Heterodimeric Fc-CLEC-1 fusion molecules and uses thereof

Abstract

Heterodimeric Fc-CLEC-1 fusion molecules and uses thereof The present invention relates to heterodimeric Fc-CLEC1 fusion molecules comprising an immunoglobulin fragment comprising at least one binding moiety comprising at least a portion of the extracellular domain of CLEC-1 and a heterodimeric Fc region comprising two different Fc polypeptide chains. The present invention provides heterodimeric Fc-CLEC-1 fusion molecules that have improved ability to bind to CLEC-1 in terms of toxicity, half-life, and bioavailability compared to prior art fusion molecules. The present invention also relates to the use of the fusion molecules in therapy.

Description

[Technical Field]

[0001] The present invention relates to heterodimeric Fc-CLEC1 fusion molecules comprising at least one binding moiety comprising at least a portion of the extracellular domain of CLEC-1 and an immunoglobulin fragment comprising a heterodimeric Fc region comprising two different Fc polypeptide chains. The present invention provides heterodimeric Fc-CLEC-1 fusion molecules that have improved capabilities in terms of toxicity, half-life, and bioavailability when compared to prior art fusion molecules for binding to CLEC-1. The present invention also relates to the use of heterodimeric Fc-CLEC-1 fusion molecules in therapy. [Background technology]

[0002] One of the challenges in cancer treatment is destroying tumor cells without damaging healthy tissue. Antitumor immunotherapy, which redirects a patient's immune system toward the destruction of cancer cells, has attracted increasing interest over the past decade. It has been clearly demonstrated that tumor cells exploit immune checkpoints to evade immune defenses. The crucial role of these inhibitory checkpoints in thwarting antitumor immunity has been exemplified 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 and develop resistance, typically after failure of classical chemotherapy. These patients require effective novel therapies, and myeloid cells have become promising therapeutic targets. In fact, myeloid cells are the most abundant tumor-infiltrating immune cells. C-type lectins (CLECs) expressed on immune cells are environmental sensors and immune response modulators. CLEC-1 represents a potential therapeutic target for boosting myeloid cells and antitumor responses. In order to better understand CLEC-1 function and to develop compounds aimed at modulating its activity, there is a need for molecules that mimic the effects of CLEC-1 and potentially have an improved ability to enhance or stimulate signaling pathways and / or cells that express CLEC-1.

[0003] CLEC-1 (also named CLEC-1A, CLEC1, or CLEC1A) is a C-type lectin-like receptor, i.e., C-type lectin-like receptor-1, that is expressed particularly in mammalian species, more particularly 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. In these receptors, glycan recognition is mediated by Ca 2+ Many related CLRs are nevertheless capable of recognizing carbohydrates but are dependent on Ca 2+These receptors are unrelated to CLEC-1; these receptors are called C-type lectin-like receptors (CTLRs), a family of receptors that includes CLEC-1. These receptors are particularly interesting for their role in linking both innate and adaptive immunity. CTLRs are predominantly expressed by cells of the 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, and allergies. CTLR modulation appears to be a promising strategy for disease management, but efforts to identify their ligands and elucidate their role in immunity have remained inadequate to date.

[0004] CLEC-1 is known to be expressed on myeloid and endothelial cells. CLEC-1 has been described as a receptor that can be upregulated by immunoregulatory mediators and can 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 Immunol 2009; 183:3099-3108). The present inventors have demonstrated that CLEC-1 is expressed on the cell surface by conventional DCs (cDCs) and a small subset of monocytes and DCs in human blood, and that its expression is enhanced by the immunosuppressive cytokine TGFβ (see International Application No. WO2018073440). They showed that in both rodents and humans, CLEC-1 acts as an inhibitory receptor on myeloid cells, preventing IL12p40 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) pp. 557-568).

[0005] These properties make it interesting to consider CLEC-1 in the design of additional tools for the treatment of health conditions involving a patient's immune response, in particular to develop new treatments for cancer. In particular, these results call for further research to provide compounds that can bind to at least one ligand of CLEC-1 and / or capture at least one ligand of CLEC-1 with enhanced properties such as a better half-life, or enhanced functions such as complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP).

Prior Technical Literature

Charter Documents

[0006] [Patent Document 1] International application number WO2018073440 [Patent Document 2] WO 96 / 34103 [Patent Document 3] WO 94 / 04678

Non-licensed literature

[0007]

Non-patent document 1

Non-patent document 2

Non-patent document 3

[0008] The present invention is based on the association within a fusion molecule of a binding moiety comprising or consisting of at least a part of the extracellular domain of CLEC-1, which is mutated or not mutated compared to wild-type CLEC-1, and a heterodimeric Fc compound comprising at least two different Fc polypeptide chains.

[0009] As further detailed in this application, the inventors have determined that the fusion molecules of the present invention have, inter alia, the following properties: - anti-CLEC-1 compounds of the prior art, in particular heterodimeric Fc-CLEC-1 fusion molecules which have reduced in vivo toxicity compared to prior art Fc-CLEC-1 fusion proteins; heterodimeric Fc-CLEC-1 fusion molecules that do not form aggregates (e.g., accumulations of fusion molecules that aggregate to form bodies or clumps; an aggregate can be considered when more than two fusion proteins accumulate and aggregate; in the context of the present invention, dimers of fusion proteins cannot be considered aggregates); - heterodimeric Fc-CLEC-1 fusion molecules which have an increased half-life, especially compared to prior art anti-CLEC-1 compounds, in particular prior art Fc-CLEC-1 fusion proteins; - heterodimeric Fc-CLEC-1 fusion molecules with enhanced bioavailability compared to prior art anti-CLEC-1 compounds, in particular compared to prior art Fc-CLEC-1 fusion proteins; - Heterodimeric Fc-CLEC-1 fusion molecules that do not increase the secretion of pro-inflammatory cytokines, in particular IL-6. The present invention provides evidence leading to a molecule having at least one of:

[0010] As illustrated in the Examples, the heterodimeric Fc-CLEC-1 fusion molecules of the invention have lower toxicity than prior art Fc-CLEC-1 fusions, thereby reducing the risks (e.g., adverse effects, resistance) associated with the administration of the compounds in patients in need thereof and / or allowing for the administration of higher doses of the Fc-CLEC-1 fusion proteins to enhance in vivo therapeutic efficacy. Furthermore, the therapeutic potential of the heterodimeric Fc-CLEC-1 fusion molecules of the invention is demonstrated in cancer models, thereby demonstrating that these fusion molecules have potent in vivo therapeutic efficacy. Data are also provided demonstrating that the heterodimeric Fc-CLEC-1 fusion molecules of the invention can bind to a CLEC-1 ligand, a reference CLEC-1 ligand, or CLEC-1L, with the same affinity as prior art Fc-CLEC-1 fusion proteins or CLEC-1-HIS proteins. Furthermore, the heterodimeric Fc-CLEC-1 fusion molecules of the invention can bind to tumor cells expressing a CLEC-1 ligand.

[0011] The heterodimeric Fc-CLEC-1 fusion molecules of the invention have enhanced bioavailability in vivo compared to prior art Fc-CLEC-1 fusion proteins. As illustrated in the examples of the invention, the in vivo concentration of the heterodimeric Fc-CLEC-1 fusion molecules is detectable for a longer period of time than prior art Fc-CLEC-1 fusion proteins; whereas the prior art fusion proteins reach the lower limit of quantitation (LLOQ), the concentration of the heterodimeric Fc-CLEC-1 fusion molecules of the invention remains close to their initial concentration.

[0012] The heterodimeric Fc-CLEC-1 fusion molecules of the invention do not form aggregates, thereby reducing their immunogenicity. While prior art Fc-CLEC-1 fusion proteins may form aggregates in certain circumstances and thus initiate an immune response against the aggregates, the heterodimeric Fc-CLEC-1 fusion molecules of the invention do not form aggregates and, when administered, do not cause an increase in the secretion of pro-inflammatory cytokines (particularly IL-6).

[0013] In a first aspect, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: (i) at least one binding moiety comprising or consisting of at least a portion of the extracellular domain of human C-type lectin domain family 1 member A (CLEC-1), wherein at least a portion of the extracellular domain of human CLEC-1 is a functional equivalent of the extracellular domain of wild-type human CLEC-1; and (ii) a. a first Fc polypeptide chain comprising a first CH3 domain (CH3B chain or whole chain); and b. a second Fc polypeptide chain comprising a second CH3 domain (the CH3A chain or knob chain); Including, The first and second CH3 domains are different, a heterodimeric immunoglobulin or a fragment thereof, Including, One of the at least one binding moieties is fused to the C-terminus or N-terminus of the first Fc polypeptide chain, and / or one of the at least one binding moiety is fused to the C-terminus or N-terminus of the second Fc polypeptide chain. Concerning fusion molecules.

[0014] In another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: (i) at least one binding moiety comprising or consisting of at least a portion of the extracellular domain of human C-type lectin domain family 1 member A (CLEC-1), wherein at least a portion of the extracellular domain of human CLEC-1 is a functional equivalent of the extracellular domain of wild-type human CLEC-1; and (ii) a. a first Fc polypeptide chain comprising, from its N-terminus to its C-terminus: a hinge region, a CH2 domain, a first CH3 domain (a CH3B chain or a hole chain), and a linker; and b. a second Fc polypeptide chain comprising a second CH3 domain (CH3A chain or knob chain), in particular comprising from its N-terminus to its C-terminus: a hinge region, a CH2 domain, and a second CH3 domain (CH3A chain or knob chain); a hetero-dimeric immunoglobulin or fragment thereof comprising Including, at least one binding moiety is fused to the C-terminus or N-terminus of the first Fc polypeptide, particularly to the C-terminus of the first Fc polypeptide; Concerning fusion molecules.

[0015] In another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: (i) at least one binding moiety comprising or consisting of at least a portion of the extracellular domain of human C-type lectin domain family 1 member A (CLEC-1), wherein at least a portion of the extracellular domain of human CLEC-1 is a functional equivalent of the extracellular domain of wild-type human CLEC-1; and (ii) a. a first Fc polypeptide chain comprising a first CH3 domain (CH3B chain or hole chain), in particular comprising from its N-terminus to its C-terminus: a hinge region, a CH2 domain, and a first CH3 domain (CH3B chain or hole chain); b. a second Fc polypeptide chain comprising, from its N-terminus to its C-terminus: a hinge region, a CH2 domain, a second CH3 domain (the CH3B or hole chain), and a linker; a heterodimeric immunoglobulin or fragment thereof comprising Including, at least one binding moiety is fused to the C-terminus or N-terminus of the second Fc polypeptide, particularly to the C-terminus of the second Fc polypeptide; Concerning fusion molecules.

[0016] In another aspect, the present invention relates in particular to a subject suffering from cancer or an infectious disease or an inflammatory disease including sepsis, an autoimmune disease or an acute or chronic inflammatory disease, in particular a cancer listed herein, in particular a liquid cancer, a solid cancer, a cancer expressing at least one ligand of human CLEC-1, a cancer with CLEC-1-ligand positive tumors (also called cancer with CLEC-1L positive tumors), a cancer with CLEC-1L positive tumor cells, a cancer with glioma cells, breast cancer, hepatocellular carcinoma, lymphoma, more particularly B-cell lymphoma, colon cancer, 1. For use as a medicament in the treatment of a human subject having a disease selected from the group consisting of cancer, thyroid cancer, liver cancer, testicular cancer, kidney cancer, melanoma, colorectal cancer, nasopharyngeal carcinoma, adenocarcinoma, pancreatic cancer, or a disease selected from the group consisting of chronic infections, sepsis, infectious diseases, in particular viral infections caused by Coxsackie viruses or by encephalitis viruses, more particularly by Coxsackie virus B3 or Japanese encephalitis virus, mycoses, cardiovascular diseases, autoimmune diseases, in particular Sjögren's syndrome or systemic lupus erythematosus or systemic sclerosis, inflammatory diseases, (i) at least one binding moiety comprising or consisting of at least a portion of the extracellular domain of human C-type lectin domain family 1 member A (CLEC-1), wherein at least a portion of the extracellular domain of human CLEC-1 is a functional equivalent of the extracellular domain of wild-type human CLEC-1; and (ii) a. a first Fc polypeptide chain comprising a first CH3 domain (CH3B chain or whole chain); and b. a second Fc polypeptide chain comprising a second CH3 domain (a CH3A chain or a whole chain); Including, The first and second CH3 domains are distinct, Heterodimeric immunoglobulin or fragment thereof Including, One of the at least one binding moieties is fused to the C-terminus or N-terminus of the first Fc polypeptide chain and / or one of the at least one binding moiety is fused to the C-terminus or N-terminus of the second Fc polypeptide chain. Concerning fusion molecules.

[0017] In a particular embodiment of the invention, at least the binding portion of the fusion molecule of the invention comprising or consisting of a part of the extracellular domain of human CLEC-1 comprises at least 50 amino acid residues, in particular at least 100 amino acid residues, more particularly at least 200 amino acid residues and shares at least 70% identity, in particular at least 80% identity, more particularly at least 90% identity, even more particularly at least 95% identity to the extracellular domain of human CLEC-1 of SEQ ID NO: 2.

[0018] In a particular embodiment of the invention, the portion of the extracellular domain of human CLEC-1 comprises at least the amino acid sequence shown in SEQ ID NO:2.

[0019] In certain embodiments of the invention, the CH3 domain of the first Fc polypeptide chain and the second Fc polypeptide chain comprise K iH, K iH S-S , HA-TF, ZW1, 7.8.90, DD-KK, EW-RVT, SEED and A107.

