PTPS-BASED VACCINES AGAINST CANCER

Abstract

The present invention relates to the field of medicine. It more particularly relates to peptides, microvesicles containing such peptides, compositions containing same, in particular vaccine, and methods for stimulating an immune response in a subject.

Claims

1-15. (canceled)

16. A vaccine composition comprising a first Pioneer Translation Product (PTP), said PTP consisting of a peptide having 7 to 50 amino acids, a microvesicle and a pharmaceutically acceptable carrier or excipient.

17. The vaccine composition according to claim 16, wherein the microvesicle comprises a second PTP consisting of a peptide having 7 to 50 amino acids, said second PTP presenting at least one MHC class I epitope and/or at least one MHC class II epitope.

18. The vaccine composition according to claim 16, wherein the composition further comprises the full-length protein corresponding to the first PTP.

19. The vaccine composition according to claim 17, wherein the microvesicles express both the first and at least second PTP, optionally together with at least one third distinct PTP.

20. The vaccine composition according to claim 19, wherein microvesicles are CD8+ T cells activating microvesicles.

21. The vaccine composition according to claim 16, wherein the composition comprises PTPs activating CD4+ T cells and/or CD8+ T cells.

22. The vaccine composition according to claim 16, wherein the vaccine is a cancer vaccine.

23. The vaccine composition according to claim 22, wherein the composition comprises PTPs and microvesicles both derived from the cancerous tumor of the subject to be vaccinated.

24. The vaccine composition according to claim 22, wherein the cancer is a sarcoma or a melanoma.

25. A vaccine composition comprising a nucleic acid sequence encoding a Pioneer Translation Product (PTP) consisting of a peptide having 7 to 50 amino acids and a pharmaceutically acceptable carrier or excipient, wherein the nucleic acid sequence is selected from an intron, a 3 or 5 untranslated region (UTR), a LncRNA (Long non coding RNA), a miRNA (microRNA), an intergenic sequence and a combination thereof.

26. A microvesicle comprising a Pioneer Translation Product (PTP) consisting of a peptide having 7 to 50 amino acids expressed from a sequence selected from an intron, a 3 or 5 untranslated region (UTR), a LncRNA (Long non coding RNA), a miRNA (microRNA), an intergenic sequence and a combination thereof, said PTP presenting at least one MHC class 1 epitope and/or at least one MHC class II epitope.

27. A method of inducing an immune response in a subject comprising administering a vaccine composition according to claim 16 to said subject.

Description

FIGURES

[0068] FIG. 1: Role of the Pioneer Translation Products (PTPs) in tumor rejection.

[0069] A) Mice were injected subcutaneously with either MCA205 WT tumor cells or MCA205 transfected by the plasmid coding for glob-intron-SL8, the plasmid coding for glob-exon-SL8 or Ovalbumin. Half of the mice from each group received intravenous OT1 cells at Day 6 or Day 4. Tumor size was assessed through time until day 20. Data are given as mean?SEM. * p<0.05 (unpaired student t test).

[0070] B) Mice were injected subcutaneously with B16F10 WT tumor cells or B16F10 transfected by the plasmid coding for glob-intron-SL8, the plasmid coding for glob-exon-SL8 or Ovalbumin. At Day 3, half of the mice from each group received intravenous OT1 cells. Tumor size was assessed through time until day 19. Data are given as mean?SEM. * p<0.05 (unpaired student t test).

[0071] C) Mice were injected intraveinously with 2.Math.10.sup.6 OT1 cells marked with CFSE. After 3 h, 5.Math.10.sup.6 Hek cells WT or transfected by the plasmid glob-intron-SL8 or glob-exon-SL8 or Ova were injected intraperitoneally. After 3 days, cells from the lymph nodes and the spleen were collected and the CFSE expression in CD8 cells was analyzed. The dot plots are representative of the results obtained in the different mice.

[0072] FIG. 2: All PTPs: source of peptides for cancer-vaccines.

[0073] Groups of 6 mice were vaccinated with 125 ?g (PTPs?1), 64 ?g (PTPs?1/2), 32 ?g (PTPs?1/4) of PTPs or with 8 ?g (SIINFEKL 1/25) of SIINFEKL epitope (positive control for the MCA-205-Ova and negative control for the MCA-205 WT cells) emulsified in CpG+poly I:C (negative control). 15 days later, mice were challenged subcutaneously with 50.10.sup.3 MCA-205 living cells expressing Ovalbumin in the right flank (A) and with 50.10.sup.3 MCA-205 WT living cells in the left flank (B). The tumor growth was measured every 7 days for each tumor cell lines. Each line represents the tumor size in area (mm.sup.2) of the 6 mice in each group.

[0074] FIG. 3: specific PTPs from sarcoma cell lines: source of peptides for cancer-vaccines.

[0075] Groups of 6 mice were vaccinated with 125 ?g (PTPs-his ?1), 64 ?g (PTPs-his ?1/2), 32 ?g (PTPs-his ?1/4) of PTPs or with 8 ?g (SIINFEKL 1/25) of SIINFEKL epitope (positive control) emulsified in CpG+poly I:C (negative control). 15 days later the mice were challenged subcutaneously with 50.Math.10.sup.3 MCA-205 living cells expressing Ovalbumin in the right flank (A) and with 50.Math.10.sup.3 MCA-205 WT living cells in the left flank (B). The tumor growth was measured every 7 days for each tumor cell lines. Each line represents the tumor size in area (mm.sup.2) of the 6 mice in each group.

[0076] FIG. 4: PTPs plus exosomes: a new cancer-vaccine.

