combined with gemcitabine on mouse lung cancer LLC, and explored the combined effect of interferon and gemcitabine. Through experiments, it was found that intermittent administration of PEG-mIFN- ⁇ 4 and simultaneous administration of gemcitabine can also achieve significantly better therapeutic effects in the treatment of lung cancer LLC, compared with single administration of gemcitabine or intermittent administration of PEG-mIFN- ⁇ 4 alone .
  • PEG-mIFN- ⁇ 4 intermittently (administered once every 48 hours, administered 4 times continuously, and discontinued for 192 hours; lasted for 5 rounds), continuous administration (administered once every 48 hours, administered continuously for 32 times) ), combined with gemcitabine administration (PEG-mIFN- ⁇ 4 administered once every 48 hours for 4 consecutive times, drug withdrawal for 360 hours, continued for 4 rounds; gemcitabine administered once a week) on mouse transplanted lung cancer LLC. compare research.
  • C57BL/6N mice were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.
  • mouse lung cancer LLC cell lines were purchased from Peking Union Cell Resource Center, China.
  • mice 6-8 weeks old, 18-22g SPF grade C57BL/6N mice were subcutaneously inoculated with lung cancer LLC (1 ⁇ 10 6 cells/mL, 0.2 ml/mice) under the axilla of the right forelimb.
  • the patients were randomly divided into groups on the day of tumor receiving.
  • the mode of administration of PEG-mIFN- ⁇ 4 was subcutaneous injection on the back of the neck, and the dose was 1 microgram per animal. The administration started on the same day after the grouping; the intermittent administration group of PEG-mIFN- ⁇ 4 was administered once every 48 hours for 4 consecutive administrations, and the drug was discontinued for 192 hours; it continued for 5 rounds.
  • the PEG-mIFN- ⁇ continuous administration group was administered once every 48 hours.
  • the PEG-mIFN- ⁇ 4 combined with gemcitabine group was administered once every 48 hours for 4 consecutive times, and the drug was discontinued for 360 hours for 4 rounds; gemcitabine was administered once a week.
  • the normal saline control group was given an equal volume of normal saline. Gemcitabine was injected intraperitoneally at a dose of 60 mg/kg.
  • the mortality of each group is shown in Figure 10, and the statistical comparison results are shown in Table 5.
  • the mortality rate of the PEG-mIFN- ⁇ 4 continuous administration group was 100.0%, and that of the intermittent administration group was 34.6%, and the difference was significant (P ⁇ 0.001). Mortality was significantly lower than in the continuous dosing group.
  • the intermittent administration of PEG-mIFN- ⁇ 4 showed significantly better efficacy than the continuous administration.
  • Example 7 Study on intermittent administration of interferon combined with gemcitabine in the treatment of colorectal cancer
  • the inventors have studied the tumor suppressive effect of intermittent administration of PEG-mIFN- ⁇ 4 combined with gemcitabine on mouse colorectal cancer CT26, and explored the combined effect of interferon and gemcitabine. Through experiments, it was found that intermittent administration of PEG-mIFN- ⁇ 4 and concurrent administration of gemcitabine, compared with single administration of gemcitabine or intermittent administration of PEG-mIFN- ⁇ 4 alone, can also achieve significantly better treatment of colorectal cancer CT26. treatment effect.
  • PEG-mIFN- ⁇ 4 was intermittently administered (administered once every 48 hours, administered 3 times in a row, and discontinued for 144 hours; lasting 11 rounds), continuous (administered once every 48 hours, administered 55 times in a row), Combined administration of gemcitabine (PEG-mIFN- ⁇ 4 administered once every 48 hours, 3 consecutive administrations, discontinued for 240 hours for 8 rounds); gemcitabine administered once a week) on the effect of transplanted colorectal cancer CT26 in mice.
  • BALB/c mice were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.
  • CT26 mouse colorectal cancer cell line was purchased from the Cell Resource Center of Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences.
  • the anticancer agent was gemcitabine (Jiangsu Hansoh Pharmaceutical).
  • mice 6-8 weeks old, 18-22g SPF grade BALB/c mice were subcutaneously inoculated with CT26 cells (1 ⁇ 10 6 cells/mL, 0.2ml/mice) in the axilla of the right forelimb.
  • the patients were randomly divided into groups on the day of tumor receiving.
  • the mode of administration of PEG-mIFN- ⁇ 4 was subcutaneous injection on the back of the neck, and the dose was 1 microgram per animal.
  • Gemcitabine was injected intraperitoneally at a dose of 60 mg/kg. The administration started on the same day after the grouping; the PEG-mIFN- ⁇ 4 intermittent administration group was administered once every 48 hours for 3 consecutive administrations, and the drug was discontinued for 144 hours for 11 rounds.
  • the PEG-mIFN- ⁇ continuous administration group was administered once every 48 hours.
  • PEG-mIFN- ⁇ 4 combined with gemcitabine group PEG-mIFN- ⁇ was administered once every 48 hours for 3 consecutive times, and the drug was discontinued for 240 hours for 8 rounds; gemcitabine was administered once a week.
  • the gemcitabine monotherapy group was dosed once a week.
  • the normal saline control group was given an equal volume of normal saline.
  • the survival curve is shown in Figure 11, and the statistical comparison results are shown in Table 6.
  • the PEG-mIFN- ⁇ 4 continuous administration group was 53.5 days, and the intermittent administration group was 89.0 days, which were both greater than 49.5 days of the normal saline control group.
  • the survival time of the PEG-mIFN- ⁇ 4 intermittent administration group was significantly longer than that of the continuous administration group.
  • intermittent administration of PEG-mIFN- ⁇ 4 showed significantly better efficacy than continuous administration.
  • the median survival time of the PEG-mIFN- ⁇ 4 intermittent administration group was 89.0 days, and the survival rate at the end of the observation period (110 days after receiving the tumor) in the PEG-mIFN- ⁇ 4 intermittent administration group combined with gemcitabine was 75.0%.
  • Example 8 Study on intermittent administration of interferon combined with gemcitabine in the treatment of melanoma
  • PEG-mIFN- ⁇ 4 intermittently (administered once every 48 hours, administered 3 times in a row, and discontinued for 240 hours; lasted for 4 rounds) and continuously administered (administered once every 48 hours, administered 24 times in a row) , Combined administration of gemcitabine (PEG-mIFN- ⁇ 4 administered once every 48 hours for 3 consecutive times, drug withdrawal for 240 hours for 4 rounds; gemcitabine administered once a week) on transplanted melanoma B16 in mice compare research.
  • the mode of administration of PEG-mIFN- ⁇ 4 was subcutaneous injection on the back of the neck, and the dose was 1 microgram per animal.
  • Gemcitabine was injected intraperitoneally at a dose of 60 mg/kg. The administration started on the same day after the grouping; the PEG-mIFN- ⁇ 4 intermittent administration group was administered once every 48 hours for 3 consecutive administrations, and the drug was discontinued for 240 hours for 4 rounds.
  • the PEG-mIFN- ⁇ 4 continuous administration group was administered once every 48 hours.
  • PEG-mIFN- ⁇ 4 was administered once every 48 hours for 3 consecutive times, and the drug was discontinued for 240 hours for 4 rounds; gemcitabine was administered once a week.
  • the gemcitabine monotherapy group was dosed once a week.
  • the normal saline control group was given an equal volume of normal saline.
  • the present inventors studied the tumor suppressive effect of intermittent administration of PEG-mIFN- ⁇ 4 combined with gemcitabine on mouse transplanted breast cancer 4T1, and explored the combined effect of interferon and gemcitabine. Experiments have found that intermittent administration of PEG-mIFN- ⁇ 4 and concurrent administration of gemcitabine can also achieve significantly better treatment in breast cancer 4T1 treatment than gemcitabine alone or intermittent administration of PEG-mIFN- ⁇ 4 alone Effect.
  • intermittent administration of PEG-mIFN- ⁇ 4 once every 48 hours, 3 consecutive administrations, and withdrawal for 144 hours; continuous 5 rounds
  • continuous administration of PEG-mIFN- ⁇ 4 once every 48 hours, continuous administration of 25 times
  • gemcitabine administration administered once every 48 hours for 3 consecutive times, drug withdrawal for 240 hours, continued for 4 rounds; gemcitabine administered once a week
  • BALB/c mice were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd., and the mouse breast cancer 4T1 cell line was purchased from China Center for Type Culture Collection (CCTCC).
  • CTCC China Center for Type Culture Collection
  • mice 6-8 weeks old, 18-22g SPF grade BALB/c mice were subcutaneously inoculated with breast cancer 4T1 (1 ⁇ 10 6 cells/mL, 0.2ml/mice) in the axilla of the right forelimb. 4 days after receiving the tumor, the patients were randomly divided into groups.
  • the mode of administration of PEG-mIFN- ⁇ 4 was subcutaneous injection on the back of the neck, and the dose was 1.5 micrograms per animal. The administration started on the same day after the grouping; the PEG-mIFN- ⁇ 4 intermittent administration group was administered once every 48 hours for 3 consecutive administrations, and the drug was discontinued for 144 hours; it continued for 5 rounds.
  • the PEG-mIFN- ⁇ 4 continuous administration group was administered once every 48 hours.
  • PEG-mIFN- ⁇ 4 combined with gemcitabine group, PEG-mIFN- ⁇ 4 was administered once every 48 hours for 3 consecutive times, and the drug was discontinued for 240 hours for 4 rounds; gemcitabine was administered once a week.
  • the normal saline control group was given an equal volume of normal saline. Gemcitabine was injected intraperitoneally at a dose of 60 mg/kg.
  • the mortality of each group is shown in Figure 13, and the statistical comparison results are shown in Table 8.
  • the mortality rate of the PEG-mIFN- ⁇ 4 continuous administration group was 87.5%, and that of the intermittent administration group was 58.3%, which were lower than 100.0% of the normal saline control group.
  • intermittent administration of PEG-mIFN- ⁇ 4 showed significantly better efficacy than continuous administration.
  • the mortality rate of the PEG-mIFN- ⁇ 4 intermittent administration group alone was 58.3%, and the mortality rate of the PEG-mIFN- ⁇ 4 intermittent administration combined with gemcitabine group was 4.2%, and the difference in mortality between the two groups was significant. (P ⁇ 0.0001), the mortality rate of PEG-mIFN- ⁇ 4 intermittent administration combined with gemcitabine group was significantly lower than that of PEG-mIFN- ⁇ 4 intermittent administration group alone.
  • Table 8 The experimental mortality results of PEG-mIFN- ⁇ 4 continuous, intermittent and combined administration of gemcitabine in the treatment of mouse transplanted breast cancer 4T1
  • Example 10 Intermittent administration of interferon combined with anticancer agent epirubicin in the treatment of transplanted liver cancer H 22 in mice
  • the anti-cancer agent used in this example is epirubicin, an anti-tumor antibiotic in chemotherapeutic drugs, and the effect of intermittent administration of interferon combined with anti-cancer agent epirubicin in the treatment of transplanted liver cancer H 22 in mice was investigated.
  • BALB/c mice were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.
  • mouse liver cancer H 22 cell line was purchased from China Collection of Type Cultures (CCTCC), and the manufacturer of epirubicin was Peking Union Medical College pharmacy.
  • mice 6-8 weeks old, 18-22 g SPF grade BALB/c mice were inoculated subcutaneously with hepatoma H 22 tumor cells (1 ⁇ 10 6 cells/mL, 0.2 ml/mice) in the axilla of the right forelimb; There were 28 animals in each group, half male and half female; the experimental groups included the normal saline control group, the epirubicin single-use group, and the PEG-mIFN- ⁇ 4 combined epirubicin group.
  • the administration started on the same day after the grouping and was administered once a week; PEG-mIFN- ⁇ 4 was subcutaneously injected into the back of the neck, and the dose was 1 ⁇ g each time; epirubicin was intraperitoneally injected, and the dose was 3.5 mg per kilogram each time. ; Normal saline control group was given an equal volume of normal saline.
  • the survival curve of each group of mice is shown in Figure 14, and the median survival time is shown in Table 9.
  • the median survival time of the saline group was 13.5 days
  • the median survival time of the epirubicin group was 15 days
  • the survival rate of the PEG-mIFN- ⁇ 4 combined epirubicin group during the observation period was 57.1%.
  • the survival curve of PEG-mIFN- ⁇ 4 combined with epirubicin group was significantly different from that of normal saline group (P ⁇ 0.0001); Epirubicin alone could significantly prolong the survival of tumor-bearing mice (P ⁇ 0.0001).
  • Example 11 Intermittent administration of interferon combined with anticancer agent oxaliplatin in the treatment of transplanted liver cancer H 22 in mice
  • the anticancer agent used in this example is the chemotherapeutic drug oxaliplatin, and the effect of intermittent administration of interferon combined with the anticancer agent oxaliplatin in the treatment of transplanted liver cancer H 22 in mice was investigated.
  • BALB/c mice were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.
  • H 22 mouse liver cancer cell line was purchased from China Collection Center for Type Cultures (CCTCC); the manufacturer of oxaliplatin was Qilu Pharmaceutical Co., Ltd. (Hainan) Co., Ltd.
