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WO2022206962A1 - Virus oncolytique recombiné et son utilisation médicale - Google Patents

Virus oncolytique recombiné et son utilisation médicale Download PDF

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WO2022206962A1
WO2022206962A1 PCT/CN2022/084842 CN2022084842W WO2022206962A1 WO 2022206962 A1 WO2022206962 A1 WO 2022206962A1 CN 2022084842 W CN2022084842 W CN 2022084842W WO 2022206962 A1 WO2022206962 A1 WO 2022206962A1
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oncolytic virus
virus
tumor
recombinant oncolytic
prodrug
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段建新
孟繁英
齐天阳
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Ascentawits Pharmaceuticals Ltd
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Ascentawits Pharmaceuticals Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof

Definitions

  • the present invention relates to recombinant oncolytic virus and its medicinal use.
  • Oncolytic viruses rely on their own specificity to replicate in tumor cells to lyse tumor cells, and the viruses or toxins released after cell lysis can further infect surrounding tumor cells without damaging or affecting normal cells and tissues. smaller.
  • Oncolytic viruses are generally divided into two categories: one is a wild-type virus and a naturally mutated attenuated virus strain, which naturally has an affinity for some tumor cells, such as reovirus, Newcastle disease virus, and self-replicating small viruses. Viruses, etc., these viruses can reproduce and lyse cells in some tumor cells, and have natural specific oncolytic activity; the other is viruses that can only replicate in tumor cells after the virus genome is modified.
  • adenovirus is a relatively early oncolytic virus, and the oncolytic mechanism is relatively clear, and adenovirus type 5 is more clearly studied.
  • Adenovirus was used to treat head and neck malignant tumors soon after its discovery. The tumor was shrunk to varying degrees after adenovirus injection, but the tumor was easy to recur after treatment, and the effect was difficult to last; it was not until 1996 that Bischoff et al. Recombinant adenovirus Onyx-015 can selectively replicate in tumor cells with abnormal p53 and cause tumor killing.
  • Oncolytic adenovirus research has received widespread attention again and has developed rapidly. Therefore, many new types of oncolytic adenovirus have emerged. Characteristic has been committed to the development and marketing of oncolytic viruses into therapeutic drugs.
  • Adenovirus H101 (Ankerui) for nasopharyngeal carcinoma, which has been marketed in China, and Imlygic (talimogene laherparepvec), a herpes simplex virus for melanoma approved by the US FDA for marketing, etc.
  • Imlygic titaniumimogene laherparepvec
  • a herpes simplex virus for melanoma approved by the US FDA for marketing, etc.
  • there are multiple clinical trials of oncolytic virus therapy in progress as shown in the table in Figure 8.
  • the oncolytic virus As a foreign body of the immune system, will be discovered by the immune system and eliminated or eliminated after entering the solid tumor tissue. Therefore, the oncolytic virus has a limited time to act in the body. Or a larger amount is administered by injection at the tumor site.
  • oncolytic viruses For blood cancers, since T lymphocytes or B lymphocytes are in the circulating blood system, oncolytic viruses must circulate in the blood system before they can take effect. However, as foreign bodies in the immune system, oncolytic viruses are bound to be attacked by the immune system. Therefore, its action time is necessarily shorter than that of solid tumors. Because of this, there is no oncolytic virus for the treatment of blood cancer on the market, and there is no related clinical research and development project.
  • one of the current strategies for using oncolytic viruses to treat tumors is to genetically modify the oncolytic viruses to carry genes of therapeutic significance, and to introduce such genes into specific tumor cells. expression of related proteins.
  • the tumor suppressor properties of the expressed protein or antibody are used to treat tumors in combination.
  • a key factor in the design of oncolytic virus constructs is to consider the expression time of the transfected gene and the rupture time due to viral replication.
  • the inventor envisages that the targeted protein can be highly expressed before the tumor ruptures caused by virus replication by using early promoter gene transcription, and then administering the targeted protein or enzyme-activated chemotherapeutic drugs, so that the chemotherapeutic drugs can enter the oncolytic virus after infection. Under the activation of highly expressed targeting proteins or enzymes, it exerts its medicinal effect and releases cytotoxins to directly kill cancer cells. In this way, cancer cells are infected by oncolytic viruses integrated with specific targeting protein genes, and then the targeting proteins are administered.
  • oncolytic viruses only need to infect cancer cells without eventually lysing cancer cells to kill cancer cells under the action of targeted proteins or enzyme-activated chemotherapy drugs, so the administration of oncolytic viruses The amount can be greatly reduced, and the impact on the human immune system is also smaller.
  • the final result is that the combination or compound of an oncolytic virus with a specific targeted protein gene and a targeted protein or enzyme-activated chemotherapeutic drug can greatly improve the immune system. Anticancer effects of oncolytic viruses.
  • Chemotherapy drugs have a long history and their efficacy has been verified for a long time, but they have the disadvantage of large side effects. It is an important direction to reduce toxic and side effects by redesigning chemotherapeutic drugs as prodrugs.
  • the prodrugs obtained by the structural modification of chemotherapeutic drugs are a kind of compounds which have little or no activity in vitro, and release active substances through enzymatic or non-enzymatic action in vivo to exert pharmacological effects. In most cases, prodrugs are simple chemical derivatives that can be converted to the active parent drug in one or two steps of chemical or enzymatic catalysis.
  • chemotherapeutic drugs targeting protein or enzyme activation include macromolecular antitumor prodrugs targeting enzyme activation and small molecule antitumor prodrugs targeting enzyme activation.
  • Macromolecular antitumor prodrugs targeting enzyme activation in ADEPT (Antibody-Directed Enzyme Prodrug Therapy), the administered enzyme antibody and antigen present on the surface of target cells. Subsequently, a prodrug activated by this enzyme is administered, resulting in the formation of a toxic drug. In most cases, toxic drugs must penetrate the cell membrane to kill the cell.
  • the drug can cause cell death without penetrating cell membranes (eg, the anemone toxin palytoxin) (Rooseboom M, Commandeur J N M, Vermeulen N P E. Enzyme-Catalyzed Activation of Anticancer Prodrugs[J].Pharmacological Reviews, 2004, 56(1):53-102, DOI: 10.1 124/pr.56.1.3).
  • penetrating cell membranes eg, the anemone toxin palytoxin
  • Small molecule anti-tumor prodrugs activated by targeting enzymes which can be combined with exogenous enzymes introduced into tumor sites or enzymes expressed in tumor cells (GDEPT and VDEPT, Gene-Directed Enzyme Prodrug Therapy and Virus directed enzyme prodrug therapy) namely Targeted enzymatic interactions for translation into cytotoxic drugs.
  • Small molecule antitumor prodrugs activated by targeting enzymes can improve the targeting of antitumor drugs, and the expression of targeting enzyme genes integrated in oncolytic viruses can enable cancer cells to express higher levels of targeting enzymes that can activate prodrugs , so as to better exert the anti-tumor effect of small molecule anti-tumor prodrugs activated by targeting enzymes and reduce the toxic and side effects.
  • prodrugs that are activated by specific enzymes. These enzymes include endogenous enzymes as well as exogenous enzymes.
  • Endogenous enzymes include oxidoreductases, transferases, transferases, hydrolases, and lyases.
  • Oxidoreductases include aldehyde dehydrogenase Aldehyde oxidase, amino acid oxidase Amino acid oxidase, Cytochrome P450 reductase, DT-Diaphorase DT-Diaphorase, Cytochrome P450 Cytochrome P450, Tyrosinase Wait.
  • Transferases include Thymidylate synthase, Thymidine phosphorylase, Glutathione S-Transferase, Deoxycytidine kinase, etc.
  • Hydrolases hydrolases include carboxylesterase, alkaline phosphatase, Alkaline phosphatase, ⁇ -glucuronidase and ⁇ -Glucuronidase.
