WO2024062098A1 - Virus de la paraviccine recombinant codant pour l'interleukine-12 - Google Patents

Virus de la paraviccine recombinant codant pour l'interleukine-12 Download PDF

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WO2024062098A1
WO2024062098A1 PCT/EP2023/076224 EP2023076224W WO2024062098A1 WO 2024062098 A1 WO2024062098 A1 WO 2024062098A1 EP 2023076224 W EP2023076224 W EP 2023076224W WO 2024062098 A1 WO2024062098 A1 WO 2024062098A1
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pcpv
nucleic acid
composition
cancer
amino acid
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PCT/EP2023/076224
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Karola Rittner
Nathalie Silvestre
Caroline TOSCH
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Transgene
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    • 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
    • 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
    • A61K35/768—Oncolytic viruses not provided for in groups A61K35/761 - A61K35/766
    • 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
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5434—IL-12
    • 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
    • A61K2039/58—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation
    • A61K2039/585—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation wherein the target is cancer
    • 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
    • C12N2710/24011—Poxviridae
    • C12N2710/24211—Parapoxvirus, e.g. Orf virus
    • C12N2710/24241—Use of virus, viral particle or viral elements as a vector
    • C12N2710/24243—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present invention relates to the field of viral immunotherapy.
  • the invention concerns a novel recombinant pseudocowpox virus (PCPV) comprising a nucleic acid molecule inserted in its genome and encoding an interleukine-12, composition thereof as well as their therapeutic use for preventive or treating diseases, and, notably, proliferative diseases like cancers.
  • PCPV pseudocowpox virus
  • the present invention also provides methods for generating and amplifying such a PCPV and a method for eliciting or stimulating and/or re-orienting an immune response using such a PCPV.
  • the recombinant PCPV according to the invention further encodes for a heterologous antigenic polypeptide.
  • the invention may be largely used for the therapeutic vaccination.
  • Immunotherapy seeks to boost the host's immune system to help the body to eradicate pathogens and abnormal cells. Widely used in traditional vaccination, immunotherapy is also being actively investigated as a potential modality for treating severe, chronic, or life-threatening diseases in an attempt to stimulate specific and innate immune responses. A vast number of immunotherapeutics have been described in the literature for decades.
  • VV vaccinia virus
  • TG4001 is an investigational therapeutic vaccine based on a non-propagative, highly attenuated Vaccinia vector, the modified vaccinia virus Ankara (MVA), which is engineered to express HPV16 antigens (E6 & E7) and an adjuvant (IL-2).
  • MVA modified vaccinia virus Ankara
  • IL-2 an adjuvant
  • TG4001 is designed to have a two-pronged antiviral approach: to alert the immune system specifically to cells presenting the HPV16 E6 and E7 antigens, that can be found in HPV16-related tumors, and to further stimulate the infection-clearing activity of the immune system through interleukin 2 (IL-2).
  • IL-2 interleukin 2
  • TG4001 has been administered to more than 300 individuals, demonstrating good safety and promising efficacy results.
  • TG4001 Its mechanism of action and good safety profile make TG4001 an excellent candidate for combinations with other therapies in HPV-mediated solid tumors.
  • This therapeutical cancer vaccine is part of a phase lb/ll trial evaluating the combination of TG4001 and avelumab in patients with HPV-16 positive recurrent or metastatic malignancies (ClinicalTrials.gov identifier NCT03260023).
  • TG4050 Another MVA-based viral vector is the TG4050 product.
  • This virus-based therapeutic vaccine is an individualized immunotherapy that encodes neoantigens (patient-specific mutations).
  • TG4050 is designed to stimulate the immune system of patients in order to induce a T-cell response that is able to recognize and destroy tumor cells based on their own neoantigens.
  • This individualized immunotherapy is developed and produced for each patient (WO2018/234506).
  • TG4050 entered into a phase 1-2 clinical trial for patients with ovarian or head and neck cancers (ClinicalTrials.gov Identifiers NCT04183166 and NCT0839524 ; Malone et al.
  • Parapoxviruses represent different candidates that can be used in vector vaccines.
  • Parapoxvirus belongs to the family Poxviridae and the subfamily Chordopoxvirinae. Parapoxviruses are commonly known as causative agents of dermal diseases in ruminants, leading to papular stomatitis and contagious pustular dermatitis, especially in the regions of the lips, nostrils, oral mucosa, and teats. Like other members of the Poxviridae family, parapoxvirus are relatively large and enveloped double-stranded DNA viruses with ovoid geometries that can infect vertebrates including a wide selection of mammals and humans.
  • Parapoxviruses have a unique spiral coat that distinguishes them from other poxviruses.
  • viral replication of parapox is cytoplasmic. Entry into the host cell is achieved by attachment of the viral proteins to host glycosaminoglycans (GAGs) that mediates endocytosis of the virus into the host cell. Fusion with the plasma membrane permits to release the core into the host cytoplasm.
  • GAGs host glycosaminoglycans
  • Early genes are transcribed in the cytoplasm by viral RNA polymerase. Early expression begins at 30 minutes postinfection. Intermediate phase triggers genomic DNA replication at approximately 100 minutes post- infection. Late genes are then expressed from 140 min to 48 hours post-infection, producing all structural proteins.
  • IMV virion intracellular mature virion
  • EEV enveloped virion
  • the virus exits the host cell by existing in occlusion bodies after cell death and remains infectious until it finds another host. Replication-competent as well as inactivated Parapoxviruses are known for their immunomodulating properties (Schulze et al., 2009, Vet Microbiol. 137: 260-7).
  • Parapoxvirus ovis (ORFV), the prototype species of the parapoxvirus genus, has been used successfully in veterinary medicine for increasing general resistance in animal chronically persistent viral infections (see e.g. US6,365,393; WO97/32029 and US2003/0013076) as well as in human medicine for treating HIV (W02006/005529) and considered as oncolytic (Rintoul et al., 2012, Mol. Ther.20(6): 1148-57).
  • ORFV Parapoxvirus ovis
  • ORFV canine distemper virus
  • W02012/01145 canine distemper virus
  • pseudorabies virus in pigs Rost al., 2010, Vaccine 28(7): 1808-13.
  • Zylexis® formerly known as Baypamune®, which is a preparation of chemically inactivated ORFV derived from strain D1701 is used for the prophylaxis and therapeutic treatment of infectious diseases and for preventing stress- induced diseases in animals.
  • Inactivated ORFV was shown to induce plasmacytoid dendritic cells (pDC) probably through the engagement of a TLR-9 dependent pathway (Von Buttlar et al., 2014, PLOS One 9(8): el06188). More recently, it has been reported potent cytotoxic activities of a chimeric parapoxvirus (referred as CF189) as an oncolytic immunotherapy in triple negative breast cancer (TNBC) tumors (Choi et al. Surgery. 2018 Feb;163(2):336-342). Active replication of the chimeric ORF virus was detected in the tumor tissues 1 week after its injection and natural killer (NK) cell infiltration was observed in the periphery of virus treated tumor tissues.
  • NK natural killer
  • ORF virus has an effective monotherapy in a murine model of advanced-stage epithelial ovarian cancer, and support the translational potential of ORFV as a NK stimulating immunotherapeutic for treatment of such cancer (Van Vloten et al. J Immunother Cancer 2022;10:e004335). It was also shown that ORFV infection enhances CXCL16 secretion and causes oncolysis of lung cancer cells through immunogenic apoptosis (Wang et al. Front Cell Infect Microbiol. 2022 Jul 25;12:910466).
  • PCPV Pseudocowpox virus
  • W02019/170820 discloses a Pseudocowpox virus (PCPV) encoding a heterologous nucleic acid inserted in its genome and its therapeutic use for preventing or treating proliferative diseases like cancers (Ramos et al. Clin Transl Immunology. 2022 May 8;ll(5):el392).
  • PCPV-infected peripheral blood mononuclear cells induced the secretion of very high levels of IFN-alpha in a MOI-dependent way (MOI: Multiplicity of infection).
  • MOI Multiplicity of infection
  • the levels secreted by the PCPV- infected PBMC are well above the moderate secretion levels of IFN-alpha observed with Swine pox virus (SWPV) and Parapoxvirus ovis (ORFV).
  • SWPV Swine pox virus
  • ORFV Parapoxvirus ovis
  • PCPV induced a 1000-fold higher expression of IFN-alpha in human PBMCs whereas SWPV and ORFV displayed a lower 10 to 100-fold induction.
  • Copenhagen Vaccinia Virus and other oncolytic vectors did not raise the IFN alpha level.
  • Parapoxvirus ovis (ORFV) and Yaba-like disease virus (YLDV) were particularly toxic on infected PBMCs: 90% of cells infected at the MOI of 5 died within 16 hours, while at least 50% of living cells were observed for all other viruses, including PCPV.
