Identification of CLEC-1 ligands and uses thereof

Abstract

The present invention relates to the use of compounds that reduce signalling pathways induced by CLEC-1 / TRIM21 interaction, in particular compounds that antagonise the interaction, in particular the binding between CLEC-1 and TRIM21, the use of the identified ligand TRIM21 in identifying cells suspected of interacting with cells expressing CLEC-1, for designing treatments in patients and their use in therapy.

Description

[Technical field]

[0001] The present invention arises as a result of the identification of ligands for the CLEC-1 protein, in particular TRIM21. The invention relates to the use of compounds which reduce signalling pathways induced by CLEC-1 / TRIM21 interaction, in particular compounds which antagonise the interaction, in particular the binding between CLEC-1 and TRIM21, the use of the identified ligand TRIM21 in identifying cells suspected of interacting with cells expressing CLEC-1, for designing treatments in patients, and their use in therapy.

[0002] The present invention also relates to methods which allow the detection of the interaction between CLEC-1 and TRIM21 to assess the antagonist capability of compounds for the CLEC-1 / TRIM21 signalling pathway or to screen for compounds capable of binding to CLEC-1 or TRIM21, in particular compounds capable of competing with the binding between CLEC-1 and TRIM21. [Background technology]

[0003] One of the problems in the treatment of cancer is to destroy tumor cells without damaging healthy tissues. In recent years, the main problem in the treatment of cancer is to target tumor cells without damaging healthy tissues. Antitumor immunotherapy has gained increasing interest in the past decade, as it redirects the patient's immune system to destroy cancer cells. It has been clearly shown that tumor cells exploit immune checkpoints to evade immune defenses. The important role of these inhibitory checkpoints in thwarting antitumor immunity is illustrated by the remarkable success of antibody-driven blockade of T cell immune checkpoints. However, a significant proportion of patients do not respond to these checkpoint inhibitor (CKI) therapies or develop resistance to them, generally after failure of typical chemotherapy. These patients need effective new treatments, and myeloid cells represent a promising therapeutic target. In fact, myeloid cells are the most abundant tumor-infiltrating immune cells. C-type lectins (CLECs) expressed on immune cells are sensors of the environment and regulators of the immune response. CLEC-1 represents a potential therapeutic target to boost myeloid cells and antitumor responses. In order to better decipher the function of CLEC-1 and develop compounds aimed at modulating its activity, there is a need to identify its ligands, particularly in the context of the tumor environment.

[0004] CLEC-1 (also called CLEC-1A, or CLEC1, or CLEC1A) is a C-type lectin-like receptor, or C-type lectin-like receptor 1, that is particularly expressed in mammalian species, more specifically in humans. More precisely, CLEC-1 belongs to the DECTIN-1 cluster of C-type lectin-like receptors (CTLRs), which also includes CLEC-2, DECTIN-1 (CLEC7-A), CLEC-9A, MICL, MAH, and LOX-1 (Colonna M, Samaridis J, Angman L. Molecular characterization of two novel C-type lectin-like receptors, one of which is selectively expressed in human dendritic cells. Eur J Immunol. 2000;30(2):697-704). Functionally, C-type lectin receptors (CLRs) are a large family of transmembrane and soluble receptors that contain one or more carbohydrate recognition domains that can recognize a wide variety of glycans on pathogens or on self-proteins. For these receptors, glycan recognition is mediated by Ca 2+ Nevertheless, many related CLRs are dependent on Ca 2+These receptors are termed C-type lectin-like receptors (CTLRs). These receptors are of particular interest for their role in linking both innate and adaptive immunity. CTLRs are mainly expressed by cells of myeloid lineage, such as monocytes, macrophages, dendritic cells (DCs), and neutrophils. CTLRs not only serve as antigen uptake receptors for internalization and presentation to T cells, but also trigger multiple signaling pathways leading to NF-κB, type I interferon (IFN), and / or inflammasome activation. Due to their ability to present antigens and ensure a balance between cellular activation and inhibition, CTLRs have emerged as challenging pharmacological targets for treating a wide variety of diseases, including cancer, autoimmune diseases, or allergies. CTLR modulation appears to represent a promising strategy for disease management, but efforts to identify ligands and clarify their role in immunity have been inadequate to date.

[0005] CLEC-1 is known to be expressed in myeloid cells and endothelial cells. CLEC-1 has been described as a receptor that can be upregulated by immunomodulatory mediators and moderate T cell activation (Thebault P. et al The C-Type Lectin-Like Receptor CLEC-1, Expressed by Myeloid Cells and Endothelial Cells, Is Up-Regulated by Immunoregulatory Mediators and Moderates T Cell Activation. J Immonol 2009; 183:3099-3108). The inventors have shown that in human blood, CLEC-1 is expressed on the cell surface by conventional DCs (cDCs) and by a small subset of monocytes and DCs, and is enhanced by the immunosuppressive cytokine TGFβ (see International Application No. WO2018073440). In both rodents and humans, CLEC-1 has been shown to act as an inhibitory receptor on myeloid cells, preventing IL-12p40 expression and downstream Th1 and Th17 responses in vivo (Lopez-Robles MD et al., Cell-surface C-type lectin-like receptor CLEC-1 dampens dendritic cell activation and downstream Th17 responses Blood Adv. 2017;Mar 22;1(9) 557-568).

[0006] These properties provide the benefit of considering CLEC-1 in the design of further means for the treatment of health conditions involving the immune response of patients, especially to develop new treatments against cancer. In particular, these results call for further studies to identify expressed compounds that may be endogenous ligands of the receptor. Endogenous ligands refer to mammalian ligands, especially human ligands, by comparison with exogenous ligands emanating from non-mammalian species. Indeed, an exogenous ligand of CLEC-1 has been identified in a pathogenic organism, namely Aspergillus Fumigatus, where the ligand is the naphthalene diol unit of 1,8-dihydroxynaphthalene (DHN)-melanin (Stappers MHT et al Recognition of DHN-melanin by a C-type lectin receptor is required for immunity to Aspergillus Nature 2018 Mar 15;555(7696):382-386). This exogenous ligand has been shown to be present in mice or humans under conditions of infection with the pathogenic fungus. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Application No. WO2018073440 [Patent Document 2] WO 96 / 34103 [Patent Document 3] WO 94 / 04678 [Non-patent literature]

[0008] [Non-Patent Document 1] Colonna M, Samaridis J, Angman L. Molecular characterization of two novel C-type lectin-like receptors, one of which is selectively expressed in human dendritic cells. Eur J Immunol. 2000;30(2):697-704 Non-Patent Document 2 Thebault P. et al The C-Type Lectin-Like Receptor CLEC-1, Expressed by Myeloid Cells and Endothelial Cells, Is Up-Regulated by Immunoregulatory Mediators and Moderates T Cell Activation. J Immonol 2009; 183:3099-3108 Non-Patent Document 3 Lopez-Robles MD et al., Cell-surface C-type lectin-like receptor CLEC-1 dampens dendritic cell activation and downstream Th17 responses Blood Adv. 2017;Mar 22;1(9) 557-568 Non-Patent Document 4 Stappers MHT et al Recognition of DHN-melanin by a C-type lectin receptor is required for immunity to Aspergillus Nature 2018 Mar 15;555(7696):382-386 Non-Patent Document 5 Brauner et al, Journal of Internal Medicine, Vol. 278(3), September 2015, pages 323-332 Non-Patent Document 6 Ding, Q., He, D., He, K. et al. Downregulation of TRIM21 contributes to hepatocellular carcinoma carcinogenesis and indicates poor prognosis of cancers. Tumor Biol. 36, 8761-8772 (2015). https: / / doi.org / 10.1007 / s13277-015-3572-2 Summary of the Invention [Problem to be solved by the invention]

[0009] There is a need to identify CLEC-1 ligands, particularly endogenous ligands, and determine the binding of CLEC-1 thereto, in order to identify pathways involved in the modulation of the immune response or pathways that can be used to design / identify compounds for modulating the immune response. [Means for solving the problem]

[0010] The present invention is based on the identification of a ligand of CLEC-1 endogenous to human cells. The inventors have identified that TRIM21 (tripartite motif-containing protein 21) is a ligand of CLEC-1, at least in humans. The inventors have been able to observe that TRIM21 and CLEC-1 can interact (i.e. bind to each other) in vitro. Starting from lysates of the RAJI cell line, the inventors have been able to purify and identify proteins that are able to interact (i.e. bind to) the functional region of CLEC-1 (Fc-CLEC1). The inventors have also observed that some anti-TRIM21 and anti-CLEC-1 antibodies are able to inhibit the interaction between TRIM21 and CLEC-1 and are therefore antagonists of the binding between CLEC-1 and TRIM21. In particular, the inventors have observed that recombinant or fusion molecules, including in particular recombinant or fusion polypeptides, which comprise the extracellular domain of CLEC-1, in particular the extracellular domain of human CLEC-1 (represented in the present specification by Fc-CLEC-1), can bind to TRIM21, and that this binding is disrupted by several anti-TRIM21 and anti-CLEC-1 antibodies.

[0011] As further detailed herein, the inventors provide evidence that antagonism of the CLEC1 / TRIM21 signalling pathway significantly leads to an increased phagocytic capacity of myeloid cells, in particular macrophages and / or dendritic cells, in particular increased phagocytosis of TRIM21 positive cells by myeloid cells, in particular increased phagocytosis of tumour cells and / or secondary necrotic cells, more particularly increased phagocytosis of TRIM21 positive tumour cells and / or secondary necrotic cells, by dendritic cells and / or macrophages.

[0012] All triplicate motif-containing (TRIM) proteins contain a structurally conserved N-terminus with a Really Interesting New Gene (RING) motif followed by one or two zinc finger domains called B-boxes and a coiled-coil (CC) domain. Like other family members, TRIM21 is an E3 ligase via the RING domain. As a member of the TRIM family, TRIM21 is composed of five domains: RING, B-box, coiled-coil, PRY and SPRY (these last two domains are often associated with the PRY-SPRY domain). Human TRIM21 may have the amino acid sequence of SEQ ID NO:2, and the RING, B-box, coiled-coil and PRY-SPRY domains may be located between amino acid residues 16 and 55, 92 and 123, 128 and 238, and 268 and 465, respectively. TRIM21 controls innate immune signaling through its E3 ligase function. It is an autoantigen for autoimmune diseases such as systemic lupus erythematosus and Sjogren's syndrome. Previous studies (Brauner et al, Journal of Internal Medicine, Vol. 278(3), September 2015, pages 323-332) have suggested that TRIM21 may play a role in tumorigenesis, and recent studies have investigated the relationship between TRIM21 expression and tumor prognosis (Ding, Q., He, D., He, K. et al. Downregulation of TRIM21 contributes to hepatocellular carcinoma carcinogenesis and indicates poor prognosis of cancers. Tumor Biol. 36, 8761-8772 (2015). https: / / doi.org / 10.1007 / s13277-015-3572-2). High and low levels of TRIM21 expression are usually associated with good and poor prognosis, respectively.Following the present discovery that TRIM21 is a ligand for CLEC-1, and due to the involvement of CLEC-1 in the immune response, TRIM21 could be considered as a target for the development of new therapeutic agents, a new diagnostic tool, a prognostic factor and a therapeutic factor, particularly in the treatment of cancer or autoimmune diseases, and the TRIM21 / CLEC1 pathway, particularly the interaction.

[0013] In a first aspect, the present invention relates to a compound that inhibits the C-type lectin-like receptor 1 (CLEC-1) / tri-element motif-containing protein 21 (TRIM21) signaling pathway for use as a medicament in the treatment of a subject, particularly a human subject, having a disease selected from the cancers mentioned herein, such as breast cancer, hepatocellular carcinoma, lymphoma, more particularly B-cell lymphoma, colon cancer, thyroid cancer, liver cancer, renal cancer, melanoma, colorectal cancer, nasopharyngeal cancer, pancreatic cancer, in particular cancers with TRIM21-positive tumor cells, chronic infections, sepsis, infections, viral infections, in particular infections with Coxsackieviruses or encephalitis viruses, more particularly infections with Coxsackievirus B3 or Japanese encephalitis virus, fungal infections, cardiovascular diseases, autoimmune diseases, in particular Sjögren's syndrome or systemic lupus erythematosus or systemic sclerosis, inflammatory diseases.

[0014] In another aspect, the present invention relates to compounds that bind to TRIM21, that are antagonists of the binding between CLEC-1 and TRIM21, in particular between human CLEC-1 and human TRIM21, for use as a medicament for the treatment of a subject, in particular a human subject, with a disease selected from the cancers mentioned herein, such as breast cancer, hepatocellular carcinoma, lymphoma, more particularly B-cell lymphoma, colon cancer, thyroid cancer, liver cancer, renal cancer, melanoma, colorectal cancer, nasopharyngeal cancer, pancreatic cancer, in particular cancers with TRIM21-positive tumor cells, chronic infections, sepsis, infections, in particular infections with Coxsackieviruses or encephalitis viruses, more particularly infections with Coxsackievirus B3 or Japanese encephalitis virus, cardiovascular diseases, autoimmune diseases, in particular Sjögren's syndrome or systemic lupus erythematosus or systemic sclerosis, inflammatory diseases. In a preferred embodiment, the patient has cancer, sepsis or chronic infection.

[0015] In another aspect, the present invention also relates to the use of compounds that inhibit the C-type lectin-like receptor 1 (CLEC-1) / tripartite motif-containing protein 21 (TRIM21) signaling pathway to increase phagocytosis of cells, particularly TRIM-21 positive cells, particularly tumor cells and / or secondary necrotic cells, more particularly TRIM-21 positive tumor cells and / or secondary necrotic cells, by myeloid cells, particularly macrophages and / or dendritic cells. Compounds that may be used include antibodies, antigen-binding fragments thereof, antigen-binding antibody mimetics, peptides or polypeptides that are functional equivalents of TRIM21 or CLEC-1, such as but not limited to Fc-CLEC-1, particularly Fc-CLEC-1 comprising the amino acid residues set forth in SEQ ID NO: 3, nucleic acid molecules, particularly oligonucleotide molecules, more particularly RNA molecules, siRNA molecules or miRNA molecules, that reduce the expression of TRIM21 and / or CLEC-1.

[0016] In another aspect, the present invention also relates to methods, wherein the interaction between CLEC-1 and TRIM21 is used to assess the likely efficacy of treatment with anti-cancer and / or antagonist anti-human CLEC-1 antibodies or antigen-binding fragments thereof, or anti-TRIM21 antibodies or antigen-binding fragments thereof, to screen antagonist compounds, to treat diseases.

[0017] definition As used herein, the terms "CLEC-1" and "CLEC-1A" relate to a CLEC-1A protein from a mammalian species, preferably human CLEC-1 or CLEC-1A. The reference sequence of the human CLEC-1A receptor corresponds to the sequence associated with accession number Q8NC01 Uniprot. Preferably, the term "human CLEC-1" refers to the protein of amino acid sequence referenced by Q8NC01 Uniprot accession number and encoded by the CLEC-1 gene referenced by NCBI accession number 51267. As used herein, the terms CLEC-1A, CLEC-1, CLECA, CLEC-1, Clec1, Clec-1, CLEC-A1 and Clec-1A are used interchangeably and all refer to the mammalian CLEC1 receptor, its orthologue protein, or its homologue protein, which corresponds to the human CLEC-1A receptor, characterized by the amino acid sequence associated with accession number Q8NC01 Uniprot.

[0018] Preferably, the term "human CLEC-1" refers to a protein of amino acid sequence referenced by the Q8NC01 Uniprot accession number and encoded by the CLEC-1 gene referenced by the NCBI accession number 51267. CLEC-1 may be characterized by the amino acid sequence shown in SEQ ID NO: 1. In a particular embodiment, the amino acid sequence of the extracellular domain of human CLEC-1 is:

[0019] [ka]

[0020] The sequence comprises or is in the sequence:

[0021] As used herein, a "functional equivalent of CLEC-1", in particular a "functional equivalent of human CLEC-1", is in particular a polypeptide, peptide, oligopeptide, protein comprising at least a fragment (or part) of the extracellular domain of CLEC-1, possibly comprising the extracellular domain of CLEC-1, with the proviso that it is not the entire CLEC-1 polypeptide. Such a fragment of the extracellular domain of CLEC-1 or the entire extracellular domain of CLEC-1 may be combined with another molecule, such as a peptide or a fragment of another protein, such as an antibody, to stabilize the structure of the extracellular domain of CLEC-1 or its fragment, leading to the provision of a fusion protein comprising at least a fragment of the extracellular domain of CLEC-1. Such a functional equivalent of CLEC-1, in particular human CLEC-1, may alternatively be a polypeptide, peptide, oligopeptide, protein, whose amino acid sequence has at least 80%, in particular at least 90% identity to the aligned amino acid sequence shown in SEQ ID NO: 3 (so-called variants of SEQ ID NO: 3).

[0022] In particular, a functional equivalent of CLEC-1 comprises the extracellular domain of CLEC-1 or a sequence which has at least 80%, in particular more than 80%, in particular at least 85%, in particular more than 85%, at least 90%, in particular more than 90%, at least 95%, in particular more than 95% identity to the extracellular domain of CLEC-1, in particular the sequence shown in SEQ ID NO: 3.

