ARENAVIRUSES FOR USE IN THE TREATMENT AND/OR PREVENTION OF TUMORS AND METHOD FOR PRODUCING ARENAVIRUSES WITH (IM-PROVED) TUMOR-REGRESSION PROPERTIES

Abstract

The invention relates to arenaviruses for use in the treatment and/or prevention of tumors and also a method for preparing arenaviruses with (improved) tumor-regressive properties.

Claims

1. A method of treating and/or preventing a tumor comprising administering to a subject in need thereof an arenavirus capable of effecting tumor regression by activating innate (congenital) and/or adaptive immune cells.

2. The method of claim 1, wherein the method comprises activating innate (congenital) or adaptive immune cells and thereby effecting tumor regression.

3. The method of claim 1, wherein the method comprises effecting increased secretion of anti-tumoral cytokines by immune cells by activating said immune cells.

4. The method of claim 4, wherein the anti-tumoral cytokine is interferon-, interferon- or interferon-.

5. The method of claim 1, wherein the arenavirus is a wild-type arenavirus.

6. The method of claim 1, wherein the arenavirus is a natural mutant of a wild-type arenavirus.

7. The method of claim 1, wherein the arenavirus has been obtained starting from its wild-type form by serial passage in a host animal and/or a host cell.

8. The method of claim 1, wherein the tumor is selected from the group consisting of carcinoma, melanoma, blastoma, lymphoma, and sarcoma.

9. The method of claim 1, wherein the arenavirus is an Old World arenavirus.

10. The method of claim 1, wherein the arenavirus is lymphocytic choriomeningitis virus.

11. The method of claim 10, wherein the arenavirus comprises a nucleic acid comprising an S-ribonucleic acid sequence according to SEQ ID No. 5 or ambisense sequence thereof, or a nucleic acid comprising an L-ribonucleic acid sequence according to SEQ ID No. 6 or ambisense sequence thereof.

12. The method of claim 10, wherein the lymphocytic choriomeningitis virus is a lymphocytic choriomeningitis virus strain WE or a natural mutant thereof.

13. The method of claim 1, wherein the arenavirus is a New World arenavirus.

14. The method of claim 1, wherein the arenavirus is a Junin virus.

15. The method of claim 14, wherein the arenavirus comprises a nucleic acid comprising an S-ribonucleic acid sequence according to SEQ ID No. 1 or 3 or ambisense sequence thereof, or a nucleic acid comprising an L-ribonucleic acid sequence according to SEQ ID No. 2 or 4 or ambisense sequence thereof.

16. The method of claim 14, wherein the Junin virus is strain Candid #1.

17. The method of claim 1, wherein the subject is a human.

18. A pharmaceutical composition comprising an arenavirus of claim 1 and, optionally a pharmaceutically acceptable carrier.

19. A method for generating an arenavirus with tumor-regressive or improved tumor-regressive properties, in particular an arenavirus according to claim 1, comprising: a) infecting dendritic cells or tumor cells with an arenavirus, b) culturing the arenavirus in the infected dendritic cells or infected tumor cells and c) isolating the cultured arenavirus or a subset of the cultured arenavirus from the infected dendritic cells or infected tumor cells.

20. The method of claim 19, wherein the sequence of steps a) to c) is repeated once to 1000 times, particularly 10 times to 100 times, preferably 30 times to 60 times, wherein new tumor cells, preferably of the same type, are used for each repetition.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0113] FIG. 1 shows LCMV (WE strain) replication results in immortalized macrophages (tumour) and macrophages (primary) generated from primary bone marrow.

[0114] FIG. 2 shows the effect of LCMV (WE strain) administration on MOPC (mouse plasmacytoma cell) tumor growth in C57BL/6 mice.

[0115] FIG. 3 shows the effect of LCMV (WE strain) administration on MC38 (mouse colon adenocarcinoma cell) tumor growth in C57BL/6 mice.

[0116] FIG. 4 shows the effect of LCMV (WE strain) administration on spontaneous liver carcinoma tumor growth in LoxP-TAg mice.

[0117] FIG. 5 shows the effect of LCMV (WE strain) on interferon- secretion in MOPC tumor-bearing C57BL/6 mice.

[0118] FIG. 6 shows the effect of LCMV (WE strain) administration on MOPC tumor growth in Map3k14.sup.aly/aly mice compared to corresponding wild type mice.