  • mice 6-8 weeks old, 18-22 g SPF grade BALB/c mice were inoculated subcutaneously with hepatoma H 22 tumor cells (1 ⁇ 10 6 cells/mL, 0.2 ml/mice) in the axilla of the right forelimb; There were 28 animals in each group, half male and half female; the experimental groups included the normal saline control group, the oxaliplatin single-use group, and the PEG-mIFN- ⁇ 4 combined with oxaliplatin group.
  • the administration started on the day after the grouping and was administered once a week; PEG-mIFN- ⁇ 4 was subcutaneously injected on the back of the neck, with a dose of 1 ⁇ g each time; oxaliplatin was intraperitoneally injected, with a dose of 10 mg/kg each time; physiological
  • the saline control group was given an equal volume of normal saline.
  • the survival curve of each group of mice is shown in Figure 15, and the median survival time is shown in Table 10.
  • the median survival time was 11 days in the saline group, 12.5 days in the oxaliplatin group, and 31 days in the PEG-mIFN- ⁇ 4 combined with oxaliplatin group.
  • Example 12 Intermittent administration of interferon combined with anticancer drug paclitaxel in the treatment of transplanted liver cancer H 22 in mice
  • the anticancer agent used in this example is the chemotherapeutic drug paclitaxel, and the effect of intermittent administration of interferon combined with the anticancer agent paclitaxel in the treatment of transplanted liver cancer H 22 in mice was investigated.
  • BALB/c mice were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.
  • H 22 mouse liver cancer cell line was purchased from China Type Culture Collection (CCTCC)
  • the manufacturer of paclitaxel was Jiangsu Osaikang Pharmaceutical Co., Ltd. Co., Ltd.
  • mice 6-8 weeks old, 18-22 g SPF grade BALB/c mice were inoculated subcutaneously with hepatoma H 22 tumor cells (1 ⁇ 10 6 cells/mL, 0.2 ml/mice) in the axilla of the right forelimb; There were 28 animals in each group, half male and half female; the experimental groups included the normal saline control group, the paclitaxel single group, and the PEG-mIFN- ⁇ 4 combined paclitaxel group.
  • the administration started on the same day after the grouping and was administered once a week; PEG-mIFN- ⁇ 4 was subcutaneously injected on the back of the neck, and the dose was 1 microgram each time; Paclitaxel was intraperitoneally injected, 10 mg per kilogram each time.
  • the survival curve of each group of mice is shown in Figure 16, and the median survival time is shown in Table 11.
  • the median survival time of the saline group was 15 days, and that of the paclitaxel group was 17 days; during the observation period, the survival rate of the PEG-mIFN- ⁇ 4 combined with paclitaxel group was 82.1%.
  • interferon after administering interferon for a period of time to achieve a certain curative effect, stop the administration of interferon for a period of time before partial immune suppression and depletion of immune cells, so that immune cells can recover as soon as possible, and then re-administration of interferon can still achieve better results.
  • therapeutic efficacy A better therapeutic effect can be achieved by intermittently administering the therapeutic agent of interferon in a rational combination with other drugs.
  • SEQ ID NO: 1 is the amino acid sequence of interferon alpha 2a (e.g. rosin or peroxin)
  • SEQ ID NO: 2 is the amino acid sequence of interferon alpha 2b (eg Gannron or PegIntron or Pegbine)
  • SEQ ID NO: 3 is the amino acid sequence of recombinant integrated interferon (such as Ganfujin)
  • SEQ ID NO: 4 is the amino acid sequence of interferon alpha-2 (eg derived from probiotics)
  • SEQ ID NO:6 is the amino acid sequence of murine interferon ⁇ 4
  • Li L, et.al.Anti-HBV response to toll-like receptor 7agonist GS-9620 is associated with intrahepatic aggregates of T cells and B cells.J Hepatol.2018 May;68(5):912-921.doi: 10.1016/j.jhep.2017.12.008.Epub 2017Dec 14.
  • Zeng Y, et.al.Toll-like receptors, long non-coding RNA NEAT1, and RIG-I expression are associated with HBeAg-positive chronic hepatitis B patients in the active phase.J Clin Lab Anal.2019Jun;33 (5):e22886.doi:10.1002/jcla.22886.Epub 2019Mar 29.

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Abstract

本发明属于生物医药领域，公开了一种治疗癌症的药物组合，包括基于干扰素的治疗剂和额外的抗癌剂。本发明还公开了该药物组合物的施用方法。

Description

一种基于干扰素的癌症治疗方法和药物组合 发明领域
本发明涉及生物医药领域，公开了一种基于干扰素的癌症治疗方法和药物组合。具体而言，本发明涉及一种基于干扰素的癌症治疗方法，包括给对象i)间歇性施用基于干扰素的治疗剂，和ii)施用额外的抗癌剂，优选吉西他滨。本发明还涉及用于所述方法的药物组合。
发明背景
干扰素(IFN)是一类具有多种功能的活性蛋白质，是由单核细胞和淋巴细胞产生的细胞因子。它们具有广谱的抗病毒、影响细胞生长，以及分化、调节免疫功能等多种生物活性，是目前最主要的抗病毒感染和抗肿瘤生物制品。
目前，干扰素被分成I、II、III型干扰素。I型干扰素包括IFN-α与IFN-β等。IFN-α主要由单核-巨噬细胞产生，此外B细胞和成纤维细胞也能合成IFN-α；IFN-β主要由成纤维细胞产生。IFN-α/β二者结合相同受体，分布广泛，包括单核-巨噬细胞、多形核白细胞、B细胞、T细胞、血小板、上皮细胞、内皮细胞与肿瘤细胞等。已知IFNα有23个以上的亚型。IFNβ仅有一种亚型。II型干扰素即γ干扰素，主要由活化的T细胞(包括Th0、Th1细胞和几乎所有的CD8+T细胞)和NK细胞产生，是所谓的淋巴因子的一种。IFN-γ可以以细胞外基质相连的形式存在，故通过旁邻方式控制细胞生长，其可以分布在除成熟红细胞以外的几乎所有细胞表面，IFNγ仅有1个亚型。III型干扰素主要指干扰素λ。
干扰素、干扰素突变体和干扰素衍生物已经被广泛批准用于各类的治疗。目前已批准用于人体临床治疗的干扰素类及突变体包括干扰素α2a，干扰素α2b、干扰素α1b、复合干扰素(干复津)、干扰素突变体(如乐复能)、干扰素β、干扰素γ(1b)等。干扰素衍生物包括聚乙二醇干扰素α2a，聚乙二醇干扰素α2b，集成干扰素等。
此外，近年来随着TLRs、RLRs、STING等信号通路的发现和阐明，一系列作用于上述信号通路以产生干扰素的激动剂被发现和阐明，利用该类激动剂也成为未来以干扰素类物质为治疗基础的新方向，目前该类激动剂已被用于HBV及相关肿瘤治疗研究。