  • PCPV was also shown to be superior to MVA and Vaccinia Virus to trigger the expression of CD86 in primary human monocyte-derived dendritic cells (moDCs).
  • PCPV-E7 As a recombinant vector, assessment of immunogenicity was shown in a model of a recombinant PCPV encoding for the non-oncogenic form of the E7 protein (deleted for amino acids 21-26) of human papillomavirus type 16 (HPV16), thereafter referred as PCPV-E7.
  • PCPV-E7 vaccinated mice showed a significant increase in the number of E7-specific T cells.
  • a significantly higher percentage of antigen-specific CD8+ T cells was observed in the PCPV-E7 group compared to MVA-E7.
  • PCPV-E7 as a therapeutic vaccine candidate was also challenged in a translational setting in HPV16+ infected HNSCC (head and neck squamous cell carcinoma) cancer patients, with MVA-E7 vaccine as control.
  • HNSCC head and neck squamous cell carcinoma
  • MVA-E7 vaccine as control.
  • TDLN tumor-draining lymph nodes
  • IFN-a is able to induce rapid differentiation of monocytes into highly activated dendritic cells called IFN-DCs, which are particularly effective in inducing B and T cell immunity (Rizza et al. Autoimmunity. 2010 Apr;43(3):204-9).
  • the Inventors designed a novel recombinant pseudocowpox virus (PCPV) comprising inserted in its genome at least one heterologous gene encoding for an interleukin 12 (IL-12), and methods of generating and using such recombinant PCPV.
  • PCPV pseudocowpox virus
  • PCPV recombinant PCPV induced both an upregulation of NK cells (natural killer cells) and an upregulation of activated CD8 T cells (CD8+ T cells) when injected to a patient suffering from a cancer, as it will be further described in the present disclosure.
  • the Inventors designed a novel recombinant PCPV comprising inserted in its genome: at least one heterologous gene encoding for an interleukin 12 (IL-12); and at least one heterologous gene encoding for an antigen, preferably a tumor antigen, or a viral antigen.
  • IL-12 interleukin 12
  • an antigen preferably a tumor antigen, or a viral antigen.
  • the Inventors have identified that treatment of a tumor with a recombinant PCPV encoding for an IL-12 and a tumor antigen increased survival of the patient and reduced the tumor growth, and an upregulation of antigen-specific CD8+ T cells, compared to a treatment of a tumor with a recombinant PCPV encoding for the same tumor antigen only (to be understood: without encoding for an IL-12).
  • PCPVs described herein are expected to stimulate or improve immune response, especially in cancer immunotherapy.
  • the present invention thus relates to a recombinant pseudocowpox virus (PCPV) comprising at least one heterologous nucleic acid inserted in its genome; wherein one of the at least one heterologous nucleic acid inserted in its genome is a nucleic acid encoding for an interleukin 12 (IL-12).
  • PCPV pseudocowpox virus
  • said PCPV is obtained from the wild-type TJS strain as identified by ATCC reference number ATCC VR-634TM or from a virus strain of the same or similar name or functional fragments and variants thereof.
  • said PCPV is further defective for a viral function encoded by the PCPV genome and preferably is defective for a non-essential viral function and, more preferably for a viral gene function encoded at the insertion site of said heterologous nucleic acid.
  • said heterologous nucleic acid encodes a polypeptide selected from the group consisting of a suicide gene product; an immunomodulatory polypeptide; an antigenic polypeptide; an antibody; a functional derivative of an antibody; a functional fragment of an antibody; and any combination thereof.
  • the immunomodulatory polypeptide is selected from the group consisting of cytokines, such as interleukins, chemokines, interferons, tumor necrosis factor, colony-stimulating factors, polypeptides having an anti-angiogenic effect and agonists or antagonists of immune checkpoints, an any combination thereof.
  • cytokines such as interleukins, chemokines, interferons, tumor necrosis factor, colony-stimulating factors, polypeptides having an anti-angiogenic effect and agonists or antagonists of immune checkpoints, an any combination thereof.
  • an immunomodulatory polypeptide that is an interleukin or a colony-stimulating factor and, in particular GM-CSF or is an agonist OX40-directed antibody; or the immunomodulatory polypeptide is a tumor necrosis factor, in particular a CD40L, an IL-4, an IL-21 or a BAFF.
  • the antigenic polypeptide is a tumor antigen, selected from the group consisting of tumor-associated antigens (TAA), tumor-specific antigens (TSA) and oncoviral antigens.
  • TAA tumor-associated antigens
  • TSA tumor-specific antigens
  • oncoviral antigens said tumor antigen is a class I or class II epitope derived from a tumor antigen, or a combination of class I epitopes, or a combination of class II epitopes, or a combination of class I and class II epitopes.
  • said antigenic polypeptide is a full-length antigen.
  • the TAA is selected from the group consisting of WT1, MAGE-A3 MUC1, HER2/Neu and NY-ESO-1; or a tissue specific TAA, preferably a tissue specific TAA selected among Mesothelin, PSA, and gplOO.
  • the TSA is selected from the group consisting of viral oncogenes; preferably a viral oncogene inducing virus selected among EBV antigens, HPV antigens; or a mutated self TSA, preferably a mutated self TSA selected among EGFRvlll, KRAS, p53 and BRAF; or neo-epitopes resulting from non-synonymous somatic mutations.
  • the oncoviral antigens is selected among viral oncogene inducing virus selected among EBV antigens, HPV antigens.
  • the at least one heterologous nucleic acid is operably linked to suitable regulatory elements for expression in a desired host cell or subject.
  • the at least one heterologous nucleic acid is placed under the control of a poxvirus promoter, preferably, a vaccinia virus promoter and more preferably one selected from the group consisting of the p7.5K, pH5R, pllK7.5, pSE, pTK, pB2R, p28, pll, pF17R, pA14L, pSE/L, pA35R, pCIR and pKIL promoter, synthetic promoters and early/late chimeric promoters.
  • a poxvirus promoter preferably, a vaccinia virus promoter and more preferably one selected from the group consisting of the p7.5K, pH5R, pllK7.5, pSE, pTK, pB2R, p28, pll, pF17R, pA14L, pSE/L, pA35R, pCIR and pKIL promoter, synthetic promoters and early/late chimeric promoter
  • the at least one heterologous nucleic acid is inserted in the VEGF locus.
  • the at least one heterologous nucleic acid inserted in its genome is a nucleic acid encoding for an interleukin 12 (IL-12), wherein the IL-12 is human IL-12.
  • IL-12 interleukin 12
  • the IL-12 is a fusion protein comprising an IL-12 p40 subunit and an IL-12 p35 subunit.
  • the IL-12 p40 subunit is N-terminal to the IL-12 p35 subunit.
  • the IL-12 p40 subunit comprises the amino acid sequence of SEQ ID NO:
  • amino acid sequence 1 or an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the amino acid sequence of SEQ. ID NO: 1.
  • the IL-12 p35 subunit comprises the amino acid sequence of SEQ ID NO:
  • the IL-12 p40 subunit and the IL-12 p35 subunit are fused in a single polypeptide via an amino acid linker.
  • said amino acid linker is about 5 to about 10 amino acids in length.
  • said amino acid linker is 7 amino acids in length.
  • the amino acid linker is a glycine-serine linker.
  • the amino acid linker comprises the amino acid sequence of SEQ ID NO: 3.
  • the IL-12 comprises the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the amino acid sequence of SEQ ID NO: 4.
  • the IL-12 p40 subunit and the IL-12 p35 subunit are directly fused in a single polypeptide.
  • the nucleic acid sequence encoding the IL-12 comprises a nucleotide sequence at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to the nucleotide sequence of SEQ ID NO: 5.
  • the nucleic acid sequence encoding the IL-12 comprises the nucleotide sequence of SEQ ID NO: 5.
  • the nucleic acid sequence encoding the IL-12 is inserted within the VEGF locus.
  • the nucleic acid sequence encoding the IL-12 is inserted within the VEGF locus; and said PCPV further comprises a heterologous nucleic acid encoding for an antigenic polypeptide which is also inserted within the VEGF locus.
  • the expression cassette comprising the nucleic acid sequence encoding the IL-12 and the expression cassette comprising the nucleic acid encoding the antigenic polypeptide are in a back-to-back orientation in relation to each other within the VEGF locus.
  • the nucleic acid sequence encoding the IL-12 is placed under control of a pSE/L promoter, and the nucleic acid encoding the antigenic polypeptide is placed under control of a p7.5K promoter.
