TLR3 ligands that activate both epithelial and myeloid cells

Abstract

The invention relates to a composition comprising a double-stranded RNA (dsRNA) having two complementary strands, comprising at least one block of poly A and the complementary block of poly U, each strand having a length of between 50 and 200 bases, preferably between 55 and 200 bases, and a pharmaceutically acceptable vehicle, carrier or excipient, for use in a method of treating a cancer expressing a TLR3 receptor.

Claims

1. A double-stranded RNA (dsRNA) having two complementary strands, wherein the dsRNA comprises at least one block or homopolymer of poly A and the complementary block or homopolymer of poly U, each block comprising at least 15 Å, or U, and each strand having a determined length of between 50 and 200 bases, the dsRNA being of formula (III):
[P]a[A]b[R]c
[Y]a[U]b[Z]c, wherein P and R are independently chosen among I and C, and Y and Z are the complementary bases, b is an integer between 20 and 100, and a and c independently are between 5 and 50.

2. A method of treatment of cancer, comprising administering to a patient in need thereof an efficient amount of the double-stranded RNA (dsRNA) according to claim 1.

3. The method according to claim 2, wherein the cancer is a cancer expressing a TLR3 receptor.

4. The method of claim 2, wherein the cancer is bladder cancer.

5. The method of claim 2, wherein the cancer is Non Muscle Invasive Bladder Cancer, Muscle Invasive Bladder Cancer, or Metastatic Muscle Invasive Bladder Cancer.

6. The method of claim 2, wherein the cancer is an epithelial cancer.

7. The method of claim 2, wherein the cancer is Lung cancer, Non-Small-Cell Lung cancer, or Breast cancer.

8. The method of claim 2, further comprising administering to said patient an efficient amount of an Immune Check Point Inhibitor.

9. The method of claim 8, wherein the Immune Check Point Inhibitor is an anti-PD-1 or an anti-PD-L1 monoclonal antibody.

10. The dsRNA according to claim 1, wherein the dsRNA is selected from: 5′ (I)10-(U)50-(I)10 3′ 5′ (I)10-(A)50-(I)10 3′ 5′ (I)5-(A)60-(I)5 3′ 5′ (I)15-(A)40-(I)15 3′ 5′ (I)20-(A)30-(I)20 3′ 5′ (I)25-(A)20-(I)25 3′ 5′ (I)5-(A)50-(I)15 3′ 5′ (I)13-(A)64-(I)13 3′; and 5′ (I)10-(A)70-(I)10 3′.

11. The dsRNA according to claim 1, wherein the A, U, I, or C are modified nucleotides comprising O-methylated nucleotides or phosphorothioate nucleotides.

12. A composition comprising the dsRNA of claim 1 and a pharmaceutically acceptable vehicle, carrier or excipient.

13. The dsRNA according to claim 1, wherein a+b+c is at least 50.

14. The dsRNA according to claim 1, wherein a+b+c is at least 60.

15. The dsRNA according to claim 1, wherein a+b+c is at least 100.

16. The dsRNA according to claim 1, wherein a+b+c is between 70 and 150.

17. The dsRNA according to claim 1, wherein b is an integer between 35 and 100.

18. The dsRNA according to claim 1, wherein b is an integer between 40 and 100.

19. The dsRNA according to claim 1, wherein b is an integer between 50 and 100.

20. The dsRNA according to claim 1, wherein b is an integer between 50 and 90.

Description

FIGURES

(1) FIG. 1: dsRNA profile on a 8% acrylamide gel (TBE 1×) of 50 bp dsRNAs and commercial Poly(A:U). 5 μg of dsRNA (dsRNA ID #s 345, 397, 398, 415, 418, 405, 411, 412, 413, as defined in Table 1) and commercial Poly(A:U) were loaded on 8% acrylamide gel and stained with BET. Data are representative of 9 out of 17 50 bp dsRNA tested.

(2) FIG. 2: Secretion of TNF alpha by mouse macrophages RAW264.7 cells in response to 50 bp dsRNA alone. RAW264.7 cells were treated for 24 hours with 10 μg/mL of each dsRNA, as defined in Table 1, and mTNF alpha secretion was measured with ELISA. Data are representative of two independent assays using two different batches of the 50 bp dsRNA ID #412.

(3) FIG. 3: Secretion of IL6 by human non-small cell lung cancer cells NCI-H292 after treatment with 9 50 bp dsRNA alone. NCI-H292 cells were treated for 24 hours with 10 μg/mL of each dsRNA, as defined in Table 1, and hIL6 secretion was measured with ELISA. Data are representative of three independent assays.

(4) FIG. 4: 50 bp dsRNA ID #412 does not trigger death in human non-small cell lung cancer cells NCI-H292. NCI-H292 cells were treated for 24 hours with 50 μg/mL of the dsRNA. AnnexinV positives cells were measured by chemiluminescence (kit Annexin V-Glo, Promega). Data are representative of three independent assays using two different batches of the 50 bp dsRNA ID #412.

(5) FIG. 5: dsRNA profile on a native 6% acrylamide gel (TBE 1×) of the dsRNA ID #s 412, 432, 452 and commercial Poly(A:U). 1 μg of dsRNA ID #s 412 (Poly(A:U) 50pb), 432 (Poly(A:U) 70pb), 452 (Poly(A:U) 90pb) (as defined in Tables 1 and 2) and commercial Poly(A:U) were loaded on 6% acrylamide gel and stained with BET. Data representative of two independent experiments.

(6) FIG. 6: Secretion of TNF alpha by mouse macrophages RAW264.7 cells in response to Poly(A:U) of increasing size alone. RAW264.7 cells were treated for 24 hours with 10 μg/mL of each dsRNA, as defined in Tables 1 and 2, and mTNF alpha secretion was measured with ELISA. Data are representative of three independent assays.

(7) FIG. 7: Poly(A:U) of increasing sizes alone activate the ISRE-reporter gene in mouse macrophages RAW264.7. RAW264.7 cells were treated for 24 hours with 50 μg/mL with the indicated dsRNA, as defined in Tables 1 and 2. ISRE-driven bioluminescence was measured (QUANTI-luc, Invivogen). Data are representative of three independent assays.

(8) FIG. 8: Poly(A:U) of increasing sizes alone trigger the TLR3-dependant secretion of IL6 by human non-small cell lung cancer cells NCI-H292. WT or TLR3 KO NCI-H292 cells were treated for 24 hours with 10 μg/mL of each dsRNA, as defined in Tables 1 and 2, and hIL6 secretion was measured with ELISA. Data are representative of three independent assays.

(9) FIG. 9: Poly(A:U) of increasing sizes alone trigger the TLR3-dependant death of human non-small cell lung cancer cells NCI-H292. NCI-H292 cells were treated for 24 hours with 50 μg/mL of the indicated dsRNAs, as defined in Tables 1 and 2. AnnexinV positives cells were measured by chemiluminescence (kit Annexin V-Glo, Promega). Data are representative of three independent assays.

(10) FIG. 10: dsRNA profile on a native 6% acrylamide gel (TBE 1×) of the dsRNA ID #422, 432, 442, 532, 533, 534, 535, and commercial Poly(A:U). 1 μg of each one of these dsRNA and commercial Poly(A:U) were loaded on 6% acrylamide gel and stained with BET. Data representative of two independent experiments.

(11) FIG. 11: Secretion of TNF alpha by mouse macrophages RAW264.7 cells in response to commercial Poly(A:U) and dsRNA ID #422, 432, 442, 532, 533, 534, 535. RAW264.7 cells were treated for 24 hours with 10 μg/mL of each dsRNA and mTNF alpha secretion was measured with ELISA. Data are representative of two independent assays.

(12) FIG. 12: TLR3-dependant secretion of IL6 by human non-small cell lung cancer cells NCI-H292, in response to commercial Poly(A:U) and dsRNA ID #422, 432, 442, 532, 533, 534, 535. WT or TLR3 KO NCI-H292 cells were treated for 24 hours with 10 μg/mL of each dsRNA and hIL6 secretion was measured with ELISA. Data are representative of at least two independent assays.

(13) FIG. 13: TLR3-dependant death of human non-small cell lung cancer cells NCI-H292, in response to commercial Poly(A:U) and dsRNA ID #422, 432, 442, 532, 533, 534, 535. NCI-H292 cells were treated for 24 hours with 50 μg/mL of the indicated dsRNAs. AnnexinV positive cells were measured by chemiluminescence (kit Annexin V-Glo, Promega). Data are representative of two independent assays.

(14) FIG. 14: Secretion of TNF alpha by mouse macrophages RAW264.7 cells in response to dsRNA ID #532 made from two different chemical manufacturing technologies. RAW264.7 cells were treated for 24 hours with a dose-response from 1 to 100 μg/mL of the indicated dsRNA ID #532 and mTNF alpha secretion was measured with ELISA. Data are the mean of at least three independent assays and are representative from at least six independent assays.

(15) FIG. 15: Secretion of TNF alpha by mouse macrophages RAW264.7 cells in response to dsRNA ID #532 made from two different manufacturers companies. RAW264.7 cells were treated for 24 hours with a dose-response from 1 to 100 μg/mL of the indicated dsRNA ID #532 and mTNF alpha secretion was measured with ELISA. Data are the mean of at least five independent assays.

(16) FIG. 16: dsRNA ID #532 made from two different chemical synthesis technologies or from two different manufacturers companies trigger the same level of death of human non-small cell lung cancer cells NCI-H292. NCI-H292 cells were treated for 24 hours with a dose-response from 1 to 100 μg/mL of the indicated dsRNAs. AnnexinV positives cells were measured by chemiluminescence (kit Annexin V-Glo, Promega). Data are representative of at least four independent assays.

(17) FIG. 17: dsRNA ID #532 made from two different chemical synthesis technologies trigger the same level of cell viability reduction of human non-small cell lung cancer cells NCI-H292. NCI-H292 cells were treated for 24 hours with a dose-response from 1 to 100 μg/mL of the indicated dsRNAs. Cell viability assays were measured by MTS (kit Cell Titer Aqueous solution, Promega). Data are representative of at least four independent assays.

(18) FIG. 18: dsRNA ID #532 made from two different manufacturers companies trigger the same level of cell viability reduction of human non-small cell lung cancer cells NCI-H292. NCI-H292 cells were treated for 24 hours with a dose-response from 1 to 100 μg/mL of the indicated dsRNAs. Cell viability assays were measured by MTS (kit Cell Titer Aqueous solution, Promega). Data are representative of at least five independent assays.

