WO2022179527A1 - 治疗胰腺癌的组合物及其用途 - Google Patents

治疗胰腺癌的组合物及其用途 Download PDF

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WO2022179527A1
WO2022179527A1 PCT/CN2022/077475 CN2022077475W WO2022179527A1 WO 2022179527 A1 WO2022179527 A1 WO 2022179527A1 CN 2022077475 W CN2022077475 W CN 2022077475W WO 2022179527 A1 WO2022179527 A1 WO 2022179527A1
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pancreatic cancer
oncolytic virus
pfu
nab
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French (fr)
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梁廷波
白雪莉
沈艺南
宋巍
杨子帆
梁兴梅
郦宇炜
林丹妮
丁隽
贾为国
赵荣华
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浙江大学医学院附属第一医院
深圳复诺健生物科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A61K35/763—Herpes virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/643—Albumins, e.g. HSA, BSA, ovalbumin or a Keyhole Limpet Hemocyanin [KHL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present application belongs to the field of biomedicine, and relates to the application of oncolytic virus in the treatment of pancreatic cancer, in particular to the application of the oncolytic virus in combination with gemcitabine and albumin paclitaxel to treat pancreatic cancer.
  • Herpes simplex virus is one of the most studied oncolytic viruses with the best antitumor effect. In addition to the approved T-Vec, nearly ten herpes simplex oncolytic viruses have entered the clinical research stage.
  • the human body's anti-tumor immunity is mainly composed of acquired immunity and innate immunity. In order to open up all the links of the body's acquired immunity and activate the innate immunity, and give full play to the characteristics that oncolytic viruses can carry multiple genes, more and more A new generation of oncolytic viruses is trying to carry two or more foreign genes with immunostimulatory or other tumor-killing functions.
  • Representative products are LOAd 703 (carrying two genes CD40L and 4-1BB), NG348 (carrying two genes CD80 and CD3) and recombinant herpes oncolytic virus T3011 carrying PD-1 antibody and IL-12.
  • the armed genes carried by the above viruses are all limited to activating the acquired immune response, without taking into account the effector cells that stimulate the body's innate immune system, and neither carrying cytokine genes that can activate adoptive anti-tumor effects at the same time. Clinical development and application are limited. .
  • the GEM+Nab-PTX regimen is the first-line treatment regimen for pancreatic cancer, but so far there is no more effective method for pancreatic cancer.
  • the rise of immunotherapy undoubtedly brings hope for the treatment of advanced pancreatic cancer.
  • Oncolytic virus therapy is a new type of immunotherapy, and its combination with traditional chemotherapy is expected to break through the current efficacy bottleneck of pancreatic cancer.
  • this application provides a new medical use of oncolytic virus VG161 in the treatment of pancreatic cancer, aiming at the characteristics of pancreatic cancer (eg, advanced pancreatic cancer), and also relates to the combination of oncolytic virus with GEM+Nab-PTX in the treatment of pancreatic cancer. application on.
  • the inventors of the present application conducted research on the anti-tumor immunity principle of the oncolytic virus VG161 in the early stage, and confirmed that VG161 has the effect of changing the tumor microenvironment, inducing specific anti-tumor immune memory and systemic anti-tumor immune effects.
  • the principle diagram is shown in Figure 1. .
  • the first aspect of the present application provides the use of the oncolytic virus VG161 in the preparation of a drug for the treatment of pancreatic cancer.
  • VG161 has a good killing effect on pancreatic cancer cells and 3D tumor-like models ( Figure 2 and Figure 4), and has superior replication ability in different pancreatic cancer cell lines ( Figure 3); in vivo experiments , by constructing three sets of CDX models of different pancreatic cancer cells in 4-week-old male nude mice, after injection of VG161, they all showed good therapeutic effects compared with the negative control group, confirming the effectiveness of VG161 in vivo ( Figure 5 ).
  • drugs with oncolytic virus VG161 as the only active ingredient can be used to treat pancreatic cancer.
  • the second aspect of the present application provides the use of the oncolytic virus VG161 in combination with gemcitabine and nab-paclitaxel or other drugs for treating pancreatic cancer in preparing a drug group for treating pancreatic cancer.
  • VG161 The inventors first explored the optimal combination concentration of VG161: two sets of CDX models bearing pancreatic cancer cell line BxPC3 were constructed on 4-week-old male nude mice; then five consecutive injections and a single injection of different concentrations were administered respectively. After the effect observation was carried out, a single injection of VG161 at a concentration of 1 ⁇ 10 5 pfu was finally selected as the optimal combination concentration ( FIG. 6 ).
  • VG161 and GEM+Nab-PTX regimen construct a CDX model of pancreatic cancer cell line BxPC3 on 4-week-old male nude mice; GEM (50mg/ kg), nad-PTX (30 mg/kg) and VG161 (1 ⁇ 10 5 pfu), and it was finally found that the VG161 injection followed by the GEM+Nab-PTX regimen achieved the best effect (Figure 7). It was confirmed that the VG161 combined with GEM+Nab-PTX regimen can achieve good effect in pancreatic cancer, and further enhance the efficacy of the original single drug regimen.
  • the active ingredients of the drug group are oncolytic virus VG161, gemcitabine and nab-paclitaxel.
  • the oncolytic virus VG161 was the first drug, and gemcitabine and nab-paclitaxel were the follow-up drugs.
  • the drug group can be an injection, VG161 is injected first, and then GEM+Nab-PTX is injected.
  • a third aspect of the present application provides a pharmaceutical composition for treating pancreatic cancer, which is composed of oncolytic virus VG161 and pharmaceutically acceptable excipients.
  • a fourth aspect of the present application provides a drug group for treating pancreatic cancer, comprising an advance drug and a subsequent drug, the advance drug is composed of oncolytic virus VG161 and pharmaceutically acceptable excipients, and the subsequent drug is composed of gemcitabine and It consists of nab-paclitaxel or other drugs for the treatment of pancreatic cancer.
  • VG161 single drug can effectively kill pancreatic cancer cells.
  • VG161 single drug can effectively treat transplanted tumors in pancreatic cancer mouse models; while VG161 is used in combination with GEM+Nab-PTX regimen After that, the therapeutic effect was further enhanced.
  • the oncolytic virus VG161 is the first drug, gemcitabine and nab-paclitaxel are the follow-up drugs, and the combination regimen of VG161 injection first has the best effect.
  • VG161 is a drug that is currently undergoing clinical trials.
  • Gemcitabine and nab-paclitaxel are drugs that have been clinically used. Their pharmacological effects are clear, the toxic and side effects are small, and the drug safety has been clinically recognized. Therefore, the drug group of this application can be compared with Rapid clinical translation for the neoadjuvant treatment of patients with advanced pancreatic cancer who have failed first-line therapy and patients with borderline resectable pancreatic cancer.
  • Figure 1 shows the schematic diagram of the anti-tumor immunity of the oncolytic virus VG161.
  • FIG. 2 shows the effect of VG161 on pancreatic cancer cell lines in vitro: BxPC3 (A), MIAPaCa-2 (B), PANC-1 (C), SW 1990 (D) are all common human sources Pancreatic cancer cell line, reflecting the effect of different concentrations of VG161 (MOIs of 0.01, 0.1, 1, 10) on the proliferation of pancreatic cancer cell lines. The lower the OV value, the stronger the inhibitory ability on pancreatic cancer cells. The vehicle in the figure refers to the blank control group.
  • Figure 3 shows the results of the replication ability of VG161 in different pancreatic cancer cell lines and the expression ability of exogenous genes in BxPC3 pancreatic cancer cell lines:
  • the results of replication ability test in the figure Titer (pfu/ml) refers to the virus concentration, and the higher the virus replication ability, the stronger the virus replication ability;
  • Figure 3B is the result of VG161 expression ability of exogenous genes in BxPC3 and KPC pancreatic cancer cell lines,
  • Figure 3 Medium Target gene expression refers to the exogenous gene expression concentration, the higher the expression, the more.
  • Figure 4 shows the result of the effect of VG161 on the pancreatic cancer 3D cancer-like model:
  • Figure 4A is the result of the killing effect of VG161 in the 3D model of pancreatic cancer. The more obvious the spheroid is broken under the microscope, the stronger the killing effect is;
  • Figure 4 4B is a resulting plot of VG161's ability to replicate in 3D models.
  • Figure 5 shows the effect of VG161 on different tumor pancreatic cancer cell tumor-bearing mouse models:
  • Figure 5A is the therapeutic effect of VG161 in the BxPC3 cell line tumor-bearing mouse model;
  • Figure 5B is VG161 in the SW 1990 cell line
  • Figure 5C shows the therapeutic effect of VG161 in the MIAPaCa2 cell line tumor-bearing mouse model.
  • the Tumor volume (mm 3 ) in the figure refers to the size of the tumor, and the smaller the volume, the better the effect.
  • the treatment in the figure refers to the injection time of VG161.
