METHOD FOR OBTAINING TUMOR PEPTIDES AND USES THEREOF

Abstract

The present invention refers to a method for obtaining a cell culture supernatant or a fraction thereof having a specific tumor antigen peptide repertoire having the steps of: a) exposing in suitable conditions a tumor cell culture expressing the specific tumor antigen peptide repertoire to a Pattern Recognition Receptor (PRR) agonist and/or to one inflammatory cytokine to increase the opening of connexin-hemichannels (CxH); b) collecting the cell culture supernatant; and c) optionally obtaining a fraction of the supernatant, wherein the supernatant has MHC class I and MHC class II peptides and non-classical MHC molecules (HLA-E).

Claims

1. A method for obtaining a cell culture supernatant or a fraction thereof comprising a specific tumor antigen peptide repertoire comprising the steps of: a) exposing in suitable conditions a tumor cell culture to a Pattern Recognition Receptor (PRR) agonist and/or to one inflammatory cytokine to increase the opening of connexin-hemichannels (CxH); b) collecting the cell culture supernatant; and c) optionally obtaining a fraction of said supernatant, wherein said supernatant comprises MHC class I and MHC class II peptides and non-classical MHC molecules (HLA-E).

2. The method for obtaining a specific tumor antigen peptide repertoire loaded and/or activated dendritic cell comprising the steps of: a) exposing in suitable conditions a tumor cell culture to a Pattern Recognition Receptor (PRR) agonist and/or to one inflammatory cytokine to increase the opening of connexin-hemichannels (CxH); b) collecting the cell culture supernatant; c) culturing dendritic cells with the collected cell culture supernatant, or a fraction thereof or with a purified peptide from said cell culture supernatant, to get specific tumor antigen peptide repertoire loaded and/or activated dendritic cells; and d) optionally purifying said specific tumor antigen peptide repertoire loaded and/or activated dendritic cells.

3. The method for obtaining an activated tumor antigen-specific CTL comprising the steps of: a) exposing in suitable conditions a tumor cell culture to a Pattern Recognition Receptor (PRR) agonist and/or to one inflammatory cytokine to increase the opening of connexin-hemichannels (CxH); b) collecting the cell culture supernatant; and c) co-culturing dendritic cells and CTLs with the cell culture supernatant, or a fraction thereof or with a purified peptide from the cell culture supernatant, to get activated tumor antigen-specific CTLs.

4. The method according to claim 2 wherein dendritic cells are autologous or HLA-compatible or semi-compatible allogenic dendritic cells.

5. The method according to claim 1 wherein in step a) the tumor cell culture is incubated for at least 30 minutes with a Pattern Recognition Receptor (PRR) agonist and/or to one inflammatory cytokine to increase the opening of connexin-hemichannels (CxH).

6. The method according to claim 1, wherein the tumor cell culture is incubated at a temperature of 25-50? C. with a Pattern Recognition Receptor (PRR) agonist and/or to one inflammatory cytokine to increase the opening of connexin-hemichannels (CxH).

7. The method according to claim 1 wherein in step a) the tumor cell culture is incubated for 1 hour and half at 37? C. with a Pattern Recognition Receptor (PRR) agonist and/or to one inflammatory cytokine to increase the opening of connexin-hemichannels (CxH).

8. The method according to claim 1 wherein said cell culture supernatant is obtained by centrifugation of cells.

9. The method according to claim 8 wherein after centrifugation the supernatant is filtered.

10. The method according to claim 1 wherein the supernatant comprises a peptide comprising an amino acid sequence selected from the group consisting of: SEQ ID NO:3, SEQ ID NO:2, SEQ ID NO: 4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO:17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ IS NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32 or orthologues, variants or fragments thereof.

11. The method according to claim 10 wherein the supernatant comprises peptides comprising an amino acid sequence selected from the group consisting of: SEQ ID NO:3, SEQ ID NO:2, SEQ ID NO: 4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO:17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ IS NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32 or orthologues, variants or fragments thereof.

12. The method according to claim 1 wherein the inflammatory cytokine is gamma-IFN.

13. The method according to claim 1, wherein the tumor cell is an established tumor cell line, or a combination of tumor cell lines expressing a specific tumor antigen peptide repertoire or a tumor cell isolated by a tumor affected subject.

14. The method according to claim 1, wherein the tumor cell derives from solid or non-solid tumors, including melanoma, lung carcinoma, ovarian cancer, pancreatic cancer, glioma, glioblastoma, hepatocellular carcinoma, bladder cancer, stomach cancer, colorectal adenocarcinoma, prostate adenocarcinoma, sarcoma, osteosarcoma, leukemia and T cell-lymphoma and the said specific tumor antigen peptide repertoire is specific for said tumor.

15. The method according to claim 1, wherein the PRR agonists are Gram-negative, or Gram-positive bacteria or components thereof.

16. The method according to claim 15 wherein Gram negative bacteria components are LPS and/or flagellin or wherein Gram positive bacteria component is Lipoteichoic acid (LTA).

17. A supernatant or a fraction thereof obtainable by the method according to claim 1.

18. The supernatant or a fraction thereof according to claim 17 comprising peptides characterized through mass spectrometry analysis by at least one of the pics selected from the pics represented in FIGS. 3 and/or 12.

19. The supernatant or fraction thereof according to claim 17 comprising a peptide comprising the amino acid sequence selected from the group consisting of: SEQ ID NO:3, SEQ ID NO:2, SEQ ID NO: 4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO:17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ IS NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32 or orthologues, variants or fragments thereof.