(19) FIG. 19: dsRNA profile on a native 6% acrylamide gel (TBE 1×) of the Poly(A:U) and dsRNA ID #532 after digestion with either RNAse I or RNAse III. 1 μg of dsRNA and commercial Poly(A:U) were loaded on 6% acrylamide gel and stained with BET. Data are representative of at least two independent assays.

(20) FIG. 20: dsRNA Tm calculation profile of the Poly(A:U) and the dsRNA ID #532. 1 μg of dsRNA were mix with a SyBr green Q-PCR mix and melting curve analysis were performed. Data are representative of at least three independent assays. In abciss, temperature in ° Celsius. In ordinate, −d(RFU)/dT. The Melt peak is indicated.

(21) FIG. 21: DsRNA ID #532 (TL #532) has no toxicity but induces antitumor inflammation and over expression of TLR3 within normal urothelial bladder primary cells (UBPC). (A) Toxicity of TLR3 ligands on UBPC: UBPC were treated for 24 hours with a dose-response from 8 to 2000 μg/mL of the indicated dsRNAs. Cell viability assays was measured by MTS (kit Cell Titer Aqueous solution, Promega). Data are the mean of two independent assays. (B) TL #532 induces antitumor inflammation on normal UBPC: Supernatants of UPBC either mock treated of treated with TL #532 were collected at 24 h post-treatment and submitted to Multiparametric ELISA from Mesoscale Diagnostics. Results are shown as single dot per independent experiment on 2 different healthy donors. (C) TL #532 induces TLR3 overexpression on normal UBPC: UPBC were treated for 24 hours using the indicated dsRNAs without transfection reagent at final concentrations of 160 ug/ml before Flow cytometry was performed using FACS-Calibur.

(22) FIG. 22: TL #532 and dsRNA ID #533 (TL #533) induces bladder tumor cells death through apoptosis. RT4 (left panel) and J82 (right panel) bladder cancer cell lines from ATCC (Cat #HTB-2 and #HTB-1), were treated for 24 hours with the indicated dsRNAs, at final concentrations ranging from 16 μg/mL to 500 ug/ml. (A) Cell viability was measured 24 h post-treatment using MTS Cell Titer Aqueous solution from Promega. (B) Apoptosis was measured with AnnexinV+ luminescence read-out using the Real-Time Glo AnnexinV kit from Promega. Data are the mean of three independent experiments.

(23) FIG. 23: TL #532 and TL #533 induces immunogenicity and inflammation. RT4 (left panel) and J82 (right panel) bladder cancer cell lines from ATCC (Cat #HTB-2 and #HTB-1) were treated for 24 hours with the indicated dsRNAs, without transfection reagent, at final concentrations ranging from 5 μg/mL to 500 ug/ml. (A) Early active ATP release was measured using CellTiter-Glo Luminescent Assay from Promega (Cat #G7570). Data are the mean of two independent experiments with Standard Deviation. (B) Supernatants of RT4 and J82 either were collected at 24 h post-treatment and submitted to Multiparametric ELISA from Mesoscale Diagnostics. Results are shown as single dot per independent experiment. Out of range values are indicated by red dots.

(24) FIG. 24: Experiments on freshly resected bladder tumors treated ex-vivo demonstrates that TL #532 induces cell death through apoptosis and proinflammatory antitumor response. (A) Representative tissues sections (HPS staining) of two tumors (bladder metastasis—left panel and high grade of NMIBC—right panel) from two patients, after 24 h of treatment with either saline (mock) or TL #532 (final concentration 500 μg/ml). Cell death is demonstrated by the partial disruption of cancer tissue and the appearance of late apoptotic bodies (circled in black). (B) The supernatants of the same tumor tissues section cultures contained several inflammatory cytokines and chemokines measured with the Multiparametric Elisa MSD (Meso Scale Discovery). Mean values are presented and statistics were performed using unpaired t-test (*=p≤0.05, **=p≤0.01, ***=p≤0.001).

(25) FIG. 25: TLR3-Ligands induces cell death through apoptosis, is specific of TLR3 activation, in murine syngeneic cell line MTB2 #63. (A) MBT2 #63 murine cell line model express murine TLR3. MBT2 cells were fixed and permeabilized before flow cytometry was performed. Results are shown for 10.000 cells per treatment as dot plot of autofluorescence versus FITC-fluorescence intensity and a merged histogram where isotype and mTL3 are represented in dark-grey and light-grey respectively. (B) TLR3 cell death specificity was monitored by pretreating MBT2 cells with small interferent RNA for TLR3 expression. The transfection of si-scramble or si-mTLR3 of MBT2 cells was performed before treatment using the indicated dsRNAs without transfection reagent, at 250 ug/ml. (C) Acidic endolysosomal conditions are detrimental for TLR3 activation. MBT2 cells were pretreated with Bafilomycin-A1 before MBT2 cells were treated with the indicated dsRNAs, without transfection reagent, at the final concentrations of 250 ug/ml. (B and C upper panel) Apoptosis was measured with AnnexinV+ luminescence read-out using the Real-Time Glo AnnexinV kit from Promega. (B and C Lower panel) Cell viability was measured 24 h post-treatment using MTS Cell Titer Aqueous solution from Promega. Data are the mean of two independent experiments with SD.

(26) FIG. 26: TL #532 induces equal or better anti tumoral effect than Poly(I:C) on syngeneic bladder cancer model MBT2 in vivo, with a better tolerance for the TL #532 treatment. Mouse model C3H/HeN from Charles River, is an immunocompetent species from which MBT-2 #63 bladder cancer model is derived. 6 weeks old female mice were used to perform subcutaneous grafts of 1,000,000 MBT-2 #63 cells on the right flank. The first day of treatment is designated as day zero (d0). Tumor inclusion window was between 75 and 125 mm3. In order to best mimic the route of clinical administration, one half is injected via the intra-tumoral route and the remainder via the subcutaneous peri-tumoral route. Three groups of ten mice were treated either with Poly(I:C) or TL #532, without transfection reagent, at final dose of 200 and 500 ug respectively. Treatments were administered 3 times per week, up to the end point or the total regression of the tumor or the end of treatment at three months. The weight of the animals and tumor volumes were monitored three times a week. Tumor growth of the mock-treated (A), or treated with either Poly(I:C) (B) or TL #532 (C) groups, are shown. We observed 1/10 mice and 3/10 complete responder mice in Poly(I:C) and TL #532 treated groups respectively. (D) Effect of TL #532 is confirmed by the tumor growth delay (TGD). TGD of the Poly(I:C) group is extended by 13 days (×2,6) and the TGD TL #532 by 18 days (×3,25), as compared to Mock-treated group. (E) A significant improvement is observed in the survival of the mice when they are treated with TLR3-ligands. (F) TL #532 is better tolerated compared with Poly(I:C) as shown by the weight curve. CR: mice in complete responders/total treated. Kaplan Meier results were analyzed using Mann-Whitney U test and Log rank statistical methods. Tumor growth monitoring was analyzed using an unpaired t-test with p value 0.05 considered as significant.

(27) FIG. 27: TL #532 induces vaccinal effect for all the three complete responders when rechallenged on the opposite flank 3 months after remission. All the complete responders shown in FIG. 26 (1 from Poly(I:C) group and 3 from TL #532 group) were rechallenged on the opposite flank three months after their remission, using the same protocol. The TGD of the rechallenged mice treated with TL #532 is greater than the rechallenged mouse treated with Poly (I:C), with two TL #532 rechallenged mice displaying a complete vaccine response effect.

(28) FIG. 28: TL #532 induces better anti tumoral effects than Immune-checkpoint inhibitor anti-PDL1 and their combo-therapy induces synergistic effects in vivo.

(29) MBT2/C3H/HeN syngeneic model was used and grafted as described in FIG. 27. Mice were included when they developed a homogeneous spheroid tumour measuring between 75 and 125 mm3. 6 groups were sub-divided: group I was Mock-NaCl without monoclonal antibodies (mAb) treatment, group II was (Mock-NaCl+Isotype-control mAb), group III was (Mock-NaCl+Anti-PDL1 mAb), group IV was TL #532 group, group V was (TL #532+Isotype-control mAb) and the group VI was (TL #532+Anti-PDL1 mAb). A treatment, for a maximum period of 3 months, was chosen for this “proof of concept” experimentation. Tumor growth for mock-treated mice versus TL #532 treated mice are shown either without mAb combination treatments (A), in combination with isotype control mAb (B) or with anti-PDL1 mAb (C). Among all of the groups, only the TL #532 treated groups were able to reach complete responses, with a more pronounced complete response rate in the group VI where the combination is tested. The growth curves (A, B and C) and the TGD curves (D), demonstrate a strong synergy when anti-PDL1 is combined with TL #532. (E) Kaplan Meier curves show a very significant increase in survival in the groups treated with TL #532 as compared to the control groups and a slight improvement in survival in the “TL #532+anti-PDL1 combo”. (F) Evolution of the weights of the mice in the groups IV, V and VI. The weights of the mice remain homogeneous and comparable between the three groups. Kaplan Meier results were analyzed using Mann-Whitney U test and Log rank statistical methods. Tumor growth monitoring was analyzed using an unpaired t-test with p value 0.05 considered as significant. CR: mice in complete responders versus total treated.

