US20180117137A1 - Arenaviruses for use in the treatment and/or prevention of tumors and method for producing arenaviruses with (improved) tumor-regression properties. - Google Patents

Arenaviruses for use in the treatment and/or prevention of tumors and method for producing arenaviruses with (improved) tumor-regression properties. Download PDF

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US20180117137A1
US20180117137A1 US15/567,343 US201615567343A US2018117137A1 US 20180117137 A1 US20180117137 A1 US 20180117137A1 US 201615567343 A US201615567343 A US 201615567343A US 2018117137 A1 US2018117137 A1 US 2018117137A1
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arenavirus
virus
tumor
carcinoma
cells
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Karl Sebastian Lang
Halime Kalkavan
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
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    • A61K2039/5254—Virus avirulent or attenuated
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
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    • C12N2760/10011—Arenaviridae
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    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/10011—Arenaviridae
    • C12N2760/10021—Viruses as such, e.g. new isolates, mutants or their genomic sequences
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    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/10011—Arenaviridae
    • C12N2760/10032—Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent
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    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/10011—Arenaviridae
    • C12N2760/10033—Use of viral protein as therapeutic agent other than vaccine, e.g. apoptosis inducing or anti-inflammatory
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to arenaviruses for use in the treatment and/or prevention of tumors and also methods for preparing arenaviruses with (improved) tumor-regressive properties.
  • Arenaviruses belong to the family of human pathogenic, pleomorphic RNA viruses. Diseases with these viruses belong to the zoonoses due to their natural reservoir in animals, predominantly rodents. Zoonoses refer to diseases that can be transferred from the animal to humans and vice versa from humans to the animal.
  • At least eight arenaviruses are known to cause illness in humans. Typical are aseptic meningitis and haemorrhagic fever.
  • Known viruses which can trigger a disease in humans are the lymphocytic choriomeningitis virus (LCMV), Guanarito virus (GTOV), Junin virus (JUNV), Lassa virus (LASV), Lujo virus (LUJV) Machupo virus (MACV), Sabia virus (SABV) and the Whitewater Arroyo virus (WWAV).
  • Arenaviruses are generally divided into two groups, namely the Old World arenaviruses and the New World arenaviruses. These groups differ geographically and genetically.
  • Old World arenaviruses such as the lymphocytic choriomeningitis virus
  • New World arenaviruses have been found in countries of the western hemisphere, such as Argentina, Cambodia, Venezuela, Brazil and the United States of America, for example.
  • the name of the virus family is derived from the Latin arenosus (sandy) and arena (sand) to describe the sandy ribosomal structure within the virions.
  • the virions of the arenaviruses have a round to irregular shape and have a diameter, depending on species and preparation of the test material, from 50 nm to 300 nm, usually between 110 nm and 130 nm.
  • Club-shaped glycoprotein spikes 8 nm to 10 nm long, are embedded in the virus envelope.
  • the individual spikes consist of a tetramer of the viral envelope protein.
  • the virions also comprise two closed-ring capsids with helical symmetry.
  • the length of the capsids varies between 450 nm and 1300 nm.
  • One molecule of the viral RNA (ribonucleic acid) polymerase (L-protein) is attached to each of them.
  • Each capsid comprises one molecule of a single-stranded RNA with mixed (i.e. ambisense, +/ ⁇ ) polarity.
  • the two single-stranded RNA molecules represent the viral genome. They are referred to as L (large) and S (small) and are about 7.5 kb (kilobases) or 3.5 kb (kilobases) large.
  • L large
  • S small
  • 7.5 kb kilobases
  • 3.5 kb kilobases
  • Very exceptional morphologically is the presence of an alternating number of cellular ribosomes within the virions, which give the viral particles their “sandy” appearance.
  • cellular RNAs also including ribosomal RNA
  • viral mRNAs messenger ribonucleic acids bound to the ribosomes
  • complete complementary strands of the virus genome are found in purified virus preparations.
  • arenaviruses used as vaccination vectors.
  • a prominent example is the vaccination virus Candid #1 used against Argentinian hemorrhagic fever. This is a vaccination variant of the Junin virus.
  • WO 2009/083210 A1 Known from WO 2009/083210 A1 is the use of replication defects, i.e. genetically modified arenavirus particles (virions), inter alia, for the treatment of neo-plastic diseases such as, for example, melanoma, prostate carcinoma, breast carcinoma and lung carcinoma.