[0077] A) Analysis of the expression of CD9 and CD81 in exosomes purified from MCA205-glob-intron-SL8 cells by FACS. In pale grey the unstained exosomes, in dark grey the WT exosomes and in black the glob-intron-SL8-exosomes.

[0078] B) Left Panel: BMDCs (bone marrow dendritic cells) were pulsed by exosomes purified from MCA205 WT or MCA205-glob-intron-SL8. They were collected and cultured with OT1 cells. An ELISA to detect IL-2m was performed. Data are given as mean?SEM. Right panel: Exosomes were added to OT1 cells in absence of BMDCs. The quantity of mIL-2 produced in the supernatant after at least 18 h was evaluated by ELISA. The data are expressed as mean?SEM.

[0079] C) FACS analysis of the expression of SIINFEKL using the 25D1 antibody on the MCA205 cells and exosomes. Left panel, in dashed line the unstained MCA205 cells, in pale grey the WT MCA205 and in white MCA205 cells expressing the Glob-intron-SL8 construct. Right panel, in pale grey the unstained exosomes, in dark grey exosomes purified from MCA205 cells and in black the exosomes purified from MCA205 cells expressing the Glob-intron-SL8 construct.

[0080] D) Groups of 6 mice were vaccinated with 64 ?g (PTPs-his ?1/2), 32 ?g (PTPs-his ?1/4) of tumor-derived PTPs or with 64 ?g (PTPs-his ?1/2), 32 ?g (PTPs-his ?1/4) of tumor-derived PTPs plus 15 ?g of tumor-derived exosomes containing PTPs, or as positive control 8 ?g (SIIN 1/25) of SIINFEKL epitope emulsified in CpG+poly I:C. 15 days later, the mice were challenged subcutaneously with 50.Math.10.sup.3 MCA-205 living cells expressing Ovalbumin in the right flank. The tumor growth was measured for each tumor cell lines every 7 days. Each line represents the tumor size in area (mm.sup.2) of the 6 mice in each group.

[0081] FIG. 5: Addition of CD4 epitope to improve the PTPs cancer-vaccine.

[0082] Groups of 6 mice were vaccinated with 64 ?g (PTPs-his ?1/2) of tumor-derived PTPs or with 64 ?g (PTPs-his ?1/2) of tumor-derived PTPs plus 1 mg of purified Ovalbumin, or as positive control 8 ?g (SIIN 1/25) of SIINFEKL epitope emulsified in CpG+poly I:C. 15 days later, the mice were challenged subcutaneously with 50.Math.10.sup.3 MCA-205 living cells expressing Ovalbumin in the right flank and with 50.Math.10.sup.3 MCA-205 WT living cells in the left flank. The tumor growth was measured every 7 days for each tumor cell lines. Each line represents the tumor size in area (mm.sup.2) of the 6 mice in each group.

[0083] FIG. 6: Figure illustrating the different positions of the SL8 antigenic epitope in the Ovalbumin cDNA and in the introns sequence of the ?-Globin gene.

[0084] FIG. 7: specific PTPs from melanoma cell lines: source of peptides for cancer-vaccines.

[0085] Groups of 6 mice were vaccinated with 32 ?g or 16 ?g of PTPs-His or with 8 ?g of SIINFEK1 epitope (positive control) emulsified in CpG+Poly I:C (negative control). Fifteen days later the mice were challenged subcutaneously with 30.Math.10.sup.3 B16F10 living cells expressing Ovalbumine in the right flank along with matrigel (A) and with 30.Math.10.sup.3 B16F10 WT living cells in the left flank (B). The tumor growth was measured every 3-4 days for each tumor cell lines. Each line represents the average tumor size in area (mm.sup.2) of the 6 mice in each group.

[0086] FIG. 8: PTPs plus exosomes from melanoma cell lines.

[0087] Groups of 6 mice were vaccinated with 16 ?g of PTPs-His, with 15 ?g of exosomes derived from B16F10 cells or with PTPs-His 16 ?g along with 15 ?g exosomes emulsified in CpG+Poly I:C (negative control). Fifteen days later the mice were challenged subcutaneously with 30.Math.10.sup.3 B16F10 living cells expressing Ovalbumine in the right flank along with matrigel (A) and with 30.Math.10.sup.3 B16F10 WT living cells in the left flank (B). The tumor growth was measured every 3-4 days for each tumor cell lines. Each line represents the average tumor size in area (mm.sup.2) of the 6 mice in each group.

[0088] FIG. 9: PTPs plus melanosomes from melanoma cell lines.

[0089] A) BMDCs were pulsed by melanosomes purified from B16F10-glob-intron-SL8 cells. The BMDCs were then co-culture with the SL8-specific CD8+ T-cell hybridoma (B3Z) for 16 h and T-cell activation was estimated by measuring ?-galactosidase. B and C) Groups of 6 mice were vaccinated with 32 ?g of PTPs-His or with 30 ?g of melanosomes derived from B16F10 cells emulsified in CpG+Poly I:C (negative control). Fifteen days later the mice were challenged subcutaneously with 30.Math.10.sup.3 B16F10 living cells expressing Ovalbumine in the right flank along with matrigel (B) and with 30.Math.10.sup.3 B16F10 WT living cells in the left flank (C). The tumor growth was measured every 3-4 days for each tumor cell lines. Each line represents the average tumor size in area (mm.sup.2) of the 6 mice in each group.

[0090] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

[0091] Other characteristics and advantages of the invention are given in the following experimental section (with reference to FIGS. 1 to 6), which should be regarded as illustrative and not limiting the scope of the present application.

