METHODS AND COMPOSITIONS FOR TREATING CANCERS

Abstract

The invention relates to a method for treating cancers. Many cancers harbour sternness signature to de-differentiate into immature progenitors confer to tumor clones the re-expression of genes from fetal development. Inventors have obtained mice per group which received two boosts of vaccine 7 and 14 days with 2×106 irradiated hESCs cells that were mixed with 3 different adjuvants: 500 μg of TLR3, 50 μg of TLR9 agonist or 50 μg/ml of Quil Saponin vaccine adjuvant. After 14 days 5×104 4T1 cells were injected into the mammary fat pad of the mice and Valproic acid added in the drinking water at the dose of 4 mg/ml. They have shown that in contrast to the non-vaccinated mice, the mice vaccinated with hESC combined with a TLR3 agonist have generated the highest reduction of breast tumor volume (p<0.001) compared to the use of a TLR9 agonist or to Quil-A® Saponin vaccine adjuvant. Accordingly, the invention relates to a method for treating a subject suffering from a cancer with i) an agent that induces MHC-I presentation of antigens, ii) a vaccine composition containing an immunogenic element and iii) an adjuvant.

Claims

1. A method for treating a subject suffering from a cancer, comprising administrating simultaneously, separately, or sequentially to said subject a therapeutically effective amount of: (i) an agent that induces MHC-I presentation of antigens, wherein such agent is a histone deacetylase inhibitor (HDACi), (ii) a vaccine composition containing an immunogenic element, wherein the vaccine composition comprises inactivated pluripotent or fetal stem cells and (iii) an adjuvant, wherein the adjuvant is an agonist of toll-like receptor (TLR) 3.

2. (canceled)

3. The method of claim 1, wherein the vaccine composition comprises a population of inactivated fetal stem cells containing a multiplicity of fetal antigens.

4. The method of claim 1, wherein cells of the vaccine population express one or more antigen(s) also expressed by the cancer cells of the subject.

5. The method of claim 1, wherein the vaccine composition comprises a population of inactivated fetal cells expressing neo-fetal antigens.

6. The method of claim 1, wherein the vaccine composition comprises a population of inactivated pluripotent cells expressing neo-fetal antigens.

7. The method of claim 5, wherein the fetal stem cells have been obtained by a process comprising: a. differentiation of a population of pluripotent cells towards the pathway pertaining to the specific cancer of the patient, b. expansion of the cells thus differentiate, c. optionally, exposition to a mutagenic agent during expansion, to induce mutagenesis of genes in cells of said population, d. verification that at least 70% of the cells of the population express fetal markers, e. optionally, verification that the cells of the population express at least one tumor associated antigen (TAA) or neo-antigen that is present in the subject's cancer cells, and f. inactivation of the cells, in order for the cells to lose their ability to divide.

8. The method of claim 6, wherein the stem cells have been obtained by a process comprising: a. expanding pluripotent cells, in the presence of such conditions as to maintain the pluripotent ability of the cells, optionally in the presence of an agent that induces MHC-I presentation of antigens in said population during the expansion, and b. exposing the expanded cells to an inactivating agent that will inactivate the cells, while maintaining the cell envelope integrity.

9. The method of claim 1, wherein the histone deacetylase inhibitor is selected from the group consisting of Valproic acid (VPA), Vorinostat, Panobinostat, Givinostat, Belinostat, Levetiracetam, Entinostat, Mocetinostat, Practinostat, Chidamide, Quisinostat, and Abexinostat.

10. The method of claim 1, wherein the TLR3 agonist is Poly(A:U) or poly(I:C).

11. The method of claim 1, wherein an initial administration of the vaccine composition containing an immunogenic element and the agonist of TLR 3 is performed and multiple administrations of the histone deacetylase inhibitor are performed after the initial administration.

12. The method of claim 1, wherein the cancer is selected from the group consisting of liver cancer, bladder carcinoma, breast carcinoma, cervical carcinoma, cholangiocarcinoma, colorectal carcinoma, gastric sarcoma, glioma, glioblastoma, lung carcinoma, lymphoma, acute and chronic lymphoid and myeloid leukemias, melanoma, multiple myeloma, osteosarcoma, ovarian carcinoma, pancreatic carcinoma, prostate carcinoma, stomach carcinoma, renal carcinoma, head and neck tumor, and all sub-type of solid tumor, hematopoietic malignancies, and RET-mutated endocrine tumors including medullary thyroid cancer.