  • neo-plastic diseases such as, for example, melanoma, prostate carcinoma, breast carcinoma and lung carcinoma.
  • neo-plastic diseases such as, for example, melanoma, prostate carcinoma, breast carcinoma and lung carcinoma.
  • neo-plastic diseases such as, for example, melanoma, prostate carcinoma, breast carcinoma and lung carcinoma.
  • neo-plastic diseases such as, for example, melanoma, prostate carcinoma, breast carcinoma and lung carcinoma.
  • cancer immunotherapy is mentioned as a potential area of application for such viral particles.
  • WO 2006/008074 A1 discloses the use of packaging cells, which produce retroviral virions pseudotyped with arenavirus glycoprotein, for gene therapy of solid tumors.
  • the present invention is therefore based on the object of providing a simpler and, in particular, more efficient therapeutic solution for tumors, in particular carcinomas and sarcomas, compared to the prior art.
  • the invention relates to an arenavirus for use in the treatment and/or prevention of a tumor, preferably a malignant tumor, in humans or animals.
  • the arenavirus is preferably characterized in that it is free of genomic foreign RNA, i.e. it does not comprise any genomic foreign RNA.
  • the genome of the arenavirus is preferably free of foreign RNA or preferably comprises no foreign RNA.
  • genomic foreign RNA is intended to mean an RNA (ribonucleic acid) or RNA sequence which does not occur or is not present in the genome of a wild-type arenavirus or in the genome of a mutant of a wild-type arenavirus (mutated arenavirus), in particular in the genome of a natural mutant of a wild-type arenavirus (naturally mutated arenavirus).
  • foreign RNA are artificial or synthetic RNA molecules, RNA of organisms and RNA from other viruses.
  • wild-type arenavirus is understood to mean an arenavirus whose genome is the genetically normal form occurring in nature.
  • mutant of a wild-type arenavirus or “mutated arenavirus” is understood to mean an arenavirus whose genome comprises a spontaneous, i.e. naturally-induced, modification, or modification induced by mutagens, compared to the wild-type genome.
  • the expression “natural mutant of a wild-type arenavirus” or “naturally mutated arenavirus” is understood to mean an arenavirus whose genome comprises a spontaneous, i.e. naturally-induced, modification, compared to the wild-type genome.
  • a naturally mutated arenavirus can be produced preferably by passage, in particular serial passage, which will be discussed in more detail below.
  • the invention is based on the surprising finding that arenaviruses without genomic foreign RNA are able to effect tumor regression.
  • Tumor regression is due to an activation or stimulation of congenital and adaptive immune cells caused by the arenaviruses.
  • the activated immune cells secrete increased antitumoral cytokines such as interferon- ⁇ and interferon- ⁇ , thereby counteracting or repelling the tumor.
  • a further surprising finding is the realization that the arenaviruses cause a significantly increased secretion of antitumoral cytokines in the case of a tumor manifestation.
  • Arenaviruses without genomic foreign RNA are thus suitable for use in tumor treatment. This has been successfully verified by the applicant by means of animal experiments. For this purpose, mice were used, inter alia, in which growth of human tumors is possible.
  • the arenavirus is also free of non-genomic foreign RNA.
  • non-genomic foreign RNA is intended to mean an RNA or RNA sequence, apart from the arenavirus genome, which does not occur or is not present in a wild-type arenavirus or a mutant of a wild-type arenavirus (mutated arenavirus), in particular of a natural mutant of a wild-type arenavirus (naturally mutated arenavirus).
  • the arenavirus does not comprise overall any foreign RNA, i.e neither genomic foreign RNA nor non-genomic foreign RNA.
  • the arenavirus is a wild-type arenavirus.
  • the arenavirus is a natural mutant of a wild-type arenavirus, i.e. a naturally mutated arenavirus.
  • the natural mutant or the naturally mutated arenavirus is preferably produced by passage, in particular multiple passage, in host animals and/or host cells.
  • the natural mutant or the naturally mutated arenavirus is particularly preferably produced by serial passage in host animals and/or host cells.
  • the arenavirus provided according to the invention is thus preferably an arenavirus which is produced starting from its wild-type form by passage, preferably serial passage, in host animals and/or host cells.
  • the host animals mentioned in the preceding paragraphs are preferably rodents, particularly mice.
  • the host cells mentioned in the previous paragraphs, on the other hand, are preferably dendritic cells or tumor cells.