  • Figure 6 shows the effect of VG161 on the adenocarcinoma tumor-bearing mouse model under different injection times:
  • Figure 6A is the administration scheme of VG161
  • Figure 6B is the case of 5 consecutive injections
  • Figure 6C shows the effect of VG161 on an adenocarcinoma-bearing mouse model under a single injection.
  • Figure 7 shows the effect of VG161 combined with GEM+Nab-PTX regimen on adenocarcinoma tumor-bearing mouse model.
  • Figure 7A shows 5 nude mice in each group implanted subcutaneously with 2 ⁇ 10 6 BxPC3 human pancreatic cancer cells into the left lower abdomen.
  • mice with too small or too large tumor volumes were excised, and the remaining 25 mice were randomly divided into 5 groups according to the tumor volume, including Vehicle group, VG161 (1.0 ⁇ 10 7 pfu/mouse) ) group, GEM(50mg/kg)+Nab-PTX(30mg/kg) group, GEM+Nab-PTX+VG161 combination therapy group, VG161+GEM+Nab-PTX combination therapy group.
  • Figure 7B shows the growth of injected tumors.
  • Figure 7C is tumor growth of a single injected tumor.
  • Figure 8 shows the results of flow cytometry experiments 48 hours after VG161 with MOI of 1 infected BxPC-3, MIA PaCa-2, PANC-1, SW 1990, A-498, C918, J82 and MCF7.
  • Figure 9 shows mice injected intratumorally with 1 x 10 7 pfu/mouse of VG161, euthanized at different time points from liver, spleen, lung, heart, kidney, injected tumor, uninjected tumor Quantification of viral copies by qPCR with the codon-optimized IL-15R gene isolated from genomic DNA (Figure 9A), serum markers of organ toxicity alanine aminotransferase (ALT), creatinine (Cr), and lactate dehydrogenase ( LDH) detection results (FIG. 9B), and HE staining results of tissues of heart, lung, liver, spleen and kidney (FIG. 9C).
  • Figure 9A shows mice injected intratumorally with 1 x 10 7 pfu/mouse of VG161, euthanized at different time points from liver, spleen, lung, heart, kidney, injected tumor, uninjected tumor Quantification of viral copies by qPCR with the codon-optimized IL-15R gene isolated from genomic
  • Figure 10 shows that foreign genes expressed by VG161 inhibit the growth of pancreatic cancer in the C57BL/6 mouse model by stimulating anti-tumor immunity.
  • A 5 C57BL/6 mice in each group bearing bilateral KPC tumors were injected intratumorally into the left side, 1 ⁇ 10 7 pfu/mouse of VG160, mVG161 or vehicle control.
  • B growth of injected and distant tumors
  • C tumor growth of single injected tumors and uninjected distant tumors.
  • D Expression levels of IFN- ⁇ in spleen detected by ELISpot
  • E Tumor infiltration of immune cells in injected and uninjected distant tumors was assessed by flow cytometry.
  • FIG 11 shows that the foreign genes expressed by VG161 have strong immunostimulatory activity.
  • Tumor-injected tissues were taken from the C57BL/6 mouse model and treated with VG160 or mVG161, respectively.
  • AC Single-cell RNA-seq data of 4000 CD45 sorted cells were extracted on days 3, 7 and 15.
  • D t-distributed random neighborhood embedding (t-SEN) analysis and quantification of VG160 and mVG161 treated mice. Population percentage was determined from the percentage of total CD45 + cells.
  • other drugs for the treatment of pancreatic cancer include fluorouracil (abbreviation: 5-Fu), Seggio, capecitabine, oxaliplatin, irinotecan and other single drugs or combination drugs , can also be used in combination with the oncolytic virus VG161 for the treatment of pancreatic cancer.
  • VG161 As used herein, the singular forms “a” and “the” generally include plural referents unless the context clearly dictates otherwise.
  • the terms “oncolytic virus VG161” or “VG161” are used interchangeably and generally refer to a virus capable of expressing the exogenous gene products IL-12, IL-15, IL15 receptor alpha subunit (IL- 15R ⁇ ) and PD-L1 blocking peptide (TF-Fc) HSV-1 virus that lacks the ICP34.5 gene, resulting in reduced neurotoxicity, VG161 has an intact ICP47 protein that enhances viral persistence and prolongs efficacy Time window for load transfer.
  • VG161 There is a synergistic immunostimulatory effect among the exogenous genes IL-12, IL-15 and PD-L1 blockade expressed by VG161.
  • the foreign gene of VG161 is of human origin, and the virus that replaces the human IL-12 of VG161 with murine IL-12 is called "mVG161". Examples and more information on the construction methods of VG161 and mVG161 can be found in Chouljenko DV, Ding J, Lee IF, Murad YM, Bu X, Liu G, et al. Induction of Durable Antitumor Response by a Novel Oncolytic Herpesvirus Expressing Multiple Immunomodulatory Transgenes . Biomedicines. 2020; 8(11).
  • the term "prior drug” generally refers to a drug administered first in a drug group
  • the term “subsequent drug” generally refers to a drug administered later in a drug group, eg, in a drug group, the first drug is administered first , and then administer the follow-up drug
  • the administration time interval between the advance drug and the follow-up drug can be 1 hour, 2 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 4 days, 6 days , 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days or more.
  • the term "effective amount” generally refers to an amount of the oncolytic virus VG161 and/or gemcitabine and nab-paclitaxel sufficient to affect the treatment of the target cancer, eg, an amount effective to reduce the size or load of the target tumor, or to hinder the target tumor The amount of cell growth rate. More specifically, it refers to an amount of oncolytic virus effective to achieve the desired result, administered at the necessary dose and during the treatment period.
  • an effective amount of a composition described herein is an amount that causes remission, reduces tumor burden, and/or prevents tumor spread or cancer growth.
  • the effective amount may vary depending on various factors, such as the disease state, age, sex, and weight of the subject, as well as the pharmaceutical formulation, route of administration, etc., but can be routinely determined by those skilled in the art.
  • the therapeutic composition is administered to a subject diagnosed with or suspected of having cancer.
  • a subject can be a human or a non-human animal.
  • drug group generally refers to a combination comprising two or more active ingredients or drugs.
  • Active ingredients or drugs may include oncolytic viruses and/or chemotherapeutic agents.
  • Two or more active ingredients or drugs may be administered simultaneously or sequentially, and when administered sequentially, the drug administered first may be referred to as the first drug and the drug administered later may be referred to as the subsequent drug.
  • Two or more active ingredients or drugs can be placed in the same container or in separate containers.
  • treatment generally refers to a process for obtaining beneficial or desired results, including clinical results.
  • beneficial or desired clinical outcomes may include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions of detectable or undetectable disease, reduction in disease severity, stabilization of disease (ie, no worsening) state, preventing the spread of the disease, delaying or slowing the progression of the disease, remission or amelioration of the disease state, reduction in the recurrence of the disease, and control (in part or in whole).
  • treatment can also mean prolonging survival as compared to expected survival if not receiving treatment.
  • pancreatic cancer usually refers to malignant tumors originating from the pancreas, which can include pancreatic adenocarcinoma originating from pancreatic ducts and tumors originating from pancreatic hormone-secreting cells, such as pancreatic endocrine tumors, insulinomas, etc. .
  • the pancreatic cancer may comprise early, intermediate and/or advanced pancreatic cancer.
  • the pancreatic cancer can metastasize to other sites.
  • the present application provides the use of the oncolytic virus VG161 in the preparation of a medicament for the treatment of pancreatic cancer.
  • the drug has oncolytic virus VG161 as the only active ingredient.
  • the application provides a kit comprising the oncolytic virus VG161 as the sole active substance, and a delivery device or instructions.
  • the kit can treat pancreatic cancer.
  • the application provides the use of oncolytic virus VG161, gemcitabine and nab-paclitaxel, and oncolytic virus VG161 and other drugs for treating pancreatic cancer in preparing a drug group for treating pancreatic cancer.
  • the active ingredients of the pharmaceutical group are oncolytic virus VG161, gemcitabine, and nab-paclitaxel.
  • oncolytic virus VG161 is the first drug, and gemcitabine and nab-paclitaxel are the subsequent drugs.
  • the oncolytic virus VG161 and gemcitabine and nab-paclitaxel are administered at intervals of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, or 21 days.
  • the oncolytic virus VG161 and gemcitabine and nab-paclitaxel were administered at a time interval of 7 days.
  • the oncolytic virus VG161 and gemcitabine and nab-paclitaxel were administered at a time interval of 14 days.
  • the time interval between administration of the oncolytic virus VG161 and gemcitabine and nab-paclitaxel is 21 days.
  • the pharmaceutical group is an injection.
  • the present application provides a pharmaceutical composition for treating pancreatic cancer, the pharmaceutical composition comprising the oncolytic virus VG161 and a pharmaceutically acceptable excipient.
  • the present application provides a drug group for treating pancreatic cancer, the drug group includes an advance drug and a subsequent drug, the advance drug includes an oncolytic virus VG161 and a pharmaceutically acceptable excipient, and the subsequent drug includes Gemcitabine along with nab-paclitaxel or other medicines for pancreatic cancer.
