WO2018060513A1 - Méthodes et compositions pour le traitement de lésions précancéreuses ou d'un cancer comprenant des agonistes de tlr/tlr ou tlr/clr - Google Patents

Méthodes et compositions pour le traitement de lésions précancéreuses ou d'un cancer comprenant des agonistes de tlr/tlr ou tlr/clr Download PDF

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WO2018060513A1
WO2018060513A1 PCT/EP2017/074946 EP2017074946W WO2018060513A1 WO 2018060513 A1 WO2018060513 A1 WO 2018060513A1 EP 2017074946 W EP2017074946 W EP 2017074946W WO 2018060513 A1 WO2018060513 A1 WO 2018060513A1
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agonist
tlr
composition
compound
composition according
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Fabienne Anjuere
Feriel HACINI-RACHINEL
Emmanuel Vial
Carmelo LUCI
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Galderma Research & Development
Centre National De La Recherche Scientifique
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2851—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the lectin superfamily, e.g. CD23, CD72
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants

Definitions

  • 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).
  • TLR Toll-like Receptor
  • CLR C-Type Lectin Receptor
  • 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.
  • CTL cytotoxic T lymphocytes
  • 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.
  • DCs also called “professional Antigen Presenting cells”
  • NK natural killer
  • 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).
  • 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).
  • DC PRRs Pattern Recognition Receptors
  • TLRs Toll Like Receptors
  • PAMPS pathogen-associated molecular patterns
  • RLR RNA helicase RIG-I-Like Receptor
  • CLR C-Type Like Receptor
  • NLR NOD-like Receptor
  • 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.
  • TLR-based cancer therapy strategies are known in the art.
  • 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.
  • 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).
  • 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).
  • TLR Toll-like Receptor
  • CLR C-Type Lectin Receptor
  • 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.
  • 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.
  • composition for enhancing anti-tumor immunity comprises:
  • a first compound said first compound being an agonist of a first Toll-like Receptor (TLR), and
  • 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).
  • TLR Toll-like Receptor
  • CLR C-Type Lectin Receptor
  • the first compound of the composition is a TLR9 agonist.
  • the TLR9 agonist is an oligodeoxynucleotide sequence containing one or more CpG (CpG ODN).
  • the TLR9 agonist is a Class C CpG ODN.
  • the TLR9 agonist is selected from ODN 2395, ODN M362 and DSL03.
  • the second compound present in the composition of the invention is a TLR7 and/or a TLR8 agonist.
  • the TLR7 and TLR8 agonist is Resiquimod (R848).
  • the TLR7 agonist is selected from Imiquimod (R837), CL075, CL097, CL264, CL307, GardiquimodTM, Loxoribine and Poly(dT).
  • the TLR8 agonist is selected from ssRNA40, ORN02, ORN06, ssPolyU Naked, ssPolyU, ssRNA40, ssRNA41, ssRNA-DR, CL075, CL097, Poly(dT) and TL8-506.
  • the second compound is a Dectin-1 agonist.
  • the Dectin-1 agonist is selected from a Cell wall preparation of S. cerevisiae (Zymozan), ⁇ (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.
  • composition according to the present invention for use as a drug or medicament.
  • composition according to the present invention is preferably for local administration.
  • composition according to the present invention is for intratumoral and/or peritumoral administration.
  • 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.
  • composition according to the present invention is for use for preventing or treating skin carcinoma.
  • 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).
  • SCC skin squamous cell carcinoma
  • BCC skin basal cell carcinoma
  • composition according to the present invention is for use for preventing or treating actinic keratosis and/or Bowen disease.
  • 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.
  • kits including a composition according to the present invention, typically a combination of the first and second compounds herein described, and uses thereof.
  • a method for preventing or treating a precancerous lesion and/or a cancer comprising 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).
  • SCC skin squamous cell carcinoma
  • BCC skin basal cell carcinoma
  • composition for enhancing anti-tumor immunity comprises:
  • a first compound said first compound being an agonist of a first Toll-like Receptor (TLR), and
  • 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.
  • the first TLR agonist is a TLR7, TLR8 and/or TLR9 agonist. More preferably, the first TLR agonist is a TLR9 agonist.
  • 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.
  • the second TLR agonist is a TLR7 and/or TLR8 agonist.
  • TLR1 agonist suitable for the invention is Pam3Csk4.
  • 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.
  • lipoproteins such as Pam2Csk4, Pam3Csk4, FSL-1 (Pam2CGDPKHPKSF), CL429; zymozan; lipopolysaccharide from Porphyromonas gingivalis (LPS-PG); lipoglycans such as Lipoarabinomannan from Mycobacterium smegmati
  • 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.
  • 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).
  • HKAL Heat Killed Acholeplasma laidlawii
  • HKEB Heat
  • 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 ).
  • 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 Lipopolysaccharide
  • LPS-EK LPS from Escherichia coli K12
  • LPS- B5 LPS from Escherichia coli 055 :B5
  • LPS-EB LPS from Escherichia coli 0111 :B4
  • LPS-PG LPS from Salmonella minnesota R595 (LPS-SM); Monophosphoryl Lipid A
  • MLA Salmonella minnesota R595
  • HKST HKST
  • 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.
  • TLR6 agonists suitable for the invention are FSL-1 and Pam2Csk4.
  • TLR7 agonists suitable for the invention are CL075, CL097, CL264, CL307,
  • TLR8 agonists suitable for the invention are ssRNA40, ORN02, ORN06, ssPolyU Naked, ssPolyU, ssRNA41 and ssRNA-DRCL075, CL097, Poly(dT) and TL8-506.
  • 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 LyoVecTM.
  • TLR7 and TLR8 agonist suitable for the invention is Resiquimod (R848).
  • TLR11 agonist suitable for the invention is Toxoplasma gondii Profilin.