I型IFN是FDA批准临床用于癌症的首批免疫治疗药物。其中，疗效最好的为血液系统肿瘤，淋巴系统肿瘤次之，实体瘤最差，例如恶性黑色素瘤、卵巢瘤、结肠直肠癌等疗效最高不超过20％。此外，干扰素与化疗，放疗及免疫治疗等联合应用是目前干扰素抗肿瘤的常用治疗方法，例如干扰素联用5-氟尿嘧啶治疗肝癌、结直肠癌，干扰素联合顺铂、卡铂等治疗肺癌，这些研究虽然取得一些成效，但是总体效果一般，预后较差。此外，干扰素在临床上一般采用大剂量、连续的施用方式，不可避免带来药物相关 的毒性大、患者的依从性和耐受性差等问题。
综上，干扰素或干扰素联合抗癌剂或放疗等治疗手段，在临床应用上取得了一些进展，但总体上看，疗效并不理想，预后差，毒副作用大。
因此，本领域仍然需要对基于干扰素的疗法进行改进，以获得更佳的治疗效果。发明简述
在一方面，本发明提供一种在对象中治疗癌症的方法，其包括给所述对象
i)间隔地施用多个连续疗程的基于干扰素的治疗剂；和
ii)施用额外的抗癌剂。
在一些实施方案中，所述基于干扰素的治疗剂包含干扰素或其突变体或衍生物，或包含编码干扰素或其突变体或衍生物的核酸分子，或包含促进内源干扰素产生的物质。
在一些实施方案中，所述干扰素是I型、II型或III型干扰素，例如干扰素α、干扰素β、干扰素γ或干扰素λ，优选干扰素α。
在一些实施方案中，所述基于干扰素的治疗剂包含干扰素α2a、干扰素α2b、干扰素α1b、干扰素λ，或其突变体或衍生物。
在一些实施方案中，所述干扰素或其突变体或衍生物是PEG化修饰的。
在一些实施方案中，所述基于干扰素的治疗剂选自P1101、派格宾、派罗欣、佩乐能、干复津、乐复能、甘乐能、罗荛素、运德素、派益生和聚乙二醇干扰素λ。
在一些实施方案中，所述基于干扰素的治疗剂包括TLRs、RLRs及STINGs信号通路的激动剂。
在一些实施方案中，所述基于干扰素的治疗剂选自GS-9620、GS-9688、RO7020531、RO6864018、TQ-A3334、JNJ-4964、SB9200、MIW815、DMXAA、MK-1454和diABZI。
在一些实施方案中，在所述连续疗程中，所述基于干扰素的治疗剂的施用使得基本上在整个疗程期间，对象体内新喋呤的浓度高于首次施用前的新喋呤浓度，例如是首次施用前新喋呤浓度的大约110％、大约120％、大约130％、大约140％、大约150％、大约200％、大约250％或更高。
在一些实施方案中，所述连续疗程的长度为该疗程中第一次施用至最后一次施用的时间，再加上所述治疗剂的大约5个体内半衰期。
在一些实施方案中，所述多个连续疗程各自的长度为大约1周-大约24周，优选大约1周-大约12周，更优选大约1周-大约8周，更优选大约2周-大约6周。
在一些实施方案中，每个连续疗程的长度为大约1周-大约12周，且其中各疗程之间间隔大约1周-大约12周。
在一些实施方案中，各连续疗程之间间隔大约1周-大约24周，优选大约1周-大约12周，更优选大约1周-大约8周，更优选大约2周-大约6周。
在一些实施方案中，每个连续疗程的长度为大约1周-大约8周，且其中各疗程之间间隔大约1周-大约8周。
在一些实施方案中，每个连续疗程的长度为大约2周-大约6周，且其中各疗程之间间隔大约2周-大约6周。
在一些实施方案中，所述基于干扰素的治疗剂施用2个-25个或以上的连续疗程。
在一些实施方案中，所述多个连续疗程的长度基本上相同。
在一些实施方案中，所述各疗程之间具有基本上相同的时间间隔。
在一些实施方案中，所述癌症选自白血病(如急性淋巴细胞白血病(ALL)、急性髓细胞白血病(AML)、慢性髓细胞白血病(CML)、慢性淋巴细胞白血病、多毛细血管白血病)、肝癌、肺癌、结直肠癌、皮肤癌、胃癌、乳腺癌、前列腺癌、非霍奇金淋巴瘤、黑色素瘤、多发性骨髓瘤、喉乳头状瘤、滤泡性淋巴瘤、艾滋病相关卡波氏肉瘤和肾细胞癌，优选是肝癌、肺癌、乳腺癌、结直肠癌或黑色素瘤。
在一些实施方案中，基于干扰素的治疗剂的施用与所述额外的抗癌剂的施用不重叠。
在一些实施方案中，在所述多个连续疗程之间施用所述额外的药剂。
在一些实施方案中，所述“基于干扰素的治疗剂”的施用与所述额外的药剂的施用重叠。
在一些实施方案中，在所述多个连续疗程期间和所述多个连续疗程之间施用所述额外的药剂。
在一些实施方案中，所述额外的抗癌剂按照其常规方案施用。
在一些实施方案中，所述抗癌剂是
i)化疗剂，例如烷化剂：尼莫司汀、卡莫司汀、洛莫司汀、环磷酰胺、异环磷酰胺、甘磷酰芥、司莫司汀；抗代谢药：去氧氟鸟苷、多西氟鸟啶、5-氟尿嘧啶、巯嘌呤、硫鸟嘌呤、阿糖胞苷、氟鸟苷、替加氟、吉西他滨、卡莫氟、羟基脲、甲氨蝶呤、优福定、安西他滨、卡培他滨；抗肿瘤抗生素：放线菌素D、多柔比星、柔红霉素、表柔比星、丝裂霉素、培洛霉素、平阳霉素、吡柔比星；抗肿瘤动植物成分药：伊立替康、三尖杉酯碱、羟基喜树碱、长春瑞宾、紫杉醇、白蛋白紫杉醇、泰索帝、拓扑替康、长春新碱、长春地辛、长春酰胺、长春碱、替尼泊苷、依托泊苷、榄香烯；抗肿瘤激素类化疗药物：阿他美坦、阿那曲唑、氨鲁米特、来曲唑、福美坦、甲他孕酮、他莫昔芬；化疗药物杂类：门冬酰胺酶、卡铂、顺铂、达卡巴嗪、奥沙利铂、乐沙定、可铂奥沙、米托蒽醌、丙卡巴肼；或
ii)免疫检查点抑制剂，例如PD-1、PD-L1、CTLA4的抑制剂，例如选自以下的抗体：Nivolumab、Pembrolizumab、Atezolizumab、Durvalumab、Avelumab；或
iii)小分子靶向药物，例如伊马替尼、吉非替尼、硼替佐米、厄洛替尼、索拉非尼、来那度胺、舒尼替尼、达沙替尼、尼洛替尼、拉帕替尼、帕唑帕尼、依维莫司、凡德他尼、克唑替尼、威罗菲尼、ruxolitinib、阿西替尼、vismodegib、卡非佐米、瑞戈非尼、博舒替尼、托法替尼、卡博替尼、帕纳替尼、泊马度胺、曲美替尼、达拉菲尼、阿法替尼、埃克替尼、依鲁替尼、色瑞替尼、艾代拉里斯、阿帕替尼、帕布昔利布、乐伐替尼、阿西替尼、埃克替尼、阿帕替尼、sonidegib、cobimetinib、osimertinib、alectinib、ixazomib； 或
iv)肿瘤相关抗原特异性抗体类，如美罗华、赫赛丁；
优选地，所述抗癌剂选自奥沙利铂、表柔比星、紫杉醇和吉西他滨，更优选是吉西他滨。
在另一方面，本发明提供一种用于对象中治疗癌症的药物组合，其包含基于干扰素的治疗剂和抗癌剂。
在一些实施方案中，所述基于干扰素的治疗剂包含干扰素或其突变体或衍生物，或包含编码干扰素或其突变体或衍生物的核酸分子，或包含促进内源干扰素产生的物质。
在一些实施方案中，所述干扰素是I型、II型或III型干扰素，例如干扰素α、干扰素β、干扰素γ或干扰素λ，优选干扰素α。
在一些实施方案中，所述基于干扰素的治疗剂包含干扰素α2a、干扰素α2b、干扰素α1b、干扰素λ，或其突变体或衍生物。
在一些实施方案中，所述干扰素或其突变体或衍生物是PEG化修饰的。
在一些实施方案中，所述基于干扰素的治疗剂选自P1101、派格宾、派罗欣、佩乐能、干复津、乐复能、甘乐能、罗荛素、运德素、派益生和聚乙二醇干扰素λ。
在一些实施方案中，所述基于干扰素的治疗剂包括TLRs、RLRs及STINGs信号通路的激动剂。
在一些实施方案中，所述基于干扰素的治疗剂选自GS-9620、GS-9688、RO7020531、RO6864018、TQ-A3334、JNJ-4964、SB9200、MIW815、DMXAA、MK-1454和diABZI。
在一些实施方案中，所述抗癌剂是
i)化疗剂，例如烷化剂：尼莫司汀、卡莫司汀、洛莫司汀、环磷酰胺、异环磷酰胺、甘磷酰芥、司莫司汀；抗代谢药：去氧氟鸟苷、多西氟鸟啶、5-氟尿嘧啶、巯嘌呤、硫鸟嘌呤、阿糖胞苷、氟鸟苷、替加氟、吉西他滨、卡莫氟、羟基脲、甲氨蝶呤、优福定、安西他滨、卡培他滨；抗肿瘤抗生素：放线菌素D、多柔比星、柔红霉素、表柔比星、丝裂霉素、培洛霉素、平阳霉素、吡柔比星；抗肿瘤动植物成分药：伊立替康、三尖杉酯碱、羟基喜树碱、长春瑞宾、紫杉醇、白蛋白紫杉醇、泰索帝、拓扑替康、长春新碱、长春地辛、长春酰胺、长春碱、替尼泊苷、依托泊苷、榄香烯；抗肿瘤激素类化疗药物：阿他美坦、阿那曲唑、氨鲁米特、来曲唑、福美坦、甲他孕酮、他莫昔芬；化疗药物杂类：门冬酰胺酶、卡铂、顺铂、达卡巴嗪、奥沙利铂、乐沙定、可铂奥沙、米托蒽醌、丙卡巴肼；或
ii)免疫检查点抑制剂，例如PD-1、PD-L1、CTLA4的抑制剂，例如选自以下的抗体：Nivolumab、Pembrolizumab、Atezolizumab、Durvalumab、Avelumab；或
iii)小分子靶向药物，例如伊马替尼、吉非替尼、硼替佐米、厄洛替尼、索拉非尼、来那度胺、舒尼替尼、达沙替尼、尼洛替尼、拉帕替尼、帕唑帕尼、依维莫司、凡德他尼、克唑替尼、威罗菲尼、ruxolitinib、阿西替尼、vismodegib、卡非佐米、瑞戈非尼、 博舒替尼、托法替尼、卡博替尼、帕纳替尼、泊马度胺、曲美替尼、达拉菲尼、阿法替尼、埃克替尼、依鲁替尼、色瑞替尼、艾代拉里斯、阿帕替尼、帕布昔利布、乐伐替尼、阿西替尼、埃克替尼、阿帕替尼、sonidegib、cobimetinib、osimertinib、alectinib、ixazomib；或
iv)肿瘤相关抗原特异性抗体类，如美罗华、赫赛丁；
优选地，所述抗癌剂选自奥沙利铂、表柔比星、紫杉醇和吉西他滨，更优选是吉西他滨。
在一些实施方案中，所述药物组合用于通过本发明的方法在对象中治疗癌症。
附图说明
图1.mIFN-α4发酵上清液SP Sepharose Fast Flow洗脱图谱。
图2.mIFN-α4 SP Sepharose Fast Flow纯化目的样非还原型SDS-PAGE(分离胶浓度14％，银染显色)电泳结果。
图3.去除糖基mIFN-α4Q Sepharose Fast Flow纯化洗脱图谱。
图4.去除糖基mIFN-α4Q Sepharose Fast Flow纯化原液非还原型SDS-PAGE(分离胶浓度14％，银染显色)电泳结果。
图5.PEG-mIFN-α4 SP Sepharose Fast Flow纯化洗脱图谱。
图6.PEG-mIFN-α4原液非还原型SDS-PAGE(银染显色)电泳结果。
图7.BALB/c小鼠单次皮下注射1μg/只PEG-mIFN-α4的血药浓度-时间曲线。
图8.PEG-mIFN-α4连续、间歇给药治疗小鼠移植性肝癌H 22疗效比较实验生存曲线。
图9.PEG-mIFN-α4连续给药、间歇给药、联用吉西他滨给药治疗小鼠移植性肝癌H22疗效比较实验生存曲线(A)和死亡率曲线(B)。
图10.PEG-mIFN-α4连续给药、间歇给药、联用吉西他滨给药治疗小鼠移植性肺癌LLC疗效比较实验死亡率曲线。A：接瘤至45天；B：接瘤至64天。
图11.PEG-mIFN-α4连续给药、间歇给药、联用吉西他滨给药治疗结直肠癌CT26比较实验生存曲线(A)和死亡率曲线(B)。
图12.PEG-mIFN-α4连续给药、间歇给药、联用吉西他滨给药治疗黑色素瘤B16比较实验死亡率曲线。
图13、PEG-mIFN-α4连续给药、间歇给药、联用吉西他滨给药治疗乳腺癌4T1比较实验死亡率曲线。
图14.间歇施用干扰素联合抗癌剂表柔比星治疗小鼠移植性肝癌H22的生存曲线。
图15.间歇施用干扰素联合抗癌剂奥沙利铂治疗小鼠移植性肝癌H22的生存曲线。
图16.间歇施用干扰素联合抗癌剂紫杉醇治疗小鼠移植性肝癌H22的生存曲线。
具体实施方式
本发明人意外地发现，相比于在一个固定疗程中连续施用干扰素，通过多个疗程间 歇性地施用干扰素可以取得显著更优的治疗效果，例如在施用基本上相同的剂量，施用基本上相同的次数，或在基本上相同的治疗时间内取得显著更优的治疗效果。不受任何理论约束，认为长时间地连续施用干扰素，将会造成免疫细胞的消耗且难以恢复。干扰素的疗效依赖于免疫系统，因此基于干扰素的治疗在免疫细胞仍然充足的治疗前期能够取得很好的治疗效果例如很高的肿瘤抑制，然而，在治疗后期由于长期施用干扰素导致的免疫细胞消耗，治疗效果将大幅下降，并且即使继续施用干扰素也可能无法提高治疗效力。而通过间歇性施用干扰素，可以避免这一问题。例如，施用一段时间的干扰素取得一定疗效后，在免疫部分抑制和免疫细胞耗竭前停止干扰素的施用一段时间，使得免疫细胞能够尽快恢复，之后再次重新施用干扰素，仍然能够取得较好的治疗效力。在间歇性施用干扰素的基础上，联用抗癌剂能够进一步提高治疗效力。
因此，在一方面，本发明提供一种在对象中治疗癌症的方法，其包括给所述对象
i)间隔地施用多个连续疗程的基于干扰素的治疗剂；和
ii)施用额外的抗癌剂。
“干扰素”可以是人干扰素，例如可以是I、II或III型干扰素，例如干扰素α、干扰素β、干扰素γ或干扰素λ，优选干扰素α。
如本文所用，“基于干扰素的治疗剂”指的是能够产生天然干扰素的至少部分效果的治疗剂。
