Methods for promoting t cells response by administering an antagonist of human c-type lectin-like receptor 1 (CLEC-1)

Abstract

The present invention relates to methods for promoting T cells response. The inventors examined the expression and function of CLEC-1 in human DCs and demonstrated for the first time a cell-surface expression. They investigated its functional role following triggering on orchestration of T-cell responses. The inventors showed in vitro and in vivo with CLEC-1 deficient rats and rat CLEC-1 Fc fusion protein that disruption of CLEC-1 signalling enhances in vitro Th17 activation and in vivo enhances T cell priming and Th17 and Th1 activation. In particular, the present invention relates to CLEC-1 antagonists for promoting T cells response in a subject in need thereof.

Claims

1. A method of promoting T cells response in a human subject suffering from cancer, comprising administering to the subject a therapeutically effective amount of an antagonist of human CLEC-1, wherein the antagonist is selected from the group consisting of an antibody, an antigen-binding fragment thereof, and a polypeptide that is a functional equivalent of human CLEC-1 having at least 90% identity with the amino acid sequence set forth in SEQ ID NO: 1.

2. The method of claim 1 wherein the subject suffers from a cancer selected from the group consisting of bile duct cancer, bladder cancer, bone cancer, brain and central nervous system cancer, breast cancer, Castleman disease cervical cancer, colorectal cancer, endometrial cancer, esophagus cancer, gallbladder cancer, gastrointestinal carcinoid tumors, Hodgkin's disease, non-Hodgkin's lymphoma, Kaposi's sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, liver cancer, lung cancer, mesothelioma, plasmacytoma, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oral cavity and oropharyngeal cancer, ovarian cancer, pancreatic cancer, penile cancer, pituitary cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, skin cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, vaginal cancer, vulvar cancer, and uterine cancer.

3. The method of claim 1, wherein the antibody or antigen-binding fragment is selected from the group consisting of chimeric antibodies, humanized antibodies and fully human monoclonal antibodies.

4. The method of claim 1, wherein the antibody or antigen-binding fragment thereof specifically binds to the extracellular domain of human CLEC-1.

5. The method according to claim 1, wherein the polypeptide that is a functional equivalent of human CLEC-1 is fused to an immunoglobulin constant domain.

6. The method according to claim 1, wherein the antagonist of human CLEC-1 is used in combination with a conventional treatment.

7. The method according to according to claim 1, wherein the antagonist of human CLEC-1 is used in combination with a chemotherapeutic agent, a targeted cancer therapy, an immunotherapeutic agent or radiotherapy.

8. A method of treating cancer by promoting a T cell response, in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an antagonist of human CLEC-1 selected from the group consisting of an antibody, an antigen-binding fragment thereof, and a polypeptide that is a functional equivalent of human CLEC-1 having at least 90% identity with the amino acid sequence set forth in SEQ ID NO: 1.

9. The method according to claim 8, wherein the antagonist of human CLEC-1 is used in combination with a conventional treatment.

10. The method according to according to claim 8, wherein the antagonist of human CLEC-1 is used in combination with a chemotherapeutic agent, a targeted cancer therapy, an immunotherapeutic agent or radiotherapy.

Description

FIGURES

(1) FIG. 1A-C: Expression and regulation of CLEC-1 mRNA and protein in human organs and cells.

(2) FIG. 1A) CLEC-1 mRNA expression was assessed by quantitative RT-PCR in peripheral blood leucocytes (PBL), neutrophils (neutro), monocytes (mono), monocytes derived dendritic cells (moDC), Human aortic endothelial cells (HAECs), T and B cells.

(3) FIG. 1B) Representative dot and histograms plots of IgG1 isotype or CLEC-1 staining evaluated by flow cytometry on human blood i) CD45+CD14-CD11c+ HLA-DR.sup.high DCs, ii) CD45+CD14+ monocytes iii) SSC.sup.high CD16+ neutrophils and on and in iiii) HAECs (extracellular and intracellular), with mAb anti-hCLEC-1. Histogram plot with the overlay image of CLEC-1 matching the histogram plot of cells stained with control isotype IgG1.

(4) FIG. 1C) CLEC-1 expression on human moDCs. i) Representative dot plots of % of moDCs expressing cell-surface CLEC-1 (APC) and HLA-DR (FITC) evaluated by flow cytometry in unstimulated (US) cells and cells stimulated with LPS (TLR4-L), Poly I:C (TLR3-L), R848 (TLR7-L) and TGF-β as described in Material and Methods with mAb anti-hCLEC-1. IgG1 isotype was performed as control. ii) Histogram represents Mean fluorescence intensity (MFI)±SEM of CLEC-1 staining in 6 independent experiments. Statistical analysis of CLEC-1 MFI staining was performed between US and stimulated cells.

(5) FIG. 2A-C: Effect of CLEC-1 triggering on human moDC maturation and downstream T cell activation.

(6) FIG. 2A) Human moDC incubated with nothing or with plate-bound anti-CLEC-1 mAb or IgG1 isotype control were alternatively stimulated simultaneously with LPS (1 μg/ml) for 24 hours, harvested and CD80, CD86, CD83 and HLA-DR were assessed by flow cytometry and FIG. 2B), TNF-α, IL-12p70, IL-6, IL-23 and IL-10 were assessed in supernatants by ELISA.

(7) FIG. 2C) Then, cells were washed and subjected to MLR with allogeneic T cells for 5 days. Proliferation was assessed by flow cytometry by CFSE dilution in allogeneic T cells and IL-17 and IFN-γ were assessed in supernatant by ELISA.

(8) FIG. 3A-B: Effect of CLEC-1 deficiency (CLEC-1 KO rats) on rat BMDC maturation, cytokine secretion and T-cell activation properties.