  • the present invention further provides a method for generating the PCPV of the invention, by homologous recombination between a transfer plasmid comprising the heterologous nucleic acid flanked in 5' and 3' with PCPV sequences respectively present upstream and downstream the insertion site and a PCPV genome, wherein said method comprises a step of generating said transfer plasmid and a step of introducing said transfer plasmid into a suitable host cell, notably together with a PCPV virus comprising the flanking sequence present in the transfer plasmid.
  • the site of insertion of the at least one heterologous nucleic acid in the PCPV genome is in a viral gene, with a preference for a non-essential viral gene, in an intergenic region, in a portion of the PCPV genome which does not encode gene products or in a duplicated locus.
  • the method for generating the PCPV comprises a step wherein upon insertion of the heterologous nucleic acid in the PCPV genome the viral locus at the insertion site is deleted at least partially, resulting in a defective PCPV virus for said virus function.
  • said at least one heterologous nucleic acid is inserted in the VEGF locus.
  • the transfer plasmid further comprises one or more selection and/or detectable gene to facilitate identification of the recombinant PCPV.
  • the transfer plasmid is introduced into the host cell in the presence of an endonuclease capable of providing a double-stranded break in said selection or detectable gene.
  • the selection gene used in the method for generating the PCPV is the GPT gene encoding a guanine phosphoribosyl transferase permitting growth in a selective medium and/or said detectable gene encodes GFP, e-GFP or mCherry.
  • the suitable host cell is selected from Bovine Turbinate (BT) cell or HeLa cell.
  • the present invention further provides a method for amplifying the PCPV according to the invention, or generated by the method according to the invention, comprising the steps of a) preparing a producer cell line, b) transfecting or infecting the prepared producer cell line, c) culturing the transfected or infected producer cell line under suitable conditions so as to allow the production of the virus, d) recovering the produced virus from the culture of said producer cell line and optionally e) purifying said recovered virus.
  • the producer cell is Bovine Turbinate (BT) or HeLa.
  • the present invention further provides a composition comprising a therapeutically effective amount of the PCPV according to the present disclosure, or amplified by the method according to the present disclosure, and a pharmaceutically acceptable vehicle.
  • the composition is thus preferably a pharmaceutical composition.
  • said composition is formulated in individual doses comprising from approximately 10 3 to approximately 10 12 pfu, advantageously from approximately 10 4 pfu to approximately 10 11 pfu, preferably from approximately 10 5 pfu to approximately IO 10 pfu; and more preferably from approximately 10 s pfu to approximately 10 9 pfu of PCPV.
  • composition is formulated for intravenous, intramuscular, intradermal, intranasal, subcutaneous or intratumoral administration.
  • said composition is for use for treating or preventing diseases or pathological condition caused by a pathogenic organism or an unwanted cell division, or for inhibiting tumor cell growth.
  • composition according to the invention for the manufacture of a drug for treating or preventing diseases or pathological condition caused by a pathogenic organism or an unwanted cell division, or for inhibiting tumor cell growth.
  • composition according to the invention for treating or preventing diseases or pathological condition caused by a pathogenic organism or an unwanted cell division, or for inhibiting tumor cell growth.
  • composition according to the invention comprising administering the composition according to the invention to a subject in need thereof in an amount sufficient to treat or prevent a disease or a pathological condition caused by a pathogenic organism or an unwanted cell division. It is also disclosed a method for inhibiting tumor cell growth comprising administering the composition of the present invention to a subject in need thereof.
  • composition for use, the use or the method according to the present disclosure comprising 2 to 6 weekly administrations possibly followed by 2 to 15 administrations at 3 weeks interval of the PCPV composition comprising 10 s to 10 9 pfu.
  • composition for use, the use or the method according to the present invention wherein said method or use is for treating a cancer selected from the group consisting of renal cancer, prostate cancer, breast cancer, bladder cancer, colorectal cancer, lung cancer, liver cancer, gastric cancer, bile duct carcinoma, endometrial cancer, pancreatic cancer, ovarian cancer, head and neck cancer, melanoma, glioblastoma, multiple myeloma, or malignant glioma cells.
  • a cancer selected from the group consisting of renal cancer, prostate cancer, breast cancer, bladder cancer, colorectal cancer, lung cancer, liver cancer, gastric cancer, bile duct carcinoma, endometrial cancer, pancreatic cancer, ovarian cancer, head and neck cancer, melanoma, glioblastoma, multiple myeloma, or malignant glioma cells.
  • composition for use, the use or the method according to the present invention which is used in conjunction with one or more other therapeutic agents selected from the group consisting of surgery, radiotherapy, chemotherapy, cryotherapy, hormonal therapy, toxin therapy, immunotherapy, cytokine therapy, targeted cancer therapy, gene therapy, photodynamic therapy and transplantation.
  • composition for use, the use or the method according to the present invention which is carried out according to a prime boost approach which comprises sequential administrations of a priming composition(s) and a boosting composition(s).
  • composition for use, the use or the method according to the invention wherein the priming composition is a PCPV composition, and the boosting composition is a MVA composition or a PCPV composition.
  • composition for use, the use or the method according to the present disclosure whereas the priming composition is a PCPV composition administered by intratumoral route and the boosting composition is a MVA composition administered by intravenous route.
  • composition according to the invention is for use for eliciting or stimulating and/or re-orienting an immune response in a subject in need thereof.
  • composition according to the invention for the manufacture of a drug for eliciting or stimulating and/or re-orienting an immune response in a subject in need thereof.
  • composition according to the invention for eliciting or stimulating and/or re-orienting an immune response in a subject in need thereof.
  • composition according to the present invention preferably results in at least one the following properties:
  • FIG. 1 IFN-a and IL12-p70 secretion by PBMCs from cancer patients. IFN-a secretion in PBMC cultures from 10 cancer patients. The patients, between 31 and 78 years of age, had different indications (breast, lung, bladder, and head and neck cancer) and were untreated when blood was drawn. PBMCs were infected at MOI 0.3 with PCPV-GFP, MVA-GFP or VACV-GFP. IFN-a and IL12p70 secretion was measured in the cell culture supernatant the next day. Shown is the mean IFN-a and IL12p70 secretion in 10 individuals +/- SEM.
  • FIG. 1 Schematic representation of recombinant PCVPs according to the invention.
  • FIG. 1 A) Western Blot analysis: detection of epitope expression with anti-Flag HRP (horse radish peroxidase) antibody.
  • PCPTG19873 shows around 5-fold higher gene expression than PCPTG19874
  • FIG. 4 Murine splenocytes were infected at MOI 0.3 and 1 with PCPV-mlL12 (filled triangle; code number PCPTG19818) or PCPV-ctr (filled quadrangle) or remained uninfected (empty circle). Next day, supernatant was harvested, and IFN-gamma determined by Luminex analysis. Splenocytes were stained with antibodies and Live Dead before being analyzed on a MacsQuant cytometer. Gating strategies using the Kaluza 2.1.1 software (Beckman Coulter): SSC vs FSC. Dot plot (LiveDead vs SSC) to determine live cells.
  • Dot plots of live cells were created to identify the CD3+CD8+, CD3+CD4+ or the NK cells CD49b+CD3-. From each of these populations, dot plots CD69 vs PDL1 were created to phenotype the activation state of these lymphocytes. Double positive populations were shown. A representative experiment is shown.
  • PBMCs were infected at MOI 0.3 with PCPV-mlL12 (code number PCPTG19818) or a control PCPV (PCPV-ctr). Next day, supernatant was harvested, and IFN-gamma determined by Luminex analysis. Cells were stained with antibodies and Live Dead before being analyzed on a MacsQuant cytometer. Gating strategies using the Kaluza 2.1.1 software (Beckman Coulter): SSC vs FSC. Dot plot (LiveDead vs SSC) to determine live cells. Dot plots of live cells were created to identify the CD3+CD8+, CD3+CD4+ or the NK cells CD56+CD3-. From each of these populations, dot plots with CD69 vs PDL1 were created to phenotype the activation state of these lymphocytes. Double positive populations were shown. A representative experiment is shown.
  • PCPV-mlL12 (code number PCPTG19818) controls tumor growth better than PCPV-ctr and increases survival frequencies:
  • FIG. 7 Nine days after MC38 tumor cell grafting, 5xl0 5 pfu of PCPV-ctr, PCPV-mlL12 or the empty MVA vector MVA-ctr were injected into the growing tumors (5 mice per group). The day after, mice were sacrificed, axillary and inguinal draining lymph nodes (DLN) were harvested and analyzed by flow cytometry. Shown are the mean frequencies of activated NK cells, and activated CD8+ and CD4+ T cells in 5 individuals +/- SEM.
