MODIFIED VESICULAR STOMATITIS VIRUS GLYCOPROTEIN AND USES THEREOF FOR THE TREATMENT OF BRAIN TUMORS

Abstract

A vaccine for treating and/or preventing a brain tumor. More particularly, a modified vesicular stomatitis virus glycoprotein (VSV-G) including at least one tumor antigen, or a fragment thereof, for use in preventing and/or treating a brain tumor in an individual in need thereof, when administered before a surgery intended to remove all or part of the tumor, such as, a tumor resection. The inventors have shown that vaccination of individual with a brain tumor with a vaccine including a nucleic acid sequence encoding a modified VSV-G may be combined to a tumor resection in order to ameliorate the prognostic of the individuals.

Claims

1-19. (canceled)

20. A method for preventing and/treating a brain tumor in an individual in need thereof, said method comprising the steps of: a) administering to said individual a therapeutically effective amount of the modified vesicular stomatitis virus glycoprotein (VSV-G) comprising at least one tumor antigen, or a fragment thereof, a nucleic acid encoding the same, a vector containing said nucleic acid, a dendritic cell population transfected by said nucleic acid, a pharmaceutical composition comprising any of the foregoing, or a vaccine comprising any of the foregoing; and b) performing a surgery in said individual as to remove all or part of the tumor.

21. The method according to claim 20, wherein said surgery is a brain tumor resection.

22. The method according to claim 20, further comprising the step c) of administering to said individual a second therapeutically effective amount of a modified VSV-G, or fragment thereof, nucleic acid, vector, dendritic cell population, pharmaceutical composition or vaccine.

23. The method according to claim 20, wherein said at least one tumor antigen is selected in (i) a group of antigens comprising ALK, GALT3, NA17-A, HSD3B7, BCAN, CHI3L2, CSPG4, FABP7, IGF2BP3, NLGN4X (Neuroligin 4, X-linked), NRCAM, PTPRZ1, TNC, AIM2, gp100, MAGE, TRP2, HER2, IL13Rα2, MAGE A11, SSX5, NOL4, MAGE C2, EPHA2, YKL-40, VEGFR1, VEGFR2, SURVIVIN, pp65, IE1, MART-1, SART-1, HER2/NEU, GNT-V, Tyrosinase, hTERT, B-CYCLIN, IDH1, EGFRvIII, WT-1, HSPPC-96, HB-EGF, EGFR, PCNA, ITGAV, STAT-3, IQGAP-1, HO-1, BSG, SEC61G and PIK3R1, or (ii) a group of neoantigens comprising PAPPA2, NF1, ATP8B3, HOXA1, OR4C3, FAM20B, INSM2, GOLGA6L22, TMEM241, POTEJ, PRKRA, C9orf57, LILRB3, MYLK, ABCA2, ATP1A2, LINC00273, CDH7, ELL, NCAN, TTN, GPR50, LCE1F, GOLGA6L1, GOLGA6L2, LOC645752, DSPP, CRHBP and TENM3.

24. The method according to claim 20, wherein said at least one tumor antigen is selected in (i) a group of antigens comprising gp100, TRP2, pp65 and EGFRvIII, or (ii) a group of neoantigens comprising PAPPA2, NF1, ATP8B3, HOXA1, OR4C3, FAM20B, INSM2, GOLGA6L22, TMEM241, POTEJ, PRKRA, C9orf57, LILRB3, MYLK, ABCA2, ATP1A2, LINC00273, CDH7, ELL, NCAN, TTN, GPR50, LCE1F, GOLGA6L1, GOLGA6L2, LOC645752, DSPP, CRHBP and TENM3.

25. The method according to claim 20, wherein said at least one tumor antigen is gp100 and/or TRP2.

26. The method according to claim 20, wherein said at least one tumor antigen comprises an epitope selected in the group of epitopes of sequences SEQ ID NO: 60 to SEQ ID NO: 104 and of neoepitopes of sequences SEQ ID NO: 105 to SEQ ID NO: 136.