[0020] In certain embodiments of the present invention, (i) at least one binding moiety comprising or consisting of at least a portion of the extracellular domain of human C-type lectin domain family 1 member A (CLEC-1), wherein at least a portion of the extracellular domain of human CLEC-1 is a functional equivalent of the extracellular domain of wild-type human CLEC-1; and (ii) a. a first Fc polypeptide chain comprising a first CH2 domain (CH2B chain or whole chain); and b. a second Fc polypeptide chain comprising a second CH2 domain (CH2A chain or knob chain); Including, The first and second CH2 domains are different, Heterodimeric immunoglobulin or fragment thereof Including, One of the at least one binding moieties is fused to the C-terminus or N-terminus of the first Fc polypeptide chain and / or one of the at least one binding moiety is fused to the C-terminus or N-terminus of the second Fc polypeptide chain. Fusion molecules are provided. According to this embodiment, the fusion molecule may further comprise one (on a single Fc polypeptide chain) or two CH3 domains (each on a single Fc polypeptide chain), which CH3 domains are different or identical, particularly different. The Fc polypeptide chains may further comprise a hinge region and / or a linker region as disclosed herein.

[0021] In certain embodiments of the invention, the single binding moiety is present within the fusion protein, and the binding moiety is fused to the C-terminus or N-terminus of the first Fc polypeptide chain, particularly the C-terminus of the first Fc polypeptide chain.

[0022] In certain embodiments of the invention, a binding moiety is fused to the C-terminus or N-terminus of a first Fc polypeptide chain, particularly to the C-terminus of the first Fc polypeptide chain, where a binding molecule different from the binding moiety associated with the first Fc polypeptide chain is fused to the C-terminus or N-terminus of a second Fc polypeptide chain. A "binding molecule" may be a functional equivalent of a protein (including, but not limited to, a portion of the extracellular domain of a protein of interest) or an antigen-binding molecule. In particular, when at least one binding moiety is present at one end of the same Fc polypeptide chain, the binding molecule is at the opposite end of the Fc polypeptide chain.

[0023] In a particular embodiment of the invention, a binding moiety is fused to the C-terminus or N-terminus of a first Fc polypeptide chain, in particular the C-terminus of the first Fc polypeptide chain, and another binding moiety is fused to the C-terminus or N-terminus of a second Fc polypeptide chain, in particular the C-terminus of the second Fc polypeptide chain, and the binding moieties are identical, in which case the two binding moieties are functional equivalents of human CLEC-1 and comprise or consist of at least a portion of the extracellular domain of human CLEC-1.

[0024] In certain embodiments of the invention, at least one binding moiety is fused to the Fc polypeptide chain through a linker peptide, more particularly through the linker peptide of SEQ ID NO:13.

[0025] In certain embodiments of the invention, the first Fc polypeptide chain, or the second Fc polypeptide chain, or both the first and second Fc polypeptide chains, particularly comprise a CH2 domain N-terminal to the CH3 domain.

[0026] In certain embodiments of the invention, the first Fc polypeptide chain, or the second Fc polypeptide chain, or both the first and second Fc polypeptide chains, particularly comprise a hinge region at the N-terminus of the Fc polypeptide chain, more particularly at the N-terminus of the CH2 domain, if present.

[0027] In certain embodiments of the invention, the fusion molecule comprises: (iii) further comprising at least one antigen-binding domain comprising or consisting of an antibody variable domain or antigen-binding fragment thereof, said antigen-binding domain fused to a first Fc polypeptide chain or a second Fc polypeptide chain, or a first antigen-binding domain fused to a first Fc polypeptide chain and a second antigen-binding domain fused to a second Fc polypeptide chain. In certain embodiments, the antigen-binding domain binds to an antigen or epitope expressed by macrophages and / or lymphocytes, particularly B cells and / or T cells, and / or tumor cells. In a preferred embodiment, the antigen binding domain is selected from the group consisting of SIRP alpha, SIRP beta, SIRP gamma, CD47, CTLA-4, CD86 (B7.2), CD28, CD40, CD40L, ICOS, ICOS-L, OX40L, GITR, HVEM, BTLA, CD160, LIGHT, TNFRSF25, 2B4, CD48, Tim1, Tim3, Tim4, Gal9, LAG-3, CD40, CD40L, CD70, CD27, VISTA, B7H3, B7H4 (B7x), TIGIT, CD112, HHLA2 (B7-H7), TMIGD2 (CD28H), butyrophilin-like 2 (BTNL2), SIGLEC, AXL, B7.1, B The antibody binds to an antigen or epitope selected from the group consisting of 7-DC, B7-H1, B7-H2, B7-H3, B7-H4, CD19, CD20, CD22, CD24, CD137(4-1BB), CD137L(4-1BBL), CEA, CXCR3, CXCR4, EGFR, EGFRvIII, ELTD1, EMR1, EMR2, EMR3, EMR4P, ENG, EPCAM, EPHR, PD-L1, TLR1, TLR10, TLR2, TLR3, TLR4, VEGFR, VEGFR2, VIPR1, VIPR2, CD101, CD3, CD30, CD38, CD39, CD44, DR3, LFA-1, NKG2D, PD-1, and PDL2. In particular, if at least one binding moiety is present at one end of the same Fc polypeptide chain, the antigen-binding domains are present at opposite ends of the Fc polypeptide chain.

[0028] In certain embodiments, the antigen-binding domain is fused to the N-terminus of the first Fc polypeptide chain or the second Fc polypeptide chain, or the first antigen-binding domain is fused to the N-terminus of the first Fc polypeptide chain and the second antigen-binding domain is fused to the second Fc polypeptide chain, particularly to the N-terminus of the CH2 domain or, if present, the hinge region.

[0029] In certain embodiments, two antigen-binding domains are present within the fusion molecule, each associated with a single Fc polypeptide chain, and the first and second antigen-binding domains bind to two different epitopes, in particular two different antigens, or to the same epitope or the same antigen.

[0030] In a particular embodiment of the invention, a dimeric molecule is provided comprising two fusion molecules according to the invention.

[0031] In certain embodiments of the present invention, there are provided soluble fusion molecules according to the invention.

[0032] In a particular embodiment of the invention, a soluble dimeric molecule is provided comprising two fusion molecules according to the invention.

[0033] In certain embodiments of the present invention, bispecific fusion molecules are provided.

[0034] In certain embodiments of the present invention, monospecific fusion molecules are provided.

[0035] In a particular embodiment of the invention, a fusion molecule is provided which comprises or consists of the amino acid sequences set forth in SEQ ID NO:10 and SEQ ID NO:12, or SEQ ID NO:3 and SEQ ID NO:5.

[0036] In another aspect, the present invention relates to a fusion molecule which is an antagonist of the binding between CLEC-1 and one of its ligands, in particular between human CLEC-1 and human CLEC-1L, for its use as a medicament for the treatment of a subject, in particular a human subject, having a disease selected from the group consisting of cancers listed herein, in particular cancers with CLEC-1L-positive tumor cells, cancers with glioma cells, breast cancer, hepatocellular carcinoma, lymphoma, more particularly B-cell lymphoma, colon cancer, thyroid cancer, liver cancer, testicular cancer, kidney cancer, melanoma, colorectal cancer, adenocarcinoma, nasopharyngeal carcinoma, pancreatic cancer, chronic infection, sepsis, infectious diseases, in particular infections caused by Coxsackieviruses or by encephalitis viruses, more particularly infections caused by Coxsackievirus B3 or Japanese encephalitis virus, cardiovascular diseases, autoimmune diseases, in particular Sjögren's syndrome or systemic lupus erythematosus or systemic sclerosis, inflammatory diseases.

[0037] The structures of different fusion molecules according to the invention are illustrated in FIG.

[0038] definition As used herein, the terms "CLEC-1" and "CLEC-1A" refer to a CLEC-1A protein derived from a mammalian species, preferably human CLEC-1 or CLEC-1A. The reference sequence for 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 designated by the Q8NC01 Uniprot accession number and the amino acid sequence encoded by the CLEC-1 gene designated 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 designate mammalian CLEC1 receptors corresponding to the human CLEC-1A receptor characterized by an amino acid sequence associated with accession number Q8NC01 Uniprot, its orthologous proteins, or its homologous proteins.

[0039] Preferably, the term "human CLEC-1" refers to the protein having the amino acid sequence designated by the Uniprot accession number Q8NC01 and encoded by the CLEC-1 gene designated by the NCBI accession number 51267. CLEC-1 may be characterized by the amino acid sequence set forth in SEQ ID NO: 1. In a particular embodiment, the amino acid sequence of the extracellular domain of human wild-type CLEC-1 is:

[0040] [ka]

[0041] It comprises or consists of an array of

[0042] A fusion molecule is a "fusion protein" comprising all or part of a (typically biologically active) functional equivalent of CLEC-1 operably linked to at least one heterologous polypeptide (i.e., a polypeptide other than the same polypeptide) issued from, derived from, or selected from an Fc polypeptide. Within the fusion protein, the term "fused to" is intended to indicate that the functional equivalent of CLEC-1 and the heterologous polypeptide are fused in-frame to each other. The heterologous polypeptide can be fused to the N-terminus or C-terminus of the functional equivalent of CLEC-1 of the present invention. As detailed in the description of the Fc polypeptide domains, the domains present on the first and second Fc polypeptide chains are not fused because the first and second Fc polypeptide chains associate via sub-nM affinity and, if present, via disulfide bonds in the hinge region.

[0043] Certain embodiments of the present invention provide fusion molecules featuring, inter alia, a functional equivalent of CLEC-1 through at least a portion of its extracellular domain. Thus, at least a fragment of the extracellular domain of CLEC-1 is fused to a constant domain or fragment thereof of a human immunoglobulin comprising at least two Fc polypeptide chains, referred to as "heterodimeric Fc polypeptides" or "Fc polypeptide chains" or "Fc fragments." The combination of the immunoglobulin and the fragment of the extracellular domain of CLEC-1 forms a fusion molecule that can possess many of the beneficial chemical and biological properties of human antibodies. The Fc polypeptide chains exhibit spontaneous pairing interactions between a first Fc polypeptide chain and a second Fc polypeptide chain, and the first and second Fc polypeptides differ at least in their CH3 and / or CH2 domains, particularly at least in their CH3 domains, and particularly only within their CH3 domains, more particularly in their CH2 and CH3 domains. Thus, the first and second Fc polypeptide chains of the fusion molecule undergo heterodimerization to form a heterodimer.

[0044] In particular embodiments of the fusion molecules of the invention, the Fc fragment of a human immunoglobulin comprised in the Fc-CLEC-1 fusion molecule is an Fc fragment of human IgG1 or IgG4, and the Fc fragment is fused to the N-terminus or C-terminus of a mammalian CLEC-1 domain, in particular to the extracellular domain of a human CLEC-1 domain.

[0045] As used herein, the term "polypeptide" refers to a polymer of amino acids having any or 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, etc.

[0046] Heterodimeric Fc polypeptide chain or portion thereof The fragment crystallizable region (Fc region or Fc) is the tail region of an antibody that interacts with cell surface receptors called Fc receptors and certain proteins of the complement system. Typically, the Fc region of an antibody isotype is composed of two identical protein fragments derived from the constant domains of the antibody's two heavy chains (referred to as CH2 and CH3, and, if present, CH4). IgA, IgD, and IgG Fc regions contain two heavy chain constant domains, while IgM and IgE Fc regions contain three heavy chain constant domains in each polypeptide chain. In the context of IgA, IgD, and IgG antibodies, IgA, IgD, and IgG isotypes each have three CH regions, two of which belong to the Fc region. For example, the "CH" domains in the context of IgG are as follows: "CH1" refers to positions 118-215 according to the EU index as defined by Kabat; "hinge" refers to positions 216-230 according to the EU index as defined by Kabat. "CH2" refers to positions 231-340 according to the EU index in Kabat, and "CH3" refers to positions 341-447 according to the EU index in Kabat.

[0047] The Fc region of IgG consists of two paired CH3 domains and, in contrast, two separate, non-interacting CH2 domains with two oligosaccharide chains inserted between them. Wild-type Fc homodimerization is mediated with sub-nM affinity by a large, tightly packed interface between two identical CH3 domains, followed by disulfide bonding in the hinge region. Heterodimeric Fc molecules have been engineered primarily by replacing homodimer-favoring interactions at the CH3 domain interface with heterodimer-favoring interactions. This is achieved by introducing asymmetric mutations into each CH3 domain of each Fc chain, which facilitates assembly of Fc chains from two different antibodies.

[0048] A functional equivalent of the extracellular domain of CLEC-1 (i.e., at least one binding moiety present in the fusion molecule of the invention) is fused to an immunoglobulin constant domain (Fc region) to form an immunoadhesin. Immunoadhesins possess many of the beneficial chemical and biological properties of human antibodies. Because immunoadhesins can be constructed from human protein sequences with the desired specificity, linked to appropriate human immunoglobulin hinge and constant domain (Fc) sequences, the desired binding specificity can be achieved using entirely human components. Such immunoadhesins are minimally immunogenic to patients and safe for chronic or repeated use. In some embodiments, the Fc polypeptide is derived from a native-sequence Fc region. In some embodiments, the Fc polypeptide is a variant Fc region. In yet another embodiment, the Fc polypeptide 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, including native-sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fc region is usually defined to stretch from the amino acid residue at position Cys226, or from Pro230, to the carboxyl-terminus. The adhesive moiety and the immunoglobulin sequence portion of the immunoadhesin can 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 IgG1 or IgG4. In some embodiments, the extracellular domain of CLEC-1 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 connects the polypeptide of the invention to the immunoglobulin sequence portion. Such a linker can 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, or 30 amino acid residues. However, the upper limit is not critical and may be selected for reasons of convenience, for example, with respect to biopharmaceutical production of such polypeptides. The linker sequence may be the linker reference herein (G4S)3. The linker sequence may be a naturally occurring or non-naturally occurring sequence. When used for therapeutic purposes, the linker is typically non-immunogenic in the subject to which the immunoadhesin is administered.