  • the application provides a kit comprising the oncolytic virus VG161, gemcitabine and nab-paclitaxel, and a delivery device or instructions.
  • the kit can treat pancreatic cancer.
  • the application provides methods of treatment and pancreatic cancer comprising administering to a subject in need thereof an effective amount of the oncolytic virus VG161.
  • the administration concentration of the oncolytic virus VG161 was 1 ⁇ 10 5 pfu-1 ⁇ 10 7 pfu.
  • the oncolytic virus VG161 is administered at a concentration of 1 ⁇ 10 5 pfu, 5 ⁇ 10 5 pfu, 1 ⁇ 10 6 pfu, 5 ⁇ 10 6 pfu, or 1 ⁇ 10 7 pfu.
  • the oncolytic virus VG161 may be administered at a concentration of 1 ⁇ 10 5 pfu.
  • the oncolytic virus VG161 may be administered at a concentration of 1 ⁇ 10 6 pfu.
  • the oncolytic virus VG161 may be administered at a concentration of 5 ⁇ 10 6 pfu.
  • the oncolytic virus VG161 may be administered at a concentration of 1 ⁇ 10 7 pfu.
  • the administration is by injection.
  • the injection administration is intratumoral injection.
  • the application provides a method of treating pancreatic cancer, the method comprising administering to a subject in need thereof an effective amount of the oncolytic virus VG161 in combination with gemcitabine and nab-paclitaxel or other drugs for treating pancreatic cancer.
  • the method of treating remission of pancreatic cancer comprises concurrently administering to a subject in need thereof a combination of oncolytic virus VG161 and gemcitabine and nab-paclitaxel, and administering oncolytic virus VG161 first and then gemcitabine and paclitaxel A combination of protein paclitaxel.
  • the method comprises following administration of the oncolytic virus VG161 (eg, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 2 weeks or more thereafter) later) gemcitabine along with nab-paclitaxel, or other drugs for pancreatic cancer.
  • the oncolytic virus VG161 is administered on day 1, and gemcitabine and nab-paclitaxel, or other drugs for pancreatic cancer, are administered on day 7.
  • the oncolytic virus VG161 and gemcitabine and nab-paclitaxel or other drugs for treating pancreatic cancer are administered at intervals of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days or 21 days.
  • the oncolytic virus VG161 and gemcitabine and nab-paclitaxel were administered at a time interval of 7 days.
  • the oncolytic virus VG161 and gemcitabine and nab-paclitaxel were administered at a time interval of 14 days.
  • the oncolytic virus VG161 and gemcitabine and nab-paclitaxel were administered at an interval of 21 days.
  • the oncolytic virus VG161 is administered in a single dose. In certain embodiments, the oncolytic virus VG161 is administered multiple times in succession (eg, 2, 3, 4, 5, or more).
  • the administration dose of the oncolytic virus VG161 is 1 ⁇ 10 5 pfu-1 ⁇ 10 7 pfu. In certain embodiments, the oncolytic virus VG161 is administered at a dose of 1 ⁇ 10 5 pfu, 5 ⁇ 10 5 pfu, 1 ⁇ 10 6 pfu, 5 ⁇ 10 6 pfu or 1 ⁇ 10 7 pfu. For example, in certain embodiments, the oncolytic virus VG161 is administered at a dose of 1 x 105 pfu.
  • the gemcitabine is administered at a dose of 50 mg/kg. In certain embodiments, the nab-paclitaxel is administered at a concentration of 30 mg/kg.
  • the method of treating pancreatic cancer remission comprises concurrently administering to a subject in need thereof 1 ⁇ 10 5 pfu of oncolytic virus VG161, and 50 mg/kg of gemcitabine and 30 mg/kg of nab-paclitaxel , and the combination of 50 mg/kg of gemcitabine and 30 mg/kg of nab-paclitaxel after administration of 1 ⁇ 10 5 pfu of the oncolytic virus VG161.
  • the VG161 and/or gemcitabine and nab-paclitaxel are administered by injection.
  • the injection is intratumoral.
  • the different components of the pharmaceutical group or pharmaceutical composition can be packaged separately (eg, not mixed with each other prior to administration) or premixed and packaged in the same packaging unit.
  • compositions of the present application may be pharmaceutical compositions, which may further include pharmaceutically acceptable excipients.
  • pharmaceutically acceptable excipients include, but are not limited to, inert solid diluents and fillers, diluents, sterile aqueous solutions and various organic solvents, penetration enhancers, solubilizers and adjuvants.
  • oncolytic viruses VG161 and mVG161 can be found in Chouljenko DV, Ding J, Lee IF, Murad YM, Bu X, Liu G, et al. Induction of Durable Antitumor Response by a Novel Oncolytic Herpesvirus Expressing Multiple Immunomodulatory Transgenes. Biomedicines.2020 ; 8(11).
  • Example 1 VG161 effectively kills pancreatic cancer cells and promotes apoptosis in vitro
  • VG161 single drug can kill and promote apoptosis of pancreatic cancer cell lines, which were detected by CCK8 and cytometry.
  • immune cells were stimulated with Leukocyte Activation Cocktail (550583, BD Biosciences) for 4 hours at 37°C and then stained with the following antibodies and a fixable viability kit (423106, BioLegend): anti-CD16/32 ( 156604, BioLegend), anti-CD45 (103128, BioLegend), anti-CD3 (100204, BioLegend), anti-CD4 (100434, BioLegend), anti-CD25 (102016, BioLegend), anti-CD8a (10744, BioLegend), anti-NK1.1 (108749, BioLegend), anti-FOXP3 (320008, BioLegend) and anti-PD1 (135221, BioLegend). All samples were collected on a BD LSRFortessa instrument and data were analyzed using FlowJo V10 software.
  • CCK8 stands for Cell Counting Kit-8, which can be used for simple and accurate analysis of cell proliferation and toxicity.
  • the basic principle is: the reagent contains WST-8 [chemical name: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4 - Benzene disulfonate)-2H-tetrazolium monosodium salt], which is absorbed by cells in the cell under the action of the electron carrier 1-methoxy-5-methylphenazine dimethyl sulfate (1-Methoxy PMS). Reduction by dehydrogenase gives a highly water-soluble yellow formazan product (Forzazan dye). The amount of formazan produced is proportional to the number of viable cells. This property can therefore be used directly for cell proliferation and toxicity assays.
  • WST-8 chemical name: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4 - Benzene disulfonate)-2H-t
  • VG161 The ability of oncolytic virus to replicate normally in host cells is the key to its anti-tumor effect.
  • the exogenous gene carried by VG161 is its main feature different from other oncolytic viruses, and whether it can be expressed normally in pancreatic cancer is also one of the keys. Plaque test is often used to check the replication ability of virus, and enzyme-linked immunosorbent assay (ELISA) can be used to detect the expression of exogenous gene.
  • ELISA enzyme-linked immunosorbent assay
  • the principle of the virus plaque test is to inoculate the virus solution of each dilution into the monolayer cell culture environment. After 2 hours of adsorption, the monolayer cells are covered with agarose, and the virus infects the cells and proliferates in the cells, causing the cells to rupture. die. Due to the limitations of the solid medium, the released virus can only spread around from the initially infected cells. After several proliferation cycles, a localized diseased cell area, the viral plaque, is formed. After staining with neutrophil live cell dye, the living cells were red, while the cells in the plaque area were not stained, forming an unstained area. Viral plaques such as plaques formed by phage infection of bacteria.
  • each plaque obtained is derived from a virus particle that originally infected the cell, that is, the virus in the plaque is the reproductive progeny strain of a virion, thus achieving the purpose of virus purification.
  • the plaque assay can also be used for interferon, antibody neutralization experiments of virus reproduction ability. Therefore, by counting the number of plaques, we can count the replication capacity of the virus in this experiment, thus quantifying its replication ability.
  • ELISA refers to a qualitative and quantitative detection method that binds a soluble antigen or antibody to a solid phase carrier such as polystyrene, and uses the specific binding of antigen and antibody to carry out an immune reaction.
  • the supernatant of the above BxPC3 or KPC was selected for enzyme-linked immunosorbent assay experiment, and the expression of the inserted VG161 endocrine factor IL12, IL15/15RA and PD-L1 blocking peptide was calculated.
  • VG161 can replicate effectively in all pancreatic cancer cell lines, and generally reaches a replication peak at 48 hours to 72 hours (Figure 3A).
  • This example also detected the expression of exogenous genes after VG161 infected mouse pancreatic cancer cells (KPC) and human pancreatic cancer cells (BxPC-3).
  • ELISA results showed that the exogenous genes IL-12, IL-15/IL-15R ⁇ and PD-L1 blocker carried by VG161 could be stably expressed in both cell lines, among which PD-L1 antagonist peptide (PD-L1 blocker ) had the highest expression (Fig. 3B).
  • the effect of VG161 was evaluated by observing the external structural stability of the 3D model under a microscope.
  • the method of plaque experiment was used to calculate the replication ability of VG161 in the 3D model.
  • Example 2 VG161 effectively kills pancreatic cancer cells in vivo and has safety
  • VG161 in solid tumors was verified by constructing a pancreatic cancer subcutaneous tumor model in nude mice and performing intratumoral injection.