例如，“基于干扰素的治疗剂”可以包括天然分离的或重组产生的干扰素，例如I型干扰素，优选干扰素α。合适的干扰素α包括但不限于干扰素α2a、干扰素α2b或干扰素α1b。
“基于干扰素的治疗剂”还可以包括干扰素突变体，例如干复津(重组集成干扰素，Infergen)。
“基于干扰素的治疗剂”还可以包括干扰素衍生物，例如PEG化修饰的干扰素或其突变体、白蛋白化的干扰素或其突变体、以及其它蛋白和有机物修饰的干扰素及其突变体等。PEG化修饰的干扰素或其突变体的实例包括但不限于聚乙二醇干扰素α2a(例如派罗欣，40Kd双分支UPEG-NHS修饰)、聚乙二醇干扰素α2b(例如佩乐能，12Kd线型PEG-SC修饰)、聚乙二醇干扰素α2b(例如派格宾，Y型40Kd PEG修饰)、培集成干扰素α-2(例如派益生)、聚乙二醇干扰素λ、P1101等。
在一些实施方案中，所述“基于干扰素的治疗剂”包含长效干扰素，例如PEG化修饰的干扰素或其突变体。在一些实施方案中，所述“基于干扰素的治疗剂”包含短效干扰素。
在一些实施方案中，所述“基于干扰素的治疗剂”包含多种不同类型的干扰素或其突变体或衍生物。
术语“基于干扰素的治疗剂”涵盖了已经获批上市的各种包含干扰素或其突变体或衍生物的治疗剂。在一些实施方案中，所述“基于干扰素的治疗剂”是派格宾(Y型40Kd PEG修饰，聚乙二醇干扰素α2b，特宝生物)。在一些实施方案中，所述“基于干扰素 的治疗剂”是派罗欣(聚乙二醇干扰素α2a，Roche)。在一些实施方案中，所述“基于干扰素的治疗剂”是佩乐能(聚乙二醇干扰素α2b注射剂，Schering-Plough)。在一些实施方案中，所述“基于干扰素的治疗剂”是干复津(Infergen，重组集成干扰素，美国安进)。在一些实施方案中，所述“基于干扰素的治疗剂”是甘乐能(重组人干扰素α2b，Schering-Plough)。在一些实施方案中，所述“基于干扰素的治疗剂”是罗荛素(干扰素α2a，Roche)。在一些实施方案中，所述“基于干扰素的治疗剂”是运德素(重组人干扰素α1b，北京三元基因工程有限公司)。在一些实施方案中，所述“基于干扰素的治疗剂”是派益生(培集成干扰素α-2注射液，北京凯因科技股份有限公司)。在一些实施方案中，所述“基于干扰素的治疗剂”是聚乙二醇干扰素λ(Nanogen Pharmaceutical biotechbology)。
在一些优选实施方案中，所述基于干扰素的治疗剂包含干扰素α2b。在一些优选实施方案中，所述基于干扰素的治疗剂包含聚乙二醇修饰的干扰素α2b。在一些优选实施方案中，所述基于干扰素的治疗剂是派格宾。
在一些实施方案中，所述“基于干扰素的治疗剂”包含干扰素或其突变体或衍生物，所述干扰素或其突变体或衍生物包含SEQ ID NO:1-5中任一项的氨基酸序列，或者所述干扰素或其突变体或衍生物包含与SEQ ID NO:1-5中任一项具有至少60％、至少65％、至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％序列相同性的氨基酸序列。
在一些实施方案中，所述“基于干扰素的治疗剂”也包括编码干扰素或其突变体或衍生物的核酸分子例如重组核酸表达载体。合适的表达载体特别是适于治疗应用的表达载体是本领域技术人员能容易确定的。
“基于干扰素的治疗剂”还可以包括能够促进内源干扰素产生的物质，例如TLRs、RLRs及STINGs信号通路的激动剂。促进内源干扰素产生的物质的实例包括但不限于GS-9620、GS-9688、RO7020531、RO6864018、TQ-A3334、JNJ-4964、SB9200、MIW815、DMXAA、MK-1454、diABZI等 [1]-[34]。
如本文所用，“连续疗程”指的是在该疗程中，所述治疗剂的施用使得持续维持患者体内干扰素(外源或内源)的起效浓度(例如起效血液浓度)或者维持干扰素类物质的主要药效学标志物新喋呤(NPT)的体内浓度(例如血液浓度)高于未使用所述治疗剂时的浓度(初始浓度或基线浓度)。由于干扰素类物质的主要药效学标志物新喋呤与干扰素类物质施用具有良好的相关性，因此特别优选地采用新喋呤的体内浓度(如血液浓度)变化作为“连续疗程”的指标。例如，“连续疗程”可以定义为，在该疗程中，施用一或多次基于干扰素的治疗剂，使得基本上在整个疗程期间，对象体内新喋呤的浓度高于首次施用前的新喋呤浓度(基线浓度)，例如是首次施用前新喋呤浓度的大约110％、大约120％、大约130％、大约140％、大约150％、大约200％、大约250％或更高。体内新喋呤浓度的浓度可以通过本领域公知的方法测定。
在所述连续疗程中，基于干扰素的治疗剂的施用方案通常取决于所选治疗剂本身的特性，例如其体内半衰期。例如，对于长效干扰素(体内半衰期通常为30-120小时)，在该连续疗程中可以约每周、约每两周施用一次，或者在提高剂量情况下可每月甚至更长时间施用一次；而对于短效干扰素(体内半衰期通常为2-5小时)，则可以每天或每两天施用一次或每周施用三次，或者在提高剂量(例如9-36MIU或更高剂量)的情况下，可每周施用一次，或者在降低剂量的情况下，可每天施用多次。在所述连续疗程中，基于干扰素的治疗剂的施用次数并没有特别限制，只要其能满足上述连续疗程的定义。本领域技术人员能够根据基于干扰素的治疗剂的药效学标志物例如新喋呤的体内浓度(如血液浓度)确定所述连续疗程。
在一些实施方案中，在所述“连续疗程”中，“基于干扰素的治疗剂”可以以其常规方案施用。例如，干复津(重组集成干扰素，Infergen)、干扰素α2b(如甘乐能)、干扰素α2a(如罗荛素)、干扰素α1b(如运德素)在3-18MIU的剂量区间内可以每日或每两日施用一次或每周施用三次；聚乙二醇干扰素α2a(如派罗欣)、聚乙二醇干扰素α2b(如佩乐能或派格宾)、培集成干扰素α-2(如派益生)或聚乙二醇干扰素λ可以在45-270μg的剂量区间内每周施用一次。P1101每两周约400μg施用一次。白蛋白化干扰素α2b可以每两周施用约900-约1800μg或每4周施用约1200μg。
所述多个连续疗程各自的时间长度应当允许治疗剂实现一定治疗效果，但是应当避免使免疫细胞过度消耗。疗程中免疫细胞的消耗通常能够通过相关治疗指标的变化来鉴别。例如，当相关治疗指标显示所述治疗剂的疗效变差，则表示免疫细胞可能已经过度消耗。
在一些实施方案中，所述多个连续疗程各自的长度为至少大约1周。
在一些实施方案中，所述多个连续疗程各自的长度为至多大约24周。
在一些实施方案中，所述多个连续疗程各自的长度为大约1周-大约24周，例如大约1周、大约2周、大约3周、大约4周、大约5周、大约6周、大约7周、大约8周、大约9周、大约10周、大约11周、大约12周、大约13周、大约14周、大约15周、大约16周、大约17周、大约18周、大约19周、大约20周、大约21周、大约22周、大约23周、大约24周。
在一些优选实施方案中，所述多个连续疗程各自的长度为大约1周-大约12周。在一些更优选的实施方案中，所述多个连续疗程各自的长度为大约1周-大约8周。在一些进一步优选的实施方案中，所述多个连续疗程各自的长度为大约2周-大约6周。在一些进一步优选的实施方案中，所述多个连续疗程各自的长度为大约2周。
在一些实施方案中，各个连续疗程的终点(也即连续疗程之间的间隔时间的起点)可以是该连续疗程中最后一次施用基于干扰素的治疗剂再加上所述治疗剂的大约5个体内半衰期的时刻。也就是说，所述连续疗程的长度为第一次施用至最后一次施用的时间，再加上所述治疗剂的大约5个体内半衰期。据认为，在治疗剂施用5个半衰期后，将不再产生实质性的治疗效果。
在本发明的方法中，所述多个连续疗程之间的间隔时间可取决于免疫细胞的再生周期。所述间隔时间长度应当允许患者体内由于治疗而降低的免疫细胞恢复至能够有效实现治疗的水平。通常认为，免疫细胞增殖需要大约1-2周，因此，所述多个连续疗程之间的间隔时间最短可以为大约1周。
在一些实施方案中，其中各连续疗程之间至少间隔大约1周。
在一些实施方案中，所述各连续疗程之间至多间隔大约24周。
在一些实施方案中，各连续疗程之间间隔大约1周-大约24周，例如大约1周、大约2周、大约3周、大约4周、大约5周、大约6周、大约7周、大约8周、大约9周、大约10周、大约11周、大约12周、大约13周、大约14周、大约15周、大约16周、大约17周、大约18周、大约19周、大约20周、大约21周、大约22周、大约23周、大约24周。
在一些优选实施方案中，各连续疗程之间间隔大约1周-大约12周。在一些更优选的实施方案中，各连续疗程之间间隔大约1周-大约8周。在一些进一步优选的实施方案中，各连续疗程之间间隔大约2周-大约6周。
在一些实施方案中，其中每个连续疗程的长度为大约1周-大约24周，且其中各疗程之间间隔大约1周-大约24周。
在一些实施方案中，其中每个连续疗程的长度为大约1周-大约12周，且其中各疗程之间间隔大约1周-大约12周。
在一些实施方案中，其中每个连续疗程的长度为大约1周-大约8周，且其中各疗程之间间隔大约1周-大约8周。
在一些实施方案中，其中每个连续疗程的长度为大约2周-大约6周，且其中各疗程之间间隔大约2周-大约6周。
在一些实施方案中，其中每个连续疗程的长度为大约2周，且其中各疗程之间间隔大约2周。
在一些实施方案中，所述“基于干扰素的治疗剂”施用至少2个、至少3个、至少4个、至少5个、至少6个、至少7个、至少8个、至少9个、至少10个、至少11个、至少12个、至少13个、至少14个、至少15个、至少20个、至少25个或更多个连续疗程。
在一些实施方案中，其中所述多个连续疗程的长度基本上相同。
在一些实施方案中，所述各疗程之间具有基本上相同的时间间隔。
在一些具体实施方案中，所述基于干扰素的治疗剂是派格宾，每个连续疗程的长度为大约5周-大约24周，各疗程之间间隔大约2周-8周。
在一些实施方案中，所述癌症包括但不限于白血病(如急性淋巴细胞白血病(ALL)、急性髓细胞白血病(AML)、慢性髓细胞白血病(CML)、慢性淋巴细胞白血病、多毛细血管白血病)、肝癌、肺癌、结直肠癌、皮肤癌、胃癌、乳腺癌、前列腺癌、非霍奇金淋巴瘤、黑色素瘤、多发性骨髓瘤、喉乳头状瘤、滤泡性淋巴瘤、艾滋病相关卡波氏肉瘤、 肾细胞癌等。在一些实施方案中，所述疾病是骨髓增殖性肿瘤(MPN)。在一些优选实施方案中，所述疾病是肝癌、肺癌、乳腺癌、结直肠癌或黑色素瘤。
如本文所用，“额外的抗癌剂”可以是化疗剂包括但不限于，如烷化剂：尼莫司汀、卡莫司汀、洛莫司汀、环磷酰胺、异环磷酰胺、甘磷酰芥、司莫司汀；如化疗药物抗代谢药：去氧氟鸟苷、多西氟鸟啶、5-氟尿嘧啶、巯嘌呤、硫鸟嘌呤、阿糖胞苷、氟鸟苷、替加氟、吉西他滨、卡莫氟、羟基脲、甲氨蝶呤、优福定、安西他滨、卡培他滨；如化疗药物抗肿瘤抗生素：放线菌素D、多柔比星、柔红霉素、表柔比星、丝裂霉素、培洛霉素、平阳霉素、吡柔比星；如化疗药物抗肿瘤动植物成分药：伊立替康、三尖杉酯碱、羟基喜树碱、长春瑞宾、紫杉醇、白蛋白紫杉醇、泰索帝、拓扑替康、长春新碱、长春地辛、长春酰胺、长春碱、替尼泊苷、依托泊苷、榄香烯；如化疗药物抗肿瘤激素类：阿他美坦、阿那曲唑、氨鲁米特、来曲唑、福美坦、甲他孕酮、他莫昔芬；如化疗药物杂类：门冬酰胺酶、卡铂、顺铂、达卡巴嗪、奥沙利铂、乐沙定、可铂奥沙、米托蒽醌、丙卡巴肼。
“额外的抗癌剂”还可以是小分子靶向药物，包括但不限于伊马替尼、吉非替尼、硼替佐米、厄洛替尼、索拉非尼、来那度胺、舒尼替尼、达沙替尼、尼洛替尼、拉帕替尼、帕唑帕尼、依维莫司、凡德他尼、克唑替尼、威罗菲尼、ruxolitinib、阿西替尼、vismodegib、卡非佐米、瑞戈非尼、博舒替尼、托法替尼、卡博替尼、帕纳替尼、泊马度胺、曲美替尼、达拉菲尼、阿法替尼、埃克替尼、依鲁替尼、色瑞替尼、艾代拉里斯、阿帕替尼、帕布昔利布、乐伐替尼、阿西替尼、埃克替尼、阿帕替尼、sonidegib、cobimetinib、osimertinib、alectinib、ixazomib。
“额外的抗癌剂”还可以是肿瘤相关抗原特异性抗体类如美罗华、赫赛丁等。
“额外的抗癌剂”还可以是免疫检查点抑制剂，例如PD1、PDL1、CTLA4等的抑制剂如特异性抗体。免疫检查点抑制剂的实例包括但不限于Nivolumab、Pembrolizumab、Atezolizumab、Durvalumab、Avelumab等。
在一些具体实施方案中，所述化疗剂是奥沙利铂。在一些具体实施方案中，所述化疗剂是表柔比星。在一些具体实施方案中，所述化疗剂是紫杉醇。