[0049] The present invention provides a fusion molecule comprising at least two different protein fragments derived from the constant domain of an antibody heavy chain. These two different protein fragments are referred to as a first Fc polypeptide chain and a second Fc polypeptide chain, respectively. In particular, each of the first and second Fc polypeptide chains comprises a CH3 domain and / or a CH2 domain, and are referred to as a first CH3 (or CH2) domain or a second CH3 (or CH2) domain, respectively. In particular, the first CH3 (or CH2) domain and the second CH3 (or CH2) domain are different, i.e., they do not share the same amino acid residue sequence (e.g., they are not 100% identical). Such CH3 (or CH2) domains may differ by one amino acid residue, two amino acid residues, three amino acid residues, four amino acid residues, or five or more amino acid residues. In a preferred embodiment of the invention, the first CH3 (or CH2) domain and the second CH3 (or CH2) domain are capable of interacting to allow pairing of the first and second Fc polypeptide chains.

[0050] In certain embodiments of the invention, the two Fc polypeptide chains originate from, are derived from, or are selected from human immunoglobulin heavy chains, in particular IgG immunoglobulin heavy chains, more particularly from the Fc region of an IgG1, IgG2, IgG3, or IgG4 immunoglobulin heavy chain. More particularly, the Fc polypeptide chains originate from, are derived from, or are selected from an IgG4 Fc region. Even more particularly, the Fc polypeptide chains originate from, are derived from, or are selected from an IgG4 Fc region having the substitution S228P, which enhances the stability of the Fc region.

[0051] In an embodiment of the invention, each Fc polypeptide chain comprises a CH3 domain.

[0052] The CH3 domain may originate from or be selected from an immunoglobulin heavy chain, in particular a human immunoglobulin heavy chain, more particularly from an IgG heavy chain, e.g., it may be derived from an IgG1, IgG2, IgG3, IgG4 heavy chain.

[0053] In embodiments of the invention, each Fc polypeptide chain comprises a truncated Fc region or a fragment of an Fc region comprising a CH3 domain.

[0054] In embodiments of the invention, the heterodimeric Fc domain comprises a first Fc chain and a complementary second Fc chain based on the "knob and hole" technique. By way of example, the first Fc chain is a "knob" or K chain, meaning that it contains substitutions that characterize knob chains, and the second Fc chain is a "hole" or H chain, meaning that it contains substitutions that characterize hole chains. Vice versa, the first Fc chain is a "hole" or H chain, meaning that it contains substitutions that characterize hole chains, and the second Fc chain is a "knob" or K chain, meaning that it contains substitutions that characterize knob chains. In a preferred aspect, the first Fc chain is a "hole" or H chain and the second Fc chain is a "knob" or K chain.

[0055] In a preferred embodiment of the invention, the first Fc polypeptide chain and the second Fc polypeptide chain correspond to the pairs listed in the table below.

[0056] [Table 1]

[0057] In a preferred embodiment, the "hole" Fc polypeptide chain comprises the following substitutions: T366S, L368A, Y407V and Y349C. The whole Fc chain may further comprise additional substitutions.

[0058] In a preferred embodiment, the "knob" Fc polypeptide chain comprises the following substitutions: T366W and S354C. The "knob" Fc chain may further comprise additional substitutions.

[0059] In a preferred embodiment, the "hole" Fc polypeptide chain comprises the following substitutions: T366S, L368A, Y407V, and Y349C, and the "knob" Fc polypeptide chain comprises the following substitutions: T366W and S354C. The two Fc chains may further comprise additional substitutions.

[0060] In another embodiment, each Fc polypeptide chain comprises a CH3 domain as disclosed herein above and at least a portion of a CH2 domain, more particularly a complete CH2 domain, which may be derived from or selected from an immunoglobulin heavy chain, particularly a human immunoglobulin heavy chain, more particularly an IgG heavy chain, e.g., an IgG1, IgG2, IgG3, or IgG4 heavy chain.

[0061] In another embodiment, each Fc polypeptide chain comprises a CH3 domain and a hinge region as disclosed hereinabove. The hinge region is a short sequence of an antibody heavy chain that links the Fab (fragment antigen-binding) region to the Fc region. The hinge region may be derived from or selected from an immunoglobulin heavy chain, particularly a human immunoglobulin heavy chain, more particularly an IgG heavy chain, e.g., an IgG1, IgG2, IgG3, or IgG4 heavy chain. One useful group of hinge sequences is derived from the hinge regions of heavy chain antibodies described in WO 96 / 34103 and WO 94 / 04678. Another example is a polyalanine linker sequence. The hinge region may correspond to the amino acid sequence set forth in SEQ ID NO: 20.

[0062] In another embodiment, each Fc polypeptide chain comprises a constant domain generated or derived from an IgG1 isotype or an IgG4 isotype.

[0063] In embodiments, the fusion molecule of the invention comprises the amino acid residues set forth in SEQ ID NO:3, which is the KIH-Fc-G1 knob chain.

[0064] In embodiments, a fusion molecule of the invention comprises the amino acid residues set forth in SEQ ID NO:4, which is the KIH-Fc-G1 hole chain.

[0065] In embodiments, a fusion molecule of the invention comprises the amino acid residues set forth in SEQ ID NO:3 and the amino acid residues set forth in SEQ ID NO:4.

[0066] In embodiments, the fusion molecule of the invention comprises the amino acid residues set forth in SEQ ID NO:5, which is the KIH-Fc-G1-CLEC-1 hole chain.

[0067] In embodiments, a fusion molecule of the invention comprises the amino acid residues set forth in SEQ ID NO:5 and the amino acid residues set forth in SEQ ID NO:3.

[0068] In embodiments, a fusion molecule of the invention comprises an Fc polypeptide chain that is an Fc-G1 chain, which originates from or is derived from the amino acid residues set forth in SEQ ID NO: 6. The mutations disclosed herein (see Table 1) may be present in this chain.

[0069] In embodiments, a fusion molecule of the invention comprises an Fc polypeptide chain that is an Fc-G1e3 chain, which is generated or derived from the amino acid residues set forth in SEQ ID NO: 7. The mutations disclosed herein (see Table 1) may be present in this chain.

[0070] In embodiments, a fusion molecule of the invention comprises an Fc polypeptide chain that is the FcG1-N297A chain, which is generated or derived from the amino acid residues set forth in SEQ ID NO: 8. The mutations disclosed herein (see Table 1) may be present in this chain.

[0071] In embodiments, a fusion molecule of the invention comprises an Fc polypeptide chain that is the FcG4-S228P chain, which is generated or derived from the amino acid residues set forth in SEQ ID NO: 9. The mutations disclosed herein (see Table 1) may be present in this chain.

[0072] In embodiments, a fusion molecule of the invention comprises an Fc polypeptide chain that originates from or is derived from the amino acid residues set forth in SEQ ID NO:10, which is the KIH-FcG4 knob chain.

[0073] In embodiments, a fusion molecule of the invention comprises an Fc polypeptide chain that originates from or is derived from the amino acid residues set forth in SEQ ID NO:11, which is the KIH-FcG4 hole chain.

[0074] In embodiments, a fusion molecule of the invention comprises an Fc polypeptide chain formed from or derived from the amino acid residues set forth in SEQ ID NO:10 and the amino acid residues set forth in SEQ ID NO:11.

[0075] In embodiments, a fusion molecule of the invention comprises an Fc polypeptide chain that is a KIH-FcG4-CLEC-1 hole chain, which is generated or derived from the amino acid residues set forth in SEQ ID NO: 12.

[0076] In embodiments, a fusion molecule of the invention comprises or consists of an Fc polypeptide chain formed from or derived from the amino acid residues set forth in SEQ ID NO:10 and the amino acid residues set forth in SEQ ID NO:12.

[0077] In embodiments, the fusion molecule comprises: (i) a single binding moiety comprising or consisting of at least a portion of the extracellular domain of human C-type lectin domain family 1 member A (CLEC-1), wherein at least a portion of the extracellular domain of human CLEC-1 is a functional equivalent of the extracellular domain of wild-type human CLEC-1; and (ii) a. a first Fc polypeptide chain comprising a first CH3 domain (CH3B chain or whole chain); and b. a second Fc polypeptide chain comprising a second CH3 domain (the CH3A chain or knob chain); Including, The first and second CH3 domains are different, the binding moiety is fused to the C-terminus of the first Fc polypeptide chain or the second Fc polypeptide chain; a heterodimeric immunoglobulin or a fragment thereof, and (iii) an antigen-binding domain comprising or consisting of an antibody variable domain or an antigen-binding fragment thereof, in particular an antigen-binding domain that binds to PD-1, wherein the antigen-binding domain is fused to the N-terminus of an Fc polypeptide chain that is fused to a binding moiety; Includes.

[0078] In certain embodiments, the binding moiety and the antigen binding domain are both fused to a whole Fc polypeptide chain.

[0079] CLEC-1 binding part The term "functionally equivalent fragment" as used herein may refer to any fragment or collection of fragments of CLEC-1. Accordingly, the present invention provides polypeptides, in particular functional equivalents, that are capable of decreasing / reducing or inhibiting the binding of CLEC-1 to at least one of its ligands, which polypeptides comprise constituent amino acids having a sequence that is the sequence of at least a part of the extracellular domain of CLEC-1, which represents a functional equivalent of CLEC-1, which in a preferred embodiment is a portion that binds to at least one ligand of CLEC-1, in particular human CLEC-1L.

[0080] In a particular embodiment of the invention, the extracellular domain of CLEC-1 is a polypeptide comprising at least 20, particularly at least 25, particularly at least 30, particularly at least 40, particularly at least 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 within SEQ ID NO:2. Alternatively, the extracellular domain of CLEC-1 comprises 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 at least 50 amino acid residues, particularly at least 80 amino acid residues, particularly at least 100 amino acid residues, particularly at least 150 amino acid residues, particularly at least 180 amino acid residues, particularly at least 200 amino acid residues, particularly at least 210 amino acid residues, and is a peptide or polypeptide having at least 70% identity, particularly 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, or sharing 100% identity, with the extracellular domain of CLEC-1 of SEQ ID NO: 2. The extracellular domain of CLEC-1 may be a polypeptide fused to a linker sequence, which polypeptide comprises or consists of an amino acid sequence 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, or sharing 100% identity, with the extracellular domain of CLEC-1 of SEQ ID NO:2.

[0081] The percentage of identity referred to in the present disclosure is determined based on a comprehensive alignment of the sequences to be compared, i.e., based on the alignment of the sequences over their entire length, for example using the algorithm of Needleman and Wunsch 1970. This sequence comparison can be carried out using the Needle software, for example, by using the parameters "gap open" equal to 10.0, the parameter "gap extend" equal to 0.5, and the matrix "BLOSUM62". Software such as Needle is available on the website ebi.ac.uk worldwide under the name "Needle".

[0082] As used herein, a "functional equivalent" of CLEC-1 is a compound that is capable of binding to at least one CLEC-1 ligand, in particular CLEC-1L, thereby preventing its interaction with CLEC-1. The term "functional equivalent" includes fragments, mutants, and muteins of CLEC-1. The term "functional equivalent" therefore includes any equivalent of CLEC-1 obtained by altering the amino acid sequence, for example, by one or more amino acid deletions, substitutions, or additions, so that the protein analog retains the ability to bind its ligand. Amino acid substitutions may be made, for example, by point mutations in the DNA encoding the amino acid sequence.

[0083] Functional equivalents of CLEC-1 include, but are not limited to, molecules that bind to at least one ligand of CLEC-1, in particular CLEC-1L, and comprise all or part of the extracellular domain of CLEC-1 so as to form a molecule that can bind to at least one ligand of CLEC-1, in particular CLEC-1L (in particular so as to form a soluble receptor that can capture at least one ligand of CLEC-1). Functional equivalents include soluble forms of CLEC-1. Suitable soluble forms of these proteins or functional equivalents thereof may include, for example, mutants, in particular truncated forms, of the proteins in which the transmembrane domain has been removed by chemical, proteolytic or recombinant methods. In particular, functional equivalents consist of an amino acid sequence that has at least 70%, in particular 80% identity to the corresponding protein over the entire length of the corresponding protein, 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. As used herein, the term "corresponding protein" refers to a protein with which the 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 comprehensive alignment of the sequences to be compared, i.e., based on an alignment of the sequences over their entire length, for example, using the algorithm of Needleman and Wunsch 1970. This sequence comparison can be performed using the Needle software, for example, by using the parameters "Gap open" equal to 10.0, "Gap extend" equal to 0.5, and the matrix "BLOSUM62." Software such as Needle is available at the website ebi.ac.uk worldwide under the name "Needle."

[0084] In an embodiment of the invention, the portion of the extracellular domain of human CLEC-1 corresponds to a truncated portion of the extracellular domain of CLEC-1, in particular, the N-terminus of the extracellular domain of CLEC-1, in particular, SEQ ID NO: 2, deleted. The N-terminus may correspond to the first 10, or the first 11, or the first 13, or the first 14, or the first 15, or the first 16, or the first 17, or the first 18, or the first 19, or the first 20, or the first 21, or the first 22, or the first 23, or the first 24, or the first 25 amino acid residues of SEQ ID NO: 2. Alternatively, the N-terminus of the extracellular domain of CLEC-1 may correspond to at least the first amino acid residue located at the N-terminus of the extracellular domain of CLEC-1, up to a maximum of 10% of the amino acid residues of the extracellular domain of CLEC-1 located at the N-terminus. In a preferred embodiment, the truncated portion of the extracellular domain of CLEC-1 may have the sequence of amino acid residues shown in SEQ ID NO: 19.