  • Exogenous enzymes include nitroreductase Nitroreductase, purine nucleoside phosphorylase Purine-nucleoside phosphorylase, thymidine kinase Thymidine kinase, alkaline phosphatase Alkaline phosphatase, ⁇ -glucuronidase ⁇ -Glucuronidase, carboxypeptide Enzymes Carboxypeptidase, Penicillin amidase, ⁇ -Lactamase, Cytosine deaminase, Methionine ⁇ -lyase, etc.
  • antitumor prodrugs activated by exogenous targeted enzymes that are not intervened in humans by interventional means are shown in Table 2 below.
  • IPdR i.e. Ropidoxuridine
  • IPdR i.e. Ropidoxuridine
  • Menadione, menadione alias vitamin K3
  • Tirapazamine i.e. tirapazamine
  • AQ4N namely banoxantrone
  • Chinese name is banoxantrone
  • CAS number is 136470-65-0
  • 4-S-CAP namely 4-S-cysteaminylphenol
  • Chinese name is 4-S-cysteinyl phenol
  • GHB namely ⁇ -L-glutaminyl-4-hydroxybenzene
  • Chinese name is ⁇ -L-glutamine-4-hydroxybenzene
  • NB1011 i.e. (E)-5-(2-bromovinyl)-2-deoxy-5-uridylphenyl-L-methoxyalaninylphosphoramidate
  • 5'-DFUR namely 5-deoxy-5-fluorouridine, also known as Doxifluridine
  • Chinese name is deoxyfluridine
  • PTA namely cis-3-(9H-purin-6-ylthio)acrylic acid
  • PTA namely cis-3-(9H-purin-6-ylthio)acrylic acid
  • Chinese cis-3-(9H-purine-6-thio)acrylic acid
  • Pentostatin i.e. pentostatin
  • Fludarabine i.e. fludarabine
  • Ancitabine or Ancitabine
  • Epirubicin-glucuronide that is, epirubicin-glucuronide conjugate.
  • HMR 1826 the CAS number of this compound is 148580-25-0, which is a conjugate of glucuronic acid and doxorubicin
  • DNR-GA3 namely N-[4-daunorubicin-N-carbonyl-(oxymethyl)-phenyl]O- ⁇ -glucuronyl carbamate, also known as glucuronidated daunorubicin, is a conjugate of daunorubicin and gibberellin;
  • DOX-GA3 namely N-[4-doxorubicin-N-carbonyl(oxymethyl)phenyl]O-beta-glucuronyl carbamate, also known as glucuronidated doxorubicin, is a conjugate of doxorubicin and gibberellin;
  • Paclitaxel glucuronide Glucuronic acid-derived paclitaxel compound
  • Chinese translation is glucuronic acid-derived paclitaxel compound
  • 5-FU glucuronide the glucuronide derivative of 5-FU.
  • SeCys conjugate namely selenocysteine Se conjugates
  • Chinese translation is selenocysteine selenium conjugate
  • 5-FU namely 5-Fluorouracil
  • Chinese name is 5-fluorouracil
  • Hydrogen peroxide that is, hydrogen peroxide.
  • Nitroxide radical the nitric oxide free radical
  • Isophosphamide mustard the structure of ifosphamide mustard
  • BQ i.e. dihydro-1,4-benzothiazine-6,7-dione
  • GBQ ⁇ -l-glutaminyl-3,4-benzoquinone
  • Aziridinium agent the aziridine cation salt structure reagent, has the following chemical structure
  • S-CPHC-glutathione namely S-(N-p-chlorophenyl-N-hydroxycarbamoyl) glutathione
  • Chinese can be translated as S-(N-p-chlorophenyl-N-hydroxycarbamoyl) glutathione
  • 6-MP or 6-mercaptopurine, or 6-mercaptopurine
  • Paclitaxel or paclitaxel
  • Carboxylic acids and amines that is, carboxylic acids and amines
  • N,N-di-(2-chloroethyl)-p-hydroxyaniline mustard can be translated as N,N-di(2-chloroethyl)-p-hydroxyaniline mustard
  • Doxorubicin i.e. doxorubicin
  • MeP-dR namely 9-( ⁇ -2-deoxyerythropentofuranosyl)-6-methylpurine (6-methylpurine-2-deoxyribonucleoside)
  • Chinese translation is 6-methylpurine-2′-deoxyribonucleoside
  • Ganciclovir also known as ganciclovir
  • Mitomycin C phosphate namely p-[N,N-bis(2-chloroethyl)amino]phenyl phosphate, mitomycin C phosphate
  • N-(4-phosphonooxy)-phenylacetyl)doxorubicin which can be translated as N-(4-phosphonooxy)-phenylacetyl)doxorubicin
  • Glucuronidated Nornitrogen mustard the structure of glucuronidated nitrogen mustard
  • Glucuronide mustard the structure of nitrogen mustard glucuronide
  • CMDA 4-[N-(2-chloroethyl)-N-[2-(mesyloxy)ethyl]amino]benzoyl-L-glutamic acid, CAS number 122665-73-0
  • DPO i.e. doxorubicin-N-p-hydroxyphenoxyacetamide
  • doxorubicin-N-p-hydroxyphenoxyacetamide can be translated as doxorubicin-N-p-hydroxyphenoxyacetamide
  • NHPAP or N-(4'-hydroxyphenylacetyl)palytoxin, can be translated as N-(4,-hydroxyphenylacetyl) anemone toxin
  • C-DOX also known as BMY46633
  • cephalosporin doxorubicin prodrug is cephalosporin doxorubicin prodrug, and its structural formula is (See Evans LE, Krishna A, Ma Y, et al. Exploitation of Antibiotic Resistance as a Novel Drug Target: Development of a ⁇ -Lactamase-Activated Antibacterial Prodrug. J Med Chem. 2019;62(9):4411-4425. doi:10.1021/acs.jmedchem.8b01923)
  • CM 7-(phenylacetamido)-cephalosporin mustard
  • CM 7-(phenylacetamido)-cephalosporin mustard
  • CCM, 7-(4-carboxybutanamido)-cephalosporin mustard can be translated as 7-(4-carboxybutanamido)-cephalosporin mustard structure
  • Cephalosporin-DCCP namely cephalosporin and 4'-carboxyphthalato(1,2-cyclohexanediamine)platinum
  • 4'-carboxyphthalato(1,2-cyclohexanediamine)platinum can be translated as 4,-carboxyphthalic acid(1,2-cyclohexanediamine)platinum conjugated compound, see literature Hanessian,Stephen,and J.Wang.”Design and synthesis of a cephalosporincarboplatinum prodrug activatable by a-lactamase.”Canadian Journal of Chemistry(1993), the specific structure is
  • PROTAX the structural formula is (See Evans LE, Krishna A, Ma Y, et al. Exploitation of Antibiotic Resistance as a Novel Drug Target: Development of a ⁇ -Lactamase-Activated Antibacterial Prodrug. J Med Chem. 2019;62(9):4411-4425 .doi:10.1021/acs.jmedchem.8b01923)
  • 5-Fluorocytosine i.e. 5-Fluorocytosine
  • Trifluoromethionine or trifluoromethionine
  • Ganciclovir-triphosphate nucleotide that is, ganciclovir triphosphate nucleotide
  • Etoposide i.e. etoposide
  • POM i.e. p-(N,N-bis(2-chloroethyl)amino)phenol
  • Doxorubicin i.e. doxorubicin
  • Methotrexate which is aminopterin
  • DAVLBHYD 4-desacetylvinblastine-3-carboxylic acid hydrazide
  • DAVLBHYD 4-desacetylvinblastine-3-carboxylic acid hydrazide
  • DACCP namely 4'-carboxyphthalato(1,2-cyclohexanediamine)platinum
  • Taxol or paclitaxel
  • antineoplastic prodrugs activated by aldo-keto reductase 1C3, AKR1C3 (EC: 1.1.1.188) are disclosed in the following patents:
  • DNA alkylating agent corresponding to PCT application number PCT/US2016/021581, publication number WO2016/145092A, corresponding to Chinese application number 2016800150788, publication number CN107530556A;
  • Nitrobenzyl derivative anticancer reagent corresponding to PCT application number PCT/US2016/025665, publication number WO2016/161342, corresponding to Chinese application number 2016800200132, publication number CN108136214A;
  • Prodrug compounds activated by AKR1C3 and their use in treating hyperproliferative disorders corresponding to PCT application number PCT/NZ2019/050030, publication number WO2019190331, corresponding to Chinese application number 201980023423.6, publication number CN111918864A.
