Immuno-modulated replication-efficient vaccinia virus strain

Abstract

The invention refers to new immuno-modulated replication-efficient Vaccinia virus strain (IOVA) and its derivatives for the use in medicine.

Claims

1. A genetically modified, immuno-modulating Vaccinia virus, wherein the virus is identified by the presence of SEQ ID No.: 1, has a functional active K1L, is replication competent in mammalian cells, and induces calreticulin transiocation to the membrane of an infected HeLa cell.

2. The Vaccinia virus according to claim 1, wherein the virus has, additionally, a functionally inactivated A56R.

3. The Vaccinia virus according to claim 1, wherein the virus has a functionally inactivated A26R.

4. The Vaccinia Virus according to claim 1, wherein the virus additionally comprises one or more functionally inactivated immune-evasion genes selected from the group of open reading frames consisting of B21R, C10L, C9L, C4L, M1L, A51R, A52R, A55R, and B13R/B14R.

5. The Vaccinia virus according to claim 1, wherein the virus comprises, additionally, at least one functionally inactivated, partially deleted or fully deleted gene selected from the group consisting of J2R, C11R, and F4L.

6. The Vaccinia virus according to claim 1, wherein the virus is replication-competent and lytic in cell-cycle-activated cells and/or tumour cells.

7. The Vaccinia virus according claim 1, wherein the virus, upon infection, causes the formation of syncytia.

8. A nucleic acid sequence or fragment thereof encoding the Vaccinia virus according to claim 1.

9. A viral vector comprising the nucleic acid sequence according to claim 8.

10. The Vaccinia virus according to claim 1, characterised by carrying one or more insertion sites with at least one insertion of one or more transgenes.

11. The recombinant Vaccinia virus according to claim 10, wherein the transgene is selected from the group comprising genes encoding tumour antigens, tumour associated antigens, disease associated antigens, and pathogen-derived antigens.

12. A method of treating a subject, the method comprising administering to the subject the Vaccinia virus according to claim 1.

13. A method of treating cancer in a subject, the method comprising administering to the subject the Vaccinia virus according claim 1.

14. A pharmaceutical composition comprising the Vaccinia virus according to claim 1 and a pharmaceutically acceptable carrier, diluent, or excipient.

Description

SHORT DESCRIPTION OF THE FIGURES

(1) FIG. 1. Syncytia formation in cancer cells after infection with IOVA viruses. Fluorescent photomicrographs (40×) are shown after infection of (a) human or (b) mouse cancer cell lines (24 hours post-infection). HeLa and CT26 cells were infected with an MOI of 0.5. 143B, MCF-7, and LLC1 were infected with an MOI of 5. mCherry is expressed from all the viruses under the P11 promoter. Massive fusion of cells (syncytium) can be observed when cells were infected with IOVA/A56−.

(2) FIG. 2. Viral production of IOVA viruses in human and mouse tumour cells. (a) Human and (b) mouse tumour cell lines were infected with WR/TK−, IOVA/A56−, or IOVA/A56+ at an MOI of 5, and progeny was measured by plaque-assay at different time points. Viral yield was evaluated in quadruplicate for each cell line, by carrying two independent experiments. Means+SD are plotted. *, significant p<0.05 compared with WR/TK−. #, significant p<0.05 compared with IOVA/A56+.

(3) FIG. 3. IOVA viruses present increased cytotoxicity to tumour cells. Cancer cells were infected with WR/TK−, IOVA/A56−, or IOVA/A56+ at doses ranging from 100 to 0.0005 PFU/cell. At day 3 after infection, viability of cells was determined. Both human (a) and mouse (b) cancer cells were tested. Four different replicates were quantified for each cell line and mean±SD of each MOI is depicted.

(4) FIG. 4. Induction of Immunogenic Cell Death by IOVA viruses. (a) Analysis of Calreticulin expression on the surface of infected cells. Indicated tumour cell lines were infected with WR/TK−, IOVA/A56−, or IOVA/A56+ with an MOI of 5, and 24 hours after infection Calreticulin+ cell populations were determined by flow cytometry. Uninfected cells (Mock) and Staurosporin 1 μM were used as negative and positive controls, respectively. (a) Percentage of Calreticulin+ cells. Values of individual replicates and means±SEM of the different treatments are plotted. (b-c) Concentration of HMGB1 (b) and ATP (c) in cell supernatant after infection with IOVA viruses. ELISA assays and ENLITEN ATP assay system were utilized, respectively, to determine such concentrations at 24 hours after infection (MOI of 5) of indicated tumour cell lines. Data were obtained in quadruplicate and are plotted as fold change versus WR/TK−+SD. *, significant p<0.05 compared with WR/TK−. #, significant p<0.05 compared with Mock.

