WO2024005211A1 - Vecteur d'adénovirus modifié par hexon - Google Patents

Vecteur d'adénovirus modifié par hexon Download PDF

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WO2024005211A1
WO2024005211A1 PCT/JP2023/024534 JP2023024534W WO2024005211A1 WO 2024005211 A1 WO2024005211 A1 WO 2024005211A1 JP 2023024534 W JP2023024534 W JP 2023024534W WO 2024005211 A1 WO2024005211 A1 WO 2024005211A1
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amino acid
acid sequence
seq
modified
amino acids
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裕之 水口
文教 櫻井
春彦 鎌田
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国立大学法人大阪大学
国立研究開発法人医薬基盤・健康・栄養研究所
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Publication of WO2024005211A1 publication Critical patent/WO2024005211A1/fr

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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
    • A61K35/761—Adenovirus
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/01—DNA viruses
    • 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
    • C12N15/86—Viral vectors
    • C12N15/861—Adenoviral vectors
    • 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 an adenovirus vector in which the hexon region of human adenovirus type 5 has been modified, a recombinant adenovirus derived from the vector, and uses thereof.
  • Ad vectors conventionally used for gene therapy and vaccines are mainly based on human type 5 Ad, but human type 5 Ad has a preexisting antibody retention rate of 60 to 70%. exceed. So far, for example, clinical trials of Ad vector vaccines against HIV have revealed that vaccine efficacy cannot be expected in subjects who have existing antibodies against human Ad type 5 (for example, non-patented (See Reference 1). In order to avoid such problems, vaccines based on monkey (chimpanzee) Ad (see, for example, Non-Patent Document 2) and vaccines based on human Ad type 26 (see, for example, Non-Patent Document 3) have been developed. (The retention rate of existing antibodies for these Ads is very low at 10-20% or less). However, human type 5 Ad vectors have a moderate ability to induce innate immunity, can be obtained in large quantities, and are advantageous in terms of production, so there is a need for their use as base viruses for vaccines, etc. There is a current situation.
  • the present invention was made in consideration of the above situation, and provides the following hexon-modified adenovirus vectors, recombinant adenoviruses derived from the vectors, and their uses (vaccines, pharmaceutical compositions, etc.). This is what we provide.
  • a hexon-modified adenovirus vector The virus is based on human adenovirus type 5, The amino acid sequence of at least one of the hypervariable regions (HVR) HVR1 to HVR7 present in the hexon region of the virus has been modified.
  • the adenovirus vector [2] The adenovirus vector according to [1] above, wherein among HVR1 to HVR7 of the virus, HVR6 has an unmodified amino acid sequence.
  • the modified amino acid sequence has the following (a) to (f): (a) (i) DEAATALEIXLEEGGGGSEDEVDEQAEQQK (SEQ ID NO: 1; where X is N or E), (ii) An amino acid sequence in which one or more amino acids are deleted, substituted, or added to the amino acid sequence of SEQ ID NO: 1 (however, the 14th to 18th amino acids in the amino acid sequence of SEQ ID NO: 1 are retained) ), or (iii) an amino acid sequence having 70% or more identity with the amino acid sequence of SEQ ID NO: 1 (however, the 14th to 18th amino acids in the amino acid sequence of SEQ ID NO: 1 are retained).
  • a modified amino acid sequence of HVR1 that is; (b) (i) VEGQTAA (SEQ ID NO: 2), (ii) An amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 2 (however, the 6th and 7th amino acids in the amino acid sequence of SEQ ID NO: 2 are retained) ), or (iii) an amino acid sequence having 70% or more identity with the amino acid sequence of SEQ ID NO: 2 (however, the 6th and 7th amino acids in the amino acid sequence of SEQ ID NO: 2 are retained).
  • a modified amino acid sequence of HVR2 that is; (c) (i) YETAINHAA (SEQ ID NO: 3), (ii) an amino acid sequence in which one or more amino acids are deleted, substituted, or added to the amino acid sequence of SEQ ID NO: 3 (however, the fourth amino acid in the amino acid sequence of SEQ ID NO: 3 is retained), or (iii) An amino acid sequence having 70% or more identity with the amino acid sequence of SEQ ID NO: 3 (however, the fourth amino acid in the amino acid sequence of SEQ ID NO: 3 is retained).
  • a modified amino acid sequence of HVR3 which is; (d) (i) GILVKQAAGKL (SEQ ID NO: 4), (ii) An amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 4 (however, the 7th and 8th amino acids in the amino acid sequence of SEQ ID NO: 4 are retained) ), or (iii) an amino acid sequence having 70% or more identity with the amino acid sequence of SEQ ID NO: 4 (however, the 7th and 8th amino acids in the amino acid sequence of SEQ ID NO: 4 are retained).
  • a modified amino acid sequence of HVR4 that is; (e) (i) STTEATAGAGDNLT (SEQ ID NO: 5), (ii) An amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 5 (However, the 3rd, 4th, and 9th amino acids in the amino acid sequence of SEQ ID NO: 5 are ), or (iii) an amino acid sequence having 70% or more identity with the amino acid sequence of SEQ ID NO: 5 (provided that the 3rd, 4th, and 9th (Amino acids are preserved.) and (f) (i) GGVGNNSTYTKVKPKTGQENGWEKDATEFSDKXE (SEQ ID NO: 7; where X is N or I), (ii) An amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 7 (However, the 4th, 6th, 7th, and 9th amino acids in the amino acid sequence
  • a vaccine comprising a recombinant adenovirus derived from the adenovirus vector according to any one of [1] to [6] above.