20. The supernatant or fraction thereof according to claim 19 comprising peptides comprising an amino acid sequence selected from the group consisting of: SEQ ID NO:3, SEQ ID NO:2, SEQ ID NO: 4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO:17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ IS NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32 or orthologues, variants or fragments thereof.

21. An isolated peptide comprising an amino acid sequence selected from the group consisting of: SEQ ID NO:3, SEQ ID NO:2, SEQ ID NO: 4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO:17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ IS NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32 or orthologues, variants or fragments thereof.

22. An isolated nucleic acid encoding the peptide or orthologues, variants or fragments thereof according to claim 21.

23. An expression vector capable of expressing a nucleic acid according to claim 22.

24.-29. (canceled)

30. A specific tumor antigen peptide repertoire loaded and/or activated dendritic cell obtainable by the method according to claim 2.

31.-34. (canceled)

35. A tumor antigen-specific CTL obtainable by the method according to claim 3.

36.-42. (canceled)

Description

FIGURE LEGENDS

[0139] FIG. 1. Cx43 expression in B16 cells. - are cells not infected. AT is the attenuated strain of Salmonella thyphimurium SL3261AT. Vinculin is used as loading control.

[0140] FIG. 2. Measure of ATP extracellular concentration from uninfected or infected B16 (AT) and B16 OVA cells treated or not with the gap junction blocker heptanol.

[0141] FIG. 3. Identification of murine endogenous peptide release into the SN by Mass Spectrometry

A) IFN-gamma secretion by OT-I CD8 T cells activated by D1 dendritic cells loaded with the indicated SN derived fractions
B) Full nLC-ESI spectrum [300-1650 Da] at 53.3 min of B16 OVA-derived SUP. JUNG (sequence: SIINFEKL [SEQ ID NO:1]) is mostly detected as double charge m/z=482.28 z=2. C) nLC-ESI-MS/MS spectrum confirmed JUNG identity (m/z 482.28, z=+2).

[0142] FIG. 4. Evaluation of stability of HLA molecules on the surface of T2 cells incubated with supernatant obtained from uninfected or infected SK-mel 24 and HT29 cells treated or not with heptanol. Stabilization of MHC class I complex on the surface of T2 cells indicate the presence of exogenous peptides in supernatant obtained from uninfected or infected SK-mel 24 and HT29 cells treated or not with heptanol. Control: T2 incubated with peptide Mart-1 (26-35); MFI is the mean of fluorescence intensity; *p<0.05, **p<0.01, ***p<0.001.

[0143] FIG. 5. IL-2 production after coculture of OVA-specific T cells with murine DCs loaded with supernatant (SN) obtained from infected (AT) or not B16 or B16 OVA cells. Negative Control: DCs (D1) alone and OVA-specific T cells alone (B3Z). Positive control: DCs loaded with OVA peptide (257-264).

[0144] FIG. 6. Cx43 expression in SK-mel24. TY means Salmonella TY21a-infected cells.

[0145] FIG. 7. a) IFN-gamma production after coculture of CTLs Mart-1 specific with moDCs loaded with supernatant obtained from SK-mel24 or 624.38 treated as indicated. Control: moDCs loaded with Mart-1 (26-35). b) Same as in a) but dendritic cells are purified ex-vivo (PDC, Plasmacytoid dendritic cells).

[0146] FIG. 8. Mice were immunized in CFA (complete Freund's adjuvant) with the indicated products. as indicated. In figure A immunization was performed with OVA protein, B16 OVA extract, supernatant (SN) from B16 OVA cells in absence or presence of the gap junction inhibitor heptanol. In figure B immunization was performed with OVA protein, SN from B16 OVA or SN from Salmonella infected B16 OVA (B16 OVA AT). After 7-9 days lymph nodes cells were re-stimulated in vitro with PPD (Tuberculin purified protein derivate) or OVA protein and IFN-gamma secretion was measured after 72 hours of culture. *p<0.05.

[0147] FIG. 9. Antibody response to Salmonella SL3261AT peptides (A) and B16 tumor peptides (B). MG132 is a proteasome inhibitor; IFA is Freund's incomplete adjuvant.

[0148] FIG. 10. Tumor growth in mice vaccinated with DCs loaded with supernatant from uninfected (B16) or Salmonella infected B16 cells (B16 AT). DCs loaded with supernatant vaccine are protective against melanoma. Control: DCs not loaded (D1 alone). *p<0.05.

[0149] FIG. 11. Tumor released peptides combined with CpG boost a stronger antitumor response. Kaplan-Meier survival curves of C57/6J mice (n=5 animals per group) vaccinated with: (A-C) increasing doses of peptides derived by Salmonella treated B16 cells (0.5?10.sup.6 cells (Vax1), 2?10.sup.6 cells (Vax2), 5?10.sup.6 cells (Vax3)) combined with IFA Aldara; (D) dendritic cells loaded with Vax2; (E) Vax2 mixed up with CpG-ODN1826; (F) IFA Aldara Vax2 following gavages at day ?4 and ?1 of 109 cfu of 1.plantarum. *P<0.05, versus adjuvant control. (G) Difference (calculated as %) between AUC derived by the mean-tumor-growth curve of Vax-treated mice and AUC derived by the mean tumor growth curve of the linked adjuvant control (H) Specific T cell degranulation (CD8+CD107a+) in a CD107a mobilization assay. Data are mean?s.e.m. of three replicates (n=5 animals per group). ***P<0.001.

[0150] FIG. 12. Mass Spectrometry analysis of murine endogenous peptides differentially released into SN of B16 Salmonella infected cells. A) Full MS spectrogram of a fraction of bacteria treated B16 cells-derived-supernatant. B) Venn Diagram resulted by the analysis of the protein associated to the detected peptides comparing bacteria treated versus untreated B16 cells-derived-supernatant.