(5) FIG. 5. PCR Assay for Virus identification. FIG. 5a: Sequence of the PCR product employed in the PCR Assay. FIG. 5b: Results of the PCR assay clearly identify the sequence of IOVA strain as the assay generates a unique pattern of two bands (2320 and 920 bp) for IOVA, in contrast to all the other Vaccinia virus strains, which present a single band of 3300 bp for WR, COP and CVA. MVA strain generates no amplified product due to a C2L deletion.

(6) FIG. 6. Number of nuclei in syncytia after infection with IOVA viruses. Human tumour cell lines were infected with WR/TK−, IOVA/A56−/A26−, or IOVA/A56+/A26− at an MOI of 5. At 16 hours post-infection, cultures were dyed with Hoechst 33342 and the number of nuclei in one syncytium were counted under the microscope. Values of individual replicates and means±SEM are plotted. ***, significant p<0.0001 compared with WR/TK−.

(7) FIG. 7. Size of the plaques in cancer cells. Cancer cell monolayers were infected with indicated viruses for 1 hour at an MOI of 0.0001 and cultivated for 4 days covered with a 1:1 mixture of culture media and 1% carboxymethylcellulose. After fixation and staining with crystal violet, the diameter of the plaques was determined. Values of individual replicates and means±SEM are plotted. *, significant p<0.05 compared with WR/TK−. #, significant p<0.05 compared with IOVA/A56−.

EXAMPLES

Example 1: Deletions in IOVA Genome

(8) A novel Immune-Oncolytic Vaccinia virus (IOVA) strain is generated incorporating a deletion in the thymidine kinase (TK, J2R) gene in order to confer selective replication in cancer cells. Additionally, the mCherry gene has been cloned into the TK site under the control of the Vaccinia virus-specific promoter P11 in order to monitor virus replication.

(9) In addition, the newly generated IOVA contains several deletions or functional inactivations among genes considered to be immune modulators and selected from the ORF of B21R*, C10L, C9L, C4L, C2L, N1L, N2L, M1L, A26R, A51R, A52R, A55R, A56R, and B13R/B14R. The functions of the proteins coded for these genes are summarized in Table 1 above.

(10) The IOVA genome is further characterised by the inclusion of a mutated version of the A26R and/or the A56R gene. The presence of A26 protein in the virion prevents direct virus-cell fusion mechanism and its deletion has been associated with induction of syncytia. A56R encodes a viral regulatory protein with haemagglutination activity, and its inactivation in Vaccinia virus is believed to result in viruses with a fusogenic phenotype. All gene deletions or partial deletions as well as all functional inactivation or gene insertions have been confirmed by sequencing.

Example 2: Syncytia Formation Induced by IOVA

(11) In order to evaluate the pros and cons of syncytia formation for tumour destruction, the inventors restored by homologous recombination the wild-type Vaccinia virus A56R gene sequence in the novel IOVA genome. The resulting virus was named IOVA/A56+, in comparison to the IOVA with the truncated A56R version, which was named IOVA/A56−.

(12) Upon infection, it was observed (FIG. 1), that cells infected with the IOVA/A56− virus fuse with neighboring cells, and a formation of huge syncytia could be clearly observed in both human and mouse tumour cell lines traced by the mCherry expression. As hypothesized, the expression of wild-type A56 in IOVA/A56+ restored a phenotype very similar to Vaccinia virus WR strain and did not lead to syncytia formation.

(13) Additionally, it was observed that The expression of wild-type A56 in IOVA/A56−/A26− may be described also as only partially blocking the formation of syncytia, as still a fusion of up to 10 cells could be observed after infection with IOVA/A56+/A26− (FIG. 6).

Example 3: Replication Competence of IOVA

(14) We tested the replication competence of IOVA in comparison with the standard strain Vaccinia virus WR in a wide panel of human and mouse cancer cell lines.

(15) For monitoring the replication of the newly generated IOVA, the replication capacity of two IOVA virus isolates (A56− and A56+) and WR/TK− as a control was tested in several human and mouse cancer cell lines.

(16) For this, human and mouse tumour cell lines were infected with WR/TK−, IOVA/A56−, or IOVA/A56+ at an MOI of 5, and progeny was measured by plaque-assay at different time points. Viral yield was evaluated in quadruplicate for each cell line, by carrying two independent experiments (previously described in: Rojas J J et al., Cell Rep. 2016).

(17) As shown in FIG. 2, both IOVA viruses (A56− and A56+) performed a growth curve very similar to control strain WR/TK− in most cell lines tested, with only a slight reduction of the yield at early time points for the syncytia-forming IOVA.