  • the vaccine according to [8] above which is for the treatment or prevention of infectious diseases, allergies, cancer, Alzheimer's disease, diabetes, or hypertension.
  • a pharmaceutical composition comprising a recombinant adenovirus derived from the adenovirus vector according to any one of [1] to [6] above.
  • hexon-modified human adenovirus type 5 (Ad) vector that can avoid immune reactions caused by existing antibodies.
  • the hexon-modified Ad vector according to the present invention can provide vaccines, pharmaceutical compositions, etc. containing recombinant adenoviruses derived from the Ad vector, which can avoid immune reactions caused by existing antibodies. , highly practical and useful.
  • FIG. 3 is a diagram showing a construction scheme of Ad vectors having each modified HVR.
  • FIG. 3 is a diagram showing a construction scheme of Ad vectors having each modified HVR.
  • FIG. 2 is a diagram showing a construction scheme of a human type 5 Ad vector plasmid in which hexons HVR1 to HVR5 and HVR7 are modified.
  • FIG. 2 is a diagram showing a construction scheme of luciferase expression Ad vectors having each modified HVR.
  • FIG. 2 is a diagram showing a construction scheme of ⁇ -galactosidase (LacZ) or luciferase expression Ad vectors with modified hexons HVR1 to HVR5 and HVR7.
  • FIG. 3 depicts the results of transduction efficiency of Ad vectors in human tumor cell lines. Panc-1 cells (A) and SK HEP-1 cells (B) were transduced with Ad vectors at 60 and 300 vector particles (VP)/cell, and 48 hours after transduction, X-gal staining was performed, and then , shows the results (photo) of observing cells under a microscope.
  • FIG. 3 shows the results of transduction efficiency of Ad vectors in human tumor cell lines.
  • FIG. 3 is a diagram showing the results of gene expression efficiency of various Ad vectors in the presence of human serum.
  • Various Ad vectors were incubated with human serum at various concentrations for 1 hour at room temperature. Thereafter, various Ad vectors were allowed to act on H1299 cells at 200 VP/cell. Luciferase activity was measured 72 hours after acting on cells. The percentage in each graph indicates a relative value when the luciferase activity in the absence of human serum is taken as 100%.
  • Hexons and fibers which are major proteins on the Ad surface, are the main antigenic sites of antibodies against Ad (anti-Ad antibodies). It is known that the main antigenic site within the hexon region is the hypervariable region (HVR; there are seven sites (HVR1 to HVR7) within one hexon) consisting of 6 to 41 amino acids.
  • HVR hypervariable region
  • the present inventors have developed a hexon-modified Ad vector that can evade anti-Ad antibodies by partially modifying the amino acid sequence of this HVR.
  • Non-patent Document 4 (page 239, right column, second paragraph) states that when HVR1 and all HVR1-7 are replaced with those derived from human type 48 Ad, the effect is 3 to 5 times lower than that of the parent strain, but still sufficient.
  • a hexon-modified Ad vector that can evade anti-Ad antibodies will become a basic vector for the development of effective vaccines against emerging and re-emerging infectious diseases by using it as a non-propagating Ad vector.
  • it becomes a base virus for oncolytic viruses (viral therapy) that kill cancer cells.
  • human type 5 Ad vectors have a moderate ability to induce innate immunity and are advantageous in terms of production since they can yield large amounts of virus, so if the problems with existing antibodies (anti-Ad antibodies) are solved, they can be used to develop vaccines and vaccines. It has excellent basic properties for use in viral therapy.
  • the hexon-modified Ad vector of the present invention and the recombinant adenovirus derived therefrom can solve the above problems and are extremely practical and useful.
  • Hexon-modified Ad vector The hexon-modified Ad vector according to the present invention is based on human type 5 adenovirus, and the amino acid sequence of at least one HVR among HVR1 to HVR7 in the hexon region has been modified. . From the viewpoint of production amount and production efficiency of the modified Ad vector, one in which the amino acid sequence of HVR6 among HVR1 to HVR7 is not modified (that is, one in which the amino acid sequence of at least one HVR among HVR1 to HVR5 and HVR7 is Modified versions) are preferred. In addition, from the viewpoint of stronger evasion from anti-Ad antibodies, it is more preferable to have the amino acid sequences of all HVRs HVR1 to HVR5 and HVR7 modified.
  • the amino acid sequence of the wild-type hexon region in human type 5 adenovirus is shown in SEQ ID NO: 8.
  • the region consisting of amino acids 136th to 165th (30 residues) is HVR1
  • the region consisting of the 188th to 194th amino acids (7 residues) is HVR2
  • the region consisting of the 212th to 220th amino acids (9 residues) is HVR3
  • the region consisting of the 248th to 258th amino acids (11 residues) is HVR4
  • the region consisting of amino acids 268th to 281st (14 residues) is HVR5
  • the region consisting of amino acids 305th to 310th (6 residues) is HVR6,
  • the region consisting of amino acids 418th to 451st (34 residues) is HVR7.
  • the full-length genome nucleotide sequence information of wild-type human type 5 adenovirus can be found, for example, on the GenBank website (http://www.ncbi.nlm.nih) of the National Center for Biotechnology Information (NCBI). .gov/ ) has been published as "Accession number: M73260.1".