  • the term “passage” is understood to mean a multiple, regular introduction of the arenavirus into host animals and/or host cells.
  • the expression “serial passage” is understood to mean a multiple, regular introduction of the arenavirus into different host animals, preferably of the same type, and/or different cells, preferably of the same type. Due to the multiple changes of environment (host animal and/or host cell), the arenavirus is subject to an increased adaptation pressure or mutational pressure, thereby increasing the likelihood of advantageous mutations occurring in the genome of the arenavirus from the perspective of tumor regression.
  • the tumor is selected from the group comprising or consisting of carcinoma, melanoma, blastoma, lymphoma and sarcoma.
  • carcinoma is intended to mean malignant neoplasia of epithelial origin.
  • sarcoma is intended to mean malignant neoplasia of mesodermal origin.
  • melanoma is intended to mean malignant neoplasia of melanocytic origin.
  • lymphocytic origin In the context of the present invention, (in accordance with the understanding of those skilled in the art), the term “lymphoma” is intended to mean malignant neoplasia of lymphocytic origin.
  • blastoma is intended to mean malignant neoplasia of embryonic origin.
  • the carcinoma is selected from the group comprising or consisting of anal carcinoma, bronchial carcinoma, lung carcinoma, endometrial carcinoma, gallbladder carcinoma, hepatocellular carcinoma, testicular carcinoma, colorectal carcinoma, laryngeal carcinoma, oesophogeal cancer, gastric carcinoma, breast carcinoma, renal carcinoma, ovarian carcinoma, pancreas tumor, pharyngeal carcinoma, prostate carcinoma, thyroid carcinoma and cervical carcinoma.
  • the sarcoma is selected from the group comprising or consisting of angiosarcoma, chondrosarcoma, Ewing's sarcoma, fibrosarcoma, Kaposi's sarcoma, liposarcoma, leiomyosarcoma, malignant fibrous histiocytoma, neurogenic sarcoma, osteosarcoma and rhabdomyosarcoma.
  • the arenavirus is an Old World arenavirus which is preferably selected from the group comprising or consisting of Ippy virus (IP-PYV), Lassa virus (LASV), lymphocytic choriomeningitis virus (LCMV), Mobala virus (MOBV) and Mopeia virus (MOPV).
  • IP-PYV Ippy virus
  • Lassa virus Lassa virus
  • LCMV lymphocytic choriomeningitis virus
  • MOBV Mobala virus
  • MOPV Mopeia virus
  • the arenavirus is the lymphocytic choriomeningitis virus, preferably a strain which is selected from the group comprising or consisting of WE, Armstrong, Clone 13 and Docile.
  • the arenavirus is a New World arenavirus, which is preferably selected from the group comprising or consisting of Allpahuayo virus (ALLV), Amapari virus (AMAV), Bear Canyon virus (BCNV), Chapare virus, Cupixi virus (CPXV), Flexal virus (FLEV), Guanarito virus (GTOV), Junin virus (JUNV), Latino virus (LATV), Machupo virus (MACV), Oliveros virus (OLVV), Parana virus (PARV), Pichinde virus (PICV), Pirital virus (PIRV), Sabia virus (SABV), Tacaribe virus (TCRV), Tamiami virus (TAMV) and Whitewater Arroyo virus (WWAV).
  • ALLV Allpahuayo virus
  • AMAV Amapari virus
  • BCNV Bear Canyon virus
  • Chapare virus Cupixi virus
  • CPXV Cupixi virus
  • CPXV Flexal virus
  • GTOV Guanarito virus
  • JUNV Junin virus
  • the arenavirus is a Junin virus, in particular the strain Candid #1 (Candid No. 1).
  • the Junin virus in particular the strain Candid #1 (Candid No. 1), has a nucleic acid sequence, in particular an S-ribonucleic acid sequence or ambisense sequence, according to SEQ ID No. 1 (according to sequence listing).
  • the Junin virus in particular the strain Candid #1 (Candid No. 1), has a nucleic acid sequence, in particular an L-ribonucleic acid sequence or ambisense sequence, according to SEQ ID No. 2 (according to sequence listing).
  • the Junin virus in particular the strain Candid #1 (Candid No. 1), has a nucleic acid sequence, in particular an S-ribonucleic acid sequence or ambisense sequence, according to SEQ ID No. 3 (according to sequence listing).
