METHOD FOR PRODUCING AN ANTITUMORAL ARENAVIRUS AS WELL AS ARENAVIRUS MUTANTS

Abstract

The invention relates to a mutant of an arenavirus having improved antitumoral properties. The invention also relates to a method of generating such an arenavirus mutant, related pharmaceutical compositions, medical uses, methods of treatment, and isolated proteins and nucleic acids.

Claims

1. A mutant of lymphocytic choriomeningitis virus, wherein the mutant is capable of undergoing a stronger propagation in a tumor cell as compared to the wild type lymphocytic choriomeningitis virus strain WE, wherein the mutant comprises a nucleic acid encoding a glycoprotein, wherein said glycoprotein comprises at least one of the mutations Arg 185.fwdarw.Trp and Ile 181.fwdarw.Met as compared to the wild type glycoprotein sequence set forth in SEQ ID NO: 10.

2. The mutant of claim 1, wherein the mutant comprises a nucleic acid encoding a glycoprotein that has at least about 95%, preferably at least about 96%, preferably at least about 97%, preferably at least about 98% preferably at least about 99%, preferably at least about 99.1%, preferably at least about 99.2%, preferably at least about 99.3%, preferably at least about 99.4%, preferably at least about 99.5%, preferably at least about 99.5%, preferably at least about 99.7% sequence identity, to the wild type glycoprotein sequence set forth in SEQ ID NO: 10.

3. The mutant of claim 1 or 2, wherein the mutant comprises a nucleic acid encoding a glycoprotein, wherein said glycoprotein comprises the mutations Arg 185.fwdarw.Trp and Ile 181.fwdarw.Met as compared to the wild type glycoprotein set forth in SEQ ID NO: 10.

4. The mutant any one of the preceding claims, wherein the mutant comprises a nucleic acid encoding a glycoprotein, wherein said glycoprotein has at least about 95%, preferably at least about 96%, preferably at least about 97%, preferably at least about 98% preferably at least about 99%, preferably at least about 99.1%, preferably at least about 99.2%, preferably at least about 99.3%, preferably at least about 99.4%, preferably at least about 99.5%, preferably at least about 99.5%, preferably at least about 99.7% sequence identity, or is preferably identical, to a sequence set forth in any one of SEQ ID NOs: 18, 26, 34, 42, 50, and 58.

5. The mutant of any one of the preceding claims, wherein the mutant comprises a nucleic acid encoding a glycoprotein, wherein said nucleic acid comprises a sequence that has at least about 95%, preferably at least about 96%, preferably at least about 97%, preferably at least about 98% preferably at least about 99%, preferably at least about 99.1%, preferably at least about 99.2%, preferably at least about 99.3%, preferably at least about 99.4%, preferably at least about 99.5%, preferably at least about 99.6%, preferably at least about 99.7% sequence identity, or is preferably identical, to a sequence set forth in SEQ ID NOs: 17, 25, 33, 41, 49, and 57.

6. The mutant of claim 1, wherein the mutant comprises a nucleic acid encoding a L-protein, wherein said L-protein has at least about 95%, preferably at least about 96%, preferably at least about 97%, preferably at least about 98% preferably at least about 99%, preferably at least about 99.1%, preferably at least about 99.2%, preferably at least about 99.3%, preferably at least about 99.4%, preferably at least about 99.5%, preferably at least about 99.6%, preferably at least about 99.7% sequence identity to the wild type L-protein sequence set forth in SEQ ID NO: 16.

7. The mutant any one of the preceding claims, wherein the mutant comprises a nucleic acid encoding a L-protein, wherein said L-protein has at least about 95%, preferably at least about 96%, preferably at least about 97%, preferably at least about 98% preferably at least about 99%, preferably at least about 99.1%, preferably at least about 99.2%, preferably at least about 99.3%, preferably at least about 99.4%, preferably at least about 99.5%, preferably at least about 99.6%, preferably at least about 99.7%, preferably at least about 99.8%, preferably at least about 99.9% sequence identity, or is preferably identical, to a sequence set forth in any one of SEQ ID NOs: 24, 32, 40, 48, 56, and 64.

8. The mutant of any one of the preceding claims, wherein the mutant comprises a nucleic acid encoding a L-protein, wherein said L-protein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the following mutations: Lys 253.fwdarw.Arg; Lys 1512.fwdarw.Met; Lys 1513.fwdarw.Glu; Ser 1758.fwdarw.Phe; Phe 1995.fwdarw.Ser; Ile 2094.fwdarw.Val; Lys 2115.fwdarw.Glu; Thr 2141.fwdarw.Ala; Arg 2175.fwdarw.Lys; Thr 2185.fwdarw.Ala as compared to the wild type L-protein sequence set forth in SEQ ID NO: 16.

9. The mutant of any one of the preceding claims, wherein the mutant comprises a nucleic acid encoding a L-protein, wherein said L-protein comprises one of the following sets of mutations: (a) Ser 1758.fwdarw.Phe; (b) Phe 1995.fwdarw.Ser; optionally Ile 2094.fwdarw.Val; and optionally Thr 2141.fwdarw.Ala; (c) Lys 1513.fwdarw.Glu; Phe 1995.fwdarw.Ser; and optionally Arg 2175.fwdarw.Lys; (d) Lys 253.fwdarw.Arg; Lys 1512.fwdarw.Met; Lys 2115.fwdarw.Glu; optionally Thr 2141.fwdarw.Ala; Thr 2185.fwdarw.Ala (e) Phe 1995.fwdarw.Ser; or (f) Lys 2115.fwdarw.Glu, as compared to the wild type L-protein sequence set forth in SEQ ID NO: 16.