  • GenBank website http://www.ncbi.nlm.nih
  • NCBI National Center for Biotechnology Information
  • .gov/ The 18442nd to 21700th nucleotide sequence in the published full-length genome nucleotide sequence (SEQ ID NO: 15) is the nucleotide sequence encoding the wild-type hexon region (SEQ ID NO: 8) in human type 5 adenovirus. It is.
  • the modified amino acid sequence of HVR1 in the hexon-modified Ad vector of the present invention is not limited, but preferably includes, for example, the amino acid sequence of SEQ ID NO: 1 below.
  • DEAATALEIXLEEGGGGSEDEVDEQAEQQK SEQ ID NO: 1; where X is N or E.
  • the modified amino acid sequence of HVR2 is not limited, but preferably includes, for example, the amino acid sequence of SEQ ID NO: 2 below.
  • VEGQTAA SEQ ID NO: 2
  • the modified amino acid sequence of HVR3 is preferably, but not limited to, the amino acid sequence of SEQ ID NO: 3 below.
  • the modified amino acid sequence of HVR4 is not limited, but preferably includes, for example, the amino acid sequence of SEQ ID NO: 4 below.
  • GILVKQAAGKL SEQ ID NO: 4
  • the modified amino acid sequence of HVR5 is not limited, but preferably includes, for example, the amino acid sequence of SEQ ID NO: 5 below.
  • STTEATAGAGDNLT SEQ ID NO: 5
  • the modified amino acid sequence of HVR7 is preferably, but not limited to, the amino acid sequence of SEQ ID NO: 7 below.
  • GGVGNNSTYTKVKPKTGQENGWEKDATEFSDKXE SEQ ID NO: 7; where X is N or I
  • modified amino acid sequences of HVR1 to HVR5 and HVR7 include, for example, One or more (preferably one or several (for example, 1 to 10, more preferably 1 to 5)) amino acids are deleted, substituted, or added in the amino acid sequences of SEQ ID NOS: 1 to 5 and 7.
  • amino acid sequence 70% or more (preferably 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, Mutant amino acid sequences, such as those having an identity of 99% or more, are also preferred.
  • the modified amino acid sequence of HVR1 preferably retains the 14th to 18th amino acids in the amino acid sequence of SEQ ID NO: 1
  • the modified amino acid sequence of HVR2 preferably retains the 6th and 7th amino acids in the amino acid sequence of SEQ ID NO: 2
  • the modified amino acid sequence of HVR3 it is preferable that the fourth amino acid in the amino acid sequence of SEQ ID NO: 3 is retained
  • the modified amino acid sequence of HVR4 it is preferable that the 7th and 8th amino acids in the amino acid sequence of SEQ ID NO: 4 are retained
  • the modified amino acid sequence of HVR5 it is preferable that the third, fourth, and ninth amino acids in the amino acid sequence of SEQ ID NO: 5 are retained
  • the modified amino acid sequence of HVR7 it is preferable that the 4th, 6th, 7th, and 9th amino acids in the amino acid sequence of SEQ ID NO: 7 are retained.
  • the amino acid sequences of the mutant types similar to the amino acid sequences of SEQ ID NOS: 1 to 5 and 7, contain modified HVRs 1 to 5 and 7 that exhibit the effect of avoiding the immune reaction by existing antibodies against human type 5 Ad. It is preferable that the amino acid sequence is a constituting amino acid sequence.
  • the amino acid sequence of HVR6 is preferably the wild-type amino acid sequence shown in SEQ ID NO: 6, but is not limited to it, and the amino acid sequence may be modified as appropriate. It's okay.
  • each modified HVR in the hexon-modified Ad vector of the present invention and the amino acid sequence of those mutants are based on the gene sequence of the underlying human type 5 adenovirus (specifically, within the hexon region).
  • appropriate mutations are introduced so that the gene (codon) encodes the desired modified amino acid sequence, and the mutated virus gene is transfected into cultured cells such as HEK293 cells. It is produced by expressing and multiplying the virus.
  • the amino acid sequence of the fiber region is replaced with the amino acid sequence of a fiber protein that binds to CD46.
  • the adenovirus fiber protein that binds to CD46 also contains cells that do not express or express a small amount of CAR (Coxsackievirus and adenovirus receptor), which is a receptor for human type 5 adenovirus (CAR-negative cells). It can infect almost all cells.
  • CD46 is expressed in almost all human cells except red blood cells, and is also expressed in highly malignant cancer cells (eg, cancer cells with high invasion, metastasis, and proliferation ability).
  • adenoviruses containing fiber proteins that bind to CD46 can infect even CAR-negative and highly malignant cancer cells.
  • human adenovirus type 34 and human adenovirus type 35 bind to CD46 as a receptor and infect cells.
  • adenovirus fibers consist of a knob region, a shaft region, and a tail region, and since adenoviruses infect cells by binding the fiber knob region to a receptor, at least the fiber knob region of the fiber protein is By replacing the virus derived from type adenovirus with that derived from human type 34 or human type 35 adenovirus, the substituted virus can infect CAR-negative cells via CD46.
  • fiber proteins that bind to CD46 are preferably fiber proteins derived from adenoviruses belonging to group B, and specifically, human types 3, 7, 34, 35, and 11. , 16, 21 and 50 adenoviruses are more preferred, human 34 and 35 adenoviruses are even more preferred, and human 35 adenoviruses are particularly preferred.