13. The method of claim 1, wherein the cancer is a hormone-dependent cancer.

14. The method of claim 13, wherein the cancer is selected from a breast cancer, a prostate cancer, a uterus cancer, and an ovary cancer.

15-28. (canceled)

Description

FIGURES

[0411] FIG. 1: Vaccination study with hESC combined with Valproic acid (VPA), Poly(A:U), ODN 2395 or Quil-A® adjuvant on 4T1 mice model. Vaccination study with hESC combined with Valproic acid (VPA), Poly(A:U), ODN 2395 or Quil-A® adjuvant on 4T1 mice model. Blab/c mice two boosts of vaccine containing 3 different adjuvants the day of tumor challenge. Tumor growth was assessed until 26 days to follow the tumor size usingcalipers permitting to measure both the longitudinal (a, mm) and transverse (b, mm) diameters. Tumor volume was calculated and plotted using calculation formula 1/611 (a+b/2)3 mm3.

[0412] FIG. 2: Valproic acid treatment increases MHC I expression in breast tumor cells. Variation in MHC I expression during VPA treatment at 2 mM on 4T1 adherent cells and on 4T1-derived mammospheres.

[0413] FIG. 3: HDACi up-regulates MHC1 in Lung cancer cell lines. Effects of VPA, Leviteracetam, Vorinostat and Entinostat on the LLC1 immunogenicity. MHCI and MHCII expression was quantified by flow cytometry analysis measuring the Relative Fluorescence Intensity (RFI). LLC1 were treated for 48 hours with Valproic Acid (A), Entinostat (B) Levetiracetam (C) and Vorinostat (D) at the dose corresponding to the IC50.

EXAMPLES

Example 1

[0414] The experiment was carried out as followed: Five mice per group received two boosts of vaccine 7 and 14 days with 2×10.sup.6 irradiated hESCs cells that were mixed with 3 different adjuvants: 500 μg of TLR3, 50 μg of TLR9 agonist or 50 μg/ml of Quil-A® Saponin vaccine adjuvant. After 14 days 5×10.sup.4 4T1 cells were injected into the mammary fat pad of the mice and Valproic acid added in the drinking water at the dose of 4 mg/ml. Five mice receiving no treatment were used as control.

[0415] Adjuvants used in the vaccine product: Three different adjuvants were tested:

1/ A TLR3-based adjuvant: Poly(A:U) (ref # tlrl-pau, InvivoGen) Polyadenylic-polyuridylic acid (poly(A:U)) is a synthetic double stranded RNA molecule that specifically signals through TLR3. Poly(A:U) is known to induce the activation of dendritic cells and T lymphocytes, to promote antigen-specific Th1-immune responses and to boost antibody production. The potent adjuvant activity of poly(A:U) has been exploited and approved in the treatment of breast cancers that express TLR3 (Conforti R. et al., 2010. Opposing effects of toll-like receptor (TLR3) signaling in tumors can be therapeutically uncoupled to optimize the anticancer efficacy of TLR3 ligands. Cancer Res. 70(2):490-500).
2/ TLR9-based adjuvant: ODN 2395 VacciGrade™ CpG ODN, type C (ref # vac-2395-1. InvivoGen) ODN 2395 is a type C CpG ODN composed by a synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs (CpG ODNs) that are mainly found present in bacterial DNA. CpG ODNs are recognized by murine TLR9, which is expressed exclusively on human B cells and dendritic cells, thereby inducing Th1-dominated immune responses. ODN 2395 that activate specifically mouse TLR9 is a potent inducer of IFN-α from dendritic cells and a strong B cell activators.
3/ Quil-A® Saponin vaccine adjuvant (ref # vac-quil InvivoGen). Quil-A® adjuvant is a saponin adjuvant containing the water-extractable fraction of saponins from the South-American tree, Quillaja saponaria Molina. Saponins induce a strong adjuvant response to T-dependent as well as T-independent antigens and induce strong cytotoxic CD8+ lymphocyte responses. When combined with cholesterol and phospholipids to form immunostimulatory complexes. Quil-A® adjuvant can activate both the cell-mediated and the antibody-mediated immune responses to a broad range tumor antigens.