10. The mutant of any one of the preceding claims, wherein the mutant comprises a nucleic acid encoding an L-protein, wherein said nucleic acid is complementary to a sequence that has at least about 95%, preferably at least about 96%, preferably at least about 97%, preferably at least about 98% preferably at least about 99%, preferably at least about 99.1%, preferably at least about 99.2%, preferably at least about 99.3%, preferably at least about 99.4%, preferably at least about 99.5%, preferably at least about 99.6%, preferably at least about 99.7%, preferably at least about 99.8%, preferably at least about 99.9% sequence identity, or that is preferably identical, to a sequence set forth in SEQ ID NOs: 17, 25, 33, 41, 49, and 57.

11. The mutant any one of the preceding claims, wherein the mutant comprises a nucleic acid encoding an nucleoprotein, wherein said nucleoprotein has at least about 95%, preferably at least about 96%, preferably at least about 97%, preferably at least about 98% preferably at least about 99%, preferably at least about 99.1%, preferably at least about 99.2%, preferably at least about 99.3%, preferably at least about 99.4%, preferably at least about 99.5%, preferably at least about 99.6%, preferably at least about 99.7%, sequence identity, or that is preferably identical, to a nucleoprotein set forth in any one of SEQ ID NOs: 12, 20, 28, 36, 44, 52, and 60.

12. The mutant any one of the preceding claims, wherein the mutant comprises a nucleic acid encoding an nucleoprotein, wherein said nucleic acid is complementary to a sequence that has at least about 95%, preferably at least about 96%, preferably at least about 97%, preferably at least about 98% preferably at least about 99%, preferably at least about 99.1%, preferably at least about 99.2%, preferably at least about 99.3%, preferably at least about 99.4%, preferably at least about 99.5%, preferably at least about 99.6%, preferably at least about 99.7%, sequence identity, or that is preferably identical, to a nucleoprotein set forth in any one of SEQ ID NOs: 11, 19, 27, 35, 43, 51, and 59.

13. The mutant any one of the preceding claims, wherein the mutant comprises a nucleic acid encoding a Z-protein, wherein said Z-protein has at least about 95%, preferably at least about 96%, preferably at least about 97%, preferably at least about 98% preferably at least about 99%, preferably at least about 99.1%, preferably at least about 99.2%, preferably at least about 99.3%, preferably at least about 99.4%, preferably at least about 99.5%, preferably at least about 99.6%, preferably at least about 99.7%, sequence identity, or that is preferably identical, to a sequence set forth in any one of SEQ ID NOs: 14, 22, 30, 38, 46, 54, and 62.

14. The mutant any one of the preceding claims, wherein the mutant comprises a nucleic acid encoding a Z-protein, wherein said nucleic acid comprises a sequence that has at least about 95%, preferably at least about 96%, preferably at least about 97%, preferably at least about 98% preferably at least about 99%, preferably at least about 99.1%, preferably at least about 99.2%, preferably at least about 99.3%, preferably at least about 99.4%, preferably at least about 99.5%, preferably at least about 99.6%, preferably at least about 99.7%, sequence identity, or that is identical, to a sequence set forth in any one of SEQ ID NOs: 13, 21, 29, 37, 45, 53, and 61.

15. A method of producing an antitumoral arenavirus, in particular an arenavirus, which has improved antitumor properties over an original arenavirus, the process comprising the following steps: a) plating primary tumor cells or cells of the cell line H1975, C643 or Tramp-C2 onto and/or into a nutrient medium, b) inoculating the plated primary tumor cells or plated cells of the cell line H1975, C643 or Tramp-C2 with an original arenavirus, c) incubating the inoculated primary tumor cells or inoculated cells of the cell line H1975, C643 or Tramp-C2 under conditions which are suitable for infecting at least a portion of the inoculated primary tumor cells or the inoculated cells of the cell line H1975, C643 or Tramp-C2 with the original arenavirus, d) extracting an arenavirus-containing cell culture supernatant from a cell culture containing the incubated primary tumor cells or the incubated cells of the cell line H1975, C643 or Tramp-C2, wherein the step sequence a) to d) is repeated a plurality of times, wherein the primary tumor cells or the cells of the cell line H1975, C643 or Tramp-C2 when performing the first repetition of step sequence a) to d), for performing step b) is inoculated with the arenavirus-containing cell culture supernatant or a part thereof extracted when performing step d) before the first repetition of the step sequence a) to d), and wherein the primary tumor cells or the cells of cell line H1975, C643 or Tramp-C2 when performing each further repetition of the step sequence a) to d), for performing step b) is inoculated with the arenavirus-containing cell culture supernatant or a part thereof extracted when performing step d) of a previous repetition of the step sequence a) to d).

16. The method of claim 15, characterized in that the primary tumor cells are passaged at most 1000 times, in particular at most 100 times, preferably at most 10 times, before performing step a).

17. The method of claim 15 or 16, characterized in that step c) is performed within a period from 1 hour to 1000 hours, in particular 3 hours to 300 hours, preferably 12 hours to 96 hours, and/or at a temperature of 4° C. to 50° C., in particular 20° C. to 42° C., preferably 34° C. to 39° C. and/or in a nutrient medium selected from the group consisting of RPMI-1640, DMEM and IMDM, wherein the nutrient medium in particular additionally comprises serum, such as fetal calf serum or human serum, and/or amino acids, such as glutamate and/or glutamine, and/or antibiotics, and/or under a carbon dioxide atmosphere of 0% to 20%, in particular 2% to 10%, preferably 4% to 6%.

18. The method of any one of claims 15 to 17, characterized in that the sequence of steps is repeated 3 to 1000 times, in particular 10 to 100 times, preferably 20 to 50 times.