[0151] FIG. 13. HeatMap that shows 31 identified peptides significantly more abundant in Vax supernatants; analysis were performed with MaxQuant software on the identified peptides. P value<0.05. Red color: up-regulated. Green color: down-regulated.

[0152] FIG. 14. Canine osteosarcoma cell culture A) OSA cells analyzed by optical microscope, B) OSA cells stained red for phalloidin (cytoplasmic staining) and blue with DAPI (nuclear staining), C) alkaline phosphatase staining.

[0153] FIG. 15. Cx43 expression in canine osteosarcoma cells (OSA), infected or not with Salmonella Ty21a.

EXAMPLE

Material and Methods

Mice, Cell Lines and Bacterial Strain

[0154] Six-week-old female C57BL/6J mice were purchased from Charles River and maintained in specific pathogen-free animal house. The cell lines used in present study were murine melanoma B16F10 (called throughout B16) were cultured in RPMI 1640 medium supplemented with 10% fetal serum bovine, 2 mM Glutamine, 100 U/ml Penicillin, 100 ?m/mL Streptomycin and 50 ?M 2-Mercaptoethanol (complete RPMI).

[0155] The murine melanoma B16F10-OVA (called throughout B16 OVA) were cultured in Iscove's Modified Dulbecco's Medium (IMDM) supplemented with 10% fetal serum bovine, 2 mM Glutamine, 100 U/ml Penicillin, 100 ?g/mL Streptomycin and 50 ?M 2-Mercaptoethanol, 100 ?g/mL Hygromycin B. Primary canine osteosarcoma cells were obtained from dissociation of dog's osteosarcoma specimens. Tissues were minced with a scalper in a cell strainer. Cells were washed with DMEM (supplemented with 10% FBS, 2 mM L-Glutamine, 100 U/mL Penicillin, 100 mg/mL Streptomycin) and filtered with a cell strainer (100 ?M).

[0156] The cellular suspension was centrifuged at 300 g for 10 minutes and resuspendend with 2 mL of Lysis Buffer in order to lyse red blood cells; then the cells were washed twice with complete medium and cultured. Murine dendritic cells (D1), derived from bone marrows of C57BL/6J mice were cultured in IMDM containing 10% FBS, supplemented with 30% supernatant from granulocyte macrophage colony-stimulating factor-producing NIH-3T3 cells.

[0157] The B3Z T-cells hybridoma specific for the H-2Kb restricted OVA peptide were grown in Iscove's modified Dulbecco's medium (IMDM) supplemented with 5% FBS.

[0158] C57BL/6J OT-I mice contain transgenic T cell receptor designed to recognize OVA(.sub.257-264) in the context of H2Kb. Purified CD8 OT-I T cell were re-stimulated in vitro by D1 dendritic cell line previously loaded with OVA(.sub.257-264) or SN of B16 OVA infected (AT) or not, in RPMI 1640 medium supplemented with 10% fetal serum bovine, 2 mM Glutamine, 100 U/ml Penicillin, 100 ?g/mL Streptomycin.

[0159] T2 cells are an HLA-A0201 hybrid human cell line lacking TAP-2 and express low amount of MHC class I on their surface. These cells were cultured in RPMI 1640 medium supplemented with 10% fetal serum bovine, 2 mM Glutamine, 100 U/ml Penicillin, 100 ?g/mL Streptomycin.

[0160] The bacteria, Salmonella typhimurium SL3261AT is an aroA metabolically defective strain on SL1344 background and is grown at 37? C. in Luria broth (LB).

[0161] Vivofit? (Thyphoid vaccine live oral Ty21a) is a live attenuated vaccine containing the attenuated strain of Salmonella enteric serovar thyphi Ty21a and is grown at 37? C. in Luria broth (LB).

In Vitro Infection with Bacteria and Supernatant Production

[0162] Single bacterial colonies were grown overnight and restarted the next day to reach an absorbance at 600 nm ranging between 0.550 and 0.650 corresponding to 0,550-0,650?10.sub.9 colonyforming units (CFUs)/mL. Tumor cells were incubated with or without Gap Junctions blocker, Heptanol (1 mM) for 1 hour and half at 37? C. Then, cells were incubated with bacteria for 1 hour and half at 37? C. at a cell-to-bacteria ratio of 1:50, in the appropriate medium without antibiotics. After incubation, the cells were washed and incubated at 37? C. in medium supplemented with gentamicin (50 ?m/mL) for 2 hours. The cells were washed twice and incubated overnight with medium supplemented only with gentamicin in order to kill extracellular bacteria. The next day, the cells were centrifuged at 2000 rpm for 5 minutes; the supernatant was collected and filtered with 70 ?M cell strainer. The supernatant was freeze-dried.

Mart-1-Specific CTL Generation

[0163] To generate peptide-specific CTLs from PBMC, 106 PBMC HLA-A2+ were cultured in 1 mL of RPMI 1640 medium (supplemented with 5% human serum, 2 mM L-Glutamine, 100 U/mL Penicillin, 100 ?g/mL Streptomycin, 10 ?g/mL Gentamicin, 10 ?g/mL beta-mercaptoethanol and 1% nonessential amino acids) in 24-well-plates. To this, 2?106 Mart-1-pulsed and irradiated (10 Gy) PBMC (HLA-A2) were added as antigen presenting cells in the same medium supplemented with 100 U/mL IL-2. To pulse PBMC they were incubated for 90 minutes at 37? C. in RPMI supplemented with 50 ?M of peptide. After incubation, cell were washed twice and irradiated before mixing with the responding cells. The cells were stimulated at intervals of 10 days with irradiated peptide-pulsed autologous PBMC and 100 U/mL IL-2.