  • Tricyclic ARK1C3-dependent KARS inhibitor corresponding PCT application number PCT/IB2020/057285, publication number WO2021005586A1, corresponding Chinese application number CN202080053804.1, publication number CN114206870A.
  • the compounds disclosed in the above seven patent applications are all anti-tumor/anti-cancer prodrugs activated by AKR1C3 enzyme.
  • AKR1C3 aldehyde-ketone reductase 1C3 in English, EC: 1.1.1.188
  • the above-mentioned compounds in the form of prodrugs are reduced through the catalysis of AKR1C3 in the special microenvironment of cancer cells to obtain cytotoxic toxins and play a role in killing cancer cells.
  • the Chinese name is (S)-1-(3-(3-N,N-dimethylaminocarbonyl)phenoxy-4-nitrophenyl)-1-ethyl-N,N'-bis (Ethylene) phosphoramidate, also known as AST-3424, OBI-3424, TH-3424, AST-106), CAS number is 2097713-69-2, and its structure is as follows:
  • Anti-tumor prodrugs activated by cytochrome P450 reductase are represented by TH-302, PR-104, etc., which are activated under the combined action of hypoxia and cytochrome P450 reductase , so the following hypoxia-activated anticancer prodrugs (HAP, Hypoxia Activated Prodrugs) patent applications are disclosed in the following patents:
  • the compounds disclosed in the above two patent applications are all anti-tumor/anti-cancer prodrugs activated by cytochrome P450 reductase (EC: 1.6.2.4).
  • Antitumor prodrugs activated by cytochrome P450 reductase should meet the following conditions:
  • the cancer cell proliferation inhibitory effect of a certain compound detected is greater than the cancer cell proliferation inhibitory effect of the compound detected in a normoxia environment.
  • the cancer cell proliferation inhibition effect is quantified by IC50, if a certain compound If the IC50 of a certain cancer cell line detected in a normoxic environment is greater than the IC50 detected in a hypoxic environment, it can be determined that the compound is an anticancer drug activated by hypoxia. The larger the difference, the higher the specificity of hypoxia activation.
  • NTR nitroreductase
  • alkylating agents based on nitroaniline and their purposes as prodrugs corresponding PCT application number PCT/NZ2003/000225, publication number WO2004/033415A1, corresponding Chinese application number 200380102812.7, publication number CN1711236A;
  • Novel nitrophenyl mustards and nitrophenyl aziridines and their corresponding phosphates and uses as targeting cytotoxic agents corresponding to PCT application number PCT/NZ2004/000275, publication number WO 2005/ 042471, corresponding to Chinese application number 200480039430.9, publication number CN1902159A.
  • the above-mentioned compounds in the form of prodrugs will be in the special microenvironment of cancer cells (due to the rapid proliferation of tumors, hypoxia will occur in the cancer cells of the tumor due to insufficient oxygen supply, that is, the cancer cells of the tumor will be in a hypoxic environment) in the absence of oxygen. Reduction occurs catalyzed by oxygen and cytochrome P450 reductase (EC: 1.6.2.4) (see Meng et al, Molecular and Cellular Pharmacology of the Hypoxia-Activated Prodrug TH-302, MCT, 2012(11):740 ; DOI: 10.1158/1535-7163.MCT-11-0634)), to obtain a cytotoxic toxin and play a role in killing cancer cells.
  • cytochrome P450 reductase EC: 1.6.2.4
  • Glufosfamide the chemical name is ⁇ -D-glucopyranosyl-N, N'-bis(2-chloroethyl) phosphoramide, the English name is ⁇ -D-Glucopyranosyl-[N,N'- bis[(2-chloroethyl)]phosphoric acid diamide, a new type of alkylating agent antitumor drug, is formed by linking a molecule of isophosphoramide mustard with direct alkylation and a molecule of glucose through a glycosidic bond. of. Glufosfamide is transported into tumor cells by the sodium-dependent glucose transmembrane transporter SAAT1, and then activated by ⁇ -glucuronidase (EC: 3.2.1.31) hydrolysis to release isophosphoramide mustard.
  • SAAT1 sodium-dependent glucose transmembrane transporter
  • ⁇ -glucuronidase EC: 3.2.1.31
  • Threshold obtained the fast-track approval of the drug from the U.S. FDA in 2004 for the treatment of unresectable locally advanced or metastatic pancreatic cancer previously treated with gemcitabine (W.Steve Ammons, Jin-Wei Wang y , Zhijian Yang y, George F. Tidmarshz and Robert M. Hoffmany, Neoplasia, 2007.8,9(8):625-633), but in 2007 the company announced a phase III clinical trial for patients with metastatic pancreatic cancer as a second-line treatment did not significantly increase overall survival (Tudor E. Ciuleanua, Alexander V. Pavlovskyb, Gyorgy Bodokyc, Avgust M. Garind, Virginia K. Langmuire, Stewart Krolle, George T.
  • the anti-tumor prodrugs activated by the above targeted enzymes are less cytotoxic when they are not activated (the IC50 value of inhibiting cancer cell proliferation is greater), but they are more cytotoxic when the active ingredients are obtained after being activated by the targeted enzymes ( The IC50 value of inhibiting the proliferation of cancer cells is smaller)
  • the therapeutic effect of these prodrugs on the patient is related to the level of the targeted enzyme expressed by the tumor tissue or cancer cells in the patient.
  • tumor prodrugs that is, for tumors or cancer patients who may have low expression levels of targeted enzymes in tumor tissues or cancer cells, intervention to selectively increase the expression levels of targeted enzymes in tumor tissues or cancer cells It is expected to improve the therapeutic effect of targeted enzyme-activated antitumor prodrugs and expand the scope of clinical applications.
  • the present application obtains a recombinant oncolytic virus by transforming an oncolytic virus, integrating the coding sequence of RNA or DNA capable of expressing targeting enzymes in tumor cells into the genome of the oncolytic virus, and combining the recombinant oncolytic virus with the target Enzyme-activated anti-tumor prodrugs are used to improve the anti-tumor effect of oncolytic viruses.
  • a recombinant oncolytic virus characterized in that the recombinant oncolytic virus is a selective replication oncolytic virus, and the genome of the recombinant oncolytic virus is integrated with RNA or DNA capable of expressing targeting enzymes in tumor cells.
  • the coding sequence, the targeting enzyme is an enzyme capable of activating an anti-tumor prodrug.
  • the targeting enzyme here can be an in vivo human enzyme, or an exogenous enzyme that is not in the human body.
  • the activated targeting enzyme and enzyme classification number EC in Table 1 above please refer to the activated targeting enzyme and enzyme classification number EC in Table 1 above.
  • the specific types of non-human exogenous enzymes can refer to the activated targeted enzymes in Table 2.
  • the enzymes listed in the above Table 1/2 are only the enzymes reported in the literature that can activate anti-tumor prodrugs. Not an exhaustive list.
  • any one or more of the following RNA or DNA coding sequences for targeting enzymes are integrated into the genome of the recombinant oncolytic virus:
  • Thymidine phosphorylase (EC: 2.4.2.4).
  • the genome of the recombinant oncolytic virus is integrated with two kinds of said genes capable of simultaneously expressing deoxycytidine kinase (EC: 2.7.1.74) and deoxyguanosine kinase (EC: 2.7.1.113) in tumor cells
  • deoxycytidine kinase EC: 2.7.1.74
  • deoxyguanosine kinase EC: 2.7.1.113
  • aldo-keto reductase is a human aldo-keto reductase family 1, and further preferably, the human aldo-keto reductase is a human aldo-keto reductase family 1 member C3 (AKR1C3).