19. The method of any one of claims 15 to 18, characterized in that the primary tumor cells or the cells of the cell line H1975, C643 or Tramp-C2 and/or the original arenavirus and/or the arenavirus contained in the extracted cell culture supernatant or a part thereof are treated with at least one chemotherapeutic agent and/or subjected to a radiation treatment before performing step d) and/or primary tumor cells or cells of the cell line H1975, C643 or Tramp-C2 which are resistant to at least one chemotherapeutic agent are used, and/or primary tumor cells or the cells of the cell line H1975, C643 or Tramp-C2 and/or the original arenavirus and/or the arenavirus contained in the extracted cell culture supernatant or a part thereof before performing step d) are treated with at least one antiviral compound, in particular with alpha-interferon and/or gamma-interferon.

20. The method of any one of claims 15 to 19, characterized in that the primary tumor cells are selected from the group consisting of primary choroidal melanoma cells, primary anal carcinoma cells, primary angiosarcoma cells, primary astrocytoma cells, primary basal cell carcinoma cells, primary cervical carcinoma cells, primary chondrosarcoma cells, primary chorionic carcinoma cells, primary dermal squamous cell carcinoma cells, primary small intestine carcinoma cells, primary endometrial carcinoma cells, primary Ewing sarcoma cells, primary fibrosarcoma cells, primary gallbladder carcinoma cells, primary bile duct carcinoma cells, primary glioblastoma cells, primary bladder carcinoma cells, primary ureter carcinoma cells, primary urethral carcinoma cells, primary hepatocellular carcinoma cells, primary testicular tumor cells, primary hypopharyngeal carcinoma cells, primary pituitary carcinoma cells, primary Kaposi sarcoma cells, primary small-cell bronchial carcinoma cells, primary colon carcinoma cells, primary colorectal carcinoma cells, primary laryngeal carcinoma cells, primary leiomyosarcoma cells, primary liposarcoma cells, primary gastric carcinoma cells, primary malignant fibrous histiocytoma cells, primary breast carcinoma cells, primary medulloblastoma cells, primary melanoma cells, primary oral floor carcinoma cells, primary sinus carcinoma cells, primary nasopharyngeal carcinoma cells, primary adrenocortical carcinoma cells, primary parathyroid carcinoma cells, primary neurogenic sarcoma cells, primary non-small-cell bronchial carcinoma cells, primary renal carcinoma cells, primary oropharyngeal carcinoma cells, primary osteosarcoma cells, primary ovarian carcinoma cells, primary pancreatic tumor cells, primary penile carcinoma cells, primary pheochromocytoma cells, primary pleural mesothelioma cells, primary prostate carcinoma cells, primary rectal carcinoma cells, primary retinoblastoma cells, primary rhabdomyosarcoma cells, primary thyroid carcinoma cells, primary salivary gland carcinoma cells, primary esophageal carcinoma cells, primary tonsil carcinoma cells, primary vaginal carcinoma cells, primary vulvar carcinoma cells, primary Wilms tumor cells, primary cells of neuroendocrine tumors, and primary tongue carcinoma cells.

21. The method of any one of claims 15 to 20, characterized in that as original arenavirus a wild-type arenavirus is used, in particular a wild-type lymphocytic choriomeningitis virus.

22. A lymphocytic choriomeningitis virus mutant comprising a glycoprotein with at least one mutation, wherein the at least one mutation is an amino acid substitution of the isoleucine at position 181 of the glycoprotein by another amino acid, preferably methionine, and/or an amino acid substitution of the arginine at position 185 of the glycoprotein by another amino acid, preferably tryptophan, and/or an L-protein having at least one mutation, wherein the at least one mutation is an amino acid substitution of the lysine at position 1513 of the L-protein by another amino acid, preferably glutamate, and/or an amino acid substitution of the phenylalanine at position 1995 of the L-protein by another amino acid, preferably serine, and/or an amino acid substitution of isoleucine at position 2094 of the L-protein by another amino acid, preferably valine, and/or an amino acid substitution of threonine at position 2141 of the L-protein by another amino acid, preferably alanine, and/or an amino acid substitution of arginine at position 2175 of the L-protein by another amino acid, preferably lysine.

23. A lymphocytic choriomeningitis virus mutant, in particular according to claim 22, characterized in that the lymphocytic choriomeningitis virus mutant comprises a protein or peptide, in particular a glycoprotein or L-protein, having or consisting of an amino acid sequence according to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 18, SEQ ID NO: 26, SEQ ID NO: 34, SEQ ID NO: 42, SEQ ID NO: 50, SEQ ID NO: 58, SEQ ID NO: 24, SEQ ID NO: 32, SEQ ID NO: 40, SEQ ID NO: 48, SEQ ID NO: 56, or SEQ ID NO: 64.

24. The lymphocytic choriomeningitis virus mutant, in particular according to claim 22 or 23, characterized in that the lymphocytic choriomeningitis virus mutant comprises a nucleic acid which is coding for a protein or peptide comprising or consisting of an amino acid sequence SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 18, SEQ ID NO: 26, SEQ ID NO: 34, SEQ ID NO: 42, SEQ ID NO: 50, SEQ ID NO: 58, SEQ ID NO: 24, SEQ ID NO: 32, SEQ ID NO: 40, SEQ ID NO: 48, SEQ ID NO: 56, or SEQ ID NO: 64, and/or the lymphocytic choriomeningitis virus mutant comprising a nucleic acid which has a nucleic acid sequence or consists of a nucleic acid sequence which is or is complementary to a nucleic acid sequence according to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 17, SEQ ID NO: 25, SEQ ID NO: 33, SEQ ID NO: 41, SEQ ID NO: 49, SEQ ID NO: 57, SEQ ID NO: 23, SEQ ID NO: 31, SEQ ID NO: 39, SEQ ID NO: 47, SEQ ID NO: 55, or SEQ ID NO: 63.

