METHODS AND VACCINE COMPOSITIONS TO TREAT CANCERS

Abstract

The present invention relates to a method for obtaining a population of oncogenic cells modified comprising the following steps: i) obtaining a population of oncogenic cells from a subject suffering from a cancer; and ii) treating said cells with a fusion protein comprising an AAC-11 leucine-zipper (LZ) derived peptide which is fused to at least one heterologous polypeptide. Inventors have evaluated here the antileukemic efficacy of RT53, an anticancer peptide with potential immunological properties. Their results indicate that RT53 possesses a direct antileukemic effect, even at late stage. They also demonstrated that single injection of a vaccine consisting of leukemic blasts exposed to RT53, which induces the hallmarks of immunogenic cell death, was highly effective in preventing leukemia development in both prophylactic and therapeutic settings. The vaccine comprising RT53-treated APL cells generated long-term antileukemic protection and depletion experiments indicated that CD4+ T cells were of crucial importance for vaccine efficacy. Combined, their results provide the rational for the exploration of RT53-based therapies for the treatment of cancer, such as acute leukemia.

Claims

1. A method for treating a cancer in a subject in need thereof comprising the following step: i. obtaining a population of oncogenic cells from a subject suffering from a cancer; ii. treating said oncogenic cells with a fusion protein comprising an AAC-11 leucine-zipper (LZ) derived peptide which is fused to at least one heterologous polypeptide; and iii. administering to the subject a therapeutically effective amount of the population of the oncogenic cells modified in step ii).

2. The method for treating according to claim 1, wherein said population of oncogenic cells is obtained from blood, bone marrow or biopsy.

3. The method for treating according to claim 1, wherein said fusion protein comprises and/or consists of a sequence selected from the group consisting of SEQ ID NO:4, 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:13; and SEQ ID NO:14; SEQ ID NO:16; SEQ ID NO:17; SEQ ID NO:18; and SEQ ID NO:19.

4. The method for treating according to claim 1, wherein said fusion protein comprises and/or consists of a sequence SEQ ID NO: 18.

5. The method for treating according to claim 1, wherein said fusion protein comprises and/or consists, of a sequence SEQ ID NO: 19.

6. A vaccine composition comprising a population of oncogenic modified cells obtained according to the following steps: a. obtaining a population of oncogenic cells from a subject suffering from a cancer; and b. treating said cells with a fusion protein comprising an AAC-11 leucine-zipper (LZ) derived peptide which is fused to at least one heterologous polypeptide.

7. The vaccine composition according to claim 6 for use in the treatment of a cancer in a subject in need thereof.

8. The vaccine composition according to claim 6, wherein the cancer is selected from the following group but is not limited to: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous; adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; and roblastoma, malignant; Sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malig melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.

9. The vaccine composition according to claim 8 wherein the cancer is resistant.

10. The method for treating according to claim 1, wherein the population of oncogenic cells modified is combined with a classical treatment.

11. A kit comprising the vaccine composition according to claim 6 for use in the treatment of a cancer and/or resistant cancer, wherein the population of oncogenic modified cells is obtained according to the following steps: a. obtaining a population of oncogenic cells from a subject suffering from a cancer; and b. treating said cells with a fusion protein comprising an AAC-11 leucine-zipper (LZ) derived peptide which is fused to at least one heterologous polypeptide.

12. The method for treating according to claim 1, wherein the cancer is selected from the following group but is not limited to: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous; adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; and roblastoma, malignant; Sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malig melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.