(9) BMDC were generated from WT or KO rats for 8 days as described in Material and Methods, and incubated for 4 days in MLR with allogeneic purified CD4+ T cells from naïve rats. FIG. 3A Histogram and representative staining of proliferation assessed in CD4+ T cells by CFSE dilution by flow cytometry and FIG. 3B Histogram and representative dot plots of percentage of IL-17+ and IFN-γ+ cells among gated CD4+ T cells assessed by flow cytometry. Data were expressed in histograms as mean±SEM of 4 independent experiments.

(10) FIG. 4A-B: Effect of CLEC-1 signalling blockade with CLEC-1 Fc fusion protein on rat BMDC-mediated T-cell activation.

(11) BMDCs from WT rats were incubated for 4 days in MLR with allogeneic purified CD4+ T cells from naïve rats together with CLEC-1-Fc or irrelevant hSEAP-Fc fusion proteins (produced and purified under the same conditions) (10 μg/ml). FIG. 4A Proliferation of Foxp3+ and Foxp3− cells were assessed in CD4+ T cells by CFSE dilution by flow cytometry and FIG. 4B IL-17 and IFN-γ cytokine productions were assessed in supernatants of MLR by ELISA. Data were expressed in histograms as mean±SEM of 4 independent experiments.

(12) FIG. 5A-B: Effect of CLEC-1 deficiency (CLEC-1 KO rats) on in vivo DC-mediated CD4+ T-cell priming and Th polarization fate.

(13) FIG. 5A) CLEC-1 mRNA expression was assessed by quantitative RT-PCR in different cell subtypes of rat CD103+CD4−(cross-presenting) and CD4+(non-cross-presenting) conventional DCs from secondary lymphoid organs (SLO) (n=5, *p<0.05).

(14) FIG. 5B) WT and CLEC-1 KO rats were immunized subcutaneously in the footpad with CFA plus KLH protein (100 μg/ml). At day 10 after immunization, popliteal lymph nodes were harvested and cultured in the presence of KLH or control OVA (25 μg/ml) for 3 days. Histograms and representative plots of i) Proliferation in CD4+T assessed by CFSE dilution and ii) proportion of IL-17+, IL-17+IFN-γ+ and IFN-γ+ cells among gated CD4+ T cells assessed by flow cytometry. Data were expressed in histograms as mean±SEM of 4 independent experiments.

(15) FIG. 6A-C: Effect of potential antagonist of CLEC-1 on MLR activation (moDCs+ Allo T).

(16) MLR was consisted of purified T cells isolated from peripheral blood (5×10.sup.4) mixed with allogenic monocytes derived dendritic cells (12.5×103) expressing high level of CLEC-1. Isotype control (IgG1) or anti-human CLEC-1 antibody were added at doses of 0.5 to 10 μg/ml for 5 days. Proliferation of T cells was then assessed by carboxyfluorescein succinimidyl ester dilution and IFN-γ expression assessed by flow cytometry in T cells and by ELISA in supernatants (FIGS. 6A, B and C, histograms and representative plots).

(17) FIG. 7a-d: Inhibitory checkpoint CLEC-1 in cancer (proof of concept in rodent).

(18) FIG. 7a) Rapid and long-lasting expression of CLEC-1 in solid tumors in rodent (hepa 1.6 hepatocarcinoma tumor cells intraportally injected in liver of b6 mice, Clec1a expression was assessed by Q-PCR).

(19) FIG. 7b) CLEC-1 deficient rodents struggle better against tumor than wild-type animals mice: hepa 1.6 hepatocarcinoma tumor cells intraportally injected in liver of b6 WT or Clec1a deficient mice, data are expressed in % of micesurvival Wilcoxson test ***p<0.001

(20) FIG. 7c) rats: C6 glioma cells (1 million) were subcutaneously injected in the flank of sprague dawley (spd) WT or Clec1a deficient rats and tumor volume was monitored until d35.

(21) FIG. 7d) C6 glioma cells (1 million) were subcutaneously injected in the flank of sprague dawley (spd) WT or Clec1a deficient rats and tumor and lymph nodes were harvested at day 18 and subjected to Q-PCR for inos, ifng and tnfa. Data represent histograms of expression of inos, ifng and tnfa relative to hprt and expressed in arbitrary unit (fold change compared to the expression in littermate WT rats, value of 1) for tumors (n=3) or in transcrit ratio for lymph nodes (n=6,* p<0.05)

(22) FIG. 8a-d: CLEC-1 is expressed by M2-type pro-tumoral macrophages and is expressed by myeloid cells from pleural effusion mesothelioma and from ovarian tumor ascites.

(23) Human monocytes were cultured with M-CSF to generate M0 macrophages and then with IFNγ, LPS or IL-4 to generate M1 or M2 macrophages as described by Zajac, Blood, 2013). CLEC-1 expression was assessed by Q-PCR (FIG. 8a) and by flow cytometry (FIG. 8b), n=4 *p<0.05. Pleural effusion from mesothelioma were collected and subjected to flow cytometry to assess CLEC-1 expression on CD45.sup.+ HLA DR.sup.+ CD16.sup.+ myeloid cells after the blockade of Fc receptors with FcBlock and human AB positive serum (isotype control) (FIG. 8c). Ascites are collected during the routine care of ovarian carcinoma patient and mononuclear cells are isolated after a Ficoll gradient. Human CD14.sup.+ cells are isolated using CD14 microbeads and positive selection with AutoMACS. After the blockade of Fc receptors with FcBlock and human AB positive serum, CD14.sup.+ cells were stained with either an isotype control monoclonal antibody or an anti-human CLEC-1 (FIG. 8d).