  • the Junin virus in particular the strain Candid #1 (Candid No. 1), has a nucleic acid sequence, in particular an L-ribonucleic acid sequence or ambisense sequence, according to SEQ ID No. 4 (according to sequence listing).
  • the lymphocytic choriomeningitis virus (LCMV virus) mentioned above has a nucleic acid sequence, in particular an S-ribonucleic acid sequence or ambisense sequence, according to SEQ ID No. 5 (according to sequence listing).
  • the lymphocytic choriomeningitis virus (LCMV virus) mentioned above has a nucleic acid sequence, in particular an L-ribonucleic acid sequence or ambisense sequence, according to SEQ ID No. 6 (according to sequence listing).
  • the arenavirus is isolated from tumor lysates, organ lysates, urine or blood.
  • the arenavirus is isolated from a cell culture medium, in particular from a human tumor cell line.
  • the arenavirus is for administration in the form of virions, i.e. in the form of arenavirus particles, which are outside a cell.
  • the arenavirus is provided, preferably prepared, for local, in particular intramuscular, intraperitoneal or subcutaneous administration.
  • the arenavirus is used in a further embodiment for local, in particular intramuscular, intraperitoneal or subcutaneous administration.
  • the arenavirus is provided, preferably prepared, for local administration at a dose of 1 PFU (Plaque Forming Unit)/kg body weight to 10 12 PFU/kg body weight, particularly 10 2 PFU/kg body weight to 10 6 PFU/kg body weight, preferably 10 3 PFU/kg body weight to 10 5 PFU/kg body weight.
  • the arenavirus is used, preferably for local administration, at a dose of 1 PFU (Plaque Forming Unit)/kg body weight to 10 12 PFU/kg body weight, particularly 10 2 PFU/kg body weight to 10 6 PFU/kg body weight, preferably 10 3 PFU/kg body weight to 10 5 PFU/kg body weight.
  • the arenavirus is provided, preferably prepared, for systemic, in particular intravenous, administration.
  • the arenavirus is used in an alternative embodiment for systemic, in particular intravenous, administration.
  • the arenavirus is provided, preferably prepared, for systemic administration at a dose of 1 PFU/kg body weight to 10 12 PFU/kg body weight, particularly 10 2 PFU/kg body weight to 10 6 PFU/kg body weight, preferably 10 3 PFU/kg body weight to 10 5 PFU/kg body weight.
  • the arenavirus is used, preferably for systemic administration, at a dose of 1 PFU/kg body weight to 10 12 PFU/kg body weight, particularly 10 2 PFU/kg body weight to 10 6 PFU/kg body weight, preferably 10 3 PFU/kg body weight to 10 5 PFU/kg body weight.
  • the invention relates to a medicament for use in the treatment and/or prevention of a tumor, in particular a malignant tumor.
  • the medicament is characterized in particular by the fact that it has an arenavirus according to the first aspect of the invention.
  • the medicament preferably further comprises a pharmaceutically acceptable carrier.
  • the carrier may in particular be selected from the group comprising or consisting of water, saline solution, buffer solution and cell culture medium.
  • the medicament also comprises an active ingredient.
  • the active ingredient can in particular be a cytostatic agent, an antibody and/or a cytokine.
  • the invention relates to a method for producing an arenavirus with tumor-regressive, i.e. tumor-repelling/counteracting properties or improved tumor-regressive properties.
  • the tumor is preferably a malignant tumor, preferably a carcinoma, melanoma, blastoma, lymphoma or sarcoma.
  • the method is preferably a method for producing an arenavirus with (improved) carcinoma-, melanoma-, blastoma-, lymphoma- or sarcoma-regressive properties.
  • the method comprises the following steps:
  • sequence of steps a) to c) may also be referred to as a (single) passage of the arenavirus in the dendritic cells or tumor cells.
  • the arenavirus is an arenavirus which has been subjected to a serial passage in host animals prior to carrying out step a).
  • the dendritic cells or tumor cells are in the form of a cell culture (dendritic cell culture or tumor cell culture).
  • the tumor cells are preferably malignant tumor cells, in particular carcinoma, melanoma, blastoma, lymphoma or sarcoma cells.