13. The method for treating according to claim 12 wherein the cancer is resistant.

Description

FIGURES

[0214] FIG. 1: RT53 treatment increases APL mice survival. (A) The indicated cells were left untreated or exposed to increasing concentrations of RT53 for 20 h. Cell death induced by peptide treatment was measured by lactate dehydrogenase (LDH) release. Data are means±s.e.m. (n=3). (B) APL spleen cells were exposed to 5 μM of RT53 in the presence or absence of 50 μM zVAD-fmk or 50 μM Necrostatin-1 (Nec-1) for 3 h. Necrotic cell death was monitored by lactate dehydrogenase (LDH) release from cells into the culture medium. The obtained values were normalized to those of the maximum LDH released (completely lysed) control. Data are means±s.e.m. (n=3). (C) 104 APL blasts were inoculated intravenously (i.v.) into FVB/N mice at day zero. Mice were then either left untreated (n=6), treated with ATRA (5 mg, subcutaneous implantation of 21-day release pellets, n=6) at day 6 or injected intraperitoneally (i.p.) with RT53 (2.4 mg/kg in normal saline) at day 10 every day for a total of seven doses (n=6). Survival curves were analyzed with the Mantel-Cox test. (D) APL mice obtained as in (D) were either left untreated (n=6), injected i.p. with RT53 (2.4 mg/kg in normal saline) at day 10 every other day for a total of seven doses (D10 Q2D schedule, n=6) or at day every day for a total of seven doses (D20 schedule, n=4). Survival curves were analyzed with the Mantel-Cox test.

[0215] FIG. 2: Inhibition of APL progression by prophylactic vaccination with RT53-treated APL blasts. (A) APL blasts in basal RPMI medium were left untreated or treated with either 2.5 μM of RT53 for 6 h (CRT exposure analysis) or 10 μM of RT53 for 1 h (HMGB1 and ATP release analysis). Extracellular HMGB1 (left) and ATP (middle) were then measured in the culture supernatant by ELISA and ATP-bioluminescence assays, respectively, and surface exposure of CRT (right) detected by FACS analysis. (B) APL blasts were exposed to 30 μM RT53 in basal RPMI medium for 3 h for cell death induction and the whole suspension was injected subcutaneously (2×106 cells) into the left flanks of FVB/N mice. Twelve days later, the vaccinated (n=8) or control mice (n=7) were injected i.v. with live 104 APL blasts. Survival curves were analyzed with the Mantel-Cox test. The schematic protocol used is illustrated (right).

[0216] FIG. 3: Tumor specificity and long-lasting effect of prophylactic vaccination with RT53-treated APL blasts. (A) FVB/N mice were vaccinated with RT53-treated APL blasts (n=10), RT53-treated spleen cells from healthy mice (n=5), or the indicated RT53-treated cells (n=5 per group) using the same protocol as in FIG. 2B. Twelve days later, the vaccinated or control mice were injected i.v. with live 104 APL blasts. Survival curves were analyzed with the Mantel-Cox test. (B) Surviving mice from FIG. 2B were challenged with 104 live APL spleen blasts 107 days (group 1, n=5) or 226 days (group 2, n=4) after initial APL engraftment. Survival curves were analyzed with the Mantel-Cox test. The schematic protocol used is illustrated (lower panel).

[0217] FIG. 4. Requirement of CD4+ and CD8+ T cells for prolonged survival induced by vaccination with RT53-treated APL blasts. FVB/N mice were depleted of either CD4+, CD8+ or both T cell populations by bi-weekly i.p. injection of 0.2 mg of T cell-type specific monoclonal antibodies starting 2 weeks before experiments. Injections were then performed 2 times per week during the study period. The efficacy of depletion was monitored by flow cytometric analysis (right panel). Depleted (n=5 per group) or naive mice (n=10) were then vaccinated with RT53-treated APL blasts and injected i.v. with live 104 APL blasts (left, upper panel). Survival curves were analyzed with the Mantel-Cox test. The schematic protocol used is illustrated (left, lower panel).

[0218] FIG. 5: Therapeutic efficacy of RT53-treated APL blasts vaccination in well-established leukemia. 104 APL blasts were inoculated i.v. into FVB/N mice at day zero. Mice were then vaccinated with RT53-treated APL blasts 3 or 10 days after leukemia engraftment (n=5 per group). Survival curves were analyzed with the Mantel-Cox test. The schematic protocol used is illustrated (right panel).

[0219] FIG. 6: RT39 triggers calreticulin exposure as well as the release of HMGB1 and ATP. ((A) U2OS cells were left untreated or exposed to increasing concentrations of RT39 for 3 h. Extracellular HMGB1 was then measured in the culture supernatant by an ELISA assay. Data are means±s.e.m. (n=3). (B) U2OS cells were left untreated or exposed to increasing concentrations of RT39 for 3 h. Extracellular ATP was then measured in the culture supernatant using an ATP-bioluminescence assay. Data are means±s.e.m. (n=3).