27. The method according to claim 20, wherein said at least one tumor antigen is inserted in a VSV-G comprising SEQ ID NO: 1.

28. The method according to claim 20, wherein said at least one epitope is epitope gp10044-59 of sequence SEQ ID NO: 71 and/or epitope TRP2180-188 of sequence SEQ ID NO: 73.

29. The method according to claim 20, wherein epitope gp10044-59 of sequence SEQ ID NO: 71 is inserted at VSV-G amino acid positions 18 of SEQ ID NO: 1 and/or epitope TRP2180-188 of sequence SEQ ID NO: 73 is inserted at VSV-G amino acid positions 191 of SEQ ID NO: 1.

30. The method according to claim 20, wherein the brain tumor is selected in the group consisting of glioblastoma, anaplastic astrocytoma, meningioma, and oligodendroglial tumor.

31. The method according to claim 20, wherein the brain tumor is a glioblastoma.

32. The method according to claim 20, wherein said modified VSV-G, nucleic acid sequence, vector, dendritic cell population, pharmaceutical composition, or vaccine is administered to the individual by intramuscular injection, intradermal injection, intra-tumoral injection, peritumoral injection, gene gun, electroporation or sonoporation.

33. The method according to claim 20, wherein said pharmaceutical composition comprises at least one pharmaceutically acceptable excipient.

34. The method according to claim 20, wherein said vaccine comprises at least one adjuvant.

35. The method according to claim 20, wherein said vaccine is a nucleic acid vaccine or a protein vaccine.

36. The method according to claim 20, wherein said modified VSV-G, nucleic acid sequence, vector, dendritic cell population, pharmaceutical composition, or vaccine is administered in combination with a further tumor treatment.

37. A method for ameliorating the prognostic of an individual with a brain tumor, said method comprising the steps of: a) administering to said individual a therapeutically effective amount of the modified vesicular stomatitis virus glycoprotein (VSV-G) comprising at least one tumor antigen, or a fragment thereof, a nucleic acid encoding the same, a vector containing said nucleic acid, a dendritic cell population transfected by said nucleic acid, a pharmaceutical composition comprising any of the foregoing, or a vaccine comprising any of the foregoing; and b) performing a surgery in said individual as to remove all or part of the tumor.

38. The method according to claim 37, wherein said surgery is a brain tumor resection.

39. The method according to claim 37, wherein the brain tumor is selected in the group consisting of glioblastoma, anaplastic astrocytoma, meningioma, and oligodendroglial tumor.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0414] FIGS. 1A-C is a combination of plots showing a therapeutic immunization with pTOP vaccines in a murine glioblastoma model. FIG. 1A: Schematic protocol. C57BL/6 mice were first subcutaneously injected with GL261 tumor cells (2×10.sup.6 cells). pTOP7 vaccine (1 μg) was intramuscularly electroporated 2, 9 and 16 days after the injection of tumor cells. FIG. 1B: Evolution of tumor volume expressed in mm.sup.3 (ordinate), as a function of time expressed in day (abscissa). FIG. 1C: Survival curves, as the percent of survival (ordinate) as a function of time expressed in day (abscissa). The errors bars represent mean±SEM; n=6-7. Statistical analysis: Two-way ANOVA with Bonferroni post-tests or Mantel-Cox test for comparison of survival curves. *p value<0.05 as compared to naive.

[0415] FIGS. 2A-G is a combination of plots showing a therapeutic immunization and resection in an orthotopic glioblastoma model and evaluation of the systemic immune response. FIG. 2A: Schematic protocol. C57BL/6 mice first received an intracranial injection of 5×10.sup.4 cells of GL261 at day 0. MRI was used to monitor brain tumor at day 10 and 27. pTOP7 vaccine (1 μg) was intramuscularly electroporated 16, 23 and 29 days after the injection of tumor cells and resection of the tumor was performed at day 17, n=9-12. FIG. 2B: Survival curves for the therapeutic immunization.

