Field of the invention:

The present invention relates to methods and compositions for use for preventing or treating precancerous lesions, more particularly skin precancerous lesions, and/or cancers, more specifically, skin carcinomas. More particularly, a composition of the invention comprises the combination of a first compound, which is an agonist of a first Toll-like Receptor (TLR), and of a second compound, which is an agonist of a second Toll-like Receptor (TLR) different from the first TLR and/or an agonist of a C-Type Lectin Receptor (CLR).

Background of the invention:

The skin is an immune competent tissue. Tumor progression in epithelial tissues is controlled by immune mechanisms in place within the epithelium itself (Hagerling et al, Trends Cell Biol. 2015, 25:214-220). Indeed, it has been well established that a strong lymphocytic infiltration with cytotoxic T lymphocytes (CTL) is associated with a good clinical outcome (Pages et al, N Engl J Med. 2005, 353:2654-2666). Dendritic cells (DCs) are key actors of antitumoral immunity as they are in charge of educating CTL. Nevertheless, antitumoral DCs are rare cells in progressing tumors, as factors expressed by the tumoral environment inhibit their function and favor the recruitment or the local shaping of immunosuppressive DCs and other immunosuppressive immune cells. Dysfunctional DCs are a cause of immune system failure to detect and reject transformed cells and tumor rejection.

In particular, DCs, also called "professional Antigen Presenting cells", have strong ability at antigen presentation and thus at inducing T cell adaptive immunity (Banchereau J and Palucka AK, Nat Rev Immunol. 2005, 5:296-306). DCs are also crucial actors for controlling humoral immunity and can interact with innate immune cells, including natural killer (NK) cells, phagocytes and mast cells.

DCs appear like key targets for the generation of protective anti-tumor immunity because DCs allow the activation of the adaptive immunity (cellular and humoral) and also the innate immune system (Steinman and Banchereau, Nature 2007. 449:419-26).

In the art, therapeutic strategies based on DCs are known for treating cancer. As example, DC-based vaccination approaches have been developed regarding the noteworthy ability of these cells in coordinating innate and adaptive immune responses. DC vaccination promotes tumor-specific effector T cells to induce tumor mass reduction and promotes immunological memory for controlling tumor relapse (Sabado and Bhardwaj, Immunotherapy. 2010, 2: 37- 56).

Another promising cancer therapy based on DCs relies on targeting specific innate sensors expressed by DCs named Pattern Recognition Receptors (PRRs) in order to activate them. Importantly, DC PRRs that can be targeted for cancer immunotherapy include receptors that recognize microbes and more particularly members of the Toll Like Receptor (TLR) family. Ten different TLRs have been identified in humans and thirteen in mice (Beutler, Nature 2004, 430:257-263). TLRs represent the best-characterized family of PRRs, which recognize pathogen-associated molecular patterns (PAMPS) derived from bacteria or virus, expressed by many cell types, and that induce signaling cascades leading to expression of proinflammatory gene (West et al, Ann Rev Cell Dev Biol. 2006, 22:409-37).

In parallel to TLR family, other key PRRs families that can be targeted for cancer immunotherapy comprise: the RNA helicase RIG-I-Like Receptor (RLR) family, the C-Type Like Receptor (CLR) family and the NOD-like Receptor (NLR) family.

PRRs expression profile, including TLRs and CLRs appears to be one of the guidelines for diagnosing and treating tumors. The analysis of the PRRs distribution including TLRs and CLRs in different types of tumor and of the cellular response to PRRs including TLR and CLR agonists or antagonists in different types of tumor is herein revealed by inventors.

Using a stepwise mouse model of skin carcinogenesis that recapitulates the human pathology, it has been demonstrated that DCs infiltrate pre-cancerous skin lesions and carcinomas but display altered phenotype and functions. They also express specific PRRs at pre-cancerous and cancerous stages that offer the opportunity to identify rationale targets for the local restoration of their antitumoral function (Figure 1). Consequently, PRRs including TLRs and CLRs represent potential candidates to stimulate the antitumor immunity locally at the site of carcinogenesis and skin lesions.

TLR-based cancer therapy strategies are known in the art.

As example, WO2007063421 relates to methods for modulating an immune response by administering a composition comprising a Toll-like receptor agonist and an immune mediator which, in particular, downregulates the expression of the anti- inflammatory cytokine IL10. These methods can be used to provide therapeutic treatment for cancerous conditions and infectious diseases. TLR9 agonists such as CpG ODN are mentioned but no specific CpG- ODN, in particular Class C CpG ODN, is identified. Cancers may be treated in accordance with the invention disclosed in this document, in particular squamous cell carcinoma and basal cell carcinoma, but the treatment of precancerous lesions is not suggested. WO03045431 relates to methods for treating disease states, including cancer, by activating dendritic cells from the host which are rendered hyporesponsive to activation stimuli by the disease. These methods comprise a step of administering the host with an effective amount of a tumor-derived DC inhibitory factor antagonist in combination with an effective amount of a Toll-like receptor (TLR) agonist. TLR9 agonists are specifically cited such as CpG ODN but, the specific use of Class C CpG-ODN is not suggested. Types of cancer which could be treated include but are not limited to melanoma. Other skin cancers such as squamous cell carcinoma and basal cell carcinoma or skin conditions such as precancerous lesion are not identified.

WO2007063421 and WO03045431 both use a similar therapeutic approach which consists of a stimulation of anti-tumor immunity with a TLR agonist and of a blockade of pro tumor immunosuppression (by targeting a specific factor such as IL-10).