  • the carcinoma cells may be selected from the group comprising or consisting of anal carcinoma cells, bronchial carcinoma cells, lung carcinoma cells, endometrial carcinoma cells, gallbladder carcinoma cells, hepatocellular carcinoma cells, testicular carcinoma cells, colorectal carcinoma cells, laryngeal carcinoma cells, oesophogeal carcinoma cells, gastric carcinoma cells, breast carcinoma cells, renal carcinoma cells, ovarian carcinoma cells, pancreas tumor cells, pharyngeal carcinoma cells, prostate carcinoma cells, thyroid carcinoma cells and cervical carcinoma cells.
  • anal carcinoma cells bronchial carcinoma cells, lung carcinoma cells, endometrial carcinoma cells, gallbladder carcinoma cells, hepatocellular carcinoma cells, testicular carcinoma cells, colorectal carcinoma cells, laryngeal carcinoma cells, oesophogeal carcinoma cells, gastric carcinoma cells, breast carcinoma cells, renal carcinoma cells, ovarian carcinoma cells, pancreas tumor cells, pharyngeal carcinoma cells, prostate carcinoma cells, thyroid carcinoma cells and cervical carcinoma cells.
  • the sarcoma cells may be selected from the group comprising or consisting of angiosarcoma cells, chondrosarcoma cells, Ewing's sarcoma cells, fibrosarcoma cells, Kaposi's sarcoma cells, liposarcoma cells, leiomyosarcoma cells, malignant fibrous histiocytoma cells, neurogenic sarcoma cells, osteosarcoma cells and rhabdomyosarcoma cells.
  • the tumor cells are immortalized immune cells, in particular immortalized macrophages.
  • the dendritic cells or tumor cells are infected according to step a) preferably by adding the arenavirus to the cells.
  • the sequence of steps a) to c) is repeated with new, in particular non-infected, dendritic cells, preferably of the same type, or with new, in particular non-infected, tumor cells, preferably of the same type (the same tumor type).
  • the sequence of steps a) to c) is repeated many times.
  • new, in particular non-infected, dendritic cells preferably of the same type, or new, in particular non-infected, tumor cells, preferably of the same type, are used for each repetition.
  • the sequence of steps a) to c) is preferably repeated once to 10000 times, particularly 10 times to 1000 times, preferably 30 times to 60 times, wherein new, in particular non-infected, dendritic cells, preferably of the same type, or new, in particular non-infected, tumor cells, preferably of the same type, are used for each repetition.
  • the arenavirus is constantly forced to adapt to a new environment, i.e. to new dendritic cells or tumor cells.
  • This permanent adaptation pressure favors the occurrence of mutations, which can produce or improve the tumor-regressive properties of the arenavirus.
  • a replication of the arenavirus genome and a propagation of the arenavirus occur within the dendritic cells or tumor cells.
  • the arenavirus according to step b) is preferably cultured under standard cell culture conditions.
  • the arenavirus is preferably cultured in the dendritic cells or tumor cells for a period of 1 minute to 1 year, in particular 10 hours to 1 month, preferably 24 hours to 72 hours.
  • the cultured arenavirus according to step c) is preferably isolated from a cell culture supernatant.
  • the dendritic cells or tumor cells are sorted according to specific properties, preferably by means of a cell sorting device, and subsequently cultured, prior to isolating according to step c).
  • the sorted cells are preferably cultured over a period of 24 hours.
  • the method further comprises the following steps:
  • the invention relates to a method for producing an arenavirus with tumor-regressive, i.e. tumor-repelling/counteracting, properties or improved tumor-regressive properties.
  • the tumor is preferably a malignant tumor, preferably a carcinoma, melanoma, blastoma, lymphoma or sarcoma.
  • the method is preferably a method for producing an arenavirus with (improved) carcinoma-, melanoma-, blastoma-, lymphoma- or sarcoma-regressive properties.
  • the method comprises the following steps:
  • sequence of steps a) to c) may also be referred to as a (single) passage of the arenavirus in the host animal.
  • tumor tissue is transplanted to the host animal prior to carrying out step a).
  • a genetically modified host animal which spontaneously develops the tumor.
  • Rodents in particular, preferably mice can be used as host animals.
  • NOD SCID mice or LoxP-Tag mice can be used as host animals.
  • the SCID (Severe Combined Immunodeficiency) mutation is combined with a NOD (non-obese diabetic) type.
  • NOD non-obese diabetic
  • mice that are homozygous for the SCID mutation no functional T-cells or B-cells are formed.
  • these animals are exceptionally suitable for tolerating foreign body cells, for example transplanted tumors.