25. A lymphocytic choriomeningitis virus mutant of any one of claims 1 to 14 and 22 to 24 for use in medicine, in particular for use in the treatment and/or prevention of a tumor, preferably a tumor selected from the group consisting of anal carcinoma, bronchial carcinoma, lung carcinoma, endometrial carcinoma, gallbladder carcinoma, bladder carcinoma, hepatocellular carcinoma, testicular carcinoma, colon carcinoma, colorectal carcinoma, colorectal carcinoma, hepatocellular carcinoma, testicular carcinoma, colon carcinoma, tumor of the bladder, bladder carcinoma, tumor of the bladder, hepatocellular carcinoma, lung carcinoma, lung carcinoma, endometrial carcinoma, colorectal carcinoma, rectal carcinoma, laryngeal carcinoma, esophageal carcinoma, gastric carcinoma, breast carcinoma, renal carcinoma, ovarian carcinoma, pancreatic carcinoma, pharyngeal carcinoma, opharyngeal carcinoma, prostate carcinoma, thyroid carcinoma, Cervical cancer, angiosarcoma, chondrosarcoma, Ewing sarcoma, fibrosarcoma, Kaposi sarcoma, liposarcoma, leiomyosarcoma, malignant fibrosis histiocytoma, lymphoma, leukemia, neurogenic sarcoma, osteosarcoma and rhabdomyosarcoma.

26. A medicament or pharmaceutical composition comprising a lymphocytic choriomeningitis virus mutant of any one of claims 1 to 14 and 22 to 24.

27. The medicament according to claim 26, characterized in that the medicament further comprises a checkpoint blocker, such as PD-1, and/or an apoptosis modulator, in particular an apoptosis inhibitor, such as SMAC mimeticum.

28. An isolated protein or peptide, in particular glycoprotein, L-protein, nucleoprotein, or Z-protein, comprising or consisting of an amino acid sequence according to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 18, SEQ ID NO: 26, SEQ ID NO: 34, SEQ ID NO: 42, SEQ ID NO: 50, SEQ ID NO: 58, SEQ ID NO: 24, SEQ ID NO: 32, SEQ ID NO: 40, SEQ ID NO: 48, SEQ ID NO: 56, or SEQ ID NO: 64, SEQ ID NO: 12, SEQ ID NO: 20, SEQ ID NO: 28, SEQ ID NO: 36, SEQ ID NO: 44, SEQ ID NO: 52, SEQ ID NO: 60, SEQ ID NO: 14, SEQ ID NO: 22, SEQ ID NO: 30, SEQ ID NO: 38, SEQ ID NO: 46, SEQ ID NO: 54, or SEQ ID NO: 62.

29. An isolated nucleic acid, in particular encoding for a glycoprotein, L-protein, nucleoprotein, or Z-protein, wherein the nucleic acid has a nucleic acid sequence or consists of a nucleic acid sequence which is or is complementary to a nucleic acid sequence according to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 17, SEQ ID NO: 25, SEQ ID NO: 33, SEQ ID NO: 41, SEQ ID NO: 49, SEQ ID NO: 57, SEQ ID NO: 23, SEQ ID NO: 31, SEQ ID NO: 39, SEQ ID NO: 47, SEQ ID NO: 55, SEQ ID NO: 63, SEQ ID NO: 11, SEQ ID NO: 19, SEQ ID NO: 27, SEQ ID NO: 35, SEQ ID NO: 43, SEQ ID NO: 51, SEQ ID NO: 59, SEQ ID NO: 13, SEQ ID NO: 21, SEQ ID NO: 29, SEQ ID NO: 37, SEQ ID NO: 45, SEQ ID NO: 53, or SEQ ID NO: 61.

Description

EXAMPLES

[0205] 1. Methods and Materials

[0206] 1.1 Mice

[0207] For in vivo anti-tumoral analyses WT animals (on C57BL/6 background) or NOD.SCID mice without an adaptive immune systrain WEre used. OT-1 mice carry an ovalbumin-specific MHC-I restricted T cell receptor as transgene.

[0208] 1.2 Cell Lines

[0209] MC57 (CVCL_4985) is a murine fibroblast cell line in which the arenavirus LCMV can multiply well. C643 (CVCL_5969) is a human anaplastic thyroid carcinoma cell line. H1975 is a human lung carcinoma cell line (CVCL_1511, ATCC, CRL-5908, adenocarcinoma). TrampC2 is a murine adenocarcinoma cell line (CVCL_3615). MOPC is a murine oropharyngeal cell line. CMT167 (CVCL_2405) is a murine lung carcinoma cell line. B16F10-OVA is a murine melanoma cell line (CVCL_0159) expressing ovalbumin as a model antigen. UKE-Mel-13a are primary tumor cells isolated from a human melanoma metastasis and have been passeged less than 100×. 511950 are primary tumor cells isolated from a transgenic murine pancreatic carcinoma (Mazur P K et al Nat Med. 2015 October; 21(10):1163-71) and have been passaged less than 20×. 511950R originate from 511950 cells and have been passaged less than 100× under treatment with the MEK inhibitor trametinib.

[0210] 1.3 Viruses

[0211] The LCMV strain WE was obtained from the laboratory of Prof. Zinkernagel (Experimental Immunology, Zurich, Switzerland) and has been propagated in L929 cells or BHK cells since 2008. The clones LCMV-P42, LCMV-P52 and LCMV-P91 were isolated and sequenced after different passages.