  • the BxPC3 cell suspension was injected into the right armpit of nude mice, and the tumor formed after 2 weeks.
  • the tumor size reached 800 mm 3
  • intratumoral injection of VG161 was performed, and the vehicle was used as a control.
  • Observation indicators included tumor volume change and mouse body weight, which were measured every 3 days, and the mouse survival curve was recorded.
  • Observational endpoints included mouse death or mouse tumor volume exceeding 2000 mm 3 .
  • the BxPC3 nude mouse model was injected with different concentrations of VG161 for 5 consecutive times and a single injection to observe the therapeutic effect of VG161 under different administration modes.
  • the BxPC3 cell suspension was injected into the right armpit of nude mice, and the tumor formed after 2 weeks.
  • intratumoral injection of VG161 was performed, and the vehicle was used as a control.
  • 4 concentrations were taken, which were 5 ⁇ 10 5 pfu, 1 ⁇ 10 6 pfu, 5 ⁇ 10 6 pfu and 1 ⁇ 10 7 pfu, respectively.
  • the single injection group took 5 concentrations, respectively 1 ⁇ 10 5 pfu, 5 ⁇ 10 5 pfu, 1 ⁇ 10 6 pfu, 5 ⁇ 10 6 pfu and 1 ⁇ 10 7 pfu.
  • Observation indicators included tumor volume change and mouse body weight, which were measured every 3 days, and the mouse survival curve was recorded.
  • Observational endpoints included mouse death or mouse tumor volume exceeding 2000 mm 3 .
  • FIG. 5 three pancreatic cancer nude mouse models of BxPC3, SW 1990 and MIAPaCa-2 were established respectively.
  • BxPC3 model a single injection of 1 ⁇ 10 7 pfu virus was used for 5 consecutive injections, and the observation found that the effect was significant, and the tumors in most experimental groups completely disappeared (Fig. 5A); in the SW 1990 and MIA PaCa-2 tumor-bearing models
  • Fig. 5A the SW 1990 and MIA PaCa-2 tumor-bearing models
  • IL-15R ⁇ expression levels were measured in mice injected with a single dose of VG161 at various time points. The results showed that IL-15R ⁇ expression was significantly elevated only in tumor-injected lesions and peaked at 48 hours (Fig. 9A). Indices of organ damage (eg, alanine aminotransferase [ALT], creatinine [CR], and lactate dehydrogenase [LDH]) were measured in the peripheral blood of mice and all of the above were found to increase transiently after injection, reaching at 48 hours peaked and then quickly returned to baseline (Figure 9B). Finally, all organs were collected for HE staining and observed under a microscope. Histological analysis showed that VG161 did not cause damage to tissue cells (Fig. 9C). Therefore, VG161 has a good safety profile in naive mice and exhibits anticancer ability by virtue of its oncolytic activity.
  • organ damage eg, alanine aminotransferase [ALT], creatinine [CR], and lactate dehydrogenas
  • Example 3 The foreign gene carried by VG161 has the ability of immune activation
  • the VG160 backbone virus (without the genes encoding the foreign genes IL-12, IL15, IL-15R ⁇ and PD-L1 blocking peptides, the virus Backbone modifications were the same as VG161) as a control.
  • VG161 the VG160 backbone virus
  • 4 x 105 KPC cells were implanted into C57BL/6 mice. After tumor formation, the tumor on the left side was selected as the injection foci for injection therapy.
  • the results showed that both the mVG161 and VG160 groups significantly inhibited tumor growth on the injected side. Furthermore, the tumor suppressive ability of mVG161 was greater than that of VG160 (Fig. 10B).
  • the mVG161 group showed significant inhibitory ability on the growth of the uninjected tumor on the other side (ie, distant effect) (Fig. 10B).
  • the partial response (PR) on the injected side reached 100% in the mVG161 and VG160 groups (Fig. 10C).
  • the PR in the mVG161 group also reached 100%, while the PR in the VG160 group was only 60% (Fig. 10C).
  • Complete remission (CR) was not observed in either group.
  • spleen samples were obtained for ELISpot assays. The results showed that interferon gamma (IFN- ⁇ ) was significantly up-regulated in the mVG161 group (FIG. 10D).
  • Example 4 VG161 affects the immune and metabolic microenvironment