  • amino acid sequences of the fiber proteins of various adenoviruses belonging to the above group B can be obtained as appropriate from known genetic information databases such as NCBI's GenBank.
  • the amino acid sequence of the fiber protein includes one or more (preferably one or several (for example, 1 to 10, more preferably 1 to 5)) amino acids in the amino acid sequence obtained from the above database. is deleted, substituted or added, or 70% or more (preferably 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96%) of the amino acid sequence obtained from the above database.
  • Variant amino acid sequences such as amino acid sequences having an identity of 97% or more, 98% or more, or 99% or more are also included.
  • the base sequences of genes encoding the fiber proteins of various adenoviruses belonging to the above group B can also be obtained as appropriate from publicly known genetic information databases such as NCBI's GenBank. .
  • the base sequence may be hybridized under stringent conditions with DNA consisting of a complementary base sequence to DNA consisting of the base sequence, or DNA consisting of a base sequence and 70% or more (preferably 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more)
  • nucleotide sequences encoding a protein having the same identity as , and having binding activity to CD46 are also included.
  • the binding activity to CD46 can be evaluated by measuring the infectivity of a recombinant adenovirus having DNA containing the base sequence to CD46-expressing cells.
  • the measurement can be performed using a known method, such as detecting GFP expressed by a virus that has infected CD46-expressing cells using a fluorescence microscope, flow cytometry, or the like.
  • the technical idea of the present invention is not limited to fiber proteins as long as it binds to CD46, and extends to various proteins capable of binding to CD46 and proteins having motifs capable of binding to CD46. .
  • Recombinant adenovirus The present invention also includes recombinant Ad derived from the hexon-modified Ad vector described above.
  • a desired base sequence such as a foreign gene is introduced (integrated) into the gene (viral vector genome) of the hexon-modified Ad vector described above using conventionally known gene recombination technology. ), it is produced by transfecting cultured cells such as HEK293 cells with the introduced viral gene, and expressing and propagating the virus.
  • a desired base sequence such as a foreign gene is preferably introduced (integrated) into the E1 region, E3 region, or fiber C-terminal region of the gene (viral genome) of the hexon-modified Ad vector, although it is not limited thereto.
  • the recombinant Ad according to the present invention can be used for various purposes depending on what kind of base sequence is introduced as the desired base sequence.
  • the recombinant Ad according to the present invention can be used as an active ingredient in, for example, vaccines, various pharmaceutical compositions, and various reagents, but is not limited thereto.
  • the vaccine according to the present invention contains the recombinant Ad according to the present invention.
  • it can be used as a vaccine for the treatment or prevention of various diseases, symptoms, or pathological conditions.
  • various diseases, symptoms, or pathological conditions include, but are not limited to, infectious diseases, allergies, cancer (or tumors), Alzheimer's disease, diabetes, and hypertension.
  • the vaccine according to the present invention contains a recombinant Ad into which a gene encoding a predetermined antigen protein is incorporated so that antibodies effective for treating or preventing various diseases, symptoms, or pathological conditions are produced in vivo.
  • the infectious disease is not limited and includes all infectious diseases for which vaccine therapy has conventionally been used or is likely to be used in the future, but for example, the new coronavirus infection (COVID-19) -19), seasonal influenza, Haemophilus influenzae infection (Hib infection), new type influenza, hepatitis A, hepatitis B, tuberculosis, diphtheria, chickenpox (varicella), Japanese encephalitis, pneumococcal infection, tetanus, pertussis , rubella/congenital rubella syndrome, measles, mumps, rotavirus infection (infectious gastroenteritis), yellow fever, rabies, postherpetic neuralgia, HIV, monkeypox, Preferred examples include Zika virus infection and dengue virus infection.
  • Cancers or tumors include, for example, solid cancers or hematological tumors, and specifically include brain tumors, cervical cancers, esophageal cancers, tongue cancers, lung cancers, breast cancers, pancreatic cancers, stomach cancers, Cancer of the small intestine, duodenal cancer, colon cancer, bladder cancer, kidney cancer, liver cancer, prostate cancer, uterine cancer, cervical cancer, ovarian cancer, thyroid cancer, gallbladder cancer, Examples include pharyngeal cancer, sarcoma, melanoma, leukemia, lymphoma, and multiple myeloma (MM).
  • MM multiple myeloma
  • Alzheimer's disease examples include all types of Alzheimer's dementia, including early-onset Alzheimer's disease. Diabetes includes, for example, type 1 diabetes, type 2 diabetes, and gestational diabetes.
  • hypertension examples include essential hypertension and secondary hypertension. Secondary hypertension includes endocrine hypertension, vascular hypertension, renal parenchymal hypertension, renovascular hypertension, and hereditary hypertension. These include hypertension, drug-induced hypertension, and hypertension due to brain/central nervous system disease.
  • the vaccine according to the present invention is expected to be useful and practical as a vaccine against various infectious diseases, and is particularly suitable for preventing new coronavirus (SARS-CoV-2) infection (COVID-19). It is preferable to use it as a vaccine (SARS-CoV-2 vaccine).
  • SARS-CoV-2 vaccine When used as a vaccine for SARS-CoV-2, the recombinant Ad according to the present invention includes genetic DNA encoding a non-structural protein region or a structural protein region of the entire gene region of SARS-CoV-2.
  • a recombinant Ad is used, which has been integrated with a promoter capable of expressing the DNA.