Inventors herein disclose a new therapeutic approach. Inventors have discovered that compositions comprising the combination of a first compound, said first compound being an agonist of a first Toll-like Receptor (TLR) and of a second compound, said second compound being an agonist of a second Toll-like Receptor (TLR) different from the first TLR and/or an agonist of a C-Type Lectin Receptor (CLR), are effective for treating precancerous lesions such as those observed in actinic Keratosis or Bowen disease, and cancers such as skin squamous cell carcinoma (SCC) and/or skin basal cell carcinoma (BCC). This therapeutic approach relies on an enhanced stimulation of anti-tumor immunity obtained thanks to a combination of two specific TLR agonists or a combination of a TLR agonist and of a CLR agonist. Contrary to available methods of the art, this therapeutic approach does not require a blockade of pro tumor immunosuppression.

This therapeutic strategy is neither described nor suggested in the art and presents noteworthy advantages. Combination of two different TLR agonists is an interesting alternative because based on the combined use of two small molecules easier to deliver, to formulate and to produce than large molecules. Moreover, use of small molecules avoids the variability of large molecule due to modification such as glycosylation during the manufacturing process. The avoidance of such modification variability is interesting for safety and stability concerns. Furthermore, using this strategy for treating precancerous lesions and more particularly skin precancerous lesions is not known in the art and appears as an original approach. Skin precancerous lesions are skin conditions in need of treatment. Targeting precancerous lesions enables to treat cancer at an early stage before the tumor becomes aggressive. Moreover, inventors have shown that such strategy enhances the stimulation of both adaptive immunity and innate immunity contrary to the strategy known in the art with Aldara which only restores anti-cancer innate immunity. Consequently, this new therapeutic approach has a large spectrum of action on immunity actors for carcinoma therapy.

Dendritic cells from most solid tumors are inflammatory DCs, but nevertheless there is a specific imprinting of each tissue (the skin is different from the lung as an example) that probably influences their exact phenotype and responsiveness. The present invention is in particular efficient for preventing or treating skin cancers such as skin carcinoma, and skin pre-cancerous lesions.

Summary of the invention:

According to a first aspect of the present invention a composition for enhancing anti-tumor immunity is herein provided. This composition comprises:

(i) a first compound, said first compound being an agonist of a first Toll-like Receptor (TLR), and

(ii) a second compound, said second compound being an agonist of a second Toll-like Receptor (TLR) different from the first TLR targeted in (i) and/or an agonist of a C-Type Lectin Receptor (CLR).

In one aspect, the first compound of the composition is a TLR9 agonist.

Preferably, the TLR9 agonist is an oligodeoxynucleotide sequence containing one or more CpG (CpG ODN).

More preferably, the TLR9 agonist is a Class C CpG ODN.

Most preferably, the TLR9 agonist is selected from ODN 2395, ODN M362 and DSL03. In a preferred embodiment, the second compound present in the composition of the invention is a TLR7 and/or a TLR8 agonist.

In another aspect, the TLR7 and TLR8 agonist is Resiquimod (R848).

In another aspect, the TLR7 agonist is selected from Imiquimod (R837), CL075, CL097, CL264, CL307, Gardiquimod™, Loxoribine and Poly(dT).

In another aspect, the TLR8 agonist is selected from ssRNA40, ORN02, ORN06, ssPolyU Naked, ssPolyU, ssRNA40, ssRNA41, ssRNA-DR, CL075, CL097, Poly(dT) and TL8-506. In another aspect, the second compound is a Dectin-1 agonist.

More particularly, the Dectin-1 agonist is selected from a Cell wall preparation of S. cerevisiae (Zymozan), β(1 ^→ 4,H 3, l→6)-glucan peptide (BGP) from Trametes versicolor; Beta-l,3-glucan from Alcaligenes faecalis (Curdlan AL); Heat-killed preparation of Candida albicans; Heat-killed S. cerevisiae; Soluble β(1 ^→ 3, l ^→ 6)-glucan from Laminaria digitata; β(1 →3, l→4)-glucan from Cetriana islandica; P(l→6)-glucan from Lasallia pustulata; β(1→3, 1 →6)-glucan from Schizophyllum commune; and β(1→3, l→6)-glucan from Sclerotium rolfsii.

Also herein described is a composition according to the present invention for use as a drug or medicament.

The composition according to the present invention is preferably for local administration. In a particular aspect, the composition according to the present invention is for intratumoral and/or peritumoral administration.

In another particular aspect, the first compound and the second compound are for simultaneous co-administration in a separate vehicle or in a same vehicle.

The invention also relates to a composition as herein described for use for preventing or treating a precancerous lesion, in particular a skin precancerous lesion, and/or a cancer, in particular a skin cancer.

In a preferred embodiment, the composition according to the present invention is for use for preventing or treating skin carcinoma.

In a more preferred embodiment, the composition according to the present invention is for use for preventing or treating skin squamous cell carcinoma (SCC) and/or skin basal cell carcinoma (BCC).

In another embodiment, the composition according to the present invention is for use for preventing or treating actinic keratosis and/or Bowen disease.

Also herein described is the use of a first compound as herein described and of a second compound as herein described for preparing a composition, in particular a pharmaceutical composition, preferably a composition for use as a medicament, in particular a composition for preventing or treating a skin disease, a cancer and/or a precancerous lesion as herein described.