  • RNA coding sequence of AKR1C3 enzyme is shown in SEQ ID No.1; the amino acid coding sequence of human aldo-ketone reductase is shown in SEQ ID No.2.
  • the anti-tumor prodrug is selected from the following formula ( Compounds of 1), (2), (3), (4), (5), (6), (7) or their salts, esters, solvates, and isotopic isomers:
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 8 , R 9 , R 10 are defined as in patent application PCT/CN2020/089692, publication number WO2020228685A1 (corresponding to Chinese application number CN202080035889.0, publication number CN113853379A) in the claims record;
  • R 100 , R 101 and R 102 are each independently hydrogen, C1-C8 alkyl, C6-C12 aryl; or R 101 and R 102 together with the nitrogen atom to which they are attached form a 5-7 membered heterocycle;
  • alkyl and aryl groups are each substituted with 1-3 halo groups or 1-3 C1-C6 alkyl groups;
  • R 1 and R 2 are each independently phenyl or methyl
  • X, Y and Z are each independently hydrogen or halo
  • R is hydrogen or C1-C6 alkyl or halogen substituted alkyl
  • n, H, Z, R 1 , R 2a , R 2b , R 3 , R 4 , R 5 are as in patent application PCT/IB2020/057285, publication number WO2021005586A1 (corresponding to Chinese application number CN202080053804.1, publication number CN114206870A) described in the claims.
  • antitumor prodrug of formula (1) is selected from the following structural compounds:
  • the antitumor prodrug of formula (2) is selected from the following structural compounds:
  • the antitumor prodrug of formula (3) is selected from the following structural compounds:
  • the antitumor prodrug of formula (4) is selected from the following structural compounds:
  • the antitumor prodrug of formula (5) is selected from the following structural compounds:
  • the antitumor prodrug of formula (6) is selected from the following structural compounds:
  • the antitumor prodrug of formula (7) is selected from the following structural compounds:
  • the recombinant oncolytic virus is selected from the group consisting of adenovirus, pox virus, herpes simplex virus, measles virus, Semliki forest virus, vesicular stomatitis virus, poliovirus, retrovirus, reovirus with oncolytic action , Seneca Valley Virus, Echo Enterovirus, Coxsackie Virus, Newcastle Disease Virus and Maraba Virus.
  • the oncolytic viruses described in the present invention include gene-mutated viruses with oncolytic effects and wild-type viruses with oncolytic effects.
  • the genetically mutated virus with oncolytic effect includes selected from: adenovirus (adenovirus), pox virus (also called vaccinia virus), herpes simplex virus (herpes simplex virus, HSV), measles virus (measles virus), Semliki Forest virus, vesicular stomatitis virus, poliovirus and retrovirus.
  • adenovirus adenovirus
  • pox virus also called vaccinia virus
  • herpes simplex virus herpes simplex virus
  • measles virus measles virus
  • Semliki Forest virus Semliki Forest virus
  • vesicular stomatitis virus poliovirus and retrovirus.
  • the wild-type virus with oncolytic effect is selected from the group consisting of: reovirus, vesicular stomatitis virus, poliovirus, Seneca Valley Virus, Ecotype Enterovirus (echo enterovirus), Coxsackie virus (Coxsackie virus), Newcastle disease virus (Newcastle disease virus) and Maraba virus (maraba virus).
  • the adenovirus is selected from: human adenovirus type 5 or human chimeric adenovirus; specifically including (for example): Onyx-015 (available from Onyx Pharmaceuticals), H101 (available from Shanghai Sunway Biotechnology Co., Ltd.) , Ad5-yCD/mutTKSR39rep-hIL12 (available from Henry Ford Health System Company), CG0070 (available from Cold Genesys Company), DNX-2401 (available from DNAtrix Company), OBP-301 (available from Oncolys BioPharma Company) ), ONCOS-102 (available from Targovax Oy company/Oncos Therapeutics company), ColoAd1 (available from PsiOxus Therapeutics company), VCN-01 (available from VCN Biosciences company), ProstAtakTM (available from Advantagene company) and the like.
  • Onyx-015 available from Onyx Pharmaceuticals
  • H101 available from Shanghai Sunway Biotechnology Co., Ltd.
  • the poxvirus is selected from: Pexa-vac (available from Jennerex Biotherapeutics), JX-963 (available from Jennerex Biotherapeutics), JX-929 (available from Jennerex Biotherapeutics), VSC20 (for preparation methods, see Science and Technology Literature: "McCart, JA, et al. Systemic cancer therapy with a tumor-selective vaccinia virus mutant lacking thymidine kinase and vaccinia growth factor genes. Cancer Res (2001) 61:875
  • the herpes simplex virus includes (but is not limited to): HSV-1, HSV-2 type herpes simplex virus; specifically includes (for example): (available from Amgen Corporation), G207 (available from Medigene Corporation), HF10 (available from Takara Bio Corporation), Seprehvir (available from Virttu Biologics Corporation), OrienX010 (available from Beijing Aoyuan Heli Bio Corporation) , NV1020 (available from Catherax Company) and the like.
  • tumors and cancers include lung cancer, non-small cell lung cancer, liver cancer, pancreatic cancer, gastric cancer, bone cancer, Esophagus, breast, prostate, testicular, colon, ovary, bladder, cervix, melanoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma , papillary adenocarcinoma, cystic adenocarcinoma, cystic carcinoma, medullary carcinoma, bronchial carcinoma, bone cell carcinoma, epithelial carcinoma, cholangiocarcinoma, choriocarcinoma, embryonic carcinoma, spermatogonial carcinoma, Wilms carcinoma, glial cell carcinoma, astrocytoma, medulloblastoma, craniopharyngioma
  • the active metabolites corresponding to the anti-tumor prodrugs activated by the targeted enzymes are all cytotoxic compounds, they are generally classified into alkylating agents, anti-metabolites, anti-cancer antibiotics, and plant-based drugs according to the source and chemical structure of the drugs. Drugs, etc., are divided into cell cycle specific drugs and cell cycle non-specific drugs according to the effect of drugs on cell proliferation kinetics. Most of the above alkylating agents and anticancer antibiotics are cell cycle nonspecific drugs, while most anti-cancer drugs are Metabolic and plant anticancer drugs are both cell cycle specific drugs, which can inhibit the proliferation of cancer cells in a broad spectrum.
  • prodrugs activated by different targeted enzymes may have different effects on different tumors or cancers.
  • AKR1C3 the anti-tumor prodrug AST-3424 activated by AKR1C3 as an example, the expression levels of AKR1C3 in different tumor tissues are quite different. According to literature (Harvey D J, Singleton R S, Dachs G U, et al.
  • the Bioreductive Prodrug PR-104A Is Activated under AerASTc Conditions by Human Aldo-Keto Reductase 1C3[J].Cancer Research,2010,70(4):1573.)
  • Statistical analysis of 2700 tumor tissue samples shows that AKR1C3 is in liver cancer, gastric cancer, esophageal cancer, Bladder cancer has higher expression, while small cell lung cancer, breast cancer, leukemia, prostate cancer have lower expression. Therefore, AST-3424, an anti-tumor prodrug activated by AKR1C3 alone, may have better effects in the treatment of cancer patients with high expression of AKR1C3 such as liver cancer, gastric cancer, esophageal cancer, and bladder cancer.
  • prostate cancer and other cancer patients with low expression of AKR1C3 should be combined with the above-mentioned recombinant oncolytic virus or the above-mentioned recombinant oncolytic virus integrated with the AKR1C3 enzyme gene for better therapeutic effect.
  • a pharmaceutical composition is also provided, wherein the pharmaceutical composition comprises the above-mentioned recombinant oncolytic virus as an active ingredient, and a pharmaceutically acceptable excipient.
  • compositions refer to the excipients and additives used in the production of drugs and the formulation of prescriptions; they are substances other than active ingredients, which have been reasonably evaluated in terms of safety and are included in pharmaceutical preparations.
  • pharmaceutical excipients also have important functions such as solubilization, solubilization, slow and controlled release, and are important components that may affect the quality, safety and effectiveness of drugs.