  • the non-structural protein region is a region consisting of ORF1a, ORF1b, ORF3a, ORF6, ORF7a, ORF7b, ORF8, and ORF10 regions
  • the structural protein region is a region consisting of spike (S), envelope (E), and integral membrane. (M) and nucleoprotein (N) regions.
  • S spike
  • E envelope
  • M nucleoprotein
  • S spike
  • N nucleoprotein
  • information on all genes encoding SARS-CoV-2 proteins, genes encoding the outer shell protein region, and genes encoding nonstructural protein regions involved in replication includes: Gene sequence information of SARS-CoV-2 virus strain: hCoV-19/Japan/AI/I-004/2020 is registered in NCBI's GenBank as accession number: LC521925. Therefore, a gene containing the nonstructural protein region or structural protein region of SARS-CoV-2 can be obtained by common genetic engineering techniques. For example, a nucleic acid synthesis method using a DNA synthesizer, which is commonly used as a genetic engineering technique, can be used.
  • a PCR method in which specific primers are designed for each gene and the gene sequence is amplified using a PCR device, or a gene amplification method using a cloning vector.
  • the above method can be carried out by those skilled in the art in accordance with "Molecular cloning 4th Edt. Cold Spring Harbor Laboratory Press (2012)” and the like. Known methods can be used to purify the obtained PCR product.
  • the vaccine according to the present invention can be inoculated and introduced into a living body by any known method, such as intramuscular, intraperitoneal, intradermal, or subcutaneous injection, or inhalation through the nasal cavity, oral cavity, or lungs, or oral administration.
  • the vaccine according to the present invention includes known pharmaceutically acceptable carriers such as excipients, fillers, binders, lubricants, buffering agents, tonicity agents, chelating agents, coloring agents, preservatives, It can be mixed with fragrances, flavoring agents, sweeteners, etc.
  • the vaccine according to the present invention can be administered orally, such as tablets, capsules, powders, granules, pills, liquids, syrups, etc., or parenterally, such as injections, external preparations, suppositories, eye drops, nasal drops, etc. Depending on the form of the drug, it can be administered orally or parenterally. Preferred examples include local injection into the skin, muscle, abdominal cavity, etc.
  • the dose of the vaccine can be appropriately selected depending on the type of active ingredient, administration route, subject, age, weight, sex, symptoms and other conditions of the patient, and the dose range in the pharmaceutical composition of the present invention described below can be selected as appropriate. You can also refer to it.
  • Vaccines are usually administered once a day, but can also be administered multiple times over an appropriately set period.
  • the present invention includes administering the above-described vaccine according to the present invention to patients to treat various diseases, symptoms, or pathological conditions (e.g., infectious diseases, allergies, cancer (or tumors), Alzheimer's disease, diabetes, etc.).
  • the present invention also provides a method for treating or preventing diseases such as hypertension and high blood pressure.
  • the present invention also provides the use of the recombinant Ad of the present invention to produce a vaccine (eg, a vaccine for the treatment or prevention of the various diseases, symptoms, or pathologies mentioned above).
  • composition according to the present invention contains the recombinant Ad according to the present invention.
  • it can be used as a pharmaceutical composition for treating or preventing various diseases, symptoms, or pathological conditions.
  • various diseases, symptoms, or pathological conditions include, but are not limited to, cancer (or tumor), and the like.
  • the type of cancer or tumor targeted is not limited, and can include any type of cancer or tumor.
  • Examples include solid cancers or blood tumors, specifically brain cancer, cervical cancer, esophageal cancer, tongue cancer, lung cancer, breast cancer, pancreatic cancer, stomach cancer, small intestine cancer, duodenal cancer, Colorectal cancer, bladder cancer, kidney cancer, liver cancer, prostate cancer, uterine cancer, cervical cancer, ovarian cancer, thyroid cancer, gallbladder cancer, pharyngeal cancer, sarcoma, melanoma, leukemia , lymphoma and multiple myeloma (MM).
  • solid cancers or blood tumors specifically brain cancer, cervical cancer, esophageal cancer, tongue cancer, lung cancer, breast cancer, pancreatic cancer, stomach cancer, small intestine cancer, duodenal cancer, Colorectal cancer, bladder cancer, kidney cancer, liver cancer, prostate cancer, uterine cancer, cervical cancer, ovarian cancer, thyroid cancer, gallbladder cancer, pharyn
  • telomeres eukaryotic chromosomes
  • the recombinant Ad according to the present invention may include, but is not limited to, "human telomerase reverse transcription".
  • Preferred is a recombinant Ad in which a replication cassette containing a polynucleotide containing, in this order, "enzyme (hTERT) promoter, E1A gene, IRES sequence and E1B gene” is integrated into the E1 region of the Ad genome.
  • the recombinant Ad can specifically proliferate in cancer cells (cancer cells expressing hTERT) due to the function of the polynucleotide.
  • the recombinant Ad exhibits the effect of destroying cancer cells (ie, oncolysis) by specifically proliferating in cancer cells.
  • hTERT human telomerase reverse transcriptase
  • E1A gene E1A gene
  • E1B gene E1B gene
  • IRES sequence for example, the explanation in Patent Document 1 (WO 2013/027427 A1) mentioned above can be referred to as appropriate. .
  • the pharmaceutical composition of the present invention can be introduced into a living body by any known method, such as intramuscular, intraperitoneal, intradermal or subcutaneous injection, inhalation through the nasal cavity, oral cavity or lungs, or oral administration.