Results

[0416] We discovered that in contrast to the non-vaccinated mice, the mice vaccinated with hESC combined with a TLR3 agonist have generated the highest reduction of breast tumor volume (p<0.001) compared to the use of a TLR9 agonist or to Quil-A® Saponin vaccine adjuvants (FIG. 1).

[0417] The reduction rate of tumor growth, with statistically significant differences in the average Tumor size are shown in table 1.

TABLE-US-00001 TABLE 1 Percentage of tumor reduction at day 26 compared to the control group receiving only PBS and statistical test (Student test). % of reduction P value VPA 24 p < 0.05  ODN2395 22 p < 0.05  Quil-A 20 p < 0.05  Poly(A:U) 54 p < 0.001

[0418] 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.

Example 2: Ability for a Compound to Increase MHC I

[0419] A compound can be assessed for its ability to increase MHC I on tumor cells (and thus suitable in the context of the present methods) on tumor cells.

[0420] Valproic Acid Increases MHC I on Breast Cancer Cells

[0421] 4T1 cells are a triple negative breast cancer cell line that was incubated with increased doses (0.2 and 2 mM) of valproic acid (VPA). After 4 days of treatment, the MHC I surface marker was quantified by flow cytometry analysis. VPA has the propriety of increasing MHC I levels in a dose-dependent manner highlighting that VPA can enhance an anti-tumor immune response by improving T cell tumor recognition. Furthermore, Relative Fluorescence Intensity (RFI), measured with flow cytometry analysis, revealed an increase of MHC I expression by 2.1-and 2.7-fold in 4T1 and mammosphere-derived 4T1 cells respectively when treated with 2 mM of VPA (FIG. 2). These results suggest that VPA treatments efficiently enhance MHC I expression in both baseline and “stem-cell-like” contexts.

[0422] In addition, when 4T1 cells were treated with 2 mM of VPA, we also observed a 2.1-fold increase in PDLL expression, contrasting with PDL2 expression which was only weakly upregulated after VPA treatment (FIG. 2)

[0423] HDAC Inhibitor Increases MHC1 Expression in Lung Tumor Cells

[0424] Non-small cell lung cancer (NSCLC) cell line (lewis lung carcinoma, LLC) expressing a particularly aggressive metastatic phenotype was used. Four HDAC inhibitors molecules were evaluated: Entinostat, Levetiracetatm, Vorinostat (Zolinga®) and valproic acid (VPA) (Depakine®), for the expression of Major Histocompatibility Complex (MHC) Class I and II using Flow cytometry analysis.

[0425] The half maximal inhibitory concentration (IC50) was first determined for each HDACi using an MTT cell proliferation assay. LLC1 was treated with an increasing dose of HDCAi and proliferation was estimated measuring the absorbance after the incorporation by the cell of MMT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) substrate. The IC50 (levels) for Vorinostat, Entinostat and Levetiracetam were between 2 and 4 μM and around 2 mM for valproic acid.

[0426] LLC1 treated with the 4 HDACi with the dose corresponding to IC50 were shown to modify the MHC class I expression for all HDACi tested. Vorinostat, Levetiracetam, Entinostat and VPA have the propriety of significantly increasing the expression of MHC1 on the cell membrane by 2-fold compared to untreated cells in contrast to MHC class 2 expression that was not modulated at with dose (FIG. 3). VPA was shown to overexpress MHC1 10 folds more than non-treated LLC1 cells (FIG. 3A).

[0427] It is also to be noted that some HDACi increased PDL-1 on the surface of the cells (data not shown).

Example 3: 4T1 Cells Treated with Valproic Acid (VPA) Induced an Upregulation of the Immune Response

[0428] To determine the effect of VPA on 4T1 cells, a transcriptome analysis on cells treated with 0.5 mM of VPA for 10 days was performed and compared with that of non-VPA treated cells. These analyses allowed for the identification of the set of genes implicated in TNF-α signaling and in the response of IFN-α and IFN-γ. In addition, the use of the SAM algorithm allowed for the discrimination of 44 immune-related genes between the VPA-treated samples and their control counterparts, which were validated by principal component analysis (p-value=3.3×10.sup.−4). Among these 44 immune-related genes, CD74, CCL2 and TNFRSF9 were found to be overexpressed with a fold change greater than 2. These three molecules are known to contribute to the clonal expansion, survival, and development of T cells and regulate CD28 co-stimulation to promote Th1 cell responses. Consequently, this HDACi improves the immune response against the cancer cells.