  • Oncolytic virus For recombinant oncolytic virus, it is mostly developed as a dosage form for injection administration. Injection administration generally requires isotonicity and isotonicity. The corresponding dosage form can be ready-to-use injection, concentrated injection or lyophilized powder. Different dosage forms of medicines have different types of pharmaceutical excipients.
  • Animal glue is selected from gelatin, bone glue or skin glue, etc.
  • sugar is selected from one or more of sucrose, mannitol, trehalose, lactose, maltose
  • inorganic salt is selected from sodium chloride, potassium chloride, potassium dihydrogen phosphate , one or more of disodium hydrogen phosphate, calcium chloride, magnesium chloride
  • amino acid medium balanced salt is selected from commercially available basic amino acid medium (Dulbecco's Modified Eagle Medium, referred to as D-MEM) balanced salt.
  • D-MEM basic amino acid medium
  • the animal glue and sugar used in the freeze-dried preparation protective agent can play a role as a scaffold. If trehalose is added, the thermal stability of the live virus preparation can be improved; the inorganic salt used is to adjust the pH value and isotonic point of the preparation to meet the physiological needs. Adaptability requirements and the requirements of drug safety, stability and effectiveness; the balanced salt of amino acid medium with pH value of 7.2-8.0 is used as a co-solvent to increase the solubility of adenovirus and enhance stability.
  • ingredients can also be added to the protective agent: cosolvents such as urea, monosodium glutamate, etc.; antioxidant synergists such as L-cysteine, etc.; to further improve the stability of the preparation.
  • cosolvents such as urea, monosodium glutamate, etc.
  • antioxidant synergists such as L-cysteine, etc.
  • the preferred final weight volume (w/v) concentration of each component of the protective agent is animal glue 0.5-2.0%, sugar 1.5-21.5%, inorganic salt 0.1-12%, and amino acid culture balanced salt 0.5-2%.
  • D-MEM basic amino acid medium
  • the protective agent used in the above preparation is a pharmaceutical excipient, which is composed of animal glue, sugar, inorganic salt, and amino acid culture medium balanced salt, wherein the animal glue and sugar can play the role of a scaffold, and the inorganic salt is used to adjust the pH value of the preparation And isotonic point, amino acid medium balanced salt is a cosolvent to increase the solubility of adenovirus and enhance stability.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a recombinant oncolytic virus and an anti-tumor prodrug activated by a targeting enzyme, and the genome of the recombinant oncolytic virus is integrated with RNA capable of expressing the targeting enzyme in tumor cells Or the coding sequence of DNA, and the targeting enzyme is an enzyme capable of activating anti-tumor prodrugs.
  • the pharmaceutical composition has two types: the recombinant oncolytic virus and the anti-tumor prodrug activated by the targeted enzyme form a compound formula, or the recombinant oncolytic virus and the anti-tumor prodrug activated by the targeted enzyme are individually packaged to form a kit.
  • the recombinant oncolytic virus and the targeted enzyme-activated anti-tumor prodrug exist independently in the pharmaceutical composition without being mixed with each other, that is, the recombinant oncolytic virus and the targeted enzyme-activated anti-tumor prodrug are packaged separately Constitutes a pillbox, which is similar to the one listed on the Chinese market (Bayer Health Care Co., Ltd.
  • the drug with tumor virus as the active ingredient preferably the injection dosage form
  • the drug with the above-mentioned targeted enzyme-activated antitumor prodrug as the active ingredient preferably the injection dosage form or the oral and sublingual dosage form
  • a kit preferred oncolytic virus injections can be dispensed using prefilled syringes with instructions for administration.
  • Oncolytic viruses are administered by intratumoral injection or intravenous injection, and antitumor prodrugs are administered intravenously or orally.
  • the injection method can be considered, and the recombinant oncolytic virus and the anti-tumor prodrug activated by the targeted enzyme can be used as the active ingredients of the drug. After physical mixing, it is integrated into a compound injection through formulation technology.
  • the compound injection can be an emulsion (water-in-oil or oil-in-water) or a suspension, etc., and the recombinant oncolytic virus can enter the cancer cells by using a specific formulation technology. After the onset of action, the anti-tumor prodrug that targets the activation of the enzyme can enter the cancer cell to take effect.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a recombinant oncolytic virus and an anti-tumor prodrug activated by a targeted enzyme, wherein the recombinant oncolytic virus is integrated into the genome of the recombinant oncolytic virus capable of expressing the targeted enzyme in tumor cells
  • the coding sequence of RNA or DNA, the targeting enzyme is an enzyme capable of activating an anti-tumor prodrug
  • a tumor patient or cancer patient is a patient with low expression of the targeting enzyme in the tumor tissue or cancer cell.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a recombinant oncolytic virus and an anti-tumor prodrug activated by a targeted enzyme, wherein the recombinant oncolytic virus is integrated into the genome of the recombinant oncolytic virus capable of expressing the targeted enzyme in tumor cells
  • the coding sequence of RNA or DNA, the targeting enzyme is an enzyme capable of activating anti-tumor prodrugs, and the inhibitory ability of the anti-tumor prodrug activated by the targeting enzyme on cancer cell proliferation is related to the effect of the targeting enzyme in tumor patients or cancer patients.
  • the level of expression in tumor tissue or cancer cells is positively correlated.
  • the therapeutic effect of anti-tumor prodrugs on patients is closely related to the level of the targeted enzyme expressed in the tumor tissue or cancer cells in the patient, the catalytic activation of the targeted enzyme, and the sensitivity to the release of toxins.
  • the level is high or low, that is, when the concentration of the targeted enzyme in cancer cells or tumor tissue is positively correlated with the reaction rate of catalyzing the conversion of prodrugs into active pharmaceutical ingredients, it can be better promoted by increasing the expression level of the targeted enzyme, that is, the concentration.
  • the anti-tumor effect of the tumor prodrug therefore, when the inhibitory ability of the anti-tumor prodrug activated by the targeting enzyme on the proliferation of cancer cells (the IC50 value of cancer cell proliferation inhibition) is related to the effect of the targeting enzyme in the tumor patient or the tumor of the cancer patient
  • the level of expression in tissue or cancer cells the content of targeted enzymes contained in tumor tissue or cancer cells
  • the recombinant oncolytic virus and the anti-tumor prodrug activated by the targeted enzyme are combined or administered after the compound preparation
  • the activation of the targeted enzyme can be better improved in patients with low expression levels of the targeted enzyme in the tumor tissue or cancer cells.
  • the drug response of anti-tumor prodrugs that is, for tumors or cancer patients with low levels of expression of targeted enzymes in tumor tissues or cancer cells
  • the intervention of recombinant oncolytic viruses can selectively increase the tumor tissue or cancer cells.
  • the expression level of the targeted enzyme can improve the therapeutic effect of the anti-tumor prodrug activated by the targeted enzyme.
  • the applicant has conducted an assay study on tumor cell lines with different expression levels of AKR1C3 enzyme for the anti-tumor prodrug AST-3424 activated by AKR1C3 enzyme, and the results are shown in Tables 3/4/5 below.