Example 4: Cytotoxicity of IOVA

(18) We examined the cytotoxic effect of an IOVA infection in comparison with the standard strain Vaccinia virus WR in a wide panel of human and mouse cancer cell lines.

(19) For this, various cancer cell lines were infected with WR/TK−, IOVA/A56−, or IOVA/A56+ at doses ranging from 100 to 0.0005 PFU/cell. At day 3 after infection, viability of cells was determined. Both human and mouse cancer cells were tested. Four different replicates were quantified for each cell line and mean±SD of each MOI is depicted.

(20) Interestingly, infections with the IOVA viruses resulted in clearly enhanced levels of cytotoxicity in cancer cell compared to Vaccinia virus WR (FIG. 3). Vaccinia virus WR was able to kill around 70-80% of the cancer cells in culture, even when at the highest multiplicity of infection (MOI). On the contrary, IOVA/A56− virus was able to kill between 95-100% of the cultured tumour cells and reduced the EC50 (amount of virus necessary to kill 50% of the cells).

(21) Surprisingly, IOVA/A56− decreased the amount of virus required to reduce 50% of cell culture viability of HeLa cells by more than 40 fold compared to Vaccinia virus WR (WR/TK−). IOVA/A56+ also presented a phenotype with enhanced cytotoxicity for cancer cell in vitro; the A56-restored virus killed HeLa, CT-26, and LLC1 cells at similar rates as IOVA/A56−. Yet in 143B and MCF-7 cells the cytotoxicity was very similar to that obtained with WR/TK− infection, suggesting that virus-mediated big syncytia formation may contribute to enhanced destruction of tumours.

Example 5: Large Plaque Phenotype of IOVA Viruses in Cancer Cells

(22) An increased size of the plaques in cancer cells has been associated to a better spread of the virus throughout the tumour and to a higher antitumor activity. For testing the plaque size of IOVA viruses, a panel of cancer cell lines were infected at a MOI of 0.0001 and, after 1 hour of infection, the infected cells were cultured with a carboxymethylcellulose overlay. For 4 after the infection, the cultures were fixed and dyed with crystal violet and the diameter of the plaques were determined.

(23) As shown in FIG. 7, both IOVA/A56− and IOVA/A56+ induced larger plaques in all cancer cell lines tested compared to WR/TK− virus control. In HeLa cells, plaques after infection with IOVA viruses were as a mean 40% larger compared to WR/TK−. Impressively, very large plaques could be observed after infection of 143B and MCF-7 with IOVA viruses, with plaques 2 times the diameter of plaques generated by WR/TK− in the case of 143B cells and plaques 2.6 time larger in the case of MCF-7.

(24) With regard to plaque size, the generation of big syncytia by IOVA/A56− do not have a significant influence except in the case of MCF-7 breast cancer cell line, were IOVA/A56+ virus generated plaques 1.4 times smaller compared to IOVA/A56−.

Example 6: IOVA Viruses Induce Presentation of Calreticulin on Cellular Membrane and Immunogenic Cell Death of Cancer Cells

(25) In order to test whether the IOVA viruses may be able to elicit and potentiate an immune response against cancer cells, the inventors initially analysed by flow cytometry the exposure of calreticulin (CRT) on the surface of human cancer cells after infection with IOVA/A56−, IOVA/A56+, or the control virus WR/TK−.

(26) For this, cells were infected with an MOI of 5 and, 24 hours after virus infection, detached using a non-enzymatic cell dissociation solution. Calreticulin was detected by incubating the cells for 1 hour at 4° C. with a human anti-calreticulin-AlexaFluor405 antibody (Abcam, Ref N° ab210431). Uninfected cells and staurosporin (1 μM) were used as negative and positive controls, respectively.

(27) Upon infection of HeLa cells, WR/TK− induced a surface-exposure of CRT on around 15% of the cells (FIG. 4a); on the contrary, surprisingly high levels of more than 80% of the cells expressed CRT on the surface upon infection with both IOVA viruses. Similarly, exposure of CRT increased from around 35% (with WR/TK−) to almost 90% (with IOVA viruses) in 143B cells, and from 3% to more than 72% in MCF-7 cells.

(28) In order to further investigate the possible induction of a pronounced immunogenic cell death upon infection with IOVA viruses, the release of HMGB1 and ATP was determined using an ELISA assay and a luciferase-mediated ATP assay system, respectively.

(29) In all cell lines tested and with both IOVA/A56− and IOVA/A56+, significantly higher concentrations of HMGB1 could be detected in the supernatant of infected cells compared with cells infected with WR/TK− (FIG. 4b), with an increase ranging from 1.23 times (143B cells, IOVA/A56−) to 1.68 times (MCF-7 cells, IOVA/A56+). ATP concentration on the supernatant of infected cells was also increased when infected with the IOVA viruses compared to the levels after infection with WR/TK− (FIG. 4c), with an increase ranging from 1.12 times (143B cells, IOVA/A56+) to 2.27 times (HeLa, IOVA/A56−).