[0220] FIG. 7: Inhibition of APL progression by prophylactic vaccination with RT39-treated APL blasts. APL blasts were exposed to 30 μM RT39 in basal RPMI medium for 3 h for cell death induction and the whole suspension was injected subcutaneously (2×10.sup.6 cells) into the left flanks of FVB/N mice. Twelve days later, the vaccinated (n=8) or control mice (n=8) were injected i.v. with live 10.sup.4 APL blasts. Survival curves were analyzed with the Mantel-Cox test. The schematic protocol used is illustrated (right).

EXAMPLE

[0221] Material & Methods

[0222] Peptides

[0223] Peptides were synthesized by Proteogenix (Strasbourg, France) and were >95% pure as verified by HPLC and mass spectrographic analysis.

[0224] Peptides sequence of RT53 and RT39 are the following:

TABLE-US-00002 RT53:  SEQ ID NO: 18 RQIKIWFQNRRMKWKKAKLNAEKLKDFKIRLQYFARGLQVYIRQLRLAL QGKT RT39:  SEQ ID NO: 19 RQIKIWFQNRRMKWKKLQYFARGLQVYIRQLRLALQGKT

[0225] The penetratin sequence is underlined.

[0226] Cell Lines and Chemicals

[0227] NB4 (purchased from ATCC), UF-1 (provided by Dr. Y. Ikeda, Tokyo, Japan), HUT-78 (provided by Dr. A. Marie-Cardine, INSERM U976, Paris, France) and B16F10 (provided by Dr. M. Dutreix, CNRS UMR3347, INSERM 1021, Paris, France) were used for the experiments. Cells were grown in RPMI 1640 medium supplemented with 10% foetal calf serum, L-glutamine (2 mM), 1 mM Hepes and 200 ug/ml penicillin/streptomycin antibiotics (Gibco). All cells were maintained at 37° C. in humidified 5% C02 atmosphere. All chemicals were purchased from Sigma.

[0228] Lactate Dehydrogenase, ATP and HMGB1 Release Assays

[0229] Release of lactate dehydrogenase (LDH) and ATP in the culture medium were assessed with the CytoTox 96 Non-Radioactive Cytotoxicity Assay and Enliten ATP Assay, respectively (Promega, Madison, WI, USA). HMGB1 release in the culture medium was assessed with the HMGB1 ELISA kit (IBL International, Hamburg, Germany).

[0230] Electronic Microscopy

[0231] Samples were fixed in 3% glutaraldehyde in phosphate buffer, pH 7.4 for 1 hour, washed, post-fixed with 1% osmium tetroxide in 0.1 M phosphate buffer and then gradually dehydrated in 70, 90 and 100% ethanol. After 10 min in a 1:2 mixture of epoxy propane and epoxy resin and 10 min in epon, samples were embedded in epoxy resin and polymerized at 60° C. for 24 h. After polymerisation, ultrathin sections of 90 nm were cut with an ultra-microtome (Reichert ultracut S), stained with uranyl acetate and Reynold's lead and observed with a transmission electron microscope (JEOL 1011). Acquisition was performed with a Gatan Orius 1000 CCD camera.

[0232] Determination of Surface-Exposed CRT

[0233] CRT exposure was assessed by surface immunostaining and flow cytometry. In brief, APL blasts cells (106 cells per mL) in RPMI 1640 medium supplemented with 10% foetal calf serum, L-glutamine (2 mM), 1 mM Hepes and 200 ug/ml penicillin/streptomycin antibiotics plated in 24 well plate were treated overnight with increasing concentrations of RT53 or RT39 peptides. Cells were washed with PBS (Phosphate-Buffered Saline), harvested and plated in 96-well round bottomed microtiter plates and incubated in blocking solution for 45 min (Blocking Solution Image-iT® Fixation/Permebilization Kit Cat. #R37602, Thermofischer Scientific). After 1×wash with PBS, cells were stained with anti-calreticulin primary antibody (Calreticulin (D3E6) XP® Rabbit mAb #12238, Cell Signaling). Goat anti-rabbit Alexa Fluor 488 was used as secondary antibody after another PBS wash (Alexa Fluor® 488 goat anti-rabbit IgG secondary antibody Cat. #A11034, Thermofischer Scientific). Cells were then analyzed with a CytoFLEX Flow Cytometer (Beckman and Coulter).