  • the following combinations of recombinant oncolytic viruses and the anti-tumor prodrugs activated by the targeting enzymes are preferred:
  • aldo-keto reductase 1C3 Activated by recombinant oncolytic virus with aldo-keto reductase 1C3 (EC: 1.1.1.188) integrated into its genome coding sequences for RNA or DNA capable of expressing aldo-keto reductase 1C3 (EC: 1.1.1.188) in tumor cells antineoplastic prodrugs in combination; or
  • cytochrome P450 reductase Activated by a recombinant oncolytic virus with cytochrome P450 reductase (EC: 1.6.2.4) integrated into its genome a coding sequence for RNA or DNA capable of expressing cytochrome P450 reductase (EC: 1.6.2.4) in tumor cells antineoplastic prodrugs in combination; or
  • a recombinant oncolytic virus and thymidine phosphorylase (EC: 2.4.2.4) that integrates into its genome a coding sequence for RNA or DNA capable of expressing thymidine phosphorylase (EC: 2.4.2.4) in tumor cells antineoplastic prodrugs in combination; or
  • deoxycytidine kinase EC: 2.7.1.74
  • deoxyguanosine kinase EC: 2.7.1.113
  • a combination of antineoplastic prodrugs co-activated by kinase EC: 2.7.1.74
  • deoxyguanosine kinase EC: 2.7.1.113
  • the anti-tumor prodrug activated by the aldo-keto reductase 1C3 is a small molecule anti-tumor prodrug, preferably AST-3424;
  • the anti-tumor prodrug activated by the ⁇ -glucuronidase (EC: 3.2.1.31) is a small molecule anti-tumor prodrug, preferably glufosfamide;
  • the anti-tumor prodrug activated by the cytochrome P450 reductase (EC: 1.6.2.4) is a small molecule anti-tumor prodrug, preferably TH-302;
  • the anti-tumor prodrug activated by deoxycytidine kinase (EC: 2.7.1.74) is a small molecule anti-tumor prodrug activated by deoxycytosine kinase, preferably cytarabine, amcitabine, enoctabine, cytaridine Citabine, fludarabine, azacitidine, gemcitabine, pentostatin, cladribine;
  • the anti-tumor prodrug activated by thymidine phosphorylase (EC: 2.4.2.4) is a small molecule anti-tumor prodrug, preferably capecitabine;
  • the antitumor prodrugs co-activated by the deoxycytidine kinase (EC: 2.7.1.74) and deoxyguanosine kinase (EC: 2.7.1.113) are deoxycytidine kinase (EC: 2.7.1.74), deoxyguanosine kinase (EC: 2.7.1.74) EC: 2.7.1.113) co-activated small molecule anti-tumor prodrug, preferably nerabine.
  • anti-tumor prodrugs and prodrugs are selected from marketed drugs or experimentally verified drugs in the clinical trial stage with broad-spectrum anti-tumor effects.
  • the present invention also provides a method for treating tumor and/or cancer, administering the above-mentioned pharmaceutical composition to a tumor and/or cancer patient, including the following steps performed accordingly:
  • the recombinant oncolytic virus capable of selectively replicating in tumor cells
  • the targeted enzyme-activated anti-tumor prodrug is administered to the tumor and/or cancer patient some time after administration of the recombinant oncolytic virus.
  • the recombinant oncolytic virus capable of selectively replicating in tumor cells
  • the targeted enzyme-activated anti-tumor prodrug is administered to the tumor and/or cancer patient some time after administration of the recombinant oncolytic virus.
  • the present invention also provides a method for treating tumor and/or cancer by administering the above-mentioned pharmaceutical composition.
  • the tumor patient is ineffective or unresponsive to the anti-tumor prodrug activated by the targeting enzyme alone, the following steps are performed accordingly. :
  • the targeted enzyme-activated anti-tumor prodrug is administered to the tumor and/or cancer patient some time after administration of the recombinant oncolytic virus.
  • the present invention also provides a kind of pharmaceutical use corresponding to above-mentioned treatment method:
  • the present invention provides a kit for treating tumors and/or cancers with a synergistic combination drug, comprising:
  • composition comprising a recombinant oncolytic virus and the anti-tumor prodrug activated by the targeting enzyme;
  • Figure 1 is a schematic diagram showing the correlation between the IC 50 value of the inhibition rate of AST-342 on different hepatocytes and the protein level of AKR1C3 in the hepatocytes;
  • Fig. 3 is FV184 vector (virion plasmid) DNA information and map schematic diagram
  • Figure 4 shows the electrophoresis results of the OVAD-hAKR1C3-3Flag-OE plasmid: the left side is the plasmid electrophoresis result, the OVAD-hAKR1C3-3Flag-OE plasmid under the 17 characters corresponds to the Marker protein, and the right side is the OVAD-hAKR1C3-3Flag-OE plasmid. Marker's ruler diagram;
  • Figure 5 shows the fluorescence and brightfield photomicrographs of oncolytic virus at different times after infection of HEK293 cells.
  • the upper left photo is the photomicrograph taken at 500x magnification under brightfield conditions after the first day (24 hours), and the upper right photo is Photomicrographs taken at 500x magnification under fluorescent conditions after the first day (24 hours), photomicrographs taken at 500x magnification under brightfield conditions after the second day (48 hours), and the top right image at 500x magnification Photomicrographs taken at 500X magnification under fluorescent conditions after two days (48 hours);
  • Figure 6 is a schematic diagram of immunoblotting results, the left picture is the electrophoresis result of Marker protein, M represents Marker protein, the middle picture is the immunoblot electrophoresis result of FLAG tag antibody as a control, the right picture is the immunoblot electrophoresis result of Actin as a control, wherein, 1 is Hacat CON control cell sample, 2 is HepG2CON control cell sample, 3 is 1 ⁇ L OVAD-hAKR1C3-3Flag-OE, protein sample after infection of Hacat cells, 4 is 1 ⁇ L OVAD-hAKR1C3-3Flag-OE, protein after infection of HepG2 cells Sample, M represents the electrophoresis result of protein Marker;
  • Figure 7 is the relative content of the target protein AKR1C3 compared with Actin;
  • Figure 8 is a table of multiple ongoing clinical trials of oncolytic virotherapy.
  • Patient and “individual” are used interchangeably and refer to a mammal in need of cancer treatment.
  • the patient is a human.
  • the patient is a human diagnosed with cancer.
  • patient or “individual” may refer to non-human mammals, such as non-human primates, dogs, cats, rabbits, pigs, mice, for use in screening, characterizing and evaluating drugs and therapies or rats.
  • prodrug refers to a compound that, after administration or administration, is metabolized or otherwise converted into a biologically active or more active compound (or drug) with respect to at least one property. Relative to the drug, the prodrug is chemically modified in such a way that it is less active or inactive relative to the drug, but the chemical modification results in the production of the corresponding drug through metabolism or other biological processes after administration of the prodrug.
  • Prodrugs may have altered metabolic stability or delivery characteristics, less side effects or less toxicity, or improved flavor relative to the active drug. Prodrugs can be synthesized using reactants other than the corresponding drug.
  • Solid tumor refers to solid tumors including, but not limited to, metastases in bone, brain, liver, lung, lymph nodes, pancreas, prostate, skin, and soft tissues (sarcomas).
  • a “therapeutically effective amount" of a drug refers to a drug that, when administered or administered to a patient with cancer, will have the desired therapeutic effect (eg, alleviation, amelioration, amelioration, or elimination of the clinical manifestations of one or more cancers in the patient) amount.
  • a therapeutic effect does not necessarily have to occur by administering or administering one dose, and may only occur after the administration or administration of a series of doses. Thus, a therapeutically effective amount can be administered or administered one or more times.
  • Treatment of a condition or patient refers to taking steps to obtain beneficial or desired results (including clinical results).
  • beneficial or desired clinical outcomes include, but are not limited to, alleviation or amelioration of one or more symptoms of cancer; attenuation of disease extent; delay or slowing of disease progression; improvement, remission or stabilization of disease state; or other beneficial results.
  • treatment of cancer can result in a partial response or stabilize the disease.
  • Tumor cell refers to a tumor cell of any suitable species (eg, mammalian, eg, murine, canine, feline, equine, or human).
  • suitable species eg, mammalian, eg, murine, canine, feline, equine, or human.
  • This example is to illustrate the correlation between the expression level of AKR1C3 enzyme and the IC50 value.
  • Example 1-1 Correlation between AKR1C3 protein level and IC 50 value in liver cancer cell line.
  • test compound AST-3424 Exponentially growing cells were seeded, and 24 hours later, the test compound AST-3424 was added. After addition of the test compound AST-3424, the plates were incubated in a standard tissue culture incubator at 37°C for the indicated hours. At the end of the experiment, the CellTiter Glo (CTG) assay kit was used. Drug concentrations (IC50) resulting in 50 % growth inhibition relative to untreated controls were calculated using XLfit (IDBS, Boston, MA) or Prism 6 (GraphPad, San Diego, CA).