  • FIG. 8 Naive C57BL/6 mice were repeatedly (days 0, 7 and 14) vaccinated (sc) with lxlO 7 pfu of PCPV-ctr, PCPTG19873or PCPTG19874. Blood was taken at days 7, 14 and 45 (10 mice per group). SlINFEKL-specific T cells were identified by flow cytometry using SlINFEKL-pentamer staining. The day after, mice were sacrificed, axillary and inguinal draining lymph nodes (DLN) were harvested and analyzed by flow cytometry. Shown are the mean frequencies of SlINFEKL-specific CD8+ T cells in 10 individuals +/- SEM. Figure 9.
  • FIG. 10 Detection of murine IL12 in extracellular vesicle (EV) fraction isolated from infected PBMCs: Suspensions of PCPV-mlL12, incubated o/n with or without PBMCs at MOI 1, were cleared by centrifugation, concentrated, filtered, and ultracentrifuged. mlL12 was quantified after concentration and in EV fraction by Luminex analysis.
  • EV extracellular vesicle
  • SP is a signal peptide from the rabies glycoprotein: amino acid 1-23 (P32550.1)
  • Msln is an epitope from Mus musculus Mesothelin: amino acid 396-424 (NP_001343215)
  • SIINFEKL is a reference for an amino acid sequence known as a pure CD8 epitope
  • ISQAVHAAHAEINEAGR is a reference for an amino acid sequence known as a pure a pure CD4 epitope
  • CB100 is an epitope from Pyrophorus plagiophtalamus luciferase: amino acid 91-119 (Uniprot S29353)
  • CB441 is an epitope from Pyrophorus plagiophtalamus luciferase: amino acid 430-458 (Uniprot S29353)
  • Flag is a FLAG tag (Flag HRP (horse radish peroxidase))
  • DMSO Dimethyl sulfoxide
  • KNGENAQAI referenced as CTRL on Figure 12
  • Figure 14 Figure 14 ( Figure 14A and Figure 14B). Comparison of tumor growth PCPV-OVA-IL12 (PCPTG19874), PCPV-OVA (PCPTG19873), MVA-OVA-IL-12 (MVATG20014) and MVA-OVA (MVATG20022) versus their respective control vectors PCPV ctr and MVA ctr in E.G7-OVA tumor model.
  • Figure 14A Measured tumor volume in mm 3
  • Figure 14B Graphical representation of an estimated tumor diameter expressed in mm 2 based on mixed model built with measured tumor volume according to Figure 15a and accorded to the method described in Bastogne et al, 2010.
  • a recombinant pseudocowpox virus comprising at least one heterologous nucleic acid inserted in its genome, wherein one of the at least one heterologous nucleic acid inserted in its genome is a nucleic acid encoding for an interleukin 12 (IL-12), induced an improve immune response.
  • PCPV pseudocowpox virus
  • IL-12 interleukin 12
  • the vectorization of IL-12 into PCPV is therefore a solution to such problem of a vaccination approach for minimizing toxicities and providing antitumor efficacy.
  • the recombinant PCPV according to the invention showed interesting capacity to produce secreted IL-12 within extracellular vesicles (EVs). It is known in the art that exosome therapeutic that displays functional IL-12 exhibit prolonged tumor retention and greater antitumor activity than a recombinant IL-12 (Lewis et al. Mol Cancer Ther. 2021 Mar;20(3):523-534). Such prolonged retention could increase the effects of IL-12 on tumor-resident NK and T cells, leading to their activation or reinvigoration.
  • a and “an” are used in the sense that they mean “at least one”, “at least a first”, “one or more” or “one or a plurality” of the referenced compounds or steps, unless the context dictates otherwise.
  • a cell includes a plurality of cells, including mixtures thereof.
  • one or more refers to either one or a number above one (e.g. 2, 3, 4, etc.).
  • amino acids amino acids
  • amino acid analogs e.g. non-natural, synthetic and modified amino acids, including D or L optical isomers.
  • a polypeptide “comprises” an amino acid sequence when the amino acid sequence might be part of the final amino acid sequence of the polypeptide.
  • Such a polypeptide can have up to several hundred additional amino acids residues (e.g.
  • polypeptide refers to polymers of amino acid residues comprising at least nine amino acids covalently linked by peptide bonds.
  • the polymer can be linear, branched or cyclic and may comprise naturally occurring and/or amino acid analogs and it may be interrupted by non-amino acids. No limitation is placed on the maximum number of amino acids comprised in a polypeptide. As a general indication, the term refers to both short polymers (typically designated in the art as peptide) and to longer polymers (typically designated in the art as polypeptide or protein).
  • This term encompasses native polypeptides, modified polypeptides (also designated derivatives, analogs, variants or mutants), polypeptide fragments, polypeptide multimers (e.g. dimers), recombinant polypeptides, fusion polypeptides among others.
  • nucleic acid refers to any polymer of at least 9 nucleotide residues in either deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) or mixed polyribo-polydeoxyribonucleotides.
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • mixed polyribo-polydeoxyribonucleotides encompass single or double-stranded, linear or circular, natural or synthetic, unmodified or modified versions thereof (e.g. genetically modified polynucleotides; optimized polynucleotides), sense or antisense polynucleotides, chimeric mixture (e.g.
  • RNA-DNA hybrids include without limitation, complementary DNA (cDNA), genomic DNA, plasmid DNA, vectors, viral DNA (e.g. viral genomes, viral vectors), oligonucleotides, probes, primers, coding DNA, non-coding DNA, or any fragment thereof etc.
  • exemplary RNA nucleic acids include, without limitation, messenger RNA (mRNA), precursor messenger RNA (pre-mRNA), coding RNA, non-coding RNA, etc.
  • Nucleic acid sequences described herein may be synthesized by standard methods known in the art, e.g., by use of an automated DNA synthesizer (such as those that are commercially available from Biosearch, Applied Biosystems, etc.) or obtained from a naturally occurring source (e.g. a genome, cDNA, etc.) or an artificial source (such as a commercially available library, a plasmid, etc.) using molecular biology techniques well known in the art (e.g. cloning, PCR, etc).
  • an automated DNA synthesizer such as those that are commercially available from Biosearch, Applied Biosystems, etc.
  • a naturally occurring source e.g. a genome, cDNA, etc.
  • an artificial source such as a commercially available library, a plasmid, etc.
  • the percent identities referred to in the context of the disclosure of the present invention are determined after optimal global alignment of the sequences to be compared, which optimal global alignment may therefore comprise one or more insertions, deletions, truncations and/or substitutions.
  • the alignment is global, meaning that it includes the sequences to be compared taken in their entirety over their entire length.
  • the alignment is "optimal", meaning that the number of insertions, deletions, truncations and/or substitutions is made as low as possible.
  • the optimal global alignment may be performed and the percent identity calculated using any sequence analysis method well-known to the person skilled in the art. In addition to manual comparison, it is possible to determine global alignment using the algorithm of Needleman and Wunsch (1970).
  • the sequence comparison may be performed using any software well-known to a person skilled in the art, such as the Needle software.
  • the parameters used may notably be the following: “Gap open” equal to 10.0, “Gap extend” equal to 0.5, and the EDNAFULL matrix (NCBI EMBOSS Version NUC4.4).
  • the sequence comparison may be performed using any software well-known to a person skilled in the art, such as the Needle software.
  • the parameters used may notably be the following: “Gap open” equal to 10.0, “Gap extend” equal to 0.5, and the BLOSUM62 matrix.
  • variants can be used interchangeably to refer to a component (polypeptide, nucleic acid, virus, etc) exhibiting one or more modification(s) with respect to the native counterpart. Any modification(s) can be envisaged, including substitution, insertion and/or deletion of one or more nucleotide/amino acid residue(s).
  • variants that retain a degree of sequence identity of at least 75%, advantageously at least 80%, desirably at least 85%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 98% identity after optimal global alignment with the sequence of the native counterpart, i.e.
  • identity means 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.
  • identity refers to an amino acid to amino acid or nucleotide to nucleotide correspondence between two polypeptides or nucleic acid sequences.
  • the percentage of identity between two sequences is a function of the number of identical positions shared by the sequences, considering the number of gaps which need to be introduced for optimal alignment and the length of each gap.
  • Various computer programs and mathematical algorithms are available in the art to determine the percentage of identity between amino acid sequences, such as for example the Blast program available at NCBI or ALIGN in Atlas of Protein Sequence and Structure (Dayhoffed, 1981, Suppl., 3: 482-9).
  • Emboss Needle software available at https://www.ebi.ac.uk/Tools/psa/emboss_needle/.
  • This software reads two input sequences and writes their optimal global sequence alignment to file. It uses the Needleman-Wunsch alignment algorithm to find the optimum alignment (including gaps) of two sequences along their entire length. The algorithm uses a dynamic programming method to ensure the alignment is optimum, by exploring all possible alignments and choosing the best. A scoring matrix is read that contains values for every possible residue or nucleotide match.