[0023] In particular, a functional equivalent of CLEC-1 is a functional equivalent of human CLEC-1 and comprises the extracellular domain of human CLEC-1 or a sequence which has at least 80%, particularly more than 80%, particularly at least 85%, particularly more than 85%, at least 90%, particularly more than 90%, at least 95%, particularly more than 95% identity to the extracellular domain of human CLEC-1, more particularly a sequence which has at least 80% (particularly at least 85% or more, at least 90% or more, at least 95% or more) identity to the amino acid sequence shown in SEQ ID NO:3.

[0024] A functional equivalent of CLEC-1, in particular human CLEC-1, may for example comprise a fragment of the extracellular domain of CLEC-1, or the entire extracellular domain, or a variant thereof having at least 80% or more, in particular at least 90% or more, in particular at least 95% or more, identity with the aligned amino acid sequence of the sequence shown in SEQ ID NO: 3, further comprising a linker peptide, a tag, the Fc part of an antibody. Fc-CLEC-1 may for example comprise the amino acid sequence shown in SEQ ID NO: 3. This functional equivalent of CLEC-1 may for example be a fusion polypeptide. In a particular embodiment, the compound of the invention is a polypeptide which is a functional equivalent of CLEC-1, in particular human CLEC-1, fused to an immunoglobulin constant domain.

[0025] According to certain embodiments of the invention, fusion molecules are provided that feature a functional equivalent of CLEC-1 as defined herein. Thus, the extracellular domain of CLEC-1 may be fused to a tag (such as a tag comprising an amino acid sequence), to a moiety suitable to stabilize the extracellular domain of CLEC-1 when used to form a fusion molecule that may have many of the valuable chemical and biological properties of human antibodies and / or linkers as defined herein, targeting ligands and / or immunoglobulin constant domains (Fc regions), in particular the constant domains of human immunoglobulins.

[0026] In a particular embodiment of the fusion molecule, the Fc fragment of a human immunoglobulin comprised in the Fc-CLEC-1 fusion molecule is an Fc fragment of human IgG, and the Fc fragment is fused at its N-terminus to the extracellular domain of a mammalian CLEC-1, in particular human CLEC-1.

[0027] As used herein, the term "TRIM21" has its general meaning in the art and specifically refers to tripartite motif-containing protein 21 from mammalian species, more specifically human TRIM21.

[0028] Preferably, the term "human TRIM21" refers to a protein of amino acid sequence encoded by the TRIM21 gene referenced by Uniprot accession number P19474 and referenced by Gene ID 6737 (NCBI accession number). In a particular embodiment, TRIM21 has the amino acid sequence shown in SEQ ID NO: 2. In particular, TRIM21 is a ligand for CLEC-1 and can bind to CLEC-1, in particular the extracellular domain of CLEC-1, in particular the CLEC-1 of SEQ ID NO: 1 (e.g. when cells expressing TRIM21 are permeabilized, such as permeabilized RAJI cells, or such as certain tumor cells).

[0029] As used herein, the terms "TRIM21-positive cell," "TRIM21-positive tumor," and "TRIM21-positive tumor cell" refer to a cell or group of cells that express TRIM21 protein, either by constitutive expression (e.g., by normal expression by a cell of the gene encoding the TRIM21 protein) or by induced expression (e.g., by an external stimulus, or by a cell undergoing a stress condition or an apoptotic pathway, etc.).

[0030] Antagonists of the CLEC-1 / TRIM21 signaling pathway In a first aspect, the present invention relates to compounds that are antagonists of the CLEC-1 / TRIM21 signaling pathway, i.e. compounds that inhibit the CLEC-1 / TRIM21 signaling pathway. In this specification, the expressions "compounds that are antagonists of the CLEC-1 / TRIM21 signaling pathway" and the term "antagonist compound" are used interchangeably. The CLEC-1 / TRIM21 signaling pathway includes any compound that is involved in a chemical reaction in a cell to control a cellular function following the interaction between CLEC-1 and TRIM21. Upon interaction between CLEC-1 and TRIM21, the cell receives a signal from its environment and a CLEC-1 or TRIM21 compound is expressed by the cell. After receiving the signal, the first molecule of the pathway activates another molecule. This process is repeated throughout the signaling pathway until the last molecule is activated to perform the cellular function. An antagonist of the CLEC-1 / TRIM21 signaling pathway, i.e. a compound that inhibits the CLEC-1 / TRIM21 signaling pathway, may correspond to one of the following molecules: - a molecule that binds, in particular specifically binds, to TRIM21 or CLEC1 and antagonizes the binding between TRIM21 and CLEC-1, or - a compound that reduces the expression of a functional TRIM21 or CLEC1 molecule in a cell, such as, but not limited to, a nucleic acid molecule that can inhibit the expression or translation of TRIM21 or CLEC1 into a functional protein, such as an oligonucleotide molecule, more particularly an RNA molecule, an siRNA molecule or an miRNA molecule, which reduces the expression of a functional TRIM21 or CLEC1 molecule in a cell; or - a functional equivalent of TRIM21 or CLEC-1, or - a compound that reduces or inhibits the ability of the compound to participate in a chemical reaction induced by binding between CLEC-1 and TRIM21, and is capable of activating or inhibiting another different compound involved in the same signaling pathway.

[0031] Other compounds capable of antagonizing signaling pathways that are activated or inhibited following interaction between CLEC-1 and TRIM21 are contemplated for use in the present invention.

[0032] The antagonist effect of a compound on the CLEC-1 / TRIM21 pathway may be assessed by measuring increased phagocytosis of cells, particularly TRIM21 positive cells, more particularly tumour cells and / or secondary necrotic cells, more particularly TRIM21 positive tumour cells and / or secondary necrotic cells, by myeloid cells, particularly dendritic cells and / or macrophages (compared to a negative control).

[0033] In one embodiment the invention relates to a compound which binds to TRIM21, particularly which binds specifically to TRIM21, which is an antagonist of the binding between CLEC-1 and TRIM21, particularly between human CLEC-1 and human TRIM21, for use as a medicament, particularly in the treatment of a subject, particularly a human subject, suffering from a disease selected from cancer, particularly cancer with TRIM21 positive tumour cells, such as breast cancer, hepatocellular carcinoma, lymphoma, more particularly B-cell lymphoma, colon cancer, thyroid cancer, liver cancer, renal cancer, melanoma, colorectal cancer, nasopharyngeal cancer, pancreatic cancer, chronic infection, sepsis, infectious disease, particularly infection by Coxsackievirus or encephalitis virus, more particularly infection by Coxsackievirus B3 or Japanese encephalitis virus, cardiovascular disease, autoimmune disease, particularly Sjogren's syndrome or systemic lupus erythematosus or systemic sclerosis, inflammatory disease.

[0034] As used herein, a compound that is an antagonist of the binding between CLEC-1 and TRIM21 (also referred to herein as "antagonist" or "antagonist compound") may refer to a compound that binds to TRIM21, in particular specifically binds to TRIM21, and may be selected from the group consisting of antibodies, antigen-binding fragments of antibodies, antigen-binding antibody mimetics, macromolecules including antigen-binding fragments of antibodies or whole antibodies, small organic compounds, polypeptides, peptides, oligopeptides, proteins. Alternatively, the compound may correspond to a functional equivalent of TRIM21, in particular human TRIM21. Such a functional equivalent may in particular be a polypeptide, peptide, oligopeptide, protein comprising at least a fragment of the extracellular domain of TRIM21. Such a fragment may be combined with another molecule, such as a peptide or a fragment of another protein, such as an antibody, to stabilize the structure of the fragment of the extracellular domain of TRIM21, leading to the provision of a fusion protein comprising at least a fragment of the extracellular domain of TRIM21. Such a fusion protein may for example comprise a fragment of the extracellular domain of TRIM21 and a linker peptide, a tag, the Fc part of an antibody. In a particular embodiment, the compound may be a functional equivalent of CLEC-1, in particular human CLEC-1. Such a functional equivalent may be a polypeptide, peptide, oligopeptide, protein, in particular comprising at least a fragment of the extracellular domain of CLEC-1, or may be a polypeptide, peptide, oligopeptide, protein (so-called variant) whose amino acid sequence has at least 80% or more, in particular at least 85% or more, in particular at least 90% or more, identity with the established amino acid sequence shown in SEQ ID NO: 3. Such a fragment or variant may be combined with another molecule, such as a peptide, or a fragment of another protein, such as an antibody, to stabilize the structure of the fragment of the extracellular domain of CLEC-1, leading to the provision of a fusion protein comprising at least a fragment of the extracellular domain of CLEC-1. Such a fusion protein may, for example, comprise a fragment of the extracellular domain of CLEC-1 and a linker peptide, a tag, the Fc portion of an antibody. Fc-CLEC-1 may, for example, comprise the amino acid sequence shown in SEQ ID NO:3.A compound that is an antagonist of the binding between CLEC-1 and TRIM21 is a compound that is capable of reducing, inhibiting or blocking the interaction between CLEC-1 and TRIM21 in vitro, in vivo and / or ex vivo. The antagonist effect on the binding between CLEC-1 and TRIM21 can be determined using a method as described in the Examples herein. In other words, a compound that is an antagonist and binds to TRIM21 adversely interferes with the interaction between CLEC-1 and TRIM21, thereby downregulating, reducing or inhibiting the signaling pathway induced by the interaction between CLEC-1 and TRIM21. The presence or absence of such antagonist ability on the binding between CLEC-1 and TRIM21 can be evaluated according to the Examples of the present invention, in particular Example 3, and can be illustrated in Figures 5 and 6, where a competition test for measuring the binding of TRIM21 to Fc-CLEC-1 protein in the presence of an anti-TRIM21 antibody is disclosed. In a preferred embodiment, the antagonist compounds of the invention compete with CLEC-1 or Fc-CLEC-1 for binding to TRIM21 as disclosed herein. The presence of antagonistic potential in a competition assay between TRIM21 on the one hand and an antagonist compound of the invention and CLEC-1 or Fc-CLEC-1 on the other hand can be determined if in the presence of an antagonist compound of the invention, the binding of TRIM21 to CLEC-1 or Fc-CLEC-1 is less than 90%, more preferably less than 80%, even more preferably less than 50% and most preferably less than 20% of that of TRIM21 to CLEC-1 or Fc-CLEC-1 under the same experimental conditions but in the absence of the antagonist of the invention being evaluated. Alternatively, antagonistic potential for CLEC-1 / TRIM21 binding can be determined according to the methods described in the Examples of the present invention.In particular, a compound that binds to TRIM21 may be considered an antagonist of the interaction between CLEC-1 and TRIM21, particularly between human CLEC-1 and human TRIM21, if it reduces the binding of the extracellular domain of CLEC-1 to TRIM21, particularly if it reduces the binding of a fusion protein comprising the extracellular domain of the human CLEC-1 receptor fused to the Fc fragment of human immunoglobulin, particularly human IgG, to TRIM21, compared to the same binding experiment in the absence of the compound being evaluated. A reduction is observed when binding is reduced by at least 1-log, more particularly at least 2-log, most preferably at least 3-log, compared to a control experiment.

[0035] In one aspect of the invention, the antagonist compound binds to TRIM21, in particular binds specifically to TRIM21, in particular to human TRIM21. In other words, the antagonist compound of the invention exhibits specific affinity for its target TRIM21. In another aspect of the invention disclosed herein, the antagonist compound binds to CLEC-1, in particular to human CLEC-1. In other words, the antagonist compound of the invention exhibits specific affinity for its target CLEC-1. The terms "specifically binds" and "specifically binds to" and "specific affinity for" refer to the fact that the antagonist compound used according to the invention, in particular an antibody, antigen-binding fragment thereof, antigen-binding antibody mimetic or modified antibody of the invention, binds to TRIM21 or CLEC-1, respectively, at least 1×10 -6 M, 1×10 -7 M, 1×10 -8 M, 1×10 -9 M, 1×10 -10 M, 1×10 -11 M, 1×10 -12M or greater, and / or the ability to bind to TRIM21 or, respectively, CLEC-1 with an affinity at least twice as great as its affinity for a non-specific target (i.e., another protein of TRIM21, such as CLEC-1, or, respectively, another protein of CLEC-1, such as TRIM21). Affinity can be assessed according to various methods well known to those skilled in the art. These methods include, but are not limited to, Biacore analysis, BLItz analysis, and biosensors such as Scatchard plots.

[0036] The antagonist compound may be an antibody, an antigen-binding fragment of an antibody, an antigen-binding antibody mimetics, a small organic compound, a peptide, a polypeptide, an oligopeptide, a protein, in particular a peptide or a polypeptide comprising a part of the TRIM21 amino acid sequence, including consecutive amino acid residues, such as the sequence of SEQ ID NO: 2. The antagonist compound may also be an RNA molecule, in particular an RNA molecule that reduces the expression of the TRIM21 protein, such as, but not limited to, small interfering RNA, double-stranded RNA, microRNA (miRNA), and has the ability to interfere with the RNA interference (RNAi) pathway. Such RNA molecules interfere with the expression of the gene encoding TRIM21 with a complementary nucleotide sequence by degrading the mRNA after transcription of the trim21 gene, thereby preventing its translation into the TRIM21 protein. The most preferred antagonist compound is an anti-TRIM21 antibody.

[0037] In certain embodiments of the invention, the compound is an antibody, an antigen-binding fragment of an antibody, or an antigen-binding antibody mimetic that binds to an epitope sequence located within the coiled-coil domain of TRIM21, in particular the coiled-coil domain located between amino acid residues 128 and 238 of TRIM21 of SEQ ID NO: 2 or the coiled-coil domain corresponding to the amino acid residues of SEQ ID NO: 4, and / or binds to an epitope sequence located within the PRY-SPRY domain of TRIM21, in particular the PRY-SPRY domain located between amino acid residues 268 and 465 of TRIM21 of SEQ ID NO: 2 or the PRY-SPRY domain corresponding to the amino acid residues of SEQ ID NO: 5. In certain embodiments, the compound is an antibody, an antigen-binding fragment of an antibody, that binds to an epitope sequence located between amino acid residues 141 and 280 of TRIM21 of SEQ ID NO: 2, or an epitope located within the amino acid sequence shown in SEQ ID NO: 7. In certain embodiments, the compound is an antibody, or an antigen-binding fragment of an antibody, that binds to an epitope sequence located between amino acid residues 195 and 293 of TRIM21 of SEQ ID NO: 2, or an epitope located within the amino acid sequence set forth in SEQ ID NO: 8. In certain embodiments, the compound is an antibody, or an antigen-binding fragment of an antibody, that binds to an epitope sequence located between amino acid residues 254 and 475 of TRIM21 of SEQ ID NO: 2, or an epitope located within the amino acid sequence set forth in SEQ ID NO: 6. In certain embodiments, the compound is an antibody, or an antigen-binding fragment of an antibody, that binds to an epitope sequence located between amino acid residues 463 and 475 of TRIM21 of SEQ ID NO: 2, or an epitope located within the amino acid sequence set forth in SEQ ID NO: 9.

[0038] In a particular embodiment of the invention, the compound is an antibody, an antigen-binding fragment thereof or an antigen-binding antibody mimetic that binds to TRIM21, in particular binds specifically to TRIM21 and recognizes the binding between CLEC1 and TRIM21, in particular between human CLEC-1 and human TRIM21, said antibody, antigen-binding fragment thereof or antigen-binding antibody mimetic being selected from the list consisting of the anti-TRIM21 antibodies sc-25351, PA5-22294, PA5-18147 and MAB-62191, and a competing antibody for binding to TRIM21, in particular a coiled-coil domain of TRIM21, in particular a coiled-coil domain located between amino acid residues 128 and 238 of TRIM21 of SEQ ID NO: 2. and / or competes for binding to the PRY-SPRY domain of TRIM21, particularly the PRYSPRY domain located between amino acid residues 268 and 465 of TRIM21 of SEQ ID NO: 2 or consecutive amino acid residues located within the PRY-SPRY domain corresponding to the amino acid sequence of SEQ ID NO: 5, and the antibody, antigen-binding fragment thereof, or antigen-binding antibody mimetic enhances phagocytosis of cells, particularly TRIM21 positive cells, particularly tumor cells and / or secondary necrotic cells, by myeloid cells, particularly dendritic cells and / or macrophages, compared to a competing antibody.

[0039] A cross-blocking assay (e.g., a competitive ELISA assay) may be performed to determine whether a test antibody can compete for binding to the same target bound by at least one competing antibody selected from the list consisting of anti-TRIM21 antibodies sc-25351, PA5-22294, PA5-18147, and MAB-62191. In an exemplary competitive ELISA assay, a polypeptide comprising or consisting of a coiled-coil domain located between amino acid residues 128 and 238 of TRIM21 of SEQ ID NO:2 or a coiled-coil domain corresponding to amino acid residues of SEQ ID NO:4, and / or binding to an epitope sequence located within the PRY-SPRY domain of TRIM21, in particular the PRY-SPRY domain located between amino acid residues 268 and 465 of TRIM21 of SEQ ID NO:2 or the PRY-SPRY domain corresponding to amino acid residues of SEQ ID NO:5, may be coated on the wells of a microtiter plate and pre-incubated with or without a candidate competing antibody, and then a biotin-labeled candidate antibody is added. The amount of labeled candidate antibody bound to the polypeptide is measured using an avidin-peroxidase conjugate and an appropriate substrate. The antibody may be labeled with a radioactive or fluorescent label, or with some other detectable and measurable label. The amount of labeled candidate antibody bound to the polypeptide has an indirect correlation with the ability of the candidate antibody to compete for binding to the same epitope, i.e., the higher the affinity of the candidate antibody for the target, the less labeled competing antibody will bind to the polypeptide-coated well. A candidate antibody is considered an antibody of the present invention that competes for binding to the same polypeptide as the competing antibody if it is able to block the binding of the competing antibody by at least 20%, preferably at least 20-50%, and even more preferably at least 50%, compared to a control run in parallel in the absence of the candidate antibody (but which may be in the presence of a known non-competing antibody). It will be understood that variations of this assay may be performed to arrive at the same quantitative value.