Adenosine 5-triphosfate (ATP) Bioluminescent Assay

[0164] Adenosine 5-triphosphate (ATP) Biolumescent Assay (CellTiter-Glo Luminescent cell viability Assay, Promega) allow to measure the quantity of ATP containing in the samples. ATP is consumed and light is emitted when firefly luciferase catalyzes the oxidation of D-luciferin.

[0165] Briefly, 100 ?L of Assay Mix solution were added to a reaction vial for 3 minutes at room temperature; then rapidly were added 100 of sample diluent, mixed and immediately measured the amount of light produced. The final value is proportional to the amount of ATP in the sample.

SN Preparation, Functional Assay and Mass Spectrometry

[0166] Supernatant (SN) from B16 or B16-OVA untreated or Salmonella treated cells were treated as follow. Proteins were removed from the SN through a Trichloroacetic acid (TCA)-based protein precipitation procedure. In detail, samples were incubated with TCA (13% final concentration) for 5 min at ?20? C. and then for 5 hours at 4? C. Samples are then ultra-centrifuged at 15000 g for 15 min; peptides-enriched SN was collected and the pellet was discarded.

[0167] Products were loaded on the C18 matrix and washed with 0.1% Formic Acid. Finally, peptides are eluted with increasing percentages of Acetonitrile (5%, 10%, 20%, 50%, 80%).

[0168] Acetonitrile was removed by speed vacuum centrifuge.

[0169] Fractions were analyzed by matrix-assisted laser desorption/ionisationtime of flight mass spectrometry (MALDI-TOF MS). Full nLC-ESI spectrum [300-1650 Da] at 53.3 min was analyzed to confirm SIINFEKL (SEQ ID NO:1) identity.

[0170] The presence of functional SIINFEKL (SEQ ID NO:1) peptide, OVA(.sub.257-264), was assessed by a functional assay using OT-I CD8 cells as describe in Materials and Methods.

T2 Assay for Peptide Binding

[0171] The T2 binding assay is based upon the ability of peptides to stabilize the MHC class I complex on the surface of T2 cells.

[0172] T2 cells were incubated overnight at 37? C. at 2?105 cells/well in FCS-free RPMI medium with 100 ?L of supernatant or MART-1 peptide (1 ?M and 10 ?M) as a positive control.

[0173] The next day, the cells were washed with FACS buffer (PBS, 0.1% sodium azide, 5% fetal bovin serum) and incubated for 10 min with blocking buffer (200 ?g/mL mouse IgG in FACS buffer). Then, the cells were incubated for 20 minutes at +4? C. with BB7.2, an HLA-A2 conformation specific mouse antibody. The cells were washed twice with FACS buffer and fixed in paraformaldehyde for later acquisition by Accuri C6 Flow Cytometer (BD).

Immunofluorescence Staining

[0174] OSA cells were washed twice with PBS and incubated for 30 minutes at room temperature with blocking buffer (PBS+0.03% of Tryton+2% FBS). Then, cells were incubated with Ab anti-phalloidin for 30 minutes at room temperature. The cells were washed twice with PBS and examined by fluorescent microscope.

Alkaline Phosphatase Staining

[0175] OSA cells were washed twice with PBS and incubated with paraformaldeide (1%) for 10 minutes at room temperature. The cells were washed and incubated with NBT/BCIP solution for 1 hour at room temperature. Then, the cells were washed with PBS and examined microscopically.

In Vivo Immunization with Supernatant

[0176] Mice were immunized subcutaneusly with 100 ?L of emulsion with supernatant containing released peptides and either Freund's Complete Adjuvant (CFA) or Freund's Incomplete Adjuvant (IFA). Nine days after the immunization, mice were killed, popliteal lymph nodes were smashed and cells were plated in flat-bottom 96-well plates and stimulated with PPD (3 ?g/mL), OVA protein (30 ?g/mL), OVA peptide 323-339 (3 ?g/mL) or OVA peptide 257-264 (3 ?g/mL). After 72 hours, the supernatant was collected and IFN-? production was measured by ELISA.

[0177] To evaluate antibody response mice were killed four weeks after immunization, and serum was collected. The antibody titer to Salmonella and B16 antigens was evaluated by ELISA.

In Vivo Vaccination with DC Loaded Supernatant

[0178] DC1 dendritic cells, matured with LPS (1 ?g/mL), were loaded with supernatant containing released peptides for 4 hours at 37? C. After incubation, the cells were washed twice and subcutaneously injected in the right flank of mice (on days 0 and 4). On 21 day 10.sup.5 B16 cells were subcutaneously injected in the left flank.

Results

Opening of Connexin Hemichannels by Salmonella Infection of Melanoma Cells

[0179] In the work of Saccheri et al (Saccheri, Pozzi et al. 2010) and in WO 2012/017033, it was shown that Salmonella induces, in melanoma cells, the up-regulation of connexin 43 (Cx43), a ubiquitous protein that forms gap junctions and that is often lost during carcinogenesis.

[0180] FIG. 1 confirmed that Cx43 expression was up-regulated in B16 melanoma cells after infection with the attenuated strain of Salmonella thyphimurium SL3261AT, as evaluated by Western blot analysis.

[0181] As described in the introduction, the single hemichannel that forms a gap junction in the plasma membrane is closed under resting conditions but can be induced to open under the influence of different stimuli (Saez, Retamal et al. 2005).

[0182] In order to assess whether Salmonella is able to stimulate the opening of connexin hemichannels on the surface of melanoma cells, inventors measured ATP extracellular concentration using an adenosine 5-thriphosfate (ATP) bioluminescent assay (as described in Materials and Methods) in Salmonella treated B16 cells.