EXAMPLES

Abreviations

(30) TLR3: Toll-like Receptor 3 (CD283)

(31) WT: Wild Type

(32) KO: Knock Out

(33) TBE: Tris Borate EDTA

(34) APS: Ammonium Persulfate

(35) TEMED: Tetramethylethylenediamine

(36) BET: Bromure Ethidium

(37) DNA: Desoxyribonucleic Acid

(38) RNA: Ribonucleic Acid

(39) dsRNA: double stranded RNA

(40) ssRNA: double stranded RNA

(41) Poly(A:U): PolyAdenylic PolyUridylic acid

(42) PAU: high molecular weight commercial Poly(A:U)

(43) Poly(I:C): Polylnosinic PolyCytidylic acid

(44) PIC: Poly(I:C)

(45) bp: base pair

(46) mTNFa: mouse Tumor Necrosis Factor alpha

(47) hIL6: human Interleukin-6

(48) Å: Angstrom

(49) 5′ppp: 5′ tri-phosphate

(50) ISRE: Interferon Stimulated Response Element

(51) TABLE-US-00001 TABLE 1  dsR SEQ NA ID ID# Sequence 5′.fwdarw.3′ NO: 1 Sense UACIUCAUAUAIIIACCUAUIUUAUCUICIUIUCCAACCUUAIIAUUCAC 1 Antisense IUIAAUCCUAAIIUUIIACACICAIAUAACAUAIIUCCCUAUAUIACIUA 2 3 Sense UCIIUCIACICAAICIAUUACACUCCUIUCACAUCAUAAUCIUUUICUAU 3 Antisense AUAICAAACIAUUAUIAUIUIACAIIAIUIUAAUCICUUICIUCIACCIA 4 4 Sense AAUIAAIUAUUIICAIACAUUIAIUICCIAACAAIACCUIACCUAACIIU 5 Antisense ACCIUUAIIUCAIIUCUUIUUCIICACUCAAUIUCUICCAAUACUUCAUU 6 5 Sense CICUIUUUUCIAAAUUACCCUUUAUICICIIIUAUUIAACCACICUUAUI 7 Antisense CAUAAICIUIIUUCAAUACCCICICAUAAAIIIUAAUUUCIAAAACAICI 8 6 Sense IIAAIUIUIICUAIAUCUUAICUUACIUCACUAIAIIIUCCACIUUUAIU 9 Antisense ACUAAACIUIIACCCUCUAIUIACIUAAICUAAIAUCUAICCACACUUCC 10 9 Sense AAIAIAIUCUCAUAAUACIUCCIICCICAUICICAIIIUAUAUUUIIACA 11 Antisense UIUCCAAAUAUACCCUICICAUICIICCIIACIUAUUAUIAIACUCUCUU 12 10 Sense AUAIAAACUACAIIACUAACCUUCCUIICAACCIIIAIIUIIIAAUCCIU 13 Antisense ACIIAUUCCCACCUCCCIIUUICCAIIAAIIUUAIUCCUIUAIUUUCUAU 14 13 Sense IAIIAIIAIUCIUCAIACCAIAUAICUUUIAUIUCCUIAUCIIAAIIAUC 15 Antisense IAUCCUUCCIAUCAIIACAUCAAAICUAUCUIIUCUIACIACUCCUCCUC 16 14 Sense IIAUACIAIAUCCIUAIAUUIAUAAIIIACACIIAAUAUCCCCIIACICA 17 Antisense UICIUCCIIIIAUAUUCCIUIUCCCUUAUCAAUCUACIIAUCUCIUAUCC 18 16 Sense ACIUUCUAAIAIUUIIACIAAAUIUUUCICIACCUAIIAUIAIIUCICCC 19 Antisense IIICIACCUCAUCCUAIIUCICIAAACAUUUCIUCCAACUCUUAIAACIU 20 17 Sense UACIUAICAAIIUIACACAAICACAIUAIAUCCUICCCICIUUUCCUAUI 21 Antisense CAUAIIAAACICIIICAIIAUCUACUIUICUUIUIUCACCUUICUACIUA 22 18 Sense CUAIUUIUIIAUUIIAUUICCAUUCUCCIAIUIUAUUACCIUIACIICCI 23 Antisense CIICCIUCACIIUAAUACACUCIIAIAAUIICAAUCCAAUCCACAACUAI 24 19 Sense CACIIIUCCCAUIUAAUICAIUCIUAICCUACCUIACUIUACUUIIAAIU 25 Antisense ACUUCCAAIUACAIUCAIIUAIICUACIACUICAUUACAUIIIACCCIUI 26 20 Sense IACCIIACIAACCACAIAICICUIIAAIAAUCUCUAICUICUUUACAAAI 27 Antisense CUUUIUAAAICAICUAIAIAUUCUUCCAICICUCUIUIIUUCIUCCIIUC 28 21 Sense UUUCCCACUICCUUAAICCIICUUICCCUUUCUICCUIUAIAUCCAUUII 29 Antisense CCAAUIIAUCUACAIICAIAAAIIICAAICCIICUUAAIICAIUIIIAAA 30 22 Sense ICAACUUCIAIIACCUAAUIUIACCIACCUAIAUUCIICAUUIUIIICAI 31 Antisense CUICCCACAAUICCIAAUCUAIIUCIIUCACAUUAIIUCCUCIAAIUUIC 32 23 Sense IAUCUAUIICIUIAIACCCIUUAUICUCCAUUACIIUCAIUIIIUCACAI 33 Antisense CUIUIACCCACUIACCIUAAUIIAICAUAACIIIUCUCACICCAUAIAUC 34 24 Sense ACUICIACIUUCUAAACIUUIIUCCIUCAIAAICICCAUCCAIIAUCACI 35 Antisense CIUIAUCCUIIAUIICICUUCUIACIIACCAACIUUUAIAACIUCICAIU 36 25 Sense ACUIIUICCAACICICAIICAUAIUUCIAIIAIAAUUAUCCIIIIICAAU 37 Antisense AUUICCCCCIIAUAAUUCUCCUCIAACUAUICCUICICIUUIICACCAIU 38 26 Sense IACAACCAICAUCUCIIIUCUUICCCAACCCIUCUACACICUIUUAUAIC 39 Antisense ICUAUAACAICIUIUAIACIIIUUIIICAAIACCCIAIAUICUIIUUIUC 40 27 Sense CAUICUAICIUICIIIIUACACUUICUAACCAUUUIIIACACIIIACACU 41 Antisense AIUIUCCCIUIUCCCAAAUIIUUAICAAIUIUACCCCICACICUAICAUI 42 28 Sense AUAIACIIACAICUUIIUAUCCUIAICACAIUCICICIUCCIAAUCUAIC 43 Antisense ICUAIAUUCIIACICICIACUIUICUCAIIAUACCAAICUIUCCIUCUAU 44 29 Sense UACCCAUACUCCACCIUUIICAIIIIIAUCICAUIUCCCACIUIAAACAU 45 Antisense AUIUUUCACIUIIIACAUICIAUCCCCCUICCAACIIUIIAIUAUIIIUA 46 30 Sense AIUACAAIACUAICCUUICUAICAACCICIIICUIIIAICCUAAIIUAUC 47 Antisense IAUACCUUAIICUCCCAICCCICIIUUICUAICAAIICUAIUCUUIUACU 48 31 Sense UUCAICICICAIICUUIIIUCIAIAUAAAAUCUCCAIUICCCAAIACCAC 49 Antisense IUIIUCUUIIICACUIIAIAUUUUAUCUCIACCCAAICCUICICICUIAA 50 32 Sense ICAACIIAACIUCCUUAICUCCIICAIICAAUUAAIIIIAACICAAICAU 51 Antisense AUICUUICIUUCCCCUUAAUUICCUICCIIAICUAAIIACIUUCCIUUIC 52 33 Sense IUAUCAUUIUICACCUICCIIUIACCACUCAACIAUIUIIIIACICCIUU 53 Antisense AACIICIUCCCCACAUCIUUIAIUIIUCACCIICAIIUICACAAUIAUAC 54 34 Sense IUUACCCAUAUIIUCCACAIIACACUCIUCICUUCCIIICUUICCCUCUA 55 Antisense UAIAIIICAAICCCIIAAICIACIAIUIUCCUIUIIACCAUAUIIIUAAC 56 36 Sense ACICUIUCUCUIICACIUIIIUIICCUAIAIIAAUCACAUCCAAICCUII 57 Antisense CCAIICUUIIAUIUIAUUCCUCUAIICCACCCACIUICCAIAIACAICIU 58 37 Sense IUCIUIICAAUIUUCIUCUIIIUIUIIUCUACACAAUICIIICIIUICIU 59 Antisense ACICACCICCCICAUUIUIUAIACCACACCCAIACIAACAUUICCACIAC 60 38 Sense UIICAIACACACCIUIACCCCICCUCUCCAUUIAUICCACIICIAAUIUC 61 Antisense IACAUUCICCIUIICAUCAAUIIAIAIICIIIIUCACIIUIUIUCUICCA 62 40 Sense AICCCUUCUCCCCUICIICCACICCCIUAIAIAUCACICCUUUIACCCUC 63 Antisense IAIIIUCAAAIICIUIAUCUCUACIIICIUIICCICAIIIIAIAAIIICU 64 41 Sense ACICUICAIIACUUICAACCIIICAIACUCIICIICAIIUCCUAIUICAI 65 Antisense CUICACUAIIACCUICCICCIAIUCUICCCIIUUICAAIUCCUICAICIU 66 42 Sense IICIAAIICCCUAACIIIAIAUACICICCCACAACUCIICICIAAUACII 67 Antisense CCIUAUUCICICCIAIUUIUIIICICIUAUCUCCCIUUAIIICCUUCICC 68 44 Sense ICACCAIAUCUIUAAIIUCCICCACICAIACIAIICCIIICIIAIACCAC 69 Antisense IUIIUCUCCICCCIICCUCIUCUICIUIICIIACCUUACAIAUCUIIUIC 70 45 Sense UCCUIIAIIAIIIICIIAUAICCUCUUACCCIUICCCCACCIUUIICIIU 71 Antisense ACCICCAACIIUIIIICACIIIUAAIAIICUAUCCICCCCUCCUCCAIIA 72 47 Sense UICICCIIUCCCCAICCICICUCAUICUCIICACCICCAUAACCAIACCI 73 Antisense CIIUCUIIUUAUIICIIUICCIAICAUIAICICIICUIIIIACCIICICA 74 48 Sense UAICCICCCCUIIICCICIIUCCICUACCUUICAIIAAUCIAIICCIUCC 75 Antisense IIACIICCUCIAUUCCUICAAIIUAICIIACCICIICCCAIIIICIICUA 76 345 Sense ICUICUUCIICICCCCIIICICACCCCUICCICIIIIICIIIAUCICCCI 77 Antisense CIIICIAUCCCICCCCCICIICAIIIIUICICCCIIIICICCIAAICAIC 78 397 Sense IUCIICICCCIICCCCCCIICCCCICAICIIICUCCCCICCCIIICCICC 79 Antisense IICIICCCIIICIIIIAICCCICUICIIIICCIIIIIICCIIICICCIAC 80 398 Sense IIIIIICCCACICIICIICICCICCIICICCCCCIIIICICCCCICIUCI 81 Antisense CIACICIIIICICCCCIIIIICICCIICIICICCICCICIUIIICCCCCC 82 415 Sense CIIICCIICIIIIIICIIICIIIIICCCCUIICCCICCCIICICCCCICI 83 Antisense CICIIIICICCIIICIIICCAIIIICCCCCICCCICCCCCCICCIICCCI 84 418 Sense IIICCIIIICIICCCCIIICIICIICCICCIICCCCIICIICIIIICICC 85 Antisense IICICCCCICCICCIIIICCIICIICCICCICCCIIIICCICCCCIICCC 86 405 Sense IIAUAIIIICIICCCCIIICIICIICCICCIICCCCIICIICIIIICIUU 87 Antisense AACICCCCICCICCIIIICCIICIICCICCICCCIIIICCICCCCUAUCC 88 413 Sense AUUUAAAAAAUAAAUAUAAUAAAAUAUAAUUUAAUUAAUUAUUUAUUAAU 89 Antisense AUUAAUAAAUAAUUAAUUAAAUUAUAUUUUAUUAUAUUUAUUUUUUAAAU 90 411 Sense IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII 91 Antisense CCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCC 92 412 Sense AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 93 Antisense UUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUU 94

