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WO2018185110A1 - Nouveaux vecteurs d'expression et utilisations associées - Google Patents

Nouveaux vecteurs d'expression et utilisations associées Download PDF

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Publication number
WO2018185110A1
WO2018185110A1 PCT/EP2018/058504 EP2018058504W WO2018185110A1 WO 2018185110 A1 WO2018185110 A1 WO 2018185110A1 EP 2018058504 W EP2018058504 W EP 2018058504W WO 2018185110 A1 WO2018185110 A1 WO 2018185110A1
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Prior art keywords
expression vector
vector
protein
dna sequence
integrating expression
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PCT/EP2018/058504
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English (en)
Inventor
Andres Männik
Mart Ustav
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Fit Biotech Oy
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Publication of WO2018185110A1 publication Critical patent/WO2018185110A1/fr

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    • 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
    • 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
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/20011—Papillomaviridae
    • C12N2710/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/20011—Papillomaviridae
    • C12N2710/20041—Use of virus, viral particle or viral elements as a vector
    • C12N2710/20043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present invention consequently also relates to said non-integrating expression vector for use in expressing a protein or fragment(s) thereof binding an antigen associated with cancer, tumor(s) and/or solid tumor(s) and/or cell signaling associated with said cancer, tumor(s) and/or solid tumor(s) as well as to a gene therapeutic containing said vector, to methods for the preparation of the vector and to therapeutic uses of said vector, in particular for non-intergrating gene therapy for cancer treatment.
  • Such vectors have in particular been used for vaccine purposes. Since 1990, DNA immunization has become a standard method to induce immune responses to foreign proteins in experimental animal and human studies. Such vectors are called DNA vaccines.
  • the DNA vectors used in vaccines contain a cloning site for the gene of interest, a strong viral promoter, such as the immediate early promoter of the CMV virus, in order to drive the expression of the gene of interest, a polyadenylation region, and an antibiotic resistance gene and a bacterial replication origin for the propagation of the DNA vector (plasmid) in bacterial cells.
  • a strong viral promoter such as the immediate early promoter of the CMV virus
  • the vectors described above it is possible to obtain a detectable level of expression of the gene of interest after administering the vector to experimental animals or to humans, either by a direct injection to muscle or to skin with a particle bombardment technique or by applying the vector in a solution directly to mucous membranes.
  • the expression obtained by these vectors is short lived: the vectors tend to disappear from the transfected cells little by little and are not transferred to daughter cells in a dividing cell population.
  • the short-term expression of the gene of interest and lim- ted number of cells targeted are probably the major reasons, why only temporary immune responses are observed in subjects immunized with DNA vaccine vectors described above.
  • the replication, episomal maintenance, expression of the late genes and virus assembly are tightly coupled to the differentiation of the epithelial tissue: the papilloma virus DNA episomal replication takes place during the initial amplificational replication and the second, i.e. latent, and the third, i.e. vegetative, replications in the differentiating epithelium.
  • the DNA binding-dimerization domain of E2 has a structure of a dyad-symmetric eight- stranded antiparallel beta barrel, made up of two identical "half-barrel" subunits
  • expression vectors which carry (A) an expression cassette of a gene of a nuclear-anchoring pro- tein that binds both to (i) a specific DNA sequence and (ii) to a suitable nuclear component and (B) a multimerized DNA binding sequence for said nuclear-anchoring protein, are capable of spreading in a proliferating cell population.
  • nuclear- anchoring proteins include, but are not limited to, chromatin-anchoring proteins, such as the Bovine Papilloma Vims type 1 E2 protein (BPV1 E2).
  • BPV1 E2 Bovine Papilloma Vims type 1 E2 protein
  • Viral vectors are clearly suitable for both therapeutic application and as tools for biological studies; however, their delivery properties can be challenging to engineer and improve.
  • the most frequently used have been based on adenovirus, retrovirus, vaccinia virus, herpesvirus, and AAV.
  • Said encoded protein or fragment(s) thereof binding an antigen can be selected from the group consisting of an antibody or fragment(s) thereof, a monoclonal antibody or fragment(s) thereof, and a protein or fragment(s) thereof which bind(s) an antigen via one or more epitope(s) associated with said cancer, tumor(s) and/or solid tumor(s) and/or cell signaling associated with said cancer, tumor(s) or solid tumor(s).