  • VG161-infected tumors also had a proportionate loss of dysfunctional or suppressor cells, such as Tregs (days 3 and 7) and tumor-associated macrophages (days 7 and 15), compared to VG160-infected tumors days) (Fig. 11D).
  • dysfunctional or suppressor cells such as Tregs (days 3 and 7) and tumor-associated macrophages (days 7 and 15)
  • the BxPC3 nude mouse model was established again to explore the therapeutic effect of VG161 combined with the first-line chemotherapy regimen for pancreatic cancer (GEM+Nab-PTX regimen).
  • GEM+Nab-PTX regimen that is, gemcitabine combined with nab-paclitaxel regimen
  • nab-paclitaxel regimen is currently a commonly used first-line neoadjuvant chemotherapy regimen for pancreatic cancer.
  • Vehicle was set as the negative control group, and VG161 single drug and GEM+Nab-PTX regimen were set as the positive control group to explore the therapeutic effect of the combined regimen of VG161+GEM+Nab-PTX in pancreatic cancer.
  • different sequences of administration of VG161 followed by GEM+Nab-PTX and GEM+Nab-PTX followed by VG161 were set up in order to explore the best combination strategy.
  • both the positive control group and the combined use group have better effects than the negative control group (Vehicle group).
  • the effects of the two combination groups were better than the GEM+Nab-PTX group.
  • the effect of GEM+Nab-PTX+VG161 group was not better than that of VG161 single-agent group, while the effect of VG161+GEM+Nab-PTX group was significantly better than other groups.
  • VG161 and GEM+Nab-PTX regimen will further improve its therapeutic effect in pancreatic cancer, and VG161 administration first, followed by GEM+Nab-PTX regimen administration is the best choice.

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Abstract

提供了溶瘤病毒VG161在制备治疗胰腺癌药物中的用途，还提供了溶瘤病毒VG161联合吉西他滨以及白蛋白紫杉醇在制备治疗胰腺癌药物组中的用途。进一步，还提供了治疗胰腺癌的药物组合物、试剂盒和方法。

Description

治疗胰腺癌的组合物及其用途 技术领域
本申请属于生物医药领域，涉及溶瘤病毒在治疗胰腺癌中的应用，尤其涉及其与吉西他滨和白蛋白紫杉醇联合治疗胰腺癌的应用。
背景技术
单纯疱疹病毒是目前研究最多且抗肿瘤作用最好的溶瘤病毒之一。除去已批准上市的T-Vec外，目前有近十个单纯疱疹溶瘤病毒进入临床研究阶段。人体的抗肿瘤免疫主要由获得性免疫和固有免疫两大部分组成，为了打通机体获得性免疫的所有环节并激活固有免疫，充分发挥溶瘤病毒可以搭载多个基因的特点，越来越多的新一代溶瘤病毒正在尝试携带两个或两个以上具有免疫刺激或其它肿瘤杀伤功能的外源基因。代表性产品有LOAd 703(携带CD40L和4-1BB两个基因)、NG348(携带了CD80和CD3两个基因)以及携带PD-1抗体和IL-12的重组疱疹溶瘤病毒T3011。然而，以上病毒所携带的武装基因全部局限于激活获得性免疫反应，没有兼顾刺激机体固有免疫系统的效应细胞，均未同时携带能够激活过继性抗肿瘤效应的细胞因子基因，临床发展应用受到限制。
GEM+Nab-PTX方案是胰腺癌的一线治疗方案，然而至今仍未有更有效的胰腺癌手段出现。免疫治疗的崛起无疑为晚期胰腺癌治疗带来希望。而溶瘤病毒疗法作为一种新型免疫疗法，其与传统化疗的联用有望突破胰腺癌目前的疗效瓶颈。
发明内容
本申请在当前研究的基础上，针对胰腺癌(例如，胰腺癌晚期)的特点，提供溶瘤病毒VG161治疗胰腺癌的新医药用途，同时涉及溶瘤病毒联合GEM+Nab-PTX在治疗胰腺癌上的应用。
本申请的发明人前期对溶瘤病毒VG161的抗肿瘤免疫原理进行研究，证实VG161具有改变肿瘤微环境，诱导特异性抗肿瘤免疫记忆和系统性抗肿瘤免疫效应的作用，作用原理图参见图1。
本申请的第一方面，提供了溶瘤病毒VG161在制备治疗胰腺癌药物中的用途。
体外实验中，VG161对胰腺癌细胞以及3D仿癌模型均具有良好的杀伤效果(图2和图4)，同时在不同的胰腺癌细胞系中具备优越的复制能力(图3)；体内实验中，通过在4周龄雄性裸鼠上构建三套不同胰腺癌细胞的CDX模型，分别注射VG161后与阴性对照组相比均呈现出良好的治疗效果，证实了VG161在体内的有效性(图5)。
因此，以溶瘤病毒VG161作为唯一活性成分的药物，能够用于治疗胰腺癌。
本申请的第二方面，提供了溶瘤病毒VG161联合吉西他滨以及白蛋白紫杉醇或其他治疗胰腺癌药物在制备治疗胰腺癌药物组中的用途。
发明人首先探索了VG161的最佳联用浓度：在4周龄雄性裸鼠上构建了两套以胰腺癌细胞系BxPC3荷瘤的CDX模型；而后分别予不同浓度连续5次注射和单次注射后进行效果观察，最终选定单次注射1×10 5pfu浓度的VG161为最适联用浓度(图6)。
在获得最适联用浓度后，进一步探索VG161与GEM+Nab-PTX方案的联用效果：在4周龄雄性裸鼠上构建以胰腺癌细胞系BxPC3荷瘤的CDX模型；予GEM(50mg/kg)、nad-PTX(30mg/kg)以及VG161(1×10 5pfu)注射，最终发现先注射VG161后注射GEM+Nab-PTX方案获得了最佳效果(图7)。证实了VG161联合GEM+Nab-PTX方案可以在胰腺癌中取得不错的效果，且在原有单药方案上进一步增效。
由上可知，药物组的活性成分为溶瘤病毒VG161、吉西他滨以及白蛋白紫杉醇。在药物组中，溶瘤病毒VG161为先遣药物，吉西他滨以及白蛋白紫杉醇为后续药物。
药物形式上，药物组可以为注射剂，先注射VG161，后注射GEM+Nab-PTX。
本申请的第三方面，提供了一种治疗胰腺癌的药物组合物，由溶瘤病毒VG161以及药学上可接受的辅料组成。
本申请的第四方面，提供了一种治疗胰腺癌的药物组，包括先遣药物以及后续药物，所述先遣药物由溶瘤病毒VG161以及药学上可接受的辅料组成，所述后续药物由吉西他滨以及白蛋白紫杉醇或其他治疗胰腺癌药物组成。
发明的作用与效果
通过体外细胞试验，VG161单药可有效对胰腺癌细胞进行杀伤，通过小鼠体内实验，VG161单药可有效治疗胰腺癌小鼠模型中的移植瘤；而VG161与GEM+Nab-PTX方案联用后，治疗效果进一步增强。此外，溶瘤病毒VG161为先遣药物，吉西他滨以及白蛋白紫杉醇为后续药物，先进行VG161注射的联用方案效果最优。
此外，VG161属于当前已在进行临床试验的药物，吉西他滨以及白蛋白紫杉醇为已 经临床应用的药物，其药理作用明确、毒副作用小，药物安全性已得到临床认可，因此本申请的药物组可较快实现临床转化，用于一线治疗失败的中晚期胰腺癌患者以及边界可切除胰腺癌患者的新辅助治疗。
附图说明
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图的简要说明如下：
图1显示的是溶瘤病毒VG161抗肿瘤免疫原理图。
图2显示的是VG161在体外实验中对胰腺癌细胞系的影响结果图：BxPC3(A)、MIAPaCa-2(B)、PANC-1(C)、SW 1990(D)均为常见的人源胰腺癌细胞系，反映了不同浓度VG161(MOI分别为0.01、0.1、1、10)作用下对胰腺癌细胞系增殖影响，OV value越低表明对胰腺癌细胞的抑制能力越强，图中vehicle指空白对照组。