[0119] FIG. 7 shows the effect of LCMV (WE strain) administration on MOPC tumor growth in Irf3Ir7.sup./ mice compared to corresponding wild type mice.

[0120] FIG. 8A shows effect of LCMV (WE strain) administration on tumor microvessel density (MDV) and vessel-vessel spacing in MOPC tumor-bearing wild type mice.

[0121] FIG. 8B shows effect of LCMV (WE strain) administration on tumor hypoxic regions in MOPC tumor-bearing wild type mice.

[0122] FIG. 9 shows the effect of LCMV (WE strain) administration (ipsilateral, contralateral, and intravenous) on MOPC tumor growth in C57BL/6 mice.

[0123] FIG. 10 shows effect of LCMV (WE strain) administration on survival in A431 (squamous carcinoma) tumor-bearing NOD.SCID mice.

[0124] FIG. 11 shows effect of LCMV (WE strain) administration on tumor growth in A431 (colon adenocarcinoma) tumor-bearing NOD.SCID mice.

[0125] FIG. 12 shows effect of Candid #1 administration on tumor growth in HeLa cell-bearing NOD.SCID mice.

[0126] FIG. 13 shows effect of Candid #1 administration on tumor growth in HepG2 (hepatocellular carcinoma) tumor-bearing NOD.SCID mice.

[0127] FIG. 14 shows LCMV (MOI 1) replication results in primary hepatocytes, colon epithelial cells, and melanocytes) and in tumour cells from the same tissue source.

[0128] FIG. 15A shows the effect of LCMV administration on metastatic MOPC tumor growth in C57BL/6 mice.

[0129] FIG. 15B shows the effect of LCMV administration on survival in MOPC metastatic tumor-bearing C57BL/6 mice.

[0130] FIG. 16 shows the effect of LCMV administration on B16F10 (melanoma) tumor growth in C57BL/6 mice.

[0131] FIG. 17 shows the effect of LCMV administration on spontaneous melanoma tumor growth in MT/ret mice.

[0132] FIG. 18A shows the effect of LCMV administration on Sw872 (fibrosarcoma) tumor growth in NOD.SCID mice.

[0133] FIG. 18B shows the effect of LCMV administration on survival in Sw872 (fibrosarcoma) tumor-bearing NOD.SCID mice.

[0134] FIG. 19A shows the effect of LCMV administration on FaDu (pharyngeal carcinoma) tumor growth in NOD.SCID mice.

[0135] FIG. 19B shows the effect of LCMV administration on survival in FaDu (pharyngeal carcinoma) tumor-bearing NOD.SCID mice.

[0136] FIG. 20 shows the effect of LCMV on T-cell expression of PD-1, IL2 receptor, and IL7 receptor in B16F10 tumor-bearing mice.

[0137] FIG. 21A shows the effect of LCMV administration on EL4 (lymphoma) tumor growth in C57BL/6 mice.

[0138] FIG. 21B shows the effect of LCMV administration on survival in EL4 (lymphoma) tumor-bearing C57BL/6 mice.

[0139] FIG. 22 shows the effect of LCMV administration on survival in MOPC tumor-bearing C57BL/6 and Pdcd1.sup./ mice.

EXPERIMENTAL SECTION

1. Methods and Materials

1.1 Mice

[0140] Unless mentioned otherwise, the mice used were from a C57BL/6 background. Map3k14.sup.aly/aly mice lack NF-kB signals and are therefore highly immunosuppressed. Irf3Ir7.sup./ 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.

1.2 Cell Lines and Reagents

[0141] 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/l L-glutamine and 100 U/ml penicillin. All cells were cultured in 5% CO2.

1.3 Viruses

[0142] 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).

1.4 Tumor Growth and Treatments

[0143] Approximately 510.sup.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 210.sup.4 PFU LCMV-WE or Candid #1 (in 100-200 microL).

1.5 Morphometric Analysis of Tumor Vessels

[0144] Morphometric analyses were performed with successive frozen sections, in which the endothelial cell marker CD31 was stained. Quantification of the microvessel density (MVD) was calculated using the mean of three tumor sections. MVD was calculated as the number of vessels per tumor area.

1.6 Detection of Hypoxia

[0145] 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.).