  • the pharmaceutical composition of the present invention also includes known pharmaceutically acceptable carriers such as excipients, fillers, binders, lubricants, buffering agents, tonicity agents, chelating agents, coloring agents, and preservatives. , flavorants, flavoring agents, sweeteners, etc.
  • the pharmaceutical composition of the present invention can be used for oral administration such as tablets, capsules, powders, granules, pills, liquids, and syrups, and for parenteral administration such as injections, external preparations, suppositories, eye drops, and nasal drops. Depending on the form of the drug, it can be administered orally or parenterally. Preferred examples include subcutaneous, intradermal, intramuscular, intraperitoneal, intratumor, etc. local injection, and intravenous injection.
  • the dosage of the pharmaceutical composition depends on various conditions such as the type of recombinant Ad according to the present invention as an active ingredient, administration method/route, administration target, age, sex, weight and symptoms of the patient, therapeutic effect, and treatment time. is selected as appropriate.
  • the daily dosage is preferably about 10 6 to 10 11 PFU (plaque-forming unit), preferably about 10 9 to 10 11 PFU, and can be administered once a day or divided into several doses. It can also be administered. Moreover, it can also be administered multiple times over an appropriately set period.
  • PFU plaque-forming unit
  • the pharmaceutical composition of the present invention is used, for example, for the treatment or prevention of cancer (or tumor), it is not prohibited to use it in combination with known drugs and therapies such as known anticancer agents and radiotherapy.
  • the present invention provides a method for treating or preventing the various diseases, symptoms, or pathological conditions (e.g., cancer (or tumor), etc.) described above, which includes administering the pharmaceutical composition according to the present invention to a patient.
  • the present invention provides the use of the recombinant Ad of the present invention for producing a drug for the treatment or prevention of the various diseases, symptoms, or pathological conditions described above (e.g., cancer (or tumor), etc.).
  • the various diseases, symptoms, or pathological conditions described above e.g., cancer (or tumor), etc.
  • Hexon-modified adenovirus (Ad) vector plasmid Unique restriction enzyme site ClaI, XbaI, Csp45I (BstBI) recognition sequences were introduced into the hexon gene, and the hexon-modified Ad vector plasmid "pAdHM309- Hex1-F35' was produced (see Figure 1 (especially the 'mHexon3' region)).
  • the plasmid pHM5-mHexon which contains the nucleotide sequence 18324th to 21790th of the full-length adenovirus genome (SEQ ID NO: 15) containing the hexon portion, was cleaved with ApaI (cutting the 19173rd site)/PstI (cutting the 19173rd site).
  • ApaI cutting the 19173rd site
  • PstI cutting the 19173rd site
  • plasmid pmHexon3 which introduced unique restriction enzyme sites ClaI, XbaI, and Csp45I (BstBI) recognition sequences into the hexon gene.
  • pmHexon3 was produced by total synthesis (Genewiz Inc.) of the base sequence shown in SEQ ID NO: 16.
  • a fragment (3453 bp) obtained by cutting pHM5-mHexon3 with HindIII/BglII and a fragment obtained by cutting pAdHM309-E1-F35 with AsiSI were recombined using the homologous recombination method using E. coli (strain BJ5183) to create "pAdHM309-Hex1-F35". Obtained.
  • the above pAdHM309-E1-F35 is a plasmid related to pAdHM309-E1 used in the aforementioned non-patent document 5 (Anticancer Res., 42, 1719-1727 (2022)), and is a plasmid related to the pAdHM309-E1 used in the aforementioned non-patent document 6 (Gene, 285, 69-77 (2002)) in which the fiber region was replaced with one derived from human type 35.
  • Csp45I and BstBI are isodizomers, and although their enzyme names are different, they are restriction enzymes that cleave the same recognition sequence (hereinafter referred to as Csp45I). Further, the cut ends of ClaI and Csp45I can be combined with each other, but once combined, they cannot be re-cleaved with ClaI or Csp45I. Utilizing this property, HVR modified fragments are ligated in the production of HVR modified Ad vector plasmids described in (2) to (4) below.
  • Modification strategy for hexon HVR1, hexon HVR2 to HVR3, hexon HVR4 to HVR5, and hexon HVR6 Contains DNA encoding the amino acid sequence of modified hexon HVR1, hexon HVR2 to HVR3, hexon HVR4 to HVR5, or hexon HVR6 , HVR-modified human type 5 Ad vector plasmids (pHVR1m, pHVR2/3m, pHVR4/5m, and pHM8-HVR6m, respectively) were created.
  • Modified HVR1, modified HVR2, modified HVR3, modified HVR4, and modified HVR5 have the amino acid sequences shown in SEQ ID NO: 1, 2, 3, 4, and 5, respectively (however, Xaa in SEQ ID NO: 1 is glutamic acid (E)). (The same applies to section (4) below).
  • the modified HVR6 had the 6th asparagine (N) in the amino acid sequence of SEQ ID NO: 6 replaced with alanine (A).
  • pAdHM309-Hex1-F35 prepared in (1) above was digested with ClaI and XbaI, and then the fragments obtained by digesting each of the above HVR-modified human type 5 Ad vector plasmids with Csp45I and XbaI were added to this. was ligated. At this time, the ClaI cleavage site of pAdHM309-Hex1-F35 and the Csp45I cleavage site of the above fragment are combined.