[0416] FIG. 2C: Representative axial T2-weighted MRI image of an untreated mouse brain before (day 10) and after tumor resection (day 27). The white arrows indicate the GL261 primary and recurrent tumor, respectively. FIG. 2D-G: Analysis of immune cells in the spleen, 29 days after GL261 inoculation. The percentage of splenic CD8 is shown for all the groups (FIG. 2D) and the production of IFNγ from splenocytes stimulated with TRP2 peptide is assessed by ELISPOT (FIG. 2E). The percentage of MDSC (FIG. 2F) and the ratio of M1/M2 macrophages (FIG. 2G) are displayed. The errors bars represent mean±SEM; n=7-9. Statistical analysis: One-way ANOVA with Tukey multiple comparisons test or Mantel-Cox test for comparison of survival curves. *p value<0.05 and **p value<0.01 as compared to naive or to the specified group.

[0417] FIGS. 3A-G are a combination of plots showing the evaluation of immune cells and immunosuppressive cells in the brain, 29 days after GL261 inoculation. FIG. 3A-B: Total number of CD8 and ratio of IFNγ secreting CD8/total CD8, respectively. FIG. 3C-D: Total number of CD4 and ratio of IFNγ secreting CD4/total CD4, respectively. FIG. 3E-F: percentage of MDSC and ratio of M1/M2 macrophages in the brain, respectively. FIG. 3G: Number of Treg in the brain. The errors bars represent mean±SEM; n=7-9. Statistical analysis: One-way ANOVA with Tukey multiple comparisons test. *p value<0.05, **p value<0.01, ***p value<0.001 as compared to naive or to the specified group.

EXAMPLES

[0418] The present invention is further illustrated by the following examples.

Example 1

[0419] 1. Materials and Methods

1.1—Materials

[0420] a) Plasmids and Primers

[0421] pTOP refers to the plasmids encoding VSV-G (of sequence SEQ ID NO: 1) in which the foreign epitopes were inserted. Codon-optimized gene sequences were designed using GeneOptimizer and obtained by standard gene synthesis from GeneArt® (Thermo Fisher Scientific®, US). The sequences were subcloned in the pVAX2 vector using cohesive-end cloning. To allow easy modifications of the epitopes, several restriction sites were added. Digestion by BamHI and HindIII or by SpeI and EcoRI allows insertion in position (18) or (191), respectively. The inserted epitopes are detailed in Table 4.

TABLE-US-00003 TABLE 4 Plasmids details Name Position (18) Position (191) pVAX2-VSVG — — pTOP7.sup.1 gp100.sub.44-59 (WNRQLYPEWTEAQRLD.sup.2) TRP2.sub.180-188 (SVYDFFVWL.sup.3) .sup.1The resulting construct, VSVG-gp100.sub.44-59-TRP2.sub.180-188, has a nucleic acid sequence SEQ ID NO: 137; and an amino acid sequence SEQ ID NO: 138; .sup.2Amino acid sequence SEQ ID NO: 71; .sup.3Amino acid sequence SEQ ID NO: 73.

[0422] The positions into which the epitopes are inserted are defined by the amino acid residue directly after the insertion site. In other words, insertion position (18) corresponds to the region between amino acid residues 17 and 18. For the epitopes inserted at the N terminus (e.g., position (18), just after the signal peptide), an additional lysine residue was included. Overlapping phosphorylated oligonucleotides that encoded the restricted epitope (IDT-DNA®, Belgium) were incorporated in the digested vector using cohesive-end cloning. The plasmids were prepared using the EndoFree Plasmid Mega or Giga Kit (Qiagen®, Germany) and diluted in PBS. The quality of the purified plasmid was assessed by the ratio of optical densities and by 1% agarose gel electrophoresis. Plasmids were sequenced by Sanger DNA sequencing (Genewiz®, UK) and stored at −20° C.