图3是VG161在不同胰腺癌细胞系中复制能力以及在BxPC3胰腺癌细胞系中外源性基因表达能力的结果图：图3A是VG161在MOI＝1的情况下，在不同胰腺癌细胞系中的复制能力检测结果图，图中Titer(pfu/ml)指病毒浓度，越高表明病毒复制能力越强；图3B是VG161在BxPC3和KPC胰腺癌细胞系中外源性基因表达能力的结果图，图中Target gene expression指外源性基因表达浓度，越高表明表达越多。
图4显示的是VG161在胰腺癌3D仿癌模型上作用效果的结果图：图4A是VG161在胰腺癌3D模型中杀伤效果的结果图，镜下球体破碎越明显，证明杀伤效果越强；图4B是VG161在3D模型中复制能力的结果图。
图5显示的是VG161在不同肿瘤胰腺癌细胞荷瘤的小鼠模型上作用效果图：图5A是VG161在BxPC3细胞系荷瘤小鼠模型中治疗效果图；图5B是VG161在SW 1990细胞系荷瘤小鼠模型中治疗效果图；图5C是VG161在MIAPaCa2细胞系荷瘤小鼠模型中治疗效果图。其中，图中Tumor volume(mm 3)指肿瘤大小，越小说明效果越好，图中treatment是指VG161注射时间。
图6显示的是不同注射次数的情况下，VG161在腺癌细胞荷瘤的小鼠模型上作用效果图：图6A是VG161施用方案，图6B是连续5次注射的情况下，VG161在腺癌细胞荷瘤的小鼠模型上作用效果图；图6C是单次注射的情况下，VG161在腺癌细胞荷瘤的小鼠模型上作用效果图。
图7显示的是VG161联合GEM+Nab-PTX方案在腺癌细胞荷瘤的小鼠模型上作用效果图。图7A为每组5只裸鼠皮下植入2×10 6BxPC3人胰腺癌细胞到左下腹。当小鼠平均肿瘤体积达到150mm时，切除肿瘤体积过小或过大的小鼠，其余25只小鼠根据肿瘤体积随机分为5组，包括Vehicle组、VG161(1.0×10 7pfu/小鼠)组、GEM(50mg/kg)+Nab-PTX(30mg/kg)组、GEM+Nab-PTX+VG161联合治疗组、VG161+GEM+Nab-PTX联合治疗组。图7B为注射肿瘤的生长。图7C为单个注射肿瘤的肿瘤生长。
图8显示的是MOI为1的VG161感染BxPC-3、MIA PaCa-2、PANC-1、SW 1990、A-498、C918、J82和MCF7后48小时的流式细胞实验结果。
图9显示的是用1×10 7pfu/只小鼠的VG161瘤内注射小鼠，在不同时间点对小鼠实施安乐死，从肝脏、脾脏、肺、心脏、肾脏、注射肿瘤、未注射肿瘤中分离基因组DNA，用密码子优化的IL-15R基因进行qPCR定量病毒拷贝的结果(图9A)，器官毒性的血清标志物丙氨酸转氨酶(ALT)、肌酐(Cr)和乳酸脱氢酶(LDH)的检测结果(图9B)，以及心脏、肺、肝脏、脾脏和肾脏的组织的HE染色结果(图9C)。
图10显示的是VG161表达的外源基因通过刺激抗肿瘤免疫抑制C57BL/6小鼠模型中胰腺癌的生长。其中，A：每组携带双侧KPC肿瘤的5只C57BL/6小鼠被肿瘤内注射到左侧，1×10 7pfu/小鼠的VG160、mVG161或载体对照。B：注射和远处肿瘤的生长，C：单个注射肿瘤和未注射远处肿瘤的肿瘤生长。D：ELISpot检测脾脏中IFN-γ的表达水平，E：通过流式细胞术评估注射肿瘤和未注射远处肿瘤中免疫细胞的肿瘤浸润。
图11显示的是VG161表达的外源基因具有强大的免疫刺激活性。注射肿瘤的组织取自C57BL/6小鼠模型，分别用VG160或mVG161处理。其中，A-C：在第3、7和15天提取了4000个CD45分选细胞的单细胞RNA-seq数据。D：VG160和mVG161处理的小鼠的t-分布随机邻域嵌入(t-SEN)分析和定量。群体百分比由总CD45 +细胞的百分比确定。
具体实施方式
在描述本申请的实施例之前，应理解，这些实施例仅以举例的方式提供，在本文中描述的本申请实施例的各种替代方案可以用于实践本申请。本领域的技术人员将想到不脱离本申请的众多变化形式、改变和替换。
本申请中，除了吉西他滨以及白蛋白紫杉醇外，其他治疗胰腺癌药物包括氟尿嘧啶 (简称：5-Fu)、替吉奥、卡培他滨、奥沙利铂、伊立替康等单药或联合用药，亦可与溶瘤病毒VG161联用用于对胰腺癌进行治疗。
除非另有定义，否则本文中使用的所有技术和科学术语具有与本申请所属领域的普通技术人员通常所理解相同的含义。虽然与本文中所描述的方法和材料类似或等效的方法和材料可以用于实践或测试本申请，但下文描述了合适的方法和材料。以防冲突，将以本专利说明书(包含定义)为准。另外，材料、方法和实例仅为说明性的且没有限制性的意图。本领域的技术人员将想到不脱离本申请的众多变化形式、改变和替换。
除非上下文另有清晰指示，否则本文所使用的单数形式“一”和“所述”通常包括复数是指物。在本申请中，术语“溶瘤病毒VG161”或“VG161”可以互换使用，通常是指一种能够表达外源性基因产物IL-12、IL-15、IL15受体α亚基(IL-15Rα)和PD-L1阻断肽(TF-Fc)的HSV-1病毒，其缺失ICP34.5基因，从而具有降低的神经毒性，VG161具有完整的ICP47蛋白，可以增强病毒的持久性并延长有效载荷传递的时间窗口。VG161表达的外源基因IL-12、IL-15和PD-L1阻断剂之间具有协同免疫刺激作用。VG161的外源基因为人源，将VG161的人源IL-12替换为鼠源IL-12的病毒称为“mVG161”。关于VG161和mVG161的构建方法的示例和更多信息可参见Chouljenko DV,Ding J,Lee IF,Murad YM,Bu X,Liu G,et al.Induction of Durable Antitumor Response by a Novel Oncolytic Herpesvirus Expressing Multiple Immunomodulatory Transgenes.Biomedicines.2020；8(11)。
在本申请中，术语“先遣药物”通常是指在药物组中先施用的药物，术语“后续药物”通常是指在药物组中后续施用的药物，例如，在药物组中，先施用先遣药物，后施用后续药物，所述先遣药物和所述后续药物的施用时间间隔可以为1小时、2小时、6小时、12小时、1天、2天、3天、4天、4天、6天、7天、8天、9天、10天、11天、12天、13天、14天、15天、16天、17天、18天、19天、20天、21天或更长。
在本申请中，术语“有效量”通常是指足以影响目标癌症治疗的溶瘤病毒VG161和/或吉西他滨以及白蛋白紫杉醇的量，例如，有效减少目标肿瘤尺寸或载荷的量、或者妨碍靶标肿瘤细胞生长速率的量。更具体地是指以必要剂量和治疗期间施用，有效达到期望结果的溶瘤病毒的量。例如，在治疗癌症的情形中，本文所述组合物的有效量是引起缓解、减小肿瘤负荷和/或防止肿瘤扩散或癌症生长的量。有效量可以根据各种因素变化，例如受试者的疾病状态、年龄、性别和体重，以及药物配方、施用途径等等，但能够由本领域技术人员常规地确定。向诊断患有癌症或疑似患有癌症的受试者施用该治疗组合 物。受试者可以是人或非人动物。
在本申请中，术语“药物组”通常是指包含两种或多种活性成分或药物的组合。活性成分或药物可以包括溶瘤病毒和/或化疗剂。两种或多种活性成分或药物可以同时施用或以先后顺序施用，当以先后顺序施用时，先施用的药物可以称为先遣药物，后施用的药物可以称为后续药物。两种或多种活性成分或药物可以置于同一个容器中，也可以分别置于不同的容器中。
在本申请中，术语“治疗”通常是指用于获得有益或期望结果的过程，包括临床结果。有益的或期望的临床结果可以包括，但不限于，可检测的或不可检测的疾病的一种或多种症状或病况的缓解或改善、疾病程度的减轻、疾病的稳定(即，未恶化)状态、防止疾病扩散、延迟或减慢疾病发展、疾病状态的缓解或减轻、疾病复发的减少以及控制(部分或全部)。术语“治疗”也可以意指相比于不接受治疗的预期存活期而言，存活期延长。
在本申请中，术语“胰腺癌”通常是指起源于胰腺的恶性肿瘤，可包括起源于胰腺导管的胰腺腺癌和起源于胰腺有分泌激素功能细胞的肿瘤，如胰腺内分泌瘤、胰岛素瘤等。在本申请中，所述胰腺癌可以包含早期、中期和/或晚期胰腺癌。在本申请中，所述胰腺癌可以转移至其他部位。
一方面，本申请提供了溶瘤病毒VG161在制备治疗胰腺癌药物中的用途。
在某些实施方式中，所述药物以溶瘤病毒VG161作为唯一活性成分。
另一方面，本申请提供了一种试剂盒，其包括溶瘤病毒VG161作为唯一活性物质，以及递送装置或说明书。所述试剂盒可以治疗胰腺癌。
另一方面，本申请提供了溶瘤病毒VG161和吉西他滨以及白蛋白紫杉醇，以及溶瘤病毒VG161和其他治疗胰腺癌的药物在制备治疗胰腺癌药物组中的用途。
在某些实施方式中，所述药物组的活性成分为溶瘤病毒VG161、吉西他滨以及白蛋白紫杉醇。
在某些实施方式中，在所述药物组中，溶瘤病毒VG161为先遣药物，吉西他滨以及白蛋白紫杉醇为后续药物。在某些实施方式中，施用溶瘤病毒VG161和吉西他滨以及白蛋白紫杉醇的时间间隔为1天、2天、3天、4天、5天、6天、7天、8天、9天、10天、11天、12天、13天、14天、15天、16天、17天、18天、19天、20天或21天。例如，施用溶瘤病毒VG161和吉西他滨以及白蛋白紫杉醇的时间间隔为7天。例如，施用溶瘤病毒VG161和吉西他滨以及白蛋白紫杉醇的时间间隔为14天。例如，施用溶 瘤病毒VG161和吉西他滨以及白蛋白紫杉醇的时间间隔为21天。
在某些实施方式中，所述药物组为注射剂。
另一方面，本申请提供了一种治疗胰腺癌的药物组合物，所述药物组合物包括溶瘤病毒VG161以及药学上可接受的辅料。
另一方面，本申请提供了一种治疗胰腺癌的药物组，所述药物组包括先遣药物以及后续药物，所述先遣药物包括溶瘤病毒VG161以及药学上可接受的辅料，所述后续药物包括吉西他滨以及白蛋白紫杉醇或其他治疗胰腺癌的药物。
另一方面，本申请提供了一种试剂盒，其包括溶瘤病毒VG161，吉西他滨以及白蛋白紫杉醇，以及递送装置或说明书。所述试剂盒可以治疗胰腺癌。
另一方面，本申请提供了治疗和胰腺癌的方法，所述方法包括向有需要的受试者施用有效量的溶瘤病毒VG161。