EXAMPLE 1

(24) Material & Methods

(25) Animals

(26) Rats were purchased from the “Centre d'ElevageJanvier” (Genest, Saint-Isle, France) and experimental procedures were carried out in strict accordance with the protocols approved by the Committee on the Ethics of Animal Experiments of Pays de la Loire and authorized by the French Government's Ministry of Higher Education and Research. Clec-1 knock out (KO) were generated by the Transgenic Rats and Immunophenomics Platform facility (SFR-Santé-Nantes) with the zinc finger nucleases (ZFN) technology in the inbred RT1a Lewis background. Absence of CLEC-1 protein at the expected size of 32 kDa was confirmed by western blot.

(27) Antibodies

(28) Anti-human CLEC-1 monoclonal Abs (mAb) was generated by lymphocyte somatic hybridization (Biotem, Apprieu, France) by immunisation of Balb/c mice with a peptide encoding the extracellular domain of hCLEC-1 and selected by screening on recombinant human CLEC-1 protein (RD system) by ELISA and then purified by chromatography on protein A. Anti-human CLEC1 mAb (IgG-D6) was from Santa Cruz Biotechnologies (Dallas, Calif.). Purified anti-rat-β-actin, CD3 (G4.18); anti-rat TCRαβ-A647 or -A488 (R73), CD4-PECy7 (OX35), CD8-A488 (Ox8), IL-17-APC (ebio17B7), Foxp3-APC, IFNγ-FITC, CD11b-PerCP-Cy5.5 (WT.5), CD103 (αE Integrin)-FITC and anti-human phosphotyrosines (p-Tyr) (4G10), CD4-PE, CD3-APC, CD45-PercP, CD3-FITC, CD19-PE, CD16-PE, CD14-FITC HLA-DR-APC/Cy7, HLA-DR-FITC, CD11c-PECy7, CD11b-FITC CD80-FITC, CD86-FITC, CD83-FITC and IgG1 isotype control were from BD Biosciences (Franklin Lakes).

(29) Generation of Clec-1.sup.−/− Knock-Out (KO) Rats by Zinc Finger Nuclease (ZFN) Technology.

(30) In vitro-transcribed mRNA-encoding ZFN-targeted sequences specific for rat clec-1 (Sigma-Aldrich, St Louis, Mo.) were microinjected in fertilized one-cell stage embryos as previously described. Mutations in newborn were detected by PCR. One of the founders that presented a 7 bp deletion leading to a premature stop-codon at the 114 amino-acid of CLEC-1 lacking most of the extracellular domain. Heterozygotes were subjected to breeding to generate KO and wild-type (WT) littermates.

(31) Generation of Rat CLEC-1 Fc Fusion Protein.

(32) The cDNA encoding the extracellular domain of CLEC-1 (ADK94891 amino acids 74-261), was amplified by PCR and the 5′ and 3′ ends tagged with ECORI BglII restriction sites, respectively. Following digestion, cDNA products were cloned and insert in-frame into pFUSE-mIgG2Ae1-Fc2 v10 [Fab] (Invivogen, San Diego, Calif.) vector containing IgG2a Fc fragment mutated on 3 amino-acid to prevent FcγRI binding. Plasmids were transfected in eukaryote cells with lipofectamine according to the manufacturers' instructions (ThermoFisher). CLEC-1 Fc was purified from supernatant with HiTrap g affinity column (GE Healthcare Bio-sciences, Pittsburgh, Pa.), dialysed using a Slide-A-Lyzer dialysis cassette (ThermoFisher) and quantified using BCA Protein Assay Reagent Kit (Pierce). Purity and protein structure was confirmed by SDS-PAGE followed by Coomassie staining and western blot analysis with anti-mouse IgG or anti-rat CLEC-1 antibody as described in western blot section of supplemental Materials and Methods. A control recombinant secreted truncated form of a human embryonic alkaline phosphatase (hSEAP Fc) was generated (pFUSE-SEAP-hFc, Invivogen) and purified under the same conditions than CLEC-1 Fc.

(33) KLH Immunization

(34) Rats were immunized sc. in the footpad with keyhole limpet hemocyanin (KLH) protein (Sigma) (100 μg) emulsified (v:v) in 100 μl of Complete Freund Adjuvant (CFA) (Difco) and the popliteal lymph nodes were harvested 10 days after immunization. Carboxyfluoresceinsuccinimidyl ester (CFSE) (Molecular Probes/Invitrogen)-labelled (5 μM) total cells (1×10.sup.5) or purified CD4.sup.+ T cells (1×10.sup.5) plus T-cell depleted splenocytes (1×10.sup.5) from naïve WT rats were subjected to in vitro secondary challenge with KLH or irrelevant protein OVA (25 μg/ml) for 3 days.

(35) Flow Cytometry and Cell Sorting

(36) Before staining, cells were subjected to Fc block (BD Biosciences) as described by Manufacturer instructions. For intra-cellular cytokine staining, cells were stimulated for 4 hours with PMA and ionomycin (50 ng/ml and 1 μg/ml respectively) in the presence of the protein transport inhibitor GolgiStop (2 μl/well) and subjected to fixation and permeabilization (Facspermeabilizing solution) (all reagents from BD Biosciences). Fluorescent labelling of stained cells (2.5 μg/ml) was measured using a FACS LSR II (BD Biosciences) and analyzed with FlowJo® software (Tree Star, Inc., Ashland).

(37) For cell sorting, rat CD4.sup.+ T cells and CD11b.sup.+CD103.sup.+CD4.sup.− and CD11b.sup.+CD103.sup.+CD4.sup.+ DCs were purified from spleen of naïve rats by positive selection using a FACSAria flow cytometer (BD Biosciences) by TCR.sup.+ and CD4.sup.+ and by CD11b, CD103 and CD4 staining respectively and for human cells by SSC.sup.lowCD45.sup.+CD3.sup.+ or CD19.sup.+ for T and B cells respectively, SSC.sup.highCD45.sup.+CD16.sup.+ for neutrophils, SSC.sup.lowCD45.sup.+CD14.sup.+ for monocytes and SSC.sup.lowCD45.sup.+HLA-DR.sup.+CD11c.sup.+, for cDCs. Purity was >99%.