[0423] Primers used herein are depicted in Table 5 below:

TABLE-US-00004 TABLE 5 Primers used herein Gene length Primer sequence (5′.fwdarw.3′) Amplicon TRP2 For-CCAGGATGACCGTGAGCAA (SEQ ID NO: 139) 171 bp Rev-GGGCAGTCAGGGAATGGAT (SEQ ID NO: 140) Murine- For-GGAGCTTCCTTCCCGTGCTT (SEQ ID NO: 141) 321 bp gp100 Rev-GCTCCCATTGATGATGGTGT (SEQ ID NO: 142) Human- For-ATAGGTGCTTTGCTGGCTGT (SEQ ID NO: 143) 263 bp gp100 Rev-ACCTGCCCATCTGGCAATAC (SEQ ID NO: 144) VSV-G For-AACTGGCACAACGACCTGAT (SEQ ID NO: 145) 144 bp Rev-GATGTACTTGGGGCCGTACC (SEQ ID NO: 146) b-actin For-ACTCCTATGTGGGTGACGAG (SEQ ID NO: 147) 206 bp Rev-CATCTTTTCACGGTTGGCCTTAG (SEQ ID NO: 148)

[0424] b) Cell lines

[0425] GL261 tumor cells were cultured in DMEM. Media were supplemented with 10% FBS, 100 μg/mL streptomycin, and 100 U/mL penicillin (Gibco®, Life Technologies®, USA). Cells were sub-cultured in 75 cm.sup.2 culture flasks (Corning® T-75, Sigma-Aldrich, USA) and incubated at 37° C. and 5% CO.sub.2.

[0426] c) Animals

[0427] Six- to eight-week-old C57BL/6NRj, Balb/c and DBA/2 female mice were obtained from Janvier Labs® (France) and housed in an air-conditioned animal facility with ad libitum access to food and water. Temperature and humidity were monitored daily. For tumor implantation and electroporation, the mice were anaesthetized with a 150 to 200 μL intraperitoneal injection of 10 mg/mL ketamine and 1 mg/mL xylazine. All in vivo experiments were performed following the Belgian national regulation's guidelines in accordance with EU Directive 2010/63/EU, and were approved by the ethical committee for animal care of the faculty of medicine of the Université Catholique de Louvain (2011/UCL/MD/007, 2014/UCL/MD/004 and 2016/UCL/MD/001).

1.2—Methods

[0428] a) Immunization

[0429] Intramuscular electroporation—After the mouse hair was removed using a rodent shaver (AgnTho's, Lidingö, Sweden), 30 μL of a PBS solution containing 1 μg of plasmid was injected into the tibial cranial muscle. The leg was placed between 4-mm-spaced plate electrodes, and 8 square-wave electric pulses (200 V/cm, 20 ms, 2 Hz) were delivered. For prophylactic immunizations, two boosts were similarly applied two and four weeks after priming. For therapeutic immunizations, the vaccine was administered 2, 9 and 16 days after subcutaneous tumor injection or 16, 23 and 29 days after orthotopic injection.

[0430] For all electroporation protocols, electric pulses were generated by a Gemini System generator and delivered with BTX Caliper Electrodes (BTX; both from VWR International, Belgium). A conductive gel was used to ensure electrical contact with the skin (Aquasonic 100; Parker Laboratories®, Inc., USA).

[0431] b) Subcutaneous Tumor Implantation and Tumor Measurement

[0432] A total of 2×10.sup.6 GL261 cells diluted in 100 μl of PBS were injected subcutaneously into the right flank of C57Bl/6 mice. The tumor cells were inoculated before the plasmid treatment for the therapeutic experiments and two weeks after complete immunization for the prophylactic studies. Tumor size was measured three times a week with an electronic digital caliper. Tumor volume was calculated as the length×width×height (in mm.sup.3). Mice were sacrificed when the tumor volume was greater than 1500 mm.sup.3 or when they reached the end points (behavior changes e.g. lack of grooming and clinical signs of distress e.g.: paralysis, arched back, lack of movement plus 10% body weight loss and/or 20% body weight loss).