所述溶瘤病毒VG161的施用浓度为1×10 5pfu-1×10 7pfu。在某些实施方式中，所述溶瘤病毒VG161的施用浓度为1×10 5pfu、5×10 5pfu、1×10 6pfu、5×10 6pfu或1×10 7pfu。例如，所述溶瘤病毒VG161的施用浓度可以为1×10 5pfu。例如，所述溶瘤病毒VG161的施用浓度可以为1×10 6pfu。例如，所述溶瘤病毒VG161的施用浓度可以为5×10 6pfu。例如，所述溶瘤病毒VG161的施用浓度可以为1×10 7pfu。
在某些实施方式中，所述施用为注射施用。
在某些实施方式中，所述注射施用为瘤内注射。
另一方面，本申请提供了治疗胰腺癌的方法，所述方法包括向有需要的受试者施用有效量的溶瘤病毒VG161，和吉西他滨以及白蛋白紫杉醇或其他治疗胰腺癌的药物的组合。
在某些实施方式中，所述治疗缓解胰腺癌的方法包括向有需要的受试者同时施用溶瘤病毒VG161和吉西他滨以及白蛋白紫杉醇的组合，以及先施用溶瘤病毒VG161之后施用吉西他滨以及白蛋白紫杉醇的组合。
在某些实施方式中，所述方法包括在施用溶瘤病毒VG161之后(例如，之后1天、2天、3天、4天、5天、6天、7天、10天、2周或更久之后)施用吉西他滨以及白蛋白紫杉醇，或其他治疗胰腺癌的药物。例如，第1天施用溶瘤病毒VG161，第7天施用吉西他滨以及白蛋白紫杉醇，或其他治疗胰腺癌的药物。在某些实施方式中，施用溶瘤病毒VG161和吉西他滨以及白蛋白紫杉醇或其他治疗胰腺癌的药物的时间间隔为1天、2天、3天、4天、5天、6天、7天、8天、9天、10天、11天、12天、13天、14天、 15天、16天、17天、18天、19天、20天或21天。例如，施用溶瘤病毒VG161和吉西他滨以及白蛋白紫杉醇的时间间隔为7天。例如，施用溶瘤病毒VG161和吉西他滨以及白蛋白紫杉醇的时间间隔为14天。例如，施用溶瘤病毒VG161和吉西他滨以及白蛋白紫杉醇的时间间隔为21天。
在某些实施方式中，所述溶瘤病毒VG161为单次给药。在某些实施方式中，所述溶瘤病毒VG161为连续多次(例如，2次、3次、4次、5次或更多次)给药。
所述溶瘤病毒VG161的给药剂量为1×10 5pfu-1×10 7pfu。在某些实施方式中，所述溶瘤病毒VG161的给药剂量为1×10 5pfu、5×10 5pfu、1×10 6pfu、5×10 6pfu或1×10 7pfu。例如，在某些实施方式中，所述溶瘤病毒VG161的给药剂量为1×10 5pfu。
在某些实施方式中，所述吉西他滨的给药剂量为50mg/kg。在某些实施方式中，所述白蛋白紫杉醇的施用浓度为30mg/kg。
在某些实施方式中，所述治疗缓解胰腺癌的方法包括向有需要的受试者同时施用1×10 5pfu的溶瘤病毒VG161，和50mg/kg的吉西他滨以及30mg/kg的白蛋白紫杉醇的组合，以及先施用1×10 5pfu的溶瘤病毒VG161之后施用50mg/kg的吉西他滨以及30mg/kg的白蛋白紫杉醇的组合。
在某些实施方式中，注射施用所述VG161和/或吉西他滨以及白蛋白紫杉醇。在某些实施方式中，所述注射为瘤内注射。
药物组或药物组合物的不同组分可独立地包装(例如，在施用之前并不彼此混合)或预先混合且包装在同一包装单元中。
本申请的组合物可为药物组合物，可进一步包括药学上可接受的赋形剂。药学上可接受的赋形剂的实例包含但不限于惰性固体稀释剂和填充剂、稀释剂、无菌水溶液以及各种有机溶剂、促渗剂、增溶剂和佐剂。
虽然本文中已展示和描述本申请的各种实施方案，但本领域的技术人员将显而易见，这些实施方案仅仅通过实例的方式提供。本领域的技术人员可想到不脱离本申请的众多变化形式、改变和替换。应理解，可以采用本文所描述的本申请实施方案的各种替代方案。
实施例
溶瘤病毒VG161以及mVG161的构建示例可参见Chouljenko DV,Ding J,Lee IF,Murad YM,Bu X,Liu G,et al.Induction of Durable Antitumor Response by a Novel Oncolytic  Herpesvirus Expressing Multiple Immunomodulatory Transgenes.Biomedicines.2020；8(11)。
实施例1 VG161体外有效杀伤胰腺癌细胞并促进凋亡
一、体外细胞杀伤及促凋亡实验
在体外实验中证实VG161单药对胰腺癌细胞系具有杀伤及促进凋亡作用，采用CCK8及细胞流式实验对此进行检测。在流式实验中，免疫细胞在37℃下用白细胞活化混合物(550583,BD Biosciences)刺激4小时，然后用以下抗体和可固定活力试剂盒(423106，BioLegend)进行染色：抗-CD16/32(156604，BioLegend)、抗-CD45(103128，BioLegend)、抗CD3(100204，BioLegend)、抗CD4(100434，BioLegend)、抗CD25(102016，BioLegend)、抗CD8a(10744，BioLegend)、抗NK1.1(108749，BioLegend)、抗FOXP3(320008，BioLegend)和抗PD1(135221，BioLegend)。所有样品均在BD LSRFortessa仪器上采集，并使用FlowJo V10软件分析数据。
CCK8即Cell Counting Kit-8，该试剂盒可用于简便而准确进行细胞增殖和毒性分析。其基本原理为：该试剂中含有WST-8【化学名：2-(2-甲氧基-4-硝基苯基)-3-(4-硝基苯基)-5-(2,4-二磺酸苯)-2H-四唑单钠盐】，它在电子载体1-甲氧基-5-甲基吩嗪鎓硫酸二甲酯(1-Methoxy PMS)的作用下被细胞中的脱氢酶还原为具有高度水溶性的黄色甲瓒产物(Formazan dye)。生成的甲瓒物的数量与活细胞的数量成正比。因此可利用这一特性直接进行细胞增殖和毒性分析。
选取4株常见的人胰腺癌细胞系BxPC3、MIAPaCa-2、PANC-1以及SW 1990，加入MOI分别为0.01、0.1、1以及10的VG161，以vehicle为空白对照，随后进行CCK8检测。
结果如图2所示：CCK8结果显示，VG161可显著抑制胰腺癌细胞系的增殖，且作用随着病毒浓度(MOI)的提升而增强。
此外，流式细胞术结果显示VG161可以促进多种胰腺癌细胞系的凋亡(图8)。
二、病毒复制和外源基因表达能力检测
溶瘤病毒在宿主细胞中能够得以正常复制才是其发挥抗瘤作用的关键。而VG161所携带的外源性基因是其不同于其他溶瘤病毒的主要特点，是否可以在胰腺癌中正常表达也是关键之一。空斑试验常用于检查病毒的复制能力，而酶联免疫吸附测定(Enzyme linked immunosorbent assay，ELISA)可用于检测外源性基因的表达。
病毒空斑试验的原理是将各稀释度的病毒液接种到单层细胞培养环境中，吸附2小 时后，在单层细胞上覆以琼脂糖，病毒感染细胞并在细胞中增殖，使细胞破裂死亡。由于固体介质的限制，释放的病毒只能由最初感染的细胞向四周扩展。经过几个增殖周期，便形成一个局限性病变细胞区，即病毒蚀斑。经中性红活细胞染料着色后，活细胞显红色，而蚀斑区细胞而不着色，形成不染色区域。病毒蚀斑如噬菌体感染细菌形成的噬菌斑。理论上，当病毒液充分稀释后，获得的每个蚀斑均源于最初感染细胞的一个病毒颗粒，即蚀斑中的病毒为一个病毒体的繁殖后代品系，由此达到纯化病毒的目的。除了可用于纯化病毒以外，空斑实验还可用于干扰素、抗体中和病毒繁殖能力的实验。因此，通过计数空斑形成数，我们即可在本实验中计数出病毒的复制量，从而使其复制能力量化。
ELISA指将可溶性的抗原或抗体结合到聚苯乙烯等固相载体上，利用抗原抗体特异性结合进行免疫反应的定性和定量检测方法。
选取4株常见的人胰腺癌细胞系BxPC3、MIAPaCa-2、PANC-1以及SW 1990，加入MOI＝1的VG161，培养箱培养48h后收集上清和细胞，随后进行病毒空斑检测并计算病毒滴度。选取上述BxPC3或KPC的上清进行酶联免疫吸附测定实验，计算得出插入VG161内分泌型因子IL12、IL15/15RA和PD-L1阻断肽的表达情况。
空斑实验结果如图3显示：VG161在各胰腺癌细胞系内均可进行有效的复制，一般在48小时-72小时时达到复制高峰(图3A)。
本实施例还检测了VG161感染小鼠胰腺癌细胞(KPC)和人胰腺癌细胞(BxPC-3)后的外源基因表达。ELISA结果显示，VG161携带的外源基因IL-12、IL-15/IL-15Rα和PD-L1阻断剂在两种细胞系中均能稳定表达，其中PD-L1拮抗肽(PD-L1 blocker)的表达量最高(图3B)。
三、3D仿癌模型构建
本实施例通过镜下观察3D模型外在结构稳定性的办法来评估VG161的作用效果。同时，采用空斑实验的方法计算VG161在3D模型中的复制能力。
选取人胰腺癌细胞系BxPC3进行细胞3D成球实验。在96孔板中铺上100ul琼脂糖并冷却，每孔加入3000个左右BxPC3胰腺癌细胞，恒温箱培养3天。镜下确认成球后加入MOI＝1的VG161，以vehicle为空白对照，分别在24h、96h、168h时显微镜下观察并拍摄细胞3D球形态。随后选取上述VG161感染组细胞进行病毒空斑检测并计算病毒滴度。
根据图4，镜下观察3D模型发现，实验组球体结构受VG161影响逐渐破坏，而对 照组即便在168h仍然保持球体完整，证实VG161可对3D胰腺癌模型进行有效杀伤(图4A)。提取3D球体，打散后取上清液进行空斑实验，提示VG161可在3D模型中进行有效复制(图4B)。
实施例2 VG161体内有效杀伤胰腺癌细胞并具有安全性
本实施例通过在裸鼠上构建胰腺癌皮下瘤模型并进行瘤内注射，从而验证VG161在实体瘤中的治疗效果。