[0085] Linker peptide and hinge domain In certain embodiments of the invention, any binding moiety included in the fusion molecule may be fused to the Fc polypeptide chain via a linker peptide, which typically ensures that the attached binding moiety is connected to the Fc polypeptide in a conformation that allows each to perform its function.

[0086] In certain embodiments, each binding moiety is preferably linked to the Fc polypeptide chain via a linker peptide. In certain embodiments, when a binding moiety is fused toward the C-terminus of the Fc polypeptide chain, the binding moiety may be fused to the Fc polypeptide chain via a linker peptide. As used herein, the term "linker peptide" refers to a sequence of at least one amino acid residue. Linkers are typically 1 to 44 amino acid residues in length. Preferably, linkers have 3 to 30 amino acid residues. In certain embodiments, linker peptides have 2, 3, 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, or 30 amino acid residues.

[0087] The linker peptide may be a naturally occurring or non-naturally occurring sequence. In certain embodiments, the linker peptide is non-immunogenic in the subject to which the fusion molecule will be administered. Another example is a polyalanine linker sequence. Further preferred examples of linker sequences are Gly / Ser linkers of different lengths, including (Gly4Ser)4, (Gly4Ser)3, (Gly4Ser)2, Gly4Ser, Gly3Ser, Gly3, Gly2Ser, and (Gly3Ser2)3, in particular (Gly4Ser)3. Preferably, the linker is selected from the group consisting of (Gly4Ser)4, (Gly4Ser)3, and (Gly3Ser2)3. More preferably, the linker is (GGGGS)3. In certain embodiments, the linker peptide included in the fusion molecule is selected from the group consisting of (Gly4Ser)4 (SEQ ID NO: 14), (Gly4Ser)3 (SEQ ID NO: 13), (Gly4Ser)2 (SEQ ID NO: 15), Gly4Ser (SEQ ID NO: 16), Gly3Ser (SEQ ID NO: 17), Gly3, Gly2ser, and (Gly3Ser2)3 (SEQ ID NO: 18), preferably (Gly4Ser)3. Preferably, the linker is selected from the group consisting of (Gly4Ser)4, (Gly4Ser)3, and (Gly3Ser2)3. In certain embodiments, the linker peptide is (Gly4Ser)3 of SEQ ID NO: 13. In certain embodiments, the binding moiety may be fused to an Fc chain via a hinge sequence (commonly referred to as a "hinge") naturally found in immunoglobulin heavy chains to connect the VH domains, and in particular to connect the CH1 domain to the CH2 domain. In certain embodiments, when the binding moiety is fused towards the N-terminus of the Fc polypeptide chain, the binding moiety may be fused to the Fc polypeptide chain via a hinge domain. One useful group of hinge domains are derived from the hinge regions of heavy chain antibodies described in WO 96 / 34103 and WO 94 / 04678.

[0088] In an embodiment of the invention, the fusion molecule comprises at least one binding moiety, particularly a single binding moiety, fused to the C-terminus of a first Fc polypeptide chain. In a preferred embodiment, the at least one binding moiety or single binding moiety is fused to the C-terminus of the first Fc polypeptide via a linker peptide, particularly a linker peptide comprising or consisting of SEQ ID NO: 13. In a specific embodiment, the binding moiety is a mutated extracellular domain of CLEC-1, particularly a truncated portion of the extracellular domain of CLEC-1 having the sequence of amino acid residues set forth in SEQ ID NO: 19. As illustrated in the examples of the invention, such constructs are less likely to aggregate. In an embodiment of the invention, the fusion molecule comprises at least one binding moiety, particularly a single binding moiety, fused to the C-terminus of a second Fc polypeptide chain. In a preferred embodiment, the at least one binding moiety or single binding moiety is fused to the C-terminus of the first Fc polypeptide via a linker peptide, particularly a linker peptide comprising or consisting of SEQ ID NO: 13. In a particular embodiment, the binding moiety is a mutated extracellular domain of CLEC-1, in particular a truncated portion of the extracellular domain of CLEC-1 having more particularly the sequence of amino acid residues set out in SEQ ID NO: 19. As illustrated in the examples of the present invention, such constructs are less likely to aggregate.

[0089] In embodiments of the invention, the fusion molecule comprises a heterodimeric immunoglobulin comprising or consisting of an Fc KIH region, a linker region, and a CLEc-1 domain region. In a preferred embodiment, the fusion molecule comprises a heterodimeric immunoglobulin comprising or consisting of an Fc KIH region selected from the left column of Table 2, a linker region selected from the middle column of Table 2, and a CLEC-1 domain selected from the right column of Table 2.

[0090] [Table 2]

[0091] SEQ ID NO: 3 corresponds to the Fc-KIH (knob) described in the examples of the present invention, which is in particular an IgG1 domain with T366W and S354C.

[0092] SEQ ID NO: 4 corresponds to the Fc-KIH(hole) described in the examples of the present invention, which is in particular an IgG1 domain with T366S, L368A, Y407V and Y349C.

[0093] SEQ ID NO: 10 corresponds to Fc-KIH(Whole), which is an IgG4 domain with, among other things, S228P, T366W and S354C.

[0094] SEQ ID NO: 11 corresponds to the Fc-KIH (knob), which is in particular an IgG4 domain with S228P, T366S, L368A, Y407V and Y349C.

[0095] SEQ ID NO: 20 corresponds to the hinge domain, found for example in the Fc-KIH (knob or hole) described in the examples of the present invention.

[0096] SEQ ID NO: 21 corresponds to the CH2 domain, found for example in the Fc-KIH (knob or hole) described in the examples of the present invention.

[0097] SEQ ID NO: 22 corresponds to the CH3 domain, which is found, for example, in Fc-KIH(hole) described in the examples of the present invention.

[0098] The linker is a linker as detailed herein above.

[0099] The CLEC-1 domain of SEQ ID NO: 2 corresponds to the extracellular domain of CLEC-1.

[0100] The CLEC-1 domain of SEQ ID NO: 19 corresponds to the truncated extracellular domain of CLEC-1 detailed above.

[0101] In an embodiment of the invention, the fusion molecule comprises the following domains: - the Fc-KIH domain of SEQ ID NO: 3; and - a linker of SEQ ID NO: 13; and - a CLEC-1 domain of SEQ ID NO: 2, and optionally in a preferred embodiment, the first Fc polypeptide chain comprises or consists of an Fc KIH domain of SEQ ID NO: 4, and a heterodimeric immunoglobulin comprising a second Fc polypeptide chain comprising or consisting of:

[0102] In an embodiment of the invention, the fusion molecule comprises the following domains: - the Fc-KIH domain of SEQ ID NO: 3; and - a linker of SEQ ID NO: 13; and - a CLEC-1 domain of SEQ ID NO: 19, and optionally in a preferred embodiment, the first Fc polypeptide chain comprises or consists of an Fc KIH domain of SEQ ID NO: 4, and a heterodimeric immunoglobulin comprising a second Fc polypeptide chain comprising or consisting of:

[0103] In an embodiment of the invention, the fusion molecule comprises the following domains: - the Fc-KIH domain of SEQ ID NO: 4; and - a linker of SEQ ID NO: 13; and - a CLEC-1 domain of SEQ ID NO: 2, and optionally in a preferred embodiment, the second Fc polypeptide chain comprises or consists of an Fc KIH domain of SEQ ID NO: 3, and a heterodimeric immunoglobulin comprising a first Fc polypeptide chain comprising or consisting of:

[0104] In an embodiment of the invention, the fusion molecule comprises the following domains: - the Fc-KIH domain of SEQ ID NO: 4; and - a linker of SEQ ID NO: 13; and - a CLEC-1 domain of SEQ ID NO: 19, optionally in a preferred embodiment, the second Fc polypeptide chain comprises or consists of an Fc KIH domain of SEQ ID NO: 3; and a heterodimeric immunoglobulin comprising a first Fc polypeptide chain comprising or consisting of:

[0105] In an embodiment of the invention, the fusion molecule comprises the following domains: - a hinge domain of SEQ ID NO: 20; and - the CH2 domain of SEQ ID NO: 21; and - a CH3 domain of SEQ ID NO: 22; and - a linker of SEQ ID NO: 13; and - a CLEC-1 domain of SEQ ID NO: 2; and optionally, in a preferred embodiment, the second Fc polypeptide chain comprises or consists of an Fc KIH domain of SEQ ID NO: 3, and a heterodimeric immunoglobulin comprising a first Fc polypeptide chain comprising or consisting of:

[0106] In an embodiment of the invention, the fusion molecule comprises the following domains: - a hinge domain of SEQ ID NO: 20; and - the CH2 domain of SEQ ID NO: 21; and - a CH3 domain of SEQ ID NO: 22; and - a linker of SEQ ID NO: 13; and - a CLEC-1 domain of SEQ ID NO: 19; and optionally, in a preferred embodiment, the second Fc polypeptide chain comprises or consists of an Fc KIH domain of SEQ ID NO: 3, and a heterodimeric immunoglobulin comprising a first Fc polypeptide chain comprising or consisting of:

[0107] In an embodiment of the invention, the fusion protein comprises a first Fc polypeptide chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 4 and a second Fc polypeptide chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 3, and at least a binding moiety comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 2. In particular, at least one binding moiety is linked to the C-terminus of SEQ ID NO: 4 or SEQ ID NO: 3, in particular via a linker of SEQ ID NO: 13.

[0108] In an embodiment of the invention, the fusion protein comprises a first Fc polypeptide chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 4 and a second Fc polypeptide chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 3, and at least a binding moiety comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 19. In particular, at least one binding moiety is linked to the C-terminus of SEQ ID NO: 4 or SEQ ID NO: 3, in particular via a linker of SEQ ID NO: 13.

[0109] In an embodiment of the invention, the fusion protein comprises a first Fc polypeptide chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:5 and a second Fc polypeptide chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:3, wherein the binding moiety of SEQ ID NO:2 is present within the first Fc polypeptide chain.

[0110] In an embodiment of the invention, the fusion protein comprises a first Fc polypeptide chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 4 and a second Fc polypeptide chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 23, wherein the binding portion of SEQ ID NO: 23 is present within the second Fc polypeptide chain.

[0111] Other binding molecules (antigen-binding domains and other binding molecules different from at least one binding moiety of the fusion protein) In certain embodiments of the invention, the fusion molecule further comprises at least an antigen-binding domain or another binding moiety different from the at least one binding moiety comprising or consisting of at least a portion of the extracellular domain of human C-type lectin domain family 1 member A.

[0112] The fusion molecule may contain at least one binding moiety different from the at least one binding moiety comprising or consisting of at least a portion of the extracellular domain of human CLEC-1, but which is not an antigen-binding domain derived from an antibody or related compound. Such a binding moiety may correspond to the extracellular domain of a protein or part thereof, in particular a protein known to be involved in immune responses, such as, but not limited to, PD1, PDL1, CTLA4, 4-1BBL, CD80, CD137. The binding moiety should retain the binding ability of the native protein for at least one of its ligands.

[0113] Where the fusion molecule comprises an antigen-binding domain, this domain comprises or consists of an antibody variable domain or antigen-binding fragment thereof, and said domain is fused to a first Fc polypeptide chain or a second Fc polypeptide chain, or the first antigen-binding domain is fused to the first Fc polypeptide chain and the second antigen-binding domain is fused to the second Fc polypeptide chain.

[0114] In certain embodiments, when an antigen-binding domain is present on an Fc polypeptide chain associated with a binding moiety at one end, the antigen-binding fragment or binding molecule is associated with the opposite end of the Fc polypeptide chain. For example, when a binding moiety is associated with the C-terminus of an Fc polypeptide chain, the antigen-binding domain or binding molecule is associated with the N-terminus of the Fc polypeptide chain. When a binding moiety is associated with the N-terminus of an Fc polypeptide chain, the antigen-binding domain or binding molecule is associated with the C-terminus of the Fc polypeptide chain.

[0115] As used herein, an antigen-binding domain refers to a portion or fragment of an antibody, i.e., a molecule corresponding to a structural portion of an antibody, which, presumably in its native form, exhibits antigen-binding ability to its target; such fragments in particular exhibit the same or substantially the same antigen-binding specificity to said antigen as compared to the antigen-binding specificity of the corresponding four-chain antibody. Advantageously, the antigen-binding fragment has a binding affinity similar to that of the corresponding four-chain antibody. However, antigen-binding fragments with reduced antigen-binding affinity relative to the corresponding four-chain antibody are also encompassed for use within the present invention. Antigen-binding ability can be determined by measuring the affinity between the antibody and the target fragment of the antibody. These antigen-binding fragments may also be termed "functional fragments" of antibodies.

[0116] Antigen-binding fragments of antibodies are fragments containing the hypervariable domains, termed CDRs (complementarity-determining regions), of a reference antibody or a portion thereof, which contain the recognition site for the antigen. Antigen-binding fragments of antibodies containing variable domains containing the CDRs of the antibody include Fv, dsFv, scFv, Fab, Fab', and F(ab')2. In a specific embodiment of the invention, the antigen-binding domain is a Fab domain.