  • the present invention relates to a non-integrating expression vector for use according to the present invention, wherein said DNA sequence of interest encodes an anti-VEGF antibody or fragment(s) thereof, comprising the variable re- gion:
  • gtGTU® expression vector For the design and construction of the herein for the first time presented gtGTU® expression vectors, whole sequences of the prior disclosed GTLT* gene vaccination vector (GTU8) were completely redesigned to avoid mouse and/or human ISSs.
  • GTU8 GTLT* gene vaccination vector
  • a gtGTU® expression vector is produced in a particularly compact form as a minicircle plasmid.
  • a non-integrating expression vector with reduced ability to activate an innate immune response in a mammalian cell comprises (a) a DNA sequence encoding an ⁇ 2 protein of Bovine Papilloma Virus type 1 (BPV1), comprising a DNA- binding dimerization domain, said E2 protein being operatively linked to a heterologous promoter, and (b) an oligomerized DNA sequence, forming a binding site for an E2 protein of Bovine Papilloma Virus type 1 (BPV1), wherein said vector lacks an origin of replication functional in any mammalian cell, and wherein said vector is at least 50% devoid of immunostimulatory sequences (ISSs) from the plasmid backbone and from the promoter, and wherein at least 10% of the immunostimulatory sequences (ISSs) of said one or more expression cassette(s) of a DNA sequence of interest are removed and/or modified in such a way that they do not activate an innate immune response in a mammalian cell.
  • a non-integrating expression vector according to the present invention comprises one or more expression cassette(s) of a DNA sequence encoding at least one of
  • a non-integrating expression vector according to according to the present invention comprises one or more expression cassette(s) of a DNA sequence of inter- est, wherein at least 10% of all human ISSs as shown in SEQ.ID.NO.: 16 and/or at least 10% of all mouse ISSs as shown in SEQ.ID.NO.: 17 are removed and/or modified in such a way that they do not activate an innate immune response in a mammalian cell.
  • a non-integrating expression vector with reduced ability to activate an innate immune response in a mammalian cell according to the present invention can comprise a heterologous promoter which is ubiquitous and/or a heterologous promoter which is tissue specific.
  • a non-integrating expression vector with reduced ability to activate an innate immune response in a mammalian cell comprises a heterologous promoter which is tissue specific for any one or more of the group of tissues or cells consisting of Langerhans cells, epidermis, keratino- cytes, lung cells, muscle cells (including but not limited to smooth muscle, skeletal muscle and cardiac muscle cells), melanocytes, Merkel cells, liver cells (including but not limited to hepatocytes) , Langerhans cells, epithelial cells (including but not limited to alveolar type epithelial cells), endothelial cells, stroma cells, bone marrow cells (including but not limited to hematopoietic cells).
  • tissue specific for any one or more of the group of tissues or cells consisting of Langerhans cells, epidermis, keratino- cytes, lung cells, muscle cells (including but not limited to smooth muscle, skeletal muscle and cardiac muscle cells), melanocytes, Merkel cells, liver cells (including
  • a non-integrating expression vector with reduced ability to activate an innate immune response in a mammalian cell according to the present invention is typically intended for a use selected from the group consisting of; treating and/or preventing an inherited or acquired genetic defect, for use in monoclonal antibody therapy, for use in enzyme replacement therapy, for use in delivery of an acute medicament, for use in cancer treatment, for production of a therapeutic macromolecular agent in vivo and for treat- ment of a viral infection(s).
  • Said method can further comprise cultivating a host cell containing said expression vector; and recovering the expression vector, and/or before this, transforming said host cell with said vector, wherein said host cell is a pro- karyotic or a eukaryotic cell, such as but not limited to an Escherichia coli cell.
  • the present invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a non-integrating expression vector with reduced ability to activate an innate immune response in a mammalian cell according to the present invention, and a suitable pharmaceutical carrier.
  • immunostimulatory sequences in the present context relates to manipulation of DNA sequences in order to reduce the im- munostimulatory profile of the plasmid DNA.