在一些优选实施方案中，所述化疗剂是抗代谢类化疗药，如吉西他滨、卡培他滨、安西他滨。在一些最优选实施方案中，所述化疗剂是吉西他滨。
在一些实施方案中，所述“基于干扰素的治疗剂”的施用与所述额外的抗癌剂的施用不重叠。
在一些实施方案中，在所述多个连续疗程之间施用所述额外的抗癌剂。例如，所述额外的抗癌剂可以在“基于干扰素的治疗剂”施用间隔期内施用一段时间，所述一段时间短于或等于所述间隔期。
在一些实施方案中，所述“基于干扰素的治疗剂”的施用与所述额外的抗癌剂的施用重叠。
在一些实施方案中，在所述多个连续疗程期间和所述多个连续疗程之间施用所述额 外的抗癌剂，即在整个治疗期间施用所述额外的抗癌剂(全部重叠)。
在一些实施方案中，所述额外的抗癌剂按照常规方案施用，例如，其针对所治疗的具体癌症的常规施用方案。
在一些实施方案中，所述额外的抗癌剂是吉西他滨，所述吉西他滨的施用方案可以是每四周施用三次：每周一次，施用三周，然后停药一周；每三周施用两次：每周一次，施用两次，然后停药一周；或者每两周施用一次。示例性的吉西他滨的每次剂量为1000mg/m 2表面积。
在一些实施方案中，本发明的方法导致对象中肿瘤消退或肿瘤体积减小或延长生存期。特别地，本发明的方法导致：减少癌细胞的数目、减少肿瘤体积、抑制(即减缓或停止)癌细胞浸润到外周器官中、抑制(即减缓或停止)肿瘤转移、抑制肿瘤生长、和/或缓解与癌症相关的一种或多种症状。
在一方面，本发明提供一种药物组合物，其包含基于干扰素的治疗剂，所述药物组合物用于通过本发明的方法在对象中治疗癌症。在一些实施方案中，所述药物组合物还包括额外的抗癌剂。所述基于干扰素的治疗剂和抗癌剂如上所定义。
在一方面，本发明提供基于干扰素的治疗剂在制备药物组合物中的用途，所述药物组合物用于通过本发明的方法在对象中治疗癌症。在一些实施方案中，所述药物组合物还包括额外的抗癌剂。所述基于干扰素的治疗剂和抗癌剂如上所定义。
在一方面，本发明提供基于干扰素的治疗剂在制备药物组合物中的用途，所述药物组合物用于与额外的抗癌剂组合在对象中治疗癌症。所述基于干扰素的治疗剂和抗癌剂如上所定义。所述基于干扰素的治疗剂和/或额外的抗癌剂可以根据本发明所述的方法施用。
在一方面，本发明提供基于干扰素的治疗剂在制备用于增强额外的抗癌剂的疗效的药物组合物中的用途。所述基于干扰素的治疗剂和抗癌剂如上所定义。所述基于干扰素的治疗剂和/或额外的抗癌剂可以根据本发明所述的方法施用。
在一方面，本发明提供一种药物组合，其包含基于干扰素的治疗剂和额外的抗癌剂，所述药物组合用于通过本发明的方法在对象中治疗癌症。所述基于干扰素的治疗剂和抗癌剂如上所定义。
在一方面，本发明提供一种药盒，其包含基于干扰素的治疗剂、额外的抗癌剂以及使用说明，所述使用说明提供关于所述基于干扰素的治疗剂和所述额外的抗癌剂可以根据本发明的方法或根据本发明的方法施用以在对象中治疗癌症的描述。所述基于干扰素的治疗剂和抗癌剂如上所定义。
实施例
下文实施例仅仅是为了更好地阐释本发明，而不意在以任何方式限制本发明。
实施例1、重组小鼠干扰素α4(mIFN-α4)的制备
干扰素同样广泛用于癌症治疗，但是效果有待改进。为了研究干扰素是否的确由于 连续施用导致免疫部分抑制和免疫细胞耗竭，进而导致后续治疗效果的不佳，在小鼠上进行了干扰素间隙施用和连续施用的效力比较。
大量抗肿瘤效应的研究是以裸鼠(缺乏正常胸腺)为宿主进行实施研究的，而裸鼠的免疫状态弱于正常鼠，难以体现正常状态的免疫反应。而基于正常鼠在抗肿瘤模型中，可部分实现干扰素对免疫系统的效应，体现施用所述干扰素的抗肿瘤疗法的效果。从功能的实现途径上，干扰素应依托宿主的免疫系统才能更完整发挥抗病毒、抗肿瘤作用。由于干扰素具有较强的种属特异性，因此实施例中以正常小鼠为研究对象时，使用鼠干扰素或其衍生物更能反映出其生理学效应及实施效果。在本发明使用小鼠作为研究模型中，采用了聚乙二醇化重组小鼠干扰素α4(PEG-mIFNα4)作为干扰素类治疗药物及其衍生物的代表。
根据mIFN-α4的氨基酸序列(GenBank NP_034634)，按毕赤酵母偏好密码子进行编码并优化设计出mIFN-α4的cDNA序列，委托金斯瑞生物科技有限公司合成该cDNA。将编码mIFN-α4的cDNA同源重组插入pPIC9K质粒，转化TOP10感受态细胞，涂布LB固体培养基，37℃培养过夜。挑取单克隆接种LB液体培养基，37℃培养过夜；提取质粒，XhoI、NotI双酶切，核酸电泳鉴定出阳性克隆，并进一步经核酸测序确认。阳性克隆质粒SalI酶切线性化，电转化转入毕赤酵母GS115，涂布RD平板，28-30℃培养3天。挑取阳性转化子接种YPD液体培养基，28-30℃培养过夜；按OD600nm终浓度为约1转接BMMY培养基，28-30℃培养约24小时；补加甲醇至甲醇终浓度为约1％，28-30℃继续培养约24小时。取培养液离心收集上清液，SDS-PAGE电泳检测mIFN-α4表达量。根据SDS-PAGE电泳结果，筛选出表达量较高且表达稳定的工程菌株进行后续上罐发酵。
发酵罐规模为30L，参照“Pichia Fermentation Process Guidelines”进行发酵培养及甲醇诱导，诱导时长约30h；离心收集发酵上清液，5kD中空纤维膜管超滤浓缩3～5倍，并替换缓冲体系为20mM磷酸盐缓冲液-20mM精氨酸盐酸盐-50mM氯化钠缓冲溶液(pH6.5)；然后上样SP Sepharose Fast Flow层析柱(GE Healthcare，柱床Φ38mm×160mm)，20mM磷酸盐缓冲液-20mM精氨酸盐酸盐(pH6.5)(溶液A)清洗约3个柱床体积；用溶液A和20mM磷酸盐缓冲液-20mM精氨酸盐酸盐–1M氯化钠溶液(pH6.5)(溶液B)进行梯度洗脱，收集mIFN-α4目的样品，取样进行非还原型SDS-PAGE(分离胶浓度为14％，银染显色)检测。洗脱图谱见图1，电泳结果见图2。
mIFN-α4 SP Sepharose Fast Flow纯化目的样用5K超滤膜包超滤浓缩并替换缓冲液为20mM磷酸盐缓冲液-50mM精氨酸盐酸盐-10mM甲硫氨酸-20mM氯化钠(pH7.0)，调整浓度为约1.0mg/ml；按mIFN-α4蛋白与酶质量比为约20：1加入糖苷酶，25℃酶切约20h去除糖基。酶切样品用5mM硼酸缓冲液-10mM精氨酸盐酸盐(pH9.0)稀释约6倍，上样Q Sepharose Fast Flow层析柱(GE Healthcare，柱床Φ50mm×154mm)，20mM硼酸缓冲液-20mM精氨酸盐酸盐-10mM甲硫氨酸(pH9.0)(溶液C)清洗约3个柱床体积；用溶液C和20mM硼酸缓冲液-20mM精氨酸盐酸盐-10mM甲硫氨酸-0.3M氯化钠 (pH9.0)(溶液D)进行梯度洗脱，收集去除糖基mIFN-α4目的样品，并用10％乙酸调pH至约pH5.0；用5K超滤膜包超滤浓缩并替换缓冲液为5mM乙酸/乙酸钠缓冲液-50mM精氨酸盐酸盐-100mM氯化钠(pH5.0)，所得样品即为去糖基化mIFN-α4原液。取样送检，余样-70℃冻存备用。洗脱图谱见图3，SDS-PAGE电泳结果见图4。
去糖基化mIFN-α4原液鲎试剂法测定细菌内毒素含量为＜60EU/mg；以商品化mIFN-α4(R&D，货号12115-1)为标准品，小鼠成纤维细胞/脑心肌炎病毒(L929/EMCV)细胞病变抑制法测定比活性为5.4×10 8U/mg。
实施例2、PEG化重组鼠干扰素(PEG-mIFN-α4)的制备
去糖基化mIFN-α4原液用5kD超滤膜包超滤替换缓冲液为5mM乙酸/乙酸钠缓冲液-50mM氯化钠(pH5.0)；取约333ml样品(去糖基化mIFNα4含量为约500mg)，加入约22ml 0.8M硼酸/氢氧化钠缓冲液(pH9.4)，搅拌均匀；按蛋白与40kD Y型聚乙二醇琥珀酰亚胺酯(YPEG-NHS)质量比为约1:8加入YPEG-NHS，快速搅拌，室温反应10分钟；加入0.2M甲硫氨酸约20ml终止反应，10％乙酸调pH至5.0。加入550ml纯水，再加入600ml 20mM乙酸/乙酸钠缓冲液-20mM精氨酸盐酸盐-10mM甲硫氨酸(pH5.1)(溶液E)，混匀；上样SP Sepharose Fast Flow层析柱(GE Healthcare，柱床Φ50mm×194mm)，溶液E清洗约5个柱床体积；然后用溶液E和20mM乙酸/乙酸钠缓冲液-20mM精氨酸盐酸盐-10mM甲硫氨酸-600mM氯化钠(pH5.1)(溶液F)进行梯度洗脱，收集PEG-mIFN-α4目的样品；5kD超滤膜包超滤替换缓冲液为：20mM磷酸盐缓冲液-123mM氯化钠(pH6.5)并适当浓缩，添加0.5％Tween80至Tween80终浓度为约0.005％，所得样品即为聚乙二醇化鼠干扰素α4原液(PEG-mIFN-α4)；取样送检，余样-70℃冻存备用。洗脱图谱见图5，SDS-PAGE电泳结果见图6。
PEG-mIFN-α4原液鲎试剂法测定细菌内毒素含量为＜15EU/mg；以商品化mIFN-α4(R&D，货号12115-1)为标准品，小鼠成纤维细胞/脑心肌炎病毒(L929/EMCV)细胞病变抑制法测定比活性为6.1×10 6U/mg。
实施例3、健康BALB/c小鼠皮下注射PEG-mIFN-α4的药代动力学研究
本实施例中，BALB/c小鼠购自北京维通利华实验动物技术有限公司。PEG-mIFN-α4血药浓度采用双抗体夹心ELISA法检测。检测试剂盒采用Mouse IFN alpha Platinum ELISA kit(货号BMS6027/BMS6027TEN，Thermo)，但将其报告抗体替换成抗PEG抗体3.3biotin(中央研究院生物医学科学研究所，台湾)，以避免内源性mIFN-α对测定结果的干扰。
6-8周龄、SPF级BALB/c小鼠(N＝60，雌雄各半)于颈背部皮下单次注射1μg/只PEG-mIFN-α4，分别采集药前(0h)和药后6h、15h、24h、36h、48h、72h、96h、120h、168h、216h、264h、312h的血浆样本，测定血药浓度。采用Phoenix WinNonlin 6.4软件计算药代动力学参数。
血药浓度测定结果见表1，血药浓度-给药时间曲线见图7，药代动力学参数结果见表2。BALB/c小鼠颈背部皮下单次注射1μg/只PEG-mIFN-α4，血浆药物浓度达峰时间(T max)为24h，达峰浓度(C max)为268ng/ml，消除半衰期(T 1/2)为28.3h。
表1、BALB/c小鼠单次皮下注射1μg/只PEG-mIFN-α4血药浓度检测结果(N＝5，ng/ml)
Figure PCTCN2021073191-appb-000001
注：在药代动力学分析时，血药浓度低于定量下限的样品，血药浓度达峰前样品按0计，达峰后样品以BLQ表示。
表2、BALB/c小鼠单次皮下注射1μg/只PEG-mIFN-α4的药代动力学参数汇总
Figure PCTCN2021073191-appb-000002
实施例4、PEG-mIFN-α4间歇给药和连续给药对小鼠移植性肝癌H 22的疗效比较研究。
本实施例中，BALB/c小鼠购自北京维通利华实验动物技术有限公司，小鼠肝癌H 22细胞株购自中国典型培养物保藏中心(CCTCC)。
6-8周龄、18-22g SPF级BALB/c小鼠右前肢腋下皮下接种H 22细胞(0.5×10 6个/mL，0.2ml/只)。接瘤当天随机分组，实验分组包括PEG-mIFN-α4连续给药组(N＝15，雌7雄8)、PEG-mIFN-α4间歇给药组(N＝15，雌7雄8)及生理盐水对照组(N＝10，雌5雄5)等。
PEG-mIFN-α4施用方式为颈背部皮下注射给药，剂量为1微克每只。PEG-mIFN-α4间歇给药组每48小时给药一次、连续给予3次，停药144小时；连续4轮。PEG-mIFN-α 连续给药组每48小时给药一次、连续给予21次。生理盐水对照组给予等体积的生理盐水。
所有组均进行生存分析，比较组间差异。统计分析采用SAS 9.4、office2010软件，统计检验均为双侧检验。
生存曲线如图8所示，统计比较结果如表3所示。至接瘤40天，生理盐水对照组的中位生存期为19.5天，PEG-mIFN-α4连续给药组为38天；PEG-mIFN-α4间歇给药组的生存率为93.3％，尚无法计算中位生存期。PEG-mIFN-α4连续给药组、间歇给药组的生存曲线与生理盐水对照组的差异均显著；PEG-mIFN-α4连续给药组与间歇给药组的差异也显著(P＝0.0035)，PEG-mIFN-α4间歇给药组的生存期相对连续给药组显著延长。针对小鼠移植性肝癌H 22的治疗，PEG-mIFN-α4间歇性施用相对连续施用方式体现出显著更优的疗效。