  • Needle finds the alignment with the maximum possible score where the score of an alignment is equal to the sum of the matches taken from the scoring matrix, minus penalties arising from opening and extending gaps in the aligned sequences.
  • the substitution matrix and gap opening and extension penalties are user-specified.
  • the Emboss Needle software may be used with default parameters, i.e. :
  • originating or “originate” is used to identify the original source of a component (e.g. polypeptide, nucleic acid molecule) but is not meant to limit the method by which the component is made which can be, for example, by chemical synthesis or recombinant means.
  • a component e.g. polypeptide, nucleic acid molecule
  • the term "host cell” should be understood broadly without any limitation concerning particular organization in tissue, organ, or isolated cells. Such cells may be of a unique type of cells or a group of different types of cells such as cultured cell lines, primary cells and dividing cells. This term also includes cells that can be or has been the recipient of the non-propagative viral vector for use in the invention, as well as progeny of such cells.
  • subject generally refers to a vertebrate organism for whom any of the product or methods disclosed herein is needed or may be beneficial.
  • the organism is a mammal, particularly a mammal selected from the group consisting of domestic animals, farm animals, sport animals, and primates (human and non-human).
  • subject and patient may be used interchangeably when referring to a human organism and covers male and female as well as a fetuses, newborn, infant, young adult, adult and elderly.
  • tumor may be used interchangeably with any of the terms “cancer”, “malignancy”, “neoplasm” and encompasses any disease or pathological condition resulting from uncontrolled cell growth and spread. These terms are meant to include any type of tissue, organ or cell, any stage of malignancy (e.g. from a prelesion to stage IV). Typically, tumors, especially malignant tumors, show partial or complete lack of structural organization and functional coordination as compared to normal tissue and generally show a propensity to invade surrounding tissues (spreading) and/or metastasize to farther sites.
  • the present invention is preferably designed for the treatment of solid tumors as described herein.
  • a “neoplastic cell”, “cancer cell” or “tumor cell” can be used interchangeably to refer to a cell that divides at an abnormal (i.e. increased) rate.
  • treatment refers to therapy.
  • therapy refers to a pathological condition with the purpose to improve at least one clinical or biochemical symptom (size of tumor, expression level of associated biomarker%), to slow down or control the progression of the targeted pathological condition, symptom(s) thereof, or a state secondary to the pathological condition in the subject treated in accordance with the present invention.
  • prevention and any form of the term such as “preventing”, “prevent”, etc.,) and “prophylaxis” are used interchangeably and refer to preventing, delaying the onset or decreasing the severity of the first occurrence or relapse of at least one clinical or biochemical symptom (size of tumor, expression level of associated biomarker, stage progression).
  • administering refers to the delivery to a subject of a component (e.g. the fusion polypeptide according to the invention) according to the modalities described herein.
  • fusion polypeptide refers to any arrangement possible of various components (e.g. the fusion polypeptide according to the invention and another treatment). Such an arrangement includes mixture of said components as well as separate combinations for concomitant or sequential administrations.
  • the present invention encompasses combinations comprising equal molar concentrations of each component as well as combinations with very different concentrations. It is appreciated that optimal concentration of each component of the combination can be determined by the artisan skilled in the art.
  • virus refers to viral particles that are formed when the nucleic acid vector is transduced into an appropriate cell or cell line according to suitable conditions allowing the generation of viral particles.
  • viral vector has to be understood broadly as including nucleic acid vector (e.g. DNA viral vector) as well as viral particles generated thereof.
  • infectious refers to the ability of a viral vector to infect and enter into a host cell or subject.
  • Viral vectors can be replication-competent or -selective (e.g. engineered to replicate better or selectively in specific host cells), or can be genetically disabled so as to be replicationdefective or replication-impaired.
  • viral vector refers to a nucleic acid vector that includes at least one element of a virus genome and may be packaged into a viral particle or to a viral particle.
  • Naturally occurring is used to describe a biological molecule or organism that can be found in nature as distinct from being artificially produced by man.
  • a naturally occurring, native or wild-type virus refers to a virus which can be isolated from a source in nature or obtained from specific collections (e.g. ECCAC, ATCC, CNCM, etc).
  • a biological molecule or an organism which has been intentionally modified by man in the laboratory is not naturally occurring.
  • Representative examples of non- naturally occurring viruses include, among many others, recombinant viruses engineered by insertion of one or more nucleic acid(s) of interest in the viral genome and/or defective virus resulting from one or more modification(s) in the viral genome (e.g. total or partial deletion of a viral gene).
  • recombinant indicates that the virus has been modified by the introduction of at least one foreign nucleic acid (also called recombinant gene or nucleic acid, or heterologous gene or heterologous nucleic acid), notably a nucleic acid of therapeutic interest as described herein.
  • the "heterologous nucleic acid” that is inserted in the PCPV genome is not found in or expressed by a naturally occurring PCPV genome. Nevertheless, the heterologous nucleic acid can be homologous or heterologous to the subject into which the recombinant PCPV is introduced. More specifically, it can be of human origin or not (e.g.
  • said heterologous nucleic acid encodes a polypeptide or is a nucleic acid sequence capable of binding at least partially (by hybridization) to a complementary cellular nucleic acid (e.g., DNA, RNA, miRNA) present in a diseased cell with the aim of inhibiting a gene involved in said disease.
  • a polypeptide is understood to be any translational product of a polynucleotide regardless of size, and whether glycosylated or not, and includes peptides and proteins.
  • Such a foreign nucleic acid may be a native gene or portion(s) thereof (e.g.
  • the heterologous nucleic acid encodes a polypeptide which is capable of providing a therapeutic or prophylactic activity when administered appropriately to a subject (i.e. a polypeptide of therapeutic interest), leading to a beneficial effect on the course or a symptom of the pathological condition to be treated.
  • a polypeptide of therapeutic interest i.e. a polypeptide of therapeutic interest
  • the heterologous nucleic acid encodes a polypeptide selected from the group consisting of polypeptides that compensate for defective or deficient proteins in a subject, polypeptides that act through toxic effects to limit or remove diseased cells from the body (e.g.
  • suicide gene products polypeptides capable of potentiating anti-tumor efficacy (e.g. armed gene products); and polypeptides capable of inducing or activating an immune response (such as immunomodulatory and antigenic polypeptides).
  • a heterologous nucleic acid encoding a detectable gene product may also be useful in the context of the invention.
  • immunomodulatory polypeptide is used to describe a polypeptide capable of modifying or regulating one or more immune functions (up or downregulation).
  • tumor antigen or "tumoral antigen”, or “tumoral specific antigen” is used to describe a protein of peptide that can be recognized by cellular or humoral components of the immune system to target tumor tissue.
  • Tumor is to be understood as abnormal neoplastic tissular formations that might have or not a malignant behaviour.
  • a malignant behaviour is usual associated with spreading and invasion of the nearby tissue and throughout the body. Malignancy is associated with cancer and many non-malignant tumors have the potential to evolve toward a malignant tumor.
  • the antigen can be a full-length antigen, or an epitope derived from an antigen or an association of epitopes, an epitope being defined as the shortest sequence from an antigen necessary and sufficient to induce a class I or a class II response.
  • TAA tumor-associated antigens
  • TSA tumor-specific antigens
  • antibody is used in the broadest sense and encompasses naturally occurring antibodies and engineered antibodies; including synthetic, monoclonal, polyclonal antibodies as well as full length antibodies and fragments, variants or fusions thereof provided that such fragments, variants or fusions retain binding properties to the target protein.
  • Such antibodies can be of any origin; human or non-human mammal (e.g. rodent or camelid antibody), or chimeric.
  • a nonhuman antibody can be humanized by recombinant methods to reduce its immunogenicity in human.
  • the antibody may derive from any of the well-known isotypes (e.g.
  • antibody also includes an antigen-binding fragment of any of the aforementioned antibodies and includes a monovalent and a divalent fragment and single chain antibodies.
  • the term antibody also includes multi-specific (e.g. bispecific) antibody so long as it exhibits the same binding specificity as the parental antibody. It is within the skill of the artisan to screen for the binding properties of a candidate antibody.
  • full length antibodies are glycoproteins comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds.
  • Each heavy chain comprises a heavy chain variable region (VH) and a heavy chain constant region which is made of three CHI, CH2 and CH3 domains (optionally with a hinge between CHI and CH2).
  • Each light chain comprises a light chain variable region (VL) and a light chain constant region which comprises one CL domain.
  • the VH and VL regions comprise three hypervariable regions, named complementarity determining regions (CDR), interspersed with four conserved regions named framework regions (FR) in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • a "humanized antibody” refers to a nonhuman (e.g. murine, camel, rat, etc) antibody whose protein sequence has been modified to increase its similarity to a human antibody (i.e. produced naturally in humans).