[0433] c) Orthotopic GL261 Brain Tumor (Glioblastoma) Syngeneic Model

[0434] Mice were anesthetized by intraperitoneal injection of ketamine/xylazine (100 mg/kg and 13 mg/kg, respectively) and fixed in a stereotactic frame. A surgical high-speed drill (Vellman®, Belgium) was used to perform a hole in the right frontal lobe and 5×10.sup.4 GL261 cells were slowly injected using a Hamilton syringe fitted with a 26S needle. To obtain cortical tumors, the injection coordinates were 0.5 mm posterior, 2.1 mm lateral from the bregma and 2.2 mm deep from the outer border of the cranium. The presence, volume and location of the tumors were determined by magnetic resonance imaging (MRI), which was performed for all mice included in the study before the surgical resection of the tumor. Animals presenting GL261 tumors were randomly divided into four groups.

[0435] d) Magnetic Resonance Imaging

[0436] MRI was performed using a 11.7 T Bruker Biospec MRI system (Bruker®, Germany) equipped with a 1H quadrature transmit/receive surface cryoprobe after anesthetizing animals with isoflurane mixed with air (2.5% for induction, 1% for maintenance). Tumor was visualized using rapid acquisition with relaxation enhancement (RARE) sequence (repetition time=2500 ms; effective echo time=30 ms; RARE factor=8; field of view=2×2 cm; matrix 256×256; Slice thickness=0.3 mm; 25 contiguous slices were acquired, N average=4).

[0437] e) Surgical Resection of the Tumor Mass

[0438] At day 17 post-tumor inoculation, the tumor mass was surgically removed using the biopsy-punch resection technique. Briefly, animals were anaesthetized with ketamine/xylazine and immobilized in a stereotactic frame. An 8 mm incision was made in the midline along the previous surgical scar and a 2.1 mm diameter circular cranial window was created around the previous burr hole using fine tip tweezers (Dumont®, Switzerland) to expose the brain. A 2 mm diameter biopsy punch (Kai Medical®, Germany) was then inserted 3 mm deep and twisted for 15 s to cut the brain region surrounding the tumor. Once withdrawn, the tumor and brain tissues were aspired using a diaphragm vacuum pump (Vaccubrand® GBMH+CO KG, Germany) connected to a Pasteur pipette and a 200 μl tip. Residual blood was removed from the surgical cavity using a hemostatic triangle (Fine Science Tools®, Germany) The cranial window was then sealed with a 4×4 mm square piece of Neuro-Patch® (Aesculap®, Germany) impregnated with a reconstituted fibrin hydrogel (25 mg/mL fibrin, 10 IU/mL thrombin, equal volumes; Baxter Innovations®, Austria).

[0439] All animals were monitored daily and an MRI follow-up was performed 27 days after surgery. Eight to nine animals per group were sacrificed 29 days post-tumor inoculation for immunological analysis (FACS and PCR). The spleen and the brain of the animals were collected for further analysis. The remaining animals were sacrificed when they reached the end points.