[0212] 1.4 Reagents

[0213] LCL161 (Selleckchem) and anti-PD1 (BioXcell) were tested in combination with LCMV and LCMV-P52, respectively, for their anti-tumor activity.

[0214] 1.5 Determination of LCMV Infected Cells by Immunofluorescence

[0215] Immunofluorescence was used to detect LCMV in cells. Cells were seeded in 24 well plates, each containing a cover glass. After 24 hours the cells were infected with LCMV and stained 24 hours later with a fluorochrome-labelled anti-LCMV-NP antibody (clone VL4), visualized with a fluorescence microscope and photographed with an integrated CCD camera.

[0216] 1.6 Determination of LCMV in Supernatant by Plaque Assay

[0217] To determine the LCMV production of cells, cells were sown in 24-well plates and infected with LCMV after 24 hours. The supernatant was extracted after 24 hours. The supernatant was titrated in 24-well plates and MC57 cells (150,000 cells/hole) were added. Methyl cellulose was added after 4 hours. After another 48 hours, the cell lawn was analyzed for LCMV plaques with anti-LCMV-NP antibodies (clone KL53). The plaques were counted to determine the number of infectious particles/ml in the supernatant.

[0218] 1.7 Passages of Viruses

[0219] In order to adapt viruses to tumors, different primary tumor cells or tumor cell lines were infected with LCMV-WE. Cells were plated in 24-well plates (approx. 100,000 cells/well in 1 mL medium). After 24 hours viruses with a Multiplicity of Infection (MOI)=1 in 100 μL were added. Depending on the setup, the initial inoculum was removed between 1-30 minutes and new medium was added. After 24 or 48 or 72 hours the cell culture supernatant was extracted and frozen for further analysis. Newly plated cells were infected with 100 microL of the extracted supernatant. This procedure was repeated between 30 and 100 times.

[0220] 1.8 Sequencing

[0221] After reverse transcription of the viral RNA, the cDNA was amplified with sequence specific primer pairs (oligonucleotides) in the polymerase chain reaction (PCR). The PCR products were purified, sequenced by cycle sequencing (modified singer method), the products separated by capillary electrophoresis and the sequence recorded as an electropherogram. The nucleic acid sequence was translated to obtain the protein sequence.

[0222] 1.9 Innate Immune Activation

[0223] The ability of LCMV to activate the innate immune system was determined by the biomarker IFN-alpha using murine IFN-alpha ELISA (ThermoFisher).

[0224] 1.10 Adaptive Immune Activation

[0225] The ability of LCMV to activate the adaptive immune system was tested by tetramer staining (NIH, Tetramer Facility) of activated lymphocytes.

[0226] 1.11 Tumor Growth and Treatments

[0227] To measure the anti-tumoral effect, C57BL/6 or NOD.SCID mice (6-12 weeks old) 5×10.sup.5 tumor cells (in 100 μL) were injected subcutaneously into the right or left flank. After a visible tumor was formed, the animals were treated and the mean tumor diameter or tumor volume was determined. For a metastasis model, B16F10-OVA cells were applied intravenously.

[0228] 1.12 Isolation and Transfer of Ovalbumin (Tumor)-Specific CD8+ T Cells

[0229] For the analysis of tumor-specific CD8.sup.+ T cells, cells from the spleen of OT-1 mice were transferred into C57BL/6 mice carrying ovalbumin-expressing tumor (B16F10-OVA) cells. Spleens were mechanically crushed. After filtration, 10.sup.7 spleen cells per mouse were injected intravenously.

[0230] 1.13 Measurement of Tumor-Specific CD8+ T Cells

[0231] To analyze the number of tumor-specific CD8.sup.+ T cells, cells from the blood were incubated with fluorescent ovalbumin tetramers (four coupled H-2 Kb MHC-I molecules carrying the peptide SIINFEKL, NIH Tetramer Facility) and fluorochrome-coupled anti-CD8 antibodies (eBioscience) and analyzed after washing in a flow cytometer. For the analysis of T cell function, spleen cells were mechanically crushed and incubated after filtration with Brefeldin A and with or without SIINFEKL peptide. After six hours the cells were fixed, permeabilized and stained with fluorescent antibodies specific for CD8 (anti-CD8, eBioscience) and intracellular interferon-gamma (anti-interferon-gamma, eBioscience). The frequency of IFN-gamma producing cells was analyzed by flow cytometry.

[0232] 1.14 Statistical Analysis

[0233] The mean values were compared using an unpaired two-sample Student's t-test. The data are presented as mean±SEM. The level of statistical significance was determined to be p<0.05.

[0234] 1.15 Generation of LCMV-P42:

[0235] LCMV-WE was passaged 42 times in primary tumor cell cultures (UKE-Mel-13a). After 42 passages, a functional mutation was detected in the new virus (LCMV-P42). Nucleic acid and amino acid sequences of LCMV-WE and mutant P42 are shown in FIG. 25 (A and B).