[0234] Ethics Statement

[0235] This study has been carried out in accordance with the EC Directive 86/609/EEC for animal experiments and was approved by the Committee for Experimental Animal Studies of the University of Paris 7 Institute Board Ethics (Protocol Number: 2303.01). Animals were housed and bred at our animal facility (Institut de Recherche Saint-Louis, Saint Louis Hospital, Paris, France) in vented animal cabinets under controlled temperature (22° C.) and 12 h light-dark cycle under pathogen-free conditions and were allowed food and water ad libitum.

[0236] Preclinical Acute Promyelocytic Leukemia-Transplantable Mice Model

[0237] APL blasts (provided by Drs. M. Bishop and S. Kogan, UCSF, USA) origin from the spleen of mice bearing the human PML-RARA cDNA construct driven by a myeloid linage specific promoter (hMRP8) in the FVB/N inbred strain of mice. For amplification, cells (1×105 or 1×106) were suspended in PBS and transplanted by intravenous (i.v) tail injection (200 uL) into female syngeneic recipient mice (5-6 weeks old). Establishment of leukemia was assessed by a decrease in blood platelet counts approx. 3 weeks after graft. Spleen cells from a primary recipient were collected, washed, re-suspended in PBS and injected (104 cells/mouse; 200 uL) into the tail veins of male FVB/N mice (7-8 weeks old) for experiments. For direct treatment experiments, mice were treated daily or every other day (i.p) with normal saline, RT53 or RT39 at 2.4 mg/kg in normal saline starting from day 10 or day 20 for a total of 7 injections.

[0238] RT53 or RT39-Treated APL Blasts Vaccination Assay

[0239] 2×106 live cells from primary recipients' spleens or the indicated cells were washed in PBS and resuspended in 200 μl of serum-free RPMI medium. The cells were then exposed to 30 μM RT53 for 3 h for cell death induction and the whole suspension of RT53 or RT39-treated cells was injected subcutaneously (2×106 cells) into the left flanks of FVB/N syngeneic mice. For leukemia induction, the mice were injected i.v. with 1×104 blast cells from primary recipients' spleens at the indicated time.

[0240] T Cell Depletion

[0241] Mice were depleted of either CD4+, CD8+ or both T cell populations by bi-weekly i.p. injection of 0.2 mg of ascites fluids containing an anti-CD4 or -CD8 antibody starting 2 weeks before experiments. Injections were then performed 2 times per week during the study period. Blood was collected by submandibular bleeding. PBMC were labelled with a mix of anti-CD3E-APC (MACS), anti-CD4-PE (MACS) and anti-CD8-APC-cy7 (BD Biosciences) (2.5 μl, 30 min, 4° C.). Red blood cells were then lysed in ACK buffer for 7 min at RT. The efficacy of depletion was monitored using Canto II (BD Biosciences) cytometer and data analyzed with FlowJo software.