Further herein described is a kit including a composition according to the present invention, typically a combination of the first and second compounds herein described, and uses thereof. Also herein described is a method for preventing or treating a precancerous lesion and/or a cancer, wherein the method comprises a step of administering a composition according to the invention to a subject in need thereof, the precancerous lesion being preferably selected from actinic keratosis and Bowen disease, and the cancer being preferably a skin carcinoma, preferably a skin carcinoma selected from a skin squamous cell carcinoma (SCC) and a skin basal cell carcinoma (BCC).

Detailed description of the invention:

According to a first aspect of the present invention, a composition for enhancing anti-tumor immunity is herein provided. This composition comprises:

(i) a first compound, said first compound being an agonist of a first Toll-like Receptor (TLR), and

(ii) a second compound, said second compound being an agonist of a second TLR different from the first TLR targeted in (i) and/or an agonist of a C-Type Lectin Receptor

(CLR).

In one aspect, the first compound of the composition is a first TLR agonist selected from a TLR1 agonist, TLR2 agonist, TLR3 agonist, TLR4 agonist, TLR5 agonist, TLR6 agonist, TLR7 agonist, TLR8 agonist, TLR9 agonist, TLR11 agonist, TLR12 agonist, TLR13 agonist and a combination thereof. Preferably, the first TLR agonist is a TLR7, TLR8 and/or TLR9 agonist. More preferably, the first TLR agonist is a TLR9 agonist.

In another aspect, the second compound of the composition is a TLR agonist selected from a TLR1 agonist, TLR2 agonist, TLR3 agonist, TLR4 agonist, TLR5 agonist, TLR6 agonist, TLR7 agonist, TLR8 agonist, TLR9 agonist, TLR11 agonist, TLR12 agonist and TLR13 agonist. Preferably, the second TLR agonist is a TLR7 and/or TLR8 agonist.

An example of a TLR1 agonist suitable for the invention is Pam3Csk4.

Examples of TLR2 agonists suitable for the invention are lipoproteins such as Pam2Csk4, Pam3Csk4, FSL-1 (Pam2CGDPKHPKSF), CL429; zymozan; lipopolysaccharide from Porphyromonas gingivalis (LPS-PG); lipoglycans such as Lipoarabinomannan from Mycobacterium smegmatis (LAM-MS), Lipomannan from Mycobacterium smegmatis (LM- MS); peptidoglycans ( PGN) such as PGN from Bacillus subtilis (PGN-BS); PGN from E. coli 0111 :B4 (PGN-EB), PGN from Escherichia coli K12 (PGN-EK), PGN from Staphylococcus aureus (PGN-SA); lipoteichoic acid (LTA) such as LTA from Bacillus subtilis (LTA-BS), LTA from Staphylococcus aureus ; Heat Killed Bacteria.

Example of Heat Killed Bacteria are Heat Killed Acholeplasma laidlawii (HKAL), Heat Killed Escherichia coli 0111 :B4 (HKEB), Heat Killed Helicobacter pylori(UKEP), Heat Killed Listeria monocytogenes(HKLM); Heat Killed Legionella pneumophila (HKLP), Heat Killed Lactobacillus rhamnosus (HKLR), Heat Killed Mycoplasma fermentans (HKMF), Heat-killed Mycobacterium tuberculosis (HKMT), Heat Killed Pseudomonas aeruginosa (HKPA), Heat Killed Porphyromonas gingivalis (HKPG), Heat Killed Staphylococcus aureus (HKSA), Heat Killed Staphylococcus epidermidis (HKSE), Heat Killed Streptococcus pneumonia (HKSP), Heat Killed Salmonella typhimurium (HKST). Examples of TLR3 agonists suitable for the invention are Polyadenylic-polyuridylic acid( Poly(A:U)), Polyinosinic-polycytidylic acid of high molecular weight with an average size of 1.5-8 kb (Poly(LC)-HMW); and Polyinosinic-polycytidylic acid of low molecular weight with an average size of 0.2-1 kb (Poly(LC)-LMW ).

Examples of TLR4 agonists suitable for the invention are Lipopolysaccharide (LPS), for example LPS from Escherichia coli K12 (LPS-EK), LPS from Escherichia coli 055 :B5 (LPS- B5), LPS from Escherichia coli 0111 :B4 (LPS-EB), LPS from Porphyromonas gingivalis

(LPS-PG), LPS from Salmonella minnesota R595 (LPS-SM); Monophosphoryl Lipid A

(MLA), more particularly MLA from Salmonella minnesota R595 (MLA-SM); and HKST.

Examples of TLR5 agonists suitable for the invention are Heat Killed Salmonella typhimurium (HKST); and Flagellin and for example flagellin from Salmonella typhimurium (FLA-ST), Flagellin from Bacillus subtilis (FLA-BS), or Flagellin from Pseudomonas aeruginosa (FLA-PA). The flagellin is either recombinant or non-recombinant.

Examples of TLR6 agonists suitable for the invention are FSL-1 and Pam2Csk4.

Examples of TLR7 agonists suitable for the invention are CL075, CL097, CL264, CL307,

GardiquimodTM, Imiquimod (R837), Loxoribine and Poly(dT).

Examples of TLR8 agonists suitable for the invention are ssRNA40, ORN02, ORN06, ssPolyU Naked, ssPolyU, ssRNA41 and ssRNA-DRCL075, CL097, Poly(dT) and TL8-506.