  • gtGTU® vectors are designed based on minimizing the plasmid vector size by removal of dispensable components (e.g. bacterial origin, antibiotic resistance marker) which in itself improves the vector outcome in gene therapy applications in vivo. Higher and more prolonged expression of the transgene can be achieved. Further, the bulk of immunostimulatory sequences (ISSs), i.e. at least 50% of all ISSs, preferably at least 55% of all ISSs, are also removed, striving to completely remove all ISSs in gene therapy vectors.
  • ISSs immunostimulatory sequences
  • a minicircle plasmid is a small circular plasmid devoid of prokary- otic vector elements and derived from a parental and bacterial plasmid with eukaryotic inserts.
  • this can be achieved by removing and/or modifying, in such a way that they do not activate an innate immune response in a mammalian cell, at least 10% of all human ISSs as shown in SEQ.ID.NO.: 16 and/or at least 10% of all mouse ISSs as shown in SEQ.ID.NO.: 17, in said expression cassette(s) of a DNA sequence of interest.
  • DNA from bacteria can induce a non-specific immune response, possibly at least in part due to un- methylated CpG nucleotide duplexes found in the bacterial genome.
  • the unmethylated CpG dinucleotides are present at a higher frequency in bacterial DNA or vector DNA compared to its frequency in mammalian DNA.
  • the CpG dinucleotides contain cytosine triphosphate deoxynucelotide ( " C " ) followed by a guanine triphosphate deoxynucleotide f ' G " ).
  • the deoxynucleotides are linked together by a phosphodiester bond ( " p " ).
  • the unmethylated CpG sequences act as immunostimu- lants and are recognized by the pattern recognition receptor (PPR); Toll-Like Receptor 9.
  • nuclear-anchoring protein refers to a protein, which binds to a specific DNA sequence and is capable of providing a nuclear compartmentalization function to the vector, i.e., to a protein, which is capable of an- choring or attaching the vector to a specific nuclear compartment.
  • the nuclear-anchoring protein is a natural protein.
  • nuclear compartments are the mitotic chromatin or mitotic chromosomes, the nuclear matrix, nuclear domains like ND10 and POD etc.
  • nuclear- anchoring proteins are the Bovine Papilloma Virus type 1 (BPV1) E2 protein, EBNA1 (Epstein-BarrVirus Nuclear Antigen 1), and High Mobility Group (HMG) proteins etc.
  • the gene of a natural nuclear- anchoring protein which contains a DNA binding domain capable of binding to a spe- cific DNA sequence and a functional domain capable of binding to a nuclear component
  • a viral protein such as the E2 protein of Bovine Papilloma Virus or the EBNA1 (Epstein-Barr Virus Nuclear Antigen 1) of the Epstein-Barr Virus, a eu- karyotic protein such a one of the High Mobility Group (HMG) proteins or a like protein, or a prokaryotic protein.
  • HMG High Mobility Group
  • the multimerized DNA binding sequence of a vector of the present invention can contain any one of known 17 different affinity E2 binding sites as a hexamer or a higher oligomer, as a octamer or a higher oligomer, as a decamer or higher oligomer. Oligomers containing different E2 binding sites are also applica- ble. Specifically preferred E2 binding sites useful in the vectors of the present invention are the BPV1 high affinity sites 9 and 10, affinity site 9 being most preferred. When a higher oligomer is concerned, its size is limited only by the construction circumstances and it may contain from 6 to 30 identical binding sites. Preferred vectors of the invention contain 10 BPV-1 E2 binding sites 9 in tandem.
  • a non-integrating expression vector according to the present invention is e.g. as shown in SEQ.ID.Nos:1-21.
  • the vectors contain any DNA sequence coding for a protein that is defective in any hereditary and/or single gene hereditary disease.