[0242] Results

[0243] RT53 Possess Direct Antileukemic Properties

[0244] To test the antileukemic properties of RT53, we first exposed human all-trans retinoic acid (ATRA)-sensitive (NB4) and ATRA-resistant (UF-1) acute promyelocytic leukemia (APL) cells as well as mouse APL spleen blast cells derived from hMRP8-PML-RARA transgenic mice9 to increasing concentrations of RT53. As shown in FIG. 1A, RT53 decreased viability of all the tested cells through the rapid loss of plasma membrane integrity, as detected by the release of the intracellular enzyme lactate dehydrogenase (LDH). Importantly, no cell death was detected in spleen cells from healthy mice even at the highest concentration tested, indicating that RT53 exhibits selective cytotoxicity toward leukemic cells, but not normal cells. The observed LDH release upon RT53 treatment indicates that leukemic cells death is associated with loss of outer cell membrane integrity and cytoplasmic leakage, which are characteristic features of necrosis. In line with this hypothesis, ultrastructural analysis of APL spleen cells exposed to RT53 revealed an obvious necrotic morphology of the leukemic blasts, with loss of plasma membrane integrity and cytoplasmic swelling without morphological signs of nuclear apoptosis (data not shown). No changes in cellular integrity or ruptured cells could be detected in the normal spleen cells, confirming that RT53 exhibit high specificity towards leukemic cells (data not shown). Finally, the killing activity of RT53 was not hampered by inhibitors of apoptosis or necroptosis (FIG. 1B), confirming that, as previously observed in other settings.sup.6, 8, RT53-induced leukemic blasts death occurs through a non-regulated form of necrosis.

[0245] To explore the in vivo therapeutic potential of RT53 as a treatment of acute leukemia, we used a well-characterized preclinical APL model bearing the human PML-RARA oncogene which mimics human APL, both in its characteristics and its response to conventional therapeutic drugs such as ATRA and arsenic trioxide.sup.9, 10. In this model, 100% of the syngeneic mice (FVB/N) transplanted with 104 primary recipients' spleen blasts developed an APL and succumbed within 30 days (FIG. 1C). APL mice treated with RT53 (2.4 mg/kg) for 7 days starting on day 10 after leukemia engraftment had a significantly extended survival compared to control mice (P<0.0001). Survival of APL mice treated with RT53 was significantly (P<0.0001) superior to that of mice treated by ATRA (FIG. 1D). Similar survival advantage was obtained when RT53 was administered every other day for a total of seven administrations (D10 Q2D schedule, FIG. 1D). RT53 treatment starting on day 20 after leukemia engraftment, when leukemia is fully established.sup.11 as shown by standardized minimal residual disease (MRD) monitoring (high level of PML-RARα transcripts in PBL, bone marrow and spleen), also prolonged the survival of leukemic mice (D20 schedule, FIG. 1D). To our knowledge, no therapeutic approach has demonstrated such a pronounced effect on survival of mice with comparable advanced disease stage in this preclinical model. No organ toxicity (macroscopic or microscopic) was noted with either treatment schedule (data not shown). Therefore, these results indicate that RT53 possesses robust antileukemic activity both in vitro and in vivo.

[0246] A Vaccine Comprising RT53-Treated APL Cells Induces Long-Term Survival

[0247] Anticancer chemotherapies are particularly effective when they induce immunogenic cell death (ICD), thus eliciting an antitumor immune response.sup.12. We therefore investigated whether RT53 treatment of APL spleen cells would be able to induce the key known biomarkers of ICD, which include the endoplasmic reticulum (ER) chaperone calreticulin (CRT) surface exposure and the release of the chromatin protein high mobility group box1 protein (HMGB1) as well as ATP13. As showed in FIG. 2A, RT53 treatment triggered the release of both HMGB1 and ATP in the culture medium, detected by ELISA and ATP-bioluminescence assays, respectively, as well as surface exposure of CRT, detectable by FACS analysis, indicating that RT53 can induce all tested characteristics of ICD. Similar data were obtained with the human APL cells NB4 (not shown). To further investigate the capacity of RT53 to induce an antileukemic response, we took advantage of the APL preclinical model, which is based on immunocompetent FVB/N mice, to develop a prophylactic tumor vaccination model (FIG. 2B). Interestingly, 7 out of 8 mice vaccinated subcutaneously with RT53-exposed APL spleen blast cells did not develop disease after APL engraftment (FIG. 2B), indicating prophylactic effect of RT53-exposed APL cells. Eight months after APL engraftment, surviving animals were found disease-free by MRD monitoring. These data indicate that a simple vaccine constituted by RT53-treated APL cells triggered a very effective prophylaxis, protecting against the development of leukemia.