[0440] f) Flow Cytometry Analysis of Immune Cells

[0441] TAM, MDSC, CD4 and CD8 T cell populations in brains and spleens removed 29 days after GL261 orthotopical cell injection were analyzed by FACS. Cells were passed through a 70 μm cell strainer (BD Falcon®, New Jersey), collected, counted using an automatic cell counter (Invitrogen®, California) and washed with PBS, before adding the blocking solution with anti-CD16/CD32 antibody for 10 minutes on ice (clone 93, Biolegend®, San Diego, Calif.). Cells were washed and incubated for 60 minutes at 4° C. with the following antibodies: anti-CD3-APC-Cy7 (Biolegend®, San Diego, Calif.), anti-CD4-PE (BD Bioscience®, United Kingdom), anti-CD8-BV421 (Biolegend®, San Diego, Calif.) for CD4 and CD8 T cell detection; with anti-CD11b-FITC (BD Bioscience®, United Kingdom), anti-F4/80-AF647 (BD Bioscience®, United Kingdom), anti-CD206-BV421 (Biolegend®, San Diego, Calif.) and anti-Grl-PE (BD bioscience, United Kingdom) for TAMs and MDSCs; with anti-CD3-APC-Cy7, anti-CD8-FITC (Proimmune®, United Kingdom) and Pentamers-TRP2-PE (Proimmune®, United Kingdom) for the detection of TRP-2-specific CD8 T cells. For staining with antiFoxP3-AF488 (BD Bioscience®, United Kingdom) or anti-IFNg-APC (Biolegend®, San Diego, Calif.), cells were previously incubated overnight at 4° C. with a permeabilization/fixation solution (eBioscience™ Foxp3/Transcription Factor Staining Buffer Set, Thermo Fisher, Waltham, Mass.). Cells were then incubated with anti-CD16/CD32 antibody for 10 minutes on ice (Biolegend®, San Diego, Calif.), washed and incubated for 60 minutes at 4° C. with anti-IFNg-APC or antiFoxP3-AF488 diluted in the permeabilization/fixation solution. Samples were washed with PBS fixed for 10 minutes with 4% formalin and, then, suspended in PBS. Sample data were acquired with FACSVerse (BD Bioscience®, Franklin Lakes, N.J.) and analyzed with FlowJo software (FlowJo® LLC, Ashland, Oregon).

[0442] g) Enzyme-Linked ImmunoSpot (ELISpot)

[0443] ELISpot was performed according to the manufacturer's instruction (Immunospot, the ELISPOT source, Germany) Briefly, 3×10.sup.5 fresh splenocytes diluted in 100 μl CTL-Test medium (Immunospot, the ELISPOT source) were cultured overnight at 37° C. in anti-IFNg-coated 96 well plate. For stimulation, 10 ng/μl of TRP2.sub.180-188 peptide (SVYDFFVWL; SEQ ID NO: 73) was added to the splenocytes and incubated for 2 days. As positive control for splenocyte activation, Cell Stimulation Cocktail (Invitrogen®, California) was used; PBS and a P815 irrelevant peptide (LPYLGWLVF; SEQ ID NO: 149) were used as negative control. The development of the ELISpot plate followed the manufacturer's instruction and pots were counted by using an ELISPOT reader system (the ELISPOT source).

[0444] h) RT-PCR Analysis

[0445] GL261 cells were analyzed by RT-PCR to verify the presence of TRP2 and gp100 expression. Total RNA was isolated using TRIzol reagent (Thermo Fisher Scientific®, Waltham, Mass.) and phenol separation. The quality and quantity of RNA were evaluated using a nano-spectrophotometer (NanoDrop 2000, Thermo Fisher Scientific®, Waltham, Mass.). One microgram of RNA was reverse transcribed using a first-strand synthesis system (SuperScript™, Thermo Fisher Scientific®, Waltham, Mass.) and oligo(dT) primers according to the supplier's protocol. The resulting cDNA was used as template for 30 cycles of PCR amplification. The PCR products were individualized to electrophoresis on a SYBR Safe (Thermo Fisher Scientific®)-stained 1.5% agarose gel.

[0446] i) Statistical Analysis

[0447] Statistical analyses were performed using GraphPad Prism 7® for Windows®. P-values lower than 0.05 were considered statistically significant.