EXPERIMENTAL PART

Example 1Pioneer Translation Products (PTPs) in Combination with Exosomes: A New Cancer Vaccine

Materials and Methods

Cell Culture

[0092] MCA 205 mouse sarcoma cell line were cultured at 37? C. under 5% CO.sub.2 in RPMI 1640 medium (Life Technologies) in the presence of 1% glutamine, 1% pyruvate, 1% non-essential amino-acids and 10% FBS (Life Technologies) under standard conditions. B16F10 (syngeneic from C57BL/6J mice) were cultured at 37? C. under 5% CO.sub.2 in DMEM containing 10% FCS, 2 mM L-glutamine and 100 IU/ml penicillin/streptomycin.

[0093] MCA 205 and B16F10 cells were transfected with YFP-globine-intron-SL8-his plasmid using JetPrime according to the manufacturer's protocol (Ozyme) for the purification of PTPs. For the tumor rejection experiment, stable MCA 205-Ova and stable B16F10-Ova cells were prepared. Stable MCA 205-Ova are cultured in RPMI 1640 under standard conditions. Stable B16F10-Ova cells stably expressing the Ovalbumin protein are cultured in DMEM under standard conditions.

Animal Studies.

[0094] C57B1/6J mice were obtained from Harlan. OT1 C57B1/6J mice were generously provided by the CERFE (C. Daviaud) and bred at Gustave Roussy animal facility. 7 weeks C57BL/6J mice were inoculated with 0.1?10.sup.6 MCA205 or B16F10 tumor cells subcutaneously in the right flank. For MCA205, when the tumors reached a size around 20 mm.sup.2, the mice were injected with 0.1?10.sup.6 OT1 cells intravenously. In the B16F10 model, 0.2?10.sup.6 OT1 cells were inoculated intravenously three days after the tumor inoculation. All animal experiments were carried out in compliance with French and European laws and regulations.

PTPs-his Purification

[0095] Transfected MCA 205 or B16F10 tumor cells were sonicated in 10 mL of 6M guanidium-HCl, 0.01M Tris/HCl, pH 8.0, 5 mM imidazole and 10 mM ?-mercaptoethanol. Then, the lysate was incubated and rotated with Ni.sup.2+-NTA-agarose beads (Qiagen) for 4 h at RT. The beads were washed successively for 5 min at RT with 8 mL of each of the following buffers: 6M guanidium-HCl, 0.01M Tris/HCl, pH 8.0 and 10 mM ?-mercaptoethanol; 6M urea, 0.01M Tris/HCl, pH 8.0 and 10 mM ?-mercaptoethanol; 6M urea, 0.01M Tris/HCl, pH 6.8, 10 mM ?-mercaptoethanol and 0.2% Triton X-100; 6M urea, 0.01M Tris/HCl, pH 6.8 and 10 mM ?-mercaptoethanol; 6M urea, 0.01M Tris/HCl, pH 6.8, 10 mM ?-mercaptoethanol and 0.1% Triton X-100. PTPs were then eluted by incubating the beads for 20 min at RT in 400 mM imidazole, 0.15M Tris/HCl, pH 6.8, 30% glycerol, 0.72M ?-mercaptoethanol and 5% SDS. The eluate was dialyzed in PBS using a dialysis tubing MWCO 0.5 kD (VWR) overnight at RT. Finally, the eluate was quantified by a Bradford assay (ThermoFisher).

All PTPs Purification

[0096] MCA 205 tumor cells were lysed then sonicated in 10 mL of 6 M guanidium-HCl, 0.01 M Tris/HCl, pH 8.0, 5 mM imidazole and 10 mM ?-mercaptoethanol. The lysates were purified and the polypeptides were concentrated using a 3 kDa centrifugal filter (Merck Millipore). This column was centrifugated for 90 min. at 3000 g which allow us to purify small polypeptides, the definition of PTPs. The lower part was dialyzed in PBS using a dialysis tubing MWCO 0.5 kDa (VWR) overnight at RT. Finally, the eluate was quantified by a Bradford assay (ThermoFisher).

Peptides Extraction from Solid Tumor

[0097] Solid tumor disintegration was performed on ice by crushing material with a 0.22 ?m cell strainer. Solubilization was done by the addition of 1?SDS buffer (0.125 M Tris-HCl (pH 6.8), 2% sodium dodecyl sulfate, 10% glycerol, 5% 2-mercaptoethanol) ten times the weight of the tissue. The disintegrated tissue was incubated at 70? C. and was shacked at 1 400 rpm for 10 min. Then, it was centrifuged at 13 200 g for 5 min at RT in order to sediment and eliminate solid tissue. The D-Tube? Dialyzers (MerckMillipore) was used to purify and concentrate peptides with a molecular weight cut-offs of 5 kDa. A centrifugation was performed at 3 000 g for 1 h30. Finally, the peptide concentration was measured using BCA Protein Assay kit (Pierce).