[0117] These basic antigen-binding fragments for use according to the present invention can be combined together to obtain multivalent antigen-binding fragments such as diabodies, tribodies or tetrabodies, which are also part of the present invention when the Fab domains are associated into fusion molecules.

[0118] Antigen-binding antibody mimetics are organic compounds that specifically bind to antigens but are structurally unrelated to antibodies. These mimetics are typically artificial peptides or small proteins with molar masses of approximately 3-20 kDa. Nucleic acids and small molecules are also sometimes considered antibody mimetics, but artificial antibodies, antibody fragments, and fusion proteins constructed from them are not. Their common advantages over antibodies include better solubility, tissue penetration, heat and enzymatic stability, and relatively low production costs. Antibody mimetics are being developed as therapeutic and diagnostic agents. Antigen-binding antibody mimetics can be selected from groups including affibodies, affilins, aptamers, anticalins, affimers, affitins, DARPins, and monobodies.

[0119] In certain embodiments of the present invention, a fusion molecule comprises two antigen-binding domains, each associated with a single Fc polypeptide chain, wherein the first and second antigen-binding domains bind to the same epitope, the same antigen, two different epitopes, or two different antigens. An epitope is the portion of an antigen molecule to which an antibody binds. Thus, when a fusion molecule comprises two antigen-binding domains, the two antigen-binding domains may bind to the same epitope, two different epitopes located within the same antigen, or two different epitopes located within two different antigens. In other words, when a fusion molecule comprises two antigen-binding domains, these molecules may bind to the same antigen or two different antigens.

[0120] In certain embodiments of the invention, at least one antigen-binding domain specifically binds to an antigen expressed by macrophages, and / or lymphocytes, in particular B cells or T cells, and / or tumor cells.

[0121] In certain embodiments of the invention, at least one antigen binding domain is selected from the group consisting of SIRP alpha, SIRP beta, SIRP gamma, CD47, CTLA-4, CD86 (B7.2), CD28, CD40, CD40L, ICOS, ICOS-L, OX40L, GITR, HVEM, BTLA, CD160, LIGHT, TNFRSF25, 2B4, CD48, Tim1, Tim3, Tim4, Gal9, LAG-3, CD40, CD40L, CD70, CD27, VISTA, B7H3, B7H4 (B7x), TIGIT, CD112, HHLA2 (B7-H7), TMIGD2 (CD28H), butyrophilin-like 2 (BTNL2), SIGLEC, AXL, Specifically binds to an antigen selected from the list consisting of B7.1, B7-DC, B7-H1, B7-H2, B7-H3, B7-H4, CD19, CD20, CD22, CD24, CD137(4-1BB), CD137L(4-1BBL), CEA, CXCR3, CXCR4, EGFR, EGFRvIII, ELTD1, EMR1, EMR2, EMR3, EMR4P, ENG, EPCAM, EPHR, PD-L1, TLR1, TLR10, TLR2, TLR3, TLR4, VEGFR, VEGFR2, VIPR1, VIPR2, CD101, CD3, CD30, CD38, CD39, CD44, DR3, LFA-1, NKG2D, PD-1, and PDL2.

[0122] In certain embodiments of the invention, the fusion molecule comprises two antigen-binding domains, each associated with a single Fc polypeptide chain, and both antigen-binding domains are selected from the group consisting of SIRP alpha, SIRP beta, SIRP gamma, CD47, CTLA-4, CD86 (B7.2), CD28, CD40, CD40L, ICOS, ICOS-L, OX40L, GITR, HVEM, BTLA, CD160, LIGHT, TNFRSF25, 2B4, CD48, Tim1, Tim3, Tim4, Gal9, LAG-3, CD40, CD40L, CD70, CD27, VISTA, B7H3, B7H4 (B7x), TIGIT, CD112, HHLA2 (B7-H7), TMIGD2 (CD28H), butyrophilin and B7-like 2 (BTNL2), SIGLEC, AXL, B7.1, B7-DC, B7-H1, B7-H2, B7-H3, B7-H4, CD19, CD20, CD22, CD24, CD137 (4-1BB), CD137L (4-1BBL), CEA, CXCR3, CXCR4, EGFR, EGFRvIII, ELTD1, EMR1, EMR2, EMR3, EMR4P, ENG, EPCAM, EPHR, PD-L1, TLR1, TLR10, TLR2, TLR3, TLR4, VEGFR, VEGFR2, VIPR1, VIPR2, CD101, CD3, CD30, CD38, CD39, CD44, DR3, LFA-1, NKG2D, PD-1, and PDL2.

[0123] In certain embodiments of the invention, the fusion molecule comprises two antigen-binding domains, each of which binds to an antigen, and the two antigens recognized by each of the antigen-binding domains are selected from the group consisting of SIRP alpha, SIRP beta, SIRP gamma, CD47, CTLA-4, CD86 (B7.2), CD28, CD40, CD40L, ICOS, ICOS-L, OX40L, GITR, HVEM, BTLA, CD160, LIGHT, TNFRSF25, 2B4, CD48, Tim1, Tim3, Tim4, Gal9, LAG-3, CD40, CD40L, CD70, CD27, VISTA, B7H3, B7H4 (B7x), TIGIT, CD112, HHLA2 (B7-H7), TMIGD2 (CD28H), and butyroglobulin. and / or independently selected from the list consisting of philin-like 2 (BTNL2), SIGLEC, AXL, B7.1, B7-DC, B7-H1, B7-H2, B7-H3, B7-H4, CD19, CD20, CD22, CD24, CD137 (4-1BB), CD137L (4-1BBL), CEA, CXCR3, CXCR4, EGFR, EGFRvIII, ELTD1, EMR1, EMR2, EMR3, EMR4P, ENG, EPCAM, EPHR, PD-L1, TLR1, TLR10, TLR2, TLR3, TLR4, VEGFR, VEGFR2, VIPR1, VIPR2, CD101, CD3, CD30, CD38, CD39, CD44, DR3, LFA-1, NKG2D, PD-1, and PDL2.

[0124] In certain embodiments, the antigen binding domain is present at the N-terminus of the first Fc polypeptide chain or the second Fc polypeptide chain.

[0125] In a specific embodiment, an antigen binding domain is present at the N-terminus of a first Fc polypeptide chain and a second antigen binding domain is fused to a second Fc polypeptide chain.

[0126] In certain embodiments, the antigen binding domain is N-terminal to the CH2 domain or, if present, the hinge region.

[0127] Use of fusion molecules The fusion molecules are provided for use as pharmaceuticals. In particular, the fusion molecules are provided for use in treating disease in a subject (i.e., a patient, particularly a human patient), particularly a subject with cancer, or an infectious disease, or sepsis, or an autoimmune disease, or an inflammatory disease, including an acute or chronic inflammatory disease. In another embodiment, the fusion molecules may be provided for use in preventing disease in a subject. As used herein, "treatment" or "treating" is an approach for obtaining 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 the disease; curing the disease; reducing the extent of the disease; stabilizing the disease (e.g., preventing or slowing the worsening of the disease); preventing or slowing the spread of the disease; preventing or slowing the recurrence of the disease; slowing or slowing the progression of the disease; ameliorating the disease state; causing remission (partial or total) of the disease; reducing the dose of one or more other drugs required to treat the disease; delaying the progression of the disease; improving quality of life; and / or prolonging survival. According to embodiments, the term "treatment" relates to prophylactic treatment. As used herein, the term "preventing" refers to reducing the risk of acquiring or developing a given condition.

[0128] In certain embodiments of the present invention, fusion molecules are provided for use in treating patients, where modulation of phagocytosis of target cells, such as cells expressing at least one ligand of CLEC-1, particularly CLEC-1L, by myeloid cells, particularly dendritic cells and / or macrophages, improves disease outcome. Accordingly, in certain embodiments, fusion molecules are provided that increase phagocytosis of cells, particularly CLEC-1 ligand-positive cells, particularly CLEC-1L-positive cells, more particularly tumor cells and / or secondary necrotic cells, even more particularly CLEC-1L-positive tumor cells and / or CLEC-1L-positive secondary necrotic cells, by myeloid cells, particularly dendritic cells and / or macrophages. Thus, CLEC-1L-positive tumors (i.e., tumors comprising at least one cell that expresses, particularly aberrantly expresses, CLEC-1L) can be targeted by the fusion molecules of the present invention. Indeed, it has been shown that antagonists of the CLEC-1L-CLEC1 signalling pathway (e.g. compounds that bind to CLEC-1L or CLEC1 which inhibit the binding between CLEC-1 and CLEC-1L or a functional equivalent of CLEC-1L or CLEC1, or compounds that reduce the expression of functional CLEC-1L or CLEC1, or compounds that reduce or inhibit the signalling pathway induced by the binding between CLEC-1L and CLEC-1) are able to modulate, in particular enhance, phagocytosis of target cells, such as CLEC-1-expressing tumour cells, by myeloid cells, in particular dendritic cells and / or macrophages, thereby leading to their use in therapy or disease treatment, whereby modulating phagocytosis of target cells, such as tumour cells or cells expressing CLEC-1L, by myeloid cells, in particular dendritic cells and / or macrophages, improves the health of the patient. Thereby, the use of the fusion molecules of the present invention, which at least reduce the interaction of CLEC-1 (expressed on the cell surface of dendritic cells and macrophages) with its ligand CLEC-1L (e.g., expressed at least by tumor cells), is believed to be useful in regulating the phagocytosis of cells, such as tumor 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 CLEC-1L, phagocytosis by these macrophages or dendritic cells can be inhibited or reduced. For example, it has been shown that tumor cells expressing CLEC-1L (i.e., Raji cells) escape phagocytosis exerted by macrophages. By using antagonist compounds according to the present invention that modulate the interaction between CLEC-1A-expressing myeloid cells and CLEC-1L-expressing cells, particularly CLEC-1L-positive tumor cells, the reduction or inhibition of phagocytosis of CLEC-1L-expressing cells by myeloid cells, particularly dendritic cells or macrophages, is reduced. Thus, phagocytosis of CLEC-1L-expressing cells, such as tumor cells, is enhanced in the presence of the fusion molecules of the present invention.

[0129] More particularly, the present invention relates to the use of fusion molecules in the treatment of a condition or disease in a patient, wherein said condition or disease is or is related to a cancer listed herein, in particular a cancer having CLEC1 ligand-positive tumor cells, in particular a cancer having CLEC-1L-positive tumor cells, a cancer having glioma cells, breast cancer, hepatocellular carcinoma, lymphoma, more particularly B-cell lymphoma, colon cancer, thyroid cancer, liver cancer, testicular cancer, rectal cancer, melanoma, colorectal cancer, adenocarcinoma, nasopharyngeal carcinoma, pancreatic cancer, chronic infection, sepsis, in particular an infection caused by a Coxsackievirus or by an encephalitis virus, more particularly an infection caused by Coxsackievirus 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.

[0130] The term "cancer" has its common meaning in the art and refers to a group of diseases involving abnormal cell growth that can 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, liquid cancers, solid cancers, cancers that express at least one ligand of human CLEC-1, cancers with CLEC-1 ligand-positive tumors, cancers with CLEC-1L-positive tumor cells, cancers with CLEC-1L-positive tumors, cancers originating in the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal tract, gums, head, kidney, liver, lung, nasopharynx, cervix, ovary, prostate, skin, stomach, testis, tongue, or uterus. Furthermore, cancer may specifically be of the following histological types: neoplasm, malignant; cytoma; cytoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; malignant pilomatricoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; mixed hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid tumor; carcinoid tumor, malignant; bronchioloalveolar adenocarcinoma; papillary adenocarcinoma; chromophobe eosinophilic cell carcinoma; eosinophilic adenocarcinoma; basophilic cell carcinoma; clear cell adenocarcinoma; granulocytoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; non-encapsulating sclerosing adrenocortical carcinoma; endometrioid carcinoma; skin adnexal carcinoma; apocrine adenocarcinoma; sebaceous carcinoma; auditory canal adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; invasive ductal carcinoma; medullary carcinoma; lobular carcinoma; inflammatory breast carcinoma; Paget's disease, breast; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma with squamous metaplasia w / squamous metaplasia); thymoma, malignant; ovarian stromal tumor, malignant; theca cell tumor, malignant; granulosa cell tumor, malignant;and blastoma, malignant; Sertoli cell tumor; Leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extramammary paraganglioma, malignant; pheochromocytoma; glomus sarcoma; malignant melanoma; amelanotic malignant melanoma; superficial spreading melanoma; malignant melanoma in giant pigmented nevi; epithelioid cell melanoma melanoma); blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mixed Müllerian tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymal cell tumor, 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; solitary fibrous tumor, malignant; Lymphangiosarcoma; Osteosarcoma; Parosteal osteosarcoma; Chondrosarcoma; Chondroblastoma, malignant; Mesenchymal chondrosarcoma; Giant cell tumor of bone; Ewing's sarcoma; Odontogenic tumor, malignant; Ameloblasticodontosarcoma; Ameloblastoma, malignant; Ameloblastic fibrosarcoma; Pinealoma, malignant; Chordoma; Glioma, malignant; Ventriculoepithelioma; Astrocytoma; Protoplasmic astrocytoma; Fibrillar astrocytoma; Astroblastoma; Glioblastoma; Oligodendroglioma; Oligodendroglioma; Primitive neuroectodermal tumor; Cerebellar sarcoma sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; schwannoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin's lymphoma; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphocytic leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia The cancer may be, but is not limited to, myeloid leukemia, basophilic leukemia, eosinophilic leukemia, monocytic leukemia, mast cell leukemia, megakaryoblastic leukemia, myeloid sarcoma, and hairy cell leukemia.