[0248] The Prophylactic Effect Generated by the RT53-Treated APL Cells Vaccine is Tumor Type Specific and Long-Lasting

[0249] To demonstrate the specificity of RT53-exposed APL cells prophylactic effect, mice were vaccinated subcutaneously with various human (NB4, HUT78) or mouse (B16F10) cancerous cells treated with RT53. As shown in FIG. 3A, none of the tumor cells generated protection against leukemia development, indicating that the protection induced by RT53-treated APL cells is tumor specific. Moreover, the absence of protection observed following vaccination with RT53-treated NB4 cells suggests that immune clearance of leukemic cells in RT53-exposed APL cells vaccinated animals does not rely on the recognition of the unique PML-RARα fusion protein, which is also expressed in NB4 cells. Finally, vaccination with RT53-treated spleen cells from healthy FVB/N mice generated no protection (FIG. 3A), indicating that the prophylactic effect against APL was exclusively triggered by the RT53-treated leukemic APL cells.

[0250] We next determined whether surviving mice developed long-lasting antileukemic protection. At 107 or 226 days after initial leukemia engraftment, survivors from FIG. 2B or control mice were challenged with 104 live APL spleen blast cells, in the absence of any further therapy. Strikingly, all vaccinated animals that received vaccination were protected from APL cells challenge, whereas all control mice succumbed to leukemia within 40 days (FIG. 3B). These results indicate that single vaccination with RT53-exposed APL cells induces eradication of a rapidly fatal tumor burden and evokes effective, long-lasting prophylaxis capable of preventing leukemia. Moreover, when we inoculated 107 spleen cells originating from long-term survivors vaccinated with RT53-treated APL blasts into secondary recipients, none of the injected mice developed APL (followed up >200 days; not shown), suggesting that this vaccine leads to eradication of APL-initiating cells. Injection of 104 spleen cells from unvaccinated APL mice was sufficient to establish APL and all recipients died (not shown).

[0251] CD4+ T Cells are Critical for the Induction of Prolonged Survival Induced by the RT53-Treated APL Cells Vaccine

[0252] To determine the cells involved in the prophylactic effect of RT53-treated APL cells vaccination, mice were depleted of CD4+ T, CD8+ T or both T cell populations using cell type-specific antibodies. Depletion of CD4+ T cells notably reduced vaccine-induced protection, whereas depletion of CD8+ T cells had no effect on vaccine efficacy (FIG. 4), demonstrating the essential role of CD4+ T cells in the induction of effective antileukemic immunity. As CD4+ T cells are crucial in the establishment of immune memory.sup.14, our results might explain the protective effect observed in FIG. 3B. However, complete loss of protection was observed in mice that were depleted of both T-cell populations (FIG. 4), indicating that the antileukemic response generated by RT53-treated APL cells vaccination required the presence of both CD4+ and CD8+ T cells. Although the precise mechanisms involved in vaccination-induced protection remain to be defined, the observation that leukemia development is effectively contained in mice depleted for CD8+ T cells is suggestive of the induction of innate immune cells, such as macrophages and natural killer cells, which can be activated by CD4+ T cells.sup.15. A cytotoxic activity of CD4+ T cells toward the leukemic cells is also possible, as witnessed in different experimental tumor settings.sup.1618. Of note, depletion of either CD4+, CD8+ or both T cell populations in long-term survivors from FIG. 2B did not result in APL (not shown), indicating that CD4+ and/or CD8+ T cells were not necessary for the maintenance of the antileukemic effects of RT53-treated APL cells vaccination and suggesting strongly that this vaccination leads to a cure of the mice.

[0253] The RT53-Treated APL Cells Vaccine is Effective in Mice with Well-Established Leukemia

[0254] Having shown that RT53-treated APL cells vaccination can elicit an efficient prophylactic antileukemic effect, we next tested the therapeutic benefit of the vaccine in mice with well-established leukemia. For that purpose, mice received the RT53-treated APL cells 3 (rising disease) or 10 (well-established disease) 11 days after APL cell engraftment and the onset of the disease were compared to that of non-vaccinated controls. Very interestingly, 100% of the vaccinated mice were protected from leukemia development and remained disease-free through 80 days of observation, irrespective of the immunization schedule (FIG. 5, lower panel). These data indicate that therapeutic administration of the RT53-treated APL cells vaccine resulted in eradication of leukemic cells in all the tested mice, even when vaccination was delayed until 10 days after tumor inoculation, indicating the effectiveness of this approach.