表3、PEG-mIFN-α4连续、间歇给药治疗小鼠移植性肝癌H 22疗效比较实验生存分析结果
Figure PCTCN2021073191-appb-000003
注：1、与“生理盐水对照组”比较。2、与“PEG-mIFN-α4间歇给药组”比较。3、*：统计学差异显著。
实施例5、间歇式施用干扰素联合吉西他滨治疗小鼠肝癌的研究
本发明人研究了间歇式施用PEG化重组小鼠干扰素α(PEG-mIFN-α4)联合吉西他滨对小鼠肝癌H22的抑瘤作用，探索干扰素与吉西他滨的联合疗效。本发明人惊奇地发现，通过间歇性地施用PEG-mIFN-α4并同时施用吉西他滨，相比于单独施用吉西他滨或单独间歇式施用PEG-mIFN-α4，在H22肿瘤治疗中可以获得显著更优的治疗效果。
在本实施例中，进行了PEG-mIFN-α4间歇(每48小时给药一次、连续给予3次，停药144小时；持续8轮)、连续给药(每48小时给药一次，连续给予42次)、联用吉西他滨给药(PEG-mIFN-α4每48小时给药一次、连续给予3次，停药240小时，持续6轮；吉西他滨每周给药一次)对小鼠移植性肝癌H22的疗效比较研究。
本实施例中，BALB/c小鼠购自北京维通利华实验动物技术有限公司，小鼠肝癌H22细胞株购自中国典型培养物保藏中心(CCTCC)。
6-8周龄、18-22g SPF级BALB/c小鼠右前肢腋下皮下接种H22细胞(1×10 6个/mL，0.2ml/只)。接瘤当天随机分组，实验分组包括PEG-mIFN-α4连续给药组(N＝24，雌12雄12)、PEG-mIFN-α4间歇给药组(N＝24，雌12雄12)、生理盐水对照组(N＝24，雌12雄12)、吉西他滨单用组(N＝24，雌12雄12)、以及PEG-mIFN-α4联用吉西他滨组(N＝24，雌12雄12)。
PEG-mIFN-α4施用方式为颈背部皮下注射给药，剂量为1微克每只。吉西他滨为腹 腔注射，剂量为60mg/kg。分组后当天开始给药；PEG-mIFN-α4间歇给药组每48小时给药一次、连续给予3次，停药144小时，持续8轮。PEG-mIFN-α连续给药组每48小时给药一次。PEG-mIFN-α4联用吉西他滨组为PEG-mIFN-α4每48小时给药一次、连续给予3次，停药240小时，持续6轮；吉西他滨每周给药一次。吉西他滨单用组每周给药一次。生理盐水对照组给予等体积的生理盐水。
所有组均进行生存分析，比较组间差异。统计分析采用SAS 9.4、office2010软件，统计检验均为双侧检验。
生存曲线如图9所示，统计比较结果如表4所示。至接瘤108天，PEG-mIFN-α4连续给药组的中位生存期为37.0天，间歇给药组为64.0天，均大于生理盐水对照组的17.5天。PEG-mIFN-α4连续给药组与间歇给药组的生存曲线差异显著(P＝0.0245)，PEG-mIFN-α4间歇给药组的生存期相对连续给药组显著延长。针对小鼠移植性肝癌H22的治疗，PEG-mIFN-α4间歇性施用相对连续施用方式体现出显著更优的疗效。
吉西他滨单用组的中位生存期为58.5天，PEG-mIFN-α4间歇给药联用吉西他滨组观察期终点(接瘤108天)时的生存率为75.0％，两组的生存曲线差异显著(P<0.0001)，PEG-mIFN-α4间歇给药联用吉西他滨组的生存期相对于吉西他滨单用组显著延长。
PEG-mIFN-α4间歇给药组的中位生存期为64.0天，PEG-mIFN-α4间歇给药联用吉西他滨组观察期终点(接瘤108天)时的生存率为75.0％，两组的生存曲线差异显著(P<0.0001)，PEG-mIFN-α4间歇给药联用吉西他滨组的生存期相对于PEG-mIFN-α4间歇给药单用组显著延长。
结果显示，针对小鼠移植性肝癌H22的治疗，间歇式施用聚乙二醇化鼠干扰素α优于连续施用聚乙二醇化鼠干扰素α；间歇式施用聚乙二醇化鼠干扰素α联合吉西他滨的抗癌活性，显著优于单独施用吉西他滨及单独间歇式施用聚乙二醇化鼠干扰素α。
表4、PEG-mIFN-α4连续、间歇、联用吉西他滨给药治疗小鼠移植性肝癌H22疗效比较实验生存分析结果
Figure PCTCN2021073191-appb-000004
注：1、与“PEG-mIFN-α4间歇给药组”比较。2、与PEG-mIFN-α4间歇施用联合吉西他滨组”比较。3、*：统计学差异显著。
实施例6、间歇式施用干扰素联合吉西他滨治疗小鼠肺癌的研究
本发明人研究了间歇式施用PEG-mIFN-α4联合吉西他滨对小鼠肺癌LLC的抑瘤作 用，探索干扰素与吉西他滨的联合疗效。通过实验发现，间歇性地施用PEG-mIFN-α4并同时施用吉西他滨，相比于单独施用吉西他滨或单独间歇式施用PEG-mIFN-α4，在肺癌LLC治疗中同样也可以获得显著更优的治疗效果。
本实施例中，进行PEG-mIFN-α4间歇(每48小时给药一次、连续给予4次，停药192小时；持续5轮)、连续给药(每48小时给药一次，连续给予32次)、联用吉西他滨给药(PEG-mIFN-α4每48小时给药一次、连续给予4次，停药360小时，持续4轮；吉西他滨每周给药一次)对小鼠移植性肺癌LLC的疗效比较研究。
本实施例中，C57BL/6N小鼠购自北京维通利华实验动物技术有限公司，小鼠肺癌LLC细胞株购自中国北京协和细胞资源中心。
6-8周龄、18-22g SPF级C57BL/6N小鼠右前肢腋下皮下接种肺癌LLC(1×10 6个/mL，0.2ml/只)。接瘤当天后随机分组，实验分组包括PEG-mIFN-α4连续给药组(N＝26，雌13雄13)、PEG-mIFN-α4间歇给药组(N＝26，雌13雄13)、生理盐水对照组(N＝26，雌13雄13)、吉西他滨单用组(N＝26，雌13雄13)、以及PEG-mIFN-α4联用吉西他滨组(N＝26，雌13雄13)。
PEG-mIFN-α4施用方式为颈背部皮下注射给药，剂量为1微克每只。分组后当天开始给药；PEG-mIFN-α4间歇给药组每48小时给药一次、连续给予4次，停药192小时；持续5轮。PEG-mIFN-α连续给药组每48小时给药一次。PEG-mIFN-α4联用吉西他滨组为每48小时给药一次、连续给予4次，停药360小时，持续4轮；吉西他滨每周给药一次。生理盐水对照组给予等体积的生理盐水。吉西他滨为腹腔注射，剂量为60mg/kg。
采用SAS 9.4软件比较组间死亡率的差异，统计检验均为双侧检验。
各组死亡率如图10所示，统计比较结果如表5所示。至接瘤45天，PEG-mIFN-α4连续给药组的死亡率为100.0％，间歇给药组为34.6％，两者差异显著(P<0.001)，PEG-mIFN-α4间歇给药组的死亡率相对连续给药组显著降低。针对小鼠移植性肺癌LLC的治疗，PEG-mIFN-α4间歇性施用相对连续施用方式体现出显著更优的疗效。
至接瘤64天，吉西他滨单用组的死亡率为80.8％，PEG-mIFN-α4间歇给药联用吉西他滨组的死亡率为53.8％，两者差异显著(P＝0.0385)，PEG-mIFN-α4间歇给药联用吉西他滨组相对于吉西他滨单用组的死亡率显著降低。
至接瘤64天，PEG-mIFN-α4间歇给药单用组的死亡率为84.6％，PEG-mIFN-α4间歇给药联用吉西他滨组的死亡率为53.8％，两组的死亡率差异显著(P＝0.0162)，PEG-mIFN-α4间歇给药联用吉西他滨组的死亡率显著低于PEG-mIFN-α4间歇给药单用组。
结果显示，针对小鼠移植性肺癌LLC的治疗，间歇式施用聚乙二醇化鼠干扰素α优于连续施用聚乙二醇化鼠干扰素α；间歇式施用聚乙二醇化鼠干扰素α联合吉西他滨的抗癌活性，显著优于单独施用吉西他滨及间歇式单独施用聚乙二醇化鼠干扰素α。
表5、PEG-mIFN-α4连续、间歇、联用吉西他滨给药治疗小鼠移植性肺癌LLC疗效比较实验死亡率结果(接瘤至64天)
Figure PCTCN2021073191-appb-000005
注：1、于接瘤45天时与“PEG-mIFN-α4间歇给药组”比较；2、于接瘤64天时，与PEG-mIFN-α4间歇施用联合吉西他滨组”a比较。3、*：统计学差异显著。
实施例7、间歇式施用干扰素联合吉西他滨治疗结直肠癌的研究
本发明人研究了间歇式施用PEG-mIFN-α4联合吉西他滨对小鼠结直肠癌CT26的抑瘤作用，探索干扰素与吉西他滨的联合疗效。通过实验发现，间歇性地施用PEG-mIFN-α4并同时施用吉西他滨，相比于单独施用吉西他滨或单独间歇式施用PEG-mIFN-α4，在结直肠癌CT26治疗中同样也可以获得显著更优的治疗效果。
本实施例中，进行PEG-mIFN-α4间歇(每48小时给药一次、连续给予3次，停药144小时；持续11轮)、连续(每48小时给药一次，连续给予55次)、联用吉西他滨给药(PEG-mIFN-α4每48小时给药一次、连续给予3次，停药240小时，持续8轮)；吉西他滨每周给药一次)对小鼠移植性结直肠癌CT26的疗效比较研究。
本实施例中，BALB/c小鼠购自北京维通利华实验动物技术有限公司，CT26小鼠结直肠癌细胞株购自中国科学院上海生科院细胞资源中心。抗癌剂为吉西他滨(江苏豪森药业)。
6-8周龄、18-22g SPF级BALB/c小鼠右前肢腋下皮下接种CT26细胞(1×10 6个/mL，0.2ml/只)。接瘤当天随机分组，实验分组包括PEG-mIFN-α4连续给药组(N＝24，雌12雄12)、PEG-mIFN-α4间歇给药组(N＝24，雌12雄12)、生理盐水对照组(N＝24，雌12雄12)、吉西他滨单用组(N＝24，雌12雄12)、以及PEG-mIFN-α4联用吉西他滨组(N＝24，雌12雄12)。
PEG-mIFN-α4施用方式为颈背部皮下注射给药，剂量为1微克每只。吉西他滨为腹腔注射，剂量为60mg/kg。分组后当天开始给药；PEG-mIFN-α4间歇给药组每48小时给药一次、连续给予3次，停药144小时，持续11轮。PEG-mIFN-α连续给药组每48小时给药一次。PEG-mIFN-α4联用吉西他滨组为PEG-mIFN-α每48小时给药一次、连续给予3次，停药240小时，持续8轮；吉西他滨每周给药一次。吉西他滨单用组每周给药一次。生理盐水对照组给予等体积的生理盐水。
所有组均进行生存分析，比较组间差异。统计分析采用SAS 9.4、office2010软件，统计检验均为双侧检验。
生存曲线如图11所示，统计比较结果如表6所示。至接瘤110天，PEG-mIFN-α4 连续给药组为53.5天，间歇给药组为89.0天，均大于生理盐水对照组的49.5天。PEG-mIFN-α4连续给药组与间歇给药组的生存曲线差异显著(P＝0.0150)，PEG-mIFN-α4间歇给药组的生存期相对连续给药组显著延长。针对小鼠移植性结直肠癌CT26的治疗，PEG-mIFN-α4间歇性施用相对连续施用方式体现出显著更优的疗效。
吉西他滨单用组的中位生存期为99.0天，PEG-mIFN-α4联用吉西他滨组观察期终点(接瘤110天)时的生存率为75.0％(相同观察期内，未达中位生存期)，两组的生存曲线差异显著(P＝0.0102)，PEG-mIFN-α4间歇给药联用吉西他滨组的生存期相对于吉西他滨单用组显著延长。
PEG-mIFN-α4间歇给药组的中位生存期为89.0天，PEG-mIFN-α4间歇给药联用吉西他滨组观察期终点(接瘤110天)时的生存率为75.0％，两组的生存曲线差异显著(P＝0.0048)，PEG-mIFN-α4间歇给药联用吉西他滨组的生存期相对于PEG-mIFN-α4间歇给药单用组显著延长。
结果显示，针对小鼠移植性结直肠癌CT26的治疗，间歇式施用聚乙二醇化鼠干扰素α优于连续施用聚乙二醇化鼠干扰素α；间歇式施用聚乙二醇化鼠干扰素α联合吉西他滨的抗癌活性，显著优于单独施用吉西他滨及间歇式单独施用聚乙二醇化鼠干扰素α。
表6、PEG-mIFN-α4连续、间歇、联用吉西他滨给药治疗小鼠移植性结直肠癌CT26疗效比较实验生存分析结果
Figure PCTCN2021073191-appb-000006
注：1、与“PEG-mIFN-α4间歇给药组”比较。2、与PEG-mIFN-α4间歇施用联合吉西他滨组”比较。3、*：统计学差异显著。
实施例8、间歇式施用干扰素联合吉西他滨治疗黑色素瘤的研究
本发明人研究了间歇式施用PEG-mIFN-α4联合吉西他滨对小鼠黑色素瘤B16的抑瘤作用，探索干扰素与吉西他滨的联合疗效。通过实验发现，间歇性地施用PEG-mIFN-α4并同时施用吉西他滨，相比于单独施用吉西他滨或单独间歇式施用PEG-mIFN-α4，在黑色素瘤B16治疗中同样也可以获得显著更优的治疗效果。