[0131] In certain embodiments, the subject has 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, colorectal cancer, adenocarcinoma, endometrial cancer, esophageal cancer, gallbladder cancer, gastrointestinal carcinoid tumor, Hodgkin's disease, non-Hodgkin's lymphoma, Kaposi's sarcoma, kidney cancer, pharyngeal and hypopharyngeal cancer, liver cancer, lung cancer, mesothelioma, plasmacytoma, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oral cavity and oropharyngeal cancer, ovarian cancer, pancreatic cancer, penile cancer, pituitary cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, skin cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, vaginal cancer, vulvar cancer, and uterine cancer.

[0132] CLEC-1A is also known to be involved in various pathological conditions, including sepsis, infections, and allergic inflammation. Tone et al. (Frontiers in Immunology, 2021), Gao et al. (iSciences, 2020), and Zhu z et al. (Invest. Ophthalmo. Vis. Scis., 2021) emphasize that blocking CLEC-1A signaling is associated with regulating immune responses (CLEC-1 is an immunosuppressant of myeloid cells), which may enhance patient outcomes.

[0133] The present invention also relates to the use of heterodimeric fusion molecules in the treatment of infectious diseases.

[0134] The present invention also relates to the use of heterodimeric fusion molecules in the treatment of sepsis.

[0135] The present invention also relates to the use of the heterodimeric fusion molecules in the treatment of autoimmune diseases.

[0136] The present invention also relates to the use of the heterodimeric fusion molecules in the treatment of inflammatory diseases, particularly acute or chronic inflammatory diseases.

[0137] The present invention also relates to the use of heterodimeric fusion molecules in the treatment, including prophylactic treatment, of deleterious conditions or diseases, particularly where dendritic cells and / or T cells are involved, and where stimulation of T cell proliferation and / or phagocytosis by myeloid cells, particularly dendritic cells and / or macrophages, may ameliorate or treat the condition or disease. In certain embodiments, the disease or condition is selected from the group consisting of cancer, particularly the cancers listed herein above, more particularly CLEC-1L-positive tumor cancer, liquid cancer, solid cancer, lymphoma, colorectal cancer, adenocarcinoma, mesothelioma, or hepatocellular carcinoma.

[0138] Pharmaceutical Composition The present invention also relates to pharmaceutical compositions for use in treating patients with a disease, comprising a fusion molecule as defined herein as a therapeutic agent, alone or in combination with a second therapeutic agent, together with a pharmaceutically acceptable, pharmaceutically suitable solvent for the formulation, which can be administered to a patient in need thereof. These formulations may in particular be isotonic, sterile, saline (such as mono- or di-sodium phosphate, sodium, potassium, calcium or magnesium chloride, or mixtures of such salts), or a dried, in particular lyophilized, composition that, upon addition of sterile water or saline, constitutes an injectable solution, as the case may be.

[0139] nucleic acid molecule The present invention also relates to a nucleic acid molecule or combination of nucleic acid molecules that encodes at least a portion of the fusion proteins described herein.

[0140] To this end, the present invention also relates to a nucleic acid molecule or combination of nucleic acid molecules encoding a fusion protein according to any one of the definitions disclosed herein. In particular, the nucleic acid molecule encodes at least a first Fc polypeptide chain and / or a second Fc polypeptide chain and / or at least one binding moiety disclosed herein. Preferably, the nucleic acid molecule encodes a first Fc polypeptide chain, a second Fc polypeptide chain, and at least one binding moiety. In particular, the nucleic acid molecule may encode a hinge portion, a linker, and / or a CH2, if these elements are present.

[0141]

[82] The present invention may also relate to a combination of a first nucleic acid molecule and a second nucleic acid molecule, wherein the first nucleic acid molecule encodes at least a first Fc polypeptide chain and the second nucleic acid molecule encodes at least a second Fc polypeptide chain.

[0142] Any nucleic acid molecule according to the invention may be inserted into an expression vector, such as a plasmid, suitable for expression of the encoded sequence in a host cell.

[0143] Compound combinations The present invention also relates to compound combinations comprising a first therapeutic agent and at least one additional therapeutic agent.

[0144] A combination of compounds is a formulation containing at least two different products, drugs, or compounds, which may be packaged together or separately, and which are prepared for simultaneous, co-administration, or coordinated (or sequential) administration. In particular, the amount of each compound in the combination may range from 1 μg / kg to 100 mg / kg of the patient's body weight.

[0145] The first therapeutic agent is a fusion molecule (i.e., a molecule comprising or consisting of a binding moiety comprising or consisting of at least a portion of the extracellular domain of CLEC-1, and a heterodimeric Fc region) as defined in any embodiment of the invention disclosed herein.

[0146] The at least one further therapeutic agent 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 CLEC-1L by myeloid cells, in particular by dendritic cells and / or macrophages, and even more particularly alemtuzumab, atezolizumab, bevacizumab, cetuximab, herceptin, panitumumab, rituximab, trastuzumab, A monoclonal antibody selected from the group consisting of anti-PDL-1 antibody and anti-CD47 antibody, or another antibody or monoclonal antibody selected from the group consisting of anti-PD1 antibody and anti-SIRPα antibody; and / or a chemotherapeutic agent, particularly a cytotoxic agent with antiproliferative, pro-apoptotic, cell cycle arresting, and / or differentiation-inducing effects, 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, and cyclophosphamide. Tumor-targeting antibodies may be defined as therapeutic monoclonal antibodies that recognize tumor-specific membrane proteins, block cell signaling, and induce tumor killing via an Fc-driven innate immune response. Chemotherapeutic agents may also be conventional cytotoxic agents, i.e., compounds that induce irreversible lethal damage following exposure through interference with DNA replication, mitosis, etc. These drugs may have antiproliferative, proapoptotic, cell cycle arresting and differentiation-inducing effects. These drugs are preferentially selected from the group consisting of alkylating agents (cisplatin, chlorambucil, procarbazine, carmustine), anthracyclines and other cytotoxic antibiotics, antimetabolites (i.e., methotrexate, cytarabine, gemcitarabine), 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 DNA strands).The combination may include more than one second therapeutic agent selected from the list. The combination may also include additional therapeutic agents not listed, and / or further components such as, but not limited to, excipients or administration vehicles.

[0147] In certain embodiments, the therapeutic agents may be administered simultaneously, separately or sequentially in the treatment of disease, particularly in the treatment of cancer.

[0148] In certain embodiments, the fusion molecules defined in any of the embodiments of the invention disclosed herein are provided for the treatment of liquid cancers, particularly leukemia, lymphoma or myeloma, or for the treatment of solid cancers, particularly colorectal cancer or adenocarcinoma.

[0149] In certain embodiments, a fusion molecule as defined in any embodiment of the invention disclosed herein is provided in combination with cyclophosphamide.

[0150] In certain embodiments, a fusion molecule as defined in any embodiment of the invention disclosed herein is provided in combination with cyclophosphamide for the treatment of cancer, particularly liquid cancer, more particularly leukemia, lymphoma or myeloma, or particularly solid cancer, more particularly colorectal cancer.

[0151] In a particular embodiment of the present invention there is provided a fusion molecule as defined in any embodiment of the invention disclosed herein which inhibits the C-type lectin-like receptor-1 (CLEC-1) signalling pathway, which compound binds to CLEC1 or reduces the expression of functional CLEC1 or a functional equivalent of CLEC1, in particular a fusion protein that is an antagonist of the binding between CLEC-1 and CLEC-1L, more particularly CLEC-1L, which binds to human CLEC-1, for use in treating a subject, particularly a human subject, suffering from a cancer having CLEC-1L-positive tumour cells or CLEC-1L-positive tumour cells, preferably wherein the fusion protein increases the phagocytic capacity of myeloid cells, in particular dendritic cells and / or macrophages, in particular wherein the fusion protein increases the phagocytosis of CLEC-1L-positive cells, more particularly CLEC-1L-positive tumour cells and / or secondary necrotic cells, by dendritic cells and / or macrophages, in particular wherein the fusion protein increases the phagocytosis of tumour cells and / or secondary necrotic cells by dendritic cells and / or macrophages.

[0152] In particular embodiments of the present invention, the present invention is directed to cancer, or an infectious disease, or an inflammatory disease including sepsis, an autoimmune disease, or an acute or chronic inflammatory disease, more preferably cancer, in particular liquid cancer or solid cancer, in particular cancer having CLEC-1L-positive tumor cells and / or CLEC-1-positive tumor cells, cancer having glioma cells, breast cancer, hepatocellular carcinoma, lymphoma, more particularly B-cell lymphoma, colon cancer, thyroid cancer, liver cancer, testicular cancer, rectal cancer, melanoma, colorectal cancer, adenocarcinoma, nasopharyngeal carcinoma, pancreatic cancer, chronic infection, sepsis, in particular caused by a coxsackie virus or an encephalitis virus, more particularly coxsackievirus B3 or Japanese encephalitis virus. The present invention provides a fusion molecule for use in treating patients suffering from an infectious disease, a cardiovascular disease, an autoimmune disease, particularly Sjögren's syndrome or systemic lupus erythematosus or systemic sclerosis, or an inflammatory disease caused by a myeloid leukemia, wherein the fusion molecule enhances the phagocytic ability of bone marrow cells, particularly dendritic cells and / or macrophages, particularly the phagocytosis of CLEC-1L-positive cells or CLEC-1-positive cells, more particularly CLEC-1L-positive tumor cells and / or secondary necrotic cells and / or CLEC-1-positive tumor cells, by dendritic cells and / or macrophages, and more particularly the phagocytosis of tumor cells and / or secondary necrotic cells by dendritic cells and / or macrophages.

[0153] Methods for Treating Patients In a further aspect, the present invention therefore relates to a method for treating a human patient diagnosed with cancer, or an infectious disease, or an inflammatory disease, including sepsis, an autoimmune disease, or an acute or chronic inflammatory disease, more preferably cancer, in particular a liquid or solid cancer, comprising administering to the subject a therapeutically effective amount of a fusion molecule according to any of the embodiments disclosed herein. The fusion molecule may be administered in combination with conventional treatments, for example, cyclophosphamide.

[0154] In a further aspect, the present invention therefore provides a method for treating a human patient diagnosed with cancer, comprising: - assessing whether the patient has tumor cells that express CLEC-1L and / or CLEC-1; - administering a fusion molecule according to any embodiment disclosed herein if the patient has CLEC-1L-positive tumor cells and / or CLEC-1-positive tumor cells; The present invention relates to the method comprising the steps of:

[0155] In a further aspect, the present invention therefore relates to a method for treating cancer in a human patient in need thereof, comprising administering to the subject a therapeutically effective amount of a fusion molecule as defined herein. The fusion molecule may be administered in combination with conventional treatments.

[0156] As used herein, the term "standard or conventional treatment" refers to any cancer treatment (drugs, radiation therapy, etc.) that is typically administered to subjects with cancer. In particular, the fusion molecules of the present invention are used in combination with chemotherapeutic agents, radiation therapy agents, immunotherapeutic agents (such as tumor-targeting monoclonal antibodies), cellular therapy agents (such as CAR-T cells), immunosuppressants, proapoptotic agents, antibiotics, targeted cancer therapies, and / or probiotics.

[0157] In another aspect, there is provided a fusion molecule according to any embodiment disclosed herein for the treatment of a patient having CLEC-1 ligand-positive tumor cells (i.e., cells expressing a ligand for CLEC-1).

[0158] In another aspect, there is provided a fusion molecule according to any embodiment disclosed herein for the treatment of a patient having tumor cells recognized by CLEC-1, in particular by an anti-CLEC-1 antibody or Fc-CLEC-1, more particularly by an Fc-CLEC-1 fusion molecule according to the invention.

[0159] In another aspect, there is provided a fusion molecule according to any embodiment disclosed herein for the treatment of a patient undergoing or who has undergone treatment with conventional therapies for cancer.

[0160] In another aspect, there is provided a fusion molecule according to any embodiment disclosed herein for the treatment of a patient having cancer and undergoing or having undergone treatment with an agent selected from the group consisting of a chemotherapeutic agent, a targeted cancer therapy, an immunotherapeutic agent, or a radiotherapeutic agent.

[0161] In another aspect, there is provided a fusion molecule according to any embodiment disclosed herein for the treatment of a patient having cancer and undergoing or having undergone treatment with an agent selected from the group consisting of a cytotoxic drug, an angiogenesis inhibitor, an anti-cancer agent, a cell cycle / control apoptosis regulator, an anti-cancer antibody, and a hormone regulator.

[0162] In another aspect, there is provided a fusion molecule according to any embodiment disclosed herein for use in the manufacture of a medicament for treating a patient having cancer, or an infectious disease, or an inflammatory disease including sepsis, an autoimmune disease, or an acute or chronic inflammatory disease, particularly a liquid cancer, more particularly a leukemia, lymphoma, or myeloma, or a solid cancer, more particularly a colorectal cancer or adenocarcinoma. In another aspect, there is provided a fusion molecule according to any embodiment disclosed herein for use in the manufacture of a medicament for treating cancer, particularly a liquid cancer, more particularly a leukemia, lymphoma, or myeloma, or a solid cancer, more particularly a colorectal cancer or adenocarcinoma.

[0163] Further aspects and features of the present invention will be found in the following examples and in the drawings.