  • Known gene targets for hereditary diseases are in the present context e.g. selected from the non-exclusive list consisting of ABCA4 Retinitis Pigmentosa, ABCC9 Dilated Cardiomyopathy 10, ABCD1 X-Linked Adrenoleukodystrophy, ACADVL Very Long Chain Acyl-Coenzyme A Dehydrogenase Deficiency, ACTA2 Thoracic Aortic Aneurysms and Aortic Dissections, ACTC1 Familial Hypertrophic Cardiomyopathy, ACTN2 Dilated Cardiomyopathy 1AA, ADA Severe Combined Immunodeficiency, AIPL1 Leber Congenital Amaurosis, AIRE Autoimmune Polyendocrine Syndrome, AKAP9 Long QT Syndrome, Autosomal Dominant, AKR1 B1 Androgen Insensitivity Syndrome
  • b a light chain, of a virus specific antibody.
  • a non-integrating expression vector according to the invention which comprises one or more expression cassette(s) of a DNA sequence encoding both a heavy chain and a light chain, of a monoclonal an- tibody gives rise to a rapid high MAb level (see example 5) in blood in all animals treated.
  • the vectors of the invention are used to treat and/or pre- vent diseases and disorders caused by pathologically high activity of a protein.
  • the DNA sequence of interest encodes an antagonist of the overactive protein.
  • antagonists include, but are not limited to, an- tisense RNA molecules, ribozymes, antibodies, and dominant negative proteins.
  • the DNA sequence of interest encodes an in- hibitor of an oncogene.
  • the vector of the invention comprises one or more expression cassettes comprising a DNA sequence of interest.
  • the DNA sequence of interest can encode a protein and/or a biologically active RNA molecule.
  • the DMA sequence is inserted into the vector of the invention for expression in recombinant cells or in cells of the host in the case of gene therapy.
  • An expression cassette refers to a DNA sequence of interest opera- bly linked to one or more regulatory regions or enhancer/promoter sequences which enables expression of the protein of the invention in an appropriate host cell.
  • "Opera- bly-linked” refers to an association in which the regulatory regions and the DNA sequence to be expressed are joined and positioned in such a way as to permit transcription, and in the case of a protein, translation.
  • a number of selection systems may be used for mammalian cells, including but not limited to the Herpes simplex virus thymidine kinase, hypoxanthine-guanine phos- phoribosyltransferase, and adenine phosphoribosyltransferase, genes can be em- ployed in tk ' , hgprf or aprf cells, respectively.
  • a vector of the invention comprising an expression cassette comprising DNA sequences of interest is administered to treat, or prevent various diseases.
  • the DNA sequence of interest may encode a macromolecule, such as, but not limited to, a protein and/or a biologically active RNA molecule.
  • Gene therapy refers to therapy performed by the administration to a subject of an expressed or expressible DNA sequence, i.e., the DNA sequence produces its encoded protein or RNA molecule that mediates a therapeutic effect.
  • the expression vector(s) of the present invention do(es) not activate an innate immune response in a mammalian cell, as is demonstrated in the experimental section, wherein in particular experiments 6 and 7 clearly disclose that the expression vector(s) of the present invention do(es) not induce humoral responses in an animal model. It is therefore concluded that the expression vector(s) of the present invention will not activate a significant induction of antigen specific lgG1 response in the patient treated with the gene therapy.
  • Some embodiments of the present invention relate to a non-integrating expression vector according to the present invention, for a use selected from the group consisting of treating and/or preventing an inherited or acquired genetic defect, for use in antibody and/or monoclonal antibody therapy, for use in enzyme replacement therapy, for use in therapy with antibody or monoclonal antibody fragments, for use in delivery of an acute medicament, for use in cancer treatment, for production of a therapeutic macromolecular agent in vivo and for treatment of viral infections.
  • Any of the methods for gene therapy available in the art can be used according to the present invention. .Exemplary methods are described below.
  • antibody molecules can thus be provided with new functions in unexpected scenarios: e.g. expression of antibody domains in precise intracellular locations and grafting of new binding activities to engineered cells. In particular, this is applicable for antibody- based cancer therapy.
  • four main types of CmAbs murine, chimeric, humanized and human CmAbs.
  • Murine CmAbs derived exclusively from mouse, were the first to be applied in cancer chemo- therapeutics. Utilization, however, was rapidly revoked because of inability to effectively interact with components of the human immune system due to their foreign na- ture and subsequent limited recognition by the host immune system.
  • Chimeric CmAbs typically comprise variable regions derived from a murine source and constant regions (65%) derived from a human source.