  • TLR12 agonist suitable for the invention is Toxoplasma gondii Profilin.
  • TLR13 agonist suitable for the invention is ORN SA19/c.
  • TLR9 agonists suitable for the invention are oligodeoxynucleotides sequences containing one or more CpG (CpG ODN).
  • CpG ODNs are Class C CpG
  • Class C CpG ODNs suitable for the invention are ODN 2395, ODN
  • 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.
  • 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 3 ⁇ or 5 ⁇ group, modification of a nucleotide base, modification of the sugar component, and modification of the phosphate ring.
  • modification such as a modification of the 3 ⁇ or 5 ⁇ 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).
  • 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.
  • 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.
  • the CLR agonist is Dectin-1 agonist. More preferably, the Dectin-1 agonist is Zymozan.
  • Dectin 1 agonists suitable for the invention are ⁇ (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.
  • 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.
  • compositions for use as a drug or medicament are provided.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the administration is at the same region or a different region of the subject's body.
  • the first compound and the second compound are simultaneously co-administered in a same vehicle and peritumorally.
  • 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.
  • slow release formulations or a slow release apparatus may be used for continuous administration of the herein described composition.
  • compositions for use for treating skin precancerous lesions for example a composition for use for treating actinic keratosis and/or Bowen disease lesions.
  • the skin precancerous lesions are actinic keratosis.
  • Skin precancerous lesions targeted by the invention may also be actinic cheilitis or premalignant cutaneous horn.
  • 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
  • a method for preventing or treating a precancerous lesion and/or a cancer comprising 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).
  • SCC skin squamous cell carcinoma
  • BCC skin basal cell carcinoma
  • 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.
  • FIG. 1 shows the TLR (A) and CLR (B) transcripts levels expressed by skin-associated dendritic cells and intralesional dendritic cells.
  • 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.
  • FIG. 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.
  • 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.
  • 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.
  • 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.
  • Figure 8 shows the impact of local treatments on the proportion of tumor-associated CD4 + T cells, CD8 + T cells and NKp46 + ILC.
  • Figure 9 shows the impact of local treatments on the proportion of tumor-associated FoxP3 + CD4 + T cells.
  • 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.
  • DCs dendritic cells
  • 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.
  • agonists include acetylated lipopeptides (a TLR1/2 agonist, Pam2Csk4 used at ⁇ g/mL; a TLR2/6 agonist, Pam3Csk4 used at 2 ⁇ g/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 2 ⁇ g/mL (TLR13 agonist) and protozoan profilin- like proteins such as toxoplasma gondii profilin protein used at 3 ⁇ g/mL (recognized by TLR11 and TLR 12).
  • TLR1/2 agonist a TLR1/2 agonist
  • Pam2Csk4 used at ⁇ g/mL
  • TLR2/6 agonist Pam3Csk4 used at 2 ⁇ g
  • CpG ODNs TLR9 agonists
  • the CpG ODN 2395 has been used in the following experiments at a concentration of 5 ⁇ .
  • 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 Tehalose-6,6-dimycolate from M. tuberculosis
  • Mincle agonist was used at l( ⁇ g/mL.
  • TLR ligands or dectin-1 agonists were 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 ⁇ g/mL; Pam2CSK4 used at 1 and O. ⁇ g/mL; ORNSA19 used at 2 and O ⁇ g/mL; Profilin used at 15 ⁇ g/mL and 3 ⁇ g/mL; Curdlan used at 100 and 10 ⁇ g/mL). Secondly, their immunostimulatory potential was evaluated using intratumoral CD45 + leukocytes as illustrated in Figure 2.
  • 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).
  • 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.
  • TLR7L Imiquimod
  • Resiquimod TLR7L/TLR8L
  • Class C CpG ODN ODN 2395
  • ORN ORN SA19
  • TLR13L ORN and its ORN control
  • Pam2Csk4 TLR2/6L
  • Pam3Csk4 TLR1/2L
  • Toxoplasma gondii profilin TLR11/12L
  • ultrapure E. coli LPS strain 0111 :B4
  • TLR4L zymosan
  • TLR2 and Dectin-1 agonist used at optimal concentration.
  • 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).
  • intratumoral DCs produced huge amounts of IL10 in response of most stimuli used contrary to control DC ( Figure 3A).
  • 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).
  • 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).
  • CTL cytotoxic T lymphocytes
  • 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.
  • 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.
  • ODN2395/Resiquimod induced lower production of the immunosuppressive cytokine IL-10 that ligands alone or other combinations of TLR ligands.
  • TLR9L stimulation in combination with another TLR or with a TLR2/Dectin-1 agonist increased the production of IL12 by intratumoral leukocytes.
  • ODN2395 and Resiquimod is the one that induced the lower production of the IL-10 immunosuppressive cytokine
  • 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.
  • 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.
  • 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.
  • lymphocyte types were quantified by flow cytometric analysis using classical markers:
  • 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.

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Abstract

L'invention concerne des méthodes et des compositions permettant d'améliorer l'immunité antitumorale ou de traiter une lésion précancéreuse et/ou un cancer, ladite composition comprenant : (i) un premier composé, ledit premier composé étant un agoniste d'un premier récepteur de type Toll (TLR), et (ii) un second composé, ledit second composé étant un agoniste d'un second TLR différent du premier TLR visé au (i) et/ou un agoniste d'un récepteur de lectine de type C (CLR).
PCT/EP2017/074946 2016-09-30 2017-10-02 Méthodes et compositions pour le traitement de lésions précancéreuses ou d'un cancer comprenant des agonistes de tlr/tlr ou tlr/clr WO2018060513A1 (fr)

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