[0183] Briefly, inventors used the attenuated strain of Salmonella thyphimurium SL3261AT to infect mouse melanoma cell line B16 and the same cells expressing ovalbumin (B16 OVA), previously treated or not with the gap-junction blocker, heptanol.

[0184] As shown in FIG. 2, Salmonella infection induces the release of extracellular ATP from both cell lines and this effect is significantly reduced by the gap junction blocker heptanol. This result indicates that a cytoplasmatic molecule can be released by connexin hemichannel (CxH) in a Salmonella dependent manner, demonstrating the role of bacteria to stimulate the opening of hemichannels, in a condition where it induces the up-regulation of Cx43.

Identification of Released Endogenous Peptides Functional Assay and Mass Spectrometry

[0185] SIINFEKL (SEQ ID NO:1) H-2Kb restricted OVA octapeptide (OVA .sub.257-264) is known to be processed and presented by B16-OVA MHC class I molecules. To identify this prototype endogenous peptide, released in the supernatant, inventors used the attenuated strain of Salmonella thyphimurium SL3261AT (SL) to infect mouse melanoma cell line B16 and the same cells expressing ovalbumin (B16 OVA). B16 OVA derived SN treated as described in material and methods were analyzed both functionally and biochemically to detect the presence of (OVA .sub.257-264) in the derived fractions.

[0186] As shown in FIG. 3a CD8 OT-I T cells activation measured, as IFN-gamma released, is mainly present in SN fractions 20% and 50%, which were further analyzed by mass spectrometry.

[0187] Full MS, FIG. 3b, and full nLC-ESI, FIG. 3c, spectra [300-1650 Da] at 53.3 min of B16 OVA-derived SN was performed and analysis of nLC-ESI spectrogram confirm the SIINFEKL (SEQ ID NO:1) identity demonstrating the release of a processed endogenous peptide in SN.

Gap-Junction Hemichannel Dependent Tumor Peptides Release by Salmonella-Infected Tumor Cells

[0188] Inventors continued their investigation asking whether cytoplasmic peptides could be transferred in a CxH dependent manner by Salmonella infected tumor cells to the extracellular milieu.

[0189] In order to investigate the release of MHC class I peptides by Salmonella infected tumor cells, inventors exploited the ability of exogenous peptides to stabilize the MHC class I complex on the surface of T2 cells. T2 are an HLA-A0201 hybrid human cell line lacking TAP-2 (transporter-associated with antigen processing) and consequently are defective in loading class I molecules with antigenic peptides generated in the cytosol. This leads to very unstable MHC class I molecules on the cell surface. The association of exogenously added peptides stabilizes surface expression of HLA molecules, recognizable by specific anti-HLA-A0201 antibody.

[0190] Briefly, inventors infected human melanoma cells SKmel-24 and colorectal adenocarcinoma cells HT29, with Vivofit?, an oral typhoid vaccine that contains live, attenuated cells of the bacteria Salmonella enterica serovar Thyphi (TY21a), and inventors collected the supernatant as described in Materials and Methods. T2 cells were incubated with the supernatant obtained from uninfected or infected cells treated or not with the gap junction blocker (heptanol). Surface expression of HLA-A0201 was evaluated using a conformation-specific mouse antibody, as described in Materials and Methods.

[0191] FIG. 4 shows that T2 cells incubated with Mart-1 (26-35) peptide, as positive control, display a level of HLA-A0201 mean fluorescence intensity (MFI) significantly higher than that of unloaded T2 cells (none), indicating that the presence of the exogenous peptides is capable of stabilizing MHC class I complexes on the surface of T2 cells. Incubation of T2 cells with supernatant obtained from infected tumor cells increases the MFI that is abolished by the gap junction blocker. These results demonstrate that peptides from Salmonella infected tumor cells are released in a CxH-dependent manner and they can bind to MHC class I molecules.

[0192] Similar results were obtained using supernatants collected from another tumor cell line (colorectal adenocarcinoma cells) suggesting that this phenomenon could be widely applied to other type of cancer cells.

[0193] Since infected tumor cells are able to release peptides in a CxH-dependent manner, inventors tested whether antigen-specific T cells could recognize those peptides. Murine dendritic cells (D1), previously incubated with the supernatant obtained from B16 cells expressing ovalbumin (B16-OVA) infected or not with Salmonella SL3261AT, were cocultured with the OVA specific-B3Z hybridoma T cells. After 72 hours, the amount of IL-2 secretion was assessed by ELISA as a measure of OVA peptide recognition.

[0194] In FIG. 5 it is shown that dendritic cells incubated with supernatant obtained from B16-OVA cells treated with Salmonella alone or in combination with IFN-? activate OVA-specific T cells as shown by the increase of IL-2 secretion. This result demonstrates that the antigenic peptides released by Salmonella infected tumor cells are recognized and are able to activate antigen-specific T cells.

[0195] By contrast, when dendritic cells loaded with supernatant obtained from B16 cells, were co-cultured with OVA specific T cells there was no production of IL-2, demonstrating the absence of OVA peptide and the specificity of the assay.

[0196] Based on this evidence, inventors decided to investigate the release of tumor peptides by Salmonella infected human tumor cell lines.

[0197] Inventors analysed, by Western blot analysis, the effect of Salmonella infection on Cx43 expression in human melanoma cells, SK-mel 24. As shown in the FIG. 6, Cx43 expression is high already in resting conditions and after Salmonella infection is slightly up-regulated.