[0448] 2. Results

2.1—Insertion of Tumor Epitopes in the pTOP Plasmid Allows Therapeutic Vaccinations Against Subcutaneous GL261 Tumors

[0449] We evaluated the efficacy of pTOP against a brain tumor. pTOP7 was obtained by inserting two tumor epitopes (TRP2.sub.189-188 and gp100.sub.44-59) in the VSV-G sequence and evaluated as a therapeutic vaccine delivered at days 2, 9 and 16 after subcutaneous tumor cell injection (FIG. 1A). The expression of TRP2 and gp100 in GL261 cells was verified by RT-PCR. Tumor growth of vaccinated mice was significantly delayed compared to the untreated group (FIG. 1B) and 6 out of 7 mice were considered long-term survivors (FIG. 1C). Thus, pTOP7 can be effective against GL261 tumors.

2.2—in a GL261 Orthotopic Model, Tumor Resection and pTOP Vaccination Significantly Prolonged Mice Survival

[0450] As pTOP7 was highly efficient against the subcutaneously implanted GL261 tumors, we checked whether this vaccine could prevent recurrences in a murine orthotopic GBM model when the first dose of vaccine was administered just before surgical resection. Tumoral lesions of GL261-bearing mice were observed between the cortex and the striatum in all implanted animals at day 10 post-inoculation, by MRI. Mice were vaccinated at day 16, 23 and 29 and the tumor was resected 17 days after the GL261 inoculation (see FIG. 2A). In the control groups (naive, resection or pTOP7), most of the mice showed signs of discomfort and pain starting from day 27-30 after the tumor injection and their median survival time was less than 40 days. However, when resection was combined to therapeutic immunization with pTOP7, 78% of the mice were able to survive for at least 250 days and thus considered as long-term survivors (FIG. 2B). MRI performed 27 days after tumor inoculation confirmed the presence of infiltrative and aggressive recurrences in control groups (FIG. 2C). Due to the infiltrative patterns of the GL261 tumors, we are unable to provide adequate volume estimation of the tumors at the designated time points but the presence of the tumor and of its infiltrative nature was confirmed post-mortem by hematoxylin and eosin staining (H/E).

2.3—pTOP Induced Systemic Antigen-Specific Immune Response and Modulated the Number of Immune Cells in the Spleen

[0451] Next, we evaluated the systemic immune activity after resection and/or immunization with pTOP7. To this end, splenocytes were collected 29 days after the tumor challenge and analyzed by flow cytometry and ELISpot. When resection and pTOP7 vaccine were combined, the CD8 infiltration was significantly higher compared to the untreated mice and those that underwent the resection alone (FIG. 2D). The activation of TRP2-specific T cells was assessed by IFNγ ELISpot. In the absence of vaccination, almost no spot has been detected whereas the splenocytes from pTOP7 treated mice showed a significantly higher number of spots (FIG. 2E). In addition, the number of myeloid-derived suppressor cells (MDSC) was lower when mice were treated with pTOP7 (FIG. 2F) and a modulation (albeit not statistically significant) of the M1/M2 macrophage ratio was observed for mice treated with pTOP7 with or without tumor resection (FIG. 2G).

2.4—pTOP and Tumor Resection Enhanced the Activity of Immune Cells and Reduced the Number of Infiltrated Immunosuppressive Cells in the Brain

[0452] To study the mechanism underpinning the synergy between pTOP7 and the resection of GL261 tumors and their contribution in prolonging mice survival, the infiltration of different immune cells was assessed in the mice brains 29 days after tumor inoculation. A decreased infiltration of CD8 T cells in the treated groups, especially in the combination group (FIG. 3A). However, when analyzing the activity of those cells, we observed that almost 40% of infiltrated CD8 produced IFNγ as compared to only 10-15% for the other groups (FIG. 3B). The same trend was observed for the number of CD4 (FIG. 3C) and IFNγ-secreting CD4 T cells but the slight increase observed in the combination group was not significantly different to the other groups (FIG. 3D). The flow cytometry analysis also revealed a significant decrease of the infiltrated immunosuppressive cells for all the groups compared to the untreated group. This effect was seen for MDSC (FIG. 3E), M1/M2 macrophage ratio (FIG. 3F) and regulatory T cells (FIG. 3G). The greatest on infiltrated immunosuppressive cells was observed when pTOP7 was used alone or in combination with the resection. The M1/M2 macrophage ratio was significantly higher for mice treated with the combination as compared to untreated mice or mice with resected tumors.