[0040] Of course, depending on the competing antibody, the polypeptide coating the plate may be altered to correspond more closely to the domain recognized by the competing antibody.

[0041] In a particular embodiment of the invention, the compound is an antibody, antigen-binding fragment thereof, or antigen-binding antibody mimetic that binds to TRIM21, particularly specifically binds to TRIM21 and antagonizes the binding between CLEC1 and TRIM21, particularly between human CLEC-1 and human TRIM21, which antibody, antigen-binding fragment thereof, or antigen-binding antibody mimetic competes with the competing antibody PA5-22294 for binding to the amino acid sequence set forth in SEQ ID NO: 6, and which antibody, antigen-binding fragment thereof, or antigen-binding antibody mimetic enhances phagocytosis of cells, particularly TRIM21 positive cells, particularly tumor cells and / or secondary necrotic cells, by myeloid cells, particularly dendritic cells and / or macrophages, compared to the competing antibody.

[0042] In a particular embodiment of the invention, the compound is an antibody, antigen-binding fragment thereof, or antigen-binding antibody mimetic that binds to TRIM21, particularly specifically binds to TRIM21 and antagonizes the binding between CLEC1 and TRIM21, particularly between human CLEC-1 and human TRIM21, wherein the antibody, antigen-binding fragment thereof, or antigen-binding antibody mimetic competes with the competing antibody sc-25351 for binding to the amino acid sequence set forth in SEQ ID NO:7, and the antibody, antigen-binding fragment thereof, or antigen-binding antibody mimetic enhances phagocytosis of cells, particularly TRIM21 positive cells, particularly tumor cells and / or secondary necrotic cells, by myeloid cells, particularly dendritic cells and / or macrophages, compared to the competing antibody.

[0043] In a particular embodiment of the invention, the compound is an antibody, antigen-binding fragment thereof, or antigen-binding antibody mimetic that binds to TRIM21, particularly specifically binds to TRIM21 and antagonizes the binding between CLEC1 and TRIM21, particularly between human CLEC-1 and human TRIM21, and the antibody, antigen-binding fragment thereof, or antigen-binding antibody mimetic competes with the competing antibody MAB6219 for binding to the amino acid sequence set forth in SEQ ID NO:8, and the antibody, antigen-binding fragment thereof, or antigen-binding antibody mimetic enhances phagocytosis of cells, particularly TRIM21 positive cells, particularly tumor cells and / or secondary necrotic cells, by myeloid cells, particularly dendritic cells and / or macrophages, compared to the competing antibody.

[0044] In a particular embodiment of the invention, the compound is an antibody, antigen-binding fragment thereof, or antigen-binding antibody mimetic that binds to TRIM21, particularly specifically binds to TRIM21 and antagonizes the binding between CLEC1 and TRIM21, particularly between human CLEC-1 and human TRIM21, wherein the antibody, antigen-binding fragment thereof, or antigen-binding antibody mimetic competes with the competing antibody PA5-18147 for binding to the amino acid sequence set forth in SEQ ID NO:9, and the antibody, antigen-binding fragment thereof, or antigen-binding antibody mimetic enhances phagocytosis of cells, particularly TRIM21 positive cells, particularly tumor cells and / or secondary necrotic cells, by myeloid cells, particularly dendritic cells and / or macrophages, compared to the competing antibody.

[0045] The term "small organic compounds" includes molecules of similar size to organic molecules commonly used in pharmaceuticals. The term excludes biopolymers (e.g., proteins, nucleic acids, etc.). Preferred small organic molecules range in size up to about 5000 Da, more specifically up to 2000 Da, and most specifically up to about 1000 Da.

[0046] As used herein, the term "antibody" includes polyclonal, monoclonal or recombinant antibodies. As used herein, the term "monoclonal antibody" is intended to refer to a preparation of antibody molecules to obtain antibodies sharing a common heavy chain and a common light chain amino acid sequence, as opposed to a "polyclonal" antibody preparation, which contains a mixture of antibodies of different amino acid sequences. Monoclonal antibodies can be produced by several known techniques, such as phage, bacterial, yeast or ribosome display, and the typical method is exemplified by antibodies derived from hybridomas. They can also be synthesized using the disclosed amino acid sequences as a reference. Thus, the term "monoclonal" is used to refer to all antibodies derived from one nucleic acid clone.

[0047] As used herein, the term "antibody" further refers to an antibody that has been modified compared to a wild-type antibody, and includes chimeric antibodies, humanized antibodies, fully human antibodies, de-immunized antibodies, modified antibodies, and antigen-binding antibody mimetics. In a particular embodiment of the invention, the compound is an antibody or antigen-binding fragment thereof that binds to TRIM21, in particular specifically binds to TRIM21, and comprises a human IgG1, IgG2, IgG3 or IgG4 constant region.

[0048] The antibody of the present invention includes recombinant antibodies. As used herein, the term "recombinant antibody" refers to an antibody produced, expressed, generated or isolated by recombinant means, such as an antibody expressed using a recombinant expression vector transfected into a host cell, an antibody isolated from a recombinant combinatorial antibody library, an antibody isolated from an animal (e.g., a mouse) transgenic with human immunoglobulin genes, or an antibody produced, expressed, generated or isolated by any other method in which a particular immunoglobulin gene sequence (such as a human immunoglobulin gene sequence) is assembled with other DNA sequences. Recombinant antibodies include, for example, chimeric and humanized antibodies.

[0049] As used herein, the term "chimeric antibody" refers to an antibody in which variable domain sequences derived from the germline of a mammalian species, such as mouse, have been grafted onto constant domain sequences derived from the germline of another mammalian species, such as human.

[0050] As used herein, "humanized antibody" refers in a first embodiment to an antibody in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human, or in particular humanized, framework sequences. In a further embodiment, "humanized antibody" refers to an antibody in which at least one CDR and all or some of the framework sequences have been humanized.

[0051] As used herein, "antigen-binding fragment of an antibody" refers to a part of an antibody, i.e. a molecule corresponding to a part of the structure of an antibody of the invention, that exhibits antigen-binding ability, possibly in its native form, to TRIM21 or CLEC-1, and such fragments in particular exhibit the same or substantially the same antigen-binding specificity for the antigen compared to the antigen-binding specificity of the corresponding four-chain antibody. Advantageously, the antigen-binding fragment has a similar binding affinity to the corresponding four-chain antibody. However, antigen-binding fragments with reduced antigen-binding affinity relative to the corresponding four-chain antibody are also included within the scope of the invention. Antigen-binding ability can be determined by measuring the affinity between the antibody and the target fragment. These antigen-binding fragments may also be referred to as "functional fragments" of antibodies.

[0052] Antigen-binding fragments of antibodies are fragments that contain their hypervariable domains, called CDRs (complementarity determining regions), or parts thereof that contain the recognition site for the antigen, i.e. TRIM21. Antigen-binding fragments of antibodies that contain the variable domains that contain the CDRs of the antibody include Fv, dsFv, scFv, Fab, Fab', F(ab')2. These basic antigen-binding fragments of the invention may be combined to obtain multivalent antigen-binding fragments, such as bispecific antibodies, trispecific antibodies (tribodies) or tetraspecific antibodies (tetrabodies). These multivalent antigen-binding fragments are also part of the invention.

[0053] Antigen-binding antibody mimetics are organic compounds that specifically bind to antigens but are not structurally related to antibodies. Antigen-binding antibody mimetics are usually artificial peptides or small proteins with a molar mass of about 3-20 kDa. Although nucleic acids and small molecules may also be considered antibody mimetics, artificial antibodies, antibody fragments, and fusion proteins composed of these are not considered antibody mimetics. General advantages over antibodies are higher solubility, tissue penetration, heat and enzyme stability, and relatively low production costs. Antibody mimetics are being developed as therapeutic and diagnostic agents. Antigen-binding antibody mimetics may also be selected from the group including Affibody, Affilin, Aptamer, Anticalin, Affimer, Affitin, DARPin, and Monobody.

[0054] As used herein, the term "polypeptide" refers to a polymer of amino acids having any length of amino acid residues. Thus, peptides, oligopeptides, and proteins are included within the definition of "polypeptide," and these terms are used interchangeably throughout the specification and in the claims. The term "polypeptide" does not exclude post-translational modifications, including, but not limited to, phosphorylation, acetylation, glycosylation, and the like.

[0055] The term "functionally equivalent fragment" as used herein may refer to any fragment or collection of fragments of TRIM21 or of CLEC-1 that binds to TRIM21, in particular specifically binds to TRIM21. Thus, the present invention provides polypeptides, in particular functional equivalents, that are capable of decreasing / reducing or inhibiting the binding of CLEC-1 to TRIM21, said polypeptides comprising consecutive amino acids having a sequence which, for a functional equivalent of TRIM21, is the sequence of at least a part of a domain of TRIM21 that binds to CLEC-1, or, for a functional equivalent of CLEC-1, is the sequence of at least a part of an extracellular domain of CLEC-1 that binds to TRIM21, in particular specifically binds to TRIM21. Functional equivalents of CLEC-1 are defined above and include in particular fragments of the extracellular domain of CLEC1 of the amino acid sequence of SEQ ID NO: 3 or variants thereof which are polypeptides having at least 80% or more, in particular at least 85% or more, in particular at least 90% or more, identity with an aligned amino acid sequence in the sequence of SEQ ID NO: 3.

[0056] In some embodiments, the functional equivalent is fused to a heterologous polypeptide to form a fusion protein. As used herein, a "fusion protein" comprises all or a portion (typically biologically active) of the functional equivalent of the present invention operably linked to a heterologous polypeptide (i.e., a polypeptide other than the same polypeptide). Within the fusion protein, the term "operably linked" is intended to indicate that the functional equivalent of the present invention and the heterologous polypeptide are fused in frame to each other. The heterologous polypeptide may be fused to the N-terminus or C-terminus of the functional equivalent of the present invention.

[0057] In some embodiments, the functional equivalent is fused to an immunoglobulin constant domain (Fc domain) to form an immunoadhesin. Immunoadhesins can have many of the valuable chemical and biological properties of human antibodies. Immunoadhesins can be constructed from human protein sequences with the desired specificity linked to appropriate human immunoglobulin hinge and constant domain (Fc) sequences, so that the desired binding specificity can be achieved using entirely human components. Such immunoadhesins are minimally immunogenic to patients and are safe for long-term or repeated use. In some embodiments, the Fc region is a native sequence Fc region. In some embodiments, the Fc region is a variant Fc region. In yet another embodiment, the Fc region is a functional Fc region. As used herein, the term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain and includes native sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain may vary, the human IgG heavy chain Fc region is usually defined to extend from an amino acid residue at position Cys226 or Pro230 to its carboxy-terminus. The adhesive portion and the immunoglobulin sequence portion of the immunoadhesin may be linked by a minimal linker. The immunoglobulin sequence is typically, but not necessarily, an immunoglobulin constant domain. The immunoglobulin portion in the chimera of the invention may be derived from IgG1, IgG2, IgG3 or IgG4 subtypes, IgA, IgE, IgD or IgM, but typically from IgG1 or IgG4. In some embodiments, the functional equivalent of CLEC-1 or TRIM21 and the immunoglobulin sequence portion of the immunoadhesin are linked by a minimal linker. As used herein, the term "linker" refers to a sequence of at least one amino acid that links the polypeptide of the invention to the immunoglobulin sequence portion. Such linkers may be useful to prevent steric hindrance. In some embodiments, the linker has 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 amino acid residues.However, the upper limit is not critical, but is selected for reasons of convenience, for example, for biopharmaceutical production of such polypeptides. The linker sequence may be a sequence of natural origin or a sequence of non-natural origin. When used for therapeutic purposes, the linker is typically non-immunogenic in the subject to which the immunoadhesin is administered. One useful group of linker sequences are linkers derived from the hinge region of heavy chain antibodies, as described in WO 96 / 34103 and WO 94 / 04678. Another example is a polyalanine linker sequence.

[0058] In a particular embodiment of the invention, the antagonist compound is a functional equivalent of TRIM21. A functional equivalent of TRIM21 includes, but is not limited to, a molecule that binds to CLEC-1 and contains all or part of TRIM21 to form a soluble receptor that can bind to CLEC-1. Such a functional equivalent of TRIM21 can be a peptide or polypeptide. In particular, such a functional equivalent of TRIM21 can be a peptide or polypeptide that comprises at least 20, particularly at least 25, particularly at least 30, particularly at least 40, particularly 50 amino acid residues, particularly at least 80 amino acid residues, particularly at least 100 amino acid residues, particularly at least 200 amino acid residues, particularly at least 300 consecutive amino acid residues in SEQ ID NO:2. Alternatively, the antagonist is a peptide or polypeptide comprising at least 20 amino acid residues, particularly at least 25 amino acid residues, particularly at least 30 amino acid residues, particularly at least 40 amino acid residues, particularly 50 amino acid residues, particularly at least 80 amino acid residues, particularly at least 100 amino acid residues, particularly at least 200 amino acid residues, particularly at least 300, of TRIM21 of SEQ ID NO: 2 and having at least 80% identity, more particularly at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% and even more particularly at least 99% identity with TRIM21 or a corresponding protein, particularly TRIM21 of SEQ ID NO: 2, or sharing 100% identity.The antagonist compound may be a polypeptide fused to an immunoglobulin constant domain, comprising or consisting of an amino acid sequence that has at least 80% identity with TRIM21 or a corresponding protein, in particular TRIM21 of SEQ ID NO: 2, more specifically at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% and even more specifically at least 99%, or shares 100% identity. As used herein, the term "corresponding protein" refers to a protein whose functional equivalent of the present invention has a similar function as defined herein. The percentage of identity referred to in the present presentation is determined based on a global alignment of the sequences to be compared, i.e., an alignment over the entire length of the sequences, for example using the algorithm of Needleman and Wunsch 1970. This sequence comparison can be performed using needle software, for example, by using a "gap open" parameter equal to 10.0, a "gap extend" parameter equal to 0.5, and the "BLOSUM 62" matrix. Software such as needle is available at the website ebi.ac.uk worldwide under the name "needle".

[0059] Functional equivalents of CLEC-1 include, but are not limited to, molecules which bind to TRIM21 and comprise all or part of the extracellular domain of CLEC-1 to form a soluble receptor capable of binding to TRIM21. Thus, functional equivalents include soluble forms of CLEC-1. Suitable soluble forms of these proteins, or functional equivalents thereof, may include, for example, truncated forms of proteins in which the transmembrane domain has been removed by chemical, proteolytic or recombinant methods, and may comprise or consist in the extracellular domain of CLEC-1 or a fragment thereof, as defined herein. Specifically, a functional equivalent is at least 80% or greater, more specifically at least 85% or greater, at least 86% or greater, at least 87% or greater, at least 88% or greater, at least 89% or greater, at least 90% or greater, at least 91% or greater, at least 92% or greater, at least 93% or greater, or at least 93% greater, of the corresponding protein over the entire length of the corresponding protein. The term "corresponding protein" as used herein refers to a protein whose functional equivalent of the present invention has a similar function. The percentage of identity referred to in the present disclosure is determined based on a global alignment of the sequences to be compared, i.e., an alignment over the entire length of the sequences, for example using the algorithm of Needleman and Wunsch 1970.This sequence comparison can be performed using needle software, for example, by using a "gap open" parameter equal to 10.0, a "gap extend" parameter equal to 0.5, and the "BLOSUM 62" matrix. Software such as needle is available at the website ebi.ac.uk worldwide under the name "needle".

[0060] The antagonist compounds are provided for use as medicines. In particular, the antagonist compounds are provided for use in the treatment of disease in a subject (i.e., a patient, particularly a human patient), particularly a subject with cancer, sepsis, or infection. In another embodiment, the antagonist compounds may be provided for use in the prevention of disease in a subject. As used herein, "treatment" or "treating" is an approach to obtain a beneficial or desired result. Beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating, preventing, or eliminating one or more symptoms resulting from a disease, curing a disease, reducing the extent of a disease, stabilizing a disease (e.g., preventing or slowing the progression of a disease), preventing or slowing the spread of a disease, preventing or slowing the recurrence of a disease, slowing or slowing the progression of a disease, improving a disease state, providing remission (partial or complete) of a disease, reducing the dose of one or more other medications required to treat a disease, slowing the progression of a disease, improving quality of life, and / or prolonging survival. According to one embodiment, the term "treatment" relates to a prophylactic treatment. As used herein, the term "prevent" refers to a reduction in the risk of acquiring or developing a given condition.