(38) Dead cells were excluded by gating on 4′,6-diamidino-2-phenylindole (DAPI)-negative cells.

(39) Cells Generation, In Vitro Stimulation and Mixte Leukocyte Reaction (MLR)

(40) Human monocyte-derived DCs (moDCs) were generated from elutriated monocytes cultured for 7 days in complete RPMI 1640 medium (10% endotoxin-free FCS (Perbio Sciences), 2 mM L-glutamine (Sigma), 1 mM sodium pyruvate (Sigma), 1 mMHepes (Sigma), and 5×10.sup.−5 M 2-mercaptoethanol (Sigma)), supplemented with IL-4 (40 ng/ml; AbCys, Paris, France) and GM-CSF (1000 IU/ml; AbCys). Then, cells were stimulated for 24 h (1×10.sup.6/ml) with LPS (0.5 μg/ml) (Sigma), Poly I:C (2 μg/ml) (Invivogen, San Diego, Calif.), R848 (2.5 μg/ml) (Invivogen), recombinant human TGFβ1 (20 ng/ml) (R&D systems) alternatively in the presence of 10 μg/ml of anti-CLEC-1 mAb or IgG1 isotype control previously coated to plates) and were subjected to flow cytometry or cultured (12.5×10.sup.3) with 5×10.sup.4 allogeneic human T cells (Pan T Cell Isolation kit (Mylteni)) for 5 days (MLR). Recombinant CLEC-1-His tag (R&D system) or irrelevant recombinant pig alpha1,3GT-6-His proteins were added at 10 μg/ml in MLR. Proliferation was measured by flow cytometry by CFSE profile in CD3.sup.+CD4.sup.+ cells and IL-17 and IFN-γ cytokines assessed in supernatants by ELISA.

(41) Human endothelial cells (ECs) from aorta (HAEC) or from Umbilical Vein (HUVEC) were isolated and cultured and were alternatively stimulated with 1000 units/ml of recombinant human TNFα (eBiosciences) for 12 hours. Human monocytes were cultured with M-CSF to generate M0 macrophages and then with IFNγ, LPS or IL-4 to generate M1 or M2 macrophages as described by Zajac, Blood, 2013). Pleural effusion from patients with mesothelioma were collected and subjected to flow cytometry Ascites are collected during the routine care of ovarian carcinoma patient and mononuclear cells are isolated after a Ficoll gradient. Human CD14.sup.+ cells are isolated using CD14 microbeads and positive selection with AutoMACS.

(42) Rat CD4.sup.+ T cells were stimulated with plate-bound anti-CD3 (Clone G4.18) (5 μg/ml). Bone Marrow-derived DCs (BMDCs) from naïve, CLEC-1 WT and KO LEW.1A (RT1a) littermates' rats were obtained by culturing cells for 8 days in complete RPMI medium supplemented with rat IL-4 (4 ng/ml) and murine GM-CSF (1.5 ng/ml). Then, BMDCs were stimulated with LPS (1 μg/ml) (Sigma) or zymozan (20 μg/ml) (Invivogen) for 6 hours for transcript analysis and for 24 hours for expression of maturation markers and for MLR (co-culture of 5 days with purified allogeneic CD4.sup.+ T cells from mesenteric lymph nodes of LEW.1W (RT1u) naïve rats labelled with 5 μM CFSE). Alternatively, rat CLEC-1 Fc fusion protein and control hSEAP-Fc (10 μg/ml) were added in MLR in the presence of endotoxin inhibitor polymyxine B (10 μg/ml)(Invivogen).

(43) RNA Extraction and Real-Time Quantitative RT-PCR

(44) Total RNA from tissues, tumors or cells was prepared using Trizol (Invitrogen) according to the manufacturer's instructions. cDNA from pooled human organs were from Human Immune System and MTC Panel I from male or female Caucasians (Clontech Mountain View). Real-time quantitative PCR was performed using the ViiA 7 Real-Time PCR System and SYBR® Green PCR Master mix (Applied Biosystems). Hypoxanthine-guanine phosphoribosyltransferase (HPRT) was used as an endogenous control gene to normalize for variations in the starting amount of RNA. Relative expression was calculated using the 2.sup.−ΔΔCt method and expressed in arbitrary units.

(45) Immunoprecipitation and Western Blot

(46) Human moDCs were plated on anti-CLEC-1 or control IgG1 isotype (Invitrogen) mAb coated plates (10 μg/ml) for 5 or 20 minutes in medium in conjunction or nor with zymozan (20 μg/ml). Human moDCs, HAEC, HUVEC and HEK were lyzed in Nonidet P-40 1% lysis buffer with protease inhibitors cocktail (Sigma Aldrich). CLEC-1 immunoprecipitation was performed with 4 μg of anti-human CLEC1 mAb (D6) followed by incubation with protein G-Sepharose beads. Proteins were then treated overnight with PNGase F (Sigma Aldrich), eluted and dissolved by boiling for 5 min in Laemmli sample buffer. Protein concentration was determined using the BC assays kit with BSA as standard (Interchim, San Pedro). Nitrocellulose membranes were blocked with Tween-20-Tris buffered saline and 5% milk and incubated with 0.5 μg/ml of antiphosphotyrosine (4G10) or 2 μg/ml of anti CLEC1 mAbs, followed by horseradish peroxidase-conjugated secondary Abs (Jackson immunoresearch, West Grove, Pa.). Proteome Profiler Human NF-κB Pathway Array Kit was performed as described by Manufacturer's instruction (R&D System). Detection by chemiluminescence was performed using West Pico chemiluminescence substrate (Thermofisher scientific, Waltham, Mass.) and protein expression assessed by Las 4000 (Fuji).