(30) These results indicated that IOVA virus, but not Vaccinia virus WR, induces an immunogenic cell death of infected human cancer cells. Thus, IOVA can be suggested as a particularly promising candidate virus that may represent a huge benefit in terms of anti-tumour activity in clinical trials.

Example 7: PCR Assay for the Identification of IOVA Viruses or their Derivatives

(31) In order to identify IOVA strain, the DNA of the virus is isolated by digesting the cell extract of infected cells with proteinase K, and by using a QIAamp genomic DNA kit (QIAGEN) following manufactured instructions. A unique sequence of IOVA covering the C2L-C1L-N1L-N2L fragment (FIG. 5a; SEQ ID No.1) is amplified by PCR using the following oligos: Forward 5′-ATGTTATCCTGGACATCGTAC-3′ (SEQ ID No. 2) and Reverse 5′-TCATGACGTCCTCTGCAATGG-3′ (SEQ ID No. 3). The PCR product using these two primers is 50 bp larger than the unique SEQ ID No.1; for stability reasons, 50 more bp were included in design of the PCR reaction. The PCR product (SEQ ID No.4) is purified by using a QIAquick PCR purification kit and is digested with BstXI restriction enzyme.

(32) By using this assay, IOVA strain can be clearly identified as it generates a specific and unique pattern of two DNA bands (2361 and 923 bp) visualized by electrophoresis in 1% agarose, in contrast to all the other Vaccinia strains, which present a single band of 3384 bp. PCR of MVA genomic DNA generates no PCR product due to the absence of the C2L sequence (FIG. 5b).

REFERENCES

(33) D. Kirn, T. Hermiston, F. McCormick, ONYX-015: clinical data are encouraging. Nature medicine 4, 1341 (December 1998).

(34) C. J. Breitbach et al., Oncolytic vaccinia virus disrupts tumour-associated vasculature in humans. Cancer research 73, 1265 (Feb. 15, 2013).

(35) S. H. Thorne et al., Targeting localized immune suppression within the tumour through repeat cycles of immune cell-oncolytic virus combination therapy. Molecular therapy: the journal of the American Society of Gene Therapy 18, 1698 (September 2010).

(36) C. J. Breitbach et al., Intravenous delivery of a multi-mechanistic cancer-targeted oncolytic poxvirus in humans. Nature 477, 99 (Sep. 1, 2011).

(37) J. Heo et al., Randomized dose-finding clinical trial of oncolytic immunotherapeutic vaccinia JX-594 in liver cancer. Nature medicine 19, 329 (March 2013).

(38) H. J. Zeh et al., First-in-man study of western reserve strain oncolytic vaccinia virus: safety, systemic spread, and antitumour activity. Molecular therapy: the journal of the American Society of Gene Therapy 23, 202 (January 2015).

(39) A. Volz, G. Sutter, Protective efficacy of Modified Vaccinia virus Ankara in preclinical studies. Vaccine 31, 4235 (Sep. 6, 2013).

(40) H. Meyer, G. Sutter, A. Mayr, Mapping of deletions in the genome of the highly attenuated vaccinia virus MVA and their influence on virulence. The Journal of general virology 72 (Pt 5), 1031 (May 1991).

(41) R. T. Zhang, S. D. Bines, C. Ruby, H. L. Kaufman, TroVax((R)) vaccine therapy for renal cell carcinoma. Immunotherapy 4, 27 (January 2012).

(42) I. Marigo, L. Dolcetti, P. Serafini, P. Zanovello, V. Bronte, Tumour-induced tolerance and immune suppression by myeloid derived suppressor cells. Immunol Rev 222, 162 (April 2008).

(43) S. J. Gardai et al., Cell-surface calreticulin initiates clearance of viable or apoptotic cells through trans-activation of LRP on the phagocyte. Cell 123, 321 (Oct. 21, 2005).

(44) G. L. Smith et al., Vaccinia virus immune evasion: mechanisms, virulence and immunogenicity. The Journal of general virology 94, 2367 (November 2013).

(45) Goebel et al. The complete DNA sequence of vaccinia virus. Virology 1990 November; 179(1): 247-66, 517-63.

(46) L. Galluzzi, A. Buque, O. Kepp, L. Zitvogel, G. Kroemer, Immunogenic cell death in cancer and infectious disease. Nat Rev Immunol 17, 97 (February 2017).

(47) Rojas J J et al., Manipulating TLR signaling increases the anti-tumour T cell response induced by viral cancer therapies. Cell Rep., 2016 Apr. 12; 15(2): 264-73.