Vaccination

[0098] Vaccines for the MCA205 cells were prepared according to the following groups: PTPs-his ?1 (128 ?g), PTPs-his ?1/2 (64 ?g), PTPs-his ?1/4 (32 ?g) ?/+exosomes, exosomes (purified from MCA transfected cells (15 ?g)), PTPs-his ?1/2 (64 ?g) ?/+(1 mg/50 ?L/mouse) Ovalbumin protein (Calbiochem), all PTPs ?1 (128 ?g), all PTPs ?1/2 (64 ?g), all PTPs ?1/4 (32 ?g), CpG (20 ?g) (Invivogen) and Poly(I:C) (50 ?g) (Invivogen), PBS (up to 300 ?L). Vaccines were prepared 2 h before injection and kept on ice. Prior to vaccination, C57BL/6 mice were anesthetized with 3% isoflurane. The vaccines were injected subcutaneously in the legs (150 ?L/leg) and in footpad (50 ?L/foot). Two weeks later, subcutaneous injections of 50*10.sup.3 MCA 205 tumor cells (right flank) and MCA 205 OVA tumor cells (left flank) were given. Once a week, tumors size was measured until they reached 300 mm.sup.2.

[0099] Vaccines for the B16F10 cells were prepared according to the following groups: 32 ?g or 16 ?g of PTPs-His, exosomes (purified from B16F10 transfected cells, 15 ug), melanosomes (purified from B16F10 cells, 30 ?g) or with 8 ?g of SIINFEKL epitope (positive control), CpG (20 ?g) (Invivogen) and Poly(I:C) (50 ?g) (Invivogen). Vaccines were prepared 2 h before injection and kept on ice. Prior to vaccination, C57BL/6 mice were anesthetized with 3% isoflurane. The vaccines were injected subcutaneously in the legs (150 ?L/leg) and in footpad (50 ?L/foot). Two weeks later, subcutaneous injections of 30?10.sup.3 B16F10 tumor cells (right flank) and B16F10 OVA tumor cells (left flank) were given. Once a week, tumors size was measured until they reached 300 mm.sup.2.

Results

Role of the Pioneer Translation Products (PTPs) in Tumor Rejection.

[0100] In the last decade, PTPs and DRiPs have been proposed to be the major source of peptides for the endogenous MHC class I pathway. To precisely define the role of PTPs in mediating a specific CD8+ T cells anti-tumor immune response, inventors inoculated individual C57BL/6 mice with two different tumor models: the MCA sarcoma model and the B16F10 melanoma model stably expressing their different constructs (see FIG. 6). For the MCA model, 10.sup.5 tumor cells were subcutaneously injected in C57BL/6 mice. Then tumors were allowed to grow to approximately 20 mm.sup.2. At this point, 10.sup.5 na?ve Ova-specific TCR-transgenic CD8+ OT-1 T cells were adoptively transferred to the mice. Then tumor growth was monitored and recorded every two days. After 14 days, inventors observed that adoptive transfer of OT-1 T cells prevent the development of MCA tumors stably expressing independently the SIINFEKL/SL8 epitope in the Glob-intron or in the Glob-exon setting (FIG. 1A, down and up panels). And, as expected, adoptive transfer of OT-I T cells do not prevent the growth of SL8-negative MCA tumors (FIG. 1A, down and up panels) confirming the specific recognition of the CD8+ T cells for the antigen that expressed the tumor cell line and confirming the specific role of the PTPs in inducing an anti-tumor response. For the B16F10 model, 10.sup.5 tumor cells were subcutaneously injected in C57BL6 mice. Then, 3 days later, 10.sup.5 na?ve Ova-specific TCR-transgenic OT-1 cells were adoptively transferred to the mice. Similarly to the MCA model, adoptive transfer of OT-I T cells prevents on one hand the development of B16F10 tumors stably expressing the SIINFEKL/SL8 epitope in the intron or exon sequences (FIG. 1B, up and down panels), and on another hand do not prevents the growth of SL8-negative B16F10 tumors (FIG. 1B, up and down panels) supporting again the idea that PTPs are inducing a specific anti-tumor response.

[0101] Moreover, to finally conclude that PTPs can contribute to cross priming in an in vivo model, HEK-293 cells were transfected with the different constructs and injected subcutaneously into CD45.1 congenic C57Bl/6 mice that received, 3 h earlier, na?ve OT-I CD8+ T cells stained with CFSE. If PTPs, expressed from exon and/or intron sequences contribute to cross priming then they expected to see a diminution over time of the CFSE fluorescence, demonstrating a proliferation of the CD8+ OT-I T cells. As seen in FIG. 1C, after 3 days of inoculation, the PTPs induced a CD8+ OT-I T cell division as compared to the negative control where HEK-293 cells were only transfected with an empty vector and where CD8+ OT-1 T cells did not proliferated over the same time of inoculation. Since HEK-293 cells are of human origin, they cannot directly present antigen that come from the PTPs directly to the murine CD8+OT-1 T cells. Therefore, the proliferation of the CD8+ T cells can only occur via the cross priming of the PTPs, supporting the tumor rejection results.

[0102] These results demonstrate that PTPs can induce a specific immune response in vivo by promoting a specific antigen tumor rejection. Furthermore, these results show that PTPs, in addition to being used as a major source of antigenic peptides for the endogenous pathway, might be also a source of exogenous peptides for the MHC class I exogenous pathway.

Tumor Polypeptides: Source of Peptides for Cancer-Vaccines

[0103] In parallel and to confirm the specific role of polypeptides carrying MHC class I epitopes as being a major source for a cancer vaccine, inventors have purified PTPs from WT tumor cell lines. For that purpose, the MCA205 WT tumor cell lines were lysed and all polypeptides of 5 kDa or smaller than 5 kDa, the definition of a PTP, were purified and used as vaccine in mice, as described previously with PTPs coming from inventors' constructs of interest. Different groups of 6 mice were vaccinated with different concentration of PTPs, or with the adjuvant itself (negative control). After 2 weeks, 50.Math.10.sup.4 MCA205 tumor cell line, expressing or not the Ovalbumin construct were subcutaneously injected in the right flank (MCA-205 Glob-intron-SL8) and left flank (MCA-205 WT) of mice. Inventors' data indicate that polypeptides of 5 kDa or smaller than 5 kDa purified from the nuclear compartment of a tumor cell lines can induce a defect of the same tumor independently of whether or not the tumor expresses inventors' specific model epitope (FIGS. 2A and 2B).