(47) Immunohistochemistry

(48) Neutrophils, moDCs, adherent transfected HEK293T cells and HUVEC cells (cultured on a coverslip overnight) were fixed in 4% paraformaldehyde (Electron Microscopy Science, Hatfield, Pa., USA) and permeabilized except for moDCs with Triton ×100 (0.1%). Cells were stained with anti-CLEC1 mAb (D6) or IgG1 isotype control (Invitrogen) (4 μg/ml) for 1 h at room temperature in PBS 1% FCS, 1% BSA and Fc Block for moDCs and then with secondary Alexa Fluor 488 or Alexa Fluor 568 anti-mouse IgG1 antibodies, 1 h. After 10 min incubation in PBS containing 1% DAPI, slides were mounted using Prolong Antifade Reagent (Invitrogen) and observed by fluorescence microscopy (Nikon A1 R Si Confocal microscope). Images were obtained (X60 Plan Apo N.A: 1.4, zoom 2) with sequential mode and analyzed by using ImageJ program.

(49) In Vivo Tumor Models

(50) hepa 1.6 hepatocarcinoma tumor cells were intraportally injected in liver of b6 WT or Clec1a deficient mice. C6 glioma cells (1 million) were subcutaneously injected in the flank of sprague dawley (spd) WT or Clec1a deficient rats.

(51) Statistical Analysis

(52) All statistical analyses were performed using Graphpad Prism software (La Jolla) with two-tailed unpaired nonparametric Student's t test (Mann-Whitney). Results were considered significant if p values were <0.05.

(53) Results

(54) Human Myeloid DCs Express CLEC-1 at the Cell-Surface.

(55) Only limited information has been published so far on CLEC-1 expression in human. Very few has been described so far on human CLEC-1 protein expression, regulation and function. There is solely one publication about CLEC-1 expression which discloses that human CLEC-1 could only be detected intracellularly in endothelial cells with a staining pattern resembling endoplasmic reticulum proteins and that neither TGF-β nor inflammatory stimuli could promote significant translocation to the cell surface (The human C-type lectin-like receptor CLEC-1 is upregulated by TGF-β and primarily localized in the endoplasmic membrane compartment. Sattler et al., ScandJImmunol. 2012 March; 75(3):282-92). Thus, the only information available in the state of the art on hCLEC-1 (ie, intracellular localisation in endothelial cells) is contrary to what was known in the rat (i.e; rCLEC-1 localised on the surface of endothelial and myeloid cells).

(56) The inventors observed by quantitative RT-PCR a strong expression of CLEC-1 transcripts in lung and placenta and a more moderate expression in lymphoid organs such as thymus, lymph nodes, spleen and tonsils (data not shown). In human cell subtypes, abundant CLEC-1 transcripts were found in neutrophils, monocytes, moDCs and HAECs (FIG. 1A) and no transcript was detected in T and B cells. Presence of CLEC-1 protein in moDCs and ECs was confirmed by CLEC-1 immunoprecipitation followed by western blot (data not shown) and contrasts with the low quantity observed in epithelial HEK cells. The inventors generated a mouse anti-human CLEC-1 mAb directed against the extracellular domain and observed a low ectopic expression of CLEC-1 at the cell-surface of transfected HEK cells corroborating what was previously described in the literature (data not shown). As for other CLRs, CLEC-1 may require other adaptor chains, other PRRs or sufficient glycosylation for efficient expression, transport and cell-surface stability. Cell-surface expression of CLEC-1 protein was confirmed by immunohistochemistry in transfected cells (data not shown).

(57) With the generated mAb, they demonstrated by flow cytometry for the first time to our knowledge the cell-surface expression of CLEC-1 on a subpopulation of human blood circulating myeloid CD16.sup.− DCs (CD45.sup.+CD14.sup.−HLA-DR.sup.highCD11c.sup.+) and on CD14.sup.+CD16.sup.+ monocytes (CD45.sup.+CD14.sup.+CD16.sup.+). Neither cell-surface expression was observed on BDCA3.sup.+ myeloid DC subpopulation (BDCA3.sup.+CD45.sup.+HLA-DR.sup.highCD11c.sup.low) nor on CD123.sup.+ plasmacytoid DCs (CD123.sup.+CD11c.sup.−HLA-DR.sup.high) (data not shown). Low expression of CLEC-1 was observed at the cell-surface of neutrophils or HAECs for which expression is as previously reported mostly intra-cellular (FIG. 1B). The same localization of CLEC-1 protein was observed by immunohistochemistry with an intra-cellular CLEC-1 staining in human neutrophils and ECs and a cell-surface expression on DCs (data not shown). Importantly, similarly to what we previously described in rat, the inventors observed that CLEC-1 expression in human is down-regulated on DCs by inflammatory stimuli such as TLR ligands and up-regulated by TGFβ (representative dot plots and MFI histogram, FIG. 1C respectively).

(58) CLEC-1 Triggering on Human moDCs Suppresses In Vitro Downstream Allogeneic Th17 Activation.

(59) As CLEC-1 natural ligands have not yet been identified, the inventors used anti-human CLEC-1 mAb to mimic the ligand and cross-link CLEC-1 at the cell-surface of moDCs. Following CLEC-1 immunoprecipitation in low stringent conditions, they observed by western blot no tyrosine phosphorylation at the expected size of CLEC-1 (32 kDa) after CLEC-1 triggering suggesting that tyrosine motif in the cytoplasmic tail is not directly phosphorylated (data not shown). Nevertheless, we observed several changes in tyrosine phosphorylation patterns with enhanced or decreased phosphorylation of several bands around 40-50 kDa in size strongly suggesting that CLEC-1 is a functional receptor that signal via binding partners remaining to be identified.