[0236] 1.16 Generation of LCMV-P91, LCMV-P52 and It's Subclones:

[0237] LCMV-WE was passaged 52 times in the tumor cell lines H1975. After 52 passages, the mutations I181M, R185W were detected in the glycoprotein of the new virus. In addition some, most likely irrelevant and/or oligoclonal mutations (quasispecies) were found. This virus is named: “LCMV-P52”. To determine the stability and importance of the mutations I181M and R185W, the supernatant of passage P52 was passaged 39 more times. The virus derived from this passage is named: “LCMV-P91” and still contained the mutations I181M and R185W. The virus LCMV-P52 was subcloned by limiting dilution. This clonal virus is named: “LCMV-P52.1”. The mutations I181M, R185W remained stable. The as irrelevant considered and/or oligoclonal mutations (quasispecies) showed some changes. To determine the role of the single mutations I181M and R185W some earlier passages were analyzed. The passage P29 contained viruses with individual I181M and R185W mutations. Viruses from passage P29 were subcloned by limiting dilution. One subclone with the singular mutation R185W was named: “LCMV-P52-1.3”; one subclone with the singular mutation I181M was named: “LCMV-P52-2.1”. Nucleic acid and amino acid sequences of LCMV mutants P52, P92, P52-1, P52-1.3, and P52-2.1 are shown in FIG. 25 (C-G). Alignments of nucleic acid and amino acid sequences LCMV-WE and mutants P42, P52, P92, P52-1, P52-1.3, and P52-2.1 are shown in FIG. 26 (A-H).

[0238] 2. Investigations

[0239] 2.1 Tumor cell lines MC57, C643, H1975 and primary tumor cell cultures (UKE Mel-13a) were infected with LCMV (strain WE, MOI 1). Replication was measured after 24 hours (n=3).

[0240] Thereby it was proven that LCMV (strain WE) spread differently. In comparison to the tumor cell line MC57, the spread was reduced in the cell lines C643 and H1975 as well as in primary tumor cell cultures (UKE-Mel-13a). The obtained results are shown graphically in FIG. 1.

[0241] In white the LCMV infected cells in the cultures with MC57, C643, H1975 and UKE-Mel-13a can be seen. Ø=without infection, LCMV=LCMV infected.

[0242] 2.2 Tumor cell line MC57 and primary tumor cell cultures 511950 were infected with LCMV (strain WE, MOI 0,1) (left graph). Tumor cell lines MC57, Tramp-C2 and primary tumor cell cultures (511950 and 511950R) were infected with LCMV (strain WE, MOI 1) (right graph). The number of infectious virus particles was measured after 24 hours in the supernatant.

[0243] Thereby it was proven that LCMV (strain WE) proliferate less in comparison to the MC57 tumor cell line in the Tramp-C2 cell line as well as in the primary tumor cell cultures (511950 and 511950R). The results are shown in FIG. 2.

[0244] FIG. 2 has the following legend:

[0245] Ordinate: Infectious LCMV particles in supernatant (PFU/mL),

[0246] Abscissa: Treatment groups; MC57, Tramp-C2, 511950, 511950R.

[0247] 2.3 LCMV-WE was passaged 42 times in primary tumor cell cultures (UKE-Mel-13a). After 42 passages, a functional mutation was detected in the new virus (LCMV-P42).

[0248] Thereby it could be proven that primary tumor cells are suitable for modifying arenaviruses by passaging. The experimental procedure is shown in FIG. 3.

[0249] 2.4 LCMV-WE was passaged 52 times in tumor cell lines H1975. After 52 passages, functional mutations were detected in the new virus LCMV-P52.

[0250] Thereby, using the example of H1975, it was proven that that tumor cell lines with reduced LCMV replication (compared to cell line MC57) are suitable for altering arenaviruses by passaging. The experimental procedure is illustrated in FIG. 4.

[0251] 2.5 LCMV-P52 was passaged 39 times in tumor cell lines H1975. After 39 passages further functional mutations were detected in the new virus LCMV-P91.

[0252] Thereby it was proven that H1975 cells are capable of altering arenaviruses by passaging. The experimental procedure is shown graphically in FIG. 5.

[0253] 2.6 Cell lines H1975 were infected with LCMV-WE and LCMV-P52 (MOI 1). Virus was determined after 12 hours in the supernatant (n=6).

[0254] Thereby it was proven that LCMV-P52 replicate better than LCMV-WE in H1975 cells. The results obtained are shown graphically in FIG. 6.

[0255] FIG. 6 has the following legend:

[0256] Ordinate: Infectious LCMV particles in the supernatant (PFU/mL),

[0257] Abscissa: Treatment groups; LCMV-WE or LCMV-P52

[0258] 2.7 Murine bone marrow-derived dendritic cells were infected with LCMV-WE and LCMV-P52 (MOI 1). Virus was determined after 24 hours in supernatant (n=3).

[0259] Thereby it was proven that LCMV-P52 replicate better than LCMV-WE in antigen-presenting cells. The results obtained are shown graphically in FIG. 7.

[0260] FIG. 7 has the following legend:

[0261] Ordinate: Infectious LCMV particles in the supernatant (PFU/mL),

[0262] Abscissa: Treatment groups; LCMV-WE or LCMV-P52

[0263] 2.8 Tumor cell line H1975 was infected with LCMV-WE or LCMV-P52 (MOI 1). Virus spread was measured by immunofluorescence after 24 hours.

[0264] Thereby it was proven that LCMV-P52 can spread better in tumor cell lines compared to LCMV WE using the example of H1975. The results obtained are shown graphically in FIG. 8.

[0265] Shown in white are the LCMV infected cells for a culture infected with LCMV-WE or LCMV-P52.

[0266] 2.9 Primary tumor cells (UKE-Mel-13a) were infected with LCMV-WE or LCMV-P52 (MOI 1). The spread of the viruses was measured by immunofluorescence after 24 hours.

[0267] Thereby it was proven that LCMV-P52 can spread better in primary tumor cells compared to LCMV-WE. The obtained results are shown graphically in FIG. 9.

[0268] The LCMV infected cells for cultures infected with LCMV-WE or LCMV-P52 are shown in white.

[0269] 2.10 C57BL/6 mice were infected with LCMV-WE or LCMV-P52 (2×10.sup.4 PFU intravenously). One day after infection the activation of the innate immune system was determined by measuring IFN-alpha in serum.