  • the host animal can be infected according to step a), for example by systemic, particularly intravenous, or local, for example subcutaneous, administration of the arenavirus.
  • the host animal can be infected by administration, preferably injection, of the arenavirus into the tumor of the host animal.
  • sequence of steps a) to c) is repeated with a new, in particular non-infected, host animal, preferably of the same type.
  • the sequence of steps a) to c) is repeated many times.
  • a new, in particular non-infected host animal, preferably of the same type, is preferably used for each repetition.
  • the arenavirus is subjected to serial passage in host animals, preferably of the same type.
  • the sequence of steps a) to c) is preferably repeated once to 1000 times, particularly 10 times to 100 times, preferably 30 times to 60 times, wherein a new, in particular non-infected, host animal, preferably of the same type, is used for each repetition.
  • the arenavirus is constantly forced to adapt to a new environment, i.e. to a new host animal.
  • This permanent adaptation pressure favors the occurrence of mutations, which can produce or improve the tumor-regressive properties of the arenavirus.
  • a replication of the arenavirus genome and a propagation of the arenavirus occur within the host animal.
  • the arenavirus is preferably cultured in the host animal for a period of 1 minute to 500 days, in particular 10 minutes to 100 days, preferably 1 hour to 30 days.
  • the arenavirus is isolated from urine, blood, the tumor, or organ lysates of the host animal.
  • the method further comprises the following steps:
  • sequence of steps d) to f) may also be referred to as a (single) passage of the arenavirus in the dendritic cells or tumor cells.
  • the dendritic cells or tumor cells are infected according to step d) preferably by adding the arenavirus to the cells.
  • the sequence of steps d) to f) is repeated with new, in particular non-infected, dendritic cells, preferably of the same type, or with new, in particular non-infected, tumor cells, preferably of the same type (the same tumor type).
  • the sequence of steps d) to f) is repeated many times.
  • new, in particular non-infected, dendritic cells preferably of the same type, or new, in particular non-infected, tumor cells, preferably of the same type, are used for each repetition.
  • the arenavirus is additionally subjected to a serial passage in dendritic cells, preferably of the same type, or tumor cells, preferably of the same type.
  • the sequence of steps d) to f) is preferably repeated once to 1000 times, particularly 10 times to 100 times, preferably 30 times to 60 times, wherein new, in particular non-infected, dendritic cells, preferably of the same type, or new, in particular non-infected, tumor cells, preferably of the same type, are preferably used for each repetition.
  • the combination of a serial passage of the arenavirus in host animals, preferably of the same type, with a serial passage of the arenavirus in dendritic cells, preferably of the same type, or tumor cells, preferably of the same type, is suitable, because of the additionally increased adaptation pressure or mutation pressure in a particular manner, for producing arenaviruses with (improved) tumor-regressive properties.
  • the arenavirus is preferably cultured in the dendritic cells or tumor cells for a period of 1 minute to 500 days, in particular 10 minutes to 100 days, preferably 1 hour to 30 days.
  • the dendritic cells or tumor cells are sorted according to specific properties, preferably by means of a cell sorting device, and subsequently cultured, prior to isolating according to step f).
  • the sorted cells are preferably cultured over a period of 24 hours.
  • the method further comprises the following steps:
  • the arenavirus isolated according to step c) is cloned and subsequently sequenced.
  • mice used were from a C57BL/6 background.
  • Map3k14 aly/aly mice lack NF-kB signals and are therefore highly immunosuppressed.
  • Irf3 ⁇ Ir7 ⁇ / ⁇ mice cannot produce any interferon.
  • NOD.SCID mice have no adaptive immune system. Therefore, it is possible to grow human tumors in these mice.
  • LoxP-Tag mice spontaneously develop liver tumors.
  • MOPC cells are murine oropharynx carcinoma cells. Mc38 are murine colon carcinoma cells. Raw cells are immortalized macrophages. A431 are human lung carcinoma cells; Sw40 are human colon carcinoma cells, Hela are human cervical carcinoma cells. Primary macrophages were cultured from bone marrow precursor cells by means of M-CSF. Cells were maintained in Dulbecco's modified Eagle's medium with 10% fetal bovine serum (Sigma-Aldrich), 2 mmol/1 L-glutamine and 100 U/ml penicillin. All cells were cultured in 5% CO2.