  • the process of humanization is well known in the art and typically is carried out by substituting one or more residue of the FR regions to look like human immunoglobulin sequence whereas the vast majority of the residues of the variable regions (especially the CDRs) are not modified and correspond to those of a non-human immunoglobulin.
  • a “chimeric antibody” comprises one or more element(s) of one species and one or more element(s) of another species, for example, a non-human antibody comprising at least a portion of a constant region (Fc) of a human immunoglobulin.
  • the antibody is preferably a monoclonal antibody, preferably humanized or chimeric.
  • antibody fragments and/or regions are known in the art, including heavy (H) chain, light (L), heavy chain variable region (VH), heavy chain constant region, CH domain, light chain variable region (VL), light chain constant region, CL domain, complementarity determining regions (CDR), constant region (Fc), Fab, Fab', F(ab')2, dAb, Fd, Fv, scFv, ds-scFv, diabody, sdAb, etc.
  • antigen-binding fragments include Fab, Fab', F(ab')2, dAb, Fd, Fv, scFv, ds-scFv, and diabody.
  • a particularly useful antibody fragment is a single chain antibody (scFv) comprising the two domains of a Fv fragment, VL and VH, that are fused together, eventually with a linker to make a single protein chain.
  • APC antigen presenting cell
  • APC antigen presenting cell
  • APC-exposed proteins any antigenic protein displayed by an APC on its surface and that may be presented to T-cells in order to trigger an adaptative immune response.
  • PCPV pseudocowpox virus
  • Pseudocowpox virus is a virus of the genus Parapoxvirus that causes pseudocowpox in humans and cattle.
  • Pseudocowpox occurs as a common enzootic infection of cattle in most countries of the world. The infection is most frequent in milking herds, affecting the teats and udder of cows and the muzzles and mouths of nursing calves.
  • the lesions of pseudocowpox are characterized by "ring" or "horseshoe"-shaped scabs, the latter being characteristic of the disease. Infection is transmitted by cross-suckling of calves, improperly disinfected teat clusters of milking machines, and probably by the mechanical transfer of virus by flies.
  • PCPV possesses a linear and double-stranded DNA genome, typically of 130-150 kilobases.
  • the present invention encompasses naturally occurring forms of pseudocowpox virus of any strain as well as variants thereof which may be modified for various purposes including those described herein.
  • PCPV strains for use herein include, without limitation, YG2828 (Genbank accession number LC230119), F07.801 R (Genbank accession number JF773693), F10.3081C (Genbank accession number JF773695), F07.798R (Genbank accession number JF773692), F99.177C (Genbank accession number AY453678), IT1303/05 (Genbank accession number JF800906), F00.120R (Genbank accession number GQ329669; Tikkanen et al., 2004, J. Gen. Virol.
  • TJS also called VR634; Genbank accession number GQ.329670; Friedman- Kien et al., 1963, Science 140: 1335-6; available at ATCC under accession number VR634).
  • Such strains may have morphological, structural and/or genetic differences each other, e.g., in terms of ITR length, number of predicted genes and/or G C rich content (see e.g. Hautaniemi et al., 2010, J. Gen. Virol.91 : 1560-76).
  • the PCPV virus of the present invention is obtained from the wild-type TJS strain as identified by ATCC reference number ATCC VR-634TM or from a virus strain of the same or similar name and functional fragments and variants thereof.
  • a variant maintains at least 75% identity at the nucleotide or amino acid level with at least a segment of 10 kilobase (e.g. a continuous sequence of lOkb) in the wild-type TJS pseudocowpox virus genome.
  • Exemplary modifications that are appropriate in the context of the present invention include without any limitation insertion(s), substitution(s) and/or deletion(s) of one or more nucleotide(s) within the PCPV genome with the goal of modulating (e.g. increase or decrease) expression of one or more viral gene or virus infectivity when compared to the wild-type pseudocowpox virus (e.g. TJS strain) or increasing its therapeutic efficacy (e.g. increase the ability of the PCPV virus to differentially express in diseased cells relative to healthy cells) or insertion of one or more foreign nucleic acid(s) of therapeutic interest or generating a chimeric virus containing PCPV genomic fragment(s) with ones obtained from a different virus origin.
  • modulating e.g. increase or decrease expression of one or more viral gene or virus infectivity when compared to the wild-type pseudocowpox virus (e.g. TJS strain) or increasing its therapeutic efficacy (e.g. increase the ability of the PCPV virus to differential
  • the PCPV of the invention comprises inserted in its genome at least one foreign nucleic acid (e.g. resulting in a recombinant PCPV virus), wherein one of the at least one heterologous nucleic acid inserted in its genome is a nucleic acid encoding for an interleukin 12 (IL-12).
  • IL-12 interleukin 12
  • the PCPV virus may be defective for a viral function encoded by the PCPV genome (e.g. a non-essential viral function), preferably for a viral gene function encoded at the insertion site(s) of the foreign nucleic acid.
  • a viral function encoded by the PCPV genome e.g. a non-essential viral function
  • a viral gene function encoded at the insertion site(s) of the foreign nucleic acid e.g. a non-essential viral function
  • PCPV pseudocowpox virus
  • recombinant indicates that the virus has been modified by the introduction of at least one foreign nucleic acid (also called recombinant gene or nucleic acid), notably a nucleic acid of therapeutic interest as described herein.
  • the "foreign nucleic acid” that is inserted in the PCPV genome is not found in or expressed by a naturally-occurring PCPV genome.
  • the foreign nucleic acid can be homologous or heterologous to the subject into which the recombinant PCPV is introduced. More specifically, it can be of human origin or not (e.g. of bacterial, yeast or viral origin except PCPV).
  • said foreign nucleic acid encodes a polypeptide or is a nucleic acid sequence capable of binding at least partially (by hybridization) to a complementary cellular nucleic acid (e.g., DNA, RNA, miRNA) present in a diseased cell with the aim of inhibiting a gene involved in said disease.
  • a polypeptide is understood to be any translational product of a polynucleotide regardless of size, and whether glycosylated or not, and includes peptides and proteins.
  • Such a foreign nucleic acid may be a native gene or portion(s) thereof (e.g. cDNA), or any variant thereof obtained by mutation, deletion, substitution and/or addition of one or more nucleotides.
  • the foreign nucleic acid encodes a polypeptide which is capable of providing a therapeutic or prophylactic activity when administered appropriately to a subject (i.e. a polypeptide of therapeutic interest), leading to a beneficial effect on the course or a symptom of the pathological condition to be treated.
  • a polypeptide of therapeutic interest may be envisaged.
  • the foreign nucleic acid encodes a polypeptide selected from the group consisting of polypeptides that compensate for defective or deficient proteins in a subject, polypeptides that act through toxic effects to limit or remove diseased cells from the body (e.g. suicide gene products); polypeptides capable of potentiating anti-tumor efficacy (e.g. armed gene products); and polypeptides capable of inducing or activating an immune response (such as immunostimulatory and antigenic polypeptides).
  • a foreign nucleic acid encoding a detectable gene product may also be useful in the context of the invention.
  • PCPV pseudocowpox virus
  • Interleukin-12 has been considered as a potential candidate for anti-cancer therapy and has been introduced into viral vectors such as adenoviral vectors for evaluation.
  • IL-12 is a cytokine with immune-modulating and anti-angiogenic functions.
  • IL-12 acts as a key regulator of cell-mediated immune responses through the induction of T helper 1 differentiation, and it induces cellular immunity by promoting IFN-y production, proliferation, and cytolytic activity of natural killer and T cells.
  • the multi-functionality of IL-12 has led to investigation of this cytokine as an anti-cancer agent (Manetti et al. J Exp Med. 1993 Apr l;177(4):1199-204).
  • the human IL-12 encoded into the recombinant PCPV is a fusion protein as a single chain IL-12 protein expressed from PCPV in which the full length p40 subunit was fused, via a G6S linker, to the p35 subunit truncated of its leader sequence (Lieschke et al. Nat Biotechnol. 1997 Jan;15(l):35-40). It is to be understood that such embodiment has a purpose of being an illustrative nucleotide sequence encoding an IL-12 and not to be considered as a limiting embodiment of the present invention.
  • PCPV pseudocowpox virus
  • the recombinant pseudocowpox virus comprising at least one heterologous nucleic acid inserted in its genome; wherein one of the at least one heterologous nucleic acid inserted in its genome is a nucleic acid encoding for an interleukin 12 (IL-12); and wherein one of the at least one heterologous nucleic acid inserted in its genome is a nucleic acid encoding for an antigenic polypeptide which is a tumor antigen.