(60) The inventors then investigated whether CLEC-1 triggering modulates TLR-induced maturation of moDCs as it is described for other activating or inhibitory CLRs. They observed that CLEC-1 triggering neither induces by itself nor potentiates or suppress the LPS-induced maturation state of moDCs according to the expression of the activation markers CD80, CD86, CD83 and HLA-DR (FIG. 2A) and to the production of TNFα, IL-12, IL-6, IL-23 or IL-10 (FIG. 2B). Similar results were observed with other TLR ligands (data not shown).

(61) Then, the inventors evaluated the effect of CLEC-1 triggering on the capacity of moDCs to polarize a downstream allogeneic T cell response. No difference in the subsequent allogeneic T cell proliferation was observed following CLEC-1 triggering alone (FIG. 2C) or in combination with TLRs (data not shown). However, they denoted significantly less of IL-17 and more of IFNγ secreted by allogeneic T cells suggesting that CLEC-1 triggering on moDCs have reduced subsequent allogeneic Th17 activation and skewed the response toward a Th1 one (FIG. 2C).

(62) Given that CLR signaling led to activation of NF-κB, they investigated the level and activation of NF-κB pathway related proteins by Proteome Profiler following CLEC-1 triggering alone or in conjunction with zymosan, an agonist of both DECTIN-1 and TLR signaling pathways. The inventors observed that in contrast to zymozan, CLEC-1 triggering does not induce by itself activation of the NF-κB pathway evaluated by the degradation of the NF-κB inhibitor, IκBα, and by the phosphorylation of the RelA p65 (Ser529) subunit (data not shown). However, they denoted that conjunction of CLEC-1 triggering have reduced the degradation of IκBα induced by zymozan. However, no significant reduction on phosphorylation of the p65 subunit was observed. Since phosphorylation at the Ser529 is IKKβ independent, these data suggest that CLEC-1 may inhibit particularly the IKKβ activation pathway. Collectively, these data suggest that CLEC-1 triggering on human moDCs is functionally active and that although we observed no significant effect on cytokine production, may regulate NF-κB signaling pathways induced by PRRs to finely modulate their activation state and suppress downstream Th17 response.

(63) Disruption of CLEC-1 Signaling in Rat BMDCs Enhances In Vitro T Cell Responses.

(64) To gain insight into the function of CLEC-1, the inventors generated CLEC-1 deficient rats. CLEC-1 deficient rats were viable, healthy and were born from heterozygote breeding with the expected Mendelian frequency. At steady-state, CLEC-1 deficient rats exhibited regular myeloid and lymphoid immune cell compartments in blood and peripheral lymphoid organs (data not shown).

(65) The inventors generated BMDCs from CLEC-1 deficient rats and observed that these cells differentiate and maturate normally in response to LPS stimulation (CD80, CD86, Class I and II MHC) (data not shown). Interestingly, they observed that following activation by LPS or zymosan, CLEC-1 deficient BMDCs expressed higher level of IL-12p40 transcripts than BMDCs from wild-type rats (data not shown). No significant difference was observed for IL-6, IL-23, TGFβ and IL-10 expression. However, impressively, CLEC-1 deficient BMDCs induced an enhanced proliferation of allogeneic CD4.sup.+ T cells that was associated with an increased activation of Th17 T cells (FIG. 3).

(66) To further confirm these data, the inventors generated rat CLEC-1 Fc fusion protein (data not shown). This fusion protein consisted of the extracellular domain of rat CLEC-1 fused to IgG Fc fragment mutated on 3 amino-acid to prevent FcγRI binding, should block CLEC-1 interactions on BMDCs with its putative ligands and thus mimic CLEC-1 deficiency. Similarly, they observed in the presence of CLEC-1 Fc fusion protein in the in vitro MLR, a more prominent proliferation of non-Foxp3 allogeneic effector T cells and more Th17 activation (FIG. 4). Importantly, no induction of Th-17 was observed with CLEC-1 Fc on purified T cells following anti-CD3 polyclonal activation (data not shown) demonstrating that this effect was specific to CLEC-1 signaling disruption in BMDCs and not due to ligation of CLEC-1 Fc and agonist effect on a putative ligand on T cells.

(67) Taken collectively, these data suggest that the absence of CLEC-1 signaling in myeloid cells, notably in DCs enhanced their activation state required for in vitro efficient T cell proliferation and activation.

(68) CLEC-1 Deficiency Enhances In Vivo DC-Mediated T Cell Response.

(69) The inventors then investigated the potential function of CLEC-1 in vivo in DC-mediated Th differentiation following immunization by subcutaneous injection with the foreign antigen keyhole limpet hemocyanin (KLH) and complete Freund adjuvant. First, they evaluated CLEC-1 transcripts expression in different subtypes of cDCs CD103.sup.+ CD11b.sup.+ in lymph nodes. Interestingly, they observed that CLEC-1 expression is restricted to CD4.sup.− DCs corresponding to CD8α.sup.+ DC in mice specialized for the phagocytosis of dead cells and that exhibit cytotoxic activity (FIG. 5A).

(70) Following immunization, they observed after in vitro secondary challenge of draining lymph node, an increased proliferation of KLH-specific CD4.sup.+ T cells from immunized CLEC-1 deficient rats that was associated with an increased number of IL-17.sup.+, IL-17.sup.+IFNγ.sup.+ and IFNγ.sup.+ CD4.sup.+ T cells (histogram and representative dot plots, FIG. 5B). Importantly, enhanced recall response to KLH was also observed with purified CD4.sup.+ T cells from immunized CLEC-1 deficient rats in the presence of DCs from wild-type rats demonstrating a specific increase in the in vivo T cell priming in the absence of CLEC-1 (data not shown).

(71) These data demonstrate in vivo that the deficiency of CLEC-1 signaling in cDCs exacerbates downstream T cell-mediated immune response.