(52) TABLE-US-00002 TABLE 2 Sequences of the Poly(A:U) dsRNAs ranging from 60 to 90 pb (dsRNA ID 422, 432, 442, 452), and sequences of the dsRNA 70 pb family with increasing amount of Poly(Inosinic acid:Cytidylic acid) (dsRNAs ID #532, 533, 534, 535) with respect to the dsRNA ID #432 sequence and tested for their capacity to activate the inflammatory response in the mouse macrophages RAW264.7 cells, and apoptosis with inflammatory response in the human lung cancer NCI-H292 cell. dsRNA ID# 422 Sense 60 bases A (SEQ ID NO: 103) Antisense 60 bases U (SEQ ID NO: 104) 432 Sense 70 bases A (SEQ ID NO: 105) Antisense 70 bases U (SEQ ID NO: 106) 442 Sense 80 bases A (SEQ ID NO: 107) Antisense 80 bases U (SEQ ID NO: 108) 452 Sense 90 bases A (SEQ ID NO: 109) Antisense 90 bases U (SEQ ID NO: 110) 532 Sense 70 bases : 10 I - 50 A - 10 I (SEQ ID NO: 111) Antisense 70 bases: 10 C - 50 U - 10 C (SEQ ID NO: 112) 533 Sense 70 bases : 35 A-35 I (SEQ ID NO: 113) Antisense 70 bases : 35 U - 35 C (SEQ ID NO: 114) 534 Sense 70 bases: 10 A-50 I - 10 A (SEQ ID NO: 115) Antisense 70 bases: 10 U - 50 C - 10 U (SEQ ID NO: 116) 535 Sense 70 bases I (SEQ ID NO: 117) Antisense 70 bases C (SEQ ID NO: 118)

(53) The following other strands and their complementary strands may be synthesized the same way and then hybridized:

(54) 5′ (I)10-(U)50-(I)10 3′ (SEQ ID NO: 95)

(55) 5′ (A)60-(I)10 3′(SEQ ID NO: 119)

(56) 5′ (A)50-(I)20 3′(SEQ ID NO: 120)

(57) 5′ (A)20-(I)50 3′(SEQ ID NO: 121)

(58) 5′ (A)10-(I)60 3′(SEQ ID NO: 122)

(59) 5′ (I)5-(A)60-(I)5 3′(SEQ ID NO: 96)

(60) 5′ (I)15-(A)40-(I)15 3′(SEQ ID NO: 97)

(61) 5′ (I)20-(A)30-(I)20 3′(SEQ ID NO: 98)

(62) 5′ (I)25-(A)20-(I)25 3′(SEQ ID NO: 99)

(63) 5′ (I)30-(A)10-(I)30 3′(SEQ ID NO: 123)

(64) 5′ (I)5-(A)50-(I)15 3′(SEQ ID NO: 100)

(65) 5′ (I)13-(A)64-(I)13 3′(SEQ ID NO: 101)

(66) 5′ (I)10-(A)70-(I)10 3′(SEQ ID NO: 102)

Example 1 and FIG. 1: Analysis of 50 bp dsRNAs and Commercial Poly(A:U) on Native 8% Acrylamide Gel (TBE 1×)

(67) Lyophilized dsRNAs obtained from DHARMACON™ (Colorado, USA) were resuspended in sterile RNAse-free physiological water (INVIVOGEN, France) according to the manufacturer protocol. After addition of nucleic acid loading buffer (Invitrogen, Cat #AM8556), 5 μg of dsRNAs was loaded in a 8% acrylamide gel prepared as follow: 9.3 mL of sterile RNAse-free water (Sigma-Aldrich where, catalogue #W4502), 1.5 mL of TBE 10× (Sigma-Aldrich), 4 mL acrylamide-bis 30% (Merck Chemicals, Germany, Cat #1.00639.1000), 0.2 mL APS (Sigma-Aldrich), 20 μL TEMED (Sigma-Aldrich). RNA ladder was obtained from Invitrogen (cat #SM1833). Samples migration was set at 100V for 1 h before BET (Sigma-Aldrich) staining at 1 μg/mL. Gel was then visualized using the GelDoc XR+ analyzer (BIORAD, California, USA). Data are representative of 9 out of 17 50 bp dsRNA.

Example 2 and FIG. 2: Secretion of TNF-Alpha by Mouse Macrophages RAW264.7 Cells in Response to 50 bp dsRNA Alone

(68) Lyophilized dsRNAs were obtained from IDT or DHARMACON. 5.Math.10.sup.4 RAW264.7 cells were seeded in a final volume of 200 μL in 96-wells plates (CORNING, USA, Cat #353072). 24 hours later, cells were treated with dsRNA without transfection reagent at a final concentration of 10 μg/mL for 24 hours and supernatants were harvested to measure mouse TNF-alpha secretion by ELISA (BioLegend, USA Cat #430903). PAU=high molecular weight commercial Poly(A:U). Data are representative of two independent assays using two different batches of the 50 bp dsRNA ID #412.

Example 3 and FIG. 3: Secretion of IL6 by Human Non-Small Cell Lung Cancer Cells NCI-H292 after Treatment with PAU or with 50 bp dsRNAs Alone

(69) 3.Math.10.sup.3 NCI-H292 WT or TLR3 KO cells were seeded in a final volume of 300 μL/well of p48-well plates (CORNING, USA, Cat #353078). 24 hours later, culture medium was replaced with fresh one and cells treated with dsRNAs without transfection reagent at a final concentration of 10 μg/mL. 24 h later, supernatants were harvested to measure human IL-6 secretion by ELISA (BioLegend, USA Cat #430501). PAU=high molecular weight commercial Poly(A:U). Data are representative of three independent assays.

Example 4 and FIG. 4: 50 Bp dsRNA ID #412 does not Trigger Death in Human Non-Small Cell Lung Cancer Cells NCI-H292

(70) 1.Math.10.sup.4 NCI-H292 WT or TLR3 KO cells were seeded in a final volume of 100 μL/well of p96-well plates (GREINER, USA, Cat #655098). 24 hours later, cells were treated with dsRNAs without transfection reagent at a final concentration of 50 μg/mL for 24 h later before apoptosis was measured with AnnexinV.sup.+ luminescence read-out using the Real-Time Glo AnnexinV kit from Promega (France, Cat #JA1000). PAU=high molecular weight commercial Poly(A:U). Data are representative of three independent assays using two different batches of the 50 bp dsRNA ID #412.

Example 5 and FIG. 5: Analysis of dsRNA ID #412, 432, 452 and Poly(A:U) on a Native 6% Acrylamide Gel (TBE 1×)

(71) Lyophilized dsRNAs obtained from DHARMACON were resuspended in sterile RNAse-free physiological water (INVIVOGEN) according to the manufacturer protocol. After addition of RNA loading buffer (Invitrogen), 1 μg of dsRNA was loaded in a 6% acrylamide gel prepared as follow: 10.3 mL of sterile RNAse-free water (Sigma-Aldrich), 1.5 mL of TBE 10× (Sigma-Aldrich), 3 mL acrylamide-bis 30% (Merck Chemicals), 0.2 mL APS (Sigma-Aldrich), 20 μL TEMED (Sigma-Aldrich). RNA ladder was purchased from Invitrogen. Samples migration was set at 100V for 1 h before BET (Sigma-Aldrich) staining at 1 μg/mL. Gel was then visualized using the Gel Doc analyzer (BIORAD).

Example 6 and FIG. 6: Secretion of TNFalpha by Mouse Macrophages RAW264.7 Cells in Response to Poly(A:U) of Increasing Size Alone

(72) 5.Math.10.sup.4 RAW264.7 cells were seeded in a final volume of 200 μL in 96-well plate (CORNING, USA, Cat #353072). 24 hours later, cells were treated with dsRNA without transfection reagent at a final concentration of 10 μg/mL for 24 hours and supernatants were harvested to measure mouse TNF-alpha secretion by ELISA (BioLegend). PAU=high molecular weight commercial Poly(A:U). Data are representative of three independent assays.

Example 7 and FIG. 7: Poly(A:U) of Increasing Sizes Alone Activate the ISRE-Reporter Gene in Mouse Macrophages RAW264.7

(73) 5.Math.10.sup.4 RAW264.7 cells (Invivogen, France, Cat #rawl-isg) were seeded in a final volume of 100 μL per well in 96-wells plates (CORNING, USA, 353072). 24 hours later, cells were treated with dsRNA without transfection reagent at a final concentration of 50 μg/mL for 24 hours before ISRE-driven bioluminescence assay using QUANTI-Luc kit (Invivogen, Cat #rep-old), according to manufacturer's protocol. PAU=high molecular weight commercial Poly(A:U). Data are representative of three independent assays.

Example 8 and FIG. 8: Poly(A:U) of Increasing Sizes Alone Trigger the TLR3-Dependant Secretion of IL6 by Human Non-Small Cell Lung Cancer Cells NCI-H292

(74) 3.Math.10.sup.4 NCI-H292 WT or TLR3 KO cells were seeded in a final volume of 300 μL per well in 48-wells plates (CORNING, USA, Cat #353078). After 24 hours, culture medium was replaced with fresh one and cells treated with dsRNAs without transfection reagent at a final concentration of 10 μg/mL. 24 h later, supernatants were harvested to measure human IL-6 secretion by ELISA (BioLegend). PAU=high molecular weight commercial Poly(A:U). Data are representative of three independent assays.