  • HVR-modified Ad vector plasmids containing DNA encoding each modified HVR (respectively, "pAdHM309-HVR1m-F35”, “pAdHM309-HVR2/3m-F35”, “pAdHM309-HVR4/5m-F35”, and “pAdHM309-HVR6m-F35”) (see FIG. 2A).
  • each of the above fragments (that is, fragments containing DNA encoding each modified HVR) consists of the base sequences shown in SEQ ID NOs: 9 to 12, as shown below.
  • -Fragment containing DNA encoding modified HVR1 SEQ ID NO: 9 (In this fragment, the DNA encoding HVR2-6 is wild type.)
  • -Fragment containing DNA encoding modified HVR2 and modified HVR3 SEQ ID NO: 10 (In this fragment, the DNA encoding HVR1, 4 to 6 is wild type.)
  • -Fragment containing DNA encoding modified HVR4 and modified HVR5 SEQ ID NO: 11 (In this fragment, the DNA encoding HVR1-3,6 is wild type.)
  • -Fragment containing DNA encoding modified HVR6 SEQ ID NO: 12 (In this fragment, the DNA encoding HVR1-5 is wild type.)
  • the above fragments of SEQ ID NOs: 9 to 12 do not contain DNA encoding wild-type HVR7 or modified HVR7.
  • pHVR1m, pHVR2/3m, and pHVR4/5m were produced by total synthesis (Genewith Inc. or Twist Bioscience Inc.) of the base sequences of SEQ ID NOs: 9, 10, and 11, respectively.
  • the pHM8-HVR6m was produced by synthesizing SEQ ID NO: 12 using the PCR method using pHM5-mHexon3 as a template, and inserting this into pHM8.
  • pAdHM309-Hex1-F35 prepared in (1) above was digested with Csp45I and XbaI, and then the fragment obtained by digesting the above HVR7 modified human type 5 Ad vector plasmid with ClaI and XbaI was added to it. , ligated. At this time, the Csp45I cleavage site of pAdHM309-Hex1-F35 and the ClaI cleavage site of the above fragment are combined. As a result, an HVR7 modified Ad vector plasmid ("pAdHM309-HVR7m-F35") containing DNA encoding the modified HVR7 was produced (see FIG. 2B).
  • the above fragment ie, the fragment containing the DNA encoding modified HVR7 consists of the base sequence shown in SEQ ID NO: 13.
  • the fragment of SEQ ID NO: 13 does not contain DNA encoding wild-type HVR1-6 or modified HVR1-6.
  • the pHVR7m2 was produced by total synthesis (Genewith Inc.) of the base sequence of SEQ ID NO: 13.
  • pAdHM309-Hex1-F35 prepared in the above (1) was digested with Csp45I and XbaI, and then the above HVR7 modified human type 5 Ad vector plasmid (pHVR7m2) was digested with ClaI and XbaI. The fragments were ligated. Furthermore, after digesting the plasmid (pAdHM309-HVR7.1-F35) obtained after the above ligation with ClaI and XbaI, the above HVR1-5 modified human type 5 Ad vector plasmid (pHVR1-5m) was added to it with Csp45I. The fragments obtained by digestion with and XbaI were ligated.
  • HVR1-5,7 modified Ad vector plasmid (“pAdHM309-HVR1-5,7-F35") containing DNA encoding modified HVR1 to HVR5 and modified HVR7 was produced (see FIG. 2C).
  • each of the above fragments consists of the base sequences shown in SEQ ID NOs: 14 and 13 as shown below.
  • ⁇ Fragment containing DNA encoding modified HVR1 to HVR5 SEQ ID NO: 14 (In this fragment, the DNA encoding HVR6 is wild type.)
  • -Fragment containing DNA encoding modified HVR7 SEQ ID NO: 13
  • the fragment of SEQ ID NO: 14 does not contain DNA encoding wild-type HVR7 or modified HVR7.
  • the pHVR1-5m was produced by total synthesis (Genewith Inc.) of the base sequence of SEQ ID NO: 14.
  • the pHVR7m2 is as described in section (3).
  • HVR-modified Ad vector plasmids i.e., "pAdHM309-HVR1m-F35-L1", “pAdHM309-HVR2/3m-F35-L1", “pAdHM309-HVR4/5m" carrying the firefly luciferase gene expression cassette were obtained.
  • -F35-L1'', ⁇ pAdHM309-HVR6m-F35-L1'', and ⁇ pAdHM309-HVR7m-F35-L1'') see FIG. 2D).
  • HVR1-5,7 modified Ad vector plasmid (pAdHM309-HVR1-5,7-F35) prepared in (4) above was After digestion with CeuI and PI-SceI, a plasmid carrying a foreign gene (specifically, ⁇ -galactosidase (LacZ) or firefly luciferase (Luc)) expression cassette is digested with I-CeuI and PI-SceI. The resulting fragments were ligated. This allows HVR1-5,7 modified Ad vector plasmids carrying foreign gene expression cassettes (i.e. "pAdHM309-HVR1-5,7m-F35-LacZ", "pAdHM309-HVR1-5,7m-F35-Luc”) was obtained (see Figure 2E).
  • HVR-modified Ad vectors prepared in (5) and (6) of Example 1 were virus-amplified using HEK293 cells (10 150 mm dishes), and various HVR-modified recombinant Ads were collected. After purification by the various recombinant Ad ultracentrifugation methods, the virus titer (Virus particle (VP)/ml) was calculated based on the OD260 value. The results are shown in Table 1 below.