  • these studies utilize controls that distinguish between antisense gene inhibition and nonspecific biological effects of oligonucleotides. It is also preferred that these studies compare levels of the target RNA or protein with that of an internal control RNA or protein. Additionally, it is envisioned that results obtained using the antisense DNA sequence are compared with those obtained using a control DNA sequence. It is preferred that the control DNA sequence is of approximately the same length as the test oligonucleotide and that the DNA sequence of the oligonucleotide differs from the antisense sequence no more than is necessary to prevent specific hybridization to the target sequence.
  • Methods for use with the invention include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. Methods for use with the invention further include administration by any con- venient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.).
  • compositions may take the form of tablets or lozenges formulated in conventional manner.
  • the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellent, e.g., dichlorodi- fluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellent e.g., dichlorodi- fluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • the dosage unit may be de- termined by providing a valve to deliver a metered amount.
  • the "non-GTU” vector was identical to the epi-GTUK14, but contained disrupting mutations in the E2 coding sequence, thus the E2 protein is not produced from the mutated gene of the "non-GTU".
  • the transfected region was visually identified by EGFP expression using blue flashlight with appropriate filters. Thereafter, the EGFP positive region in each site was collected using 8mm punch biopsies. The biopsies were lysed in IxCCLR buffer (Promega) and luciferase activities were measured from the lysates using Luciferase Assay System kit (Promega).
  • FIG 6 shows the outcome of an Ichor experiment.
  • the GTU® and control regular plasmid plMS-431 were used, expressing a secretable alkaline phosphatase (mSEAP).
  • mSEAP secretable alkaline phosphatase
  • the enzymatic activity of the SEAP in the blood was meas- ured in time after DNA delivery, as shown in the horizontal axis.
  • IM is most likely intramuscular injection and in the TDS-IM groups, the injection is assisted by electro- poration, using an Ichor device.
  • GTU® and non-GTU® control vectors were designed and constructed. All vectors contained a tricistronic GEF markergene, expressing three marker proteins from a single cDNA:
  • the markergene and E2 expression were controlled by muscle specific promoter 3xUSE/Tnl (human Slow Troponin I promoter (Tnl) and its oligomerized upstream enhancer elements (USE), described in Blain et al. 2010 [PMID: 19719387]).
  • the "non- GTU” was a regular vector containing the same expression unit for GEF but no E2 cassette or E2 BSs. Equimolar amounts (40 pg GTU® or 29 ug non-GTU) were delivered to muscle tibialis anterior of Swiss Webster mice (22-25 g), using 5 mice in both group:
  • the gtGTU® concept was specifically created for the usage of GTU* in gene therapy applications. It includes minimizing the vector elements, e.g. incorporating the E2BSs into the intron, removal of dispensable components, e.g. bacterial origin, antibiotic resistance marker ensures more efficient delivery/expression.
  • the gtGTU® concept also includes removing a bulk of immunostimulatory sequences (ISSs) from the GTU* DNA sequences by intelligent mutagenesis. ISS removal reduces the ability of activation of innate immune responses and thus decreases the risk of the vector itself to work as a DNA vaccine, which can induce immunity against the product of interest, especially when administered repetitively.
  • ISS-free variants of several promoters are designed (based on RSV LTR, CMV, for example), as well as polyadenylation signals, introns etc. for their usage in gtGTU® vectors.
  • the general gtGTU® concept is disclosed in figure 11.
  • the gtGTU® concept includes minimizing the plasmid vector size and removing potentially immunostimulatory se- quences.
  • whole sequences of GTU* gene vaccination vector (GTU8) were completely redesigned to avoid mouse or human ISSs.
  • the final gtGTU® products were produced in most compact form as minicircle plasmids.
  • three different gtGTU® configurations were designed and constructed that express the Glue markergene.
  • the first configuration (gtGTU 1 on figure 12) generally mimics the structure of the latest generation of GTU® gene vaccination vector (GTU8).
  • cAMP response elements CRE
  • CRE cAMP response elements
  • the fifth 19-bp element is a variant of CRE (AF-1/CRE) and it does not contain ISS (Keller et al., J Virol 77, 6666).