DISCUSSION

(72) In this study, the inventors demonstrated for the first time to our knowledge, that human CLEC-1 is a functional DC cell-surface regulatory receptor that suppresses subsequent effector Th17 response. Moreover, CLEC-1 deficient rats revealed an in vivo role for CLEC-1 in prevention of excessive DC-mediated CD4.sup.+ T cell priming. As in rat, they observed that CLEC-1 expression in human moDCs is decreased by inflammatory stimuli and is up-regulated by TGFβ. This profile of expression in DCs with a decrease following inflammatory stimulation represents a classic response observed for other inhibitory receptors such as MICL or DCIR that have also been shown to suppress in vivo T cell responses and inflammation (Uto T. et al. Clec4A4 is a regulatory receptor for dendritic cells that impairs inflammation and T-cell immunity. Nat Commun. 2016; 7:11273) (Redelinghuys P, et al. MICL controls inflammation in rheumatoid arthritis. Ann Rheum Dis. 2015). Interestingly, the inventors found that CLEC-1 is expressed in rat by the CD4.sup.+ subpopulation of cDCs in lymphoid organs. This subpopulation of DCs correspond to CD8α.sup.+ DCs mice counterparts involved in cytotoxic activity and phagocytosis of dead cells and that is described to be the main producers of IL-12 and involved in cross-presentation of tumor antigens. This pattern of expression contrast with the inhibitory receptor DCIR-2 that has been shown to be restricted to CD8α.sup.−cDCs. Nevertheless, in human blood, CLEC-1 was observed to be expressed at cell-surface on a subpopulation of CD16.sup.−CD14.sup.− myeloid DCs and not on BDCA3.sup.+ DCs, the human counterparts of CD8α.sup.+ DCs. This discrepancy between human and rodent warrants further investigation. The inventors observed that disruption of CLEC-1 signaling enhances particularly DC-mediated Th17 activation in vitro but both Th1 and Th17 responses were increased following immunization in vivo. This suggests that CLEC-1 may differently suppress Th1 and Th17 responses according to co-engagement of PRRs. Conversely, DECTIN-1 that acts as an activating receptor in DCs, differently promotes the Th17/Th1 balance according to the ligands and PRR co-engagement by finely regulating the secretion of the polarizing cytokines IL-12 and IL-23 (Gringhuis S I, et al. Dectin-1 directs T helper cell differentiation by controlling noncanonical NF-kappaB activation through Raf-1 and Syk. Nat Immunol. 2009; 10(2):203-213) (LeibundGut-Landmann S, et al. Syk- and CARDS-dependent coupling of innate immunity to the induction of T helper cells that produce interleukin 17. Nat Immunol. 2007; 8(6):630-638) (Lee E J, et al. Mincle Activation and the Syk/Card9 Signaling Axis Are Central to the Development of Autoimmune Disease of the Eye. J Immunol. 2016; 196(7):3148-3158). For example, in response to Aspergillus Fumigatus challenge, DECTIN-1 was shown in mice to potentiate Th17 differentiation notably by decreasing IFN-γ and IL12p40 expression thereby decreasing Th1 polarization. Intriguingly, the inventors did not observe that CLEC-1 signaling in DCs suppress the PRR-induced expression of Th17 polarizing cytokines and noted only an effect on IL-12p40 production on rat KO BMDCs. These results suggest that CLEC-1 in DCs may shape the Th17/Th1 balance by other mechanisms than the expression of polarizing cytokines. For example, DECTIN-1 signaling has been shown to influence T cell polarization fate by also modulating the expression of the costimulatory molecules Ox40 ligand on DCs (Joo H, et al. Opposing Roles of Dectin-1 Expressed on Human Plasmacytoid Dendritic Cells and Myeloid Dendritic Cells in Th2 Polarization. J Immunol. 2015; 195(4):1723-1731). Interestingly, the inventors observed that CLEC-1 triggering on human DCs prevent the IκBα degradation induced by DECTIN-1 signaling. Therefore, CLEC-1 may also prevent the Card9 signaling pathway mediated by activating CLRs and known to specifically sustain Th17 response.

(73) They have not been able to detect with CLEC-1 Fc fusion protein the cells expressing endogenous ligands. Nevertheless, their data suggest that CLEC-1 ligands may be expressed by hematopoietic cells or released “naturally” or in particular context of tissue or cell damage. Ligands may be expressed by the CLEC-1 expressing cells themselves as it is the case for DCIR-2 or alternatively on T cells. Endogenous DECTIN-1 ligand has been reported to be expressed by T cells that in contrast to CLEC-1 acts as a costimulatory molecule enhancing T cell proliferation (Ariizumi K, et al. Identification of a novel, dendritic cell-associated molecule, dectin-1, by subtractive cDNA cloning. J Biol Chem. 2000; 275(26):20157-20167).

(74) These findings establish the relevance of CLEC-1 in DCs in the tight control of the degree and quality of downstream T cell activation and as a cell-surface receptor may provide a therapeutic tool to manipulate T cell response.

(75) Therefore, CLEC-1 as a cell-surface inhibitory receptor in myeloid cells highlights a potential target for therapeutic intervention and new treatment paradigm in cancer.

(76) Several experimental studies demonstrate that CLRs contribute to cancer progression and metastatic spread by their function in cell-adhesion or in T-cell response shaping ((Yan, Kamiya et al. 2015; Ding, Yao et al. 2017). For example, the immunomodulatory receptors DC-SIGN, MINCLE, DCIR and BDCA-2 have been shown to inhibit myeloid cell activation, inflammation and be critical to drive Foxp3.sup.+ CD4.sup.+ CD25.sup.+ Tregs expansion (Yan, Kamiya et al. 2015; Ding, Yao et al. 2017). DC-SIGN recognize carcinoembryonic antigen overexpressed on almost all human carcinoma (Nonaka, Ma et al. 2008) and promotes the secretion of the immunosuppressive cytokines IL-10 and IL-6 by myeloid cells. Besides, polymorphisms in DC-SIGN gene promoter were found to be associated with increased risk in colorectal cancer patients (Lu, Bevier et al. 2013). MINCLE was shown to be enhanced in tumor infiltrating leukocytes in pancreatic ductal adenocarcinoma and especially by myeloid suppressive cells (MSCs). Ligation of MINCLE with SAP130 (a subunit of the histone deacetylase complex) released from dying cells induces strong peri-tumoral suppression (Seifert, Werba et al. 2016). Similarly, the CLR LOX-1 has been shown to be specifically enhanced at the cell surface of blood or tumor-infiltrating neutrophils (15 to 50%) in cancer patients whereas is nearly undetectable in blood of healthy donors (Condamine, Dominguez et al. 2016). In this study, they showed that endoplasmic reticulum stress induces LOX-1 expression and convert neutrophils to MSCs with strong suppressive function.