[0061] In certain embodiments of the present invention, antagonist compounds are provided for use in the treatment of patients, where the outcome of the disease is improved by modulation of phagocytosis of target cells, such as, in particular, cells expressing TRIM21, more particularly, TRIM21-positive tumor cells, by myeloid cells, in particular, dendritic cells and / or macrophages. Thus, in certain embodiments, antagonist compounds are provided for increasing phagocytosis of cells, in particular, TRIM21-positive cells, more particularly, tumor cells and / or secondary necrotic cells, even more particularly, TRIM21-positive tumor cells and / or TRIM21-positive secondary necrotic cells, by myeloid cells, in particular, dendritic cells and / or macrophages. Thus, TRIM21-positive tumors (i.e. tumors comprising at least one cell that expresses, in particular aberrantly expresses, TRIM21) may be targeted by the compounds of the present invention. Indeed, it has been shown that antagonists of the TRIM21-CLEC1 signalling pathway (e.g. compounds which inhibit the binding between CLEC-1 and TRIM21, which bind to TRIM21 or CLEC1, or functional equivalents of TRIM21 or CLEC1, or compounds which reduce the expression of functional TRIM21 or CLEC1, or compounds which reduce or inhibit the signalling pathway induced by the binding between TRIM21 and CLEC-1) are able to modulate, in particular enhance, the phagocytosis of target cells, such as tumour cells expressing TRIM21, by myeloid cells, in particular dendritic cells and / or macrophages, thereby leading to their use in therapies or treatment of diseases in which the health of a patient is improved by modulation of phagocytosis of target cells, such as tumour cells or cells expressing TRIM21, by myeloid cells, in particular dendritic cells and / or macrophages. The use of TRIM21 antagonist compounds thereby at least reduces the interaction of CLEC-1 (expressed on the cell surface of dendritic cells and macrophages) with its ligand TRIM21 (e.g. expressed at least by tumour cells) and may be useful in regulating phagocytosis of cells, such as tumour cells, by myeloid cells, particularly dendritic cells and / or macrophages.When CLEC-1A expressing myeloid cells, particularly macrophages or dendritic cells, interact with cells expressing TRIM21, phagocytosis by these macrophages or dendritic cells can be inhibited or reduced. As an example, it has been shown that tumor cells expressing TRIM21 (i.e. Raji cells) escape phagocytosis exerted by macrophages. By using the antagonist compounds of the present invention that modulate the interaction between CLEC-1 expressing myeloid cells and TRIM21 expressing cells, particularly TRIM21 positive tumor cells, the reduction or inhibition of phagocytosis of TRIM21 expressing cells by myeloid cells, particularly dendritic cells or macrophages, is reduced. As a result, phagocytosis of TRIM21 expressing cells, such as tumor cells, is enhanced in the presence of the antagonist compounds of the present invention.

[0062] More specifically, the present invention relates to the use of antagonist compounds in the treatment of a condition or disease in a patient, wherein the condition or disease is or relates to the cancers mentioned herein, such as breast cancer, hepatocellular carcinoma, lymphoma, more specifically B-cell lymphoma, colon cancer, thyroid cancer, liver cancer, renal cancer, melanoma, colorectal cancer, nasopharyngeal cancer, pancreatic cancer, in particular cancers having TRIM21-positive tumor cells, chronic infections, sepsis, infections, viral infections, in particular Coxsackieviruses or encephalitis viruses, more specifically infections by Coxsackievirus B3 or Japanese encephalitis virus, cardiovascular diseases, autoimmune diseases, in particular Sjogren's syndrome or systemic lupus erythematosus or systemic sclerosis, inflammatory diseases.

[0063] The term "cancer" has its general meaning in the art and refers to a group of diseases involving abnormal cell proliferation that may invade or spread to other parts of the body. The term "cancer" further encompasses both primary and metastatic cancers. Examples of cancers that may be treated by the methods and compositions of the present invention include, but are not limited to, cancers with TRIM21-positive tumor cells, cancers with TRIM21-positive tumors, cancers of bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal, gingiva, head, kidney, liver, lung, nasopharynx, cervix, ovary, prostate, skin, stomach, testis, tongue, or uterus origin. Cancers may also be of, but are not limited to, the following histological types in particular: Neoplasm, malignant;Carcinoma;Carcinoma, undifferentiated;Giant cell and spindle cell carcinoma;Small cell carcinoma;Papillary carcinoma;Squamous cell carcinoma;Lymphoepithelial carcinoma;Basal cell carcinoma;Hair stromal carcinoma;Transitional cell carcinoma;Papillary transitional cell carcinoma;Adenocarcinoma;Gastrinoma, malignant;Choledochocarcinoma;Hepatocellular carcinoma;Mixed hepatocellular cholangiocarcinoma;Tractular adenocarcinoma;Adenoid cystic carcinoma;Adenomatous intrapolypoid adenocarcinoma;Familial polyposis coli;Solid tumor;Carcinoid tumor, malignant;Bronchioloalveolar adenocarcinoma;Papillary adenocarcinoma;Agonist Pigmented carcinoma; eosinophilic carcinoma; eosinophilic adenocarcinoma; basophilic carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary follicular adenocarcinoma; non-encapsulated sclerosing carcinoma; adrenocortical carcinoma; Mucoepidermoid carcinoma; Cystic carcinoma; Papillary cystic carcinoma; Papillary serous cystic carcinoma; Mucinous cystic carcinoma; Mucinous carcinoma; Signet ring cell carcinoma; Invasive ductal carcinoma; Medullary carcinoma; Lobular carcinoma; Inflammatory carcinoma; Paget disease, breast; Acinic cell carcinoma; Adenosquamous carcinoma;Adenocarcinoma with squamous metaplasia;Thymoma, malignant;Ovarian stromal tumor, malignant;Theca cell tumor, malignant;Granulosa cell tumor, malignant;Roblastoma, malignant;Sertoli cell carcinoma;Leydig cell tumor, malignant;Lipocytoma, malignant;Paraganeuroma, malignant;Extramammary paraganglioma, malignant;Pheochromocytoma;Glomus angiosarcoma;Malignant melanoma;Amelanotic melanoma;Superficial spreading melanoma;Malignant in giant pigmented nevus Melanoma;epithelioid cell melanoma;blue nevus, malignant;sarcoma;fibrosarcoma;fibrous histiocytoma, malignant;myxosarcoma;liposarcoma;leiomyoma;rhabdomyoma;embryonal rhabdomyosarcoma;alveolar rhabdomyosarcoma;stromal sarcoma;mixed tumor, malignant;mixed Müllerian tumor;nephroblastoma;hepatoblastoma;carcinosarcoma;mesenchymoma, malignant;Brenner tumor, malignant;phyllodes tumor, malignant;synovial sarcoma;mesothelioma, malignant;dysgerminoma;embryonal carcinoma;teratoma, malignant;ovarian goiter, malignant;Choriocarcinoma;Mesonephroma, malignant;Angiosarcoma;Hemangioendothelioma, malignant;Kaposi's sarcoma;Hemangiopericytoma, malignant;Lymphangiosarcoma;Osteosarcoma;Paracortical osteosarcoma;Chondrosarcoma;Chondronblastoma, malignant;Mesenchymal chondrosarcoma;Giant cell tumor of bone;Ewing's sarcoma;Odontogenic tumor, malignant;Ameloblastic odontosarcoma;Ameloblastoma, malignant;Ameloblastic fibrosarcoma;Pinealoma, malignant;Chordoma;Glioma, malignant;Ependymoma;Astrocytoma;Protoplasmic astrocytoma;Fibrillary astrocytoma;Astroblastoma;Glioblastoma;Oligodendroglioma;Oligodendroglioma;Primitive neuroectodermal tumor;Cerebellar sarcoma;Ganglioneuroblastoma;Neuroblastoma;Retinoblastoma;Olfactory neurogenoma tumors;meningiomas, malignant;neurofibrosarcomas;schwannoma, malignant;granular cell tumors, malignant;malignant lymphomas;Hodgkin's disease;Hodgkin's lymphoma;paragranulomatous;malignant lymphoma, lymphocytic;malignant lymphoma, large cell, diffuse;malignant lymphoma, follicular;mycosis fungoides;other specified non-Hodgkin's lymphomas;malignant histiocytosis;multiple myeloma;mast cell sarcoma;immunoproliferative small bowel disease;leukemia;lymphocytic leukemia;plasma cell leukemia;erythroleukemia;lymphosarcoma cell leukemia;myeloid leukemia;basophilic leukemia;eosinophilic leukemia;monocytic leukemia;mast cell leukemia;megakaryoblastic leukemia;myeloid sarcoma;and hairy cell leukemia.;

[0064] In certain embodiments, the subject is suffering 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's disease, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, gallbladder cancer, gastrointestinal carcinoid, Hodgkin's disease, non-Hodgkin's lymphoma, Kaposi's sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, liver cancer, lung cancer, mesothelioma, plasmacytoma, nasal and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oral 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.

[0065] The present invention also relates to the use of the compounds in the treatment, including prophylactic treatment, of adverse conditions or diseases, in particular those in which dendritic cells and / or T cells are involved and where the condition or disease is ameliorated or treated by stimulation of T cell proliferation and / or phagocytosis by myeloid cells, in particular dendritic cells and / or macrophages. In a particular embodiment, the disease or condition is selected from the group consisting of cancer, in particular the cancers listed herein above, more particularly cancers with TRIM21 positive tumor cells, liquid cancer, solid cancer, lymphoma, colon cancer, mesothelioma or hepatoma.

[0066] In certain embodiments of the invention there are provided compounds which are antagonists of the binding between CLEC-1 and TRIM21, in particular antagonists of the binding between human CLEC-1 and human TRIM21, for use in treating a human subject suffering from a cancer having TRIM21-positive tumour cells.

[0067] In other words, in a particular embodiment, the invention relates to compounds which are antagonists of the binding between CLEC-1 and TRIM21, in particular antagonists of the binding between human CLEC-1 and human TRIM21, for use in the treatment of cancers having TRIM21-positive tumour cells, particularly in human subjects suffering from such cancers having TRIM21-positive tumour cells.

[0068] In particular embodiments, such compounds for use in the treatment of a human subject suffering from a cancer having TRIM21-positive tumour cells or for use in the treatment of a cancer having TRIM21-positive tumour cells bind to CLEC-1, in particular human CLEC-1, in particular specifically bind to CLEC-1, in particular specifically bind to human CLEC-1 or are a functional equivalent of CLEC-1, in particular human CLEC-1 as defined above, in particular are a polypeptide.

[0069] In a particular embodiment, the present invention relates to such antagonist compounds which are antagonists of the binding between CLEC-1 and TRIM21 for use in the treatment of a human subject suffering from a cancer having TRIM21-positive tumor cells or for use in the treatment of a cancer having TRIM21-positive tumor cells, particularly by increasing phagocytosis of TRIM21-positive tumor cells by dendritic cells and / or macrophages.

[0070] In certain embodiments, the compounds for use in the treatment of cancer having TRIM21 positive tumor cells in patients suffering from cancer are particularly for the treatment of a cancer selected from breast cancer, hepatocellular carcinoma, lymphoma, more particularly B-cell lymphoma, colon cancer, thyroid cancer, liver cancer, renal cancer, melanoma, colorectal cancer, nasopharyngeal carcinoma, pancreatic cancer.

[0071] The present invention also relates to the use of a compound which is an antagonist of the binding between CLEC-1 and TRIM21, in particular an antagonist of the binding between human CLEC-1 and human TRIM21, for the manufacture of a medicament for the treatment of a human subject suffering from a cancer having TRIM21-positive tumor cells.

[0072] The invention also relates to the use of a compound which is an antagonist of the binding between CLEC-1 and TRIM21, in particular an antagonist of the binding between human CLEC-1 and human TRIM21, for the manufacture of a medicament for the treatment of a cancer having TRIM21-positive tumor cells, in particular in a human subject suffering from such a cancer having TRIM21-positive tumor cells, the compound being as defined in any of the embodiments disclosed herein.

[0073] The present invention also relates to a method of treating a cancer having TRIM21-positive tumor cells in a human subject in need thereof, comprising administering to said subject an effective amount of a compound which is an antagonist of the binding between CLEC-1 and TRIM21, in particular an antagonist of the binding between human CLEC-1 and human TRIM21, wherein the compound is as defined in any of the embodiments disclosed herein.

[0074] The present invention also relates to a method of treating a human subject suffering from a cancer having TRIM21-positive tumor cells, comprising administering to said subject an effective amount of a compound which is an antagonist of the binding between CLEC-1 and TRIM21, in particular an antagonist of the binding between human CLEC-1 and human TRIM21, the compound being as defined in any of the embodiments disclosed herein.

[0075] The present invention also relates to a method for increasing the phagocytosis of TRIM21 positive cells, more particularly TRIM21 positive tumor cells and / or secondary necrotic cells, in particular by dendritic cells and / or macrophages, in a patient in need thereof, comprising the administration of an effective amount of a compound of the invention, in particular wherein said compound is an antagonist of the binding between CLEC-1 and TRIM21. The compound is as defined in any of the embodiments disclosed herein.

[0076] In another particular embodiment of the present invention there is provided a compound which inhibits the C-type lectin-like receptor 1 (CLEC-1) / tripartite motif-containing protein 21 (TRIM21) signalling pathway, said compound binding to CLEC1, in particular specifically binding to CLEC-1 or reducing the expression of functional CLEC1 or being a functional equivalent of CLEC1, in particular a polypeptide which is a functional equivalent of CLEC-1, in particular said compound binding to CLEC1, more particularly binding to human CLEC-1, in particular specifically binding to human CLEC-1, in a more particular embodiment specifically binding to the extracellular domain of human CLEC-1, an antagonist compound of the binding between CLEC-1 and TRIM21, for use in the treatment of a subject, in particular a human subject, suffering from a cancer having TRIM21 positive tumour cells. Preferably, the compound increases the phagocytic capacity of myeloid cells, in particular dendritic cells and / or macrophages, in particular increases the phagocytosis of TRIM21-positive cells, more particularly TRIM21-positive tumor cells and / or secondary necrotic cells, by dendritic cells and / or macrophages, in particular increases the phagocytosis of tumor cells and / or secondary necrotic cells by dendritic cells and / or macrophages. In particular, the compound is selected from antibodies, antigen-binding fragments thereof, and antigen-binding antibody mimetics which bind to CLEC-1, in particular bind to human CLEC-1 and therefore particularly specifically bind to CLEC-1, which competes with an anti-CLEC-1A antibody having a heavy chain variable domain of SEQ ID NO: 10 and a light chain variable domain of SEQ ID NO: 11 for binding to CLEC-1, in particular human CLEC-1, and the compound is an antagonist of the binding between CLEC-1 and TRIM21, in particular between human CLEC-1 and human TRIM21, which enhances the phagocytic ability of myeloid cells, in particular dendritic cells and / or macrophages, in particular the phagocytosis of TRIM21-positive cells, more particularly TRIM21-positive tumor cells and / or secondary necrotic cells, by dendritic cells and / or macrophages, more particularly the phagocytosis of tumor cells and / or secondary necrotic cells by dendritic cells and / or macrophages.

[0077] In a particular embodiment of the invention there is provided a compound selected from antibodies, antigen-binding fragments thereof, antigen-binding antibody mimetics which bind to CLEC-1, particularly human CLEC-1, and therefore particularly specifically binds to CLEC-1, which competes with an anti-CLEC-1 antibody having a heavy chain variable domain of SEQ ID NO: 10 and a light chain variable domain of SEQ ID NO: 11 for binding to CLEC-1, particularly human CLEC-1, and in particular which compound is an antagonist of the binding between CLEC-1 and TRIM21, particularly between human CLEC-1 and human TRIM21, which enhances the phagocytic capacity of myeloid cells, particularly dendritic cells and / or macrophages, in particular the phagocytosis of TRIM21 positive cells, more particularly TRIM21 positive tumor cells and / or secondary necrotic cells, by dendritic cells and / or macrophages, more particularly the phagocytosis of tumor cells and / or secondary necrotic cells by dendritic cells and / or macrophages, for use in the treatment of patients suffering from cancer, particularly cancer having TRIM21 positive tumor cells. Cancers that may be treated include, by way of example, breast cancer, hepatocellular carcinoma, lymphoma, more specifically B-cell lymphoma, colon cancer, thyroid cancer, liver cancer, renal cancer, melanoma, colorectal cancer, nasopharyngeal carcinoma, pancreatic cancer.

[0078] In a particular embodiment of the invention there is provided a compound selected from antibodies, antigen-binding fragments thereof or antigen-binding fragment mimetics which bind to CLEC-1, particularly human CLEC-1, and therefore particularly specifically binds to CLEC-1 (particularly its extracellular domain), which is an antagonist of the binding between CLEC-1 and TRIM21, particularly between human CLEC-1 and human TRIM21, which competes with an anti-CLEC-1 antibody having a heavy chain variable domain of SEQ ID NO: 10 and a light chain variable domain of SEQ ID NO: 11 for binding to CLEC-1, particularly human CLEC-1, and in particular which compound enhances the phagocytic ability of myeloid cells, particularly dendritic cells and / or macrophages, in particular the phagocytosis of TRIM21 positive cells by dendritic cells and / or macrophages, more particularly the phagocytosis of TRIM21 positive cells, more particularly TRIM21 positive tumor cells and / or secondary necrotic cells, more particularly the phagocytosis of tumor cells and / or secondary necrotic cells by dendritic cells and / or macrophages, for use in the treatment of patients with cancer having TRIM21 positive tumor cells.