When the oligonucleotides ssRNA40, ORN06, ssPolyU, ssRNA41 and ssRNA are used, they can be in a form complexed with a transfection reagent as for example the cationic lipid known under the name LyoVec™.

An example of a TLR7 and TLR8 agonist suitable for the invention is Resiquimod (R848).

An example of TLR11 agonist suitable for the invention is Toxoplasma gondii Profilin.

An example of TLR12 agonist suitable for the invention is Toxoplasma gondii Profilin.

An example of TLR13 agonist suitable for the invention is ORN SA19/c.

Examples of TLR9 agonists suitable for the invention are oligodeoxynucleotides sequences containing one or more CpG (CpG ODN). Preferably, the CpG ODNs are Class C CpG

ODNs. Examples of Class C CpG ODNs suitable for the invention are ODN 2395, ODN

M362 and ODN D-SL03. Other examples of Class C CpG ODNs suitable for the invention can be found in Jurk et al, 2004, Vollmer et al, 2004, and Yang L. et al., 2013. As used herein, the terms "Class C CpG ODNs" consists of a stimulatory hexameric CpG motif positioned at or near the 5' end and linked by a T spacer to a GC-rich palindromic sequence.

CpG ODN suitable for the invention can be of any length greater than 18 nucleotides and can contain modification, such as a modification of the 3ΌΗ or 5ΌΗ group, modification of a nucleotide base, modification of the sugar component, and modification of the phosphate ring. The prior list of modifications only gives examples and is not limitative to appreciate the scope of the invention. CpG ODN may be single or double stranded DNA sequence or other modified polynucleotides. CpG ODNs sequences may or may not include one or more palindromic sequence.

The CpG ODN can be isolated using conventional polynucleotides isolation procedures, or can be synthetized using techniques and nucleic acid synthesis equipment which are well known in the art including, but not limited to, enzymatic methods, chemical methods and the degradation of larger oligonucleotide sequences (see, for example, Ausubel et al, 1987 and Sambrook et al, 1989).

The TLR agonists suitable for the invention listed herein above can be in a modified form. Further suitable agonists to the TLR agonists listed above may also be used, wherein said agonists function to activate at least one of the Toll-Like Receptors listed above.

In another aspect, the second compound of the composition of the invention is a CLR agonist selected from a Clec7a/dectinl agonist, Clecl0a/MGL/CD301 agonist, Clec4a/DCIR agonist, Clec4b/DCAR agonist, Clec4d/dectin3 agonist, Clec4e/Mincle agonist, Clec4g agonist, Clec4n/Dectin2 agonist, DC-SIGN/CD209 agonist, MRC1/CD206 agonist. Preferably, the CLR agonist is Dectin-1 agonist. More preferably, the Dectin-1 agonist is Zymozan.

Examples of Dectin 1 agonists suitable for the invention are β(1 ^→ 4,H 3, l ^→ 6)-glucan peptide (BGP) from Trametes versicolor; Beta-l,3-glucan from Alcaligenes faecalis (Curdlan AL); Heat-killed preparation of Candida albicans; Heat-killed S. cerevisiae; Soluble β(1→3, 1 ^→ 6)-glucan from Laminaria digitata; β(1→3, l ^→ 4)-glucan from Cetriana islandica; β(1→6)- glucan from Lasallia pustulata; β(1→3, l→6)-glucan from Schizophyllum commune; β(1→3, l ^→ 6)-glucan from Sclerotium rolfsii; and Cell wall preparation of S. cerevisiae (Zymozan). An example of Dectin-2 agonist suitable for the invention is Malassezia furfur cell wall preparation (furfurman). Examples of Mincle agonists suitable for the invention are heat killed Mycobacterium tuberculosis, Trehalose-6,6-dibehenate (TDB), and a major synthetic immunostimulatory component of Mycobacterium tuberculosis.

TLR or CLR agonists listed herein above can be in a modified form. Further suitable agonists different to the TLR or CLR agonists listed above may also be used, wherein said agonists function to activate at least one TLR or CLR listed above.

In the further aspect, herein described is a composition for use as a drug or medicament.

The composition is preferably delivered to the subject in need thereof by local administration. The subject is typically a mammal, in particular a human being. The subject is for example a human patient.

Examples of local administration routes suitable for the invention are, but not limitatively, transdermal, subcutaneous, intradermal, intratumoral, and topical administration. Preferably, the composition according to the invention is delivered by peritumoral and/or by intratumoral administration(s). More preferably, the composition is administered by peritumoral or intratumoral administration in small lesions. But, a person having ordinary skilled in the art can easily adapt the composition for another administration route such as non-local administration route. Examples of non-local administration routes are, but not limitatively, intramuscular, intravenous and transmucosal.

The optimal dose(s) of the first and/or second compounds of the composition according to the invention will be adjusted according to several parameters including age, sex, weight, stage of the condition to treat, the nature of the active molecule(s) and the route of administration. A broad range of doses may be used between 0,1 mg and 2 mg per kilogram of body per day. Doses will be adjusted to give an optimal therapeutic response.

In a further aspect, the first compound and the second compound of the composition of the invention are simultaneously co-administered in a separate vehicle or in a same vehicle. When the first compound and the second compound of the composition of the invention are simultaneously co-administered in a separate vehicle, they can be administered according to a same administration route or according to a different administration route as identified herein above. When the first compound and the second compound are simultaneously co- administered in separate vehicles according to a same administration route, the administration is at the same region or a different region of the subject's body. Preferably, the first compound and the second compound are simultaneously co-administered in a same vehicle and peritumorally. In another aspect, the first compound and the second compound of the composition of the invention are administered according to a sequential administration. More particularly, either the first compound is administered before the second compound or the second compound is administered before the first compound. They can be administered according to a same administration route or according to a different administration route as identified herein above.