  • Human cell extracts were prepared and protein concentrations were determined. Proteins were detected using antibodies recognizing human AKR1C3 and tubulin or ⁇ -actin. Band densities of AKR1C3 and tubulin or ⁇ -actin were scanned and quantified using the Odyssey Laser Imaging System and Software (LI-COR Biosciences, Lincoln, NE), and the effect of AKR1C3 on tubulin or ⁇ -actin was calculated ratio of kinesin.
  • IC50 values were determined using an in vitro proliferation assay, and the expression of AKR1C3 protein in the hepatoma cell lines was determined using immunoblotting (tubulin was used as a loading control), The results are shown in Table 3.
  • liver cancer cell lines with high AKR1C3 expression both at the protein level were more sensitive to AST-3424 with IC50 values in the low nanomolar range.
  • cells expressing low AKR1C3 were less sensitive to AST-3424 with IC50 values above 1000 nM.
  • Example 1-2 Correlation between AKR1C3 protein levels and IC50 values in leukemia cell lines
  • the nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse model of childhood acute lymphoblastic leukemia reveals intrinsic differences in biologic characteristics at diagnosis and relapse. Blood 2002;99:4100–8.).
  • the development of lentivirally transduced ALL-11 PDX [empty vector (EV) and AKR1C3 overexpression] has been described previously (Jamieson SM, Gu Y, Manesh DM, El-Hoss J, Jing D, Mackenzie KL, et al.
  • a novel fluorometric assay for aldo-keto reductase 1C3 predicts metabolic activation of the nitrogen mustard prodrug PR-104A in human leukaemia cells. Biochem Pharmacol 2014;88:36–45.).
  • Leukemia cell lines were suspended in RPMI medium supplemented with FBS (Biosera), while ALL PDX cells were grown in either Flt-3 ligand (20 ng/mL, BioNovus Life Sciences) or IL7 (10–20 ng/mL, Jomar Life Research) ) in QBSF medium (Quality Biological Inc,). Cells were seeded according to the optimal cell density and incubated for 3 hours or overnight (37°C, 5% CO2 ). PDX cells and leukemia cell lines were treated with AST-3424 (10 mmol/L–1 pmol/L) or vehicle control for 48 or 72 hours, respectively. Viability was determined using the Cell Titer-Glo Luminescent Cell Viability Assay (Promega). Half-maximal inhibitory concentrations ( IC50 ) were calculated by GraphPad Prism 7 software for interpolation of nonlinear regression curves.
  • Cryopreserved leukemia cells were thawed and lysed in RIPA lysis buffer and protein concentrations were quantified by BCA assay. Each sample was loaded with 20 ⁇ g of protein lysate in a NuPAGE 4-12% Bis-Tris protein gel, then electrophoresed at 120 V and transferred to a polyvinylidene fluoride membrane for 1 h at 30 V.
  • Anti-mouse anti-AKR1C3 (#A6229, Sigma-Aldrich, St.
  • AKR1C3-related AST-3424 In vitro cytotoxicity of AKR1C3-related AST-3424 was observed in nine T-ALL cell lines, one B-ALL cell line transfected with granulocyte colony-stimulating factor, and one BCP-ALL cell line. The expression level of AKR1C3 protein was determined using immunoblot analysis. The in vitro cytotoxicity of AST-3424 was determined using the CellTiter-Glo assay and calculated as the 50% maximal inhibitory concentration ( IC50 ).
  • AST-3424 showed in vitro cytotoxicity with IC50 ranging from 3.0-30.0 nM in 6 cell lines expressing high (strong) levels of AKR1C3. For cell lines with moderate AKR1C3 expression levels, IC50s ranged from 3.0 to 84.0 nM (Table 4).
  • G-CSF granulocyte colony stimulating factor
  • BCP-ALL B cell precursor ALL
  • AST-3424 has been demonstrated to treat T-ALL, B-ALL, acute myeloid leukemia, acute promyelocytic leukemia (APL) and erythroleukemia.
  • Table 5 In vitro cytotoxicity of AST-3424 against leukemia cell lines after 72 hours of exposure to leukemia cell lines
  • AKR1C3 expression was assessed by immunoblotting and expressed relative to control ⁇ -tubulin. High, AKR1C3/ ⁇ -tubulin>5.0; moderate, AKR1C3/ ⁇ -tubulin 2.0-5.0; low, AKR1C3/ ⁇ -tubulin ⁇ 2.0.
  • AST-3424 exerted potent cell killing effects on all leukemia cell lines.
  • the cytotoxicity of AST-3424 against 6 B-ALL cell lines is presented in Figure 2a
  • the cytotoxicity of AST-3424 against 7 T-ALL cell lines is presented in Figure 2b.
  • AKR1C3-dependent activation of AST-3424 as a DNA alkylating agent is presented in Figure 2a.
  • 5x, 10x means 5-fold, 10-fold dilution, and so on for the rest.
  • Part 1 The hAKR1C3 gene was constructed on the oncolytic virus plasmid to obtain the recombinant oncolytic adenovirus OVAD-hAKR1C3-3Flag-OE integrated with the hAKR1C3 gene
  • the pLenti6.3-CMV-MSC lentiviral vector was connected with the known hAKR1C3 gene sequence using a conventional method to obtain the pLenti6.3-CMV-MSC-hAKR1C3 lentiviral vector plasmid, and the human AKR1C3 gene (hAKR1C3 gene , NM_003739.6) CDS sequence as a template, using PCR amplification method to obtain the target gene sequence, and then using seamless cloning method, the PCR-amplified target fragment was constructed on the oncolytic virus vector PV184 ( Figure 1).
  • the oncolytic virus vector includes the following DNA original, hTERT-455-E1A-IRES-E1B-mCMV-MCS-3Flag-SV40-EGFP, with a FLAG tag on it, for subsequent western blot detection of hAKR1C3 overexpressed protein, when FLAG tag When the protein is detected to be expressed, the corresponding hAKR1C3 protein is also expressed accordingly.
  • the oncolytic virus is based on type 5 adenovirus.
  • the E1 region gene is conditionally activated, and the E3 gene is deleted or modified. It has the ability to infect both dividing cells and non-dividing cells.
  • Body-mediated endocytosis enters the cell and then transfers the oncolytic adenovirus genome into the nucleus, where it remains extrachromosomal and does not integrate into the host cell genome. It can be replicated and amplified in HEK293 cells and some tumor cells, and is a relatively safe gene therapy vector.
  • EGFP is a gene sequence corresponding to green fluorescent protein, which can be used to express EGFP protein to display fluorescence after infection of cancer cells.
  • Figure 3 shows FV184 vector (oncolytic adenovirus plasmid) information and map.
  • Vector size 8.9kb; Prokaryotic resistance: AmpR; Selection marker: EGFP; Framework structure: hTERT-455-E1A-IRES-E1B-mCMV-MCS-3Flag-SV40-EGFP; MCS region forward sequencing primer: 5'- GGTATAAGAGGCGCGACCAG-3'; MCS region reverse sequencing primer: 5'-TTCCACACCCTAACTGACAC-3'; commonly used restriction sites: SalI, BamHI, AgeI.
  • the CDS region sequence of the gene that is, the DNA gene sequence of hAKR1C3 is shown in SEQ ID No.1; the corresponding amino acid sequence, that is, the amino acid sequence of hAKR1C3 is shown in SEQ ID No.2.
  • the equipment used in the experiment are all conventional commercially available equipment and instruments.
  • Upstream primer (ie SEQ ID No. 3): cgactctagaggatccgccaccatggattccaaacaccagtgtgtaaag
  • Downstream primer ie SEQ ID No.4: tgtagtccataccggtatattcatctgaatatggataattagggtggctag
  • Double restriction site 5'BamHI, 3'AgeI
  • the enzyme digestion reaction conditions were 37°C/30min.
  • the system that completed the digestion reaction was added to the sample well for electrophoresis, and the electrophoresis condition was 220V/30min. After electrophoresis, place the gel block on the gel cutting table, cut out the target fragment and put it into a sterilized 1.5mL EP tube.