  • Ad-L2 conventional Ad vector expressing firefly luciferase
  • virus yields comparable to or slightly lower than that of the control "Ad-L2" (conventional Ad vector expressing firefly luciferase) could be recovered.
  • AdF35HVRm-LacZ and AdF35HVRm-Luc substantially the same amount of virus as Ad-L2 could be recovered.
  • Ad-LacZ conventional Ad vector that expresses ⁇ -galactosidase
  • AdF35HVRm-LacZ conventional Ad vector that expresses ⁇ -galactosidase
  • Hexon-modified Ad vector was allowed to act at 60VP/cell and 300VP/cell.
  • ⁇ -galactosidase expression was evaluated by X-gal staining. No significant difference was observed between Ad-LacZ and AdF35HVRm-LacZ in terms of ⁇ -galactosidase expression (see Figure 3).
  • Ad-L2 conventional Ad vector expressing firefly luciferase
  • AdF35HVRm-Luc conventional Ad vector expressing firefly luciferase
  • Panc-1 panc-1
  • SK HEP-1 SK HEP-1
  • H1299 cells hexon-modified Ad vector
  • Firefly luciferase expression was measured 48 hours after Ad vector action. No significant difference was observed in the expression of firefly luciferase between Ad-L2 and AdF35HVRm-L2 (see Figure 4).
  • Ad-L2 conventional Ad vector that expresses firefly luciferase
  • AdF35-L2 hexon is wild type, has type 35 adenovirus fiber, expresses firefly luciferase
  • AdF35HVRm-Luc vector expressing firefly luciferase with modified hexon and type 35 adenovirus fiber
  • human serum No. 4-9 diluted at various dilution ratios. It was incubated with a medium (without Fetal bovine serum) at room temperature for 1 hour. Thereafter, it was allowed to act on H1299 cells at 200 VP/cell.
  • Firefly luciferase activity was measured 72 hours after Ad vector action. The results are shown in FIG. In FIG. 5, the percentage on the graph bar indicates the firefly luciferase activity when the firefly luciferase activity by the Ad vector incubated with a medium containing no human serum is taken as 100%.
  • the firefly luciferase activity induced by Ad-L2 and AdF35-L2 decreased in a human serum concentration-dependent manner, whereas the firefly luciferase activity induced by AdF35HVRm-Luc did not decrease significantly even in the presence of human serum.
  • the present invention in order to avoid antibodies (existing antibodies) against human type 5 Ad that inhibit gene transfer activity contained in human serum (avoidance from immune reactions caused by existing antibodies), the present invention was used. It was demonstrated that modification of the hexon region (particularly each HVR region) is important as in the hexon-modified Ad vector, and modification of the fiber region is not substantially involved.
  • hexon-modified human adenovirus type 5 (Ad) vector that can avoid immune reactions caused by existing antibodies.
  • the hexon-modified Ad vector according to the present invention can provide vaccines, pharmaceutical compositions, etc. containing recombinant adenoviruses derived from the Ad vector, which can avoid immune reactions caused by existing antibodies. , highly practical and useful.

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Abstract

L'invention concerne un vecteur Ad 5 humain, etc, capable d'éviter une réponse immunitaire par un anticorps existant. La présente invention est basée sur un adénovirus humain de type 5 et concerne un vecteur d'adénovirus modifié par hexon dans lequel la séquence d'acides aminés d'au moins une région hypervariable (HVR) parmi HVR1 à HVR7 présente dans la région d'hexon a été modifiée.
PCT/JP2023/024534 2022-06-27 2023-06-27 Vecteur d'adénovirus modifié par hexon WO2024005211A1 (fr)

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Publication number Priority date Publication date Assignee Title
WO2021078735A1 (fr) * 2019-10-21 2021-04-29 Universität Ulm Adénovirus comprenant une protéine hexon d'adénovirus modifiée

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021078735A1 (fr) * 2019-10-21 2021-04-29 Universität Ulm Adénovirus comprenant une protéine hexon d'adénovirus modifiée

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
MIZUGUCHI, H. ; HAYAKAWA, T.: "Adenovirus vectors containing chimeric type 5 and type 35 fiber proteins exhibit altered and expanded tropism and increase the size limit of foreign genes", GENE, ELSEVIER AMSTERDAM, NL, vol. 285, no. 1-2, 20 February 2002 (2002-02-20), NL , pages 69 - 77, XP027353633, ISSN: 0378-1119 *
ROBERTS DIANE M, ET AL: "Hexon-chimaeric adenovirus serotype 5 vectors circumvent pre-existing anti-vector immunity.", NATURE, vol. 441, no. 7090, 11 May 2006 (2006-05-11), pages 239 - 243, XP002385300, DOI: 10.1038/nature04721 *
SHINYA ABE, KENJI OKUDA, TAKEHIRO URA, ASAMI KONDO, ATSUSHI YOSHIDA, SHINJI YOSHIZAKI, HIROYUKI MIZUGUCHI, DENNIS KLINMAN, MASARU : "Adenovirus type 5 with modified hexons induces robust transgene-specific immune responses in mice with pre-existing immunity against adenovirus type 5", THE JOURNAL OF GENE MEDICINE, JOHN WILEY & SONS, vol. 11, no. 7, 1 July 2009 (2009-07-01), pages 570 - 579, XP055092320, ISSN: 1099498X, DOI: 10.1002/jgm.1332 *

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