(77) Conversely, triggering signaling of activating CLR such as DECTIN-1, has been shown to mount anti-tumor immunity and to decrease Tregs and MSCs (Tian, Ma et al. 2013). Administration of beta-glucans, a ligand of DECTIN-1 inhibits tumor growth in murine carcinoma models (Li, Cai et al. 2010; Masuda, Inoue et al. 2013; Tian, Ma et al. 2013), in human melanoma, neuroblastoma, lymphoma xenograft models (Modak, Koehne et al. 2005) as well as in human ovarian and gastric cancer (Inoue, Tanaka et al. 1993; Oba, Kobayashi et al. 2009).

(78) Therefore, enhancing DC or more broadly myeloid cell activation by CLEC-1 antagonist may represent an immune checkpoint target to modulate downstream effector T-cell immune that could have important clinical implication in cancer.

EXAMPLE 2

(79) The inventors previously showed in rat that CLEC-1 blockade with CLEC-1 Fc fusion protein enhance T cell proliferation in a mixed leukocyte reaction (MLR) (see EXAMPLE 1). They have generated several mAbs directed against the extra-cellular part of human CLEC-1 and they show that one mAbs appears antagonist of CLEC-1 signalling and thus enhances T cell proliferation and IFN-γ production in mixte leukocyte reaction (MLR). MLR was consisted of purified T cells isolated from peripheral blood (5×10.sup.4) mixed with allogenic monocytes derived dendritic cells (12.5×10.sup.3) expressing high level of CLEC-1. Isotype control (IgG1) or anti-human CLEC-1 antibody were added at doses of 0.5 to 10 μg/ml for 5 days. Proliferation of T cells was then assessed by carboxyfluorescein succinimidyl ester dilution and IFNγ expression assessed by flow cytometry in T cells and by ELISA in supernatants (FIGS. 6A and 6B, 6C histograms and representative plots).

EXAMPLE 3

(80) CLEC-1 is Highly Expressed in Tumors and Plays a Functional Role in Tumor Immunity

(81) In a mouse hepatocarcinoma model (intraportal injection of hepa 1.6 tumor cells), the inventors observed an increased and long-lasting expression of CLEC-1 in tumors (FIG. 7a). Importantly, in this model, mice deficient in CLEC-1 (kindly gifted by Derrick J. Rossi, Harvard Stem Cell Institute, Cambridge) are better resistant to tumor growth and exhibit an increased survival rate (median survival 28 d in KO versus 21 d in WT) (FIG. 7b). Similarly, in a subcutaneous (sc.) rat glioma model (C6), the inventors observed total regression of tumors in 3 out of 5 of CLEC-1 deficient rats (generated by ZFN technology in our lab) (FIG. 7c). Importantly, by Q-PCR in tumors harvested at day 18 after tumor cell inoculation, a higher mRNA expression of inos, tnfa and ifng were assessed in tumor from CLEC-1 deficient rats (FIG. 7d). In addition, a higher mRNA expression of ifng was detected in lymph nodes (FIG. 7d). These data demonstrate a better anti-tumor response in CLEC-1 deficient rats and suggest that the absence of CLEC1 have induced more anti-tumoral M1 macrophages and more cytotoxic and Th1 T cells.

(82) CLEC-1 Expression is Restricted to cDCs Specialized in Cross Presentation

(83) Interestingly, the inventors observed in both rat and mice that CLEC-1 expression by cDCs from secondary lymphoid organs is restricted to the specific subset of DCs specialized in the cross presentation of antigens (CD4+ in rat, CD8α+ in mice) (FIG. 5A). These cells were shown to secrete high level of IL-12 and to be responsible for the activation of anti-tumor cytotoxicCD8+ T lymphocyte (CTL) response. Therefore, by acting as an inhibitory receptor in the specialized subsets of DCs, CLEC-1 triggering may prevent IL-12 production, efficient cross presentation and CTL response towards tumor antigens.

(84) CLEC-1 is Expressed by M2-Type Pro-Tumoral Macrophages and is Expressed by Myeloid Cells from Pleural Effusion Mesothelioma and from Ovarian Tumor Ascites.

(85) The inventors observed higher CLEC-1 expression at both transcripts (a) and protein level (b) on human M2-type protumoral macrophages compared to M1-type anti-tumoral macrophages or compared to M0 macrophages (FIG. 8). Furthermore, they observed CLEC-1 cell-surface protein expression on human myeloid CD45+ HLADR+ CD16+ cells from pleural effusion mesothelioma (c) and on CD14+ myeloid cells from ovarian carcinoma ascites (d) compared to control isotype (FIG. 8). These data demonstrate that CLEC-1 expression is enhanced on myeloid cells in tumor microenvironment and could play a critical role in tumor immune escape.

(86) In conclusion, in view of the results above (in particular results showing that CLEC-1 Fc fusion protein and antibodies directed against the extra-cellular part of human CLEC-1 enhance T cell proliferation), it appears credible that CLEC-1 antagonists may be used for the treatment of cancer.

REFERENCES

(87) Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.