[0164] The following figures and examples are presented to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are the following figures and examples intended to represent all or the only experiments performed. While the present invention has been described with respect to specific embodiments thereof, those skilled in the art will recognize that various modifications 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, process, process steps, objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto. [Brief explanation of the drawings]

[0165] Some of the figures referenced in this application are in color. This application as filed will contain color printouts of the figures in its archives, which may be accessed by viewing the application files at the Patent Office. [Figure 1]Schematic diagrams of different Fc-CLEC-1 fusion molecules according to the invention. A represents a pre-existing Fc-CLEC-1 comprising two identical Fc chains (blue squares) and two binding moieties comprising the extracellular domain of CLEC-1 (green spheres). B represents a heterodimeric Fc-CLEC-1 of the invention comprising two different Fc chains (blue squares with red triangles) and a single binding moiety comprising the extracellular domain of CLEC-1 (green sphere). C represents a heterodimeric Fc-CLEC-1 of the invention comprising two different Fc chains (blue squares with red triangles), a single binding moiety comprising the extracellular domain of CLEC-1 (green sphere), and another binding moiety corresponding to the extracellular domain of another protein other than CLEC-1 (blue sphere). D represents a heterodimeric Fc-CLEC-1 of the invention comprising two different Fc chains (blue squares with red triangles), a single binding moiety comprising the extracellular domain of CLEC-1 associated with the C-terminus of one Fc chain (green sphere), and an antigen-binding domain associated with the N-terminal domain of the Fc chain (blue and cyan squares). E represents a heterodimeric Fc-CLEC-1 of the invention comprising two different Fc chains (blue squares with red triangles), a single binding moiety comprising the extracellular domain of CLEC-1 (green sphere), another binding moiety corresponding to the extracellular domain of another protein other than CLEC-1 (blue sphere), and a single antigen-binding domain associated with the single N-terminal domain of the Fc chain (blue and cyan squares). F represents a heterodimeric Fc-CLEC-1 of the invention comprising two different Fc chains (blue square with red triangles), a single binding moiety comprising the extracellular domain of CLEC-1 (green sphere), and two antigen-binding domains (blue and cyan squares), each associated with a single N-terminal domain of the Fc chain. G represents a heterodimeric Fc-CLEC of the invention comprising two different Fc chains (blue square with red triangles), a single binding moiety comprising the extracellular domain of CLEC-1 (green sphere), another binding moiety corresponding to the extracellular domain of another protein other than CLEC-1 (blue sphere), each associated with the C-terminus of the Fc chain, and two antigen-binding domains (blue and cyan squares), each associated with a single N-terminal domain of the Fc chain.H represents a heterodimeric Fc-CLEC of the invention comprising two different Fc chains (blue square with red triangle), a single binding moiety comprising the extracellular domain of CLEC-1 (green sphere), another binding moiety corresponding to the extracellular domain of another protein other than CLEC-1 (blue sphere), each associated with the C-terminus of an Fc chain, and two different antigen-binding domains (blue, cyan, and red squares), each associated with a single N-terminal domain of an Fc chain. [Figure 2] Antitumor effect of Fc CLEC-1 treatment. A. Tumor volume and B. survival of MC38 colon adenocarcinoma-implanted mice following combined treatment with cyclophosphamide (CPA) and Fc control or Fc CLEC-1 fusion proteins. [Figure 3] Figure 1. Toxicity scores following intravenous injection of different Fc CLEC-1 formats. A. Toxicity scores were assessed over time following intravenous injection of either human Fc CLEC-1 (mouse protein), mouse Fc CLEC-1 (human protein), and two different Fc KIH CLEC-1 (Fc-KIH G1(hole)-(G4S)3-CLEC1 and Fc-KIH-G1(knob)-(G4S)3-CLEC1) (human proteins) according to the invention, according to the table shown in B. [Figure 4] Analytical SEC profiles (evaluation of monomeric, dimeric and aggregate forms) of different Fc CLEC-1 or control recombinant proteins (A) and different formulations (B). [Figure 5] Elisa binding to CLEC-1 ligand (CLEC-1L) Binding of human Fc CLEC-1, Fc-KIH-CLEC1 recombinant protein and Fc-KIH control to human CLEC-1 ligand [Figure 6] Pharmacokinetic profiles of Fc CLEC-1 and three Fc-KIH-CLEC1 recombinant proteins according to the invention in mice [Figure 7] ELISA binding assay of anti-CLEC-1A antibodies using three different Fc KIH CLEC-1 constructs of the invention. Binding of anti-CLEC-1A antibodies to immobilized Fc KIH CLEC-1 and isotype control. [Figure 8]Inflammatory Cytokine Secretion Pro-inflammatory / tumor-promoting murine IL-6 secretion following intravenous injection of Fc CLEC-1 and Fc-KIH-CLEC1 recombinant proteins as measured by ELISA in mouse serum. [Figure 9] Example of binding of Fc KIH CLEC-1 constructs versus control Fc KIH to tumor cells (here MC38 murine colon adenocarcinoma cells). [Figure 10] Binding of Fc-CLEC-1 to different tumor cells. Human non-small cell lung cancer (NSCLC) (A549), triple-negative breast cancer (SK-BR3), colorectal cancer (CRC) (DLD-1, HT-29), ovarian cancer (HeLa), hepatocellular carcinoma (HCC) (HepG2, Huh7), osteosarcoma (U2OS), glioblastoma (U373), T-ALL (HPB-ALL, DND41, Jurkat), lymphoma (T2), B-ALL (Raji, Ramos, RPMI8866), myeloma (U266, RPMI8226), and AML (THP-1, U937) were stimulated with UV-C light (CL-1000 UV Crosslinker, Analytik Jena) (150 mJ / cm2), X-ray (Faxitron CP 160, Fa ... Cells were killed by treatment with 10 Gy of cisplatin (X-Ray Corp., Wheeling) or the chemotherapy drugs C. cisplatin (20 μM Merck) or D. staurosporine (1 μM Sigma-Aldrich), followed by incubation at 37°C for 18 hours under culture conditions. Cells were then bound and stained with Fixable Viability Dye (eBioscience) and Fc-CLEC-1 (or Fc-Ctrl) at 100 nM for 30 minutes at 4°C. The percentage of Fc-CLEC1-positive cells was determined after reading on a hemocytometer. DETAILED DESCRIPTION OF THE INVENTION

[0166] Materials and Methods Tumor Models and Treatments: MC38 colon adenocarcinoma cells (ATCC, Manassas, Virginia, USA) were cultured at 37°C and 5% CO2 in 1640 RPMI medium (Life Technologies) supplemented with 10% endotoxin-free fetal bovine serum (Thermo Fisher), 2 mM L-glutamine (Sigma-Aldrich), 100 U / ml penicillin, and 100 μg / ml streptomycin (Life Technologies, Carlsbad, USA) according to the manufacturer's instructions. One million cells per mouse were injected subcutaneously into the left flank of C57 / BI6 mice (Janvier labs, Le Genest-Saint-Isle, France). Mice received 100 mg / mL endothelial growth factor (EGF) and 100 mg / mL endothelial growth factor (EGF). Tumors were 65 mm in diameter and 100 μg / mL endothelial growth factor (EGF). The tumors were then grafted onto the grafted mouse. ... 3 Once tumor size reached 100% (days 7–12), mice were injected with cyclophosphamide (CPA) (150 mg / kg, Sigma-Adrich, St. Louis, Missouri, USA) and co-injected with Fc control or Fc CLEC-1 fusion protein (5 mg / kg, twice weekly for 3 weeks). Tumor growth was measured blindly using calipers and expressed as an area based on the two-way product sum ((length × width) 1.5) × π / 6. Mice were euthanized when tumor size reached acceptable size.

[0167] Pharmacokinetic and in vivo toxicity profiling of Fc CLEC-1 recombinant protein: Fc CLEC-1 recombinant protein was intravenously injected at 10 mg / kg into the tail vein of naive Balb-c mice. Blood was collected following injection at different time points, as listed in Figure 7. Careful monitoring for signs of toxicity was performed over time based on the scoring method detailed in Figure 3B. Pharmacokinetic profiling was performed using a sandwich ELISA assay. In the ELISA assay, goat anti-human Fc (Jackson Immunoresearch, reference 109-005-098) was immobilized on plastic at 1 μg / ml, and serum or standard recombinant protein was added to quantify serum concentrations. After incubation and washing, peroxidase-labeled donkey anti-human IgG (Jackson Immunoresearch, reference 709-035-149) was added and revealed by conventional methods.

[0168] Analytical SEC profiling of Fc CLEC-1 recombinant proteins: SEC profiles were performed by gel filtration chromatography (GeHealthcare Superdex200 10 / 300GL column) using the AktaPure system. Antibodies were injected into a loop (100 μl) with PBS buffer and analyzed using AktaPure software.

[0169] ELISA IL6 assay: Mouse IL-6 detection in mouse serum was performed using a mouse IL-6 ELISA set (BD Biosciences, Franklin Lakes, New Jersey, USA, OptEIA - 555240) according to the manufacturer's instructions.

[0170] ELISA binding CLEC-1L-CLEC-1 assay: Binding of different Fc CLEC-1 recombinant proteins or an Fc control to human CLEC-1L recombinant protein was performed using a CLEC-1L binding ELISA. For the activity ELISA assay, recombinant hCLEC-1L-His (Biotech, reference 18010-H07B) was immobilized at 2 μg / ml on plastic in P96 polysorbate plates, purified recombinant protein was added, and binding was measured. After incubation and washing, peroxidase-conjugated donkey anti-human IgG (Jackson Immunoresearch, reference 709-035-149) was added and revealed by conventional methods.

[0171] ELISA binding assay anti-CLEC-1A using coated Fc KIH CLEC-1A In the binding ELISA assay, human Fc KIH CLEC-1A molecules were immobilized on plastic at 1 μg / ml, and binding was measured by adding a mouse anti-CLEC-1A monoclonal antibody (OSE Immunotherapeutics) at 5 μg / ml. After incubation and washing, peroxidase-conjugated donkey anti-human IgG (Jackson Immunoresearch, reference 715-036-151) was added and revealed by conventional methods.

[0172] Cell binding assay: Fc KIH or Fc KIH CLEC-1 constructs were conjugated to AF647 using the A30009 Alexa Fluor™ 647 Microscale Protein Labeling Kit (FISHER, Hampton, NH, USA). Tumor cells were stained with 200 nM of fusion protein in PBS BSA 1% azide 0.02% solution at pH 6 for 45 min on ice. [Example]

[0173] Antitumor effects of Fc-CLEC-1 fusion molecules Figure 2 illustrates the results obtained in the treatment of cancer models highly relevant to oncology. As shown in the figure, Fc-CLEC-1 fusion molecules enhance the antitumor effects of chemotherapy. For example, in a syngeneic mouse model of MC38 colon adenocarcinoma, Fc CLEC-1 treatment, but not control Fc recombinant treatment, enhances the positive effects of cyclophosphamide (CPA) on tumor regression (Figure 2A) and mouse survival (Figure 2B). [Example]

[0174] In vivo toxicity through adverse effect analysis To select the most potent or safest Fc CLEC-1 fusion protein format for oncology applications, prior art and Fc KIH CLEC-1 fusion proteins were evaluated in vivo. Prior art Fc CLEC-1 molecules induced transient signs of toxicity (adverse effects) in animals treated with these molecules (using mouse or human CLEC-1 proteins) by intravenous administration, as monitored by careful scoring of the global mouse status (Figure 3B), whereas the Fc KIH CLEC-1 format did not induce any signs of toxicity in vivo (Figure 3A). Fc-KIH G1(hole)-(G4S)3-CLEC1 and Fc-KIH-G1(knob)-(G4S)3-CLEC1 represent two embodiments of the present invention in which the binding moiety is fused to the C-terminus of the hole or knob polypeptide. Thus, while administration of prior art Fc-CLECs cannot be considered toxic when appropriate doses are administered (adverse effects may occur when the prescribed drug is administered to a patient in need thereof), the heterodimeric Fc-CLEC-1 fusion molecules of the invention cause few adverse effects, which is a clear advantage over prior art compounds that cause adverse effects as illustrated for the Fc-CLEC-1 compound in Figure 3A. [Example]

[0175] Aggregation of Fc-CLEC fusion molecules While analytical SEC profiling of prior art Fc CLEC-1 fusion proteins revealed the presence of large aggregates, analysis of the Fc KIH CLEC-1 fusion proteins of the present invention surprisingly revealed that the molecules form dimers without aggregates. A summary table of the different isotypes fused to proteins evaluated by analytical SEC is shown in Figure 4, highlighting the fact that the Fc KIH CLEC-1 format of the present invention (gray) is the only Fc fusion format that allows the absence of aggregates. This aggregative nature of Fc CLEC-1 was unique to recombinant CLEC-1 proteins, since other targets, such as Fc-Dectin-1R, did not induce the same degree of dimerization / aggregation. Furthermore, and as shown in the figures, several different Fc KIH CLEC-1 according to the invention (Fc-KIH-G1(hole)-(G4S)3-CLEC-1 comprising or consisting of a chain comprising or consisting of SEQ ID NO:5 and a chain comprising or consisting of SEQ ID NO:3; Fc-KIH-G1(knob)-(G4S)3-CLEC-1 comprising or consisting of a chain comprising or consisting of SEQ ID NO:23 (or SEQ ID NO:3 + SEQ ID NO:13 + SEQ ID NO:2) and a chain comprising or consisting of SEQ ID NO:4; and Fc-KIH-G1(hole)-(G4S)3-mutant CLEC-1 comprising a chain comprising or consisting of SEQ ID NO:4 + SEQ ID NO:13 + SEQ ID NO:19 and a chain comprising or consisting of SEQ ID NO:3) are the only ones that do not form aggregates. Indeed, heterodimeric Fc-IL-15 fusion molecules (i.e., in which the binding moiety derived from CLEC-1 is replaced by IL-15 in the fusion molecule) form aggregates (see the bottom row of the table in Figure 4). Thus, the inability to form aggregates is only achieved with the heterodimeric Fc-CLEC-1 fusion molecules of the invention. As mentioned in Figure 4, different classical formulations were tested to prevent aggregation, but any buffer may be efficient in reducing aggregation in Fc CLEC-1. The absence of aggregates of the heterodimeric Fc-CLEC-1 fusion molecules of the invention may underlie the lack of in vivo toxicity observed particularly following treatment with said molecules. As illustrated in Figure 4, dimers of heterodimeric Fc-KIH-CLEC fusion proteins were obtained.As expected by the prior art (see, e.g., Ha JH, 2016, Front. Immunol. 7:394), the authors expected to obtain a monomer of the heterodimeric Fc-based monomeric protein; indeed, when only one monomer is associated with the heterodimeric immunoglobulin, natural homodimeric proteins such as Dectin-1 or IL-15 lead to a monomer of the heterodimeric Fc monomeric protein, as illustrated in Figure 4A: the Fc-Dectin1R and Fab anti-PD1-KIH-Fc-IL15 fusion proteins are almost exclusively monomeric or monomeric. In the constructs of the present invention described in this example, the authors associated a single CLEC-1 domain with the heterodimeric immunoglobulin. Thus, although the CLEC-1 domain is present in only one of the Fc polypeptide chains of the constructs of the present invention, the dimers of the fusion proteins of the present invention are dimers, not monomers. Contrary to other formats of Fc CLEC-1 or other Fc proteins, these dimers did not form large aggregates: Fc CLEC-1, Fc-Dectin1R and Fab anti-PD1-KIH-Fc-IL15 fusion proteins aggregate. The fusion proteins of the present invention form dimers, which is unexpected and does not form large aggregates. [Example]