[0079] In the examples of the present invention, the referenced antibody, anti-CLEC-1 mAb #5 (also called αCLEC-1 #5), has a heavy chain variable domain of SEQ ID NO: 10 and a light chain variable domain of SEQ ID NO: 11. Competition for binding to CLEC-1 can be assessed by cross-blocking assays (e.g., competitive ELISA assays). In an exemplary competitive ELISA assay, a polypeptide comprising or consisting of at least the extracellular domain of CLEC-1 can be coated on a well of a microtiter plate and pre-incubated with or without a candidate competing antibody, and then a biotin-labeled candidate antibody is added. The amount of labeled candidate antibody bound to the polypeptide is measured using an avidin-peroxidase conjugate and an appropriate substrate. The antibody can be labeled with a radioactive or fluorescent label, or with some other detectable and measurable label. The amount of labeled candidate antibody bound to the polypeptide has an indirect correlation with the ability of the candidate antibody to compete for binding to the same epitope, i.e., the higher the affinity of the candidate antibody for the target, the less labeled competing antibody will bind to the polypeptide-coated well. A candidate antibody is considered an antibody of the present invention that competes for binding to the same polypeptide as the competing antibody if it is able to block binding of the competing antibody by at least 20%, preferably at least 20-50%, and even more preferably at least 50%, compared to a control run in parallel in the absence of the candidate antibody (but which may be in the presence of a known non-competing antibody). It will be understood that variations of this assay may be performed to arrive at the same quantitative value.

[0080] The compound which binds to CLEC-1, in particular human CLEC-1, in particular specifically binds to human CLEC-1, is an antibody, which may be a chimeric antibody, a humanized antibody, a recombinant antibody, a fully humanized antibody, a monoclonal antibody, a deimmunized antibody, in particular a humanized monoclonal antibody.

[0081] In a particular embodiment, the invention relates to compounds which are polypeptides which are functional equivalents of CLEC-1, in particular human CLEC-1, as disclosed herein, in particular such polypeptides fused to immunoglobulin constant domains.

[0082] Pharmaceutical Compositions The present invention also relates to pharmaceutical compositions for use in treating patients with a disease, comprising an antagonist of the CLEC-1 / TRIM21 signaling pathway, in particular an antagonist compound that binds to TRIM21, in particular specifically binds to TRIM21, or a CLEC-1A antagonist that is an antibody or an antigen-binding fragment thereof, or an antigen-binding antibody mimetic, that binds to CLEC-1, and thus in particular specifically binds to CLEC-1, according to any embodiment disclosed herein, together with a suitable vehicle of a pharma- ceutically acceptable medicine for a formulation that can be administered as a therapeutic agent, alone or in combination with a second therapeutic agent, to a patient in need thereof. These formulations can in particular be isotonic and sterile saline (monosodium phosphate, disodium phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride, etc., or mixtures of their salts), or a dried, in particular lyophilized composition, which allows the constitution of an injectable solution upon the addition of, optionally, sterile water or saline.

[0083] Compound combinations The present invention also relates to combinations of compounds comprising a first therapeutic agent and at least one additional therapeutic agent.

[0084] A combination of compounds is a formulation that includes at least two different products, or agents, or compounds, which may be packaged together or packaged separately and are prepared for simultaneous administration, co-administration, or coordinated administration (or sequential administration). In particular, the amount of each compound in the combination may be comprised between 1 μg / kg and 100 mg / kg of patient weight.

[0085] The first therapeutic agent is an antagonist as defined in any embodiment of the invention disclosed herein (i.e., an antagonist that binds to TRIM21, in particular specifically binds to TRIM21, such as an antibody, antigen-binding fragment thereof, or antigen-binding antibody mimetics as disclosed in embodiments of the present invention, a compound that reduces or inhibits a signal transduction pathway activated following interaction between TRIM21 and CLEC-1, a compound that reduces expression of TRIM21, etc.).

[0086] At least one of the further therapeutic agents is a tumor-targeting antibody or antigen-binding fragment thereof, in particular a tumor-targeting monoclonal antibody or antigen-binding fragment thereof, more particularly a tumor-targeting monoclonal antibody or antigen-binding fragment thereof that activates and / or enhances phagocytosis of tumor cells or cells expressing TRIM21 by myeloid cells, in particular dendritic cells and / or macrophages, and even more particularly alemtuzumab, atezolizumab, bevacizumab, cetuximab, herceptin, panitumumab, rituximab, trastuzumab, anti-PDL-1 antibodies and anti-CD4 7 antibodies, or another antibody or monoclonal antibody selected from the group consisting of anti-PD1 antibodies and anti-SIRP antibodies, and / or chemotherapeutic agents, in particular cytotoxic drugs with antiproliferative, proapoptotic, cell cycle arresting, and / or differentiation-inducing effects, more particularly selected from the list consisting of cytotoxic drugs selected from the group consisting of cytotoxic antibodies, alkylating agents, anthracyclines, antimetabolites, microtubule inhibitors, topoisomerase inhibitors, alkaloids, bleomycin, antineoplastic agents, cyclophosphamide. Tumor-targeting antibodies may be defined as therapeutic monoclonal antibodies that recognize tumor-specific membrane proteins, block cell signaling, and induce tumor damage through an innate immune response driven by Fc. Chemotherapeutic agents may be conventional cytotoxic drugs, i.e. compounds that induce irreversible lethal lesions after exposure through interference with DNA replication, mitosis, etc. These agents may have antiproliferative, proapoptotic, cell cycle arresting, and differentiation-inducing effects. These agents are preferentially selected from the group consisting of alkylating agents (cisplatin, chlorambucil, procarbazine, carmustine), anthracyclines and other cytotoxic antibodies, antimetabolites (i.e. methotrexate, cytarabine, gemcitabine), microtubule inhibitors (i.e. vinblastine, paclitaxel, docetaxel), topoisomerase inhibitors (i.e. etoposide, doxorubicin), alkaloids (i.e. vincristine, vinblastine, vinorelbine, camptothecin) or bleomycin (which inhibits the incorporation of thymidine into the DNA strand). The combination may include more than one second therapeutic agent selected from the list.The combinations may also include additional therapeutic agents not recited in the lists, and / or further components such as, but not limited to, pharmaceutical excipients or administration vehicles.

[0087] In certain embodiments, the therapeutic agents may be administered simultaneously, separately or sequentially in the treatment of a disease.

[0088] Identification of TRIM21 as a ligand for CLEC-1 To the applicant's knowledge, previous methods for identifying ligands of CLEC-1 have made it possible to identify the interaction of CLEC1 with ligands overexpressed by necrotic tumor cells, but failed to identify the ligand. The applicant has developed an inventive method leading to TRIM21 as a key endogenous ligand, which method involves the use of specific non-denaturing conditions.

[0089] CLEC-1 is part of the C-type lectin family. Compounds belonging to this family recognize a wide variety of exogenous and endogenous ligands, such as carbohydrates, proteins, lipids, in nucleic acids. Previous attempts to identify the endogenous ligand of CLEC1 revealed that at least one ligand of proteic nature is found in secondary necrotic cells. However, several approaches have been developed to identify at least the ligand of CLEC-1 that did not show any valuable ligands.

[0090] First, limitations were due to the non-specific nature of the ligands. This challenge requires the use of different experimental conditions and different starting materials. Technical fine-tuning was required to obtain and maintain the interaction of the dead sensor to the potentially labile ligand released by secondary necrotic cells. After testing several approaches to identify CLEC-1 ligands without success, a solution was worked out, among which: 1) a double-hybrid screening of a leukemia / activated monocyte cDNA library; 2) a ligand capture trial with a panel of different CLEC-1 fusion proteins immobilized on a surface plasmon resonance (Biacore) chip from different cell extracts (MoDC, PBMC, K562 cell line) expected to contain CLEC-1 ligands, combined with mass spectrometric identification of the retained ligands; 3) immunoprecipitation of CLEC-1 in cellulo from cell extracts obtained after binding of CLEC-1 to its ligands, cross-linking and extraction of cellular proteins.

[0091] Despite considerable efforts, none of these approaches led to the identification of candidate ligands that could be further confirmed by ELISA or BLItz for selective interaction with CLEC-1. There was a need to develop a sensitive and selective method to capture, isolate and identify CLEC-1 ligands, which are known to be expressed in cells undergoing secondary necrosis and in dendritic cells.

[0092] Applicants have developed an unusual, chemically gentle protein extraction method based on low detergent concentrations, combined with rapid freeze-thaw based mechanical restraint to maximize cellular compartment disruption to extract proteins from cells while preserving post-translational modifications of the proteins.

[0093] We focused on cells in which CLEC-1 binding was observed following loss of membrane integrity (e.g., the RAJI cell line). In order to maximize CLEC-1 binding to endogenous ligands while maintaining low non-specific binding, we: 1) Direct binding of Fc-CLEC-1 or Fc-control to protein G beads; without the use of any intermediate antibodies; 2) the use of magnetic beads to minimize non-specific binding; 3) Interactions in PBS alone, without additional detergents except for the low detergent concentrations present in cell extracts; 4) gentle detergent-free washing with increased stringency only at high salt concentrations (preferably 500 mM NaCl) to remove non-specifically bound contaminants; 5) Elution of bound ligands with DTT denaturation only at room temperature to avoid heat-induced protein aggregation, and especially glycoprotein aggregation. The present invention provides an immunoprecipitation method comprising:

[0094] This original method led to the isolation of a discrete band from the SDS-PAGE eluate. Mass spectrometry analysis led to the identification of TRIM-21 as an endogenous CLEC-1A ligand, confirmed by ELISA for selective interaction with CLEC-1A.

[0095] Methods for screening antagonists and agonists of the binding between CLEC-1 and TRIM21 In another aspect, the invention relates to a method of screening for compounds, including but not limited to small organic compounds, antibodies or antigen-binding fragments thereof, polypeptides (particularly functional equivalents of human CLEC-1 or human TRIM21), particularly therapeutic agents that bind to TRIM21 or CLEC-1 and target (i.e. disrupt, reduce, inhibit or enhance) the CLEC-1 / TRIM21 signaling pathway, particularly compounds that compete or antagonize the binding between CLEC-1 and TRIM21, the method comprising: a) providing at least one compound and assessing its (their) ability to interfere with the CLEC-1 / TRIM21 signalling pathway, in particular its ability to interfere with the interaction between CLEC1 and TRIM21, more particularly its ability to compete or antagonise the binding between CLEC-1 and TRIM21, in particular said interaction being determined between an Fc-CLEC-1 fusion molecule and at least one domain of TRIM21, in particular full length TRIM21, or between CLEC-1 and at least one domain of TRIM21, in particular full length TRIM21, b) measuring the activity of the CLEC1 / TRIM21 signalling pathway and / or the binding between CLEC-1 and TRIM21 in the presence of the compound; c) identifying compounds that modulate the activity of the CLEC1 / TRIM21 signaling pathway and / or the binding between CLEC-1 and TRIM21 if the compound enhances or reduces the activity and / or binding of the CLEC1 / TRIM21 signaling pathway measured in step b) compared to a negative control, in particular identifying compounds that enhance or reduce the interaction between CLEC-1 and TRIM21. Includes.

[0096] The measurement in step b) may be assessed by measuring the phagocytic capacity of myeloid cells, in particular dendritic cells and / or macrophages, in the presence of the compound and comparing the results with those observed in the absence of the compound. The phagocytic capacity of myeloid cells, in particular dendritic cells and / or macrophages, may be assessed with respect to TRIM21-positive cells, in particular TRIM21-positive tumor cells or secondary necrotic cells, in particular tumor cells or secondary necrotic cells.

[0097] In another aspect, the invention relates to a method of screening for compounds, including but not limited to small organic compounds, antibodies or antigen binding fragments thereof, polypeptides (particularly functional equivalents of human CLEC-1 or human TRIM21), particularly therapeutic agents that bind to TRIM21 or CLEC-1 and target (i.e. disrupt, reduce or inhibit) the binding between CLEC-1 and TRIM21, particularly compounds that compete or antagonize or affect the binding between CLEC-1 and TRIM21, the method comprising: a) providing at least one compound and assessing its (their) ability to interfere with the binding between CLEC-1 and TRIM21, in particular to compete or antagonize the binding between CLEC-1 and TRIM21, in particular the interaction being determined between at least one domain of CLEC-1 and TRIM21, b) isolating and / or identifying compounds which modulate the interaction (e.g. binding) between CLEC-1 and TRIM21, in particular isolating and / or identifying compounds which enhance and / or decrease the interaction between CLEC-1 and TRIM21, c) optionally testing the binding ability of the compound to TRIM21, in particular human TRIM21, and / or to CLEC-1, in particular human CLEC-1, modulating the interaction (e.g. binding) between CLEC-1 and TRIM21. Includes.

[0098] The present invention also relates to a method for selecting a compound useful for treating a cancer having TRIM21-positive tumor cells, comprising carrying out a method for screening compounds as described herein, wherein a compound that reduces the activity of the CLEC1 / TRIM21 signaling pathway and / or the binding between CLEC-1 and TRIM21 is selected.

[0099] In particular, a method for selecting antagonists which bind to TRIM21, in particular which specifically bind to TRIM21 and antagonize the binding between CLEC-1 and TRIM21, in particular between human CLEC-1 and human TRIM21, comprises the following steps: a) providing a TRIM21 protein or a part thereof comprising at least one domain of TRIM21 selected from the group consisting of RING, B-box, coiled-coil, PRY, PRYSPRY and SPRY, and a CLEC-1 protein or a part thereof comprising the extracellular domain of CLEC-1, in particular Fc-CLEC-1, more particularly Fc-CLEC-1 comprising SEQ ID NO: 3, b) providing a compound to be tested for its ability to antagonize binding between CLEC-1 and TRIM21; c) measuring the binding between CLEC-1 and TRIM21 in the presence of the compound; d) optionally measuring the binding of the compound to TRIM21, in particular human TRIM21, and / or measuring the binding of the compound to CLEC-1, in particular human CLEC-1, where the compound is an antagonist of the binding between CLEC-1 and TRIM21 if it reduces the binding measured in step c) compared to a negative control, and the compound further specifically binds to TRIM21 if the binding to TRIM21 measured in step d) is higher than the binding to CLEC-1, in particular if the affinity of the compound for TRIM21 is at least twice as high as its affinity for CLEC-1. may include.

[0100] The negative control may correspond to a compound of similar nature (e.g. an antibody if the test compound is an antibody, an organic molecule if the compound being tested is an organic molecule) that is known not to interfere with the binding between CLEC-1 and TRIM21, for example because the control does not bind to or interfere with either or both of TRIM21 and CLEC-1.

[0101] The present invention also relates to a method for selecting a compound which binds to TRIM21, in particular which binds specifically to TRIM21 and which is an antagonist of the binding between CLEC-1 and TRIM21, in particular between human CLEC-1 and human TRIM21, said method comprising the steps of: a) providing at least one compound for testing its (their) ability to bind to TRIM21 and to interfere with the interaction between CLEC-1 and TRIM21, in particular to compete or antagonize the binding between CLEC-1 and TRIM21, in particular said interaction being determined between a polypeptide or peptide comprising the extracellular domain of CLEC-1 and at least one domain of TRIM21, in particular full length TRIM21, or an Fc-CLEC-1 fusion molecule and TRIM21, in particular at least one domain of full length TRIM21, in particular said interaction being determined between CLEC-1 and at least one domain of TRIM21, in particular full length TRIM21, b) measuring the interaction (e.g. binding) between CLEC-1 and TRIM21 in the presence of the compound; c) identifying compounds that modulate the binding between CLEC-1 and TRIM21, in particular compounds that enhance or reduce the interaction between CLEC-1 and TRIM21, if the compound enhances or reduces the binding measured in step b) compared to a negative control. Includes.

[0102] Antagonists of the present invention, in particular antagonists that bind to TRIM21, in particular antagonists that specifically bind to TRIM21 and antagonize the binding between CLEC-1 and TRIM21, may be identified by the following method, which comprises at least the following steps: a) providing a plurality of cells expressing TRIM21 or a fragment thereof comprising at least one domain of TRIM21 selected from the group consisting of RING, B-box, coiled-coil, PRY and SPRY; b) incubating said cells, in particular a lysate of said cells, with a candidate compound to be tested for its ability to antagonize the binding between CLEC-1 and TRIM21, in the presence and / or absence of a polypeptide comprising at least a part of the extracellular domain of CLEC-1, c) determining whether the candidate compound binds to, blocks, inhibits or reduces the binding between CLEC-1 and TRIM21; and d) selecting candidate compounds which bind to, block, inhibit or reduce the binding between CLEC-1 and TRIM21.

[0103] Methods for assessing the likelihood that a treatment will be effective In another aspect, the invention relates to a method for assessing the likely efficacy of treatment with an anti-cancer agent and / or an antagonist of the CLEC-1 / TRIM21 signalling pathway in a human patient diagnosed with cancer, in particular a TRIM21 positive tumour or a cancer having TRIM21 positive tumour cells, such as, in particular, breast cancer, hepatocellular carcinoma, lymphoma, more particularly B-cell lymphoma, colon cancer, thyroid cancer, liver cancer, renal cancer, melanoma, colorectal cancer, nasopharyngeal cancer, pancreatic cancer, etc., a chronic infection, sepsis, in particular an infection with a Coxsackie virus or an encephalitis virus, more particularly Coxsackie virus B3 or Japanese encephalitis virus, a cardiovascular disease, an autoimmune disease, in particular Sjögren's syndrome or systemic lupus erythematosus or systemic sclerosis, an inflammatory disease, in particular cancer, said method comprising: - determining the expression of TRIM21 in a biological sample, in particular in a biological sample comprising tumor cells, the sample having been previously obtained from a human patient; - if the sample is positive for TRIM21 expression, concluding that the patient is likely to benefit from treatment with an anti-cancer drug and / or antagonist compound of the invention, in particular an antibody or antigen-binding fragment thereof that is an anti-TRIM21 antagonist. Includes.