Example 9 and FIG. 9: Poly(A:U) of Increasing Sizes Alone Trigger the TLR3-Dependant Death of Human Non-Small Cell Lung Cancer Cells NCI-H292

(75) 1.Math.10.sup.4 NCI-H292 WT or TLR3 KO cells were seeded in a final volume of 100 μL/well of p96-well plate (CORNING, USA, Cat #655098). 24 hours later cells were treated with dsRNAs without transfection reagent at a final concentration of 50 μg/mL for 24 h before apoptosis was measured with AnnexinV+ luminescence read-out using the Real-Time Glo AnnexinV kit from Promega (France, Cat #JA1000). PAU=high molecular weight commercial Poly(A:U). Data are representative of three independent assays.

Example 10 and FIG. 10: Analysis of dsRNA ID #422, 432, 442, 532, 533, 534, 535, and Poly(A:U) on a Native 6% Acrylamide Gel (TBE 1×)

(76) Lyophilized dsRNAs obtained from DHARMACON were resuspended in sterile RNAse-free physiological water (INVIVOGEN) according to the manufacturer protocol. After addition of RNA loading buffer (Invitrogen), 1 μg of dsRNA was loaded in a 6% acrylamide gel prepared as follow: 10.3 mL of sterile RNAse-free water (Sigma-Aldrich), 1.5 mL of TBE 10× (Sigma-Aldrich), 3 mL acrylamide-bis 30% (Merck Chemicals), 0.2 mL APS (Sigma-Aldrich), 20 μL TEMED (Sigma-Aldrich). RNA ladder was purchased from Invitrogen. Samples migration was set at 100V for 1 h before BET (Sigma-Aldrich) staining at 1 μg/mL. Gel was then visualized using the Gel Doc analyzer (BIORAD). Gel is representative of two independent assays.

Example 11 and FIG. 11: Secretion of TNFalpha by Mouse Macrophages RAW264.7 Cells in Response to Poly(A:U) and dsRNA ID #422, 432, 442, 532, 533, 534, 535

(77) 5.Math.10.sup.4 RAW264.7 cells were seeded in a final volume of 200 μL per well using p96-well plate (CORNING, USA, Cat #353072). 24 hours later, cells were treated with dsRNA without transfection reagent at a final concentration of 10 μg/mL for 24 hours and supernatants were harvested to measure mouse TNF-alpha secretion by ELISA (BioLegend). PAU=high molecular weight commercial Poly(A:U). Data are representative of two independent assays.

Example 12 and FIG. 12: Poly(A:U) of Increasing Sizes and dsRNA ID #422, 432, 442, 532, 533, 534, 535 Alone Trigger the TLR3-Dependant Secretion of IL6 by Human Non-Small Cell Lung Cancer Cells NCI-H292

(78) 3.Math.10.sup.4 NCI-H292 WT or TLR3 KO cells were seeded in a final volume of 300 μL per well using p48-well plate (CORNING, USA, Cat #353078). 24 hours later, cells were treated with dsRNAs without transfection reagent at a final concentration of 10 μg/mL for 24 h and supernatants were harvested to measure human IL-6 secretion by ELISA (BioLegend). PAU=high molecular weight commercial Poly(A:U). Data are representative of at least two independent assays.

Example 13 and FIG. 13: Poly(A:U) of Increasing Sizes and dsRNA ID #422, 432, 442, 532, 533, 534, 535 Alone Trigger the TLR3-Dependant Apoptosis of Human Non-Small Cell Lung Cancer Cells NCI-H292

(79) 1.Math.10.sup.4 NCI-H292 WT or TLR3 KO cells were seeded in a final volume of 100 μL per well using p96-well plate (GREINER, USA, Cat #655098). 24 hours later, cells were treated with dsRNAs without transfection reagent at a final concentration of 50 μg/mL for 24 h before apoptosis was measured with AnnexinV.sup.+ luminescence read-out using the Real-Time Glo AnnexinV kit from Promega (France, Cat #JA1000). PAU=high molecular weight commercial Poly(A:U). Data are representative of at least two independent assays.

Example 14 and FIG. 14: Secretion of TNF-Alpha by Mouse Macrophages RAW264.7 Cells in Response to dsRNA ID #532 Made with Two Different Chemical Manufacturing Technologies

(80) Lyophilized dsRNAs were obtained from DHARMACON using two different chemical manufacturing technologies: TBDMS or 2′ACE. 5.Math.10.sup.4 RAW264.7 cells were seeded in a final volume of 200 μL per well of p96-wells plates (CORNING, USA, Cat #353072). 24 hours later, cells were treated with dsRNA without transfection reagent at a final concentration ranging from 1 to 100 μg/mL for 24 hours and supernatants were harvested to measure mTNF-alpha secretion by ELISA (BioLegend, USA Cat #430903). Data are the mean from at least three different assays and are representative from at least six independent assays. Statistical analysis is performed using two-tailed unpaired t-student test; NS=Not Significant with p>0.05.

Example 15 and FIG. 15: Secretion of TNF-Alpha by Mouse Macrophages RAW264.7 Cells in Response to dsRNA ID #532 Made from Two Different Manufacturers Companies

(81) Lyophilized dsRNAs were obtained either from DHARMACON or from BIOSPRING. 5.Math.10.sup.4 RAW264.7 cells were seeded in a final volume of 200 μL per well of p96-wells plates (CORNING, USA, Cat #353072). 24 hours later, cells were treated with dsRNA without transfection reagent at a final concentration ranging from 1 to 100 μg/mL for 24 hours and supernatants were harvested to measure mTNF-alpha secretion by ELISA (BioLegend, USA Cat #430903). Data are the mean from at least five different experiments. Statistical analysis is performed using two-tailed unpaired t-student test; NS=Not Significant with p>0.05.

Example 16 and FIG. 16: dsRNA ID #532 from Different Chemical Manufacturing Synthesis and from Different Manufacturers Companies Trigger the Same Apoptosis Level of Human Non-Small Cell Lung Cancer Cells NCI-H292

(82) Lyophilized dsRNAs were obtained from DHARMACON using two different chemical manufacturing technologies: TBDMS or 2′ACE; in addition, lyophilized dsRNAs were obtained from BIOSPRING. 1.Math.10.sup.4 NCI-H292 WT cells were seeded in a final volume of 100 μL per well using p96-well plate (GREINER, USA, Cat #655098). 24 hours later, cells were treated with dsRNAs without transfection reagent at a final concentration ranging from 1 to 100 μg/mL for 24 h before apoptosis was measured with AnnexinV+ luminescence read-out using the Real-Time Glo AnnexinV kit from Promega (France, Cat #JA1000). Data are representative of at least four independent assays.

Example 17 and FIG. 17: dsRNA ID #532 from Different Chemical Manufacturing Synthesis Trigger the Same Cell Viability Reduction Level of Human Non-Small Cell Lung Cancer Cells NCI-H292

(83) Lyophilized dsRNAs were obtained from DHARMACON using two different chemical manufacturing technologies: TBDMS or 2′ACE. 1.Math.10.sup.4 NCI-H292 WT cells were seeded in a final volume of 100 μL per well using p96-well plate (GREINER, USA, Cat #655098). 24 hours later, cells were treated with dsRNAs without transfection reagent at a final concentration ranging from 1 to 100 μg/mL for 24 h before cell viability was measured with Cell Titer Aqueous solution from Promega (France, Cat #G3580). Data are representative of at least four independent assays. Statistical analysis is performed using two-tailed unpaired t-student test; NS=Not Significant with p>0.05.

Example 18 and FIG. 18: dsRNA ID #532 from Different Manufacturers Companies Trigger the Same Cell Viability Reduction Level of Human Non-Small Cell Lung Cancer Cells NCI-H292

(84) Lyophilized dsRNAs were obtained either from DHARMACON or from BIOSPRING. 1.Math.10.sup.4 NCI-H292 WT cells were seeded in a final volume of 100 μL per well using p96-well plate (GREINER, USA, Cat #655098). 24 hours later, cells were treated with dsRNAs without transfection reagent at a final concentration ranging from 1 to 100 μg/mL for 24 h before cell viability was measured with Cell Titer Aqueous solution from Promega (France, Cat #G3580). Data are representative of at least five independent assays.

Example 19 and FIG. 19: Analysis of Poly(A:U) and the dsRNA ID #532 Sensitivity to the RNAse I and to the RNAse III and Analysis on a Native 6% Acrylamide Gel (TBE 1×)

(85) RNAse I is a RNAse that displays high preference for single stranded RNA over double stranded RNA independently of the sequence, while RNAse III is a RNAse that cleaves double stranded RNA into short 12-30 bases dsRNA. Lyophilized dsRNAs obtained from DHARMACON were resuspended in sterile RNAse-free physiological water (INVIVOGEN) according to the manufacturer protocol. 1 μg of dsRNA was first incubated with either 1 unit of RNAse I (ThermoFischer AMBION, Cat #AM2294) or 1 unit of RNAse III (ThermoFischer AMBION, Cat #AM2290) for 10 or 30 min at 37° C. before addition of RNA loading buffer (Invitrogen) and loading in a 6% acrylamide gel prepared as follow: 10.3 mL of sterile RNAse-free water (Sigma-Aldrich), 1.Math.5 mL of TBE 10× (Sigma-Aldrich), 3 mL acrylamide-bis 30% (Merck Chemicals), 0.2 mL APS (Sigma-Aldrich), 20 uL TEMED (Sigma-Aldrich). RNA ladder was purchased from Invitrogen. Samples migration was set at 100V for 45 minutes before BET (Sigma-Aldrich) staining at 1 μg/mL. Gel was then visualized using the Gel Doc analyzer (BIORAD). PAU=high molecular weight commercial Poly(A:U). Data are representative from at least two independant assays.

Example 20 and FIG. 20: Analysis of RNA Melting Curve Profile Using Q-PCR Machine Analysis

(86) Lyophilized dsRNAs obtained from DHARMACON were resuspended in sterile RNAse-free physiological water (INVIVOGEN) according to the manufacturer protocol. 1 μg of dsRNA was mixed with SyBr green PCR mix (Biorad, Cat #1725270) before melting curve analysis using Q-PCR machine from Biorad (Biorad, CFX Connect). Bold line corresponds to the named dsRNA above the respective graph. PAU=high molecular weight commercial Poly(A:U). Data are representative from at least three independant assays.