[0104] In parallel, polypeptides from solid tumor that have grown in mice for few weeks were purified. The solid tumors were disintegrated and then the polypeptides containing PTPs were purified with a cut-off of 5 kDa. The purified polypeptides were used as a vaccine in mice challenged two weeks after with the same tumor cell lines from which these polypeptides have been purified. Inventors' data indicate that polypeptides purified from solid tumors can induce a defect of the same tumor independently of whether or not the tumor expresses inventors' specific model epitope.

[0105] These experiments shed light the specific effect as a vaccine composed of tumor-derived polypeptides of different lengths, on the growth of the tumor, and, support the idea that PTPs can be used as vaccine to elicit a specific anti-tumor-T-cell response.

PTPs: Source of Peptides for Cancer-Vaccines

[0106] In this study, before being used as a vaccine, PTPs purified from sarcoma MCA205 and melanoma B16F10 cell lines were analyzed by mass spectrometry to look more closely at the nature of the different polypeptides that compose the vaccine. As shown in Table 1, the vaccine consists of different polypeptides of different length.

TABLE-US-00001 TABLE1 Massspectrometryanalysisofpeptidesderived fromexosomesproducedbyMCA205cells.The peptidecorrespondingtotheSIINFEKLpeptide derivedfromanintronsequenceishighlighted. Peptide Peptide Peptidesequence length,a.a. origin VNVDEVGGEALGR 13 YFP-globin (SEQ01) SAMPEGYVQER 11 YFP-globin (SEQ02) FEGDTLVNR 9 YFP-globin (SEQ03) FSVSGEGEGDATYGK 15 YFP-globin (SEQ04) SIINFEK 7 Chicken (SEQ05) Ovalbumin LEYNYNSHNVYIMADK 16 YFP-globin (SEQ06) GEELFTGVVPILVELDGDVNGHK 23 YFP-globin (SEQ07)

[0107] The SL8 epitope is the epitope that will be recognized at the cell surface by the na?ve Ova-specific TCR-transgenic CD8+ OT-1 T cells and will have for consequence to induce a proliferation of specific CD8+ T cells and a tumor rejection.

[0108] In the previous part of the study, inventors were looking at tumor rejections of tumor cell lines that were expressing their PTPs with the help of specific CD8+ T cells, whereas in this part of the study they aimed to demonstrate that mice vaccinated with their tumor-derived PTPs in a prophylactic manner show a defect in tumor growth when compared to mice that have not been vaccinated, supporting the hypothesis that PTPs can induce as vaccine a tumor growth defect and a specific CD8+ T cells immune anti-tumor response.

[0109] For that purpose, different groups of 6 mice were vaccinated with different concentration of PTPs, or with the adjuvant itself (negative control), or with the SL8 epitope emulsified in the same adjuvant (positive control). These PTPs were purified from mice tumor cell lines that were previously transfected by the Glob-intron-SL8-His construct. After 2 weeks, 50.Math.10.sup.4 cells from the transfected MCA205 tumor cell line expressing PTPs identical to the purified PTPs, were subcutaneously injected in the right flank of mice. In the left flank of the mice 50.Math.10.sup.4 wild-type MCA205 tumor cells were similarly inoculated. Inventors' data indicate that PTPs can induce a defect in the tumor growth from the tumor cell line that expresses the PTPs but not from the wild-type (WT) tumor cell lines (FIGS. 3A and 3B), demonstrating the specific anti-tumor effect of the PTPs vaccine.

[0110] All those experiments shed light the specific effect of PTPs on tumor growth, and, support the concept that PTPs can be used as vaccine in mice to elicit a specific anti-tumor-T-cell response in prophylactic and therapeutic manners.

PTPs and Exosomes: A New Cancer-Vaccine

[0111] Inventors have recently demonstrated that PTPs are a better source of peptide for the MHCclass I cross presentation pathway than full length protein. Inventors are now reporting that PTPs allows a better cross presentation when stored in vesicles. In fact subcellular fraction that can be released by most of the cells, when smaller than 400 nm, are called microvesicles or exosomes (30-100 nm). To follow this idea, inventors hypothesized that the PTP transfer is mediated by exosomes secreted from the donor cells and internalized by bone marrow dendritic cells (BMDCs). The exosomes from the MCA 205 cell lines were purified according to previous reports. To confirm that the purified materials are exosomes, a FACS analysis was conducted. FIG. 4A reveals the presence of the different surface proteins CD9 and CD81, usual markers of exosomes, confirming that the purified microvesicles from the different cell lines were exosomes. Then, these exosomes were pulsed directly on BMDCs. FIG. 4B (left panel) shows that BMDCs, that have engulfed the MCA 205 tumor-derived exosomes, are capable to activate the CD8+ OT-1 T cells. Since the exosomal purified fraction was from MCA tumor cell lines, a cell line that expressed endogenously the K.sup.b molecules, inventors were wondering if the exosomes from this cell lines could have activated directly the CD8+ OT-1 T cells. The derived-MCA exosomes were pulsed directly on the CD8+ OT-1 T cells. No activation of the T cells was seen after addition of the exosomes (FIG. 4B, right panel). In fact when they looked at the expression of the MHC class I K.sup.b molecules by FACs analysis, using the anti-K.sup.b antibody, they could detect K.sup.b molecules, as expected, on the cells surface of the mouse cell lines and in the same time they could not detect K.sup.b molecules at the cell surface of the exosomes (FIG. 4C), supporting the fact that MCA exosomes could not by themselves activate CD8+ OT-1 T cells.