  • the DNA was recovered according to the instructions of the TIANGEN ordinary agarose gel DNA recovery kit. The recovered DNA was measured for its concentration and 260/280 value, and stored at -20°C for later use.
  • the seamless cloning method was used to connect the oncolytic virus vector and the target gene fragment.
  • the seamless cloning method also known as the exchange recombination cloning method, is different from the traditional PCR product cloning (TA cloning).
  • TA cloning traditional PCR product cloning
  • the ligation product was transformed into TOP10 competent Escherichia coli, and evenly spread on the LB culture plate with ampicillin resistance, and the plate was inverted and placed in a 37°C bacterial incubator for overnight cultivation. Monoclonal colonies were picked from the above plates, cultured in LB liquid medium, and plasmid extraction was performed. Plasmid sequencing and identification, the sequencing results were compared with the target gene sequence for identification. The results showed that the sequencing results were completely consistent with the DNA sequence of the target gene, indicating that the OVAD-hAKR1C3-3Flag-OE plasmid (ie, the viral plasmid integrated with hAKR1C3) was successfully constructed (Figure 2).
  • positions 1032-1104 are the 3Flag DNA gene, and its specific sequence is shown in SEQ ID No.6.
  • the hAKR1C3 gene has been constructed on the oncolytic virus plasmid to obtain the recombinant oncolytic adenovirus OVAD-hAKR1C3-3Flag-OE integrated with the hAKR1C3 gene.
  • Oncolytic virus packaging cells HEK293 are anchorage-dependent epithelioid cells grown in DMEM medium containing 10% FBS. The cells contain and express the E1 region of the oncolytic virus that initiates replication to amplify the oncolytic virus in large quantities.
  • CPE cytopathic effect
  • the cells were collected by low-speed centrifugation and resuspended in 2ml DMEM, freeze-thawed at -70°C/37°C repeatedly, shaken 3 times, and centrifuged at 4°C, 7000g for 5min , the virus supernatant was collected and stored at -70°C.
  • the well-grown HEK293 cells in one T25 cell culture flask were 4-fold diluted and transferred to another T25 cell culture flask, and cultured in DMEM medium containing 10% FBS at 37°C and 5% CO 2 (below). Cells were cultured under this condition in all expansions). When the cells reach 60% confluence, discard the old culture medium, add 2 mL of the crude extract harvested after the successful recombination of the OVAD-hAKR1C3-3Flag-OE virus into the culture flask, and place the culture flask in a cell incubator for 90 min. Add 3 mL of complete culture solution to the culture flask and continue to culture.
  • All the HEK293 cells in a good growth state in a T25 cell culture flask were transferred into a T75 cell culture flask, and the culture was continued with complete medium.
  • the cells reach 90% confluence, discard the old culture medium, add 2 mL of the virus solution obtained in the first round of amplification to the culture flask, place the culture flask in a cell incubator for 90 minutes, and finally add to the culture flask.
  • the cells were collected by low-speed centrifugation and resuspended in 10ml DMEM, freeze-thawed at -70°C/37°C, shaken 3 times, and centrifuged at 7000g at 4°C. After 5 min, the virus supernatant was collected and stored at -70°C.
  • Figure 5 shows fluorescence and brightfield micrographs of oncolytic virus at different times after infection of HEK293 cells. As shown in Figure 5, it was observed that in the fluorescent environment, HEK293 cells emitted fluorescence, and the comparison showed that after 48 hours, the viral DNA was replicated and the EFGP fluorescent protein was expressed.
  • the virus solution after the second round of amplification was concentrated by an ultrafiltration concentration column, and then divided into 1.5mL centrifuge tubes in equal volumes and stored at -70°C.
  • Dilution of the virus solution to be tested Take 10Ll of the oncolytic virus stock solution and add it to a 990 ⁇ L Ep tube for 1:100 dilution (1E-2); then take this as a starting point and add 100 ⁇ L of the dilution to a 900 ⁇ L Ep tube Do a 1:10 dilution (1E-3), and dilute in sequence until the dilution reaches (1E-13).
  • Viral titer 10 (x+0.8) (PFU/ml)
  • the negative control has no CPE and growth inhibition
  • virus titer 1E+10 (PFU/mL).
  • Figure 12 Schematic diagram of oncolytic virus titer detection data
  • X is the sum of the positive rates of CPE at successive dilutions from 1E-1 to 1E-13.
  • Hacat cells are human immortalized epidermal cells, a non-tumor derived human normal skin immortalized keratinocyte cell line, as experimental control cells.
  • HepG2 cells are human hepatoma cells, derived from a 15-year-old white man's liver cancer tissue, the cells secrete a variety of plasma proteins: albumin, ⁇ 2-macroglobulin, plasminogen, transferrin and so on.
  • the OVAD-hAKR1C3-3Flag-OE oncolytic virus carries the hTERT promoter (human telomere reverse transcriptase), it can be greatly amplified in tumor cells with high expression of this protein, but in non-tumor cells with low expression of hTERT protein Can't be copied in bulk. Therefore, the purpose of this experiment is to detect whether the constructed oncolytic virus can replicate in tumor cells in large quantities and lead to the overexpression of the target protein hAKR1C3. At the same time, in non-tumor cells, because the oncolytic virus cannot replicate, the target protein hAKR1C3 is underexpressed.
  • hTERT promoter human telomere reverse transcriptase
  • Part 3 Verification that for cancer cells or tumors with low expression of AKR1C3 (hAKR1C3) enzyme, the combination of recombinant oncolytic adenovirus with hAKR1C3 gene and AKR1C3 enzyme-activated anticancer drug AST-3424 can improve AST-3424 or oncolysis The effect of virus alone in inhibiting the proliferation of cancer cells or tumors
  • nitroreductase-activating prodrug PR-104 was used in combination with the oncolytic virus ONYX-411 NTR with the nitroreductase gene fragment inserted, and the combined effect was better than that of PR-104 and ONYX- 411.
  • the anti-cancer prodrug activated by a specific enzyme protein is used in combination with the oncolytic virus, and its anti-tumor effect is stronger than that alone.
  • Anticancer prodrugs activated by oncolytic viruses and individual specific enzymatic proteins are used in combination with the oncolytic virus, and its anti-tumor effect is stronger than that alone.
  • the AKR1C3 gene is inserted into the oncolytic virus, and the oncolytic virus can express the AKR1C3 protein after infecting the tumor cell HepG2, so those skilled in the art can predict the anticancer prodrug AST-
  • the combination of 3424 and the oncolytic virus inserted with the AKR1C3 gene has a stronger antitumor effect than the oncolytic virus alone and the anticancer prodrug AST-3424 activated by the AKR1C3 enzyme alone;
  • the oncolytic virus inserted with the AKR1C3 gene is administered first, and then the prodrug AST-3424 activated by the AKR1C3 enzyme is administered, which will have a better therapeutic effect.

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Abstract

La présente invention concerne un virus oncolytique recombiné obtenu par l'intégration d'une séquence codante capable d'exprimer l'ARN ou l'ADN d'une enzyme ciblée dans les cellules tumorales dans le génome d'un virus oncolytique, le virus oncolytique recombiné étant combiné avec un promédicament antitumoral activé par l'enzyme ciblée pour améliorer l'effet antitumoral du virus oncolytique. La présente invention concerne également un virus oncolytique recombiné, caractérisé en ce que le virus oncolytique recombiné est un virus oncolytique de type à réplication sélective, une séquence codante capable d'exprimer l'ARN ou l'ADN d'une enzyme ciblée dans les cellules tumorales est intégrée dans le génome du virus oncolytique recombiné, et l'enzyme ciblée est une enzyme capable d'activer un promédicament antitumoral. La présente invention concerne également une composition pharmaceutique, comprenant le virus oncolytique recombiné et un promédicament antitumoral activé par une enzyme ciblée.
PCT/CN2022/084842 2021-04-02 2022-04-01 Virus oncolytique recombiné et son utilisation médicale Ceased WO2022206962A1 (fr)

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