(87) TABLE-US-00003 TABLE 3 Effects of the lengths and base composition of dsRNAs, as defined in Tables 1 and 2, on RAW264.7 and NCI-H292 cells activation. Molecules Inflammation in murine myeloid Human epithelial non-small macrophage cell lung cancer NCI-H292 RAW264.7 cells Inflammation Apoptosis Commercial Poly(A:U) ++ ++ ++ PAU dsRNA ID #411 − − − dsRNA ID #535 − ++ ++ dsRNA ID #412 ++ − − dsRNA ID #432 ++ + + dsRNA ID #532 ++ ++ ++ dsRNA ID #533 ++ ++ ++ dsRNA ID #534 − ++ ++ Legend : − = Negative Effect, + to ++ = Positive Activation

(88) TABLE-US-00004 TABLE 4 Effects of the lengths and base composition of dsRNAs, as defined in Tables 1 and 2, as defined in on RAW264.7 and NCI-H292 cells activation. Inflammation in murine myeloid Human epithelial macrophage non-small cell Bases RAW264.7 lung cancer NCI-H292 composition Molecules cells Inflammation Apoptosis (A:U) > Commercial +++ +++ +++ 50% Poly(A:U) PAU dsRNA ID #413 − − − dsRNA ID #412 +++ − − dsRNA ID #422 +++ + + dsRNA ID #432 +++ ++ ++ dsRNA ID #442 +++ ++ +++ dsRNA ID #452 +++ ++ ++ dsRNA ID #532 +++ +++ +++ (A:U) = dsRNA ID #533 +++ +++ +++ (I:C) = 50% (I:C) > 50% dsRNA ID #411 − − − dsRNA ID #535 − +++ +++ dsRNA ID #534 − +++ +++ Legend : − = Negative Effect, + to ++ = Positive Activation

(89) TABLE-US-00005 TABLE 5 Effects of the lengths and base composition of dsRNAs, as defined in Tables 1 and 2, on RAW264.7 and NCI-H292 cells activation. Inflammation in murine myeloid Human epithelial non- macrophage small cell lung Bases RAW264.7 cancer NCI-H292 composition Molecules cells Inflammation Apoptosis (A:U) > Commercial +++ +++ +++ 50% Poly(A:U) PAU dsRNA ID #413 − − − dsRNA ID #412 +++ − − dsRNA ID #422 +++ +/− +/− dsRNA ID #432 +++ + + dsRNA ID #442 +++ + ++ dsRNA ID #452 +++ ++ ++ dsRNA ID #532 +++ +++ +++ (A:U) = dsRNA ID #533 +++ +++ +++ (I:C) = 50% (I:C) > 50% dsRNA ID #411 − − − dsRNA ID #535 − +++ +++ dsRNA ID #534 − +++ +++ Legend : − = Negative Effect, +/− = Border line, + to +++ = Increase Positive Activation.

(90) Application of the Invention to Bladder Cancer and Combined Therapy with an Anti-PD-L1

(91) Bladder cancer is ranked fifth among all cancers in humans in Europe and accounts for 3% of deaths caused by cancer. Despite the efficacy of the different immunotherapies, Bacillus Calmette-Guerin (BCG) treatment and immune checkpoint inhibitors, a significant proportion of patients are non-responders either related to sub-optimal reprogramming of the immunosuppressive tumour microenvironment by BCG, or to the absence of cytotoxic T lymphocytes at the tumour site during treatment with anti-PD1/PD-L1.

(92) It is shown that the ligands of the invention enable the stimulation of Toll like receptor 3 (TLR3) that induce cancer cell-specific extrinsic apoptosis, the invasion of tumour antigen specific cytotoxic T lymphocytes and immunostimulatory reprogramming of the tumour microenvironment, thus making it possible to overcome the limitations of available immunotherapies. The preclinical efficacy of the dsRNA ID #532 ligand (TL #532) has been studied in the human bladder tumour cell lines J82 and RT4 as also the toxicity thereof on human urothelial bladder primary cells (UBPC). Its efficacy has also been assessed in freshly resected human bladder cancer (ex vivo) as well as ectopic syngeneic murine (mouse) models of bladder cancer cells (MBT-2). The in vitro results show an apoptosis induction in the bladder tumour cell lines RT4 and J82 by TL #532 with the absence of toxicity on UBPC. The ex vivo results show TLR3 (IHC) expression in all the samples (7/7) and apoptosis of bladder cancer cells triggered by TL #532 (microscopic analysis on HES staining), on 4/4 of the samples. Reprogramming the tumour microenvironment is also observed, with stimulation of the secretion of 10 pro-inflammatory cytokines (IL6, CCL5, CXCL9, CXCL10, CD253, IL 1 beta, IFN-α2a, IFNγ, IFN lambda, CX3CL1). In the ectopic (subcutaneous) murine model of bladder cancer cells, the intra tumour injection of TL #532 leads to a very significant slowing of tumour growth in the majority of mice (tumour growth delay multiplied by approximately 2.5) and a total regression of tumours in one third of cases (10/31). In addition, all the mice that were cured with TLR3 ligand rejected MBT-2 cells reimplanted 3 months after their recovery, demonstrating that an anti-tumour autovaccination had occurred. The preclinical project provides encouraging data for TLR3 ligand development in the treatment of urothelial carcinoma.

(93) Non-Muscle-Invasive Bladder Cancer (NMIBC) is the most commonly occuring form of urothelial tumor and accounts for 70 to 80% of cases. 60 to 70% of patients experience recurrence or recidivism in the first year and 10 to 20% progress to either the muscle-invasive stage, or to a metastatic stage. Given a high risk of recurrence or recidivism, resection of the tumour is insufficient for intermediate and high risk NMIBCs. As a consequence, several complementary treatments associated with trans urethral resection of bladder tumour (TURBT) are recommended: either intra-vesical chemotherapy such as epirubicin, mitomycin or even gemcitabine, or immunotherapy with BCG therapy. Currently, BCG treatment with at least one year of maintenance treatment is the most effective complementary treatment and the only one that has shown a reduction in the risk of progression on to muscle-invasive stage. However, 30% of patients, mainly patients at very high risk, do not respond to this treatment. Currently, the Association Francaise d'Urologie (AFU)/French Association of Urology, European Association of Urology (UAE) and American Urology Association (AUA) recommend the undertaking of maintenance treatment with BCG for a period of 1 year for intermediate risk NMIBCs and for a period of 3 years for high risk NMIBCs. Intra-vesical instillation of Bacillus Calmette-Guérin (BCG) is a prototypical immunotherapy and has been used to treat NMIBC, making it the first treatment therapy to reprogram the immune microenvironment of the bladder tumour. It induces an innate immune response for several weeks that leads to a durable anti-tumour adaptive immune response. This mechanism is mainly mediated by Toll Like Receptors (TLRs), such as TLR2, 4 and 9. However, it appears that the BCG's ability to reprogram the cancer-associated cytokine environment remains suboptimal, limiting its therapeutic activity and therefore its effectiveness. Moreover, there is significant toxicity with a rate of severe adverse events at around 10%. As a result, only 16% of patients complete the maintenance treatment.

(94) Muscle-Invasive Bladder Cancer (M IBC): 20 to 30% of patients are initially diagnosed with a muscle-invasive bladder carcinoma (MIBC), usually treated with neoadjuvant cisplatin-based chemotherapy followed by cystectomy. Despite these intensely demanding approaches, the 5-year survival rate is about 50%. In addition, 5% of these patients are diagnosed to be at a metastatic stage with poor prognosis in spite of chemotherapy. For several decades, no major progress had been achieved in respect of bladder cancer treatment. Since 2016 however, a very promising new option, consisting of an antibody from the “immune checkpoint inhibitors” (ICI) family, has been approved by the FDA. These are monoclonal antibodies targeting the ICI, anti-PD1 and anti-PD-L1. Despite remarkable results with anti PD1/PD-L1 antibodies, the main problem lies in the low proportion of response in patients with locally advanced or metastatic disease with overall response rate of only 20 to 30% and complete response rate of approximately 10%. As in other types of cancer, failure has often been attributed to the absence of cytotoxic CD8+T cells at the tumour site, which would be required in order to kill the cancer cells and/or to the immunosuppressive environment of the tumor.

Example 21 and FIG. 21: TL #532 has No Toxicity but Induces Antitumor Inflammation and Over Expression of TLR3 within Normal Urothelial Bladder Primary Cells (UBPC)

(95) (A) Toxicity of TLR3-Ligands on normal urothelial bladder primary cells (UBPC): UPBC from different healthy donors (Cell-Applications-INC, Cat #938-05a), were seeded at 7.Math.10.sup.3 cells in a final volume of 100 μL per well of synthetic BEC-GM medium (Cell-applications-INC, Cat #217-500), using p96-well plate (GREINER, USA, Cat #655098). 24 hours later, cells were treated using either Poly(A:U) from Invivogen (Cat #tlrl-pau); Poly(I:C) from Invivogen (Cat #tlrl-pic); or TL #532 from Dharmacon, without transfection reagent, at final concentrations ranging from 8 μg/mL to 2000 μg/ml. Cell viability was measured 24 h post-treatment using MTS Cell Titer Aqueous solution from Promega (France, Cat #G3580). Data are the mean of two independent experiments using UBPC from different healthy donors, each in triplicates. Statistical analysis is performed using confident intervals for p<0.05.

(96) (B) TL #532 induces antitumor inflammation on normal UBPC: Supernatants of UPBC either mock treated of treated using 62 μg/ml or 500 μg/ml of TL #532 from previous experiment were collected at 24 h post-treatment and submitted to Multiparametric ELISA from Mesoscale Diagnostics, against eleven cytokines and chemokines. A threshold of 1 μg/ml was arbitrary determined. Results are shown as single dot per independent experiment on 2 different healthy donors.