1.7 IFN- ELISA

[0146] Serum IFN- levels were determined by ELISA according to the manufacturer's data (Research Diagnostics RDI, Flanders, N.J.).

1.8 Statistical Analysis

[0147] The mean values were compared using an unpaired two-sided student t-test. The data are shown as meanSEM. The level of statistical significance was set at p<0.05.

2. Investigations

[0148] 2.1 Immortalized macrophages (tumour cells) and macrophages (primary) generated from primary bone marrow were infected with LCMV (WE strain). Replication was measured after 24 hours (n=3).

[0149] It could be shown that LCMV (WE strain) replicates both in immortalized and healthy cells. The results obtained are shown graphically in FIG. 1. [0150] FIG. 1 has the following legend: [0151] Ordinate: LCMV (log.sub.10 PFU/ml) [0152] Abscissa: Tumor cells/healthy macrophages (primary)

[0153] 2.2 WT C57BL/6 mice were treated with 510.sup.5 MOPC cells (day 3). One group of mice was additionally treated peritumorally with 210.sup.4 PFU LCMV (WE strain) (day 0). Tumor growth was observed. [0154] It could be shown that the treatment with LCMV caused almost complete tumor regression. The results obtained are shown graphically in FIG. 2. [0155] FIG. 2 has the following legend: [0156] Ordinate: Tumor diameter (cm) [0157] Abscissa: Time (days)

[0158] 2.3 WT C57BL/6 mice were treated with 510.sup.5 MC38 cells (day 3). One group of mice was additionally treated peritumorally with 210.sup.4 PFU LCMV (WE strain) (day 0). Tumor growth was observed. [0159] It could be shown that the treatment with LCMV caused a significant tumor regression. The results obtained are shown graphically in FIG. 3. [0160] FIG. 3 has the following legend: [0161] Ordinate: Tumor diameter (cm) [0162] Abscissa: Time (days)

[0163] 2.4 About nine month old LoxP-TAg mice with spontaneously developed liver carcinomas were infected intravenously with 210.sup.6 PFU LCMV or left untreated. The tumor nodes (diameters>=3 mm) were quantified macroscopically on day 6 (n=3) and day 20 (n=4-5). [0164] It could be shown that the treatment with LCMV significantly reduced the number of carcinomatous liver nodes. The results obtained are shown graphically in FIG. 4. [0165] FIG. 4 has the following legend: [0166] Ordinate: Liver nodes (number) [0167] Abscissa: Time

[0168] 2.5 WT C57BL/6 mice (n=4/group) were injected subcutaneously with 510.sup.5 MOPC cells (day 3) or LCMV (WE strain) 210.sup.4 PFU (day 0) or both 510.sup.5 MOPC cells (day 3) and 210.sup.4 PFU LCMV (day 0). Serum samples were collected on day 3 and an IFN--ELISA was performed. [0169] It could be shown that the LCMV caused a drastically increased secretion of interferon- in experimental animals which were simultaneously administered carcinoma cells. The results obtained are shown graphically in FIG. 5. [0170] FIG. 5 has the following legend: [0171] Ordinate: IFN- (pg/ml)

[0172] 2.6 Map3k14.sup.aly/aly mice and WT mice were treated with 510.sup.5 MOPC cells (day 3). One group of mice was additionally treated peritumorally with 210.sup.4 PFU LCMV (WE strain) (day 0). Tumor growth was observed. [0173] It could be shown that the treatment with LCMV caused tumor regression. The results obtained are shown graphically in FIG. 6. [0174] FIG. 6 has the following legend: [0175] Ordinate: Tumor diameter (cm) [0176] Abscissa: Time (days)

[0177] 2.7 Irf3Ir7.sup./ mice and WT mice were treated with 510.sup.5 MOPC cells (day 3). One group of mice was additionally treated peritumorally with 210.sup.4 PFU LCMV (WE strain) (day 0). Tumor growth was observed. [0178] It could be shown that the treatment with LCMV caused tumor regression. The results obtained are shown graphically in FIG. 7. [0179] FIG. 7 has the following legend: [0180] Ordinate: Tumor diameter (cm) [0181] Abscissa: Time (days)