[0104] The present invention also relates to methods for assessing the likely effectiveness of treatment with anti-cancer agents and / or compounds which are antagonists of the CLEC-1 / TRIM21 signaling pathway, such as the above-mentioned antibodies, or antigen-binding fragments thereof, or antigen-binding antibody mimetics, functional equivalents of CLEC-1 or TRIM21, compounds which reduce expression of TRIM21, and, if the sample is positive for expression of human TRIM21, the administration of a therapeutic amount of an anti-cancer agent and / or anti-human TRIM21 or anti-CLEC-1, such as an antibody and / or antigen-binding fragment thereof.

[0105] The present invention also relates to an in vitro or ex vivo method for determining whether treatment of a patient having a disease with an anti-TRIM21, particularly an anti-human TRIM21 compound, an anti-CLEC-1, particularly an anti-human CLEC-1 compound, or a functional equivalent of TRIM21, particularly human TRIM21, or a functional equivalent of CLEC-1, particularly human CLEC-1, is likely to be effective, which method comprises detecting expression of TRIM21 in a biological sample previously obtained from the patient, and if TRIM21 is detected on the surface and / or in the cytosol of cells, particularly tumor cells, present in the biological sample previously obtained from the patient, then treatment is likely to be effective.

[0106] In one aspect the invention relates to an in vitro or ex vivo method for determining whether a patient having a disease is likely to benefit from treatment with an anti-TRIM21, particularly an anti-human TRIM21 compound, an anti-CLEC-1, particularly an anti-human CLEC-1 compound, or a functional equivalent of TRIM21, particularly human TRIM21, or a functional equivalent of CLEC-1, particularly human CLEC-1, wherein the disease is cancer, particularly a cancer having TRIM21 positive tumour cells, such as breast cancer, hepatocellular carcinoma, lymphoma, more particularly B cell lymphoma, colon cancer, thyroid cancer, liver cancer, renal cancer, melanoma, colorectal cancer, nasopharyngeal cancer, pancreatic cancer, or the disease is chronic infection, sepsis, , particularly an infection with a Coxsackievirus or an encephalitis virus, more particularly Coxsackievirus B3 or Japanese encephalitis virus, a cardiovascular disease, an autoimmune disease, particularly Sjogren's syndrome or systemic lupus erythematosus or systemic sclerosis, an inflammatory disease, and a method for determining that treating a patient for this disease with the above-mentioned antagonist compound of the present invention is likely to be effective comprises detecting expression of TRIM21 in a biological sample previously obtained from the patient, and if TRIM21 is detected on the surface and / or in the cytosol of cells, particularly tumor cells, present in the biological sample previously obtained from the patient, then there is a likelihood of treatment being effective.

[0107] Thus, in a further aspect, the present invention relates to a method for treating a human patient diagnosed with cancer, particularly cancer with TRIM21 positive tumor cells, such as breast cancer, hepatocellular carcinoma, lymphoma, more particularly B-cell lymphoma, colon cancer, thyroid cancer, liver cancer, renal cancer, melanoma, colorectal cancer, nasopharyngeal cancer, pancreatic cancer, etc., chronic infection, sepsis, infection, particularly infection with Coxsackievirus or encephalitis virus, more particularly infection with Coxsackievirus B3 or Japanese encephalitis virus, cardiovascular disease, autoimmune disease, particularly Sjögren's syndrome or systemic lupus erythematosus or systemic sclerosis, inflammatory disease, said method comprising: - determining the expression of TRIM21 in a biological sample, in particular in a biological sample comprising tumor cells, the sample having been previously obtained from a human patient, and - administering to the human patient, if TRIM21 expression is positive, a drug, in particular an anti-cancer drug, and / or an anti-human TRIM21 antagonist, such as, but not limited to, an antibody or an antigen-binding fragment thereof. Includes.

[0108] In a further aspect, the present invention relates to a method for enhancing the phagocytic capacity of myeloid cells, in particular dendritic cells and / or macrophages, in particular for enhancing the phagocytosis of TRIM21 positive cells, in particular tumor cells and / or secondary necrotic cells, in particular TRIM21 positive tumor cells and / or TRIM21 positive secondary necrotic cells, by myeloid cells, said method comprising the steps of: - selecting a patient having a disorder associated with TRIM21-positive cells, in particular a disorder associated with TRIM21-positive tumor cells and / or TRIM21-positive secondary necrotic cells, and - administering to the patient an effective amount of a compound of the invention that inhibits the CLEC-1 / TRIM21 signaling pathway. Includes.

[0109] In a further aspect, the present invention relates to a method for enhancing the phagocytic capacity of myeloid cells, particularly dendritic cells and / or macrophages, in particular for enhancing the phagocytosis of TRIM21-positive cells, particularly tumour cells and / or secondary necrotic cells, in particular TRIM21-positive tumour cells and / or TRIM21-positive secondary necrotic cells, by myeloid cells, which method comprises the administration to a patient in need thereof of an effective amount of a compound of the present invention which inhibits the CLEC-1 / TRIM21 signalling pathway.

[0110] In a further aspect, the present invention relates to the use of compounds that inhibit the C-type lectin-like receptor 1 (CLEC-1) / tripartite motif-containing protein 21 (TRIM21) signaling pathway, in particular antagonizing the binding between CLEC-1 and TRIM21, more particularly between human CLEC-1 and human TRIM21, which compounds are useful in treating diseases, in particular cancers such as breast cancer, hepatocellular carcinoma, lymphoma, more particularly B-cell lymphoma, colon cancer, thyroid cancer, liver cancer, renal cancer, melanoma, colorectal cancer, nasopharyngeal cancer, pancreatic cancer, in particular cancers having TRIM21 positive tumor cells, chronic infections, sepsis, infectious diseases, viral infections, in particular Coxsackievirus or encephalitis viruses, more particularly Coxsackievirus B3 or is an antagonist compound of the binding between CLEC-1 and TRIM21 which binds to TRIM21, particularly specifically binds to TRIM21, or binds to an intracellular molecule involved in the CLEC-1 / TRIM21 signaling pathway, or reduces the expression of functional TRIM21, or a functional equivalent of TRIM21, particularly binds to TRIM21, more particularly binds to human TRIM21, particularly specifically binds to TRIM21, for the manufacture of a medicament for the treatment of a patient having a disease selected from the group consisting of infection with Japanese encephalitis virus, cardiovascular disease, autoimmune disease, particularly Sjogren's syndrome or systemic lupus erythematosus or systemic sclerosis, inflammatory disease.

[0111] In particular, methods for treating a human patient diagnosed with cancer such as breast cancer, hepatocellular carcinoma, lymphoma, more particularly B-cell lymphoma, colon cancer, thyroid cancer, liver cancer, renal cancer, melanoma, colorectal cancer, nasopharyngeal cancer, pancreatic cancer, in particular cancers with TRIM21-positive tumour cells, chronic infection, sepsis, infection, viral infection, in particular infection with a Coxsackievirus or an encephalitis virus, more particularly infection with Coxsackievirus B3 or Japanese encephalitis virus, cardiovascular disease, autoimmune disease, in particular Sjögren's syndrome or systemic lupus erythematosus or systemic sclerosis, inflammatory disease, in particular cancer, may comprise the step of determining whether a biological sample obtained from the patient, in particular a biological sample comprising pathological cells, in particular tumour cells, comprises cells which express TRIM21, before administration of an agent, in particular an anti-cancer agent, or during treatment with such an agent, in particular such an anti-cancer agent, or after treatment with a first agent or a further agent, in particular an anti-cancer agent. If the sample may be considered TRIM21 positive, particularly if the patient has TRIM21 positive tumor cells, then treatment is administered.

[0112] Determining the expression of human TRIM21 may involve the use of chimeric molecules comprising the extracellular domain of CLEC-1 as described herein, in particular the use of Fc-CLEC-1 as described herein.

[0113] The compounds of the invention may be administered as therapeutic agents, in particular anti-cancer agents, in particular to patients with CLEC-1 positive tumour cells and / or TRIM21 positive tumour cells, or may be added as a further active ingredient in a treatment with another drug, in particular an anti-cancer drug.

[0114] Thus, the invention provides TRIM21 expressed in cells of a biological sample previously taken from a patient, such as a sample comprising tumour cells or apoptotic or necrotic cells, for use as a positive marker for the selection of patients for treatment with an immunotherapeutic or chemotherapeutic anti-cancer agent, optionally including quantification of the marker.

[0115] Thus, in a further aspect, the present invention relates to a method for treating a human patient diagnosed with cancer, a human patient having a TRIM21-positive tumor. A TRIM21-positive tumor may correspond to a tumor in which at least one cell expresses TRIM21. Thus, the present invention also relates to a method in which the presence of TRIM21 or the abnormal expression of TRIM21 in tumor cells is determined in a biological sample (previously) obtained from the patient, and if the tumor cells express TRIM21 or show abnormal expression of TRIM-21, a compound inhibiting the CLEC-1 / TRIM21 signaling pathway, as detailed herein, is administered to the patient. The abnormal expression of TRIM21 may be assessed by comparing the expression of TRIM-21 in healthy cells obtained from the same patient or in healthy cells obtained from a different patient.

[0116] The present invention also relates to the use of TRIM21, in particular human TRIM21, as a biomarker in in vitro tests to assess the likelihood that treating a patient with a disease, in particular cancer, more particularly cancer having TRIM21-positive tumor cells, with a compound that binds to CLEC-1 or TRIM21, which is an antagonist of the binding between CLEC-1 and TRIM21, will be effective.

[0117] Further aspects and features of the present invention are seen in the following examples and drawings.

[0118] The following figures and examples are set forth to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the invention, and are not intended to limit the scope of what the inventors consider to be their invention, nor are they intended to represent that all or only the experiments described below have been performed. Although the invention has been described with reference to specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, method, method step or steps, to the objective, spirit and scope of the invention. All such modifications are intended to be within the scope of the claims appended hereto. [Brief description of the drawings]

[0119] [Figure 1] Figure 1 shows SDS-PAGE of immunoprecipitation of Fc-CLEC-1 with contents of RAJI cell line lysates. A: Immunoprecipitation elution products were separated by SDS-PAGE on stain-free 4-15% gels. Protein G magnetic beads were coated with Fc only (lanes 1 and 2), no Fc protein (lane 3), and Fc-CLEC-1 protein (lanes 4 and 5). Fc-CLEC-1A immunoprecipitation of RAJI WCE (whole cell extract) proteins (lane 5) was compared to several controls, Fc only (lane 1), Fc+RAJI WCE (lane 2), RAJI WCE (lane 3), and Fc-CLEC-1A only (lane 4). Grey and white triangles indicate a discrete band present only in the Fc-CLEC-1A+RAJI WCE condition (lane 5). Asterisks indicate monomeric* (i.e. partially denatured) and dimeric** Fc-fusion recombinant proteins. B: Immunoprecipitation elution products were separated by SDS-PAGE on 4-15% gels stained with colloidal Coomassie. Immunoprecipitation was performed as in A. Boxes indicate areas excised from the gel for mass spectrometry analysis and correspond to the major bands specific for Fc-CLEC-1-RAJI WCE co-immunoprecipitation (boxes in lanes 7 and 8 correspond to the white triangles in A and originate from the same extracts as lane 5). Boxes in lanes 6 and 9 and 10 correspond to controls at apparently the same size levels (lanes 6: Fc-CLEC only, lanes 9 and 10: RAJI WCE only). [Diagram 2] Figure 2: Analysis of potential ligands for CLEC-1A. Molecular analysis of the contents of the 50 kDa band extracted from SDS-PAGE and of the total eluate obtained after cell protein extract interaction with beads coupled with Fc-CLEC-1 as illustrated in Figure 1. RAJI corresponds to extracts generated from RAJI cells. HPB-ALL corresponds to extracts generated from a leukemic cell line called HPB-ALL (DSMZ No. ACC 483). [Diagram 3]Figure 1: Co-immunoprecipitation of TRIM21 with CLEC-1A. Immunoprecipitation of WCE proteins from RAJI cells (lanes 5-8) or MoDCs (lanes 9-12) was performed with magnetic beads carrying Fc only (lanes 2, 6 and 10), Fc-CLEC-1A (lanes 3, 7 and 11), Fc-Dectin-1A (lanes 4, 8 and 12) or no Fc fusion protein (lanes 1, 5 and 9). Lanes 1-4 are controls made without cell extract. A: Western blot of co-immunoprecipitated (IP) proteins with anti-TRIM21 antibody revealed. TRIM21 is evident in samples with Fc-CLEC-1A (band at 52 kDa). B: Western blot of unretained extracts (samples depleted from IP supernatant). Fc-CLEC-1a is also depleted (lanes 7 and 11). C: Co-immunoprecipitation of RAJI (top) or MoDC (bottom) from whole cell extracts was performed with fixed Fc-CLEC in the presence of 50-fold molar excess of various antibodies. A: negative control antibody, B: anti-TRIM21 antibody (PA5-22294, Invitrogen). Western blot revealed bound TRIM21. [Figure 4] Figure 1: ELISA binding of human Fc-CLEC1 and His-CLEC1 to TRIM21. Recombinant human TRIM21 protein was immobilized on plates. Plates were bathed and dose response of biotinylated Fc-CLEC1-A protein was captured with TRIM21. Fc-CLEC1-biot and His-CLEC1-biot represent CLEC1 protein as described herein with either a His tag or Fc protein, biotinylated. Hum Fc ctrl OSE represents the homemade human receptor domain. [Diagram 5]Figure 1 shows ELISA measurements of the antagonistic activity of anti-TRIM21 antibodies against the binding between CLEC-1 and TRIM21. (A) Localization of epitopes targeted by anti-TRIM21 antibodies in full-length TRIM21 protein. Boxes represent different domains of TRIM21 and arrows represent the regions recognized by the antibodies associated with each arrow. Numbers correspond to the position of amino acid residues in full-length TRIM21. (B) Anti-TRIM21 antibodies used in the experiments are listed to the right of the graph. Recombinant human TRIM21 protein was immobilized on a plate. The plate was bathed in anti-TRIM21 antibody and the dose response of biotinylated Fc-CLEC1 protein was captured. [Figure 6] Figure 1 shows the interaction between TRIM21 and CLEC-1 in the presence of different anti-CLEC1 antibodies (αCLEC-1 mAb #3 and αCLEC-1 mAb #5), an anti-TRIM21 antibody (Fisher PA5-22295) and a control antibody that does not bind either CLEC-1 or TRIM21. (A) represents the percentage of interaction between TRIM21 and CLEC1 compared to the baseline corresponding to the experiment in the presence of the control antibody. (B) SDS-PAGE illustrating the co-immunoprecipitation of TRIM21 with Fc-CLEC or Fc-control in the presence of the above mentioned antibodies. (C) BIACORE. [Figure 7] FIG. 1 shows the phagocytosis index of HPB-ALL cells by human macrophages in the presence of increasing concentrations of Fc-CLEC-1A or Fc-control peptide compared to untreated HPB-ALL cells. [Figure 8-1]Figure 8A shows phagocytosis of TRIM-21 positive tumor cells (Raji cells) in the presence of rituximab (10 ng / ml) and various anti-TRIM21 or anti-CLEC1 or control antibodies. The anti-TRIM21 antibody corresponds to the anti-TRIM21 antibody designated herein as MAB-62191, which binds to an amino acid sequence located between amino acid residues 195 and 293 of SEQ ID NO:2. Figure 8B shows phagocytosis of TRIM-21 positive tumor cells (Raji cells) in the presence of rituximab (10 ng / ml) and various anti-TRIM21 or anti-CLEC1 or control antibodies. The anti-TRIM21 antibody corresponds to the anti-TRIM21 antibody designated herein as MAB-6219(1), which binds to an immunogen corresponding to the amino acid sequence shown in SEQ ID NO:8. [Figure 8-2] Figure 8C shows phagocytosis of TRIM-21 positive tumor cells (Raji cells) in the presence of rituximab (10 ng / ml) and various anti-TRIM21 or anti-CLEC1 or control antibodies. The anti-CLEC1 antibody corresponds to the anti-CLEC1 antibody designated herein as αCLEC-1 mAb #5, having a heavy chain of SEQ ID NO: 10 and a light chain of SEQ ID NO: 11. Figure 8D shows phagocytosis of TRIM-21 positive tumor cells (Raji cells) in the presence of rituximab (10 ng / ml) and various anti-TRIM21 or anti-CLEC1 or control antibodies. Western blot of anti-TRIM21 on Raji cell lysates. Raji cell lysates (50 μg samples) and control recombinant TRIM21 protein (0.010 μg) were transferred on SDS-Page stain-free gels 10% (Biorad), transferred to nitrocellulose membranes, incubated with anti-TRIM21 antibody (clone AB01 / 1G5 (Biorad, #VMA00730)) and then revealed with peroxidase-conjugated anti-mouse antibody (Jackson Immunoresearch #715-036-151). TRIM21 protein was detected at 50 KDa in Raji lysates and control proteins. [Figure 9]Figure 1 shows the positive effect of antagonist monoclonal antibodies that inhibit CLEC-1 interaction with TRIM21 on macrophage-mediated phagocytosis. A: In the context of tumor cell lines lacking CLEC-1 expression (but expressing TRIM21), macrophage-mediated phagocytic activity is not enhanced by treatment with αCLEC-1 mAb #5, which inhibits CLEC-1 interaction with TRIM21. B: In the context of tumor cell lines expressing both CLEC-1 and TRIM21, enhancement of macrophage-mediated phagocytic activity by treatment with αCLEC-1 #5, which inhibits CLEC-1 interaction with TRIM21. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0120] Materials and Methods Generation of recombinant soluble human Fc-CLEC1 protein Soluble Clec1-human IgG Fc chimera (Fc-Clec-1) was generated by cloning the extracellular domain of human Clec-1 (Q74-D280) fused to the N-terminus of human IgG1e3 Fc sequence into pcDNA3.1. The IgG1e3 Fc sequence contains the following mutations E233P / L234V / L235A / ΔG236+A327G / A330S / P331S (substitutions into IgG2 and IgG4 residues that reduce FcγRI binding and ADCC / CDC). The detailed sequence is available in technical report pFuse-hIgG1e3-Fc1 (Invitrogen #pfc1-hg1e3). Synthesis of the Fc-Clec sequence was ordered from GenScript with 5' XhoI and 3' XbaI restriction sites for cloning into pcDNA3.1. For expression in mammalian cells, a Kozak sequence (GCCACC - SEQ ID NO: 10) and an IgK leader signal peptide (METDTLLLWVLLLWVPGSTGD - SEQ ID NO: 9) were added to the beginning of the sequence. Midipreps of plasmid pcDNA3.1-Fc-Clec were made with the Nucleobond Xtra Midi EF kit (Macherey Nagel #740420.50). Recombinant Fc-Clec protein was produced in Hek FreeStyle cells (Thermo Fisher #R79007) by transient transfection using 293fectin transfection reagent (Thermo Fisher #12347019). 6Hek FreeStyle cells were transfected with 60 μg of plasmid pcDNA3.1-Fc-Clec and 120 μl of 293fectin in 60 ml of FreeStyle293 medium (Thermo Fisher #12338018). Production was maintained for 5 days, then the supernatant was clarified by centrifugation at 3000G for 30 minutes and filtered through 0.22 μM Stericup (Merck Millipore #SCGPU05RE). The clarified supernatant was purified on a HiTrap Protein A column (GE #17040201) with elution in 0.1 M citric acid pH 3. The eluate was concentrated on a Microsep Advance 10 kDa (Pall #MCP010C41) and filtered through a 0.22 μM filter.