(97) (C) TL #532 induces TLR3 overexpression on normal UBPC: UPBC from healthy donors-2, were seeded at 3.Math.10.sup.4 cells in a final volume of 300 μL per well of synthetic BEC-GM medium, using p24-well plate (Falcon, Cat #353047). 24 hours later, cells were treated using either Poly(A:U) from Invivogen; Poly(I:C) from Invivogen; or TL #532 without transfection reagent, at final concentrations of 160 μg/ml. Cells were harvested 24 h post-treatment, fixed and permeabilized using Cytofix/Cytoperm as preconized by manufacturer (BD, Cat #554714). Cells were incubated with either TLR3.Math.1 primary antibody mouse anti-human from Dendritics or Isotype IgG1 from R&D, at the final concentration of 5 μg/ml for 30 minutes at 4° C. Secondary antibody Goat anti-mouse-Alexa-488 from Thermofisher (Cat #A21235) was used as preconized by manufacturer. Flow cytometry was performed using FACS-Calibur (BD). Results are shown for 10.000 cells per treatment as dot plot of autofluorescence versus FITC-fluorescence intensity.

Example 22 and FIG. 22: TL #532 and TL #533 Induces Bladder Tumor Cells Death Through Apoptosis

(98) RT4 (left panel) and J82 (right panel) bladder cancer cell lines from ATCC (Cat #HTB-2 and #HTB-1), were seeded at 1.Math.10.sup.4 cells in a final volume of 100 μL per well of MacCoy's 5A and MEMalpha medium respectively (Gibco, Cat #26600-023, #32581-029), supplemented with 10% FBS from Dominique Dutscher (Cat #51810-500), using p96-well plate (GREINER, USA, Cat #655098). 24 hours later, cells were treated using either Poly(A:U) from Invivogen (Cat #tlrl-pau); Poly(I:C) from Invivogen (Cat #tlrl-pic); TL #532 or TL #533 from Dharmacon, without transfection reagent, at final concentrations ranging from 16 μg/mL to 500 ug/ml.

(99) (A) Cell viability was measured 24 h post-treatment using MTS Cell Titer Aqueous solution from Promega (France, Cat #G3580).

(100) (B) Apoptosis was measured with AnnexinV+ luminescence read-out using the Real-Time Glo AnnexinV kit from Promega (France, Cat #JA1000). Data are the mean of three independent experiments. Statistical analysis is performed using confident intervals for p<0.05.

Example 23 and FIG. 23: TL #532 and TL #533 Induces Immunogenicity and Inflammation

(101) RT4 (left panel) and J82 (right panel) bladder cancer cell lines from ATCC (Cat #HTB-2 and #HTB-1), were seeded at 1.Math.10.sup.4 cells in a final volume of 100 μL per well of MacCoy's 5A and MEMalpha medium respectively (Gibco, Cat #26600-023, #32581-029), supplemented with 10% FBS from Dominique Dutscher (Cat #51810-500), using p96-well plate (GREINER, USA, Cat #655098). 24 hours later, cells were treated using either Poly(A:U) from Invivogen (Cat #tlrl-pau); Poly(I:C) from Invivogen (Cat #tlrl-pic); TL #532 or TL #533 from Dharmacon, without transfection reagent, at final concentrations ranging from 5 μg/mL to 500 μg/ml. (A) Early active ATP release is a biomarker of immunogenicity inducing T-cells activation and promoting cross presentation with dendritic cells. ATP release was measured 3 h45 post-treatment using CellTiter-Glo Luminescent Assay from Promega (Cat #G7570). Data are the mean of two independent experiments with Standard Deviation. (B) Supernatants of RT4 and J82 either mock treated or treated using 50 μg/ml or 500 μg/ml of TL #532 from previous experiment were collected at 24 h post-treatment and submitted to Multiparametric ELISA from Mesoscale Diagnostics, against eleven cytokines and chemokines. A threshold of 1 μg/ml was arbitrary determined. Results are shown as single dot per independent experiment. Out of range values are indicated by “#” dots.

Example 24 and FIG. 24: Experiments on Freshly Resected Bladder Tumors Treated Ex-Vivo Demonstrates that TL #532 Induces Cell Death Through Apoptosis and Proinflammatory Antitumor Response

(102) Freshly resected bladder tumors were sliced in sections of 250-300 μm thickness using a vibratome (Thermo Scientific Microm HM650V). Tumors sections were then prepared and cultured in 1 ml of synthetic BEC-GM medium (Cell-applications-INC, Cat #217-500), and treated treated using either Poly(A:U) from Invivogen (Cat #tlrl-pau); Poly(I:C) from Invivogen (Cat #tlrl-pic); or TL #532 from Dharmacon, without transfection reagent, at a final concentration of 500 ug/ml for 24 hours at 37° C. and 5% CO.sub.2. The tissue sections were harvested at 0 and 24 h and fixed in 4% formalin for a period of 24 hours and embedded in paraffin. Paraffin sections of 4 μm were generated for morphological (HPS) and immunohistochemical (IHC) analyses. Supernatants were harvested at 24 hours and stored at −80° C. for subsequent cytokine assays.

(103) (A) Representative HPS sections of two different tumors (bladder metastasis—left panel and high grade of NMIBC—right panel), from different patients, either mock-treated (upper panels) or treated using 500 μg/ml of TL #532 (lower panels). TL #532 induces cell death as demonstrated by the partial disruption of cancer tissue and the appearance of late apoptotic cores (circled in black).

(104) (B) The reprogramming of the tumour microenvironment was studied by measuring cytokines via Multiparametric Elisa MSD (Meso Scale Discovery) Kits that assay 12 soluble biomarkers (Ref MSD U-Plex and R-Plex), some pro-inflammatory such as IL6, CCL5 (RANTES), CXCL9 (MIG), CXCL10 (IP10), CD253 (TRAIL), IL 1 beta, IFNα2a, IFNγ, IFN lambda, CX3CL1 and other anti-inflammatory agents such as CCL22 (MDC) and sFas. Grey circles and black circles represents mock treated bladder cancer tissues and tissues treated with 500 ug/ml of TL #532 respectively. Mean value is represented and statistics were performed using unpaired t-test and are represented by asterisks: * means p≤0.05; ** means p≤0.001; *** means p≤0.000.1; “ns” means Not Significant.

Example 25 and FIG. 25: TLR3-Ligands Induces Cell Death Through Apoptosis, is Specific of TLR3 Activation, in Murine Syngeneic Cell Line MTB2 #63

(105) (A) MBT2 #63 murine cell line model express murine TLR3. MBT2 cells were seeded at 1.Math.10.sup.5 cells in a final volume of 300 μL per well of RPMI 1640 medium from Gibco (Cat #21875-034), supplemented with 10% FBS from Dominique Dutscher (Cat #51810-500), using p24-well plate (Falcon, Cat #353047). Cells were harvested, fixed and permeabilized using Cytofix/Cytoperm as preconized by manufacturer (BD, Cat #554714). Cells were incubated with either with Rat-anti-mouse-TLR3-PE or rat-IgG2a-PE isotype (Biolegend), at the final concentration of 1.Math.25 μg/ml for 30 minutes at 4° C. Flow cytometry was performed using FACS-Calibur (BD). Results are shown for 10.000 cells per treatment as dot plot of autofluorescence versus FITC-fluorescence intensity and a merged histogram where isotype and mTL3 are represented in dark-grey and light-grey respectively.

(106) (B) TLR3 cell death specificity was monitored by pretreating MBT2 cells with small interferent RNA for TLR3 expression (or its scramble control). Briefly, MBT2 cells were transfected using JetPrime transfection reagent from PolyPlus (Cat #114-07) with 30 nM of either si-mTLR3 or si-scramble from GeneCopoeia (Cat #SR421090 and #S-4001-2), according to the manufacturer protocols. The transfection was performed twice, 72 h and 24 h prior to treatment. MBT2 cells were then treated using either Poly(I:C) from Invivogen (Cat #tlrl-pic) or TL #532 from Dharmacon, without transfection reagent, at the final concentration of 250 ug/ml.

(107) (C) Acidic endolysosomal conditions are detrimental for TLR3 activation. In order to demonstrate the specificity of the TL #532 to TLR3, we pretreated MBT2 cells 45 minutes prior to TLR3-Ligands, using 200 nM of Bafilomycin-A1, an inhibitor of vacuolar H(+)-ATPase (Sigma-Aldrich Cat #B1793), or with the same amount of DMSO. MBT2 cells were then treated using either Poly(I:C) from Invivogen (Cat #tlrl-pic) or TL #532 from Dharmacon, without transfection reagent, at the final concentrations of 250 μg/ml. (B and C upper panel) Apoptosis was measured with AnnexinV+ luminescence read-out using the Real-Time Glo AnnexinV kit from Promega (Cat #JA1000). (B and C Lower panel) Cell viability was measured 24 h post-treatment using MTS Cell Titer Aqueous solution from Promega (France, Cat #G3580). Data are the mean of two independent experiments with SD.

Example 26 and FIG. 26: TL #532 Induces Equal or Better Anti Tumoral Effect than Poly(I:C) on Syngeneic Bladder Cancer Model MBT2 In Vivo, with a Better Tolerance for the TL #532 Treatment

(108) Mouse model C3H/HeN from Charles River, is an immunocompetent species from which MBT-2 #63 bladder cancer model is derived (syngeneic model). 6 weeks old female mice (the age at which the immune system is fully mature) were used at the time of tumor graft. Ectopic grafts of the cells MBT-2 #63 were performed subcutaneously on the right flank using 1,000,000 cells in 100 μl of RPMI 1640 medium from Gibco (Cat #21875-034), without serum nor antibiotics. The first day of treatment is designated as day zero (d0). Tumour inclusion window was between 75 and 125 mm.sup.3. In order to best mimic the route of clinical administration (submucosal injections and/or instillation), one half (in a volume of 10 μl) is injected via the intra-tumoral route and the remainder via the subcutaneous peri-tumoral route. Three groups of ten mice were treated either with Poly(I:C) from Invivogen (Cat #tlrl-pic), or TL #532 from Dharmacon, without transfection reagent, at final dose of 200 and 500 μg respectively, while mock treatment was performed using the same volume of molecule diluent NaCl 0.9%. Note that 200 μg is the maximal injectable dose of Poly(I:C) due to its solubility. Treatments were administered 3 times per week, up to the end point or the total regression of the tumor or the end of treatment. A treatment, for a maximum period of 3 months, was chosen for this “proof of concept” experimentation. The weight of the animals was noted at the time of inclusion and follow-up, at least three times a week. The tumor volume was assessed by means of three-dimensional caliper gauge measurements, calculated according to the spheroidal volume formula: π/6×L×W×H (L=Length, W=width, h=height). The animals were monitored for pain, signs of suffering and any signs of abnormal behavior. In accordance with animal welfare regulations, the following ethical sacrifice criteria are applicable to all the mice, regardless of their experimental status: size of tumor >1500 mm.sup.3, weight loss >20%, reduced mobility, signs of pain and/or discomfort, prostration, ascites, necrosis inducing ulcer or anemia.