[0270] Thereby it was proven that LCMV-P52 causes a stronger innate immune activation than LCMV-WE. The obtained results are shown graphically in FIG. 10.

[0271] FIG. 10 has the following legend:

[0272] Ordinate: IFN-alpha concentration (ng/mL serum)

[0273] Abscissa: Treatment groups; LCMV-WE or LCMV-P52 infected animals.

[0274] 2.11 C57BL/6 mice were infected with LCMV-WE or LCMV-P52 (2×10.sup.4 PFU intravenously). Activation of the adaptive immune system was determined by flow cytometry using tetramer (measures virus-specific CD8+ T cells).

[0275] Thereby it was proven that LCMV-P52 induces a stronger adaptive immune activation than LCMV-WE. The obtained results are shown graphically in FIG. 11.

[0276] FIG. 11 has the following legend:

[0277] Ordinate: Virus-specific CD8+ T cells (% of total CD8+ T cells in blood),

[0278] Abscissa: Time (days after infection)

[0279] 2.12 NOD.SCID mice were subcutaneously treated with 5×10.sup.5 H1975 cells (day −7). The mice were additionally infected with 2×10.sup.4 PFU LCMV-WE or LCMV-P52 intratumorally (day 0). Tumor growth was analyzed.

[0280] Thereby it was proven that treatment with LCMV-P52 had a stronger anti-tumoral effect than treatment with LCMV-WE. The results obtained are shown graphically in FIG. 12.

[0281] FIG. 12 has the following legend:

[0282] Ordinate: mean tumor diameter (cm),

[0283] Abscissa: Time (days after LCMV treatment)

[0284] 2.13 C57BL/6 mice were subcutaneously treated with 5×10.sup.5 MOPC cells (day −7). A group of mice was additionally infected with 2×10.sup.4 PFU LCMV-P52 intravenously (n=3, day 0). Tumor growth was analyzed.

[0285] Thereby it was proven that treatment with LCMV-P52 had a strong anti-tumoral effect. The results are shown graphically in FIG. 13.

[0286] FIG. 13 has the following legend:

[0287] Ordinate: tumor volume (mm.sup.3),

[0288] Abscissa: Time (days after LCMV treatment)

[0289] 2.14 C57BL/6 mice were subcutaneously treated with 5×10.sup.5 CMT167 cells (day −7). One group of mice was additionally infected with 2×10.sup.4 PFU LCMV-P52 intravenously (n=3, day 0). Tumor growth was analyzed.

[0290] Thereby it was proven that treatment with LCMV-P52 had a strong anti-tumoral effect. The results are shown in FIG. 14.

[0291] FIG. 14 has the following legend:

[0292] Ordinate: tumor volume (mm.sup.3),

[0293] Abscissa: Time (days after LCMV treatment)

[0294] Thereby it was proven that treatment with LCMV-P52 had a strong anti-tumoral effect. The results obtained are shown graphically in FIG. 15.

[0295] In black tumor cells in the lung are shown; Ø=without infection

[0296] 2.15 C57BL/6 mice were treated intravenously with 5×10.sup.5 B16F10-OVA cells (day −7). Tumor-specific CD8.sup.+ T cells isolated from the spleen of an OT-1 mouse were transferred (day −4). A group of animals were additionally treated with LCMV-P52 intravenously (2×10.sup.4 PFU, n=4). On day 9 the lungs were removed and photographed.

[0297] Ø=Control animals, 4 lungs each; LCMV-P52=treated with LCMV-P52, 4 lungs each.

[0298] 2.16 C57BL/6 mice were treated intravenously with 5×10.sup.5 B16F10-OVA cells (day −7). Tumor-specific CD8.sup.+ T cells isolated from the spleen of an OT-1 mouse were transferred (day −4). One group of animals were additionally treated with LCMV-P52 intravenously (2×10.sup.4 PFU, n=4). On day 3, the frequency of tumor-specific CD8.sup.+ T cells in the blood was determined.

[0299] Thereby it was proven that treatment with LCMV-P52 increases the expansion of tumor-specific CD8+ T cells. The results obtained are shown graphically in FIG. 16.

[0300] FIG. 16 has the following legend:

[0301] Ordinate: Frequency of tumor-specific CD8+ T cells in the blood (% of total CD8+ T cells),

[0302] Abscissa: Treatment groups Ø=without infection LCMV-P52=treated with LCMV-P52

[0303] 2.17 C57BL/6 mice were intravenously treated with 5×10.sup.5 B16F10-OVA cells (day −7). Tumor-specific CD8.sup.+ T cells isolated from the spleen of an OT-1 mouse were transferred (day −4). One group of animals were additionally treated with LCMV-P52 intravenously (2×10.sup.4 PFU, n=4). On day 9, the function of tumor-specific CD8.sup.+ T cells in the spleen was determined by in vitro restimulation.

[0304] Thereby it was proven that treatment with LCMV-P52 increases the function of tumor-specific CD8+ T cells. The obtained results are shown graphically in FIG. 17.

[0305] FIG. 17 has the following legend:

[0306] Ordinate: Frequency of IFN-gamma-producing tumor-specific CD8+ T cells (% of total CD8+ T cells),

[0307] Abscissa: Treatment groups; Ø: Without LCMV-P52 treatment; LCMV-P52: Treatment with LCMV-P52; Legend: −: Without antigen; +: Restimulation with antigen (SIINFEKL peptide).

[0308] 2.18 C57BL/6 mice were subcutaneously treated with 5×10.sup.5 B16F10-OVA cells (day −7). One group of animals was not further treated (n=3). One group of animals was treated with the inhibitor LCL-161 (oral 50 mg/kg body weight) twice a week from day 0. One group was treated intratumorally with LCMV-WE on day 0 (2×10.sup.4 PFU, n=4). One group was treated with LCL-161 and LCMV. Tumor growth was analyzed.