[0198] To evaluate the release of antigenic peptides inventors infected human melanoma cells SKmel24 and 624.38 with Salmonella TY21a in presence or not of heptanol and the supernatant was harvested as described in Materials and Methods.

[0199] Human monocyte derived DCs differentiated in vitro from monocytes with GM-CSF and IL-4 (moDCs) were incubated with the supernatant, produced as indicated above, and cocultured with Mart-1 (26-35) specific CTLs (obtained as described in Materials and Methods). After 72 hours of coculture, the amount of IFN-gamma secretion was measured by ELISA.

[0200] FIG. 7a and FIG. 7b show respectively that moDCs or PDC incubated with supernatant of melanoma infected tumor cells, activate Mart-1 (26-35) specific human CTLs and the gap junction blocker significantly inhibits this effect. Exogenous addition of Mart-1 (26-35) peptide restored completely the response.

[0201] This result demonstrates a CxH-dependent release of tumor peptides by Salmonella infected human melanoma cell lines and their ability to activate tumor antigen-specific human CTLs.

Release of Tumor Peptides by Salmonella Infected Tumor Cells Induces an In Vivo Antitumor Immune Response

[0202] To investigate whether the tumor peptides released from infected tumor cells could induce an in vivo immune response, C57BL/6J mice were immunized in the footpad with supernatant, obtained from B16 OVA cells infected or not with Salmonella SL3261AT, emulsified with Freund's complete adjuvant (CFA).

[0203] Briefly, nine days after immunization, the popliteal lymph nodes were removed and cells were stimulated in vitro with PPD (Tuberculin purified protein derivated), as a positive control, and OVA protein. After 72 hours of incubation, the amount of IFN-gamma secretion was assessed by ELISA to evaluate the activation of immune cells.

[0204] As shown in FIG. 8A, immunization with the supernatant obtained from B16 OVA cells induced IFN-gamma production after in vitro recall with OVA protein that was reduced by heptanol. This response is similar to that induced after immunization with B16 OVA extract. Instead, as shown in FIG. 8B, when the mice were immunized with the supernatant obtained from Salmonella infected B16 OVA (SN B16 OVA AT), the IFN-gamma secretion was increased. These results demonstrate that peptides released by Salmonella infected tumor cells can induce an in vivo immune response and such response is CxH-dependent.

[0205] In order to identify the type of released tumor antigens, C57BL/6J mice were immunized subcutaneously with supernatant obtained from Salmonella infected or uninfected B16 cells treated or not with the gap-junction blocker (heptanol) or the proteasome inhibitor (MG132), emulsified with Freund's Incomplete adjuvant (IFA). Four weeks later inventors evaluated the amount of total IgG specific to Salmonella SL3261AT and to mouse melanoma cell line B16 by ELISA.

[0206] FIG. 9A shows that immune sera from mice immunized with the supernatant obtained from infected B16 cells recognize Salmonella antigens and this response is reduced by heptanol and MG132. This data suggests that Salmonella after infection is processed and peptides are released in the supernatant through CxH, as shown by the effect of heptanol. Importantly, these peptides are partly generated by proteasome processing because the antibody response is reduced by MG132 treatment.

[0207] FIG. 9B shows that Salmonella infection increased the release of B16 proteasome processed tumor peptides as indicated by the presence of B16 specific antibody in the serum of mice immunized with supernatant obtained from infected B16 cells.

[0208] This effect is reduced by heptanol and MG132, underlining the release of proteasome-processed tumor peptides by CxH.

[0209] Moreover, in FIG. 9B, it was shown that an antibody response against tumor peptides was induced also in mice immunized with supernatant obtained from uninfected B16 cells. However, the titer of antibody response is significantly lower than that of Salmonella infection's group, probably due to the lower concentration of tumor peptides release in the supernatant.

[0210] These results indicate the induction of an in vivo immune response by pre-processed tumor peptides released by Salmonella infected tumor cells.

[0211] In order to investigate the effect on tumor growth induced by the released pre-processed tumor peptides, inventors decided to use dendritic cells as adjuvant.

[0212] Briefly, inventors vaccinated C57BL/6J mice with murine dendritic cells (D1) previously loaded with supernatant obtained from non infected or infected B16 cells, twice (on days 0 and 4) before the challenge with B16 cells (day 21).

[0213] In FIG. 10, it is shown a statistically significant delay of tumor growth in mice vaccinated with DCs loaded with supernatant obtained from Salmonella infected B16 cells (red line) and the effect was lost when DCs were loaded with supernatant obtained from uninfected B16 cells (black line). The tumor peptides released by Salmonella infected tumor cells were captured and cross-presented by dendritic cells inducing an in vivo antitumor immune response.

[0214] These data confirm that tumor peptides released by Salmonella infected tumor cells are able to induce an in vivo anti-tumor immune response.

[0215] The inventors also assessed the effect of a three increasing doses of supernatant not in association with dendritic cells, to evaluate whether they could induce an immune response without exogenous dendritic cells but targeting endogenous cells so to use the peptides as a vaccine. They combined the peptides with different adjuvants: Incomplete Freund's adjuvant (IFA) plus Aldara (Imiquimod: a Toll-like receptor (TLR)-7 agonist, or ODN1826 (CpG Vax). Mice vaccinated with an increasing dose of supernatant equivalent to 0.5?10.sup.6 cells (Vax1), 2?10.sup.6 cells (Vax2), 5?10.sup.6 cells (Vax3) showed a positive correlation between dose and antitumor response in terms of overall survival (FIG. 11A-C). Mice vaccinated with CpG-Vax had a high cytotoxic T lymphocyte degranulation in their peripheral blood mononucleated cells (PBMCs); a similar trend was observed in PBMCs isolated from mice immunized with the higher dose of peptides-based vaccine combined with IFA and Aldara (IFA Aldara Vax3) (FIG. 11 H). Consistently, they observed that both CpG Vax and IFA Aldara Vax3 mice had a significant prolonged survival compared with their control group (respectively CpG and IFA Aldara, FIGS. 11 C and E). Interestingly, vaccination mediated by dendritic cells loaded with peptides (DC Vax2) did not induce any increased antitumor response (FIG. 11D) suggesting that targeting endogenous DCs may be even more efficient. The inventors also tested the adjuvant role of a L. plantarum administered to mice before the immunization procedure (FIG. 11F). Unexpectedly, the oral administration of L. plantarum did not augment the effect of the immunization.