本实施例进行了PEG-mIFN-α4间歇(每48小时给药一次、连续给予3次，停药240小时；持续4轮)、连续给药(每48小时给药一次，连续给予24次)、联用吉西他滨给药(PEG-mIFN-α4每48小时给药一次、连续给予3次，停药240小时，持续4轮；吉西他滨每周给药一次)对小鼠移植性黑色素瘤B16的疗效比较研究。
6-8周龄、18-22g SPFC57BL/6N小鼠右前肢腋下皮下接种B16细胞(1×10 5个/mL，0.2ml/只)。接瘤当天随机分组，实验分组包括PEG-mIFN-α4连续给药组(N＝28，雌14雄14)、PEG-mIFN-α4间歇给药组(N＝28，雌14雄14)、生理盐水对照组(N＝28，雌14雄14)、吉西他滨单用组(N＝28，雌14雄14)、以及PEG-mIFN-α4联用吉西他滨组(N＝28，雌14雄14)。
PEG-mIFN-α4施用方式为颈背部皮下注射给药，剂量为1微克每只。吉西他滨为腹腔注射，剂量为60mg/kg。分组后当天开始给药；PEG-mIFN-α4间歇给药组每48小时给药一次、连续给予3次，停药240小时，持续4轮。PEG-mIFN-α4连续给药组每48小时给药一次。PEG-mIFN-α4联用吉西他滨组为PEG-mIFN-α4每48小时给药一次、连续给予3次，停药240小时，持续4轮；吉西他滨每周给药一次。吉西他滨单用组每周给药一次。生理盐水对照组给予等体积的生理盐水。
采用SAS 9.4软件比较组间死亡率的差异，统计检验均为双侧检验。
各组死亡率如图12所示，统计比较结果如表7所示。至接瘤46天，PEG-mIFN-α4连续给药组的死亡率为100.0％，间歇给药组为39.3％，两者差异显著(P<0.0001)，PEG-mIFN-α4间歇给药组的死亡率相对连续给药组显著降低。针对小鼠移植性黑色素瘤B16的治疗，PEG-mIFN-α4间歇性施用相对连续施用方式体现出显著更优的疗效。
至接瘤48天，吉西他滨单用组的死亡率为89.3％，PEG-mIFN-α4间歇给药联用吉西他滨组的死亡率为21.4％，两者差异显著(P<0.0001)，PEG-mIFN-α4间歇给药联用吉西他滨组的死亡率显著低于吉西他滨单用组。
至接瘤48天，PEG-mIFN-α4间歇给药单用组的死亡率为46.4％，PEG-mIFN-α4间歇给药联用吉西他滨组的死亡率为21.4％，两者差异显著(P＝0.0482)，PEG-mIFN-α4间歇给药联用吉西他滨组的死亡率显著低于PEG-mIFN-α4间歇给药单用组。
结果显示，针对小鼠移植性黑色素瘤B16的治疗，间歇式施用聚乙二醇化鼠干扰素α优于连续施用聚乙二醇化鼠干扰素α；间歇式施用聚乙二醇化鼠干扰素α联合吉西他滨的抗癌活性，显著优于单独施用吉西他滨及间歇式单独施用聚乙二醇化鼠干扰素α。表7、PEG-mIFN-α4连续、间歇、联用吉西他滨给药治疗小鼠移植性黑色素瘤B16疗效比较实验死亡率结果
Figure PCTCN2021073191-appb-000007
注：1、于接瘤46天时与“PEG-mIFN-α4间歇给药组”比较；2、于接瘤48天时，与PEG-mIFN-α4间歇施用联合吉西他滨组”比较。3、*：统计学差异显著。
实施例9、间歇式施用干扰素联合吉西他滨治疗乳腺癌的研究
本发明人研究了间歇式施用PEG-mIFN-α4联合吉西他滨对小鼠移植性乳腺癌4T1的抑瘤作用，探索干扰素与吉西他滨的联合疗效。通过实验发现，间歇性地施用PEG-mIFN-α4并同时施用吉西他滨，相比于单独施用吉西他滨或单独间歇式施用PEG-mIFN-α4，在乳腺癌4T1治疗中同样也可以获得显著更优的治疗效果。
本实例进行了PEG-mIFN-α4间歇(每48小时给药一次、连续给予3次，停药144小时；持续5轮)、连续(PEG-mIFN-α4每48小时给药一次，连续给予25次)、联用吉西他滨给药(每48小时给药一次、连续给予3次，停药240小时，持续4轮；吉西他滨每周给药一次)对小鼠移植性乳腺癌4T1的疗效比较研究。
本实施例中，BALB/c小鼠购自北京维通利华实验动物技术有限公司，小鼠乳腺癌4T1细胞株购自中国典型培养物保藏中心(CCTCC)。
6-8周龄、18-22g SPF级BALB/c小鼠右前肢腋下皮下接种乳腺癌4T1(1×10 6个/mL，0.2ml/只)。接瘤4天后随机分组，实验分组包括PEG-mIFN-α4连续给药组(N＝24，雌12雄12)、PEG-mIFN-α4间歇给药组(N＝24，雌12雄12)、生理盐水对照组(N＝24，雌12雄12)、吉西他滨单用组(N＝24，雌12雄12)、以及PEG-mIFN-α4联用吉西他滨组(N＝24，雌12雄12)。
PEG-mIFN-α4施用方式为颈背部皮下注射给药，剂量为1.5微克每只。分组后当天开始给药；PEG-mIFN-α4间歇给药组每48小时给药一次、连续给予3次，停药144小时；持续5轮。PEG-mIFN-α4连续给药组每48小时给药一次。PEG-mIFN-α4联用吉西他滨组为PEG-mIFN-α4每48小时给药一次、连续给予3次，停药240小时，持续4轮；吉西他滨每周给药一次。生理盐水对照组给予等体积的生理盐水。吉西他滨为腹腔注射，剂量为60mg/kg。
采用SAS 9.4软件比较组间死亡率的差异，统计检验均为双侧检验。
各组死亡率如图13所示，统计比较结果如表8所示。至接瘤53天，PEG-mIFN-α4连续给药组的死亡率为87.5％，间歇给药组为58.3％，均低于生理盐水对照组的100.0％。PEG-mIFN-α4连续给药组与间歇给药组的死亡率差异显著(P＝0.0230)，PEG-mIFN-α4间歇给药组的死亡率相对连续给药组显著降低。针对小鼠移植性乳腺癌4T1的治疗，PEG-mIFN-α4间歇性施用相对连续施用方式体现出显著更优的疗效。
至接瘤53天，吉西他滨单用组的死亡率为87.5％，PEG-mIFN-α4间歇给药联用吉西他滨组的死亡率为4.2％，两组差异显著(P<0.0001)，PEG-mIFN-α4间歇给药联用吉西他滨组相对于吉西他滨单用组的死亡率显著降低。
至接瘤53天，PEG-mIFN-α4间歇给药单用组的死亡率为58.3％，PEG-mIFN-α4间歇给药联用吉西他滨组的死亡率为4.2％，两组的死亡率差异显著(P<0.0001)，PEG-mIFN-α4间歇给药联用吉西他滨组的死亡率显著低于PEG-mIFN-α4间歇给药单用组。
结果显示，针对小鼠移植性乳腺癌4T1的治疗，间歇式施用聚乙二醇化鼠干扰素α 优于连续施用聚乙二醇化鼠干扰素α；间歇式施用聚乙二醇化鼠干扰素α联合吉西他滨的抗癌活性，显著优于独施用吉西他滨及间歇式单独施用聚乙二醇化鼠干扰素α。
表8、PEG-mIFN-α4连续、间歇、联用吉西他滨给药治疗小鼠移植性乳腺癌4T1疗效比较实验死亡率结果
Figure PCTCN2021073191-appb-000008
注：1、与“PEG-mIFN-α4连续给药组”比较。2、与PEG-mIFN-α4间歇施用联合吉西他滨组”比较。3、*：统计学差异显著。
实施例10、间歇施用干扰素联合抗癌剂表柔比星治疗小鼠移植性肝癌H 22的研究
本实施例中采用的抗癌剂为化疗药物中抗肿瘤抗生素类药物表柔比星，考察间歇施用干扰素联合抗癌剂表柔比星治疗小鼠移植性肝癌H 22的实施效果。
本实施例中，BALB/c小鼠购自北京维通利华实验动物技术有限公司，小鼠肝癌H 22细胞株购自中国典型培养物保藏中心(CCTCC)，表柔比星厂家为北京协和药厂。
6-8周龄、18-22g SPF级BALB/c小鼠右前肢腋下皮下接种肝癌H 22瘤细胞(1×10 6个/mL，0.2ml/只)；接瘤3天后，随机分组，每组28只，雌雄各半；实验分组包括生理盐水对照组、表柔比星单用组、PEG-mIFN-α4联用表柔比星组。
分组后当天开始给药，每周给药一次；PEG-mIFN-α4为颈背部皮下注射，每次施用剂量为1微克每只；表柔比星为腹腔注射，剂量为每次3.5毫克每千克；生理盐水对照组给予等体积的生理盐水。
所有组别均进行生存曲线分析，并比较中位生存期；采用SAS 9.4、office2010软件进行统计分析。统计检验均为双侧检验。
各组小鼠的生存曲线如图14所示，中位生存期如表9所示。生理盐水组的中位生存期为13.5天，表柔比星组的中位生存期为15天，PEG-mIFN-α4联用表柔比星组在观察期内的生存率为57.1％。PEG-mIFN-α4联用表柔比星组的生存曲线与生理盐水组的差异具有显著性(P<0.0001)；间歇性施用PEG-mIFN-α4同时联用表柔比星，相比于单独单独施用表柔比星可以显著延长荷瘤小鼠的生存期(P<0.0001)。
该实施例提示，在小鼠移植性肝癌H 22的治疗中，间歇性地施用PEG-mIFN-α4同时联合表柔比星的疗效显著优于单独施用表柔比星。
表9.实施例12生存分析结果
Figure PCTCN2021073191-appb-000009
Figure PCTCN2021073191-appb-000010
注：P值 1为与“生理盐水(0.2ml/w)”比较，P值 2为与“表柔比星(3.5mg/kg/w)”比较，*具有统计学差异。
实施例11、间歇施用干扰素联合抗癌剂奥沙利铂治疗小鼠移植性肝癌H 22的研究
本实施例中采用的抗癌剂为化疗药物奥沙利铂，考察间歇施用干扰素联合抗癌剂奥沙利铂治疗小鼠移植性肝癌H 22的实施效果。
本实施例中，BALB/c小鼠购自北京维通利华实验动物技术有限公司，H 22小鼠肝癌细胞株购自中国典型培养物保藏中心(CCTCC)；奥沙利铂厂家为齐鲁制药(海南)有限公司。
6-8周龄、18-22g SPF级BALB/c小鼠右前肢腋下皮下接种肝癌H 22瘤细胞(1×10 6个/mL，0.2ml/只)；接瘤3天后，随机分组，每组28只，雌雄各半；实验分组包括生理盐水对照组、奥沙利铂单用组、PEG-mIFN-α4联用奥沙利铂组等。
分组后当天开始给药，每周给药一次；PEG-mIFN-α4为颈背部皮下注射，每次施用剂量为1微克每只；奥沙利铂为腹腔注射，每次10毫克每千克；生理盐水对照组给予等体积的生理盐水。
所有组别均进行生存曲线分析，并比较中位生存期；采用SAS 9.4、office2010软件进行统计分析。统计检验均为双侧检验。
各组小鼠的生存曲线如图15所示，中位生存期如表10所示。生理盐水组的中位生存期为11天，奥沙利铂组的中位生存期为12.5天，PEG-mIFN-α4联用奥沙利铂组的中位生存期为31天。PEG-mIFN-α4联用奥沙利铂组的生存曲线与生理盐水组的差异显著(P＝0.0001)，与单独施用奥沙利铂组的差异也显著(P＝0.0014)；间歇性施用PEG-mIFN-α4同时联用奥沙利铂可以显著延长荷瘤小鼠的生存期。
该实施例提示，在小鼠移植性肝癌H 22的治疗中，间歇性地施用PEG-mIFN-α4并同时联合施用奥沙利铂，相比于单独施用奥沙利铂，可以获得显著更优的治疗效果。
表10.实施例13生存分析结果
Figure PCTCN2021073191-appb-000011
注：P值 1为与“生理盐水(0.2ml/w)”比较，P值 2为与“奥沙利铂(10mg/kg/w)”比较，*具有统计学差异。
实施例12、间歇施用干扰素联合抗癌剂紫杉醇治疗小鼠移植性肝癌H 22研究
本实施例中采用的抗癌剂为化疗药物紫杉醇，考察间歇施用干扰素联合抗癌剂紫杉 醇治疗小鼠移植性肝癌H 22的实施效果。
本实施例中，BALB/c小鼠购自北京维通利华实验动物技术有限公司，H 22小鼠肝癌细胞株购自中国典型培养物保藏中心(CCTCC)，紫杉醇厂家为江苏奥赛康药业股份有限公司。
6-8周龄、18-22g SPF级BALB/c小鼠右前肢腋下皮下接种肝癌H 22瘤细胞(1×10 6个/mL，0.2ml/只)；接瘤3天后，随机分组，每组28只，雌雄各半；实验分组包括生理盐水对照组、紫杉醇单用组、PEG-mIFN-α4联用紫杉醇组。
分组后当天开始给药，每周给药一次；PEG-mIFN-α4为颈背部皮下注射，每次施用剂量为1微克每只；紫杉醇为腹腔注射，每次10毫克每千克。
所有组别均进行生存曲线分析，并比较中位生存期；采用SAS 9.4、office2010软件进行统计分析。统计检验均为双侧检验。
各组小鼠的生存曲线如图16所示，中位生存期如表11所示。生理盐水组的中位生存期为15天，紫杉醇组为17天；在观察期内，PEG-mIFN-α4联用紫杉醇组的生存率为82.1％。PEG-mIFN-α4联用紫杉醇组的生存曲线与生理盐水组的差异显著(P＝0.0001)，与单独施用紫杉醇组的差异也显著(P<0.0001)；间歇性施用PEG-mIFN-α4同时联用紫杉醇可以显著延长荷瘤小鼠的生存期。