  • PCPV pseudocowpox virus
  • Said tumor antigen may be selected from the group consisting of tumor-associated antigens (TAA), tumor-specific antigens (TSA) and oncoviral antigens.
  • TAA tumor-associated antigens
  • TSA tumor-specific antigens
  • Said tumor antigen may be selected from the group consisting of tumor-associated antigens (TAA), tumor-specific antigens (TSA) and oncoviral antigens.
  • PCPTG19873 is a PCPV expressing five epitopes (see Figure 11), among them one sequence known to be a pure CD8 OVA epitope of amino acid sequence [SIINFEKL] and CD4 OVA epitope one sequence known to be a pure CD4 epitope of amino acid sequence [ISQAVHAAHAEINEAGR], Epitopes are separated by GSG linkers, the polyepitopes are preceded by a signal peptide for the endoplasmic reticulum (ER) and terminated by a subsequent FLAG tag (Flag HRP (horse radish peroxidase)).
  • Said polyepitopes encoded into PCPV is of nucleotide sequence as disclosed as SEQ ID NO: 9.
  • the polyepitopes expressed amino acid sequence is disclosed as SEQ. ID NO: 8.
  • the expression cassette is under control of the poxviral promoter p7.5K (SEQ ID NO: 7) and inserted in the VEGF loci.
  • PCPTG19818 is a PCPV expressing a murine IL-12 (mlL-12) of nucleotide sequence disclosed as SEQ ID NO: 11 under the control of the pSE/L promoter (SEQ ID NO: 6) and inserted in the VEGF loci.
  • the mlL-12 amino acid sequence as expressed is disclosed as SEQ ID NO: 10.
  • PCPTG19874 is a PCPV expressing a murine IL-12 (mlL-12) of nucleotide sequence disclosed as SEQ ID NO: 11 under the control of the pSE/L promoter (SEQ ID NO: 6) and inserted in the VEGF loci.
  • PCPTG19874 is also expressing the same five epitopes (polyepitopes) as PCPTG19873.
  • Said polyepitopes encoded into PCPV is of nucleotide sequence as disclosed as SEQ ID NO: 9.
  • the polyepitopes expressed amino acid sequence is disclosed as SEQ ID NO: 8.
  • the mlL-12 expression cassette and the five epitopes (polyepitopes) expression cassette are inserted into the PCPV genome in a back-to-back orientation in relation to each other, each under separate regulatory control (see Figure 2).
  • the expression cassette of the polyepitopes is under control of the poxviral promoter p7.5K (SEQ ID NO: 7).
  • the expression cassette of the mlL-12 is under control of the pSE/L promoter (SEQ ID NO: 6).
  • MVATG20022 is a MVA expressing four epitopes fused to the C-terminal part of the protein coding for the eGFP. Two epitopes were issued from OVA, one sequence known to be pure CD8 OVA epitope of amino acid sequence [SIINFEKL] and one CD4 OVA epitope of amino acid sequence [ISQAVHAAHAEINEAGR] known to be pure CD4 epitope.
  • the two other epitopes are specific for the male antigen H-Y, one is the Dby epitope of amino acid sequence [NAGFNSNRANSSRSS] and the other one is Uty epitope of amino acid sequence [WMHHNMDLI], eGFP and polyepitopes are separated by a (G3S)a linker.
  • Said polyepitopes fusion encoded into MVA is of nucleotide sequence as disclosed as SEQ ID NO: 12.
  • the polyepitopes expressed amino acid sequence is disclosed as SEQ ID NO: 13.
  • the expression cassette is under control of the poxviral promoter pH5R (SEQ ID NO: 14) and inserted in the deletion III locus.
  • MVATG20014 is a MVA expressing a murine IL-12 (mlL-12) of the nucleotide sequence disclosed as SEQ ID NO: 11 under the control of the pllK7.5 promoter (SEQ ID NO: 15) and inserted in the deletion II locus.
  • MVATG20014 is also expressing the same fusion eGFP-polyepitopes as MVATG20022.
  • the expression cassette of the fusion is under the control of the poxviral promoter pH5R (SEQ ID NO: 14) and is inserted in the deletion III locus.
  • MVA control or MVA ctr
  • MVA ctr is a wild type MVA.
  • Said wild type MVA is accessible via culture collections, like ATCC (e.g.: VR-1508TM).
  • PCPV control or PCPV ctr
  • PCPV ctr is a wild type PCPV (wild-type TJS strain as identified by ATCC reference number ATCC VR-634TM).
  • EXAMPLE 1 IL-12 not induced by Poxviruses in human primary cells
  • Cytokine secretion profiles notably of IFN alpha and Interleukin-12 (IL-12), were analysed in the supernatant of Human Peripheral Blood PBMCs cryopreserved from Cancer Donors (purchased from NeoBiotech via CliniSciences), infected with the human Orthopoxviruses VACV, the MVA, or the bovine Parapoxvirus PCPV (MOI of 0.3, incubation period 16 h).
  • Luminex analysis showed that PCPV induced significantly higher secretion of IFN-alpha than MVA, and that VACV had no effect on IFN-alpha secretion.
  • PCPV was considered a novel vector to enhance the therapeutic efficacy of antitumor vaccination (see Ramos et al. 2022).
  • the absence of a clear IL-12 signals in infected PBMCs suggest that poxviral infection block this signalling pathway (compare effect of R848 in PBMCs).
  • PCPTG19818 Schematic representation of the three different recombinant PCPVs (PCPTG19818; PCPTG19873; PCPTG19874) are shown on Figure 2.
  • PCPTG19873 encodes five epitopes, among them one sequence known to be a pure CD8 epitope [SIINFEKL] and one sequence known to be a pure CD4 epitope [ISQAVHAAHAEINEAGR], Epitopes are separated by GSG linkers, the polyepitopes are preceded by a signal peptide for the endoplasmic reticulum (ER) and terminated by a subsequent FLAG tag (Flag HRP (horse radish peroxidase)).
  • the expression cassette is under control of the poxviral promoter p7.5K and inserted in the VEGF loci.
  • PCPTG19874 contains mlL12 and epitope encoding cassettes (polyepitopes) in a back-to-back orientation in relation to each other within the VEGF locus.
  • PCPVs were generated and produced in bovine Bos Taurus Turbinate (BT) cells (ATCC CRL-1390) and purified as described in Ramos et al., 2022 and W02019/170820.
  • HeLa cells were infected in triplicates at MOI 1 with the indicated virus. After 48 hours, supernatants were harvested for Luminex analysis (murine I L12 simplex assay, ThermoFisher), and cell extracts were prepared for Western Blot analysis (Figure 3).
  • PCPV-encoded mlL12 (PCPTG19818)
  • splenocytes were prepared from C57BL/6 mice and infected at MOI 0.3 and 1 in duplicates with a PCPV vaccine encoding for the murine IL-12 (PCPTG19818) or not encoding for IL-12 (PCPV Ctr).
  • PCPTG19818 PCPV vaccine encoding for the murine IL-12
  • PCPV Ctr PCPV Ctr
  • Activated NK, CD4+T and CD8+T cells were detected after infection with both PCPV vectors, which confirms earlier results about PCPV-induced activation of NK and T cells (Ramos et al., 2022).
  • PCPV- mlL12 caused an incremental increase of activated NK cells (1,4-fold), of CD8+ T cell (1,8- fold) and of CD4+ T cells (2,5-fold) compared to PCPV ctr.
  • IFN gamma was only detected in supernatant from splenocytes infected with PCPV-mlL12.
  • PBMCs were isolated from leukocyte concentrates by density gradient centrifugation using Ficoll-Paque PLUS (GE Healthcare) and infected at MOI 0.3 in duplicate, or treated with recombinant mlL12 (PeproTech 210-12) between 5 and 0,5 ng/ml. On the next day, supernatant was harvested, and cells were analyzed by flow cytometry (MACSQuant) to detect activated NK, CD4+T and CD8+ T cells.
  • FCSQuant flow cytometry
  • PCPV has shown efficacy in the murine tumor model MC38: PCPV primed specific T cell response without encoding a tumor antigen (Ramos et al, 2022).
  • PCPV-mlL12 PCPTG19818
  • PCPV-ctr At day 0, the colon carcinoma cell line MC38 (2x10 s cells), were grafted subcutaneously (sc) in the flank of 10 C57BL/6 mice. On day 2, 9 and 16, viruses were injected at the suboptimal dose of 5x10 s pfu into the tumors ( Figure 6).
  • EXAMPLE 7 Activation of immune cells in draining lymph nodes day 1 after sc/itu injection
  • DLN draining lymph nodes
  • DLN from tumor bearing mice were collected and scratched through a cell strainer into culture medium.