In the context of the present invention, slow release formulations or a slow release apparatus may be used for continuous administration of the herein described composition.

Also herein described is a composition for use for treating skin precancerous lesions, for example a composition for use for treating actinic keratosis and/or Bowen disease lesions. Preferably, the skin precancerous lesions are actinic keratosis. Skin precancerous lesions targeted by the invention may also be actinic cheilitis or premalignant cutaneous horn.

In another aspect, a composition as disclosed herein above is described for use in the treatment of cancer. Both primary and metastatic cancers can be treated with the composition of the invention. More specifically, a cancer which can be treated with the composition of the invention can be selected for example from adrenal cancer, anal cancer, appendix cancer, bile duct cancer, bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, eye cancer, gallbladder cancer, glioblastoma, head and neck cancer, hemangioendothelioma, hodgkin lymphoma, Intestinal cancer, kidney cancer, leukemia, liver cancer, lung cancer, melanoma, mesothelioma, multiple myeloma, neuroendocrine cancer, non-Hodgkin lymphoma, oral cancer, ovarian cancer, pancreatic cancer, paranaseal sinus cancer, pelvic cancer, penile cancer, pineoblastoma, prostate cancer, sinus cancer, skin cancer, small intestine cancer soft tissue carcinoma, spinal cancer, stomach cancer, testicular cancer, throat cancer, thymus cancer, thyroid cancer, tubal cancer, uterine cancer, urethral cancer, carcinoma and metastatic carcinoma. Preferably, the composition is for use for treating skin carcinoma. More preferably, the composition is for use for treating squamous cell carcinoma (SCC) and/or basal cell carcinoma (BCC).

Also herein described is a method for preventing or treating a precancerous lesion and/or a cancer, wherein the method comprises a step of administering a composition according to the invention to a subject in need thereof, the precancerous lesion being preferably selected from actinic keratosis and Bowen disease, and the cancer being preferably a skin carcinoma, preferably a skin carcinoma selected from a skin squamous cell carcinoma (SCC) and a skin basal cell carcinoma (BCC). Further herein described is a kit including a therapeutic combination according to the invention, that-is-to-say a composition comprising a first compound, said compound being an agonist of a first TLR, and a second compound, said second compound being an agonist of a second TLR different from the first TLR and/or a CLR agonist.

Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by a person who is skilled in the art in the field of the present invention.

The invention is further illustrated below by the following Examples, which are not intended to limit its scope.

Brief description of the drawing

Figure 1 shows the TLR (A) and CLR (B) transcripts levels expressed by skin-associated dendritic cells and intralesional dendritic cells. Transcriptomic analyses were performed on sorted dendritic cells from skin of control skin (naive skin, n=2), PMA treated-mice (PMA skin, n=3), and from skin surrounding papillomas (Papilloma skin, n=4), skin surrounding tumors (Tumor skin, n=4), papillomas (n=6) and tumors (n=5) of pooled DMBA/PMA treated FVB/N mice. The data correspond to the mean values of raw expression of selected TLR and CLR transcripts in the different DC samples.

Figure 2 shows the in vitro experimental approach disclosed in example 1.

Figure 3 shows the stimulation of dendritic cells sorted from TCI cutaneous tumors with different TLR agonists. The TLR9 agonist, the ODN CpG class C 2395 as well as other nucleic acids types including Imiquimod and Resiquimod (TLR7), ORNSA19 (TLR13L) and zymozan (TLR2/Dectinl agonists) induce the production of IL-12p40 by intratumoral DCs contrary to other TLR ligands that induce the secretion of IL-6 and TNF but no or low levels of IL-12p40. The intratumoral DCs also produce high levels of IL-10 compared to FLT3L- derived DCs from naive C57/B6 mice after stimulation with several TLR agonists. Cytokine concentration (IL10, IL-6, TNF and IL-12p40) were measured in culture supematants of intratumoral DCs and control FLT3L-derived DCs after in vitro stimulation during 22 hours with indicated TLR and CLR agonists using the CBA technology. Bars represent the mean value of cytokine concentration ± SEM for tumor DC (n= between 4 and 6), control DC (n=l). The cytokine production by non-stimulated intratumoral DCs was substracted.

Figure 4 shows the cytokine pattern secreted by total intratumoral CD45 ⁺ leukocytes and leukocytes from draining lymph nodes after in vitro stimulation. CD45 ⁺ Leukocytes were isolated from 14 days and 21 days tumors (A) or from the lymph nodes draining 21 days tumors or draining wild-type control mice (B). 100 000 CD45 ⁺ leukocytes were stimulated with PMA/ionomycin for 4 hours. Culture supernatants were collected and frozen. Cytokines measured using the CBA technology.

Figure 5 shows the cytokine pattern produced by intratumoral CD45 ⁺ leukocytes after in vitro stimulation with several TLR and CLR agonists or combination of two agonists. Intratumoral leukocytes were isolated from 14 days tumors and CD45 ⁺ cells sorted. 100 000 CD45 ⁺ intratumoral leukocytes were stimulated with the indicated ligands or combination of ligands. Culture supernatants were collected after 72h and cytokines measured using the CBA technology.

Figure 6 shows the impact of local treatments on tumor sizes and leukocyte infiltration.