  • AF-1/CRE cAMP response elements
  • A673 Ewing sarcoma tumors were induced 30 days after DNA delivery according to the timeline represented in Fig 25. This was performed essentially as described in Gerber et al., 2000: Human A673 rhabdomyosarcoma cells were trypsinized and trypsin was inactivated with fetal bovine serum containing medium. Then the cells were precipitated and re-suspended in ice-cold PBS. After second precipitation, the cells were re-suspended in DMEM medium with high glycose, glutamax, pyruvate (Thermo Fisher Scientific 11995073).
  • Figure 40 summerizes that all plasmid DMA received animals showed similar and low IL6 serum levels in 4h time-point.
  • mice were immunized with ovalbumin (OVA) as antigen alone or mixed with GTU®, gtGTU® or CpG oligodeoxynucleotide (ODN) as adjuvant; and OVA specific lgG1 and lgG2a response was analyzed at different time points.
  • OVA ovalbumin
  • Class C CpG oligodeoxynucleotides ODN 2395 (Invivogen, tlrl-2395)
  • mice origin Charles River Laboratory, further breeding's are made in University of Tartu, Laboratory Animal Centre (generation 6), 6-7 weeks old:
  • bleed time points (indicated in Timeline above, 0 days means base sample collected before 1. immunization), 20-times diluted total blood samples were collected: 5 ⁇ of peripheral blood (from tail tip bleeding) was mixed with 95 ⁇ of 1%BSA-PBS; and the samples were stored at -20 °C up to analyses.
  • OVA specific lgG1 and lgG2a antibodies are detectable not earlier than 17 and 22 days after initial immunization (or after 4. and 5. immunization, respectively).
  • lgG2a (Th1) response was induced mainly in positive control group G3 immunized with CpG ODN adjuvant (figures 45 and 47).
  • G4 gtGTU
  • DIMA can be highly immunogenic and therapies based on DMA should prevent the de- velopment of adaptive immune responses for long term efficacy.

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Abstract

La présente invention concerne des vecteurs d'expression viraux non intégrants destinés à être utilisés dans des schémas d'administration répétée pour une thérapie génique non intégrative, telle que, mais non limitée, au traitement du cancer, qui n'induisent pas de réponse immunitaire adaptative à long terme chez le patient. La présente invention concerne les nouveaux vecteurs, ainsi que leurs utilisations médicales. Les vecteurs d'expression non intégrants comprennent une séquence d'ADN codant pour une protéine E2 du virus du papillome bovin de type 1 (BPV1), comprenant un domaine de dimérisation de liaison à l'ADN, ainsi qu'une séquence d'ADN oligomérisé, formant un site de liaison pour ladite protéine E2. De plus, facultativement, au moins 50 % de toutes les séquences immunostimulantes (ISS) provenant du squelette plasmidique, du promoteur et/ou de la cassette d'expression dudit vecteur peuvent être éliminées et/ou modifiées de telle sorte que les vecteurs n'activent pas une réponse immunitaire innée dans une cellule de mammifère. La présente invention concerne en outre ledit vecteur d'expression non intégrant destiné à être utilisé dans l'expression d'une protéine ou d'un(de) fragment(s) de cette dernière se liant à un antigène et/ou à une molécule de signalisation cellulaire associée à un cancer, à une(des) tumeur(s) et/ou à une(des) tumeur(s) solide(s).
PCT/EP2018/058504 2017-04-03 2018-04-03 Nouveaux vecteurs d'expression et utilisations associées WO2018185110A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
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US11066465B2 (en) 2015-12-30 2021-07-20 Kodiak Sciences Inc. Antibodies and conjugates thereof
US11155610B2 (en) 2014-06-28 2021-10-26 Kodiak Sciences Inc. Dual PDGF/VEGF antagonists
EP3953484A4 (fr) * 2019-04-12 2023-04-12 University of Massachusetts Thérapie génique à médiation par aav de la leucinose
US11912784B2 (en) 2019-10-10 2024-02-27 Kodiak Sciences Inc. Methods of treating an eye disorder
US12071476B2 (en) 2018-03-02 2024-08-27 Kodiak Sciences Inc. IL-6 antibodies and fusion constructs and conjugates thereof

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