[0453] 3. Discussion

[0454] The inventors have demonstrated that pTOP7, i.e. a plasmid encoding a modified VSV-G protein comprising inserted defined T cell epitopes (originating from gp100 and TRP2), was able to generate a specific and long-lasting immune response against GL261 GBM and to target residual GBM cells in mice that had undergone surgical resection. Resection induces immunological changes that could contribute to the vaccine activity such as the induction of excessive healing response, production of inflammatory cytokines and recruitment of both M1 and M2 macrophages. Combined resection and vaccination induced many immunological changes, both systemically and locally. In the brain, the ratio IFNγ-producing CD8/total CD8 was significantly higher, indicating the presence of active infiltrated CD8 T cells. This may indicate that the infiltrated CD8 T cells in the combination group, even if low in number, are not exhausted and still able to recognize the antigen and produce IFNγ. Furthermore, all the vaccinated groups showed higher levels of antigen-specific T cells in the spleen. In addition, less MDSC, M2 macrophages and Treg were observed in the brain suggesting that the immunosuppressive activity was reduced, especially when the vaccine was combined with the resection, thus permitting a higher CD8 T cell activation. The combination of DNA vaccination and surgical resection drastically increased mice survival, due to a decreased infiltration of immunosuppressive cells and the concomitant activity and antigen-specificity of T cells in the brain. The strength of this combination could overcome the limits of each single treatment: from one side the tumor resection reduces the number of tumor cells and induces a local inflammation that could strengthen the adaptive immunity activated by the vaccine. From the other side, the vaccine activates the host adaptive immune system against the residual tumor cells, thus avoiding the GBM recurrences. To the inventors' knowledge, this is the first study reporting the combination between GBM surgical resection and vaccine immunotherapy being performed before the tumor debulking. The vaccine administration prior to surgery might take advantage of the acute inflammatory response induced by the resection to activate specific antitumor immune response acting on residual GBM cells and on the tumor resection microenvironment, thus avoiding the on-set of tumor recurrences.

Example 2: Assessment of the Combined Efficacy of pTOP Vaccine and Immune Checkpoint Blockade ICB

[0455] For orthotopic GBM tumor grafting, C57BL/6 mice were anesthetized and fixed in a stereotactic frame. A surgical high-speed drill (Vellman®, Belgium) was used to perform a hole in the right frontal lobe and 5×10.sup.4 GL261 cells were slowly injected using a Hamilton syringe fitted with a 26S needle. To obtain cortical tumors, the injection coordinates were 0.5 mm posterior, 2.1 mm lateral from the bregma and 2.2 mm deep from the outer border of the cranium. The presence, volume and location of the tumors were determined by MRI. Animals presenting GL261 tumors were randomly divided into four groups (naive, vaccine alone, ICB alone and combined treatment).

[0456] pTOP vaccine—After the mouse hair was removed using a rodent shaver (AgnTho's, Lidingo, Sweden), 30 μL of a PBS solution containing 1 μg of plasmid was injected into the tibial cranial muscle. The leg was placed between 4-mm-spaced plate electrodes, and 8 square-wave electric pulses (200 V/cm, 20 ms, 2 Hz) were delivered. The vaccine was administered 16, 23 and 29 days after GL261 orthotopic injection.

[0457] ICB—Immune checkpoint blockade antibodies directed against CTLA4 (clone 9D9) and PD1 (clone 29 F.A12) were purchased from Bioconnect® (Netherlands) and mice were injected intraperitoneally with 100 μg of each antibody in 100 μl of PBS 17, 20 and 23 days after tumor injection.