[0121] Introduction of monocyte-derived dendritic cells (MoDCs) MoDCs were generated from monocytes obtained after magnetic PBMC separation with a typical monocyte kit Miltenyi (130-117-337) or CD14 beads (130-050-201). Monocytes underwent differentiation for 6-7 days in culture in complete RPMI medium (10% FCS, 1% PS, 1% L-Gln, 1x Na-pyruvate, 1x NEAA, 1mM HEPES) supplemented with 50ng / ml GM-CSF (Miltenyi-130-095-372) and 20ng / ml IL-4 (Miltenyi-130-093-917). Immature dendritic cells (iDCs) were further transformed into fully differentiated MoDCs by 2 days of culture in the presence of 20ng / ml TGFb.

[0122] Generation of whole cell protein extracts (WCE) from cells Lymphoblastoid cell line RAJI and MoDC cells were washed extensively with cold PBS. Cell pellets were then resuspended in 5 volumes of ice-cold mild RIPA-IP lysis buffer composed of PBS (Corning), 1% NP-40, 0.25% Na deoxycholate, 1x protease inhibitor cocktail (PIC) (all from Sigma Aldrich). To complete cell lysis, samples were subjected to a quick freeze at -150°C and then thawed in ice-cold water. The cycle was repeated twice. Cell lysates were clarified by centrifugation at 14000g for 15 min at 4°C. Protein concentration was assessed by BCA assay.

[0123] Fc fusion protein immunoprecipitation and candidate band isolation Protein G SureBeads magnetic beads (Biorad) were prepared according to the manufacturer's recommendations and resuspended in PBS + 0.1% Tween-20 (Sigma Aldrich). 30 pmol of Fc fusion protein (Fc only, Fc-CLEC-1, or Fc-hDectin-1A (Invivogen)) was added to beads in an original volume of 30 ul per IP. Fc fusion protein was allowed to bind to the beads for 1 hour on a rotating wheel at +4°C. After 3 washes in PBS-tween followed by one in PBS, 1 mg of WCE extract was added in a final volume of 600 ul of cold PBS + 1x PIC. The interaction was allowed to proceed for 2-3 hours on a rotating wheel at +4°C. The supernatant was collected and the magnetic beads were subjected to 5 min washing in 600 ul on a wheel followed by 2 min magnetic capture, all at +4°C; once in PBS, twice in PBS with 500 mM NaCl and twice in PBS. Bound proteins were recovered after 10 min incubation at room temperature in 15 ul elution buffer consisting of 1x Laemmli buffer (Biorad) with 250 mM DTT (Sigma Aldrich). The eluate was collected and proteins were separated by SDS-PAGE. SDS-polyacrylamide gels were stained with colloidal Coomassie: 0.08% Coomassie Brilliant Blue G250, 10% citric acid, 8% ammonium sulfate (all Sigma Aldrich), 20% methanol (Fisher) for 6 h to overnight with gentle shaking. The gel was destained with distilled water until the background was clear. Bands of interest were excised and studied by mass spectrometry analysis.

[0124] In-gel digestion, extraction and mass spectrometry analysis After extrusion of the bands, the gel plugs were washed four times with 50 μL of 25 mM ammonium bicarbonate (NH4HCO3) and 50 μL of acetonitrile. Cysteine ​​residues were reduced with 50 μL of 10 mM dithiothreitol at 57 °C and alkylated with 50 μL of 55 mM iodoacetamide. After dehydration with acetonitrile, proteins were excised from the gel with 200 ng of modified porcine trypsin (Promega) in 25 mM NH4HCO3. Digestion was carried out overnight at 37 °C. The resulting peptides were extracted with 60% acetonitrile in 0.1% formic acid, followed by a second extraction with 100% acetonitrile. Acetonitrile was evaporated under vacuum and peptides were resuspended in 25 μL of H2O and 0.1% formic acid prior to mass spectrometry analysis. NanoLC-MS / MS analysis of tryptic peptides was performed on a nanoACQUITY Ultra-Performance_LC system (Waters, Milford, MA) coupled to a TripleTOF 5600 mass spectrometer (AB Sciex) equipped with a nanoelectrospray ion source.

[0125] Samples were captured on a Symmetry C18 precolumn (20 x 0.18 mm, 5 μm, Waters) and peptides were separated on an ACQUITY UPLC® BEH130C18 column (75 μm x 250 mm, 1.7 μm particle size, Waters). The solvent system consisted of 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). Capture was performed with 99% solvent A and 1% solvent B at 5 μL / min for 3 min. Elution was performed at a flow rate of 300 nL / min using a 3-25% gradient (solvent B) over 60 min followed by a 25-40% gradient over 20 min, then 80% (solvent B) over 10 min at 60 °C.

[0126] The mass spectrometer was operated in positive mode with the following settings: ion spray float voltage (ISVF) 2300 V, curtain gas (CUR) 25 psi, interface heater temperature (IHT) 75 °C, ion source gas 1 (GS1) 2 psi, declustering voltage (DP) 100 V. Information-dependent acquisition (IDA) mode was used with the top 5 MS / MS scans. MS scans had a 50 min accumulation time in the m / z [400–1250] range, and MS / MS scans had a 50 min m / z [150–1600] range in high sensitivity mode. Switching criteria were set to ions with charge states 2–4, abundance thresholds >150 counts, and exclusion time of 12 s. The IDA rolling collision energy script was used to automatically adapt the CE. Mass calibration of the spectrometer was achieved using peptides from digested BSA. The complete system was fully controlled by AnalystTF 1.6 (AB Sciex).

[0127] The collected raw data was processed and converted into “.mgf peak list” format.

[0128] Data analysis and protein identification The search engine MASCOT 2.6.2 algorithm (Matrix Science) was used. A search was performed against the Human database linked to the same reversed sequence ('Target / Decoy' database). Trypsin was selected as the cleavage enzyme, a maximum of 1 missed cleavage was allowed, and a variable modification (oxidation of methionine residues) of 1 and a fixed modification (carbamidomethylation of cysteine) of 1 were specified.

[0129] Spectra were searched at 20 ppm for MS and a mass tolerance of 0.07 Da for MS / MS data.

[0130] For validation, Mascot results were loaded into Proline software (http: / / proline.profiproteomics.fr). For validation, a false discovery rate (FDR) of 1% was applied to the protein set (at the protein level).

[0131] Western blot Proteins separated by SDS-PAGE were transferred to nitrocellulose membranes, followed by blocking in PBS-tween + 5% BSA (Sigma). Membranes were incubated with anti-TRIM-21 (SCBT sc-25351 1 / 1000e) overnight (O / N) at 4°C and successively with anti-mouse-HRP conjugate (Jackson) and revealed with Clarity ECL substrate (Biorad).

[0132] ELISA The interaction between Fc-CLEC-1A and TRIM21 was measured by ELISA. PolySorp 96-well plates (Nunc) were coated with 50 μl per well of TRIM21-his (Sino 18010-H07B) at 2 μg / ml in carbonate buffer (pH 9.2) overnight at 4°C. The plates were submerged in SuperBlock buffer (#37515, Thermo Fisher). Fc and Fc-CLEC-1A biot binding proteins were captured at 30 μg / ml in 50 μl of PBS-Tween 0.05%-BSA 1% for 2 h at 37°C. After extensive washing, bound proteins were detected with 50 μl / well of 1 / 1000 diluted streptavidin-PO (JIR 016-030-084) in PBS-Tween 0.05% for 1 h at 37° C. and TMB (Sigma T8665) was used for development.

[0133] The antagonistic effect of commercial anti-TRIM21 antibodies on the CLEC1 / TRIM21 binding interaction was measured by ELISA. Recombinant human TRIM21 protein (#18010-H07B, Sino Biological) was immobilized on 96-well PolySorp plates at 2 μg / ml in carbonate buffer (pH 9.2) overnight at 4°C. The plates were submerged in SuperBlock buffer (#37515, Thermo Fisher) and commercial anti-TRIM21 antibodies were added in excess (20 μg / ml). Biotinylated human Fc-CLEC1 (#07 / 08 / 20, OSE IMMUNO) or His-CLEC1 (#1704-CL, Biotechne) proteins were added at 30 μg / ml in a dose response manner and binding was measured. After incubation and washing, streptavidin-peroxidase (#016-030-084, Jackson Immunoresearch, USA) was added and revealed by conventional methods.

[0134] CLEC / TRIM21 interactions in Biacore Analysis was performed on a T200 (Cytiva, France) by platform PP2I (Montpellier, France). Recombinant human TRIM21 protein (produced in baculovirus, Sino Bio #18010-H07B) was immobilized on a CM5 sensor chip (Cytiva) at 20 μg / ml in acetate 10 mM pH 5 buffer. Immobilized TRIM-21 recombinant protein was injected with His-CLEC (produced in NSO cell line, Biotechne #1704-CL) at 1 μM alone or at 5 μM after co-incubation with competing anti-CLEC mAbs.

[0135] Antagonism assay by co-IP Volumes were adjusted and co-IP was performed as in the Fc-fusion protein immunoprecipitation method described above with the following modifications: 5 pmol of Fc-fusion protein, 150ug WCE, and 250 pmol of the antibody being tested for antagonistic activity were used in each reaction. Antibodies were incubated for 30 min at +4°C with their target (WCE) for anti-TRIM antibodies; with Fc-CLEC fusion protein bound to beads for anti-CLEC antibodies, before loading the extracts onto the beads carrying the Fc-CLEC fusion protein. Eluted proteins were separated by SDS-PAGE and revealed by Western blot. Quantification was performed through a ChemiDoc imaging system (Biorad).

[0136] Phagocytosis assay 2.5 × 10 cells labeled with CellTracker Green (Thermo Fisher, Waltham, MA, USA, 1 / 2000, 20 min at 37°C) in serum-free RPMI. 4 Human M1 macrophages and 5 × 10 labeled with CPD (Thermo Fisher, 1 / 2000, 10 min at 37 °C) 4In vitro phagocytosis assays were performed by co-culture of 1 h of leukemic HPB-ALL or Raji cells. Phagocytosis was analyzed by analysis using a CytoFLEX flow cytometer (Beckman, Brea, CA, USA) and FlowJo software (TreeStar, BD Life Sciences, Franklin Lakes, NJ, USA). The phagocytosis index was calculated as follows: the fold change of the percentage of CPD+ cells in CTG+ macrophages compared to that detected in control wells (no treatment) multiplied by the fold change of the geometric mean of APC fluorescence (CPD) in CTG+ macrophages compared to that detected in control wells (no treatment). Treatments applied were Fc control (IgG1-E3) or FcCLEC1A (IgG1-E3) at the concentrations indicated in Figure 9, or anti-TRIM21 antibodies called MAB-6219(1) and MAB-62191 at a concentration of 10 μg / ml or an anti-CLEC1 antibody called αCLEC-1 mAb #5 (Figure 8). The anti-CLEC1 antibody called αCLEC-1 mAb #5 has a heavy chain variable domain according to SEQ ID NO: 10 and a light chain variable domain according to SEQ ID NO: 11.

[0137] Primary human macrophage-mediated phagocytosis of TRIM21+ / CLEC1+ versus TRIM21+ / CLEC1- tumor cells. 2.5 × 10 cells labeled with CellTracker Green (Thermo Fisher, Waltham, MA, USA, 1 / 2000, 20 min at 37°C) in serum-free RPMI for 15 min. 4 Human M1 macrophages and 5 × 10 labeled with CPD (Thermo Fisher, 1 / 2000, 10 min at 37 °C) were 4In vitro phagocytosis assays were performed by co-culture with tumor cells. The tumor cells used in these assays were THP1 cells expressing TRIM21 but not CLEC-1, or THP1 cells expressing both TRIM21 and CLEC-1 (CLEC-1 expression introduced by lentiviral transduction of the human full-length sequence of the CLEC-1 gene). Phagocytosis was analyzed by analysis using a CytoFLEX flow cytometer (Beckman, Brea, CA, USA) and FlowJo software (TreeStar, BD Life Sciences, Franklin Lakes, NJ, USA). The phagocytosis index was calculated as follows: the expression ratio of the percentage of CPD+ cells in CTG+ macrophages compared to that detected by treatment with the isotype control multiplied by the expression ratio of the geometric mean of APC fluorescence (CPD) in CTG+ macrophages compared to that detected by the isotype control. The antagonist monoclonal antibodies used in this assay in FIG. 9 are αCLEC-1 mAb #5 and the corresponding isotype control (evitria, Schlieren, Switzerland). EXAMPLES

[0138] Identification of TRIM21 as a ligand for CLEC-1 Previous attempts to capture CLEC-1A ligands from libraries or from endogenous cellular proteins obtained under strong ionic detergent conditions were unsuccessful. It was shown that CLEC-1A ligands are found in abundance in permeabilized cells derived from the RAJI cell line. These cells are easily expandable in cell culture, allowing the preparation of large amounts of cellular protein extracts from which the ligands are available. Previous attempts using strong detergents did not allow the capture of the more abundant intracellular ligands, and the problem was the quality and integrity of the ligands extracted from the cells. To obtain such integrity, applicants have investigated certain suitable conditions, focusing on milder detergent extraction and interaction conditions.

[0139] RAJI cells were lysed in RIPA-IP buffer with low anionic detergent content and subjected to mechanical disruption of cell compartments by repeated freeze-thaw cycles. To identify ligands of CLEC-1 from the resulting whole cell extracts (WCE), a fusion protein consisting of a dimeric form of the human CLEC1A extracellular domain fused to the Fc portion of human IgG was used. CLEC7a (Dectin-1) fusion protein and a dimeric Fc fragment were used as controls. First, Fc, Fc-CLEC1A or no capture protein were directly bound to Protein G magnetic SureBeads through the Fc fragment. RAJI WCE was subjected to interaction with these beads bound to proteins, carefully washed under low detergent but high salt conditions, and eluted with 250 mM DTT without heating. SDS-PAGE separation of the eluted proteins revealed only a few bands specific for Fc-CLEC-1A (Fig. 1A, lane 5) and clear background in control conditions (Fig. 1A, lanes 1-4). The low background confirmed our hypothesis that mild interaction conditions are required to capture the ligand without denaturation. For mass spectrometry analysis, the experiment was repeated in duplicate and the prominent specific band of Fc-CLEC1A around 50 kDa was excised (Fig. 1B).