[0182] 2.8 WT mice were treated with 510.sup.5 MOPC cells (day 3). One group of mice was additionally treated peritumorally with 210.sup.4 PFU LCMV (WE strain) (day 0). On day 9 after the tumor graft, the tumors were analyzed histologically with CD31 staining. The microvessel density (MDV) and the vessel-vessel spacing were quantified. [0183] It could be shown that the treatment with LCMV caused a decrease in tumor vessel density. The results obtained are shown graphically in FIG. 8A. [0184] FIG. 8A has the following legends:

TABLE-US-00001 Left side: Right side: Ordinate: MVD [mm.sup.3] Ordinate: Vessel-vessel spacing (m) Abscissa: Tumor/Tumor LCMV Abscissa: Tumor/Tumor LCMV

[0185] 2.9 WT mice were treated with 510.sup.5 MOPC cells (day 3). One group of mice was additionally treated peritumorally with 210.sup.4 PFU LCMV (WE strain) (day 0). On day 9, the animals were injected with pimonidazole, and the tumors were then analyzed histologically for hypoxic regions. [0186] It could be shown that the treatment with LCMV caused oxygen deficiency in the carcinoma tissue. The results obtained are shown graphically in FIG. 8B. [0187] FIG. 8B has the following legend: [0188] Ordinate: Hypoxic regions/tumor (%) [0189] Abscissa: Tumor/Tumor LCMV

[0190] 2.10 WT C57BL/6 mice were injected subcutaneously with 510.sup.5 MOPC cells in the right flank (day 3). On day 0, a group of animals were treated with 210.sup.4 PFU LCMV (WE strain) in the right flank (ipsilateral), left flank (contralateral) or intravenously. Tumor growth was observed. [0191] It could be shown that the treatment with LCMV caused tumor regression even with systemic administration. The results obtained are shown graphically in FIG. 9. [0192] FIG. 9 has the following legend: [0193] Ordinate: Tumor diameter (cm) [0194] Abscissa: Time (days)

[0195] 2.11 NOD.SCID mice were injected subcutaneously with 510.sup.5 A431 cells (day 3) and then either left untreated or treated with 210.sup.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. [0196] It could be shown that the treatment with LCMV increased the survival rate in the experimental animals. The results obtained are shown graphically in FIG. 10. [0197] FIG. 10 has the following legend: [0198] Ordinate: Survival (%) [0199] Abscissa: Time (days)

[0200] 2.12 NOD.SCID mice were treated with 510.sup.5 Sw40 cells (day 0). A group of mice was additionally treated peritumorally with 210.sup.4 PFU LCMV (WE strain) or 210.sup.4 PFU Candid #1 (day 0). Tumor growth was observed. [0201] It could be shown that the treatment with LCMV and Candid #1 caused tumor regression. The results obtained are shown graphically in FIG. 11. [0202] FIG. 11 has the following legend: [0203] Ordinate: Tumor diameter (cm) [0204] Abscissa: Time (days)

[0205] 2.13 NOD.SCID mice were treated with 510.sup.5 Hela cells (day 0). A group of mice was additionally treated peritumorally with 210.sup.4 PFU LCMV (WE strain) (day 3). Tumor growth was observed. [0206] It could be shown that the treatment with LCMV caused tumor regression. The results obtained are shown graphically in FIG. 12. [0207] FIG. 12 has the following legend: [0208] Ordinate: Tumor diameter (cm) [0209] Abscissa: Time (days)

[0210] 2.14 NOD.SCID mice were treated with 510.sup.5 HepG2 cells (day 10) and then additionally treated peritumorally with or without 210.sup.4 PFU Candid #1 (day 0). Tumor growth was observed. [0211] It could be shown that the treatment with Candid #1 caused tumor regression with this tumor type. The results obtained are shown graphically in FIG. 13. [0212] FIG. 13 has the following legend: [0213] Ordinate: Tumor diameter (cm) [0214] Abscissa: Time (days)

[0215] 2.15 Primary human cells (hepatocytes, colon epithelial cells, melanocytes) and tumour cells from the same tissue source were infected with LCMV (MOI 1). The amount of virus was measured in the supernatant after 1, 2 and 3 days. [0216] In this experiment it was shown that arenaviruses are replicated in tumor cells in comparison to healthy tissue. [0217] FIG. 14 has the following legend: [0218] Ordinate: Infectious virus in cell culture supernatant (logarithmic plaque forming units) [0219] Abscissa: Time (days)