该实施例提示，在小鼠移植性肝癌H 22的治疗中，间歇性地施用PEG-mIFN-α4并同时联合施用紫杉醇，相比于单独施用紫杉醇，可以获得显著更优的治疗效果。
表11.实施例14生存分析结果
Figure PCTCN2021073191-appb-000012
注：P值 1为与“生理盐水(0.2ml/w)”比较，P值 2为与“紫衫醇(10mg/kg/w)”比较，*具有统计学差异。
本发明上述结果提示，通过间歇性地施用PEG-mIFN-α4并同时联合化疗剂例如施用吉西他滨、紫杉醇、奥沙利铂、表柔比星等，相比于单独施用化疗剂，可以获得显著更优的癌症治疗效果。
上述结果提示，长时间地连续施用干扰素，将会造成免疫抑制和免疫细胞消耗且难以恢复。干扰素的疗效依赖于免疫系统，因此基于干扰素的治疗在免疫细胞仍然充足的治疗前期能够取得很好的治疗效果例如很高的肿瘤抑制，然而，在治疗后期由于长期施用干扰素导致的免疫细胞消耗或免疫抑制，治疗效果将大幅下降，并且即使继续施用干扰素也可能无法持续保持较高的治疗效力。而通过间歇性施用干扰素，可以避免这一问题。例如，施用一段时间的干扰素取得一定疗效后，在免疫部分抑制和免疫细胞耗竭前停止干扰素的施用一段时间，使得免疫细胞能够尽快恢复，之后再次重新施用干扰素， 仍然能够取得较好的治疗效力。通过间歇性施用干扰素的治疗剂合理联合其他药物，可取得更好的治疗效果。
序列及说明
>SEQ ID NO:1是干扰素α2a的氨基酸序列(例如罗荛素或派罗欣)
Figure PCTCN2021073191-appb-000013
>SEQ ID NO:2是干扰素α2b氨基酸序列(例如甘乐能或佩乐能或派格宾)
Figure PCTCN2021073191-appb-000014
>SEQ ID NO:3是重组集成干扰素氨基酸序列(例如干复津)
Figure PCTCN2021073191-appb-000015
>SEQ ID NO:4：是培集成干扰素α-2氨基酸序列(例如派益生)
Figure PCTCN2021073191-appb-000016
>SEQ ID NO:5：是干扰素λ氨基酸序列
Figure PCTCN2021073191-appb-000017
>SEQ ID NO:6：是鼠干扰素α4氨基酸序列
Figure PCTCN2021073191-appb-000018
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Claims (35)

  1. 一种在对象中治疗癌症的方法，其包括给所述对象
    i)间隔地施用多个连续疗程的基于干扰素的治疗剂；和
    ii)施用额外的抗癌剂。
  2. 权利要求1的方法，其中所述基于干扰素的治疗剂包含干扰素或其突变体或衍生物，或包含编码干扰素或其突变体或衍生物的核酸分子，或包含促进内源干扰素产生的物质。
  3. 权利要求1或2的方法，其中所述干扰素是I型、II型或III型干扰素，例如干扰素α、干扰素β、干扰素γ或干扰素λ，优选干扰素α。
  4. 权利要求1-3中任一项的方法，其中所述基于干扰素的治疗剂包含干扰素α2a、干扰素α2b、干扰素α1b、干扰素λ，或其突变体或衍生物。
  5. 权利要求1-4中任一项的方法，其中所述干扰素或其突变体或衍生物是PEG化修饰的。
  6. 权利要求1-5中任一项的方法，其中所述基于干扰素的治疗剂选自P1101、派格宾、派罗欣、佩乐能、干复津、乐复能、甘乐能、罗荛素、运德素、派益生和聚乙二醇干扰素λ。
  7. 权利要求1-3中任一项的方法，其中所述基于干扰素的治疗剂包括TLRs、RLRs及STINGs信号通路的激动剂。
  8. 权利要求7的方法，其中所述基于干扰素的治疗剂选自GS-9620、GS-9688、RO7020531、RO6864018、TQ-A3334、JNJ-4964、SB9200、MIW815、DMXAA、MK-1454和diABZI。
  9. 权利要求1-8中任一项的方法，其中在所述连续疗程中，所述基于干扰素的治疗剂的施用使得基本上在整个疗程期间，对象体内新喋呤的浓度高于首次施用前的新喋呤浓度，例如是首次施用前新喋呤浓度的大约110％、大约120％、大约130％、大约140％、大约150％、大约200％、大约250％或更高。
  10. 权利要求1-9中任一项的方法，其中所述连续疗程的长度为第一次施用至最后一次施用的时间，再加上所述治疗剂的大约5个体内半衰期。
  11. 权利要求1-10中任一项的方法，所述多个连续疗程各自的长度为大约1周-大约24周，优选大约1周-大约12周，更优选大约1周-大约8周，更优选大约2周-大约6周。
  12. 权利要求1-11中任一项的方法，其中每个连续疗程的长度为大约1周-大约12周，且其中各疗程之间间隔大约1周-大约12周。
  13. 权利要求1-12中任一项的方法，其中各连续疗程之间间隔大约1周-大约24周，优选大约1周-大约12周，更优选大约1周-大约8周，更优选大约2周-大约6周。
  14. 权利要求1-13中任一项的方法，其中每个连续疗程的长度为大约1周-大约8 周，且其中各疗程之间间隔大约1周-大约8周。
  15. 权利要求1-14中任一项的方法，其中每个连续疗程的长度为大约2周-大约6周，且其中各疗程之间间隔大约2周-大约6周。
  16. 权利要求1-15中任一项的方法，所述基于干扰素的治疗剂施用2个-25个或以上的连续疗程。
  17. 权利要求1-16中任一项的方法，其中所述多个连续疗程的长度基本上相同。
  18. 权利要求1-17中任一项的方法，所述各疗程之间具有基本上相同的时间间隔。
  19. 权利要求1-18中任一项的方法，其中所述癌症选自白血病(如急性淋巴细胞白血病(ALL)、急性髓细胞白血病(AML)、慢性髓细胞白血病(CML)、慢性淋巴细胞白血病、多毛细血管白血病)、肝癌、肺癌、结直肠癌、皮肤癌、胃癌、乳腺癌、前列腺癌、非霍奇金淋巴瘤、黑色素瘤、多发性骨髓瘤、喉乳头状瘤、滤泡性淋巴瘤、艾滋病相关卡波氏肉瘤和肾细胞癌，优选是肝癌、肺癌、乳腺癌、结直肠癌或黑色素瘤。
  20. 权利要求1-19中任一项的方法，所述基于干扰素的治疗剂的施用与所述额外的抗癌剂的施用不重叠。
  21. 权利要求20的方法，在所述多个连续疗程之间施用所述额外的药剂。
  22. 权利要求1-19中任一项的的方法，所述“基于干扰素的治疗剂”的施用与所述额外的药剂的施用重叠。
  23. 权利要求22的方法，在所述多个连续疗程期间和所述多个连续疗程之间施用所述额外的药剂。
  24. 权利要求1-23中任一项的方法，其中所述额外的抗癌剂按照其常规方案施用。
  25. 权利要求1-24中任一项的方法，其中所述抗癌剂是
    i)化疗剂，例如烷化剂：尼莫司汀、卡莫司汀、洛莫司汀、环磷酰胺、异环磷酰胺、甘磷酰芥、司莫司汀；抗代谢药：去氧氟鸟苷、多西氟鸟啶、5-氟尿嘧啶、巯嘌呤、硫鸟嘌呤、阿糖胞苷、氟鸟苷、替加氟、吉西他滨、卡莫氟、羟基脲、甲氨蝶呤、优福定、安西他滨、卡培他滨；抗肿瘤抗生素：放线菌素D、多柔比星、柔红霉素、表柔比星、丝裂霉素、培洛霉素、平阳霉素、吡柔比星；抗肿瘤动植物成分药：伊立替康、三尖杉酯碱、羟基喜树碱、长春瑞宾、紫杉醇、白蛋白紫杉醇、泰索帝、拓扑替康、长春新碱、长春地辛、长春酰胺、长春碱、替尼泊苷、依托泊苷、榄香烯；抗肿瘤激素类化疗药物：阿他美坦、阿那曲唑、氨鲁米特、来曲唑、福美坦、甲他孕酮、他莫昔芬；化疗药物杂类：门冬酰胺酶、卡铂、顺铂、达卡巴嗪、奥沙利铂、乐沙定、可铂奥沙、米托蒽醌、丙卡巴肼；或
    ii)免疫检查点抑制剂，例如PD-1、PD-L1、CTLA4的抑制剂，例如选自以下的抗体：Nivolumab、Pembrolizumab、Atezolizumab、Durvalumab、Avelumab；或
    iii)小分子靶向药物，例如伊马替尼、吉非替尼、硼替佐米、厄洛替尼、索拉非尼、来那度胺、舒尼替尼、达沙替尼、尼洛替尼、拉帕替尼、帕唑帕尼、依维莫司、凡德他尼、克唑替尼、威罗菲尼、ruxolitinib、阿西替尼、vismodegib、卡非佐米、瑞戈非尼、 博舒替尼、托法替尼、卡博替尼、帕纳替尼、泊马度胺、曲美替尼、达拉菲尼、阿法替尼、埃克替尼、依鲁替尼、色瑞替尼、艾代拉里斯、阿帕替尼、帕布昔利布、乐伐替尼、阿西替尼、埃克替尼、阿帕替尼、sonidegib、cobimetinib、osimertinib、alectinib、ixazomib；或
    iv)肿瘤相关抗原特异性抗体类，如美罗华、赫赛丁；
    优选地，所述抗癌剂选自奥沙利铂、表柔比星、紫杉醇和吉西他滨，更优选是吉西他滨。
  26. 一种用于对象中治疗癌症的药物组合，其包含基于干扰素的治疗剂和抗癌剂。
  27. 权利要求26的药物组合，其中所述基于干扰素的治疗剂包含干扰素或其突变体或衍生物，或包含编码干扰素或其突变体或衍生物的核酸分子，或包含促进内源干扰素产生的物质。
  28. 权利要求26或27的药物组合，其中所述干扰素是I型、II型或III型干扰素，例如干扰素α、干扰素β、干扰素γ或干扰素λ，优选干扰素α。
  29. 权利要求26-28中任一项的药物组合，其中所述基于干扰素的治疗剂包含干扰素α2a、干扰素α2b、干扰素α1b、干扰素λ，或其突变体或衍生物。
  30. 权利要求26-29中任一项的药物组合，其中所述干扰素或其突变体或衍生物是PEG化修饰的。
  31. 权利要求26-30中任一项的药物组合，其中所述基于干扰素的治疗剂选自P1101、派格宾、派罗欣、佩乐能、干复津、乐复能、甘乐能、罗荛素、运德素、派益生和聚乙二醇干扰素λ。
  32. 权利要求26-28中任一项的药物组合，其中所述基于干扰素的治疗剂包括TLRs、RLRs及STINGs信号通路的激动剂。
  33. 权利要求32的药物组合物，其中所述基于干扰素的治疗剂选自GS-9620、GS-9688、RO7020531、RO6864018、TQ-A3334、JNJ-4964、SB9200、MIW815、DMXAA、MK-1454和diABZI。
  34. 权利要求26-33中任一项的药物组合，其中所述抗癌剂是
    i)化疗剂，例如烷化剂：尼莫司汀、卡莫司汀、洛莫司汀、环磷酰胺、异环磷酰胺、甘磷酰芥、司莫司汀；抗代谢药：去氧氟鸟苷、多西氟鸟啶、5-氟尿嘧啶、巯嘌呤、硫鸟嘌呤、阿糖胞苷、氟鸟苷、替加氟、吉西他滨、卡莫氟、羟基脲、甲氨蝶呤、优福定、安西他滨、卡培他滨；抗肿瘤抗生素：放线菌素D、多柔比星、柔红霉素、表柔比星、丝裂霉素、培洛霉素、平阳霉素、吡柔比星；抗肿瘤动植物成分药：伊立替康、三尖杉酯碱、羟基喜树碱、长春瑞宾、紫杉醇、白蛋白紫杉醇、泰索帝、拓扑替康、长春新碱、长春地辛、长春酰胺、长春碱、替尼泊苷、依托泊苷、榄香烯；抗肿瘤激素类化疗药物：阿他美坦、阿那曲唑、氨鲁米特、来曲唑、福美坦、甲他孕酮、他莫昔芬；化疗药物杂类：门冬酰胺酶、卡铂、顺铂、达卡巴嗪、奥沙利铂、乐沙定、可铂奥沙、米托蒽醌、 丙卡巴肼；或
    ii)免疫检查点抑制剂，例如PD-1、PD-L1、CTLA4的抑制剂，例如选自以下的抗体：Nivolumab、Pembrolizumab、Atezolizumab、Durvalumab、Avelumab；或
    iii)小分子靶向药物，例如伊马替尼、吉非替尼、硼替佐米、厄洛替尼、索拉非尼、来那度胺、舒尼替尼、达沙替尼、尼洛替尼、拉帕替尼、帕唑帕尼、依维莫司、凡德他尼、克唑替尼、威罗菲尼、ruxolitinib、阿西替尼、vismodegib、卡非佐米、瑞戈非尼、博舒替尼、托法替尼、卡博替尼、帕纳替尼、泊马度胺、曲美替尼、达拉菲尼、阿法替尼、埃克替尼、依鲁替尼、色瑞替尼、艾代拉里斯、阿帕替尼、帕布昔利布、乐伐替尼、阿西替尼、埃克替尼、阿帕替尼、sonidegib、cobimetinib、osimertinib、alectinib、ixazomib；或
    iv)肿瘤相关抗原特异性抗体类，如美罗华、赫赛丁；
    优选地，所述抗癌剂选自奥沙利铂、表柔比星、紫杉醇和吉西他滨，更优选是吉西他滨。
  35. 权利要求26-34中任一项的药物组合，其用于通过权利要求1-25中任一项的方法在对象中治疗癌症。
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