Mass Spectrometry Analysis Identify Pool of Endogenous Peptides Differentially Released by B16 Salmonella Infected Cells

[0216] In order to assess whether endogenous peptides are differentially released in the SN by SL infection, B16 cell were infected with SL and SN recovered and treated as describe in material and methods. FIG. 12a showed full MS spectra of B16 infected SN, and Venn diagram FIG. 12b highlight 399 protein associated to the detected and sequence peptides, that are differentially released in the SN of B16 SL-infected cells.

[0217] These data indicate that the ability to induce an in vivo anti-tumor immune response reside in a pool of immunogenic tumor peptides released by Salmonella infected tumor cells.

[0218] The inventors assessed the nature of the differentially produced peptides.

[0219] Owing to MaxQuant software that quantitatively analyze the sequenced and identified peptides they found that 31 peptides were significantly more abundant inside Vax samples (FIG. 13) of which 9 were selected based on their capacity to bind the MHC class I molecule (Table I). Importantly one of the statistically-selected Vax peptides is a known tumor antigen and nine peptides were predicted to be good MHC binders by Immuno Epitopes Data Base (IEDB) (SEQ ID Nos:2-10), suggesting that they could be potential novel epitopes (Table I).

Translational Study of Canine Osteosarcoma

[0220] Starting from these preliminary results, inventors then investigated whether this strategy could be translatable to other types of tumors, testing our approach in an experimental veterinary study for the treatment of a deadly form of spontaneous canine osteosarcoma.

[0221] Tumor specimens were obtained from a Veterinary clinic and primary osteosarcoma cell lines were generated as described in Materials and Methods.

[0222] Experimental vaccine containing the supernatant collected after infection of canine osteosarcoma cells with the vaccine strain Vivofit? of Salmonella enterica serovar thyphi (TY21a) (as described in Materials and Methods) was produced for all dog patients.

[0223] In order to induce the shrinkage of the tumor and afterwards a long lasting anti-tumor immune response, the treatment schedule includes the association of standard chemotherapy (4 cycles of carboplatin every 21 days) with experimental vaccination. Vaccination started after 2 cycle of standard chemotherapy. Two cycle of vaccination were administered intradermically, after topical application of 5% Imiquimod cream (Aldara?) on the injection site with the following modality: first cycle of 2 vaccinations at 21 days intervals and second cycle of 4 vaccinations at 30 days intervals. The patients will remain under observation for the following 24 h in the veterinary clinic. FIG. 14 shows primary canine osteosarcoma cells obtained from dog's osteosarcoma specimen. Panel A shows OSA cells analyzed by optical microscope: the cells appear adherent, mostly elongated of varying size or large pentagonal and polyhedral. There are numerous characteristic cytoplasmatic granules and vacuoles in most cells.

[0224] Panel B shows OSA cells stained with anti-phalloidin antibody to visualize actin filaments and with DAPI for nuclear counterstain. Moreover, inventors identified OSA cells by staining them for alkaline phosphatase activity (panel C).

[0225] Finally, inventors characterized the effect of Salmonella on connexin 43 (Cx43) in canine osteosarcoma cells. As evaluated by Western blot analysis (FIG. 15), Salmonella Ty21a is able to up-regulate Cx43 expression in osteosarcoma cells.

[0226] Inventors enrolled twenty osteosarcoma or high grade sarcoma dog patients and eight of these started the experimental therapy.

[0227] Prognosis for dogs suffering from osteosarcoma, undergoing surgery and chemotherapy is approximately 235-360 days after diagnosis. Since from one dog inventors could not obtain the specimen to generate the cell line, inventors decided to treat it using the vaccine generated from another dog in heterologous fashion.

[0228] Present results (Table II) indicate that three osteosarcoma patients died before completing the vaccination (OSA 1, 8 and 23), two are under vaccination (OSA 25 and OSA 29) and one patient (OSA 0) had a long overall survival, dying after 653 days for reasons not related to the tumor. With regard to the sarcoma affected dogs (SA), one patient is alive but still within the life expectancy period (SA 19), and the second patient is alive after more than 1073 days and survived also to a recurrence of disease (SA 5) that was again treated with vaccination, showing that a marked antitumor response has been promoted.