(A) The mice were sacrificed at day 22 and the tumoral volumes measured after the indicated in vivo treatment using the elipsoid formula 7i/6*H*W*D. (B) Tumors were dissociated by enzymatic digestion and the percentage of tumor-associated CD45 ⁺ leukocytes determined by flow cytrometric analysis.

Figure 7 shows the impact of local treatments on dendritic cells and neutrophils infiltration and on DC maturation. The mice were sacrificed at day 22 and the tumors were dissociated by enzymatic digestion. (A) Gating strategy for flow cytometric analysis of myeloid cell subsets.

(B) Percentages of tumor-associated immature and mature dendritic cells and neutrophils and (C) expression of the maturation markers CD40 and CD86 by mature dendritic cells (black bars) and immature dendritic cells (grey bars) were determined by flow cytrometric analysis. Figure 8 shows the impact of local treatments on the proportion of tumor-associated CD4 ⁺ T cells, CD8 ⁺ T cells and NKp46 ⁺ ILC. (A) Gating strategy for lymphoid cell subsets analysis by flow cytometry. (B) Mean values ± SD of the frequency of the indicated subsets.

Figure 9 shows the impact of local treatments on the proportion of tumor-associated FoxP3 ⁺ CD4 ⁺ T cells. A) Gating strategy used for the flow cytometric analysis of FoxP3 CD4 ⁺ T cells. (B) Mean values ± SD of the frequency of the indicated subsets in one experiment. Figure 10 shows the impact of in vivo local treatments on T cell polarization. Tumors and draining lymph nodes from control and treated mice were collected and dissociated 3 days post-treatment. Sorted CD45 ⁺ tumor cell suspensions (100 000/w) (A) and CD45 ⁺ lymph nodes cells (100 000/w) (B) were stimulated in vitro with anti CD3/CD28 coated Dynabeads and culture supernatants collected after 24h. The cytokines produced were measured by the CBA technology. Data correspond to mean values ± SD (n=2). Example 1: In vitro consequences of TLRs stimulation on DCs phenotype and polarization of intratumoral leukocytes in mice.

The aim of this example is to determine the in vitro consequences of the stimulation of several TLRs or the Dectin-1 CLR on dendritic cells (DCs) phenotype/function and on the cytokines secreted by intratumoral leukocytes in mice.

• Methodology

Specific TLRs or CLRs were shown to be expressed by the dendritic cells sorted from papillomas and carcinomas (Figure 1). Among them, several agonists of selected TLRs and CLRs were used to stimulate sorted intratumoral DCs. These agonists include acetylated lipopeptides (a TLR1/2 agonist, Pam2Csk4 used at ^g/mL; a TLR2/6 agonist, Pam3Csk4 used at 2μg/mL), Imiquimod ^® used at l(^g/mL (a TLR7 agonist) and Resiquimod ^® used at l(^g/mL (recognized by TLR7 in mice and TLR7 and TLR8 in humans), 23 S rRNA oligoribonucleotide from salmonella used at 2μg/mL (TLR13 agonist) and protozoan profilin- like proteins such as toxoplasma gondii profilin protein used at 3μg/mL (recognized by TLR11 and TLR 12). CpG ODNs (TLR9 agonists) including class A, B and C CpG. The CpG ODN 2395 has been used in the following experiments at a concentration of 5μΜ. CpG ODN 2395 is a CpG ODN of class C with the broadest range of applications and usable in both mice and humans. Zymosan and Curdlan directed against the CLR dectin-1 were used at l(^g/mL. TDB (Trehalose-6,6-dimycolate from M. tuberculosis), a Mincle agonist, was used at l(^g/mL.

The effect of these TLR ligands or dectin-1 agonists was first tested using mouse dendritic cells freshly isolated from skin tumors by cell sorting. These DCs were isolated from skin TCI tumors obtained by the syngeneic grafting of the epithelial TCI tumor cell line by intradermal injection in C57BL/6 mice). Subsets of Flt3-ligand derived DC subsets (CDl lb ⁺ FLDC and CD24+ FLDC) were used as control DCs. TLR and CLR agonists from Invivogen were used at the optimal concentration recommended by the manufacturer (CpG ODN, LPS, Imiquimod, Resiquimod, Zymosan and TDB) or were titrated on DC to determine the optimal concentration in dose/response experiments (Pam3Csk4 used at 20, 2 and 0.2 μg/mL; Pam2CSK4 used at 1 and O. ^g/mL; ORNSA19 used at 2 and O^g/mL; Profilin used at 15μg/mL and 3μg/mL; Curdlan used at 100 and 10μg/mL). Secondly, their immunostimulatory potential was evaluated using intratumoral CD45 ⁺ leukocytes as illustrated in Figure 2. Functions of DC subsets after in vitro TLR/CLR stimulation was assessed by measuring the cytokine levels produced by sorted intratumoral DCs (100 000 DCs/well) cultured in vitro during 22 hours with the proposed TLR/CLR ligands. The following cytokines were quantified in DCs culture supernatants using the CBA technology (BD Biosciences): IL6, IL10, TNF, ILlb, IL12p40, IL12p70 and IL23pl9.