(109) Tumor growth of the mock-treated (A), or treated with either Poly(I:C) (B) or TL #532 (C) groups, are shown in fold increase compared to the inclusion date with significant effect of TL #532 and Poly(I:C). In particular, we observed 1/10 mice and 3/10 mices complete responders in Poly(I:C) and TL #532 treated groups respectively.

(110) (D) Effect of TL #532 is also confirmed by the tumor growth delay (TGD), in the treated groups as compared to the control group. TGD is objectified by the difference in the averages of the tumor volumes of the different groups when the mice therein are alive. TGD of the Poly(I:C) group is extended by 13 days (×2.6) and the TGD TL #532 by 18 days (×3.25), as compared to Mock-treated group.

(111) (E) A significant improvement is observed in the survival of the mice when they are treated with TLR3-ligands.

(112) (F) Eventually, it should be noted that the study of the evolution of weight gain in the mice, corresponding to the two relatively homogeneous groups of treatment with Poly(I:C) and TL #532, shows a clear disfavor for the group of Poly(I:C) as compared to the group of TL #532, meaning a better tolerance of the TL #532 compared with Poly(I:C). CR: mice in complete responders/total treated. Kaplan Meier results were analyzed using Mann-Whitney U test and Log rank statistical methods. Tumor growth monitoring was analyzed using an unpaired t-test with p value 0.05 considered as significant.

Example 27 and FIG. 27: TL #532 Induces Vaccinal Effect for all the Three Complete Responders when Rechallenged on the Opposite Flank 3 Months after Remission

(113) All the complete responders (1 from Poly(I:C) group and 3 from TL #532 group), of the previous experiment shown in FIG. 27 were rechallenged on the opposite flank three months after their remission, using the same protocol. For this experiment, five naive mice were included as a control group. No new treatment were introduced. The tumor growth was measured 3 times a week. Mouse treated with Poly (I:C) displays a very short tumor growth delay after rechallenge (about 35-40 days to reach 1500 mm.sup.3) as compared to the naïve mice (around 20-30 days), showing a weak vaccine effect of this complete responder mouse treated with Poly (I:C). Similarly, we observed a tumor growth for the complete responders mouse treated with TL #532 after rechallenge. However, the TGD observed was more pronounced (D70) than that for the mouse treated with Poly(I:C), demonstrating an effect of immunity, even though the latter was not sufficient to contain the tumor growth. Finally, we observed a complete vaccine response effect (CVR) in the other two mice treated with TL #532, with no visible or palpable tumor growth.

Example 28 and FIG. 28: TL #532 Induces Better Anti Tumoral Effects than Immune-Checkpoint Inhibitor Anti-PDL1 and their Combo-Therapy Induces Synergistic Effects In Vivo

(114) MBT2/C3H/HeN syngeneic model was used and grafted as described in FIG. 27. Sixty-five mice were included when they developed a homogeneous spheroid tumour measuring between 75 and 125 mm3. The latter were divided into 6 groups: 11 mice in the control group (Mock-NaCl without monoclonal antibodies (mAb) treatment=group I), 10 mice in the group II (Mock-NaCl+Isotype-control mAb), 11 mice in the group III (Mock-NaCl+Anti-PDL1 mAb), 11 mice in the TL #532 group (group IV), 10 in the group V (TL #532+Isotype-control mAb) and 12 mice in the group VI (TL #532+Anti-PDL1 mAb). Monoclonal antibody (mAb) therapies were administered through intra-peritoneal route using 400 μg of either anti-PDL1 mAb or isotype-control mAb from BioXCell (respectively Cat #BE0101 and #LTF-2), in 200 μl of NaCl 0.9%, once a week until ethical end points are reached, or if the total regression of the tumor is achieved. TL #532 treatments were administered as described in FIG. 27. A treatment, for a maximum period of 3 months, was chosen for this “proof of concept” experimentation.

(115) Tumor growth for mock-treated mice versus TL #532 treated mice are shown either without mAb combination treatments (A), in combination with isotype control mAb (B) or with anti-PDL1 mAb (C). Among all of the groups, only the TL #532 treated groups were able to reach complete responses. It should be noted that the “TL #532+anti-PDL1 combo” increased in a very significant manner the rate of mice in total remission (36% in “TL #532 monotherapy” and 30% in “TL #532+isotype”, as opposed to 50% in “TL #532+anti-PDL1 combo”).

(116) The growth curves (A, B and C) and the TGD curves (D), demonstrate a weak anti tumoral effect in monotherapy of anti-PDL1 but a strong synergy when combined with TL #532. Despite a pro-tumor effect of the control isotype in monotherapy, the “TL #532 monotherapy” and “combo TL #532+control isotype” treatments are found to be relatively similar. As a result, the tumor growth delay (TGD) was multiplied by 2.1 for the “TL #532” group (group-IV versus I). This anti-tumor effect is even more pronounced for the “TL #532+isotype combo” (group-V versus II), with a TGD multiplied by 3. Finally, despite the weak effect of the anti-PDL1 on tumor growth as monotherapy, the “TL #532+anti-PDL1 combo therapy” turns out to be synergistic. The tumor growth delay is multiplied by 3.Math.8 (group-VI versus III) against 2.1 for TL #532 in monotherapy.

(117) (E) Kaplan Meier curves show a very significant increase in survival in the groups treated with TL #532 as compared to the control groups and a slight improvement in survival in the “TL #532+anti-PDL1 combo”.

(118) (F) Evolution of the weights of the mice in the groups IV, V and VI. Contrary to the results of the proof of concept (where a clear difference in weight gain was observed between TL #532 and Poly(I:C)), here the weights of the mice remain homogeneous and comparable. Kaplan Meier results were analyzed using Mann-Whitney U test and Log rank statistical methods. Tumor growth monitoring was analyzed using an unpaired t-test with p value 0.05 considered as significant. CR: mice in complete responders versus total treated.

CONCLUSIONS

(119) The results show that TL #532 has an anti-tumour activity on an ectopic model of murine urothelial carcinoma, that is comparable to that induced by Poly (I:C), but showing less adverse effects, in particular with respect to the weight gain. In vivo, tumor growth delay was significantly increased. Although all the tumors of the mice on the control group progressed, one-third of the TL #532-treated mice were complete responders (with a complete disappearance of the tumor). The “rechallenge” of the complete responders using the same murine bladder cancer cells, at 3 months, prevented the tumor proliferation in ⅔ of the mice. These data favorably support the development of an anti-tumor immune memory, resulting to an anti-tumor vaccination. This effect could be explained by a maturation and invasion of memory T cells specific to the tumor site.

(120) The ex-vivo experiments demonstrated on freshly resected tumors of patients, TL #532 induces a strong secretion of cytokines and chemokines involved in the attraction of CTL (in particular RANTES, MIG and IP10) and decreases the chemokines involved in the recruitment of T-reg and MDSC (in particular MDC and sFas). These results are correlated with the in-vivo experiments, in which a synergistic effect between the ligand TLR3 and anti-PDL1 was observed by the extension of the tumor growth delay (TGD), as compared to the TLR3 ligand or anti-PDL1 alone. Especially, the rate complete responders increased to 50% when the two treatments were combined, as opposed to about 30% when TL #532 was in monotherapy.

(121) Both in-vivo and in-vitro data demonstrate the absence of toxicity of the TL #532. The mice were found to maintain a better growing weight curve compared to the mice treated with Poly (I:C), demonstrating an enhanced tolerance of the TL #532 compared with Poly(I:C). This result is consistent with in-vitro experiments demonstrating the absence of toxicity of TL #532 on primary human bladder cells at 24 hours up to very high doses compared with toxic effects of Poly(I:C) at very low doses. This difference in toxicity observed between the different ligands TLR3, Poly(I:C) and TL #532, is explained by the Polyvalent effect of Poly (I:C). Indeed, Poly (I:C) will act on several pattern recognition receptors such as TLR3, but also MDA5 (Melanoma Differentiation-Associated Protein 5) and RIG-I (retinoic acid-inducible gene-I-like receptors) inducing several inflammatory pathways as well as high cytokinemia thus explaining its high toxicity (Matsumoto M, et al. Biochem. Biophys. Res. Commun. 2002 and Kato H, et al. Nature. 2006). In contrast, TL #532 has a specific activity on TLR3 by means of experiments using bafilomycin or siRNAs. Currently the standard medical treatments in human urothelial carcinoma, BCG therapy in NMIBC and chemotherapy in MIBC, are poorly tolerated therapies with many adverse effects (Lamm D L, et al. J. Urol. 2000; Colombel M, et al. J. Urol. 2006; Alfred Witjes J, et al. Eur Urol. 2017). The absence of toxicity of TL #532 is a real advantage for its development in the treatment of bladder cancer.

(122) In addition, it has been demonstrated on freshly resected tumors treated ex-vivo using TL #532 that its mechanism of action involves apoptosis of bladder cancer cells, showing apoptotic bodies as early as from 24 hours of treatment. Finally, a reprogramming of tumor microenvironment, with secretion of pro-inflammatory cytokines (including CCL5, CXCL9, CXCL10, IFN gamma, IFN lambda and TRAIL) was observed on bladder tumors when they were treated with TL #532.

(123) The ex vivo results show that 100% of bladder tumors express TLR3. Recently, Ayari et al showed that TLR3 receptors are expressed and functional on all their normal urothelial samples (11 samples), in most of the NMIBC tissues (11 samples), and to a lesser extent in the MIBC tumors (15 samples) (Ayari C, et al. J. Urol. 2011). Another team showed in 60 bladder cell samples (24 urothelial carcinomas and 46 normal tissues), the expression of the family of TLRs (TLR-1 to 9), but did not provide accurate data related to TLR3 (Sabah-Ozcan S, et al. Urol. Oncol. 2017).

(124) The ligands of the invention are characterized by efficacy as a monotherapy, synergism with the anti-PDL1s, a vaccine effect and low toxicity, in addition to being synthesizable.