[0112] So the next step in the vaccin design has been to include in the PTPs-based cancer vaccines, the exosomes of the same tumor cell lines where the PTPs have been purified. For that purpose, inventors have incubated purified PTPs from MCA-205-Glob-intron-SL8 with exosomes from the same tumors for few hours in an adjuvant. Then different groups of 6 mice were vaccinated with different concentration of PTPs, with or without exosomes (15 ?g) or with the adjuvant itself (negative control), or with the SL8 epitope emulsified in the same adjuvant (positive control). Inventors' data indicate that the vaccine composed of tumor-derived PTPs with tumor-derived exosomes induce a better defect in the tumor growth (FIG. 4D, cross lines) from the tumor cell line that expresses the SL8 epitope as PTPs (MCA Ova tumor cells) than the vaccines composed only by the tumor-derived PTPs (FIG. 4D, square lines).

Addition of CD4 Epitope to Improve the PTPs Cancer Vaccine

[0113] From above results, inventors have shown that MHC class I peptides incorporated in PTPs and found in exosomes induce a specific anti-tumor response in mice. Nevertheless the main goal of vaccination and especially in cancer treatment is to avoid the relapse of it. And to avoid this relapse, it is necessary to induce a long lasting immunity. It is well established that CD4+ T cells can initiate and extend the life of specific anti-tumor CD8+ T cells and furthermore to induce an accumulation of professional antigen presenting cells (pAPCs) at the tumor sites. This accumulation can be beneficial as PTPs produced by the tumor are a better source for the MHC class I pathway presented by pAPCs than full length proteins. For all those reasons, a vaccine composed of PTPs in combination with the full length protein from the same gene was used. Different groups of 6 mice were vaccinated respectively with PTPs alone, or in combination with the protein Ovalbumin, or with the adjuvant itself (negative control), or with the SL8 epitope emulsified in the same adjuvant (positive control). Inventors data indicate that the vaccine composed of tumor-derived PTPs in combination with the full length protein induce a better defect of the tumor growth (FIG. 5, cross line) from the tumor cell line that expresses the SL8 epitope as PTPs (MCA Ova tumor cells) than the vaccine composed only by the tumor-derived PTPs (FIG. 5, square line) and no effect can be seen of the growth of the WT tumor cell lines, demonstrating the specific anti-tumor effect of the PTPs-full length protein vaccine and supporting the idea that for a better immune response against transformed cells it is preferred to combine peptides activating CD8.sup.+ and CD4.sup.+ T cells in the vaccine to induce a better and long-lasting anti-tumor immune response.

Example 2Vaccines Against Melanoma

PTPs-Based Vaccines Against Melanoma:

[0114] Inventors have shown in example 1 that PTPs purified from sarcoma cell lines such as MCA205 can be used as vaccine in mice to elicit a specific anti-tumor-T-cell response in prophylactic manner. To expend their idea that PTPs are suitable as anti-cancer vaccine they looked at other types of cancer. For that purpose inventors have purified PTPs from melanoma cell lines such as the murine B16F10 cell line. Then, different groups of 6 mice were vaccinated with different concentration of PTPs, with the adjuvant itself (negative control), or with the SL8 epitope emulsified in the same adjuvant (positive control). These PTPs were purified from mice B16F10 tumor cell lines that were previously transfected with inventors' Glob-intron-SL8-His construct. After 2 weeks, 50.Math.10.sup.4 cells from the transfected B16F10 tumor cell line, expressing PTPs identical to those which have been purified, were subcutaneous injected in the right flank of mice. In the left flank of the mice 50.Math.10.sup.4 wild-type B16F10 tumor cells were similarly inoculated.

[0115] Inventors' data indicate that PTPs can induce a defect of the tumor growth from the melanoma tumor cell line that expresses the PTPs but not from the wild-type (WT) melanoma-tumor cell lines, demonstrating the specific anti-tumor effect of the PTPs vaccine (FIGS. 7A and 7B).

[0116] All those experiments shed light the specific effect of PTPs on any tumor growth subtypes, and, support the idea that PTPs can be used as vaccine in mice to elicit a specific anti-tumor-T-cell response in prophylactic and therapeutic strategies.

PTPs-Exosomes Based Vaccines Against Melanoma:

[0117] Inventors have previously reported that tumor-derived exosomes contain PTPs, and that these exosomes can be associated with PTPs, themselves purified from tumor cell lines, to be used as a cancer vaccine. The exosomes from the B16F10 cell lines expressing the Glob-intron-SL8 construct were purified according to previous reports. The inventors have incubated purified PTPs from B16F10-Glob-intron-SL8 with exosomes from the same tumor for few hours in an adjuvant. Then different groups of 6 mice were vaccinated with different concentration of PTPs, with or without exosomes (15 ?g) or with the adjuvant itself (negative control), or with the SL8 epitope emulsified in the same adjuvant (positive control). Inventors' data indicate that the vaccine composed of tumor-derived PTPs with tumor-derived exosomes induce a better defect in the tumor growth (FIG. 8A, square line) from the tumor cell line that expresses the SL8 epitope as PTPs (B16F10 Ova tumor cells) than the vaccines composed only by the tumor-derived exosomes (FIG. 8A, circle black line). From these results the tumor-derived exosomes from melanoma cell lines containing PTPs are stimulating a weak specific immune response, compare to what inventors have seen with the MCA 205-derived exosomes. In fact, the combination of tumor-derived PTPs and tumor-derived exosomes is more potent in inducing a tumor growth defect even if this effect is not enough to induce a complete tumor rejection. Inventors' data indicate also that combination of PTPs and exosomes purified from melanoma cell lines can induced a weak defect in the tumor growth from the tumor cell line that expresses the PTPs but not from the wild-type (WT) tumor cell lines (compare FIGS. 8A and 8B), demonstrating the specific anti-tumor effect of the PTPs-exosomes based vaccine.