[0255] Inventors have also demonstrated an inhibition effect of APL progression by a prophylactic vaccination with RT39-treated APL blasts (FIGS. 6 & 7).

[0256] Conclusion

[0257] By using a well-established, aggressive APL model, inventors showed that single vaccination with a vaccine comprising whole leukemic cells exposed to a fusion peptide comprising an AAC-11 leucine-zipper (LZ) derived peptide with penetratin (such as RT53 or RT39), shown here to induce immunogenic cell death, protected against the development of leukemia in vivo both prophylactically and therapeutically. Cure of the tumor was observed and the vaccinated animals were protected against subsequent leukemia challenge in the absence of any further boosts, through the generation of long lasting, protective tumor specific responses involving CD4+ T cells. Indeed, the use of whole tumor cells lysates, obtained after in vitro treatment of cancerous cells with the peptides of the invention allows, upon injection, to reach sufficient antigen concentration in dendritic cells (DCs) to allow their activation. DCs cells are known to drive both CD4+ and CD8+ T cell responses. CD4+ T cells are needed for optimal and sustained effector CD8+ T cell responses as well as induction and maintenance of CD8+ memory.sup.26-27. The inventors clearly show that the anticancer response generated by peptide-treated APL cells vaccination required the presence of both CD4+ and CD8+ T cells, indicating activation of both T cell populations. One explication for this activation is that peptide-induced whole cell lysates, as tumor lysates, should contain all relevant major histocompatibility complex class I and class II epitopes capable of stimulating CD8+ and CD4+ T cells, respectively.sup.28. In addition, whole cell lysates such as the ones obtained with the peptide of the invention could greatly diminish the chance of tumor escape compared to using single epitope vaccines. Furthermore, the use of whole tumor cells eliminates the need to define, test and select for immunodominant epitopes. The tumor cells could be autologous, i.e. obtained from the patients, or allogeneic “off-the-shelf”. Tumor cells from each patient potentially carry gene mutations encoding for unique tumor associated antigens (TAAs) that are important in stimulating effective and long-lasting anti-tumor responses in the patient.

Such vaccine-based approach is practical, as it does not require the knowledge of specific tumor antigens, is not limited by the HLA phenotype and is safe because the antitumor effect is obtained without the use of potentially toxic immunostimulatory adjuvants in the vaccine. Indeed in view of the overwhelming importance of activated DCs in the initiation of therapeutic CD4+ and CD8+ T cell responses, therapeutic cancer vaccines must activate DCs with adjuvants in order to increase the immunogenicity of whole-cell tumor vaccines. Appropriate adjuvants include haptens, TLR (toll-like receptor) or CD40 agonists, cytokines, or activators of IFN genes.sup.25. Although adjuvants show promising results to favor therapeutic vaccines efficacy, at this time, only few immunostimulants have been approved for human use. Herein, surprisingly, the inventors do not use conventional adjuvants for the vaccine preparation: the lytic peptides used to prepare the herein described whole cell vaccines act as adjuvant, as they are still present when the vaccines are injected. As no toxicity nor adverse side effects were detected upon vaccination, these peptides could therefore be of great interest as innovative adjuvants for human clinic.

[0258] Because leukemic blasts can be easily obtained from the blood or bone marrow of patients at diagnosis, yielding sufficient material for clinical use, fusion peptide comprising an AAC-11 leucine-zipper (LZ) derived peptide with penetratin (such as RT53 or RT39) treated leukemic cells vaccines are a workable and effective strategy for immunotherapy of leukemia. Further, inventors demonstrated the single agent efficacy of a fusion peptide comprising an AAC-11 leucine-zipper (LZ) derived peptide with penetratin (such as RT53 or RT39) for the treatment of established leukemia, even at late stages, suggesting that a fusion peptide comprising an AAC-11 leucine-zipper (LZ) derived peptide with penetratin (such as RT53 or RT39) constitutes a therapeutic solution for patients who experienced multiple relapse because of resistance to approved therapies.

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