[0220] 2.16 Tumor diameter (A) and survival (B) of C57BL/6 mice bearing a metastasis in the shoulder and a metastasis in the flank (MOPC cells), which were left untreated or had been treated intravenously with 210.sup.6 PFU LCMV. [0221] It could be shown in this experiment that intravenous therapy of LCMV acts very efficiently on two local metastases and thus prolongs survival. [0222] FIG. 15A has the following legend: [0223] Ordinate: Tumor diameter of both metastases (cm) [0224] Abscissa: Time (days)
FIG. 15B has the following legend: [0225] Ordinate: Survival in percent [0226] Abscissa: Time (days)

[0227] 2.17 Tumor diameter of C57BL/6 mice bearing a melanoma (B16F10 cells) which were left untreated or were treated intratumorally with 210.sup.4 PFU LCMV. [0228] It could be shown in this experiment that local therapy with LCMV is very efficient in melanoma. [0229] FIG. 16 has the following legend: [0230] Ordinate: Tumor diameter of the melanoma (cm) [0231] Abscissa: Time (days)

[0232] 2.18 Number of melanomas of MT/ret mice (develop endogenous melanomas), which were left untreated or were treated intravenously with 210.sup.6 PFU LCMV. [0233] It could be shown in this experiment that systemic therapy with LCMV is very efficient in melanoma. [0234] FIG. 17 has the following legend: [0235] Ordinate: Number of melanomas

[0236] 2.19 Tumor diameter (A) and survival (B) of NOD.SCID mice bearing a human fibrosarcoma (Sw872 cells), which were left untreated or were treated intratumorally with 210.sup.6 PFU Candid #1. [0237] It could be shown in this experiment that Candid #1 acts very efficiently also in the case of fibrosarcoma and thus prolongs survival. [0238] FIG. 18A has the following legend: [0239] Ordinate: Tumor diameter (cm) [0240] Abscissa: Time (days)
FIG. 18B has the following legend: [0241] Ordinate: Survival in percent

[0242] Abscissa: Time (days)

[0243] 2.20 Tumor diameter (A) and survival (B) of NOD.SCID mice bearing a human pharyngeal carcinoma (FaDu cells), which were left untreated or were treated intratumorally with 210.sup.6 PFU LCMV. [0244] It could be shown in this experiment that LCMV acts very efficiently also in the case of pharyngeal carcinoma and thus prolongs survival. [0245] FIG. 19A has the following legend: [0246] Ordinate: Tumor diameter (cm) [0247] Abscissa: Time (days)
FIG. 19B has the following legend: [0248] Ordinate: Survival in percent [0249] Abscissa: Time (days)

[0250] 2.21 Expression of receptors on tumor-specific T cells (PD-1, IL2 receptor, IL7 receptor), which influence the function of T cells. Tumor-specific T cells are derived from the blood of mice with B16F10 tumors, which were additionally treated intratumorally with or without LCMV. [0251] It could be shown in this experiment that LCMV positively influences the tumor-specific T cells. [0252] FIG. 20 has the following legend: [0253] Ordinate: Potency of the expression of the different receptors (mean fluorescence intensity)

[0254] 2.22 Tumor diameter (A) and survival (B) of C57BL/6 mice bearing a murine subcutaneous lymphoma (EL4 cells) which were treated with or without tumor-specific T cells (OT1 cells) and additionally intratumorally with or without LCMV (210.sup.6 PFU). [0255] It could be shown in this experiment that LCMV acts synergistically with T cell therapy. [0256] FIG. 21A has the following legend: [0257] Ordinate: Tumor diameter (cm) [0258] Abscissa: Time (days)
FIG. 21B has the following legend: [0259] Ordinate: Survival in percent [0260] Abscissa: Time (days)

[0261] 2.23 Survival of C57BL/6 mice and PD-1 deficient mice (Pdcd1.sup./ mice) bearing a murine pharyngeal carcinoma (MOPC cells) and which were treated intratumorally with LCMV (210.sup.4 PFU). [0262] It could be shown in this experiment that LCMV acts synergistically with a PD-1 blockade. [0263] FIG. 22 has the following legend: [0264] Ordinate: Survival in percent [0265] Abscissa: Time (days)

[0266] The 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.