The polarization of intratumoral leukocytes was measured in vitro by analyzing the cytokines produced by sorted CD45 ⁺ leukocytes (100 000 leukocytes/well) isolated from TCI tumors after 4h stimulation with a mix of PMA (100 ng/mL; Sigma- Aldrich) and ionomycin (^g/mL; Sigma- Aldrich) or after 24h or 72 hours of stimulation with the above mentioned TLR or CLR ligands. Cytokines including Thl (IFNY, IL12), Th2 (IL4, IL5 and IL13), Thl7 (IL17A), regulatory (IL10) and pro -inflammatory (TNF, ILip, IL6) cytokines were quantified in culture supernatants using the CBA (technology). In some experiments, the intratumoral leukocytes were stimulated with the indicated TLR ligand in presence of the neutralizing CD210/IL10R1 antibody (BioXcell, clone lB1.3a) or its isotypic control at the indicated concentrations.

• Results

DC functional maturation through TLR/CLR stimulation

Phenotypic analyses revealed that most intratumoral DCs expressed intermediate levels of MHCII as well as CD40 and CD86 co-stimulatory molecules when freshly isolated from tumors. Their in vitro stimulation with the different TLR agonists increased MHCII and CD86 expression without notable differences between agonists indicating that TLR stimulation induced the maturation of intratumoral DCs. The TLR agonists used included Imiquimod (TLR7L), Resiquimod (TLR7L/TLR8L), Class C CpG ODN (ODN 2395) (TLR9L) and its control, Staphylococcus A. ORN (ORN SA19), (TLR13L) and its ORN control, Pam2Csk4 (TLR2/6L), Pam3Csk4 (TLR1/2L), Toxoplasma gondii profilin (TLR11/12L), ultrapure E. coli LPS (strain 0111 :B4) (TLR4L) and zymosan (TLR2 and Dectin-1 agonist) used at optimal concentration.

In a second step, sorted intratumoral DCs were stimulated in vitro with the above mentioned TLR and CLR agonists and the cytokines produced in culture supernatant were quantified (Figure 3). Importantly, intratumoral DCs produced huge amounts of IL10 in response of most stimuli used contrary to control DC (Figure 3A). Furthermore, intratumoral DCs produced substantial levels of IL12p40 in response to ODN2395 (TLR9L), as well as in response to other nucleic acids types including Imiquimod and Resiquimod (TLR7L), ORNSA19 (TLR13L) and in response to zymozan (TLR2/Dectinl agonists). They did not secrete any IL-12 after stimulation with TLR1/2 and TLR2/6 agonists (Pam2Csk4 and Pam3Csk4, respectively) or LPS contrary to control non tumoral CD24 Flt3L-derived DC (Figure 3B). Both intratumoral DCs and Flt3L-derived DCs secreted huge amounts of inflammatory cytokines IL-6 and TNF in response of most stimuli (Figure 3C and 3D).

In summary, the biased cytokine responses of intratumoral DCs after in vitro stimulation with stimuli that activate control DCs indicate they have a specific cytokine pattern related to a protumoral profile. Such profile is expected to be associated with low efficacy to activate anti-tumoral CD8 ⁺ cytotoxic T lymphocytes (CTL). As several TLR agonists induce the production of IL12p40, one can expect that the combination in vivo of these TLR agonists increase the antitumoral response.

Reprogramming of the antitumoral immunity

Sorted CD45 ⁺ intratumoral leukocytes have been used to screen more ligands and combination of ligands. Such CD45 based assay has the advantage to take into account the impact of stimulation on all the immune cells present in the tumoral environment and consequently reflects more the in vivo situation.

As illustrated in Figure 4 A, tumor leukocytes produced huge amount of IL10 and TNF, as well as low amounts of IFN-Y (also herein identified as IFNg) and IL-17A in response to broad unspecific PMA/ionomycin stimulation. Furthermore, the leukocytes from lymph nodes draining the tumor produced less IFN-Y than leukocytes from a wild type mouse (Figure 4B). This indicates that the tumoral environment is immunosuppressive and that the IL-10 cytokine contributes to the repression of the protective antitumoral Thl immunity.

The next step was to determine whether selected TLR/CLR agonists alone or in combination were able to induce the IL-12 antitumoral cytokine as well as Thl cytokines (IFNy) in the tumoral environment.

As shown in Figure 5, the stimulation with a Class C CpG ODN (ODN2395) with another TLR ligand (Imiquimod, resiquimod) or the TLR2/dectin-l CLR agonist zymosan increased the amount of IL-12p40, suggesting that such combinations of two agonists improves antitumoral immunity in vivo. In comparison, the combination of Imiquimod and Resiquimod did not improve IL12 production. Except for the combination of ODN2395 with zymosan (TLR2/dectin-l agonist), inventors did not observe increased productions of IL-6, IL-10 and TNF. Interestingly, the ODN2395/Resiquimod induced lower production of the immunosuppressive cytokine IL-10 that ligands alone or other combinations of TLR ligands. In summary, TLR9L stimulation in combination with another TLR or with a TLR2/Dectin-1 agonist increased the production of IL12 by intratumoral leukocytes. As the combination of ODN2395 and Resiquimod is the one that induced the lower production of the IL-10 immunosuppressive cytokine, inventors tested this combination in an in vivo assay of mouse skin carcinoma. Example 2: In vivo validation of ODN2395/Resiquimod combination validated in vitro using an orthotopic model of carcinoma based on the intradermal grafting of TC-1 epithelial tumor cells.

The aim of this example is to validate the molecules selected in vitro using a mouse model of carcinoma based on the intradermal grafting of TC-1 epithelial tumor cells.