PTPs-Melanosomes Based Vaccines Against Melanoma:

[0118] Since exosomes from melanoma cell lines induce a weak defect in tumor growth, the inventors hypothesize that another vesicles released by the melanoma cell lines could have the same effect as the sarcoma exosomes on tumor growth. Melanoma cell lines have the ability to secrete not only exosomes but also melanosomes. In fact melanocytes are specialized in the production of melanin pigment that is stored in organelles called melanosomes (Raposo and Marks, 2007). Melanosomes are a tissue-specific lysosome-related organelle (Raposo and Marks, 2007), classified into two main maturation stages based on morphology and pigmentation level (Watabe, Kushimoto et al., 2005). Immature (stage I and II) melanosomes lack pigment and are located in the central cytoplasm; these are termed pre-mature melanosomes. Mature, heavily pigmented melanosomes (stage III and IV) or mature melanosomes predominate at distal dendrites, the main site of their secretion.

[0119] To follow the idea that the PTP transfer could be mediated by melanosomes secreted from the melanoma donor cells and internalized by bone marrow dendritic cells (BMDCs) as inventors have reported from the sarcoma exosomes, the secreted melanosomes from the B16F10 cell lines were purified according to previous reports. Then these melanosomes were pulsed directly on BMDCs. FIG. 9A shows that BMDCs, that have engulfed the B16F10 tumor-derived melanosomes, are capable to activate the B3Z hybridoma cell lines, which are specific to recognize the MHC class I Kb/SIINFEKL complex at the cell surface. Inventors next tested if inventors could detect the corresponding PTPs inside these secreted melanosomes. For that purpose inventors expressed a construct in B16F10 cells in which the 6?His-tag was inserted next to the SL8 epitope in the intron. Inventors then enriched the PTPs from purified and sonicated secreted stage IV melanosomes using nickel agarose beads and subjected these fractions to LC-MS/MS mass spectrometry analysis. Table 2 shows different peptide fragments carrying, or not, the SL8 epitope. It is worth pointing out that the enrichment step was required in order to yield a sufficient enough concentration of intron-derived PTPs to be detected by MS analysis. This is in line with previous observations showing that despite being an excellent substrate for the endogenous pathway, the PTPs are rare products.

TABLE-US-00002 TABLE2 Massspectrometryanalysisofpeptidesderived frommelanosomesproducedbyB16F10cells transfectedbyGlob-inron-SL8construct.Thepep- tidecorrespondingtotheSIINFEKLpeptide derivedfromanintronsequenceishighlighted. Peptide Peptide Peptidesequence length,a.a. origin LEYNYNSHNVYIMADK 16 YFP-globin (SEQ06) AGYTMVHLTPEEK 13 YFP-globin (SEQ12) SAMPEGYVQER 11 YFP-globin (SEQ02) SAVTALWGK 9 YFP-globin (SEQ13) VNVDEVGGEALGR 13 YFP-globin (SEQ01) DHMVLLEFVTAAGITLGMDELYK 23 YFP-globin (SEQ14) FEGDTLVNR 9 YFP-globin (SEQ03) GEELFTGVVPILVELDGDVNGHK 23 YFP-globin (SEQ07) AEVKFEGDTLVNRIELK 17 YFP-globin (SEQ15) GIDFKEDGNILGHK 14 YFP-globin (SEQ16) TIFFKDDGNYK 11 YFP-globin (SEQ17) SIINFEK 7 Chicken (SEQ05) Ovalbumin FSVSGEGEGDATYGK 15 YFP-globin (SEQ04) SAVTALWGKVNVDEVGGEALGR 22 YFP-globin (SEQ18) KAGYTMVHLTPEEK 14 YFP-globin (SEQ19) YQTSLYK 7 YFP-globin (SEQ20) FSVSGEGEGDATYGKLTLK 19 YFP-globin (SEQ21) FEGDTLVNRIELK 13 YFP-globin (SEQ22) AEVKFEGDTLVNR 13 YFP-globin (SEQ23)

[0120] Moreover, the inventors included in the PTPs-based cancer vaccines as herein described above, the purified secreted stage IV melanosomes from the B16F10 melanocytes. For that purpose, different groups of 6 mice were vaccinated respectively, with 30 ?g of secreted stage IV melanosomes, with 16 ?g of PTPs purified from B16F10-Glob-intron-SL8 and with the adjuvant itself (negative control). Inventors' data indicate that the vaccine composed of tumor-derived melanosomes of stages IV induce a better defect of the tumor growth from the tumor cell line that expresses the SL8 epitope as PTPs (B16F10 Ova tumor cells) than the vaccines composed only by the tumor-derived PTPs (FIG. 9B). Inventors' data indicate also that melanosomes can induce a defect in the tumor growth from the tumor cell line that expresses the PTPs but not from the wild-type (WT) tumor cell lines (compared FIGS. 9B and 9C), demonstrating the specific anti-tumor effect of the melanosome-containing PTPs based vaccine.