[0140] Mass spectrometry analysis was performed on this band or on the entire eluate obtained after interaction of RAJI or HPB-ALL WCE with Fc-CLEC bound to beads versus Fc alone. Raw data was filtered to eliminate typical impurities (e.g. skin proteins) and proteins bound to Fc. The final short list contains proteins that were identified with at least seven specific peptides (Figure 2). TRIM21 E3 ubiquitin protein ligase stands out from this short list, being the only one identified with sequence coverage above 50% in three conditions for RAJI cells and a simple condition for HPB-ALL cells. The other candidates had lower scores and were mainly cytoskeletal proteins and abundant cellular proteins. EXAMPLES

[0141] Interaction between TRIM21 and CLEC-1 Since TRIM21 is known as an intracellular Fc receptor, it is important to verify that binding to Fc-CLEC-1A is mediated through CLEC-1A itself and not its Fc portion. We repeated this immunoprecipitation using Fc and Fc-Dectin as a control, and anti-TRIM21 Western blot revealed bound protein. With RAJI cell extracts, this confirmed that TRIM21 from RAJI WCE strongly co-immunoprecipitated (co-IP) with Fc-CLEC-1A (Figure 3A, lane 7), but the signal was undetectable with Fc-Dectin (lane 8) and much lower with Fc alone (lane 6). This weak signal is indeed mainly due to background signal generated by Fc alone in the absence of cell extracts, and possibly due to detection of Fc cross-reactivity by a secondary HRP-conjugated anti-mouse Fc antibody. The specific interaction between Fc-CLEC-1A and TRIM21 was further maintained by the depletion of TRIM21 from the supernatant after IP reaction containing proteins that were no longer retained (Figure 3B) only by magnetic beads carrying Fc-CLEC-1A (compare lane 7 with lanes 5, 6 and 8) but not by beads carrying Fc only, beads carrying Fc-Dectin or beads without Fc fusion protein. The experiment was repeated using monocyte-derived dendritic cell (MoDC) protein extract as the starting endogenous protein mixture. The same specific co-IP of TRIM21 was observed (Figure 3A, lane 11) and an even stronger interaction signal and a very clear depletion of TRIM21 from the protein extract with CLEC-1A (Figure 3B, lane 11). Taken together, these experiments demonstrate a strong and specific interaction between Fc-CLEC-1A and endogenous TRIM21 that is not mediated through the Fc portion of the fusion protein but through CLEC-1A itself. The reality of this interaction is supported by our previous observation of CLEC-1A binding to permeabilized RAJI and MoDC cells.In Figure 3C it is illustrated that the anti-TRIM21 antibody prevented the binding of TRIM21 to immobilized CLEC-1 (lane 4) compared to the negative control antibody (lane 3), thus indicating an antagonist effect of the anti-TRIM21 antibody on the CLEC-1 / TRIM21 interaction.

[0142] Co-immunoprecipitation reveals an interaction between CLEC-1A and endogenous TRIM21 in the cellular protein mixture. However, to prove a direct interaction without any intervening partners in the cell extracts, ELISA experiments were performed with recombinant Fc-CLEC1A, His-CLEC1A and TRIM21 proteins. ELISA dose-dependence curves of Fc- or His-binding proteins bound to TRIM21 revealed selective binding of CLEC-1A to TRIM21 starting at a concentration of 1 μg / ml and no binding to any of the Fc fusion protein controls (Figure 4A). Interestingly, there was no binding to Fc alone (Fc-biot control), suggesting that at these concentrations the interaction between CLEC-1A and TRIM21 was not mediated through the Fc fragment. Furthermore, no interaction was observed between TRIM21 and another C-type lectin receptor, Dectin-1 (Figure 4B), supporting a specific and selective TRIM21-CLEC-1A direct interaction. Human His-CLEC1 and human Fc-CLEC1 bind to human TRIM21 with an EC50 of 13 and 15 μg / ml, respectively. This is further illustrated in FIG. 4B, where at a fixed concentration of 10 μg / ml (CLEC1A or control), Fc-CLEC1A is the only protein that interacts with the coated TRIM21 protein. In the absence of coated TRIM21, no non-specific binding was observed with either Fc fusion protein. As illustrated in FIG. 4C, at a concentration of 10 μg / ml, Fc-CLEC-1 binds to surfaces coated with TRIM21 and is unable to bind to surfaces not coated with TRIM21. It is therefore clear that TRIM21 and CLEC-1 interact. Compounds that are able to bind to TRIM21 or CLEC1A and antagonize the interaction between these two molecules therefore represent potential therapeutic agents, particularly for treating diseases involving TRIM21 and / or CLEC-1, specifically the TRIM21 / CLEC-1 signaling pathway. This ELISA interaction validation confirms the co-IP results and validates the direct interaction of endogenous TRIM21 from lymphoma or dendritic cells with CLEC-1A, thus identifying the first endogenous CLEC-1A ligand.

[0143] In summary, biotinylated His-CLEC-1A and Fc-CLEC1A bind to TRIM21. The binding between CLEC-1 and TRIM21 is not associated with the Fc domain and these experiments demonstrate a direct interaction between CLEC-1 and TRIM21. To further demonstrate this direct interaction between CLEC-1 and TRIM21, biotinylated Fc-CLEC-1 and His-CLEC-1 were added to surfaces that were either coated or uncoated with human TRIM21. EXAMPLES

[0144] Antagonist compounds of the binding between CLEC-1 and TRIM21 Five commercially available anti-TRIM21 antibodies were tested for their antagonistic activity against the TRIM21 / CLEC1 interaction.

[0145] [Table 1]

[0146] Anti-TRIM21 antagonist antibodies sc-25351, PA5-22294, PA5-18147 and MAB-62191, MAB-621911 are particularly promising antagonists of the interaction between CLEC1A and TRIM21. In FIG. 5A, the domains of interaction between each anti-TRIM21 antibody used in this example and the TRIM21 protein are localized. As can be seen in FIG. 5B, the interaction between Fc-CLEC1 and TRIM21 is reduced by more than 50% by these antibodies. Thus, several different anti-TRIM21 antibodies specifically antagonize the binding between CLEC-1 and TRIM21.

[0147] Anti-CLEC-1 and anti-TRIM21 antagonist antibodies were also tested to evaluate their respective effects on TRIM21 / CLEC-1 interaction in corresponding assays. In these experiments, the antagonist effects of two in situ anti-CLEC-1 antibodies (αCLEC-1 mAb #3 and #5) and anti-TRIM21 antibody Fisher PA5-22295 were compared; these three antibodies reduce the binding between CLEC-1 and TRIM21 by 40%, 55% and 65%, respectively (Figure 6A). The same effect was measured in an antagonist assay with co-IP of cell-intrinsic TRIM-21 with Fc-CLEC (Figure 6B). These results illustrate that anti-TRIM21 antibodies may be useful to reduce or inhibit the interaction between CLEC-1 and TRIM21. EXAMPLES

[0148] Phagocytosis of TRIM21-positive cells by myeloid cells in the presence of compounds that block the CLEC-1 / TRIM21 signaling pathway The results of phagocytosis of HPB-ALL cells, a model of T-cell leukemia, in the presence of Fc-CLEC1A, a compound that disrupts the binding between CLEC-1A and TRIM21, are illustrated in Figure 7. We found that treatment of the T-ALL cell line HPB-ALL, a TRIM-21 positive cell, with FcCLEC1A, which prevents the interaction of CLEC1A with its ligand TRIM21, induced an increase in the phagocytic activity of human macrophages against HPB-ALL cells, a phenomenon that is not detected upon treatment of the tumor cells with the Fc control.

[0149] In FIG. 8, the results obtained with the combined treatment of TRIM21-positive tumor cells (Raji cells) with rituximab and various anti-TRIM21 antibodies (FIG. 8A-FIG. 8B) or anti-CLEC-1 antibodies (FIG. 8C), all of which are antagonists of the binding between CLEC-1 and TRIM21, are illustrated. As illustrated in FIG. 8D, Raji cells are TRIM21-positive tumor cells. When the two types of antibodies are administered, the phagocytosis of TRIM21-positive tumor cells (RAJI cells) is approximately 40% of the total TRIM21-positive tumor cells (Raji cells), compared to a phagocytosis percentage of 25% in the absence of anti-TRIM21 antibodies. Thus, the phagocytosis of TRIM21-positive tumor cells (RAJI cells) is significantly improved when the CLEC-1 / TRIM21 signaling pathway is disrupted.

[0150] Figure 9 illustrates the positive effect of an anti-CLEC-1 antibody, an antagonist of the binding between CLEC-1 and TRIM21, on macrophage-mediated phagocytosis. Macrophage-mediated phagocytic activity was not enhanced by the anti-CLEC-1 antibody in the context of a tumor cell line lacking CLEC-1 expression (but expressing TRIM21). However, an anti-CLEC-1 antibody, an antagonist of the binding between CLEC-1 and TRIM21, enhanced macrophage-mediated phagocytic activity in the context of a tumor cell line expressing both CLEC-1 and TRIM21. All of these data provide functional applications for the inhibition of the interaction between CLEC1A and its ligand, TRIM21.

Claims

1. A pharmaceutical composition comprising a compound that is an antagonist of the binding between CLEC-1 and TRIM21, in particular an antagonist of the binding between human CLEC-1 and human TRIM21, for the treatment of cancer having a TRIM21-positive tumor in a human subject in need of treatment.

2. The pharmaceutical composition of claim 1, wherein the compound is a polypeptide that binds to CLEC-1, particularly human CLEC-1, or is a functional equivalent of CLEC-1, particularly human CLEC-1.

3. A pharmaceutical composition according to claim 1 or 2, wherein the compound is a polypeptide that inhibits the CLEC-1 / TRIM21 signalling pathway, binds to CLEC1, particularly human CLEC-1, or reduces the expression of functional CLEC1, or is a functional equivalent of CLEC1.

4. A pharmaceutical composition described in claim 2 or 3, wherein the compound binds to the extracellular domain of CLEC-1, particularly human CLEC-1, more specifically human CLEC-1, and is an antibody, an antigen-binding fragment thereof, or an antigen-binding antibody mimetic.

5. The pharmaceutical composition of claim 4, wherein the compound is a chimeric antibody, a humanized antibody, a recombinant antibody, a fully humanized antibody, a monoclonal antibody, a deimmunized antibody, in particular a humanized monoclonal antibody.

6. A pharmaceutical composition described in claim 4 or 5, wherein the compound competes with an anti-CLEC-1A antibody having a heavy chain variable domain of sequence number 10 and a light chain variable domain of sequence number 11 for binding to CLEC-1, particularly human CLEC-1.

7. A pharmaceutical composition according to claim 2 or 3, wherein the compound is a polypeptide that is a functional equivalent of human CLEC-1, in particular the polypeptide is fused to an immunoglobulin constant domain.

8. A pharmaceutical composition described in any one of claims 1 to 7, wherein the compound increases the phagocytic ability of myeloid cells, particularly dendritic cells and / or macrophages, and in particular increases the phagocytosis of TRIM21-positive cells, more particularly TRIM21-positive tumor cells and / or secondary necrotic cells, by dendritic cells and / or macrophages, and in particular increases the phagocytosis of tumor cells and / or secondary necrotic cells by dendritic cells and / or macrophages.

9. A pharmaceutical composition according to any one of claims 1 to 8, wherein the compound increases phagocytosis of TRIM21-positive tumor cells.

10. The pharmaceutical composition of claim 1 or 2, wherein the compound binds to TRIM21, particularly human TRIM21.

11. A pharmaceutical composition as described in claim 10, wherein the compound increases the phagocytic ability of myeloid cells, particularly dendritic cells and / or macrophages, particularly increases the phagocytosis of TRIM21-positive tumor cells by dendritic cells and / or macrophages, particularly increases the phagocytosis of tumor cells and / or secondary necrotic cells, more particularly TRIM21-positive tumor cells and / or TRIM21-positive secondary necrotic cells, by dendritic cells and / or macrophages.

12. The pharmaceutical composition described in claim 10 or 11, wherein the human subject is suffering from cancer having TRIM21-positive tumor cells.

13. A pharmaceutical composition described in claim 11 or 12, wherein the compound is an antibody, an antigen-binding fragment thereof, an antigen-binding antibody mimetic, or a peptide or polypeptide that is a functional equivalent of TRIM21, particularly wherein the polypeptide is fused to an immunoglobulin constant domain.

14. A pharmaceutical composition described in any one of claims 11 to 13, wherein the compound binds to an epitope sequence located within the coiled-coil domain of TRIM21, particularly the coiled-coil domain located between amino acid residues 128 and 238 of TRIM21 of SEQ ID NO: 2, and / or binds to an epitope sequence located within the PRY-SPRY domain of TRIM21, particularly the PRY-SPRY domain located between amino acid residues 268 and 465 of TRIM21 of SEQ ID NO:

2.

15. A pharmaceutical composition described in any one of claims 11 to 14, wherein the compound is a chimeric antibody, a humanized antibody, a recombinant antibody, a fully humanized antibody, a monoclonal antibody, a deimmunized antibody, in particular a humanized monoclonal antibody.

16. The compound, wherein the compound is capable of binding to at least one competing antibody selected from the list consisting of the anti-TRIM21 antibodies sc-25351, PA5-22294, PA5-18147 and MAB-62191 for binding to TRIM21, particularly to consecutive amino acid residues located within the coiled-coil domain of TRIM21, particularly the coiled-coil domain located between amino acid residues 128 and 238 of TRIM21 of SEQ ID NO: 2, and / or to consecutive amino acid residues located within the PRYSPRY domain of TRIM21, particularly the PRYSPRY domain located between amino acid residues 268 and 465 of TRIM21 of SEQ ID NO:

2.

16. The pharmaceutical composition of claim 11, wherein the antibody, antigen-binding fragment, or antigen-binding antibody mimetic competes for binding to consecutive amino acid residues present in the antibody, and wherein the antibody, antigen-binding fragment, or antigen-binding antibody mimetic enhances the phagocytic ability of myeloid cells, particularly dendritic cells and / or macrophages, and in particular increases the phagocytosis of TRIM21-positive cells, more particularly TRIM21-positive tumor cells and / or secondary necrotic cells, by dendritic cells and / or macrophages, and in particular increases the phagocytosis of tumor cells and / or secondary necrotic cells by dendritic cells and / or macrophages.

17. A pharmaceutical composition according to any one of claims 11 to 16, wherein the compound increases phagocytosis of TRIM21-positive cells.

18. Use of a compound that is an antagonist of the binding between CLEC-1 and TRIM21, particularly an antagonist of the binding between human CLEC-1 and human TRIM21, in the manufacture of a medicament for the treatment of cancer having a TRIM21-positive tumor in a human subject in need of treatment.

19. A combination of (i) the pharmaceutical composition of any one of claims 1 to 17 and (ii) at least one further therapeutic agent for the treatment of cancer having a TRIM21-positive tumor in a subject in need of treatment, wherein the pharmaceutical composition is administered to the subject simultaneously, separately or sequentially with the at least one further therapeutic agent, wherein at least one of the further therapeutic agents is a tumor-targeting antibody or antigen-binding fragment thereof, particularly a tumor-targeting monoclonal antibody or antigen-binding fragment thereof, a tumor-targeting monoclonal antibody or antigen-binding fragment thereof that activates and / or enhances phagocytosis of tumor cells by myeloid cells, particularly macrophages, or more specifically alemtuzumab, atezolizumab, bevacizumab, serotonin-3 ... A combination selected from the list consisting of a monoclonal antibody selected from the group consisting of tuximab, herceptin, panitumumab, rituximab, trastuzumab, anti-PDL-1 antibody and anti-CD47 antibody, and / or another antibody or monoclonal antibody selected from the group consisting of anti-PD1 antibody and anti-SIRPa antibody, and / or a chemotherapeutic drug, in particular a cytotoxic drug having antiproliferative, pro-apoptotic, cell cycle arresting and / or differentiation-inducing effect, more particularly a cytotoxic drug selected from the group consisting of cytotoxic antibodies, alkylating agents, anthracyclines, antimetabolites, microtubule inhibitors, topoisomerase inhibitors, alkaloids, bleomycin, antineoplastic agents, cyclophosphamide.

20. A combination as described in claim 19, wherein the compound that is an antagonist of the binding between CLEC-1 and TRIM21 as defined in any one of claims 1 to 17 is selected from the group consisting of antibodies, antigen-binding fragments thereof, antigen-binding antibody mimetics, or peptides or polypeptides that are functional equivalents of TRIM21, particularly human TRIM21, that bind to TRIM21.

21. A combination as described in claim 19, wherein the compound that is an antagonist of the binding between CLEC-1 and TRIM21 as defined in any one of claims 1 to 17 is selected from the group consisting of antibodies, antigen-binding fragments thereof, antigen-binding antibody mimetics, or peptides or polypeptides that are functional equivalents of CLEC-1, particularly human CLEC-1, that bind to CLEC-1.

22. A combination described in claim 21, wherein the compound that is an antagonist of the binding between CLEC-1 and TRIM21 defined in any one of claims 1 to 17 is selected from the group consisting of antibodies, antigen-binding fragments thereof, and antigen-binding antibody mimetics that bind to CLEC-1 and compete with an anti-CLEC-1A antibody having a heavy chain variable domain of SEQ ID NO: 10 and a light chain variable domain of SEQ ID NO: 11 for binding to CLEC-1, particularly human CLEC-1.

23. A combination as described in claim 21, wherein the compound that is an antagonist of the binding between CLEC-1 and TRIM21 as defined in any one of claims 1 to 17 is a polypeptide that is a functional equivalent of CLEC-1, in particular said polypeptide is fused to an immunoglobulin constant domain.

24. A method for selecting a compound which is an antagonist of binding between CLEC-1 and TRIM21, in particular between human CLEC-1 and human TRIM21, said method comprising: a) providing at least one compound and assessing its ability to interfere with the interaction between CLEC-1 and TRIM21, in particular its ability to compete or antagonize the binding between CLEC1 and TRIM21, in particular said interaction being determined between CLEC-1 and at least one domain of TRIM21, b) measuring the binding between CLEC-1 and TRIM21 in the presence of said compound; c) identifying a compound that reduces the interaction between CLEC-1 and TRIM21 if the compound reduces the binding between CLEC1 and TRIM21 measured in step b) compared to a negative control; A method comprising:

25. 25. A method for selecting a compound useful for treating cancer having a TRIM21-positive tumor, comprising performing the method according to claim 24, wherein a compound that reduces binding between CLEC-1 and TRIM21 is selected.

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