• Methodology

A mouse model of carcinoma based on the intradermal grafting of the epithelial TCI tumor cell line was used. 20 000 tumor cells were injected intradermally and tumoral volumes were measured 22 days after grafting using the ellipsoid formula (tumoral volume=7i/6.L.W.D). In absence of treatment, mice developed tumors with volumes comprised between 500 and 600 mm ³ . When the tumors reached a volume comprised between 30 and 50 mm ³ (day 7 post- injection), mice were homogeneously distributed in the indicated groups (between n= 3 to 7 mice per group) for treatments at D7, D14 and D18 by peritumoral injection: PBS and ODN control (control groups), Imiquimod, Resiquimod, ODN 2395 alone and ODN2395+Resiquimod. A group of mice was treated with the reference treatment used for BCC which is Aldara® cream (imiquimod containing cream). Aldara® was applied topically every other day as recommended in published setting. The impact of the different treatments on tumor growth, immune cell infiltrates and T cell immunity was analyzed as detailed in the result section.

• Results

First, the impact of local treatments as detailed above on tumor outgrowth was assessed. As shown in Figure 6 A, ODN2395 treatment significantly delayed the tumor growth and is more efficient that Aldara® but these two treatments were not sufficient to induce complete tumor regression. The most potent treatment was the one combining ON2395 (TLR9L) and Resiquimod that led to a total regression of tumors in 2 of the 4 treated mice (50% efficacy) and the substantial regression of tumors in the 2 other mice. As shown in Figure 7B with the analysis of the frequency of infiltrating leukocytes after treatment, the regressing tumor of the ODN2395+Resiquimod group had a 50% increase in infiltrating leukocytes in comparison to the control group which suggests that the regression is associated with in vivo stimulation of the antitumoral immunity.

Secondly, the impact of local treatments on the frequency of infiltrating myeloid cells and on DCs maturation was analyzed. Tumors were resected at day 22 and cells dissociated enzymatically. Cell suspensions were stained with fluorescent antibodies to quantify the frequency of DCs and neutrophils as well as DCs maturation based on MHCII, CD40 and CD86 expression by flow cytometric analysis (Figure 7). As shown in Figure 8B, all the treatments increased the frequency of mature DCs and reduced the proportion of neutrophils that are supposed to be immunosuppressive cells in the tumor environment. Interestingly, the TLR9L agonist and the combined ODN2395/resiquimod treatments also upregulated the surface expression of the co-stimulatory molecule CD86 on dendritic cells in comparison to the control group. This supports that tumor regression is associated with improved stimulation of dendritic cells and consequently increased adaptive immune response.

In a third step, the impact of local treatments on frequency and phenotypes of tumor- associated lymphocytes was assessed. To that end, the following lymphocyte types were quantified by flow cytometric analysis using classical markers:

CD4 ⁺ CD3 ⁺ and CD8 ⁺ CD3 ⁺ T lymphocytes as well NKp46+CD3neg innate lymphoid cells including natural killer cells (Figure 8);

Foxp3+ regulatory CD4 ⁺ T lymphocytes (Figure 9)

As shown in Figure 8, the treatment with ODN2395 alone or combined with Resiquimod increased the proportion of CD8 ⁺ T lymphocytes. These treatments did not reduce the frequency of FoxP3+ regulatory CD4 ⁺ T lymphocytes (Figure 9). Altogether, these data strongly suggest that the combined ODN2395/resiquimod treatment and to a lesser extent the ODN2395 treatment induced the reprogramming of anti CD8 antitumor immunity by a mechanism that did not involve the control of regulatory CD4 ⁺ T cells. The ODN2395 -based treatment did not seem to modulate the proportion of NKp46 innate lymphoid cells at the time point studied.

Finally, the impact of local treatments on in vivo T cell polarization was studied. To that end, the intratumoral CD45 ⁺ leukocytes were isolated from 22-days tumors after the indicated treatments and sorted tumoral CD45 ⁺ cells or cells from draining lymph nodes (100 000 cells per well) were stimulated with anti CD3/CD28 coated Dynabeads. Cell supernatants were collected 24 hours after stimulation for cytokine assessment.

As shown in Figure 10A, ODN2395 treatment and ODN2395 together with Resiquimod strongly increase the production of intratumoral IFN-Y compared control group and Aldara group, suggesting a reprogramming of the adaptive antitumoral immunity. Nevertheless, in order to have an idea of the immunity induced in ODN2395/Resiquimod experimental mice in which tumors had completely regressed, the cytokine profiles from draining lymph nodes leukocytes were measured after anti CD3/CD28 stimulation (Figure 10B). Interestingly, inventors observed huge amounts of IFNy and TNF in all experimental mice treated with ODN2395 and Resiquimod as well as in the mice treated with ODN2395 demonstrating the impact of such treatments on the restoration of an efficient antitumoral immunity.

In summary, in vivo peritumoral treatments with ODN2395 and Resiquimod were able to induce the total regression of existing tumors in a mouse model of grafted skin tumor. In comparison, the topical treatment with Aldara® alone (imiquimod containing cream) induced only a delay in tumor growth like the peritumoral administration of ODN2395 alone.

The originality of this approach relied on the peritumoral delivery of a two TLR ligands that potentiates IL12 production and the restoration of the adaptive antitumoral immunity.

Finally, these results are in accordance with the in vitro data disclosed in example 1. From inventors' in vitro study other combinations of TLR or CLR agonists have antitumoral properties in vivo, in particular the combination of a TLR9 agonist with the TLR2/Dectin- 1 agonist zymosan.