[0176] Binding of Fc-CLEC-1 fusion molecules according to the invention to CLEC-1L, a known ligand of CLEC-1 Human CLEC-1L is a ligand for human CLEC-1 (unpublished results). To confirm the functionality of several different Fc KIH CLEC-1 fusion proteins according to the present invention, we tested their binding ability to CLEC-1L by ELISA assay (Figure 5) and found that binding of different Fc KIH CLEC-1 to CLEC-1's natural ligand (CLEC-1L) was conserved. It is important to emphasize that this ability was observed regardless of the attachment of the CLEC-binding moiety to the hole or knob chain and regardless of the presence of the complete extracellular domain of CLEC-1 or a mutated version thereof. Next, the pharmacokinetic profiles of the prior art Fc CLEC-1 versus Fc KIH CLEC-1 formats were investigated. The Fc KIH CLEC-1 format was cleared less rapidly in mouse serum following intravenous injection at 10 mg / kg than Fc CLEC-1 (Figure 6). The half-life of the Fc KIH CLEC-1 formats of the invention is at least twice as long as the half-life of Fc CLEC-1. Furthermore, while in vivo concentrations of Fc CLEC-1 decline after 72 hours, the Fc KIH CLEC-1 constructs of the invention remain at high concentrations in the serum of treated mice for a longer time, even 6 days after injection, at the same time point that Fc CLEC-1 is completely eliminated by the aggregated Fc CLEC-1 format. [Example]

[0177] Binding of anti-CLEC-1 antibodies to Fc KIH CLEC-1 constructs To assess the structural conservation of CLEC-1 when fused to an Fc KIH domain, anti-CLEC-1 mouse antibodies were used to target several different immobilized Fc KIH CLEC-1 proteins according to the invention. As illustrated in Figure 7, the anti-CLEC-1 mouse antibodies recognize CLEC-1 regardless of its binding to the hole or knob chain. Furthermore, the anti-CLEC-1 mouse antibodies also recognize mutant versions of CLEC-1, particularly truncated versions of CLEC-1 in which the N-terminal end of the CLEC-1 extracellular domain is deleted. These results demonstrate that CLEC-1 maintains its structural conformation when fused to an Fc KIH, allowing in vivo or in vitro interactions between the Fc KIH CLEC-1 of the invention and its ligands. [Example]

[0178] Absence of inflammatory effects caused by the heterodimeric Fc-CLEC-1 fusion proteins of the invention To assess the undesirable tumor-promoting / pro-inflammatory effects of Fc CLEC-1 molecules in vivo, we investigated the effect of Fc CLEC-1 injection on IL-6 secretion in mouse serum. We found that prior art aggregated Fc CLEC-1 fusion proteins induced IL-6 secretion in serum, whereas the heterodimeric Fc KIH CLEC-1 protein of the present invention did not (Figure 8), thereby confirming that this format is safer for oncology applications. [Example]

[0179] Ability of the heterodimeric Fc-CLEC-1 fusion proteins of the invention to bind to tumor cells expressing a ligand for CLEC-1 To evaluate the ability of the heterodimeric Fc KIH CLEC-1 constructs of the present invention to target tumor cells, we performed a cell binding assay of Fc KIH (control) versus heterodimeric Fc KIH CLEC-1 on MC38 murine colon adenocarcinoma cells (Figure 9), thereby confirming that this format binds to CLEC-1 ligand-positive tumor cells. [Example]

[0180] Binding of Fc-CLEC-1 fusion molecules As illustrated in Figure 10, Fc-CLEC-1 can bind to several different tumor cells (more than 21 tumor cell lines tested) that are treated by conventional methods for cancer. These results demonstrate the ability of Fc-CLEC-1 fusion molecules to bind to different tumor cells, thereby supporting the broad use of the fusion molecules of the invention in the treatment of different types of cancer.

Claims

1. (i) at least one binding moiety comprising or consisting of at least a portion of the extracellular domain of human C-type lectin domain family 1 member A (CLEC-1), wherein said at least a portion of the extracellular domain of human CLEC-1 is a functional equivalent of the extracellular domain of wild-type human CLEC-1; and (ii) a. a first Fc polypeptide chain comprising a first CH3 domain (CH3B chain or whole chain), and b. a second Fc polypeptide chain comprising a second CH3 domain (the CH3A chain or knob chain); wherein the first and second CH3 domains are different, A fusion molecule comprising: one of said at least one binding moiety is fused to the C-terminus or N-terminus of said first Fc polypeptide and / or one of said at least one binding moiety is fused to the C-terminus or N-terminus of said second Fc polypeptide chain; fusion molecule.

2. the CH3 domains of the first Fc polypeptide chain and the second Fc polypeptide are S-S , HA-TF, ZW1, 7.8.90, DD-KK, EW-RVT, SEED and A107, and more particularly, the first Fc polypeptide has the amino acid residues set forth in SEQ ID NO: 3 and the second Fc polypeptide chain has the amino acid residues set forth in SEQ ID NO: 5, or the first Fc polypeptide has the amino acid residues set forth in SEQ ID NO: 10 and the second Fc polypeptide chain has the amino acid residues set forth in SEQ ID NO:

12.

3. 3. The fusion molecule of claim 1 or 2, wherein a single binding moiety is present and the binding moiety is fused to the C-terminus of the first Fc polypeptide chain or the C-terminus of the second Fc polypeptide chain.

4. 4. The fusion molecule of claim 1, wherein a binding moiety is fused to the C-terminus or N-terminus of the first Fc polypeptide chain, in particular to the C-terminus of the first Fc polypeptide chain, and another binding molecule, different from the binding moiety associated with the first Fc polypeptide chain, is fused to the C-terminus or N-terminus of the second Fc polypeptide chain, in particular, when the at least one binding moiety is present at one end of the same Fc polypeptide chain, the binding molecule is at the opposite end of the Fc polypeptide chain.

5. 5. The fusion molecule of claim 1, wherein the at least one binding moiety is fused to the Fc polypeptide chain via a linker peptide, more particularly via a linker peptide of SEQ ID NO:

13.

6. 6. The fusion molecule of claim 1, wherein the first Fc polypeptide chain, or the second Fc polypeptide chain, or both the first and second Fc polypeptide chains comprise a CH2 domain, particularly N-terminal to the CH3 domain, and particularly wherein the CH2 domains are different.

7. 7. The fusion molecule of claim 1, wherein the first Fc polypeptide chain, or the second Fc polypeptide chain, or both the first and second Fc polypeptide chains comprise a hinge region, particularly at the N-terminus of the Fc polypeptide chain, more particularly at the N-terminus of a CH2 domain, if present.

8. the first Fc polypeptide chain comprises, from its N-terminus to its C-terminus: a hinge region, a CH2 domain, and a first CH3 domain (a CH3B chain or a hole chain), respectively; and the second Fc polypeptide chain comprises, from its N-terminus to its C-terminus: a hinge region, a CH2 domain, and a second CH3 domain (a CH3B chain or a hole chain), respectively; 8. The fusion molecule of claim 1, wherein the at least one binding moiety is fused to the C-terminus of the first CH3 domain or the second CH3 domain via a linker.

9. 9. The fusion molecule of claim 1, wherein the at least one binding moiety is fused to the C-terminus of the first Fc polypeptide chain, in particular via a linker peptide of SEQ ID NO:

13.

10. 9. The fusion molecule of claim 1, wherein the at least one binding moiety is fused to the C-terminus of the second Fc polypeptide chain, in particular via a linker peptide of SEQ ID NO:

13.

11. Fusion molecule according to any one of claims 1 to 10, wherein the part of the extracellular domain of human CLEC-1 corresponds to a mutated extracellular domain of CLEC-1, in particular of SEQ ID NO:

2.

12. The fusion molecule of claim 11, wherein the mutant extracellular domain of CLEC-1 has the sequence of amino acid residues shown in SEQ ID NO:

19.

13. 13. The fusion molecule of any one of claims 1 to 12, comprising a first Fc polypeptide chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 4 and a second Fc polypeptide chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:

3.

14. 14. The fusion molecule of any one of claims 1 to 13, comprising a first Fc polypeptide chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 5 and a second Fc polypeptide chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:

3.

15. 14. The fusion molecule of any one of claims 1 to 13, comprising a first Fc polypeptide chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 4 and a second Fc polypeptide chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:

23.

16. (iii) at least one antigen-binding domain comprising or consisting of an antibody variable domain or an antigen-binding fragment thereof, wherein the antigen-binding domain is fused to the first Fc polypeptide chain or the second Fc polypeptide chain, or wherein the first antigen-binding domain is fused to the first Fc polypeptide chain and the second antigen-binding domain is fused to the second Fc polypeptide chain, particularly when the at least one binding moiety is present at one end of the same Fc polypeptide chain, the antigen-binding domain is at the opposite end of the Fc polypeptide chain.

16. The fusion molecule of claim 1, further comprising:

17. The antigen-binding domain may bind to antigens expressed by macrophages, and / or lymphocytes and / or tumor cells, in particular SIRP alpha, SIRP beta, SIRP gamma, CD47, CTLA-4, CD86 (B7.2), CD28, CD40, CD40L, ICOS, ICOS-L, OX40L, GITR, HVEM, BTLA, CD160, LIGHT, TNFRSF25, 2B4, CD48, Tim1, Tim3, Tim4, Gal9, LAG-3, CD40, CD40L, CD70, CD27, VISTA, B7H3, B7H4 (B7x), TIGIT, CD112, HHLA2 (B7-H7), TMIGD2 (CD28H), butyrophilin-like 2 (BTNL2), SIGLEC, 17. The fusion molecule of claim 16, which specifically binds to an antigen selected from the group consisting of AXL, B7.1, B7-DC, B7-H1, B7-H2, B7-H3, B7-H4, CD19, CD20, CD22, CD24, CD137(4-1BB), CD137L(4-1BBL), CEA, CXCR3, CXCR4, EGFR, EGFRvIII, ELTD1, EMR1, EMR2, EMR3, EMR4P, ENG, EPCAM, EPHR, PD-L1, TLR1, TLR10, TLR2, TLR3, TLR4, VEGFR, VEGFR2, VIPR1, VIPR2, CD101, CD3, CD30, CD38, CD39, CD44, DR3, LFA-1, NKG2D, PD-1, and PDL2.

18. 18. The fusion molecule of claim 16 or 17, wherein the antigen-binding domain is fused to the N-terminus of the first Fc polypeptide chain or the second Fc polypeptide chain, or wherein the first antigen-binding domain is fused to the N-terminus of the first Fc polypeptide chain and the second antigen-binding domain is fused to the second Fc polypeptide chain, particularly to the N-terminus of the CH2 domain or, if present, the hinge region.

19. 18. The fusion molecule of claim 16 or 17, wherein there are two antigen-binding domains, each associated with a single Fc polypeptide chain, and wherein the first antigen-binding domain and the second antigen-binding domain bind to two different epitopes, in particular two different antigens.

20. 20. The fusion molecule of any one of claims 1 to 19 for use in the treatment of a patient with a disease selected from cancer, in particular a solid or liquid cancer, or an infectious disease, or an inflammatory disease, including sepsis, an autoimmune disease, or an acute or chronic inflammatory disease.

21. 21. The fusion molecule of any one of claims 1 to 20 for use in the treatment of a patient having cancer and who is undergoing or has undergone treatment with a conventional treatment for cancer, in particular with an agent selected from the group consisting of chemotherapeutic agents, targeted cancer therapies, immunotherapeutic agents or radiotherapeutic agents, more particularly with an agent selected from the group consisting of cytotoxic drugs, angiogenesis inhibitors, anti-cancer agents, cell cycle / control apoptosis regulators, anti-cancer antibodies and hormone regulators, wherein the fusion molecule and the agent are administered simultaneously, separately or sequentially.

22. 22. A nucleic acid molecule or combination of nucleic acid molecules encoding a fusion protein according to any one of claims 1 to 21.

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