TABLE-US-00001 TABLEI ListoftheVax-specificpeptidesselectedfortheirMHC-bindingcapability(predicted byIEDBinsilicotoolaspercentilerank). MHCbinding Sequence Proteinnames prediction (SEQIDNO:2)PTDAQGSASGNHSV Poimin 0.7 (SEQIDNO:3)YDATYETKESKKEDL Cofilin 0.8 (SEQIDNO:4)REQAGGDATENF Cytochromeb5 0.8 (SEQIDNO:5)EEHPGGEEVL Cytochromeb5 0.9 (SEQIDNO:6)AVDKKAAGAGKVTKSAQKA Elongationfactor1-alpha 1.5 (SEQIDNO:7)ARPREEVVQKEQE Eukaryotictranslationinitiationfactor4H 1.6 (SEQIDNO:8)YDQTVSNDLEEH RasGTPase-activatingprotein-bindingprotein1 1.8 (SEQIDNO:9)KEQIQKSTGAP Cleavagestimulationfactorsubunit2 2.1 (SEQIDNO:10)PTPQDAGKPSGPG Adisintegrinandmetalloproteinasewith 2.2 thrombospondinmotifs1

TABLE-US-00002 TABLE II Treatment schedule Survival Patient Breed Diagnosis VAX (from diagnosis) OSA0 Penelope Terranova osteosarcoma Dec. 15, 2012 Heterologous Mar. 11, 2013 Completed dead after 653 OSA1 Kira Amstaff osteosarcoma Jan. 18, 2013 Autologous Apr. 3, 2013 5 out of 6 dead after 224 OSA8 Zorro Meticcio osteosarcoma Dec. 1, 2013 Autologous Mar. 27, 2014 5 out of 6 dead after 229 days OSA23 Lacy Rottweiler osteosarcoma Nov. 24, 2015 Autologous 5 out of 6 dead after 273 days OSA 25 Balti Rottweiler osteosarcoma Feb. 16, 2016 Autologous on going 236 OSA29 Margot Tosa Inu osteosarcoma Autologous On going SA 5 Luna Pitbull sarcoma November 2013 Autologous Feb. 17, 2014 Completed alive after 1073 days sarcoma relapse November 2015 Autologous Feb. 20, 2016 Completed SA 19 Shary Rottweiler sarcoma Aug. 25, 2015 Autologous Oct. 20, 2015 completed 411

TABLE-US-00003 TABLEIII ListofthepeptidesidentifiedbyMassspectrometryasdifferentiallypresentinthe supernatantofbacteria-treatedversusuntreatedmelanomacellsinFIG.13 Gene Sequence Proteins name Proteinname AKADGIVSKNF(SEQIDNO:11) Q9CQR2 Rps21 40SribosomalproteinS21 AKAPTKAAPKQ(SEQIDNO:12) Q8BP67 Rpl24 60SribosomalproteinL24 AKEAAEQDVEKK(SEQID P47963 Rpl13 60SribosomalproteinL13 NO:13) AKEAAEQDVEKKK(SEQID P47963 Rpl13 60SribosomalproteinL13 NO:14) ARPREEVVQKEQE(SEQIDNO:7) Q9WUK2 Eif4h Eukaryotictranslationinitiationfactor4H AVDKKAAGAGKVTKSAQKA(SEQ Q58E64 Eef1a1 Elongationfactor1-alpha IDNO:6) DNEYGYSNR(SEQIDNO:15) S4R1W1 Gm3839 Glyceraldehyde-3-phosphatedehydrogenase EEHPGGEEVL(SEQIDNO:5) G5E850 Cyb5a Cytochromeb5 GQVINETSQHHDDLE(SEQID Q5FWJ3 Vim Vimentin NO:16) HLDKAQQNNVE(SEQIDNO:17) F6SVV1 Gm9493 40SribosomalproteinS7 IGDSGVGKSN(SEQIDNO:18) G3UY29 Rab11b Ras-relatedproteinRab-11A IPSDSTRRKG(SEQIDNO:19) P62264 Rps14 40SribosomalproteinS14 KEQIQKSTGAP(SEQIDNO:9) A2AEK1 Cstf2 Cleavagestimulationfactorsubunit2 KKVAPAPAVVKKQEAK(SEQID NO:20) Q58ET1 Rpl7a 60SribosomalproteinL7a KNLQTVNVDEN(SEQIDNO:21) Q5M9K9 Rpl31 60SribosomalproteinL31 LQDSGEVRED(SEQIDNO:22) J3QPS8 Eif5a Eukaryotictranslationinitiationfactor5A-1 NKSTESLQANVQR(SEQID P47963 Rpl13 60SribosomalproteinL13 NO:23) PDPAKSAPAPKKGSKK(SEQID Q64525 Hist2h2bb HistoneH2Btype2-B NO:24) PEPAKSAPAPKKGSK(SEQID Q6ZWY9 Hist1h2bc HistoneH2Btype1-C/E/G NO:25) PTDAQGSASGNHSV(SEQID D6REH0 Tmem123 Porimin NO:2) PTPQDAGKPSGPG(SEQID P97857 Adamts1 Adisintegrinandmetalloproteinasewith NO:10) thrombospondinmotifs1 PVVQPSVVDRVA(SEQID Q9DBG5 Plin3 Perilipin-3 NO:26) RDGQVINETSQ(SEQIDNO:27) Q5FWJ3 Vim Vimentin REQAGGDATENF(SEQIDNO:4) G5E850 Cyb5a Cytochromeb5 RLSSLRASTSKSESSQK(SEQID Q5BLK1 Rps6 40SribosomalproteinS6 NO:28) RSAVPPGADKKAEAGAGSATE Q5M9K7 Rps10 40SribosomalproteinS10 (SEQIDNO:29) TEEEKNFK(SEQIDNO:30) P47963 Rpl13 60SribosomalproteinL13 TVETRDGQVINETSQ(SEQID Q5FWJ3 Vim Vimentin NO:31) TVGGDKNGGTRVVKLR(SEQID Q3UCH0 Rpl6 60SribosomalproteinL6 NO:32) YDATYETKESKKEDL(SEQID F8WGL3 Cfl1 Cofilin-1 NO:3) YDQTVSNDLEEH(SEQIDNO:8) P97855 G3bp1 RasGTPase-activatingprotein-bindingprotein 1

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