  • the LCMV strain WE was obtained from the laboratory of Prof. Zinkernagel (Experimental Immunology, Zurich, Switzerland) and was propagated in L929 cells. Candid #1 was obtained from Professor Paula Cannon (University of Southern California).
  • mice Approximately 5 ⁇ 10 5 tumor cells (in 100 microL) were injected subcutaneously into the right flank of 6 to 12 week old mice. The longest tumor diameter was measured by. Mice were treated by peritumoral injections of 2 ⁇ 10 4 PFU LCMV-WE or Candid #1 (in 100-200 microL).
  • MVD microvessel density
  • hypoxic tumor regions were detected by the formation of pimonidazole adducts after injection of pimonidazole into tumor-transplanted animals for 30 min.
  • the tumor sections were stained using the Hypoxyprobe-1 Plus kit according to the manufacturer's instructions (Pharmacia Natur International, Inc.).
  • Serum IFN- ⁇ levels were determined by ELISA according to the manufacturer's data (Research Diagnostics RDI, Flanders, N.J.).
  • the mean values were compared using an unpaired two-sided student t-test. The data are shown as mean ⁇ SEM. The level of statistical significance was set at p ⁇ 0.05.
  • mice 2.2 WT C57BL/6 mice were treated with 5 ⁇ 10 5 MOPC cells (day 3). One group of mice was additionally treated peritumorally with 2 ⁇ 10 4 PFU LCMV (WE strain) (day 0). Tumor growth was observed.
  • mice were treated with 5 ⁇ 10 5 MC38 cells (day 3).
  • One group of mice was additionally treated peritumorally with 2 ⁇ 10 4 PFU LCMV (WE strain) (day 0). Tumor growth was observed.
  • Map3k14 aly/aly mice and WT mice were treated with 5 ⁇ 10 5 MOPC cells (day 3).
  • One group of mice was additionally treated peritumorally with 2 ⁇ 10 4 PFU LCMV (WE strain) (day 0). Tumor growth was observed.
  • mice and WT mice were treated with 5 ⁇ 10 5 MOPC cells (day 3).
  • One group of mice was additionally treated peritumorally with 2 ⁇ 10 4 PFU LCMV (WE strain) (day 0). Tumor growth was observed.
  • mice 2.8 WT mice were treated with 5 ⁇ 10 5 MOPC cells (day 3).
  • One group of mice was additionally treated peritumorally with 2 ⁇ 10 4 PFU LCMV (WE strain) (day 0).
  • PFU LCMV WE strain
  • the tumors were analyzed histologically with CD31 staining.
  • the microvessel density (MDV) and the vessel-vessel spacing were quantified.
  • mice were treated with 5 ⁇ 10 5 MOPC cells (day 3).
  • One group of mice was additionally treated peritumorally with 2 ⁇ 10 4 PFU LCMV (WE strain) (day 0).
  • the animals were injected with pimonidazole, and the tumors were then analyzed histologically for hypoxic regions.
  • mice 2.11 NOD.SCID mice were injected subcutaneously with 5 ⁇ 10 5 A431 cells (day ⁇ 3) and then either left untreated or treated with 2 ⁇ 10 4 PFU LCMV (WE strain). The tumor size (longest diameter) was measured on the specified day. The mice were sacrificed when the tumor size reached 12 mm.
  • mice 2.12 NOD.SCID mice were treated with 5 ⁇ 10 5 Sw40 cells (day 0). A group of mice was additionally treated peritumorally with 2 ⁇ 10 4 PFU LCMV (WE strain) or 2 ⁇ 10 4 PFU Candid #1 (day 0). Tumor growth was observed.
  • mice 2.13 NOD.SCID mice were treated with 5 ⁇ 10 5 Hela cells (day 0). A group of mice was additionally treated peritumorally with 2 ⁇ 10 4 PFU LCMV (WE strain) (day 3). Tumor growth was observed.
  • Tumor-specific T cells are derived from the blood of mice with B16F10 tumors, which were additionally treated intratumorally with or without LCMV.
  • EL4 cells murine subcutaneous lymphoma
  • OT1 cells tumor-specific T cells
  • LCMV LCMV
  • mice and PD-1 deficient mice bearing a murine pharyngeal carcinoma (MOPC cells) and which were treated intratumorally with LCMV (2 ⁇ 10 4 PFU).
  • nucleic acid sequences SEQ ID No. 1 to SEQ ID No. 6 mentioned in the general description correspond to the nucleic acid sequences disclosed in the following sequence listing.

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