Discussion

[0121] If the main goal of a vaccine is to reduce the chance of transformed cells to escape the host immune system, inventors are demonstrating in this study that i) polypeptides (PTPs) produced earlier by a translation event distinct from the canonical event giving rise to full length proteins can be used as a specific and robust cancer-vaccine, ii) that the combination of such polypeptides and exosomes-carrying similar polypeptides can be an even more powerful combination as a cancer vaccine to trigger a broad T cell repertoire against transformed cells, and that iii) for a long lasting immune response a combination of CD8 and CD4 PTPs is required.

[0122] A class I binding synthetic epitope derived from the MAGE-1 protein has been already tested as a single peptide based vaccine in a clinical trial. Then other short peptides directed against different cancer have been used after that. Nevertheless in all of these studies, using single synthetic epitopes as vaccines the expected results were not as good as hoped since they were able to see any beneficial clinical responses in melanoma patients. These results can be explained by the fact that short peptides can bind directly to numerous types of cells and not only to pAPCs that could activate specific CD8+ T cells. Even worse when short peptides bind to MHC class I molecules to non-professional cells, they might induced a tolerance immune response. Also since these peptides are short they might be having any tertiary structure and so being subjected to rapid degradation. For all those reasons, inventors' PTPs-based cancer vaccine seems to be a better strategy than using short peptide. The different reasons are i) inventors' PTPs have been shown to be composed of peptides of different length, longer than 6 amino acids, preferably of at least 7 or 8 amino acids, ii) they have been shown to be the major source of peptides for the endogenous but also the exogenous MHC class I pathway iii) they need to be taken by pAPCs and being properly processed to reach the MHC class I pathway and being presented at the cell surface, iv) they can be composed of MHC class I epitopes but also of MHC class II epitopes. This last reason is very important if the main goal of the vaccine is to induce a long lasting immune response against cancer. In fact, inventors are designing a cancer vaccine to avoid any relapse of any type of cancer. Their vaccine is a therapeutic vaccine where PTPs and exosomes will need to be purified from a patient that has developed already a cancer. The goal of inventor's vaccine is to have a complete tumor rejection and no relapse. For these purposes their vaccine require to induce a quick immune response base on the activation of cytotoxic CD8+ T cells but for a long lasting response the vaccine require also to induce a memory response. In that particular case the memory response will be based on the role of CD4+ T cells. In fact when inventors purified PTPs from WT tumor cell lines (FIG. 2A) they observe a better inhibition of the tumor growth than when they used only the PTPs that come specifically from their engineered model construct where only one MHC class I epitope is used (FIG. 3A). The explanation is that they believe that in the purified PTPs from the WT tumor cell lines not only polypeptides containing MHC class I epitopes are purified but also polypeptides that contain MHC class II epitopes. This observation is supported by the fact that when they mixed PTPs purified from their engineered construct with the full length Ovalbumin, the effect of this combined vaccine is much more potent that when they used only PTPs itself (FIG. 5). CD4.sup.+ T cells have been shown to be essential for the maintenance of memory CD8+ T cells though the CD40-CD40L interaction between the CD4.sup.+ T cells and the pAPCs, this again demonstrating the important and specific role of the pAPCs in the success of a vaccination.

[0123] According to a series of reports, tumor-derived exosomes have been found to be immunosupressor inducing tumor immune evasion by acting on different pathways, for example by inhibiting the differentiation of DCs or by negatively regulating the NK cells, but they were also reported to have an immunostimulatory effect by inducing a specific tumor-immune response. They have been shown to usually contain tumor antigens and therefore been used as a novel source of tumor antigens for cancer vaccines. From inventors results the tumor-exosomes containing PTPs are stimulating a specific immune response. In fact, the combination of tumor-derived PTPs and tumor-derived exosomes is more potent in inducing a tumor rejection than tumor-derived PTPs themselves (FIG. 4). This result can be explained by the fact that they have succeeded to purify PTPs, produced from their engineered construct, inside the exosomes and that exosomes may also contain MHC class II epitopes coming from the tumor itself.

[0124] According to series of reports, melanosomes can be transfer from melanocyte to keratinocytes. In fact series of studies have reported that e.g. the melanosomes are the vesicles that are responsible for the transfer of melanin from the melanocytes to the neighboring keratinocytes. But more importantly for inventors it has been also shown recently that melanoma cells can acquire an MHC class II antigen by intercellular transfer with the help of secreted melanosomes. Here, inventors report that not only MHC class II epitope can be transferred but also MHC class I epitope. In fact inventors have discovered that secreted melanosomes contain PTPs that can be transferred from melanoma cell line to BMDCs with for consequence an activation of specific CD8.sup.+ T cells. Furthermore, inventors also report that melanosome can be a base for a melanoma cancer vaccine. Inventors showed that injected melanosomes in mice that have been inoculated with melanoma cell lines can induce an important tumor growth defect, supporting the idea that PTPs in combination with melanosomes can be used as a proper melanoma cancer vaccine. Taking into account that an appropriate tumor immune response is dependent on the recruitment and activation of specific CD8 cytotoxic T cells, and the fact that CD4.sup.+ T cells are necessary to these processes, and the fact that a proper anti-CD8 tumor immune response fail to established long lasting T cell memory in the absence of antitumoral-CD4.sup.+ T cell, their results show the importance of using melanosomes in a PTPs-based melanoma cancer vaccine, where secreted melanosomes contain MHC class II but also MHC class I epitopes.

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