在裸鼠右侧腋下注射BxPC3细胞悬液，一般于2周后成瘤，待瘤体大小达到800mm 3时进行VG161瘤内注射，以vehicle作为对照。观察指标包括肿瘤体积变化、小鼠体重，每3天测量1次，记录小鼠生存曲线。观察终点包括小鼠死亡或小鼠肿瘤体积超过2000mm 3。
为了探索VG161与其他药物联用的最佳浓度，在BxPC3裸鼠模型上分别以不同浓度VG161连续5次和单次注射，观察不同给药模式下VG161的治疗效果。
在裸鼠右侧腋下注射BxPC3细胞悬液，一般于2周后成瘤，待瘤体大小达到800mm 3时进行VG161瘤内注射，以vehicle作为对照。在连续5次注射组，取4个浓度，分别为5×10 5pfu、1×10 6pfu、5×10 6pfu以及1×10 7pfu。单次注射组取5个浓度，分别为1×10 5pfu、5×10 5pfu、1×10 6pfu、5×10 6pfu以及1×10 7pfu。观察指标包括肿瘤体积变化、小鼠体重，每3天测量1次，记录小鼠生存曲线。观察终点包括小鼠死亡或小鼠肿瘤体积超过2000mm 3。
根据图5，分别建立BxPC3、SW 1990、MIAPaCa-2三种胰腺癌裸鼠模型。在BxPC3模型中，单次注射1×10 7pfu病毒，连续5次注射，观察中发现效果显著，大多数实验组肿瘤完全消失(图5A)；在SW 1990和MIA PaCa-2荷瘤的模型中，我们予以病毒减量，单次注射1×10 5pfu，且仅一次注射，观察中发现尽管实验组肿瘤并未消失，但VG161仍然达到了非常理想的治疗效果(图5B和图5C)。
还采用4个浓度连续5次注射的方法进行最佳浓度探索，结果发现即便是最低浓度(5×10 5pfu)，VG161的作用依旧显著，大部分肿瘤完全消失(图6B)。将注射次数改为单次，同时再降低一个浓度梯度，即取5个浓度单次注射的方式进行探索，结果发现1×10 5pfu可作为比较理想的联用浓度(图6C)。
本实施例还测量了在不同时间点注射单剂量VG161的小鼠的IL-15Rα表达水平。结果表明，IL-15Rα表达仅在肿瘤注射病灶中显著升高，并在48小时达到峰值(图9A)。 在小鼠的外周血中测量器官损伤指标(例如，丙氨酸转氨酶[ALT]、肌酐[CR]和乳酸脱氢酶[LDH])，发现上述所有指标在注射后瞬时增加，在48小时达到峰值，然后迅速回到基线(图9B)。最后收集所有器官进行HE染色并在显微镜下观察。组织学分析表明VG161不会对组织细胞造成损伤(图9C)。因此，VG161在未免疫小鼠中具有良好的安全性，并凭借其溶瘤活性表现出抗癌能力。
实施例3 VG161携带的外源基因具有免疫激活的能力
为了证实VG161携带的外源基因具有激活抗肿瘤免疫的能力，本实施例以VG160骨架病毒(不包含编码外源基因IL-12、IL15、IL-15Rα和PD-L1阻断肽的基因，病毒骨架修饰与VG161相同)作为对照组。为此，将(4×10 5)KPC细胞植入C57BL/6小鼠体内。肿瘤形成后，选择左侧肿瘤作为注射灶进行注射治疗。结果表明，mVG161和VG160组均显着抑制了注射侧的肿瘤生长。此外，mVG161的肿瘤抑制能力大于VG160(图10B)。并且mVG161组对另一侧未注射肿瘤的生长(即远隔效应)显示出显着的抑制能力(图10B)。关于缓解率,mVG161和VG160组在注射侧的部分反应(PR)达到100％(图10C)。在非注射侧，mVG161组的PR也达到了100％，而VG160组的PR仅为60％(图10C)。两组均未观察到完全缓解(CR)。在给药后的第3、7和15天，获取脾脏样本用于ELISpot检测。结果显示干扰素γ(IFN-γ)在mVG161组中显著上调(图10D)。在给药后第3、7和15天，还从小鼠身上收集了双侧肿瘤，用于流式细胞术分析(图10E)。在第3天和第7天，VG160和mVG161组的双侧肿瘤中均观察到CD8 +T细胞明显浸润，并且mVG161组的浸润水平略高于VG160组。此外，给药后15天，mVG161组未注射肿瘤中CD8 +T细胞浸润明显高于其他组。此外，mVG161组的NK细胞在给药后第3天明显减少，并逐渐升高至略高于其他组的水平，说明IL-15在体内发挥了效果(图10E)。
实施例4 VG161影响免疫和代谢微环境
为了确认VG161携带的外源基因的作用，在给药后第3、7和15天对KPC细胞植入的C57BL/6小鼠注射侧的肿瘤进行了单细胞测序。首先使用unsupervised clustering数据分析将CD45 +细胞分成不同的免疫群体组(图11A和11B)。然后根据每个群体的已知标记的表达对这些免疫群体进行分类(图11C)。结果表明，与VG160相比，mVG161引起更积极的免疫反应(图11D)。此外，感染mVG161的肿瘤显示出大量 新的效应样CD8 +T细胞(第7天和第15天)、NK细胞(第7天)和单核细胞(第3天)。与VG160感染的肿瘤相比，VG161感染的肿瘤中，功能失调或抑制性细胞也有一定比例的损失，例如Tregs(第3天和第7天)和肿瘤相关巨噬细胞(第7天和第15天)(图11D)。
还评估了OV治疗后肿瘤组织代谢微环境的变化，在接受VG160和VG161三天后，使用AFADSI-MSI技术测试小鼠皮下肿瘤样本。结果表明，与载体组相比，VG160或VG161引起的代谢变化相对相似。
实施例5 VG161与吉西他滨以及白蛋白紫杉醇联用
基于实施例2获得了VG161最适联用浓度后，再次建立BxPC3裸鼠模型，探索VG161联合胰腺癌一线化疗方案(GEM+Nab-PTX方案)的治疗效果。
GEM+Nab-PTX方案即吉西他滨联合白蛋白紫杉醇方案，是目前常用的胰腺癌一线新辅助治疗化疗方案。已有多项研究证实，两药联合有利于延长胰腺癌患者的生存时间。设置Vehicle为阴性对照组，VG161单药和GEM+Nab-PTX方案为阳性对照组，以探索VG161+GEM+Nab-PTX的联用方案应用于胰腺癌的治疗效果。同时，设置先给药VG161后给药GEM+Nab-PTX方案、先给药GEM+Nab-PTX方案后给药VG161两组不同顺序的给药方式，以探索最佳的联用策略。
从图7可以看到，无论是阳性对照组还是联用组的效果均优于阴性对照组(Vehicle组)。而联用组与阳性对照组的比较中，我们发现两个联用组的效果均优于GEM+Nab-PTX组。但是，GEM+Nab-PTX+VG161组的效果并未优于VG161单药组，而VG161+GEM+Nab-PTX组的效果显著优于其他组。
由上可得，VG161与GEM+Nab-PTX方案联用将进一步提高其在胰腺癌中的治疗效果，而且先进行VG161给药，后进行GEM+Nab-PTX方案给药是最优选择。
以上显示和描述了本申请的基本原理、主要特征和本申请的优点。本行业的技术人员应该了解，本申请不受上述实施例的限制，上述实施例和说明书中描述的只是说明本申请的原理，在不脱离本申请精神和范围的前提下本申请还会有各种变化和改进，这些变化和改进都落入要求保护的本申请范围内。本申请要求保护范围由所附的权利要求书及其等同物界定。

Claims (21)

  1. 溶瘤病毒VG161在制备治疗胰腺癌药物中的用途。
  2. 根据权利要求1所述的用途，其中所述药物以溶瘤病毒VG161作为唯一活性成分。
  3. 溶瘤病毒VG161和至少一种治疗胰腺癌的药物在制备治疗胰腺癌药物组中的用途。
  4. 根据权利要求3所述的用途，其中所述至少一种治疗胰腺癌的药物为吉西他滨以及白蛋白紫杉醇。
  5. 根据权利要求3所述的用途，在所述药物组中，溶瘤病毒VG161为先遣药物，吉西他滨以及白蛋白紫杉醇为后续药物。
  6. 根据权利要求3所述的用途，其中所述药物组为注射剂。
  7. 一种治疗胰腺癌的药物组合物，其由溶瘤病毒VG161以及药学上可接受的辅料组成。
  8. 一种治疗胰腺癌的药物组，其包括先遣药物以及后续药物，所述先遣药物由溶瘤病毒VG161以及药学上可接受的辅料组成，所述后续药物由吉西他滨以及白蛋白紫杉醇组成或由其他治疗胰腺癌的药物组成。
  9. 治疗和/或缓解胰腺癌的方法，包括向有需要的受试者施用溶瘤病毒VG161。
  10. 根据权利要求9所述的方法，其中所述溶瘤病毒VG161的施用浓度为1×10 5pfu、5×10 5pfu、1×10 6pfu、5×10 6pfu或1×10 7pfu。
  11. 根据权利要求9或10所述的方法，其中所述施用为注射施用。
  12. 根据权利要求11所述的方法，其中所述注射施用为瘤内注射。
  13. 治疗和/或缓解胰腺癌的方法，其包括向有需要的受试者施用溶瘤病毒VG161，和吉西他滨以及白蛋白紫杉醇的组合。
  14. 根据权利要求13所述的方法，其包括在施用溶瘤病毒VG161之后，施用吉西他滨以及白蛋白紫杉醇。
  15. 根据权利要求13所述的方法，其中施用溶瘤病毒VG161和吉西他滨以及白蛋白紫杉醇的时间间隔为1天、2天、3天、4天、5天、6天、7天、8天、9 天、10天、11天、12天、13天、14天、15天、16天、17天、18天、19天、20天或21天。
  16. 根据权利要求13-15中任一项所述的方法，其中所述溶瘤病毒VG161的给药剂量为1×10 5pfu、5×10 5pfu、1×10 6pfu、5×10 6pfu或1×10 7pfu。
  17. 根据权利要求16中所述的方法，其中所述吉西他滨的给药剂量为50mg/kg。
  18. 根据权利要求18中所述的方法，其中所述白蛋白紫杉醇的施用浓度为30mg/kg。
  19. 根据权利要求13-15中任一项所述的方法，其中所述溶瘤病毒VG161为单次给药。
  20. 根据权利要求13-15中任一项所述的方法，其中所述施用为注射。
  21. 根据权利要求20所述的方法，其中所述注射为瘤内注射。
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