  • Cells were washed with phosphate-buffered saline (PBS) by centrifugation before the staining with antibodies including CD45 VioGreen (Miltenyi #130-110-665), CD3 PE Cy7 (BD Pharmingen #552774), FVS575V (BD Horizon #565694), CD8 FITC (BD Pharmingen #553030), CD4 APC Vio770 (Miltenyi #130-118-957), NKp46 R718 (BD Pharmingen #751865), PDL1*BV421 (BD Pharmingen #564716) and CD69**APC (Miltenyi #130-115-461).
  • PBS phosphate-buffered saline
  • Rat lgG2a Isotype control BV421 (BD Pharmingen #562965) and Control REA293 APC (Miltenyi #130-113-434) for 30 min at +4°C.
  • Cells were washed with PBS and suspended in 100 pL MACSQuant Running Buffer for flow cytometry analysis. The following strategy of gating was used using the Kaluza 2.1.1 software (Beckman Coulter). Cells were identified by side scatter forward scatter (SSC vs FSC). From this gate, a dot plot (FVS757V vs SSC) was created to determine the Live cells (FVS575V negative).
  • PCPV vectors increased the frequencies of activated NK and T cells.
  • PCPV-mlL12 PCPTG19818
  • MVA-ctr had little effects on NK cells and no effects on T cells.
  • PCPV-mlL12 (PCPTG19818) augments survival and tumor control compared to PCPV-ctr. Cases of complete tumor rejection were observed.
  • lxlO 7 pfu of a PCPV-ctr vector, or the polyepitopes encoding vectors PCPTG19873 and PCPTG19874 were injected days 0, 7 and 14 into the flank (subcutaneously) of C57BL/6 mice. At days 7, 14 and 45, blood was taken quantify CD8+ OVA-specific T cells. Spleens were harvested at the end of the experiment (day 45).
  • a MHC-pentameric assay was used. Whole blood samples were collected into heparinized tubes. Whole blood was diluted in PBS EDTA 2mM and layered on lympholyte M separation cell media (TEBU BIO #CL5031), then centrifuged 1500xg 20 min at room temperature without brake. The enriched cell fraction of splenocytes at interphase was collected and transferred into 96 wells plate to proceed with the surface staining. Cells were washed with wash buffer (0.1% BSA in PBS) by centrifugation 5 min at 400xg.
  • wash buffer (0.1% BSA in PBS
  • Pellets were resuspended in wash buffer containing 10 pL/test of R-PE Pro MHC Pentamer H-2Kb SIINFEKL (Prolmmune #F093-2B-E) and incubated at +4°C for 30 min. Cells were washed with wash buffer (0.1% BSA in PBS) by centrifugation 5 min at 400xg. Pellets were resuspended in wash buffer containing CD8 APC (BD Pharmingen #553035) and CD19 V450 (BD Pharmingen #560376) and incubated for 30 min at +4°C. Cells were washed with wash buffer (0.1% BSA in PBS) by centrifugation 5 min at 400xg.
  • PCPTG19873 PCPV encoding tumor epitopes
  • PCPTG19874 PCPV encoding tumor epitopes and mlL12
  • E.G7-OVA murine T lymphoblast cell line
  • PCPTG19873 and PCPTG19874 vectors C57BL/6 mice were grafted with 3xl0 5 cells sc. Day 5, 12 and 19, lxlO 7 pfu of PCPV-ctr, PCPTG19873 or PCPTG19874 were injected into the growing tumors (10 mice per group). Tumor growth and survival proportions were monitored.
  • EXAMPLE 10 Detection of murine IL12 in extracellular vesicle (EV) fraction isolated from infected PBMCs.
  • PBMCs from a healthy donor were cultivated in RPMI, supplemented with 1% glutamine and 1% gentamicin, and 10% fetal bovine serum (FBS).
  • FBS fetal bovine serum
  • the serum was pretreated by ultracentrifugation o/n at 100 000 g to eliminate serum inherent EVs.
  • 1X10 8 PBMCS were infected in 35 ml with PCPV-mlL12 (PCPTG19818) at the MOI of 1, or virus was added to cell-free medium. Next day, the cultures were centrifuged at 300 g, then 2000 g. The cleared supernatants were concentrated using a Centricon 70-Plus, membrane Ultracel-PL, 10 kD (Merck Millipore) to a volume of 2 ml. This solution was filtered using Ultrafree-CL Centrifugal Filter 0.1 pm pore size (Merck Millipore).
  • mlL12 was quantified in various fractions of the EV isolation procedure by Luminex (mlL12 Simplex) ( Figure 10). Compared to the cell-free control, mlL12 increased in the various fractions. This observation suggests that virus infection and mlL12 gene expression occurred in infected PBMC cultures.
  • mlL12 was readily detected in the EV fraction. This suggests that mlL12 was associated with EVs and such modified EVs associated with IL-12 can exert activation of target cells such as, but not limited to, pDCs, NK, B cells.
  • PCPV-OVA vectors encoding IL12 (PCPTG19874) or not (PCPTG19873), by intracellular cytokine staining (ICS): lxlO 7 pfu of a PCPV ctr vector, wherein said polyepitopes encoding vectors PCPTG19873 and PCPTG19874 were injected days 0, 7 and 14 into the flank (subcutaneously) of naive C57BL/6 mice. After 45 days, mice were sacrificed, and splenocytes were isolated. Frozen splenocytes were thawed, cultivated, and stimulated for 5 hours in the presence of OVA specific peptide SIINFEKL or appropriate controls. The percentage of live CD8+ T cells secreting IFN-gamma is shown in Figure 12.
  • Splenocytes (lxlOE 6 cells), harvested from three different immunized mice were incubated for 4-6 hours with lpg/ml OVA peptide (ProteoGenix NH2-SIINFEKL-COOH) or irrelevant peptide (ProteoGenix NH2-KNGENAQAI-COOH) in RPMI 1640 media containing 10 % FBS.
  • Golgi Stop (BD #554724)
  • Golgi Plug (BD #555029) were added to block cytokine release.
  • splenocytes were stained with surface antibodies CD3 PerCP Cy5.5 (BD Pharmingen #551163) and CD8 V500 (BD Horizon #560776) and with Live Dead Violet (Invitrogen #L34958) for viability, in phosphate- buffered saline (PBS) 20 min at +4°C.
  • Splenocytes were then treated with fixation buffer and permeabilization buffer following the manufacturer's protocol (Becton Dickinson Cytofix/CytopermTM Fixation/Permeabilization Solution Kit # 554714) and intracellularly stained for interferon gamma (IFNy-FITC (BD Pharmingen #554411) 30 min at +4°C.
  • fixation buffer and permeabilization buffer following the manufacturer's protocol (Becton Dickinson Cytofix/CytopermTM Fixation/Permeabilization Solution Kit # 554714) and intracellularly stained for interferon gamma (IFNy-FITC (BD Pharmingen #
  • results show that the highest percentage IFN-gamma secreting CD8+ T cells were detected after stimulation with the OVA-peptide SIINFEKL in splenocytes from mice vaccinated with PCPTG19874 (PCPV-OVA-IL12).
  • PCPTG19874 PCPV-OVA-IL12 induces an IFN-gamma secreting CD8+ T cells response at least as twice superior.
  • EXAMPLE 12 Comparison of therapeutic vaccination with PCPV-OVA and PCPV-OVA-IL12 versus MVA-OVA and MVA-0VA-IL12 after repeated subcutaneous injections
  • PCPV and MVA-based therapeutic vaccines were compared in an OVA-specific tumor model E.G7- OVA.
  • C57BL/6 mice were grafted with 3xl0 5 cells sc.
  • PCPV and MVA backbones were comparable. Tumor growth was better controlled after subcutaneous treatment with PCPV-OVA-IL12 compared to PCPV-OVA. Same observation was made for MVA-0VA-IL12 and MVA-OVA.

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Abstract

La présente invention relève du domaine de l'immunothérapie virale. En particulier, l'invention concerne un nouveau virus de la paraviccine recombinant (PCPV) comprenant une molécule d'acide nucléique insérée dans son génome et codant pour une interleukine-12, une composition de celui-ci ainsi que leur utilisation thérapeutique pour prévenir ou traiter des maladies, et, notamment, des maladies prolifératives comme les cancers. La présente invention concerne également des procédés de génération et d'amplification d'un tel PCPV et un procédé de déclenchement ou de stimulation et/ou de réorientation d'une réponse immunitaire à l'aide d'un tel PCPV. Dans un mode de réalisation, le PCPV recombinant selon l'invention code en outre pour un polypeptide antigénique hétérologue. L'invention peut être largement utilisée pour la vaccination thérapeutique.
PCT/EP2023/076224 2022-09-23 2023-09-22 Virus de la paraviccine recombinant codant pour l'interleukine-12 WO2024062098A1 (fr)

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