[0309] Thereby it was proven that treatment with LCMV-WE has a synergistic effect with LCL-161. The obtained results are shown graphically in FIG. 18.

[0310] FIG. 18 has the following legend:

[0311] Ordinate: Tumor volume (mm.sup.3),

[0312] Abscissa: Time (days after LCMV administration)

[0313] 2.19 C57BL/6 mice were subcutaneously treated with 5×10.sup.5 B16F10-OVA cells (day −7). One group of animals was not further treated (n=3). One group of animals was treated with the inhibitor LCL-161 (oral 50 mg/kg body weight) twice a week from day 0. One group was treated intratumorally with LCMV-WE on day 0 (2×10.sup.4 PFU, n=4). One group was treated with LCL-161 and LCMV. Survival of the animals was analyzed.

[0314] It could be shown that the treatment with LCMV-WE has a synergistic effect with LCL-161. The results obtained are shown graphically in FIG. 19.

[0315] FIG. 19 has the following legend:

[0316] Ordinate: survival of animals (%),

[0317] Abscissa: Time (days after LCMV administration)

[0318] 2.20 C57BL/6 mice were subcutaneously treated with 5×10.sup.5 B16F10-OVA cells (day −9). One group of animals was not further treated (n=3). One group was treated intratumorally with LCMV-P52 on day 0 (2×10.sup.4 PFU, n=6-8). A group of animals was treated with the checkpoint blocker anti-PD-1 (200 μg. intraperitoneal) on days 1, 5 and 8. One group was treated with checkpoint blocker anti-PD-1 and LCMV-P52. Tumor growth was analyzed.

[0319] Thereby it was proven that the treatment with LCMV-P52 has a synergistic effect with checkpoint blockers (e.g. anti-PD-1). The results obtained are shown graphically in FIG. 20.

[0320] FIG. 20 has the following legend:

[0321] Ordinate: tumor volume (mm.sup.3),

[0322] Abscissa: Time (days after LCMV treatment)

[0323] The amino acid and nucleic acid sequences mentioned in the present description correspond to the amino acid and nucleic acid sequences disclosed in the following sequence listing.

[0324] 2.21 10.sup.5 H1975 cells were seeded in 24 well plates. After 24 hours cells were infected with the viruses LCMV-WE, LCMV-P52.1 (I181M, R185W), LCMV-P52-1.3 (R185W) and LCMV-P52-2.1 (I181M) with a multiplicity of infection (MOI) of 0.1. Virus was analyzed in the supernatant after 24 hours. The results are shown in FIG. 21 (mean+SEM, n=6, *p<0.05, t-test).

[0325] The data show that the mutations I181M and R185W increase the viral propagation in tumor cells separately.

[0326] FIG. 21 has the following legend:

[0327] X-axis: Different viruses

[0328] Y-axis: LCMV in the supernatant (log.sub.10 PFU/ml)

[0329] 2.22 2.5×10.sup.5 HCC1954 cells were seeded in 24 well plates, followed by infection of LCMV-WE, LCMV-P52.1 (I181M, R185W), LCMV-P52-1.3 (R185W) and LCMV-P52-2.1 (I181M) with a multiplicity of infection (MOI) of 0.001. Virus was analyzed in the supernatant after 48 hours. The results are shown in FIG. 22 (error bars show SEM, n=4, **p<0.001, n.s. indicates not significant, one-way ANOVA with an additional Tukey post-test was used).

[0330] The data show that either the mutation I181M or the mutation 185W increase viral propagation in HCC1954 tumor cells.

[0331] FIG. 22 has the following legend:

[0332] X-axis: Different viruses

[0333] Y-axis: LCMV in the supernatant (log.sub.10 PFU/ml)

[0334] 2.23 Murine Pancreatic cancer cells (511950, 4×10.sup.5 cells), human Melanoma cells (UKE118b, 4×10.sup.5 cells) or (UKE118c, 4×10.sup.5 cells) were seeded in 24 well plates, followed by infection with LCMV-WE (white bars) or LCMV-P42 (I181M, black bars) with a multiplicity of infection (MOI) of 0.1. Virus was analyzed in the supernatant after 24 hours. The results are shown in FIG. 23 (mean+SEM, n=3, *p<0.05, t-test).

[0335] The data show that the mutation I181M leads to increase viral propagation in tumor cells.

[0336] FIG. 23 has the following legend:

[0337] X-axis: Different cell types

[0338] Y-axis: LCMV in the supernatant (log.sub.10 PFU/ml)

[0339] 2.24 C57BL/6 mice were infected intravenously with 2×10.sup.4 PFU of either LCMV-WE, LCMV-P52.1 (I181M, R185W), LCMV-P52-1.3 (R185W) and LCMV-P52-2.1 (I181M). On day 8 (white bars) and day 10 (black bars) blood was analyzed for the frequencies of LCMV-GP33-specific CD8.sup.+ T cells by using tetramers in flow cytometry. The results are shown in FIG. 24 (mean+SEM, n=6-12, pooled from four separate experiments, *p<0.05, t-test).

[0340] The data show that the mutations I181M and R185W increase the capacity of LCMV to stimulate specific T cells. The combination of the mutations I181M and R185W might have synergistic properties on early T cell stimulation.

[0341] FIG. 24 has the following legend:

[0342] X-axis: Different viruses

[0343] Y-axis: Frequency of tetramer-GP33-binding CD8.sup.+ T cells (% of total CD8.sup.+ T cells).

[0344] The invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by exemplary embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[0345] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.